key: cord- -ff zadol authors: zhao, rongmao; cui, shujuan; guo, li; wu, chao; gonzalez, richard; paranhos-baccalà, gláucia; vernet, guy; wang, jianwei; hung, tao title: identification of a highly conserved h subtype-specific epitope with diagnostic potential in the hemagglutinin protein of influenza a virus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ff zadol subtype specificity of influenza a virus (iav) is determined by its two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na). for ha, distinct subtypes (h –h ) exist, while nine exist for na. the epidemic strains of h n iav change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious influenza pandemic. the recent introduction of pandemic a/h n iav (h n pdm virus) into humans re-emphasizes the public health concern about h n iav. several studies have identified conserved epitopes within specific ha subtypes that can be used for diagnostics. however, immune specific epitopes in h n iav have not been completely assessed. in this study, linear epitopes on the h n pdm viral ha protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from h n pdm patients. one epitope, p (aa – ) was found to be immunodominant in patients and to evoke high titer antibodies in mice. multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza h ha [with a coverage of . % ( , / , )] and almost completely absent in other subtypes [with a coverage of . % ( / , )]. this previously unidentified linear epitope is located outside the five well-recognized antigenic sites in ha. a peptide elisa method based on this epitope was developed and showed high correlation (χ( ) = . , p< . , pearson correlation coefficient r = . ) with a hemagglutination inhibition test. the highly conserved h subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against h n iavs. influenza a viruses (iavs), members of the orthomyxoviridae family, are highly contagious to a variety of avian and mammalian species. iavs cause seasonal influenza epidemics annually and recurring pandemics with severe consequences for public health and global economy [ , ] . at least three iav-pandemics emerged in the last century ( a/h n , a/h n , and a/h n ). the spanish flu was the most serious influenza pandemic that killed over million people worldwide [ ] . the latter two pandemics, although mild compared to the incidence, resulted in significant mortality, with close to million and million deaths, respectively [ ] . the latest pandemic influenza, and newest global health challenge, occurred in due to the emergence of an a/ h n pandemic iav (h n pdm virus). the h n pdm virus has been detected in more than countries and territories and has caused , deaths as of july , [ ] . the viral genome of iav consists of eight single-stranded negative sense rna segments that encode at least viral proteins, including two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) [ ] . based on the antigenic properties of ha and na, iavs have been classified into ha subtypes and na subtypes [ ] . all ha subtypes have been identified in avian species, while only ha subtypes (h , h , h , h , h and h ) are known to infect human beings [ , , ] . h , h and h subtypes have caused pandemics, while h and h also dominate seasonal epidemics together with influenza b virus. ha, encoded by segment of the iav genome, is a glycoprotein of approximate amino acid. the biologically active ha is a homologous trimeric molecule that is attached to the virion membrane through its carboxy terminus [ ] . ha plays a critical role in the pathogenesis of iavs. ha mediates iavs' binding to the cellular receptor n-acetylneuraminic (sialic) acid as well as the subsequent membrane fusion process [ ] . ha also stimulates host protective immunities, specifically the production of neutralizing antibodies. the generation of anti-ha neutralizing antibodies has been the major target for influenza vaccine development [ , ] . due to its specificity in immune response, ha is also an important target for iav subtyping using immunoassays [ , ] . active serological surveillance for viral antibodies is of great importance for influenza control and prevention. several iav subtype-specific serological tests have been developed. at present, subtyping of iav mainly relies on a hemagglutination inhibition (hi) test using ha and na subtype-specific reference sera [ ] . however, there are a number of drawbacks to hi testing. this assay is ) relatively laborious; ) low in sensitivity; ) requires preparation of antigen from viable viruses which are potentially hazardous and ) contains low signal to noise ratio, e.g. the assay exhibits inter-variability and subtype cross-reactivity [ , ] . moreover, the hi test can be confounded by steric hindrance from na antibodies, leading to nonspecific inhibition and misidentification [ ] . microneutralizing test is an alternative method to type and subtype influenza viruses. however, due to the needs of cell culture process, this method is labor-intensive and requires biological safety containments (particularly for high pathogenic strains). as such, it is not suitable for large scale investigations [ , ] . recently, subtyping of iav antibodies using different categories of elisa assays have also been reported [ , , ] . however, present elisa assays mainly rely on an ha antigen, which can lead to nonspecific detection to some extent due to the possible cross-reaction of different subtypes [ , ] . virus-derived epitopes are useful tools to accurately evaluate immune response and to differentiate which responses are specific or due to cross-reactivity [ , , ] . several studies have reported the existence of ha subtype-specific as well as inter subtypeconserved epitopes [ , , ] . elisa assays based on epitopes that are highly conserved and specific for one certain ha subtype will be useful for rapid and simple subtyping of iavs. such epitopes in iavs have not been fully addressed although many studies have been performed. in the present study, we report the successful identification of a new epitope, which is highly conserved among the majority of iav strains of h subtype. moreover, we developed an elisa assay for h antibody subtyping based on this epitope. results derived from this new assay correlate with results obtained through the use of hi test. to identify the immunodominant epitopes in the ha protein, a peptide scanning assay was performed. a set of peptides with five residues overlapping with the adjacent peptides spanning the ectodomain sequences of the ha protein of the h n pdm virus strain a/california/ / were synthesized. the binding between these peptides and the convalescent serum samples from h n pdm patients were examined by elisa using these peptides as coating antigens. five of these peptides (p , p , p , p and p ) were found to react well with the sera tested. these peptides corresponded to the sequences of amino acid (aa) residues - , - , - , - , and - in the ha molecule, respectively ( fig. a and table ). among them, the p peptide reacted with . % ( / ) of the sera, the p and p peptides reacted with . % ( / ) of the sera, while the p and p peptides reacted with % ( / ) of the sera. these data indicated that these peptides may contain h n pdm virus b cell epitopes. to visualize the location of the peptides on the ha protein, we mapped the peptides on the crystal model of this protein (fig. b) . the various colors in figure b represent the different peptides. although p (residues - , indicated by blue) and p (residues - , indicated by red) are parts of ha in primary sequence, they are located in the middle of helix a and b in the trimeric structure and are partially surface exposed. p (residues - , indicated by magenta) seems to be a dispatch that links the stem region and the globular region and is fully surface exposed (fig. b) . p and p (residues - and - , indicated by orange) are located in the receptor binding domain [ ] . to confirm the immunogenicity of these peptides in vivo, we analyzed sera derived from peptide-immunized mice. the five positive peptides and two control peptides (p and p ) were coupled with keyhole limpet hemocyanin (klh) and were used to immunize balb/c mice ( table ). the antisera were collected five days after the third immunization and titrated by elisa using corresponding peptide as a coating antigen. our results showed that all of the peptide conjugates except p induced potent antibody titers. the endpoint titers of antisera in elisa from mice immunized with p , p , p , p , p , and p peptides were : , , : , , : , , : , , : , , and : , , respectively ( fig. a) . these data indicate that most of the positive peptides elicite humoral immunity and are highly immunogenic in mice. to confirm that these antibodies can recognize the ha antigen, the reactivity of the anti-peptide sera were evaluated by western blot and elisa against the purified ha protein of h n pdm virus. our data demonstrate that sera against p , p , and p but not those against p and p (controls) react to the ha protein ( fig. b and c ). the anti-p sera did not react to the ha protein, although it exhibited a high elisa reactivity to the ha protein ( fig. b and c ). taken together, our results demonstrate that p , p , and p peptides contain dominant epitopes of h n pdm virus. we then characterized these three peptides in the following studies. to determine if the epitopes identified in this study can stimulate neutralizing antibodies, a ha-pseudotype neutralization test was performed against the anti-peptide sera using the h n pdm pseudotyped lentivirus. none of the sera against p , p , p , and p could efficiently inhibit ( % inhibition [ ] ) the entry of h n pdm ha pseudotypes ( figure d ), indicating that these epitopes do not contain neutralizing activity. western blot analysis was used to determine the specificity of the epitopes present in the peptides p , p , and p . the h -h recombinant ha proteins were obtained by transient expressions of corresponding genes by the pcaggs vector in t cells. the lysates of these cells were used to examine the specificity of antibodies elicited by peptide-conjugates. as shown in fig. a , the anti-p serum reacted with h (including h and h viruses), h , h , and h ha proteins, while anti-p and anti-p sera only reacted with the h ha proteins. these findings indicated that p and p may contain h -subtype specific epitopes. to evaluate the subtype-specificity of epitopes in p and p further, additional ha proteins of three epidemic human strains from different years ( , and ) as well as a swine strain were expressed by pcaggs vector in t cells. the reactivity of anti-p and p sera with the cell lysates was determined by western blot analysis. our results showed that the anti-p serum strongly reacted with all of the six h ha proteins in a manner similar to an antibody against the ha of h n pdm virus (fig. b ). we found that the anti-p sera reactivity was weak against ha proteins from the and virus strains (fig. b ). these data indicated that the epitopes in p and p peptides are relatively conserved among h -subtype iavs though these viruses have circulated in the world for almost a century. to test if anti-p sera cross-react with influenza type b virus, the reactivity of anti-p sera with two representative influenza type b virus strains (b/hubeiwujiagang/ / ,yamagata lineage and b/heilongjianghulan/ / , victoria lineage) and an influenza type a virus strain (a/h n /pr / ) was examined by western blot analysis. the results showed that anti-p serum reacted well with a/h n /pr / virus but not with influenza type b virus strains (fig. c) , further confirming the specificity of the epitope in p . to determine the conservation of the identified epitopes among iavs, the aa sequences of p , p , and p were aligned with the corresponding aa sequences of all the subtype has available in the genbank. fig. is a representative of the alignment analysis, showing that p is identical to ha of h subtype strains. p is identical to ha of the h subtype, as well as highly identical to ha of the h , h , and h subtypes. these data are consistent with the specificity analysis by western blot (fig. a) . although anti-p antibody only recognizes the h -subtype ha, it is similar to multiple subtypes. to assess the identity levels of p and p sequences among the known iav strains, in silico coverage analysis was performed. this analysis showed that the p peptide sequence could be identified in . % ( / ) of the h -subtype ha sequences available in the influenza research database (http://www.fludb.org) ( table ) . notably, this sequence scarcely presented ( . % / ) among the has of h -h subtype iavs. however, despite a high identity in the h ha proteins ( . %), the peptide sequence of p also presented among the has of h -h viruses ( . %). taken together, these findings indicate that the p peptide is h -subtype specific and is conserved among h virus strains. to define the epitope contained in p precisely, a peptideinhibition elisa was performed. this experiment is reliable and is a standard methodology to determine the fine specificity of antigen-antibody reactions [ , ] . a panel of short peptides derived from p (n -n , with c-terminus truncation; and c -c , with n-terminus truncation) were used to block the binding of anti-p antibody to coated p . as shown in fig. , antibody induced by the p -klh conjugate was inhibited by peptide n -n and the parental peptide p to similar extents, whereas peptides n -n only showed inefficient inhibition even at high molar concentrations. a similar pattern of inhibition was observed with the c-terminal conservative derivatives. peptides c , c , c , and the parent peptide p demonstrated comparable and efficient inhibition, whereas only slight inhibition was observed in peptides c -c (fig. b) . since the amino acid sequence lrgvapl overlapped both peptides n and c (fig. ) , we speculate that this sequence met the minimum requirements of binding to the anti-p antibody. however, the synthetic peptide lrgvapl did not block the binding between p peptide and its antibody, nor did it directly bind to the p antibody (fig. ). as p ( , , , , ) and a swine h n strain. t cells transfected with an empty vector was used as a control (ctrl). b-actin was used as a loading control. for the backgrounds of various subtype iav strains, see inspired by the high specificity of p among the h -subtype viruses, we developed an indirect elisa assay using the p peptide to evaluate its performance as a diagnostic tool for h antibodies. the hi test was used as a reference method. as shown in table , the overall agreement of these two methods was %, showing that the two methods have good correlation (pearson correlation coefficient r = . ). the sensitivity and specificity of peptide-elisa versus hi test was . % and . %, respectively, indicating the potential of the peptide-elisa method in detecting antibody against h -subtype iavs. in the present study, we identified immunodominant linear b cell epitopes on the h n pdm virus ha protein by a peptide scanning approach using h n pdm patients sera. we confirmed that an unidentified epitope was highly conserved among h subtypes viruses and showed a good correlation with results obtained using the hi test. these findings demonstrate the potential of epitope-based antibody detection in iav diagnosis and surveillance. iav escapes the human immune system by continuous antigenic drifts and occasional antigenic shifts [ ] . attempts to develop universal vaccines and reliable diagnostic tools based on conserved epitopes of iav are big challenges. several epitopes that can elicit broad spectrum neutralizing antibodies have been identified recently. for example, sui et al. identified a universal neutralizing epitope for group ha [ ] . yoshida et al. reported a universal epitope in antigenic site b shared by h , h , h , h , h , and h subtypes [ ] . all these epitopes are conformationdependent. in this study, we identified two epitopes (p and p ) which have not been identified previously (fig. s ). the p (aa - ) seems to be a dispatch that links the stem region and the globular region and is fully exposed on the surface, while p (aa - d) is located in the middle of helix a and b on ha . in contrast to previous studies, we found p to be a linear b cell epitope. our data demonstrate that this epitope is highly conserved among h viruses ( / , . %). because viral mutants that are resistant to conformational epitopes are more easily generated, the conserved linear epitope is more suitable for differentiating subtypes than conformational epitopes [ ] . hence, the epitope in p provides a new target for reliable diagnostics of h -subtype iavs. antigenic sites in iav ha proteins of h , h , and h subtypes had previously been characterized by sequence analysis on antigenic variants and amino acid substitutions. these previously identified antigenic sites were mainly located in the globular head in the three-dimensional structure of the ha subunit of the ha molecule [ , , , ] . for instance, five antigenic sites have been identified in ha of influenza virus a/pr/ / , a well-known reference strain of h n iav [ ] . recently, several epitopes were identified in the ha unit [ , , ] . together with these reports, our results indicate that there are more epitopes than what we have imaged and the epitopes of iav need to be further characterized. the difference between our findings and previously identified epitopes can be explained by the difference of screening method used between our study and those of others. in previous studies, monoclonal antibodies from murine hybridoma cells were used to identify antigenic sites, while in this study we used a peptide scanning approach, which involves overlapping peptide library and human convalescent antisera-a strategy that is widely used for viral epitope identification [ , ] . given the fact that viral antigen can be recycled and presented as short peptides with different conformation during humoral immune response and these short peptides can be selected by b cell clones [ ] , and that convalescent sera from patients were much more complex than monoclonal antibodies from mice and can reflect the real immune responses during viral infection [ ] , our approach adds to the available techniques currently being used to identify linear epitopes in serologic tests. because ha pseudotyped lentivirus has been widely applied in the study on neutralizing antibodies against iavs [ ] , we used this method to evaluate if the epitopes identified in this study could stimulate neutralizing antibodies. our data showed that these epitopes could not elicit neutralizing antibodies in pseudovirion neutralizing assays due to their linear nature. previous studies have shown that most neutralizing epitopes are conformation dependent [ , ] . the length of b cell epitopes can vary from to amino acids [ , ] . to map the epitope contained in p , we performed a peptide-inhibition elisa using a series of n-terminal and cterminal truncated peptides. however, we found that the fulllength p ( aa in length) rather than truncated peptides showed strongest binding to the corresponding antibody ( fig. and fig. ). as peptides n and c are the shortest truncated p that can bind to anti-p antibody and share a core sequence of lrgvapl, we tested whether this sequence could be the epitope. however, the synthetic peptide lrgvapl did not block p -antibody interactions nor bind p antibody (fig. ) . thus, we speculate that adjacent amino acids to this sequence are also involved in the binding of antibody elicited by p -klh conjugates. the concept of using linear epitopes in influenza virus diagnostics and control has not been extensively investigated. in a recent study, an epitope-blocking elisa, which can universally detect antibodies to human h n virus, has been developed [ ] . our results show that a peptide-elisa based on the highly conserved h subtype-specific epitope can also be used for the detection of h antibodies, displaying good correlativity with the hi test. our results indicate the potential of the p epitope in h subtype iav diagnosis. however, the performance of this assay needs to be further evaluated in studies with large scale samples. in conclusion, our data provide evidence that the h subtype ha harbors more epitopes than what has been found previously. the conservation of an epitope (p , aa - ) in the h -subtype ha of iav and its near complete absence in other subtypes indicate that this epitope meets the critical requirements for diagnosis of h subtype influenza virus infections. the peptide-elisa developed in our study may be applicable for serodiagnosis and may serve as a universal diagnostic tool for h subtype iav surveillance. to screen the h -subtype specific epitopes, a set of peptides spanning the amino acid sequences of the ha protein ectodomain of pandemic a/h n (h n pdm) influenza virus strain a/ california/ / were synthesized. each peptide is amino acids in length with five residues overlapping with the adjacent peptides [ ] (fig. ) . the peptides selected for immunization experiments are shown in table . these peptides were conjugated with a carrier protein, the keyhole limpet hemocyanin (klh; sigma, st. louis, mo), to improve their immunogenicity [ ] . as the water solubility of peptides p , p , p and p were too low to conjugate with klh directly, these peptides were first linked to -aminocaproic acid and then to the tripeptide kkc prior to being conjugated with klh [ ] . in addition, a family of short peptide homologs to the p peptide was also synthesized to fine map the epitope contained in the p peptide (fig. ) . all of the peptides and their conjugates used in this study were synthesized by sangon (shanghai) biotechnol co., ltd. (shanghai, china). each peptide was purified to homogeneity (. % purity) by highperformance liquid chromatography and verified by mass spectrometry. the reference influenza virus strains a/pr / (h n ) (abbreviated pr ), b/hubeiwujiagang/ / (yamagata lineage, abbreviated by) and b/heilongjianghulan/ / (vic-toria lineage, abbreviated bv) were kindly provided by the beijing center for disease control and prevention. the viruses were grown in mdck cells as described elsewhere [ ] . the titers of virus strains were determined by hemagglutination tests and expressed as hemagglutinating units (hau). for western blot analysis, the inactivated viruses were lyzed in a lysis buffer ( mm tris-hcl, mm nacl, mm edta, % triton-x, ph . ) supplemented with a protease inhibitor cocktail (roche, indianapolis, in). serum samples were collected from convalescent patients during the early h n pandemic in beijing. the diagnostic criteria for h n influenza virus infection of these patients fully followed the who's descriptions. sera from healthy blood donors were used as negative controls. in addition, serum samples collected from blood donors were recruited to evaluate the peptide-elisa assay developed in this study. all these samples were kindly provided by the beijing municipal center for disease control and prevention (beijing, china) and written informed consent was obtained from all participants. all samples were coded prior to analysis to ensure anonymity and the procedures were approved by the institutional medical ethic review board of the institute of pathogen biology, chinese academy of medical sciences (beijing, china). the full-length cdna fragments corresponding to h -h ha subtypes of iav were inserted into the pcaggs vector (purchased from addgene) to express entire ha proteins (unpublished data). for h proteins, ha gene representing human iav strains from different years ( , , , and ) and a swine influenza virus strain were expressed by inserting the corresponding cdna fragments into the pcaggs vector in a similar manner. for the details of these influenza virus strains, please refer to fig. . recombinant plasmids were transfected into t cells (atcc number crl- ) using lipofectamine (invitrogen, carlsbad, ca) according to the manufacturer's instructions. the cells were harvested and lyzed hours after transfection. the expression of ha proteins was verified by western blot analysis using murine antibodies against corresponding ha proteins (unpublished data). the reactivities of the synthetic ha peptides or purified ha protein (eenzyme llc, montgomery village, md) with the convalescent-phase sera from h n pdm patients and the serum samples from mice immunized with peptide conjugates were determined by elisa. briefly, each peptide ( mg/well) or protein ( . mg/well) was used to coat -well microtiter plates (corning costar, acton, ma) in . m carbonate buffer (ph . ) at uc overnight. after blocking with % bovine serum albumen (bsa), the plates were incubated with indicated diluted serum samples (human or mouse) at uc for hr, then washed four times with pbs containing . % tween (pbs-t). bound igg antibodies were detected with horseradish peroxidase (hrp)-conjugated goat anti-human igg or anti-mouse igg (sigma) at uc for hr. after four washes with pbs-t, the reaction was visualized by addition of the substrate , , , -tetramethylbenzidine (tmb, sigma). color development was stopped by the addition of ml/well of m sulphuric acid after min. the optical density at nm (od nm ) was measured by an elisa plate reader (molecular devices, sunnyvale, california). to evaluate the reactivity of the p -derived short peptides with the anti-p antibody, peptide-inhibition elisa assays were performed by adding dilutions of the peptides to a constant amount of the antibody elicited by the p -klh conjugates ( : dilution). maximum binding to antigen-coated wells was observed in the absence of a peptide inhibitor. the antibody bound was expressed as a percentage of the maximum level of binding. female balb/c mice of - weeks old were immunized subcutaneously with various peptide-klh conjugates mixed with freund's complete adjuvant (sigma) at mg per injection. boost injections were given at -week intervals with mg antigen in freund's incomplete adjuvant (sigma) [ ] . the antibodies were collected five days after the third boost. all the animal experiments were carried out in the facilities of the institute of laboratory animal sciences (ilas), chinese academy of medical sciences (cams). all the experimental procedures were approved (permit number slxkj - ) and supervised by the animal protection and usage committee of ilas, cams. at hr post-transfection, the cells transfected with haexpressing plasmids were harvested, pelleted, and lyzed in a lysis buffer ( mm tris-hcl, mm nacl, mm edta, % triton-x, ph . ) supplemented with a protease inhibitor cocktail (roche, indianapolis, in). aliquots of cell lysates ( mg) or virus lysates were blotted after % sds-page onto nitrocellulose membranes (pall, port washington, ny). the membranes were blocked with % non-fat milk and then incubated with the primary antibodies indicated in the figures at uc overnight. this was followed by incubation with the goat anti-mouse irdyeh fluor -labeled igg secondary antibody ( : , ) (li-cor, lincoln, ne). after washing, the membranes were scanned by the odysseyh infrared imaging system (li-cor) and analyzed with odyssey software. the molecular sizes of the developed proteins were determined by comparison with the pre-stained protein markers (fermentas, maryland, ca). hi test was carried out as described elsewhere [ ] . rde treated serum samples were inactivated at uc for min and two-fold serially diluted at an initial dilution of : . twenty five ml of the diluted serum were incubated with ml of the four hemagglutination units from reference influenza strains for min at room temperature. the reference h n iav strains for hi test were a/ tianjinjinnan/ / (h n ) and a/california/ / (h n ), respectively. ml of % chicken erythrocyte suspension was added to each well and incubated for min at uc. positive reactions were recorded when the hi antibody titer was equal to or greater than . h n pdm virus pseudotyped lentiviruses were produced in t cells co-transfected with pnl . -r e , ha and na constructs using a polyethylenimine (pei)-based transfection protocol [ ] . cell culture supernatants were collected hr post-transfection, filtered through a . mm-pore size filter (millipore, billerica, ma ) and used in pseudotype neutralization test. serum samples, heat inactivated at uc for min, were diluted -fold in culture medium and mixed with an equal volume of diluted h n pdm influenza pseudovirions. after incubation at uc for hr, ml of pseudovirions (containing ng/ml of hiv p gag protein) and serum mixtures were added into -well plates that contained t cells grown for hr at initial cells. infectivity was evaluated at hr post-infection by luciferase assay. the percentage of infectivity of pseudovirions treated by tested serum to that of negative serum (as control) was calculated. % reduction in infectivity by serum addition is considered to be neutralizing activity [ ] . tests were run at least as duplicates. to assess the identity of the ha epitopes in iavs, in silico analysis was performed by utilizing bioinformatics tools at the influenza research database (http://www.fludb.org) [ ] . the two programs used in this study were search for protein sequences and identify short peptides in flu proteins. the former program was used to define the number of total sequences in ha proteins according to the subtype parameter (h or h -h ). the latter program defined the number of hits (p or p ) in the h or h -h total sequences. because there are no standards for evaluating short peptide sequence homology, we chose the fuzzy match analysis to represent the identical level of a peptide sequence to ha proteins. the analysis type was chosen as fuzzy match, which meant . % of characters were identical to the searched aa string. for example, entering gilgfvftl may also find ailgfvfti but not aligfvfsi. the pearson correlation coefficient was calculated by pearson chi square test for crosstab tables using spss software. the sensitivity and specificity of the peptide-elisa versus hi test was determined by roc curve analysis using spss software. figure s localization comparison between the identified peptides and the classical five antigenic sites in stereo view. the ha monomer surface view of influenza virus a/pr/ / (pdb id: ru ) is shown and colored to illustrate the five antigenic sites (sa, sb, ca , ca , and cb) and the identified peptides. from most membrane distal to proximal: p (blue), p (red), p (black), cb (green), ca (magenta), ca (rainbow), sa (yellow), and sb (cyan). (tif) influenza: lessons from past pandemics, warnings from current incidents influenza: old and new threats the origins of pandemic influenza-lessons from the virus the influenza a (h n ) pandemic: what have we learned in the past months fields virology: orthomyxoviridae. th edn characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gulls continuing evolution of h n influenza viruses in southeastern china avian influenza a virus (h n ) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome avian influenza a (h n ) receptor binding and membrane fusion in virus entry: the influenza hemagglutinin the structure and function of the hemagglutinin membrane glycoprotein of influenza virus influenza vaccines for the future characterization of a novel influenza hemagglutinin, h : criteria for determination of influenza a subtypes manual of diagnostic tests and vaccines for terrestrial animals development of blocking elisa for detection of antibodies against avian influenza virus of the h subtype development of epitope-blocking elisa for universal detection of antibodies to human h n influenza viruses a laboratory manual for the isolation and identification of avian pathogens comparison of the hemagglutination-inhibiting and neutralizing antibody responses of volunteers given chick cellagglutinating units of influenza a/new jersey/ split-virus vaccine role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics development of rha -elisa for specific and sensitive detection of h subtype influenza virus prokaryotic expression and purification of ha and ha polypeptides for serological analysis of the pandemic h n influenza virus utility of pandemic h n influenza virus recombinant hemagglutinin protein-based enzymelinked immunosorbent assay for serosurveillance ab and t cell epitopes of influenza a virus, knowledge and opportunities epitope analysis for influenza vaccine design quantifying influenza vaccine efficacy and antigenic distance antigenic characterization of recombinant hemagglutinin proteins derived from different avian influenza virus subtypes epitope mapping of the hemagglutinin molecule of a highly pathogenic h n influenza virus by using monoclonal antibodies evaluation of the subtype specificity of monoclonal antibodies raised against h and h subtypes of human influenza a virus hemagglutinins establishment of retroviral pseudotypes with influenza hemagglutinins from h , h , and h subtypes for sensitive and specific detection of neutralizing antibodies minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin one step closer to universal influenza epitopes structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses crossprotective potential of a novel monoclonal antibody directed against antigenic site b of the hemagglutinin of influenza a viruses antigenic structure of the haemagglutinin of human influenza a/h n virus structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution structural identification of the antibodybinding sites of hong kong influenza haemagglutinin and their involvement in antigenic variation the antigenic structure of the influenza virus a/pr/ / hemagglutinin (h subtype) a simple slot blot for the detection of virtually all subtypes of the influenza a viral hemagglutinins using universal antibodies targeting the fusion peptide monoclonal antibodies against the fusion peptide of hemagglutinin protect mice from lethal influenza a virus h n infection identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus the who, how and where of antigen presentation to b cells screening of random peptide library of hemagglutinin from pandemic a(h n ) influenza virus reveals unexpected antigenically important regions predicting flexible length linear b-cell epitopes b cell epitope mapping using synthetic peptides approaches to augmenting the immunogenicity of melanoma gangliosides: from whole melanoma cells to ganglioside-klh conjugate vaccines universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza a viral hemagglutinins production of antipeptide antisera analysis of hemagglutinin-mediated entry tropism of h n avian influenza biohealthbase: informatics support in the elucidation of influenza virus host pathogen interactions and virulence understanding sensitivity and specificity with the right side of the brain we thank dr. fang huang for her assistance in serum sample collection. key: cord- -lwirkob authors: yan, liping; hu, jianhua; lei, jing; shi, zhiyu; xiao, qian; bi, zhenwei; yao, lu; li, yuan; chen, yuqing; fang, an; li, hui; song, suquan; liao, min; zhou, jiyong title: novel protein chip for the detection of antibodies against infectious bronchitis virus date: - - journal: bmc vet res doi: . /s - - -x sha: doc_id: cord_uid: lwirkob background: infectious bronchitis (ib) caused by the ib virus (ibv) can cause acute damage to chickens around the world. therefore, rapid diagnosis and immune status determination are critical for controlling ibv outbreaks. enzyme-linked immunosorbent assays (elisas) have been widely used in the detection of ibv antibodies in the early infection and continuous infection of ib because they are more sensitive and quicker than other diagnostic methods. results: we have developed two indirect microarray methods to detect antibodies against ibv: a chemiluminescent immunoassay test (cit) and a rapid diagnostic test (rdt). ibv nonstructural protein (nsp ) was expressed, purified from escherichia coli, and used to spot the initiator integrated poly(dimethylsiloxane), which can provide a near “zero” background for serological assays. compared with the idexx ibv ab test kit, cit and rdt have a sensitivity and specificity of at least . % and . %, respectively. no cross-reaction was detected with antibodies against avian influenza virus subtypes (h , h , and h ), newcastle disease virus, marek’s disease virus, infectious bursal disease virus, and chicken anemia virus. the coefficients of variation of the reproducibility of the intra- and inter-assays for cit ranged from . to . %. the reproducibility of rdt was consistent with the original results. the application of the ibv nsp protein microarray showed that the positive rate of the cit was . %, that of the nsp elisa was . %, and that of the rdt was . %. furthermore, the rdt, which was visible to the naked eye, could be completed within min. our results indicated that compared with nsp elisa, the cit was more sensitive, and the rdt had similar positive rates but was faster. furthermore, the two proposed methods were specific and stable. conclusions: two microarray assays, which were rapid, specific, sensitive, and relatively simple, were developed for the detection of an antibody against ibv. these methods can be of great value for the surveillance of pathogens and monitoring the efficiency of vaccination. infectious bronchitis (ib) is an acute, highly contagious, and economically important respiratory disease in chickens; it is caused by the ib virus (ibv), which is a significant respiratory pathogen that causes considerable economic losses in the commercial poultry industry worldwide [ ] . the ibv genome is a single-stranded, positive-sense rna that is . kb in size [ ] . it encodes four major structural proteins, namely, glycosylated spike protein (s), membrane protein (m), phosphorylated nucleoprotein (n), and envelope protein (e) [ ] , and nonstructural proteins (nsp -nsp ). generally, nonstructural proteins are present in infected cells but not in the virus, and they only play a role in the process of virus infection and replication [ ] . chickens immunized with an inactivated vaccine will produce no antibodies or low levels of antibodies against viral nonstructural proteins. thus, nonstructural proteins have the potential application in differentiating natural infection from inactivated vaccine immunity [ ] . ib diagnosis is complicated due to the continual emergence of new serotypes [ ] and the difficulty in differentiating ib from other upper respiratory diseases [ ] . virus isolation is regarded as the gold standard for the diagnosis of ibv infection, but it is time-consuming and costly [ ] . the agar gel precipitation test is used in ibv antibody detection; however, this method has low sensitivity. hemagglutination inhibition (hi) assays are suitable for the rapid diagnosis of ib, which requires a series of methods to treat the antigen; however, the hi titer is not related to protection. the virus neutralization test correlates with protection and has the highest specificity among ib diagnostic methods, but it is tedious and laborious [ ] . compared with these methods, enzyme-linked immunosorbent assay (elisa) has been widely used for testing ibv early infection and continuous infection, and this technique can be used for both antigenic and antibody detection. the immunogenicity of the coating antigen is one of the crucial factors when performing an elisa test for antibody detection. an inactivated whole virus is the most commonly used coating antigen in commercial diagnosis kits for ibv diagnosis. recombinant antigenic protein expressed using prokaryotic, yeast, or baculovirus systems has been widely used in preparing specific coating antigens for elisa kits [ ] [ ] [ ] [ ] . elisas based on purified recombinant protein may have higher specificity and sensitivity as the target antigen is immune-dominant and devoid of any nonspecific immune responses [ ] . elisas based on whole virus particles as well as recombinant s (spike protein subunit) and n proteins (nucleoproteins) can provide a rapid and large-scale detection method for ibv infection. however, few ibv detection methods have been developed based on nonstructural proteins (nsps). our laboratory has established an nsp elisa to detect ibv infection [ ] . the nsp antibodies detected are likely to be non-neutralizing and exist in lower numbers than the ones generated by other proteins. based on previous studies, we developed a rapid, highly sensitive protein microarray and a visible detection method to detect ibv nsp antibodies for epidemiological investigation and antibody level monitoring. initiator integrated poly(dimethylsiloxane) (ipdms) membrane ( in this study, clinical serum samples were collected from a chicken farm. forty-two negative sera were obtained from different ages of specific-pathogen-free (spf) chickens raised in spf isolators in zhejiang university. three-month-old spf chickens, which were purchased from shennong company (zhejiang, china) and reared in spf isolators, were used to prepare negative serum and positive serum. we prepared standard positive serum samples from chickens infected with h , h , and h avian influenza virus (aiv); newcastle disease virus (ndv); ibv; infectious bursal disease virus (ibdv); and chicken anemia virus (cav). a microarray was prepared in a , -grade clean room. proteins were first dissolved with % acetonitrile solution (v/v, in milli-q water) to mg/ml stock solution and then diluted into the optimized concentration ( μg/ml) with printing buffer ( . m phosphate buffer, . % glycerin, . % triton x- , and . % mannitol) for further printing. ipdms membranes were first activated with . m edc and . m nhs mixtures for min, rinsed with milli-q water, and immediately used for printing. to determine the optimal antigen concentration, the protein was diluted with . m phosphate buffer to different concentrations. each dilution of protein was printed on ipdms using a protein microarray (scienion, germany). once the antigen concentration was determined, the optimized concentration of nsp was achieved by dilution with printing buffer and printed on ipdms for subsequent experiments in triplicate. the protein microarray was prepared using the smartarrayer contact printer (capitalbio, china) with approximately . nl of printing solution for each sample. each subarray had a positive control with chicken-igy at a concentration of . mg/ml and negative control with printing buffer. the procedure for the cit is shown in fig. . serum samples were first diluted with serum-dilution buffer ( % bovine serum albumin, % casein, . % sucrose, . % polyvinylpyrrolidone, . % tween in . m phosphate-buffered saline, ph = . ). in total, μl of the diluted serum samples was then added into each protein microarray and incubated for min on a shaker (thermo fischer, usa) at rpm and °c. microarrays incubated with serum-dilution buffer were used as negative controls. each microarray was then rinsed thrice with washing buffer and incubated with μl of mg/ml hrp-igg diluted : , in peroxidase conjugate stabilizer/diluent for another min on the shaker ( rpm, °c), followed by the same washing steps described above. a total of μl of the chemiluminescent substrate was added to the microarray, and images were taken at a wavelength of nm with the amersham imager (ge, usa). chemiluminescent signals were acquired using genepix pro . software, and the signal-to-noise ratio (snr) was calculated. the purified recombinant nsp was printed on the ipdms membrane to form a microarray with a concentration of . , . , . , and . mg/ml. subsequently, the serum samples were added to microplates at the following dilutions: : , : , : , : , : , : , : , and : . to identify the optimal time of exposure, the images were taken at an exposure time of s, min, min, min, and min. to determine the cit threshold, a total of serum samples, including positive samples and negative samples, identified by the idexx ibv ab test kit were tested according to the optimal working conditions. results were then compared with those obtained using the idexx ibv ab test kit. finally, receiver operating characteristic (roc) curve analysis was conducted to determine the accuracy of the ibv protein microarray test. the specificity of the cit was evaluated by detecting the positive sera against aiv (h , h , and h ), ndv, mdv, ibdv, and cav. the evaluation of the cit reproducibility within and between runs was carried out as described by jacobson [ ] . thirteen field serum samples (nine idexx positive samples and four idexx negative samples) were selected for the reproducibility experiments. for intra-assay reproducibility, three replicates of each serum sample were analyzed within the same plate. for inter-assay reproducibility, three replicates of each sample were run in different plates. the mean snr, standard deviation (sd), and coefficient of variation (cv) were then calculated. the procedure of the rdt is also shown in fig. . serum was first diluted : with serum-dilution buffer, and μl of the diluted serum sample was added into each protein microarray and incubated for min on a shaker ( rpm, °c). the microarray incubated with serum-dilution buffer was used as a negative control. the microarray was then rinsed thrice with washing buffer and incubated with μl of mg/ml hrp-igg diluted : in peroxidase conjugate stabilizer/diluent for another min on a shaker ( rpm, °c), followed by the same washing steps described above. a total of μl of tmb was added to the microarray and incubated for min in the dark; then, the results were observed. to confirm the concentration of the nsp protein in the rdt, the purified recombinant nsp was printed on an ipdms membrane to form a microarray with concentrations of . , . , . , and . mg/ml. the specificity of the rdt was evaluated by detecting the positive sera against aiv (h , h , and h ), ndv, mdv, ibdv, and cav. the sensitivity experiments of the rdt were conducted by detecting the ibv positive serum with different titers. then, the results were observed, and the detection limit was determined. to further evaluate the cit and rdt, clinical serum samples were detected by the cit, rdt, and nsp elisa antibody test kit [ ] . subsequently, the positive rate of each method was determined. step , the prepared chip was rinsed thrice with pbst; step , μl of diluted serum was added and incubated on a constant temperature oscillator and then washed with pbst thrice; step , μl of goat anti-chicken igy conjugated to hrp was added, and the plate was incubated on a constant temperature oscillator and washed with pbst thrice; step , for chemiluminescence, μl of chemiluminescent substrate was added to each well, and images were taken at a wavelength of nm with amersham imager ; step , for rdt, μl of tmb was added to each well and incubated for min in a dark place; then, the results were observed chemiluminescent signals were acquired using gene-pix, and the snr was calculated as follows: snr = (signal intensity − background)/background. graph-pad prism and microsoft excel were used for the statistical analysis of all data, including the determination of the threshold and the calculation of the snr value, means, sds, and cvs. the roc curve was obtained using graphpad prism . sensitivity and specificity were calculated according to the following formulas: sensitivity = true positive/(true positive + false negative) × %; specificity = true negatives/(false positives + true negatives) × %. the area under the curve (auc) was used to validate the diagnostic application of the cit. the area under the roc curve quantifies the overall ability of the test to discriminate between those individuals with the disease and those without the disease. a truly useless test (one no better at identifying true positives than flipping a coin) has an auc of . , whereas a perfect test (one that has zero false positives and zero false negatives) has an auc of . for the cit, the optimal antigen concentration was . mg/ml (fig. a, b) , and the dilution for the serum samples was : (fig. c, d) , on the assumption that the snr between the positive and the negative sera was the highest. the dilution of the hrp-conjugated goat anti-chicken antibody was defined as : , . when the exposure time was more than min, the snr of the negative serum rose rapidly; thus, we set the exposure time to min (fig. ) . roc analysis showed that the ibv nsp microarray had high selectivity (p < . ) between the positive and the negative samples, and the auc was . (fig. a) . based on the roc analysis of the ibv nsp microarray, the snr value of the idexx-negative serum samples varied from a minimum of . to a maximum of . , whereas the snr value of the idexx-positive serum samples was from a minimum of . to a maximum of . (fig. b) . a threshold snr value of for ibv nsp microarray was found to provide optimal results, with a (table ) . thus, the samples with snr < were considered negative, whereas those with snr ≥ were considered positive. the specificity of the cit was evaluated by detecting the cross-reactivity of the antibodies against aiv (h , h , and h ), ndv, mdv, ibdv, and cav. the snrs of all sera from the previously mentioned viruses were all below the threshold of . these data revealed that no cross-reactivity occurred between the ibv gst-fused nsp antigen and antibodies against other avian viruses. this result demonstrated that the antigen has a high specificity. the reproducibility of the cit detection was determined by comparing the snr value of each clinical serum sample from the below tests. the within-plate cvs of nine positive and four negative serum samples tested ranged from . to . % (table ) , whereas the between-run cvs of these serum samples ranged from . to . % (table ). these results showed that the cit detection results were reproducible and had low and acceptable variation. one hundred and forty-four clinical serum samples ( samples were positive for antibodies against ibv, and samples were negative as confirmed by the idexx ibv ab test kit) were subjected to visual rapid detection following the procedure described above. the data showed that serum samples were positive for antibodies against ibv, and samples were negative, similar to the results of the idexx ibv ab test kit with the nsp concentration of . mg/ml (table ). if ibv antibodies exist in the serum, the spot with the ibv antigen turns blue, thereby allowing us to determine the concentration of nsp as . mg/ml. the specificity of the rdt was evaluated by detecting the cross-reactivity of antibodies against aiv (h , h , and h ), ndv, mdv, ibdv, and cav. the specific experiments of the rdt showed that no cross-reaction fig. a distribution of the snrs of the idexx-positive (n = ) and idexx-negative (n = ) serum samples of the clinical sera obtained from the ibv protein microarray. the threshold was defined as . the diagnostic sensitivity and specificity of the assay were greater than %. b roc curve obtained with graphpad prism software with positive (n = ) and negative (n = ) samples. the auc was . , indicating that the ibv protein microarray is a reliable test occurred between the ibv gst-fused nsp antigen and the antibodies against other avian viruses. the sensitivity experiments demonstrated that when the positive serum was diluted : , the spot still turned blue (fig. ) . (table ). most serological assays, including the idexx elisa kit, use viral particles of ibv as an antigen for the detection of antibodies against ibv. however, the preparation of purified virions for use as an antigen is time-consuming and expensive. in the present study, recombinant nonstructural proteins expressed in escherichia coli antigen-based protein microarray was evaluated for the first time in the serological diagnosis of ib [ ] . protein microarrays have high sensitivity and good reproducibility in quantitative and qualitative assays, and they are a valuable asset when analyzing complex biological samples [ ] . in clinical sample testing, many factors, including time, cost, accuracy, sensitivity, and throughput, determine the performance and usefulness of an immunoassay. in this study, a new solidly supported material, ipdms membrane, which has a near "zero" background for identification, was used. it achieved high sensitivity in detecting antibodies in serum [ ] . these unique features of ipdms not only simplify data analysis but also reduce nonspecific interactions [ ] . elisa detection has been widely used in the detection of ibv antibodies in early infection and continuous infection of ib and vaccine-immune, and no diagnosis method is more sensitive and quicker than elisa. in this study, two microarray methods (cit and rdt) were established. except for the method of the observation, the reaction processes of the two methods are akin to the detection process of elisa. however, unlike elisa, the established methods only require ng of antigen coating on each spot, and the amount of hrp-igg required for each reaction well is only ng. the antigen and hrp-igg used in both methods were less than those used in elisa, thereby reducing the cost of detection. in addition, the cit can detect antibodies against ibv nsp quantitatively and is more sensitive than the ibv nsp elisa kit. the rdt was developed to detect antibodies against ibv visually, and the results can be obtained within min with great sensitivity and specificity. compared with elisa, rdt has a shorter detection time and better detection efficiency. in this study, we only used one antigen of ibv for testing and verification. in the future, we will apply antigens of different diseases to ipdms to achieve high-throughput test results. for the establishment of the ibv nsp protein chip, we first optimized the procedure and determined the cit threshold as with the idexx ibv antibody detection kit. with the threshold of , the cit showed high sensitivity ( . %), specificity ( %), and accuracy ( . %) in the antibody detection of the samples compared with those of other thresholds ( table ). the rdt demonstrated a high success rate compared with the commercial idexx ibv ab test kit, suggesting that the rdt is a reliable assay for the detection of ibv infection. clinical serum samples were also subjected to rapid detection. furthermore, the rdt has higher sensitivity than the commercial idexx ibv ab test kit. it is also simpler and faster than elisa methods. to further evaluate ibv nsp protein chip, clinical serum samples were detected by the ibv nsp protein chip and the nsp elisa antibody test kit. the positive rates of the cit, nsp elisa, and rdt were . %, . %, and . %, respectively. compared with nsp elisa, the cit was more sensitive, and the rdt had similar positive rates but was faster. protein chips are a high-throughput monitoring system that monitors the interaction among protein molecules through the interaction between a target molecule and a capture molecule. although protein chips have been produced in the context of proteomics research, its application is not limited to proteomics alone. with the development of protein chip technology, researchers have gradually applied this technology to other fields, such as food inspection, disease diagnosis, drug screening, agriculture, forestry, animal husbandry, and forensic science. at present, this technology is rarely studied and applied in veterinary medicine. high throughput is an [ , ] and enable their use for determining antibody responses to infectious diseases [ ] . in the future, we will print recombinant antigenic proteins of different avian viruses to achieve high-throughput detection results with the same serum. the nsp protein chips were developed for the detection of antibodies against ibv. these assays are comparable to the commercial idexx ibv ab test kit in terms of sensitivity and specificity. the rdt can generate results within min and may be a suitable alternative to screen for the presence of ibv in chickens. raw data is available from the corresponding author on reasonable request. authors' contributions lpy, jhh, and jl performed the experiments, interpreted the results, and drafted the manuscript. lpy, zys, and qx analyzed the data. zwb, ly, yl, yqc, af, and hl collected the clinical samples. sqs and ml analyzed the data and revised the manuscript. lpy and jyz designed the study, analyzed the data, and revised the manuscript. all authors reviewed the results and approved the final version of the manuscript. this study was performed in accordance with the recommendations in the guide for the care and use of laboratory animals of the ministry of health, china. the protocol of the current study was reviewed and approved by the institutional animal care and use committee of nanjing agricultural university (approval no. syxk - ). written informed consent to use clinical serum samples, which were collected from a chicken farm, were obtained from the owner of the animals. all efforts were made to minimize animal suffering during sample collection. not applicable vaccination against infectious bronchitis virus: a continuous challenge completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus the molecular biology of coronaviruses development and application of nsp -elisa for the detection of antibody to infectious bronchitis virus the longevity of anti nsp antibodies and the sensitivity of a abc elisa -a years follow up of repeatedly vaccinated dairy cattle infected by foot and mouth disease virus an emerging recombinant cluster of nephropathogenic strains of avian infectious bronchitis virus in korea fine level epitope mapping and conservation analysis of two novel linear b-cell epitopes of the avian infectious bronchitis coronavirus nucleocapsid protein antigenic variation in strains of avian infectious bronchitis virus. archiv fur die gesamte virusforschung evaluation of four enzyme linked immunosorbent assays for the detection of antibodies to infectious bursal disease in chickens development and application of a saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus application of purified recombinant antigenic spike fragments to the diagnosis of avian infectious bronchitis virus infection recombinant lipl antigen-based single serum dilution elisa for detection of canine leptospirosis validation of serological assays for diagnosis of infectious diseases overview of protein microarrays integrated poly(dimethysiloxane) with an intrinsic nonfouling property approaching "absolute" zero background in immunoassays initiator integrated poly(dimethysiloxane)-based microarray as a tool for revealing the relationship between nonspecific interactions and irreproducibility the analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays protein microarrays: potentials and limitations antigen microarrays for serodiagnosis of infectious diseases the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - r g p i authors: wang, yu; wang, gang; duan, wei-tong; sun, ming-xia; wang, meng-hang; wang, shang-hui; cai, xue-hui; tu, ya-bin title: self-assembly into virus–like particles of the recombinant capsid protein of porcine circovirus type and its application on antibodies detection date: - - journal: amb express doi: . /s - - - sha: doc_id: cord_uid: r g p i pcv capsid protein (cap) is an important antigen for diagnosis and vaccine development. to achieve high-level expression of recombinant pcv cap in escherichia coli (e. coli), the gene of wild-type entire cap (wt-ecap) was amplified from clinical samples, and three optimized entire cap (opti-ecap) and one optimized cap deleted nuclear location signal (nls) (opti-dcap) gene fragments encoding the same amino acid sequence with wt-ecap were synthesized based on the codon bias of e. coli. those gene fragments were inserted into the pet a expression vector. one recombinant strain with the highest expressed soluble ecap from four entire cap (one wt-ecap and three opti-ecap) and one recombinant strain expressed opti-dcap were selected for further purification. the purified ecap and dcap were identified by transmission electron microscopy (tem), a large number of round hollow particles with a diameter of nm virus-like particles (vlps) were observed in ecap, whereas irregular aggregation of proteins observed in dcap. after formation the vlps were applied as a coating antigen to establish an indirect elisa (i-elisa) for detection of pcv -specific antibody in swine serum. clinical swine serum samples from china collected in were tested utilizing the vlp-based i-elisa method under optimized conditions. to the best of our knowledge, this is the first report of self-assembly into vlps of pcv recombinant cap. our results demonstrated that the vlp-based i-elisa will be a valuable tool for detecting the presence of pcv antibodies in serum samples and will facilitate screening of large numbers of swine serum for clinical purposes. porcine circovirus (pcv) has three strains with the following nomenclature pcv , pcv and pcv . pcv was first isolated from a contaminated porcine kidney pk- cell line and has no pathogenicity for pigs. pcv can cause clinical symptoms including postweaning multisystemic wasting syndrome (pmws), pneumonia, porcine dermatitis, nephropathy syndrome (pdns), and reproductive failure (madson and opriessnig ) . pcv was identified as a new member of pcv in (palinski et al. ) , additional studies showed that pcv appeared in other countries (china, poland, brazil, korea, denmark, italy and spain) (franzo et al. a; kim et al. b; shen et al. ; stadejek et al. ; tochetto et al. ) . specifically, the existence of pcv in pigs can be traced back to as early as in china according to early studies . although the pathogenicity of pcv is currently debated, studies have shown that it has the potential to threaten the swine open access as well as to enhance pig susceptibility to other microorganisms (chen et al. ; li et al. ) . currently, the common difficulty for the study of pcv is that the virus cannot be isolated from cell lines. however, unraveling the characteristic of the virus is still ongoing by constructing virus-like particles (vlps) based on genetic engineering technology which can be achieved without the virus. these vlpbased studies may provide new ideas leading to a better understanding of the pcv virus which will lead to potential treatments and diagnotics. vlps are self-assembled macromolecules composed of viral structural proteins but lacking of genetic materials. due to the increased safety and ease of use vlps are preferable for development of serologic diagnostic tests. the enzyme-linked immunosorbent assay (elisa) based on vlps is widely used to measure antibodies or neutralizing epitopes (almanza et al. ; chao et al. ) . for example, pcv vlps are used as coating antigens to be able to detect serum neutralizing antibodies (snabs) in elisa (nainys et al. ; zhang et al. ) . currently, pcv cap proteins harvested from insect cells or e. coli have been used as antigens to detect pcv -specific antibodies in an elisa (deng et al. ; zhang et al. ). there is still lacking a vlp-based serological diagnosis method for pcv . in this study, one recombinant strain with the highest expressed soluble opti-ecap was selected and successfully defined conditions for the protein to self-assemble into vlps. the vlps were purified through a two-step chromatographic technology. additionally, a highly specific, sensitive and reproducible vlp-based indirect elisa (i-elisa) has been established. this new pcv elisa is a valuable tool for detecting the presence of pcv antibodies in serum samples and will facilitate the screening of large numbers of swine serum for clinical purposes. positive sera was collected from clinical swine serum samples. to assess the specificity of the assay, samples were screened for only pcv infection, excluding other pathogens such as pcv and prrsv by pcr and elisa. the samples with the highest concentration were selected as positive serum and used to coat elisa plates for the pcv vlp elisa and used for western blot and elisa optimization. negative serum samples were collected from forty pathogen-free (spf) piglets which were obtained from the experimental animal center at the veterinary research institute (harbin, china). clinical serum samples including wild boar serum samples were collected from china in for testing using the pcv vlp elisa. the wt-ecap was amplified by pcr using dna from lymph nodes of postweaning multisystemic wasting syndrome-suffering pigs. opti-ecap- was fully optimized for the full-length gene of pcv cap protein based on factors such as codon bias and gc content, while opti-ecap- and opti-ecap- were partially optimized. meanwhile, one optimized cap deleted nuclear location signal (nls) (opti-dcap) gene fragments encoding the same amino acid sequence with wt-ecap were synthesized, and a × his-tag was fused to the nh -terminal end of the dcap to aid protein purification. sequence alignment of the four entire cap (one wt-ecap and three opti-ecap) and opti-dcap is provided in fig. . one wt-ecap, three opti-ecap and one opti-dcap gene fragments were subcloned into the pet a expression vector via the ndei/xhoi sites. the recombinant fusion proteins ecap and dcap were obtained by transforming the corresponding plasmid into escherichia coli (e. coli) bl (de ) under conditions of rpm shaking speed at °c until the od reached . , at which time . mm isopropyl-β-d-thiogalactopyranoside(iptg) was added and the bacteria were incubated at °c for h. bacteria were harvested by centrifugation at ×g for min at °c. the cell pellet was resuspended in ml of mm tris-hcl buffer (ph . ) and sonicated on ice for cycles of s pulses at s intervals using a cell ultrasonic crusher (cole parmer, usa) at % amplitude. lysates were divided into supernatant and pellet by centrifugation at , ×g for min at °c. pellets were resuspended in pbs at a volume equal to the supernatant. expression and solubility of ecap and dcap were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and western blot. briefly, equal molar amounts of each recombinant protein were separated using % sds-page. separated proteins were transferred electrophoretically onto a polyvinylidene difluoride (pvdf) membrane. unbound sites on the membrane were blocked with blocking buffer at overnight. the membrane was incubated with specific pig positive serum for h, and was washed in pbst (pbs containing . % of tween ) three times. next, anti-pig igg (whole molecule)-fitc antibody produced in goat (sigma, usa, : , ) was added and incubated for h. the final colorimetric reaction was developed at room temperature using the infrared imaging systems (ge, usa). the highest expressed soluble opti-ecap- was chosen and purified by anion-exchange chromatography as the first step. the supernatant was loaded on a deae bestarose fast flow column (bestchrom, china) in an automated fplc system (akta, ge-healthcare life sciences, usa). after the column had been washed with mm tris-hcl buffer (ph . ), ecap was eluted and collected with buffer b ( mm tris and mm nacl, ph . ). the purity of the ecap protein was assessed by sds-page. formation of pcv vlps was verified with tem (h , hitachi, japan). then, the product was subjected to size-exclusion chromatography equipped with a prepacked sepharose ff / column (bestchrom, china) in buffer b. the flow rate was set to . ml/min and the first peak was collected, and vlps were detected by sds-page and tem. in addition, the recombinant dcap was purified by ni-nta affinity (ge, usa) and also detected by tem. purified pcv vlps were used as antigens for development of an indirect elisa (i-elisa) to detect anti-pcv antibodies in swine serum. the optimal dilutions of antigen and serum were determined by a checker board titration with positive and negative swine sera. the concentration of pcv vlps were measured by bca (thermo, usa). the prepared antigen was used to coat -well elisa plates (biofil, china) ranging from . , , . , , . to μg/ml and μl per well diluted in carbonate-bicarbonate buffer (ph . ). sera diluted in pbs ranging from : , : , : to : (v/v) was used and tested to determine the optimal serum dilution. the optimal dilutions of antigen and serum were determined on the basis of the maximum difference in absorbance at nm between positive and negative serum (p/n) were selected on a larger scale. in addition, the reaction temperature, time and other conditions were also optimized. after optimization, μl of μg/ml pcv vlps in carbonate-bicarbonate buffer was coated onto a -well elisa microplate (corning, usa) at °c overnight. the antigen-coated plate was washed three times with pbst and blocked with pbst containing % (w/v) skim milk at °c for h. after washing three times, μl of diluted serum samples were added and incubated at °c for h. the plates were washed three times with pbst, followed by incubation for h at room temperature with μl diluted hrp-labeled goat anti-pig igg (solarbio, china, : ). after being washed three times, the plates were incubated with μl tetramethylbenzidine (tmb, solarbio) in the dark for min at room temperature, which was used as a chromogenic substrate. reactions were stopped by adding μl of abts stop solution ( m hcl) and the absorbance ( nm) was measured using an elisa plate reader (pe, usa). forty negative sera were used to determine the cut-off value. all sera were subjected to pcv vlp-elisa three times to abate deviation. the mean od nm value (x) and standard deviation (sd) were calculated, and the cutoff value was defined as x + sd. a total of serum samples were selected to evaluate the reproducibility of the pcv vlp-elisa. for each sample, the coefficient of variation (cv) was calculated between plates (inter-assay variation) and within the same plate (intra-assay variation). each sample was tested in five different plates on different occasions to determine the inter-assay cv and five replicates within each plate were used to calculate the intra-assay cv. for cross-reactivity assay, standard positive sera of pcv , prrsv, pegv, tegv, prv and csfv were tested in triplicate according to pcv vlp-elisa procedure and the od values were calculated. in addition, six pcv positive and one negative serum were used to determine assay sensitivity. for gene optimization, optimum-gene ™ algorithm was used to produce a single gene which is highly expressed. all sequences have been uploaded to genbank, wt-ecap (gene accession number mn ), opti-ecap- (gene accession number mn ), opti-ecap- (gene accession number mn ), opti-ecap- (gene accession number mn ) and opti-dcap (gene accession number mn ). the recombinant expression plasmids ecap-pet a and dcap-pet a were constructed for efficient expression of target proteins in e. coli. recombinant cap protein was harvested by induction with . mm iptg and incubation at °c for h with shaking. the results of sds-page showed wt-ecap (molecular weight ~ kda) was hardly expressed in e. coli, while opti-ecap (molecular weight ~ kda) can be expressed in a considerable level and opti-ecap- was dominant (fig. ) . for the future research, the highest expressed soluble opti-ecap was selected and it can also specifically react with positive serum (fig. a) . due to the complex composition of the supernatant, host proteins or subcellular organelles, it is impossible to see clear viral particles directly under the tem (data not shown). therefore, purification by chromatography is necessary. during the purification process, higher purity was preferred over yield rate. after the purification by iec, sds-page results showed little heteroprotein bands (fig. b) , nm particles can be clearly observed under the tem (fig. c) . however, there were still some special-shaped impurities detected in the particles. in order to make the results more clear and useful for subsequent elisa establishment, the purest sample had been obtained from the next sec (fig. a) . uniform particles without any impurities were observed (fig. b) . a large fraction of the particles were in an aggregate state. therefore, we can confirm the expression of the opti-ecap successfully self-assembled into virus-like particles. furthermore, the recombinant ecap(molecular weight ~ kda) also had a high level of expression in e. coli (fig. ) but there were only irregular aggregation of proteins observed in tem (fig. c) . it means that removing the nsl may lose the ability of vlps forming. optimization conditions of the vlp-based i-elisa were determined by od nm and p/n values. mm carbonate-bicarbonate buffer (ph . ) and % skimmed milk was chosen for the optimal coating and blocking buffers, respectively. the checkerboard titration tests indicated that optimal antigen concentration for plate coating was µg/ml with a serum dilution of : in a maximal p/n ratio, and all experiments were performed in triplicates (table ) . negative serum samples were employed for deciding cut-off value, the mean od value and sd was . and . , respectively using in the result (fig. a) . hence, the cutoff value of the vlp elisa was calculated as . (mean + sd). any serum with od greater than or equal this threshold was regarded as positive, otherwise it was determined to be pcv antibody negative. all non-pcv serum samples (ppv, pcv , prv, tegv, pedv, csfv, and prrsv) were negative as the result of vlp elisa (fig. a) . the od nm values decreased with successive dilutions of six positive serum samples indicating high sensitivity (fig. b) . swine serum from china collected in were tested. in total, samples ( . %) tested positive by vlp elisa. it was worth noting that the positive rate of swine ( . %) is much higher than the wild boar ( . %) (fig. ). this suggests that differences between species and feeding conditions may affect pcv infection. in conclusion, the vlp elisa has high application value in the future epidemiology research of pcv . vlps, composed of viral structural proteins without genetic materials, are self-assembled macromolecules. because of their ability in stimulating strong immune response and plentiful antibody production, they are regarded as candidates of novel vaccines. (garcea and gissmann ) . vlps provide more safety in use and the possibility of large scale production of vaccines with reproducible high quality results compared with traditional live-attenuated or inactivated virus vaccines (noad and roy ; raghunandan ) . some vlps, such as hepatitis b surface antigen (hbsag) vlps, human papillomavirus (hpv) vlps and malaria vlpbase vaccines have already been clinically used for the prevention of infectious diseases (kim and kim a) . in recent years, the technology of expressing capsid protein (cap) of pcv and self-assembly into virus-like particles (vlps) has advanced, and can be used in multiple recombinant protein expression systems including baculovirus, yeast and e. coli (liu et al. ; nainys et al. ; wu et al. ) . many commercial vaccines based on vlps have effectively prevented the infection of pcv (beach and meng ). similar to pcv , there are two major open reading frames (orfs), orf and orf , in pcv genome. orf encode the immunogenic cap which is the sole structural protein of the viral coat (palinski et al. ) . whether the idea of forming recombinant cap (rcap) into vlps is equally applicable to pcv is the main purpose in our research. in this study, the e. coli expression system has been employed successfully for expression of pcv vlps due to its relative simplicity, low cost, and fast high-density cultivation, which is significant in future diagnosis and vaccine development. however, the n-terminal nls domain of the cap protein is abundant in arginine residues and contains several rare codons for e. coli that impedes a foreign gene expression, which is disadvantageous for full-length cap expression (wu et al. ). using codon optimization can overcome the difficulty of high-level expression of full-length cap. removing of nls has also been utilized to improve the expression efficiency and stability of expressed protein in e. coli but has failed to self-assemble into vlps. the diameter of the circovirus is around nm, similar to beak and feather disease virus (bfdv), bat circovirus (btcv) and pcv . the morphological study of pcv has not be conducted, whether nm particles is the true size of the virus remains to be determined. previous studies have shown that expressing the full-length pcv cap gene and nls domains presenting within the n-terminal arginine rich motif (arm) may cause misfolding of the protein and induce formation of circular virus complexes of - nm (sarker et al. ) . other groups have published that, different sizes of vlps appear in different expression systems (bucarey et al. ; kim et al. a; wu et al. ), a number of factors including storage conditions (ion-strength, ph and etc.) as well as the process design also influence the characteristics of vlps (effio and hubbuch ; fernandes et al. ; kim and kim b) . nm particles can be used as a preliminary evaluation standard before the real virus morphology of pcv is discovered. hydrophobic interaction chromatography (hic) has also been used as a step in the purification process. but ecap cannot be eluted under any conditions even if the least hydrophobic filler was selected. therefore, it is suspected that pcv vlps are highly hydrophobic. this conclusion can also be derived from genetic analysis as the entire cap gene contains many hydrophobic amino acids. therefore high-purity particles exist in an aggregated form under the tem, which also explained the observation that the target protein eluted at the first peak of sepharose ff column. theoretically nm particles should elute at the end of this gel. virions have properties that tend to aggregate (gerba and betancourt ) , the aggregate status may be changed by adjustment of the ph or salt concentration and type of cationic salt in suspension (mattle et al. ) . vlps may also encounter the problem of aggregation due to their large size and complex structure, this can occur during the process such as during separation, purification and storage (chen et al. ; jezek et al. ; kissmann et al. ; shi et al. ) . vlps are preferable for development of serologic diagnostic tests, as they mimic the structure of virus with repetitive surface epitopes of viral antigens in a proper conformation compared to monomeric viral candidate antigens. the enzyme-linked immunosorbent assay (elisa) based on vlps is widely used to measure antibodies or the neutralizing epitopes (almanza et al. ; chao et al. ) . pcv vlps as coating antigens can detect serum neutralizing antibodies (snabs) in elisa (nainys et al. ; zhang et al. ) . some reports show that pcv infection in pigs do not present any significant clinical signs or symptoms and wild boars may also have susceptibility (franzo et al. b; klaumann et al. ; zheng et al. ) , thus it is particularly important to establish an effective antibody detection method to assess the pcv infection in pigs. the isolation and cultivation of pcv is a recognized problem in the world. there is a report that the virus can be rescued from an infectious pcv dna clone ). our lab is actively trying to rescue the virus through the method reported in this article. without virus it is impossible to carry out immunization and challenge experiments to prove whether the vlps have protective effect on pcv infection as a vaccine because the virus has not been successfully isolated to date. to provide another solution here we describe the establishment of a vlp-based i-elisa for antibody detection. in this process, the choice of positive serum was extremely important. in addition to pcv , the specific positive serum cannot be detected with other pathogens by pcr and elisa. active viremia indicates that the virus is in the infection stage. the vlps can react specifically with such serum as the result of western and elisa experiments, which fully demonstrates the immunogenicity for pcv . as a novel swine virus, positive results either through pcr or elisa, are required since they indicate recent infection with pcv . elisa has more advantages due to its less operation labor and higher throughput, which can rapidly confirm the pcv infection status in pig populations. the vlp elisa showed good sensitivity to positive serum samples and had no cross reactivity with other swine viral pathogens including pcv . subsequently, the vlp elisa was successfully applied to the antibodies detection of clinical serum samples from china collected in , indicating that this method can be more widely applied to the epidemiological research of pcv . in conclusion, this is the first report of the ability of pcv vlps to self-assemble which were successfully expressed in e. coli and applied in the development of an elisa for testing the specific antibodies of clinical pig serum. the vlp elisa was highly specific, sensitive and reproducible, it is a valuable tool to monitor the prevalence of the pcv virus. the invention of virus-like particles will play a significant role in providing a new tool for the study of pcv . virus-like particles as vaccines and vessels for the delivery of small molecules viral aggregation: impact on virus behavior in the environment a heat-stable hepatitis b vaccine formulation induction of porcine dermatitis and nephropathy syndrome in piglets by infection with porcine circovirus type current status and future prospects for human papillomavirus vaccines yeast as an expression system for producing virus-like particles: what factors do we need to consider? comparison of the size distributions and immunogenicity of human papillomavirus type l virus-like particles produced in insect and yeast cells the prevalence and genetic characteristics of porcine circovirus type and in korea physical stabilization of norwalk virus-like particles porcine circovirus is highly prevalent in serum and tissues and may persistently infect wild boar (sus scrofa scrofa) a duplex real-time pcr assay for the simultaneous detection of porcine circovirus and circovirus efficient production of type porcine circovirus-like particles by a recombinant baculovirus effect of porcine circovirus type (pcv ) infection on reproduction: disease, vertical transmission, diagnostics and vaccination impact of virus aggregation on inactivation by peracetic acid and implications for other disinfectants generation in yeast of recombinant virus-like particles of porcine circovirus type capsid protein and their use for a serologic assay and development of monoclonal antibodies virus-like particles as immunogens a novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure virus-like particles: innate immune stimulators structural insights into the assembly and regulation of distinct viral capsid complexes genome characterization of a porcine circovirus type in south china stabilization of human papillomavirus virus-like particles by nonionic surfactants first detection of porcine circovirus type on commercial pig farms in poland retrospective study of porcine circovirus infection in china full-genome sequence of porcine circovirus type recovered from serum of sows with stillbirths in brazil characterization of porcine circovirus type (pcv ) capsid particle assembly and its application to virus-like particle vaccine development efficient expression and purification of porcine circovirus type virus-like particles in escherichia coli generation of e. coli-derived virus-like particles of porcine circovirus type and their use in an indirect igg enzyme-linked immunosorbent assay development and application of a baculovirus-expressed capsid protein-based indirect elisa for detection of porcine circovirus igg antibodies the occurrence of porcine circovirus without clinical infection signs in shandong province publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions y-bt and yw designed the work and drafted the manuscript; x-hc and gw took part in review and editing; w-td and s-hw performed the research study; m-xs and m-hw analyzed the data. all authors read and approved the final manuscript. key: cord- -tr ageky authors: gaikwad, satish s.; lee, hyun-jeong; kim, ji-ye; choi, kang-seuk title: expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of newcastle disease virus nucleoprotein date: - - journal: clin exp vaccine res doi: . /cevr. . . . sha: doc_id: cord_uid: tr ageky purpose: the aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (diva) strategy accompanying the marker vaccine lacking an immunodominant epitope (ide) of nucleoprotein of newcastle disease virus (ndv). materials and methods: recombinant epitope-repeat protein (rerp) gene encoding eight repeats of the ide sequence (etqfldlmravansmr) by tetra-glycine linker was synthesized. recombinant baculovirus carrying the rerp gene was generated to express the rerp in insect cells. specificity and sensitivity of an indirect enzyme-linked immunosorbent assay (elisa) employing the rerp was evaluated. results: the rerp with molecular weight of kda was successfully expressed by the recombinant baculovirus in an insect-baculovirus system. the rerp was antigenically functional as demonstrated by western blotting. an indirect elisa employing the rerp was developed and its specificity and sensitivity was determined. the elisa test allowed discrimination of ndv infected sera from epitope deletion virus vaccinated sera. conclusion: the preliminary results represent rerp elisa as a promising diva diagnostic tool. n-and c-terminal regions of the np serve as b-cell epitopes in host [ , ] . in particular, c-terminal immunological region that forms alpha helical structure is permissible for modification. thus this epitopic region can serve as negative marker if replaced with a foreign epitope [ ] . for example, amino acid sequence etqfldlmravansmr (aa - ) on ndv np is an epitope, which serves as linear immunodominant epitope (ide) and can be replaced or deleted [ ] . this antigen should facilitate differentiation of vaccinated animal to that of infected during serosurveillance in the case of the marker vaccine applying vaccination program. a multiepitope protein as a vaccine candidate [ ] [ ] [ ] [ ] [ ] [ ] or as diagnostic reagent [ ] [ ] [ ] has been explored in multiple instances. multiepitope recombinant proteins are advantageous as they can be designed rationally and offers higher specificity and sensitivity [ ] . the aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (diva) strategy accompanying an ndv marker vaccine lacking the np ide. for this purpose, an indirect enzyme-linked immunosorbent assay (elisa) format employing a recombinant protein, which carries multimers of ide, was developed. the recombinant protein was expressed in baculovirus system. specificity and sensitivity of recombinant protein was evaluated. epitope repeat protein (erp) gene encoding eight repeats of an ide sequence containing etqfldlmravansmr (c-terminal ide aa - ) on ndv np separated by tetra-glycine linker was synthesized commercially using codon optimization for baculovirus expression. the recognition enzyme sites of ecori and hindiii were built in the upstream and downstream of coding sequence, respectively. the synthetic gene cloned in vector backbone pmk-rq (kanr) was received as μg of lyophilized plasmid preparation. the conceptual translated sequence was submitted to i-tasser server for homology modeling [ ] and top five predicted structures were visualized with pymol software [ ] . synthetic gene was subcloned into pfastbac ht b vector using ecori and hindiii restriction sites. the recombinant plasmid ( ng) was transformed in escherichia coli dh b cells. transformed cells were plated onto the luria-bertani agar containing kanamycin ( μg/ml), gentamicin ( μg/ml), tetracycline ( μg/ml), bluo-gal ( μg/ml), and isopropylthio-β-galactoside (iptg, μg/ml) and incubated at °c for to hours. the high molecular weight bacmid dna was isolated from the overnight cultures by alkaline lysis purification according to the manufacturer's manual of bac-to-bac baculovirus expression system (invitrogen, carlsbad, ca, usa). spodoptera frugiperda (sf ) cells were cultured at °c in sf- ii serum free medium (invitrogen), supplemented with % heat-inactivated fetal bovine serum (fbs) (invitrogen), u/ml penicillin and μg/ml streptomycin. sf cells were transfected with recombinant bacmid dna using cellfectin ii, a cationic lipid for the transfection of the baculovirus particles according to the manufacturer's instructions. briefly, for each transfection, ml of grace's insect medium, unsupplemented (without antibiotics and serum) was added in each well, × cells per well were seeded in a -well plate and allowed to attach for hours. the bacmid dna and cellfectin ii ( μl of reagent) were diluted separately in μl of grace's medium, unsupplemented (without antibiotics and serum), then mixed and incubated for minutes at room temperature to form lipid-dna complexes. the cells were washed with fresh medium, and incubated with lipid-dna complex at °c for hours. the transfection solution was removed and ml supplemented sf- ii sfm containing % fbs was added. transfected sf cells were incubated at °c for hours for baculovirus production. recombinant baculovirus production was monitored daily by visualization of the cytopathic effects. three to four days after transfection, recombinant baculovirus was harvested from the cell culture medium and stored at °c. recombinant viruses were identified by polymerase chain reaction using gene vector specific primers (invitrogen). the resulting baculovirus was passaged three times by infecting more sf cells. sf suspension cultures ( × cells/ml) grown in l of sf ii medium supplemented with % of fbs were infected with recombinant baculovirus at multiplicity of infection of plaque-forming unit/cell. cells were grown for hours and cell pellets were washed in phosphate buffered saline (pbs it was purified by using ni-nta spin columns under denaturant condition with m urea according to ni-nta spin handbook (qiagen, hilden, germany). the collected cells were lysed in ml buffer b ( mm nah po , mm nacl, m urea, ph . ) and centrifuged the lysate at , ×g for minutes and collecting supernatant. loading up to μl of the cleared lysate supernatant containing the × his-tagged protein onto a pre-equilibrated ni-nta spin column, centrifuged minutes at ×g and washing the column with μl buffer c ( mm nah po , mm nacl, m urea, ph . ) and centrifuged minutes at ×g, the recombinant protein was finally eluted with buffer e ( mm nah po , mm nacl, m urea, ph . ). the recombinant protein was allowed to refold using refolding buffer ( mm tris ph . , . m nacl, . % chaps, mm dtt, % glycerol). rerp was dialyzed against refolding buffer ( mm chaps and mm dtt). proteins extracted from infected sf cells were fractionated by % sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. the separated proteins were blotted onto an immun-blot polyvinylidene fluoride membrane (bio-rad, richmond, ca, usa) using a wet transfer system (bio-rad). the membrane was blocked with % skim milk in pbs containing . % tween (pbst) at room temperature for hour. after washing times with pbst, the blocked membrane was subsequently incubated with ndv chicken antiserum (diluted : ) for hour, rinsed in pbst, and incubated with alkaline phosphatase-conjugated goat anti-chicken immunoglobulin (diluted : , , pierce, rockford, il, usa) for hour. protein bands were visualized by enhanced chemiluminescence using nbt/bcip solution (sigma-aldrich, st. louis, mo, usa). optimal dilutions of rerp and sera were determined by a checkerboard titration test with ndv positive and negative sera previously confirmed by hemagglutionation inhibition (hi) test. the antigen was coated in -well elisa plates (polysorb, nunc, roskilde, denmark) ranging in concentration from μg/ml to . μg/ml in mm carbonate/bicarbonate buffer (ph . ). reference positive and negative sera were both diluted serially from : - : , and tested to determine the optimal serum dilution. the dilutions that gave the optimum difference in absorbance at nm between posi-tive and negative sera were selected to test the sera panel. the working dilution of goat anti-chicken hrp-igg (sigma-aldrich), the reaction temperature, time and other conditions also were optimized. ten ndv negative serum samples from specific pathogen free (spf) chickens were used to determine a cutoff value for the elisa assay. the mean negative serum optical density (od) value plus three standard deviations (sd) was used as the cutoff value. each sample was repeated in triplicate wells and the mean value was calculated. the experiment was repeated two more times. in addition to spf chicken serum, different hyper-immune chicken sera to ndv, h n avian influenza virus (aiv), infectious bronchitis virus (ibv), and infectious bursal disease virus (ibdv) were tested for specificity of rerp using an indirect elisa assay. to test the sensitivity of the rerp, the chicken anti-ndv serum was diluted serially ( : - : , ) and the serum with each dilution was used to react with the expressed rerp in an indirect elisa. a total of chicken sera comprising three groups with each group of were used in the study. these sera were kept at the oie reference laboratory for newcastle disease, animal and plant quarantine agency (apqa), korea. first group were sera taken from mock-infected spf chicken (ndv antibody negative). second and third groups were sera taken dpi from spf chickens vaccinated with ide deleted recombinant newcastle disease virus (rndv) [ ] and ndv lasota strain [ ] , respectively, which kindly supplied by the oie reference laboratory for nd, korea. all vaccinated sera were proved to be serologically positive by a hi test using ndv antigen. all sera were available with the laboratory. microtiter plates were coated with μl of μg/ml rerp protein in mm carbonate/bicarbonate buffer ph . , and incubated overnight at °c. plates were washed three times with pbst and blocked with μl per well pbst with % skim milk powder solution at °c for hours. the chicken anti-ndv serum ( : ) was added to each. after -hour incubation at °c, plates were washed three times with pbst. one hundred microliters horseradish peroxidase-conjugated goat anti-chicken igg (pierce) at : , dilution was added to each well and plates were incubated at °c for hour. then plates were washed three times with pbst and μl tmb ( , , , -tetramethylbenzidine) was added to each well. tekan, männedorf, switzerland). mean elisa titer and sds of each group were calculated using microsoft excel (microsoft, redmond, wa, usa). differences in elisa absorbance between groups were analyzed by the one-way analysis of variance (anova). a p-value of < . was considered statistically significant. the construct encoding erp on ndv np was designed as shown in fig. a . the codon optimized gene ( bp) was designed to have repeats of amino acids (etqfldlm-ravansmr) mimicking c-terminal ide (aa - ) on ndv np. rerp gene insert was excised from the plasmid after digestion with ecori and hindiii enzymes. resulting fragment ( bp) was subcloned into the pfastbac ht b vector using above mentioned restriction sites. recombinant baculovirus containing the recombined erp gene insert (bac-rerp) was generated by transposition of recombinant vector into the sf cells by transfection. the codon optimized gene was commercially synthesized from geneart (regensburg, germany) and subcloned into baculovirus transfer vector. alignment of obtained nucleotide and deduced amino acid sequences of the rerp gene insert revealed % match with the designed construct (data not shown). i-tasser server predicted models, all of which showed optimal spacing in epitopes separated by linkers (fig. b) . graphic visualization of tertiary structure of top predicted models for rerp protein suggests that ide reactive antibodies would be freely accessible to epitopes separated by tetra-glycine linkers in tertiary dimensional space. bac-to-bac expression system was used to produce a baculovirus encoding n-terminally × his tagged rerp protein un- der the transcriptional control of polyhedrin promoter. recombinant baculovirus bac-rerp infected cells were lysed under denaturing conditions and rerp was purified using his tag purification. the protein was allowed to refold using refolding buffer. to identify the expressed rerp, the purified protein was analyzed by western blot assay. the chicken anti-ndv serum raised by infecting spf chickens with la sota strain of ndv [ ] was used as the primary antibody in the analysis. the results showed purified rerp with molecular weight of approximately kda reacted specifically (lanes to in fig. ) to the ndv positive serum. no specific band was detected with negative controls (lanes n and n in fig. ) , which indicated that the rerp is expressed correctly, and has good reaction ability with chicken specific anti-ndv serum. the optimal concentration of the rerp ( μg/ml) and the dilution rate of sera ( : ) for indirect elisa assays to evaluate the specificity and sensitivity of the expresses rerp were determined. to set up a cutoff value for indirect elisa assay, spf chicken serum samples were analyzed. the mean od value of these samples as measured by elisa was . with a sd of . . for a % confidence interval, the cutoff value determined was . (mean od of spf sera samples+ sd). based on the cutoff value, the reactivity of rerp with chicken anti-aiv, anti-ibv, anti-ibdv, and normal sera was measured. the result showed that od nm values of non-ndv sera were lower than cutoff value (fig. a) . the minimum dilution titer of chicken anti-ndv serum detected was : , according to cutoff value of . (fig. b ). a battery of chicken sera samples divided in ten chickens per group in three was tested. samples in groups of mock infected chicken sera and chicken vaccinated with the ide deleted rndv showed od values below cut-off value. the od value for samples from wildtype ndv lasota vaccinated chicken group were in the range of . to . , which was above determined cut-off value (fig. ) . the indirect elisa showed marker ndv vaccines in conjugation with serological diagnostic test has substantial value in poultry industry for demonstration of the freedom of nd. a serologic marker antigen is a viral protein or an epitope that is absent from the vaccine strains but consistently present in the corresponding wildtype viruses [ ] . therefore, only animals that have been infected with wild-type virus will develop antibodies against the serologic marker antigen while the vaccinated animals will not. the marker antigen should be conserved and immunodominant to ensure that the companion diagnostic test based on this marker antigen will produce reliable diagnostic results [ ] . in addition, the marker antigen should be dispensable for the viral life cycle but should not contribute significantly to the overall immunogenicity of the vaccine. subunit vaccines using f and hn protein [ ] can serve as marker vaccines but are less effective than whole virus vaccines [ ] . this necessitates ndv marker vaccine based on chimeric live virus. ndv np is known to be carry three antigenic regions, two of which are located at n terminal [ ] . c terminal ide (aa res-idues - ) etqfldlmravansmr is a dispensable epitope on np protein, which can be replaced with foreign epitope [ ] . in preliminary study, when chickens were immunized with viable recombinant ndv lacking this ide with reverse genetics technique, monomeric ide peptide based elisa showed poor sensitivity most likely due to small hapten size of aa and immunosteric hindrance to bovine serum albumin conjugated peptide (data not shown). to address this issue, this problem can be solved by using multimers of ide in the form of recombinant protein antigen. in the study, we designed, expressed and characterized the rerp in baculovirus expression system. this protein carried tandem repeats of ide on ndv np. the protein carried n terminal his tag allowing one-step purification under ni nta agar column [ , ] . the construct designed to express recombinant protein was codon optimized for sf cells. codon optimization involves codon adaptability, mrna structure, trna usage. it significantly increases protein expression [ ] [ ] [ ] . we used tetra-glycine linker as linker in between epitopes as it provides flexibility due to lack of β-carbon and is preferred linker in multi-epitope proteins [ ] . computer modeling showed that all the epitopes are freely accessible in three-dimensional space. this rerp was expressed at high concentration in insect cells when titrated by elisa. we purified pro- tein under denaturing condition on a ni-nta matrix with a high degree of purity [ ] . western blot analysis showed the purified rerp was recognized by chicken anti-ndv serum with a specific band at approximately kda (lanes - in fig. ) , while no specific band was observed when using negative control serum. this indicates that the expressed rerp has the molecular weight as expected. we employed the indirect elisa to evaluate the specificity and sensitivity of the expressed rerp using confirmed positive and negative sera samples. rerp could react specifically with the anti-ndv serum and showed no reactivity towards anti-ibv, anti-ibdv, anti-aiv, and normal sera. it suggests that rerp can specifically recognize anti-ndv antibodies. the potential of rerp elisa to serve as discriminatory test in combination with the ide deleted marker vaccine was investigated using three panels of sera. the test correctly classified the marker vaccinated and spf chicken sera as antibody negative but sera from wildtype ndv immunized chickens as antibody positive. these results revealed that rerp possessed good reactivity, specificity and sensitivity. future work involves testing sera samples under field trials. if proven satisfactory, the recombinant protein represents a promising candidate as companion diagnostic test in ndv diva vaccination program. this strategy of epitope-based recombinant proteins as elisa antigens against modification permissible regions of viruses makes it a highly effective approach in diva vaccination. newcastle disease virus detection and differentiation from avian influenza genetic diversity of avian paramyxovirus type : proposal for a unified nomenclature and classification system of newcastle disease virus genotypes recombinant haemagglutinin neuraminidase antigen-based single serum dilution elisa for rapid serological profiling of newcastle disease virus newcastle disease virus (ndv) marker vaccine: an immunodominant epitope on the nucleoprotein gene of ndv can be deleted or replaced by a foreign epitope localization of the antigenic sites of newcastle disease virus nucleocapsid using a panel of monoclonal antibodies strategies for differentiating infection in vaccinated animals (diva) for foot-and-mouth disease, classical swine fever and avian influenza a custom-designed recombinant multiepitope protein as a dengue diagnostic reagent cellular immunogenicity of a multi-epitope peptide vaccine candidate based on hepatitis c virus ns a, ns b and core proteins in hhd- mice design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach safety and immunogenicity of a ctl multiepitope peptide vaccine for hiv with or without gm-csf in a phase i trial design of immunogenic and effective multiepitope dna vaccines for melanoma the immunoreactivity of a chimeric multi-epitope dna vaccine against ibv in chickens a recombinant multiepitope protein for hepatitis b diagnosis evaluation of multiple antigenic peptides based on the chikungunya e protein for improved serological diagnosis of infection evaluation of a novel fusion protein antigen for rapid serodiagnosis of tuberculosis recombinant multiepitope protein for diagnosis of leptospirosis i-tasser: fully automated protein structure prediction in casp the pymol molecular graphics system. palo alto: delano scientific protective efficacy of commercial inactivated newcastle disease virus vaccines in chickens against a recent korean epizootic strain classical swine fever in pigs: recent developments and future perspectives protective immune response of chickens against newcastle disease, induced by the intranasal vaccination with inactivated virus protective effect of individual glycoproteins of newcastle disease virus expressed in insect cells: the fusion protein derived from an avirulent strain had lower protective efficacy challenges and recent advances in affinity purification of tag-free proteins overview of affinity tags for protein purification a critical analysis of codon optimization in human therapeutics effects of codon optimization on the mrna levels of heterologous genes in filamentous fungi expression of codon optimized genes in microbial systems: current industrial applications and perspectives effect of linker flexibility and length on the functionality of a cytotoxic engineered antibody fragment easy expression of the c-terminal heavy chain domain of botulinum neurotoxin serotype a as a vaccine candidate using a bi-cistronic baculovirus system key: cord- -grndfx b authors: koopmans, m.; van den boom, u.; woode, g.; horzinek, m.c. title: seroepidemiology of breda virus in cattle using elisa date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: grndfx b two direct blocking enzyme linked immunosorbent assays (elisa) for the detection of antibodies to breda virus in sera of cattle were compared. an elisa with consecutive addition of antigen and test serum to an antibody-coated plate gave higher positive: negative absorbence ratios than an elisa in which antigen and test serum were added simultaneously. sera collected from breeding and fattening herds in the netherlands (n = ) and the f.r.g. (n = ) were tested, and antibodies to breda virus were demonstrated in % of adult cattle. ninety percent of newborn calves had high levels of maternal antibodies, which waned until the age of months. active seroconversion occurred between and months in most animals. during an outbreak in of neonatal calf diarrhea in a beef herd in breda, ia, u.s.a., a new virus was detected. this "breda virus" (brv) is morphologically and antigenically different from known bovine viruses and causes diarrhea in gnotobiotic and colostrum-deprived calves (woode et al., ) . an antigenic relationship of brv with berne virus (bev) was discovered (weiss et al., ) ; bev has been extensively characterized (weiss and horzinek, ; horzinek et al., a,b) and proposed as prototype of the new family toroviridae horzinek et al., a,b) . other breda viruses were found in the feces from a colostrum-deprived calf in ames, ia (woode et al., ) and in - -month-old diarrheic calves in ohio (saif et al., ) . all viruses shared antigens as determined by enzyme-linked im-munosorbent assay (elisa), with - -fold differences between the homologous and heterologous reactions. for this reason and on the basis of hemagglutination inhibition tests and immune electron microscopy the three isolates were assigned to two serotypes: breda virus serotype i (brv ) is represented by the original breda, ia isolate, and serotype (brv ) comprises the ohio isolate and the second iowa isolate (woode et al., ) . so far, all attempts to grow brv in vitro have failed (woode et al., ) ; recent observations by lamouliatte et al. ( ) need to be confirmed. for this reason, only limited studies on the characterization of the virus (koopmans et al., ) , its diagnosis, pathology and epidemiology (woode, ) were done. in neonatal calves, brv infections spread rapidly with diarrhea commencing as early as - days after birth (woode, ) . large amounts of virus ( viral hemagglutinating units ml- ) were detectable in feces - days after infection. in experimentally infected calves incubation periods ranged from to h (woode et al., ) . the role of older cattle and of other animal species in brv epidemiology is not known. in the present communication we describe the distribution of brv antibodies in the netherlands and the f.r.g. and some epidemiological aspects of the infection; the data were obtained using two modifications of the elisa method. we chose to use brv in our studies because it is relatively stable as compared with brv (koopmans et al., ) . a gnotobiotic calf (gc ) was fed a suspension of brv (iowa isolate at passage in gc) at day of age; diarrheic fecal samples were collected at and h. no other virus was observed to be present, by electron microscopy, hemagglutination and tissue-culture methods, including bovine coronavirus, rotavirus, astrovirus, parvovirus and bovine pestivirus (bvdv). a % suspension in phosphate buffered saline (pbs) was clarified by low speed sedimentation ( ×g for min) and virus from the supernatant was spun through a % (w/w) sucrose solution onto a cushion of % (w/w) sucrose. the interphase was collected, diluted fold in pbs, layered on top of a linear - % sucrose gradient and centrifuged to equilibrium at xg for h. the peak fractions ( . g m - ) were used as antigen in elisa. a gnotobiotic calf (gc ) was fed brv (iowa isolate at passage in gc) at day of age. breda virus was extracted from the diarrheic feces of this calf and semipurified by ultracentrifugation through % sucrose. at and days post-infection, the calf was vaccinated intramuscularly with brv with freund's incomplete adjuvant and at days post-infection with brv without adjuvant. the calf was bled days later. the hyperimmune serum was shown to be free of antibodies to rotavirus, bovine coronavirus, astrovirus and bovine pestivirus (bvdv) and by electron microscopy and tissue-culture isolation methods, no other virus was observed to be present in the feces. immunoglobulins were precipitated from the serum by salt fractionation (hudson and hay, ) and quantified using the lowry method. a fraction of the purified igg was conjugated with horseradish peroxidase as described by nakane and pierce ( ) . for the serosurvey, bovine sera were obtained from veterinary diagnostic institutes in arnsberg (n-- ), niirnberg (n-- ) and neumiinster (n-- ) and from the virology department of the veterinary school in hannover, f.r.g. (n-- ); additional samples were collected at farms in hessen and baden-wiirtemberg, f.r.g. (n= ). the sera representing the dutch cattle population were obtained from animal health services in overijssel (n= ), gelderland (n= ), friesland (n-- ) and west/midden nederland (n-- ), the central veterinary institute (cvi), lelystad (n-- ), and the department of large animal medicine of the veterinary faculty in utrecht (n-- ). the sera obtained from the dutch animal health services and the german veterinary diagnostic institutes were collected for regular screening purposes. the cvi sera included sera from specified pathogenfree (spf) calves and paired samples from milking cows from farms where a tentative diagnosis of winter dysentery was made, based on the following signs: outbreak of rapidly spreading scours in milking herds with a morbidity rate approaching %, lasting - days and resulting in a sharp drop in milk production (hoyer et al., ) . the sera from the veterinary faculty in utrecht included paired samples from an outbreak of acute respiratory disease in month-old calves and six sera from newborn calves with blood protein levels below g l- as a result of insufficient colostrum intake. the sera were coded and stored at - ° c until testing was done. in addition, colostrum samples from healthy dairy cows were tested. a direct blocking elisa was used for mass serology. advantages of this test are that antibodies of different ig classes and from different animal species can be detected, and that antibodies are detected also at low concentrations (ellens, ) . a modification of the method described by brown et al. ( ) was em-ployed. in short, the wells in a polystyrene plate were coated with purified bovine anti-brv immunoglobulin (capture antibody, babrv ig) diluted : in . m na cojnahco ph . by overnight incubation at ° c; all subsequent incubation steps were done in -zl volumes at ° c for i h unless indicated otherwise. to remove excess immunoglobulin, the plates were washed with a solution of . m nac containing . % tween . from this point onward, two different techniques were followed. in the first approach (consecutive method) a : dilution of the brv antigen preparation, purified from calf feces was added to the coated wells in elisa buffer (pbs supplemented with . m nac , mm edta ph . and . % tween ). the plates were washed and the field serum to be tested was added at a : dilution in elisa buffer (pbs supplemented with . m nac , i mm edta ph . and . % tween ). after another rinse, purified bovine anti-brv immunoglobulin conjugated to horseradish peroxidase (babrv ig-po) diluted : in buffer was added, and bound conjugate was visualized after adding the substrate ( . m citric acid, ph , containing . % h and . mm , '-azinodi-( -ethylbenzthiazoline sulfonic acid). high absorbence is seen in the absence of anti-brv antibodies whereas the reaction is blocked when the ~serum contains antibodies. hyperimmune sera against brv and cryptosporidia or rotavirus, prepared similarly to ba-brv ig, served as positive ( % blocking) and negative ( % blocking) reference preparations. all the sera from the netherlands were tested in this system. in the other type of test (simultaneous method), antigen and test serum were mixed in a separate microtiter plate. after i h of incubation, the mixture was pipetted into the babrv ig-coated plates. the remaining steps were similar to the consecutive test method. all sera from germany were tested in this way. a comparison was made between both methods by testing serum samples in parallel. the blocking results were grouped into one of the following clusters: - %, - %, >~ % inhibition of binding of babrv ig-po. positive to negative (p/n) ratios were determined for both elisa modifications by testing a known positive (gc ) and negative (gc ) serum times in parallel. the optimal combination of babrv ig concentration and brv dilution was determined by checkerboard titration: a microtiter plate was coated with serial -fold dilutions of babrv ig in coating buffer and serial -fold dilutions of the antigen in elisa buffer were added. we used a combination of dilutions giving p/n ratios of >/ in our routine testing. the optimal conjugate concentration was determined by testing serial -fold dilutions in elisa buffer at constant antigen and ig-coating concentrations. a dilution with a p/n ratio of >i was used in all subsequent experiments. the results are expressed as blocking percentages; they were calculated using the formula: (ane~ -at) percentage blocking-(aneg --apos) x at = mean absorbence at nm of the test serum; aneg----mean absorbence of the negative reference serum; apos = mean absorbence of the positive reference serum. to detect a possible correlation between percentage of blocking and antibody titer the sera were assigned to groups according to the percentage of blocking ( - %, - %, etc. ) at a dilution of : . from each group, sera were selected and titrated in serial -fold dilutions in the consecutive elisa. titers were calculated from the highest serum dilution resulting in >/ % blocking when compared with the positive and negative reference sera. the correlation coefficient was calculated using pearson's test (dixon and massey, ) . when using the consecutive method more animals were found negative for antibodies to brv or had low blocking percentages (table ) . a higher p/n ratio was also noted between positive and negative reference sera, when tested in the consecutive elisa method ( . + . ) as compared with the simultaneous method ( . + . ); this difference is significant (p< . , n-- , student's t-test). the correlation coefficient between the blocking percentages and the serum titers in the consecutive elisa was . (p< . , pearson's test; fig. ). as can be seen from fig. , the sera with blocking percentages of < and > do not follow the normal distribution pattern. expressed in serum titers, distributions of blocking percentages in sera from the f.r.g. and the netherlands are shown in fig. a and b ; since most sera came from the dutch provinces gelderland and overijssel we show the results obtained with these samples separately (fig. c and d ) . when disregarding the - % columns, a normal distribution can be recognized; median values are situated at - % blocking in fig. b and c (the netherlands and gelderland), and at - % in fig. a and d (germany and overijssel) . in fig. , the percentages of animals with antibodies to brv ( >~ % blocking, corresponding to a serum titer of/> ) are shown for different age groups (n-- ). ninety percent of calves < month old had antibodies, % at months of age; then a gradual decline was noted until at approximately - weeks of age most calves were seronegative. when the animals had reached the age of about months, seroconversion took place. from this age onward an increasing fraction of animals had antibodies to brv, with . % of adult animals detected in our populations; . % of adult animals had blocking percentages of >~ %, corresponding to titers of >/ . six sera from adult bulls of a closed artificial insemination center as well as sera from spf calves and six sera of hypoproteinemic newborn calves had blocking percentages of ~< %. all colostrum samples had high levels of brv antibodies ( - % blocking). in view of the regular occurrence of winter dysentery in the netherlands, we were interested to know whether seroconversion to breda virus antigens would occur. paired serum samples from adult milking cows were, therefore, collected during outbreaks and were tested. seroconversion was defined as > % rise in percentage blocking between sera collected as soon as possible after the onset of disease, as compared with sera collected - weeks later. antibody rises occurred in out of farms, with % (n= ), % (n= ), % (n = ) and % (n = ) seroconversion (average % ). in another group of paired sera, from -month-old calves with a history of acute respiratory disease, seroconversion was seen in four out of animals. three of these animals had low starting titers ( ~< % blocking). in our hands, the simultaneous elisa resulted in comparatively more animals being grouped into the high blocking percentage columns. partially antibody-covered brv antigen (as should be found after preincubation in the simultaneous elisa) will probably not bind as efficiently to the antibody-coated polystyrene plate as brv alone does. during the subsequent washing cycles these complexes may be rinsed away and no binding sites for the conjugate will be left, resulting in a false-positive blocking reaction. in combination with the higher p/n ratios determined for the consecutive elisa we decided to use this method in all further experiments. at this point all german sera had already been assayed in the simultaneous test. when the antibody concentration in a test serum is at equilibrium with the antigen, % blocking will occur. at higher antibody concentrations, however, the elisa results will be the same, leading to an accumulation of animals in the - % blocking group. this is likely to occur when tests are performed at a constant serum dilution ( : in our case ). when disregarding these groups in the frequency distribution of blocking percentages, a normal distribution can be recognized. the graph for the sera from germany has a maximum at lower values as compared with the graph from the netherlands. the different test system used for the german sera does not explain this lower median value: a higher percentage of seropositive animals should be expected, as discussed above, leading to a right shift of the distribution of blocking percentages. a similar difference was observed within the dutch sera between the provinces gelderland and overijssel. one reason for this finding may be a lower incidence of brv infections in farms in overijssel and germany, resulting in less frequent booster infections. a second possibility is that different sampling strategies between the animal health services would give similar results: if a relatively high percentage of animals between and months of age is tested, when most calves are seronegative, lower overall values may be calculated; the age was unknown for many animals. third, a torovirus serotype, different from that in our test, may exist in cattle in europe. when studying our elisa results per animal, lower average blocking percentages are found in the overijssel area; therefore, the presence of a different serotype in this province is a possibility. ninety percent of newborn calves had antibodies to brv. antibodies were absent in specified pathogen-free calves from the central veterinary institute, lelystad, and in -day-old diarrheic calves from the department of large animal medicine with low blood protein levels as a result of insufficient colostrum intake. this indicates that the brv antibodies in young calves are maternally derived as has been found for antibodies to other pathogens (snodgrass and wells, ) ; indeed all colostrum samples tested had high levels of antibodies to brv. the rise in percentage of seropositive animals at months of age indicates an infection in the -month age group. for rotaviruses, enteric infection in the presence of circulating antibodies has been proven (moerman et al., ; snodgrass and wells, ) . we have preliminary data indicating that a similar situation exists for brv. the sudden seroconversion of most calves at the age of - months coincides with the time they are stabled after the grazing period; this leads to closer contact with older cattle and exposure to infection. crouch and acres ( ) have shown that up to % of adult cows excrete rotavirus and % coronavirus with their feces. the role of fecal spread from seropositive cows in brv epidemiology remains to be studied. our findings of . % positive serum samples are in agreement with those of other authors reporting . % by elisa in the u.s.a. (woode et al., ) and % by neutralization test in switzerland ; in gt. britain, % of cattle was found seropositive (brown et al., ) . in that study, however, a serum was considered to contain brv-specific antibodies at > % blocking. when using the same threshold in the interpretation of our results a comparable % of dutch cattle (n-- ) would be found positive. the higher percentages, however, are more likely to reflect the true distribution, since they have also been found in neutralization tests with their intrinsically higher sensitivity. both brv serotypes have been isolated from outbreaks of diarrhea and are pathogenic under experimental conditions (woode, ) . the pathogenicity under farm conditions, however, remains to be proven. with this intention we have tested paired serum samples from field outbreaks of winter dysentery. in four out of the five tested farms, a varying percentage of animals ( - %) showed a distinct antibody rise at the time of the second testing. the seroconversion seen in cattle aged - months (fig. ) does not account for this high number of seroconversions, since only milking cows were tested. further studies are needed to examine the role of brv in this disease. serological evidence for infection with bovine coronavirus in outbreaks of winter dysentery in japan has been presented (takahashi et al., ) ; in the netherlands no such evidence was found (p.w. de leeuw, personal communication, ) . the seroconversions seen in sera from four out of calves, aged months and with a history of acute respiratory disease, may be coincidental although in experimental infections with brv , ocular discharge and hyperpnea were seen (woode et al., ) . detection of breda virus antigen and antibody in humans and animals by enzyme immunoassay prevalence of rotavirus and coronavirus antigens in the feces of normal cows introduction to statistical analysis. mcgraw-hill kogakusha laboratory diagnosis in neonatal calf diarrhoea toroviridae: a taxonomic proposal a new family of vertebrate viruses: toroviridae. intervirology toroviridae, a proposed new family of enveloped rna viruses practical immunology surface proteins of breda virus further studies on breda virus prevalence and significance of viral enteritis in dutch dairy calves enzyme labelled antibodies: preparation and application for localisation of antigens studies on an enteric "breda" virus in calves. nd passive immunity in rotaviral infections bovine epizootic diarrhea resembling winter dysentery caused by bovine coronavirus the proposed family toroviridae: agents of enteric infections purification and partial characterization of a new enveloped rna virus (berne virus) antibodies to berne virus in horses and other animals breda and breda-like viruses: diagnosis, pathology and epidemiology studies with an unclassified virus isolated from diarrheic calves diagnostic methods for the newly discovered "breda" group of calf enteritis inducing viruses comparative studies on three isolates of breda virus of calves we would like to thank the animal health centers of friesland, overijssel, gelderland, west/midden nederland (the netherlands), arnsberg, niirnberg, neumiinster (f.r.g.), the virology department of the veterinary school in hannover and the dutch central veterinary institute, lelystad, for sending us sera. key: cord- -r wkx ml authors: jacobs, sophie; wavreil, fanny; schepens, bert; gad, hans henrik; hartmann, rune; rocha-pereira, joana; neyts, johan; saelens, xavier; michiels, thomas title: species specificity of type iii interferon activity and development of a sensitive luciferase-based bioassay for quantitation of mouse interferon-λ date: - - journal: j interferon cytokine res doi: . /jir. . sha: doc_id: cord_uid: r wkx ml the type iii interferon (ifn-λ) family includes ifn-λ subtypes in man. in the mouse, only the genes coding for ifn-λ and -λ are present. unlike mouse and human type i ifns (ifn-α/β), which exhibit strong species specificity, type iii ifns were reported to act in a cross-specific manner. we reexamined the cross-specificity and observed that mouse and human ifn-λ exhibit some species specificity, although much less than type i ifns. mouse ifn-λ displayed clear species specificity, being -fold less active in human cells than the closely related mouse ifn-λ . this specificity likely depends on amino acids in α helices a and f that diverged from other ifn-λ sequences. human ifn-λ , in contrast, retained high activity in mouse cells. we next developed a firefly luciferase-based reporter cell line, named fawa-λ-luc, to detect ifn-λ in biological fluids with high specificity and sensitivity. fawa-λ-luc cells, derived from mouse epithelial cells that are responsive to ifn-λ, were made nonresponsive to type i ifns by inactivation of the ifnar gene and strongly responsive to ifn-λ by overexpression of the mouse ifnlr . this bioassay was as sensitive as a commercially available enzyme-linked immunosorbent assay in detecting mouse ifn-λ in cell culture supernatant, as well as in serum and bronchoalveolar lavage samples of virus-infected mice. the assay also enabled the sensitive detection of human ifn-λ activity, including that of the divergent ifn-λ with a bias, however, due to variable activity of ifn-λ subtypes. t ype i and iii interferons (ifns) are typically produced in response to viral infection, and these cytokines induce an antiviral state in target cells (isaacs and lindenmann ; kotenko and others ; sheppard and others ) . type i ifns consist of ifn-a subtypes and ifn-b, -o (human) , and -z (mouse) -e and -k. the type iii family of ifns in humans comprises functional genes that express ifn-l (il- ), ifn-l (il- a), and ifn-l (il- b) (kotenko and others ; sheppard and others ) . a fourth ifn-l subtype (ifn-l ) is present in a part of the human population, depending on a dinucleotide frameshift polymorphism upstream of the ifnl gene, which creates or disrupts the open reading frame (orf) encoding ifn-l (prokunina-olsson and others ) . in the mouse, ifnl is a pseudogene, and only ifn-l and ifn-l are expressed. unlike mouse and human type i ifns that exhibit strongly species-specific activity (veomett and veomett ) , type iii ifns were reported to act on cell types from both origins (lasfar and others ; hermant and others ) . the type i ifn family members signal through a unique heterodimeric receptor (ifnar), composed of the ifnar and ifnar subunits. type iii ifns engage a distinct receptor (ifnlr), composed of chains: the ifn-l-specific ifnlr , and il rb which is shared by other il -related cytokines (kotenko and others ; sheppard and others ) . while ifnar is ubiquitously expressed, ifnlr is expressed by a restricted range of cell types and mostly acts at mucosal surfaces. epithelial cells are well-established targets of ifn-l in vivo (sommereyns and others ) , although some immune cells such as neutrophils and dendritic cells have been characterized as ifn-l responders (koltsida and others ; blazek and others ; broggi and others ; espinosa and others ) . although type i and type iii ifns act on distinct receptors, they activate a similar jak-stat transduction pathway, leading to the phosphorylation of stat and stat that associate with irf to form the isgf complex. type i and type iii ifn signaling leads to the transcription of an overlapping set of ifn-stimulated genes (isgs) (dumoutier and others ; ank and others ) . type i ifn in biological samples can be measured by a variety of bioassays, but efficient techniques for type iii ifn quantification are largely lacking. enzyme-linked immunosorbent assay (elisa) for ifn-l detection is time and cost intensive and fails to detect ifn-l . conventional cytopathic effect reduction bioassays to quantify antiviral activity as a proxy for the presence of ifn require the manipulation of infectious virus. luciferase reporter cells that have previously been used for recombinant human ifn-l detection were still responsive to type i ifn and would not allow specific ifn-l detection from biological samples (uze and monneron ) . in this work, we reexamined the species specificity of ifn-l activity, and we developed a very sensitive luciferase-based bioassay specific for type iii ifn detection. the lkr cell line (kind gift from guido bommer, de duve institute, brussels, belgium) is derived from lung adenocarcinoma tissues from a k-rasla mouse ( johnson and others ) . a cells (atcc) were kindly provided by pierre coulie, balb/ t fibroblasts (aaronson and todaro ) those cells, and derivatives, were maintained in dulbecco's modified eagle's medium (dmem) (lonza, vervier, belgium) containing . g/l glucose, supplemented with % fetal calf serum (fcs) (sigma-aldrich, overijse, belgium). african green monkey kidney (vero) cells (atcc) were cultured in dmem supplemented with % fcs, nonessential amino-acid, l-glutamine, and sodium pyruvate. bhk- cells (atcc) were cultured in glasgow's minimum essential medium (gibco; thermo fisher scientific, asse, belgium) supplemented with % newborn calf serum and . g/l tryptose phosphate broth. all media were supplemented with u/ml penicillin and mg/ml streptomycin (lonza). expression vectors used in this study are listed in table . plasmids psj and psj are pcdna derivatives (invitrogen; thermo fisher scientific) used for mouse ifn-l and ifn-l expression in t cells. these plasmids were constructed by cloning the orf encoding ifn-l (psj ) and between the bamhi and xbai sites of the vector. coding sequences were polymerase chain reaction (pcr)-amplified from liver cdna samples of a c bl/ mouse infected with influenza turh (h /n ) strain (hermant and others ) , using the following primers: forward (fw): bamhi-agei-kozak-mifn-l / , ¢-aaa agg atc cac cgg tgc cac cat gct cct cct gct gtt gcc tct g- ¢; reverse (rev): mifn-l / -stop-xbai, ¢-aaa atc tag att atc aga cac act ggt ctc cay tgg c- ¢. the sequence of psj matches the genomic ifnl sequence but diverges from a few nucleotides from that of the previously constructed pcs plasmid (sommereyns and others ) . both plasmids encode functional ifn-l . pph was obtained by pcr-cloning the ifna orf from hela-m cell cdna, between the ecori and noti sites of pcdna (fw: ecori-kozak-ifna , ¢-aaa aga att cac cat ggc ctt gac ctt tgc ttt- ¢; rev: ifna - noti, ¢-aaa agc ggc cgc tca ttc ctt act tct taa act tt- ¢). pmk is a lentiviral vector, derived from ptm , that carries the mouse mx gene promoter driving the expression of the firefly luciferase gene. it is derived from pcclsin. cppt.hpgk.gfp.pre (follenzi and others ) in which a cloning polysite was first inserted to replace the coding sequences, yielding ptm . the firefly luciferase gene from pgl . (promega) was inserted between the xhoi and xbai sites of the vector, and the mx promoter was then cloned from pbsk-mx , a gift from peter staeheli (freiburg university, freiburg, germany), as a bamhi/bsabi fragment. the lentiviral vector psj , used for expression of the mouse ifn-l receptor, was obtained by cloning the ifnlr orf between the bamhi and xbai sites of ptm , a lentiviral vector allowing the coexpression of the cloned gene and of mcherry (hermant and others ) . the ifnlr orf sequence was subcloned in this plasmid from pcr . -licr , kindly provided by laure dumoutier (de duve institute, brussels, belgium). gene inactivation was done with px plasmid (pspcas n- a-gfp) coding for the cas nickase and green fluorescent protein (gfp) (ran and others a). the single guide rnas (sgrnas) were designed using the mit clustered regularly interspaced short palindromic repeats (crispr) design tool website (http://crispr.mit.edu). the selected sgrna pair (sgrna -fw: ¢-cac cgt caa att ctg gcg gct caa g- ¢; rev: ¢-aaa cct tga gcc gcc aga att tga c- ¢; sgrna -fw: ¢-cac cga gac cac ata aac gtg acg a- ¢; rev: ¢-aaa ctc gtc acg ttt atg tgg tct c- ¢) was targeting exon of the mouse ifnar gene (genbank: y . ) and exhibited no expected off-target cleavage site. the sgrnas were cloned into px to form the plasmids psj and psj . these constructs were cotransfected in lkr cells, using transit Ò -lt transfection reagent (mirus bio llc, madison), according to the manufacturer's instructions. after h, gfp-positive cells were sorted by fluorescence activated cell sorting (facs aria iii; bd biosciences) and cloned in -well plates. clones were screened for loss of type i ifn response with an antiviral assay. genome editing of the targeted exon was further confirmed by sequencing (genewiz). isg expression was measured by reverse transcriptionquantitative pcr. ifnar -knockout (ko) and wild-type (wt) lkr cells were treated with u/ml mouse ifn-aa, pg/ml mouse ifn-l , or control supernatant (mock) for h before rna extraction. total rna extraction, reverse transcription, and sybr green quantitative pcr for mrna encoding mouse b-actin, oasl , and usp were performed as previously described (paul and michiels ) . for infection analysis, cells were dissociated with trypsin-edta and suspended in phosphate-buffered saline (pbs) containing % of filtered fcs and . % of paraformalde-hyde. data acquisition was done with an lsrfortessa flow cytometer (bd biosciences) using facsdiva software. data were analyzed using flowjo . . . the rate of infection was defined as the percentage of mcherry-positive cells h postinfection with . pfu/cell tm . for cell sorting, transduced cells were suspended in pbs containing % fcs and mm edta. mcherry-or gfppositive cells were cloned at cell per well in -well plates using the facs aria iii (bd biosciences). immunoblotting stat , p-stat , and b-actin were detected by western blot using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing % acrylamide and run in a tris-glycine buffer. blots were probed with anti-stat polyclonal (sc- ; santa cruz biotechnology, heidelberg, germany), p-stat monoclonal (# ; cell signaling technology, leiden, the netherlands), and anti-b-actin monoclonal (a ; sigma-aldrich) antibodies. mouse ifn-aa, ifn-b, ifn-l , ifn-l , and human ifn-a were produced as described previously from t cells transfected with pcdna -ifn-aa, pcdna -ifn-b (van pesch and others ), pcdna -ifn-l (psj ), and pcdna -ifn-l (psj ). human ifn-a was produced similarly, using pcdna -ifn-a (pph ). supernatant collected from t cells transfected in parallel with the empty pcdna vector was used for control treatment (mock) of cells and diluted as for ifns. recombinant mouse ifn-l ( -ml- ) and human ifn-l ( -il- ), ifn-l ( -il- ), and ifn-l ( -il- ) were purchased from r&d (r&d systems, minneapolis). recombinant human ifn-l was produced as described (hamming and others ) . mouse (ref - ) and human (ref - ) ifn-g were purchased from pe-protech (london, united kingdom). genbank accession numbers for mouse and human ifn-l are listed in table . ifns were diluted in culture medium for cell treatment or in reagent diluent for elisa. mouse ifn-l and ifn-l were quantified by elisa. mouse ifn-aa and human ifn-a antiviral activities were quantified, as described previously, by a cytopathic effect reduction assay in mouse balb- t and human hela-m cells, respectively, using mengovirus (van pesch and michiels ) . similar assays were conducted in human a cells and mouse lkr cells for quantification of ifn-l cross-species activity. ifn- l was quantified by a cytopathic effect reduction assay in a cells, using recombinant human ifn-l as a standard, which was reported to have a similar specific activity (hamming and others ). mouse ifn-l / measurement by elisa was performed using the mouse ifn-l / duoset (r&d systems), according to the manufacturer's protocol. mouse sera were diluted -to -fold, and bronchoalveolar lavage fluids (balfs) were diluted -fold. when required (in case of samples derived from infected cells or mice), infectious virus present in biological samples was ultraviolet light (uv)-inactivated. for uv inactivation, . mm thick fluid samples were exposed at fixed uv dose ( . - . - - j/cm ) with the uv irradiation system biolink (vilber lourmat, eberhardzell, germany), either in -( ml) or in -well ( ml) plate. uv-exposed samples were titrated on bhk- cells by a standard plaque assay to confirm virus inactivation. to confirm that the uv irradiation procedure that was established for picornaviruses effectively inactivates respiratory syncytial virus (rsv), samples of ml containing . · pfu of rsv were exposed to j/cm with the uv irradiation system bio-link at room temperature in a -well plate (uv irradiated samples). as controls similar samples of ml containing . · pfu of rsv were incubated at room temperature in a -well plate (untreated samples). the titers of replicating rsv in the uv irradiated and untreated samples were determined by plaque assay using vero cells. cells were seeded at , cells per well in -well plates for cell supernatant and recombinant ifn analysis or at , cells per well in -well plates for mouse serum and bal analysis. forty-eight hours after seeding, cells were treated with ifn-l / or mock-treated or incubated with uv-treated mouse serum ( -and -fold dilutions) or bal ( -fold dilutions). samples were diluted in culture medium. recombinant mouse ifn-l provided in the elisa kit was used as a standard. firefly luciferase activity was measured h after treatment using the luciferase assay system (promega). twenty microliters and -ml lysis buffer were used in -and -well plates, respectively; ml of the lysate was mixed with ml of substrate for detection. luminescence was measured with a glomax / luminometer (promega). the limit of detection (lod) for each test was established based on the mean of mock-treated samples, plus standard deviations, in all assays. tm is a theiler's murine encephalomyelitis virus (tmev) (strain da) derivative that carries a capsid adapted to infect l cells and the orf encoding the mcherry fluorescent protein cloned as a xbai/bsiwi fragment to replace codons - of the leader protein coding region, in the pkj vector ( jnaoui and michiels ) . mengovirus (a strain of encephalomyocarditis virus) used for ifn bio-assay is an attenuated variant that has been described previously (hermant and others ) . those viruses were quantified by plaque assay on bhk cells. rsv propagation and enrichment were performed as described in schepens and others (schepens and others ) . mouse experiments were conducted according to the national (belgian law / / and / / , belgian royal decree / / ) and european (eu directives / /eu, / /eeg) animal regulations. animal protocols were approved by the ethics committee of ghent university (permit no. la , approval id - ). all efforts were made to avoid or ameliorate suffering of animals. specific pathogen-free female balb/c mice at the age of - weeks were purchased from charles river. the mice were housed in a specific-pathogen-free temperature controlled environment with -h light/ -h dark cycles and given water and food ad libitum. mice were used at weeks of age after adaptation in the animal room for week. for challenge, the mice were sedated with isoflurane and infected intranasally with · pfu of human rsv. mock infection of the control group was performed with hank's balanced salt solution. five days postinfection, bal was isolated under anesthesia with an intraperitoneal injection of avertin ( . % in pbs). a gauge cannula was inserted into the trachea, and cells were collected by washing the airway lumen twice with . ml pbs containing . mm edta. after removing the cells by centrifugation ( min at g), the balf was stored at - °c. serum samples from norovirus-infected and plasmidelectroinjected mice were reused from a previously published experiment (rocha-pereira and others ) to minimize animal experimentation. statistical analysis was performed using prism software (graphpad software, san diego, ca). [***p < . , **p < . , *p < . , and ns no statistically significant difference (p ‡ . )]. unlike mouse and human type i ifns (ifn-a/b) that exhibit strong species specificity, type iii ifns were reported to act in a cross-species manner. to confirm whether a mouse cellbased bioassay would permit the detection of ifn-l subtypes from different mammalian species, the species specificity of mouse and human type iii ifns was reexamined. crossreactive antiviral activity was systematically compared in a cytopathic effect reduction assay, using epithelial cell lines from mouse (lkr ) and human (a ) origin. as expected, type i human (ifn-a ) and mouse (ifn-aa) ifns were extremely species-specific in the antiviral assay, with an activity difference higher than , -fold when applied to mouse and human cells. ifn-l exhibited some level of species specificity although much less than type i ifns (fig. a) . mouse ifn-l displayed the most pronounced species specificity. for a quantity that yielded the same antiviral activity in mouse cells, ifn-l was times less active in human cells than the closely related mouse ifn-l . human ifn-l , ifnl- and, to a lesser extent, ifnl- exhibited significant antiviral effect on mouse epithelial cells. as ifn-l has been shown to have a specific activity similar to that of human ifn-l in human cells, it was quantified accordingly using the antiviral assay (hamming and others ) . remarkably, human ifn-l displayed much more antiviral activity on mouse cells than equivalent amounts of human ifn-l . accordingly, treatment of lkr cells with concentrations of human ifn-l and ifn-l that yielded equivalent antiviral activities in human cells resulted in clear stat phosphorylation after ifn-l but not after ifn-l treatment (fig. b) . in line with their epithelial origin, mouse lkr cells respond to ifn-l. to ensure a selective detection of type iii ifns, the gene coding for the ifnar subunit of the type i ifn receptor was inactivated using the double nickase crispr/crispr-associated (cas) technology (ran and others b). an lkr -ifnar -ko clone was selected for the absence of response to type i ifn and intact response to type iii ifn. sequencing of the targeted exon revealed frameshift mutations in both ifnar alleles, resulting in premature stop codon appearance. in contrast to ifn-l treatment, ifn-a treatment of this clone failed to induce the expression of the ifn-stimulated genes, oasl ( fig. a) and usp (fig. b) , and to protect against infection with tm , a tmev derivative expressing mcherry (fig. c) . to develop ifn-l reporter cells, the lkr -ifnar -ko clone was transduced with a lentiviral vector encoding the firefly luciferase gene under the control of the mx pro-moter. among the clones tested for luciferase activity induction after ifn-l treatment, the best one displayed not more than -fold luciferase activity induction. to try to boost the reporter gene responsiveness to ifn-l, this clone was transduced with sj , a lentiviral vector that coexpresses the mouse ifnlr and mcherry. transduced cells were sorted, based on mcherry expression, and cloned. a selected clone, named ''fawa-l-luc,'' responded vigorously to ifn-l . this clone displayed increased constitutive stat expression and strong stat phosphorylation after ifn-l but not after ifn-a treatment (fig. d) . a significant luciferase activity increase was measured in fawa-lluc cells as soon as h after treatment with pg/ml (quantified by elisa) of mouse ifn-l . maximal induction was reached within h and lasted up to h posttreatment (fig. a) . the sensitivity of the fawa-l-luc assay was compared to that of an available elisa test for mouse ifn-l. mouse ifn-l and - produced by transfected t cells and recombinant mouse ifn-l were quantified in parallel by elisa and the fawa-l-luc assay. in the elisa, responses were linear between , and . pg/ml, the experimental lod of this test (fig. b ). in the fawa-l-luc assay, linearity was reached in a narrower concentration range ( . - . pg/ml for mouse ifn-l ; . - pg/ml for mouse ifn-l ), but a higher sensitivity was observed with the detection limit being below . pg/ml and . pg/ml, for ifn-l and - , respectively (fig. c, d) . based on elisa quantification, mouse ifn-l bioactivity in this assay was -fold higher than that of mouse ifn-l . importantly, after up to passages, the intensity of the luciferase signal in the fawa-l-luc cells tended to decrease, but the sensitivity of the bioassay was preserved because table showing the relative antiviral activity of mouse and human ifn-l on mouse lkr and human a cells. species specificity index was calculated as the ratio between the relative antiviral activity on cells of homologous to nonhomologous species (eg, for mouse ifn: relative activity in mouse cells/relative activity in human cells). relative activities in a given cell line were calculated as the ifn dilutions (starting from ng/ml) that yielded similar antiviral activities. *due to the low amount of ifn-l available, the initial concentration of this ifn was estimated by comparison with human ifn-l antiviral activity that was reported to have a similar specific activity. (b) western blot showing stat phosphorylation in mouse lkr cells min after treatment with control medium (mock) or ifn-l. concentrations of human (hu) ifn-l ( pg/ml) and ifn-l that yielded the same antiviral activity on human a cells were used. as a control, mouse ifn-l was used at a concentration ( . ng/ml) showing equivalent antiviral activity as human ifn-l on mouse cells. results are representative of experiments. the background activity also decreased with high passage numbers. to confirm the specificity of fawa-l-luc cells for ifn-l, we also evaluated any possible luciferase induction after treatment with type i and type ii (ie, ifn-g) ifns. as expected, no signal was observed after treatment with up to , iu/ml of either mouse ifn-aa or ifn-b. human ifn-g also failed to induce a detectable luminescent signal in the fawa-l-luc cells in the tested concentration range ( - ng/ml). the lod for mouse ifn-g was . ng/ml, a concentration at least -fold higher than what is observed in mouse fluids in infectious contexts (pomeroy and others ; claser and others ) . ifn-g is thus not expected to interfere with ifn-l detection in biological samples. we tested the sensitivity of our bioassay for human ifn-l detection. responses were analyzed individually for all human ifn-l subtypes (fig. a-d) . as their cross-species activity differed, different detection sensitivities were reached with our mouse cell-based reporter assay: . pg/ml for ifn-l , pg/ml for ifn-l , . pg/ml for ifn-l , and . pg/ml for ifn-l . the bioassay turned out to be extremely sensitive for human ifn-l detection. it is, however, worth noting that the recombinant ifn-l stock concentration was determined by comparison of its activity with that of a human ifn-l standard. when testing biological samples, potential occurrence of infectious virus may interfere with the viability of reporter cells. it is therefore important to inactivate infectious viruses in such samples. as ifn-l had previously been shown to be acid labile (kugel and others ) , uv inactivation of the virus was preferred. we thus tested whether virucidal uv doses would affect ifn-l activity. to this end, tm virus, mouse ifn-l , and mouse ifn-l were irradiated with increasing uv doses. mock-and uv-exposed samples were then analyzed with the bioassay for mouse ifn-l and ifn-l detection and by plaque assay to determine viral titers. a j/cm uv exposure was sufficient to ensure a virus titer reduction of more than , -fold ( . · pfu to < pfu). at this uv dose, mouse ifn-l activity was not significantly reduced and mouse ifn-l showed a bioactivity reduction of less than % (fig. a) . similarly, ifn-l and ifn-l bioactivities were reduced by %, while human ifn-l and ifnl- were slightly more sensitive to irradiation, with about -fold activity decrease (fig. b) . we then compared the sensitivities of the fawa-l-luc and of the elisa assays to detect ifn-l in mouse serum and bal samples. ifn-l was first quantified in the serum of mice that were either injected intramuscularly with an ifn-l expressing plasmid (pcs ), thus expected to produce circulating ifn-l, or infected with mouse norovirus (rocha-pereira and others ). a preliminary test revealed that the minimal serum dilution that did not interfere with spiked-in ifn-l detection was fold for the bioassay and -fold for the elisa (fig. c, d) with both the fawa-l-luc and elisa assays. ifn-l was clearly detected at day and in the serum of mice that were electroinjected with the ifn-l expressing plasmid, as well as at day postnorovirus infection (fig. a, b) . at day after a single ifn-l injection, the cytokine was detectable in out of mice by elisa and in out of mice by the bioassay. the detection limit was higher for the elisa ( pg/ml) than for the bioassay ( . pg/ml), showing the higher sensitivity of the luciferase-based bioassay (fig. a, b) . next, we compared ifn-l detection by the fawa-l-luc assay and by elisa in balf of balb/c mice that were mock infected or infected with rsv. balf and lungs were collected days postinfection. plaque assay revealed that balf of all infected mice contained between . · and . · pfu/ ml of live replicating virus. the used uv irradiation procedure proved to readily inactivate rsv (rsv titer reduction of more than , -fold; . · pfu to < pfu). five-fold diluted control balf did not modify ifn-l / detection in any of the assays. using -fold dilutions of the balf as in the bioassay, we failed to detect any ifn-l by elisa (fig. c ). in contrast (fig. d) , ifn-l was detected by fawal-luc assay at a very low concentration in the balf of the rsv infected mice ( / ) and not detected in the control group. percentage of ifn-l activity (mean and sd) remaining after uv treatment (n = ) at j/cm . ifn activity was measured in fawa-l-luc cells for ifn concentrations that yielded equivalent luciferase activities ( , rlu) before uv treatment ( pg/ml mifn-l and mifn-l , pg/ml human ifn-l , ng/ml huifn-l , pg/ml huifn-l , and . pg/ml huifn-l ). (c, d) influence of serum dilution on ifn-l detection by fawa-l-luc cells (c) and elisa (d). (c) fawa-l-luc cells were treated in triplicate with fixed doses of and pg/ml mifn-l supernatant and with -fold serial dilutions ( - -fold) of control mouse serum. (d) pg/ml recombinant mifn-l was mixed with -fold serial dilutions of control mouse serum ( . - -fold) before detection by elisa. (a-c) reporter cells were exposed to ifn for h before luciferase assay. student's t-test: */**/***denotes a significant difference in signal compared to no uv exposure (a) or the absence of serum (c). uv, ultraviolet light. we conclude that the fawa-l-luc-based assay described in this study allows to quantify ifn-l from biological samples in a highly sensitive way. we reexamined the species specificity of ifn-l and highlighted a previously overlooked difference in bioactivity between mouse and human type iii ifns. mouse ifn-l displayed a surprising species specificity, with a -fold difference in relative antiviral activity when applied to lkr (mouse) and a (human) cells. this contrasted strongly with the closely related mouse ifn-l ( % amino acid sequence identity), which displayed little species specificity and was times more active in human cells than mouse ifn-l . in contrast, human ifn-l displayed a strikingly strong activity on mouse cells compared to human ifn-l . although ifn-l is not expressed in mouse and rat, it is highly conserved in many mammalian species (key and others ) where it was shown to be cross-reactive. the nonhuman ifn-l orthologs were reported to activate ifnlr in human cells, sometimes with a higher efficacy than human ifn-l (eg, dog ifn-l ) (paquin and others ). the mouse cell response to human ifn-l is thus in line with the ability of ifn-l to be cross-reactive among mammalian species, although we do not have any physiological explanation for this phenomenon. ifn-l proteins follow the typical class ii cytokine structure, consisting of secondary structure elements (a-f) (pestka and others ) . helix a is involved in both ifnlr and il rb binding, while helix d binds il rb and helix f binds ifnlr (gad and others ). among amino acid residues that have been characterized as crucial for ifnlr binding and activation (gad and others ), a few divergences are observed between sequences from mouse and human ifn-l (fig. ) . notably, the more species-specific mouse ifn-l has amino acid residues in a helices a and f that diverge from the other ifn-l sequences, including the related mouse ifn-l . in helix a, gly uniquely replaces the asp residue present in the other sequences. in helix f, residues are unique to mouse ifn-l : asp (ala / asp), gln and bioassay (b) in the serum of ag mice or days after electroinjection of mouse ifn-l (mifn-l ) expressing (pcs ) or empty (pcdna ) plasmids, days after injection of pcs , or days after infection with mouse norovirus. ifnl detection in the serum by elisa was performed without uv exposure to keep maximal sensitivity. (c, d) ifn-l / detection by elisa (c) and bioassay (d) in the bronchoalveolar lavage of balb/c mice, days postinfection with rsv, compared to control mice (mock). balf were uv-exposed before testing. (a-d) the horizontal dotted line represents the lod. mann-whitney: */**indicates a significant difference compared to pcdna group at days or (a, b) or compared to mock (d). balf, bronchoalveolar lavage fluid; rsv, respiratory syncytial virus. (arg / gln), and leu (thr / leu). future site-directed mutagenesis studies could help defining the key residues involved in the species specificity. we designed a highly sensitive bioassay named ''fawa-lluc'' based on luciferase reporter cells, specific for ifn-l detection and quantification. the bioassay is based on a cell line that is naturally responsive to ifn-l in which the type i ifn receptor gene was inactivated. overexpression of the mouse ifnlr receptor in those cells led to increased induction of the reporter gene after ifn-l treatment. unlike previously described bioassays, such as hl- fibroblasts stably transfected with the human ifnlr (uze and monneron ), fawa-l-luc cells permit the selective detection of type iii ifn because they lack the type i ifn receptor. they also fail to respond to ifn-g concentrations that are reached in infected mouse serum. this novel bioassay is efficient in measuring ifn-l from mouse bal and serum. ifn-l activity was hardly affected by uv exposure at virucidal doses, thus allowing detection in infected biological fluids. the high sensitivity of the assay permits sparing biological material as -fold more diluted serum samples could be used for bioassay detection ( - -fold dilution) compared to the elisa ( - -fold). when cell toxicity of the tested samples is suspected, cell viability may independently be confirmed with nonlytic viability assays such as resazurin-based tests, which are compatible with luciferase detection. importantly, our bioassay offers a physiologic analysis, attesting of the functionality of the ifn-l present in the samples, irrespective of specific activity. the bioassay also allows to detect human ifn-l. given the divergent cross-species activity and the higher uv lability of the human type iii ifns, the bioassay might be used for individual subtype analysis, but would not fit for their detection in complex biological samples. finally, it offers a sensitive detection of the divergent ifn-l , for which no efficient commercial test is available. development of t -like lines from balb-c mouse embryo cultures: transformation susceptibility to sv lambda interferon (ifn-lambda), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo ifn-lambda resolves inflammation via suppression of neutrophil infiltration and il- beta production ifn-lambda suppresses intestinal inflammation by non-translational regulation of neutrophil function host resistance to plasmodiuminduced acute immune pathology is regulated by interleukin- receptor signaling basis for regulated rna cleavage by functional analysis of rnase l and ire p role of the interleukin (il)- receptor tyrosine residues for antiviral and antiproliferative activity of il- /interferon-lambda : similarities with type i interferon signaling type iii interferon is a critical regulator of innate antifungal immunity gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by hiv- pol sequences the structure of human interferon lambda and what it has taught us interferon lambda signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses ifn-l sequence alignments. sequences of human and mouse ifn-l regions implicated in receptor binding were aligned. key amino acid residues involved in receptor activation that differs between mouse and human ifn-l and ifn-l sequences are indicated in bold in mouse sequences. residues unique to mouse ifn-l in helices a and f are indicated in bold red. *indicates identical amino acids between human ifn-l and ifn-l human but not mouse hepatocytes respond to interferon-lambda in vivo ifnepsilon is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines virus interference. i. the interferon adaptation of theiler's virus to l cells: mutations in the putative receptor binding site on the capsid map to neutralization sites and modulate viral persistence somatic activation of the k-ras oncogene causes early onset lung cancer in mice selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda (ifnl ) il- a (ifn-lambda ) modulates lung dc function to promote th immune skewing and suppress allergic airway disease ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex novel nonviral bioassays for mouse type i and type iii interferon characterization of the mouse ifn-lambda ligand-receptor system: ifn-lambdas exhibit antitumor activity against b melanoma comparative functional analysis of mammalian ifn-lambda orthologs cardiovirus leader proteins are functionally interchangeable and have evolved to adapt to virus replication fitness interferons, interferonlike cytokines, and their receptors role of interferon-gamma in murine cytomegalovirus infection a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus double nicking by rna-guided crispr cas for enhanced genome editing specificity genome engineering using the crispr-cas system interferon lambda (ifn-lambda) efficiently blocks norovirus transmission in a mouse model protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein il- , il- and their class ii cytokine receptor il- r ifnlambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo il- and il- : newcomers to the interferon family characterization of the murine alpha interferon gene family characterization of interferonalpha , a novel constitutive murine interferon-alpha subtype species specificity of interferon action: maintenance and establishment of the antiviral state in the presence of a heterospecific nucleus the authors are grateful to nicolas dauguet for his help in flow cytometry (ludwig institute for cancer research). work was supported by the eos joint programme of fonds de la recherche scientifique-fnrs and fonds wetenschapellijk onderzoek-vlaanderen-fwo (eos id: ), by interuniversitary attraction poles program initiated by the belgian science policy office (iap-p / belvir), by actions de recherche concertées (arc), and by a pdr grant (t. . ) of fnrs to t.m., and b.s. is a doctoral assistant at the department of biomedical molecular biology of ghent university. no competing financial interests exist. key: cord- - mzsaz a authors: wium, martha; jonker, hester isabella; olivier, adriaan jacobus; bellstedt, dirk uwe; botes, annelise title: dna vaccines against mycoplasma elicit humoral immune responses in ostriches date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mzsaz a in ostriches, the population densities resulting from intensive rearing increases susceptibility to pathogens such as mycoplasmas. in addition to good management practices, vaccination offers an attractive alternative for controlling mycoplasma infections in food animals, instead of using antibiotics, which often leave unacceptable residues. the use of live attenuated vaccines, however, carry the concern of reversion to virulence or genetic recombination with field strains. currently there are no commercially available vaccines against ostrich-infecting mycoplasmas and this study therefore set out to develop and evaluate the use of a dna vaccine against mycoplasma infections in ostriches using an oppa protein as antigen. to this end, the oppa gene of “mycoplasma nasistruthionis sp. nov.” str. ms was cloned into two dna vaccine expression vectors after codon correction by site-directed mutagenesis. three-months-old ostriches were then vaccinated intramuscularly at different doses followed by a booster vaccination after weeks. the ability of the dna vaccines to elicit an anti-oppa antibody response was evaluated by elisa using the recombinant oppa protein of ms as coating antigen. a statistically significant anti-oppa antibody response could be detected after administration of a booster vaccination indicating that the oppa protein was successfully immunogenic. the responses were also both dose and vector dependent. in conclusion, the dna vaccines were able to elicit an immune response in ostriches and can therefore be viewed as an option for the development of vaccines against mycoplasma infections. the ostrich (struthio camelus var. domesticus) is the largest living, flightless bird species belonging to the ratite group ( ) . although ostriches are traditionally endemic to africa, parts of arabia and the middle east ( ), they are reared in several countries across the world. south africa, however, is currently the largest producer of ostrich products (meat, leather, and feathers) on international markets ( ) . here, the majority of ostriches destined for slaughter are reared in free-range systems, but when environmental conditions are unfavorable for grazing, they are reared in grow-out camps from about months of age ( ) . mycoplasma, a genus of bacteria that belongs to the prokaryotic class mollicutes, is known to be highly infectious in these intensive rearing systems ( , ) . characteristic features of mycoplasma include the lack of a cell wall, small at-rich genome and a minimal set of genes ( , ) . the species found to specifically infect ostriches are "mycoplasma struthionis sp. nov." str. ms , mycoplasma sp. str. ms , and "mycoplasma nasistruthionis sp. nov." str. ms ( , ) . they are typically associated with upper respiratory tract infections ( ) , reduced growth rates, downgrading of carcasses and in extreme cases, chick mortalities ( , ) . the severity of infections increases with exposure to environmentally induced stress, other bacterial and viral infections. of the three mycoplasma species, "mycoplasma nasistruthionis sp. nov." str. ms was found to have the highest prevalence ( ) amongst ostriches and therefore was the focus of this study. mycoplasma infections are currently managed using a combination of biosecurity systems and antibiotics. with antibiotics there is the risk of resistance ( ) as well as an accumulation of residues in meat with concomitant risks for consumers ( ) . development of whole organism vaccines for use in ostriches is limited by the slow-growing nature of these organisms and the cost of medium required to sustain growth. dna vaccines, on the other hand, do not require large scale cultivation of the pathogen, can be produced at relatively low cost and are more temperature stable than other vaccines ( , ) . it is therefore an attractive alternative for use in ostriches where the vaccine market is small, relative to e.g., poultry, and ostriches are usually farmed in semi-arid or arid regions where temperatures are frequently high and cold storage of vaccines can be problematic. similar to live vaccines, they are also able to stimulate humoral and cellular immunity as indicated by measuring antibody, t-helper cell and cytotoxic t-lymphocyte responses ( ) . but unlike live vaccines cannot revert back to an infectious form ( ) . el gazzar et al. ( ) has reported the reversion to virulence of a commonly used live mycoplasma vaccine in poultry, ts- , whilst another has reported possible genetic recombination between live vaccines and field strains upon long term use ( ) . this exchange or transfer of genes could have a negative impact on either the pathogenicity or transmissibility of field strains. chromosomal integration of dna vaccines is of some concern, but preclinical and clinical studies have shown the rate of integration of plasmid dna into the host genome to be lower than that of spontaneous mutations ( , ) . to date, dna vaccines have not been tested in ostriches or any other ratites. the aim of this study was to use the oppa protein of "m. nasistruthionis sp. nov." str. ms as antigen in the development and evaluation of dna vaccines in ostriches at different doses and using different vectors. a dna vaccine is a plasmid expression vector containing a gene that codes for a protein antigen. as a result of their parasitic lifestyle ( ), mycoplasmas possess, and rely on, a wide range of transmembrane transport systems for their survival. the extracellular components of these transporters are ideal targets for vaccine development ( , ) . in this study the extracellular oppa domain of an oligopeptide permease (opp) transporter was chosen as protein antigen ( , ) . using mouse models, oppa has to date been evaluated as antigen in subunit vaccines against brachyspira pilosicoli ( ) , moraxella catarrhalis ( ) , and yersinia pestis ( ) , as well as against haemophilus parasuis in pigs ( ) . oppa has, however, not been evaluated in any organism as part of a dna vaccine. in this study we report, for the first time, that a dna vaccine can elicit a humoral immune response in ostriches using oppa as antigen. this response was both dose and vector dependent. site-directed mutagenesis of the oppa gene cultures of "m. nasistruthionis sp. nov." str. ms (genbank: km . ) were obtained from mr j.j. gouws (faculty of veterinary science, onderstepoort, university of pretoria). genomic dna (gdna) was isolated from these cultures using a method described by hempstead ( ) . the type a oppa gene ( ) pci-neo (promega) and vr (vical inc.) were chosen as vaccine vectors (supplementary figure ) . the pci-neo vector is a mammalian expression vector that contains a cmv enhancer/promoter region, chimeric intron and sv late polyadenylation signal sequence. the vr vector also contains a cmv enhancer/promoter region, but in combination with a cmv intron, tissue plasminogen activator (tpa) signal peptide sequence and a bovine growth hormone polyadenylation signal sequence. the mutated oppa gene was sub-cloned into each vector using restriction digestion. for pci-neo, acci and mlui (fastdigest, thermo scientific) and for vr , bamhi (fermentas) restriction enzymes were used according to the manufacturer's instructions. cultures of the pci-neo_oppa and vr _oppa vaccine plasmid were prepared and the respective plasmids purified with an endotoxin-free plasmid dna purification kit (nucleobond r xtra midi plus ef, macherey-nagel, germany). yields were determined using a nanodrop spectrophotometer. prior to large scale plasmid isolation for vaccination, the nucleobond kit's ability to yield supercoiled pci-neo_oppa and vr _oppa plasmids was evaluated by digesting plasmids with the acci and bamhi restriction enzymes, respectively, followed by electrophoresis on a % (w/v) agarose gel. to prepare a sufficient amount of plasmid for vaccination, the plasmids were prepared in batches over several days. the purified plasmids were again evaluated as before to confirm the integrity and supercoiling of the plasmids prior to being used for vaccination. the isolated plasmids were diluted to , , and , µg/ml with sterile pbs ( mm nacl, . mm kcl, mm na hpo , and . mm kh po , ph . ), respectively, a day before use. dilutions were prepared under aseptic conditions, in sterile serum glass bottles, closed with inert silicone stoppers and sealed with a tear away center aluminum cap (sigma-aldrich). ethical approval was obtained from the stellenbosch university animal ethics committee (su-acum - ) as well as the south african department of agriculture, forestry and fisheries (reference: / / / / ) in terms of section of the animal disease act (act no. of ). a group of ostrich chicks of ± -months-old were randomly selected from a chick rearing unit in the fraserburg district (northern cape province, south africa). since fraserburg is outside the major commercial ostrich production region, this limits pathogen exposure during the critical - months-old rearing phase ( ) . as per standard practice, upon reaching a weight of about kg (± months of age), trial ostriches were transported back (± km) to a commercial ostrich farm in the oudtshoorn district (western cape province, south africa). three-month-old chicks were chosen for vaccination to allow sufficient time for a primary immune response to develop before being transported to a higher stress environment with concomitant increase in pathogen load and increased risk of exposure to mycoplasma. the chicks were quarantined for days after relocation and random testing was performed for avian influenza. this is required by avian influenza control measure before ostriches can be released and allowed to mix with other populations on any farm to which they have been transported. at the start of the trial, each ostrich was tagged with a unique number for identification under the right wing, in accordance with standard ostrich farming practices. the trial was conducted under similar conditions to which a commercial vaccine would be administered and therefore trial ostriches were at all times housed and treated in the same manner as non-trial ostriches on the farms. in fraserburg, the chick rearing units were open air camps ( , m ) with - chicks per camp. in oudtshoorn, ostriches were kept in open air grow-out camps ( , m ) with - ostriches per camp. the ostriches received food and water ad libitum and were only handled by trained and experienced farm personnel. trial birds were not kept separately but grouped with other ostriches of the same weight and age. the ostriches were randomly allocated to seven treatment groups of ostriches each. the pci-neo_oppa and vr _oppa vaccine groups were vaccinated at a dose of , , and , µg, respectively. the last group was the control group that did not receive any vaccine. the dose typically administered to poultry ranges from . to µg when injected intramuscularly, with a booster dose after - weeks ( - ). dunham ( ) indicated that larger animals may require a larger dose of - , µg, but that this needs to be optimized for individual vaccines since the dose required is influenced by the target species, its size and the efficiency of plasmid delivery. the different doses used in our study were therefore selected to compensate for the increase in weight of the ostriches during the course of the trial and the fact that no adjuvant was used in the vaccine formulation to limit possible skin reactions which can impact hide quality. vaccine doses were administered in a single ml volume at week and a booster injection administered at week by intramuscular injection in the upper thigh. blood samples ( ml) were drawn from the jugular vein using gx / ′′ needles (vacuette r ) and collected in vacuette r z serum sep clot activator tubes at week , , , and . serum was separated by centrifugation at low speed for min and stored at − • c. week and samples were collected in fraserburg, and week and samples in oudtshoorn. the ostriches were moved from fraserburg to oudtshoorn days prior to the week sampling and booster injections were therefore administered during the quarantine period. ostriches were monitored after vaccination for any adverse reactions. for analysis of anti-oppa antibody responses, recombinant oppa protein was produced for use as coating antigen in an enzyme-linked immunosorbent assay (elisa). to this end, the sdm corrected oppa gene was pcr amplified and sub-cloned into a pgex- t- vector (ge healthcare life science, uk) using restriction digestion. primer sequences with bamhi and noti restriction sites are shown in supplementary table . for protein expression, the pgex- t- _oppa plasmid was transformed into escherichia coli bl (de )plyss cells (promega) and freezer stocks prepared. an overnight culture (from a freezer stock) and expression cultures were prepared using terrific-broth (tb) medium. expression of the glutathione s-transferase (gst) oppa fusion protein was induced at an od between . and . by the addition of . mm iptg, and cells harvested at and h by centrifugation ( , × g at • c). the cells were resuspended in xten extraction buffer [ mm tris-hcl, mm edta, mm nacl, . % triton x- , . m dithiothreitol and % glycerol (v/v)] at µl extraction buffer per ml culture used for centrifugation. protease inhibitor was also added to the ten buffer ( tablet per ml extraction solution of complete ultra tablets, mini, easypack tablet, roche). the gst-oppa protein was isolated using glutathioneagarose chromatography (sigma-aldrich) under gravity flow at • c according to the manufacturer's instructions. a sample ( ml) was prepared for loading of the column by treating the resuspended pellets with three freeze-thawing cycles ( min at • c followed by min at − • c) and five cycles of s sonication followed by min on ice. the samples were then triturated three times through a gx / ′′ needle (avacare) into a ml injekt-f syringe (bbraun). this was repeated using a gx / " needle (nipro) followed by centrifugation at , × g for min ( • c) to obtain a clear supernatant which was loaded onto the column under gravity flow at • c. fractions ( ml) were collected and their protein concentration determined using a modified bradford assay ( ) . expression and isolation products were analyzed using sds-page ( ) and western blot analysis ( ) . microtiter plates (maxisorp, nunc) were coated overnight at • c with µl of recombinant gst-oppa protein diluted to µg/ml in carbonate buffer ( mm nahco , ph . ). to block non-specific binding, µl/well casein buffer ( mm tris-hcl, mm nacl, . % casein and . % thiomersal, ph . ) was added and the plate incubated for h at • c. serum samples were diluted : with casein-tween [casein buffer containing . % (v/v) tween r ] and µl/well added to the plate in triplicate before incubation for h at • c. biotinylated rabbit anti-ostrich ig polyclonal antibodies, prepared as previously described ( ), were next added at a dilution of : in casein-tween and incubated for h at • c. a streptavidin horseradish peroxidase (hrp) conjugate mixture [ ml streptavidin hrp (invitrogen), ml . % casein buffer, and ml % glycerol], diluted : with casein-tween, was then added ( µl/well) and the plate incubated for h at • c. finally, µl/well substrate solution ( . mg/ml abts, . µl/ml h o , . m citrate buffer, ph ) was added and absorbance measured at nm with a thermo scientific multiskan ex plate reader after min incubation at • c. after each incubation step the content of the plate was first decanted followed by washing five times with pbs-tween [ mm nacl, . mm kcl, mm na hpo and . mm kh po , ph . , . % (v/v) tween r ] and washing three times with milli-q r water. between the coating and blocking steps washing was not applied. a first-row column blank (containing all components except ostrich serum) was used on each plate to blank absorbance values. all plates contained controls to monitor plate to plate variation. the controls were serum samples representing the week , , , and sampling points of a single ostrich, randomly selected from the pci-neo_oppa , µg group based on high titers produced after vaccination. prior to the analysis of samples, the elisa was optimized with regard to coating concentration ( - µg/ml), serum dilution ( : - : ) as well as number of washing cycles between steps. non-specific binding of anti-oppa antibodies during elisa analysis was evaluated by coating the wells with carbonate buffer ( µl/well) containing no capture antigen and serum from a vaccinated bird that gave a high absorbance value at a dilution of : when using the gst-oppa protein as capture antigen. the absorbance values obtained are referred to as elisa titres, which are a measurement of the antibody levels produced when using a serum dilution of : in the elisa. the weight of trial birds was monitored and recorded at weeks , , and . to determine the possible influence of existing mycoplasma infections on immune response data, trachea swabs were collected at weeks , , , and and tested for the presence of mycoplasma infections by pcr. birds were, however, not excluded from the trial if they tested positive since ostriches are often naturally infected with mycoplasmas. swab samples were collected using dry sterile swabs (copan) which were then rinsed in µl sterile pbs buffer. the eluate was tested by pcr using a generic primer pair specific for the mycoplasma genus ( ). positive samples were further evaluated for the presence of the ostrich-infecting mycoplasmas ms , ms , and ms ( ). statistical analysis of the elisa titer data was performed using the general linear model (glm) procedure in the agrobase generation ii r (agronomix software inc.) software. analysis of variance (anova) and least significant difference (lsd) values were calculated at a significance level of . . the bp oppa gene was successfully amplified from the ms gdna and cloned into a pgem r -t easy vector as confirmed by sequencing. using sdm, all mycoplasma tga codons in this plasmid were successfully mutated to universal tgg codons in seven consecutive steps of sdm as confirmed by sequencing (supplementary figure ) . the mutated oppa gene was successfully sub-cloned into the pci-neo and vr vaccine vector as confirmed by sequencing (supplementary figure ) . large scale production of the dna vaccines was achieved and approximately mg of plasmid was isolated for each of pci-neo_oppa and vr _oppa. the / absorbance ratio of the isolated plasmids ranged from . to . . the integrity of the isolated plasmids was confirmed with agarose electrophoresis and most of the pdna was in the required supercoiled conformation. a supercoiled conformation has a higher transfection rate into mammalian cells, is less susceptible to intracellular degradation and is more effective at inducing an immune response than other plasmid conformations ( , ) . ostriches were vaccinated with the prepared dna vaccines and no adverse reactions were observed at the injection sites (redness, swelling, inflammation, or allergic reaction) during and after the trial. ostriches resumed normal behavior such as eating, walking around and exploring immediately after vaccination (no lameness or loss of appetite). sub-cloning of the mutated oppa gene into the pgex- t- vector was successful as confirmed by sequencing frontiers in immunology | www.frontiersin.org (supplementary figure ) . sds-page and western blot analyses confirmed that the recombinant oppa protein was expressed successfully as an n-terminal gst-fusion protein with the predicted size of kda ( kda due to the gst tag) (figures a,b) . bradford analysis indicated that the oppa protein was eluted between fraction and with the highest concentration obtained in the th fraction. ostriches with more than two missing sampling points were excluded from elisa and subsequent statistical analysis. there was no non-specific binding of the anti-oppa antibodies in ostrich serum and the plate control samples indicated limited plate to plate variation. the titer values resulting from vaccination with the pci-neo_oppa and vr _oppa vaccines are shown in figures a,b , respectively. the titer values of the control group, which received no vaccine, did not show significant variation over time. however, there was a slight increase in the average titer at week . vaccination with the pci-neo_oppa vaccine (figure a ) resulted in significant treatment x time interactions (p = . ), but only the and µg doses resulted in average elisa titers that differed significantly from the control. this was also only at week after a booster vaccination was administered. the average titers of the different doses were, however, not significantly different from one another. vaccination with the vr _oppa vaccine (figure b ) resulted in a significant treatment × time interaction (p < . ) and all three doses resulted in average elisa titers that differed significantly (lsd = . ) from the control. this was again only at week after a booster vaccination was administered and all the doses differed significantly from one another. the vaccinated groups as well as the control group gained weight from weeks to , but from weeks to there was an average weight loss of . kg ( table ) . there was, however, no statistically significant difference between the treatment groups and the control group over the weeks period (p = . for pci-neo_oppa and p = . for vr _oppa). the presence of mycoplasma infections was monitored with pcr during the trial ( table ) . mycoplasma infections could not be detected in any of the groups at weeks , , and . infections were, however, detected at week . at this point the ostriches had been in oudtshoorn for days, of which days were out of quarantine. in the groups that received the vr _oppa vaccine the highest percentage of infections at week was due to ms ( . %) followed by ms ( . %) and ms ( . %) ( table ) . compared to this, the groups that received the pci-neo_oppa vaccine had fewer mycoplasma infections. amongst these the highest percentage of infections was again due to ms ( . %) followed by ms ( . %) and ms ( . %). for both vaccine groups, some of the birds were infected by more than one of these mycoplasma species. the only group that had no pcr-detectable mycoplasma infections at week was the group that had received , µg of the vr _oppa vaccine and the control group. in this study, dna vaccines were developed for ostriches using the oppa gene of an ostrich-infecting mycoplasma (ms ) as vaccine antigen. the expression vectors used (pci-neo and vr ), were selected based on dna vaccine studies in other birds, and on their immunostimulatory characteristics ( ) ( ) ( ) . after vaccination of ± -month-old chicks a statistically significant anti-oppa antibody response could not be detected at week , although a general trend of increases in the average titer values was observed from week to for groups that received the vr _oppa vaccines. based on previous studies using inactivated vaccines in ostriches ( , ) , we expected a primary immune response about weeks after the first vaccination (i.e., between weeks and ). after administering a booster vaccination, both dna vaccines were able to elicit a statistically significant anti-oppa antibody response. as these responses against oppa were significant in comparison to the negative responses following the first vaccinations, this is evidence of a secondary immune response as a result of immune memory. if memory cells are produced after the initial contact with an antigen, subsequent exposure to the same antigen will allow the host to recognize the antigen faster, and with greater magnitude ( , ) . the responses elicited by the dna vaccines were, however, dose dependent as well as vector dependent since different results were produced by each vaccine when using the same dose. this highlights a possible role of the vaccine vector in the observed antibody responses. the vr _oppa vaccine, on average, elicited higher titer values compared to the pci-neo_oppa vaccine, which might be due to the tpa signal sequence of the vr plasmid, which is situated upstream of the oppa gene, and assists with protein expression in mammalian cells and export of the protein out of the cell. this would in turn increase the immunogenicity of the antigen ( ) . in addition to this, sequences in the plasmid backbone have intrinsic immunostimulatory activity which enhance the immune system's response to the expressed protein antigen ( , ) . over the whole vaccination trial, the health of the ostriches was monitored by recording weight gain. all groups gained weight up to week , but toward week their weight gain decreased. given that this trend was also observed for the control group ostriches, the weight loss cannot be ascribed to vaccination. the decrease in weight did, however, coincide with the movement of the birds to a new location. ostriches are known to be severely affected by stress and during this period of adapting to changes in social dynamic and housing environment, reduction in weight gain and even weight loss is normal amongst farmed ostriches ( ) . the presence of existing mycoplasma infections at the start of the trial, as well as changes in infection status during the trial, were also monitored by pcr analysis of trachea swabs. mycoplasma infections could only be detected at week , which was after they were moved and exposed to the farm environment in oudtshoorn for more than days. the absence of detectable mycoplasma infections at the earlier time points may be ascribed to fraserburg being outside of the commercial ostrich production region. the oudtshoorn district, on the other hand, has a higher incidence of mycoplasma infections especially during seasonal changes and our trial was conducted during late autumn. amongst the vaccinated groups, the number of detected ms and ms infections were low compared to ms , with only the µg vr _oppa and control group having no detectable ostrichinfecting mycoplasmas. in future trials, careful consideration should be given to the timing of the primary and booster vaccination relative to the time of introduction to growout conditions. it is possible that the mycoplasma infections had an influence on the observed antibody responses. in a study done by yang et al. ( ) using recombinant oppa protein as subunit vaccine against m. catarrhalis in mice, it was found that the elisa titer values were already raised at the start of the vaccination trial which they ascribed to the presence of existing systemic antibodies to oppa as a result of existing infections. despite the possible presence of antibodies due to mycoplasma infections, this did not mask the detection of a dose dependent antibody response to the oppa protein of ms during vaccination. in conclusion, this is the first study to show that dna vaccines are able to elicit an antibody response in ostriches against a mycoplasma antigen in spite of ostriches being prone to environmentally induced stress conditions that can suppress immune function. the mycoplasma oppa protein was sufficiently immunogenic to induce an anti-oppa antibody response and this response was firstly, dose dependent and secondly, required a booster vaccination. further trials are required to determine the extent of protection provided by this mycoplasma antigen relative to the timing of the primary and booster vaccination before introduction to grow-out conditions. this study (field trial) was carried out in accordance with the recommendations of the south african department of agriculture, forestry and fisheries (reference: / / / / ) in terms of section of the animal disease act (act no. of ) . the protocol was approved by the stellenbosch university animal ethics committee (su-acum - ). mw performed the sdm, prepared the dna vaccine and expression plasmids, optimized the expression of the recombinant oppa protein, and the initial optimization of the elisa. hj further optimized the elisa and evaluated the anti-oppa antibody responses during the vaccination trial. ao assisted with the vaccine trial. mw wrote the manuscript with support from ab. ab supervised the project with the assistance of db and ao. ab and db conceived the original idea. all authors provided critical feedback and helped shape the research, analysis and manuscript. this work was supported by the technology and human resources for industry programme (thrip), south africa (project reference tp ) and south african ostrich business chamber. we would like to thank vical inc., usa for supplying the vr vector for research purposes and mr. j. j. gouws (faculty of veterinary science, onderstepoort, university of pretoria) for the culturing of the mycoplasma bacteria. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material ratite nonmonophyly: independent evidence from novel loci molecular genetic relationships of the extinct ostrich, struthio camelus syriacus: consequences for ostrich introductions into saudi arabia profile of the south african ostrich market value chain bird handling, transportation, lairage, and slaughter: implications for bird welfare and meat quality ostrich diseases investigations into mycoplasma infections in south african ostriches. the third international ratite science symposium molecular biology and pathogenicity of mycoplasmas a systematic review of mycoplasma and ureaplasma in urogynaecology identification of three novel mycoplasma species from ostriches in south africa proposal for molecular tools for the epidemiology of contagious bovine pleuro pneumonia and classification of unknown mycoplasma sp. isolated from struthio camelus mycoplasmas and their host: emerging and re-emerging minimal pathogens antibiotic residues in food: the african scenario dna-antiviral vaccines: new developments and approaches-a review immunization of animals: from dna to the dinner plate dna vaccines: an historical perspective and view to the future dna vaccines: ready for prime time? characterization of a ts- -like mycoplasma gallisepticum isolate from commercial broiler chickens the development and application of a mycoplasma gallisepticum sequence database detection of integration of plasmid dna into host genomic dna following intramuscular injection and electroporation biodistribution of dna plasmid vaccines against hiv- , ebola, severe acute respiratory syndrome, or west nile virus is similar, without integration, despite differing plasmid backbones or gene inserts atp-binding cassette transporters are targets for the development of antibacterial vaccines and therapies bacterial surface proteins and vaccines oppa, the substrate-binding subunit of the oligopeptide permease, is the major ecto-atpase of mycoplasma hominis the identification of oppa gene homologues as part of the oligopeptide transport system in mycoplasmas evaluation of recombinant brachyspira pilosicoli oligopeptide-binding proteins as vaccine candidates in a mouse model of intestinal spirochaetosis characterization and evaluation of the moraxella catarrhalis oligopeptide permease a as a mucosal vaccine antigen the abc transporter protein oppa provides protection against experimental yersinia pestis infection immune response to oligopeptide permease a (oppa) protein in pigs naturally and experimentally infected with haemophilus parasuis an improved method for the rapid isolation of chromosomal dna from mycoplasma spp ostrich manual. elsenburg: western cape department of agriculture cross-protection among lethal h n influenza viruses induced by dna vaccine to the hemagglutinin protective efficacy of dna vaccines against duck hepatitis b virus infection turkeys are protected from infection with chlamydia psittaci by plasmid dna vaccination against the major outer membrane protein dna vaccination in the avian a dna prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge protection of chickens against infectious bronchitis virus with a multivalent dna vaccine and boosting with an inactivated vaccine induction of a protective response in ducks vaccinated with a dna vaccine encoding engineered duck circovirus capsid protein sequential dna immunization of chickens with bivalent heterologous vaccines induce highly reactive and cross-specific antibodies against influenza hemagglutinin the application of nucleic acid vaccines in veterinary medicine evaluation of dna vaccines against mycoplasma nasistruthionis sp. nov. str. ms infections in ostriches and the production of iga heavy chain proteins. master's dissertation the development of a dna vaccine against mycoplasma nasistruthionis sp. nov. for use in ostriches antibody responses to la sota strain vaccines of newcastle disease virus in ostriches (struthio camelus) as detected by enzyme-linked immunosorbent assay impact of plasmid supercoiling on the efficacy of a rabies dna vaccine to protect cats transgene expression of transfected supercoiled plasmid dna concatemers in mammalian cells vaccinia expression of mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of m. tuberculosis ag . microbes infect measuring the effects of an ever-changing environment on malaria control comparison of five expression vectors for the ha gene in constructing a dna vaccine for h n influenza virus in chickens growth rate and hatching date in ostrich chicks reflect humoral but not cellmediated immune function generating memory with vaccination vaccination to gain humoral immune memory effect of plasmid backbone modification by different human cpg motifs on the immunogenicity of dna vaccine vectors cpg dna as a vaccine adjuvant the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © wium, jonker, olivier, bellstedt and botes. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -t hoox e authors: dearden, c. j.; al‐nakib, w. title: direct detection of rhinoviruses by an enzyme‐linked immunosorbent assay date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: t hoox e this paper describes the first enzyme‐linked immunosorbent assay for the detection of rhinovirus antigens in clinical specimens (nasal washings), either directly or following overnight cell culture amplification. the assay takes approximately hours to perform and utilizes the same rabbit antirhinovirus hyperimmune serum as both the capture and detecting antibody. the latter has been biotin‐labelled and is detected via a streptavidin ‐galactosidase preformed complex. this new assay has been found to be very sensitive, detecting human rhinovirus (hrv)‐el and hrv‐ at titres as low as ( . ) tcid( ) μl(− ) and < ( ) tcid( ) μl(− ), respectively. furthermore, when different human rhinovirus serotypes were tested in both the hrv‐el and hrv‐ elisa systems a total of ( %) were found to be cross‐reactive. of clinical specimens tested by virus isolation, cell‐culture‐amplified (cca) elisa, and direct elisa, were positive by isolation, by cca‐elisa, and by direct elisa. the overall correlation of the cca and direct elisa techniques with virus isolation was found to be . % and . %, respectively. the present study demonstrates that the elisa system developed is a sensitive technique for the diagnosis of rhinovirus infections. rhinoviruses are the major causative agent of the common cold and could be responsible for at least half of all acute respiratory infections in humans [mogabgab, ; higgins et al, . most rhinovirus infections are trivial and self-limiting in that only a minor upper respiratory tract illness of some - days duration occurs. however, in a proportion of cases, particularly in immunocompromised individuals and children with a history of bronchitis and allergy, infection can be more serious [craighead et al, . in addition, there is some evidence to suggest that rhinovirus infections are associated with exacerbations of bronchitis and asthma [gregg, . furthermore, krilov et a have recently detected rhinoviruses in out of ( %) specimens from infants with lower respiratory tract infection [krilov et al, . rhinoviruses are therefore appropriate targets for treatment with antiviral chemotherapeutic agents. rapid diagnosis of rhinovirus infection will be necessary so that antiviral treatment can be initiated. however, at present, virus isolation and identification, in an appropriate cell-culture system, may take up to weeks to complete. we have therefore developed an enzyme-linked immunosorbent assay (elisa) so that this time interval can be reduced to approximately hours. in this test the capture and the detecting antibody are both prepared from the same rabbit hyperimmune antirhinovirus serum. the latter, however, is labelled with biotin and is then detected using a streptavidin- -galactosidase preformed complex. this system was proven to be both sensitive and specific. in this paper we describe an assay which can be used to detect rhinoviral antigens both directly in nasal washings and following overnight cell culture amplification of the same specimen. the results obtained were compared with those yielded by conventional virus isolation procedures. the rhinovirus used to establish the elisa procedure was the untyped human rhinovirus el. although a member of the rhinovirus genus according to its physical and biochemical characteristics, hrv-el has not yet been assigned a serotype number in the collaborative scheme [kapikian et al, ; kapikian et al, . hrv-el was grown to high titre by infecting confluent monolayers of ohio human epithelial carcinoma (hela) cells, in -oz medical flats, overlaid with -ml of hela maintenance media (bme containing % foetal calf serum, . % tryptose phosphate broth, . % nah co,, mm mgcl,, and antibiotics). after the cytopathic effect had developed, the virus was released from the cells by three cycles of freezing and thawing. the tissue culture fluid containing the virus was then clarified by low-speed centrifugation ( , rpm for minutes) and stored at - ooc. control antigen was prepared in the same way except that the cells were not infected. all the other rhinoviruses used in this study were passaged once from laboratory stocks. ohio hela cells, grown to confluence in -cm plastic tissue culture flasks (sterilin), were infected with virus and overlaid with -ml hela maintenance media. the tissue culture fluids were then harvested as for hrv-el and stored at - ooc. the titre of all the rhinovirus stocks were estimated by titration in ohio hela cells. the high titre hrv-el, prepared as an antigen, was further purified by fluorocarbon extraction. one volume of arklone (ici) was shaken, at room temperature, with two volumes of antigen; and the mixture was then centrifuged at , rpm for minutes. the aqueous layer was harvested and centrifuged at , rpm for hours. the virus pellet was then resuspended in a small volume at . m nacl/o. m tris buffer (ph . ) and mixed with an equal volume of freund's incomplete adjuvant. rabbits were immunized with the purified antigen by inoculating . ml of the emulsion intramuscularly in each hind leg followed by ml intravenously weeks later. the animals were bled week later, and the hyperimmune serum was found to have a neutralisation titre of : , against tcid,, of the homologous virus. a hyperimmune serum to hrv- was prepared following the same schedule and was found to have a neutralisation titre of : against tcid,, of the homologous virus. a small aliquot of the hyperimmune serum was dialyzed overnight at °c against three changes of . m nahc (ph . ) (b.d.h.). in ml of dimethylsulphoxide (sigma) . mg of biotin (n-hydroxy-succinimidobiotin) (sigma) was dissolved. two hundred microlitres of the biotin preparation was added to each millilitre of the hyperimmune sera, and the mixture was allowed to react for hours, mixing slowly on a laboratory stirrer, at room temperature. the mixture was then dialyzed overnight at °c against three changes of . m phosphate-buffered saline (ph . ) (difco) [gary et al, . eighteen volunteers inoculated intranasally with hrv-el or saline, who took part in a separate trial at the common cold unit [zerial et al, . provided samples for this study. nasal washings were collected on days after virus challenge by instilling millilitres of hanks' balanced salt solution into each nostril. the expelled fluid was mixed with an equal volume of nutrient broth and stored at - °c. virus in nasal washings was isolated in ohio hela cells. the streptavidin wash (amersham) was supplied as a mg ml-' solution in mm phosphate buffer (ph . ) containing . % w/v sodium azide and was diluted to a working concentration ( / ) with phosphate buffered saline (ph . ) (difco) for the test. the streptavidin preformed complex was supplied as streptavidin-biotinylated p-galactosidase complex (amersham) and was diluted to a working concentration ( / ) with phosphate buffered saline (ph . ) (difco) for the test. ortho nitrophenyl-p-d-galactosidase (onpc) (sigma), a water-soluble yellow product that absorbs light at nm, was used as the basis of the substrate and prepared for the test as follows: . mg ml-i onpg, mm mgcl, (b.d.h.), and . m -mercaptoethanol (sigma) in phosphate buffered saline (ph . ) (difco) [craven et al, . all assays were performed in rigid, nonsterile, u-bottomed, -well polystyrene plates (cibco/nunc). wells were coated overnight at oc with pl of rabbit hyperimmune antirhinovirus-el serum diluted / , with . m carbonate-bicarbonate buffer (ph . ) (elisa coating buffer, don whitley scientific ltd). plates were emptied and pl of % v/v bovine serum albumin (bsa) (sigma) in pbs was added for hours at °c in order to block any nonspecific binding. the hrv-el antigen, which had a titre of approximately lo tcid,, pl-l, was diluted in log,, steps in pbs-tween + . % v/v bsa + % v/v control antigen. the control antigen (uninfected tissue culture fluid) was also diluted in the same way. plates were washed three times with pbs-tween ( p per well) using a -outlet minimicrowash (skatron) and p of hrv-el or control antigen was added to a set of wells and plates were then incubated overnight at °c. after washing as before, pl of the streptavidin wash, diluted / with pbs, was added to the wells in order to block any nonspecific binding to endogenous biotin in the specimens. the plates were incubated at room temperature for minutes, followed by five washes with p per well of pbs-tween. one hundred microlitres of biotinylated antiserum, diluted to its optimal working dilution with pbs-tween + . % v/v bsa + % v/v control antigen, was added and the plates were incubated at oc for hours. after three washes as before with pbs-tween, pl of streptavidin / -galactosidase preformed complex, diluted / with pbs-tween + . % v/v bsa, was added to the plates, which were again incubated at °c for hours. plates were then washed five times with pbs-tween, pi per well, and pl of substrate (prepared approximately minutes prior to use) was added to all the test wells plus a row of wells, not used in the test, as a blank. plates were left covered at oc and read hourly at nm using an automatic plate reader (titertek, multiscan, flow laboratories) until the colour intensity exceeded its maximum. as each dilution of hrv-el or control antigen was added to a duplicate set of wells, the mean optical density of each set was calculated. a positive result was recorded when the mean optical density of the test wells was . times the mean optical density of a similar dilution of control angiten. this "cutoff" value was set after experiments were performed in which a series of negative and positive specimens at various dilutions were assayed. mean optical densities recorded for the negative specimens never exceeded . times the mean optical density of the control antigen (undiluted), whereas the mean positive specimen optical densities were always > . times the mean control antigen optical density. a similar assay for hrv- was also developed using the same procedure as described for hrv-el. the optimal dilutions of the reagents used in the elisa systems were determined by checkerboard titrations. experiments to determine optimal incubation times and temperatures were performed once the reagent concentrations had been standardized. cross-reactivity experiments were performed with a total of different human rhinoviruses and four other control viruses (influenza a/eng/ / , coronavirus , echovirus , and coxsackie a ) plus control antigen (uninfected tissue culture fluid) as controls. the titre of all the viruses was adjusted to approximately lo .' tcid ml-' by diluting with pbs-tween + . % v/v bsa + % v/v control antigen, whereas the control antigen was used undiluted in the test. the antigens were then tested in both the hrv-el and hrv- elisa systems according to the described protocol. the "cutoff" value was calculated as stated in the elisa procedure. therefore, a positive result was again recorded when the mean optical density (od) of a duplicate set of sample wells was . times the mean optical density calculated for the control antigen (undiluted). nasal washings, taken on consecutive days, from the volunteers challenged with hrv-el or saline were tested for the presence of hrv-el antigen in the hrv-el elisa system. initially a cell culture amplification procedure was employed in which p of each nasal washing was inoculated, in duplicate, into monolayers of ohio hela cells grown in microtitre plates (nunc microwell plate f with lid). after minutes of adsorption at oc, the nasal washings were removed and pl hela maintenance media was added to each well. the plates were incubated at oc overnight. after one cycle of freezing and thawing pl from each test well was removed and placed in the corresponding well of a precoated elisa plate. the plates were then incubated overnight at "c, and the test continued according to the elisa protocol. secondly, a direct elisa system was developed in which the nasal washings or control antigen (uninfected tissue culture fluid) were added directly to each of a set of duplicate elisa plate wells coated with either pre-or postchallenge rabbit anti hrv-el hyperimmune serum. plates were then incubated overnight at °c and the test continued as described above. test results were calculated by subtracting the mean optical density of a specimen tested in duplicate pre-serum-coated wells from the mean optical density of the same specimen tested in duplicate post-serum-coated wells. results for the control uninfected tissue culture fluid were also calculated in this way. a nasal washing was considered positive when the test result was . times that of the control uninfected tissue culture fluid. this calculation was found to be the most appropriate method of controlling the considerable variation in background optical densities obtained with different nasal wash specimens. the "cutoff" value was set after a series of positive and negative specimens had been assayed using the direct elisa as described in the elisa procedure. a % bsa block and a streptavidin wash stage were incorporated in the test in order to block unoccupied sites on the surface of the elisa plate and endogenous biotin in clinical specimens, respectively. experiments were performed in which each stage of the test was omitted in turn and the results compared with that obtained from a complete test. figure shows that the % bsa block and streptavidin wash were necessary stages in the elisa test in order to keep background levels to a minimum. with the optimal test conditions established, experiments were performed in order to determine the limits of detection of the assay for both hrv-el and hrv- antigen detection. the optical density, recorded at nm, decreased with increasing dilutions of the hrv-el and hrv- stocks and remained approximately the same for all dilutions of control antigen. at a titre of lo'.' tcidso pl-' and <lo' tcid,, pl-' hrv-el and hrv- , respectively, the mean optical density was approximately equal to . times that recorded for control antigen. therefore the limits of detection of the assay were concluded to be tcid,, pl-' and <lo' tcid,, pl-' for hrv-el and hrv- , respectively. studies on the cross-reactivity between the various rhinovirus serotypes are summarised in table . briefly, of the different human rhinoviruses tested, ( . %) showed strong cross-reactivity, ( . %) showed a lesser degree of cross-reactivity, ( . %) were weakly cross-reactive, whilst ( . %) showed no reaction at all with hrv-el. all four control viruses (echovirus type , coronavirus e, coxsackie a and influenza a/eng/ / ) were not cross-reactive. control antigen also showed no reaction. in the hrv- elisa system, ( . %) of the rhinoviruses tested (including hrv- ) showed strong cross-reactivity. twelve ( . %) showed a lesser degree of cross-reactivity, ( . %) were weakly cross-reactive, whilst ( . %) showed no reaction. of the controls, coronavirus e, coxsackie a , influenza a/eng/ / , and control antigen did not cross-react with hrv- . however, echovirus type reacted strongly in the test, thus suggesting a degree of cross-reactivity with hrv- . these results were reproduced several times. figure shows the results obtained with nasal washings, collected from three volunteers on consecutive days following hkv-el or saline challenge, tested in both the cell-culture-amplified (cca)-elisa and direct elisa systems. the figure also illustrates the good correlation obtained between cca-elisa, direct elisa, and virus isolation in detecting positive or negative specimens from these three volunteers. nasal washings collected or days after inoculation with hrv-el or saline from volunteers who participated in a trial were investigated by all three methods strong" moderateb weak' negatived , , , el , , , , , , , , . , la, ib, , , , , , , , , , , , , , , , , , , , , , , , , echo i, , , . , coronavirus e, influenza , , , , , , , , , , , , , , , , , la, ib, , , , , , , , , . , , , , , , , , , , , , , , , , , , , , , , , , , . x the od of control antigen. "weak = od of test sample . - x the od of control antigen. dnegative = od of test sample i l . x the od of control antigen. (table ). samples from of ( . %) volunteers gave concordant results by all three procedures for both days. in two cases (ad and ag) specimens were positive by isolation but negative by cca-elisa. however, overall there was . % correlation between the cca-elisa and virus isolation. the direct elisa failed to detect viral antigen in specimens that were positive by isolation and specimens that were positive by both isolation and cca-elisa. however, viral antigen was detected in specimens that were negative by isolation and cca-elisa. hence, the direct elisa showed . % agreement with either virus isolation or it is noteworthy that all the control nasal washings collected from the four volunteers who were challenged with saline instead of hrv-el (n.o., a.c., j. ram, j.r.) were negative by all three procedures (table ii) , thus indicating the high specificity of the elisa techniques. cca-elisa. in this paper we describe the first elisa system for rhinovirus detection. the assay was found to be simple, rapid, and capable of detecting a large number of human rhinovirus serotypes. it is a reliable method for the detection of hrv-el antigen in clinical specimens, and results have suggested that the limits of detection were as low as lo -' tcid,, loopl-' and <lo tcid,, pl-l for hrv-el and hrv- antigen, respectively. the assay offers three distinct advantages over virus isolation in cell culture. firstly, the test is rapid, requiring only hours to complete once antibody coated plates are available whereas virus isolation and serological identification may take as long as weeks. secondly, the elisa, as demonstrated in this study, is capable of detecting noninfectious as well as infectious virus (some volunteers showed r -fold rises in antibody titre, were elisa positive yet did not excrete virus); hence, the need to preserve infectivity of virus in the clinical specimen may not be essential. thirdly, as tests are read spectrophotometrically evaluation of results is objective rather than subjective. a biotin-avidin elisa utilising the enzyme ,f -galactosidase was developed in this study because of the following advantages over conventional elisa. the high affinity of avidin for biotin (affinity constant = mol-') [green, ensures that the biotinavidin complexes are not easily dissociated by the washing steps in the test and hence this may result in enhanced sensitivity [guesdon et al, . furthermore; one molecule of avidin is capable of binding four molecules of biotin and biotin can be covalently linked to antibody without affecting the antigen binding capacity [guesden et al, . it is more specific as the enzyme p-galactosidase prepared from escherichia coli and used in this assay has characteristics different from those found in human tissues under the conditions of the assay. therefore by employing a -galactosidase-based substrate (onpg), high background readings-as seen with alkaline phosphatase or horseradish peroxidase-were avoided. the data presented in this paper demonstrate that a large number of human rhinovirus serotypes are cross-reactive in both the hrv-el and hrv- elisa systems. this would suggest that an antirhinovirus serum is capable of binding not only its homologous virus but also a number of heterologous viruses. however, the degree of cross-reactivity depends on how close the serological relationship is between the test virus and that against which the hyperimmune serum was prepared. nevertheless, our data clearly suggest that it may be possible to prepare a hyperimmune serum capable of detecting many rhinoviruses by including a number of rhinovirus serotypes in the initial inoculum. indeed, in this study we demonstrated that by using the two elisas (for hrv-el and hrv- ) we were able to detect a large number of the human rhinoviruses investigated (od x od of control antigen). alternatively, it may be possible to develop hyperimmune sera or monoclonal antibodies with c-type reactivity capable of detecting an even larger number of rhinovirus serotypes. although we would like to emphasise that our data are preliminary, both the direct and cca-elisas gave a good correlation with virus isolation when used to detect rhinovirus antigens in nasal washings (obtained from volunteers challenged with either hrv-el or saline). however, the cca-elisa showed better correlation ( . %) with virus isolation than the direct assay ( . %). this was not totally unexpected as both virus isolation and the cca-elisa depend on the presence of viable virus rather than antigen. the inclusion of an amplification stage in the elisa system may be necessary when virus is present at a low concentration (<lo tcid,, ml-i), as is often found with nasal washings obtained from adults. the addition of an amplification stage in the assay does not, in our opinion, increase the work or time load of the test, as whilst the elisa plates are precoated overnight with the capture antibody nasal washings are inoculated into monolayer cell cultures. recent studies with cytomegalovirus (cmv) and influenza virus have suggested that cell culture amplification followed by immunofluorescent staining was necessary for the detection of virus present at low concentration in urine [griffiths et al, ; alpert et al, or throat swabs [espy et al, , respectively. since rhinoviruses are also excreted at low concentrations, particularly by adults, and as nasal washings (although the optimal method of collecting a rhinovirus specimen for virus isolation) contribute further to the dilution of the excreted virus, we feel that the cca-elisa may be the method of choice for the detection of rhinoviruses when present at low concentrations. it is noteworthy that the time required to investigate serial specimens from volunteers using conventional virus isolation and neutralisation techniques is some to weeks. however, when our elisas are employed this time interval is reduced to hours. in addition, by reacting the same clinical specimen with both a pre-immunisation and a hyperimmune rabbit serum as capture antibodies, as in the direct method, the elisa can offer an in-built confirmation stage. it is therefore our aim to further modify the elisas described in this paper in order to improve sensitivity and decrease the time required to obtain results to within a few hours [same day diagnosis]. these developments will be essential if antiviral chemotherapy becomes available and a rapid diagnosis is therefore required in order to initiate treatment in individuals who may be at risk of developing more serious lower respiratory tract complications following rhinovirus infection. rapid detection of human cytomegalovirus in the urine of rapid detection of influenza virus by shell vial assay detection of norwalk virus antibodies with a ): . biotin-avidin immunoassay provocation of airflow limitation by viral infection: implication for treatment rapid diagnosis of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci the use of avidin-biotin interaction in immunoenzymatic techniques viruses associated with acute respiratory infections in royal air force personnel rhinoviruses: a numbering system a collaborative report: rhinoviruses-extension of a numbering system the association of rhinoviruses with lower respiratory tract disease in hospitalized patients acute respiratory illness in university ( - ), military and industrial ( - ) populations studies on r.p., a new antirhinovirus compound, in cell cultures and in volunteers. antimicrobial agents and chemotherapy we are very grateful to dr. d.a.j. tyrrell for helpful discussions and advice. we thank dr. r.j. phillpotts for preparing the rabbit hyperimmune sera, and mrs. n. bailey and mrs. l. treagust for performing virus isolation and neutralisation tests. key: cord- -nn l rai authors: garcia-sanchez, j.; corral, c.; halaihel, n.g.; simon, m.c.; alonso, j.l.; muzquiz, j.l.; ortega, c.; girones, o. title: survey of rotavirus infection in a dairy herd: comparison between polycrylamide gel electrophoresis and two commercial tests date: - - journal: vet microbiol doi: . / - ( ) -e sha: doc_id: cord_uid: nn l rai a survey of rotavirus infection in a dairy herd with a history of neonatal diarrhoea was carried out. faecal samples taken from cows before and after calving as well as faeces taken from their calves daily from birth to two weeks of life were tested for rotavirus by polyacrylamide gel electrophoresis (page) and compared with an elisa and a latex agglutination commercial test. rotavirus excretion was not detected in faeces from cows around parturition by any of the three tests. however, all of their calves shed rotaviruses during the observation period. the onset of rotavirus excretion determined by page ranged from day to day of life (day . ± . on average) and lasted for to days ( . ± . days on average). chi-square test showed a significant association (p= . ) between the presence of rotavirus and the altered consistency of calves faeces. all the three tests showed similar results (overall agreement . %) but discrepancies were detected mainly at the beginning or at the end of the rotavirus excretion period. results obtained with both commercial kits closely paralleled each other and parameters other than sensitivity, specificity, diagnostic accuracy or predictive values have to be considered as selection criteria. neonatal calf diarrhoea is a major threat to the cattle industry due to mortality, medical costs and poor growth of calves in both dairy and beef herds. the syndrome has a great aetiological complexity and numerous pathogens have been reported in association with the disease (woode and bridger, ; woode et at., ; moon et at., ; tzipori, accepted to be an important cause of neonatal diarrhoea in calves throughout the world with its greatest frequency and severity within the first two weeks of life (acres et al., ; de leeuw et al., ) . large numbers of rotavirus particles are excreted in faeces of diarrheic calves and are highly resistant to inactivation. thus, the main source of rotavirus in an outbreak results from environmental contamination and the spread of infection can temporarilly be broken by thorough cleaning and disinfection of the premises (mcnulty and logan, ) . however, subclinically infected older calves and adult cows may play a role in the spread of infection to young calves as well as in the perpetuation of infection within a herd (goto et al., ) . various methods have been developed for rapid detection of rotaviruses in faecal samples; these include electron microscopy (considered for a long time as the reference method), immune electron microscopy, counter immuno-electrophoresis, complement fixation, fluorescent antibody tests, different immunoassays using antibodies labelled with radioisotopes or enzymes (elisa), latex agglutination and polyacrylamide gel electrophoresis (page). some of these tests are commercially available. this paper presents the results of a longitudinal survey of rotavirus infection from birth to two weeks of life in conventionally reared calves using the page technique and analyzes the relationship between rotavirus shedding and the consistency of faeces. faecal shedding of rotavirus in their dams around calving is also studied. the page results are compared with those obtained by two commercial kits, one using elisa and the other latex agglutination. the survey was carried out on a friesian dairy farm located in northeastern spain containing approximately heifers and cows. the herd had a history of recurrent neonatal diarrhoea for previous years. pregnant cows were vaccinated with a bacterin against e. coli k + (imocolibov, rhone-merieux laboratories) approximately three weeks before the expected calving date and were usually moved to a communal house one week before calving. the calves were separated from their dam a few hours after birth and moved to a communal pen next to the cows where they remained for at least days and then they were transferred to fattening units with - calves per unit. each calf was bucket-fed its mother's colostrum during the first day of life ( feedings, litres per feeding), the first feeding being usually within hours after birth, and pooled cows colostrum during the second day of life in the same conditions. thereafter, they were fed milk replacers from an automatic feeder. fifteen cows, chosen on the basis of their predicted calving dates, were included in the survey. calvings took place over the period january- february . faeces samples were collected from cows before calving (days , , and between days - pre-partum; more samples were taken but only those cited were tested), the day of calving and after calving (days , and post-parturn). faeces samples were also obtained from the rectum of the calves daily from birth (considered as day ) to days of life. at each sampling the faeces were recorded as being normal, abnormal (if not obviously diarrheic but changed with respect to consistency and/or presence of blood or mucus ) or diarrheic (if fluid or profuse ). faecal score was determined by the same person for reasons of uniformity of interpretation. none of the calves with diarrhoea was treated at all or isolated from other calves. all the faeces samples ( from cows and from calves) were tested for the presence of rotaviruses by polyacrylamide gel electrophoresis (page), a commercial elisa kit and a commercial latex agglutination kit. page. the extraction of viral rna, its resolution and staining was carried out as described by herring et al. ( ) with minor modifications. briefly, faecal specimens were diluted : by weight with extraction buffer containing % sodium dodecyl sulfate, an equal volume of : phenol-chloroform was added and the mixture was vortexed and centrifuged for min at g. the aqueous phase was removed. for electrophoresis, pl of the clear supernatant were mixed with # of blue marker ( % sucrose containing . % bromophenol blue) and loaded onto a discontinuous polyacrylamide gel ( % concentration for the stacking gel and . % for the running gel). the gels were assembled using a mini-protean ii cell (biorad laboratories, richmond, california, usa) and ran with a ma running current and constant voltage for . h using a / . model power supply (biorad). after that, gels were fixed, developed and silver-stained. a positive control sample was always included in each gel for comparison of the segmented viral rna migration pattern. commercial elisa. the enzygnost rotavirus (ag) monoclonal test (behring institute, behringwerke ag, marburg, germany) was performed according to the manufacturer's instructions. briefly, faeces taken using the dispenser provided were transferred to . ml of the dilution buffer and throughly mixed. then, . ml of the supernatant was transferred to the test plate wells coated with rabbit sa- rotavirus antiserum. following incubation for min at °c the plates were washed times and . ml of antirotavirus conjugate consisting of a mouse monoclonal antibody conjugated with peroxidase were added to each well. the plates were incubated and washed again in the same conditions, . ml of the substrate-chromogen were added per well and were incubated for min at room temperature protected from light. finally, the reaction was stopped and the plates were read at nm wavelength using a multiskan spectrophotometer (flow laboratories). the cut-off value was calculated by adding the mean value of the negative control samples to a threshold value of . .d. units. the retest limit was calculated by adding . o.d. units to the cut-off value. in this way results could be considered as negative (if the absorbance of the sample was less than the cut-off value ), positive (if it was greater than the retest limit) and gray zone (if it was between the cut-off and retest limits ). commercial latex agglutination. the slidex rota-kit monoclonal test (biomrrieux, marcy-l'etoile, france) was carried out according to the instructions enclosed with the kit. briefly, . gr of the faecal specimen were diluted in ml of the buffer supplied and mixed. the emulsions were centrifuged for min at g and then a drop of supematant from each specimen was placed in each of two circles on an agglutination card. a drop of reagent (latex coated with monoclonal antirotavirus antibody) was added to one and a drop of reagent (latex coated with rotavirus negative serum) to the other. following mixing, the cards were rotated gently for min. a positive reaction was considered when clear agglutination of the test latex with no agglutination of the control was seen. agglutination of both control and test latex indicated a non-specific reaction. all samples were read by the same person. in order to study the association between the variable "presence of rotavirus" and the variable "consistency of faeces", a × contingency table was done and the chi-square test was performed. diagnostic test results were analyzed in two ways: first, with page as a gold standard and secondly, on a comparative basis with each other. in the first case, results of the commercial tests were compared with those of page by using sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy. in the abscence of a standard, the kappa statistic was used for comparison. the comparative measures are defined as follows: sensitivity (true-positive rate ): is the proportion of samples in which the test is positive when page is positive. specificity (true-negative rate ): is the proportion of samples in which the test is negative when page is negative. positive predictive value: is the proportion of samples in which page is positive when the test is positive. negative predictive value: is the proportion of samples in which page is negative when the test is negative. diagnostic accuracy: was determined by dividing the number of positive and negative specimens in both the commercial kit and page by the total number of specimens tested. kappa statistic is an overall measure of agreement between two tests and is useful for measuring agreement in the absence of a standard. it compares the observed proportion of samples in which the tests agree with the proportion that would be expected to agree by chance. kappa calculation was carried out as described by goodman and kruskal ( ) . for interpreting kappa values the following benchmarks can be considered: a result between . and . indicates a slight agreement, between . and . is a fair agreement, between . and . is a moderate agreement, between . and . is a substantial agreement and between . and . is an almost perfect agreement. rotavirus shedding was not detected in any sample taken from cows before or after parturition. all samples were also negative for rotavirus when cold-ethanol precipitation of rna was performed. the results of rotavirus shedding in faecal samples taken from the calves are shown in table . during the first two weeks of life rotavirus was detected in the faeces of all calves. the presence of rotavirus was determined in out of samples ( %). thirteen out of calves showed a continuous shedding pattern and only two calves (calves no. and ) showed a discontinuous one. the onset ofrotavirus excretion in faeces ranged from day to day of life (day . +_ . as a mean) and the duration of that excretion ranged from to days (mean . _+ . days). only one electropherotype was found in the survey and it was characteristic of group a rotaviruses. all calves passed abnormal and diarrheic faeces along the two week observation period but no fatal cases were observed during that time (table ) . the onset of rotavirus excretion in faeces coincided with the onset of diarrhoea or of excretion of abnormal faeces in some of the animals (clearly in calves no. , , , and ) but that association was quite variable. however, when every sample was considered independently of the animal and of the chronological sequence, the chi-square test showed a high association be- tween the two variables in such a way that, when a sample was positive for rotavirus, the faeces tended to be diarrheic and when a sample was negative for rotavirus it tended to be normal (p= . ) ( table ) . as observed with page, rotavirus shedding was not detected in cows faeces taken around calving by any of the commercial tests. when calves faeces were tested, results obtained by elisa and latex agglutination commercial kits closely coincided with those by page: the agreement between page and latex agglutination was . %, % between page and elisa, . % between latex agglutination and elisa and the overall agreement between the three tests was . % (table ). the discrepant resuits fall into four categories and are shown in table . most of the discrepancies ( samples) were observed in the first or in the last sample positive for rotavirus by page in the calves rotavirus excretion period, that's to say, when the shedding pattern changed from negative to positive or vice versa and corresponded to samples negative by latex agglutination while negative (or gray zone) by elisa ( samples) or positive by elisa ( samples). another three discrepancies were detected in which the samples were positive by page and negative by both elisa and latex agglutination and all of them corresponded to two calves with a discontinuous shedding pattern (calf no. , days and ; calf no. , day ). the other two discrepant results corresponded to samples positive by elisa and negative by both page and latex agglutination; one of them was detected in a calf with a discontinuous shedding pattern (calf no. , day ) and the other one corresponded to the first positive sample in the rotavirus shedding pattern determined by elisa (calf no. , day ). in table sensitivity, specificity, positive and negative predictive values and diagnostic accuracy of both latex agglutination and elisa kits are shown considering page as a gold standard. latex agglutination showed a lower sensitivity ( %) and a higher specificity ( %) than elisa ( . % and . %, respectively) but the predictive values and the diagnostic accuracy were quite similar for both tests. the kappa statistic for each pair of tests (when no gold standard was considered) is given in table . it shows an almost perfect agreement between the three tests (kappa values between . and . ). all calves monitored in our survey were shown to be infected and shed rotaviruses in faeces within the first two weeks of life. this result seems to indicate that bovine rotavirus is enzootic at this farm and it could explain its history of recurrent outbreaks of neonatal diarrhoea. in the present study, rotavirus was detected in the faeces of some animals as early as - days of age with an increasing number of excretors thereafter. experimental infection of calves has shown that the period between infection and detection of excretion of the virus in faeces is about two days (de leeuw et al., ; logan et al., ) . that indicates that most calves were exposed to infection very soon after birth. excretion of rotavirus in calves faeces has been reported before hours of age but such an early detection is infrequent (acres and b abiuk, ; de leeuw et al., ) . the delay in excretion of the virus under field conditions is probably because of the presence of specific colostral antibody within the gut lumen and the elimination of this antibody renders the calf susceptible to overt infection as suggested by de leeuw et al. ( ) . so, the early detection of rotavirus in faeces detected in the present study could be due to an inadequate feeding of colostrum. in our study, rotavirus shedding in faeces started at . days of life on average and showed a mean duration of . days. in a similar survey, mcnulty et al. ( ) reported an onset of rotavirus excretion at . days old on average in an endemically infected dairy herd and similar excretion periods have been described in both beef and dairy herds by others (acres and babiuk, ; de leeuw et al., ; goto et al., ) . nevertheless, the exact onset and duration of rotavirus shedding is difficult to establish in all these studies because they lack a daily sampling design. rotavirus excretion coincided with abnormal or diarrheic faeces in some animals and chi-square test showed a statistically significant association (p= . ) between the presence of rotavirus and the altered consistency of faeces. this suggests that rotaviruses are involved in the aetiology of this outbreak of neonatal diarrhoea although no other agents were studied. de leeuw et al. ( ) described that rotavirus infection was associated with cases of "late diarrhoea" (diarrhoea from the th to the th day of life) whereas other agents appeared to be of minor importance, and schusser et al. ( ) reported that, although the correlation between rotavirus excretion and clin-ical diarrhoea in calves was poor during the first weeks of life, the severest form of watery diarrhoea was limited to the first three weeks of life, where rotavirus excretion peaked. the association between rotavirus infection and abnormal or diarrheic faeces has also been reported by several authors (mcnulty and logan, ; goto et al., ; bellinzoni et al., ; murakami et al., ) . the role that adult animals may play as a source of infection for calves has been a matter of controversy. in the present study, we did not detect the presence of rotavirus in any sample taken from cows around parturition by any of the three tests. our results agree with those of several authors that have also failed to detect rotaviruses in faeces of adult animals (dagenais et al., ; scherrer et al., ; sihvonen and miettinen, ) . on the contrary, there have been several reports describing the excretion of rotavirus in faeces of a small number of animals with or without diarrhoea and usually over a short period around parturition (mcnulty and logan, ; bellinzoni et al., ; murakami et al., ) and it has been suggested that adult carriers might be important in the epizootiology of the infection (woode, ; crouch and acres, ) . recently, kodituwakku and harbour ( ) reported an intermitent excretion of rotavirus in asymptomatic cows throughout pregnancy although rotavirus excretion and diarrhoea were detected in only one of their calves and goto et al. ( ) have indicated that the perpetuation of rotavirus infection within a herd may result from persistent or chronic infections in older calves and adult animals. however, it is generally accepted that once an outbreak is underway, young calves act as amplifiers of the infection and that environmental contamination is the major source of infection for newborn calves, whereas maternal infection is of minor significance (dagenais et al., ; mcnulty and logan, ; goto et al., ; murakami et al., ) . the rotavirus shedding pattern obtained with the three tests was quite similar, the overall agreement being . % among the three tests. most discrepant results were positive by page while negative by latex agglutination ( samples ) and negative (or gray zone ) by elisa ( samples ) and all of them except three corresponded to the first or last positive sample in the page shedding pattern, when the viral content in faeces was probably low. these results seem to indicate a higher sensitivity of the page test compared with that of latex agglutination and elisa. the same explanation may be given for the discrepant results observed in calf no. (days and ) and calf no. (day ) in which a discontinuous shedding pattern was detected by page but we have no certainty. the results observed in two samples (calf no. , day and calf no. , day ), in which rotaviruses were detected only by elisa, are more difficult to explain. they could be false-positive results but if the shedding patterns of that calves are considered, the true-positive result cannot be ruled out. latex agglutination for detection of rotavirus in faecal specimens quantitative observations on experimental reo-like virus (rotavirus) infection in colostrum-deprived calves an assessment of the sensitivity of three methods for the detection of rotavirus longitudinal survey of rotavirus infection in calves comparison of six commercial kits for the diagnosis of rotavirus infection in man and calves pathogenic relationships of rotavirus, escherichia coli and other agents in mixed infections in calves comparison of six methods for detecting human rotavirus in stools a survey on bovine rotavirus type l-associated neonatal calf diarrhea in a beef herd direct appraisal of latex agglutination testing, a convenient alternative to enzyme immunoassay for the detection of rotavirus in childhood gastroenteritis, by comparison of two enzyme immunoassays and two latex tests elisa for the detection of rotavirus and rotavirus/antibody complexes in faeces a follow-up study on bovine rotavirus dissemination among calves of a large dairy herd rotavirus and enterotoxigenic escherichia coli infections of calves on a closed finnish dairy farm evaluation of seven immunoassays for detection of rotavirus in pediatric stool samples the aetiology and diagnosis of calf diarrhoea epizootology of bovine rotavirus infection viral enteritis of calves morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals key: cord- -ovjdzyxv authors: pan, qing; wang, jing; gao, yulong; cui, hongyu; liu, changjun; qi, xiaole; zhang, yanping; wang, yongqiang; li, kai; gao, li; wang, xiaomei title: development and application of a novel elisa for detecting antibodies against group i fowl adenoviruses date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: ovjdzyxv since , outbreaks of hepatitis-hydropericardium syndrome (hps) caused by a novel genotype of fowl adenovirus (fadv- ) infection have created serious economic losses in china. given that other serotypes of hypervirulent fadvs have also been reported in poultry around the world, a common elisa for all serotypes within the group i fowl adenoviruses (fadv-i) is urgently needed, especially for clinical epidemic serotypes. in this study, we used high purity and concentration virions of fadv- and developed a common elisa for detecting antibodies against fadv-i serotypes. the developed elisa was able to distinguish between antibodies against fadv-i, fadv-iii, and other heterologous viruses without any cross-reaction. furthermore, the elisa showed higher sensitivity than the fadv- -based elisa to the novel fadv- found in china. moreover, since there are no commercial vaccines against fadvs in china, the elisa was applied to detect sera samples from specific pathogen-free chickens inoculated with inactivated fadv- , fadv- , and fadv- a. the assay showed high sensitivities for all three detected serotypes within fadv-i. in conclusion, a novel, common elisa for fadv-i was developed in this study and could be a powerful tool for seroepidemiological investigations and fadvs vaccine development. fowl adenoviruses are divided into three groups, group i fowl adenovirus (fadv-i) contains five species (a to e) with serotypes ( - a, b- ) (hess ) isolated from fowls, group ii (fadv-ii) includes the hemorrhagic enteritis virus (hev) of turkeys and the marble spleen disease virus (msdv) of pheasants (domermuth et al. ) , and group iii (fadv-iii) is mainly associated with egg drop syndrome virus (edsv) (huang et al. ) . inclusion body hepatitis (ibh), hydropericardium syndrome (hps), and gizzard erosion (ge) associated with fadv-i infection have been reported worldwide, e.g., in pakistan (khawaja et al. ; mansoor et al. ), chile (toro et al. ) , korea (choi et al. ) , canada (dar et al. ) , hungary (kaján et al. ) , india (mittal et al. ) , japan (mase and nakamura ) , south africa (joubert et al. ) , mexico (vera-hernández et al. ) , poland (niczyporuk ) , and china , resulting in significant economic losses to the poultry industry. hps is caused by fadv- (pan et al. a ), whereas ibh is always related to fadv- , fadv- , fadv- a, or fadv- b (morshed et al. ) and ge is mainly caused by fadv- (matczuk et al. ) . notably, severe hps, with a high mortality rate of - %, caused by a novel genotype fadv- has been widespread in china since (li et al. ; pan et al. a ) and is a major threat to the poultry industry. in recent years, a variety of fadvs detection methods have been developed for large-scale seroepidemiological investigations and hps vaccine evaluation. for antigen detection, polymerase chain reaction (pcr) (günes et al. ) , quantitative pcr (qpcr) (pan et al. c ), high-resolution melting (hrm) curve analysis (steer et al. ), loop-mediated qing pan and jing wang contributed equally to this work. * xiaomei wang wangxiaomei@caas.cn isothermal amplification zhai et al. ) , and sandwich enzyme-linked immunosorbent assays (elisa) (shao et al. a; shao et al. b ) have been developed. the traditional methods for fadvs serological diagnosis are agar gel precipitation (agpt) and virus neutralization (vn) tests. while agpt is generally less sensitive, the process of vn is not conducive to rapid and large-scale diagnosis. thus, a simple, rapid, and sensitive diagnostic method is urgently required for fadvs detection, for which elisa, which is widely used in large-scale serological investigation, is simple to operate, and has high sensitivity, is an excellent candidate technology. prevention and control of hps have been attempted, mainly through the use of inactivated virus pan et al. b) or subunit vaccines schachner et al. ; shah et al. ; wang et al. ) , for some fadv serotypes. the immune response to vaccines is generally monitored by the presence of an antigen-specific antibody and the neutralization antibody. in one recent study, recombinant fiber-based indirect elisa was used to detect serum samples from chickens experimentally inoculated with different fadv- or fadv- strains (feichtner et al. ) . a recombinant hexon-based single serum dilution elisa was also developed to measure the hexon-specific antibodies against fadv- in sera of chickens (rajasekhar and roy ) . both of the elisas mentioned above were fadv serotypespecific and based on variable recombinant proteins. however, some other fadv serotypes, such as fadv- and fadv- a, have also been shown to cause serious economic losses to the poultry industry. unfortunately, there is currently no commercial fadv-i elisa kit in china, thus a common elisa for all fadv-i serotypes is urgently needed. in this study, we developed a group-specific and sensitive elisa based on the novel genotype of fadv- for detecting antibodies against twelve fadv-i serotypes. the common elisa provides a powerful tool for the seroepidemiological investigations and vaccine development. as we reported previously (pan et al. ) , the hljfad strain (genbank no. ku ) was isolated from layers and identified as serotype (fadv- ). the fadv- celo (genbank no. u ) and fadv- a (a clinical isolate) strains were deposited at the state key laboratory of veterinary biotechnology, harbin veterinary research institute (harbin, china). chicken leghorn male hepatocellular (lmh) cells were kindly gifted by prof. guozhong zhang (china agricultural university, beijing, china) and were cultured in dulbecco's modified eagle's medium (dmem) (thermo fisher scientific, waltham, ma, usa) supplemented with % fetal bovine serum (fbs) (gibco, san diego, ca, usa), iu/ml penicillin, and μg/ml streptomycin. following hljfad (fadv- ) strain propagation and purification, the fbs concentration in confluent cultures was reduced to % for maintenance. the lmh cells were infected with . multiplicity of infection (moi) of fadv- and incubated at °c in a % co atmosphere for days. formaldehyde was added to the culture medium for virus inactivation, and viral particles were harvested from virusinfected lmh cells at a concentration of . % in the final product (kim et al. ) . culture fluids were collected and, after cycles of freezing and thawing, the mixture was centrifuged at ×g for min to remove cellular debris. supernatants were transferred to %, % (w/w) sucrose solution and centrifuged at , rpm for h using a beckman sw rotor in a model optima xpn- ultracentrifuge (beckman coulter, brea, ca, usa). virus pellets were collected and suspended in phosphate-buffered saline (pbs). culture suspensions were collected, purified in . g/ml cesium chloride (cscl ) (amresco, solon, usa), and centrifuged at , rpm for h using a beckman sw rotor. two discrete bands were formed following final ultracentrifugation. the bands were aspirated with a syringe by puncturing the side of the tube, suspended in pbs, centrifuged at , rpm for h using a beckman sw rotor, and culture fluids were collected (pan et al. ) . the morphology of fadv- preparations was verified by electron microscopy. carbonate buffer (ph = . ), tris-hcl buffer (ph = . ), and phosphate buffer (ph = . ) were used as coating buffers. pbst containing % skim milk, pbs containing % bovine serum, and pbs containing % gelatin were used as blocking buffers. fadv- stocks with a concentration of . mg/ml, as measured by micro-volume spectrophotometer (implen, munchen, germany), were obtained and prepared into working dilutions ( μg/ml, μg/ml, and μg/ml) using coating buffer. the working dilutions were added into microtitre plates ( μl/well) and incubated at °c for , , or h. after incubation with the coating antigen, the plates were washed three times with pbs containing . % tween- (pbst) and then incubated with blocking solution at °c for , , or h. after three washes, serum samples were diluted : , : , : , : , : , : , : , , and : and incubated at °c for . , , and h, respectively. following incubation, samples were washed three times and incubated at °c for . , , and h with hrp-conjugated rabbit anti-mouse antibodies (sigma, missouri, usa) diluted : , : , and : , , respectively. after washing, μl tetramethylbenzidine (tmb) substrate (amresco, solon, usa) was added to each well and the plates were incubated in the dark for , , and min. the enzymatic reaction was quenched by hydrofluoric acid and the optical density (od) was determined at nm. ods presented represent the mean from duplicate wells. the optimal conditions were determined by evaluating the od values and the positive/negative ratio (p/n) of the samples. the cut-off was determined according to the sample/positive (s/p) ratio by calculating the arithmetic mean plus three times the standard deviation (sd). forty specific antibody-negative chickens were divided into four groups: fadv- immunization group (n = ), fadv- immunization group (n = ), fadv- a immunization group (n = ), and a control group of age matched unvaccinated chickens (n = ). the formaldehyde inactivated antigen solution was emulsified with oil adjuvant at a ratio of : (w/w). the immunization group was immunized intramuscularly with . ml inactivated viruses containing tcid fadv- , fadv- , fadv- a antigens per chicken at the age of days. five serum samples of chickens in each group were collected weekly post inoculation until the termination of the experiment. in total, clinical serum samples were examined by the developed elisa for the presence of fadv antibodies. these samples were collected from breeder flocks of unknown disease and vaccination status in jiangsu, heilongjiang province. fadv antibodies were detected by the common elisa and results were compared with a commercial kit (biochek, reeuwijk, the netherlands) for fadv-i. anti-fadvs antibodies were detected by indirect immunofluorescence assay (ifa). an ifa was carried out on lmh cells plated in well plates and infected with . moi of the fadv- . after -day incubation at °c in a % co atmosphere, the wells were fixed with iced ethanol for min at room temperature and washed three times with pbs. clinical serum samples diluted : in pbs were used as primary antibodies for h at °c. after washing three times, alexa fluor tm goat anti-mouse igg (h+l) secondary antibody ( : ) (invivogen, san diego, california, usa) was added, followed by incubation for h at °c. after washing three times, the wells were investigated by evos f inverted fluorescence microscope. differences between the two groups were evaluated by student's t test and considered significant at ** p < . . purified intact virus particles were used as an optimal coating antigen to develop a common elisa in this study. highpurity virions were collected by sucrose and cscl density gradient centrifugation. subsequently, fadv- stocks with a concentration of . mg/ml, as measured by micro-volume spectrophotometer, were obtained. the fadv- virions were verified by electron microscopy, with negatively stained preparations showing nearly round surface with a diameter of - nm. as shown in fig. , the purified virions were intact and of high purity, and the preparation had good dispersion and was evenly distributed throughout the field. fig. the morphology of the purified virus particles from cell-cultured hljfad (genbank no. ku ) by electron microscopy the optimum working range of antigen and serum were decided by checkerboard titration and determined by the greatest p/n ratio. antigen concentrations of μg/ml were added to microtitre plates coated with μl in carbonate buffer (ph . ). following incubation for h at °c, the plates were washed three times with pbst and blocked with pbst containing % non-fat milk for h at °c. serum samples diluted : in pbs were incubated for min at °c. after washing three times, secondary antibodies were incubated for min at °c before a final washing step. subsequently, μl tmb substrate was added to each well and the plates were incubated in the dark for min. the enzymatic reaction was quenched with μl . m hydrofluoric acid and the od values were determined at nm. based on the arithmetic mean s/ p value of the clinical negative samples ( . ) and the sd value ( . ), the cut-off for the elisa was determined to be . . serum samples with s/p value greater or less than . were determined to be positive or negative, respectively. in the specificity assay, the negative control serum collected from non-treated spf chickens was confirmed to be negative by the common elisa developed in this study, and the positive control serum obtained from spf chickens inoculated with hljfad was detected to be positive. as shown in fig. , the s/p values of other heterologous viruses, including h and h aiv, ndv, ibv, and iltv positive sera, were below the established cut-off value (ranging from . to . ) and were hence determined to be negative by the developed elisa. in group-specificity and serotype-specificity assays, edsv (fadv-iii)-positive serum tested by the common elisa was negative (fig. a) . different serotypes of fadv-i including fadv- , fadv- gy, fadv- a, fadv- , fadv- , and fadv- positive serums ranged from . to . (fig. b) , above the elisa cut-off value, were determined to be positive. sensitivity analyses comparing the fadv- elisa and fadv- elisa using serum samples collected from chickens infected with hps showed a significantly higher sensitivity of the elisa developed based on fadv- in this study than the fadv- -based elisa. as shown in fig. , the limit of detection (lod) of the common elisa developed for hljfad (fadv- ) positive sera was : , while the elisa based on fadv- was : . the common elisa showed higher s/p values than the fadv- -based elisa detecting antibodies against fadv- positive sera with each dilution assayed. kinetic analyses of specific antibody responses following inoculation of all three fadv hypervirulent serotypes were assessed by the common elisa. fadv-i antibodies in the fig. fadv-specificity assay for the common elisa using different heterologous viruses, including h and h aiv, ndv, ibv, iltv. spf chicken serum as negative control; fadv- positive serum as positive control fig. group-specificity and serotype-specificity assay for the common elisa using edsv (fadv-iii) (a) and different serotypes of fadv-i, including fadv- , fadv- gy, fadv- , fadv- , fadv- , and fadv- a, respectively (b). spf chicken serum as negative control; fadv- positive serum as positive control negative control group were all negative when tested by the common elisa. as shown in fig. , serum antibody titres of the experimentally inoculated chickens increased with the time of immunization. in chickens inoculated with inactivated fadv- (fig. a) , fadv- (fig. b) , or fadv- a (fig. c) , the earliest detection of antibodies occurred weeks after inoculation (p < . ). two weeks after immunization with inactivated virus, the positive rate of antibodies against all three detected serotypes within fadv-i reached %. the antibodies against the emergent novel fadv- showed higher s/p values than antibodies against fadv- and fadv- a. in field sera, the common elisa detected . % ( / ) positives, whereas the commercial fadv-i elisa kit test detected . % ( / ) positives (table ). the positive coincidence rate between the commercial kit and the developed elisa was . % and the negative coincidence was . %; the total coincidence was . %. anti-fadvs antibodies were detected by ifa. the common elisa and ifa detected . % ( / ) and . % ( / ) positives, respectively, and the total coincidence between the common elisa and ifa was % (data not shown). hps induced by fadv- has emerged across several different areas in china since (niu et al. ; zhao et al. ) and caused serious economic losses and poses a great threat to the poultry industry. the pathogenic fadv- strain has been isolated and characterized as a novel genotype (ye et al. ) . however, there are currently no commercial fadv-i or fadv- elisa kits in china for detection of antibodies against the pathogenic fadvs or fadv- specifically. many studies have been conducted on fadvs antibody detection using elisa based on fadv- . in one study, celo (fadv- ) virus was used as a coating antigen to establish an elisa method for detecting fadv- and avian adenovirus-associated virus (dawson et al. ) . as reported, fadv- showed higher cross-reactivity among serotypes than did fadv- (calnek et al. ) . given that homologous elisa reactions were stronger than heterologous reactions in many cases, we evaluated fadv- as a coating antigen to develop a common elisa for detecting all twelve fadv-i serotypes. the elisa developed based on fadv- in this study showed considerable cross-reaction within the twelve fadv-i serotypes and higher sensitivity than the fadv- -based elisa for detecting antibodies against the novel emergent fadv- . ultimately, we successfully established a common elisa for specifically detecting antibodies against all twelve fadv-i serotypes, which could be a powerful tool for seroepidemiological investigation of ibh, ge, and, especially, the emergent hps in china. unfortunately, there is no commercial vaccine against fadvs, including the fadv- strain, and a common detection method is urgently needed to facilitate vaccine development and evaluation. though several recombinant protein-based elisas have been used to detect antibodies in chickens inoculated with subunit vaccines, such as rfiber- , rfiber- , and rhexon protein, these elisa were able to detect antibodies against fadv- -specific (feichtner et al. ; he et al. ; rai et al. ; rajasekhar and roy ) . furthermore, prokaryotic expressed proteins need to be renatured to restore tertiary and quaternary spatial structures, which may lead to false-negative results from assays using recombinant proteins. considering that fadv- and some other serotypes within fadv-i are pathogenic to chickens worldwide (grgić et al. ; lim et al. ; morshed et al. ), a common elisa should be more suitable for fadvs vaccine development. the common elisa developed in our study was applied to detect serum samples from spf chickens inoculated with inactivated fadv- , fadv- , and fadv- a, and showed high sensitivity for all three fadv hypervirulent serotypes. these results suggest that the common elisa developed in our study is capable of monitoring antibodies against all serotypes in variable fadv-i vaccine development and evaluation. in conclusion, the common elisa developed in this study showed considerable cross-reactivity among all fadv serotypes and was capable of detecting specific antibodies against fadv-i. furthermore, the elisa showed higher sensitivity in detecting serum samples of hps caused by the novel fadv- genotype that has recently emerged in china. our elisa could be a powerful tool for seroepidemiological investigations and development of vaccines for different fadv-i serotypes. serological crossreactivity of avian adenovirus serotypes in an enzyme-linked immunosorbent assay epidemiological investigation of outbreaks of fowl adenovirus infection in commercial chickens in korea pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in saskatchewan chickens pronovost ad ( ) an enzyme-linked immunosorbent assay for detection of antibodies to avian adenovirus and avian adenovirus-associated virus in chickens incidence and distribution of "avian adenovirus group ii splenomegaly of chickens development of sensitive indirect enzyme-linked immunosorbent assays for specific detection of antibodies against fowl adenovirus serotypes and in chickens pathogenicity and complete genome sequence of a fowl adenovirus serotype isolate real-time pcr assay for universal detection and quantitation of all five species of fowl adenoviruses (fadv-a to fadv-e) recombinant fiber- protein-based indirect elisa for antibody detection of fowl adenovirus serotype detection and differentiation of avian adenoviruses: a review egg drop syndrome virus enters duck embryonic fibroblast cells via clathrin-mediated endocytosis molecular differentiation and pathogenicity of aviadenoviruses isolated during an outbreak of inclusion body hepatitis in south africa molecular typing of fowl adenoviruses, isolated in hungary recently, reveals high diversity isolation of an adenovirus from hydropericardium syndrome in broiler chicks an inactivated oil-emulsion fowl adenovirus serotype vaccine provides broad cross-protection against various serotypes of fowl adenovirus fowl adenovirus species c serotype is attributed to the emergence of hepatitishydropericardium syndrome in chickens in china identification and virulence characterization of fowl adenoviruses in korea molecular characterization of fowl adenovirus serotype (fav- ) isolate associated with fowl hydropericardiumhepatitis syndrome in pakistan phylogenetic analysis of fowl adenoviruses isolated from chickens with gizzard erosion in japan whole genome sequencing of fowl aviadenovirus a -a causative agent of gizzard erosion and ulceration, in adult laying hens identification, pathogenicity of novel fowl adenovirus serotype sdjn in shandong, china and immunoprotective evaluation of the newly developed inactivated oil-emulsion fadv- vaccine characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens fowl adenoviruses d and e cause inclusion body hepatitis outbreaks in broiler and broiler breeder pullet flocks phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in poland molecular epidemiology and phylogenetic analysis of fowl adenoviruses caused hydropericardium outbreak in china during characterization of a hypervirulent fowl adenovirus with the novel genotype newly prevalent in china and establishment of reproduction infection model of hydropericardium syndrome in chickens an inactivated novel genotype fowl adenovirus protects chickens against the hydropericardium syndrome that recently emerged in china different dynamic distribution in chickens and ducks of the hypervirulent, novel genotype fowl adenovirus serotype recently emerged in china the natural large genomic deletion is unrelated to the increased virulence of the novel genotype fowl adenovirus recently emerged in china induction of immune response in chicken vaccinated with a plasmid dna encoding fowl adenovirus hexon gene recombinant hexon antigen based single serum dilution elisa for rapid serological profiling against fowl adenovirus- causing hydropericardium syndrome in chickens a subunit vaccine based on fiber- protein provides full protection against fowl adenovirus serotype and induces quicker and stronger immune responses than an inactivated oil-emulsion vaccine recombinant fadv- fiber- protein protects chickens against hepatitishydropericardium syndrome (hhs) fowl adenovirus: history, emergence, biology and development of a vaccine against hydropericardium syndrome two novel monoclonal antibodies against fiber- protein of fadv- and their application in detection of fadv- / a novel monoclonal antibodies-based sandwich elisa for detection of serotype fowl adenovirus classification of fowl adenovirus serotypes by use of highresolution melting-curve analysis of the hexon gene region characterization of fowl adenoviruses from outbreaks of inclusion body hepatitis/hydropericardium syndrome in chile clinicopathological characterization and genomic sequence differences observed in a highly virulent fowl aviadenovirus serotype immune protection efficacy of fadv- surface proteins fiber- , fiber- , hexon and penton base outbreaks of serotype fowl adenovirus with novel genotype lamp real-time turbidity detection for fowl adenovirus a loop-mediated isothermal amplification coupling with a lateral flow dipstick for rapid and specific detection of fowl adenovirus serotype- pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in china key: cord- -gpz gkv authors: weiss, m.; steck, f.; kaderli, r.; horzinek, m.c. title: antibodies to berne virus in horses and other animals date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: gpz gkv after inoculation into foals, berne virus induced neutralizing antibody, but did not cause clinical symptoms. in a horizontal study of seropositive mares and their offspring, a decline of maternal antibodies and a sudden synchronous seroconversion in all foals were observed, again without clinical symptoms. the virus is widespread in the swiss horse population and has been so during the last decade; rises in antibody titers were noted in % of paired sera sampled at random. positive reactions were also obtained in serum neutralization tests and elisa using small numbers of horse sera from germany, france and the u.s.a. the results of neutralization tests and elisa were correlated in % of random samples tested; % were neutralization-positive and elisa-negative and in % the inverse was observed. neutralizing activity was found in the sera of other ungulates (cattle, goat, sheep and pig), laboratory rabbits and species of wild mice (clethrionomys glareolus and apodemus sylvaticus). inconclusive results were obtained with feline and human sera; those from dogs and foxes (vulpes vulpes) were consistently negative. the probable occurrence of antigenic variants in berne-type viruses is discussed. the purification and partial characterization of a new enveloped rna virus isolated during routine diagnostic work from a horse in berne, switzerland has been reported (weiss et al., ) . 'berne virus' measures -- nm in its largest diameter and consists of a peplomer-bearing envelope and an elongated core which is usually bent into an open torus within the membrane. the core is tubular in appearance and has a morphology indicative of helical nucleocapsid symmetry. the virus possesses an rna genome since its growth is not affected by dna nucleotide analogues; actinomycin d and alpha-amanitin on the other hand do inhibit replication, as does uv irradiation of the cells before infection. a unique pattern of viral polypeptides was reported (horzinek et al., ) . berne virus was shown to be serologically unrelated to known equine viruses as well as to representatives of the main antigenic clusters of the coronaviridae family (infectious bronchitis, mouse hepatitis and transmissible gastroenteritis viruses), which it resembles superficially in negatively stained preparations. however, an antigenic relationship was detected with the breda group of viruses recently discovered by workers in ames, ia (woode et al., ) . in this paper, results of seroepidemiological studies in horses and other animal species are presented, with the aim of showing the distribution and possible significance of this virus in the domestic and wild animal population. berne virus (strain p / ) was originally isolated in secondary horse kidney cells and our neutralization tests early in this study (before ) were also performed in these cells. later on, virus propagation and assay was done in embryonic mule skin cells (ems line) grown in eagle's minimum essential medium supplemented with non-essential amino acids ( %), lglutamine ( mm), sodium bicarbonate, antibiotics and -- % foetal calf serum; the serum had been pre-screened for the absence of antibody against berne virus by neutralization tests. a total of randomly selected adult horses from various regions of switzerland were sampled two times, in order to show seroconversion. the period between the bleedings ranged between and days, with about % of the second samples taken -- days after the first ones. additional ungrouped random serum samples were obtained from berlin (germany), the lyon area (france) and from various regions of the usa. in the swiss "haras federal avenches" mares and their foals were followed serologically over a period of months (table i) . a limited serosurvey was attempted using random samples from cattle, goats, sheep, pigs, dogs, cats and laboratory rabbits; most sera were from switzerland, supplemented with some cat and rabbit specimens from the netherlands. in addition, red fox (vulpes vulpes)sera were tested. from the same source, mouse sera from wild-caught specimens of the species clethrionomys glareolus, apodemus sylvaticus and a. flavicollis were obtained. infectious doses (ids ) were routinely determined in flat bottom microtiter trays (greiner and soehne, nuertingen, germany) by adding /a volumes of serial -fold virus dilutions to wells containing a monolayer of × ems cells. the spearman-kaerber formula was applied for the calculation of infectivity titers after reading the plates days after infection. neutralization titers were determined after reacting serial -fold dilutions of the inactivated serum with ids units of native berne virus for min at °c. subsequently, ems cell monolayers (in microtiter wells) per dilution were inoculated and the plates were examined for cpe days after infection. an indirect elisa procedure (engvall and perlmann, ) was used for monitoring antibody in sera from horses, cattle and goats. antigen semipurified by ammonium sulphate precipitation and subsequent interphase centrifugation ( / % sucrose; weiss et al., ) was adsorbed to polystyrene dynatech immulon flat bottom micro-elisa plates in the presence of a . m carbonate/bicarbonate buffer, ph . . prediluted horse sera ( / or / ) and an anti-horse total igg preparation conjugated to horse radish peroxidase (sigma, st. louis, mo, u.s.a.)by a -step glutaraldehyde coupling procedure (avrameas et al., ) were employed. in the substrate mixture . '-azinodi-( -ethyl-benzthiazolin-sulphonate( )) (abts, boehringer, mannheim west germany) was used at a concentration of mm in . m acetate buffer, ph . , containing mm nah po and mm h as described previously (horzinek et al., ) . absorption measurements were made at nm using a titertek multiscan recording photometer (flow labs., glasgow, u.k.). since berne virus had been isolated from a horse with pseudomembraneous enteritis and miliary granulomas with necrosis in the liver (weiss et al., ) , the pathogenic properties of the agent were studied in foals. the animals, which were about months old, received approximately ~ ids units of p / material by the intravenous route. they were examined daily for disease symptoms. serum samples taken from the foals before inoculation of berne virus had neutralization titers of ~< (fig. ) . a distinct rise in titer could be detected on days and post-infection (p.i.) and maximum values were reached at days and p.i., respectively; afterwards the titers tended to decline. neither clinical signs nor abnormal laboratory data were observed in the animals during the whole experiment. it can be seen in table i that the antibody titers in foals initially reflected those of their mothers with whom they shared separate boxes; when they were to weeks old the titers had dropped below detection level. in december--january, when the animals were between and months of age a sudden seroconversion was noted in the whole herd which had been housed together in one stable months previously (beginning of october). endemic infection had obviously taken place; however, enteric disease symptoms were not reported for this period. the animals remained seropositive at subsequent tests. the activity of berne virus in the swiss adult horse population was eval- positive reactions in serum neutralization of small numbers of randomlycollected equine sera from germany ( / ) and the usa ( / ) and in elisa of samples from southern france ( / ) and the usa ( / ) were recorded. the correspondence between the results of the serum neutralization test and elisa was studied. one hundred randomly-selected horse sera were assayed in both tests and correlation of the results was obtained in samples. in cases, the neutralization test was positive (titers approaching ) whereas elisa was negative. the inverse was observed in instances where neutralization-negative sera were weakly ( cases) or distinctly positive ( case) by elisa (table iii) . as shown in table iv , high percentages of serum samples with elevated neutralization titers were encountered in all ungulates studied (horse, cattle, goat, sheep and pig). no antibodies were detected in dogs and foxes; in cats, one sample from the netherlands and one from switzerland reproducibly showed titers of and , respectively. low, but reproducible titers, were also obtained with sera from laboratory rabbits. all sera of the wild mouse species apodemus flavicollis had titers < whereas the ( ) clethrionomys samples had tlters of and , respectively. of the samples from a. sylvaticus, only one had a titer value below . testing of the human sera at low dilutions resulted in neutralization values of <= in all samples; of these samples showed values of < upon re-examination. erratic inhibition of virus growth was observed with sera (calculated "titers" between and ). our study shows that: a high percentage of the adult horses in switzerland has experienced an infection with berne virus; in the very few experimental and natural cases observed by us, the infection was not accompanied by overt clinical symptoms; berne virus is not "new" since antibody incidence has not changed significantly during the last years; berne virus is present in other european countries and in north america. however, it is obvious that the horse, from which the virus was originally isolated, is not the only host species of berne virus or antigenically related viruses. the high prevalence of antibody in other ungulates and the previously demonstrated serological relationship of berne virus with the breda viruses and lyon- virus (weiss et al., ) demonstrated in cattle with diarrhea (woode et al., ) indicate that agents of this new taxonomic cluster may be of significance as pathogens. we have preliminary evidence (from neutralization tests using randomly collected cattle sera) that a berne-related virus is active also in the dutch cattle population; in only samples titers were < and in sera values of > were recorded. also, cattle sera from austria ( / ) and the u.s.a. ( / ) showed neutralization in a titer range of to > (m. weiss and m.c. horzinek, ) . earlier, steck et al. ( ) have reported similar results from switzerland. virus neutralization is characterized by its high sensitivity and specificity. these properties should be kept in mind when interpreting findings of antibody in different species of animals. in ruminants and the pig where high neutralization titers occur, the causative viruses are expected to be closely related to our equine isolate. the marginal values and erratic inhibitions obtained with feline, rabbit and human sera may reflect low affinity antibody induced by a more distant serotype; murine sera would assume an intermediate position. using hemagglutination-inhibition tests, woode et al. ( ) compared isolates (iowa and ) with another strain from ohio (saif et al., ) and arrived at the conclusion that serotypes exist in breda virus. in its exclusivity (recognition of determinants on the virion membrane surface), hemagglutination-inhibition is similar to neutralization; elisa, however, would also detect internal (nucleocapsid) antigens which tend to be evolutionary more conserved and broadly cross-reactive. the discrepancies observed (table iii) in the sense that elisa-negative horse sera gave positive results in neutralization are explained by the superior sensitivity of the latter test. it can be assumed that in those cases where neutralization-negative sera have been found elisa-positive, internal virion antigens are detected. experiments are under way to examine sera from different animal species using elisa and radioimmune precipitation. berne virus did not induce overt disease symptoms in the horse in the few experimental and natural infections observed. it cannot be excluded, however, that the virus may cause inconspicuous or chronic degenerative changes after an apparent primary infection. the pathogenic significance of related viruses has been established in cattle (saif et al., ; woode et al., ) and electron microscopic evidence of coronavirus-like particles in association with enteric disease (see e.g., pass et al., ) should alert the investigator to this new family of viruses (weiss et al., ; horzinek et al., ) . for the time being, however, berne virus in horses must be considered as a "virus in search of disease". coupling of enzymes to antibodies and antigens enzyme-linked immunosorbent assay, elisa. ill. quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigencoated tubes antigenic relationships among homologous structural polypeptides o~ porcine, feline, and canine coronaviruses berne virus is not 'coronavirus-like intestinal coronavirus-like particles in sheep with diarrhoea studies of an enteric "breda" virus in calves. nd immune responsiveness in cattle fatally affected by bovine virus diarrhea-mucosal disease purification and partial characterization of a new enveloped rna virus (berne virus) studies with an unclassified virus isolated from diarrheic calves key: cord- -n rgqyv authors: schuh, amy j.; amman, brian r.; sealy, tara s.; flietstra, timothy d.; guito, jonathan c.; nichol, stuart t.; towner, jonathan s. title: comparative analysis of serologic cross-reactivity using convalescent sera from filovirus-experimentally infected fruit bats date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: n rgqyv with the exception of reston and bombali viruses, the marburgviruses and ebolaviruses (family filoviridae) cause outbreaks of viral hemorrhagic fever in sub-saharan africa. the egyptian rousette bat (erb) is a natural reservoir host for the marburgviruses and evidence suggests that bats are also natural reservoirs for the ebolaviruses. although the search for the natural reservoirs of the ebolaviruses has largely involved serosurveillance of the bat population, there are no validated serological assays to screen bat sera for ebolavirus-specific igg antibodies. here, we generate filovirus-specific antisera by prime-boost immunization of groups of captive erbs with all seven known culturable filoviruses. after validating a system of filovirus-specific indirect elisas utilizing infectious-based virus antigens for detection of virus-specific igg antibodies from bat sera, we assess the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigens. this data is then used to generate a filovirus antibody fingerprint that can predict which of the filovirus species in the system is most antigenically similar to the species responsible for past infection. our filovirus igg indirect elisa system will be a critical tool for identifying bat species with high ebolavirus seroprevalence rates to target for longitudinal studies aimed at establishing natural reservoir host-ebolavirus relationships. unlike the spillover events leading to marburgvirus disease, those for ebolavirus disease have been difficult to associate with a specific environment, such as caves. furthermore, the evidence linking ebolavirus spillover to a particular species of bat is largely circumstantial. for example, the and index cases of sudan virus disease both worked in a cotton factory in sudan, where a retrospective investigation eight to nine months after the initial outbreak identified a large roof colony of trevor's free-tailed bats (mops trevori) directly over the working area of the index case . the putative index case of the ebola virus disease outbreak in the kasaï-occidental province of the drc was said to have purchased bats for consumption following a reported annual migration of hammer-headed bats (hypsignathus monstrosus) and franquet's epauletted fruit bats (epomops franquetti) . lastly, the presumed index case of the ebola virus disease outbreak that started in guinea was reported to have played in a tree hollow, where dna traces of mops condylurus were later identified . the majority of research directed towards the identification of the natural reservoir hosts of the ebolaviruses has consisted of cross-sectional surveillance of the sub-saharan african and asian bat population for evidence of active and past ebolavirus infection. the predominance of cross-sectional studies can be attributed to the large number of bat species residing within the geographic range of ebolavirus circulation that require investigation, as well as the challenges associated with obtaining diagnostic specimens from a mammal that is difficult to capture due to its elusive nocturnal nature and ability to fly. collectively, ebola virus rna has been detected in three bat species captured in the republic of the congo and gabon (hypsignathus monstrosus, epomops franquetti and myonycteris torquata) and reston virus rna has been reportedly detected in four bat species captured in the philippines (chaerephon plicatus, cynopterus brachyotis, miniopterus australis and miniopterus schreibersii) . yet, infectious virus was not isolated from any of these species , , indicating that they are either dead-end virus hosts, had cleared virus infection prior to sampling, or that current filovirus isolation techniques lack the sensitivity to recover infectious virus from specimens with low viral loads. serological reactivity of bat sera with ebolavirus antigens using indirect elisas, indirect fluorescent tests, bead-based multiplex assays and/or western blots has been reported in bats representing at least species throughout sub-saharan africa and asia [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, interpretation of this data has been exceedingly difficult due to multiple reasons that include: ( ) the absence of a panel of positive and negative bat sera for initial serological assay validation and continuing quality control, ( ) the use of various uncharacterized recombinant filovirus antigens, and ( ) the application of different statistical methods for establishing cutoff values for seropositivity. nonetheless, ebolavirus serosurveillance offers several distinct advantages compared to surveillance for active virus infection including a longer diagnostic window (i.e., length of time a diagnostic test can detect evidence of infection), no knowledge of virus-tissue tropism and no requirement for destructive sampling. although erbs are natural reservoir hosts of the marburgviruses only, they develop a robust virus-specific igg antibody response following experimental inoculation with the ebolaviruses , . in this study, we generate high quality, virus-specific antisera by prime-boost immunization of captive erbs with marburg virus, ravn virus, ebola virus, bundibugyo virus, taï forest virus, sudan virus or reston virus. these virus-specific antisera, along with a set of filovirus-naïve bat sera, are then used to validate a series of indirect elisas that utilize non-recombinant, infectious-based filovirus antigens for the detection of virus-specific igg antibodies from bats. we then assess the level of serological cross-reactivity between each virus-specific antiserum and viral antigen and use this information to generate a filovirus antibody fingerprint that is able to predict which of the six filovirus species in our system is most antigenically similar to the species responsible for past infection. filovirus-specific antisera generated from erbs. the bats used to generate the filovirus-specific antisera in this study originated from an erb breeding colony . a total of erbs were divided into seven groups (table ) (table ) , prepared in . ml of sterile dulbecco's modified eagle's medium (thermo fisher scientific inc., waltham, ma, usa). after eight weeks, the bat groups were subcutaneously challenged with log tcid of homologous virus. the bats were euthanized two to four weeks thereafter by cardiac exsanguination under isoflurane anesthesia, followed by an overdose of isoflurane. whole blood collected into serum separator tubes (fisher scientific, grand island, ny, usa) at euthanasia was allowed to clot and then centrifuged at , g for min. the sera were transferred into polypropylene cryoelite wheaton vials (dwk life sciences, millville, nj, usa), and then temporarily stored under liquid nitrogen vapors in the biosafety level four laboratory (bsl- ) until they were double bagged, passed through a dunk tank containing % micro-chem plus (national chemical www.nature.com/scientificreports www.nature.com/scientificreports/ laboratories incorporated, inc., philadelphia, pa, usa) and transported to the gamma-cell irradiator. the doublebagged serum aliquots were sandwiched between layers of dry ice and then exposed to × rads of gamma-cell irradiation using a cobalt- source. filovirus-naïve sera collected from erbs. sera obtained from filovirus-naïve bats from the breeding colony and negative control bats from previous experimental studies , [ ] [ ] [ ] were used as filovirus-naïve sera. these sera were collected and processed in the same manner as outlined above. husbandry. all bats were group-housed according to filovirus inoculum in a climate controlled bsl- animal area, with a h day/ h night cycle. bats received a quantity of fresh fruit, supplemented with protein/vitamin powder, equal to their body mass daily and water ad libitum. (table ) and uninfected control antigen lysate for the filovirus igg indirect elisas were prepared as previously described [ ] [ ] [ ] . briefly, roller bottles of vero e cells were inoculated with virus and the cultures were harvested when ≥ % of cells exhibited evidence of infection by immunofluorescence assay. filovirus antigen lysates were prepared from the cultures by detergent basic buffer extraction of infected cells. uninfected control antigen lysate was generated in the absence of virus, but otherwise prepared in the exact same manner. filovirus igg indirect elisas. each filovirus-specific antiserum (n = ) and filovirus-naïve serum (n = ) was tested for reactivity against each filovirus antigen lysate (n = ) and uninfected control antigen lysate (n = ; supplementary fig. s ). wells of -well elisa plates were coated ( µl) with the dilution of filovirus antigen lysate (diluent: pbs containing % thimerosal) that was found to result in optimal reactivity with sera pooled from each of the filovirus-infected bat groups ( : for the ebola, bundibugyo, taï forest and reston virus lysates, and : for the marburg, ravn and sudan virus lysates) and corresponding wells were coated with an equivalent dilution of uninfected control lysate ( : or : ). after incubation overnight at °c, the plates were washed with pbs containing . % tween- (pbs-t) and µl of serum diluent (pbs containing % skim milk and . % tween- ) was added to each well of the plate. after min, µl of a : dilution of gamma-irradiated bat serum pre-diluted in masterplate diluent (pbs containing % skim milk powder, . % tween- and % thimerosal) was added to the first well of the plate and four-fold serial dilutions were www.nature.com/scientificreports www.nature.com/scientificreports/ performed. final bat serum concentrations were : , : , : and : . following a hr incubation at °c, the plates were washed with pbs-t and µl of a : , dilution of goat anti-bat igg conjugated to horseradish peroxidase (bethyl laboratories, montgomery, tx, usa, cat#: a - p, lot#: a - p- ) in serum diluent was added to the plates. the manufacturer notes that this antibody reacts specifically with bat igg and with light chains common to other immunoglobulins. after incubation for hr at °c, the plates were washed with pbs-t, µl of the two-component abts peroxidase system (kpl, gaithersburg, md, usa) was added, and the plates were allowed to incubate for min at °c. the plates were then read on a microplate spectrophotometer set at nm. the optical density (od) values of each four-fold serial dilution were visually inspected to ensure linearity. to negate non-specific background reactivity, adjusted od values were calculated by subtracting the ods at each four-fold dilution of wells coated with uninfected control antigen lysate from ods at corresponding wells coated with filovirus antigen lysate. the adjusted sum od value was determined by summing the adjusted od values at each four-fold serial dilution. the average adjusted sum od of duplicate runs was reported. notably, performing each indirect elisa (seven filovirus antigen lysates and two dilutions of uninfected control antigen lysate) in duplicate required only µl of serum. influence of sex and age on the reactivity of antisera with homologous antigen. the breeding colony was founded from ugandan wild-caught, filovirus-naïve adult (≥ yr) erbs . for founder bats, age in years was determined by subtracting the date of capture from the date of euthanasia and then adding one year. for bats born in captivity, age in years was determined by subtracting the date of birth from the date of euthanasia. the bats were then classified into three age categories: < yr, ≥ yr-≤ yrs and > yrs. a two-way anova was performed to determine if serological reactivity of bat sera with homologous antigen was significantly influenced (p < . ) by sex, age category, or the interaction between sex and age category (spss statistics , ibm software, armonk, ny, usa). for each filovirus-specific igg indirect elisa, quadratic discriminant analysis was used to determine a cutoff value for seropositivity by considering: ( ) the reactivity of the filovirus-specific antisera group with homologous antigen, ( ) the reactivity of the filovirus-naïve sera group with that same antigen, and ( ) the prior probability of the filovirus-specific antisera group (vba for microsoft access , microsoft office professional plus , redmond, wa, usa). the prior probability of each filovirus-specific antisera group was set at . . this value represents the overall population seroprevalence of marburgvirus in its natural reservoir host, the erb . we assume that the seroprevalence of the ebolaviruses in their respective natural reservoir bat hosts approximates this value. however, the prior probability has relatively little influence on determining the magnitude of the cutoff value for seropositivity. the cutoff value for each filovirus-specific indirect elisa represents the value where the posterior probabilities for the reactivity of the filovirus-specific antisera with homologous antigen and the reactivity of filovirus-naïve sera with this same antigen are equal. sensitivity and specificity of the indirect elisas. for each indirect elisa at its specified seropositivity cutoff value, the filovirus-specific antiserum and filovirus-naïve serum samples were classified as true positive, false positive, false negative or true negative. the sensitivity of each assay was then calculated by dividing the number of individuals with a positive test result by the number of individuals with a history of past infection (experimentally inoculated with that particular virus), and the specificity of each assay was calculated by dividing the number of individuals without a history of past infection (filovirus-naïve group) by the number of individuals with a negative test result. level of serological cross-reactivity. the level of serological cross-reactivity between each group of filovirus-specific antisera and each heterologous virus antigen was calculated by dividing the number of antisera positive for reactivity with a virus antigen by the total number of antisera tested against that antigen (e.g., number of marburg virus antisera positive for reactivity with ravn virus antigen/number of marburg virus antisera tested for reactivity against ravn antigen), and was expressed as a percentage. after the nucleotide sequences of the filovirus isolates used to inoculate the bats in this study were retrieved from genbank, the coding regions were translated into amino acids and aligned using the muscle algorithm (geneious . . , biomatters limited, auckland, new zealand). percent identity values were then calculated from pairwise amino acid comparisons generated from the virus protein alignment. a pearson's product-moment correlation was performed to assess the relationship between percent serological cross-reactivity and percent amino acid identity (spss statistics , ibm software, armonk, ny, usa). antibody fingerprint analysis. quadratic discriminant analysis was used to predict which one of the seven filoviruses in the system was most antigenically similar to the filovirus responsible for past infection by considering the relative reactivity of each filovirus-specific antiserum and filovirus-naïve serum with each filovirus antigen, as well as their covariance (visual basic for microsoft access). first, prior probabilities were calculated for the eight classes used in the analysis (seven filovirus antigen classes and one negative class). second, classification was performed by calculating posterior probabilities for the inclusion of an antisera/sera sample in each class and then assigning the sample to the class with the highest probability. evaluating the performance and discriminatory ability of the filovirus igg indirect elisa system. to evaluate the ability of our system comprising seven filovirus-specific indirect elisas to predict the filovirus species most antigenically similar to the species responsible for past infection, we tested seven marburg virus convalescent serum or whole blood samples collected from experimentally inoculated erbs. five of these samples www.nature.com/scientificreports www.nature.com/scientificreports/ were collected four weeks post primary marburg virus inoculation , , while two of the samples were collected at and weeks post primary inoculation following a "natural" boost (i.e., marburg virus-specific antibody levels waned in these bats and then increased following contact with infectious cagemates) . serological reactivity with homologous filovirus antigen is not influenced by age or sex. a total of erbs were used in this study that was performed over a two-year time span. the total number of bats dedicated to this study, as well as the size and composition of each group was dependent on the reproductive capacity of the erb breeding colony, as well as the characteristics and number of bats needed for other experimental studies. the bats were divided into seven groups, with group sizes ranging from - individuals (table ) . two groups of bats had approximately equal sex ratios (ravn and taï forest), while two groups were comprised of mostly female individuals (bundibugyo and sudan), and the remaining five groups were comprised of all male (reston) or all female (marburg and ebola) individuals. two groups of bats were comprised solely of individuals < yr of age (bundibugyo and taï forest), while the majority of individuals in three groups were ≥ -≤ yrs of age (marburg, ebola and reston) and the majority of individuals in two groups were > yrs of age (ravn and sudan). despite the observed variability in group composition, a two-way anova revealed that serological reactivity with homologous filovirus antigen was not significantly (p > . ) influenced by sex (f( , ) = . ), age (f( , ) = . ), or the interaction between sex and age (f( , ) = . ). filovirus-specific indirect elisas are highly sensitive and specific. with the exception of the igg antibody indirect elisa using marburg virus antigen ( % sensitivity and % specificity; fig. a and table ), all of the assays exhibited % sensitivity and specificity ( fig. and table ). the mean group adjusted sum od values ranged from . (± . sd) for the igg antibody indirect elisa using marburg virus antigen to . (± . sd) for the igg antibody indirect elisa using ebola virus antigen. strong positive correlation between filovirus serological cross-reactivity and filovirus protein amino acid identity. figure and table show the level of serological cross-reactivity between each group of bat filovirus-specific antisera and the six heterologous filovirus antigens. the consistent magnitudes of serological reactivity of the individual bat antisera across all six heterologous filovirus antigens highlights the robust performance of our filovirus igg indirect elisa system. strong serological cross-reactivity was observed between marburg virus antisera and ravn virus antigen ( %), and ravn virus antisera and marburg virus antigen ( %) (fig. a,b) . (fig. e-g) . very limited cross-reactivity was observed between marburgvirus antisera and ebolavirus antigen ( %: majority of antisera-antigen combinations; %: marburg virus antisera versus taï forest virus antigen, ravn virus antisera versus bundibugyo and taï forest virus antigens; %: ravn virus antisera versus reston virus antigen), and ebolavirus antisera tested against marburgvirus antigen ( . %: sudan virus antisera versus marburg virus antigen; . % sudan virus antisera versus ravn virus antigen) (fig. ) . a pearson's product-moment correlation revealed that there was a statistically significant, strong positive correlation between percent filovirus serological cross-reactivity and percent filovirus amino acid identity (r = . , n = , p < . ). filovirus serological cross-reactivity dataset used to develop an antibody fingerprint classification system. we used the adjusted sum od data (each serum tested against each antigen) generated from bats intentionally infected with each of the seven known culturable filoviruses to develop a classification system (fingerprint) to predict which filovirus elicited the antibody response. supplementary table s shows posterior probability support values for classification of each filovirus-specific antiserum or filovirus-naïve serum sample into eight pre-defined classes (seven filovirus antigen classes and one negative class), with probabilities of each row in the table summing to . . each sample was assigned to the class with the highest posterior probability (i.e., highest degree of certainty). overall, all of the samples ( / ) were correctly classified, with posterior probabilities in support of the true class (i.e., filovirus used to prime-boost each bat) ranging from . to . . this indicates that this system, using seven independent filovirus igg indirect elisas, has the ability to establish an antibody fingerprint from filovirus convalescent bat sera that can be used to predict which of the filovirus species in the system is the most antigenically similar to the species responsible for past infection (see supplementary note for an example of how the antibody fingerprinting was performed in this study). to further assess the performance and predictive ability of our filovirus igg indirect elisa system, we tested five marburg virus convalescent sera or whole blood samples collected four weeks post primary marburg virus experimental inoculation and two whole blood samples collected at and weeks post primary experimental inoculation following a "natural" boost (i.e., marburg virus-specific antibody levels waned and then increased following contact with infectious cagemates). as expected, marburgvirus-specific igg antibody levels were markedly lower in these seven convalescent serum and whole blood samples compared to marburgvirus-specific igg antibody levels in the serum samples from the prime-boosted bats in this study (fig. a) . however, all samples collected after primary marburg virus inoculation or following a "natural" boost were classified as marburgvirus igg antibody positive. the antibody fingerprint analysis predicted that three of these bats were previously infected with marburg virus (sample ids , and with marburg posterior probability values of . , . , and . , respectively) and four were previously infected with ravn virus (sample ids , , and with ravn posterior probability values of . , . , . and . , respectively; fig. b ). www.nature.com/scientificreports www.nature.com/scientificreports/ in this study, we generated filovirus-specific antisera from prime-boosted erbs and collected filovirus-naïve sera to validate and characterize an indirect elisa system that utilizes non-recombinant, infectious-based filovirus antigens for the detection of virus-specific igg antibodies from bats. initial validation of the system revealed that the individual filovirus-specific igg indirect elisas exhibited high sensitivity ( - %) and specificity ( %). similar to previous studies using human sera, we observed limited to no serological igg cross-reactivity between the filovirus genera ( - %) [ ] [ ] [ ] , varying levels of serological igg cross-reactivity between the ebolavirus species table for statistics on the reactivity of each filovirus antigen lysate with homologous antisera and filovirus-naïve sera, as well as for the sensitivity and specificity of each of the filovirus igg indirect elisa. www.nature.com/scientificreports www.nature.com/scientificreports/ ( - %) , and strong serological igg cross-reactivity within the species marburg marburgvirus ( %). the low level of serological igg cross-reactivity between some of the ebolavirus species underscores the necessity of performing all assays within the system to avoid false negative results. although significant levels of serological igg cross-reactivity were observed between the prime-boost filovirus-specific antisera and some of the filovirus antigens, when the overall covariance of the seven-individual indirect elisas in the system were considered, we were able to predict the filovirus species responsible for past infection % of the time using as little as μl of sera (each serum was tested against each antigen in duplicate). further evaluation of the performance and predictive ability of our system using seven marburg virus convalescent serum or whole blood samples collected after primary experimental infection with marburg virus (or primary experimental infection with marburg virus plus a "natural" boost) confirmed that the system was able to predict the filovirus species most likely responsible for past infection. as expected, the filovirus igg indirect elisa system was not able to correctly predict whether past infection was due to marburg virus or ravn virus % of the time. this finding supports the current classification of marburg and ravn viruses into a single virus species, marburg marburgvirus . the robustness and discriminatory power of our system results from its underlying components and various control points. we employed infectious-based filovirus antigens, rather than recombinant virus protein antigens, as a strategy to increase both the sensitivity (i.e., ability of the filovirus igg indirect elisa system to correctly identify those with past exposure to a filovirus as filovirus igg antibody positive) and specificity (i.e., ability of the filovirus igg indirect elisa system to correctly identify those with no past exposure to a filovirus as filovirus igg antibody negative) of the system. although recombinant filovirus antigens can be generated in large quantities and do not require a bsl- laboratory for production, the use of single recombinant virus proteins for antibody detection can lead to false negative results if an individual's antibody repertoire is not directed against the particular recombinant virus protein that was employed , [ ] [ ] [ ] [ ] [ ] [ ] . alternatively, false positive serological results can occur when recombinant virus antigens unknowingly share similar epitopes with other virus antigens for which the study population possesses antibodies against , . false positive results can also arise from failing to account for non-specific serological reactivity resulting from the presence of non-virus contaminants in an antigen preparation. here, we negated non-specific serological reactivity to vero e cell components by subtracting the reactivity of wells coated with uninfected antigen lysate from the reactivity of corresponding wells coated with filovirus antigen lysate. while the majority of previously published ebolavirus serosurveys of bats used recombinant filovirus antigens generated in bacterial expression systems, not all of the studies implemented procedures to negate non-specific reactivity between residual bacterial epitopes in the antigen preparation and antibacterial antibodies in bat sera , . like the majority of published ebolavirus serosurveys of bats, we performed serial serum dilutions to assess for linearity and specificity , , , [ ] [ ] [ ] [ ] . most importantly, we included pooled filovirus-specific bat antisera as a positive control and pooled filovirus-naïve bat sera as a negative control in every run to ensure that our filovirus igg indirect elisa system continually performed as expected. using filovirus-specific antisera collected from bats that were prime-boosted at the same time not only allowed us to thoroughly examine the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigen, but also provided the opportunity to use this knowledge to evaluate previously published ebolavirus serosurveys of bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . four out of the previously published ebolavirus serosurveys of bats used ebola virus antigen only to detect ebolavirus igg antibodies , [ ] [ ] [ ] and another four of these serosurveys used ebola and reston antigens only to detect ebolavirus igg antibodies , [ ] [ ] [ ] . based on the low serological cross-reactivity between ebola virus antigen and sudan virus antisera ( . %) and reston virus antigen and sudan virus antisera ( . %) observed in our study, we predict that the serosurveys using ebola virus antigen or ebola and reston virus antigens only would have missed the opportunity to detect bats previously infected with sudan virus. likewise, due to the high level of serological cross reactivity between ebolavirus-specific antisera and heterologous ebolavirus antigen that we observed in our study, it is possible that the serological reactivity of bat sera with ebola or reston virus antigens in the previous serosurveys was due to past infection with one of the other known ebolaviruses or an undescribed ebolavirus. although we successfully used quadratic discriminant analysis to predict which of the filovirus species in the system was most antigenically similar to the species responsible for past infection, only nucleotide sequence data can be used to ascertain infection with a particular filovirus. furthermore, incrimination of an ebolavirus-natural reservoir host relationship requires consistent detection of both active (rna and virus isolation from tissues/bodily fluids suggestive of a transmission mechanism) and past infection with a particular ebolavirus through space and time. www.nature.com/scientificreports www.nature.com/scientificreports/ while our filovirus igg indirect elisa system enables the robust detection of virus-specific igg antibodies from bats and was able to predict the filovirus species most likely responsible for past infection, it does have some limitations. first, this system was validated using virus-specific antisera from prime-boosted bats and sera from table for statistics on the cross-reactivity between each filovirus-specific bat antisera and heterologous filovirus antigen. www.nature.com/scientificreports www.nature.com/scientificreports/ filovirus-naïve bats. although all seven of the marburg virus convalescent whole blood and serum samples collected from erbs after primary experimental marburg virus inoculation or following a "natural" boost were marburgvirus igg antibody positive, these samples had markedly lower marburgvirus-specific igg antibody levels compared to samples collected from the bats in this study that were prime-boosted with one of seven filoviruses. this suggests that the sensitivity of the individual filovirus igg indirect elisas may be lower than what was reported here ( - %) using filovirus-specific antisera from prime-boosted bats and filovirus naïve bat sera. however, statistical determination of seropositivity cutoff values for the indirect elisas were largely driven by the variance surrounding the mean reactivity of the filovirus-naïve sera with each of the virus antigens. it is likely that circulation and long-term maintenance of the ebolaviruses in their natural reservoir hosts leads to multiple virus exposures throughout the lifetime of an individual, resulting in boosting of virus-specific igg antibody levels. "natural" boosting of virus-specific igg antibody levels in erbs that had been previously infected with marburg virus was observed during a recent experimental transmission study shortly after documenting virus infection and seroconversion in naïve contact bats . others have reported periodic boosting of rabies virus neutralizing antibody titers in a wild colony of big brown bats (eptesicus fuscus) . second, we used a goat anti-bat igg conjugated to horseradish peroxidase as the secondary antibody in our system. this antibody was generated using www.nature.com/scientificreports www.nature.com/scientificreports/ sera from erbs, as well as sera from nine other bat species comprising four chiropteran families (manufacturer product datasheet). while this antibody has been confirmed to react with sera from these chiropteran species and has been used with success to detect igg antibodies in sera from > bat species comprising seven chiropteran families [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it is possible that its reactivity with igg antibodies from divergent bat species might diminish. if this is the case, the concentration of secondary antibody may need to be optimized prior to testing a new species of bat or an alternative secondary antibody may need to be used. third, our filovirus igg indirect elisa system includes seven virus antigens that represent all of the reported culturable filoviruses. given the recent discoveries of filovirus rna from divergent bat families in geographically distant locations ( ), we expect that there are a number of undiscovered ebolaviruses that continue to circulate in nature. however, we believe that the genetic diversity of ebolavirus antigens (up to . %) included in our system will allow for the detection of past infection with a novel ebolavirus, and prediction of the ebolavirus species that is most antigenically similar to the species responsible for past infection. we intend to use our filovirus igg indirect elisa system to screen archived and incoming specimens from cross-sectional serosurveys of the bat population of sub-saharan africa and asia for evidence of past filovirus infection. if other specimen types exist for bat species identified as filovirus seropositive, they will be tested for evidence of active infection using virus-specific qrt-pcr, pan-filovirus rt-pcr and/or virus isolation techniques. bat species positive for evidence of active filovirus infection or species exhibiting a seroprevalence equivalent to or larger than that of marburgvirus in erbs (~ % total population seroprevalence) will be targeted for longitudinal studies aimed at collecting a wide range of specimens for evidence of active and past ebolavirus infection. while most of the virus-specific antisera generated in this study will be pooled to use as positive controls in our indirect filovirus elisa system, some will be reserved for investigations aimed at determining the mechanisms by which bats clear and control filovirus infections through virus neutralization and cross-neutralization assays, as well as epitope mapping studies. any remaining filovirus-specific bat antisera may be used to develop a pan-ebolavirus indirect elisa that utilizes a mixture of non-recombinant, infectious based antigens from culturable ebolaviruses, as well as validate the performance of indirect elisas that use recombinant filovirus proteins to detect filovirus antibodies. ecology of filoviruses taxonomy of the order mononegavirales: second update epidemiology of ebola (subtype reston) virus in the philippines the discovery of bombali virus adds further support for bats as hosts of ebolaviruses field aspects of the marburg virus outbreak: . primate supply marburg-virus disease in kenya report of a who/international study team. ebola haemorrhagic fever in sudan report of an international commission. ebola haemorrhagic fever in zaire human infection due to ebola virus, subtype cote d'ivoire: clinical and biologic presentation newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda epidemiologic investigation of marburg virus disease marburg hemorrhagic fever associated with multiple genetic lineages of virus characterization of a new marburg virus isolated from a fatal case in kenya studies of reservoir hosts for marburg virus isolation of genetically diverse marburg viruses from egyptian fruit bats response to imported case of marburg hemorrhagic fever, the netherlands imported case of marburg hemorrhagic fever -colorado seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection ch. approaches towards studies on potential reservoirs of viral haemorrhagic fever in southern sudan ( ) human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo investigating the zoonotic origin of the west african ebola epidemic fruit bats as reservoirs of ebola virus molecular evidence of ebola reston virus infection in philippine bats spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus long-term survival of an urban fruit bat seropositive for ebola and lagos bat viruses ebola virus antibodies in fruit bats serological evidence of ebolavirus infection in bats, china ebola virus antibodies in fruit bats seroepidemiological prevalence of multiple species of filoviruses in fruit bats (eidolon helvum) migrating in africa serologic evidence of fruit bat exposure to filoviruses survey of ebola viruses in frugivorous and insectivorous bats in guinea, cameroon, and the democratic republic of the congo experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera experimental inoculation of egyptian fruit bats (rousettus aegyptiacus) with ebola virus oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) modelling filovirus maintenance in nature by experimental transmission of marburg virus between egyptian rousette bats egyptian rousette bats maintain long-term protective immunity against marburg virus infection despite diminished antibody levels clinical virology of ebola hemorrhagic fever (ehf): virus, virus antigen, and igg and igm antibody findings among ehf patients in kikwit, democratic republic of the congo elisa for the detection of antibodies to ebola viruses serologic cross-reactivity of human igm and igg antibodies to five species of ebola virus enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies serological evidence of ebola virus infection in indonesian orangutans human survivors of disease outbreaks caused by ebola or marburg virus exhibit cross-reactive and long-lived antibody responses development of a sensitive and specific serological assay based on luminex technology for detection of antibodies to zaire ebola virus false-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid enzyme-linked immunosorbent assay due to hcov-oc and hcov- e rectified by western blotting with recombinant sars-cov spike polypeptide cross-reactivity between caprine arthritisencephalitis virus and type human immunodeficiency virus variability in seroprevalence of rabies virus neutralizing antibodies and associated factors in a colorado population of big brown bats (eptesicus fuscus) serologic survey of eptesicus fuscus from georgia, usa for rickettsia and borrelia and laboratory transmission of a rickettsia by bat ticks coronavirus antibodies in african bat species immunogenicity difference between the sars coronavirus and the bat sars-like coronavirus spike (s) proteins bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family. hepeviridae hibernating little brown myotis (myotis lucifugus) show variable immunological responses to white-nose syndrome isolation and characterization of a novel alphaherpesvirus in fruit bats a novel rhabdovirus isolated from the straw-colored fruit bat eidolon helvum, with signs of antibodies in swine and humans evidence of hantavirus infection among bats in brazil identification of secreted and membrane-bound bat immunoglobulin using a microchiropteran-specific mouse monoclonal antibody antibodies against henipa-like viruses in brazilian bats. vector borne zoonotic dis discovery of an ebolavirus-like filovirus in europe re-emergence of lloviu virus in miniopterus schreibersii bats characterization of a filovirus (měnglà virus) from rousettus bats in china the authors declare that all data supporting the findings of this study are available within the article or from the corresponding author upon request. we thank the diagnostics team from the viral special pathogens branch at the centers for disease control and prevention (cdc) for preparing the filovirus antigen lysates used in this study. we would also like to thank peter eworonsky, lester slough and eddie jackson from the comparative medicine branch at the cdc for providing care to the bats during this study. this study was partially funded by dtra grant hdtra- - - , subaward s- - . the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. supplementary information accompanies this paper at https://doi.org/ . /s - - -z. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -mpuovmeb authors: bratcher, preston e.; gaggar, amit title: factors influencing the measurement of plasma/serum surfactant protein d levels by elisa date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: mpuovmeb background: extensive variations in human surfactant protein d (sp-d) levels in circulation as measured by elisa exist in the published literature. in order to determine the source of these variations, factors influencing the measurement by elisa were explored. materials and methods: peripheral blood from healthy individuals was collected into various vacutainers during the same blood draw. recombinant sp-d was diluted into different matrices and used for a standard curve. samples were analyzed by capture elisa using one of two distinct detection antibodies. results: the type of matrix had some effects on detection of recombinant sp-d. the type of anticoagulant used and dilution factor had very little effect, except for in plasma collected in edta vacutainers. the extent of variation in published values seemed to be due to the elisa configuration employed, and, in agreement with this, we found that by switching the detection antibody, there was a % decrease in the extrapolated sp-d value of serum and plasma samples. storage of samples resulted in slight changes in measured sp-d levels. conclusions: the elisa configuration employed to measure circulating levels of sp-d has a significant effect on the extrapolated values. in both configurations tested, the use of edta as a coagulant resulted in inconsistent values, and we, therefore, suggest the avoidance of this anticoagulant when assaying for sp-d by elisa. while the demonstrated effects of several factors on measurement of sp-d may not account for all the disparities amongst the previous studies, they stress that variations in methodologies for measuring the same protein can result in very inconsistent results. surfactant protein d (sp-d) is a pulmonary collectin involved in regulation of inflammation, innate immune defense, and surfactant homeostasis. it is expressed by clara cells and alveolar type ii cells in the lung. sp-d has a multimeric structure which gives it the ability to agglutinate pathogens, as well as aid in the clearance of apoptotic cells, cellular debris, and foreign particles in the lung [reviewed in [ ] ]. circulating levels of sp-d have been examined for their potential use as a biomarker in various diseases including dermatitis [ , ] , acute lung injury (ali)/acute respiratory distress syndrome (ards) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , periodontitis [ ] , interstitial pulmonary fibrosis (ipf) [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , chronic obstructive pulmonary disease (copd) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , emphysema [ ] , cystic fibrosis (cf) [ , , ] , coronary disease [ , ] , sclerosis [ ] [ ] [ ] [ ] [ ] , cancer [ , ] , sarcoidosis [ , ] , allergies [ , [ ] [ ] [ ] , rheumatoid arthritis [ , ] , and respiratory infections [ , [ ] [ ] [ ] [ ] [ ] [ ] . sp-d levels have also been proposed to correlate with genetic elements [ ] [ ] [ ] , body mass index (bmi) [ ] [ ] [ ] [ ] [ ] , age [ ] , circadian rhythm [ ] , and with particle exposure [ , ] and cigarette smoking habits [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] . in addition, there have been studies examining the levels of sp-d in subjects with turner syndrome [ ] , paraquat intoxication [ ] , swimming in variably treated waters [ ] , lung transplant patients [ ] , patients undergoing neurosurgical operations [ ] , drowning victims [ ] , polymyositis/dermatomyositis [ ] , dementia [ ] , lupus [ ] , and sleep apnea [ ] . similarly to cc- and kl- , sp-d is thought to be a marker of pulmonary leak into the vasculature [ ] , and therefore alveolar destruction would result in an increase in levels of these pulmonary proteins in the blood. however, protein levels in lung do not always correlate with protein levels in blood [ ] , suggesting the possibility of alternative mechanisms affecting sp-d levels in circulation. various commercially available kits and non-commercially available elisa configurations have been used to compare sp-d levels in plasma and serum from normal, healthy controls and the various disease states described above. interestingly, there is a very substantial discrepancy between the reported values of the healthy control populations between studies, as well as in the magnitude of the range of values in this population. while the elisa configuration used to measure sp-d seemed to have a large impact on the values reported, there are significant variations in the healthy control sp-d levels and range amongst reports using the same configuration. the purpose of this study is to determine factors affecting the measurement of sp-d by elisa that may, therefore, explain the variations of serum and plasma sp-d levels reported in the published literature. all human studies were approved by the university of alabama at birmingham institutional review board for human use with all subjects providing written consent. peripheral blood was collected from healthy volunteers by venipuncture into serum, heparin sulfate, k edta, and sodium citrate vacutainers (bd biosciences) during a single draw. samples were kept at room temperature until blood in the serum tube was coagulated. afterwards, samples were centrifuged at xg for minutes to separate blood cells from serum or plasma. samples were either directly aliquoted and stored at uc or given an additional round of centrifugation at xg for minutes to separate platelets from serum or plasma. all serum and plasma sample values depicted in figures were free of platelets. all experiments had samples per group except for the experiments depicted in mouse antihuman sp-d capture antibody, biotinylated mouse antihuman sp-d detection antibody, streptavidin conjugated to horse radish peroxidase, and recombinant human sp-d standard were purchased as a kit from r&d systems (catalog # dy ) and used according to the manufacturer's protocol. for experiments testing the effects of different matrices on the detection of recombinant human sp-d, a concentrated stock was made in pbs with % bsa from separately purchased recombinant human sp-d expressed in nso cells (r&d systems, catalog # -sp- ). for studies comparing detection antibodies, polyclonal goat antihuman sp-d antibody (r&d systems, catalog # af ) was used instead with a monoclonal mouse antigoat conjugated to horse radish peroxidase (sigma) used as a secondary. all sample values were extrapolated from a second order polynomial curve fit of concentrations of the standard (two-fold dilutions with a high concentration of ng/ml) diluted in % bsa pbs. statistical analyses were performed as described in the figure legends using prism (graphpad software), and all reported p values are twotailed. all error bars represent standard deviation. in order to determine the mean and range of sp-d levels in the serum/plasma of normal, healthy individuals, we performed a non-exhaustive search of the literature which revealed more than publications in which these values were described. interestingly, an unexpected amount of variation on these reported values was observed. when grouping according to the elisa configuration used, one configuration consistently resulted in significantly higher values than the two other configurations for which multiple references were obtained (figure a ). for each configuration, a substantial range of means/medians was observed in the healthy control population. in addition to the variations seen in the averages, the spectrum of values in all populations ranged from . pg/ml [ ] to . mg/ml [ ] , representing a more than , fold difference. when examining the range of values seen in healthy subjects from each individual study and grouping according to the elisa configuration used, a large variation in range was observed ( figure b) . overall, the study with the smallest range for any population (as a percentage of the average) reported a % confidence interval of . - . [ ] , while the study containing the population with the largest range reported a standard deviation of % of the mean value [ ] . based on the above observations, we can infer that the largest difference in the measured levels of sp-d is due to the elisa configuration employed, but that there are still significant differences between the averages of healthy individuals from studies using the same configurations. in order to compare the results of various studies in another manner, we examined the fold change from healthy populations for the four diseases/conditions for which a substantial number of publications were available (ipf, cf, cigarette smoking, and systemic sclerosis). this would allow us to control for elisa configurations employed, technical protocol followed for the elisa as well as for sample collection and processing, and any differences in the populations (as controls have been appropriately matched). the expectation was that the fold change from healthy would be very similar for each disease/ condition. however, large differences in the extent of this change were observed (figure c ). in order to determine if the matrix used to generate the serial dilutions of recombinant human standard (rhsp-d) had any effects upon measurement, we compared values produced by dilutions in pbs, solutions of bsa (in pbs), and solutions of fbs (diluted in pbs) (figure ). at higher concentrations of protein (i.e. % bsa, % fbs, and % fbs), a decrease in the amount of rhsp-d detected was observed, with the amount measured in samples containing ng/ml rhsp-d being the most variable of any samples measured in this assay. using the manufacturer's recommended matrix ( % bsa) produced results very similar to a % fbs matrix, with both giving very consistent measurements and the greatest values. the values for pbs matrix gave similarly consistent measurements, but at a slightly lower value. this effect may be due to adsorption of rhsp-d without a carrier protein to the tubes used for serial dilutions [ ] . serum matrix (millipore), which had background levels of sp-d, also inhibited detection of recombinant sp-d at higher concentrations. all further standard curves were established by serial dilutions of recombinant sp-d in % bsa in pbs. one factor that was found to differ between studies and could, therefore, be a source of variation of reported healthy population values, was the type of anticoagulant (or lack thereof) in the collection container. when blood was simultaneously collected in various vacutainers, serum and heparin plasma gave similar measurements of sp-d, while citrate plasma gave values significantly lower than serum values (figure ). edta plasma gave the most inconsistent results, and values were also significantly lower than serum values. in order to determine if calcium concentration in the sample had a significant effect upon the measurement of sp-d, rhsp-d was assayed in serially diluted cacl or edta. the detection of rhsp-d was only slightly effected by the changes in calcium concentration in the sample (figure a ). additionally, we examined the effects of calcium concentration in the human samples by adding calcium to edta plasma samples and by adding edta to serum samples. while reconstituting the free calcium in the edta samples had little effect, the addition of edta to the serum samples had a dramatic effect that was inconsistent amongst the patient samples. while many elisas employ the use of pbs lacking divalent cations as a buffer during the detection of antibody-captured antigens, the elisa configuration employed by holmskov et al. uses a tris-buffered saline solution containing mm calcium chloride, as the monoclonal detection antibody binds to sp-d in the presence of calcium [ ] . in addition, it was previously demonstrated that sp-d has the ability to bind to various immunoglobulins in a calcium-dependent manner [ ] . in order to determine the effects of calcium in the context of sp-d detection by elisa, elisas were performed with antibodies diluted in buffer with or without calcium. we examined these effects on both recombinant sp-d and sp-d in plasma with either the elisa kit's detection antibody or a polyclonal goat anti-sp-d antibody produced by the same manufacturer. in all cases but one, the addition of mm calcium to the dilution and wash buffer resulted in a small (, %) but significant increase in sp-d concentration relative to detection in the absence of calcium (figure b ). it is important to note that with the kit reagents, since the relative increase in detection of recombinant sp-d and sp-d in plasma in the presence of calcium is very similar, the extrapolated sp-d concentration in plasma when using a standard curve of recombinant sp-d should not significantly change. interestingly, while the inclusion of calcium increased the recognition of native sp-d by the polyclonal antibody, it had no effect on the detection of recombinant sp-d by this antibody. although it is beyond the scope of this study, future work will explore whether this effect is due to an increase in antibody recognition of antigen, increase in non-specific binding of antibodies by captured sp-d, or an increase through another mechanism. given that some of the variation seen in the published literature might be explained by the use of different antibodies, we detected sp-d in serum and plasma samples using either the elisa kit's detection antibody or the polyclonal goat anti-sp-d antibody. while there is no significant difference between detection by the [ , [ ] [ ] [ ] [ ] ] , cf [ , , ] , cigarette smoking [ , , [ ] [ ] [ ] [ ] , or sclerosis [ ] [ ] [ ] ] was different between publications. an asterisk (*) denotes p# . by one-way anova with tukey's multiple comparison test. doi: . /journal.pone. .g kit antibody versus the polyclonal goat antibody with regard to the type of collection tube used, overall, there is a , % reduction in the extrapolated value produced by detection with the polyclonal goat antibody compared to the kit antibody ( figure ). in both cases, the same capture antibody was employed, and it is therefore possible that using a different capture antibody could have a similar effect on varying the extrapolated sp-d concentration in the sample. another factor that fluctuated from study to study was whether the sample was assayed neat or the sample was prediluted (and the amount the sample was diluted). given this fact and that diluting recombinant sp-d into % fbs had some influence in the detection of said sp-d, we compared sp-d values detected in undiluted and diluted samples. there was a very minimal difference between values produced by undiluted and fold diluted serum, citrate plasma, and heparin plasma ( figure ). ten fold diluted edta plasma, however, produced a significantly higher extrapolated sp-d value when compared to the same sample undiluted. this same effect was seen when the polyclonal goat antibody tested above was used for detection (data not shown). while the most common processing technique involved separating the blood cells (but not platelets) from serum and plasma and storing this sample at uc until assayed, some variations in processing and storage were present. to address this, we assayed serum and heparin plasma without platelets immediately after processing or after storage at uc, uc, or uc ( figure ) ; there were no significant differences between conditions. additionally, there were only subtle differences between a sample stored at uc and uc, and depending on whether the sample went through a freeze/ thaw (ft) cycle, contained platelets or not, or was spun after thawing to separate any precipitates/debris (data not shown). sp-d concentrations of samples stored at uc and uc for two weeks were not different from the same samples at one week (data not shown). this study provides experimental evidence that variations in the anticoagulant used and elisa configuration can have dramatic effects upon the measured sp-d level. storage and processing of samples as well as diluent used for the standard have minor effects on the level of sp-d extrapolated by elisa. although these results may partially explain the variations seen in reported sp-d values in the blood, it is important to note the caveats associated with this review of the literature. for the comparison of healthy control groups amongst various studies, it is possible that differences in ethnic background and/or geographical location from which subjects were recruited may contribute to the variations seen when the same elisa configuration was employed, as could the type of anticoagulant. however, we would expect the range of samples between healthy populations of different studies to be similar. in addition, assuming that an appropriately matched control population was used for comparison to the disease population, the fold change between healthy and disease should be more similar than what was observed in the literature. measuring sp-d by elisa presents a problem due to its multimeric structure. for each molecule (a complete dodecamer) of multimerized protein, there are at least of the same epitopes present. due to this property, it is not necessary to use discrete antibodies in a sandwich elisa format to capture and then detect sp-d; even the same monoclonal antibody could be used for both. however, this may cause a difference in read out compared to using antibodies that detect different epitopes, due to the fact that the degree of multimerization has been shown to vary in the lungs during different disease states [ ] and using the same monoclonal antibody would fail to detect a monomer. even using two monoclonals that recognize distinct epitopes to measure sp-d in its native state would be confounded by differences in the degree of multimerization. for example, if an antibody against the carbohydrate recognition domain of sp-d was used to capture and an antibody against the neck region of sp-d was used to detect, a single, captured monomer of sp-d would be able to bind a single anti-neck antibody. however, a single, captured dodecamer would be able to bind anti-neck antibodies. in order to accurately establish absolute concentrations by elisa, it would be important that the multimeric structure of the standard was equivalent to that of the samples, as there could be differences in antigen binding in higher order multimers due to steric hinderance. unfortunately, we did not have the necessary resources to examine the influence of the degree of multimerization on elisa measurements. in addition to the possible effects caused by its multimeric nature, there is evidence in the literature to variable posttranslational modifications of sp-d in native samples. these modifications may, in fact, mask the epitope recognized by an elisa antibody. this may account for one of the largest differences seen in the reported elisa measurements of sp-d in serum/plasma, as the highest concentrations of sp-d came from the non-commercially available elisa which used purified, native sp-d as a standard; this is opposed to almost all others, which used recombinantly expressed sp-d as a standard. regardless of whether the antibodies used were raised against recombinant sp-d or purified native, because the post-translational modifications can vary from one patient to the next, this presents a significant obstacle to accurate measurements of natural sp-d using antibodies. the results of this study demonstrate the influence that the antibody may have on the sp-d concentration as measured by elisa. in our experiments, it is important to note that the same recombinant standard and human serum/plasma samples were analyzed. given the confounders mentioned above, it is possible that the antibody from the kit recognizes an epitope that is not post-translationally modified or altered in human serum/plasma sp-d, while several of the epitopes recognized by the polyclonal antibody are modified in human serum/plasma sp-d, but were unmodified in the recombinantly expressed sp-d (which was used as the immunogen). this would result in the observed relative decrease in binding of the native sp-d from the blood compared to the recombinant sp-d. accuracy of sp-d measurements is critically important to the validation of this protein as a biomarker in pulmonary disease. standardization of sample processing and storage, including the avoidance of edta as an anticoagulant, is necessary to ensure consistent results. although absolute values may vary greatly due to the elisa configuration employed, relative differences in sp-d concentrations amongst various disease groups should be consistent throughout the published literature. we are currently exploring alternative methods to quantify sp-d levels in the blood, as we believe this is necessary in order to establish the absolute concentration. surfactant proteins sp-a and sp-d: structure, function and receptors surfactant protein d (sp-d) and systemic scleroderma (ssc) surfactant protein d in atopic dermatitis and psoriasis plasma surfactant protein levels and clinical outcomes in patients with acute lung injury effects of leukoreduced blood on acute lung injury after trauma: a randomized controlled trial surfactant phospholipids, surfactant proteins, and inflammatory markers during acute lung injury in children prognostic and pathogenetic value of combining clinical and biochemical indices in patients with acute lung injury plasma cc levels are associated with development of ali/ards in patients with ventilator-associated pneumonia: a retrospective observational study plasma levels of surfactant protein d and kl- for evaluation of lung injury in critically ill mechanically ventilated patients plasma biomarker profiles in acute exacerbation of idiopathic pulmonary fibrosis biomarkers of lung epithelial injury and inflammation distinguish severe sepsis patients with acute respiratory distress syndrome serum surfactant proteins-a and -d as biomarkers in idiopathic pulmonary fibrosis serial changes in surfactant-associated proteins in lung and serum before and after onset of ards increased plasma concentration of surfactant protein d in chronic periodontitis independent of sftpd genotype: potential role as a biomarker effect of single vs bilateral lung transplantation on plasma surfactant protein d levels in idiopathic pulmonary fibrosis pulmonary surfactant protein d in sera and bronchoalveolar lavage fluids enzymelinked immunosorbent assay using f(ab') fragment for the detection of human pulmonary surfactant protein d in sera comparative study of kl- , surfactant protein-a, surfactant protein-d, and monocyte chemoattractant protein- as serum markers for interstitial lung diseases increased circulating levels of soluble fas ligand are correlated with disease activity in patients with fibrosing lung diseases monitoring markers of disease activity for interstitial lung diseases with serum surfactant proteins a and d clinical importance of bronchoalveolar lavage fluid and blood cytokines, surfactant protein d, and kerbs von lungren antigen in idiopathic pulmonary alveolar proteinosis significance of serum vascular endothelial growth factor level in patients with idiopathic pulmonary fibrosis pneumocyte biomarkers kl- and surfactant protein d reflect the distinct findings of high-resolution computed tomography in nonspecific interstitial pneumonia integrating lung and plasma expression of pneumo-proteins in developing biomarkers in copd: a case study of surfactant protein d ageing and smoking contribute to plasma surfactant proteins and protease imbalance with correlations to airway obstruction inflammatory biomarkers improve clinical prediction of mortality in chronic obstructive pulmonary disease budesonide/formoterol enhances the expression of pro surfactant protein-b in lungs of copd patients exhaled metallic elements and serum pneumoproteins in asymptomatic smokers and patients with copd or asthma circulating surfactant protein d as a potential lung-specific biomarker of health outcomes in copd: a pilot study the effects of fluticasone with or without salmeterol on systemic biomarkers of inflammation in chronic obstructive pulmonary disease serum surfactant protein d is steroid sensitive and associated with exacerbations of copd serum surfactant protein d during acute exacerbations of chronic obstructive pulmonary disease comprehensive characterisation of pulmonary and serum surfactant protein d in copd serum surfactant protein d: biomarker of chronic obstructive pulmonary disease value of serum and induced sputum surfactant protein-d in chronic obstructive pulmonary disease surfactant protein d, soluble intercellular adhesion molecule- and highsensitivity c-reactive protein as biomarkers of chronic obstructive pulmonary disease the role of matrix metalloproteinase- in cigarette smoke-induced emphysema surfactant protein d in serum from patients with allergic bronchopulmonary aspergillosis serum-surfactant sp-d correlates inversely to lung function in cystic fibrosis circulating surfactant protein-d and the risk of cardiovascular morbidity and mortality the utility of lung epithelium specific biomarkers in cardiac surgery: a comparison of biomarker profiles in on-and off-pump coronary bypass surgery can serum surfactant protein d or cc-chemokine ligand predict outcome of interstitial lung disease in patients with early systemic sclerosis? serum levels of surfactant proteins a and d are useful biomarkers for interstitial lung disease in patients with progressive systemic sclerosis clinical significance of surfactant protein d as a serum marker for evaluating pulmonary fibrosis in patients with systemic sclerosis comparative study of serum surfactant protein-d and kl- concentrations in patients with systemic sclerosis as markers for monitoring the activity of pulmonary fibrosis surfactant protein d and kl- as serum biomarkers of interstitial lung disease in patients with scleroderma clinical significance of serum pulmonary surfactant proteins a and d for the early detection of radiation pneumonitis diagnostic significance of surfactant proteins a and d in sera from patients with radiation pneumonitis study of clara cell , kl- , and surfactant protein-d in serum as disease markers in pulmonary sarcoidosis serum surfactant protein d is elevated in allergic patients involvement of eicosanoids and surfactant protein d in extrinsic allergic alveolitis functional and biological characteristics of asthma in cleaning workers circulating surfactant protein d is decreased in early rheumatoid arthritis: a -year prospective study circulating surfactant protein -d is low and correlates negatively with systemic inflammation in early, untreated rheumatoid arthritis elevated plasma surfactant protein d (sp-d) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific igg antibody in sars patients analysis of plasma surfactant protein d levels in lung transplant recipients clinical significance of the serum surfactant protein d and kl- levels in patients with measles complicated by interstitial pneumonia surfactant protein d (sp-d) serum levels in patients with community-acquired pneumonia small star, filled serum sp-d levels as a biomarker of lung injury in respiratory syncytial virus bronchiolitis serum kl- and surfactant protein d in children with pandemic h n influenza infection serum kl- and surfactant proteins a and d in pediatric interstitial lung disease polymorphisms in the human surfactant protein-d (sftpd) gene: strong evidence that serum levels of surfactant protein-d (sp-d) are genetically influenced surfactant protein d, a marker of lung innate immunity, is positively associated with insulin sensitivity circulating levels of clara cell protein but not surfactant protein d identify and quantify lung damage in patients with multiple injuries blood biomarkers and measures of pulmonary function-a study from the swedish twin registry surfactant protein d of the innate immune defence is inversely associated with human obesity and sp-d deficiency infers increased body weight in mice long-term stability and circadian variation in circulating levels of surfactant protein d a common polymorphism in the sftpd gene influences assembly, function, and concentration of surfactant protein d plasma surfactant protein d levels and the relation to body mass index in a chinese population plasma c d levels of young farmers correlate with respirable dust exposure levels during normal work in swine confinement buildings respiratory effects associated with wood fuel use: a cross-sectional biomarker study among adolescents roles of serum clara cell protein and surfactant protein-d in the early diagnosis and progression of silicosis serum surfactant protein-a, but not surfactant protein-d or kl- , can predict preclinical lung damage induced by smoking biomarkers of early respiratory effects in smoking adolescents circulating markers of interstitial lung disease and subsequent risk of lung cancer inhaled lps challenges in smokers: a study of pulmonary and systemic effects lung epithelium injury biomarkers in workers exposed to sulphur dioxide in a nonferrous smelter the effects of gh and hormone replacement therapy on serum concentrations of mannan-binding lectin, surfactant protein d and vitamin d binding protein in turner syndrome plasma surfactant d in patients following acute paraquat intoxication respiratory function and changes in lung epithelium biomarkers after a short-training intervention in chlorinated vs. ozone indoor pools a panel of lung injury biomarkers enhances the definition of primary graft dysfunction (pgd) after lung transplantation the sitting position during neurosurgical procedures does not influence serum biomarkers of pulmonary parenchymal injury a sandwich enzyme immunoassay for pulmonary surfactant protein d and measurement of its blood levels in drowning victims clinical significance of serum surfactant protein d (sp-d) in patients with polymyositis/ dermatomyositis: correlation with interstitial lung disease serum surfactant protein d is correlated to development of dementia and augmented mortality circulating surfactant protein d is decreased in systemic lupus erythematosus comparison of biomarkers of subclinical lung injury in obstructive sleep apnea lung epithelium-specific proteins: characteristics and potential applications as markers the association between bmi and plasma cytokine levels in patients with acute lung injury characterization and prevention of the adsorption of surfactant protein d to polypropylene collectin surfactant protein d binds antibodies and interlinks innate and adaptive immune systems increased prevalence of low oligomeric state surfactant protein d with restricted lectin activity in bronchoalveolar lavage fluid from preterm infants the authors would like to thank tarek abdalla for his assistance with sample collection and processing. conceived and designed the experiments: peb. performed the experiments: peb. analyzed the data: peb. contributed reagents/materials/ analysis tools: peb ag. wrote the paper: peb ag. key: cord- -k wfyj b authors: paweska, janusz t.; moolla, naazneen; storm, nadia; msimang, veerle; conteh, ousman; weyer, jacqueline; van vuren, petrus jansen title: evaluation of diagnostic performance of three indirect enzyme-linked immunosorbent assays for the detection of igg antibodies to ebola virus in human sera date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: k wfyj b filovirus serological diagnosis and epidemiological investigations are hampered due to the unavailability of validated immunoassays. diagnostic performance of three indirect enzyme-linked immunosorbent assays (i-elisa) was evaluated for the detection of igg antibody to ebola virus (ebov) in human sera. one i-elisa was based on a whole ebov antigen (wag) and two utilized recombinant nucleocapsid (np) and glycoproteins (gp), respectively. validation data sets derived from individual sera collected in south africa (sa), representing an ebov non-endemic country, and from sera collected during an ebola disease (ebod) outbreak in sierra leone (sl), were categorized according to the compounded results of the three i-elisas and real time reverse-transcription polymerase chain reaction (rt-pcr). at the cut-off values selected at % accuracy level by the two-graph receiver operating characteristic analysis, specificity in the sa ebov negative serum panel (n = ) ranged from . % (gp elisa) to . % (wag elisa). diagnostic specificity in the sl ebov negative panel (n = ) was % by the three elisas. the diagnostic sensitivity in rt-pcr confirmed ebod patients was dependent on the time when the serum was collected after onset of disease. it significantly increased weeks post-onset, reaching % sensitivity by wag and np and . % by gp i-elisa. high-mortality and occurrence of ebola disease (ebod) outbreaks almost each year in the last three decades [ , ] are of great public health concern. the unprecedented and first epidemics of ebod in west africa from to [ ] [ ] [ ] [ ] and the large-scale re-emergence of ebod in the democratic republic of the congo in and [ , ] exemplify the devastating health, humanitarian, and socio-economic impacts and challenges in containing ebod outbreaks in resource-poor and politically conflicted settings [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the increasing incidence, severity and size of ebod outbreaks highlight the importance of developing and standardizing diagnostic tools for ebod rapid diagnosis and post-epidemic surveillance. the most devastating ebod outbreak to date, the west african outbreak, prompted determining seroprevalence rates, infection risk population studies, and assessing occurrence of asymptomatic infections [ ] . the purpose of this study was to evaluate and compare the diagnostic performance of ebov igg-indirect elisas based on antigens produced by classical virological and recombinant protein expression methods using human serum panels from ebov non-infected and ebov infected humans. a total of individual banked sera collected in south africa (sa) and sierra leone (sl), were used. sa sera (n = ) were originally submitted for various routine diagnostic testing to the centre for emerging zoonotic and parasitic diseases of the national institute for communicable diseases (nicd), johannesburg. these sera represented specimens collected from individuals in ebov non-endemic country and are regarded as igg ebov negative reference serum panel. ethics clearance for using sa human banked sera in the development and validation of diagnostic assays was obtained from human ethics committee, university of the witwatersrand, johannesburg, sa, clearance certification no. m , february . sl blood specimens were originally submitted for ebov rt-pcr testing to the sa modular high-biosafety field ebola diagnostic laboratory (fedl) established in august near freetown, in international response to the rapidly increasing number of ebod cases in sl [ ] . selected aliquots of processed sera were shipped on dry ice from fedl to the nicd's biosafety level (bsl- ) in total, sera obtained from sl patients suspected of having ebod between august and march [ ] were used. of those sera were from ebov rt-pcr confirmed cases for whom date of disease onset was recorded on ebod case submission form. ribonucleic acid from blood was extracted using the qiaamp viral rna kit (qiagen, hilden, germany) according to the manufacturer's instructions. rt-pcr was performed using the qiagen one-step rt-pcr kit (qiagen, hilden, germany) as per the manufacturer's instructions using previously described primers and probes targeting the ebov l gene [ ] . specimens with ct values below were considered positive for ebov rna [ ] and thus confirming ebov infectious status of a patient. serum specimens from the sl ebov rt-pcr positive patients were regarded as sl reference positive serum panel. the remaining specimens were from ebov rt-pcr negative individuals whose sera tested negative for anti-ebov igg by all igg elisas evaluated in this study using cut-offs derived from sa igg ebov negative reference serum panel. this serum panel was regarded as sl reference negative serum panel. the source of positive control serum (c++) was the imported ebod case from gabon to sa [ , ] . the case was a gabonese physician who had been brought from libreville, gabon on october and admitted to a private hospital in johannesburg where a nurse died after being infected through exposure to his blood. negative control serum (reference no. cezpd svpl / ) was obtained from south african blood service. it tested negative for anti-ebov igg and igm antibodies by in-house elisa using procedures published by ksiazek et al. [ ] . internal quality control (iqc) data were generated as described previously [ ] . upper and lower control iqc limits together with coefficients of variations ≤ % for replicates of positive internal control serum and test sera were applied as an assay acceptance criteria. diagnostic performance of three indirect elisas (i-elisa) for the detection of anti-ebov igg antibody in human sera was evaluated. these elisas were based on antigens prepared from infected cell lysate, or on recombinant antigens produced in a bacterial or mammalian expression system. the ebov whole antigen (wag) was produced as previously described with some modifications [ ] . vero c cells (atcc, manassas, va, usa) were infected with the spu / isolate of ebov ( th passage in vero cells) isolated from the serum of a nurse who contracted a fatal infection from a gabonese physician admitted to a private hospital in south africa in [ ] . after incubation at • c when cytopathic effect was observed, cells and supernatant were collected and snap freeze-thawed at − • c and • c. lysed cells and supernatant were separated by centrifugation ( , × g, • c, min). the collected supernatant was gamma irradiated with , gy to inactivate the virus. saturated ammonium sulphate solution ( %) (sigma aldrich, merck, kenilworth, nj, usa) was slowly added with constant stirring to the irradiated supernatant to a final concentration of %, and the mixture was incubated at • c overnight. the antigen-containing pellet precipitate was collected by centrifugation at , × g for min at • c and resuspended in one tenth of the original supernatant volume of phospate buffered saline (pbs) ph . . the antigen was further dialysed against pbs to remove the ammonium suphate ( - buffer changes). the wag preparation was aliquoted and stored at − • c until use. uninfected vero c cells were prepared in the same way and used as control antigen. the codon optimized nucleotide sequence for ebov nucleocapsid (np) (genbank accession number af . ) was synthesized with a c-terminal glycine linker to a × histidine tag (genscript, piscataway, nj, usa) and subcloned into the ncoi and xhoi restriction sites of the pet- b expression vector (novagen, merck, usa). the sequence verified plasmid (pet- b zebov np) was used to transform competent bl star (de ) e. coli. a starter culture was grown overnight at • c, then diluted fold and allowed to reach exponential-phase growth (od of between . - . ) before protein expression was induced using mm iptg (sigma aldrich, merck, usa) for h at • c with vigorous shaking. cells were harvested by centrifugation, resuspended in sodium phosphate buffer ( mm nah hpo , mm nacl, ph . ) and lysed using a combination of bugbuster and lysonase (novagen, merck, usa) treatment, freeze-thaw cycles and sonication. the recombinant np protein-containing soluble phase was collected by high speed centrifugation ( , × g, min, • c) and loaded onto profinity imac (biorad, hercules, ca, usa) cobalt-charged resin. the protein was left to bind overnight with gentle shaking at • c, using a ratio of ml resin to ml soluble protein fraction. contaminating proteins were removed by washing the resin twice with packed-resin volumes of sodium phosphate buffer containing . mm imidazole (ph . ), using gentle centrifugation ( × g, min, • c). the recombinant np was then eluted overnight with gentle shaking at • c in packed-resin volumes of sodium phosphate buffer containing mm imidazole (ph . ). eluted protein was dialysed into . m carbonate/bicarbonate buffer, ph . (sigma aldrich, merck, usa). to remove any residual contaminating proteins, the purified protein was passed through a sec size exclusion column (biorad, hercules, ca, usa) and the fractions containing the np were collected. collected fractions were pooled and concentrated using amicon ultra filters (millipore, merck, usa) with a kd cut-off. the purified np was quantified using the bradford concentration assay kit (pierce, thermofisher scientific, waltham, ma, usa) and aliquots were stored at − • c for later use. for the control antigen, the same process was followed using an expression vector (pet- b) without an insert. recombinant ebov glycoprotein (gp) antigen expressed in human embryonic kidney t cells was obtained from integrated biotherapeutics (rockville, md, usa). optimal immunoreagents concentrations/working dilutions for i-elisa were determined using standard checkerboard titration procedures [ ] . microtiter -wells plates (maxisorb immunoplates, nunc, roskilde, denmark) for wag, np, and gp i-elisas were respectively coated with wag and corresponding control antigen, np and corresponding control antigen or with gp. for wag and np i-elisas virus and control antigens were added to rows of the top half of the plate (rows a-d: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the bottom half of the plate (rows e-h: - ), respectively. for gp i-elisa all rows (rows a-h: - ) were coated with the commercial gp for which control antigen was not available. the np and corresponding control antigen were diluted : (stock concentration . mg/ml) in carbonate-bicarbonate buffer, ph . . all the other antigens were diluted in phosphate pbs without mg and ca, ph . ; wag and uninfected vero c cell culture antigen was diluted : and the gp was diluted : (stock concentration . mg/ml). each -well plate had four replicates of positive serum control (c++), two replicates of negative serum control (c−) and two replicates of conjugate control (cc). for wag and np i-elisa c++ was added to wells a - and b - coated with virus antigen and to corresponding wells e - and f - coated with a control antigen. accordingly, c− was added to c - and g - , and cc (diluent buffer) was added to d - and h - . the remaining wells (a-d - and e-h - ) were used for testing test sera in duplicate with test serum no. added to wells a and b and corresponding e and f ; adding of the remaining test sera followed the same layout principle. for gp i-elisa, not having control antigen, internal controls were placed as follows: c++ in wells a - and b - ; c− in wells c - ; cc in wells d - . the remaining wells (a-d - and e-h - ) were used for testing test sera in duplicate with test serum no. added to wells a and b and accordingly test serum no. added to wells g and h . all reagents were added to the immunoplates at a volume of µl/well unless otherwise stated. passive adsorption onto elisa plates was performed at • c overnight and all subsequent incubations (except for substrate addition) were performed at • c in a humidified chamber for h. following coating, plates were washed times with µl of pbs containing . % tween ; the same washing procedure followed each subsequent stage of i-elisas. plates were blocked with µl % non-fat milk powder in pbs. after incubation, plates were washed, and control and test sera diluted : in % non-fat milk powder in pbs (diluent buffer) were added. each test serum, negative control serum and conjugate control was tested in duplicate, and positive control was tested in quadruplicate. following incubation with the sera, the plates were washed and goat anti-human igg hrpo conjugate (invitrogen, thermofisher scientific, usa), diluted : , in diluent buffer, was added to the wells. after incubation, plates were washed and , '-azinodiethylbenzothiazoline sulfonic acid (abts) peroxidase (seracare lifesciences, milford, ma, usa) substrate added to wells. plates were incubated in the dark at room temperature (± • c) for min. reactions were stopped by the addition of % sodium dodecyl sulphate (sds) and the optical densities (od) readings were measured at nm. od readings were converted into a percentage of the positive internal control serum (pp) using the equation as previously described [ ] . briefly, a specific activity of each serum (net od) was calculated by subtracting the non-specific background od in the wells with control antigen from the od in wells with virus antigen (for np and wag i-elisa only). the mean net od readings were converted to pp using the formula: pp = (mean net of test serum/mean net od of positive control) × [ ] . cut-off values were determined as mean + standard deviations (sd) of pp values recorded in sa and sl igg ebov negative serum panels and also selected at % accuracy level by the misclassification cost term (mct) option of the two-graph receiver operating characteristics (tg-roc) analysis available from microsoft excel (redmond, wa, usa) [ , ] using sl reference igg ebov negative and positive serum panels. coefficient of variation (cv) values were calculated to measure relative variability using the formula cv = (sd of replicates/mean of replicates) × . optimisation of cut-off values by mct tg-roc was based on the following equation: to compare the diagnostic performance of the assays and agreements between results of matched test samples, data were analyzed in stata using mcnemar's test and cohen's kappa statistic (κ) [ ] . to evaluate the effect of serum inactivation on the levels of the detectable anti-ebov igg by wag, np and gp i-elisas, laboratory protocols previously shown to completely inactivate ebov virus in human serum were used [ , ] . briefly, the c++ was first diluted : in pbs containing either . % triton x- or . % tween- (sigma-aldrich, taufkichen, germany) and heated at • c for min. then, two-fold log dilutions (from : to : , ; log −log . ) of untreated and treated serum was tested by each of the i-elisas. each dilution was tested in duplicate on separate runs. a serum titer was considered the highest sample dilution at which its pp value was ≥ the i-elisa cut-off for at least % of six replicates. both within and between the runs, the c++ od readings for wag, np and gp i-elisas were within the iqc lower (lcl) and upper (ucl) control limits. within the runs, the average cv ranged between . ± . (np i-elisa) and . ± . (gp i-elisa). between the runs, the average cv ranged between . ± . (np i-elisa) and . ± . (gp i-elisa) ( table ) . both within and between the runs, the c− and cc od readings were within the iqc lcl and ucl. for wag, np, and gp i-elisa, the iqc c− od control limits ranged from − . to . , − . to . , . to . , and the iqc cc− od control limits ranged from − . to . , − . to . , and . to . , respectively. the distribution of i-elisa pp values and determination of cut-offs as mean plus three standard deviations of pp values recorded by each test in sa and sl ebov igg negative serum panels are shown in figure . selection and optimization of cut-offs by the mct tg-roc in sl serum panels is shown in figure . mct tr-roc was based on the non-parametric program option due to departure from a normal distribution of data sets analyzed. pp threshold values for each of the assays were similar irrespective of serum panels analyzed and the methods used for the determination of cut-offs: . , . ( figure a ) and . (figure a) for wag i-elisa; . , . ( figure b ) and . ( figure b ) for np i-elisa; . , . ( figure c ) and . ( figure c ) for gp i-elisa, respectively. the higher cut-off values for gp i-elisa were likely due to higher elisa noise resulting from not including a control antigen in this assay. irrespective of data set analyzed and cut-offs used, all the three assays evaluated in this study had high estimates of d-sp, ranging from . % to . % in sa, and from . % to % in sl negative serum panels. however, d-sp for gp i-elisa was generally lower in the sa negative serum panel ( table ). mct tr-roc was based on the non-parametric program option due to departure from a normal distribution of data sets analyzed. pp threshold values for each of the assays were similar irrespective of serum panels analyzed and the methods used for the determination of cut-offs: . , . ( figure a figure c ) and . ( figure c ) for gp i-elisa, respectively. the higher cut-off values for gp i-elisa were likely due to higher elisa noise resulting from not including a control antigen in this assay. irrespective of data set analyzed and cut-offs used, all the three assays evaluated in this study had high estimates of d-sp, ranging from . % to . % in sa, and from . % to % in sl negative serum panels. however, d-sp for gp i-elisa was generally lower in the sa negative serum panel ( table ) . individual results yielded by wag, np and gp i-elisas in sera from rt-pcr confirmed ebod cases at different times post disease onset and using different cut-off values are given in table . irrespective of the cut-offs used, the results were similar for the same assay, but the np i-elisa was more sensitive in detecting igg antibody during the first two weeks post disease onset compared to wag and gp i-elisas, the latter being the least sensitive. for example, when using the tg-roc derived threshold, of ebod patients bled during the two weeks post onset, ( . %), ( %), and ( . %) were positive by wag, np, and gp i-elisa, respectively. mc nemar test indicate a disagreement of diagnostic capacity (combined dse and dsp) between np and wag (p = . ) or gp i-elisa (p = . ) respectively using all sierra leone data (n = ). agreement was found between wag and gp i-elisa (p = . ; . % κ = . ± . ). most of the discrepant (non-matching) results were recorded during the first two weeks post disease onset. after two weeks post onset, detection of igg antibody and agreement between assays significantly improved. of sera tested - days post onset, all tested positive by wag and np i-eliss irrespective of the cut-off used, and depending on the cut-off, or were positive by gp i-elisa (table ) . mc nemar test indicate agreement between np, wag and gp i-elisas ranging from . % (κ = . ± . ) to % using data from non-ebod and diseased patients bled on day or later post-onset (n = ), except for gp and wag or np i-elisas with the cut-offs of respectively . , . , . (p < . ). estimates of d-se in sera collected at different times post disease onset are given in table . mean levels of igg responses measured by wag, np, and gp i-elisas in ebod rt-pcr confirmed cases bled at different times after disease onset are shown in figure . on average, the first seroconversions were detectable by np on days - , then on days - by np and wag, and from day post onset by all i-elisas. the mean levels of igg measured by all the i-elisas were not significantly different. estimates of d-se in sera collected at different times post disease onset are given in table . between - days post onset, the d-se ranged from . % (gp i-elisa) to . (np i-elisa). between - days post onset, the d-se was % for both wag and np i-elisa irrespective of the cut-off used, and ranged from . % to . % for the gp i-elisa. table . diagnostic sensitivity of a whole antigen (wag), nucleocapsid (np), and glycoprotein (gp) i-elisas for the detection of anti-igg ebov antibody in humans. cut-off pp wag i-elisa . / . ( . - . ) / ( . - ) wag i-elisa . / . ( . - . ) / ( . - ) wag i-elisa . mean levels of igg responses measured by wag, np, and gp i-elisas in ebod rt-pcr confirmed cases bled at different times after disease onset are shown in figure . on average, the first seroconversions were detectable by np on days - , then on days - by np and wag, and from day post onset by all i-elisas. the mean levels of igg measured by all the i-elisas were not significantly different. the different inactivation protocols used did not have an adverse effect on the kinetics and the detectable levels of the anti-ebov igg in c++ by either i-elisa evaluated in this study. the titers ( table ) as well as the kinetics of dose-response curves ( figure ) were similar before and after inactivation in each assay. the diagnostic decision limit or cut-off represents a serological assay test value used to dichotomize negative and positive results, and by inference, to define the infection status of an individual against a specific pathogen of disease. the relevance of data used for the determination of cut-off consequently impacts on estimates of d-se and d-sp and other measures of test performance [ ] . important consideration in determining a serological assay cut-off is to select sera from unequivocally infected individuals and sera from individuals who have never been infected with the agent in question. also, in order to account for the distribution of covariate factors (genetic, nutritional, geographical, and stage of infection) that may influence the estimates of d-se and d-sp, the target population should preferably be sampled using simple random, systematic or stratified sampling methods [ ] . these ideal conditions could not be applied during this study. an assay validation data should preferably be derived not only from testing samples from reference individuals of known history and infection status but also from the country or region in which the test is to be used. traditionally, gold standards for selection of truly infected and uninfected subjects include isolation of the agent or pathognomonic histopathological criteria. because a true gold standard is difficult to accomplish, relative standards of comparison are often necessary, and include results from other serological assays [ ] . in order to ensure ebov true infection status of individuals whose sera were used in our study, rt-pcr negative sera that tested negative for anti-ebov igg by all igg elisas using cut-offs derived from sa igg ebov negative reference serum panel were regarded as sl reference negative serum panel. serum specimens from the sl ebov rt-pcr positive patients with known dates of disease onset were regarded as sl reference positive serum panel. various statistical analyses used in our study for the selection of the cut-off values provided similar results. a cut-off value determined as two or three sd above the mean in uninfected individuals is frequently used for the interpretation of serodiagnostic tests. however, this assumes a normal distribution of the test values in population targeted by an assay, and provides only an estimate of d-sp [ ] . deviations from normality are often recorded in serological data and should be addressed in the selection of threshold values [ ] . therefore, we also used the tg-roc analysis for the selection and optimization of cut-off values to account for parametric versus nonparametric distribution of test values. all i-elisas evaluated in our study had high estimates of d-sp, but the d-se was dependent on the time when the serum was taken post disease onset. high detection rate the diagnostic decision limit or cut-off represents a serological assay test value used to dichotomize negative and positive results, and by inference, to define the infection status of an individual against a specific pathogen of disease. the relevance of data used for the determination of cut-off consequently impacts on estimates of d-se and d-sp and other measures of test performance [ ] . important consideration in determining a serological assay cut-off is to select sera from unequivocally infected individuals and sera from individuals who have never been infected with the agent in question. also, in order to account for the distribution of covariate factors (genetic, nutritional, geographical, and stage of infection) that may influence the estimates of d-se and d-sp, the target population should preferably be sampled using simple random, systematic or stratified sampling methods [ ] . these ideal conditions could not be applied during this study. an assay validation data should preferably be derived not only from testing samples from reference individuals of known history and infection status but also from the country or region in which the test is to be used. traditionally, gold standards for selection of truly infected and uninfected subjects include isolation of the agent or pathognomonic histopathological criteria. because a true gold standard is difficult to accomplish, relative standards of comparison are often necessary, and include results from other serological assays [ ] . in order to ensure ebov true infection status of individuals whose sera were used in our study, rt-pcr negative sera that tested negative for anti-ebov igg by all igg elisas using cut-offs derived from sa igg ebov negative reference serum panel were regarded as sl reference negative serum panel. serum specimens from the sl ebov rt-pcr positive patients with known dates of disease onset were regarded as sl reference positive serum panel. various statistical analyses used in our study for the selection of the cut-off values provided similar results. a cut-off value determined as two or three sd above the mean in uninfected individuals is frequently used for the interpretation of serodiagnostic tests. however, this assumes a normal distribution of the test values in population targeted by an assay, and provides only an estimate of d-sp [ ] . deviations from normality are often recorded in serological data and should be addressed in the selection of threshold values [ ] . therefore, we also used the tg-roc analysis for the selection and optimization of cut-off values to account for parametric versus nonparametric distribution of test values. all i-elisas evaluated in our study had high estimates of d-sp, but the d-se was dependent on the time when the serum was taken post disease onset. high detection rate of anti-ebov igg in rt-pcr ebov confirmed cases was only recorded after two weeks post disease onset. results of previous study in a small number of the ebod patients in kikwit, democratic republic of congo, suggested that many patients do not have antibody early in the course of their illness, and that many may die without developing detectable antibodies to ebov. therefore, measurement of igg antibody is of rather limited use in the diagnosis of acute ebod cases [ ] . i-elisa, represents one of the simplest elisa formats, but can be difficult to validate because of signal amplification of both specific and non-specific components [ ] . for these reasons, to determine the specific binding of antibody, sera should be tested with both a specific viral antigen and its corresponding control or comparison antigen to account for possible non-specific background activity. the gp i-elisa evaluated in our study was based on a commercially available recombinant gp for which a negative control could not be obtained. compared to wag and np i-elisas, which included control antigens, the higher gp i-elisa test values in ebov negative serum panels and consequently the higher pp cut-off values derived for this assay are likely due to not having a control antigen. due to inherent differences amongst assay systems, binding-antibody levels should be expressed in relative rather than absolute terms. one of the advantages of using pp values as a measure of antibody activity in the i-elisa is that this method of od readings conversion does not assume a uniform background activity, and therefore it is also more suitable for inter-laboratory standardization [ ] . the first elisas for the detection of antibodies to ebov were based on whole antigen prepared from infected cell lysate [ ] , and variations of this assay are still widely used in diagnostic and research laboratories [ ] . while whole filovirus antigens can be relatively easily produced in large volumes, their preparation pose health risks, restricting their production to biosafety level (bsl- ) facilities. these facilities are not only very expensive to construct and operate, but also are not easily available or accessible for countries where fatal filoviruses are endemic. in addition, the binding of antibodies to cellular contaminants present in ebov-infected cell lysates may lead to cross-reactivity, resulting in reduced specificity [ ] . high quality filovirus recombinant protein antigens can be safely prepared without the need for high bsl- biocontainment facilities and outside ebov endemic areas. their use in elisa has potential to reduce the risk of false positive results and allows for better standardization [ , ] . testing clinical specimens potentially containing a bsl- viral agent presents a serious biohazard. while a number of inactivation methods were shown to completely inactivate ebov [ , ] , they also markedly alter the protein components in human blood, e.g., enzymes and coagulation factors [ ] . results of viral inactivation protocols evaluated in our study indicate that they do not alter detectable levels of anti ebov-igg, thus together with recombinant antigen based elisas, provide a safe and reliable testing platform. it has previously been reported that the use of single filoviral proteins as antigens is disadvantageous for filovirus serology [ , , ] . sera from patients infected with ebov contain antibodies to several viral proteins and therefore might display reduced activity or later seroconversion in elisas based on a single recombinant antigen [ ] . results of our study indicate that anti-ebov np igg is detectable earlier then anti-ebov gp igg antibody during the first week post disease onset. this is likely due to higher abundance of this highly immunogenic protein in infected cells [ ] . long-lasting persistence of igg antibody in humans after infection with ebov [ , , ] , renders igg detection elisa a suitable tool for epidemiological investigations. evaluation of the efficacy of filovirus vaccines and therapeutics require monitoring of immune responses that correlate with protection and survival using reliable and reproducible serological methods. anti-ebov gp igg elisa was recently shown to reproducibly quantify levels of anti-ebov igg antibodies in sera from ebod survivors and immunized individuals [ ] , thus offering an important laboratory tool for assessing immunogenicity of candidate ebov vaccines. the np igg i-elisa evaluated in this study has a potential to be used for testing immunity and evaluating protection against natural ebov exposure in individuals immunized with ebov gp-based vaccines or receiving anti-ebov gp passive immunization. as highly accurate, robust and safe tests, the elisas based on recombinant ebov antigens have a potential to replace traditional serological diagnostic methods which pose health risks thus necessitating their use only in high biocontainment facilities. practically simple viral inactivation protocols evaluated in our study do not alter measurable levels of anti ebov-igg, thus together with recombinant antigen based elisas provide a safe testing application. long-lasting igg antibody makes their detection useful for epidemiological investigations. new filovirus disease classification and nomenclature ebola virus outbreaks in africa: past and present emergence of zaire ebola virus disease in guinea the international ebola emergency who ebola response team. ebola virus disease in west africa-the first months of the epidemic and forward projections world health organization-ebola virus disease-democratic republic of the congo drc ebola outbreak crisis update unnecessary ebola-related deaths: building citizen trust in health systems the economic impact of the ebola epidemic: short-and medium-term estimates for west africa health-care worker mortality and the legacy of the ebola epidemic a retrospective and prospective analysis of the west african ebola virus disease epidemic: robust national health systems at the foundation and an empowered who at the apex the link between the west african ebola outbreak and health systems in guinea, liberia and sierra leone: a systematic review institutional trust and misinformation in the response to the - ebola outbreak in north kivu, dr congo; a population-based survey comparative evaluation of the diagnostic performance of the prototype cepheid genexpert ebola assay establishing ebola virus disease (evd) diagnostics using genexpert technology at a mobile laboratory in liberia: impact on outbreak response, case management and laboratory systems strengthening lateral flow immunoassays for ebola virus disease detection in liberia comparative performance of four rapid ebola antigen-detection lateral flow immunoassays during the - ebola epidemic in west africa serologic cross-reactivity of human igm and igg antibodies to five species of ebola virus serological investigation of laboratory-confirmed and suspected ebola virus disease patients during the late phase of the ebola outbreak in sierra leone production of antigens for elisa analysis of linear b-cell epitopes of the nucleoprotein of ebola virus that distinguish ebola virus subtypes sequence analysis of the ebola virus genome: organization, genetic elements, and comparison with the genome of marburg virus enzyme-linked immunosorbent assays for detection of antibodies to ebola and marburg viruses using recombinant nucleoproteins human survivors of disease outbreaks caused by ebola or marburg virus exhibit cross-reactive and long-lived antibody responses enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies protective efficacy of neutralizing antibodies against ebola virus infection recombinant ebola virus nucleoprotein and glycoprotein (gabon strain) provide new tools for the detection of human infections serological reactivity of baculovirus-expressed ebola virus vp and nucleoproteins nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect elisa) for detecting antibodies specific to ebola virus and marburg virus quantitative serology assays for determination of antibody responses to ebola virus glycoprotein and matrix protein in nonhuman primates and humans recombinant protein expression in escherichia coli: advances and challenges engineering n-glycosylation pathways in the baculovirus-insect cell system asymptomatic infection and unrecognised ebola virus disease in ebola-affected households in sierra leone: a cross-sectional study using a new non-invasive assay for antibodies to ebola virus standardization of the filovirus plaque assay for use in preclinical studies south african ebola diagnostic response in sierra leone: a modular high biosafety field laboratory diagnostic reverse-transcription polymerase chain reaction kit for filoviruses based on the strain collections of all european biosafety level laboratories world health organization. ebola hemorrhagic fever-south africa unexpected ebola virus in a tertiary setting: clinical and epidemiological aspects elisa for the detection of antibodies to ebola viruses validation of igg-sandwich and igm-capture elisa for the detection of antibody to rift valley fever virus in humans elisa theory and practice two-graph receiver operating characteristics (tg-roc)-a microsoft-excel template for the selection of cut-off values in diagnostic tests two-graph receiver operating characteristics (tg-roc)-a microsoft-excel template for the selection of cut-off values that minimize overall misclassification costs application of diagnostic tests in veterinary epidemiological studies the measurement of observer agreement for categorical data physicochemical inactivation of lassa, ebola and marburg viruses and effect on clinical laboratory analyses effective chemical inactivation of ebola virus validation of serological assays for diagnosis infectious diseases principles of validation of diagnostic assays for infectious diseases determination of the optimum cut-off value of a diagnostic test clinical virology of ebola hemorrhagic fever (ehf): virus, virus antigen, and igg and igm antibody findings among ehf patients in kiwit, democratic republic of the congo standarization and validation of enzyme-linked immunosorbent assay techniques for the detection of antibody in infectious disease diagnosis development of an antibody capture elisa using inactivated ebola zaire makona virus profiling the native specific human humoral immune response to sudan ebolavirus strain gulu by chemiluminescence enzyme-linked immunosorbent assay antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate transmissable gastroenteritis virus from porcine respiratory coronavirus antibodies clinical, virologic and immunologic follow-up of convalescent ebola hemorrhagic fever patientsand their household contacts immunoglobulin g in ebola outbreak survivors development, qualification, and validation of the filovirus animal nonclinical group anti-ebola virus glycoprotein immunoglobulin g enzyme-linked immunosorbent assay for human serum samples this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -heqsfzkm authors: blanco vázquez, cristina; alonso-hearn, marta; juste, ramón a.; canive, maría; iglesias, tania; iglesias, natalia; amado, javier; vicente, fernando; balseiro, ana; casais, rosa title: detection of latent forms of mycobacterium avium subsp. paratuberculosis infection using host biomarker-based elisas greatly improves paratuberculosis diagnostic sensitivity date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: heqsfzkm bovine paratuberculosis (ptb) is a chronic granulomatous enteritis, caused by mycobacterium avium subsp. paratuberculosis (map), responsible for important economic losses in the dairy industry. current diagnostic methods have low sensitivities for detection of latent forms of map infection, defined by focal granulomatous lesions and scarce humoral response or map presence. in contrast, patent infections correspond to multifocal and diffuse types of enteritis where there is increased antibody production, and substantial mycobacterial load. our previous rna-seq analysis allowed the selection of five candidate biomarkers overexpressed in peripheral blood of map infected holstein cows with focal (abca and mmp ) and diffuse (fam a, sparc and des) lesions vs. control animals with no detectable ptb-associated lesions in intestine and regional lymph nodes. the aim of the current study was to assess the ptb diagnostic potential of commercial elisas designed for the specific detection of these biomarkers. the ability of these elisas to identify animals with latent and/or patent forms of map infection was investigated using serum from naturally infected cattle (n = ) and non-infected control animals (n = ). roc analysis revealed that the abca -based elisa showed the highest diagnostic accuracy for the detection of infected animals with focal lesions (auc . , sensitivity . % and specificity . %) and with any type of histological lesion (auc . , sensitivity . % and specificity . %) improving on the diagnostic performance of the popular idexx elisa and other conventional diagnostic methods. sparc and mmp showed the highest diagnostic accuracy for the detection of animals with multifocal (auc . ) and diffuse lesions (auc . ), respectively. in conclusion, our results suggest that quantification of abca , sparc and mmp by elisa has the potential for implementation as a diagnostic tool to reliably identify map infection, greatly improving early detection of map latent infections when antibody responses and fecal shedding are undetectable using conventional diagnostic methods. a a a a a bovine paratuberculosis (ptb) is a chronic granulomatous enteritis caused by mycobacterium avium subsp. paratuberculosis (map), that is responsible for important economic losses due to reduced milk production, premature culling, reduced slaughter value and continued spread of infection [ , ] . furthermore, map has a clear zoonotic potential since it has been postulated as a possible trigger factor in several autoimmune diseases in humans such as crohn's disease (cd) [ , ] , type i diabetes (t d) [ ] , multiple sclerosis (ms) [ , ] or rheumatoid arthritis (ra) [ , ] . one mechanism that has been suggested to cause the onset and/or exacerbation of autoimmune disease is molecular mimicry, whereby map antigens share sequences or structural similarities with self-antigens [ ] , so the immune response against map antigens could also induced undesirable immune responses against host proteins in genetically predisposed individuals [ ] . two forms of infection, latent or patent, can be distinguished in map infected cattle [ ] . the disease typically progresses from a latent form with low or moderate frequency of microbiological or humoral immunological evidence of infection, characterized by the presence of focal histological lesions in their intestinal tissues to more severe forms of the disease with a high frequency of microbiological or humoral immunological evidence of infection, in which the granulomatous lesions are patent (multifocal and diffuse lesions readily detected upon microscopic examination of the intestine and associated lymph nodes). a latent form would represent a form of silent ptb that causes no direct losses, but that maintains a hidden map reservoir in a herd, while a patent form often corresponds with a visibly clinical disease. transmission of map primarily occurs by the fecal-oral route through the ingestion of map contaminated feces, colostrum, or milk. infection usually occurs within the first months of life of the animal but remains subclinical for an average of - years before becoming clinical in a few cases. spread of ptb is mainly due to its extremely long latent period during which map can be shed intermittently into the environment through feces. thus, early detection and removal of animals in that stage from the herd is critical for ptb control. most ptb control programs are based on testing and culling test-positive cows combined with good management practices [ ] . several diagnostic techniques are used to detect map infected cattle; however, their performance vary widely depending on the stage of map infection [ ] [ ] [ ] . currently available diagnosis methods have low sensitivities and specificities for the detection of latent infection, as the bacteria is excreted in low numbers and animals have low titers of specific antibodies. fecal culture has been considered the gold standard for diagnosis of map but its sensitivity varies from % in cattle with ptb-associated clinical signs to - % in infected cattle with no detectable clinical signs [ ] . pcr offers a rapid method of assessing map status in cultures, feces, tissue and milk [ ] . the sensitivity and specificity of fecal pcr were estimated to be % and . %, respectively [ ] . recently, taniguchi et al. [ ] have established an association between the amount of map dna in feces, determined by real-time quantitative pcr and the histopathological classification of ileocecal valve lesions. elisas, used to detect anti-map antibodies, provide rapid results and offer a cheaper alternative to fecal culture and pcr. however, the appearance of an antibody response detectable by elisa is late as it is associated with disease progression, high shedding, clinical signs and the appearance of severe lesions in gut tissues [ , [ ] [ ] [ ] . thus, the sensitivity of serum specific antibody elisa varies depending on the stage of infection, being - % in cattle with clinical signs, - % in cattle with no clinical signs but shedding map and - % in infected cattle with no clinical signs and no shedding [ ] . in general, elisa specificity varies between and % [ ] and depends on the particular test, exposure to environmental mycobacteria, concurrent infection with mycobacterium bovis, previous intradermal tuberculosis (tb) test and map vaccination. early stage diagnostics primarily target pro-inflammatory cellular immune responses, the interferon-gamma (ifn-γ) release assay (igra) uses a sandwich elisa to detect whether a t-cell mediated immune response has been elicited in response to map by measuring the difference in ifn-γ signals for whole blood samples activated by a map-specific antigenic extract and a non-specific antigen (m. phlei extract) (id vet, grabels, france) [ ] . however, the igra only reflects map exposure, and thus cannot discriminate between individuals with controlled infection from those with sub-clinical disease, indicating that this method might lead to culling healthy individuals. this means that, even though detection of patent forms can be readily done through different methods, there is not an affordable, sensitive and specific method for detection of latent forms. therefore, novel diagnostic tools with high sensitivity and specificity are needed to detect latent map infections. the potential of emerging -omic approaches to complement and enhance the diagnosis of map infection in cattle has been previously reviewed [ ] . host biomarkers, identified using transcriptomics and proteomics, have been proposed as tools to develop novel diagnostic methods for ptb [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the application of those biomarkers for ptb diagnosis has yet to be developed and properly validated for naturally infected cattle in various stages of infection [ ] . park et al. [ ] developed a real-time pcr method based on measuring the gene expression levels of bovine biomarkers (timp , hp, serpine , tfrc, mmp , defb , defb , and s a ) in whole blood of cows that might be useful for the diagnosis of ptb disease, including subclinical stage cases. we previously identified several potential biomarkers for the diagnosis of map-infected animals [ ] . whole rna-sequencing (rna-seq) was used to identify host genes differentially expressed in peripheral blood samples collected from animals with focal, or diffuse lesions in gut tissues vs. control animals without detected lesions nor bacterial load in gut tissues. genes encoding for bovine proteins mmp (matrix metallopeptidase ) and abca (atp binding cassette subfamily a member ) showed significantly higher expression in animals with focal lesions, while genes encoding for fam a (family with sequence similarity member a), sparc (secreted protein acidic and cysteine rich) and desmin (des) were up-regulated in animals showing diffuse lesions when compared with the control animals (table ). in the present study, these five proteins were selected for their validation as biomarkers for the different ptb forms of infection by elisa according to their high expression levels in peripheral blood, their cellular location (extracellular proteins, present in sera) and commercial availability of elisa kits for its specific detection. the aim of this study was to evaluate the diagnostic potential of commercial elisas based on detection of these five bovine biomarkers to detect latent and patent forms of map infection in naturally infected cattle, using reference serum samples from well characterized animals with focal, multifocal and diffuse histological lesions in their intestinal or associated lymphoid tissues [ ]. two groups of animals (n = ) were included in this field study: slaughtered group) ninety-four holstein friesian cows (ranging from . to . years of age) came from farms located in the principality of asturias (northwest of spain). in asturias, . % of the herds and . % of the animals were positive by serum elisa in (unpublished data from the regional government). specifically, fifty-six animals came from a dairy farm with a mean herd size of cows ( - ) and a mean prevalence of ptb of . % in the sampling period, based on serum elisa test (idexx laboratories, hoofddorp, the netherlands). the infection status of these animals was also determined once a year by fecal culture and real time pcr ( - ). another thirty-four animals were randomly selected from cows slaughtered in a local abattoir (coming from different farms) and four more cows were culled from the serida's friesian cow farm ( % mean prevalence of ptb in the period - ). the ptb infection status of these animals at the time of slaughter was determined by histopathology, specific antibody serum elisa test, and bacteriological culture and specific real-time pcr of tissues and feces; and ptb-free group) sixty-one animals (ranging from . to . years of age) came from a farm in asturias with % prevalence of ptb. the ptbfree status of this farm was verified yearly by repeatedly negative idexx serum elisa results and lack of a history of clinical ptb in the period - , as well as by bacteriological culture and specific fecal real-time pcr in . samples of serum and feces included in this field study were collected from all animals while tissue samples were only taken from the slaughtered animals in situ at the local abattoir after evisceration and split for microbiological and histopathological processing. experimental procedures were approved by the serida animal ethics committee and authorised by the regional ministry of agro-livestocks and indigenous resources of the principality of asturias (authorization codes proae / and proae / ). all the procedures were carried out in accordance with directive / /eu of the european parliament (guidelines for the care and use of animals for research purposes). peripheral blood and fecal samples were collected by trained personnel and in accordance with good veterinary practice. tissue sections of distal jejunum, ileocecal valve (icv) and, jejunal and ileal lymph nodes were collected from the slaughtered cows, fixed in % neutral buffered formalin, sliced and embedded in paraffin wax using standard procedures. afterwards, μm sections were assessed by haematoxylin-eosin (he) and ziehl-neelsen (zn) staining for specific acid-fast bacteria (afb) detection. slices were observed using an olympus bh- light microscope (olympus, tokyo, japan) and photographed using an olympus dp- digital camera (olympus, tokyo, japan). the stained sections were examined by light microscopy for afb and evidence of map disease-specific pathological lesions, and were classified in four groups: focal, multifocal, diffuse and with no detectable ptb-associated lesions [ ] . briefly, the focal lesions consist of granulomas, mainly located in the jejunal and ileal lymph nodes, and not affecting the intestinal lamina propia. the multifocal lesions consist of well-demarcated granulomas in the intestinal lymphoid tissue and also in the intestinal lamina propia. the diffuse lesions were characterized by severe and diffuse granulomatous enteritis and lymphadenitis, which markedly altered the normal histological structure. according to the inflammatory cell type present in the infiltrate and the amount of acid-fast bacilli (afb), diffuse lesions were subdivided into diffuse lymphoplasmocytic or paucibacillary, diffuse intermediate and diffuse histiocytic or multibacillary lesions [ ] . the focal, multifocal, diffuse and with no detectable ptb-associated lesions groups correspond to the previously described latent (focal), patent (multifocal and diffuse) and apparently free (no lesions) forms of infection [ ]. blood ( for bacteriological culture, a pool ( gr) of ileocecal lymph nodes, distal jejunal lymph node, icv, and distal jejunum were decontaminated with ml of hexa-decyl pyridinium chloride at a final concentration of . % (sigma, st. louis, mo, usa) and homogenized in a stomacher blender. after min of incubation at room temperature, a ml aliquot of the suspension was transferred to a new tube and left overnight for decontamination and sedimentation. approximately, μl of the suspension was taken from the layer right over the sediment and inoculated into two slants of herrolds egg yolk medium (heym; becton dickinson, sparks, md, usa) and into two slants of lowenstein-jensen medium (lj; difco, detroit, mi, usa), both supplemented with mg/l of mycobactin j (id.vet innovative diagnostics, grabels, france). feces were taken from the rectum of each animal, maintained at ˚c and processed within h after arrival at the laboratory. the fecal samples ( g each) were decontaminated, blended in a stomacher, and cultured in heym and lj, as previously described for tissue culture. isolation of genomic dna from tissues and feces was performed using the magmax total nucleic acid isolation kit according to the manufacturer's instructions ( the concentration of the selected biomarkers in the serum of each animal were measured using commercially available elisas according to the manufacturers´instructions (mybio-source, san diego, ca. usa). quantitative sandwich elisa kits bovine matrix metalloproteinase (detection range . - ng/ml); bovine protein fam a elisa kit (detection range . - pg/ml), bovine sparc elisa kit (detection range . - ng/ml), and competitive bovine atp-binding cassette sub-family a member elisa kit (detection range - pg/ml) and bovine desmin elisa kit (detection range - ng/ml) were used for specific detection of mmp , fam a, abca , sparc and des, respectively. a standard curve was used to determine the concentration of each biomarker in the serum samples (average od of each standard was plotted on the vertical axis against the concentration on the horizontal axis and the best fit drawn to generate a regression curve). standards and samples were tested in duplicate. the mean value of the blank control was subtracted from mean raw od values before result interpretation. the concentration of the biomarkers in each sample was interpolated from the standard curve. for optimization various dilutions of the serum were tested (for instance: undiluted, : , : and : ) and the dilution which showed a larger number of samples with measurement values included within the range of the standard curve was considered optimal. regarding the repeatability and reproducibility of these elisas the supplier indicates that both the intra-plate and inter-plate variability expressed as coefficient of variation (cv% = standard deviation/mean of replicates x ) for fam a and mmp are less than %, the intra-plate cv is � . % and the inter-plate cv� . % for sparc and both the intra-lot and inter-lot cv are less than % for des and abca . data obtained from biomarkers quantification was analyzed using the proc, optimalcutpoints and caret packages of r program statistical environment version . . (http://www.rproject.org/), with confidence intervals stated at % for the final results. the auc (area under the curve) and optimal cut off value for each biomarker-based elisa was determined individually by receiver operator characteristic (roc) curve analysis. the optimal cut off values for sensitivity and specificity were based on maximum youden index (j = se+sp- ). the discriminatory power of each biomarker to discern between the different histopathological groups and the control group was determined as follows: biomarker-based elisas with auc values � . were considered to have excellent discriminatory power; . �auc< . good discriminatory power; . �auc < . fair discriminatory power; and auc < . , poor discriminatory [ , ] . multivariate binary logistic regression models (caret package of r) were used to assess the diagnostic capacity of the simultaneous use of several biomarkers providing auc, sensitivity and specificity values for the different biomarker combinations. comparison of roc curves to test the statistical significance of the difference between the areas under roc curves (derived from the same cases) was performed for the biomarkers with fair, good and excellent auc values (auc� . ) within each histopathological group using the delong method [ ] . the proc freq of the sas statistical package (sas inc., cary, nc, usa) was used for agreement analysis between pairs of diagnostic assays. the coefficient of agreement (kappa (κ)) was interpreted as follows: κ = . - . , poor; κ = . - . , fair; κ = . - . , moderate; κ = . - . , good; and κ = . - . , excellent agreement. the histopathological, immunological and microbiological characteristics of the animals included in this study, slaughtered animals (n = ) and control animals from a ptb-free farm (n = ), are summarized in table . the complete dataset is available in s table. pathological examination of intestinal tissue sections allowed the classification of the animals in four groups according to the type and extension of the histopathological lesion: focal (n = , . %), multifocal (n = , . %), diffuse (n = , . %) and with no detectable histological lesions (n = , . %). in the group of animals with focal lesions, . % were positive by one or more diagnostic methods (zn, fecal and tissue real-time pcr, fecal or tissue bacteriological culture or serum elisa). specifically, . % were positive by zn, . % by both fecal bacteriological culture (low bacterial load, < cfu/gr), and serum elisa, . % by fecal real-time pcr, and . % by both tissue real-time pcr and by tissue bacteriological culture. none of the animals in the focal group ( out of animals with known clinical status) showed clinical signs associated with ptb. in the group of animals with multifocal lesions, % of the animals were positive for at least one of the following techniques: zn, fecal and tissue real-time pcr, fecal and tissue bacteriological culture and serum elisa. specifically, . % of the animals were positive by zn, . % by serum elisa, . % by fecal real-time pcr, . % by both tissue real-time pcr and tissue culture and . % by fecal culture (one animal with low and two with heavy bacterial load). in this group . % of the animals ( out of with known clinical status) showed clinical signs. in the diffuse group, % of the animals were positive by zn. serum elisa and both fecal and tissue real-time pcr analysis showed, in each case . % positives. fecal and tissue culture analysis revealed . % and . % positives, respectively, both with heavy bacterial loads (> cfu/gr). in this group, . % ( out of with known clinical status) of the animals had ptb-associated clinical signs. only out of the animals analyzed by histopathology did not show any detectable histological lesion in their intestinal tissues and associated lymph nodes. these animals were the diagnostic accuracies of each biomarker-based elisa to discriminate between the different histopathological groups and the control group was investigated by roc analysis ( table ). the aucs, sensitivities, and specificities values were estimated for each biomarker individually and in combination based on optimal cut off values. the diagnostic performance of the biomarker-based elisas was also compared to that of the specific anti-map antibody elisa (idexx elisa). roc analysis of the elisas for the detection of biomarkers (fam a, des, mmp and spacr) and the idexx elisa was performed using serum samples from animals with focal, with multifocal and with diffuse lesions while roc analysis of the abca -based elisa was performed using serum samples from animals with focal, with multifocal and with diffuse lesions. the expression levels of each biomarker in the serum of every single holstein friesian cattle within the different histopathological groups (focal, multifocal, and diffuse) and in the noninfected control group are shown in fig . roc curves of each selected biomarker for the different histopathological groups when compared to the non-infected control group are shown in fig . . . detection of animals with focal histological lesions. three biomarkers had fair (mmp , . �auc< . ) or good (abca and sparc, . �auc< . ) discriminatory power between the focal and control groups, while the rest of the biomarkers had poor discriminatory power (auc< . ). the abca -based elisa showed the most accurate diagnostic performance with an auc value of . ( % confidence interval [ci]: . - . , p< . ), a sensitivity of . % and a specificity of . %. comparison of the roc curves of biomarker-based elisas with aucs� . (abca , mmp and sparc) showed that there were no significant differences between the three roc curves (p> . in all cases). we also tested whether using a combination of biomarker-based elisas increases sensitivity of the method. logistic regression analysis indicated that biomarkers fam a and mmp were excluded and des, abca and sparc were included in the diagnostic model for discrimination of animals with focal lesions and control animals. the auc value of this diagnostic model reached . ( % ci: . - . ), and the model exhibited . % sensitivity and . % specificity. diagnostic performance of abca , mmp and sparc for the detection of animals with focal lesions was better than that of the idexx elisa which had an auc value of . ( % ci: . - . , p> . ), a sensitivity of . % and a specificity of . %. two biomarkers had fair (mmp , . �auc< . ) or good (sparc, . �auc< . ) discriminatory power between the multifocal and control groups, while the rest of the biomarkers had poor discriminatory power. the sparc-based elisa showed the most accurate diagnostic performance with an auc value of . ( % ci: . - . , p< . ), a sensitivity of . % and a specificity of . %. comparison of the roc curves of mmp and sparc-based elisas (aucs� . ) showed that there were no significant differences between their roc curves (mmp vs. sparc, p = . ). logistic regression analysis indicated that abca and mmp were excluded and biomarkers fam a, des and sparc were included in the diagnostic model for discrimination of animals with multifocal lesions and control animals. the auc of the diagnostic model was . ( % ci: . - . ) with a . % sensitivity and . % specificity. diagnostic performance of mmp and sparc-based elisas for detection of animals with multifocal lesions was better than that of the idexx elisa which had an auc value of . ( % ci: . - . , p> . ), a sensitivity of . % and a specificity of . %. abca and mmp -based elisas had good discriminatory power between the diffuse and control groups ( . �auc< . ), while the rest of the biomarkers had poor discriminatory power (auc< . ). the mmp -based elisa showed the most accurate diagnostic performance with an auc value of . (( . - . , % ci), p< . ) a sensitivity of . % and a specificity of . %. the diagnostic performance of the idexx elisa for the detection of animals with diffuse lesions was better than that of the biomarker-based elisas with an auc value of . ( % ci: . - . , p< . ), a sensitivity of . % and a specificity of . %. comparison of the roc curves of elisas with aucs� . (abca , mmp and idexx elisa) showed that there were no significant differences between the three roc curves (p> . in all cases). logistic regression analysis did not find any significant diagnostic model for the discrimination of animals with diffuse lesions and control animals. abca , mmp and sparc-based elisas had fair ( . �auc< . ) discriminatory power between animals with multifocal and diffuse lesions and the non-infected control group. the mmp -based elisa showed the most accurate diagnostic performance with an auc value of . ( % ci: . - . , p< . ) a sensitivity of . % and a specificity of . %. diagnostic performance of the mmp -based elisa for detection of animals with multifocal and diffuse lesions was better than that of the idexx elisa which had an auc value of . ( % ci: . - . , p< . ), a sensitivity of . % and a specificity of . %. comparison of the roc curves of elisas with aucs� . (abca , mmp , sparc and idexx) showed that there were no significant differences between the four roc curves (p> . in all cases). logistic regression analysis indicated that biomarkers des, abca , mmp and sparc-based elisas can be included in a diagnostic model for discrimination of animals with multifocal and diffuse lesions and the control animals. the auc value of the diagnostic model reached . ( % ci: . - . ), and the model had % sensitivity ( %) and . % specificity. for the detection of animals with any type of histological lesions (focal, multifocal or diffuse) we compared these animals with the control group. it must be taken into account that in this analysis the three different histopathological groups are not equally represented (focal n = , multifocal n = and diffuse n = ), however, this is a reflection of the real situation on farms (n = ) . the number of animals with focal, multifocal, diffuse or with any type of lesions represented in the plot for the abca -based elisa was , , and , respectively. biomarkers were detected and quantified in bovine serum by specific elisas supplied by mybiosource, san diego, ca, usa. fam a, bovine family with sequence similarity member a; des, bovine desmin; abca , bovine atp binding cassette subfamily a member ; mmp , bovine matrix metallopeptidase ; sparc, bovine secreted protein acidic and cysteine rich; idexx elisa. the data are represented as scatter plots with each dot representing a single animal. the mean of each histopathological group is represented by a gross black point and the standard deviation by a vertical line. the asterisks indicate if the differences between each histopathological group and the control are or not significant ( � , p< . ; �� , p< . ; ��� , p< . ). https://doi.org/ . /journal.pone. .g [ ]. abca , mmp and sparc-based elisas had fair ( . �auc< . ) discriminatory power between animals with any type of lesion and the control group. the abca -based elisa showed the most accurate diagnostic performance with an auc value of . ( % ci: . - . , p< . ), a sensitivity of . % and a specificity of . %. however, comparison of the roc curves of biomarker-based elisas with aucs� . (abca , mmp and sparc) showed that there were no significant differences between the three roc curves (p> . in all cases). logistic regression analysis indicated that biomarker fam a was excluded and biomarkers des, abca , mmp and sparc-based elisas included in the diagnostic model for discrimination of infected animals and non-infected control animals. the auc value of the model reached . ( % ci: . - . ) with an . % sensitivity and . % specificity. the diagnostic performance of the abca , mmp and sparc-based elisas was better than that of the idexx elisa which had an auc value of . ( %ci: . - . , p< . ), a sensitivity of . % and a specificity of . %. the coefficients of agreement (κ values) between the histopathological classification of lesions and the results of the elisas are also included in table . the best coefficients of agreement between the focal, multifocal and diffuse histopathological groups and the biomarker-based elisa results were obtained for biomarker abca (good agreement, κ = . ), sparc (good agreement, κ = . ) and mmp -based elisa (moderate agreement, κ = . ), respectively. the agreement between the histopathological findings and the biomarker-based elisa results was worse when the multifocal and diffuse lesion groups were grouped. in this case, the best result was obtained for the mmp -based elisa which showed a moderate agreement (κ = . ). the best coefficient of agreement between the group of animals with any type of lesion and the biomarker-based elisa results was obtained for abca -based elisa with a moderate κ value of . . an excellent agreement (κ = . ) was obtained between the histopathological classification of animals with diffuse lesions and the idexx elisa results. in the rest of the histopathological groups, the coefficient of agreement of the biomarker-based elisas was better than that of the idexx elisa. the agreement coefficients were consistent with the auc values obtained by roc analysis. the diagnostic performance of the abca , mmp and sparc-based elisas for the focal and any type of lesion groups was compared with the idexx elisa and additionally with other conventional ptb diagnosis methods such as specific fecal and tissue real-time pcr and bacteriological culture ( table ). the biomarker-based elisas showed a better diagnostic value than the other diagnostic methods tested for the detection and discrimination of animals with focal lesions. for instance, the abca -based elisa detected as positive out of animals with focal lesions ( . % sensitivity) and as negative out of the controls ( . % the number of animals with focal, multifocal, diffuse or with any type of lesions represented for abca was , , and , respectively. fam a, bovine family with sequence similarity member a; des, bovine desmin; abca , bovine atp binding cassette subfamily a member ; mmp , bovine matrix metallopeptidase ; sparc, bovine secreted protein acidic and cysteine rich; idexx, idexx serum elisa. https://doi.org/ . /journal.pone. .g specificity). specifically, the idexx elisa was % specific but only detected out of animals with focal lesions ( . % sensitivity) when the cut off used was the one established by the supplier ( %) or out of ( . %) when the cut-off point established by roc analysis was used ( . %). ante-mortem assays like fecal culture and pcr were % specific but only detected out of ( . %) and out of ( . %) animals with focal lesions, respectively. post-mortem assays like tissue culture and pcr were % specific and detected a higher percentage of animals with focal lesions, out of ( . %) and out of ( . %), respectively. bovine biomarker-based elisas also showed a higher sensitivity and diagnostic value than the other diagnostic methods tested for overall detection of animals with any type of histological lesions ( table ). the abca -based elisa was able to detect out of infected animals ( . %) and as negative out of ( . %) while the idexx elisa was % specific but only detected as positive out of animals with lesions ( . %) when the cut off was % or out of ( %) when the cut off used was . %. ante-mortem assays like fecal culture and pcr were % specific but only detected out of ( . %) and out of ( %) animals with any type of lesions, respectively. post-mortem assays like tissue culture and pcr were % specific and detected a higher percentage of animals with lesions, auc, area under the curve; p-value, it is the p-value of the auc area, indicates whether the discrimination between animals with focal, multifocal, diffuse or any type of lesions and controls is significant; the cut-off point is expressed as ng/ml for the biomarkers and as a % of the positive reference sample for the idexx elisa; se, sensitivity; sp, specificity; dv, diagnostic value (semi-sum of the sensitivity and specificity); the control group consists of animals, animals with no lesions detected and from a ptb-free farm; abca , bovine atp binding cassette subfamily a member ; a, indicate that the number of animals with focal, multifocal, diffuse or with any type of lesions analysed for abca was , , and , respectively; b, the cut off value used to estimate the sensitivity and specificity of the assay was calculated by roc analysis; c, the cut off value used to estimate the sensitivity and specificity of the assay was the one established by the supplier � , the estimation of the specificity for this methods is based on the analysis of animals with no lesions, there is not availability of tissues for the live animals from the ptb-free farm. the diagnostic methods with the best diagnostic value are shown in bold face. https://doi.org/ . /journal.pone. .t out of ( . %) and out of ( . %), respectively. all the tested methods had lower sensitivity than the biomarker-based elisas for the detection of animals with focal lesions and for the overall detection of animals with any type of histological lesion. the goals of ptb control programmes may vary from eradication in areas of low prevalence, control in areas with high prevalence or increased surveillance in an area with no prior history of disease [ ] . currently, the control of ptb at the herd level is based mainly on the identification and withdrawal of infected animals, especially map-shedding animals, to suppress sources of infection and maximize the productive life of the animals [ ] . therefore, the effectiveness of these control programs is strongly conditioned by the diagnostic methods used for the detection of infected animals. host biomarkers may provide improved diagnostics for ptb increasing the effectiveness of control programmes. to the best of our knowledge this is the first study where the detection of biomarkers in serum samples by specific elisas has been used and validated as a diagnostic tool for detection of map-infected animals. our results indicate that the abca , sparc and mmp -based elisas have higher auc values and sensitivities than the idexx elisa and other current diagnostic methods for detection of animals with focal, multifocal and any type of histopathological lesions, respectively. specifically, the abca -based elisa had the best diagnostic performance for detection of animals with any type of lesions, it was able to detect . % of these animals while the idexx elisa, fecal culture and fecal pcr detected . %, . % and %, respectively. therefore, the abca -based elisa greatly improves overall detection of animals with ptb-specific histological lesions. this is due to the fact that the biomarker-based elisas have better diagnostic accuracies for the focal and multifocal groups than the other diagnostic methods. in fact, the abca -based elisa showed the most accurate diagnostic performance for detection of animals with focal lesions. it had a . % sensitivity vs. the . %, . % and . % sensitivities for the idexx elisa, fecal culture and fecal pcr, respectively. likewise, the sparc-based elisa showed the best discriminatory power between the multifocal and control animals. it identified . % of animals with multifocal lesions while the idexx elisa, fecal culture and fecal pcr identified . %, . % and . %, respectively. the mmp -based elisa showed the highest diagnostic accuracy for detection of animals with focal and multifocal lesions when they were grouped, it has a sensitivity of . % vs. the . %, . % and . % of the idexx elisa, fecal culture and fecal pcr, respectively. these results indicate that the biomarker-based elisas consistently show higher sensitivity values than the current diagnostic methods. the specificity of the idexx elisa and the other conventional diagnostic methods (fecal and tissue culture and pcr) was higher (> %) than that observed for the biomarker-based elisas in each histopathological group. the specificity values obtained for the best biomarker-based elisas in each histopathological group ranged from . % of the mmp -based elisa for detection of the grouped multifocal and diffuse animals to . % of the sparc-based elisa for detection of the multifocal group. lower specificity values could be explained by the presence of infected animals that have not been recognized as such in the control group. this could be due to the low sensitivity of the diagnostic methods (idexx elisa, and fecal culture and pcr) used to determine the ptb-status of the ptb-free farm [ ] . there was a lack of control animals with no ptb-associated histopathological lesions detected. but even using histopathology to determine the ptb-status it is possible that some animals with focal lesions were treated as negative. the sections of intestine examined by histopathology correspond to the areas where lesions are most consistently found but the actual fraction of intestine analysed is very small which is not representative of what can be in the entire intestine. another possible hypothesis to explain the lower specificity values obtained for the biomarker-based elisas is the presence in the ptb-free farm of animals infected by non-tuberculous mycobacteria that cause an increase in the levels of the selected biomarkers. however, this possibility needs to be explored before drawing any definitive conclusions. these considerations highlight the difficulty in establishing ideal control groups for the diagnosis of this disease. logistic regression analysis indicated that combination of biomarker-based elisas could improve the diagnostic performance (auc values) of the elisas used individually for detection of some histopathological groups. on the whole, the diagnostic models had higher auc values, increased sensitivities and decreased specificities. it will be necessary to assess whether the changes in sensitivity and specificity produced by the use of combined biomarkers are worthwhile as the use of such combined biomarkers would raise testing costs and complicate the diagnostic procedure. the sample size used in this study for validation of the biomarkerbased elisas and the diagnostic models was moderate (n = ) so further studies with a larger collection of samples need to be performed to validate that these biomarkers can be used to accurately diagnose all manifestations of map infection. up-regulation of these genes is a host response to infection of map. the abca gene is a member of the abc gene subfamily a. abc proteins facilitate translocation of heterogeneous substrates (lipids, peptides, proteins, ions, etc) across the cell membrane using energy acquired by the hydrolysis of atp. gene ontology annotations related to this gene include atpase and cholesterol transporter activity. in humans, the expression of abca is elevated in subjects with several pathologies as leukemia, prostate tumor, colorectal cancer, and tumor cell lines in central nervous system [ , ] . several studies have associated overexpression of abca with poor prognosis of cancer [ , ] . abca is also considered a useful marker for predicting lymph node metastasis in resected gastric cancer patients in early stage [ ] . disruption to abc transporter activity results in lipid accumulation and elevated levels of inflammatory cytokines in lung tissue [ ] . therefore, increased values of abca protein might be related to the chronic inflammation observed in the small intestine of map-infected animals. there is evidence suggesting that map can induce the expression of matrix metallopeptidases (mmps), which are the main proteases in the pathogenesis of mucosal ulcerations such inflammatory bowel disease [ ] . tissue inhibitors of mmp (timsps) have been suggested as potential biomarkers for tb. timps- , - and - facilitate remodeling and repair of tissue following destruction by mmps. for instance, the concentration of mmp- (or collagenase- ) has been demonstrated to decrease rapidly during tb treatment [ ]. mmp and timp are known to be up-regulated in tuberculosis infection and have been proposed as biomarkers for diagnosis of tuberculosis [ ] . mmp is an mmp mainly produced by neutrophils and associated with many inflammatory conditions [ , ] . up-regulation of mmp- expression in peripheral blood of map infected animals may indicate that mmp- plays an important role in the inflammation and destruction of tissue observed in the development of ptb. sparc, also known as osteonectin, is a matrix protein that binds collagen, and is required for the development of granuloma-like structures during chronic infections [ ] . in our previous rna-seq study [ ] , the protein-protein interaction analysis revealed a col a centered network containing a coli a -sparc functional interaction. sparc and col a were upregulated in the peripheral blood gene expression profiles from the cows with diffuse lesions which suggests that the expression of both proteins could lead to a bad prognosis in mapinfected cows. a sparc-centered network was also expressed more strongly in mycobacterium bovis-challenged monocyte-derived macrophages from bovine tb infected cows than in healthy cows [ ] . currently, it is impossible to identify all infected animals which makes it difficult to improve control and eradicate ptb. even though, as we have confirmed here, the map specific antibody elisa performs well for patent forms of infection, latent forms are mostly overlooked. for this reason, sensitive biomarker-based diagnostic assays that widen the detection range could be used: ) in eradication campains by identifying cows before they commence fecal shedding of the pathogen; ) to prevent the purchase of infected cattle that escape current detection methods avoiding rapid spread of ptb between herds; and ) to reduce the potential of a herd to transmit infection to other domestic and wild ruminants. that is especially relevant since ptb is a disease that affects not only cattle but also other domestic (sheep, goats) [ ] and wild ruminants (deer, fallow-deer) [ , ] , and other species like camelids [ ] , that have similar digestive characteristics although they are not true ruminants [ ] . improvement of the identification of map-infected animals through sensitive biomarker-based elisas could also help to study and confirm the potential role of map-infection as a common pathogenetic contributor to various autoimmune diseases [ ] . in conclusion, biomarker-based elisas with good diagnostic performance could be used to improve ptb management. this is especially relevant for early detection of animals during latent stages of infection which are currently escaping detection. individual or combined biomarker-based elisas could provide significant improvements in current ptb control programs when used together with traditional diagnostic methods. supporting information s economic losses due to paratuberculosis in dairy cattle relationship between antibodies against mycobacterium avium subsp. paratuberculosis in milk and shape of lactation curves mycobacterium avium suespecies paratuberculosis and crohn's disease: a systematic review and meta-analysis the consensus from the mycobacterium avium ssp type diabetes at-risk children highly recognize mycobacterium avium subspecies paratuberculosis epitopes homologous to human znt and proinsulin anti-mycobacterial antibodies in paired cerebrospinal fluid and serum samples from japanese patients with multiple sclerosis or neuromyelitis optica spectrum disorder epstein-barr virus and mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of ms patients interferon regulatory factor is a potential target of autoimmune response triggered by epstein-barr virus and mycobacterium avium subsp. paratuberculosis in rheumatoid arthritis: investigating a mechanism of molecular mimicry rheumatoid arthritis patient antibodies highly recognize il- in the immune response pathway involving irf and ebv antigens antibody response to homologous epitopes of epstein-barr virus, mycobacterium avium subsp. paratuberculosis and irf in patients with different connective tissue diseases and in mouse model of antigen-induced arthritis association of mycobacterium avium subsp. paratuberculosis with multiple sclerosis in sardinian patients paratuberculosis control: a review with a focus on vaccination strategies for time of culling in control of paratuberculosis in dairy herds elisa and fecal culture for paratuberculosis (johné s disease): sensitivity and specificity of each method evaluation of three elisas for mycobacterium avium subsp. paratuberculosis using tissue and fecal culture as comparison standards age-specific characteristics of elisa and fecal culture for purpose-specific testing for paratuberculosis ante mortem diagnosis of paratuberculosis: a review of accuracies of elisa, interferon-γ assay and faecal culture techniques development and evaluation of a novel multicopy-element-targeting triplex pcr for detection of mycobacterium avium subsp. paratuberculosis in feces evaluation of a rapid fecal pcr test for detection of mycobacterium avium subsp. paratuberculosis in dairy cattle the association between detection of mycobacterium avium subsp. paratuberculosis dna in feces and histopathological classification evaluation of a commercial elisa for diagnosis of paratuberculosis in cattle detection of mycobacterium avium subsp. paratuberculosis: comparing fecal culture versus serum enzyme-linked immunosorbent assay and direct fecal polymerase chain reaction potential application of emerging diagnostic techniques to the diagnosis of bovine johne´s disease (paratuberculosis) composition and potency characterization of mycobacterium avium subsp. paratuberculosis purified protein derivatives biomarker discovery in subclinical mycobacterial infections of cattle proteomic analysis of plasma from holstein cows testing positive for mycobacterium avium subsp. paratuberculosis (map) analysis of transcriptional profiles to discover biomarker candidates in mycobacterium avium subsp. paratuberculosis-infected macrophages, raw . gene-expression profiling of calves and months after inoculation with mycobacterium avium subsp. paratuberculosis whole-blood gene-expression profiles of cows infected with mycobacterium avium subsp. paratuberculosis reveal changes in immune response and lipid metabolism host transcriptional profiles and immunopathologic response following mycobacterium avium subsp. paratuberculosis infection in mice transcriptional profiling of ileocecal valve of holstein dairy cows infected with mycobacterium avium subsp. paratuberculosis responses of bovine innate immunity to mycobacterium avium subsp. paratuberculosis infection revealed by changes in gene expression and levels of microrna application of transcriptomics to enhance early diagnostics of mycobacterial infections, with an emphasis on mycobacterium avium ssp. paratuberculosis mycobacterium tuberculosis extracelular vesicle-associated lipoprotein lpqh as a potential biomarker to distinguish paratuberculosis infection or vaccination from tuberculosis infection gene expression profiles during subclinical mycobacterium avium subspecies paratuberculosis infection in sheep can predict disease outcome identification of micrornas in bovine faeces and their potential as biomarkers of matrix metalloproteinase (mmp ) gene polymorphisms in chronic periodontitis contrasting roles of sparcrelated granuloma in bacterial containment and in the induction of anti-salmonella typhimurium immunity transcriptome changes upon in vitro challenge with mycobacterium bovis in monocyte-derived macrophages from bovine tuberculosis-infected and healthy cows paratuberculosis in sheep and goats paratuberculosis in free-ranging wildlife in north america histopathological classification of lesions observed in natural cases of paratuberculosis in free-ranging fallow deer (dama dama) paratuberculosis in small ruminants, deer, and south american camelids evolutionary history and differences between camelids and ruminants we would like to acknowledge the expert technical assistance from the statistical advice unit of the scientific-technical services of the university of oviedo, our collaborating farms and the astega veterinary services for their collaboration in the sampling work. finally, we would like to acknowledge the daily work of serida´s farm operators in the care and maintenance of animals. we gratefully acknowledge kevin p. dalton for proofreading the manuscript. conceptualization: marta alonso-hearn, rosa casais. key: cord- - bsixsgd authors: chirnside, e.d.; francis, p.m.; de vries, a.a.f.; sinclaira, r.; mumford, j.a. title: development and evaluation of an elisa using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus date: - - journal: j virol methods doi: . / - ( ) -u sha: doc_id: cord_uid: bsixsgd a recombinant glutathione-s-transferase fusion protein expressing amino acids – of equine arteritis virus (eav) g(l) (rg(l) – ) was tested in an elisa for its ability to detect serum antibodies to eav. host antibodies induced following eav infection bound the recombinant antigen by elisa. the elisa specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. a good correlation existed between eav neutralizing antibody titers and elisa absorbance values (r = . ). the sensitivity and specificity of the elisa were . and . %, respectively, compared with the eav neutralization test and the recombinant antigen did not crossreact in elisa with equine sera directed against other common equine respiratory viruses. three post-eav infection equine sera raised against different eav isolates reacted strongly in the elisa, as did two equine sera raised against eav vaccines, indicating that the viral epitope was conserved between the viruses tested. following vaccination with an inactivated whole virus vaccine, antibody detected with the recombinant antigen elisa preceded the development of a virus-neutralizing response. the study demonstrates the potential application of rg(l) – as a diagnostic antigen. of virologicul methodc ( ) equine viral arteritis (eva) has to date only been reported to infect horses (doll et al., ; chirnside, ) although seropositive donkeys have been found (paweska and barnard, ) . the disease has been known for some years and manifests itself with widely varying clinical signs. in its most severe form equine arteritis virus (eav) infection causes abortion (doll et al., ) and foal death (golnik et al., ; vaala et al., , although it is more usual for this virus to cause mild respiratory disease (chirnside, ) . disease outbreaks are identified infrequently due to clinically inapparent infection and field isolates of the virus are rare. the causative agent, eav, is a small positive single-stranded rna virus with a genome organization, mode of replication and gene expression strategy similar to that of corona-and toroviruses (de vries et al., (de vries et al., , den boon et al., ; snijder et al., ) . eav has recently been classified in the genus arterivirus (cavanagh et al., ) along with lactate dehydrogenase-elevating virus of mice (ldv) (godeny et al., ; kuo et al., ) , simian hemorrhagic fever virus (shfv) (plagemann and moennig, ) , and the virus causing porcine respiratory and reproductive syndrome (prrsv) (conzelmann et al., ) lelystad virus (lv) (meulenberg et al., ) . eav is transmitted by the respiratory and venereal routes, with a % carrier state existing in seropositive stallions (timoney et al., ) making the latter route a particular cause for concern, as these stallions shed virus in their semen and may consequently infect broodmares neu et al., ) . eva is geographically widespread (chirnside, ) and most european and north american countries have eav-seropositive animals. some outbreaks have occurred following the importation of persistently infected animals causing primary transmission of eav by the venereal route, followed by secondary respiratory spread (wood et al., ) . in the light of the potential economic importance of this disease to the horse breeding and racing industry, a requirement exists for both prophylactic treatment and reliable serodiagnosis of infection. laboratory-based serological tests include eav virus neutralization test (nt), complement fixation (cf) and enzyme-linked immunosorbent assay (elisa) (senne et al., ; fukunaga and mccollum, ; cook et al, ) . the elisa has a relatively low specificity when applied to sera from horses previously vaccinated against equine influenza and herpesviruses; this is primarily due to host antibody induced by tissue culture contaminants of these vaccines reacting with cell culture-derived antigen present in the whole-virus elisa antigen. the cf test has limited temporal sensitivity and is consequently only useful for serodiagnosis up to weeks postinfection. at present, nt results from designated laboratories are internationally accepted for import/export testing. infection with eav elicits a virus-neutralizing antibody response within - days. stallions which have previously been exposed to eav, and therefore may be shedders of infectious virus, can be detected by the nt as a discernible level of circulating neutralizing antibody persists for many years after recovery (gerber et al., ) . the nt is sensitive, but suffers from the disadvantage that it requires laboratory testing to maintain cell cultures and stocks of infectious virus. in addition, the nt takes several days to complete. laboratories use varied test protocols and different reagents which can lead to disparate results. moreover, the nt does not differentiate between the host serological response induced by vaccination and that resulting from natural infection. it has recently been demonstrated that neutralizing monoclonal antibodies recognize a determinant on the large envelope glycoprotein, g, (balasuriya et al., ; deregt et al., ) . this paper describes an indirect elisa using a recombinant glutathione-stransferase fusion protein (smith and johnson, ) as an antigen to screen equine sera for the presence of antibodies to eav, and its evaluation as a diagnostic test with large numbers of equine serum samples. plasmid dna manipulations were carried out as described previously (sambrook et al., ) . transformations with plasmid pgex- x (smith and johnson, ) were carried out in escherichia coli tgl and recombinant clones selected by antibiotic resistance. clones were subsequently screened for fusion protein expression after iptg induction by analysis in sds- . % polyacrylamide gels, and the eav-specific insert size, orientation and in-frame insertion confirmed by restriction endonuclease digestion analysis and dsdna sequencing of purified plasmid dna (chirnside et al., ) . the expression construct srsal, expressing rg, - , comprised nucleotides , - , of eav open reading frame (orf) fused in-frame to the carboxyl-terminus of the glutathione-s-transferase gene in the expression vector pgex- x. fusion protein was affinity purified using glutathione sepharose b (pharmacia). a -ml culture of bacterial cells expressing recombinant antigen was harvested by centrifugation for min at g and °c resuspended in ml mm tris-hcl (ph . , mm edta, mm nacl (ste) and recentrifuged to pellet the cells. the supernatant was discarded and the cells resuspended in ml ste, ~ lysozyme ( mg/ml) added and the tube incubated at °c for min. the tube was then frozen at - °c overnight followed by a short immersion in a boiling water bath to thaw the frozen suspension. dnase ( pg) and ~ of m mgcl, were then added to the thawed cells and the tube incubated at °c for min. triton x- was then added to a final concentration of l%, the mixture kept on ice for min and centrifuged at , g and °c for min. glutathione sepharose b gel was added to the supernatant and the solution mixed by gentle agitation on a rotary mixer for min. the gel was washed times with pbs and the recombinant antigen eluted from the gel with reduced glutathione ( mm glutathione, mm tris-hcl (ph . )). eluate was collected in l-ml fractions and analyzed by sds-polyacrylamide gel electrophoresis prior to pooling fractions containing purified protein. pooled protein was dispensed into -~ aliquots and frozen at - °c until used. protein concentration was determined with a modified lowry reagent solution from a protein assay kit (sigma). optimal concentrations of reactants were determined by checkerboard titration. immulon microtiter plates (dynatech) were coated with gst or fusion protein diluted in mm carbonate buffer (ph . ) and kept overnight at °c. plates were then washed times with pbs containing . % tween (pbst) and ,ul of pbst containing % goat serum (pbstg) added to each well. following incubation at °c for h and washes with pbst, ~ of horse sera diluted lo-* in pbstg was added to each well and the plates incubated for min at °c. plates were then washed times with pbst, ~ of a lo-" dilution of affinity-purified biotin-labeled goat anti-y chain specific horse igg (kpl) was added to each well and the plates incubated for min at °c. the plates were then washed times with pbst, and ~ of a -j dilution of streptavidin-peroxidase (kpl) in pbstg added to each well and the plate incubated at room temperature for min. after a final washes with pbst, substrate solution ( . mg/ ml o-phenylenediamine dihydrochloride dissolved in mm phosphate-citrate buffer (ph . ), containing . % sodium perborate) was added to each well and the plate incubated at room temperature for min, after which the reaction was stopped by the addition of ~ m h,so, to each well and the absorbance read at nm. each serum sample was assayed in duplicate wells against both purified gst and fusion protein and the mean absorbance value taken as the a,,,, reading to each antigen. each elisa was validated by the inclusion of nt-/elisam control sera. the mean absorbance value for these control sera was < . in each individual elisa test, with a grand assay mean over all tests of . . in addition, nt+/elisa+ control sera were also run as standards in each test to confirm the sensitivity of the assay. immunoblots were carried out with equine sera diluted o-'. using a modified version of the elisa protocol after electrophoretic transfer of proteins from sds-polyacrylamide gels onto nitrocellulose membrane. non-specific binding of equine antibody to the nitrocellulose membrane was first blocked by overnight incubation at °c in pbstg. the membrane was then washed times in pbst and the elisa protocol for binding and washing the components of the antibody sandwich performed as above with washes, each of min between steps. to develop a signal from the immunoblots the ecl detection system (amersham) was employed using the manufacturer's protocol. the eav neutralization test was carried out according to the method of senne et al. ( ) with minor modifications. all sera were initially screened at a : dilution in replicate wells of a microtiter plate. virus neutralizing sera were subsequently titrated in replicate wells from an initial dilution of : to : . each individual nt included positive control sera, a virus control to ensure that lootcid,, of the bucyrus strain of eav was added to each well, and a series of rk- cell controls. individual wells were scored for > % cytopathic effect (cpe) after h incubation ( °c % co,) and titers calculated according to the formula of karber ( ) . a serum was considered seropositive when it had an nt titer > : (> log,, . ) and a -fold rise in titer regarded as a seroconversion following virus infection or vaccination. throughout the study, the same batch of the bucyrus strain of eav and of guinea pig complement was used, and rk- cells were used at passage numbers - . this ensured excellent comparability between eav neutralization tests. the field sera used for this study were submitted to the diagnostic service of the animal health trust. the virus type-specific sera were provided by dr. yoshio fukunaga of the equine research institute, japan, and the panels of european and american sera obtained from dr. margaret lucas, central veterinary laboratories, uk. fusion proteins derived from eav g, have been shown to react with postinfection, eav neutralizing horse sera (chirnside et al., ) . the glutathione-s-transferase of fusion protein expressed by plasmid construct rsal contains amino acid residues - of eav g, derived from the bucyrus isolate. this fusion protein (rg,, - ) was expressed at high levels in iptg-induced cells and purified readily by affinity chromatography on glutathione sepharose b; the purified rg, - contained few contaminating bacterial proteins (fig. la) . in immunoblots to equine sera (fig. lb-d) rg, - was very strongly recognized by eav neutralizing sera (fig. lb) , faintly by the antiserum against gst (fig. id) and only very slightly by the non-neutralizing equine serum tested (fig. lc) . purified gst was recognized by equine sera raised specifically to gst and also by both virus neutralizing and non-neutralizing equine sera. the optimal antigen and antibody concentrations in elisa were determined by checkerboard titration. a serum dilution of l/ and antigen concentration of . pg per well were selected to give maximum discrimination between nt+ and nt-sera. the majority of the equine sera had some reactivity to gst, in the absorbance range o- . , at these reagent concentrations. consequently the absorbance value to gst was subtracted from the absorbance value to the recombinant protein to give an eav-specific figure for each test sample. equine sera raised specifically against equine herpesvirus types - , equine rhinovirus types and and equine adenovirus did not recognize rg, - in elisa; the recombinant antigen was only bound by eav neutralizing equine sera. in order to evaluate the potential of rg, - as antigen for a diagnostic elisa, and to compare results directly with the eav neutralization test, the elisa absorbances of equine sera were compared with their nt titers (fig. ) . from the data plotted in fig. an a, ,,] > . (the y intercept plus two standard deviations as determined by linear regression analysis in fig. ) was taken as the cut-off point determining an elisa seropositive value. table shows a numerical break-down of the results using these seropositive/seronegative cut-off values. virus neutralizing sera were clearly distinguishable in elisa from samples seronegative in the nt. the elisa sensitivity in detecting nt seropositives was . % ( detected from ). only one virus-neutralizing sample, with a low antibody titer (log,, . , was elisa-(a,,, = . ). the specificity of the elisa to detect nt seronegative samples correctly was . % ( detected from ). this was due to the detection of samples which had absorbance readings > . but which were negative by nt (log,, < . ). this nt-/elisa+ group included blood samples from horses held on equine premises affected during the eav outbreak in england (wood et al., , diagnostic samples from assorted horses submitted for serological screening which included an eav nt, and blood samples from horses which had previously been vaccinated with either one or two doses of an inactivated eav vaccine. the anomalous equine sera were subjected to further investigation by: ( repeating the nt; ( ) repeating the elisa with a fresh batch of rg, - ; ( ) elisa nt-samples were elisa' to all eav-specific antigens; this number comprised serum samples from vaccinated horses, from outbreak-associated animals and two from sera submitted for general serodiagnosis. paired sera originating from horses during the course of the uk eav outbreak (wood et al., ) seroconverted in both the nt and elisa tests from nt-/ elisato nt+/elisa+. a further paired sera from horses associated with the eav outbreak did not seroconvert in either diagnostic test. paired sera from horses were tested by nt and elisa both prior to, and - weeks after administration of two doses of an inactivated eav vaccine. among the vaccinees l/ ( %) was nt-/elisa-, / ( %) were nt+/elisa+ and / ( %) had an elisa antibody response detectable in the absence of any vaccine-induced neutralizing antibody. the possible effect of variation in g, between viruses was investigated by comparing elisa absorbance readings and nt titers of two post vaccination sera raised against a live and killed vaccine derived from the bucyrus strain, and postinfection eav isolate specific equine sera (fig. ) . although differences between absorbance values were evident between the sera, all were positive by elisa. the homologous sera to rg, - , derived from infection with the bucyrus isolate (bucyrus) or vaccination with inactivated virus (kill bucyrus) had higher absorbance values than equine sera raised to the heterologous virus isolates ( -ky-al, wroclaw- ) and the live attenuated vaccine (arvac; fort dodge laboratories) although little variation in nt antibody titer to the bucyrus isolate of eav (from which the recombinant antigen is derived) was detectable between isolates. to evaluate the utility of rg, - two further panels of equine sera were assayed by elisa. the panels, one of european and one of american origin, comprised a mixture of postinfection and eav-negative sera and have been used as reference sera to monitor the standard of virus neutralization testing between laboratories (unpublished). with these two serum panels (tables and ) the elisa sensitivity was % and the specificity %; / nt+ sera tested elisa', / nt-sera tested elisa-and / nt-sera tested elba+. the serological reactivity of a recombinant eav g, fusion protein is reported and its use is described for the detection of equine antibodies to eav in elisa. the assay proved to be highly sensitive and specific for the detection of eav antibodies; it correlated well with results from the eav microneutralization test and detected postinfection seroconversions in horses following natural infection and vaccination. in the elisa, field sera diluted : reacted with gst causing a variable background absorbance of o-os. it should be possible to reduce this background absorbance by cleaving the gst moiety from the g, fusion protein, or by cloning eav g, - into a different expression vector. however in its present format, the correlation between the rg, - elisa and nt is high (r = . ) with the elba detecting additional seropositives to the nt. the elisa+/ nt results could be due to differences in sensitivity between the two tests or because the elisa detects equine antibodies that bind eav g,, of which those capable of neutralizing virus in a nt are a subpopulation. alternatively, the detection of elisa+/nt-samples, including vaccinated horses, poses questions about the specificity of the elisa and its biological relevance when compared with the nt. however, following one or two doses of an inactivated vaccine, it was possible to demonstrate elisa+/nt-results in % of vaccinees. if a similar situation exists following natural infection with eav, then the additional seropositive field samples detected by elisa may have originated from horses previously infected with eav, but in which a virus neutralizing antibody response was not induced or in which the response has dropped below detectable levels. this observation requires further investigation since the presence of circulating virus neutralizing antibody has to date been accepted as evidence of prior exposure to eav, either through infection or vaccination. the rg, . - elisa results suggest this may not be the case. the membrane topology of eav g, remains to be established. however, the hydropathy profile (de vries et al., ) and position of the sole n-glycosylation site (den boon et al., ) both determine that the protein ectodomain is likely to encompass residues - . by testing > equine sera in elisa to g, - , we have demonstrated that amino acid residues - of the bucyrus strain of eav g,_ encompass a highly immunoreactive antigen which correlates closely with the host virus neutralizing response. eav g, has been shown to contain a major antigenic site (chirnside et al., ) and virus neutralizing murine monoclonal antibodies react with a protein of a molecular size ( kda) equivalent to g,, by western blots (balasuriya et al., ; deregt et al., ) . eav strain variation has been demonstrated at the nucleotide level by rnase tl fingerprinting (murphy et al., (murphy et al., , and by direct sequencing of the eav n-and m-genes (chirnside et al., ) and antigenic variation inferred by cross-neutralization studies (fukunaga et al., ) . the equine immune response to eav induced by different eav isolates was detectable in elisa with rg, - derived from the bucyrus isolate, implying a high degree of conservation of this epitope. to substantiate this finding sequencing studies of eav g, from different isolates is currently being undertaken. changing from reliance solely on the nt, to an initial rapid screen by elisa followed by neutralizing antibody testing of elisa+ samples would improve the speed of diagnosis for eav seronegative horses, lower the cost of initial testing, and potentially eradicate differences in test results between laboratories. additionally, the enhanced detection of seropositive animals allied to a vaccination policy and selective breeding practices would allow the better control and possible eradication of eav from the breeding population. the expression and purification of g, - is simple and the elisa is easily standardized between different testing laboratories. since the recombinant protein is not infectious it affords the opportunity to undertake eav serodiagnostic assays rapidly and safely without virus containment facilities. in addition, the use of a purified protein expressed in a bacterial system removes the problem of non-specific antibody crossreactivity to cell culture-derived antigen which has plagued previous eav elisa tests (cook et al., ) . a k envelope glycoprotein of equine arteritis virus expresses neutralisation determinants recognised by murine monoclonal antibodies revision of the taxonomy of the coronavirus, torovirus and arterivirus genera equine arteritis virus: an overview comparison of m and n gene sequences distinguishes variation amongst equine artcritis virus isolates equine arteritis virus neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein g molecular characterisation of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group the effects of vaccination with tissue culture-derived viral vaccines on detection of antibodies to equine arteritis virus by enzyme-linked immunosorbent assay all subgenomic rnas of equine arteritis virus contain a common leader sequence the structural proteins of equine arteritis virus monoclonal antibodies to equine arteritis virus proteins identify the g, protein as a target for virus neutralisation an outbreak of abortion caused by the equine arteritis virus complement fixation reactions in equine viral artcritis induction of immune response and protection from equine viral artcritis (eva) by formalin inactivated-virus vaccine for eva in horses an attempt to protect against persistent infection of equine viral arteritis in the reproductive tract of stallions using formalin inactivated-virus vaccine y ) use of the serum neutralisation test for equine viral arteritis with different virus strains serological investigations on equine viral arteritis complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (ldv) yxl) natural equine viral arteritis in foals beitrag zur kollektiven behandlung pharmakologischer reihenversuche. naunyn-schmiedebergs arch lactate dehydrogenaseelevating virus (ldv): subgenomic mrnas, mrna leader and comparison of 'terminal sequences of two ldv isolates the recovery of virus from horses with experimental cases of equine arteritis using monolayer cell cultures of equine kidney responses of vaccinated and non-vaccinated mares to artificial insemination with semen from stallions persistently infected with equine arteritis virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav analysis of genetic variation among strains of equine arteritis virus genomic variability among globally distributed isolates of equine arteritis virus persistent infection of the reproductive tract of stallions persistently infected with equine arteritis virus serological evidence of equine arteritis virus in donkeys in south africa lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus: a new group of positive-strand rna viruses molecular cloning: a laboratory manual equine viral arteritis: a standard procedure for the virus neutralisation test and comparison of results of a proficiency test performed at five laboratories single-step purification of polypeptides expressed in escherichia coli as fusion proteins with glutathiones-transferase the coronaviruslike superfamily demonstration of the carrier state in naturally acquired equine arteritis virus infection in the stallion fatal, congenitally acquired infection with equine arteritis virus in a neonatal thoroughbred the first recorded outbreak of equine viral arteritis virus in the united kingdom this work was funded by the animal health trust and maff contract research award csa . patent no. gb . covers the use of rg,. key: cord- - ggaqf authors: nan title: abstracts of the papers presented in the xix national conference of indian virological society, “recent trends in viral disease problems and management”, on – march, , at s.v. university, tirupati, andhra pradesh date: - - journal: indian j virol doi: . /s - - - sha: doc_id: cord_uid: ggaqf nan patients showed rashes on face, hand and foot. ev detection carried out in vesicular fluid, stool, serum and throat swab specimens by rt-pcr of ncr gene. serotyping was carried out by using rt-pcr of viral protein of vp / a junction region followed by sequencing and phylogenetic analysis using neighbor-joining-algorithm and kimura- parameter model of mega- software. overall ev positivity detected in hfmd patients from kerala, tamil nadu, west bengal and orissa states was found to be . %, . %, . % and . % respectively. typing of vp gene sequences indicated presence of ca- , ev- , echo- strains in kerala and ca- in west bengal, orissa and tamil nadu. phylogenetic analysis indicated ca- , ev- , echo- strains showed . - . % and - . % homology with japanese, australian and french strains. however, ca- strains were closer to malaysian strains with . - . % nucleotide homology. the present study documents the association of multiple types of ev's i.e., ca- , ev- , echo- and ca- strains contributing as prime viral pathogens in hfmd epidemics in the reported regions with new emergence of ca- circulating strain in kerala, india. tasgaon september . sera were collected from suspected hepatitis cases and there contacts and tested for anti hev igm/igg antibodies (elisa) and liver enzymes like alanine aminotransferase (alt). anti hev igm antibodies were detected in . % ( / ) of the suspected cases. the overall attack rate was . %. male to female ratio was : . majority ( . %) of the cases were in the age group - years and recovered without any clinical complications. weekly distribution of cases showed that the majority ( . %, / ) cases occurred between nd and rd week of june. dark urine ( . %), jaundice ( . %), fatigue ( . %), abdominal pain ( . %), anorexia ( . %), vomiting ( . %), fever ( . %), giddiness ( . %), diarrhoea ( . %) and arthalgia ( . %) were the prominent symptoms. sera collected from antenatal cases (ancs) showed anti hev igm antibody in . affected pregnant women had a normal outcome. a death of year, male hepatitis e case was reported during the outbreak period that had cirrhosis of liver with oesophageal varices. sanitary survey revealed that water pipelines were laid down in close proximity of sewerage system, and water posts were without tap. these are the likely sources of faecal contamination of water supplies. among water samples collected from various places, were found to be unfit for drinking based on the routine bacteriological tests conducted at state public health laboratory, pune. no case occurred after the pipelines were repaired. this typical outbreak of hepatitis e re-emphasizes need for proper water supply/sewage disposal pipelines and adequate maintenance measures. jayanthi shastri, nilima vaidya, sandhya sawant, umesh aigal department of molecular biology, kasturba hospital for infectious diseases, mumbai, india dengue and dengue haemorrhagic fever are amongst the most important challenges in tropical diseases due to their expanding geographic distribution, increasing outbreak frequency, hyperendemicity and evolution of virulence. the gobal prevalence of dengue has grown dramatically in recent decades. who estimates - million cases of dengue virus infections worldwide every year resulting in , to , cases of dhf and , deaths each year. public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. laboratory diagnosis of dengue virus infection can be made by the detection of specific virus, viral antigen, genomic sequence and/or antibodies. currently basic methods used by laboratories for diagnosis of dengue virus infection are virus isolation and characterisation, detection of genomic sequence by nucleic acid amplification technology assay and detection of dengue virus specific antibodies/antigen. molecular diagnosis based on reverse transcription (rt)-pcr s.a. one step or nested pcr, nucleic acid sequence based amplification (nasba), or real time rt-pcr, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute phase serum samples. several pcr protocols for detection have been described that vary in the extraction method, genomic location of primers, specificity, sensitivity and the methods to determine the products and the serotype. pcr-based dengue tests, due to the specificity of amplification, enable a definitive diagnosis and serotyping of the virus. in addition dna sequencing of the amplification product enables the virus to be genotyped, providing important information on the sources of infection. more recently tests have incorporated flurogenic probe, so called taq man technology for the specific real time detection of dengue - amplicons. product is detected by a specific oligodeoxy nucleotide probe that is labelled with carboxy-fluorescein (fam). this technology offers the advantage of being both rapid and potentially quantitative. second, the detection of product by hybridisation of flurochrome labelled probes increases specificity. third, as the product is detected without the need to open the reaction tube, the risk of contamination by product carry over is minimised. the advantages of speed, contamination minimisation and reduced turn around time justify application of this assay over the currently used nested pcr assay. during the period january to october , molecular laboratory received samples from patients presenting with acute onset fever for dengue . %) samples were tested positive by this method. the disease peaks in the monsoon season with a percentage of . %. rapid tests, igm and igg capture elisa are popularly used tests for diagnosis of dengue infection. its utility is limited for diagnosing dengue in convalescecce ( - days) . specificity is also compromised due to infections with flaviviruses: japanese encephalitis and chikungunya. dengue ns ag elisa with its cost effectiveness, specificity and sensitivity should be considered as the test of choice for diagnosing dengue in the acute phase of illness in the developing countries. molecular diagnosis enables confirmatory diagnosis of dengue in the acute phase of the illness and is suitable for further typing methods. assistant general manager and r&d coordinator, division of quality control and r&d, bharat immunologicals and biologicals corporation ltd., village chola, bulandshahr, up vaccine development in india, though slow to start, has progressed by leaps and bounds in the past years. it was dependent on imported vaccines but now it is not only self-sufficient in the production of vaccines conforming to international standards with major supplier of the same to unicef. the role of drug authorities is to enhance the public health by assuring the availability of safe and effective a indian j. virol. (september ) (suppl. ):a -a vaccines, allergenic extracts, and other related products. vaccine development is tightly regulated by a hierarchy of regulatory bodies. guidelines provided by the indian council of medical research (icmr) set the rules of conduct for clinical trials from phase i to iv studies as well as studies on combination vaccines. these guidelines address ethical issues that arise during a vaccine study. a network of adverse drug reaction (adr) monitoring centers along with the adverse events following immunization (aefi) monitoring program provide the machinery for vaccine pharmacovigilance. genetic modifications have been developed to develop effective and cheaper vaccines by the use of recombinant technology. to ensure safety of consumers, producers, experimental animals and environment, governments all over the world are following regulatory mechanisms and guidelines for genetically modified products. as with other industrializing countries undergoing rapid shifts, india clearly recognizes the need to restructure its regulatory system so that its biopharmaceutical industry can compete in international markets. genetic engineering approval council (geac), recombinant dna advisory committee (rdac), review committee on genetic manipulation (rcgm), institutional biosafety committees (ibsc) are responsible for development, commitment for parameters and commercialization of recombinant vaccines. to centralize and coordinate the whole system, government has taken to form two agencies to regulate the regulation laws to develop recombinant pharmaceuticals products including vaccines. the first is the creation of the national biotechnology regulatory authority (nbra), under the department of biotechnology (dbt), as part of india's long-term biotech sector development strategy. the second major initiative will affect the entire indian pharmaceutical industry. this is the replacement of most state, district, and central drug regulatory agencies with a single, central, fda-style agency, the central drug authority (cda). the cda is expected to have separate, semi-autonomous departments for regulation, enforcement, legal, and consumer affairs; biotechnology products; pharmacovigilance and drugs safety; medical devices and diagnostics; imports; quality control; and traditional indian medicines. it will set up offices throughout india and will be paid for inspection, registration, and license fees. its enforcement powers will be strengthened by a new law increasing the criminal penalties for illegal clinical trials. in the manufacturing area, though, the country has been tightening the rules and enforcement. an amendment to the regulations, ''schedule m'' of the drug and cosmetics act, now specifies the good manufacturing practice (gmp) requirements for factory premises and materials. these requirements were modeled after us fda regulations, to improve regulatory coordination between indian and us regulators. india has realized the importance of regulations in pharmaceutical specially in vaccine field but it will take several years to implementation of these. india has coordinated some of its regulatory functions with western organizations. the us pharmacopoeia established an office in hyderabad in . a representative of the indian pharmaceutical lobby also recently has expressed openness to an expansion of the fda's oversight of indian manufacturing. as india expands its global drug and biologicals production, us and europe, as the world's largest drug importers, will likely expand their regulatory support in the development of the country's regulatory systems. rapid diagnosis of japanese encephalitis virus (jev) infections is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. however, the speed and accuracy of diagnosis must be balanced against test cost and availability, especially in developing countries. an antigen capture enzyme-linked immunosorbent assay (elisa) for detection of circulating jev specific nonstructural protein (ns ) was developed by using monoclonal antibodies (mabs) specific to recombinant (ns ). the applicability of this jev ns antigen capture elisa for early clinical diagnosis was evaluated with acute phase serum/ cerebrospinal fluid (csf) specimens collected from different epidemics during [ ] [ ] [ ] . jev ns antigen was detected in circulation from day to . the sensitivity and specificity of jev ns detection in serum/csf specimens with reference to reverse transcriptase pcr was %, and . % respectively. no crossreactions with any of the other closely related members of the genus flaviviruses (dengue, westnile, yellow fever and saint louis encephalitis (sle) viruses) were observed when tested with either clinical specimens or virus cultures. these findings suggested that the reported jev specific mab-based ns antigen capture elisa will be a rapid and reliable tool for early confirmatory diagnosis as well as surveillance of je infections in developing countries. manmohan parida the recent emergence of a novel human influenza a virus (h n ) poses a serious global health threat. the h n virus has caused a considerable number of deaths within a short duration since its emergence. a two-step single tube accelerated rapid real-time and quantitative swine flu virus specific h rtlamp assay is reported by targeting the h gene of the novel h n hybrid virus. the feasibility of swine flu h rtlamp for clinical diagnosis was validated with a panel of suspected throat wash samples comprising confirmed positive and confirmed negative cases of ongoing epidemic. the comparative evaluation of h specific rtlamp assay with real-time rt-pcr demonstrated exceptionally higher sensitivity by picking up all the h n positive and additional positive cases amongst the negatives that were sequence confirmed as h n . none of the real-time rtpcr positive samples were missed by rtlamp system. the comparative study revealed that rtlamp was -fold more sensitive than rtpcr with a detection limit of copy number. these findings suggested that rtlamp assay is a valuable tool for rapid, real-time detection as well as quantification of h n virus in acute phase throat swab samples without requiring any sophisticated equipments. because of its recurrent nature. despite considerable progress in understanding of the virus at cellular and molecular levels, the proper management of the disease in its different stages is still a dilemma particularly whether to use antiviral or steroids or both. the risk of using steroids with its attendant complications has to be weighed against the risk of progression of the disease if avoiding the use of steroids. this dilemma can be reduced to a considerable extent if basic principles of virology and pathogenesis are kept in mind. this article reviews current concepts of virological and clinical aspects of hsv keratitis to enable a broad understanding of the disease process. it is recognized several influential host factors including the fact that hsk is more common in men than women. it is observed that the ability of hsv to establish latent infection in sensory neurons and possibly cornea, but have as yet been unable to use this knowledge to prevent the disease limitations. acknowledging limitations may further stimulate application of laboratory knowledge in coping with hsk which constitutes to present major challenge in terms of management. mvo- study on effect of human bhsp in immunity of hcv core protein and hbv hbsag there are more than million individuals with hepatitis b and c in the world. in spite of vaccination in the different areas there are several reports about patients who got vaccine before. also there is not efficient vaccine against of hepatitis c and one of the important problems in vaccine project is development of effective and suitable adjuvant in human vaccines. at present research we applied human bhsp protein as adjuvant and chaperon. this protein injected to balbc mice as adjuvant together with recombinant proteins of hcv core and hbv hbsag. then humoral and cellular immune systems of the mice were studied. core and hbsag genes were cloned into petduet- vector and thermal vector of pgp - was used for human heat shock protein expressions. the different combination of these three proteins was injected to mice and we evaluated the total igg and igg a of mice serums after a week. two weeks after booster injection, we studied the proliferation and cytokine secretion of spleen, inguinal and popliteal lymph nodes lymphocytes in vitro and ex vivo conditions. so the core/hbsag + hsp and core + hbsag + hsp complexes induced total igg and igg a secretion. the spleen lymphocytes proliferation were increased equal to serum igg a level that was constant in second time bleeding with significant different to complexes with freund's adjuvant. at first il- and il- cytokines were increased and then decrease of il- meaned no hypersensitivity. the chaperon effect of hsp on structure of core and hbsag proteins was studied by cd and flourometer. it could fold the proteins after heating and unfolding. hepatitis b virus (hbv) infection is vaccine preventable global public health problem. all commercially available vaccines contain one or more of the recombinant hepatitis b envelope protein or surface antigen (hbsag). measurement of antigen responsible for immunogenicity of vaccine is central to quality assessment. the problems associated with the use of a polyclonal antibody in an assay with regard to its poorly defined nature and batch-to-batch variation has been mitigated by the use of mabs as described in this paper. the initial capture of hbsag by the mab could orientate it such that the same antibody could bind to it as a detection antibody after labeling with out steric hindrance. the development of an immuno-capture elisa (ic-elisa) to measure the hbsag content using a monoclonal antibody (mab) specific to determinant ''a'' of hbsag in the experimental vaccine formulations is being discussed. murine mabs developed against hbsag, subtype adw were found to cross-react with the other subtypes viz. ad and ay too. the mabs have been characterized following which, one mab hbs was chosen for developing ic-elisa format for the quantification of the hbsag in the final algel adsorbed vaccines. the unadsorbed hbsag was used to establish the standard curve of hbsag/a. the elisa had a sensitivity of ng/ml of hbsag. the recovery rate of hbsag/a was found to be around % in the vaccines treated to desorb the antigen from algel. twenty seven experimental batches of monovalent hepatitis b vaccines were analyzed for the hbsag content, both by ic-elisa and a commercial kit (axsym kit, abbott laboratories, usa). the statistical analysis of ic-elisa results indicated that an experimental equation f(x) = . (x) + . , could precisely estimate the amount of hbsag in the adsorbed vaccines. the amounts of hbsag recovered from the adsorbed vaccines as estimated by the ic-elisa format had a good correlation with the estimates derived from a commercial kit, which is being used by several vaccine manufacturers in india for the quality control of vaccine antigen. the varying amounts of vaccine antigens that could be recovered seemed to depend on the quality of the hbsag and the methods of hbsag adsorption to the alum gel during vaccine manufacture. epidemiology of the spread of h n virus. children of school going age have become victim of this deadly virus as evident from the reporting data generated in the past few weeks. the mortality rate has also been slightly increased. the disease spread in wave pattern and presently the world is passing through the second wave of pandemic with more severity in young and otherwise health people with a predilection for lungs leading to viral pneumonia and respiratory failure. now the pandemic gained hold in the developing world affecting more severely as millions of people live under deprived conditions having multiple health problems, with little access to basic health care. current data about the pandemic from developed counties need to be very closely watched in relation to shift in virus sub type, shift of the highest death rate to younger populations, successive pandemic waves, higher transmissibility than seasonal influenza, and demographic differences etc. presently the world appears to be better prepared. vaccine is available in market in many countries. even vaccine trials are actively going on in indian population. effective antivirals are available. although till now h n diagnostic centers worked with cdc/who recommended h n specific primer, probes with taqman chemistry by real time pcr, efforts on the development of indigenous diagnostics, vaccines and chemoprophylaxis is going on to have a better combat against this highly infectious virus. were positive for rotavirus infection by either page or elisa methods. the available data highlights the importance of rotavirus as a cause of diarrhea in children, which is severe enough to deserve specialized care. the observed proportion of . % of all diarrhea cases being associated with rotavirus falls within the range of values reported by other workers. the reported positivity varies from . to . %. in our study a complete concordance of elisa and page results were observed in ( %) of the tested specimens. this finding closely correlates with the findings of other authors who found a . - . % concordance results between elisa and page methods. some authors found rna-page method that is as sensitive and rapid as elisa for detecting rotavirus in stool samples of cases of diarrhea and some others proposed elisa is more sensitive than page method fond to be % specific. the remaining ( %) samples showed conflicting results. in a lone sample in which the od value of elisa test was . , this value was almost at the cutoff level, the possibility of this sample being positive by elisa test is doubtful. negative result of the same sample in page method is difficult to explain, the possibility of presence of lot of empty virus particles or due to low concentration of viral rna in the fecal specimen and insufficient extraction of viral rna could be possible. on the other hand, of the samples which gave positive results by page method were negative by elisa test. these samples had a typical - - - rna pattern. the reason for their being elisa negative thus remains unexplained, however blocking factors or the presence of inhibitory substance in stools might have been responsible. the samples containing predominantly complete particles can also give false negative results. since, the group antigen is not exposed. earlier studies have also reported page to be the most sensitive technique although some are of view that it is laborious procedure. how ever, the page system used in this study was very simple to perform and the results were available on the same day. the main requirement was of trained personnel and proper standardization of the technique. most reports states that the greatest advantage of page and silver stain method are its lack of ambiguity and the fact that it provides information about viral electropherotypes. the modified page system was thus found to be reliable, simple and rapid, no expensive reagents were required. locally available reagents from hi media were used. the cost of the chemical for page per specimen was rs. approximately as compared to rs. per test by confirmatory elisa. a locally produced slab gel electrophoresis system with power pack was the only equipment required. this method could be used for the routine diagnosis of rotavirus infection in the laboratory. vaccine, rapid diagnosis plays an important role in early management of patients. in this study a qc-rt-pcr assay was developed to quantify chikungunya virus rna by targeting the conserved region of e gene. a competitor molecule containing an internal insertion was generated, that provided a stringent control of the quantification process. the introduction of -fold serially diluted competitor in each reaction was further used to determine sensitivity. the applicability of this assay for quantification of chikungunya virus rna was evaluated with human clinical samples and the results were compared with real-time quantitative rt-pcr. the sensitivity of this assay was estimated to be rna copies per reaction with a dynamic detection range of to copies. specificity was confirmed using closely related alpha and flaviviruses. the comparison of qc-rt-pcr result with real-time rt-pcr revealed % concordance. these findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of chikungunya virus in acute-phase serum samples. in india, measles vaccine was introduced as part of expanded programme of immunization in . measles, mumps and rubella (mmr) vaccine is still not part of the national immunization schedule of india. the indian association of paediatrics (iap) recommends measles vaccine at months of age and mmr vaccine at - months. however, in a recent policy update, iap committee on immunisation opined that there is a need for a second dose of mmr vaccine for providing adequate immunity against mmr. the aim of the present study was to assess the extent of sero-protection against mmr at - years of age in children who have received one dose of mmr between and months of age. an attempt has also been made to assess the sero-response to the second dose of mmr vaccine in - years old children. a total of consecutive children between the ages of - years who had received mmr vaccine between and months of age and attending the immunization clinic of gtb hospital, delhi were enrolled. the vaccination status, anthropometry and physical examination findings were recorded. three ml of venous sample was again withdrawn for estimation of post vaccination antibody titre. it was observed that . %, . % and . % children were seroprotected for mmr respectively after . - . year of receiving first dose of mmr vaccine. seroprotection rose to . %, % and % for mmr respectively after - weeks of receiving second dose of mmr vaccine. geometric mean concentration of antibody also rose significantly in all three diseases. in view of low seroprevalence of mmr and hence high susceptibility to infection at - years of age, who have already received mmr vaccine, there is need to boost the immune responses against these three diseases by giving a second dose of mmr vaccine. baseline information on the epidemiology of viral agents causing stis and types of risk behaviour of affected persons are essential for any meaningful targeted intervention. the present study documents the pattern of viral stis in patients attending a tertiary care hospital, correlating the syndromic approach and the laboratory investigations to determine the aetiology. three hundred consecutive patients attending the sti clinic were diagnosed and categorized according to the syndromic approach of the who along with detailed history and demographic data. majority of the patients were men ( . %) with a mean age of years. men received education up to middle school. half of the female subjects were illiterate. sixty percent of the patients were married and among these, % were regular condom users. first sexual contact at or before years of age was more in men ( % vs. . % in women). promiscuity was more among male patients who had contact with csw. genital herpes was the commonest viral sti ( / ) followed by genital wart ( / ). concomitant infection with more than one virus was seen in % of patients. hiv was prevalent in . % of sti patients. hepatitis b, hepatitis c, herpes simplex type and molluscum contagiosum were the other viral agents seen in sti clinic attendees at our centre. this disease currently prevalent in more than countries world wide and annually - million people are infected with dengue virus among which . - lakhs cases were dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) which are serious forms of dengue virus infection and due to this condition , deaths might occur annually world wide and approximately million children were hospitalized for the fast decades. this disease is characterized by sudden onset of high fever with sever headache, pain in the back and limbs, lymphadenopathy macuolo-papulur rash over the skin and retro-bulbar pain. early diagnosis can be established with simple and rapid lgg/ gm antibodies detection in the blood samples of the patients based on the bi-directional immunoassay system for its management and control to reduce morbidity and mortality. details will be presented. myocarditis and dilated cardiomyopathy (dcm) are common causes of morbidity and mortality both in children and adults. the most common viruses involved in myocarditis are coxsackievirus b or adenovirus. recently, the coxsackievirus and adenovirus receptor (car), a common receptor for coxsackieviruses b , b and adenoviruses , has been identified. increased expression of car has been reported in patients with dcm suggesting utilization of car by these viruses for cell entry. the present study was designed to study the expression of car in myocardial tissue of patients with dcm. formalin fixed myocardial tissues were obtained from autopsy cases. a total of cases of dcm and cases of controls which included non-cardiac (group-a) and cardiac disease other than dcm (group-b) were included in the study. expression of car was studied by immunohistochemical staining of myocardial tissue using car specific rabbit polyclonal antibody and biotin conjugated secondary antibody. the tissue sections were considered positive when[ % of the cell showed brown color staining by immunohistochemistry (ihc). the car positivity in dcm cases was found to be % ( / ) as compared to % in control group a and % in control group b respectively. the car positivity was significantly higher in the test group as compared to both the control groups. further car positivity in all the cellular types (myocytes, endothelial cells and interstitial cells) was found significantly higher in test group as compared to both the control groups. the expression of car was significantly higher in myocytes as compared to both endothelial and interstitial cells in all the groups. however, no significant difference was observed in car positivity between endothelial and interstitial cells. the present study highlights the increased expression of car in dcm cases with further significance of car expression in myocytes and endothelial cells. this may help further in understanding the tropism of viruses or cellular susceptibility, which in turn will help in appropriate diagnostic and therapeutic approach in management of viral myocarditis and dcm cases. food security and safety vary widely around the world, and reaching these goals is one of the major challenges, raising public concern for the wellbeing of mankind, in particular. industrialized production and processing as well as improper environmental protection have clearly shown severe limitations such as worldwide contamination of the food chain and water. contaminated water and food during the processes of production, processing and handling are essentially responsible for food and water borne viral infections/diseases. the cases of viral food borne outbreaks are on the rise, creating a threat to human health. recent researches indicate that epidemiological studies are meager to focus the frequently contaminated foods and food borne viral diseases. current paper projects the etiology of select food borne viral diseases, probable reasons for non availability of appropriate methods to detect the viruses responsible for the diseases, routes of water and food borne transmission of enteric viral infections, currently available methods of detection of select viruses and bio safety measures to prevent food borne viral infections. dietary/nutritional management in food borne viral diseases is crucial to control weakness and gastro enteric intolerance due to disease condition and antibiotic therapy. it will principally improve food intake, resulting in better nutritional status leading to optimum immune response. food borne viruses are mainly belong to rotaviruses, enteropathogenic viruses, astroviruses, adenoviruses and caliciviruses, causes acute gastroenteritis (ag) which is an important health problem. the frequency of rotavirus as a cause of sporadic cases of ag ranges between . % and . %. astroviruses cause ag, with a frequency ranging between and %: outbreaks have been described in schools and kindergartens, but also in adults and the elderly. the frequency of identification of adenoviruses and as causes of sporadic ag in non-immuno suppressed children ranges between . % and . %, although there is probably underreporting because the sensitivity of conventional techniques is low. caliciviruses are separated phylogenetically into two genera: norovirus and sapovirus. norovirus is frequently associated with food-and water-borne outbreaks of ag. it is estimated that % of cases of ag due to norovirus are food borne. in sweden and some regions of the united states, norovirus is the first cause of outbreaks of food borne diseases. sapovirus outbreaks due to person-to-person and food borne transmission affecting both children and adults have recently been reported in countries such as canada and japan. it has been predicted that the importance of diarrhoeal disease, mainly due to contaminated food and water, as a cause of death will decline worldwide. evidence for such a downward trend is limited. this prediction presumes that improvements in the production and retail of microbiologically safe food will be sustained in the developed world and, moreover, will be rolled out to those countries of the developing world increasingly producing food for a global market. sustaining food safety standards will depend on constant vigilance maintained by monitoring and surveillance but, with the rising importance of other food-related issues, such as food security, obesity and climate change, competition for resources in the future to enable this may be fierce. in addition the pathogen populations relevant to food safety are not static. food is an excellent vehicle by which many pathogens (bacteria, viruses/prions and parasites) can reach an appropriate colonization site in a new host. although food production practices change, the well-recognized food-borne pathogens, such as salmonella spp. and escherichia coli, seem able to evolve to exploit novel opportunities, for example fresh produce and even generate new public health challenges, for example antimicrobial resistance. in addition, previously unknown food-borne pathogens, many of which are zoonotic, are constantly emerging. awareness and surveillance of viral food-borne pathogens is generally poor but emphasis is placed on norovirus, hepatitis a, rotaviruses and newly emerging viruses such as sars. it is clear that one overall challenge is the generation and maintenance of constructive dialogue and collaboration between public health, veterinary and food safety experts, bringing together multidisciplinary skills and multi-pathogen expertise. such collaboration is essential to monitor changing trends in the well-recognized diseases and detect emerging pathogens. it is also necessary to understand the multiple interactions between these pathogens and their environments during transmission along the food chain in order to develop effective prevention and control strategies. to analyse the effectiveness of these sirnas targeting rabies virus l gene, the bhk- cells expressing sirnas in shrna form were produced by transduction of cells with radv-l. the transduced bhk- cells expressing sirna were infected with rabies virus pv- strain. there was reduction in rabies virus multiplication as analysed by reduction in fluorescent foci forming unit (ffu) count by . % ( ffu in bhk- cells expressing sirna-l compared to ffu in bhk- cells expressing negative sirna). the expression of l gene mrna was reduced by . fold in rabies virus infected radv-l transduced cells compared to radv-neg transduced cells (negative control) as detected using real-time pcr. after analyzing the effectiveness of radv-l in vitro, its effectiveness was also evaluated in vivo in mice after virulent rabies challenge. the mice were inoculated with plaque forming units (pfu) of radv-l in masseter muscle (i/m route) and challenged with ld rabies virus challenge virus standard (cvs) strain. the results indicated % protection with improved median survival from to days compared with group of mice treated with radv-neg. the results of this study indicated that sirnas targeting rabies virus polymerase (l) gene delivered through adenoviral vector inhibited rabies virus multiplication in vitro and in vivo. and were successfully produced and purified from the infected spodoptera frugiperda (sf- ) cells using these recombinant baculovirus. the morphology of the vlps was validated by electron microscopy in comparison to the authentic bt virions. the vlps produced here were stable and were highly immunogenic with intact outer layer which is rapidly lost during normal infection of btv. these btv-vlps elicited long lasting protective immunity in vaccinated sheep against virulent virus challenge. with the use of btv-vlps it was also possible to differentiate the infected and vaccinated animals (diva). vlp-based btv vaccine has potential advantages with regard to controlling the spread of btv with multiple serotypes. it is possible to produce milligram quantities of correctly folded and processed protein complexes using this baculovirus expression system and hence it is a more promising system for producing new generation vaccines like vlp subunit vaccine against any viral diseases in large scale. peste des petits ruminants (ppr), goatpox and orf are oie notifiable diseases of small ruminants especially goat and sheep. these diseases are economically important, in enzootic countries like india and cause significant loss and are major constraints in the productivity. considering the geographical distribution of ppr, goat pox and orf infections and prevalence of mixed infection, in the present study, safety and potency of the experimental triple vaccine comprising attenuated strains of thermostable-ppr virus (pprv jhansi, p- ) grown at °c, high passaged goat poxvirus (gtpv uttarkashi, p ) and attenuated orf virus (orfv mukteswar, p ) was evaluated in sub-himalayan local hill goats. goats simultaneously immunized with ml of vaccine consisting of either tcid or tcid of each of pprv, gtpv and orfv were monitored for clinical and serological responses for a period of - weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to pprv, gtpv and orfv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, snt, respectively following immunization. all the immunized animals resisted infections when challenged with virulent strains of either gtpv or pprv or orfv on day dpi, while in contact control animals developed characteristic signs of respective disease. further, ppr viral antigen could be detected by using ppr sandwich elisa kit in the excretions (nasal, ocular and oral swab materials) of unvaccinated control animals after challenge but not from any of the immunized goats. triple vaccine was found safe at dose as higher as tcid and induced protective immune response even at lower dose ( tcid ) in goats, which was evident from sero-conversion as well as challenge studies. the study indicated that these viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this triple vaccine in combating these infections simultaneously. toll like receptors (tlrs), primary sensors of microbial origin, plays a crucial role in the innate immunity. till now mammalian tlrs have been identified, while there is no information available on tlrs of yak. this study is part of world bank funded-icar project. yak, named bos grunniens for its distinctive vocalization and relationship with cattle, is natural habitant of extremely cold environment. when these animals comes to a lower altitude grazing land, adjacent to villages, become susceptible to the diseases of cattle, buffalo etc. thus, present study was undertaken to with genetic characterization and evolutionary lineage analysis of yak tlrs. we worked on tlr gene, which plays an important role in recognition of ssrna viruses. total rna was extracted from mitogen stimulated pbmcs of yak. the rt-pcr conditions were standardized for full length amplification of tlr gene using specific self designed primers. the expected amplicon of bps was obtained. it was cloned in pgemt-easy vector followed by transformation in e. coli top strain. the recombinant clones were screened, picked up for plasmid isolation and release of tlr was confirmed by restriction digestion. the cloned tlr product was sequenced and analyzed for the nucleotide and deduced amino acid sequences, and d structure analysis. the results revealed that yak shows more than % sequence homology with other bos indicus breeds and bos taurus breeds. however, identity was less than % with other animal species (equine, murine, feline, canine etc.). the evolutionary lineage findings cluster yak more closely with bovine species. point mutations revealed changes at nucleotide positions with corresponding amino acid change at positions. smart analysis of yak protein domain architecture revealed toll-interleukin i receptor (tir), leucine rich repeats (lrr) and signal peptide region. the variations in yak mainly lie in the lrr region. homology modeling revealed horse shoe shaped structure with alpha helix. the additional alpha helix present in bos indicus was not detected in yak. the present study shows existence of genetic variability in tlr gene of yak, in particular the lrr region, which plays an important role in the pathogen recognition and the evolutionary lineage analyses shows its closeness with other bovine species. a.p. aquaculture and fisheries, tirupati in this new millennium, aquatic animal health management strategies in asia expanded and adjusted to the current disease problems faced by the aquaculture sector. this presentation will briefly discuss some of the most serious trans-boundary pathogens affecting asian aquaculture including a newly emerging disease and highlight recent regional and national efforts on responsible health management for mitigating the risks associated with aquatic animal movement. a regional approach is fundamental since many countries share common social, economic, industrial, environmental, biological and geographical characteristics. capacity and awareness building on aquatic animal epidemiology, science-based risk analysis for aquatic animal transfers, surveillance and disease reporting, disease zoning and establishment of aquatic animal health information systems to support development of national disease control programs and emergency response to disease outbreaks are needed. molecular diagnostics with emphasis towards standardization and harmonization, inter-calibration exercises and quality assurance in laboratories, accreditation program and utilization of regional resource centres on aquatic animal health will also be needed. whilst most of these strategies are directed in support of government policies, implementation will require pro-active involvement, effective cooperation and strategic networking between governments, farmers, researchers, scientists, development and aid agencies, and relevant private sector stakeholders at all levels. their contributions are essential to the health management process. generally, aquaculture plays an important role in economy as harvests from natural waters have declined or, at best, remained static in most countries. fish and shrimp, the main aquaculture product sources, have gained the most attention. many factors can cause losses in yields of fish products and infectious disease in fish and shrimp is the biggest threat to the fishery industry. shrimp and fish aquaculture has grown rapidly over several decades to become a major global industry that serves the increasing consumer demand for seafood and has contributed significantly to socio-economic development in many poor coastal communities. however, the ecological disturbances and changes in patterns of trade associated with the development of shrimp and fish farming have presented many of the pre-conditions for the emergence and spread of disease. shrimp and fish are displaced from their natural environments, provided artificial or alternative feeds, stocked in high density, exposed to stress through changes in water quality and are transported nationally and internationally, either live or as frozen product. these practices have provided opportunities for increased pathogenicity of existing infections, exposure to new pathogens, and the rapid transmission and trans boundary spread of disease. not surprisingly, a succession of new viral diseases has devastated the production and livelihoods of farmers and their sustaining communities. this review examines the major viral pathogens of farmed shrimp and fish, the likely reasons for their emergence and spread, and the consequences for the structure and operation of the shrimp farming industry. in addition, this review discusses the health management strategies that have been introduced to combat the major pathogens and the reasons that disease continues to have an impact, particularly on poor, smallholder farmers in asia. btv isolates from the same geographic region have been termed as 'topotypes' and initial observation on segment nucleotide sequences identified a correlation between topotypes and genetic information. later topotyping was proposed based on segment , on the premise that the encoding protein ns , which is involved in virus egress from insect cells, would lead to evolutionary fitness in parallel with the geographic distribution of the different culicoides species. further studies attempted to extend this to nucleotide sequence homology in segments and , but failed to identify clear cut correlations or any evidence for positive selection. for example, south african isolates were found not to cluster into separate african lineage. in this study, we carried out a more extensive analysis of segment sequences. our analysis showed no segregation of isolates into topographically distinct groups. instead we observed topological clustering of the clades, and we attribute this to genetic bottleneck resulting in genetic drift and founder effect leading to homogenous gene pool in a geographical area. we hypothesize that when a new virus enters a geographical area where local btv strains are already circulating, the new genes/segments would enter into a bigger gene pool. consequently, the newer incursions into a heavily endemic area tend to get diluted and disappear from the population because the rate of drift is inversely proportional to the population size, unless they are positively selected. use of live attenuated vaccine in israel, europe, south africa and usa also led to more homogenous population similar to the vaccine strains due to continuous infusion of the vaccine type genes into the gene pool. we conclude that restriction of specific strains to certain geographical areas could generate uniquely imprinted genotypes which would not only indicate origin but also predict movement of viral strains to new areas. vvo- viral diseases of zoonotic importance: indian context k. prabhudas pd-admas, ivri, campus, hebbal, bangalore zoonoses are generally defined as animal diseases that are transmissible to humans. they continue to represent an important health hazard in most parts of the world, where they cause considerable expenditure and losses for the health and agricultural sectors. the emergence of these zoonotic diseases are very distinct, hence their prevention and control will require unique strategies, apart from traditional approaches. such strategies require rebuilding a cadre of trained professionals of several medical and biologic sciences. the article discusses virus infections that have significant zoonotic implications for india. buffalopox is a contagious viral disease affecting milch buffaloes and rarely, cows, with a morbidity rate up to % in the affected herd. although the disease is not responsible for high mortality, it adversely affects the productivity of the animals, resulting in large economic losses. furthermore, the disease has zoonotic implications, as outbreaks are frequently associated with human infections, particularly in the milkers. the causative agent, buffalopox virus (bpxv), is closely related to vaccinia virus. the outbreaks of febrile rash illness among humans and buffaloes were investigated in the villages of districts solapur and kolhapur of western maharashtra. clinico-epidemiological investigations of humans and buffaloes were carried out and representative clinical samples were collected respectively. the samples include vesicular fluid, scab, and blood. laboratory investigations for buffalo-pox virus (bpxv) was done by pcr on blood samples, scabs and vesicular fluid. in vitro virus isolation attempts were carried out by using vero e- cells. negative staining electron microscopy was also employed for detection of virus particles. a total of human cases with pox lesions on hand and other body parts from village kasegaon, district-solapur and cases from different villages of kolhapur district were reported. besides pox lesions patients were having fever, malaise, pain at site of lesion and axillary and inguinal lymphadenopathy. in kasegaon village, attack rate in human cases was . % and in buffaloes . % ( / ). whereas in kolhapur area attack rate in buffaloes was . % ( / ). bpxv was confirmed in blood, vesicular fluid and scab specimens from human cases and scab specimen from buffalo by polymerase chain reaction (pcr) method. the bpxv was also isolated from different clinical specimens and further identified by pcr and electron microscopy. clinical manifestation of the disease in buffaloes from solapur district was as reported earlier like common pox lesions on teats and udders whereas the buffaloes from kolhapur district had lesions on hairless parts of ears and on the eyelids with purulent discharge. bpxv from human and buffalo cases showed similarity. vaccines have been made against several diseases and used for controlling the afflictions. however a few of them were not effective for successfully controlling the disease. the reasons for the failure are many, the major being, either the pathogen is not completely cleared from the vaccinated animal or it reemerges after changing its antigenic structure, thus making the vaccination programme less effective. in addition to this, emergences of newer diseases such as hiv the development of suitable vaccines have become a challenging task. this is especially true in the case of viral diseases. these challenges have warned the researchers ''that protection by vaccination is not that simple and strait forward approach'', and lot need to be understood in terms of host virus interaction and role of environment in perpetuating the disease. so the immediate step that was considered was the environmental safety by way using non infectious materials as vaccines. with the understanding that has been developed in molecular immunology and molecular biology and with the availability of molecular tools that have been developed through recombinant dna technology the field of vaccinology has changed dramatically to emerge as modern vaccinology. this presentation deals with the modern approaches that are being used to produce effective vaccines in the case of foot and mouth disease of cloven footed animals. the similar approach may be worked out for other viral diseases also. despite the availability of an inactivated vaccine that is noted to provide solid immunity against the disease over a short period of time, the search for an ideal vaccine, the criteria for which are; safety of the vaccine for environment, easy in its preparation, does not require a cold chain for its storage, provides longer lasting immunity, economically viable and may be able to clear the virus in case of persistent infection is on. the advent of recombinant dna technology together with the information available on the molecular biology of viruses has enabled to design the development of newer vaccines that can induce strong cellular and humoral responses. the underlying principal in the present vaccine development strategy world over is the virus antigen gene has to be expressed in the tissue and the vaccine backbone has to trigger the immune system for eliciting desired immune response. bangalore campus of ivri has been vigorously pursuing research to develop ideal vaccines for foot and mouth disease keeping above principal in mind to achieve the previously mentioned criteria. the approaches selected are to see that the virus antigen/s replicate transiently in the host. the self replicating vaccines that have been developed are pox virus vectored vaccines, alpha virus replicase based vaccines and fmdv vectored vaccines. the approach and the result obtained so far will be discussed. silkworm, bombyx mori is affected with various diseases caused by viruses viz., nuclearpolyhedrosis (bmnpv), densosnucleosis (bmdnv) and infectious flacherie (bmifv). silkworm viral diseases form major constraints for the silk cocoon production in all the sericultural countries. the losses due to silkworm diseases is estimated about - % and among them viral diseases are most common. in sericulture, prophylactic measures play a vital role in the management of silkworm diseases. these include disinfection of silkworm rearing house and appliances, rearing area, rearing surroundings, silkworm egg and body, and rearing bed disinfection associated with maintenance of general hygiene and personnel hygiene. all these activities are generally carried out as rituals by using general disinfectants often with partial success. recent trends in complete management of silkworm diseases include development of silkworm hybrids evolved from disease resistant/tolerant breeds, effective eco-and user-friendly disinfectants, anti-microbial feed-supplements and use of transgenic silkworms. biotechnological breakthrough in this regard is through rna interference (rnai) approach involving dsrna mediated nuclear polyhedrosis management and this is presently pursued by apssrdi, hindupur in collaboration with centre for dna fingerprinting and diagnostics (cdfd), hyderabad. nadu and karnataka. the disease appears to be more severe in rural flocks than organized farms. our investigations revealed the morbidity, mortality and case fatality rates among rural and organised farms as . %, . %, . % and . %, . %, . % respectively. higher morbidity and mortality in rural areas may be due to stress factors like poor nutrition, parasitic burden, fatigue due to long walks and non availability of veterinary aid. kulkarni et al. also reported the severe bt outbreaks in rural areas of maharashtra with overall morbidity, mortality and case fatality of %, % and % respectively. all the south indian sheep breeds were found to be susceptible and clinical farm of the disease is evident in all of them though saravanabava ( ) reported variations in susceptibility among the indigenous sheep. trichy black and ramnad white sheep were found to be more susceptible than the vambur and mecheri sheep of tamil nadu. prevalence of bluetongue in sheep, goat and cattle appears to be high in the region. serological surveys conducted in andhra pradesh during revealed the prevalence of btv antibodies in sheep ( . %) goats ( . %) cattle ( %) and buffaloe ( %). similar high prevalence of btv antibodies in sheep and goats were also reported from the other states in the region. clinical disease has not been recorded in kerala though btv antibodies were recorded in sheep ( . %) and goats ( . %) (ravi sankar ) . culicoides are the known biological vectors of btv. all the culicoides species are not capable of transmitting the btv. the occurrence of the disease is related to the presence of the competent vectors in the area. jain et al. ( ) established the involvement of the culicoides in transmitting the btv by isolating the virus from culicoides at haryana, the north indian state. c. imicola and c. oxystoma were found to be prevalent in andhra pradesh and tamil nadu. narladakar et al.( ) reported the presence of c. schultzei, c. perigrinus and c. octoni in marathwada region of maharastra. culicoid vectors are significantly affected by the climate and annual variations in the climate reflects the outcome of the disease. the monsoon season (june to dec) with the temperature ranging from . to . °c appears to be favourable period for the multiplication of culicoides. the maximum no of outbreaks were recorded during the north east monsoon period (oct-dec) followed by south west monsoon period (june to sep) in the region. however, details on the distribution of the competent vectors, feeding habits and their dynamics in the region is lacking multiple btv serotypes were found to be circulating in the region. (kulkarni and kulkarni ; janakiraman etal. ; mehrotra et al. ) a total of serotypes viz. - , , , , , and were identified based on the virus isolations. sreenivasulu et al. isolated btv serotype from an outbreak of bt in native sheep of andhra pradesh. btv serotype , and were also isolated from the outbreaks occurred in andhra pradesh. some of the isolates need to be serotyped. deshmukh and gujar ( ) isolated btv type from maharashtra. following is the summary of the distribution of btv serotypes in this region. clinical picture of bt in native sheep appears to be slightly different, the major difference being that swelling of lips and face was less conspicuous. mucocutaneous borders appeared to be very sensitive to touch and bleed easily upon handling. the classical signs of cyanosis of tongue and reddening of coronary band are not the common features of the disease in native sheep. the disease was also confirmed by the virus isolation and identification. clinical disease has not been reported in cattle, buffaloes and goats in spite of high seroprevalence. in conclusion bt is established in native sheep and causes severe economic losses to the farmers. the disease is concentrated in the southern peninsula of the country. the disease is seasonal and is associated with the rain fall. multiple serotypes appear to be circulating in this region. the btv serotypes were of virulent in nature as evident by severe outbreaks. s. janardana reddy*, d. c. reddy department of fishery science and aquaculture, sri venkateswara university, tirupati in less than three decades, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of u.s. dollars in revenue, and augmentation of the world's food supply with a high value crop. concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. in india viral diseases have become an important limiting factor for growth of shrimp aquaculture industry. although more than different viral pathogens have been identified in different species of shrimp world wide, only a few viruses have identified which are causing disease problems in cultured tiger shrimps in india, east coast of andhra pradesh, in particular. diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, and transmission electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. while tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. the need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. the industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like infectious hypodermal and hematopoietic necrosis (ihhnv), hepatopancreatic virus (hpv), taura syndrome virus (tsv), white spot syndrome virus (wssv), monodon baculo virus (mbv), and bp. highly sensitive detection methods for some pathogens that employ dna amplification methods based on the polymerase chain reaction (pcr) now exist, and more pcr methods are being developed for additional agents. these advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease. within this field, shrimp immunology is a key element in establishing strategies for the control of diseases in shrimp aquaculture. research needs to be directed towards the development of assays to evaluate and monitor the immune state of shrimp. the establishment of regular immune checkups will permit the detection of shrimp immunodeficiencies but also to help monitor and improve environment quality. for this, immune effectors must be first identified and characterised. in the end, however, the assumption may be made that the sustainability of aquaculture will depend on the selection of disease-resistant shrimp, i.e. to develop research in immunology and genetics at the same time. the development of strategies for prophylaxis and control of shrimp diseases could be aided by the establishment of a collaborative network to contribute to progress in basic knowledge of penaeid immunity. however, to improve efficiency, it appears essential also to open this network to complementary research areas related to shrimp pathology, physiology, genetics and environment. bluetongue is an important viral disease of sheep causing severe economic losses to the farmers. lack of effective vaccine is the major impediments in controlling the disease. multiple serotypes were found to be circulating in the state. attempts are being made to develop the vaccine employing the available serotypes to control the disease. hence, it is essential to identify the antigenic relationship among the serotypes to identify the candidate vaccine strains to be incorporated in the preparation of vaccine. reciprocal cross neutralization test was employed to find out the r% values between btv- , - and - which indicated the extent of antigenic relationship between the serotypes. r% value between btv- and btv- was recorded as . r% value of . and . were observed between btv- and - and btv- and - respectively. the r% values recorded in the present study revealed a weak antigenic relationship between the btv serotypes. the extent of antigenic relationship between the btv serotypes was also determined by multiple sequence alignment of the nucleotide and amino acid sequences of the reference btv serotypes , and . the sequence analysis of the vp gene revealed a homology of - % and - % at the nucleotide and amino acid levels respectively. r% values obtained using reciprocal cross neutralization test with the btv- , and serotypes isolated in native sheep of andhra pradesh and the genomic analysis of the reference serotypes of btv- , and revealed very weak antigenic relationship and were highly divergent. diseases especially those by viral pathogens cause greater economic losses in most horticultural crop species throughout the world as compared to agricultural crops. non-genetic methods of management of these diseases include quarantine measures, eradication of infected plants and weed hosts, crop rotation, use of certified virus-free seed or planting stock and use of pesticides to control insect vector populations implicated in transmission of viruses. however, none of these measures is likely to provide an enduring solution against these diseases especially those caused by viruses due sometimes to the huge expenditure involved, but mostly to the questionable effectiveness and reliability of those methods. as key control pesticides are getting increasingly abandoned, development of alternative methods to control diseases has been a felt-need in the recent past. though breeding for disease resistance generally provides a reliable security in a long run, introgression of host plant resistance did not materialise in most important crops. non-availability of an appropriate source of resistance in inter-fertile relatives, linkage to undesirable traits, or often times polygenic nature of such sources of resistance are the stumbling blocks in breeding programs. the limitations of conventional breeding and routine cultural practices prompted the need for the development of other approaches of virus control that could be fully incorporated into traditional methods. in this perspective, the concept of pathogen-derived resistance offers an attractive strategy to evolve newer methods of virus management, by transforming crop plants with nucleotide sequences derived from the pathogen's genome. an increasing number of molecular characterisation of plant virus genomes and the stable transformation of a number of horticultural crop species have in fact opened an avenue for molecular breeding against virus pathogens. successful field-testing of genetically modified crop cultivars renders proof of their supremacy over existing cultivars. it also contributes to demonstrate their capability with regard to environmental safety with a view to winning over public concern and scepticism. in general, the eventual commercialisation transgenic lines expressing virus resistance will rely upon a host of factors including their field performance, genetic stability, public acceptance and the resolution of environmental concerns and patent related issues. as such, elaborate field trials and allied studies are now required to adapt genetically engineered horticultural crops expressing virus resistance for their implementation into practical agriculture. a few examples from current research at tnau, in india or elsewhere will be discussed in this presentation. virology unit, division of plant pathology, iari, new delhi in recent times there has been greater emphasis on vegetatively propagated crops in india to help diversify the indian agriculture. fruit, flower, spice and plantation crops are important vegetatively propagated horticultural crops, which have become a driving force for economic development in several parts of india. however, most of the vegetatively propagated crops are threatened by biotic stress caused by plant pathogens in general and plant viruses in particular. plant viruses produce specific and non specific symptoms and in some cases no symptoms are produced. correct identification and diagnosis of viral diseases is first step in the management of any disease including viral diseases. there have been two major breakthroughs in virus diagnostics during last four decades. the first one was serological assay using monoclonal or polyclonal antibodies in enzyme linked immunosorbent assay (elisa) and the other one was the use of in vitro amplification of dna in polymerase chain reaction (pcr). a significant development in serological assays has been its simplification in form of user's friendly quick strip/dip stick method. the one-step lateral-flow (lf) tests have been developed for the on-site detection and identification of several plant viruses. rapid advancement in virus genome characterization has led to the development of novel approaches of nucleic acid based diagnostics which include conventional pcr, real time pcr, multiplex pcr, micro/macro arrays and biochips. pcr protocols already exist for many plant viruses of citrus, banana, apple, papaya, vegetables, ornamental and spice crops. a further advancement has led to development of realtime pcr assay which is relatively easy but requires training for diagnosticians. in real-time pcr assays, results can be available within min. the nucleic acid template preparation in pcr has been simplified. membrane based dna template protocol and co-isolation of nucleic acid template preparation are novel approaches in pcr detection of virus and virus like pathogens. since many of the horticultural crops are often infected by more than one virus, their individual detection by pcr is not only expensive but also time consuming. therefore, multiplex pcr has been developed where in genome of more than one virus could be amplified and detected in the same reaction mixture. development of nucleic acid based chip is now one of the fastest and recent growing areas in the field of pathogen detection. these nucleic acid based chips have been named as dna/rna chips, biochips, genechips, biosensors or dna arrays. when it comes to applications of microarray technology for plant viruses, it is not too difficult to see the value of a method that could potentially detect a whole range of viruses using a single test. however, microarrays are unlikely to become the only method in use in a diagnostic laboratory. processing of germplasm including transgenic planting material imported for research purposes into the country. during the last two decades, a total of , samples of wheat including transgenics were imported from cimmyt (mexico), icarda (syria) and many other countries. these were grown in post-entry quarantine nursery each year at nbpgr, new delhi and the transgenic samples were grown in national containment facility of level- (cl- ) since its inception to ensure that no viable biological material/pollen/pathogen enters or leaves the facility during quarantine processing of transgenics. in addition, post-entry quarantine inspections of the transgenic wheat grown by indenters are also undertaken by nbpgr quarantine scientists. virus-induced gene silencing (vigs) is a technique in which viral genomes are used, usually after appropriate modifications, for transient gene silencing in plants. the mechanism behind vigs is the phenomenon called rna-interference (rnai), which is widespread in many organisms and is believed to be form of inherent defence system against intracellular pathogens, such as viruses and transposons. double-stranded rna or rna containing strong secondary structures, commonly produced during viral infections, are believed to cause triggering of rnai, which employs a battery of proteins and nucleoprotein complexes to identify and degrade specific viral transcripts. in vigs, viral genomes not causing severe symptoms, but which can accumulate and spread efficiently in the host plant are used as vectors in which a host gene is cloned and introduced into the plant. upon replication, the viral vector triggers rnai response in the host plant, which also targets the host gene, leading to its silencing and subsequently, the silenced phenotype revealing gene function in vivo. vigs has been used extensively to study gene functions in dicot plants, such as tobacco, tomato, pea, soybean, etc., using vectors derived from reference genes are commonly used as an/the endogenous normalisation measure for the relative quantification of target genes. the expression (characteristics) of seven potential reference genes was evaluated in tissues of healthy, physiologically stressed and barley yellow dwarf virus (bydv) infected cereal plants. these genes were tested by rt-qpcr and ranked according to the stability of their expression (characteristics) using three different methods (two-way anova, genorm and normfinder tools). in most cases, the expression (characteristics) of all genes did not depend on the abiotic stress conditions or on the virus infections. all the genes showed significant differences in expression (characteristics) among plant species. glyceraldehyde- -phosphate dehydrogenase (gapdh), beta-tubulin (tubb) and s ribosomal rna ( s rrna) always ranked as the three most stable genes. on the other hand, elongation factor- alpha (ef a), eukaryotic initiation factor a (eif a), and s ribosomal rna ( s rrna) for barley and oat samples; and beta-tubulin (tubb) for wheat samples were consistently ranked as the less reliable controls. the bydv titre was determined in two oat varieties by rt-qpcr by three different quantification approaches. statistically, there were no significant differences between the absolute and the relative quantification, or between quantification using gapdh + tubb + tuba + s rrna and ef a + eif a + s rrna. the geometric average of gapdh, s rrna, tuba and tubb is suitable for normalisation of bydv quantification in barley and oat tissues. for wheat samples, a combination of gapdh, s rrna, tubb, eif a and e fa is recommended. department of microbiology, yogi vemana university, vemanapuram, kadapa large scale production and import of propagative material poses potential risk of introducing several destructive pathogens particularly viruses and mycoplasma like organisms in our country. this demands adequate quarantine safe guards such as growing them under approved post entry quarantine facility for specific period so as to facilitate virus detection, thereby curtailing risk. when such facilities are coupled with propagation by tissue culture will ensure virus free propagative plant material. the requirement of nationwide network of post entry quarantine facility working in close collaboration with crop institutions are very much emphasized for considering import of high risk plant genera for agriculture development. present paper discusses about virus disease of quarantine importance affecting ornamental and fruit plants such as chrysanthimum, dahlia, dianthus, rosabengalensis, cattleya, cymbidium, dendrobium, lilium, citrus, vitis etc. the paper also discusses on immunodiagnostic methods of detection and methods of obtaining virus free propagative material. rice tungro occurs as epidemics in regular cycles and has been reported in the last years from all the major rice growing regions of india, especially prevalent in the southern and eastern states. development of the durable resistant varieties to tungro is crucial for the management of the disease. molecular breeding, involving the use of dna markers linked to the resistant gene(s) for selection, can overcome the difficulties encountered in conventional resistant breeding programs. for successful marker-assisted selection (mas), the identification of closely linked markers through the process of gene tagging and mapping is a prerequisite. attempts have been initiated for identification of tungro resistance genes through molecular mapping and their introgression into the target varieties using marker-assisted selection at drr, hyderabad. the inheritance of resistance to rice tungro virus disease was studied in seven resistant rice cultivars with field evaluation at hot spot locations. the microsatellite markers linked to rice tungro resistance in utri merah was studied and found that resistance genes were linked to rm on chromosome . through molecular mapping two qtl were identified controlling rtv resistance on chromosomes and in 'utri rajapan' explaining . % and . % of the phenotypic variance. in variety 'vikramarya', another two qtl for rtv resistance were detected on chromosomes and explaining . % and . % of the phenotypic variance. the closely linked markers identified in this study flanking the gene of interest through mapping will improve the efficiency and precision of introgression programs in marker assisted breeding for rtv resistance. functional characterization of these qtl for rtv resistance is under progress. there is only a limited pool of natural virus resistance in cassava against cassava mosaic geminiviruses and cassava brown streak ipomovirus hence the development of transgenic resistance in this significant crop might present an option. rna mediated resistance through the expression of inverted-repeat dsrna sequences derived from the virus genome and the modification of plant microrna to produce antiviral artificial microrna are strategies that have recently been proven very effective for induction of virus resistance (immunity) against a number of rna viruses. results from rna interference strategies against geminiviruses never resulted in immunity of transgenes. however, it suggest that viral mrna are targets of rna silencing and that the success of the strategy depends on the relevance of the target gene in the systemic spread of the virus. we have generated a number of rna silencing constructs to induce resistance against cbsv and the indian cassava mosaic viruses icmv and slcmv. due to the serious problems inherent with transformation of cassava and subsequent resistance screening, these constructs were tested for efficiency either by transient-or by transgenic expression in n. benthamiana. complete immunity was reached in transgenic n. benthamiana against cbsv using inverted repeat or amirna constructs. using different species of cbsv for resistance screening, immunity was broken, to show the minimum context for broad spectrum resistance. similarly, highly specific resistance was reached in expression of amirna. in contrast, virus resistance against icmv/ slcmv using single amirna constructs was not successful. results from the experiments to generate virus resistance against cbsv and icmv/slcmv will be shown; methods to evaluate efficiency of rnai gene constructs by transient gene expression in n. benthamiana and strategies to develop efficient resistance against rna and dna viruses in cassava will be discussed. bitter gourd (momordica charantia l.) which is also called bitter melon, balsam apple and balsam pear belongs to family cucurbitaceae. it is an important traditional vegetable of nutritive and medicinal value that is cultivated in tropical and sub-tropical asia, but is considered as a weed host reservoir for viruses in jamaica. viral disease-like symptoms were observed occurring naturally on the crops of bitter gourd grown in the fields of northern india during - . an incidence of . % of diseased plants was recorded which showed chlorotic spots and mosaic ranging from mild mottling to green blisters along with leaf smalling, leaf and fruit deformations, bud necrosis and stunted growth whereas . % plants exhibited leaf curling alone or in combination with mosaic-type disease. a reduction of . % in fruit yield was recorded in mosaic-like disease which could be attributed to lesser fruit setting due to bud necrosis, smaller fruit size and stunted plant growth. such plants produced deformed, notched, irregularly shaped fruits wherein pre-mature yellowing and necrosis on the anterior and posteriors ends made . % fruits unfit for marketability. the dwindling yield and production of unmarketable fruits posed a major constraint for profitable cultivation of this economically important crop, thus warranting for studies on etiology and management of these diseases. the mosaic-like disease was transmitted to healthy seedlings of bitter gourd at -leaves stage by sap inoculation as well as by aphid viz., myzus persicae sulz. and aphis gossypii glov. initially studies were carried out to optimize protocols for efficient plant regeneration and agrobacterium-mediated transformation for nagpur sweet orange, which is a popular and elite citrus cultivar in india. organogenesis was induced in etiolated epicotyl explants of one-month-old axenically raised polyembryonic seedlings by culturing them in mt medium supplemented with g/l sucrose with varying concentrations of plant hormones. it was found that bap at mg/l without auxin was best for efficient shoot regeneration in citrus using epicotyl explants. a % regeneration frequency was obtained and multiple shoot formation was obtained from both the cut ends of all the explants. an average of . well-differentiated shoots per explant were obtained, all of which rooted normally under the influence of mg/l iba. this improved regeneration protocol was utilized in standardizing agrobacterium-mediated transformation of citrus using a. tumefaciens strain eha , containing binary plasmid pcambia that harbors gus reporter gene and npt-ii plant selection marker gene. one-month-old epicotyl explants infected with over-night grown agrobacterium (a . - . ) for min and co-cultured for days were found to be optimum for transformation as assessed on the basis of pcr analysis and gus activity displayed by the stem and leaf sections of putative transgenics. overall transformation frequency ranged from to %. current study focuses on the generation of citrus transgenics for ctv resistance using a. tumefaciens strain eha containing binary plasmid pbinar harboring portion of coat protein gene of ctv and npt-ii gene employing the standardized protocols. several putative transgenic shoots were recovered on selection medium and they are being utilized for molecular analyses and resistance against ctv. work is also in progress on the generation of citrus transformants using rnai construct harboring ctv cp and p genes, singly and in conjunction. our lab was also involved in developing rice transgenics for resistance against rice tungro disease, which is one of the most important and widespread virus diseases of rice in south and southeast asia, causing an annual estimated loss in crop yield of economic losses worth millions of rupees are caused due to these diseases annually. virus diseases are frequently less conspicuous than those caused by other plant pathogens and last for much longer. this is especially true for perennial crops and those that are vegetatively propagated. one further problem with attending to assess losses due to various diseases on a global basis is that what most of the data are from small comparative trials rather than wide scale comprehensive surveys, even the small trials do not necessarily give data that can be used for more global estimates of losses. this is for several reasons, including: ( ) variation in losses by a particular crop from year to year; ( ) variation from region to region and climatic zone to climatic zone: ( ) differences in loss assessment methodologies; ( ) identification of the viral etiology of the disease; variation in the definition of the term 'losses' and ( ) chilli is the major vegetable and spice crop grown in thar desert areas of rajasthan. leaf curl disease (chlcd) is one of the major constrains in chilli cultivation faced by farmers and cause yield loss up to %. a survey was conducted in major chilli growing areas of thar desert; bikaner, nagur, jodhpur and jalore districts of rajasthan during november, to understand the present status of leaf curl disease in chilli. among the four district surveyed for chlcd, the disease incidence was recorded maximum (up to %) in jodhpur district followed by jolore district (up to %). no relation was found between the disease incidence and varieties. the major varieties grown in these area are; mehsana, rch (mandoria), haripur raipur, mathania and local cultivars. the number of whitefly was also counted in top, middle and bottom leaf of chilli grown in these areas. the average number of whitefly per plant ranged from . to . . more number of whitefly ( . ) was recorded in jodhpur district and lowest ( . ) in jalore district. total dna was extracted from three leaf curl infected samples from each district and tested for the presence of begomovirus using coat protein (cp) and dna-b specific primers. all the samples were positive for cp and dna-b amplifications by pcr. the cloning and sequencing of selected cp gene and dna-b fragments are in progress. the preliminary investigations shows that the leaf curl disease of chilli is widespread in the arid region of rajasthan and may be caused by begomovirus associated with satellite dna-b. bittergourd (momordica charantia) is an important vegetable crop of kerala. the crop is affected by several diseases of which mosaic is a prominent one. a field experiment was conducted to evaluate the efficacy of potentised resistance inducing substances (ris) viz., mosaic affected bittergourd plant tissue, ash of mosaic affected bittergourd plant tissue, plumbago and salicylic acid for control of bittergourd mosaic in march . ris were applied as drench and foliar spray at three potency levels twice, before flowering of the crop. the experimental crop was grown as per the package of practice recommendations in split plot design with five replications per treatment. the disease incidence, disease severity and yield of the crop were recorded. the result of the experiment shows that spraying was more effective than drenching of treatments for reducing mosaic incidence and severity. among treatments, infected plant extract at potency was the most effective one for reducing mosaic incidence and it showed the maximum incubation period and minimum disease severity. the spray application of treatments produced significantly higher yield than drenching. among the treatments, ash of infected plant at and potency and infected plant extract at potency were on par and produced comparatively higher yield. elephant foot yam (amoprhophallus paeoniifolius), colocasia (colocasia esculenta) and tannia (xanthosoma sagittifolium) are the major edible aroids cultivated in india. the elephant foot yam cultivation is gaining importance due to its high production potential, nutritional and medicinal values and good economic returns. all these aroids are vegetatively propagated and viral diseases are spreading through planting materials. ctcri has the mandate of producing healthy planting materials of these edible aroids. accurate diagnosis and identification of the virus is essential for production of healthy planting material and effective management of the disease. though occurrences of viral diseases on edible aroids in india were known in s, not much attention was given for detection and identification of the virus involved. in case of elephant foot yam - % mosaic incidence was observed with varying symptoms of mosaic, puckering, filiformy etc. in colocasia and tannia, - % incidence was noticed. rt-pcr amplification with potyvirus group specific primers and subsequent cloning and sequencing of the amplified product has confirmed the association of dasheen mosaic virus (dsmv) with all the three edible aroids cultivated in india. the complete full length coat protein gene of dsmv infecting elephant foot yam was cloned in pgem-t vector and sequenced. further sequence analysis revealed that the cp of dsmv consisted of nucleotides and the utr comprised of nucleotides. blast and phylogenetic analysis showed highest similarity of % with that of dsmv isolate af , reported from usa. the deduced amino acid sequence of cp had . - . % identity with other dsmv isolates. blast analysis of the partial cp gene sequences of colocasia and tannia also confirmed that the virus involved is dsmv. rt-pcr analysis of large number of samples from all the three crops confirmed that the potyvirus group specific primers (mj and mj ) are good for rapid detection of dsmv in these crops. dsmv specific biotinylated cdna and digoxigenin labelled crna probes were also prepared and dsmv in elephant foot yam was detected through nucleic acid spot hybridization. yellow leaf disease (yld) caused by sugarcane yellow leaf virus (scylv) is a recently recorded disease in india and is found wide spread throughout country. in popular varieties, the disease incidence varied from to . % and attained epidemic levels under field conditions. detailed studies on the impact of yld on sugarcane revealed that the virus infection significantly reduces various cane growth parameters, cane yield and juice quality. sequence comparisons of the coat protein (cp) and movement protein (mp) of scylv isolates from india and database sequences showed a significant variation between indian isolates and the database sequences both at nt and aa level in the cp/mp coding regions. the significant variation in our isolates with the database isolates, even in the least variable region of the scylv genome showed that the population existing in india is different from rest of the world. further, comparison of partial sequences encoding for orf and revealed that yld in sugarcane in india is caused at least by three genotypes viz., cub, ind and bra-per, of which a majority of the samples were found infected with cuban genotype (cub). the genotype ind was identified as a new genotype and this was found to have significant variation with the reported genotypes. we have identified specific primers from cp region of the virus and optimized rt-pcr conditions to diagnose the virus. this assay has been found efficient in detecting the virus in asymptomatic plants and tissue culture derived seedlings. elimination of the virus through meristem culture has been demonstrated to purify the virus from the infected planting materials and this technique needs to be adopted to supply disease-free planting materials for effective management of the disease. studies are also in progress to identify the yld-resistant sources in sugarcane germplasm to initiate breeding for yld-resistance in sugarcane. mycoviruses are viruses that infect fungi. they have been identified in all major fungal families. in the present scenario, mycoviruses are the important means of biocontrol of plant fungal pathogens. most identified fungal viruses have double stranded rna genomes, often with more than one dsrna present per virus particle, and have been spherical in shape. these viruses are mostly vesicle bound, as other viruses have protein coatings. to be a true mycovirus, they must demonstrate an ability to be transmitted-in other words be able to infect other healthy fungi through anastomosis and spores. mycoviruses lead 'secret lives', reduce the ability of their fungal hosts to cause disease in plants. this property, known as hypovirulence (hypovirulence is a term used to describe reduced virulence found in strains of pathogens), this phenomenon was first observed in cryphonectria (endothia) parasitica (chestnut blight fungus) on european castanea sativa in italy, where naturally occuring hypovirulent strains were able to reduce the effect of virulent ones. these slower growing hypovirulent strains of c. parasitica contain a single cytoplasmic element of double-stranded rna (ds rna) similar to that found in mycoviruses that was transmitted by anastomosis in compatible strains through natural virulent populations of c. parasitica. hypovirulence has also been reported in many other fungal plant pathogens, including rhizoctonia solani, gaeumannomyces gramini var. tritici, ophiostoma ulmi, sclerotinia homoeocarpa, diaporthe ambigua alternaria alternata, and fusarium sp. etc. hypovirulence has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. it reduces the use of toxic fungicides which also affect the plant growth. the symptoms resulted by the mycoviruses are reduction in growth, reduction in pigmentation and sporulation, excessive sectoring and aerial mycelial collapse. these are the consequences of alteration in complex physiological and biochemical processes involving interaction between host and virus. cassava (manihot esculenta crantz.) is the major tuber crop in peninsular india, it is grown in an area of . lakh hectares with the annual production of . million tonnes both for direct consumption and the starch grain (sago) producing industries, mainly in the southern states of tamil nadu, kerala and andhra pradesh (fao ) . in tamil nadu, cassava primarily produced for sago producing industries where it is considered as an industrial crop rather than food crop, so the resource rich farmers are cultivating the cassava as irrigated crop in their fertile land and the poor farmers are raising the crop under rainfed conditions. in south india in addition to cassava there is a practice of intercropping important vegetable crops like, tomato, brinjal, legumes and gourds in cassava fields since all the above mentioned crops are short duration and are money spinners for the farmers. unfortunately, the major production constraint in these vegetable crops including cassava is the geminiviruses belonging to the family of in recent years there has been growing concern regarding the standard of scientific researches in india. the strengths, weaknesses, opportunities and threats (swot) analysis on indian scientific research reviewed the progress of science during the last six decades. although the 'strengths' were highlighted in good measure, it was the list of 'weaknesses' that called for attention to upgrade the standard of research and 'opportunities' that provide scope for overall scientific growth. a comparison between india and other countries in terms of research papers published revealed that india's contribution to science has come down enormously. what ails indian science? should we compare the growth of indian science with other developed countries? what criteria should be adopted to judge the quality and standard of scientific research? how to motivate the scientists to improve their scientific output? how do motivate the scientists to improve their scientific output? how do indian journals perform in maintaining quality? this paper analyses critically the scientific journals around the world, based on the scores allotted by the national academy of agriculture sciences (naas) in and for and journals respectively. in general, the indian journals performed poorly irrespective of the disciplines with only - % in the high standard. the paper dealt with the reasons for low impact factor, the anomalies in the allotment of scores to wide spectrum of the journals and the disadvantages the scientists face with the scoring system. a case study was presented of an institute with over scientists whose publications were analyzed to discuss the merits and demerits of the system. the performance of the journals published by prestigious academics, societies and councils was also projected. the paper concluded with the need for enhancing the image of the country through research publications in high standard journals and the role of various scientific bodies with shore and long term measures. poster session herpes simplex virus (hsv) keratitis is a leading cause of corneal blindness throughout the world. the infection can be diagnosed by clinical manifestations but in case of atypical ocular cases, laboratory diagnosis is more helpful in timely management of disease. collection of corneal scrapings in all cases of stromal and epithelial keratitis may not be possible, but collecting tear fluid is a convenient procedure causing less discomfort to the patients. therefore, the present study was intended to evaluate the suitability of tear specimens for detecting hsv by polymerase chain reaction (pcr) and immunofluorescence (ifa). tear fluid and corneal scrapings were collected from patients of suspected herpetic keratitis. hsv- antigen was detected by ifa using rabbit anti-hsv antibodies. pcr was performed to amplify bp region of thymidine kinase (tk) coding gene and bp region from dna polymerase coding gene of hsv. out of patients hsv antigen was detected in ( . %) of corneal scrapings and ( . %) of tear specimens and in ( . %) patients from both the specimens. hsv gene could be amplified in ( . %) of corneal scrapings and ( . %) of tear fluids and in ( . %) patients from both the specimens. although, corneal scraping seemed to be marginally superior material for detection of hsv, tear fluid may also serve as an appropriate alternative clinical specimen, due to ease of collection and least discomfort to the patients. in either cases pcr detected higher number of hsv cases than ifa. therefore if and when feasible, both ifa and pcr should be used simultaneously on each specimen to obtain best results. cytokines play a key role in the regulation of immune responses. in hepatitis c virus infection (hcv), the production of inappropriate cytokine levels appears to contribute to viral persistence and to affect response to therapy. il- is produced by a variety of cells including t cells, phagocytes and fibroblast. cytokine genes are polymorphic at specific sites, and certain mutations located within coding/regulatory regions have been shown to affect the overall expression and secretion of cytokines in patients with hcv infection. to correlate the serum levels and polymorphism of il- gene in chronic hepatitis c patients and healthy controls. forty patients positive for hcv rna attending the medicine out patient department and wards of lok nayak hospital, new delhi as well as forty healthy controls were enrolled for the study. the serum level of il- was detected by using elisa. genomic dna was extracted from whole blood of hcv infected patients and healthy controls by using accuprep genomic dna extraction kit according to manufacture's instruction. the genotyping of il- promoter (- variant) was carried out by pcr and direct sequencing using the method of patricia woo et al. . the serum level of il- was significantly down regulated in hcv infected chronic patients as compared to the healthy controls. genotyping of - promoter variant of il- was performed by pcr and direct sequencing. il- polymorphism in the g/g, g/c and c/c allele was non significant when compared to hcv patients and healthy controls. the il- serum levels were significant among hcv infected patients when compared to healthy controls. the polymorphism in the promoter region of il- (- ) was found nonsignificantly associated in hcv patients compared to healthy controls. in conclusion, the present study suggests that the host il- polymorphism alone may not play a significant role in the outcome of hcv infection. acute gastroenteritis (age) is a global health problem and has been associated with multiple etiological agents, which include bacteria, protozoa and viruses. viral gastroenteritis is considered as the second most common illness in children after upper respiratory tract infection. among enteric viruses, rota, noro, enteric adeno, astro and enterovirus are found to be associated with gastroenteritis. although, association of enteric viruses has been established in children hospitalized for age no such data is available from hospitalized children other than enteric infections. to determine the prevalence of enteric viruses circulating in hospitalized children. fecal samples, n = ( symptomatic and asymptomatic for age) were collected from children \ year of age from three different hospitals across the city of pune from june to feb. . detection of group a rotavirus was carried out by using antigen captured elisa. rt-pcr and pcr was carried out for the detection of norovirus, enterovirus, astrovirus and enteric adenovirus detection by using primers targeted to rdrp gene, ncr gene and consevered gene for serine protease and hexon gene respectively. out of fecal samples tested for enteric viruses in age cases, the prevalence of rota, entero, noro, enteric adeno and astrovirus were . % ( ), . % ( ), . % ( ), . % ( ) and . % ( ) respectively. however, the presence of these viruses in the asymptomatic cases (n = ) was detected at . % ( ), . % ( ), . % ( ), . % ( ) and . % ( ) levels respectively. mixed infections of enterovirus and rotavirus were found in both symptomatic . % ( ) and asymptomatic cases . % ( ). however, mixed infection of enterovirus with adenovirus were found only in asymptomatic cases . % ( ). no marked difference was observed in the seasonal pattern of all viruses in the patients with or without gastroenteritis. the findings of this study document highest circulation of rotaviruses in patients symptomatic and asymptomatic for age. the entero and noroviruses remain second most important enteric viruses in these patients. influenza in humans is a major public health concern and the understanding of its evolution in the light of its ''antigenic drift'' helps prediction of epidemics and update of yearly influenza vaccine. to antigenically characterize influenza a (h n ) isolates and study antigenic drift during to in pune city. patients with influenza like illness were identified using a strict case definition from dispensaries located in different areas in pune and clinical samples (ns/ts) were collected after obtaining informed consent. these clinical samples were processed in vivo (in fertile eggs) and in vitro ( overall, an additional ( . %) positive cases of dengue could be detected when ns antigen assay was also used in the study. highest ns antigen positivity was encountered among the samples collected on the rd day of fever whereas mac elisa for anti igm antibody was positive after th day and gradually there was an increase in the positivity towards the convalescent phase of the disease. the results of this study indicate that ns antigen based elisa test can be an useful tool to detect the dengue virus infection in patients during the early acute phase of disease since appearance of igm antibodies usually occur after fifth day of the infection. concurrent use of both diagnostic assays namely ns antigen as well as mac elisa will improve the overall detection of dengue infection. early detection of acute dengue virus infection is crucial to provide timely information for the management of patients. human parvovirus b , a member of the parvoviridae family, is a pathogen associated with a wide variety of diseases. most commonly, it causes childhood rash erythema infectiosum, but in some cases more serious symptoms such as persistent arthropathy, critical failures of red cell production causing transient aplastic crisis, this infection in pregnancy causes hydrops fetalis and myocarditis. traditional immunosuppressive therapy being unsuccessful, anti-viral therapy might be worthy of consideration. functional annotation would provide role of viral proteome in its survival and pathogenic mechanisms. svmprot functional family annotations of vp protein had deciphered its zincbinding, coat protein, outer membrane, chlorophyll biosynthesis, dna repair and calcium-binding nature. vp protein is having a key role in viral assembly of b virus and being non-homologous to human proteome, it was identified as an attractive molecular target for structure based drug discovery. the vp protein crystal structure was energy minimized using charmm. a structure based virtual screening method was applied using ligandfit to identify potential inhibitors of vp protein from chembank database and ten potential human parvovirus b vp inhibitors were proposed. prism genetic analyzer. the drafting of the sequences was performed using bioedit software and were submitted in genbank. for phylogenetic interpretation denv representing the full extent of genetic diversity in denv- , denv- and denv- were collected from genbank. neighbor joining algorithm was implemented with bootstrap value of , replicates for phylogenetic inference using mega . . . the genomic region to (c-prm gene junction) of denv were amplified directly from patient serum. twelve of samples were positive for dengue viral rna. of these were dengue type , was dengue type and were dengue type . for molecular epidemiological survey and genotyping of the sequences more than sequences from different geographical areas including sequences form previously reported north indian isolates were compared with our present data set. the critical analysis of the sequences revealed: dengue type sequences were clustered within sub-type of genotype iii and all the sequences of den- clustered along with genotype iii. thus, among the dengue types , and currently circulating in north india, dengue type , genotype iii, being the predominant one followed by, genotype iii of dengue type . although there is no specific treatment or vaccine available currently, the confirmative rapid diagnosis based on detection of viral nucleic acid or igm antibodies in serum, an indication of recent infection, helps in epidemiological monitoring, symptomatic treatment of patients and determining prognosis. serological detection of anti-cgv igm antibodies was performed using rapid immuno-chromatographic assay (rica) and igm-antibody capture enzyme linked immunosorbant assay (mac-elisa). eighty convalescent sera were tested by rica and of them were found positive for anti-cgv igm antibodies. twenty-five anti-cgv igm antibody rica positive sera were further assayed using mac-elisa. more sera from the patients are currently being tested to compare the sensitivity of these two serological assays in anti-cgv igm antibody based early serological diagnosis of cgv infection and the findings will be presented. thus the present study was designed to evaluate the utility of multiplex pcr (mpcr) for simultaneous and rapid detection of dengue and chikungunya viral infections. seventy-two acute phase blood samples from clinically suspected dengue cases were subjected for dengue and chikungunya uniplex pcr using dengue genus specific primers and e gene specific primers for chikungunya virus as well as multiplex pcr was developed for simultaneous detection of dengue and chikungunya infection. standard strains of dengue and chikungunya virus were used as controls. of the clinically suspected dengue samples were found to be positive for dengue viral rna by dengue uniplex pcr as well as dengue chikungunya mpcr whereas none of the samples were positive for chikungunya virus infection by both uniplex chikungunya pcr and dengue chikungunya mpcr. the result of dengue and chikungunya uniplex pcr was found to be % concordant with dengue chikungunya multiplex pcr. dengue chikungunya multiplex pcr was found to be a potential rapid test to detect dengue and chikungunya viral infections simultaneously in clinical samples. sheetal malhotra, neelam marwaha, karan saluja, ratti ram sharma department of transfusion medicine, pgimer, chandigarh transmission through blood and blood products can be reduced to a great extent by efficient and reliable testing of the blood. the newer fourth generation elisa assays simultaneously detect antibodies against hiv- and and the presence of p antigen and thus shorten the window period to about days, as compared to days with third generation elisa. to compare the hiv seroprevalance among blood donors using fourth generation elisa (antigen-antibody) versus third generation elisa (antibody) assay. this was a prospective study involving blood donors of which were voluntary donors ( being students and being non students) and were replacement donors. sex workers are one of the core group for transmission of sti/hiv and as a ''bridge group'' to the general population. accordingly, highest priority is given to this group in targeted intervention for prevention of hiv/aids. here we are describing one such female sex worker who was harbouring concomitant sti including viral sti. a year old female sex worker was brought to the sti clinic of a tertiary care hospital by ngo with complaint of genital discharge for days. on per speculum examination, cervix was slightly erythematous, tender with mucopurulent discharge. there was no vaginal discharge or ulcer in anogenital area. however, there was a wart at lateral wall of vagina. as per naco syndromic management guideline, treatment was given for n. gonorrhoeae, c. trachomatis and hpv. cervical swab was taken and subjected to various microbiological investigation for the detection of sti viz n. gonorrhoeae, c. trachomatis, t. pallidum, candida spp., t.vaginalis, hsv- , hsv- , hiv, hbv, hcv, hpv and m. contagiosum. saline wet mount showed pus cells, but no yeast cells or trophozoite of trichomonas vaginalis. gram stained smear showed more than four polymorphonuclear leucocytes in the absence of gramnegative intracellular diplococci and a presumptive diagnosis of non gonococcal urethritis was made. no organism was isolated on any culture media after appropriate incubation. cervical swab was negative for antigen of c. trachomatis. serum was tested positive for hbv, hcv, hsv- and t. pallidum though it was seronegative for hiv. in the present case, the female sex worker was harbouring four viral sti viz hsv- , hbv, hcv and hpv alongwith t. pallidum. however clinically she was diagnosed and treated accurately only for genital wart while cervical discharge due to hsv- was misdiagnosed. it is necessary to try to test alternative approaches such as periodic presumptive therapy of viral sti, because this will not only boost up the efforts of sti control in the target group but also help in hiv control. alternatively, regular clinical and laboratory screening for viral sti may be tried. densonucleosis viruses (dnv) belong to parvoviridae family. they are the etiological agents of insect's disease known as densonucleosis, which leads to death or loss of vital functions of the infected insect. densonucleosis virus of mosquitoes has generated lot of scientific interests because of its tremendous potential in biological control and its application as a transducing vector. earlier, we have reported the isolation and characterization of a dnv from aedes aegypti mosquitoes and its prevalence among different ae. aegypti populations from india. there are reports suggesting that when aedes albopictus mosquitoes co-infected with dengue- and dnv, the multiplication of den- is suppressed. the present study focus on the effect of coinfection of ae. aegypti mosquitoes with dnv and chikungunya virus (chik). the first instar mosquito larvae were infected with dnv and the emerging dnv infected females were then infected with chikv by oral feeding. thus obtained chik infected female mosquitoes were analyzed by real time pcr for both dnv and chikv on alternate days post-infection, up to the th day. the data showed no significant difference in the multiplication of either of the viruses after co-infection. results suggest that chikv neither stimulates the replication of dnv nor is its own replication suppressed due to co-infection. this study forms an initial step in understanding the role played by such endogenous viruses on the vector dynamics. chandipura virus pathogenesis is manifested as encephalitis in young children with a very high mortality rate. this damage could be due to direct replication of the virus in brain parenchymal tissue or immune system mediated. this study aims at elucidating the role of brain infiltrating lymphocytes in pathogenesis using mice as the model system. mice were inoculated intracerebrally with the virus and the perfused brain tissue was used to isolate the lymphocytes. control mice were inoculated with an equal amount of media. in order to standardize the procedure for isolation of lymphocytes from brain tissue, splenocytes were processed to isolate the lymphocytes using histopaque density gradient method. methods to isolate lymphocytes from brain tissue as described by earlier workers were tested for the ease and efficiency of procedure using known suspension of lymphocytes from spleen. percoll density gradient method provided optimum yield of lymphocytes with an ease of handling. in this, brain cell suspension used to prepare % percoll is layered over % percoll prepared using media in : ratio. density gradient centrifugation is carried out at g for min at °c to obtain lymphocyte layer at the interface. leishman staining was performed to analyze the morphological characteristics of isolated lymphocytes. normal lymphocytes showed dark blue stained nucleus. some bigger sized cells with diffused nucleus characteristic of atypical lymphocytes were observed and some of the cells were surrounded by hair like structures. phenotypic characterization was carried out using flow cytometry. the presence of cd + , cd + and cd + cells was observed. the percentages of cd + , cd + and cd + cells was found to be . %, . % and . % respectively in the lymphocytes isolated from infected animal and . %, . % and . % respectively from control animal. hence, cd + cells showed maximum infiltration after infection. (santosh et al. ; pradeep et al. ). in the present study chikv suspected blood samples were collected and the acute phase samples were subjected to rt-pcr for the presence of virus specific rna by using the primer pair dvrchk-f/dvrchk-r as described by us earlier (naresh kumar et al. ). the convalescent phase samples were screened for chikv specific antibodies by using sd bioline chikungunya igm rapid test. six sets of primers were designed to amplify the complete nsp and complete structural genes of chikungunya virus. the products were further gel purified, cloned in ptz r/t vector and the recombinant clones were sequenced and submitted to the genbank. the complete ns gene and structural genes were compared with other available sequences in the genbank. sequence analysis results will be presented. the present study discusses these aspects in detail. . some of these phages (viz. v , v ) showed plaques at °c but not at °c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v , v , v , v and v . also, phage dilution medium (pdm) as described by chaterjee et al. ( ) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p . the various passages of the phage isolates v , v , v , v and v (i.e. original, p , p and p ) were stored at - °c. the four major routes of transmission are unsafe sex, contaminated needles, transmission from an infected mother to her baby at birth (vertical transmission) and breast milk. screening of blood products for hiv has largely eliminated transmission through blood transfusions or infected blood products in the developed world. in , globally, about million people died of aids, . million were living with hiv and . million people were newly infected with the virus. hiv infections and aids deaths are unevenly distributed geographically and the nature of the epidemics vary by region. more than % of people with hiv are living in the developing world. there is growing recognition that the virus does not discriminate by age, race, gender, ethnicity, socioeconomic status-everyone is susceptible. however, certain groups are at particular risk of hiv, including men who have sex with men (msm), injecting drug users (idus), and commercial sex workers (csws). the present study indicates the prevalence of hiv infection among the people residing in the northern region of india predominantly among the foothills of the himalayas. the study was carried out on the patients visiting herbertpur christian hospital (a unit of emmanuel hospital association) under the integrated counselling and testing centre scheme at the respective hospital during the - . the study indicates the screening of people groups residing in the respective area through community health schemes. the diagnosis of the hiv infection is done by three types of assays namely. the tridot method which is the rapid method of diagnosis followed by the. hiv coombs test which involves the dot immunoassay principle. the third assay is the enzyme linked immunosorbent assay (elisa). the number of patients screened during the period of september to march is which include patients coming from four different states namely haryana uttarakhand uttarpradesh and himachal pradesh. the number of people who were tested positive are and the number of people who were tested negative are . the people tested positive are sent to the higher centre for other confirmatory tests such as pcr and western blot analysis. these patients are sent for treatment and prophylaxis at a respective recognised centre in dehradun. the present study determines a consistent community hiv screening and treatment approach through diagnostics counselling and awareness programmes. classical swine fever (csf) also known as hog cholera is a highly contagious and fatal disease of swine. csf became rapidly a major issue of pig industries. it still causes important economical losses worldwide. it is considered as a major health problem of swines in india. during the month of august to october there was an outbreak of classical swine fever in bihar. from three districts darbhanga, patna and supol, total numbers of different infected tissue samples like kidney, spleen and lymphnode were collected from the dead morbid/pigs. total rna was isolated from % homogenate of infected tissues in sterile pbs by tri-reagent (sigma, usa) according to the manufacturer's instructions and cdna was prepared by using commercial available kit. the cdna was stored frozen at - °c until used. for the molecular detection of classical swine fever virus specific nested pcr amplification of e and ntr was done along with ns b and e rns amplification. primarily these samples were found positive with these primers. further confirmation by sequencing was done by cloning of these pcr products in pgem-t easy vector. e and ntr sequences were considered for phylogentic analysis along with complete available sequences of csfv. nucleotide sequence alignments were carried out using the clustalw program (dnastar) and phylogenetic tree analysis (dnastar) showed that ntr have close proximity with taiwan strain (accession no. ay ) and e shows close proximity with chinese isolate csfv- (accession no. af ). peste des petits ruminants (ppr) and sheeppox are oie notifiable diseases of small ruminants especially sheep and goat. both the diseases are economically important, in enzootic countries like india and are major constraints in the productivity of animals. considering the geographical distribution of both ppr and sheep pox infections and prevalence of mixed infection, in the present study, safety and potency of the experimental duel vaccine comprising attenuated strains of thermostable-ppr virus (pprv-revati, p- ) grown at °c and attenuated sheep poxvirus (sppv-srinagar, p ) was evaluated in local non-descript sheep. experimental animals were grouped into four groups and each group was comprising six animals, received doses ( tcid ), dose ( tcid ) and / th dose of vaccines and normal saline as control in ml volume subcutaneously, respectively. serum samples were collected on , , , and th day post vaccination. sheep simultaneously immunized with ml of vaccine consisting of either or doses of each of pprv and sppv were monitored for clinical and serological responses for a period of - weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to both pprv and sppv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, respectively following immunization. all the immunized animals' resisted infection when challenged with virulent strain of sppv (srinagar isolate at p- ) on day dpi, while in contact control animals developed characteristic signs of sheeppox. the challenge of the sheep against ppr was not carried out, however, the antibody titre after immunization determined by snt and elisa, indicated that protective titre, as per earlier report on the goats. dual vaccine was found safe at higher dose and induced protective immune response even at lower dose ( tcid ) in sheep, which was evident from sero-conversion as well as challenge study with sppv. the study indicated that both the viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this dual vaccine in combating both infections simultaneously. goatpox is one of the highly contagious, oie notifiable and economically important viral diseases of goats. the disease is caused by goatpox virus (gtpv) is classified of the genus capripoxvirus in the family poxviridae. the disease incurs severe economic losses in terms of high morbidity in adults and heavy mortality in young kids and is a major constraint in goat farming in india. considering the enzotic nature and economic impact of the disease, it is all important to control the infection by developing an effective vaccine. recently, vero cell based a live attenuated goat pox vaccine; using gtpv uttarkashi isolate (p ) has been developed in authors' laboratory and evaluated in goats. the vaccine was found safe, potent and immunogenic experimentally and even at field trials. the vaccine has been evaluated at large-scale at different regions of the country and found suitable for mass vaccination. however, the longevity of potency was not evaluated. therefore, a long term potency trials were studied for a period of years with annual challenge by using virulent goatpox virus and sero-monitoring. a sufficient number of hill goats has been vaccinated with dose of vaccine ( . tcid /ml) and monitored for clinical and serological response. every year, significant number of vaccinated (n = ) and control animals (n = ) were used for challenge with virulent strain ( . srd /ml, gtpv mukteswar). sera of pre-and post-challenged ( dpc) animals including controls have been collected and monitored for serological response in the form of specific antibody production by snt and indirect elisa. all the vaccinated animals were protected on challenge, whereas, all unvaccinated controls developed infections. the same has been reflected in sero monitoring of collected sera. so the developed live attenuated goat pox vaccine was found safe, immunogenic and potent for a period of years of immunization and suitable for mass scale vaccination in control and eradication of goat pox along with a/are suitable diagnostic tool/s in goatpox enzootic country like india. rotavirus infection in avian species varies from subclinical infections to outbreaks of diarrhea. the economic significance of rotaviral enteritis to the poultry industry has not yet been defined, but by analogy to the situation in mammals, it is likely to be significant. unlike the extensive studies performed on rotavirus infection in humans and animals, limited studies have been carried out to determine the extent of exposure of poultry birds to rotaviruses. to determine the prevalence of avian rotavirus antibodies in commercial broiler chickens. a total of chicken serum samples were collected from the lairage of a poultry slaughter house where birds from four different broiler farms in and around pune city were supplied to. the serum samples were tested by an igg antibody capture elisa wherein purified chicken rotavirus ch was used as coating antigen. sera from specific pathogen free (spf) chick (n = ) served as negative control in the test. cut off was calculated as mean negative control ? sd (standard deviation). s/co (mean sample od /cut off) values above ( . - . ) in % ( / ) serum samples were indicating positivity to rotavirus antibodies. the result of the study indicates exposure of the birds to avian rotavirus or similar agent that is circulating in pune city. bluetongue has become established in south india causing regular outbreaks in sheep. btv serotypes , , and were isolated from native sheep of andhra pradesh. the other serotypes circulating in the state need to be identified. however the major constraint is the serotype identification. to overcome the difficulties of traditional serotyping methods (neutralization tests), nucleic acid based tests are being tried. rt-pcr for serotyping was standardized using primers specific to vp gene of btv- , and serotypes. rt-pcr resulted in bp product of btv- , bp product of btv- which was defined by specific primers. however non specific amplification at two different sites i.e. bp and bp was noticed for btv- . specificity of rt-pcr was evaluated. btv- and btv- specific primers could amplify only btv- and btv- respectively where as btv- type specific primers amplified not only btv- but also btv- and btv- . nucleic acid sequence data obtained from btv- pcr product and btv- cloned products were specific to vp gene of btv- and btv- respectively. however, and bp products of btv- were identical to vp gene of btv- , , , , and and vp gene of btv- , and respectively, indicating the non specific amplification of btv- . foot and mouth disease is the most contagions and highly economically impotent disease of cloven footed animals. the disease is controlled by regular vaccination using the vaccine produced from the virus grown in the cell culture. the vaccine strain used for vaccine production is selected from the field isolates based on the adaptability and growth kinetics in bhk cells and antigen coverage. however the field viruses need to be passaged several times to adapt in tissue culture. passage of field viruses in tissue culture may results in development of mutants whose genetic makeup may differ from the field samples also some of the field strains may fail to adapt or may grow poorly in the tissue culture, thus the efficiency of the vaccine gets affected. structural proteins of fmdv carry the sequences which determine the serotype specificity and immunogenicity. thus one may replace the gene coding for structural proteins from the full length cdna copy of the vaccine strain that has been adapted to the tissue culture with the poly-structural protein gene (pi) so that the chimeric virus gets the serotype specificity of the field strain besides retaining the other characteristics that are needed for a vaccine virus. we have made replication competent fmdv asia i full length genome and cloned under t and cmv promoter separately in plasmid vectors. bam h sites were created for inserting pi- a gene of other field strains. the p - a of type 'o' vaccine strain was amplified directly from the cattle tongue material, cloned in plasmid vector and studied the specificity by sequence analysis and gene expression. we have introduced 'o' p - a gene into the full length construct devoid of asia structural protein gene, p - a. the in vitro transcribed rna in case of t promotered construct and plasmid dna in case of cmv promotered construct were transfected into the bhk cells. after the passaging the virus obtained was studied for the speciality. this approach may be used not only for rapid selection of vaccine strain and also as a repository of the cdna copy of the virus. the p is composed of a, b, c and d (vp , vp , vp , and vp ) respectively of which the vp is the most immunogenic and subunit vaccine produced with vp alone was able to induce high level of neutralising antibodies. thus to control the disease in india polyvalent vaccine consisting of the inactivated virus of all the three serotypes are in use. however the conventional vaccines have several drawbacks which include safety and temperature sensitivity. hence alternatively sub-unit vaccines consisting of vp protein has been tried. however this showed limited success due to the antigenic variations occurring in the field viruses thus escaping the neutralization from the antibodies generated from single cloned protein. hence the present study was undertaken with an objective to include all the neutralizing epitopes present in the three serotypes by linking vp ( d) genes and produce a poly valent protein for using as poly subunit vaccine. in this study we have constructed a cassette by linking the genes of three serotypes 'o' ( bp), 'a' ( bp) and 'asia ' ( bp). these genes were cloned individually in commercially pbsk vector and confirmed by sequence analysis before linking in pc dna vector. the linked gene construct was sub-cloned in pet expression vector. the expression of the protein gene from the pet vector was induced with iptg and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). a fusion protein of size kda was observed in page gels. since the protein contains his residues from the vector at the n-terminal end, affinity purification was carried out using nickel nitrilo-tri-acetic-acid (ni-nta) agarose matrix. the immunoreactivity of the purified protein was assayed by western blot with the anti fmdv type 'o' and 'asia ' specific sera. the may be used as a subunit vaccine. silkworm diseases caused by viruses, bacteria, fungi and protozoans form major constraints for the silk cocoon production in all the sericultural countries and among these silkworm viral diseases viz., nuclear polyhedrosis and infectious flacherie caused by bmnpv and bmifv cause severe crop loss. the traditional disease management strategies include prophylactic measures and use of disease free silkworm eggs. the prophylactic measures such as disinfection of silkworm rearing house and appliances, egg surface, silkworm bed disinfection and rearing surroundings. the disinfectants used presently in sericulture are either formaldehyde or chlorine based products, but these chemicals are neither eco-nor user-friendly. the awareness about health hazards caused by formaldehyde and environmental pollution caused by cl necessitated the development of eco-and user-friendly disinfectant products for use in sericulture. these include alternative disinfectant products developed using biodegradable chemicals and plant based ingredients by apssrdi, hindupur and central silk board for the management of silkworm diseases in india. the ideal disinfectant for sericulture would be the one which can inactivate silkworm pathogens of diverse origin and economical for sericulture. the paper discusses on the disadvantages of hcho and cl based disinfectants and advantages of eco-and user-friendly disinfectant for the management of silkworm diseases especially the ones caused by viruses. the baculovirus expression vector system (bevs) is widely used for the production of high levels of properly post-translationally modified, biologically active and functional recombinant proteins and has facilitated basic biomedical research on protein structure, function, drug discovery and the roles of various proteins in disease. bevs is based on the introduction of a foreign gene into nonessential for viral replication genome region via of homologous recombination with a transfer vector containing target gene. the resulting recombinant baculovirus lacks one of nonessential gene (polh, v-cath, chia etc.) replaced with foreign gene encoding heterologous protein which can be expressed in cultured insect cells and insect larvae. insect cell-bev system is widely used to produce recombinant proteins. bevs also eliminates concerns regarding pathogens that could potentially be transmitted to humans as it is non-infectious to vertebral animals. these features make silkworm system an ideal expression and delivery package for producing proteins of medicinal importance. the efficiency, low cost and large-scale production of proteins using bevs represents breakthrough technology that is facilitating highthroughput proteomic studies. the bevs has become a core technology for cloning and expression of genes for study of protein structure, processing and function; production of biochemical reagents; study of regulation of gene expression; commercial exploration, development and production of vaccines, therapeutics and diagnostics; drug discovery research; exploration and development of safer, more selective and environmentally compatible biopesticides. utilization of silkworm larvae and pupae as bioreactor with recombinant bmnpv producing foreign proteins extends the usages of silkworms. due to its large-size and high protein synthesis ability as well as the expediency in mass culture, silkworm is considered as good candidate for producing recombinant proteins. wssbv is the causative agent of a disease, which has recently caused high shrimp mortalities and severe damage to shrimp culture. wssbv has been found across different penaeid shrimp species. in order to develop a effective diagnostic tool, a wssbv genomic library was constructed by cloning wssbv genomic dna extracted from purified virions. in the present study wssv disease free (confirmed by pcr analysis) were collected from hatcheries from different areas of guntur and prakasam districts and analysed to study the effect of various physical parameters like temperature, p h , salinity and turbidity on the prevalence of above disease. the studies on the surface water temperature revealed fluctuations in the ponds ranging between to . °c in diseased ponds and . to . °c in healthy ponds. these results show definite influence of temperature on the prevalence of wssv. present day strategy in vaccine development is to include marker facility that helps in distinguishing antibody response due to vaccination vis-à-vis infection in vaccinated animals. such information becomes relevant for effective disease control programmes especially when using inactivated virus vaccines like foot and mouth disease (fmd). the antibodies generated in the animals, only through vaccination, is the measure of vaccine efficacy and safety. presently inactivated fmd virus (fmdv) vaccines are used to control the disease in the endemic countries like india. the quality assurance of the vaccine depends on the efficacy of the vaccine in generating protective antibody without causing subclinical disease due to improper inactivation. since protective antibody response in vaccinated animals can not be distinguished from that of infected animals one needs to assay the antibody response against non structural proteins (nsps) and the vaccine must be free of contaminated nsps. production of vaccine free of nsps requires the cumbersome method of virus purification which adds to the cost of the vaccine. alternatively one may develop a positive marker vaccine by including a foreign protein or epitope which is not expected to be present in the vaccine and the antibodies generated against which helps in detecting the vaccine related response. here we report a molecular approach by which we introduced a immuno-dominant epitope of green fluorescent protein (gfp) into the structural protein gene of foot and mouth disease virus vaccine strain asia ( / ). our laboratory has produced a mini-genome of fmdv asia that lacks structural protein gene (p - a) coding for all the structural proteins (vp - ) of fmdv asia as a vector (pcfl dasia ). the p - a of the asia vaccine strain was cloned separately into a plasmid vector and by successive pcr mutagenesis and cloning we have introduced nucleotide sequence corresponding to amino acid epitope of gfp into p - a gene. gfp epitope was inserted by replacement at n-terminal region of vp- which is not immunogenic. the modified p - a was expressed in e. coli and studied. the modified p - a gene with gfp epitope was inserted into the pcfl dasia to get full length replication competent cdna cloned under cmv promoter in pcdna (pcflasiagfp). this can be used to produce synthetic virus with gfp epitope that can generate antibodies not only against neutralizing epitopes but also against gfp epitope. presence of antibody against gfp epitope in the vaccinated animal will reveal vaccine efficacy. elisa against gfp can be used as a companion test not only for safety evaluation but also for quick evaluation of efficacy. further absence of nsp antibodies in the serum may reveal the quality of the vaccine in respect of safety. self replicating dna vaccines are developed to achieve robust immune response through enhanced antigen production and gamma interferon expression in vaccinated animals. since self replicating dna vaccines induce gamma interferon expression which helps in viral clearance such vaccines are expected to be useful to cure even the carrier and persistently infected animals. understanding the events that help in the elicitation of both the arms of immune response in vaccinated animals is necessary to understand the effectiveness of the vaccine. the work presented here deals with the immunological evaluation of a sindbis virus replicase based dna vaccine carrying linked fmdv vp genes in vaccinated guinea pigs. we have constructed self replicating dna vaccine vector and to the down stream of a sub genomic promoter we have inserted secretory signal followed by linked-vp genes of fmdv serotypes (o-a-asia ) with glycine and proline bridge in between. guinea pigs were vaccinated with the construct and the sera at days post vaccination were evaluated both for cellular response by studying the cd levels and by mtt and cytokine profiles by real time assays. the humoral response was evaluated by studying cd levels in the whole blood by facs analysis and serum antibody levels by snt and elisa. the animals were challenged with gp infective dose of fmdv type 'o' virus lesions were scored. further the replicative efficiency of the challenge virus was studied by ab elisa. the results showed that all the assays except antibodies against ab protein have positive correlation with the protection. as expected the titre of the antibodies against ab protein was lower indicating that the challenge virus replication was inhibited in the vaccinated animals. the limited studies conducted by us showed that self replicating vaccine has a potentiality to emerge as potent vaccine for fmd. ganjam virus (ganjv) belongs to the genus nairoviruses (family bunyaviridae). these viruses cause diseases in livestock. it has been isolated from different animal hosts and tick vectors from india. genus nairoviruses includes a total of tick-borne viruses, classified into serogroups. the important serogroups are crimean congo hemorrhagic fever (cchf) and the nairobi sheep disease (nsd). the main members of the nsd group are nsd and dubge viruses. their genome consists of three segments of single stranded rna, viz. s, m and l that encodes viral nucleocapsid protein, viral glycoprotein g and g and the viral polymerase respectively. ganjv is very closely related to (nsdv). nsdv is found in east and central africa, causes very high morbidity and mortality in livestock. the present study involves phylogenetic comparison of ganjv isolates from india with other nairoviruses based on complete n gene. it will help to understand the kind of nucleotide (nt) and amino acid (aa) changes that have occurred in ganjv strains from different geographical areas. eight strains of ganjv isolated at niv during - from different parts of india were used in this study. virus stocks were prepared in vero e cell line these were used as the source of viral rna. the n gene was amplified either as a complete gene in one reaction or in fragments whenever necessary. thus obtained sequences were analyzed; annotated to get a consensus sequence, aligned against the sequence of prototype strain of ganjv and other representative nairoviruses. the nt sequences were converted to aa sequences and analysis was done at both nucleotide and amino acid levels. based on what nt or aa phylogenetic tree was constructed and compared with other nairoviruses (cchf, dugv, hazv, kupv and nsdv) where complete s segment sequences were available gen-bank database (ncbi). the phylogenetic data at both the nt and aa levels showed that all the strains of ganjv form monophyletic lineage with the nsdv. cchfv and hazv together formed another clade, whereas dugv and kupv made a separate branch in the tree. the different ganjv strains showed - % difference with nsdv at the nucleotide level and - % difference at the amino acid level. hazv showed - % difference at the nt level and % difference at the aa level with ganjv as well as nsdv. the present data obtained suggests that ganjv and nsdv are minor variants of the same virus. diarrhoeal syndrome is one of the major concerns of the livestock industry. most of the diarrheic cases in animals go unnoticed and limited attention is paid on viral etiology. presence of large amount of fecal matter in animal shed acts as a source of infection for calves via drinking water, feed, or contaminated soil. keeping this in view, investigation was planned to detect the association of rotaviruses with diarrhea in dairy calves and to observe the genomic diversity among the circulating viruses in tarai area of uttarakhand. a total of diarrheic fecal samples collected from instructional dairy farm, nagla, pantnagar, uttarakhand were screened during the study. samples were collected from both cow calves and buffalo calves in - months of age. for the diagnosis of rotavirus, all the fecal samples were subjected to rna-electrophoresis after nucleic acid extraction. viral genome segments were visualized by silver staining. out of the total samples tested, seven were found positive in rna-page showing typical genome segments migration pattern of bovine rotavirus. in the given samples prevalence of bovine rotavirus was . % and % in cow and buffalo calves, respectively. on the basis of migration patterns of rotavirus in rna-page, group a were identified with typical : : : pattern. variation within movement of various genome segments among isolates of bovine rotaviruses was observed during the study that may be indicative of emergence of mutants in the circulating isolates. the vp gene based group a specific rt-pcr was standardized and all the isolates in this area were confirmed to be of group a type. work is in progress to genotype the bovine rotaviruses of this region based on vp and vp genes. this study emphasizes the need to explore the prevalence of bovine group a rotaviruses in different places of uttarakhand and their genetic characterization which could help in selection of control strategies for rotavirus infections. foot-and-mouth disease (fmd) is endemic in india causing enormous economic loss to the animal keepers and trade embargo with fmd free countries in livestock and animal products. rapid diagnosis of fmd is of immense importance in prevention and control of the disease. fmd is initially diagnosed clinically and confirmed by laboratory tests. virus isolation in cell culture and sandwich elisa for antigen detection are commonly practiced in laboratories. the virus isolation though is very sensitive but it can be slow and analytical sensitivity of the elisa is lower and can not be used with certain sample types. the use of molecular techniques in the diagnostic laboratory has greatly increased the speed, specificity and sensitivity of fmd diagnostic tests. molecular techniques like rt-pcr, pcr-elsa and dot hybridization can be used with more success for detecting carrier animals and animals harboring sub-clinical infection and can be applied in a wide range of clinical sample types. these techniques can be used as genus and serotype specific test including detection of particular lineage/genotypes with in the serotype. multiplex pcr has been used to differentiate serotypes of fmdv and the technique is sensitive, experimentally simpler, cost effective and less time consuming. the assay can be used for serotyping on elisa negative samples. the molecular techniques not only help in diagnosis but also useful for epidemiological studies. lineage differentiating rt-pcr has been useful in identifying different lineages of serotype asia (lineage b, c and d) before proceeding with sequencing of d region. similarly genotype differentiating rt-pcr has been developed and used in differentiating two different genotypes of serotype a (genotype vi and vii). these assays have the potential to be applied on clinical samples directly, thereby saving much time needed for sample processing and nucleotide sequencing. recent development of real time rt-pcr methodology has allowed the diagnostic potential of molecular assays to be realised. advancement in real time pcr technology made it possible to combine several assays within a single tube which is in the progress in our laboratory. integration of these assays onto automated high throughput platforms provides diagnostic laboratories with the capability to test large numbers of samples. microarray technology was provided greater screening capabilities for pathogen detection. the microarray allows the addition of large number of oligonucleotide probes for identification of mutant pathogen and also for subtype determination. the combined properties of high sensitivity and specificity, low contamination risk, and speed has made realtime pcr and microarray technology a highly attractive alternative to conventional methods in increasing percentage of outbreaks confirmed and analyzed and for tracing the origin of fmd virus responsible for outbreaks. dna vaccines are expected to elicit both humoral and cellular responses, cellular response being long lasting. however the approach has several limitations like poor stability of dna, poor expression and risk of integration. poor expression becomes the major limitation in the case of fmd as fmdv proteins are poor immunogens. also dna vaccine vectors carrying only eukaryotic promoters elicit strong cmi response and weak humoral response. the methodology to achieve humoral response involves the expression and secretion of the expressed protein so that the antigen presenting cells will be able to process the antigen and produce humoral response. in case of fmd humoral response is as important as cellular response. the present project aims at addressing these issues; achieving higher expression and getting the protein secreted out by constructing self replicating gene vaccines for fmd and studying their efficacy. the vector for humoral immune response contains eef promoter, sindbis virus polymerase gene and secretory and anchoring signals. the integrity of the vectors was confirmed by sequence analysis. the linked polyvalent protein genes of fmdv serotype a, o and asia were cloned into the vectors and the presence of the insert was confirmed by restriction enzyme digestion. the functionality of the constructed dna vaccine vector (pvac self rep ) was assayed by transfecting the dna into bhk cell monolayer and studying the s labeled proteins in immuno-precipitation assays. the studies showed high level of expression in case of constructed vector as compared to infected virus for the specific protein. the secretion of the expressed protein was assayed by immuno-fluorescence assay and found to be positive. encouraged with these studies the preliminary studies were conducted on vaccine efficacy studies in guinea pig model. the immunized guinea pigs showed high antibody titres by snt and elisa, as compared to conventional dna vaccines (pup cd) even at / th of the dose. this approach of constructing self replicating dna vaccine for humoral response is the first report. genetically engineered microorganisms are important sources of industrial and medicinal proteins. over the past decade, plant host system has been investigated as potential host system for expressing proteins of therapeutic and diagnostic use. however concerns regarding the stability and environmental safety need to be addressed. chloroplast engineering is expected to resolve some of these issues since, plastids/chloroplasts are inherited maternally and are not disseminated through pollen. this makes plastid transformation a valuable tool for transgenic creation besides offering biological containment. since foot and mouth disease (fmd) of cloven footed animals is a major concern in the world over. foot and mouth disease (fmd) is the most feared, viral disease of the cloven footed animals causing heavy losses to the live stock industry. the disease is enzootic in many parts of the world including asia. the conventional vaccines for fmd have several limitations which include safety, temperature sensitivity and duration of immunity. attempts have been made to overcome these limitations using recombinant dna technology. amongst the newer vaccines, edible vaccines are cost effective and easy to administer. since the stability of the gene of interest is the major concern in the case of plant transgenics, marker genes are used for regular selection. the detection methods based on the available marker proteins like b glucoronidase (gus) protein/antibiotic selection are cumbersome and cost intensive. however selection based on herbicide resistance is much simpler and easy. hence in the present study, the -enolpyruvylshikimate- -phosphate synthase (epsp) gene was used as a marker along with the immunogen gene of fmdv. epsp is the key enzyme in the shikimate biosynthesis pathway necessary for the aromatic amino acids production. in order to investigate the mechanism of long term immunity and the effect of protective immunity induced by cationic plg micro particle coated dna vaccination. we constructed the expression plasmid containing a foot-and-mouth disease virus (fmdv) id gene sero type a. intramuscular vaccination of guinea pigs with the micro particles coated plasmid dna induced a strong antibody response and neutralization antibodies, cellular mediated immune response which lasted year. we further analyzed the persistence and expression of id gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction and quantitative pcr. the results showed that id gene was present and expressed in the muscle cells up to year after days post vaccination. furthermore, guinea pigs vaccinated with micro particles coated plasmid dna were protected against a challenge with fmdv virus. therefore the micro particles coated plasmid dna vaccination dose induce a protective immunity and long term humoral, cellular immuno responses against fmdv, which could be maintained by persistent expression of id gene in muscle cells. foot and mouth disease virus (fmdv) causes a highly contagious viral disease of cloven hoofed animals, which has a considerable socioeconomic impact on the countries affected. interleukin- (il- ) enhances the il- driven th immune response that is important in immunity against intracellular pathogens. the multiple roles of il- in many physiological and pathological processes have generated a great deal of interest in recent years. antiviral effects of il- have been reported. we evaluated the effects interleukin- (il- ) on the replication of fmdv in vitro in bhk- cells. bovine il- mature protein coding sequence was amplified from the bovine pbmc cells and cloned into prokaryotic expression vector pet a. protein expressed was purified and specificity was confirmed by immunoblotting. bhk- cells were treated with purified expressed il- protein with ( lg/ml) h prior to fmd infection. cell culture supernatants were collected at h post infection were subjected for elisa and virus titration assay. rna extracted from the cells was subjected to real time pcr for viral rna quantification. log titer reduction was observed in the fmd virus titer in il- treated cells compared to the untreated cells where as virus antigen quantified by elisa has shown a reduction of -folds. -fold reduction in the fmd viral rna copy number was observed in the il- treated cell compared to the untreated measured by qpcr. current study demonstrated the potent anti viral activity of il- on fmdv by inhibiting the viral replication. these results further suggests that il- has the potential role of il- as molecular adjuvant in fmd vaccine development and development of therapeutic for fmd. foot and mouth disease is the most contagious viral disease of farm animals. control of the disease in animals is by vaccination and slaughtering of infected animals. conventional oil adjuvant vaccine has its own limitation. alternate to this genetic vaccines where the dna encoding viral antigen may be a promising approach. naked dna vaccine has limitations like poor uptake of dna by cells and more importantly by nucleus. as a result delivery of naked dna through calcium phosphate nanoparticle was attempted. calcium phosphate nanoparticle is a potential delivery agent which proved to enhance the immune response. fmdv p - cd ''o'' vaccine gene constructs in pcdna . ? entrapped by the nanoparticles was prepared by using different molarity of calcium chloride and disodium hydrogen orthophosphate. the nanoparticles entrapping fmdv p - cd ''o'' and naked dna were presented to the guinea pigs through intramuscular injection to study the mrna expression of antigen by rt-pcr. animals were sacrificed at defined time to collect different organs and total rna was extracted. each time blood was collected to analyse the fmdv specific serum antibodies. dna vaccines presented through calcium phosphate produced transcripts in the injected muscle up to days whereas naked dna up to days. serum antibody levels of naked dna vaccine showed antibody titre till days. whereas nanoparticle injected animals showed serum antibody till days. serum neutralization titres of . were observed in calcium phosphate dna vaccines at about - days, where as naked dna sn titers were observed for short period of - days. the study clearly showed calcium phosphate nanoparticle entrapping fmdv vaccine dna may be a better delivery system for dna vaccines as it confirms availability of the antigen and persistence of antibody for longer duration than naked dna. capripox is highly infectious, contagious, and oie notifiable disease of small ruminants, caused by sheeppox and goatpox viruses which are members of capripoxvirus genus of the family poxviridae. in the present study, we analyzed the partial gene sequences of p protein, an immunogenic envelope protein of capripox viruses (capv) to assess the genetic relationship among different sheep pox and goat pox virus isolates from different geographical areas of the country. product of this gene has been shown to be important in attachment of capv to host cell surface receptors during viral entry and host immune response. the following virus isolates have been used in the analysis: gtpv-uttarkashi, p , vaccine virus; gtpv mukteswar, p , challenge virus; gtpv (akola), gtpv bareilly/ , gtpv ladakh/ and gtpv sambalpur/ , field isolates and sppv srinagar, p ; sppv ranipet, p ; sppv-rf, p , vaccine viruses and sppv makdhoom/ , sppv cirg/ , sppv pune/ , sppv bareilly, sppv / and sppv / , field isolates. in this study, all virus isolates were confirmed by pcr amplification and analysed in pcr-restriction fragment length polymorphism (pcr-rflp) using ecori enzyme to confirm their specificity. further, the amplicons were cloned and sequenced commercially. nucleotide and the deduced amino acid (aa) sequences were compared with published sequences of the members of the genus capripox virus. sequence analysis of partial bp sequence has shown high sequence identity among all indian sppv and gtpv isolates at both nt and aa levels. it revealed a . - % and . for gtpv field isolates where as, % for sppv field isolates at both the nt and aa levels. in general, capv isolates in this study shown . - . and . % homology between gtpv and sppv at nt and aa levels as reported earlier. further, it revealed a unique change of g a in all gtpv isolates resulting in formation of drai site in place of ecori and possible development of restriction enzyme specific pcr-rflp for differentiation of sppv and gtpv from field isolates. orf or contagious ecthyma is considered as non-contagious, proliferative disease and is caused by orf virus of the genus parapox virus of the family poxviridae. it is reported most commonly in sheep and goats and also a zoonotic agent. camels are also infected by orf virus and reported in camel rearing countries as a mixed infection with camel pox, the later is caused by an orthopox virus. in india, there are few reports of the orf virus infection in camels and identified by clinical signs and pcr. in this study, we identified the presence of orf virus from clinical samples of suspected case of sporadic infection in camels by serological and molecular techniques. viral dna isolated from processed scabs used initially in nested polymerase chain reaction as diagnostic pcr which successfully amplified bp fragments and also sequenced to check the fidelity of the product. after confirming the infection by pcr, some of the structural and non-structural genes were amplified for sequence analysis. out of the five genes characterized, the major important one selected for sequence and phylogenetic analysis is b l gene which is homologous to a major envelope protein p k of vaccinia virus. full open reading frame of bp from orf b l was amplified by pcr, cloned and sequenced commercially. nucleotide and deduced amino acid sequences of b l were compared with other published sequences of the members of the genus papapox virus. sequence analysis shows a maximum percent identity of . and (indian orf virus isolates); . and . (other orf isolates); . and . (orf-camel/jodhpur/ ); and . (bovine popular stomatitis virus) and finally . and . (pseudo cowpox virus) respectively at nt and aa levels. phylogenetic analysis of the isolate was also performed using the neighbour joining method in mega program to know the phylogeny relatedness of the virus, which revealed that the isolate is well grouped with the jodhpur isolate and closely related to pseudo cowpox virus. it warrants further analysis of other potential genes to confirm the causative agent of the contagious ecthyma in camels as pseudo cowpox virus. chikungunya an arboviral disease is transmitted through the bite of an infected aedes mosquito. it causes a self limited febrile illness along with arthralgia and myalgia. in some cases neurological and severe hemorrhagic manifestations has been observed. chikv epidemic has been reported in africa, india, south east asian countries and during the current out break imported cases of chikv has been encountered in most of the european countries. the causative agent belongs to the genus alphavirus family togaviridae. human beings serve as the chikungunya virus reservoir host during epidemic periods. outside these periods the main reservoirs are monkeys, rodents, birds, and other unidentified vertebrates. antibodies to chikv have been detected in domestic animals. in the present study we surveyed madanapalli, palamaner, b. kotta kota and tirupati and collected a total of rodent samples, bovine samples; sheep samples and canine samples. total rna was isolated from all these samples and subjected to rt-pcr using a primer pair dvrchk-f/dvrchk-r which could amplify a bp e gene product specific to chikungunya virus (naresh kumar et al. ). all the serum samples were further screened for chikv specific igm antibodies using commercially available ctk biotech strips. none of the samples were found positive either for chikv specific rna or chikv specific igm antibodies. more number of samples from domestic animals as well as rodents are being screened to study their possible role if any in the maintenance of chikv in nature and during the inter epidemic periods. the present study discusses these aspects in detail. petunia hybrida is widely used as experimental host plant for begomovirus identification and its characterization. hitherto, natural infection of begomovirus on petunia has not been reported in india. recently, petunia hybrida grown in and around ludhiana were found to be depicting typical symptoms caused by begomovirus. the symptoms include severe reduction in leaf size, downward curling and distorted leaves. severely infected plant became bushy, stunted and produces no flower. total genomic dna was extracted from the plants showing symptoms of begomovirus, by ctab method. the presence of virus was confirmed by using degenerated primers, designed to identify all the begomovirus prevailing in the world. to identify the strain associated with the disease, the positive samples along with healthy control were tested against different strain specific primers of tomato leaf curl virus, so far reported in india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus. among these, only tomato leaf curl new delhi virus specific primer was able to give the desired amplicon of * bp. hence, it is confirmed that the leaf curl disease of petunia hybrida is associated with tomato leaf curl new delhi virus. this disease of petunia can become a sever production constraint in coming years. from last years ( and ) it was observed that some varieties of brinjal grown in rainy season, showed typical leaf curl type of symptoms. the symptoms include upward curling of the leaves, cupping, vein thickening, reduction in leaf size and distortion of leaves. the severely infected plant remains stunted and bushy, became unproductive or produces only few fruits. the disease was experimentally transmitted from naturally infected brinjal to healthy seedlings by whiteflies (bemisia tabaci) and grafting, but not by mechanical or aphid transmission. to detect the begomovirus associated, total genomic dna was extracted from the plants showing disease symptoms. the presence of virus was confirmed by using pcr based begomovirus geneus-specific primers designed by deng et al., wyatt and brown and rojas et al. these degenerated primers give the expected product size of * , * and * bp, respectively. core coat protein (cp) gene and dna-b was also amplified in the samples using specific primers. to identify the strain associated with leaf curl virus, dna was subjected against primers of different indian tomato leaf curl virus strain i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl new delhi virus primer was able to show the desired product size of * bp. therefore, it was confirmed that leaf curl disease of brinjal is caused by tomato leaf curl new delhi virus in association with satellite b-dna. to identify the strain associated with the disease, all samples were further subjected to the specific primers, designed to amplify all the tomato leaf curl virus strains, so far reported from india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl palampur virus specific primer was able to give the expected product size of * bp. this shows the association of tomato leaf curl palampur virus with leaf curl disease of calendula and marigold. thus, calendula and marigold can act as a reservoir for the tomato leaf curl palampur virus and may cause severe constrain in the production of these important ornamental plants. groundnut bud necrosis virus (gbnv) belongs to serogroup iv of the genus tospovirus in bynayaviridae family and infects several economically important crops all over india. the nucleocapsid protein (np) encoded by the small rna of gbnv encapsidates the viral rnas. apart from this structural role, the np has also been implicated in the replication, transcription, maturation and cell to cell movement. with a view to study the structure and function, the np of gbnvtomato isolate from karnataka was over expressed in e. coli and purified by ni-nta chromatography. the purified np was present as ribonucleoprotein complex and as heterogeneous mixture containing monomers, tetramers and higher order multimers. in order to determine the regions involved in oligomerization and nucleic acid binding, mutational approach was taken. n-and c-terminal deletion clones were generated (n np, n np, c np and c np), over expressed in e. coli, and were purified by a procedure identical to that used for the wild type protein. initial studies on oligomeric status suggested that in addition to n-and c-terminal regions there may be additional regions or residues which contribute to multimerization of np. the amount of rna bound to the truncated proteins was reduced in case of n np, n np and c np. interestingly removal of amino acid residues (natively unfolded region) from the c terminus resulted in complete loss of nucleic acid binding suggesting that the rna binding domain was located in c-terminal region of np. further np was observed to get phosphorylated in in vitro kinase assays by a kinase present in the soluble fraction of tobacco plant sap. both atp and gtp were utilized as phosporyl donors and mn ? was the preferred metal ion which suggests that np might be phosphorylated by a ck -like protein kinase. phosphorylation studies with n-and c-terminal truncated proteins revealed that the site of phosphorylation lies within the amino acid residues - . by mass spectrometric analysis of the protein threonine- and serine- were identified as possible phosphorylation sites. a naturally occurring isolate of virus infecting gherkin (cucumis anguira l.) showing mosaic symptoms of mosaic, leaf distortion and dark green islands in the lamina was identified in the export cultivars of gherkin grown in commercial fields of kuppam rural, chittoor district, andhra pradesh. the virus infection was deadly prevalent among the field that caused a lot of economic damage to the crop that resulted in yield losses and reduced quality of fruits meant for export. symptoms of the infected fruit included blistering and malformation of the fruit. the virus infected leaf samples were collected and initial host range tests were conducted with different cucurbit species showed that the host range include propagation hosts like cucumis anguira (gherkin), cucumis sativus, cucurbita pepo, cucumis melo, langeneria vulgaris, momordica charantia and local assay host like chenopodium amaranticolor. the virus host range was only restricted to cucurbit species and chenopodium. the virus was maintained for further studies on cucurbita pepo by sap or mechanical inoculation. the virus induced mosaic, vein clearing symptoms on pumpkin. electron microscopy of the leaf dip preparations stained with % uranyl acetate from the pumpkin leaves showing symptoms revealed the presence of a long flexuous filamentous particle measuring nm. the virus positively reacted to the polyclonal antisera of papaya ringspot virus-w, potato virus y, tobacco etch virus and also strongly reacted with the polyclonal antiserum of zucchini yellow mosaic virus in direct antigen coated-enzyme linked immunosorbent assay (dac-elisa). because of very strong reaction to polyclonal antisera of zucchini yellow mosaic virus, we tried to amplify the partial nib and cp genes of the virus along with the utr by using two primers zy gctccatacatagctgag acagc and zy taggctttttgcaaacggagtcta at c . total rna from gherkin infected leaves was isolated using trizol ls reagent (sigma). rt-pcr was performed to obtain an amplicon of * . kbp, cloned into fermentas ptz r/t vector and sequenced at mwg biotech, bangalore. sequence analysis revealed that the virus was isolate of zucchini yellow mosaic virus and was showing % of homology to that of the zucchini yellow mosaic virus strain b genome ay and zucchini yellow mosaic virus nat genome ef which were strains reported from israel. the sequence of the present study was submitted to the genbank gq . the results state a suspicion that the virus could have been mobilized by some infected source brought by the commercial israeli based companies into india due to poor quarantine regulations as the gherkin cultivation in these regions is chiefly supported, purchased, exported and marketed by these private companies that are based from israel. this is the first report on molecular characterisation of zucchini yellow mosaic virus infecting cucumis anguira (gherkin) from india. they also exhibited synergism with other virus which was region specific. fifty percent of the total symptomatic plant population was found be positive only for carla while remaining showed mixed infection of carla with tospo in some regions while in others carla virus was found to be associated with cmv. presence of only carlavirus was up to - % incidence, without association of tospo, cmv, poty or tobamo viruses was also observed in some fields. avijit tarafdar, raju ghosh, k. k. biswas plant virology unit, division of plant pathology, indian agricultural research institute, new delhi citrus tristeza virus (ctv), a brown citrus aphid (toxoptera citricidus) transmitted closterovirus under family closteroviridae, is one of the major limiting factors in cultivation of citrus worldwide. ctv is a longest known plant virus having flexuous particle of nm in size. ctv genome is a positive sense ssrna of about kb nucleotide containing open reading frames (orfs) encoding proteins. several biological as well as genetic variants of ctv are reported in all the citrus growing countries in the world. ctv causes decline and death of millions of citrus trees in the world. in india, ctv is a century old problem, and has killed an estimated one million citrus trees till today. in molecular and genetic level, ctv isolates from india were not fully characterized. genetic diversity and sequence divergence in ctv isolates of india are not fully established. further, evidence of recombination and causes of evolution of ctv variants in india have not been studied till date. therefore, in the present study, effort has been made to characterize several indian ctv isolates in genetic level, examine their genetic diversity, identify recombination events and analyze evolution of divergent ctv. a total number of ctv isolates from different regions of india ( from darjeeling hills, five from bangalore, from delhi and from vidarbha) were under taken for genetic study. two genomic regions of ctv, i.e., entire cp gene (cpg) ( nt) and a gene fragment of orf a (orf a) ( nt) were amplified, cloned, sequenced and nucleotides were analyzed. based on cpg, indian isolates shared - % nucleotide identity, and based on orf a they shared - % identity, among them. incongruence of phylogenetic relationship was observed as on sequence analysis five phylogenetic clades based on cpg, and eight clades based on orf a, were generated suggesting the recombination events have been occurred between the sequences of indian ctv isolates. thus, to identify the potential recombination events, and determine the parental sequences in ctv isolates, six recombination detecting algorithms, namely, rdp, genconv, bootscan, maxchi, chimera and siscan were used. out of indian ctv, cpg of and orf a of isolates of ctv showed recombination events suggesting orf a was more prone and fragile to rna recombination as compared to cpg. this findings indicated that high degrees of genetic diversity and incongruent relationships of indian ctv isolates are due to genetic recombination occurred, which may be the important factors in driving evolution ctv variants in india, that was also supported by a splittree decomposition analysis. b. v. bhaskara reddy, y. sivaprasad, k. rekha rani, k. raja reddy department of plant pathology, regional agricultural research station, acharya n.g. ranga agricultural university, tirupati, andhra pradesh sunflower (helianthus annus l.) is one of the most important oil seed crops in the world which ranks third in area after soyabean and groundnut. the sunflower necrosis disease (snd) is characterized by necrosis of leaves, necrosis streaks on petioles, stem, floral parts and stunted growth. the causal agent of the disease has been identified as tobacco streak virus (tsv) which belongs to genus ilarvirus of the family bromoviridae. the suspected tsv infected sunflower samples collected from chittoor district in andhra pradesh were found positive for tsv-dac elisa. total rna was extracted from sunflower using rnaeasy isolation kit (qiagen). the tsv coat protein (cp) gene, movement protein (mp) gene and replicase (rep) gene were amplified by rt-pcr with specific primers, cloned in ptz r/t vector, sequenced and deposited in genbank (gu , gu and gu ). the size of cloned cp gene was bp and codes for amino acids. the cp gene sequence analysis revealed that the tsv-tpt infecting sunflower has - % homology at nucleotide level with soybean, tagietus-tpt and okra-tn isolates and - % homology at amino acid level. the movement protein gene was bp and codes for amino acids. the mp gene sequence analysis showed that it has - % homology at nucleotide level and - % at aminoacid level. chilli (capsicum annuum), the important commercial vegetable/spice of himachal pradesh, is affected by several viral diseases; of them cucumo, tospo, poty and gemini viruses are the most common genera. however, these viruses are not identified clearly and characterized fully, which are foremost needed to formulate the management strategy. therefore, in the present study, effort has been made to identify and characterize the important viruses causing diseases in chilli. in this study, several farms in major chilli growing areas of bilaspur and kangra districts in himachal pradesh were surveyed and infected plant samples were collected randomly. virus infection in these samples were detected by das-elisa using antisera to cucumber mosaic virus (cmv) and potyvirus (group specific) and through slot-blot hybridization (sbh) using cmv, iris severe mosaic poty virus (ismv), tomato spotted wilt tospo virus (tswv) and chilli leaf curl gemini virus (clcuv). based on das-elisa and sbh, the incidence of disease was estimated and ranged from . to . % by cmv and . to . % by potyvirus. to detect tospo and geminivirus in the infected chilli, sbh test was carried out. infected samples showed maximum virus titer in both das-elisa and sbh test were further confirmed by pcr using specific primers. desired sizes of amplicons; * bp, * bp, * bp and * bp of cmv, poty, gemini and tospo viruses, respectively, were obtained. as the present study clearly indicated that cmv appeared as a major one among the viruses infecting chilli in the hilly region of himachal pradesh, two isolates of cmv were characterized in genetic level. thus the amplified products (* bp) of cmv, palampur and palampur were cloned in pgemt cloning vector, sequenced and the sequences were submitted to ncbi database (palampur : acc-fm and palampur : acc-fm ). the sequences were then analyzed and compared with other sequences available in the data base. based on sequence analysis, it was found that present cmv isolates shared % nucleotide identity between them, are closely related with australian cmv isolate cmv-ly (acc-af ) by % nucleotide identity. in phylogenetic tree analysis, it was observed that indian cmv isolates formed same cluster along with cmv-ly. as it is known that cmv subgroup ii comprises cmv-ly, it is concluded that the cmvs of this hilly region of himachal pradesh belong to subgroup ii. chilli is essentially a crop of the tropics and grows better in hotter regions. chlii (capsicum annuum), a member of family solanaceae is an important vegetable and spice crop of immense commercial importance. the pungency in pepper is due to an alkaloid known as capsaicine and peppers are characterized as sweet, hot or mild depending on capsaicine content. the present investigation were conducted to find out the highly resistant cultivars of capsicum annuum against cmv and tylcv among ten cultivars of chilli in agroclimatic condition of aligarh. the highest ( and ) percentage of infection was observed in hc- and kalyanpur type- by showing the positive reaction to cmv by elisa test. no symptoms was recorded in case of bc- , lca- and jca- and showed negative reaction to cmv by elisa. bc- and lca- also showed negative reaction to tylcv by elisa and these were symptomless. maximum infection ( and ) was registered in hc- and c , cultivar. so, the bc- , lca- and jca- has proved highly resistant varieties against cmv and tylcv and these may be used in breeding programmes against viruses. cotton leaf curl virus belongs to the family geminiviridae, genus begomovirus. the members of this family contain circular single stranded dna molecules as their genomes. there are two kinds of begomoviruses-bipartite viruses with genomes consisting of two dna molecules designated dna-a and dna-b and the monopartite viruses which contain only dna-a but not dna-b. in monopartite viruses, the dna-a is accompanied by a small circular dna molecule called dna-b which is essential for the development of typical disease symptoms. cotton leaf curl virus is a monopartite virus and causes the cotton leaf curl disease which has emerged as a major disease of cotton in the indian subcontinent. the non-structural protein ac of cotton leaf curl kokhran virus-dabawali isolate (clcukv-dab) was cloned into pgex x vector and overexpressed in bl (de )plyss e. coli cells. the overexpressed gst-ac protein was purified by glutathione sepharose chromatography. the purified gst-ac protein was found to possess atpase activity. the optimum temperature and ph for the activity were °c and . respectively. the atpase activity was inhibited in presence of edta, showing that it is dependent on divalent metal ions. the activity was supported by magnesium, manganese and zinc ions but inhibited in presence of calcium ions. it was also inhibited by the non-hydrolyzable atp analogue adenosine-b, c-imido triphosphate and in the presence of other nucleotides like ctp and gtp. the k m and the v max of the reaction for atp as the substrate are . mm and . nmol/min/ mg of the protein respectively. the enzyme could also utilize gtp as the substrate. the fact that ac is specifically an ntpase and not a general phosphatase is revealed by the finding that it does not hydrolyze p-nitrophenyl phosphate to yield yellow colour while a similar reaction carried out in parallel with alkaline phosphatase readily yields the colour. it has been suggested earlier that ac may be involved in cell to cell movement of the virus (rojas et al. ) . it is possible that by its ability to hydrolyze atp, ac serves to power viral movement in the plant. thirteen sugarcane yellow leaf virus isolates causing yellow midrib and irregular yellow spot pattern from six states of india were characterized by rt-pcr assays. scylv- f and scylv- r primers were used as forward and reverse primer pairs and the amplified products were cloned and sequenced. comparative coat protein sequence analysis confirmed that all the scylv-indian isolates were clustered into two major groups confirming the existence of two strains of scylv affecting sugarcane crops of india. in a separate experiment, the member of both of the phylogenetic groups were found to be transmitted by the sugarcane aphid, melanaphis sacchari from infected to healthy sugarcane suggesting its secondary spread in nature. the symptoms produced by the virus causing cotton mosaic disease were little bit different in both sap inoculation and under natural field condition. in natural field condition it has shown clear chlorosis type of symptoms on major leaves of plants but in sap inoculated plants veinal chlorosis and mosaic type of symptoms are found to be common. in field conditions infected plants grows erect and have less boll formation. there is no effect found on seed shape or seed size. the initial symptoms produced on cotton leaves after inoculation were wonderful. local lesions observed in second week from inoculation and then they changes to chlorotic type of symptoms and some are necrotic symptoms also. the plants at early stage are found to be affected, has less lateral branch development and hence reduction in yield production. the naturally field infected plants showing good symptoms are also difficult to identify in lateral stage of plant. because they disappear with time. the virus is very easily sap transmissible. the virus is found to be transmitted by thrips palmi and thrips tobacci in persistent manner. no seed transmission is observed. virus showed same physical properties as it shows in stem necrosis of peanut or sunflower necrosis disease. the physical properties are found to be thermal inactivation point (tip) - °c, dilution end point (dep) - to - and longevity in vitro (liv) h, virus infecting nineteen different host plants are identified belonging to five different types of families viz. malvaceae, chenopodiaceae, compositae, leguminaceae and solanaceae. however they found to produce same types of symptoms as in most of the host that have been tested before. in elisa test report it is found that the virus showing positive test only with anti serum of tsv of a cowpea and cotton but negative reaction with pbnv of cowpea and cotton which clearly denied possibility of presence of pbnv in cotton producing these kinds of symptoms elisa report clearly shows that tsv antiserum of cowpea is showing positive results with clear chlorotic types of symptoms. a powerful approach to functional genomics, and an alternative to the massive generation of transgenic plants, is the use of the recently described virus induced gene silencing (vigs) process, which allows viral vectors to knock out the function of a gene-of-interest. vigs is based on a silencing mechanism that regulates gene expression by the specific degradation of rna. as a tool for reverse genetics, vigs has many advantages over other common ways to study gene function because of the ability of viruses to replicate and move systemically within a plant. vigs can generate a phenocopy of a mutant without all the troubles of traditional methods of mutagenesis. geminiviruses with their small dna genomes and ease of inoculation through agrobacterium, are excellent candidates for vigs vector development. as a first step, the geminivirus bhendi yellow vein mosaic virus, characterized in our lab (jose and usha, virology : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) has been chosen. the satellite b dna associated with this virus has a single open reading frame (bc ). bc is essential for symptom development but not for replication. therefore, bc has been replaced by a multiple cloning site harbouring sali, xbai, bamhi, bsrgi and xhoi, initially in a cloning vector and then in the binary vector containing the partial tandem repeat of the b dna. in the place of the bc orf, the plant phytoene desaturase gene has been cloned and the resulting construct was used for agroinfiltration along with the partial tandem repeat clone of the begomovirus (dna a component chilli (capsicum annuum l.) plants exhibiting prominent symptoms of begomovirus like: leaf curl, vein swelling, shortening of petioles, crowding of leaves and stunting of plants were collected from rorkee, uttarakhand and dhaulpur, rajasthan, india. total genomic dna was isolated from naturally infected chilli samples and pcr was carried out with coat protein (located in dna-a) gene specific primers. as expected to the primers, * bp dna fragments were amplified from the infected chilli samples. to know the bipartite nature of the virus isolates, nuclear shuttle protein (located in dna-b) gene specific primers were employed which also resulted in positive amplification of * bp dna bands with all the coat protein tested positive samples. to ascertain the association of dnab component with the virus isolates, a set of dna-b specific primers were used which resulted in positive amplification of full length (* . kb) dna bands in the chilli samples collected from rorkee, uttarakhand, however, multiple sizes bands were resulted with the samples collected from dhaulpur, rajasthan. these findings confirmed that both the virus isolates under study are bipartite begomovirus associated with dna-b satellite. the sequencing of the pcr products is under progress which analysis will be discussed. groundnut bud necrosis virus (gbnv) belonging to the genus tospovirus, which is a unique member of the family bunyaviridae, infects several economically important crops. the virus has three genomic ssrna segments namely s (ambisense), m (ambisense) and l (negative sense). the s rna codes for nucleoprotein (np) and non-structural protein (nss) from viral complimentary and viral strands respectively. many viral nonstructural proteins such as ns of hepatitis c virus, yellow fever virus, dengue virus, sv large t antigen and cytoplasmic inclusion protein of tamarillo mosaic potyvirus are known to exhibit rna/dna stimulated ntpase, dntpase and helicase activity. nss of gbnv does not have any sequence similarity with any of the above mentioned viral rna/dna helicases but has a ntp binding domain. however, it has been implicated as suppressor of gene silencing in vivo. with a view to elucidate the mechanism by which nss could act as a suppressor of gene silencing and examine the other potential roles of nss in the life cycle of the virus, the gbnv (to) nss was over-expressed in e. coli and purified by ni-nta chromatography. in vitro studies with the purified rnss suggest that it exhibits an rna stimulated ntpase activity. many of the proteins that possess the rna/ dna stimulated ntpase and datpase activity, are also shown to have atp dependent nucleic acid unwinding activity. it was therefore of interest to examine whether nss has the nucleic acid unwinding activity. the helicase assays revealed that nss has dna/rna helicase activity. helicase activity of nss was absolutely dependent on atp and mg ? ion. nss could unwind dsdna substrate with overhang, or overhang. mutation of the crucial lysine in walker motif a (k ) severely affected the unwinding activity where as mutation of aspartate residue in walker motif b (d ) resulted in only % loss of activity. in this regard, rnss is a unique enzyme which does not have the canonical helicase motifs but can catalyze dsdna/dsrna unwinding in an atp and mg ? dependent manner. the rnss might act as a suppressor of by unwinding the dsrna, the substrate for dicer. in addition to being a suppressor of ptgs, nss may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. this new research finding on nss might pave way for further studies on its role in viral replication and transcription. yellow vein mosaic disease of pumpkin (cucurbita moschata) poses a serious threat to the cultivation of this crop in india. the disease was found to be associated with whitefly-transmitted bipartite begomoviruses were detected in varanasi field using polymerase chain reaction (pcr) with primer design through coat protein conserved region of begomoviruses from ncbi database. all plant samples showing symptoms were infected with begomovirus. the virus species were provisionally identified by sequencing * bp of the viral coat protein gene (av ageratum conyzoides is commonly known as billygoat-weed, chick weed, goatweed and whiteweed. in india it is popularly known as bill goat weed. it is an annual herbaceous plant with a long history of traditional medicinal uses in several countries of the world and also reputed to possess varied medicinal properties including the treatment of wounds and burns. in cameroon and congo, it is used traditionally to treat fever, rheumatism, headache, and colic. during survey in and around gorakhpur in , ageratum plants were found affected with the symptoms of leaf curling, mosaic mottling and leaf yellows. the infected leaf samples were processed for virus identification and association with pcr assays. total dna was extracted and pcr were performed with begomovirus specific primers (tlcv-cp). a * bp band was consistently amplified on % agarose. the pcr products were directly sequenced and sequence was submitted in genbank with the accession no. gq . the blast search analysis showed highest similarity of % with the ageratum enation virus. vernonia cinerea leaves with yellow vein symptoms were collected around crop fields in madurai. a bp product amplified from total dna extracted from symptomatic leaves with degenerate primers designed to amplify a part of the av gene from begomoviral dna a component was cloned and sequenced. based on the above sequences, specific primers were designed and the full length dna a of nucleotides with typical genome organization of begomoviral dna a was obtained and was submitted to embl data base (acc no: am ). the sequence comparison with other begomoviruses revealed the closest identity ( %) with emilia yellow vein virus from china and less than % with all known begomoviruses. the international committee on taxonomy of viruses (ictv) has therefore recognized vernonia yellow vein virus (vyvv) as a distinct begomovirus species. conventional pcr could not amplify the dna b or dna b from the infected tissue. however, the b dna ( bp) associated with the disease was obtained (acc no: fn ) by the rolling circle amplification-restriction fragment length polymorphism method (rca-rflp) using phi dna polymerase. sequence analysis shows that dna b of vyvv has the highest identity ( %) with dna b of ageratum leaf curl disease and - % with the b dna associated with other begomoviruses. infectious clones of vyvv dna a and dna b as dimers were made using the products of rca-rflp. these infectious clones will be used for agroinfection of vernonia and the results will be discussed. this is the first report of the molecular characterization of vernonia yellow vein virus (vyvv) from vernonia cinerea in india. production of bulb and seed crop of onion (allium cepa l.) is hampered by onion yellow dwarf virus (oydv) and iris yellow spot virus (iysv) with an incidence of . % and . % in bulb crop and . % and . % in seed crop, respectively in the popularly grown cv. hisar- . four symptom-based variants of oydv designated as grade a, b, c and d produced varied types of symptoms in onion crop incurring heavy losses in bulb and seed production. iysv caused tiny hay coloured spots of different shapes and sizes on leaves and scapes which later coalesced and led to drying and lodging of scapes. the plant height, bulb weight and bulb size were . cm, . g and . cm in plants infected with oydv, . cm, . g and . cm in iysv infection, . cm, . g and . cm due to their combined infection, as compared to . cm, . g and . cm respectively, in healthy plants of bulb crop. in plants infected with oydv grade a the plant height was minimum ( . cm) whereas the number of umbels was maximum ( . umbels/pl.) but other yield parameters viz., weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were recorded to be the lowest. the minimum reduction in plant height ( . cm), weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were recorded in oydv grade d. the plant height was . cm with . umbels per plant, . g weight/umbel, seeds/umbel, . g seed weight/umbel and . g seed yield/plant in iysv infected plants. the plant height ( . cm), umbels/plant ( . ), weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were found to be the lowest in combined infection of oydv and iysv diseases in comparison to higher values in healthy controls ( . cm, . , . g, , . g, . g, respectively). a minimum reduction in the test weight, germination and seed vigour index were found ( . g, . % and ) due to oydv grade a infection, whereas these were . g, . % and in iysv disease infected plants and . g, . % and in combined infection of oydv and iysv diseases in comparison to . g, . % and in healthy plants. the maximum hampering of seed vigour parameters was recorded due to iysv infection. lodging of scapes caused by this disease was responsible for heavy losses in seed production and seed quality. cotton leaf curl disease is one of the major threats to cotton cultivation from northern india. survey conducted during , observed the disease incidence ranged from to % from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar. in order to study genetic variability in the virus, twelve clcuv isolates were partially characterized ( bp common region, full length av gene and partial sequences of ac and av gene). full length characterization of representative isolates from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar is under progress. partial sequence analysis of clcuv isolates revealed that, the virus isolates collected during cropping season are closely related to cotton leaf curl burewala virus from pakistan and results were discussed. pratibha singh, h. s. savithri department of biochemistry, indian institute of science, bangalore tospoviruses, belonging to the family bunyavirideae, infect economically important plants such as groundnut, tomato, watermelon etc. they have a tripartite genome, with l, m and s segments of rna, in pseudo circular (panhandle) form. the viral genomes encode four structural proteins (l, n, g and g ) in the antisense orientation, and two non structural proteins nss and nsm in the sense orientation. the nsm is the only protein unique to tospoviruseses that infect plants in the bunyaviridae family and hence is proposed to be important for cell to cell movement. ground nut bud necrosis virus (gbnv), a member of the tospovirus genus, is the most prevalent virus infecting several species of leguminosae and solanaceae plants in india. total rna was isolated from gbnv infected tomato leaves and rt-pcr was performed using appropriate primers to amplify the nsm gene. the pcr product was cloned in pgex x vector. the recombinant nsm clone was transformed into bl (de ) e. coli cells and over-expressed by induction with . mm iptg. sds-page analysis of induced and uninduced fraction revealed the presence of overexpressed protein of expected size. the soluble gst-nsm was purified by gsh sepharose affinity chromatography. purified gst-nsm was shown to interact with in vitro transcribed rna transcript by electrophoretic mobility shift assay. further nsm was shown to interact with viral encoded proteins np and nss using elisa and yeast two hybrid system. nsm was also shown to be phosphorylated in vitro by pellet fraction of plant sap. thus the recombinant gbnv nsm possesses the characteristic features of a movement protein such as nucleic acid binding, interaction with nucleocapsid protein, and ability to undergo posttranslational modification. solanum melongena, commonly called as egg plant is one of the most important vegetable crop in the world. it is cultivated widely in the tropical and sub tropical regions. several viruses such as cucumber mosaic cucumo virus (cmv), potato virus-y (pvy), potato virus-x (pvx) and tobacco ring spot virus (trsv) infect egg plant under natural conditions. in india major crop losses due to cmv infection in brinjal is % (fao stat- ) . in the present study the infected leaf samples were collected from local fields of ramapuram, chandamama palli, chandragiri, madanapalli, yadhamari, durgasamudram villages in and around tirupati, were tested for cmv infection by dac-elisa with cmv antisera. the resulting positive samples were further inoculated to the raised brinjal seedlings of selected varieties through mechanical sap inoculation. different varieties of brinjal like mullabadhine, ankhur, ravya, mattigulla, casper and easter egg were used for monitoring the susceptibility to cmv infection. the mosaic symptoms were observed after weeks of inoculation in all varities of brinjal except mullabadhina. among all these susceptible varities ankhur variety is selected to study induced biochemical changes such as chlorophylls, carbohydrates, proteins, nucleic acids and polyphenol oxidases in cmv infected brinjal leaves. in the infected leaves considerable reduction in chlorophyll and starch and increase in total proteins, sugars, rna and polyphenol oxidases was observed when compared to healthy leaves. the amount of total starch, protein and dna decreased to about , and lg/g respectively in infected leaves, where as sugars ( lg/g), rna content ( lg/g) and polyphenol oxidase activity was increased as compared to healthy leaves. the above results suggests that there is an altered concentrations of chlorophyll, proteins, nucleic acids, carbohydrates and polyphenol oxidase activity in the brinjal leaves due to the effect of cucumber mosaic cucumo virus infection. leaf analysis was found to be used as widely accepted diagnostic tool to assess the nutritional status of the vegetables. the present study deals with these aspects in detail. the total rna and dna was isolated from infected leaf samples. rt-pcr assays were performed using sugarcane yellow leaf virus (scylv) specific primers (scylv- f and scylv- r). the infection of scylv was detected in all the collected samples, which showed the expected size (* bp) amplicon during rt-pcr. in another experiment with nested pcr analysis, a phytoplasma characteristic . kb rdna pcr product were amplified from dnas of all infected samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs p /p and fu /ru . dna extracts from plants with yellow mid rib and leaf yellows produced products of bp, which gave typical phytoplasma profiles when digested with hae iii and hha i. no pcr amplifications were produced using dna from symptomless plants. our results suggest that the yellow mid rib and leaf yellows symptoms on sugarcane varieties in uttar pradesh and uttarakhand states of india exhibiting midrib yellowing and leaf yellows symptoms is mainly caused by mixed infection of scylv and scylp. the affected clumps showed reduction in stalk height as compared to healthy fields. thirty-one sugarcane mosaic isolates belonged to sugarcane mosaic virus (scmv) and sugarcane streak mosaic virus (scsmv were collected from china and india), confirmed in indirect elisa and rt-pcr amplification with scmv and scsmv-specific primers. the amplicons ( . kb) from the coding region of coat protein (cp) were cloned, sequenced and compared to each other as well as to the sequences of scmv isolates from sugarcane (australia, usa, china, brazil, mexico and south africa), maize (australia, china, iranian) and one scsmv isolate from sugarcane (india) in genbank. maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the scmv subgroup that included isolates from china and only isolates from india, and the scsmv subgroup that contained all isolates from india. maize dwarf mosaic virus (mdmv) and johnsongrass mosaic virus (jgmv) were not detected in any of the samples tested. a strong correlation was observed between the sugarcane groups and the geographical origin of the scmv isolates. the millable sugarcane samples from china contained a virus tentatively described as sorghum mosaic virus (srmv). three isolates from nine chewing canes in fujian, yunnan and guizhou provinces of china also contained srmv, and the other samples including five isolates from india was found infected with scmv. no srmv infection has been detected in sugarcane mosaic samples from india. sequence comparisons and phylogenetic analysis indicated that srmv can be considered as the most common and prevalent potyvirus infecting sugarcane in china, however in india sugarcane streak mosaic virus is dominant in causing mosaic symptoms on sugarcane. dig-labeled dna probe complementary to coat protein (cp) region of tobacco streak virus (tsv) sunflower isolate was designed for the sensitive and broad-spectrum detection of tsv isolates, the most devastating virus in india. dot-blot and tissue print hybridizations with the digoxigenin labeled probe were performed for the tsv detection at field levels. here, dot-blot hybridization was used to check a wide number of tsv isolates with a single probe and sensitivity with different sample extraction methods. the probe with cp conserved region prepared from sunflower pcr amplicon was hybridized with the tsv field isolates of gherkin, pumpkin, sunflower, marigold and globe amaranth samples because of highly conserved with little variability in cp region. the sensitivity limits were decreased from total nucleic acid to partially purified and crude extract preparations. in particular, tissue blot hybridization offers a simple, reliable procedure as dot-blot, but requires no sample processing. because there is minimal sample preparation, tissue-print hybridization could be an important component of tsv management programs. thus, the above non-radioactive labeled probe techniques can facilitate in screening the samples during tsv outbreaks and in quarantine services. savita patil, rupali sawant*, k. banerjee virology group, agharkar research institute, macs, g.g. agarkar road, pune two mycobacterium smegmatis strains (ari lab nos. v and v ) were employed for the isolation of mycobacteriophages from soil and sewage samples. mycobacteriophages were isolated from soil samples collected from an area surrounding the tuberculosis (tb) ward, naidu hospital, pune, against m. smegmatis strain v . these were numbered as v , v and v and were isolated by using washed-cell preparation method. the bacteriophages against the other m. smegmatis strain, i.e. v , were isolated from soil samples (collected from around tb ward, sassoon hospital, pune). some of these phages (viz.v , v ) showed plaques at °c but not at °c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v , v , v , v and v . also, phage dilution medium (pdm) as described by chaterjee et al. ( ) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p . the various passages of the phage isolates v , v , v , v and v (i.e. original, p , p and p ) were stored at - °c. pvp- effect on pigments due to geminivirus infection on cowpea (vigna unguiculata) shail pande*, naveen pandey, k. shukla mahatma gandhi p. g. college gorakhpur, d.d.u. gorakhpur university, gorakhpur geminiviruses are one of the most important group of viruses causing economic losses in tropics. the symptom produced are yellowing of leaves which directly affect the pigments of diseased plants it in turn affects productivity and yield of diseased plant. cowpea vigna unguiculata is one of the important crop cultivated throughout india for its green pods which are used as vegetables and seeds are used as pulse. cowpea is affected by many viruses amongst them geminiviruses are one of the important virus on the cowpea plant. in the present study total chlorophyll content was studied in leaf of cowpea of diseased and healthy plants using arnon's method. carotenoids were also studied using ikan's method. it was found that chlorophyll content in diseased plants were lower compared to healthy plant similar results were found with carotenoids so the geminivirruses infection lowers the chlorophyll and carotenoid content in diseased plants which reduces yield of diseased cowpea plant. shweta sharma , amrita banerjee , j. tarafdar , r. rabindran , indranil dasgupta * department of plant molecular biology, university of delhi, south campus, new delhi; bidhan chandra krishi vishwavidayalaya, kalyani, nadia, west bengal ; tamil nadu agricultural university, coimbatore, tamil nadu rice tungro disease is an important disease of rice, caused by a joint infection by two viruses: rice tungro spherical virus (rtsv) and rice tungro bacilliform virus (rtbv) in south and southeast asia. the complex of rtbv and rtsv is transmitted by an insect vector green leaf hopper (glh). previously we reported complete genomic sequences of two geographically distinct isolates of rtbv; rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) collected from the field in mid- s. both the sequences showed high homology all along the genome but showed divergence from previously reported southeast asian isolate i.e. rtbv-phil (philippines). to check whether a time period of a decade has resulted into variability in the genomic sequence of different isolates of rtbv in india, we cloned and sequenced the complete genome of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari collected from the field in . the complete nucleotide sequence of the dna fragments covering the whole genome of rtbv was determined using universal primers m f and m r and by primer walking, without any ambiguities remaining. the nucleotide sequences of overlapping clones were assembled and analyzed using the dna analysis software generunner and blastn program of ncbi. homology search at the nucleotide and amino acid level were performed using the blastn and blastp (respectively) programs of ncbi. multiple sequence alignments were performed using clus-tal-w software. sequence analysis results thus obtained showed that both the recently obtained complete genomic sequences of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari showed very high homology (both at the nucleotide and amino acid levels) with the two previously reported rtbv isolates from india i.e. rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) all along the genome. as observed earlier both the sequences diverged significantly from the southeast asian isolates. this suggests that even after the spatial and temporal difference (a time gap of approx years) between the two previously reported rtbv isolates and the recently reported one, there is very little sequence variability between them. this further strengthens the earlier reports that the rtbv genomes in india are highly conserved. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of % with the rtbv west bengal isolate and % with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. complete genomic rna sequences of two geographically distinct isolates of rice tungro spherical virus (rtsv), a member of the genus waikavirus, family sequiviridae, were determined from india. out of the two previously reported sequences, the indian isolates were closer to the resistance breaking strain rtsv-[vt ] than rtsv- [phila] . between them, the indian sequences showed nucleotide as well as amino acid identities of %. a moderate homology was observed between the leader peptide and a putative helper component protein involved in insect transmission of the maize chlorotic dwarf virus, a closely related waikavirus, indicating its possible transmission-related function. unlike rice tungro bacilliform virus, which causes rice tungro disease jointly with rtsv, and is significantly different between isolates from india and philippines, rtsv genomes were observed to be much more conserved between isolates from the two countries. rice tungro bacilliform virus (rtbv) are believed to be the joint causative agents for the devastating tungro disease of rice prevalent in south and southeast asia [ ] . rice tungro disease has become the major cause of production losses in rice during last three decades in several rice growing states of india. here, we report, for the first time the complete sequence analysis of two geographically distinct indian isolates of rtsv. we analyze the deduced protein sequences and their phylogenetic relationship with the two complete rtsv sequences from philippines as well as with other members of sequiviridae family. we provide molecular evidence that the indian isolates of rtsv are closely related to those from the philippines. we had earlier reported that rtbv isolates between india and philippines differ significantly from each other [ ] . this study was undertaken in order to see whether rtsv isolates from india also show similar difference from those reported from the philippines. frequent outbreaks of tungro were reported near kanyakumari in the last - years. the present work was undertaken to clone and sequence the full-length rtbv and rtsv genomes from the infected rice plants collected from above region and to analyze the similarity of its genetic material with the existing indian isolates of rtbv and rtsv. a . kb dna fragment encoding the reverse transcriptase gene of rtbv genome was amplified and cloned in t/a vector and was sequenced commercially. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of % with the rtbv west bengal isolate and % with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. similarly, the cp region of rtsv was amplified by rt-pcr and was cloned in t/a vector. recently, rice tungro disease has been reported from kanyakumari district of tamil nadu. it is important to determine the genetic nature of this isolate in order to develop resistance strategies. it is thus necessary to clone and characterize the viruses from kanyakumari and to determine the mechanism of virus resistance in transgenic lines. rice tungro disease is an important viral disease of rice. rice tungro is caused by infection by two viruses: rice tungro bacilliform virus (rtbv) and rice tungro spherical virus (rtsv). rtsv is a plant picornavirus with a kb single stranded rna genome. it belongs to genus waikavirus in the family sequiviridae and is necessary for transmission of the two viruses by the leafhopper vector nephotellix virescens. rtsv rna is translated to form a large polyprotein, which is then self cleaved to form the viral proteins, including the three coat proteins, replicase, protease. studies have been conducted on rtsv from philippines. correct information of sequence variability of viral isolates to check whether different geographical conditions like those present in india select for genotypically variable strain and to design for transgenic resistance strategy, information on rtsv from india is absolutely essential. the objective of this study was to clone rtsv isolates from india and compare the genetic diversity of indian isolates from other southeast asian isolates and amongst each other. also develop strategy to impair the attack of virus-complex on rice. the achieve this, complete genomes of two isolates from india were cloned by amplifying different genes by rt-pcr and subsequently cloned in ta vectors, followed by sequencing. subsequently constructs containing cp - , antisense replicase, sense replicase and double stranded replicase were cloned in plant transformation vector. these constructs were used to transform aromatic rice variety from indian-pusa basmati (pb ). pcr analysis of the above plants was done to check the stable insertion of insert in the transgenics. jatropha (jatropha curcas) of the family euphorbiaceae is being grown in india as a major commercial fuel (bio-diesel) crop. jatropha is cultivated in districts of potential states of india. unfortunately, the cultivation of jatropha is limited by the severe mosaic disease. recently, a severe mosaic disease with significant disease incidence was observed in - on j. curcas grown in experimental plots of nbri and j. gossypifolia, a weed growing road side around lucknow and kathaupahadi, madhya pradesh. the disease consisted of the symptoms of severe mosaic, blistering, leaf distortion and stunting of whole plant and no fruit/seed production in severely affected plants. symptomatology and whitefly population observed on them suggested the occurrence of begomovirus infection. to detect the begomovirus infection, the total dna from leaf samples of infected jatropha plants was extracted and polymerase chain reaction (pcr) were performed using three sets of begomovirus genus specific (cpit-i/cpit-t, paliv /paric and paliv /palic ) primers and the expected size * bp, . kb and . kb amplicons were obtained which confirmed the begomovirus infection. further to identify the begomovirus/es and investigate the genetic diversity among them exists if any, the * . kb amplicons were cloned and sequenced. the sequence data were deposited in the genbank database under accession nos.: gq and fj (from j. curcas) and eu and fj (from j. gossypifolia). during blast analysis gq and fj shared highest % sequence identity with each other and - %% with sri lankan cassava mosaic virus (aj , aj , aj , aj and aj ) and indian cassava mosaic virus from india (ay ) therefore, designated as two strains of jatropha mosaic india virus-lucknow. blast analysis of eu showed maximum % similarities with croton yellow vein mosaic virus (aj ), % with tomato leaf curl new delhi virus (dq ) and - % with papaya leaf curl virus (aj and y ), therefore, identified as strain of croton yellow vein mosaic virus. blast analysis of the virus isolate (fj ) showed highest % identities with tomato leaf curl virus-bangalore ii (tolcv-b ii-u ) and - % with tomato leaf curl karnataka virus (tol-ckv, ay , fj ), therefore, considered as new begomovirus species ''jatropha yellow mosaic india virus''. the phylogenetic analysis of gq and fj (from j. curcas) and eu and fj (from j. gossypifolia) was performed along with some selected isolates of begomovirus which showed [ % sequence identities during blast analysis. the isolate eu showed closest relationship with croton yellow vein mosaic virus while fj showed separate clustering of all the four begomovirus from jatropha species. during phylogenetic analysis these isolates formed three separate clusters, therefore, they were considered as three distinct begomoviruses. the above data clearly show that some genetic diversity exists among the begomoviruses infecting jatropha species in india. bitter gourd (momordica charantia l.) of the family cucurbitaceae, also known as bitter melon is extensively cultivated in north eastern region of uttar pradesh, india. it is regarded as one of the world's major vegetable crops and has great economic importance. a severe yellow mosaic disease on bitter gourd (momordica charantia) with a significant disease incidence was observed during the survey of different locations of eastern up, india in the year . the whitefly (bemisia tabaci) population was also observed in the vicinity. the characteristic disease symptoms and whitefly population indicated the possibility of begomovirus infection. total dna were isolated from infected as well as healthy leaf samples. two primer pair (tlcv-cp and roja's primer) were used to study, which resulted * bp with tlcv-cp in / samples and * . kb amplicons with roja's primer in / samples. for further identification of the begomovirus, the pcr amplicons were cloned and sequenced (genbank accession no. eu and eu , respectively). the blastn search analysis of eu indicated - % identity with several isolates of tomato leaf curl new delhi virus (tolcndv). the phylogenetic analysis also showed closest relationships of the isolate (eu ) with tolcndv isolates. based on highest sequence identity and closed relationships with tolcndv the virus isolated from bitter gourd was considered as an isolate of tomato leaf curl new delhi virus. while, blastn search analysis of eu isolate, shared highest - % identites with pepper leaf curl bangladesh virus (peplcbv) isolates. the phylogenetic analysis of the virus isolate with selected begomovirus isolates revealed a closest relationship with peplcbv. these results confirmed the association of peplcbv on bitter gourd. study revealed the variability of viruses on bitter gourd in eastern up, india. tobacco streak virus groundnut isolate was characterized biologically by taking six cultivars (jl , tmv , k , k , k ) and one pre-release culture (k ) using seedlings of - days old under glasshouse conditions. there were clear differences were observed among cultivars tested regarding incubation period, percent seedling wilt and time taken to death of seedlings. k- was least susceptible among all the cultivars tested and it supported least virus titer (a nm: . - . ). both localized (necrotic lesions on leaf, veinal necrosis, leaf yellowing, wilting) and systemic (petiole necrosis, necrotic lesions on young leaves, death of top growing buds not only on main stem but also on all primaries (side shoots), followed by stem necrosis, stunted growth, axillary shoot proliferation with small leaves having general chlorosis, peg necrosis, pod necrosis, pod size reduction, wilt of plants) symptom were observed in all cultivars tested. biological differentiation of tsv and gbnv was made by sap inoculation of both viruses separately using susceptible groundnut cultivar jl under glasshouse conditions. there were certain similarities and differences were observed between these viruses infecting groundnut. seed infection of tsv ranged from . to . % in seeds collected from naturally infected and sap inoculated groundnut cultivars/pre-releases (jl , tmv , k- , k- , k- and k- ) belonging to spanish and virginia types. tsv was detected both in pod shell and seed testa from pod samples produced by sap inoculation under glasshouse conditions. however, seed transmission of tsv was not observed in groundnut. coat protein (cp) gene of three groundnut tsv isolates (gn-ap- - ; gn-ap - ; gn-ap - ) were sequenced and all the three isolates contained a single open reading frame (orf) of bp nucleotide and could potentially code for amino acids (aa). cp gene of tsv isolates originating from different hosts shared high degree of sequence identity both at nucleotide ( . - %) and amino acid ( . - %) levels respectively. tones grown in an area of . . ha (fao stat ). in india papaya is grown in nearly , ha with an annual production of , , tones (fao stat ) and occupies fourth place in the world. the crop is severely affected by a number of viruses. papaya ring spot virus (prsv-p) is the most important virus. the detection of virus infection in plants has traditionally involved either bioassay on indexing plants and or immunological methods (hill , torrence and jones ) . use of nucleic acid probes has improved the detection and sensitivity of viruses. the most common non-radioactive probes are biotynilated probes, which are very specific and sensitive. papaya ring spot virus (prsv-p) is a positive sense ssrna virus belonging to the genus potyvirus family potyviridae and transmitted by aphids. prsv-p coat protein gene region was used as template cdna for probe preparation. dot-blot hybridization with the biotin labeled probe were performed for prsv-p detection. the clarified sap of healthy and infected plants were serially diluted and spotted onto the nitrocellulose membrane, hybridized to biotin labeled probe. biotin labeled rna's are employed as probes, with a subsequent detection based on streptavidin-alkaline phosphatase conjugates. the sensitivity for viral detection of the biotin labeled probe was found to be sensitive than enzyme linked immunosorbent assay (elisa). in recent years tospovirus is causing devastating damage to the yield of vegetables in india. it infects economically important crops viz., tomato, chilli, peppers, groundnut, watermelon and various legumes. now it is emerging as severe disease in brinjal also. in order to monitor the natural occurrence and distribution of tospovirus in vegetable, surveys were conducted in the predominant brinjal growing areas of gujarat, karnataka, maharashtra and andhra pradesh during - incidence ranging from to %, to %, to %, and to . % respectively. samples collected from different places of india were found positive to pbnv in direct antigen coating-enzyme linked immunosorbent assay (dac-elisa). pbnv infected brinjal plants showed mosaic mottling of leaves with leaf distortion, longitudinal streaks on the stem and necrotic rings on leaves and fruits. early infection led to severe stunting and abnormal fruiting. biological and molecular characterization of pbnv-brinjal isolates were compared with other isolates and results are discussed. for identification of virus causing mosaic symptoms on soybean various host plants were tested. plants species belonging to the different families viz. caricaceae, graminae, leguminosae, malvaceae and solanaceae were tested. the virus produced symptoms on diagnostic plant species like chenopodium album, c. quinoa, helianthus anus, phaseolus vulgaris and vigna ungiculata. among tested families the leguminosae that were the host of virus included arachis hypogea, the virus causing mosaic symptoms in soybean is inactivated between and °c and between dilution of - to - . all the inoculated plants of assay host showed the symptoms at °c but not at °c. similarly local lesions produced at - but not at - . the virus in crude sap was infectious up to h but not at h at room temperature. however, the percentage infectivity decreased progressively as the aging of the sap was increased at room temperature. on the basis of reactions on diagnostic hosts pvp- identification and characterization of potyvirus infected chilli (capsicul annum l the virus under study caused mild mosaic and severe mottling symptom in leaves of infected plants. the dilution end point (dep) of the virus was found to be - to - , longevity in vitro (liv) - days at room temperature ( °c), thermal inactivation point (tip) - °c. electron microscopy of purified virus preparation revealed the presence of flexuous particle of size nm long and nm in width with characteristic cytoplasmic inclusions: pinwheels and scrolls. the virus was transmitted by sap and by aphid myzus persicae. the host range study revealed that the host species were restricted to family chenopodiaceae and solanaceae. on the basis of above characteristic, the virus under study was identified as potyvirus associated with mild mosaic and severe mottling symptom in capsicum. phytoplasma causing grassy shoot disease and sugarcane yellow leaf viruses are important pathogens of sugarcane. these pathogens are causing severe losses in sugarcane productivity. with a view to producing virus and phytoplasma free planting material of sugarcane, experiments were undertaken using infected varieties of sugarcane growing at the farms of sugarcane research institute. apical meristems measuring about mm in length, were dissected out, surface sterilized and cultured on agar gelled murashige and skoog's (ms) medium containing growth regulators for shoot induction. the established shoot cultures were multiplied through repeated subcultures on fresh media at - days interval. elimination of gsd and scylv was confirmed through molecular analysis of regenerated plants using specific primers of scylv and gsd. results revealed that apical meristem culture technique is effective in eliminating the pathogens like scylv and phytoplasma (gsd) from the infected clones. this is probably the first report on elimination of grassy shoot disease in sugarcane through meristem culture. papaya ringspot virus (prsv), which causes the most widespread and devastating disease in papaya, isolates originating from different geographical regions in south india were collected and maintained on natural host papaya. the entire coat protein (cp) gene of papaya ringspot virus-p biotype (prsv-p) was amplified by reverse transcription-polymerase chain reaction (rt-pcr). the amplicon was inserted into pgem-t vector by t-a cloning method, sequenced and sub cloned into a bacterial expression vector prset-a using directional cloning strategy. the prsv coat protein was over expressed as fusion protein in e. coli. sds-page gel revealed that cp expressed as a * kda protein. the recombinant coat protein (rcp) fused with his-tag was purified from e. coli using ni-nta resin. the antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified prsv. the purified rcp was used as an antigen to produce high titer prsv specific polyclonal antiserum. the resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (ic-rt-pcr) assay and compared its sensitivity levels with elisa based assays for detection of prsv isolates. ic-rt-pcr was shown to be the most sensitive test followed by dot-blot immunobinding assay (dbia) and plate trapped elisa. key: cord- - cyz o c authors: barclay, wendy s.; callow, kathleen a.; sergeant, marianne; ai‐nakib, widad title: evaluation of an enzyme‐linked immunosorbent assay that measures rhinovirus‐specific antibodies in human sera and nasal secretions date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: cyz o c rhinovirus‐specific antibodies have traditionally been detected by their ability to neutralise the homologous rhinovirus serotype in tissue culture. recently, however, we have described an enzyme‐linked immunosorbent assay that detects rhinovirus‐specific antibodies in sera and nasal secretions [barclay and al‐nakib, ]. here we describe an evaluation of the elisa in a study involving adult volunteers inoculated intranasally with human rhinovirus type (hrv‐ ). pre‐and post‐inoculation serum samples and pre‐inoculation nasal washings were tested for the presence of hrv‐ ‐specific antibodies by elisa. such antibodies were associated with protection against infection when present locally in nasal secretions, but when also present in the serum they were associated with protection against both infection and the development of illness. the antibody concentrations showed strong correlation with each other and with that of antibodies detected by the neutralisation test. following hrv‐ infection, rises in hrv‐ ‐specific iga in sera detected by elisa occurred more frequently than rises in neutralizing antibody. these results suggest that the elisa is a sensitive and reliable indicator of recent infection, as well as a predictor of homologous immune status. rhinoviruses are the major causative agents of the common cold [couch, . earlier studies suggested that, following infection, rhinovirus serotype-specific antibodies appear in both the serum and nasal secretions and that the presence of such antibodies confers long-term protection against re-infection by the homologous rhinovirus serotype [doggett, . these antibodies have traditionally been detected by neutralisation tests [hamparian, . recently, however, we have established a sensitive enzyme-linked immunosorbent assay that detects rhinovirus-specific antibodies in sera and unconcentrated nasal washings. this new assay is simple, rapid, and can differentiate between classes of rhinovirus-specific immunoglobulins. in a preliminary study involving volunteers inoculated intranasally with human rhinovirus el (hrv-el), we showed that the presence of circulating rhinovirus-specific iga, rather than igg, protected volunteers from infection [barclay and al-nakib, . in the present study we describe a more extensive analysis of the anti-human rhinovirus type (anti-hrv- ) immunoglobulin responses in both sera and nasal washings measured by elisa. thus volunteers were inoculated intranasally with hrv- , and the course of infection was monitored both clinically and virologically . our data confirm the importance of iga anti-rhinovirus immunoglobulins in protection against re-infection and/or illness; we provide evidence that a rise in specific serum iga following infection is a good indicator of a recent infection. samples were obtained from volunteers taking part in a separate study to test the therapeutic effect of zinc gluconate lozenges on colds caused by hrv- [al-nakib et al., . they were housed in isolation according to standard procedures at the common cold unit [beare and reed, . serum samples were collected before the trial and weeks after rhinovirus inoculation. these were stored at - °c. nasal washings for antibody measurement were collected before the trial by instilling ml of phosphate-buffered saline (pbs) into each nostril. the volunteer then forcibly expelled the liquid into a pot containing glass beads. after vortexing for sec to break up mucus, the effluents were centrifuged at , rpm in a microcentrifuge, and the supernatants were stored at - °c. nasal washings for virus isolation were taken from to days after inoculation using standard methods [barclay and al-nalub, . these were added to an equal volume of nutrient broth and stored at - °c. viruses were isolated by inoculation of ohio hela cells. samples of . ml nasal washing were inoculated into confluent test tube cultures, these were rolled at °c. after - days they were examined for the presence of virus cytopathic effect (cpe). colds were assessed by a combination of tissue count and subjective and objective evaluation of severity of symptoms, that is, clinical score [beare and reed, . on the basis of virus isolation and these criteria, volunteers were divided into three groups for analysis as follows: ) colds group: those from whom virus was isolated and who had clinical colds; ) subclinically infected group: those from whom virus was isolated but who did not have colds; ) not infected group: those from whom no virus was isolated and who did not have colds. human rhinovirus type (hrv- ) was grown in ohio hela cells and crude antigen preparations were made as previously described for hrv-el [barclay and al-nakib, . control antigens was prepared in the same way from uninfected monolayers . fifty millilitres of crude hrv- antigen with a titre of approximately lo .' tcid /ml was concentrated by ultracentrifugation and resuspending the virus pellet in ml of pbs; . ml of this antigen was then emulsified in an equal volume of freund's complete adjuvant (fca) and used to inoculate one rabbit subcutaneously. six weeks later, the rabbit was inoculated intramuscularly with an emulsion of . ml hrv- and . ml of freund's incomplete adjuvant (fica). fourteen days later, the animal was bled out. the resulting hyperimmune serum had a neutralisation titre of : , against lo tcidso hrv- . this was stored at - °c. the elisa for hrv-zspecific immunoglobulins in sera and nasal secretions was an adaptation of that previously described for hrv-el-specific immunoglobulins [barclay and al-nakib, . that is, polystyrene round-bottomed micro elisa plates (nunc immunoplates u type ) were incubated overnight at °c with pl per well of a : ,ooo dilution of rabbit anti-hrv- hyperimmune serum in carbonate/bicarbonate buffer, ph . . wells were drained and washed three times in pbs containing . % tween (pbs-t). then pl of hrv- or control antigen (cag) diluted : in pbs-t containing % bovine serum albumin (bsa) was added to alternate rows of wells. plates were incubated for hr at "c, then drained and washed as before. one hundred microlitres of standard or test sample diluted in pbs-t containing % bsa and % cag was added in duplicate to both hrv- -and cag-coated wells. as standard, a pool of known hrv- -antibody-positive sera or nasal washings was titrated on each plate in one-half loglo dilution steps. samples were diluted to - . and respectively, for the assay of specific iga and igg in sera and for the detection of specific iga in nasal washings. plates were then incubated for hr at °c. after washing as before, p of either anti-human iga alkaline phosphatase conjugate (sigma) or anti-human igg alkaline phosphatase conjugate (icn) was added, diluted : ,ooo in pbs-t with % bsa, and incubated overnight at °c. plates were finally washed four times with pbs-t and p substrate (sigma p-nitrophenol phosphate tablets dissolved at mg/ml in % diethanolamine buffer) was added to each well. plates were incubated for hr and optical densities (od) at nm were read in a dynatech mr elisa reader using a reference filter of nm. mean ods from duplicate wells were corrected by subtracting the mean od of cag-coated wells. the serum standards were arbitrarily allotted . loglo units and the nasal washing standards, . loglo units. using corrected ods, from the titrations of these on each elisa plate a standard curve was calculated. corrected ods for each sample were then read off on this standard curve. thus each sample was awarded a number of loglo units, which related it to the known standards. antibody units for hrv- specific iga in nasal washings were further corrected by normalising for total iga in the sample. the elisa for total iga in nasal washings was performed as previously described by callow [ . this normalisation resulted in a more significant difference between the specific iga concentrations in nasal secretions for the three groups. to establish the size of a significant rise in hrv- -specific antibody following hrv- infection, paired sera from volunteers who had received an intranasal inoculation of saline (no virus), and who had been housed in isolation exactly as the other volunteers, were tested for hrv- antibodies. a significant rise in hrv- specific antibody was then set as the mean difference between these paired sera plus sd of that mean. thus a significant rise for elisa hrv- -specific iga in sera was a difference of . loglo units, and for igg serum-specific antibody this value was . log lo units. a rise in hrv- -neutralising antibody was considered significant if the difference was fourfold (i.e., . loglo steps) or greater. neutralising antibodies in sera were detected by microneutralisation tests against a challenge of tcidso homologous rhinovirus as previously described [barclay and al-nakib, . the statistical analyses were performed on an ibm xt computer, using the programme, statistical package for personal computers (spp, patrick royston, clinical research centre, northwick park hospital, harrow, england). for comparing the frequencies of antibody rises in the three volunteer groups, a x test was used, with yates' correction. for examining the significance of the differences in antibody concentration between the groups, a rank analysis of variance was used; for comparing antibody concentrations of different types, or the magnitude of antibody rises, spearman's rank correlation coefficient was calculated. of the volunteers who were intranasally inoculated with hrv- , ( . %) had colds, ( . %) had subclinical infections, and ( %) did not become infected. figure shows a) the mean hrv- -neutralising antibody, b) specific igg and c) iga in the serum, and d) specific iga in nasal secretions (normalized for total iga) in pre-inoculation samples from the three groups of volunteers. the presence of hrv- -specific antibody in the serum appears to confer protection against both infection and symptoms. thus volunteers who did not get infected had more hrv- -specific antibody in their sera (iga and igg antibodies) than those who had subclinical infections or those who were infected and developed colds. those who developed colds had the lowest amount of hrv-zspecific antibody in their sera. these differences in amounts of hrv-zspecific serum antibody between the three groups were highly significant for elisa-specific iga and neutralising antibody ( p < .ool) but were not significant for elisa-specific igg. volunteers volunteers were divided into these three groups according to their subsequent response to hrv- inoculation, as described in the text. who were not infected had significantly more hrv- -specific iga in pre-inoculation nasal washings ( ' < . ) than those who became infected whether or not they had colds. however, the differences between the concentrations of this antibody when the three groups were considered individually, or between the colds group and the subclinically infected group were not significant. therefore, although the presence of local specific antibodies appears to protect against infection, they do not appear to prevent illness caused by the infection. table i shows the number of volunteers in each of the three groups who had a significant rise in hrv- -specific serum antibody following hrv- intranasal inoculation. the frequencies of rises in neutralising antibody and elisa-serum-specific iga were significantly different in the three groups (p < . ), the proportion of rises being highest in the colds group, and least in the not infected group. the difference in frequency of rises in specific igg in the three groups was not significantly different. thirty-six of the volunteers showed rises in both elisa-serum-specific iga and neutralising antibody, but only two volunteers showed rises by neutralisation and not by elisa. in contrast, nine volunteers showed rises by elisa and not by neutralisation (table ) . thus the rises in elisa-serum-specific iga were slightly more frequent than for neutralising antibody ( of or . % vs. of or . %), suggesting that the former might be a more sensitive test for detecting such rises. the concentrations of the various hrv- -specific antibodies were tested for correlations with each other and with the size of antibody rises. substantial correlations were found between the concentrations of serum antibodies and between rises in these antibodies, as shown in figure a . generally the presence of, or a rise in, neutralising or elisa serum igg or iga antibody was significantly associated with the presence of, or rise in, the other rhinovirus-specific antibodies. there was also a positive correlation (p < . ) between the presence of hrv- -specific iga in nasal secretion and that in the serum. in addition, a negative correlation was found between the concentration of specific iga in nasal secretion and the size of the rise in specific iga in serum ( p < .ol) (fig. b ). thus volunteers with low amounts of pre-inoculation hrv- specific iga in nasal secretions were those most likely to show a significant rise in serum-specific iga. similarly, there was also a negative correlation between the concentration of specific iga in pre-inoculation sera and rises in rhinovirus-neutralising antibody and vice versa ( p < .ool). this suggests that a low specific iga or neutralising antibody concentration in pre-inoculation sera was associated with subsequent large rises in both neutralising antibody and specific iga following inoculation with hrv- . we have found a significant association between the presence of neutralising antibody and elisa-serum-specific iga and protection against re-infection and illness. thus, volunteers who had high levels of hrv-zspecific iga or neutralising antibody in their sera did not get infected or develop symptoms of colds when inoculated with hrv- . in contrast, those who had lower levels of specific iga or neutralising antibody had subclinical infections, and those who had little or no antibody became infected and developed colds. interestingly, the presence of hrv- specific iga in nasal secretions was only associated with protection against infection but not illness. our findings are in general agreement with those for another virus that causes common colds-coronavirus e [callow, . however, in that study, serum-specific igg appeared to be at least as effective in preventing infection and disease as serum-specific iga. furthermore, coronavirus-specific iga in nasal secretion, while significantly shortening the period of virus shedding, also appeared to reduce symptoms. recently, a similar study involving samples obtained from volunteers inoculated with another rhinovirus serotype (hrv- ) and tested by elisa for homologous antibodies, appears to confirm the association between elisa-serumspecific iga and protection against reinfection (callow and sergeant, unpublished data). the standard serological test for a recent rhinovirus infection is the detection of a rise in neutralising antibody. this study showed that rises in circulating rhinovirusspecific iga were at least as reliable for this purpose. in fact, the present elisa detected rises in iga in three subclinically infected and four uninfected volunteers, in whose sera the neutralisation test failed to detect such rises. these elisa antibody rises may have been "false," since the "uninfected" volunteers did not excrete virus. in a separate study in our laboratory, however, several nasal washings from these hrv- -inoculated volunteers from which no virus was isolated (i.e., the not infected group) contained hrv- antigen detectable by a biotidstreptavidin bridge elisa system. thus, although they were not excreting infectious virus that could be isolated in ohio hela cells, they were actively producing viral antigens, which suggests that viral replication may have been taking place. the size of rises in hrv- -specific iga in the serum after virus inoculation showed a significant negative correlation with the concentration of specific iga in the pre-inoculation nasal secretions and with the presence of serum neutralising antibody, indicating that volunteers who were most susceptible to infection and illness, in fact, showed the largest antibody response. the small number of serum-specific igg rises detected by the elisa and the weak correlation between the presence of pre-inoculation igg and protection against re-infection might be explained by the fact that, generally, the elisa detects large amounts of specific igg in all volunteer pre-inoculation sera so far tested. we suggested previously that this may be due to the use of crude tissue culture fluid harvests as antigens in the elisa that contain a mixture of bothjkll or native virus particles and empty capsids. the latter have been shown to cross-react with antisera against heterologous serotypes [lonberg-holm and yin, . thus, igg persisting from previous infections with other rhinovirus serotypes could cross-react in the elisa. we have recently obtained evidence that purification of the homotypic full virus particles on sucrose density gradients for use as antigens in the assay improves the specificity of the test (barclay, unpublished data) . this cross-reactivity does not appear to be a problem when an iga class-specific conjugate is used, presumably because iga antibody responses are of short duration and do not persist as long as those of the igg class. in conclusion, the elisa described in this study is an accurate predictor of the jmmune status of an individual to the homologous rhinovirus and can be a reliable diagnostic assay detecting recent rhinovirus infection by measuring significant rises in specific iga. thus it could be useful in evaluating the efficacy of any novel rhinovirus vaccine or antiviral compound in the future by measuring the incidence of infection following challenge with the homologous virus. we are now studying the possibility that this elisa system can be modified to detect rises to other rhinovirus serotypes. prophylaxis and treatment of rhinovirus colds with zinc gluconate lozenges an elisa for the detection of rhinovirus specific antibody in serum and nasal secretion chemoprophylaxis and virus infections of the respiratory tract effect of specific humorai immunity and some non-specific factors on resistance of volunteers to respiratory coronavirus infection the common cold: control? the journal of infectious diseases prevention of colds by vaccination against a rhinovirus: a report by the scientific committee on common cold vaccines diagnostic procedures for viral, rickettsia and chlamydia infections antigenic determinants of infective and inactivated human rhinovirus type we are very grateful to dr. d.a.j. tyrrell for helpful discussion and advice. we thank miss morag forsyth, miss caroline dearden, and mrs. patricia chadwick for performing virus isolations, and mrs. nicola bailey for collecting samples. miss wendy barciay is supported by an mrc studentship. miss marianne sergeant is supported by nih grant no. r a - . key: cord- -kxa zbc authors: bolton, jessica s.; chaudhury, sidhartha; dutta, sheetij; gregory, scott; locke, emily; pierson, tony; bergmann-leitner, elke s. title: comparison of elisa with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination date: - - journal: malar j doi: . /s - - - sha: doc_id: cord_uid: kxa zbc background: profiling immune responses induced by either infection or vaccination can provide insight into identification of correlates of protection. furthermore, profiling of serological responses can be used to identify biomarkers indicative of exposure to pathogens. conducting such immune surveillance requires readout methods that are high-throughput, robust, and require small sample volumes. while the enzyme-linked immunosorbent assay (elisa) is the classical readout method for assessing serological responses, the advent of multiplex assays has significantly increased the throughput and capacity for immunoprofiling. this report describes the development and assay performance (sensitivity, linearity of detection, requirement for multiple dilutions for each sample, intra- and inter-assay variability) of an electro-chemiluminescence (eclia)-based multiplex assay. methods: the current study describes the development of a multiplex eclia-based assay and characterizes the sensitivity, linear range, and inter- and intra-assay variability of the eclia platform and its agreement with the traditional elisa. special emphasis was placed on potential antigenic competition when testing closely related antigens in the multiplex format. results: multiplexing of antigens in eclia provides significant practical benefits in terms of reducing sample volume requirements and experimental time. beyond the practical advantages of multiplexing, the eclia provides superior assay performance when compared to the elisa. not only does eclia show good agreement with the elisa assay, but the linear range of eclia is also sufficiently wide to permit single-dilution measurements of concentration without the need to do serial dilutions. the lack of antigenic competition allows the simultaneous testing of closely related antigens, such as plate antigens representing different alleles of the same protein, which can inform about cross-reactivities—or lack thereof—of serological responses. conclusion: the advantages of the newly developed tool for assessing the antigen profiles of serological responses may ultimately lead to the identification of biomarkers associated with various disease stages and or protection against disease. serological measures have long been used as either correlates of protection induced by a wide range of licensed vaccines targeting pathogens such as yellow fever, tetanus, polio, hepatitis a and b, measles, pertussis, rubella (reviewed in [ ] ), or as markers of exposure to a variety of pathogens [ ] . testing sera from preclinical and clinical studies has also been used to determine the potency of vaccine formulations as well as their potential to induce cross-species or cross-serotype reactive antibodies. enzyme-linked immunosorbent assay (elisa) have been the standard readout method to answer these aforementioned questions. the advent of multiplex testing platforms, such as the electro-chemiluminescence immunoassays (eclia), and bead-based flow cytometric assays enables the simultaneous detection for different antibody specificities and significantly increases the throughput of testing. the nature of multiplex platforms is ideal for sample sparing, enabling more in-depth analyses compared to single-plex assays such as the elisa. depending on the serological assay platform, antigens are either simply coated onto assay plates as in the case of the elisa or they require modifications such as biotinylation or chemical linkage to fluorescent beads. in the case of the eclia, antigens require biotinylation to complex with proprietary linkers that allow targeted binding to specific regions in the assay well. the eclia technology tested here allows up to ten antigens to be coated in a single assay well. the eclia follows the same logistics as the elisa: assay plates are coated with antigens, then non-specific binding is reduced by a blocking step to exhaust remaining antigen-binding sites in the well, and finally, samples are added to the assay wells. antibody binding to the plate antigens is detected by adding a secondary antibody specific for the immunoglobulin heavy chain of the antibodies in the test sample. for use in the eclia, the polyclonal secondary antibody is coupled with a proprietary sulfo-tag as the reporter molecule. lastly, the substrate for the sulfo-tag is added. upon inserting the specialized eclia plates into the reader, an electric pulse initiates the substrate conversion, resulting in chemiluminescence. a high-resolution camera quantifies the eclia signal in the various sectors of the well and reports the luminescence signal in each well sector. one of the significant advantages of eclia is that the substrate is activated by the reader thus eliminating any variability as result of timing associated with the addition of the substrate to the wells and the plates, which can be an issue in the elisa. a wide range of reagents are available for both the elisa and the eclia, and several kits are available for clinical indication [ ] . the results from the two platforms are typically reported as titres (od titre or endpoint titre for elisa) or mean luminescence signal for eclia. quantitative data can be generated if a standard curve using purified immunoglobulins of a known concentration is run in parallel with the test samples for both assay platforms. the present study describes a newly established eclia-based readout for malarial antigens using a model system in which human and nonhuman primate sera reactive to the circumsporozoite protein (csp), the lead antigen for malaria vaccine development, were used as test sera. the plate antigens were either the full-length csp [ ] , or peptides representing the central csp-repeat region or c-terminal end of the csp. plate antigens with significant epitope-overlap were chosen deliberately to address potential antigenic competition when simultaneously testing sera for reactivity with different epitopes. the performance of the new eclia-based readout was compared to that of a qualified, malaria-specific elisa performed in an international serology reference center, since the elisa is a commonly employed serological readout in malaria (due to the relatively basic requirement for hardware), and historical comparison to earlier results within our program spanning close to years [ , ] . the elisa requires testing of several replicates of a serially-diluted sample to either determine the od titre or endpoint titre. in the case of a quantitative elisa, several sample dilutions need to be tested to ensure that the od of the sample falls within the linear range of the standard curve. the objective of this study was to identify the serological assay platform that has the highest sensitivity, specificity, and linear range. furthermore, the current study sought to determine whether simultaneous testing of closely related antigens in the same well of the assay plate was subject to antigenic competition. the antigens used for this study were derived from the sequence of the circumsporozoite protein (csp, strain d ), the main surface protein of the plasmodium falciparum parasite. the pfcsp-fl protein is comprised of tyr - asp linked to pro - ser [ ] ; "repeat" is a keywords: serology, vaccine, antigen, multiplex, antigenic competition, elisa, electro-chemiluminescence -mer peptide representing the central repeat region (nanp ); c-term is a recombinant protein representing the c-terminal fragment (aa - ); pf is an epitope within the c-terminus that has been used as a functional marker when evaluating anti-csp antibodies induced by vaccination [ , , ] . to characterize the eclia platform and compare it to the classical elisa, pre-existing cspimmune nonhuman primate (nhp) samples (n = ) [ ] and a de-identified human csp-immune serum pool were used. commercial human pooled serum (gemini biosciences, sacramento, ca) was used as negative (malaria-naïve) control serum. two mouse monoclonal antibodies, one specific for the c-terminus of the csp (clone e , path/mvi), and one specific for the csprepeat region of the csp (clone a , path/mvi), were used as assay controls. the pfcsp-fl was biotinylated using the lightning-link rapid biotin conjugation kit (expedeon, san diego, ca) according to manufacturer's instructions. the peptides were synthesized with a biotin-tag (atlantic peptides, concord, nh). the elisa assay was performed in the malaria serology laboratory (usmmrp, wrair silver spring, usa) employing full-length csp, nanp peptide and c-terminal peptide (pf ) as plate antigens as previously described [ , ] . the coating concentrations of the plate antigens were nm for csp-fl, and nm for the nanp repeat and pf peptides. elisa titres are listed as endpoint dilution at an optical density (od) of . the described multiplex eclia methodology is based on the mesoscale u-plex platform and -spot eclia plates (msd, gaithersburg, md). an overview of the eclia platform regarding setup, assay logistics and data acquisition is given in additional file : fig. s . biotinylated proteins were diluted to desired concentrations using coating diluent ( . % bsa, xpbs). all calculations were done based on molarity. µl of each biotinylated protein ( nm) was combined with µl of a unique u-plex linker provided by the u-plex platform (msd), vortexed, and then incubated at room temperature (rt) for min. post incubation, µl of stop solution (msd) was added to the biotinylated proteins and linker mix, vortexed, and incubated at rt for min, resulting in a × coating concentration. all u-plex-coupled protein solutions for the multiplexing were combined into one tube ( µl each of the eight, u-plex-coupled protein solution). the u-plex-coupled protein solutions were brought up to ml with stop solution, creating a × multiplex coating solution. fifty µl of the × multiplex coating solution was added to each well of the u-plex -assay plates. plates were sealed with sealing tape (thermo scientific, waltham, ma) and incubated at rt for h on a titramax plate shaker (heidolph, schwabach, germany), shaking at rpm. coated plates can be stored for up to seven days at - °c, based on manufacturer information. after incubation, the plates were washed with a working solution of × msd wash buffer (msd) three times ( µl/well). sera were diluted to desired concentration with diluent (msd) and added to each well ( µl/well). the plates were sealed and incubated at rt for h on a plate shaker ( rpm). plates were washed three times with × msd wash buffer ( µl/well). the detection antibody, sulfo-tag goat anti-human antibody was diluted to µg/ml in diluent (msd) and added to the wells ( µl/well). plates were sealed and incubated at rt for h on a plate shaker ( rpm). after washing, µl a working solution of × read buffer t (r tc- ; msd) was added to each well and the plates were read on the meso quickplex sq (msd), per manufacturer's instructions. for the elisa assay, antibody titres for all four antigens were calculated using the linear extrapolation based on antibody dilutions closest to an od of , to estimate the titre at od = , as is standard practice in the wrair malaria serology lab. for the eclia data, antibody titres were calculated using a -parameter logistic ( pl) fit model [ ] . the pl model is to fit data from the entire titration curve, providing a more robust estimate of the titre at a particular signal intensity. eclia titres were calculated for a luminescence intensity of , intensity units (iu). the antibody titres for the elisa and eclia assays were assessed for agreement using the bland-altman analysis for three antigens: csp-fl protein, nanp csp-repeat peptide, and c-term protein. the bland-altman analysis compares the difference in the eclia and elisa titres (y-axis) with the average of the eclia and elisa titres (x-axis). the shapiro-wilk test was applied to determine whether the differences between the two assays were normally distributed, using an alpha value of . . if the differences were determined to be normally distributed, the standard deviation of the differences was used to determine the limits of agreement between which % of the differences would be expected to fall. to determine whether there was a systematic trend in the difference between eclia and elisa titres (eclia titre-elisa titre) as a function of antibody concentration ((eclia titre + elisa titre)/ ), a linear regression analysis using lm function was carried out in the r statistical package. a linear fit was performed, then the % confidence interval of that linear fit estimated, and the statistical significance of whether the slope of that fit was non-zero determined. a non-zero slope would indicate a systematic trend in the discrepancy between the eclia and elisa titres as a function of serum concentration. the linear range of an instrument is the antibody concentration range where the read-out of a sample is proportional to the concentration. the linear range of the eclia assay was assessed in two ways. first, the correlation of the eclia luminescence intensity was measured at single-point dilutions with the antibody titres calculated using all the dilutions, across all samples. second, to assess linearity directly, the change in signal intensity (intensity, i) was calculated as a result of a change in antibody concentration (concentration, c), or Δintensity/Δconcentration, across the range of antibody concentrations and dilutions measured. then the Δi/Δc curve was estimated by first plotting the Δintensity and Δconcentration from consecutive data points in the correlation plot and then by applying a loess smoothing function using the loess function in r statistical package. the dilution and concentration span at which Δi/Δc ≈ is indicative of the linear range, while a Δi/Δc ≈ indicates that the eclia instrument is either below its sensitivity limit (at low concentrations) or saturated (at high concentrations), and the readout is unresponsive to differences in antibody concentrations. to establish a multiplex assay using an eclia platform, several parameters (i.e., antigen coating concentration, antigenic competition between closely related antigens, sample dilutions) were optimized and the performance of the assay determined in regards to specificity, linearity, and throughput. four different, closely related antigens were tested to simulate potential field applications where either different epitopes of a given antigen or different alleles of the same antigen may be tested. the first step was to determine the optimal coating concentration for the eclia plates. based on the manufacturer's suggestions, the range of concentrations for the csp-fl protein vs. csp-derived peptides was based on the molecular weight (fig. ) . the coating conditions for subsequent experiments were nm for the csp-fl protein and nm for the peptides as these concentrations represented the upper end of the linear titration curves. a potential drawback of the eclia compared to the elisa may be that antigens have to be biotinylated to enable coating of the assay plates. to demonstrate the impact of biotinylation on the reactivity of antibodies to the antigens, competition assays were set up to demonstrate specificity and epitope accessibility of the biotinylated, sector-specific and linker-coupled antigens (fig. ) . eclia plates were coated with the biotinylated/sector-specific linker-coupled pf peptide using u-plex linker . malaria naïve pooled human serum (specificity control) and the csp-immune serum pool were tested at a : , dilution. unlinked, non-biotinylated pf peptide was used as competitor at different concentrations (two-fold dilutions starting at nm, which is the concentration of the linked plate antigen). competing equal concentrations of plate-bound vs. soluble pf peptide results in a roughly % competition. this could be due to different orientations and valencies when providing the peptide in a monomeric form vs. a format that may resemble multimers (due to the closer spatial arrangement of the peptide on the eclia well spot). in conclusion, biotinylation of the tested antigens does not alter the reactivity with csp-immune antibodies. the next step in optimizing the assay conditions for a quantitative multiplex assay that is able to test closely related antigens in parallel was to determine whether the various u-plex linkers were equivalent and did not introduce a bias in the analysis. biotinylated protein aliquots were complexed with u-plex linkers , , , , , and plates were coated with the different u-plexcoupled antigen solutions. a wide range of dilutions ( : , - : , , ) of a human csp-immune serum pool was tested to determine potential quantitative differences in the luminescence signal (fig. ) . the results demonstrated that the u-plex linkers were equivalent and differences in signal strength only reflect differences in the fine specificity of test samples. to enable multiplexing of closely related antigens, it was important to determine whether such antigens compete with each other for binding to antibodies in the sample (fig. ) . the multiplexed eclia experiment tested the full length antigen (csp-fl), its central repeat region (nanp), its c-terminal fragment protein (c-term) and the smaller c-terminal peptide pf . the optimal coating concentrations ( nm for csp-fl, nm for the fragments/protein subunits) were applied to coating either wells with one antigen only (singleplex) or a cocktail consisting of all peptides (multiplex). csp-immune serum and malaria-naïve human pooled serum (negative control) were used at : dilution to determine whether multiplexing resulted in lower luminescence signal, which would indicate antigenic competition (fig. a) . to demonstrate specificity of the response, mouse monoclonal antibodies (mabs), specific for the c-terminus or the csp-repeat region, were tested against all plate antigens (fig. b, c) . titrations of these mouse mabs were performed to demonstrate that no antigenic competition occurs at any antibody concentration and to further establish the peptide was linked with the five different, randomly selected linkers and then eclia plate wells coated in a singleplex format. csp-immune pool was used at dilutions indicated on x-axis to detect potential differences in the equivalency of the linkers. the signal with the negative control serum (specificity control) did not exceed mls < . data expressed as mean luminescence signal (mls) (± sd) of two independent experiments). % cv was less than % for all tests specificity of the responses in the single-vs. multiplexed format. the c-terminus specific mab e did not react with the csp-repeat peptide (fig. b) and the csp-repeat -specific mab a did not react with the c-terminal fragments of csp (fig. c) . it is noteworthy that the titrations of the mabs showed a different dose response curve; mab e yielded a typical response curve with a linear portion and a saturation point for both the csp-fl and the c-terminal fragments. in contrast, the titration of the csp-repeat-specific mab a did not reach saturation despite a wide range of concentrations. both mabs responded stronger with their respective fragment than with the csp-fl. in summary, no antigenic competition was detected when using either csp-immune human serum or mouse monoclonal antibodies as evidenced by comparable signal strength in the singleplex and the multiplex assay format. the variability of the elisa platform has been well documented [ ] . the accepted %cv in the malaria serology laboratory (wrair) has been ≤ % for the plate antigens described in the current study. to complete the characterization of the eclia platform, human cspimmune and control serum pools were repeatedly tested over the course of eight months and by two operators to determine the robustness of the data obtained with this assay platform (fig. ) . using csp-immune human serum, there was a clear hierarchy in the reactivity to the different plate antigens: the highest reactivity was against the csp-fl followed by the c-terminal fragment, the fig. testing of closely related antigens to identify antigenic competition. human csp-immune serum pool ( : dilution) tested for reactivity against either singleplexed antigens (sp) or multiplexed antigens. a in addition, titrations of csp c-terminus specific mouse mab e b and csp-repeat-specific mab a c were tested reactivity against either singleplexed antigens (sp) or multiplexed antigens. data are expressed as mean luminescence signal (mls) of two independent experiments for each panel. % cv was less than % for all tests. the luminescence signal with malaria-naïve serum (specificity control) did not exceed mls < c-terminal protein (pf ), and the csp-repeat peptide. this may reflect the number of epitopes that are available for antibody binding or may indicate the need for some conformation. overall, the minor variability (≤ . % cv for csp-fl, ≤ . % cv for csp-repeat, and ≤ . % cv for both c-terminus antigens) in the results indicate that this assay was highly reproducible and significantly lower than the variability of the elisa. a sample set of nonhuman primates immunized with a particle-based csp vaccine [ ] were tested in parallel by an established, qualified elisa [ , ] vs. the multiplex eclia assay. samples were serially diluted (range of : to : for elisa and : to : , for eclia) and simultaneously tested in both assays. for the elisa data, antibody titres were calculated using linear extrapolation, as is common practice. for the eclia data, antibody titres were calculated using a -parameter logistic fit fig. a correlation plots of the elisa and eclia titres for csp-fl (left), csp-repeat (nanp; center), and c-term (right) antigens from samples from nhp animals immunized with a particle-based csp vaccine. slope and correlation coefficient are shown. b bland-altman plots comparing elisa and eclia assays for the aforementioned antigens. the mean difference between the elisa and eclia titres (black line) and the % confidence interval of the difference (dotted black line) are shown. the systematic trend in the difference in elisa and eclia titres as a function of antibody concentration is represented by the slope (dashed red line), along with the % confidence interval of the slope (solid red line). the slope is labelled along with a corresponding p-value (*p < . , **p < . ) model [ ] , which better accommodates the wide dilution range used in this assay. a correlation analysis was carried out comparing the elisa and eclia titres for three antigens: csp-fl, csp-repeat, and csp c-term (fig. a) , followed by a bland-altman analysis to assess the level of agreement between the two assays (fig. b) . for the csp-fl antigen, there was a high level of agreement between the elisa and eclia titres. the titres from the two assays had an r of . , and the bland-altman analysis showed that the eclia titre was within . -fold of the elisa titre with % confidence. furthermore, although there was an absolute bias in the eclia titres relative to the elisa titres, which is to be expected as titres are a relative measure of concentration, there was no systematic trend in the discrepancy between eclia and elisa titres across the concentration ranges measured here. for the csp-repeat antigen, there was also good agreement between the two assays, with an r of . , and eclia titres found to be within . -fold of the elisa titres with % confidence. however, unlike in the case of the csp-fl antigen, there was a systematic trend of increasing difference between eclia and elisa titres at lower antibody concentrations, indicated by a slope of . in the bland-altman plot (p < . ). this suggests that the eclia assay may be more sensitive than the elisa assay at these low concentrations. for the c-term antigen, there was moderate agreement between the elisa and eclia titres, with a r of . , and eclia titres found to within . -fold of the elisa titres with % confidence. as for the csp-repeat antigen, the bland-altman plot revealed a systematic trend of increasing difference between the eclia and elisa titres at lower antibody concentrations (slope . , p < . ), again suggesting that the eclia assay may be more sensitive at lower concentrations. one important question for high-throughput screening is whether sample testing needs to be done at multiple dilutions. although time consuming and resource intensive, multiple dilutions are often necessary to ensure that all the samples are measured at least once by the instrument within its range of linearity-that is, the concentration range where the readout is linearly related to the concentration. outside of this range, for example, below the sensitivity of the instrument or above the concentration where the signal is saturated, the readout no longer reliably reflects antibody concentrations. therefore, the next step was to assess the linear range of the two assay platforms eclia and elisa. towards that end, serially diluted human csp-immune serum pool was tested across twofold dilutions, from : to : , (fig. a ) and the serum samples were measured against three antigens in the elisa: csp-fl, csp c-term, and csp-repeat (nanp). the readout showed linear behavior over a serum concentration range of approximately -fold (six twofold dilutions. the change in elisa signal intensity was calculated as a function of a change in antibody concentration (Δi/Δc) for the fifteen dilutions to assess the degree of linearity (fig. b ). an estimated Δi/Δc near . would indicate perfect linearity, while a Δi/Δc of . would indicate either being below the sensitivity limit of the instrument (at low concentrations), or saturation of the instrument (at high concentrations). at dilutions of : to : for csp-fl, and : to : for c-term and csp-repeat, the Δi/Δc was near . , indicating some degree of linearity. this corresponds to a linear range of approximately tenfold concentrations. to measure the linear range of eclia, serially diluted human csp-immune serum pool was tested across eight fivefold dilutions, from : to : , , (fig. c ) and the serum samples were measured against three antigens: csp-fl, csp c-term, and csp-repeat (nanp). the eclia assay readout showed linearity for a range of five fivefold dilutions for all three antigens, encompassing a -fold range in antibody concentrations. the signal intensity showed robust linearity with relation to concentration, achieving Δi/Δc greater than . , and close to . , across this wide range of concentrations for all three antigens (fig. d) . these findings not only demonstrate the wide linear range of the eclia assay platform, but also highlight its high sensitivity even at very low antibody concentrations. for csp-fl-and csp-repeatspecific antibodies, the highest dilution still exceeded the sensitivity limit of the instrument, while for csp c-term, the three highest dilutions did appear to be below the sensitivity limit. given the wide linear range of eclia, the next step was to determine whether single-dilution measurements of the biological samples would be sufficient to accurately determine serum concentration across all the samples against the csp-fl antigen. the biological samples tested here were from thirty csp-immune nonhuman primates. towards that end, the single dilution read-out was compared across four dilutions ( : , , : , : , , : , ) with the titre calculated from the serial dilutions as the 'gold standard' (fig. a) . for the four single-point dilutions analysed, r values as high as . for the highest dilution were observed, over a serum antibody concentration range of approximately tenfold. the : and : dilutions did show evidence of saturation for samples with higher antibody concentrations. at the : , and : , dilutions, the relationship between signal intensity and concentration was highly linear, with Δi/Δc of close to . across most of the serum antibody concentration range (fig. b) . by contrast, : and : showed Δi/Δc approaching . for samples with higher antibody concentrations, indicating some degree of instrument saturation. these findings suggest that testing the thirty csp-immune nhp serum samples at a single dilution ( : , ) was sufficient to accurately determine the antibody titres by eclia. the present study describes the features of a newly developed serological panel that is based on a multiplex eclia-assay platform. comparing some of the features with the classic elisa demonstrated the advantages of fig. linearity of elisa vs eclia based assay for the assessment of csp-specific antibodies. a elisa readout for single sample (human csp-immune pool) across fifteen twofold dilutions from : to : , , compared against the relative serum concentration, expressed as the log dilution − for the csp-fl, csp c-term, and nanp antigens. dilution points that were found to be in the linear range are marked (solid circles), with corresponding r values. b Δod/Δc values calculated between adjacent dilutions against relative serum concentration for the elisa. Δod/Δc curve estimates are shown as well. c eclia assay readout for a single sample (human csp-immune serum pool) across eight fivefold dilutions against csp-fl, csp c-term, and nanp against the relative serum concentration, expressed as the log dilution − . five dilution points (solid circles) were found to be in the linear range for each antigen, with corresponding r values shown. d Δi/Δc values calculated between adjacent dilutions against relative serum concentration for the eclia. Δi/Δc curve estimates are shown as well the eclia based assay for assays that establish the antigenic profile of humoral immune responses in either vaccinated individuals or residents of malaria-endemic areas (summarized in table ). special emphasis was placed on determining whether closely related antigens could be tested simultaneously without impacting the quantification of such antibodies. to this end, a single malarial antigen, csp, and its fragments were used as plate antigens. csp is one of the leading malaria vaccine antigens [ ] ; the magnitude of antibody responses to either full length csp [ ] or its fragments has been identified as a potential biomarker of protection [ , ] . while the role of csp-repeat-specific antibodies has been well documented [ , , , ] , there are conflicting data on the role of c-terminus-specific antibodies [ , , ] and their ability to contribute to protection against infection. the method described here enables high-throughput testing and permits profiling of large samples sets even when sample volumes are limited to determine the role of epitope specificity of csp-specific antibodies. the assay development report evaluates crucial parameters for a sensitive and reproducible assay: ( ) the optimal coating concentrations for the csp protein, as well as the derived peptides that represent important functional elements in efficacious immune responses induced by vaccination; ( ) the equivalency of the u-plex linkers to ensure that no bias is introduced by assigning test antigens to linkers that are not capable of delivering the same signal strength; ( ) the impact of biotinylation on immunoreactivity with specific antibodies. in this study, biotinylation did not notably change the interaction between antibodies and the antigens. however, it should not be assumed that this will be the case for all antigens. one cannot exclude the possibility that biotinylation may impact access to select epitopes by a subset of antibodies due to steric hindrance. the assay described here is intended for the characterization of polyclonal responses where steric hindrance would only have a negligible impact on the overall signal considering the wide range of epitope specificities and the high sensitivity of the assay platform. other multiplex platforms such as bead-based flow cytometry also requires modification of the antigens by either biotinylation or chemical linkage. the latter assay platform has been used in some field studies for the profiling of antibody specificities and their contribution to clinical outcomes [ , ] . alternatively, protein arrays are available that allow an in-depth profiling of the antibody responses in vaccinees or residents of malaria endemic areas with special emphasis on polymorphic antigens where several alleles have been included into the array chips [ ] . the eclia method described here could be considered complementary to protein microarrays. while microarrays typically contain hundreds of antigens printed onto chips and can be costly, they are instrumental in profiling serological responses and are invaluable in identifying biomarkers of protection [ ] . once specific markers of protection or disease have been identified, they could be applied to the eclia assay platform, thus streamlining the testing process and reducing the overall cost of assay performance and analysis. the present study demonstrates the superiority of the eclia based serological assay over the conventional elisa. the two assays show strong quantitative agreement. however, because of the extremely wide linear range of the eclia, a simple single-point measurement is sufficient to determine antibody titres. by contrast, in the elisa, a much narrower linear range means that multiple dilution points are necessary for each sample to be in the linear range of the instrument, and then serial dilutions are required to create a titration curve from which a titre can be calculated. furthermore, the eclia can be multiplexed to measure responses to multiple antigens simultaneously from a single sample. equally important, no antigenic competition could be detected when testing closely related antigens in the eclia. these characteristics make the eclia the preferred platform for serological immunoprofiling, which is crucial for the identification of biomarkers of exposure or correlates of immunity. complex immune correlates of protection in hiv- vaccine efficacy trials use of pathogen-specific antibody biomarkers to estimate waterborne infections in population-based settings elisa in the multiplex era: potentials and pitfalls igg antibodies against a clinical grade plasmodium falciparum csp vaccine antigen associate with protection against transgenic sporozoite challenge in mice fractional third and fourth dose of rts, s/as malaria candidate vaccine: a phase a controlled human malaria parasite infection and immunogenicity study a preliminary evaluation of a recombinant circumsporozoite protein vaccine against plasmodium falciparum. malaria rts, s malaria vaccine evaluation group the biological function of antibodies induced by the rts, s/as malaria vaccine candidate is determined by their fine specificity delayed fractional dose regimen of the rts, s/as malaria vaccine candidate enhances an igg response that inhibits serum opsonophagocytosis safety, toxicity and immunogenicity of a malaria vaccine based on the circumsporozoite protein (fmp ) with the adjuvant army liposome formulation containing qs (alfq) randomized, double-blind, phase a trial of falciparum malaria vaccines rts, s/as b and rts, s/as a in malaria-naive adults: safety, efficacy, and immunologic associates of protection use of coefficient of variation in assessing variability of quantitative assays vaccines against malaria the relationship between rts, s vaccine-induced antibodies, cd (+) t cell responses and protection against plasmodium falciparum infection concentration and avidity of antibodies to different circumsporozoite epitopes correlate with rts, s/as e malaria vaccine efficacy structural basis for antibody recognition of the nanp repeats in plasmodium falciparum circumsporozoite protein diverse antibody responses to conserved structural motifs in plasmodium falciparum circumsporozoite protein rare pfcsp c-terminal antibodies induced by live sporozoite vaccination are ineffective against malaria infection correlation between malaria-specific antibody profiles and responses to artemisinin combination therapy for treatment of uncomplicated malaria in western kenya identifying immune correlates of protection against plasmodium falciparum through a novel approach to account for heterogeneity in malaria exposure seroreactivity to a large panel of field-derived plasmodium falciparum apical membrane antigen and merozoite surface protein variants reflects seasonal and lifetime acquired responses to malaria convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year ready to submit your research ? choose bmc and benefit from antibody biomarkers associated with sterile protection induced by controlled human malaria infection under chloroquine prophylaxis springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank dr supplementary information accompanies this paper at https ://doi. org/ . /s - - - .additional file : figure s . overview of eclia assay platform. (panel a) experimental steps for assay setup. biotinylated antigens are coupled with proprietary u-plex linkers in separate tubes. once coupling is complete, u-plex-coupled antigens are combined into a cocktail and the assay plates coated. each u-plex linker can only bind to its respective spot (color coded in figure) ; up to antigens can be coated per well (top view plate well). (panel b) overview of all steps to complete the assay: antigen-coated wells are incubated with diluted serum or plasma. antigen-specific antibodies will bind to the antigen and the binding visualized by adding a sulfo-tag-labeled secondary antibody and substrate. (panel c) data acquisition. plate is inserted into reader which will deliver an electric pulse that activates the substrate. a high resolution camera measures the luminescence above each spot. the data and detailed protocol can be made available upon request from the corresponding author. the serum sample use was reviewed by the wrair human subjects' protection branch which determined that the research does not involve human subjects (nhsr protocol wrair# ) as the samples used were de-identified and no link between samples and subjects exists. not applicable. the authors declare that they have no competing interests. key: cord- -neowfhwg authors: liu, weixiao; liu, xuri; liu, chao; zhang, zhe; jin, wujun title: development of a sensitive monoclonal antibody-based sandwich elisa to detect vip aa in genetically modified crops date: - - journal: biotechnol lett doi: . /s - - - sha: doc_id: cord_uid: neowfhwg objectives: to develop a sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (elisa) to detect vip aa in genetically modified (gm) crops and their products. results: vegetative insecticidal proteins (vips) are secreted by bacillus thuringiensis (bt) and are known to be toxic to lepidoptera species. vip aa family proteins, vip aa and vip aa , were successfully applied in gm crops to confer an effective and persistent insecticidal resistance. a sensitive monoclonal antibody-based sandwich elisa was developed to detect vip aa in gm crops and their products. two monoclonal antibodies were raised against the overexpressed and purified his-vip aa , were purified from mouse ascites and characterized. a sandwich elisa method was developed using the g - d monoclonal antibody for capture and the biotin-labeled f - f monoclonal antibody for detection of vip aa . the linear detection range of the method was found to be approximately . – pg/ml, with a sensitivity of . pg/ml. conclusions: the established elisa was effective for detecting vip aa family proteins other than vip aa , and was successfully applied in the detection of vip aa and vip aa expressed in transgenic maize and cotton. some insect pathogenic microbiology synthesizes a large number of insecticidal proteins. these insecticidal proteins form inclusion bodies (such as cry and cyt proteins) or are secreted into the cultural medium (such as vip and sip proteins). cry proteins are widely used in agricultural pest control (ashouri ; clive ; de maagd et al. ; estruch et al. ; shelton ; tabashnik et al. ) . however, some important pests, such as agrotis ipsilon and diabrotica spp., exhibit high tolerance to cry proteins and are seriously harmful to crops (chakroun et al. ; chattopadhyay and banerjee ) . in , vip proteins were screened from the culture supernatant of bacillus thuringiensis (estruch et al. ) and found to be toxic to a wide range of lepidoptera species, some of which show tolerance or low susceptibility to cry proteins. vip proteins are divided into four families based on the homology of their amino acid sequences: vip , vip , vip and vip (https://www.btnomenclature. info/). among them, the vip aa protein has been found to be toxic to most lepidoptera insects. notably, vip aa is highly toxic to agrotis, which is resistant to cry. vip aa has also been applied against species of spodoptera, which are insensitive to cry toxicity (donovan et al. ; liu et al. ; selvapandiyan et al. ) . furthermore, vip aa genes have been successfully transferred into cotton and maize, as patented in the united states in (adamczyk and mahaffey ; kurtz et al. ) . the rapid development of genetically modified (gm) crops has resulted in growing public concern that gm crops may lead to unexpected food and environmental safety issues. thus, accurate detection of foreign genes and their products in gm crops is becoming increasingly important and urgent. there are numerous methods for detecting foreign genes in transgenic crops (kamle et al. ; salisu et al. ) , and the most direct detection method is based on the gene-encoded protein. elisa is a specific, sensitive, and convenient method for protein detection. furthermore, the method is precise, reproducible and employs stable reagents and inexpensive equipment. therefore, elisa is applicable for routinely detecting foreign gene-encoded proteins in gm crops and their products (albright et al. a (albright et al. , b kamle et al. b kamle et al. , . based on this information, we used overexpressed his-vip aa as an immunogen to generate mouse monoclonal antibodies (mabs) that recognize certain surface components of the protein. these antibodies were then employed to develop a sandwich elisa for sensitive, direct and convenient measurement of the vip aa concentration in gm crops and their products. to the best of our knowledge, this is the first quantitative monoclonal antibody-based elisa method reported for the sensitive detection of vip aa proteins. reagents, strains and animals dna markers, pfu polymerase and escherichia coli (trans and bl ) chemically competent cells were obtained from transgen biotech. ni-resin, superdex and protein a-sepharose columns were purchased from ge healthcare. a protein marker was purchased from fermentas. hyclone dmem and fetal calf serum (fcs) were purchased from thermo. all reagents were of analytical grade. complete and incomplete freund's adjuvant, % polyethylene glycol (peg), hypoxanthine/aminopterin/thymidine (hat) and hypoxanthine/thymidine (ht) were obtained from sigma-aldrich and proteinase inhibitor cocktails from roche. goat anti-mouse immuno-globulin horseradish peroxidase conjugate was obtained from univ-bio (shanghai, china) and avidin-horseradish peroxidase conjugate from invitrogen. babl/c mice were obtained from sbf company (beijing, china). the vip aa gene was amplified from gm maize mir genomic dna and subcloned into the pet a plasmid. pet a-vip aa / and pet a-vip aa / were generated by site-directed mutagenesis (palma et al. ). the vip aa and vip aa genes were chemically synthesized and subcloned into the pet a plasmid. the reconstituted plasmids were transformed into e. coli bl competent cells. overexpressed recombinant his-vip aa , his-vip aa / , his-vip aa / , his-vip aa and his-vip aa proteins were purified with his-affinity purification (native or denature) and further fractionated by size-exclusion chromatography using a super-dex column with mm tris hcl, ph . , mm potassium chloride and % glycerol. the immunization protocol followed conventional subcutaneous (s.c.) injection, with slight modifications (li et al. ). eight-week-old male balb/c mice were immunized with immunogen [his-vip aa dissolved in l l of sterile phosphate-buffered saline (pbs) adjuvant with complete freund's adjuvant or incomplete freund's adjuvant] at the nape of the neck at weeks , , and . the immunizing dose was fixed at lg by subcutaneous multi-point injection per mouse each time. three days prior to cell fusion, the mice were boosted with lg of adjuvant-free immunogen. blood samples were collected from the mouse tail, and the titers were determined by elisa. fusion, hybridoma screening and mab generation seven days after the final booster immunization, a single-cell suspension was aseptically prepared in rpmi- medium from mouse spleen samples. the cell suspensions were mixed with murine myeloma cells sp / (genecreate biological engineering co.) at a ratio of : . the mixed cell suspension was centrifuged, and the supernatant was completely removed. the combined splenocyte suspension and sp / cells were fused using a modification of the method described by galfre et al. (galfre and milstein ) . one milliliter of % peg was added dropwise to the mixed cell pellet in a -ml centrifuge tube over s. the mixture was gently stirred while peg was added, and the mixture was then allowed to incubate for min. fusion was stopped by dropwise addition of ml of rpmi- to the mixture. the temperature was maintained at °c throughout the entire procedure. the cell suspension was then centrifuged at rpm for min. the precipitated cells were gently resuspended in ml of hat complete liquid medium, and the fusion suspension was gently mixed with ml of semi-solid complete medium containing g of methyl cellulose. finally, . ml of the fusion cell suspension was distributed into -well plates and incubated in % co at °c. after the cells were cultured for - days, many white dots suggestive of monoclonal hybridomas had formed on the semi-solid selective complete medium. these white dots were transferred to -well culture plates to enable screening for hybridoma cell lines secreting the anti-vip aa antibody. next, the absorbance of the culture supernatant of anti-vip aa antibody-secreting hybridoma cell lines was evaluated at nm (a ). anti-vip aa antibody-secreting hybridoma cell lines with an a value higher than . were injected into the abdomens of -week-old balb/c mice (daginakatte et al. ; dong et al. ; esch et al. ; narat et al. ) , and the ascitic fluid was collected approximately seven days later. the mabs were purified from the ascitic fluid using the saturated ammonium sulfate precipitation method and subsequently purified using protein a-sepharose columns (groopman et al. ) . antibody titers were determined via indirect elisa. the antibody titer was defined as the highest antibody dilution that gave an absorbance greater than . -fold of the background absorbance of pbs (negative control) (martin et al. ) . subclass assessments of the mabs were performed by direct elisa using a commercially available kit from sigma (azimzadeh and van regenmortel ; beatty et al. ) . protein sample preparation mir maize standard substance (aocs -a, usa) and negative maize samples and cot cotton standard substance (aocs -c, usa) and negative cotton samples were ground in liquid n , resuspended in lysate buffer ( mm tris, ph . , mm nacl, mm edta, % glycerol, . % sds, protease cocktail and mm pmsf) at °c for min and then centrifuged at , rpm and °c for min. the supernatants were collected and mixed with loading buffer for sds-page/western blotting analysis or diluted to an appropriate concentration for elisa. the his-vip aa protein, mir maize standard substance and negative maize samples, and gradient concentrations of his-vip aa , his-vip aa / , his-vip aa / , his-vip aa and his-vip aa were electrophoresed on a - % nupage gel (invitrogen, usa) and transferred onto polyvinylidene fluoride (pvdf) membranes (millipore, us). the membranes were incubated with lg/ml his-vip aa mabs at °c overnight after nonspecific sites were blocked with % skim milk. the membranes were washed three times and incubated with alexa fluor tm goat anti-mouse igg (h?l) (invitrogen, usa) for h at room temperature. the membranes were processed using an odyssey scanner (li-cor, usa). the wells of an elisa plate were coated with l l of lg/ml capture antibody and incubated overnight at °c. three washes with pbst were performed to remove unbound antibody. each well was blocked with l l of % skim milk and incubated at °c for min. the washing step was repeated, and ll of his-vip aa protein serially diluted in pbs was added to each well. the plate was incubated for h at °c. next, lg/ml of biotin-labeled anti-his vip aa mab was added to each well and incubated for h at °c. the plates were washed and incubated with avidin-hrp for min at °c. the plate was then washed five times, and l l of tmb solution was added to each well. the reaction was terminated by the addition of ll of m h so , and the absorbance at nm was measured. detection of vip aa by the sandwich elisa one hundred microliters of vip aa protein, vip aa / , mir , cot or negative sample was added to each antibody-coated well. the plate was covered with an adhesive strip and incubated for h at °c. next, the liquid in each well was removed, and ll of biotin-labeled antibody was added. the wells were incubated for h at °c and then washed three times with pbst (phosphate buffer solution tween- ). after the last wash, any remaining liquid was removed by aspiration. next, ll of avidin-hrp was added to each well. the plate was covered with a new adhesive strip and incubated for h at °c, followed by five washing steps. next, l l of tmb solution was added to each well, the reaction was terminated by the addition of ll of m h so , and absorbance at nm was measured. data are expressed as the average value and standard deviation (sd). the limit of detection (lod) was calculated using the standard formula, with a slight modification (dixit et al. ; vashist ; vashist et al. ). sequences of vip aa family proteins were downloaded from https://www.btnomenclature.info/ and aligned using dnaman software. the vip aa gene was amplified from gm mir genomic dna and subcloned into the pet a expression vector for overexpression and purification. his-vip aa was successfully expressed in e. coli bl cells. after nickel affinity purification, the protein was further fractionated by size-exclusion chromatography on a superdex column (fig. a) . the molecular weight of the overexpressed his-vip aa is approximately kd (fig. b) . purified his-vip aa was used to immunize -week-old male balb/c mice. anti-his-vip aa mabs were prepared according to antibody preparation techniques. more than hybridomas were screened using indirect elisa. hybridomas with a values greater than . were selected for further subcloning (fig. a) . finally, two anti-his-vip aa antibody-secreting hybridoma clones (named f - f and g - d ) were screened, expanded and injected into the abdomens of -weekold balb/c mice for ascitic fluid preparation. the mabs f - f and g - d were purified from ascitic fluid using saturated ammonium sulfate precipitation and a protein a-sepharose column. sds-page results demonstrated that the purified antibodies f - f and g - d are -kda (heavy chain) and -kda (light chain), as expected, and that extraneous proteins were eliminated (fig. b) . the titers of the purified mabs f - and g - d were : , , and : , , , respectively (fig. c, d) . the isotypes of the two mabs were determined to be igg and igg a (table ) . his-vip aa and vip aa in mir standard substance were successfully recognized by the mabs (fig. e) . several proteins to which insects exhibit resistance, such as cry c, cry a and cry a, which have been widely and successfully applied in transgenic crops, were used to test the cross-reactivity of the g - d and f - f mabs (fig. f, g) . these mabs specifically recognized the vip aa protein but not the other proteins assessed. these g - d and f - f mabs were used for subsequent analyses. the mab f - f served as the capture antibody, and the biotin-labeled mab g - d served as the detection antibody. standard dilutions were obtained by serial dilution with an initial his-vip aa protein concentration of ng/ml (fig. a) and used to construct a standard curve, with the equation y = . x- . . the working range of the assay was defined as the part of the curve with a linear coefficient of r [ . . the linear range included concentrations of . pg/ml to . ng/ml, with an lod of . pg/ml. g - d was also used as the capture antibody, with biotin-labeled mab f - f as the detection antibody. standard dilutions of an initial his-vip aa protein concentration of ng/ml (fig. b) were used to construct a standard curve, with y = . x- . . the working range of the assay was defined as above, and the linear range included concentrations of . pg/ml to pg/ml, with an lod of . pg/ml. due to its higher sensitivity, this second elisa method was selected for further analysis. the variability of this elisa method was examined using the coefficient of variation (cv). as shown in table , the intra-and interassay variation values of this elisa were . % and . %, respectively. these data indicate that the sandwich elisa developed for vip aa detection is a convenient and sensitive assay. the sequences of vip aa family proteins were downloaded from https://www.btnomenclature.info/ and analyzed using dnaman (fig. ) . two proteins with the highest (vip aa / and vip aa / ) and lowest (vip aa and vip aa ) sequence consistency were expressed and purified. with the exception of his-vip aa , which has a molecular weight of approximately kda, the molecular weights of the other proteins were identical to that of his-vip aa , which is kda (fig. a) . his-vip aa / , his-vip aa / and his-vip aa were successfully identified by the purified mabs f - and g - d , whereas was not his-vip aa (fig. b, c) . because vip aa and vip aa have been applied in transgenic crops, his-vip aa and his-vip aa were diluted in a concentration gradient and detected by the elisa (fig. d) . the results showed the elisa method to also be suitable for detecting vip aa family proteins other than vip aa . vip aa proteins secreted by bacillus thuringiensis have been screened for lepidoptera toxicity and applied in gm crops (such as maize and cotton). however, with the rapid development of gm crops, there is heightened concern that these crops may lead to unexpected food safety and environmental safety issues. thus, sensitive detection of exogenous proteins has become increasingly important. numerous methods for detecting and monitoring transgenic crops have been established. the most popular techniques and g - d . c western blotting analysis of vip aa and vip aa against lg/ml mabs f - f and g - d . d elisa analysis of a gradient dilution of vip aa methods for monitoring cry ab, cry ac and cry ie were established (walschus et al. ; wang et al. ; zhang et al. ) . in this study, we describe a sensitive monoclonal antibody-based elisa method for the detection of vip aa in gm crops and their products. compared with the previously developed triple antibody sandwich elisa for vip a (kumar ) , the elisa method developed in the present study is more sensitive. furthermore, we clearly indicate the scope of the application of this elisa method, which is suitable for detecting vip aa family proteins other than vip aa . specifically, our results indicate that the c-terminal sequences of vip aa family proteins are recognized by the two mabs screened, f - f and g - d (fig. b, c) . the newly developed elisa method is a sensitive technique for determining the vip aa content in gm crops and their products. this report describes a sensitive monoclonal antibodybased elisa for the detection of vip aa in gm crops. the titers of the screened mabs g - d and f - f were : , , and : , , , respectively. this sensitive elisa method was developed to detect vip aa family proteins (other than vip aa ), with a working range of . - pg/ml and an lod of . pg/ml. the elisa method performed well in recovery tests and can be used for quantitative and convenient detection of vip aa proteins in maize and cotton samples. efficacy of vip a and cry ab transgenic traits in cotton against various lepidopteran pests assessing copy number of mon integrations in commercial seed maize varieties by ' event-specific real-time pcr validated method coupled to (-delta delta ct) enzyme-linked immunosorbent assay detection and bioactivity of cry ab protein fragments a review of cry protein detection with enzyme-linked immunosorbent assays transgenic-bt potato plant resistance to the colorado potato beetle affect the aphid parasitoid aphidius nigripes measurement of affinity of viral monoclonal antibodies by elisa titration of free antibody in equilibrium mixtures measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay bacterial vegetative insecticidal proteins (vip) from entomopathogenic bacteria recent advancement on chemical arsenal of bt toxin and its application in pest management system in agricultural field the global status of the commercialized biotechnological/genetically modified crops production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of elk coronavirus structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria multisubstrate-compatible elisa procedures for rapid and high-sensitivity immunoassays production and characterization of monoclonal antibody broadly recognizing cry toxins by use of designed polypeptide as hapte gene knockout demonstrates that vip a contributes to the pathogenesis of bacillus thuringiensis toward agrotis ipsilon and spodoptera exigua production and characterization of monoclonal antibodies to estrogen-related receptor alpha (err alpha) and use in immunoaffinity chromatography transgenic plants: an emerging approach to pest control vip a, a novel bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects preparation of monoclonal antibodies: strategies and procedures high-affinity monoclonal antibodies for aflatoxins and their application to solid-phase immunoassays current perspectives on genetically modified crops and detection methods development of multiplex and construct specific pcr assay for detection of cry ab transgene in genetically modified crops and product development of an enzyme linked immunosorbant assay for the detection of cry ab protein in transgenic plants development of enzymelinked immunosorbent assay for the detection of bt protein in transgenic cotton development of elisa for the detection of transgenic vegetative insecticidal protein in gm crops/ produce insect resistance management for syngenta's vipcot (tm) transgenic cotton development of a class-specific monoclonal antibody-based elisa for aflatoxins in peanut identification of vip a-type genes from bacillus thuringiensis strains and characterization of a novel vip a-type gene standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections production, characterization and use of monoclonal antibodies recognizing igy epitopes shared by chicken, turkey, pheasant, peafowl and sparrow the vip ag insecticidal protoxin from bacillus thuringiensis adopts a tetrameric configuration that is maintained on proteolysis molecular approaches for high throughput detection and quantification of genetically modified crops: a review front toxicity analysis of nand c-terminus-deleted vegetative insecticidal protein from bacillus thuringiensis genetically engineered vegetables expressing proteins from bacillus thuringiensis for insect resistance: successes, disappointments, challenges and ways to move forward dual mode of action of bt proteins: protoxin efficacy against resistant insects a sub-picogram sensitive rapid chemiluminescent immunoassay for the detection of human fetuin a one-step antibody immobilization-based rapid and highly-sensitive sandwich elisa procedure for potential in vitro diagnostics development of monoclonal antibodies against cry ab protein from bacillus thuringiensis and their application in an elisa for detection of transgenic bt-maize development of elisa for the determination of transgenic bt-cottons using antibodies against cry ac protein from bacillus thuringiensis hd- development of monoclonal antibody-based sensitive elisa for the determination of cry ie protein in transgenic plant publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we thank dr. liang li for critical reading of the manuscript. we are grateful to dr. si chen (genecreate biological engineering co., ltd., wuhan, china) for technical assistance with monoclonal hybridoma screening. this work was supported by fundamental research funds for non-profit scientific institution (grant no. ) and national transgenic major program of china (no. zx ). author contributions wl and wj conceived of and designed the experiments. xl, cl and zz performed the experiments. wl analyzed the data and wrote the paper. all authors reviewed the manuscript. conflict of interest the authors declare that they have no conflict of interest.ethical approval all of the animal experiments were performed according to approved institutional animal care and use committee (iacuc) protocols (# - ) of the institute of zoology, chinese academy of sciences.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. key: cord- -rifr uub authors: deng, junhua; jin, yipeng; liu, yuxiu; sun, jie; hao, liying; bai, jingjing; huang, tian; lin, degui; jin, yaping; tian, kegong title: serological survey of sars‐cov‐ for experimental, domestic, companion and wild animals excludes intermediate hosts of different species of animals date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: rifr uub the pandemic sars‐cov‐ has been reported in countries with more than , patients died from it. however, the original and intermediate hosts of the virus remain unknown. in this study, , serum samples from animal species were used for detection of sars‐cov‐ ‐specific antibodies using double‐antigen sandwich elisa after validating its specificity and sensitivity. the results showed that no sars‐cov‐ ‐specific antibodies were detected in above samples which excluded the possibility of animal species as intermediate host for sars‐cov‐ . more importantly, companion animals including pet dogs (including one dog the sars‐cov‐ patient kept and two dogs which had close contact with it) and cats, street dogs and cats also showed serological negative to sars‐cov‐ , which relieved the public concerns for the pets as sars‐cov‐ carriers. deng et al. giant panda, masked civet, porcupine, bear, yellow-throated marten, weasel, red pandas and wild boar). the results showed that no sars-cov- -specific antibodies were detected in above species of animals including pangolin which has been reported as an intermediate host of sars-cov- (kangpeng xiao, ) . more importantly, we found companion animals including dogs and cats were serologically negative to sars-cov- including one dog kept by the sars-cov- patient and two dogs with close contact with it during the quarantine. the sars-cov- double-antigen sandwich elisa was purchased from luoyang putai biotechnology co., ltd. the coating was based on s protein of sars-cov- . the same antigen was linked to horseradish peroxidase (hrp) to function as conjugate. the serum samples were tested according to the manufacture manual instructions. briefly, µl serum sample was added into each well of elisa plate and incubated at °c for min. after washing the plate with washing buffer for five times, hrp-labelled antigen was added into the wells at °c for min before μl of the substrate solution was added to each well and incubated at °c for min to stop the reaction. the optical density (od) was measured at nm. to test the specificity of elisa kit, the serum samples of spf chicken ( ), duck ( ), mouse ( ), rat ( ) and pig ( ) were applied. the final value of od of samples ranged from . to . (median . ), . to . (median . ), . to . (median . ), . to . (median . ) and . to . (median . ) for chicken, duck, mouse, rat and pig, respectively. serum samples from other species of experimental animals including guinea pig ( ), rabbits ( ), beagle dogs ( ) and rhesus monkeys ( ) were also tested. there were no sars-cov- antibodies detected in above animals (data not shown). next, the potential cross-reaction with other coronavirus including ibv ( ) we next tested the sensitivity of elisa kit. the sars-cov- experimental-infected ferret positive sera were tested. as shown in table , the neutralizing antibody titres of infected ferret (f -f ) were between : and : at days post-infection (dpi). by contrast, the neutralizing antibody titres of placebo ferrets (c -c ) were all negative at dpi. in the line with the results of neutralizing antibodies, the final od of positive sera detected by elisa was all above , which indicated strongly serological positive to sars-cov- . to further test the dynamic changes of elisa titre of infected ferret, serum samples from one ferret were collected from , , , and dpi, respectively. the positive elisa results were shown at dpi and lasted until dpi when the ferrets were humanely euthanized (table ) . the above results showed that the elisa has good specificity and sensitivity and suitable for different species of animals. after confirming the specificity, sensitivity and suitability of sars-cov- elisa kit for different species of experimental animals, clinical serum samples from domestic livestock (pig, cow, sheep, horse), poultry (chicken, duck, goose), experimental animal (mice, rat and rhesus monkey), companion animal (dog and cat) and wild animals (camel, fox, mink, alpaca, ferret, bamboo rat, peacock, eagle, tiger rhinoceros, pangolin, leopard cat, jackal, giant panda, masked civet, porcupine, bear, yellow-throated marten, weasel, red pandas and wild boar) were used for antibody detection. as shown in table unknown. since sars-cov- is genetically close to sars-cov, it has been proposed that bat could be the natural host (phan, ) . snake is also presumed as wildlife animal reservoir for sars-cov- based on the virus relative synonymous codon usage (rscu) bias (ji, wang, zhao, zai, & li, ) . however, there is no report of sars-cov- isolation or molecular and serological confirmation of infection from snake samples. pangolins recently was suggested to be direct animal source of sars-cov- for humans since the sars-cov- -related coronaviruses were isolated from malayan pangolins which shared . % similarity with sars-cov- in virus receptor-binding domain in s gene (kangpeng xiao, ) . in our study, we did not detect sars-cov- antibodies in pangolin serum samples. consistent with our results, li et al., ( ) reported the coronavirus carried by pangolins did not have the rrar motif, a unique peptide insertion in the human sars-cov- virus. the rrar motif may be involved in the proteolytic cleavage of spike protein and host range and transmissibility which suggests human sars-cov- virus did not come directly from pangolins . masked civet and camel were confirmed to be natural hosts for sars-cov and mers-cov, and no specific sars-cov- antibodies were detected in masked civets and camels in this study. to the sars-cov- has been major concern for the public. one pet dog was reported to be sars-cov- -positive detected by rt-pcr in hongkong (https://www.news.gov.hk/eng/ / / / _ _ .html). later, the serological result of the dog showed negative after quarantine of days. in our study, cats including pet cats and street cats showed serological negative to sars-cov- (table ) . at the same time, dogs including beagle dogs, pet dogs and street dogs during the outbreak of sars-cov- were also tested serological negative. among them, pet dog and street dog sera were collected from wuhan city. it should be noted that one pet dog from confirmed sars-cov- -infected patient showed serologically negative, and other two dogs which had close contact with this dog also tested to be negative. however, we cannot rule out of susceptibility of cats and dogs to sars-cov- , which need to be tested by experimental infections. molecular techniques such as reverse-transcriptase pcr tests and viral genome sequencing are widely used for the confirmation of human infection. these techniques are also used to explore the potential hosts of sars-cov- (pfefferle, reucher, norz, & lutgehetmann, ) . compared to these molecular methods, serological test such as elisa has several advantages. first, the host generates sars-cov- -specific antibodies after infection which could last longer than the viraemia. it provides a wider detection window for elisa than rt-pcr. second, rna extraction from susceptive infected samples has to be performed in a bsl- laboratory. by abbreviation: dpi, days post-infection. *the neutralizing antibody titre of positive samples was ≥ . contrast, elisa can be performed in a safety level laboratory and does not require high containment facilities after the serum samples were inactivated at °c for min. third, double-antigen sandwich elisa based on recombinant s protein could detect both igm and igg antibodies and is not limited to species. to find the host of sars-cov- , the screening of other wild animals using elisa is undergoing in our laboratory. we want to thank dr. zhigao bu, director of harbin veterinary research institute, chinese academy of agricultural sciences for providing inactivated sars-cov- -negative and sars-cov- positive ferret serum samples. this study was supported by luoyang heluo talent plan (kegong tian). we declare that ethical statement is not applicable. there was no conflict of interest with others. the data that support the findings of this study are available from the corresponding author upon reasonable request. kegong tian https://orcid.org/ - - - three emerging coronaviruses in two decades cross-species transmission of the newly identified coronavirus -ncov the emergence of a novel coronavirus (sars-cov- ), their biology and therapeutic options evolutionary history, potential intermediate animal host, and cross-species analyses of sars-cov- evaluation of a quantitative rt-pcr assay for the detection of the emerging coronavirus sars-cov- using a high throughput system genetic diversity and evolution of sars-cov- . infection a novel coronavirus outbreak of global health concern novel coronavirus , an emerging public health emergency serological survey of sars-cov- for experimental, domestic, companion and wild animals excludes intermediate hosts of different species of animals key: cord- -lsksys authors: goto, keiko; yamaoka, yutaro; khatun, hajera; miyakawa, kei; nishi, mayuko; nagata, noriko; yanaoka, toshikazu; kimura, hirokazu; ryo, akihide title: development of monoclonal antibodies and antigen-capture elisa for human parechovirus type date: - - journal: microorganisms doi: . /microorganisms sha: doc_id: cord_uid: lsksys human parechovirus type (hpev ) is an etiologic agent of respiratory diseases, meningitis, and sepsis-like illness in both infants and adults. monoclonal antibodies (mabs) can be a promising diagnostic tool for antigenic diseases such as virus infection, as they offer a high specificity toward a specific viral antigen. however, to date, there is no specific mab available for the diagnosis of hpev infection. in this study, we developed and characterized mabs specific for hpev capsid protein vp . we used cell-free, wheat germ-synthesized viral vp protein for immunizing balb/c mice to generate hybridomas. from the resultant hybridoma clones, we selected nine clones producing mabs reactive to the hpev -vp antigen, based on enzyme-linked immunosorbent assay (elisa). epitope mapping showed that these mabs recognized three distinct domains in hpev vp . six mabs recognized hpev specifically and the other three mabs showed cross-reactivity with other hpevs. using the hpev -specific mabs, we then developed an elisa for viral antigen detection that could be reliably used for laboratory diagnosis of hpev . this elisa system exhibited no cross-reactivity with other related viruses. our newly developed mabs would, thus, provide a useful set of tools for future research and ensure hpev -specific diagnosis. human parechoviruses (hpevs) belong to the parechovirus genus of the picornaviridae family [ , ] . the two serotypes (hpev and hpev ) of parechoviruses were initially isolated in from children with diarrhea, and were assigned to the enterovirus genus. however, it became evident that they were genetically distinct from the enteroviruses, and reclassified as the parechovirus genus in . at present, hpevs are reported and categorized, based on the nucleic acid sequences of the vp gene, not on the classification as enteroviruses serotype. hpev was first identified in japan. hpev was isolated from a stool sample provided by a -year-old infant who was experiencing fever, gastritis like symptoms, and transient lower extremity paralysis [ ] . hpev types to are the common identified strains; among them, hpev type , , and account for the majority of infectious strains worldwide [ ] . infection with hpevs is associated with a broad spectrum of clinical manifestations, ranging from respiratory symptoms and mild gastrointestinal illness to sepsis-like diseases, meningitis, and encephalitis in children [ ] . while most hpevs cause mild symptoms in children between and years of age, human parechovirus (hpev ) is clinically the most important genotype, owing to its association to severe diseases in younger infants under months of age [ ] [ ] [ ] . hpev infection in infants can trigger a sepsis-like dysregulated host response involving the central nervous system [ ] [ ] [ ] [ ] . in cases of acute meningitis or encephalitis, patients might develop abnormal white matter lesions and neurological sequelae, and even death might occur [ ] [ ] [ ] [ ] [ ] . apnea can occur in children regardless of encephalitis [ ] . hpev is known to cause myalgia and myositis in adult patients and a similar pattern is also sporadically seen among pediatric patients. [ , ] . an epidemic of hpev occurs every to years in japan. as respiratory disease or meningitis cases due to hpev are not subject to notifiable disease surveillance in japan, the actual number of the patients is not known [ , , [ ] [ ] [ ] [ ] . the appropriate diagnosis tool for hpev detection might be able to rule out infectious etiology and avoid unnecessary antibiotics use, which is a given because hpev leads to septic shock-like symptoms. for these reasons, the establishment of a method to detect hpev plays a vital role in healthcare fields. importantly, hpev s epidemic cycle occurs in summer time and is concurrent with enteroviral infection. thus, it is essential to develop a detection method that does not cross-react with enterovirus. hpev contains a small, non-enveloped, single-stranded positive-sense rna genome of approximately . -kb nucleotides [ , ] . the hpev virion is composed of copies of three structural proteins (vp , vp , and vp ) that fit together to form a -nm-diameter icosahedral shell around the viral genome [ ] . the genome encodes a single polyprotein that, during infection, is subsequently cleaved into all essential capsid components and non-structural proteins [ ] . vp is an important protein for stabilizing the surface of the viral capsid, and the assembly of hpev is controlled by multiple interactions of the genome with the capsid, through conserved amino acids in vp and vp [ ] . although rt-pcr-based diagnostic tests targeting -utr of the hpev genome were developed for hpev detection in clinical samples, there is currently no diagnostic method for detecting the viral antigens. recently, chen et al. generated polyclonal antibodies for hpev vp , and proposed an immunofluorescence-based diagnostic assay [ ] . however, this method requires virus isolation by cell culture and takes several weeks for the identification of viral genotype/serotype. abed et al. developed a serological enzyme-linked immunosorbent assay (elisa), using a synthetic peptide from the vp protein of hpevs [ ] . although it can provide a definitive diagnosis, serological test requires paired serum samples from acute and recovery phases, which makes it difficult to diagnose immediately as a point of care testing (poct). to develop a rapid and effective diagnostic strategy, there is an urgent need to produce highly specific monoclonal antibodies (mabs) toward hpev antigens. in this study, we sought to generate mabs specific to the capsid protein vp of hpev . we prepared the viral vp antigen using the wheat germ cell-free system, which has the advantage of producing properly folded functional proteins [ , ] , to immunize mice. as a result, we obtained nine mab clones for characterization, and thereafter, generated an elisa system that is specifically able to detect the hpev vp antigens. complementary dna encoding hpev -vp (genbank no. ab ) was used to generate the expression vector for antigen production with the wheat germ cell-free system. the hpev -vp open reading frame was amplified by pcr, using the corresponding primer pairs. the amplified fragment was cloned into vector peu-e -his-tev-mcs-n (cellfree sciences, yokohama, japan), using restriction enzymes xhoi and spei. in vitro wheat germ cell-free protein synthesis was carried out as previously described [ , ] . for cell-free protein synthesis, wepro h wheat extract (cellfree sciences, yokohama, japan) was used in the bilayer translation reaction, as previously described. synthesized proteins were confirmed by immunoblotting. the his-hpev -vp (full length, - ) protein was synthesized using a proteomist xe robotic protein synthesizer (cellfree sciences, yokohama, japan) for mouse immunization. the cell-free translation reaction mixture was separated into soluble and insoluble fractions by centrifugation at , × g for min. the soluble fraction was mixed with ni-sepharose high performance beads (ge healthcare, waukesha, wi, usa) in the presence of mm imidazole. the beads were washed thrice with a washing buffer [ mm tris-hcl (ph . ), mm nacl] containing mm imidazole. his-hpev -vp was then eluted in another washing buffer containing mm imidazole. amicon ultra centrifugal filters (millipore, bedford, ma, usa) were used to concentrate the purified his-hpev -vp . protein concentration was determined using the bradford method, with bovine serum albumin (bsa) as a protein standard. immunization of balb/c mice and generation of anti-hpev -vp mab-producing hybridomas were carried out as previously described [ , ] . briefly, his-tagged full-length hpev -vp protein was injected into the footpad of the balb/c mice, using keyhole limpet hemocyanin as an adjuvant. four weeks later, the spleen cells were isolated and fused to the myeloma cell line, sp /o, using polyethylene glycol (peg ). monoclonal antibodies in the hybridoma culture supernatant were tested using elisa with his-tagged recombinant hpev -vp protein. isotype determination was performed using isostrip mouse monoclonal antibody isotyping kit, following the manufacturer's instructions (roche diagnostics, basel, switzerland). vero cells were grown in dmem containing % fbs. hpev was provided by dr. masaki takahashi (iwate prefectural institute of public health). hpev was propagated in vero cells and quantified by qrt-pcr. the sequence information was as follows: -gtaacaswwgcctctgggs ccaaaag- (forward primer), -ggccccwgrtcagatccayagt- (reverse primer), and -vic-cctrygggtacctycwgggcatccttc-bhq- (probe). wheat germ-synthesized recombinant his-tagged hpev -vp proteins were separated by % sds-page in running buffer ( mm glycine, mm tris, . % sds). the separated proteins were transferred to a pvdf membrane (millipore). the membranes were washed with blotting buffer tbst (tbs containing . %-tween ), and then blocked for h at room temperature in % non-fat powdered milk in tbst. thereafter, the membranes were incubated overnight with generated hybridoma supernatant ( : dilution in tbst) at • c. next, after washing thrice in tbst, the membranes were incubated for h at room temperature with anti-mouse igg-hrp secondary antibody ( : dilution in tbst). finally, after washing thrice in tbst, the target protein was detected with the immobilon western chemiluminescence detection system (ge healthcare) using fluor chem fc (alpha innotech corp. tokyo, japan). for epitope mapping, we prepared deletion mutants of hpev -vp using pcr mutagenesis with template vector peu-his-hpev -vp , followed by wheat germ cell-free protein synthesis. the proteins were analyzed by immunoblotting using our generated mabs. to examine the amino acid variability among the vp proteins of other hpev genotypes, vp sequences for hpev - , , and - were accessed from the genbank and aligned with the hpev -vp sequence using the mega software. k d values were determined by bio-layer interferometry (bli) using octet red (fortebio, usa). anti-mouse igg capture biosensor tips (amc, fortebio) were loaded with µg/ml of mab # and # for min, in pbs containing . % bsa and . % tween . the association of recombinant vp at concentrations of , , , and nm for # mab, and , , . , and . nm for # mab, was measured for min, followed by a -min-long dissociation phase. all measurements were corrected for baseline drift by subtracting a reference well. the operating temperature was maintained at • c. data were analyzed using a : binding model with global fitting algorithms in the fortebio data analysis software. each mab was diluted in mm of carbonate buffer (ph . ) to a concentration of µg/ml, and then added to an elisa plate (agc techno glass, shizuoka, japan). to immobilize the antibodies, the plate was incubated overnight at • c. wells were blocked with pbs containing % (w/v) skim milk for h at room temperature (rt). after three washes with pbs containing . % (v/v) tween- (pbs-t), µl of antigen protein ( ng/ml) diluted with pbs-t or blank (pbs-t alone) was added, and the mixture was incubated for min at rt. after three washes with pbs-t, µl of each mab, conjugated with horseradish peroxidase (hrp), was added into each well and incubated for min at rt. antibody labeling was performed using the peroxidase labeling kit-nh (dojindo laboratories, kumamoto, japan). after three washes with pbs-t, µl of abts substrate solution (kirkegaard and perry laboratories, washington, dc, usa) was added and the mixture was incubated for min at rt. absorbance at / nm was measured on glomax discover system (promega), and the signal-to-noise ratio (s/n) was calculated. for generation of mabs, we produced n-terminal his-tagged full-length vp protein of hpev as an antigen. as a result, hpev -vp protein was produced with high aqueous solubility (figure a) . the protein was subsequently purified using ni-sepharose beads, followed by elution with imidazole. balb/c mice were then immunized with the purified protein. after weeks of immunization, the mice splenocytes were isolated, fused with myeloma cells, and hybridomas were produced. as a result, stable hybridomas were generated and designated as # to # . among these clones, nine were selected (# , # , # , # , # , # , # , # , and # ), based on the reactivity in elisa to the target antigens vp proteins derived from hpev and hpev (figure b) . isotype analysis revealed that # mab belongs to igg , kappa isotype, # and # mabs belong to igg a, kappa isotype, while the others belongs to igg b, kappa isotype ( figure c) . to demonstrate the antibody-binding sites within the antigen, we next performed epitope mapping. for epitope mapping, we produced five deletion mutants of vp and performed immunoblotting analysis. we found that our newly developed mabs recognized three distinct domains in hpev vp : # and # mabs bind to - amino acid (aa), # , # , and # mabs bind to - aa, and remaining four mabs (# , # , # , and # ) bind to - aa within c-terminal region of the vp protein of hpev (figure a) . we further created deletion mutants for a more precise epitope determination for these mabs, and found that # and # mabs bind to - aa, # , # , and # mabs bind to - aa, and # , # , # , and # mabs bind to - aa (figure b) . we next examined whether the antigenic epitopes were located on the surface of hpev -vp . the ucsf chimera software revealed that, except for # and # , the binding regions of all mabs were located on the molecular surface of vp protein (figure c) . the binding region of mabs # and # was relatively conserved among the analyzed hpevs (figure d ). to demonstrate the antibody-binding sites within the antigen, we next performed epitope mapping. for epitope mapping, we produced five deletion mutants of vp and performed immunoblotting analysis. we found that our newly developed mabs recognized three distinct domains in hpev vp : # and # mabs bind to - amino acid (aa), # , # , and # mabs bind to - aa, and remaining four mabs (# , # , # , and # ) bind to - aa within c-terminal region of the vp protein of hpev (figure a) . we further created deletion mutants for a more precise epitope determination for these mabs, and found that # and # mabs bind to - aa, # , # , and # mabs bind to - aa, and # , # , # , and # mabs bind to - aa (figure b ). we next examined whether the antigenic epitopes were located on the surface of hpev -vp . the ucsf chimera software revealed that, except for # and # , the binding regions of all mabs were located on the molecular surface of vp protein (figure c) . the binding region of mabs # and # was relatively conserved among the analyzed hpevs (figure d ). we next investigated the specificity of our newly developed mabs. for this, we created vp proteins encoded by the six different hpev genotypes and vp -vp proteins derived from enteroviruses and d . immunoblotting analysis showed that six mabs (# , # , # , # , # , and # ) react specifically with hpev vp , while three mabs (# , # , and # ) showed some crossreactivity to other hpevs (figure ) . no mabs showed cross-reactivity to enterovirus vp -vp proteins. we next investigated the specificity of our newly developed mabs. for this, we created vp proteins encoded by the six different hpev genotypes and vp -vp proteins derived from enteroviruses and d . immunoblotting analysis showed that six mabs (# , # , # , # , # , and # ) react specifically with hpev vp , while three mabs (# , # , and # ) showed some cross-reactivity to other hpevs (figure ) . no mabs showed cross-reactivity to enterovirus vp -vp proteins. sandwich elisa systems with highly specific matched antibody pairs are commonly used to detect and quantify viral antigens in immunoassay. hence, we next determined the optimal pair of mabs for antigen-capture using elisa, by evaluating all possible combinations of immobilized and labeled mabs. among the possible pair combinations, only the # and # mab pair represented a combination of antibodies specific for hpev -vp (figure a ). therefore, we selected this combination for further analysis. sandwich elisa systems with highly specific matched antibody pairs are commonly used to detect and quantify viral antigens in immunoassay. hence, we next determined the optimal pair of mabs for antigen-capture using elisa, by evaluating all possible combinations of immobilized and labeled mabs. among the possible pair combinations, only the # and # mab pair represented a combination of antibodies specific for hpev -vp (figure a ). therefore, we selected this combination for further analysis. to characterize the equilibrium dissociation constant (k d ) of the selected antibodies and the target vp antigen, we used the bli octet assay system. k d for antibody-antigen binding in mabs # and # was calculated as < × − and . × − , respectively, suggesting that both mabs showed high binding affinity to the hpev -vp (figure b) . we next performed antigen-capture elisa with recombinant vp protein and virions released into the cell-culture supernatant of the hpev -infected cells. using the optimal antibody pair (# and # mabs) identified above, we determined the detection threshold for antigen recognition by antigen-capture elisa. our results revealed that our system was highly sensitive to the recombinant antigen, capable of detecting the protein at a concentration of ng/ml (figure c, left) . in parallel, we investigated the detection limit of elisa for the hpev virion. this elisa system could detect heat-treated hpev , but not non-heated virions, and its detection limit of the system was × copies/ml (figure c, right) . we also found that this elisa system exhibited no cross-reactivity with enteroviruses ( figure d ) to characterize the equilibrium dissociation constant (kd) of the selected antibodies and the target vp antigen, we used the bli octet assay system. kd for antibody-antigen binding in mabs # and # was calculated as < × − and . × − , respectively, suggesting that both mabs showed high binding affinity to the hpev -vp (figure b) . we next performed antigen-capture elisa with recombinant vp protein and virions released into the cell-culture supernatant of the hpev -infected cells. using the optimal antibody pair (# and # mabs) identified above, we determined the detection threshold for antigen recognition by antigen-capture elisa. our results revealed that our system was highly sensitive to the recombinant antigen, capable of detecting the protein at a concentration of ng/ml (figure c, left) . in parallel, we investigated the detection limit of elisa for the hpev virion. this elisa system could detect heattreated hpev , but not non-heated virions, and its detection limit of the system was × copies/ml hpev is increasingly being highlighted as a potentially severe viral infection in neonates and young infants. therefore, there is an urgent need to develop assays for early diagnosis of hpev infection for reducing inappropriate antimicrobial use, unnecessary investigations, and prolonged hospitalization. it is also likely to lead to follow-up for potential complications in infants who are severely affected [ ] . in this context, a real-time pcr-based molecular test to detect virus from patients was recently developed [ ] . however, pcr tests are extremely sensitive and need extensive controls, whereas antigen detection by mabs has the advantage of the relative ease of sample handling and the use of less stringent procedures. specific mabs could then be used to develop a rapid test such as elisa. in this study, we sought to generate specific mabs and develop an elisa test for the detection of hpev vp antigen. hpev genome encodes for three structural proteins, namely vp , vp , and vp , which assemble to create the virus particle [ ] . among these, vp was identified as an antigenic determinant and it might be relatively more useful for diagnostic purposes, due to a higher level of sequence conservation [ ] . furthermore, it possesses high immunogenicity [ ] . therefore, the selection of vp as an antigen is both practical and reasonable. in addition, the mab quality was determined mostly by preparation of high-quality antigen. here, we used a wheat germ, cell-free protein production system for synthesizing recombinant vp proteins, as this system produces properly folded, soluble, and biologically active native proteins similar to those expressed in mammalian cells [ , , ] . in our current study, we newly produced nine different mabs that recognized hpev -vp as an antigen. we then performed epitope mapping of our generated mabs, as identification of the epitope is a key step in the characterization of monoclonal antibodies [ ] . based on the epitope analysis, the mabs were able to recognize three different areas of hpev -vp and specify - aa length epitopes within the hpev -vp . interestingly, two mab clones (# and # ) exhibiting cross-reactivity to vp proteins of other hpevs, bound a distinct epitope ( - aa), which partially overlapped with the recognition site for the polyclonal antibody ( - aa) created by chen et al. [ ] . nevertheless, we obtained mabs specific to hpev , which recognized sites - aa and the - aa site of vp , which was not reported earlier. moreover, we also developed an elisa for detecting hpev antigen using two of our newly generated hpev -specific mabs (# and # ), as mab-based elisa is highly specific and sensitive towards viral antigen detection [ ] . our octet assay suggested that both mabs show a high binding affinity to the full-length hpev -vp recombinant protein. vp protein can be folded into the correct native structure and is likely to form the capsid-like structure via oligomerization. in this situation, # mab could bind various sites on polymerized vp proteins, as a result of an allosteric effect or "avidity", owing to which the k d value of # mab was calculated to be much lower than that of # mab. our newly developed elisa system requires heat treatment of hpev to detect vp antigen. based on previous studies [ ] , the binding area of mab # was estimated to localize to the interface between vp and vp . on the other hand, three-dimensional modeling of viral particles revealed that the antigen-recognizing sites of mabs # and # in vp protein are proximally located. moreover, a previous study showed that glu (in the epitope region of # ) and ser (in the vp protein of hpev ) bind together by a hydrogen bond [ ] . thus, a possible explanation for our observation is that heat treatment helps antigen retrieval for the interface between vp and vp , resulting in efficient access of mabs to the epitopes. according to a previous study [ ] , an increase in antibody-binding capacity was exhibited when glycosylation of capsid protein was removed. based on this finding, we carried out pretreatment by resecting the connection between o-linked and n-linked glycosylation of hpev . however, there was no obvious effect on antibody recognition in our sandwich elisa assay (data not shown), indicating that glycosylation might not affect the antigenicity for mab recognition. the detection limits of our newly developed sandwich elisa were ng/ml for recombinant vp protein and × copies/ml for viral particles, respectively. assuming that one viral particle contains a single copy of a viral genome, there are copies of vp protein per viral particle. for a viral particle detection sensitivity of × copies/ml, the vp protein detection sensitivity is presumed to be ng/ml. therefore, we conclude that the detection sensitivity for the recombinant vp protein and the virus particles was almost equivalent, suggesting the feasibility of our method for use in actual clinical settings. other than the elisa method, several antigen-detecting tests using an antigen-antibody interaction are now available. for instance, the multi-array technology using electrochemiluminescence immunoassay (eclia) and chemiluminescent enzyme immunoassay (cleia) can provide several hundred times more sensitivities than the conventional elisa method [ ] . another example is an influenza-testing kit using a highly sensitive immunochromatographic detection method based on silver amplification [ ] . the limitation of sensitivity might be overcome by utilizing these sophisticated technologies combined with our monoclonal antibodies for hpev vp antigen. furthermore, we can potentially improve the detection sensitivity by altering the second antibody to recognize the poly-hrp complex [ ] or by adding other antibodies, which can target vp or vp proteins. in summary, we utilized the wheat germ cell-free protein production system to synthesize the hpev vp protein and produced mabs that could specifically detect hpev but not other hpevs. we further explored the feasibility of these mabs in terms of their utility in various immunological applications. to the best of our knowledge, the hpev vp antibody as well as the elisa-based viral detection system reported here is the first of its kind ever reported. with the implementation of more sophisticated applications, our newly developed mabs could be useful for further development of diagnostic methods for hpev infection. human parechovirus: an increasingly recognized cause of sepsis-like illness in young infants detection and characterization of a novel human parechovirus genotype in thailand isolation and identification of a novel human parechovirus identification and molecular characterization of the first complete genome sequence of human parechovirus type enterovirus and parechovirus infection in children: a brief overview sepsis-like disease in infants due to human parechovirus type during an outbreak in australia strain-dependent neutralization reveals antigenic variation of human parechovirus human parechovirus causing sepsis-like illness in children from midwestern united states human parechovirus and neonatal infections human parechovirus- infection: emerging pathogen in neonatal sepsis prevalence and characteristics of human parechovirus and enterovirus infection in febrile infants comparison of diagnostic clinical samples and environmental sampling for enterovirus and parechovirus surveillance in scotland infant deaths associated with human parechovirus infection in wisconsin human parechovirus causes encephalitis with white matter injury in neonates lyall, h. characteristics and outcomes of human parechovirus infection in infants human parechovirus type infection: an emerging infection in neonates and young infants epidemic myalgia and myositis associated with human parechovirus type infections occur not only in adults but also in children: findings in yamagata epidemic myalgia associated with human parechovirus type infection the human parechoviruses: an overview epidemic myalgia associated with human parechovirus type infection among adults occurs during an outbreak among children: findings from yamagata severe epidemic myalgia with an elevated level of serum interleukin- caused by human parechovirus type : a case report and brief review of the literature a . -angstrom-resolution cryo-electron microscopy structure of human parechovirus in complex with fab from a neutralizing antibody multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis genomic rna folding mediates assembly of human parechovirus parechovirus a detection by a comprehensive approach in a development of a serological assay based on a synthetic peptide selected from the vp capsid protein for detection of human parechoviruses development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen production and characterization of monoclonal antibodies specific for major capsid vp protein of trichodysplasia spinulosa-associated polyomavirus clinical relevance of positive human parechovirus type and pcr in stool samples strategies to improve detection and management of human parechovirus infection in young infants wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type for generation and characterization of monoclonal antibody monoclonal antibody-based capture elisa in the diagnosis of previous dengue infection identification of two n-linked glycosylation sites within the core of the simian immunodeficiency virus glycoprotein whose removal enhances sensitivity to soluble cd measurement of sirolimus concentrations in human blood using an automated electrochemiluminescence immunoassay (eclia): a multicenter evaluation development of a highly sensitive immunochromatographic detection kit for h influenza virus hemagglutinin using silver amplification improving the sensitivity of traditional western blotting via streptavidin containing poly-horseradish peroxidase (polyhrp) we thank naohito nozaki and satoko matsunaga for antibody production and technical assistance, and masaki takahashi (iwate prefectural institute of public health) for providing regents.conflicts of interest: y.y. is a current employee of kanto chemical co., inc. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -dvp luxe authors: jones, r. d; offutt, d. m; longmoor, b. a title: capture elisa and flow cytometry methods for toxicologic assessment following immunization and cyclophosphamide challenges in beagles date: - - journal: toxicology letters doi: . /s - ( ) - sha: doc_id: cord_uid: dvp luxe abstract the purpose of this subacute -day study was to evaluate methods for canine circulating immunoglobulins (igm, igg, and ige) and select b- and t-lymphocyte populations (cd -helpers, cd -suppressors, pan-t and pan-b) for immunotoxicity testing using an organ system (concordance) approach. the challenge substance for immunoglobulin testing was repeated immunization with six-way distemper vaccination (dhlapp), while the challenge substance for leukocyte subpopulations was treatment with cyclophosphamide. immunoglobulin measurements were made by capture enzyme-linked immunosorbent assay (elisa), and leukocyte immunophenotyping by fluorescein isothiocyanate/phycoerythrin conjugation (flow cytometry). a battery of parameters that would be used in a typical regulatory study were taken to aid interpretation of the data generated by these methods. body weights, food consumption, clinical observations, complete clinical chemistry and urinalysis measurements were taken. gross pathology and micropathology of sternal bone marrow, spleen, mesenteric and retropharyngeal lymph nodes, thymus, liver and kidney were completed. the elisa method demonstrated acceptable intra-assay reproducibility for igm, igg and ige, with values in good agreement as reported for radial immunodiffusion. the immunologic challenge demonstrated a biological trend of an increase in igm that preceded an increase in igg with no discernible trend in ige response, and no abnormalities in lymphocyte subpopulations. principle flow cytometry findings related to cyclophosphamide were that the relative percent of b cells decreased dramatically and progressively after compound administration; being statistically decreased in males on day compared with day − . the relative percent cd and cd contribution increased, but the cd /cd ratio remained relatively unchanged as total white blood cells decreased progressively. the increase in relative percent cd (males only) was statistically significant according to a two-sample t-test on days , and when compared with the pre-treatment day − . there was a relative percent increase in cd -pant, but absolute numbers were dramatically decreased. we conclude that an organ system approach to assessment of the immune system which incorporates humoral antibody, enumeration of lymphocyte populations and pathologic evaluation of the lymphoreticular organs assists in the interpretation of an adverse toxicological response. the elisa method for measurement of igs detected the expected levels of igg, igm and ige due to repeated vaccinations and to cyclophosphamide treatment. the flow cytometry method was acceptable for measuring select canine lymphocyte populations and detecting the expected decrease in b cells due to cyclophosphamide treatment. both methods may be added to a testing battery for assessing immunotoxicity in canine regulatory studies. the dog is an important immunological model for mechanism work for diseases such as atopy, rheumatoid arthritis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune thyroiditis, autoimmune dermal conditions and systemic lupus erythematosis (pedersen and pool, ) . imunodeficient dwarfism is a relevant model for elucidating the endocrine role of the thymus in its relationships between the neuroendocrine and immune systems in pre-pubertal dogs (roth et al., ) . moreover, the predictivity of pharmaceutical agent human toxicities by preclinical animal species is almost % from dog or primate studies, but very few toxicities are identified from rat studies alone (olson et al., ) . therefore, the scientific and medical rationale for using the dog as an alternative immunologic model to rodent species extends beyond being the nonrodent alternative in regulatory studies. the use of albino rodent models is often justified on the basis of reduced maintenance costs and ease of reagent development, but recent progress to transgenic models (burns et al., ) seem more conducive to specific hypothesis testing. in the dog, nonspecific serum immunoglobulin levels are considered one of the most common assessments of immunocompetence in clinical disease (german et al., ) . nonspecific immunoglobulin changes have been successfully used in human occupational medicine to investigate possible immunotoxic potential of pesticide mixtures, air pollutants and to screen for possible atopic immunoglobulin (ig)e-mediated disease (stiller-winkler et al., ) . when the specific antigen exposure is known, it can be injected or exposed to the test subject, to enhance detection of an immunologic response. some examples of measuring antigen specific response in the dog are vaccination (barthold et al., ) , diagnosis of atopic conditions, parasitisms (hammerberg et al., ) , autoimmune diseases (jones et al., ) and immunologic challenge from injected sheep red blood cells. the capture enzyme-linked immunosorbent assay (elisa) method was chosen because it has the advantage of enhanced immunologic sensitivity and accuracy compared with existing radial immunodiffusion (rid) methods (ginel et al., ) . flow cytometry for lymphocyte phenotyping in the dog has been useful in quantifying lymphocyte subpopulations and, coupled with cytochemical methods, the ontogeny and therapeutic sensitivity of myeloproliferative disorders (modiano et al., ) . cyclophosphamide (cy) is a drug that interferes with cell proliferation by cross-linking dna (calabresi and chabner, ) , useful as a standard challenge substance in immunotoxicology investigations (burns et al., ; dean et al., ) , and as anti-neoplastic therapy in the dog and humans (medleau et al., ; calabresi and chabner, ) . in general, cy is known to suppress both humoral and cell-mediated immune responses in rodents and in the rat model, and causes reduced cd , b-cell numbers and srbcspecific serum igm levels (ladics et al., ; burns et al., ; dean et al., ) . the preferential inhibition of b-cell response may possibly be due to decreased production and surface expression of immunoglobulins (burns et al., ) . in the dog, medleau et al. ( ) reported that cy reduces leukocytes, but that antibody response and lymphocyte blastogenesis were not affected. the objective of an organ system approach to immunotoxicology assessment is detecting adverse effects of suppression, hypersensitivity, endocrine disruption (pre-pubertal thymus) that are directly or indirectly related to exposure of a drug or chemical directed toward components of the immune system (burns et al., ; us-epa-tsca, ) . the purpose of this study was to evaluate immunoglobulins (igm, igg, ige) by elisa and lymphocytes (cd -helpers, cd -suppressors, pan-t cd and pan-b cd ) by flow cytometry for detecting adverse effects in dogs administered positive control challenges to the immune system. the reagents used for elisa consisted of: capture antibody-sheep anti-dog igg-affinity purified, goat anti-dog ige-affinity purified, goat anti-dog igm, affinity purified (bethyl laboratories, montgomery, tx), standard canine immunoglobulin reference serum (bethyl laboratories), secondary antibody-sheep anti-dog igg horeseradish peroxidase (hrp), goat anti-dog ige hrp, and goat anti-dog igm hrp conjugate (bethyl laboratories). the materials and chemicals used were: immulon ® flat-bottom plates (dynatech labs, burlington, ma), blocking buffer of mm phosphate-buffered saline (ph . ) with . % tween , wash buffer of ml tween and l highperformance liquid chromatography (hplc) water, phosphate-citrate buffer with urea hydrogen peroxide tablets, , %-azino-bis( -ethylbenzthiazoline -sulfonic acid) diammonium (abts), all supplied by sigma chemical (st. louis, mo), and u-bottom -well microplates. flow cytometry was performed using a fac-scalibur flow cytometer (becton dickinson, san jose, ca). the reagents used were: phycoerythrin (pe) anti-human cd , fluorescein isothiocyanate (fitc) rat igg , fitc rat igg a ; pe rat igg (pharmingen, san diego, ca), and rat anti-canine cd :fitc, rat anti-canine cd :pe; rat anticanine cd :fitc (serotec, raleigh, nc), dulbecco's phosphate-buffered saline (pierce, rockford, il), and fetal calf serum (sigma). the materials included: hplc grade water, ammonium chloride, sodium azide, potassium bicarbonate, ethylenediamine tetraacetic acid (edta)-tetrasodium (sigma), falcon test tubes, and ml centrifuge tubes with caps. positive control substances for testing biological specificity were duramune da pp+ cvk/ lci: canine distemper, adenovirus, parainfluenza, parvovirus, leptospira and coronavirus (fort dodge laboratories, fort dodge, ia) and cyclophosphamide ( -bis( -chloroethyl)amino- oxo- -aza- -oxaphosphoridin) from (sigma), considered as challenge substances a and b, respectively. the animal ration fed was purina mills lab canine diet - , analyzed for contaminants by pmi feeds, inc. (st. louis, mo). immunoglobulins in dog serum were measured by an antibody capture elisa method (immunotox, ) . serum samples were taken from control animals to conduct the method testing. serum test samples taken were aliquotted and stored at − °c or below, then thawed and brought to room temperature before being analyzed. prior to running samples, optimal concentrations of antigen coating (usually mg/ml) and conjugate were determined. optimal dilutions for standards and test sera were also determined. in this procedure, immulon flat bottom plates were prepared by adding ml of the optimal concentration of capture antibody in mm phosphate-buffered saline (ph . ) to all test wells in columns - of the microplate. column of the plate was used to determine any nonspecific binding and orientation of the plate, and column served as the blank. after the addition of capture antibody, plates were sealed to prevent evaporation and stored overnight at - °c. the capture antibody binding step was followed by a wash step using a bio-tek microplate washer with the following settings: ml wash buffer fill volume, s soak time, and three cycles. this step was used between each incubation of the procedure. test wells were then blocked with the addition of ml of mm phosphate-buffered saline (ph . ) with . % tween for h at room temperature. after incubation, plates were washed as previously described, then ml blocking buffer was added to each well in columns - . next, appropriate test samples and stan-dard were diluted in blocking buffer, and ml each were added to a u-bottom plate. all samples and standard were performed in duplicate on each plate. the addition of ml standard and test sera from the u-bottom plate to column of the immulon plate was performed using a multichannel pipette. serial dilutions were prepared from columns to . plates were incubated for h at room temperature. following the wash step, ml secondary antibody was added to each test well and incubated for h at room temperature. preparation of the citrate buffer (one tablet per ml distilled water) was performed approximately min prior to use. the peroxidase substrate was prepared by adding one abts tablet for every ml citrate buffer. after the wash step, ml substrate was added to all wells of the plate. plates were incubated until the standard on the first plate reached an optimal optical density at nm on the bio-tek ceres microplate reader. subsequent plates were read using this timing as the read point. data were collected from the microplate reader and entered into a corel quattro pro worksheet. the mean absorbance values were calculated for each dilution of standard and sample. the linear portion of the standard curve was identified, using a log -log curve fit. the standard curve concentration and absorbance values were converted to log scale for calculation of the regression equation. once the curve was established, absorbance values of the unknown were compared with the standard to determine gravimetric units of the unknown sample. gravimetric units close together were averaged and used to obtain the final gravimetric unit. log values were converted to base , and the mean, standard deviation and coefficient of variation were obtained for the gravimetric units of the samples. fresh whole blood samples were collected from control animals by cephalic venipuncture to conduct the method testing. the flow cytometer was used to analyze fitc-or pe-conjugated monoclonal antibodies to cell antigens. optimal antibody titers had been previously determined. antibodies were diluted in working buffer with protein carrier ( . ml fetal calf serum in . ml working buffer), prepared fresh daily and protected from light. the lysing buffer × stock solution (ph . ) was prepared by dissolving . g ammonium chloride, . g potassium bicarbonate, and . g edta-tetrasodium in ml hplc grade water. a × working solution of lysing buffer was prepared and maintained at room temperature. a working buffer was prepared from dulbecco's phosphatebuffered saline (dpbs) containing no calcium or magnesium, with the addition of . g sodium azide to ml dpbs. the cold buffer was used for diluting and washing cells. the procedure was as follows: ml whole blood was added to ml of × lysing buffer at room temperature, the tube capped and mixed by inversion, then left to stand at room temperature for - min. the mixture was centrifuge at × g for min at room temperature. the supernatant was aspirated, the remaining contents mixed gently and ml cold working buffer was added, mixed and centrifuged again at × g for min at - °c. the supernatant was aspirated and the pellet resuspended in . - . ml working buffer with protein carrier. confirmation of cell viability may be performed at this point. it was our experience that the optimal concentration should be approximately ( . - . )− /ml, but lower concentrations were found to be acceptable. for single color staining, ml fitc-or pe-labeled antibody were added to the appropriately labeled falcon test tube, then ml cell preparation pipetted into each tube, and the mixture incubated in a dark ice bath for at least min. an auto and isotype control were analyzed with each sample. after incubation, approximately ml working buffer was added to each tube, and if unable to analyze immediately, were stored in a dark ice bath to be analyzed within h. results obtained from the facscalibur were entered into a spread sheet and appropriate statistics generated. the limited number of published canine values by capture elisa and flow cytometry, caused a robust set of acceptance criteria to be applied prior to use in good laboratory/good automated laboratory practices regulatory studies (taylor, ) . the acceptance criteria for elisa included: linearity, intra-assay reproducibility (system precision), inter-assay reproducibility (method precision), accuracy (% observed/expected), and immunological specificity. the acceptance criteria for flow cytometry was intra-assay reproducibility (system precision), while biological specificity for both assays was determined by use of positive control challenge substances in the target species. purebred male and female (nulliparous and nonpregnant) beagle dogs, canis familiaris, were obtained from white eagle laboratories (doylestown, pa). the-study design required eight animals (four males and four females) who were approximately months of age that had been immunized with a similar modified-live vaccine weeks previous to the start of the study. the beagle was selected as the test species because of its acceptance as the nonrodent species of choice for regulatory testing, the availability of a large historical database on the strain, and because the dog is an important mechanistic model for several autoimmune diseases. the study protocol was reviewed and approved by the institutional animal care and use committee. upon receipt, animals were given complete physical examinations by a veterinarian, placed into individual pens, then acclimated and quarantined prior to pre-treatment measurements. the animals were housed in an accredited facility, where the environment was regulated and continuously monitored to maintain a room temperature range of - °c, a relative humidity range of - % and a daily photoperiod of approximately h light and h darkness. a treatment-by-study design (bruning and kintz, ) was chosen to reduce the number of animals needed by using each animal as its own control to minimize inter-subject variability and for similar statistical power as used in a regulatory study. the rationale was to utilize an organ system (concordance) approach to assess the immune system, meaning an incorporation of humoral antibody, enumeration of lymphocyte populations and pathologic evaluation of the lymphoreticular organs to aid in interpretation of a potential adverse toxicological response. clinical chemistries, urinalysis, complete blood count (including differentials) and flow cytometry phenotyping were performed on all animals twice prior to administration of challenge substance a. serum samples were retained for measurement of immunoglobulins. the combination vaccine was given on day , and . the next day following each immunization, serum samples were taken for analysis of igg, igm and ige, along with clinical chemistry, hematology, urinalysis, and flow cytometry on day , to evaluate possible influences of repeated immunization. on day , cy was administered as challenge substance b. prior to dosing, a single pre-treatment clinical chemistry, urinalysis, complete blood count (including differentials), and flow cytometry phenotyping with serum retained for immunoglobulins was done on each dog. the band t-lymphocyte subpopulations measured were cd -helpers, cd -suppressors, pan-t (cd ) and pan-b (cd ). each dog was given a single mg/kg intraperitoneal injection of cy daily for days (day , and ). a clinical chemistry, hematology, urinalysis, flow cytometry phenotyping and retained serum profile was done on each animal on days , and . statistical analysis was performed using num-ber cruncher statistical software (ncss, kaysville, ut). the continuous data were analyzed by the mann -whitney u-test or paired t-test. a probability value of p b . was accepted as significant. the data were visually inspected for trends or with the aid of orthogonal polynomial linear trend analysis. the acceptance criteria results for the elisa igg, igm and ige method are given in table . all three immunoglobulin assays had acceptable linearity. the intra-assay coefficient of variation was acceptable with , and % for igg, igm and ige, respectively. as expected when conducted on different days, the method precision (inter-assay) coefficient of variation was greater, but considered acceptable (table ). the accuracy, defined as percent observed/expected, averaged , and % for igg, igm and ige, respectively. the immunological specificity for canine was parallel with respect to the standard curve for the rat for all three immunoglobulins. the detection limit was , and ng/ml for igg, igm and ige, respectively ( table ). the linear range for igg ( - ng/ml), igm ( - ng/ml) and ige ( - ng/ml) was considered acceptable based on the subsequent biological specificity testing (days − and in fig. a,b and fig. ). the intra-assay reproducibility for measurement of lymphocyte subpopulations was found to be acceptable ( table ). the percent coefficients of variation for cd , cd , cd and cd were , , and %, respectively. the biological specificity was based on treatment with cy as the positive control challenge substances in the target species (fig. a,b ). the predominant basal immunoglobulin at pretreatment was igg ( - mg/dl), followed by igm ( - mg/dl), and the lowest basal concentration was ige ( . - . mg/dl) according to the capture elisa method (fig. a,b and fig. ) . serum samples taken after the previous day's immunologic challenge on days , and demonstrated a biological trend (nonstatistical, physiologically relevant) in the humoral response of an increase in igm that preceded an increase in igg in both sexes. there was no discernible trend in ige response due to immunization (fig. ) , which was consistent since ige functions primarily in type i hypersensitivity and release of vasoactive agents. statistically, there were no differences on day due to multiple vaccination when compared with pretreatment day − . as expected, food consumption and body weight were not affected by immunization, nor were there clinical signs attributed to this immunological challenge. there was no evidence of clinical chemistry, hematology or urinalysis alterations, nor was there discernible influence on select lymphocyte parameters attributed to the immunization challenge. cyclophosphamide treatment on days - , contributed to a trend of decreased igm that preceded an increase in igg in both sexes, possibly influenced by the normal decline of the anamnestic response to hyper-immunization (fig. a,b) . the ig response occurred despite a more than % decreased in relative percent of (relative%) b cells and total leukocyte count. immunoglobulin levels appeared little changed by cy treatment until day , when all three immunoglobulins decreased dramatically as a nonstatistical biological trend. principle flow cytometry findings related to cy were that the relative% b-cell decreased dramatically and progressively after compound administration; being statistically decreased in males on day compared with day − (fig. a,b) . the relative% cd and cd contribution increased, but the cd /cd ratio remained relatively unchanged as total white blood cells (wbc) decreased progressively. the increase in relative% cd (males only) was statistically significant according to a two-sample t-test on days , and when compared with pre-treatment day − . there was a relative% increase in cd -pant, but absolute numbers were dramatically decreased. as expected (data not presented), the principle hematologic findings noted were that the rela-tive% segmented neutrophils were increased and relative% lymphocytes were decreased days after cy, suggesting a stress leukogram. however, beginning days after cy, relative segmented neutrophils were markedly decreased and relative lymphocytes increased, along with a reduced m/e ratio that suggested granulopoietic hypoplasia. the relative% monocyte and eosinophil decreased progressively, and there was evidence of a moderate nonregenerative anemia and marked thrombocytopenia. the principle gross pathology observations considered due to cy treatment included: inflammation of the urogenital tract, gray discolored tonsil, and thymic atrophy. principle micropathology findings consisted of lymphoid depletion of the lymph nodes (mesenteric and retropharyngeal), thymus, spleen and tonsil, along with erythrophagocytosis in both lymph nodes. in addition, there was diffuse, marked hypocellularity of the sternal bone marrow in all animals. food consumption and body weight were dramatically reduced. clinical signs and observations attributed to cy treatment included: abdominal ascites, dark/mucoid feces, decreased activity, red penis (males) and vaginal opening (females), hematuria, hematochezia, vomiting, dehydration, and thin body condition. additional systemic effects of cy included decreased tsh and t , increased alp and bile acids, along with proteinuria and hematuria and days after initiation of cy challenge. the dog represents animal welfare challenges with regard to numbers of subjects that can be used in a regulatory study, as well as scientific challenge, as the common nonrodent species for extrapolation to man for risk assessment. therefore, this study design approached scientific and clinical validation from the limitations of a regulatory study, and evaluated the immune system based on a concordance or holistic (keil et al., ) assessment of humoral antibody response, enumeration of lymphocyte populations and functionality of lymphoreticular organs (burns et al., ) . the elisa and flow cytometry assessments met current industry objectives for tier i and ii immunologic testing designed for rodents (dean et al., ; pallardy et al., ) . however, the application of these methods to toxicology testing in dog studies conducted under good laboratory practices has been minimal. the elisa method was chosen because it has the advantage of enhanced immunologic sensitivity and accuracy compared with existing rid methods (tizard, ) . nonspecific immunoglobulin changes have been successfully used in human occupational medicine to detect possible immunotoxic potential when the antigen exposure is a pesticide mixture or pollutant, and cannot be injected or exposed directly to the test subject of interest. (stiller-winkler et al., ) . this study found that when multiple vaccinations were administered, a nonadverse humoral antibody response was detectable. the general immunoglobulin response pattern due to administration of a vaccine challenge was typical of an initial igm response that preceded igg then, upon subsequent challenge, an enhanced anamnestic response of both immunoglobulins was observed (tizard, ) . immunoglobulin e in normal dogs was found in extremely low concentrations, but is of major importance in type i hypersensitivity reactions, atopy, and parastisms (hammerberg et al., ; ginel et al., ) . the coefficient of variation was largest for igg but all were of similar magnitude as previously reported (german et al., ) . this vaccination challenge demonstrated a normal functional response of increased igg and igm, and a lack of ige response that did not translate into an adverse toxicological response. neither the immunization or cy challenges revealed a clear immunoglobulin response difference attributed to sex of the animal in this small study. however, given the thymus involution and pubertal influences that occur in dogs used in regulatory testing (age range, - months), it seems appropriate to account for variation in age and sex when measuring immunoglobulins, as has been reported for ige (racine et al., ) , igg, igm and iga (schreiber et al., ; german et al., ) . the decline in igm and igg after cy testing may have reflected the half-life of circulating immunoglobulins, considered to be approximately days (burns et al., ) . additionally, immunoglobulin levels during cy challenge may have been influenced by secondary systemic effects on protein homeostasis and albumin concentrations, known to correlate with igg (german et al., ) . previous work with cy in the dog (single dose, mg/kg, intravenously) found decreased lymphocyte numbers but no effect on antibody response or lymphocyte blastogenesis (medleau et al., ) . flow cytometry is becoming a critical cell biology tool for quantifying and sorting specific phenotypes, and characterizing structure and functional attributes to meet the increased demands for mechanistic work in regulatory studies (gossett et al., ) . the use of flow cytometry demonstrated that a relative% b-cell decrease was attributed to cy treatment, but the increased cd , cd and cd % contributions, when considered with the dramatic decreased in total wbc and unchanged cd /cd ratio, indicates absolute values were dramatically decreased. the preferential inhibition of b-cell response may possibly be due to decreased production and surface expression of immunoglobulins (burns et al., ) . adverse reactions in humans associated with clinical use of cyclophosphamide include vomiting, anorexia, hemorrhagic colitis, leukopenia, hemorrhagic ureteritis and cystitis (calabresi and chabner, ) , most of which were observed clinically or at necropsy in this study. it has previously been demonstrated that neurological and ophthalmological investigations are appropriate to conduct within the framework of existing guideline studies as a nested hypothesis, benefiting from the battery of tests for an organ system assessment of drugs and chemicals toxicodynamic profiles (jones et al., ) . multivariant statistical analysis of data gathered from assessment of the immune system as a whole, as described by keil et al. ( ) , would seem a useful tool toward attempting to characterize the complexity of immunotoxicities. we conclude that a concordance approach to assessment of the immune system that incorporates humoral antibody, enumeration of lymphocyte populations and pathologic evaluation of the lymphoreticular organs assists in the interpretation of an adverse toxicological response. the elisa method for measurement of igs detected a nonspecific biological response following immunization and after cy treatment. the flow cytometry method detected adverse changes in lymphocyte subpopulations after cy treatment. both methods are acceptable and may be added to a screening battery, if needed for assessing tier ii immunotoxicity, within the framework of existing guidelines for chronic nonrodent regulatory studies. serologic responses of dogs naturally exposed to or vaccinated against borrelia burgdorferi infection computational handbook of statistics casarett & doull's toxicology -the basic science of poisons goodman and gilman's the pharmacological basis of therapeutics immunotoxicity assessment in the pharmaceutical industry measurement of igg, igm and iga concentrations in canine serum, saliva, tears and bile concentrations of plasma immunoglobulins in the dog as determined by laser nephelometry. comparison with radial immunodiffusion and enzyme-linked immunosorbent assay evaluation of a commercial elisa test for the detection of allergen-specific ige antibodies in atopic dogs flow cytometry in the preclinical development of biopharmaceuticals auto igg, anti-ige and igg times ige immune complex presence and effects on elisa-based quantitation of ige in canine atopic dermatitis, demodectic acariasis and helminthiasis capture elisa for total igg levels in rat serum use of a direct enzyme-linked antiglobulin test for laboratory diagnosis of immune-mediated hemolytic anemia in dogs absence of neurovisual effects due to tissue and blood cholinesterase depression in a chronic disulfoton feeding study in dogs evaluation of multivariate statistical methods for analysis and modeling of immunotoxicology data possible incorporation of an immunotoxicogical functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study immunosuppressive effects of cyclophosphamide, vincristine, and l-asparaginase in dogs the use of cytochemistry, immunophenotyping, flow cytometry, and in vitro differentiation to determine the ontogeny of a canine monoblastic leukemia the predictivity of the toxicity of pharmaceuticals in humans from animal data -an interim assessment testing strategies in immunotoxicology the canine as a biomedical research model: immunological, hematological, and oncological aspects. department of energy influence of sex and age on serum total immunoglobulin e concentration in beagles immunodeficient dwarfism in dogs: a model for neuroimmunomodulation concentrations in serum of igg, igm and iga and their age-dependence in beagle dogs as determined by a newly developed enzyme-linked-immunosorbent-assay (elisa) immunological parameters in humans exposed to pesticides in the agricultural environment principles of measurement veterinary immunology immunotoxicity testing guidelines, cfr section key: cord- -uib h lb authors: mawle, alison c.; nisenbaum, rosane; dobbins, james g.; gary, howard e.; stewart, john a.; reyes, michele; steele, lea; schmid, d. scott; reeves, william c. title: seroepidemiology of chronic fatigue syndrome: a case-control study date: - - journal: clin infect dis doi: . /clinids/ . . sha: doc_id: cord_uid: uib h lb we performed serological testing for a large number of infectious agents in patients from atlanta who had chronic fatigue syndrome (cfs) and in controls matched by age, race, and sex. we did not find any agent associated with cfs. in addition, we did not find elevated levels of antibody to any of a wide range of agents examined. in particular, we did not find elevated titers of antibody to any herpesvirus, nor did we find evidence of enteroviral exposure in this group of patients. we performed serological testing for a large number of infectious agents in patients from atlanta who had chronic fatigue syndrome (cfs) and in controls matched by age, race, and sex. we did not find any agent associated with cfs. in addition, we did not find elevated levels of antibody to any of a wide range of agents examined. in particular, we did not find elevated titers of antibody to any herpesvirus, nor did we find evidence of enteroviral exposure in this group of patients. chronic fatigue syndrome (cfs) is an illness of unknown etiology, characterized by debilitating fatigue lasting longer than months and a variety ofnonspecific symptoms, including myalgia, arthralgia, lymphadenopathy, low-grade fever, sleep disorders, and inability to concentrate [ ] . an infectious etiology has been suggested for cfs, although the evidence is not compelling [ ] . many patients report the sudden onset of a flulike illness that presaged their fatiguing illness, and a number of infectious agents are known to cause postinfection fatigue. reports that viral antibody titers are elevated in cfs cases has led to the speculation that latent viruses may be reactivated in this illness as a result of an underlying perturbation of immune function, and that elevated titers of antibody to common agents may be a reflection of this disturbance. we conducted serological tests for a large number of infectious agents as part of a case-control study assessing risk factors for cfs. our goals were ( ) to determine whether we could detect a common exposure history among cfs patients who met a stringent research case definition and ( ) to determine whether levels of antibody to any of the agents examined were elevated in cfs cases. patients with cfs were recruited from the atlanta component of the centers for disease control and prevention's (cdc's) surveillance study ofcfs [ ] . cases ofcfs in atlanta received who currently met the cfs research case definition [ ] and who had been sick for~ years were eligible for the study; cases were recruited to participate in the study. we selected two controls for each case. they were matched for age (± years), race, and sex. controls were selected by random-digit dialing in the five-county area of atlanta covered by the surveillance system. all controls were screened to eliminate those with medical conditions that could bias the results (reyes et al., unpublished data). we collected blood and stool samples from each participant. serum and stool specimens were stored at - °c. each laboratory was requested to perform serological testing of samples as a single batch to minimize interassay variation. if that was not possible, samples from a case and two matched controls were tested in the same batch. all samples were coded so that the tests were performed in a blinded fashion. retroviruses. serological testing for hiv- and hiv- was performed with use of a u.s. food and drug administration (fda)-licensed elisa kit (genetic systems, redmond, wa). confirmatory testing was performed with an fda-licensed western blot test (cambridge biotech, rockville, md). antibodies against human t-lymphotrophic virus types i and ii were detected with an elisa, and confirmatory testing was performed by western blotting [ ] . to detect the presence of a retrovirus in peripheral blood lymphocytes (pbls) from cases, we cultured x pbls with allogeneic phytohemagglutinin-stimulated pbls for weeks. cultures were fed weekly and culture supernatants were screened weekly for reverse transcriptase activity [ ] . igm with use of a monoclonal antibody-capture elisa similar to that described previously [ ] . to detect active infection, we prepared rna from stool samples and performed a reverse transcriptase pcr to detect enteroviral sequences [ ] . for four cases and three controls, materials were insufficient for enteroviral rna testing. arboviruses. sera were screened with a hemagglutinationinhibition assay [ ] against a panel of arboviruses. antibodies against colorado tick fever virus and vesicular stomatitis virus were detected by means of a cf assay [ ] . titers of : were considered positive. herpesviruses. titers of antibody to cytomegalovirus, human herpesvirus , and varicella zoster virus were determined by means of an elisa against the whole virus. optical density readings were converted into antibody units with use of known standards [ ] . cytomegalovirus and varicella zoster virus values were considered positive at~ antibody units, and human herpesvirus values were considered positive at~ antibody units. epstein-barr virus (ebv) early antigen antibody titers were determined with a commercial elisa (gull laboratories, salt lake city), and a titer of~i: was considered positive. antibodies specific for ebv viral capsid antigen (vca) and nuclear antigen were titrated by indirect immunofluorescence assay (ifa) [ ] . a titer of~ : was considered positive. type-specific herpes simplex virus serology was performed with a glycoprotein g-based western blot assay [ ] . respiratory viruses. sera were tested for the presence of antibodies to adenovirus; parainfluenza viruses i, , and ; and respiratory syncytial virus. testing was performed by elisa with a : serum dilution [ ] . antibodies to coronavirus subtypes oc and sn e were detected with a microneutralization assay [ ] . hepatitis viruses. sera were screened for antibody to hepatitis b or hepatitis c by elisa, with use of commercially available kits (abbott laboratories, abbot park, il). elisapositive sera were subjected to confirmatory testing by western blotting (abbott laboratories). other viruses. antibody to measles nucleoprotein [ ] , rubella (rubestat g, bio whittaker, walkersville, md), and parvovirus b [ ] was detected by elisa. measles neutralization titers were also determined [ ] . rickettsiaceae. serological testing for antibodies to rickettsia typhi, rickettsia ricketts ii, coxiella burnetii, and ehrlichia chaffeensis was performed with use of ifa against fixed whole organisms [ ] . a titer of~ : was considered positive. bartonella (formerly rochalimaea species). sera were screened for antibodies to bartonella henselae, b. quintana, and b. elizabethae by means ofifa with fixed whole organisms [ ] . a titer of~ : was considered positive. borrelia burgdorferi. sera were screened by elisa for antibodies against flagellin. a positive result was confirmed by western blotting (robbins et ai., manuscript in preparation). candida albicans. precipitating antibodies were detected by both latex agglutination and immunodiffusion. the result was considered positive if either the immunodiffusion assay was positive or the latex agglutination titer was~ : . we also screened for the circulating candida antigens mannan a and mannan b [ ] . chlamydia. sera were screened by ifa at : dilution against chlamydia trachomatis, serotype l . this contains the lipopolysaccharide antigen specific for the genus chlamydia and thus detects antibodies to all chlamydia species [ ] . we analyzed our data using matched analysis procedures. we used a nonparametric test (cochran-mantel-haenzel statistic [ ] with modified ridit scores [ ] ) to assess differences in continuous variables between cases and controls. the logarithm in base of the titers of antibody to ebv-vca, ebvearly antigen, ebv -nuclear antigen, and coronavirus oc as well as the measles neutralization titer were used in the analysis, since they have a natural interpretation when titers are considered (each dilution represents unit in the log base scale). in all statistical testing we carried out, a p value of~. was considered significant. since the design of the study was exploratory and not hypothesis-testing, we did not correct for multiple comparisons [ ] . statistical analyses were performed with the statistical software sas (sas institute, cary, nc). we recruited patients with cfs ( female and male) and matched controls. two controls withdrew, leaving cases with only matched control. all participants were white (this finding is consistent with those of other studies). the median duration of illness for the cases was . years (range, . - . years), and the median age at onset of illness was . years (range, - years). occupation, income, and education were comparable between cases and controls. agents infrequently detected in or absent from cfs cases. reverse transcriptase pcr analysis of stool specimens revealed the presence of enteroviral rna in three cases ( . %) and four controls ( . %). there was no overlap between those who had enterovirus-specific igm and those who had detectable viral rna in their stool. there were no statistically significant differences between cases and controls for any of these agents. agents frequently detected in cfs cases. all other agents tested were detected in ;;::: % of cfs cases, and antibody levels were compared between cases and controls. these agents included the herpesviruses cytomegalovirus, varicella zoster virus, human herpesvirus , ebv (early antigen, nuclear antigen, and vca), and herpes simplex virus ; the respiratory viruses adenovirus, respiratory syncytial virus, coronavirus, and parainfluenza viruses , , and ; measles virus; parvovirus b ; and rubella (tables and ). the seropositivity rates in both cases and controls were ;;::: %. there were no differences between the two groups in terms of seropositivity rates or virusspecific antibody levels, as measured by titer, by antibody units, or by optical density in an elisa. we have screened a well-characterized group of patients with cfs for evidence of infection with a wide range of agents and have compared percentages of infection with age-, race-, and sex-matched controls. we found no evidence of a common infectious agent in the patients studied. there were no differ-ences in prevalence of current enteroviral infection between cases and controls, as determined by levels of circulating enterovirus-specific igm, nor in the frequency of detectable enterovirus in stool samples. thus, in the group of patients from atlanta, enteroviral infection was not more common in cfs cases. we were unable to detect evidence of infection with any known human retrovirus, and our inability to detect reverse transcriptase activity in cocultures suggests that there is no common, unknown retrovirus in these patients. this is consistent with other studies that have been unable to detect retroviruses in cfs [ , ] . we found no evidence of infection withb. burgdorferi, the causative agent oflyme disease. infection with b. burgdorferi can lead to a cfs-like illness, and in areas where lyme disease is endemic, it can contribute to cfsrelated estimates [ ] . in this group of patients from atlanta, there was no evidence that lyme disease is a factor in cfs. other agents that had < % seroprevalence in these patients included arboviruses, hepatitis b and c, rickettsiaceae, bartonella species, chlamydia species, and precipitating antibodies to c. albicans. we also looked for circulating candida antigens, since these are frequently found in severely immunodeficient patients. none of our patients with cfs had detectable mannan a or mannan b, which suggests that these patients were not immunodeficient. the lack of precipitating antibodies to c. albicans argues against the occurrence of a chronic candidal infection in patients with cfs. herpesviruses-in particular, ebv and human herpesvirus -have been associated with cfs. although elevated titers to ebv-vca and ebv early antigen were initially described as occurring in patients with cfs [ , ] , subsequent studies made it clear that these were not specific to cfs [ ] [ ] [ ] . in this study, we found no differences in titers to ebv-vca or ebv nuclear antigen between cases and controls. however, the titer of early antigen was slightly elevated in the cases compared with that in controls, although the difference was not statistically significant. in conclusion, we were unable to find evidence of a single infectious agent associated with cfs in this patient population, nor did we find evidence of elevated antibody titers consistent either with reactivation of a latent virus or with generalized immune activation. the chronic fatigue syndrome: a comprehensive approach to its definition and study is chronic fatigue syndrome an infectious disease? epidemiology of chronic fatigue syndrome: the centers for disease control study chronic fatigue syndrome: a working case definition simultaneous confirmation and differentiation of human t-lymphotropic virus types i and ii infection by modified western blot containing recombinant envelope glycoproteins isolation, characterization, and transmission of human t-lymphotrophic virus types i and ii in culture echovirus infection and aseptic meningitis in parents of children attending a child care center enteroviral rna sequences detected by polymerase chain reaction in muscle of patients with postviral fatigue syndrome techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses adaptation oflecf microtechnique in standardized diagnostic complement fixation method and adaptation to micro test prior herpes simplex type infection as a risk factor for hiv infection immunofluorescence in cells derived from burkitt's lymphoma evaluation of a test based on baculovirus-expressed glycoprotein g for detection of herpes simplex virus type-specific antibodies viral antibody screening system that uses a standardized single dilution immunoglobulin g enzyme immunoassay with multiple antigens antigen detection in human respiratory coronavirus infections by monoclonal time resolved fluoroimmunoassay baculovirus expression of the nucleoprotein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays human parvovirus bi -specific igg, iga, and igm antibodies and dna in serum specimens from patients with erythema infectiosum evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus a comparison of the complement fixation, indirect immunofluorescent antibody, and microagglutination tests for the serological diagnosis of rickettsial diseases serological response to rochalimaea henselae antigen in suspected cat-scratch disease serodiagnosis of fungal diseases single antigen fluorescence test for chlamydia antibodies average partial association in threeway contingency tables: a review and discussion of alternative tests on the combination of independent two-sample tests of wilcoxon no adjustments are needed for multiple comparisons search for retrovirus in the chronic fatigue syndrome lack of evidence for infection with known human and animal retroviruses in patients with chronic fatigue syndrome jennekens fgl chronic muscle weakness caused by borrelia burgdorferi meningoradiculitis evidence for active epstein-barr virus infection in patients with persistent unexplained illness: elevated anti-early antigen antibodies persisting illness and fatigue in adults with evidence of epstein-barr virus infection frequency of chronic active epstein-barr virus infection a cluster of patients with a chronic mononucleosis-like syndrome: is epstein-barr virus the cause? antibodies to epstein-barr virus in patients with chronic fatigue the authors gratefully acknowledge drs. l. schonberger, w. gunn, a. khan, and g. holmes (cdc), whose initial work on cfs led to this study. they are indebted to drs. w. bellini, c. black, r. craven, d. erdman, t. folks, . hierholzer, n. karabatsos, r. lal, s. lambert, m. pallansch, r. regnery, e. reiss, and t. tzianabos (cdc), in whose laboratories many of the reported assays were performed, and to dr. . gow (institute of neurological science, scotland) for performing the enteroviral rtpcr. they also acknowledge d. connell, b. randall, and r. jacobs (abt associates) as well as e. parris (cdc), who were responsible for study logistics and data processing. key: cord- -a gkath authors: leung, danny tze ming; chi hang, tam frankie; chun hung, ma; sheung chan, paul kay; cheung, jo lai ken; niu, haitao; tam, john siu lun; lim, pak leong title: antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: a gkath the recent outbreak of severe acute respiratory syndrome (sars) provided an opportunity to study the antibody response of infected individuals to the causative virus, sars coronavirus. we examined serum samples obtained from patients with sars, patients with non-sars pneumonia, and healthy individuals, by use of western blotting (wb), enzyme-linked immunoassay (elisa), and immunofluorescence assay, using both native and bacterially produced antigens of the virus. we found a highly restricted, immunoglobulin g-dominated antibody response in patients with sars, directed most frequently ( % by elisa) and predominantly at the nucleocapsid. almost all of the subjects without sars had no antinucleocapsid antibodies. the spike protein was the next most frequently targeted, but only % of the patients (by elisa) responded. other targets of the response identified by use of wb included antigens of and kda. several nonstructural proteins cloned were not antigenic, and the culture-derived nucleocapsid appeared to be specifically degraded. peptidase n [ ] . after this initial attachment, the viral e protein fuses with the plasma membrane of the host cell, and a cascade of intracellular events follows, including interaction between the m and n proteins. this interaction is important for packaging of the progeny virions [ ] . after infection, patients with sars develop antibodies to the virus, and % of them experience seroconversion within days [ ] . however, little else is known about the antibody responses in these patients. the conventional antibody test, which detects antibodies to antigens present in virus-infected cells by use of immunofluorescence assay (ifa) [ ] , does not reveal which viral antigens are targeted by the immune response. knowing the viral targets is important for several reasons. first, this allows the development of cell-free antibody tests that are less cumbersome and subjective than ifa and that can be used for mass screening in times of epidemics. second, this enhances our understanding of the immunopathology of the disease, and moreover, helps in design of a vaccine. both the humoral and cellular arms of the adaptive immune response are presumed to be important in controlling or preparation u reacted with a serum sample from a patient with sars showing the highly reactive antigens-nucleocapsid (n) , n , and n -in the former. mw, molecular weight markers (in kda). c, wb results of patients with sars (s) and patients with non-sars pneumonia (p), showing strong reactivities of the n -n antigens and lesser reactivities of the spike and the -and -kda proteins or the partial reactivity (n ) or total lack of reactivity. the infection. as with other covs, antibodies can function by blocking the adsorption or entry of the virus to the target cell [ ] [ ] [ ] or by interfering with viral transcription [ ] . in the present study, we identify the viral antigens to which our patients with sars had responded. patients and control subjects. we diagnosed sars according to the world health organization criteria [ ] , on the basis of the following symptoms: acute onset of fever ( Њc) accompanied by frequent chills or rigor, dyspnea, headache, myalgia, hypoxemia, and radiological evidence of pneumonia. in addition, the patients with sars had anti-sars cov igg antibodies detected by use of an ifa [ ] , which showed seroconversion or a -fold increase in titer. in this assay, which uses virus-infected monkey kidney (vero) cells and which is performed routinely in our laboratory, a serum titer of у : was considered to be positive. all patients ( males and females; age range, - years; age, years) had contact with mean ‫ע‬ sd . ‫ע‬ . patients with confirmed sars within days of the onset of their symptoms and were admitted to the prince of wales hospital, hong kong, during the sars outbreak that started in early march . two of the patients died, required intensive care but eventually recovered, and the rest had a mild clinical course. because methods of detection were not available early during the outbreak, the sars cov was cultured from only patient, and viral rna was detected by use of reverse-transcription polymerase chain reaction (pcr) [ ] from the respiratory or stool samples from patients. the serum samples used in the present study were obtained - days ( , days) mean ‫ע‬ sd . ‫ע‬ . after the onset of fever. control subjects included inpatients ( males and females; age range, - years; mean age, . years) who had presented at the prince of wales hospital with clinical features of atypical pneumonia and were treated accordingly at the hospital in before the emergence of sars and healthy individuals (blood donors and students). institutional approval for both human and animal ethics was obtained for the study, and the institutional guidelines were followed. viral culture and crude native antigens. vero cells (atcc crl- ) were grown in dulbecco's modified eagle medium containing % fetal calf serum at Њc in a % co humidified incubator. the cuhk-w sars cov strain (genbank accession no. ay ) used was isolated from a patient in our hospital. ). after incubation for h on ice, the supernatant obtained from the cell lysate by centrifugation was heated for min at Њc, to inactivate any live virus present, and was stored at Ϫ Њc until use. control cell lysate was similarly prepared from uninfected vero cells. recombinant viral antigens. total rna extracted from sars cov-infected vero cells by use of a qiamp viral rna kit (qiagen) was reverse-transcribed with random hexamers (applied biosystems), and the cdna obtained was used to generate the various gene segments. the following forward (f) primers (containing a bamh site, underlined) and reverse (r) primers (containing an ecor site, underlined) were used in pcrs described elsewhere [ ] for the corresponding gene segments (sizes the pcr conditions used were min at Њc, cycles of min at Њc, min at Њc, min at Њc, and min at Њc. the gel-and affinity-purified fragments were cloned into the bacterial expression vector pgex- t (amersham bioscience), which is fused to the bacterial glutathione s-transferase (gst) gene. the ligated vector was transfected to escherichia coli bl . recombinant antigens were recovered from selected transformants induced with isopropyl-b-d-thiogalactopyranoside. in brief, the cell pellet was resuspended in ice-cold lysis buffer ( mmol/l tris-hcl, mmol/l nacl, . % triton x- , mmol/l pmsf, and ϫ protease inhibitor cocktail [ph . ; sigma]) and was sonicated ( mm). the recombinant antigen was recovered from the supernatant of the lysate by use of affinity chromatography using glutathione-coupled agarose (amersham) and, for elution, mmol/l tris-hcl buffer (ph . ) containing mmol/l reduced-form glutathione, mmol/l nacl, . % triton x- , and mmol/l ddt. the eluted antigen was examined on sds-page gels stained with coomassie blue (expected size and good purity were observed in all cases; data not shown). in sds-page, the antigen preparation was heated for min at Њc in loading buffer ( . mol/l tris-hcl [ph . ], % -mercaptoethanol, % glycerol, % sds, and . % bromophenol blue) and electrophoresed ( v for min at room temperature [rt]) on % polyacrylamide. antigens of interest (pn , pn , and ps) were located in the unstained gel by use of a parallel gel stained with coomassie blue and were recovered with an eluter (harvard bioscience) at v overnight in mmol/l tris buffer (ph . ) containing mmol/l glycine, % methanol, and . % sds. in other experiments, the whole gel was electroblotted onto a . -mm polyvinylidene fluoride membrane (bio-rad) and was used for protein sequencing (protein facility, iowa state university) or for wb. in the latter assay, the membrane was blocked with skim milk and incubated (for h at rt) with the unknown human or mouse serum (diluted : in ml of pbs containing % skim milk) [ ] . after washing, the blot was incubated (for h at rt) with peroxidase-labeled goat anti-human igg or anti-mouse ig (all classes) (bd biosciences) and later with the chemiluminescence substrate ecl (amersham). the assay was developed by exposure to hyperfilmb max (amersham). in inhibition wb, the unknown serum ( ml) was preincubated with the inhibiting antigen ( mg/ml) in ml of pbs containing % skim milk overnight at Њc. elisa. the native viral antigens, either crude or purified (pn , pn , and ps; : stock dilution), or the recombinant antigens (rna, rsa, rsb, rsc, rnsp a, rnsp b, rnsp , and rnsp ; mg/ml) were coated onto -well immunon- plates (dynex) in bicarbonate buffer (ph . ) overnight at Њc, and the assay was performed as described elsewhere [ ] . in brief, ml of the unknown human serum diluted : in pbs (containing . % bovine serum albumin, . % casein, and . % tween- ) were added to the wells and incubated for min at rt. the plates were washed and incubated (for min at rt) with horseradish peroxidase-labeled goat anti-human ig (igg, igm, or iga specific) (bd biosciences). after washing, substrate ( , , , -tetramethylbenzidine) was added, and reaction was allowed for min at rt. the results were read at nm in a dynex mrx ii reader. mouse immunization. balb/c mice ( mice/group) were injected intraperitoneally with the affinity-purified, alum-precipitated antigen ( mg/mouse) in complete freund's adjuvant and received booster injections with mg of the antigen in incomplete freund's adjuvant weeks later. blood was obtained from the retro-orbital plexus days after administration of the booster injection. statistics. the relationships of the (rna) elisa with other immunoassays or with sampling times were examined by use of regression analysis (graphpad prism ; graphpad software). we first examined the antibody response of the patients with sars by use of wb. figure b shows the gel-separated crude extract of the culture-grown virus. several virus-specific antigens are discernible by comparing the control (uninfected) extract with the viral extract, particularly ones at , , and kda molecular weight. the antigenicity of these proteins, labeled n , n , and n , respectively, is shown by immunoblotting with serum samples obtained from patients with sars ( figure b) . the relative abundance of n -n varied among batches of the extracts, but n , which is actually a doublet, was consistently lowest in quantity. the wb results from representative patients with sars and patients with non-sars pneumonia are shown in figure c . these results were based on the igg response; the igm reactions were either weak or absent. the n -n antigens were the most reactive antigens found in the patients with sars but were absent in patients with non-sars pneumonia. these antigens always appeared as a triplet. one of the non-sars serum samples reacted weakly with an antigen at the position of n . lessreactive antigens found at , , and kda were not seen in the non-sars serum samples. the molecular size of n suggests that it is the nucleocapsid, but n and n could not be identified in this way. protein sequencing of these antigens was not successful, but it confirmed the -kda "s" antigen (sdldr) to be the s protein. on the basis of wb results for a total of patients with sars, patients with non-sars pneumonia, and healthy subjects, most ( %) of the patients with sars produced antibodies to the n -n antigens, but only % produced antibodies to the s protein ( figure ). in addition, %, %, %, and % of the patients with sars produced antibodies to the -, -, -, and -kda antigens, respectively (data not shown). none of the control subjects produced antibodies to any of these antigens, although % of the serum samples from patients with non-sars pneumonia showed weak reactivity with the (presumably) n antigen (see below). the sars serum samples were positive for viral antibodies when examined by use of an ifa using infected cells, although some ( %) had low titers (! : ) only (figure ). none of the serum samples from the control cohorts examined were positive by either ifa or wb. using a bigger study group ( patients with sars, patients with non-sars pneumonia, and healthy individuals), we examined the antibody responses further by use of elisa. first, when the crude viral extract was used as antigen, % of the patients with sars were positive for antibodies, compared with ! % of the combined control subjects (figure ). all patients with sars who were negative for antibodies had low ifa titers (! : ). all serum samples were not reactive with the control antigen, gst (data not shown). the results are based on the igg response. the igm response, in contrast, was less robust and less frequent ( %) among the patients with sars (figure ) and less discriminatory between the sars and non-sars cohorts. second, we made a recombinant antigen of the n-terminal half of the n protein (rna), and, when it was used in an igg elisa, we found results almost identical to those found with the crude viral extract ( % sensitivity and %- % specificity), including negative cases in common ( figure ) . the igm responses were similarly low and infrequent among the patients with sars (data not shown). we investigated the possibility that the % of serum samples that were negative or weakly positive in the elisa but were ifa positive might have antibodies directed to antigens other than the nucleocapsid or those present in the crude extract. we thus examined the recombinant antigens made from several nsps of the virus. however, although nsp (both subunits) and nsp were found to be nonantigenic, nsp showed only weak reactivities with some ( %) of the serum samples from patients with sars (data not shown). native s antigen that was purified and used in an elisa detected antibody responses in % of the patients with sars (figure ), slightly more than those detected by use of wb. however, most of the responses were weak. using recombinant s antigens, we found that only the c-terminal end (rsc) of the protein was antigenic and that only a small number ( %) of serum samples from patients with sars were reactive, similar to results for serum samples from patients with non-sars pneumonia (figure ). we purified the native n and n antigens together from the gel-separated crude extract and used these in an elisa. the results obtained were very similar to those of the rna elisa, particularly with respect to the negative or positive cases ( figure ). this suggests that n or n or both might be antigenically related to rna. to investigate this possibility, we performed wb experiments using rna as inhibitor. rna was indeed found to reduce the reactivity of not only n , but also of n and n (figure a). in fact, inhibition was greatest with n and least with n . of interest, there appeared to be a fourth, minor n fragment (n ) with a molecular weight of kda (figure a) that was seen with some serum samples (e.g., s ; figure ). similar results were obtained when the purified n and n antigens were used as the inhibitor in the wb analysis ( figure a) . in contrast, when the s antigen, rsc, was used as inhibitor, reactivity at the -kda region ("s"), but not that of n -n , was affected (i.e., abolished) ( figure a) . we proved further that n , n , n , and n are all n antigens. mouse serum made against the recombinant antigens rna, rsa, and rsc (rsb was not available at the time) were used in wb against the crude viral extract. all mice immunized with rna produced antibodies that reacted specifically with n , n , n , and n , but not with other antigens (figure b). in figure . comparison of the efficiency of various detection assays for severe acute respiratory syndrome (sars). individual serum samples from each group ( patients with sars, patients with non-sars pneumonia, and healthy individuals) were examined by use of immunofluorescence assay (ifa), western blotting (wb), or elisa. antigens used included native spike (s), native nucleocapsid (n), crude antigen, crude viral extract, recombinant nucleocapsid (rna), gel-purified native n and n antigens (pn , ), gel-purified native spike (ps), and recombinant spike (rsc). except where marked "igm", all tests are based on detection of igg. in each test, the cutoff for positivity is shown by the shaded bar; for elisa, this is based on the sd value of the combined cohorts of patients with non-sars pneumonia and healthy individuals. in wb, the intensity of the mean + reaction was arbitrarily scored by eye ( , strongest) . serum samples that were not examined because of a lack of antigen or serum or because the results were not readable are shown by a dot. contrast, neither of the s antigens produced any antibodies against the crude extract (figure b). the n antigen of sars cov stands out as the most important diagnostic antigen of the virus. the majority of our patients who developed sars ( % by elisa) produced antibodies to this antigen. when present, these antibodies are also the most abundant of the antibodies made to the virus. it is not clear why the n antigen is so immunogenic, but we have found that even a bacterially produced fragment of it induced good production of antibodies in mice. the n antigen also has been found to be highly immunogenic in the elk cov [ ] and in the infectious bronchitis virus [ ] . we have found the n antigen to be the predominant antigen in the crude viral extract. this is shown by the good correlation between the rna and crude antigen elisas (figures and a). in contrast, there was only weak correlation between the rna elisa and the ifa performed on these serum samples ( figure b ). this result was not due to the fact that we used a singledilution measurement in the elisa, rather than titrating the serum ( figure c ). it is possible that some antigens cannot be extracted from the cell or become degraded during the extraction, but this was not the case with nsp or nsp . it is noteworthy that the serum samples from the patients with non-sars pneumonia, which reacted with the (presumably) n component by wb, were negative by the rna elisa. this result suggests that the reactive epitope is located elsewhere in the n antigen or in a nonviral (vero) antigen. as expected, detection of the anti-n antigen antibodies in patients with sars by use of the rna elisa improved with increasing intervals between the time samples were obtained and the onset of fever ( figure d ). thus, in of the negative cases, the serum was obtained within days after the onset of fever. on the other hand, other serum samples obtained during the same period were found to be positive. although this means that more than half of the early sars cases (and all of the late [ days] cases) could be detected, it is important to note that we used serum samples that were ifa selected. of note with regard to the anti-n antibody response in the patients with sars is the predominance of igg antibodies over igm antibodies, even early (second week) during the course of the disease. this result implies that there was strong t helper cell involvement. an exaggerated t cell response probably also accounts for the pneumonia-associated immunopathology seen in patients with sars [ ] . the n antigen may be important here. it may indeed be both a potent b cell and a potent t cell immunogen; that is, an important candidate for a vaccine. although antibodies to the nucleocapsid are nonneutralizing, whether such antibodies can be protective in other ways, as observed for mouse hepatitis virus [ ] and rotavirus [ ] , needs to be addressed formally. the latter phenomenon may be due to the ability of the iga antibodies to enter the infected cell by the iga-transcytosis pathway, thereby interfering with viral transcription [ ] . we found evidence that the n antigen is degraded in cell cultures of the virus. the degradation is specific, since the major n antigens, n -n , were found in every preparation of the crude extract that we used. a minor antigen, n , was also sometimes seen. n is presumably the full-length protein, and the others are fragments of n that lack varying lengths of the c-terminal end. because of the high specificity seen, we do not consider the degradation to be an artifact of the antigen-extraction procedure. rather, we suspect that n is cleaved by caspases in the vero cells as the cells undergo apoptosis, similar to what eleouet et al. [ ] observed for the n antigen from the transmissible gastroenteritis cov. after the n antigen, the s antigen is the one most often targeted by the immune response of the patients with sars. however, ! % (by elisa) of the patients responded, and their responses were generally weak. it is possible, on the other hand, that the responses were underestimated because the s antigen contained in the crude extract used for wb or elisa was degraded or underrepresented and the recombinant antigen used for elisa that was produced in bacteria lacked the necessary glycosylation normally associated with the native antigen (see: http://www.cbs.dtu.dk). other antigens of the virus targeted by the immune response are presumably minor. examples are the -and -kda antigens. the latter could be nsp , recently postulated to be an mrna cap- methyltransferase [ ] . the -kda antigen to which some patients responded could be the m protein. both this and the e protein of the virus (the latter was too small [ . kda] to assess in our wb gels) are presumed to be important in protection. identification of severe acute respiratory syndrome in canada coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov- e characterization of the coronavirus m protein and nucleocapsid interaction in infected cells clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein protection from lethal coronavirus infection by immunoglobulin fragments interference of coronavirus infection by expression of immunoglobulin g (igg) or iga virus-neutralizing antibodies inhibition of rotavirus replication by a non-neutralizing, rotavirus vp -specific iga mab world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) human metapneumovirus detection in patients with severe acute respiratory syndrome a human and a mouse anti-idiotypic antibody specific for human t + anti-dna antibodies reconstructed by phage display production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of elk coronavirus recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice protective effect of rotavirus vp -specific iga monoclonal antibodies that lack neutralizing activity the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase- and - during tgev-induced apoptosis mrna cap- methyltransferase in the sars genome we thank peggy fung for excellent secretarial help. key: cord- -i on authors: nan title: abstracts dgrh-kongress date: - - journal: z rheumatol doi: . /s - - - sha: doc_id: cord_uid: i on nan im namen der dgrh, der dgorh und der gkjr begrüßen wir sie ganz herzlich zu unserem diesjährigen kongress visualisierung therapeutischer effekte von vasodilatantien beim sekundären raynaud-syndrom mittels fluoreszenzoptischer bildgebung di. stellenwert der gelenksonographie bezüglich diagnose, behandlung und therapiekontrolle der bursitis intermetatarsalis -einer häufig übersehenen differenzialdiagnose. fünf fallbeispiele wegen der deutlich eingeschränkten nierenfunktion konnten therapeutisch keine nsar angewandt werden. wir haben mit × , mg colchicin täglich behandelt. die anfänglich schwerkranke bettlägerige patientin konnte innerhalb von h mobilisiert werden. um eine abschließende sicherung der diagnose einer uratinduzierten sakroiliitis erreichen zu können ist die patientin mit einem dual-energy-ct (dect) untersucht worden. ergebnisse. mit dieser methode konnten gichttophi in beiden sakroiliakalgelenken dargestellt werden, ebenso an beiden mtp -gelenken. schlussfolgerung. aktuell liegen bisher noch keine weiteren berichte vor, dass diese methode auch für die diagnostik einer gicht im bereich der sakroiliakalregion zuverlässige ergebnisse liefern kann. zudem zeigt dieser krankheitsverlauf, dass sich die gicht durchaus primär im bereich des achsenskeletts manifestieren kann und nicht in erster linie an den peripheren gelenken zu entsprechenden beschwerden führen muss. a. glimm , s. werner , s. ohrndorf , c. schwenke , g. schmittat , g. burmester einleitung. typische pathologische veränderung bei der rheumatoiden arthritis (ra) ist die synovialitis. auch bei der osteoarthrose (oa) lassen sich entzündliche veränderungen der gelenke finden. diese können mittels fluoreszenzoptischer bildgebung (foi) und dem gelenkultraschall (us) sichtbar gemacht werden. ziel der studie: vergleich der foi mit dem us bei patienten mit ra und oa. methoden. es wurde bei patienten ( ra, oa) die foi beider hände sowie die us des handgelenks (hg) und der fingergelenke (mcp, pip, dip) der klinisch beschwerdeführenden hand von dorsal und palmar sowohl im b-bild (b-us) als auch mit power-doppler (pd-us) durchgeführt. synovialitis und tenosynovitis im us sowie die intensität des fluoreszenzsignals im bereich der gelenke in der foi wurden qualitativ als auch semiquantitativ nach standardisierten verfahren für den primavistamode (pvm) und drei verschiedene phasen (p - ; [ ] ) bewertet. in der statistischen analyse wurden anschließend sensitivitäten und spezifitäten für die foi bei der ra und oa getrennt für synovialitis und tenosynovitis, dorsal und palmar jeweils für b-us und pd-us als referenzmethode berechnet. ergebnisse. in abhängigkeit von der betrachteten phase zeigen sich für die ra und oa moderate sensitivitäten und spezifitäten. für die ra wurden in der phase des foi die höchsten sensitivitäten mit % für b-us und % für pd-us berechnet. auch bei der oa ergaben sich die höchsten sensitivitäten in der phase des foi mit % für b-us und % für pd-us als referenzmethode. die höchsten spezifitäten für beide diagnosen wurden in der foi in phase erreicht. hierbei lag die spezifität bei der ra für b-us bei % und für pd-us bei %. der höchste spezifitätswert bei der oa sowohl für b-us als pd-us war % (. tab. background. pet is a nuclear imaging technique that depicts functional processes within the body with high sensitivity by detecting annihilation radiation from radioactive decay of a positron-emitting radionuclide that was labeled to a biologically active molecule (tracer) and introduced into the body. f-fluoride ( f) can be used for pet as a bone-seeking agent reflecting bone perfusion and remodeling. we inaugurated a pilot study with simultaneous pet/mr to examine whether addition of pet provides different and additional information in comparison to mri in axspa patients. methods. eleven axspa patients, median age y, disease duration range . - y, mean basdai . , were examined by pet/ -tesla mri minutes after injection of a mean dose of mbq of f using a integrated whole-body pet/mr scanner (siemens biograph mmr®). t-mris were scored blinded to patient's clinical characteristics by two readers ( rheumatologist and radiologist/nuclear medicine specialist) using the berlin mri score and also by recording inflammatory lesions on a vertebral edge (ve) level. in a second step pet/mris were read blindly by the same readers also based on the ve involvement of individual vertebral bodies. results. the procedure was successful in all patients. the resulting mean effective radiation dose per patient was . msv. co-registration of pet/mri fusion images was highly accurate, allowing a precise comparison of mri and pet. in the direct comparison of the mri and pet signal the two readers saw consistent signals in almost % of the sites studied. however, there were areas where signals differed, e.g. within existing syndesmophytes where pet signal was increased but conventional mris showed no signal, or the sternum area and lateral or posterior spinal elements such as facets and spinous processes. conclusion. the new technique of integrated pet/mri provides similar imaging signals as conventional mri. however, we observed differences between the two modalities in areas with less inflammatory activity but where bone metabolism seemed to be active or in areas with blurred resolution on conventional mri. the possibility that pet detects osteoblastic activity in areas where no inflammatory signal is detected with mri seems to be of interest. einleitung. sensitiven bildgebenden verfahren wie der hochauflösenden arthrosonographie kommen bei der detektion initial entzündlicher veränderungen im rahmen der frühdiagnostik der psoriasisarthritis (psa) eine große bedeutung zu. die vorliegende prospektive studie untersucht die diagnostische und prognostische wertigkeit der sonographischen befunde im vergleich zur klinischen untersuchung auf ebene einzelner gelenke bei früher psoriasis-arthritis (psa). methoden. rekrutierung von patienten mit therapienaiver früher psa. sonographie von gelenken mit semiquantitativer graduierung (grad - ) von b-bild (gsus) und power-doppler-aktivität (pdus; baseline, monate). klinische parameter: anzahl druckschmerzhafter und geschwollener gelenke (tjc , sjc ), visuelle analogskala, das -crp, health assessment questionnaire haq. für jede followup-visite erfolgte eine kategorisierung des klinischen ansprechens nach eular-response-kriterien und der für die psa validierten minimal-disease-activity(mda)-kriterien (coates et al.). ergebnisse. baseline patienten, nach monate patienten, erkrankungsdauer ( ± , monate). patienten ohne therapie ( ), mit nsar ( ), steroid i.a. ( ) , dmards ( ), biologicals ( ). bei diagnosestellung zeigte sich eine signifikante korrelation zwischen dem us synovitis score und folgenden klinischen parametern: tjc (r= , ), scj (r= , ), das -crp (r= , ). nach monaten zeigte sich eine gute korrelation zwischen der relativen veränderung des us synovitis scores und der relativen veränderung folgender klinischer parameter: tjc (r= , ), haq (r= , ), pasi (r= , ), das -crp (r= , ). zu baseline waren von gelenken sonographisch auffällig, davon zeigten kein klinisches korrelat (subklinisch). nach monaten zeigten % der initial subklinischen gelenke einen unveränderten befund, % waren sonographisch nicht mehr auffällig und % wurden klinisch manifest. bei den klinischen respondern war der rückgang deutlicher ausgeprägt. schlussfolgerung. der ultraschall-synovitis-score korreliert mit klinischen aktivitätsparametern sowohl zum zeitpunkt der diagnosestellung als auch im krankheitsverlauf unter immunsuppressiver therapie. die subklinischen veränderungen bilden sich unter immunsuppressiver therapie zu einem großen teil zurück, deutlicher bei klinischen respondern. ein geringer anteil der initial subklinischen gelenke wird im verlauf klinisch manifest, in höherem maße bei klinischen non-respondern. t. diekhoff , k. hermann charité -universitätsmedizin berlin, radiologie, berlin einleitung. die gicht ist mit einer prävalenz von , - , % insbesondere in den industrieländern eine häufige erkrankungen, die mit gelenkschmerzen einhergeht. bei typischer symptomatik und laborkonstellation ist die diagnose der arthritis urica oft einfach zu stellen, ein atypisches beschwerdebild kann jedoch gelegentlich die abgrenzung zu anderen erkrankungen erschweren. besonders die kalziumpyrophosphat-kristallarthropathie (cppd oder pseudogicht), die selbst mit sehr variabler symptomatik auftreten kann, ist eine relevante differenzialdiagnose, besonders bei älteren patienten. methoden. mit der dual-energy-computertomographie (de-ct) steht ein modernes, innovatives verfahren zur verfügung, das eine detektion von harnsäurehaltigen weichteilverkalkungen ermöglicht und darüber hinaus eine sichere abgrenzung zu kalziumhaltigen verkalkungen gewährleisten kann. das prinzip der de-ct ist relativ simpel und seit längerem bekannt: die messung des untersuchungsvolumens mit zwei unterschiedlichen röhrenspannungen macht es möglich, einen schwächungskoeffizienten zu errechnen, der spezifisch für das untersuchte material ist. allerdings ermöglichten erst moderne cts mit zwei röntgenröhren die klinische anwendung. in jüngster zeit werden jedoch anstrengungen unternommen, die de-ct auch für ein-röhren-systeme verfügbar zu machen. ergebnisse. mit der de-ct können gichttophi sicher vom knochen aber auch von anderen verkalkungen getrennt und zum beispiel farblich kodiert dargestellt werden. im gegensatz zum konventionellen röntgenbild verspricht die de-ct jedoch nicht nur eine höhere sensitivität für tophöse veränderungen, sondern als schnittbildverfahren auch eine bessere abgrenzung und einordnung von anderen morphologischen veränderungen wie zum beispiel von erosionen. schlussfolgerung. dieser vortrag fasst die vor-und nachteile der de-ct in der detektion und abgrenzung von weichteilverkalkungen bei kristallarthropathien zusammen und gibt darüber hinaus einen ausblick auf zukünftige entwicklungen in diesem gebiet. background. anionic glycosaminoglycans interact with a variety of soluble and membrane bound molecules. chondroitin sulfate was shown to have anti-inflammatory properties but its role in arthritis is controversial. methods. we have analyzed the effect of chondroitin sulfate on collagen induced arthritis starting treatment before and after induction of arthritis and in mice with established arthritis. results. in all of these settings chondroitin sulfate significantly reduced the severity of arthritis. it prevented joint destruction, diminished the inflammatory infiltrate and reduced proinflammatory cytokines in joints and plasma. splenocytes restimulated with collagen produced less il- and more il- and il- . the beneficial effects of chondroitin sulfate were transient and closely correlated to the suppression of the collagen-specific humoral immune response. chondroitin sulfate, but not other glycosaminoglycans induced a direct btk and syk-dependent proliferation of b cells and markedly expanded the number of plasma cells in the spleen. in immunized mice chondroitin sulfate reduced the number of antigen specific plasma cells in the bone marrow and was able to suppress established humoral immune responses. conclusion. displacement of disease inducing plasma cells from the bone marrow might contribute to the beneficial effects of chondroitin sulfate and could be an attractive strategy to suppress antibody mediated autoimmunity. background. in rheumatoid arthritis a functional deterioration of the hpa-axis in form of inadequately low secretion of glucocorticoids in relation to severity of inflammation can be detected. the reasons for this phenomenon are not known. the purpose of this study was to find possible reasons responsible for adrenal insufficiency during arthritis. methods. da rats were immunized with type ii collagen in incomplete freund adjuvant to induce arthritis. plasma corticosterone was evaluated by ria and plasma acth by elisa. adrenal cholesterol was quan-titatively studied by sudan-iii staining and scavenger receptor class bi (sr-bi, the hdl receptor) by immunohistochemistry. fluorescent nbd-cholesterol uptake kinetics were analysed by flow cytometry. ultrastructural morphology of adrenocortical mitochondria and lipid droplets was studied by electron microscopy. results. initially increased corticosterone and acth levels were reduced to baseline levels in the later phase of the disease. serum levels of corticosterone relative to il- β were markedly lower in arthritic than control animals (inadequacy). cholesterol storage in adrenocortical cells and expression of sr-bi did not differ between immunized and control rats. however, number of impaired mitochondria largely increased during the course of arthritis (maximum on day ), and this was paralleled by reduced numbers of activated cholesterol droplets (inhomogenous droplets relevant for generation of glucocorticoids). in addition, number of normal mitochondria positively correlated with serum corticosterone levels. conclusion. this first study on adrenal reasons for inadequate glucocorticoid secretion in arthritis demonstrated impaired mitochondria and altered cholesterol breakdown paralleled by low corticosterone levels in relation to ongoing inflammation. justus-liebig universität gießen, kerckhoff-klinik gmbh, rheumatologie u. klinische immunologie, osteologie, physikalische therapie, bad nauheim, agaplesion markus krankenhaus, akademisches lehrkrankenhaus der johann wolfgang goethe-universität, klinik für orthopädie und unfallchirurgie, frankfurt/main, universitätsklinikum gießen und marburg, orthopädische klinik, labor für experimentelle orthopädie, gießen, universitätsklinikum gießen und marburg, orthopädie und orthopädische chirurgie, gießen, universitätsklinikum erlangen, medizinische klinik , rheumatologie und immunologie, erlangen background. obesity is a risk factor in osteoarthritis (oa), but there is limited information about the interaction between bone formation and adipose tissue-derived factors, the so-called adipokines. adipokines such as adiponectin, resistin or visfatin are associated with the pathogenesis of rheumatoid arthritis (ra) and oa. adipokines are produced also by other cell types than adipocytes in ra and oa joints, for example osteoblasts, osteoclasts or chondrocytes. however, in contrast to their joint-destructive role in ra, their role in oa joint remodeling is unclear. therefore, adipokine expression in osteophyte development and bone forming cells as well as their effect on these cells was analyzed. methods. osteophytes and bone were obtained from oa patients during joint replacement surgery. serial sections of bone tissue were stained (masson trichrome, trap) and scored from grade one (no ossification, mainly connective tissue and cartilage) to five (ossified, mineralized osteophyte, < % connective tissue, ossified remodeling zones). immunohistochemistry against alkaline phosphatase, collagen-type ii, adiponectin, resistin, and visfatin was performed. oa osteoblasts were stimulated with adiponectin and measurements of il- , il- and mcp- were performed in cell culture supernatants. results. adiponectin, resistin and visfatin were detectable in osteoblasts and all osteophyte grades. in non-ossified osteophytes (grade ), especially adiponectin and to a lower extend resistin and visfatin were localized in connective tissue fibroblasts. in ossified osteophytes (grade - ), resistin, visfatin and to a lower extend adiponectin protein expression was co-localized with osteoblasts. resistin and visfatin were expressed by osteoclasts. visfatin was found in chondrocytes of all osteophyte grades ( % of chondrocytes) and adiponectin was detectable in blood vessels. osteoblast stimulation with adiponectin increased the release of the inflammatory mediators il- ( . -fold), il- ( . -fold), and mcp- ( . -fold). zeitschrift für rheumatologie suppl · | conclusion. the expression of adiponectin and visfatin expression in osteophyte connective tissue and cartilage suggests their involvement in early osteophyte formation. resistin and visfatin expression by osteoblasts and osteoclasts in ossified osteophytes indicates a role in bone remodeling of osteophytes at later stages. osteoblasts respond to adiponectin stimulation with the release of inflammatory mediators. therefore, adipokines are most likely involved in osteophyte formation at different stages affecting different cell types of bone remodeling. free fatty acids contribute to promotion of arthritis k. frommer , a. schäffler , s. rehart , a. sachs , u. müller-ladner , e. neumann justus-liebig universität gießen, kerckhoff-klinik gmbh, rheumatologie u. klinische immunologie, osteologie, physikalische therapie, bad nauheim, universitätsklinikum regensburg, klinik und poliklinik für innere medizin i, regensburg, agaplesion markus krankenhaus, akademisches lehrkrankenhaus der johann wolfgang goethe-universität, klinik für orthopädie und unfallchirurgie, frankfurt/main background. obesity is a known risk factor for several arthritic diseases and mechanical stress has been shown not to be the only factor. due to increased levels of free fatty acids (ffa) in obese compared to nonobese individuals and due to the involvement of ffa in inflammatory cardiovascular and metabolic diseases, we hypothesized that ffa play a role in the promotion of arthritic diseases. therefore, we therefore investigated the effect of ffa on various effector cells of arthritis. methods. rheumatoid (ra) synovial fibroblasts (sf), osteoarthritis (oa) sf, psoriatic arthritis (psa) sf, human primary chondrocytes (hch), human osteoblasts (ob), human macrovascular (huvec) and microvascular (hbdmec) endothelial cells were stimulated in vitro with different ffa within their physiological range of concentrations. immunoassays were used to quantify ffa-induced protein secretion. sulfosuccinimidyl oleate sodium (sso) was used to inhibit fatty acid translocase (fat). results. ffa dose-dependently increased the secretion of the proinflammatory factors (il- , il and mcp- ) as well as matrix-degrading enzymes (mmp- and mmp- ) in rasf (e.g. for lauric acid [ µm] with rasf/il- : . -fold increase; il : . fold increase; mcp- : . fold increase; pro-mmp : . -fold increase; mmp- : . fold increase). saturated and unsaturated ffa had similar effects on rasf. however, saturated ffa induced strong secretion of il- in chondrocytes, while unsaturated ffa only had a weaker effect on this cell type. at µm, both saturated and unsaturated ffa significantly increased il- secretion by osteoblasts to a similar degree as for sf. a high concentration of ffa ( µm) significantly induced il- secretion in huvec and hbdmec, whereas a low concentration of ffa ( µm) did not have a significant effect (p> . ) on human endothelial cells. blocking ffa transport into rasf by using sso almost completely abolished the effect of palmitic acid on il- secretion. conclusion. ffa are not only metabolic substrates but can also directly contribute to articular inflammation and degradation mediated by various effector cells of arthritis. our data also show that ffa transport into the cell is required for ffa-induced effects in sf. background. chronically inflamed tissues in ra are characterized by local hypoxia and enhanced angiogenesis. the hypoxia inducible factor (hif)- and (hif)- serve as key regulators of adaptation to hypoxia thereby promoting both angiogenesis and metabolic adaptation of endothelial cells. to investigate the impact of hif- /hif- on the angiogenic and metabolic transcriptome under hypoxia ( % o ) versus normoxia ( % o ) we performed a knockdown of either hif- α or hif- α in human microvascular endothelial cells (hmec). methods. specific knockdown of either hif- α or hif- α was achieved using shrna-technology. angiogenic and metabolic transcriptome of hmecs was studied by performing an agilent human whole genome microarray under normoxia vs hypoxia. significantly regulated genes were allocated to angiogenic and metabolic processes using panther database. results. in comparison to normoxia the incubation of untransduced hmecs under hypoxia resulted in regulated angiogenesis related genes and regulated cellular metabolism related genes. in both hif- α and hif- α knockdown cells, hypoxia was still capable of inducing a differential gene expression pattern, but with a much less pronounced effect compared to control cells. analysis of angiogenesis related processes (vegf-pathway, hif-activation, egfr-pathway) showed that % of the differentially expressed genes are controlled by both hif- and hif- . another % of the regulated genes are controlled by hif- . the remaining % of regulated genes are under control of hif- . the differentially regulated genes involved in the cellular metabolism (atpsynthesis, glycolysis, tca-cycle) were found to be to % controlled by both hif- and hif- . the remaining % are dependent on the presence of hif- . conclusion. hif- and hif- are both key regulators of the adaptation of endothelial cells towards hypoxia with overlapping functions. however, they do differ in their capacity to regulate cellular energy metabolism and angiogenesis. this leads us to conclude that hif- affects angiogenesis via indirect effects on cellular energy metabolism as indicated by the regulation of metabolic transcriptome to one fifth. hif- does more influence angiogenesis directly via regulating the synthesis of proangiogenic factors (as has been previously shown).these findings provide new insights into the divergent regulation of angiogenesis in inflamed (hypoxic) tissues by hif- and hif- and are, therefore, considered to be of clinical relevance in ra. background. membrane bound glucocorticoid receptors (mgr) play a pivotal role in pathogenesis of chronic inflammatory diseases as indicated by clinical observations. patients with sle show high frequencies of mgr positive monocytes, sometimes even higher than found in patients with active ra. with increasing glucocorticoid dosages expression of mgr on monocytes of sle-patients is downregulated, suggesting a negative feedback loop to control glucocorticoid action. these receptors represent an effective target for diagnosis and monitoring of different inflammatory diseases, but a feasible detection method is still necessary. objectives. we compare two methods of high-sensitive immunofluorescence staining -the well established liposome procedure with the commercialized faser-technique. methods. hek t cells were cultured for h with/without µg/ml brefeldin a in a humidified incubator at °c. human cd positive t cells and cd positive monocytes were isolated via magnetic-activated cell sorting and subsequently cultured in rpmi . monocytes were incubated for h with/without µg/ml lps. for liposome based highsensitivity immunofluorescence staining cells were incubated with the monoclonal (digoxigenin conjugated) anti-gr antibody, followed by incubation with anti-digoxigenin/anti-biotin matrix. subsequently biotinylated cy liposomes were added. faser technique was performed as described by the manufacturer (miltenyi biotec). dead cells were excluded by adding pi before cell acquisition, using a bd facs calibur flow cytometer. the acquired data were analyzed using flowjo . . software. results. the human mgr, which cannot be reliably detected with conventional staining methods, is detectable with the liposome procedure as well as with the commercialized faser-apc technique. furthermore, the faser-apc-procedure is more sensitive ( . % vs . %) and more specific ( . % vs. . %) compared to the liposome technique. additionally, minor changes of mgr expression can also be demonstrated with the faser technique. the faser procedure shows technical advantages: the commercially available faser-apc-kit is performed according to a standarized protocol and is less time consuming compared to the liposome procedure. conclusion. the human mgr is easily detectable with the commercialized faser kits, which represent an alternative due to a consistent quality and a standardized production. this method facilitates the analysis of the role that mgr play in the pathogenesis of chronic inflammatory diseases and perhaps provoke new insights in glucocorticoid therapy. background. in previous studies we detected th-positive, catecholamine-producing cells in inflamed hypoxic synovial tissue. therefore, the aim of our study was to investigate the influence of hypoxia induced catecholamines on inflammatory responses in arthritis. methods. synovial cells of rheumatoid arthritis (ra) and osteoarthritis (oa) patients were isolated and cultivated under normoxia or hypoxia with/without stimulating enzyme cofactors of th and inhibitors of th. expression of th and release of cytokines and catecholamines was analyzed. the effect of th+ cells was tested by adoptive transfer into dba/ mice with collagen type ii-induced arthritis (cia). th+ cells were generated from mesenchymal stem cells by defined dopaminergic factors. results. hypoxia increased th protein expression and catecholamine synthesis and decreased release of tnf in oa/ra synovial cells compared to normoxic conditions. this inhibitory effect on tnf was reversed by th inhibition with alpha-methyl-para-tyrosine (αmpt). incubation with specific th cofactors (tetrahydrobiopterin and fe +) increased hypoxia-induced inhibition of tnf, which was also reversed by αmpt. adoptive transfer of th+ cells reduced cia in mice, and hydroxydopamine, which depletes th+ cells, reversed this effect. conclusion. in summary, this study presents that th-dependent catecholamine synthesis exhibits anti-inflammatory effects in human ra synovial cells in vitro, which can be augmented under hypoxic condi-tions. in addition, the anti-inflammatory effect of th+ cells has been presented the first time in experimental arthritis in mice. background. previously, we demonstrated that long-lived plasma cells contribute to the pathogenesis of antibody-mediated diseases and should therefore be considered as a promising therapeutic target in systemic lupus erythematous (sle). in bone marrow stromal cells expressing the chemokine cxcl organize these niches that provide for the plasma cell survival. cxcl is the ligand of cxcr expressed on plasma cells. in this study we investigated the contribution of cxcl -cxcr interaction to the longevity of plasma cells in the murine model of lupus. methods. plasma cells purified from spleens of nzb/w mice were incubated with the cxcr blocker amd and then adoptively transferred to immunodeficient rag −/− mice. after days we analyzed the number of plasma cells in bone marrow. furthermore, ova immunized nzb/w mice were treated intraperitoneally with amd after boost; anti-ova secreting plasma cells in bone marrow were checked on day and after boost. the effect of plasma cell depletion was investigated in nzb/w mice using amd alone or combined with bortebomib for two weeks. results. two weeks after adoptive transfer the number of plasma cells treated with amd was lowered by % in bone. after secondary immunization with ova the amd treatment resulted in a significant reduction of anti-ova secreting plasma cells in bone marrow by % on day and by % on day . after days the number of mhc class ii negative anti-ova secreting plasma cells significantly decreased by % in bone marrow of treated mice. amd efficiently depleted plasma cells including long-lived. after two weeks treatment, total plasma cell number was decreased by % in spleen and % in bone marrow; long-lived plasma cells were reduced by % in spleen and % in bone marrow. the combination of bortezomib with amd in nzb/w significantly enhanced the depletion of long-lived plasma cells compared to monotherapy. conclusion. cxcr blockade with amd can reduce the homing of plasma cells to the bone marrow and the survival of long-lived plasma cells. the combination of bortezomib with amd shows synergistic effects on plasma cell depletion. the findings highlight the importance of the cxcr -cxcl axis for the plasma cell niche. zeitschrift für rheumatologie suppl · | er. tnfr expression defines synovial tissue infiltrating cd + t cells in patients with rheumatoid arthritis k background. one hallmark of rheumatoid arthritis (ra) is the infiltration of the synovial membrane by cd + t cells. it has previously been shown that infiltrating cd + t cells differ from non-infiltrating ones in their increased expression of tnfr . furthermore, tnfr is expressed on a fraction of circulating cd + t cells from ra patients, but not from healthy controls. aim of the study was the characterization of tnfr + cd + t cells in patients with rheumatoid arthritis. methods. peripheral tnfr + cd + t cells from ra patients were analyzed by flow cytometry. the expression of naive and memory t cell markers (cd ra and cd ro), markers for t cell activation (cd , cd and cd ) and of icam- as well as the frequencies of the positive cells were determined. to identify the t helper cell signature of tnfr + cd + t cells, intracellular staining of the th , th and th master transcription factors t-bet, gata- and ror-γt, respectively, was performed. results. peripheral tnfr + cd + t cells have neither a preferential naive nor a memory phenotype, but showed higher expression of the activation markers cd , cd and cd than tnfr -cd + t cells. tnfr + cd + t cells express higher frequencies of the t-bet and rorγt than tnfr -cd + t cells. there is no difference in gata- expression between tnfr positive and negative cd + t cells. functionally, it has been shown that the cytokine tnf acts as chemokine to attract cd + t cells to the rheumatoid joints. beside this direct effect of tnf, there are known indirect effects of tnf including the upregulation of cell adhesion molecules like icam- . therefore, icam- expression of migrating tnfr + t cells was investigated. the results show, that migrating tnfr + t cells recovered from synovial tissue are more frequently icam- positive than non-migrating ones. conclusion. tnfr + expression characterizes cd + t cells functionally capable of infiltrating the rheumatoid synovium in an icam- dependent manner. the results show, that tnfr expression defines a pathogenic subset of activated cd + t cells with th and/or th signature in patients with rheumatoid arthritis. hypoxia increased the production of interleukin- β in lps-primed human monocytes n background. monocytes are major players in the innate immune system and are recruited to sites of inflammation, where the environmental conditions vary extremely compared to the interstitium under physiological conditions. for example, in rheumatoid arthritis the inflamed joints are severely hypoxic. this decreased oxygen level could be a triggering factor for the activation and survival of monocytes. aim of the study was to analyze the influence of hypoxia on lipopolysaccharide (lps)-induced cytokine production in primary human monocytes methods. immunomagnetically separated monocytes from the blood of healthy donors were cultured for h under hypoxic conditions ( % oxygen). results. cytokine measurement in the supernatant with elisa showed increased concentrations of interleukin- β ( . ng/ml vs. . ng/ ml, p= . ) and interleukin- ( . ng/ml vs. . ng/ml, p= . ), but not of tnf, after hypoxia and lps-stimulation. cleavage of the il- β proform to its active form is dependent on the assembly of the inflammasome and the recruitment of caspase- followed by their activation. when inflammasome assembly was blocked with high extracellular k+-buffer or by inhibiting intracellular ca-signalling with the ca-chelator bapta-am, hypoxia induced il- β release was abrogated. similarly, il- β release after culture under hypoxia was also abolished in monocytic thp -cells, which are genetically made deficient for the inflammasome components nlrp and asc. one activating signal for the inflammasome was shown to be the release of reactive oxygen species (ros), since mitochondrial ros staining with mitosox revealed an increased mitochondrial ros release under hypoxic conditions. accordingly, the induction of mitochondrial ros through decoupling of the electron transport chain with rotenone also triggered an increase of il- β release under normoxic conditions. analysis of blood monocyts from ra patients showed no difference in lps and hypoxia induced il- β release compared to healthy controls ( . ng/ml vs. . ng/ml). conclusion. this study shows, that hypoxia leads to the activation of the inflammasome, the recruitment of caspase- and the subsequent cleavage and release of interleukin- β in human primary monocytes. intracellular calcium mobilization and mitochondrial ros production were shown to be essential mechanisms triggering inflammasome assembly. background. cell-derived membrane-coated microparticles have been identified as important mediators in intercellular communication. during the process of apoptosis, dying cells start to dynamically release microparticles. polymorphonuclear neutrophils are the most abundant type of leukocytes, representing - % of all white blood cells. due to their very short lifespan, they are the source of massive amounts of apoptotic cell-derived microparticles (admps). while the interaction between neutrophils and t lymphocytes has been focus of extensive research, the influence of neutrophil-derived microparticles on t cells has not been analysed yet. in this study, we investigated the effect of membrane-coated microparticles released by apoptotic neutrophils on different t helper cell subsets. methods. different cd + t cell subtypes were sorted according to the expression of cd , cd , cd ra and cd ro and co-cultured with admps or apoptotic cell remnants purified from uv-irradiated neutrophils isolated from the peripheral blood of healthy donors. t cells were stimulated by okt and anti-cd antibodies and cell proliferation was measured by h-thymidine incorporation or pkh -staining. secretion of cytokines was quantified by elisa. results. admps released by neutrophils selectively suppressed the proliferation of cd +cd -cd + tc in a dose-dependant manner and prevented the upregulation of cd on the t cell surface, while maintaining the expression of cd . the secretion of tumor necrosis factoralpha (tnfα) by t cells stimulated in the presence of admps was significantly reduced. interestingly, in contrast to admps, the apoptotic cell remnants of neutrophils exerted no effect on t cells. the suppressive effect of admps could be completely abrogated by the addition of interleukin(il)- or il- or by the presence of cd +cd +cd + t cells. conclusion. neutrophil admps suppress the proliferation of cd +cd -cd + t cells under conditions of limiting il- and il- concentrations. this could represent an important mechanism to prevent inappropriate activation and expansion of resting t helper cells in the absence of sufficient stimulation and cytokine production. t. alexander background. recent reports have shown dysregulated micrornas in murine lupus models, among them increased expression of mirna- , which has been demonstrated to target the transcription factor foxo in activated cd + t cells. the loss of foxo activity in t cells is associated with spontaneous t cell activation, clonal expansion and autoantibody production, all of which are present in systemic lupus erythematosus (sle). methods. expression levels of microrna- and foxo were analyzed with rt-pcr in magnetic purified peripheral blood cd + t cells from patients with sle and healthy controls (hc). multicolor flow cytometry was performed to analyze cd + t cell expression for ccr , cd ra, ki- , foxp , the interleukin- receptor-α and phosphorylated stat- a (pstat ). analysis of serum il- levels was performed with elisa in sle patients and hc (r&d systems). results. mirna- was significantly upregulated in cd + t cells from sle patients compared to hc (median expression . × e- vs. . × e- , p= . ) while foxo mrna levels were decreased, yet without reaching statistical significance. analysis of ki- expression revealed an increased percentage of proliferating cd + t cells in sle ( . % vs . %, p= . ). overall, cd + t cellular proliferation in sle was associated with increased frequencies of cd ra-ccr -effector memory t cells and enhanced basal pstat levels (median mfi . vs . , p= . ), suggesting a recent stimulation with common gamma chain(γc)-signaling cytokines. in this regard, tcons from sle samples displayed decreased expression levels for the foxo target gene cd (mfi vs. , p= . ) and serum il- levels were significantly higher in sle compared to hc ( . pg/ml vs. . pg/ml, p= . ). conclusion. mir- expression has been shown to be dependent on stat activation and to promote clonal expansion of activated cd + t cells. our data suggest that enhanced il r/stat signaling mediates induction of mir expression, which in turn promotes the proliferation of tcons in sle. the relative contribution of il r/mir- /foxo axis on the enhanced proliferative capacity of sle tcons remains elusive and merit further investigation. collectively, our data provide new insights in the pathophysiology of t cell hyperactivity in sle and identifies mir- as a candidate target for future therapeutic approaches. background. cell activation and apoptotic cell death leads to the formation of membrane-coated vesicles (mcvs). mcvs have previously been identified as mediators of cell-to-cell communication and carriers of microrna. an impaired clearance of apoptotic debris caused by an increased rate of apoptosis or a defect in phagocytic-cell clearance has been observed in sle patients. in this study, we analyzed the microrna content of activated and apoptotic lymphocytes and their corresponding mcvs from both normal healthy donors (nhds) and sle patients. further we investigated the immunomodulatory effect of mcv uptake by monocytes. methods. microrna content of activated and apoptotic lymphocytes and corresponding mcvs of nhds and sle patients were compared in an agilent microrna array and validated by qpcr. apoptosis was induced by uvb-irradiation. mir- expression in monocytes after uv-mcvs engulfment was determined by qpcr. expression of mir- target protein tab- was analyzed by western blot. results. mir- * levels were decreased after apoptosis induction in lymphocytes and apoptotic mcvs compared to their viable correlates. mir- , mir- a and mir- b were decreased in apoptotic lymphocytes compared to viable ones but increased or not significantly changed in apoptotic mcvs compared to viable mcvs, indicating a directional transport of microrna into mcvs. mir- a was expressed at higher levels in viable sle lymphocytes and mcvs compared to nhds. mir- b expression was decreased in uv-lymphocytes and uv-mcvs of sle patients. functional assays confirmed higher mir- levels and consecutively decreased target protein levels in monocytes after engulfment of uv-mcvs. conclusion. within this study we could show an unequal distribution of distinct microrna into mcvs released by activated or apoptotic lymphocytes. further the microrna content was regulated in whole apoptotic cells after uvb-irradiation. this suggests a directional transport rather than a random distribution. thus, cells regulate their microrna as well as the microrna content within released mcvs. we could show a microrna and protein expression change in phagocytes after mcv engulfment. hence, our results suggest mcvs could serve as a transport vehicle for microrna to mediate cell-to-cell communication and influence intracellular processes in phagocytes. disturbances of this system might contribute to the pathogenesis of sle. results. we found in the spleens of nzb mice -times higher numbers of long-lived plasma cells and megakaryocytes compared to wildtype, in nzw mice equal numbers and in nzb/w mice numbers between those for nzb and nzw or wildtype. moreover, in the spleen a fraction of plasma cells clustered around megakaryocytes. we also detected a missense mutation in the c-mpl gene of nzb mice leading to an amino acid replacement within the essential tpo-binding site. upon tpo stimulation of splenocyte and bone marrow cultures nzb cultures responded significantly stronger resulting in the double amount of megakaryocytes compared to nzw cultures. conclusion. in summary, our data indicate that augmented megakaryopoiesis enables the accumulation of a greater number of autoreactive plasma cells in lupus prone nzb/w mice. thus, we assume that enhanced megakaryopoiesis and higher megakaryocyte numbers are contributing to the development and/or pathogenesis of sle. background. baff is a cytokine important for the stimulation and survival of autoreactive b cells and therefore might play a role in several autoimmune diseases, e.g. autoimmune arthritis. in psoriasis arthritis, baff correlates with disease activity and testosterone, but only in male patients, suggesting a role for sex hormones in the regulation of baff. therefore, we wanted to determine if baff production in rheumatoid arthritis and osteoarthritis fibroblasts was regulated by neuroendocrine mediators. methods. fibroblasts were isolated from synovial tissue of ra (n= ) and oa (n= ) patients and cultured in vitro under different conditions. baff was determined by elisa. results. isolated fibroblasts were cultured in the presence or absence of interferon-gamma (ifn-γ), il- , lipopolysaccharide (lps), tumor necrosis factor (tnf), cpg, poly i:c, and cortisol in different combinations for and hours to determine the optimal stimulation strategy for induction of baff production (measured by elisa in supernatants) in fibroblasts. ifn-γ best induced baff in ra and oa fibroblasts. ifn-γ-induced baff production in fibroblasts was decreased by dihydrotestosterone in a concentration dependent manner. the effect was specifically inhibited by nilutamid, a testosterone receptor antagonist. furthermore, stimulation of beta-adrenoceptor increased, whereas stimulation of alpha-adrenoceptors did not change inf-γ-induced baff in synovial fibroblasts. in general the effects were more pronounced in ra as compared to oa fibroblasts. conclusion. taken together, inf-γ-induced baff production in synovial fibroblasts is decreased by testosterone and increased by betaadrenergic stimuli. therefore, neuroendocrine regulation of inflammation in the inflamed joint might be in part mediated by regulating baff production in synovial fibroblasts. a. grützkau , c. kyogoku , b. smiljanovic , j. grün , r. biesen , t. alexander , f. hiepe , a. radbruch , t. häupl deutsches rheuma-forschungszentrum (drfz), berlin, charité -universitätsmedizin berlin, medizinische klinik mit schwerpunkt rheumatologie und klinische immunologie, berlin background. gene expression profiling experiments using peripheral blood mononuclear cells (pbmcs) revealed a crucial role of type i interferon (ifn) in the pathogenesis of systemic lupus erythematosus (sle). however, it is almost unknown how particular leukocyte subsets contribute to the overall type i ifn signature described for pbmcs. furthermore, a detailed analysis of how ifn signatures differ in autoimmune disease from that observed after viral infection is missing so far. therefore, we compared expression levels of ifn signature genes in peripheral cd + t helper cells and monocyte (mo) subsets isolated from patients with sle, healthy donors (nd) and nd vaccinated against yellow fever by global gene expression profiling. methods. peripheral blood from patients with sle and nd were recruited. same nd were examined before and after immunization by yellow fever vaccine. after sorting cells, isolated rna were applied to affymetrix human genome u plus . array. data analysis was done using bioretis database, genesis software and ingenuity pathway analysis (ipa). results. comparing gene expression profiles of yellow fever immunized individuals and active sle patients it was possible to identify a "common" and an "autoimmune-specific" ifn signature. although major ifn signature genes were commonly expressed in cd + t cells and mo of patients with sle and immunized nd, expression magnitudes of them were higher in patients with sle compared to immunized nd. in sle, in addition to the typical "viral-induced" ifn signature, genes that are involved in apoptosis signaling, antiviral pkr signaling, fcγ receptor-mediated phagocytosis and il- -/il- -/il- -mediated jak/ stat signaling pathways were identified by ipa. conclusion. this study demonstrated that ifn signature in autoimmunity and that in viral infection are quite different in the number of ifn-related genes activated and their expression magnitudes. autoimmunity is characterized by a much stronger expression of ifn signature genes and is obviously modulated by a separate set of co-regulated genes defining the "autoimmune-specific" ifn signature. in summary, "common" and "autoimmune-specific" ifn signature genes are of potential interest as clinical biomarkers in sle diagnostics to differentiate between a disease flare and a viral infection. of peripheral blood lymphocytes (pbl). there is currently no data available about nk cells in gpa. the aim of this study was to evaluate the presence of nk cells in gpa granulomas and their proportions in pbl as a basis for a potential role in gpa. methods. paraffin sections of granulomas of gpa, sarcoidosis and tuberculosis patients were stained with a cd monoclonal antibody. nk cell (cd -cd +) proportions of pbl in gpa patients and healthy controls (hc) were analysed by facs analysis. clinical data was extracted from medical records. results. contrary to granulomas from tuberculosis and sarcoidosis which showed a considerable infiltration by cd positive cells, there was not a single cd positive cell in granulomas from gpa patients. therefore, the tissue destructive character of gpa granulomas is associated with a lack of nk cells. gpa patients with inactive disease [birmingham vasculitis activity score (bvas) = , n= ] possessed a significantly higher nk cell proportion in pbl (mean ± standard deviation: . ± . %) than both gpa patients with active disease (bvas> , n= , mean= . ± . %) (p= . ) and hc (n= , mean= . - . %, p= . ). thus, clinical remission is accompanied by an increase in the nk cell proportion in pbl. interestingly, patients with inactive disease that had "normal" nk cell proportions of less than % of pbl (n= ) showed a more severe disease course than those with more than % of pbl. conclusion. nk cells might, therefore, be helpful to limit granulomatous inflammation. whether nk cell proportion in pbl might be a useful biomarker in gpa, e.g. as predictor for relapses, will be further evaluated in our future studies. v. gerl background. plasmacytoid dendritic cells (pdcs) are considered a crucial element in sle pathogenesis due to their potency to produce high levels of ifn-α. this innate immunological function of pdcs is lost by terminal differentiation into a professional antigen-presenting cell (pdc-derived dc), thereby upregulating costimulatory molecules and downregulating innate characteristics, e.g. bdca- and ifn-α expression. pdc-derived dcs have not been described in vivo yet, probably due to the fact that they lose their specific markers during differentiation. furthermore, pdcs can differentiate into myeloid dcs by various stimuli. in sle, where low expression of bdca- is commonly seen, this differentiation could be relevant and point to such a lineage switch as well as to an activated state of pdcs. aim. to characterize pdc subsets of differentiation/activation in human peripheral blood and to study their impact on autoimmune inflammation in sle. methods. -color-flowcytometric analyses were performed on whole blood of healthy donors and sle patients. pdcs were identified by cd -/cd -/cd -/cd high//bdca- +/hla-dr+ expression and characterized for cd c, bdca- and the macrophage-associated siglec- , expressed on monocytes of active sle patients in an ifn-α dependent manner. cd and cd expression were measured in parallel. results. we found a small subpopulation of siglec- expressing pdcs in human peripheral blood. compared to siglec- negative pdcs, siglec- positive pdcs express significantly lower bdca- and cd , higher hla- dr background. agonistic autoantibodies against the angiotensin ii receptor type (at r) and the endothelin receptor type a (etar) have been identified in patients suffering from systemic sclerosis (ssc). here we examined the expression of at r and etar in human immune cells and pathological effects mediated through these receptors by corresponding autoantibodies (aabs). methods. at r and etar protein expression on peripheral blood mononuclear cells (pbmcs) from healthy individuals and ssc patients was analyzed using flow cytometry, mrna expression was examined by real-time pcr in pbmcs from healthy donors. in addition, pbmcs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin g (igg) fractions from ssc patients positive for at rand etar-aabs, and with igg from healthy donors serving as control. alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, elisa, and chemotaxis assays, respectively. results were correlated with characteristics/clinical findings of the igg donors. results. both at r and etar were expressed on human peripheral lymphocytes and monocytes. protein expression of both receptors was decreased in ssc patients when compared to healthy donors and correlated negatively with disease duration. in addition, igg fractions of ssc patients induced t cell migration in an anti-at r and anti-etar aab level-dependent manner. moreover, igg of ssc patients was capable of stimulating pbmcs to produce more il- and ccl than igg of healthy donors. all effects could be significantly abrogated by the application of selective at r and etar antagonists. statistical analysis revealed a negative correlation between ssc igg-induced il- concentrations and disease duration, between ssc igg-induced ccl concentrations and time since onset of lung fibrosis as well as an association of ccl concentrations with vascular complications of the corresponding ssc igg donors. conclusion. we demonstrated the expression of both, at r and etar, on human peripheral t cells, b cells and monocytes and found signs for a chronic receptor activation in ssc patients. the inflammatory and profibrotic effects upon aab stimulation in vitro, and their associations with clinical findings suggest a role for autoantibody-mediated activation of immune cells mediated through the at r and etar in the pathogenesis or even the onset of the disease. the bioenergetic role of hif- and hif- during angiogenesis of human microvascular endothelial cells background. hypoxia and angiogenesis are features of inflamed and injured tissues. the transcription factors hypoxia inducible factor (hif)- and (hif)- regulate the cellular and metabolic responses to reduced oxygen tensions thereby promoting angiogenesis with implications on the pathogenesis of ra. we investigated the effects of a knockdown of either hif- α or hif- α in human microvascular endothelial cells (hmec) on angiogenesis and bioenergetics under hypoxia ( % o ) versus normoxia ( % o ). methods. specific knockdown of either hif- α or hif- α was conducted by shrna-technology. to assess angiogenesis of hmecs both tubuli and node formation under hypoxia versus normoxia were investigated. expression of hypoxia driven genes involved in the metabolic response to hypoxia (gapdh/pgk/glut /ldha) was quantified by realtime rt-pcr. the bioenergetic status of the cells was quantified via atp/adp measurements. results. knockdown of hif- α/hif- α resulted in a loss of hypoxia induced angiogenesis. focusing on bioenergetic aspects, we found hypoxia to significantly induce pgk, ldha and gapdh in control cells. knockdown of hif- α and hif- α, respectively, did not affect the hypoxic induction of pgk and ldha. in hif- α and hif- α knockdown-cells, hypoxia was still capable of inducing gapdh, with a less pronounced effect in hif- α knockdown-cells. hypoxia did not significantly up-regulate glut , neither in control nor in hif- α or hif- α knockdown-cells. the knockdown of hif- α resulted in significantly decreased expression of glut under hypoxia. we also found the atp/ adp ratio to be similar in control, hif- α and hif- α knockdown-cells under normoxia. under hypoxic conditions hif- α knockdown-cells showed significantly reduced atp/adp ratios -indicating that less atp is available -compared to hif- α knockdown-cells. conclusion. hif- α and hif- α are both key regulators of angiogenesis. however, they do differ in their potency to regulate cellular energy metabolism. this leads us to conclude that hif- α does directly influence angiogenesis via regulating the synthesis of proangiogenic factors (as previously shown), whereas hif- α affects angiogenesis via effects on cellular energy metabolism as indicated by the reduced expression of gapdh and the diminished atp/adp ratio. these findings provide new insights into regulation of angiogenesis in inflamed (hypoxic) tissues and are, therefore, considered to be of clinical relevance in ra. low baseline complement levels, autoantibody persistence and delayed thymic reactivation are risk factors for development of relapses after hematopoietic stem cell transplantation for refractory sle background. our previous research has provided the evidence that an autoreactive immune system can be "reset" into a healthy, tolerant state by immunoablative treatment to eradicate pathogenic effector cells, followed by transplantation of hematopoietic progenitor cells (hsct). nevertheless, disease flares may occur in a subset of these patients posttransplantation. here, we longitudinally analyzed the immune reconstitution of these patients to identify markers for favorable long-term responses. methods. since , patients with refractory sle received a cd +selected autologous stem cell transplantation after immunoablation with antithymocyte-globulin (atg) and cyclophosphamide as part of a monocentric phase i/ii clinical trial. autoantibody titers were evaluated with elisa, peripheral t-and b lymphocyte subsets immunophenotyped using multicolor flow cytometry. results. clinical remission (sledai ≤ ) could be achieved in all patients, despite immunosuppressive drug withdrawal, associated with disappearance of anti-dsdna antibodies and marked reduction of protective antibodies in serum. unfortunately, two patients died due to transplant-related infections. from the remaining eight patients, five patients are in long-term clinical remission for up to years after hsct, while three patients suffered a relapse of sle at , and months post-transplantation, respectively. patients with early relapses (≤ months) had decreased baseline complement levels, showed persistence of antinuclear antibodies (ana), less significant reduction in protective antibody levels and had slower repopulation of cd + cd ra+ thymic-derived cd + t cells after hsct (< /µl at months) when compared to long-term responders. in addition, flow cytometric analyses revealed an expansion of circulating plasmablasts and increased coexpression of siglec- on monocytes (as surrogate marker for type-i interferon signature), preceding the clinical flares by ~ months. conclusion. low baseline complement levels, persistence of ana and delayed thymic reactivity post-transplantation could be identified as risk factors for development of lupus flares after hsct. since atg-mediated cell lysis is complement-dependent, we conclude that low serum complement is directly associated with incomplete depletion of immunologic memory cells in these patients, which provides a rationale for complement substitution before immunoablation. moreover, lupus flares may be predicted individually by flow cytometry with plasmablast expansion and recurrence of type-i interferon signature. background. systemic lupus erythematosus (sle) is a chronic autoimmune disease characterized by the generation of pathogenic antibodies directed against a variety of autoantigens. we have previously shown that long-lived autoreactive plasma cells can contribute to chronicity and refractoriness of sle. our study is aimed to develop new methods for depletion of long-lived plasma cells in nzb/w mice, a model of sle. methods. we studied different treatment protocols on plasma cell survival: irradiation-based and more selective depletive treatments. - week-old nzb/w f mice were exposed to three different irradiation doses ( , , and gy in two splitted doses with a -h interval). the following protocols were also investigated: ) two bortezomib (bz) injections ( , mg/kg, i.v.) combined with anti-mouse cd ( mg/kg, i.v.), ) three bortezomib injections combined with anti-mouse cd , ) three bortezomib injections combined with anti-lfa- and anti-vla- antibodies (affecting directly the plasma cell niche; µg, i.p.) in a -d interval, plus anti-mouse cd and anti-b ( µg, i.v.). the plasma cells were analyzed in spleen and bone marrow by facs and elispot. results. the frequency of remaining plasma cells in bone marrow after , and gy irradiation were , and , % respectively, and in spleen were almost , and , %. short-term treatments with agents that affect plasma cells (bortezomib, anti-lfa plus anti-vla ) effectively deplete plasma cells including long-lived plasma cells in spleen and bone marrow of nzb/w mice. because of the b cell hyperactivity in nzb/w mice, we observe a rapid regeneration of autoreactive plasma cells in spleen and bone marrow. therefore, plasma cell depletion protocols were combined with b cell depletion. especially, the combination of plasma cell targeting with bortezomib, anti-lfa and anti-vla with b cell targeting (anti-cd plus anti-b ) interrupted the repopulation of autoreactive plasma cells in spleen and bone marrow. conclusion. very high doses of irradiation result in effective depletion of long-lived plasma cells but lower doses not. depletion of long-lived plasma cells can be achieved by the proteasome inhibitor bortezomib and by targeting both adhesion molecules lfa and vla . the combination with b cell depletion is needed to prevent regeneration of autoreactive plasma cells. varicella-zoster-virus(vzv)-specific lymphocytes and igg antibody avidity in patients with juvenile idiopathic arthritis or rheumatoid arthritis background. varicella zoster virus (vzv) is a herpes virus that establishes a life-long latent infection with risk of reactivation (shingles) particularly in immunosuppressed patients with autoimmune disorders. patients with rheumatoid (ra) or juvenile idiopathic arthritis (jia) have a high risk for disseminating varicella zoster virus (vzv) infection or herpes zoster. this study was aimed to investigate the humoral and cellular immune response to vzv including assessment of igg-anti-vzv avidity and vzv-specific reactivity of lymphocytes in ra (n= ) or jia patients (n= ) on different treatments, including biologic agents, such as anti-tumor-necrosis-factor(tnf)-alpha or anti-interleukin- (il- ) receptor inhibition (tocilizumab), compared to healthy adults (ha) and children (hc). methods. igg-anti-vzv concentrations and avidities were quantified by an adapted elisa. vzv-specific interferon-gamma-producing lymphocytes (spot forming units, sfu/ , , cells) were analyzed by elispot. results. no significant differences in the vzv-igg concentrations or avidities were found between the groups. however, lower igg-anti-vzv concentrations were found in tocilizumab-treated ra compared to ha and ra without biologic agents. ra showed lower median sfu ( / , , cells) than ha ( / , , cells), with lowest sfu in adalimumab-treated ra ( / , , cells). sfu were not altered in tocilizumab-treated ra and after incubation with anti-il- in vitro. no differences regarding igg-anti-vzv concentrations, rai and cellular reactivity were found between jia and hc. conclusion. our study demonstrated that ra and jia patients are still able to maintain humoral and cellular immune responses to vzv despite immunosuppressive therapy or biologic agents. in ra, the role of lower cellular reactivity for risk of herpes zoster has to be considered for recommendations on vaccination. cmv-specific cd + t cells from ra patients contribute to autoimmune disease zeitschrift für rheumatologie suppl · | . increased frequency of lir- (also called cd j or ilt ) on cd + t cells has been associated with autoimmune disease. furthermore, it has been shown that latent cytomegalovirus (cmv) infection contributes to the expansion of cd − t cells. hence we were interested in the influence of cmv infection on the lir expression on t cells in ra patients. methods. we were interested in the role of lir + t cells in ra patients, which potentially contribute to the autoreactive t cell pool, especially in cmv+ patients. therefore, we investigated the expression and function of lir- on cd + t cells in peripheral blood mononuclear cells (pbmc) from patients with rheumatoid arthritis by flow cytometry and cytotoxicity assay. results. flow cytometry analysis revealed higher frequencies of lir- + cd + t cells in cmv seropositive ra (n= , mean%: . ) compared to cmv+ hd (n= , mean%: . , p= . ). using hla-a* /cmvpp dextramers we analyzed cmv-specific cd + t cells. patients with ra had higher frequencies of cvm specific cd + t cells (n= ; mean%: . ) compared to healthy individuals (n= ; mean%: . , p= . ). phenotypically, cmv-specific cd + t cells are mainly cd negative and express lir- . analysis of the cytolytic potential by cd a expression revealed higher numbers of cd a+cd + t cells in ra patients (n= , mean%: , ) compared to healthy donors (n= , mean%: , ). importantly, we found a significant correlation (p= . ) of high numbers of cd +lir- + t cells with high disease activity score (das ) in ra patients without immunosuppressive treatment (n= , r= , ) . tab. . conclusion. this is the first demonstration of significantly increased frequencies of lir- +cd + t cells and of cmv-specific cd + t cells in patients with rheumatoid arthritis. these cells are characterized by a terminally differentiated phenotype. the higher cytolytic potential of cmv-specific t cells likely can be attributed to their function in containing latent cmv infection and to prevent cmv disease, but might potentially contribute to disease severity in ra patients. background. systemic lupus erythematosus (sle) is an autoimmune disease characterized by an acquired il- deficiency, which leads to a homeostatic imbalance between regulatory t cells (treg) and effector t cells (tcon; humrich et al. ). we recently demonstrated that treg homeostasis in lymphoid organs of diseased (nzbxnzw) f mice can be restored by treatment with recombinant il- (il- ) resulting in an amelioration of kidney disease. the aim of this study was to investigate the impact of il- therapy on intrarenal foxp + treg and kidney infiltrating conventional cd + t cells (tcon) in the (nzbxnzw) f mouse model of lupus nephritis. methods. (nzbxnzw) f mice with active nephritis were treated with recombinant il- either for a short period of days or for a longer period of days in total. absolute numbers, phenotype and proliferation of kidney infiltrating cd + t cell subsets were determined by flow cytometry at different time points. results. short-term il- treatment resulted in an enhanced proliferation and increased numbers and frequencies of intrarenal cd +foxp + treg compared to untreated control mice. on the other hand, long term il- treatment did not result in a persistent expansion of the intrarenal foxp + treg population. however, total numbers of kidney infiltrating cd + tcon with a memory/effector phenotpye were diminished and cd + tcon showed markedly reduced signs of cellular activation. conclusion. our data indicate that short term il- treatment is able to expand the size of the intrarenal treg pool. in contrast, long term il- treatment decreases the numbers of kidney infiltrating memory/ effector t cells and reduces cellular hyperactivity suggesting that treg suppress the activation and expansion of infiltrating tcon. these results may in part explain the amelioration of disease induced by treatment with il- and underline the important role of intrarenal treg for the suppression of kidney disease in lupus mice. these results also provide additional important rationales for an il- based immunotherapy of human disease. from transcriptome to protein biomarkers in ra: joint compartment and monocytes outperform serum and whole blood background. a main challenge in disease-management of ra is to establish objective criteria relevant for diagnosis and therapeutic stratification of patients. this study focused on global approaches in dissecting inflammation in ra including transcriptome analyses of synovial tissue and blood monocytes and proteome analyses of synovial fluid and serum. methods. gene-expression profiles from synovial tissues and blood monocytes of ra and osteoarthritis (oa) patients were generated by affymtetrix arrays. elisa and multiplex immunoassays were used for validation of candidate markers at the protein level in synovial fluid (sf) from ra and oa patients and in serum from the same group of patients and healthy donors. results. transcriptome analyses of synovial tissues from ra and oa revealed more than differentially expressed genes. to avoid difficulties in sampling synovial tissue and to avoid fluctuation in cellular composition of various cell types in blood, the transcriptome analyses from peripheral blood was focused on a specific cell population. monocytes were selected as the favourable cell type involved in the production of cytokines, which are often considered as therapeutic targets in ra. comparisons between ra and oa monocytes disclosed differential expression of more than genes. in total, genes that were up-regulated in synovial tissues and/or monocytes were used for validation at the protein level as potential biomarkers for ra. among these biomarkers, chemokines (cxcl , ccl , ip ), adhesion molecules (vcam , icam , e-and p-selectins), proteolytic enzymes (mmp , a at), and the shedding form of cell surface molecules (cd , cd ) background. idiopathic membranous nephropathy (imn) is a common cause of nephrotic syndrome in adults and has recently been identified as an autoimmune-mediated disease [ ] . autoantibodies directed towards the m-type phospholipase a receptor (pla r) are fairly specific for idiopathic mn and only found to a small percentage in sera from patients with secondary mn [ ] . the outcome of patients with imn is quite diverse: about one third of patients have spontaneous remission, another third progress to require dialysis and the last third continue to have proteinuria without progression to renal failure. we performed serological profiles of imn patients in order to compare antibody profiles to antibody frequencies found in the normal healthy population and to hopefully identify factors that help to predict disease course in imn. methods. serum samples of patients with imn were assayed for a variety of autoantibodies by elisa, addressable laser bead immunoassay (albia) and to dsdna by crithidia luciliae assay. results. the prevalence of autoantibodies found in our imn cohort is summarized in tab. . anti-pla r antibodies were found in about % of imn patients whereas the frequency of other antibodies was mostly below %. the one exception is anti-dfs that was found in . % of imn patients. conclusion. the prevalence of anti-pla r positive patients in our imn cohort matches what has been previously described [ ] . the frequency of the other antibodies that we determined is comparable to what has been reported in the normal healthy population. it is important to note that anti-dfs antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases (sard; [ ] ) whereas anti-ro reactivity is often regarded as a marker for sard. the absence of anti-ro and the high prevalence of anti-dfs confirms that imn is a rather organ specific autoimmune disease. background. activity and the quality of movement belong to the most fundamental diagnostic parameters for neurobehavioural analysis but in the past it has been difficult to include this information into pre-clinical murine disease models. here we tested the applicability of a radiofrequency identification (rfid) based automated tracking system in the experimental murine model of ovalbumin induced arthritis. methods. c bl/ mice were immunized twice with cationized ovalbumin in freund's complete adjuvant and onset of arthritis was induced two weeks after the last immunization by direct injection of cationized ovalbumin into the knee joint of the right hind leg. severity of arthritis was assessed through measurement of joint swelling and evaluation of histological changes. additionally mice were implanted with a rfid transponder and throughout the experiment their activity level was monitored by an id-grid sensor plate placed underneath the homecage. results. the joint inflammation in the ovalbumin induced arthritis model showed a quantifiable impact on the activity levels of the mice. our experiments could also show that movement activity correlates with disease severity as evaluated by clinical and immunological parameters. in the past employing behavioral methods was often limiting by group size, observation time and reproducibility and the stress of handling and new surroundings made results difficult to interpret. in our experiments a rfid-based automated tracking system allowed us to monitor individual activity long-term without removal of the mice from their homecage environment. this allowed for the correlation of clinical parameters to behavioral factors and adds another level of analysis to an established murine model. progranulin antibodies in a wide spectrum of autoimmune diseases results. autoantibodies against progranulin, a secreted and direct inhibitor of tnf-α receptors & were frequently identified in primary vasculitides. in detail, progranulin-antibodies were found during the course of disease in giant cell arteritis/polymyalgia rheumatica ( / ), takayasu's arteritis ( / ), classical panarteritis nodosa ( / ), behcet's disease ( / ), in granulomatosis with polyangiitis ( / ), churg-strauss syndrome ( / ) and in microscopic polyangiitis ( / ). in extended screenings progranulin-antibodies were also frequently detected in autoimmune connective tissue disorders, in rheumatoid and psoriatic arthritis and in inflammatory bowel disorders. in contrast progranulin-antibodies were only detected rarely in healthy controls ( / ), patients with obesity ( / ), residents of nursing homes ( / ), not in patients with cutaneously limited psoriasis ( / ), not in patients undergone sepsis ( / ), and not in patients with melanoma ( / ). a significant association of progranulin-antibodies with active disease states in granulomatosis with polyangiitis suggested a pro-inflammatory activity of progranulin-antibodies. this was supported by an observed neutralizing effect of progranulin-antibodies on the levels of circulating progranulin in elisa and western-blot. moreover, functional assays revealed, that progranulin-antibody containing sera render wehi-s cells far more sensitive to effects of administrated tnf-α, providing evidence for the suspected pro-inflammatory effect of progranulin-antibodies. conclusion. progranulin-antibodies occur in a widespread spectrum of autoimmune diseases and have a pro-inflammatory effect by neutralizing the physiologic tnf-blocker progranulin. background. flow cytometry (fcm) is widely used in research for molecular characterization at single cell level. conventional analysis is a semiautomated process of user defined gating and investigation in -d projections. for multiple parameter analysis with hundreds of marker combinations, this manual process is most limiting and impedes high throughput analysis. therefore, we developed a new algorithm for automated and standardized analysis of multiplex fcm data. methods. automation included asinh-transformation of data, cell grouping, population detection and population feature extraction. for grouping of cells, an unbiased unsupervised model based t-mixture approach with expectation maximization (em)-iteration was applied. populations were identified by meta-clustering of several experiments according to position and extension of cell-clusters in multi-dimensional space and by including a general procrustes analysis (gpa) step. for validation, peripheral leukocytes from healthy donors and patients with rheumatoid arthritis (ra) were prepared by hypoosmotic erythrocyte lysis and stained with different sets of lineage-specific antibodies. in parallel, different leukocyte samples were depleted of one of these populations by magnetic beads. qualitative and quantitative characteristics of major populations were compared with conventional manual analysis. results. whole blood leukocytes stained simultaneously with up to markers were correctly distinguished in all major populations including granulocytes, t cells and their subpopulations, monocytes, b cells, and nk-cells. the result was comparable to the "gold standard" of manual evaluation by an expert. the new technology is able to detect subclusters and to characterize so far neglected smaller populations based on the new parameters generated. automated clustering did not require fluorescence compensation of data. cell-grouping is applicable even for large fcm datasets of at least parameters and more than million events. comparing the cell-clusters between ra and healthy controls, differences were detectable in several cell (sub-)populations, stable enough to perform correct classification into controls and disease. conclusion. this new approach reveals promising results for automated and time-saving analysis of large datasets from multiplex fcm. the algorithm avoids operator-induced bias, is able to detect unexpected sub-clusters and to characterize so far neglected populations. this may reveal not only new markers for disease activity but also for therapeutic stratification. background. lasp- localizes at focal adhesions, along stress fibres and leading edges of migrating cells regulating metastatic dissemination of different tumors. since rasf have been implicated in the spreading of disease by leaving cartilage destruction sites, migrating via the bloodstream and re-initiating the destructive process at distant articular cartilage surfaces, the underlying mechanisms are of special interest. therefore, we investigated the role of lasp- in sf migration and its effects on ra. methods. to identify lasp- expression and its sub-cellular distribution in human sf as well as in hind paws of wt and htnftg mice, we performed western blots and immunofluorescence. the migration of sfs derived from wt, lasp- -/-, htnftg and lasp -/-/htnftg mice was studied in a modified scratch assay as well as in live cell imaging studies. furthermore, a transmigration assay using sf from all four genotypes and murine endothelioma cells (bend. ) as an endothelial barrier was carried out. sf transmigration under inflammatory conditions was also evaluated by tnf-alpha stimulation of the endothelial cells in vitro. results. lasp- expression was up regulated in rasf and in sf from htnftg mice compared to healthy controls and was found at structures of cell adhesion and invasion. the scratch assay as well as the live cell imaging studies showed a significantly reduced migration of lasp- ( ng/ml) was applied. in parallel, il- stimulation significantly amplified the expression of anti-apoptotic bcl- in sle treg but not tcon. conclusion. in analogy to our previous findings in lupus-prone mice, treg from sle patients show the classical hallmarks of il- deficiency with loss of cd expression and a homeostatic imbalance between treg and tcon. these findings could be associated with a reduced il- expression by cd + t cells in sle patients. on the other hand, low-dose il- stimulation in vitro could restore these defects, underlying the potential of il- as a novel therapeutic option in sle. the glucocorticoid-dependent modulation of immune-mediated inflammatory arthritis by osteoblasts in mice is t cell independent background. at present, the role of adiponectin in rheumatoid arthritis is still controversial. there is some evidence indicating anti-inflammatory effects, for example adiponectin reduces the tnf release by macrophages. in contrast to its anti-inflammatory role, adiponectin also exerts pro-inflammatory effects locally in joints, inducing for example pro-inflammatory factors and matrix-degrading enzymes in ra synovial fibroblasts. moreover, our immunohistochemical analysis of ra bone tissue showed a co-localization of adiponectin with key cells of bone remodelling (osteoblasts, osteoclasts). however, the role of adiponectin in bone remodelling of ra still needs to be defined. in this study, we therefore focussed on adiponectin and its immunomodulatory properties on ra osteoblasts and osteoclasts. methods. human osteoblasts and osteoclasts were isolated from bone tissue and blood samples of ra patients. immunocytochemistry and rt-pcr were used to analyze the expression of adiponectin and its receptors in osteoblasts and osteoclasts. osteoblasts and osteoclasts were treated with adiponectin ( µg/ml). adiponectin-mediated effects on the cytokine expression in osteoblasts and osteoclasts were analyzed using elisa. results. the expression of adiponectin and its receptors (adipor , adipor , and paqr ) by cultured ra osteoblasts and osteoclasts could be confirmed on translational and transcriptional level. stimulation of primary ra osteoblasts and osteoclasts with adiponectin resulted in an alteration of cytokine release. osteoblasts showed a time-and dose-dependent increase in il- production. furthermore, adiponectin induced the secretion of il- and gro-alpha and significantly increased the il- and mcp- production (il- : -fold, p= . ; mcp- : -fold, p= . ). stimulation with adiponectin resulted in an increase in il- production in pre-osteoclasts ( -fold) but not in osteoclasts. the secretion of il- was increased in pre-osteoclasts ( -fold) and osteoclasts ( -fold). the results of the present study confirm the pro-inflammatory potential of adiponectin in ra. the cytokines released after adiponectin treatment by osteoblasts and osteoclasts promote osteoclastogenesis or the migratory potential of osteoclasts and monocytes. together with the finding that adiponectin is present in the bone compartment of ra suggest an involvement of adiponectin in articular destruction. acknowledgement: funded by the german research society (spp , immu-nobone, ne / - ). novel mechanisms of glucocorticoid therapy in arthritis anti-inflammatory acting glucocorticoids (gcs) are an important component of rheumatoid arthritis (ra) therapy. but their beneficial usefulness, especially in ra therapy, is hampered by severe side effects like glucocorticoid-induced osteoporosis (gio). until now the molecular mechanisms underlying the beneficial and side effects of gc therapy are poorly understood. gcs exert their actions via the glucocorticoid receptor (gr) that alters gene expression by either binding as a dimer to gc response elements in the promoter region of target genes or by interacting with and thus interfering with other transcription factors. for a long time gr dimerization was considered as the molecular base of side effects. interference of pro-inflammatory transcription factors, such as ap- and nf-κb by the gr monomer was believed to contribute to the therapeutic effects of gcs. in a model of gio we previously showed that unexpectedly interaction of the gr monomer with ap- , but not nf-kb in osteoblasts is decisive for bone loss (cell metabolism : ). in contrast, in antigen-induced arthritis (aia), we could demonstrate that gcs act in the acute inflammation of ra via the dimerized gr. particularly, gc therapy suppressed th and th cell derived pro-inflammatory cytokines in a dimerization dependent manner. furthermore th , rather than th cells seem to be the most crucial targets for an efficient gc therapy since il- -/-mice were resistant to gc therapy whereas ifnγ-/-mice responded as efficient as wild type mice to steroid treatment (pnas : ). in a more chronic arthritis model, the k/bxn serum transfer induced arthritis, we demonstrate now that, unexpectedly, dimerization of the gr in non-hematopoietic cells also contributes to the anti-inflammatory effect of gcs. thus, for immunosuppression of arthritis the gr is required in distinct cell types. taken together, for anti-inflammatory actions the gr dimerization dependent gene regulation is decisive in ra, whereas gio depends on the suppression of ap- dependent gene expression. intriguingly for anti-inflammatory activities of gcs immune and non-immune cells are involved. our approaches give new insights into gc action on arthritis and bone that can be translated into new concepts for anti-inflammatory therapies preventing gio. background. new bone formation and ankylosis are a hallmark of ankylosing spondylitis (as). the impact of cytokines and different mechanisms of new bone formation (endochondral vs. membranous) to syndesmophyte formation and joint ankylosis in as are still poorly understood. in order to analyze cartilage hypertrophy -as a potentially important element of endochondral bone formation -and to assess the possible influence of cytokines, we performed an immunohistochemi-cal study of the hyaline articular cartilage of facet joints of as patients in comparison to autopsy controls and patients with osteoarthritis (oa). methods. the cytokines interleukin(il)- , il- and il- , as well as the marker of cartilage hypertrophy runt-related transcription factor (runx ), matrix metalloproteinase (mmp ) and collagen type (col ) were determined in facet joints from patients with as (undergone correction surgery of rigid hyperkyphosis), oa patients (undergone surgery of the lumbar spine, because of neurological deficits) and controls (autopsies without spinal diseases). immunohistochemistry was performed and the entire cartilage area was analyzed for the frequency of positively stained chondrocytes. background. immunization with glucose- -phosphate isomerase (g pi) induces arthritis in susceptible strains of mice. depletion of regulatory t cells (tregs) prior to immunization switches the usually acute, self-limiting course to a non-remitting, destructive arthritis. this provides a possibility to study molecular switches for the transition from acute, self-limiting to chronic, destructive arthritis within one mouse model. to examine the role of fibroblast-like synoviocytes (fls), which are known to modulate immune responses via the production of pro-and anti-inflammatory mediators, the phenotype and function of fls from mice with either acute, self-limiting or non-remitting, destructive arthritis was studied. methods. fls from dba/ mice that developed either the acute or the chronic form of arthritis were isolated from joints over a time course of days. to investigate the phenotype of fls elisa studies as well as zymography have been performed. for the functional clarification of those cells the matrix-associated transepithelial resistance invasion (matrin) assay and a cartilage attachment assay have been used. furthermore, fls have been transferred in vivo into the knee joints of immunodeficient mice and the joints have been scored histologically. results. fls from treg-depleted mice produced significantly more cytokines (e.g. interleukin (il- )) upon stimulation with other cytokines, growth factors and tlr ligands. this increased susceptibility to cytokine stimulation in chronic animals compared to acute ones is observable throughout the disease course ( days). furthermore, the secretion and activity of matrix metalloproteases (mmps) was enhanced in the fls from chronic mice compared to samples from acute ones. additional functional differences include the collagen-destructive potential and the potential to attach and eventually invade wild type cartilage. here, fls from treg-depleted chronic arthritic mice showed a higher invasive and destructive potential. ultimately, fls from treg-depleted mice were able to destroy cartilage in immunodeficient mice. conclusion. our results are compatible with the hypothesis that uninhibited inflammation in the early phase of treg-depleted mice causes the acquisition of an autonomously aggressive phenotype of synoviocytes which contribute to the switch from acute to chronic arthritis even in the absence of late support from t and b lymphocytes. collagen-induced arthritis modulates reactivity to sympathetic neurotransmitter stimuli during osteoclastogenesis of bone marrow-derived macrophages from da rats background. osteoclast(oc)-mediated bone destruction contributes to increased disease burden in rheumatoid arthritis. simultaneously, changes in synovial tissue innervation occur, leading to a reduction in catecholaminergic nerve fibres. studies on sweat gland innervation revealed that catecholaminergic fibres are capable of phenotypic transition to cholinergic nerves. the sympathetic neurotransmitters norepinephrine (ne) and acetylcholine (ach) affect osteoclastogenesis oppositely prompting us to study osteoclastogenesis at different phases of collagen-induced arthritis (cia) in an altered neurotransmitter microenvironment. methods. for induction of experimental arthritis, da rats were immunized with bovine collagen type ii while controls received isotonic nacl solution. to generate oc, bone marrow-derived macrophages (bmm) were isolated and differentiated with recombinant m-csf and rank ligand. the influence of ne and ach stimulation on osteoclast differentiation and activity was compared between arthritic and control animals at the acute ( days post immunization, pi) and the chronic ( days pi) disease state. as the nicotinic α ach receptor subunit is involved in the cholinergic anti-inflammatory reflex, we also applied a specific agonist, arr- . additionally, the gene expression profile for ne and ach neurotransmitter receptors was analyzed. results. ach stimulation generated significantly more osteoclasts in controls ( days pi). arr- mediated effects were similar to ach. ne decreased osteoclastogenesis via β-adrenoceptors and enhanced via α-adrenoceptor stimulation. cells from arthritic animals were less affected by ne and ach stimulation.oc from arthritic animals showed tendentially decreased activity in an enzymatic cathepsin k activity assay. ach and arr- stimulation decreased cathepsin k activity days pi, but the effect disappeared days pi, representing the chronic arthritis state. ne stimulation significantly inhibited enzyme activity days pi, but has little effect under chronic conditions. the receptor gene expression profile changed in the time course of arthritis. days past immunization muscarinic ach receptors m and m were significantly upregulated whereas after days adrenoceptors α d and α b were significantly downregulated. conclusion. we conclude that cia differentially modulates neurotransmitter influence during oc differentiation and activation but the underlying processes remain still unknown. the observed time pointdependent changes in neurotransmitter receptor gene expression may constitute a regulatory mechanism to counteract alterations in the local neurotransmitter composition. background. the generation of memory t lymphocytes allows effective and fast immune responses during antigen re-challenge and represents a hallmark of adaptive immunity. previous work from our group has demonstrated that murine memory cd + t cells reside in specific bone marrow niches and are characterized by the high expression of cd and ly- c. these cells were designated as resting in the context of gene expression and proliferation. here, we aimed to phenotypically and functionally characterize human memory t lymphocytes in peripheral blood and bone marrow of healthy individuals. methods. mononuclear cells were isolated from paired blood and bm samples from individuals undergoing hip replacement surgery. phenotypic analysis and cytokine profile of distinct memory t cell subsets were assessed by flow cytometry. proliferation and cell cycle status were analyzed using ki- and propidium iodide (pi) staining, respectively. results. distinct populations of cd -expressing cd +cd ra-and cd +cd ra-t cells were detected in bone marrow but not in the periphery. ccr and cd l expression was reduced on bone marrow cd +cd +cd ra-and cd +cd +cd ra-t cells compared to their cd -counterparts in bone marrow and peripheral blood. cell cycle analysis and ki- expression levels demonstrated the nonproliferative state of bone marrow memory t cells. furthermore, bone marrow resident memory t cells showed reactivity against various pathogenic agents, such as tetanus, measles and cmv. conclusion. we have identified a population of cd -expressing cd +cd ra-and cd +cd ra-t cells in the bone marrow. despite cd expression, which is generally regarded as early activation marker, the cells were resting in terms of proliferation. bone marrow cd + memory t cells have downregulated ccr and cd l indicating reduced homing capacity to secondary lymphoid organs. our data underline the role of the bone marrow as a major reservoir for resting memory t lymphocytes. therapeutic methods exerted an influence on satisfaction and future expectations in patients with rheumatoid arthritis (ra). methods. when visiting their rheumatologist, patients with ra were asked to complete a questionnaire at home after the consultation and then return it to an independent opinion research centre, where the data was collected and analysed. the form comprised various areas, namely demography, aspects of the diagnosis, medical care, therapeutic measures, and the illness in a personal context. results. patients ( females/ males) from the whole of austria with a particular emphasis on lower austria, upper austria and tyrol completed the questionnaire (of distributed), resulting in a response rate of %. % of the patients lived in settlements of under , inhabitants; a further % in settlements of under , inhabitants.. the rheumatologist attended could be reached within one hour for % of the patients and within minutes for %. in slightly fewer than % of the respondents the diagnosis was made within three months, in % within six months. in %, the diagnosis was made by a rheumatologist. after experiencing the first symptoms, % contacted their general practitioner. a high degree of satisfaction appears to originate from the information supplied by the rheumatologist attended. most patients felt they were involved in decisions regarding their therapy. % of the respondents were employed prior to their illness; as a consequence of the disease % had to leave their jobs. conclusion. the majority of the respondents came from rural areas. the correct diagnosis was made within six months for almost half of the patients questioned. most patients felt well informed by their rheumatologists and involved in therapeutic decision-making. , combinational therapy of synthetic dmards ( . ; . - . ), and biological-monotherapy ( . ; - ). all of these differed significantly on later observation periods. comparing the prescriptions separately by sequential treatments there were no differences in retention rates for the individual dmard classes. regarding retention, in the first treatment synthetic dmards showed the longest retention, but from the second on this was the case for tnfi-combinational treatment and non-tnfi-biologicals (. abb. ) conclusion. traditional dmards are the starting point of therapy and mtx is the anchor drug for the first and all subsequent courses. already at the second sequential course, the combination therapies of mtx+tnfi become numerically more relevant, and their retention is better than mtx. therapie der rheumatoiden arthritis im letzten jahrzehnt -was hat sich verändert? abb. | ev. anzahl eingeschlossener patienten nach einschlussjahren und therapie sowie zeitlichen entwicklungen im das und der eular-response cal features of ra (erosive arthritis with classical radiological features) plus specific laboratory markers (ccp-antibodies). the third patient, a -year-old female presented initially with features of ra and sle simultaneously (alopecia, subcutaneous nodules, leucopenia, positive ccp-antibodies, high titres of ana and dna-antibodies). the fourth patient, a -year-old patient presented with severe polyarthritis in the upper and lower limbs, subcutaneous nodules, fever and cervical lymphadenopathy, she had high titres of ccp-antibodies ( u/ml by a normal range of less than iu/ml), ana of (normal less than ), and dna of iu/ml (normal less than iu/ml). conclusion. the take home massage of this presentation is to be aware of rhupus if the sle patient develops erosive arthritis or subcutaneous nodules, or if the ra patient develops features of sle like leucopenia, active urine sediment, or clinically significant serositis. rhupus seems to be a distinctive entity and should be kept in mind while dealing with patients having ra or sle as it can affect the treatment and outcome. vorgeschichte. bei der abklärung eines akuten thoraxschmerzes sind auch seltene ursachen, so zum beispiel der einbezug thorakaler organe in eine entzündliche systemerkrankung, zu bedenken. anhand eines besonderen falles möchten wir den weg zur diagnose bei einem schwer kranken notfallpatienten zeigen. leitsymptome bei krankheitsmanifestation. ein -jähriger mann wird bereits zum dritten mal mit akuten thoraxschmerzen in die notaufnahme aufgenommen, jeweils war eine kardiale ischämie ausgeschlossen und ambulante diagnostik empfohlen worden. nach der schmerzcharakteristik, der vorgeschichte von rezidivierenden thoraxschmerzen und entsprechenden risikofaktoren wird bei massiver symptomatik jetzt von einem akuten koronarsyndrom ausgegangen und die invasive diagnostik durchgeführt. eine akute koronare ischämie kann jedoch ausgeschlossen werden. fieber, hohe entzündungszeichen, hinfälligkeit und gewichtsverlust von mehr als kg in den letzten wochen lassen dann eine infektbedingte oder tumorbedingte ursache vermuten, abdomensonographie und röntgen-thorax sowie ausführliches labor samt immunologie führen nicht weiter. wegweisende weitere organbefunde finden sich nicht. die behandlung mit antibiotika hat keinerlei effekt auf klinik oder entzündungsparameter. nach tagen wird der krankheitsverlauf kritisch evaluiert, eine nichtinfektiöse ursache der beschwerden wird in betracht gezogen, eine transösophageale echokardiographie wird zum ausschluss einer endokarditis und zur beurteilung der aorta (leitsymptome thoraxschmerzen und fieber!) durchgeführt. dabei zeigen sich die klappen sämtlich unverdächtig, die aorta ascendens weist eine massive echoarme wandverdickung auf. zum sicheren ausschluss eines intramuralen hämatoms wird sofort eine ct der thorakalen aorta durchgeführt, die eine ausgeprägte zirkuläre wandverbreiterung ohne hinweise auf dissektion zeigt. diagnose. riesenzellarteriitis mit aortitis. therapie. steroide, einleitung einer remissionsinduzierenden therapie mit cyclophosphamid boli. weiterer verlauf. innerhalb von tagen ist der patient beschwerdefrei, die entzündungsparameter sind halbiert, der bettlägerige patient lässt sich mobilisieren und verlässt nach tagen die klinik. bei der diffe-renzialdiagnose des akuten thoraxschmerzes sollten eine unklare entzündungsserologie und eine b-symptomatik frühzeitig an eine aortitis denken lassen. in diesem fall fand sich bereits in tee und ct ein ausgeprägter befund, der sich am ehesten durch den langen verlauf vor diagnosestellung erklären lässt. einleitung. die progressive familiär intrahepatische cholestase (pfic) gehört zu einer heterogenen gruppe seltener, autosomal rezessiv vererbter erkrankungen der gallensäurenexkretion mit intrahepatischer cholestase, hohem risiko der leberzirrhose bereits im kindesalter und hepatozellulärem karzinom. das auftreten eines sle bei pfic ist bisher in der literatur nicht beschrieben. methoden. wir berichten über eine jetzt -jährige patientin mit genetisch gesicherter pfic- (defekt der atp abhängigen gallensalz-exportpumpe bsep), biliostomaanlage im kindesalter, therapie mit udca und kontinuierlicher hepatologischer betreuung. / manifestierte sich ein sle mit az-minderung, florider polyarthritis, polyserositis, splenomegalie, anämie und leukozytopenie. die ana waren mit > : homogen erhöht, die dns-ak im elisa mit u/ml, ena und antiphospholipid antikörper waren negativ. eine steroidtherapie wurde mit prednisolon mg/tag begonnen. bei guter verträglichkeit und stabilen cholestaseparametern wurde die therapie um chloroquin mit mg an tagen der woche ergänzt. die betreuung wurde interdisziplinär fortgesetzt. ergebnisse. unter der steroidtherapie waren gelenkbeschwerden und serositis gebessert, das crp, die leukozytopenie und anämie normalisiert. die dns-antikörper fielen auf u/ml, c und c stiegen um %. es persistierten lediglich physiotherapeutisch behandelte muskuläre schmerzen und schonatmung nach pleuritis. bei steroidreduktion unter mg traten die gelenkbeschwerden wieder auf, die dns-antikörper stiegen auf u/ml und c /c fielen um %, das crp blieb normal. die steroiddosis wurde auf mg/tag und die chloroquindosis auf × mg/woche erhöht. neue organkomplikationen im rahmen des sle sind nicht aufgetreten. die gallensäuren waren mit µmol/l (norm < ) im jahr (letzte messung vor manifestation des sle) deutlich erhöht, bei manifestation des sle mit µmol/l bereits deutlich gebessert und unter hoch dosierter steroidtherapie mit , µmol/l nach woche sowie , µmol/l nach monat normalisiert. unter prednisolon mg/tag stiegen sie bei inaktiviertem sle auf µmol/l nach monaten an, bei steroidreduktion unter mg auf µmol/l. nach erneuter steroiddosiserhöhung auf mg prednisolon/tag fielen sie auf µmol/l ab. schlussfolgerung. die pfic- schützt nicht vor der manifestation eines sle. eine behandlung mit steroiden und chloroquin ist auch bei pfic sicher und wirksam. der floride sle und die höher dosierte steroidtherapie senken trotz bestehenden genetischen defekts unabhängig voneinander und synergistisch die gallensäurenspiegel im serum bei genetischer störung der atp-abhängigen sekretionspumpe bsep. augenentzündungen. eine ausgeprägte b-symptomatik mit fieber, nachtschweiß und abgeschlagenheit seit ca. zwei jahren hatte sich jeweils unter der therapie der hörstürze verflüchtigt. die mr-angiographie der aortenwand zeigte den typischen befund eines ausgeprägten wandenhancements der thorakalen aortenwand sowie der supraaortalen gefäße, duplexsonographisch fand sich eine zirkuläre wandverdickung der proximalen und mittleren acc beidseits ohne relevante stenosen. bei fehlendem nachweis zerebraler vaskulitischer oder embolischer veränderungen im kraniellen mrt und bei normalem eeg wurde der cerebrale krampfanfall als gelegenheitsanfall dd im rahmen der hochaktiven grunderkrankung interpretiert. diagnose. takayasu-arteriitis mit assoziiertem cogan-syndrom und rezidivierender polychondritis. therapie. steroidstoß, darunter rasche reduktion der entzündungszeichen und promptes ansprechen der b-symptomatik, beginn einer steroidsparenden therapie mit mtx. bei anhaltender krankheitsaktivität und hohem steroidbedarf wird der patient aktuell mit tocilizumab behandelt. schlussfolgerung. der präsentierte fall zeigt, dass mittels neuer bildgebender verfahren gelegentlich eine großgefäßvaskulitis detektiert werden kann, obwohl das klinische bild (hier: kopfklinik) eine kleingefäßvaskulitis vermuten lässt. das cogan-syndrom ist häufig mit einer großgefäßvaskulitis assoziiert, in diesem fall auch mit einer polychondritis. umgekehrt erweitert sich unser wissen um das befallsmuster der großgefäßvaskulitiden, die zwar überwiegend große, aber auch mittelgroße und kleine gefäße einbeziehen können. in unserem zentrum ist dies der zweite fall einer takayasu-arteriitis mit assoziiertem cogan-syndrom in einem kollektiv von fällen. diagnostik. im rahmen einer vorstellung in unserer rheumatologischen ambulanz zur abklärung eines möglichen sjögren-syndroms, konnte in der lungenfunktionsdiagnostik eine leichte restriktion und Überblähung festgestellt werden. in einem auswärtig durchgeführten mrt der halsweichteile zeigten sich die glandula parotis und submandibularis beidseits inhomogen und kräftig kontrastiert, ebenfalls mehrere grenzwertig große lymphknoten. aufgrund der erneut pathologischen lungenfunktion und dem verdacht auf eine lungenbeteiligung bei sjögren-syndrom erfolgte ein ct-thorax. hier zeigte sich eine zysti-sche rarefizierung vor allem der zentralen lungenanteile, differentialdiagnostisch mit einer langerhans-zell-histiozytose vereinbar. auch erschien der diabetes insipidus passend zur histiozytose. die immunologischen untersuchungen waren unauffällig, insbesondere konnten keine ssa-/ssb-antikörper oder eine hypergammaglobulinämie nachgewiesen werden. somit erschien ein sjögren-syndrom unwahrscheinlich. zur weiteren abklärung erfolgte eine bronchoskopie, in den biopsaten konnte immunhistochemisch eine langerhans-zell-histiozytose nachgewiesen werden. in einer pathologischen nachuntersuchung auswärtiger parotis-biopsate bestätigte sich diese, zudem konnte eine mutation des braf-proto-onkogens nachgewiesen werden. im abschließend durchgeführten pet-ct konnte eine vermehrte kontrastmittelaufnahme des hypophysenstiels, eine vermehrte stoffwechselaktivität der parotis beidseits sowie kutan axillär links diagnostiziert werden. abschließend lag somit eine langerhans-zell-histiozytose mit befall von hypophyse, lunge, parotis, haut, lymphknoten sowie einem fraglichen befall des darms vor. therapie. in absprache mit der adulten langerhans-zell-histiozytose studiengruppe erfolgt nun eine therapie mit cytarabin. weiterer verlauf. der weitere verlauf nach geplantem therapiebeginn bleibt abzuwarten. einleitung. die anti-gbm-erkrankung (goodpasture-syndrom) ist eine prototypische autoimmunerkrankung mit ernster prognose, wenn sie als "pulmorenales syndrom" mit der trias rapid-progressive glomerulonephritis, alveoläre hämorrhagie und nachweis von autoantikörpern gegen glomeruläre basalmembranen (anti-gbm-ak) auftritt. Über weniger aggressive verläufe ist wenig bekannt. in der literatur wird die bedeutung der frühen diagnosestellung für die prognose der patienten betont. wir berichten über eine -jährige patientin, die sich mit abgeschlagenheit, müdigkeit und blutbeimengungen im sputum vorstellt. sie betreibt einen nikotinabusus. methoden. wir sehen eine blasse patientin (hb , mmol/l, normochrom, normozytär), die keine weiteren auffälligen befunde in der körperlichen untersuchung zeigt. die apparative diagnostik mittels endoskopie und sonographie ergibt keine befunde, die die anämie erklären können; in der bronchoskpie zeigt sich das bild einer alveolären hämorrhagie ohne aktive blutungszeichen. neben der ausgeprägten anämie bestehen eine milde proteinurie mit mg/d sowie eine geringe erythrozyturie. die immunologische diagnostik ergibt gering erhöhte anti-gbm-ak, negative ana und anca. die nierenbiopsie zeigt eine rapid-progressive glomerulonephritis mit halbmondbildung in einem glomerulum, zusätzlich können lineare igg-ablagerungen in der immunfluorenz dargestellt werden. es ist ein glomerulum in der biopsie betroffen, die anderen glomeruli sind unauffällig. es bestehen einzelnen erythrozytenzylinder, diffuse entzündungszeichen lassen sich nicht nachweisen. die nachträglich durchgeführte immunfluoreszenz in der lungenbiopsie bestätigt den befund linearer igg-ablagerungen. ergebnisse. in unserem fall sehen wir eine junge patientin in einem relativ guten allgemeinzustand mit gering erhöhten anti-gbm-antikörpern und einer gering ausgeprägten nierenschädigung mit einem befallenen glomerulum in der biopsie. es wird angesichts des jungen alters der patientin und der geringen ausprägung der erkrankung eine therapie mit cyclophosphamid nach dem "euro-lupus-protokoll" von f. hossiau eingeleitet. im verlauf steigt der hb auf , mmol/l, die alveoläre hämorrhagie sistiert und die proteinurie ist rückläufig. schlussfolgerung. das goodpasture-syndrom mit einer inzidenz von , - , / mio. einwohner und jahr ist eine sehr seltene autoimmunerkrankung mit ernster prognose. möglicherweise spielen umweltfaktoren (hier: nikotinabusus) eine rolle für die manifestation der erkrankung. interessant ist, dass die erstbeschreibung im rahmen einer influenza-epidemie -wie auch in diesem jahr bei unserer patientinerfolgte. Über die therapie gibt es wenige informationen. die meisten empfehlungen gibt es zum pulmorenalen-syndrom, über weniger aggressiv verlaufende manifestationen gibt es nur wenige informationen. einleitung. wir berichten über einen -jährigen raucher mit akut aufgetretener polyarthritis und neu aufgetretenem raynaud-phänomen. auffallend war die diskrepanz zwischen nur geringer systemischer entzündungskonstellation und einer hochaktiven polyarthritis mit schwerem raynaud-phänomen. im verlauf kam es zu einer spontanen thrombophlebitis der v. cephalica. methoden. pathologische werte: crp maximal , mg/dl (norm < , ), zellzahl im gelenkpunktat . leukozyten/µl. im normbereich lagen: bsg mm/ h, ana, ena, ds-dns-ak, rheumafaktoren, anca, kälteagglutinine, kryoglobuline, tumorsuche initial ohne befund (ct-thorax, bronchoskopie, ct abdomen und becken, coloskopie, gastroskopie, skelettszintigraphie), fdg pet-ct: zwei suspekte lymphknoten rechts cervical sowie suspekte rechte tonsille. ergebnisse. selbst unter mg prednisolon weiterhin hochaktive polyarthritis. nach unauffälliger initialer tumorsuche veranlassten wir ein fdg-pet ct mit nachweis zweier suspekter lymphknoten rechts cervical sowie einer suspekten rechten tonsille. die biopsie der klinisch sich nur in der palpation diskret induriert darstellenden region ergab ein gering differenziertes, gering verhornendes plattenepithel mit %iger sequenzhomologie mit hpv typ . nach resektion des tumors und radiatio sistierten sowohl die polyarthritis als auch das raynaud-phänomen ohne weiteres rezidiv auch nach absetzen der glukokortikoide. schlussfolgerung. eine akut auftretende hochaktive polyarthritis mit hohem glukokortikoidbedarf in kombination mit einem raynaud-syndrom, auffallend niedriger systemischer entzündungsaktivität und fehlendem autoantikörpernachweis sollte insbesondere bei einem langjährigen raucher anlass zu einer intensiven tumorsuche geben. ein fdg-pet-ct kann bei okkulten tumoren zielführend sein. bemerkenswert ist hier der nachweis von hpv- im tumor als weiterer risikofaktor neben dem zigarettenrauch. methoden. bei der klinischen untersuchung fielen ein beidseits positives menell-zeichen und deutlicher klopfschmerz über dem lumbosakralen Übergang auf. labordiagnostisch konnte ein erhöhtes c-reaktives protein und ein positiver hla-b nachgewiesen werden. der bath ankylosing disease activity (basdai) index betrug , . die beckenübersichtsaufnahme zeigte eine definitive bilaterale sakroiliitis grad gemäß den modifizierten new-york-kriterien. der befund der kontrastmittelunterstützten mrt der iliosakralgelenke bewies das vorliegen einer bilateralen floriden sakroiliitis. bei gesicherter hla-b positiver ankylosierender spondylitis wurde die indikation zur einleitung einer biologikatherapie mit einem tnfα-inhibitor gestellt. während der abklärung von kontraindikationen wurde in der konventionellen röntgen-thorax-aufnahme eine rundliche, glatt begrenzte zystische läsion mit flüssigkeitsspiegeln im rechten mittellappen entdeckt. die weitere abklärung mittels nativer thorax-ct, bronchoskopie und biopsieentnahme bestätigte den verdacht auf eine bronchogene zyste. die erregerdiagnostik in der bronchoalveolären lavage zeigte lediglich eine kontamination mit der residenten standortflora. ergebnisse. in anbetracht der geplanten immunsuppressiven therapie, die mit einem erhöhten infektionsrisiko einhergeht, wurde die bronchogene zyste im september operativ entfernt. zur linderung der beschwerden erhielt die patientin eine schmerztherapie mit nsar. als sich die patientin sechs monate später erneut zur einleitung der biologikatherapie vorstellte, berichtete sie, dass die schmerzen etwa einen monat nach der operativen resektion praktisch verschwunden seien. die klinische untersuchung war unauffällig, der basdai-index lag bei , . auch das zur verlaufskontrolle angefertigte mrt der isg zeigte im vergleich zur voruntersuchung einen deutlichen rückgang der floridität. schlussfolgerung. es handelt sich um den ersten fall einer kompletten klinischen und radiologischen remission einer hla-b -positiven ankylosierenden spondylitis nach operativer entfernung einer bronchogenen zyste als potenziellen entzündlichen fokus. bei der systemischen verlaufsform der erkrankung treten neben kutanen erscheinungen zusätzlich muskuloskeletale, hämatopoetische ( - %), renale (ca. %), kardiale, cerebrale und pulmonale ( - %) manifestationen auf. eine polyserositis ( - %) ist häufig. mit - % werden im sle-schub abdominelle schmerzen beschrieben. nur in seltenen fällen (amerika , %, asien , - , % aller sle-patienten) kommt es zum bild einer lupusassoziierten mesenterialen vaskulitis (lmv). die Ätiologie der lmv ist weitestgehend unklar, eine genetische präsidsposition sowie auslösende faktoren (bakterielle darminfektionen, medikamente wie nsar, phosphodiesterasehemmer) werden diskutiert. pathogenetisch wird eine mesenteriale ischämie durch eine mikroangiopathie (arteriolen, venolen) bei inflammatorischer immunkomplexpräzipitation sowie thrombembolischen ereignissen angenommen. radiologisch/sonographisch zeigt sich ein segmentales darmwandödem mit darmdilatation. endoskopisch dominieren oberflächliche ulzerationen, perifokale hämorrhagien bis hin zur gangrän. eine erhöhte perforationsneigung wird beschrieben. mikroskopische befunde zeigen eine fibrinoide nekrose subseröser gefäße mit leukozytoklasie der gefäßwand bzw. ein submuköses Ödem mit nur diskreter invasion mononukleärer zellen. die prognose der lmv scheint abhängig von genetischer prädisposition, raschem beginn einer immunsuppression sowie restriktivem einsatz operativer interventionen und wird je nach literaturquelle mit einer letalität bis zu % angegeben. background. we report a patient with tma in the context of sle treated successfully with the c inhibitor eculizumab. the patient had sle with lupus nephritis (ln). before she developed tma with renal failure and neurologic manifestations, she was treated with various immunosuppressive regimens for mucocutaneous and musculoskeletal manifestations and later for ln. the diagnosis of atypical hemolytic uremic syndrome (ahus) was made based on the presence of coombs-negative hemolytic anemia, thrombocytopenia, renal failure, seizures due to cerebral ischemia and signs of tma in the renal biopsy. plasma exchange and hemodialysis were started immediately and could stabilize her condition. six weeks after the beginning of plasmapheresis but still severely compromised renal function and thrombocytopenia, complement inhibition with eculizumab became a therapeutic option. after the first infusions, renal function, anemia and thrombocyte counts markedly improved. dialysis could be stopped. extensive genetic testing of mutations associated with the overactivation of the alternative complement pathway was negative. after months, when the patient was still in remission, eculizumab infusion intervals were widened and it was finally stopped after months of treatment. since then, renal function remained stable with nearly normal glomerular filtration rates. background. il- signaling plays an important role in inflammation but is restricted by different regulatory mechanisms. these mechanisms include the decreased availability of gp , the signal transducing chain of the il receptor, on the cell surface. the aim of this study was to determine whether the inflammatory environment in the arthritic joint has an impact on monocytic gp surface expression and the extent to which regulatory processes in the synovial fluid (sf) can be transferred to an in vitro model. flow cytometry and live cell imaging were used to measure the cell surface expression and internalization of gp . stat phosphorylation was monitored by flow cytometry and western blotting. results. the level of cell surface gp expression on sf monocytes was reduced compared to peripheral blood (pb) monocytes from patients with juvenile idiopathic arthritis (jia). this reduction could be reproduced by stimulating pb monocytes from healthy donors with sf and was dependent on p mapk. the induction of p by il- β in pb monocytes interfered with il- signaling due to the reduced cell surface expression of gp . the results suggest that p -mediated pro-inflammatory stimuli induce the downregulation of gp on monocytes and thus restrict gp mediated signal transduction. this regulatory mechanism could be relevant in the inflamed joints of patients with jia. kr. sjia patient characteristics of those who successfully discontinued corticosteroids during canakinumab treatment: secondary analysis from a pivotal phase trial background. interleukin- β (il- β) is a key driver in the pathogenesis of systemic juvenile idiopathic arthritis (sjia). canakinumab (can), a selective fully human anti-il- β monoclonal antibody, has been shown to be efficacious in the treatment of sjia [ ] . corticosteroids (cs) are a mainstay of therapy for sjia, however due to the well-known long-term side effects, reduction of cs dosage is desirable. objectives. to assess patient features associated with cs discontinuation during can therapy. methods. patients ( - years of age) with active sjia received s.c. can ( mg/kg to mg max) every four weeks during the maximum week cs-tapering phase [ ] . cs tapering was to be initiated when at least an adapted acr was achieved and no fever. a -year-old boy presented with nocturnal tingling paresthesia affecting his feet and his calves. no excessive leg movements were noted at night. within a few months, his symptoms worsened. the paresthesia occurred both during the day and at night. moreover, the paresthesia came to be triggered by merely standing up. affecting a sharply demarcated area not corresponding to dermatomes, symptoms resolved promptly with movement. the paresthesia was associated with local skin erubescence in spots that slowly began spreading all over the affected area. symptoms did not occur while the patient was seated. mild painless swelling around both of the ankles was noticed in the evenings. approximately one and a half years after the initial manifestation, painful triphasic color changes of all fingers and toes triggered by cold or stress occurred. the family history was positive only for psoriasis. extensive laboratory studies excluded inflammatory and hematological conditions as well as occlusive arterial diseases known to be associated with secondary raynaud's phenomenon. polyneuropathy and other neurological disorders were excluded as well. inflammatory joint disease suspected from the initial imaging with magnetic resonance of the feet and ankles was not confirmed by repeated investigations and scintigraphy. the only consistent abnormality was a reduced pulse amplitude corresponding to vasospasm, which was revealed by photoplethysmography of toe vessels. additionally, paradoxical amplitude reduction after application of nitroglycerine was seen in finger vessels. placing his hands or feet in cold water did not trigger raynaud's phenomenon. initial treatment with non-steroid anti-inflammatory drugs, topical isosorbiddinitrate and local steroid instillation (suspicion of inflammation of tibialis posterior tendon) was ineffective. systemic therapy with the calcium channel blocker amlodipine was initiated. the initial dosing of mg ( . mg/kg/day) was slowly increased to mg ( . mg/kg/day) which lead to complete resolution of the patient's ailments. after three years of pharmacotherapy and . years in remission, a weaning off the treatment is planned. based on the patient's positive response to calcium channel blocker, we conclude that the lower-leg paresthesia was of vascular origin and can be considered an atypical presentation of raynaud's phenomenon. background. the initial treatments of choice for jdm are high-dose corticosteroids and methotrexate. however, no consensus exists about second line therapeutic options in refractory or recurrent cases. results. we present a -year-old boy who was diagnosed with jdm due to severe proximal muscle weakness, dysphagia, a heliotrope rash, gottron's sign, nail teleangiectasia and a characteristic muscle biopsy. creatine kinase levels were within normal range and no antinuclear antibodies were present. over a period of seven years, the patient was treated with high-dose corticosteroids, methotrexate, intravenous immunoglobulins, oral steroids, mycophenolate mofetil, rituximab and infliximab. despite all treatment efforts, skin and muscle inflammation persisted and the boy developed severe subcutaneous calcifications, rendering him wheelchair-bound. as il- production correlates with disease activity in adult and juvenile dm, treatment with tocilizumab ( mg/kg every weeks) was initiated, leading to a complete resolution of skin inflammation within months. within months of treatment, the disease activity score (das) decreased from to (out of ), the childhood myositis assessment scale (cmas) increased from to (out of ) and the kendall manual muscle test (mmt) increased from to (out of ). in daily life the wheelchair was no longer necessary. treatment was well tolerated but accompanied by a moderate increase in liver transaminase activities. interestingly, therapy with rituximab was associated with a decline in igm levels only, whereas igg and iga stayed markedly elevated. in contrast, following initiation of tocilizumab treatment, igg levels rapidly declined to normal range, emphasizing the role of the humoral immune system in the pathogenesis of dm. conclusion. taken together, treatment of a severely affected jdm patient with tocilizumab was safe, well tolerated and led to a significant improvement in disease activity. further investigations of il- -blocking agents as a treatment option in otherwise therapy-resistant jdm patients are warranted. functional capacity of jia patients with an initial adjustment to an anti-tnf-alpha therapy background. thirty three percent of patients with polyarticular jia are treated with biologics [ ] . despite substantial improvement achieved by anti-tnf-α treatment according to disease activity [ ] patients have joint-specific impairments. this factor should be considered when analyzing the functional effects on joint limitations while performing daily activities. methods. in a prospective study on polyarticular jia patients treated with anti-tnf-α therapies plus functional therapies we study the longitudinal effects on joint function. the measurements include -d gait analysis (eight vicon f cameras, omg, london, balance control (s -check, tst, großhoeflein), pedobarography (emed plate ( sensors/ cm², novel, munich), daily activity assessment (step watch, orthocare innovations, ok usa), acr pedi and joint mobility testing. we here present the cross-sectional data of the first patients ( . ± . yrs, . ± . cm, . ± . kg, pain-level: . ± . / vas, active joints: . ± . , chaq: . ± . ). results. preliminary results demonstrate that the ability to walk is slightly limited. patients have a reduced push off power generation within the ankle joint (patients: . ± . w/kg; healthy controls: . ± . w/ kg). further on they show limited sensory motor control and stability in comparison to patients with an inactive disease status while performing balance tests (patients: sensory index: . ± . , stability-index: . ± . , patients with inactive status: sensory-index: . ± . , stability index: . ± . (p< . ). note: lower indices values are better results. conclusion. it is one of the first studies which show functional joint-specific deficits during every day activities in patients who receive an initial anti-tnfα-therapy. the limited stability and motor control might be due to limited joint integrity in the ankle joint. this is supported by the impaired push off function while walking. the next study step will show possible effects of the anti-tnfα-therapy. background. the role of sport as a therapeutic tool in treating patients with jia is becoming more important recently [ ] . effects of exercise therapy are reviewed beneath others by takken et al. [ ] . they state that short-term effects look promising but the effects of long-term studies remain unknown. methods. the preventive mobility workout (pmw) is a whole body home-exercise-therapy ( min each day) for patients with an inactive diseases status. it counteracts the deficits which were observed during functional studies in the past [ ] . it consists of exercises for muscular strengthening (squads: hamstring to quadriceps ratio), hamstring flexibility (lift and raise), core stability (prone bridge -time-to-failure), shoulder griddle mobility (horizontal extension) and ankle joint integrity (mechanical power while walking . for statistical analysis an anova was calculated and the level of statistical significance was set to p< . . results. preliminary results show a group effect of the pmw for the hamstring to quadriceps ratio (h-q-r) for the right side (p< . ) and a tendency for the left side (p= . ). the h-q-r for the right side has changed in the tg and cg from . ± . to . ± . and from . ± . to . ± . , respectively. it has changed for the left side in the tg and the cg from . ± . to . ± . and from . ± . to . ± . , respectively. all other parameters regarding flexibility or joint integrity show low or no effects. we have re-tested n= out of so far and the pmw training show little training-effects. the preliminary results might be a reasonable proof for long-term effects. a possible reason for the little effects might be that patients are supposed to train every day but the training diaries show that they exercise approximately three times a week. the authors would like to thank the "deutsche kinder-rheumastiftung" for supporting the study. conclusion. we will validate these proposed definitions prospectively in a jia associated uveitis cohort. based on the results, we will weight these measures to develop an overall scoring system. background. minute walk is a primary outcome measure in studies in pulmonary hypertension. currently we have a two of sets of data [ , ] regarding test results in the minute walk test ( mwt) in healthy children with a large span in the norm values in the different age groups. aim of the study was to establish norm values for healthy german children for the minute walk test. methods. the team of an occupational therapist and a study nurse were visiting schools. permission from the parents was give before the test. always just probands from one class were invited to participate. the test were performed according the international guidelines [ ] . the demographic data of the probands were collected and the parents filled out a short survey regarding the physical activity and the health condition. children with chronic diseases, which decrease the stamina were excluded. up till now probands participated from the age to years. of them were female. the mean minute walk continuously increased with age (. tab background. juvenile idiopathic arthritis (jia) is the most common chronic disease in pediatric rheumatology which often results in foot impairments [ ] . patients with jia are reported to have smaller pressure loads underneath the foot while walking [ ] . the aim of the study was to analyze the peak plantar pressure distribution of a well described cohort of jia patients with an active symmetrical ankle joint arthritis and no history of foot involvement. [ ] ) wurden in bezug auf therapie und outcome anhand der wallace-kriterien beurteilt: "active disease" (ad), "inactive disease" (id), "clinical remission under medication" (crm), "clinical remission off medication" (crom; [ ] background. familial mediterranean fever (fmf) is one of the most common autoinflammatory diseases (aid). pathogenomic relevant mutations in the mefv gene show autosomal recessive inheritance, but co-dominant mutations have been described. we aimed to evaluate correlations between ethnic origin, phenotype and genotype for fmf patients in the german aid-net-registry. methods. we used two common scoring systems modified for children (mor et al., pras et al.) to assess disease severity in fmf patients of the aid-net-registry. for the five most frequent mutations, we tested for a correlation of the genotype with the phenotype, mean crp and ethnic origin, respectively. furthermore, we evaluated the applicability of the two severity scores for children. results. among the patients, we detected a total of pyrin mutations and different sequence variants, including one new mutation (p.gly asp). the five most frequent alterations were p.met val ( %, n= ), p.met lle ( %, n= ), p.val ala ( %, n= ), p.glu gln ( %, n= ) and p.met ile ( . %, n= ). ethnic origin could be determined in cases; the prevailing ancestry was turkish ( %, n= ), % (n= ) were lebanese. p.met val in homozygous form ( %, n= ) was correlated with a more severe disease activity, based on the score by mor, as well as with a higher mean crp ( mg/l, n= , mg/l, n= ) compared to patients without this mutation (p= . and p< . , respectively). the score suggested by pras did not yield a significant genotype-phenotype correlation; indeed, the two scoring systems were inconsistent with each other (κ< . ). although a typical distribution of mutations in different ethnic populations was obvious, this trend was not statistically significant, probably due to the divergent number of cases. conclusion. the homozygous p.met val substitution was associated with a more severe disease activity. there was no origin-genotype correlation in this fmf population. the well-known severity scores for children (mor, pras) are inconsistent. the aid-net is working on a new scoring system. . all patients with rp should be investigated by capillaroscopy. capillaroscopy will be classified into "normal", "aspecific changes" or "scleroderma pattern". . all patients who have additional symptoms pointing to a definite connective tissue disease should be evaluated according to disease specific guidelines. . ana-negative and capillaroscopy-negative patients should be followed-up at least every months. . ana positive patients without disease-specific antibodies and with negative capillaroscopy findings should be followed-up at least every months. . ana and disease-specific antibody positive patients should have organ specific evaluation according to symptoms, examination and relevant to that particular disease e.g. patients who are ana and scl- positive may need organ specific evaluation for jssc as per the juvenile systemic sclerosis inception cohort protocol (www.juvenilescleroderma.com). . ana-positive patients, who have no disease specific antibody but have positive capillaroscopy results, should be followed-up at least every months. . ana-negative patients with positive capillaroscopy result should be followed-up at least every months. . the group could not reach an agreement regarding treatment, due to a lack of data for the paediatric age group. the group agreed that implementation of adult recommendations conclusion. the group made a suggestion for a standard of good clinical practice for rp in children. our aim is that this will facilitate a large multicentre prospective follow-up study of children with rp. background. chronic non-bacterial osteomyelitis (cno) is an inflammatory disorder of the skeletal system of unknown etiology. long-term follow-up and response to treatment data have rarely been reported. the aim of the study was to characterize the clinical, radiological, histological and laboratory data at juvenile cno onset, and to analyze the long term treatment response. methods. the course of disease of juvenile patients with non-bacterial inflammatory bone lesions was evaluated retrospectively. clinical, radiological, histological and laboratory data were assessed at disease onset and for a median time of disease of months. results. the mean age at disease onset was . years, the mean time between the first symptoms and the diagnosis of cno was months. % of the patients had multifocal bone lesions. biopsy was performed in patients. only when bone biopsy was taken within months of symptom onset, cellular infiltrates could be observed. at later time points, fibrosis, hyperostosis and bone edema predominated. the initial treatment consisted of non-steroidal anti-inflammatory drugs (nsaids). % of the patients required second line therapy consisting of sulfasalazine and short term oral corticosteroids, % of the patients required bisphosphonates or tnf-blocking agents. the number of clinical lesions decreased to % within . months and reached . % after months of treatment. the number of radiological lesions, however, declined to only . % after months of treatment. in detail analysis of the tre-atment response revealed that initiation of sulfasalazine treatment in nsaid non-responders led to a significant and sustained decline of the clinical, as well as the radiological number of lesions. conclusion. the rapid clinical improvement in cno, following initiation of therapy with nsaids, is not accompanied by a likewise decrease of the number of radiological lesions. treatment with sulfasalazine is effective in childhood cno. background. exercise has a wide variety of beneficial health effects. it stimulates bone formation and maintains bone strength as well as decreases the risk of falls. moreover, exercise at regular intervals is also assumed to positively affect immune functions. conversely, in more than % of the astronauts during/after space flight and under simulated weightlessness immune functions are suppressed. to assess the effects of simulated weightlessness during the nd berlin bedrest study (bbr- ) on immunological parameters. furthermore, to compare the effects of two different exercise performances (resistive vibration exercise and resistance exercise without vibration). methods. physically and mentally healthy male volunteers ( - y) experienced days of six degree head down tilt bed rest. they were randomized to groups: resistive vibration exercise (n= ), resistance exercise without vibration (n= ), inactive controls (n= ). blood samples were taken days before bed rest, on day and after beginning of bed rest. composition of immune cells was analyzed by flow cytometry. cytokines and neuroendocrinologic parameters were analyzed by a multiplex suspension array/ elisa in plasma. general changes over time were identified by paired t-test, exercise-dependent effects by -group repeated measurements anova. results. for all cases pooled, the number of granulocytes (p< . ), nkt cells (p< . ) and hematopoietic stem cells (p< . ) increased during the study; the concentrations of dhea (p< . ) and eotaxin (p< . ) decreased. different impacts of the specific types of exercise on the change over time were shown for lymphocytes, nk cells, nkt cells, tcell subpopulations and the concentrations of ip- and rantes. conclusion. we found immobilization/simulated weightlessness to significantly impact immune cell populations, and cytokine and neuroendocrine factor concentrations. exercise was able to specifically influence immunologic parameters. interestingly, these changes resemble those found during the aging process. background. novel therapies have made remission and low disease activity (lda) achievable goals in ra. we assessed the impact of treatment with subcutaneous abatacept or adalimumab on these goals and on functional and radiographic outcomes in ample (abatacept versus adalimumab comparison in biologic-naïve ra subjects with background methotrexate), the first head-to-head trial of biologics in ra patients with inadequate response to mtx (mtx-ir). methods. ample is a -year, phase iiib, randomized, investigator-blinded study. biologic-naïve ra patients with mtx-ir were randomized to receive mg abatacept weekly or mg adalimumab biweekly, combined with a stable dose of mtx [ ] and radiographic non-progression (defined as change in modified total sharp score ≤ . ) were analysed in patients achieving or not achieving remission or lda at days or . results. baseline clinical characteristics of abatacept and adalimumab treatment groups were balanced, as was clinical, functional and radiographic efficacy and safety at day [ ] . the proportions of patients meeting each of the remission criteria or lda at day were similar for both groups, but significantly more patients achieved das (crp) remission compared to cdai, sdai or rapid remission, and the smallest proportion achieved boolean remission. compared to remission, a higher proportion of patients achieved lda. across all definitions of remission or lda, > % of the patients achieving remission at days and were haq responders at day . more than % of patients achieving remission or lda at days and were radiographic nonprogressors at day . improvement in physical function and radiographic outcomes were consistent between the two treatment groups in both remission and lda populations (. tab. ). conclusion. through year, patients treated with subcutaneous abatacept or adalimumab in ample achieved comparable rates of remission and lda. similar improvements in physical function and radiographic outcomes were observed. these data help to illustrate the relationship between remission, lda and functional and radiographic outcomes independent of treatment with subcutaneous abatacept or adalimumab. background. previous small studies suggest that responses to some immunizations may be attenuated by intravenous abatacept but remain clinically meaningful [ , ] . we investigated the magnitude of response to pneumococcal and influenza vaccination in a larger number of patients receiving subcutaneous (sc) abatacept therapy. the objective of the study was to evaluate the antibody response to the standard -valent pneumococcal polysaccharide vaccine and the - seasonal influenza trivalent vaccine in adult patients with ra on sc abatacept and background dmards. these multicentre, open-label sub-studies of the -valent pneumococcal polysaccharide vaccine and seasonal influenza vaccine enrolled patients in the acquire (pneumococcal and influenza) or attune (pneumococcal) studies. patients were enrolled at any point during their sc abatacept treatment cycle after completion of ≥ months' abatacept treatment. all patients received fixed-dose sc abatacept mg/week with background dmards. a pre-vaccination blood sample was collected and vaccines administered, while continuing background sc abatacept and dmards. after ± days, a post-vacci- background. in real life, dosage increases are common with biologic agents [ ] . intravenous abatacept is administered by patient body weight ( mg/kg) and weeks after the first infusion and every weeks the-reafter [ ] totalling infusions over the first months. no adjustments to this schedule are recommended. abatacept retention rates, efficacy and safety over months in action (abatacept in routine clinical practice) have been reported previously [ , ] this study was designed to assess adherence to recommended dosing of abatacept over the first months in action. methods. action is an ongoing, -year, international, non-interventional, prospective cohort of ra patients treated with intravenous abatacept. all patients on abatacept treatment for ≥ months, and with infusion data available at initiation and at months, were considered in this analysis. good adherence was defined as correct dose by patient body weight and number of actual-to-recommended infusions within the range - % (i.e. - infusions). results. in total, / ( . %) patients received abatacept ≥ months and had the infusion data available. most had established ra and failed ≥ anti-tnf agent ( . %). of patients with body weight data available at initiation, . % received the recommended initial dose, . % a lower dose and . % a higher dose than recommended. good adherence to the abatacept treatment schedule was found in / ( . %) patients. over months, . % of patients received infusions, . % received infusions and . % had infusions. change in dosage over time was assessed in / patients with data available at both time points. the majority of patients ( . %) maintained the recommended dosage. / ( . %) patients received abatacept at the recommended dose for body weight and at the recommended treatment schedule over months. conclusion. in the real-world action study, adherence to the recommended abatacept treatment regimen over months was good. few patients received changes in dose and/or frequency of administration over this time period. background. in rheumatoid arthritis (ra), synovial fibroblasts (sf) secrete large amounts of il- , il- and matrix metalloproteinases (mmps) which are crucial for cartilage destruction. rasfs are sensitive to the action of cannabinoids and they express cannabinoid receptors type i and ii (cb and cb ), the vanilloid receptor (trpv ) as well as endocannabinoid degrading enzymes. cannabinoids are regarded as antiinflammatory and since anandamide (aea) is found in ra synovial fluid we investigated how this endocannabinoid affects adhesion, proliferation, and production of inflammatory mediators of rasf. methods. adhesion was assessed by the xcelligence system. proliferation was quantified by the amount of incorporated fluorescent dye into cellular dna. mmp- and cytokines were detected by elisa. in oasf, aea dose-dependently decreased the il- β induced production of mmp- (by %) in a trpv -mediated manner. il- and il- levels were only weakly modulated. in rasf however, aea decreased il- β induced production of il- ( %), il- ( %) and mmp- ( %). the effects of aea were not inhibited by cb , cb or gpr antagonists but were blocked by the trpv antagonist capsazepine. the inhibitory capacity of aea was enhanced by cyclooxygenase- inhibition in rasfs and oasfs, but was unaltered or even slightly reduced by faah inhibition. aea was even more potent in reducing above mentioned mediators when rasfs but not oasfs were incubated under hypoxic conditions and treated with tnf. furthermore aea increased adhesion of oasfs and rasfs to fibronectin. adhesion was modulated by cb , gpr , and trpv antagonists. combined faah and cyclooxygenase- blocked the stimulatory effect of aea on adhesion. proliferation was decreased by aea in rasfs and oasfs via a cyclooxygenase- but not via cb , cb or trpv dependent mechanism. conclusion. in conclusion, aea promotes an antiinflammatory phenotype of rasfs and oasfs by activating trpv . cb , trpv , and gpr act in concert to modulate adhesion of sfs and this is highly dependent on the intracellular concentration of aea. additionally, cyclooxygenase- metabolites of aea exert their anti-proliferative effects independent of cb and cb . fc. it has been reported that lower levels of czp, compared to ada or ifx, are transferred from treated mothers to the neonate [ ] . this discrepancy may be due to active transport of antibodies across the placenta thought to be mediated by the neonatal fc receptor (fcrn). however, anti-tnf binding to fcrn, and fcrn-mediated transcytosis have not been studied. the objective of this study is to quantify binding of czp, ifx, ada and eta to fcrn and to measure fcrn-mediated transcytosis. a biacore™ assay was used to determine binding of czp, ada and ifx to human fcrn. anti-tnfs were passed over an fcrn-coated chip .mdck-ii cells transfected with human fcrn were used to measure fcrn mediated transcytosis. the anti-tnfs and the control antibody (p ), which possessed a fc modified to prevent binding to fcrn, were biotinylated to allow visualization. the amount of each anti-tnf transcytosed across the cell layer over hours was measured by msd assay. results. ifx ( nm) and ada ( nm) had high binding affinity to fcrn while the binding affinity of eta to fcrn was - -fold lower ( nm). in contrast, czp did not bind to the fcrn with any measurable affinity. the levels of transcytosis seen with ifx and ada were . ng/ml and . ng/ml, respectively (mean of experiments). transcytosis of eta ( . ng/ml) was lower than that of ada and ifx. in contrast, the level of czp transcytosis was significantly lower, at . ng/ml, than that observed with the other anti-tnfs and comparable to the control p ( . ng/ml). conclusion. czp didn't bind to fcrn and thus no fcrn-mediated czp transcytosis was detected. in contrast, ada and ifx had a relatively high binding affinity to fcrn and were actively transcytosed. eta showed lower binding affinity and transcytosis, but fcrn-mediated transport could still be measured. these results explain the previously observed active transport of anti-tnfs across the placenta seen in patients treated with ifx and ada, whereas only low levels were observed with czp [ ]. background. anti-cyclic citrullinated peptide (ccp) status was reported previously as predictive of abatacept response [ ] . predictors of retention with abatacept have not been published previously. this study was designed to identify predictors of abatacept retention after failing ≥ biologic agent. in routine clinical practice) is an ongoing, -year, international, non-interventional, prospective cohort including patients with ra treated with intravenous abatacept [ , ] . patients from canada, germany, greece and italy, where patient numbers were sufficient to explore between-country effects, were included. at data cut-off (february ), all patients had -year follow-up (interim analysis). abatacept discontinuations were reported by the investigator at any time point during follow-up. socio-demographics, disease characteristics and medical history at abatacept initiation, and previous and concomitant treatments were deemed potential predictive variables. clinically relevant variables and those with p≤ . (univariate analysis) were entered into a multivariate cox proportional-hazards regression model, adjusted for clustered data from one investigator. using backwards selection, variables with p≤ . were retained in the final model. . there were no interactions or effects of c-reactive protein level, rheumatoid factor status, type of previous anti-tnf failure, infection at initiation and abatacept monotherapy. sensitivity analysis, including all variables significant in univariate analysis, was consistent. conclusion. in this first report of real-world predictors of abatacept patient retention, anti-ccp positivity and failing < prior anti-tnf agents were associated with higher retention. differences in retention between some countries may reflect specificities in healthcare systems and populations. abatacept, a biologic agent with no contraindications or special warnings for cardiac comorbidity, seems to be a good option for these patients. weekly subcutaneous abatacept confers comparable onset of treatment response and magnitude of efficacy improvement over months when administered with or without an intravenous abatacept loading dose zeitschrift für rheumatologie suppl · | methods. patients from the intent-to-treat populations of the acqui-re [ ] and ample [ ] studies randomized to subcutaneous abatacept plus mtx were included. all patients received fixed-dose subcutaneous abatacept mg/week; in acquire but not ample, patients also received an intravenous loading dose (~ mg/kg based on weight range) on day . for this post-hoc analysis, assessments included acr and haq-di response (improvement ≥ . ) over months, with patients who discontinued considered non-responders. mean changes from baseline over months in das (crp) were assessed in patients with das > . at baseline (last observation carried forward) to account for differences in baseline disease activity between the two studies. results. all patients were biologic naïve at baseline, with mean disease duration of . and . years, das (crp) . and . , and haq-di . and . in acquire and ample, respectively. efficacy was compared throughout the study. for patients treated with subcutaneous abatacept with and without an intravenous loading dose, acr response rates were similar (. tab. ). haq-di response rates were also similar with and without the intravenous loading dose (. tab. ). for the overall populations, mean (standard deviation [sd]) changes from baseline to day in das were − . ( . ) and − . ( . ) in acquire and ample, respectively. for patients with baseline das > . , mean (sd) changes in das from baseline to day were − . ( . ) and − . ( . ) in acquire and ample, respectively. conclusion. time to onset and magnitude of acr and haq-di responses and das improvements were generally similar with subcutaneous abatacept with or without intravenous loading in patients with ra and an inadequate response to mtx. the findings from this posthoc analysis suggest that subcutaneous abatacept can be given effectively without an intravenous abatacept loading dose. background. ra is associated with pain and impairment of physical function, significantly impacting a patient's health-related quality of life (hrqol) and ability to perform daily activities. patient-reported outcomes (pros) related to hrqol and daily activity have become an essential part of assessment in ra. we continue to report here comparative findings from pros assessed with subcutaneous abatacept or adalimumab on background mtx in the first head-to-head study, ample. we compared changes in pros at year in patients with ra treated with abatacept or adalimumab, both on background mtx. methods. ample is a phase iiib, randomized, investigator-blinded study of months' duration. biologic-naïve patients with active ra and inadequate response to mtx were randomized to either mg abatacept weekly or mg adalimumab biweekly in combination with mtx. pros evaluated through day included: hrqol, assessed using short form- (sf- ; including physical and mental component summary subscores [pcs and mcs]); activity limitation over the previous days, using the activity limitation questionnaire (alq; [ ] ); productivity, using the work productivity and activity impairment questionnaire for ra [ ] ; physical and psychosocial independence, captured using items from haq, sf- score; and alq [ ] . other pros previously reported from ample include: patient pain, patient global assessment, fatigue, and physical function [ ] . all efficacy analyses were done using the intent-to-treat population, which included all patients who were randomized and received at least one dose of study drug. baseline characteristics were analysed descriptively and changes in pros from baseline were assessed using ancova. results. baseline demographic and clinical characteristics of the abatacept and adalimumab treatment arms were similar. improvements in all domains of the sf- , including pcs and mcs observed at day parameter baseline woche woche itt-gesamt das , mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) vas da pat., mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) sjc , mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) vas schmerz, mw ± sd zielsetzung der ole-studie beinhaltete die beurteilung der verträglichkeit und der wirksamkeit von czp. die retentionsraten sowie die wirksamkeit wurden bis woche und die verträglichkeitsdaten bis woche beobachtet. in die verträglichkeitsanalyse wurden alle pat einbezogen, die in die ole-studie eintraten und czp erhielten (n= ; n= kombitherapie; n= monotherapie), einschließlich der plazebo/czp-patienten, die die ausgangsstudien erfolgreich abgeschlossen/abgebrochen haben. bezüglich der wirksamkeit wurden folgende analysen vorgenommen: ) czp pat, die die ausgangsstudien erfolgreich beendet haben und zu irgendeinem zeitpunkt während der ausgangsstudien oder ole-studie andere dmards eingenommen haben (n= ; kombitherapie completer); ) czp pat, die die fast ward studie erfolgreich beendet haben und zu keinem zeitpunkt andere dmards eingenommen haben (n= ; monotherapie completer). ergebnisse. verteilung und häufigkeit der unerwünschten ereignisse (ue), einschließlich der reaktionen an der injektionsstelle (ereignisse/ patientenjahre: monotherapie , , kombitherapie , ) und der schwerwiegenden unerwünschten ereignisse (sue) waren mit dem vergleichbar, was bisher für czp berichtet wurde (. tab. ) . das auftreten von schwerwiegenden infektionen (si) und malignitätsraten war niedrig. es wurden todesfälle berichtet: kardiovaskuläre ereignis-se, infektionen, unfall und tumorerkrankung. die retentionsraten der pat, die die ausgangsstudien erfolgreich beendet haben, waren zur w in der czp monotherapie-( / , %) und der czp kombitherapie-gruppe ( / ; %) vergleichbar. der durchschnittliche das - (crp)-wert und dessen abweichung vom baseline-wert der ausgangsstudien zum zeitpunkt des eintrittes in die ole-studie und nach w der monotherapie-completer und der kombitherapie-completer, sowie die zugehörigen haq-werte sind in . tab. dargestellt. schlussfolgerung. vorliegende ole-studie konnte das günstige risiko-nutzen-profil der czp-monotherapie bestätigen. die langzeitwirksamkeitsdaten zeigten keine unterschiede zwischen pat, die czp als monotherapie erhielten und pat, die czp in kombination mit anderen dmards erhielten. background. rheumatoid arthritis (ra) is the most common disease of joints that non-or deficiently treated leads to functional loss and premature cardiovascular death within years. but nearly % of the ra patients fail to treatment with tnfα-inhibitor (tnfi) indicating a switch to rituximab (rtx). the urgency of personalized promising treatment in time presupposes predictive parameter. rheumatoid factor (rf) and anti-citrullinated protein antibodies (acpas; especially accp) are shown to be better diagnostic than less theranostic biomarkers. in that context we investigated the role of antibody subtypes against mutated citrullinated vimentin (amcv) that determine response outcome in rtx-treatment. methods. a cohort of only amcv igg positive ra patients was tested for amcv subtype igm and iga (additionally for rf igg, igm, iga and accp igg) by elisa at baseline (after failure to first approach with tnfi) and at week (after first rtx cycle). responders were characterized by a difference in their das of ≥ . (eular good-response) between baseline and week . the cohort comprises responders (rr) and non-responders to rtx (nrr). results. amcv igg, igm and iga showed higher treatment related decreases compared to rf and accp ig subtypes and additionally even diverged in both groups depending on response outcome: especially amcv iga exhibited a higher mean titer decline of rr by % at lo- wer baseline titers ( . to . u/ml) and a mean titer increase of nrr by nearly % at higher baseline titers ( . to . u/ml). at baseline rr displayed relatively more negative iga titers ( %; n= / ) than nrr, who in return showed more iga positive titers ( %; n= / ). amcv iga positive patients were more likely to show positively for rf iga ( %) and igm ( %), what could be inversely detected for iga negative patients with seronegativity of rf iga ( %) and igm ( %). conclusion. amcv immunoglobulin subtypes showed treatment dependent changes contrary to already known antibodies (accp). especially amcv iga reflects response outcome: amcv iga negativity at baseline and decreasing titers during treatment are predictive for good eular-response to rtx. einleitung. im rahmen der abklärung eines unter tocilizumab-therapie aufgetretenen arzneimittelexanthems erfolgte die bestimmung von c c und c -beide parameter waren erniedrigt. bei recht geringer und nur kurz andauernder ausprägung des exanthems wurde die therapie komplikationslos fortgeführt. die komplementfaktoren wurden im verlauf bestimmt und blieben erniedrigt. im weiteren verlauf erfolgte die konsekutive messung bei weiteren patienten. methoden. nephelometrische bestimmung von c c und c im serum vor und während der therapie mit tocilizumab (jeweils vor der nach wochen anstehenden infusion) bei patienten mit gesicherter rheumatoider arthritis (rf+, ccp+). ergebnisse. c c-und c -komplement wurden bei konsekutiven patienten mit rheumatoider arthritis vor und unter tocilizumab-therapie bestimmt. bei allen patienten fielen sowohl c c, als auch c unter der therapie mit tocilizumab ( mg/kg kg) ab. / patienten hatten eine c c-erniedrigung (bestimmter wert unterhalb des laborinternen normbereichs). / patienten hatten eine c -erniedrigung (bestimmter wert unterhalb des laborinternen normbereichs). drei patientinnen entwickelten unter der therapie ein exanthem, davon hatten eine komplementerniedrigung. keine "offensichtlich" erhöhte infektneigung in abhängigkeit von komplementspiegeln. bei verlängerten infusionsintervallen aufgrund von infekten zeigte sich, dass der effekt von tocilizumab auf die komplementspiegel reversibel ist. durch blockade des il- -rezeptors tocilizumab kann ein erworbener komplementmangel induziert werden. Ähnliche daten wurden im rahmen einer pilotstudie an sle-patienten erhoben, die mit tocilizumab behandelt wurden. der effekt ist bei der rheumatoiden arthritis nicht vorbeschrieben. der genaue umfang des komplementmangels ist bisher nicht untersucht (andere bestandteile der kaskade?) wurde in der sle-studie ausführlicher untersucht. da die verschiedenen komplementbestandteile erniedrigt waren wurde auf eine synthesestörung und nicht auf einen gesteigerten verbrauch geschlossen, was auch in dieser kohorte der fall zu sein scheint. der erworbene komplementmangel könnte einen teil der infektiösen komplikationen unter der therapie erklären. eine korrelation ist aber aufgrund der geringen fallzahl nicht möglich. schlussfolgerung. anhand dieser studie konnte eine exzellente korrelation zwischen den parametern der dxr und des bx verifiziert werden. mittels der neu entwickelten voll digitalisierten bx-technik ist somit eine quantifizierung der periartikulären demineralisation möglich und als surrogatparameter der radiologischen progression bei einer ra eingesetzt werden. jahre, die ra bestand im median seit , jahren. , % der patienten waren mit tnf-alpha-blockern vortherapiert, , % ausschließlich mit dmards. der mittlere das lag zur baseline bei , . zur woche zeigten , % der patienten eine das remission (< , ) und , % bzw. , % der patienten ein gutes bzw. moderates ansprechen gemäß eular-kriterien. Über den beobachtungszeitraum stieg der anteil der tcz-monotherapiepatienten von , % auf , %. die mtx-komedikation sank im gleichen zeitraum um , %. , % der patienten, die tcz zunächst zusammen mit einem dmard erhalten haben, konnten dieses absetzen. tcz zeigte in der mono-und kombinationstherapie eine vergleichbare wirksamkeit: , % bzw. , % der patienten erreichten eine cdai remission (≤ , ). der anteil von patienten ohne glucocorticoid(gc)-begleittherapie stieg über den beobachtungszeitraum um , % auf , % an, der anteil mit einer tagesdosis ≤ mg auf , %. bei , % war eine reduktion der gc-dosis möglich, nur bei , % war eine erhöhung notwendig. bei , % der patienten, die zur bl mit gc behandelt wurden, konnten diese komplett abgesetzt werden. die mittlere gc-tagesdosis verringerte sich kontinuierlich von , (bl) auf , mg/d (w ). schlussfolgerung. diese interimsanalyse der nichtinterventionellen studie ichiban zeigt bei den ersten patienten mit mittelschwerer bis schwerer ra über die bisherige beobachtungsdauer von wochen deutliche verbesserungen der aktivitätsparameter, sowie eine reduktionen der begleitenden dmard-therapien und des bedarfs von glucocorticoiden unter behandlung mit tcz. vergleichbar mit den kontrollierten studien ist die tcz-monotherapie auch unter praxisbedingungen der kombination mit dmards ebenbürtig. diese anhaltende wirksamkeit wird erstmals in rheumatologischen praxisdaten für den langzeitverlauf von , jahren gezeigt. zeitschrift für rheumatologie suppl · | einleitung. die arteriosklerose (as) steht als häufigste todesursache im besonderen fokus der medizinischen forschung. neuere erkenntnisse weisen auf einen starken zusammenhang zwischen parametern der systemischen entzündung und der pathogenese der as hin. patienten mit rheumatoider arthritis (ra) haben daher ein stark erhöhtes kardiovaskuläres risiko. ziel: untersuchung des zusammenhangs zwischen verschiedenen ra-krankheitsspezifischen risikofaktoren und dem auftreten einer arteriosklerose bei ra-patienten. methoden. ra-patienten, davon % weiblich, ± , jahre alt, wurden hinsichtlich der krankheitsaktivität (krankheitsdauer , ± , ; das , ± , ; serum-crp , ± , mg/dl,; anti-ccp-antikörper , ± , u/ml, radiologisches stadium , ± , ; davon , % mit erosionen), sowie klassischer kardiovaskulärer risikofaktoren der as erfasst, welche durch den score-wert (systematic coronary risk evaluation) zusammengefasst wurden. zur as darstellung wurde eine carotis-duplexsonographie mittels eines mhz-schallkopfes (ge vivid pro) durchgeführt. die mittlere intima-media-dicke (imd) der a. carotis communis wurde durch ein softwaregestütztes messverfahren ermittelt. ergebnisse. plaques waren bei patienten ( %) nachweisbar. diese korrelierten mit einer erosiven form der ra (p= , ), einer längeren krankheitsdauer (p= , ) und höheren anti-ccp-antikörpern (p= , ). die mittlere imd betrug , ± , mm. je ausgeprägter die radiologischen veränderungen sind, umso höher war die wahrscheinlichkeit der plagues (p= , ). mittels altersadjustierter partieller korrelationsanalyse wurde der das als altersunabhängiger einflussfaktor auf die imd ermittelt (p= , ). mittel-und hochgradige stenosen zeigten sich bei fünf ra-patienten ( , %), welche ausnahmslos eine erosive verlaufsform aufwiesen. normalbefunde stehen in zusammenhang mit einem crp-wert unter mg/dl (p= , ). auch die traditionellen kardiovaskulären risikofaktoren haben signifikanten einfluss auf as. der score-wert erwies sich als äußerst verlässlicher prädiktor für plaques (p< , ), imd-verdickung (p= , ) und stenosen (p< , ). durch elimination der traditionellen risikofaktoren mittels partieller score-adjustierter korrelationsanalyse bestätigte sich erneut die assoziation von pathologischen ultraschallbefunden mit dem das (p= , ). schlussfolgerung. die erhebung klassischer risikofaktoren bei ra-patienten ist unerlässlich. die nutzung des score-werts als screening-parameter ist besonders effektiv. zusätzlich sollten parameter der krankheitsaktivität von ra zum management von arteriosklerose herangezogen werden. besonders aussagekräftig hierfür sind der das , ein erosiver krankheitsverlauf, die crp-werte und die erkrankungsdauer background. apremilast, an oral small molecule specific inhibitor of phosphodiesterase- , works intracellularly to modulate inflammatory mediators. the palace - trials compared the efficacy and safety of apremilast vs placebo in patients with active psa despite prior dmards and/or biologics. the overall safety and tolerability of apremilast was assessed in a pooled analysis of the palace , and placebo-controlled phases. methods. safety data was pooled from phase , randomized, placebo-controlled studies; patients with active psa despite prior dmards and/or biologics were randomized : : to placebo, apremilast mg bid (apr ), or apremilast mg bid (apr ) stratified by baseline dmard use. at week , patients with < % reduction in swollen and tender joint counts were required to be re-randomized (early escape) to apr or apr (placebo group) or remained on initial apremilast dose through week . stable concurrent dmard therapy was allowed (mtx, sulfasalazine, leflunomide, or combination). the analysis comprises data from the placebo-controlled periods (weeks - ). results. patients were randomized to placebo (n= ), apr (n= ), or apr (n= ) and included in the safety population. baseline demographic and disease characteristics and prior/concurrent therapy were comparable across treatment groups; . % had prior biologic exposure. adverse events (aes) occurred in . % (placebo), . % (apr ), and . % (apr ) of patients. aes occurring in ≥ % of any treatment group were diarrhea, nausea, headache, and urti (. tab. ); most occurred within the first weeks of treatment and nearly half resolved within weeks. of patients with these aes, most ( - %) were mild or moderate. rates of discontinuation due to aes were low: . % (placebo), . % (apr ), and . % (apr ). serious aes occurred in . % (placebo), . % (apr ), and . % (apr ) of patients. one death occurred (apr ) due to multiorgan failure not suspected to be treatment-related. no cases of serious systemic opportunistic infections, lymphoma, vasculitis, or reactivation/de novo tb were reported. there were no clinically meaningful differences between apremilast and placebo in terms of major cardiovascular aes, changes in blood pressure, malignancies, or effects on laboratory measurements. conclusion. apremilast was generally well-tolerated with no new safety concerns identified compared with the known profile. the aim of the current study was to investigate the relationship between worsening of functional status, clinical disease parameters and radiographic spinal progression over two years in patients with early axial spondyloarthritis (axspa). methods. in total, patients with early axspa ( with as and symptom duration ≤ years, and with non-radiographic axspa (nr-ax-spa) and symptom duration ≤ years) from the german spondyloarthritis inception cohort (gespic) were included in the current analysis based on the availability of radiographic data and data on the functional status at baseline and after years of follow-up. spinal radiographs were scored according to the modified stoke ankylosing spondylitis spinal score (msasss). functional status was assessed by the bath ankylosing spondylitis functional index (basfi), and clinical disease activity by the bath ankylosing spondylitis disease activity index (basdai). results. basfi worsening in ≥ point after years (n= , . %) was significantly associated only with higher basdai worsening over years in comparison to those without functional worsening: . ± . vs - . ± . , p< . . basfi worsening by ≥ points (n= , %) was, however, associated not only with basdai change ( . ± . vs - . ± . , p< . ), but also with a higher rate of radiographic spinal progression measured by the proportion of patients with msasss worsening by ≥ units ( . % vs. . % in patients without basfi worsening, p= . ), or with new syndesmophyte formation ( . % vs. . %, p= . ). importantly, in the multivariate analysis both basdai increase and progression of structural damage in the spine remained statistically significantly associated with basfi worsening. no other disease-related parameters (e.g. sex, hla-b positivity, symptom duration etc) were found to be significantly associated with basfi worsening over two years. conclusion. in this prospective study we could demonstrate that only factors were significantly associated with worse functional outcome over two years in patients with early axspa: ) increase of disease activity and ) progression of structural damage. elevated serum vascular endothelial growth factor is highly predictive for radiographic spinal progression in patients with axial spondyloarthritis who are at high risk for progression background. vascular endothelial growth factor (vegf) is an essential mediator of the endochondral ossification and, therefore, might play a pathogenetic role in the process of syndesmophyte formation in axial spondyloarthritis (axspa). the aim of the study was to investigate the role of serum vegf as a predictor of radiographic spinal progression in patients with axspa. methods. altogether patients with definite axspa [ with ankylosing spondylitis (as) and with non-radiographic axspa] from the german spondyloarthritis inception cohort (gespic) were included in the current study. radiographic spinal progression was defined as ) worsening of the modified stoke ankylosing spondylitis spine score (msasss) by ≥ units after years, and ) development of a new syndesmophyte or progression of existing syndesmophytes after years. serum vegf levels were detected at baseline. results. mean baseline vegf values were significantly higher in patients with msasss worsening by ≥ units after years (n= ) as compared to those without progression ( ± vs. ± pg/ml, respectively, p= . ) and in patients with syndesmophyte formation/ progression (n= ) as compared again to those without progression ( ± vs. ± pg/ml, respectively, p= . ). area under the curve (auc) was . , p= . for the msasss worsening ≥ units and . , p= . for syndesmophyte formation/progression. importantly, the performance of vegf as a predictor of radiographic spinal progression was clearly in patients who were already at high risk for such a progression due to the presence of syndesmophytes at baseline (n= ): auc was . , p= . , and . , p= . , respectively. vegf serum level of > pg/ml in high-risk patients had a sensitivity of %, a specificity of %, and an odds ratio (or)= . ( %ci . - . ) as a predictor of msasss worsening by ≥ units over years. the same serum level of results. immediately after the second session of plasmapheresis, therapy with infliximab mg/kg resumed. after weeks of hospitalization with repeated administration of infliximab had good dynamics (bas-dai . , asdas (crp) = . , basfi . ), significantly reduced pain in the joints and spine, stiffness, increased mobility in the joints and spine. the treatment continued: holding plasmapheresis followed by infliximab. after infusions patient experienced a good effect -basdai . , asdas (crp)= . , basfi . . conclusion. plasmapheresis in some patients could be effective by reducing activity and dealing with secondary tnf inhibitors failure, since this procedure deletes the macromolecular blood proteins, including tnf-, igg antibodies, and circulating immune complexes results. there were still signs of osteitis in sacroiliac joints in patients at week , in patient the mri-determined sacroiliitis has resolved completely. the patient has improved clinically and fulfilled asas improvement criteria. there was a minor decrease in sparcc sacroiliitis score (from to ) in patients at week , indicating reduction of inflammation in sacroiliac joints. sparcc sacroiliitis score stayed the same in the remained patient. conclusion. rituximab may be of some benefit in decreasing mri-evident sacroiliitis in patients with highly active as, even in patients in whom tnf-α inhibitors have failed. background. despite the differences in the pathogenesis of ra and as, neck pain is a frequent clinical symptom in both diseases. we evaluated the correlation between subjective reports of neck pain and objective signs of inflammation as assessed by f bone marrow edema (bme) on mri in ra and as patients. methods. stir-mri of the cervical spine of patients ( ra, as) were included. mris were scored by two blinded readers using a recently published mri scoring system, with quantification of the extension of bme in the atlantoaxial region, corpus, facet joints and processus spinosus of all cervical vertebrae, ranging from - points. the presence or absence of degenerative changes was also recorded. conclusion. the majority of patients with ra and as had objective signs of bme but also degenerative changes on mri at different cervical locations. assessment of bme in the atlantoaxial region is important in clinical practice, in addition to degenerative changes, since its presence seems to influence the intensity of neck pain reported by these patients. x. baraliakos , having mri data at w . of these , ten were treated with secukinumab and with placebo in the core study. mris were rescored for this study. asspimri-a scores and the occurrence of vertebral edges (ve) inflammatory and fatty lesions were evaluated by an independent blinded reader. results. all pts completed this exploratory mri substudy. in pts receiving × mg/kg secukinumab followed by × mg/kg (n= ) secukinumab, spinal inflammation was reduced compared to bl at w -similar to the results of the core study -and this reduction was sustained up to w (abb. ). also in pts who had initially received placebo switching to secukinumab at w , mri inflammation at w was reduced. of the ves evaluated, the proportion of ves with inflammatory lesions was reduced from . % (n= ) at bl to . % (n= ) at w and . % (n= ) at w . in contrast, the proportion of fatty lesions at bl ( . %, n= ) remained largely unchanged at w ( . %, n= ) and w ( . %, n= ). secukinumab reduced mri inflammation at w and w . conclusion. mri analysis suggests that the il- a inhibitor secukinumab can reduce spinal inflammation and this effect may be sustained for up to years. unlike reports with tnf blockers, secukinumab appeared to leave the proportion of fatty lesions unchanged. the potential impact of these preliminary findings on radiographic progression under secukinumab therapy will be studied in larger trials. schlussfolgerung. es zeigen sich keine signifikanten unterschiede in der krankheitsaktivität der beiden gruppen (vor einleitung der ada-therapie nach dmard-und nach anti-tnf-versagen). auch bei der patientengruppe mit mehreren vortherapien mit tnf-inhibitoren können keine signifikanten unterschiede in der ausprägung der erkrankung nachgewiesen werden. im trend wurde ein früheres einsetzen der haut-und gelenkmanifestation sowie eine stärkere systemische entzündungsreaktion in den patienten mit vorheriger tnf-therapie festgestellt werden, während die dauer der erkrankung und der bmi mit den charakteristika der patienten mit ausschließlicher dmard-vortherapie vergleichbar sind. long-term ( -week) results of a phase , randomized, controlled trial of apremilast, an oral phosphodiesterase inhibitor, in patients with psoriatic arthritis (palace ) background. apremilast, an oral phosphodiesterase inhibitor, works intracellulary to modulate a network of pro-and anti-inflammatory mediators. the palace study assessed the efficacy and safety of apremilast in patients with active psoriatic arthritis (psa) despite prior dmards and/or biologics. methods. patients were randomized : : to placebo, apremilast mg bid (apr ), or apremilast mg bid (apr ). at week , patients with < % reduction from baseline in swollen/tender joint counts were required to be re-randomized (early escape) to apr or apr (placebo group), or remained on their initial apremilast dose. at week , all remaining placebo patients were re-randomized to apr or apr through week . results. patients were randomized. at week , significantly more apr ( . %; p= . ) and apr patients ( . %; p< . ) achieved an acr vs placebo ( . %). at week , all patients had a minimum weeks of apremilast exposure. response to apremilast was generally maintained over the treatment period. at week , acr was achieved by . % (apr ) and . % (apr ) of patients (table) . exposure-adjusted incidence rates for adverse events (aes), severe aes, and serious aes were comparable between - and - weeks. the proportion of patients remaining on apremilast to week who first reported the most common gi disturbances (e.g., diarrhea, nausea, and vomiting) after week was low (ranging from . - % for apr and - . % for apr ). there were no clinically meaningful laboratory findings with exposure up to weeks. no deaths beyond the previously reported in the - week period were observed in the - week period. no safety signals with respect to major cardiac events, malignancies, and opportunistic infections were observed, consistent with the - week period. no cases of lymphoma, tuberculosis, or tuberculosis reactivations were reported for the -week period (. tab. ). conclusion. apremilast administered to patients with psa beyond weeks continued to demonstrate meaningful clinical response. for patients who completed weeks of the study, acr response rates up to % were observed. apremilast continued to be well tolerated with an acceptable longer-term safety profile. methods. to identify how conventional cd + and cd + t cells and regulatory t cells are recruited into the inflamed kidneys in ln, serum and urine samples of sle patients were analyzed for chemokines using multiplex assays. based on the assay's results a group of corresponding chemokine receptors (ccr - , cxcr and cxcr ) was chosen, whose frequencies on urinary t cells were subsequently determined in patients with acute ln by flowcytometry. results. chemokines (ccl , ccl , ccl , ccl , ccl , ccl , ccl , ccl , cxcl , cxcl , cxcl and cx cl ) were significantly elevated in the urine of patients with active ln when compared to the control group. the other chemokines (ccl , ccl , ccl , cxcl , cxcl ) and cxcl showed no significant differences between the groups. ccr and cxcr were the most prominent receptors on both urinary cd + and cd + t cells, although cd + t cells also expressed high amounts of ccr and ccr . however, when compared to t cells in the blood, urinary cd + t cells showed significantly higher expression of all examined chemokine receptors but ccr while urinary cd + t cells only had higher expression of ccr and ccr . the chemokine receptor expression on cd +foxp +cd -regulatory t cells (treg) differed from conventional cd + t cells as well. treg expressed significantly more ccr and significantly less cxcr . conclusion. ccr and cxcr are the primary receptors in the mechanism of recruiting t cells into the inflamed kidney. key chemokines are ccl , ccl , ccl and ccl as well as cxcl and cxcl . however, at least for cd + t cells, there are secondary pathways of recruitment involving ccr /ccl and ccr /ccl . also, treg recruitment seems to rely more on ccr than that of conventional cd + t cells. methods. observe is a multicenter, retrospective medical chart review study. rheumatologists from german academic and non academic centers who treat > sle patients annually and have > years of practice experience were randomly recruited. physicians identified consecutively all their adult sle patients who had received belimumab as part of usual-care. index date was the first belimumab infusion date. the primary outcome was the change in overall sle disease manifestations months after index date based on physician judgment. the overall response rates as well as reasons for early treatment discontinuation within months were assessed. changes in formal disease area indices, e.g. selena-sledai if available and changes in oral steroid dose are also reported. results. previous analyses from us patients treated with belimumab have described significant clinical improvement across relevant organ systems based on clinical judgment and formal disease activity indices and marked reductions in corticosteroid use in patients that received at least infusions of belimumab. the current study is the first description of patient characteristics and outcomes after months of therapy with belimumab outside of the us. it is also the first time overall responder rates and reasons for discontinuation with belimumab have been described in a real world setting. the study provides insights into the effectiveness and safety of belimumab in an ex-us clinical setting. larger, prospective observational studies are needed to confirm the results. commercial support grant disclosure: research funded glaxosmithkline. background. toll-like receptor (tlr- ) signaling is considered to play an important role in b cell hyperreactivity in sle. b cells from slepatients express significantly more tlr- than those from healthy donors (hd), especially if patients have positive dsdna-antibodies and high disease activity. tlr- stimulation of b cells is tightly linked to their differentiation into plasma blasts and memory cells. the objective of this study was to analyze in a comprehensive manner the effect of tlr- signaling on cytokine production by b cells from sle-patients, in comparison to b cells from hd, and in relation with disease activity. methods. b cells from sle-patients and hd were stimulated in vitro using cpg for hours, and culture supernatants were then tested for cytokines and chemokines (bio-plex). the cytokine responses were compared between both groups. in addition, within sle patients, the patterns of cytokines produced by b cells were compared with indices of disease activity. results. cpg-stimulation significantly increased cytokine production ( out of parameters; p< . ) compared to baseline. striking increases were found for il- ra ( ± pg/ml), il- ( ± pg/ml), il ( ± pg/ml) and ip- ( ± pg/ml; p< . ). there was no significant difference between both groups. remarkably, production of il- , il- , il- , il- p , il , il- , il- a, eotaxin, basic fgf, g-csf, gm-csf, ifn-γ, ip- , mip- α, and vegf correlated inversely with the sledai (p< . ) and even more (additionally il- β, il- ra, mip- β and tnf-α) with anti-dsdna antibody titers. the frequency of cd + memory b cells showed a positive correlation between the production of ip- and tnf-α in sle, whereas the levels of il- β, il- , mip- α, and mip- β showed a positive correlation with cd + b cells in hd. conclusion. the current data indicate hitherto unknown perturbations of cytokine/chemokine production by b cells in active sle. the inverse correlation of cytokines/chemokines produced by b cells from sle patients with sledai and anti-dsdna titer suggests that the known enhanced b cell proliferation and differentiation upon tlr -stimulation possibly diminishes cytokine production. background. several cytokines, including ifn-γ, il- , il- , and il- have been implicated in the pathophysiology of autoimmune disease. il- , a potent inducer of ifn-γ, enhances th responses that are thought to be synergistic and dependent on il- . we tested the hypothesis that intra-renal il- mediates kidney and systemic disease in mrl-faslpr mice. methods. by constructing il- p /il- -/-mrl-faslpr mice and using an ex-vivo gene transfer to deliver il- intra-renally, we determined that il- , independent of il- and/or il- , incites kidney disease in mrl-faslpr mice. moreover, we provide the novel finding that local intra-renal il- mediates systemic disease (lung pathology, systemic auto-abs). results. thus, our data indicate that il- is a potential therapeutic target for immune mediated kidney and systemic disease in mrl-faslpr mice. using a caspase- inhibitor, that inhibits the release of active il- and il- β, we successfully treated kidney (improved renal function, pathology) and systemic disease (skin lesions, lymphadenopathy, and splenomegaly) in mrl-fas lpr mice, while administration of an il- receptor antagonist did not influence disease progression. probing further we found that inhibition of il- activation results in an amelioration of lupusnephritis by a reduction of intra-renal infiltrating leukocytes (macrophages and t cells) and reduced activation of these leukocyte populations. moreover, caspase- inhibition resulted in decreased inf-y and il- production, indicating an altered balance of th and th cell responses in this model. conclusion. taken together, our findings indicate that il- , independent of il- β, il- and/or il- , is the major mediator of kidney and systemic disease mrl-faslpr mice. therefore, caspase- inhibition is a potential therapeutic target for autoimmune disease in the mrl-faslpr mice. background. in the treatment of giant cell arteritis (gca) glucocorticoid-related adverse effects occur frequently, particularly in patients with relapsing disease. a -year-old woman presented with a month history of fever, chills, arthralgias and cephalgias and markedly elevated serum inflammatory markers. whereas further evaluation including ultrasound of the temporal arteries was unremarkable, a positron emission tomography-computed tomography (pet-ct) demonstrated an intense fluorodeoxyglucose uptake of the aorta, the subclavian, carotid and femoral arteries. gca was diagnosed and treatment with high dose prednisone was begun. results. because of disease flares at prednisone dosages below mg/ day and the occurrence of vertebral fractures, cyclophosphamide and methotrexate (mtx) were added as glucocorticoid-sparing agents. as these treatments had to be stopped because of intolerance and mtxpneumonitis, respectively, we started tcz infusions ( mg/kg body weight). the clinical status rapidly improved. after infusions of tcz follow-up pet scan showed resolution of the previously seen uptake and we were able to taper the daily dose of prednisone to mg. treatment was well tolerated. however, the patient developed mild hyperthyroidism with a rapid rise of the initially normal levels of anti-thyroid peroxidase and anti-thyroid antibodies, anti-tsh receptor antibodies remained normal. thyroid function normalized and the antibody-levels fell without further treatment in the following months. in conclusion, this case demonstrates the successful treatment of a patient with relapsing giant cell arteritis with tcz. for the first time, we report the occurrence of a transient autoimmune thyreoiditis possibly induced by tcz. klinik für pädiatrie mit schwerpunkt pneumologie und immunologie, sektion rheumatologie, berlin, vestische kinder-und jugendklinik der universität witten/herdecke deutsches zentrum für kinder-und jugendrheumatologie organización médica de investigación arthr care res ar&t in press sp division of rheumatology diagnosesicherung: a + b) mr-morphologisch myositistypische veränderungen (os) histologie + c) generalisierte myalgien und laborchemisch dtl. elevierter ck, sowie positivem nachweis von ana und jo- -ak, pulmonales ct mit diffusen milchglasinfiltraten, in bronchoalveolärer lavage neutrophile alveolitis. ergebnisse. vormedikationen: a) glukocorticoidmonotherapie, mtx-monotherapie, mtx in kombination mit etanercept, cyclophosphamidboli, und zuletzt intravenöse immunglobuline (ivig) in kombination mit mycophenolatmofetil . b) mtx-monotherapie, mtx in kombination mit glukokortikoiden, cyclophosphamidboli, intermittierend intravenöse immunglobuline, cyclophosphamid per os (fau-ci). c) cyclophosphamidboli jeweils gutes ansprechen des ck-wertes auf jeweilige rituximabgaben mit ebenfalls ansprechen des klinischen bildes mit guter regredienz des aus myalgien resultierenden schmerzniveaus. im fall a keine beatmung mehr notwendig. im fall von c) auch gute regredienz subjektiver dyspnoesymptomatik und besserung wichtiger lungenfunktionsparameter, regredienz ctmorphologischer milchglasinfiltrate, im verlauf fehlender nachweis neutrophilie in bal. weitere rituximabgaben bei a, b und c im verlauf zum remissionserhalt nach jeweiligem klinischem befund production of cytokines by b cells in response to tlr stimulation inversely correlates with disease activity in sle-patients berlin zeitschrift für rheumatologie suppl · | das muskuloskeletale system, eines der am häufigsten betroffenen organsysteme bei sle (bei - % der sle-patienten). das ziel dieser analyse war es um diejenigen parameter zu identifizieren, die zu diesem effekt beigetragen hatten, wurde jeder der einzel-parameter zur untersuchung und symptom-erfassung innerhalb des muskuloskeletalen bilag-organsystems analysiert. die post-hoc-analyse umfasste nur patienten, bei denen ein parameter zu studienbeginn als vorhanden gewertet wurde, und jeder parameter erforderte ≥ patienten-beobachtungen pro kohorte um einen vergleich zu erstellen dadurch wurde die zahl der patienten mit einer initialen beteiligung des muskuloskeletalen systems aufgedeckt, die eine in woche auflösung der manifestation aufwiesen auch im selena-sledai-score war die rate der verbesserung bei dem arthritis-parameter in der belimumab-gruppe mit mg/ kg ( , %; n= ) und mg/kg ( , %; n= ) signifikant höher als die daten weisen darauf hin, dass mg/kg belimumab effektiv auf muskuloskeletale organmanifestationen sind akzeptiert als posterbeitrag auf dem eular klinische forschergruppe für rheumatologie (kfr), freiburg i. br., universitätsklinikum ulm, klinik für dermatologie und allergologie die physikalische therapie (pt) ist ein wesentlicher bestandteil der medizinischen versorgung von ssc-patienten patientenregister des dnss erfasst prospektiv, jährlich klinische verlaufsdaten zur organbeteiligung und therapie von patienten mit systemischer sklerodermie. die mittels freitext erfassten angaben zur verordneten pt wurden ausgewertet hivamat n= ( , %) und hylase n= ( , %) anwendung. die anzahl der verfahren, die die patienten zeitgleich erhielten, variierte zwischen mind. und max. . Über % der patienten erhielten anwendungen gleichzeitig. insgesamt wurden therapiearten genannt. , % der patienten mit gelenkkontrakturen zeigten nach einem jahr physikalischer therapie eine signifikante verbesserung der symptomatik (p= , ) gegenüber den patienten die keine physikalische therapie erhielten. nach drei jahren waren es , % der patienten (p= , ). bei den patienten mit muskelschwäche zeigten % der patienten eine signifikante symptomverbesserung (p= , ) dieser studie kann erstmals gezeigt werden, dass pt-symptome wie gelenkkontrakturen und muskelschwäche bei ssc-patienten signifikant verbessern kann. dennoch erhält weniger als die hälfte der ssc-patienten eine physikalische therapie punkten zur kontrolle einer mmf-therapie in der klinischen praxis zu untersuchen bei patienten ( -mal sle, je -mal systemische sklerose, sharp-syndrom und primäres sjögren-syndrom) die mmf erhielten, wurde , und min nach einnahme von mmf die mpa-konzentrationen im serum per hplc bestimmt. die mpa-auc wurde durch die mathematische methode der bayes %) und in der standarddosis von g/tag bei von patienten ( %) eine mpa-auc von > µg.h/ml. bei zwei patienten wurde nach der messung die dosis adjustiert: eine patientin mit einem sle mit diffus-proliferativer lupusnephritis hatte trotz einer mmf-dosis von g/tag nur eine mpa-auc von , µg.h/ ml. die dosis wurde daraufhin auf g/tag erhöht. der mpa-auc stieg danach auf max. , µg.h/ml und die krankheitsaktivität nahm ab (sledai von auf , proteinurie von auf mg/ h und prednisondosis von auf mg/tag) pharmakokinetic study of mycophenolate mofetil in patients with systemic lupus erythematosus and design of bayesian estimator using limited sympling strategies mycophenolic acid area under the curve correlates with disease activity in pupus patients treated with mycophenolate mofetil colony stimulating factor- (csf- ) -neuer aktivitätsmarker der lupusnephritis? brigham and wome's hospital, boston, renal division the authors would like to thank pfizer for supporting the study. furthermore the authors would like to thank the "deutsche kinder-rheumastiftung". einleitung. bei patienten mit früher axialer spondyloarthritis (spa) mit einer krankheitsdauer von< jahren und nachweis von akut-entzündlichen veränderungen in der ganzkörper-magnetresonanztomographie (mrt) in der wirbelsäule und/oder den sakroiliakalgelenken (sig) zu baseline [ ] untersuchten wir die langzeit-effektivität über vier jahre. methoden. in der esther-studie wurden patienten mit etanercept (eta, n= ) vs. sulfasalazin (n= ) behandelt [ ] . ab dem zweiten studienjahr wurden alle patienten mit eta behandelt (einige patienten unterbrachen zwischenzeitlich die therapie (n= ) zur untersuchung der biologika-freien remission und wurden dann (erneut) mit eta behandelt) [ ] . klinische, laborchemische und mrt-daten der patienten, die zu den jeweiligen studienzeitpunkten vorhanden waren, wurden im vierten studienjahr analysiert (as-observed-analyse). ergebnisse. von patienten, die zu baseline eingeschlossen wurden, erreichten , % das ende von jahr (n= ). in der gesamtgruppe zeigte sich ein gutes bis sehr gutes ansprechen, wobei etwa % eine asas partielle remission und etwa - % eine asdas inaktive erkrankung erreichten (. tab. ). der anteil der patienten mit normalem crp ("crp-remission") stieg von , % zu screening auf , % zu woche , während der anteil der patienten mit negativem mrt ("mrt-remission" definiert als fehlen akut-entzündlicher veränderungen in den sig und der wirbelsäule gemäß beider scorer) auf von % auf , % anstieg. , % der patienten zu woche waren sowohl in asas-remission, im status einer asdas inaktiven erkrankung als auch in mrt-remission. das ansprechen nach vier jahren war sehr ähnlich in den gruppen unabhängig davon, ob im ersten jahr sulfasalazin gegen wurde oder die therapie im jahr unterbrochen worden war (ergebnisse werden nicht gezeigt).schlussfolgerung. es zeigte sich ein konstantes und anhaltendes ansprechen bei patienten mit früher axialer spa, die mit etanercept behandelt wurden. das ansprechen scheint besser zu als bei patienten mit etablierter ankylosierender spondylitsi mit einer langen krankheitsdauer (> jahren; [ ] ). einleitung. in einer -wöchigen placebokontrollierten studie mit -wöchiger offener verlängerung bei patienten mit aktiver nichtröntgenologischer axialer spondyloarthritis (nr-axspa) wies adalimumab eine gute effektivität auf [ ] . bei patienten, bei denen es zum wiederauftreten der krankheitsaktivität nach absetzen des medikaments in woche kam, wurde die therapie wiederbegonnen ziel der studie war es, die langzeiteffektivität nach wiederaufnahme der therapie von adalimumab nach stopp über jahre zu evaluieren. methoden. bei ursprünglich in die studie eingeschlossenen patienten wurde die therapie nach wochen beendet und patienten ( % männlich, mittleres alter jahre, range - , mittlere krankheitsdauer vor therapiebeginn jahre, range - , % positiv für hla-b ) hatten, definiert durch erreichen eines % ansprechens gemäß der assessments in spondyloarthritis society-kriterien (asas ), gut auf die therapie angesprochen. bei wiederauftreten von krankheitsaktivität (definiert durch nicht mehr erreichen von asas ) wurde adalimumab mg alle wochen über jahre (woche r ) weitergeführt. die asas kriterien und der bath ankylosing spondylitis disease activity index (basdai) wurden in form einer completer-analyse berechnet. ergebnisse. der patienten mussten wiederbehandelt werden: / ( %) erreichten jahr , / ( %) erreichten jahr und / ( %) der patienten erreichten jahr der wiederbehandlung. nach jahren wiederbehandlung mit adalimumab erreichten / ( %) wieder asas und / ( %) erreichten partielle remission gemäß der asas-kriterien. nach jahren erreichten / ( %) und nach jahren / ( %) asas . asas partielle remission wurde nach jahren von / ( %) und nach jahren von / ( %) patienten erreicht. in der completer analyse fiel der mittlere basdai von , ± , zum zeitpunkt der wiederbehandlung auf , ± , im jahr (p< , ), , ± , (p= , ) im jahr und auf , ± , (p= , ) im jahr der wiederbehandlung ab. schlussfolgerung. in dieser gruppe von patienten mit aktiver nr-axspa, die ein gutes therapieansprechen über wochen mit adalimumab erreicht hatten und die bei wiederauftreten von krankheitsaktivität nach stopp der therapie in woche weiterbehandelt werden mussten, sprach die mehrheit der patienten, die in der studie verblieben, gut und anhaltend auf das fortsetzen der therapie an. tab. | sp- langzeit-effektivität über jahre etanercept-therapie bei patienten mit früher axialer spondyloarthritis. daten zu baseline (bl), jahr (w ), jahr (w ), jahr (w ) und jahr (w ). daten background. secukinumab (ain ) is a new fully human monoclonal antibody (mab) targeting il- a for the treatment of inflammatory diseases. administration of mabs can be associated with immunogenicity via the induction of anti-drug antibodies (adas). adas can lead to unwanted clinical consequences, such as loss of exposure, loss of efficacy due to altered pharmacokinetics and/or functional neutralization and, in the worst case, anaphylactic reaction and immune complex diseases. the assessment of ada formation is therefore a critical component in the assessment of biotherapeutic safety. methods. the immunogenicity assessment strategy for secukinumab follows a three-tiered approach. first, samples are analyzed for presence of ada in a screening assay which takes a % false-positive rate into account. in a second step, screening assay positive samples are tested in a confirmatory assay that identifies true positive responses. finally, true immunogenicity-positive samples are quasi-quantified via titration. a biacore-based assay was used during the early stages of the secukinumab program, and an msd-based bridging assay was applied during the later stages of the program. in addition, pharmacokinetics and clinical efficacy as well as safety data are also evaluated. samples to assess immunogenicity were obtained from individual subjects encompassing clinical studies in different indications during treatment and during follow-up. dosing regimens included single doses such as mg subcutaneously in psoriasis patients as well as multiple × mg/kg doses intravenously in ms patients over a six-month period.results. none of the subjects tested for immunogenicity developed sustained adas. in total, subjects met the definition of treatment-related, transient positive immunogenicity showing low ada titers. none of these subjects had evidence of loss of efficacy, deviating pk behavior or reported anaphylactic reaction or immune complex disease.conclusion. based on the available data, secukinumab appears to carry a low risk of immunogenicity. in the very few transient immunogenicitypositive patients identified so far, there has been no indication of altered pharmacokinetics or loss of efficacy, and no adverse event that could be linked to immunogenicity has been detected. more data from the ongoing phase studies are required to strengthen this encouraging finding in a larger patient population. risikofaktoren für eine aa-amyloidose bei entzündlich-rheumatischen erkrankungen und bei der idiopathischen aa-amyloidose methods. we report a case of an -year-old woman suffering from ulceration and signs of infection of the ulnar aspect of the right forearm due to subcutaneous calcification in association with crest syndrome.results. this case presents an unusual case of extensive subcutaneous calcification in crest syndrome requiring surgical excision due to secondary ulceration, inflammation and infection. while a surgical approach has already been described for calcification in different connective tissue diseases, only scant data of massive subcutaneous calcification related to a forearm in crest syndrome followed by surgical excision exist. conclusion. in crest syndrome, extensive subcutaneous calcification related to the forearm can occur. surgical excision followed by primary wound closure can lead to an excellent postoperative result. background. the whole blood interferon signature (wbifns) is measured in several clinical trials studying inhibitors of interferon alpha (ifn-α) in sle, but failed repeatedly -in contrast to the less sensitive ifnα -to reflect longitudinal changes in lupus activity and to guide dosage finding of rontalizumab. therefore, better ifn biomarkers reflecting disease activity over time and individual response to the inhibition of ifnα are needed to optimize the risk-benefit ratio of ifn-inhibitors. here, we show that the highly sensitive monocyte restricted ifnα response protein siglec- , also known as sialoadhesin or cd , is a useful biomarker to monitor longitudinal changes in disease activity of sle patients. methods. ifn-α and siglec- were measured by delfia and flow cytometry, respectively, in accurately characterized lupus patients over a period of up to months (overall visits). changes of biomarker and changes of disease activity (bilag ) were correlated using spearman rank test (srt). disease courses of selected sle patients were plotted to demonstrate in detail the relations of ifn-biomarkers with disease activity, sle medication and clinical manifestations. background. a -year-old woman was admitted because of sudden attack of convulsion and somnolence situation with positive canca and myeloperoxidase antibodies. cerebral magnetic resonance imaging (mri) showed thickening and marked progression of the dura-meningeal enhancement and edematous changes at pre and post central gyrus left side. based on these findings, it was diagnosed as hypertrophic cranial pachymeningitis related to anca-associated vascultis as unusual presentation. there was only temporarily und partial responce to a -month therapy with cyclophosphamide mg i.v and oral glucocorticosteroids . taking into consideration the severe, life-threatening course of the disease in the case of our patient, the decision was made to use rituximab, a chimeric, monoclonal igg antibody directed against cd , leads to destruction of b cells via complement mediated lysis and antibody dependent cellular cytotoxicity. the first administration of the medication was performed according to the pattern for rheumatoid arthritis patients treated with rituximab, i.e. infusions for mg in -day intervals in combined therapy with glucocorticosteroids. a follow-up mri at months after start with rituximab showed significant regression of the meningeal pathology at temporo-occipatel aspects (pachymeningitis) and completely resolution of edematous changes at pre and post central gyres. the complete clinical remission was achieved by introducing rituximab. conclusion. rituximab seems to be successful therapie for the induction and maintenance of remission in patients with anca-associated vasculitis (aav) with cns involvement (hypertrophic cranial pachymeningitis ) , who had previously failed to respond to standard treatment with cyclophosphamide and steroids and a range of alternative treatments [ , ] . antikörperdiagnostik. mit prednisolon-therapie ( mg/kgkg, mg/ tag) und zusätzlich methotrexat mg wöchentlich war keine anhaltende normalisierung der entzündungsserologie zu erzielen. infliximab ( mg/kg) -wöchentlich i.v. erbrachte nur kurzzeitig eine normalisierung der entzündungswerte, dann trotz weiterer infusionen einen erneuten anstieg der bsg bis auf mm, crp mg/l. nach umstieg auf tocilizumab ( mg / mg/kgkg) alle wochen konnte nach wochen eine bislang anhaltende normalisierung der entzündungsserologie erzielt werden (crp , mg/l, bsg mm . std.). der allgemeinzustand der patientin besserte sich deutlich, der hb-wert normalisierte sich auf , mmol/l. in der kontrastverstärkten sonographie fand sich ein abfall in der kontrastmittelaufnahme der a. carotis communis. die maximale intima-media-dicke reduzierte sich bislang auf , mm. schlussfolgerung. die bisherige standardtherapie der takayasu-arteriitis mit prednisolon und mtx führte auch im vorliegenden fall nicht zur remission. für infliximab fanden wir ein frühzeitiges therapieversagen des sonst erfolgreich beschriebenen ansatzes einer tnf-α-blockade bei riesenzellarteriitis. dennoch gelang mit tocilizumab eine bislang über monate andauernde klinische, sonomorphologische und serologische remissionsinduktion bei monatlicher fortführung der il- -blockierenden therapie. background. cd is the prototypic nk receptor that is also expressed on a unique population of effector cd + cells. these cd -expressing t cells are expanded in rheumatoid arthritis patients and had features of senescent cells. nkg d is another nk receptor over expressed on effector cd + cells in aav patients. cd + as well as nkg d + t cells seem to be involved in tissue injury as they are capable of mediating tcr-independent immune activation. it is hypothesized that il- is able to up regulate the expression of nk cell receptors. interleukin- (il- ) is a proinflammatory cytokine that is over expressed in aav and is linked to the expansion of cd + effector memory t cells (tem). in aav in remission a persistent expansion of these cd + effector memory t cells has been observed. in the present study we assessed the expression cd on cd + t cells of aav and if expression of these molecules was influenced by il- . methods. the distribution of cd + tem and the proportion of cd +cd + t cells and nkg d+ cd + t cells were analysed in aav-patients and hcs by facs. in vitro effects of il- on the expansion of cd + tem and up regulation of cytotoxic markers were assessed in the same way. in addition il- serum levels were measured in patients and hc by elisa. results. we observed an increased proportion of circulating cd +cd + t cells in aav as well as nkg d+ cd + t cells in patients in remission compared to hc ( . vs . p< . and vs . p< . ). % to % of these cells were cd + effector memory t cells. the percentages of the cd +cd + t cells and nkg d+ cd + t cells were constant over time. we also observed elevated il- serum levels in patients in remission compared to hc (p= . ). in vitro stimulation of pbmcs with il- increased not only the proportion of cd + memory cells (cd ro+) but also the expression of cd and nkg d on these cells. conclusion. the driving force behind the persistent expansion of a cytotoxic subset of cd + effector memory t cells expressing cd and nkg d+ and being tcr -independent is likely the increased il- expression in aav patients . ergebnisse. unabhängig von regime der remissionsinduktion und der primären erhaltungstherapie lag am ende der nachbeobachtungsperiode bei % der patienten eine renale remission vor ( %; % pr). % hatten eine persistierende proteinurie von > , g/tag bei stabiler nierenfunktion, % eine persistierende niereninsuffizienz mit erhöhtem kreatinin bei inaktivem sle, bei % wurden eine persistierende aktive ln und/oder renale rezidive beobachtet. vier patienten verstarben. patienten mit langzeit-cr waren gekennzeichnet durch einen niedrigeren tubulointerstitiellen chronizitätsindex in der initialen nierenbiopsie ( , ± , vs. , ± , ; p= , ), eine hochsignifikant geringere proteinurie nach cyc-pulsen ( , ± , vs. , ± , g/tag; p= , ) und niedrigere dsdna-ak ( ± vs. ± u/ml; p< , ) zum zeitpunkt des beginns der erhaltungstherapie. eine proteinurie von < , g/tag nach pulsen cyc zeigte eine sensitivität von % und eine spezifität von % für eine langzeit-cr. schlussfolgerung. eine proteinurie von < . g/tag nach remissionsinduktion mit pulsen cyc sowie ein geringer tubulointerstitieller chronizitätsindex in der nierenbiopsie sind prädiktoren einer anhaltenden kompletten renalen remission bei ln. background. to evaluate and compare clinical efficacy of three biomarkers for interferon activity (measured directly and indirectly) and six traditional biomarkers to indicate current disease activity in sle. methods. ifn-α (delfia), ip- (elisa) and siglec- (flow cytometry) was measured in accurately characterized lupus patients and compared to serum titres of anti-dsdna (elisa and ria), anti-dsdna-ncx elisa, anti-nuc elisa, c and c . disease activity was evaluated using bilag- and a modified sledai- (msle-dai- k). additionally, clinically quiescent patients were monitored for flares over the course of days. results. increased levels of ifn-α, ip- and siglec- were found in %, % and % of active sle patients. ifnα (r= . ; p< . ) and siglec- (r= . ; p< . ) correlated better with bilag- than ip- (r= . ; p= . ), farr assay (r= . ; p= . ), anti-dsdna-ncx elisa (r= . ; p= . ), anti-dsdna elisa (r= . ; p= . ), anti-nuc elisa (r= . ; p= . ), c (r=- . ; p< . ) and c (r=− . ; p= . ). predictors of sle flares were disease duration ≤ months, mild clinical activity (in contrast to no activity), complement c ≤ mg/dl and ifn-α ≥ pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. conclusion. ifn-α, ip- and siglec- emerged as beneficial biomarkers for disease activity in lupus patients. therefore, implementation of ifn biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-ifn-directed therapies. . ) and carried significantly more often other antibodies ( . %; p< . ), which were separated into u rnp-( . %), ro-( . %), pmscl-( . %) antibodies, followed by . % with rheumatoid factors, . % with la-, . % with dsdna-and . % with jo- -and . % with ku-antibodies. the kaplan-meier analysis of the onset of organ involvement revealed a clear inclined position of overlap patients between patients suffering from lcssc and dcssc, especially regarding lung fibrosis and heart involvement. patients suffering from pah, oesophagus involvement and kidney involvement, overlap and lcssc patients showed nearly similar curve progression (log rank < . ). furthermore musculoskeletal involvement was significantly more frequent and more progressive in patients with overlap disease, followed by patients with dcssc and lcssc (log rank < . ). conclusion. these data support the current concept, that ssc-overlap syndromes should be regarded as a separate ssc subset, distinct from lcssc and dcssc, due to a different course of the disease, different proportional distribution of specific autoantibodies and skin/organ involvement. methoden. patienten mit gpa ( mit aktiver und mit in remission befindlicher gpa) wurden durchflusszytometrisch analysiert und mit gesunden verglichen. eine färbung für cd , cd , cd , igd, iga, cd , mhcii, wurde mittels flowjo-software analysiert. die statistische auswertung erfolgte mit "graph pad prism" und p-werte< , wurden als signifikant angesehen. die studie wurde von der ethik-kommission der charité genehmigt. ergebnisse. deutliche unterschiede (p= , ) wurden sowohl für die absolute zahl als auch die frequenz der plasmazellen im peripheren blut der patienten mit gpa mit krankheitsaktivität ( , ± , /µl) im vergleich zu denen mit einem bvas von ( , ± , /µl) oder gesunden ( , ± , /µl) gefunden, ähnlich wie bei sle. bei patienten mit gpa ist außerdem eine signifikante erhöhte anzahl der plasmazellen igapositiv (p= , ). die anzahl der plasmazellen sowie die frequenz der plasmazellen an den b-zellen im blut korrelieren mit dem bvas (r= , ; p< , ). interessanterweise zeigte sich keine expansion der doppelt negativen memory-zellen, die zum beispiel beim sle beschrieben ist. für die naiven b-zellen fand sich ebenfalls ein signifikanter unterschied zwischen patienten mit aktiver erkrankung im vergleich zu gesunden. bei den t-zellen fanden sich nur diskrete veränderungen. schlussfolgerung. die anzahl der plasmazellen ist bei patienten mit aktiver gpa deutlich erhöht, was eine rolle von plasmazell-vermittelten mechanismen in der pathogenese nahelegt. ein großteil dieser plasmazellen ist iga-positiv, diese könnten eine rolle bei der hno-beteiligung spielen. key: cord- -a a of authors: gupta, varsha; sengupta, manjistha; prakash, jaya; tripathy, baishnab charan title: molecular diagnostics date: - - journal: basic and applied aspects of biotechnology doi: . / - - - - _ sha: doc_id: cord_uid: a a of effective and early management of diseases requires record of the history, behavioral parameters, and travel information. these are helpful for the diagnosis, prevention, and control of the disease. there have been several advancements in the methods for diagnosing infectious diseases. the wide spectrum of tests such as biochemical evaluation, microbiological tools, immunological and molecular biology techniques, etc., is available. each type of diagnostic technique is strong and reliable in its own sense but poses certain limitations. these limitations may be complemented by using a combination of tests. older techniques such as microscopy and culturing of organisms from clinical specimens are error-free but are very labor intensive and extremely time consuming. there is a need to develop rapid and sensitive tests that can be used in both high- and low-resource settings. molecular diagnostics such as western blot, elisa, pcr, dna, and protein microarrays are revolutionizing the clinical practice of infectious diseases. their effects are significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. effective and early management of diseases requires record of the history, behavioral parameters, and travel information. these are helpful for the diagnosis , prevention, and control of the disease. there have been several advancements in the methods for diagnosing infectious diseases. the wide spectrum of tests such as biochemical evaluation, microbiological tools, immunological and molecular biology tech-niques, etc., is available. each type of diagnostic technique is strong and reliable in its own sense but poses certain limitations . these limitations may be complemented by using a combination of tests. older techniques such as microscopy and culturing of organisms from clinical specimens are error-free but are very labor intensive and extremely time consuming. there is a need to develop rapid and sensitive tests that can be used in both high-and low-resource settings. molecular diagnostics such as western blot , elisa , pcr , dna, and protein microarrays are revolutionizing the clinical practice of infectious diseases. their effects are signifi cant in acutecare settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. the diagnosis of these agents is done by using many tests either alone or in combination. these are serology-based diagnostic tools. they are more sensitive and specifi c than microscopic tests. there are two categories of these diagnostic tools that are based on antigen-detection assays and antibody-detection assays. these assays include the western blotting, enzyme-linked immunosorbent assay (elisa) , and all its derived tests such as the falcon assay screening test-elisa ( fast-elisa ), dot-elisa , hemagglutination (ha) test, indirect or direct immunofl uorescent antibody ( ifa or dfa) tests, complement fi xation (cf) test, and immunoblotting and rapid diagnostic tests (rdts) . in a western blot , the proteins present in a sample are separated according to their molecular weight by gel electrophoresis. a nitrocellulose membrane is placed on the gel, and with the help of electrical current, the proteins are transferred from the gel to the membrane where they adhere. the pattern of protein separation is maintained in the membrane after transfer. the membrane is then probed with specifi c antibodies (primary antibodies) to determine the presence of the protein. often a secondary antibody conjugated to biotin or a reporter enzyme is used to enhance the signal and detect the binding of the primary antibody . this procedure is used mainly to determine the presence of an antigen in biological sample with simultaneous determination of the molecular weight of a protein and measure relative amount of a protein present in different samples ( fig. . ). elisa is a diagnostic tool that is used in medicine and other industries to detect and quantify specifi c antigens . the sample with an unknown amount of antigen is immobilized on a solid support, usually a microtiter plate. this is done either nonspecifi cally by adsorption or specifi cally by capture by another antibody specifi c to the same antigen , in a "sandwich" elisa. after the antigen is immobilized, the detection antibody is added, forming a complex with the antigen . the detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme. the plate is developed by adding an enzymatic substrate to produce a visible signal which indicates the quantity of the antigen in the sample ( fig. . ). both western blot and elisa are used to detect hiv infection in the blood. they are called indirect tests as they measure the immune system's response to an infectious agent rather than looking for the components of the agent itself. since elisa detects hiv antibodies which the body starts to produce between and weeks after becoming infected with hiv, one should wait for at least months to confi rm for hiv aids . western blot is the most common method of testing to confi rm positive results from elisa test. it is used more as a confi rmatory test as it is diffi cult to perform and requires high skills. one advantage of western blot is that it is less likely to give false-positive results as it can effectively distinguish between hiv antibodies and other antibodies. this test uses synthetic and recombinant peptides to evaluate antibody responses to an antigen . however, this technique is subjected to the same drawbacks as most serology-based tests. antibodies raised against a peptide from one protein may cross-react with proteins from other species. moreover, antibodies raised against a antibody specifi c for a particular epitope of antigen are coated on well followed by the addition of the sample containing antigen . this results in antigen-antibody binding. binding of another antigen-specifi c antibody linked with enzyme results in color formation upon addition of the substrate . diagnosis of bacterial, viral, and parasitic diseases peptide may react in some assays but not in others as some regions of a peptide may be more immunogenic than others. in the past, the method has been applied to the study of malaria , fasciolosis, schistosomiasis, and taeniasis. lately it is not used regularly. the main difference between the regular elisa and the dot-elisa lies in the surface used to bind the antigen of choice. in the dot-elisa, the plastic plate is replaced by a nitrocellulose or other paper membrane onto which a small amount of sample volume is applied. the principle is similar to that of immunoblotting. the dotted membrane is incubated fi rst with an antigen-specifi c antibody followed by an enzyme-conjugated anti-antibody (secondary antibody ). the addition of a precipitable, chromogenic substrate causes the formation of a colored dot on the membrane which can be visually read. it is convenient to use, gives rapid results that are fairly easy to interpret, is fast and costeffective, and hence can be used in the fi eld (e.g., as a dipstick ). for all these reasons, the dot-elisa is extensively used in the detection of human and animal parasitic diseases, including amebiasis, babesiosis, fascioliasis, cutaneous and visceral leishmaniasis, cysticercosis, echinococcosis, malaria , schistosomiasis, toxocariasis, toxoplasmosis, trichinosis, and trypanosomiasis [ ] . this test is based on immunochromatographic antigen detection and has been implemented in many diagnostic laboratories as an adjunct to microscopy for the diagnosis of malaria . rdts consist of capturing soluble proteins by complexing them with capture antibodies embedded on a nitrocellulose strip. a drop of blood sample is applied to the strip and eluted from the nitrocellulose strip by the addition of a few drops of bufer containing a labeled antibody . the antigen-antibody complex can then be visualized directly from the membrane. rdts are now rapid, stable at temperatures up to °c, easy to use, and costeffective, thereby providing many advantages over traditional microscopic methods. this is a modifi ed elisa -based assay in which serum containing antigen-specifi c antibodies can be identifi ed by measuring light production. basically, an antigen of choice is fused to the enzyme reporter renilla luciferase (ruc) and expressed as a ruc-fusion in mammalian cell s to allow for mammalian-specifi c posttranslational modifi cations. the crude protein extract is then incubated with the test serum and protein a/g beads. during the incubation, the ruc-antigen fusion becomes immobilized on the a/g beads, which allows the antigen-specifi c antibody to be quantitated by washing the beads and adding coelenterazine substrate and measuring light production. some of the advantages of the lips technology include its rapidity and accuracy in detecting infected patients. sensitivity is improved in part by the use of mammalian cells which produce fusion antigens free of contaminating bacterial proteins. in addition, low backgrounds are produced compared to the elisa. monoclonal antibodies (mab) are derived from identical immune cells that are clones of unique parent cells and can bind to a specifi c epitope (for further details, refer to chap. ). they have been extensively used in biomedical and microbiological research as tools for diagnosis of diseases such as hepatitis , aids , infl uenza, herpes simplex virus infection, chlamydial infection, and treatment of cancer [ , ] . the monoclonal antibodies being directed against single epitopes are homogeneous and highly specifi c and can be produced in unlimited quantities. monoclonal antibodies have tremendous applications in the fi eld of diagnostics, therapeutics, and targeted drug delivery systems , not only for infectious diseases caused by bacteria, viruses, and protozoa but also for cancer and metabolic and hormonal disorders. in , kohler and milstein invented the hybridoma technology . the key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, fuse these cells with healthy antibody-producing b cells, and select for the successfully fused cells. in hybridoma technology , a myeloma cell rendered drug sensitive through mutation in a growth essential gene, hypoxanthine guanine phosphoribosyl transferase ( hgprt ), is chemically fused with immune cells from a host immunized with the antigen of interest, and the resulting cells are grown in medium containing the selective drug. since the immune cells have a short life span in tissue culture and the myeloma cells are drug sensitive, the only cell that will survive are those myeloma cells which obtained a normal hgprt gene from the immune cells. such cells also have a high likelihood of carrying the immune cell's antibody gene resulting in the generation of a hybridoma that can grow continuously in vitro and secrete a single monoclonal antibody ( fig. . ). the diagnosis of any infectious disease often requires the demonstration of the causative organism or presence of a specifi c antibody . specifi c antibody-based tests identify the pathogens associated with the disease. mabs recognizing unique antigenic determinants on pathogens are developed. this restricted reactivity allows for precise identifi cation of the organism of interest which is the major advantage of mabs over polyclonal antisera. in case of a pathogen occurring as subtype defi ned by unique antigenic differences, specifi c mabs can be used, whereas conventional antisera needs laborious absorption to remove cross-reactive antibodies. because of the specifi city, homogeneity, and unlimited availability of the mabs, vast amount of work has been carried out on the production/development the immuno-diagnoses of protozoan and parasitic diseases have signifi cantly been improved by mab technology because the tests involving mab as diagnostic reagents overcome the limitations of polyclonal antibodies. mabs were found to be extremely useful in the rapid outbreak of east coast fever (ecf). mabs of diagnostic value have also been developed against trichomonas vaginalis , leishmania donovani , trypanosoma congolense , and babesia bovis . development of monoclonal antibodies for the detection of mycoplasma pneumonia and plum pox virus has been reported . nucleic acid-mediated tests pcr is the most well-developed molecular technique that has not only been successfully applied for several wide-ranged clinical diagnoses but also has great potential for clinical applications, including specifi c or broad-spectrum pathogen detection, evaluation of emerging novel infections, surveillance, early detection of biothreat agents, and antimicrobial resistance profi ling. pcr-based methods may also be cost-effective relative to traditional testing procedures. further advancement of technology is needed to improve automation, optimize detection sensitivity and specifi city, and expand the capacity to detect multiple targets simultaneously (multiplexing). pcr is the most sensitive and rapid method of detecting pathogens in clinical samples. it is very useful as some of the microorganisms are not easily culturable in vitro or has a very long incubation time. under these conditions, the diagnostic value of pcr is very important [ ] . traditional pcr procedure includes amplification of specifi c genes ( fig. . ) of the microorganisms and running the product on a gel. the presence of a microbe is confi rmed by the presence of a band of appropriate size. nested, multi-plexed, and real-time pcr ( rt-pcr ) are used for effi ciency and quantitation. multiplexed pcr allows the detection of multiple sequences in the same reaction tube proving useful in the diagnosis of several infections simultaneously ( fig. . ) . rt-pcr system, unlike conventional pcr, allows for the quantifi cation of the original template's concentration through the use of various fl uorescent dyes and primers. the concentration is measured through comparison to standard curves. this eliminates the need to visualize the amplicons by gel electrophoresis, thereby greatly reducing the time, risk of contamination, and the introduction of false-positives. pcr is used to diagnose the presence of several opportunistic pathogens in the cerebrospinal fl uid of hiv patients or multiple sclerosis patients [ , ] . the viral infections that can be determined by this method are herpes simplex virus (type and ), varicella zoster virus , cytomegalovirus , epstein-barr virus , and japanese encephalitis virus. bacterial infection such as chlamydia pneumoniae is also identifi ed. mycoplasma sp. is very diffi cult to cultivate in laboratory; hence, pcr method is the only reliable method to identify the presence of the samples [ ] . dna probes consisting of cloned ribosomal rna genes, cdna to mycoplasmal rrna, synthetic s rrna oligonucleotide sequences, or cloned mycoplasmal protein genes have been developed and applied as diagnostic tools in a variety of human and animal mycoplasma infections . is a unique amplifi cation method with extremely high specifi city and sensitivity able to discriminate between a single nucleotide differences. it is characterized by the use of four different primers specifi cally designed to recognize six distinct regions on a target gene, with amplifi cation only occurring if all primers bind and form a product ( fig. . ). the reaction occurs at a constant temperature using strand displacement activity of dna polymerase [ ] . amplifi cation and detection takes place in a single step at a constant temperature ( °). it does not require expensive thermo cyclers. the corresponding release of pyrophosphate causes turbidity that is detected visually. sometimes dna-intercalating dye is also used. this has been applied for rapid detection of several dna and rna viruses such as west nile and sars virus. it has also been used for the identifi cation of several parasites. molecular-based approaches based on nucleic acids offer greater sensitivity and specifi city over the existing diagnostic tests. they permit the detection of infections from very low titer samples including those from asymptomatic patients. luminex technology is a bead-based fl owcytometric assay that allows the detection of various targets simultaneously. the microsphere beads can be covalently bound to antigens, anti-bodies, or oligonucleotides that will serve as probes in the assay. up to microspheres are available, each emitting unique fl uorescent signals when excited by laser, therefore allowing the identifi cation of different targets. this method has been successfully used for detecting cryptosporidium species. c. hominis and c. parvum has a single nucleotide difference in the microsatellite- region (ml- ) that can be identifi ed only by sequencing which is very time consuming and labor intensive. they can be detected and distinguished by this technology. however, there are several drawbacks of these methods regarding clinical samples, as pcr is susceptible to inhibitors, contamination, and experimental conditions. the sensitivity and specifi city of a pcr assay is dependent on target genes, primer sequences, pcr techniques, dna extraction procedures, and pcr product detection methods. these might not be optimal in clinical specimens such as blood, urine, sputum, cerebrospinal fl uid (csf), and others. the pcr conditions need to be carefully evaluated and the single nucleotide polymorphisms or snps (pronounced as snips) are tiny variations in an individual's genetic code. snps occur when a single nucleotide (a, t, g, or c) is substituted for another between the members of the same species or between two chromosomes of the same person. when the dna sequence of a gene differs by only one nucleotide between two individuals, they are called as alleles. snp analysis can be done in a single step by using genomic dna and pcr method ( fig. . ) . a single snp analysis can be done by using a specifi c primer attached to a fl uorescence marker, also known as a quenching probe or q-probe. when the primer binds with a specifi c dna sequence, the fl uorescence is quenched due to association with guanine residue. when it disso-ciates, the fl uorescence is acquired. when the primer binds to the wild-type allele, the dissociation occurs at a higher temperature, whereas in a mutant allele, the binding is weak and dissociation takes place at a lower temperature. this change in dissociation curve is analyzed. two different colors can be used for multiplex analysis . snps occur due to mutation, recombination, and natural selection . snps may occur in coding region of genes, in noncoding regions of genes, or in intergenic regions. they are classifi ed into different categories. due to degeneracy of genetic code, the amino acid sequence of the polypeptide might not change. these are known as synonymous polymorphism. when the changes produce different polypeptides, they are known as non-synonymous or replacement polymorphism. this may result in missense mutation, where a different amino acid is produced, or nonsense mutation, where there is a premature stop codon. lot of disease mutations are caused by replacement polymorphism. snps occurring in noncoding region might affect gene splicing, transcription factor binding, m-rna degradation, or mutate noncoding rna. about . % of dna sequences are identical between individuals of same species. out of . % variation, around % is due to snps. thus they bring about diversity among individuals. this trait is used for dna fi ngerprinting in forensic science. several diseases are caused by genetic variations in an individual. genetic factors are responsible for susceptibility and disease progression. snp profi le or haplotype associated with a disease trait may reveal relevant genes associated with a disease state. it provides understanding of many polygenic diseases. in future there are chances that by viewing the snps profi le of an individual, the physicians might be able to fi nd out the risks associated and plan a personalized medicine. snps help in determining the likelihood of a person to develop a particular disease. one of the genes associated with alzheimer's disease in apolipoprotein e or apoe . it contains two snps that result in three possible alleles for this gene: e , e , and e . each allele differs by one dna base, and the protein product of each gene differs by one amino acid. each individual inherits one maternal copy of apoe and one paternal copy of apoe . a person who inherits at least one e allele has a greater chance of developing alzheimer's disease, whereas inheriting the e allele reduces the likelihood of developing alzheimer's. snps are not absolute indicators of disease development. apoe is just one gene that has been linked to alzheimer's. like most common chronic disorders such as heart disease, diabetes , or cancer, alzheimer's is a disease that can be caused by variations in several genes. the polygenic nature of these disorders is what makes genetic testing for them so complicated. protein microarrays are tools that can be used in both translational as well as basic research. protein chips can be used for a variety of applications including identifi cation of protein-protein interactions, protein-phospholipid interactions, and substrates for protein kinase. they are used for clinical diagnosis and disease state progression. they can be used to phenotype leukemia cells, identify new protein-protein interactions, screen entire proteomes, and profi le hundreds of patient samples. several arrays are available for specifi c use. they have been graphically represented in fig. . . some of them are discussed here: these are high-density arrays and are used to identify novel proteins and protein-protein interactions ( fig. . a ). the array library is usually a high-density expression library and the probes are either directly labeled with fl uorophores or are tagged with labeled antibodies. these are used for quantitative profi ling of protein expression in clinical samples and cell culture ( fig. . b ). these are low-density arrays. known antibodies are arrayed to capture antibodies from unknown samples. the antigens are either labeled directly or are attached to a secondary antibody . the latter gives a sandwich assay similar to elisa . it consists of multiple known antibodies arrayed on a solid surface (fig. . c ). it is used to profi le the presence of known antigens from a pool of samples. normal and disease samples are used. they are either labeled directly or with haptens . these are used to detect autoantibodies in clinical and research samples. these are low-density arrays and are probed with serum or plasma ( fig. . d ). reverse arrays are used to probe hundreds of samples to detect the presence of few antibodies. cell lysates, plasma, and serum are arrayed and are probed with few known antibodies. this is an alternative strategy where samples containing several proteins are arrayed on slide and probed with labeled antibodies. level of number of proteins can be measured simultaneously. this is used to identify novel protein-binding motifs and protein-protein interactions (fig. . e ) . engineered proteins and peptides proteomic studies can provide substantial information about clinical state of a disease as they are the fi nal molecular machines of biological processes. they can be used as biomarkers for disease states. diagnostics use protein and peptide biomarkers from body fl uids. all proteomicbased diagnostic efforts seek to identify biomarkers that, alone or in combination, can distinguish between "case" and "control" groups. this can be done in several ways. profi ling and identifi cation of the protein this is a method to identify proteins and peptides in their natural form. here the proteins are resolved in the fi rst dimension based on ph (a process called isoelectric focusing) and in the second dimension by their molecular weight. this technique is labor intensive. this is an analytical technique where mass-tocharge ratios of particles are measured. it is used to determine the composition of peptides. proteins from body fl uids can be proteolytically cut into small pieces. they are ionized usually to cations by removal of electron. these charged particles are then separated according to their charge and mass. the separated ions are measured and displayed. the resulting spectra can be compared with other peptides in the data base ( fig. . ). but in this approach it is diffi cult to quantitate and study the protein modifi cations. it is a relatively novel technique in which a coprecipitate of a uv light-absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. the ionized biomolecules are accelerated in an electric fi eld and enter the fl ight tube. during the fl ight in this tube, different molecules are separated according to their mass-tocharge ratio and reach the detector at different fig. . the usage of proteomics approach for diagnostics or profi ling. ( ) first dimensional isoelectric focusing (ief) gel is used to separate the sample components according to their isoelectric point. ( ) second dimensional sds-page is used which further separates the proteins according to their molecular mass. sample spots obtained are isolated and prepared for application in mass spectrometer (ms). ms consists of ionization device as maldi or esi and mass sorting device as tof or quad and detection is done by a detector. after peptide mass fi ngerprint is obtained, it is analyzed through comparing the experimentally determined peptide mass fi ngerprint with known and virtual mass fi ngerprints using bioinformatics tools times. in this way each molecule yields a distinct signal. the method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides, and oligonucleotides, with molecular masses between and , da. maldi-tof is used for identifying bacterial strains in clinical microbiology laboratories. the development of automated, highthroughput proteomic technologies such as ms and maldi-tof has enabled large numbers of clinical samples to be analyzed simultaneously in a short time. these platforms have made "population-based proteomics" feasible for the fi rst time. with the use of naat , it is now possible to have many copies of target dna and the technique has advantage of being sensitive, specifi c, and rapid. it targets the conserve region of the target species. the naat test may be planned which would be able to detect single species, strain, or resistance-inducing mutation. using broadspectrum probes, the broad categories of the organism may be detected. naat has been successfully used in the diagnosis of infective endocarditis as compared to culture technique even when culture reports were negative. in patients with negative sputum smears, the tests based upon naat were quiet useful in the clinical diagnosis of tuberculosis . [ ] . it might cause potential discrimination regarding social acceptability, job or employment availability, and health insurance coverage. prenatal testing for genetic disorder may lead to abortion of a fetus. carriers of genetic mutations ethically should disclose the fact to their life partner or their siblings. but he or she might face social isolation. he or she might not be able to marry and start a family. similarly if a person is at risk of a late onset of a genetic disorder, the employer might not be willing to hire him or her. the health insurance companies would not want to pay for the medical expenses or might increase the premium [ , ] . one should also keep in mind that genetic testing cannot give all the answers. for example, it cannot tell about the exact time of onset, penetrance, or person to person variation of a disorder. there are several issues regarding the ethical consideration of genetic testing. until and unless there are clear laws to protect the individuals, privacy and confi dentiality of genetic information should always be protected and individuals wish to be tested or not should be respected [ ] . • biotechnology has played a very important role in diagnosis and treatment of various bacterial, fungal, viral, and parasitic diseases. it . chapter end summary has also helped in identifi cation of early stages of cancer. • the advancement in molecular techniques has helped in identifi cation of biomarkers that signifi es early development and progress of a disease. • the various tests like serology-based tests and nucleic acid-based tests are diagnostics, but preliminary data from traditional microbiology-based methods are also helpful. q . how has the advancement in biotechnological techniques helped in diagnosis of the diseases? q . discuss a few serological tests for the diagnosis. q . what is the importance of pcr in pathogen detection? q . how are the proteomic assays helpful in aiding diagnostics? q . discuss dna microarray technology. q . what is maldi-tof? bioethics in india, proceedings of the international bioethics workshop in madras: biomanagement of biogeoresources polymerase chain reaction on cerebrospinal fl uid for diagnosis of virus-associated opportunistic diseases of the central nervous system in hiv-infected patients ethical issues in genetic testing diagnosis of parasitic diseases: old and new approaches. interdisciplinary perspectives on infectious diseases diagnostic testing and the ethics of patenting dna ethics of genetic testing: medical insurance and genetic discrimination monoclonal antibodies in clinical diagnosis: a brief review application dna probes and pcr in diagnosis of mycoplasma infections monoclonal antibodies as diagnostics; an appraisal loopmediated isothermal amplifi cation (lamp) of gene sequences and simple visual detection of products pcr in diagnosis of infection: detection of bacteria in cerebrospinal fl uids pcr-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings key: cord- -mgegswvn authors: callebaut, p.; debouck, p.; pensaert, m. title: enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea date: - - journal: veterinary microbiology doi: . / - ( ) - sha: doc_id: cord_uid: mgegswvn abstract an enzyme-linked immunosorbent assay (elisa) was developed for the detection of the coronavirus-like agent in feces of pigs naturally affected with porcine epidemic diarrhea (ped) or experimentally infected with the cv isolate. the assay was specific and more sensitive than electron microscopy. an elisa blocking assay is described for the detection and titration of antibodies. specific antibody formation was demonstrated in pigs experimentally infected with cv and in swine naturally affected with ped. a coronavirus-like agent (cvla) is associated with diarrhea affecting swine of all ages. this type of diarrhea has been called "porcine epidemic diarrhea" (ped) (pensaert, ) . based on its electron microscopic appearance, the agent (isolate cv ) has been proposed as a member of the coronaviridae family (pensaert and debouck, ) . to date, infections by this agent have been diagnosed by the demonstration of the virus in intestinal epithelium using immunofluorescence (if) on gut sections and in feces using electron microscopy (em) (debouck et al., b) . antibodies to cv have been demonstrated by indirect if staining and by immunoelectron microscopy . all these methods are rather cumbersome and time consuming for large scale diagnostic and serological purposes. the epizootiology of cvla has, therefore, not been examined in detail. the elisa has found wide application in the detection of antibodies and small amounts of antigen and seems to be particularly suitable for widespread epizootiological studies. the assay has been used for the detection of bovine coronavirus in feces (ellens et al., a) and for the serology of several coronaviruses, including feline infectious peritonitis virus (osterhaus et al., ) , murine hepatitis virus (peters et al., , kraaijeveld et al., a and human coronavirus strain e {kraaijeveld et al., a, b) . in the present report an elisa is described for the detection of cvla antigens in crude fecal suspensions and the results are compared with those of em. it will also be shown that elisa is useful for the detection of antibodies against cv in swine sera. specimens, to be examined for cvla antigens by elisa, consisted either of fecal material, obtained directly from the rectum of live animals, or of contents collected from the caecum at the time of killing. the specimens originated not only from experimentally infected pigs but also from naturally infected animals and from controls. after collection all samples were stored at - °c until further processing. intestinal contents were obtained from two uninoculated, cesarean-derived colostrum-deprived (cdcd) piglets and from cdcd piglets, orally inoculated at the age of days to weeks with approximately pig-infectivedoses (pid) of cvla isolate cv (debouck and pensaert, ; debouck et al., a) . specimens were collected from three piglets that were killed during the incubation period ( and days after inoculation), from twelve piglets killed during the phase of severe diarrhea ( to days after inoculation) and from two pigs killed during the stage of recovery ( to days after inoculation). thirty-six samples of fecal material were collected from seven cdcd piglets (nos. to ) during an day observation period following oral administration of pid of cv at the age of weeks. from each piglet, a sample was collected prior to inoculation and during the incubation period day after inoculation. further samples were collected during the stage of severe diarrhea ( to days after inoculation) and towards the end of diarrhea ( to days) as indicated in table ii . fecal specimens were also obtained from an uninoculated conventional fattening pig and from two conventional fattening pigs during the acute phase of diarrhea, days after oral inoculation with pid of cv . a fecal specimen was collected from each of conventional pigs of varying ages originating from four different farms on which a natural outbreak of ped had been diagnosed by if staining on gut sections of sacrificed animals. at the time of collection, of these pigs had diarrhea while the remaining five pigs had been convalescent for more than week. control fecal samples, used to determine the limit between positive and negative absorbance values in elisa, consisted of non-diarrheal specimens obtained from conventional pigs of all ages. control samples, to test the specificity of elisa, consisted of fecal specimens from piglets with diarrhea, caused by transmissible gastroenteritis virus (tgev)or porcine rotavirus. a total of serum samples, to be examined for antibody by elisa blocking, were collected from four cdcd piglets and seven conventional experimental fattening pigs prior to inoculation with cv and at various intervals between and days thereafter. additionally, serum specimens were obtained from conventional pigs of all ages on five different farms. the latter samples were collected from to days after the onset of an outbreak of ped. control serum specimens consisted of a convalescent serum from two conventional pigs recovered from tge-and rotavirus infection and of three hyperimmune pig sera prepared respectively against a virulent belgian strain of tgev, hemagglutinating encephalomyelitis virus (hev) strain vw (pensaert and callebaut, ) and porcine rotavirus strain rv (debouck and pensaert, ) . these hyperimmune sera were prepared as described elsewhere . all sera were stored at - °c until used. for the detection of cvla antigen by elisa, the "double antibody sandwich" form of the assay was used. it was conducted essentially as described by ellens et al. ( ,b) in flat-bottomed microplates (cooke microtitre m b), using pl volumes of reagents per well. plates were coated with a / dilution of anti-cv globulin, prepared according to the method of purcell et al. ( ) from a hyperimmune porcine antiserum, obtained as described elsewhere (debouck and pensaert, ) . samples to be examined were first homogenized by shaking for h at °c in four volumes of phosphate buffered saline, ph . (pbs), containing . % tween , and each was then added to two coated wells. controls included pb and a series of twofold dilutions of a standard cv antigen preparation. the latter preparation was intestinal perfusate (debouck and pensaert, ) . briefly, a cdcd piglet was inoculated orally with a bacteria-free filtrate of a % intestinal homogenate containing cv . after h, when the diarrhea started, the small intestinal lumen of the anesthetized piglet was perfused with ml eagle's minimal essential medium during h at °c. the perfusate was subsequently centrifuged at x g at °c for h. the supernatant, containing cv antigen, was known to contain elisa units (e.u.) in previous titrations. specificity testing was performed by a blocking assay. the first sample well was treated with a porcine negative control serum, diluted / ; to the second sample well a / dilution of porcine anti-cv serum was added. both sera were obtained from pigs, different from that which provided the serum used for preparation of the anti-cv globulin-fraction. the conjugate used was the anti-cv globulin preparation, labelled with horseradish peroxidase (grade i, boehringer) according to the method described by wilson and nakane ( ) . the optimal working dilution was / . the amount of conjugate bound was determined by adding the enzyme substrate solution, containing mg/ml of recrystallized -aminosalicylic acid, . % hydrogen peroxide, mm na edta and . m sodium phosphate, final ph . . after overnight reaction at °c, the absorbance of each well was measured at nm against the pbs blank using a multiskan colorimeter (flow laboratories). a sample was scored positive if the absorbance in the well treated with negative control serum was equal to or higher than that of the / dilution of the standard cv preparation and if the absorbance value was reduced by > % in the well treated with anti-cv serum. antibody to cv in serum was measured by an elisa blocking assay, performed in a manner similar to the blocking assay used to control the specificity of the cv antigen elisa. the same anti-cv globulin preparation, diluted / , was used to coat the plates. antigen for all assays was the standard cv preparation, diluted / in pbs supplemented with . % fetal bovine serum and . % tween ; this amount of antigen represented e.u. test sera were serially diluted in twofold series, starting from a / dilution, in pbs containing . m nac , . % tween and % porcine negative control serum, and were added to the plates to which antigen had been bound. diluent alone served as a control. the plates were incubated for h at °c and thereafter overnight at °c before the conjugate was added. the anti-cv antibody, used for the preparation of this conjugate, was obtained from a pig other than that which provided the coating antibody in order to decrease the chance for aspecific reactions. the optimal dilution was / in test serum diluent. the amount of conjugate bound to each well was determined as above. a given dilution of test serum was considered to be positive if it reduced the absorbance by at least % when compared with the buffer control. titers are expressed as the reciprocal of the highest positive dilution. to compare the sensitivity of em and elisa for the detection of cvla, the specimens of fecal material and intestinal contents obtained from experimentally infected cdcd piglets and experimental fattening pigs as described in the section "specimens from experimental pigs", were examined for coronavirus by em. the samples were processed as described elsewhere (pensaert and debouck, ) . the absorbance profile obtained with serially diluted perfusate in the antigen assay is shown in fig. a . all control non-diarrheal fecal specimens, used to determine the limit between positive and negative absorbance values, showed values in the range of . to . and these values were not reduced in the blocking assay. the mean absorbance of . plus times the standard deviation resulted in an absorbance value of . . therefore, an absorbance value of ~ . was considered evidence of the presence of cv antigen in a sample. using this criterion, the standard cv antigen preparation in fig. a was positive up to a dilution of / . this preparation was, therefore, considered to contain e.u. in the specificity tests all fecal specimens from tgev-or rotavirus positive pigs were elisa negative for cv antigen. additionally, there was a good correlation between the elisa results and clinical data in the experimentally inoculated swine. as shown in table i , all preinfection fecal or intestinal samples, obtained from experimental piglets and fattening swine, were elisa negative. all the fecal samples obtained during the incubation period were elisa negative. however, all three samples of intestinal contents collected during the same period were elisa positive. the twelve samples of intestinal contents and of the specimens of fecal material collected during the acute phase of diarrhea were elisa positive. only three out of ten fecal specimens and none of two samples of intestinal contents, obtained during the stage of recovery, were elisa positive for cv antigen. of the fecal samples, collected from naturally infected pigs of all ages, were elisa positive, all of which were obtained from diarrheic animals. the remaining five samples, in which no cvla antigen could be demonstrated, were collected from animals that had convalesced for more than week. the elisa positive samples had absorbance values in the range of . to . {median . ). after blocking by incubation of the specimens with cv antiserum, the absorbance values were reduced in every instance by more than %, ranging from - . to . (median . ). as shown in table ii , the experimental piglets nos. , , and were found to excrete the coronavirus-like agent in their feces both by em and elisa. in the remaining piglets, nos. , and , virus shedding was detected by elisa only. in total, nine fecal specimens found negative for coronaviruslike particles by em were elisa positive, whereas only one em positive specimen. was negative by elisa. all the samples collected during the acute stage of diarrhea at days after inoculation, when coronavirus was regularly but not always detected by em, were elisa positive. towards the end of the diarrhea ( and days after inoculation} some em negative samples were still elisa positive. the fecal samples of the two experimentally inoculated fattening pigs were negative for cvla by em, but positive by elisa. in intestinal contents of experimentally infected piglets, cvla was detected at various times after inoculation by both methods. in all three specimens, collected during the incubation period, cvla was detected by both em and elisa. nine out of twelve specimens, obtained during the stage of diarrhea, were also positive by both methods. however, the three remaining specimens, obtained on day , and after inoculation respectively, were negative for coronavirus by em, but positive by elisa. finally, the two samples collected at the time of recovery were negative both by em and elisa. the absorbance profile obtained with a serially diluted porcine serum sample in the elisa blocking test is shown in fig. b. by comparison with the buffer control, having an absorbance value of . , the titer of this sample was estimated to be . the sensitivity and specificity of the elisa blocking assay in detecting a sero-conversion to cv infection was first established by testing pre-and post-inoculation sera obtained from experimentally infected piglets and fattening swine. as shown in table iii , upper part, all preinfection and early postinfection sera from the cdcd piglets were negative (titer ~ ). starting from days after inoculation, the elisa blocking assay detected cv antibodies in all the piglet sera and titers ranged from to . of the seven conventional experimental fattening pigs (table iii, sera, but in the remaining animals preinfection titers varying from to were found. significant antibody rises (>/fourfold rise in antibody titer) were detected in the sera from the latter animals days after inoculation, when the elisa blocking titers ranged from to . the highest titers were found on day after inoculation. the results of the serological examination of the pigs of all ages naturally infected in the field are given in table iv . fourteen sera were collected between and days after the onset of the diarrheal outbreak and were elisa negative. all the sera except , collected days or later after the start of the disease, contained antibodies detectable by elisa blocking. the titers of these positive sera varied from to . finally, the specificity of the elisa blocking assay was indicated by the negative results obtained with convalescent and hyperimmune antisera directed towards other viruses, including tgev, rotavirus and hev. the results obtained with intestinal contents and fecal material of experimentally and naturally infected swine demonstrate the efficacy of elisa for the detection of cvla antigen. the specificity of the test was proven by the results on control material containing other viruses. further evidence of the specificity is obtained by the correlation of the elisa results with the clinical data: virus shedding is detected with high consistency during the acute phase of disease, but much less consistently during the incubation period and during the recovery phase. the elisa results on samples collected during the latter two periods appear to depend upon the type of material tested. all samples of intestinal contents collected the first and second day after inoculation were positive, whereas all fecal samples collected day after inoculation were negative. during the phase of recovery on the contrary, some fecal samples were positive, while all intestinal samples were negative. it is likely that this is a reflection of the time delay between the production and release of virus progeny in the intestine and its excretion in the feces. however, as only a small number of specimens have been tested, more extensive examination is needed to corroborate the present findings. the sensitivity of elisa compares favourably to that of em. more fecal and intestinal specimens from experimentally infected pigs were positive by elisa than by em. elisa is more reliable than em as shown by the finding that during the acute phase of diarrhea, virus excretion could be demonstrated in the feces of all experimentally infected piglets by elisa but not by em. the finding that viral antigen is still occasionally detected by elisa in feces towards the end of diarrhea, but not by em, further substantiates the previous statement. for use as a diagnostic tool elisa is to be preferred above em because it is a specific test for cvla, whereas em would have to be followed by a confirmatory test such as iem (debouck et al., b) . in order to detect cvla antigen in fecal and intestinal suspensions the standard "double antibody sandwich" elisa, as originally described by ellens and de leeuw ( ) for bovine rotavirus, has been modified in this study by incubating the specimens with porcine serum free of antibody to cv , prior to determination of the extent of conjugate binding. this was required to eliminate false-positive results that otherwise would have been encountered during this study in of a total of fecal specimens. similar experiences have been reported with solid-phase immunoassays for detection of human rotavirus (yolken et al., ; cukor et al., ; brandt et al., ) and norwalk enteritis virus (greenberg et al., ) in feces. as suggested by brandt et al. ( ) , these false-positive reactions may be due to the presence in the test samples of intestinal bacteria or their products, which bind nonspecifically to the antibodies used as reagents in elisa. the elisa blocking assay, using crude virus-containing intestinal perfusate as antigen, is an efficient assay for the detection of an antibody response to cvla, as shown by the clear-cut seroconversion found in sera of experimentally and naturally infected swine. the specificity of the assay is demonstrated because the monospecific sera directed against other viruses failed to react in the cv elisa blocking assay. the finding that in sera of experimental piglets and of naturally infected pigs of all ages, antibodies to cv were only detectable by elisa at weeks after the onset of diarrhea, may be caused by a low sensitivity of the elisa blocking assay or by a weak immunogenicity of cvla antigen. the high antibody titers found in sera of experimental fattening pigs, very soon after inoculation, probably represent a secondary response because several animals had a low titer prior to inoculation. to date, the routine detection of cvla antigen in diarrheal feces and its antibody in sera from recovered swine in field outbreaks has been hampered by the lack of suitable diagnostic and serological techniques. this was related to the inability to cultivate the agent in vitro. attempts to isolate cv from intestinal perfusates in porcine organ cultures and in cultures of primary and continuous cells, usually of porcine but also of bovine, simian and human origin, have failed. pancreatic enzymes, used for treatment of cell cultures or inoculum, remained ineffective as a means to promote viral growth (callebaut and debouck, ) . the development of an elisa will, therefore, facilitate further study of this virus. extensive epizootiological research now becomes possible, and will reveal the importance of the coronavirus-like agent as a cause of ped in field outbreaks of enteritis in pigs of all ages. comparison of direct electron microscopy, immune electron microscopy and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children some characteristics of a new porcine coronavirus and detection of antigen and antibody by elisa simplified radioimmunoassay for detection of human rotavirus in stools experimental infection of pigs with belgian isolates of the porcine rotavirus experimental infection of pigs with a new porcine enteric coronavirus, cv the pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, cv the diagnosis of coronavirus-like agent (cvla) diarrhea in suckling pigs enzyme-linked immunosorbent assay for diagnosis of rotavirus infections in calves diagnosis of bovine coronavirus infections with hemadsorption-elution-hemagglutination assay (heha) and with enzyme-linked immunosorbent assay (elisa) comparison of five diagnostic methods for the detection of rotavirus antigens in calf faeces solid-phase microtiter radioimmunoassay for detection of the norwalk strain of acute nonbacterial epidemic gastroenteritis virus and its antibodies enzyme-linked immunosorbent assay for coronaviruses hcv and mhv enzyme-linked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus strain e elisa for the serology of fip virus porcine epidemic diarrhea characteristics of a coronavirus causing vomiting and wasting in pigs a new coronavirus-like particle associated with diarrhea in swine an immunoelectron microscopic and immunofluorescent study on the antigenic relationship between the coronaviruslike agent, cv , and several coronaviruses enzyme-linked immunosorbent assay for detection of antibodies to routine hepatitis virus microtiter solid-phase radioimmunoassay for hepatitis b antigen recent developments in the periodate method of conjugating horseradish peroxidase (hrpo) to antibodies elisa for rotavirus acknowledgements these studies were supported by the institute for the encouragement of scientific research in industry and agriculture (iwonl), brussels, belgium.the technical assistance of mrs. b. frijling and mrs. a. bauwens is gratefully acknowledged. key: cord- -yc xtw s authors: lappin, michael r. title: microbiology and infectious disease date: - - journal: small animal clinical diagnosis by laboratory methods doi: . /b - - - - . - sha: doc_id: cord_uid: yc xtw s nan infectious agents may be identified directly by cytologic analysis, histopathologic evaluation, culture, viral isolation, antigen detection, or nucleic acid amplification techniques. polymerase chain reaction (pcr) assays are the most commonly used nucleic acid amplification techniques in small animal infectious diseases. detection of antibodies against infectious agents provides indirect evidence of prior exposure or current infection. this chapter describes methods for obtaining specimens, outlines currently used testing procedures for some of the more common infectious diseases, and discusses interpretation of results from the various procedures and tests.* infectious diseases should be on the differential list for most problems, especially those with fever or other signs of inflammation. history, physical examination findings, and routine clinical pathologic testing are seldom pathognomonic for an infectious cause, but they help the clinician rank differential diagnoses and develop a logical diagnostic plan. history can increase the degree of suspicion for infectious disease. exposure to other infected animals or contaminated fomites is important for agents with direct transmission, such as those inducing respiratory disease (e.g., feline herpesvirus , canine bordetellosis) or gastroenteritis (e.g., canine and feline giardiasis, canine and feline parvovirus infection). potential exposure to vectors (e.g., mosquitoes for dirofilariasis; ticks for lyme borreliosis [ixodes spp.], ehrlichiosis [rhipicephalus sanguineus], rickettsia rickettsii [rocky mountain spotted fever; dermacentor spp.], and babesia canis [r. sanguineus]) or appropriate travel history (e.g., coccidioidomycosis in the southwest; blastomycosis in the mississippi, missouri, and ohio river valleys) can also suggest infectious disease. vaccination history, deworming history, and determination of whether other animals or people in the environment are affected can aid in ranking infectious diseases on a differential diagnoses list. physical examination findings may suggest an infectious cause. infectious agents can induce fever. lymphadenomegaly as a result of reactive lymphoid hyperplasia can be infectious in origin. hepatosplenomegaly can be caused by immunologic stimulation induced by chronic intracellular infections (e.g., ehrlichiosis, brucellosis). endogenous uveitis commonly occurs after infections by feline immunodeficiency virus (fiv), feline infectious peritonitis (fip) virus, toxoplasmosis, and systemic mycoses. mucopurulent discharges can suggest primary or secondary bacterial infections. certain infectious diseases cause specific abnormalities such as dendritic ulcers (feline herpesvirus ), chorea myoclonus (canine distemper virus), or testicular swelling plus pain (canine brucellosis). finally, clinicopathologic abnormalities can suggest disease caused by infectious agents. neutrophilic leukocytosis, particularly if a left shift or degenerative neutrophils (see chapter ) are also present, is consistent with an infectious cause of disease. gram-negative sepsis is suggested by leukopenia with a degenerative left shift. monocytosis or lymphocytosis can be induced by persistent infection with a number of intracellular agents that result in persistent infection. examples include ehrlichiosis, toxoplasmosis, and bartonellosis. polyclonal (e.g., multiple infectious causes) or monoclonal (e.g., usually induced by neoplasia, rarely associated with canine ehrlichiosis) gammopathies may suggest chronic immune stimulation. neutrophils in aqueous humor, cerebrospinal fluid (csf), synovial fluid, or urine may indicate inflammation induced by infectious agents. morulae of ehrlichia spp. are rare in the cytoplasm of mononuclear cells (ehrlichia canis), neutrophils (ehrlichia ewingii; anaplasma phagocytophila [previously e. equi]), or platelets (ehrlichia platys). mycoplasma haemofelis (cats only), "candidatus m. haemominutum" (cats only), "candidatus m. turicensis" (cats only), m. haemocanis (dogs only), "candidatus m. haematoparvum" (dogs only), cytauxzoon felis (cats only), and babesia spp. sometimes will be identified cytologically on the surface or within canine or feline erythrocytes. for demonstration of cheyletiella spp., a piece of transparent adhesive tape is gently pressed against areas with crusts or dandruff and then placed on a microscope slide. next the hair is clipped, mineral oil is placed on the skin and on a microscope slide, and the skin is scraped using a blunt no. scalpel blade. for skin scrapings to look for demodex spp., the skin should be immobilized and mites expressed from follicles by pinching and scraping the extruded material. for scrapings to look for sarcoptes spp. or cheyletiella spp., the scraping is continued more superficially (inducing a mild capillary ooze) over a larger surface area. after transfer of the scraping, the microscope slide field is scanned at × for mites. for identification of dermatophytes, hairs are plucked from the periphery of a lesion, placed on a microscope slide, and covered with % to % potassium hydroxide to clear debris. the slide is then heated (not boiled) and examined under the × or × objective to search for hyphae, spores, conidia, budding yeasts, and fungusinduced damage (e.g., swollen or broken hair shafts). the × objective is used to identify arthrospores (dense aggregates of spherical structures that may cover the hair shaft [ figure - ]). failure to find arthrospores does not rule out dermatomycosis. culture is more sensitive for diagnosis of dermatophytosis (see fungal culture). romanowsky-type stains (e.g., wright) are used in preference to wet mount preparations and ink when looking for fungi other than dermatophytes (see chapter ) . romanowsky-type stains are also useful in advantages • cytology is inexpensive and readily available and may allow rapid confirmation and identification of an infectious agent. it assists in establishing normal flora and contaminants versus infection (e.g., interpretation of relative numbers of bacteria and yeasts in the ear canal). cytologic examination also permits visualization of relative numbers of organisms at the time of collection (culture results may be misleading in terms of fast-or slow-growing bacteria). disadvantages • infectious agents cannot always be found (e.g., ehrlichiosis, haemoplasmosis, infections with numbers of organism that are below sensitivity level of cytology). sometimes a presumptive cytologic diagnosis must be confirmed by other methods (e.g., histopathology, culture, pcr assay), and cytology is of limited value in detecting viral inclusions except in brief viremic stages of canine distemper. see chapter for discussion of cytologic techniques and cytologic conclusions. discharges from animals with suspected bacterial disease should be placed on a microscope slide, air dried, fixed, and stained with both gram and romanowsky-type stains (see chapter ) . the examination is started on low power ( × magnification), with oil immersion ( ×) used for inspection of bacterial morphologic features (i.e., rods, cocci) and gram stain characteristics (i.e., gram-positive [blue] or gram-negative [pink] ). the primary disadvantage of gram staining is that gramnegative bacteria may be difficult to find because background material stains pink. it is easier to find bacteria (dark-blue stain) and easier to study morphologic detail of other cells (i.e., inflammatory cells) using romanowsky-type stains. gram staining may be variable; organisms in body fluids may stain differently from those grown on a blood agar plate. gram stain demonstrates the gram-positive, branching filaments of actinomyces spp. and nocardia spp. (see . acid-fast stains can be used for mycobacterium spp. and to help differentiate nocardia spp. (acid-fast) from actinomyces spp. some bacteria have characteristic morphologic features. large rod-form bacteria containing spores found on fecal cytology of dogs or cats with diarrhea suggest clostridium perfringens (figure - ) . bipolar-staining, gramnegative coccobacilli found in aspirates of inflamed cervical lymph nodes from cats in the southwest or west suggest yersinia pestis. short spirochetes found on fecal cytology of animals with diarrhea suggest but do not prove campylobacteriosis. spirochetes found on cytology of gastric mucosa of vomiting animals suggest helicobacteriosis. to demonstrate inclusion bodies in acute feline chlamydial conjunctivitis, conjunctival scrapings are obtained with a flat spatula, smeared on a slide, stained with romanowsky-type stains, and examined for intracytoplasmic aggregations of chlamydophila felis (previously chlamydia). been resistant to medications. remember: skin and mucosal surfaces have a resident microflora (box - ); therefore care must be taken to avoid contamination. advantages • culture and antimicrobial susceptibility usually allows the most effective treatment to be administered. disadvantages • culture requires time for agents to grow; also, some organisms are fastidious or have special culture requirements. other disadvantages are the expense and the ease of contaminating or making inactivate cultures, rendering results worthless. body cavities • the site of skin puncture should be prepared as for blood culture (see discussion in cardiovascular system). if pyothorax or peritonitis seems likely but fluid cannot be aspirated, lavage (see chapter ) is indicated. because mixed infections are common and pure anaerobic infections may occur, aerobic and anaerobic cultures should be performed. cardiovascular system • blood cultures are indicated in suspected bacterial endocarditis or septicemia. a large vein prepared surgically with sequential iodine and alcohol scrubs is used for three blood culture specimens obtained during a febrile episode over a -hour period in dogs with suspected endocarditis. culture of fewer than three specimens significantly decreases the chance of positive results. at least ml of blood is placed directly into a transport medium that will support the growth of aerobic and anaerobic bacteria, and it is incubated at ° c for hours. clotted blood or blood containing ethylenediaminetetraacetic acid (edta) or citrate are unacceptable because this decreases isolation of organisms (bartonella spp. are exceptions; they can be cultured from edta tubes; see bartonellosis sections). if a patient is critically ill and sepsis is suspected, three cultures should be obtained over to hours before antimicrobial therapy is instituted. because the urinary system is a common portal of entry for bacteria into the body, urine identifying yeasts such as blastomyces dermatitidis, histoplasma capsulatum, sporothrix schenckii, coccidioides immitis, or cryptococcus spp. (see figure - ) in exudates, csf, lymph node aspiration cytology, or transtracheal aspiration cytology. canine distemper virus inclusions in lymphocytes, neutrophils, or erythrocytes ( figure common indications • culture and antimicrobial susceptibility are indicated in most suspected bacterial diseases (box - ), especially when clinical syndromes have eye • conjunctival culture should be performed before topical anesthesia or application of fluorescein stain by rolling a moistened sterile swab over the conjunctiva. ocular paracentesis is necessary for intraocular culture. gastrointestinal tract • primary bacterial gastroenteritis occasionally occurs. salmonella spp., campylobacter spp., c. perfringens, and escherichia coli are agents that can be involved. these organisms can also be isolated from normal animals, however. salmonella spp. and campylobacter spp. can cause small or mixed bowel diarrhea; c. perfringens is usually associated with large bowel diarrhea. approximately to g of fresh feces should be submitted to the laboratory for optimal results. if delayed transport of feces to the laboratory is expected, the clinician should is often cultured in patients when the source of septicemia or bacterial endocarditis is unknown. central nervous system • bacterial infection of the central nervous system (cns) is uncommon. even when infection occurs, low numbers of organisms make cytology and culture low-yield procedures. if increased numbers of neutrophils and increased protein are detected in csf (see chapter ) , however, aerobic and anaerobic bacterial culture and antimicrobial susceptibility testing are indicated. csf samples should be placed in transport media and delivered to the laboratory as soon as possible. aerobic and anaerobic bacterial culture should be performed when bacterial infection of the cns is suspected. culture of the third fraction of an ejaculate (preferred) or prostatic massage is recommended for prostatic culture. culture of the second fraction of an ejaculate is recommended for testicular culture. culture of prostatic or testicular material retrieved by aspiration or biopsy can also be performed. prostatic massage and closed prostatic aspiration or biopsy should be avoided in dogs with suspected prostatic abscesses. obtaining distal urethral specimens for quantitative culture before and after ejaculation may help avoid confusion caused by urethral contamination. anaerobic culture of urine or prostatic fluid is rarely useful. integument and ear • in superficial pyoderma, hair is clipped from the surrounding area, but disinfection is not attempted. a pustule is ruptured with a sterile fine-gauge needle, and a swab of pus is cultured. in deep pyoderma, hair surrounding the lesion is clipped and the area is disinfected with an antiseptic. the lesion is squeezed to express exudate, which is collected on a swab. gloves should be worn. for culture of ears, a sterile otoscope cone is inserted to the level of the horizontal canal and the ear is swabbed through the cone. when middle-ear infection is suspected, the animal is anesthetized and material for culture is retrieved by myringotomy by penetration of the tympanum with a sterile csf needle placed through a sterile otoscope cone. musculoskeletal system • no normal flora exists in musculoskeletal tissues. dogs with radiographic evidence of discospondylitis should be evaluated for brucella canis and bartonella spp. infections (see diagnostic tests for select bacterial infections). intervertebral joints can be cultured after fluoroscopically guided aspiration or when decompressive spinal surgery is required. most cases of discospondylitis develop after hematogenous spread of bacteria from an extravertebral source. blood and urine are commonly cultured from patients with discospondylitis; staphylococcus spp. are commonly involved. dogs or cats with suppurative arthritis (with or without cytologic visualization of bacteria) should have the synovial fluid cultured for aerobes and mycoplasma spp. (see chapter ) . likelihood of positive culture results increases if the synovial fluid contains degenerative neutrophils. l-form bacteria usually cannot be grown from joint fluid via routine culture techniques. synovial biopsy for culture plus histopathologic evaluation for l-form bacteria is more sensitive than only culture of fluid. borrelia burgdorferi is almost never isolated by routine culture from joints of dogs with lyme disease. use of pcr assays on synovial fluid to amplify dna of mycoplasma spp., b. burgdorferi, a. phagocytophilum, and ehrlichia spp. may prove to be an effective way of documenting the presence of the organisms in the joints of affected animals, but objective data are lacking. in osteomyelitis, culture of fistulous tracts is less sensitive than culture of affected bone. culture for infectious myositis is seldom performed unless suspicion for an anaerobic infection (e.g., clostridium spp.) is based on foul odor, subcutaneous (sc) emphysema, or empyema. the clinician can better evaluate for other infectious myopathies (e.g., toxoplasmosis, leptospirosis) using serologic testing or pcr assays. respiratory system • lower airway specimens are best obtained by transtracheal aspiration or bronchoalveolar lavage during bronchoscopy. fine-needle pulmonary aspiration biopsy can be used but carries more risk (see chapter ) . bacteria can be isolated from the trachea in some clinically healthy dogs. these bacteria are probably transient; common isolates are listed in box - . because many organisms isolated from normal dogs have also been associated with lower respiratory tract inflammation, all transtracheal aspiration samples should be evaluated by culture, antimicrobial susceptibility, and cytology. with cytology, the clinician should look for squamous cells coated with bacteria (which indicate oropharyngeal contamination) (see figure - ). bacteria should not be considered significant unless accompanied by neutrophilic inflammation. mycoplasma spp. have been isolated in pure culture from lower airways of patients with clinical signs of respiratory disease. , culture for mycoplasma spp. should be performed on all transtracheal aspiration samples; these samples need to be transported to the laboratory in amies medium or modified stuart bacterial transport medium. mycoplasma spp. culture should be specifically requested. amplification of mycoplasma spp. dna from airway secretions by pcr assay can also be used to document infection. nasal specimens are best obtained from nasal lavage or core biopsy, or by passing a swab through a sterile otoscope cone (see chapter ) . the clinician can best obtain pharyngeal specimens using a guarded swab taken during pharyngoscopy. nasal and pharyngeal cultures can be difficult to interpret because of extensive normal flora in the nasal cavity and nasopharynx (see . for aerobic culture, no special transport medium is required if the swab remains moist and can be inoculated onto the culture medium within hours. swabs containing liquid or gel transport media are frequently used, a disk containing a fixed amount of antibiotic. the procedure is qualitative and allocates organisms to the sensitive (susceptible), intermediate (indeterminate), or resistant category. advantages • advantages of the disk diffusion test are its simplicity and suitability for most routine cultures, that it can be performed in the office, and its applicability for rapidly growing organisms (e.g., enterobacteriaceae, staphylococcus aureus). disadvantages • this test is not suitable for slowgrowing organisms and anaerobes. in addition, there is inaccuracy in predicting susceptibility of poorly diffusing antibiotics (e.g., polymyxins), and factors that influence the test (e.g., ph and thickness of the medium, concentration of organisms, incubation time) must be standardized. it is imperative that proper procedures be followed to avoid errors in diagnosis. artifacts • artifacts result from improper sample collection (i.e., wrong sample, contamination), improper sample transport, failure to notify the laboratory of suspected pathogens (e.g., salmonella spp., anaerobic bacteria, campylobacter spp., mycoplasma spp.), recent antibiotic treatment, and culture for a secondary rather than a slowgrowing primary pathogen (i.e., insufficient duration of culture). failure to grow fastidious anaerobes may be caused by short, seemingly insignificant exposure to oxygen or failure to use prereduced culture media. interpretation • recognizing normal flora (see box - ) is necessary for correct interpretation. preliminary identification is expected in to hours, and antibiotic sensitivity is reported in to hours. most aerobic and facultative organisms are identified within days; identification of anaerobic organisms or mycoplasma spp. may require an additional to days. bacterial pathogens commonly isolated from various body systems are listed in box - . the overlap between resident and pathogenic organisms should be noted. staphylococcus pseudintermedius is the major pathogen isolated from the skin of dogs with pyoderma. gramnegative organisms are likely to be contaminants in superficial pyoderma and secondary to s. pseudintermedius in deep pyoderma. primary bacterial rhinitis is rare in dogs and cats but can result from infection with bordetella bronchiseptica, mycoplasma spp., and chlamydophila felis (cats). primary bacterial pneumonia can result from b. bronchiseptica or mycoplasma spp., whereas other organisms are usually secondary to viral infections or aspiration. bacterial growth from urine obtained by cystocentesis is significant because the bladder is normally sterile. urine cultures, however, are best interpreted in conjunction with a urinalysis. if growth occurs despite absence of significant pyuria (see chapter ), sample contamination, improper sample transport, or diseases causing immune suppression (e.g., hyperadrenocorticism, diabetes mellitus, fiv infection) must be considered. in quantitative culture of urine obtained by catheterization or midstream voiding, greater than or equal to , colonies/ml is significant. lower concentrations may however. routine cultures can be safely stored in transport media at room temperature for up to hours. after this time, overgrowth is a potential problem because of various growth rates of different organisms. refrigerated, routine specimens can be stored in transport media for at least days. tissue samples can be refrigerated for up to days. fluids (e.g., urine) can be safely stored at room temperature for to hours, refrigerated for hours, and refrigerated in transport media for hours. quantitative culture is not accurate for fluids stored in transport media because of artifactual dilution. for anaerobic culture, fluid should be aspirated into a syringe, the needle capped with a rubber stopper, and the sample inoculated onto culture medium within minutes of collection. transport media that support the growth of anaerobic bacteria are available but are not ideal for all fastidious organisms. with these limitations, samples can be refrigerated for days in an appropriate transport medium. analysis • blood agar plates grow most routine bacterial pathogens. a biplate containing blood agar and macconkey agar is frequently used. the common anaerobic culture medium is thioglycolate. the decision to perform in-office testing instead of using a commercial laboratory is based on case load, available equipment, and expertise. sensitivity testing • sensitivity testing gives an in vitro estimation of suitability of a given concentration of an antimicrobial agent. two techniques are used: ( ) the dilution test and ( ) the disk diffusion test. dilution test • this test is quantitative and determines the least amount of antimicrobial agent needed to prevent growth of a microorganism (minimum inhibitory concentration [mic]). quantitative susceptibility testing is indicated when antimicrobial dosing schedules need to be monitored closely (e.g., gentamicin) or when disk test results are inapplicable, equivocal, or unreliable (e.g., slow-growing organisms, confirmation of susceptibility to polymyxins, confirmation of susceptibility or resistance to given doses of aminoglycosides). other indications include anaerobes and testing for synergism or antagonism between antimicrobials. advantages • the dilution test may be effective even though disk diffusion techniques suggest otherwise (e.g., antibiotics concentrated in urine). disadvantages • disadvantages of the dilution test include expense, inability to perform the test in the office, and need to determine if required concentrations of a certain antibiotic are feasible. ideally, blood concentrations of drugs should be more than times the mic and urine concentrations to times the mic. mic sensitivity for topically administered antimicrobials is seldom determined because these methods are based on blood or urine concentrations. disk diffusion test • this is the most widely used method in clinical practice (i.e., kirby-bauer technique). a zone of inhibition of bacterial growth is noted around these assays prove previous exposure to a bartonella spp., but as serologic cross-reaction occurs between b. henselae, b. clarridgeaie, and b. koehlerae, assays based on b. henselae antigens cannot differentiate between the agents. current bartonella spp. infection is proven by culture or pcr assay results. results of some pcr assays or genetic sequencing can be used to identify the species of bartonella involved with the infection. cats can be infected with more than one bartonella spp. concurrently. detection of local antibody production by the eye and documentation of bartonella spp. dna in aqueous humor has been used to document uveitis as a result of bartonellosis. (see appendix i for laboratories for infectious diseases.) up to % of healthy cats exposed to c. felis can be seropositive, and so antibody tests results alone cannot be used to prove clinical bartonellosis. between % and % of seronegative cats are bacteremic, and so antibody test results cannot be used to exclude bartonella spp. infection from the differential list. positive blood culture or pcr results prove current infection but do not document clinical illness. repeated bacteremia has been detected in experimentally inoculated and naturally infected cats; therefore a single negative blood culture or pcr result does not exclude infection. because of these findings, it is currently recommended to combine serologic test results with those of blood culture or pcr assay results when evaluating clinically ill cats for bartonellosis. (see appendix i for laboratories for infectious diseases.) if test results are positive and there is no better explanation for the cause of illness, treatment may be indicated. to date, there has been no proven clinical benefit to following bartonella spp. assay results after positive response to therapy. because serologic test results do not accurately correlate with presence of bacteremia and individual culture or pcr assay results can be falsely negative, there is no indication for testing healthy cats for bartonella spp. infection. , the centers for disease control and prevention recommends maintaining flea control and avoiding bites and scratches to avoid bartonellosis. healthy cats used for blood donors should be seronegative and culture or pcr negative and should be maintained on flea control products. occasional indications • dogs from endemic areas or with an appropriate travel history with unexplained myocarditis, endocarditis, granulomatous lymphadenitis, cutaneous vascular disease, hemolytic anemia, polyarthritis, idiopathic effusions, granulomatous meningoencephalitis, granulomatous rhinitis, or thrombocytopenia should be considered for bartonella vinsonii testing. , the full spectrum of b. henselae-associated illnesses in dogs has not been determined but appears to be similar to that for b. vinsonii. , based on seroprevalence studies, rural dogs with fleas or ticks are most likely to be exposed to bartonella spp. analysis, artifacts, and interpretation • circulating antibodies against b. vinsonii and b. henselae are most commonly detected by ifa. infection can be documented be significant in chronic infections or in females. in samples of prostatic fluid obtained by ejaculation, infection is diagnosed if the specimen contains greater than or equal to times more bacteria than the urethral sample. culture of prostatic aspirates may be more accurate. blood cultures can be difficult to interpret. falsepositive results are caused by contamination with normal cutaneous microflora, including corynebacterium spp., bacillus spp., coagulase-negative staphylococci, anaerobic diphtheroids, streptococci, and clostridium spp. isolation of the same organism from two or more cultures strongly suggests that it is pathogenic, whereas growth in only one culture is less certain unless it is a pathogenic bacterium unlikely to be a contaminant. csf and synovial fluid are normally sterile; any growth in an aseptically obtained sample is significant. for dermatophyte culture, hair is clipped from the lesion periphery; hair shafts are plucked with forceps and cultured on dermatophyte test medium (dtm) or derm duet. sc and deep fungal infections are best diagnosed by cytologic or histopathologic evaluation, with or without serology. if organisms cannot be identified, cutaneous lesions can be cultured, but these are rarely useful owing to overgrowth by resident bacteria and fungi. the lesion is prepared as for dermatophytes, and a swab is cultured onto sabouraud and mycose medium. systemic and sc fungi may require weeks' cultivation on sabouraud medium for growth to occur. bartonellosis, feline (bartonella henselae, b. clarridgeaie, b. koehlerae) occasional indications • to date, most cats with clinical bartonellosis have been infected with bartonella henselae. however, b. clarridgeaie, b. koehlerae, and b. quintana have been identified in some clinically ill cats. , bartonella henselae, b. clarridgeaie, and b. koehlerae are transmitted among cats by ctenocephalides felis and so cats with a history of flea infestation are more likely to be infected. most cats exposed to bartonella spp. maintain subclinical infections; however, fever, uveitis, lymphadenopathy, endocarditis, myocarditis, hematuria, and hyperglobulinemia have been documented convincingly in experimentally infected or naturally exposed cats. results have been equivocal for a link between bartonella spp. infection and gingivitis or stomatitis. bartonella testing may be indicated in cats with one or more of these clinical syndromes for which another explanation is not readily apparent. analysis, artifacts, and interpretation • circulating antibodies are detected by immunofluorescent antibody assay (ifa), enzyme-linked immunosorbent assay (elisa), and western blot immunoassay. , results of laboratories for infectious disease); however, clinical utility of this assay has not been documented. definitive diagnosis requires demonstration of the organism by culture, histopathologic evaluation of tissue, or pcr assay. presumptive diagnoses of clinical lyme disease in dogs can be based on appropriate clinical, historic, and laboratory evidence of disease combined with positive serologic testing and response to therapy. occasional indications • dogs with reproductive tract abnormalities, lymphadenomegaly, hyperglobulinemia, discospondylitis, or uveitis should be suspected of having brucellosis and be screened for antibodies against brucella canis. analysis, artifacts, and interpretation • circulating antibodies are most commonly detected in serum by rapid slide agglutination test (rsat), tube agglutination test (tat), agar gel immunodiffusion (agid), and elisa. , the rsat and tat are screening procedures; an rsat for point-of-care use is commercially available. both assays should be performed with -mercaptoethanol ( -me) to eliminate heterologous igm agglutinins responsible for most false-positive reactions. false-positive reactions in the -me tat may be the result of autoagglutination in hemolyzed samples. agid can be performed using cell wall antigens or cytoplasmic antigens. agid performed with cytoplasmic antigens is the most specific antibody assay; agid performed with cell wall antigens is the most sensitive. because of nonspecific precipitin reactions, positive results in agid with cell wall antigens are difficult to interpret. minimal time between infection and a positive test result varies with the test, but most infected dogs are seropositive in the -me tat and agid by week to after infection. -me tat titers from different laboratories cannot be meaningfully compared; however, a titer of : to : is generally suspicious, whereas a titer greater than or equal to : usually correlates with isolation of b. canis from blood culture. after cessation of bacteremia, -me tat titers rapidly decrease to less than : within a few weeks and remain low ( : to : ) for months or longer. in agid, antibodies to external antigens persist for a few weeks, whereas antibodies to internal (i.e., cytoplasmic) antigens persist up to months after cessation of bacteremia. although these animals are abacteremic, b. canis can be isolated from selected organs (e.g., epididymis, prostate). when the -me rsat or tat is used as a screening test and results are positive, a tentative diagnosis of brucellosis is made; positive blood culture or agid should be used to confirm results. if blood culture or agid is negative, brucellosis is unlikely. if -me rsat or tat results are negative in a dog strongly suspected of having brucellosis, the test should be repeated in weeks to preclude the possibility of early infection. definitive diagnosis requires isolation of b. canis, although this is not always achieved. although blood culture is ideal, it is inconvenient and expensive. culture of urine or an ejaculate may also be performed in males. occasional indications • dogs from areas endemic for ixodes ticks or with an appropriate travel history and fever, lameness, glomerulonephritis, or nonseptic, suppurative polyarthritis should be suspected of having lyme disease (borreliosis) and screened for antibodies against b. burgdorferi. , , analysis, artifacts, and interpretation • circulating antibodies are detected in serum by ifa, elisa, and western immunoblot. the organism can be documented in tissues by culture, histologic techniques, or pcr assay result. (see appendix i: select laboratories for infectious disease.) serum antibodies are generally used to screen for exposure to b. burgdorferi in dogs. immunoglobulin m (igm) and immunoglobulin g (igg) antibodies against b. burgdorferi can be detected in canine serum. titers considered significant vary by laboratory and assay. both antibody classes can persist in serum for months after exposure. depending on the assay used, cross-reactivity with b. burgdorferi antigens used in ifa and elisa can occur with other spirochetes; thus a positive titer does not always document exposure to b. burgdorferi. b. burgdorferi vaccines induce antibodies that are detected by some ifa and elisa. western immunoblot can be used to differentiate vaccine-induced antibodies from antibodies resulting from natural infection. antibodies against the c peptide of b. burgdorferi are rarely induced by vaccination; point-of-care assays using this peptide are commercially available. because b. burgdorferi migrates through the tissues, most dogs with borreliosis are positive for antibodies by the time illness is detected. healthy dogs develop the same antibody responses as clinically ill dogs, however. because of these factors, interpretation of positive serum antibody titers is difficult. serum antibodies against b. burgdorferi only document exposure to b. burgdorferi (or a similar antigen), not clinical disease. an assay to quantify antibodies against the c peptide is commercially available (see appendix i: select or oral ulceration, particularly if tick exposure, rabbit ingestion, or potential for human infection is confirmed. tularemia is a direct zoonosis from clinically ill cats to people. analysis, artifacts, and interpretation • clinicians measure antibodies in serum using a microscopic agglutination (ma) assay. , (see appendix i: select laboratories for infectious disease.) time between acquisition of infection and a positive titer is not known. in dogs, titers of : to : are commonly detected in acute infections. in cats with tularemia, ma titers are generally greater than : . development of a positive titer or a fourfold increase in titer between acute and convalescent sera ( weeks later) is presumptive evidence of infection. definitive diagnosis is obtained by isolation of the bacterium in a culture of a blood specimen or by identification in tissue by immunofluorescence. occasional indications • dogs and cats with nasal or pulmonary disease can be serologically screened for antibodies against aspergillus fumigatus; cats are affected less frequently than dogs. results are generally interpreted in conjunction with cytology, radiology, histopathology, and culture. analysis, artifacts, and interpretation • agid, counterimmunoelectrophoresis (ciep), and elisa have been used to detect circulating antibodies against a. fumigatus in serum. presence of serum antibodies can represent either exposure or infection, and some dogs with nasal aspergillosis are falsely negative for serum antibodies. in one recent study of dogs with and without nasal aspergillosis, the sensitivity, specificity, and positive and negative predictive values for serum anti-aspergillus antibody results were %, %, %, and %, respectively. owing to persistence of titers in some treated dogs (i.e., months), monitoring titers to assess therapeutic response is not recommended. dogs or cats infected with penicillium spp. will be seronegative if assessed only in assays using a. fumigatus antigens. radiographic demonstration of nasal turbinate destruction suggests aspergillosis or nasal neoplasia. cytologic analysis (see figure - ) and culture of canine nasal exudate alone are not diagnostic because fungal elements may be nondetectable in affected dogs whereas they may be found in noninfected dogs (including dogs with nasal tumors). the organism is sometimes difficult to culture from an aspergilloma (fungal ball). nasal lavage is a low-yield procedure for demonstration of the organism; nasal biopsy is suggested (see chapter ) . definitive diagnosis should be based on three factors: ( ) histopathologic evidence of tissue invasion, ( ) an aspergilloma combined with serologic and culture evidence of infection, or ( ) serologic and radiographic evidence of infection (i.e., bone lysis). in rare cases with growth usually occurs within days, but cultures should be held for to weeks before being discarded. at least three cultures from specimens obtained several days apart are recommended. occasional indications • serologic testing for antibodies against leptospira spp. should be considered in dogs with undiagnosed fever, ecchymoses, vomiting, diarrhea, muscle pain, uveitis, coughing, dyspnea, renal pain, thrombocytopenia, renal failure (particularly acute), or increased activities of hepatic enzymes. , the most common pathogenic serovars in dogs include leptospira canicola, l. icterohaemorrhagiae, l. grippotyphosa, l. bratislava, l. autumnalis, and l. pomona. analysis, artifacts, and interpretation • circulating antibodies are detected in serum by the microscopic agglutination test (mat), elisa (igm, igg), and microscopic microcapsular agglutination test (mcat). most diagnostic laboratories use mat. the primary disadvantage of serologic testing is that it is difficult to determine whether positive titers are caused by active infection, previous infection, or vaccination. in addition, when results from different laboratories are compared, results commonly vary. a laboratory that assesses for antibodies against multiple serovars and participates in the international leptospirosis society proficiency program should be used. antibodies are detected by mat days to weeks after infection. acutely infected dogs are often mat negative; dogs with suggestive clinical signs of disease but negative mat results should be retested in to days; development of a positive titer confirms recent infection. a fourfold increase in antibody titer also can confirm recent infection. vaccination can induce positive mat titers. because of the presence of cross-reactive antibodies, one cannot assume that the serovar inducing the highest titer during acute infection is the serovar causing infection. the combination of increasing antibody titers with appropriate clinical pathologic abnormalities and clinical findings suggests clinical leptospirosis. definitive diagnosis requires demonstration of the organism by urine dark-field microscopy, phase-contrast microscopy, culture, or pcr assay. examination of urine for leptospires is a low-yield procedure. demonstration of spirochetes by histopathologic evaluation of renal tissue leads to a presumptive diagnosis, which may be confirmed by tissue culture. culture or pcr assay can be of most benefit early in the course of infection when mat results are negative. the organism is in high levels in blood for the first days of infection and then is highest in urine. repeated culture may be needed because of intermittent shedding. administration of antimicrobial therapy can result in false-negative results. rare indications • testing for tularemia (i.e., rabbit fever) should be considered in animals from endemic areas developing fever, lymphadenomegaly, weight loss, organism is not demonstrated by cytology (figure - ) , histopathology, or culture. feline disease is rare but has been associated with nodular or ulcerative skin lesions, pulmonary interstitial disease, osteomyelitis, uveitis, and cns disease. an antigen test has been evaluated for use with samples from humans but has not been validated for use with samples from dogs or cats. analysis, artifacts, and interpretation • circulating antibodies are detected in serum by complement fixation (cf), agid, elisa, latex agglutination (la), and tube precipitin (tp) tests. tp detects igm antibodies; cf and agid detect igg antibodies. false-negative results in tp occur in early infections (< weeks), chronic infection, rapidly progressive acute infection, and primary cutaneous coccidioidomycosis. false-positive results in the cf test are caused by anticomplementary serum, which may be caused by bacterial contaminants or immune complexes. finally, cross-reactions in patients with histoplasmosis and blastomycosis may occur with all tests. after resolution of disease, cf titers decrease over weeks but remain positive at a low titer (e.g., : ) for months. definitive diagnosis requires demonstration of the organism on smears, aspirates, histopathologic evaluation, or culture. the organism is often difficult to demonstrate. wet mount examination of unstained or stained (periodic acid-schiff) smears or aspirates is more suitable than dry mounts, which may distort the spherules. common thoracic radiographic findings are mixed interstitial, bronchial, and alveolar pulmonary patterns and hilar lymphadenomegaly. positive serologic test results and characteristic radiographic changes allow tentative diagnosis. disseminated disease, cytologic evaluation of aspirates of affected tissue may be useful. if the organism cannot be demonstrated by biopsy samples obtained through the nares, positive serologic test results may support exploratory surgery. occasional indications • dogs from endemic areas with fever, weight loss, pulmonary interstitial disease, lymphadenomegaly, uveitis and blindness, ulcerative or draining skin lesions, undiagnosed prostatic or testicular disease, intracranial disease, osteomyelitis, or (rarely) renal disease can be serologically screened for antibodies against blastomyces dermatitidis and b. dermatitidis antigens if the organism is not demonstrated by cytology (see figure - ), histopathology, or culture. , in endemic areas, screening for antibodies against b. dermatitidis should be considered in cats with pulmonary interstitial disease, intracranial disease, lymphadenomegaly, ulcerative or draining skin lesions, or uveitis and blindness. analysis, artifacts, and interpretation • circulating antibodies are most commonly detected in serum by agid. blastomyces antigens in urine and blood of dogs have been measured using the mvista blastomyces antigen eia (miravistalabs.com). because subclinical canine infections are unusual, positive serum antibody test results are considered significant. false-negative results occur in animals with peracute infection or with advanced cases overwhelming the immune system. in dogs, the sensitivities of antigen testing of urine, antigen testing of serum, and agid serum antibody testing were . %, . %, and . %, respectively. thus dogs with a high index of suspicion for blastomycosis that are negative for serum antibodies should be screened for urine or serum antigens. many cats with blastomycosis are serum antibody negative. whether serum or urine antigen testing will aid in the diagnosis of blastomycosis in cats remains to be determined. definitive diagnosis requires identification of the yeast by cytology, histopathology, or fungal culture. impression smears from skin lesions and aspirates from enlarged lymph nodes frequently reveal organisms; recovery of organisms from transtracheal aspiration, pulmonary aspiration biopsy samples, or urine is less consistent. culture requires to days and is of lower yield than cytology or biopsy. diffuse nodular interstitial pulmonary disease and hilar lymphadenomegaly are common radiographic findings. positive serologic results combined with appropriate clinical signs and radiographic abnormalities allow presumptive diagnosis. occasional indications • dogs from endemic areas with pulmonary interstitial disease, fever of undetermined origin, hilar lymphadenomegaly, osteomyelitis, uveitis, pericarditis, and nodular or ulcerative skin lesions can be screened for antibodies against c. immitis if the occasional indications • cats and rarely dogs with undiagnosed respiratory (especially nasal), cns, eye (especially uveal tract), and skin (especially nodular or ulcerative lesions) infections can be screened for cryptococcus neoformans and c. gattii antigens if the organism is not demonstrated by cytology, histopathology, or culture. , analysis, artifacts, and interpretation • measurement of antibodies against c. neoformans or c. gattii is not clinically useful. cryptococcal antigen is detected in serum, aqueous humor, or csf using latex agglutin ation. negative serum la titers may occur in early disease or uncommonly in chronic low-grade infections, in chemotherapy-induced remission, or in nondisseminated disease. specificity of the serum la is high. a titer of greater than : in serum or csf is positive; very high titers are commonly detected. in some animals, decreases in serum titer parallel response to therapy. positive titers occur in some animals after apparently successful clinical responses, suggesting persistent low-grade infection or false-positive results. , cryptococcal encephalitis may cause a positive csf la titer despite a negative serum la. definitive diagnosis is based on cytologic, histopathologic, or culture demonstration of the organism or a positive la test result. cytology is commonly positive (see figure - ) because there are usually numerous yeasts found in affected tissues (i.e., nasal and cutaneous lesions, aqueous and vitreous humor). the organism can occasionally be recovered from nasal washings of normal animals. csf may contain the yeast, but concentration techniques (i.e., cytocentrifugation) should be used. routine cytology stains (e.g., wright) are adequate to demonstrate the organism. large numbers of organisms are usually visible despite little or no inflammation. culture is seldom necessary. serologic testing is used if the yeast cannot be demonstrated cytologically or to monitor response to treatment. a pcr assay has been used to amplify the organism dna from tissue but has not been assessed extensively to date. rare indications • animals with weight loss, pulmonary interstitial disease, uveal disease, diarrhea, or lymphadenomegaly can be serologically screened for antibodies against histoplasma capsulatum if the organism is not demonstrated by cytology, histopathology, or culture. analysis, artifacts, and interpretation • primarily, agid is used to detect circulating antibodies in serum. presence of serum antibodies confirms exposure but not clinical illness because of infection. agid has questionable clinical usefulness because titers persist longer than year after resolution of disease in some animals, and both false-positive and false-negative results occur. antibody testing is even less rewarding in cats. definitive diagnosis requires demonstration of the organism by cytology (see figure - a), biopsy, culture, or pcr assay. an enzyme immunoassay test used to detect histoplasma antigen in the urine of people has not been validated for use with samples from dogs and cats. the organism is more difficult to demonstrate than b. dermatitidis; however, cytologic examination of rectal scrapings in dogs with colonic histoplasmosis is often diagnostic. fine-needle aspiration of other organs may demonstrate the organism. in most cats with systemic histoplasmosis, the organism is identified on bone marrow cytology. thoracic radiographs are indicated if pulmonary histoplasmosis is suspected; a nodular interstitial pattern is expected. culture of h. capsulatum is of lower yield than biopsy. rare indications • babesia canis vogeli and b. gibsoni infect dogs in the united states, and diagnostic tests can be performed in dogs from endemic areas or in those with an appropriate travel history that have fever, anemia, icterus, splenomegaly (i.e., acute babesiosis), or intermittent fever and weight loss (i.e., chronic babesiosis). although babesiosis can cause anemia in cats, species infecting cats are not found in the united states. exposure to r. sanguineus ticks (b. canis) or pit bull terriers (b. gibsoni) are risk factors for exposure to the two agents in dogs. analysis, artifacts, and interpretation • circulating antibodies are detected in serum by ifa. (see appendix i: select laboratories for infectious disease.) in most laboratories, titers greater than : are considered positive. experimentally infected dogs develop detectable igg titers approximately weeks after infection. false-negative results can occur in immature dogs, in peracute cases, or in dogs with concurrent immunosuppression. antibodies against b. gibsoni and b. canis may or may not cross-react, depending on the antigen source used by a particular laboratory, and so specific ifa should be used for both organisms. dna of b. canis and b. gibsoni can be amplified by pcr assay, and positive results indicate current infection. , however, both antibodies and dna can be detected in dogs that are healthy and those that are clinically ill. antibody titer magnitude does not correlate to the presence or absence of disease. a titer of greater than : was suggested for b. gibsoni, but not all infected dogs achieve a titer of this magnitude. it is important to determine which species are involved in a case because response to treatment varies. duration of positive titers after resolution of disease is unknown. in untreated experimentally infected dogs, titers remained high for at least months. untreated, seropositive dogs should be considered carriers of the infection. treatment is indicated only for seropositive, clinically ill dogs. definitive diagnosis requires demonstration of the organism in blood smears stained with romanowskytype preparations (e.g., wright and giemsa) (figure - ) or pcr assay. organisms are best found in blood (particularly in acute disease) from a microcapillary system (e.g., ventral surface of ear or toenail). note: shape of the organism may be distorted in old blood. infection, not clinical disease. however, if antibodies are detected in an animal with appropriate clinical signs of disease, treatment may be indicated in an attempt to slow progression. definitive diagnosis is based on demonstration of the organism in tissues. the organism can be differentiated from t. gondii structurally, by immunohistochemistry, and by pcr assay. n. caninum oocysts are found in the feces of some dogs. occasional indications • healthy cats • t. gondiispecific antibodies form in serum, aqueous humor, and csf of healthy and diseased cats or dogs. antibodies do not directly correlate with clinical toxoplasmosis. no serologic test is currently available that accurately predicts when a seropositive cat previously shed oocysts. a seropositive cat is less likely than a seronegative cat to shed the organism if re-exposed. elisa, ifa, and western blot immunoassay can be adapted to detect various antibody classes; igm and igg are those usually assessed. t. gondii-specific igm is detectable in serum by elisa in approximately % of subclinically ill cats to weeks after experimental induction of toxoplasmosis; these titers generally are negative less than weeks after infection. detectable igm titers were present in the serum of . % of cats in a study of clinical toxoplasmosis; igg titers were detected in %. igm titers persist in some clinically ill cats for greater than weeks; these cats are frequently co-infected with fiv or have ocular toxoplasmosis. after repeat inoculation with t. gondii, primary inoculation with the petaluma isolate of fiv, and administration of glucocorticoids, some cats with chronic toxoplasmosis experience short-term recurrence of detectable igm titers. healthy and clinically ill dogs occasionally develop detectable igm titers. kinetics of postinfection igm titers in dogs is unknown. after experimental induction of infection in subclinically ill cats, t. gondii-specific igg can be detected by elisa in serum from most cats by weeks. positive igg antibody titers generally persist for years after infection. single high igg titers have been suggested to indicate recent or active infection. the author, however, has demonstrated igg antibody titers greater than : , in subclinically ill cats years after experimental induction in chronic disease or asymptomatic carriers, demonstration of organisms is unreliable, and a tentative diagnosis is based on clinical signs and a positive titer. dogs with babesiosis are often coombs positive (see chapter ). rare indications • serology for neospora caninum can be performed in dogs with clinical evidence of polyradiculomyositis, including progressive ascending rigid paralysis, dysphagia, muscle atrophy, and (rarely) myocardial dysfunction or pneumonia. analysis, artifacts, and interpretation • circulating antibodies are detected in serum by ifa. (see appendix i: select laboratories for infectious disease.) a presumptive diagnosis of neosporosis can be made by combining appropriate clinical signs of disease and positive serology or presence of antibodies in csf with the exclusion of other causes inducing similar clinical syndromes, in particular, toxoplasma gondii. igg antibody titers greater than or equal to : have been detected in most dogs with clinical neosporosis; minimal serologic cross-reactivity exists with t. gondii at titers greater than or equal to : . because the organism is a tissue protozoan, seropositivity may correlate with permanent infection. circulating antibodies against neospora caninum only document base an antemortem diagnosis of clinical toxoplasmosis on these tests alone. antemortem diagnosis of clinical toxoplasmosis can be tentatively based on the combination of the following: • demonstration of serologic evidence of infection • clinical signs of disease referable to toxoplasmosis • exclusion of other common causes • positive response to appropriate treatment t. gondii was detected by pcr in aqueous humor of . % of cats with uveitis. the organism also can be detected transiently in aqueous humor and blood of healthy, experimentally inoculated cats, however, making the positive predictive value of the pcr for clinical disease less than %. rare indications • serologic testing for antibodies against trypanosoma cruzi should be considered in dogs from endemic areas and those with generalized lymphadenomegaly, neurologic signs, or myocardial dysfunction (especially second-or third-degree heart block or ventricular tachycardia). analysis, artifacts, and interpretation • ifa, direct hemagglutination, and cf usually detect circulating antibodies in canine sera. dogs are generally seropositive weeks after infection. a positive titer documents exposure to the organism, not clinical disease. positive titers vary by assay. definitive diagnosis requires demonstration of the organism on blood smear, lymph node impression, or buffy coat and plasma interface smear. t. cruzi is occasionally found in peripheral blood without demonstrable organisms in tissue. a standard workup for myocardial disease, including chest radiographs, electrocardiogram, electrolytes, and echocardiography (if available), is indicated. alternatively, t. cruzi amastigotes can be demonstrated in tissues. pcr can be used to amplify organism dna. indications • dogs living in ixodes spp. endemic areas with acute fever or polyarthritis should be screened for antibodies against anaplasma phagocytophilum (previously ehrlichia equi) or for a. phagocytophilum dna in blood by pcr assay. , , the role this organism plays in chronic disease syndromes in dogs is unknown. analysis, artifacts, and interpretation • antibodies against a. phagocytophilum in serum can be measured by ifa or commercially available elisa (snap dx, table - , idexx corporation, westbrook, me). the antibodies have only variable cross-reactivity with other anaplasma spp. ehrlichia spp., or neorickettsia spp. and so positive test results likely indicate exposure to a. phagocytophilum. infected dogs can be seronegative when clinical signs of of toxoplasmosis. a positive igg antibody titer in a single serum sample only documents exposure, not recent or active disease. demonstration of an increasing igg titer can document recent or active disease. unfortunately, the time span from the first detectable positive igg titer to the maximal igg titer is approximately to weeks, leaving a very narrow window for documenting an increasing titer. many cats with clinical toxoplasmosis have chronic low-grade signs, and they are not tested until their igg antibody titers have reached maximal values. in humans with reactivation of chronic toxoplasmosis, igg titers only rarely increase; cats appear to be the same. several agglutination tests have been evaluated using cat serum. an la and an indirect hemagglutination assay (iha) are commercially available. these assays are not species specific and potentially detect all classes of serum immunoglobulins directed against t. gondii. unfortunately, la and iha rarely detect antibody in feline sera when positive for only igm by elisa. modified agglutination using formalin-fixed tachyzoites is the most sensitive procedure for detection of t. gondii antibodies in cat sera, but it is generally unavailable commercially. local production of t. gondii-specific igg in csf and aqueous humor occurs in experimentally inoculated, subclinically ill cats and in cats and dogs with clinical disease because of toxoplasmosis. local igm production has only been detected in csf and aqueous humor of animals with clinical disease. most cats with uveitis and production of t. gondii-specific antibodies in aqueous humor have responded to administration of anti-toxoplasma drugs, suggesting that aqueous humor antibody testing aids in diagnosis of clinical ocular feline toxoplasmosis. (see appendix i: select laboratories for infectious disease.) fecal examination • fecal oocysts can be demonstrated using flotation techniques with various solutions with specific gravities from . to . . sugar solution centrifugation is probably the optimal technique. oocysts of t. gondii are to µm in diameter, approximately oneeighth the size of toxocara cati eggs. focusing on only one plane of the microscope slide or coverslip can result in oocysts being overlooked. the oocysts cannot be distinguished grossly from hammondia hammondi or besnoitia darlingi (nonpathogenic coccidians infecting cats). sporulated oocysts isolated from feces can be inoculated into mice or tissue cultures for definitive identification. because oocyst shedding has rarely been documented in cats with subfatal, clinical toxoplasmosis, the diagnostic usefulness of fecal examination is limited. cats with clinical signs referable to t. gondii should undergo fecal evaluation, however, because of potential zoonotic risk. interpretation • exposure to t. gondii is suggested by finding antibodies in serum, aqueous humor, or csf. recent or active toxoplasmosis is suggested by finding an igm titer greater than : or a fourfold or greater increase in igg titer, or documenting local antibody production in aqueous humor or csf. because t. gondii-specific antibodies can also be detected in the serum, csf, and aqueous humor of healthy, infected animals, one cannot assay results in clinically ill dogs, e. canis appears to be the organism in this group that is most commonly associated with clinical illness. serologic testing or pcr assay for e. canis is indicated for dogs from endemic areas or with an appropriate travel history and thrombocytopenia, anemia, leukopenia, hyperglobulinemia, proteinuria, polyarthritis, fever, uveitis, lymphadenomegaly, hepatosplenomegaly, or inflammatory cns disease, particularly if the animal has a history of exposure to rhipicephalus ticks. analysis, artifacts, and interpretation • circulating antibodies against e. canis are detected in serum by ifa or elisa; they do not cross-react with rickettsia rickettsii or anaplasma platys (see canine thrombocytotropic anaplasmosis later). serologic cross-reactivity between e. canis antibodies and those against a. phagocytophilum (previously ehrlichia equi; see canine granulocytotropic anaplasmosis earlier), e. chaffeensis, e. ewingii (see canine granulocytotropic ehrlichiosis earlier), and neorickettsia risticii (previously e. risticii) is variable. multiple serologic tests are needed to exclude all of the ehrlichia spp., anaplasma spp., and neorickettsia spp. from the differential list. thus pcr assays are often combined with serologic tests. in addition, pcr assay results may be positive before seroconversion in some dogs. in experimentally infected dogs, antibodies against e. canis can be detected as early as days and are almost always present by days after inoculation. antibody titers continue to increase for weeks to months after inoculation in untreated, experimentally infected dogs. e. canis titers of less than : are suspect and should be rechecked in approximately to days; a titer of : or higher is diagnostic. initial positive results in a recently marketed point-of-care test occur at approximately : . positive titers revert to negative to months after resolution of infection; persistence of titers for greater than or equal to months suggests a carrier state. however, positive antibody titers have been detected for months after apparently successful therapy in some naturally infected dogs. clinically ill, seropositive dogs should be treated a minimum of days and until clinical and laboratory abnormalities have resolved. whether to treat healthy, seropositive dogs is controversial; the issues involved in this decision were recently reviewed. , the clinician can make a definitive diagnosis of e. canis infection by demonstrating morulae (i.e., clusters of the organism) in mononuclear cells, culture, or pcr assay. morulae are rarely found on routine blood smear or bone marrow aspiration cytology unless the dog has been immunosuppressed. cytology and pcr assay results can be falsely negative in dogs that have been treated. ehrlichia spp. can be isolated by tissue culture of heparinized infected canine blood or bone marrow aspiration samples, but culture is of limited availability, expensive, and of low yield. ehrlichia spp. can be detected in whole blood by pcr, which has potential benefit for use in monitoring treatment. (see appendix i: select laboratories for infectious disease.) the consensus statement on ehrlichial disease of small animals from the infectious disease study group of the american college of veterinary internal medicine (acvim) states the following: disease first occur and can be immediately assessed by pcr assay or have repeat serology performed in approximately weeks to evaluate for seroconversion. alternately, both assays can be performed at the same time. antibiotic therapy can lead to falsely negative pcr assay results. a. phagocytophilum dna and antibodies can be detected in both healthy and clinically ill dogs, and so positive test results do not document clinical illness. morulae are only rarely documented cytologically in clinical specimens. indications • cats living in ixodes spp. endemic areas with fever, mild thrombocytopenia or clinical evidence of polyarthritis should be evaluated for a. phagocytophilum dna in blood by pcr assay. , antibodies can be detected in serum by ifa, but a standardized test is not available. (see appendix i: select laboratories for infectious disease.) analysis, artifacts, and interpretation • dna of a. phagocytophilum has been amplified from several cats with clinical signs of anaplasmosis. some of the cats were seronegative when first assayed but had seroconverted when assayed at a later date. whether the point-of-care assay used to detect a. phagocytophilum antibodies in canine serum is valid for use with cat serum is unknown. antibiotic therapy can lead to falsely negative pcr assay results. a. phagocytophilum dna and antibodies can be detected in both healthy and clinically ill cats, and so positive test results do not document clinical illness. morulae are only rarely documented cytologically in clinical specimens. untreated healthy cats can be pcr positive for weeks after tick exposure. indications • dogs living in the midwest united states with evidence of fever or clinical evidence of polyarthritis should be evaluated for ehrlichia ewingii dna in blood by pcr assay. , specific antibodies can be detected in serum, but a standardized test is not currently available. analysis, artifacts, and interpretation • there is variable cross-reactivity between e. canis antigens and e. ewingii antigens, and so serologic tests for e. canis will not always be positive when the infecting agent is e. ewingii. pcr assays that amplify the dna of e. ewingii are available and should be performed on blood from dogs with suspected acute infection. antibiotic therapy can lead to falsely negative pcr assay results. healthy dogs can be pcr positive for e. ewingii dna in blood. common indications • ehrlichia canis, e. chaffeensis, and neorickettsia risticii all infect monocytes of dogs and can be associated with clinical illness. based on pcr occasional indications • serologic testing for rickettsia rickettsii antibodies (rocky mountain spotted fever [rmsf]) is indicated for dogs from endemic areas or with an appropriate travel history and acute onset of fever, lymphadenomegaly, petechiae, neurologic signs, stiff gait, peripheral edema, dyspnea, or scleral congestion. history of tick exposure is inconsistent. exposed dogs either develop acute disease with approximately a -day clinical course or are subclinically infected. the primary tick vectors are active from spring to fall in most of the united states; therefore rmsf should only be considered a principal differential diagnosis for clinically ill dogs during this time span. the majority of cases are diagnosed in southeastern states. analysis, artifacts, and interpretation • antibodies against r. rickettsii in canine serum can be measured by ifa, elisa, and la. elisa or ifa can detect igm and igg antibodies against rmsf. la is not antibody class-specific. cutoffs for positive antibody titers, as well as specificity and sensitivity, vary by assay. antibodies against the nonpathogenic spotted fever group rickettsia (r. belli, r. montana, r. rhipicephali) cross-react with r. rickettsii antigens. in dogs with clinical illness because of rmsf, igm antibody titers are generally positive. because igm has short duration in serum, false-negative results may occur with igm testing. false-positive results are most common in the igm elisa. positive igg titers are detectable to days after infection. serum samples with igg titers greater than or equal to : are generally considered positive. if igg or igm antibodies are not detected in a patient with clinical and laboratory evidence of rmsf, a convalescent igg titer to weeks later is recommended. timing of the second titer is not critical because igg antibody titers do not decrease for at least to months after infection. documentation of seroconversion or a fourfold increase in igg titer is consistent with recent infection. a presumptive diagnosis of canine rmsf can be based on the combination of appropriate clinical, historic, and clinicopathologic evidence of disease; serologic test results; exclusion of other causes of the clinical abnormalities; and response to anti-rickettsial drugs. documentation of seroconversion or an increasing titer to weeks after initial serologic testing suggests recent infection. diagnostic criteria used in one recent study included a fourfold rise in antibody titer or a single titer of greater than or equal to : if the initial titer was submitted week or more after initial onset of clinical abnormalities. positive serum antibody test results alone do not prove rmsf because subclinical infection is common. in addition, positive serum antibody tests do not document infection by r. rickettsii because infection with nonpathogenic spotted fever group agents induce cross-reacting antibodies. demonstration of r. rickettsii by inoculating affected tissues or blood into susceptible laboratory animals or by documenting the organism in endothelial cells using direct fluorescent antibody staining leads to a definitive diagnosis of rmsf, but these techniques are not clinically practical. pcr can be used to document the if pcr is used to monitor treatment, the pcr assay should be repeated after antimicrobial therapy has been discontinued for weeks. if pcr results are positive, an additional weeks of treatment should be given with the pcr assay repeated after antimicrobial therapy has been discontinued for weeks. if pcr results are positive after treatment cycles, use of an alternate antiehrlichial drug should be considered. if pcr results are negative the test should be rechecked in months; if still negative therapeutic elimination is likely. however, the organism may be sequestered in other tissues like the spleen. rare indications • cats with thrombocytopenia, anemia, leukopenia, hyperglobulinemia, proteinuria, polyarthritis, fever, or lymphadenomegaly should be evaluated for ehrlichia spp. dna in blood by pcr assay if no other obvious cause exists. , , to date, e. canis is the monocytotropic strain amplified from naturally exposed cats. antibodies can be detected in serum by ifa, but a standardized test is not available. (see appendix i: select laboratories for infectious disease; protatek reference laboratories.) analysis, artifacts, and interpretation • while serum antibodies against e. canis have been detected in the serum of some cats, a number of cats with e. canis dna amplified from blood were seronegative. , thus while ifa testing is available for e. canis antibodies in feline serum, it should be combined with pcr assay. antibodies against e. canis can be detected in serum from healthy cats and therefore cannot be used alone to make a definitive diagnosis of ehrlichiosis. a tentative diagnosis of feline ehrlichiosis is based on the combination of clinical signs, positive serologic or pcr assay results, exclusion of other known causes, and response to tetracyclines. occasional indications • testing for anaplasma platys infection is indicated for dogs from endemic areas or with appropriate travel history and thrombocytopenia or endogenous uveitis. , analysis, artifacts, and interpretation • circulating igg antibodies against a. platys are detected in serum by ifa. antibodies against a. platys react with a. phagocytophilum antigen used in a commercially available kit. experimentally infected dogs become antibody positive to days after infection. infected dogs can be seronegative when clinical signs of disease first occur and can be immediately assessed by pcr assay or have repeat serology performed in approximately weeks to evaluate for seroconversion. alternately, both assays can be performed at the same time. antibiotic therapy can lead to falsely negative pcr assay results. a. platys dna and antibodies can be detected in both healthy and clinically ill dogs, and so positive test results do not document clinical illness. morulae are only rarely documented cytologically in clinical specimens. analysis, artifacts, and interpretation • determining serum antibodies to feline or canine parvoviruses or coronaviruses is rarely performed clinically because positive results do not correlate with clinical disease. a point-ofcare assay for detection of canine parvovirus antibodies is available. vaccinated dogs seropositive in this assay probably do not need to be boosted. detecting fecal shedding of canine parvovirus viral antigen by electron microscopy, virus isolation, fecal hemagglutination, fecal la, or elisa can be used to confirm current infection. in-office elisa for canine parvovirus in feces seems to accurately detect fecal shedding of parvovirus in acute cases (see chapter ) . the specificity of the assays is good, but they cannot differentiate vaccine strains of parvovirus and virulent strains. falsenegative reactions can occur. these assays also detect feline parvovirus. virus isolation, electron microscopy, or molecular assays can be used to document coronaviruses in feces, but results do not correlate with the presence of illness. rare indications • fip is an appropriate differential diagnosis in cats with fever; uveitis; retinal hemorrhage; nonseptic abdominal or pleural exudates or modified transudates; anemia; hyperglobulinemia; and renal, hepatic, or neurologic abnormalities. results of currently available serum or blood tests cannot be used alone to definitively diagnose fip. analysis, artifacts, and interpretation • circulating antibodies against coronaviruses can be detected by ifa and elisa in feline serum. antibody to coronavirus indicates prior exposure to either enteric coronaviruses or fip-inducing coronaviruses. a positive titer does not diagnose fip or protect against disease. feline vaccines containing bovine serum occasionally cause false-positive results. cats with fip can rarely have negative results because of rapidly progressive disease with a delayed rise in titer, disappearance of antibody in terminal stages of the disease, or immune complex formation. a positive coronavirus antibody titer does not predict whether a cat will ever develop fip. titer magnitude cannot distinguish between exposure to enteric coronaviruses or fip-inducing strains. rarely, positive titers can be induced by vaccination for coronavirus. kittens can be seropositive because of colostrumderived antibodies until weeks of age. if adult cats in the environment infect kittens, antibodies can be detected again to weeks later. current coronavirus infections can be detected by fecal virus isolation, electron microscopy of feces, or rt-pcr of feces. however, positive test results do not indicate fip because antibody-positive, healthy cats can pass coronaviruses. definitive diagnosis of fip requires histopathologic evaluation of tissues. lesions visible by light microscopy are generally pathognomonic, but immunohistochemistry can be used to confirm coronavirus particles. rt-pcr can amplify coronavirus rna from effusions, tissues, and blood and usually correlates with the presence of fip. amplification of coronavirus by pcr in effusions and tissues predicts fip, but detection in presence of rickettsial agents in blood, other fluids, or tissues and will likely be clinically useful in the future. rare indications • dogs with appropriate signs of cns disease can have antibodies in csf and serum against canine distemper virus. analysis, artifacts, and interpretation • the clinician can measure csf and serum igg antibodies against canine distemper virus by serum virus neutralization, ifa, or elisa. elisa can be used to measure serum igm antibodies. csf antibodies to distemper virus are increased in some dogs subsequently diagnosed histopathologically as having distemper encephalitis. false-positive results can occur in csf samples contaminated with blood. concurrent measurement of serum antibody concentrations can be helpful; if csf concentrations are greater than serum concentrations, the antibody in csf had to be produced locally and suggests cns distemper. detection of serum igg antibodies is of minimal diagnostic value because a positive titer could develop secondary to vaccination or previous exposure. a fourfold increase in serum igg titer over a -to -week period suggests recent infection. detection of circulating igm antibodies is consistent with recent infection but not clinical disease. a point-of-care assay for detection of canine distemper antibodies is available. vaccinated dogs that are seropositive in this assay probably do not need to be boosted. a presumptive diagnosis of distemper encephalitis can be made with increased csf protein and leukocytes (lymphocytes predominating) plus a positive csf antibody titer in a sample not contaminated with peripheral blood. definitive diagnosis of canine distemper infection requires demonstration of viral inclusions by cytologic examination (see figure - a and b), direct fluorescent antibody staining of cytologic or histopathologic specimens, histopathologic evaluation, or reverse transcriptase-pcr (rt-pcr) documentation of distemper viral rna in peripheral blood, csf, or conjunctival scrapings. (table - ; see appendix i: select laboratories for infectious disease.) positive rt-pcr test results can be induced by modified live vaccination. viral inclusions can rarely be found in erythrocytes, leukocytes, and leukocyte precursors of infected dogs. inclusions are generally present for only to days after infection and therefore often are not present when clinical signs occur. inclusions may be easier to find in smears made from buffy coats or bone marrow aspirates than those made from peripheral blood. viral particles can be detected by immunofluorescence in cells from the tonsils, respiratory tree, urinary tract, conjunctival scrapings, and csf for to days after infection. indications • viral enteritis induced by parvoviruses, coronaviruses, and other viruses should be suspected in young animals with fever and diarrhea, particularly if neutropenia is present (i.e., parvoviruses). neoplastic, reproductive, immunologic, or hematologic disease, as well as in clinically normal cats exposed to felv-positive cats. analysis, artifacts, and interpretation • viral antigen (p ) is detected by ifa in neutrophils and platelets from blood or bone marrow, or in blood, plasma, serum, saliva, or tears by elisa. nucleic amplification assays can also be used to assess the stage of infection. , when evaluating for antigen, testing of serum or blood gives the best results; tears and saliva should not be tested. several point-of-care elisa tests are available in the united states. other assays are also available in other countries. antibody titers to felv envelope antigens (neutralizing antibody) and against virustransformed tumor cells (feline oncogenic cell membrane antigen, or focma, antibody) are available in some laboratories, but the prognostic significance of the results is currently unknown; therefore the tests are not used clinically. felv infection has four major outcomes. cats with inappropriate immune responses develop progressive infection and usually develop felv-associated diseases. cats with regressive infection can be transiently positive for p antigen in blood or serum but ultimately become negative. abortive exposure occurs in cats with good immune responses and infection never occurs. rarely, focal infection of tissues such as the spleen, lymph nodes, small intestine, or mammary glands can occur. cats with regressive infection, abortive exposure, or focal infection rarely become ill. elisa test results generally become positive within days of exposure to felv and can revert to negative in cats that develop regressive infection. cats suspected to have regressive infection can be isolated from other cats and retested by elisa in several weeks or be tested by ifa or pcr assay. positive ifa test results prove the bone marrow has been infected and has % correlation with virus isolation. these cats generally develop progressive infection. false-negative ifa reactions may occur when leukopenia or thrombocytopenia prevents evaluation of an adequate number of cells. false-positive reactions rarely occur from nonspecific staining of eosinophils. a positive ifa indicates that the cat is viremic and contagious. virtually all ifa-positive cats are elisa-positive. finding an ifa-positive but elisa-negative cat suggests technique-related artifact. a negative elisa result is approximately % correlated with negative ifa and an inability to isolate felv. cats that are elisa-positive but ifa-negative are called discordant. discordant results are usually caused by false-positive elisa results, falsenegative ifa results, or early stages of regressive infections. use of pcr assays to detect viral rna or cell-associated dna (provirus) can be performed on blood, bone marrow, or tissues and be used to evaluate cases with discordant elisa and ifa results. some cats with focal infection localized to bone marrow have positive bone marrow ifa results. the most reliable means of identifying focal felv infections is virus isolation or pcr assay. a cat with focal infection may become viremic after extreme stress or administration of glucocorticoids. blood does not. , hyperproteinemia and polyclonal gammopathy (detected by electrophoresis; see chapter ) can occur, particularly in the noneffusive form. monoclonal gammopathy rarely occurs. classic nonseptic pyogranulomatous exudate or modified transudate with high protein and relatively low cell count (see chapter ) is commonly used for presumptive diagnosis. electrophoresis can also be performed on body fluids. a gamma globulin fraction greater than or equal to % is highly suggestive of fip, whereas an albumin:globulin ratio in body fluid greater than . probably rules out fip. in another study, an albumin:globulin ratio of . had a positive predictive value of % and an albumin:globulin ratio of . had a negative predictive value of %. common indications • cats with chronic weight loss, fever, rhinitis, conjunctivitis, gingivitis, dermatitis, diarrhea, uveitis, recurrent abscessation, clinical toxoplasmosis, any chronic infectious disease, chronic renal failure, or lymphadenomegaly should be evaluated for fiv infection. analysis, artifacts, and interpretation • igg antibodies are detected in serum by elisa, ifa, and western blot immunoassay. , there are many different in-clinic kits available depending on the country. western blot immunoassay is performed in some commercial laboratories. in the united states, one in-office elisa is available to detect fiv antibodies and feline leukemia virus (felv) antigen combined. this assay is available with and without dirofilaria immitis antigen assay. seroconversion occurs to weeks after inoculation in experimentally infected cats. seropositive cats are probably infected with fiv for life. false-positive reactions can occur in the elisa but are thought to be rare. positive elisa results should be confirmed via western blot immunoassay or ifa, particularly in healthy cats unlikely to have been exposed to fiv. detection of circulating antibodies only confirms infection, not clinical illness. kittens can have detectable colostrum-derived antibodies until to weeks. because many clinical syndromes associated with fiv infection are caused by opportunistic infections, further diagnostic procedures may determine treatable causes. for example, many fiv-seropositive cats with endogenous uveitis are co-infected by t. gondii. virus isolation and rt-pcr are available in some laboratories and can be used to confirm infection. a recently marketed fiv vaccine induces serum antibodies that are indistinguishable from antibodies induced by natural exposure, at least by use of currently available antibody tests. the ability of virus isolation or rt-pcr to accurately differentiate naturally exposed and vaccinated cats is currently unknown and varies between laboratories. common indications • because of diverse manifestations of felv infection, testing is indicated in all clinically ill cats, especially those with evidence of infectious, in dogs or cats with clinical signs, laboratory abnormalities, or thoracic radiographic changes consistent with dirofilariasis. the test can also be used to assess efficacy of adulticide treatment. analysis, artifacts, and interpretation • elisa can detect circulating heartworm antigen in serum; several kits are commercially available. there is greater sensitivity when compared with microfilaria detection. in dogs, d. immitis antigen tests may be positive as early as to months and are usually positive to months after infection. false-negative results usually occur in early stages of infection and may occur in single-sex infections (male only) or in dogs or cats with low worm burdens (< to worms). retesting in to months should be performed to detect dogs in which results were negative in early stages of infection. after successful adulticide treatment, test results become negative in approximately weeks. in experimental infections, cats testing positive did so about months after infection. however, single-sex or low worm burden infections can lead to false-negative results. therefore a positive antigen test result is specific for infection, but a negative result does not rule out dirofilariasis. in cats, the combination of serum antigen test results with serum antibody test results is more sensitive than performing either test alone (see heartworm antibody titer, next). heartworm antibody titer (feline) rare indications • a heartworm antibody titer should be obtained in cats with coughing, unexplained vomiting, syncope, or radiographic evidence of heartworm disease. analysis, artifacts, and interpretation • several elisas detect antibodies to d. immitis in feline sera. the assays are more sensitive than microfilaria demonstration techniques. the assays are very specific; no cross-reactivity exists with d. reconditum. the positive predictive value for heartworm disease is less than %, however, because circulating antibodies can be present in cats that have cleared the infection naturally. false-negative antibody test results also occur; therefore serum antibody and antigen tests should be performed in concert in cats with suspected dirofilariasis. common indications • cytologic evaluation for microfilaria is indicated in dogs with signs consistent with heartworm disease (right-sided heart disease, coughing, dyspnea, eosinophilia, polyclonal hyperglobulinemia, protein-losing nephropathy [pln] ), in dogs about to begin prophylactic therapy (with diethylcarbamazine, ivermectin, or milbemycin), and rarely in cats with signs consistent with heartworm disease (i.e., dyspnea, cardiomegaly, unexplained vomiting). analysis, artifacts, and interpretation • cytologic testing for dirofilaria immitis is very specific (microfilaria morphology differentiates d. immitis microfilaria from those of dipetalonema reconditum), quick, and inexpensive; all concentration techniques (knott's and filter tests) are much more sensitive than examination of fresh blood smears and are reasonably sensitive in dogs that have not been treated with filaricidal drugs. up to % of dogs have spontaneous occult dirofilariasis and must be diagnosed by serologic testing and radiographic examination. all cytology tests have poor sensitivity in cats. thus, microfilaria techniques should no longer be used as stand-alone diagnostic tests and should only be used conconcurrently with antigen tests.* , † a positive test result diagnoses heartworm disease, except in juveniles less than months of age that could have received the microfilaria by transplacental transfer. common indications • a heartworm antigen titer should be included in annual screening of dogs to evaluate for exposure to d. immitis, and should be performed continued geographic distribution of babesiosis among dogs in the united states and association with dog bites: cases babesia gibsoni infections in dogs from north carolina feline granulocytic ehrlichiosis-a report of a new clinical entity and characterization of the new infectious agent clinical ehrlichiosis in a cat prevalence and geographic distribution of dirofilaria immitis, borrelia burgdorferi, ehrlichia canis, and anaplasma phagocytophilum in dogs in the united states: results of a national clinic-based serologic survey a study of naturally occurring feline coronavirus infections in kittens interpretation and misinterpretation of serological test results american trypanosomiasis post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on patients treated primarily with tetracycline and/or doxycycline clinicopathological findings in dogs seroreactive to bartonella henselae antigens infectious diseases of the dog and cat infectious diseases of the dog and cat comparison of latex agglutination, indirect immunofluorescent antibody, and enzyme immunoassay methods for serodiagnosis of rocky mountain spotted fever in dogs infectious diseases of the dog and cat coccidioidomycosis in cats: a retrospective study ( - ) experimental canine leptospirosis caused by leptospira interrogans serovars pomona and bratislava detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis comparison of different tests to diagnose feline infectious peritonitis quality of different in-clinic test systems for feline immunodeficiency virus and feline leukaemia virus infection clinical relevance of annual screening using a commercial enzymelinked immunosorbent assay (snap dx) for canine ehrlichiosis how molecular methods change our views of felv infection and vaccination cryptococcal infection in cats: factors influencing treatment outcome, and results of sequential serum antigen titers in cats rapid identification and differentiation of bartonella species using a single-step pcr assay laboratory diagnosis of bacterial infections pcr detection of the cryptococcus neoformans caps gene from a biopsy specimen from a case of feline cryptococcosis guidelines for prevention and treatment of opportunistic infections in hiv-infected adults and adolescents relapsing bacteremia following blood transmission of bartonella henselae in cats feline toxoplasmosis: interpretation of diagnostic test results prevalence of bartonella species dna in the blood of cats with and without fever molecular and serological evidence of anaplasma phagocytophilum molecular evidence supporting ehrlichia canis-like infection in cats bartonella vinsonii subsp. berkhoffii and related members of the alpha subdivision of the proteobacteria in dogs with cardiac arrhythmias, endocarditis, or myocarditis bartonellosis: an emerging infectious disease of zoonotic importance to animals and human beings association of feline practitioners panel report on diagnosis, treatment, and prevention of bartonella spp. infections the detection of feline coronaviruses in blood samples from cats by mrna rt-pcr canine brucellosis: a diagnostician's dilemma babesia canis canis, babesia canis vogeli, babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal rna genes mycoplasmal respiratory infections in small animals: cases ( - ) performance of a commercially available in-clinic elisa for the detection of antibodies against anaplasma phagocytophilum, ehrlichia canis, and borrelia burgdorferi and dirofilaria immitis antigen in dogs experimental transmission of bartonella henselae by the cat flea accuracy of polymerase chain reaction assays for diagnosis of feline immunodeficiency virus infection in cats morphologic, immunohistochemical, and ultrastructural characterization of a distinctive renal lesion in dogs putatively associated with borrelia burgdorferi infection: cases ( - ) association of bartonella species, feline calicivirus, and feline herpesvirus infection with gingivostomatitis in cats toxoplasmosis and neosporosis a combined approach for the enhanced detection and isolation of bartonella species in dog blood samples: pre-enrichment liquid culture followed by pcr and subculture onto agar plates clinical characteristics and predictors of mortality for cryptococcus gattii infection in dogs and cats of southwestern british columbia clinical and serologic findings in cats with cryptococcosis serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test canine rocky mountain spotted fever: a retrospective study of cases seroprevalences of antibodies against bartonella henselae and toxoplasma gondii and fecal shedding of cryptosporidium spp, giardia spp, and toxocara cati in feral and pet domestic cats evaluation of peptide-and recombinant protein-based assays for detection of anti-ehrlichia ewingii antibodies in experimentally and naturally infected dogs suspected ehrlichial infection in five cats from a household comparison of serologic evaluation via agar gel immunodiffusion and fungal culture of tissue for diagnosis of nasal aspergillosis in dogs prevalence of mycoplasmal and ureaplasmal recovery from tracheobronchial lavages and prevalence of mycoplasmal recovery from pharyngeal swab specimens in dogs with or without pulmonary diseases comparative strain analysis of anaplasma phagocytophilum infection and clinical outcomes in a canine model of granulocytic anaplasmosis protein electrophoresis on effusions from cats as a diagnostic test for feline infectious peritonitis performance of serologic tests used to detect heartworm infection in cats antigen and antibody testing for the diagnosis of blastomycosis in dogs feline ehrlichiosis: literature review and serologic survey acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention re-examination of feline leukemia virus: host relationships using real-time pcr wanke mm: canine brucellosis prevalence of and risk factors for leptospirosis among dogs in the united states and canada: cases ( - ) canine and feline blood donor screening for infectious disease bartonella spp. antibodies and dna in aqueous humor of cats infectious diseases of the dog and cat american association of feline practitioners' feline retrovirus management guidelines effect of vaccination against feline immunodeficiency virus on results of serologic testing in cats utility of an in-office c elisa test kit for determination of infection status of dogs naturally exposed to borrelia burgdorferi quantitative measurement of c antibody following antibiotic treatment of borrelia burgdorferi antibody-positive nonclinical dogs ehrlichiosis and anaplasmosis in dogs and cats acvim small animal consensus statement on lyme disease in dogs: diagnosis, treatment, and prevention novel chemically modified liquid medium that will support the growth of seven bartonella species detection of antibodies to francisella tularensis in cats infectious diseases of the dog and cat canine nasal aspergillosis/ penicilliosis clinical presentation of anaplasma phagocytophilum-seropositive dogs residing in an endemic area dogs are definitive hosts of neospora caninum pcr detection of acute ehrlichia canis infection in dogs consensus statement on ehrlichial disease of small animals from the infectious disease study group of the acvim key: cord- -q k krnm authors: w. quimby, fred; h. luong, richard title: clinical chemistry of the laboratory mouse date: - - journal: the mouse in biomedical research doi: . /b - - / - sha: doc_id: cord_uid: q k krnm the frontier of clinical chemistry in the mouse has advanced and expanded because of two major events such as, the increasing reliance on mice in biomedical research, and increasing availability of practical yet sophisticated techniques and instrumentations that have allowed for the detection of a wider variety of biomarkers of disease. the progression of these two events is partially driven by the increasing regulatory demands related to safety/toxicity assessment of novel drug development. the availability of inbred strains has led to major breakthroughs in cancer, biology, and immunology. in addition, outbred stocks continue to be utilized in a wide variety of studies but particularly in the fields of toxicology and pharmacology. the power of these models to elucidate the genetic basis of disease cannot be overemphasized. this provided complete nucleotide sequences for each genome allowing investigators to quickly develop the equivalent murine model for many of the inherited human diseases. transgenic and knockout mice have helped clarify disease pathogenesis in virtually every area of medicine and often elucidated biochemical pathways, previously unknown, which are now subject to testing and quantification. it has been more than years since publication of the first edition of the mouse in biomedical research, and since that time new emphasis has been placed on the mouse as a model for the pathophysiology and treatment of human diseases. during this time, the frontier of clinical chemistry in the mouse has advanced and expanded because of two major events: the increasing reliance on mice in biomedical research, and increasing availability of practical yet sophisticated techniques and instrumentations that have allowed for the detection of a wider variety of biomarkers of disease. the progression of these two events has been partially driven by the increasing regulatory demands related to safety/toxicity assessment of novel drug development. abbreviations used in this chapter are summarized in appendix - . for the last quarter century, mice have been the most frequently used mammal in biomedical research, rising from % of all mammals used in to % in (national center for research resources . the nature of their use has changed dramatically during this time. during the first half of the th century, a great deal of emphasis was placed on the development of inbred strains. the availability of inbred strains has led to major breakthroughs in cancer, biology, and immunology (quimby ) . by there were approximately inbred strains available. in addition, outbred stocks continue to be utilized in a wide variety of studies but particularly in the fields of toxicology and pharmacology. during the second half of the th century, investigators developed congenic lines and recombinant inbred strains, each having an impact on elucidating the role of individual genes and assigning new traits to linkage groups respectively (paigen a ). however, beginning in the early s scientists began to genetically engineer mice by either adding a new gene (transgenic) or deleting a normal mouse gene (knockout, ko) (paigen b) . the power of these models to elucidate the genetic basis of disease cannot be overemphasized. over the past years, publications citing transgenic and ko mice have increased exponentially and at the time of this writing the mutant mouse resource of the jackson laboratory listed more than , such unique lines. during this same period the results of both the human and mouse genome projects were published (international human genome sequencing consortium ; mouse genome sequencing consortium ) . this provided complete nucleotide sequences for each genome allowing investigators to quickly develop the equivalent murine model for many of the inherited human diseases. transgenic and ko mice have helped clarify disease pathogenesis in virtually every area of medicine and often elucidated biochemical pathways, previously unknown, which are now subject to testing and quantification. as a result, clinical chemistry in the mouse has grown from evaluation of - analytes found in plasma (or urine), to hundreds of biomarkers that can monitor disease status at the cellular level. due to the massive amount of new information characterizing each of the standard strains and mutant lines of mice, a mouse phenome database has been developed to manage data and provide researchers with the ability to explore raw phenotypic data (including clinical chemistry) and summary analyses. the resource allows investigators to select the appropriate murine model for physiology testing, drug discovery, toxicology studies, mutagenesis, modeling human disease, quantitative trait loci (qtl) analyses, identification of new genes, and unraveling the influence of environment on genotype (bogue and grubb ) . by far, the most important new technology in the rapidly growing and changing field of clinical chemistry is the discipline of proteomics. proteomics combines the disciplines of molecular biology, biochemistry, and genetics to the analysis of the structure, function, and interactions of proteins produced by the genes of a particular cell, tissue, or organism. the field of proteomics has grown mainly due to the development of new instruments that are based on sophisticated techniques, yet amenable to practical implementation. for example, the development of surface-enhanced laser desorption/ionization (seldi) platform time-of-flight (tof) mass spectroscopy (ms) allowed petricoin et al. ( a) to identify five peptides in the sera of women with ovarian cancer that were not found in women without ovarian cancer, even though the structure and function of some of these peptides were not actually known. building on these findings, zhang et al. ( ) combined the seldi-tof ms with protein separation procedures to develop a multianalyte immunoassay for rapidly screening potential cancer patients. this process of identifying proteins (biomarkers) that are predictive for a disease is known as plasma protein profiling. similar technology has been used to develop plasma protein profiles for neoplastic conditions in humans, such as prostate cancer petricoin et al. b; qu et al. ) and bladder cancer (adam et al. ; vlahou et al. ) , as well as a variety of non-neoplastic conditions, such as ischemic versus hemorrhagic stroke , severe acute respiratory syndrome (kang et al. ), alzheimer's disease (carrette et al. ) , and creutzfeldt-jakob disease (sanchez et al. ) . plasma protein profiling has also begun in mouse models. xiang et al. ( ) used a combination of two-dimensional gel electrophoresis ( d-ge) and matrix-assisted laser desorption/ionization (maldi) tof ms to quantify serum protein profiles of c bl/ mice harboring the lewis carcinoma with and without treatment using acetazolamide. they found upregulation of many peptides associated with tumor growth and metastasis, and many of these same peptides were modified during treatment. two specific targets of acetazolamide antitumor activity were subsequently identified as histone h b and croci (a ubc-like peptide). park et al. ( ) used similar technology to determine plasma protein profiles for high (c bl/ ) and low (c h) atherosclerosis prone strains of mice on normal or atherogenic diets. they identified proteins in which the levels had changed after eating an atherogenic diet. of these, were differentially changed in c bl/ mice and an additional were changed in both strains. in addition, proteins were differentially expressed between the two strains regardless of diets. four of these proteins were upregulated in c bl/ and were upregulated in c h. nine of those protein markers were definitively identified and their roles in the pathogenesis of atherosclerosis discussed in the "atherosclerosis" section. plasma protein profiling in mice has the potential to become an important discovery tool for translational research, particularly in identifying cancer-associated plasma biomarkers in humans. for example, juan et al. ( ) also used d-ge and maldi tof ms to identify plasma biomarkers in balb/c-nude mice harboring human xenotransplanted tumors. in mice bearing a human stomach cancer cell line, serum amyloid a (saa) was elevated. the authors then went back to screen the sera of human stomach cancer patients and were able to demonstrate that, when compared to controls, humans with stomach cancer also had significantly elevated levels of saa. all of these examples illustrate the power of proteomics. proteomic pattern diagnostics offers a means to look at molecular diagnostic information in human or mouse serum, without preconceived assumptions about the existence or identity of the biomarkers. this allows for the development of sensitive and specific peptide assays for identified biomarkers of disease. practical applications of proteomics have been facilitated by the creation of instrumentation that can analyze multiple analytes from small sample volumes with fast through-put (such as multiplex technology, which are discussed in the "multiplex technology" section). in an attempt to manage all the experimental data arising from the fields of proteomics, metabolomics and transcriptomics (gene array), the chemical effects in biological systems (cebs) knowledge base was developed (xirasagar et al. ). this is a useful, searchable database that integrates experimental findings from all three disciplines, by biosource identification (animal, test article, genotype, and investigator). by making the cross correlation from data arising from three different streams it is hoped that combination groups of predictive markers for disease and toxicity will emerge. the cost of bringing a new drug to market is estimated at $ . - . billion (food and drug administration ), of which a large proportion is spent on safety/toxicity assessments during the preclinical phase of the development of a drug. although expensive, safety/toxicity regulations are necessary, as illustrated by the fact that a new drug entering phase i clinical trials has only an % chance of reaching the marketplace due to toxicity issues. the ability to decrease the costs of drug development due to safety/toxicity issues is becoming increasingly reliant on the use of the laboratory mouse as a model for human disease and the identification of sensitive biomarkers for toxicity and disease. the underlying basis of using laboratory mice as models for human disease was demonstrated by everett and harrison ( ) , who showed that the predictive reliability of mice for quantitative toxicity of five chemotherapeutic drugs in humans was at least as good, if not better, that the predictive reliability demonstrative by dogs and monkeys. more recently, newell et al. ( ) presented data on predictive performance of rats and mice to demonstrate qualitative human toxicity when given novel cancer chemotherapeutic agents. they claim that nonrodent species are unnecessary for identification of a safe phase starting dose for human trials. furthermore, the two rodent species (rats and mice) used gave similar quantitative and qualitative results. the arrival of genetically engineered mice has further enhanced the relevance of using mice in research, especially in terms of basic mechanistic research and applied screening for genotoxicity and carcinogenicity. transgenic mice carrying a human gene and expressing the protein are said to be "humanized." these animals are invaluable in drug assessment especially when the drug interacts only with the human protein (bolon ) . zambrowicz and sands ( a) showed that the ko phenotype of mice correlated well with the molecular targets of the best selling drugs available to the u.s. market. zambrowicz et al. ( b) compared the physiology of ko mice, where the deleted gene was known to produce a novel target for each of the new drugs in the developmental pipeline of the largest pharmaceutical companies, and found that % of these targets demonstrated a sound biologic rationale for the selected disease. transgenic and ko mice have been used successfully to screen new drug candidates for safety and to elucidate basic mechanisms of toxicity. a large number of drug metabolizing enzyme (dme) ko lines have been employed in safety screening. dmes may be involved in the safe metabolism of a drug or they may generate toxic intermediates. removal of the dme gene may result in the animal being more sensitive to the test article, and it may provide protection against toxic effects of the drug. for instance, ko mice lacking the nqo- gene have increased menadione toxicity, and mice lacking the cyp e gene are resistant to acetaminophen hepatotoxicity (henderson and wolf ) . murine models are also available for evaluation of chemical mutagenicity (big blue mouse; stratagene, la jolla, ca and muta mouse; covance research products, denver, pa). these marker genes are present in mice of different genetic backgrounds (bolon ) . ko lines have been created with the increased sensitivity to chemically induced carcinogenesis. mice carrying a single p allele, or over expressing ha-ras, or having a complete deletion of the xpa (xeroderma pigmentosum) gene, have all been used for screening xenobiotics in vivo (bolon ) . these same transgenic and ko models are useful in screening environmental chemicals for toxicities (jacobson-kram et al. ) . it is clear that the mouse will continue to be essential for discovery and in evaluating the safety of new drugs. equally relevant is the development of clinical chemistry assays needed to study the associated metabolic events in mice (especially in transgenic and ko lines) and assess serum/plasma biomarkers. these biomarkers should either be quantitative measures of the biologic effects (which provide informative links between mechanism of action and clinical effectiveness) or surrogate markers (which are quantitative measures that predict effectiveness). in this arena, the field of proteomics is likely to make a dramatic impact on clinical chemistry. although we hope all readers of this chapter will benefit from this section on assays and instruments, the primary purpose of this chapter is to briefly introduce the reader to areas where methods in clinical chemistry are changing and provide sources of information for services, reagents (including test kits), and instrumentation, specifically for testing biomarkers (including traditional analytes) in mouse serum, plasma, or urine. those seeking a detailed description of clinical chemistry methods and instruments should refer to the fundamentals of clinical chemistry (burtis and ashwood ) . some dramatic changes have occurred over the past two decades that have revolutionized the discipline of clinical chemistry. historically, most analytes were measured by a colorimetric end point assay (based on the binding of an analyte to another molecule creating a new substance that absorbs light of a specific wavelength). some of these analytes and the newer biomarkers are now measured by enzymatic tests that have higher specificities and sensitivities. another change has been the substitution of electrochemical assays, such as ion-selective electrodes, for flame photometry used in the quantitation of sodium and potassium. perhaps the change that has had the largest impact on clinical chemistry is the development of monoclonal antibodies and their use as reagents in immunoassays (to be discussed later). many commercial companies now offer services that measure analytes and biomarkers in the blood of mice using a combination of colorimetric-, enzymatic-, electrochemical-, and immunologic-based methods, and many of the instruments used are capable of running multiple assay types simultaneously. the use of laboratories that offer validated assays specifically for mouse blood is important to ensure accurate and precise results. a summary of a few laboratories that provide validated murine assays is presented in table - . the increasing availability of mouse-specific reagents has resulted in many new assay techniques that provide a high degree of sensitivity and specificity. growth in the number of reagents capable of quantitating analytes in mouse serum is illustrated in table - . techniques employing nonisotopic labels for detection such as enzyme cascade, fluorescence, chemiluminescence, and electrochemiluminescence, are rapidly replacing older radioimmunoassay (ria) techniques. enzyme immunoassays, such as enzyme-linked immunosorbent assay (elisa), enzyme-multiplier immunoassay technique (emit), and cloned enzyme donor immunoassay (cedia), provide quantitative results based on photometric methods. enzyme immunoassay is popular because it generates compounds that can be quantitated photometrically. typical enzymatic labels include ~-galactosidase, horseradish peroxidase, alkaline phosphatase, and glucose- -dehydrogenase. in addition these elisa test kits are compact, easy to use, and quantitated with inexpensive instruments. these kits (usually in a well format) are available for quantitation of a wide range of mouse serum and plasma biomarkers (table - ) . fluoroimmunoassay (fia), which utilizes a fluorescent molecule as an indicator label, was previously subject to problems associated with background fluorescence. today this problem has been largely resolved by using chelates of lanthanide as a label. modifications of fia have eliminated the need to separate free from bound label (homogenous assay). chemiluminescent and electrochemilumiscent immunoassays are similar to enzyme immunoassays, except that quantitation of results is based on the emission of light after a chemical label is exposed to an oxidation reaction (chemiluminescence) or to an electrochemical reaction (electrochemiluminescence). the number of biomarkers that can be quantified by commercially available test kits based on immunoassays is likely to grow at unparalleled rates as a result of proteomics research where mice are the favored model. mulitplex immunoassay is a unique technology that combines four distinct components in a manner that allows for simultaneous analysis of up to different analytes from ktl of serum/plasma. the core component is an inexpensive, consumable, . ~tm diameter polystyrene microsphere that are encoded into different fluorescent color sets using two fluorophores (red and infrared) at multiple concentrations. the second component is a biologic assay (combined with an mmp- (matrix metalloproteinases) pro-mmp- mmp- tissue inhibitor of metalloprotease (timp- ) orange fluorescent reporter molecule) that is built onto the surface of the microspheres. a diverse range of biologic assays can be built onto the microsphere surface, including immunoassays, nucleic acid assays, enzymatic reaction assays, or receptor-ligand analysis assays. the third component is a flow cytometer that focuses the microspheres into a single file in front of a two interrogating lasers, which allow for high throughout. one laser is a red diode emitting at nm, which illuminates each microsphere. the resulting red and infrared fluorescence provides classification information for that particular microsphere set. the other laser is a green yag diode emitting at nm that excites the orange fluorescent reporter molecules of the surface of the microsphere, providing a quantitative signal for that particular biologic assay. the last component is digital signal processing data acquisition hardware that provides the speed necessary to read the microspheres at up to per second. because multiplex technology can analyze a wide range of biomarkers simultaneously, dynamic reference results (plasma profiles) can be developed based on the changing concentrations of the biomarkers during the course of a disease. the known and potential applications of developing plasma profiles of diseases are powerful. plasma profiles can be used to screen and identify diseases (especially during the early development of a disease) and characterize the efficacy of drugs targeted against these diseases. multiplex technology can help elucidate new biomarkers of disease. furthermore, plasma profiles could also potentially be developed to monitor the blood concentrations of drugs as well. investigators seeking additional information on availability of reagents and test kits should refer to "clinical laboratory reference" (nelson ) and the linscott's directory of immunologic and biologic reagents (linscott ) . sections of these two references provide the names and addresses of sources. the catalogs of individual companies may be fruitful, but many reagents for use in human blood and have not been validated for use in other species (including the mouse). each of the various instruments mentioned in this section may be found in the th annual analyzers buyer's guide (advance for administrator of the laboratory ). this guide lists all manufacturers and gives detailed information on each instrument including technology platforms, methods used, through-put capability, purchase price, maintenance costs, reagent package cost, as well as a variety of options available. contact information, including web site, is available for each manufacturer. additional information regarding clinical chemistry on animal blood is available on the web site of the division of animal clinical chemistry at the american association of clinical chemistry (www.aacc.org/divisions/animal). with a mean body size of g, adult mice have approximately . ml of blood volume, allowing ~tl to be removed weekly without consequence to health and welfare (loeb ; loeb and quimby ) . four blood collection sites for acquiring ~tl of blood repetitively from adult mice include the orbital plexus, the tail vein, the jugular vein, and cardiac puncture (quimby b) . general anesthesia is recommended for orbital plexus and cardiac puncture, although terril- robb et al. ( ) claim that topical application of proparacaine hydrochloride is an acceptable procedure for collecting orbital plexus blood. the jugular vein is punctured using a lancet directed at the rear of the jaw, exposing the jugular vein and its tributaries and the submandibular and facial veins (golde et al. ) . collection of blood from any of these veins at this site works well, and the animal does not require anesthesia or a restraint device. the blood should be collected with a small centrifuge tube or capillary tube. nerenberg and zedler ( ) describe a vacuum apparatus used to collect larger volumes of blood from the tail vein. lewis et al. ( ) found that heparinization of mice before tail bleeding increases the yield. prewarming the mouse under a lamp or through immersion in warm water facilitates tail bleeding. the use of heparinized microhematocrit tubes, during tail or orbital plexus bleeding can maximize the plasma volume with minimal hemolysis (quimby b) . hem and smith ( ) recommend a saphenous vein prick method over the orbital plexus method for collecting repeated small volumes from conscious mice. macleod and shapiro ( ) used indwelling fight atrial catheters for repetitive bleeding of conscious, unstressed mice. disadvantages of specific procedures have been described. sakaki et al. ( ) reported sympathetic nervous system stimulation associated with tail bleeding and the mixing of venous and arterial blood with the orbital plexus method. patrick et al. ( ) found that, compared to jugular vein collection, cardiac puncture was associated with higher plasma glucose concentrations and lower creatine kinase (ck) activity. plasma glucose concentrations are higher in blood collected from the orbital sinus compared to the tail vein. differences associated with the method of anesthesia are also important. halothane, methoxyflurane, isoflurane, and pentobarbital sodium have been widely used and are considered safe. however, caution should be exercised in the interpretation of certain chemistry values. higher plasma glucose levels are reported with tail vein sampling in mice anesthetized with pentobarbital or methoxyflurane compared with conscious mice (chuang and luo ) . plasma glucose levels are higher after collection from the orbital plexus if pentobarbital or proparacaine hydrochloride is administered. methoxyflurane has no glucose elevating effect on samples collected from the orbital plexus. cunliffe-beamer ( ) claims that carbon dioxide narcosis provides sedation and analgesic for - minutes and is an appropriate anesthetic for orbital plexus bleeding. when greater volumes of blood are required, four collection sites can be used that require anesthesia and subsequent euthanasia of the mouse. these sites include the jugular vein (ambrus et al. ) , the abdominal aorta (lushbough and moline ) , the brachial artery (young and chambers ) , and the heart (cubitt and barrett ; mitruka and rawnsley ) . blood collection from a newborn mouse can be accomplished by decapitation. injection of two units of heparin subcutaneously several minutes before decapitation may allow collection of up to ~tl of blood ( ~tl of plasma). although collection of urine is possible in mice, it is impractical due to anatomic and physiologic restrictions. the total urinary bladder volume is less than . ml, and total urinary output per day is less than ml (jung et al. ) , meaning that mice frequently micturate. this restricts the amount urine that can be collected at any one time point and, therefore, will also restrict the number of analytes that can be assessed from any one urine sample. to be able to collect enough urine for assessment from a single live mouse, mice can be placed in a plastic cage without absorbent bedding material and urine then collected from the bottom of the cage after - h. however, a disadvantage of this technique is that the urine is exposed to the contamination by feces and other environmental contaminants and organisms. pooled urine samples collected from a group of live mice using the same technique can increase the total amount of urine collected but will not allow specific results to be related back to specific individuals from that group. mice removed quickly from their cage and held over a piece of parafilm will frequently void and the parafilm helps prevent the urine from spreading. urine can also be collected directly from the urinary bladder from mice after euthanasia. this technique allows for a sterile sampling, but the volume that can be collected is restricted by total urinary bladder volume ( . ml). often, the volume is much less because mice typically void urine from the urinary bladder at the time of death. reference ranges refer to the range of an analyte or biomarker in a population that has not been selected for the presence of disease or abnormality. reference ranges are usually generated from a large number of individuals from a population, so reference ranges for the same analyte or biomarker can vary between two different populations. table - lists the reference ranges for selected analytes in two inbred strains and an outbred stock. in some situations, reference ranges that have been published or generated by laboratories cannot be relied on to accurately some variables known to adversely affect the host include environmental factors, pathogens, and shipment. in addition, nutrition, time of sample collection, and storage techniques may all contribute to variability. age has an effect on analytes in the mouse. in an early study, barrett et al. ( ) found that serum calcium levels in -monthold c h/fg and a/fg inbred mice were significant higher than -month-old mice of the same strains. more recent and more comprehensive analyses completed by loeb et al. ( ) reinforces the effect that age has on clinical chemistry parameters in five inbred strains and two f hybrids, including serum protein. the effect sex has on chemistry parameters is most demonstrable with sex hormones, including follicle-stimulating hormone (fsh), luteinizing hormone (lh), and progesterone. table - illustrates these differences between male and female mice, and between female mice in estrus and out of estrus. strain-associated changes appearing in healthy animals have been documented for complement components (goldman and goldman ) , cholesterol (dunnington et al. ; meade and gore ) , testosterone (ivanyi et al. ) , cortisol-binding protein (goldman et al. ) , and serum protein (borovkov and svirdov ; loeb ) . cyclic biorhythms, whether circadian or ultradian, influence blood levels of various analytes in mice. blood levels of adrenocorticotropic hormone (acth), corticosterone, growth hormone (gh), and lh may peak one or more times daily, and thus special attention must be given to both the time of collection and the order of sampling individuals between groups if between-group comparisons are to be made (loeb ) . the degree of hydration, exposure to noise, degree of confinement, and environmental temperatures has all been shown to affect serum chemistry analytes (quimby b) . diet is known to influence the blood levels of many analytes. perhaps the best studied is the effect of atherogenic diets on serum cholesterol. similarly, significant differences in both serum cholesterol and urea nitrogen are seen in mice maindetermine the presence or absence of disease or abnormality, tained on a semipurified (ain- ) diet. the mouse is unique these situations include evaluating analytes and biomarkers in j among mammals because murine muscles do not contain transgenic and ko mouse populations (especially in experiments in which population sizes are small) and evaluating certain analytes and biomarkers (such as those assessing immune function). instead, baseline data compiled from controls (the population of wild-type mice from which the transgenic and ko mice were derived from) should be used. the usefulness of compiled baseline data depends on controlling a large number of variables known to influence chemistry determinations. several studies have demonstrated significant differences in selected serum analyte concentrations with age difference in the same strain, between sexes in the same strain, and among strains (everett and harrison ) . carnosine or anserine. carnosine serves as a source of histidine when histidine is restricted. unlike other mammals, mice on histidine-free diets show signs of histidine deficiency. fasting may also affect the levels of certain analytes (quimby b) . the presence or absence (axenic mice) of intestinal microbial flora is associated with dramatic changes in immunoglobulin (ig) levels and may be associated with changes in other analytes as well. for example, axenic mice have significantly lower levels of iga compared to conventional mice (moreau et al. ) . the presence of pathogens may be associated with dramatic changes in various analytes, even with subclinical infection. for instance, infection with lactic dehydrogenase-elevating virus (ldv) is associated with major elevations in serum lactic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, aspartate aminotransferase, and glutathione reductase activities (quimby b ). asummarized from loeb and quimby ( ) . alterations in clinicopathologic parameters can be attributed to stress in mice. landi et al. ( ) found that plasma corticosterone concentrations in mice tested within h after arrival (by plane or truck transport) were significantly higher than tested after h post arrival. mice sensitized on arrival with sheep red cells as an antigen had significantly lower antibody titers, fewer plaque-forming cells, and a decreased delayed type of hypersensitivity reaction when compared to normal mice allowed to acclimate to the facility for h before being sensitized. elevated serum corticosterone levels can arise from excessive handling of mice prior to blood collection. the effect of sample storage at various temperatures and for varying periods has been described. falk et al. ( ) evaluated the effect of storage time (after freezing) on serum analytes in six laboratory species. they found that in the mouse, only creatine kinase activity changed significantly with storage up to days. hemolysis is a common problem during blood collection in mice. hemolyzed samples are associated with changes in various enzymes, such as increased ck activity and decreased lipase activity. to minimize this, everett and harrison ( ) recommend heparinized plasma and careful selection of collection sites for routine chemical determinations. the effect of lipemia, various anticoagulants, and pharmacologic agents on clinical chemistry values has been reviewed for domestic animals (meyer and harvey ) . using several methods. regardless of the distribution of data, it is generally useful to describe the limits that include % of the test results in a disease-free population. for values exhibiting a gaussian distribution, parametric methods (such as mean and standard deviation) are appropriate. for gaussian distributed data, this is the range that includes two standard deviations above and below the mean. certain murine analytes have non-gaussian distributions and must be evaluated using nonparametric methods. a variety of methods are available, including the percentile method and logarithm-transformed data analyzed with parametric methods. the method of percentile estimates is more vulnerable to bias due to extreme values (outliers) than is the log-transformed parametric method. boyd ( ) asserts that a sample size of at least is required to give % confidence intervals using the percentile method, whereas a sample size of may give reliable ranges if parametric analyses are used. neither statistical method just described will replace raw data in certain situations, such as when assessing analytes for immune function, nor when using data derived from wild-type mice as comparison baseline data for transgenic and ko mice. the issue of quality control and test validity has been thoroughly discussed for common domestic animals and is applicable to chemistry determinations in mice (meyer and harvey ) . quality assurance in clinical chemistry determinations is important to ensure that consistently accurate and precise results are achieved. everett and harrison ( ) stressed the importance of a clinical pathology quality assurance program that includes regular assays of pooled and commercially prepared pre-assayed sera. in addition, they encourage participation in a subscription quality assurance program, such as with a veterinary laboratory association. the within-day and day-to-day coefficient of variation should be known for each analyte measured. due to the tremendous number of variables known to influence clinical chemistry values in mice, it is often prudent to test adequate numbers of control specimens along with the experimental samples. this technique is often impractical when tests are being conducted strictly for diagnostic purposes, and in those situations, compiled values that are controlled for as many variables as possible may be sufficient. the values that define a reference range for a particular analyte or biomarker in normal or healthy mice may be described this section is intended to serve as a review of specific tests for routine analytes. additionally, a more comprehensive discussion is presented for analytes and biomarkers involved in two areas of translational research of intense interest (obesity/diabetes and atherosclerosis) and novel biomarkers of disease (such as immune function tests). only analytes and biomarkers for which there are currently available commercial tests for mice serum, plasma, and/or urine are discussed; certain analytes and biomarkers for which commercially available tests are not available for the mouse (such as calcitonin) are not covered. for more complete information for the routine analytes, please refer to loeb and quimby ( ) . glucose is the main source of energy in mice. blood glucose concentrations depend on the rates of entry and the removal rate from the blood. the rate of entry is dependent on intestinal absorption of dietary sources of glucose, the breakdown of body glycogen stores (glycogenolysis), and synthesis from gluconeogenic metabolites (gluconeogenesis). the removal rate is mainly dependent on insulin, which is released from [ -cells of the pancreatic islets. insulin promotes cellular uptake of blood glucose (mainly in muscle, liver, and fat) by stimulating the translocation of glucose transporters, glut- to glut- to the cell membrane. when removed from circulation, blood glucose may either be utilized (to maintain cell function) or converted to fat and glycogen in liver and muscle as an energy store. however, the effect of insulin is modulated by other hormones (such as glucagon, corticosterone, gh, epinephrine, somatostatin, and amylin) that ultimately result in the tight control the levels of blood glucose, depending on tissue demands for energy. glucagon is released from t~-cells of the pancreatic islets in response to low circulating glucose, stimulating the liver to increase circulating glucose through glycogenolysis and gluconeogenesis. corticosterone and gh antagonize the action of insulin. epinephrine suppresses insulin release and stimulates glucagon release and glycogenolysis. somatostatin suppresses both insulin and glucagon secretion, whereas amylin increases blood glucose, blood insulin, and insulin resistance (burtis and ashwood ; kaneko ) . glucose determinations are generally conducted on fresh serum. however, plasma glucose determination is acceptable if delay of greater than minutes before separation of erythrocytes is anticipated. in this case, fluoride should be used as the anticoagulant because it inhibits glycolysis by erythrocytes. blood glucose in mice is measured using the hexokinase or glucose oxidase. these tests may be performed as an analytical method or by using glucose oxidase coated test strips (for urine glucose), or small portable analyzers (seidelmann et al. ) . assessment of long-term average blood glucose levels in mice is also available by rias measuring glycosylated hemoglobin and glycosylated serum proteins (collectively known as fructosamines) (gould et al. ). the mouse phenome database lists serum glucose levels of strains with blood collected after a -h fast on - week old males and females on a standard laboratory diet. for all mice, the overall mean was + . mg/dl (naggert et al. ) , but blood glucose levels varied with age, sex, and strain. females of a strain tended to have lower levels than males, with lp/j mice showing the lowest values (f = + . and m = _+ . mg/dl) and c b / j mice showing the highest values (f = _+ . ; m = _+ . mg/dl). in mice serum glucose levels decrease between the rd and th month of age and in c bl/ and balb/c strains the glucose level rises again after months (loeb ) . serum or plasma glucose levels may also vary depending on the site of collection and anesthetic used (quimby b) . patrick et al. ( ) found that compared to jugular vein collection, cardiac puncture was associated with higher blood glucose. differences associated with the method of anesthesia are also important. higher plasma glucose levels are reported after tail vein sampling under either pentobarbital or methoxyflurane compared to sampling conscious mice (chuang and luo ) . plasma glucose levels were higher after orbital plexus sampling of mice if pentobarbital or proparacaine hydrochloride was provided. methoxyflurane had no glucose elevating effect on samples collected from the orbital plexus; however, in conscious animals, plasma glucose levels were higher in blood collected by retro-orbital sinus compared to tail bleeding. hyperglycemia (increased blood glucose levels) in the mouse can be due to increased peripheral resistance of tissues to insulin (such as with exogenous corticosteroid administration, increased endogenous corticosterone release, glucagons administration, and gh administration), and diabetes (see discussion later). hypoglycemia (decreased blood glucose levels) can be due to excessive circulating insulin (such as with excessive insulin administration and transgenic mice with [ -cell tumors of the pancreatic islets), reduced glycogen stores (such as with advanced liver disease), excessive glucose use (such as with pregnancy, septicemia, and neoplasia), and reduced glucose intake (such as with starvation and diseases of malabsorption). diabetes in mice is defined as persistent blood glucose levels of greater than mg/dl in fasting mice. this level also corresponds to the renal threshold for glucose urine excretion in mice, so urine glucose assessment is useful in this regard for mice. nonobese diabetic mice (a model of type diabetes) have nonfasting plasma glucose levels in young prediabetic mice between - mg/dl, which rises to > mg/dl between - weeks of age (leiter ) . genetically modified mice have been identified for virtually every ligand and receptor regulating glucose metabolism and, depending on the gene mutation, may exhibit altered levels of plasma glucose. for example, adenosine monophosphate-activated protein kinase (ampk) is a critical enzyme in energy metabolism (including cellular glucose uptake and fatty acid oxidation in muscle, and fatty acid synthesis and gluconeogenesis in hepatocytes). using ko mice, shaw et al. ( ) demonstrated that kinase lkb is an important activator of ampk in the liver under energy-stress conditions, and that ko mice deficient in kinase lkb were persistently hyperglycemic. the authors also demonstrated that kinase lkb is the target of the type diabetic therapeutic drug, metformin. glucose tolerance tests (gtt) have been performed in mice. one-, -, and -h tests have been conducted. in the -h test, glucose concentrations are evaluated before and h following the administration of mg/g glucose administered intraperitoneally (ip) (oldstone et al. ) . the -h test compares pre-injection serum to serum collected at , , , and minutes following administration of mg/g glucose given ip (hotamisligil et al. ) . a sensitive -h gtt has also been described in which mice are given ml/kg of % glucose orally (gates et al. ) . the hypoglycemic response to insulin in mice has also been described (hotamisligil et al. ) . kaku et al. ( ) measured glucose, glycosylated hemoglobin and insulin levels in six inbred strains undergoing gtt (either in fed or fasted mice). to estimate the number of genes involved in phenotypic differences in glucose tolerance, the least glucose tolerant strain (c bl/ ) was bred to the most tolerant strain (c h/hej) and f hybrids and backcross animals tested. the authors concluded that glucose tolerance in six commonly used inbred strains is a polygenic trait. thrifty genes have been hypothesized to give early human hunter-gatherers a survival advantage by providing an economic mechanism to store energy during periods of famine (zimmet and thomas ) . because the most efficient mechanism for energy storage is through promotion of adipose tissue, it seems reasonable that at least some of these "thrifty" genes may function in this manner. another hypothesis holds that during periods of famine it is essential to conserve glucose for use by the brain and that the mechanism responsible involves insulin resistance in peripheral tissues (neel ) . should both hypotheses be true it is easy to envision an association between obesity and type diabetes, especially where sedentary lifestyle and unrestricted access to food occur together (lazar ) . one candidate thrifty gene encodes the hormone leptin. leptin is produced by adipose tissue, and its absence leads to obesity and insulin resistance. during times of high adipose storage blood levels of leptin are high and it promotes energy metabolism and inhibits food intake. the opposite occurs during starvation. but leptin is only one of a number of adipokines, secreted by adipose tissue, which aid in the regulating appetite and metabolism. in fact the location of the adipose tissue, the size of average adipocytes, and adipocyte metabolism of glucose and corticosteroids each modify the endocrine function of adipose tissue (lazar ) . among the proteins secreted by adipose tissue are adiponectin, adipsin, resistin, visfatin, tumor necrosis factor-a (tnf-a), interleukin- (il- ), macrophage chemoattractant protein- (mcp- ), plasminogen activator inhibitor- (pai- ), angiotensinogen, saa, and a-acid glycoprotein. like adipocyte-derived free fatty acids, which have been shown to contribute to insulin resistance in liver and muscle, most of these proteins are capable of modulating glucose metabolism and insulin action. adiponectin and visfatin work synergistically with insulin to enhance glucose uptake by muscle and block glucose synthesis by the liver (hug and lodish ) . blood levels of visfatin increase in obesity and the cytokine can bind to and stimulate the insulin receptor (fukuhara et al. ) . blood levels of adiponectin negatively correlate with body mass and are lower in obese humans and mice, suggesting that reduced mrna expression of the adiponectin gene may be involved with obesity (masaki et al. ) . increases in adiponectin downregulate the hepatic expression of tnf-a. it mediates its antidiabetogenic effects via receptors on peripheral tissues, especially liver. tnf-a, resistin, and il- each induce resistance to insulin. however tnf-a also suppresses expression of adipocyte specific fred w. quimby and richard h. luong t genes; resistin maintains glucose during fasting; and il- production increases in the obese. the cytokines tnf-~ and il- are proinflammatory, are also produced by monocytes, and act on the liver to produce acute phase reactants. they also induce suppressor of cytokine signaling- (socs- ), an intracellular signaling molecule that impairs neuronal signaling by leptin and insulin, and thus causes resistance to the central actions of both hormones (schwartz and porte ) . resistin mediates its effects principally by decreasing the expression of gluconeogenic enzymes in the liver (banerjee et al. ). certain cytokines, increased endoplasmic reticulum stress, chronic hyperglycemia, chronic hyperlipidemia, and oxidative stress may all induce apoptosis of insulin producing ~l-cells in the islets of the pancreas (rhodes ) . insulin receptor substrate- (irs- ) is a key molecule promoting ~-cell growth and survival. these molecules act immediately downstream from surface receptors for insulin and insulin-like growth factor- and inhibition of irs- has been shown to lead to insulin resistance. inhibition may result from accumulation of reactive oxygen species in ~-cells chronically exposed to increased glucose metabolism or from chronic exposure to elevated fatty acid (which through production of long chain acyl-coa active protein kinase c-isoforms degrade irs- ). leptin has been shown to modulate interleukin-l~ (il-i~), a potent inducer of apoptosis. tnf-t~ and il- induce ~-cell apoptosis by activating the transcription factor nuclear factor k:i] (nf-~:[ ). centrally the actions of both insulin and leptin take place in the mediobasal hypothalamus, where the neurons exert potent effects on food intake and energy expenditure. here neurons co-express neuropeptide y (npy) and agouti-related peptide (agrp), which stimulate food intake and reduce energy expenditure. leptin and insulin inhibit these neurons. under conditions of reduced leptin and insulin signaling, npy increases, inducing hyperphagia, weight gain, insulin resistance, and glucose intolerance. the anabolic effects of agrp arise from its antagonism of the melanocortin receptors mc r and mc r, which serve to limit food intake. blockage of mc r and mc r leads to weight gain and insulin resistance. precursor proopiomelanocortin (pomc) and pomc neurons in the arcuate nucleus are stimulated by leptin and insulin and the resultant production of melanocortin and its binding to mc r and mc r inhibits food intake and promotes weight loss (schwartz and porte ) . however, in the absence of leptin, as seen in lep ~ mice, neurons of the arcuate nucleus of the hypothalamus are permanently disrupted and treatment in adulthood cannot reverse this defect (bouret et al. ) . orexin a (hypocretin- ) and orexin b (hypocretin- ) are neuropeptides produced in the lateral hypothalamus by neurons with axonal projections to many sites including those that control feeding behavior and sleep/wakefulness (taylor and samson ) . orexin ko mice develop hypophagia with obesity and insulin-resistant diabetes (hara et al. ). ghrelin, a peptide made predominantly by the stomach, is also known to act centrally and affect food intake and increase secretion of gh (ghigo et al. ; korbonits et al. ). in the periphery leptin has been shown to specifically repress rna levels and enzymatic activity of hepatic stearoyl-coa desaturase- (sld- ), which catalyzes the biosynthesis of monounsaturated fatty acids. this effect was found to be an important metabolic action of leptin (cohen et al. ) . leptin resistance, a common feature of obesity in mice and humans, has also been shown to result, in part, from the shedding of membrane-bound hepatic leptin receptors into the plasma, where soluble receptors modulate circulating leptin levels and possibly its biologic activity (cohen et al. ) . thus the connections between factors regulating obesity and insulin resistance (diabetes) are complex and occur both centrally and peripherally. further investigations in mice will involve quantifying glucose, adipokines, insulin, leptin, soluble leptin receptor, and sld- . for further discussion of mouse models of obesity and diabetes please refer to chapter in this text. metabolism and food intake insulin, leptin, amylin (also called islet amyloid polypeptide, iapp), glucagon, ghrelin, obestatin, orexin a, orexin b, gh, and corticosterone have been measured in mice, and ria and elisa kits are commercially available (see table - ). in addition, these hormones may be measured as part of a multiplex panel (see "multiplex technology" section). leptin values in c bl/ , , and fvb/n strains range from - ng/ml, insulin values range from . - . ~t iu/ml, corticosterone levels range from - ~tg/dl, and gh ranges from - ~tg/ml. epinephrine is measured by ria, and the range for mice is - pg/dl (depaolo and masoro ). adipokines secreted entirely by adipose tissue include adiponectin, adipsin, and resistin. adiponectin and resistin have been measured in mouse serum and commercial elisa test kits are available for this species. a polyclonal antibody that cross-reacts with a conserved sequence of mouse adipsin has been created (searfoss et al. ) . tnf-ct, il- , and visfatin are adipokines that are synthesized also by macrophages and lymphocytes; as such they provide a common link between regulation of obesity, resistance to insulin and inflammation (lazar ) . both tnf-t~ and il- may be measured by commercially available elisa kits (see the "cytokines and chemokines" section). a method has also been published for measurement of visfatin (fukuhara et al. ). reagents for quantifying the soluble leptin receptor and sdl- are not commercially available at this time, although methods for measurement of these analytes in mouse serum have been published (cohen et al. ; cohen et al. ). the four main types of lipids in plasma are free cholesterol, esterified cholesterol, triglycerides, and phospholipids. lipids are derived from the diet (mainly from long-chain fatty acids), although endogenous recycling (mainly in the liver) occurs. plasma lipids have poor water solubility; thus, they require water-soluble protein molecules for their transport in plasma. the complex of plasma lipids and proteins are known as apoproteins (also known as apolipoproteins), which contain a core of nonpolar lipids surrounded by a surface layer of phospholipids, free cholesterol, and apoproteins. apoproteins are classified according to physical and chemical parameters, and include (in order of increasing density) chylomicrons, very low-density lipoproteins (vldl), intermediate density lipoproteins (idl), low-density lipoproteins (ldl), and high-density lipoprotein (hdl). particle density is proportionally related to the amounts of phospholipid, protein, and triglycerides they contain; increasing particle density corresponds with increasing proportions of phospholipid and protein, and decreasing density corresponds with decreasing proportions of triglyceride. chylomicrons and vldl are often referred to as triglyceride-rich lipoproteins. apoproteins on the surface of the particles serve as ligands for receptors, cofactors of enzyme interaction and structural components (wagner et al. ). table - lists the mouse apoproteins and describes their known function. the largest lipoprotein particle is the chylomicron, which transports dietary lipids in the form of triglycerides from the intestines. chylomicrons contain apoproteins a and b and are absorbed into the lymphatics from the intestine, eventually entering the blood. they deliver fatty acids to the tissues with assistance from lipoprotein lipase (lpl) and release glycerol into the blood. as chylomicrons lose triglycerides they become smaller and are called remnants. these remnants acquire apoprotein e from plasma hdl and are rapidly cleared by the liver by the apoprotein e receptor or chylomicron remnant receptor. vldl is the second most prevalent lipoprotein particle in normal mouse blood (after hdl) and it transports triglyceride from liver to extrahepatic tissues. vldl is synthesized in the liver with apoprotein b , c, and e attached. lpl aids in the release of fatty acids (from triglycerides) to peripheral tissues. ldl, which is present in very low concentrations in normal mice, carries cholesterol to extrahepatic tissues. ldl contains apoprotein b and is the primary source of cholesterol deposited in the intima of arteries in mice. apoprotein b binds to the ldl receptor (ldlr). hdl is synthesized in the liver and contains large amounts of free cholesterol and apoproteins a, c, and e. during metabolism hdl free cholesterol is esterified by the action of lecithin:cholesterol acyltransferase (lcat). the exchange of cholesterol ester for triglycerides (from vldl) results in a less dense hdl subfraction that is completely removed from the circulation by the liver. exchange of esterified cholesterol for triglycerides is mediated by cholesteryl ester transfer protein (cetp) in humans; however, this activity is absent in mice. phospholipid transfer from vldl to hdl is mediated by phospholipids transfer protein (pltp). high levels of soluble hepatic lipase are found in mouse plasma (lusis, ; wagner et al. ) . hdl is thought to transport cholesterol from peripheral tissue to liver by a process known as reverse cholesterol transport (rct). this process is believed to be facilitated by the adenosine triphosphate (atp)binding cassette transporter a (abca ), which transports phospholipids and cholesterol to the acceptors apoprotein a- and apoprotein e (aiello et al. ) . hdl also serves as a reserve of apoprotein c and apoprotein e necessary for vldl metabolism. table - lists the apolipoprotein receptors found in mice and describes their ligands and functions. atherosclerosis is a major factor in heart disease, stroke, and peripheral vascular disease in humans, and as such, is the principal cause of death in western countries. the etiology is complex, involving both genetic and environmental components. although there are some examples of single gene defects in humans that lead to atherosclerosis, these conditions are rare and do not explain disease prevalence. atherosclerosis is characterized by the formation of plaques in the intima of large and medium size arteries, usually in locations where blood flow is disturbed. the plaques contain a variety of cells including endothelial cells, monocytes or macrophages, smooth muscle cells, and lymphocytes, which secrete products that modulate progression of plaque formation. in addition plaques contain a complex mix of collagen, proteoglycans, occasional cartilaginous tissue with calcification and lipoprotein (primarily ldl). disease risk correlates directly with elevated circulating levels of ldl cholesterol and risk is indirectly correlated to levels of hdl. thus, although ldl promotes atherosclerosis, hdl protects against it (national cholesterol education program ). the combination of high ldl and low hdl is termed dyslipidemia and is found in human patients with insulin resistance syndrome. mice do not develop spontaneous atherosclerosis, because they have low circulating levels of ldl and lack cetp activity. however, genetically engineered mice that over-or under-express genes involved in lipid metabolism have contributed greatly to our understanding of lipoprotein metabolism (marschang and herz ) . in particular, transgenic mice have been developed with plasma lipid profiles that are similar to those of humans with atherosclerosis (breslow (breslow , . in humans and transgenic mice with elevated ldl (or low hdl), ldl passively diffuses across the endothelium, especially in areas where endothelial cell morphology has been altered by the sheer forces generated by blood turbulence. once ldl is within the intima there is an interaction between ldlapoprotein b and matrix proteoglycans resulting in ldl trapping. ldl undergoes modifications associated with oxidation, lipolysis, proteolysis, and aggregation that contribute to inflammation and uptake of ldl by tissue macrophages. lipoxygenases (los) insert molecular oxygen into polyenoic fatty acids producing hydroperoxyeicosatetraenoic acid (hete). endothelial cells release hete into the vessel wall, where it initiates oxidation of ldl. further oxidation of ldl is aided by myeloperoxidase and sphingomyelinase. mice lacking los have diminished atherosclerosis. macrophages and monocytes recognize oxidized ldl via their surface scavenger receptors and become loaded with cholesterol ester, forming a fatty streak appearing along the vessel wall. hdl, on the other hand, removes excess cholesterol from peripheral tissue and inhibits lipoprotein oxidation. hdl carries paraoxonase, which degrades oxidized phospholipids (lusis ) and protects against subsequent oxidative damage. oxidized ldl stimulates endothelial cells to express adhesion molecules (such as icam, vla- , and vcam) and monocyte colony-stimulating factor (m-csf) on their luminal surface, which attracts additional monocytes and lymphocytes. oxidized ldl also inhibits nitric oxide production. infiltrating thymic-derived lymphocytes (t cells) and macrophages initiate migration of smooth muscle cells from the media to the intima, where they synthesize matrix components. mcp- is also released by macrophages, inducing monocyte differentiation, migration, and scavenger receptor expression. the accumulation of excess free cholesterol can be inhibited by activation of acyl coa:cholesterol acyltransferase (acat)mediated cholesterol esterification and cellular cholesterol effiux. one mechanism responsible for cholesterol effiux is secretion of apoprotein e by macrophages that promotes effiux via hdl. if this fails, as in the case of cholesterol-laden macrophages in plaques, apoptosis of the macrophages ensues. loading of the endoplasmic reticulum with free cholesterol activates er resident protein kinase and the unfolded protein response (upr) that initiates apoptosis through activation of caspase- (feng et al. a) . as macrophages undergo apoptosis they create a necrotic core in the plaque, promoting extracellular cholesterol cleft formation. smooth muscle cells create a fibrous cap over the plaque near the luminal surface. the lesion advances as t cells, activated by cd -cd l ligation, release cytokines such as interferon- , (ifn-y) and tnf-ct. these substances activate matrix degrading proteases and adhesion molecules, promoting additional inflammation. as the inflammation progresses the fibrous cap becomes compromised, leading to physical rupture and the generation of a thrombogenic surface. oxidized ldl increases the production of tissue factor that, on the thrombogenic surface, initiates the coagulation cascade and thrombus formation. each of these details have been elucidated using genetically modified mice (auerbach et al. ; choudhury et al. ; feng et al. b; lusis ; reardon and getz ; tailleux et al. ; trigatti et al. ) . for a further discussion of these models readers are urged to read chapter in this book. for measurements of plasma lipids, serum is the preferred sample. serum can be stored at ~ for - days without adverse effect on measurements. a. serum cholesterol and triglycerides serum cholesterol in mice can be measured using the enzymatic oxidation method of roschlay (meade and gore ) , the lipid research clinic program protocol (morrisett et al. ) , or the abell technique (mitruka and rawnsley ) . the most common enzymatic method employs cholesterol ester hydrolase (to convert cholesterol ester to cholesterol), cholesterol oxidase (which oxidizes cholesterol to hydrogen peroxide and cholest- -en- -one) and peroxidase (which catalyzes a reaction involving hydrogen peroxide, phenol and -aminoantipyrine to form the dye quinonelmine). all of these components of this test are combined in a single reagent mix. the dye absorbance is measured at nm (choudhury et al. ) . triglyceride (tg) levels, which are mainly reflective of the tg content of chylomicrons and vldl in the mouse, are quantified using a variety of enzymatic methods. the most popular method combines lipoprotein lipase, glycerol kinase, and glycerol-phosphate oxidase with peroxidase and -aminoantipyrine (usually in two reagents). in this method, serum tgs are converted to glycerol by lpl via hydrolysis. next the glycerol is phosphorylated in an atp-dependent reaction catalyzed by glycerol kinase. glycerol-phosphate is catalyzed to dihydroxyacetone and hydrogen peroxide by glycerophosphate oxidase and peroxidase catalyzes the final reaction in which peroxide and -aminoantipyrine are converted to a stable dye and absorbance can be read at nm (tsimikas et al. ) . reagents for both total cholesterol and total tgs may be used in an automated analyzer or purchased directly from a chemical company with instructions for manual laboratory analysis. total serum cholesterol and tg levels vary by strain, gender, age, length of fast, and diet. jiao et al. ( ) studied total plasma cholesterol (tpc) and tg levels of various inbred strains fed a standard diet. after - h of fasting, tpc levels ranged from mg/dl (akr/j) to mg/dl (nzb/b nj), and tg levels ranged from mg/dl (c bl/ ) to mg/dl c h/hej; each much lower than seen in normal humans. albers and piagen ( ) quantified total cholesterol levels in strains give a standard laboratory diet and fasted for h before blood collection. levels (in -to -week-old mice) varied greatly between strains with c blks/j females having the lowest level ( mg/dl) and nzb/b nj males having the highest levels ( mg/dl). for a given strain, males always had higher levels than females. trends in total hdl levels in the same strains paralleled total cholesterol levels. triglyceride levels also varied greatly among strains but did not parallel cholesterol levels. among the strains tested, c bl/ j mice had the lowest tg levels ( - mg/dl) and c h/hesnj the highest (f, mg/dl; m, mg/dl). differences between genders of the same strain were less pronounced, compared to cholesterol and males generally had higher levels than females. a transient cause of plasma lipid elevation (hyperlipidemia) in the mouse is related to recent feeding (postprandial hyperlipidemia), especially on a high-fat diet. causes of persistent hyperlipidemia include diabetes, sustained feeding of a high-fat diet, and nephrotic syndrome. b. hdl, ldl, idl, and vldl levels of individual apolipoproteins can be measured by density gradient ultracentrifugation combined with either electrophoretic, immunologic, chemical, or morphologic analyses. although published reference ranges are not available, the distribution and characterization of murine apoproteins has been reported (camus et al. ). similar methods were used to evaluate plasma apoproteins in mice consuming atherogenic diets (moltisett et al. ) . more recently the profile of murine plasma lipoprotein cholesterol has been determined by fast protein liquid chromatography with on-line post-column analysis of superose gel-filtration eluates (sehayek et al. ; strauss et al. ) . in contrast to humans, mice normally have low levels of ldl in their plasma ( mg/dl), with the major plasma lipoprotein being hdl. the low ldl concentration is due to editing of % of apoprotein b mrna transcripts in mouse liver leading to apoprotein b containing particles that are cleared much faster than ldl in humans (lusis ) . in contrast, human apoprotein b mrna transcript editing only occurs in the intestine. a much simpler methodology has been developed to measure hdl and non-hdl lipoproteins in mice, based on precipitation and enzymatic analysis. when phosphotungstate and magnesium salt is added to serum (or plasma), all lipoprotein, except hdl, is precipitated and can be removed. the remaining solution is then tested for cholesterol as described previously. the non-hdl-cholesterol component is calculated by subtracting hdl-cholesterol from total cholesterol in the nonprecipitated sample (sehayek et al. ) . some strains develop elevated cholesterol levels associated with increases in non-hdl levels after consuming high fat diets (breslow ) . c. mouse apoproteins commercial antibodies against mouse apoprotein a and apoprotein a are available and elisas have been described for the measurement of these apoproteins in plasma and in hdl fractions of plasma (dansky et al. ) . plasma apoprotein a levels can also be measured by multiplex analysis. an assay for mouse apoprotein j (apoj or clusterin) has also been described (navab et al. ) . apoj, which is a ubiquitous glycoprotein postulated to have multiple functions, is associated with hdl and is the amyloid-associated protein associated with amyloid plaque formation in alzheimer's disease in humans. d. other analytes associated with lipid metabolism and atherosclerosis in mice elisa kits are commercially available for the quantitation of many mouse coagulation proteins including: fibrinogen, factor vii, d-dimer, tissue factor, and von willebrand's factor antigen. elisa kits are also available to quantify many murine products of arachidonic acid metabolism including hete. reagents, elisa kits, and multiplex assays are available for murine inflammatory cytokines and chemokines (see the "cytokines and chemokines" section), as well as monocyte colony stimulating factor. dansky et al. ( ) have described assays for the quantitation of murine aryl esterase and paraoxonase. table - lists the mouse enzymes involved in lipid metabolism with references that describe their measurement. the innate and adaptive immune responses have been extensively studied (see volume of this series) and therefore we will not attempt to describe all the features of mouse immunity in detail here. readers are directed to volume , molecular and cellular immunology of the mouse, for a general overview, and the chapters that follow for a more detailed description. this section describes the growing number of quantifiable soluble serum proteins and lipids associated with immunity and inflammation in the mouse (see table - ). furthermore, in addition to immunodeficiency, autoimmunity, and allergy, investigations of atherosclerosis, obesity, diabetes, cancer, as well as infectious diseases, each have immunologic and inflammatory components. . immunoglobulins (ig) as in humans, mouse immunoglobulins (ig) are molecules composed of four polypeptide chains; two of lower molecular weight called light (l) chains, and two with higher molecular weight called heavy (h) chains. disulfide bonds link one l chain to one h chain, and the two h chains to each other. immunoglobulin h chains are composed of four to five domains, including an n-terminal variable region domain and four constant-region domains. in addition, structural differences in the constant-region domains of the heavy chain are used to classify the five different classes of immunoglobulin, igm, igg, iga, ige, and igd. l chains are composed of only two domains and structural differences in these domains are used to classify l chains as either kappa or lambda type. in the mouse, % of serum ig has kappa l chains. any individual antibody secreting b cell (or plasma cell) will make a single ig molecule composed of two identical h chains and two identical l chains. mice make four subtypes of igg: igg , igg a, igg b, and igg . certain strains, c bl/ , c b / , sjl, and nod, do not make igg a but rather make a novel igg c. the igg subtypes in mice are not exact homologues of human subtypes (mestas and hughes ) . prenatal (transplacental) transfer of maternal ig as well as postnatal transfer across the intestinal epithelium is limited to the igg a, b, and subclasses of immunoglobulin and is mediated by neonatal fc receptors (fcrn) located in the placenta or on the intestinal brush border of the proximal small intestine (bankert and mazzaferro ) . mouse igg b fixes complement by the classical pathway and igg and igg a fix complement by the alternative pathway. ige and igg are homocytotropic antibodies capable of binding to receptors on mast cells and basophils and mediate immediate hypersensitivity reactions. although igg and ige circulate in the mouse as monomers, igm circulates as a pentamer and iga circulates as a polymeric molecule. normally very little igd can be detected in serum. serum levels of igm, iga, igg, and ige are influenced by the rate of synthesis and rate of catabolism. like humans, the rate of igg catabolism in mice is directly proportional to the serum concentration of the subclass. the average half-life of murine igg is . days. the catabolic rate of iga is independent of serum concentration. mice synthesize from - mg/kg/day of total ig, although this is dependent on strain and level of antigenic stimulation. certain strains have a propensity to develop specific t-helper (th, cd +) cell subclasses in response to antigenic stimulation, and these strains are referred to as having principally a th or th - ike phenotype. typically cd + lymphocytes modulate immune responses by the cytokines they secrete. those secreting il- , ifn-y, and lymphotoxin are generally referred to as th (and are favored responses for immunity against viruses and intracellular pathogens) and those secreting il- , il- , il- , and il- are referred to as th (and enhance humoral immunity while suppressing cell mediated immunity). when confronted with the same antigen, balb/c mice exhibit a th -dominant response, and c bl/ mice exhibit a thl-dominant response. il- participates in immunoglobulin (antibody) class switching (see volume , chapter ). consequently, th strains are the models of choice for investigations of allergic inflammation because they produce higher concentrations of il- induced immunoglobulin classes (ige and igg ). the cba/n strain is deficient in its ability to produce igm and igg . immunoglobulin levels are also greatly reduced in germ-free mice, offspring of mice on zinc-deficient diets, and mice on protein-deficient diets (quimby b) . quantifying the various classes and subclasses of murine ig can be done using various immunoassays including: radial immunodiffusion, ria, or enzyme immunoassay. both ria and enzyme immunoassay have the higher degrees of sensitivity that are needed to accurately quantitate levels of ige and igd in murine serum. enzyme immunoassay has become the favored assay to avoid isotope handling and disposal. the reported concentrations of normal balb/c mice are: igg ( . mg/ml), igg a ( . mg/ml), igg b ( . mg/ml), igg ( . - . mg/ml), iga ( . mg/ml), igm ( . mg/ml), and ige and igd are both less than . mg/ml (bankert and mazzaferro ) . in addition, there are many commercial sources of enzyme-linked antibodies that supply reagents (and instructions) for developing assays to measure antigen-specific antibody concentrations by subclass of antibody. the complement system is composed of or more chemically and immunologically distinct proteins capable of interacting with antibodies, certain bacterial products, and cell membranes. a brief summary of this system is described later. please refer to two recent publications (quimby a; turnberg and botto ) for more details about the structural and functional aspects of each protein of the complement system. the role of the complement system in mouse immunity is described in volume (overview) of this series. the sequential activation of individual complement proteins from inactive to active substances is a dynamic event called the complement cascade. the ability of the first component of complement, c , to bind specific sites on the heavy chain of mouse igg b and activate a sequence of reactions leading to production of a molecular unit capable of lysing a target cell membrane has established the complement system as the primary mediator of antibody-antigen reactions. recent findings suggest that the pentraxins, c-reactive protein (crp), serum amyloid protein (sap), and pentraxin (ptx ) can bind to complement component clq and activate the classical pathway. this may be an important mechanism for removal of apoptotic cells that might otherwise predispose to autoimmune disease (nauta et al. ) . each protein of the complement system is normally present in the circulation as an inactive molecule. although the complement cascade may be activated by any of four separate pathways, the central event for each is activation of c to c b yielding a small c cz chain fragment. the two c convertases are c b a (for the classical, lectin, or pentraxin pathways) and c bbb for the alternative pathway. each c convertase cleaves c and adds the c b fragment to the convertase complex forming c convertase. cleavage of c leads to the membrane attack complex (mac) common to all activation pathways (goldsby et al. ) . assemblage of the mac on the surface of a target cell leads to the formation of a large channel enabling ions and other small molecules to diffuse out leading to cell death. the activated components of complement also participate in chemotaxis, phagocytosis, cell adhesion, and b-cell differentiation. there are many notable differences between mice and humans regarding expression of complement components and regulation of cascade activation. mice have both an active and inactive form of circulating c and the genes (both on chromosome ) are designated ss and slp, respectively. many inbred strains do not synthesize active c (e.g., dba/ and a/j strain) due to a post-translational defect and therefore they cannot generate a mac. some strains are deficient in the production of c (e.g., dba/ j strain). complement component exists as two allelic forms in mice with and kda molecular weights. similarly there are several notable differences in the regulators of complement activation. most of these regulators are found in the regulator of complement activation (rca) locus on murine chromosome and, as in humans, restrict assembly and stability of convertase enzymes. in mice, decay acceleration factor (daf) is encoded by two genes. however, only the product of daf- is widely dispersed on all tissues. this membrane-anchored protein inhibits c cleavage by accelerating decay of c convertases. in mice it is the major regulator of c in the skin (not kidney) and is a ligand for cd which, on cross-linking, leads to lymphocyte activation. cd is a membrane-anchored inhibitor of c b- formation (mac) and prevents c from binding the c b- complex. deficiencies of cd in humans lead to hemolytic anemia but not in mice. membrane cofactor protein (mcp) is a major cofactor for factor i in humans causing cleavage of c b and c b deposited on self-tissue. in mouse mcp is only expressed in the testis. complement receptors and are encoded by separategenes in humans but are produced by alternative splicing of a single gene in mouse. mice have a complement receptor (cr- ) related gene/protein y (crry) not found in humans, which is a membrane-anchored c inhibitor. it is the major regulator of c in mouse kidney and has some of the functions as mcp and cr in humans. crry has daf activity and serves as a cofactor for factor i (which enzymatically cleaves c b). the mouse has been widely used in studies on the biosynthesis and molecular biology of individual components of complement. the genes encoding components, subcomponents, receptors, and inhibitors have been identified in mice. the complement components may be quantified by assays designed to measure the functional properties of these proteins or their antigenic properties. tests designed to measure antigenic properties of complement are generally simpler, less subject to error, and less expensive; however, they have the disadvantage of measuring both active and inactive forms of their proteins and therefore may not correlate well with functionally active protein. functional assays measure the ability of the entire classical or alternative pathway, or individual components of pathways to lyse (hemolyze) antibody-coated (sensitized) or noncoated (for the alternative pathway) red cells in suspension or in agarose gel. these assays are precise and sensitive (quimby a) . antigenic assays include radial immunodiffusion, electroimmunodiffusion (rocket electrophoresis), automated immunoprecipitation, crossed immunoelectrophoresis, and more recently elisa (quimby a) . elisa kits are commercially available for quantifying murine c lq, c , c , c a (desarg), and c a. in addition antibodies are commercially available for these as well as murine c a, c d, c , and c . complement components c , c , c , c , and factor b may be quantified using functional assays (quimby a) . membrane-bound complement regulators, crry, cd , and daf, can all be detected using previously published antibodies (lin et al. (lin et al. , . the principle mediator of the lectin activation pathway is mannose-binding lectin (mbl). after binding to mannose residues on the surface of microorganisms, two mbl-associated serine proteases (masps), masp- and masp- , bind to mbl. this complex causes cleavage and activation of c and c (masp and mimic the activities of c and cls). commercially available elisa kits are available for murine mbl-a and mbl-c quantitation. other activators of complement include members of the pentraxin family. in mice this may include crp or sap, although the concentration of crp in normal mice is very low. immunologic reagents are available to quantify both pentraxins in mice (gentry ; quimby b ). circulating immune complexes (cic) are multimolecular substances composed of antigen, antibody, and activated complement components. in mice, igm, igg , igg a, and igg b have complement activation regions. although cic may form following exposure to circulating foreign antigens, such as those associated with microorganisms, they are also common manifestations of spontaneous autoimmune disease such as that seen in (nzb x nzw)f , mrl/lpr, bxsb, and krn strains. cic are cleared from the circulation by both cr- , cr- , and fcr. tissue deposition of immune complexes may lead to vasculitis. cic have been quantified in mice by capitalizing on their binding to various complement receptors, by precipitation of clq with polyethylene glycol, or by immunoassay. commercially available elisa kits are commonly employed today (quimby b ). more than inbred strains or mutant lines spontaneously develop autoimmune disease or are susceptible to autoimmune disease induction. the details of many of these lines may be found in volume , chapters l l and in this series. autoimmune diseases in mice include: thyroiditis, rheumatoid arthritis, sj gren's syndrome (ss), hemolytic anemia, lupus erythematosus, type diabetes mellitus, experimental allergic encephalitis, oophoritis, orchitis, gastritis, ulcerative colitis, and polyendocrine disease (boyton and altmann ; ravirajan and isenberg ; sakaguchi ) . antibodies directed to self-antigens in the mouse have been quantified using a wide range of methods from immunofluorescence to elisa. table - lists the commercially available elisa kits used to quantify murine autoantibodies. those antigenic targets associated with systemic lupus erythematosus include: cardiolipin, double-stranded deoxyribonucleic acid (dsdna), single-stranded deoxyribonucleic acid (ssdna), histone, [~- glycoprotein, proliferating cell nuclear antigen (pcna), neutrophil cytoplasmic antibody (canca), ribosomal p, and smith. antigenic targets for arthritis include: rheumatoid factor (rf), collagen type , collagen type , and ssdna. antibodies against insulin and glutamic acid decarboxylase are seen in type (juvenile) diabetes. mixed connective tissue diseases are characterized by antibodies to ribonuclearprotein (rnp). antibodies to myeloperoxidase (mpo) may be observed in vasculitis. mice with ss develop autoantibodies to ss antigens a and b (ssa and ssb, respectively). panels containing autoantigens are available as multiplex assays for quantifying murine autoantibodies. a. interleukins (il) (ils are cytokines that are secreted by leukocytes and act on other leukocytes. interleukins have been classified based on the secretory cell type (i.e., monokines vs. lymphokines), and they have been classified based on whether they are primarily involved in innate (il- , il- , il- , tnf-t~, ifn-t~), or adaptive (il- , il- , il- , il- ) immunity. commercially available elisa kits are available to quantify most murine interleukins, as demonstrated in table - . i. interleukin- (il- ) il- is a name for two proteins, il-lt~ and il- [ , that are encoded by separate genes. along with il- receptor antagonist, il- , il- , and tnf-t~, these proteins modulate acute inflammation. the effects of il- are pleotrophic and involve bone remodeling, insulin secretion, appetite regulation, fever induction, neuronal development, and many others. both il- o~ and il- [ are secreted as - amino acid (aa) pro-cytokines that are enzymatically cleaved into bioactive -kda segments. unlike il- [ , the intact procytokine of il- ct is also bioactive, both within the cytoplasm and on the cell surface, where it is anchored to the cell membrane via a mannose glycosylation residue that attaches to the membrane-associated lectin. there is % sequence identify between mouse and human il-i~ genes and % identity between mouse and human il- t~ genes. a third gene encodes il- receptor antagonist, a soluble -kda molecule with % sequence homology to il-lt~ and % homology with il- . mouse il-lra is % homologous with human il-lra. there are two il- receptors, types i and ii, but only il- ri is capable of signal transduction. il-lra inhibits the action of il-lt~ and il- b by binding il- ri. a -kda form of il- ri has also been described that is soluble and preferentially binds il-lra. il- rii has no signal transducing associated protein and serves to modulate levels of il- t~, il-i[ , and il-lra by binding them on the cell surface. it can also occur as a soluble receptor. with the recent finding of six new members in the il- ligand family (in humans), a revised nomenclature for both il- ligands fred w. quimby and richard h. luong and il- /il- receptor families has been developed (sims ) . further details may be found in volume (overview and chapter ) of this series. ii. interleukin- (il- ) il- is a lymphokine secreted by activated t-helper cells. it acts in an autocrine fashion to induce the expression of il- receptor on t cells, resulting in t-cell proliferation (cogoli-greuter et al. ) . il- also acts in a paracrine fashion modulating the activities of b cells, natural killer (nk) cells, and lymphocyte activated killer (lak) cells. il- is a glycoprotein of amino acids (in humans) with % homology between mouse and human. the il- receptor (il- r) is a multisubunit cellular receptor belonging to the class cytokine receptor family (hematopoietin receptor family). the il- r has a-, [~-, and y-chains. [ -and y-chains interact to transduce the il- r signal and the y-chain is shared with receptors for il- , il- , il- , and il- . iii igg in mice and is responsible for the downstream events leading to differentiation and activation of th cells. il- also induces expression of adhesion molecules like vcam, th cytokines such as il- , il- , and il- and chemokines like eotaxin- and- . il- primes mast cells and basophils leading to enhanced activation during allergic challenge (mueller et al. ) . homology between mouse and human molecules is low ( %) and each is species-specific in its biologic activity. it induces the growth of b- progenitors and igm production by b- cells. il- induces class switch, favoring production of iga, igg , and ige. on eosinophils, il- induces iga and igg receptors and stimulates leukotriene (lt), c , and paf secretion, in addition to inducing eosinophil growth and maturation. the receptors for il- consist of a ligand binding tx-subunit and a non-ligand binding (common) signal transducing ~-subunit that is shared by receptors for il- and granulocyte-monocyte colony-stimulating factor (gm-csf) (sato and miyajima ) . vi. interleukin- (il- ) il- is secreted by a wide variety of cells including t cells, b cells, monocytes, fibroblasts, hepatocytes, keratinocytes, astrocytes, and endothelial cells. it has broad pleiotropic effects on host defense, acute phase responses, immune responses, and hematopoiesis. il- is classified as an inflammatory cytokine and based on a helical cytokine structure and subunit makeup, il- is the prototypic member of a family of molecules that includes leukemia inhibitory factor (lif), oncostatin m (osm), ciliary neurotrophic factor (cntf), cardiotrophin (ct- ), and il- . mouse il- is kda and contains four cysteines and contains o-glycosylation sites and shares % homology with the human molecule (van snick ). the il- receptor has two subunits, a nonsignal transducing subunit binding with low affinity (~-subunit), and a signal transducing subunit (~-subunit) that does not bind il- by itself but participates in high-affinity binding. the soluble il- r~ chain binds il- and the complex induces expression of mcp- , which attracts monocytes into areas of inflammation (kaplanski et al. ) . vii. interleukin- (il- ) il- , previously called lymphopoietin- , is expressed by stromal cells, especially in the bone marrow and thymus, where it promotes thymopoiesis of t cells and the differentiation of pro-b cells into pre-b cells (aspinall et al. ; goldsby et al. ) . mouse il- has % amino acid sequence homology with human il- and both proteins exhibit cross-species activity. viii. interleukin- (il- ) il- is not expressed in the mouse; however, another protein, kc, is secreted and has many properties of the human chemokine, gro, which is known to bind the il- receptor (see the "cytokines and chemokines" section). ix. inrelclevlcln-io (il-io) il- is the prototypic member of the il- cytokine family comprising ill , il- , il- , il- , il- (fisp), and il- . il- is a amino acid protein with an amino acid signaling sequence. both mouse and human il- s have two intrachain disulfide bonds and form non-disulfide linked homodimers. mouse and human il- are % homologous. il- is a th cytokine, which inhibits ifn-y and gm-csf production by th cells. additionally it induces cd + t-cell chemotaxis, inhibits t-cell apoptosis, participates in iga class switch in b cells, induces histamine release from mast cells, and promotes tnf-tx and gm-csf production by nk cells. il- inhibits secretion of the neutrophil chemokines mip-lt~ and mip-i~ and blocks production of il- ~ and tnf-ct by neutrophils. it is immunosuppressive to dendritic cells and induces the differentiation of a subset of regulatory cd + t cells (trl) (grouz and cottrez ; morel et al. ). x. il- , also known as adipogenesis inhibitory factor (agif), is a pleiotropic cytokine with effects that overlap that of il- . il- is a member of the il- cytokine family and as such has a four-helix bundle fold motif. it binds to the multimeric il- receptor that shares the promiscuous gpl signaling ~-subunit with other receptors in this family. the il- r ~ chain is unique and binds il- but does not have a cytoplasmic domain; instead binding leads to homodimerization of the ~-chain that activates the janus kinases. il- stimulates proliferation and differentiation of monocytes and megakaryocytes causing thrombopoiesis. it also activates osteoclasts and enhances bone resorption, decreases new bone formation, and stimulates chondrocyte and synoviocyte production. il- protects small intestinal epithelial cells from chemotherapy and radiation injury and ameliorates inflammatory bowel disease (schwertschlag et al. ) . il- inhibits adipogenesis, regulates neuronal differentiation, and regulates t-cell function (enhances th and inhibits th cytokine production). overexpression of il- in the lung causes airway remodeling, fibrosis, and mononuclear nodules analogous to the clinical picture in chronic asthma. il- is also required for the uterine decidualization response (robb et al. ; zheng et al. ) . xi. il- , also known as natural killer cell stimulatory factor (nksf), is a heterodimeric cytokine composed of a -kda (p ) subunit and a kda (p ) subunit. the p subunit is shared by il- , a cytokine with similar activities. macrophage, monocytes, and dendritic cells produce il- after activation of toll-like receptors (tlr) on these cells by bacterial ligands. il- induces production of ifn- by th and nk cells and intact il- skews the balance between th subsets in favor of th cells. il- binds to the il- receptor that is composed of two subunits, [ and ~ , on the surface of nk and th cells. il- p interacts with il- r[ , and il- p binds il- r[ . negative feedback regulation of il- production involves down regulation of tlr signaling by phosphoinositide -kinases (piks). thus il- is centrally involved at the interface of innate and adaptive immunity (fukao and koyasu " ottenhoff et al. ) . xit, il- , along with il- and il- , is a member of the type- cytokine family and as such is involved in inflammation, mucus production, tissue remodeling, and fibrosis. this single-chain protein shares % amino acid sequence homology with human il- . il- is produced by activated t cells, mast cells, and nk cells and promotes th responses including synthesis of ige. signaling is mediated by the type- il- receptor which consists of il- r~ and il- rc~i chains. another il- binding protein, il- r~ , strongly inhibits the activity of il- (goldsby et al. ; mentink-kane and wynn, ) . xiii. il- , also known as cytotoxic t lymphocyte-associated antigen- (ctla- ), is produced by t cells and is pleiotropic in activity. il- is a amino acid residue polypeptide with a amino acid signal sequence and a mature polypeptide of amino acids. it is a disulfide-linked homodimer. based on the presence of spatially conserved cysteine residues in the il- family of proteins, there are six family members in humans and mice, il- , il- b, il- c, il- d, il- e, and il- f (aggarwal and gurney ) . like il- itself, several of the family members appear to modulate immune function. produced by mouse cd § t cells, il- induces il- , mcp- , prostaglandin-e (pge ) and granulocyte colony-stimulating factor (g-csf) by fibroblasts, keratinocytes, epithelial cells, and endothelial cells. it induces icam- surface expression, proliferation of t cells, and the differentiation of cd + marrow progenitors into neutrophils (fossiez et al. ). the ubiquitously distributed receptor is a type transmembrane glycoprotein of amino acids in length. xiv. il- is a -kda, nonglycosylated polypeptide that lacks a classical signaling sequence. its structure resembles il- and the propeptide undergoes proteolytic cleavage by interleukin- [ -converting enzyme (ice) or another caspase to produce an -kda bioactive molecule. there is % sequence homology between mouse and human il- . il- induces the production of ifn- by t cells and nk cells and the expression of fas ligand (fasl) on a variety of all types. il- activates nf-~:[ and the induction of various chemokines. il- plays an important role in the early antibacterial host response (weijer et al. ) . xv. and il- induces keratinocyte differentiation and proliferation. il- is a four-helix-bundle cytokine similar in structure to il- and sharing sequence homology with il- and il- . murine il- is % homologous to human il- . the il- receptor utilizes the common -chain. il- is produced by the th cells. the actions of il- are pleiotropic and seen on b cells, t cells, nk cells, and dendritic cells. il- induces apoptosis in resting and activated b cells, an effect counteracted by activation of cd . it also upregulates production of igg and inhibits ige, in fact it inhibits many il- activities. il- also inhibits dendritic cell differentiation. il- enhances the activity of activated nk cells and mediates the proliferation and expansion of t-cell subsets. il- induces acute phase reactants by hepatocytes and reduces il- production by th (mehta et al. ) . recombinant antigens and antibodies are commercially available for murine il- , il- , and il- . b. the transforming growth factor-~ superfamily (tgf-~sf) of cytoi~nes members of tgf-~ family share - % sequence homology with tfg-~i and a monomeric structure that consists of two antiparallel pairs of [ -strands forming a flat curved surface, a separate long cz-helix, and a disulfide rich core with a characteristic cysteine knot. most tgf-~sf members are disulfide-linked homodimers; however, three members lack the seventh conserved cysteine residue and are not covalent homodimers. members of the tgf-[ sf include tgf-[ , tgf- , tgf- , activins, inhibins, bone morphogenic proteins (bmp), growth differentiation factors (gdf), glial-derived neutrophic factors (gdnf), and mtillerian inhibiting substance (mis). tgf-[ is stimulatory for cells of mesenchymal origin and inhibitory for cells of epithelial or neuroectodermal origin. in the murine immune system tgf-[ is the mediator of immune suppression via cd +cd +tr cells and, at least in the case of suppressing cd + effector t cells, involves tgf-[ receptor ii on these cells (powrie ; von boehmer ) . in addition tgf-[ is known to inhibit b-cell proliferation and it promotes isotype switch to iga. oral tolerance to th responses (against food allergins) is mediated by tgf-~i (mucida et al. ). tgf-[ has a wide range of effects on cell growth differentiation and malignant transformation (letterio ) . murine tgf-~i may be quantified in serum or plasma using commercially available elisa kits. antibodies are also available which specifically bind murine bmp, activin a, activin c, and gdf- ,- ,- ,- , and- , although they are not recommended for elisa development. c. the tumor necrosis factor superfamily (tnfsf) tnf-related ligands share many features but high amino acid sequence homology is not one of them. with the exception of nerve growth factor and tnf-~, all ligands are type ii tramsmembrane proteins (extracellular c-terminus) that contain a short cytoplasmic segment and a long extracellular region. tnf-~ is fully secreted and has a nonfunctional transmembrane segment. tnfsf members form trimeric structures and their monomers are composed of ~-strands oriented into a twosheet structure. receptors for the tnfsf ligands also belong to a superfamily, tnfrsf (gruss and dower ) , and are characterized as type i transmembrane proteins (with their amino termini outside of the cell), with extracellular cysteine-rich structural motifs. tnfrsf members exist both as membrane and soluble forms. commercially available elisa kits or matched antibody for development of assays are available to quantify the murine receptors and ligands of the tnfsf discussed in this section. i. tumor necrosis factor-ix (tnf-ix) tnf-tx is expressed as a -kda membrane glycoprotein and the soluble glycoprotein is generated by proteolytic cleavage via tnf-ct converting enzyme (tace). the -kda homotrimer cleavage product circulates. mouse tnf-tx has % sequence homology with human. tnf-tx is expressed widely on tissues throughout the body (goetz et al. ) . tnf-tx is a strong mediator of inflammation and immune function, or regulates on growth and differentiation, and is cytotoxic for many transformed cells. ii iii. cd l cd l (tnfsf , cd ) is a -kda, type ii, transmembrane glycoprotein that can be proteolytically cleaved to -to -kda soluble forms with full biologic activity. it forms natural trimeric structures and the mouse cd l shares % sequence identity with human cd l. cells expressing cd l include b cells, cd + and cd § t cells, monocytes, nk cells, and y t cells (toubi and shoenfeld ) . on binding cd , the complex initiates signals important for cell proliferation or apoptosis. cross-linkage between t and b cells allows cd to transduce the tyrosine kinases lyn and syk, and activate phospholipase c, ip , and dag. when combined with other cytokines, ligation of b-cell cd provides the second signal allowing differentiation of b cells to plasma cells (goldsby et al. ) . iv. cd l cd l (tnf f , cd ) is a -kda glycoprotein with % sequence homology between murine and human molecules. cd l is expressed on monocytes, macrophages, b cells, activated t cells, neutrophils, megakaryocytes, resting cd § t cells, erythroid precursors, and eosinophils. ligation to cd can induce either proliferation or apoptosis. v. fas ligand (fasl) fasl (also known as tnfsf ), is a -kda glycoprotein that, after cleavage, forms a -kda homotrimer that is active only in membrane form in the mouse. polymorphisms in fasl also exist and a single amino acid substitution in position (phe to leu) results in the generalized lymphoproliferative disease (gld) mutation. fasl is expressed on cells of the adaptive and innate immune systems, as well as cells of the lung and intestine. there is % sequence homology between murine fasl and human fasl (lynch et al. ) . ligation of fas by fasl on mature t cells leads to activation of the caspase cascade and apoptosis. this is a major homeostatic mechanism regulating the size of the t-cell pool and for eliminating t cells that repeatedly encounter self-antigens (goldsby et al. ) . vi. tnf-relateo activation-induced cytokines (trance) trance, also called rank ligand and osteoprotegerin ligand (opgl), is an osteoclast differentiation factor. mouse and human share % sequence homology, and trance is expressed on t cells and t-cell rich organs such as thymus and lymph nodes. vii. tnf-receeror superfamily (tnfrsf) tnfrsf members mediate the cellular effects of tnfsf members. tnfri and tnfrii bind tnf-~ and it appears tnf-~ complexes with lt-[ and the complex binds to tnfri or lt-[~ receptor. cd is associated with b-cell proliferation but is expressed on many cells throughout the body. mouse cd shares % sequence homology with human cd ; however, the mouse molecule has a amino acid extension of its cytoplasmic tail. cd has amino acid residues and a amino acid deletion in the extracellular region compared to human. it is expressed on cd + and cd + t cells and ligation results in production of il- . murine fas lacks amino acid residues found in human and shares only % sequence homology. soluble forms result from alternative gene splicing and circulate as dimers or trimers. fas is expressed by cd stem cells, fibroblasts, nk cells, keratinocytes, hepatocytes, b and t cells (and their precursors), and eosinophils. osteoprotegerin (opg) inhibits the action of osteoclasts and is a secreted member of the tnfrse although it has no transmembrane segment and circulates as a disulfide-linked homodimer. murine troy, also named toxicity and jnk inducer (taj) and tnfrsf , shares % homology with human in its extracellular domains. d. interferons interferons are a group of related but distinct proteins that share more than % amino acid sequence homology. members of the type i interferon family share a common cell surface receptor composed of two subunits. commercially available elisa kits may be used to quantify murine io interferon-~ (ifn-~) ifn-~ is induced in a wide variety of cells, including monocytes and macrophages, in response to viral infection. one known inducer is double stranded ribonucleic acid (dsrna). induce resistance to viral replication by binding the ifn-~/~ receptor, which activates the jak-stat pathway, inducing several genes. one of those genes is ribonuclease, which degrades viral rna. binding of ifn-~ to nk cells enhances their lytic activity for virally infected cells. ifn-~ is secreted by leukocytes. iii inreturelcon-'t(ifn-y) ifn-y is secreted by th cells, nk cells, and cytotoxic t cells (tc), which activates macrophages to secrete tnf-ct, express class ii major histocompatibility complex (mhc) molecules, and produce antimicrobial activities. ifn-y secretion by th also induces antibody-class switch to igg a, which supports phagocytosis and complement fixation. ifn-y promotes differentiation of tc from cd + precursors that will be involved in the effector response to viral infections and intracellular pathogens. ifn-y also inhibits the expansion of th cells. ifn-y secretion is induced by successful stimulation of t cells by antigen presenting cells. e. chemo~s chemokines, along with adhesion molecules, are the principle controllers of leukocyte migration and as such directly affect leukocyte retention and relocation during hematopoiesis and at sites of immune defense and inflammatory disease (moser et al. ) . chemokine-induced signaling is via g-protein coupled cell surface receptors. although most chemokines are secreted proteins, two chemokines, cxcl and cx cl , are membrane bound. two primary subfamilies are recognized based on the arrangement of two nh -terminal cysteine residues that are either located adjacent to each other (cc) or are separated by a single amino acid (cxc). two minor subfamilies include chemokines with a single cysteine resides (xcl , xcl ) and a chemokine with three amino acids separating the cysteine residues (cx cl ). functionally conserved regions of the n-terminus of each member mediate receptor binding and extracellular matrix fixation (or binding cell surface glycosaminoglycans). many chemokines are designated with a name given at the time of their identification; all have also been assigned a name based on their structural motif (cc, cxc, xc, cx c) followed by l for ligand. receptors are heterotrimers and their activated g-protein subunits stimulate phospholipase c[ , piks, and c-src tyrosine kinases (moser et al. ) . receptors are designated by the type of chemokine they bind (i.e., cxc, cc, xc, or cx c), followed by r. mice lack the cxcr family of chemokines and receptors found in humans. table - lists the murine chemokine receptors and the known ligands. table - lists the murine chemokines for which there are either commercial test kits or matched antibodies for kit development. two main functional groups define chemokines. inflammatory chemokines recruit effector leukocytes to sites of infection, inflammation, and repair. homeostatic chemokines control the navigation of leukocytes during hematopoiesis in the bone marrow and thymus, control homing of cells to spleen, lymph nodes, and peyer's patches during the adaptive immune response and control immune surveillance in peripheral tissues. some chemokines participate in both inflammation and homeostasis and are called dual-function chemokines. many dual function chemokines are highly selective for the recruitment of t cells. other chemokines have ill-defined functions regarding homeostasis and inflammation but participate in other vital activities such as the role of pf (cxcl ) in thrombosis and the role of cxcl in gut epithelial cell turnover (cliffe et al. ) . most inflammatory chemokines are thought to be induced and the variety of stimuli that induce their expression is broad. by contrast most homeostatic chemokines are thought to be constitutively expressed. an exception is the inducing effect of lymphotoxin and tnf-~ on b-cell attracting chemokine- (bca-i, cxcl ), ccl , and secondary lymphoid tissue chemokine (slc, ccl ), which also participate in inflammation. leukocytes also release several kinds of proteases that degrade chemokines at their n-terminus, resulting in the loss of receptor binding, antagonist generation, or enhancing their biologic function. leukocyte cd , dipeptidyl peptidase iv is known to act on cxcl , cxcl , cxcl , and cxcl . murine sulphostin inhibits the action of cd and stimulates g-csf and granulopoiesis (abe et al. ) . matrix metalloproteases (mmps) are enzymes that degrade extracellular matrix proteins, including stromal cell derived factor- (sdf- , cxcl ) and mcp- . table - lists the murine mmps that can be quantified by commercially available elisa kits. in addition to mmps, cathepsins have been shown to modify chemokines. table - lists the murine chemokines and the proteases that degrade them and stimuli that induce them. certain chemokines may antagonize the activity of other chemokines; for instance, three agonists for the receptor cxcr are also antagonists for receptor ccr (which is agonized by eotaxin [ccl ]). because cxcr and ccr are differentially expressed on th and th cells, these chemokines [monokine induced by ifn-y (mig/cxcl ), ifninducible protein- (ip- /cxcl ), and ifn-inducible t-cell chemoattractant (i-tac/cxcl )] modulate the th subpopulation allowed to enter tissue sites favoring th immune polarization (moser et al. ) . table - summarizes some known effects of murine chemokines. because chemokines and their receptors are known to modulate many inflammatory diseases including the autoimmune diseases, they have become target for new therapeutics. the chemokine antagonist, met-rantes, has been shown to be an effective inhibitor of allergic airway disease in mice. likewise, deficiencies of chemokines and their receptors in mice have modified disease progression in atherosclerosis, autoimmunity, and also prolong allograft survival (mackay ). in addition, many viral genomes are known to encode structural genes for chemokine antagonists, which appears to be a principal mechanism used by many viruses to evade the host immune system. these present another target for drug intervention for viral infections. finally, pepducins, derived from the intracellular loops of cxcr , cxcr , and cxcr , specifically inhibit receptor g-protein signaling in mice and prevent fatal sepsis and disseminated intravascular coagulation (kaneider et al. ). certain growth factors (see table - ) and cytokines activate phospholipases after binding their cell surface receptors. these phospholipases act on membrane phospholipids to release arachidonic acid, a precursor for several eicosanoids. arachidonic acid is metabolized by any one of three biochemical pathways: the cyclooxygenase (cox) pathway, which forms pgs and thromboxane, the lo pathway, which forms hetes and leukotrienes (lts), and the cytochrome p- monooxygenase pathway, which forms epoxides and hetes. the cox enzymes, cox- and cox- , catalyze the first step in the synthesis of pgs by converting arachidonic acid to prostaglandin h (pgh ). pgh is chemically unstable and is the precursor for enzymatic and nonenzymatic production of pgd , pge , and pgfza. thromboxane synthetase converts pgh to thromboxane a (txa ) that is quickly converted to thromboxane b (txb ). in vascular tissue, pgh is converted to pgi or prostacyclin by prostacyclin synthetase (natarajan and nadler ; reimers ) . although cox- is constitutively expressed in most tissues, cox- is induced by bacterial lipopolysaccharides, il-i~, il-ll~, and tnf-~ (see the "cytokines and chemokines" section). in the circulation, pge and pgi cause vasodilation, whereas pgfza and txb are potent vasoconstrictors. in the kidney, pge , pge , pgd , pgg , pgi , and pgh produce vasodilation, increased renal blood flow, and urinary excretion of sodium. renal production is increased by pgd , pge , and pgi . pgg , pgh , and txa modulate platelet aggregation and, following platelet adhesion, they release catecholamines, serotonin, and adenosine diphosphate, which enhance platelet aggregation. pgi is a potent inhibitor of platelet aggregation. pge increases, whereas pge decreases, the ability of red cells to pass through capillaries. pge and pgfza inhibit the activities of t and b cells and the production of ils and chemokines, which attract monocytes. pgd is a potent inducer of chemotaxis for th cells and plays a major role in allergic airway disease. through the varied activities of vasodilation, vascular permeability, and leukocyte migration, the pgs are potent modulators of inflammation. in addition pge inhibits mackay rossi et al. niess et al. mackay huang and xiang yamaguchi et al. insulin secretion and release after glucose challenge (see the "glucose and carbohydrate metabolism" section), and it inhibits the lipolytic effects of glucagons, adrenocorticotropic hormone, and epinephrine (see the lipid metabolism section) and inhibits the secretion of corticosterone, prolactin (prl), gh, thyroid stimulating hormone (tsh), and lh. in fact, pgs have a multitude of effects associated with female reproduction (reimers table under the action of -lo, arachidonic acid is converted to -hydroxyperoxyeicosatetraenoic acid ( -hpete) and then leukotriene a (lta ), which is unstable. lta is metabolized to ltb by lta hydrolase or to cysteinyl leukotrienes (ltc , ltd h, and ltc ) by ltc synthase. -lo expression is limited to neutrophils, eosinophils, monocytes, and mast cells; however, lta hydrolase is expressed in erythrocytes, t cells, platelets, airway and intestinal epithelial cells, fibroblasts, heart, kidney, and adrenal cortex. because the latter cells and tissues do not express -lo, lta must be delivered to them via myeloid cells by a process known as transcellular biosynthesis (maclouf ) . receptors for ltb (blt and blt ) have been identified and are either widely expressed (blt ) or are confined to myeloid cells, t cells, and lung cells (blt ). in addition to attracting myeloid cells to sites of inflammation, ltb is a potent inducer of th and th t effector cell chemotaxis (as potent as cxcl in the mouse) and cd +t-effector cell chemotaxis (as potent as rantes). like pgd , ltb plays a major role in allergic airway disease (luster and tager ) . additional information on murine prostanoids and their receptors is available in recent reviews (jala and haribabu ; kobayashi and narumiya ) . all the arachidonic acid metabolites discussed in this section may be quantified using commercially available elisa kits (see table - ). e. enzymes ap is an inducible enzyme in which serum activity is increased due to increased synthesis. the exact physiologic function of ap is not known but is thought to transport metabolites across cell membranes. quantification of serum ap is based on a reaction between ap and a suitable phosphorylated substrate that is susceptible to ap activity. as in other species, there are two major forms of ap in mice: intestinal ap (lap) and tissue nonspecific ap (tnap). unlike other domestic animal species, iap activity contributes to serum ap activity, in addition to tnap. lap is located along the brush-border of the enterocytes. intestinal ap activity was shown to vary four-fold between two strains of swiss mice (nayudu and moog ) . this difference in activity was under polygenic control and influenced by a strain-specific factor in milk. tnap is found in various tissues, and in each location, post-translation modifications may result in a different isoenzyme. hoshi et al. ( ) used immunohistochemistry to localize these isoenzymes in mice to the following locations: bone tissue (specifically the entire cell surface of preosteoblasts and the basolateral cell membrane of osteoblasts), cartilage (mostly in chondrocytes of the proliferative and hypertrophic zones), the incisors (particularly the cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts), kidneys (on the brush borders of proximal renal tubules in kidney), liver (on cell membrane of the biliary canaliculi), and the placenta (on trophoblasts). serum ap activity can vary due to the type of assay used, age, sex, and strain. different ap assays vary in the type of substrate used, the ph of the reaction, and the incubation temperature of the reaction, all of which can affect quantitation of ap activity. for example, the hausamen technique can detect renal and intestinal ap activity, but not hepatic ap activity (hausamen et al. ). loeb et al. ( ) and frith et al. ( demonstrated that serum ap activity decrease significantly after months of age in balb/c and c bl/ mice of both sexes but increase again in very old ( month old) mice. the high levels of serum ap seen in juveniles is related to increases in the bone ap isoenzymes, which is associated with osteoblastic activity due to rapid growth. picketing and picketing ( ) showed that serum ap activity is lower in males than in females. quantification of serum ap measures the total ap activity from all sources, and therefore elevations in ap activity can be a nonspecific determinant of tissue dysfunction, depending on the tissue (and therefore ap isoenzyme) involved. for example, changes in the serum activity of ap due to liver disease only occur if cholestasis is concurrently involved. mice lacking the gene that modifies tnap into the bone isoenzyme suffer from skeletal hypomineralization (anderson et al. ). alt is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. alt (also known as glutamic pyruvic transaminase [gpt]) reversibly catalyzes the conversion of alanine to pyruvate. quantification of serum alt is based on a reaction between alt and a suitable substrate (such as alanine). in mice, alt is found in the highest concentrations in the liver, although activity has also been demonstrated in intestine, kidney, heart, muscle, and brain (clampitt and hart ) . despite its widespread tissue distribution, alt is mostly used as an analyte to assess hepatocellular damage (masaki et al. ; taieb et al. ). an , % increase in serum alt activity has been reported following infection with mouse hepatitis virus, and significant increases follow infection by helicobacter hepaticus (mccathey et al. ). ast is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. ast (also known as glutamic oxaloacetate transaminase [got]) reversibly catalyzes the conversion of aspartate to oxaloacetate. quantification of serum ast is based on a reaction between ast and a suitable substrate (such as aspartate). in mice, ast is found in a variety of tissues, including liver, skeletal muscle, cardiac muscle, erythrocytes, blood vessels, brain, intestine, kidney, lung, testes (papadimitriou and van duijn ) . the highest specific activity of ast is found in mouse cardiac muscle and lowest in skeletal muscle (herzfeld and knox ) . in the liver, ast is found mainly in periportal hepatocytes based on histochemical studies (papadimitriou and van duijn ) . activity in lung, kidney, intestine, and skeletal muscle is very low when measured by the technique of bergmeyer and bernt ( ) . loeb et al. ( ) demonstrate significant age-associated increases in serum ast activity in two inbred and two f hybrid strains. although widely distributed, alt is mainly used to assess hepatocellular damage, cardiac muscle damage (naraoka et al. ; ray et al. ) , and testicular injury (santos et al. ) . alt activity has been used as an indicator of hepatic injury of mice infected with mouse hepatitis virus (fassati et al. ). ldh is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. ldh reversibly catalyzes the conversion of pyruvate to lactate. quantification of serum ldh is based on a reaction between ldh and a suitable substrate (such as pyruvate or lactate). as in other species, ldh in the mouse is a tetrameric enzyme consisting of either a or b subunits. there are five isoenzymes of ldh (based on differences subunit a and b composition), and these are ldh- (b ), ldh- (a b ), ldh- (a b ), ldh- (a b ), and ldh- (a ). specific isoenzyme distribution depends on differential expression of either the a and b subunits (quimby b) . for instance, all embryonic murine tissues contain ldh- because the a subunit is only expressed during early fetal development. as the embryo matures, subunit expression can involve both a or b subunits in different tissues, meaning that by birth and sexual maturity, each tissue contain a characteristic ldh subunit profile. in adult mice, the heart and erythrocytes contain ldh- and ldh- , whereas most other tissues have ldh- . skeletal muscle and hepatocytes fail to express subunit b and therefore are composed predominantly of ldh- . serum ldh activity can vary due to age and sex. ldh levels have been shown to be higher in males versus females of the balb/c strain (frith et al. ) . serum ldh levels increase with age in balb/c and c bl/ mice of both sexes (frith et al. ) . serum ldh can also be elevated falsely by hemolysis. quantification of serum ldh measures the total ldh activity from all sources. however, damage to a particular tissue will result in increased activity of that isoenzyme in serum. in general, the highest activity of ldh in the mouse is in skeletal muscle, with decreasing activity in the heart, liver, kidney, and intestine. serum ldh- activity rises within h after inoculating mice with the mouse hepatitis virus (fassati et al. ) . mice infected with the ldh virus (ldv) exhibit increased serum concentrations of ldh, isocitric dehydrogenase, malic dehydrogenase, phosphohexase isomerase, and ast (notkins ) . along with ast and ck, ldh is considered an excellent marker for cardiac injury (naraoka et al. ; ray et al. ) . otc is a mitochondrial enzyme in which serum activity is increased due to leakage across damaged mitochondrial membranes. otc is found primarily in the liver of mice, and there increases in serum activity reflect severe injury to hepatocytes. abnormal otc activity has been described in mice having the sparse-fur (spf/y) mutation. they serve as a model for the most common inborn error of urea synthesis in humans. assays for mouse otc have been developed to monitor activity levels following gene transfer or liver transplantation (batshaw et al. ; ye et al. ). ck is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. ck reversibly catalyzes the phosphorylation of adenosine diphosphate (adp) to ate using creatine phosphate as the donor for the phosphate group. quantification of serum ck is based on a reaction between ck and a suitable substrate (such as creatine phosphate). as in other species, cytoplasmic ck is a dimeric enzyme consisting of either m or b subunits. there are three isoenzymes of ck (based on differences subunit m and b composition), and these are ck- (bb), ck- (mb), and ck- (mm). brain contains ck- , skeletal muscle contains ck- (mm), and cardiac muscle contains ck- , ck- , and ck- (mb) (quimby b ). in the mouse, the greatest ck activity is found in skeletal muscle, with much less activity found in the heart and brain. mitochondrial ck (ck-mt) is found in mitochondria of many tissues. serum ck activity is affected by age, sex, and method of collection and anesthesia. patrick et al. ( ) found that compared to jugular vein collection, cardiac puncture was associated with lower ck activity. levels of serum ck activity have been reported for balc/cann mice and c bl/ mice of varying ages and sex (sub et al. ) . serum ck activity is a useful and specific marker enzyme of muscle injury, because ck in central nervous tissue does not cross the blood-brain barrier. sub et al ( ) compared normal c bl/ mice with heterozygous male and homozygous female mice carrying the mdx (mutant dystrophin) allele, and found that homozygous females have -to -fold increases and heterozygous males (mdx/y) have fold increases in plasma ck compared to wild-type mice. plasma ck levels correlated with skeletal muscle necrosis in these dystrophic mice. mice have been engineered that lack cytoplasmic ck and ck-mt . mitochondria from heart or skeletal muscle from double kos had higher adp concentrations compared to wild-type animals, suggesting the higher concentrations contribute to the control of the reduced cytosolic atp free energy potentials seen in double kos. aldolase is a cytosolic enzyme that can alter its distribution between soluble and particulate forms, according to the metabolic status of the tissue. in adult mice, nine aldolase isoenzymes are known to occur in tissues with significant activities in the muscle, brain, liver, kidney, and spleen. in the mouse liver aldolase, together with fructokinase and triokinase, metabolize fructose (hagopian et al. ) . everett and harrison ( ) report no apparent advantages in mice in the measurement of aldolase over other enzymes known to have specific liver or muscle activity. sdh is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. sdh (also known as iditol dehydrogenase [idh]) reversibly catalyzes the conversion of fructose to sorbitol. quantification of serum sdh is based on a reaction between sdh and a suitable substrate (such as fructose). sdh is located primarily liver, kidney, and seminal vesicles. the activity of sdh is usually low in the serum and rises during hepatic injury. however, the labile nature of sdh during handling makes is less suitable overall as a indicator of hepatic dysfunction compared to over liver-specific enzymes (such as ast). mice with the gene for sdh knocked out have been used to study the role of sorbitol accumulation in diabetic albuminuria (ii et al. ). amylase is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. amylase hydrolyzes complex carbohydrates to form maltose and glucose in the presence of free calcium ions. quantification of serum amylase is based on a reaction between amylase and a suitable substrate (such as starch). similar to humans and pigs (but not dogs, cats, cattle, and horses), expression of amylase in mice is related to two distinct but closely linked loci (meisler et al. ) . salivary amylase is the gene product of amy- (salivary), and appears to be a single enzyme. pancreatic amylase is the gene product of amy- , and based on electrophoretic studies in inbred mice, there appear to four isoenzyme classes: a , a , b , and b . similar to other domestic animal species, pancreatic amylase is filtered through the glomerulus, but unlike other domestic species, pancreatic amylase is not resorbed by renal tubular epithelial cells and is excreted rapidly in the urine. therefore normal serum amylase activity in mice consists mainly of salivary amylase (mackenzie and messer ) . despite this, elevations in serum amylase activity is usually considered a reliable marker for pancreatitis in mice (nathan et al. ) . ross et al. ( ) reported two-to three-fold increases in serum amylase activity in mice infected with coxsackievirus of salivary and pancreatic trophism. alterations in the activity of specific pancreatic isoenzymes have been shown in streptozotocin-induced diabetes in mice (quimby b) . there are two pancreatic lipase isoenzymes in mice. serum lipase activity has been used to monitor cerulean-induced acute pancreatitis in mice (cuzzocrea et al. ) . recently a new colipase-dependent lipase has been described in suckling mice (d'agostino and lowe ) . the enzyme '-nucleotidase was measured in the serum of normal mice using a simple one-step kinetic method (dooley and racich ) . a reference range of . + . (sd) u/ has been recorded in mice, and it is thought but not proven to be a good indicator of hepatic injury (clampitt and hart ) . glutamate dehydrogenase (gdh) has been measured in the tissues and serum of mice. gdh is known to play a key role in insulin secretion (carobbio et al ) . the activity of gdh is fivefold greater in the liver than in the kidney and brain, and the authors speculated that its measurement in serum would be a sensitive indicator of hepatic cell injury. serum gdh activity is also elevated in mice on caloric restriction (hagopian et al. ). corticosterone is the major glucocorticoid secreted by the adrenal cortex of mice. it functions as a regulator of carbohydrate, protein, and fat metabolism and modifies the host response to stress. the male mouse has a well-defined diurnal concentration pattern, with a maximum concentration of ktg/dl at the start of the dark cycle and a minimum concentration of btg/dl shortly before the end of the dark cycle (ottenweller et al. ) . in contrast, female mice have a minimum concentration of . ktg/dl at the beginning of the dark cycle and a maximum of ~tg/dl well into the dark period (scheving et al. ) . the length of the dark cycle was different in each study. it may be measured using radioimmunoassay, elisa, or fluorometric assay in mouse serum or plasma. corticosterone circulates in both free and protein bound forms. in the mouse, greater than % circulates bound to cortisol-binding globulin (cbg) and albumin. the diurnal variation in corticosterone levels is paralleled by the diurnal pattern of cbg. urinary and salivary corticosterone is derived only from the free plasma fraction. corticosterone synthesis and secretion may be influenced by many drugs (woodman ) . measurements of corticosterone in unrestrained mice using indwelling catheters have elucidated the necessity of eliminating stress for accurate interpretation of data (macleod and shapiro ) . both handling and crowding laboratory mice can cause elevations in corticosterone (balcombe et al. ; fullwood et al. ) . c bl/ ob/ob mice have elevated corticosterone levels that increase markedly after weeks of age, preceding elevations in serum glucose (garthwaite et al. ) . lean c bl/ controls had lower serum concentrations of corticosterone that declined between and weeks of age. food restriction (to % of ad libitum) profoundly affects the diurnal increase in plasma corticosterone in mice. at : hours ( p.m.), the daily maximum in this study, food restricted balb/c mice had % more corticosterone compared to controls. these mice also had significantly reduced thymic weight and inflammation in response to the injection of carrageenan subcutaneously. the authors report that the magnitude of carrageenan-induced inflammation fluctuates with a diurnal trough, which coincides with peak corticosterone levels (klebanov et al. ) . corticosterone is synthesized in response to acth, which is made in the anterior pituitary. acth release is episodic (not at fixed intervals) and does not involve steroid feedback. acth concentration peaks in the early evening in mice and can be reversed by reversing the light cycle. acth is highly conserved with the mouse acth differing from human by only two amino acid residues. ria may be used to measure acth in the mouse (daily levels vary from . - . ng/ml) and to demonstrate rhythmic cycling. samples must be collected at -to -minute intervals. commercial elisa kits for acth are also available (quimby b) ko mice incapable of synthesizing corticotrophin releasing hormone or acth may require corticosterone supplementation in drinking water and are particularly susceptible to hypoglycemia during fasting. fetuses of homozygous ko crh null mothers must be supplemented from gestational day to weaning with mg/ml corticosterone in drinking water (mugila et al. ) . oxytocin gene ko mice respond to a psychogenic stressor with more anxiety-related behavior and more corticosterone production (amico et al. ). lh promotes follicular development, increases estradiol secretion in the preovulatory follicle, causes follicular rupture, converts the preovulatory follicle into a corpus luteum, increases progesterone production by the corpus luteum, and, in the male, increases production of testosterone from leydig cells (woodman ) . lh is a glycoprotein composed of t~-and ~-chains. the amino acid sequence of the t~-chain is identical to that of follicle-stimulating hormone (fsh); however, the ~l-chain is specific and confers the receptor binding properties on the hormone. a homologous ria for rat lh using antisera to rat lh and purified rat lh has been employed to measure lh in mice (quimby b) . lh and folliclestimulating hormone (fsh) are secreted by the same anterior pituitary cell in response to stimulation by gonadotrophinreleasing hormone (gnrh), which is synthesized in the hypothalamus. gnrh is identical in mouse and humans, and its differential stimulatory effect on gonadotropes, producing fsh or lh is controlled through gnrh pulse frequency. pulsatile secretion of lh leads to great variations in blood concentration. in female mice, there is a -to -fold increase in basal lh levels in the afternoon of proestrus. in a study comparing young cycling c bl/ female mice to old females ( % of which not cycling), flurkey et al. ( ) found lower lh levels in the older group and a more rapid rise in lh among younger cycling mice. these authors found that reproductive failure in male mice correlated with the loss of episodic release of lh. female homozygous ko mice lacking the gene for the immediate early transcription factor ngfi-a were infertile secondary to lh-[ deficiency. although ovaries from these mice had a similar number of follicles, they lacked corpora lutea (lee et al. ) . basal levels of serum progesterone were lower in ngfi-a females ( . + . ng/ml) compared to wild type ( . +_ . ng/ml), whereas serum estradiol levels were similar in deficient ( . + . pg/ml) and wild-type ( . _+ . pg/ml) mice. decreased amounts of mrna encoding lh-[ , but not fsh-[ , were observed in ngfi-a-deficient mice; no changes were observed in mrna levels for prl or gnrh receptor between deficient and wild-type mice. an ovariectomy normally removes gonadal feedback inhibition, allowing for increased amounts of lh-~ and fsh-[ in the pituitary; however, ovariectomy of ngfi-a deficient female mice lead to an increase in fsh-~ only. homozygous ngfi-a deficient males did make lower amounts of lh-~ compared to wild-type males, but they made significantly more lh-[ than did ngfi-a-deficient females. the levels of lh in males appeared to be sufficient for fertility, and they had normal serum testosterone levels. these results suggest that ngfi-a acts synergistically with transcription factor sf- to regulate the promoter for lh-[ gene expression. the simultaneous activation of both sf- and ngfi-a by gnrh appears to confer the specificity of lh-~ synthesis. female transgenic mice overexpressing lh are anovulatory, develop granulosa cell tumors, and undergo precocious puberty (mann et al. ) . excellent reviews detailing the use of transgenic and ko mice in elucidating the secretion and function of lh have been published (bums and matzuk ; huhtaniemi et al. ; wells and murphy ) . fsh stimulates the growth and maturation of ovarian follicles in the female and promotes the latter stages of spermatogenesis in males. it acts principally on the sertoli cells of the seminiferous tubules of the testis and induces the secretion of androgen-binding protein and inhibin. in females, fsh and lh have distinct secretory patterns that are synchronized to the estrous cycle; mouse estrous cycles have a -to -day periodicity. in addition to estrous cycle dependent rhythms, both lh and fsh have ultradian pulses and show circadian periodicity, with highest levels occurring during the dark period of the light cycle. testosterone, estrogen, and progesterone all provide feedback regulation of lh and fsh secretion (woodman ) . because rat and mice share considerable sequence homology for the fsh-~ chain, the rat ria kit from national institute of diabetes and digestive and kidney diseases (niddk) can be used to measure fsh in the mouse (dalterio et al. ) . a mouse specific ria is commercially available. in cycling female mice, the plasma levels of fsh increase from to ng/ml during proestrus and - ng/ml during estrus. although bronson and desjardins ( ) found age associated decreases in serum lh and testosterone in male cbf mice experiencing decreased sperm production, no similar decreases were observed in fsh concentration. no significant differences in the twofold increased fsh levels were seen among young versus old males following castration. this appears to differ from results obtained from young versus old rats (finch et al. ) . the regulation and function of fsh has been studied in transgenic and ko mice (cooke and saunders ; wells and murphy ) . testosterone promotes spermatogenesis and provides feedback regulation of gonadotrophin synthesis. through its action on the epididymis, proteins necessary for spermatozoal maturation are synthesized. dihydrotestosterone (dht) promotes the growth and differentiation of accessory sex organs (depaolo and masoro ) . in addition to their effects on reproductive organs, testosterone and dht have physiologic functions on the central nervous system, cardiac tissue, and the liver. in the mouse, testosterone stimulates the growth of kidneys and synthesis of erythropoietin (woodman ) . lh stimulates synthesis of testosterone from cholesterol by the interstitial leydig cells of the testis. in target tissues, the enzyme r converts testosterone to dht. in the fetal testes, leydig cells first become identifiable during the rise of testosterone. in most mammalian species, leydig cells disappear when testosterone levels fall during gestation. however, the mouse is an exception and leydig cells do not undergo regression postnatally (aoki ) . testosterone and dht circulate in both free and protein bound forms. ninety-eight percent of total testosterone is bound to protein, mainly albumin. mice, unlike humans, dogs, monkeys, and cats, do not have circulating sex hormone-binding globulin. in mice, a pulse release pattern may be seen within their diurnal rhythm of testosterone. testosterone can be measured in mice using ria or elisa. plasma testosterone was not found to change during the average lifespan of c bl/ and dba/ j mice (finch et al. ) . this is in contrast to cbf mice, wistar and long-evans rats, and humans, which experience greater than a % decrease in testosterone at midlife (bronson and desjardins ) . in contrast to testosterone levels in older mice, castration-induced elevation of lh was impaired in -month-old c bl/ j mice compared to -month-old mice. androgen receptor ko mice have been described (cooke and saunders ) . . estradiol (e ) the granulosa cells of the mature graafian follicle are the main source of [ -estradiol (e ) in mammals. fsh stimulates the activation of aromatase in follicles, which is responsible for the conversion of androgens to e . e is also synthesized by the testis, adrenal, liver, and skin, although in much lower amounts than by the ovary. e provides negative feedback control over lh secretion, and it stimulates prl secretion in mice. e promotes the growth and development of the female reproductive tract, external genitalia, and the ductal system of the mammary glands. in addition, e sensitizes the follicle to fsh. e is measured in mice using ria and elisa. high circulating levels of e precede the preovulatory surge in lh. ryan and schwartz ( ) reported basal levels of e in mice of to pg/ml. holinka et al. ( ) studied circulating levels of progesterone and estradiol in young and old c bl/ female mice during gestation. they found that the old multiparous females have delayed and reduced preparturitional rise is plasma e compared to young females. because preparturitional e is thought to regulate uterine progesterone levels, the decline in e seen in older females coupled with elevated progesterone may delay the onset of labor and lead to prolonged gestation. when sex steroid hormones such as e bind their receptor, they induce a conformational change that allows the complex to bind dna response elements on nuclear target genes and associate with coactivators and transcription factors to form an active transcriptional complex. this complex is responsible for initiating the transcription of the target gene. one coactivator that associates with the active transcriptional complex involving sex hormones is steroid receptor coactivator- (src- ). to better understand its physiologic role during sex hormone-receptor binding, ko mice that were deficient in src- were created (xu et al. ). deficient male mice had a % reduction in testosterone-stimulated prostate growth and small testes compared to wild-type mice. deficient females had a half-normal uterine growth response to exogenous e and only a partial ductal growth response (in the mammary gland) following exogenous e and progesterone. serum concentrations of e and testosterone were elevated . to . times wild-type levels, indicating an abnormality in the endocrine feedback control system. although src- was shown to clearly be necessary for optimal sex hormone-induced cellular activation, the lesion created in this ko mouse was not nearly as profound as that seen in mice with disrupted e receptors (korach et al. ) . mice with the genes for the e receptors and aromatase knocked out have been described (simpson et al. synthesized by the corpus luteum before implantation and by both corpus luteum and placenta following implantation, progesterone is necessary for preparing the uterus for implantation and maintaining pregnancy. in addition, a small amount of progesterone is synthesized by the adrenal gland. activation of the corpus luteum to secrete progesterone requires both lh and prl. progesterone provides negative feedback control over lh. in mice, progesterone is measured using ria or elisa. in cycling mice, the levels of progesterone increased from to ng/ml on proestrus and returned back to baseline on estrus. flurkey et al. ( ) compared the plasma levels of lh, progesterone, and prl in cycling young ( -week) and old ( month) female c bl/ j mice. they showed age-related deficits in the preovulatory levels of all three hormones. the lh elevations during the ascending and descending portions of the preovulatory surge were smaller; the slower rises in lh during the ascending phase of the surge correlated with decreased progesterone in older females. there was no correlation between lh or progesterone level and length of estrus cycle. holinka et al. ( ) observed changes in plasma progesterone levels in young and old female mice during gestation. older mothers had a much slower decline in circulating progesterone than younger mothers between gestational days - . because a major decrease in plasma progesterone is thought to be essential for the onset of uterine contractions and parturition in mice, the authors hypothesized that the attenuated decline in older mothers may account for their prolonged gestation. surgimoto et al. ( ) engineered mice lacking the pgfzareceptor (fp) and found that deficient female mice had normal estrous cycles, ovulation, fertilization, and implantation but did not undergo parturition. these pregnant females were characterized by a decline in preparturition progesterone levels due to delayed luteolysis and failure to develop labor. close examination revealed that near term there was no expression of the oxytocin receptor in the uterus of fp-deficient mice. when the ovaries of fp-deficient pregnant females were removed on day of pregnancy, progesterone levels fell, uterine oxytocin receptors were expressed, and pups were born alive within h. this study demonstrated that the role of pgf ~ is to initiate luteolysis, resulting in an immediate decline in progesterone levels. the subsequent induction of oxytocin receptors and their activation by bound oxytocin initiate labor. it is feasible that the age-associated prolongation of gestation in old female mice may involve a defect in pgfz,~-induced luteolysis. this mechanism for the induction of labor also explains the well-known observation that aspirin-like drugs (that inhibit cox metabolism) may delay parturition in women. prl, a -kda single-chain polypeptide, is principally made by the pituitary gland but may be also be found in brain, thymus, spleen, lymph nodes, mammary gland, myometrium, sweat gland, lacrimal gland, and bone marrow. more than functions have been attributed to prl, involving reproduction and lactation, growth and development, endocrinology and metabolism, brain and behavior, immunomodulation, and electrolyte balance. it mediates effects by endocrine, paracrine, and autocrine mechanisms. in mice, prl is of major importance for maintenance of the structure, life span, and function of the corpora lutea and the development and maintenance of the mammary gland and lactation. however, prl-receptor ko males have no major defect in fertility. prl plays a role in anxiety-related behaviors, bone development, and abdominal fat deposition in both sexes (kelly et al. ) . its role in immune function is more controversial, although it appears to modulate immunity during times of stress (dorshkind and horseman ) . prl mediates its effects by binding the prl receptor (prlr). mice have two major forms of prlr that are created via alternative splicing of a single gene. the prlrs are members of the class i cytokine receptor superfamily. although most functions of prl are mediated by the unmodified -kda peptide, post-translational modification allows variants of prl to bind its receptor eliciting transcription of genes necessary for tissue specific changes (harris et al. ) . prl secretion is under inhibitory control by dopamine and among its secretagogues are thyrotropin-releasing hormone (trh), vasoactive intestinal peptide, gastrin, serotonin, ~-endorphin, oxytocin, angiotensin ii, gnrh, and arginine vasopressin. prl in mice is quantified using ria using the nih rp- reference standard. male mice measured during the light phase have < ng/ml whereas the range during the dark phase was - ng/ml (depaolo and masoro ). oxytocin, a -kda nonapeptide, is synthesized as a prohormone in neurons whose bodies are located in the supraoptic and paraventricular nuclei at the base of the hypothalamus. once synthesized, the prohormone is packaged into neurosecretory granules and transported down the axons of these neurons transport synthesized oxytocin to the pars nervosa of the pituitary gland. during transport, the molecule is cleaved to yield the -kda carrier proteins, neurophysin i, and the l l-kda oxytocin. the primary stimulus for oxytocin release is mechanical stimulation of the mammary gland and distention of the reproductive tract (reimers ) . there is a single oxytocin receptor (otr). investigations using ko mice have shown that oxytocin is required for milk let down by the mammary gland as well as for postpartum alveolar proliferation. in males, oxytocin is required for normal spermiation and sperm transfer. in both genders, oxytocin helps control normal blood pressure and salt intake. kos display reduced aggression and a striking deficit in the ability to recognize a previously encountered mouse (young and gainer ) . oxytocin may be quantified in mice using commercially available elisa kits or by species-specific ria. values in one recent publication showed plasma oxytocin at . _+ . pg/ml in male c bl/ mice ). avp is synthesized and released by the same neurons previously described for oxytocin, although the two secretory granules rarely colocalize in the same neuron. the carrier protein for avp is neurophysin ii. avp is released from neuron secretory granules by electrical signals from osmoreceptors measuring the osmolarity of extracellular fluid. action potentials, generated in the receptors, cause calcium influx into axon terminals and exocytosis of ave avp is transported in the blood to the kidneys, where it binds receptors in the distal segment of the nephron and collecting ducts leading to increased resorption of water (reimers ) . there are three avp receptors, v l ar, vlbr, and v r; and these are all g-protein coupled receptors. nonsense mutations of the avp gene (as seen in the brattleboro rat) or kd mice develop neurohypophysial (central) diabetes insipidus. mice with the v r knocked out develop nephrogenic diabetes insipidus characterized by polyuria, polydipsia, and failure to concentrate urine. when the v ar gene is knocked out, mice develop an enhanced proliferation of splenic b cells and enhanced igg and igg b production to thymicdependent antigens. when v lbr is knocked out the mice display a marked reduction in social aggression and a deficit in social recognition (young and gainer ) . avp can be measured by ria or elisa and the reported values in normal male c bl/ mice are . + . pg/ml in plasma ). tsh is a -kda glycoprotein made by the anterior pituitary gland on stimulation by thyrotropin-releasing hormone (trh). trh is made in the hypothalamus. tsh is secreted in a pulsatile manner, similar to acth. tsh secretion is regulated by triiodothyronine (t ), which acts on the hypothalamus to inhibit secretion of trh and acts on the pituitary gland to inhibit tsh synthesis (reimers ) . tsh plays a role in stimulating the thyroid gland to produce thyroxine and it influences the outcome of t-cell development in thymus and intestine. in addition to the thyroid gland, tsh receptors are found on many different populations of hematopoietic cells in bone marrow, subsets of dendritic cells, monocytes, and lymphocytes in the spleen and lymph nodes (klein ; scofield et al. ) . ko mice have been created in which genes for trh (wells and murphy ; yamada et al. ) and tsh receptors (biesiada et al. ) have been deleted. in addition, spontaneous mutations in the pitl gene lead to deficiencies of gh, prl, and tsh (yeap and leedman ) . tsh is measured by ria and reference ranges established around a . u/mg reference standard were - ng/ml (depaolo and masoro ). . thyroxine (t ) and triiodothyronine (t ) t is synthesized entirely in the thyroid gland, whereas t is produced primarily by conversion of t to t in extrathyroidal tissues. in the thyroid, thyroglobulin is synthesized and stored, and later hydrolyzed to form t . the tyrosine residues, held in peptide linkage within thyroglobulin, are first iodinated and later iodotyrosines are chemically coupled to form hormonally active t and t . production of t depends on adequate supplies of iodine. both t and t circulate in the blood primarily bound to albumin and m-globulin; however, mouse transthyretin does not bind t . approximately % of t , the active thyroid hormone, in blood is made through the monodeiodination of the outer ring of t in a variety of tissues. certain drugs, food deprivation, and illness can all effect monodeiodinase activity (reimers ) . t actions are mediated via two t receptors, tr~x and tr[ , which act as hormone-inducible transcription factors and belong to a large superfamily of nuclear hormone receptors including the steroid hormone, vitamin d, retinoic acid, and peroxisomal proliferator receptors (yen ) . in mouse and human tr~ and tr[ encode nine mrna isoforms through alternative splicing; four isoforms tr~i, trc~ , tr[ , and tri] , are expressed as proteins. trc~ ~ ko mice, which lack all products from the tr~ locus, are fertile and have normal basal thyroid status but have increased sensitivity to thyroid hormones in the pituitary and liver following provocative testing with increasing doses of t . tr~x -/-have a disruption of the first coding exon in the tr~x locus, which prevents transcription of tr~i and try mrnas but not tra~i or tra~ . these pups die shortly after weaning unless supplemented with t for the first . months of life, after which they develop normal thyroxine levels. however, both genders are infertile. try -/-overexpresses tr~i and are hypothyroid but have inappropriately normal tsh levels. they exhibit some signs of hyperthyroidism, such as increased heart rate, weight loss, and elevated body temperature. tr~ -/-lack all tr[ isoforms and display resistance to thyroid hormones demonstrating the key role of tr~ in set-point control of the pituitary-thyroid feedback axis. tr[ -/-ko mice have resistance to thyroid hormones and elevated t , t , and tsh. they have defective tsh suppression by t . several double kos (both tr~ and tri]) have been developed with profound resistance to t . these targeted mutants have helped to elucidate the full function of thyroid hormones involving: bone formation and mineralization, abnormal development of skeletal muscle, disrupted development of the cones in the retina, abnormalities in the auditory system, cochlear and vestibular structures, delayed small intestine development, impaired thermogenesis, and altered development of the central nervous system and immune system (o'shea and williams ). both total t and t can be measured by ria in mice, and reference ranges vary greatly by strain and age. although t is the active hormone, its serum levels are so low that it is a less reliable indicator of thyroid status. total t ranges in swiss webster and icr mice are . _+ . and . + . jag/ml, respectively. total t levels range from - ng/ ml in both sw and icr stocks (depaolo and masoro ). many factors, including autoantibodies, are known to interfere with thyroid hormone assays (despres and grant ; reimers ) . . parathyroid hormone (pth) pth is synthesized by the chief cells of the parathyroid gland and secreted as an amino acid peptide. circulating levels of ionized calcium induce synthesis of pth. low calcium stimulates pth synthesis, and high calcium inhibits secretion. the primary function of pth is to control calcium concentrations in extracellular fluid and prevent hypocalcemia by increasing calcium resorption from bone, glomerular filtrate, and intestines (reimers ) . pth mediates its effect via its g-protein coupled receptor, pth r. interestingly a second protein, pth related protein (pthrp), which is secreted by a variety of tissues and acts by autocrine and paracrine signaling, uses the same pth r. pthrp modulates a wide range of physiologic and developmental responses (goltzman and white ) . mice with targeted mutations of the pth, pthrp, and pth r genes have demonstrated the critical role of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes and their replacement by bone cells (schipani and provost ) . serum levels of pth may be quantified in mice using a commercially available elisa kit, which can also be used to quantify the protein in rats. . other hormones elisa kits are commercially available to quantify many additional hormones in the serum of mice, including insulin, leptin, gh, epinephrine, orexin a, orexin b, adiponectin, adipsin, and resistin. each of these hormones have been briefly discussed in the "glucose and carbohydrate metabolism" section. the liver has many complex functions including protein metabolism, carbohydrate metabolism, and lipid metabolism. the liver is involved with the synthesis of many plasma proteins (including albumin), the conversion of ammonia to urea, and the production of glucose from glucogenic amino acids). the liver is implicated with the regulation of blood glucose levels (via glycogenolysis and gluconeogenesis, as discussed in the "glucose and carbohydrate metabolism" section), the removal of glucose from the blood via glut- and glut- membrane transporters, and storage in the form of glycogen. the liver is also involved in cholesterol and triglyceride synthesis, the formation of lipoproteins (discussed in the "lipid metabolism" section), and the synthesis of carbohydrates from fats. there are also other more specific functions that the liver facilitates, including the synthesis of heme, the synthesis of many coenzymes, detoxification/biotransformation of exogenous and endogenous substances via the cytochrome p- microsomal enzymes, and the synthesis of bile. the role of the liver in these functions for various animals has been reviewed (tennant ; woodman ). the elevation of plasma or serum enzymes usually confined to the cytosol or mitochondria of hepatocytes is helpful in elucidating liver injury. many factors influence the duration of elevated serum enzyme levels including molecular size, intracellular location, rate of plasma clearance, rate of enzyme inactivation, and hepatic production. hepatic necrosis is associated with elevations of alt, ast, sd, and otc in mice, and extrahepatic cholestasis is associated with elevated ap (tennant ) . please refer to the "enzymes" section for a description of each enzyme. bilirubin is a pigment that is produced by the degradation of the hemoglobin by cells of the mononuclear phagocyte system. there are two main types of bilirubin. unconjugated bilirubin is a non-water soluble molecule that is transported in blood bound to albumin. hepatocytes uptake unconjugated bilirubin, where it undergoes glucuronidation to produce the water-soluble form, conjugated bilirubin. conjugated bilirubin is then excreted via the hepatobiliary system and excreted in bile. quantification of serum bilirubin levels is based on the van den bergh or diazo reaction. diazo reacts directly with conjugated bilirubin, and with unconjugated bilirubin only after addition of alcohol to the reaction. therefore, measuring serum bilirubin involves the following steps. first, the level of conjugated bilirubin is measured. second, alcohol is added to the reactants, which allows quantification of total bilirubin levels. finally, the level of unconjugated bilirubin is determined by subtracting the conjugated measurement from the total measurement. the excretory capacity of the liver may be assessed by measuring serum bilirubin. elevated levels of unconjugated bilirubin are usually observed in situations of increased erythrocyte breakdown, such as in hemolytic diseases. unconjugated hyperbilirubinemia is also seen in disease in which hepatic uptake, conjugation, and secretion of bilirubin are diminished. increased levels of conjugated bilirubin are usually associated with intrahepatic cholestasis or extrahepatic bile duct obstruction. conjugated bilirubin in blood is normally filtered by the glomerulus in small amounts. conjugated hyperbilirubinemia may result in bilirubinuria (tennant ) . bilirubin clearance from blood and the role of the constitutive androstane receptor in this process has been studied in normal and transgenic mice (huang et al. ; . studies documenting the bilirubin-metabolism/detoxifying enzymes, their regulatory nuclear receptors, and lipid transporters in mice have been reported (wagner et al. ) . bile acids are cholesterol breakdown products that are secreted by hepatocytes into the hepatobiliary system, and ultimately into the intestinal tract. bile acids aid digestion by emulsifying dietary lipid aggregates, and solubilizing and transporting lipids in an aqueous environment (in particular fat-soluble vitamins). in the liver, bile acids also play a role regulating the secretion of apolipoprotein b (elzinga et al. ) . extensive enterohepatic recirculation results in almost % return of bile acids secreted by the liver from the intestinal tract. absorption occurs mainly in the ileum under the influence of the apical na § bile acid transporter on epithelial cells (kida et al. ) . some absorption also takes place in the large intestine. absorbed conjugated bile acids (from ileum) pass unaltered to the portal circulation where they are removed by na+-taurocholate cotransporting polypeptide located on the basolateral hepatocyte membrane (jung et al ) . there are two types of bile acids, cholic acid and chenodeoxycholic acid. in mice, they are conjugated with taurine before secretion by the liver. the liver-specific enzymes, bile acid coa ligase and bile acid-coa:amino acid n-acyltransferase, are responsible for conjugation (inoue et al. ). stedman et al. ( ) described normal serum bile acid levels in normal and transgenic mice. elevated serum bile acid levels in mice is usually due to decreased bile acid recycling by the liver, and mainly includes diseases associated with decreased hepatic functional mass and cholestasis (tennant ) . hoda and green ( ) measured increased levels of serum bile acids after bile duct ligation. albumin is a nonglycosylated protein and is the most abundant protein in plasma. it serves as the most important determinant of plasma oncotic pressure, and it is a major transport protein for both endogenous metabolites and xenobiotics. it is made exclusively by the liver. serum albumin may be measured by radial immunodiffusion, dye binding reactions (using bromcresol green or bromcresol purple), electrophoresis, and elisa. serum albumin levels decrease with age in many inbred strains (quimby b) . hypoalbuminemia is usually reflective of decreased hepatic synthesis (due to hepatic disease, inflammation, and malabsorptive/maldigestive diseases), increased loss due to hemorrhage or via the intestinal tract (protein-losing enteropathies) and kidneys (protein-losing nephropathies) (tennant ) . most of the proteins associated with coagulation are synthesized by the liver (except factor viii), and measurement of fibrinogen or prothrombin time has served as a marker for decreased synthesis due to hepatic injury (tennant ) . prothrombin time will also be increased due to consumption of clotting components and in vitamin k deficiency. fibrinogen is also an acute phase reactant, and plasma elevations are seen during inflammation (tennant ) . fibrinogen may be quantified in mouse plasma by elisa. ceruloplasmin is copper-containing acute phase reactant and iron transport protein made exclusively in hepatocytes of mice. its serum levels may be decreased during hepatic injury and increased in response to inflammatory stimuli (min et al. ; shim and harris ) . many of the complement components are also made in the mouse liver, especially c , c , c , and factor b. decreased circulating levels may be seen due to hepatic injury or due to consumption during activation. please refer to the "complement" section for further details on complement function and measurement. two dyes, sulfobromophthalein and indocyanine green, have been used to assess hepatocellular or bile tract function. following an intravenous bolus, these dyes are rapidly cleared from the plasma by hepatocytes and excreted into the bile. both dyes have been used to assess liver function in mice (hurwitz et al. ; huang and vore ) . the kidney plays a complex role in maintaining homeostasis in the body and is involved in such functions as water and electrolyte balance, nutrient conservation, maintenance of blood ph, and removal of the end products of nitrogen metabolism (such as urea, creatinine, and allantoin). in addition, the kidney produces and responds to a variety of hormones. however, assessing renal function is complicated by the large functional reserve of the kidneys. for instance, increases in urea nitrogen do not occur until - % of renal mass has become functionally compromised. additionally, assessment of analytes in urine is difficult due to the small size of mice (as discussed in the "sampling" section). urea is produced in the liver as a waste product of protein catabolism (specifically a breakdown product of ammonia). urea is a small molecule that freely diffuses across cell membranes, and therefore the urea concentration is the same in blood, serum, and plasma. traditionally, urea concentrations is measured in terms of urea nitrogen (the amount of nitrogen contained within urea). determination of urea nitrogen is usually made from serum, but whole blood can be used (hence the term blood urea nitrogen [bun] ). quantification of urea nitrogen is based on spectrophotometry assays, measuring the amount of urea that is hydrolyzed by urease. frith et al. ( ) investigated urea nitrogen levels in balb/c and c bl/ mice. urea nitrogen levels decreased after months of age but increased again after months in both sexes. in balb/c mice, levels were higher in males than females, whereas in c bl/ mice, females had significantly higher levels than males. elevated urea nitrogen (azotemia) can be caused by prerenal, renal, and postrenal conditions. prerenal causes include increased protein catabolism (such as with inflammation, starvation, and high-protein diets). renal causes usually are associated with conditions that compromise more than - % of functional renal mass and include conditions such as renal amyloidosis, glomerular immune complex disease, and polycystic disease. postrenal include any cause that results in obstruction of the lower urinary system. decreased urea nitrogen is found with disease associated with hepatic insufficiency and low-protein diets. creatinine is a degradation product of creatine and creatine phosphate and represents an end-product of muscle metabolism. quantification of creatinine is based on spectrophotometry assays. baseline serum levels are directly related to muscular conditioning and total muscular mass, which varies between individuals. pathologically elevated serum creatinine levels are caused by the same prerenal, renal, and postrenal causes that elevate urea nitrogen in serum. therefore, quantification of serum creatinine offers no interpretative advantage over urea nitrogen (everett and harrison ) . proteinuria is a common finding in normal mice, and includes rodent-specific proteins such as uromucoid, small quantities of c~-and [ -globulins, and a family of prealbumins known as major urinary protein (mup). the mup has three electrophoretic variants, designated as mup- , mup- , and mup- , that are under both genetic and hormonal control. a regulatory locus designated mup- with codominantly expressed alleles a and b (located on chromosome ) controls the urinary levels of the three variants. the mup is synthesized in the liver, secreted into blood, and excreted into urine. males are have higher levels of proteinuria than are females, with levels of mg/ml, and age-related increases are seen in mice of both sexes. increases in other urinary proteins have been associated with a variety of renal diseases in mice. the concentration of urine (amount of solutes dissolved in urine) can be measured by urine specific gravity (usg) or osmolality. urine concentration in healthy mice is very high. watts ( ) determined the usg of healthy cba mice, which ranged from . to . . the author also found the usg increased from to days of age. the urine concentrating ability of transgenic mice expressing human sickle cell hemoglobin (kos for mouse hb) was assessed by ryan et al. ( ) . they found that sickled cells caused vascular, tubular, and glomerular changes, as well as corresponding hyposthenuria ( -h water deprived osmolality of affected mice was + mosm compared to + mosm in controls). i. electrolytes . sodium, potassium, chloride, and phosphorus sodium and potassium levels are easily measured in murine serum using flame photometry (lithium reference) or ion-specific electrodes. serum sodium levels are slightly higher in mice than in most other mammalian species, with reported values of + (sd) meq/ (finch and foster ) , + (sd) meq/l (everett and harrison ) , and - meq/ (loeb et al. ) . no differences in serum sodium were seen during aging, between sexes, or among strains. serum chloride has been measured using mercuric thiocyanate and the chloridimetric or ion-specific electrode techniques, and inorganic phosphorus in mice has been measured using the phosphomolybdate technique. serum inorganic phosphorus levels decreased as mice aged between and months. this change was documented in balb/c and c bl/ strains, as well as in outbred mice (loeb et al. ) . total serum calcium reflects both ionized (active calcium) and protein-bound calcium (mainly bound to albumin). ionized calcium is biologically active in bone formation, neuromuscular activity, cellular biochemical processes, and blood coagulation. decreased serum albumin levels are also expected to decrease total calcium levels, by decreasing the amount of protein-bound calcium. however, hypoalbuminemia does not result in clinical signs of hypocalcemia. in mice, total serum calcium has been measured using the sodium alizarin sulfonate technique or atomic absorption spectrometry. two reports, using different techniques, list similar reference ranges of + (sd) mg/dl, whereas a third report lists reference ranges of . + . (sd) mg/dl for male and . + . (sd) mg/dl for female albino mice. the latter values reported for male mice were significantly lower than those for six inbred strains of mice in two different age-groups using the same techniques (quimby b) . likewise, no differences associated with sex were reported by everett and harrison ( ) , who also examined random bred albino mice. however, bonella et al. ( ) demonstrated significantly higher levels in female swiss albino mice. serum calcium levels appear to decline between and months of age in the balb/c and c bl/ strains as well as in outbred mice . frith et al. ( ) found that female c bl/ mice had lower calcium levels than males. bonella et al. ( ) demonstrated significantly elevated calcium levels when blood was collected by orbital puncture versus cardiac puncture. hypercalcemia in mice can results as a response to increased levels of pth (such as with hyperparathyroidism) or pthrp (such as with certain neoplasms like multiple myeloma). in mice, the response of parathyroid chief cells to hypercalcemia appears different from other species. grone et al. ( ) studied this response in nude mice with pthrp secreting tumor cells or infusions of pthrp, and found prominent membranous whorls and increased cytoplasmic area of chief cells in mice with hypercalcemia ( . + . mg/dl), which marked these cells as distinctly different from parathyroid chief cells of other species. hypocalcemia can be due to hypoalbuminemia (as previously discussed), renal disease, pancreatitis, and hypomagnesemia. alcock and shils ( ) found that mice fed magnesium-deficient diets developed hypocalcemia, as did mice receiving intramuscular injections of heparin. total serum protein has been evaluated in mice using either the lowry or biuret methods, although the lowry method is preferred for analysis of small volume samples. hypoproteinemia is seen in hepatic injury (also see the "liver function tests" section) or during protein-losing enteropathies or nephropathies. hyperproteinemia is seen during dehydration. age-related changes have been described in a number of strains, such as increased total serum protein in older balb/c and b mice and lower levels in dba/ mice (loeb et al. ) . each of the major classes of serum protein ((xl-, (x -, ~-, and y-globulins) in mice can be distinguished by electrophoresis. changes in serum levels have been associated with a wide variety of diseases in mice. for instance, hypergammaglobulinemia was found in scid mice injected with human cag multiple myeloma cells . the acute phase reactants ceruloplasmin and fibrinogen have been discussed in the "serum proteins" section and crp and sap in the "complement" section. saa is an acute phase reactant synthesized by the liver and may be induced by the inflammatory cytokines il- or il- (see the "cytokines and chemokines" section). normally saa is quickly cleaved to a small molecular weight product, but during chronic infection or failure in the degradation pathway, tissue deposition of amyloid may occur. differences in serum concentrations have been described in various mouse strains and during disease. commercial ria and elisa kits are available for its quantitation in mice (quimby b) . alpha- -fetoprotein (afp) is encoded by the afp gene which is nearly identical in its organization to the mouse albumin gene, albl. it is likely afp arose due to gene duplication. afp is present in fetal serum but the synthesis declines shortly after birth. serum levels are regulated by two loci, afr- and afr- , which are not linked to afp. the action of these genes may be modified by tgf-~ and p (wilkinson et al. ) . levels in mouse serum have been quantified by immunoelectrophoresis (zizkovsky ) and both polyclonal antibodies and recombinant dna-derived mouse afp are available for assay development (boismenu et al. ) and are elevated in mice with certain neoplasms and during hepatic regeneration (jin et al. ; quimby b ). sulphostin, a novel inhibitor of dipeptidyl peptidases iv (dppiv) that stimulates hematopoiesis in mice proteomic approaches to biomarker discovery in prostate and bladder cancers th annual analyzers buyer's guide il- : prototype member of an emerging cytokine family increased atherosclerosis in hypeflipidemic mice with inactivation of abca- in microphages diet-induced changes in plasma phospholipids transfer protein activity, lipids, and lipoproteins. mpd: , , . mouse phenome database web site comparison of magnesium deficiency in the rat and mouse apoc- and apoc-iii as potential plasmatic markers to distinguish between ischemic and hemorrhagic stroke comparison of methods for obtaining blood from mice anxiety and stress responses in female oxytocin deficient mice impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice hormonal control of leydig cell differentiation interleukin- an interleukin for rejuvenating the immune system a spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid mast cell lineage development and phenotypic regulation laboratory routines cause animal stress regulation of fasting blood glucose by resistin biochemistry of immunoglobulins variations in serum calcium between strains of inbred mice correction of ureagenesis after gene transfer in an animal model and after liver transplantation in humans with ornithine transcarbamylase deficiency biology of the congenitally hypothyroid hyt/hyt mouse the chemokine stromal cell-derived factor- regulates the migration of sensory neutron progenitors methods of enzymatic analysis the mouse phenome project purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in escherichia coli genetically engineered animals in drug discovery and development: a maturing resource for toxicologic research effects of heparin on serum calcium in mice albumin content in blood of inbred mice strains lentiviral shrna silencing of murine bone marrow cell ccr leads to persistent knockdown of ccr function in vivo trophic action of leptin on hypothalamic neurons that regulate feeding the interpretation of serum biochemistry test results in domestic animals transgenic models of autoimmune disease lipoprotein metabolism and atherosclerosis susceptibility in transgenic mic mouse models of atherosclerosis reproductive failure in aged cbf male mice: interrelationships between pituitary gonadotropic hormones, testicular function, and mating success stromal cell derived factor /cxcl selectively counteracts inhibitory effects of myelosuppressive chemokines on hematopoieteic progenitor cell proliferation in vitro minireview: genetic models for the study of gonadotropin actions recruitment of cxcr and ccr t-cells and production of interferon-gamma inducible chemokines in rejecting human arteries tietz fundamentals of clinical chemistry distribution and characterization of the serum lipoproteins and apolipoproteins in the mouse, mus musculus disparate lymphoid chemokine expression in mice and men: no evidence for ccl synthesis by human high endothelial venules insulin secretion profiles are modified by over expression of glutamate dehydrogenase in pancreatic islets a panel of cerebrospinal fluid biomarkers for the diagnosis of alzheimer's disease normal, benign, preneoplastic, and malignant prostate cells have distinct protein expression profiles resolved by surface enhanced laser desorption/ionization mass spectrometry chemokine-cytokine cross talk. the elrt cxc chemokine lix (cxcls) amplifies a proinflammatory cytokine response via a phosphatedy linositol -kinase-nf-kappa b pathway rapid neurosecretory and cardiovascular response to osmotic stimulation in conscious mice high density lipoproteins retard the progression of atherosclerosis and favorably remodel lesions without suppressing indices of inflammation or oxidation influence of blood collection sites and use of anesthesia on plasma glucose concentration in mice the tissue activities of some diagnostic enzymes in ten mammalian species accelerated intestinal epithelial cell turnover: a new mechanism of parasite expulsion signal transduction in t cells: an overview role of stearoyl-coa desaturase- in leptinmediated weight loss induction of leptin receptor expression in the liver by leptin and food deprivation mouse models of male infertility the primary germinal center response in mice a comparison of serum calcium levels obtained by two methods of cardiac puncture in mice biomethodology and surgical techniques reduction in the development of cerulean-induced acute pancreatitis by treatment with m , a new selective superoxide dismutase mimetic pancreatic lipase-related protein is the major colipase-dependent pancreatic lipase in suckling mice effects of cannabinoids and female exposure on the pituitary testicular axis in mice: possible involvement of prostaglandins genetic background determines the extent of atherosclerosis in apoe-deficient mice endocrine hormones in laboratory animals antibody interference in thyroid assays: a potential for clinical misinformation a new kinetic determination of serum '-nucleotidase activity, with modifications for a centrifugal analyzer the roles of prolactin, growth hormone, insulin-like growth factor-l, and thyroid hormones in lymphocyte development and function; insights from genetic models of hormone and hormone receptor deficiency selection for serum cholesterol, voluntary physical activity, -day body weight, and feed intake in random bred mice. ii. correlated response ccr expression by brain microvascular endothelial cells critical for macrophage transendothelial migration in response to ccl inhibition of apolipoprotein b secretion by taurocholate is controlled by n-terminal end of the protein in rat hepatoma mcardle-rh cells clinical biochemistry the effect of freezing on various serum chemistry parameters of common laboratory animals alkaline phosphatase, lactate dehydrogenase, and aspartate aminotransferase and their isoenzymes as indicators of the development of experimental hepatitis in mic the endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages niemann-pick c heterozygosity confers resistance to lesional necrosis and macrophage apoptosis in murine atherosclerosis hematologic and serum electrolyte values of the c bl/ j male mouse in maturity and senescence hormone production by the pituitary and testes of male c bl/ j mice during aging age effects of luteinizing hormone, progesterone, and prolactin in proestrus and acyclic c bl/ j mice innovation~stagnation: challenge and opportunity on the critical path to new medical products interleukin- hematologic and clinical chemistry findings in control balb/c and c bl/ mice pi k and negative regulation of tlr signaling visfatin: a protein secreted by visceral fat that mimics the effects of insulin floor space needs for laboratory mice: c bl/ males in solid-bottom cages with bedding a longitudinal hormonal profile of the genetically obese mouse return to normal of blood-glucose, plasma insulin, and weight gain in new zealand obese mice after implantation of islets of langerhans acute phase proteins ghrelin: more than a new frontier in neuroendocrinology tumor necrosis factors a rapid, simple, and humane method for submandibular bleeding of mice using a lancet palatal cytosol cortisol-binding protein associated with cleft palate susceptibility and h- genotype relationship in the functional levels of early components of complement to the h- complex of mice developmental and tissue-specific regulation of parathyroid hormone (pth)/pth-related peptide receptor gene expression ccl -ccr interactions: an axis mediating the recruitment of t-cells and langerhans-type dendritic cells to sites of atopic skin inflammation measurement of glycosylated haemoglobins and glycosylated plasma proteins in animal models with diabetes or inappropriate hypoglycaemia effects of humoral hypercalcemia of malignancy on the parathyroid gland in nude mice the complex role of interlukin- in autoimmunity tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas caloric restriction increases gluconeogenic and transaminase enzyme activities in mouse liver fructose metabolizing enzymes from mouse livers influence of age and caloric restriction genetic ablation of orexin neurons in mice result in narcolepsy, hypophagia, and obesity prolactin and the prolactin receptor: new targets of an old hormone optimal conditions for the determination of serum alkaline phosphatase by a new kinetic method blood collection in mice using the saphenous vein: an alternative to retroorbital collection transgenic analysis of drug-metabolizing enzymes: pre-clinical drug development and toxicology the distribution of aspartate aminotransferases in normal and neoplastic rat and mouse tissues hepatic canalicular membrane transport of bile salt in c l/j and akr/j mice: implications for cholesterol gallstone formation prolonged gestation, elevated preparturitional plasma progesterone, and reproductive aging in c bl/ j mice mild dyslipidemia in mice following targeted inactivation of the hepatic lipase gene immunolocalization of tissue non-specific alkaline phosphatase in mice uncoupling of obesity from insulin resistance through a targeted mutation in a p , the adipocyte fatty acid binding protein synergistic effect of lymphotactin and interferongamma-inducible protein- transgene expression in t-cell localization and adoptive t-cell therapy of tumors multidrug resistance p-glycoprotein is essential for the biliary excretion of indocyanine green induction of bilirubin clearance by the constitutive androstane receptor a traditional herbal medicine enhances bilirubin clearance by activating the nuclear receptor car visfatin: a new adipokine transgenic and ko mouse models for the study of luteinizing hormone and luteinizing hormone reception function loperamide effects on hepatobiliary function, intestinal transit, and analgesia in mice redox state-dependent and sorbitol accumulationindependent diabetic albuminaria in mice with transgene-derived human aldose reductase and sorbitol dehydrogenase deficiency hepatocyte nuclear factor alpha is a central regulator of bile acid conjugation genetic association between h- gene and testosterone metabolism in mice use of transgenic mice in carcinogenicity hazard assessment leukotrienes and atherosclerosis: new roles for old mediators endothelial lipase and hdl metabolism targeted mutation of plasma phospholipids transfer protein gene markedly reduces high density lipoprotein levels genetic heterogeneity of lipoproteins in inbred strains of mice: analysis by gel permeation chromatography afp gene expression after acute diethylnitrosamine intoxication is not afr regulated identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human role of liver enriched transcription factors and nuclear receptors in regulating the human, mouse, rand rat ntcp gene expression of urea transporters in potassium-depleted mouse kidney genetic analysis of glucose tolerance in inbred mouse strains. evidence for polygenic control reversing systemic inflammatory response syndrome with chemokine receptor pepducins carbohydrate metabolism proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome il- : a regulator of the transition from neutrophil to monocyte recruitment during inflammation implications of multiple phenotypes observed in prolactin receptor ko mice. front neuroendocrino vectorial transport of bile acids in immortalized mouse bile duct cells hyperadrenocorticism, attenuated inflammation, and the life prolonging action of food restriction in mice physiological relevance of thyroid stimulating hormone and thyroid stimulating hormone receptor in tissues other than the thyroid genetic modifiers of atherosclerosis in mice function of prostanoid receptors: studies in ko mice estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes ghrelin--a hormone with multiple functions effects of shipping on immune functions of mice how obesity causes diabetes: not a tall tale luteinizing hormone deficiency and female infertility current protocols in immunology disruption of the tgf-i] pathway and modeling human cancer in mice a new technique for obtaining blood from mice decay-accelerating factor confers protection against complement-mediated podocyte injury in acute nephrotoxic nephritis tissue distribution of products of the mouse decay-accelerating factor (daf) genes: exploitation of a daf- knockout mouse and site-specific monoclonal antibodies linscott's directory of immunological and biological reagents clinical biochemistry of laboratory rodents and rabbits. in clinical biochemistry of domestic animals clinical biochemistry the clinical chemistry of laboratory animals improved terminal bleeding method t-cell trafficking in asthma: lipid mediators grease the way the mouse fas-ligand gene is mutated in gld mice and is part of a tnf family gene cluster chemokines: immunology's high impact factors studies on the origin and excretion of serum alpha-amylase in the mouse repetitive blood sampling in unrestrained mice using a chronic indwelling right atrial catheterization apparatus transcellular biosynthesis of arachidonic acid metabolites: from in vitro investigations to in vivo reality consequences of elevated luteinizing hormone on diverse physiological systems: use of the lh beta ctp transgenic mouse as a model of ovarian hyperstimulation-induced pathophysiology mouse models as tools for dissecting disorders of lipoprotein metabolism ccl and ccl induce a potent proinflammatory differentiation program in licensed dendritic cells the role of histamine hi receptor and h receptor in lps-induced liver injury adiponectin protects lps-induced liver injury through modification of tnf-t~ in kk-ay obese mice in vivo administration of helicobacter hepaticus cytotoxin is associated with hepatic inflammation and necrosis an h- -associated difference in murine serum cholesterol levels tissue-specific expression of the human gene for lecithin: cholesterol acyltransferase in transgenic mice alters blood lipids, lipoproteins, and lipases towards a less atherogenic profile biology of il- and the il- receptor multiple genes encode mouse pancreatic amylases opposing roles for il- and il- receptor ct in health and disease of mice and not men: differences between mouse and human immunology veterinary laboratory medicine: interpretation and diagnosis induction of hepatic metallothionein by non-metallic compounds associated with the acute phase response in inflammation clinical biochemistry and hematological reference values in normal experimental animals relationship between the development of the intestinal iga immune system and the establishment of microbial flora in the digestive tract of young holoxenic mice split activity of interleukin- on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step genetic susceptibility and resistance to diet-induced atherosclerosis and hyperlipoproteinemia chemokines; multiple levels of leukocyte migration control initial sequencing and comparative analysis of the mouse genome oral tolerance in the absence of naturally occurring tregs structure, binding, and antagonists in the il- /il- receptor system corticotropinreleasing hormone deficiency reveals major fetal, but not adult, glucocorticoid need diet effects on bone mineral density and content, body composition, and plasma glucose, leptin and insulin levels. mpd: . mouse phenome database web site evaluation of h-fabp as a marker of ongoing myocardial damage using hgh transgenic mice lipid inflammatory mediators in diabetic vascular disease transgenic expression of pancreatic secretory trypsin inhibitor- ameliorates secretagogue-induced pancreatitis in mice summary of the second report of the national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults recognition and clearance of apoptotic cells: a role for complement and pentraxins mildly oxidized ldl induces an increase apolipoprotein j/paraoxonase ration the genetic control of alkaline phosphatase activity in the duodenum of the mouse diabetes mellitus: a "thrifty" genotype rendered detrimental by "progress"? cx crl-mediated dendritic cell access to the intestinal lumen and bacterial clearance clinical laboratory reference sequential blood samples from the tail vein of rats and mice obtained with modified liebig condenser jackets and vacuum the cancer research uk experience of pre-clinical toxicology studies to support early clinical trials with novel cancer therapies lactic dehydrogenase virus insight into the physiological actions of thyroid hormone receptors from genetically modified mice ccr governs skin dendritic cell migration under inflammatory and steady state conditions viral pertubation of endocrine function: disordered cell function leads to disturbed homeostasis and disease targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity genetics, cytokines, and human infectious disease: lessons from weekly pathogenic mycobacteria and salmonellae circadian rhythms of plasma corticosterone binding activity in the rat and mouse one hundred years of mouse genetics: an intellectual history. i. the classical period one hundred years of mouse genetics: an intellectual history. ii. the molecular revolution the ultrastructural localization of the isoenzymes of aspartate aminotransferase in murine tissues proteomic analysis of diet-induced hypercholesterolemic mic clinical chemistry values of the n:nih(s) mice and parameter variations due to sampling techniques use of proteomic patterns in serum to identify ovarian cancer lessons from kitty hawk: from feasibility to routine clinical use for the field of proteomic pattern diagnostics alkaline phosphatase activity of the mouse immune regulation in the intestine boosted decision tree analysis of surface enhanced laser desorption/ionization mass spectral serum profiles discriminates prostate cancer from non-cancer patients animal models in biomedical research complement the mouse transgenic models of tolerance and autoimmunity: with special reference to systemic lupus erythematosus short-term and long-term in vivo exposure to an ephedra-and caffeine-containing metabolic nutrition system does not induce cardiotoxicity in b c f mice model models of atherosclerosis hormones type diabetes: a matter of beta-cell life and death leukemia inhibitory factor and interleukin-ll: cytokines with key roles in implantation virus induced pancreatic disease; alterations in concentrations of glucose and amylase in blood lungkine, a novel cxc chemokine, specifically expressed by lung bronchoepithelial cells changes in serum hormone levels associated with male-induced ovulation in group housed adult female mice ko transgenic mouse model of sickle cell disease animal models of autoimmunity and their relevance to human diseases hematological comparison of the mouse blood taken from the eye and tail cystatin c as a potential cerebrospinal fluid marker for the diagnosis of creutzfeldt-jakob disease efficacy of , -dimercapto-l-propanesulfonic acid (dmps) and diphenyl diselenide on cadmium induced testicular damage in mice multimeric cytokine receptors: common versus specific functions bilateral lesions in the suprachiasmatic nuclei affect circadian rhythms and h-thymidine incorporation into deoxyribonucleic acid in mouse intestinal tract, mitotic index of corneal epithelium, and serum corticosterone pthrp, pth, and the pth/pthrp receptor in endochondral bone development diabetes, obesity, and the brain hematopoietic, immunomodulatory and epithelial effects of interleukin- intestinal tsh production is localized in crypt enterocytes and in villus 'hotblocks' and is coupled to il- production: evidence for involvement of tsh during acute enteric virus infection adipsin, a biomarker of gastrointestinal toxicity mediated by a functional gamma-secretase inhibitor loci controlling plasma non-hdl and hdl cholesterol levels in a c bl/ j x casa/rk intercross quantitative trait loci mapping of genetic modifiers of metabolic syndrome and atherosclerosis in low-density lipoprotein receptor-deficient mice the kinase lkb mediates glucose homeostasis in liver and therapeutic effects of metformin genetic defects in copper metabolism estrogen: the good, the bad, and the unexpected il- and il- receptors, and their extended family b-cell-and monocyte-activating chemokine (bmac), a novel non-elr ct-chemokine feed-forward regulation of bile acid detoxification oby cyp a : studies in humanized transgenic mice adenovirus-mediated rescue of lipoprotein lipase-deficient mice breeding of the gad-mdx mouse: influence of genetically induced denervation on dystrophic muscle fibers failure of parturition in mice lacking the prostaglandin f receptor inactivation of stress protein p increases murine carbon tetrachloride hepatotoxicity via preserved cyp e activity murine models to investigate pharmacological compounds acting as ligands of ppars in dyslipidemia and atherosclerosis the other side of the orexins: endocrine and metabolic actions assessment of hepatic function mitochondrial affinity for adp is twofold lower in creatine kinase ko muscles. possible role in rescuing cellular energy homeostasis evaluation of proparacaine hydrochloride as a topical anesthetic for the collection of blood from the orbital sinus of mice mitochondrial affinity for adp is twofold lower in creatine kinase knockout muscles. possible role in rescuing cellular energy homeostasis the role of cd -cd interactions in autoimmunity and the benefit of disrupting this pathway scavenger receptor class b type in high-density lipoprotein metabolism, atherosclerosis, and heart disease: lessons from gene targeted mice cxc chemokine receptor cxcr is essential for protective innate host response in murine pseudomonas aeruginosa pneumonia in vivo uptake of radiolabeled mda , an oxidation-specific monoclonal antibody, provides an accurate measure of atherosclerotic lesions rich in oxidized ldl and is highly sensitive to their regression the regulation of the complement system: insights from genetically-engineered mice interleukin- : an overview development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine mechanisms of suppression by suppressor t-cells lipids and lipoproteins car and pxr agonists stimulate hepatic bile acid and bilirubin detoxification and elimination in mice pneumocystis carinii activates the nf-kappa[~ signaling pathway in alveolar epithelial cells a simple capillary tube method for the determination of the specific gravity of and micro quantities of urine interleukin- facilitates the early antimicrobial host response to escherichia coli peritonitis transgenic studies on the regulation of the anterior pituitary gland function by the hypothalamus cxcr and its ligand participate in the host response to bordetella bronchiseptica infection of the mouse respiratory tract but are not required for clearance of bacteria from the lung a direct intersection between p and transforming growth factor beta pathways targets chromatin modification and transcription repression of the alpha-fetoprotein gene assessment of hepatic function and damage in animal species laboratory animal endocrinology investigation of antitumor effects of synthetic epothilone analogs in human myeloma models in vitro and in vivo the protein profile of acetazolamidetreated sera in mice bearing lewis neoplasm cebs object modes for systems biology data, sysbio-om partal hormone resistance in mice with disruption of the steroid receptor coactivator- (src- ) gene platelet factor gene transfection into tumor cells inhibits angrogenesis tumor growth and metastasis mice lacking the thyrotropin-releasing hormone gene: what do they tell us? differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors combined pituitary hormone deficiency: lessons from the murine models molecular basis of resistance to thyroid hormone a mouse bleeding technique yielding consistent volume with minimal hemolysis transgenesis and the study of expression, cellular targeting, and function of oxytocin, vasopressin and their receptors kos model the best-selling drugs: will they model the next ? predicting drug efficacy: kos model pipeline drugs of the pharmaceutical industry three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer cxc chemokine ligand stromal cell-derived factor alpha and cxcr -dependent migration of ctls toward melanoma cells in organotypic culture il-ii: insights in asthma from overexpression transgenic modeling genotype, obesity, and cardiovascular disease: has technical and social advance outstripped evolution? specific fetal serum proteins of mammalian species t t tace taj tc tg tgf-i tgf-~isf th tlr tnap tnf tnfrsf tnfsf tof tpc trance trh tsh tx upr usg vldl serum amyloid a serum amyloid protein standard deviation stromal cell-derived factor- sorbitol dehydrogenase surface-enhanced laser desorption/ionization secondary lymphoid tissue chemokine stearoyl-coa desaturase- suppressor of cytokine signaling- steroid receptor coactivator- sj gren's syndrome single-strand deoxyribonucleic acid triiodothyronine thyroxine tumor necrosis factor-a converting enzyme toxicity and jnk inducer cytotoxic t-cells triglyceride transforming growth factor-j transforming growth factor- superfamily t-helper toll-like receptors tissue nonspecific alkaline phosphatase tumor necrosis factor tumor necrosis factor receptor superfamily tumor necrosis factor superfamily time-of-flight total plasma cholesterol tumor necrosis factor-related activation-induced cytokines thyrotropin-releasing hormone thyroid-stimulating hormone thromboxane unfolded protein response urine specific gravity very low-density lipoprotein key: cord- -pbcr qm authors: pignatelli, j.; jimenez, m.; luque, j.; rejas, m.t.; lavazza, a.; rodriguez, d. title: molecular characterization of a new ptov strain. evolutionary implications date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: pbcr qm toroviruses are emergent viruses, belonging to the nidovirales order, that remain mostly ignored, despite they are able to infect different species of domestic animals and humans, causing enteric diseases and diarrhea. thus far, only five variants of porcine torovirus (ptov) have been identified. in this report we describe the identification and partial characterization of a new strain of porcine torovirus (ptov-bres) that was detected by rt-pcr in a swine faecal specimen from a farm in brescia (italy). the complete genes coding for the nucleocapsid (n), hemagglutinin-esterase (he) and membrane (m) proteins were amplified, and sequence analysis showed that ptov-bres is a new ptov strain that, based on the he gene sequence, is phylogenetically related to p strain, that was up to now the only member of a distinct ptov lineage. the nucleocapsid protein from ptov-bres was expressed in insect cells as a his-tagged protein, purified by affinity chromatography and used to develop an elisa method to detect antibodies against ptov. this assay was evaluated using a serum collection including samples from three commercial farms from spain. high antibody prevalence against ptov was observed in the three farms, both in adult animals and in piglets, which could suggest that ptov might be endemic in spanish porcine population. the elisa method developed in this work could be useful in future epidemiological surveys about toroviruses. toroviruses have been described as potential gastroenteritis causing agents in horses, cows, pigs and humans. they were classified as a new genus within the family coronaviridae, from the order nidovirales (cavanagh et al., ) , although the possibility of establishing two subfamilies, coronavirinae and torovirinae within the family coronaviridae has been proposed and is currently under consideration (gonzalez et al., ; coronaviridae study group, ) . torovirus genome consists of a single rna molecule of about - kb. genome organization is similar to that of coronavirus: the two-thirds contain two large and overlapping open reading frames, orf a and orf b, that code for the replication machinery. the last third of the genome contains four open reading frames, orfs - , coding respectively for the structural proteins: spike (s), membrane (m), hemagglutinin-esterase (he) and nucleocapsid (n). torovirus particles have a characteristic torus-shape nucleocapsid formed by the n protein interacting with the viral rna. the nucleocapsid is surrounded by an envelope that contains the triple spanning m protein, and the s and he proteins that conform the large and short spikes, respectively. the first torovirus was identified in after examining a diarrheic faecal sample from a horse in berne (switzerland), and thus it was named berne virus or bev (weiss et al., ) . a morphologically related virus was later found in a cattle farm from breda (iowa), and this bovine torovirus (btov) was designated brv (woode et al., ) . over the years, there have been a few reports describing the presence of toroviral particles in faecal samples from humans (htov) (beards et al., ; duckmanton et al., ; koopmans et al., ; krishnan and naik, ; jamieson et al., ; uziel et al., ; lodha et al., ) and pigs (ptov) (scott et al., ; woode, ; durham et al., ; penrith and gerdes, ; lavazza et al., ) . ptov has later been detected by rt-pcr in swine faecal specimens from farms in the netherlands, belgium, hungary and italy (kroneman et al., ; matiz et al., ) . moreover, a partial genomic characterization of five european ptov isolates has been reported (smits et al., ) . epidemiological surveys about toroviruses have been largely concentrated on btov, and they showed that this virus is distributed worldwide, having been detected in united states, japan, south korea, india, and in different european countries like united kingdom, germany, belgium, france, switzerland, and italy (liebler et al., ; koopmans et al., ; ito et al., ; park et al., ; van kruiningen et al., ; krishnan and naik, ; koopmans et al., ; weiss et al., ; lavazza, ) . moreover, high seroprevalences against btov have been reported in cattle from united kingdom (brown et al., ) , the netherlands and germany (koopmans et al., ) . although there are few reports about ptov epidemiology, high seroprevalences, similar to those of btov, have also been reported in swine populations from switzerland (weiss et al., ) and the netherlands (kroneman et al., ) . despite this extensive geographical distribution and the broad host range, these viruses have attracted little attention. this is likely due in part to the fact that torovirus infection has not been associated with disease causing important losses in livestock, but also to the lack of an "in vitro" system to work with most of these viruses, that has precluded the development of specific tools for their diagnosis. for a long time only the equine isolate bev could be grown in cell cultures (weiss et al., ) , and, it has only been very recently reported the ability of a btov variant isolated in japan to grow in cells derived from a human rectal adenocarcinoma (kuwabara et al., ) . in addition, btov can be propagated in experimentally infected gnobiotic calves (woode et al., ) . thus, there have been a few reports where indirect elisa using partially purified btov or bev particles were used for torovirus serodiagnosis, but purification procedures are not affordable by all laboratories and, in addition, this assay would also provide low sensitivity for detection of antibodies to human and porcine toroviruses (brown et al., ) . torovirus serodiagnosis has also been performed by analyzing the ability of clinical serum samples to neutralize bev infectivity (weiss et al., ) . the virus neutralization test is "a priori" a very sensitive assay that provides also information about the existence of a protective humoral immune response in the animal under study, however, since bev, a non-homologous virus, would be used to search for antibodies to torovirus in cattle, human or swine sera, the sensitivity of the assay would be reduced. in addition seroneutralization test is a time-consuming and difficult assay that cannot be used to perform extensive studies. thus, alternative strategies have to be undertaken to generate torovirus specific antigens for immune detection. a frequently used alternative is the expression of immunologicaly relevant viral proteins in recombinant expression systems (chirnside et al., ; denac et al., ; pelosi et al., ; farkas et al., ; bogdanova et al., ; hou et al., ) . for this purpose the nucleocapsid proteins from many viruses have proven to be very useful. specifically, the n proteins of different coronaviruses including sars have been expressed either in bacteria or through the baculovirus expression system and have been used for serodiagnosis (timani et al., ; woo et al., ; wang et al., ) . moreover, the n protein is the most abundant protein in torovirus particles (horzinek et al., ) , and strong responses against it have been reported from experimental infections of gnobiotic calves with btov . therefore, n protein from ptov appears as an interesting viral antigen to develop diagnostic methods. in addition, since the nucleocapsid proteins are usually highly conserved, serodiagnosis tests based on the use of ptov n protein may prove useful for detection of antibodies against other toroviruses. the aim of this work was to characterize a new ptov strain that was detected in a faecal sample from italy, which was previously considered as torovirus positive by electron microscopy diagnosis, and to develop an elisa diagnostic system to detect antibodies to torovirus. the elisa was evaluated by analyzing pig serum samples from three different spanish farms. our results, although obtained with a reduced number of animals, indicate a high ptov seroprevalence suggesting that ptov might be endemic in spanish farms. equine dermal (e. derm) (atcc ® ccl- tm ) cells were kindly provided by r.j. de groot (utrecht university. utrecht. the nether-lands). they were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % foetal calf serum (fcs), units/ml of penicillin, and mg/ml of streptomycin. high five tm (highfive) insect cells were cultured at • c in tc medium supplemented with antibiotics and % fcs. the equine torovirus strain berne (p / ) (bev) was propagated in e. derm cells as described previously (weiss and horzinek, ) , and bev particles were purified by sucrose gradient centrifugation. porcine respiratory and reproductive virus (prrsv) and transmissible gastroenteritis virus (tgev), both provided to us by l. enjuanes (cnb-csic, spain), were propagated in monkey-derived ma (atcc ® crl- . tm ) cells and in swine testis fibroblasts (st), respectively. both viruses were partially purified by centrifugation over a % sucrose cushion. three porcine faecal specimens ( / , / , / ), taken in brescia (italy) in , and one bovine faecal sample ( / ) taken at the same location in , were described to contain torovirus-like particles when examined by electron microscopy, but, with the exception of / , all samples were also shown to contain other viral particles that could correspond to coronavirus, enterovirus and circovirus. swine blood samples were collected from three commercial farms located in different regions in the northern part of spain. ten blood samples were collected from sows in a farm located in navarra, where diarrhea and gastroenteritis symptoms in piglets had been reported previously, but they were not observed at the time of serum collection. twenty sow blood samples were collected from a farm located in aragon where most newborn piglets were suffering from diarrhea at the moment of collection. fifteen blood samples were collected from to week-old healthy piglets from a farm with high sanitary conditions located in galicia. blood samples were centrifuged at × g during min at • c, and the obtained sera were stored at − • c until their use. a commercial porcine serum obtained from healthy pigs was purchased from abd serotec and has been named as cps throughout the paper. a serum specimen from a piglet infected under field conditions with ptov, previously described (antiserum / ; lavazza et al., ) was used as ptov positive serum control, and was referred to as ␣bres. an anti-bev hyperimmune polyclonal serum (␣-bev) was generated in our laboratory (garzon et al., unpublished results). s. van gucht (gent university, belgium) provided us three serum samples from caesarean-derived, colostrum-deprived (cd/cd) pigs kept under germ free conditions (spf) that were used as negative control, and sera from two other pigs that were reared under these same conditions, but that had subsequently been inoculated with either porcine respiratory coronavirus (prcv) (␣prcv) or prrsv (␣prrsv), and that were used as cross-reactivity controls. in addition, serum samples from cd/cd piglets taken at - days of age, were provided by j. segales (cresa. barcelona, spain) and were also used as negative controls. a rabbit hyperimmune serum against the n protein from ptov-bres was raised by immunizing a white new zeeland rabbit with chromatography purified n chimerical protein fused to a histidine tag, and expressed in insect cells by a baculovirus recombinant (see below). the faecal samples containing toroviral particles were diluted : in sterile phosphate buffered saline (pbs), homogenized by vortexing and centrifuged at × g for min at • c. for toroviral particle aggregation the supernatants were mixed (v:v) with a convalescent serum sample from a torovirus infected pig (␣bres), previously diluted : in pbs. after a min incubation period at • c, viral particles were adsorbed to collodion-carbon coated copper grids by ultacentrifugation in a beckman airfuge, at psi for min as previously described (lavazza et al., ) . for detection of bound antibodies, particles adsorbed to the surface of the grids were incubated with protein-a conjugated with nm colloidal gold particles as previously described (rodriguez et al., ) . after washing in distilled water, samples were negatively stained with % sodium phosphotungstate (pta) in water for min, allowed to dry, and observed in a jem electron microscope (jeol, japan). pictures were taken with a bioscan digital camera (gatan). the faecal samples containing toroviral particles were diluted : in pbs, homogenized by vortexing and centrifuged at × g for min at • c. the supernatant was aliquoted and kept at − • c. l of each supernatant were used for viral rna extraction using a commercial kit (high pure rna isolation kit, roche applied science) following the manufacturer's recommendations. the rna was recovered in l of dnase, rnase free water, aliquoted and maintained at − • c. all specimens were extracted in a dedicated class laminar flow hood using dedicated pipettes and aerosol resistant tips. reverse transcription (rt) reaction was performed using superscript ii reverse transcriptase (invitrogen, corp.) following the manufacturer's instructions with minor modifications. briefly, l of the rna sample were mixed with a mm dntps mix (roche applied science) and mm random hexamer primer (roche applied science) and incubated for min at • c and min on ice. reverse transcription reaction was completed by adding l of reaction mix containing x rt buffer, mm dtt, mm mgcl , u ribonuclease inhibitor (fermentas) and u of superscript ii reverse transcriptase and incubating for min at • c, followed by min at • c and finally min at • c. to table oligonucleotide primers: location within torovirus genome and sequences. . primer sequence a polarity location minimize cross-contamination reverse transcription reaction was carried out in an independent room using dedicated pipettes with aerosol resistant pipette tips. the cdna preparation was aliquoted and conserved at − • c until use. sequences from etov, berne strain (bev), btov, strains brv- , brv- , b , b , b , b and ptov, strains markelo, p , p , p and p were retrieved from genbank database (http://www.ncbi.nlm.nih.gov) and aligned using megalign program (dnastar, inc.). primers tovm and tovm were designed based on a conserved region from the m gene (table ) . besides, primers and previously described (kroneman et al., ) were used to amplify a ptov specific fragment from the n gene. to amplify the full coding sequences for the structural proteins m, n and he from ptov, sets of primers were designed using ptov conserved sequences at both and regions from each gene (table ) . tails containing recognition sites for bamhi and xbai restriction enzymes (underlined in the table) were added to each forward and reverse primer, respectively. as described below, to amplify the entire he gene a new primer, ptov-bres-he , had to be designed. diagnostic pcr reactions were carried out using l of cdna and a reaction mix containing × pcr reaction buffer, . mm of each dntp, . mm mgcl , . m of each primer, tov-m and tov-m or and , and . u of taq platinum (invitrogen, corp.) in a final volume of l. the mixture was subjected to an initial denaturation step of min at • c, followed by cycles of s at • c, s at • c and min at • c. a final extension step of min at • c was added at the end. to amplify sequences of m and n genes using the primer pairs ptov-m -m and ptov-n -n , respectively, we used the same thermal cycling as described above. in the case of the he gene we could not obtain amplification from the cdna using the designed primer pair, ptov-he -he . however, using primers tov-m and ptov-he a bp fragment of ptov genome comprising the -end of m gene and the complete he gene was obtained. this fragment was amplified using a high fidelity polymerase (triplemaster polymerase mix from eppendorf), and was sequenced to determine the end region of he gene from ptov-bres, and thus, the primer ptov-bres-he was designed accordingly. to amplify just the complete he gene, a second amplification reaction was performed using ptov-bres-he and ptov-he primers and cdna as template. briefly, l of ptov-bres cdna were added to a reaction mix containing × high fidelity buffer, m of each dntp, nm of each primer and . u/l of triplemaster polymerase mix. thermal cycling conditions for both reactions were as follows: cycle of • c; cycles of denaturation at • c for s, annealing at • c for s and elongation at • c for min. all pcr reactions were performed in physically separated rooms and dedicated pipettes and filter tips were used. several negative controls of distilled water for each step of the procedure, rna isolation, reverse transcription and pcr were included in each assay. pcr reaction products were visualized in . - % agarose gels and extracted from agarose using the commercial gel extraction kit (qiagen) following instructions provided by the manufacturer. for sequencing analysis, all rt-pcr products corresponding to the n, m and he genes were cloned in the commercial vector pgem-t ® -easy (promega) and the resulting plasmids, pgt-ptov-m, pgt-ptov-n and pgt-ptov-he, respectively, were used to transform e. coli dh ␣ competent bacteria. at least independent clones were used for sequencing in the sequencing facility of the national centre of biotechnology (madrid, spain). obtained sequences were analyzed using seqman program (dnastar, inc.) and aligned using clustalx method with torovirus sequences available from ncbi database using megalign software (dnastar, inc.). the rt-pcr amplified products and the corresponding sequences from genbank were compared using the program megaling (dnastar, inc.) employing clustalw method. this program was used to carry out phylogenetic analysis from gene sequences. phylogenetic analyses were performed using the neighbour-joining method, and statistical parameters of phylogenetic trees were determined by bootstrap analysis using replicates. the nucleotide sequences described in this paper have been submitted to the ddbj nucleotide sequences database and are retrievable from genbank. the accession numbers for the complete n, m, and he genes of ptov-bres are fj , fj , and fj , respectively. a baculovirus recombinant that contains the nucleotide coding sequence for ptov n protein fused at its -end to a sequence encoding a -histidine tag was generated by using the bac-to-bac system (invitrogen, corp.) . for this, the pgt-ptov-n vector clone containing the consensus sequence for the n gene from ptov-bres isolate was used to subclone this gene into the baculovirus transfer vector pfastbac tm b (invitrogen, corp.). the n gene was extracted from pgt-ptov-n by enzymatic restriction with bamhi and xbai and cloned into pfastbac tm b, previously digested with the same restriction enzymes, giving rise to pfb-ptov-n vector. competent e. coli dh bac cells, containing a bacmid (baculovirus shuttle vector plasmid) and a helper plasmid, were used to generate recombinant bacmids according to the manufacturer's instructions. the obtained recombinant bacmid, bac-ptov-n, was transfected into monolayers of highfive insect cells using lipofectin (invitrogen, corp.). culture medium containing recombinant baculovirus rbac-ptov-n was collected at h post-transfection, clarified by centrifugation and stored at • c as virus stock. transfected cells were collected and recombinant ptov-n protein expression was analyzed by western blot using a commercial polyclonal antibody against the histidine tag (␣his) and the ␣bres serum. the recombinant n protein fused to a histidine tag at its n terminus was purified using bd talon tm imac resin (bd biosciences clontech). highfive cells were infected with rbac-ptov-n at a multiplicity of infection (moi) of plaque forming units per cell (pfu/cell) and maintained for h at • c. infected cells were harvested, pelleted by centrifugation at × g min and resuspended in lysis buffer ( mm h napo , ph . , mm nacl, . % np- ) containing protease inhibitors (complete mini tablets from roche applied science). cell suspensions were then incubated on ice for min and sonicated. the insoluble fraction, which contains the n protein, was recovered by centrifugation at × g min and solubilized with denaturing buffer containing mm h napo , ph . , mm nacl, and m guanidine. after a second centrifugation at × g for min, the supernatant was taken and mixed with the talon tm resin equilibrated with denaturing buffer, and incubated for h at • c in a rotating shaker. unbound proteins were removed by centrifugation at × g for min and resin was washed with volumes of denaturing buffer three times, and two times with a buffer containing mm h napo , ph . , mm nacl, m urea. the n protein was eluted from the resin with successive elution steps with equal resin volume of elution buffer ( mm h napo , ph . , mm nacl, m urea and m imidazol). concentration and purity of ptov-n protein was analyzed by sds-page and western blot. protein concentration was determined according to bca assay (pierce) using a : dilution of the eluted protein in distilled water and bovine serum albumin (sigma) as standard prepared similarly. aliquots of the purified recombinant n protein were electrophoresed on sds-page gels ( ng/lane). the protein was transferred to a nitrocellulose membrane that was cut into strips corresponding to each gel track. western blots were carried out with porcine serum samples diluted : . first, non-specific binding of antibodies to the nitrocellulose membrane was blocked by overnight incubation at • c in pbs containing . % tween (pbst) and % skim milk. the membranes were then incubated with porcine serum samples diluted in pbst with % skim milk. after extensive washings, the membranes were incubated with secondary goat anti-porcine igg antibodies coupled to horseradish peroxidase. western blots were developed by incubating the nitrocellulose membranes with a solution containing . mg/ml chloro- -naphtol (sigma) in pbs and . % h o , during min at room temperature, and then overnight at • c, in distilled water. the presence of neutralizing antibodies against torovirus in swine sera was analyzed by plaque reduction test using bev virus. this method, that is a conventional procedure to determine neutralizing antibody titers for many viruses, has not been previously used for bev. the reason being that bev virus titers have always been determined by establishing the % tissue culture infectious dose (tcid ) and neutralization assays were performed accordingly. instead, we titrated bev stocks by determining the number of pfu/ml (garzon et al, unpublished results). thus, bev neutralization assay was performed as follows: l of heat inactivated diluted serum (two-fold serial dilutions in dmem, from : to : ) were incubated with an equal volume of a viral suspension containing pfu of bev for h at • c. virus-serum mixtures were added to confluent monolayers of e. derm cells grown in -well plates. after h of virus adsorption at • c, the infection medium was removed and ml per well of × dmem medium mixed (v/v) with . % agar and containing . mg/ml of deae-dextrane and % fcs was added, and the cells were maintained at • c for days. after removing the agar overlay, cells were stained with crystal violet and lysis plaques were counted. the virus-neutralizing titer (nd ) of each serum sample corresponds to the reciprocal of the highest serum dilution giving % reduction in the number of plaques with respect to that obtained with the virus without serum. serum samples were tested by duplicate and negative (spf) and positive (cps) control sera were included in each test. neutralization titers were calculated using spearman-kärber formula. serum samples with neutralizing antibody titers equal to or higher than (or . that is the equivalent value in logarithmic scale) were considered as positive. an indirect elisa was established in our laboratory to test for the presence of antibodies against porcine torovirus in pig serum samples using ptov-n protein as antigen. optimal concentration of reagents was established by checkerboard titration. paired rows of a -well microtiter plate (maxisorp, nunc) were coated with ng/well of purified n protein diluted in mm carbonatebicarbonate buffer (ph . ) by overnight incubation at • c. after coating and at each washing step, plates were rinsed times with l/well of pbst. subsequently, plates were blocked for h at • c with l/well of pbst- % bsa. then l of each serum sample diluted : in pbst- % bsa was added in paired wells and the plates were incubated h at • c. next, commercial goat anti-pig igg antibodies conjugated to horseradish peroxidase (sigma) diluted in pbst- % bsa was added and the plates were incubated h at • c. the enzymatic reaction was developed using o-phenylenediamine dihydrochloride (opd fast tm , sigma). after min at room temperature the reaction was stopped by adding l/well of n sulphuric acid, and absorbance at nm was measured with a multichannel spectrophotometer (titertek multiscan mcc/ ). torovirus-like particles were observed by electron microscopy analysis in three porcine ( / , / , / ) and one bovine ( / ) faecal samples, in the course of a diagnostic examination of animals suffering diarrhea in brescia, italy. in some of these samples ( / , / and / ), other viral particles were also observed that could correspond to coronavirus, circovirus and enterovirus (data not shown). moreover, in two of the porcine samples ( / and / ), the torovirus-like particles were not well preserved and were difficult to detect. to enhance the efficacy and specificity of the electron microscopy method for toroviral particle detection, faecal suspensions were incubated with convaslescent serum from a torovirus infected pig (␣-bres) facilitating particle aggregation, as previously described (lavazza et al., ) . samples were then ultracentrifuged onto a grid, and adsorbed viral particles were incubated with protein a-gold for antibody detection, and negatively stained with % pta. fig. shows immunogold-labeled toroviral particles in one of the porcine faecal samples ( / ), which will later be identified as ptov-bres. the results obtained with the other three samples were not conclusive. to detect toroviral genomic rna in these samples we designed a new primer set based on a region from the m gene that is conserved in all available torovirus sequences (tovm and tovm described in table ). optimal rt-pcr conditions were established using serial dilutions of a titrated bev stock and using primers tovm and tovm we were able to detect up to pfu/ml (data not shown). these primers were then used for rt-pcr detection of ptov and btov in the faecal samples from three pigs and one calf described above. the expected bp fragment was amplified from one of the porcine specimens ( / ) ( fig. a, lane ) and also from the bovine stool sample (fig. b, lane ) . sequence of the ptov fragment showed a % similarity with respect to other available ptov sequences and % with respect to btov sequences. conversely, the fragment amplified from btov showed a % similarity with respect to other btov isolates, but only an % identity with respect to ptov isolates. both fragments showed a - % similarity with respect to bev. a second diagnostic pcr was carried out to amplify specifically a fragment from ptov n gene, using ptov specific primers and (table ) . a bp fragment was obtained only from the / sample ( fig. a, lane ) , but not from the other two porcine samples (not shown), nor from the bovine one. the amplified fragment was of the expected size and sequence of this second fragment showed a homology of - % with respect to ptov strains, % with respect to btov strains and only % with bev. negative water controls were analyzed in parallel with each primer set to discard sample cross-contamination ( fig. a and b, lanes and ) . thus, a new ptov strain has been identified in this work and it has been named as ptov-bres throughout this report. to establish the phylogenetic relationship of ptov-bres with other ptov strains, we amplified the sequences corresponding to the genes coding for m, he and n proteins from ptov-bres cdna. the amplicons were analyzed by agarose gel electrophoresis, purified from the gel, and cloned into pgemt ® -easy vector and at least independent clones were sequenced in both directions for each gene. the n gene of ptov-bres was amplified as bp fragment (fig. c, lane ) that showed . - . % homology with respect to other ptov strains, . - . % with respect to btov and % with bev (fig. a) . the bp dna fragment corresponding to m gene (fig. c, lane ) showed . - . % homology with other ptov strains, . - . % with respect to btov strains, and . % with respect to the bev isolate (fig. c) . the he gene from ptov-bres was amplified using a two-step strategy. first, we amplified a bp (fig. c, lane ) . this fragment was sequenced and it showed to contain the end from m gene, the intergenic region containing the canonical translation regulatory sequence (trs) and the complete he gene. a new primer, ptov-bres-he , was designed to amplify only the complete he gene without extra sequences ( . kb fragment) using the cdna as template (fig. c, lane ) . consensus sequence for ptov-bres he gene shows % homology with the previously described p strain of ptov, and - % homology with the other ptov strains. with respect to btov strains a - % identity was obtained. only % homology was observed with respect to the htov-he gene sequence and % with respect to the he gene fragment conserved in bev (fig. b) . these results strongly suggest that ptov-bres is a new strain of ptov not detected previously. we have generated a recombinant baculovirus that expressed the nucleocapsid protein from ptov-bres fused at its n-terminus to a histidine tag (rbac-ptov-n). highfive insect cells infected with rbac-ptov-n showed a prominent kda protein band when analyzed by sds-page and coomassie brilliant blue staining at and hpi (data not shown), and this protein was specifically recognized by ␣his (fig. a) and ␣bres (fig. b) antibodies in western blot. the estimated size of this main product was in agreement with the predicted size for the recombinant fusion protein. other protein bands of lower molecular weight, likely corresponding to degradation products, and a slower migrating protein were also observed by western blot. the recombinant n protein was solubilized by treatment of the infected cells with m guanidine, and was purified by affinity chromatography using a cobalt-coated resin. the protein was recovered by three rounds of elution with imidazole in presence of m urea. purity of the protein was analyzed by sds-page and coomassie brilliant blue staining. fig. c shows the analysis by sds-page of the different steps of the purification procedure. after elution, only the bands corresponding to the recombinant n protein were observed (fig. c, lanes - ) , indicating that the protein was highly purified. protein was stored in m urea to keep it soluble. to generate an anti-ptov n protein polyclonal serum (r␣ptov-n) the purified protein was dialysed against distilled water, freeze-dried and resuspended in pbs before being inoculated in a rabbit. specificity of antibodies was analyzed by western blot using extracts from rbac-ptov-n infected and non-infected high-five insect cells (data not shown). purified recombinant n protein was used to develop an elisa to detect antibodies against ptov in porcine serum samples. optimal concentrations of reagents was determined by checkerboard titration using the above described polyclonal serum, r␣ptov-n, two porcine serum samples, ␣bres and cps, as positive controls, and five serum samples from spf animals that were used as negative controls. the optimal protein concentration that provided higher discrimination between negative and positive sera was ng/well, and the optimal porcine serum dilution was : (data not shown). since there are no reference sera available for porcine torovirus, elisa cut-off value was established using spf serum samples and serum samples from cd/cd animals that had been collected between and days after birth. all these serum samples were analyzed for igg antibodies in five individual assays performed on different days. these control serum samples showed a mean elisa reactivity of . ± . . therefore, elisa cut-off values were established as the mean value obtained from control animals plus three times the standard deviation (o.d. nm = . ). none of these control serum samples showed elisa o.d. values above the cut off. lack of cross-reactivity between ptov and antibodies to other related viruses infecting pigs was confirmed by elisa, western blot and virus neutralization tests using ␣prrsv, ␣prcv, and ␣bres serum samples, purified ptov n protein or bev particles as toroviral antigens, and purified prrsv and tgev virions as related viruses (data not shown). in order to evaluate the potential use of the ptov-n protein as antigen for diagnostic elisa to detect antibodies against porcine torovirus, serum samples were collected from three spanish farms located in galicia, navarra and aragon and were analyzed by elisa, virus neutralization assay and/or western blot. all serum samples analyzed by elisa were positive against torovirus and these results were confirmed by western blot and/or serum neutralization test in cases ( %). most serum samples from the farm in navarra showed moderate elisa values (between . and . ) and low neutralizing antibodies titers (log nd < . ) (fig. a) . one sample showed positive elisa value (o.d. nm = . ) and a neutralizing titer close to the cut-off (log nd = . ) (fig. a , arrow), however, it was positive by western blot (fig. a, strip ) . from the farm located in galicia, more dispersed results were obtained by elisa (o.d. nm between . and . ), neutralization test (log nd between . and . ) and western blot (fig. b ). all serum samples were elisa positive, and that result was confirmed by western blot in samples and by neutralization test in samples. the only sample that was positive by elisa (o.d nm = . ) but negative by the other two tests (fig. b , strip and arrow) had a neutralizing titer that was close to the cut-off value (log nd = . ). serum samples from aragon showed high titers of antibodies against ptov-n (o.d. nm between . and . ), and accordingly, they were highly neutralizing (log nd between . and > . ), and for some of them we could not determine the neutralizing titer since the highest serum dilution used, : , still reduces the number of viral plaques to more than %, and they were all positive by western blot (fig. c ). the results obtained by the elisa test using ptov n protein as antigen are in good agreement with those obtained by neutralization assay, indicating that the elisa could be used instead for detection of antibodies to torovirus, providing a simpler and more feasible diagnostic system. toroviruses have been described as potential gastroenteritis causing agents that appear to infect many animal species, including humans. most torovirus epidemiological information comes from studies on btov performed in different countries, and they indicate that torovirus are broadly dispersed around the world, with high prevalences in animal populations. however, few studies have been reported about ptov, despite they indicate an important prevalence of ptov at least in european countries. the lack of a growing system for ptov has hindered its study and the development of diagnostic tools. this work reports the identification and partial characterization of a new ptov strain and the development of an elisa assay to detect antibodies against ptov in porcine serum samples. first, we describe a new primer pair, tovm and tovm , whose sequences are highly conserved between torovirus species. using this primer set we were able to detect a new strain, ptov-bres. in addition, primer set tovm and tovm was able to amplify bev and btov genomes from cell culture medium and a stool sample, respectively. moreover, although htov detection using tovm and tovm primer pairs has not been tested yet, these primers might also amplify htov since they were designed in conserved regions from all torovirus m gene available sequences. hence, we propose that this new primer set could be used for torovirus epidemiological trials. at present, little information about ptov genome sequence is available since only five ptov strains have been described (kroneman et al., ; matiz et al., ; smits et al., ) . genomic information about other ptov variants is needed to understand torovirus evolution and to develop new diagnostic tools. in this work we describe the amplification and sequencing of m, he and n genes from ptov-bres. interestingly, phylogenetic analysis of he gene showed a high sequence homology ( %) with the he gene from strain p , but only - % with other ptov strains. two ptov lineages had previously been described based on he gene sequences, which are represented by p and markelo strains. it has been suggested that these two ptov lineages were generated by recombination events into the he gene between a parental ptov and an unknown donor (smits et al., ) . ptov-p was detected in italy in , during an enteritis outbreak (lavazza et al., ) , and since then no other strains have shown a phylogenetic relationship with it. therefore, p was considered as the likely ptov parental strain, and markelo-like strains (markelo, p , p and p ) could then be considered as recombinant progeny originated from it. our finding of a new ptov strain phylogenetically related to p in a sample collected in suggests that at least these two ptov lineages might be currently circulating in porcine populations. antigenic differences between he proteins from both ptov lineages have not been determined but this possibility should be considered. in this regard, antigenic differences between the known btov lineages have been previously described (smits et al., ) . in this work, we also describe the expression of the n protein of ptov-bres and its use for the development of an elisa method to detect antibodies to torovirus in swine serum samples. to our knowledge, no specific serodiagnostic assay for ptov has been developed to date. antibodies against ptov were previously analyzed by bev serum neutralization test (weiss et al., ; liebermann, ; kroneman et al., ) , however, this method is time consuming and its sensitivity could be compromised by the fact that it is based on cross-reactivity between torovirus species. besides, two elisa assays to detect antibodies against btov and bev have been previously reported. both methods were developed using sucrose gradient purified virus, obtained either from faeces from gnobiotic calves infected with btov (brown et al., ; van kruiningen et al., ) or from culture medium from bev infected cells (weiss et al., ) . however, these approaches cannot be applied to porcine torovirus, since ptov cannot be propagated in cell cultures yet, and no experimental infection protocol has been reported. therefore, alternative strategies to obtain specific ptov antigens are needed. expression of viral proteins in insect cells to develop diagnostic assays have been reported for other viruses (pelosi et al., ; sestak et al., ; guo et al., ; deng et al., ; timani et al., ; farkas et al., ; shin et al., ) . the torovirus nucleocapsid protein is the most abundant protein in the virions, and is highly antigenic as shown by the strong antibody response developed against it in gnobiotic calves experimentally infected with btov. the n and he proteins from btov strain brv- were expressed previously in e. coli and in insect cells (duckmanton et al., ) , respectively, and both proteins showed immunoreactivity to specific antisera and field serum samples by western blot. however, these proteins were not used to develop any diagnostic assay. therefore, we sought to use n protein from ptov as antigen to develop diagnostic methods. thus, recombinant nucleocapsid protein from ptov-bres isolate was expressed in insect cells and purified by affinity chromatography. using this approach, large amounts of highly purified protein were achieved. recombinant ptov-bres-n protein was used to develop an elisa assay to detect antibodies against torovirus in swine serum samples. moreover, given the high degree of n protein conservation, the developed assay may also be useful to study the seroprevalence to bovine and even human toroviruses. elisa optimization was carried out using a serum sample from a pig naturally infected by ptov (␣bres), a commercial serum mixture from healthy pigs (cps) and serum samples from spf pigs inoculated or not with prrsv or prcv. the assay prove to be very reproducible since very little variation was observed in the o.d. values of positive and negative control sera among assays performed in different days. cross-reactivity of ptov with other related viruses infecting pigs has been discarded in the three assays tested (elisa, western blot and virus neutralization) and these results confirm that these viruses are serologically unrelated, and are in agreement with previous results described by other authors (woode et al., (woode et al., , lamouliatte et al., ) . however, cps antibodies recognized both ptov-n and bev-n proteins (not shown), indicating that the animals used to obtain the serum had been infected by, or, exposed to ptov. similar observations had previously been described with different batches of commercial newborn calf serum that were shown to react with bev (vanopdenbosch et al., b) . due to the lack of reference sera for ptov, elisa cut-off value was empirically determined using serum samples from spf and cd/cd animals. overall, elisa results from adult animals from different spanish farms located in aragon (n = ) and navarra (n = ) suggest a high prevalence of antibodies in spanish pig population, since all adult pigs analyzed from these farms were positive by elisa, and these results were confirmed by neutralization test and western blot. on the other hand, the high prevalence of antibodies in serum samples from piglets from the farm from galicia suggests that ptov infection occurs early in piglets. this prevalence rate is higher than others previously reported for ptov in the netherlands ( %) using neutralization assay (kroneman et al., ) and for btov (brown et al., ; koopmans et al., ) . however, these ptov prevalence rate data can only be considered as preliminary, since both, our, and the study performed in the netherlands, were carried out with a short number of animals. further epidemiological studies are needed to obtain definitive information regarding ptov prevalences in spain and in other countries such as italy, where ptov strains seem to normally circulate. pathogenic potential of ptov as diarrhea causing agent remains unclear. in this regard, pigs from the farm in aragon, where an acute gastroenteritis outbreak was reported without an identified causing agent, show high levels of antibodies against torovirus. more studies are needed to understand the potential pathogenesis of ptov. in conclusion, we have developed an elisa for torovirus diagnosis that could be very useful for future epidemiological studies, to determine seroprevalence to porcine torovirus, but also to bovine and even human toroviruses. moreover, the nucleocapsid protein expressed by the baculovirus expression system could also be evaluated in immunization protocols to study its potential application for vaccination purposes against toroviruses. in this regard, protective responses elicited against the nucleocapsid protein have been reported for different coronaviruses (wasmoen et al., ; seo et al., ; cavanagh, ) . in addition, we have reported a new ptov strain and three genes coding for structural proteins have been fully sequenced providing information about torovirus evolution. this work contributes to increase our knowledge about this scarcely studied, but highly prevalent group of viruses, provides important tools for future studies, and presents, for the first time, preliminary evidences on the presence of antibodies to torovirus in spanish livestock. preliminary characterisation of torovirus-like particles of humans: comparison with berne virus of horses and breda virus of calves enzyme-linked immunosorbent assay for detection of antibodies to porcine reproductive and respiratory syndrome virus, by using recombinant nucleocapsid protein n detection of breda virus antigen and antibody in humans and animals by enzyme immunoassay severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus revision of the taxonomy of the coronavirus, torovirus and arterivirus genera development and evaluation of an elisa using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus xith international symposium on nidoviruses an indirect elisa for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen studies of epidemiology and seroprevalence of bovine noroviruses in germany characterization of torovirus from human fecal specimens the novel hemagglutininesterase genes of human torovirus and breda virus bovine torovirus: sequencing of the structural genes and expression of the nucleocapsid protein of breda virus coronavirusassociated diarrhea (winter dysentery) in adult cattle seroprevalence of noroviruses in swine a comparative sequence analysis to revise the current taxonomy of the family coronaviridae detection and molecular characterization of cultivable caliciviruses from clinically normal mink and enteric caliciviruses associated with diarrhea in mink the nucleocapsid of berne virus development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (pedv) antibodies epidemiological analysis of bovine torovirus in japan human torovirus: a new nosocomial gastrointestinal pathogen seroepidemiology of breda virus in cattle using elisa association of diarrhea in cattle with torovirus infections on farms association of torovirus with acute and persistent diarrhea in children electronmicroscopic evidence of torovirus like particles in children with diarrhoea identification and characterization of a porcine torovirus first isolation of cytopathogenic bovine torovirus in cell culture from a calf with diarrhea studies on bovine breda virus electron microscopic identification of torovirus-like particles in post-weaned piglets with enteritis diarrea neonatale dei vitelli: identificazione al m.e. in colorazione negativa di particelle virali morfologicamente riferibili al bredavirus new types of virus infections of domestic animals in the german democratic republic. . serologic survey studies of the distribution of equine torovirus infections in the gdr the significance of bredavirus as a diarrhea agent in calf herds in lower saxony human torovirus: a new virus associated with neonatal necrotizing enterocolitis torovirus detection in faecal specimens of calves and pigs in hungary: short communication molecular epidemiology of bovine toroviruses circulating in south korea the seroepidemiology of genogroup and genogroup norwalk-like viruses in italy breda virus-like particles in pigs in south africa inducible expression of the vaccinia virus a l gene provides a synchronized system to monitor sorting of viral proteins during morphogenesis porcine torovirus? the carboxyl-terminal -residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protects chickens from acute infection evaluation of the baculovirusexpressed s glycoprotein of transmissible gastroenteritis virus (tgev) as antigen in a competition elisa to differentiate porcine respiratory coronavirus from tgev antibodies in pigs antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: the effect of phosphorylation on immunoreactivity and specificity nidovirus sialate-o-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events cloning, sequencing, expression, and purification of sars-associated coronavirus nucleocapsid protein for serodiagnosis of sars torovirus gastroenteritis presenting as acute abdomen a serologic investigation for coronavirus and breda virus antibody in winter dysentery of dairy cattle in the northeastern united states bovine torovirus: cell culture propagation of a respiratory isolate and some epidemiological data profiles of igg antibodies to nucleocapsid and spike proteins of the sars-associated coronavirus in sars patients protection of cats from infectious peritonitis by vaccination with a recombinant raccoon poxvirus expressing the nucleocapsid gene of feline infectious peritonitis virus purification and partial characterization of a new enveloped rna virus (berne virus) antibodies to berne virus in horses and other animals morphogenesis of berne virus (proposed family toroviridae) longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus breda and breda-like viruses: diagnosis, pathology and epidemiology studies with an unclassified virus isolated from diarrheic calves comparative studies on three isolates of breda virus of calves we thank raquel blanco and juan ramón rodríguez for critical reading of the manuscript. we also want to thank r.j. de groot (utrecht university, utrecht, the netherlands) for providing the e. derm cells and bev virus, s. van gucht (gent university, belgium) for providing the serum samples from spf piglets, j. segales for providing the serum samples from cd/cd animals and l. enjuanes for supplying tgev and prrsv viruses.this work was supported by grants cicyt-bio - and consolider-porcivir csd - to d.r. j.p. was recipient of fpi fellowship from the spanish ministry of education and science. key: cord- -efmozngq authors: nan title: infectious diseases other than cmv ( st section) date: - - journal: bone marrow transplant doi: . /sj.bmt. sha: doc_id: cord_uid: efmozngq nan performed after culturing cd + cells for days, we observed the same effect as described above. however, when cd + cells were cultured for days prior to infection, a comparable suppression of cell expansion could be found for both hhv- variants a and b. our results suggest, that hhv- a may influence the expansion of more immature progenitor cells, whereas hhv- b displays a suppressive effect only on more mature, differentiated cells in vitro. w.h. krü ger, n. krö ger, m. abromeit, h. renges, f. tö gel, j. schrum, p. schafhausen, r. erttmann, h. kabisch, a.r. zander (hamburg, d) patients with a positive history of systemic fungal infection undergoing allogeneic sct are highly endangered by reactivation of their infection. for such patients some attempts of antimycotic prophylaxis with low dose conventional amphotericin-b (am-b) have been reported. however, the major side effect of conventional am-b is nephrotoxicity. here, we report data of patients grafted under the protection of liposomal (lip) am-b. patients (w/m: / ) with a median age of ( - ) years underwent allogeneic mrd (n= ), mud (n= ), syngeneic (n= ) or autologous sct (n= ) for treatment of al (n= ), cml (n= ), mds (n= ), saa (n= ), and granulomatosis (n= ). all patients had a history of fungal pneumonia with evidence for aspergillus spp. as infectious agent in cases. three patients had suffered from aspergillus sinusitis (n= ) and aspergillosis of the bone, respectively. fungal infection was culture-documented in cases. x-ray examination or clinical parameters led to the diagnosis in cases. therapy with lip-amb was started either primarily (n= ) or after a short course of conventional am-b (n= ) or itraconazole (n= ). lip-amb was initiated on day + (- - ) for a median duration of days ( - ). the start dose of , ( , - ) mg/kg was increased to , ( , - , ) mg/kg in some patients. lip-amb was excellently tolerated, fever and chills occurred in two patients. creatinine showed a slight increase to % ( %- %) of the base line probably not related to lip-amb. after a median follow-up of ( - ) days ( %) patients were discharged without evidence for fungal infection. / ( %) of patients with history of culture-documented mycosis died from relapse of c. krusei septicaemia (n= ), from cns bleeding probably related to relapsed aspergillosis and from multi-organ failure without culture-proven mycosis. patients have died from aspergillosis (n= ) or candidosis. one of these patients had to be grafted during active aspergillosis of the lung for disease-specific reasons. the remaining seven patients have died from multi-organ failure (n= ), gvhd and septicaemia (n= ) and pneumonia (n= ) related to bacterial infection in two cases. patients with a history of preceding mycotic infection are at high risk to acquire potentially fatal infections under sct. however, our data clearly show that only / ( %) of our patients developed culture-positive fungal infection during severe immunosuppression. we conclude that a history of preceding fungal infection is no contraindication for stem cell transplantation. between / and / , out of pts manifested proven or possible txo after allogeneic sct. during this period / patients died from txo as primary cause of death. since / we started a prospective randomized monocentric study comparing tmp/smx with pym/dps to prevent reactivation in patients at risk. risk was defined as a positive serology for txo in recipients or donors. additionally, immunosuppression with a daily dose of more than mg prednisolone was requested for inclusion. randomization was executed when hematopoiesis appeared stable at the end of hospitalization. arm a received tmp/smx x / mg twice a week (a). arm b was treated with pym mg once a week and dps mg once a day (b). folinic acid was given x mg twice a week to all pts. / pts were at risk, / were randomized and evaluable. patients with drug related side effects discontinued prophylaxis and switched to the alternative arm after resolution. during the study, pt developed lethal cerebral txo prior to randomization. the principal reason for exclusion was cytopenia ( / pts), immunosuppression with pdn Ͻ mg/d (n= ), and non signed informed consent (n= ). prophylaxis associated side effects were equally distributed in the two study arms (fisher exact test p= , ). main reason for discontinuation was cytopenia. interestingly, the covered interval by prophylaxis was significantly longer for arm a: versus days, respectively (t-test: p= . ). at a first glance, arm a seems to be better tolerated in view of the longer covered period of prophylaxis. however, longer follow up and further analysis will show whether this is due to side effects itself or to the practice of the physician to take arm b side effects more serious than those of arm a. to evaluate the influence of the helicobacter pylori (h.p.) infection on gastrointestinal complications after high-dose chemotherapy and stem cell transplantation (stx) we tested patients ( female, male) by the c-urea breath test prior to initiation of conditioning therapy. at the initial testing patients (solid tumors: pts., hematological malignancies: pts.) showed a negative result and patients (solid tumors: pts., hematological malignancies: pts.) a positive result without any gastrointestinal complaints. compared to the overall incidence of h.p. infection of % in these patients, the subpopulation of breast cancer-patients had a higher infection rate ( %). these patients with a median age of years ( - ) underwent an autologous (n= ) or allogeneic transplantation (n= ). beside gut decontamination they all received prophylaxis with intravenous ranitidin ( mg/d) which was replaced by a proton pump inhibitor (ppi) at a dose of mg/d i.v. if clinical symptoms of gastritis appeared. no main differences between both groups according to the occurrence of nausea/ vomitus, enteritis and mucositis could be seen. however, the incidence of severe mucositis whoiii/iv as well as enteritis who iii/iv was higher in h.p. positive patients ( % and %) in comparison with h.p. negative patients ( % and %). especially in breast cancer patients (n= ) mucositis whoiii/iv was more pronounced in the h.p. positive group ( % vs. % respectively). furthermore, the occurrence of clinical signs of gastritis could not be related to the helicobacter infection: % of the h.p. positive vs. % of the h.p. negative patients felt epigastrical pain. % vs. % needed treatment with a proton pump inhibitor. in conclusion helicobacter pylori infection seems not to influence the incidence of gastrointestinal complications but possibly the severity of mucositis and enteritis. low-dose amphotericin b lipid complex (ablc) is safe and effective as empiric anti-fungal therapy in immunocompromised patients with hematologic malignancies r. powles, b. sirohi, j. mehta, s. kulkarni, k. murphy, b. cheung, a. conway, r. saso, a. riggs, s. singhal, d. cunningham, j. treleaven (sutton, uk) ablc (abelcet, the liposome company) is a ribbon-shaped liposomal formulation of amphotericin b consisting of dimyristoylphosphatidylcholine and dimyristoyl-phosphatidylglycerol in a : molar ratio which is especially concentrated in pulmonary tissue. it is known to be safe and effective in presumed and confirmed fungal infections in immunocompromised patients at the dose of mg/kg. there are limited data on its use at lower doses. we explored low-dose ablc in immunocompromised patients with hematologic malignancies who had fever of unknown origin which had failed to respond to combination antimicrobials, and were presumed to have fungal sepsis. immunocompromised patients ( - y, median ; leukemia, myeloma, lymphoma) received courses of ablc at the median daily dose of mg/kg rounded off to the nearest vial size (range, . - . mg/kg) after autologous (n= ) or allogeneic (n= ) stem cell transplantation or chemotherapy (n= ). the median neutrophil count at start of ablc therapy was . x /l (range, - . ). courses of ablc had been preceded by conventional amphotericin b and by fluconazole without response. the median days of therapy with ablc was (range, - ). courses of doses (n= ) were considered evaluable for efficacy and all courses were evaluable for toxicity. courses resulted in complete response, in partial response, and in failure (overall response rate %). the failures were switched after and days of ablc to and . mg/kg ambisome for and days respectively, grew candida and aspergillus from the sputum respectively, and died without responding. the change in serum creatinine from the beginning to the end of therapy was - to + micromol/l (median - ). treatment courses had been premedicated with chlorpheniramine/hydrocortisone for the first few days. infusion-related toxicities comprised rigors (n= ), pyrexia (n= ), hypertension (n= ), and hypotension (n= ); at least one of these toxicities was seen in ( %). these data suggest that low-dose ablc is very effective empiric anti-fungal therapy in immunocompromised patients with hematologic malignancies. viral pneumonia is an important cause of morbidity and mortality in patients undergoing allogeneic stem cell transplantation. a previous study from the usa has found viral pathogens such as respiratory syncytial virus (rsv), influenza a and b (flu a & b), parainfluenza (paraflu) and adenovirus emerging as significant causes of upper and lower respiratory tract infections (clin inf dis ; : - ) . a retrospective analysis of the incidence of community respiratory viral infections in our population was carried out for the allogeneic bm and pbsc transplants performed on patients between january and december . rsv, flu a & b, paraflu, adenovirus and picornavirus were detected using direct immunofluorescence testing of nasopharyngeal aspirate or bronchoalveolar lavage fluid as clinically indicated. the overall incidence of proven community respiratory viral infection was % in the first year post allograft ( of transplants). these consisted of cases of rsv ( %), of flu a ( %), of flu b ( %), of paraflu iii ( %), of adenovirus ( %) and of picornavirus ( %). these results are comparable to previous us studies. six of these patients ( %) had upper respiratory signs only, received specific anti-viral therapy with nebulised ribavirin for rsv ( ), paraflu iii ( ) and flu a ( ). the other patients ( %) had lower respiratory tract signs on examination and had chest x-ray changes. of this group, were treated with nebulised ribavirin for rsv ( ), paraflu iii ( ) and flu a ( ) and went on to receive iv ribavirin for rsv, one received zanamivir and amantidine for flu a and had no specific antiviral therapy for flu a ( ), paraflu iii ( ) and picornavirus ( ). overall of the proven infections, patients made a full recovery, died of rsv pneumonitis, two patients with paraflu iii died of superimposed infection, one bacterial and one fungal; neither received specific antiviral therapy and three died of unrelated causes. to conclude, prompt treatment of community respiratory viral infections may reduce morbidity and mortality in adult allogeneic bmt and pbsct patients but respiratory viral infection was still the primary cause of death in % of our patients and probably contributed significantly in a further %. a.j. ullmann, k. weise, p. brandt, c. huber (mainz, d) objective: patients post stem cell transplantation are at very high risk for reactivating varicella-zoster virus [vzv] . herpes zoster infection is frequent and has the potential to disseminate. atypical generalized zoster is associated with an increased mortality rate. methods: retrospective analysis of blood samples originally collected from patients for cmv-monitoring was performed. period of analysis was approximately one year with a minimum of months. thirty-seven of the patients received an allogeneic transplantation and underwent a cd -selected autologous transplantation. pcr-primers were selected from the orf of the vzv-genome. confirmation of the positive results was performed by southern blot analysis. the patients' histories were available from their medical record and/or telephone interview. results: none of the patients with negative blood pcr-results developed vzv-disease. seven of the allogeneic transplanted patients with at least one positive pcr-result had a vzv-disease. three of the positive patients developed a disseminated disease. four of the patients with a positive result and no vzv-disease received at that time point polyvalent immunoglobulins. none of the five received antiviral medications. all (n= ) of the autologous transplanted patients with a positive pcr-result developed a vzv-disease. conclusion: this retrospective analysis reveals that the pcr testing of the blood for vzv has a high sensitivity and suggests that a vzv-viremia occurs even during localized disease. a prospective study to evaluate the possibility to predict a disseminated vzv-disease is warranted. antimicrobial prophylaxis in ebmt centers: a report from the ebmt infectious diseases working party h. akan, c. cordonnier for the infectious diseases working party of the ebmt (ankara, tr; creteil, f) in order to know about the anti-infectious prophylactic policies used in the ebmt centers, the idwp run a mail-in survey regarding antibacterial, antifungal, antiviral, and immunoglobulin (ivig) prophylaxis. the survey consisted of questions on indications and modalities (drugs, doses, duration) of prophylaxis in autologous and allogeneic sct. seventy-four centers from countries responded. . allogeneic transplant centers / centers give antibacterial prophylaxis during neutropenia, mostly by quinolones ( %) and / centers ( %) continue prophylaxis after the neutropenic phase, mostly until discharge. / ( %) centers give antifungals, at least during neutropenia, with fluconazole ( %), oral ampho b ( . %) or itraconazole ( . %). three centers give prophylaxis until discharge. prolonging prophylaxis in case of gvhd is a common approach. the main selection criteria for antifungal prophylaxis are secondary prophylaxis ( %) and unrelated transplants ( %). / ( %) centers give antiviral prophylaxis during a mean duration of days. centers use ganciclovir and use valaciclovir instead of aciclovir for selected pts (unrelated sct, cmv positivity). / ( %) centers give ivig to all allogeneic pts and half of them give . - . g/kg/w. / ( %) centers restrict ivig prophylaxis to selected pts (unrelated transplants, hypogammaglobulinemia, cmv positivity). . autologous transplant centers / ( %) centers give prophylactic antibacterials, mainly quinolones ( %) during neutropenia, and centers continue prophylaxis after the first month. / ( %) centers give prophylactic antifungals mainly fluconazole ( %), itraconazole ( %) and oral ampho b ( %). / ( %) centers give antiviral prophylaxis mainly aciclovir ( %) during a mean duration of days. / ( %) centers give ivig to all pts and / ( %) only to selected pts. our survey indicate that although antimicrobial prophylaxis is common in sct centers, there are great discrepancies in the policies used in the ebmt centers, with different regimens, selection criteria, and durations of prophylaxis. these findings indicate the need for guidelines. this survey will serve as a basis for future discussions and proposals of recommendations from the idwp. radiologically guided fine needle lung biopsies in the evaluation of focal pulmonary lesions in allogeneic stem cell transplant recipients e. jantunen, a. piilonen, l. volin, p. ruutu, t. parkkali, p. koukila-kahkola, t. ruutu (kuopio, helsinki, fin) lung problems are common in allogeneic stem cell transplant (sct) recipients. we have retrospectively evaluated feasibility and usefulness of radiologically guided fine needle lung biopsies(fnlb) in the evaluation of focal pulmonary lesions in this patient population. during a -year period, altogether fnlbs in patients were performed ( - biopsies/patient) guided by either ultrasound (n= ) or computed tomography (n= ). the median time from sct to the first fnlb was days ( - d). in addition to complications of fnlb, also biopsy findings in relation to the final diagnosis were assessed. prophylactic platelet transfusions were given in procedures ( %). complications of fnlb included clinically insignificant pneumothorax in four procedures ( %) and hemoptysis in one case. the first fnlb was suggestive of invasive pulmonary aspergillosis (ipa) in six patients ( %). additional clinically useful findings of fnlb included pseudomonas aeruginosa (two patients) and nocardia (one patient). the final diagnosis was ipa in patients, immunological lung problems in three patients and other in four patients. fnlb is feasible in allogeneic sct recipients with a low complication rate. the diagnostic yield is relatively high especially in suspicion of ipa. however, re-biopsy or other diagnostic methods are often needed in patients with non-confirmatory findings on fnlb. among chronic carriers of hepatitis b virus receiving cht for nhl, a reactivation of virus replication has been often observed and this may give rise to hepatitis, hepatic failure and death, and may prevent from performing further therapy including trasplantation procedures. herein we present our experience in four patients with nhl where a hepatitis flare-up was observed after (in three patients) and (in one patient) cycles of standard-dose chemotherapy. the patients were affected by mantle cell, follicle centre grade iii, peripheral t-cell unspecified and diffuse large b-cell lymhoma respectively, were male aged , , and years respectively, hcv and hiv negative. in all patients, pretreatment hbv serology was as follow: hbsag positive, hbeag negative, total anti-c ab positive, igm anti-c negative, anti-e ab positive and anti-s ab negative. three of them were treated with f-machop regimen, the oldest was treated with the chop regimen. hbv-dna, was negative before starting chemotherapy and became positive in all four patients. after spontaneous recovery they were treated with lamivudine mg once daily and this allowed resuming and completing the chemotherapy program without another reactivation of hepatitis b. in two patients high-dose chemotherapy and autologous stem cell tranplantation was also performed under lamivudine as a part of our program for high-risk nhl. antiviral treatment was stopped to months after the last chemotherapy given. during the follow-up period they were monitored with twice-monthly blood counts, transaminases'levels and hbv-dna: all these parameters remained normal/negative all throughout the period. currently, patient b.s. is in cr months from diagnosis and months from the end of chemotherapy; patient d.f.a. is in cr months from diagnosis and months from the end of chemotherapy, patient v.e is in cr months from diagnosis and months from trasplantation; patient c.g. is in cr months from diagnosis and months from transplantation. this data suggests a possible role of lamivudine in preventing hepatitis b reactivation during administration of chemotherapy and asct to chronic carriers of hepatitis b virus. background: severe acute graft-versus-host-disease (agvhd) of the gut is still a major complication after allogeneic stem-cell transplantation (sct) as response rates to treatment (tx) of intestinal gvhd (igvhd) are lower than those observed for gvhd of the skin. since tnf-alpha (tnfa) is one of the key cytokines in agvhd, murine monoclonal antibodies (mab) against tnfa were used in gvhd-prophylaxis and also in the therapy of severe igvhd. infliximab, a chimeric human and mouse mab against tnfa has been introduced recently as a new tx option for pts with progressive crohn's disease. this antibody showed also promising results in the tx of steroid-resistant (sr) agvhd achieving response rates up to % in pts with igvhd. in previous studies of our own, the combination of okt and a murine mab against tnfa (mak ) showed potent synergistic effects without major side effects in the tx of pts with sr igvhd. thus it seemed reasonable to combine okt and infliximab in the tx of severe sr agvhd of the gut. results: we report the results of a pilot study using the combination of okt and infliximab as second or third-line-tx in pts who developed sr agvhd grade iii to iv with severe involvement of the gut after allogeneic sct. infliximab was given in a dose of mg/kg weekly for two weeks (i.e. three doses) combined with okt mg daily for seven days. all three pts responded well showing marked improvement of diarrhea and abdominal pain. but several days to weeks after the tx all three pts died due to severe infectious complications. one patient developed histologically proven aspergillosis of the liver, in another patient invasive aspergillosis of the gut was demonstrated at autopsy and finally the third patient developed hepatic dysfunction and pneumonia which clinically presented as fungal pneumonia and of which he died despite of antibacterial and antifungal tx. conclusions: t cell-depletion and blocking of tnfa is an effective tx of refractory intestinal agvhd but provokes life-threatening fungal infections. inhibiting the inflammatory t cell response by t cell depletion and blocking the effector functions of neutrophils and macrophages by tnfa-suppression gives way to breakthrough-infections of aspergilli as could be dramatically demonstrated in our small series of three pts. we therefore conclude that pts who are really at need to receive both, t cell depleting and tnfa blocking antibodies should receive antifungal tx with substances effective against aspergilli not only in prophylactic but in full therapeutic doses. objective: toxoplasmosis is a rare but serious infectious complication after allogeneic bmt. by pcr, parasitemia can be detected in the peripheral blood before symptomatic disease develops. the risk factors for reactivation of toxoplasmosis, however, are unknown. methods: we prospectively studied consecutive patients seropositive for t. gondii with pcr at least fortnightly for the presence of t. gondii dna in the peripheral blood. the following potential risk factors were evaluated: gender, age, type of preparative regimen (myeloablative or non-myeloablative), presence or absence of gvhd before parasitemia, receiving methylprednisolone before parasitemia, underlying disease (cml versus other malignancies), cmv reactivation, amount of specific igg against t. gondii before bmt, and donor (family donor versus matched unrelated donor). results: of the patients ( %) showed t. gondii dna in the peripheral blood at a mean of days after bmt ( % ci, to days). of those patients, four had a persistent parasitemia which could be treated successfully in two patients. the other two patients died of fulminant toxoplasmosis despite treatment. of the patients demonstrated only a transient parasitemia on one or two occasions. patients without parasitemia were compared to the parasitemic patients. there was no statistically significant difference between the two groups with respect to age, gender, length of follow-up, amount of specific igg against t. gondii before bmt, preparative regimen (myeloablative versus non-myeloablative), underlying disease (cml versus other malignancies), presence of gvhd, occurrence of cmv reactivation, or donor (family donor versus mud). only the use of methylprednisolone was observed more frequently in patients with t. gondii parasitemia ( of patients) versus the control group ( of patients); this, however, just missed statistical significance (p = . by the fisher exact test). conclusions: in this series of patients, no risk factor for t. gondii parasitemia could be detected. until a population at risk can be identified in larger series, regularly monitoring allogeneic bmt patients seropositive for t. gondii may be justified for the presence of t. gondii dna, especially if the patients receive steroid medication. abcd consists of amphotericin and sodium cholesteryl sulfate in a : molar ratio. between / and / , immunocompromised/neutropenic patients ( - y, median ) with hematologic malignancies ( acute leukemia, myeloma, lymphoma, other) received courses of abcd at daily dose of mg/kg for presumed (n= ) and - mg/kg for proven/strongly suspected (n= ; microbiologic, radiologic) fungal infections. abcd administration followed allogeneic (n= ), autologous (n= ), or syngeneic (n= ) stem cell transplantation, or chemotherapy (n= ). the major indications for the use of abcd were renal dysfunction or potassium requirements Ͼ mmol/kg/d on conventional amphotericin. in view of known problems with infusion-related toxicity of abcd, each dose was administered over - h after premedication comprising g paracetamol, mg chlorpheniramine, - mg pethidine, mg hydrocortisone and mg nefopam in various combinations. courses comprising doses were evaluable for efficacy, and all courses were evaluable for toxicity. the median neutrophil count at initiation of treatment was and the median baseline creatinine was micromol/l ( - ). courses in patients ( - doses, median ) were evaluable for efficacy. courses in proven infections resulted in complete and partial responses, and failures ( % response). courses in presumed infections resulted in complete and partial responses, and failures ( % response). the overall response rate was %. the change in serum creatinine from the beginning to the end of therapy was - to + micromol/l (median - ). despite premedication, significant infusion-related toxicity was seen: ( %) rigors, ( %) pyrexia, ( %) bronchospasm, ( %) rash, and ( %) anaphylaxis. at least of these toxicities was seen in ( %). tachyphylaxis developed to infusion-related reactions with subsequent doses. ( %) patients experienced hepatotoxicity which could not be definitely attributed to abcd. the underlying intervention (type of transplant or chemotherapy) did not affect efficacy or toxicity. we conclude that despite its efficacy, the high incidence of infusion-related side effects seen in abcd-treated patients despite extensive premedication is likely to be a limiting factor in the usefulness of this drug. the frequent use of corticosteroids mandated by abcd may be detrimental in patients who are already immunocompromised and/or neutropenic. human herpes virus (hhv- ) is ubiquitous through the human population, with more than % of adults being seropositive. the incidence of hhv- reactivation and its impact on morbidity in allografted recipients is poorly known. we have conducted in the last six months a prospective pcr screening of hhv- in o consecutive patients allografted in our institution ( cml, cll, hd, aml, all, mds) with hla-identical ( related and unrelated) and haplo-identical donors. acyclovir was used in all as prophylaxis. pcr screening for hhv- was performed twice a week on peripheral blood (pb) and on bone marrow or organs biopsies (liver, gut or skin) when available. a nested pcr using primers recognizing type a and b hhv- early antigen gene was used. out of the patients, five became pcr-positive, four of which displayed clinical symptoms. p (matched unrelated t-cell depleted transplant) engrafted normally, with grade ii skin gvhd after the second dli. hhv- was initially detected in a liver biopsy (day ) performed for hepatitis associated with viral encephalopathy. he was first successfully treated with foscavir but reactivated months later with the same clinical pattern. p (t-cell depleted haploi-identical transplant) engrafted normally without agvhd. he was successfully treated with foscavir after detection of hhv- in pb at month associated with encephalopathy confirmed by mri. at month , pcr became positive again in pb and marrow without clinical symptoms and it was decided not to treat. p (matched related transplant) engrafted normally, and developed limited cgvhd after early cyclosporin withdrawal for lack of molecular response. he presented with mild hepatitis at month . a liver biopsy was performed and revealed positive for hhv- , with marrow and pb screening remaining negative throughout the follow-up. foscavir treatment led to resolution of hepatitis. p (matched related transplant), engrafted normally without gvhd. he then became pcr positive for both cmv and hhv- in pb and marrow and was treated with ganciclovir with negativation of both pcr tests. we conclude that hhv- reactivation may be an underestimated factor of morbidity in allogeneic transplant and should therefore monitored carefully. however, pcr screening in pb does not appear as a sensitive marker as in cmv reactivation, some patients showing positivity only in the involved organ tissue and the role of pb pcr screening remains to be better defined. j. olson, j. adler-moore (pomona, usa) systemic candidiasis is difficult to completely eradicate using conventional treatment regimens, especially in continuously suppressed animals. it was hypothesized that with the unique pharmacokinetics of ambi (i.e., sustained bioavailability in tissues and reduced toxicity at high doses), an intermittent, high dose treatment regimen could be designed that would be effective in clearing the infection from the kidneys of both ic and is candida albicans infected mice. by monitoring the kidneys for clearance at various times during treatment, it could also be determined if the immune status of the animals effected the length of treatment needed. groups of ic or is mice (cyclophosphamide mg/kg ip, x/wk throughout study)(n= /group), were challenged iv with . log cfu (ic) or . log cfu (is). beginning d post-challenge, ic or is mice received iv, either mg/kg ambi or % dextrose, x/wk. after , or weeks of treatment, the amount of candida in the kidney tissue was assessed h post-treatment. an additional five ic and is mice received no further treatment after wk . at the end of wk , their kidneys were examined for the presence of fungi. both is and ic mice had a dose dependent, significant (pϽ . ) reduction in median introduction viral respiratory infections are thought to complicate treatment of hematological malignancies frequently, but current techniques do not always yield a causative agent. therefore, we evaluated the additional value of pcr in the detection of viral infections in haematologic cancer patients with pulmonary abnormalities on x-ray, of whom broncho-alveolair lavage (bal) samples had been stored. methods between october and june , we collected bal sample from patients (m/f: / ; median age: ). in of these patients nose/troat (nt) swabs were collected within week as well. after initial examination of these samples by virus culture, they were stored at - degrees c. pcr was done on both bal and nt samples for the following respiratory viruses: parainfluenza virus , , and , respiratory syncytial virus (rsv), rhinovirus, influenzavirus a and b, enterovirus and coronavirus. we compared the results of pcr detection with those of virus culture and paired serology specimens (n= ). results twenty-eight patients had been treated with stem cell transplantation (sct) for their underlying diseases (allo-sct: , auto-sct: ), and patients had been treated with cytotoxic agents for leukaemia (n= ) or nhl (n= ). on presentation with respiratory symptoms patients were neutropenic and patients were on immunosuppressive medication. virus cultures of bal showed respiratory viruses in patients, that were also detected by pcr. in addition, pcr was able to detect respiratory viruses in another patients. interestingly, when from a patient both bal and nt samples were available (n= ) pcr on nt swabs yielded the same results as pcr on bal samples. serological examination only detected a rsv infection in patients, and had no additional value over pcr. two of viral infections were acquired nosocomially. four of ( %) of patients treated with cytotoxic therapy and of ( %) sct recipients developed respiratory diseases related to respiratory viruses. six patients had a respiratory illness related to both respiratory virus and other cause (bacterial and fungal pneumonia: n= , bronchiolitis: n= ). conclusion pcr is more sensitive compared to direct culture and serology for the detection of respiratory viruses in patients with haematologic malignancies. examination of nt swabs by pcr appears as sensitive as on bal samples. introduction : the diagnosis of invasive aspergillosis (ia) in neutropenic patients affected by hematological neoplasms is cumbersome, due to the difficulty of obtaining adeguate bioptic and cultural specimens, very scarce results of radiological detection of early lesions, inadeguate results of serologic tests. rt-pcr detection of dna is still under study, and detection of galactomannan by elisa is sensible, but not particularly specific. patients and methods : we performed seriated elisa assays twice a week in auto/allobmt and neutropenic patients at risk for invasive mycoses (neutropenia, gvhd, high-dose chemotherapy, immunosuppressive treatment): allobmt ( anll, mm, bc cml), autobmt ( mm, nhl, hd), anll, saa, bc/ap cml, cll, hd, nhl. antifungal prophylaxis included fluconazole, itraconazole or i.v. low-dose amphotericin b (am-b). the results of elisa assay was considered positive in a single patient, if at least two consecutive positive tests were obtained. all febrile patients entered a program of radiological survey by weekly chest ct scan. results : assays out of were negative. there were positive results for affected (positive) patients, developing ia as follows : proven cases, and probable according the revised eortc mycoses study group criteria; in all the patients positivity of elisa assay was sustained and durable, and decreased with the efficacy of antifungal treatment. at the onset of elisa assay positivity the chest rx was negative; while the chest ct scan detected only subtle pulmonary changes, suggestive of fungal lesions, and thereafter became diagnostic of ia. the remaining patients (including with borderline single positive assay and one with only a single positive test) never developed ia. sensitivity and specificity of elisa assay (considering proven and probable ia diagnoses) were %. at onset of elisa assay positivity the patients were given - . mg/kg/day am-b, eventually changed to - mg/kg/day liposomal am-b. in anll patients, refractory to liposomal am-b, voriconazole determined improvement of pulmonary cavitations in and regression in the other patient, enabling him to undergo allobmt. mortality was high, reaching % in allobmt and . % in autobmt recipients. conclusion : we suggest that combination of routinary, twice a week, elisa assay and weekly chest ct scan could enable an early diagnosis of pulmonary ia, for targeted antifungal treatment. respiratory virus infections in adult t-cell depleted allograft recipients: risk factors and response to anti-viral therapy s. chakrabarti, k. collingham, k. holder, c. fegan, t. gentle, d. milligan (birmingham, uk) we prospectively evaluated respiratory virus infections in allograft recipients conditioned with conventional myeloablative (n= ) or non-myeloablative treatment and t-cell depleted with campath antibodies. throat samples were obtained weekly for days. upper and lower respiratory symptoms were evaluated by naso-pharyngeal aspirates and bal. parainfluenza (piv), rsv, influenza (if)b and adenovirus infections were treated with ribavirin (oral, inhaled or iv) in a dose escalation regimen. ifa infections were treated with amantadine or zanamivir if unresponsive. episodes of respiratory virus infection were detected in patients ( %). all infections were associated with upper respiratory illness and had lower respiratory involvement. piv (piv ; , piv ; ), and rsv( ) were the commonest isolates. there were four episodes of influenza (ifa ; ifb ) and episodes of rhinovirus and a single episode of adenovirus. six patients had infection with multiple viruses and the median number of respiratory virus isolated was (range - ). the median time to the onset of the infection was days (range to ). the duration of treatment ranged from to days (median days). two episodes of rhinovirus and one of ifb were self-limited and were not treated with antivirals. the adenovirus infection responded to donor lymphocytes. two patients each with piv and rsv required oral or iv ribavirin at mg/kg/day. the other episodes of piv and episodes of rsv responded to inhaled ribavirin. ifa infection responded to amantadine in one patient and another patient needed zanamivir. there was one death ( %) related to respiratory virus infection (rsv and ifa). on analysis of the risk factors, the dose of campath antibody was the only significant factor ( infections in patients receiving mg campath versus / in patients receiving a lower dose p= . , or . ( . - ). on comparing the immunological recovery, the median cd cell count at - months was lower in the infected group ( ± /mm ), compared to the non-infected group ( ± /mm ); p= . . in conclusion, multiple respiratory virus infections are frequent after t-cell depleted transplants, the dose of t-cell antibody being a significant risk factor. infection correlated with a poor cd recovery at - months post-transplant. although mortality was low, prolonged antiviral therapy added to the morbidity and cost. screening for aspergillus spp. by a pcr of whole blood samples from patients with haematological disorders c. lass-flö rl, e. gunsilius, d. nachbaur, h. einsele, m. dierich (austria, germany) we performed a screening for aspergillus spp. by polymerase chain reaction of whole blood samples in patients with haematological disorders. in a two-year study, patients admitted to the university hospital of innsbruck for cancer chemotherapy without clinical signs of fungal infection were prospectively screened for aspergillus spp. in of ( %) patients aspergillus dnaemia was detected. of these patients ( %) were positive only once for aspergillus dna but positivity was never associated with invasive aspergillosis. pcr positive episodes were short and resolved without antifungal treatment. five patients ( %) had intermittent pcr positive results. seven ( %) patients presented at least two consecutive positive pcr results, one of these patients developed invasive aspergillosis and another two were highly suspected of having aspergillosis. based on the criteria of eortc case definitions sensitivity and specificity of serial pcr monitoring were % and %. positive pcr results became negative shortly after commencement of antifungal treatment but the changes did not correlate with clinical responsiveness to treatment in three patients. our results indicate the potential usefullness of pcr for screening for aspergillus spp. in patients at risk yet without antifungal treatment. r. herbrecht, a. thiebaut, a. vekhoff, f. isnard-grivaud, p. moreau, g. michel, s. dupuis, f. bastides, a. datry, o. lortholary (strasbourg, lyon, paris, nantes, marseille, tours, bobigy, f) a pharmaco-epidemiology study to evaluate current therapeutic practice in treating invasive fungal infections (ifi) was carried out in haematology departments between november and october . two hundred fifty two ifi were recorded, leading to an incidence of . %. therapeutic practice was analysed in the last patients treated in each center for a total of cases. the average age of patients was years (range week - years). one hundred and nine ( %) had a haematological malignancy. eighty one ( %) had aspergillosis, ( %) candidiasis and ( %) another ifi. according to the criteria of the eortc/msg, aspergillosis was retrospectively graded as "proven" in % of the cases, as "probable" in % and as "possible" in %. factors associated with ifi were antibiotherapy ( %), chemotherapy ( %), presence of a central veinous catheter ( %), neutropenia Ͻ anc/µl ( % of which % were severe Ͻ anc/µl), haematopoetic stem cells transplant ( % with two thirds of allografts), corticosteroid therapy ( %), immunosuppressive therapy ( %), radiotherapy ( . %), prior ifi ( %) and diabetes ( %). eighty eight percent of patients received nephrotoxic agents and % prophylactic antifungal therapy. fifty six percent of the aspergillosis and % of the candidiasis were treated with more than lines of antifungal treatment. first line treatments were amphotericin b ( %), azoles ( %), amphotericin b mixed in intralipid ( %), liposomal amphotericin b ( . %), lipid complex of amphotericin b ( %) with a cure rate of %, %, %, % and % respectively. in haematopoietic stem cells transplant recipients, antifungal first line treatments were %, %, %, % and % respectively. the overall mortality rate was % and was higher in aspergillosis ( %) than in candidiasis ( %). death was attributed to the ifi in % of the aspergillosis and in % of the candidiasis. in transplant patients, cure rate was % and an overall mortality was %. in conclusion, this first pharmaco-epidemiological study evaluates the incidence of ifi in haematology departments in france and reflects the variety of treatments used as first line antifungal therapy. the overall incidence of adenovirus infection following bone marrow transplant (bmt) has been reported in two comprehensive studies at % and %, with rates of disease of % and . %, respectively. the clinical manifestations of adenovirus infection in bmt recipients range from fever with gastroenteritis or cystitis to disseminated disease with multi-organ involvement and high mortality. although there are several case-reports which support either the use of ribavirin with or without immunoglobulins, or ganciclovir, no conclusive evidence of benefit or clear treatment recommendations has been defined. the antiviral drug cidofovir ((s)- -( -hydroxy- -phosphonylmethoxypropyl) cytosine, hpmpc) is an acyclic nucleoside phosphonate with broadspectrum activity in vitro against several dna viruses, including adenovirus. we report the use of intravenous (iv) cidofovir for the treatment of adenovirus infection from june to october in five haematology patients: four children and one adult (age range: years - years). in four patients, the infection occurred post-allogeneic transplantation ( hla-identical related; haplotype mismatched) between days + and days + ; one patient had adenovirus infection before transplantation. in three patients, adenovirus was isolated only from faeces, but two patients had disseminated infection with isolates from multiple sites (oropharynx, urine, faeces, and conjunctiva). all patients received iv cidofovir at a dose of mg/kg once a week (and thereafter every fortnight) for at least weeks or until negative for adenovirus by viral culture. four patients cleared adenovirus within one month after commencing treatment and one patient died because of a fungal infection while still on cidofovir, and no follow-up sample was available. cidofovir was well tolerated, and no severe side effects were observed. conclusion: cidofovir has been used for the treatment of adenovirus infection in pre-/post-bmt patients. based on these preliminary results further randomised/multicentre studies are warranted to establish the efficacy of cidofovir in the treatment of adenovirus infection. prevalence of hepatitis c after bone marrow transplantation for solid tumours in children included in a randomised study from to comparing prophylactic versus therapeutic transfusion of platelet concentrates there was still no recoverable fungus in the is mice, and only one ic mouse had . log cfu/g. in conclusion, both is and ic mice responded similarly to x/wk treatment with mg/kg ambi. there was a significant as the risk of post-transfusion hepatitis c was high during this period, we tested in , post-bmt sera of children ( in ppt and in tpt group) to determine whether either of these two strategies was associated with a higher risk of hepatitis c. children were screened for anti-hcv using a rd generation enzym linked immunosorbent assay (elisa)(elysis, ortho diagnostic) and we investigated the presence of viral rna by pcr (test cobas amplicor hcv v . , roche diagnostic). the median interval between the last blood products transfusion and the serum sample was months (range: - ). pcr analysis of rna was positive in children ( %): / in group ppt versus / in group tpt (p=ns). the mean total number of pc units transfused during the trial was not significantly different between the ppt and tpt groups: . +/- . versus . +/- . respectively. this mean total number of transfused pc units in relation to the weight and the total duration of neutropenia was . in ppt group versus . units in tpt group (p= . ). anti-hcv antibodies were detected by the elisa assay in / pcr-positive serum rna samples (elisa sensitivity= . ). all the pcr-negative serum rna samples were negative by the elisa assay (elisa specificity= ). the interval between the last blood product transfusion and the serum sample one of pre-bmt serum rna samples was pcr-positive and / were pcr negative. in conclusion, the prevalence of hepatitis c after bmt in children transfused with pooled random pc between and was % in our study. the elisa test used has a poor sensitivity in this population and pcr analysis of rna must be preferred to test hepatitis c after bmt one other patient treated with immunosuppressive therapy for saa has developed ebv-lpd at the same period. diagnosis of ebv-lpd was confirmed by pcr and several immunological studies at the time of first clinical symptoms in three. cause of death in one was not clear until autopsy. diagnosis of ebv-lpd was confirmed on autopsy in all three patients who died , , and days after first clinical symptoms. lpd as a cause of death still cannot be excluded in one other patient. first signs of lpd appeared , and days after last dose of atg ( mg/kg) in transplant patients, days after last dose of atg ( mg/kg) in patient treated with atg+csa for relapsing saa. signs of ebv-lpd included fever, pancytopenia, lymphadenopathy, splenomegaly, lymphocytosis, elevated igg. serological studies done prior exposure to atg were positive for latent ebv infection in all of them. all affected patients got same batch of rabbit-atg as a gvhd prophylaxis pre-transplant in three, as an is therapy in one. thirteen other transplanted patients got the same dose and batch of r-atg, which was followed with other unusual and otherwise less frequent complications (encephalitis in two, severe and relapsing bkv hemorrhagic cystitis in one, autoimmune cytopenia in two and unexplained anemia in two). transplant related mortality days after transplant in our cohort of patients who got the same batch of r-atg was extremely high ( %) compare to % trm over last two years in fully comparable cohort of patients receiving r-atg ( % alternative donors) transplanted at the same center gonzá lez-fraile, m.c. del cañ izo women with a median age of years ( - ) diagnosed of breast cancer were treated with autologous peripheral stem-cell support using two different conditioning regimens: in cases stamp-v regimen and, in patients stamp-i. the median number of mnc infused was . x /kg and a median of . x /kg cd + cells. granulocyte colony-stimulating factors were used in all patients febrile episodes were classified: ) microbiologically confirmed with bacteriemia in cases ( %), (gram-positive infections in cases ( . %), and only ( . %) gram-negative, ) microbiologically confirmed infections without bacteriemia in cases ( . %), with a similar proportion of gram-positive and gram-negative agents, ) clinically documented infections in cases ( . %), of which were pneumoniae, ) fever of unknown origin in ( %), % responded to first-line antimicrobial therapy aim:to present a fungal pcr assay for detection and identification of fungal pathogens in blood and bronchoalveolar lavage (=bal) number of patients analysed were six, ( males and females, ages ranged between months and years). all had received allogeneic stem cell transplantation because of all (n= ),scid (n= ), cml (n= ), nhl (n= ) and aml (n= ) ultrasound confirmed with typical hepato-splenic candida lesions. blood cultures were negative.one patient was positive for c.parapsilosis in pcr (we get our samples after first blood culture was positive). nine blood cultures were positive for c.parapsilosis. one patient was positive in bal for c.albicans in pcr. culture of bal confirmed c.albicans. one patient was positive for fungi in pcr (detected by electrophoresis only ,negative for candida and aspergillus spp. in elisa) days after blood culture revealed phichia etchellsii. conclusion: fungal pcr performed in blood and bal for detection and identification of fungal pathogens is a rapid and sensitive method ebv-infection/disease in allogeneic stem cell transplantation and cidofovir (cdf) treatment this study was a retrospective survey among ebv-infection/disease and the efficacy of cdf. cdf applications were evaluated. of cdf-treated pts tested concommitantely positive for cmv, for hhv and for vzv reactivation/infection. pt received previous antiviral therapy with ganciclovir and foscarnet, pts received cidofovir combined with foscarnet and ganciclovir. pts. received cdf as first line therapy. the dosage of cdf was - mg/kg/week. the duration was from application to times. all patients received probenecid and prehydratation. pts suffered from presumed side effects of cdf (vomiting and nausea) key: cord- -cug fnf authors: hollingshead, melinda g.; westbrook, louise; ross, martha j.; bailey, jean; qualls, k.jeanine; allen, lois b. title: an elisa system for evaluating antiretroviral activity against rauscher murine leukemia virus date: - - journal: antiviral res doi: . / - ( ) -i sha: doc_id: cord_uid: cug fnf a system for evaluating the activity of antiviral agents against rauscher murine leukemia virus (r-mulv) has been developed using an enzyme linked immunosorbent assay technique. the activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the uv-xc plaque reduction assay, which has been used historically for evaluating anti-r-mulv compounds. the assay is based upon detection of r-mulv encoded p protein production in virus infected murine cells. the assay reagents are readily available and the assay system is amenable to automated data collection systems. cytotoxicity evaluations are conducted in parallel to the rauscher mulv elisa assay in order to assess drug-induced reductions in cell viability. cytotoxicity evaluations are important to interpretation of the elisa results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. this system is less sensitive than the classical uv-xc plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay. rauscher murine leukemia virus (r-mulv) (rauscher, ) infection of mice has been used as a model for evaluating anticancer (chirigos et al., a; chirigos et al., b) and, more recently, anti-hiv compounds (ruprecht et al., ; ruprecht et al., ) . generally, in vitro assays are conducted prior to in vivo evaluations to assess the potential of the drugs to inhibit virusinduced disease. historically, these evaluations have been performed using the uv-xc plaque reduction assay (shannon et al., ) . this assay system is labor-intensive and requires experienced personnel to quantitate plaque formation. several workers have reported alternate assay systems for measuring mulv. generally these assays have relied upon immunofluorescence (decl ve et al., ) , in situ hybridization (rein et al., ) , purothionin a cytotoxicity (tahara et al., ) or radioimmunoassay (scolnick et al., ) for virus quantitations. more recently, focus forming assays have been described which use immunofluorescent (sitbon et al., ) or immunoperoxidase (nexo, ) labelling to aid in detection of mulvinduced foci. while these systems improve the visual counting efficiency of the assays, they are not amenable to automated data collection and analysis like the newer elisa methodologies have proven to be. thus, we have established an assay system which allows automated data collection. this system is similar in concept to assay systems described for quantitating antiviral activity or antiviral antibody responses to herpesviruses (rabalais et al., ) , dengue (fiqueiredo and shope, ) , bovine viral diarrhea (justewicz et al., ) and coronaviruses (smith and winograd, ) . for these assays, virus-infected monolayer cultures serve as the antigen source in an indirect elisa assay. multiwell ( ) culture plates were seeded with sc- cells (hartley and rowe, ) in eagle's minimum essential medium (emem) supplemented with % heat-inactivated fetal bovine serum (fbs), u penicillin/ml and #g streptomycin/ml [tissue culture medium]. following overnight incubation ( % co , °c) for cell adherence, the medium was removed and deae-dextran solution ( #g/ml in pbs) was added to each well. following incubation ( min) and deae-dextran removal, the wells were rinsed with phosphatebuffered saline (pbs). serial dilutions of the test compounds, prepared in tissue culture medium at . x the desired final concentration, were added to each of replicate wells ( #l/well). six virus control wells received # of tissue culture medium and cell control wells received # of tissue culture medium. each of the virus-challenged wells (test and virus controls)then received # of viral inoculum prepared at x the desired final concentration ( pfu/well). after days of incubation ( °c, % co ), the medium was aspirated from the plates and the wells were rinsed with pbs. the cells were fixed with cold ethanol:acetone ( : ) at °c for min. the fixative was decanted and the plates were allowed to air-dry overnight at room temperature (rt). for the elisa assay, the wells were blocked ( h, rt) with % fbs prepared in pbs. the primary antibody, consisting of a : dilution of goat anti-rauscher p antibody (nci/bcb repository), was added to each well and incubated for h at °c. the wells were rinsed times with wash solution ( . % tween- ® in pbs) and the second antibody, affinity-purified horseradish peroxidase-labelled anti-goat igg (tago immunologicals, burlingame, ca) was added. following incubation for h at °c, the wells were rinsed times with wash solution and , '-azino-di-[ -ethylbenzthiazoline sulfonate ( )] (abts/h ) substrate (kirkegaard and perry laboratories, inc., gaithersburg, md) was added. the optical density at nm (od o ) was determined spectrophotometrically on a microplate reader following incubation at °c for h. the average od was calculated for each set of replicates and for the virus and cell controls. the percent reduction in p protein was calculated as: the percent reduction was plotted and the % inhibitory concentration (ic ) was determined. the uv-xc plaque reduction assay has been described previously (shannon et al., ) . briefly, -well tissue culture plates were seeded with sc- cells in tissue culture medium. after overnight incubation ( °c, % co ), the medium was aspirated from the wells and ml of the drug solution ( . x ) and . ml of the virus inoculum were added to replicate ( ) wells. controls included cell controls (tissue culture medium only, no drug or virus) and virus controls (virus in medium, no drug). on day post-inoculation, the medium was decanted and the cultures were irradiated with ultraviolet light. each well then received xc cells (svoboda, ) . on day post-irradiation, the cultures were rinsed with pbs, fixed with °/ buffered formalin and stained with . o/ crystal violet. after air-drying, the plaques were enumerated with the aid of a dissection microscope. antiviral activity was expressed as the reduction in the mean number of plaques counted in the drug-treated, virus-infected cultures compared with the mean number of plaques in the untreated, virus-infected control cultures. the drug concentration which reduced the mean number of plaques by % (ic ) was calculated using regression analysis for semi-log curve fitting. cytotoxicity was determined in parallel by the mtt assay performed in well plates. cytotoxicity determinations were performed by a -( , -dimethylthiazol- yl)- , -diphenyltetrazolium bromide (mtt) dye reduction assay (mosmann, ) . for this, sc- cell monolayers were prepared in multiwell ( ) tissue culture plates in parallel to the elisa or uv-xc plates. for cytotoxicity assays performed in parallel to the elisa assay, the cells were pretreated with deae-dextran. the test compounds were added to each well at the desired final concentration in a volume of # . controls included cells incubated in tissue culture medium alone (cell controls) and color controls which contain no cells but received all the remaining reagents. the plates were incubated in parallel with the elisa ( days) or uv-xc ( days) plates. following incubation, each well received # of a mg/ml solution of mtt prepared in tissue culture medium. after a -h incubation at °c, /~ of a % sodium dodecyl sulfate (sds): . n hc solution were added to each well and the plates were incubated overnight at °c. the optical density at nm was determined spectrophotometrically on a microplate reader. the average od was calculated for each set of replicates and the percent of cell control was calculated for each drug dilution as: (mean drug treated od --color control od ) (mean cell control od -medium control od ) x these data were plotted in conjunction with the percent reduction in virus production. the minimum toxic concentration was defined as that concentration which reduced the cell viability to less than % of the cell control. a series of compounds have been assayed in the elisa and uv-xc systems. the icso and mtc results of compounds are presented in table . the results obtained with the two systems correlate well although the drug concentrations required to suppress p protein production, as measured in the elisa system, are generally higher than those required to suppress uv-xc plaque formation. one compound, diarylsulfone , demonstrated antiviral activity in the uv-xc plaque reduction assay; however, no activity was detected by the p protein reduction assay (elisa). this compound was inactive in a virus yield reduction assay (data not shown) thus, the elisa assay may be more indicative of the activity for some compounds. in the case of this compound, the activity detected by the uv-xc plaque reduction assay was believed to result from cytotoxicity to the xc cells used as an overlay. graphic presentation of the uv-xc and elisa data for ' ' dideoxythymidine (azt) is shown in fig. . reductions in virus production, either as percent plaque reduction or as percent reduction in p protein, are graphed on the left y-axis as the virus-infected cells. cytotoxicity, presented as the percent of cell control, is graphed on the right y-axis as the toxicity control cells. data presented in this form is readily interpreted visually without the need for extensive analysis of the numerical values. we have assayed the positive control drug, azt, multiple times. the average ids for azt in these assays ( ) was . #m with a median ics of . ~m and a standard deviation of . . we feel that this indicates that the results are reproducible from assay to assay. the primary difference between the-elisa assay and the uv-xc plaque reduction assay is in the drug concentrations required to suppress the virus production endpoint. the uv-xc plaque reduction assay measures the capacity of an antiviral compound to reduce production of infectious virus. when rat xc cells are placed into contact with mulv-infected cells, syncytia are formed. when these xc cells are overlaid onto mulv-infected sc- cell monolayers, they fail to infiltrate the sc- cell sheet in the mulv-infected areas resulting in lightly stained areas (plaques). mulv infected sc- cells that are killed by exposure to uv light are still capable of inducing syncytia. the xc cell overlay grows on the dying sc- cells as a uniform monolayer except where mulv infection is present. in these areas, there are 'holes' in the xc cell monolayer which contain multiple giant cells or focal masses of giant cells. production of these syncytia is presumably a function of the gp glycoprotein as typified by other retroviruses (i.e. gp in hiv). in contrast, the elisa system measures the capacity of a compound to reduce production of the virus protein, p , which forms part of the virion core similar to the p protein in hiv. this protein is encoded by the viral nucleic acid. reductions in p protein production will occur if a compound interferes with translation of the viral nucleic acid. thus, compounds which interfere with virus attachment, penetration, integration, transcription and translation will reduce p protein production. a compound which interferes with late stages in the virus production cycle (i.e. virus budding) would not necessarily reduce p protein levels. the only cases in which the elisa assay does not give results compatible with those found in the uv-xc assay are: ( ) assays of compounds which have toxic effects on the xc cells and ( ) assays of compounds which are active late in the virus replication cycle or which act by interfering with the infectivity of the progeny virus so that p protein is produced but the progeny virus is unable to infect new target cells• while the system described here is not as sensitive to antiviral drugs as the uv-xc plaque reduction assay, it does offer an alternative method for antiviral drug evaluations. we have found compounds that produce toxicity to the xc cell overlay which could be evaluated in this test system. also, smaller quantities of compound are required for the elisa since it is performed in well plates rather than the -well plates used in the uv-xc plaque reduction assay. we have used this system to evaluate analogs of known positive compounds for selecting the optimal drug for in vivo testing. further, the system can be modified at various steps including ( ) increasing the virus replication phase (i.e., -day incubation), ( ) altering the virus inoculum dose, ( ) altering the elisa reagent concentrations and incubation times and ( ) collecting supernatants for yield reduction assays. thus, this system can be adjusted to meet the desired goals. studies with the murine leukemogenic rauscher virus. i. chemotherapy studies with in vivo and in vitro assay systems studies with the murine leukemogenic rauscher virus. ii. chemotherapy of virus-induced lymphoid leukemia an improved murine leukemia virus immunofluorescence assay an enzyme immunoassay for dengue antibody using infected cultured mosquito cells as antigen clonal cell lines from a feral mouse embryo which lack hostrange restrictions for murine leukemia viruses bovine viral diarrhea virusinfected mdbk monolayer as antigen in enzyme-linked immunosorbent assay (elisa) for the measurement of antibodies in bovine sera rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays a plaque assay for murine leukemia virus using enzyme-coupled antibodies a virus-induced disease of mice characterized by erythrocytopoiesis and lymphoid leukemia in situ hybridization: general infectivity assay for retroviruses rapid herpes simplex virus susceptibility testing using an enzyme-linked immunosorbent assay performed in situ on fixed virus-infected monolayers suppression of retroviral propagation and disease by suramin in murine systems suppression of mouse viraemia and retroviral disease by '-azido- '-deoxythymidine radioimmunoassay of mammalian type c viral proteins i. species-specific reactions ofmurine and feline viruses inhibition of gross leukemia virus-induced plaque formation in xc cells by -deazauridine use of a focal immunofluorescence assay on live cells for quantitation of retroviruses: distinction of host range classes in virus mixtures and biological cloning of dual-tropic murine leukemia viruses two enzyme immunoassays for the detection of antibody to rodent coronaviruses a new method for titration of murine leukemia virus using purothionin a this work was supported by contract n -cm - from the national cancer institute. key: cord- - vr cm s authors: nguyen, n. n.; mutnal, m. b.; gomez, r. r.; pham, h. n.; nguyen, l. t.; koss, w.; rao, a.; arroliga, a. c.; wang, l.; wang, d.; hua, y.; powell, p. r.; chen, l.; mccormack, c.; linz, w. j.; mohammad, a. a. title: correlation of elisa based with random access serologic immunoassays for identifying adaptive immune response to sars-cov- date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: vr cm s public health emergency of sars-cov- has facilitated diagnostic testing as a related medical countermeasure against covid- outbreak. numerous serologic antibody tests have become available through an expedited federal emergency use only process. this paper highlights the analytical characteristic of an elisa based assay by anshlabs and three random access immunoassay (raia) by diasorin, roche, and abbott that have been approved for emergency use authorization (eua), at a tertiary academic center in a low disease-prevalence area. the anshlabs gave higher estimates of sero-prevalence, over the three raia methods. for positive results, anshlabs had . % and % concordance with diasorin or abbott and roche respectively. for negative results, anshlabs had . % and . % concordance with diasorin and roche or abbott respectively. all discrepant samples that were positive by anshlabs and negative by raia tested positive by all-in-one step sars-cov- total (cov t) assay performed on the automated siemens advia centaur xpt analyzer. none of these methods, however, are useful in early diagnosis of sarscov- . the sars-cov- virus outbreak that began in late in wuhan, has a mortality rate of approximately . % worldwide [ ] [ ] [ ] . diagnostic testing is necessary for identifying and isolating infected individuals to limit spread of disease. molecular testing such as reverse-transcriptase polymerase chain reaction (rtpcr) detects active infection; and serology testing helps identify those who were previously infected (including asymptomatic infections) and have recovered [ , ] . nucleic acid detection using rtpcr has become the confirmation test, due to its % specificity and - % sensitivity within days of exposure [ ] but is faced with numerous supply challenges [ ] . the united states food and drug administration (fda) issued an emergency use authorization (eua) approval for antibody testing as complementary to rtpcr, leading to an explosion of new antibody methods, including rapid diagnostic test (rdt), enzyme-linked immunosorbent assay (elisa), virus neutralization assay (vna), and chemiluminescent immunoassay one hundred and fifty-two serum samples were from patients who tested negative by rtpcr, of these were collected on same day as rtpcr testing. for the remaining samples, the interval between rtpcr and sample collection ranged from - days. to avoid degradation, the specimens were tested by four methodologies within - h of each other. only samples having sufficient serum volume and rtpcr test results were included in the evaluation project. table summarizes the characteristics of the four serologic assays we investigated. peroxidase. after a second incubation and washing step, the wells are incubated with the substrate tetramethylbenzidine (tmb) chromogen solution to induce color change. an acidic stopping solution is all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . https://doi.org/ . added and the degree of enzymatic turnover of the substrate is determined by wavelength absorbance measurement, with nm as the primary filter and nm as the reference filter. the intensity of color change corresponds to arbitrary units of antibody-antigen complex concentration present in the specimen. the analyzer calculates antibody concentration in arbitrary concentration units (au/ml). samples with au/ml of > , - , and < are considered positive, indeterminate and negative for igg respectively. it is the only test that uses a three-point calibration curve. the sensitivity and specificity are . % and . % respectively [ ] . the abbott sars-cov- igg assay was run on the abbott architect i sr analyzer that measures igg antibodies to the nucleocapsid protein. the automated, two-step immunoassay uses chemiluminescent microparticle immunoassay (cmia) technology for qualitative detection of igg antibodies in human serum. the sample, sars-cov- antigen-coated paramagnetic microparticles, and diluent are combined and incubated. the antibodies bind to the antigen-coated microparticles. the mixture is washed and anti-human igg acridinium-labeled conjugate is added. following incubation, the pre-trigger is added. the resulting chemiluminescent reaction is measured as a relative light unit (rlu). the presence or absence of igg antibodies is determined by dividing the sample rlu by the stored calibrator rlu to find the igg assay index (s/c), with a positive cutoff of ≥ . . the sensitivity and specificity are % and . % respectively at ≥ days post onset of symptoms [ ] . the liaison sars-cov- s /s igg is a chemiluminescent immunoassay (clia) for detection of anti- s and anti-s spike glycoprotein specific to sars-cov- in human serum or plasma on the diasorin xl analyzer (stillwater, mn). specimen, calibrator, control, coated magnetic particles and diluent are incubated in reaction cuvettes. the antibodies bind to the solid phase through the recombinant s and s antigens. a second incubation links recombinant s and s antigens to an isoluminol-antibody conjugate. the starter reagents are then added, and a flash chemiluminescence reaction induced. the light signal, and hence the amount of isoluminol-antibody conjugate, is measured by a photomultiplier and result converted to arbitrary concentration, au/ml. samples with au/ml of ≥ are considered positive for all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint igg antibodies. the sensitivity and specificity are - % and % respectively ≥ days post onset of symptoms [ ] . the elecsys anti-sars-cov- assay is performed on the roche cobas e analyzer for total antibodies specific for igg, igm and iga which target nucleocapsid protein, in human serum or plasma. in order to rule out non-specific binding, samples that tested positive by elisa assay were diluted using sample diluent provided in the anshlabs assay kit. we made and reran samples for a : , : and : dilution, and calculated percent recovery. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . the specificities of the validated in-house anshlabs sars-cov- -igg and igm are listed in table . the cross reactivity to anti-influenza b igg ( samples), anti-respiratory syncytial virus igg ( samples were positive for these analytes. no cross-reactivity was noted for either sars-cov- -igg or igm. the clinical sensitivity and specificity using rtpcr results as the gold standard were found to be . and . % respectively. all samples used for the sensitivity and specificity evaluation were collected from symptomatic patients, either hospitalized inpatients or treated in emergency department. the interval between rtpcr confirmation and serology testing ranged from - days. the specificity toward common cold coronavirus is shown in table . three of prepandemic samples tested positive for igg by elisa and none tested positive by raia methods, thereby giving a calculated specificity of % and % for elisa and raia respectively. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint table shows the concordance between elisa and raia results for samples that were confirmed positive for sars-cov- by rtpcr. these samples were collected from symptomatic patients > days post rtpcr confirmation. elisa assay correlated best with total antibody assay on roche elecsys e analyzer. this could possibly be attributed to the measurement of igg antibodies directed towards multiple antigenic proteins (nucleocapsid & spike) by elisa or measurement of total antibodies (igg, igm, and iga) on roche elecsys e analyzer. table . concordance of rtpcr positive samples between a) elisa and raia systems and b) among three raia platforms anshlabs igg vs architect i . % anshlabs igg vs elecsys e % all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . the concordance of elisa and raia results with rtpcr is shown in table . all patient tested positive by rtpcr also tested positive by elisa and elecsys e total antibody. architect i igg and liaison xl were unable to detect antibodies in one sample. all raia methodologies showed high correlation with nucleic acid test for patient samples that tested negative by rtpcr, with all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . the non-specific binding dilution data of the anshlabs assay showed five samples with various concentration levels of igg were serial diluted to : , : : : and : . all samples gave a consistent dilution pattern and expected - % recovery of neat sample in au/ml units (fig ) . all raia methods correlated well with elisa and rtpcr for samples collected > days post rtpcr confirmation. there were no significant differences among the methods which tested for igg targeted to one or both nucleocapsid and spike proteins, or tested for total antibodies. elisa detected higher sero-prevalence in rtpcr negative samples than the raia methods. this may be due to i) higher analytical sensitivity or a lower cutoff by elisa, which triggered more positive results; ii) cross reactivity to other coronavirus; iii) non-specific binding of other antibodies, for example autoimmune antibodies or deposition of detection antibody on the microtiter well which led to increased absorbance causing false positives all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . https://doi.org/ . elisa assays are generally known for low detection limits in sub ng/ml to low pg/ml because of their increased incubation time thereby allowing antigen-antibody to reach reaction equilibrium and extra washing steps [ , ] . the dynex dsx analyzer used for elisa assay provided optimization flexibility and automation, which is not available on raia due to throughput constraint. cross-reactivity to other coronavirus was evaluated by testing prepandemic samples and found to be % and % for elisa and raia respectively. the differences in cross-reactivity may account for one or two false positive results, but not for all and positives picked up by elisa. non-specific deposition of other antibodies in patient samples or detection antibody was ruled out by dilution studies for elisa. recovery of - % ruled out non-specific binding as a possible cause for false positives (fig ) . the difference in results for positive and negative samples by raia methods may also be due to a higher threshold for positivity. the rtpcr assay is used as the gold standard in maximizing analytical sensitivity and specificity during method development which is the most accurate in the early days of the infection when antibody development is low and results in the reported sensitivity of - % on samples collected < days post rtpcr confirmation [ ] [ ] [ ] . we believe that higher rate of positivity observed for elisa i.e. versus by architect, by liaison was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . https://doi.org/ . our quality assurance project has some notable limitations. at this stage of the disease, true clinical sensitivity and specificity for different methodologies is difficult to determine because of our limited understanding of the disease process and kinetics. secondly, our assumption that elisa has better limits of detection is based on circumstantial evidence, as certified standards quantifying limits of detection on different platforms are not available. third, the cutoffs provided by manufacturers were relied on which may not have undergone extensive validation. establishing laboratory specific cut-off is akin to establishing reference ranges, which is highly dependent on prevalence of disease in local population. all of the assays we investigated would work well for epidemiological sero-prevalence studies. among rtpcr negative patients, elisa gave higher estimates of sero-prevalence in our dataset and would probably do so in population-based epidemiological surveys using serological testing. raia methods could however offer other advantages over elisa which includes i) faster turnaround time; ii) random access to allow immediate testing; iii) longer calibration stability, obviating the need to perform daily calibration as required by elisa; iv) the ability to perform other immunoassay testing concurrently; and v) higher test throughput and walk away capabilities. however in conclusion, no serological method tested has sensitivity and specificity greater than or equal to % for one to days post exposure, limiting their use in early diagnosis. the authors would like to thank ms. briget da graca for editorial comments and manuscript revision. we also acknowledge ms. laura gonazales and her team from health texas provider network (dallas, tx) for correlation of anshlabs results using siemens centaur total antibody assay. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint non-specific binding in anshlabs elisa assay all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint novel coronavirus in the united states . world health organization. who coronavirus disease (covid- ) dashboard the new england journal of medicine specific antibody responses in covid- patients antibody responses to sars-cov- in patients of novel coronavirus disease infectious diseases society of america recent advances and perspectives of nucleic acid detection for coronavirus european centre for disease prevention and control. novel coronavirus disease (covid- ) pandemic: increased transmission in the eu/eea and the uk -sixth update - evaluation of nine commercial sars-cov- immunoassays neutralizing antibody responses to sars- korean center for disease control and prevention. findings from investigation and analysis of re-positive cases sars-cov igg elisa il- ivd use for dynex analyzer . diasorin. liaison® sars-cov- s /s igg cov- seroconversion in humans: a detailed protocol for a serological assay current protocols in microbiology predicting detection limits of enzyme-linked elisa) and bioanalytical techniques in general clinical performance of two sars-cov- serologic assays clinical performance of the roche sars-cov- serologic assay comparison and development of two different solid phase chemiluminescence elisa or the determination of albumin in urine key: cord- -k uvqcmp authors: xia, hongyan; liu, lihong; nordengrahn, ann; kiss, istván; merza, malik; eriksson, ronnie; blomberg, jonas; belák, sándor title: a microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k uvqcmp this study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (bvdv). the intra- and inter-assay variability are . % and less than %, respectively, and variability of bead conjugations is less than . %. the diagnostic performance of the assay was evaluated by testing a total of serum samples. based on a negative/positive cut-off value of . %, the assay has a sensitivity of . % and a specificity of . % relative to elisa. the new microsphere immunoassay provides an alternative to conventional elisa systems and can be used for high-throughput screening in the bvd control programmes. the genus pestivirus of the family flaviviridae consists of four approved species: bovine viral diarrhoea virus (bvdv- ), bovine viral diarrhoea virus (bvdv- ), classical swine fever virus (csfv), border disease virus (bdv); and a tentative species pestivirus of giraffe (thiel et al., ) . bvdv infections are usually very mild or inapparent clinically (baker, ) , but severe hemorrhagic syndrome is also observed in acute infection (perdrizet et al., ; corapi et al., ; ridpath et al., ) . diaplacental infection can lead to birth of persistently infected animals that serve as a reservoir for further spreading of the virus (sandvik, ) . bvd is considered as one of the major diseases with a worldwide economic impact in the cattle industry. for example, the cost of bvdv infection has been estimated about $ per cow in dairy herd in new zealand (reichel et al., ) , and may exceed $ per cow when infected with a high virulent strain or co-infected with other pathogens (pritchard et al., ; carman et al., ; houe, ) . sweden is one of the first countries to implement national bvdveradication programme since (lindberg and alenius, ) , and now the country is almost free of bvdv (ståhl et al., ; lindberg et al., ) . serological tests, including virus neutralization and elisa, have been used widely for detection of bvdv antibodies. the recent advance in microsphere-based flow cytometric technology has provided the possibility to develop multiplex diagnostic assays on a single platform. these assays can be designed more sensitive than conventional immunoassays due to the uses of small beads ( m) that leads to better reaction kinetics approaching liquid-phase conditions, and of chromophore phycoerythrin, an exceptionally bright reporter dye (krishhan et al., ) . the microspheres used in luminex xmap technology (luminex corp., austin, tx) are coded with unique combinations of fluorescent dyes, and can be immobilized with capture molecules, e.g. antibody. immunoassays can be developed in a similar way as elisa, but signals are detected and processed by luminex analyzer. by using xmap technology, a range of diagnostic assays has been developed in the recent years for the improved serological detection of viruses, e.g. respiratory syncytial virus that causes maladies (jones et al., ) , human immunodeficiency virus (faucher et al., ) , human papillomaviruses (dias et al., ) , equine arteritis virus (go et al., ) , and avian influenza virus (watson et al., ) . the objectives of this study were to develop a blocking microsphere-based immunoassay (bmia) for detection of antibodies against bvdv, and to compare the performance of the assay with a commercial elisa kit. a total of serum samples were evaluated in this study. these included clinical samples from sweden, where only bvdv- is present; and samples (including sera against bvdv- ) from svanova biotech ab, uppsala, sweden. bovine turbinate (tb) cells were maintained in f-dmem with % foetal calf serum, % l-glutamine, and % antibiotics. cells at % confluence were infected with oregon c v (kindly provided by prof. martin beer, institute of diagnostic virology, friedrich-loeffler-institut (fli), greifswald-insel riems, germany). at h post-infection, cultures were frozen at − • c. after thawing, . % nonidet p- (np- ) was added to the cell lysate and incubated at • c for h. following centrifugation at × g for min, the supernatant was taken as viral antigen. microplex microspheres were purchased from luminex corp (austin, tx). the coupling reaction was performed according to the manufacturer's instructions. briefly, million microspheres were resuspended in l of activation buffer ( mm monobasic sodium phosphate, ph . ). the microspheres were activated by l of mg/ml of n-hydroxysulfosuccinate (sulfo-nhs, pierce, rockford, il), followed by l of mg/ml -ethyl- - -dimethylaminopropyl carbodiimide (edc, pierce, rockford, il). after incubation at room temperature on an end-over-end rotator for min, the microspheres were washed twice with l of mm morpholineethanesulfonic acid (mes, ph . ) and resuspended in l of mm mes. two mabs wb and wb , which were described previously in blocking elisa (paton et al., ; kramps et al., ) , were added to the activated microspheres, respectively. after a -h incubation, the microspheres were washed twice by pbs-tbn (pbs, . % bsa, . % tween- , . % azide, ph . ) and resuspended in l of the buffer pbs-tbn and stored at • c in dark. the coupling reaction was confirmed by measuring fluorescent intensity on a luminex analyzer after incubation of microspheres with twofold serial dilutions ( . - g/ml) of r-phycoerythrin conjugated anti-mouse igg (sigma-aldrich co, st. louis, mo) for min. biotin labelling reaction was performed according to the manufacturer's instruction (pierce, rockford, il). briefly, g of wb or wb was incubated on ice with l of mm sulfo-nhs-lc-biotin for h. after removal of excess biotin using a desalting column, the biotinylated mabs were stored at − • c until use. the wb -conjugated microspheres ( microspheres/l), viral antigen, and diluted serum samples were mixed in equal volume ( l) and incubated for min on a plate shaker in dark. after addition of l of the biotinylated mab wb and incubation for another min, l of g/ml of r-phycoerythrin-conjugated streptavidin (prozyme, san leandro, ca) were added and followed by a further incubation for min. each sample was tested in duplicate if not indicated specifically. fluorescence intensity of each reaction was measure on the luminex analyzer, and median fluorescence intensity (mfi) was calculated based on the measurement of beads per sample. the results were expressed as inhibition percentage and calculated as the following: × (mfi negative control − mfi sample )/mfi negative control . the reproducibility of the assay was assessed by determining the level of both intra-assay and inter-assay variations. intra-assay variation within a plate was calculated as the mean percentage of coefficient of variation (cv%) for samples determined in triplicate. inter-assay variation was assessed by testing a panel of samples ( positive sera and negative sera) in three separate runs. elisa was performed with a p blocking elisa kit (svanova biotech ab, uppsala, sweden), according to the manufacturer's instruction. the kit had a sensitivity of . % and specificity of . % compared with virus neutralization test (svanova biotech ab, uppsala, sweden). receiver operating characteristics (roc) curve was generated to assess the diagnostic performance of the bmia. an roc curve is a plot of a test's true positive fractions (tpf) versus falsepositive fractions (fpf) for each possible cut-off value of the test and could achieve the best relationship between diagnostic sensitivity and specificity (detilleux et al., ; greiner and gardner, ) . sensitivity and specificity were calculated as the following formula: sensitivity = [true positives/(true positives + false negatives)] × ; specificity = true negatives/(true negatives + false positives) × . the analysis was performed by software medcalc (medcalc software, mariakerke, belgium). the coupling efficiency of each mab to microspheres was compared at different amount of mabs in coupling reaction with dilutions of a phycoerythrin-labelled anti-mouse igg (pe-igg). as shown in fig. , both wb and wb had a very low mfi value when g of mab was used in coupling reaction. at g/ml of pe-igg, g of wb gave an mfi value of , whereas wb gave an mfi value of . this indicated that, for the same amount of mab, wb had a higher coupling efficiency than wb . further increasing wb from g to g had less effect on the mfi values. therefore, wb ( g) was selected finally as the capture antibody for coupling to microspheres, and wb was used as detection antibody in this study. two key factors in the bmia were determined in a sandwich immunoassay: optimal serum dilution factor and the amount of detection antibody. a twofold dilution of serum sample gave the maximum difference in mfi values between positive and negative sera (fig. ) . titration of biotinylated detection antibody (wb bio) showed that the mfi values reached the plateau at about g/ml of wb -bio (fig. ) . to maximize the sensitivity of the assay, g/ml of wb were used in the assay, which corresponds to % of the maximum plateau mfi value. sensitivity and specificity of the bmia were compared with a commercial blocking elisa. a total of samples were tested in parallel by the two assays, and the results are presented in table . based on the nonparametric roc analysis of all samples, the cut-off value (percentage of inhibition) of the bmia was determined as . %. under this cut-off value, the bmia classified samples as positive (including bvdv- serum samples) and samples as negative (table ). comparing to blocking elisa, the bmia had a sensitivity of . % and a specificity of . %. fig. . selection of capture antibody according to its coupling efficiency. two mabs wb and wb at indicated amounts were coupled to million microspheres in a -l reaction volume, respectively. the median florescence intensity (mfi) was determined at various dilutions of anti-mouse igg-pe conjugate. the reproducibility of the bmia was evaluated by intra-and inter-assay variability, and variations in different preparations of conjugated microspheres. the mean cv% of samples tested in triplicate in one plate was . %. inter-assay variation was determined by testing samples in three separate runs. the mean cv% was below %. the reproducibility of the different preparations of conjugated microspheres was also investigated. the percentage of coefficient of variation (%cv) within three preparations of beads was less than . %. this study describes the development and evaluation of a microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies. the diagnostic performance of the new bmia was compared to that of a commercial blocking elisa system, by testing a large panel of bovine sera. in general, the two assays worked consistently for detection of bvdv antibodies in samples. six samples were positive in bmia but negative in elisa. these samples had a percentage of inhibition value between % and % in blocking elisa. one reason for this discrepancy could be that these sera blocked binding of the viral antigen to the detection antibody in the bmia, leading to false-positive results. but more likely it was due to a slight higher sensitivity of the mia than elisa. only one sample was negative by the bmia but positive by elisa with a percentage of inhibition value of . % (just above the cut-off value of %). the observed discrepancies are in line with another comparison of elisa with a luminex assay for detection of human papillomavirus : six out of samples reacted differently in the two assays (faust et al., in press) . the bmia utilises two mabs against highly conserved ns protein of bvdv. as demonstrated, the assay is capable of detection antibodies against both bvdv- and bvdv- . it would be very interesting to detect antibodies, when available, against novel pestiviruses th/ khonkaen (liu et al., a) and sva/cont- (liu et al., b) , which have been proposed as a new species bvdv- (liu et al., c) . another direction would be to extend the assay for detection of antibodies against other viruses, e.g. bovine herpesvirus type , bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza- virus, thus allowing a robust diagnosis of bovine respiratory diseases in a multiplex format. the power of a luminex assay lies in its multiplex capacity. khan et al. ( ) reported a multiplex microbead immunoassay for simultaneous detection of antibodies against six nonhuman-primate viruses. multiplexing can bring down the high cost of the bmia dramatically by reducing the number of elisa kit for each pathogen, labour input and hands-on time. this will, in turn increase the applicability of the assay. in summary, the bmia described above is an alternative to elisa for detection of antibodies against bvdv. the assay is reliable and sensitive, providing a powerful tool for the bvd surveillance programmes. in addition, this assay can be extended to a multiplex format allowing a robust, high-throughput diagnosis of bovine respiratory diseases. bovine viral diarrhea virus: a review severe acute bovine viral diarrhea in ontario severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus methods for estimating areas under receiver-operating characteristic curves: illustration with somatic-cell scores in subclinical intramammary infections optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses , , , and protein bead array for the detection of hiv- antibodies from fresh plasma and dried-blood-spot specimens validation of multiplexed human papillomavirus serology using pseudovirions bound to heparin coated beads development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test application of diagnostic tests in veterinary epidemiologic studies economic impact of bvdv infection in dairies multiplex assay for detection of strain-specific antibodies against the two variable regions of the g protein of respiratory syncytial virus simultaneous detection of antibodies to six nonhuman-primate viruses by multiplex microbead immunoassay a simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (bvdv) specific antibodies in cattle serum, plasma and bulk milk multiplexed microbead immunoassays by flow cytometry for molecular profiling: basic concepts and proteomics applications the control of bovine viral diarrhoea virus in europe: today and in the future principles for eradication of bovine viral diarrhoea virus (bvdv) infections in cattle populations virus recovery and full-length sequence analysis of the atypical bovine pestivirus th/ khonkaen maximum likelihood and bayesian analyses of a combined nucleotide sequence dataset for genetic characterization of a novel pestivirus sva/cont- phylogeny, classification and evolutionary insights into pestiviruses an elisa detecting antibody to conserved pestivirus epitopes bovine virus diarrheaclinical syndromes in dairy herds severe disease in a dairy herd associated with acute infection with bovine virus diarrhoea virus, leptospira harjo and coxiella burnetii does control of bovine viral diarrhoea infection make economic sense? multiple outbreaks of severe acute bvdv in north america occurring between and linked to the same bvdv strain selection and use of laboratory diagnostic assays in bvd control programmes molecular epidemiology of bovine viral diarrhoea during the final phase of the swedish bvd-eradication programme family flaviviridae a multiplexed immunoassay for detection of antibodies against avian influenza virus we thank ms. pia fällgren, the national veterinary institute (sva), uppsala, sweden for providing the serum samples. we are grateful to dr. siamak zohari (sva) for his invaluable comments. the work was supported by the award of excellence (excellensbidrag) provided to sb by the swedish university of agricultural sciences (slu). key: cord- -p pns r authors: malik, yashpal singh; verma, atul; kumar, naveen; deol, pallavi; kumar, deepak; ghosh, souvik; dhama, kuldeep title: biotechnological innovations in farm and pet animal disease diagnosis date: - - journal: genomics and biotechnological advances in veterinary, poultry, and fisheries doi: . /b - - - - . - sha: doc_id: cord_uid: p pns r the application of innovative diagnostic technologies for the detection of animal pathogens at an early stage is essential in restricting the economic loss incurred due to emerging infectious animal diseases. the desirable characteristics of such diagnostic methods are easy to use, cost-effective, highly sensitive, and specific, coupled with the high-throughput detection capabilities. the enzyme-linked immunosorbent assay (elisa) and polymerase chain reaction (pcr) are still the most common assays used for the detection of animal pathogens across the globe. however, utilizing the principles of elisa and pcr, several serological and molecular technologies have been developed to achieve higher sensitivity, rapid, and point-of-care (poc) detection such as lateral flow assays, biosensors, loop-mediated isothermal amplification, recombinase polymerase amplification, and molecular platforms for field-level detection of animal pathogens. furthermore, animal disease diagnostics need to be updated regularly to capture new, emerging and divergent infectious pathogens, and biotechnological innovations are helpful in fulfilling the rising demand for such diagnostics for the welfare of the society. therefore, this chapter primarily describes and discusses in detail the serological, molecular, novel high-throughput, and poc assays to detect pathogens affecting farm and companion animals. livestock, poultry, and aquaculture are among the fastest growing and expanding agriculture sectors to fulfill the need of the growing population of humans. however, the growth in this sector is under the continuous increasing threats of infectious diseases worldwide. this menace is further aggravated by globalization in animal trade for various purposes. the sudden entry of an infectious disease in a new country or geographical location could lead to delayed diagnosis and rapid spread into the susceptible animal population. in response to climate change, vector-borne diseases are also increasing worldwide. to prevent the spread of infectious diseases, one of the basic and critical requirements as prescribed by the world organization of animal health (oie) is the application of rapid, accurate, and highly sensitive identification of infectious agents. though the term "biotechnology" was coined in the year by karl ereky, the tangible biotechnological advancements in improving the human and animal health were started in the late th century. since then, biotechnological applications have been making significant contributions in the development of novel powerful diagnostic assays for the efficient diagnosis and control of animal infectious diseases. importantly, biotechnology has made the availability of pen-side tests for use at the field level to detect the causative infectious agent during a disease outbreak. in this chapter, we primarily describe and discuss the innovative biotechnological advancements made in the animal disease diagnosis in a step-wise manner. the impact of infectious diseases is immense and is felt all across the world. infectious diseases have affected the whole society, economy, and political system. vital sectors are under continuous economic loss and unrelenting development. the infectious diseases have taken a huge physical toll on animals and humans. this has pressed on humanity and has caused substantial economic, social, and mental losses. thus, it is a matter of animal health and economic interest to invest in strategies to give a blow to infectious diseases and put them under control. elimination of the pathogens and/or their vectors from their natural reservoirs would always be a first thought, but the removal is not easy, as they are constantly emerging and it is always very difficult to predict the emergence of infectious agents. evolution of pathogens is putting extra challenges, pressing on humanity to look at the new strategies and forcing the researchers to look for innovative ones. the newly evolved pathogens are always more advanced and deadly from the previous ones and put up a strong resistance. "from the evolutionary perspective, they [viruses and bacteria] are 'the fittest' and the chances are slim that human ingenuity will ever get the better of them." with the increase in the knowledge of infectious diseases and science, the degree of pace in pathogen discovery has increased. to keep pace and for better diagnosis, new tools and techniques need to keep on evolving. this is not only to quickly detect the pathogens, but also to make predictions with probable and possible outbreaks. to understand the scenario and to reach a definite conclusion, knowledge of epidemiology and pathogenic etiology also needs to be studied. this will facilitate in understanding the ancestry of the pathogen, and provide an insight and a mechanism for the epidemic, endemic, and even pandemic outbreaks. this system will also assist in understanding the interface transmission between and the directional flow of the zoonotic infectious diseases. thus, phylogenetic analysis and epidemiology would aim toward strategizing the challenges during pathogen surveillance and discovery. sars, a coronavirus was pandemic in . but epidemiology and microbiology mediated to stem its disastrous results and also the causative agent of sars was identified. bacteria, viruses, and parasites, present in feces, contaminate foodstuffs and cause disease in humans and animals, affecting the social set up and consumer demands. to increase the productivity and for the maintenance of good health of animals, antibiotics are frequently administered resulting in the growth and emergence of antibiotic-resistant bacteria. this further aggravates the condition, and makes the situation more appalling. overseas imported pets were also found to transmit and carry over the diseases to humans (smith et al., ) . even aquaculture is at risk of contaminants with the virus directly affecting the marine lives, as in the case of the new virus discovered in salmon (finstad et al., ) . even honeybees and other pollinators are transmitting pathogens like fungi, bacteria, and viruses through the contaminated food items (cox-foster et al., ) . an array of classical and conventional techniques have been developed and used for the laboratory diagnosis of infectious agents or pathogens. the techniques include serological, cell culture, and electron microscopyÀbased methods, which are either time-consuming or labor-intensive or both. however, with the advancement in the biotechnology field, new and robust diagnostic techniques are continuously evolving and taking over the conventional methods (caliendo et al., ) . presently, molecular detection-based methods such as polymerase chain reaction (pcr) or its variants, and serological methods such as enzyme-linked immunosorbent assay (elisa), are being used worldwide for the accurate diagnosis of many animal diseases. however, point-of-care (poc) and high-throughput novel assays have been developed recently. furthermore, we discuss the pros and cons of frequently used diagnostics assays for animal diseases in accordance with the following sections: serological methods were introduced in the early s for the diagnosis of pathogens. various serological diagnostics have been developed such as complement fixation, counter-immunoelectrophoresis, immunofluorescence in cell culture, elisa, radio immunoassay, immune adherence haemagglutination assay, reverse passive hemagglutination assay, latex agglutination (la), chemiluminescent immunoassay, and immunochromatography test (ict). among these, ict and elisa especially sandwich elisa and competitive elisa are used frequently in the commercial diagnostic kits for animal diseases worldwide. ict assays mostly utilized mammalian igg in commercial diagnostic kits, however, avian igy antibodies, with added advantages over the mammalian igg, have been employed for the detection of norovirus, rotavirus, and astrovirus in the fecal samples with good sensitivity and specificity ranging between % and % (khamrin et al., ) . modifications in elisa format or combinations with other diagnostic methods have proved novel ways to detect pathogens more efficiently and accurately. for example, recently a novel elisa for the detection of group a rotavirus antigen in the fecal samples of multiple host species has been developed . this assay utilizes the potential use of synthetic peptides and is based on the detection of conserved vp protein using anti-recombinant vp antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immune-dominant epitopes within vp protein) antibodies as detector antibodies. another assay, which is simple to perform without the requirement of laboratory facilities, is dot-elisa. a highly sensitive and specific dot-blot assays for rapid detection of staphylococcal enterotoxin-a in food has been reported (singh et al., ) . dot-elisa has been employed in diagnosing various important poultry diseases (alam et al., ; dhama et al., ; he et al., ; majumder et al., ; manoharan et al., ) . immuno-pcr is another powerful assay that has been used for the immunodetection of viral nucleic acids. by combining elisa with pcr, sensitivity of detection can be increased up to times and is especially useful in detecting low quantity viruses in the stool samples (bonot et al., ) . the major advantage of immune-pcr is that several viral nucleic acids can be detected simultaneously. recently, a combination of nanoparticles with the immuno-pcr, also known as nanoparticle amplified immune pcr (npa-ipcr), has been reported which increases the sensitivity -folds compared to elisa and several folds to rt-pcr. antigen detection using an antibody bound to gold nanoparticle cofunctionalized with thiolated dna complementary to a hybridized dna has been developed (perez et al., ) . here, the presence of antigen/virus particles activates the formation of a "sandwich" complex of gold nanoparticle construct, virus, and an antibody functionalized nanoparticles used for extraction. now, this complex is heated to c, thus releasing dna tags followed by the detection through real-time pcr. npa-ipcr offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection. nucleic acid-based detections are used through the amplification methods, hybridization methods, which could be in situ, in vitro, and in vivo. the most common and widely used hybridization-based method is in situ hybridization, which could utilize fluorescent (fish) or chromogenic (cish) molecules. the cish-based assays for the rapid characterization of microorganisms, such as mycobacterium species and the dimorphic fungi in positive culture samples have been described (louro et al., ; scarparo et al., ) . recently, a fish-based assay has been developed for the identification and differentiation of mycobacterium tuberculosis complex from nontuberculous mycobacteria (baliga et al., ) . nucleic acid amplification methods are amongst the best in detecting pathogens with high sensitivity and specificity in the clinical samples. various modifications in nucleic acid amplification methods have provided collectively robust methods to yield better and accurate results. these modifications could be categorized into two amplification methods viz pcr and its variants, and isothermal amplification methods. these are the most common tools used for the pathogen detection worldwide. the three basic variants include ( ) real-time pcr, which is a modified version of conventional pcr, where quantification of dna sequence is possible without any further step of running the amplified product on agarose gel; ( ) multiplex pcr, where multiple sequences can be detected in a single reaction mixture, and ( ) reverse transcriptase pcr (rt-pcr) where rna is transcribed to cdna and this cdna is used in the amplification as template. the real-time pcr can utilize different fluorescence chemistries such as sybr green, taqman, or molecular beacon probes. recently, a taqman real-time rt-pcr assay has been developed for rapid detection and quantification of japanese encephalitis virus in swine blood and mosquito vectors (pantawane et al., ) . in isothermal amplification, a number of target dna copies increase at a constant temperature in just one cycle without the need of a thermocycler. various techniques have been developed using isothermal amplification methods viz nucleic acid sequence-based amplification (nasba), transcription-mediated amplification (tma), signal-mediated amplification of rna technology (smart), strand displacement amplification (sda), rolling circle amplification (rca), loopmediated isothermal amplification (lamp), isothermal multiple displacement amplification (imda), helicase-dependent amplification (hda), circular helicasedependent amplification (chda), single primer isothermal amplification (spia), and strand invasion-based amplification (siba). in all these methods, isothermal temperature amplified products can be visualized on gel through the various structures visible on gel or by the incorporation of dyes in the special structures formed on amplification serving in real-time detection. among all these techniques, lamp is the most widely used isothermal amplification method which is in fact an autocycling strand displacement dna synthesis, which deploys four primers forming a stem-loop dna by self-primed dna synthesis and a dna polymerase with strand displacement activity (malik et al., ; parida et al., ) . recently, an improved strategy using a double-labeled probe to overcome the problem of false positivity of lamp together with target gene real-time quantification is devised for detection of avian orthoreovirus (kumar et al., ) and salmonella spp. (mashooq et al., ) . other potential isothermal amplification techniques are recombinase polymerase amplification (rpa) and nasba. the former has brought a breakthrough in the detection of nucleic acids as it does not require denaturation of the template. rt-rpa is an extension of the above method, in which bacterial rt is used in the amplification of rna. rt-rpa was developed to study the outbreak of footand-mouth disease (fmd) disease in egypt (abd el wahed et al., ) . high degree of fidelity, portability, cost efficiency, simplicity, sensitivity, and tolerance to inhibitors, put this method into the category of resounding techniques, and implementation is quite easy at quarantine stations (moore and jaykus, ); while the latter one requires initial denaturation of the template followed by temperature labile polymerase dependent isothermal amplification and was designed specially to detect rna (compton, ) . a multiplex real-time nucleic acid sequence-based amplification (qnasba) system for the simultaneous detection of rotavirus a, norovirus genogroup ii/astrovirus has recently been developed (mo et al., a) . rt-nasba proved as more efficient than the conventional rt-pcr and taqman rt-pcr assays (mo et al., b) . a microarray is a multiplex lab-on-a-chip test. it is an arrangement of the large amount of biological materials for high-throughput screening on a solid support generally a glass slide, through the detection-based assays. microarray has done wonders in the high-throughput screenings and for the breakthrough causes of the outbreaks. simultaneous detections of coinfections and other more phenomenal changes during the outbreaks are the crucial developments to study the infectious diseases in endemic regions. their easiness has brought the working systems onto the platform on global diagnostics. multiple diagnostics with hybridizing ability put it at more ease to strategize the control management programs. but, this technique comes at high expenditures. data management skills and their interpretations need off to the most important tasks to be worked on. with the advent, new kits for point-of-care detections, bioelectric arrays, and liquid microarrays are in the development process. this would be an easy and an improved hybridization method for individual probe and target combinations with accurate detections. this will reduce the effort from clinical diagnosis to the personal level. these all will help in understanding the proper and common pathogens with scaling down the time. peptide nucleic acids (pnas) are highly versatile synthetic oligonucleotides, in which the native sugar-phosphate backbone of dna is replaced with amino acids. pnas bind to complementary dna strands with higher specificity and strength. furthermore, they are resistant to nucleases and proteases, making them a highly stable diagnostic reagent. the pna-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid (ray and nordén, ) . the potential diverse uses of pna have been exhaustively described in a recent review (gambari, ) . a rapid label-free visual pna-based assay for detection and pathotyping of newcastle disease virus has also been reported (joshi et al., ) . similarly, pna-based beacons have also been used in hiv genotyping with high specificity (zhang and apella, ) . aptamers are artificial nucleic acid ligands that are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery, and reamplification. dna aptamers in particular have many advantages over antibodies (brody and larry, ) . aptamers, first reported in , are attracting interest in the areas of diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors (o'sullivan, ) . the aptamers have proved to be potential diagnostic assays, especially in the detection of toxins such as brevetoxin- , potent marine neurotoxins (shimaa et al., ) , marine biotoxinpalytoxin (shunxiang et al., ) , β-bungarotoxin (β-butx), and a neurotoxin from the venom of bungarus multicinctus (ye et al., a) . furthermore, aptamers have been used for the serological detection of mycobacterium bovis (fu et al., ) , cryptosporidium parvum (iqbal et al., ) , and prion disease (saijin et al., ) . biosensors are portable, easy to handle, ultrasensitive, quick, and may be quite specific with less probability of a false positive. biosensors work on various principles viz detecting the changes in the ph, the ion concentrations, mass by specific hybridization, enzymatic reaction, loss of functionality, change in the electrical potential, change in color, and temperature. based on these principles, many biosensors have been devised for the detection of animal pathogens; for example, an extended-gate field-effect transistor for the direct potentiometric serological diagnosis of the bhv- (tarasov et al., ) , nanowire-based immunosensor for bovine viral diarrhea virus (bvdv) (montrose et al., ) , luminescence resonance energy transferÀbased biosensors for the ultrasensitive detection of the h strain (ye et al., b) , quartz crystal microbalance (qcm)Àbased immunosensors to detect h n (li et al., ) , and spectrosenstm optical microchip sensors for foot-and-mouth disease virus (fmdv) (bhatta et al., ) . the limited methods for detection of microbial signatures and the advent of new technology for quick and parallel gene expression capacities have eased in the detection of microbial disease. next-generation sequencing (ngs) is now being increasingly applied in understanding the molecular epidemiology, transmission, and characterization of animal pathogens. instead of gene-by-gene analysis, large deposits of genes available in the clinical sample can be detected in a single test. applications of ngs are considered as more resourceful. thus, it is widely accepted as a diagnostic tool and speedily is being replaced with most other molecular diagnostic technologies and has brought revolution in the diagnosis of pathogens. various modifications and improvements have brought a huge change in the sequencing and identification of genomes. it all started with pyrosequencing on roche , with small read lengths and less efficiency. roche was followed by ion-torrent illumina platform. the ngs has made it possible to sequence the complete viral genomes of many viruses cost-effectively such as including an avian influenza virus (croville et al., ) , classical swine fever virus (leifer et al., ) , and bluetongue viruses (rao et al., ) . recently, nanopore technology, with the promising improvement has brought a wonderful efficiency with emerging science and technology (goodwin et al., ) . nanopore systems can sequence both dna and rna viral genome in real time. this technology is based on the principle that when a strand of dna/rna is allowed to pass through a nanopore, the current is changed as the based a, t, c; and c passes through the pore in different combinations. using these systems, sequencing can be performed on the portable minion device, the benchtop gridion and the high-throughput, high-sample number promethion. recently, nanopore sequencing has proved a revolutionary diagnostic tool in detecting the ebola virus (hoenen et al., ) , influenza viruses (keller et al., ; wang et al., ) and porcine viral enteric disease complexes (theuns et al., ) . overall, the biotechnological innovations have equipped us now to have high-resolution sequencing tools that are revolutionizing the ability of veterinary diagnostic laboratories to detect emerging animal pathogens. with the advent of many biotechnological advances in veterinary diagnostics, point-of-care diagnostics (pocd) are now available for economically important animal diseases. the pocd is basically a simple, rapid, and portable diagnostic device that can be applied at the field level in effective monitoring the disease status. most of the commercially available pocds utilize either antigen/antibody or nucleic acid detection technologies. the former is usually available in the format of lateral flow assays or immunochromatographic strip tests. these assays are simple to use, rapid, inexpensive, disposable, and thus make them the ideal assay for pocd for animal pathogens. the commercially available immunochromatographic strip tests for economically important animal diseases are summarized in table . . these assays are equally sensitive as compared to elisa (ferris et al., ) . furthermore, combining these assays with smartphones has made the increased sensitivity and quick reporting of results possible (yeo et al., ) . therefore, these assays offer a novel herd level surveillance tool, and provide immediate results to the farmers. however, these assays have less analytical sensitivity as compared to nucleic acid-based pocd. the real-time pcr (qpcr) is a well-established tool with high sensitivity of pathogens detection and recently, qpcr has been transitioned into pocd platform. these platforms are fully automated combining nucleic acid extraction, thermal cycling, and reporting of results on-site. for example, minilab (enigma diagnostics) is a platform ( À kg) which can be easily carried to field level and it combines silica paramagnetic-bead-based nucleic acid extraction with lyophilized qpcr reagents in a single cartridge. this platform has been validated for aiv, asfv, csfv, and fmdv (goldenberg and edgeworth, ) . however, this platform is still not available commercially. there are other platforms that do not include nucleic acid extraction step (need to be done separately), such as genesig (primerdesign ltd, united kingdom), genedrive (epistem ltd, manchester, united kingdom), cepheid smartcycler (cepheid), t-cor (tetracore), and r.a.p.i.d. (idaho technologies) (takekawa et al., (takekawa et al., , . the genesig is now supplying lyophilized qpcr assay kits for bovine, equine, porcine, avian, canine, and feline different pathogens. however, these kits are not yet licensed for diagnosis of animal pathogens and are for research purposes only. as per the agreement on trade related aspects of intellectual property rights (trips) under paragraph of article , many countries have excluded diagnostic, therapeutic, and surgical methods of humans or animals from the scope of patentable systems. however, the important patented technologies that are being used in the various formats of diagnostic assays are provided in table . . a high-speed reagent system for qpcr, full velocity technology has been developed by the stratagene which saves time in addition to highly reproducible results. this technology has been used for infectious diseases, cancer, and drug sensitivities testing and already granted five us patents, us , us , us , us , and us . besides, a pocd product, dual path platform (dpp) has been developed by the chembio diagnostic systems, on which tests to detect hiv and syphilis have already been developed. this company in collaboration with national institutes of health and the infectious disease research institute, united states is working constantly to use this platform for the detection of infectious diseases of humans and animals. farm animals reared all over the world for major agricultural and production purposes majorly include cattle, buffalo, sheep, and goats. since the last few decades, a number of infectious diseases have been found associated with farm animals, causing colossal loss to the livestock rearing community and few of them being zoonotic in nature, becoming a problem for the public health. highly contagious livestock diseases such as fmd, hemorrhagic septicemia, peste-des-petits ruminants, and surra cause irreparable economic losses. several other infectious diseases of dairy cows such as bvd, johne's disease, tuberculosis, infectious bovine rhinotracheitis, and liver fluke infestations are generally regarded as being widespread and endemic. the best known and arguably most important discovery of farm animal diseases in the last few decades is bovine spongiform encephalopathy (bse), and others include digital dermatitis, neosporosis and bovine abortion, bovine neonatal pancytopenia, arcanobacterium pluranimalium, and schmallenberg virus. among all, the world organization for animal health classifies fmd and bse as diseases of major interest in cattle. these diseases are known to have a significant effect on dairy production either directly due to death or indirectly due to effects on fertility or milk production, and subsequently, culling. the disease conditions are usually identified based on history and clinical profile of the affected population, but for affirmative diagnosis of the pathogens responsible, the identification of the causal agent is done on the samples or clinical specimens for submission to diagnostic labs. the clinical profiles of several diseases overlap, making diagnosis a little tricky and cumbersome, so initially, the isolation of the infectious agent in pure form using cell culture systems or growth on specific and selective medium became a chosen method for the diagnosis of many pathogenic diseases in farm animals. albeit their usefulness as most sensitive method of detection, they are not used routinely due to time-lapse in confirming illness (bursle and robson, ) . these methods may take hours to several weeks to obtain a confirmatory result. therefore, other approaches based on morphology/biochemical properties of pathogens took the lead and were favored for pathogen detection and identification. several infectious viral disease agents viz astrovirus, adenovirus, rotavirus, etc. were identified through electron microscopy (ong and chandran, ) . but, these also have some drawbacks like more time consuming, less sensitivity, and costly instrumentation. apart from these techniques, approaches like detection of pathogen-specific antibodies or detection of antigenic proteins of pathogens were adopted and categorized under serological assays. serological assays measure antigenÀantibody interactions for diagnostic purposes. these assays are continuously being improved with technologies like rapid strip detection, thus becoming the most preferred tools and are broadly referred to as immunoassays. enzyme immunoassays (elisa) have always been the field applicable diagnostic methods in the detection of various farm animal diseases caused by fmdv, clostridium perfringens, m. bovis, and escherichia coli. hitherto reports have shown the problem of false negative results and cross-reactivity in some of the serological methodologies. innovations including the use of synthetic biology by making highly reactive peptides help to increase the sensitivity and avoid the cross-reactivity issues to some extent. with the more recent advances in diagnostics with the availability of sequences, nucleic acid-based methods have complemented the established techniques as more specific and sensitive methods in detection of pathogens with lesser false positive results in comparison to serological-based methods (bursle and robson, ) . pcr and real-time pcr methods are regularly used in the detection of campylobacter, shigella, bovine respiratory syncytial virus, eimeria, salmonella species, and many other pathogens. likewise, seminested and nested pcr have been developed for the detection of babesia bovis and babesia bigemina. these nucleic acid-based techniques are amongst the standard detection methods and are routinely used for testing in diagnostic laboratories. to improve the efficacy and promote the simplicity, modifications in the form of isothermal amplifications, like lamp and polymerase spiral reaction (psr) have been adopted and are better in the application process, as these are easy to perform, portable, specific, sensitive, and most importantly, quick and cost-effective. lamp is established to be an apex, leading diagnostics for the detection of many farm animal-related diseases like fmd, brucellosis, bovine popular stomatitis, sheep pox, and goat pox (dukes et al., ; song et al., ; zhao et al., ) . likewise, psr has been developed for detection of brucella spp. (das et al., ) , bovine herpesvirus- (malla et al., ) , and canine parvovirus (gupta et al., ) . to further increase the sensitivity and specificity, the combination of elisa and pcr-like immune-pcr, proximity ligation assay, pcr-elisa, have been successfully discovered making the detection -fold more sensitive. the pathogen detected with these combinations includes low pathogenic strains of campylobacter (ding et al., ) . nasba, restriction fragment length polymorphism, amplified fragment length polymorphism, and random amplification of polymorphic dna, are various biotechnological tools that have been further advancing the diagnosis of various infectious diseases. in addition, ngs has brought a revolution in the diagnosis of many pathogens. it appears helpful in the identification of many pathogens, especially viruses in the fecal matter and those that could not be isolated in cell culture system. mining of sequences in samples gives varied genome sequences providing the clues of not only pathogens, but also new strains, genotypes, new viruses, and even the zoonotic efficiencies of the viruses. anis and coworkers demonstrated that targeted bovine ngs is a specific and cost-effective tool for diagnosis of major bovine pathogens in clinical samples (anis et al., ) . even pathogens with low pathogenicity such as bovine enteroviruses (bev), adenoviruses in wild captive animals, and hepatitis e viruses have been revealed in the mining of sequences in the sample. one of the approaches to disease diagnosis is the development of biosensors. assays based on biosensors uses the transducers to convert the biological interaction of pathogen with its specific antibodies to measurable signals. biosensors have been quite useful in the diagnosis in poc detection. biosensors with specific biochemical recognition helped in the identification of e. coli in cattle (dharmasiri et al., ) . colibacillosis is also seen in a variety of farm animals like cattle, pigs, and goats. in c. perfringens detection, epsilon-toxin-specific monoclonal antibody was immobilized onto single-walled carbon nanotubes and adjusted to detect relevant concentrations of toxin in nanomolars and were comparable to elisa-based results. many other methods like mass spectrometry, microarrays, and maldi-tof are also under employment for the detection of many farm animals-associated pathogens, like francisella tularensis, staphylococcus aureus, enterococcus faecalis, e. coli (demirev and fenselau, ; lundquist et al., ; van baar, ) . companion animals are the domesticated animals kept for company of human beings or for utilitarian purposes, that is, guarding, herding, military/police activity. they have grown along with the human civilization and evolution and have developed a good bond with humans. although there is a variety of species which are suitable as companion animals (dogs, cats, rabbit, ferrets, caged birds, fishes, and guinea pigs), dogs and cats are the most common companion species. their physical, behavioral, social, and emotional needs can be easily met at home. on the other hand, dogs and cats play a fundamental role in the life of human beings with many physiological and psychological benefits (wood et al., ) . also, living with companion animals makes human surroundings happier and prosperous. as these animals enrich our lives, it becomes our responsibility to take care of the companion animals and to protect them from any kind of harm. there is a spectrum of infectious diseases that occur in companion animals. lyme disease, psittacosis, hookworms, and salmonella are amongst the most common diseases in pet animals. (lembo et al., ; palatnik-de-sousa et al., ). since these animals share a close environment and are in direct contact with humans, hence they have a potential to spread these infections to human beings. chances of introduction of new diseases also arise on the import of animals from foreign lands. thus, maintenance of strict trade rules and regulations and hygienic conditions becomes a necessity. once the disease occurs, it is important to identify the causative agent to improve the effectiveness of treatment and to control the disease. since the clinical observations are not sufficient and can overlap with other diseases leading to misdiagnosis, several validated laboratory assays are used for confirmatory diagnosis. until years ago, these laboratory tests exploited cell culture (for isolation of specific pathogen) and serological assays (for detection of antibodies generated against a specific pathogen or antigenic proteins). few examples composed of vero cells and recombinant vero-slam cells used for culturing rickettsia rickettsia, and toxoplasma gondii, madin-darby canine kidney cells for canine adenovirus and canine herpesvirus. similarly, serological methods include a long list. immunodiffusion testing is most often used to detect antibodies to fungal pathogens in dogs, such as aspergillus fumigatus, coccidioides immitis, and blastomyces dermatitidis. agglutination tests include the microscopic agglutination test for serologic diagnosis of leptospirosis (agglutination of live leptospires) and the cryptococcal antigen la test (agglutination of antibody-coated latex beads). hemagglutination inhibition is used to determine antibody titers to cpv and canine influenza virus, and it evaluates the ability of serum to inhibit erythrocyte agglutination by these viruses. elisa is commonly used for the detection of feline retroviral, heartworm, giardia, leishmania, and tick-borne infections. indirect ifa for serologic testing in dogs and cats include quantitative serology for some tick-borne infectious diseases (e.g., ehrlichia canis, anaplasma spp.). direct immunofluorescence assay in veterinary medicine include diagnosis of giardia oocysts, felv within monocytes in peripheral blood or bone marrow, or canine distemper virus within epithelial cells from a conjunctival scraping. many of these assays involve the use of polyclonal antibodies, or, more commonly, monoclonal antibodies-dependent diagnosis. with the recent boom in the database of sequences for pathogens, new diagnostic tools like pcr, real-time pcr, and multiplex pcr have almost replaced the established techniques and are adopted as routine diagnostics for testing clinical samples. canine respiratory coronavirus, canine adenovirus- , canine herpesvirus, feline herpesvirus- , canine distemper virus, west nile virus, and encephalitis viruses are some of the examples, which are routinely diagnosed using these techniques. other biotechnological tools cover hybridization assays, pna, nanoparticles-based assays, etc. hybridization-based methods have also been found to be compatible with the diagnosis of many diseases and composed of taqman-based probes, molecular beacons, and fret-based probes. although not yet widely used for veterinary applications, pna probes are now increasingly available to detect target dna. fluorescent pna probes, followed by signal amplification were used to differentiate between m. tuberculosis complex and nontuberculous mycobacterium spp. (zerbi et al., ) . in another example, ngs has also been used for the comparison of the oral microbiome of canines with their owners as they are in direct contact with their pets and many diseases might get transmitted to them (oh et al., ) . gold nanoparticle-based immunochromatographic strip test using a combination of mab and pab was developed as an alternative for on-site and cost-effective diagnosis of cpv infection . another use of biotechnology has been observed for rapid and early detection of cpv using a qcm biosensor (kim et al., ) . also, for genotyping of cpv- , conventional methods are time consuming, therefore, a probe-based duplex fluorescence melting curve analysis (fmca) for genotyping six different cpv- variants (original cpv- , cpv- a, cpv- b, cpv- c, and vaccine strains of cpvpf and cpvint) using only two taqman probes has been developed (liu et al., ) . despite the fact that a wide range of diagnostic tools are available, there is a considerable chance for better advancement in diagnostics, in terms of speed and accuracy, to control and eradicate economically important diseases. in the near future, use of new biotechnological tools like biosensors and nanotechnology will pave the way. further, ngs platforms like minion (a portable, real-time ngs sequencer) coupled with nanopipe analysis are promising tools to perform bacterial and viral disease investigation in low throughput laboratories and specifically in the field (beato et al., ; shabardina et al., ) . although, yet not been adopted for animal disease diagnosis, but novel platforms such as smartphonebased diagnosis (which expands nucleic acid-based detection assays toward pocd) like rt-lamp and fluorescent lateral flow immunoassay (already developed for zika virus and dengue virus) provide exciting opportunities for veterinary diagnostics in the near future (rong et al., ) . biotechnological innovations have brought new generation diagnostic methods for rapid and sensitive diagnosis of various diseases of livestock and pet animals. infectious diseases entail remarkable economic loss, weak food production system, food insecurity, and high maintenance cost of the agriculture sectors including farm animals, poultry, and aquaculture. besides, these diseases carry a huge risk of transmission to humans as sporadic and endemic zoonoses. classical and conventional diagnostic methods are labor intensive, time consuming, less sensitive, and difficult to meet the needs of the emerging pathogen diagnostics. thus, new innovations have to be worked on and need to be practiced. over the long term, innovations will be helping in the diagnosis of pathogens with accurate, sensitive and specific detections. ngs, biosensors, and advanced amplification techniques will persist for longer periods in their constant modified forms. innovations will always be bringing the new applications in the diagnostics for the improved versions of techniques. new technique applications come with the cost and unbroken funding will be putting new prospective techniques into the trials. these techniques should be simplified in the innovations for their easy practices at the field itself, without looking for any skilled personnel/highly equipped laboratories. there is no conflict of interest. a portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus dot elisa for newcastle disease, infectious bursal disease and mycoplasmosis evaluation of targeted next-generation sequencing for detection of bovine pathogens in clinical samples rapid method for detecting and differentiating mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with dna probes identification and genetic characterization of bovine enterovirus by combination of two next generation sequencing platforms rapid detection of foot-and-mouth disease virus with optical microchip sensors detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time pcr assay with classical tools aptamers as therapeutic and diagnostic agents non-culture methods for detecting infection better tests, better care: improved diagnostics for infectious diseases a metagenomic survey of microbes in honey bee colony collapse disorder field monitoring of avian influenza viruses: whole-genome sequencing and tracking of neuraminidase evolution using pyrosequencing rapid visual isothermal nucleic acid-based detection assay of brucella species by polymerase spiral reaction mass spectrometry for rapid characterization of microorganisms diagnostic applications of molecular tools and techniques for important viral diseases of poultry enrichment and detection of escherichia coli o : h from water samples using an antibody modified microfluidic chip an overview of control strategy and diagnostic technology for foot-and-mouth disease in china novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus development and laboratory evaluation of a lateral flow device for the detection of swine vesicular disease virus in clinical samples immunohistochemical detection of piscine reovirus (prv) in hearts of atlantic salmon coincide with the course of heart and skeletal muscle inflammation (hsmi) enzyme linked aptamer assay: based on a competition format for sensitive detection of antibodies to mycoplasma bovis in serum peptide nucleic acids: a review on recent patents and technology transfer the enigma ml fluabÀrsv assay: a fully automated molecular test for the rapid detection of influenza a, b and respiratory syncytial viruses in respiratory specimens polymerase spiral reaction (psr): a novel, visual isothermal amplification method for detection of canine parvovirus genomic dna complementary monoclonal antibody-based dot elisa for universal detection of h avian influenza virus nanopore sequencing as a rapidly deployable ebola outbreak tool detection of cryptosporidium parvum oocysts on fresh produce using dna aptamers rapid label-free visual assay for the detection and quantification of viral rna using peptide nucleic acid (pna) and gold nanoparticles (aunps) direct rna sequencing of the coding complete influenza a virus genome evaluation of a rapid immunochromatography strip test for detection of astrovirus in stool specimens a novel assay for detecting canine parvovirus using a quartz crystal microbalance biosensor peptide-recombinant vp protein based enzyme immunoassay for the detection of group a rotaviruses in multiple host species a double-stranded probe coupled with isothermal amplification for qualitative and quantitative detection of avian reovirus approaches to define the viral genetic basis of classical swine fever virus virulence the feasibility of canine rabies elimination in africa: dispelling doubts with data a nanobeads amplified qcm immunosensor for the detection of avian influenza virus h n application of duplex fluorescence melting curve analysis (fmca) to identify canine parvovirus type variants direct identification of mycobacterium avium complex and mycobacterium gordonae from mb/bact bottles using accu probe discrimination of francisella tularensis subspecies using surface enhanced laser desorption ionization mass spectrometry and multivariate data analysis development of recombinant σb protein based dot-elisa for diagnosis of avian reovirus (arv) rapid detection of human rotavirus using nsp gene specific reverse transcription loop-mediated isothermal amplification assay novel polymerase spiral reaction (psr) for rapid visual detection of bovine herpesvirus genomic dna from aborted bovine fetus and semen rapid serological profiling by an immunocomb-based dot-enzyme-linked immunosorbent test for three major poultry diseases development and evaluation of probe based real time loop mediated isothermal amplification for salmonella: a new tool for dna quantification rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification comparative detection of rotavirus rna by conventional rt-pcr, taqman rt-pcr and real-time nucleic acid sequence-based amplification novel single gold nanowire-based electrochemical immunosensor for rapid detection of bovine viral diarrhoea antibodies in serum development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses comparison of the oral microbiomes of canines and their owners using next-generation sequencing identification of gastroenteric viruses by electron microscopy using higher order spectral features aptasensors À the future of biosensing? decrease of the incidence of human and canine visceral leishmaniasis after dog vaccination with leishmune in brazilian endemic areas taqman real-time rt-pcr assay for detecting japanese encephalitis virus in swine blood samples and mosquitoes loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction deep sequenc-ing as a method of typing bluetongue virus isolates peptide nucleic acid (pna): its medical and biotechnical applications and promise for the future smartphone-based fluorescent lateral flow immunoassay platform for highly sensitive point-of-care detection of zika virus nonstructural protein aptamer-based assay for prion diseases diagnostic direct identification of mycobacteria from mb/bact alert d bottles: comparative evaluation of two commercial probe assays nanopipe-a web server for nanopore minion sequencing data analysis development and evaluation of a gold nanoparticle-based immunochromatographic strip test for the detection of canine parvovirus aptamer-based competitive electrochemical biosensor for brevetoxin- enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin development and evaluation of simple dotÀblot assays for rapid detection of staphylococcal enterotoxin-a in food establishment of loop-mediated isothermal amplification (lamp) for rapid detection of brucella spp. and application to milk and blood samples field detection of avian influenza virus in wild birds: evaluation of a portable rrtÀpcr system and freeze-dried reagents rapid diagnosis of avian influenza virus in wild birds: use of a portable rrtÀpcr and freeze-dried reagents in the field a potentiometric biosensor for rapid on-site disease diagnostics nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry minion nanopore sequencing of an influenza genome the pet connection: pets as a conduit for social capital? recognition of bungarus multicinctus venom by a dna aptamer against β-bungarotoxin upconversion luminescence resonance energy transfer (lret)-based biosensor for rapid and ultrasensitive detection of avian influenza virus h subtype smartphone-based fluorescent diagnostic system for highly pathogenic h n viruses amplified in situ hybridization with peptide nucleic acid probes for differentiation of mycobacterium tuberculosis complex and non-tuberculous mycobacterium species on formalin-fixed, paraffin embedded archival biopsy and autopsy samples advantages of peptide nucleic acids as diagnostic platforms for detection of nucleic acids in resource-limited settings development of loopmediated isothermal amplification assay for specific and rapid detection of differential goat pox virus and sheep pox virus all the authors of the chapter thank and acknowledge their respective universities and institutes. key: cord- - aclcc authors: liu, jianbo; gao, ran; shi, hongyan; cong, guangyi; chen, jianfei; zhang, xin; shi, da; cao, liyan; wang, xiaobo; zhang, jialin; ji, zhaoyang; jing, zhaoyang; feng, li title: development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific siga in colostrum date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: aclcc porcine epidemic diarrhea virus (pedv) causes very high mortality in newborn piglets. the mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. antibodies derived from the secretory immunoglobulin a (siga) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. therefore, the measurement of siga levels is an important index in evaluating pedv infections and immune status. a simple and rapid method for the detection of pedv-specific siga using an immunochromatographic test strip has been developed; incorporating a colloidal gold-labeled anti-siga secretory component (sc) mab probe for the detection of anti-pedv-specific siga in swine. on the strip, a gold-labeled anti-siga sc mab was applied to a conjugate pad; purified pedv particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. results showed that the immunochromatographic test strip had high sensitivity and specificity. when compared with enzyme-linked immunosorbent assay, kappa value suggesting that the strip could be used to detect pedv specific siga in colostrum samples. furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. we found that the immunochromatographic test strip was a rapid, sensitive, and reliable method for the identification of pedv specific siga, indicating its suitability for epidemiological surveillance as well as vaccine immunity when studying pedv. porcine epidemic diarrhea virus (pedv) belongs to the family coronaviridae, first recorded in pigs in england in (pensaert and de bouck, ) , and subsequently spread to other european and asian countries (song and park, ) and north america (huang et al., ) . porcine epidemic diarrhea is a global infectious disease and is characterized by high morbidity and mortality in pre-weaned piglets, and causes serious economic losses to the swine industry in china (gao et al., ; li et al., ) . pedv is an enveloped coronavirus with a kb positive-stranded rna genome, containing at least seven open reading frames (kocherhans et al., ) . these mainly encode four major structural proteins: the spike (s), nucleocapsid, membrane, and envelope proteins. the s protein is a glycoprotein and can form homotrimers to mediate membrane fusion and gain entry into host cells (bosch et al., ) . it is known that the s protein of coronavirus plays a crucial role in the induction of neutralizing antibodies, and it has been used to prepare effective vaccines (brian and baric, ; tuboly and nagy, ) . the amino-terminal portion of the s protein of several coronaviruses has been shown to contain key antigenic sites that are responsible for eliciting humoral and cellular immune responses (gebauer et al., ) . the mucosal immune system in the gut faces the formidable task of eliminating potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. antibodies of the secretory immunoglobulin a (siga) class act as the first line of antigenspecific immunity in the gut, and can recognize both pathogens and commensals (song and park, ) . in contrast to serum iga, which is derived from plasma cells in the bone marrow, siga is generated locally by plasma cells in the lamina propria, which lies beneath the intestinal epithelium (kaetzel, ) . the protective action of siga in the infant gut is a result of many processes, including intracellular neutralization t and viral particle excretion, immune exclusion, whereby siga agglutinates bacteria and viruses, as well as prevents the binding of pathogens to mucosal surfaces, and interference with bacterial motility (van egmond et al., v) . at high endogenous concentrations of siga, infants are less likely to have experienced illness in the preceding and subsequent months (breakey et al., ) . pedv can affect the intestinal tract, and based on the above results, we can infer that the siga concentration in the colostrum and rectal swab is a better marker than iga of protection and survival rate after virulent pedv challenge. tools to monitor pedv mucosal immunity and colostral immunity could be useful for the development of preventative programs on affected farms. therefore, the aim of this study was to develop a simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled anti-siga sc mab probe for the detection of anti-pedv-specific siga in swine, and to compare its performance with an indirect siga elisa based on the whole pedv virus (cong et al., ) . vero e cells (american type culture collection no. crl- ) were used to propagate pedv (genbank accession no. kt ). virus was propagated according the method described by liu et al. ( ) with minor modifications. briefly, growth medium was removed (containing % fbs) when the cells formed a confluent monolayer and were then washed with phosphate-buffered saline (pbs, ph . ). then, pedv containing × plaque forming units (pfu) were diluted in ml of dmem (dmem, gibco brl life technologies, usa) (containing μg trypsin), and added to each culture bottle and incubated at °c. virus was harvested and purified according to the methods described by liu et al. ( ) . the purified viruses were negatively stained with % phosphotungstic acid (ph . ) for s according to previously described procedures (chen et al., ; jung et al., ) . and then identified by h- electron microscopy (hitachi, japan). spf swine were used to produce colostrum. a total of four specific pathogen free swine (supplied by experimental animal base, harbin veterinary research institute of the chinese academy of agricultural sciences) were used in this study. three swine were immunized with pedv (lnct strain), transmissible gastroenteritis virus (tgev) strain h (genbank accession no. fj ), and porcine rotavirus (porv) a (genbank accession no. jf ), respectively. all the viruses were inactivated by . % methanol solution (pbs, ph . ) at °c for h, and then emulsified with freund's complete adjuvant (sigma-aldrich, st. louis, mo, usa). and they were performed by neck intramuscular injection. the fourth animal was immunized with dmem as control and each swine was immunized twice. the first immunization was days before delivery whereas the second immunization was days before delivery. after delivery, colostrum from the four swine was collected and store at − °c. the protocols involving animal immunity in this study were approved by our institute's animal care and use committee and performed in accordance with standard ethical guidelines. the hybridoma cell line f , stably secreting mab against siga sc protein (gao et al., ) was cultured in the peritoneal cavities with pristane (sigma-aldrich, st. louis, mo, usa) primed balb/c mice to obtain ascites fluid. the ascites fluid was purified by hitrap protein g hp (ge healthcare) according the manufacturer's instruction and the purified antibody was identified by sds-page. the gel was stained by coomassie blue. colloidal gold with an average particle diameter of approximately nm was purchased from shanghai kinbio tech. co. ltd. (china). the colloidal gold-labeled mab was prepared according to the methods described by li et al. ( ) with some modifications. briefly, . ml ( mg/ml) of purified mab was incubated with ml of colloidal gold solution (ph . ) for min at °c. after addition of ml of a % casein solution, the mixture was incubated at °c for min. then, the mixture was centrifuged at , × g for min at °c. the supernatant was discarded and the pellet of the colloidal gold-labeled mab was suspended in ml of . m sodium borate buffer (containing . % nan , % bovine serum albumin (bsa), % sucrose) and stored at °c. the colloidal gold-based lateral flow test strips were generated according to the procedures described by liang et al. ( ) , with some modifications. briefly, × -mm pieces of glass fiber was immersed in phosphate-buffered saline ( . m, ph . , containing mm edta, % tween- , and % bsa) for h at °c. next, the pieces were dried for h at °c and stored for use as the sample pad. glass fiber was also immersed in phosphate-buffered saline ( . m, ph . , containing . % mycose, % bsa, % tween- ) for h at °c and then dried for h at °c and stored for use as the conjugate pad. the conjugate pad was covered with an appropriate volume of colloidal gold-labeled anti-siga sc mabs ( f ) using a xyz dispense platform (biodot, inc., irvine, ca, usa), and then dried at °c for h and stored dry. purified pedv particles were dispensed onto the nitrocellulose (nc) membrane to serve as the test line, and goat antimouse igg polyclonal antibody (pab) was dispensed onto the nc membrane, (being separated by a distance of mm) to serve as the control line. the pedv particles and goat anti-mouse igg pab were diluted with coating buffer to a final concentration of . and . mg/ ml, respectively. then, the nc membrane was dried at °c for h and stored in the dry at room temperature until use. the test strips were assembled and used as described by liang et al. ( ) . the sample pad, conjugate pad, nc membrane, absorbent pad and backing plate were assembled in sequence. the sample pad and the conjugate pad were overlapped by mm with one end of the conjugate pad; the other end of the conjugate pad was overlapped ( mm) with one end of the nc membrane underneath the conjugate pad; the absorbent pad was stuck to the other side of the nc membrane. after this, the whole plate was cut into mm-wide strips, which were assembled in the strip cassettes with desiccant. to test for colostrum, we diluted the samples : in sample buffer and mixed thoroughly then, μl of the solution was dispensed onto the sample pad well of the strip apparatus to determine the presence of pedv specific siga, as described above. with this system, the liquid migrates towards the absorption pad by capillary action. the siga in the colostrum samples will form a "gold-labeled antibody-siga" complex with the gold-labeled antibody on the conjugate pad. if the colostrum sample has pedv specific siga present, the pedv specific "gold-labeled antibody-siga" complex, while migrating to the nc membrane, will bind to pedv, thereby displaying a red line in the t line area. additional complex migrates further to react with the goat antimouse igg pab, showing another red line in the c line area. negative samples do not produce a red line in the t area, whereas the c line will always show a red line. the results can be seen within eight minutes at room temperature. . . validation of specificity and sensitivity of the immunochromatographic strip test pedv siga positive colostrum, tgev siga positive colostrum, porv siga positive colostrum, and pedv siga negative colostrum were used to test the specificity of the strip. the sensitivity of the strip was evaluated by comparing the detection of the same samples with pedv siga elisa kit. the detection limit of the strip was evaluated by using pedv siga positive-colostrum and colostrum titers determined by using an elisa kit (cong et al., ) as the reference standard with minor modifications. in brief, sample buffer was used to dilute colostrum from : to : , and μl of the solution was added into microtiter plates (costar, corning, ny, usa) incubated at °c for h. after a washing step, a horseradish peroxidase mouse anti-pig siga sc antibody : dilution was added and incubated at °c for min. after three times wash with pbst, the peroxidase reaction was visualized using , ′-azino-bis ( -ethylbenzothiazoline- -sulfonic acid) diammonium salt (bbi, shanghai, china) as the substrate for min at °c , and the reaction was stopped by adding μl of . m sulfuric acid to each well. optical densities (ods) were measured at nm using an elisa plate reader (biotek instruments, winooski, vt, usa). for the sensitivity of the elisa kit, one pedv specific siga positive colostrum was still positive when diluted at : . for the specificity, the elisa kit has no cross-reaction with tgev as well as porv specific siga positive colostrum (cong et al., ) . sixty three colostrum field samples from hebei, shandong and heilongjaing province were assayed by the strip and the pedv specific siga elisa (cong et al., ) . by comparing results obtained by the two methods, kappa statistic value was used to evaluate the effectiveness of the test strip. after removing sucrose, the bands between the % and % and % and % sucrose solutions were identified by electron microscopy. in the % and % sucrose solution band, pedv particles were observed and their diameter was approximately nm (fig. ) . ascites fluid containing mab f was purified by hitrap protein g hp and the purified antibody was identified by sds-page. as the results show (fig. ) , the purified antibody had two brands, a light chain and heavy chain. the purified antibody was stored until further use. the assay specificity was determined using anti-pedv specific siga positive colostrum, anti-tgev specific siga positive colostrum, anti-porv specific siga positive colostrum, and anti-pedv specific siga negative colostrum. as shown by the strips listed in fig. , the anti-pedv specific siga positive colostrum was positive at the test line; the other colostrum samples were negative, suggesting that the immunochromatographic strip had good specificity. anti-pedv specific siga positive colostrum was diluted to determine the sensitivity of the strip. according to the results shown in fig. , the strip had a sensitivity of up to : , while the detection limits of the elisa kit reached : dilution (table ) . all of the colostrum samples were detected by the strip and elisa mentioned above. as shown in table , colostrum samples were anti-pedv specific siga positive as determined by the strip assay, whereas colostrum samples were anti-pedv specific siga positive as detected by the elisa. the kappa statistic value for the comparison of the strip versus the elisa kit, was . . for the interpretation of agreement of the kappa statistic, a value of . to . is considered fair, fig. . identification of purified pedv by electron microscopy. the band between the % and % sucrose solutions was collected with a syringe, and after removing the sucrose the purified viruses were identified by electron microscopy. scale bar = nm. . to . is moderate, . to . is good, and . to . is very good (altman, ) . the kappa statistic value was . , suggesting that the strip could be used to detect pedv specific siga in colostrum samples. antiviral antibody detection is key for vaccine immunity surveillance and in determining pig exposure to pedv. currently, indirect elisas used to assay igg, iga, siga, have been established to monitor antibodies against pedv li et al., ; cong et al., ) . also, reverse transcription-polymerase chain reaction (rt-pcr) (ishikawa et al., ) , duplex rt-pcr (kim et al., ) , and other methods have been used to monitor pedv. although the presence of antibodies in serum is not directly related to protection of sows or piglets, serological examinations facilitate the assessment of the humoral responses to pedv, elicited either through vaccination or natural infection. however, the above-mentioned methods are time-consuming and require professional/technical personnel and are mainly limited to laboratory use. the immunochromatographic test strip used to detect anti-pedv specific siga described here is easy to operate, and sensitive. it is known that pedv is mainly transmitted by the fecal-oral route and disease is initiated following interaction with the mucosal surface lining the digestive tract. the primary defense used by this tissue is the mucosal immune system. antibodies from the siga class act as the first line of antigen-specific immunity in the gut. they prevent pathogens from binding to mucosal surfaces, and agglutinate bacteria and viruses (van egmond et al., v) . therefore, siga levels are an important index by which to evaluate pedv infection and immune states. in this study, siga, as opposed to igg, detection was developed to assess pedv infections and vaccination. here a pedv siga-specific immunochromatographic test strip was developed. the purified pedv-lnct particles, which belongs to the g genotype, were used as capture antigen. the shared amino acid identity of the pedv lnct s protein and the pedv cv (g genotype) s protein (genbank accession number kt ) is . %, whereas it is only . % for the pedv lnct s protein and the tgev s protein (purdue strain, genbank accession number dq ). reactivity using the polyclonal antibodies (pabs) revealed significant cross-reactivity between the two pedv subtypes, although there was a two-fold difference in the antigenic responses based on pab titers in the elisa and ifa (wang et al., ) . therefore, the elisa antigen based on the pedv-lnct particles could detect both g and g genotype strains. additionally, the pedv particles used in the present study had no cross-reactivity with anti-tgev positive specific colostrum or anti-porv positive specific colostrum (fig. ) . furthermore, the strip had a sensitivity of up to : , whereas the elisa kit limits for the same sample was : (table ) . based on these results, the anti-pedv specific siga immunochromatographic test strip could be used to effectively monitor siga levels. previously, researchers have used elisas to study the effect of pedv on mucosal immunity; an indirect elisa based on the s protein of pedv was used to detect iga levels in colostrum and fecal samples to evaluate pedv colostral immunity (gerber et al., and . iga plays an important role in providing protection at mucosal surfaces, and it is effective in neutralizing bacterial toxins. furthermore, polymeric iga has been shown to be more effective at neutralizing exotoxins from clostridium difficile when compared to either monomeric iga or igg with the same variable regions (stubbe et al., ) . siga consists of the sc, two iga molecules, and a linking chain. sc protects siga from proteolytic degradation (kaetzel, ) , making the siga more stable. it has been estimated that approximately g of siga is transported daily fig. . specificity of the immunochromatographic strip. lanes to : strip detection of the anti-pedv specific siga positive colostrum, anti-tgev specific siga positive colostrum, anti-porv specific siga positive colostrum, anti-pedv specific siga negative colostrum, water, milk. the samples were diluted at : in sample buffer and mixed thoroughly. then, μl of the solution was dispensed onto the sample pad. c stands for control line, t stands for test line. into the intestines of the average adult (mestecky et al., ; conley and delacroix, ) , where siga forms the first line of antigen-specific immune protection against ingested, inhaled, or sexually transmitted pathogens and antigens at mucosal surfaces (kaetzel, ) , making it more important than iga. in this study, we investigated the sc portion to detect siga in colostrum samples, as this can eliminate the effect of monomeric iga. after infection with coronaviruses, such as pedv or tgev, or after vaccination, siga is generated locally by plasma cells located in the lamina propria, which underlies the epithelium (kaetzel, ) . therefore, the anti-pedv specific siga immunochromatographic test strip established in this study could be used to detect infections and immune states. by determining the siga level, we could assess whether pig herds (unvaccinated) have been infected by pedv, as well as pigs (vaccinated) could been protected against pedv, highlighting the versatility of our method. therefore, monitoring siga titers in colostrum and milk samples could be performed to ensure that piglets receive adequate passive immunity. the anti-pedv specific siga immunochromatographic test strip for the detection of siga antibodies developed in our study provides a simple, sensitive and specific tool for monitoring passive immunity and pedv infection. null stands for not done. the cut off value of od value was . . the colostrum samples with an od value ≥ . were considered to be positive while < . were considered to be negative. practical statistics for medical research the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex illness in breastfeeding infants relates to concentration of lactoferrin and secretory immunoglobulin a in mother's milk coronavirus genome structure and replication isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states establishment and application of indirect elisa for detection of porcine epidemic diarrhea virus specific siga intravascular and mucosal immunoglobulin a: two separate but related systems of immune defense? development of an enzyme-linked immunosorbent assay for themonitoring and surveillance of antibodies to porcine epidemicdiarrhea virus based on a recombinant membrane protein phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china preparation and identification of monoclonal antibodies against porcine siga sc fragment residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein detection of immunoglobulin (ig) a antibodies against porcine epidemic diarrhea virus (pedv) in fecal and serum samples detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states direct and rapid detection of porcine epidemic diarrhea virus by rt-pcr pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in central china based on the orf gene and the main neutralization epitopes development of an indirect elisa based on a truncated s protein of the porcine epidemic diarrhea virus development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (iii) chelate microparticles-based lateral flow test strips neutralization of genotype porcine epidemic diarrhea virus strains by a novel monoclonal antibody the human iga system: a reassessment a new coronavirus-like particles associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines polymeric iga is superior to monomeric iga and igg carrying the same variable domain in preventing clostridium difficile toxin a damaging of t monolayers construction and characterization of recombinant porcine adenovirus serotype expressing the transmissible gastroenteritis virus spike gene iga and the iga fc receptor immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g and g this work was supported by grants from the national key r & d plan for the th five year plan ( yfd ), national natural science foundation of china (no. ), the natural science foundation of heilongjiang (no. qc ). all authors have carefully revised the manuscript point-by-point according to the reviewers' comments. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. key: cord- -xjbs gi authors: sozzi, enrica; luppi, andrea; lelli, davide; martin, ana moreno; canelli, elena; brocchi, emiliana; lavazza, antonio; cordioli, paolo title: comparison of enzyme-linked immunosorbent assay and rt-pcr for the detection of porcine epidemic diarrhoea virus date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: xjbs gi abstract porcine epidemic diarrhoea (ped) is a contagious enteric disease of pigs caused by a coronavirus. a double antibody sandwich enzyme-linked immunosorbent assay (das-elisa) based on the use of monoclonal antibodies was developed for the detection of porcine epidemic diarrhoea virus (pedv). the das-elisa was compared with rt-pcr in the examination of specimens collected during – from pigs originating from different farms located in the po valley. both faecal samples obtained directly from the rectum of live animals showing clinical signs and intestinal samples collected from the caecum of deceased pigs were included in the study. the correlation between the two methods was higher when testing faecal samples (k = . , % ci: . – . ) than testing intestinal samples (k = . , % ci: . – . ). the use of elisa technology provided an efficient and effective mean of evaluating the presence of coronavirus ped antigen in field samples and indicates that this procedure is a very useful tool in epidemiological studies. porcine epidemic diarrhoea virus (pedv) is a member of the order nidovirales, family coronaviridae, genus coronavirus and is part of group species. pedv was first reported in belgium and united kingdom in (pensaert and debouck, ) . since then, several outbreaks of the disease have been reported in many swineraising countries, most notably in europe and east asia (pensaert and sang-geon, ) . pedv induces an enteric disease similar to that caused by transmissible gastroenteritis virus (tgev) in pigs of all ages with high morbidity and mortality related to the age. mortality rate averages % but may be as high as % in pigs less than days of age and very low or nil in pigs older than weeks (pensaert and debouck, ; pensaert and sang-geon, ; pritchard et al., ) . since the s pedv has caused severe epidemics in some asian countries such as japan, korea and china (kweon et al., ; takahashi et al., ) . in these countries massive losses of piglets have been reported and mortality usually reached high percentages ( - %) in pigs younger than week (shibata et al., ) . between may and june , an epidemic of watery diarrhoea in pigs of all ages, including piglets, occurred in italy in the po valley, a densely pig populated area. in previous years, similar but sporadic outbreaks had been observed in growers and finishers but that was the first time that ped ''re-emerged" in epidemic form in a european country in many years (martelli et al., ) . the objective of the present study was to develop a double antibody sandwich (das) elisa, based on the use of monoclonal antibodies for pedv detection in swine intestinal and faecal samples useful for routine examinations of field samples. the diagnostic performances of the das-elisa and the correlation with rt-pcr were evaluated by testing samples collected from pig herds in the po valley during the - period. two hundred and fifteen faecal samples were obtained directly from the rectum of live animals showing clinical signs and intestinal samples were collected from the caecum of deceased pigs. these specimens were diluted with phosphate-buffered saline (pbs; . m, ph . ) to obtain a % suspension (v/v), clarified by centrifugation at g for min and then stored at À °c. the pedv reference strain (cv- ) was propagated in vero cells grown in minimum essential medium eagle (mem) in the presence of trypsin (hofmann and wyler, ) . as soon as the cytopathic effect was fully developed, the virus was released from the cells by repeated freezing/thawing. after centrifugation ( g for min), the supernatant of the infectious culture medium was kept at À °c before being used as positive control in the elisa reaction. negative control antigen was similarly prepared from non-infected cell lines. monoclonal antibodies (mabs) were prepared using standard methods (galfre and milstein, ) . mice were immunised with partially purified cv- strain. forty hybridomas were screened for secretion of desired antibodies by indirect immunoflorescence (ifi) and indirect elisa, set up using pedv infected and non-infected cells. only six mabs specific for the pedv were selected and further characterised. purified mabs from hybridomas culture supernatants or ascitic fluids conjugated with horseradish peroxidase (hrpo) using a modification of the method described by tjissen and kurstak ( ) . the different combinations of the six selected mabs were tested in das-elisa using as antigen both vero cells infected or not with pedv and cells infected with other coronaviruses or other pig viruses. the intensity and specificity of the reaction using each mab as catcher or tracer were considered as criteria for selection of the best combination, i.e. mab c as antigen catching antibody and f as conjugated mab. in the standard procedure, nunc maxisorb immunoplates were coated overnight at °c with purified mab c optimally diluted in . m carbonate-bicarbonate buffer, ph . ( lg/well). samples were dispensed to duplicate wells and, after incubation a °c for h, the hrpo-conjugated mab f was added at a pre-determined optimal dilution. positive and negative controls for pedv were included in each plate. the diluting buffer consisted of pbs (ph . ) with . % tween and % yeast extract. following incubation at °c for h, the substrate solution (orthophenylenediamine dihydrochloride . mg/ml and . % h o in mm phosphate citrate buffer, ph . ) was then added. the colorimetric reaction was stopped after min by addition of n sulphuric acid and the absorbance values were read at nm using a spectrophotometer. results were expressed as an optical density (od). fifty microliters per well of reagent were dispensed; three washings with pbs-tween were performed after each incubation. a panel of four faecal samples, that had a different degree of viral load (from high positive to negative) when examined in dilution in rt-pcr (data not shown), were analyzed by das-elisa in serial two fold dilutions (from / to / ) in order to select the optimal dilution for test samples. the titration curves based on the od values provided evidence of a clear separation between the positive and negative samples (fig. ) . the dilutions / and / provided the widest window between them and were selected as screening dilutions combined with an od cut-off value . . all the field samples were examined in parallel using das-elisa and a rt-pcr performed using primers specific for the spike protein (s-protein) as previously described (kim et al., ) . the agreement between pcr and elisa techniques was measured with the kappa statistic value (landis and koch, ) . the results obtained by examining, respectively, faecal and intestinal samples with both methods are shown in table . when examining faeces, only two samples gave discordant results, i.e. they were negative by elisa, but were identified as pedv-positive by rt-pcr. the kappa value was high ( . ), suggesting an almost perfect agreement between the two methods. the elisa test may fail to detect antigens in faecal samples with very low viral titres specially when clinical specimens are collected in the recovery phase of the disease. other reasons could be the presence in faeces of specific antibodies that form immunocomplexes, or an excessive delay between collection and examination resulting in sample degradation and loss of antigenic sites. a lower number of intestinal samples resulted pedv positive in both arrays ( vs faecal samples) and, in addition, the two methods gave more discordant results, indicating just a substantial agreement (k = . ). in particular, of the intestinal samples were positive by both methods, whereas samples resulted elisa positive, but rt-pcr negative. such disagreement could be due to the presence of pcr inhibitors and dna damaging substances in stool samples, but can also result from poor-quality specimens, e.g. extremely autolysed tissues or those stored at room temperature for prolonged periods. in addition, false-positive reactions in the elisa assay may be due to the non-specific binding of antibodies used as reagents to intestinal bacteria or their products (brandt et al., ) . however, this was probably not the case since the results did not change, even after incubation of elisa pos/rt-pcr neg samples with a negative mouse serum in order to block a potential binding between murine antibody (mabs) and pig specimens. it is already known that elisa results depend on the clinical and pathological data: in intestinal contents and faecal specimens obtained from experimentally infected pedv piglets, the virus shedding is detected with high consistency during the acute phase of disease, but much less frequently during the incubation period and the recovery phase (callebaut et al., ) . our data confirm that for a successful and correct diagnosis of pedv it is advisable to collect faecal materials at the onset of illness rather than taking intestinal contents from dead or suppressed animals with prolonged clinical signs. using das-elisa the presence of pedv was found in of ( . %) field samples of pigs with diarrhoea originating from different farms located in the po valley. a high proportion of positive results was detected in samples taken between january and june ( of examined, . %), during an epidemic of diarrhoea in the same area of italy (martelli et al., ) , from pigs of all ages. it is therefore possible that virus persisted after the acute phase in some groups of animals, for example after weaning, but this aspect was not specifically investigated by serology. during the latter part of and , the pedv was detected in field samples at a lower rate, i.e. of ( . %). the rapid decrease of the frequency of detection is probably also related to the quick development of a sufficient level of immunity in the population that caused a reduction of virus circulation. these findings are similar to those found in other countries with developed swine production (pensaert and sang-geon, ) . on the whole the results obtained by testing samples of naturally infected swine indicate that the das-elisa here described could be considered as a reliable and accurate assay for the diagnosis of pedv in clinical specimens. such data agree with previous studies in which the pedv-elisas were employed for the screening for pig herds during epizootic outbreaks (carvajal et al., ; rodak et al., ; van nieuwstadt and zetstra, ) . the advantages of the das-elisa compared with rt-pcr assay derive from the simple and rapid procedure, suitable for the screening of a large number of specimens, and from the use of mabs, that ensure standardisation and reproducibility. when taking into consideration the epidemiological characteristics of pedv, the rapid identification of the etiological agent would facilitate the implementation of effective control measures. comparison of direct electron microscopy and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhoea virus and its antibodies preparation of monoclonal antibodies, strategies and procedure propagation of the virus of porcine epidemic diarrhoea in cell culture differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by rt-pcr isolation of porcine epidemic diarrhea virus (pedv) infection in korea the measurement of observer agreement for categorical data an epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea transmissible gastroenteritis and porcine epidemic diarrhoea in britain an elisa optimized for porcine epidemic diarrhoea virus detection in faeces passive protection against porcine epidemic diarrhoea (ped) virus in piglets by colostrum from immunized cows an outbreak of swine diarrhoea of a new type associated with coronavirus-like particles in japan highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibodies conjugates for enzyme immunoassays use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhoea virus key: cord- -gv sgbk authors: shin, gu-choul; chung, yoon-seok; kim, in-soo; cho, hae-wol; kang, chun title: preparation and characterization of a novel monoclonal antibody specific to severe acute respiratory syndrome-coronavirus nucleocapsid protein date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: gv sgbk severe acute respiratory syndrome-coronavirus nucleocapsid (sars-cov n) protein has been found to be important to the processes related to viral pathogenesis, such as virus replication, interference of the cell process and modulation of host immune response; detection of the antigen has been used for the early diagnosis of infection. we have used recombinant n protein expressed in insect cells to generate mabs directed against this protein. we selected five mabs that could be used in various diagnostic assays, and all of these mabs recognized linear epitopes. three igg( b) mabs were recognized within the n-terminus of n protein, whereas the epitope of two igg( ) mabs localized within the c-terminus. these mabs were found to have significant reactivity with both non-phosphorylated and phosphorylated n proteins, which resulted in high reactivity with native n protein in virus-infected cells; however, they did not show cross-reactivity with human coronavirus. therefore, these results suggested that these mabs would be useful in the development of various diagnostic kits and in future studies of sars-cov pathology. severe acute respiratory syndrome (sars), which is caused by the sars coronavirus (sars-cov), is a newly emerging disease. sars-cov presented with high virulence and mortality, and affected countries, with more than cases and over deaths. indeed, as the clinical symptoms of sars are nonspecific compared to those of other respiratory viruses, diagnosis relies largely on laboratory tests . thus, the development of diagnostic laboratory tests for specific and early detection of sars-cov infection is of great importance for both rapid treatment of patients and control of sars outbreaks. thus far, virus isolation methods have generally been performed to determine the presence of infectious virus in samples; this process is relatively time-consuming and inefficient (keyaerts et al., ; yamashita et al., ) . rt-pcr and real time-pcr for direct detection of sars-cov rna is expen-sive and labor-intensive, relies on the availability of expertise, and may produce false-positive results due to contamination (keightley et al., ; huang et al., ) . serological methods, such as immunofluorescence assay and enzyme-linked immunosorbent assay (elisa), are not adequate for the early diagnosis of sars, because the median time to seroconversion in sars patients is - days after the onset of symptoms . therefore, the generation of monoclonal antibodies (mabs) against sars-cov antigens may provide possible diagnostic tools for early diagnosis of sars, because sars-cov can be specifically detected in the respiratory specimens, blood and stool much earlier than antibodies can be used for detection (lau et al., ; di et al., ) . the sars-cov nucleocapsid (sars-n) protein is a phosphoprotein of kda, and performs multiple functions in viral pathogenesis, such as providing a nuclear-import signal, interfering in the cell process, participating in virus replication and packaging rna (egloff et al., ; yan et al., ; surjit et al., surjit et al., , . thus, this protein may have important roles in the pathogenesis of sars. furthermore, this protein is the most abundantly expressed structural protein during infection and is highly detectable in sars patients lau et al., ; di et al., ) . therefore, this protein may serve as one of the immunodominant antigens in the early diagnosis of infection. furthermore, some researchers have suggested that antibody against the n protein could modulate cytokine responses such as il ; non-neutralizing antibodies against n protein were found to protect mice against lethal infection (nakanaga et al., ; cheng et al., ) . therefore, the development of mabs against sars-n protein may be critical in the development of drugs to treat sars-cov infection, and for further study of the pathogenesis of sars, as well as early diagnosis. here, we report the production of mabs against sars-n and the properties of the mabs, which were determined by isotyping, affinity assay, epitope mapping and reactivity with various isoforms of sars-n protein. we also suggested the applicability of the mabs in various analytical methods, such as ifa, immunoblot and antigen-capture elisa, for diagnosis and functional study of sars-cov n protein. sp / myeloma cells were kindly provided by metabolab inc. (seoul, republic of korea), and mrc- cells (atcc, ccl- ) were obtained from american type culture collection. sars-cov stock was provided from the center for disease control and prevention, and this virus was maintained in biosafety level- (bsl- ) containment laboratories in the national institute of health, korea center for disease control and prevention. the sars-cov titer was determined to be × % tissue culture infectious doses/ml (tcid /ml). the virus culture supernatants were inactivated by heating at • c for min prior to use. human coronavirus oc (atcc, vr- ; hcov oc ), which was tested to determine cross-reactivity with sars-n mab, was prepared from mrc- cells. replication of these viruses was confirmed by rt-pcr and immunofluorescence assay using mab against nucleocapsid protein of human coronavirus oc (hcov mab). the hcov oc titer was determined by hemagglutination assay (ha) using human red blood cells (rbcs). the complete coding sequence for the n protein (urbani strain, genbank accession no. ay , , - , bp) was amplified from the sars-cov genomic rna using pcr. the amplified product was digested with ecori and bamhi, and then inserted into his-tagged pentr bhrnx vector (neurogenex, seoul, republic of korea) to create pentr np . recombinant baculovirus was generated by co-transfection of sf cells with pentr np and linearized baculovirus dna using the baculogoldtm system (bd biosciences, san jose, ca). recombinant baculoviruses were harvested from sf cell culture medium h post-transfection and broken by three cycles of freezing-thawing. the × histidine-tagged recombinant n protein (brsars-n) was purified by metal-chelating affinity chromatography (merck bioscience, darmstadt, germany). the purified protein was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page), as described previously (laemmli, ) . to examine the reactivity of sars-n mabs against the phosphorylated protein and non-phosphorylated protein, rsars-n protein expressed in escherichia coli (ersars-n) was purchased as nonphosphorylated protein from biovendor laboratory medicine, inc. (heidelberg, germany). balb/c mice (samtako inc., republic of korea; weeks) were intraperitoneally injected with a mixture containing g purified brsars-n proteins in l phosphate buffered saline (pbs) and an equal volume of freund's complete adjuvant. a boost injection with the same amount of antigen in freund's incomplete adjuvant was administered at -week intervals. hybridoma fusion was performed using a method similar to that originally described (kohler and milstein, ) , with the following modifications. in brief, the splenocytes were harvested from immunized mice, mixed with sp / cells at a : ratio, and fusion was carried out with % polyethylene glycol- (roche, indianapolis, in). the fused cells were collected by centrifugation at × g for min and the cell pellet was resuspended in dmem (invitrogen, carlsbad, ca) containing % fbs and hat supplement (sigma-aldrich korea co., seoul, republic of korea). the cells were seeded in -well plates at l/well ( × cells/well) and cultured in a co incubator. antibody produced in medium was measured by indirect enzyme-linked immunosorbent assay (indirect elisa) as described below. a limiting dilution of hybridoma was carried out from putative positive individual wells, and the screening was repeated until hybridoma clones producing a strongly reactive sars-n mab were observed. selected hybridoma clones were maintained in dmem containing % fbs, × ht supplement (sigma-aldrich korea) and exchanged with fresh media once every or days. the sars-n mab of a selected hybridoma was purified using the immunopure (g) igg purification kit (pierce biotechnology inc., rochford, il) and isotyped with an immunopure monoclonal antibody isotyping kit ii (pierce biotechnology inc.), used according to the instructions of the manufacturer. the indirect elisa was carried out on a maxisorp plate (nalgen nunc international, rochester, ny), which had been coated with g of recombinant sars-ns diluted in mm carbonate buffer (ph . ) and incubated overnight at room temperature. non-specific protein binding sites were blocked with % bovine serum albumin (bsa) in pbs for h at • c. plates were washed with pbs containing . % tween (pbst). hybridoma supernatants, sars-n mab and anti-sars serum obtained from mice immunized with heat-inactivated sars-cov, or normal mouse serum as negative serum, were then added and incubated for min at • c. after washing with pbst, a : dilution of alkaline phosphatase (ap)-conjugated goat anti-mouse igg+ iga+ igm antibody (abcam) in pbst containing % bsa was added to all wells and incubated for min at • c. after a final wash, p-nitrophenyl phosphate disodium salt (pnpp, pierce biotechnology inc.) solution was added, and the plates were further incubated for min at room temperature. the color intensity was measured as the absorbance value at nm (od ) in an el elisa reader (bio-tek intstruments inc., winooski, vt). isotyping was performed using immunopure monoclonal antibody isotyping kit ii (pierce biotechnology inc.), used according to the instructions of the manufacturer. to examine whether sars-n mab recognizes the linear epitope of sars-n protein, we performed an immunoblot assay in denaturing conditions of the sars-n protein. the purified rsars-n protein were resolved by % sds-page, and were then electrotransferred onto a pvdf membrane (bio-rad, hercules, ca) and blocked with % bsa in pbst. membrane was incubated with sars-n mabs ( g/ml) or anti-his tag mab ( : dilution, abcam), as positive control, for h, and non-specific adsorption was washed away by pbst. the bound antibodies were detected by horseradish peroxidaseconjugated goat anti-mouse igg+ iga+ igm secondary antibody ( : dilution, abcam), followed by dab substrate solution (sigma-aldrich korea). a total of linear peptides ranging in size from to amino acid residues were synthesized on the basis of the fulllength nucleocapsid protein sequences of sars-cov urbani strain, genebank aap (cosmo co., seoul, republic of korea). the synthesized peptides were characterized by hplc and mass spectrometry. to identify the epitopes in sars-cov n protein that are targeted by sars-n mabs, competition elisa was performed as described previously (chiang et al., ) . the sars-n mabs ( g/ml) were incubated with the competitor peptide or brsars-n protein ( g/ml) for h at • c. the mabs were then transferred into the wells of a maxisorp plate that had been coated with . g/well of sars-n protein. the following steps were then accomplished as described in the indirect elisa procedure. affinity analysis was performed by non-competitive elisa as described previously (huang et al., ) , with the following modifications. in brief, maxisorp plate was coated overnight with g/well of brsars-n, as described in the indirect elisa procedure. the plates were blocked with % bsa, followed by incubation of sars-n mab with serial dilution ( , , , , . , . , . , . , . , . and . nm). after washing with pbst, ap-conjugated goat anti-mouse igg+ iga+ igm antibody (abcam) in pbst containing % bsa was added to all wells and incubated for min at • c. the following steps were accomplished in the same manner as that used for indirect elisa, described above. the affinity constants were presented as the concentrations (nm) of mab at . of od . reactivity of sars-n mabs with sars-cov infected cells was determined by immunofluorescence assay, performed according to the instructions of the manufacturer (euroimmun, germany). the sars-n mab ( g/ml), anti-sars serum ( : dilution) and negative serum were added to each well, and the slides were incubated at • c for min. after washing three times with pbs, fluorescein isothiocyanate-conjugated goat anti-mouse igg+ iga+ igm antibody (icn biomedical, : dilution) was added, and the slides were then incubated at • c for min. the slides were then washed three times with pbs and one time with ultrapure water and observed by fluorescence microscopy. to prepare detector mabs, the purified mabs were labeled with biotin using the ez-link sulfo-nhs-lc-biotinylation kit (pierce biotechnology inc.) according to the instructions of the manufacturer. the purified mab for the antigen-capture was immobilized on the hi-bind microplate (costar, coring incorporated, ny) by incubating g/ml antibody in mm carbonate buffer (ph . ) at • c overnight. the wells were then washed twice with pbs, followed by blocking with % bsa in pbs, ph . , for h at room temperature. after removing the blocking reagent, the wells were dried and stored at • c prior to use. virus culture supernatant or recombinant n protein diluted in % bsa in pbst or virus culture medium was added to the wells ( l/well) and incubated for h at • c. after washing, the wells were incubated for h at • c with l per well of biotin-conjugated detector mab (diluted / in pbst with % bsa or virus culture medium). after washing, the wells were incubated for h at • c with l per well of ap-conjugated goat anti-biotin antibody (diluted / in pbst with % bsa or virus culture medium, abcam). after washing, l of pnpp was added to each well. the color reaction was stopped after min with l of n naoh to each well, and the plates were examined at nm in an el elisa reader (bio-tek instruments inc.). the experiments involving the use of the inactivated sars-cov were performed in a bsl- laboratory. for the immunofluorescence assay to assess the crossreactivity of mabs with human coronavirus, spot slides were prepared by applying a suspension of cells infected with uvc-irradiated human coronavirus oc or a suspension of uninfected mrc- cells to the wells of teflon-coated slides. slides were allowed to air-dry before they were fixed in methanol. slides were stored at − • c until used for indirect fluorescence assay. the hcov mab ( : dilution, chemicon international inc., temecula, ca) as positive control, normal mouse serum as negative control, and sars-n mabs ( g/ml) were added to the slides, and the slides were incubated at • c for min. the following steps were accomplished in the same manner as that described above for sars-cov. for antigen-capture elisa, human coronavirus oc ( ha unit) supernatant that had been cultured from mrc- cells was heat-inactivated for min at • c and serially diluted two-fold. the protein samples were then extracted by three cycles of freezing-thawing before centrifugation at , rpm for min and dilution in pbs with % bsa. the preceding steps were performed in the same manner as that used for sars-cov, described above. full-length sars-n protein was produced in a recombinant baculovirus system and used in the immunization of mice. two weeks after each antigen boost dose, immunized mice were screened for sars-n specific antibody response by indirect elisa. splenocytes isolated from the mice were fused with sp / myeloma cells, resulting in ∼ proliferating hybridomas. subsequent screening of these hybridomas and single cell cloning yielded positive clones that constitutively secreted mabs that reacted to sars-n protein by indirect elisa (data not shown). further isotyping of these mabs were determined in order to facilitate future utilization. for the heavy chain subclasses, most of the mabs were found to belong to the igm subtype, two of the mabs belonged to igg , and three of the mabs belonged to igg b . the light chain of all of these mabs was of the kappa isotype (table ) . five of the mabs determined to be of the igg subclass were characterized by further analysis, because these mabs may be suitable for use as diagnostic mabs. immunoblotting was performed with brsars-n protein in order to analyze the reactivity of sars-n mabs against sars-n protein under denaturing conditions and to determine whether these mabs recognize the conformational epitope of sars-n table isotypes of the sars-n mabs generated in this study these isoforms were also observed in immunoblot analysis of total proteins extracted from baculovirus-infected cells and sars-cov-infected cells (data not shown). thus, these isoforms may be fragment cleaved by intracellular protease. the fragment of -kda that was not observed in originally purified brsars-n protein could be obtained by freezing and thawing procedures of purified protein. thus, this result indicated that the epitopes of all of the sars-n mabs are linear, and the epitopes of igg b and igg mabs are located in the n-terminus and c-terminus of sars-n protein, respectively. to more precisely analyze the epitope of sars-n protein recognized by these sars-n mabs, competitive elisa was conducted using the synthetic peptides covering the full-length sars-n protein sequence. among these synthetic peptides, the n peptide (ategalntpkdhigtr; at position - of sars-n protein) and n peptide (tfggptd-stdnnqngg; at position - ) could effectively compete in the binding of all of the igg b mabs with the sars-n protein, and the n peptide (gpeaslpygankegiv; at position - ) seems to have slightly weak activity, while the other peptides did not exhibit any effect in this assay ( fig. a) . the n and n peptides contain no apparent common epitopes, which revealed that these mabs are mixed antibodies. the n peptide (ggetalalllldrlnqleskvsgkg; at position - ) resulted in complete inhibition of the binding activity of all of the igg mabs with the sars-n protein, and the n peptide (qtvtkksaaeaskkprqkrtatkq; at position - ) seems to have slightly weak activity (fig. b ). according to the results of the immunoblot and competitive elisa, the epitopes of the igg b mabs is located in the nterminal region at aa - and - , whereas that of the igg mabs is located in the middle region at aa - (fig. c ). to examine the reactivity of the sars-n mabs against sars-cov n protein under non-denaturing conditions, the indirect elisa was performed with brsars-n protein. four of the selected mabs ( - - , - - , - - and - - ) bound slightly better to sars-n protein than - - , as shown in table . to confirm the reactivity of mabs against sars-n protein, as estimated by indirect elisa, the affinity constants of five mabs were measured by non-competitive elisa, as described in section . the affinity constants of - - , - - , - - and - - were significantly higher than that of - - (table ) . these results were similar to those of indirect elisa and indicated that the affinity levels paralleled the reactivity estimated by indirect elisa, and all sars-n mabs, except for - - mab, could be suitable for the development of sensitive methods for sars-cov diagnosis. since these mabs were generated using recombinant sars-n protein expressed in insect cells that contained phosphorylated n protein (data not shown), indirect elisa was performed to further examine the reactivity of sars-n mabs with the ersars-n expressed in e. coli as non-phosphorylated protein. although sars-n mabs showed slightly higher reactivity with the brsars-n protein than with the ersars-n protein, all of these mabs showed significant reactivity with the ersars-n protein compared with the reaction of negative serum (fig. ) . this result revealed that all sars-n mabs were effectively bound with both phosphorylated and non-phosphorylated n protein. immunofluorescence assay was performed on sars-cov infected vero cells to further assess whether the sars-n mabs recognize the native-form of endogenously synthesized n protein in sars-cov infected cells. both the negative serum and the five mabs did not show non-specific reactions with uninfected cells. all five sars-n mabs strongly reacted with sars-cov infected cells, whereas negative serum showed no reaction (fig. ) . however, - - mab showed a significantly weak reaction in affinity constants, but reacted strongly in the immunofluorescence assay; the reason for this result is unclear. the fluorescence signals of the mabs were predominantly shown in the cytoplasm of sars-cov infected cells. this indicated that all mabs were able to detect native-form n protein in sars-cov infected cells. in order to establish a sensitive and less time-consuming antigen-capture elisa for the sars-n protein, we tested each of the pairs of mabs from the five selected mabs; this allowed us to determine the highest detection sensitivity for recombinant n protein and sars-cov culture supernatant. we found that the immobilization of a mixture of - - and - - mab as capture antibody on the elisa plate, followed by the detection with biotin-conjugated - - mab, gave the best result (data not shown). to determine the sensitivity of antigencapture elisa, a serial dilution of the recombinant n protein was used to determine a standard curve (fig. ) . normal vero cell culture media were used to determine the baseline for antigencapture elisa at an optical density of . at nm (od ). therefore, the cut-off value for detection of viruses in cell culture was set to be . , which is equal to the mean + s.d. of the od for normal cell culture media. according to the cut- fig. . detection of sars-n protein in sars-cov-infected cells by immunofluorescence assay. the immunofluorescence assay was performed using the sars ifa kit according to the instructions of the manufacturer. anti-sars indicated positive serum obtained from mice immunized with heatinactivated sars-cov and negative indicated normal mouse serum. these mabs reacted with sars-n in virus-infected vero cells, whereas they did not react with uninfected cells. off threshold ( . ), a − dilution of recombinant n protein and a − dilution of the virus culture supernatant were considered positive (fig. ) . thus, it was deduced that as little as pg of recombinant n protein and tcid of virus culture supernatant could be detected. this result revealed that the sars-n mabs could be useful for detecting the n protein in virus culture supernatant and respiratory specimens from sars patients. to determine the specificity of the five sars-n mabs, immunofluorescence assays were performed in human coronavirus-infected mrc- cells. all five mabs showed no cross-reactivity with the antigens of the human coronavirus (fig. a) ; uninfected mrc- cells also showed no crossreactivity (data not shown). to further assess the specificity of the mabs, antigen-capture elisa was performed with human coronavirus-infected cell lysates and brsars-n protein as positive control (fig. b) . all of the mabs reacted only with sars-n protein, and did not react with human coronavirus, as shown by the results of immunofluorescence assays. this revealed that all of the sars-n mabs could specifically recognize the n protein of sars-cov. sars-cov is an etiological agent that causes severe acute respiratory syndrome, an infectious disease that has only recently emerged . therefore, there is an intense need for the development of sensitive and specific detection methods for sars-cov infection. many methods have been employed recently for the detection of sars-cov infection (keyaerts et al., ; yamashita et al., ; keightley et al., ; huang et al., ) . of these diagnostic methods, rt-pcr and real time-pcr have been the most widely used. however, these methods possess some general problems, as they are time-consuming and labor-intensive, require sophisticated instruments, and have high rates of false positivity. on fig. . cross-reactivity of sars-n mabs with human coronavirus antigens. (a) the immunofluorescence assay was performed by using sars-n mabs on human coronavirus oc -infected mrc- cells. as a positive control, anti-hcov was used as the mab against human coronavirus oc n protein, and normal mouse serum was used as the negative control. fm and lm indicate fluorescence and light imagery. none of these mabs showed cross-reactivity with human coronavirus. (b) cross-reactivity of sars-n mabs was examined by antigen-capture elisa using human coronavirus oc lysates ( ha unit), brsars-n protein ( ng/well) and pbst buffer with % bsa as control. the other hand, laboratory methods detecting viral antigen by mabs, including antigen-capture elisa, provide more rapid, less labor-intensive, and more convenient alternatives (lau et al., ; di et al., ) . in this study, we generated five positive clones secreting specific and highly reactive antibodies against sars-cov n protein in order to develop diagnostic methods. these mabs were available for use in detecting sars-cov n protein by various diagnostic methods, such as immunoblot assay, immunofluorescence assay and antigen-capture elisa (table ) . we also revealed the availability of these mabs in the quantification of sars-n protein by antigen-capture elisa. the detection limit of this test is pg of recombinant protein and tcid of sars-cov. this sensitivity is consistent with previous studies of sensitivity in other antigen-capture elisas (che et al., ; di et al., ) . therefore, these five mabs may be employed in the construction of various diagnostic methods for the detection of sars-cov and in quantitative analysis of viral antigen and virus titer. the major antigens of sars-cov structure proteins are the spike (s) protein and n protein (lau et al., ; di et al., ; lu et al., lu et al., , . however, recent reports have demonstrated that, because the s protein is expressed at very low levels in vivo and in cultured cells (zeng et al., ; , it is difficult to directly detect the soluble s protein from sars patients. thus, the s protein may not be suitable for use as a practical diagnostic antigen. in contrast, the n protein can be detected at significant levels in patient serum, as well as in respiratory tract samples at early stages of sars-cov infection (lau et al., ; di et al., ) . these previous reports support that the development of mabs against sars-n protein is an adequate approach for the diagnosis of sars. therefore, we generated mabs directed against sars-cov n protein and demonstrated that these sars-n mabs can successfully detect the n protein in sars-cov-infected cells; this is very useful in diagnosing sars patients. sars n proteins exist as phosphorylated forms in mature viral particles, whereas, in host cells, this protein exists in both the dephosphorylated form and the phosphorylated form (kalicharran and dales, ; surjit et al., ) . therefore, the mabs available for developing sensitive diagnostic methods have to recognize the non-phosphorylated protein as well as phosphorylated protein. a previous report suggested that, because of conformational differences between proteins, the mabs recognizing a protein expressed in insect cells cannot recognize the protein of same cdna constructs expressed in e. coli (vapalahti et al., ) . therefore, the sars-n mabs obtained in the present study were generated using recombinant sars-n protein expressed in insect cells; these mabs may not recognize the n protein expressed in e. coli. however, all of the sars-n mabs reacted significantly with the ersars-n expressed in e. coli, as well as the phosphorylated form of the brsars-n protein. these results demonstrate that these mabs can effectively detect the non-phosphorylated n protein that exists in host cells during viral replication, as well as the phosphorylated n protein in host cells and viral particles. all of these mabs could successfully detect native-form n protein in infected cells, as well as in viral particles. thus, these mabs may be useful in the development of sensitive methods used for the diagnosis of sars. epitope mapping studies of the sars-n mabs demonstrated that one of the three epitopes that were originally reported to be located in the highly immunodominant region (chen et al., ; he et al., ) is located at the middle region of sars-n protein (aa - ; igg subclass mabs). the others were newly identified at the n-terminus, which is shared with an rna binding domain (aa - and - ; igg b subclass mabs) that is the minor b cell epitope (he et al., ) . furthermore, all of these sars-n mabs were reactive in immunoblotting, which suggests that they recognized linear epitopes in the n protein. a recent report demonstrates that the n protein is easily degraded into various isoforms in the lysates of sars-cov-infected cells (zeng et al., ) . we can also suggest, as previously described, that various isoforms existed in sars-n protein expressed in insect cells and could be detected by immunoblot assay using these mabs. therefore, the blend of two mabs against the different epitopes can be used to detect various fragments from sars-n protein and enhance the sensitivity of diagnostic tools. although the sars-n protein shares low homology (approximately - %) with n proteins of other hcovs, a previous report has described that the sars-n protein has strong crossreactivity with sera against hcovs (sun and meng, ) . hence, anti-sera against sars-cov may be cross-reactive with other hcovs. however, previous reports support the idea that the sars-n mabs did not recognize the n proteins of other hcovs (che et al., ) . therefore, the issues of cross-reactivity during the detection of sars-n protein with polyclonal anti-sera can potentially be overcome by the use of a specific mab against sars-cov. in the present study, the sars-n mabs did not show cross-reactivity with n proteins of hcov, as determined by immunofluorescence assay and antigen-capture elisa. therefore, all of these sars-n mabs will be useful as mabs for the development of specific diagnostic methods to discriminate sars-cov infection from hcovs infection. sars-n protein is currently assumed to play an important role in the pathogenesis of sars, as well as in viral transcription and replication. for example, the n protein can modulate numerous intracellular mechanisms involved in apoptotic signal transduction pathways, cell cycle regulatory pathways and cellular immune response and inflammation (egloff et al., ; yan et al., yan et al., , liao et al., ; surjit et al., surjit et al., , surjit et al., , chang et al., ) . furthermore, sars-n protein is released as a soluble antigen in infected cells, and as high levels of n protein circulating in the blood vessels (che et al., ; di et al., ) . thus, sars-n protein in its soluble form may play an important role in extracellular pathogenesis of sars. however, it is totally unclear which of the intracellular and extracellular mechanisms are involved in viral replication and pathogenesis, and how the intracellular mechanisms are regulated by the n protein. therefore, these sars-n mabs may be extremely useful in providing further insight into the mechanisms of the n protein involved in the pathogenesis of sars. in conclusion, the sars-n mabs generated in the present study will be useful in providing reliable, sensitive, specific and convenient diagnostic kits for sars-cov detection; these diagnostic methods may include the immunoblot assay, immunofluorescence assay and antigen-capture elisa. furthermore, these mabs will be very useful in future studies concerning the contribution of sars-n protein in sars-cov pathology. modular organization of sars coronavirus nucleocapsid protein nucleocapsid protein as early diagnostic marker for sars antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein crossreactivity of antibody against sars-coronavirus nucleocapsid protein with il- characterization of a monoclonal antibody specific to the gag protein of porcine endogenous retrovirus and its application in detecting the virus infection monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay reveals high sensitivity of the nucleocapsid protein in acute-phase sera of severe respiratory syndrome patients the severe acute respiratory syndrome-coronavirus replicative protein nsp is a single-stranded rna-binding subunit unique in the rna virus world mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus rapid and sensitive detection of multiple genes from the sars-coronavirus using quantitative rt-pcr with dual systems development of new rabbit monoclonal antibody to estrogen receptor: immunohistochemical assessment on formalin-fixed, paraffin-embedded tissue sections dephosphorylation of the nucleocapsid protein of inoculum jhmv may be essential for initiating replication real-time nasba detection of sars-associated coronavirus and comparison with real-time reverse transcription-pcr growth kinetics of sars-coronavirus in vero e cells continuous cultures of fused cells secreting antibody of predefined specificity a novel coronavirus associated with severe acute respiratory syndrome cleavage of structural proteins during the assembly of the head of bacteriophage t detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay activation of nf-kappab by the full-length nucleocapsid protein of the sars coronavirus immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus synthetic peptides derived from sars coronavirus s protein with diagnostic and therapeutic potential protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice antigenic cross-reactivity between the nucleocapsid protein of severe acute respiratory syndrome (sars) coronavirus and polyclonal antisera of antigenic group i animal coronaviruses: implication for sars diagnosis the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos- cells in the absence of growth factors the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by - - -mediated translocation the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells early detection of antibodies against various structural proteins of the sars-associated coronavirus in sars patients the spike protein of severe acute respiratory syndrome (sars) is cleaved in virus infected vero-e cells susceptibility of human and rat neural cell lines to infection by sars-coronavirus sars coronavirus induces apoptosis in vero e cells nucleocapsid protein of sars-cov activates the expression of cyclooxygenase- by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein proteomic analysis of sars associated coronavirus using two-dimensional liquid chromatography mass spectrometry and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectrometric analysis we gratefully acknowledge w. bellini from the centers for disease control and prevention for kindly providing of the sars coronavirus. we also thank neurogenex co. for the technical assistance in the production of recombinant baculovirus and cosmo inc. for the technical assistance in the production of the sars mabs.this work was supported by the intramural research fund from the korea centers for disease control and prevention. key: cord- - posyr n authors: mohammadzadeh, sara; khabiri, alireza; roohvand, farzin; memarnejadian, arash; salmanian, ali hatef; ajdary, soheila; ehsani, parastoo title: enhanced-transient expression of hepatitis c virus core protein in nicotiana tabacum, a protein with potential clinical applications date: - - journal: hepat mon doi: . /hepatmon. sha: doc_id: cord_uid: posyr n background: hepatitis c virus (hcv) is major cause of liver cirrhosis in humans. hcv capsid (core) protein (hcvcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. although invention of transgenic (stable) tobacco plants expressing hcvcp with proper antigenic properties was recently reported, no data for “transient-expression” that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for hcvcp. objectives: the purpose of this study was to design a highly codon-optimized hcvcp gene for construction of an efficient transient-plant expression system for production of hcvcp with proper antigenic properties in a regional tobacco plant (iranian jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco. materials and methods: a codon-optimized gene encoding the kozak sequence, xhis-tag, hcvcp ( - ) and kdel peptide in tandem (from n- to c-terminal) was designed and inserted into potato virus-x (pvx) and classic pbi binary vectors in separate cloning reactions. the resulted recombinant plasmids were transferred into agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. the effect of gene silencing suppressor p protein derived from tomato bushy stunt virus on the expression yield of hcvcp by each construct was also evaluated by co-infiltration in separate groups. the expressed hcvcp was evaluated by dot and western blotting and elisa assays. results: the codon-optimized gene had an increased adaptation index value (from . to . ) and reduced gc content (from . to . ) in tobacco and removed the possible deleterious effect of “ggtaag” splice site in native hcvcp. blotting assays via specific antibodies confirmed the expression of the kda hcvcp. the expression level of hcvcp was enhanced by - times in p co-agroinfiltrated plants with better outcomes for pvx, compared to pbi vector ( . % versus . % of the total soluble protein). the plant-derived hcvcp (phcvcp) could properly identify the hcvcp antibody in hcv-infected human sera compared to escherichia coli-derived hcvcp (ehcvcp), indicating its potential for diagnostic/immunization applications. conclusions: by employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of hcvcp in tobacco with proper antigenic properties could be possible. hepatitis c virus (hcv), the major cause of blood-borne chronic hepatitis with potential progression to cirrhosis, has infected around million people globally ( ) . due to heterogeneity of hcv proteins, their high mutation rates and complex pathogenesis, no approved vaccine for human application against this viral infection is available to date ( , ) . recognition of conserved epitopes in hcv proteins and advancements in the formulation of vaccines in novel modalities, however, have led to the placement of a few hcv vaccines in the pipeline of human clinical trials in recent years ( , ) . hcv holds a single-stranded positive-sense rna genome which encodes for three structural (core protein and envelope proteins e and e ) and six nonstructural proteins ( ) . among the hcv proteins, core (hcvcp) is the most conserved hcv antigen, and hence, a good candidate to be considered for hcv vaccine formulations ( ) ( ) ( ) . accordingly, application of even isolated hcvcp-ctl epitopes ( - amino acids) in the context of synthetic multi-epitope hcv vaccines has also been reported ( ) ( ) ( ) . besides, anticore antibodies are the first to be raised after the onset of infection, a property that has provided an important diagnostic value for this protein and has located it among antibody-capturing antigens in commercially available serological assays for hcv diagnosis ( ) . in addition, nucleotide sequence of hcvcp is supposed to encode for other alternated frame-shift proteins (arfps) with important pathogenic roles in chronic hcv infection and cirrhosis ( , ) . therefore, to fulfill different diagnostic, research and therapeutic demands, production of hcvcp in various expression systems has been addressed ( ) ( ) . however, since the c-terminal hydrophobic region of hcvcp exerts immune-suppressive effects ( , ) , its first n-terminal residues, the hydrophilic region (hcvcp n- ), which contains both nuclear localization signals (nls) ( ) and most of the conserved b and t cell epitopes, is usually employed for different diagnostic ( , ) and vaccine applications ( ) ( ) ( ) . plants have an extensive potential to provide safe and inexpensive sources of biopharmaceutical and vaccination proteins ( ) . currently, several plant-derived viral proteins such as hepatitis b surface antigen ( ) and norwalk virus capsid protein ( ) are in vaccination clinical trials. recently, invention of transgenic tobacco plants expressing hcvcp stably at t and t generations ( ) or directing the expression of its -amino acid nterminal in tobacco chloroplasts ( ) were reported. although these prior reports proved the proper antigenic structure of the plant-derived hcvcp for diagnostic purposes and indicated its full functionality to react with poly/monoclonal anti-core antibodies and hcv-infected human sera, however, both studies addressed only the transgenic (stable) tobacco plant generation for hcvcp. however, transient expression in plants, compared with transgenic plant generation, is currently the method of choice due to several advantages like the simplicity and feasibility of rapid protein expression, omitting tissue culture and regeneration costs and ease of large scale industrial plans ( ) ( ) ( ) . an important example of transient expression in plants is the production of seasonal influenza vaccines in tobacco ( ) . the success in efficient plant transient expression, however, depends on several factors such as: codon-optimization of the heterologous gene according to the plant-codon adaptation index ( , ) , selection of the proper expression vector ( , ) , and efficient inhibition of the gene silencing phenomenon which suppresses the expression of foreign genes in plants ( ) . while at present knowledge and techniques for the abovementioned parameters are being improved, providing novel data on transient expression process for the regionally-adapted plant hosts in general, and overcoming the plant-based gene silencing phenomenon in specific, is of high importance. in this study, we aimed to: i) construct an efficient transient tobacco expression system for hcvcp n- by designing a highly codon-optimized gene and employment of the iranian jafarabadi-tobacco plant cultivar which is a high biomass producer ii) evaluate the expression level of hcvcp for a potato virus x-based vector (pvx) ( ) com-pared to a classic pbi binary plant vector in co-agroinfiltration with p gene silencing suppressor plasmid and iii) assess the antigenicity of this tobacco-derived hcvcp for potential clinical (diagnostic and vaccine formulation) applications. the pivex . a core plasmid encoding hcvcp n- , used as both the source of the sequence for construction of plant-based expression vectors and for the expression of n-terminally xhis-tagged hcvcp protein in bl -ai strain of escherichia coli by arabinose induction (as positive control) was previously described ( , ) . several modifications for optimized (plant) codon-usage of hcvcp nucleotide sequence were considered, including: i) codon optimization according to the codon adaptation index of nuclear-encoded genes of tobacco ( ) , ii) removal of (plant) mrna destabilizing sequences from the native hcvcp coding sequence, iii) addition of kozak (gccac-catggc) sequence ( ) and hexahistidine ( xhis)-tag for nickel affinity purification at the ′ site, iv) addition of nucleotides encoding endoplasmic reticulum retrieval signal (kdel) at the ′ end ( ) , v) addition of bamhi and saci restriction sites at both ends of the gene for directional cloning into the same sites of the plant expression binary vector pbi ( figure ). the plant-optimized hcvcp gene for recombinant expression in tobacco (also termed tr-hcvcp) was synthesized and delivered as a clone in puc plasmid by shinegene molecular biotech inc. (shanghai, china). as shown in figure a, the synthetic gene was subcloned into bamhi and saci sites of the pbi vector ( ) under the control of camv s promoter and upstream of the nopaline synthase transcriptional terminator (nos-ter). in this study, the pvx-gw vector ( ) , which was kindly gifted by dr. cristiano lacorte (embrapa recursos geneticos e biotecnologia, brasília, brazil), was used as the pvx-based viral vector. for cloning of the tr-hcvcp sequence into pvx-gw vector, the synthetic gene was pcr amplified using a forward primer, f-kozak-vx: ΄-taatatcgatctcgagccaccatggctcatcacc- ΄ and a reverse primer, r-core-vx: ΄-taatgtcgacggatcct-cagagttcgtccttctttc- ΄ harboring clai and sali restriction sites (underlined sequences). pcr amplification was performed with cycles at ˚c for seconds, ˚c for seconds, and ˚c for seconds and ˚c for minutes. the -bp amplicon was subsequently digested by clai and sali enzymes and cloned into the same sites of pvx-gw vector under the control of duplicated pvx coat protein subgenomic promoter (cpp) (figure b ). all the recombinant constructs were confirmed by restriction and sequencing analyses using f-kozak-vx and r-core-vx specific primers. all the cloning and molecular procedures were according to the standard protocols ( ). the changes to the original sequence are shown by lower case letters. location of the kozak sequence, xhis-tag, nucleotides encoding kdel and restriction sites for bamhi and saci are indicated. atg and tga denote the start and termination codons, respectively. a. tumefactions strain lb was transformed with pvxcore and pbi-core vectors ( figure a and b) in separate reactions via the standard freeze-thaw protocol ( ) . to this end, the hcvcp-coding plant constructs, pbi-core and pvx-core were introduced into agrobacterium lb (in the case of pbi-core) and into agrobacterium lb ; the latter had already harbored the helper plasmid psoup ( ) (in the case of pvx-core), which is essential for replication of the pvx-gw-based plasmids in agrobacterium species, respectively. subsequently, transformed agrobacterium cells were selected on plates containing µg/ ml rifampicin (rif) and µg/ml kanamycin (kan) and incubated for hours at °c. the transformed colonies were confirmed by gene-specific colony pcr using f-kozak-vx and r-core-vx-specific primers, as described above. all the chemicals, culture media and antibiotics were purchased from sigma (us) and merck (germany) companies. to inhibit the silencing of foreign gene expression in tobacco, the constructed expression vectors were coinfiltrated with agrobacterium strain lb , harboring the p silencing-suppressor gene ( ) from tomato bushy stunt virus (tbsv) (genbank accession number: m ), which was previously cloned into pcambia vector (cambia co., australia) in our lab (p -pcambia vector). the agroinfiltration protocol was according to kapila et al. study ( ) . in brief, the agrobacterium cultures were first adjusted to od of . with ms medium supplemented with % (w/v) sucrose, mm mes ph = . , mm mgcl , and induced for an additional hours with µm acetosyringone. the agrobacterium suspensions were subsequently mixed in equal ratios (half for p plasmid and half for each of the expression constructs, either pvx-core or pbi-core) and the n. tabacum (cultivar jafarabadi; kindly provided by dr. tohidfar, agricultural biotechnology research institute of iran) leaves were immersed in the bacterial suspension while a vacuum of . mbar was applied for two minutes. the infiltrated leaves were placed adaxial side down on wet whatman paper in glass containers and subsequently incubated at °c with a -hour photoperiod for five days. the infiltrated tobacco leaves were ground into a fine powder in liquid nitrogen, using mortar and pestle. subsequently, . -ml extraction buffer ( mm nah po , ph = , mm nacl, . % tween , mm beta-mercaptoethanol, mm pmsf, and mm edta) was added to each gram of powdered leaf tissue. the total soluble protein (tsp) was removed from cell debris by centrifugation of the leaf extracts for minutes at °c ( rpm). the e. coli-derived hcvcp (ehcvcp) and plant-derived hcvcp (phcvcp) were purified through application of nickel-nitrilotriacetic acid (ni-nta) chromatography, as previously described ( , ) . protein concentrations were determined by bradford assay ( ) using bovine serum albumin (bsa) as standard. for dot blot analysis of proteins, equal concentration of tsp ( µg- µg) from plant extracts, µg from ehcv as positive control and µg of non-agroinfiltrated plant extract were directly dotted on a nitrocellulose membrane. the membrane was dried at room temperature, blocked with % (w/v) skim milk in pbs-tween ( / v/v), and after three washing steps incubated with anti-c/n terminal xhis antibody (qiagen) : in tbs-t for two hours at room temperature. following the washing steps, the membrane was incubated with : diluted horseradish peroxidase (hrp)-conjugated goat anti-mouse antibodies (sigma, usa) for one hour. finally, - 'diaminobenzidine (dab, sigma, usa) was used for color development. for sds-page and western blotting analyses, purified hcv core from e. coli and plant and plant crude extracts of the recombinant hcv core protein expressed by p coagroinfiltrated pbi and pvx vectors were loaded onto a % sds-polyacrylamide gel; by the end of electrophoresis, the protein bands were either stained with coomassie brilliant blue (bio-rad) or transferred to the pvdf membrane. subsequently, the corresponding protein bands on the membrane were identified by biotinylated anticore polyclonal antibody (abcam, uk) ( : dilution) and streptavidin hrp conjugate (sigma, usa) ( : dilution), respectively, and were detected by tmb as substrate. elisa was performed using the streptavidin-biotin peroxidase complex (abc) assay. briefly, wells were coated overnight at room temperature with µl of µg/ml mouse monoclonal antibody against - amino acids of hcv core antigen (abcam, uk) and blocked with % bovine serum albumin (bsa) for two hours at ˚c. after several washing steps, μg of tsp from each sample or ehcvcp (for standard curve, serial dilutions in the range of . - µg/ml) were added to the coated wells in duplicates and incubated for one hour at °c. following the washing steps, biotinylated anti-core polyclonal antibodies ( : dilution, abcam, uk) were immersed into the wells and incubation continued at ˚c for one hour followed by washing steps and another similar incubation with streptavidin hrp conjugate ( : ; sigma). finally, by adding tmb and color development, the absorbance at nm was measured. to detect the immuno-reactivity (antigenicity) of tobacco-derived hcvcp, an indirect elisa for detection of anticore antibodies in human sera was developed as previously described ( ) . in brief, purified phcvcp was coated in elisa plates ( µl of µg/ml). the pooled human sera ( / dilution) from five hcv seropositive patients and one serum negative (obtained from tehran blood transfusion center) were incubated with the plate for two hours at room temperature. hrp-labeled anti-human igg ( : dilution, sigma, usa) was used for detection of bound antibodies and tmb was used for color development, as described above. prism . software (graphpad, usa) was used for data handling and statistical analysis was carried out using mann-whitney non-parametric and anova tests. statistical significance was set at p ≤ . . figure represents the nucleotide changes applied in the basal sequence of hcvcp to improve its codon utilization for efficient expression in plants. other elements (kozak, kedel, his-tag and restriction sites) were also included in hcvcp basal sequence in tr-hcvcp ( figure ). in addition, to remove the plant mrna destabilizing sequence "ggtaag" (nucleotides - ) which is present in native hcvcp sequence, it was modified ( figure ). as shown in figure a, the corresponding nucleotide alterations increased the codon adaptation index (cai) value from . to . and reduced the gc content from . to . (figure b) . results of dna sequencing (not shown) indicated that the kozak (gccaccatggc) sequence ( ) , harboring the start codon (atg) at ', kdel nucleotides at ′ end, the designed restriction sites and all nucleotide modifications, was properly located in the tr-hcvcp sequence (figure figure represents the schematic of the constructed pbi-core (figure a) and pvx-core (figure b ) plant expression vectors harboring the tr-hcvcp, located under the control of camv s promoter (in pbi-core) and cp promoter (in pvx-core), upstream of the nos-ter transcriptional terminator. the sequencing results (not shown) confirmed the presence of the cloned tr-hcvcp gene in pbi-core and pvx-core expression vectors with no alterations in the nucleotides. the resulting plasmids were transferred into a. tumefaciens lba target hosts (in the case of pvx-core, a. tumefaciens lba harboring the psoup vector). the transformed agrobacteria were screened for recombinant constructs using core-specific primers (f-kozak-vx and r-core-vx) and the colony pcr assay indicated the expected -bp fragments (figure c) . one positive clone from each construct was subsequently used for agroinfiltration of n. tabacum leaves by p silencing-suppressor gene procedure as outlined in . . to detect and analyze phcvcp, the transformed tobacco leave extracts were evaluated by dot and western blot assays ( figure ). as shown in figure a, dot blot analysis showed that hcv core was present in extracts of the tobacco leaves, transformed by each of the vectors (pbi-core and pvx-core), compared to negative controls with the same amount of loaded tsp. although dot blot is a qualitative assay rather than a quantitative one, our result tentatively implied a higher level of expression for pvx-core compared with that of pbi-core construct, while the same amounts of tsp were applied (figure a) . coomassie-stained sds-page showed that nonspecifically-bound plant proteins were eluted from ni-nta affinity chromatography besides a band with an electrophoretic mobility of kda (figure b) . the recent band was visible on a coomassie-stained sds-page gel (figure b) and was also detected using anticore polyclonal antibodies in a western blot assay. there was no degradation of phcvcp and the protein was not glycosylated because the size of the produced protein was as predicted by the protein sequence (https://www.genscript. com/ssl-bin/site /peptide_calculation.cgi). as shown in figure c, the results of western blot analysis indicated that phcvcp from pvx-core and pbi-core expressed a protein of approximately kda, while the negative control lacked the core protein and ehcvcp showed a higher molecular weight (~ kda, which is due to addition of vector-derived amino acids in pivex . a plasmid) and a few other lower-molecular weight protein bands which were presumably the results of ribosomal release and uncompleted translation, as previously reported ( ) . it should be noted that dot blotting in this study was performed based on prior studies that used this procedure in complementation of western blotting ( , ) . indeed, in western blotting, the protein will be completely denatured (by boiling in sds-page loading buffer which contains -me and sds) and therefore some information that might be related to the native/soluble structure of the protein might be lost (e.g. interaction of protein with a conformational antibody), while dot blot assay was used as a preliminary and fast test to show if the protein was expressed. the expression levels of phcvcp by each of the vectors (pbi-core as a classical plant binary vector and pvx-core as a viral-based vector) in the presence or absence of p- co-agroinfiltration were compared by elisa. sandwich elisa is the assay of choice and is usual in quantification of antigen proteins (when an exact and precise standard curve is prepared and a specific antibody against the antigen is used) and a number of diagnostic elisa kits work on this basis ( , ) . in this context and to perform an exact and precise quantitative elisa, we used a specific monoclonal antibody against the core antigen (at the bottom of elisa plates) and polyclonal anticore antibodies (at the top of the phcvcp) in a sandwich elisa-based protocol and also prepared a precise standard curve by applying serial dilution ( . - µg/ml; data not shown) of purified ehcvcp to calculate the expression levels of phcvcp. they were expressed in form of the percentage of tsp, which was determined using a bradford protein assay. the leaves transformed by pvx (vector without hcvcp) and co-agroinfiltrated with p constructs were used as negative control (figure a) . the results indicated that the expression of pbi-core and pvx-core was around . % of tsp in the absence of p co-agroinfiltration, whereas in its presence, the expression level of pbi-core and pvx-core increased to . % ( ng/g) and . % ( ng/g) of tsp fresh leaf weight, respectively, which was almost five times improvement for phcvcp expression (figure a) . the diagnosis potency of phcvcp for detection of hcvinfected human sera was also analyzed by elisa. as shown in figure b, purified phcvcp could detect hcv-infected sera in a comparable way to that of the ehcvcp. a). dot blotting; at control column: row : negative control ( μg tsp of untransformed tobacco leaves); row : μg positive control (ehcvcp). at pvx column: negative control (tobacco leaves transformed by pvx vector alone; ie, without tr-hcvcp). pvx-core and pbi-core columns: the extracts from the pvx-core and pbi-core p co-agroinfiltrated leaves, respectively. rows and in these two last columns correspond to µg and µg of tsp, respectively. b) coomassie-stained % sds-page gel, loaded with; lane : μg concentrated plant-purified hcvcp (from the pvx-core expression system). lane : μg of purified ehcvcp. lane : crude protein extract from agroinfiltrated leaves with pvx-core ( µg). lane : untransformed leaves ( µg). c) western blotting; lane : positive control ( ng purified ehcvcp). lane : purified phcvcp ( ng from the pvx-core expression system). lanes and : the extracts from p co-agroinfiltrated pvxcore and pbi-core leaves, respectively ( µg of plant tsp was applied in each lane). lane : negative control ( µg of plant tsp of untransformed tobacco leaves). lane m: prestained protein ladder (fermentas). hcvcp denote to hcv core protein, ehcvcp and phcvcp dnote to e.coli-derived and plant-derived hcvcp, respectively. in western blot and sds-page figures, the location of hcvcp under the kda molecular weight range is shown by arrows. the reason for multiple bands in the case of ehcvcp is explained in the corresponding result section of the text. the results of sds-page showed that ni-nta pull-downs of plant extract contained endogenous nonspecific plant proteins besides phcvcp. according to elisa data, the concentration of phcvcp was / of the column-purified protein. therefore, although µg ( µl of μg/ml coated) was coated, only a small amount reacted (less than ng) (figure b) . however, none of these endogenous nonspecific plant proteins reacted with anti-hcvcp in western blotting of the purified protein fraction. the primary objective of the present study was to provide an efficient transient tobacco system for expression of hcvcp in a regionally-adapted tobacco host (the iranian jafarabadi-tobacco plant cultivar). while other tobacco plant such as australian nicotiana species, n. benthamiana, is a well-known and hyper-susceptible host for plant-derived recombinant protein expression ( ) , to our knowledge there was no prior report available on expression in jafarabadi tobacco cultivar (which is one of the currently available tobacco cultivar in iran). to address this concern, the first strategy that should be considered would be the optimization of the gene sequence for efficient codon usage by the plant ( ) . in agreement, it is recently shown that the plant codonoptimized bpv- li gene product increased significantly in comparison to the unmodified counterpart ( ) . as shown in figure a, our codon optimization approach increased the cai value from . to . , which was in favor of codon utilization for n. tabacum ( ) . although increasing the cai value is usually inevitably accompanied by increase of a + t percentage (which enhances mrna instability and decreases the efficiency of translation) ( , ) , our codon optimization approach that was based on the codon usage of nuclear-encoded genes of tobacco could keep the a + t percentage at %, while reducing the gc content from . to . (figure b) , almost the perfect range desired for gc content ( %). to ameliorate the expression level, insertion of the kozak sequence (gccaccatggc) ( ) at the translation start site (atg) was also considered ( figure ). however, since the two base pairs immediately following the atg start codon were "c" and "a" and were not compatible with the optimized kozak sequence (underlined nucleotides), consistent with the approach undertaken by amani et al. ( ) , we had to insert the gct sequence that codes for alanine (a nonpolar amino acid). moreover, to improve the stability of phcvcp, kdel-encoded bases were also considered at the ' site of the tr-hcvcp sequence ( figure ). employing this strategy for expression of human epidermal growth factor in tobacco resulted in a -fold higher yield of protein expression ( ) . finally, in our codon optimization strategy, the possible deleterious splicing motif in plants "ggtaag", resulting in rna degradation and gene silencing of the gene ( ) , was removed from the native hcvcp-coding sequence (nucleotides - ). in our study, two kinds of plant vectors were employed: a classic, nonreplicative, binary vector (pbi ) and a pvxbased viral replicative vector ( , ) . our results indicated that the pvx-based vector provided higher expression levels of phcvcp compared to that of pbi (figure a and a). our result was in complete agreement with the results of a recent report comparing the expression levels by tmv and pvx replicative viral vectors which was three times of that of pbi vector ( ) . a) the expression level of pbi-core and pvx-core were assessed by elisa and compared in the presence or absence of co-agroinfiltration by gene silencing suppressor p construct. leaf control denotes the tobacco leaves transformed by pvx vector alone (ie, without tr-hcvcp). b) result of elisa assay for confirmation of plant-derived hcvcp, using hcv-positive human serum. negative control corresponds to hcv negative human sera. bsa was also used as another negative control. phcvcp (corresponding to µl of µg/ml of phcvcp from pvx-core-transformed tobacco leaves) and ehcvcp denote plant-and e. coli-derived hcvcp, respectively. statistical analysis was performed by one-way analysis of variance (anova), using the bonferroni's multiple comparison test. for obtaining the highest expression levels, it is important to prevent the silencing mechanisms that limit foreign gene expressions in plants. it is shown that coexpression of viral silencing suppressor proteins eliminates the effects of post-transcriptional gene silencing (ptgs) in a transient expression system ( ) . application of p -mediated co-agroinfiltration has already shown to improve recombinant expression in plants by several folds ( ) . our results indicated that the presence of p increased the expression levels of phcvcp by five times in both pbi and pvx expression constructs (figure a) . our data were consistent with recent reports on enhancement of the expression levels of sars-cov n ( ), antibodies ( ) and gfp ( ) proteins in the presence of p by - and even folds, respectively. the times differences in the transient expression levels of human growth hormone peptide by pbi in n. tabacum was . % of tsp ( ) to that of our study ( . % of tsp for phcvcp). the enhanced expression levels in our study compared to this prior report should be a direct result of the codon optimization and gene silencing suppression strategies undertaken for tr-hcvcp in our gene modification approaches. finally, the results of elisa in our study ( figure ) demonstrated that the transient expression of phcvcp in tobacco leaves could provide the proper protein for diagnostic purposes and indicated its full functionality to react with poly/monoclonal anticore antibodies and hcv infected human sera compared to that of ehcvcp ( ) . in addition, the proper conformation of phcvcp to interact with specific monoclonal antibody against - amino acids of the core antigen (coated at the bottom of the elisa plates) and the polyclonal anticore antibodies (at the top of the phcvcp in elisa), supported the presence of various interactive epitopes on phcvcp. the monoclonal antibody used in this study was against amino acids - on hcv core antigen. this region carries two important immunological epitopes which are conserved between hcv strains: a, b, and ( ): moreover, the amino acids of - are highly promiscuous cd + t cell epitopes and the - amino acids are hcvspecific hla-a -restricted ctl epitopes. they have been shown to be effective immunogens in the vaccine phase of two clinical trials while are present on the hcv core particle or in multi-epitope complexes ( ) . therefore, the primary results have proposed that the plant-produced phcvcp might have the promising structure to be used as a vaccine candidate too. however, these primary results should be confirmed through animal and immunization studies. although these ending results were consistent with the prior two reports on appropriate antigenicity of tobaccoderived hcvcp, both of those previous studies addressed only transgenic (stable) tobacco-plant generations for hcvcp ( , ) . however, transient expression in plants is currently the method of choice due to the simplicity and feasibility of rapid protein expression, omitting tissue culture and regeneration costs, and ease of large scale industrial plans growth ( ) ( ) ( ) . while transgenic (stable) plant generation needs longer periods of transgenic production and more deleterious effects on gene silencing ( ) which is not encountered in transient-expression approach ( ) . taken together, the present study was the first (to our best of knowledge) to address construction of a transient tobacco expression system for hcvcp. for highest possible expression efficiency, the hcvcp dna sequence was codon optimized (based on the cai for nuclear-encoded genes of tobacco), destabilizing of the ggtaag sequence in the native hcvcp coding sequence was altered and kozak and kdel sequences were included at the ' and ' ends, respectively. two types of plant expression vectors (pvx-based and classic pbi binary) for hcvcp were constructed and their levels of protein expression in the presence and absence of p co-agroinfiltration were compared. the results indicated the significant effect of our approach in enhancement of the expression levels for phcvcp. to our knowledge, all the above mentioned reports were first of their kinds for hcvcp and provided valuable information for plant-based expression studies on this antigen. the preliminary results (with five hcv positive sera) showed that plant-hcvcp has the capability to capture anti-hcvcp antibodies for potential clinical applications which should be further evaluated and confirmed by statistically acceptable numbers of positive sample sera. the next steps might be standardization of phcvcp for application in diagnostic elisa through application of positive sera with determined viral loads as well as precisely predetermined standard positive sera together with high number of optimization studies with different concentrations of antigen/antibody in elisa with precise statistical analyses and evaluation of the phcvcp for immunization studies. trends of obesity in iranian adults from s to late s; a systematic review and meta-analysis virology of hepatitis c virus infection hepatitis c virus vaccines--progress and perspectives advances in hepatitis c virus vaccines, part one: advances in basic knowledge for hepatitis c virus vaccine design advances in hepatitis c virus vaccines, part two: advances in hepatitis c virus vaccine formulations and modalities enhancement of immune responses by co-delivery of ccl /mip- beta chemokine plasmid with hcv core dna/protein immunization polytope dna vaccine development against hepatitis c virus: a streamlined approach from in silico design to in vitro and primary in vivo analyses in balb/c mice construction of hcv-polytope vaccine candidates harbouring immune-enhancer sequences and primary evaluation of their immunogenicity in balb/c mice fusion of hbsag and prime/ boosting augment th and ctl responses to hcv polytope dna vaccine the role of core antigen detection in management of hepatitis c: a critical review expression and characterization of escherichia coli derived hepatitis c virus arfp/f protein the wild-type hepatitis c virus core inhibits initiation of antigen-specific t-and b-cell immune responses in balb/c mice identification of a functional, crm- -dependent nuclear export signal in hepatitis c virus core protein collaborative team of the international pasteur network . the impact of hcv diversity on diagnosis tools for hcv infection hcv core protein immunization with montanide/cpg elicits strong th /th and long-lived ctl responses hcv core protein-expressing dna vaccine induces a strong class i-binding peptide dth response in mice immunization of mice by bcg formulated hcv core protein elicited higher th -oriented responses compared to pluronic-f copolymer expression of shigella flexneri ipab gene in tobacco overview of expression of hepatitis b surface antigen in transgenic plants bioprocessing of plant-derived virus-like particles of norwalk virus capsid protein under current good manufacture practice regulations expression of an hcv core antigen coding gene in tobacco (n. tabacum l.) a hepatitis c virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera expression of hiv- antigens in plants as potential subunit vaccines transient expression systems for plant-derived biopharmaceuticals a plant-produced influenza subunit vaccine protects ferrets against virus challenge. influenza other respir viruses a plant-based system for rapid production of influenza vaccine antigens. influenza other respir viruses modification of the coding sequence enhances plant expression of insect control protein genes in planta production of a candidate vaccine against bovine papillomavirus type magnifection--a new platform for expressing recombinant vaccines in plants plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins advances in plant molecular farming potatovirus x and tobacco mosaic virus-based vectors compatible with the gateway cloning system fcgamma receptor-like activity of hepatitis c virus core protein initiation of hepatitis c virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein the codon adaptation index--a measure of directional synonymous codon usage bias, and its potential applications the scanning model for translation: an update a c-terminal signal prevents secretion of luminal er proteins complete sequence of the binary vector pbi and its application in cloning t-dna insertion from transgenic plants molecular cloning storage of competent cells for agrobacterium transformation pgreen: a versatile and flexible binary ti vector for agrobacterium-mediated plant transformation an enhanced transient expression system in plants based on suppression of gene silencing by the p protein of tomato bushy stunt virus an agrobacterium-mediated transient gene expression system for intact leaves a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding production and secretion of human interleukin- in transgenic tobacco cell suspension culture generation of biologically active multi-sialylated recombinant human epofc in plants nicotiana benthamiana: its history and future as a model for plant-pathogen interactions effects of codon modification on human bmp gene expression in tobacco plants effect of codon optimization and subcellular targeting on toxoplasma gondii antigen sag expression in tobacco leaves to use in subcutaneous and oral immunization in mice in silico analysis of chimeric espa, eae and tir fragments of escherichia coli o :h for oral immunogenic applications expression of active human epidermal growth factor (hegf) in tobacco plants by integrative and non-integrative systems prediction of splice sites in plant pre-mrna from sequence properties plant-made trastuzumab (herceptin) inhibits her /neu+ cell proliferation and retards tumor growth boosted expression of the sars-cov nucleocapsid protein in tobacco and its immunogenicity in mice a new viral vector exploiting rna polymerase i-mediated transcription transient expression of human growth hormone in potato (solanum tuberosum), tobacco (nicotiana tobacum) and lettuce (lactuca sativa) leaves by agroinfiltration progress in the development of preventive and therapeutic vaccines for hepatitis c virus immunogenicity and safety of different injection routes and schedules of ic , a hepatitis c virus (hcv) peptide vaccine. vaccine transgene expression variability (position effect) of cat and gus reporter genes driven by linked divergent t-dna promoters it is with affection and appreciation that dr. a. jafari from the molecular biology department of pasteur institute of iran is acknowledged for her supports and help concerning this project the present study was in part to fulfill the thesis of mrs. sara mohammadzadeh in ph.d. s. m. the ph.d. candidate; she was responsible for most experimental works and prepared the predraft of the manuscript. alireza khabiri helped with elisa assays. farzin roohvand: advisor; helped with expression of ehcvcp, diagnosis potency of phcvcp by elisa and editing of the manuscript. arash memarnejadian: co-advisor; soheila ajdary and ali hatef salmanian: co-supervisors; parastoo ehsani: supervisor; design of the experimental works and editing the manuscript. this work was supported by pasteur institute of iran, tehran, iran. key: cord- - qpnlgb authors: sahu, kamal k; jindal, vishal; anderson, joseph; siddiqui, ahmad d; jaiyesimi, ishmael a title: current perspectives on diagnostic assays and anti-pf antibodies for the diagnosis of heparin-induced thrombocytopenia date: - - journal: j blood med doi: . /jbm.s sha: doc_id: cord_uid: qpnlgb heparin-induced thrombocytopenia (hit) is a recognized clinical entity in patients receiving unfractionated heparin and low–molecular weight heparin. currently, diagnosing hit includes the combination of a physician’s clinical suspicion based on a clinical scoring system and a series of laboratory tests. in the present article, we discuss challenges in suspecting and diagnosing hit in consideration of the turnaround time of available tests and recent advances in techniques and methodologies of newer immunoassays and functional assays. heparin-induced thrombocytopenia (hit) is a complication following exposure to heparin. it is most commonly seen in patients receiving unfractionated heparin (ufh) and less often with low-molecular weight heparin (lmwh). the approximate proportion of patients developing hit after receiving heparin for > days has been reported as up to %. , patients receiving ufh have a higher chance of developing hit (~ . %) than patients receiving lmwh (~ . %). more importantly, higher mortality -up to % -has been reported with untreated hit. however, with early recognition and treatment, mortality incidence has significantly reduced to < %. although hit occurs in a very small proportion of patients exposed to heparin and is unrelated to dosage, schedule, or route of administration, there is no predictor that can detect the predisposed population. since most cases of thrombocytopenia in hospitalized patients on heparin are not caused by hit, clinicians must be able to differentiate rare cases of hit from the many without hit. , similarly, overdiagnosing hit contributes to an unnecessary burden on patient-care resources, in that alternative anticoagulants are frequently more expensive. as such, a detailed understanding of the pathophysiology, evaluation, and management of hit facilitates appropriate and timely treatment. functional assays such as the c serotonin-release assay ( c-sra) and heparin-induced platelet activation (hipa) test are considered gold-standard tests for detecting pathological hit antibodies. however, the c-sra is a timeconsuming assay, expensive to perform, requires a specialized laboratory setup, and utilizes radioactive elements. a recent study has shown that absolute immature-platelet count could complement anti-pf heparin testing in evaluating patients suspected of hit. therefore, there is impetus for research focusing on alternative ways to diagnose hit. heparin is a commonly used anticoagulant in various clinical settings, and can be given either intravenously or subcutaneously. in hit, heparin interacts with pf , a small cytokine belonging to the cxc chemokine family that is also known as cxcl . this chemokine is released from α-granules of activated platelets during platelet aggregation, forming a complex with vascular endothelial heparin-like molecules and normally promoting platelet function at the site of vascular injury. with the administration of exogenous heparin, competitive binding results in the suppression of platelet function. hit is caused by an autoimmune reaction, whereby the immune system identifies the heparin-pf complex as a threat and igg antibodies are formed in response. antibody-antigen complexes (igg-heparin-pf ) bind to platelet fcγriia receptors, leading to platelet destruction. platelet destruction in turn prompts the release of more pf molecules. this can trigger a cycle that results in a rapid and significant drop (eg, % or more) in platelet numbers, ultimately leading to thrombocytopenia. however, not all patients who receive heparin will develop autoimmune antibodies, and not all patients who develop antibodies will develop thrombocytopenia. typically, thrombocytopenia results in bleeding, although up to % with hit present with thrombotic events. , it is also important to note that development and activity of these antibodies depends on the level of pf antigen in relation to heparin (or other gags). studies have shown that pf :heparin ratios of : - . : have the potential to form ultralarge complexes. , similarly, extracellular gag can mimic heparin and form a complex with pf -gag antibodies, which can indeed be pathogenic. disease burden of hit hit is more commonly triggered by treatment with ufh ( %- % of patients receiving heparin), followed by lmwh and fondaparinux ( %- %). , the production of antibodies against the pf -heparin complex is an immunologic response that can be seen in variable percentages of patients ( %- %). however, clinical sequelae of these antibodies are far less commonly noted ( . %- %). the destruction of platelets may generate cell fragments, which may predispose to the development of new or worsening thrombosis, known as hit and thrombosis (hitt). , if not detected in a timely fashion, hitt can be a catastrophic event, and mortality secondary to thrombus is generally cited as %- %, with some reporting mortality as high as % among hit patients. with advanced techniques of detection and early intervention, the mortality rate can be reduced to < %. in the following sections, we discuss in detail newer investigations related to hit diagnosis. diagnosis of hit involves a stepwise process from suspicion to screening and finally making the diagnosis ( figure ). the three most common clinical scoring systems studied are the ts, hep score (hit-expert probability) and hit score for cardiopulmonary bypass. , the t clinical scoring system is most commonly used in clinical practice and is based on typical clinical features and laboratory results of hit. this scoring system is easy to apply and has a high negative predictive value, thereby meaning a low-probability t score serves as an excellent tool to rule out hit. however, due to the low positive predictive value of this system for intermediate-and high-scoring groups, it is difficult clinically to rule out hit in these subsets of patients. therefore, laboratory testing constitutes an integral part of hit diagnosis (table ). following clinical scoring, when a clinician suspects hit the next step is evaluation of the patient with laboratory studies. currently, two classes of assays are available: immunoassays (antigenic), which detect binding of anti-pf -heparin antibodies, and functional assays (plateletactivation assays), which are more specific and detect if circulating antibodies have the capability to activate platelets. each of these assays tests separate stages in the hit pathway, with immunoassays capable of detecting initial immunoresponse and functional assays detecting specific pathogenic antibodies causing platelet activation in the presence of heparin. the various phases of hit as shown in figure are helpful to understand test results and subsequent interpretation. suspected hit is the first phase, when a patient is suspected of having hit based on clinical scenarios. acute hit constitutes the phase when a laboratory confirmation is made, and this phase lasts till the point of platelet-count recovery. following this, subacute hit is divided into two subphases namely: a (both functional assay and immunoassay are positive), and b (functional assay is negative, immunoassay is still positive). the final phase is remote hit, when immunoassays are no longer able to detect antibodies ( figure ). it is important to remember that some patients who have clinical signs of hit fail to have antibodies detected by current assays. for instance, in patients with hit diagnosis clinically, up to % could have a negative h/pf elisa result. similarly, another subset of patients with positive tests may not necessarily have clinical hit. this proportion is higher in surgical patients (up to %) than patients admitted for medical reasons (up to %). immunoassays measure the presence of anti-pf -heparin antibodies using one of several antibody-capturing platforms (eg elisa based, particle gel, turbidimetry). immunoassays have high sensitivity (> %), thereby making them ideal for use as an initial screening test for hit. the drawback is low specificity ( %- %) for a diagnosis of hit, due to asymptomatic seroconversions. , as such, they have a lower positive predictive value. in general, only %- % of patients with positive immunoassay results have platelet-activating antibodies. immunoassays have the advantage of being able to be performed by most laboratories routinely and a rapid response time. figure shows the timeline from the discovery of platelets ( ) till the recent discovery of chimeric antibodies for diagnosis of hit ( ). the first polyspecific elisa to detect hit antibodies was developed in . , , in this test, suspected patients' serum/plasma is added to pf -polyanion complex-embedded microtiter plate wells. the semiquantitative nature of the test allows assessment of intensity of color change, and measures it as optical density (od) at a specific wavelength (typically nm), which is proportional to the concentration of bound antibodies. since the development of elisa for hit, many studies have been done to assess the clinical performance of various in-house and commercially available elisas. it is important to note that there is no universally agreed-upon od value for the interpretation of the results, as elisa od units vary based on the heparin-pf elisa kit being used. therefore, it is important for physicians to connect with local reporting laboratories for the od cutoff set by the reporting laboratory, in order to interpret the elisa results. interpreting od results of elisas many laboratories report hit elisa results as positive or negative based on the prefixed od cutoff set in their laboratories. however, an absolute value is more informative than nominal results, and is helpful in anticipating the test results of an sra functional test. a study on , samples from suspected hit patients was done to correlate od results of hit elisa with functional sras. the study showed that a weakly positive od value ( . -< ) had a good correlation with a negative sra in % or more of cases. on the other hand, an od value of > correlated well with a positive sra in % or more of cases. many similar studies have evaluated the applicability and generalizability of various elisa od thresholds from special populations of patients, such as those on extracorporeal membrane oxygenation. kataria et al studied such patients. they identified an od threshold of achieved specificity and negative predictive value of % and %, respectively. they suggested that keeping the od threshold at could help clinicians in ruling out hit. a study of patients admitted with clinical suspicion of hit attempted to correlate od values of hit elisa with thrombosis. the authors reported that with every -unit rise in od value, the odds of developing other immunologic assays as discussed, once there is clinical suspicion of hit, a screening immunoassay is the first step in a diagnostic workup for hit. one of the major drawbacks of elisa is that results may be available only once a day, considering the cumbersome procedure and requirement for a specialized laboratory with skilled technicians. other drawbacks are that enzyme activity can be hampered by plasma constituents, the presence of heterophilic antibodies may generate false-positive results, and results may not be absolute. these drawbacks prompted researchers to look for newer alternatives that could have a rapid turnaround time without compromising the high sensitivity and cost-effectiveness of elisa. some other recently studied immunologic assays are particle-gel immunoassays (h-pf pagia), lateral-flow immunoassays (lfias; eg, stic; diagnostica stago, australia), chemiluminescent immunoassays (clia; eg, hemosil acustar; werfen, australia) and latex-agglutination assays. these assays exploit alternative technologies and differ in methodology and end-point measurements ( table ) . bankova et al compared elisa with the newer immunologic assays acustar hit-igg and pagia for diagnostic accuracy, reproducibility, and analytical costs. diagnostic accuracy was studied for three hit elisa od-cutoff thresholds: low ( . ), intermediate ( . ), and high ( . ). the study showed that specificity increased with the rise in od thresholds, and all three tests had specificity > % in the intermediate-and high-threshold groups. for the low-threshold group, the specificity of acustar hit-igg ( . %, p< . ) was significantly higher than pagia ( . %) and elisa ( . %). total costs per test were us$ . , $ . , and $ . for pagia, acustar hit-igg, and elisa, respectively. the study concluded that although the newer tests were . -to twofold costlier than conventional hit elisa,when implemented in a -hour service they might improve patient care, considering their rapid turnaround, similar or better specificity, and adequate reproducibility. recently, a meta-analysis by nagler et al studied the diagnostic utility of various immunologic assays. the meta-analysis included , patients from studies, and reported that the combination of high sensitivity (> %) and high specificity (> %) was found only in the following tests with specified thresholds: polyspecific elisa with an intermediate threshold (genetic testing institute, asserachrom), pagia, lfia, polyspecific clia with a high threshold, and igg-specific clia with a low threshold. in addition, they found that diagnostic accuracy was inadequate in certain tests and specified thresholds: particle-immunofiltration assay, elisa at high-dose heparin confirmation step, andigg-specific clia with high and intermediate thresholds. the authors concluded that when evaluating a suspected hit patient, physicians must utilize clinical scoring tools for pretest probabilities and estimate the posttest probabilities before running the test. recent studies have also been conducted to look for additional pathological antibodies in patients detected negative for anti-pf -heparin. bashover et al recently conducted a study on anti-pf -heparin-negative samples by using whole-cell elisa. they detected that . % (eight of ) of negative samples were found positive for antibodies against antigens on platelets or red blood cells. as immunoassays have low specificity and hence tend to overdiagnose hit, they may erroneously lead to inappropriate discontinuation of heparin anticoagulants and compel the use of costlier alternative anticoagulants. these shortcomings mandate the performance of a functional assay in patients with a positive immunoassay to reduce overdiagnosis and erroneous discontinuation of ufh/ lmwh in patients without hit. in general, functional assays have high specificity (> %) and high positive predictive values ( %- %) but lower sensitivity ( %- %). the sensitivity of this assay depends on the functional end point being studied (radioactive signal > flowbased > light-transmission detection). technically, these studies are difficult to perform and hence not routinely available at many medical centers. also, there are variables during the functional assay that need to be considered while conducting these tests and interpreting the results (table ). in clinical practice, when a functional assay is not available and if the patient has a high t score plus a strongly positive immunoassay, a functional assay may be excluded and the patient can be treated as a case of hit. similarly, a diagnosis of hit can be entertained in conditions with a negative functional assay, a strongly positive immunoassay, and a high-probability t score. functional assays detect platelet-conformation changes when a patient's serum containing hit antibodies is incubated with donor platelets and heparin. if hit antibodies are pathologically significant, they are expected to form a heparin-antibody-pf complex, which in turn binds to fcriia receptors located on the donor's platelet surfaces. this leads to donor-platelet activation with subsequent changes in platelet conformation and surface expression, including the release of dense granules, production of platelet microparticles, and translocation and surface expression of various glycoproteins (eg p-selectin or cd p). it is the capture of these changes that constitutes the end point of various functional assays. the sra end point is to detect the release of c serotonin from dense granules upon activation of donor platelets by hit antibodies present in the patient's serum. the test involves incubating radiolabeled donor platelets and the patient's serum in the presence of therapeutic or excessive heparin concentrations. a positive test result is defined as the release of c serotonin with therapeutic concentrations of heparin ( . u/ml), but not with higher heparin concentrations ( u/ml). differential serotonin-release responses to two different heparin concentrates results, since the binding of hit antibodies to antigens occurs only at a specific ratio of heparin to pf . heparin-induced platelet-activation assay in the hipa, the suspected patient's serum or platelet-poor plasma is added to a healthy donor's platelet-rich plasma with or without heparin. a positive test is indicated by visual recognition of strong platelet aggregation with low heparin concentrations (~ . - . u/ml), but no or minimal aggregation in the absence of heparin or presence of very high concentrations of heparin (~ - u/ml). the advantage of hipa is visual measurement of platelet aggregation, unlike sra, which utilizes radioactive agents. in addition to sra and hipa, there are various other functional assays available in the commercial market or clinical trials (tables and ). the goal of developing newer assays is to use simpler technologies (eg, flow cytometry, turbidometry, aggregometry, and luciferase activity) with rapid turnaround. various chemical changes in platelet surface-antigen expression (eg, p-selectin or anionic phospholipids), release of granules (eg, atp) or other platelet-derived microparticles, or physical changes like aggregation serve as end points in functional assays. the potential for wider acceptance of these alternate functional assays is encouraging, with generally good specificity ( %-> %), but variable sensitivity ( %- %), which could be due to multiple variables, as mentioned earlier (table ) . newer anti-pf antibodies and their significance initially, anti-pf -heparin antibodies were considered solely responsible for the pathogenesis of hit/hitt. however, with the discovery of the polyclonal and polyspecific nature of antibodies found in sera of hit patients, researchers are now convinced that other antibodies contribute to hit/hitt as well. following that, researchers have studied many mouse and human models using hit-mimicking monoclonal antibodies. , arepally et al developed a murine monoclonal igg b κ antibody against human pf -heparin complexes. this antibody was used recently by li et al to recognize a new second epitope named site , which was found adjacent to site in the crystal structure of the pf tetramer. similarly, vayne et al developed b , a monoclonal igg fully mimicking human hit antibodies, in their laboratory and used it to study the performance of alternative functional assays (himea, lta, and fc). they found good sensitivity for all the tested functional assays at a higher concentration of b ( µg/ml). similarly, morel et al studied the murine igg platelet-activating anti-cd antibody alb ; however, unlike b , alb was aspecific for pf -h complexes and had very poor affinity. diagnosis of hit requires a combined clinical and biochemical approach. timely suspicion of hit can essentially prevent fatal thrombotic events, but physicians should use the hit panel judiciously in order to avoid overdiagnosis, unnecessary discontinuation of heparin treatment, and financial burden on patients. since the time of the initial description of this disease almost six decades back, many novel, cheaper, and rapid confirmatory tests have been developed and shown promising results, but are not presently sensitive enough to replace sra. studies on rapid immunoassays, namely particle-gel immunoassay, lfia, and clia, are encouraging. the sensitivity and specificity of various tests depends on antibody specificities and thresholds set for the analysis. cost-wise, rapid immunoassays are costlier when compared to the elisa. however, a large-budget model study showed that availability of -hour in-hospital service for rapid immunoassays can reduce the time span of suspecion to diagnosis of hit and would reduce the amount of anticoagulant-treatment costs. therefore, an integrated diagnostic approach combining a clinical picture with immunoassays and functional assays serves as an ideal method for decision-making in suspected hit. also, clinicians should be aware of the type of tests used to diagnose hit, their antibody specificities, and the thresholds considered before interpreting the results. the authors report no conflicts of interest in this work. pmpga, platelet microparticle-generation assay; prp, platelet-rich plasma; sra, serotonin-release assay; tga, thrombin-generation assay; pagia, particle-gel immunoassay. the appropriateness of testing platelet factor /heparin antibody in patients suspected of heparin-induced thrombocytopenia risk for heparin-induced thrombocytopenia with unfractionated and low-molecular-weight heparin thromboprophylaxis: a meta-analysis the incidence of heparin-induced thrombocytopenia in hospitalized medical patients treated with subcutaneous unfractionated heparin: a prospective cohort study gender imbalance and risk factor interactions in heparin-induced thrombocytopenia thrombocytopaenia with absent radius (not radii) immune thrombocytopenia as a presenting manifestation of tuberculosis-challenge in resource constraint settings challenges for management of immune thrombocytopenia during covid- absolute immature platelet counts in the setting of suspected heparin-induced thrombocytopenia may predict anti-pf -heparin immunoassay testing results treatment and prevention of heparin-induced thrombocytopenia: antithrombotic therapy and prevention of thrombosis, th ed: american college of chest physicians evidence-based clinical practice guidelines dasatinib and dysfunction of platelets managing retroperitoneal hematoma: associated complexities and its challenges update on retroperitoneal hematoma in children additional insights regarding aortic intramural hematoma an interesting case of gluteal haematoma a spontaneous rectus sheath hematoma haemophilia-a-related haematoma: management in resource constraint settings heparin-induced thrombocytopenia: a stoichiometry-based model to explain the differing immunogenicities of unfractionated heparin, low-molecular-weight heparin, and fondaparinux in different clinical settings emphasis on the role of pf in the incidence, pathophysiology and treatment of heparin induced thrombocytopenia the role of surface pf : glycosaminoglycan complexes in the pathogenesis of heparin-induced thrombocytopenia (hit) generation of antibodies to heparin-pf complexes without thrombocytopenia in patients treated with unfractionated or low-molecularweight heparin heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin intra-cardiac thrombus in antiphospholipid antibody syndrome: an unusual cause of fever of unknown origin with review of literature cortical vein thrombosis in a case of idiopathic thrombocytopenic purpura heparin-induced thrombocytopenia (hit): identification and treatment pathways the hit expert probability (hep) score: a novel pre-test probability model for heparin-induced thrombocytopenia based on broad expert opinion diagnostic score for heparin-induced thrombocytopenia after cardiopulmonary bypass heparin-induced thrombocytopenia after cardiac surgery: an observational study of patients simple scoring system for early management of heparin-induced thrombocytopenia ten cate h, ten cate-hoek a. diagnostic value of immunoassays for heparin-induced thrombocytopenia: a systematic review and meta-analysis clinical and laboratory tests for the diagnosis of heparin-induced thrombocytopenia giulio bizzozero and the discovery of platelets the discovery of heparin the chemistry of heparin arterial embolism occurring during systemic heparin therapy heparin-a cause of arterial emboli? heparin induced thrombocytopenia with thrombotic and hemorrhagic manifestations white clot syndrome. peripheral vascular complications of heparin therapy immune endothelial-cell injury in heparin-associated thrombocytopenia platelet factor complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular pf -heparin complex as the major antigen prt- , a novel syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model atomic description of the immune complex involved in heparin-induced thrombocytopenia characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies b , a monoclonal antiplatelet factor /heparin igg with a human fc fragment that mimics heparin-induced thrombocytopenia antibodies decision analysis for use of platelet aggregation test, carbon -serotonin release assay, and heparin-platelet factor enzyme-linked immunosorbent assay for diagnosis of heparin-induced thrombocytopenia quantitative interpretation of optical density measurements using pf -dependent enzyme-immunoassays evaluation of serotonin release assay and enzyme-linked immunosorbent assay optical density thresholds for heparin-induced thrombocytopenia in patients on extracorporeal membrane oxygenation thrombosis in suspected heparin-induced thrombocytopenia occurs more often with high antibody levels rapid immunoassays for diagnosis of heparin-induced thrombocytopenia: comparison of diagnostic accuracy, reproducibility, and costs in clinical practice use of a whole-cell elisa to detect additional antibodies in setting of suspected heparin-induced thrombocytopenia functional assays in the diagnosis of heparin-induced thrombocytopenia: a review the platelet serotonin-release assay defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using kko, a murine hit-like monoclonal antibody heparin-induced multi-electrode aggregometry method for heparin-induced thrombocytopenia testing: communication from the ssc of the isth evaluation of automated immunoassays in the diagnosis of heparin induced thrombocytopenia rapid exclusion of the diagnosis of immune hit by acustar hit and heparin-induced multiple electrode aggregometry assessing the clinical and cost impact of on-demand immunoassay testing for the diagnosis of heparin induced thrombocytopenia key: cord- -eqqvwwh authors: wang, huanan; cong, feng; guan, jianchi; xiao, li; zhu, yujun; lian, yuexiao; huang, ren; chen, meili; guo, pengju title: development of a sensitive and specific xmap assay for detection of antibodies against infectious laryngotracheitis and bronchitis viruses date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: eqqvwwh background: a serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (iltv) and infectious bronchitis virus (ibv) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination. method: the microspheres coated with purified recombinant glycoprotein d (gd) of iltv or nucleocapsid (n) protein of ibv were incubated with serum samples. the simultaneous quantification of iltv and ibv antibodies were achieved through the interrogation of microspheres by luminex detection system. . results: this xmap detection demonstrated no nonspecific reactions with avian influenza virus (aiv), avian leukosis virus (alv), newcastle disease virus (ndv), and marek’s disease virus (mdv). the results also demonstrated that the xmap assay was four times more sensitive than the enzyme-linked immunosorbent assay (elisa) for iltv detection and two times more sensitive for ibv detection. a total of chicken serum samples from a chicken farm were tested by xmap and elisa assays. the results showed that the coincidence rates were . and % for iltv and ibv detection, respectively. conclusion: this study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination. infectious laryngotracheitis (ilt) and infectious bronchitis (ib) are common respiratory diseases in poultry and are currently present at epidemic levels around the world, including in china, the united states, and india. because ilt and ib have high incidence and infectivity in chickens at different ages (in days), they have caused huge economic losses to the poultry industry. both diseases have similar clinical symptoms and pathological changes, leading to significant difficulties in clinical differential diagnosis, especially in cases of mixed infection [ , ] . in addition, the use of vaccines is the main approach to control of the economically important poultry viral respiratory diseases, such as infectious laryngotracheitis and infectious bronchitis [ ] . therefore, it is important to establish a method to simultaneously detect iltv and ibv antibodies for the differential diagnosis and immune response evaluation after vaccination. infectious laryngotracheitis virus (iltv), an alphaherpes virus, possesses at least envelope glycoprotein, two main proteins of which are the glycoprotein b (gb) and gd, respectively, which are highly conserved herpesvirus structural glycoproteins [ ] . the gd of herpes virus has an important role by binding to the host receptors [ , ] . the gd protein has been demonstrated to be a candidate antigen for recombinant vaccines [ , ] . the infectious bronchitis virus (ibv) genome encodes four major structural proteins: the spike glycoprotein (s), the membrane glycoprotein (m), the nucleocapsid (n) protein, and the envelope or small membrane protein (e) [ ] . the n protein is thought to be an appropriate diagnostic reagent for antibody detection [ ] . in this study, gd and n proteins were selected as antigen molecules for the diagnosis of ilt and ib. at present, the methods used for the diagnosis and effect evaluation of vaccine immunity of avian respiratory diseases primarily include virology detection, serological detection, and molecular biological detection. traditional methods, such as virus isolation, animal inoculation experiments, elisa, hemagglutination (ha), and hemagglutination inhibition (hi) assays are characterized by complex procedures and long periods of time required for diagnosis. despite its unique, sensitive, simple features and rapidity, polymerase chain reaction (pcr) is unable to meet the requirements for high-flux quarantine and is not favorable for the urgent screening of bulk samples. therefore, it is imperative to establish a new technology for the effect evaluation of vaccine immunity and the rapid differentiation or correct identification of important avian respiratory diseases. a new high-flux detection technology, flexible xmap (x = unknown, map = multi-analyte profiling) assay, uses variously colored polystyrene beads that is carboxylated to allow covalent coupling of protein. conjugated beads can be used to capture specific antibodies in serum, and a fluorescent secondary antibody is incubated to bind to the captured serum antibodies. a red laser is used to determine the color of the bead and a green laser to detect fluorescence intensity of bound secondary antibodies through the luminex detection system [ ] . this method is applicable for the simultaneous and rapid detection of multiplex pathogen , s antibodies, with simple operation and high accuracy that are superior to conventional methods. in this study, a method employing luminex xmap technology to simultaneously detect iltv and ibv antibodies in serum was established, optimized and used for the differential diagnosis of ibv and iltv. this assay can also be used to simultaneous monitoring of ibv and iltv antibody levels for the evaluation of immunity after vaccination. a singleplex xmap for iltv was established by testing six concentrations of the iltv gd envelope protein ( . μg, μg, μg, μg, μg, and μg per × magbeads), seen in table . the p/n value is the ratio of mfi (median fluorescence intensity) value between the positive sample and negative sample. the optimal conjugation protein concentration was . μg per × magbeads for singleplex xmap detection of iltv. the mean p/n value under the condition was . . a singleplex xmap for ibv was established by testing six concentrations of the ibv n envelope protein ( μg, μg, μg, μg, μg, and μg per × magbeads), seen in table . the p/n value is the ratio of mfi value between the positive sample and negative sample. the optimal conjugation protein concentration was μg per × magbeads for singleplex xmap detection of ibv. the mean p/n value under the condition was . . based on the results in tables and , the optimum serum dilution ratio for iltv and ibv were : and : , respectively. when singleplex xmap assay was performed. to determine the optimal serum dilition ration for simultaneous detection of iltv and ibv, a serial serum dilutions from the range of : to : were prepared and were incubated with iltv and ibv antigen conjugated microspheres. from the p/n values showed in fig. , it is obvious that the optimal serum dilution ratio for duplex xmap assay was : . the threshold is considered as the sum of the average plus three times of standard deviation, which is also named cut-off value. to determine the threshold of xmap assay for iltv and ibv, special pathogen free chicken serum samples at : dilution were used to obtain the average mfi value and standard deviation. from the table , it showed that the threshold of iltv and ibv detection were . and . , respectively. the results in figs. and demonstrated that xmap duplex assay for iltv and ibv has a high specificity since there were no cross-reactions with serum positive for other avian diseases, such as avian influenza virus intra-assay repeatability experiments showed that the coefficient of variation (cv) was . % for iltv and . % for ibv. inter-assay repeatability experiments showed that the cv was . % for iltv and . % for ibv. all cvs were less than %, indicating that the method has high repeatability. the results were shown in tables and . comparison of xmap assay with elisa using clinical samples the sensitivities of xmap and elisa were compared using : to : iltv-positive sera and : to : ibv-positive sera. the results showed that xmap detected iltv-positive sera at : and ibv-positive sera at : , while elisa detected iltv-positive sera at : and ibv-positive sera at : (see table ). to compare duplex xmap assay with elisa for iltv/ibv detection, chicken serum samples from a chicken farm in huizhou, china were used and the results are shown in table . for iltv, xmap detected positive cases whereas elisa detected positive cases; among them, samples were detected positive by xmap assay and elisa. all samples were detected as positive for ibv by both methods. ib, an acute infectious disease characterized by acute watery diarrhea, kidney necrosis, and high mortality, is caused by ibv. thus far, ib has resulted in economic losses to the poultry industry in various countries [ , ] . ibv infects chickens at different ages. chicks infected with ibv are characterized by noisy breathing, panting, sneezing, nasal fluid, and other symptoms; furthermore, laying hens exhibit significantly reduced egg production, broilers exhibits low growth and bad meat quality, and breeding hens exhibit significantly reduced breeding efficiency [ ] . ilt is an acute, contact respiratory infectious disease in chickens. chickens infected with ilt are characterized by nasal fluid, conjunctivitis, panting, coughing, and the production of bloody mucus, as well as swelling, erosion, necrosis, and extensive bleeding in the throat after analysis; laying hens exhibit reduced egg production or even no production, as well as poor egg quality [ ] . the disease spreads rapidly and has a wide distribution. the high mortality rate (> %) results in huge economic losses to the poultry industry [ ] . given the similar clinical symptoms between ilt and ib, the development of diagnoses for both diseases is significant. this is the first case report of differential diagnosis of iltv and ibv with xmap assay. luminex xmap assay is an emerging technology that uses small carboxylated polystyrene microspheres that are internally dyed with both a red and an infrared fluorophore [ ] . by changing the ratio of the two fluorophores, it is possible to distinguish up to different color-coded microsphere sets, and each microsphere set may be coupled with a different biological probe. the microspheres are detected and characterized by a dedicated flow cytometer [ ] . the luminex xmap technology is useful for many different applications. one review describes the use of this technology for the multiplex detection of viral, bacterial, parasitic, and fungal agents using the microsphere-based multiplex nucleic acid assay (mbmna) and the microsphere-based multiplex immunoassay (mbmi) [ ] . mbmis are typically biochemical tests that allow the detection or measurement of the concentration of a protein in a solution via an antibody or immunoglobulin [ ] . mbmis are often used in the diagnosis of various pathogens, such as human papillomavirus and human cytomegalovirus, to detect antibodies in serum samples [ ] [ ] [ ] . the advantages of this technology are its simple operation, high accuracy, and reduction in the number this study describes the establishment and validation of single xmap and duplex xmap assays. first, to achieve effective and rapid clinical diagnosis of ilt and ibv, single xmaps were established for the separate diagnosis of these two diseases after screening to identify the best concentrations of microsphere antigens for iltv gd and ibv n ( . μg/ × magbeads and and μg/ × magbeads, respectively), with an optimal serum dilution of : . however, it is not ideal to diagnose only one type of pathogen antibody because more than one disease often affects the respiratory tract in poultry. given the similar clinical symptoms of ilt and ibv, the differential diagnosis of these two pathogens with two single xmap assays using one sample wastes time and energy. to achieve the rapid differential diagnosis of clinical samples, it is necessary to establish a duplex xmap. in addition to iltv and ibv, aiv and ndv are also common respiratory viruses for poultry. these four diseases have high incidences, and it is difficult to make a differential diagnosis based on clinical symptoms, which are all similar. figures and showed that our xmap methods was able to simultaneously detect the antibodies to iltv or ibv in the sera and had the high specificity. a duplex xmap can quickly identify iltv and ibv infection, but it does not indicate possible infection by aiv and ndv. therefore, based on this study, we propose that quadruplex xmap should be established in the future for the detection of iltv, ibv, aiv and ndv as a fast and effective differentiation tool for these four viruses. since elisa was established to detect iltv antibodies in [ ] , it has demonstrated strong advantages, as well as higher sensitivity than virus neutralization tests (vnt), making it widely applicable. elisa antibody detection of ibv is also feasible at earlier time points [ , ] and is currently broadly applied in the poultry industry. single-analyte elisa does not support the detection of multiple specific antibody responses simultaneously for a single serum sample [ ] and possesses other disadvantages, such as the requirement for a relatively large amount of sample, negligible nonspecific binding, and increased background. however, with the development of the poultry industry, requirements dictate that many samples must be detected simultaneously; thus, a new detection method for diagnosis with iltv and ibv is significant. luminex xmap assay represents an alternative for commonly employed indirect tests such as elisa. the conversion of an elisa to the xmap format is uncomplicated, efficient and cost-saving, producing an assay with superior dynamic range and sensitivity [ ] . mbmi represents increased sensitivity and the potential to quantify antibodies, antigens, and other substances (e.g., hormones, cytokines, and tumor markers), unlike conventional elisa tests [ ] . luminex xmap has also been compared with a western blot assay for antibody detection [ ] . the results obtained from both methods showed that the sensitivity of xmap was . %, while the western blot assay sensitivity was . %. therefore, xmap may be more efficient and precise than western blots for the diagnosis of diseases. although elisa provides relatively accurate results and has been used for many years, this study demonstrates that xmap is much more sensitive than elisa for the diagnosis of ilt, as indicated by the results presented in table . in addition, xmap may be used for the rapid diagnosis of many samples, saving time and effort. the results presented in table , demonstrate that xmap is highly accurate, with similar detection results to those obtained by elisa in positive clinical samples; thus, xmap is able to be widely applicable. this study describes how to determine the optimal conditions for establishing duplex xmap assay based on the singleplex xmap result, thus providing a reference for establishing multiplex xmap assay for simultaneous detection of animal pathogen , s.antibodies. the antigens used for iltv were a recombinant gd [ ] , expressed, purified, preserved in our laboratory. for the detection of antibodies against ibv, the recombinant n protein was expressed as gst fusion protein in escherichia coli bl (de ) cells [ ] . prior to coupling, the recombinant proteins was desalted by gel filtration using microbio-spin columns (bio-rad, california, usa) based on the manufacture's protocol to exchange the buffer from sodium azide or imidazole to pbs. all the antigens were quantified using pierce bca protein quantification kit (thermo scientific, usa). coupling of proteins to fluorescent microspheres was performed as described by karanikola et al. [ ] . map mc beads (luminex, usa) were coupled by gd protein of iltv and map mc beads (luminex, usa) were coupled by n protein of ibv. a certain number of beads was transferred to one coupling reaction tube, followed by μl bead wash buffer and suspended in μl bead activation buffer. then μl of mg/ml edac (sigma-aldrich) and μl of mg/ml s-nhs (sigma-aldrich, germany) were added in the reaction buffer. the reaction tube was gently vortexed for min at room temperature (rt). pbs (ph . , μl) was added twice and the recombinant protein was added. incubation was carried the xmap assays were carried out in -well polystyrene microplates using luminex detection system (luminex, usa). relevant positive and negative sera have been prepared for the establishment of the singleplex xmap assay to detect iltv antibody or ibv antibody in the serum. iltv gd protein at the following concentrations ( . μg, μg, μg, μg, μg, and μg) and ibv n protein at the following concentrations ( μg, μg, μg, μg, μg, and μg) were coupled with × magbeads to determine the optimum antigen concentration for singleplex xmap assay. a serious dilution of sera at the range from : to : have been made to determine the optimal sample dilution. cross-reactivity or specificity was evaluated by making the assay with sera from chicken infected with aiv, alv, ndv, and mdv. then, a duplex xmap assay was established based on the result of two separated singleplex xmap assay. in briefly, a μl aliquot of the simplex beads ( beads/μl) or the duplex beads ( beads/μl, beads/μl each kind) was transferred into the wells, added in μl of diluted sera. incubation was conducted on a plate shaker ( rpm) at rt for min. the centrifugation was conducted in the magnetic separator for s. the supernatant was removed from each well. beads were washed times in μl pbs. each well was added with μl of a biotinylated secondary antibody (goat anti-chicken igy, beijing solarbio science & technology, china) at μg/ml. the plate was shaken ( rpm) for min at rt and washed as described above. finally, μg/ml of streptavidin-phycoerythrin (sape, life technologies gmbh) was added to each well at μl/well. the plate was shaken and the supernatant was removed. finally, μl of assay buffer was added to each well. the plate was shaken for approximately s and analyzed according to the manufacturer's protocol. all samples were conducted in triplicates in each assay. commercial iltv and ibv elisa kit (biochek, netherlands) were used to detect presence of iltv or ibv antibodies in poultry sera. the assay was performed according to the manufacturer's protocols. herpes virus fusion and entry: a story with many characters avian infectious bronchitis virus viral respiratory diseases (ilt, ampv infections, ib): are they ever under control? molecular biology of avian infectious laryngotracheitis virus structure of herpes simplex virus glycoprotein d bound to the human receptor nectin- antibody-induced conformational changes in herpes simplex virus glycoprotein gd reveal new targets for virus neutralization newcastle disease virus (ndv) recombinants expressing infectious laryngotracheitis virus (iltv) glycoproteins gb and gd protect chickens against iltv and ndv challenges coronavirus avian infectious bronchitis virus an elisa for antibodies to infectious bronchitis virus based on nucleocapsid protein produced in escherichia coli multiplexed microbead immunoassays by flow cytometry for molecular profiling: basic concepts and proteomics applications response of young rabbits to infectious bronchitis virus (wachtel strain) review of infectious bronchitis virus around the world diseases of poultry quantitative, multiplexed detection of bacterial pathogens: dna and protein applications of the luminex labmap (tm) system report from a workshop on multianalyte microsphere assays xmap technology: applications in detection of pathogens a collection of methods and protocols for developing multiplex assays with xmap technology simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types , , , and by a multiplexed luminex assay optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses , , , and development of a multiplex bead-based assay for detection of hepatitis c virus enzyme-linked immunosorbent assay (elisa) for detecting infectious laryngotracheitis viral antibodies in chicken serum enzyme-linked-immunosorbent-assay for the detection of infectious-bronchitis virus (ibv) antigen with monoclonal-antibody a standardized enzyme-linked immunosorbent-assay for infectious-bronchitis viruscomparison with hemagglutination-inhibition and virus-neutralization assays for measuring protective antibody-levels in chickens a novel bead-based assay to detect specific antibody responses against toxoplasma gondii and trichinella spiralis simultaneously in sera of experimentally infected swine conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method validation and comparison of luminex multiplex cytokine analysis kits with elisa: determinations of a panel of nine cytokines in clinical sample culture supernatants luminex xmap combined with western blot improves hiv diagnostic sensitivity preparation of polyclonal antibodies against gd protein of iltv and application cloning and prokaryotic expression of nucleoprotein gene of infectious bronchitis virus (ibv) zz strain development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against cooperia oncophora, dictyocaulus viviparus and fasciola hepatica in cattle availability of data and materials all data generated or analyzed during this study are included in this published article. contact corresponding author for requests. all authors contributed to the interpretation of the data, critically revised the manuscript for important intellectual content, approved the final version to be published, and agree to be accountable for all aspects of the work.ethics approval and consent to participate not applicable. our manuscript does not contain any individual person's data in any form. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -i v rz x authors: chen, tsu-han; lee, fan; lin, yeou-liang; pan, chu-hsiang; shih, chia-ni; lee, ming-chang; tsai, hsiang-jung title: development of a luminex assay for the detection of swine antibodies to non-structural proteins of foot-and-mouth disease virus date: - - journal: journal of immunological methods doi: . /j.jim. . . sha: doc_id: cord_uid: i v rz x abstract foot-and mouth disease (fmd), swine vesicular disease (svd), and vesicular stomatitis (vs) are highly contagious vesicular diseases of swine but are not easy to differentiate clinically. for the purpose of instant detecting of fmd and differentiating it from the other vesicular diseases, a luminex assay was developed. sera from infected, vaccinated, and naïve pigs were tested by the luminex assay. diagnostic sensitivity of the assay was %. diagnostic specificity of the assay was . % in vaccinated pigs and . % to % in naïve pigs. agreement between the results from the luminex assay and those from a abc polypeptide blocking elisa was . % with kappa statistics of . . the luminex assay can detect the immune response to nsp- abc in swine as early as eight days post-infection. moreover, all of the vaccinated but unprotected pigs were all detected by the luminex assay. the results indicated that the luminex assay has potential with specificity in detecting antibodies to fmdv abc nsp and in distinguishing fmdv-infected pigs from with either svdv or vsv. foot-and-mouth disease (fmd), swine vesicular disease (svd), and vesicular stomatitis (vs) are serious vesicular diseases that have devastated swine populations throughout the world. fmd, caused by fmd virus (fmdv; genus aphthovirus, family picornaviridae) (clavijo et al., a; perkins et al., a perkins et al., , b oie, ; oem et al., ) , is one of the most contagious diseases in cloven-hoofed animals. species susceptible to fmdv include swine, sheep, goats, and many wild ruminants. in , a devastating fmd outbreak caused by o/tw/ strain occurred in taiwan and resulted in severe economic losses. the fmdv strain shows a porcinophilic phenotype with a deleted non-structural protein a gene (yang et al., ; beard and mason, ) . another strain o/tw/ , having a full-length a coding region, was isolated from subclinically infected cattle in (huang et al., ) . svd is caused by svd virus (svdv; genus enterovirus, family picornaviridae) (oie, ) . vs, affecting horses, cattle, and pigs, is caused by vs virus (vsv; genus vesiculovirus, family rhabdoviridae) (oie, ) . these diseases are clinically indistinguishable from fmd. to diagnose them correctly, advanced laboratory techniques are therefore required to detect and differentiate antibodies to these different viruses. when susceptible host animals are infected with fmdv, antibodies against both viral structural proteins (sps) and non-structural proteins (nsps) are elicited. in contrast, animals administered with inactivated fmd vaccines that contain no or only trace amounts of nsps are unlikely to induce nsp antibodies. therefore, for fmd diagnosis, the nsp antibodies can be used as a marker to differentiate infected from vaccinated animals. detecting nsp antibodies has an additional advantage that they are serotype independent, because the nsps are predominantly conserved between serotypes of fmdv (clavijo et al., b) . currently, many methods for detecting nsp antibodies have been used, including agar gel immunodiffusion (o'donnell et al., ) , latex bead agglutination test (sugimura et al., ) , enzyme-linked immunoelectrotransfer blot (bergmann et al., (bergmann et al., , bronsvoort et al., ) , enzyme-linked immunosorbent assay (elisa) (de diego et al., ; o'donnell et al., ; sørensen et al., sørensen et al., , shen et al., ; chen et al., ) , and chromatographic strip assay (chen et al., ). the first use of flow cytometry for analysis of microsphere immunoassay was published in (fulton et al., ) . more recently, two types of high-level multi-analyte profiling (xmap) and high-throughput diagnostic methods using microspot arrays and bead arrays have been successfully developed to complement single-analyte assays. antibodies and antigens are directly or indirectly coupled to fluorescent microspheres composed of a variety of materials, such as latex, polystyrene, polyacrylamide, and glass. luminex liquid array technology is one of the detection systems, and it employs microspheres with carboxyl, amine/hydrazide, and maleimide groups on their surfaces to which the molecules such as antibodies, proteins, oligonucleotides, polysaccharides, lipids, or small peptides can bind the microspheres and be subsequently measured by a fluorochrome-conjugated detection molecule. the platform enables simultaneous detection of numerous analytes in a single assay, and there are now many examples of the creative use of this technology (kellar and iannone, ; clavijo et al., ; perkins et al., ) . in the present study, a new application of the luminex liquid array technology was developed, and to address the limitations of existing antibody detection methods of sensitivity and specificity to fmdv, a single-signature luminex assay was developed to detect and differentiate antibodies against svdv and vsv. these tests were also compared with other methods of the same purpose. for studying the diagnostic sensitivity of the tests, fmdpositive serum samples were produced by experimental infection in pigs. sixty-four -week-old spf pigs comprised two groups of pigs. ear tag numbers in group were - . ear tag numbers in group were - , - , - , and . they were intradermally injected with tcid (in a volume of μl) of fmdv o/tw/ strain into the heel bulb of their right foot. blood samples were collected in group and group at and days post infection (dpi), respectively. a panel of sequentially sampled from group of pigs was experimentally tested during to dpi. these swine sera were used to evaluate sensitivity and specificity of the luminex assay. for studying the diagnostic specificity, sera from naïve pigs and sera from vaccinated pigs were used. the naïve pig sera comprised sera collected from spf pigs and sera collected from commercial pigs before the first fmd outbreak in taiwan in . the sera from vaccinated pigs were collected from non-infected commercial pigs that had been vaccinated twice with commercial fmd o/tw/ vaccine. for evaluating possible serological cross-reactivity between fmdv and svdv/vsd in luminex assay, six swine antisera (rs , rs , rs , rs , rs , and rs ) against svdv ukg/ / strain (eu svd reference serum batch ) and six bovine antisera against fmdv serotypes a, c, asia , sat , sat , and sat were purchased from the institute for animal health, pirbright, united kingdom. two antisera against the new jersey and indiana strains of vsv and one vsv-negative serum were purchased from the national veterinary services laboratories, united states of america. for comparing the detection ability of the luminex assay with other methods, a serum panel of group containing swine sera collected sequentially from eight-week-old spf pigs was experimentally infected with fmdv o/tw/ strain as aforementioned. the sera in the panel were sampled at , , , , , , , , , and dpi. one (ear tag numbers in group was ) and five (ear tag numbers in group were - , - , , , ) -week-old spf pigs, were intradermally injected with tcid (in a volume of μl) of fmdv o/tw/ and o/tw/ strains into the heel bulb of their right foot. blood samples were collected finally at and dpi, respectively. the no. serum sample was used as the inter-assay repeatability and the analytical sensitivity of positive control (pc), and the five o/tw/ sera were used as serotypes in identifying the developed luminex assay. the analytical sensitivity was tested through from × serial dilution of − - − to no. antiserum of positive control (pc). neutralizing antibody titers of the positive control sera ranged from : to : . one out of spf swine serum was tested negative by western blotting and elisa was used as the negative control (nc) of inter-assay of the luminex assay. to confirm the infection of the experimentally infected pigs employed for establishing a positive serum panel, the sera collected were tested by the vnt as previously described (oie, ; chen et al., ). production and purification of recombinant nsp abc were described previously (chen et al., (chen et al., , . briefly, the recombinant nsp abc was produced by expression of cloned pcr-amplified abc fragment of fmdv o/tw/ / strain (genebank accession no. aj ; nucleotides to ) within e. coli. the expressed soluble abc polypeptide was covalently coupled to a unique carboxylate bead class (luminex corp., bio-rad, hercules, california, u.s.a.). the amine coupling assay was conducted according to the instruction manual (bio-plex tm , bio-rad). for a one-fold scale coupling reaction, μl of monodisperse carboxylated polystyrene microspheres ( . × beads) (bio-plex) was transferred to one of the coupling reaction tubes. the beads were centrifuged at , ×g for min, removed and discarded the supernatant. the microspheres were resuspended in μl of bead wash buffer by vortexing and sonicating for s. the microspheres were then centrifuged at , ×g for min to remove the supernatant and were resuspended in μl of bead activation buffer. the beads were vortexed and sonicated by bath sonication for s. freshly prepared μl of a bead activation buffer solution of sulfo-n-hydroxysuccinimide (s-nhs; mg/ml) (pierce biotechnology, thermo fisher inc., rockford, illinois, u.s.a.) and n-( -dimethylaminopropyl)-n′-ethylcarbodiimide hydrochloride (edc; mg/ml) (pierce biotechnology) were added to each tube. the tubes were vortexed for s. and gently agitated at room temperature in darkness for min. after the agitation, they were centrifuged at , ×g for min to remove the supernatant and washed with μl of pbs (ph . ) once, repeating this step. following the washing step, the microspheres were centrifuged at , ×g for min to remove the supernatant, resuspended in a concentration of - μg/ μl recombinant abc protein solution by vortexing for s, and gently agitated at °c overnight in darkness, allowing the abc protein molecules to couple to the microspheres. the coupled beads were centrifuged at , ×g for min to remove the supernatant. and then the beads were washed with μl of pbs (ph . ), centrifuged at , ×g for min, and discarded the supernatant. the coupled beads were resuspended with μl of blocking buffer by pipetting, and gently agitated at room temperature in darkness for min. then, the beads were centrifuged at , ×g for min and discarded the supernatant. the abc-coupled microspheres were washed with μl pbs (ph . ) and centrifuged at , ×g for min, and then the supernatant was discarded. the abc-coupled microspheres were resuspended with μl of pbs (ph . ) for formulation to make a coupled microsphere stock and stored at °c in darkness. to prepare a working microsphere suspension, the coupled microsphere stock was diluted to a final concentration of microspheres of each set in pbs, and μl of working microsphere suspension was required for each reaction. a -well multiscreen® hts . μm filter plate (emd millipore, billerica, massachusetts, u.s.a.) was prewetted by μl/well of pbs and aspirated by vacuum manifold. a volume of μl of the working microsphere suspension was added to the appropriate wells of the wetted filter plate. blocking buffer [ % (w/v) casein; hammer-sten grade] in mm sodium phosphate ( mm nacl, ph . , containing kathon® antimicrobial agent) ( μl/well) was added to a well as the blank. in addition, μl of sera from experimentally fmdv-infected pigs (pc), spf swine serum (nc), svdv antisera and test serum samples were -fold diluted with blocking buffer and added to the appropriate wells as controls and detected test serum samples. the plate was incubated for min at room temperature on a plate shaker. for detecting swine and bovine serum samples, biotin-spconjugated goat-anti-swine igg (jackson immunoresearch laboratories inc., west grove, pennsylvania, u.s.a.; . mg/ml) and biotin-conjugated goat-anti-bovine igg (kpl, gaithersburg, md, u.s.a.; . mg/ml) as secondary antibodies, were a : and : - : diluted with pbs at optimal concentration, respectively. a volume of μl of the diluted secondary antibody was added to the wells of the filter plate with incubated controls and serum samples, after the previous -minute incubation. the filter plate was then covered with adhesive film and incubated for min at room temperature on a plate shaker. r-phycoerythrin conjugated streptavidin (jackson immuno-research laboratories, u.s.a.; . mg/ml), as a reporter reagent, were a : diluted with pbs at optimal concentration against reporter antibodies for swine and bovine, respectively. a volume of μl of the diluted reporter reagent was added to the appropriate wells of the filter plate, after the addition of secondary antibody and incubation. the filter plate was covered with adhesive film and incubated for min at room temperature on a plate shaker. after the -minute incubation, μl of reaction in each well was analyzed by luminex analyzer bio-plex tm system (bio-rad) according to the operation manual. the median fluorescence intensity (mfi) for microspheres for each specific protein was recorded for each well. to normalize the results obtained from different tests, results were expressed as a test/control (t/c) index. three commercially available elisa kits, one in-house elisa, and one chromatographic strip assay were employed to compare their detection performance with that of the developed luminex assay. the three commercially available elisa kits were priocheck fmdv-ns kit (prionics lelysted b.v., lelysted, the netherlands), which is a abc recombinant protein-based blocking elisa; ubi fmd ns eia (united biomedical inc., hauppauge, new york, u.s.a.), which is a b synthetic peptide-based indirect elisa; and idexx fmdv abc ab test (idexx laboratories inc., westbrook, maine, u.s.a.), which is a abc recombinant protein-based indirect elisa. the kits were conducted according to the manufacturers' instructions. in-house sandwich elisa for the o serotype was established to detect serum antibodies against abc nsp (chen et al., ) . shortly, -well microtiter plates were coated with abc nsp specific mab and incubated. the abc recombinant protein was then added and incubated. diluted test pig serum was added and incubated. diluted horseradish peroxidaselabeled goat anti-swine igg was added and incubated. the substrate, tmb solution, was added and incubated. finally, stop solution, sulfuric acid, was added to stop the color development. each incubation step was followed by washing. results were expressed as a test to control (t/c) index. in addition, in-house the abc-based chromatographic strip assay (chen et al., ) is described previously. the serum sample and dilution buffer were mixed, and the mixture was applied to the sample pad of the chromatographic strip. after incubation at room temperature, the strip was read by a uniscantm point-of-care testing (poct) scanner. the scanner was able to detect the color density of the developed strip and produce a corresponding analog test signal. with a signal amplifier and an analog/digital converter in the scanner, the analog signal was then amplified and converted into a digital signal. a computing unit within the scanner received the digital signal, analyzed it, and output a concentration value for the analyte. the time required to read one strip was to s. to standardize the results among different batches of strips, each value read by the scanner was divided by a modified factor supplied with the batch to be a standardized value. agreement between luminex assay and elisa kits was evaluated using kappa statistics. fifteen sera were collected from pigs that received one dose of commercial available vaccine serotype o/tw/ fmd vaccine (no. vaccine, v : fgbi "arriah"/russia; no. vaccine, v : biogenesis-bago s.a./ argentina ) at weeks old and were intramuscularly injected with tcid fmdv at weeks old. the pigs, from two farms, were assigned into four vaccinated groups. as shown in table , the digit following w indicates the farm number, and the digit following v indicates the commercial vaccine administered. additionally, two pigs in the control group, given pbs instead of vaccine, were infected as the vaccinated groups. all experimentally infected pigs demonstrated neutralizing antibody titers ranging from : to : after dpi, confirming the establishment of infection (table ) . the two-fold serial dilution of positive and negative control sera against fmdv-nsp by the single-signature luminex assay is depicted in fig. . a dilution of / - / was the best in discriminating positive from negative sera as well as minimizing false positive and false negative results. the detection limit for the pc serum (mfi n ) was / . by double duplex test, the mfi values of positive, negative, and blank were . ± . , . ± . , and . ± . , respectively (fig. ) . the analytical sensitivities of the developed luminex assay were − fold (data not shown). to differentiate infected from noninfected pigs clearly, the pc serum samples that from group and group were used in this experiment taken at and dpi, and nc sera were as illustrated in fig. , the cutoff value was determined to be t/c ratio as . (equivalent to mfi value), which equals the mean of median fluorescence intensities (mfis) of the nc the result was indicated as the virus neutralization test antibody titers over : to : after dpi and experimentally pigs were exposed in clinical syndrome. sixty-four positive control (pc) sera (group and group ) and negative control (nc) sera were tested against foot-and-mouth disease virus non-structural protein (fmdv-nsp) by the single-signature luminex assay to determine its cutoff value. sera plus three standard deviations (sd) of the nc sera. based on the cutoff, a sample was interpreted as negative when t/c ratio ≦ . , and positive when t/c ratio n . . inter-assay repeatability of the luminex assay was evaluated for the high and low controls that are run in duplicate on different plates to monitor plate-to-plate variation. the group of the fmdv o/tw/ serum panel of which the ear tag number was sampled at dpi was used as a positive control (pc) for control of standardized. one out of spf swine serum was tested and used as a negative control (nc) of the luminex assay. the mean of mfi values of pc was . ± . (coefficient of variance, or cv = . ) and the mean of mfi values of nc was . ± . (cv = . ). . . comparison of the luminex assay with the commercially available elisa and in-house methods in detecting serially sampled sera a set of serum panel of group was tested from sequentially sampled sera during the course to dpi for experimentally infected pigs by the luminex assay and the other methods. positive results were given first at dpi, and all pigs were tested positive by dpi. from dpi, positive percentage stayed over % throughout the period of the animal experiment by the luminex assay. the abc blocking elisa kit, b indirect elisa kit, in-house sandwich elisa, and the chromatographic assay gave similar results (fig. a) . the agreement between the results of our luminex assay and abc blocking elisa kit was . with kappa statistics of . ( % confidence interval: lower limit: . , upper limit: . ). the agreement between the results of our assay and those of b indirect elisa kit was . with kappa statistics of . ( % confidence interval: lower limit: . , upper limit: . ). the agreement between the results of our assay and those of the abc indirect elisa kit was . with kappa statistics of . ( % confidence interval: lower limit: . , upper limit: . ). testing vaccinated pigs by the luminex assay, out of sera ( . %) tested negative (t/c ratio ≦ . ), and all the mfi values were lower than (fig. ) . all serum samples were tested negative by the abc blocking elisa kit. with sera evaluated from group sampled at th dpi of experimentally infected pigs and from group sampled at th dpi of infection pigs, spf pigs, non-vaccinated commercial pigs, and vaccinated commercial pigs, the diagnostic sensitivity and specificity of the luminex assay are shown in table . diluted bovine antisera against fmdv serotypes a, c, asia , sat , sat , and sat gave positive results by the luminex assay. most antisera gave mfi values higher than , except that against the × dilution of secondary antibody to antiserum sat , and the dilutions of × and × of secondary antibody against the sat give the mfi below , (fig. ) . the performance of the luminex assay in detecting specific nsp antibody against either the one of the infected pigs for group serum panel to fmdv o/tw/ in dpi or the five infected pigs for group sera to fmdv o/tw/ in dpi were compared with those of the abc blocking elisa kit. it is noted that two of the five fmdv-o/tw/ sera demonstrate positive results by the luminex assay but not by the blocking elisa kit (table ) . the six antisera against svdv ukg/ / strains obtained negative results by the luminex assay (data not shown). the one (rs ) of the six antiserum was evaluated to positive control of svdv. two antisera against the new jersey and indiana strains of vsv also tested negative by the luminex assay (table ) . all of the vaccinated pigs that showed typical vesicle lesions on the feet and snout at four days post challenge tested positive by the luminex assay. all serum samples had vnt antibody titers ranging from : to ≧ : , confirming infection. the luminex assay had a highly positive correlation with those generated by chromatographic strips and elisas. however, out of the sera were tested positive by the in-house sandwich elisa. in contrast, only of the sera were positive by the commercially available abc blocking elisa kit (table ) . a microsphere immunoassay was developed and validated to detect antibodies to abc nsp of fmdv in the present study. the fmdv- abc antibody is a reliable indicator of fmdv infection for sero-epidemiological studies. luminex offers advantages over this test such as sensitivity, specificity, l a n k serotype specific positive sera and negative sera fig. . detection of non-structural protein antibodies in bovine infected with foot-and mouth disease virus serotypes other than serotype o by the luminex assay. the biotin-conjugated anti-bovine igg used as secondary antibody in the assay was diluted fold and fold, respectively. spf, specific-pathogenfree pig serum; fcs, commercially available fetal calf serum. interpreted as negative when t/c ratio ≦ . and positive when t/c ratio n . , the means of the mfi values are equal to or higher than mfi as positive. b pi: percentage inhibition obtained from priocheck fmdv-ns kit. a sample is interpreted as positive when giving a pi value equal to or higher than %. c spf: specific-pathogen-free. simplicity, reliability, multiplexing capabilities, simultaneous quantitative determination, reducing the subjectivity, less time for data acquisition, time saving and reduced cost, statistical superiority, more flexibility, faster hybridization kinetic, greater accessibility to antibody, and no-wash assay dunbar, ; oliver et al., ; vignali, ) . the assay is observed in some research, such as that for cytokines il- , - , - , - , - , - ; tnf-α; ifn-γ; antibodies to human immunodeficiency virus, avian influenza virus, cytomegalovirus, epstein-barr virus, and rubella and toxoplasma; soluble hepatitis b virus surface antigen in serum; and nucleic acid detection for single nucleotide polymorphism genotyping, genetic disease screening, gene expression profiling, infectious disease including hiv, sars-cov, hepatitis c virus, herpes simplex virus, hla dna typing, microbial detection, and other biomolecules (watson et al., ; willman et al., ; kellar and iannone, ; dunbar, ; lenhoff et al., ) . in this study, a luminex assay based on abc nsp of fmdv serotype o was developed to test serum antibody against fmdv- abc in swine. through the study, an ideal cutoff value of the assay was determined (fig. ) , early detection was validated (fig. ) , and high specificity and sensitivity were demonstrated (figs. , , and table ). the flow cytometer is capable of reading several hundred beads per second, and each analysis can be completed in as little as s. potentially up to different analyses can be performed simultaneously, thereby providing a highthroughput platform. it is also conducive to automation, employing high throughput, liquid-handling robotic platforms that minimize human resources required for running routine screening (dunbar, ; perkins et al., perkins et al., , b . it has been reported that the analytical sensitivity of a luminex assay can achieve as low as ≦ pg/ml with high-titer antibodies (kellar et al., ) . high-affinity cytokine antibody in combination with liquid-phase kinetics and the high binding capacity of three-dimensional microspheres produce a robust multiplexed immunoassay with a detection limit of these assays extending over three to four logs, as compared with one to two logs for elisas (kellar and iannone, ) . in our single-signature luminex assay, it is expected that similar results were obtained. the analytical sensitivities of the chromatographic strip assay, sandwich elisa, and the newly developed luminex assay were , , and and times higher than that of the commercial abc blocking elisa kit (diluted − fold), respectively (chen et al., ) . this revealed that the chromatographic strip assay could rapidly accomplish the detection of nsp antibodies and was more suitably applied to clinical diagnosis. however, the luminex and elisas could detect a larger number of samples at a time in a laboratory when used by skilled personnel. in this study, the cutoff value was determined to be . in t/c ratio (equivalent to of the mfi value) based on testing sera from naïve and experimentally infected pigs (fig. ) . to compare the relative sensitivities of nsp antibody assays, our laboratory generated a swine serum panel composed of samples. the experimentally infected pigs at dpi still had positive reactions of % and remained positive up to dpi. the results indicate the peaks of positive ratios appeared on dpi and dpi, respectively ( fig. a and b) . three positive reactors were obtained on and dpi in samples from the same experimentally infected pigs. these positive reactors tested negative by the virus neutralization test and the elisas, suggesting that these were false positive results. however, three of the six methods, the luminex assay, sandwich elisa, and the abc blocking elisa kit showed earliest detection of nsp antibody in sequential serum samples on dpi and gave positive ratios of %, %, and %, respectively. this is in agreement with most authors that report the appearance of specific antibodies by dpi or dpi (bergmann et al., ; chen et al., chen et al., , . in contrast, on the same time point the positive ratios given by the chromatographic strip assay and b indirect elisa were less than %. the inconsistency could be due to some differences in conformation of proteins generated by different expression systems, thus suggesting that the elisa platforms have a good capacity of detecting a small amount of antibodies in samples and are a suitable selection of serological mass screening. in fact, the abc indirect elisa can detect fmdv-infected pigs only on dpi and later whereas the other elisa assays showed high rates of positivity and earlier detection (fig. a) . thus, the performances of these elisa assays are assay-dependent. the luminex assay also detected remarkable nsp antibody responses by dpi in experimentally infected pigs, consistent with the results from the sandwich elisa, abc blocking elisa kits, and b peptide indirect elisa. it indicated the performance parameters of the luminex assay and the good correlation with the elisa recommend that we validate the luminex assay for analyzing fmdv-nsp recombinant protein profiles in immunodiagnostic reagent studies in vitro (fig. a) . there are similar other immunoassay technologies as luminex and blocking elisa and indirect elisa where the reagents are critically important (shen et al., ; sørensen et al., sørensen et al., , chen et al., ; clavijo et al., ; codorean et al., ) . currently, collaborative efforts are underway to fully optimize and validate this luminex assay for mass screening and confirmation. comparison of the performance for the detection of experimentally infected pig sera between the luminex and the abc blocking elisa, b indirect elisa, and the abc indirect elisa kits demonstrated kappa statistics of . , . , and . , respectively, demonstrating a high level of agreement against the former two. to evaluate the ability of the luminex assay to detect anti-nsp antibodies elicited by fmdvs of other serotypes, bovine sera against serotypes other than serotype o were also tested. the results suggested that our luminex assay was effective in detecting the nsp antibodies against other fmd serotypes (fig. ) , although the sera were from cattle and their numbers are very limited. the results showed that the luminex assay using the abc and anti- abc antibody derived from the fmdv o/tw/ strain could detect antibodies induced by not only o/tw/ but also o/tw/ strains. the antibody detection for two out of the five showed the pi value under % for . and as a negative reaction for the o/tw/ strain antiserum by the abc blocking elisa. indeed, both of these methods have distinct differences (table ) . as discovered through our experiments, the luminex assay demonstrated that the sensitivity was higher and the specificity was lower with cutoff values of . and . , and in contrast, sensitivity was lower and the specificity higher to the cutoff value . , but all were higher than % (data not shown). finally, evaluation of testing results was adopted based on the cutoff value in the t/c ratio of . . furthermore, with antisera against svdv and vsv, the assay proved that it could specifically recognize antibodies against fmdv and did not cross-react with antibodies against svdv and vsv (table ) , indicating that it can be used in areas where the three viruses might exist. the method was able to distinguish between antibodies elicited by naturally infected animals and those by vaccinated animals. vaccination is one of the main methods that can be implemented to control fmd, and it is currently the primary control strategy used in taiwan. with coverage through vaccination, highly sensitive methods are essential to discover virus activities, clinical and subclinical, in the field. in the present studies, we displayed vaccinated pigs by the luminex assay, and four vaccinated animals showed reactions over mfi (fig. ) , three out of four of which were closer to the threshold less than mfi value. this can be due to the presence of low levels of nsp antibody or nonspecific reactions to contaminants in the antigens or maybe actually normal. that nsp antibody in all the sera from vaccinated but unprotected pigs was able to be detected by the luminex and the chromatographic strip. however, out of the sera were positive by the sandwich elisa, and only of the sera were positive by the abc blocking elisa kit (table ). the results indicated that the former two methods were more sensitive, as confirmed by the clinical diagnosis, and worthy to be applied to the detection of nsp antibodies in vaccinated animals in the future. thus, the luminex developed here demonstrates the diagnostic application of recombinant fmdv nsp- abc in monitoring seroconversion following fmd vaccination. to sum up, as such a luminex-based immunoassay promises to be a sensitive and efficient method, we will, hopefully, be able to rapidly analyze field serum samples and effectively to diagnose the vesicular diseases in swine. therefore, we can diagnose and then cull the infected animal in a very early stage. genetic determinants of altered virulence of taiwanese foot-and-mouth disease virus diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens improvement of a serodiagnostic strategy for foot-andmouth disease virus surveillance in cattle under systematic vaccination: a combined system of an indirect elisa- abc with an enzyme-linked immunoelectrotransfer blot assay rapid serological profiling by enzyme-linked immunosorbent assay and its use as an epidemiological indicator of foot-and-mouth disease viral activity evaluation of three abc elisas for foot-and-mouth disease non-structural antibodies using latent class analysis development of a chromatographic strip assay for detection of porcine antibodies to abc non-structural protein of foot-and-mouth disease virus serotype o differentiation of foot-and-mouth disease-infected pigs from vaccinated pigs using antibody-detecting sandwich elisa developments in diagnostic techniques for differentiating infection from vaccination in foot-andmouth disease development and use of a biotinylated abc recombinant protein in a solid-phase competitive elisa for the detection of antibodies against foot-andmouth disease virus simultaneous detection of antibodies to foot-and-mouth disease non-structural proteins abc, d, a and b by a multiplexed luminex assay to differentiate infected from vaccinated cattle correlation of xmap and elisa cytokine profiles; development and validation for immunotoxicological studies in vitro the non-structural polyprotein abc of foot-and-mouth disease virus as a diagnostic antigen in elisa to differentiate infected from vaccinated cattle applications of luminex xmap technology for rapid, high-throughput multiplexed nucleic acid detection advanced multiplexed analysis with the flowmetrix system anti- ab antibodies in the chinese yellow cattle infected by the o/taiwan/ foot-and-mouth disease virus multiplexed microsphere-based flow cytometric assays multiplexed microspherebased flow cytometric immunoassays multiplexed molecular assay for rapid exclusion of foot-and-mouth disease detection of virus infectionassociated antigen and d antibodies in cattle vaccinated against foot and mouth disease simple and rapid lateral flow assay for the detection of foot and mouth disease virus multiplexed analysis of human cytokines by use of the flowmetrix system multiplexed detection of antibodies to nonstructural proteins of footand-mouth disease virus toward a multiplexed serotyping immunoassay for footand-mouth disease virus use of a standardized bovine serum panel to evaluate a multiplexed nonstructural protein antibody assay for serological surveillance of foot-and-mouth disease differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide elisa differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins d, ab and abc in elisa using antigens expressed in baculovirus differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking elisa based on baculovirus expressed fmdv abc antigen and a abc monoclonal antibody multiplexed particle-based flow cytometric assays a multiplexed immunoassay for detection of antibodies against avian influenza virus world organization for animal health (oie), . chapter . . . vesicular stomatitis, manual of diagnostic tests and vaccines for terrestrial animals epidemiological characteristics and financial costs of the foot-and-mouth disease epidemic in taiwan we are grateful to dr. t. s. huang and dr. c. wang for their technical support and recommendation in this study. this work was supported by grants as- . . -hi-h and as- . . -hi-h from the council of agriculture, taiwan. key: cord- -iesysf l authors: eshaghi, majid; tan, wen siang; chin, wai kit; yusoff, khatijah title: purification of the extra-cellular domain of nipah virus glycoprotein produced in escherichia coli and possible application in diagnosis date: - - journal: j biotechnol doi: . /j.jbiotec. . . sha: doc_id: cord_uid: iesysf l the glycoprotein (g) of nipah virus (niv) is important for virus infectivity and induction of the protective immunity. in this study, the extra-cellular domain of niv g protein was fused with hexahistidine residues at its n-terminal end and expressed in escherichia coli. the expression under transcriptional regulation of t promoter yielded insoluble protein aggregates in the form of inclusion bodies. the inclusion bodies were solubilized with m urea and the protein was purified to homogeneity under denaturing conditions using nickel–nitrilotriacetic acid (ni–nta) affinity chromatography. the denatured protein was renatured by gradual removal of the urea. light scattering analysis of the purified protein showed primarily monodispersity. the purified protein showed significant reactivity with the antibodies present in the sera of niv-infected swine, as demonstrated in western blot analysis and enzyme-linked immunosorbent assay (elisa). taken together, the data indicate the potential usefulness of the purified g protein for structural or functional studies and the development of immunoassay for detection of the niv antibodies. nipah virus (niv) is a negative sense, nonsegmented rna virus that was first isolated from cere-brospinal fluid of the human patients (chua et al., ) . it was found to be the etiologic agent for both human and swine diseases and classified to the family paramyxoviridae under a new genus henipaviruses (chua et al., ; mayo, ) . its genome encodes six structural proteins: nucleocapsid (n), phospho-(p), matrix-(m), fusion-(f), glyco-(g) and large-(l) proteins wang et al., ) . the natural reservoir of the virus is fruit bat with no signs of infection but causes fatal encephalitis in humans and a respiratory syndrome in pigs (chua et al., ) . the infected pigs can transfer the virus to other animals such as dogs, cats, and horses (chua et al., (chua et al., , paton et al., ) . most recently, guillaume et al. ( ) found that destruction of the natural habitat of fruit bats caused the migration of these bats to live closer to humans and domesticated animals, and as a result the virus can be transferred to new species. recent outbreaks of the niv and nipahlike viruses in bangladesh (who, a,b) demonstrate the existence of a time bomb that poses a major health problem worldwide, which could destroy the economies of many countries. therefore, there is a demand for rapid detection as well as serological diagnosis of the virus for monitoring the presence of the virus and its antibodies in individuals and animals in high-risk areas. currently, production of immunological reagents for these assays require biohazard level (bl ) laboratories which are limited only to a few countries worldwide. the g protein plays a central role in the viral replication process since it is responsible for the viral attachment to sialic acid-containing host cell receptors (bossart et al., ) . thus, the g protein appears to be essential for paramyxovirus replication, and as such could represent the primary target for neutralizing antibodies as well as potential targets for antiviral agents. in this study, the extra-cellular domain of the g protein of niv was cloned and expressed in the escherichia coli system. the purified product was used as the capturing antigen in an enzyme-linked immunosorbent assay (elisa) to determine the presence of the anti-niv antibodies in serum samples collected from naturally infected swine. it was found that the purified g protein reacted only with antibodies in niv positive samples, suggesting a potential replacement for currently used whole virus antigen that requires containment facilities. swine anti-niv sera with known serum neutralizing test (snt) and inactivated niv from infected cell culture medium were generous gifts from the veterinary research institute (vri), ipoh, malaysia. a vol-ume of l of niv-infected cell culture medium was used to extract total vrna using the trizol ls reagent (life technologies, usa), as recommended by the manufacturer. extracted vrna was used as a template for cdna synthesis using the superscript ii rnaseh (−) reverse transcriptase (life technologies, usa), which was subsequently used in a pcr amplification using the platinum tm high fidelity taq dna polymerase (life technologies, usa). two synthetic oligonucleotides, tgniv forward ( -gggggatcc-atggacaatcaggccgtgatc- ) and tgniv reverse primers ( -ggggggaagcttctcaacca-atgatatgcacca- ) were used to amplify the coding sequence of the niv g gene between nucleotides and . the underlined nucleotides represent bamhi and hindiii cutting sites, respectively. these two primers were designed to eliminate the transmembrane and signal regions of the g protein of niv. the pcr product was digested with bamhi and hindiii and cloned into the prset plasmid (life technologies, usa) that had been digested with the same restriction endonucleases. the resulting construction was transformed into e. coli strain bl si (life technologies, usa). the expression was confirmed with sds-page and western blot analysis before subjecting to purification and further analysis. broad range protein markers (gibco, brl) were used in sds-page and western blot analysis. swine anti-niv polyclonal antibodies ( / dilution) were used as the primary antibody. appropriate species-specific immunoglobulin conjugated to alkaline phosphatase ( / dilution) was used as the secondary antibody. the recombinant protein was extracted from bacterial cells using the bacterial protein extraction reagent (b-per) (pierce, usa), as recommended by the manufacturer with some modifications. briefly, bacterial cells from ml culture were centrifuged at × g for min and the pellet was washed with pbs ( mm sodium phosphate, . m nacl, ph . ) and centrifuged as above. the cells were resuspended in ml of b-per reagent and the mixture was gently shaken at rt for h before centrifugation at , × g for min to separate inclusion bodies from soluble proteins. after repeating the above extraction for two times, the inclusion bodies were resuspended in ml of b-per reagent containing g/ml lysozyme (sigma, usa), and the mixture was incubated at rt for min before adding ml pbs. the inclusion bodies were collected by centrifugation at , × g for min. the pellet was resuspended in ml of b-per reagent in pbs ( : dilution) and centrifuged as above. the extracted inclusion bodies were dissolved in ml of denaturing buffer ( m guanidine hcl; mm napo , ph . ; mm nacl) and loaded onto a pre-equilibrated column packed with ml of nickel-nitrilotriacetic acid (ni-nta) agarose (life technologies, usa). the protein-bound ni-nta resin was first washed with ml of denaturing binding buffer ( m urea; mm napo , ph . ; mm nacl) followed by washing with ml of denaturing wash buffer ( m urea; mm napo , ph . ; mm nacl). the column was washed with another ml of the above washing buffer at ph . the bound recombinant protein was finally eluted with ml denaturing elution buffer ( m urea; mm napo , ph . ; mm nacl) and the sample fractions were analyzed by sds-page and western blotting. the fractions containing the purified recombinant protein were pooled and first dialyzed against m urea for h. the dialysis was continued by the addition of ml of mm tris-hcl (ph . ) every h for three times and finally the dialysis solution was replaced with mm tris-hcl (ph . ), mm nacl, . % triton x- and mm ␤mercaptoethanol, and dialyzed for another h. the purity of the sample was studied with a dynamic light scattering (dls) instrument (dynapro-ms/x, proterion inc., uk) at • c, and at least measurements were taken at each experiment. all dls measurements were performed at . nm wavelength laser at • angle. the titers of the anti-g antibodies in swine sera were determined using elisa. all washing steps were carried out three times with ttb [ . % tween- in tbs ( mm tris-hcl, mm nacl; ph . )]. all antigen and antibody dilutions were done in tbs. flat-bottom polystyrene microtiter plates were used as solid-phase adsorbents (tpp immunomax high binding flat-bottomed elisa plate, usa). the plates were first coated with anti-swine igg ( / ) (kpl, usa) for overnight at rt. after washing, the plates were blocked with l of sea block blocking buffer (pierce, usa) for h at rt. subsequently, the plates were washed and incubated for h at rt with appropriate dilution ( / ) of the swine sera from infected and noninfected animals. following another round of wash-ing, l of optimum concentration ( ng/well) of the recombinant g protein in tbs was added in the wells and the plates were incubated at rt for further h. after washing with ttb, the appropriate dilution ( / ) of mouse anti-his (amersham biosciences, uk) was added. following h of incubation at rt, the plates were washed and l of the anti-mouse igg alkaline phosphatase (kpl, usa) was added and the microtiter plates were incubated further for h at rt. subsequently, the enzyme-substrate solution, containing mg/ml pnpp (p-nitrophenyl phosphate, sigma, usa) in . m diethanolamine (sigma, usa), ph . was added. the reaction was stopped after min at rt by the addition of l of n naoh to each well and the a values were measured with a microtiter plate reader (bio rad, usa). the extra-cellular domain of the niv g gene was cloned downstream of a t promoter in the prset vector system, which allows the expression proteins via transcription by t polymerase. niv g gene was expressed as a protein attached at its amino-terminus to a polyhistidine peptide. as shown in fig. a , an extra protein band with the expected m r of approximately kda appeared in the insoluble fraction of the extract of bacteria containing the recombinant plasmid and was absent in the corresponding fraction of bacteria containing the vector without an insert. the time course study showed that the highest level of expression could be achieved after h post induction (data not shown). an immunoreactive band of approximately kda was detected by the pooled anti-niv sera on a western blot (fig. b) , confirming the expression of the recombinant g protein. the inclusion bodies, which were extracted from bacterial cells using b-per reagent, were washed several times with the same reagent to remove most of the contaminants. two reasonably pure protein bands of approximately and kda were observed in a polyacrylamide gel stained with coomassie blue (fig. a) . the lower band was detected with swine anti-nipah sera but not with anti-his (data not shown), indicating that it could be either a bacterial protein or his-tag deleted proteolytic product derived from the recombinant protein. the extracted inclusion bodies were ( ) cells containing the vector without an insert, ( ) recombinant g before purification, ( ) purified inclusion bodies, ( ) purified by affinity chromatography. (b) western blot of the above samples using swine anti-niv sera. the same number of the bacterial cells was used for the first two lanes. solubilized in m guanidine hcl containing buffer and directly purified on ni-nta matrix under denaturing conditions. subsequently, the urea was gradually eliminated from the purified protein by dialysis and the protein renatured again. the concentration of the purified recombinant g protein from the total bacterial cell proteins was estimated to be approximately . - . mg g − of the wet weight of bacterial cells. light scattering of the purified protein showed monodispersity with a hydrodynamic radius (r h ) of nm, whereas the inclusion bodies were found to be polydispersed, and polymodal regression analysis in- dicated % of one species with r h nm and % of the another species with r h nm. in order to determine the specificity and sensitivity of the recombinant g protein as antigen for detecting antibodies in niv positive swine sera a quantitive immunoassay was carried out. a total of niv positive sera and negative sera from healthy swine were tested, and the results showed that the purified g protein reacted specifically with antibodies in niv positive samples (fig. ) . this reflects the reliability of the purification procedure in eliminating bacterial contaminants from the g protein. inactivated niv is currently being used as a coating antigen in elisa for detection of the anti-niv antibodies in serum samples. the process of growing the virus in cell cultures, however, requires bl laboratories. in addition, the use of niv is restricted in certain countries due to biosecurity considerations. therefore, an elisa based on a recombinant protein, as capturing antigen, is simpler to produce and perform than elisa based on inactivated virus. the latter is hazardous and not suitable for large-scale surveys. in order to assess the suitability of the g protein as an elisa antigen for the detection of specific anti-niv antibodies, a recombinant prset vector was constructed, which could be used to express the recombinant g protein in e. coli bl si. production of such recombinant proteins in e. coli as capturing antigens for diagnosis of diseases caused by viruses have been widely used including turkey coronavirus (loa et al., ) , marburg virus (saijo et al., ) , norwalklike virus (yoda et al., ) , measles virus (warnes et al., ) , african fever swine virus (freije et al., ) , lassa virus (barber et al., ) , and hepatitis b virus (stahl et al., ) . furthermore, the advantages of using a prokaryotic host to produce recombinant g protein would be considerable due to the ease of scale-up, and the low costs involved in growing bacteria. a strong specific signal was observed in western blots confirming the specific immunoreactivity of the recombinant g protein. the results of the western blot analysis and elisa test suggest that the recombinant g protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. the inclusion bodies before purification showed negligibly low background on western blot but when used as the coating antigen in elisa, a relatively high background was observed. this was probably either due to the presence of bacterial protein (s) and/or existence of some antibodies recognizing conformational epitopes in elisa. the results of the light scattering also confirmed the impurity of the inclusion body sample and monodispersity of the purified recombinant protein. based on the molecular mass of the recombinant g protein ( kda) and the assumption that it is spherical form, the calculated radius of the protein is about . whereas the r h of the purified protein detected by dls was about nm. this implies that the protein most probably exists in multimer forms (trimer or tetramer). in order to ensure a more consistent binding pattern of the coated antigen to the elisa plate and to avoid the high background, it was decided to use affinitypurified recombinant g protein as the coating antigen. the validation of the recombinant g protein elisa was carried out using samples of swine sera. the aim was to determine the variability of negative and positive sera using elisa based on recombinant g protein. the newly established assay was able to distinguish clearly the two groups of samples based on their reactivity. however, the result of the elisa should be interpreted cautiously, since proper standardization of the elisa requires sera from experimentally infected animals followed by testing a more significant number of field serum samples. unfortunately, it was not possible to obtain sera from experimentally infected animal sera due to lack of the bl laboratory throughout this study and also to the limited availability of the field sera. expression of lassa virus nucleocapsid protein segments in bacteria: purification of high-level expression products and their application in antibody detection functional expression and membrane fusion tropism of the envelope glycoproteins of hendra virus nipah virus: a recently emergent deadly paramyxovirus fatal encephalitis due to nipah virus among pig-farmers in malaysia isolation of nipah virus from malaysian island flying-foxes highlevel expression in escherichia coli of the gene coding for the major structural protein (p ) of african swine fever virus nipah virus: vaccination and passive protection studies in a hamster model molecular characterization of nipah virus: a newly emergent paramyxovirus expression and purification of turkey coronavirus nucleocapsid protein in escherichia coli a summary of taxonomic changes recently approved by ictv outbreak of nipah virus infection among abattoir workers in singapore enzyme-linked immunosorbent assays for detection of antibodies to ebola and marburg viruses using recombinant nucleoproteins hepatitis b virus core antigen: synthesis in escherichia coli and application in diagnosis molecular biology of hendra and nipah viruses expression of the measles virus nucleoprotein gene in escherichia coli and assembly of nucleocapsidlike structures outbreak of niv in bangladesh outbreak of nipah and hendra-like viruses in bangladesh expression of recombinant norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection this study was supported by the grant no. - - - from the ministry of science, technology and innovation, malaysia (mosti). m. eshaghi is supported by national science fellowship (nsf) from mosti. key: cord- - ickafd authors: kapil, sanjay; yeary, teresa; johnson, bill title: diagnostic investigation of emerging viruses of companion animals date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: ickafd in this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. veterinarians are bound to encounter emerging viruses in their practice. the problem is unavoidable, because viruses are highly mutagenic. even the immune response dictates the nature of virus that evolves in a host. if the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with the diagnostic laboratory to solve the diagnostic puzzle. c linicians and laboratorians are usually the first to detect most outbreaks of emerging diseases in animals. much attention is rightfully given to emerging diseases of commercial food animals; however, small animal practitioners also have an obligation to be vigilant to the possibility that new and devastating viral diseases might emerge that infect the companion animals in their charge. canine parvovirus (cpv) type , emerged in and spread worldwide within less than years [ ] . in , a new antigenic type, cpv- c, was reported in italy [ ] , which has since caused outbreaks in western europe, asia, south america, and the united states [ ] because current vaccines offer no protection for this type. in this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. the term emerging virus is defined broadly and includes these categories: variants of a known virus that has gained enhanced virulence or that is able to infect completely vaccinated animals a known virus that has reappeared in the population after a decline in incidence novel or previously unidentified viral agents detected for the first time because of improved diagnostic capabilities ''mystery diseases'' with large numbers of naive animals involved that are caused by previously uncharacterized viruses spread of an emerging virus among small companion animals is multifactorial and includes animal health and sanitation practices; migration of a pathogen from a wild reservoir to domestic animals because of changes in populations, trade, climate, land use, and the introduction of invasive species (eg, plant, animal, insect); and, finally, globalization, as was the case with west nile virus (wnv). emerging viral infections may take a heavy toll on the health of cats or dogs whenever they are brought into situations in which groups of animals are housed together, even temporarily, such as at greyhound racetracks, kennels, catteries, animal shelters, animal obedience training classes, dog parks, pet stores, pet day care facilities. this is especially true when pets are allowed by their owners to roam at will, commingling with ownerless feral dogs and cats and wildlife. for example, the rapid spread of cpv- , which is extremely stable in the environment and highly contagious, was caused not only by the movement of dogs by their owners but by the transfer of fecal material on shoes and clothing of travelers and, unintentionally, through national and international mail [ ] . according to the to national pet owners survey conducted by the american pet products manufacturers association, the us pet cat population is estimated to be . million and the pet dog population is estimated to be . million [ ] . municipalities throughout the united states commonly pass animal control ordinances to protect the public health and safety and general welfare of the citizens and animals residing within the city. typically, animal control codes limit the numbers of companion animals that individuals may own or keep on their private property, require that cats and dogs be licensed annually by owners and vaccinated against rabies, prevent animals from running at large, require proper disposal of animal waste, and prevent the feeding of wild or feral cats or dogs. vaccination of dogs and cats by compliant pet owners for rabies prevention has, since , dramatically reduced the occurrence of this disease; currently, most animal cases reported to the centers for disease control and prevention (cdc) now occur in wildlife [ ] . compliance with other animal control ordinances is variable, particularly among pet owners with respect to leash laws for dogs and cats and among well-intentioned individuals who maintain wild or feral colonies of cats and dogs by providing food, water, and shelter. statistics from the humane society of the united states indicate that to million companion animals are admitted to shelters each year and nearly half are adopted or reclaimed by their owners, whereas the remaining animals are euthanized [ ] . no census of ownerless dogs and cats is available. estimates of the feral cat population in the united states range from million to million animals living primarily in or near urban settings with ample opportunity to interact with pets that are allowed to roam and with wildlife [ ] . thus, ownerless, wild, or feral dog and cat populations may transmit infectious and zoonotic diseases between wildlife and companion animals. from a public health standpoint, this is of particular importance because emerging viral infections from wildlife are often transmitted to human beings by means of a pet that is allowed to stray. it is widely believed by virologists and public health epidemiologists that most viruses emerging from wildlife have an rna or single-strand dna genome [ ] because they have a high propensity for mutation. two significant canine viruses have emerged recently and meet this hypothesis: cpv and canine distemper virus (cdv). canine distemper has re-emerged in the past decade [ , ] because of antigenic and genetic drift in the surface protein (h glycoprotein). in a multicontinent study, variant cdv strains, (but not the vaccine strain of cdv virus) were the cause of illness within weeks after vaccination. in and , large outbreaks of cpv variants (cpv- c and cpv- b*) in kennels occurred in oklahoma and other states [ ] . diagnostic and molecular studies detected mutations in the parvovirus isolates that explained the failures of current commercial cpv vaccines from conferring protection and of approved commercial diagnostic kits from detecting these new viral isolates. another recent example is outbreaks of hemorrhagic symptoms associated with virulent feline calicivirus (fcv) in the united states [ ] ; however, molecular basis of gain of virulence in fcv is not yet understood. in addition to virus evolution, in some cases, the virus can be reintroduced back after the population immunity has declined after a period of disease-free status. thus, diseases that have been eradicated from developed countries but are still circulating in developing countries [ ] may re-emerge by reintroduction from trade or movement of animals. there is a major commitment by the us department of agriculture (usda) in this country and in cooperation with foreign governments and international agencies worldwide to monitor the health of food animals and certain wildlife but not of companion animals [ ] . the primary mission of the cdc is to promote and protect human health. to this end, the cdc performs surveillance for noninfectious and infectious diseases, including zoonoses [ ] ; however, the only chosen reportable viral diseases of animals that are collected by the cdc are rabies and avian influenza (h n ), and those that are reported to the cdc arbonet system are avian, animal, or mosquito wnv infections. largely, surveillance of companion animal diseases, many of which have zoonotic potential, has not been considered to be a priority until recently [ , ] . in , the cdc partnered with the purdue university school of veterinary medicine to establish a pilot surveillance system to monitor clinical syndromes and diseases of small animals [ ] to determine whether animals can serve as sentinels of health hazards to human beings. the national companion animal surveillance program (ncasp) initially drew exclusively on the database of the privately owned organization, banfield, the pet hospital, which provides medical care to approximately . million pet dogs and cats in states, and it now integrates data from antech diagnostics to detect potential emerging and zoonotic infections. a long-term goal of the ncasp is to become a national resource in veterinary public health. in the meantime, the front line of companion animal surveillance for emerging diseases is at the home front, with astute small animal clinicians playing a major role. it can be a challenge for busy and isolated veterinary practices to receive the information on emerging viruses. linking to a health-related network for companion animals might fill the gap. recently, a space-time permutation scan statistic, which was applied in the anthrax terrorist attacks in [ ] , wnv outbreaks [ ] , and enzootic raccoon rabies [ ] , has been applied to veterinary diagnostic data in the unite states and europe [ ] . this analysis provides important information about potential clusters of medical conditions and issues medical alerts about the developing situations based on mortality and confirmed diagnosis of important disease conditions. earlier and more timely notifications should lead to more thorough investigations and reduce losses, especially from emerging viral diseases. it is important to keep in mind that clinical syndromes tend to be multifactorial, and it is essential to review the entire history, including environmental factors, with the specialist in a small animal specialty practice and also with a small animal teaching hospital before arriving at a conclusion about the case. the purpose of this article is to encourage companion animal veterinarians to think outside the routine diagnostic plan when atypical cases of infectious disease are presented at their practices. detecting emerging viral diseases of companion animals requires interaction and discussion among clinicians, pathologists, and virologists, and practicing small animal veterinarians must stay engaged in communication with these specialists through their state diagnostic laboratories or nearby colleges of veterinary medicine. veterinary diagnostic medicine is rapidly progressing, and it is critical for the successful practitioner to stay abreast of new developments in small animal infectious diseases and their diagnosis through continuing education [ ] [ ] [ ] . the development of monoclonal antibody technology in the s and the advent of the polymerase chain reaction (pcr) assay in the s have reshaped veterinary diagnostic strategies, especially in the subspecialty of virology. now, these molecular techniques, which are becoming mainstream applications in routine viral diagnoses, are proving their merit in facilitating the diagnosis of emerging animal viruses. the authors offer practical information on the applications of diagnostic techniques for investigating viral disease outbreaks in companion animals. the authors provide this brief overview of diagnostic techniques in the modern virology laboratory that are used for routine diagnosis and in identifying novel and emerging viruses. every step of diagnostic investigation-history, specimen collection, transportation, and laboratory examination-has to be carefully aligned for optimal outcome. small animal clinicians are familiar with symptoms of common infectious diseases and are often the first to recognize the emergence of new disease problems. in some cases, there may be a history of vaccination compliance, yet some animals develop disease [ , ] . it is important to record the complete history, including the body system involved (eg, respiratory, gastrointestinal, reproductive tract, nervous system), clinical symptoms and their duration, the presence of lesions, and vaccination history. particularly when the case is confounding, the client must be carefully and thoroughly interviewed as to how he or she manages the pet (ie, is the pet free to roam; has the pet traveled recently and where; if this is a new pet, where and how was it obtained; are there other pets in the household). consulting a book on differential diagnoses can be useful to list the potential causes [ , ] . when a history of unusual symptoms is presented, clinicians, recognizing that these cases may be important to individual and universal animal health, should refer these cases to an accredited veterinary diagnostic laboratory. it is convenient to attach copies of all relevant hospital records to the laboratory submission form to aid the diagnostician. correct diagnosis depends on a thorough case history of the affected animal and submission of appropriate specimens that are collected and transported in a manner to preserve the integrity of the viral agent. submitting a comprehensive collection of specimens in a timely manner to the diagnostic laboratory from affected animals when the disease does not fit a familiar clinical picture, as is the case with emerging viral diseases, is of paramount importance. all the system(s) that are potentially involved and all the tissues with gross lesions should be sent to the diagnostic laboratory. it is important to check for concurrent infections. viral diagnosis depends on the quality and type of specimen collected [ ] . the best time for collection of specimens is immediately after symptoms of disease are first noticed. samples from all body systems involved in the acute stage of the disease of affected animals should be submitted to the diagnostic laboratory in a timely manner by overnight delivery. at least to g or ml of each sample should be collected. recovery of virus in cell culture depends on the condition of the specimen received by the diagnostic laboratory. freezing specimens can be detrimental to virus isolation efforts (and also to electron microscopic identification) and should only be done (À c) if it is not possible to deliver the specimen to the laboratory within hours. use wet ice for shipping virology samples, because dry ice (solid carbon dioxide gas) can inactivate many viruses, preventing isolation in cell culture. tissues intended for virus isolation should always be shipped in separate packages from specimens that are immersed in formalin to prevent fumes of formaldehyde from reaching the fresh tissues. it is imperative that tissues and organs from animals that have died be harvested as soon as possible after death. postmortem tissues should be placed in sterile containers with a small amount of transport medium ( - ml), if possible. when the clinician is unsure as to what specific organs and fluids should be retrieved, the entire carcass of the dog or cat may be delivered to the laboratory for examination. to obtain more specific details regarding specimen collection, packaging, and submission, contact the diagnostic laboratory of your choice by telephone or consult its specimen submission and fee schedule guidelines, which are often available on an internet web site. individuals who ship biologic substances for diagnostic testing are required by federal law to be in compliance with all regulations governing packaging and labeling of interstate shipments of causative agents. failure to follow the regulations results in heavy fines (fig. ) . complete instructions on appropriate packaging for laboratory specimens to be mailed or shipped by a common carrier may be accessed in several sections of the code of federal regulations (cfr). health and human service regulations define such terms as diagnostic specimen and etiologic agent and describe requirements for packaging and labeling viruses have a simple structure with a protein coat enclosed with only one type of nucleic acid (dna or rna) rather than both. thus, methods for viral diagnosis target one of the components of the virus structure. for a definitive viral disease diagnosis, four basic approaches are used: direct detection by virus isolation or direct identification, viral serology for detection of a specific antibody, viral antigen detection, and molecular-based detection of genetic material. a brief discussion of the principles of diagnostic assays representative of each approach follows. gross pathologic and histopathologic findings histologic (fig. ) and cytologic examination (fig. ) of tissues and fluids by a board-certified veterinary pathologist contributes valuable information about the pathologic signs, gross and microscopic, that distinguish infections caused by viral or bacterial pathogens and other possible etiologies. tissue tropism, mononuclear infiltrates, development of inclusion bodies (intranuclear, cytoplasmic, or both), and the formation of syncytia are some of the characteristics that differ among viruses and can sometimes distinguish different viral infections. for example, most dna viruses replicate in the nucleus, and thus tend to produce intranuclear inclusions, whereas most rna viruses form cytoplasmic inclusions, although there are exceptions. as part of the pathologist's examination, immunohistochemistry testing (figs. and ), fluorescent antibody testing, and possibly in situ hybridization (ish) studies on tissues may be ordered; these methods are considered elsewhere in this article. a complete histopathology report should include possible differentials for the lesions. the pathologist might note that some findings do not exactly fit the routine lesions he or she has observed in previously. in cases in which there are deviations in lesion type or distribution or when gross lesions and histopathologic findings suggest the involvement of a viral disease but routine virology tests do not detect the expected conventional viral agents, variant or ''emerging'' viruses or even iatrogenic infections may be suspected. in early , blue tongue virus serotype was introduced in canine populations from a commercial modified-live multivalent canine vaccine that was associated with high mortality in dogs [ , ] . in some situations, second or even third opinions from pathologists at other laboratories who have special expertise should be solicited [ ] . with the application of telepathology to veterinary case materials, networks of specialists, including veterinary pathologists, small animal clinicians, infectious disease specialists, and laboratory diagnosticians, are able to exchange patient histories, clinical data, and images (gross and microscopic) through the internet for consultation, diagnosis, and education. this allows timely access to expert opinions at other locations throughout the world [ , ] . the use of telepathology can facilitate rapid intervention through the synergy of computer technology and special pathology expertise (eg, system-and speciesspecific pathologic findings) to understand the lesions in difficult cases better. conventional virus isolation techniques are often the backbone of investigation of novel viral diseases, provided that the virus is cultivable in available cell lines or primary cell cultures. virus isolation may be relatively slow depending on the growth characteristics of the virus; however, roller culturing or centrifugation of samples onto cell monolayer(s) can enhance viral replication and recovery. in many of the recent emerging viruses from wildlife (eg, bats), the virus was first cultivated, allowing further characterization of the virus. it is important to keep in mind that virus isolation, even if the effort is successful, may have a slow turn-around time, approximately to weeks. definitive identification of virus in cell culture can only be accomplished with specific antibody nucleic acid testing, and in the case of an ''emerging'' virus, existing reagents may not be reactive with the ''new'' virus. if culture is successful, however, the viral material may be studied by electron microscopy (em) and by molecular techniques, as described in this article, to characterize the new isolate. virus isolation requires fresh tissues and cannot be done on formalin-fixed tissues. em is often used in veterinary diagnostic laboratories to detect enteric viruses in fecal samples retrieved during the course of viral diarrheal disease. additionally, em is indispensable for identification of emerging and previously unidentified viruses in clinical samples [ ] , and this method has helped in the identification of many new viruses, including, most recently, bat lyssavirus [ ] . viruses can be classified up to the virus family based on size, shape, and distinctive structural features, such as envelopes or protein spikes, particularly for parvovirus, rotavirus (fig. ) , coronavirus, astrovirus, herpesvirus, poxvirus, and picornavirus. em allows detection of multiple viruses simultaneously. application of antibodies to supplement the em diagnosis provides higher sensitivity and further confirmation of the viral diagnosis. sensitivity is the major limitation of em, and at least to virus particles per milliliter must be present in the sample being examined. because the electron microscope is an expensive piece of equipment that requires special technical skills and a high level of expertise, it is not available in many laboratories. viral components can also be determined by several basic biochemistry experiments. acridine orange (ao) staining can determine the nature of the nucleic acid of purified viral particles [ ] . differentiation as to whether the nucleic acid is single-or double-stranded in nature is based on the color developed on ao staining; double-stranded dna or rna nucleic acids stain yellow green, whereas single-stranded dna or rna acids stain flame red. nuclease susceptibility of the purified virions differentiates dna from rna. the presence of envelope on viruses can be determined by susceptibility to the virus to heat, ether, or other lipid solvents [ ] . the titrated virus preparation is treated with ether or chloroform. a decrease in virus titer of greater than log is considered to be significant to indicate the presence of envelope on the virus. the presence of envelope indicates that virus is susceptible to common disinfectants. lack of envelope indicates that the virus is resistant to the use of common disinfectants. classic serology tests indirectly determine the viral etiology of disease by detecting the presence of antibody in serum (red-topped tube) to a specific test viral antigen, and thus provide retrospective evidence of an immune response or exposure to a virus. serologic methods still provide powerful tools in the virology laboratory of today for diagnosing viral diseases that are seen routinely and for discovering and characterizing novel viral diseases. serologic tests are now used to detect antibody or antigen in serum and body fluids. typically, methods used in the virology laboratory are serum neutralization (sn), hemagglutination-inhibition (hai) test, indirect fluorescent antibody test (ifat), and elisa. serologic results require interpretation by an expert diagnostician based on critical clinical observations, confirmation by pathology examination, virus isolation, and mass screening of the populations by serology. if animals in populations that have never been exposed to or vaccinated against a given virus have specific antibodies detected in their serum, it is expected that this is most likely attributable to recent exposure to the emerging virus. paired serum samples are important to demonstrate a fourfold significant increase in antibody titers, which indicates that the diagnosis of recent exposure may be attributable to infection as opposed to previous exposure or vaccination depending on the vaccination history. serology is also useful to study the antigenic distance of the emerging virus and provides clues as to whether the newly emerged agent is or is not likely to be protected by an available vaccine(s), such as heterologous virus in another species of animal. viral hemagglutination (ha) occurs between the viral protein; hemagglutinin (hn), which is present on the viral capsid or envelope of only certain families of viruses; and specific receptors on red blood cells (rbcs) that bind to hn, causing their agglutination and precipitation from solution. this phenomenon is the basis for a powerful and sensitive assay, the hai test. when a hemagglutinating virus is mixed with serum containing antibodies specific to that virus, rbcs that are added to the mixture do not agglutinate and precipitate from solution. feline panleukopenia, cpv, influenza a, and parainfluenza antibodies may be detected by hai testing. the hai method may also be used to identify unknown virus utilizing antibodies of known specificity; however, most often, this test is applied to detect the presence of antibodies in a serum sample against specific hemagglutinating viruses. variants of cpv and feline parvovirus can differ in the hemagglutinating activity of swine erythrocytes [ , ] . sn measures the inhibitory activity of a hyperimmune serum against viral isolates in cell culture. commonly performed in a cell culture microwell format, this is a long-standing method for quantifying virus-specific antibodies, and it is usually performed to test for antibodies to viruses that typically cause cell damage (cytopathic effect [cpe] ) to the host cell culture they infect. when a virus is mixed with hyperimmune serum containing antibodies specific to that virus, the antibodies bind the virus, preventing infection of the cell culture. the sn test can diagnose current infection using acute and convalescent serum samples from individual animals. it may also be used to determine immune status conferred on vaccinated animals. vaccination antibody titers often differ from antibody titers developed in response to natural infection. usually, vaccination titers are lower relative to infection titers, and maximal titers occur approximately to days after vaccination. sn assays are commonly performed to detect antibodies to fcv, herpesvirus, enteric coronavirus, and syncytial viruses and to canine herpesvirus, cdv, coronavirus, parainfluenza virus, and adenovirus. elisa this is useful for screening large numbers of samples for the presence of antibodies against viruses. the elisa format is flexible, and it may be used to detect antibody or antigen in clinical specimens. in either case, the detection system is an antibody conjugated to an enzyme. when the enzyme-linked antibody binds to the analyte being measured, the enzyme reacts with a chromogenic substrate, causing a color change to occur that may be measured spectrophotometrically or evaluated visually. several elisa kits are available to detect antiviral antibodies in companion animals, including cpv and cdv, feline leukemia virus (felv), feline immunodeficiency virus (fiv), and feline coronavirus. the immunoglobulin m (igm) elisa is a method used to distinguish current infection from past infection. during acute disease or immediately after vaccination with modified-live viruses, igm is the first class of immunoglobulin produced in response to infection, appearing to weeks before there are detectable levels of igg in the serum. because it is short-lived, igm levels typically disappear months after infection. a single acute-phase serum test sample is sufficient to diagnose current infection with an igm elisa. testing of igm titers is available for several viral agents, including cdv and cpv among others. elisa is useful for screening naive animal populations for the presence of antibodies against viruses to track the origin and spread of emerging infections. antibodies to wnv have recently been detected in dogs and cats by igm-capture elisa [ ] . a related method known as virus neutralization can be used to identify the serotype of a newly discovered virus. western blot (wb) may be used as a supplementary test to confirm antibody elisa results for fiv testing [ ] . to perform the assay, purified virus is disrupted using detergent; the constituent proteins are then separated on the basis of molecular weight by electrophoresis in a polyacrylamide gel. the proteins are transferred (blotted) from the gel to a nitrocellulose or polytetrafluoroethylene (ptfe) membrane for stabilization. the electrophoretically separated proteins are the antigen substrates for analyzing the test sera for the presence of specific antibodies. as with the elisa format, the western immunoblot uses an enzyme-labeled antispecies antibody that binds to the test serum antibodies that have bound to the separated viral antigens. substrate reacting with the enzyme-labeled antibody in the presence of a colorless soluble benzidine derivative results in conversion to colored insoluble precipitate at the protein bands where test serum antibodies are bound. the molecular weight of the protein detected is characteristic for a particular viral component. immunoblot results of the unknown test antisera are compared with positive control test sera for interpretation. a major advantage of the immunoblot technique is that a full antibody profile of a single serum sample is made simultaneously, identifying each of the individual particulate viral antigens that patient antibodies bind. as an epidemiologic tool, wb analysis may be used to detect currently circulating viral subtypes within a population and to characterize new emerging viral subtypes. immunoblotting is also a valuable research technique for antigen detection that is often used to characterize novel viruses by comparing them with known related viral family members using standard antisera or monoclonal antibodies. immunofluorescence assays on cells from clinical samples can be applied for rapid diagnostic investigations ( - minutes), provided that the fluorescent microscope and expertise are available in a laboratory. with the pooling of primary monoclonal antibodies against potential viral agents, the assay can be used as a screening tool and the sample tested again with individual conjugates to obtain specific virus diagnosis (fig. ) . the elisa is also a means for detecting viral antigens present in clinical specimens, and it offers a relatively quick turn-around time. antigen test elisa kits are available to detect antiviral antigens in companion animals, including cpv, felv, and fiv. additionally, it is a common practice by many veterinary diagnostic laboratories to appropriate the use of some rapid antigen test kits intended for the human diagnostic market, specifically, rotavirus test kits. when monoclonal antibodies are used as capture antibodies in elisa test kits, however, they fail if there is a mutation in the epitope of the viral surface protein present in the specimen that is being tested. lateral flow immunoassay is a special application of the elisa that provides a rapid, economic, portable, sensitive, and specific technique that is convenient for performing testing outside of the laboratory. it is the technique of choice for emerging viral infections [ , ] , and it has gained attention for use in diagnosing foreign animal diseases and zoonotic and emerging viral infections of animals, such as influenza virus and wnv, in the field. the test kits are small in size (size of credit cards), extremely stable at ambient temperature ( c), and take minutes to perform. an advantage of nucleic acid-based testing is that specimens submitted for analysis do not have to have viable viral particles present to be detected by this means. there is a trend toward application of molecular or gene sequence-based techniques to routine virology testing in diagnostic laboratories, which is justified under several circumstances. first, a molecular technique may be the test of choice if conventional methods of diagnosis are technically weak, such as when a viral agent is noncultivable or there are biocontainment concerns with culturing the virus, the virus has amorphous morphology by em, antibodies are unavailable or not specific to the virus, and serologic tests result in a confounding diagnosis. second, molecular techniques may be essential to detect and classify the sequence type or genotype of a virus. third, a viral agent may be characteristically slow to replicate, such as c-herpes virus; thus, a molecular method might provide a better turn-around time for diagnosis. in this instance, a rapid diagnosis might be achieved by pan-herpesvirus pcr. finally, a novel viral isolate that cannot be definitively identified by the routine diagnostic methods described previously may merit investigation and characterization by molecular-based techniques, which are indispensable in the classification of new and emerging viruses. these advanced techniques may confirm a diagnosis of viral etiology when other tests have failed; however, they are, unfortunately, relatively expensive. furthermore, the presence of nucleic acid does not equate to infection, and infections are attributable to subclinical, latency-associated nucleic acids or defective interfering virus particles, such as in paramyxoviruses, produced in nonproductive infections in genetically resistant hosts. clients, who bear the financial burden, should be counseled as to the benefit and shortfalls of this testing before ordering molecular-based tests. an excellent review of molecular-based techniques for diagnostic testing of infectious diseases has appeared in a previous issue in this series [ ] . the most familiar nucleic acid testing technique, pcr, has been used for more than a decade; however, over the past few years, real-time pcr has taken its place, revolutionizing diagnostic virology. in this procedure, the pcr chemistry may be combined with detection using a single-stranded dna probe with a fluorescent label [ ] . moreover, the procedure may be completed within an hour, and it allows for quantitation of results. because the hands-on steps are reduced and the pcr reactions are not opened, it eliminates the chances of cross-contamination in the laboratory. real-time pcr protocols are gaining more acceptance in routine veterinary diagnosis. in situ hybridization ish involves using nucleotide probes with an attached label. non-isotopelabeled probes (digoxigenin or fluochrome) can be applied in veterinary diagnostic laboratories. diagnostic applications of ish involve identification of virus-specific sequences (dna or rna) in the tissues or cells [ ] . although uncommon in veterinary diagnostic laboratories, ish is in routine use in human diagnostic laboratories for detection of the genotype of human papilloma viruses in cervical samples. for ish, smears and tissues (fresh, unfrozen, and fixed tissues) are suitable. in electropherotyping and restriction fragment length polymorphism (rflp), double-stranded dna (rflp) or rna (electropherotypes) is purified and size-separated on agarose or acrylamide gel electrophoresis. because nucleic acids are charged and double-stranded molecules bind more ethidium bromide compared with single-stranded nucleic acids, under the electric field, the nucleic acids migrate and larger sized molecules separate out higher than smaller sized molecules. for dna molecules to be tested, the double-stranded viral dna-or pcr-amplified fragments are digested with restriction enzymes. these techniques allow quick differentiation of viral genomes (dna or rna). both techniques have applications in molecular epidemiology of rotaviruses [ ] . new generation molecular techniques viral genome sequencing technologies viral genome or mrna sequencing is a powerful molecular epidemiologic tool and has been applied for epidemiology of rabies virus [ ] . sequences of novel or emerging viruses may be derived based on known conserved sequences of previously characterized viruses within the same family. although virus sequencing is gaining more routine application in veterinary laboratories, it does add cost, and thus should be used judiciously. when these methods fail to identify a newly discovered virus, which is truly novel, metagenomic analysis, which is largely used in research laboratories, may be applied. pyrosequencing is a recent variation on sequencing short stretches of pcrgenerated dna without the need for labeled primers, labeled nucleotides, and gel electrophoresis [ ] . although this variation on pcr and nucleic acid sequencing is currently used exclusively as a research tool, it is likely to be adapted for clinical diagnostic work in future years because it has been demonstrated to detect many different unrelated viruses simultaneously in a single reaction and to identify viral serotypes and detect viral isolates that could not previously be typed by classic procedures [ , ] . a biochip or microarray is small solid support, such as a nylon membrane, silicon chip, or glass slide, on which nucleic acid fragments, antibodies, or proteins are immobilized in an orderly arrangement. thousands of different molecules, referred to as probes, may be machine-printed as spots on the support, allowing for high throughput of samples using lower volumes of analyte in less time than conventional laboratory techniques take to complete. microarrays are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions that are most commonly detected through the use of fluorophores. the fluorescent signal patterns formed by each analyte are then compared by the computer software using complex algorithms to make an identification of its contents. biochips enable researchers to screen large numbers of biologic analytes quickly for a variety of purposes, ranging from disease diagnosis to detection of bioterrorism agents. biochip technology is still relatively new and has not yet entered the mainstream of clinical diagnostics techniques, although it is widely used in research institutions. as an epidemiologic tool, the use of nucleic acid microarrays was instrumental in the rapid identification of the first severe acute respiratory syndrome (sars) coronavirus outbreak in china [ ] . coronavirus protein microarrays have been used to screen canadian sera [ ] for specific antibodies to sars and to other coronaviruses in a comparative study with the traditional elisa. scientists around the world are assessing the feasibility of using microarrays as tools for surveillance and diagnosis of influenza viruses [ , ] . once issues of sensitivity and assay validation have been addressed satisfactorily and the cost of the technology has become more affordable, microarray technology may find a place in clinical diagnosis. pathogenic virus or ''orphan'' virus or ''vaccine-source'' virus molecular methods for detecting and identifying viral pathogens are powerful. it is possible to detect a virus in a specimen, but it may have no association with the clinical condition. these types of viruses are called ''orphan viruses.'' minute virus of canine is a parvovirus, and it causes no clinical disease [ ] . as a result of the advent of sensitive molecular techniques, it is quite common to detect viral sequences of agents that may be present in a sample but not associated with the disease (orphan viral agents). it is possible to study the association of the viral agent with the pathologic findings observed to support the diagnosis. moreover, the pcr protocols targeting structural genes that are expressed only during active infection are useful and avoid the potential false-positive results attributable to latency or persistent viral infections. moreover, the sense and antisense probes offer the opportunity for resident and replication intermediates of viruses. obviously, the history of recent vaccination should be known, and the vaccine virus from the same lot of vaccine should be simultaneously included in the testing run and sequenced over critical regions to ensure that the virus in the sample is the same or different from the vaccine. when fluorescent antibody testing or immunohistochemistry testing is performed, false-negative findings result even when a related virus is present. because of changes in the sequence of the target protein epitopes, antibodybased detection methods may fail to provide the diagnosis; monoclonal antibodies used may fail to react and polyclonal antibodies may cross-react weakly when a variant strain of virus is present. thus, a sudden trend in lack of correlation between tests may signal an emerging variant of the virus. if a new variant of the virus arises, it may be associated with a change in the clinical profile and we may or may not understand the molecular basis of this shift. it is possible that the polyclonal antibodies may react weakly with the new variant of the virus. in many cases, the pcr primers may fail to amplify the new variant if the mutation occurs in the hypervariable region of the target gene amplified. for example, in the recent emergence of cpv variants, many practitioners noted clinical symptoms compatible with cpv but the commercial field tests were not working. if a new variant of virus emerges, a polyclonal antibody antiserum prepared in a heterologous species (rabbit or goat) can be used as a primary antibody against the whole virus, because it is possible that the monoclonal antibody might fail. the molecular techniques are more likely to fail compared with the antibody-based techniques because of the degeneracy of codons. it is important to keep in mind that factors other than emerging viruses can also affect the performance of usda-approved tests. for example, local anesthetic can also affect the outcome of antibody tests. in one study, the use of lidocaine was recommended over oxybuprocaine to avoid false-positive results [ ] . it should be clear to the readers that veterinarians are bound to encounter emerging viruses in their practice. the problem is unavoidable because viruses are ''perfect'' obligate parasites. even the immune response dictates the nature of virus that evolves in a host. thus, vaccines are to be viewed as preventive tools rather than as a cure for emerging viruses. in some situations, the best vaccine is bound to fail. similarly, the diagnostic methods have to be tailorfitted to keep up with the emerging viruses. if the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with diagnostic laboratory to solve the diagnostic puzzle. your state veterinary diagnostic laboratory may be the first place that issues an alert to veterinary professionals and the public at large to possible emerging viral diseases. newsletters from your state diagnostic laboratory can be a good source of information about emerging viral diseases in your area. additional sources that are dedicated to dog and cat health issues and public health are available on the internet [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . an annotated historical account of canine parvovirus evidence for evolution of canine parvovirus type in italy canine parvovirus types c and b circulating in north american dogs in and american pet products manufacturers association. industry statistics and trends: - national pet owners survey rabies surveillance in the united states during feral cat population statistic the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia canine distemper viruses circulating in north american dogs an isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus future risks from infectious diseases of animals. presented at the world association of veterinary laboratory diagnosticians linking human and animal health surveillance for emerging diseases in the united states-achievements and challenges united states department of health and human services centers for disease control and prevention animals as sentinels of environmental health hazards world health organization. future trends in veterinary public health. who technical report series purdue university-banfield national companion animal surveillance program for emerging and zoonotic diseases early statistical detection of anthrax outbreaks by tracking over-the-counter medication sales dead bird clustering: a potential early warning system for west nile virus activity spatial and temporal patterns of enzootic raccoon rabies adjusted for multiple covariates a space-time cluster investigation of an outbreak of acute respiratory disease in norwegian cattle herds methodology in diagnostic virology a primer on diagnostic virology: specimen selection and serology. a primer of veterinary diagnostic laboratory testing. compendium on continuing education for the practicing veterinarian a primer on diagnostic virology: direct and molecular-based detection of viral pathogens. a primer of veterinary diagnostic laboratory testing. compendium on continuing education for the practicing veterinarian antemortem diagnosis of cdv infection by rt-pcr in distemper dogs with neurological deficits without the typical clinical presentation clinicopathological findings in dogs with distemper encephalomyelitis presented without characteristic signs of the disease differential diagnosis in small animal medicine small animal medical differential diagnosis diagnostic virology bluetongue disease in dogs associated with contaminated vaccine iatrogenic infection of a pregnant dog with bluetongue virus, serotype interobserver variation among histopathologic evaluations of intestinal tissues from dogs and cats telepathology: its role in disease diagnosis in meat hygiene. presented at the world association of veterinary laboratory diagnosticians th international symposium static image telepathology in perspective overview of electron microscopy and its role in infectious disease diagnosis. presented at the world association of veterinary laboratory diagnosticians th international symposium diagnostic electron microscopy: historical review and future. presented at the world association of veterinary laboratory diagnosticians th international symposium acridine orange staining of purified rat virus strain x recovery and characterization of a minute virus of canines characterization of a nonhemagglutinating mutant of canine parvovirus characterization of a canine parvovirus strain isolated from an adult dog serologic survey of cats and dogs during an epidemic of west nile virus infection in humans use of western blot and radioimmunoprecipitation for diagnosis of feline leukemia and feline immunodeficiency virus infections a comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats, and mink simple rapid, on-site detection for diagnosis of animal disease update on molecular techniques for diagnostic testing of infectious disease real-time pcr in clinical microbiology: applications for routine laboratory testing in situ hybridization and its diagnostic applications in pathology the isolation of rotavirus from calves, foals, dogs and cats in new zealand clinical laboratory advances in the detection of rabies virus pyrosequencing sheds light on dna sequencing type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology identification of enterovirus serotypes by pyrosequencing using multiple sequencing primers micro-array-based detection and genotyping of viral pathogens severe acute respiratory syndrome diagnostics using a coronavirus protein microarray comparison of the mchip to viral culture, reverse transcription-pcr, and the quickvue influenza aþb test for rapid diagnosis of influenza detection of respiratory viruses and subtype identification of influenza a viruses by greenechipresp oligonucleotide microarray minute virus of canines (mvc, canine parvovirus type- ): pathogenicity for pups and seroprevalence estimate oxybuprocaine induces a false-positive response in immunochromatographic sas adeno test american association of public health veterinarians cdc: healthy pets healthy people available at national association of state public health veterinarians united states animal health association code of federal regulations (search engine) key: cord- -gm cadh authors: wang, jingqiang; wen, jie; li, jingxiang; yin, jianning; zhu, qingyu; wang, hao; yang, yongkui; qin, e’de; you, bo; li, wei; li, xiaolei; huang, shengyong; yang, ruifu; zhang, xumin; yang, ling; zhang, ting; yin, ye; cui, xiaodai; tang, xiangjun; wang, luoping; he, bo; ma, lianhua; lei, tingting; zeng, changqing; fang, jianqiu; yu, jun; wang, jian; yang, huanming; west, matthew b; bhatnagar, aruni; lu, youyong; xu, ningzhi; liu, siqi title: assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus date: - - journal: clin chem doi: . /clinchem. . sha: doc_id: cord_uid: gm cadh background: the widespread threat of severe acute respiratory syndrome (sars) to human life has spawned challenges to develop fast and accurate analytical methods for its early diagnosis and to create a safe antiviral vaccine for preventive use. consequently, we thoroughly investigated the immunoreactivities with patient sera of a series of synthesized peptides from sars-coronavirus structural proteins. methods: we synthesized peptides ranging in size from to amino acid residues of relatively high hydrophilicity. the immunoreactivities of the peptides with sars patient sera were determined by elisa. results: four epitopic sites, s , m , n , and n - , located in the sars-coronavirus s, m, and n proteins, respectively, were detected by screening synthesized peptides. notably, n and n , located at the cooh terminus of the n protein, inhibited binding of antibodies to sars-coronavirus lysate and bound to antibodies in > % of samples from sars study patients. n had the highest affinity for forming peptide-antibody complexes with sars serum. conclusions: five peptides from sars structural proteins, especially two from the cooh terminus of the n protein, appear to be highly immunogenic and may be useful for serologic assays. the identification of these antigenic peptides contributes to the understanding of the immunogenicity and persistence of sars coronavirus. the worldwide threat of severe acute respiratory syndrome (sars) becoming an epidemic creates urgent challenges for the scientific community. several laboratories have unraveled the genetic information of the sars virus ( ) ( ) ( ) ( ) . the genome size of the sars coronavirus is ϳ kb and has open reading frames, composed of a stable region encoding an rna-dependent rna polymerase with open reading frames, a variable region representing coding sequences for viral structural genes [spike (s protein), envelope (e protein), membrane (m protein), and nucleocapsid (n protein)], and putative uncharacterized proteins. its gene order is similar to that of other known coronaviruses; however, phylogenetic analyses and sequence comparisons indicate that this virus does not closely resemble any of the previously characterized coronaviruses. the incubation period of sars is usually - days, but could be as long as days. the disease progresses with unusual severity within a short time once a patient exhibits obvious clinical symptoms. therefore, an urgent task is to develop accurate and sensitive diagnostic tools for identifying sars, specifically for early diagnosis. a noninvasive diagnostic test for sars coronavirus has been reported recently that uses quantitative reverse transcription-pcr with detection of sybr green fluorescence ( ) . the pcr primer design was based on a unique region within the rna-dependent rna polymerase-encoding sequence of the virus. the amplification was very specific and showed no cross-reaction with two serogroups of human coronavirus, e and oc . in a total of sars patients and uninfected controls, the pcr assay showed positive identification for % of sars cases and negative identification for % of controls. however, this technique is limited in its clinical use. the samples for pcr were collected from nasopharyngeal aspirates of sars patients. because sars is a respiratory infection, any attempt to obtain a sample from a patient's pharynx and larynx increases the risk of infection to the healthcare worker. furthermore, % accuracy is below the level of acceptably when taking into account those in the early phase of infection, showing no symptoms, who were among the % excluded. this could certainly be cause for concern because these people could still pose a threat of transmitting the virus. serologic assays are used extensively for diagnosis of viral infection in a host ( ) . in urgent situations, a common way to perform a serologic assay is to use complex viral lysates as antigens. the use of viral lysates, however, presents several disadvantages. viral lysates consist of many viral antigens that can not be clearly purified and classified. the lysates are prepared from cells infected with the virus; thus, cellular proteins can contaminate the preparation. moreover, sars-coronavirus lysates present a considerable risk of infection to laboratory workers. to overcome these problems, the discovery of antigenic fragments in sars-coronavirus proteins is expected to lead to the rapid development of new assays for the diagnosis of sars infection. it has been shown in many cases that several epitopes located in viral proteins can be successfully mimicked with synthetic peptides ( ) . to expedite epitope mapping of the sars coronavirus, we have synthesized a group of peptides representing the most hydrophilic, as well as the most accessible, residue regions of the s, m, and n structural proteins of sars coronavirus. using sera from sars patients, we probed these peptides by elisa. sera from sars patients from eight different hospitals in beijing and from uninfected volunteers were collected for study. the clinical diagnostic criteria for sars followed the clinical description of sars released by who. confirmation of sars infection was evidenced by the presence of antibodies against sars coronavirus in the serum. the control sera were divided into two groups: samples obtained from healthy volunteers and samples obtained from patients suffering from respiratory symptoms but not infected with the sars coronavirus. on the basis of the published genome sequence of the sars coronavirus, we downloaded structural proteins s, m, and n into the protscale program at the swiss institute of bioinformatics to analyze the physical characteristics of the proteins, such as hydrophilicity, hydrophobicity, accessible residues, buried residues, molecular mass, and pi values. a total of peptides ranging in size from to amino acid residues and in molecular mass from to da were selected for synthesis ( table ). all of the peptides were synthesized commercially by chinese peptide co. the synthesized peptides were characterized by hplc and mass spectrometry. the sars coronavirus isolated from sars case bj , whose genomic sequence was determined by the beijing genomics institute, was used as a viral source and propagated in vero-e cells as described previously ( ) . after viral propagation, the cells were harvested and placed at °c for h to inactivate the virus. the inactivated infected vero-e cells were completely lysed by freeze-thaw cycles followed by centrifugation. after the cell pellet was removed, the supernatant was loaded on a sephadex g- column for virus purification. the elution fractions were collected at ml/min, and viral proteins in each fraction were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) with coomassie blue staining and confirmed by western blot using sars serum. before elisa the fractions containing virus were sonicated and concentrated with a centricon- biological filter. the reactivities of the various peptides with sars sera were determined by elisa. in brief, peptides ( mg/l, l/well) were adsorbed to duplicate wells of -well microplates in . mol/l carbonate buffer (ph . ) and incubated at °c for h. after the wells were washed with phosphate-buffered saline (pbs), they were coated with g/l bovine serum albumin at °c for h and then incubated at °c for min with l of sera in sample buffer (total volume of buffer, l). each well was then washed and incubated with peroxidase-conjugated goat anti-human igg at °c for min. finally the wells were washed again with pbs containing ml/l tween . the peroxidase reaction was visualized by use of o-phenylenediamine solution as substrate. after min of incubation at °c, the reaction was stopped by the addition of l of mol/l sulfuric acid, and the absorbance at nm (reference wavelength, nm) was measured by an automatic elisa plate reader (multiskan microplate photometer). to determine the background resulting from nonspecific binding of viral lysates or peptides to normal sera, the cutoff value was calibrated for each viral preparation or peptide by incubation with negative control serum. a panel of sera from healthy individuals was examined routinely for the calibrations. the mean absorbance was considered as the cutoff value for a viral preparation or a peptide. the cutoff values were ϳ . . western blot for each sample, g of protein was subjected to % sds-page under reducing conditions and transferred to a bio-rad pvdf membrane. the membrane was blocked with pbs containing g/l nonfat milk powder in tris-buffered saline at °c for min before incubation with diluted sars serum. bound antibodies were detected by use of an appropriate alkaline phosphatasecoupled secondary antibody with nitroblue tetrazolium chloride and -bromo- -chloro- Ј-indolyphosphate p-toluidine salt added as the substrate for visualization. estimation of binding affinities of synthesized peptides to sera from sars patients to compare the affinities of the antigenic peptides to sars-coronavirus antibodies, we estimated the binding constants by the scatchard equation. each antibody has a limited number of binding sites that become saturated as the concentration of antigen increases. this can be quantified by the following equation: where ␣ is the proportion of bound antigens per antibody based on the elisa measurement at nm; n is the number of binding sites on the antibody; k d is the dissociation constant of the antibody-antigen complex; and [p] is the antigen concentration. we used the student t-test to calculate whether the reactivities of the synthetic peptides and sars coronavirus preparations with the same serum in the elisa were significantly different. p values Ͻ . were considered statistically significant. results and discussion epitope mapping of the s, m, and n peptides to effectively develop synthetic peptide immunogens from sars-coronavirus structural proteins, we used several theoretical calculations for peptide design. for a potential epitopic peptide, we specifically looked for high local hydrophilicity, charged residues on the exposed protein surface, and accessible surface area. statistical analysis based on the linear amino acid sequences of the three proteins suggests that, in contrast to the other two viral structural proteins, the n protein contains a high percentage of accessible residues and low hydrophobicity (see figs. - in the data supplement that accompanies the online version of this article at http://www.clinchem. org/content/vol /issue /). a total of peptides spanning multiple possible epitopic sites along the s, m, and n proteins were designed and synthesized in a solid-phase peptide synthesizer. these peptides were purified by hplc, incubated with panels of sera from both controls and sars patients, and then subjected to elisa for measurement of the immunoreactivities. the correlations between the length of amino acid sequence and immunoreactivity are shown in fig. . the peptides representing the cooh terminus of the n protein, in particular n and n , had high absorbance/cutoff value ratios with the highest positive detection rate and the lowest hydrophobicity score among all of the synthesized peptides (fig. c, and fig. in the online data supplement). peptide n is located in a hydrophilic region and reacts with sars serum antibodies. however, compared with the peptide region n -n , which has a score of . for accessible residues and a hydrophobicity score of Ϫ . , peptide n has relatively low antigenicity because of a correspondingly lower score for accessible residues ( . ) and higher score for hydrophobicity (Ϫ . ). these differences may partially explain the low number of true positives ( %) that we obtained when we used n as an antigen. according to the hydrophobicity analysis (figs. and in the online data supplement), most regions in both the s and m proteins are very hydrophobic. although many factors influence antigen-antibody interactions, two major factors, charge-charge interactions and hydrogen bonding caused by hydrophilicity, have been hypothesized to be crucial for an epitopic site ( ) . of the synthesized peptides, only displayed significantly high immunoreactivity (table ) . interestingly, all five immunoreactive peptides are located in regions with a relatively high hydrophobicity score (figs. - in the online data supplement). moreover, the number of polar amino acids in each peptide correlates well with the percentage of true positive, e.g., n and n reacted with Ͼ % of the samples from sars patients and contain nine and seven polar amino acids, respectively, whereas s , m , and n reacted with Յ % of the sera from sars patients and have four, three, and three polar amino acids, respectively. our findings thus support that both hydrophobicity and peptide charge are important in determining immunoreactive sites in sarscoronavirus structural proteins. the sars coronavirus propagated in vero-e was used as an antigen to test whether sars-coronavirus antibodies were raised in sera. we collected sera from healthy controls and measured their immunoreactivities against sars coronavirus by elisa. none of the control sera reacted with the sars coronavirus. the same results were observed for other control sera obtained from patients with respiratory diseases who did not have sars. however, sera from all sars patients reacted with the sars coronavirus, with high absorbance/cutoff value ratios [mean (sd), . ( . )]. the results of the peptide screening are summarized in table . there are two key parameters listed in table , p values, which indicate whether there was a significant difference between the synthesized peptide and sars coronavirus as antigen in the elisa, and x/ , which represents the detection rate when particular peptides were used as antigen. for the s protein, peptides spanning the protein sequence were synthesized. only one peptide, s , elicited a response in the elisa that was not significantly different from the response elicited by the sars coronavirus: ϳ % ( of ) of the sera from sars patients reacted with this peptide. the other peptides reacted only slightly with the sera from sars patients and gave low detection rates, suggesting that the regions of the s protein covered by these peptides have no epitopic site. for the m protein, a relatively small viral structural protein, nine peptides were synthesized. compared with the sars coronavirus, this peptide reacted with % ( of ) of the sera (p ϭ . ), indicating that it may contain a weak epitopic site. the highest immunoreactivities were for the peptides located within the n protein. three of synthesized n-protein peptides, n , n , and n , showed reactivities comparable to that of the sars coronavirus. interestingly, n is located at the nh terminus of the n protein, whereas n and n are neighboring fragments, both at the cooh terminus. we thus reasoned that n protein has at least two epitopic regions. these three peptides showed low y/ values- / , / , and / , respectively-and high x/ values: n and n reacted with % ( of ) and % ( of ) of the sera from sars patients, respectively, whereas n reacted with ϳ % ( of ). taken together, these data suggest that the most immunoreactive epitopic site in the sars coronavirus is located at the cooh terminus of the n protein. members of the coronavirus family all have three major structural proteins, s, m, and n, but the antigenicities and roles of these viral proteins in immunity remain unclear. different viruses often cause different immunoresponses. for example, in infectious bronchitis virus ( ), the s glycoprotein is more antigenic than either the n or the m protein. because s protein is exposed to the outer viral surface, it is often used as the antigenic group for serologic testing. the m protein, embedded in the viral envelope, has highly variable sequences and is considered to exhibit weak antigenicity, but glycosylation of this protein may elicit antibody production. one common phenomenon is that the n proteins have been shown to be strong immunogens in several coronaviruses, such as murine coronavirus ( ), turkey coronavirus ( ) , and porcine reproductive and respiratory syndrome virus ( ) . in these viruses, the n protein is a highly abundant and relatively conserved antigen. importantly, the n protein may be involved in stimulating cytotoxic t lymphocytes. it has been reported that the n protein accumulates even before it is packaged in the mature virus ( ) . a cellular immune response elicited early in infection by internal viral proteins could therefore be an important defense mechanism. on the basis of the genomic sequences published to date, most of the sars-coronavirus n protein is highly conserved among strains. thus, the n protein, which has high antigenicity, is likely to have a great impact on the development of diagnostic tests for sars. the proteins extracted from vero-e cells infected by sars coronavirus were separated by sds-page ( % polyacrylamide), and the separated proteins were probed with antibodies in sera from sars patients. as shown in fig. , some immunostained bands appeared only on the membranes incubated with patient sera, suggesting that these proteins are associated with sars. on the basis of genomic data, the structural proteins of the virus have molecular masses of (s protein), (n protein), (m protein), and (e protein) kda ( ), respectively. in all of the western blots performed with sera from three sars patients as the primary antibody, no protein band Ͻ kda was immunostained, suggesting that the m and e proteins either do not elicit antibody production or generate low antibody titers in sars patients. of the protein bands Ͼ kda, most were located around two regions ( fig. ) , and - kda. the protein band slightly smaller than kda reacted consistently with all sars sera. because its location is close to the theoretical molecular mass of the n protein, kda, we believe that this protein is the n protein. the immunostained bands around - kda did not display a consistent pattern. although the theoretical value of the s protein is kda, it has been reported to be glycosylated during infection of the host cells. it therefore is not surprising that sarscoronavirus s proteins have significantly higher molecular masses than the theoretical value. we thus deduced that these immunostained proteins with high molecular masses are the s protein. to further confirm the identities of these protein bands, we conducted competition experiments to determine whether the peptides from the s or n protein would inhibit the binding of the sera from sars patients in the elisa. the patient sera preincubated with mg/l s or n gave a - % lower response in the elisa (data not shown), suggesting that the two peptides could compete with sars coronavirus for binding to the antibodies in sars serum. these observations suggest that the s and n proteins account for the antigenicity of the sarscoronavirus structural proteins. affinity of the peptides located at the cooh terminus of n protein protscale analysis suggested that the cooh terminus of the n protein has fewer buried residues and higher hydrophilicity than the other regions of this protein. we designed four peptides around the region, n , n , n , and n . of these, n , located at the end of the n protein, had low reactivity in the elisa; conversely, the other three peptides were significantly more immunoreactive in the elisa. to determine which peptides had higher affinities for antibodies from sars sera, we quantitatively measured the binding of the peptides to the antibodies in the elisa. in this experiment, the concentration of the antibodies was kept constant and the binding capacity was estimated as a function of peptide concentration (fig. ) . using the scatchard equation, we calculated the constants n and k d ; the binding curves obtained for the three peptides gave similar n values but different k d values. on the basis of the definitions of the two parameters in materials and methods, the fact that the curves give similar n numbers indicates that the peptides share similar epitopic sites, whereas the different k d values indicate that the three peptides bind to the same epitope with different affinities. of the three peptides, the k d value for n was much lower than the k d values for n and n , approximately one-fourth of the k d value for n and one-thirteenth of the k d value for n , suggesting that n is able to bind more strongly with antibodies in sars sera. thus, the region around n is most likely a strong epitopic site within the n protein. among the sars cases that were identified with use of the sars coronavirus as antigen, and cases were not detected by peptides n and n , respectively, although both were the most immunoreactive antigens of the synthesized peptides. a possible explanation for this failure may relate to the specific recognition of the antibodies raised from a sars case. in this case, the antibodies may not actively recognize the cooh terminus of the n protein. hence, combining the peptides that represent different structural proteins could improve the detection coverage. we selected n and n to use in combination with peptides s and m , respectively, and used these combinations as antigens for the elisa. contrary to our expectations, the use of peptide combinations did not improve the detection coverage, whereas single peptides displayed higher immunoreactivities in the elisa ( table in the data supplement). this unexpected behavior may be attributable to interactions between peptides n and s or between n and m . if this hypothesis is correct, then generating a chimeric peptide by linking the two peptides could be a good solution for reducing interactions. we are currently pursuing this line of investigation. in summary, the data presented indicate that (a) in three sars-coronavirus structural proteins, a total of four epitopic sites, s , m , n , and n - , were detected by screening with synthesized peptides; (b) of the five peptides with high reactivity against sars sera, n and n gave significant results compared with the sars coronavirus lysate in an elisa as well as a positive detection rate, suggesting that the cooh terminus of the n protein could be extremely antigenic and useful in clinical diagnosis based on the elisa; (c) of four peptides located at the cooh terminus of the n protein that were synthesized and tested for their binding with sars serum antibodies, n was confirmed to have the highest affinity for forming the peptide-antibody complex. taken together, these results show that synthesized peptides could be important in exploring epitopes of immunogens, specifically in urgent situations such as that presented by the emergence of the sars virus. the identification of these reactive peptides could aid in the development of diagnostic techniques for sars. the genome sequence of the sars associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection a complete sequence and comparative analysis of a sars-associated virus (isolate bj ) rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) new approaches and perspectives in cytomegagaovirus diagnosis use of peptide synthesis to probe vial antigens for epitopes to a resolution of a single amino acid isolation and identification of a novel coronavirus from patients with sars prediction of protein antigenic determinants from amino acid sequences evolution of avian coronavirus ibv: sequence of the matrix glycoprotein gene and intergenic region of several serotypes an immunodominant cd ϩ t cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis nucleocapsid protein gene sequence analysis reveals close genomic relationship between turkey coronavirus and avian infectious bronchitis virus identification of a common antigenic site in the nucleocapsid protein of european and north american isolates of porcine reproductive and respiratory syndrome virus cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry key: cord- - j n z authors: capai, l.; ayhan, n.; masse, s.; canarelli, j.; priet, s.; simeoni, m.-h.; charrel, r. n.; de lamballerie, x.; falchi, a. title: seroprevalence of sars-cov- igg antibodies, in corsica (france), april and june . date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: j n z our aim was to assess the seroprevalence of severe acute respiratory syndrome coronavirus- (sars-cov- ) infection after the lockdown in a sample of the corsican population. between th april and th june , , residual sera were collected from patients having carried out a blood analysis in one of the participating laboratories. residual sera obtained from persons of all ages were tested for the presence of anti-sars-cov- igg using the euroimmun enzyme immunoassay kit for semiquantitative detection of igg antibodies against s domain of viral spike protein (elisa-s). borderline and positive samples in elisa-s were also tested with an in-house virus neutralization test (vnt). prevalence values were adjusted for sex and age. a total of , residual sera samples were included in the study. the overall seroprevalence based on elisa-s was . % [ % confidence interval (ci) . - . ] and . % [ . - . ] after adjustment. gender was not associated with igg detection. however, significant differences were observed between age groups (p-value = e- ) and particularly for people being younger than years of age (odd ratio (or) = . % ci [ . - . ]; p-value < . *). the prevalence of neutralizing antibody titers [≥] was of % [ . - . ]. in conclusion the present study showed that a low seroprevalence for covid- in corsica in accordance with values reported for other french regions in which the impact of the pandemic was low. on december , , the municipal health commission in wuhan (hubei province, china) reports a cluster of unexplained pneumonia cases (ren et al., ) . on january, , a betacoronavirus, named the severe acute respiratory syndrome coronavirus (sars-cov- ) was identified (chang et al., ; zhou et al., ) . the disease causes by the sars-cov- was named coronavirus infectious disease . covid- is a highly infectious disease and following the first cases in china, the virus spread rapidly worldwide. reasons for the rapid spread of sars-cov- include high transmissibility of the virus (li et al., a) , asymptomatic or paucisymptomatic carriers (bai et al., ) , and the lack of any apparent cross-protective immunity from related viral infections (loos et al., ) . on january th, the world health organization (who) declared a public health emergency of international concern (li et al., b) . as of september th, , the number of sars-cov- confirmed cases exceeds million and more than , deaths had been reported. the socioeconomic impact of the covid- pandemic has also been significant, with lockdown drastically reducing the mobility and productivity of much of the world's population worldwide (who, ) . in france, the sars-cov- epidemic period ranged from w ( ) ( ) ( ) ( ) ( ) ( ) to w ( - may), with an epidemic peak at w ( - march) and a positivity rate of up to % affecting mostly the paris region and the northeastern of the country (santé publique france, ). to respond to the epidemic, on march th ( w ), france ordered all non-essential retailers and services to be closed, and the general population to remain confined. these measures have reduced the number of incident cases and the stress on the health care system. the french government announced the end of the lockdown on may th ( w ). after the epidemic period, it has been projected that in france, . million (range: . - . ) people, i.e., . % of the population, will have been infected (salje et al., ) . corsica, a french mediterranean island, was affected by the covid- pandemic like continental france, at the beginning of march. the sars-cov- , positivity rate ranged from . % ( w ) to . % ( w ), and peaked on w at % (ars, ). until may th, people died ( in south corsica, in haute-corse) of covid- in hospital (mortality rate = . % and lethality rate = . %). these epidemiological data included only a fraction of the real number of sars-cov- infections, since not all infected patients were symptomatic, required hospitalizations, or . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . provided specimens for laboratory testing (mizumoto et al., ) . the capacity to estimate the spread of sars-cov- depends on our knowledge of the immune status against sars-cov- in the population. the primary outcome of this study was to estimate for the first time the prevalence of igg antibodies against sars-cov- in corsican population in order to improve epidemiological knowledge of the virus spread and to estimate what part of the corsican population has been infected by the sars-cov- . the secondary outcome was to estimate neutralizing antibodies against sars-cov- by using an in-house virus neutralization test (vnt) currently considered as the most specific and sensitive serological assay capable of evaluating and detecting, functional neutralizing antibodies. monitoring of seroprevalence can guide public health measures to fight the pandemic. the study was conducted in the french mediterranean island of corsica ( , km ) located south-east of mainland france. the population of the island was estimated at , as of january , (insee, ) . this region is composed of two administrative departments (haute-corse and corse-du-sud) and five districts (ajaccio, bastia, calvi, corte, sartène) including counties. the age and sex distribution by age groups of the corsican population was obtained from the french national institute of statistics and economic studies (insee, ) (appendix ; fig a) . the sample size was calculated according to epidemiological calculation tools (epitools, ) . a minimum sample size of was calculated assuming an a priori % igg anti-sars-cov- seroprevalence (salje et al., ) , a confidence in the estimate of %, a maximum allowable error in the prevalence of %, and a corsican population size of , habitants based on the latest french census data (insee, ). the sampling plan was established taking into account the minimum estimated sample size and the actual distribution of the corsican population by age group and sex. information about the distribution of the studied population and the general population of corsica by age group and sex is available in the appendix ( figure a a and a b). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . between th april and thjune , , residual sera were collected from patients having carried out a blood analysis in one of the two participating laboratories (figure ). the conditions of exclusion were the following: one or more samples from the same person (only one sample included in the study), sample with missing information on age and/or gender and/or invalid serology result (insufficient serum volume or invalid result). the two private participating laboratories are located in ajaccio and corte. ajaccio is one of the two largest cities of corsica (south-west) and corresponds to the area of the island where the vast majority of covid- cases were detected during the sars-cov- epidemic. the laboratory located in ajaccio, is one of the main laboratory of the island and analyze samples coming from the south and north-western of the island. the laboratory of corte analyzes samples from the center of corsica. residual sera obtained from persons of all ages were tested for the presence of anti-sars-cov- igg using the euroimmun enzyme immunoassay kit for semiquantitative detection of igg antibodies against s domain of viral spike protein (elisa-s) (reference: in all samples with a ratio ≥ . , neutralizing antibodies were detected using a vnt as previously described (gallian et al., ) . veroe cells cultured in -well microplates, tcid of the sars-cov- strain bavpat (courtesy of pr. drosten, berlin) and serial dilutions of serum ( / - / ) were used. dilutions associated with cytopathic effect (cpe) were considered as negative (no neutralization) and those with no cpe at day post-infection were considered positive (complete neutralization), respectively. the neutralization titer referred to the highest dilution of serum with a positive result. specimens with a vnt titer ≥ were considered positive (gallian et al., ) (figure ). no nominative or sensitive data on participant people have been collected. this is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint according to french reference methodology mr- regulation on research projects concerning tube bottoms, only the age, sex, date and laboratory place of collection could be collected for each sample. the statistical analyses have been realized for these variables. descriptive statistic methods were performed for age and sex. age was described as median with interquartile ranges (iqrs). age groups were categorized as follow: - ; - ; - ; - ; - ; - ; - ; - ; - and ≥ . all categorical data were reported as percentages. igg anti-sars-cov- seroprevalences and its % exact binomial confidence intervals (cis) were estimated. associations of the presence of anti-sars-cov- igg with sex and age and location were analyzed and tested using the χ test or fisher's exact test. statistical significance was set at a p value < . . the odds ratio (ors) was used to describe the risk of different gae groups and gender in positive elisa-s serums compared with non-positive elisa-s serums. seroprevalence by age group and sex will be adjusted according to the proportions observed in the real population (insee, ) . this adjustment will be made by specifically weighting each individual. the following r packages were used for the statistical analysis: questionr; car; stats; survey; factominer and ade . all statistical analyses were performed using r software version . . (r foundation, vienna, austria). . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint a total of , serums of patients were included from the two participating private medical laboratories between late april and june . people included ranged in age from months to years including , women ( . %) and men ( . %). the median and the mean age were years (interquartile - ). the weighted population according to sex and age groups, showed that the mean age was years, including . % of women and . % of men. the table and figure described the overall seroprevalence and the seroprevalence rates estimated by age groups and gender in the included and in the weighted population. one is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint among the samples with an elisa ratio ≥ . (table ) , samples showed a ratio > . and a ratio ranging from . to . . forty-two percent (n= ) of samples had a positive neutralization antibody titer (vnt titer ≥ ) ( table ) . to the best of our knowledge this is the first study describing the prevalence of sars-cov- antibodies in a representative sample of corsican patients having carried out a blood analysis in biological laboratories after the covid epidemic period. the seroprevalence value estimated in the present study with elisa-s ( . % [ . - . ]; approximately , of people ) was in line with an estimation that . million (range: . - . ) people, i.e., . % of the french population, will have been infected during the epidemic period (salje et al., ) . the rate of seroprevalence reported in corsica was closer to the rate reported by a similar study in a french region with low proportion of covid- cases during the epidemic period ( %), than those reported in two regions with the highest rates ( - %) (carrat et al., ) .the observed seroprevalence in our population was in line with elisa-s values reported in spain, new york city and in different subcohorts of wuhan . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint (pollan et al., ) , united states (havers et al., ) and china (xu et al., ) but lower than values reported in heavily affected areas such as swiss (stringhini et al., ) , northern italy (vena et al., ) and the urban areas around madrid (soriano et al., ) . in the present study we did not observed a significantly distribution of seroprevalence values between males and females, in agreement with previous reports in spain (pollan et al., ) , dutch blood donors (slot, ) and french blood donors (gallian et al., ) . this is in line with sex-disaggregated data for covid- in several european countries showing a similar number of cases between the sexes, but more severe outcomes in aged men (gebhard et al., ) . we observed that seroprevalence values differed significantly among age groups. individuals aged less than years old showed a seroprevalence rate significantly higher than people aged more than years old. this trend was explained by high seroprevalence values observed among - ; - and - age groups ranging around - %. this is in line with results previous reported by a population-based serosurvey in geneva (stringhini et al., ) and with the values reported in three french general populations (carrat et al., ) . in our sample seroprevalence values suggest that infection was less prevalent in children than in adolescents during the epidemic period. the lower prevalence in children might be in part related to lower nasal gene expression of angiotensinconverting enzyme (bunyavanich et al., ) . because it is possible that the elisa assay could exhibit cross-reactivity with antibodies to other seasonal human coronaviruses, some results may represent false-positives leading to overestimation of seroprevalence data (takahashi et al., ) . the euroimmun assay used in this study was evaluated in different studies showing a specificity ranging from . % to % and sensitivity ranging from . % to % (beavis et al., ; kohmer et al., ; kruttgen et al., ) . the sensitivity and specificity of the elisa can vary considerably depending on the timing of the sample, in particular from samples collected ≥ days, after covid- diagnosis by pcr. the sensitivity can increase from % to % between samples analyzed at the time of pcr and them analyzed at least four days later (beavis et al., ) . virus neutralization test, currently considered as the gold standard, is the most specific serological assay capable of detecting true positive cases and functional neutralizing antibodies (algaissi a, ) . the positivity of a titer ≥ for the vnt assay used in the present study is an indicator of strong specificity ( %) (gallian et al., ) . around half of the samples showing elisa results ≥ . half showed a vnt≥ in line with a previous study (carrat et al., ) . the prevalence value based on our vnt analysis revealed that at least % of the included samples have been exposed to the virus and had . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . developed a titer of neutralizing antibodies ≥ . this value is in line with the low value of . % observed with the same vnt assay in blood donors collected during the last week of march or the first week of april in four french departments (gallian et al., ) and in the adult general population of a region with low prevalence (carrat et al., ) . seroprevalence data based on elisa assay provided information about previous exposure to sars-cov- but not about protection. in the present study we also estimated sars-cov- neutralizing antibodies titers among serum samples with dubious (≥ . and < . ) and positive (≥ . ) results. about half of these samples had neutralizing antibodies with a titer ≥ . this represents a preliminary approach to protection against re-infection, but it should be underlined that no precise correlate of protection is available yet (wu, ) . there are several limitations in our study. firstly, residual sera from screening or routine care provided by private medical biology laboratories, are more likely to come from people needing to monitor their health status for chronic medical conditions. thus data cannot be extrapolated to the general population although we have adjusted the data according to the age and sex of the general corsican population. additionally, no data concerning clinical features, chronic disease, or possible covid- exposures were available potentially biasing results. moreover, this lack of information on the covid- status of the persons included could also influence the specificity and sensitivity of the elisa test (timing of the sample in relation to the infection). as we tested by seroneutralization samples with an elisa-s test optical density ≥ . and not all samples, seoprevalence could be slightly underestimated. the strengths of this study were the size of the sample and its representativity in terms of age and gender. samples have been analysed by combining elisa and neutralization methods to strengthen results. in conclusion the present study showed that a low seroprevalence for covid- in corsica in accordance with values reported for other french regions in which the impact of the pandemic was low. this regional study is particularly important in corsica, as the island situation cannot be extrapolated from neighboring regions. . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . author contributions: conceptualization: a.f., m-h.s., j.c., r.c. and l.c.; investigation: m-h.s. and j.c.; funding acquisition: a.f.; methodology: r.c., a.f. and x.d.l, writing original draft preparation: l.c. and a.f.; writing-review and editing: x.d.l., r.c. and a.f.; laboratory analyses: s.m., n.a., s.p. and l.c.; statistical analyses: l.c.; supervision: a.f., m-h.s., j.c., r.c. and x.d.l. funding: this research received no external funding. the authors declare no conflict of interest. . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint figure a . age and sex repartition pyramid among corsican population (a a) and studied population (a b). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . neutralizing antibodies in sera using live virus microneutralization assay point épidémio régional corse spécial covid- presumed asymptomatic carrier transmission of covid- evaluation of the euroimmun anti-sars-cov- elisa assay for detection of iga and igg antibodies nasal gene expression of angiotensin-converting enzyme in children and adults seroprevalence of sars-cov- among adults in three regions of france following the lockdown and associated risk factors: a multicohort study protecting health-care workers from subclinical coronavirus infection lower prevalence of antibodies neutralizing sars-cov- in group o french blood donors impact of sex and gender on covid- outcomes in europe seroprevalence of antibodies to sars-cov- in sites in the united states pyramide des âges -régions et départements clinical performance of different sars-cov- igg antibody tests comparison of four new commercial serologic assays for determination of sars-cov- igg early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia transmission dynamics and evolutionary history of -ncov evolution of early sars-cov- and cross-coronavirus immunity estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship prevalence of sars-cov- in spain (ene-covid): a nationwide, population-based seroepidemiological study identification of a novel coronavirus causing severe pneumonia in human: a descriptive study estimating the burden of sars-cov- in france covid- herd immunity is not a realistic exit strategy during a covid- outbreak sars-cov- antibodies in adults in madrid, spain seroprevalence of anti-sars-cov- igg antibodies in geneva, switzerland (serocov-pop): a population-based study are sars-cov- seroprevalence estimates biased? prevalence of antibodies to sars-cov- in italian adults and associated risk factors who coronavirus disease (covid- ) dashboard neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications. medrxiv seroprevalence of immunoglobulin m and g antibodies against sars-cov- in china a pneumonia outbreak associated with a new coronavirus of probable bat origin cc-by-nc-nd . international license it is made available under a key: cord- -mwzz db authors: lu, guilan; gonzalez, richard; guo, li; wu, chao; wu, jiang; vernet, guy; paranhos-baccalà, gláucia; wang, jianwei; hung, tao title: large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in beijing, china date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: mwzz db background: human metapneumovirus (hmpv), a recently identified virus, causes acute respiratory tract infections (artis) in infants and children. however, studies on the seroepidemeology of hmpv are very limited in china. to assess the seroprevalence of hmpv infection in china, we tested a total of , serum specimens for the presence of anti-hmpv igg antibody in children and adults free of acute respiratory illness in beijing, china by using hmpv nucleocapsid (n) protein as an antigen. as a control, we used the human serum antibody against the n protein of human respiratory syncytial virus (hrsv), the most important viral agent responsible for aris in children. results: the seropositive rate for hmpv increased steadily with age from % at - mo to % at age . however, the rate dropped slightly between mo and yr of age. the seropositive rate for hrsv also increased steadily with age from % at - mo to % at age . in children aged six months to six years, the seropositive rates for the anti-hrsv igg antibody were significantly higher than those for hmpv. additionally, igg antibody titers to hmpv and hrsv were significantly higher in adults than in young children. consistent with the seropositive rates, the geometric mean titer of anti-hmpv igg antibody was lower than that of anti-hrsv igg antibody in children aged six months to six years. conclusions: our results indicate that similar to hrsv, exposure to hmpv is ubiquitous in the beijing population. however, the seroprevalence of anti-hmpv igg antibody is lower than that of hrsv in children between six months and six years old, which suggests a different number of repeat infections or a different response to infections. human metapneumovirus (hmpv), thought to belong to the metapneumovirus genus of the pneumovirinae subfamily, is a recently identified human respiratory pathogen first isolated from hospitalized children with acute respiratory infections (aris) in the netherlands [ ] . the viral genome, clinical manifestations, and epidemiology associated with hmpv are similar to those of human respiratory syncytial virus (hrsv), which was identified in and is the most important viral agent responsible for aris in children [ ] [ ] [ ] . since its initial identification, hmpv infections have been reported worldwide. however, fluctuating incidence of its infection has been reported by groups from different areas, varying from . % to % in respiratory tract samples from patients with aris [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . seroepidemiological investigations also showed that the prevalence of hmpv may differ between geographical locations. for example, in the netherlands, virtually all children have been exposed to hmpv by the age of five, demonstrating that hmpv infection is common in childhood [ ] . in canada, an elisa-based test using recombinant nucleocapsid protein (n protein) of hmpv produced by baculovirus revealed that more than % of patients over years of age tested seropositive for hmpv [ ] . in china, however, studies on the seroepidemeology of hmpv have been limited, and it is unclear what percentage of the population in different age groups have been infected with the virus. the n protein, about amino acids in length, is encoded by the n gene of hmpv genome. the hmpv n protein is abundantly expressed during the early replication stage of the virus and can stimulate a sustained immune response [ ] . because the amino acid identity of hmpv n is highly conserved within and between the a and b subgroups of hmpv [ ] , it has been widely applied in the immunoassay of hmpv infection and in the investigation of seroprevalence of hmpv infections [ , ] . hmpv n protein shares - % homology of amino acid sequence with hrsv [ ] . as previous studies have not shown obvious cross-reactions in immunoassays such as elisa, the hrsv n protein has been used as a reference to evaluate the seroprevalence of hmpv [ , ] . to assess the seroprevalence of hmpv infection in china, we used hmpv n protein as an antigen to test serum samples for the presence of anti-hmpv igg antibody in children and adults free of acute respiratory illness in beijing, china. the igg antibody against n protein of hrsv was tested in parallel as a control. our results indicate that exposure to hmpv is ubiquitous in the beijing population. lower seropositive rates and geometric mean titer (gmt) of anti-hmpv igg were observed in children aged six months to six years when compared to hrsv. this may reflect the divergence of infection pattern between hmpv and hrsv in children. our data will inform the evaluation of the social and economic burden of hmpv infection and enable the development of medical or public health strategies to combat hmpv infection in the population. establishment of an elisa-based detection method for seroprevalence of hmpv and hrsv igg antibody both recombinant hmpv and hrsv n proteins were effectively expressed in e. coli bl (de ) as soluble proteins. recombinant hmpv n protein was then purified from bl (de ) cell lysates by ni-chelating chromatography, and recombinant hrsv n protein was purified by anion exchange chromatography followed by ni-chelating chromatography (data not shown). the purity of both recombinant n proteins was greater than %. the purified proteins were confirmed by western blot analysis using mouse anti- ×his tag monoclonal antibody as the primary antibody and irdye goat anti-mouse monoclonal antibody as the secondary antibody ( figure ). to optimize elisa conditions, we performed chessboard titration tests using the recombinant hmpv and hrsv n proteins. hmpv-positive and negative sera samples were identified by western blot analysis (figure a , left panel). the optimal antigen (hmpv n protein) concentration for elisa under our test conditions was determined to be . μg/ml for hmpv, because at a : serum dilution, the od nm value of positive sera was approximately . and the difference between the od nm value of positive and negative sera was greatest (figure a, right panel) . similarly, we determined the optimal assay conditions for hrsv n protein-elisa to be . μg/ml hrsv n protein at : serum dilution ( figure b ). based on these elisa conditions, we then determined the cut-off value for these assays. twenty-eight anti-hmpv igg negative and anti-hrsv igg negative sera samples were identified using western blot analysis of sera samples randomly collected from children under five years of age. the od nm of each negative sample was determined by elisa for hmpv n protein or hrsv n protein. the mean value of the od nm of the hmpv negative sera was . with a standard deviation (sd) of . . the mean value of the od nm of the hrsv negative sera was . with a sd of . . based on the formula: cut-off value = mean od nm of the negative sera + three fold sds [ , ] , the hmpv and hrsv positive cut-off values were then defined as . and . . a tested sample was scored as "positive" if its od nm value was above the cut-off value. the sensitivity of these two elisa methods was determined using purified murine igg against hmpv or hrsv n proteins. the limits of detection were . μg/ml igg and . μg/ml igg, respectively. to test the specificity of the elisa methods established in this study, the reactions of mouse sera against influenza virus a (subtypes h -h ), human coronaviruses ( e, hku and nl ), and polyomavirus jc against hmpv and hrsv n protein were evaluated. there was no obvious cross-reaction between those mouse antibodies and the hmpv or hrsv n proteins (data not shown). furthermore, we did not observe cross-reaction between the hrsv n protein and hmpv n protein (data not shown). for large-scale elisa screening, each of the tested serum samples was evaluated for the presence of anti-hmpv and anti-hrsv (n protein) antibodies. our analysis indicates that among the , samples, % ( / ) of subjects aged - mo, % ( / ) of subjects aged mo- yr, % ( / ) aged - yr, % ( / ) aged - yr, % ( / ) aged - yr, and % ( / ) aged - yr were seropositive for hmpv; whereas % ( / ) of subjects aged - mo, % ( / ) aged mo- yr, % ( / ) aged - yr, % ( / ) aged - yr, % ( / ) aged - yr, and % ( / ) aged - yr were seropositive for hrsv ( figure and table ). comparison between the hmpv and hrsvpositive sera by statistical analysis (χ tests) demonstrated that the seropositive rates of hmpv were significantly lower than those of hrsv in the age groups of six months to six years, whereas no significant difference was observed in the age groups of one to six months, or six years to years ( table ) . to characterize the seroprevalence of hmpv infection in the sample used in this study further, we analyzed the titers of anti-hmpv and anti-hrsv igg antibodies in our samples. twenty randomly selected seropositive samples in each age group were analyzed. the geometric mean titers (gmts) of anti-hmpv igg and anti-hrsv igg were significantly higher in the age group of > years than in the age group of ≤ years (for gmt of anti-hmpv igg, vs , p = . ; for anti-hrsv igg, vs , p = . , mann-whitney u test) ( figure ). consistent with the seropositive rates, the gmt of anti-hmpv igg ( ) was also lower than that of anti-hrsv igg ( ) in a younger group between six months and six years of age (mann-whitney u test, p = . ). in this age group, the proportion of specimens with a low anti-hmpv igg titer (≤ : ) was significantly higher than those with a low anti-hrsv igg titer ( . % vs . %, χ test, p = . ) ( figure ). notably, for individuals over six years of age, the proportion of specimens with a high anti-hmpv titer (≥ : ) increased and was comparable to those with a high anti-hrsv igg titer ( . % vs . %, χ test, p = . ) (figures and ). in this report, we show a high seroprevalence of anti-hmpv igg antibody in the beijing area. the , serum samples included in our study were randomly collected in , from individuals receiving routine physical examinations and who were free of recent respiratory infections. thus, our data should represent the incidence of hmpv infection in beijing. our results indicate that virtually all children over the age of six years have been exposed to hmpv infection. compared to reports from the netherlands and japan in which and samples were tested [ , ], our data were derived from a larger number of subjects. consistent with those two reports, our results suggest that hmpv infection is ubiquitous. however, our results showed a higher rate of hmpv infection in infants and younger children than reported in the netherlands or japan. we found that the seroprevalence of hmpv in children aged mo to yr was % compared to % in the netherlands and . % in japan. thus, our data suggests that primary infection of children by hmpv may occur much earlier in beijing than in the netherlands or japan. this discrepancy may be caused by differences in exposure to hmpv in different geographical regions, or by other factors such as social, environmental, or climate conditions. similarly, hrsv has a high seroprevalence rate in beijing. our data correlate with previous findings that seroprevalence increases with age and that by the age of three, over % of children are seropositive for hrsv [ ] . interestingly, our results demonstrate that the seropositive rate of hmpv in children six months to six years of age are significantly lower than seropositive rates of hrsv (p < . ), suggesting that hrsv infection occurs earlier than hmpv infection. our results showed that the seropositivity of hmpv decreases during the mo- year period. maternal antibodies may be responsible for higher hmpv seropositivity in individuals - months old. the seropositivity of hmpv decreases during the mo- year period as the level of the maternal antibody decreases. the antibody titers then increase with age, after repeat hmpv infection. notably, within the age group of mo to yr, the gmts of anti-hmpv igg were significantly lower than those of anti-hrsv igg. significantly more specimens had a low anti-hmpv igg (≤ : ) titer than had a low anti-hrsv igg titer. the reasons for this disparity are unclear. it is possible that infection by hmpv occurs later than infection by hrsv, thus leading to the delayed increase in the seropositive rate and antibody titer of hmpv [ ] . it is likely that less response to hmpv or more exposure to hrsv that would boost the antibody titer, results in a higher antibody titer against hrsv than against hmpv. however, large-scale investigations using samples collected from different geographic regions are necessary to distinguish the immunological responses to the two viruses during early human life. we did not find significant differences between the seroprevalence of hmpv and hrsv infections in children less than six months of age. this reflects high levels of maternal antibody against hmpv and hrsv, which may a hmpv b hrsv figure titers of anti-hmpv and anti-hrsv igg antibodies. twenty human serum samples that were seropositive for hmpv (a) or hrsv (b) were randomly selected from each age group, for a total of samples. elisa was performed using hmpv n protein or hrsv n protein as the antigen. titers are shown for the indicated age groups. horizontal bars indicate the gmts of antibodies against hmpv or hrsv. figure and ) . to determine the specificity of our elisa method, we tested for reactions between hmpv or hrsv n protein and murine antibodies against multiple viruses, including influenza a viruses (h -h ), human coronaviruses ( e, hku , nl ) and polyomavirus jc. we found no obvious reactivity in elisa assays. additionally, we did not detect a cross-reaction between hmpv n protein and hrsv n protein. these results indicate that the n protein-based elisa specifically detects both anti-hmpv and anti-hrsv antibodies. however, we need to further evaluate the specificity of the n protein-based elisa by using antibodies against other respiratory virus antigens. the sensitivity of the elisa method for detecting hmpv and hrsv was determined by using purified murine specific anti-hmpv or hrsv n igg, respectively. to our knowledge, limits of detection comparable to the sensitivity achieved by our methods have not been reported in other serological surveys of hmpv. repeated infections of an individual by hmpv and hrsv have been reported [ ] [ ] [ ] [ ] , suggesting that immunity against hmpv and hrsv is not solid, is transient, or is incomplete against heterologous strains. the high prevalence and high titers of hmpv and hrsv that we observed in adults may suggest that re-infection by hmpv and hrsv occurs throughout life [ , ] . our results suggest that similar to hrsv infection, hmpv infection is ubiquitous in the beijing population. however, the seroprevalence of the igg antibody against hmpv is lower than that against hrsv in children between six months and six years of age, which may reflect differences in infection pattern between the two viruses. our findings provide further information to aid the development of strategies to control and prevent hmpv infection. serum specimens were collected from , subjects, including children (from the beijing children's hospital) and adults (from the beijing blood center), during routine physical examinations in . all individuals were free of acute respiratory infection for at least three months prior to sampling. all samples were collected after obtaining informed consent either from the individuals or from the individual's guardians. the sera were separated immediately after collection, stored at - °c, and inactivated at °c for minutes before use. purified recombinant proteins were separated by % sds-page and transferred onto a nitrocellulose membrane, as previously described [ ] . the membrane was blocked for two hours in % non-fat milk. mouse anti-his monoclonal antibody (sigma, munich, germany) diluted : in non-fat milk or serum samples diluted : in non-fat milk were added, and membranes were incubated for one hour at room temperature. membranes were then washed three times with pbs- . % tween and incubated for one hour at room temperature with the secondary antibody, irdye goat antimouse monoclonal antibody (li-cor biosciences, nebraska, usa) or horseradish peroxidase-conjugated goat anti-human igg monoclonal antibody (sigma, munich, germany). subsequently, membranes were washed three times with pbs- . % tween, then developed using odyssey (li-cor biosciences) or a tetramethylbenzidine (tmb) substrate (thermo scientific, rockford, usa) according to the manufacturers' instructions. chessboard titration tests were conducted using positive and negative serum samples that were randomly selected. western blot analysis was used to determine the optimal concentration of the coated antigen and serum dilution. subsequently, -well plates (costar, bethesda, usa) were coated with μl of . μg/ml purified hmpv n proteins or . μg/ml purified hrsv n proteins, in . m sodium hydrogen carbonate buffer (ph . ). plates were incubated overnight at °c, then blocked with % bsa overnight at °c and washed three times with pbs- . % tween. subsequently, μl of a : dilution of serum specimens was added and incubated at °c for one hour. the plates were then washed six times with pbs- . % tween and incubated for one hour at °c with horseradish peroxidase-conjugated goat anti-human igg (sigma) diluted : , . the plates were washed again six times with the same solution, and antibodies were detected by adding μl substrate solutions a and b (wantai biotech corp. beijing, china) followed by incubation at °c for minutes. the reactions were terminated by adding μl of m h so . optical densities (od) were read at nm (od nm ). the average od values for the hmpv-negative human sera samples (n = ), shown to be negative by western blot analysis for the hmpv-n protein, were measured. the average od values of the hrsv-negative human sera samples (n = ), shown to be negative by western blot analysis for the hrsv-n protein, were also measured. the cut-off values were defined as the mean od of the negative sera plus three standard deviations [ , ] . samples with od nm values above the cut-off value were considered positive. to determine the specificity of our elisa method, we tested mouse sera against inflenza virus ha proteins (subtype h -h ), human coronavirus ( e, hku , nl ) n proteins, and polyomavirus jc vp protein for reaction against hmpv and hrsv proteins. samples of sera were tested in serial dilutions of two-fold, starting at a : dilution. to determine the sensitivity of elisa method established in this study, mice sera against hmpv n protein and hrsv n protein were purified using protein-g sepharose column (ge healthcare) and quantified using pierce ® bca protein assay kit (thermo scientific, rockford). the purified murine igg against hmpv or hrsv were tested in serial dilutions of two-fold (starting at as μg/ml concentration). in addition, randomly selected positive serum samples in each group were subjected to anti-hmpv and anti-hrsv titer assays. to determine the endpoints of antibody titers, titers were calculated as the highest dilution of a serum showing an od nm reading of two times the mean of the negative serum control (starting at a : ) [ ] . seropositive rates were evaluated using χ tests. mean antibody titers between children and adults positive for hmpv and hrsv exposure were analyzed using the mann-whitney u test. a p value ≤ . was considered statistically significant. peking union medical college (pumc) & chinese academy of medical sciences (cams) peking union medical college & chinese academy of medical sciences, beijing , china. fondation mérieux, lyon , france. beijing centre for disease control and prevention, beijing , china. references . van den hoogen bg bronchiolitis-associated hospitalizations among us children a -year experience with human metapneumovirus in children aged < years human metapneumovirus infections in hospitalized children human metapneumovirus associated with respiratory tract infections in a year study of nasal swabs from infants in italy prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in spain human metapneumovirus as a cause of community-acquired respiratory illness human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children human metapneumovirus among children hospitalized for acute respiratory illness children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus subgroup changes and seasonality during epidemics biennial spring activity of human metapneumovirus in austria human metapneumovirus in hospitalized children in amman clinical and epidemiological comparison of human metapneumovirus and respiratory syncytial virus in seoul human metapneumovirus infection in hospital referred south afirca children human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults seroprevalence of human metapneumovirus(hmpv) in the canadian province of saskatchewan analyzed by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay development and validation of an enzyme-linked immunosorbent assay for human metapneumovirus serology based on a recombinant viral protein sequence analysis of the n, p, m and f genes of canadian human metapneumovirus strains development and evaluation of a whole virusbased enzyme-linked immunosorbent assay for the detection of human metapneumovirus antibodies in human sera seroepidemiology of human bocavirus defined using recombinant virus-like particles identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus seroprevalence of human metapneumovirus in japan seroepidemiological study of respiratory syncytial virus in sao paulo state, brazil comparison of the seroprevalence of human metapneumovirus and human respiratory syncytial virus human metapneumovirus reinfection among children in thailand determined by elisa using purified soluble fusion protein human metapneumovirus infection in japanese children age related changes in t cell mediated immune response and effector memory to respiratory syncytial virus (rsv) in healthy subjects respiratory syncytial virus infection and disease in infants and young children observed from birth in kilifi district respiratory syncytial virus and parainfluenza virus heterologous expression of full-length capsid protein of porcine circovirus in escherichia coli and its potential use for detection of antibodies protection of mice and poultry from lethal h n avian influenza virus through adenovirus based immunization submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the beijing xicheng district cdc and beijing children's hospital for their assistance with the collection of sera specimens. this study is supported in part by the national major science and technology research projects for the control and prevention of major infectious diseases in china ( zx - ). authors' contributions gl, rg, and jw designed the study. gl, lg, and cw conducted the experiments. jw was in charge of the collection of sera specimens. gl, rg, gp, gv and jw wrote the manuscript, and gv, gp, jw and th revised the manuscript. all authors have read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -scd f vk authors: pape, constantin; remme, roman; wolny, adrian; olberg, sylvia; wolf, steffen; cerrone, lorenzo; cortese, mirko; klaus, severina; lucic, bojana; ullrich, stephanie; anders-Össwein, maria; wolf, stefanie; cerikan, berati; neufeldt, christopher j.; ganter, markus; schnitzler, paul; merle, uta; lusic, marina; boulant, steeve; stanifer, megan; bartenschlager, ralf; hamprecht, fred a.; kreshuk, anna; tischer, christian; kräusslich, hans-georg; müller, barbara; laketa, vibor title: microscopy-based assay for semi-quantitative detection of sars-cov- specific antibodies in human sera date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: scd f vk emergence of the novel pathogenic coronavirus sars-cov- and its rapid pandemic spread presents numerous questions and challenges that demand immediate attention. among these is the urgent need for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. for this, sensitive, specific and quantitative serological assays are required. here we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (igg, iga and igm) of sars-cov- specific antibodies in human samples. the possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay which complements the portfolio of sars-cov- serological assays. the procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections. the recent emergence of the novel pathogenic coronavirus sars-cov- [ ] [ ] [ ] and the rapid pandemic spread of the virus has dramatic consequences in all affected countries. in the absence of a protective vaccine or a causative antiviral therapy for covid- patients, testing for sars-cov- infection and tracking of transmission and outbreak events are of paramount importance to control viral spread and avoid the overload of healthcare systems. the sequence of the viral genome became publicly available only weeks after the initial reports on covid- witnessed in the early phases of the ongoing sars-cov- pandemic. thus, complementary strategies to test for antiviral antibodies that can be rapidly deployed in situations where commercially available kits are either not yet developed or not available are an important addition to the diagnostic toolkit. immunofluorescence (if) using virus infected cells as a specimen is a classical serological approach in virus diagnostics and has been applied to coronavirus infections, including the closely related virus sars-cov [ ] [ ] [ ] . the advantages of if are (i) that it does not depend on specific diagnostic reagent kits or instruments, (ii) that the specimen contains all viral antigens expressed in the cellular context and (iii) that the method has the potential to provide high information content (differentiation of staining patterns and intensities due to reactivity against various viral proteins). a mayor disadvantage of the if approach as it is typically used in serological testing is its limited throughput capacity due to the involvement of manual microscopy handling steps and sample evaluation based on visual inspection of micrographs. furthermore, visual classification is subjective and thus not well standardized and yields only binary results. here, we address those limitations, making use of advanced automated microscopy and image analysis strategies developed for basic research. we present the establishment and validation of a semi-quantitative, semi-automated workflow for sars-cov- specific antibody detection. with its -well format, semi-automated microscopy and automated image analysis workflow it combines advantages of if with a reliable and objective semi-quantitative readout and high throughput compatibility. the protocol described here was developed in response to the emergence of sars-cov- , but it represents a general approach that can be adapted for the study of other viral infections and is suitable for rapid deployment to support diagnostics of emerging viral infections in the future. . results . setup of the if assay for sars-cov- antibody detection we decided to use cells infected with sars-cov- as samples for our if analyses, since this setup provides the best chance for detection of antibodies targeted at the different viral proteins expressed in the host cell context. african green monkey kidney epithelial cells (veroe cell line) have been used for infection with sars-cov- , virus production and if [ , ] . in preparation for our analyses we compared different cell lines for use in infection and if experiments, but all tested cell lines were found to be inferior to veroe cells for our purposes (see materials and methods and fig. s ). all following experiments were thus carried out using the veroe cell line. in order to allow for clear identification of positive reactivity in spite of a variable and sometimes high nonspecific background from human sera, our strategy involves a direct comparison of the if signal from infected and non-infected cells in the same sample. preferential antibody binding to infected compared to non-infected cells indicates the presence of specific sars-cov- antibodies in the examined serum. under our conditions, infection rates of ~ - % of the cell population were achieved, allowing for a comparison of infected and non-infected cells in the same well of the test plate. an antibody that detects dsrna produced during viral replication was used to distinguish infected from non-infected cells within the same field of view ( fig. a) . in order to define the conditions for immunostaining using human serum, we selected a small panel of negative and positive control sera. four sera from healthy donors collected before november were chosen as negative controls, and eight sera from pcr confirmed covid- inpatients collected at day or later post symptom onset were employed as positive controls. sera from this test cohort were used for primary staining, and bound antibodies were detected using fluorophore-coupled secondary antibodies against human igg, iga or igm. no difference between infected and non-infected cells in serum igg antibody binding was observed when sera collected before the onset of the sars-cov- pandemic were examined ( fig. b, fig. s ). in contrast, covid- patient sera were clearly characterized by higher serum igg antibody binding to infected compared to non-infected cells (fig. b) . all eight covid- patient serum samples yielded higher igg binding to infected compared to non-infected cells as assessed by visual inspection (fig. s ). similar results were obtained when an iga or igm specific secondary antibody was used for detection (fig. s ) . in order to allow for the parallel assessment of igg and iga or igm antibodies, we established conditions for the parallel detection of anti-igg coupled to alexafluor and anti-iga or anti-igm coupled to dylight or alexafluor secondary antibodies, respectively, without signal bleedthrough. using this approach, it was possible to implement detection of sars-cov- specific igg and iga or igm antibodies in a single experimental setup (fig. s ) . titration experiments were performed with positive control sera to determine the optimal range of serum concentration in the if experiments. all eight positive control samples showed visually detectable specific labelling of infected cells over the range of : and : , demonstrating robustness of the assay (fig. s ). serum concentrations of less than : did not yield detectable signals in all cases. we decided to employ a dilution of : in the further experiments . image analysis our next aim was to establish a semi-automated analysis workflow for image acquisition and analysis for a medium to high throughput setting. veroe cells were seeded into -well plates infected and immunostained using anti-dsrna antibody and patient serum, followed by indirect detection using a mixture of anti-igg and anti-iga/igm secondary antibodies. images were acquired using an automated widefield microscope (see materials and methods section for more detail). to obtain a measure for specific antibody binding we performed automated segmentation of cells and classified them into infected and non-infected cells based on the dsrna staining. we then measured fluorescence intensities in the serum channel per cell as a proxy for the amount of bound antibodies for both infected and non-infected cells and calculated the ratio between these values for infected and non-infected cells in a given specimen. to enable training of a machine learning approach for cell segmentation and to directly evaluate infected cell classification, we manually labelled cells and annotated them as infected/non-infected in images chosen from positive and control specimens. fig. presents a graphical overview of all analysis steps; the full description of every step can be found in materials and methods. briefly, our approach works as follows: first, we manually discarded all images that contained obvious artefacts such as large dust particles or dirt and out-of-focus images. then, images were processed to correct for the uneven illumination profile in each channel. next, we segmented individual cells with a seeded watershed algorithm [ ] , using nuclei segmented via stardist [ ] as seeds and boundary predictions from a u-net [ , ] as a heightmap. we evaluated this approach using leave-one- image-out cross-validation on the manual annotations and measured an average precision [ ] of . +- . (i.e., on average % of segmented cells are matched correctly to the corresponding cell in the annotations). combined with extensive automatic quality control which discards outliers in the results, the segmentation was found to be of sufficient quality for our analysis, especially since robust intensity measurements were used to reduce the effect of remaining errors. we then classified the segmented cells into infected and non-infected, by measuring the th percentile intensities in the dsrna channel and classifying cells as infected if this value exceeded . times the noise level, determined by the mean absolute deviation. this factor and the percentile were determined empirically using grid search on the manually annotated images (see above). using leave-one-out cross validation on the image level, we found that this approach yields an average f -score of . %. in order to make our final measurement more reliable, we then discarded whole wells, to score each sample, we computed the intensity ratio : here, is the median serum intensity of infected cells and the median serum intensity of non-infected cells. for each cell, we compute its intensity by computing the mean pixel intensity in the serum channel (excluding the nucleus area where we typically did not observe serum binding) and then subtracting the background intensity, which is measured on two control wells that did not contain any serum. we used efficient implementations for all processing steps and deployed the analysis software on a computer cluster in order to enhance the speed of imaging data processing. for visual inspection, we have further developed an open-source software tool (plateviewer) for interactive visualization of high-throughput microscopy data [ ] . plateviewer was used in a final quality control step to visually inspect positive hits. for example, plateviewer inspection allowed identifying a characteristic spotted pattern co-localizing with the dsrna staining ( fig. s ) that was sometimes observed in the iga channel upon staining with negative control serum. in contrast, sera from covid- patients typically displayed cytosol, er-like and plasma membrane staining patterns in this channel (fig. b, fig. s ). the dsrna co-localizing pattern observed for sera from the negative control cohort is by definition non-specific for sars-cov- , but would be classified as a positive hit based on staining intensity alone. using plateviewer, we performed a quality control on all iga positive hits and removed those displaying the spotted pattern colocalising with the dsrna signal from further analysis. with the immunofluorescence protocol and automated image analysis in place we proceeded to test a larger number of control samples in a high throughput compatible manner for assay validation. all samples were processed for if as described above, and in parallel analysed by a commercially available semi-quantitative sars-cov- elisa approved for diagnostic use (euroimmun, lübeck, germany) for the presence of sars-cov- specific igg and iga antibodies. as outlined above, a main concern regarding serological assays for sars-cov- antibody detection is the occurrence of false positive results. a particular concern in this case is cross-reactivity of antibodies that originated from infection with any of the four types of common cold corona viruses (cccov) circulating in the population. the highly immunogenic major structural proteins of sars-cov- nucleocapsid (n) and spike (s) protein, have an overall homology of ~ % [ ] to their counterparts in cccov and subdomains of these proteins display a higher degree homology; cross-reactivity with cccov has been discussed as the major reason for false positive detection in serological tests for closely related sars-cov and mers-cov [ ] . also, acute infection with epstein-barr virus (ebv) or cytomegalovirus (cmv) may result in unspecific reactivity of human sera [ , ] . we therefore selected a negative control panel consisting of sera collected before the fall of , comprising samples from healthy donors (n= , cohort b), patients that tested positive for cccov several months before the blood sample was taken (n= , all four types of cccov represented; cohort a), as well as patients with diagnosed mycoplasma pneumoniae (n= ; cohort z), ebv or cmv infection (n= , cohort e). we further selected a panel of sera from rt-pcr confirmed covid- patients collected at different days post symptom onset as a positive sample set (cohort c, see below). sera were employed as primary antisera for if staining using igm, iga or igg specific secondary antibodies, and samples were imaged and analysed as described above. this procedure yielded a ratiometric intensity score for each serum sample. based on the scores obtained for the negative control cohort and the patient sera, we defined the threshold separating negative from positive scores for each of the antibody channels. for this, we performed roc curve analysis [ ] [ ] [ ] on a subset of the data (cohorts a, b, c, z). using this approach, it is possible to take the relative importance of sensitivity versus specificity as well as seroprevalence in the population (if known) into account for optimal threshold definition. by giving more weight to false positive or false negative results, one can adjust the threshold dependent on the context of the study. whereas high sensitivity is of importance for e.g. monitoring seroconversion of a patient known to be infected, high specificity is crucial for population based screening approaches, where large study cohorts characterized by low seroprevalence are tested. since we envision the use of the assay for screening approaches, we decided to assign more weight to specificity at the cost of sensitivity for our analyses (see materials and methods for an in-depth description of the analysis). optimal separation in this case was given using threshold values of . , . and . for iga, igg and igm channels respectively (fig. s ). we validated the classification performance on negative control cohort e (n= ) which was not seen during threshold selection, and detected no positive scores. results from the analysis of the negative control sera are presented in fig. and table . while the majority of these samples tested negative in elisa measurements as well as in the if analyses, some positive readings were obtained in each of the assays, in particular in the iga specific analyses ( fig. and table ). since samples from these cohorts were collected between and , and donors were therefore not exposed to sars-cov- before sampling, these readings represent false positives. of note, negative control cohort e displayed a particularly high rate of false positives in elisa measurements, but not in if (table ). we conclude that the threshold values determined achieve our goal of yielding highly specific if results (at the cost of sub-maximal sensitivity). roughly . % (iga) or % (igg) of the samples were classified as positive or potentially positive by elisa ( table ). the notably lower specificity of the iga determination in a seronegative cohort observed here is in accordance with findings in other studies [ , ] accordance with other reports [ ] [ ] [ ] . consistent with other reports [ ] , sars-cov- specific igm was not detected notably earlier than the two other antibody classes in our measurements. at the earlier time points (up to day ), a similar or higher proportion of positive samples was detected by if compared to elisa for igg. although the sample size used here is too small to allow a firm conclusion, these results suggest that the sensitivity of igg detection by the semi- quantitative if approach is higher than that of an approved semi-quantitative elisa assay routinely used in diagnostic labs. in the case of iga detection at earlier time points (< day ) elisa performed slightly better ( / samples scored positive) compared to if ( / scored positive) however that came with the price of a very low specificity of elisa iga assay ( . % false negative detection) compared to if ( . %). . discussion here, we describe the development of a semi-quantitative if based assay for detection of sars-cov- specific antibodies in human samples that complements available elisa-based testing systems [ , ] . alternatives to elisa-based commercial test kits are important in situations where those kits are not available either because they are not yet developed in early days of the pandemic or due to high global demands for tests and required reagents. the microscopy-based assay described here has been developed during the early phase of the covid- pandemic to support the serological testing needs of the university hospital heidelberg, germany and is employed as a confirmatory assay in clinical studies [ ] and ongoing studies]. the assay displayed comparable or slightly better sensitivity and specificity than a commercially available semi-quantitative sars-cov- elisa approved for diagnostic use at the time. more importantly, combining two technically different serological assays, if and elisa, and classifying as "positive hits" only those that scored positive in both assays was instrumental to minimize false positive results while maintaining high sensitivity, and thus serves as a principle for serological studies or diagnostics where specificity of detection is of critical importance. specificity of detection is essential in settings of relatively low sars-cov- antibody prevalence [ ] [ ] [ ] in conjunction with high prevalence of potentially cross-reactive anti-cccov antibodies in a global population [ ] . one advantage of the if based assay presented here is that the specimens used for detection present the entire viral proteome, while elisa or chemiluminescent approaches use a single recombinantly expressed antigen. both the n and s protein of coronaviruses are highly immunogenic, and antibodies binding to the receptor binding domain on the s subunit are considered most relevant for neutralization. however, the relative importance of antibodies directed against the n protein for potential protective immunity against sars-cov- and the possible relevance of the overall breadth of the antibody response is currently unclear. other sars-cov- structural and non-structural proteins might play a role in immune response as it was shown for proteins a and b of the closely related sars-cov [ ] . in addition, expression of the viral proteome in permissive cells ensures correct protein folding and post-translational modification patterns. alterations in post-translational modifications are likely to influence the ability of serum antibodies to bind to different viral epitopes as it was shown for other viruses such as hiv- [ ] . it has to be noted that the detection of viral rna requires fixation and permeabilization of cells, which has the potential to affect epitope preservation. however, based on the high sensitivity of antibody detection and the good correlation to elisa measurements observed we conclude that this was no major concern in this case. two major disadvantages of typical if-based serological assays as applied in the past are manual microscopy acquisition steps and evaluation of samples based on a visual inspection. this procedure is incompatible with high throughput approaches and results are subjective, not quantitative and difficult to standardize. we have addressed these disadvantages by implementing automated microscopy acquisition and developing a robust software platform that is able to identify individual cells, classify infected and non-infected cells and take into account specific and non-specific background in order to generate semi-quantitative results. high-throughput application [ , ] . combining such cell lines with spectral unmixing microscopy [ ] would not only enable simultaneous determination of levels of all three major classes of antibodies (igm, igg and iga), but also identification of the viral antigens recognized, in a single multiplexed approach. the high information content of the if data (differential staining patterns) together with a machine learning-based approach [ ] and the implementation of stable cell lines expressing selected viral antigens in the if assay will provide additional parameters for classification of patient sera and further improve sensitivity and specificity of the presented if assay. the described analysis pipeline can be readily applied for serological analysis of other virus infections, provided that an infectable cell line and a staining procedure that allows differentiating between infected and non-infected cells are available. the assay described here thus offers potential as an immediate response to any future virus pandemic, as it can be rapidly deployed from the moment the first isolate of the pathogen has been obtained without requiring information on the expression of immunogenicity of viral proteins. two of our processing steps require manually annotated data: in order to train the convolutional neural network used for boundary and foreground prediction, we needed label masks for the individual cells. to determine suitable parameters for the infected cell classification, we needed a set of cells classified as being infected or non-infected. we have produced these annotations for images with the following steps. first, we created an initial segmentation following the approach outlined in the segmentation subsection, using boundary and foreground predictions from the ilastik [ ] pixel classification workflow, which can be obtained from a few sparse annotations. we then corrected this segmentation using the annotation tool bigcat (https://github.com/saalfeldlab/bigcat). after correction, we manually annotated these cells as infected or non-infected. note that this mode of annotations can introduce two types of bias: the segmentation labels are derived from an initial segmentation. small systematic errors in the initial segmentation that were not found during correction, could influence the boundary cell segmentation forms the basis of our analysis method. in order to obtain an accurate segmentation, we make use of both the dapi and the serum channel. first, we segment the nuclei on the dapi channel using the stardist method [ ] trained on data from caicedo et al. [ ] . note that this method yields an instance segmentation: each nucleus in the image is assigned a unique id. in addition, we predict per pixel probabilities for the boundaries between cells and for the foreground (i.e. whether a given pixel is part of a cell) using a d u-net [ ] based on the implementation of wolny et al. [ ] . this method was trained using the annotated images, see above. the cells are then segmented by the seeded watershed algorithm [ ] . we use the nucleus segmentation, dilated by pixels, as seeds and the boundary predictions as the height map. in addition, we threshold the foreground predictions, erode the resulting binary image by pixels and intersect it with the binarised seeds. the result is used as a foreground mask for the watershed. the dilation / erosion is performed to alleviate issues with very small nucleus segments / imprecise foreground predictions. in order to evaluate this segmentation method, we train different networks using leave-one-out cross-validation, training each network on of the manually annotated images and evaluating it on the remaining one. we measure the segmentation quality using average precision [ ] at an intersection over union (iou) threshold of . as described in https://www.kaggle.com/c/data-science-bowl- /overview/evaluation. we measure a value of . +- . with the optimum value being . . quantitation and scoring to distinguish infected cells from control cells we use the dsrna virus marker channel: infected cells show a signal in this channel while the non-infected control cells should ideally be invisible (see fig. ). we classified each cell in the cell segmentation (see above) individually, using the following procedure. first, we denoised the marker channel using a white tophat filter with a radius of pixels. to account for inaccuracies in the cell segmentation (the exact position of cell borders is not always clear), we then eroded all cell masks with a radius of pixels and thereby discard pixels close to segment boundaries. this step does not lead to information loss, since the virus marker is mostly concentrated around the nuclei. on the remaining pixels of each cell, we compute the . quantile ( ) of the intensity in the marker channel. for the pixels that the neural network predicts to belong to the background ( ), we compute the median intensity of the virus marker channel across all images in the current plate. finally, we classify the cell as infected if the . quantile of its intensity exceeds the median background by more than a given threshold: for additional robustness against intensity variations we adapt the threshold based on the variation in the background in the plate. hence, we define it as a multiple of the mean absolute deviation of all background pixels of that plate with n= . : to determine the optimal values of the parameters used in our procedure, we used the cells manually annotated as infected / non-infected (see above). we performed grid search over the following parameter ranges: in order to determine the presence of sars-cov- specific antibodies in patient sera, it was necessary to define a decision threshold r*. if a measured intensity ratio r is above a decision threshold r* than the serum would be characterized as positive for sars-cov- antibodies. for this an roc analysis was performed [ ] . each possible choice of r* for a test corresponds to a particular sensitivity/specificity pair. by continuously varying the decision threshold, we measured all possible sensitivity/specificity pairs, known as roc curves (fig. s ). to determine the appropriate r* we considered two factors [ ] : where is the prevalence or prior probability of disease. the optimal decision threshold r*, given the false-positive/false-negative cost ratio and prevalence, is the point on the roc curve where a line with slope m touches the curve. as discussed in the main text, a major concern regarding serological assays for sars-cov- antibody detection is the occurrence of false-positive results. therefore, we choose m to be larger than one in our analysis. in particular, we determine r* for the choice of m= (see fig. s ). we performed quality control of the images and analysis results at the level of wells, images and cells. the entities that did not pass quality control are not taken into account when computing the score during final analysis. we exclude wells that contain less than non-infected cells, that have a median serum intensity of infected cells smaller than times the noise level (measured by the median absolute deviation), or that have negative intensity ratios, which can happen due to the background subtraction. out of . wells, did not pass the quality control, corresponding to . % of wells. at the image level, we visually inspect all images and mark those that contain imaging artifacts using a viewer based on napari [ ] . we distinguish the following types of artifacts during the visual inspection: empty, unstained or over-saturated images, as well as images covered by a large bright object. in addition, we automatically exclude images that contain less than or more than cells. these thresholds are motivated by the observation that too few or too many cells often result from a problem in the assay. thus, of the total . images were excluded from further analysis, corresponding to . % of images. out of these, were manually marked as outliers and only a single one did not pass the subsequent automatic quality control. finally, we automatically exclude segmented cells with a size smaller than pixels or larger than . pixels that most likely correspond to segmentation errors. these limits were derived by the histogram of cell sizes investigated for several plates. two percent of the approximate . million segmented cells did not pass this quality control. in addition, we have also inspected all samples scored as positives. for the iga channel, we have found a dotty staining pattern in ten cases that produced positive hits based on intensity ratio in negative control cohorts, but does not appear to indicate a specific antibody response. we have also excluded these samples from further analysis. in order to scale the analysis workflow to the large number of images produced by the assay, we implemented an open-source python library to run the individual analysis steps. this library allows rerunning experiments for a given plate for newly added data on demand and caches intermediate results in order to rerun the analysis from checkpoints in case of errors in one of the processing steps. to this end, we use a file layout based on hdf [ ] to store multi-resolution image data and tabular data. the processing steps are parallelized over the images of a plate if possible. we use efficient implementations for the u-net [ ] , stardist [ ] and the watershed algorithm (http://ukoethe.github.io/vigra/) as well as other image processing algorithms [ ] . we use pytorch (https://pytorch.org/) to implement gpu-accelerated cell feature extraction. the total processing time for a plate (containing around images) is about two hours and thirty minutes using a single gpu and cpu cores. in addition, the results of the analysis as well as meta-data associated with individual plates are automatically saved in a centralized mongodb database (https://www.mongodb.com) at the end of the workflow execution. apart from keeping track of the analysis outcome and meta-data, a user can save additional information about a given plate/well/image in the database conveniently using the plateviewer (see below images that correspond to three different ratio scores (ratio score is indicated above the image) determined from samples stained with three different human sera, followed by staining with an anit-igg secondary antibody coupled to alexafluore . images represent overlays of three channels -nuclei (blue), igg (green) and dsrna (red). white boxes mark the zoomed area. cells in the insets are highlighted with yellow or cyan boundaries, indicating infected and non- infected cells, respectively. scale bar = m. thank embl, especially the embl it services department for providing computational infrastructure and support, as well as wolfgang huber for discussions on computing image based scores and statistical tests. we thank the patients who participated in this study berlin and the european virus archive (evag) for the provision of the sars-cov- strain bavpat . individual images used in the fig. a courtesy of medical illustrations database sb is supported by the heisenberg program (project number ) and mls is supported by the dfg (project number ). the funders had no role in study design, data collection bartenschlager microscopy development: s merle data interpretation laketa study design all authors have read and approved the final version of the manuscript miccai . miccai van den bogaard prevalence of covid- in children in baden-württemberg preliminary study report handb. open source tools we would like to thank martin weigert and uwe schmidt for their help with setting up prediction for stardist. we would like to acknowledge infectious disease imaging platform (idip) at center for integrative infectious diseases research (ciid) for microscopy support. we would like to key: cord- -rya w sh authors: kang, xiaoping; li, yuchang; fan, li; lin, fang; wei, jingjing; zhu, xiaolei; hu, yi; li, jing; chang, guohui; zhu, qingyu; liu, hong; yang, yinhui title: development of an elisa-array for simultaneous detection of five encephalitis viruses date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: rya w sh japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), and eastern equine encephalitis virus (eeev) can cause symptoms of encephalitis. establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. currently, there are still no multiple antigen detection methods available clinically. an elisa-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. an elisa-array method for the simultaneous detection of five encephalitis viruses was developed in this study. seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the elisa-array. the elisa-array assay is based on a "sandwich" elisa format and consists of viral antibodies printed directly on -well microtiter plates, allowing for direct detection of viruses. the developed elisa-array proved to have similar specificity and higher sensitivity compared with the conventional elisas. this method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. the results demonstrated that the developed elisa-array is sensitive and easy to use, which would have potential for clinical use. japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), eastern equine encephalitis virus (eeev), sindbis virus(sv), and dengue virus(dv) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [ ] . establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. currently, elisa and ifa are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [ , ] . there are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [ ] [ ] [ ] . however, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [ ] [ ] [ ] [ ] . liew [ ] validated one multiplex elisa for the detection of antigens; anderson [ ] used microarray elisa for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that elisa-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. however, the application of elisa-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. in this study, we developed an elisa-based array for the simultaneous detection of five encephalitis viruses. seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an elisa-array assay. the assay was validated using cultured viruses and inoculated chicken eggs with patient sera. the results demonstrated that this method combined the advantage of elisa and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. monoclonal antibodies were prepared from hybridoma cell lines constructed by prof. zhu et al. purification was conducted by immunoaffinity chromatography on protein g affinity sepharose [ ] . specific monoclonal antibodies ( d against jev, b against tbev, f against sv, b against serotype dv, f against serotype dv, e against eeev, and a against flavivirus) were selected for this study. all of the antibodies were raised according to standard procedures. using d , b , f , b , f , and e as capture antibodies, detection antibodies ( a , f , and e ) were coupled to biotin-nhs ester(pierce, germany) at °c for h according to the manufacturer's instructions. unincorporated biotin was removed by desalt spin column (pierce). immunologic reactions were reported by streptavidin-hrp (cwbio, beijing, china) and super signal elisa femto maximum sensitive substrate. purified goat-anti mouse antibody was used as a positive control. jev and dv were cultured in c / cells; sv, tbev, and eeev were cultured in bhk- cells. the culture of tbev and eeev was conducted in biosafety level facility, however, jev, dv and sv were conducted in biosafety level facility. viral titers were determined by the % tissue culture infectious dose (tcid ) method. all the cultures were inactivated by . % β-propionolactone at °c overnight, then °c for h to decompose β-propionolactone. antibodies were spotted using a biodot machine (bd ;california, usa) on elisa plates ( nl/dot). the plates were blocked with % bsa-pbs in °c for h, followed by washing times with pbs containing . % tween- for min each. then, the plates were dried, sealed, and stored at °c before use [ ] . when spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the elisa-array assay. the optimization was evaluated by dot morphology and signal intensity. the tested spotting buffers included × phosphate buffer saline (pbs), pbs + % glycerol, and × pbs + % glycerol+ . % triton-x . a range of monoclonal antibody concentrations ( . , . , . , . , and . mg/ml) were compared. following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, hpr-labeled avidin, and substrate, followed by signal evaluation. antigen binding was performed in pbs(containing . % tween- and % fcs) at °c for h, followed by washing times( × pbs containing . % tween- ). incubation of elisa plates with biotinylated detecting antibody cocktails was performed in pbs (containing . % tween- and % fcs) at °c for h. after washing, specific binding of the detecting antibodies was reported by streptavidin-hrp and stained with super signal elisa femto maximum sensitive substrate (thermo scientific, rockford, usa) [ , , ] . visualization of the plate was performed in ae cool ccd image analyzer(beijing bgi gbi biotech company., ltd, china). the signal intensity and background of each spot was read out and recorded with "monster"software. the positive signals were defined as a signal value > and a signal value (sample)/signal value (negative) > . the identical antibodies used in the elisa-array format were also tested in a conventional elisa format to determine the difference in sensitivity and specificity of the two methods. the conventional elisas were performed at the same time as the elisa-array assays to ensure similar reaction conditions. the conventional elisas were performed in an identical maner to the elisa-array, except that antibodies were coated at a concentration of μg/ml in pbs (ph . ), and substrate tmb was used instead of super signal elisa femto maximum sensitive substrate [ , ] . three serum samples were collected from patients with nervous system symptoms and histories of tick bites. the serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. days later, the liquid was collected and divided into two portions (one for inactivation and one for rna extraction). the rna and inactivated samples were stored at - °c before use. rna was extracted from the inoculated chicken eggs using a rneasy mini kit (qiagen inc., valencia, ca, usa) according to the manufacturer's instructions. all rna extraction procedures were conducted at bsl- facilities. the primers and probes were used as previously described [ ] . the real-time rt-pcr was conducted with a quti-teck q-rt-pcr kit (qiagen inc,). the reaction consisted of μl of × reaction buffer ( . μl reverse transcription enzyme, and nmol/l primers and probes). rna and deionized water were added to a final volume of μl. pcr was performed with a lightcycler . (roche, switzerland) [ ] . optimization of the elisa-array assay the spotted array layout is depicted in figure and the efficacy of three different spotting buffers on the quality of the printed elisa-arrays were investigated by spot morphology observation and signal intensity comparison. the spotting concentration of the capture antibodies varied from . to . mg/ml (each was serially diluted -fold). the efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. figure illustrates the array layout and figure demonstrates the result of the three spotting buffers and spot concentration of antibody b by tbe virus culture detection. cross reaction detection was also conducted by applying jev, yf, and dv cultures. spot morphology observation (figures a, b , and c) demonstrated that spotting buffer containing pbs with % glycerol produced tailed spot morphology; buffers containing pbs alone and pbs with % glycerol + . % triton-x gave good spot morphology (round and full). buffers containing pbs with % glycerol and pbs with % glycerol+ . % triton-x produced higher signal intensities than pbs alone. thus, pbs with % glycerol+ . % triton-x was adopted as the optimized spotting buffer for subsequent experiments. simultaneously, the spot concentration evaluation suggested that . mg/ml was optimal. at this concentration, the signal intensity was higher and the cross-reaction did not appear (figure d ). consequently, spotting concentration optimization of other capture antibodies ( d , f , e , and b ) demonstrated that . mg/ml was also suitable(data not shown). the optimized elisa array layout is shown in figure , which was applied in the following experiments. successful detection of viral pathogens requires a test with high sensitivity and specificity. to evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. by testing serially-diluted viral cultures, including dv- , dv- , jev, tbe, sv, and eeev, the sensitivity of elisaarray and the identical conventional elisa were compared ( table ). the detection limit of the two methods was compared and demonstrated. the cross-reactivity test was conducted using bhk- and vero cell lysate, yellow fever virus (yfv) cultures ( × tcid /ml, west nile virus(wnv) cultures( × tcid /ml), and western equine encephalitis virus( × tcid /ml). the results demonstrated that neither the elisa-array nor traditional elisa displayed cross-reactivity. equal volumes of cultured tbev, jev, dv- , dv- , sv, and eeev were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. a cocktail of biotin conjugated antibody ( a , e , and f ) was used in all tests. the results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (figures and ) . chicken eggs inoculated with infected human serum were used for validation of the elisa-array assay. all samples showed high reaction signals with capture antibody b , which was specific for tbev ( figure b ). the elisa-array assay suggested that the three patients were all infected with tbev. to verify the results tested by elisa-array, rna extracted from chicken eggs was applied to a real time-rt-pcr assay using primers and probes targeting tbev. the results were also positive (figure a) . the consensus detection results confirmed that the elisaarray assay was reliable. to be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [ , , ] . multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. thus, an elisa-array, which combines the advantages of elisa and protein array, meets the above requirements. it has been reported that an elisa-array has been used in the diagnosis of cancer and auto-allergic disease [ , ] ; however, no study has reported the detection of viral pathogens. in this study, we developed a multiplex elisa-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. the production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [ ] . cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. first, we prepared and screened monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. then, the antibodies were screened by an elisa-array with a double-antibody sandwich elisa format. the antibodies which produced cross-reactivity and low-positive signals were excluded. finally, six antibodies were selected as capture antibodies. another monoclonal antibody, a , which could specifically react with all viruses in the genus flavivirus was used for detecting antibody against dv, jev, and tbev. for the detection of eeev and sv, although the detecting and trapping antibodies were the same ( f and e , respectively), the antibodies produced excellent positive signals. the epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. as one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. in the elisa-array assay, this problem exists as well. because of the limitation of available antibodies, this assay could only detect pathogens. in the future, with increasing numbers of suitable antibodies, especially specific antibodies against flavivirus, this elisaarray might be able to test more pathogens and be of greater potential use. to make the assay more amenable to multiple virus detection, the assay protocol was optimized. in addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. heat inactivation was performed by heating the viral cultures at °c for h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. thus, β-propiolactone treatment was chosen as the virus-inactivation method. a conventional elisa is a standard method in many diagnostic laboratories. we compared the elisa-array with a conventional elisa and confirmed that the advantage of the elisa-array was evident with comparable specificity and higher sensitivity than elisa. the time required for the elisa-array is significantly less than for conventional elisa ( h vs. a minimum of h, respectively). furthermore, less igg is required for printing than for coating elisa plates. coating of a single well in microtiter plate requires μl of a μg/ml antibody solution, which is equivalent to ng of igg. for the elisa-array, only nl of a μg/ml antibody solution is required for each spot, which is equivalent to . ng of igg. with the characteristics of ease of use, sensitivity, specificity, and accuracy, the elisa-array assay would be widely accepted for clinical use. direct broad-range detection of alphaviruses in mosquito extracts multiplexed protein measurement: technologies and applications of protein and antibody arrays multiplexed sandwich assays in microarray format detection of adamantane-resistant influenza on a microarray a novel magnetic bead bioassay platform using a microchip-based sensor for infectious disease diagnosis advances in viral disease diagnostic and molecular epidemicological technologies assessment of some tools for the characterization of the human osteoarthritic cartilage proteome high-throughput microarray-based enzyme-linked immunosorbent assay (elisa) development of a sensitive microarray immunoassay and comparison with standard enzyme-linked immunoassay for cytokine analysis multiplexed sandwich assays in microarray format validating a custom multiplex elisa against individual commercial immunoassays using clinical samples application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer human neutralizing sars-cov specific fab molecules generated by phage display simultaneous multianalyte elisa performed on a microarray platform a multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis c virus infection and typing of whole blood a simple and rapid protein array based method for simultaneous detection of biowarfare agent microarray immunoassay for the detection of grapevine and tree fruit viruses development of a quantitative real-time rt-pcr assay with internal control for the laboratory detection of tick borne encephalitis virus (tbev) rna a duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses high diagnostic accuracy of antigen microarray for sensitive detection of hepatitis c virus infection the role of biosensors in the detection of emerging infectious diseases submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was supported by the national natural science foundation of china (grant no. ) and national key research special foundation of china (grant no. zx - ). authors' contributions xk: designed the study, performed the laboratory testing, analyzed the test results, and co-wrote and edited the manuscript. h l and yl performed the virus cultures. lf performed laboratory testing. xc, fl, and gc performed real time-pcr. qz and yy organized the overall project and helped edit the manuscript. all of the authors read and approved the final manuscript. state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology, beijing , china the authors declare that they have no competing interests. key: cord- -jzaeaxam authors: zhang, jian‐san; chen, jiang‐ting; liu, yu‐xuan; zhang, zhen‐shan; gao, hong; liu, yan; wang, xu; ning, ye; liu, yu‐fen; gao, qiang; xu, jian‐guo; qin, chuan; dong, xiao‐ping; yin, wei‐dong title: a serological survey on neutralizing antibody titer of sars convalescent sera date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: jzaeaxam a seroepidemiologic study was conducted in north china in to determine the neutralizing antibody titer of severe acute respiratory syndrome (sars) convalescent sera. a total of sars convalescent serum samples were collected from patients from the inner mongolia autonomous region, hebei province, and beijing – days after the onset of symptoms. the anti‐sars antibodies were detected by enzyme‐linked immunosorbent assay (elisa), neutralization assay, and western blot. eighty‐seven serum samples were confirmed to be positive for sars antibodies. the neutralizing antibody titer of the positive sera was analyzed quantitatively by neutralization assay. the geometric mean titer (gmt) of the convalescent sera was : . the kolmogorov–smirnov test showed that the neutralizing antibody titers conform to normal distribution, which suggests that the average anti‐sars antibody level in this study was representative of the convalescent antibody level of the sars population. this result could be useful for the development and quality control of sars vaccines. j. med. virol. : – , . © wiley‐liss, inc. severe acute respiratory syndrome (sars), the newly emerging severe acute respiratory syndrome caused by sars coronavirus (sars-cov), is of importance to public health kuiken et al., ] . as of july , , cases of sars had been reported and of the infected patients died. from april to may , china reported nine new laboratory-acquired sars cases. to prevent future recurrence of sars, more than a dozen sars candidate vaccines are under development [gao et al., ; bisht et al., ; marshall and enserink, ; yang et al., ] . there is an urgent need for an accurate measurement of sars convalescent serum antibody levels as a reference to evaluate the efficacy of vaccine. many efforts have been devoted to quantifying sars-cov specific antibody titer using enzyme-linked immunosorbent assay (elisa) shi et al., ; huang et al., ] , but the results are not comparable among different studies. in one report, the igg antibody titer of convalescent serum measured by indirect elisa was around : . in another report, the antibody measured was specific to recombinant sars nucleocapsid (n) protein and the titer was expressed by the logarithm [liu et al., ] . since standardized reagents for sars antibody detection have not become available, the data on sars antibody titer reported from various researchers are not applicable for sars vaccine evaluation. neutralization assay measures the ability of sera to neutralize the infectivity of sars-cov in cell culture. it is a stable method and does not involve external reagents. to gain a comprehensive understanding of the antibody to sars-cov, we report the anti-sars antibody titer of sars convalescent sera determined by neutralization assay. it provides important information for the development of sars vaccine. ninety-nine serum samples from convalescent sars patients were collected - days after the onset of symptoms from may to september . seventy-five were collected from inner mongolia autonomous region, from hebei province, and from beijing. all sars patients were confirmed to be sars cases according to the diagnosis criteria promulgated by the ministry of health of p.r. china [ ] . they all had a medical history of close contact with sars patients; symptoms of fever higher than c, coughing, difficulty in breathing; reticular change, flaky or striped infiltrative shadows on chest x-ray examination; no white blood cell (wbc) count rise; and no effect after treatment with antibiotics. a panel of serum samples were obtained from healthy adults as negative controls. all serum samples were sterilized by a . mm filter and were heatinactivated at c for min. sars-cov sino strain was isolated from pharyngeal swabs of clinically confirmed sars patients at peking union hospital. the sino strain was well-characterized biologically and was confirmed by electron microscope observation and complete genome sequencing (genbank accession no. ay ). vero cells, a line of african green monkey kidney cells, were obtained from atcc (american type culture collection). vero cells were grown at c in minimum essential medium (mem), containing % (w/v) new-born calf serum and % (w/v) glutamine. sars-cov was propagated in vero cells in culture flasks at c until % of the cells showed a cytopathic effect (cpe). after three freezethaw cycles, virus was harvested and characterized for the neutralization assay. a batch of sars-cov was inactivated by b-propiolactone in situ in flasks and then purified by centrifugation, ultrafiltration, and gel filtration. the purified sars-cov was used as antigen for western blot assay. vero cells were cultivated in -well plates at c to form a confluent monolayer. cells were inoculated with ml per well of viral suspension that was diluted serially in tenfold steps in mem containing % newborn calf serum and mg/ml penicillin, ph . ), with each dilution filling wells. the -well plates were incubated at c in a % co incubator. virus titer was expressed as cell culture infectious dose (ccid), which is the dilution of virus causing half of the cultured cells to produce cpe. the virus batch had a titer of . lg ccid /ml and was stored at À c for the neutralization assay. purified sars-cov sino strain proteins were separated by % sds-page and were transferred onto nitrocellulose membranes. the blocked membrane was incubated with the tested serum sample (diluted : ) as primary antibodies. the secondary antibody was goat anti-human igg conjugated with hrp and the color developing substrate was dab. a recombinant n protein was included in each test as positive antigen. when the n protein and a brand of purified sars-cov showing the molecular weight of about kda were detected, the serum used was considered sars antibody positive. elisa was performed using a commercial sars coronavirus igg antibody diagnostic kit (bgi-gbi biotech co. ltd., beijing, china) according to the manufacturer's instructions. the tested sera were diluted ten times and added to the elisa strip wells coated with sars-cov lysate. after the processes of washing, adding hrp-labeled anti-human igg, color developing, and stopping reaction, the absorbance of the optical density at nm (od ) was measured. the neutralization assay was conducted according to conventional procedures. briefly, ml diluted serum (twofold serial dilution from : to : , ) was added to equal volume of virus (diluted to ccid / . ml) and incubated for hr at c. then the mixture was added to confluent monolayer of vero cells in -well plates and incubated at c in a % co incubator for days. on day , the presence of viral cpe was checked. the dilution of serum that completely inhibited cpe in % of the cells was calculated using the reed-muench formula [reed and muench, ] . both positive and negative controls were included in each test. meanwhile, in each assay, the sars-cov was re-titrated for its infectivity (ccid / . ml) in parallel. the neutralization assay experiment system was validated first. a well-titrated sars in-house reference antiserum with the geometric mean titer (gmt) of : . was used as a positive control [zhang et al., ] . the test results showed that the neutralization antibody titer for negative control sera were all below : , therefore neutralization antibody titer equal to or higher than : was defined as positive. the neutralization assay was taken as valid only when the titer of the positive control was in the range of : to : , and the titer of negative serum was below : . the above experiment was repeated three times by three persons simultaneously. ninety-nine serum samples (in dilution : ) were firstly qualitatively tested by neutralization assay. those positive sera were then serially diluted to be quantitatively tested for the neutralizing antibody titer. the normal distribution analysis was performed by one sample kolmogorov-smirnov test using spss software, with the level of significance set at p < . . a total of sars convalescent sera were included in this research. all samples were tested qualitatively for antibody response to sars-cov by elisa and neutralization assay in parallel, but only samples were tested by western blot. of samples, elisa detected positive sera and neutralization assay identified positive sera. eighty-six sera were positive both for elisa and neutralization assay (table i) . neutralization assay detected one more positive serum than the result obtained by elisa and neutralization assay. this serum sample was confirmed further to be positive by the evidence of western blot, though it was negative for elisa. of neutralization positive samples, were tested by western blot and they were all positive for the anti-sars antibody (table i) . these serum samples were confirmed to be positive for anti-sars antibodies with the combination of elisa, neutralization, and western blot, so they were pooled to form a convalescent sera database for the further analysis of neutralizing antibody titer. the remaining serum samples were ruled out, as they were negative by the evidence of both neutralization assay and western blot or elisa. the anti-sars neutralizing antibody titer of positive convalescent sera was analyzed quantitatively by the neutralization assay. titers collected from to days after the onset of symptoms were tested by the neutralization assay, and the results are shown in figure . the lowest titer detected by the neutralization assay was : and the highest titer was : . the gmt of these convalescent sera was : . test results showed that sars specific neutralizing antibody level was relatively stable and persisted as long as days with a slight decline from : to : after days after the onset of the symptom (table ii) . a normal distribution test of anti-sars titer was performed by one sample kolmogorov-smirnov test using spss software after the log transformation of titers. the test result showed that the p-value was . (p ! . ), which revealed that the anti-sars neutralizing antibody titer was in normal distribution. a conclusion can be drawn that the average anti-sars neutralizing antibody level of convalescent sera obtained in our study is a representative of the convalescent neutralizing antibody level of the sars population. during the outbreak of sars in , elisa, immunofluorescent assay, western blot, and neutralization assay were used for serological diagnosis of sars cases. elisa, the most widely used assay, possesses a sensitivity and specificity of over % in some reports [wu et al., ] . the objective of this investigation was to provide a profile of anti-sars neutralizing antibody of these sera, were double positive for elisa and neutralization assay, the remaining serum was double positive for neutralization assay and western blot. titer from convalescent patients, rather than establishing neutralization assay as a diagnostic method for sars. in our laboratory, a combination of elisa, neutralization assay, and western blot were performed on sars convalescent sera. using these methods, sera were confirmed to be positive for anti-sars antibody. the results from the neutralization assay accord with those from elisa and western blot. a high level of consistency was demonstrated for elisa and the neutralization assay ( / [ . %]) for these positive samples, illustrating the high sensitivity and specificity of the neutralization assay. the gmt of anti-sars neutralizing antibody was quantitated to be : , and the normal distribution of the neutralizing antibody titer was shown by statistical analysis. the neutralization assay could, therefore, become a useful tool for the surveillance of sars serological epidemiology. it has been demonstrated in a mouse model that the neutralizing antibody elicited by primary infection of sars-cov can protect animals from re-infection. in addition, passive transfer of this immune serum to naïve mice can prevent sars-cov replication in the respiratory tract . yang et al. showed that a dna vaccine encoding the spike (s) glycoprotein of the sars-cov induced mice to generate t-cell and neutralizing antibody responses, as well as a protective immune response. they demonstrated that the protection was mediated by a humoral but not a t-celldependent immune mechanism by t-cell depletion and passive transfer of purified igg. although more investigations on humoral and cellular immunity are needed in humans, it is reasonable to conclude that human neutralizing antibody is protective against sar-cov. the development of sars vaccine entails methods that best reflect the immune response in humans to evaluate the vaccine efficacy. the neutralization assay satisfies this requirement. it is specific and correlates with the humoral immune response in sars convalescent patients. in addition, unlike some serological tests based on subunits (such as n or s protein), the neutralization assay detects all effective neutralizing antibodies having specificities for both characterized and uncharacterized epitopes. although the neutralization assay is a gold standard, several disadvantages limit its wide use. it is cumbersome, expensive, and in need of biosafety containment, because of the risk of infection. the manipulation of sars-cov might bring risk not only to the researchers but also to the public, as sars laboratorial infection cases have been reported in taiwan, singapore, and the chinese mainland [watts, ] . the average neutralizing antibody titer against sars-cov in convalescent serum is undoubtedly one of the most important indexes as a reference for sars candidate vaccine assessment. severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice identification of a novel coronavirus in patients with severe acute respiratory syndrome effects of a sars-associated coronavirus vaccine in monkeys evaluation of antibody responses against sars coronaviral nucleocapsid or spike proteins by immunoblotting or elisa newly discovered coronavirus as the primary cause of severe acute respiratory syndrome profile of specific antibodies to the sarsassociated coronavirus profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (sars) associated corona virus in probable sars patients caution urged on sars vaccines clinical diagnostic criteria for severe acute respiratory syndrome (sars) (on trial a simple method of estimating fifty percent endpoints diagnosis of severe acute respiratory syndrome (sars) by detection of sars coronavirusnucleocapsid antibodiesinanantigen-capturing enzymelinked immunosorbent assay prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice detection of the anti-sars-coronavirus antibody levels in sars patients sars under control, lab-safety questions remain serological and molecular biologic methods for sars-associated coronavirus infection a dna vaccine induces sars coronavirus neutralization and protective immunity in mice preparation and characterization of sars in-house reference antiserum we thank bameng cdc of inner mongolia autonomous region, zhangjiakou cdc of hebei province, and beijing cdc for helping with the collection of sars convalescent sera. we also thank dr. vienna l. reichert in harvard medical school for the critical review. this study was supported by the project of ''research on inactivated sars vaccine'' founded by the china plan grant aa . key: cord- -n ooziv authors: soykut, esra acar; dudak, fahriye ceyda; boyacı, İsmail hakkı title: selection of staphylococcal enterotoxin b (seb)-binding peptide using phage display technology date: - - journal: biochem biophys res commun doi: . /j.bbrc. . . sha: doc_id: cord_uid: n ooziv in this study, peptides were selected to recognize staphylococcal enterotoxin b (seb) which cause food intoxication and can be used as a biological war agent. by using commercial m phage library, single plaque isolation of phages was done and binding affinities were investigated with phage-elisa. the specificities of the selected phage clones showing high affinity to seb were checked by using different protein molecules which can be found in food samples. furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (spr) sensors. sequence analysis was realized for three peptides showing high binding affinity to seb and wwrpltpesppa, mnlhdyhrlfwy, and qhpqinqtlyrm amino acid sequences were obtained. the peptide sequence with highest affinity to seb was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide–seb interaction were determined by using isothermal titration calorimetry (itc) and compared with those of antibody–seb interaction. the binding constant of the peptide was determined as . ± . × ( ) m(− ) which indicates a strong binding close to that of antibody. in this study, peptides were selected to recognize staphylococcal enterotoxin b (seb) which cause food intoxication and can be used as a biological war agent. by using commercial m phage library, single plaque isolation of phages was done and binding affinities were investigated with phage-elisa. the specificities of the selected phage clones showing high affinity to seb were checked by using different protein molecules which can be found in food samples. furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (spr) sensors. sequence analysis was realized for three peptides showing high binding affinity to seb and wwrpltpesppa, mnlhdyhrlfwy, and qhpqinqtlyrm amino acid sequences were obtained. the peptide sequence with highest affinity to seb was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-seb interaction were determined by using isothermal titration calorimetry (itc) and compared with those of antibody-seb interaction. the binding constant of the peptide was determined as . ± .  m À which indicates a strong binding close to that of antibody. Ó elsevier inc. all rights reserved. phage display techniques have aroused interest as a new tool in protein-ligand interaction studies since described by smith [ ] . this technology has been used to detect peptides which show high affinity to target molecules required for immunological and biological studies, drug discovery, pharmacology, plant science, and also inorganic materials [ ] . also through this technology, detection of ligands which are specific target proteins is accomplished without animal or human immunization system. recombinant protein and/or peptides structures which have recognize capability of target molecules are fused on ff class (m , f , and fd) of filamentous phages. dna fragments which code peptides are added pviii, piii or pvi gen regions of phages capsid proteins. random dna sequences that coded different peptides on phage genomes supply different peptides or proteins which have different antigen specificities [ ] [ ] [ ] . the foreign dna sequences can be originated from a natural source, or it can be advisedly designed and synthesized chemically [ ] . using a process of biopanning, selection of peptides from phage library is accomplished. phage library is added to immobilized target molecules which in plates. after incubation non-binding phages washed away and selectively bound phage to target molecules are eluted and amplified in host bacteria. these amplified phages serve as input for another round of biopanning. rounds of selection are generally repeated two or three times in same way and affinity of each phage clone from eluted phages to target molecules are ascertained by phage-elisa and sequences of peptide are determined [ , ] . phage display technology has been used for selection and production of molecules that have antibody characteristics. recombinant antibodies have been selected against various analytes like staphylococcal enterotoxin b [ ] , spores of bacillus anthracis [ ] , art v glycoprotein [ ] , rotavirus nsp enterotoxin [ ] , coronavirus [ ] , clostridium botulinum neurotoxin serotype a [ ] , salmonella typhimurium [ ] , and human gonadotropin-releasing hormone promoter [ ] by phage display technology. it also has been used to design high affinity super antigens for immunochemical application and immunotherapy [ ] and to choose specific molecules to inorganic materials [ ] . in this study, we have used a peptide-phage display library to identify peptides binding to seb. seb, produced by staphylococcus aureus, is a pyrogenic toxin responsible for staphylococcal food poisoning in humans and has been an attractive choice of biological aerosol weapon due to its inherent stability and high intoxication effect [ ] . numerous immunological techniques have been applied to the detection of seb such as enzyme-linked immunosorbent assays (eli-sa) [ ] . because the traditional immunological methods are in most cases time-consuming and inconvenient, many novel and rapid immunosensors have been developed for the detection of seb based on piezoelectric crystal sensors [ , ] , surface plasmon resonance (spr) sensors [ ] [ ] [ ] , and fiber optic sensors [ ] . in this study, seb active peptide sequences were determined using phage display technique. different from the common approach, not only the affinities of selected phage clones towards seb molecule were investigated but also the interactions of clones with other molecules which can be found in real food samples were determined by phage-elisa. the affinities of selected phage clones to seb were also visualized using surface plasmon resonance system. after determining the amino acid sequences of selected peptides, the peptide with highest affinity was synthesized by solid phase peptide synthesis and the interaction of peptide with seb molecule was studied by isothermal titration calorimetry (itc). the binding constant (k) and thermodynamic binding parameters (dh, dg) were determined for the interactions of peptide-seb and antibody-seb. culture and media conditions. m phage display (phd) library and escherichia coli er , used as the host organism, were purchased from new england biolabs inc. (ipswich, ma, usa). e. coli was grown in lb medium (merck kgaa, darmstadt, germany) to determine the number of the phage titers, different dilutions of phage clones were mixed with top agar ( g tryptone, g yeast extract, g nacl, g mgcl h o, and g agarose in l di water) and were spreaded on to lb/iptg/x-gal agar. monoclonal m antibody (ge healthcare, uk) labeled with hrp (horseradish peroxidase) and polyclonal seb antibody were obtained from abcam plc. phage display protocol. a commercial m phage display (phd) library containing .  pfu ml À and has .  transformants was used to screen against seb. target molecule seb was prepared in elisa plates for screening. the plates were prepared by incubating lg ml À seb. after an overnight incubation unbound and weakly bound seb molecules were removed from the elisa plate well surface by discarding the solution in the wells. ten microliters of aliquots from phd library was added on immobilized seb molecules. after h incubation unbound and weakly bound phages were removed by washing with tbst ( . % tween ) buffer. bound phages were eluted from the seb surface by using glycine-hcl solution (ph . ). to neutralize the well content m tris-hcl (ph . ) was used. eluted phage clones were amplified by infecting liquid e. coli culture. the amplified phage clones were separated by centrifugation at ( , rpm, °c, for min). separated phage clones were purified and concentrated with peg/nacl. after incubation the phage clones were pelleted by centrifugation ( , rpm, °c, for min). the phage pellet was dissolved in . % nan in tbs buffer. stock solutions of the phage clones were prepared in % glycerol. phage-elisa protocol. phage clones from the screening were quantitatively tested for their binding affinity using phage-elisa. phage clones were incubated in seb coated elisa plates for . h. unbound and weakly bound phages were removed by washing with tbst ( . % tween ) buffer. hrp-conjugated m antibody was brought in contact with phage-seb complex and then the antibody was incubated for . h. following the incubation none specifically bound antibody conjugates were removed by washing six times with tbst. later ml abst stock solution was mixed with ll h o ( % v/v). two hundred microliters of this substrate solution was distributed to each well and the enzymatic reaction took place for min. the green color was measured at nm by bio-tek el microplate reader (biotek instruments, winooski, vt, usa). cross-specificity experiments. in order to check the specificity of the seb-binding peptides, bsa, b-glycosidase, whey, and hemoglobin were tested as target molecules for selected phage clones. for screening the affinity of clones, elisa plate wells were coated with these target molecules ( lg ml À ) and incubated for over night. phage clones were added to target molecules and incubated for . h. unbound clones were removed by washing six times with tbst ( . % tween ) buffer. phage-elisa protocol described above was followed to quantify the binding affinity of seb-binding peptides towards different proteins. surface plasmon resonance (spr) sensor measurements. the affinity of selected phage clones to seb was further analyzed by spr sensor. spreeta tm sensors (texas instruments, dallas, tx) were used for spr experiments. spreeta tm sensors are capable of monitoring ri changes between . and . refractive index units (ri) and the resolution of the system is approximately  À ri. the spr system is comprised of a spr sensor, a -channel flow cell and -bit dsp electronic control box. goldman syringe pump (biasis ltd. sti., ankara, turkey) controls the flow of reagents into the flow cell at a rate of ll min À . the changes in response unit (ru) ( ru = À ri) with respect to time are plotted in the form of sensorgrams. first, a baseline was established by injecting pbs ( . m, ph . ) to the sensor surface. following the pbs injection, ll of seb ( . mg ml À ) in pbs was injected to the flow cell and its adsorption onto the gold surface was monitored. the un-bound seb was washed off by injecting pbs and non-specific sites on the sensor surface were blocked with ll cystine ( mm) in pbs. the samples of ll containing selected phage clones ( pfu ml À ) suspended in pbs were injected to the two sensing flow cells and pbs was injected to the control flow cell. the unbound phage clones were washed with pbs and the adsorption of the phage clones to the seb immobilized sensor surface was monitored. binding of phage clones to the sensor surface was marked by the corresponding ru changes. the net response was calculated by subtracting signals of sensing and control channels. dna sequencing. phage particles purification, concentration and dna isolation were carried out according to sambrook et al. [ ] . sequencing analysis were done using by beckmann coulter ceq tm cycle sequencer with À sequencing primer (new england biolab, -gccctcatagttagcgtaacg- ) and -cagggatag caagcccaata- . isothermal titration calorimetry (itc) measurements. after dna sequencing, the peptide of phage clone number was synthesized by solid phase peptide synthesis (genscript corp., piscataway, nj, usa). itc was used to determine the thermodynamic properties of peptide-seb interaction. itc experiments were performed at °c using a tam iii microcalorimeter (ta instruments, new castle, delaware, usa). before analysis, the itc calibration was performed [ ] by diluting aqueous sucrose solution (sigma co., st. louis, mo). the dh of dilution determined was within the acceptable limits, confirming that the instrument was functioning properly. peptide and seb were suspended in pbs ( . m, ph . ) and the reference ampoule was filled with pbs solution. peptide solution ( . mm) was placed in ll syringe and ll of seb solution ( . mm) was placed in the sample ampoule. the peptide solution was injected into the stirred sample ampoule containing seb solution in injections ( ll/injection) by a computer-controlled pump. heat released with each injection was measured by the calorimeter in dynamic correction mode. blank experiments were performed by titrating peptide solution into pbs and pbs into seb solution. in order to determine the thermodynamic constants of antibody-seb interaction, seb solution ( . mm) was placed in syringe and ll of anti-seb solution ( . mm) was placed in the sample ampoule. the seb solution was injected into the sample ampoule in injections ( ll/injection). binding constants (k), the change in enthalpy (dh), the change in free energy (dg) and the change in entropy (ds) were determined by using the software package of tam assistant supplied by ta instruments. binding stoichiometry was estimated as : for both two interactions. toxins, which cause intoxications and accepted as bioterrorism agents, were usually detected by microbiological techniques. because those conventional techniques are comparatively time-consuming, lots of novel methods were developed to detect toxins in a much more rapid and easier way [ ] . in this study, phage-display technique was used to select the peptides which can be used as the recognition agents in the detection of staphylococcal enterotoxin b (seb). phage clones from the third round of biopanning were isolated and affinities of peptides to seb were determined by elisa. the results (fig. ) show that of phage clones were positive ones. phage clone number , , and showed higher seb-binding affinity and were chosen for further analysis. the specificity of the selected phage clones to seb was tested by elisa using different proteins found in food matrices frequently. the affinities of phage clones , , and to seb, albumin, whey, hemoglobin, and b-glycosidase were determined (fig. ) . phage clone showed minimal cross reactivity with other proteins when compared with seb. for phage clones and , significant cross reactivity was observed with hemoglobin and b-glycosidase. none of the phage clones bound significantly to the bovine serum albumin (bsa) and whey, although bsa is a highly hydrophobic protein. in order to investigate the affinities of selected phage clones to seb, phage clones were allowed to bind to the seb immobilized on the sensor surface. fig. a shows the immobilization of seb molecule via physical adsorption and blocking the surface by cystine. response unit (ru) was increased during the adsorption of seb and a small decrease in signal was observed after washing with pbs due to the removal of loosely bound molecules. cystine solution was used for blocking the non-specific sites on the sensor surface and a remarkable ru change was observed due to the binding of cystine molecules to the sensor surface. phage clones (phage , phage , and phage ), selected by phage-elisa technique, were injected to the sensor surface on which seb was immobilized and the change in the ru induced by the binding of phage clones is shown in fig. b . a phage clone (phage ), which showed low affinity to the seb in phage-elisa, was also injected to the sensor surface in order to investigate the selectivity of the spr sensor. the concentrations of all phage clones used in spr measurements were kept constant at pfu ml À . following the injection of phage clones, ru signal increased due to the refractive index difference between pbs and phage solution. during the period of adsorption, ru of phage clones and increased while the signal of phage clones and did not change significantly, although the concentrations of phage solutions were same. this shows the high affinity of phage clones and to the seb molecule, because when phages bind to the seb molecule, they get closer to the surface of the sensor which increases the ru. the ru difference before the injection of phage solution and after washing with pbs indicates the amount of phage clones bound to the sensor surface. the change in ru induced by phage clone was highest, followed by phage clones and which were similar to the results of phage-elisa. the change in response for non-binding phage clone ( ) was too small which shows the specificity of the spr sensor. the nucleotide sequences of the random regions of seb-binding phage clones (phage clone number , , and ) and weakly or non-binding phage clones (phage clone number and ) were determined. the nucleotide sequences and corresponding amino acid sequences of those phage clones were reported in table . the random regions of seb-binding phage clones do not share any consensus sequence. this indicates that seb-binding phage clones show affinity to different epitopes of the seb molecule. while phage clone is rich in glutamine, phage clone is rich in proline. detection of thermodynamic parameters for peptide-seb interaction by itc provides valuable information about the binding of peptide to seb. raw itc profiles obtained by the titration of peptide to the seb solution and the corrected injection heats are shown in fig. . the interaction between peptide-seb was exothermic with a binding constant (k) of . ± .  m À . an enthalpy change (dh) of - . ± . kj mol À and free energy change (dg) of À . kj mol - was obtained from the itc data. the interaction between antibody-seb was also exothermic with a higher binding constant (k = . ± .  m À ). the dh and dg values for antibody-seb interaction were À ± kj mol À and À . kj mol À , respectively. the interactions were accompanied by a large change in enthalpy and a large negative value in dg represents a high affinity [ ] . although the binding constant of the peptide is lower than that of the antibody, it still indicates a strong binding to seb molecule. by using phage display technique, three peptides with high affinities to seb molecule were selected. as the result of crossspecificity test, although there were significant amount of phages clones bound non-specifically to the other protein molecules, the selected phage clones showed higher affinity to seb than the others. the non-specific binding of phage clones may be due to the hydrophobic regions in the phage coat protein. the spr sensorgrams show that, phage and phage bind to seb with a strong affinity. after dna sequencing, the peptide of the phage clone with highest affinity was synthesized and the binding constant of peptide to seb was determined by itc. the results indicate that the selected peptides can be used as recognizing agents in biosensors. in addition to their high affinity, the small size they have, will be an important advantage in enhancing the sensitivity of biosensors like spr. in further studies, the selected peptides will be combined with spr sensors allowing the detection of seb with a sensitivity comparing favorably to that of immunosensors. filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface molecular biomimetics: nanotechnology through biology phage display phage display for detection of biological threat agents detection of biological threats. a challenge for directed molecular evolution phage-displayed peptides as biosensor reagents species-specific peptide ligands for the detection of bacillus anthracis spores production of recombinant single-chain fv antibody fragments specific for the hydroxyproline-rich domain of natural art v by phage display single-chain variable fragment (scfv) antibodies against rotavirus nsp enterotoxin generated by phage display identification of singlechain antibody fragments specific against sars-associated coronavirus from phage-displayed antibody library a cyclic peptide-polymer probe for the detection of clostridium botulinum neurotoxin serotype a biomagnetic separation of salmonella typhimurium with high affine and specific ligand peptides isolated by phage display technique selection and identification of human gonadotropin-releasing hormone promoter binding peptides by phage display-cemsa engineering high affinity superantigens by phage display trends in detection of warfare agents: detection methods for ricin, staphylococcal enterotoxin b and t- toxin highly sensitive fluorogenic enzymelinked immunosorbent assay: detection of staphylococcal enterotoxin b detection of staphylococcal enterotoxin b employing a piezoelectric crystal immunosensor piezoelectric crystal immunosensor for the detection of staphylococcal enterotoxin b detection of staphylococcus aureus enterotoxin b at femtomolar levels with a miniature integrated two-channel surface plasmon resonance (spr) sensor a miniature fiber optic surface plasmon resonance sensor for fast detection of staphylococcal enterotoxin b spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin b in milk detecting staphylococcal enterotoxin b using an automated fiber optic biosensor molecular cloning-a laboratory manual standards in isothermal microcalorimetry (iupac technical report) thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes this work was supported by a grant project k of republic of turkey prime ministry state planning organization (dpt). esra acar soykut, one of the authors of article, was financially supported by the scientific and technological research council of turkey (tubitak-bideb). key: cord- - xtiiejf authors: wang, wen-hung; takeuchi, rikiya; jain, shu-huei; jiang, yong-huang; watanuki, sonoko; ohtaki, yoshiharu; nakaishi, kazunari; watabe, satoshi; lu, po-liang; ito, etsuro title: a novel, rapid (within hours) culture-free diagnostic method for detecting live mycobacterium tuberculosis with high sensitivity date: - - journal: ebiomedicine doi: . /j.ebiom. . sha: doc_id: cord_uid: xtiiejf background: nucleic acid amplification tests (naats) are widely used to diagnose tuberculosis (tb), but cannot discriminate live bacilli from dead bacilli. live bacilli can be isolated by culture methods, but this is time-consuming. we developed a de novo tb diagnostic method that detects only live bacilli with high sensitivity within hours. methods: a prospective study was performed in taiwan from to . sputum was collected consecutively from patients with suspected tb infection. the sputum was pretreated and heated at °c for h to induce the secretion of mpt protein from live mycobacterium tuberculosis. mpt was detected with our ultrasensitive enzyme-linked immunosorbent assay (elisa) coupled with thionicotinamide-adenine dinucleotide (thio-nad) cycling. we compared our data with those obtained using a culture test (mgit), a smear test (kinyoun staining), and a naat (xpert). findings: the limit of detection for mpt in our culture-free ultrasensitive elisa was . × (− ) moles/assay. when the criterion for a positive response was set as an absorbance value ≥ mabs, this value corresponded to ca. cfu/ml in the culture method – almost the same high-detection sensitivity as the culture method. to confirm that mpt is secreted from only live bacilli, m. bovis bcg was killed using μg/ml rifampicin and then heated. following this procedure, our method detected no mpt . our rapid ultra-sensitive elisa-based method required only h to complete. comparing the results of our method with those of culture tests for specimens revealed a sensitivity of . % ( / , % ci: . – . %) and a specificity of . % ( / , % ci: . – . %). the performance data were not significantly different (mcnemar's test, p = . ) from those of the xpert tests. in addition, at a ≥ + titer in the smear test, the positive predictive value of our culture-free ultrasensitive elisa tests was in a good agreement with that of the culture tests. furthermore, our culture-free ultrasensitive elisa test had better validity for drug effectiveness examination than xpert tests because our test detected only live bacilli. interpretation: our culture-free ultrasensitive elisa method detects only live tb bacilli with high sensitivity within hours, allowing for rapid diagnosis of tb and monitoring drug efficacy. funding: matching planner program from jst (vp ), the a-step program from jst (as u), waseda university grants for specific research projects ( a- and c- ), the precise measurement technology promotion foundation to e.i. culture methods remain the "gold standard" for diagnosing tuberculosis (tb), even now in the st century [ ] . culture methods can be used to isolate live mycobacterium tuberculosis, and are more sensitive than acid-fast bacilli (afb) staining (e.g., kinyoun/ziehl-neelsen staining), which detects both live and dead bacilli, and culture methods can reliably detect mycobacteria present at a concentration of about À colony forming units (cfu)/ml of specimen [ , ] . growing cultures also permit species identification and drug effectiveness testing [ ] . the major drawback of culture techniques, however, is that it takes weeks to more than a month before a positive culture for m. tuberculosis can be identified, and the methods require at least a moderately well-equipped laboratory [ ] . on the other hand, nucleic acid amplification tests (naats) including the xpert mtb/rif (cepheid, sunnyvale, ca, usa), amplicor mycobacterium tuberculosis test (roche, basel, switzerland), and loop-mediated isothermal amplification (lamp; eiken chemical, tokyo, japan), are now widely used, even in low-and middle-income countries due to financial assistance from various global foundations [ ] [ ] [ ] . it is estimated that naats can detect m. tuberculosis in suspensions containing as few as À cfu/ml, making this technology highly sensitive [ , ] . these methods require only a few hours to provide a tb diagnosis, but they also have crucial drawbacks. naats detect both live and dead m. tuberculosis bacilli [ , ] , making them inappropriate for evaluating the effectiveness of the infection to anti-tb drugs (although the xpert mtb/rif detects resistance to rifampicin) [ ] . because live and dead bacilli cannot be discriminated by naats, the tests may provide false positive results in patients with a history of tb [ ] . in addition, hemoglobin and other factors present in the dissolved sputum may retard these tests, providing false negative results [ ] . furthermore, the implementation of these naats remains difficult in resource-limited settings (e.g., they require an air-conditioned room) [ ] . taken together, a better method for diagnosing tb is strongly needed, in which only live tubercle bacilli can be examined with high-detection sensitivity within a few hours. therefore, we developed a de novo, high-detection sensitive, culture-free, same-day tb diagnostic method based on an enzyme-linked immunosorbent assay (elisa) coupled with thionicotinamide-adenine dinucleotide (thio-nad) cycling [ ] . this ultrasensitive elisa detects trace amounts of a protein, mpt , which is specifically secreted from only live m. tuberculosis when the bacilli are heated. mpt inhibits apoptosis of host macrophages by using the cascades involving an up-regulation of bcl- , an increase in mirna and a control of nf-kb [ ] . thus, mpt is an important component for live m. tuberculosis. the feasibility of detecting mpt has already been confirmed using immunochromatography [ ] , which is less sensitive than the ultrasensitive elisa. a prospective study was performed in taiwan between from september to july . sputum was collected consecutively from patients with suspected tb infection on the basis of clinical criteria at kaohsiung medical university hospital until at least specimens were reached. this project was approved by the institutional review board of kaohsiung medical university (kmuhirb-f (i)- ). the written informed consents were obtained from the patients at kaohsiung medical university hospital. thus, the sputum experiments were performed and analyzed at kaohsiung medical university hospital, whereas the protein experiments were performed and analyzed at tauns and waseda university. the sputum was directly deposited by the patient into a sample holder and stored at °c until pretreatment. sputum specimens largely contaminated by blood and bacilli other than m. tuberculosis in culture tests were not used in the present study. specimens with an insufficient volume after pretreatment were also not used. in addition, the diagnosis for tb does not need a bsl facility. sputum ( ml) was homogenized with À ml protease solution (sputazyme; kyokuto pharmaceutical industrial, tokyo, japan), and the specimen was incubated at room temperature for at least min. the specimen was then centrifuged at £ g for min, and the evidence before this study early and accurate diagnosis of infectious diseases is required to stop their spread and increase the chances for successful treatment. for tuberculosis (tb), even now the traditional timeconsuming culture method remains the "gold standard" for testing by physicians, although the world health organization (who) recommends the use a nucleic acid amplification test (naat), the xpert mtb/rif, for diagnosing tb and rifampicin resistance. the detection sensitivity of naats is high and the detection time short (rapid communication from who in january, ). the main concern regarding naats, however, is that they detect nucleic acids, which are present in both live and dead bacilli specimens, and this may lead to false positive results in tb patients treated with anti-tb drugs. to confirm the infectiousness, physicians must still use a culture method for tb diagnosis, but this takes a long time. acid-fast bacilli staining is convenient and does not take much time, but the detection specificity is low due to the interference of nontuberculous mycobacteria. therefore, a culture-free, same-day diagnostic test for tb that detects only live bacilli with high sensitivity is strongly required. when the sputum of tb patients is heated at °c for h, a specific protein, mpt , is secreted from live mycobacterium tuberculosis. therefore, we applied a new ultrasensitive elisa coupled with thio-nad cycling to detect the trace amount of mpt . when the criterion for positive responses in our culture-free ultrasensitive elisa was set to the same detection sensitivity as that of the culture method, our method succeeded in detecting live m. tuberculosis within h. we then conducted a study to compare the results of our culture-free ultrasensitive elisa with those of culture tests for specimens; the sensitivity was . % and the specificity was . %. at a smear test titer of +, the positive predictive value of our culture-free ultrasensitive elisa tests was in good agreement with that of the culture tests. our culture-free ultrasensitive elisa for tb diagnosis revealed the same detection sensitivity as the culture method, but it enabled us to diagnose tb on the same day. our culture-free elisa for mpt can be used not only for an initial diagnosis, but also to check for drug effectiveness and drug resistance when treating tb patients. if anti-tb drugs are administered to patients for a few weeks, but mpt is detected with our method, physicians would be alerted to the possibility of resistance to the tb regimen and then a further drug susceptibility testing is warranted. if anti-tb drugs are effective and mpt is not detected in the sputum from a tb patient using our method, the doctor has a chance to decide on the same day to discontinue isolation for tb. our culture-free, same-day diagnosis for tb can be useful in several situations. precipitate was collected. for further homogenization of the sputum, the precipitate was suspended in ml of a m urea solution (tauns, shizuoka, japan), and incubated at room temperature for min. then, ml of a n-acetyl-l-cysteine (nalc) solution (cc-e supplement, japan bcg laboratory, tokyo, japan) was added to the specimen. the specimen was incubated at room temperature for min. the sample was neutralized with ml phosphate buffer ( . m, ph . ) containing . % tween was added to the suspension and centrifuged at £ g for min. the precipitate obtained was washed again and suspended in ml of heat treatment buffer comprising phosphate buffer ( . m, ph . ) and % tween . this solution was heated in an aluminum block heater at °c for h, resulting in the secretion of mpt from live bacilli [ ] . in the culture-free ultrasensitive elisa tests, we detected a specific protein for m. tuberculosis, mpt [ ] , which is secreted from only live m. tuberculosis in heated sputum specimens [ ] . an ultrasensitive elisa coupled with thio-nad cycling was originally developed by watabe and ito [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, see the supplementary data for the detailed methods of our culture-free ultrasensitive elisa. to convert the units between pg/ml and moles/ assay, the molecular mass of mpt was . kda and a single assay volume was ml. to make a calibration curve, we used recombinant his-tagged mpt antigen that was produced in e. coli. culture tests were performed according to the procedures described by garcia and isenberg [ ] and lu et al. [ ] . briefly, the mycobacteria growth indicator tube (mgit; becton, dickinson and company, franklin lakes, nj, usa) and the mgit instrument (becton, dickinson and company) were used. after the mgit-positive results were found, the immunochromatographic tests using specific antibodies for mpt (sd bioline tb ag mpt rapid kit; standard diagnostics, yongin, korea) were further performed to confirm the results. the xpert mtb/rif (xpert) assay was performed according to the manufacturer's instructions (cepheid). four of the specimens were not examined. two of the samples were found to contain non-tuberculosis mycobacteria by maldi-tof, and thus we skipped the xpert experiments, because non-tuberculosis mycobacteria do not produce mpt [ , ] . the other two samples were the same as those identified as different samples, and thus we also skipped the xpert experiments for these samples. furthermore, xpert could not be applied when the sputum volume of the specimen was too low. after processing of the sputum specimens with a nalc/naoh solution, they were stained according to the auramine-rhodamine (ar) staining method and instructions of the wescor aerospray tb afb stainer & cytocentrifuge model (discovery diagnostics, clement, ontario, canada), and visualized by fluorescence microscopy. when ar staining produced positive results, the results were confirmed using kinyoun staining ( t , tonyar biotech, taoyuan, taiwan). the results of the afb smear were graded according to the american thoracic society/center for disease control and prevention (ats/cdc) [ ] . in kaohsiung medical university hospital, the four-drug regimen (isoniazid, rifampicin, pyrazinamide, and ethambutol) was applied to all tb patients as the initial therapy recommended by the world health organization (who), usa cdc, and taiwan cdc [ ] [ ] [ ] . data are expressed as mean § standard deviation (sd). the limit of detection (lod) was estimated from the mean of the blank and the £ sd of the blank [ ] . here, the blank was measured with the heat treatment buffer only. furthermore, we attempted to find the measured lod using the low concentration of mpt . the limit of quantification (loq) was estimated by the same method as used for the lod, but with the £ sd of the blank. the % confidence interval was calculated by a clopper-pearson exact confidence interval. mcnemar's test and cochran-armitage test were performed using r version . . (http://www.r-project.org). in-vitro tests of culture-free ultrasensitive elisa tests . . . limit of detection of culture-free ultrasensitive elisa tests when we used mpt antigen and the heat treatment buffer described in the methods section, the ultrasensitive elisa coupled with thio-nad cycling yielded a linear calibration curve (y = . x + . , r = . ) in the range of À . pg/ml (fig. ) . this curve was obtained from the absorbance of the accumulated thio-nadh at a cycling reaction time of min. the statistically estimated lod was . pg/ml, corresponding to . £ À moles/ assay. this value was estimated from the mean of the blank and the £ sd of the blank as described in the methods section. furthermore, we obtained the observed lod, when we noticed that the mean of the blank and the £ sd of the blank was mabs (data not shown). in this case, the values exceeding mabs were obtained at the ratio of % ( / measurements) when we used . pg/ml of mpt antigen, and those were obtained at the ratio of % ( / measurements) when we used . pg/ml. if we set mabs as the cutoff value, which was obtained for the culture-free ultrasensitive elisa test (see below), the values exceeding mabs were obtained at the ratio of % ( / measurements) when we used . pg/ml of mpt antigen, and those were obtained at the ratio of % ( / measurements) when we used . pg/ml. therefore, we concluded that the observed lod was . pg/ml, corresponding to . £ À moles/assay. on the other hand, the statistically estimated loq was . pg/ml, corresponding to ca. . £ À moles/ assay. the coefficient of variation (cv), calculated from three replicated measurements, for . pg/ml of mpt antigen (i.e., the statistically estimated lod) was %; that for . pg/ml of mpt antigen (i.e., the observed lod) was %; and that for . pg/ml of mpt antigen (i.e., the statistically estimated loq) was %. using specimens obtained from clinical trials (see supplementary fig. and supplementary table ) , we compared the results of our culture-free ultrasensitive elisa tests and those of culture tests and produced a receiver operating characteristic (roc) curve (see supplementary fig. ). the roc curve revealed a cutoff value that was equivalent to mabs for the culture-free ultrasensitive elisa test. the data obtained from the roc curve revealed that mabs corresponded to a bcg value of cfu/ml in culture tests (fig. ) . we thus set the cut-off value at mabs in our culture-free ultrasensitive elisa tests for clinical trials. to confirm that mpt is secreted from only live bacilli, bcg was killed by rifampicin and then heated. we then measured the mpt concentration in the bcg culture medium. rifampicin at a dose of mg/ml is sufficient to kill m. tuberculosis [ ] . when we applied mg/ml rifampicin to £ cfu of bcg for h and then heated the bacilli at °c for h, the mpt concentration in the medium was À § pg/ml (mean § sd, n = ). thus, no mpt was secreted from bcg treated with rifampicin. on the other hand, when the bcg was not incubated with rifampicin for h and was heated, the mpt concentration in the culture medium was § pg/ml (mean § sd, n = ). that is, if the bacilli are killed by anti-tb drugs, the bacilli do not secrete mpt even when they are heated. clinical trials of culture-free ultrasensitive elisa tests . . . sensitivity and specificity of the culture-free ultrasensitive elisa test and smear test in comparison with the "gold-standard" culture test we used specimens in this experiment (see supplementary fig. ) . these specimens included different specimens obtained from the same patients on different dates. among these specimens, were examined before anti-tb drug administration, were examined during drug therapy, and were from patients with an unconfirmed medication history. when we compared the results of our culture-free ultrasensitive elisa test with those of culture tests using the patient sputum, the sensitivity was . % ( / , % ci: at smear titers of +, +, and +, the results of the culture tests were the same as those of the culture-free ultrasensitive elisa tests ( table ) . even if including the results of smear tests with a titer of +, the positive predictive value of the culture-free ultrasensitive elisa tests was in good agreement with that of the culture tests. for all the smear-positive specimens, the positive predictive value of culturefree ultrasensitive elisa tests was . %. we analyzed the data of table after stratifying them into smearpositive specimens and smear-negative specimens ( as described in the methods section, the number of xpert tests was different from that of culture-free ultrasensitive elisa tests due to technical issues. we thus used specimens in this experiment. fig. . cut-off value for culture-free ultrasensitive elisa tests. because the most suitable value obtained from the roc curve was mabs, its corresponding value for bcg was cfu/ml. n = each. table ). the accuracy was . %, with a positive predictive value of . % and a negative predictive value of . % (table ) . further, the relation was analyzed between the positive degrees in the results of xpert tests and those of culture-free ultrasensitive elisa tests (see supplementary fig. ). the positive correlation was found (p < . by cochran-armitage test). in this section, we compared the results of three different diagnostic methods (i.e., culture-free ultrasensitive elisa test, the smear test, and the xpert test) and the culture results for samples from patients receiving anti-tb drugs. the anti-tb treatment may result in the patients' sputa containing both live and dead bacilli. that is, the smear and xpert methods may detect dead bacilli, but the ultrasensitive elisa detects only live bacilli. the number of specimens in this experiment was and the results are presented in table . the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value for the three different diagnostic methods are listed in comparison with the culture results (table ) . when anti-tb drugs were administered to the patients, the specificity of our culture-free ultrasensitive elisa tests was much better than those of the smear and xpert tests, indicating that the smear and xpert tests detected not only live bacilli, but also dead bacilli. in the present study, we evaluated the performance of a novel culture free ultrasensitive elisa method for detecting live tubercle bacilli in sputum specimens. the sensitivity of our test was . % ( / , % ci: . À . %) and the specificity was . % ( / , % ci: . À . %). the performance of the new method was better than that of acid-fast staining and not significantly different from that of the xpert test. in addition, when the smear test titer was +, the positive predictive value of our culture-free ultrasensitive elisa test was in a good agreement with that of the culture test. furthermore, when the results of our culture-free ultrasensitive elisa test were compared with those of the culture test using sputum obtained from patients treated with anti-tb drugs, the positive predictive value was sufficiently high ( . %). given the advancements in diagnostic methods in the st century, it is remarkable that the culture method is still used as the gold standard for tb diagnosis. we believe this is because culture methods can detect live tubercle bacilli, and exclude dead bacilli, with the high detection-sensitivity required for tb diagnosis. the naats for tb, including the xpert mtb/rif ultra, recently set new records for sensitivity and specificity and produced the most reliable results for rifampicin resistance [ , ] . these methods, however, detect both live and dead bacilli, resulting in increased sensitivity due to the measurement of dead bacilli and false positives after tb treatment, because the dna of the dead bacilli can still be detected [ ] . the presence of smear-negative and naat-positive findings are currently thought to be due to differences in the sensitivity between the sputum smear and naats [ ] , but this issue requires careful re-examination. when the opposite occurs, however, i.e., the sputum smear is positive for afb and the naat is negative for m. tuberculosis dna, it produces a diagnostic dilemma as it is not clear whether the anti-tb treatments are effective [ ] . this emphasizes the need for a diagnostic method that detects only live bacilli. some other new technologies for tb diagnosis have recently emerged, such as surface plasmon resonance spectroscopy [ ] , immuno-pcr (a combination of elisa and pcr) [ , ] , voltammetric assay [ , ] , aptasensor [ , ] , immuno-nanosensor [ ] , and electrochemiluminescence immunoassay [ ] . these methods are also applied to secretory proteins (i.e., antigens) as tb markers, but culture, which takes time, is still required to obtain adequate amounts of antigen [ ] . we used heat treatment to induce the living tubercle bacilli in sputum specimens to secrete the tb biomarker mpt [ ] , and applied the ultrasensitive elisa technique [ ] to detect the mpt [ ] , therefore, only our method can detect live tubercle bacilli within a single day. more recently, a molecular bacterial load assay (mbla) has been reported as a molecular test for detection of live m. tuberculosis bacilli. it is a reverse transcriptase quantitative pcr that quantifies the m. tuberculosis load from patient sputum using the s rrna gene as a reference gene [ ] . we have not yet attempted to use mbla and thus still cannot compare the results between mbla and our ultrasensitive elisa. however, it is noted that mbla is a time-consuming method because they have two complicated processes: ( ) rna extraction and purification and ( ) the use of real-time pcr [ ] . two mpt gene mutations in the m. tuberculosis complex are reported to interfere with the detection of mpt with the anti-mpt antibodies used in the present study [ ] . one isolate from the m. tuberculosis complex had a deletion of bp from nucleotides to (amino acids position À ) and the other isolate had a deletion of bp from nucleotide in rv to nucleotide [ , ] . it is certainly a limitation of the novel detection method, but the number of unidentifiable strains is very small. the xpert mtb/rif assay was recommended by both who in and [ , ] and us food and drug administration in [ ] . the test procedure is applied directly to clinical specimens, either raw sputum specimens or sputum pellets created after decontaminating and concentrating the sputum [ ] . as shown in our study, the sensitivity of the xpert mtb/rif is very good ( %, table ). two articles by chakravorty et al. and dorman et al., however, demonstrated that lower sensitivity were obtained from smear-negative and culture-positive specimens (for xpert mtb/rif, % and % [ ] , and for xpert mtb/rif ultra, % and % [ ] , respectively). the culture-free ultrasensitive elisa tests showed that the sensitivity for the smear-negative and culture-positive specimens was % ( / ; table ). that is, the sensitivity is almost the same between the xpert mtb/rif ultra tests and the culture-free ultrasensitive elisa tests. besides the similar performance of the culture-free ultrasensitive elisa method and xpert molecular tests, the culture-free ultrasensitive elisa test is useful for monitoring the treatment response because this method detects only live bacilli [ ] . when tuberculosis cases are under effective drug therapy, the culture-free ultrasensitive elisa test may reveal negative results, whereas the molecular method might detect dna from dead bacilli. this feature of the ultrasensitive elisa test is useful for monitoring patients' treatment responses. in the near future, we should consider more the following two points. one is that our culture-free ultrasensitive elisa test depends on the quality of sputum obtained from patients. that is, the effect of blood in specimens on the tests should be excluded in our test. the other is that the examination of drug effectiveness should be promoted more [ ] . the studies according to a new design of drug monitoring for patients will be achieved. we conclude that our culture-free ultrasensitive elisa method is important for diagnosing tb and evaluating drug effectiveness, because this method detects only live bacilli without any cultures. to our knowledge, this is the first study to present a de novo, same-day (only h) diagnostic method for tb with a very high limit of detection. in the near future, we will prepare an automated apparatus to achieve our method for the practical use. anonymized data used for analysis in this study is available upon request from the corresponding author. ei had full access to all the data and pl is the guarantor. kn, sw, pl, and ei designed the study. ww, rt, sj, yj, sw, and yo performed the experiments. ww, rt, yj, kn, sw, pl, and ei analyzed the data. all authors interpreted data, drafted, and reviewed the final manuscript. all authors approved the submitted manuscript. ei had full access to all the data and pl is the guarantor. pl and ei received research funds from tauns laboratories, inc. rt, yj, sw, yo, kn, and sw are employees of tauns laboratories, inc. the other authors declare no conflict of interest. evaluation of mycotube, a modified version of lowenstein-jensen (lj) medium, for efficient recovery of mycobacterium tuberculosis (mtb) diagnosis of tuberculosis culture and molecular method for detection of mycobacterium tuberculosis complex and mycobacterium avium subsp. paratuberculosis in milk and dairy products drug resistance profile of mycobacterium tuberculosis and predictors associated with the development of drug resistance comparison of culture, microscopic smear and molecular methods in diagnosis of tuberculosis clinical value of is -based loopmediated isothermal amplification for detection of mycobacterium tuberculosis complex in respiratory specimens comparison of amplicor and genexpert mtb/ rif tests for diagnosis of tuberculous meningitis diagnostic accuracy of tb-lamp for pulmonary tuberculosis: a systematic review and meta-analysis rapid detection of mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology the new xpert mtb/rif ultra: improving detection of mycobacterium tuberculosis and resistance to rifampin in an assay suitable for point-of-care testing diagnosis of tuberculosis by amplicor mycobacterium tuberculosis test: a multicenter study comparison of a realtime polymerase chain reaction-based system and erlich-ziehl-neelsen method with culture in the identification of mycobacterium tuberculosis time to treatment for rifampicin-resistant tuberculosis: systematic review and meta-analysis evaluation of amplicor pcr for direct detection of mycobacterium tuberculosis from sputum specimens advances in tuberculosis diagnostics: the xpert mtb/rif assay and future prospects for a point-of-care test ultrasensitive enzyme-linked immunosorbent assay (elisa) of proteins by combination with the thio-nad cycling method mpt protein from mycobacterium tuberculosis inhibits apoptosis of macrophages through nf-kb-mirna -bcl- pathway immunochromatographic detection of mpb secreted from active bcg by heating: toward same-day diagnosis of tuberculosis properties of proteins mpb , mpb , and mpb of mycobacterium bovis bcg ultrasensitive detection of proteins and sugars at single-cell level immunoreactive insulin in diabetes mellitus patient sera detected by ultrasensitive elisa with thio-nad cycling subattomole detection of adiponectin in urine by ultrasensitive elisa coupled with thio-nad cycling detection of hiv- p at attomole level by ultrasensitive elisa with thio-nad cycling urinary adiponectin as a new diagnostic index for chronic kidney disease due to diabetic nephropathy ultrasensitive elisa developed for diagnosis early diagnosis with ultrasensitive elisa clinical microbiology procedures handbook evaluation of the bactec mgit system in combination with the mgit tbc identification test for detection of mycobacterium tuberculosis complex in respiratory specimens comparative evaluation of three immunochromatographic identification tests for culture confirmation of mycobacterium tuberculosis complex capilia tm tb-neo assay: a new tool for rapid distinction between tuberculous and non-tuberculous mycobacteria diagnostic standards and classification of tuberculosis in adults and children who. treatment of tuberculosis: guidelines for national programmes executive summary: official american thoracic society/centers for disease control and prevention/infectious diseases society of america clinical practice guidelines: treatment of drug-susceptible tuberculosis centers for disease control, ministry of health and welfare. th ed r. o.c. (taiwan): taiwan guidelines for tb diagnosis & treatment methods for the determination of limit of detection and limit of quantitation of the analytical methods high-dose rifampicin kills persisters, shortens treatment duration, and reduces relapse rate in vitro and in vivo xpert mtb/rif ultra for detection of mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre diagnostic accuracy study performance of xpert mtb/rif ultra: a matter of dead or alive xpert Ò mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults sputum smear-positive, xpert Ò mtb/rif-negative results: magnitude and treatment outcomes of patients in myanmar ultrasensitive immunosensing of tuberculosis cfp- based on spr spectroscopy development of an ultrasensitive polymerase chain reaction-amplified immunoassay based on mycobacterial rd antigens: implications for the serodiagnosis of tuberculosis quantitative detection of a cocktail of mycobacterial mpt and psts in tuberculosis patients by real-time immuno-pcr voltammetric immunoassay for mycobacterium tuberculosis secretory protein mpt based on a synergistic amplification strategy using rolling circle amplification and a gold electrode modified with graphene oxide, fe o and pt nanoparticles dual-aptamer-based voltammetric biosensor for the mycobacterium tuberculosis antigen mpt by using a gold electrode modified with a peroxidase loaded composite consisting of gold nanoparticles and a zr(iv)/ terephthalate metal-organic framework fullerene-doped polyaniline as new redox nanoprobe and catalyst in electrochemical aptasensor for ultrasensitive detection of mycobacterium tuberculosis mpt antigen in human serum selection, characterization, and application of dna aptamers for detection of mycobacterium tuberculosis secreted protein mpt immuno nanosensor for the ultrasensitive naked eye detection of tuberculosis sensitive electrochemiluminescence (ecl) immunoassays for detecting lipoarabinomannan (lam) and esat- in urine and serum from tuberculosis patients commercial mpt -based tests for rapid identification of mycobacterium tuberculosis complex: a meta-analysis proposal of de novo antigen test for covid- : ultrasensitive detection of spike proteins of sars-cov- molecular bacterial load assay concurs with culture on naoh-induced loss of mycobacterium tuberculosis viability mutations including is insertion in the gene encoding the mpb protein of capilia tb-negative mycobacterium tuberculosis isolates xpert mtb/rif assay for the diagnosis of pulmonary and extrapulmonary tb in adults and children who. rapid communication: molecular assays as initial tests for the diagnosis of tuberculosis and rifampicin resistance device classification under section (f)( )(de novo) evaluation of the analytical performance of the xpert mtb/rif assay ultrasensitive enzyme-linked immunosorbent assay for the detection of mpt secretory antigen to evaluate mycobacterium tuberculosis viability in sputum this study was supported by matching planner program from jst (vp ), the a-step program from jst (as u), waseda university grants for specific research projects ( a- and c- ), the precise measurement technology promotion foundation to e.i. the funder had no role in the interpretation of the data or in the decision to submit the manuscript for publication. supplementary material associated with this article can be found, in the online version, at doi: . /j.ebiom. . . key: cord- -lku cm authors: edwards, s.; chasey, d.; napthine, p.; banks, j.; hewitt-taylor, c.; cranage, m. p. title: a comparison of three rapid diagnostic methods for the detection of rotavirus infection in calves date: - - journal: veterinary microbiology doi: . / - ( ) - sha: doc_id: cord_uid: lku cm abstract three techniques for the detection of rotavirus in faecal samples from calves with neonatal gastroenteritis were compared. a preliminary study indicated that reverse passive haemagglutination (rpha) was at least as sensitive as the enzyme-linked immunosorbent assay (elisa). these two immunoassays were compared with the detection of viral rna by polyacrylamide gel electrophoresis (page) on field samples. of the samples in which at least one test gave a positive result, were positive by both rpha and page, but only were also positive by elisa, indicating a lower sensitivity for the latter test. the overall agreement between rpha and page was %. the reasons for the discrepancies between the tests are discussed. rotavirus plays a major role in the neonatal calf diarrhoea complex (mcnulty, ) , and its importance is reflected in the number of diagnostic tests available. since infected animals shed very large quantities of virus particles and associated antigenic material in the faeces, electron microscopy (almeida, ) and rapid immunoassays (reynolds, ; yolken, ) have proved particularly appropriate techniques for virus detection. the most widely exploited immunoassay is the enzyme-linked immunosorbent assay (elisa), and this has been used at the central veterinary laboratory (cvl) for the routine diagnosis of bovine rotavirus infection since . a different type of test, which identifies rotaviral nucleic acid examined by polyacrylamide gel electrophoresis, has been described recently (herring et al., ) , and preliminary studies at the cvl with this sensitive and specific method indicated that the elisa was failing to detect some of the positive samples. the present study was initiated to investigate the problem further and to explore the potential of the alternative reverse passive haemagglutination assay (rpha). a polyclonal antibody preparation to a field strain of bovine rotavirus was made by immunising a laying hen with partially purified virus and harvesting the egg yolk antibodies by the method of jensenius et al. ( ) . the yolk proteins were partitioned using dextran sulphate ( . mm), and the igg was precipitated from the aqueous fraction by % ammonium sulphate. the igg was resuspended in, and dialysed against, the appropriate buffer for the subsequent assays. murine monoclonal antibody a m (beards et al., ) to the rotavirus group antigen was kindly provided by dr t.h. flewett, who collaborating centre for reference and research on rotavirus, east birmingham hospital, birmingham, england. the elisa was developed from the method of reynolds et al. ( ) using the hen antibody both for antigen capture and, conjugated with horseradish peroxidase, as the detector system. the chromogen was o-phenylene diamine. specificity of the reaction was monitored by adding the samples to wells coated with antibody lacking any detectable anti-rotavirus activity. this was prepared from eggs from the same hen, collected prior to the rotavirus immunization. samples were classified as positive if the colour in the test well (coated with virus-specific antibody) was at least . absorbance units more intense than in the control well. the rpha was carried out as described by cranage et al. ( ) using antibody linked by chromic chloride treatment to chymotrypsin-treated sheep erythrocytes. faecal suspensions of approximately / dilution, prepared for the elisa in phosphate buffered saline (pbs) with . % tween , were further diluted / in pbs, then mixed with an equal volume of % washed sheep erythrocyte suspension. the final faecal dilution was thus / . the mixture was left to settle overnight at °c for the absorption of non-specific haemagglutinating activity from the samples. this procedure was adopted for working convenience, and shorter absorptions (e.g. min at °c) have also been found satisfactory. serial doubling dilutions of the supernatant were made in round bottom -well microtitration plates. equal volumes ( or pl) of % red cells coupled to antibody were added to all wells, and the haemagglutination reaction was read after -- h at room temperature. non-specific haemagglutination was monitored by the use of red cells coupled to rotavirus-negative antibody. this was either the pre-inoculation egg antibody or, where the monoclonal antibody was used as the detecting antibody, normal mouse igg. polyacrylamide gel electrophoresis (page) was performed essentially as described by herring et al. ( ) . viral double-stranded rna was extracted from faeces with phenol-chloroform and run for h on % polyacrylamide slab gels, in a voltage gradient of approximately v cm - . the nucleic acid bands in the gels were subsequently made visible by silver staining. a strong positive control was run on each gel and test samples were recorded as positive if the characteristic ll-segment rotavirus electropherotype could be detected visually. the samples examined were bovine faeces submitted to the central veterinary laboratory through the veterinary investigation service for the routine diagnosis of neonatal gastroenteritis. in a preliminary study, samples, collected during , were examined by elisa and rpha. a second batch consisted of all bovine faeces samples received during a -week period in march , amounting to samples after exclusion of those for which insufficient material was available for all three tests. in each case, the person interpreting the test was unaware of the results of the other two tests. the results of the preliminary comparison on samples between elisa and rpha are shown in table i . rpha detected all nine elisa-positive samples, using either monoclonal antibody a m or polyclonal hen antibody. one of the elisa-negative samples (no. ) gave a weak positive reaction in the rpha with both monoclonal and polyclonal antibodies, and the reaction could be blocked with rabbit anti-rotavirus serum. the conclusion that this was a specific reaction was supported by the fact that this sample was from the same outbreak as sample no. , which was clearly positive for rotavirus. the results of the three-way comparison between elisa, rpha (both with hen antibody) and page are shown in table ii . it became apparent during the study that non-specific haemagglutination was leading to confusion in interpreting the results of the rpha with a number of faeces samples. a positive result for rpha is recorded in table ii only where there was complete agglutination of the anti-rotavirus-coated red cells at least ~> / dilution from the faeces, with no agglutination at the same dilution with the control red cells. the agreement between rpha and page was particularly good; thus samples were positive by page and by rpha, although the two methods disagreed on seven samples. elisa detected only of the samples which were positive by either or both of the other tests. for the samples where a result was obtained from all three tests, the overall agreement between rpha and page was %, rpha and elisa %, and page and elisa %. where sufficient sample was available, discrepant results were further investigated by repeat tests, some using monoclonal antibody a m in the rpha, and electron microscopy. the elisa failed to detect samples total *unable to read rpha due to non-specific haemagglutination. which were positive by the other two tests, and gave a weak reaction in one case which was negative by rpha and page. a repeat page test on this sample was negative and the elisa was interpreted as a false-positive. two samples negative by elisa and page gave relatively strong positive reactions with both polyclonal and monoclonal antibody in the rpha. repeat tests by page were negative but the finding by electron microscopy of very small numbers of rotavirus particles in one of the two samples indicated that the low level of virus in this case was not detectable in the present gel system. rotavirus particles were not identified in the second sample and the reason for the discrepancy was not clear. one sample positive in both serological tests was consistently negative by page, but insufficient material was available for further analysis. four samples were negative by elisa and rpha but clearly positive by page. three of the genome profiles obtained were very weak and repeat tests, in which bands could not be detected, would have been scored as negative. the fourth sample in this group was examined by electron microscopy and contained large numbers of clumped rotavirus particles exhibiting poor morphology. this appearance was consistent with the presence of naturally occurring rotavirus-specific antibody which could have accounted for the negative results in both the rpha and elisa tests. an important, but small, group of samples (negative in page and elisa tests) could not be read by rpha due to non-specific agglutination. all three tests described for rotavirus detection can be effectively used at a centralised laboratory where samples can be processed in batches. the simple sandwich elisa in this study was markedly less sensitive than the rpha even though it used the same antibody preparation that was used for coating the red cells. the use in rpha of monoclonal antibody appeared to offer no significant increase in specificity or sensitivity. the sensitivity of elisa reactions can be enhanced by amplification steps in the detection system, for exampte by using the biotin avidin system (yolken et al., ) but this would be at the expense of technical and manipulative complexity with corresponding opportunities for error. page is a sensitive and relatively straightforward technique, which compares well with negative stain electron microscopy (d. chasey, unpublished observations) often taken as the standard method by which alternative assays are calibrated. since page depends on the identification of a characteristic and distinctive rotavirus electropherotype, false-positive results are unlikely. this feature, and the non-dependence on antigenicity, also enables the test to detect atypical rotavirus groups which, although uncommon in cattle, may become increasingly important (chasey and davies, ) . unlike the immunoassays, however, this use of page cannot be extended to other important agents involved in the neonatal diarrhoea syndrome, such as bovine coronavirus. the present results indicate that page and rpha have a similar sensitivity for the detection of rotavirus in faecal samples, although neither test appears to detect reliably all positive samples when low levels of virus are present. if used for screening to establish patterns of infection in groups of calves it is likely that either test would give satisfactory results. rpha requires a measure of finesse in the coupling of antibody to the erythrocytes. as the coupled cells can be stabilised with glutaraldehyde (cranage et al., ) the coupling could be carried out at a centralised laboratory and cells supplied to smaller diagnostic centres as required. the test protocol is simple and rapid, and can be applied to small numbers of samples or larger batches according to demand. the major limitation of rpha is the presence of non-specific haemagglutinating factors in a number of bovine faeces samples, although the problem in this study was not as severe as reported in previous work (bradburne et al., ) . in most cases non-specific factors can be eliminated by one or more adsorptions of the samples with normal sheep red blood cells. further improvements may be achieved by the inclusion of serum (rotavirus-antibody-free) or detergents (tween ) in the test diluent (cranage et al., ) . rpha has also been applied to bovine enteric coronavirus (sato et al., ) and it should therefore be possible to establish a simple and sensitive testing protocol using rpha for the two major enteric viral pathogens of young calves. viral diagnosis by electron microscopy enzyme-linked immunosorbent assays based on polyclonal and monoclonal antibodies for rotavirus detection a solid-phase system (space) for the detection and quantification of rotavirus in faeces atypical rotaviruses in pigs and cattle glutaraldehyde stabilisation of antibody-linked erythrocytes for use in reverse passive and related haemagglutination assays detection and quantitation of rotavirus using monoclonal antibody coupled red blood cells: comparison with elisa rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in siver-stained polyacrylamide gels eggs: conveniently packaged antibodies. methods for purification of yolk igg rotavirus infections in calves development and application of elisa for veterinary diagnosis and research on enteric and other viruses evaluation of elisa and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces detection of bovine coronavirus in feces by reversed passive hemagglutination enzyme immunoassays for detecting human rotavirus use of avidin-biotin interactions with the diagnosis of viral infections we are grateful to mr d. westcott for providing the hen's egg antibodies. key: cord- -ctx vhi authors: woo, patrick cy; lau, susanna kp; tsoi, hoi-wah; chan, kwok-hung; wong, beatrice hl; che, xiao-yan; tam, victoria kp; tam, sidney cf; cheng, vincent cc; hung, ivan fn; wong, samson sy; zheng, bo-jian; guan, yi; yuen, kwok-yung title: relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: ctx vhi background: although the genome of severe acute respiratory syndrome coronavirus (sars-cov) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. methods: we cloned and purified the nucleocapsid protein and spike polypeptide of sars-cov and examined their immunogenicity with serum from patients with sars-cov pneumonia. an elisa based on recombinant nucleocapsid protein for igg detection was tested with serum from healthy blood donors who donated years previously and with serum positive for antibodies against sars-cov (by indirect immunofluorescence assay) from patients with sars-cov pneumonia. the seroprevalence of sars-cov was studied with the elisa in healthy blood donors who donated during the sars outbreak in hong kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. all positive samples were confirmed by two separate western-blot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide). findings: western-blot analysis showed that the nucleocapsid protein and spike polypeptide of sars-cov are highly immunogenic. the specificity of the igg antibody test (elisa with positive samples confirmed by the two western-blot assays) was %, and the sensitivity was · %. three of healthy blood donors who donated during the sars outbreak and one of non-pneumonic paediatric inpatients were positive for igg antibodies, confirmed by the two western-blot assays (total, · % of our study population). interpretation: our findings support the existence of subclinical or non-pneumonic sars-cov infections. such infections are more common than sars-cov pneumonia in our locality. background although the genome of severe acute respiratory syndrome coronavirus (sars-cov) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. methods we cloned and purified the nucleocapsid protein and spike polypeptide of sars-cov and examined their immunogenicity with serum from patients with sars-cov pneumonia. an elisa based on recombinant nucleocapsid protein for igg detection was tested with serum from healthy blood donors who donated years previously and with serum positive for antibodies against sars-cov (by indirect immunofluorescence assay) from patients with sars-cov pneumonia. the seroprevalence of sars-cov was studied with the elisa in healthy blood donors who donated during the sars outbreak in hong kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. all positive samples were confirmed by two separate westernblot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide). findings western-blot analysis showed that the nucleocapsid protein and spike polypeptide of sars-cov are highly immunogenic. the specificity of the igg antibody test (elisa with positive samples confirmed by the two western-blot assays) was %, and the sensitivity was · %. three of healthy blood donors who donated during the sars outbreak and one of non-pneumonic paediatric inpatients were positive for igg antibodies, confirmed by the two western-blot assays (total, · % of our study population). severe acute respiratory syndrome (sars) has now affected countries in five continents, with more than cases and more than deaths. a novel virus, the sars coronavirus (sars-cov), is known to be the aetiological agent. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the viral genome has been completely sequenced. , we have also reported the isolation of viruses resembling sars-cov from himalayan palm civets found in a live animal market in the guangdong province of china; this finding implied that animals could be a reservoir of the virus. detection of sars-cov from a non-pneumonic case in singapore (http://www.who.int/csr/don/ _ _ /en/) suggested that non-pneumonic and subclinical sars-cov infections are possible. however, extensive seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. at present, the most widely used methods for detection of antibodies against sars-cov are indirect immunofluorescence assay and elisa with cell-culture extract. , however, antibody detection by these methods is difficult to standardise and has not been compared with recombinant-antigen-based antibody-detection tests. a recent seroprevalence study, which used the indirect immunofluorescence assay for antibody detection, did not detect sars-cov antibodies in any of health-care workers from a hospital in which a sars outbreak had occurred. an approach of elisa-based antibody-detection tests using recombinant antigens with positive results confirmed by western-blot assays that use two different antigenic proteins offers higher sensitivity, specificity, and reproducibility than indirect immunofluorescence assay and elisa with cell-culture extract and is easier to standardise. [ ] [ ] [ ] [ ] [ ] [ ] in this study, we used a sensitive and specific elisa based on the highly immunogenic nucleocapsid protein of sars-cov and confirmed positive results by western-blot assays with recombinant nucleocapsid protein and recombinant spike polypeptide (another highly immunogenic protein) of sars-cov to examine the seroprevalence of non-pneumonic sars-cov in the general population, non-pneumonic patients in hospital, and health-care workers during the sars epidemic. pellet was resuspended in l dnase-free, rnase-free double-distilled water and was used as the template for rt-pcr. cloning and purification of (his) -tagged recombinant proteins to produce a fusion plasmid of the nucleocapsid protein of the sars-cov for protein purification, primers lpw and lpw (panel) were used to amplify the gene encoding this protein by rt-pcr. the sequence coding for aminoacid residues - of the nucleocapsid protein was amplified and cloned into the bamhi and ecori sites of expression vector pet- b(+) (novagen, madison, wi, usa) in frame and downstream of the series of six histidine residues. the (his) -tagged recombinant nucleocapsid protein was expressed and purified by means of the nickel-loaded hitrap chelating system (amersham pharmacia, piscataway, nj, usa) according to the manufacturer's instructions. roughly mg purified protein was routinely obtained from l escherichia coli carrying the fusion plasmid. for the spike protein of the sars-cov, primers lpw and lpw (panel) were used to amplify the gene encoding aminoacid residues - by rt-pcr. this portion of the spike protein was used because the complete protein could not be expressed in e coli. the pcr product was cloned into the bamhi and kpni sites of vector pqe- (qiagen, hilden, germany). the resulting clone was digested by psti, and the fragment that contained the gene encoding aminoacid residues - of the spike protein was cloned into expression vector pqe- (qiagen, hilden, germany) in frame and downstream of the series of six histidine residues. expression and purification of the (his) -tagged recombinant spike polypeptide were done as described for the nucleocapsid protein. western-blot analysis was done according to our published protocols, with slight modifications. [ ] [ ] [ ] briefly, ng purified (his) -tagged recombinant nucleocapsid protein or (his) -tagged recombinant spike polypeptide was loaded into each well of a sodium dodecyl sulphate % polyacrylamide gel, then electroblotted onto a nitrocellulose membrane (bio-rad, hercules, ca, usa). the blot was cut into strips, and the strips were incubated separately with in dilution of three serum samples, obtained from three patients with sars-cov pneumonia, positive for antibodies against sars-cov detected by our indirect immunofluorescence assay. antigen-antibody interaction was detected with an ecl fluorescence system (amersham life science, buckinghamshire, uk). serum samples from three healthy blood donors were used as controls. assessment of recombinant nucleocapsid protein elisa serum samples from healthy blood donors who donated blood years previously (aged years or older) and patients with pneumonia positive for antibodies against sars-cov detected by our indirect immunofluorescence assay were used for the assessment of the elisa-based igg antibody test. the test was modified from our previous publications. , briefly, each well of a nunc immunoplate (roskilde, denmark) was coated with ng purified (his) -tagged recombinant nucleocapsid protein for h, then blocked in phosphate-buffered saline with % bovine serum albumin. l diluted human serum ( in ) was added to each well of the protein-coated plates, and they were incubated at °c for h. after five washes with washing buffer, l horseradish-peroxidase-conjugated goat antibody to human igg ( in dilution; zymed laboratories inc, south san francisco, ca, usa) was added to each well, and the plates were incubated at °c for h. after a further five washes with washing buffer, l diluted , Ј, , Ј-tetramethylbenzidine (zymed laboratories) was added to each well and incubated at room temperature for min. l · mol/l sulphuric acid was added, and the absorbance at nm of each well was measured. each sample was tested in duplicate, and the mean absorbance for each sample was calculated. the presence of specific antibodies in positive samples was confirmed by retesting of the sample by the elisa based on recombinant nucleocapsid protein and western-blot assays with recombinant nucleocapsid protein and recombinant spike polypeptide separately. using the protocol described above, we tested for the presence of igg antibodies against the nucleocapsid protein in serum samples from healthy blood donors (aged years or older) who donated blood in march-may, ; non-pneumonic paediatric inpatients (aged less than years) and nonpneumonic adult inpatients (aged years or older) admitted to queen mary hospital in may, ; and symptom-free health-care workers. the presence of specific antibodies in all positive samples was confirmed by two separate western-blot assays, one with recombinant nucleocapsid protein and the other with recombinant spike polypeptide as the antigen. the study sponsors had no role in the study design; collection, analysis, or interpretation of data; or the writing of the report. the purified (his) -tagged recombinant nucleocapsid protein and spike polypeptide were separated on denaturing polyacrylamide gels followed by western-blot analysis with serum from three patients with pneumonia positive for antibodies against the sars-cov. prominent immunoreactive protein bands of about kda were visible on the western blots that used either antigen (figure ). these sizes were consistent with the expected size of · kda for the full-length (his) -tagged nucleocapsid protein and · kda for the (his) -tagged spike polypeptide. an elisa-based sars-cov antibody test was developed for the detection of specific antibodies against the (his) -tagged recombinant nucleocapsid protein. box titration was carried out with different dilutions of (his) tagged recombinant nucleocapsid protein coating antigen and pooled serum from three patients with pneumonia positive for antibody against the sars-cov. the results identified ng purified (his) -tagged recombinant nucleocapsid protein per elisa well as the ideal amount for plate coating for igg detection (data not shown). to establish the baseline for the tests, serum samples from healthy blood donors who donated blood years previously were tested in the elisa. for these samples, the mean optical density at nm for igg detection was · (sd · ). an absorbance value of · was selected as the cut-off value (the mean value for the healthy controls plus sd; figure ) . seven of the samples from healthy blood donors had values of more than · in the igg elisa (figure ), but none of them had the specific antibody when tested by the nucleocapsid protein or the spike polypeptide westernblot assay. the specificity of the igg antibody test (elisa confirmed by western-blot assays) was %. the mean value for the samples obtained from the patients with sars-cov pneumonia, positive for igg antibodies against the sars-cov by our indirect immunofluorescence assay, was · (sd · ). samples had optical density of more than · in the igg elisa (figure ). all were confirmed to have the specific antibody by both the nucleocapsid protein and the spike polypeptide western-blot assays. the sensitivity of the igg antibody test, with the immunofluorescence assay as the gold standard, was therefore · %. ( · %) of the healthy blood donors who donated blood in march-may, , eight ( · %) of the non-pneumonic paediatric patients, eight ( · %) of the non-pneumonic adult patients, and one ( %) of the symptom-free health-care workers who had cared for the patients with sars-cov pneumonia were positive for igg antibodies by elisa ( figure ) . however, only three ( · %) healthy blood donors who donated blood in march-may, , and one ( · %) non-pneumonic paediatric patient were confirmed to have specific sars-cov antibodies by both the nucleocapsid protein and spike polypeptide western-blot assays. up to the end of may, patients from a population of about seven million in hong kong ( · %) had developed sars-cov pneumonia, compared with a rate of non-pneumonic sars-cov infections in our study population of about · % (p< · by poisson exact test of equality). previous studies in animal coronaviruses, such as infectious bronchitis virus, have shown that the nucleocapsid protein and spike protein are highly immunogenic, are abundantly expressed during infection, and can be used for serodiagnosis of animal coronavirus infections. [ ] [ ] [ ] in this study, detection of igg antibodies to both proteins was highly sensitive and specific for sars-cov infections. six ( · %) serum samples that were seropositive by the immunofluorescence assay were negative by the elisa. the reason for this discrepancy may be that the nucleocapsid protein did not elicit antibody response in this minority group of patients. conversely, of five patients with sars-cov pneumonia who were seronegative by the immunofluorescence assay but rt-pcr positive for sars-cov, two were seropositive by our elisa, with clearly positive opticaldensity values of · and · and confirmation by western-blot assay (unpublished). furthermore, in four of patients with sars coronavirus pneumonia who had serial serum samples, igg was detected earlier by the elisa than by the immunofluorescence test (unpublished). in another study that used elisa based on cell-culture extract, five of patients with sars-cov pneumonia were positive according to that elisa but negative by indirect immunofluorescence during the time when the elisa titres were low. this finding accords with the results of a previous study on human coronavirus e, which showed that three of serum samples were positive by recombinant-nucleocapsid-protein-based western blot but negative by immunofluorescence, and six of serum samples were positive by immunofluorescence but negative by recombinant-nucleocapsid-protein-based western blot. all these findings show that our elisa may be able to detect additional cases that the immunofluorescence assay has missed. with this potentially more sensitive elisa for igg antibody detection, we assessed the seroprevalence of non-pneumonic sars-cov infections in both the general population and our hospital population; all positive serum samples detected by elisa were confirmed by two separate western-blot assays, with two immunologically unrelated antigens. the spike polypeptide was used in addition to the nucleocapsid protein for western-blot confirmation to eliminate the possibility of cross-reactivity between antibodies against the nucleocapsid proteins of other human coronaviruses and that of sars-cov. however, the aminoacid identities between the nucleocapsid protein of sars-cov and those of the human coronaviruses oc and e are only · % and · %, respectively, and there were no crossreactions between pairs of oc and pairs of e human coronavirus serum samples and the sars-cov. in fact, of the individuals who were igg positive by elisa, ( %) were positive by the nucleocapsidprotein-based western-blot assay, but only four were positive by both the nucleocapsid protein and the spike polypeptide western-blot assays. this apparent high rate of false-positive non-pneumonic cases, as detected by the recombinant nucleocapsid protein elisa, is due to the use of a single antigen for screening asymptomatic or nonpneumonic infections. it is well known that the positive predictive values of serological tests are much affected when the prevalence of the infection is low, especially in clinically incompatible cases. this is the reason why an immunologically unrelated antigen, the spike protein, has to be used for confirmation of the cases detected as "positive" by the nucleocapsid-protein-based assays. cross-reactivity with human coronavirus oc or other sars-cov-like viruses remains an important issue for future studies on sars-cov serology. three of the four individuals with non-pneumonic sars-cov infections were healthy blood donors, and one was a paediatric inpatient. this patient was a -month-old girl who was admitted to hospital in may, , because of fever for days. she was also noted to have had a cough for the previous weeks and repeated vomiting before admission. there had been no breathing difficulty or diarrhoea. the child had no history of direct contact with patients with sars-cov pneumonia. however, a colleague in her mother's workplace had recently been diagnosed as having sars-cov pneumonia. physical examination of the girl revealed only shotty (palpable but too small to be measured) cervical lymph nodes and congested throat. her chest radiograph was normal. apart from mild lymphopenia (lymphocyte count · ϫ /l), her blood results were normal. nasopharyngeal aspirate was negative for influenza viruses, parainfluenza viruses, adenovirus, and respiratory syncytial virus antigens. she was given antipyretic treatment and was discharged the next day. subsequent antibody testing showed that her serum was positive for both igg and igm antibodies against the nucleocapsid protein as well as igg antibodies against the spike polypeptide of sars-cov. the difference between the rate of non-pneumonic sars-cov infections in our study population and the rate of sars in hong kong suggests that non-pneumonic infections are more common than sars-cov pneumonia and may explain cases of sars-cov pneumonia in patients who had no obvious contact with other patients with sars. contributors p c y woo and k y yuen are coprincipal investigators, jointly wrote the report, and coordinated and supervised the study. s k p lau supervised the analysis of all serological data and corrected the report. h w tsoi cloned and purified the nucleocapsid protein and did the serological assays. k h chan supervised the immunofluorescence assay. b h l wong and v k p tam did the serological assays and analysed the data. x y che cloned and purified the spike polypeptide. s c f tam coordinated the collection of clinical specimens. v c c cheng and i f n hung managed the patients' database. s s y wong, b j zheng, and y guan corrected the report. none declared. coronavirus as a possible cause of severe acute respiratory syndrome development of a standard treatment protocol for severe acute respiratory syndrome prospective study of the clinical progression and viral load of sars-associated coronavirus pneumonia in a community outbreak lung pathology of fatal severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndromeassociated coronavirus infection mp encodes an abundant and highly antigenic cell wall mannoprotein in the pathogenic fungus penicillium marneffei detection of specific antibodies to an antigenic mannoprotein for diagnosis of penicillium marneffei penicilliosis characterization of afmp : a novel target for serodiagnosis of aspergillosis detection of specific antibodies to an antigenic cell wall galactomannoprotein for serodiagnosis of aspergillus fumigatus aspergillosis enzyme-linked immunosorbent assay for detecting antibodies to borne disease virusspecific proteins an indirect elisa for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen cloning and characterization of male in burkholderia pseudomallei groel encodes a highly antigenic protein in burkholderia pseudomallei aflmp encodes an antigenic cell wall protein in aspergillus flavus localization of linear b-cell epitopes on infectious bronchitis virus nucleocapsid protein recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus an elisa for antibodies against infectious bronchitis virus using an s spike polypeptide detection of human coronavirus e-specific antibodies using recombinant fusion proteins cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients this work is partly supported by a research grant council grant (hku / m), the sars research fund, and the university sars donation fund, the university of hong kong. we thank members of the clinical microbiology laboratory, queen mary hospital, and members of the department of medicine, united christian hospital, hong kong for their assistance. key: cord- -d p u authors: abe, kento t.; li, zhijie; samson, reuben; samavarchi-tehrani, payman; valcourt, emelissa j.; wood, heidi; budylowski, patrick; dupuis, alan p.; girardin, roxie c.; rathod, bhavisha; wang, jenny h.; barrios-rodiles, miriam; colwill, karen; mcgeer, allison j.; mubareka, samira; gommerman, jennifer l.; durocher, yves; ostrowski, mario; mcdonough, kathleen a.; drebot, michael a.; drews, steven j.; rini, james m.; gingras, anne-claude title: a simple protein-based surrogate neutralization assay for sars-cov- date: - - journal: jci insight doi: . /jci.insight. sha: doc_id: cord_uid: d p u most of the patients infected with severe acute respiratory syndrome coronavirus (sars-cov- ) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. while elisa-based assays to detect and quantitate antibodies to sars-cov- in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the sars-cov- spike protein receptor binding domain (rbd) with its receptor, angiotensin-converting enzyme (ace ). the assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an elisa for the detection of antibodies against the rbd, enabling a direct comparison. the results obtained with our assay correlate with those of viral-based assays, a plaque reduction neutralization test (prnt) that uses live sars-cov- virus and a spike pseudotyped viral vector–based assay. the coronavirus s-protein (spike) is responsible for both receptor binding and fusion of the virus and host cell membranes. within the spike protein, the receptor binding domain (rbd) mediates the interaction with the host cell receptor, and sequence/structural variation in the rbd is responsible for the receptor binding specificity shown by those coronaviruses that use host proteins as receptors ( ) . most of the patients infected with severe acute respiratory syndrome coronavirus (sars-cov- ) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. while elisa-based assays to detect and quantitate antibodies to sars-cov- in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the sars-cov- spike protein receptor binding domain (rbd) with its receptor, angiotensin-converting enzyme (ace ). the assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an elisa for the detection of antibodies against the rbd, enabling a direct comparison. the results obtained with our assay correlate with those of viral-based assays, a plaque reduction neutralization test (prnt) that uses live sars-cov- virus and a spike pseudotyped viral vector-based assay. sars-cov- , like sars-cov, uses the cell surface carboxypeptidase angiotensin-converting enzyme (ace ) as a receptor for viral entry ( figure a) . the use of a common receptor is consistent with the fact that the viruses share a high degree of sequence similarity and that their rbds are ~ % identical, though the rbd of sars-cov- binds ace with higher affinity than does that of sars-cov ( ) . the spike proteins of both viruses are also both primed by the host protease, tmprss , but unlike sars-cov- , the spike protein of sars-cov does not contain a furin recognition motif that can be cleaved during viral biogenesis ( , ) . the coronavirus spike protein is also a major target of the host immune system, and antibodies directed against it play a central role in host-mediated neutralization ( ) . among neutralizing antibodies, those that block the interaction between viruses and their receptors represent the most common route to neutralization ( ) . for this reason, both the spike protein and the rbd form the basis for most of the sars-cov- vaccines currently in development. the detection and study of neutralizing antibody activity following natural infection (or vaccination) can, therefore, support research aimed at the development of novel therapeutics and vaccine candidates. it can also aid in the identification of acceptable donors for convalescent plasma therapy ( ) and, more generally, to establish immune correlates of infection. for sars-cov- , viral neutralization assays are performed using either live virus ( ) or viral vectors pseudotyped with the spike protein ( ) . however, these cell culture-based assays are challenging to implement and time-consuming to run -factors that limit scalability. the conventional plaque reduction neutralization test (prnt) that uses live sars-cov- virus is further complicated by the need for containment level (cl- ) and a specialized laboratory setup. although the pseudotyped viral vector-based assays do not require biosafety level (bsl- ) containment ( ) , they are complicated multistep procedures ( ) . by contrast, the detection and quantitation of antigen-specific antibodies in patient samples can be easily assayed by elisa ( ) . sars-cov- elisas are performed by immobilizing a recombinantly produced viral antigen (such as the spike trimer or rbd) ( figure b and supplemental figures and ; supplemental material available online with this article; https://doi.org/ . /jci.insight. ds ) (see methods) onto multiwell plastic plates that are then incubated with diluted patient serum or plasma samples. the detection of antibodies that bind to the antigen involves a second incubation with enzyme-conjugated antihuman antibodies, where the enzyme is often horseradish peroxidase (hrp). this enables the detection of a color change when an hrp substrate such as , ′, , ′-tetramethylbenzidine (tmb) is used. in direct binding assays of this type ( figure c ), the presence of patient antibodies against the viral antigen leads to a dose-dependent increase in the signal observed. elisa-based profiling has been developed by multiple groups and has been used to measure the kinetics of the antibody response in patient cohorts following sars-cov- infection. in several recent studies, including ours, this has revealed the relative stability in the igg response to the spike and rbd over several months, along with a more transient igm and iga response that wanes as patients convalesce ( ) ( ) ( ) ( ) ( ) . however, the levels of neutralizing antibodies are not typically measured in large cohorts over time (with a few notable exceptions, as seen in refs. , ) , as current assays have relatively low throughput. the relative lack of neutralizing antibody data represent a significant gap in our understanding of the immune response to sars-cov- . here, we describe a modified elisa-type assay that serves as a surrogate neutralization assay. it measures the presence of antibodies capable of blocking the rbd-ace interaction, and like the direct binding elisa, it is easily scaled to allow for the analysis of large patient cohorts over time. we show that the results obtained by this assay correlate with those of both the sars-cov- prnt and a spike pseudotyped viral vector neutralization assay in a cohort of convalescent patients and on purified antibodies. we aimed to develop a simple protein-based assay to monitor the ability of antibodies, present in the serum or plasma of patients, to block the interaction between the rbd and the host receptor ace . to do so, we elected for an elisa-type assay, since such assays are already widely used to detect antibodies that recognize sars-cov- antigens such as the spike trimer and its rbd. as with the standard direct elisa, the antigen (here, the rbd or the spike trimer) is first immobilized on multi-well plates and then incubated with patient plasma or serum ( figure b) . however, because we were interested in detecting functional antibodies that can prevent the interaction between the rbd (or spike) and ace , we replaced the hrp-conjugated secondary antibody used in the direct elisa by a detection method involving human ace . in our assay, recombinantly expressed soluble ace bearing a biotinylated c-terminal avitag is added to the antigen-bound plate after the plate has been incubated with the patient plasma or serum (see methods). bound ace is then detected by the addition of streptavidin-poly hrp and its colorimetric substrate tmb. the presence of patient antibodies that can block the rbd-ace interaction leads to a dose-dependent decrease in the signal observed, and as such, we refer to it as a surrogate neutralization elisa (snelisa; figure d ). we explored different versions of the assay: the configuration described above and one involving immobilized ace and soluble biotinylated rbd, a configuration similar to that previously reported ( ) . the assay with immobilized rbd and soluble biotinylated ace was more sensitive than its counterpart (supplemental figure ). moreover, with the rbd immobilized, the same overall protocol and colorimetric detection can be used for both the direct binding elisa and the snelisa, thereby facilitating a direct comparison. although the snelisa worked well with either the rbd or the spike ectodomain trimer immobilized ( figure e and supplemental figure a ), we focused on the rbd, since it is easier to produce and provides a simple one-to-one binding interaction with ace . using a small test set (supplemental figure b) , we first showed that the serum/plasma from positive but not negative control patients inhibited the interaction between ace and the immobilized rbd ( figure f ). the technical reproducibility of the assay was within %- % coefficient of variation (cv). the total time required to perform the assay (once the plates are coated with the antigen) is . - hours, and the assay can be performed using the same equipment and biosafety protocols as a standard elisa. using both the surrogate neutralization and direct binding (with a dilution series) elisas, we then profiled a set of serum samples acquired at the canadian blood services as part of a screen for convalescent plasma therapy donors ( table ). with reference to the direct binding results, the snelisa showed that samples with high levels of igg against the rbd were typically the most potent at blocking the rbd-ace interaction (e.g., cbs , which is included as a positive control). conversely, samples lacking detectable rbd-binding antibodies were not able to block the interaction. to more systematically evaluate the relationship between the rbd-binding antibody levels and the ability to block the rbd-ace interaction (as determined by the snelisa), we calculated the auc for both assays and plotted the rbd-binding auc versus the snelisa auc ( figure c and supplemental figure ). the plot showed a clear correlation (r = . ), with the sera containing the highest rbd-binding antibody levels being the most effective at blocking the rbd-ace interaction ( figure c ; compare figure b with figure a ; supplemental table ). nevertheless, there are samples with similar rbd-binding antibody concentrations that differ in their ability to block the rbd-ace interaction (figure d and supplemental figure ). differences in antibody isotype, affinities, and abundance, as well as the rbd epitopes bound, are all factors that could explain these outliers. while it is reasonable to expect that antibodies that block the rbd-ace interaction would be neutralizing, we validated this using cell-based viral infectivity and entry assays. fifty-seven of the samples analyzed by the snelisa were analyzed by prnt, the gold standard in the field. prnt is defined as the concentration of patient serum or plasma capable of reducing the formation of viral plaques by %; prnt is the concentration that reduces plaque formation by %. as shown in figure a , most of the samples displaying high values in the direct binding and snelisas were also positive by prnt (and those with low titers were negatives). both elisas also gave an overall agreement with the prnt titers (see supplemental figure , with a coefficient of determination of . ). we also adapted and optimized a spike-pseudotyped lentiviral-based entry assay ( ) , and we reprofiled the neutralization potential of a subset of samples. there was also a high correlation (r = . ) between the snelisa results and the titers obtained with this spike-pseudotyped lentiviral-based entry assay ( figure b and supplemental figure ). taken together, these results indicate that our snelisa is a good surrogate neutralization assay, particularly for distinguishing between samples with high versus low neutralization activity. as such, the assay should be of value in the selection of candidate donors for convalescent plasma therapy and for monitoring immune correlates of patient outcomes. future work will focus on providing a better understanding of the outliers observed across all assays. indeed, rare but potent neutralizing antibodies in patient samples with low pseudovirus neutralization titers have recently been reported ( ) . to assess whether our snelisa might also be of value for screening the neutralization potential of monoclonal antibodies, we tested it using a number of neutralizing and nonneutralizing monoclonal antibodies and compared the results with the results obtained with the pseudotyped lentiviral-based entry assay or cytopathic effect-reduction neutralization assay with sars-cov- . the llama vhh monoclonal antibody (expressed as a human fc fusion), previously shown to neutralize in a sars-cov- spike pseudotyped entry assay ( ) , blocked the rbd-ace interaction in our snelisa and viral entry in our spike pseudotyped lentivirus assay; similar results were obtained for the active motif - antibody, which was isolated from a convalescent patient and was shown to be neutralizing ( ) (figure , c and d, and supplemental figure ). in contrast, other antibodies, such as an igg derived from the monoclonal anti-sars cr or a commercial antibody (hc ) from genscript, had a much more moderate effect in the snelisa (supplemental figure ) , and the genscript antibody had no effect in the cytopathic effectreduction neutralization assay (supplemental figure ). the active motif - antibody was previously shown to be incapable of neutralizing live sars-cov- virus ( , ) . in our assays, it efficiently bound the rbd in the direct elisa but did not block the rbd-ace interaction in the snelisa. the same antibody partially prevented entry in our lentivirus entry assay. taken together, these observations suggest that our snelisa is a good complement to more complex cell-based assays for the discovery and screening of neutralizing monoclonal antibodies. in summary, we have developed a simple and safe snelisa for sars-cov- . it can be readily incorporated into existing testing platforms and may be of particular value in the selection of donors for convalescent plasma therapy and as a means of monitoring the immune response to vaccination. given that neutralizing antibody titres have recently been shown to wane fairly rapidly in some ( ) ( ) ( ) ) but not all ( , ) studies, the assay may also be useful for broad serosurveillance, especially as it should be more scalable than the approaches requiring viral infection assays. when coupled with epidemiological studies, it might also be used to assess the risk of infection/reinfection. we also note that the optimized conditions used here for the direct rbd-binding elisa are similar to those reported in ref. using rbd-expression constructs that have been widely distributed. their rbd can be obtained from bei resources ( ), and we found that it generates similar results when used with our biotinylated ace in the snelisa (supplemental figure ). this should further facilitate the broad implementation of our assay across multiple laboratories. there are limitations to the assay, however, that need to be acknowledged. first, the snelisa is limited to the detection of neutralizing antibodies that function by blocking the interaction between the rbd and ace . while by no means dominant, examples of antibodies that neutralize by other mechanisms are beginning to emerge ( ) ( ) ( ) . the snelisa, in conjunction with a neutralization assay, could be used to identify further such examples. as with those identified in this work, the outliers (e.g., those with high viral neutralization titers but low snelisa levels) provide a starting point for further work aimed at understanding the mechanisms of antibody-mediated neutralization. another limitation of our approach is that the current assay cannot directly map the epitopes targeted by the various antibodies. undoubtedly, the antibodies detected by the snelisa bind to different sites on the rbd, a suggestion supported by the structures of neutralizing antibody fragment antigen binding (fabs) in complex with the sars-cov- rbd. in one example, different neutralizing antibodies that bind to different epitopes on the rbd were found to synergistically mediate viral neutralization ( ) . while, in the current study, we simply wanted to provide evidence of antibodies that could block the rbd-ace interaction, the snelisa could be adapted to provide information on the site of antibody binding. as recently shown, a series of structure-guided point mutants in the rbd could be used to infer where on the rbd the antibodies are binding ( ) . this type of approach would likely be more important in the characterization of monoclonal antibodies, such as those presented in figure c , and would set the stage for in-depth biophysical and structural studies. while the direct binding elisa described here employed an anti-igg secondary antibody (the predominant isotype in convalescent serum), we note that the snelisa measures the ability of any antibody isotype (or even antibodies from different species or any other molecule) to block the rdb-ace interaction. in this regard, it is similar to that of a viral-based neutralization assay. while we have not performed a detailed analysis, we did show that single-point direct binding elisas performed for igm, and to a lesser extent iga, are also correlated with the results obtained by the snelisa (supplemental figure ) . the safety and simplicity of the snelisa should make it a valuable addition to the arsenal of assays for monitoring the immune response to sars-cov- infection. for all elisas, inactivation of potential infectious viruses in plasma or serum was performed by incubation with triton x- to a final concentration of % for hour before use ( ) . for the pseudotyped lentiviral assays, the serum was heat inactivated for hour at °c ( ). the expression plasmid generated is a derivative of those previously reported in our piggybac transposonbased mammalian cell expression system ( ) . two versions of the plasmid were constructed: one contains the cmv promoter (pb-cmv) and the other the tre promoter (pb-tre). the vectors are otherwise identical and can be used to generate stable cell lines for constitutive or inducible protein expression. the protein cloning region contains several optional elements separated by restriction sites as follows: an n-terminal human cystatin-s secretion signal, the protein of interest, a foldon trimerization motif ( ), a xhis purification tag, and an avitag biotinylation motif ( ) (supplemental figure ) . a woodchuck hepatitis virus posttranscriptional regulatory element (wpre) follows the orf to facilitate nuclear export of the mrna. a pair of piggybac transposon terminal repeats flank the expression cassette and an attenuated puromycin resistance marker (bioshop canada inc., pur ), thereby allowing for the generation of stable cell lines using the piggybac transposase. the human codon optimized cdna of the sars-cov- spike protein (mc_ ) was purchased from genscript. the human ace cdna was derived from mgc clone . to stabilize the soluble spike ectodomain trimer, regions of the spike protein were mutated. residues - (rrar) were mutated to ssas to remove the furin cleavage site, and residues - (kv) were each mutated to a proline residue to stabilize the prefusion form as previously described ( ) . the soluble spike protein ectodomain construct includes residues - (mfvf...qyik), followed by the foldon trimerization motif, a xhis tag, and an avitag. both the sars-cov- rbd and the human ace constructs are preceded by the human cystatin-s secretion signal and followed by the xhis and avitag. the rbd and ace constructs contain residues - (rfpn...cgpk) and - (stie...pyad), respectively. the cdna of the human cr fab was synthesized by genscript based on its previously reported sequence ( ). the light chain and heavy chains were individually cloned into the pb-tre expression plasmid. for fab production, a xhis tag was added to the c-terminal end of the fab heavy chain. an igg form was generated by fusing the human igg fc coding sequence to the c-terminal end of the fab heavy chain. freestyle -f suspension cells (thermo fisher scientific, r ) were grown in shaker flasks ( rpm) in freestyle expression medium (thermo fisher scientific, ) in a humidified °c incubator filled with % (v/v) co . the cell density and viability were monitored by manual counting using a hemocytometer and trypan blue staining. for transfection, cells of > % viability were counted and seeded at a density of approximately × cells/ml into ml freestyle medium supplemented with μg/ml aprotinin (bioshop canada inc., apr ). the pb-cmv plasmid dna ( μg) and fectin ( μl; thermo fisher scientific, ) were each added to separate tubes containing ml of opti-mem medium (thermo fisher scientific, ). the solutions were then mixed and incubated for minutes before being added to the ml cell culture. two days after transfection, the ml culture was expanded into three l shaker flasks each containing ml of culture medium. protein expression was continued for an additional days. the stable cells were scaled up in l shaker flasks containing ml freestyle medium without supplements. when the cell densities reached approximately × cells/ml, μg/ml doxycycline (milliporesigma, d ), and μg/ml aprotinin were added to initiate protein expression. during the expression phase, ml of the medium was removed, and fresh medium added every other day. the harvested expression medium was centrifuged at , g for minutes at °c to remove the cells and debris. for the xhis tagged proteins, the clarified media were passed through an ni-nta column (qiagen, ). for the spike ectodomain, ml of ni-nta resin was used for each liter of medium. for the rbd, ace , and cr fab, ml of ni-nta resin was used for each liter of medium. the ni-nta resin was washed with column volumes of phosphate buffered saline (pbs), followed by - column volumes of pbs containing mm imidazole. the protein was eluted with pbs containing mm imidazole (bio basic, ib ) and . % (v/v) protease inhibitor cocktail (milliporesigma, p- ). for the cr antibody, the harvested medium was incubated with rprotein a sepharose ff resin (ge healthcare, ). the resin was then washed with column volumes of pbs, and the antibody was eluted with mm glycine, ph . , containing mm nacl. the acid-eluted antibody was immediately neutralized by the addition of / volume of m tris, ph . . protease inhibitor cocktail was also added to a final concentration of . % (v/v). the approximate purified yields of the various proteins are as follows: rbd, mg/l; spike trimer, mg/l; ace , mg/l; cr fab, mg/l; and cr igg, mg/l. the protein samples were stored in % glycerol at - °c. shortly before use, the glycerol stocks were further purified using size-exclusion chromatography. for the rbd, ace , and cr fab/igg, a superdex increase (ge healthcare, ) column was used. for the spike ectodomain, a superose increase (ge healthcare, ) column was used (supplemental figure ). each biotinylation reaction contained μm biotin, μm atp (milliporesigma, cat # a ), μm mgcl , μg/ml bira (produced from e. coli; a gift from walid houry, university of toronto), . % (v/v) protease inhibitor cocktail, and no more than μm of the protein-avitag substrate. the mixture was incubated at °c for hours, followed by size-exclusion chromatography to remove unreacted biotin (bioshop canada inc., bio ). for the rbd, the degree of biotinylation was assessed using a band-shift assay. a total of μg of the biotinylated rbd was heated to °c for seconds in sds-page loading buffer (containing % sds, mm dtt); after cooling, μl of a mg/ml streptavidin solution was added. the mixture was then analyzed by sds-page to assess the formation of the rbd-streptavidin complex (supplemental figure ) . the llama single domain antibody vhh sequence (pdb entry waq_ ) was obtained from wrapp et al. ( ) . a cdna encoding vhh fused to an adcc-attenuated human igg fc domain (hfc x , from patent us a ) was codon optimized for expression in cho cells, synthesized by genscript, and cloned into the ptt plasmid ( ) . the ptt -vhh hfc x plasmid was transiently expressed in cho e cells ( ) using pei max transfection reagent (polysciences) and a slightly modified protocol as described previously ( ) . the cell culture was harvested at day after transfection, centrifuged minutes at g at room temperature, and filter sterilized using a . μm membrane vacuum filter (express plus, milliporesigma). filtered supernatant was loaded on a ml mabselect sure column (ge healthcare) equilibrated in pbs. the column was washed with pbs, and the antibody eluted with mm citrate buffer ph . . the fractions containing the antibody were pooled, and elution buffer was exchanged for pbs using nap- columns (ge healthcare). purified vhh h-fc x in pbs was quantified by absorbance at nm using a nanodrop spectrophotometer (thermo fisher scientific) and the calculated extinction coefficient of the protein. overall volumetric yield after protein a purification was mg/l. the purified protein was analyzed by analytical size-exclusion ultra high-performance liquid chromatography coupled to a mals detector and eluted as a major (> % integrated area) symmetrical peak of kda with less than % aggregates (not shown). an alternative source for rbd was bei resources nr- (contributors f. krammer, f. amanat, s. strohmeier; icahn school of medicine, mount sinai, new york, usa; lot ). commercial antibodies tested also included a human igg chimeric antibody from genscript (sars-cov- spike s antibody, hc ; genscript, a ) and sars-cov- spike antibodies from active motif (am , ; am , ). manual single-point elisas in -well format. for the manual single-point elisas in -well format, concentrations and incubation times were optimized to maximize the separation between anti-rbd levels in convalescent plasma or serum from that of pre-covid-era banked serum while maintaining the required levels of antigens as low as possible. a total of μl of serum or plasma was used for the detection of antibodies on -well plates coated with ng/well of recombinant purified rbd. single-point elisas are expressed as ratios to a positive control convalescent plasma sample. multipoint elisas. for the multipoint elisas, the rbd amount was fixed to ng/well to match the design of the snelisa, and -fold serial dilutions of the serum or plasma sample from μl to . μl were employed. both cases. in both cases, the rbd antigen (diluted to μg/ml in pbs) was first adsorbed to -well clear immulon hbx plates (thermo fisher scientific, ) in pbs overnight at °c and then washed times with μl pbs plus . % tween- (pbs-t; milliporesigma). plates were blocked for hour at room temperature with μl % blocker blotto (thermo fisher scientific, ) and washed times with μl pbs-t. in the single-point elisas, plate blocking was performed with % w/v milk powder (bioshop canada inc., alb . , lot h ) in pbs for - hours. patient samples (pretreated with % final triton x- for viral inactivation) diluted in pbs-t containing % w/v milk powder ( : for the single-point elisa) were then added to the plates and incubated for hours at room temperature ( μl total volume); technical duplicates were performed unless otherwise indicated. a chimeric human anti-spike antibody (sars-cov- spike s antibody, hc ; genscript, a ) was added to a set of wells on each plate as a serial dilution ( : , - : , or - . ng per well in steps) to enable cross-plate comparisons. positive (convalescent plasma from a single patient) and negative controls (pre-covid-era banked serum) were also added to each plate, at μl. wells were washed times with μl pbs-t. goat anti-human anti-igg (goat anti-human igg fcy hrp, jackson immunoresearch, - - ) at a : , dilution ( . ng/well) in % blotto was added and incubated for hour. wells were washed times with μl pbs-t, and μl of -step ultra tmb-elisa substrate solution (thermo fisher scientific, ) was added for minutes at room temperature. the reaction was quenched with μl stop solution containing . n sulfuric acid (thermo fisher scientific, n ). the plates were read in a spectrophotometer (biotek instruments inc., cytation ) at nm. for all elisa-based assays, raw od values had blank values subtracted before analysis. for the single-point direct binding assay, the average cv across cbs samples is . % (mean) and . % (median) (supplemental table ). for single-point assays, all data were normalized to the positive serum control (single point) on each plate and expressed as a ratio to this control. for the multipoint dose responses, blank-adjusted reads were used. variations. variations to this protocol included the following. (a) replacement of the rbd on the plate by the bei resources nr- . the assay was set up identically to and in parallel with our in-house produced rbd (supplemental figure ). (b) replacement of rbd ( ng) on plate by the spike trimer purified above ( ng) (supplemental figure ) . (c) performing the single-point elisas using an automated platform with chemiluminescent detection for anti-igg, -iga, and -igm, exactly as described in ( ) (supplemental figure ) . our final optimized snelisa used ng immobilized recombinant rbd on -well immulon hbx plates incubated overnight at °c ( μg/ml). all volumes added to the well were μl, unless specified otherwise. plates were washed times with μl pbs-t and blocked for - . hours at room temperature with μl % bsa (bioshop canada inc., ski . , lot h ). after washing as above, a -step, -fold serial dilution series of patient serum or plasma ( . - μl of sample) was incubated for hour. the wells were washed as above and incubated with ng biotinylated recombinant ace for hour. after washing as above, the wells were incubated with ng streptavidin-peroxidase polymer (milliporesigma, s ). the resultant signal was developed and quantified with tmb in an identical manner to the direct elisas. due to day-to-day variation in signal, all od values are normalized to the od of the well where no patient serum/antibody was added for each sample. all values are expressed in this ratio space. variations. variations of this protocol included using a different source of rbd (bei resources, nr- ) and using spike trimer as shown above ( ng/well) ( figure c and supplemental figure ). another variation of the assay was to bind nonbiotinylated ace to the plate ( ng) and to use biotinylated rbd ( ng) for detection (supplemental figure ). neutralization assays on the canadian blood services samples used in figure were performed by independent laboratories, the nml of the public health agency of canada, and the wadsworth center, new york state department of health. the cytopathic effect-reduction neutralization assay on the recombinant genscript antibody was performed in toronto. for the prnt assay at nml, sars-cov- (canada/on_on-vido- - / , epi_isl_ ) stocks were titrated ( ) for use in a prnt adapted from a previously described method for sars-cov ( ) . briefly, serological specimens were diluted -fold from : to : in dmem supplemented with % fbs and incubated with pfu of sars-cov- at °c and % co for hour. the sera-virus mixtures were added to -well plates containing vero e cells at % confluence, followed by incubation at °c and % co for hour. after adsorption, a liquid overlay composed of . % carboxymethylcellulose diluted in mem, supplemented with % fbs, l-glutamine, nonessential amino acids, and sodium bicarbonate, was added to each well; the plates were incubated at °c and % co for hours. the liquid overlay was removed, and the cells were fixed with % neutral-buffered formalin for hour at room temperature. the monolayers were stained with . % crystal violet for minutes and washed with % ethanol. plaques were enumerated and compared with controls. the highest serum dilution resulting in % and % reduction in plaques compared with controls were defined as the prnt and prnt endpoint titers, respectively. prnt titers ≥ : and prnt titers ≥ : were considered positive. for the prnt assay at wadsworth, the assay for the detection of sars-cov- neutralizing antibodies was a modified version of previously described methods ( ) ( ) ( ) . patient sera and sars-cov- (usa/wa- / , bei resources, nr- ) were diluted in vero e cell culture maintenance medium (emem, % heat-inactivated fbs, u/ml penicillin g, u/ml streptomycin). patient samples were serially diluted : - : and mixed with an equal volume of virus containing pfus. virus and serum mixtures were incubated at °c and % co for hour. following the initial incubation, . ml of each dilution was plated in a single well of a -well plate containing confluent monolayers of vero e cells (atcc, crl- ) and allowed to adsorb for hour at °c and % co . following adsorption, cell cultures were overlaid with . % agar in cell culture medium and returned to the incubator. at days after infection, a second overlay containing . % neutral red was added. monolayers were inspected for days, and plaques were counted. antibody titers were reported as the inverse of the serum dilution resulting in % (prnt ) and % (prnt ) reduction in plaques as compared with the virus inoculum control. for the cytopathic effect-reduction neutralization assay in toronto, μl of . × veroe cells/ml were seeded into a -well flat-bottom plate to adhere overnight. all plasma and serum samples were heat inactivated at °c for minutes. in a separate -well plate, the serum, plasma, or antibody ( μg/ml) samples were serially diluted -fold times in serum-free dmem starting from a dilution of : to : in a volume of μl. to all wells, μl of sars-cov- sb clone was added, ensuring that each well had a dose of issue culture infectious dose (tcid). for the cell control, μl of serum-free dmem was added. for the virus control, μl of sars-cov- sb clone was added with a dose of tcid and topped off with μl of serum free dmem. the plate was incubated for hour at °c, % co with shaking every minutes. after incubation, all the media from the veroe culture were removed, and the full μl of serum/sars-cov- coculture was layered on the cells. the plate was again incubated for hour at °c, % co , with shaking every minutes. after the incubation, the inoculum was removed, and μl of dmem containing % fbs was added. the plate was incubated for days and cytopathic effect was tracked. the assay was established using constructs previously described ( ) (constructs obtained through a gift from jesse bloom and katharine crawford, fred hutchison cancer research centre, seattle, washington, usa, and now available through bei resources) and optimized in-house. major changes to the reported protocol included: (a) use of a second-generation pspax (addgene, ) lentivirus packaging system instead of the third-generation system used by the bloom lab, (b) production of spike pseudotyped virus-like particles (vlps) at °c, (c) a neutralization assay plate layout that increases throughput, (d) adjustments to the luciferase protocol to minimize variability in readings, and (e) use of a cell line that coexpresses ace and tmprss . to generate this cell line, entry vectors for ace and tmprss coding sequences were cloned into plenti cmv puro dest (addgene, ) and plenti cmv hygro dest (addgene, ), respectively. the resulting transfer vectors were used to generate lentivirus via the second-generation pspax and vsv-g (addgene, ). hek t cells were transduced with ace lentivirus at an moi < and selected with puromycin ( μg/ml) to generate a stable population. these cells were subsequently transduced with tmprss lentivirus and selected with hygromycin ( μg/ml) in a similar fashion. for vlp generation, hek t cells were transiently cotransfected in a -well-plate format containing ml growth medium ( % fbs, % penicillin/streptomycin [pen/strep] in dmem) with . μg pspax , . μg phage-cmv-luc -ires-zsgreen-w (bei, nr- ; a gift from jesse bloom and katharine crawford; lentiviral backbone plasmid that uses a cmv promoter to express luciferase followed by an ires and zsgreen), and . μg hdm-idtspike-fixk (bei, nr- ; a gift from jesse bloom and katharine crawford; expressed under a cmv promoter a codon-optimized wuhan-hu- spike; genbank, nc_ ) using μl jetprime (polyplus-transfection sa, - )in μl jetprime buffer. after hours of transfection, the medium was replaced by ml of dmem containing % heat-inactivated fbs and % pen/strep, and the cells were incubated for hours at °c and % co ; they were then transferred to °c and % co for an additional hours. at hours after transfection, the supernatant was collected, spun at g for minutes at room temperature, filtered through a . μm filter, and frozen at - °c. the virus titers were evaluated using hek t-ace /tmprss cells at , cells per well on a poly-l-lysine-coated ( - μg/ml) -well plate using hi media ( % heat-inactivated fbs, % pen/strep), along with a virus dilution resulting in > relative luciferase units (rlu) over control (~ : virus stock dilution). for the neutralization assay, . -fold serial dilutions of the serum samples were incubated with diluted virus at a : ratio for hour at °c before being transferred to plated hek -ace /tmprss cells and incubated for an additional hours at °c and % co . after hours, cells were lysed, and bright-glo luciferase reagent (promega, e ) was added for minutes before reading with a perkinelmer envision instrument. auc values were tabulated for both the direct binding elisa and the snelisa using r version . . and r package pracma. for the snelisa, the ratios (normalized values) are used in the auc calculations. to identify outliers, we calculated the distance of each point from the regression line using total least squares and labeled points with distances > . . for the lentiviral pseudotyping assays, % inhibitory concentration or dilution (ic or id ) were calculated with nonlinear regression (log[inhibitor] versus normalized response -variable slope) using graph-pad prism (graphpad software inc.). the "variable slope" option is a parameter selected in graphpad prism for nonlinear regression that does not assume a standard slope of - . with each dose-response curve but, instead, determines the slope of the curve based on the data generated. for the extended direct binding dilution series, titres were calculated by taking the dilution of serum that produced % of the maximum response in the elisa as determined by the nonlinear regression line (sigmoidal, pl, x is log[dilution]) using graphpad prism . the assay reproducibility was estimated across experiments by comparing the auc values for those samples profiled across different batches. cbs (n = ) cv for displacement was . % and direct binding was . %; cbs (n = ) cv for displacement was . % and . % for binding; cbs (n = ) cv for displacement was . % and binding was . %. when applicable, graphical data from experiments with or more replicates are presented as mean ± sem. all samples were collected after research ethics board (reb) review. the elisas were performed at the lunenfeld-tanenbaum research institute with mount sinai hospital (msh; toronto, ontario, canada) reb approval (study no. - -e). external samples were transferred through material transfer agreements. all research has been performed in accordance with relevant guidelines and regulations. all participants have provided informed consent. the samples were deidentified before transfer to the assay laboratory. kta and acg designed the snelisa and the direct rbd antibody assay. zl and jmr designed the protein expression, biotinylation, and purification procedures. rs and pst optimized the lentiviral pseudotyping assay. ejv, hw, mad, apd, rcg, kam, pb, and mo developed, performed and analyzed the prnt and/or cytopathic effect-reduction neutralization assays. kta and br performed direct elisa experiments. yd designed the vhh hfc x construct, expression, and purification procedures. jhw and mbr implemented the automated direct binding elisa. kc helped coordinate the project. sjd provided samples, coordinated neutralization testing, and integrated prnt and snelisa data. kta and acg analyzed the snelisa data. jlg, ajm, sm, mo, and sjd contributed essential patient samples. kta, jmr, and acg wrote the manuscript with input from all authors. the order of authors for the co-first author was determined by the contribution of kta in the overall study design, as well as data analysis and manuscript preparation. coronavirus spike protein and tropism changes structure, function, and antigenicity of the sars-cov- spike glycoprotein sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor neutralizing antibodies against sars-cov- and other human coronaviruses broadly neutralizing antiviral antibodies deployment of convalescent plasma for the prevention and treatment of covid- two detailed plaque assay protocols for the quantification of infectious sars-cov- protocol and reagents for pseudotyping lentiviral particles with sars-cov- spike protein for neutralization assays pseudotype neutralization assays: from laboratory bench to data analysis a serological assay to detect sars-cov- seroconversion in humans evidence for sustained mucosal and systemic antibody responses to sars-cov- antigens in covid- patients dynamics of neutralizing antibody titers in the months after sars-cov- infection longitudinal evaluation and decline of antibody responses in sars-cov- infection neutralizing and binding antibody kinetics of covid- patients during hospital and convalescent phases sars-cov- infection induces robust, neutralizing antibody responses that are stable for at least months a sars-cov- surrogate virus neutralization test based on antibody-mediated blockage of ace -spike protein-protein interaction convergent antibody responses to sars-cov- in convalescent individuals structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies clinical and immunological assessment of asymptomatic sars-cov- infections potent neutralizing antibodies from covid- patients define multiple targets of vulnerability a neutralizing human antibody binds to the n-terminal domain of the spike protein of sars-cov- a human monoclonal antibody blocking sars-cov- infection a noncompeting pair of human neutralizing antibodies block covid- virus binding to its receptor ace . science human-igg-neutralizing monoclonal antibodies block the sars-cov- infection evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products simple piggybac transposon-based mammalian cell expression system for inducible protein production structure of bacteriophage t fibritin: a segmented coiled coil and the role of the c-terminal domain site-specific biotinylation of purified proteins using bira immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants chromosomal transposition of piggybac in mouse embryonic stem cells purification and characterization of a recombinant g-protein-coupled receptor, saccharomyces cerevisiae ste p, transiently expressed in hek ebna cells rapid protein production from stable cho cell pools using plasmid vector and the cumate gene-switch optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in cho-ebna cells assays for the assessment of neutralizing antibody activities against severe acute respiratory syndrome (sars) associated coronavirus (scv) a plaque reduction test for dengue virus neutralizing antibodies serum dilution neutralization test for california group virus identification and serology antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera we thank janet mcmanus at canadian blood services for her technical and logistical expertise and the wadsworth center media and tissue culture core. we thank joan wither for the lupus patient samples, and jesse bloom and katharine crawford for sharing protocols and reagents for the lentiviral s pseudotyping assay. key: cord- -uge uodk authors: wang, qiang; du, qin; guo, bin; mu, daiyong; lu, xiaolan; ma, qiang; guo, yangliu; fang, li; zhang, bing; zhang, guoyuan; guo, xiaolan title: a method to prevent sars-cov- igm false positives in gold immunochromatography and enzyme-linked immunosorbent assays date: - - journal: j clin microbiol doi: . /jcm. - sha: doc_id: cord_uid: uge uodk we set out to investigate the interference factors that led to false-positive novel severe acute respiratory syndrome coronavirus (sars-cov- ) igm detection results using gold immunochromatography assay (gica) and enzyme-linked immunosorbent assay (elisa) and the corresponding solutions. gica and elisa were used to detect sars-cov- igm in serum samples, including influenza a virus (flu a) igm-positive sera, influenza b virus (flu b) igm-positive sera, mycoplasma pneumoniae igm-positive sera, legionella pneumophila igm-positive sera, sera of hiv infection patients, rheumatoid factor igm (rf-igm)-positive sera, sera from hypertensive patients, sera from diabetes mellitus patients, and sera from novel coronavirus infection disease (covid- ) patients. the interference factors causing false-positive reactivity with the two methods were analyzed, and the urea dissociation test was employed to dissociate the sars-cov- igm-positive serum using the best dissociation concentration. the two methods detected positive sars-cov- igm in mid-to-high-level-rf-igm-positive sera and sera from covid- patients; the other sera were negative. at a urea dissociation concentration of mol/liter, sars-cov- igm results were positive in mid-to-high-level-rf-igm-positive serum and in covid- patient sera detected using gica. at a urea dissociation concentration of mol/liter and with affinity index (ai) levels lower than . set to negative, sars-cov- igm results were positive in mid-to-high-level-rf-igm-positive sera and in covid- patient sera detected using elisa. the presence of rf-igm at mid-to-high levels could lead to false-positive reactivity of sars-cov- igm detected using gica and elisa, and urea dissociation tests would be helpful in reducing sars-cov- igm false-positive results. t he outbreak of infections by a novel coronavirus (ncov) strain, severe acute respiratory syndrome coronavirus (sars-cov- ), that began in wuhan, china, has spread rapidly throughout the country, as well as to other countries around the world. the outlook of prevention and control of novel coronavirus infection disease (covid- ) is still grim. as of february , the number of confirmed covid- cases exceeded , , and this number has continued to rise steadily, placing enormous emphasis on the timely and accurate diagnosis and treatment of the disease. at present, diagnosis of covid- is mainly based on epidemiological history inquiry, laboratory testing, and chest radiology examination. among these examinations, detection of nucleic acid from sars-cov- represents direct evidence for covid- diagnosis ( ) ( ) ( ) . detection of sars-cov- nucleic acid should be performed in special laboratories by professional technicians, and the assays involved have the disadvantages of insufficient supply of detection kits in a public health emergency, low throughput, and time-consuming procedures. moreover, the swabs taken from the throat may not always reveal the infection of sars-cov- for patients; additional sampling is always performed for accurate diagnosis. therefore, nucleic acid detection may not the best choice for screening large-scale populations infected with sars-cov- ( ) . the detection of serum-specific igm and igg, especially the former, is routinely used in clinical laboratories to evaluate the acute phase of infection of pathogens in the serum ( , ) . in many infections, igm can be detected as early as week after infection. when the level of igm reaches the detection limit of the assay kit, the detection of igm can avoid false-negative results owing to sampling. at present, the main methods for the detection of specific antibodies in clinical laboratories are gold immunochromatography assay (gica) and enzyme-linked immunosorbent assay (elisa) ( ) ( ) ( ) ( ) ( ) , both of which have the advantages of mature methodology, high flux detection, simple operation, rapid detection, lack of the need for special equipment, and low cost. using these two methods to detect sars-cov- igm can identify or screen sars-cov- infection in suspicious and close-contact populations earlier and more quickly and effectively and can improve the accuracy of epidemiological monitoring, which is very important for patient management and epidemic prevention and control. however, in the process of using gica and elisa to detect sars-cov- igm, we found that there was interference from rheumatoid factor igm (rf-igm) in assays using the two methods. the affinity of cross-reactions between specific antigens and antibodies was lower than that of specific reactions ( ) . urea can be used as a substance for dissociation between antigen-antibody reactions to evaluate the affinity of igg, such as the evaluation of the affinity of toxoplasma gondii igg in different detection systems ( , ) . therefore, we hypothesize that the use of the urea dissociation test will help to eliminate or reduce the influence of rf-igm on the detection of sars-cov- igm antibodies. meanwhile, igm-positive sera of other pathogens were collected to evaluate the detection performance of gica and elisa for sars-cov- igm. assay. igm against flu a and flu b, m. pneumoniae, and l. pneumophila was detected by indirect immunofluorescence assay (respiratory tract joint detection kit; euroimmun, inc., germany). rf-igm was detected by rate nephelometry assay (immage , beckman coulter, inc., usa). hiv combi pertussis toxin) (pt) was detected by electrochemiluminescence assay (cobas e ; roche, inc., germany). hiv infection was confirmed by immunoblotting assay (the confirmed information was fed back by cdc). sars-cov- nucleic acid was detected using real-time pcr (rt-pcr) (kit provided by shanghai zhijiang biotechnology co., shanghai, china; detection instrument provided by shanghai hongshi biotechnology co., shanghai, china). gica and elisa were used for sars-cov- igm detection (kit provided by beijing hotgen biotechnology co., beijing, china: lot no. and for gica and lot no. and for elisa). optical density in elisa plates was measured using a microplate reader (phomo; autobio diagnostics co., zhengzhou, china). urea dissociation test of gica. sera ( l) were added into -ml sample diluents (phosphatebuffered saline [pbs], nacl, and tween ) and mixed, and then l of the diluted sample was put into the sample hole of the test card. the liquid was chromatographed upward under the control of the capillary effect; when the liquid was about to reach the upper absorbent paper, l pbs solution urea dissociation test of elisa. sera ( l) were added into -l sample diluents ( . m pbs) and mixed, and then -l volumes of the diluted sample, the negative control, and the positive control were added to the wells of plates coated with sars-cov- recombinant antigen, and the plates were incubated at °c for min. the plates were washed five times, and l of pbs solution (containing mol/liter, mol/liter, mol/liter, mol/liter, mol/liter, and mol/liter urea in different wells) was added and incubated at °c for min. after three more washes, anti-human igm horseradish peroxidase (hrp)-labeled antibody was added into the reaction system to form an indirect immune complex. following five washes to remove unbound substances, the substrate was added for the color reaction. the results were interpreted according to the ratios of the sample optical density value and the cutoff optical density value (s/co) as follows: positive, s/co equal to or greater than . ; negative, s/co less than . . the results of affinity index (ai) analyses were expressed as the ratios of the s/co values determined for different dissociated urea concentrations to that of pbs with mol/liter urea. the ai threshold value was set as the middle value between the highest ai value determined for the falsepositive sample results with the outliers removed and the lowest ai value determined for all of the sars-cov- infection samples. the results were interpreted as follows: positive, ai value of sera greater than or equal to the ai threshold; negative, ai value of sera less than the ai threshold. statistical analysis. statistical analyses were performed by the use of spss, version . (spss inc., usa). fisher's exact test was used for the specific comparisons of gica and elisa results obtained for the detection of sars-cov- igm in sera with positive rf-igm before and after urea dissociation. the specific comparisons between the gica and elisa results obtained for the detection of sars-cov- igm before and after urea dissociation in all control sera were made using pearson's chi-square test. the statistical significance of all tests was defined as a p value of Ͻ . determined by two-tailed tests. (table ; see also table ). comparison of sars-cov- igm results and detection performance before and after urea dissociation test of gica. when the dissociation concentration of urea was mol/liter, the dissociation test of gica was carried out for sera with rf-igm positive results and samples from covid- patients that were positive for sars-cov- igm in gica before urea dissociation. the results of sars-cov- igm analysis performed for serum samples with positive rf-igm turned negative (fig. ) , whereas those for the samples from the covid- patients remained positive. in the urea dissociation test, the specificity of gica after dissociation was significantly higher than before dissociation (p Ͻ . ), and the sensitivity was not affected ( table ) . comparison of sars-cov- igm results and detection performance before and after urea dissociation test of elisa. the urea dissociation test of elisa was carried positive rf-igm turned negative, whereas those of sars-cov- igm in serum samples from covid- patients remained positive (fig. ) . through the urea dissociation test, the specificity of elisa after dissociation was found to be significantly higher than that before dissociation (p Ͻ . ), and the sensitivity was unaffected (table ). sars-cov- infection patients have many clinical symptoms similar to those of patients infected with common respiratory tract pathogens such as flu a, flu b, m. pneumoniae, and l. pneumophila, including fever, fatigue, and cough. moreover, the majority of covid- patients have had preexisting diseases such as diabetes, hypertension, and other endocrine and metabolic diseases ( , ) . therefore, we fully considered the situation described above in selecting the control population for the present study. according to some reports, the bloodwork of sars-cov- infection patients mainly showed decreased lymphocyte counts. therefore, this study also included hiv infection patients with similar phenomena in the control group ( , , ) . the number of patients enrolled in each group was limited owing to insufficient amounts of diagnostic reagents and available time. at the same time, before the completion of the trial, only cases of covid- patients were recruited in our study. although the relatively small sample size inevitably shows some bias in these two methods, the improvement in specificity was clear. rf is an autoantibody against the fc segment of denatured igg, the main type of which is igm. it is the main factor that causes the interference in immune responses ( ) ( ) ( ) . when the sars-cov- igm test was carried out for all control serum and serum from covid- patients by gica and elisa, the results showed that false-positive interference occurred only in rf-igm-positive serum, and the serum results from covid- patients were all positive, indicating that the two methods had high sensitivity but that their specificity needed to be improved. the results of this study showed that when the rf-igm concentration was lower than iu/ml, there was no interference between the two methods in detecting sars-cov- igm. in the other sera with a a mid-to-high level of rf-igm can cause false-positive results of sars-cov- igm detection by gica and elisa. therefore, when serum is rf-igm positive, it is difficult to determine the real sars-cov- igm status. this study attempted to eliminate or reduce the interference by urea dissociation. the main rationale for the selection of the time point used for addition of urea solution in the gica method was as follows: first, after a certain reaction time, the specific antigen antibody may bond more firmly, thus making the procedure of urea dissociation more challenging; second, at that time point, the amount of liquid content in the sample well was small, with little change in urea concentration in the dissociation solution added later, thereby ensuring the dissociation effect. according to our previous study ( ) , the urea dissociation concentration in gica was mol/liter, with the results for of the rf-igm-positive sera that had given false-positive sars-cov- igm results turning negative, whereas the serum samples from covid- patients were not affected. in addition, when the urea dissociation concentration in elisa was mol/liter and the dissociation time was min, the results for of the with rf-igm-positive sera that had given falsepositive results for sars-cov- igm turned negative, whereas the sera from covid- patients were not affected. therefore, the improved gica and elisa outcomes not only ensured detection sensitivity but also improved the corresponding specificity and reliability. the elisa urea dissociation test showed detection performance similar to that of the gica urea dissociation test, and this may have resulted from the use of the same recombinant antigen and the fact that both methods are based on the same detection principle. in conclusion, when gica and elisa are used to detect sars-cov- igm, the level of rf-igm in the serum should be evaluated, and the urea dissociation test should be carried out to avoid the risk of false-positive results. at the same time, the results of this study suggest that the urea dissociation test cannot completely eliminate interference from rf-igm. therefore, when sars-cov- igm results are still positive after urea dissociation, rt-pcr should be used for nucleic acid detection. in addition, it should be emphasized that serological tests represent a method that is complementary to nucleic acid detection. the preferred method for detection of acute disease is via molecular testing, rather than testing for igm, precisely because of the possibility of inaccurate results. on the basis of our research results, we suggest that all of the methods mentioned above should be used to eliminate or reduce the impact of cross-reaction when using gica and elisa methods to detect sars-cov- igm, which will help in the preliminary screening of suspected and high-risk groups, as well as in the assessment, prevention, and control of the sars-cov- epidemic and the formulation of appropriate prevention systems. china novel coronavirus investigating and research team. . a novel coronavirus from patients with pneumonia in china epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study detection of novel coronavirus ( -ncov) by real-time rt-pcr evolving status of the novel coronavirus infection: proposal of conventional serological assays for disease diagnosis and infection monitoring monoclonal antibody-based capture elisa in the diagnosis of previous dengue infection validation of a khv antibody enzyme-linked immunosorbent assay (elisa) a fluorescent nanosphere-based immunochromatography test strip for ultrasensitive and point-of-care detection of tetanus antibody in human serum rapid detection of severe fever with thrombocytopenia syndrome virus via colloidal gold immunochromatography assay study and evaluation of wondfo rapid diagnostic kit based on nano-goldimmunochromatography assay for diagnosis of plasmodium falciparum sensitive detection of respiratory syncytial virus based on a dual signal amplified plasmonic enzyme-linked immunosorbent assay diagnostic laboratory immunology for talaromycosis (penicilliosis): review from the bench-top techniques to the point-of-care testing evidence for a subset of rheumatoid factors that cross-react with dna-histone and have a distinct cross-idiotype value of igg avidity commercial tests used alone or in association to date toxoplasmosis contamination comparative evaluation of three commercial toxoplasma-specific igg antibody avidity tests and significance in different clinical settings notice on the issuance of strategic guidelines for diagnosis and treatment of novel coronavirus (sars-cov- )-infected pneumonia a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster real-time tentative assessment of the epidemiological characteristics of novel corona virus infections in wuhan, china, as of capture-elisa: a new assay for the detection of immunoglobulin m isotype antibodies using chlamydia trachomatis antigen a simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal igg antibodies in plasma from arthritis patients false positive in the measurement of thyroglobulin induced by rheumatoid factor urea-mediated dissociation alleviate the false-positive treponema pallidum-specific antibodies detected by elisa we confirm that we have no conflict of interest related to the generation of the data published in this article. key: cord- -r r bc v authors: morel, noelia; lassabe, gabriel; elola, susana; bondad, mauricio; herrera, silvia; marí, carlos; last, jerold a.; jensen, oscar; gonzalez-sapienza, gualberto title: a monoclonal antibody-based copro-elisa kit for canine echinococcosis to support the paho effort for hydatid disease control in south america date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: r r bc v cystic echinococcosis is still a major concern in south america. while some regions show advances in the control of the disease, others have among the highest incidence in the world. to reverse this situation the pan american health organization (paho) has launched a regional project on cystic echinococcosis control and surveillance. an early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. under this premise, we have developed a new copro-elisa test after extensive screening of a large panel of monoclonal antibodies (mabs) and polyclonal sera, which performs with high standards of sensitivity ( . %) and specificity ( . %) as established by necropsy diagnosis of dogs. the key component of the test, mabeg has a convenient igg isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day post infection. the test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of months at room temperature. this characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion. cystic echinococcosis, caused by infections with echinococcus granulosus, is one of the main zoonoses related to dogs and is affecting most parts of the globe [ ] . different control programs aimed to decrease the impact of the disease have been instrumented in many countries. although the rate of success has been highly variable, it has become evident that a tight control of dog infections is the key element to arrest the life cycle of the parasite. this has created a demand for reliable diagnostic tests for canine echinococcosis as a tool to obtain epidemiological baseline information and help in the surveillance of hydatid control [ ] [ ] [ ] [ ] . however, accurate diagnosis of dog infection is complex and challenging, and other than careful necropsy of dogs, there is no perfect gold standard [ ] . parasitological examination of eggs is unsafe and not very useful because e. granulosus are morphologically difficult to distinguish from other taeniae eggs, and appear late (after the first month) of infection. traditionally, screening of dogs for e. granulosus has been done by arecoline purgation, followed by examination of the purge for parasites by trained personnel. the method is highly specific, but it is tedious, biohazardous, unpopular among dog owners, and its sensitivity is modest, particularly when the parasite burden is low and bowel evacuation is incomplete [ , ] . as an alternative, different laboratory tests for ante-mortem diagnosis of canine echinococcosis have been developed, including detection of antibodies in serum, polymerase chain reaction (pcr) amplification of parasite dna and immunological detection of antigens (coproantigens) in fecal samples. different studies have been carried out to explore the potential use of dog serology to diagnose the disease. however, the systemic immune response to the parasite is poor and consequently the sensitivity attained is also low [ ] . parasite dna excreted with eggs, proglottids or cells has been detected in fecal specimens by pcr amplification using specific primers derived from mitochondrial dna [ , ] . the method has provided the high specificity of pcr, but due to the presence of inhibitors the dna extraction procedure is cumbersome and the technique requires expensive reagents and specialized laboratories [ ] . in addition, the pre-patent period of the infection, when there is no egg production, is a critical time-window that challenges the sensitivity of the test. introduced at the beginning of the 's, the detection of parasite antigens in fecal samples by immunoassays became a widely accepted diagnostic test [ , ] . the major attractive features of the method include its simplicity, the possibility of detecting parasite components even in the prepatent period, and the fact that the target antigens (coproantigens) are highly stable. samples can be collected in % of % formaldehyde and kept at room temperature for extended periods of time, facilitating the logistics of large-scale studies in remote areas [ , [ ] [ ] [ ] . the study of a large group of infected dogs necropsied at the end of the pre-patent period demonstrated a superior sensitivity for the copro-elisa ( %), when compared to copro-pcr ( %) or arecoline purgation ( and % after one or two doses, respectively). most failures to detect positive infected dogs occurred when the number of worms was less than one hundred [ ] . despite its proven utility and the numerous reports of copro-elisa developments, the availability of commercial or homemade tests is often a major obstacle to the implementation and evaluation of echinococcosis control programs. the need to overcome this difficulty has been recognized as an early goal of the southern cone sub-regional project on cystic echinococcosis control and surveillance: argentina, brazil, chile and uruguay, and paho (pan american health organization), a joint and collaborative effort to battle the disease in the region. in this framework, we have developed an immunoassay for e. granulosus copro-antigen detection under the premise that in addition to performing with high standards of proven sensitivity and specificity, it had to be robust, standardized and developed in a kit format to be available for its use in regional programs for the control of the disease. all activities involving animals were performed with special care to establish high standards of biosafety and assure animal welfare. all protocols were performed according to the international guiding principles for biomedical research involving animals, (cioms) and were approved by the comisión nacional de zoonosis and the research department of the ministry of health of the province of chubut. nineteen adult dogs, of mixed breeds and sex, were vaccinated against distemper encephalomyelitis, parvovirosis, rabies, leptospirosis, hepatitis and coronavirus (merial, france), treated orally with praziquantel mg/kg, pyrantel embonate mg/kg and febantel mg/kg (disper, uruguay) to eliminate worm parasites, and topically with a solution of fipronil and methoprene (merial) to arrest the development of ectoparasites. dogs were purchase from local suppliers and maintained on commercial dog food and water ad libitum. nineteen fecal samples (n -n ), corresponding to non-infected dogs, were collected two days after deworming. then, ten dogs (identified as p -p ) were infected orally with , - , viable (. %) protoscoleces from ovine fertile hydatid cysts. two additional dogs (identified as th and th ) were infected orally with and larvae of taenia hydatigena, respectively. parasite material was obtained from sheep slaughter in local abattoirs in paysandú, uruguay. stool samples from each dog were collected daily during the experimental period, at the end which ( - days post-infection (dpi)), the animals infected with e. granulosus were euthanized for necropsy diagnosis by intramuscular injection of acepromazine maleate ( . mg/kg) and ketamine ( mg/kg) (laboratorio holliday, argentina), following by an intravenous overdose of sodium thiopental (laboratorio crusur vet, uruguay). the dogs infected with t. hydatigena were purged at dpi with arecoline bromhydrate (crescent chemical co. inc., new york, usa), following by treatment with anti-parasitic drugs. three additional samples from dogs experimentally infected with e. granulosus ( dpi) and two with t. hydatigena (about dpi) from previous studies [ , ] were obtained as a kind gift from dr. carlos carmona. in addition, unwanted dogs from the province of chubut, argentina, and unwanted dogs from junin, peru, were euthanized following the procedure described above, and the small intestines were removed post mortem, opened longitudinally and examined directly for the presence of parasites following the recommendations of the who/oie [ ] . all activities were performed following the local legislation and the international guiding principles for biomedical research involving animals (http://www.cioms.ch/). stool samples from each dog were collected daily during the experimental infection period, or from the rectum upon necropsy, in % of % formaldehyde, phosphate buffered saline (pbs), in a : v/v ratio. samples were vigorously shaken to obtain homogeneous slurries, and were then boiled for min in a water bath, to eliminate their biological risks. after centrifugation for min at , g the supernatants were aliquoted and kept frozen at uc until used. the parasite antigens were prepared essentially as described by casaravilla et al. [ ] . cystic echinococcosis, caused by infection with the larval stage of the echinococcus granulosus tapeworm, is a lifethreatening zoonosis of worldwide distribution. the adult worm parasitizes the small intestine of dogs, which become infected after eating offal of an animal contaminated with the parasite, and releases eggs into the environment that can be accidentally ingested by domestic animals or humans, maintaining the life cycle of the parasite. deworming of dogs is a major component of control programs, and simple and reliable methods are needed to monitor the base-line infection in the canine population. the lack of these tests was recognized as a major obstacle to the paho effort to control the disease in south america. this paper describes the development of a diagnostic assay that detects parasite antigens in dog feces. the key component is a monoclonal antibody carefully selected to attain high levels of sensitivity and specificity, which were established with a large panel of field fecal samples obtained from animals diagnosed by necropsy. several aspects of the long-term stability of the test were optimized to facilitate its shelf-life and transference to other laboratories. unit with an ym- membrane (millipore, usa), followed by dialysis against pbs. e. granulosus somatic antigen (sm) was obtained by sonication of adult parasites in pbs supplemented with ultra protease inhibitor tablets (roche, indianapolis) on ice, centrifugation and filtration ( . mm). antigens from t. hydatigena were similarly prepared. protein content was determined using a bca kit (pierce, rockford, illinois). to prepare polyclonal antibodies, mg of sm or e/s antigens were dissolved in ml of pbs and vigorously mixed with ml of freund's complete adjuvant (pierce, illinois) to form a thick emulsion. this emulsion was then injected subcutaneously into several points on the back of new zealand white rabbits. after and weeks, the animals were immunized intramuscularly with additional doses of mg of antigen emulsified in freund's incomplete adjuvant. ten days after the final booster the animals were bled. the igg fraction was prepared by precipitation of the sera with ammonium sulfate, affinity purified on protein g agarose (amersham, uppsala, sweden), and kept at uc until used. for monoclonal antibody (mab) preparation, balb/c mice were primed intraperitonally with mg of e. granulosus sm or e/s antigen in freund's complete adjuvant, and boosted after and weeks with the corresponding antigen using freund's incomplete adjuvant. three days after the last booster mice were sacrificed and the splenocytes fused with sp / cells using standard procedures. cultures producing mabs reactive with the corresponding antigen were selected by elisa. we initially performed a fusion experiment using mice immunized with alternate doses of e/s and sm antigens, and additional selection in a sandwich format using fecal samples. mab cpr was selected due to its convenient isotype and performance. several fusion experiments were performed to produce a large panel of monoclonal antibodies. the screening of the monoclonal antibodies was initially performed as described before, and then each of the double-positive supernatants ( clones) was tested in a sandwich format in combination with the different polyclonal antibodies. however, this time instead of using fecal samples from heavily infected dogs, each supernatant was tested against the following: i) a negative sample from a non-infected dog, ii) a sample collected from a low-burden dog at dpi (dog p , worms), iii) a sample from a heavily infected dog taken at an early stage ( dpi) of the pre-patent period (dog p , worms), and iv) a sample from dog th taken at dpi that was positive in the cpr copro-elisa. out of this complex screening clone mabeg , in combination with polyclonal antibody pabc (prepared from a rabbit immunized with e/s), was chosen because this antibody pair produced high signals with samples (ii) and (iii), and low readings with samples (i) and (iv). parasite preparations were resolved by sds-page % under reducing conditions and then transferred onto nitrocellulose sheets (bio-rad, hercules, california, usa). the nitrocellulose was blocked with % non-fat milk powder in pbs h at uc and was incubated for h at uc with a : dilution of the rabbit antisera in pbs containing . % of tween (pbs-t), % non-fat milk, or directly with the culture supernatants of the mabs antibodies. the membranes were then incubated for h at uc with alkaline phosphatase-conjugated to anti-rabbit igg or antimouse igg (pierce) (diluted / ). after extensive washing, a substrate solution containing -bromo- -chloro- -indolylphosphate p-toluidine salt (bcip) and nitro-blue tetrazolium chloride (nbt) was added according to the manufacturer's instructions (sigma). five mg/ml of polyclonal igg ( ml/well) (pab cg or pabc for the mab cpr or mabeg copro-elisa, respectively) was dispensed into microtitration plates (greiner, germany) and incubated overnight at uc. the plates were then blocked with % non-fat milk (pbs) and washed with pbs-t. alternatively, the plates were further treated with pbs, . % bovine serum albumin (bsa), % sucrose, . % sodium azide, flapped repeatedly against adsorbent paper, dried in a % relative humidity chamber for h, and kept in sealed aluminum foil bags containing adsorbent packets (sigma) until used. samples were analyzed in triplicates, after a : dilution in pbs; ml/well were incubated for h at room temperature, the wells washed and loaded ( ml/well) with a : dilution of the mab supernatants and incubated for h at room temperature. finally, the wells were incubated ( h upon necropsy at - dpi, of the experimentally infected dogs p -p dogs harbored e. granulosus worms. the number of worms recovered from each dog varied widely, from to , , and showed no correlation with the initial number of protoescoleces used for infection. no other parasites were observed in addition to the e. granulosus worms. adult worms (typically less than m long) were recovered from dogs th and th infected with t. hydatigena. after careful washing, the recovered parasites were kept in culture to prepare excretion/secretion antigens, or were sonicated as described to obtain somatic antigens. the sds-page analysis of these antigens is shown in figure a ; a protoescoleces extract was also included as a reference antigen. all preparations possessed a complex composition and the sds-page profile did not reveal any obvious similarities among the individual components of the different preparation. initially, the reactivity of polyclonal antibody pabcgb raised against the sm antigen was characterized, figure b . there was a strong response to a large number of components of the immunizing antigen, as well as to high molecular weight components of ege/s and t hydatigena. destruction of sugar epitopes by periodate treatment significantly decreased the reactivity of this antibody with the somatic antigen, as has been observed before [ , ] , but had little effect in the case of the e/s preparation. the marked cross-reactivity of the polyclonal antibodies with e/s components of t. hydatigena evidences how difficult it is to obtain a specific assay using these antibodies as reagents. for that reason we concentrated our efforts in the preparation and selection of monoclonal antibodies after initial screening against e/s and sm and further selection using pab cgb for capture and the hybridoma supernatants for detection, four clones providing the best signal/noise ratios with fecal samples were selected. among these, clone mabcpr , secreting an igg b, was chosen. this elisa was used to examine fecal samples obtained at the end of the infection, days - for e. granulosus or at selected dpi (highest cross-reactivity) for t. hydatigena ( figure ) . the assay performed with negligible reactivity with samples from noninfected dogs and moderate cross-reactivity with t. hydatigena infected animals. all e. granulosus infected dogs were positive, however, the readouts of the samples corresponding to dogs with small parasite burdens were significantly higher, but too close to the mean value of the negative dogs to give unequivocal results. the assay was then used to study the time course of coproantigen excretion during the experimental infection period ( figure ) . using a cut-off derived from the analysis of field samples (see below), we found that dogs with high parasite burdens became positive after two weeks, and in spite of some fluctuations remained so thereafter. on the other hand, dogs with less than worms at necropsy, presented low readouts throughout the experiment, becoming positives only at the end of the experimental infection. along the time course of the examined period, the dogs harboring t. hydatigena worms presented very low values (below the cut-off), showing positive values only in very few days after dpi (not shown). due to the limited sensitivity of the cpr copro-elisa, new antibodies were prepared. the best combination resulted when pab c was used as capture antibody in combination with mab eg . the reactivity of pabc with different parasite antigens in western blot was similar to that of pabcgb (figure b) and it is not shown. mabeg is an igg; its reactivity with different parasite extracts is displayed in figure c . the antibody reacted with a large number of the egsm bands, mostly in the middle to high molecular weight range. two components of and kda are the most prominent immunoreactive bands of the e/s antigens, which appear to be also present in the sm preparation. the crossreactivity with th e/s antigens was negligible (as was also the case with protoscoleces antigens, not shown). treatment with periodate had no effect upon the reactivity of mabeg with any of the antigens, which was unexpected because previous studies have shown that the sugar epitopes have a major role in the immune response against e. granulosus coproantigens [ , ] . to rule out technical artifacts, the efficacy of the periodate treatment was tested in parallel using the monoclonal antibody e /g that defines a sugar epitope highly expressed in e. granulosus protoscoleces preparations [ ] , figure d . when the experimentally-infected dog samples were analyzed with the new assay, it was evident that the eg copro-elisa produced stronger signals with samples from dogs with small numbers of worms, allowing a better discrimination between noninfected and infected dogs ( figure ) . this is an important improvement because most of the reported tests failed to detect these kind of samples. cross-reactivity with t. hydatigena coproanti-gens did not seem to be a major problem when the time course of coproantigen excretion was study with the eg copro-elisa ( figure ). using the cut-off estimated by the receiver operating characteristic (roc) curves from the analysis of field samples (see below), only on very few occasions and after dpi were some of the samples from t. hydatigena infected dogs positive. the eg copro-elisa allowed an earlier detection of infected dogs. animals with or more parasites became positive between - dpi and by dpi all infected dogs were positives and remained so thereafter. curiously, coproantigens in dogs p and p samples could be detected very early. this is more remarkable in the case of dog p that harbored a rather small number of worms ( ) at necropsy, and may be the result of the spontaneous expulsion of worms a few days after infection. the cut-off and cross-reactivity of the assays were evaluated with field samples collected from unwanted dogs in the provinces of chubut, argentina and junin, peru. upon necropsy, of dogs from chubut, and of dogs from junin were positive for e. granulosus, were infected with other parasites and were found negative, table . fecal samples from these dogs were tested using the cpr and eg copro-elisas and the cutoff selected from receiver operating characteristic (roc) curves [ ] to have a convenient balance between sensitivity and specificity as follows: cpr elisa = . au and eg elisa = . au. using these values, the sensitivity of the tests were . and . % respectively, which is in agreement with the results obtained with the experimental infection of dogs, figure . the specificity of the eg test was also superior . % versus . % for the cpr elisa. in addition, the cpr test produced readings close to the cut-off for an important number of samples, figure , which may be problematic for the use of a cutoff internal calibrator, because small inter-assay variations in the reading of the calibrator may have an important effect upon the number of samples that are classified in either category. actually, if samples that fall between % of the cut-off are considered as undetermined (a common practice), none of the samples analyzed with the eg test would fall in this category, while four of the samples analyzed with the cpr would. while it was not possible to obtain an exact counting of parasites in the dogs from the province of chubut, this was feasible in the case of the animals necropsied in junin. although most dogs had a large number of worms, two of them did not, and these two dogs were exaustively examined. we found only e. granulosus and ascaris lumbricoides, and e. granulosus and very few dipylidium caninum worms in the first and second dogs, respectively. interestingly, the average absorbance readings of the fecal samples obtained from these animal were . and . au, which represent strong positive results, indicating the sensitivity of the test and confirming the capacity of the assay to detect small numbers of parasites that had been observed in the experimentally infected dogs. in this regard, when the e. granulosus positive dogs from the experimental infection are included, the overall sensitivity of the test rises to . %. despite the fact that mabeg was selected for its reduced cross-reactivity with the thsm antigen, most of its cross-reactivity was against dogs infected with t. spp (mostly hydatigena) alone or together with other parasites. only one dog infected with t. canis ( . au) and one of the non-infected dogs ( . au) from the province of chubut were classified as positive with the eg elisa, figure . the relatively high readings of these two samples are unexpected. based on the results of the experimental infection of dogs and the fact that all other samples ( ) from dogs harboring t. canis alone or together with dipylidium caninum showed very low readings, a misdiagnosis at necropsy can not be discarded. it is worth noting that cross-reactivity was essentially against taenia. despite the fact that we could not identify to species all field samples, the great majority of samples from chubut and all from junin corresponded to t. hydatigena; hence most false positive results will be related to dogs eating offal. development of the elisa test in a kit format and interlaboratory analysis of blind samples the eg test was then formulated in a kit format to facilitate its use and transference to other laboratories. initially, we studied different conditions for coating and drying the plates and found that using sugars as additives during drying have the best effect on the stability of the capture antibody. trehalose and sucrose showed the best results and the latter was chosen because of its much lower cost. figure a shows that dried plates showed similar capacity as fresh ones to discriminate between weak positive and negative samples over a one year period of storage at uc. similar results were obtained after years of storage (not shown). to set up the value of the cut-off an internal standard was prepared by adjusting the dilution of the e. granulosus sm antigen in order to produce a readout equal to the value of the cut-off. the stability of this cut-off calibrator solution and other components of the assay (in their ready-to-use dilution) were tested using different diluent buffers at uc, room temperature, and uc, and some representative results are shown in figure b-d. different formulations of the calibrator solution were stable over a month period, even at uc; only the results for the sm antigen diluted in pbs, % glycerol, . % kathon (dow chemicals, midland, michigan) are shown. the reactivity of mab eg started to diminish after a few days at uc, but was highly stable over the month period at uc (not shown) or at room temperature, figure c . the most critical component was the secondary antibody. for several different formulations (only two shown in figure d ) all of them rapidly lost the enzymatic activity at uc, but using pbs, . % bsa, . % kathon, . mm tmb, the conjugate remained stable for up to days at room temperature (or uc, not shown). overall, we can conclude that even at room temperature the kit has a safe shelf life of months, which can surely be extended upon refrigeration, which is a convenient advantage for the shipment and use of the kit in remote places. due to the availability of commercial ready-to-use tmb substrate solutions, the preparation of this kit component was not pursued in this study. in case of high demand of the kit, it will be necessary to prepare new pab. in our experience, this is a critical component of the test, but we have selected similarly performing pabs from a panel of to immunized rabbits, after initial selection with fecal samples from dogs harboring low number of e. granulosus parasites and counter selection with samples from t. hydatigena infected dogs. the reproducibility of the results obtained with the copro-elisa kit and the feasibility of its transference were demonstrated by the analysis of blind samples. to this end, fecal samples collected in the province of junin, peru were processed as described, and aliquots were distributed to two laboratories (ins and digesa) in lima, peru, where the copro-elisa kit had been transferred, and to our laboratory in uruguay (facultad de química). the samples were independently analyzed by the three laboratories and classified as positive, negative or uncertain if their readout felt within the calibrator absorbance reading % range. twenty four samples were positive, indicating a prevalence of . % in the studied areas (canchayllo and jauja). the kit performed with excellent reproducibility and all individual samples were equally classified by the three laboratories with the exception of two of the samples with readings close to the cut-off, one of them categorized as negative by two of the labs and uncertain by the third one, and another sample classified as (weak) positive by two of the labs and uncertain by the remaining one. two tests were developed and the difference in their performance highlights the importance that careful selection of antibody pairs has to attain a high standard of sensitivity and specificity. the eg test performed with high sensitivity ( . %) and good specificity ( . %). these parameters are closely similar to previously reported values for other tests. however, it is very important to keep in mind that the only trustworthy comparison of tests is that obtained with a common panel of samples assayed under identical conditions, which highlights the need of interlaboratory studies to compare the performance of existing test. the test cross-reactivity was low, and in addition, since it was basically restricted to t. hydatigena, most false positive results will still indicate access of dogs to offal. a major contribution of our study is the use of a well-characterized mab that assures availability and batch-to-batch reproducibility, as well as the formulation of the assay in a kit format with extended shelf life. the eg test is being shared with other control programs in the region and we hope that it will help to monitor the progress of control programs and standardize the epidemiological baseline information in the region. checklist s stard checklist for the eg copro antigen test. flowchart s stard flow chart detailing the method used to assess the diagnostic performance of the eg copro antigen test. (docx) partial characterisation of carbohydrate-rich echinococcus granulosus coproantigens control of hydatidosis echinococcosis: disease, detection and transmission screening for echinococcus granulosus in dogs: comparison between arecoline purgation, coproelisa and copropcr with necropsy in pre-patent infections echinococcosis: diagnosis and diagnostic interpretation in population studies coproantigens in taeniasis and echinococcosis detection of echinococcus granulosus coproantigens in australian canids with natural or experimental infection copro-diagnosis of echinococcus granulosus infection in dogs by amplification of a newly identified repeated dna sequence polymerase chain reaction for detection of patent infections of echinococcus granulosus (''sheep strain'') in naturally infected dogs copro-dna tests for diagnosis of animal taeniid cestodes coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans detection of echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes coproantigen detection in dogs experimentally and naturally infected with echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay detection of echinococcus granulosus coproantigens in faeces from naturally infected rural domestic dogs in south eastern australia production and characterization of monoclonal antibodies against excretory/secretory products of adult echinococcus granulosus, and their application to coproantigen detection manual on echinococcosis in humans and animals:a public health problem of global concern the meaning and use of the area under a receiver operating characteristic (roc) curve echinococcus granulosus coproantigens: chromatographic fractionation and characterization modulation of the cellular immune response by a carbohydrate rich fraction from echinococcus granulosus protoscoleces in infected or immunized balb/c mice we thank dr. carmona and dr. cecilia casaravilla for their advice and provision of samples. we are also grateful to the medical veterinarians and staff of the comisión nacional de zoonosis, uruguay, for help with the experimental infection of dogs. the personnel and medical veterinarians from the dirección regional de salud de huancayo, peru, are also acknowledged for their help with the pilot study of dog infection. key: cord- -wwdqh qs authors: guzman, norberto a.; guzman, daniel e. title: a two-dimensional affinity capture and separation mini-platform for the isolation, enrichment, and quantification of biomarkers and its potential use for liquid biopsy date: - - journal: biomedicines doi: . /biomedicines sha: doc_id: cord_uid: wwdqh qs biomarker detection for disease diagnosis, prognosis, and therapeutic response is becoming increasingly reliable and accessible. particularly, the identification of circulating cell-free chemical and biochemical substances, cellular and subcellular entities, and extracellular vesicles has demonstrated promising applications in understanding the physiologic and pathologic conditions of an individual. traditionally, tissue biopsy has been the gold standard for the diagnosis of many diseases, especially cancer. more recently, liquid biopsy for biomarker detection has emerged as a non-invasive or minimally invasive and less costly method for diagnosis of both cancerous and non-cancerous diseases, while also offering information on the progression or improvement of disease. unfortunately, the standardization of analytical methods to isolate and quantify circulating cells and extracellular vesicles, as well as their extracted biochemical constituents, is still cumbersome, time-consuming, and expensive. to address these limitations, we have developed a prototype of a portable, miniaturized instrument that uses immunoaffinity capillary electrophoresis (iace) to isolate, concentrate, and analyze cell-free biomarkers and/or tissue or cell extracts present in biological fluids. isolation and concentration of analytes is accomplished through binding to one or more biorecognition affinity ligands immobilized to a solid support, while separation and analysis are achieved by high-resolution capillary electrophoresis (ce) coupled to one or more detectors. when compared to other existing methods, the process of this affinity capture, enrichment, release, and separation of one or a panel of biomarkers can be carried out on-line with the advantages of being rapid, automated, and cost-effective. additionally, it has the potential to demonstrate high analytical sensitivity, specificity, and selectivity. as the potential of liquid biopsy grows, so too does the demand for technical advances. in this review, we therefore discuss applications and limitations of liquid biopsy and hope to introduce the idea that our affinity capture-separation device could be used as a form of point-of-care (poc) diagnostic technology to isolate, concentrate, and analyze circulating cells, extracellular vesicles, and viruses. biomarkers are measurable substances or characteristics in the human body that act as indicators of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions [ ] [ ] [ ] [ ] . in practice, biomarkers include tools and technologies that have been used in clinical medicine for decades, and they play a critical role in all aspects of prevention, diagnosis, and treatment of disease [ ] . despite advances in laboratory technology and the availability of significant information on biomarkers, we are quite far from widespread clinical use of biomarkers due to several limitations. the slow, arduous, and challenging process by which biomarkers are translated into clinical use, makes them suffer from low sensitivity, specificity, and predictive value, particularly when they are applied to rare diseases in population screening programs [ ] . as a consequence, medicine in general, has long been criticized for lagging behind other industries in both innovation and adoption [ ] . however, with a new wealth of biomarker information obtained over the last decade and advancements in systems medicine, there are opportunities to make significant improvements to diagnostics and therapeutics in medicine [ ] . in this manuscript, we describe a point-of-care biomarker analyzer that has been demonstrated to identify and quantify circulating cell-free small molecules and complex biomolecules in biological fluid samples by utilizing an affinity capture-separation technology. furthermore, we will discuss the potential of this instrument to serve as a tool for detecting and analyzing cellular and subcellular entities, vesicular entities, viruses and their respective cargo contents. the ultimate goal of this biomarker analyzer is to demonstrate the potential to non-invasively and rapidly yield highly sensitive and selective tests to aid real-time clinical practice. the existence of different meanings for certain terminologies in clinical diagnostic chemistry and analytical chemistry is a source of confusion related to several laboratory assays, particularly immunoassays. in clinical diagnostic chemistry, a sensitive test refers to a test that will correctly identify almost all individuals who likely have a disease, and that will rarely yield a false-negative result. a specific test refers to a test that will almost always correctly rule out those who do not have a disease and will rarely yield a false-positive result [ ] . in analytical chemistry, sensitivity is the minimum detectable concentration of an analyte, expressed as the limit of detection (lod), that can be accurately measured. specificity is the ability to accurately assess a single analyte in the presence of components which may be expected to be present in the sample matrix, such as interferences. similar to specificity, selectivity is the ability to accurately separate out and assess multiple components present in a sample mixture or matrix [ ] . biopsy is another term that historically has referred to an invasive examination of tissue removed from a living body to discover the presence, cause, or extent of a disease [ ] . the term biopsy has evolved to include both tissue biopsy and liquid biopsy, a non-invasive or a minimally invasive procedure used for the detection and isolation of circulating tumor cells, circulating tumor dna, and exosomes circulating in biological fluids [ , ] . in turn, liquid biopsy has allowed for further prognostication and evaluation of therapeutic response in patients with malignancy. recent studies have shown that liquid biopsy has applications in non-cancerous diseases, allowing for the examination of cells, subcellular entities or vesicles, and their chemical and biochemical contents to discover the presence, cause, or extent of any disease [ , ] . for example, the use of liquid biopsy via a high-throughput metagenomic sequencing assay has recently demonstrated both detection of a diverse array of bacterial and viral pathogens and quantification of damage to host tissues. the assay implements whole-genome sequencing of cell-free dna (cfdna), small fragments of dna released by host or microbial cells into blood, urine, and other body fluids [ ] . similarly, proteins derived from the novel coronavirus (sars-cov- ) have been analyzed by high-performance liquid chromatography (hplc) coupled to mass spectrometry (ms) from samples obtained from coronavirus disease (covid- ) patients [ ] . though the concept of a miniaturized or personal laboratory has been voiced for several years [ , [ ] [ ] [ ] , the creation of a modular design that is manufactured with compact and low-weight components for easy portability has proved to be difficult. capillary electrophoresis is a separation technique that has the potential to address this limitation as it utilizes miniaturized components, including a small power supply and detectors that can fit into a small space, thereby facilitating the manufacture of an instrument no larger than the size of a lap-top computer or smaller device [ , , ] . instruments developed for ce use are manufactured in two formats: a conventional platform using fused-silica capillaries, and a microfluidic chip format consisting of microchannels etched or molded into a material made of glass, silicon, or a polymer such as polydimethylsiloxane [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . newer microfabrication techniques in particular are advancing, and microchip production using d is now under development [ , ] . overall, ce offers advantages such as reduced sample and reagent consumption, lower operating costs, shorter reaction time, better portability, higher number of theoretical plates, and higher reliability [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite many advances in the ce technology, there is still a limited number of medical applications that are routinely performed in a clinical laboratory. before it can replace existing technologies currently used in clinical laboratories, ce has its own limitations that must be addressed. for example, the internal diameters of the capillaries and micro-channels utilized in ce are usually less than micrometers, allowing the utilization of small volumes of fluids which are normally in the order of nanoliters and picoliters. consequently, the limitations of introducing small volumes of sample into the capillary column or channel results in poor concentration limits of detection, and hence risks overshadowing the many benefits of the ce technology [ , , [ ] [ ] [ ] [ ] [ ] [ ] . this is particularly concerning given that crucial biomarkers found in biological fluids are present in low concentrations which, if not detected and quantified, may lead to missing an important diagnosis. a misdiagnosis or delayed diagnosis further leads to incorrect treatment, delayed treatment, or no treatment at all, thereby worsening a patient's medical condition. unfortunately, most examples reported in the literature for the ce quantification of analytes in complex matrices are usually developed for substances found at large concentrations. while not all diagnostic errors are secondary to laboratory errors, the national academy of medicine estimates that all patients will experience one serious diagnostic error during their lifetime, and diagnostic errors are now the leading cause of medical malpractice claims [ ] . overcoming the poor analytical sensitivity of ce has therefore become the emphasis of many investigations related to life science applications. promisingly, efforts to improve the analytical sensitivity of ce thus far have led to successful improvements in the limits of detection of substances found at low concentrations. some of these methods include field-amplified sample stacking, large-volume sample stacking, ph-mediated sample stacking, on-column isotachophoresis, chromatographic preconcentration, sample stacking for micellar electrokinetic chromatography, sweeping, and derivation of analytes with fluorescent chromophores [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one technology in particular is the combination of solid-phase extraction methods, using affinity-based ligands, with ce. highly selective affinity ligands or adsorbents, such as antibodies, antibody fragments, lectins, aptamers, enzymes, metal-organic framework-based affinity sorbents, and other specially designed ligands are the cornerstone for the isolation and purification of many substances and cellular-subcellular entities found in complex matrices [ , , [ ] [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . immunoaffinity capillary electrophoresis (iace) is a disruptive technique, which combines the use of antibodies and/or other affinity ligands (e.g., lectins, aptamers, metal-organic, or others) as highly selective capture agents with the superior resolving power of capillary electrophoresis. the high-resolution analytical separation ability of iace provides the advantage of characterizing and discerning different forms of the same protein (proteoforms) biomarker and drug metabolites, which can improve insights into disease diagnosis, monitor drug efficacy, and shorten the length of clinical trials. since the introduction of iace in the early s by our laboratory, terry phillips' laboratory, and others, the technology has increased significantly in popularity, and many applications have been reported using both conventional ce and microchip ce [ , , , , [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, if iace is coupled to powerful detectors such as laser-induced fluorescence detectors or mass spectrometers, a significant improvement in limits of detection can been achieved [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . since its inception, iace technology has used a "linear or unidirectional" protocol for the introduction of samples and buffers [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a small area of the capillary known as the "analyte concentrator-microreactor" (acm) zone or device contains affinity ligands immobilized either to a matrix localized within the cavity of the acm device, or directly to the inner wall of a capillary or channel positioned near its inlet side. the term "analyte concentrator" refers to the function of isolating and concentrating a target analyte, usually present in a complex matrix, by using a "chemical or biochemical magnet" [ , , [ ] [ ] [ ] ] . the term "microreactor", on the other hand, refers to the function of performing a chemical, biochemical and/or cellular-organoid reaction, such as derivatization, chemical synthesis, enzymatic reaction, pharmacokinetic studies, or therapy response-prediction [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the linear or unidirectional iace capture-separation system aimed at performing on-line concentration and separation presents two problems: backpressure and contamination of the surface of the separation capillary or channel after repetitive uses. the backpressure is formed when there is use of a beaded matrix requiring porous frit structures, or when there is a compacted continuous bed composed of a porous micro/nanoscale monolithic structure. the contamination of the surface of the separation capillary or channel is due to the non-specific adsorption of analytes present in the sample. the fused-silica capillary or the microchip made of silica material has free silanol groups to which biomolecules such as proteins bind readily. this non-specific binding leads to changes in migration times and peak areas of the analytes after several uses of the separation capillary or channels. in turn, the altered inner surface of the capillary or microchannel yields irreproducible results and limits the use of the capillary or microchip. to solve these problems, a different approach to introduce samples and buffers was developed, which is known as an "orthogonal" iace capture-separation system. in this format or protocol, samples, buffers, or solutions are introduced into the acm in an orthogonal/perpendicular direction via capillaries: in one direction lie the transport capillaries with their entrance and exit ports, while in the other direction lie the separation capillary with its entrance and exit ports. both the transport and separation capillaries share a small section containing one or more affinity ligands immobilized to a beaded matrix localized within the cavity of the acm device, or immobilized directly to the inner wall of the capillary or channel. microvalves are used to control the direction of the passage of fluids, thus preventing contamination of the separation capillary once a sample has been suctioned or pushed through the transport capillary. the transport entrance port is connected to the transport capillary, thereby allowing samples, buffers, or other solutions to be introduced into the acm device from a container or vessel located at the inlet side of the transport capillary. for this, the microvalves localized at the transport capillary ports are open, and the microvalves localized at the separation capillary ports are closed. the transport exit port allows excess amount of sample or buffer that is passed through to be evacuated into a waste container or vessel. once a sample has been passed through the transport capillary, allowing for the target biomarker to be captured via immunoaffinity or another affinity principle, a cleaning buffer or solution is introduced through the transport capillary to remove non-specific bound materials. at this stage, the microvalves localized at the transport capillary ports are closed, and the microvalves localized at the separation capillary ports are open. a container or vessel containing a separation buffer or solution located at the inlet side of the separation capillary or channel can then be introduced. the container has a platinum-iridium electrode immersed in the buffer or solution that is connected to a high-voltage power supply, having positive and/or negative polarity. another container containing the same separation buffer or solution is localized at the outlet end of the separation capillary, having a second electrode that serves to ground the electrical system. a separation buffer is introduced from the inlet to the outlet side of the separation capillary, followed by a small plug of an elution buffer, either alone or carrying a chromophore, to release or release and derivatize the bound target biomarkers from the biorecognition affinity selectors immobilized to the acm device. a high-voltage power supply, connected to an electrode immersed into the container containing the separation buffer, is then turned on to allow the passage of the target biomarker(s) to the separation capillary exit port. separation of target biomarker(s) can be accomplished by electrophoretic mobility, electroosmotic flow, mechanical pressure, or a combination of electroosmotic flow and mechanical pressure. the system is hermetically sealed using tight fitted connectors to prevent air bubbles to enter into the capillaries. the separation capillary outlet side can be connected to one or several detectors to quantify and/or characterize the separated biomarker(s). most detectors are positioned on-line (uv-absorbance or fluorescence), in-line (amperometry), or off-line (mass spectrometry) at the outlet end of the separation capillary, usually at a single point of detection [ ] . however, when using a charge-coupled device (ccd) camera detection system, it is possible to capture the sample zones in motion during the migration through the capillary [ ] . figure depicts a schematic representation of the two models of acm devices. both containing biorecognition affinity ligands or selectors immobilized to a matrix localized within the cavity of the acm device. the acm devices are often referred to as solid-phase extractor devices or simply spe devices. the term "acm device" is more appropriate because of the dual functionality of the device: it functions as both an on-line preconcentrator and an on-line microreactor. a major advantage of the orthogonal iace design is that it permits the protection of the inner surface of the separation of the capillary or channel during the sample introduction and cleaning process. four micro-valves positioned at each entrance-exit of the acm device can be controlled manually or by a computer. when the microvalves located at the entrance-exit of the transport capillary are open and the microvalves located at the entrance-exit of the separation capillary are closed, there is no contact of the sample and cleaning the unidirectional iace design is operated without the presence of microvalves, whereas the orthogonal iace design requires the presence of microvalves, as depicted in (b) with green circles. the orthogonal iace device has four microvalves positioned at each entrance-exit port. these microvalves are crucial in controlling the path of fluids through the transport capillary or separation capillary. black arrows indicate flow direction of buffers in the separation capillary, and migration direction of the separated analytes within the separation capillary; purple arrows indicate the flow direction of sample and cleaning buffers introduced into the transport capillary. biorecognition affinity capture ligands or affinity capture selectors are immobilized to a beaded or monolithic structure positioned within the cavity of the "analyte concentrator-microreactor" (acm) devices, or immobilized directly to the inner surface of the cavity of the acm device. the affinity capture selectors can be antibodies, antibody fragments, lectins, aptamers, enzymes, phages, receptors, protein a, protein g, or a variety of substances having affinities for different kind of substances. figure modified from [ ] and [ ] . numerous immunoaffinity capillary applications employing various configurations of the acm device, using either a conventional capillary electrophoresis or a microfluidic chip capillary electrophoresis, have been reported [ , [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . figure depicts the use of an iace selective affinity capture-separation system for the quantification of brain-derived neurotrophic factor (bdnf), a neuropeptide growth factor obtained from human skin biopsies of atopic inflammatory reactions [ ] . figure a is a schematic representation of an acm device where whole tetrameric antibodies, nanobodies, antibody fragments, such as fab, or other affinity ligands, such as aptamers and lectins, can be directly immobilized to its inner surface, instead of using a beaded or monolithic matrix support as depicted in figure . since the acm device is not yet commercially available, each laboratory has made its own modifications to the original protocol published elsewhere for use in conventional or microchip capillary electrophoresis [ , , , ] . for the protocol carried out in these experiments, streptavidin was first immobilized off-line to the surface of -mm glass disks serving as a solid support. this was followed by the addition of biotinylated the unidirectional iace design is operated without the presence of microvalves, whereas the orthogonal iace design requires the presence of microvalves, as depicted in (b) with green circles. the orthogonal iace device has four microvalves positioned at each entrance-exit port. these microvalves are crucial in controlling the path of fluids through the transport capillary or separation capillary. black arrows indicate flow direction of buffers in the separation capillary, and migration direction of the separated analytes within the separation capillary; purple arrows indicate the flow direction of sample and cleaning buffers introduced into the transport capillary. biorecognition affinity capture ligands or affinity capture selectors are immobilized to a beaded or monolithic structure positioned within the cavity of the "analyte concentrator-microreactor" (acm) devices, or immobilized directly to the inner surface of the cavity of the acm device. the affinity capture selectors can be antibodies, antibody fragments, lectins, aptamers, enzymes, phages, receptors, protein a, protein g, or a variety of substances having affinities for different kind of substances. figure modified from [ , ] . numerous immunoaffinity capillary applications employing various configurations of the acm device, using either a conventional capillary electrophoresis or a microfluidic chip capillary electrophoresis, have been reported [ , [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . figure depicts the use of an iace selective affinity capture-separation system for the quantification of brain-derived neurotrophic factor (bdnf), a neuropeptide growth factor obtained from human skin biopsies of atopic inflammatory reactions [ ] . figure a is a schematic representation of an acm device where whole tetrameric antibodies, nanobodies, antibody fragments, such as fab, or other affinity ligands, such as aptamers and lectins, can be directly immobilized to its inner surface, instead of using a beaded or monolithic matrix support as depicted in figure . since the acm device is not yet commercially available, each laboratory has made its own modifications to the original protocol published elsewhere for use in conventional or microchip capillary electrophoresis [ , , , ] . for the protocol carried out in these experiments, streptavidin was first immobilized off-line to the surface of -mm glass disks serving as a solid support. this was followed by the addition of biotinylated antibody reacting with bdnf. the prepared disks containing the antibodies to bdnf were then transferred and placed in an area of the microchip termed the immunoaffinity port. extracted material obtained from frozen tissue biopsies, using a detergent-containing solution, was incubated with the immobilized antibody. the captured bdnf was then conjugated with a fluorescent dye, followed by elution and separation of the derivatized bdnf neuropeptide in the separation channel. detection was carried out using a laser-induced fluorescence detector. figure b demonstrates that the concentration of bdnf became elevated over a -h sampling period in a patient undergoing a severe reaction. figure c shows that the iace technology is able not only to distinguish differences in the severity of the lesion but could also detect different patterns in analyte concentration at different sampling sites. for example, at the center of the lesion with the highest inflammation area, elevated concentrations of bdnf were found; however, at and mm apart from the lesion, the concentration of bdnf started to decline significantly. antibody reacting with bdnf. the prepared disks containing the antibodies to bdnf were then transferred and placed in an area of the microchip termed the immunoaffinity port. extracted material obtained from frozen tissue biopsies, using a detergent-containing solution, was incubated with the immobilized antibody. the captured bdnf was then conjugated with a fluorescent dye, followed by elution and separation of the derivatized bdnf neuropeptide in the separation channel. detection was carried out using a laser-induced fluorescence detector. figure b demonstrates that the concentration of bdnf became elevated over a -h sampling period in a patient undergoing a severe reaction. figure c shows that the iace technology is able not only to distinguish differences in the severity of the lesion but could also detect different patterns in analyte concentration at different sampling sites. for example, at the center of the lesion with the highest inflammation area, elevated concentrations of bdnf were found; however, at and mm apart from the lesion, the concentration of bdnf started to decline significantly. figure . schematic representation of a fritless acm device without the presence of a matrix composed of beads, sol-gel, or monolithic materials utilized in conventional capillary electrophoresis or microchip capillary electrophoresis. immobilization of the affinity ligands occurs directly onto the inner wall of the acm device (a). the protocol for sample processing is shown in four steps: ( ) covalent immobilization of an antibody, directed against brain-derived neurotrophic factor (bdnf) neuropeptide used as affinity capture material, directly to the inner wall of a microchip channel (alternatively, -mm glass beads immobilized with streptavidin to which a biotinylated antibody against bdnf is coupled, can be inserted into the main channel of the microchip or in an immunoaffinity port of a specially designed microchip); ( ) binding of immunoreactive bdnf neuropeptide present in skin biopsies to the immobilized affinity capture antibody; ( ) cleaning excess amounts of unwanted non-specifically-bound material and equilibration of the capillary system; ( ) elution of the reversibly specifically-bound bdnf neuropeptide. (b) depicts the sequential iace electropherograms of bdnf concentrations present in skin biopsies taken at different time points in a patient with severe atopic dermatitis illustrating the concentrations of detectable bdnf over time. graph (c) illustrates the concentrations of bdnf in skin biopsy of a patient with severe atopic dermatitis and control groups measured by iace at h post-initial onset of the reaction. the green color bars represent the amount of bdnf measured within the lesion, while the orange color bars represent the background amounts of bdnf measured in tissues taken mm from the periphery of figure . schematic representation of a fritless acm device without the presence of a matrix composed of beads, sol-gel, or monolithic materials utilized in conventional capillary electrophoresis or microchip capillary electrophoresis. immobilization of the affinity ligands occurs directly onto the inner wall of the acm device (a). the protocol for sample processing is shown in four steps: ( ) covalent immobilization of an antibody, directed against brain-derived neurotrophic factor (bdnf) neuropeptide used as affinity capture material, directly to the inner wall of a microchip channel (alternatively, -mm glass beads immobilized with streptavidin to which a biotinylated antibody against bdnf is coupled, can be inserted into the main channel of the microchip or in an immunoaffinity port of a specially designed microchip); ( ) binding of immunoreactive bdnf neuropeptide present in skin biopsies to the immobilized affinity capture antibody; ( ) cleaning excess amounts of unwanted non-specifically-bound material and equilibration of the capillary system; ( ) elution of the reversibly specifically-bound bdnf neuropeptide. (b) depicts the sequential iace electropherograms of bdnf concentrations present in skin biopsies taken at different time points in a patient with severe atopic dermatitis illustrating the concentrations of detectable bdnf over time. graph (c) illustrates the concentrations of bdnf in skin biopsy of a patient with severe atopic dermatitis and control groups measured by iace at h post-initial onset of the reaction. the green color bars represent the amount of bdnf measured within the lesion, while the orange color bars represent the background amounts of bdnf measured in tissues taken mm from the periphery of the lesion. the light blue-colored bars represent the amount of background bdnf measured in tissue taken mm from the periphery of the lesion. all values are the mean ± s.e.m. figure modified from [ , ] . neuropeptides and their role in regulating inflammatory processes have become a topic of major interest in clinical medicine. bdnf has been associated with several inflammatory conditions, including asthma, hypoxic lung injury, hypersensitivity reactions, atopic dermatitis, neurological disorders, and type diabetes [ ] [ ] [ ] . in the example presented here for the analysis of bdnf in skin biopsies, iace technology shows similar results when compared to histopathologic analysis. furthermore, the example shows that results obtained with iace can be accomplished in a short period of time, whereas classical histopathology takes several days to complete [ ] . figure depicts a profile demonstrating the data obtained by the two-dimensional iace immunoassay when compared to the mono-dimensional sandwich enzyme-linked immunosorbent assay (elisa) method. two applications are shown utilizing both immunological methods: one is for the determination of alpha- -acid glycoprotein or orosomucoid, and the other is for the determination of two structurally similar exorphins, casomorphins and . the determination of immunoreactive alpha- -acid glycoprotein (agp) in serum has been a challenge due to its high carbohydrate content and the heterogeneity in its glycan structures. there are many agp isoforms or proteoforms of which about - glycoforms are detected in the serum. the variations of the agp glycoforms are of interest to the study of the binding alteration of this protein with certain drugs, and of their usefulness as diagnostic biomarkers for some systemic inflammatory diseases [ ] [ ] [ ] . the quantification of the immunoreactive exorphins derived from milk beta casein a , casomorphins and , is important as these exorphins are potential modulators of numerous regulatory processes in the body as well as potential biomarkers of several diseases. for example, opioid-like casomorphins have been associated with autism, sudden infant death syndrome, type diabetes, apnea, constipation, postpartum psychosis, schizophrenia, circulatory disorders, and food allergies [ ] [ ] [ ] [ ] [ ] [ ] . as depicted in figure , when the elisa test is used for the determination of alpha- -acid glycoprotein and casomorphins, it is unable to detect subtle chemical differences between isoforms of the two protein-peptide molecules. the result is a single measurable detection signal obtained by elisa, expressed in arbitrary units by a purple bar for alpha- -acid glycoprotein ( figure a ), and by a green bar for casomorphins ( figure c ). conversely, the results obtained by the iace technology provide more comprehensive information. figure b shows an electropherogram of the various separated isoforms of immune-reactive alpha- -acid glycoprotein extracted from serum. additionally, each peak area of the agp isoforms can be quantified. the separation of isoforms of particular proteins and their individual quantification can therefore provide significant information for disease diagnosis and prognosis. clinical proteomic applications aim to solve specific clinical problems by adopting these procedures. recent studies are now viewing protein isoforms (proteoforms) as a new class of early diagnostic biomarkers for clinical proteomics [ , ] . each individual molecular form of an expressed protein has been called a proteoform. this term captures the disparate sources of biological variation that alter primary sequence and composition at the whole-protein level [ ] [ ] [ ] . the process of identifying aberrantly expressed proteins and disease-associated proteoforms has led to a better understanding of the underlying mechanisms of diseases, particularly tumorigenesis [ ] . figure d shows an electropherogram of immunoreactive a beta casein-derived casormorphin- and casomorphin- extracted from urine and analyzed by iace. since these peptides have a very similar amino acid composition differing only by one amino acid, elisa cannot differentiate them given its lack of separation of the peptides after detection. in contract, the two-dimensional iace technology can separate the two peptides using a single antibody that cross-reacts with both peptides for capture, thereby allowing for their quantification and characterization by one or more detectors. in summary, the mono-dimensional elisa test is limited to generate a single data point, which provides information only about the sum of the two urine-extracted casomorphins and the serum-extracted isoforms of agp. the immunoreactive exorphins derived from milk beta casein a , casomorphins and , is important as these exorphins are potential modulators of numerous regulatory processes in the body as well as potential biomarkers of several diseases. for example, opioid-like casomorphins have been associated with autism, sudden infant death syndrome, type diabetes, apnea, constipation, postpartum psychosis, schizophrenia, circulatory disorders, and food allergies [ ] [ ] [ ] [ ] [ ] [ ] . . schematic representation of a comparative profile of enzyme-linked immunosorbent assay (elisa) and iace applications for the determination of alpha- -acid glycoprotein (agp) or orosomucoid, and for the determination in urine of two structurally similar exorphins, casomorphins and . the purple bar (a) depicts arbitrary units using an elisa method for the total cumulative quantitative values of the isoforms of immunoreactive agp from human serum, and the green bar (c) depicts arbitrary units using an elisa method for the total cumulative quantitative values of the similarly related immunoreactive beta-casomorphin peptides, beta-casomorphin- and beta-casomorphin- , from human urine. panel (b) represents an electropherogram profile for the determination of the isoforms of immunoreactive agp from human serum, and panel (d) represents an electropherogram profile for the determination of the isoforms of immunoreactive beta-casomorphin peptides, beta-casomorphin- and beta-casomorphin- , from human urine. the electropherograms depicted in panels (b) and (d) were carried out by on-line immunoaffinity capillary electrophoresis. the advantage of determining isoforms of a protein is that each isoform can have various biological roles. figure modified from [ , ] . despite the increase in sensitivity and specificity of immunoassay techniques over the years, analytical interference remains a major area of concern. iace overcomes many of the limitations of the sandwich elisa test, in particular the frequent incidence of false-positive and false-negative results for numerous molecules [ , [ ] [ ] [ ] [ ] . part of this problem is due to the polyreactive nature of a significant number of antibodies, which bind not only to structurally related targets but also to structurally unrelated targets [ ] [ ] [ ] . polyreactive antibodies are a major component of the natural antibody repertoire. in contrast to monoreactive antibodies, polyreactive antibodies have a low binding affinity for antigens, but the antigen-binding pocket of these antibodies is thought to be more flexible than that of the monoreactive antibodies and thereby can accommodate different antigenic configurations [ ] . substances that arise from properties of the specimen and interfere with the reaction of an intended target antigen and reagent antibodies can generate inaccurate results [ ] [ ] [ ] [ ] . consequently, an incorrectly diagnosis that is based on inaccurate testing can lead to inappropriate and, in some cases, harmful treatment. in addition to false positive results, elisa can lead to false negative results. this tends to arise from weak binding affinity of the antigen to the affinity-capture agent. a number of modifications have been recommended to avoid interferences with antigen binding in immunoassays, such as the addition of detergents, treatment of a sample at high temperatures if the analyte is thermal stable, addition of some blocking agents to a sample, and others. however, despite advances in our knowledge and understanding of the mechanisms of interference in immunoassays, there is no single procedure that can rule out all interferences with elisa or other immunoassays [ ] . the development of a portable point-of-care iace biomarker analyzer instrument that utilizes two-dimensional iace technology offers a potential solution to improve the conditions of binding between the affinity-capture molecule immobilized to the acm device and the target analyte to be tested. for example, addition of small amounts a non-ionic detergent, such as of nonidet p- , addition of certain polycations, such as polybrene, and maintenance of the optimal ionic strength of the buffer, can help to improve the binding between the target analyte and the affinity-capture biorecognition molecule. other factors are also crucial to optimize binding, such as the control of temperature, buffer ph, and time of reaction; the use of a microwave pulse or acoustic micro-mixing system; and minor sample dilution with an appropriate conditioning buffer, all of which can improve binding and reproducibility of the assay [ , , [ ] [ ] [ ] , ] . another major advantage of the design of the iace biomarker analyzer instrument is that if the biorecognition affinity ligands or selectors (e.g., antibody, antibody fragment, lectin, aptamer, others) are stable and covalently immobilized with a well-defined orientation, then they can be reutilized multiple times. the advantage of this optimization protocol is that it permits the use of multiple well-oriented affinity-capture agents with high affinity and selectivity, in order to secure the binding of the target analyte. the ability of the acm device to be reused therefore brings down the cost per assay, resulting in significant benefits to patients that need frequent analysis of biomarkers. figure shows a schematic representation of a modified version of a portable point-of-care iace biomarker analyzer instrument ( figure a ) coupled to two biological specimen collection systems: an exhaled breath/oral fluid collection system ( figure b ), and a urinary collection system composed of a urinary drainage bag connected to a foley catheter ( figure c ). the two sample collection systems can work separately and in synchronization with the point-of-care iace biomarker analyzer instrument in a sequential order. the acm device depicted in figure a is structurally positioned in order to prevent the contact of the sample with the separation capillary [ , , [ ] [ ] [ ] , ] . this task is performed with the help of four microvalves surrounding the acm device. a biological specimen containing one or more target analytes to be tested is obtained either by the exhaled breath/oral fluid collection system figure b or by the foley catheter urine collection system figure c . the corresponding specimen is then transported through a secondary or auxiliary transport capillary to the main transport passage or capillary, passing through the acm device containing three biorecognition affinity ligands ( , , and ) all the way to the outlet end of the main transport capillary to a trap or waste container. sample introduction is carried out from the outlet side of the secondary transport passage or capillary, connected to the main transport passage or capillary, by controlled suction using a vacuum pump coupled to a trap or waste container collecting the excess amount of fluid. microvalves located at the inlet side of the main transport capillary (v- ) and at the inlet and outlet side of the acm device, which is positioned at the separation capillary figure a ) coupled to two biological specimen collection systems: an exhaled breath/oral fluid collection system (figure b) , and a urinary collection system composed of a urinary drainage bag connected to a foley catheter ( figure c ). the two sample collection systems can work separately and in synchronization with the point-of-care iace biomarker analyzer instrument in a sequential order. figure . schematic representation of a modified version of a portable point-of-care iace biomarker analyzer instrument (a) coupled to two biological specimen collection systems; an exhaled breath/oral fluid collection system (b), and to a urinary collection system composed of a urinary drainage bag connected to a foley catheter (c). the two sample collection systems can work in synchronization with the point-of-care iace biomarker analyzer instrument in a separate and sequential order using control microvalves (v- to v- ) to regulate the flow of collected samples from an individual. the acm device having a staggered configuration, can have one or more biorecognition affinity ligands immobilized to its inner wall to capture one or more similar or distinct biomarkers present in the collected samples. the point-of-care iace biomarker analyzer instrument can also be equipped with one or more acm devices (depicted in the upper left side), all having a staggered configuration, with the purpose to capture, concentrate, separate, and analyze a panel of biomarkers. by generating a comprehensive set of biomarker information from the biological specimen, the instrument aims to obtain a reliable and accurate diagnosis, prognosis, and to provide an efficient method for the assessment of treatment efficacy. in addition to capturing and analyzing chemical and biochemical compounds, the acm device of the portable point-of-care iace biomarker analyzer instrument is capable of capturing circulating blood cells, subcellular entities, cell debris, viruses, exosomes, and the components of disrupted biological particles. to facilitate a smooth passage of the fluid through the tubes or capillaries, a detergent-containing solution, stored in the liquid vessel or container, is added to the system during the collection of the sample. moreover, a few filters are incorporated into the tubes or capillaries, containing digestive enzymes immobilized to their constituent matrices, with the purpose of breaking down larger biomolecules, cell aggregates, cell debris, or any material that may block the filters and may disturb the smooth flow of the biofluid throughout the transport tubes or capillaries. the integrated iace biomarker analyzer instrument can be used in remote areas, in ambulances, and in intensive care units of a medical facilities, and it can be installed under a protective and confined space for patients with infectious diseases. the data collected can be sent by a secure and codified system using web portals via internet to a supercomputer to reveal comprehensive biomarker diagnostic signatures, thereby providing accurate and effective diagnosis information to a healthcare professional, and back to the computer of the iace biomarker analyzer instrument to keep the information updated and stored. figure modified from [ , , ] . after binding occurs between the target analyte(s) present in the biological specimen and the biorecognition affinity ligands immobilized to the acm device, the microvalve (v- ), positioned at the secondary or auxiliary transport capillary is closed, and the microvalve positioned at the inlet side of the main transport capillary (v- ) is open. a cleaning buffer or solution is introduced through the container positioned at the inlet side of the main transport capillary (container- ) to remove non-specific compounds bound to the inner surface of the main transport capillary. once the binding and cleaning processes are completed, microvalves positioned at the inlet and outlet sides of the transport capillary (v- and v- ) are closed, and the microvalves positioned at the inlet and outlet sides of the separation capillary (v- , v- , and v- ) are open. a separation buffer is introduced into the separation capillary from a container positioned at the inlet side of separation capillary (container- ) to a container localized at the outlet side of the separation capillary (not shown). a plug of an elution buffer can be introduced into the inlet side of the separation capillary by pressure, using an inert gas such as argon or helium, followed by the separation buffer. electroosmotic flow can also be used for the introduction of a plug of an elution buffer. when the high-voltage power supply connected to a platinum-iridium electrode is switched on the process of elution and separation starts. at the outlet side of the separation capillary, eluted and separated target analytes are detected, quantified, and partially characterized by one or more detectors connected to the capillary in-line (amperometry), on-line (uv-absorbance or fluorescence), or off-line (mass spectrometry). as illustrated in figure a , the acm device has three types of affinity ligands ( , , and ) immobilized to its inner surface representing three different affinity-capture molecules. the biorecognition affinity ligands can be an antibody, a lectin, and an aptamer, or a combination of the three (i.e., three different antibodies). similarly, the target analytes can be three different target analytes or a single target analyte that has three different epitopes. with the advent of advanced technologies offering powerful bioengineering tools, it is possible to create new affinity-recognition-capture molecules with improved and unique binding capabilities. for example, nanobodies, or single-domain variable fragments of camelid-heavy chain-only antibodies, have been successfully engineered as highly selective reagents, with high affinity, minimal size, low cost, and great stability. nanobodies are used as research tools and in medicine [ ] [ ] [ ] . similarly, aptamers, which are short, single-stranded oligonucleotides that bind to specific target molecules, are playing an important role as affinity recognition-capture and therapeutic reagents [ ] [ ] [ ] . another important group of affinity-recognition-capture reagents are lectins, which comprise a widespread group of sugar-binding proteins occurring in all types of organisms including animals, plants, bacteria, fungi, and even viruses. they are used as an effective tool for the targeting, separation, and reliable identification of glycoprotein molecules. their importance stems from the understanding that changes in glycoprotein and glycopeptide content, altered glycosylations, and aberrant glycan structures are increasingly recognized as cancer hallmarks [ ] [ ] [ ] [ ] . the use of two or more affinity-recognition-capture reagents has been demonstrated to improve the affinity binding to a molecule with multiple epitopes, such as thrombin [ ] . two different oligomeric dna aptamers that can recognize different epitopes in thrombin were used in a potentiometric biosensor. when testing for the detection of low concentrations of thrombin, the dual aptamer-immobilized configuration yielded better results than the one with a single aptamer when tested for the detection of tiny amounts of thrombin [ ] . another application employing multiple affinity-capture agents has been reported to use six different antibodies to capture six different immunoreactive chemokines in samples of cerebral spinal fluid collected from premature babies. in such study, immunoaffinity capillary electrophoresis in microchips was used to determine the degree of brain trauma in birth traumatized premature babies. the quantification of six different chemokines was able to provide information for diagnosis and prognosis of the good and poor state of the babies with head trauma [ ] . additional examples of using multiple antibodies to capture several biomarkers have been presented in [ , , , ] . the main advantage of the acm device, having multiple affinity-recognition-capture reagents (as depicted in figure a ) is its capability to analyze a panel of biomarkers simultaneously in a single platform to generate a comprehensive data and information. for example, it is possible to obtain the following information from a single biosample analysis from a patient suffering from a chronic or an infectious disease or illness and undergoing treatment: ( ) the presence of the antigen causing the disease, such as a microorganism or its components of the microorganism if the microorganism is broken apart; ( ) the presence of one or more antibodies, if the patient's immunological system recognizes the microorganism or its components as foreign substances consequently responds by developing antibodies to the unknown antigens; ( ) the presence of pro-inflammatory substances and anti-inflammatory substances, if the patient's body is fighting the disease as it develops; and ( ) the presence of other molecular markers expressed by the body or by the cellular or subcellular entities, and secreted to the biological specimen during the development of the disease. in the case of a comprehensive collection of information from the isolation and quantification of a panel of biomarkers in the biosample(s), it should be feasible to make an accurate and reliable diagnosis of the disease, a prognosis of the disease (i.e., if it will continue its progress, or if the patient has hope for recovery), and a pathway for monitoring the effectiveness of treatment (if any). furthermore, because of the high power of resolution of capillary electrophoresis, it is achievable to isolate and characterize novel co-translational and/or post-translational modifications of biomolecules, whether during the development of the severity of the disease or as a result of other biological parameters that may serve as better and more promising biomarkers of predicting clinical outcomes. the accuracy, analytical sensitivity, and reproducibility of the tests obtained by the two-dimensional iace technology are higher than those of conventional mono-dimensional immunoassays such as elisa. most notably, there is little to no possibility of false-negative or false-positive results when using iace, whereas such results can be obtained with some frequency when using elisa. for example, if a mono-dimensional elisa test captures unrelated immunoreactive compounds, then a false-positive result may be obtained due to the polyreactivity of antibodies which are capable of interacting with related and unrelated substances. on the other hand, if a similar situation happens with the two-dimensional iace technology (wherein the test captures related and unrelated immunoreactive compounds), there still exists the possibility of confirming the identity of each separated peak using additional tools and detectors capable of providing partial or complete characterization of the separated analytes (e.g., by spiking the sample with internal standards, determining the corresponding migration time of each peak, and obtaining the absorption spectral profile and mass spectrometry profile for each separated compound). an enhancement to the features of the portable point-of-care iace biomarker analyzer instrument as described in figure a can be accomplished by coupling the instrument, whether as an integral component or as a detachable unit, to a portable-point-of-care breath/oral fluid collection system as depicted in figure b and described elsewhere [ , ] . breath analyzer systems have been reported for the determination of organic volatile compounds and ionic content in exhaled breath condensate [ , ] . however, studies of exhaled breath have demonstrated that humans generate fine particles during tidal breathing. micron and submicron particle sizes have been detected in exhaled breath of normal and pathological people [ ] . exhaled breath sampling and analysis has long attracted interest in the areas of medical diagnosis and disease monitoring [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; however, progress from laboratory settings to routine practice has been slow. one main reason is the number of technical problems encountered for sampling and analysis, and the lack of normalization and standardization leading to significant variations that exist between results of different studies [ ] . mouth-exhaled breath contains not only volatile organic compounds, but many other small and large molecules, as well as cellular and subcellular particles. these biomolecular and cellular entities originate from the airway, from the oral cavity and gut by bacterial action, and from mucus and saliva. among the substances found in exhaled breath are inflammatory cytokines. it is well-known that inflammation is part of the host's protective immune response against microorganisms; on the other hand, excessive inflammation can be detrimental to patients' health as it increases their morbidity and mortality. for example, dysregulation between pro-and anti-inflammatory cytokines has been found in exhaled breath obtained from patients with community-acquired pneumonia [ ] . the potential of the portable point-of-care iace biomarker analyzer instrument to serve as a dual-integrated and complementary unit is greater than that of traditional instrumentation, since the instrument becomes a multi-modal screening system capable of isolating, concentrating, and quantifying multiple biomarkers for a comprehensive diagnostic and prognostic test. a panel of biomarkers has the potential to detect cases missed by the use of only a few biomarkers. infectious diseases, whether bacterial, viral, or of other origins, present acute and chronic stages of inflammation. early detection remains the key challenge to the survival of patients with contagious infectious diseases that may spread rapidly from one organ to another and can cast a storm over the whole body, ending in multiple organ failure. a cytokine storm syndrome having an hyperinflammation and characterized by elevated levels of cytokines in biological fluids can be a predictor of fatality. in particular, increased levels of interleukin- , interleukin- , granulocyte-colony stimulating factor, interferon-gamma inducible protein , monocyte chemo-attractant protein , macrophage inflammatory protein -alpha, and tumor necrosis factor-alpha have been associated to mortality due to virally driven hyperinflammation, as for example, in coronavirus disease (covid- ) [ ] . figure c depicts an additional modular attachment to the portable point-of-care iace biomarker analyzer, with a different configuration from the one described in figure b . in this case, sample collection is performed from a connecting insertion to a modified urinary catheter, also called a foley catheter, as used in a catheterized patient. urinary catheters are used to help to empty the bladder, usually when a person is unable to urinate. the catheter is a sterile and flexible tube that is inserted through the urethra into the bladder. often urinary catheters are used during surgery, as the patient is unable to control their ability to urinate during anesthesia. in most cases, the catheter is intended to stay in place for a long period of time. urine drains from the bladder through the tube and into a collection bag. in addition to urinary retention and during surgery, the catheter is also used in many patients who stay in a hospital's intense care unit (icu) and who are too sick to use a bedpan. unfortunately, some complications such as urinary tract infections, may arise when catheters are used for an extended period of time [ ] . in most cases, urinary tract infections are commonly treated with empirical antibiotics, resulting in overuse of antibiotics, which promotes antimicrobial resistance. interestingly, when urine from patients suffering from urinary tract infection is cultured, approximately only one in three patients are found to have urinary tract infection as defined by positive bacterial culture [ ] . results from bacterial culture may take from one to three or more days, depending on the type of bacteria [ ] . the portable-point-of-care iace biomarker analyzer instrument can overcome this delay since it is capable of generating comprehensive information based on a panel of biomarkers analyzed in the urine collected from the modified foley catheter collection system. the panel of biomarkers may include small molecules, biomolecules, and cellular and subcellular particles. these particles can be microorganisms such as bacteria, viruses, and others, including exosomes which are considered to be a trove of biomarkers [ ] . a panel of biomarkers should therefore be able to confirm the presence or absence or a microbial infection. it has been reported that isolation and characterization of a panel of host-protein biomarkers yields significantly superior performance for diagnosing bacterial versus viral infections [ ] . there is always uncertainty on how to manage a patient who may be a potential carrier of a contagious disease, as evident in the recent coronavirus pandemic. a "patient under investigation" is often placed in conservative precautions, and healthcare teams may defer or avoid certain procedures which may have been otherwise performed in other patients [ , ] . to provide physicians with the answers they need to manage patients effectively during an outbreak setting, laboratory testing is needed on the front lines whenever it is feasible and safe to do so. in figure we describe a portable-point-of-care iace biomarker analyzer that can be placed near the bedside of a patient and be separated by a plexiglass wall, such as the biomarker analyzer instrument that can be operated from an isolated containment area if necessary. when used on a patient, the portable and modular breath/oral collection system placed on a patient can be connected to the main instrument using a long flexible tube through the plexiglass wall to avoid any contamination. a similar connection can be performed when using the modified foley tubing coupled to the main instrument. the main transport and secondary or auxiliary passages or capillaries have an internal diameter usually between and µm, permitting enough surface area for passing fluid derived from the exhaled breath and urine specimens and their respective contents. the foley tubing and the exhaled breath/oral collection system, respectively, have much larger internal diameters. the average size of most bacteria is between . and . micrometers, but others may be as large as micrometers. white and red blood cells have size ranges fluctuating between . and micrometers on average, and viruses, nanobacteria, or extracellular vesicles (evs) have size ranges primarily in the nanometer scale. the separation capillary used in conventional capillary electrophoresis instruments usually ranges between and micrometers, even though other internal diameters smaller than micrometers or larger than micrometers can be used as well. therefore, for most cases it should not be a problem to isolate and characterize cellular, subcellular, and extracellular particles when using the portable-point-of-care iace biomarker analyzer instrument. as illustrated in figure a -c, the interchangeable modular infrastructures designed for the rapid analysis of a comprehensive biomarker information, including a plexiglass wall for keeping the entire functional instrument in containment, should enable a successful robust and sustainable system which protects the patient as well as the healthcare workers. all technical operation of the biomarker analyzer instrument (devices, attachments, connectors, and valves) can be performed remotely via computer using robotic manipulators or controllers, and thus without the risk of any physical contact. robotic manipulators are capable of performing repetitive tasks at speeds and accuracies that far exceed those of human operators. modifications to the connection systems and other parts of the instrument and devices, including change of buffers and solutions, can be carried out as needed in a simple manner. the modularity of the components of the instrument and devices allows for the rapid replacement of parts. additionally, attachments of extra miniaturized detectors for improving sensitivity and characterization should facilitate significantly the detection and characterization of substances and/or particulate matters found at very low concentrations. early medical practitioners realized the importance of counting blood cells as a tool for investigation and quantitative study in healthcare [ ] . for over years, cell biologists had been using a hemocytometer (counting chambers) to quantify cells (mm). today, automated instruments such as flow cytometers are widely used in research and for management of patient diagnosis and prognosis, particularly for blood cancer cells [ , ] . significant effort has been made to analyze cellular and subcellular entities, as well as viral particles and cellular vesicles using a flow cytometer (recently dubbed "flow virometry") [ ] , but the straightforward applicability of this technique has been hampered by the small sizes of the particles and vesicles, their polydispersity, and the low refractive index [ ] [ ] [ ] . with the rise of microfluidic devices, several attempts have been made to develop cell counters using microchips. however, for microfluidic devices to supplant current cytometers, it will require more development in sample preparation and sample techniques within lab-on-a-chip microfluidic platforms or miniaturized capillary electrophoresis instruments. studies on single cellular and subcellular entities, as well as their cargo content, by using conventional capillary electrophoresis and microchip capillary electrophoresis have been reported [ ] [ ] [ ] [ ] . there are several benefits associated with using these platforms, including low-volume requirements, rapid and efficient separations, special dimensions, and versatility. however, for the isolation and separation of a heterogeneous group of cellular and subcellular entities, as well as viral particles and cellular vesicles, many parameters must be taken in consideration, and the challenge for technical improvements will continue for a few more years. we are proposing a platform, based on iace, as a complementary tool to the existing technologies for the isolation and separation of particles of small sizes, such as viruses and evs. as demonstrated in figure , capillary electrophoresis has already been used with some success for the separation of bacteria, viruses, and polymeric particles. it would be beneficial to have a sample processing method before separation, to isolate and concentrate the intended viruses or evs. immunoaffinity capillary electrophoresis has already been proven to be a useful technology to isolate, separate, and quantify cell-free molecules of biological interest based on the specificity and selectivity not only of antibody reagents, but also of lectin and aptamer reagents, quantifying molecules ranging from microgram/milliliter to femtogram/milliliter [ , , , , ] . there are several reasons why we are interested in using the iace technology to study viruses and evs, in particular exosomes. the first reason is because we are in the middle of a pandemic, and the study of sars-cov- has emerged as a priority subject of research for the medical and scientific community [ ] . the second reason to apply iace to study exosomes is because of the potential of these nanoparticles in clinical diagnosis and prognosis, as well as carriers of therapeutic drugs [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . not only are exosomes released by basically all cell types, they are also accessible in body fluids, they carry important molecular compounds of potential use as biomarkers, and they can cross many cellular barriers. as such, they play an important role in many physiological processes and have been implicated in many diseases [ , , ] . as evs encompass a heterogeneous group of cell-derived vesicles, it would be a challenge to isolate, concentrate, and separate the three groups of nanoparticle entities. in human blood serum. they are mosquito-borne flaviviruses responsible for dengue fever and severe dengue hemorrhagic fever that belong to the genus flavivirus, family flaviviridae, which contains approximately viruses. the flaviviruses are relatively small ( - nm) and spherical with a lipid-rich envelope [ ] . vaccination must protect against all four serotypes, and hence this has proved to be difficult to design. structural differences between dengue viruses produced in humans versus cell lines many be key to understanding vaccine failure and developing better models for vaccine evaluation [ ] . in the case of the coronaviruses, there are seven coronaviruses known to infect humans, but only three of them, sars-cov, mers-cov, and sars-cov- cause severe disease in humans. the seven coronaviruses are ( ) little is known about the coronavirus sars-cov- , which is responsible for the novel pneumonia known as coronavirus disease or covid- . it appears that day after day, physicians and scientists are learning new information about the transmissibility and severity of the [ ] [ ] [ ] . in view of the fact that there is a significant interest in the study of viruses and exosomes, a brief discussion of their importance is warranted. extracellular vesicles are a heterogeneous group of cell-derived membranous structures which originate from the endosomal system or which are shed from the plasma membrane, respectively. extracellular vesicles have been classified as oncosomes ( - micrometers), apoptotic bodies ( - nanometers), microvesicles ( - nanometers), and exosomes ( - nanometers) [ , ] . some even smaller vesicles known as "exomeres" have been described as having even smaller sizes [ ] . exosomes are naturally occurring nanosized vesicles and consist of natural lipid bilayers with an abundance of adhesive proteins that readily interact with cellular membranes. these vesicles' contents include cytokines and growth factors, signaling lipids, mrnas, and regulatory mirnas. exosomes have many roles and functions, and they are found in body fluids, including plasma, serum, urine, saliva, exhaled breath condensate, breast milk, tears, semen, cerebrospinal fluids, amniotic fluid, malignant ascites fluids, and cultured medium of cell cultures [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . they are involved in multiple physiological and pathological processes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these extracellular vesicles represent an important mode of intercellular communication by serving as functional vehicles that carry a complex cargo of proteins, lipids, and nucleic acids, and altering cell or tissue metabolism, influencing tissue responses to injury, infection, and disease [ ] [ ] [ ] [ ] [ ] . exosomes are heterogeneous in size, heterogeneous in composition, and enriched in membrane-associated, high-order oligomeric protein complexes. exosomes and many types of enveloped viral particles, particularly rna viruses, have a similar size making exosomes and rna viruses "close relatives" [ ] . in fact, enveloped viruses may be considered a form of ev. exosomes from virus-infected cells incorporate not only cell-encoded but also virus-encoded molecules [ ] . exosomes are nanoparticles in the range of - nm (average nm). therefore, they can be separated by capillary electrophoresis as many other biological particles have been separated. for example, figure shows the separation by capillary electrophoresis of a virus (human rhinovirus serotype ) [ ] , a bacterium (pseudomonas fluorescens, a gram-negative, rod-shaped bacterium) [ ] , and three polymeric particles (polystyrene micron-sized particles) [ ] . the highly selective affinity capture of biorecognition agents such as antibodies, lectins, and aptamers, in conjunction with the superior resolving power of capillary electrophoresis, make a perfect pair to concentrate and separate biological species or entities for potential use in diagnosis. a typical example are viruses, which usually exist as more than one type. dengue infections are caused by four closely related viruses named den- , den- , den- , and den- . these viruses are called serotypes, because each type of virus has different interactions with the antibodies in human blood serum. they are mosquito-borne flaviviruses responsible for dengue fever and severe dengue hemorrhagic fever that belong to the genus flavivirus, family flaviviridae, which contains approximately viruses. the flaviviruses are relatively small ( - nm) and spherical with a lipid-rich envelope [ ] . vaccination must protect against all four serotypes, and hence this has proved to be difficult to design. structural differences between dengue viruses produced in humans versus cell lines many be key to understanding vaccine failure and developing better models for vaccine evaluation [ ] . in the case of the coronaviruses, there are seven coronaviruses known to infect humans, but only three of them, sars-cov, mers-cov, and sars-cov- cause severe disease in humans. the seven coronaviruses are ( ) e (alpha coronavirus), ( ) nl (alpha coronavirus), ( ) oc (beta coronavirus), ( ) hku (beta coronavirus), ( ) mers-cov (the beta coronavirus that causes middle east respiratory syndrome, or mers), ( ) sars-cov (the beta coronavirus that causes severe acute respiratory syndrome, or sars), and ( ) sars-cov- (the novel coronavirus that causes coronavirus disease , or covid- ) [ ] . little is known about the coronavirus sars-cov- , which is responsible for the novel pneumonia known as coronavirus disease or covid- . it appears that day after day, physicians and scientists are learning new information about the transmissibility and severity of the epidemic it causes. the seriousness of these infections and the lack of effective, licensed treatments for sars-cov- infections underpin the need for a more detailed and comprehensive understanding of coronaviral molecular biology, with a specific focus on both coronaviruses' structural proteins as well as their accessory proteins [ ] . the known vaccines typically work by eliciting antibodies that block entry of the pathogen into cells, which in the case of enveloped viruses, involves antibody binding to the viral envelope proteins. the viral fusion protein is the key factor that induces the membrane fusion reaction that in turn allows viral entry [ ] . analyzing structural biology of the viral membrane fusion proteins and their complexes with neutralizing antibodies is thus an exceptionally powerful approach to identifying vulnerability sites and to extracting the necessary information required to develop protective vaccines to efficiently combat the emerging viral diseases threatening our planet [ ] . immunoaffinity capillary electrophoresis can serve as a platform to isolate, concentrate, and analyze various structurally related viruses, and to analyze their chemical modifications they might undergo, thereby providing significant information about a diagnostic signature classification of their serotypes. similarly, this approach can be implemented to study extravesicular vesicles and various types of circulating cells, such as immune cells and cancer cells [ , ] , as well as quantifying and analyzing their chemical and biochemical contents. capillary electrophoresis can separate molecules or entities based on the differences in the charge-mass ratio of single molecules, complexes of molecules, circulating cells, biological particles, and even man-made nanoscale quantum dots [ ] . recently, it has been proposed as a proof of concept that capillary electrophoresis can be used for the separation of bacterial extracellular vesicles [ ] . much effort has been performed during the last years to develop methods for the isolation and characterization of exosomes, which are seen as attractive candidates for clinical application as innovative diagnostic and therapeutic tools [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . lately, there has been increasing evidence which demonstrates that the positive effects of cell-based therapies are mediated by exosomes released from the administered cells, and that the micro-rna cargo in these exosomes is largely responsible for the therapeutic effects [ ] . advances in the study of exosomes have been delayed because of the lack of methods to isolate them from clinical samples. traditionally, differential ultracentrifugation, ultrafiltration, immunoaffinity capture, and size exclusion chromatography have been used as conventional methods of isolation, but these methods are generally time-consuming and labor-intensive. they also may need costly instrumentation or some special chemical reagents, and in some cases, such methods of isolation may require multiple overnight centrifugation steps [ , ] . in the last few years, several characterization and validation methods have been developed, for both research and clinical purposes, to analyze exosome purity and to quantify exosomal cargo. these methods include transmission electron microscopy (tem), scanning electron microscopy (sem), atomic force microscopy (afm), nanoparticle tracking analysis (nta), dynamic light scattering (dls), resistive pulse sensing, enzyme-linked immunosorbent assay (elisa), flow cytometry, fluorescence-activated cell sorting (facs), and microfluidics and electrochemical biosensors [ ] [ ] [ ] [ ] [ ] . the number of methods for quantifying exosomes has expanded as interest in exosomes has increased. however, consensus on proper quantification has not developed, making each study difficult to compare to another [ ] . immunologically, exosomes exhibit antigen-presenting capability [ ] . as a result, immunoaffinity studies of exosomes are becoming attractive, and iace can be an ideal platform to be used for such applications in research and in clinical diagnosis. in fact, a few laboratories are already combining immunoaffinity and microfluidic system approaches for more efficient exosome collection [ ] [ ] [ ] [ ] [ ] . given their biological activities in intercellular transportation, information communication, and cell-mediated immunity modulation after microbial infection, exosomes are considered to be of significant value for a comprehensive understanding of infectious diseases as they play a key role in clinical defense of microbial infections. furthermore, exosomes will play a significant role as immune drug carriers, both in the manufacture of biological vaccines, and as biomarkers of microbial infections [ ] . recently, it has been shown that exosomes serve as decoys, providing cellular protection against bacterially produced toxins [ ] . a consistent high-quality isolation method for exosomes, followed by characterization and identification of types exosomes and their content, is therefore crucial to distinguishing subpopulations of exosomes, other extracellular vesicles, and viral particles. a conceptual understanding of biological responses to nanoparticles is likewise needed to develop and apply safe nanoparticles in drug delivery and other uses [ ] . the future of the in-depth investigation of exosomes will rely heavily on technological advances, particularly with respect to simplifying the capture of functional microvesicles, such as exosomes, and to providing accurate quantitative measurement of their contents [ , ] . many interesting findings and hopes for advancing diagnosis and therapy for exosomes have been reported recently (table ). one such study has found that exosomes released in response to cigarette smoke might trigger chronic obstructive pulmonary disease (copd), whereas engineered versions could be used as treatment of the disease [ ] [ ] [ ] . another report has shown that exosomes from virus-infected cells modulate immune cells' responses and increase spread and infection of the virus through delivering viral nucleic acid and protein molecules to healthy cells [ ] . as discussed earlier in this paper, the two-dimensional iace technology provides many advantages when compared to other immunoassays for the analysis of cell-free molecules, such as the use of low volumes and reagents, high analytical sensitivity, speed of analysis, and the separation of all target and non-target affinity-captured analytes [ , , ] . nevertheless, there are a few technical challenges for the application of this technology to the isolation, concentration, and separation of cellular particles and vesicles which remains unresolved [ , ] . the goal of analyzing exosomes by iace is to further study the association of the cargo content of exosomes with the presence or absence of a wide range of pathogens, such as bacteria or viruses, and integrates the host injury response to infection [ ] . moreover, by primarily quantifying nucleic acids and crucial protein biomarkers which serve as indicators of early detection and evolution of diseases, exosomal analysis employing iace can further provide information about the injury of different tissues in a patient due to pathogens and/or inflammatory substances. the role of non-coding rnas (ncrnas) in evs is not fully understood, but their higher abundance in association with cancer and other diseases demonstrated their relevance. several studies are underway on micrornas, long ncrnas, and circular rna present in exosomes, and evidence exists that non-protein coding genes harbors crucial importance in terms of development, homeostasis, and disease [ ] [ ] [ ] [ ] [ ] [ ] . table . a number of publications related to the potential use of exosomes obtained from liquid biopsy or cultured cells, and their cargo contents in diagnosis, prognosis, and therapy. mesenchymal stromal cells-derived exosomes potential to improve neurological injury. understanding the effect of exosomes as mediators of the beneficial effects of cell therapy for stroke and traumatic brain injury. [ ] human hepatocellular carcinoma exosomes important role in the diagnosis and therapy for tumors. screening for biomarkers in early formation of chronic hepatitis and liver cancer. elucidation of signal pathways and their involvement in growth, metastasis, and angiogenesis; and of the significance of exosomes in the treatment of hepatocellular carcinoma. [ ] human serum exosomes symptomatic respiratory viral infections after lung transplantation. presence of lung self-antigens, s proteasome, and viral antigens implies that exosomes trigger chronic rejection. [ ] optogenetically engineered exosome system (explor) therapeutic carriers to deliver srikb (super-repressor ikb) to a therapeutic target used as sepsis model. amelioration of sepsis-induced organ injury and inhibition of secretion of proinflammatory cytokines. [ ] human placenta exosomes maternal plasma exosomes therapeutic approach for gestational diabetes mellitus. to reduce the negative impact of gestational diabetes mellitus. [ ] engineered tumor-derived exosomes therapeutic approach as anti-tumor agents. potentials usage in cancer immunotherapy. [ ] mouse adiposederived mesenchymal stem cell exosomes novel therapeutic strategy for tissue injury. proteomic analysis of exosomes found more than protein groups with a number of biological functions, implying such exosomes might be valuable as potential therapeutic targets for tissue repair. [ ] mesenchymal stem cell-derived exosomes potential role in osteoarthritis regenerative medicine. elucidation of the inflammatory and multiple pathophysiological processes in the synovium, leading to the degradation of cartilage and bone. potential role in cartilage repair and osteoarthritis therapy. [ ] vascular endothelial cells-derived exosomes vascular smooth muscle cells-derived exosomes several other cells-derived exosomes potential of exosomes in diagnosis, prognosis, and treatment of atherosclerosis. understanding of occurrence and development of cardiovascular diseases such as atherosclerosis. [ ] human urine exosomes potential of exosomes in diagnosis of nephropathies. characterization of proteolytically derived peptides that are essentially relevant to classify patients with nephropathies, cancers of the urinary tract. [ ] human urine exosomes potential of exosome gene expression assay as noninvasive test for prostate cancer. evaluation of a urinary diagnostic assay to help assess whether a prostate biopsy is warranted. [ ] human bone marrow mesenchymal stem cells-derived exosomes potential of exosome as treatment for severe covid- . understanding of the effect and capacity of exosomes to restore oxygenation, downregulate cytokine storm, and reconstitute immunity. [ ] human lungs epithelial cells-derived exosomes transduced with selected genes of the sars-cov- a new strategy to demonstrate that sars-cov- rna-containing exosomes represent an indirect route of entry into cardiomyocytes. understanding a potential cardiac dysfunction produced via sars-cov- rna containing exosomes, without the need for direct viral infection. [ ] bovine milk exosomes an alternative strategy to load hydrophilic and lipophilic small molecules, and chemotherapeutic drugs into exosomes. potential drug delivery vehicle or nanocarrier for cancer treatment. [ ] mesenchymal stem/stromal cell-derived exosomes may provide considerable advantages over their counterpart live cells, potentially reducing undesirable side effects including infusional toxicities. potential use in gene delivery, regenerative medicine, and immunomodulation. [ ] cultured third-molar pulp cell-derived exosomes potential of pulp-derived exosomes in combination with fibrin gel to fill dental hard tissues understanding the use exosomes in combination with fibrin gel in clinical translation towards improved cell-free regenerative endodontics. [ ] tumor-derived exosomes (texs) potential for circulating immune-related biomarkers, reflecting partially the genetic and molecular contents of the parent cancer cell. understanding how texs influence boost tumor growth, regulation of tumor neo-angiogenesis, premetastatic niche formation, and therapy resistance. [ ] human exosomes-based vaccines potential use of the s protein of the sars-cov, a type i transmembrane glycoprotein, incorporated into exosomes as antigen to be used in vaccines to induce high levels of neutralizing antibodies. manufacturing of highly immunogenic sars-s-based vaccines. additionally, potential use in inhibiting tumor growth. [ ] exosomal noncoding rna in glioma potential of exosomes as carriers of bioactive molecules into the brain. holds great promise in diagnostics and therapy. noncoding rnas of exosomes can be modulators of numerous hallmarks of glioma. understanding the function of exosomal noncoding rnas in cell-to-cell communication in the tumor microenvironment, tumor proliferation, invasion, angiogenesis, immune-scape, and treatment resistance. [ ] human exosomes in cardiovascular diseases potential of exosomes as more effective intervention targets on ischemic heart disease. the presence of exosomes in plaque tissue, ischemic heart, and peripheral blood can be potential biomarkers for early diagnosis and prognosis of cardiovascular diseases. understanding the participation of exosomes in the evolution of ischemic heart disease, including their role in endothelial dysfunction, lipid deposition, atheromatous plaque formation and rupture, ischemia-reperfusion, and heart failure. [ ] human urine exosomes potential source of biomarkers, pathogenic molecules, and therapeutic biologics in kidney diseases or disorders. understanding the role of exosomes in pathogenic mechanisms, biomarker discovery, and therapeutics of various kidney diseases, particularly lupus nephritis, acute kidney injury, diabetic nephropathy, renal fibrosis, kidney transplantation, and renal carcinoma. [ ] potential of exosome's cargo, such as the novel slc a transport protein, to serve as useful biomarker of inflammatory processes. understanding the significance of exosomes as carriers of inflammatory mediators involved in human pathologies. [ ] human saliva exosomes from hiv-positive people platform to demonstrate that isolated saliva exosomes from hiv-positive individuals promote kaposi's sarcoma-associated herpes virus (kshv) infectivity in human oral epithelial cells. understanding how the trans-activation response element (tar) rna in hiv-associated exosomes contribute to enhancing kshv infectivity through the epidermal growth factor receptor (egfr). [ ] human semen exosomes potential of exosome's cargo containing molecular fingerprints for a non-invasive diagnosis of prostate cancer. understanding the role of semen exosomes in prostate cancer diagnosis, and as possible agents for enhancing the transmission of sexual diseases. [ ] human serum exosomes potential role of some mirnas extracted from serum exosomes of parkinson's disease patients to serve as biomarkers. understanding the significance of the expression levels of mir b, mir , and mir as biomarkers of parkinson's disease. [ ] human milk exosomes a complementary strategy to deliver more functional insights of human milk. providing an enhanced immunological and micronutrient profile of human milk, with significant relevance to breast milk quality and the health of the mother and infant. [ ] human umbilical cord exosomes potential role of exosomes derived from akt-modified human umbilical cord mesenchymal stem cells as therapy for improving cardiac regeneration. understanding the role of why exosomes obtained from akt-modified umbilical cord mesenchymal stem cells are more effective as therapy in myocardial infarction by promoting angiogenesis via activating platelet-derived growth factor d. [ ] human bronchoalveolar lavage fluid exosomes potential role of mirna from exosomes with proinflammatory signatures in asthma and in allergic airway diseases. understanding the role of mirna obtained from bronchoalveolar lavage fluid exosomes. particularly micrornas mir- and mir- , which can modulate gene programming and promote inflammation. [ ] human cerebrospinal fluid and plasma exosomes potential use of alpha-synuclein and tau proteins obtained from central nervous system-derived exosomes that can efflux into blood can be used as biomarkers of parkinson's disease and other neurodegenerative diseases. exosomes can also serve as carriers of therapeutic substances for diseases of the central nervous system. understanding the role of the content of exosomes derived from the central nervous system in parkinson's disease. exosomes can carry and spread toxic alpha-synuclein between cells and induce apoptosis. [ ] human induced pluripotent stem cell-derived neuronal exosomes potential use as a tool to assay the capacity of exosomes to influence neuronal and circuit development. control exosomes rescue neurodevelopmental defects in a model of rett syndrome. understanding the role of exosomes in the development of neural circuits, the increase in neurogenesis, and the promotion of cell proliferation and neural differentiation. [ ] blood or cerebrospinal fluid-derived circulating circular exosomal rnas potential use of circulating exosomal circular rnas (circrnas) as biomarkers for the early detection and diagnosis of neuropsychiatric disorders. understanding the role of closed-loop structure circular rnas, a novel class of non-coding rna (ncrna), in mental diseases. some studies show that circrnas possess regulatory potential as "sponges" for target micrornas (mirnas) and rna binding proteins. [ ] ovarian cancer cells-derived exosomes potential use of exosomal proteins and lipids in the early diagnosis of ovarian cancer. understanding the role several lipid species and proteins, which significantly differ in cancer derived exosomes when compared to those from ovarian surface epithelial cells. [ ] the reliability of biomarkers in day-to-day medical practice still appears to be in question because of a lack of access to extremely sensitive and specific diagnostic testing. researchers are therefore not only discovering more comprehensive ways to determine disease activity via biomarker detection, but also linking biomarkers to risk factors that would allow for better prediction models to be created for risk of disease manifestation [ ] . clinical implementation of exosome-based diagnostic and therapeutic applications is still limited by the lack of standardized technologies that integrate efficient isolation of exosomes with comprehensive detection of relevant biomarkers. with the iace biomarker analyzer instrument and its interconnected sampling collection systems as described in this manuscript, we expect to accurately identify and quantify different morphological subpopulations of nanovesicles and their components in a single platform. we hope that this device can be affordable and readily available in a clinical setting in the near future once biomarker applications have demonstrated its clinical validity, and once analytical validation studies have demonstrated its accuracy, precision, reproducibility, and sensitivity levels. however, as in all great technological revolutions, iace has and may continue to have some limitations. the advantages of this two-dimensional affinity capture-separation technology have just begun to come into view over the past few years, and the trend in usage of iace in clinical contexts is moving consistently in the positive direction. most promisingly, the incorporation of iace into telehealth, with the intent to meet the needs of telemedicine, and precision medicine, is beginning to revolutionize the delivery of healthcare to all segments of the population. in the near future, we foresee that the use of the iace technology, in conjunction with liquid biopsy, for the study of circulating cell-free molecules, single cells, subcellular entities, viruses, exosomes, and their constituent components in biological fluids, will become a powerful tool to unravel longstanding questions in both biological research and clinical diagnostics. author contributions: both authors contributed to data analysis and manuscript writing. all authors have read and agreed to the published version of the manuscript. there was no external financial support to conduct the research projects. best (biomarkers, endpoints, and other tools) resource. maryland: food and drug administration-national institutes of health biomarker working group biomarkers: potential uses and limitations how are we going to discover new cancer biomarkers? a proteomic approach for bladder cancer making meaningful clinical use of biomarkers systems medicine: helping us understand the complexity of disease sensitivity" and "specificity" reconsidered: the meaning of these terms in analytical and diagnostic settings validation of analytical methods. in calibration and validation of analytical methods-a sampling of current approaches biopsy: its history, current and future outlook potential utility of liquid biopsy as a diagnostic and prognostic tool for the assessment of solid tumors: implications in the precision oncology advances liquid biopsy for circulating biomarker detection saliva liquid biopsy for point-of-care applications. front liquid biopsy: where did it come from, what is it, and where is it going? investig de vlaminck, i. a cell-free dna metagenomic sequencing assay that integrates the host injury response to infection biomedicines mass spectrometry identification of sars-cov- proteins from gargle solution samples of covid- patients immunoassays: a practical approach immunoassay of endogenous plasma insulin in man enzyme-linked immunosorbent assay (elisa) continuous cultures of fused cells secreting antibody of pre-defined specificity immunoassays: their history, development and current place in food science and technology a selected history and future of immunoassay development and applications in clinical chemistry antibody production and application for immunoassay development of environmental hormones: a review development of immunoassays for the determination of phthalates designs, formats and applications of lateral flow assay: a literature review development of immunoassays for multi-residue detection of small molecule compounds an emerging micro-scale immune-analytical diagnostic tool to see the unseen. holding promise for precision medicine and p medicine comparison of elisa and hplc-ms methods for the determination of exenatide in biological and biotechnology-based formulation matrices a direct comparison of liquid chromatography-mass spectrometry with clinical routine testing immunoassay methods for the detection and quantification of thyroid hormones in blood serum the pathway through lc-ms method development: in-house or ready-to-use kit-based methods? current state of bioanalytical chromatography in clinical analysis detection of -ohdg as a diagnostic biomarker high-performance liquid chromatography and enzyme-linked immunosorbent assay techniques for detection and quantification of aflatoxin b in feed samples: a comparative study lc-msms assays of urinary cortisol, a comparison between four in-house assays comparison of an hplc-ms/ms method with multiple commercial elisa kits on the determination of levels of -oxo- , -dihydro- -deoxyguanosine in human urine protein biomarker quantification by immunoaffinity liquid chromatography-tandem mass spectrometry: current state and future vision a brief history of medical diagnosis and the birth of the clinical laboratory biomedicines an historical perspective on the clinical diagnostic laboratory existing and emerging technologies for point-of-care testing the accuracy of point-of-care glucose measurements physicians'views of self-monitoring of blood glucose in patients with type diabetes not on insulin point-of-care diagnostics in low resource settings: present status and future role of microfluidics current status and future prospects of point-of-care testing around the globe the clinical and health economic value of clinical laboratory diagnostics sample preparation in microstructures devices microchips, microarrays, biochips and nanochips: personal laboratories for the st century clinically relevant advances in on-chip affinity-based electrophoresis and electrochromatography fang, q. a low-cost palmtop high-speed capillary electrophoresis bioanalyzer with laser induced fluorescence detection capillary electrophoresis technology review on the development of truly portable and in-situ capillary electrophoresis systems applications of capillary electrophoresis for the early diagnosis of cancer urinary proteomics and precision medicine for chronic kidney disease: current status and future perspectives microfluidic chip electrophoresis for biochemical analysis d printed microfluidics immunoaffinity ce for proteomics studies immunoaffinity capillary electrophoresis as a powerful strategy for the quantification of low-abundance biomarkers, drugs, and metabolites in biological matrices applications of microfluidics and microchip electrophoresis for potential clinical biomarker analysis recent advances in ce and microchip-ce in clinical applications: to mid- analysis of inflammatory mediators in newborn dried blood spot samples by chip-base immunoaffinity capillary electrophoresis recent trends in the quantification of biogenic amines in biofluids as biomarkers of various disorders: a review biocatalytic amplification of uv signal in capillary electrophoresis of microrna enhancement of concentration limits of detection in capillary electrophoresis: examples of on-line sample preconcentration, cleanup, and microreactor technology in protein characterization on-line solid-phase preconcentration for sensitivity enhancement in capillary electrophoresis on-line preconcentration methods for capillary electrophoresis lowering the concentration limits of detection by on-line solid-phase extraction-capillary electrophoresis-electrospray mass spectrometry getting the best sensitivity from on-capillary fluorescence detection in capillary electrophoresis"-a tutorial a critical retrospective and prospective review of designs and materials in-line solid-phase extraction capillary electrophoresis ten principles for conservative, care-full diagnosis sensitivity enhancement and second-dimensional information from solid-phase extraction-capillary electrophoresis of entire high-performance liquid chromatography fractions recent advances in enhancing the sensitivity and resolution of capillary electrophoresis on-capillary derivatization as an approach to enhancing sensitivity in capillary electrophoresis microfluidic chip-capillary electrophoresis device for the determination of urinary metabolites and proteins recent progress of sample stacking in capillary electrophoresis determination of sulfonamides in milk by capillary electrophoresis with peg@mos as a dispersive solid-phase extraction sorbent recent contributions for improving sensitivity in chiral ce from a central laboratory to the bedside: a point-of-care instrument to monitoring wellness and disease using two-dimensional immunoaffinity capillary electrophoresis technology recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips laser-induced fluorometry for capillary electrophoresis recent trends of capillary electrophoresis-mass spectrometry in proteomics research advances in capillary electrophoresis for the life science capillary electrophoresis-based approaches for the study of affinity interactions combined with various sensitive and nontraditional detection techniques multivalent aptamers: versatile tools for diagnostic and therapeutic applications advances in affinity ligand-functionalized nanomaterials for biomagnetic separation review on biomimetic affinity chromatography with short peptide ligands and its application to protein purification metal-organics framework-based affinity materials in proteomics modified bacteriophage tail fiber proteins for labeling, immobilization, capture, and detection of bacteria rational design of affinity ligands for bioseparation a capture and release method based on noncovalent ligand cross-linking and facile filtration for purification of lectins and glycoproteins the use of a concentration step to collect urinary components separated by capillary electrophoresis and further characterization of collected analytes by mass spectrometry automated capillary electrophoresis apparatus immunoaffinity capillary electrophoretic analysis of cyclosporin in tears new approaches in clinical chemistry: on-line analyte concentration and microreaction capillary electrophoresis for the determination of drugs, metabolic intermediates, and biopolymers in biological fluids affinity capillary electrophoresis: important application areas and some recent developments immunoaffinity ce in clinical analysis of body fluids and tissues microfluidic immunoaffinity separations for bioanalysis bioanalytical methods for food allergy diagnosis, allergen detection and new allergen discovery automated microfluidic devices integrating solid-phase extraction, fluorescent labeling, and microchip electrophoresis for preterm birth biomarker analysis capillary electrophoresis-based immunoassay and aptamer assay: a review clinical and pharmaceutical applications of affinity ligands in capillary electrophoresis: a review red diode laser induced fluorescence detection with a confocal microscope on a microchip for capillary electrophoresis capillary-scale monolithic immunoaffinity columns for immunoextraction with in-line laser induced fluorescence detection magnetic beads based immunoaffinity capillary electrophoresis of total serum ige with laser-induced fluorescence detection demonstration of a direct capture immunoaffinity separation for c-reactive protein using a capillary-based microfluidic device immunoaffinity extraction of testosterone by antibody immobilized monolithic capillary with on-line laser-induced fluorescence detection ultrasensitive on-column laser-induced fluorescence in capillary electrophoresis using multiparameter confocal detection analysis of serum transthyretin by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using magnetic beads measurement of inflammatory chemokines in micro-dissected tissue biopsy samples by chip-based immunoaffinity capillary electrophoresis immunoaffinity capture couples with capillary electrophoresis-mass spectrometry to study therapeutic protein stability in vivo confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection separation of urinary constituents by capillary electrophoresis and further characterization of collected analytes by mass spectrometry capillary electrophoresis for the analytical separation and semi-preparative collection of monoclonal antibodies affinity capillary electrophoresis: two semi-preparative approaches to concentrate samples on the capillary column and to recover microgram quantities of material fabrication of an analyte concentrator-reaction chamber containing immobilized s. aureus v protease: on-column proteolytic digestion of nanogram quantities of substrate using capillary electrophoresis enzymophoresis of nucleic acids by tandem capillary enzyme reactor-capillary zone electrophoresis determination of prolyl -hydroxylase beta-subunit at the zeptomole level using capillary electrophoresis on-line peptide mapping of antibodies by capillary electrophoresis selective preconcentration for capillary zone electrophoresis using protein g immunoaffinity capillary electrophoresis consecutive protein digestion and peptide derivatization employing an on-line analyte concentrator to map proteins using capillary electrophoresis on-line protein digestion by immobilized enzyme microreactor capillary electrophoresis-mass spectrometry on-line immobilized microreactor for evaluating inhibitory activity of phenolic acids by capillary electrophoresis and molecular docking disease detection system and method method and system for simultaneous determination of multiple measurable biomarkers during the development of a communicable disease real-time imaging through optical fiber array-assisted laser-induced fluorescence of capillary electrophoretic enantiomer separations chip-based immunoaffinity capillary electrophoresis: application to the measurement of brain-derived neurotrophic factor in skin biopsies brain-derived neurotrophic factor and its clinical implications brain-derived neurotropic factor in brain disorders: focus on neuroinflammation on-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha- -acid glycoprotein isoforms profile to facilitate its use as biomarker glycoform analysis of alpha- -acid glycoprotein by capillary electrophoresis differential glycosylation of alpha- -acid glycoprotein (agp-i) contributes to its functional diversity review on the potential health impact of ß-casomorphins and related peptides impact of milk derived ß-casomorphins on physiological functions and trends in research: a review aytekin, i. a and a bovine milk, the risk of beta-casomorphin- and its possible effects on human health: (i) a and a milk and the risk of beta-casomorphin- a /a milk and ß-casomorphins: the resurgence of controversy milk a ß-casein and health related outcomes in humans: a systematic review biological properties and other potential effects on human health of ß-casomorphin : current knowledge and concerns a method for identifying discriminative isoform-specific peptides for clinical proteomics application an isoform of aif involved in breast cancer pten proteoforms in biology and disease how many human proteoforms are there? n-terminal proteoforms in human disease origins and clinical relevance of proteoforms in pediatric malignancies false positive circumsporozoite protein elisa: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination high false positive rate of an elisa screen for the detection of anti-factor viii antibodies in congenital hemophilia a preventing intense false positive and negative reactions attributed to the principle of elisa to re-investigate antibody studies in autoimmune diseases urea-mediated dissociation alleviate the false-positive treponema pallidum-specific antibodies detected by elisa properties and function of polyreactive antibodies and polyreactive antigen-binding b cells antibody polyreactivity in health and disease: status variabilis natural antibodies-facts known and unknown. cent polyreactivity of antibody molecules interferences in immunoassay presence of anti-insulin natural autoantibodies in healthy cats and its interference with immunoassay for serum insulin concentrations cardiac troponin t quantitative assay failure as a result of antibody interference retrospective approach to evaluate interferences in immunoassay electrophoresis apparatus having staggered passage configuration immunoaffinity capillary electrophoresis: a new versatile tool for the determining protein biomarkers in inflammatory processes nanobodies and their potential applications nanobody engineering: toward next generation immunotherapies and immunoimaging of cancer the therapeutic potential of nanobodies aptamers as therapeutics analysis of aptamer discovery and technology aptamers: a review of their chemical properties and modifications for therapeutic application comprehensive list of lectins: origins, natures, and carbohydrate specificities lectin engineering: the possible and the actual glycoprotein markers of hepatocellular carcinoma-mass spectrometry based approaches use of lectin-based affinity techniques in breast cancer glycoproteomics: a review dual aptamer-immobilized surfaces for improved affinity through multiple target binding in potentiometric thrombin sensing detection of cerebral spinal fluid-associated chemokines in birth traumatized premature babies by chip-based immunoaffinity ce integrated modular unit including an analyte concentrator microreactor device connected to a cartridge-cassette single-breath analysis using a novel simple sampler and capillary electrophoresis with contactless conductometric detection breath biopsy for early detection and precision medicine in cancer origin of exhaled breath particles from healthy and human rhinovirus-infected subjected exhaled breath analysis: a review of 'breath-taking' methods off-line analysis review-non-invasive monitoring of human heath by exhaled breath analysis: a comprehensive review diagnostic potential of breath analysis-focus on volatile organic compounds critical review of volatile organic compound analysis in breath and in vitro cell culture for detection of lung cancer sensors for detecting pulmonary diseases from exhaled breath turbulent gas clouds and respiratory pathogen emissions. potential implications for reducing transmission of covid- serum and exhaled breath condensate inflammatory cytokines in community-acquired pneumonia: a prospective cohort study covid- : consider cytokine storm syndromes and immunosuppression catheter associated urinary tract infections identification of clinical and urine biomarkers for uncomplicated urinary tract infection using machine learning algorithms current and past strategies for bacterial culture in clinical microbiology urine exosomes: an emerging trove of biomarkers a host-protein signature is superior to other biomarkers for differentiating between bacterial and viral disease in patients with respiratory infection and fever without source: a prospective observational study emergence of a novel coronavirus disease (covid- ) and the importance of diagnostic testing: why partnership between clinical laboratories, public health agencies, and industry is essential to control the outbreak care for critically ill patients with covid- cell cytometry: review and perspective on biotechnological advances single cell isolation and analysis. front flow virometry: a powerful tool to functionally characterize viruses simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer detection of infectious adenoviruses in environmental waters by fluorescenceactivated cell sorting assay flow cytometry sorting to separate viable giant viruses from amoeba co-cultures supernatants microcolumn separations of single nerve cell components sampling techniques for single-cell electrophoresis high-throughput capillary-electrophoresis analysis of the contents of single mitochondria fast determination electrophoretic mobility using micro-free flow electrophoresis the emerging role of exosomes in mental disorders extracellular vesicles: composition, biological relevance, and methods of study shedding light on the cell biology of extracellular vesicles extracellular vesicles: exosomes, microvesicles and friends the biology of extracellular vesicles: the known unkowns the biology, function, and biomedical applications of exosomes fluorescence labeling of human rhinovirus capsid and analysis by capillary electrophoresis separation, identification, and characterization of microorganisms by capillary electrophoresis. microbiol using capillary electrophoresis to characterize polymeric particles dengue viruses-an overview dengue type viruses circulating in humans are highly infectious and poorly neutralized by human antibodies centers for disease control and prevention. human coronaviruses. . available online coronavirus envelope protein: current knowledge common features of enveloped viruses and implications for immunogen design for next-generation vaccines circulating immune cells in multiple sclerosis on-chip selective capture of cancer cells and ultrasensitive fluorescence detection of surviving mrna in a single living cell capillary electrophoresis method for the characterization and separation of cdse quantum dots capillary electrophoresis of bacterial extracellular vesicles: a proof of concept microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine exosome isolation and purification via hydrophobic interaction chromatography using polyester, capillary-channeled polymer fiber phase cells release subpopulations of exosomes with distinct molecular and biological properties importance of rna isolation methods for analysis of exosomal rna: evaluation of different methods rapid detection and trapping of extracellular vesicles by electrokinetic concentration for liquid biopsy on chip electrokinetic evaluation of individual exosomes by on-chip microcapillary electrophoresis with laser dark-field microscopy characteristics of exosomes and microparticles discovered in human tears microfluidic affinity separation chip for selective capture and release of label-free ovarian cancer exosomes exosomes-beyond stems cells for restorative therapy in stroke and neurological injury challenges and opportunities in exosome research-perspectives from biology, engineering, and cancer therapy the significance of exosomes in the development and treatment of hepatocellular carcinoma the methods of choice for extracellular vesicles (evs) characterization extracellular vesicle quantification and characterization: common methods and emerging approaches isolation and detection technologies of extracellular vesicles and application on cancer diagnostic. dose respon review of the isolation, characterization, biological function, and multifarious therapeutic approaches of exosomes microfluidic technology for clinical applications of exosomes quantification of exosomes advances in exosomes technology integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology detection and isolation of circulating exosomes and microvesicles for cancer monitoring and diagnostics using micro-/nano-based devices on-chip immunoelectrophoresis of extracellular vesicles released from human breast cancer cells on a chip microfluidic approaches for isolation, detection and characterization of extracellular vesicles: current status and future directions host derived exosomes-pathogens interactions: potential functions of exosomes in pathogen infection decoy exosomes provide protection against bacterial toxins drug delivery and nanoparticles: applications and hazards progress, opportunity, and perspective on exosome-based theranostics. theranostics emerging applications of exosomes in cancer therapeutics and diagnostics the therapeutic potential of exosomes the role of extracellular vesicles in covid- virus infection technical challenges of working with extracellular vesicles minimal information of extracellular vesicles (misev ): a position statement of the international society for extracellular vesicles and update of the misev guidelines exosomal micrornas and other non-coding rnss as colorectal cancer biomarkers: a review non-coding rnas in exosomes: new players in cancer biology micrornas: from mechanism to organism. front. cell dev. bio. , , exosomes and non-coding rna, the healers of the heart? (editorial) exploration of long non-coding rnas and circular rnas in porcine milk exosomes non-coding rna: what is functional and what is junk? front respiratory viral infection in lung transplantation induces exosomes that trigger chronic rejection exosomes-based delivery of super-repressor ikba relieves sepsis-associated organ damage and mortality exosomes could offer new options to combat the long-term complications inflicted by gestational diabetes mellitus engineered tumor-derived extracellular vesicles: potentials in cancer immunotheraphy proteomic analysis of exosomes from adipose-derived mesenchymal stem cells: a novel therapeutic strategy for tissue injury extracellular vesicles: potential role in osteoarthritis regenerative medicine exosomes: an emerging factor in atherosclerosis discovery of urinary biomarkers high-grade prostate cancer at initial biopsy exosomes derived from bone marrow mesenchymal stem cells as treatment for severe covid- exosomes facilitate transmission of sars-cov-genome into human induced pluripotent stem cell-derived cardiomyocytes bovine milk-derived exosomes for drug delivery mesenchymal stem cell-derived exosomes for clinical use pulp-derived exosomes in fibrin-based regenerative root filling material exosomes in cancer: circulating immune-related biomarkers exosomal vaccines containing the s protein of the sars coronavirus induce high level of neutralizing antibodies exosomal noncoding rnas in glioma: biological functions and potential clinical applications exosomes in ischemic heart disease: novel carriers for bioinformation roles for exosomes in various kidney diseases and disorders exosomes in inflammation and role as biomarkers human immunodeficiency virus-associated exosomes promote kaposi's sarcoma-associated herpesvirus infection via the epidermal growth factor exosomes in semen: opportunities as a new tool in prostate cancer diagnosis microrna biomarkers of parkinson's disease in serum exosome-like microvesicles exosomics"-a review of biophysics, biology and biochemistry of exosomes with a focus on human breast milk exosomes derived from akt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor d. stem cells transl exosomes in allergic airway diseases exosomes in parkinson's disease exosomes regulate neurogenesis and circuit assembly circular rnas in early brain development and their influence and clinical significance in neuropsychiatric disorders proteomic and lipidomic analysis of exosomes derived from ovarian cancer cells and ovarian surface epithelial cells in memoriam: we would like to dedicate this paper to the memory of five friends, colleagues, pioneers and educators, whose lives were unfortunately cut short, for their great contributions to the technology of capillary electrophoresis: csaba horváth, brian howard, craig lunte, kevin altria, and gerhardus de jong. norberto guzman is the inventor of several patents issued and pending related to the iace technology. key: cord- -w wrn lx authors: díez, josé-maría; romero, carolina; gajardo, rodrigo title: currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus antigens date: - - journal: immunotherapy doi: . /imt- - sha: doc_id: cord_uid: w wrn lx aim: there is a critical need for effective therapies that are immediately available to control the spread of covid- disease. material & methods: gamunex(®)-c and flebogamma(®) dif (grifols) intravenous immunoglobulin (ivig) products were tested using elisa techniques for antibodies against several antigens of human common betacoronaviruses that may crossreact with the severe acute respiratory syndrome coronavirus (sars-cov- ) virus. results: both ivigs showed consistent reactivity to components of the tested viruses. positive crossreactivity was seen in sars-cov, middle east respiratory syndrome-cov and sars-cov- . for sars-cov- , positive reactivity was observed at ivig concentrations ranging from μg/ml with gamunex-c to mg/ml with flebogamma % dif. conclusion: gamunex-c and flebogamma dif contain antibodies reacting against sars-cov- antigens. studies to confirm the utility of ivig preparations for covid- management may be warranted. the outbreak of a novel viral respiratory disease, covid- , is caused by infection with the severe acute respiratory syndrome (sars) coronavirus (sars-cov- ). due to its extreme transmissibility, covid- has spread dramatically within weeks since the first recognition in china in late december [ ] . increased human mobility as a global phenomenon has created favorable conditions for covid- to become a pandemic. although symptoms are typically mild, in some patient groups, covid- can progress to severe respiratory failure which is associated with significant morbidity and mortality. these patients with severe disease are straining the available critical care resources of the most affected countries [ ] . in the short term, the lack of a vaccine and therapeutic agents of proven efficacy against sars-cov- further aggravates this trend. this critical situation demands a reliable therapy that is immediately available to control the spread of the disease. historically, convalescent plasma or plasma-derived immunoglobulin (ig), either polyvalent ig (prepared from pooled plasma from thousands of healthy donors) or hyperimmune ig (prepared from the plasma of donors with high titers of antibody against a specific antigen), has been used as the fastest therapeutic option in outbreaks of emergent or re-emergent infections [ ] . coronaviruses are globally distributed [ ] and four main common human coronavirus (hcov) subtypes have been identified to date: hcov- e, hcov-nl , hcov-oc and hcov-hku . sars-cov- is a novel emerging and deadly coronavirus. it joins sars-cov, responsible for the sars outbreak in and middle east respiratory syndrome (mers)-cov, responsible for the mers outbreak in . since coronavirus infections induce virus-neutralizing antibodies, convalescent plasma therapy was successfully used in the treatment of both sars [ , ] and mers [ ] patients. common human coronaviruses are accountable for a large proportion of respiratory infections which, in most cases, are mild. because of this ubiquity, antibodies against human common coronaviruses are present in the normal population. since intravenous immunoglobulin (ivig) is a polyvalent ig prepared from plasma from thousands of donors, this product covers a large spectrum of immunity in the general population and, as expected, includes anti-coronavirus antibodies. it is important to note that coronaviruses of the same subgroup, particularly betacoronaviruses such as hcov-oc , hcov-hku , sars-cov, sars-cov- and mers-cov, show some crossreactivity in antigenic responses. crossreactivity between sars-cov and mers-cov with other common human betacoronaviruses has been reported in some neutralization assays [ ] [ ] [ ] . the fact that the new betacoronavirus sars-cov- is directly related to sars-cov (they share more than % sequence homology) [ ] suggests that antigenic crossreactivity between them is possible, at least for some proteins. to explore this potential therapeutic pathway, this study was designed to detect antibodies against common human coronaviruses in ivig products that may crossreact with the new sars-cov- virus. gamunex r -c (grifols therapeutics, inc., nc, usa) and flebogamma r dif (instituto grifols s.a., barcelona, spain) ivigs were tested for crossreactivity against several betacoronaviruses, including sars-cov, mers-cov and sars-cov- antigens, using elisa techniques. gamunex-c and flebogamma dif are unmodified human ivig products (≥ to % igg) manufactured from plasma collected from donors in the usa and/or several european countries. gamunex-c is manufactured at a concentration of mg/ml ( %) while flebogamma dif is available as and mg/ml ( and %) igg concentrations. the following kits were used for the qualitative determination of igg class antibodies against human coronaviruses: abx human coronavirus igg elisa kit (abbexa, cambridge, uk), against an undetermined antigen; mbs , hcov-hku-igg elisa kit (mybiosource, inc., ca, usa), against n protein; deia ; sars coronavirus igg elisa kit (creative diagnostics, ny, usa), against virus lysate; rv- - ; human anti-mers-np igg elisa kit (alpha diagnostic intl., inc., tx, usa), against n protein; rv- - , human anti-mers-receptor-binding domain (rbd) igg elisa kit (alpha diagnostic intl. inc.), against rbd of s subunit spike protein (s /rbd); rv- - , human anti-mers-s igg elisa kit (alpha diagnostic intl., inc.), against s subunit spike protein; rv- (formerly rv- - ); human anti-sars-cov- virus spike [s ] igg elisa kit (alpha diagnostic intl., inc.), against s subunit spike protein; ei- - -g, anti-sars-cov- igg elisa kit (euroimmun ag, luebeck, germany), against structural protein (s domain); deiasl , sars-cov- igg elisa kit (creative diagnostics), against virus lysate. in all cases, the determinations were carried out following the manufacturer's instructions. sample preparation & testing ivig samples were serially diluted using the buffer solutions provided in each igg elisa kit. with the ivig % product, the dilution series was: neat (undiluted), : , : , : , : and : . with the ivig % product, the dilution series was: neat, : , : , ; , : and : . therefore, final igg concentrations of the samples were: mg/ml, mg/ml, mg/ml, mg/ml, μg/ml, μg/ml and μg/ml. in sars-cov- tests, additional dilutions of : ( μg/ml) and : ( μg/ml) were included. reactivity against the coronavirus antigens in the different elisa kits was rated as negative (−) if no reactivity was observed even with neat ivig, or positive (+) if the lowest ivig dilution demonstrated reactivity. independent assays on the same lots were performed on different days: - for gamunex-c, - for flebogamma % dif and - for flebogamma % dif. both gamunex-c and flebogamma dif showed consistent reactivity to components of the tested viruses including a variety of virus proteins, except for the n-protein from hcov-hku . there was no reactivity to this protein even with undiluted ivig samples. as shown in table , positive reactivity was particularly apparent in sars-cov, mers-cov and sars-cov- . in the case of mers-cov, positive reactivity was observed in ivig samples down to : dilution ( μg/ml) for n protein, s -rbd protein and s protein. for sars-cov- s protein, positive reactivity ranged from an ivig concentration of μg/ml with gamunex-c using the rv- elisa kit to mg/ml with flebogamma % dif using the ei- - -g elisa kit ( table ). the sars-cov- lysate showed reactivity at half-diluted gamunex-c and undiluted flebogamma % dif. reactivity to hcov (betacoronavirus undetermined antigen) was also observed, although less consistently: negative for gamunex, but positive for flebogamma dif at low dilutions (table ) . the need for readily available effective therapies to combat sars-cov- infection is compelling. in this study, we considered whether ivig treatment could contribute to covid- disease management. to test this hypothesis, known currently available ivig products, gamunex-c and flebogamma dif, were tested for crossreactivity with sars-cov- and other coronaviruses, including sars-cov and mers-cov. in this first-time report, we found significant crossreactivity to components of all tested viruses including the s protein of sars-cov- , the protein responsible for virion attachment to the host cell and neutralization [ ] . the consistency of our crossreactivity results among the sars-cov- , sars-cov and mers-cov viruses is noteworthy. this replicates with the new sars-cov- , the crossreactivity already reported for sars-cov/mers-cov with other human betacoronaviruses [ ] [ ] [ ] . gamunex-c and flebogamma dif were confirmed to contain antibodies reacting against sars-cov- antigens, although further research is needed to prove functionality and safety for covid- . elisa results for the undetermined antigen of hcov were also mostly positive. this was in contrast to hcov-hku , which had negative reactivity. hcov-hku was discovered in in hong kong and, although it did not result in an outbreak and had only restricted spread, this virus is probably still circulating in the population [ , ] . however, the negativity of an ivig reaction using a single elisa coronavirus kit does not mean that such ivig does not contain antibodies against this pathogen. elisa sensitivity relies on factors such as the antigen used, the sequence, the organism used to produce it, and the amount of material coated. elisa results should only be compared qualitatively, since the comparison of the results between different kits is difficult based on differences in sensitivity, and there is no gold standard for quantification. this was confirmed in our results for the sars-cov- s protein subunit antigen, in which different elisa kits showed different reactivities. in addition, there is scarcity of tests for common coronaviruses. it has been observed that patients who develop a more severe clinical course of sars-cov- infection have higher plasma levels of pro-inflammatory cytokines, suggesting a possible 'cytokine storm' associated with the disease severity [ ] . ivig products have been demonstrated to be effective in the treatment of inflammatory disorders [ ] . hence, ivig is considered a therapeutic option for hyperinflammation in patients with severe covid- [ ] . to date, a number of possible mechanisms for the immunomodulatory and anti-inflammatory effects of ivig therapy have been described [ , ] , including anticomplement effects [ ] , anti-idiotypic neutralization of pathogenic autoantibodies [ ] , immune regulation via an inhibitory fc receptor [ , ] , enhancement of regulatory t cells [ ] and inhibition of th differentiation [ ] . thus, ivig may mediate a wide variety of biological and immunomodulatory effects via various types of blood cells [ ] . when considered together, these immunomodulatory effects of ivig products might be beneficial in covid- disease management, but this needs to be confirmed clinically. although ivig may not confer protection against sars-cov- , the role of antibodies crossreacting with sars-cov- has been suggested by exerting a priming effect on host immune response or through an immunomodulatory action on monocytes and tissue-resident macrophages [ ] . the antibody-dependent enhancement (ade) phenomenon associated with non-neutralizing antiviral proteins in ivig also needs to be considered. ade is often due to low-affinity antibodies and is well described for some viruses such as dengue, and it has also been mentioned for sars-cov- [ ] . ade has been only suggested for sars-cov- [ ] and, at this stage, it cannot entirely be ruled out. the positive elisa binding results reported should only be considered exploratory, and not generalized or extrapolated to indicate clinical utility. rather, the findings can be viewed as a base for further evaluation of ivig to investigate its potential in vivo activity in functional tests such as neutralization assays. even with this uncertainty, in the context of the current health emergency (pandemic), the potential of ivig as a therapy for covid- is already being evaluated in a number of studies involving patients with severe sars-cov- viral infections including pneumonia [ ] [ ] [ ] . we consistently observed crossreactivity of ivig products with sars-cov- , sars-cov and mers-cov using elisas from different manufacturers. this evidence supports the presence of anti-sars-cov- crossreacting antibodies in these ivig preparations. these results, together with the known immunomodulatory and antiinflammatory effects of ivig, suggest a potential positive contribution of currently available ivig products to covid- disease management, although it should be confirmed by antibody functionality studies. • there is an urgent need for readily available effective therapies to combat covid- disease. • this is the first time that currently available intravenous immunoglobulins have been reported to contain antibodies that crossreact against antigens of sars-cov- and other coronaviruses. • further studies to confirm the functionality of intravenous immunoglobulin antibodies may be warranted. european centre for disease prevention and control. covid- . situation update worldwide strategies for the prevention and management of coronavirus disease use of human immunoglobulins as an anti-infective treatment: the experience so far and their possible re-emerging role •• a comprehensive review on the role of intravenous immunoglobulin (ivig) as anti-infective treatment in community and emerging diseases global patterns in coronavirus diversity experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital use of convalescent plasma therapy in sars patients in hong kong feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia an outbreak of human coronavirus oc infection and serological crossreactivity with sars coronavirus. can • a report to understand the key role of antibody crossreactivity in emerging viral disease crossreactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc ( ) by both immunofluorescent and neutralizing antibody tests • a report to understand the key role of antibody crossreactivity in emerging viral disease antigenic crossreactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses e and oc • a report to understand the key role of antibody crossreactivity in emerging viral disease profiling early humoral response to diagnose novel coronavirus disease (covid- ) characterization of spike glycoprotein of sars-cov- on virus entry and its immune crossreactivity with sars-cov coronavirus hku infection in the united states characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia clinical features of patients infected with novel coronavirus in wuhan intravenous immunoglobulin a natural regulator of immunity and inflammation covid- : consider cytokine storm syndromes and immunosuppression immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin classic review on the role of pleiotropic properties of ivig beyond immunodeficiency treatment inhibitory effect of pooled human immunoglobulin on cytokine production in peripheral blood mononuclear cells post-polio syndrome patients treated with intravenous immunoglobulin: a double-blinded randomized controlled pilot study mechanisms of action of immune globulin intravenous immunoglobulin modulates the maturation of tlr -primed peripheral blood monocytes intravenous immunoglobulin g (ivig) inhibits il- -and tnf-alpha-dependent, but not chemotactic-factor-stimulated, neutrophil transendothelial migration anti-inflammatory effects of high-dose igg on tnf-alpha-activated human coronary artery endothelial cells two x-linked agammaglobulinemia patients develop pneumonia as covid- manifestation but recover molecular mechanism for antibody-dependent enhancement of coronavirus entry is covid- receiving ade from other coronaviruses? microbes infect high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease • the first clinical study to evaluate the efficacy of ivig in the treatment of severely ill covid- patients effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with covid- the efficacy of intravenous immunoglobulin therapy for severe -ncov infected pneumonia acknowledgments j bozzo, cmpp and mk james (grifols) are acknowledged for the medical writing and editorial support in the preparation of this manuscript. contribution from a páez, s fernández and e calderón (grifols) who provided their expert opinion is acknowledged.the authors acknowledge the expert technical assistance from d casals, e sala, j luque and g mercado (grifols, viral and cell culture laboratory). the authors are full-time employees of grifols, the manufacturer of gamunex-c and flebogamma dif. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.editorial assistance has been provided by content ed net spain with funding by grifols s.a. this work is licensed under the attribution-noncommercial-noderivatives . unported license. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/ . / key: cord- - pheilu authors: kostopoulou, despoina; gizzarelli, manuela; ligda, panagiota; foglia manzillo, valentina; saratsi, katerina; montagnaro, serena; schunack, bettina; boegel, annette; pollmeier, matthias; oliva, gaetano; sotiraki, smaragda title: mapping the canine vector-borne disease risk in a mediterranean area date: - - journal: parasit vectors doi: . /s - - - sha: doc_id: cord_uid: pheilu background: the aim of this study was to determine exposure to vector-borne pathogens (vbps) in populations of dogs living on greek islands in the ionian and aegean seas. methods: in total, dogs with different lifestyles and of varying ages and breeds were randomly sampled and examined for the presence of clinical signs compatible with canine vector-borne diseases (cvbds). blood was collected from each individual animal. for the detection of antibodies against leishmania spp., the witness® leishmania test was performed, and positive samples were further examined with indirect enzymatic immunoassay (elisa). antibodies to borrelia burgdorferi, ehrlichia canis or e. ewingii, as well as anaplasma phagocytophilum or a. platys were investigated using the snap® dx® plus test. positive ehrlichia spp. and anaplasma spp. samples were further examined using an indirect elisa for further identification of the species. results: in total, . % of dogs were exposed to at least one of the pathogens investigated, with seroprevalences varying regionally. of these seropositive dogs, . % displayed clinical signs suggestive of cvbds, such as cutaneous lesions, enlarged lymph nodes, pale mucous membranes, onychogryphosis and weight loss. the overall seroprevalence detected using the rapid tests was . % for leishmania spp., whereas . % of the examined dogs were found to be positive for anaplasma spp. and . % for ehrlichia spp. while b. burgdorferi was not detected. twenty-four samples positive to a. phagocytophilum by elisa were analysed by pcr for the presence of anaplasma dna. pcr and sequencing results showed the presence of a. platys dna in samples and e. canis dna in samples. the remaining samples ( . %) were negative. conclusions: in the present study, exposure of dogs to vbps was shown in the geographical areas investigated. results confirm that on greek islands vbps represent a constant health risk for both native and visiting dogs, suggesting the presence of distinct “hot-spots” of vbp infections on different islands. in order to reduce the risk of transmission and the spread to non-endemic regions, the protection of dogs through use of repellents and vaccines, together with owner education, seem to be of paramount importance. [image: see text] vector-borne diseases (vbds) represent a growing global threat, both in human and veterinary medicine, because of their constant spread from traditional geographical and temporal restraints to new areas, exposing new populations to previously unknown infectious agents and posing unprecedented challenges to veterinarians. the constantly changing epidemiology of vbds is being influenced by different factors, the most important of which are climatic changes (affecting in many ways both vector arthropods and pathogen development rates) and increasing mobility of owners and their companion pet populations, supporting in fact pathogen and vector spread [ ] [ ] [ ] [ ] . canine vector-borne diseases (cvbds) are caused by a range of pathogens transmitted to dogs by bloodfeeding arthropods, e.g. ticks, fleas, mosquitoes and sand flies. the cvbds commonly diagnosed are anaplasmosis, babesiosis, bartonellosis, borreliosis, dirofilariosis, ehrlichiosis and leishmaniosis. these diseases are characterized by a complex pathogenesis with a potentially fatal clinical course for the majority of cases with new pathogenic findings being uncovered every year [ ] . in addition, several have a zoonotic potential with possible transmission to the human population [ ] [ ] [ ] . it is important to recognize that the transmitted pathogens may also frequently originate subclinical infections that nevertheless render the host as a carrier and even a reservoir [ ] . in view of the above considerations and in the context of human and animal health protection, there is an immense need to map the presence of canine vectorborne pathogens (cvbps) in areas such as popular tourist destinations. the greek islands are amongst the most visited touristic places around the world, attracting millions of people per year (i.e. . million international airport arrivals from january to october , with . million arrivals on crete alone [ ] ), many of whom travel with their pets. overall, located in the mediterranean basin with a suitable environment for pathogen transmission and poorly managed stray dog populations, greece has been repeatedly reported as an area highly endemic for various cvbds [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, landscape and microclimate conditions are not identical throughout the country and this stresses the need for comprehensive knowledge on the distribution and abundance of various cvbps within native dog populations and across different islands, which currently is missing. the aim of this study was to gain information on (i) the health risk for both native and visiting dogs, and (ii) the risk of pathogen distribution to new areas (i.e. via dogs travelling back from holidays or stray dogs being adopted and moved to various other countries in europe), by determining the exposure of dog populations living on greek islands in different geographical locations to cvbps. a cross-sectional study was conducted in canine populations from different greek islands in order to evaluate the seroprevalence of cvbps. in total, islands were selected based on their geographical location [(i) situated in both ionian and aegean seas; (ii) covering areas west to east of the country; (iii) having different landscapes and climatic conditions], the size of their native dog population (traditionally islands like leros and paxoi despite their size are known for the high hunting dog population), and previous records (published or personal communication) of cvbd presence [ , , [ ] [ ] [ ] [ ] . altogether, dogs with different lifestyles (indoors/outdoors), irrespective of age and breed were randomly sampled and examined for the presence of clinical signs suggestive of cvbds. regarding the specific locations, dogs were enrolled from crete, from leros (located in the south aegean sea), from paxoi and from zakinthos (both located in the ionian sea) (fig. ). in crete, due to its size (both in total surface and human/dog population) sampling areas were included, representing of the cretan counties (i.e. from the west to the east: chania, rethymno and heraklion). a blood sample of approximately ml was collected from each individual animal equally divided into a gel and clot activator, and an edta tube. the samples were immediately maintained at °c. all samples were examined within days of collection. for the detection of antibodies against leishmania infantum, the witness ® leishmania test (rapid immuno migration (rim ™ ) test; zoetis diagnostics, parsippany, usa) was performed. positive samples were further examined with an indirect enzymatic immunoassay (elisa) for the detection of specific antibodies to l. infantum (inge-zim ® leishmania; ingenasa, madrid, spain). antibodies against borrelia burgdorferi, ehrlichia canis/e. ewingii, as well as anaplasma phagocytophilum/a. platys were investigated with the snap ® dx ® plus test (idexx laboratories, westbrook, usa). samples which tested positive for ehrlichia spp. and anaplasma spp. with the rapid test were further examined using an indirect elisa for the detection of antibodies specific to e. canis (ingezim ® ehrlichia; ingenasa) and a. phagocytophilum (anaplasma-elisa dog; afosa gmbh, blankenfelde-mahlow, germany) respectively. all the procedures above were performed following the manufacturers' instructions. samples found positive for the presence of anaplasma antibodies using elisa, were further investigated with molecular techniques to identify the species of anaplasma. genomic dna was extracted from positive edtablood samples using the dneasy blood & tissue kit (qiagen gmbh, hilden, germany), according to the manufacturer's instructions. pcr was performed to amplify a bp fragment of the s rrna gene [ ] of various species including e. canis, e. chaffeensis, e. muris, e. ruminantium, a. phagocytophilum, a. platys, anaplasma marginale, anaplasma centrale, wolbachia pipentis, neorickettsia sennetsu, neorickettsia risticii and neorickettsia helminthoeca, according to previously described thermal-cycling conditions [ ] . a positive (a. phagocytophilum reference dna sample) and a negative (pcr grade water) control were included in each pcr run. amplification products were visualized on . % agarose gels stained with ethidium bromide. pcr products were sent to a commercial service (cemia sa, larissa, greece) for purification and sequencing on both strands (sanger sequencing). the results were assembled with seqman . software (lasergene dnastar, madison, wi, usa). assembled sequences were aligned using the basic local alignment tool (blast) and compared with reference sequences using the megalign (lasergene dnastar). as age was deemed an important variable, dogs included were split into age groups (≤ ; > - ; > - ; > yearsold). in addition, lifestyle (indoor or outdoor) and location of the animal were used as variables to calculate proportions for each of the pathogen species identified and summarized descriptively. seroprevalence was calculated based on the rapid test results, which were also used for further analyses. analyses of the binary variables (negative or positive) were performed using a generalized linear mixed model (glimmix) and a logit link [ ] . the statistical model included lifestyle, location, age category, the interaction of lifestyle and location as fixed effects as well as the -way interactions of lifestyle * age category, and location * age category, and the -way interaction of lifestyle * location * age category as random effects. if any of the fixed factors were found to be significant (alpha = . for all analyses), then pairwise comparisons of the possible combinations were performed at the . level. sas ® statistical software version . was used for all analyses. the majority of dogs sampled were years-old or younger ( . %) and the sample size included a comparable number of dogs living indoors or outdoors (tables , and ) . during the initial screening by rapid test, the seropositivity of l. infantum, e. canis/e. ewingii and a. phagocytophilum/a. platys was recorded whereas all samples were found seronegative for b. burgdorferi. in total, . % of the animals were seropositive for at least one pathogen and . % of the animals co-seropositive for or more pathogens. of the seropositive dogs, . % displayed clinical signs suggestive of cvbds, such as cutaneous lesions, enlarged lymph nodes, pale mucous membranes, onychogryphosis and weight loss. zakinthos specifically, for the dogs tested in the different counties of crete, the percentages of samples found positive in the different tests were for: (i) chania: a total of out of serum samples tested positive for ehrlichia antibodies with the rapid test ( . %, % ci: . - . %). no statistically significant association was found in relation to lifestyle and age, even though prevalence increased with age. location was the only statistically significant factor. on the island of leros a significantly greater number of dogs were found positive (model adjusted prevalence of %) than on the other islands ( table ). all samples positive in the rapid tests for ehrlichia spp. were found to be seropositive for e. canis by elisa. twenty-six out of dogs were positive for anaplasma spp. in the rapid test ( . %, % ci: . - . %). there were no statistically significant differences in relation to lifestyle, age and location of the animals ( table ). the highest seroprevalence of anaplasma positive dogs was observed on the island of crete. all but samples positive in the rapid tests for anaplasma spp., were found to be seropositive for a. phagocytophilum by elisa. in order to further investigate the presence of a. phagocytophilum, all samples positive by elisa ( from paxoi, from zakinthos, from leros and from crete) were analysed by pcr for the presence of anaplasma dna. none of the samples tested showed the presence of in the present study, exposure of dogs to vector-borne pathogens was shown in all geographical areas investigated with the prevalence for different pathogens varying regionally. leishmania infantum was endemic on all islands, being the most prevalent agent with an overall prevalence of . % based on the rapid test and . % after specification with elisa. the prevalence was lower than results found in earlier nation-wide studies, which showed a mean prevalence as high as . % [ , ] . in these previous studies, the seroprevalence of l. infantum found in dogs in corfu, an island in the ionian sea close to paxoi, was . %, whereas in our sample from paxoi it was . % by the rapid test and . % by elisa. accordingly, the prevalence previously reported for islands located in the south aegean sea ranged from . % to % [ , , ] , rates which were to some extent higher than recorded in the present study ( % and % for crete and . % and . % for leros in the rapid test and elisa, respectively). one reason for such differences might be the diverse screening approaches used. in the present study, screening was referring to seroprevalence which was at first defined by the rapid test followed by an elisa (cut-off titre : ) for positive results. in contrast, seroprevalence using an ifat test (cut-off titre ranging from : to : ) and a questionnaire approach (based on confirmed laboratory of clinically cases of leishmaniosis) was used in previous studies [ , ] . however, the rates observed here could also be attributed to a real reduction in the abundance of leishmania, due to better awareness to the disease and better compliance to preventive measures by dog owners. it is worth mentioning that, in the meantime, the first relevant vaccine (canileish ® ; virbac, carros, france, authorized in the european union in ), and an increasing number of sand fly repellents have been introduced to the market. regarding the geographical differences, it seems that the exposure of dogs to l. infantum is higher on the ionian sea islands, especially on paxoi. this is in agreement with previous observations regarding the geographical location of leishmania-infected dogs. within crete, the highest seroprevalence was recorded in heraklion county as also previously reported [ , ] . previous records on the sand fly fauna in greece have already demonstrated a rich population of sand fly species on all of the studied islands, which includes important vectors of leishmania spp. [ , ] . however, islands in the ionian sea could represent a more favourable environment for both sand flies and leishmania due to the different climatic conditions. regardless of both areas covering a similar latitudinal range and having a typical mediterranean climate, precipitation levels are higher on the ionian islands (with mean precipitation of approximately mm annually) compared to the aegean islands (with mean precipitation of approximately mm annually), according to world clim-global climate data [ ] . as previously documented by ntais et al. [ ] animals living in areas with higher annual rainfall, and low and medium values of mean wind speed had a higher risk of exposure to leishmania. the high wind speed, influencing sand fly activity, could also be a reason for the lower seroprevalence found in leros [ ] . in general, quantitative elisa-based tests perform better than qualitative immunochromatographic rapid tests. while both tests have a proven high specificity, the sensitivity of the rapid test used in the present study (wit-ness ® leishmania, zoetis diagnostics) is significantly lower (i.e. . versus . ) compared to the elisa used (ingezim ® leishmania) [ ] . therefore, a lower number of seropositive dogs were detected when positive rapid test results were confirmed with the quantitative elisa, resulting in % lower infection rates in paxoi. this could also be due to antibody titres being below the elisa cut-off (the standard cut-off value for quantitative elisa is set at : ). it is important to mention that although the diagnostic tests used in this study were aiming to detect specific antibodies against l. infantum, they are not always distinctive between antibodies against this species and l. major and/or l. tropica [ ] . however, during clinical examinations, no clinical signs similar to those reported for l. major/l. tropica disease in dogs were recorded [ ] . due to previous records of l. tropica (and the competent sand fly vector) in greece and its capacity to infect dogs, this point should be further investigated, especially since l. major/l. tropica cause anthroponotic cutaneous leishmaniasis [ , ] . ehrlichia spp. were diagnosed only in dogs living more to the east (i.e. crete and leros), with the prevalence being significantly higher in leros. even within the island of crete there was an obvious tendency of a higher prevalence to the east (heraklion showed highest prevalence for ehrlichia and anaplasma). moreover, ehrlichia seropositive dogs were not identified on the ionian sea islands. in the present study, the overall seroprevalence recorded was lower than that found in studies recently conducted in the whole of greece (a prevalence of approximately . %) with similar sample size and methods applied [ , ] . in these studies, however, the majority, if not all, of samples originated from mainland greece. in another recent study with a remarkable number of dogs (n = ), a similar to the present data from aegean islands ehrlichia spp. seropositivity was found ( . %) [ ] . the above data further support the observation in this study that the exposure of dogs to ehrlichia spp. varies significantly within the country, a fact which could be attributed to microhabitat conditions favouring (or not) both the vector and the pathogen. it is quite interesting to note that a recent study testing ixodid ticks (i.e. rhipicephalus sanguineus (sensu lato), r. turanicus, haemaphysalis parva, and h. concinna) from mainland greece for the presence of vbp could not confirm the presence of ehrlichia dna [ ] . anaplasma spp. were detected at all collection sites. using the rapid test (snap ® dx ® plus, idexx laboratories), which detects both a. phagocytophilum and a. platys, a high number of samples were identified as a. phagocytophilum/a. platys, and later confirmed to be a. phagocytophilum by a specific quantitative elisa. this high positivity for a. phagocytophilum, especially in crete, was surprising insofar as the predominant tick species in greece is r. sanguineus (s.l.), which is the vector for a. platys [ ] . while a. phagocytophilum has been previously reported from rhipicephalus bursa ticks in greece [ ] , as well as from rhipicephalus spp. ticks from other areas, indicating that there is a potential also for these ticks to play a role in the transmission of a. phagocytophilum [ ] . the primary tick vector for a. phagocytophilum, ixodes ricinus, is only rarely found in greece. ixodes ricinus ticks are primarily associated with shrubs and forests with deciduous trees; therefore, they can be found around the forested zones in the north, but the hot and dry semi-arid environment around studied areas does not represent the optimum conditions for this species. cross-reactivity between a. phagocytophilum and a. platys in serological tests has previously been shown by testing seropositive a. phagocytophilum samples with other, more sensitive techniques like pcr [ ] . therefore, in order to further investigate the presence of a. phagocytophilum within the study population, samples which tested positive for a. phagocytophilum by elisa were further analysed by pcr. as a result, a. phagocytophilum dna was not detected at any of the samples tested positive by elisa. instead, the presence of a. platys was confirmed in four of these samples and also the presence of e. canis dna was detected in four other samples, which however, were also found to be positive for antibodies against e. canis by the species-specific elisa. the failure to detect anaplasma dna by pcr could be due to chronic infections, in which parasites are not always present in the blood, or to cross-reactions leading to false positive results in serological testing [ ] . similar to the results for ehrlichia, the overall seroprevalence recorded in the present study was lower than that in a recent study where samples originated mainly from mainland greece [ ] . likewise, in the study including samples from aegean islands, anaplasma spp. were recorded in only % ( out of samples); however, a. phagocytophilum was isolated in one of the samples by pcr [ ] . the presence of anaplasma spp. dna was confirmed in ticks collected from dogs from various locations in greece and identified at the species level [ ] as either a. platys [ ] or a. phagocytophilum and a. platys [ ] . none of the dogs examined were seropositive for borrelia spp., which is in agreement with the very low prevalences recorded in whole of greece [ , ] . within a given area, the risk of animal/human infection with b. burgdorferi (s.l.) is determined by the local abundance and infection of vector ticks and the wild vertebrate hosts. therefore, this result was expected since b. burgdorferi (s.l.) is transmitted by ticks of the i. ricinus complex. ixodes ricinus is only rarely found in greece, and recent surveys also failed to identify the presence of borrelia in ticks in greece [ , , , , ] . this study confirms that dogs living on different greek islands are largely exposed to cvbp. with different "hot-spots" of higher abundance of a specific pathogen in certain regions, more detailed knowledge on what drives the development of such "hot-spots" is necessary. unfortunately, at this current time there is neither clear and definitive information on the ectoparasite species abundance in the different locations, nor on the use of preventatives against ticks or sand flies which would contribute to the understanding of these findings. the seroprevalence of cvbps reported here represent a constant health risk for both native and visiting dogs, and in some cases may amplify the risk for human infection. additionally, a major threat is posed by the potential spread of both vectors and pathogens, endemic in southern europe to northern, non-endemic regions of europe and beyond. however, in order to define this threat, the infection pressure (i.e. as calculated by the seroprevalence of native dogs) and time of exposure to it (e.g. days in the specific environment), in order for naïve dogs to become infected, need to be better understood, since there are studies that claim the risk for infection is low especially during a limited single stay in endemic countries [ ] . in order to reduce the risk of transmission and the spread to non-endemic regions, the protection of dogs together with owner education, are of paramount importance. emerging arthropod-borne diseases of companion animals in europe imported and travelling dogs as carriers of canine vector-borne pathogens in germany arthropod-borne infections in travelled dogs in europe spread of leishmania infantum in europe with dog travelling vector-borne diseases-constant challenge for practicing veterinarians: recommendations from the cvbd world forum. parasites vectors future challenges for parasitology: vector control and 'one health' in europe: the veterinary medicinal view on cvbds such as tick borreliosis, rickettsiosis and canine leishmaniosis arthropod-borne pathogens of dogs and cats: from pathways and times of transmission to disease control the role of companion animals in the environmental circulation of tick-borne bacterial pathogens managing canine vector-borne diseases of zoonotic concern: part one insete leishmaniases in greece the use of spatial analysis to estimate the prevalence of canine leishmaniasis in greece and cyprus to predict its future variation and relate it to human disease ticks and associated pathogens in dogs from greece. parasites vectors prevalence and risk factors for selected canine vector-borne diseases in greece. parasites vectors cross-sectional serosurvey and factors associated with exposure of dogs to vector-borne pathogens in greece endoparasites and vector-borne pathogens in dogs from greek islands: pathogen distribution and zoonotic implications vector-borne pathogens affecting shelter dogs in eastern crete detection of ehrlichia platys dna in brown dog ticks (rhipicephalus sanguineus) in okinawa island detection of ehrlichia platys in dogs in australia • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research ready to submit your research ? choose bmc and benefit from phlebotomine sand flies (diptera: psychodidae) in the greek aegean islands: ecological approaches. parasites vectors performance of commercially available serological diagnostic tests to detect leishmania infantum infection on experimentally infected dogs canine leishmaniosis caused by leishmania major and leishmania tropica: comparative findings and serology. parasites vectors mucocutaneous leishmania tropica infection in a dog from a human cutaneous leishmaniasis focus leishmaniasis emergence in europe species diversity and spatial distribution of ixodid ticks on small ruminants in greece bacterial pathogens and endosymbionts in ticks seroprevalence and current infections of canine vector-borne diseases in nicaragua infectious diseases of the dog and cat molecular identification of tick-borne pathogens in ticks collected from dogs and small ruminants from greece prevalence of borrelia burgdorferi sensu lato genospecies in ixodes ricinus ticks in europe: a metanalysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank all the veterinarians in crete this study was conducted by dk, mg and pl who visualised the study, participated in the data collection and analysis and developed the original draft manuscript as well as editing all other versions. ss and go are responsible for conceptualization and supervision of the study, contributed to data collection and analysis, interpretation of findings, and reviewing and editing of the manuscript. sm was responsible for statistical analysis of the data. all other authors (vfm, ks, bs, ab and mp) played a role in data collection and analysis, and interpretation of the findings. all authors read and approved the final manuscript. the study was partly funded by bayer animal health, by the municipality of hersonissos crete and by vri own funds. all data generated or analysed during this study are included in this published article. the raw datasets used during the present study are kept in the veterinary research institute (vri) of the hellenic agricultural organization -dem-eter and are available upon reasonable request. representative sequences for a. platys and e. canis were deposited in the genbank database under the accession numbers mn -mn . the study was carried out in compliance with the national animal welfare regulations. diagnostic veterinary procedures are not within the context of relevant eu legislation for animal experimentation (directive / /ec) and may be performed in order to diagnose animal diseases and improve animal welfare. samples were collected by registered veterinarians and caused no suffering. samples were collected only after the owners' consent had been obtained. not applicable. the authors declare that they have no competing interests. veterinary research institute, hellenic agricultural organization demeter, , thermi, thessaloniki, greece. department of veterinary medicine and animal production, university of naples federico ii, naples, italy. bayer animal health gmbh, leverkusen, germany. received: february accepted: may key: cord- -pzzev hd authors: reisinger, emil c.; von possel, ronald; warnke, philipp; geerdes-fenge, hilte f.; hemmer, christoph j.; pfefferle, susanne; löbermann, micha; littmann, martina; emmerich, petra title: mütter-screening in einem covid- -niedrig-pandemiegebiet: bestimmung sars-cov- -spezifischer antikörper bei rostocker müttern mittels elisa und immunfluoreszenz-bestätigungstest date: - - journal: dtsch med wochenschr doi: . /a- - sha: doc_id: cord_uid: pzzev hd background in children, the infection with sars-cov- , the cause of covid- , tends to be clinically inapparent more often or less severe than in adults. the spread of this infection from children poses a danger to vulnerable adults. therefore, child care institutions and schools currently are widely closed. methods since the status of infection tends to be congruent in mothers and their children, we tested mothers of children between and years in the city of rostock (state of mecklenburg-westpomerania, northeast of germany), for the presence of rna of sars-cov- in throat swabs, and of antibodies against sars-cov- in serum. results in none of the mothers tested, rna of this virus was detected in the throat swab. in the elisa test, igg antibodies were positive in one serum sample, iga antibodies were positive in , and borderline in serum samples. all sera were negative in the indirect immunofluorescence test (iift) with fitc-labeled igg, iga, und igm antibodies. conclusion at the time of this study, neither sars-cov- rna, nor specific antibodies against sars-cov- were detectable in the mothers tested in rostock. tive in , and borderline in serum samples. all sera were negative in the indirect immunofluorescence test (iift) with fitc-labeled igg, iga, und igm antibodies. conclusion at the time of this study, neither sars-cov- rna, nor specific antibodies against sars-cov- were detectable in the mothers tested in rostock. in allen deutschen bundesländern wurden im märz maßnahmen (kontakt-und mobilitätseinschränkungen, schließung von kindertagesstätten und schulen etc.) ergriffen, um die ausbreitung von covid- zu verhindern. insbesondere zur infektion, zum klinischen verlauf und zur infektiosität bei kindern gibt es bislang wenig verlässliche daten, die zur entscheidung über die wiedereröffnung von kinderbetreuungseinrichtungen beitragen. kinder erkranken nach infektion mit sars-cov- meist weniger schwer als erwachsene, bei ihnen sind symptomlose oder symptomarme verläufe häufiger [ ] . daten zur prävalenz bei jüngeren kindern werden insbesondere benötigt, um aussagen zur gefahr der weitergabe der infektion an ältere und vulnerable personen, die im selben haushalt leben, zu ermöglichen. der infektionsstatus bzgl. sars-cov- ist bei kindern und deren müttern oft kongruent. die Übertragung der sars-cov- -infektion erfolgt hauptsächlich durch erwachsene, insbesondere durch haushaltskontakte [ ] . eine studie hat gezeigt, dass dem symptombeginn bei infizierten kindern eine nachgewiesene covid- -infektion bei erwachsenen familienmitgliedern vorausging [ ] . in einer weiteren studie fanden sich bei von infizierten kindern weitere erkrankte familienmitglieder [ ] . eine deutsche studie [ ] berichtet über einen deutlichen anstieg des infektionsrisikos für andere familienmitglieder, wenn ein infiziertes kind im haushalt lebt. somit kann die bestimmung der antikörper gegen sars-cov- bei müttern indirekt auskunft über den infektionsstatus bei deren kindern geben. in mecklenburg-vorpommern wurde zwischen dem . . und dem . . bei insgesamt personen sars-cov- detektiert. das entspricht einer inzidenz von erkrankungen pro einwohnern. diese relativ niedrige inzidenz kann u. a. erklärt werden durch den geringen internationalen reiseverkehr nach mecklenburg-vorpommern zu beginn der pandemie, die geringe zahl an großveranstaltungen, den frühen aufbau von abstrichzentren zur pcr-diagnostik, die rasche aufklärung von infektionsketten durch die gesundheitsämter und das distanziertere sozialverhalten der bevölkerung z. b. bei begrüßungen. da die zahl der neuinfektionen inzwischen stark abgenommen hat (▶ tab. ), stellt sich die frage, wie viele menschen die infektion bereits überstanden haben und durch antikörper geschützt sind. für den in dieser studie verwendeten elisa-test zum nachweis von sars-cov- -antikörpern (euroimmun) wird in der literatur eine hohe sensitivität und spezifität angegeben [ ] . in unserer studie wurden die positiven und grenzwertigen antikörperbefunde des elisa-tests mit einem indirekten immunfluoreszenztest (iift) als bestätigungstest überprüft. diese antikörpertests zielen nicht darauf ab, virusneutralisierende antikörper nachzuweisen. bei positivem antikörpernachweis kann jedoch von einem kontakt mit sars-cov- und einem gewissen immunschutz ausgegangen werden, zumal mehrere studien eine günstige wirkung von antikörperhaltigem rekonvaleszenten-plasma auf den verlauf von covid- nahelegen [ ] . in dieser pilotstudie wurden mütter von kindern im alter von - jahren als sentinel untersucht, um die prävalenz der infektion mit sars-cov- auch bei kindern abzuschätzen. alle mütter waren in der pcr zum nachweis von sars-cov- -rna aus dem rachenabstrich negativ. im igg-elisa-test auf das vorhandensein von sars-cov- -spezifischen antikörpern war ein serum positiv. im iga-elisa waren seren positiv und seren grenzwertig. das serum, das im igg-elisa positiv war, und die seren, die im iga-elisa positiv oder grenzwertig waren, wurden in der iift bzgl. igg-, igm-und iga-antikörper negativ getestet. somit konnten in keinem der seren spezifische antikörper gegen sars-cov- bestätigt werden. in mecklenburg-vorpommern wurden zwischen der . und . kalenderwoche für die schließung bzw. wiedereröffnung von kindergärten und schulen während bzw. nach der covid- -pandemie gibt es nur unzureichende daten in der medizinischen fachliteratur [ ] . wir haben in einer pilotstudie bei müttern nach virus-rna im rachenabstrich und nach spezifischen antikörpern im serum gesucht, um die verbreitung des virus bei müttern als sentinel für die familien zu evaluieren. gleichzeitig wurden die verwendeten antikörpertests auf ihre spezifität, d. h. den nachweis falsch positiver seren, bewertet. die sensitivität, d. h. den nachweis falsch negativer seren, konnten wir in dieser studie nicht bewerten. bei stationären covid- -patienten unserer klinik konnten wir im verlauf der erkrankung allerdings igg-und iga-antikörper im eli-sa-test nachweisen und mit dem iift für igg, iga und igm bestätigen. auch wenn die viruslast bei kindern ähnlich hoch ist wie bei erwachsenen [ ] , und bei symptomatischen kindern unter einem jahr sogar noch höher ist als bei älteren kindern, haben kleinkinder weniger schwere erkrankungen als ältere kinder [ ] . in mecklenburg-vorpommern wurden auch deutlich weniger infektionen bei kindern als bei erwachsenen gemeldet. diese niedrige infektionsrate bei kindern deckt sich mit den aussagen anderer studien [ , ] . die daten einer weiteren studie aus shanghai und wuhan legen nahe, dass die empfänglichkeit für die infektion mit sars-cov- bei kindern unter jahren nur etwa ein drittel so hoch ist wie bei -bis -jährigen, und dass die schließung von schulen und kindergärten trotzdem wahrscheinlich einen signifikanten beitrag zur verlangsamung der infektionsausbreitung geleistet hat [ ] . einige andere studien aus china treffen wiederum die aussage, dass kinder ähnlich empfänglich sind für die infektion mit sars-cov- wie erwachsene [ , ] . die infektiosität von mit sars-cov- infizierten kindern unterliegt möglicherweise starken interindividuellen schwankungen, wobei rna-viruslast nicht gleichbedeutend ist mit infektiösem virus. so hat ein mit sars-cov- infizierter -jähriger junge die infektion trotz zahlreicher sozialer kontakte nicht weitergegeben [ ] . unsere ergebnisse unterstützen die annahme, dass die virusund seroprävalenz gegen sars-cov- bei müttern in rostock sehr niedrig ist, und stehen mit der geringen zahl der an covid- erkrankten in der stadt rostock in einklang. die aussagekraft dieser studie zur prävalenz der infektion mit sars-cov- bei müttern ist durch die relativ geringe fallzahl ( % der mütter mit schulpflichtigen kindern in rostock) limitiert. weitere studien zur durchseuchung von müttern werden helfen, die prävalenz der infektion mit sars-cov- in familien genauer zu bestimmen. da in dieser studie durch die indirekte immunfluoreszenz weder das positive ergebnis des elisa für igg, noch die positiven und grenzwertigen ergebnisse des elisa für iga bestätigt wurden, empfehlen wir, positive sars-cov- -antikörperbefunde in den hier verwendeten elisa-tests mittels bestätigungstests (z. b. indirekte immunfluoreszenz) zu überprüfen. epidemiology of covid- among children in china epidemiology and clinical features of coronavirus disease in children a comparative-descriptive analysis of clinical characteristics in -coronavirus-infected children and adults clinical analysis of cases of novel coronavirus infection in children from six provinces (autonomous region) of northern china infection fatality rate of sars-cov- infection in a german community with a super-spreading event comparison of four new commercial serologic assays for determination of sars-cov- igg immunoglobulins or convalescent plasma to tackle covid- : buying time to save lives -current situation and perspectives virological assessment of hospitalized patients with covid- inactivation and safety testing of middle east respiratory syndrome coronavirus reverse elisa for igg and igm antibodies to detect lassa virus infections in africa lexikon der medizinischen laboratoriumsdiagnostik impact assessment of non-pharmaceutical interventions against coronavirus disease and influenza in hong kong: an observational study an analysis of sars-cov- viral load by patient age symptomatic infants have higher nasopharyngeal sars-cov- viral loads but less severe disease than older children coronavirus infections in children including covid- : an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children sars-cov- (covid- ): what do we know about children? a systematic review epidemiology and transmission of covid- in shenzhen china: analysis of cases and . of their close contacts changes in contact patterns shape the dynamics of the covid- outbreak in china cluster of coronavirus disease (covid- ) in the french alps key: cord- -x y xpsf authors: bachmann, p. a. title: rotavirusnachweis in faezes: erfahrungen mit dem enzyme linked immunosorbent assay (elisa) date: - - journal: j vet med b infect dis vet public health doi: . /j. - . .tb .x sha: doc_id: cord_uid: x y xpsf zusammenfassung: kälber‐, ferkel‐ und menschliche faezesproben von an durchfall erkrankten neugeborenen patienten wurden mit hilfe einer modifizierten immunelektronenmikroskopie‐technik (iem) und im enzyme‐linked immunosorbent assay (elisa) vergleichend untersucht. in kälber‐ ( %), ferkel‐ ( %) und ( %) menschlichen faezesproben konnte rotavirus oder ‐antigen nachgewiesen werden. die sicherheit der methodik wurde zusätzlich anhand der virusausscheidung von drei experimentell infizierten, kolostrumfrei aufgezogenen kälbern überprüft. der elisa, bei dem meerrettichperoxidase als enzym verwendet wurde, war spezifisch und erwies sich im vergleich zur iem als empfindlicher. bei von insgesamt positiven proben gelang der virusnachweis nur mit der iem, diese proben waren negativ im elisa. in diesen proben werden virus‐antikörperkomplexe vermutet. weitere untersuchungen sind notwendig, bevor der elisa zum rotavirusnachweis in der routinediagnostik empfohlen werden kann. summary: demonstration of rotavirus in faeces: experiences with the enzyme linked immunosorbent assay (elisa) calf, piglet and human faecal samples from newborn patients with diarrhea were investigated comparing a modified immune electron microscopy method and the enzyme linked immunosorbent assay (elisa). in ( %) calf, ( %) piglet and ( %) human samples rotavirus or its antigen could be demonstrated. the suitability of the method was also tested by studying virus shedding of three experimentally infected, colostrum‐deprived calves. the elisa, using horse‐radish peroxidase as enzyme and amino‐salicylic acid as indicator, was very specific and showed a higher sensitivity compared to iem. in out of the positive samples rotavirus could only be demonstrated by iem; these samples were negative in the elisa. it is assumed that these samples contain virus‐antibody complexes. further investigations are necessary before the elisa can be recommended as a routine diagnostic test. rÉsumÉ: mise en évidence de rotavirus dans des matières fécales: expériences avec elisa (enzyme linked immunosorbent assay) maitères fécales de veaux, de porcelets et échantillons humains de patients nouveaux‐nés atteints de diarrhée ont été examinés en comparaison avec une technique immunologique modifiée en microscopie électronique (iem) et avec elisa. rotavirus ou l'antigène ont pu être mis en évidence chez veaux ( %), porcelets ( %) et enfants la fiabilité de la méthode a été en plus testée sur la base de l'excrétion du virus chez trois veaux infectés expérimentalement et privés de colostrum. ( %). le test elisa, l'enzyme utilisée étant la peroxydase de meerrettich, fut spécifique et s'est révélé plus sensible que iem. la mise en évidence du virus a été possible dans cas sur positifs avec la méthode iem, ces échantillons s'étant révélés négatifs avec elisa. on a supposé l'existence de complexes virus‐anticorps dans ces échantillons. d'autres recherches sont nécessaires avant de pouvoir recommander le test elisa pour la mise en évidence de rotavirus dans le diagnostic de routine. resumen: identificación de virus rota en heces: experiencia con el enzyme linked immunosorbent assay (elisa) se examinaron comparativamente muestras de heces de terneros, de lechones y de niños, pacientes recién enfermos de diarrea, con ayuda de una técnica modificada de inmunomicroscopía de electrones (iem) y en el enzyme linked immunosorbent assay (elisa). en muestras de heces de terneros ( %), de lechones ( %) y de niños ( %) se pudo identificar virus o antígeno rota. la seguridad de la metodología se controló además con ayuda de la eliminación de virus por tres terneros infectados experimentalmente, criados sin calostro. el elisa, en el que se utilizó peroxidasa de rábano rusticano como enzima, era específico, resultando ser más sensible en comparación con la iem. solo en de un total de muestras positivas se logró la identificación de virus con la iem, siendo estas pruebas negativas en el elisa. se sospecha que en estas muestras había complejos de anticuerpos de virus. se precisa proseguir con las experiencias antes de poder recomendar el elisa para la identificación de virus rota en el diagnóstico de rutina. an dieser stelle möchte ich dr. d. ellens und dr. p. de leeuw, central diergeneeskundig instituut, leystad, holland, sehr herzlich für die Überlassung von referenzmaterial und die hilfe bei der entwicklung des elisa in unserem labor danken. der elisa wurde als ,,doppelte sandwich-technik" in anlehnung an die von ellens und de leeuw ( ) beschriebene methode in immunolon-flachbodenmikroplatten (flow laboratories, bonn) durchgefuhrt. die beschicbtung der platten erfolgte rnit pl der anti-rotavirus-globulinlosung, die zuvor : in , m karbonat-bikarbonatpuffer, p h , verdunnt worden war. nach einer inkubationszeit von stunden bei oc und stunden bei 'c wurden die mit folien verschlossenen platten ohne weitere behandlung bei - . ' c gelagert. aktivitatseinbuden waren wahrend einer lagerzeit von mindestens monaten nicht feststellbar. vor verwendung wurden die platten aufgetaut und dreimal mit aqua dest. mit , o/o tween grundlich gewaschen. anschlierend erfolgte die beschidtung der platten niit den aufbereiteten faezesproben in den verdunnungen : , : , : und : . bei jeder platte wurden je eine bekannte rotavirus-positive sowie -negative faezesprobe in log -verdiinnungen und eine pbs-kontrolle mitgefiihrt. nach inkubation wahrend stunden bei o c erfolgte dreimaliges waschen mit aqua dest / tween , bevor pl der konjugatgebrauchsverdiinnung in pbs mit "/o fks in jede vertiefung der mikroplatten pipettiert wurden. im anschlud an eine weitere inkubation von stunde bei 'c und dreimaligem waschen wurde das enzymsubstrat rnit dem indikator in die vertiefungen gegeben. hess, unveroff. ergebnisse, ) . bei diesen proben konnte es sich um ein gleichzeitiges vorkommen von viruspartikeln und antikorpern handeln, die als komplexe vorliegen. rotavirus-immunglobulin-g-komplexe in menschlichen stuhlproben sind bekannt ( ). derartige virus-antikorper-komplexe wurden im elisa, mit dem antigene eigenschaften festgestellt werden, nicht reagieren, mit hilfe der iem aber gut nachgewiesen werden konnen. auf einen solchen zusammenhang weisen watanabe und mitarbeiter ( ) hin. die ursachen der diskrepanz beim rotavirusnachweis, die hier zwischen der iem und dem elisa festgestellt wurde, mussen in weiteren untersuchungen abgeklart werden. unsere ergebnisse weisen jedoch darauf hin, dai beim nachweis von rotavirusantigen in faezes unter alleiniger verwendung des elisa, wie das in friiheren arbeiten empfohlen worden ist ( , ), damit gerechnet werden mui , dai eine reihe von rotavirus-enthaltenden proben derzeit nicht diagnostiziert werden konnen. eine verbesserung der unbefriedigenden resultate ware sicher moglich, wenn die proben fruhzeitig, d. h. sofort nach krankheitsbeginn entnommen wurden oder wenn von einem patienten mehrere an aufeinanderfolgenden tagen entnommene faezesproben untersucht werden. die sicherheit der methodik wurde zusatzlich anhand der virusausscheidung von drei experimentell infizierten, kolostrumfrei aufgezogenen kalbern uberpriift. der elisa, bei dem meerrettichperoxidase als enzym verwendet wurde, war spezifisch und erwies sich im vergleich zur iem als empfindlicher. bei von insgesamt positiven proben gelang der virusnachweis nur mit der iem, diese proben waren negativ im elisa. in diesen proben werden virus-antikorperkomplexe vermutet. weitere untersuchungen sind notwendig, bevor der elisa zum rotavirusnachweis in der routinediagnostik empfohlen werden kann. experiences with the enzyme linked immunosorbent assay (elisa) calf, piglet and human faecal samples from newborn patients with diarrhea were investigated comparing a modified immune electron microscopy method and the enzyme linked immunosorbent assay (elisa). in ( o/u) calf, ( o / o ) piglet and ( o / o ) human samples rotavirus or its antigen could be demonstrated. the suitability of the method was also tested 'by studying virus shedding of three experimentally infected, colostrum-deprived calves. the elisa, using horse-radish peroxidase as enzyme and amino-salicylic acid as indicator, was very specific and showed a higher sensitivity compared to iem. in out of the positive samples rotavirus could only be demonstrated by iem; these samples were negative in the elisa. it is assumed that these samples contain virus-antibody complexes. further investigations are necessary before the elisa can be recommended as a routine diagnostic test. identificacih de virus rota en heces: experiencia con el enzyme linked immunosorbent assay (elisa) se examinaron comparativamente muestras de heces de terneros, de lechones y de niiios, pacientes recikn enfermos de diarrea, con ayuda de una tkcnica modificada de inmunomicroscopia de electrones (iem) y en el enzyme linked immunosorbent assay (elisa). en muestras de heces de terneros ( o/o), de lechones ( o/o) y de nifios ( o/o) se pudo identificar virus o antigen rota. la seguridad de la metodologia se control ademas con ayuda de la eliminaci n de virus por tres terneros infectados experimentalmente, criados sin calostro. el elisa, en el que se utiliz peroxidasa de ribano rusticano como enzima, era especifico, resultando ser mis sensible en comparacidn con la iem. solo en de un total de muestras positivas se logr la identificaci n de virus con la iem, siendo estas pruebas negativas en el elisa. se sospecha que en estas muestras habia complejos de anticuerpos de virus. se precisa proseguir con las experiencias antes de poder recomendar el elisa para la identificacibn de virus rota en el diagn stico de rutina. enzyme-labelled immunosorbent assay techniques in foot-and mouth disease virus research peroxidase labelled antibody and fat, conjugates with enhanced intracellular penetration enzyme-linked immunosorbent assay for diagnosis of rotavirus infections in calves elisa for the detection of bovine coronavirus in faeces. abst. th intern. congress for virology corn arison of five diagnostic methods for the detection of rotavirus antigens in calf faeces enzyme-linked immunosorbent assay (elisa): quantitative assay of immunoglobulin g. immunochemistr rfber den einsatz der airfuge in der elektronenmikroskopischen virusdiagnostik comparison of an enzymelinked immunosorbent assay for quantitation of rotavirus antibodies with complement fixation in an epidemiological survey detection of antibodies specific for herpes simplex virus in human sera by the enzyme-linked immunosorbent assay detection of antibody to rubella virus by enzyme-linked immunosorbent assay detection of hepatitis b surface antigen (hbsag) with use of alkaline phosphatase labelled antibody to hbsag detection of adenovirus by enzymelinked immunosorbent assay solid phase enzyme-linked immunosorbent assay for detection of hepatitis a virus enzyme-linked immunosorbent assay (elisa) for detection of hepatitis a antigen in sera: comparison with solid-phase radioimmunoassay, immune electron microscopy, and immune adherence hemagglutination assay solid-phase enzyme immunoassay for herpes simplex virus sensitivity of enzymelinked immunosorbent assay, complement fixation, and hemagglutination inhibition serological tests for the detection of sendai virus antibody in laboratory mice application of enzyme-linked immunosorbeiit assay (elisa) to the detection of calf rotavirus and rotavirus antibodies immunoassay using antigenenzyme conjugates enzyme-linked immunoassays for antibodies in measles, cytomegalovirus infections and after rubella vaccination detection of soluble epstein-barr virus by enzyme-linked immunosorbent assay filter paper solid-phase radioimmunoassay for human rotavirus surface immunoglobulins human rotavirus and its antibody: their coexistence in feces of infants enzyme-linked immunosorbent assay (elisa) for detection of human reovirus-like agent of infantile gastroenteritis adresse des autors: institut fur medizinische mikrobiologie, infektions-und seuchenmedizin key: cord- -mbrw og authors: flego, michela; frau, aldo; accardi, luisa; mallano, alessandra; ascione, alessandro; gellini, mara; fanunza, elisa; vella, stefano; di bonito, paola; tramontano, enzo title: intracellular human antibody fragments recognizing the vp protein of zaire ebola filovirus inhibit the protein activity date: - - journal: bmc biotechnol doi: . /s - - - sha: doc_id: cord_uid: mbrw og background: ebola hemorrhagic fever is caused by the ebola filovirus (ebov), which is one of the most aggressive infectious agents known worldwide. the ebov pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. the multifunctional viral vp protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (ifn-α/β) response, and represents a suitable target for the development of strategies to control ebov infection. phage display technology permits to select antibodies as single chain fragment variable (scfv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. scfv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scfv is expressed as intracellular antibody (intrabody) or delivered into the cells. results: monoclonal antibodies (mab) in scfv format specific for the ebov vp were isolated from the eth- library of human recombinant antibodies by phage display technology. five different clones were identified by sequencing, produced in e.coli and expressed in cho mammalian cells to be characterized in vitro. all the selected scfvs were able to react with recombinant vp protein in elisa, one of the scfvs being also able to react in western blot assay (wb). in addition, all scfvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in a cells showed that two of the scfvs can significantly hamper the inhibition of the ifn-β-induced rig-i signaling cascade mediated by ebov vp . conclusion: five antibodies in scfv format recognize an active form of ebov vp in elisa, while one antibody also recognizes vp in wb. two of these scfvs were also able to interfere with the intracellular activity of vp in a cell system in vitro. these findings suggest that such antibodies in scfv format might be employed to develop therapeutic molecules able to hamper ebov infections. ebola hemorrhagic fever caused by the ebov is one of the most aggressive zoonoses affecting humans, leading to death within a few days of the exposure [ ] . six species of ebov are known to date, named after the geographical region in which they were first isolated: bundibugyo, reston, sudan, taï forest (formerly côte d'ivoire ebov), zaire and bombali ebov. sudan, taï forest, and zaire ebov are responsible for outbreaks in humans, whereas reston ebov infects non-human primates, and bombali virus was recently discovered in bats [ , ] . fatal ebov infections are characterized by rapid viral replication combined with an inadequate antiviral response. hallmarks of fatal cases are immune suppression with t cells levels below the normal level, no cd t cell activation, delay of antibody response in the blood, and high viremia ( genome copies/ml serum). the ebov pathogenesis starts with the subversion of both innate and adaptive immune response and the consequent induction of harmful inflammatory responses and tissue necrosis due to disseminated infections [ , ] . ebov has a linear, single-stranded, negative rna genome about , nucleotides in length. it is composed of seven genes coding for eight proteins in this order: np (encoding the nucleoprotein), vp , vp , gp (encoding the glycoproteins), vp , vp , l (encoding the polymerase). the gp gene codes for the two molecular forms gp and gp , generated by rna editing [ ] . vp is a conserved multifunctional protein which is a cofactor of the viral rna polymerase complex along with the np, vp , and l protein. its activity starts at an early stage of the ebov infection; it is also a doublestranded rna-binding protein shown to be implicated in hampering the innate immune response [ ] by blocking the ifn-mediated antiviral activity through multiple inhibitory effects which include disruption of the rig- pathway by preventing irf- phosphorylation [ , ] , and inhibition of activation of the ifn-inducible dsrna and dicer-dependent protein kinase r [ ] . antibody phage display technology makes it possible to select from an artificial immune system human antibodies in scfv format capable of specifically recognizing an antigen of interest. scfvs, consisting of the vh and vl chain regions of a whole immunoglobulin (ig), are the smaller portion still retaining the binding properties of the parental ig. this format is ideal for genetic manipulation in order to obtain antibody constructs potentially useful for diagnostic and therapeutic applications [ ] . furthermore, scfv antibodies can be expressed inside the cell as intrabodies so as to bind to their intracellular target antigen [ ] . the two main mechanisms underlying the efficacy of intrabodies are: ) knockdown of the activity of cytosolic antigens through cytosolic intrabodies [ ] [ ] [ ] ; ) diverting of antigens from their natural intracellular compartment by scfv binding due to an extra-signal for intracellular localization [ ] [ ] [ ] . intrabodies can be used to reveal the function of proteins by interfering with their function, although the possibility of targeting intracellular antigens gives them a therapeutic potential for a number of viral infections [ , ] , neurological diseases [ , ] and cancers [ , , , ] . this study reports the selection by phage display and characterization of different human scfv antibodies binding to an active form of the zaire ebov vp [ , ] . the ability of these scfvs, expressed as cytosolic intrabodies, to reverse the inhibition of type i ifn induction by intracellular expression of vp was tested by a luciferase reporter gene inhibition assay in a cells treated with dsrnas [ ] . in order to isolate antibodies specific for the recombinant vp expressed in e.coli and purified in an active form [ , ] , an approach based on the phage display technology was used. in the eth- library which we used, the diversity (about clones) has been introduced in the complementary-determining region (cdr ) of both the variable heavy chain (vh) and variable light chain (vl) domains [ ] . to recover antigen-specific antibody phages, an aliquot of the eth- antibody library containing cfu phage was used for the panning procedure as described elsewhere [ , ] . in fig. soluble scfvs derived from iptg-induced colonies were screened by elisa to find those specific for the vp protein. all the e. coli colonies corresponding to the clones exhibiting an od λ value in elisa higher than . , were grown and subjected to dna extraction and sequence analysis. several clones had identical nucleotide sequences and five different clones, namely b , a , e , f and h , were identified; the amino acid composition of the complete sequences of the vh and vl domains is shown in fig. along with the schematic representation of a scfv gene in the phagemid cassette. the scfv reactivity towards vp was further characterized by elisa (fig. , panel a) and wb (fig. , panel b) using vp recombinant antigen. the protein glucose oxidase (go) and an anti-go scfv for detection were used as negative controls. the anti-vp reactivity in elisa was confirmed for all b , a , e , f and h scfvs, while in wb the positivity was only observed for scfv a , which reacted with a kda protein identified as the recombinant his-tagged vp protein also detected by the anti-his mab. the scfvs b , e , f , h recognized their antigen in elisa but showed no reactivity in denaturing conditions of wb, suggesting that they probably recognize conformational epitopes. for its part, a is still reactive in wb and probably recognizes a linear epitope. the specificity of the anti-vp reactivity of b , a , e , f and h scfvs is confirmed by the observation that they did not react with the irrelevant go antigen either in elisa or in wb (fig. ) . in order to use the scfvs in the ebov vp luciferase reporter gene inhibition assay, the scfv genes selected were pcr amplified with opportune oligonucleotides and cloned into the ptarget vector for expression in the eukaryotic system. in view of the cytoplasmic vp localization, it was not necessary to provide the scfvs with signal sequences for expression in specific cell compartments. transfection experiments were performed in the cho cells to evaluate the functionality of the scfv ptarget constructs as described in methods. the a , h , b , f and e constructs were all able to express scfvs of the expected molecular mass in eukaryotic cells (fig. , panel a) . evaluation of the anti-ebov vp scfvs ability to restore the ifn-β activity by a luciferase reporter gene inhibition assay to evaluate the capability of the scfvs b , a , e , f and h to block the vp activity, the cell-based miniaturized luciferase reporter gene inhibition assay, previously described [ ] , was used. the assay measures the capacity of the vp expression to inhibit the ifn-β induced by dsrna treatment, in a cells. before performing the ebov vp luciferase reporter gene inhibition assay using the scfvs, it was crucial to exclude that the expression of an irrelevant scfv could influence the ifn-β induction, either in the absence or in the presence of vp expression. to this end, a cells, transfected with the pgl ifn-β luc and the pcdna -ebov-vp expression plasmid, were co-transfected with the irrelevant anti-go scfv expression plasmid. further, to exclude any non-specific effect due to the transfection procedure, the cells were co-transfected in parallel with the pgl ifn-β luc expression vector and an empty pcdna vector. the luciferase signal emitted in the case of pgl ifn-β luc and anti-go scfv concurrent expression, was comparable to that obtained with the ifn-β positive controls. also, in the case of ebov vp and anti-go scfv simultaneous expression, the measured ifn-β signal was comparable to that obtained with the ebov vp expressed alone (fig. , panel b) . the results confirmed that ifn-β induction was affected by neither the transfection procedure nor the irrelevant scfv either in the presence or in the absence of vp expression. next, we tested all the scfv ptarget constructs in the dsrna rig-i-mediated luciferase reporter gene inhibition assay. two of the five scfvs tested, f and e , showed a significant ability (p = . and p = . , respectively) to subvert the inhibition of the ifn-β production generated by dsrna, after the vp expression. by contrast, scfv h , a and b showed no ability to counteract the inhibition of ifn-β induction mediated by ebov vp in the cellular assay (fig. , panel c). competitive elisa using the scfv-expressing phage to verify whether the two different scfvs e and f , able to subvert the inhibition of the ifn-β production, targeted different epitopes, we performed a competitive elisa (fig. ). this relies on detection of the scfv-expressing phage particles that compete with soluble non-phage-fused scfvs for binding to the antigen immobilized on elisa plate. it was not possible to detect the soluble non-phage-fused scfvs using the anti-tag flag ab because the tag is also present on the phage. to determine the best scfv expressing-phage concentration for competition tests, identified as × tu/ml, we first performed a phage elisa experiment in which different scfv-expressing phage concentrations were tested on vp coated plates (data not shown). in a competitive assay, elisa plates coated with vp or with the control antigen go were blocked; they were then incubated with purified scfv-expressing phage both in the absence and in the presence of the competitor soluble non-phage-fused scfv at the maximum concentration of μg/ml. the binding of the scfv-expressing phages was measured. the detection step was via the phage coat protein using an anti-m mab conjugated to hrp. the anti-go scfv-expressing phage clone was assayed against the anti-go soluble nonphage-fused scfv as a positive control for competitive measurement (fig. , panel a) . it was found that μg/ml of soluble non-phage-fused scfv could inhibit the binding of the scfv-expressing phage in a dose-dependent manner. binding in the presence of specific anti-go soluble non-phage-fused scfv was compared: to binding in the presence of e anti-vp soluble non-phage-fused scfvs (p value = . ); to binding in the presence of f anti-vp soluble non-phage-fused scfvs (p value = . ); to binding in the absence of soluble non-phage-fused scfvs (p value = . ) using student's t-test. next, we assayed the binding of e scfv-expressing phages both in the absence and in the presence of the competitor soluble non-phage-fused scfv against itself as an intrinsic positive control, against anti-go soluble non-phage-fused scfv as a negative control and against f soluble non-phage-fused scfv for competitive measurement. the inhibiting activity was determined at a single fixed concentration of μg/ml. f soluble nonphage-fused scfv shows a clear competitive effect at the concentration used (fig. , panel b) . binding in the presence of f soluble non-phage-fused scfv was compared to the binding in the presence of anti-go soluble non-phage-fused scfv (p value = . ) and in the absence of soluble non-phage-fused scfvs (p value = . ) using student's t-test. as a further confirmation, we performed a one-shot experiment by detecting the competition suffered by the f scfv-expressing phages when co-incubated with e soluble non-phage-fused scfvs and with the control anti-go soluble non-phage-fused scfv at the concentration of μg/ml. also in this case we observed that the control anti-go soluble non-phage-fused scfv has no competitive binding effect. the e soluble non-phage-fused scfv competes with the binding of f scfv expressing-phages. binding in the presence of e soluble non-phage-fused scfvs was compared to the binding in the presence of anti-go soluble non-phage-fused scfv (p value = . ) and to binding in the absence of soluble non-phagefused scfvs (p value = . ) using student's t-test (data not shown). the urgency to find effective counter measures for the ebov disease (evd) was strengthened by the west africa outbreaks occurring in - , which resulted in , cases of ebola with , deaths [ ] , pushing the international scientific community to investigate the widest possible range of defense strategies to counteract the virus. however, the current ebola outbreak, which started in may in democratic republic of the congo (drc) and has caused cases and deaths to date [ ] , received only minor benefits from the use of new diagnostic assays, vaccines and drugs. the reason is that to control ebola outbreaks, transversal coordination between health facilities and communities is essential to achieve rapid isolation of the cases of the disease and to stop the transmission chain. detection of new cases at an early stage by rapid diagnostic tests and ring vaccination strategy with experimental vaccines on volunteers are therefore crucial for controlling ebov in the current drc outbreak [ ] . due to the variable onset of antibody response in the ebola-infected subjects, serology is not used in the acute evd diagnosis. conversely, virus and viral proteins accumulate in blood to detectable levels within a few days from disease onset. molecular tests based on the detection of viral proteins were developed and proved to be effective for diagnosis in acute infection [ ] . currently, in the case of suspected ebov infection, the world health organization (who) recommends a list of validated tests for detection of either viral rna by rt pcr or viral antigens by immunological tests [http://www.who. int/medicines/ebola-treatment/emp_ebola_diagnostics/en/]. most of the antigen-capture tests used in national reference laboratories [ ] utilize mabs generated in mice immunized with the recombinant np [ ] , vp [ ] or gp [ ] ebov proteins. during the recent outbreak, lateral flow immunoassays (lfis) emerged as powerful tools for rapid antibody-mediated antigen-capture practicable at the point of care [ ] . this confirmed the advantages of tests based on antigen-antibody reaction over rt-pcr methodology, which requires significant laboratory infrastructures often lacking in low-income countries. furthermore, in order to control the transmission-chain of the infection and limit virus spread, it is important to have tests that can be easily automated; as such, antigen capture tests meet this requirement. ebov vp is a validated drug target for which only a few small molecules have been reported to be active [ , , ] , albeit no drug has yet been approved. here, we present mabs in scfv format, which are able to react with the zaire ebov vp protein. this is a key viral protein whose action starts at an early stage of the infection and is based on interference with the host immune response by blocking the ifn-mediated antiviral activity. the antibodies presented here enrich the list of available anti-vp antibodies. they could be used individually or in combination to develop novel reagents for ebov vp detection and therapeutics. regarding therapy, pools of neutralizing antibodies have been used in passive immunization of individuals with acute infection [ ] ; nevertheless, antibodies specific for the vp would act with a different mechanism with respect to neutralizing antibodies targeting surface glycoproteins. recent studies showed that targeting vp by either nucleic acid mimics or sirnas, provides protection against the ebov infection in murine [ ] as well as in non-human primate models [ ] . however, the effectiveness of antibodies targeting the vp has not yet been demonstrated in vivo. here, we show that two out of five scfv antibodies selected against the vp significantly hindered the inhibition of the rig-i signaling cascade mediated by vp , in a cellular system. we characterized the binding of these two scfv clones to the recombinant vp antigen by competitive elisa, and found that their epitope is at least partly shared. a more detailed analysis of the binding epitopes could further define whether it is a total or partial sharing and which antigenic region of ebov vp is involved in the inhibition of the interferon pathway. regarding the other anti-vp scfvs selected, we cannot exclude that the observed lack of functionality is due to incorrect scfv folding in the cytoplasmic environment. as antibodies are usually produced in an oxidizing biochemical environment with the help of er-based chaperones, only a fraction of naïve antibodies can be folded correctly in the reducing cytoplasmic environment which prevents the formation of disulfide bridges. however, many examples of successful intrabodymediated proteins knockdown in vitro, obtained using cytosolic intrabodies, have been reported in literature [ ] . scfvs selected in the extracellular environment were previously reported to work intracellularly [ , , ] depending on intrinsic biophysical characteristics such as stability, mainly ascribable to the scaffold. however, several methods have been developed to address the issue of cytosolic intrabodies functioning [ ] . interestingly, anti-vp scfvs able to interfere with vp activity, were recently isolated from a phage library different from the eth- and were linked to a cell-penetrating peptide for intracytoplasmic delivery [ ] . therefore, although we used a different delivery system, our data strengthen the idea that intracellular antibodies in scfv format can be used to counteract ebov vp activity. scfvs against different intracellular ebov targets could be used either to develop a well-defined cocktail of antibodies with different specificities or also in combination with other drug molecules for therapeutic purposes, provided that an appropriate delivery system is developed. furthermore, the vp amino acid sequence is highly conserved among the zaire ebov isolated in several outbreaks (> . % amino acids identity). therefore, it can be hypothesized that the scfvs selected retain a broad-spectrum activity. nevertheless, the efficacy of these new antibodies should be further evaluated either in ebolavirus infected cells or in animal models of ebolavirus infection. five scfv antibodies against an active form of the zaire ebov vp were isolated and characterized. their specific reactivity in elisa and wb suggests the possibility of developing novel reagents for ebov vp detection during the virus life cycle. the two anti-vp scfvs f and e proved to be able to interfere with the vp -depending inhibition of ifn activity in a cell system, so suggesting that such antibodies also represent potential therapeutic agents. further investigations into an ebov infection system in vitro and in animal models are necessary to validate these reagents. the synthetic library of recombinant human antibodies (eth- ) consists of about scfv polypeptides displayed on the surface of the m phage. the library was built by random mutagenesis of the complementarity-determining region (cdr ) of the variable domains of both the heavy (vh) and light (vl) chain of immunoglobulins, using only three antibody germline gene segments (dp for the vh, dpk , and dpl for the vl). in the vh, diversity was created by randomizing four to six positions replacing the pre-existing positions to of the cdr ; in the vl, diversity was obtained by randomizing six positions ( to ) of the cdr [ ] . an aliquot of the eth- library, containing cfu phage, was used to isolate specific human antibodies in scfv format against the recombinant vp protein ( ) . immunotubes (nunc maxisorp; denmark) were coated overnight (on) at room temperature (rt) with purified recombinant vp protein ( μg/ml in pbs). after panning, phages were eluted with ml of mm triethylamine and the solution was immediately neutralized by adding . ml of m tris-hcl ph . . the eluted phages were used to infect an e. coli tg strain in a log phase, and amplified for the next round of selection, as described in flego et al. [ ] . three rounds of panning were performed to recover vp -specific antibody phages from the eth- library. for the preparation of soluble anti-vp scfvs, individual colonies were grown in flat bottomed wells (nunc) for h at °c in μl of . % glucose xyta medium and induced with μl of mm iptg/ xyta medium. the following day, the plates were spun down at g for min, and the supernatants containing soluble scfvs were recovered and tested for specificity with purified vp in elisa and wb, as described in flego et al. [ ] . plasmid dna from individual bacterial colonies clones was extracted using the quiaprep spin miniprep kit and subjected to enzymatic restriction; sequence analysis of the cdr regions was then performed with an automated dna sequencer (biofab, roma, italy) using the fdseq ( ′-gaa ttt tct gta tga gg- ′) and pelbback ( ′-agc cgc tgg att gtt att ac- ′) primers. the scfv gene clones reacting with the recombinant vp in elisa, were pcr amplified using the following primers: eth nco as a forward primer: ′ gcgc acc atg gcc gag gtg cag ctg ′. nhe i stop his as a reverse primer: ′ gcgc gct agc cta atg atg atg atg atg atg tgc ggc cgc gcc tag gac ′ containing the xhis tag sequence. for transient expression in eukaryotic cells, the amplimers were cloned in ptarget (promega) under the strong viral promoter (cytomegalovirus immediate-early enhancer). the clones obtained were sequenced to check for mutations possibly introduced by pcr, and used to transfect cho cells using jetpei dna transfection reagent, according to the manufacturer's instructions. transiently transfected cells were lysed after h with sds-loading buffer ( mm tris-hcl ph . , % sds, % -mercaptoethanol, % glycerol), loaded onto % sds-page, and then transferred to a nitrocellulose membrane using standard procedures. the membrane was blocked in % mpbs on at rt. blotted proteins were incubated for h in % mpbs with μg/ml of tet-ra·his antibody (qiagen), which was the only anti-his mab able to recognize the tag at the scfv cooh terminus, in our experimental conditions. after an additional incubation for h at rt in the presence of goat anti-mouse antibody hrp-conjugate ( μg/ml, dako), the reaction was developed and visualized with a chemiluminescence detection kit (pierce; il, usa). luciferase reporter gene assay ifn-β induction luciferase reporter gene assays a cells ( × per well) were transfected in -well plates with t-pro p-fect transfection reagent (t-pro biotechnology) with the construct pgl ifn-β luc, kindly provided by prof. stephan ludwig (institute of molecular virology, münster, germany). twenty-four hours after transfection, cells were additionally transfected using iav pr vrna and incubated for a further h at °c with % co . cells were harvested with lysis buffer ( mm na-mes ph . , mm tris-hcl ph . , mm dithiothreitol, . % triton x- ). the crude cell lysates were cleared by centrifugation and μl of cleared lysates were added to μl of luciferase assay buffer ( mm na-mes ph . , mm tris-hcl ph . , mm magnesium acetate, . mg/ ml atp) in a white -well plate. immediately after addition of μl of mm d-luciferin into each well, the luminescence was measured in victor luminometer (perkin elmer). the relative light units (rlu) were normalized as the fold activity of the unstimulated control. each assay was carried out in triplicate. the above described luciferase reporter gene assay was also performed for evaluating the ifn-β induction inhibition mediated by ebov vp . twenty-four hours after co-transfection with pgl ifn-β luc and pcdna or pcdna ebov wtvp expression vectors, cells were transfected with the ctrl anti-go scfv ptarget as an irrelevant scfv and with the different scfv p target vectors the next day, cells were additionally transfected with iav vrna. inhibition of luciferase expression was indicated either as the fold activity of the unstimulated control or as a percentage of the induced control. each assay was carried out in triplicate. competitive elisa using scfv-expressing phages for elisa competition assay, we used soluble nonphage-fused scfvs produced and purified as in gellini et al. [ ] . scfv-expressing phages were produced from a monoclonal bacterial culture grown to od = . - . and infected with m k helper phage in a ratio of around : phage/bacteria. one hundred ml of supernatant containing scfv-expressing phages were x concentrated by precipitation with peg and resuspended in pbs. phage titer was determined by plating of bacteria infected with phages at scalar dilutions. coating was performed with vp or go antigen as described in the elisa section. the following day, the plate was blocked with % mpbs for h at rt and washed with tpbs. twenty-five μl of soluble non-phage-fused scfvs at two-fold the desired final concentration were pre-incubated in % mpbs with the antigen for min prior to the addition of μl of the scfv-expressing phage mix at two times the desired final tu/ml in % mpbs, and incubated for h at rt. the plate was washed with tpbs and incubated with anti-m pviii coat protein mab conjugated to hrp (amhersham) diluted : , for h rt. a washing step was conducted followed by the addition of peroxidase substrate as described above. ebola haemorrhagic fever ebola virus disease the discovery of a new ebolavirus, bombali virus, adds further support for bats as hosts of ebolaviruses ebola virus: new insights into disease aetiopathology and possible therapeutic interventions how ebola and marburg viruses battle the immune system strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit the ebola virus vp protein functions as a type i ifn antagonist ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- ebola virus vp antagonizes pkr activity through its c-terminal interferon inhibitory domain production technologies for monoclonal antibodies and their fragments expression and targetingof intracellular antibodies in mammalian cells generation and functional characterization of intracellular antibodies interacting with the kinase domain of human egf receptor intracellular antibody capture technology: application to selection of intracellular antibodies recognising the bcr-abl oncogenic protein effects of intrabodies specific for rotavirus nsp during the virus replicative cycle intracellular anti-e human antibodies in single-chain format inhibit proliferation of hpv -positive cervical carcinoma cells in vivo antitumor effect of an intracellular single-chain antibody fragment against the e oncoprotein of human papillomavirus a novel intracellular antibody against the e oncoprotein impairs growth of human papillomavirus -positive tumor cells in mouse models characterization and binding of intracellular antibody fragments to the hepatitis c virus core protein generation and characterization of single-chain anti-body fragments specific against transmembrane envelope glycoprotein gp of maedivisna virus isolation of a human single chain antibody fragment against oligomeric α-synuclein that inhibits aggregation and prevents α-synuclein-induced toxicity direct in vivo intracellular selection of conformation-sensitive antibody domains targeting alzheimer amyloid-β oligomers human singledomain neutralizing intrabodies directed against etk kinase: a novel approach to impair cellular transformation a nanobody targeting the f-actin capping protein capg restrains breast cancer metastasis purification and functional characterization of the full length recombinant ebola virus vp protein expressed in e. coli dsrna binding characterization of full length recombinant wild type and mutants zaire ebolavirus vp a luciferase reporter gene assay to measure ebola virus viral protein -associated inhibition of double-stranded rna-stimulated, retinoic acid-inducible gene -mediated induction of interferon β identification of myricetin as an ebola virus vp −doublestranded rna interaction inhibitor through a novel fluorescence-based assay design and use of a phage display library. human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel design and use of phage display libraries for the selection of antibodies and enzymes generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (n) sars-cov protein using a phage display approach ebola: anatomy of an epidemic who regional office for africa. ebola virus diseases new ebola outbreak declared in democratic republic of the congo rapid diagnosis of ebola hemorrhagic fever by reverse transcription-pcr in an outbreak setting and assessment of patient viral load as a predictor of outcome identification of essential outstanding questions for an adequate european laboratory response to ebolavirus zairewest africa detection of ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein development, characterization and use of monoclonal vp -antibodies for the detection of ebola virus production of monoclonal antibodies and development of an antigen capture elisa directed against the envelope glycoprotein gp of ebola virus strategies in ebola virus disease (evd) diagnostics at the point of care antiviral agents against ebola virus infection: repositioning old drugs and finding novel small molecules insights into ebola virus vp and vp interferon inhibitory functions and their initial exploitation as drug targets, infectious disorders -drug targets a randomized, controlled trial of zmapp for ebola virus infection vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study specific in vivo knockdown of protein function by intrabodies human transbodies that interfere with the functions of ebola virus vp protein in genome replication and transcription and innate immune antagonism generation of human single-chain antibody to the cd cell surface determinant specifically recognizing ewing's sarcoma tumor cells publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank philochem for the generous permission to use of the eth- antibody phage display library. we thank martin bennett for his help in revising the manuscript for grammar and style. authors' contributions mf isolated and characterized the scfvs, participated in the design of the research and drafted the manuscript. aa and mg performed wb analyses and scfv production. am worked on the design and the genetic construction of the anti-vp scfvs in ptarget vector and their expression in cho cells. pdb and la expressed the anti-vp scfvs in the ptarget vector and critically reviewed the manuscript. af and ef produced the recombinant vp , carried out ebov vp luciferase reporter gene inhibition assay and analyzed the data. sv coordinated the study design on the basis of the scfv isolation. pdb and et conceived, coordinated and supervised the study. all authors have read and approved the manuscript. this work was supported by sardinia regional government grants lr / (crp- /f i ), and intramural fund of istituto superiore di sanità. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable the authors declare that they have no competing interests. key: cord- - d aojh authors: zou, hao; zarlenga, dante s.; sestak, karol; suo, siqingaowa; ren, xiaofeng title: transmissible gastroenteritis virus: identification of m protein-binding peptide ligands with antiviral and diagnostic potential date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: d aojh the membrane (m) protein is one of the major structural proteins of coronavirus particles. in this study, the m protein of transmissible gastroenteritis virus (tgev) was used to biopan a -mer phage display random peptide library. three phages expressing tgev-m-binding peptides were identified and characterized in more depth. a phage-based immunosorbent assay (phage-elisa) capable of differentiating tgev from other coronaviruses was developed using one phage, phtgev-m , as antigen. when the phage-elisa was compared to conventional antibody-based elisa for detecting infections, phage-elisa exhibited greater sensitivity. a chemically synthesized, tgev-m peptide (peptgev-m ; haltpikyippg) was evaluated for antiviral activity. plaque-reduction assays revealed that peptgev-m was able to prevent tgev infection in vitro (p < . ) following pretreatment of the virus with the peptide. indirect immunofluorescence and real-time rt-pcr confirmed the inhibitory effects of the peptide. these results indicate that peptgev-m might be utilized for virus-specific diagnostics and treatment. transmissible gastroenteritis (tge) is a highly contagious disease of swine characterized by up to % mortality in suckling piglets (laude et al., ) . the clinical symptoms include acute diarrhea, vomiting and dehydration. pigs of all ages and categories are susceptible. in seronegative herds, tge can cause devastating economic losses (saif and wesley, ; yin et al., ) . the virus responsible for tge (tgev) has a glycoprotein surface envelope and positive-sense rna genome of approximately . kb (ortego et al., ) . the virus consists of four structural proteins: spike (s), small membrane (sm or e), membrane (m), and nucleocapsid (n) proteins (spaan et al., ; saif and wesley, ; penzes et al., ) . the s protein is a large transmembrane surface glycoprotein that induces virus-neutralizing (vn) antibodies (jiménez et al., ; laude et al., ; suñé et al., ) ; the n protein, together with genomic rna, forms the viral nucleocapsid cavanagh, ) ; and the sm protein regulates virion assembly and release (laude et al., ) . the m protein is the most abundant component of the coronavirus particle (rottier, ) . roughly one-third of the m protein assumes a topology where part of the endo domain constitutes the fourth transmembrane segment, thereby positioning the carboxy terminus on the exterior portion of the virion (risco et al., ; masters, ) . it has been suggested that the m protein induces innate immunity including interferon production (charley and laude, ; laude et al., ) . phage display is a powerful technology that has been applied to antibody engineering (hayden et al., ; cyranka-czaja and otlewski, ) , drug discovery and manufacturing (kay et al., ; harper et al., ) , ligand identification (ehrlich and bailon, ; ladner and ley, ; yi et al., ; beer and liu, ) , and development of new diagnostics gazarian et al., ) and vaccines (lesinski and westerink, ; samoylova et al., ) . phage display yields billions of heterologous fusion peptides that are expressed on the surfaces of filamentous bacteriophage (scott and smith, ) . inasmuch as each phage expresses only one fusion peptide, the technology permits high throughput panning of a phage library with the intent of identifying single phage particles with the capacity to bind a target protein. this permits epitope mapping and easy purification at marginal costs. in the current study, three tgev m-binding phage and the concomitant peptides were identified from a phage display library. results indicate the ability of these peptides to function as tgev-specific diagnostic reagents. one peptide was further studied for its potential as an inhibitor of tgev infectivity. swine testis (st) cells were grown in dulbecco's mem with % fetal bovine serum at °c, % co . tgev (pur -mad strain) (sánchez et al., ) was propagated in st cells in the absence of serum, followed by gradient ultracentrifugation purification (krempl and herrler, ) . cytopathic effect (cpe) assays were performed as described (ren et al., b) . briefly, st cells seeded onto -well tissue culture plates (costar, usa) were inoculated with -fold serially-diluted tgev. after incubation at °c for h, cpe was recorded at  magnification using the bds microscope (optec, china). total rna was isolated from tgev-infected st cells using a commercial protocol ( , fastgene, china). sense (p : -ggggggatccatgcgctattgtgctatg) and antisense (p : -ccccgaattcttataccatatgtaataa) primers were used in rt-pcr. the amplified m gene was inserted into the bamhi-ecori site of a prokaryotic expression vector, pgex (novagen, germany), resulting in the recombinant plasmid, pgex-tgev-m. after pgex-tgev-m was transformed into rosette escherichia coli bacteria by heat shock (bowyer, ) , expression of the glutathione s-transferase (gst) tagged-tgev-m fusion protein was induced with isopropyl-b-d-thiogalactoside (iptg) and visualized by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). the recombinant protein of interest (rtgev-m) was gel purified (ren et al., a) . in parallel, rgst from pgex, was expressed and purified as well. the concentration of rtgev-m was measured spectrophotometrically using a nanodrop spectrophotometer according to manufacturer's instructions. biopanning of the phage was performed using the ph.d™- phage display peptide library kit according to manufacturer's instructions (e sc, new england biolabs, usa) with minor modifications ren et al., a) . during the first round of binding the phage library to the rtgev-m, lg/well of rtgev-m was used. in the second round, rtgev-m was replaced by rgst as a negative control to remove non-specific binding to gst. the rd- th rounds were performed under the similar conditions except for gradually decreasing the concentration of rtgev-m ( , . , , . lg/well) and increasing the concentration of tween from . % to . % to increase the stringency of binding. the phage remaining after the last round of biopanning were titered and were selected for phage amplification and further study. phages interacting positively with rtgev-m were identified by indirect elisa. the -well plates (fep , jet bio, china) were coated either with rtgev-m in . m nahco (ph . ) or with purified tgev in dmem at lg/well at °c. plates were blocked with % bsa in tbs buffer ( mm tris-hcl, ph . , mm nacl) for h at °c then washed x with tbs-tween. the selected phages derived from the last round of biopanning were added separately to the wells at concentrations of .  plaque forming units (pfu)/ml in . m nahco (ph . ) and incubated at °c for h. the remaining steps were performed as described (ren et al., a) . plates were read at od with an elx (bio-tek, usa) elisa reader. . . virus detection using elisa ten tgev-positive phage clones were amplified using an e. coli expression system ( ). the dnas of interest were extracted, purified ( , qiagen, germany), pcr amplified and sequenced to corroborate the presence of tgev-m sequences ( ). deduced amino acid sequences were determined (wu et al., ) . phage elisa and antibody-elisa were compared for their abilities to detect the presence of the virus as described (wu et al., ) . for phage-eli-sa, tgev serially-diluted in dmem was coated onto elisa plates overnight at °c. then either the specific phage clone or a nonspecific phage complex (negative control) from the phage display library was diluted in pbs ( .  pfu/ ll) and added to the wells. next, the wells were incubated with commercially-available rabbit anti-m antibody ( : in pbs) followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (garp) ( : in pbs). for antibody-mediated elisa, rabbits were immunized once with mg rtgev-m in freund's complete adjuvant followed by three additional immunizations at week intervals with the same amount of antigen in freund's incomplete adjuvant. animals were bled days after the final boost. tgev was coated onto elisa plates to which was added rabbit anti-tgev-m followed by garp secondary antibody ( : ). in all experiments, the concentrations of tgev were determined experimentally such that elisa values could be measured and where [od phage (p)/od negative control (n)]> was considered positive. the specificity of tgev-reactive phages for tgev infection was evaluated by phage-elisa. a panel of selected viruses prepared in our laboratory consisting of tgev (ren et al., ) , porcine epidemic diarrhea virus (pedv) strain hljby , porcine reproductive and respiratory syndrome virus (prrsv) strain jilintn (gao et al., ) , porcine rotavirus (prv) (ren et al., c) , porcine pseudorabies virus (prv) strain kaplan (sui et al., ) , porcine parvovirus (ppv) isolate ppv , and porcine circovirus type ii (pcv ) strain pcv -ljr (zhu and ren, ) were assembled for comparative studies. these viruses were diluted in dmem to final concentrations of lg/well and coated onto elisa plates at °c for h. elisa was performed according to standard protocols (wu et al., ) . the unpanned ph.d™- phage display peptide library (phage l) was used as a negative control. statistical significance of the od values among all viral antigens was evaluated where [od virus /od phage l]> was judged as positive and subsequently analyzed using the t-test. the amino acid sequence corresponding to the phage with the highest binding activity to tgev (peptgev-m ) was commercially synthesized (boshi, china). the peptide was diluted in sterile water to a final concentration of mg/ml. purified rtgev-m or control protein rtgev s-ad (meng et al., ) was coated onto elisa plates ( lg/well) at °c. the ll of serially-diluted peptgev-m ( , , or lg/ml) were mixed with ll of phtgev-m or ll of phtgev-s-ad ( .  pfu/ml) and incubated at °c for h. after washing, the wells were incubated with rabbit anti-m antibody ( : dilution; abcam, china) for h followed by a : dilution of garp for min; the od values were recorded. three experimental approaches were used to evaluate the antiviral activity of peptgev-m (miyazaki et al., ; ren et al., b) . first, to assess the ability of the peptide to bind tgev in vitro, tcid of tgev was pre-incubated with peptgev-m at , , , . or . lg/ml before infecting st cells. second, to determine any impact of peptgev-m on st cells, cells were treated with serially-diluted peptide at °c for h prior to tgev infection. finally, to determine the effect of peptgev-m on st cells after tgev infection, cells were first infected with tgev ( tcid ) at °c for h then washed and incubated with serially-diluted peptgev-m at °c for h. all the cells were grown to % confluency in -well plates overlaid with % methylcellulose then cultured for - h to maximize plaque development. to evaluate the effects of peptgev-m on tgev replication in vitro, indirect immunofluorescence assay (ifa) was performed. briefly, confluent st cells were incubated with peptide-treated tgev [ ll of serially diluted ( À - À ) peptide was incubated with ll of tgev ( tcid )] at °c for h then processed for ifa as previously described (meng et al., ) ; an unrelated peptide that binds the pedv-s protein, peppedv-s (diluted À ) was used as a negative control. the impact of peptgev-m on virus replication was evaluated by real time rt-pcr targeting a portion of the tgev-s gene. the tgev was treated with serially-diluted peptide ranging from to . lg/ml at °c for h; lg/ml of peppedv-s was used as negative control. after washing the st cells with pbs, confluent monolayers were infected with peptide treated tgev ( tcid ) at °c for h. then the virus-containing culture was frozen and thawed three times followed by the addition of an equal volume of % pge- at room temperature for min. the samples were centrifuged at , rpm for min and the pellets were suspended in rnase-free water. total rna was extracted according to the manufacturer's instructions (fastgene, china) and real-time rt-pcr was performed on cdna as described (ren et al., b) . it was anticipated that a detrimental effect of peptgev-m on viral infectivity would be reflected in significantly reduced numbers of tgev s gene copies. using the previously described techniques ren et al., a) , the rtgev-m protein was expressed in e. coli (fig. ) for use in biopanning the phage display library to identify peptides that bind the m protein of tgev. during the isolation process it was determined that rtgev-m was present within inclusion bodies. peak expression was observed at h post-iptg induction. the mass of the rtgev-m was $ kda (fig. ) . five rounds of biopanning were performed using the rtgev-m as the target and one additional round with gst as target. the amount of eluted phages increased between rounds and from .  to .  , respectively. ten different tgev-m protein-reactive phages were identified by elisa, when rtgev-m was used as the coating antigen (fig. a) . all ten phages exhibited better binding to rtgev-m than to other phage-expressed protein controls (p < . , fig. a ). phages m , m and m -m had higher binding affinity (p < . ) with tgev than with the other phages (fig. b) . among all ten reactive phages that bound rtgev-m with high affinity, phtgev-m exhibited the highest binding to tgev (p < . , fig. b ) followed by phtgev-m .and phtgev-m . rna samples isolated from tgev-m-reactive and control phages were amplified by rt-pcr. all samples yielded products approximately bp in length (data not shown). all deduced peptides ( aa in length) were unique (table ) . phages bearing the peptides ltfpvtttppav, mthnmhgpnsep and haltpikyippg were selected for their high-affinity binding with rtgev-m (fig. a) and tgev (fig. b) . these three peptides were named peptgev-m , peptgev-m and peptgev-m , respectively. the binding efficiencies of the selected tgev-m-reactive phages were examined by phage-elisa. virus concentrations between . - lg/well all exceeded the signal limitations of the assay. as illustrated in fig. a , titratable virus concentrations fell in the range < . ug/well. the lowest concentrations of virus detectable with phtgev-m , phtgev-m and phtgev-m where p/n p were . , . , and . lg/well, respectively. for antibody-based elisa, the rabbit antibody titers against rtgev-m where p/n p were determined to be : .  . when assaying the antibody binding to tgev, at virus concentrations serially-diluted tgev in dmem was coated into elisa plates followed by incubation with serially-diluted rabbit against tgev serum and antibody binding using garp; normal rabbit serum was used as negative control. od ratios where p/n > were considered positive. the experiment and determination of od values were derived from three independent assays. the concentration of the virus is indicated on the x axis. antibody and virus dilutions are as indicated. > . lg/ml, p/n values began to exceed the limits of detection. virus ( . - . lg/well) were reproducibly detected (p/n p ) with antibody dilutions less than / (fig. b) . the binding specificities of phtgev-m , phtgev-m , phtgev-m for non-tge viruses were examined (fig. ) . in all cases p/ n > for tgev only. demonstrable binding was not observed with any other viruses tested. further, our results showed that pept-gev-m bound to rtgev-m and did not bind other proteins (fig. ) . increasing the concentration of peptgev-m effectively competed with binding of phtgev-m for rtgev-m as evidenced by decreasing od values; however, peptgev-m did not compete with phage binding when a non-specific protein, rtgev-s-ad was used as the coating antigen (fig. ) . to determine the impact of peptgev-m on tgev infection, three separate approaches were used: ( ) tgev was treated with peptide before inoculation of st cells; ( ) st cells were treated with peptide prior to tgev inoculation, and; ( ) st cells were infected with tgev before being treated with peptide. as expected, the results showed no impact of peptgev-m on st cells already infected with tgev or on st cells pretreated with peptgev-m prior to incubation with the virus (data not shown). however, when tgev was pre-incubated with peptgev-m , its infectivity decreased in a dose-dependent manner: at lg/ml of peptgev-m , the drop in infectivity was nearly % whereas at lg/ml a % drop in virus infectivity was observed (table ) suggesting peak inhibition was reached. these data indicate that peptgev-m is able to interfere with the ability of the virus to infect st cells. the antiviral effects of peptgev-m -treated virus were further investigated by ifa. results demonstrated a dosedependent reduction in the infection of st cells in the presence of peptgev-m whereas the unrelated peptide, peppedv-s , had no effect on virus uptake (fig. ) . real-time rt-pcr was used to confirm and quantify the tgev inhibitory effects of peptgev-m (fig. ) . results showed that the amount of viral rna was lower (p < . ) when tgev was pretreated with peptgev-m than in peptide-untreated controls. at the highest concentrations ( lg/ml), virus rna levels dropped . %; however, significant (p < . ) and reproducible reductions ( %) were observed when . ug/ml of peptgev-m were used. these data corroborated the ifa studies. despite the availability of commercial vaccines, tge still poses a regional threat to the swine industry due to the high morbidity and mortality that tgev causes in suckling piglets under weeks of age (miyazaki et al., ) . although passive, lactogenic immunity induced by virulent or attenuated tgev vaccines can provide effective protection, sufficient risk remains. albeit safer, inactivated phtgev-m to rtgev m protein was monitored using anti-m antibody and garp secondary antibody which target phtgev-m only. in parallel, rtgev s-ad was screened as a negative control. concentrations of peptgev-m are shown on the y axis. statistical significance is indicated by '' ⁄ '' (p < . ) or, '' # '' ( . < p < . ) relative to controls. all experiments were performed in triplicate. vaccines provide less protection. as such, accurate diagnostic tests are needed to better effect tge prevention and therapy. phage display and biopanning are powerful methods for identifying key ligands within large target antigens that may be involved in protection and/or diagnosis (wu et al., ) . this technology has been used for identifying receptor binding domains (rbds) in tgev that may interact with its papn cellular receptor (ren et al., a) . it has also been used for identifying immunogenic proteins in pig sera from convalescent animals with a history of salmonella typhimurium infection (meyer et al., ) , and for targeting tissues like bone-marrow dendritic cells and kidney, liver, lung, spleen and visceral adipose tissues (jung et al., ) . since the m protein is one of the three major tgev structural proteins, it was used as a target in biopanning a -mer phage display peptide library to identify peptides that ultimately inhibit virus binding. to this end, the tgev-m protein was first expressed in e. coli as a gst fusion protein. given the possibility of identifying gst-binding phage, we included additional panning steps using purified recombinant gst as the target to remove phage that did not specifically target the rtgev-m protein. also, the stringency was systematically increased by panning with decreasing concentrations of rtgev-m and increasing concentrations of tween- . three of tgev-m phages identified in this study were evaluated by elisa for detecting tgev infection. results showed that the widely used tgev-m antibody-elisa and phage-elisa both could detect the virus successfully; however, phage-elsia is more costeffective because it does not involve animals and phages are in unlimited supply. further, even using serum from animals boosted peptide concentration(ug/ml) relative amplication peptgev-m peppedv-s fig. . inhibition of virus infectivity monitored by rt-pcr. real time rt-pcr was used to quantify tgev rna in st cells and therefore virus infectivity. pcr was performed on the tgev s-ad gene. tcid tgev virus was pre-incubated with -fold, serially-diluted ( - . lg/ml) peptide (peptgev-m ) then added to st cells for h. an un-related peptide peppedv-s at the maximal concentration was used as control. virus-derived cdna was amplified and dct values were measured in triplicate. the relative amplification of tgev s-ad in tgev-infected cells was normalized to beta-actin and calculated using À ct method. statistical significance is indicated by '' ⁄ '' (p < . ) relative to lg/ml peptide. x with rtgev-m, the phage-elisa appeared more sensitive. clearly, targeting the m protein rather than the whole virus could sacrifice sensitivity. although we demonstrated specificity in phage and peptide binding to rtgev-m, we have not determined how well-exposed the binding region is to the surface of the virus. as such, using the m protein as the biopanning agent is probably not as effective as using whole virus. also, this may have contributed to the lower than expected signal strength in the polyclonal ab-elisa especially when the ab titer using rtgev-m protein as antigen was so high. the use of antivirals represents one approach to treat coronavirus infections (ren et al., b) . in this study, a peptide encoded by phtgev-m that interacted with the rtgev-m protein was synthesized. the interaction was deemed specific in that it was titratable and similar results were not observed with interactions between peptgev-m and another tgev structural protein, rtgev s-ad (meng et al., ) . plaque-reduction assays showed that tgev infectivity decreased only when virus was pre-treated with the peptide. in contrast, when the peptgev-m was incubated with st cells alone or with tgev-infected st cells, no inhibitory effect was observed. this is an expected outcome given that rtgev-m was used to pan for bioreactive phage and this protein is not present on the surface of or within st cells. collectively the data presented here are consistent with peptgev-m binding to the surface of the virus and either interfering with the ability of the virus to invade the cell or generate progeny virus. neither incubating the peptide with st cells prior to or after adding virus had a demonstrable effect on replication. the virus-specific effects of peptgev-m were confirmed by ifa and rt-pcr. in summary, peptide sequences that recognize the tgev-m protein were identified in our study using phage display technology. phages bearing these peptides may be utilized in serology-based diagnosis of tge, peptgev-m had direct inhibitory effects on tgev infectivity in vitro. in future research, peptides identified in this study may be subjected to antiviral testing. panning of a phage display library against a synthetic capsule for peptide ligands that bind to the native capsule of bacillus anthracis dna-mediated transformation of fungi the coronaviridae study group of the international committee on taxonomy of viruses, revision of the taxonomy of the coronavirus, torovirus, and arterivirus genera induction of interferon alpha by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein e genome sequence of chinese porcine parvovirus strain ppv a novel, stable, helical scaffold as an alternative binder -construction of phage display libraries identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display development and evaluation of a novel reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for detection of type ii porcine reproductive and respiratory syndrome virus mimotope peptides selected from phage display combinatorial library by serum antibodies of pigs experimentally infected with taenia solium as leads to developing diagnostic antigens for human neurocysticercosis phage therapy: delivering on the promise antibody engineering critical epitopes in transmissible gastroenteritis virus neutralization identification of tissue-specific targeting peptide from peptides to drugs via phage display sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins novel frameworks as a source of high-affinity ligands sequence and n-terminal processing of the transmembrane protein el of the coronavirus transmissible gastroenteritis virus molecular biology of transmissible gastroenteritis virus single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus porcine respiratory coronavirus: molecular features and virus-host interactions novel vaccine strategies to t-independent antigens the molecular biology of coronaviruses bacterial expression of antigenic sites a and d in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection identification of immunogenic proteins and generation of antibodies against salmonella typhimurium using phage display prevalence of antibodies against transmissible gastroenteritis virus and porcine respiratory coronavirus among pigs in six regions in japan generation of a replicationcompetent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome complete genome sequence of transmissible gastroenteritis coronavirus pur -mad clone and evolution of the purdue virus cluster importance of cholesterol for infection of cells by transmissible gastroenteritis virus phages harboring specific peptides to n protein of prrsv distinguish the virus from other viruses heterologous expression of fused genes encoding the glycoprotein from prrsv: a way for producing functional protein in prokaryotic microorganism phage displayed peptides recognizing porcine aminopeptidase n inhibit transmissible gastroenteritis coronavirus infection in vitro action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus cholesterol dependence of pseudorabies herpesvirus entry membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion the coronavirusmembrane glycoprotein transmissible gastroenteritis and porcine respiratory coronavirus phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife antigenic homology among coronaviruses related to transmissible gastroenteritis virus searching for peptide ligands with an epitope library coronaviruses: structure and genome expression antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus mechanisms of transmissible gastroenteritis coronavirus neutralization phage displayed peptides to avian h n virus distinguished the virus from other viruses a phage-displayed peptide can inhibit infection by white spot syndrome virus of shrimp cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus isolation, genome phylogenetic analysis and in vitro rescue of a newly emerging porcine circovirus type . pak the research work of the authors was supported by the program for new century excellent talents at the heilongjiang provincial university ( -ncet- ). key: cord- -yfjme q authors: magtoto, ronaldo; poonsuk, korakrit; baum, david; zhang, jianqiang; chen, qi; ji, ju; piñeyro, pablo; zimmerman, jeffrey; giménez-lirola, luis g. title: evaluation of the serologic cross-reactivity between transmissible gastroenteritis coronavirus and porcine respiratory coronavirus using commercial blocking enzyme-linked immunosorbent assay kits date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: yfjme q this study compared the performances of three commercial transmissible gastroenteritis virus/porcine respiratory coronavirus (tgev/prcv) blocking enzyme-linked immunosorbent assays (elisas) using serum samples (n = ) collected over a -day observation period from pigs inoculated with tgev strain purdue (n = ), tgev strain miller (n = ), prcv (n = ), or with virus-free culture medium (n = ). elisa results were evaluated both with “suspect” results interpreted as positive and then as negative. all commercial kits showed excellent diagnostic specificity ( to %) when testing samples from pigs inoculated with virus-free culture medium. however, analyses revealed differences between the kits in diagnostic sensitivity (percent tgev- or prcv-seropositive pigs), and all kits showed significant (p < . ) cross-reactivity between tgev and prcv serum antibodies, particularly during early stages of the infections. serologic cross-reactivity between tgev and prcv seemed to be tgev strain dependent, with a higher percentage of prcv-false-positive results for pigs inoculated with tgev purdue than for tgev miller. moreover, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the elisa kit evaluated. importance current measures to prevent tgev from entering a naive herd include quarantine and testing for tgev-seronegative animals. however, tgev serology is complicated due to the cross-reactivity with prcv, which circulates subclinically in most swine herds worldwide. conventional serological tests cannot distinguish between tgev and prcv antibodies; however, blocking elisas using antigen containing a large deletion in the amino terminus of the prcv s protein permit differentiation of prcv and tgev antibodies. several commercial tgev/prcv blocking elisas are available, but performance comparisons have not been reported in recent research. this study demonstrates that the serologic cross-reactivity between tgev and prcv affects the accuracy of commercial blocking elisas. individual test results must be interpreted with caution, particularly in the event of suspect results. therefore, commercial tgev/prcv blocking elisas should only be applied on a herd basis. tality in piglets in tgev/prcv-naive herds. the virus was first described by doyle and hutchings in in the united states and subsequently reported worldwide ( ) ( ) ( ) . porcine respiratory coronavirus is a naturally occurring spike gene deletion ( to kda) mutant of tgev first isolated in belgium in ( ) . it infects the upper respiratory tract, tonsils, or lungs, with limited intestinal replication ( , ) . porcine respiratory coronavirus itself does not appear to be an important primary pathogen, with the exception of its contribution to the porcine respiratory disease complex ( ) . tgev and prcv share biological and molecular features but differ in their epidemiology, clinical presentation, and pathogenesis. real-time reverse transcription-pcr (rrt-pcr) and multiplex microarray hybridization using primers targeting the = region of the s gene spanning the deletion region in pcrv strain are commonly used for the diagnosis of tgev and differentiation of tgev and prcv ( ) ( ) ( ) ( ) . serum antibodies provide serological evidence of tgev or prcv infection, but prcv-infected pigs produce antibodies that cross-react and cross-neutralize tgev, i.e., conventional serological tests cannot differentiate between tgev-and prcv-infected animals. this presents a complication for tgev seroprevalence studies and serological surveys of sows or slaughterhouse swine tested for international trade ( ) . to address the issue of cross-reactivity, monoclonal antibodies targeting antigenic regions of tgev that have been deleted from the prcv s protein ( ) ( ) ( ) ( ) ( ) ( ) ( ) have been used to develop blocking enzyme-linked immunosorbent assays (elisas) for tgev/ prcv differential serodiagnosis ( ) ( ) ( ) ( ) . several commercial tgev/prcv blocking elisas are available, but comparative test performances have not been reported in recent publications. in this study, the diagnostic test performances of three commercial tgev/prcv blocking elisa kits were evaluated using serum samples of precisely known porcine coronavirus immune status. clinical observations and virus shedding. mild watery diarrhea was observed in pigs inoculated with tgev miller strain between to days postinoculation (dpi). no clinical signs were observed in pigs from the negative-control, tgev purdue, and prcv-inoculated groups throughout the experiment. all animals survived until the end of the study ( dpi). all fecal samples collected from pigs in the tgev strain purdue, tgev strain miller, prcv-inoculated, and negative-control groups were tested by rrt-pcr and found to be tgev and prcv negative prior to the inoculations. likewise, all oral fluid samples, with the exception of one false-positive (threshold cycle [c t ], . ) result in the tgev purdue group, were negative before inoculation. the detection of tgev (s and n genes) and prcv (n gene) in pen-based feces and oral fluids by rrt-pcr is shown in fig. . transmissible gastroenteritis virus was detected in feces between and dpi by rrt-pcr in pigs inoculated with tgev strain miller ( fig. a and c) . no significant fecal shedding was detected in pigs inoculated with tgev strain purdue or prcv throughout the study ( fig. a and c). viral shedding was specifically detected by rrt-pcr in oral fluid samples collected from pigs inoculated with tgev strain purdue ( to dpi), tgev strain miller ( to dpi), and prcv ( to dpi) ( fig. b and d) . tgev antibody response over the course of the experimental inoculation. serum samples collected from all groups of pigs prior to inoculation were antibody negative for porcine coronaviruses (tgev, prcv, porcine epidemic diarrhea virus [pedv] , and porcine delta coronavirus [pdcov]). all pigs in the negative-control group remained tgev and prcv seronegative throughout the monitoring period when tested with any of the three tgev/prcv differential blocking elisa kits evaluated in this study ( the percentages of tgev antibody-positive serum samples reported by the three commercial elisa kits evaluated over the -day study period for pigs inoculated with tgev strains purdue and miller are presented in fig. a to f, respectively. suspect results are presented as positives or negatives. the first tgev-specific antibody detection was reported between and dpi in both tgev-inoculated groups (strains purdue and miller). the number of tgev antibody-positive pigs detected increased through the study, regardless of the elisa kit used. for the tgev purdue inoculation group, no significant differences (p Ͼ . ) were found in the percentages of tgev-seropositive pigs reported by the three elisas regardless of the time postinoculation and interpretation of suspect results. in contrast, for the tgev miller inoculation group, we found that a significantly higher (p Ͻ . ) percentage of tgev-seropositive animals was detected by the swinecheck tgev/prcv recombinant elisa than by both the ingezim corona diferencial elisa (dpi ) and svanovir tgev/prcv-ab elisa ( , , and dpi) only when suspect results were interpreted as negative. a nonspecific tgev antibody response to prcv-inoculated pigs was reported by the three elisa kits between and dpi ( fig. g to i). no significant differences in the percentages of tgev false positives were found between elisas over the monitoring period when tgev suspect results were interpreted as negative (p Ͼ . ). however, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the elisa kit evaluated. moreover, the percentage of tgev-false-positive results reported by the svanovir tgev/prcv-ab elisa was significantly greater (p Ͻ . ) than those obtained with the swinecheck tgev/prcv recombinant elisa ( , , and dpi) and ingezim corona diferencial elisa ( to dpi). prcv antibody response over the course of the experimental inoculation. with the exception of one false-positive result reported ( dpi) by the ingezim corona diferencial elisa kit, the negative-control group remained seronegative for prcv by the three commercial elisas throughout the study. on prcv-inoculated animals, an early prcv-specific antibody response was first detected at to dpi by the three commercial elisas and lasted through dpi ( fig. g to i ). an analysis of the proportion of prcv-seropositive animals showed significant differences between elisa kits over time following prcv inoculation, regardless of the interpretation of suspect results (p Ͻ . ). the percentage of prcvseropositive animals detected by the svanovir tgev/prcv-ab elisa was significantly lower than those with both the swinecheck tgev/prcv recombinant elisa (dpi to ) and ingezim corona diferencial elisa (dpi to and ) (p Ͻ . ). no differences were found between the swinecheck tgev/prcv recombinant and ingezim corona diferencial elisas. on tgev-inoculated animals, a nonspecific prcv serum antibody response was detected by the three commercial elisas. this serologic cross-reactivity seemed to be tgev strain dependent, with a higher percentage of prcv-false-positive results in the tgev strain purdue-inoculated group ( fig. a to c) than in the tgev strain miller group ( fig. d to f). the cross-reactivity appeared more marked at early stages postexposure ( to dpi). no differences were found between elisa kits over the course of the study, except at dpi when the proportion of prcv-false-positive animals detected by the svanovir tgev/prcv-ab elisa was significantly greater (p Ͻ . ) than that by the ingezim corona diferencial elisa regardless of the interpretation of suspect results. comparative diagnostic performance of the tgev/prcv differential elisas. the analytical specificity and diagnostic sensitivity and specificity of the three commercial differential tgev/prcv elisa kits evaluated in this study are presented in table . diagnostic parameters are presented relative to the interpretation of suspect results as positive or negative. overall, the diagnostic sensitivity for the tgev miller inoculation group was higher than for the tgev purdue group, independent of the commercial elisa kit evaluated. the swinecheck tgev/prcv recombinant elisa kit showed the highest diagnostic sensitivity for antibody detection in pigs inoculated with tgev strain miller ( %), regardless of the interpretation of suspect results. the diagnostic sensitivity for the tgev strain purdue-inoculated group was higher with the svanovir tgev/prcv-ab elisa kit ( %) when suspect results were considered positive, while it was slightly higher with the ingezim corona diferencial elisa kit ( %) when suspect results were interpreted as negative. the diagnostic sensitivities for prcv antibody detection obtained with both swinecheck tgev/prcv recombinant elisa ( %) and ingezim corona diferencial elisa ( to %) were greater than that of the svanovir tgev/prcv-ab elisa kit ( %). with the exception of the ingezim corona diferencial elisa, the diagnostic sensitivity calculated for each elisa kit was not affected by the interpretation of suspect results (positive versus negative). the three commercial elisas showed % diagnostic specificity for both tgev and prcv detection, with the exception for one prcv-false-positive result reported by the ingezim corona diferencial elisa, which resulted in a diagnostic specificity of . % for prcv detection. the diagnostic specificity of any of the evaluated elisa kits was unaffected by the interpretation of suspect results. the ingezim corona diferencial elisa kit showed the highest analytical specificity (less cross-reactivity) for tgev-specific antibody detection ( . %). for all three commercial elisas, we found higher cross-reactivity to prcv within the tgev purdue inoculation group than in the tgev miller group. the highest analytical specificity for prcv was obtained with the ingezim corona diferencial elisa ( %) when suspect results were interpreted as positive or with the svanovir tgev/prcv-ab elisa kit ( %) when suspect results were interpreted as negative; however, the svanovir tgev/ prcv-ab elisa kit showed the lowest analytical specificity ( %) of the kits when suspect results were interpreted as positive. the greatest risk of introducing tgev is through the importation of pigs from endemically infected countries. seronegative pigs of all ages are susceptible to infection with tgev ( , , ) , with the route of infection typically being oral or oral-nasal ( , ) . to prevent tgev from entering a naive herd, it is important to introduce only animals from tgev-free and serologically negative herds, along with disciplined biosecurity. therefore, quarantine and testing for tgev-seronegative animals are requirements for export/import into a herd. moreover, the detection of tgev antibodies, particularly in reproductive animals, can assist in diagnosis and control of the disease. however, tgev serology is complicated by cross-reactivity with prcv ( ), an s protein deletion mutant of tgev with altered tissue tropism (nonenteropathogenic but respiratory) that circulates subclinically in most swine herds worldwide ( , , ) . this cross-reactivity may explain why a region endemic for prcv has less tgev-associated disease ( ) . porcine respiratory coronavirus does not cause significant losses in swine except for its contribution to the porcine respiratory disease complex. historically, prcv diagnosis was based on recognition of respiratory disease in growing pigs that have high antibody titers to tgev but have had no clinical enteric disease or lesions of atrophic enteritis. conventional serological tests, such as virus neutralization, indirect fluorescent antibody, agar-gel precipitation, indirect immunoperoxidase, radioimmunoprecipitation, and some elisas based on polyclonal tgev antibodies, cannot be used for differentiation between tgev and prcv, because the two viruses share antigenic determinants located on the spike (s), membrane (m), nucleoprotein (n), and envelope (e) structural proteins ( , ) . the exception is a large deletion ( amino acids in length) in the amino terminus of the prcv s protein that is responsible for the loss of hemagglutination activity ( ) . this deletion provided the opportunity to develop blocking enzyme-linked immunosorbent assays for differentiation of prcv and tgev infections and laid the basis for the commercial tgev/prcv differential elisas that are currently available ( ) ( ) ( ) ( ) . few field reports have described the use of tgev/prcv blocking elisas to differentiate antibodies to tgev and prcv and their utility at the herd level ( , , ( , , ) . specimens primarily used for tgev virus detection include feces and intestinal contents, and those for prcv detection include nasal swabs or lung homogenates. in this study, the verification and monitoring of the tgev or prcv viral shedding status were performed using rrt-pcr testing of pen-based feces and oral fluids. this process provided further information on the dynamics of viral shedding in feces versus oral fluids. viral shedding was detected in both feces and oral fluids. however, for pigs inoculated with prcv and, interestingly, for tgev purdue, virus shedding was only detected in oral fluids. this could be related to differences in pathogenicity and tissue tropism, which were beyond the scope of this study. these results indicated that oral fluids may replace feces as a more suitable specimen for direct detection and surveillance of tgev/prcv by rrt-pcr. overall, we observed differences in the test performances of the three commercial elisa kits evaluated in this study. however, the differences were more marked for the svanovir tgev/prcv-ab elisa kit, compared to either the swinecheck tgev/prcv recombinant elisa or the ingezim corona diferencial elisa, for which test performance was comparable. this could be due to differences/similarities in assay design. for instance, both the swinecheck tgev/prcv recombinant elisa and the ingezim corona diferencial elisa use a tgev recombinant s protein antigen on a plate, while the svanovir tgev/prcv-ab elisa claims to use noninfectious tgev antigen of an unknown source, which could detect antibodies to a variety of viral proteins. the way the antigen is presented may also affect assay performance. the antigen can either be bound directly to the plate wells (as in the swinecheck tgev/prcv recombinant elisa and svanovir tgev/prcv-ab elisa) or may be captured by antigen-specific antibodies immobilized on the plate (as in the ingezim corona diferencial elisa). in addition, the svanovir tgev/prcv-ab elisa is the only kit using unlabeled tgev and tgev/prcv mouse monoclonal antibodies (mabs), which requires an additional incubation step with anti-mouse horseradish peroxidase (hrp)-conjugated secondary antibody. undisclosed differences in buffer composition could also be involved in the differences in assay performance among the kits. all three elisa kits showed higher diagnostic sensitivity in the detection of anti-tgev antibodies in pigs inoculated with tgev strain miller ( . to . %) than in pigs inoculated with tgev strain purdue ( . to . %), regardless of the kit used. the differences in diagnostic sensitivity among tgev-inoculated groups could be due to strain-related differences in virulence, which may have impacted the magnitude of the immune response. indeed, in this study, pigs inoculated with tgev strain miller showed mild watery diarrhea between and dpi, while no clinical signs or viral shedding in feces were observed in the pigs inoculated with tgev purdue. the diagnostic specificity of the three commercial elisa kits was evaluated on pigs of precisely known negative porcine coronavirus immune status (i.e., the porcine coronavirus negative-control group) to rule out potential cross-reactivity with other porcine coronaviruses (i.e., pigs virologically and serologically negative for pedv, pdcov, porcine hemagglutinating encephalomyelitis virus [phev], tgev, and prcv). all three kits evaluated in this study showed excellent diagnostic specificity, ranging from % (svanovir tgev/prcv-ab elisa) to % (swinecheck tgev/prcv recombinant elisa and ingezim corona diferencial elisa). these results were consistent with previous reports in the field where a tgev/prcv blocking elisa (i.e., swinecheck and biovet) showed a diagnostic specificity of % in wild boar populations historically free from coronavirus disease ( ) . with the final goal of maximizing both diagnostic sensitivity and specificity (but understanding that there is a balance between the two parameters), diagnostic specificity is of paramount importance during tgev/prcv screening due to the impact of false-positive results on global pig trade operations. the false-positive rate of any diagnostic test is a function of the specificity of the test and the prevalence of the disease. the assessment of the selectivity of the antigen-antibody response (analytical specificity) was conducted on each of the three commercial blocking elisas using a panel of heterologous monotypic known-status-positive sera generated under experimental conditions. specifically, in this study, the analysis of the analytical specificity was circumscribed to the cross-reactivity between tgev (strains purdue and miller) and prcv. however, the potential cross-reactivity between tgev/prcv against other swine coronaviruses has previously been reported ( ) . a test was considered analytically specific for tgev or prcv when it did not react against heterologous positive sera. as with the diagnostic sensitivity, the overall analytical specificity varied among and across kits and groups of inoculation, even with intrakit variations depending upon the interpretation of suspect results. serological cross-reactivity between tgev and prcv was previously reported by use of an immunoblotting assay based on tgev/prcv structural proteins (s, m, and n) and polyclonal antisera ( ) . this study further confirms that the serological cross-reactivity between tgev and prcv is significant even when using tgev/prcv differential blocking elisas. overall, in this study, we observed a poor analytical specificity (i.e., high cross-reactivity) for prcv antibody detection in pigs exposed to tgev strain purdue ( to %), with no significant differences among kits. however, with the exception of the svanovir tgev/prcv-ab elisa ( to %), we found an acceptable analytical specificity for prcv antibody detection in pigs exposed to tgev miller using the swinecheck tgev/prcv recombinant elisa ( to %) and the ingezim corona diferencial elisa ( %). current tgev/prcv differential immunoassays are based on a blocking elisa format, which is inherently more specific than indirect elisas. in the blocking elisa format, the degree to which specific antibodies in the test serum sample prevent binding of an agent-specific mab is measured. therefore, the higher the levels of specific antibodies are in a serum sample, the lower the likelihood for cross-reactivity. in fact, the overall higher tgev antibody detection rate observed within the group inoculated with tgev strain miller correlates with the lower cross-reactivity against prcv. that would also explain the overall higher rate of serologic cross-reactivity (regardless of the elisa kit used) reported during the first few weeks postinoculation, when specific antibody levels are still low. previous reports indicated that the accuracy of the commercial tgev/prcv blocking elisas for differentiating between u.s. tgev and prcv strains was low ( , , ) . individual test results must be interpreted with caution, particularly in the event of "suspect" results. we have demonstrated that interpretation of suspect results can impact significantly the diagnostic performance of any of the commercial kits evaluated in this study, with differences in their robustness in response to changes in the interpretation of suspect results. while analytical specificity improved overall when suspect results were interpreted as negative, this often comes at the cost of diagnostic sensitivity. in the event of suspect, undetermined, or unexpected positive results, it is recommended that serology testing on paired serum samples be conducted, in which a second sample should be drawn to days after the first sample collection. paired serum samples should be tested together to maximize the diagnostic value of test results. moreover, false-positive results reported at early stages postexposure (when animals are actively shedding virus) could be confirmed by rrt-pcr on oral fluid specimens, as evidenced in this study. the presence of tgev remains a barrier to international livestock trade ( ) . the blocking elisa format alone was determined to be useful in large swine populations for the detection of tgev-infected herds. moreover, the ability to specifically detect prcv antibodies minimized the probability of false-positive tgev results and subsequent exclusion of those herds for trading. however, this study demonstrates that the serologic cross-reactivity between tgev and prcv at the individual pig level affects the accuracy of commercial blocking elisas. therefore, it is important to remember that the tgev/prcv blocking elisas should only be applied on a herd basis ( , , , ) . experimental design. "known-status" serum samples (n ϭ ) collected from pigs experimentally inoculated with tgev purdue (n ϭ ), tgev miller (n ϭ ), prcv (n ϭ ), or with culture medium (minimum essential medium [mem] life technologies, carlsbad, ca) (negative control; n ϭ ) were used to evaluate the diagnostic performance (diagnostic sensitivity and specificity) and antibody crossreactivity (analytical specificity) of the following three commercial tgev/prcv blocking elisas: . at % confluence of the cell monolayer, the maintenance medium was decanted, and the monolayer was washed twice with maintenance medium. thereafter, each flask of cells was inoculated with l of each virus mixed with postinoculation medium, i.e., mem supplemented with . % tryptose phosphate broth (sigma-aldrich) and . % yeast extract (sigma-aldrich). the cells were incubated at °c with % co for h to allow virus adsorption. after incubation, ml of postinoculation medium was added to each flask without removing viral inoculum. the flasks were incubated at °c with % co and subjected to one freeze-thaw cycle at Ϫ °c when % cytopathic effect (cpe) was observed. cell debris was removed by centrifugation at , ϫ g for min at °c, and the virus content was harvested by collecting the supernatant. virus titration was performed on confluent st cell monolayers grown in -well plates (costar; corning) using a method described elsewhere ( ) . animals. the animal study was conducted at the iowa state university (isu) livestock infection disease isolation facility (lidif) with the approval of the iowa state university office for responsible research. forty-eight -week-old conventional pigs were acquired from a commercial wean-to-finish farm with no history of porcine coronavirus infection. during the herd prescreening process and prior to the beginning of the study, fecal and nasal swabs from all animals were tested for tgev, prcv, porcine hemagglutinating encephalomyelitis virus (phev), porcine epidemic diarrhea virus (pedv), and porcine delta coronavirus (pdcov) by real-time reverse transcription-pcr (rrt-pcr) assays. serum samples were tested for all of the pathogens listed above using antibody-based methods, as described elsewhere ( ) ( ) ( ) . upon arrival, all animals were ear-tagged and randomly assigned to one of four inoculation groups (n ϭ per group in the tgev miller, tgev purdue, prcv, and negative-control groups). pigs within each group were housed in pens of pigs in the same room. each pen was equipped with nipple drinkers, and pigs were fed twice daily with an antibiotic-free commercial diet (heartland co-op, west des moines, ia, usa). details regarding viral dose and route of inoculation for each group are presented in table . the infectious dose used for each virus was previously determined in a pilot study (data not shown) with the objective of stimulating a humoral response effectively. pigs were evaluated clinically twice daily throughout the study. the tgev and prcv infectious status of every pig was established and monitored throughout the study by rrt-pcr tests on fecal and oral fluid samples and elisa-based tests on serum samples. sample collection. blood samples were collected on Ϫ , , , , , , , , , , and days postinoculation (dpi) from the jugular vein or cranial vena cava using a single-use blood collection system (becton dickinson, franklin lakes, nj) and serum separation tubes (kendall, mansfield, ma). serum was separated by centrifugation at , ϫ g for min, aliquoted into -ml cryogenic tubes (greiner bio-one gmbh, frickenhausen, germany), and stored at Ϫ °c until use. floor fecal samples were collected daily from each pen ( pigs per pen) within each inoculation group ( pens per group) from Ϫ to dpi. approximately ml of feces was placed in a -ml cryogenic tube (bd falcon). fecal samples ( l) from each group on the same day postinoculation were pooled into a -ml cryogenic tube (bd falcon) at the end of the study for rrt-pcr testing. oral fluid samples were collected from each pen within each inoculation group twice a day ( : a.m. and : p.m.) from Ϫ to dpi. in brief, -strand . -cm % cotton rope (web rigging supply, inc., carrollton, ga) was hung from a bracket fixed to one side of each pen for min, during which time the pigs chewed on and interacted with the rope. after min, the wet end of the rope was severed, sealed in a plastic bag, and then passed through a clothes wringer (dyna-jet, overland park, ks). the oral fluid accumulated in the bottom of the bag was decanted into -ml conical tubes (corning), aliquoted into -ml cryogenic tubes (greiner bio-one gmbh), and stored at Ϫ °c. real-time reverse transcription-pcr. feces and oral fluid samples were submitted to the isu veterinary diagnostic library (vdl) for porcine coronavirus screening by rrt-pcr. transmissible gastroenteritis virus-specific spike (s) and tgev/prcv-specific nucleocapsid (n) genes were targeted and amplified by dually labeled probe-based rrt-pcr. in brief, sample rna was extracted and eluted using the ambion magmax viral rna isolation kit (life technologies) and a kingfisher magnetic particle processor (thermo fisher scientific) following the procedures provided by the manufacturers. the tgev s gene and the tgev/prcv n gene-based rrt-pcr were modified from previous procedures ( ) and performed routinely at the isu-vdl using the taqman fast -step mastermix (thermo fisher scientific). the rt-pcrs were conducted on an abi fast instrument (life technologies) as follows: °c for min, °c for s, °c for s ( cycles), and °c for s. the rrt-pcr results were analyzed using an automatic baseline, with a threshold value of . . a quantification cycle (c t ) value of Ͻ was considered positive for both s and n gene-based rrt-pcr. samples were considered positive for tgev when both s and n genes were rrt-pcr positive. for prcv, samples were considered positive when the s gene rrt-pcr was negative and the n gene rrt-pcr was positive. transmissible gastroenteritis virus and prcv c t data were reported as "adjusted c t ," calculated as shown in the following equation: adjusted c t ϭ ͑ cutoff c t value Ϫ sample c t value ͒ tgev/prcv differential blocking elisas. serum samples were tested for tgev-and prcv-specific antibodies using the following three commercially available tgev/prcv differential blocking elisa kits: (i) swinecheck tgev/prcv recombinant (biovet, canada), (ii) ingezim corona diferencial (ingenasa, spain), and (iii) svanovir tgev/prcv-ab (svanova, sweden). all assays were performed according to the manufacturers' instructions. the pig serum samples and controls were first added to the tgev antigencoated wells for the first incubation step. in the ingezim corona diferencial elisa, plate wells are coated with a recombinant tgev s protein which is captured by a specific monoclonal antibody (mab). the swinecheck tgev/prcv recombinant elisa is also based on a recombinant tgev s protein but directly coated on plate wells, while the svanovir tgev/prcv-ab elisa uses noninfectious tgev antigen (source unknown) as a coating antigen. if anti-tgev or anti-prcv antibodies are present in the test sample, they will bind to the viral (tgev) antigen in the wells and block the antigenic sites. in contrast, if anti-tgev or anti-prcv antibodies are absent in the test sample, these sites will remain free. then, after a washing step to eliminate unbound antibodies, a horseradish peroxidase (hrp)-conjugated mouse anti-tgev mab targeting the n-terminal region of the s glycoprotein that is deleted in prcv, or an anti-tgev/prcv mab that binds to conserved regions of both tgev and prcv, is added into the first well (odd column) or second well (even column), respectively, and attaches to specific free sites of the virus. thus, when antibodies in the test sample occupy the binding sites on the antigen, the binding of the conjugated mabs is blocked. if anti-tgev or anti-prcv antibodies are absent in the test sample, these sites will remain free. the amount of antibody in the test sample that is bound to the tgev antigen is inversely proportional to the intensity of the color. antibody elisa results were expressed as the percentage of inhibition (swinecheck tgev/prcv and svanovir tgev/prcv-ab) or optical density (ingezim corona diferencial) and interpreted (positive, negative, or suspect/inconclusive) for tgev and prcv according to the manufacturers' instructions. the diagnostic sensitivity, diagnostic specificity, and analytical specificity of the three commercial elisas were calculated based on the true status of the samples compared to the specific detection of tgev (miller and/or purdue) and/or prcv antibodies. specifically, porcine coronavirus-negative serum samples (n ϭ ) were used to estimate diagnostic specificity for the three elisa kits. serum samples positive for tgev (purdue and miller strains) (n ϭ ) collected between and dpi and prcv-positive samples (n ϭ ) collected between and dpi were used to calculate the time of detection, antibody detection over time, and the overall diagnostic sensitivity of the three elisa kits for tgev and prcv ab detection, respectively. a transmissible gastroenteritis in pigs molecular basis of transmissible gastroenteritis virus epidemiology porcine coronaviruses isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis porcine respiratory coronavirus related to transmissible gastroenteritis virus polymicrobial respiratory disease in pigs respiratory and fecal shedding of porcine respiratory coronavirus (prcv) in sentinel weaned pigs and sequence of the partial s-gene of the prcv isolates development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd multiplex pcr and multiplex rt-pcr for inclusive detection of major swine dna and rna viruses in pigs with multiple infections antigenic differentiation between transmissible gastroenteritis virus of swine and a related porcine respiratory coronavirus assembly of coronavirus spike protein into trimers and its role in epitope expression antigenic homology among coronaviruses related to transmissible gastroenteritis virus active immunity and t-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins evaluation of the baculovirusexpressed s glycoprotein of transmissible gastroenteritis virus (tgev) as antigen in a competition elisa to differentiate porcine respiratory coronavirus from tgev antibodies in pigs antigenic variation among transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the s protein of tgev competition elisa, using monoclonal antibodies to the transmissible gastroenteritis virus (tgev) s protein, for serologic differentiation of pigs infected with tgev or porcine respiratory coronavirus natural infection with the porcine respiratory coronavirus induces protective lactogenic immunity against transmissible gastroenteritis a competitive inhibition elisa for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (tgev) or with the tgev-related porcine respiratory coronavirus differentiation of porcine coronavirus from transmissible gastroenteritis virus pathogenicity of an attenuated strain of transmissible gastroenteritis virus for newborn pigs coronavirus-like particles associated with diarrhea in baby pigs in quebec recovery of transmissible gastroenteritis virus from chronically infected experimental pigs intestinal protection against serologic cross-reactivity between tgev and prcv challenge with transmissible gastroenteritis virus of pigs immune after infection with the porcine respiratory coronavirus diseases of swine sero-surveillance of transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) in south korea genetic evolution and tropism of transmissible gastroenteritis coronaviruses tgev corona virus orf encodes a membrane protein that is incorporated into virions transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (nglycolylneuraminic acid) binding activity seroprevalence of porcine respiratory coronavirus in selected korean pigs field validation of a commercial blocking elisa to differentiate antibody to transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus and to identify tgev-infected swine herds detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus in situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv), in formalin-fixed paraffin-embedded tissues prevalence of and risk factors associated with viral and bacterial pathogens in farmed european wild boar antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia a simple method of estimating fifty per cent endpoints pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus this work was funded in part through the iowa pork producers association through the national pork board (pork checkoff funds, grant - ; des moines, ia) and the iowa state university veterinary diagnostic laboratory (ames, ia). data analysis. analyses were conducted using commercial statistical software (sas version . . ; microsoft corporation, usa). the percentage of results for tgev/prcv elisa were interpreted (positive, negative, or suspect) according to the manufacturer's recommendations. the percentage of seropositive animals per day postinoculation detected by each kit were analyzed in two ways, by considering suspect results to be positive or by considering suspect results to be negative. significant differences (p Ͻ . ) in elisa-positive rates relative to tgev (purdue or miller strains) and prcv were calculated using pearson's chi-square test or fisher's exact test. the diagnostic sensitivity and specificity of the three commercial elisas were evaluated based on experimental serum samples of precisely known porcine coronavirus immune status (i.e., samples collected on Ͻ dpi were negative, and serum samples collected on Ͼ dpi were positive). analytical specificity (bidirectional cross-reactivity) between tgev versus prcv was evaluated using samples (n ϭ ) collected between and dpi from animals inoculated with tgev strain purdue (n ϭ ), tgev strain miller (n ϭ ), and prcv (n ϭ ) in each given case. figures were created using commercial graphics software (sigmaplot version . ; systat software, inc.). the funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.we declare no conflicting interests with respect to authorship or the publication of this article. key: cord- -jjft den authors: rodák, l.; Šmíd, b.; nevoránková, z.; valíček, l.; smítalová, r. title: use of monoclonal antibodies in blocking elisa detection of transmissible gastroenteritis virus in faeces of piglets date: - - journal: j vet med b infect dis vet public health doi: . /j. - . . .x sha: doc_id: cord_uid: jjft den monoclonal antibodies (mab) to the transmissible gastroenteritis virus (tgev) nucleoprotein (n) and membrane protein (m) were prepared and used for the comparative assessment of three blocking elisa variants to detect tgev. the competitive blocking elisa format showed the highest sensitivity, allowing detection of ( ) tcid( ) tgev/ml in culture medium. ninety‐nine porcine field faecal samples obtained from herds affected with diarrhoea were examined, and various tgev levels were found in nine samples from six herds. however, only in three samples were significant tgev concentrations demonstrated. the relationship between incidence of tgev gastroenteritis and the spread of porcine respiratory coronavirus infection in pig farms is discussed. rotaviruses and coronaviruses rank, along with bacterial infections, among the most significant causal agents of gastroenteritis in pigs. the two coronaviruses causing transmissible gastroenteritis (tge) and porcine epidemic diarrhoea (ped) are morphologically identical, but they are antigenically different. tge gastroenteritis, occurring mostly in the enzootic form, is not such a problem in europe as it was in the past (sˇteˇpa´nek et al., ; pritchard et al., ) . although different results on the prevalence of tgev infection were given (kim and cho, ; chae et al., ) , this infection still may be topical in some countries. decreased incidence of tge gastroenteritis is largely influenced by the spread of porcine respiratory coronavirus (prcv) infection in pig herds (bernard et al., ; sˇesta´k et al., ) . high level of nucleotide sequence homology ( %) of the two viruses was proven by genome analysis (britton et al., ) with major deletions in genome coding s protein of prcv. therefore, it is presumed that prcv is a spike (s) gene deletion mutant of tgev, and minor alterations of the viral genome led to distinct viral tropism (rasschaert et al., ; sˇesta´k et al., ; ballesteros et al., ; constantini et al., ) . three major structural proteins occur in coronaviruses: the glycoprotein s (m r - k) found in the viral protrusions (corona), the nucleocapsid phosphoprotein n (m r - k) and the membrane protein m (m r - k) (saif, ; utiger et al., ) . while viral antigens n and m of tgev and prcv are identical, differences were demonstrated in glycoprotein s epitopes, which do not trigger production of neutralizing antibodies. monoclonal antibodies (mab) to those epitopes are currently used to distinguish between tgev and prcv antibodies (simkins et al., ) . however the situation is different when detection of the virus in faecal samples is considered. although the presence of prcv in faecal samples was demonstrated by extremely sensitive rt-pcr technique (constantini et al., ) , little if any intestinal multiplication of prcv was proven (cox et al., a,b,c) . it follows that the method of lower sensitivity based on the use of antibodies to both tgev and prcv antigens could be suitable for specific demonstration of tgev in faeces. therefore the objective of our study was to check such mab in modifications of blocking elisa method, and to implement the optimal one for tgev demonstration. the strain of tgev, capm v- (collection of animal pathogenic microorganisms, brno, czech republic) was propagated in the porcine kidney cell line (pk- ) in eagle-mem medium. after development of cytopathic effect and centrifugation ( g for min) of the medium, the pellet was resuspended in / of the original medium volume in phosphate-buffered saline (pbs) as crude viral antigen (v-ag). crude control antigen (c-ag) was prepared similarly from uninfected cells. purified v-ag was prepared from the supernatant of the infectious medium by ultracentrifugation on cushions of and % sucrose according to hofmann and wyler ( ) . antigens were kept at ) °c before use. culture media with known tissue culture infectious dose (tcid /ml) were used for the determination of blocking elisa method sensitivity. to exclude possible crossreactions with other agents, crude v-ag (tge, ped and rota a) were tested by all elisa methods used. the reactivities of mab with five tgev strains (v- , shizuoka, purdue, and two of our field isolates cz- and cz- ) were also examined. two hysterectomy derived, colostrum-deprived -day-old piglets were kept in sterile conditions, and orally infected with · . tcid tgev. diarrhoea appeared hpi in both piglets; samples of faeces were collected during seven consecutive days. seven weeks post-infection piglets were challenged with · . tcid tgev and killed days later by exsanguination under total anaesthesia. the titre of tgev antibodies in serum obtained (swspos.) was determined by indirect elisa ( ). tgev-negative swine blood serum (swsneg.) was obtained from -day-old uninfected piglet. the immunoglobulin fraction (swatgev) prepared from positive serum was used as a binding antibody in the blocking elisa methods. rota-and coronaviruses were detected by electron microscopic examination of faecal samples and culture media after negative staining with % ammonium molybdate solution in water, ph . (sˇmı´d et al., ) . monolayers of infected (tgev, pedv, rotavirus a) and uninfected cells were fixed with acetone min at °c. after inhibition of endogenous peroxidase (li et al., ) , the tgev was detected by mab by direct and indirect immunoperoxidase (ip) tests. the reactions were read after - min incubation in a substrate solution containing chromogen aec ( -amino- -ethylcarbazole; sigma, st louis, mo, usa). low molecular weight standard (lmw; pharmacia, uppsala, sweden) and purified tge v-ag were separated by discontinuous electrophoresis (laemmli, ) in % polyacrylamide gel (miniprotean cell; bio-rad labs, hercules, ca, usa). after transfer to a nitrocellulose (nc) membrane ( . lm; bio-rad) and separation of the nc lanes, the lane containing lmw was stained with colloid gold (moeremans et al., ) , and lanes with v-ag were used for western blot (wb) analysis. after incubation ( h/ °c) with the tested sera or mab incubations with peroxidase conjugates and subsequently in a chromogen solution dab ( , ¢-diaminobenzidine; sigma) followed. hybridomas producing mab to tgev (mabtgev) were prepared according to galfre`and milstein ( ) by fusion of splenic lymphoid cells of immunized mice of the line balb/c with cells of the myeloma line sp / . hybridomas producing mab with optimal results in indirect elisa were checked by wb and ip tests and selected mab were used for preparation of ascitic fluids. after purification by ion exchange chromatography, mabtgev obtained were stored at ) °c in % glycerol or used for the preparation of peroxidase conjugates. rabbit antibodies to swine and mouse immunoglobulins (raswig, ramoig) or swine antibodies to mouse immunoglobulins (swamoig) were purified from hyperimmune sera by affinity chromatography. these antibodies and mab were conjugated with horseradish peroxidase (hrpo, type vi-a; sigma) using the periodate method (boorsma and streefkerk, ) . stock conjugate solutions adjusted to mg of immunoglobulin/ml were further diluted for ip, wb and elisa tests · to · in pbs containing . % tween and . % lactalbuminhydrolysate (pbst-lah). indirect elisa tgev antibodies in tested sera, in culture media of hybridomas and in purified mabtgev preparations were assayed using the indirect elisa method. pairs of wells of microtitre plates (nunc-immunoplate; polysorp, roskilde, denmark), alternatingly precoated with tge crude v-ag and c-ag were incubated with diluted tested samples. after second incubation with conjugates (always h/ °c), the reactions were visualized in a substrate solution with chromogen tmb ( , ¢, , ¢tetramethyl-benzidine; sigma). after min, the reaction was stopped by addition of m h so , and absorbances were measured spectrophotometrically at nm. control wells filled with the diluent (blank) at the first incubation, or with control negative and positive serum were included in each examination. samples showing a difference in optical density of at least . (after subtraction of absorbances of blank wells) were classified as positive. three variants of elisa method with specificity checked by blocking test were compared by box titration using faeces of experimentally infected piglets hpi. two double antibody sandwich elisa variants (das-elisa) and competitive blocking elisa (cb-elisa) method were compared. microtitre plate wells (nunc-immuno plate; maxisorp, roskilde, denmark) precoated with binding antibodies was used. pairs of wells filled with ll of mixtures of faeces and swsneg. or swspos. fivefold diluted : to : or : to : , respectively, were examined. the second incubation was performed using the detection antibodies (conjugate). pbst-lah containing . m nacl and mg edta.na /ml for samples dilution at the first incubation and rinsing of wells four times with pbst between incubations at °c were used. pairs of wells filled with the diluent (blank) or the mixtures of crude v-ag (tgev, pedv, rotavirus a) and swsneg./pos. during the first incubation were included in each analysis. the following variants of the blocking elisa method were investigated. binding antibody mabtgev was used. the first incubation with antigen test samples was performed for h, the second incubation with the conjugate hrpo-mab-tgev for h. binding antibody swatgev was used. after h of incubation with the antigen test samples, the wells were supplemented with ll diluent containing . lg mabtgev/ml, and the incubation continued for another hour. the second h incubation was done using the conjugate hrpo-swamoig. binding antibody swatgev was used. the first incubation with the antigen test samples was performed for h and the second h incubation was conducted with the conjugate hrpo-mabtgev. the samples were regarded as positive if the net absorbance (na), i.e. the difference of average absorbances in the wells l. rodák et al. incubated with swsneg./pos. was > . , and the reactions were blocked by > % in the wells with swspos. blocking percentages were determined using the formula: %b ¼ ) [(a swspos. · ):(a swsneg. )]. antibodies to prcv and tgev were assessed and differentiated in blood serum samples of sows from randomly selected five herds. commercial kit (ingezim corona diferencial; ingenasa, madrid, spain) was used for examination. sample preparation and evaluation of the results was carried out according to the manufacturer's instructions. in total, faecal samples from piglets with diarrhoea on farms were examined. most samples were from piglets younger than days. after delivery, they were diluted in two to three volumes of earle's medium, centrifuged ( min, g), and the supernatants examined by electron microscopy and cb-elisa method. samples were kept at ) °c before elisa analysis. after verification of the specificity of hybridomas producing mabtgev by wb, elisa and ip tests, four preparations (d /g ; d /g ; b /f and b /f ) were selected for further use. all were of the isotype igg and reacted with the membrane protein m (d /g ) and nucleoprotein n (b /f ) in wb analysis. weak reactivity of mab d /g with epitopes present on protein m and nucleoprotein n were detected (fig. ) . conformational changes of viral antigens resulted in markedly decreased or even absent reactiveness of mab b /f in wb analysis. all mab used reacted specifically with native tgev strains in ip (fig. ) and elisa tests according to results given in table . indirect elisa titres of individual mabtgev solutions containing mg ig/ml ranged between · and · . cross reactivity of mab with other viral antigens (rotavirus a and pedv) could not be detected by any of methods used. the reactivity of individual mab with five tgev strains was checked by cb-elisa examination of infectious culture media. according to obtained positive na and %b values, the mab d /g and b /f react with all tgev strains, while mab d /g and b /f react with strains v- and cz- only. by the use of mab d /g alone or mixture of mab in cb-elisa examination of various tgev strains (table ) or positive field faecal samples (results not shown) the same results were obtained. only a little lower absorbance values with d /g were detected. therefore the mixture of equal concentrations (w/v) of all mab was selected for routine cb-elisa examination of field samples. sensitivity of the three blocking elisa variants was checked by box titration of mixtures of faces of an experimentally infected piglet hpi and swsneg./swspos. competitive blocking elisa variant (cb-elisa) was selected as optimal (table ). working dilutions of faecal samples : , swsneg./ pos. : , mabtgev mixture containing mgig/ml : and hrpo-swamoig : were used for routine cb-elisa examinations. high tgev concentration in the tested sample is connected with high absorbance values obtained, and the other way round. it follows also, that evaluation of samples with different tgev concentrations is influenced significantly both by sample and swsneg./pos. dilutions. in table results with swsneg./pos. diluted · and · only are given. the culture medium from pk- infected cells containing . tcid tgev/ml was analysed by box titration. mixtures of medium diluted : to : and porcine sera diluted : to : respectively, were examined. positive na and %b values were obtained at all dilutions of both medium and sws (results not shown). the sensitivity of the cb-elisa method was therefore estimated to exceed tcid tgev/ml. cb-elisa method specificity was confirmed by examination of crude v-ag (tgev, pedv, rotavirus a). positive results were obtained with tge v-ag only with average absorbances in the wells incubated with swsneg./pos. . / . (na ¼ . , %b ¼ . ). in the wells with crude pedv and rotavirus a the absorbances were ) . / . and . / . respectively. by examination of blood serum samples of sows from five herds, tgev antibodies were detected only in a single sample. prcv antibodies were detected in ( %) samples in all herds. percentage of prcv-positive animals in respective herds ranged between and % (results not shown). the tgev was detected by cb-elisa in all faecal samples, taken between and days after experimental infection of piglets. the propagation of tgev in the intestine thus exceeds dpi. according to absorbance values obtained, highest tgev concentrations were detected between and dpi (table ) . altogether field faecal samples from herds were examined by cb-elisa. various tgev concentrations were found in nine samples from six herds. however, high tgev concentrations confirmed by absorbances . - . (na ¼ . - . , %b ¼ . - . ) were detected in three samples only. the maximum absorbances in the remaining faecal samples indicating low tgev concentration reached . . the results obtained by em and cb-elisa were poorly correlated reaching the values of about %. similarly, as in our previous study concerning pevd detection (roda´k et al., ) corona-like particles without typical structures (corona) were frequently detected. gastroenteritis caused by tgev is less important at present in comparison with the s and s when mortality of piglets reached - % in affected herds (sˇteˇpa´nek et al., ) . in addition to possible decreased tgev virulence, it is explained by the spread of prcv infection in pig herds and by protection of piglets by crossreacting maternal antibodies (saif et al., ; lanza et al., ) . for diagnosis of gastroenteritis caused by coronaviruses elisa methods are used in addition to conventional virological procedures and molecular virology methods (paton et al., ; pritchard et al., ; kim et al., ) . the Ôdas-elisaÕ with specificity checked by blocking test has been used for both tgev and pedv demonstration in faeces (van nieuwstadt et al., ; carvajal et al., ) . therefore, the detection of tgev in faecal samples by blocking elisa method was the objective of our study. we supposed that mab against n and m antigens of various tgev strains could be useful for this purpose in spite of their crossreactivity with prcv. faecal shedding of prcv after infection was demonstrated by nested-rt-pcr (constantini et al., ) . however, the demonstration of ingested virus cannot be excluded. little if any enteric multiplication of prcv was demonstrated by immunofluorescence after experimental infection of -week-old piglets only, and it remained limited to a few unidentified cells located in or underneath the epithelial layer of the villi and/or crypts (cox et al., a,b,c) . the rt-pcr is extremely sensitive allowing rna detection of only occasional viral particles in the sample. although the sensitivity of tgev detection by cb-elisa in faecal samples is sufficient ( tcid tgev/ml), we suppose that the possibility to demonstrate traces of prcv is relatively low. nevertheless, this assumption will be further checked by application of rt-pcr in following experiments. the sensitivities of tgev detection by das-elisa and cb-elisa were compared and higher sensitivity and specificity of the last method was proven. while in das-elisa conjugated mab are used, cb-elisa method is based on the use of unconjugated mab. the results obtained also indicate (tables and ). this was confirmed by examination of field faecal samples; nine of them were tgev positive. however, high tgev concentrations, suggesting that the virus is an important causative agent of gastroenteritis, were detected in three samples only. moreover, in all tgevpositive samples, the presence of rotavirus a and/or pedv was demonstrated, indicating the importance of mixed enteric viral infections (results not shown). the demonstration of tgev antibodies in only one of randomly selected field sow serum samples indicates that the animals produce antibodies if the tgev antigenic stimulation is high enough to compete with the stimulation by other enteric pathogens. the results also correlate with the assumption that the immunity of sows to prcv leads to decreased occurrence of tgev gastroenteritis (bernard et al., ; lanza et al., ; sˇesta´k et al., ) . effectivity of virus detection in faecal samples may be affected by the time of sample collection after the first symptoms of diarrhoea had emerged and the conditions of their shipment. marked destruction of coronaviruses occurs in the gastric and intestinal contents (aynaud and bottreau, ) . it follows, that characteristic structures of coronaviruses are less frequent by em examination and cellular substructures may imitate Ôcorona-likeÕ particles. this can explain low correlation between em and cb-elisa results. the results described, confirm suitability and sufficient sensitivity of the cb-elisa for routine demonstration of tgev in field faecal samples. cb-elisa methods were also successfully applied in detection of group a rotavirus and pedv in faeces (roda´k et al., (roda´k et al., , . in spite of higher sensitivity of rt-pcr technique, the advantages of cb-elisa examinations are lower costs, the possibility to demonstrate simultaneously three most important causal agents of viral gastroenteritis by uniform method and to examine sufficient number ( - ) of samples. this can give information both on their incidence in pig herds and on the effectiveness of immunoprophylactic measures. transmissible gastroenteritis of swine: stability of coronavirus in gastric and intestinal contents two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism natural infection with the porcine respiratory coronavirus induces protective lactogenic immunity against transmissible gastroenteritis periodate or glutaraldehyde for preparing peroxidase conjugates? the cloning and sequencing of the virion protein genes from a british isolate of porcine respiratory coronavirus: comparison with transmissible gastroenteritis virus genes evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs respiratory and fecal shedding of porcine respiratory coronavirus (prcv) in sentinel weaned pigs and sequence of the partial s-gene of the prcv isolates sites of replication of a porcine respiratory coronavirus related to transmissible gastroenteritis virus intestinal replication of a porcine respiratory coronavirus closely related antigenically to the enteric transmissible gastroenteritis virus sites of replication of a porcine respiratory coronavirus in -week-old pigs with or without maternal antibodies preparation of monoclonal antibodies: strategies and procedures enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera transmissible gastroenteritis of pigs in southeastern korea in - : prevalence and factors associated with the infection differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr cleavage of structural proteins during the assembly of the head of bacteriophage t lactogenic immunity and milk antibody isotypes to transmissible gastroenteritis virus in sows exposed to porcine respiratory coronavirus during pregnancy use of azide and hydrogen peroxide as an inhibitor for endogeneous peroxidase in the immunoperoxidase method sensitive colloidal (gold or silver) staining of protein blots on nitrocellulose membranes comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus transmissible gastroenteritis and porcine epidemic diarrhoea in britain porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions verification of sensitivity and specificity of group a rotavirus detection in piglets faeces with monoclonal blocking elisa methods an elisa optimized for porcine epidemic diarrhoea virus detection in faeces coronavirus immunogens immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs competition elisa, using monoclonal antibodies to the transmissible gastroenteritis virus (tgev) s protein, for serologic differentiation of pigs infected with tgev or porcine respiratory coronavirus electron microscopic demonstration of porcine epidemic diarrhea virus in the czech republic physicochemical and biological properties of strains isolated in czechoslovakia identification of proteins specified by porcine epidemic diarrhoea virus this work was supported by projects qf and mze of the ministry of agriculture of the czech republic. the authors wish to thank mrs farnı´kova´, mrs lickova´, miss hoydenova´and miss bacˇinska´for their skilful technical assistance. key: cord- - xkt u authors: petrini, stefano; righi, cecilia; iscaro, carmen; viola, giulio; gobbi, paola; scoccia, eleonora; rossi, elisabetta; pellegrini, claudia; de mia, gian mario title: evaluation of passive immunity induced by immunisation using two inactivated ge-deleted marker vaccines against infectious bovine rhinotracheitis (ibr) in calves date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: xkt u different types of vaccines against infectious bovine rhinotracheitis (ibr) are commercially available. among these, inactivated glycoprotein e (ge)-deleted marker vaccines are commonly used, but their ability to induce passive immunity is poorly known. here, we evaluated the passive immunity transferred from dams immunised with commercial inactivated ge-deleted marker vaccines to calves. we vaccinated pregnant cattle devoid of neutralising antibodies against bovine alphaherpesvirus (bohv- ) and divided them into two groups with animals each. both groups were injected with a different inactivated ge-deleted marker vaccine administrated via intranasal or intramuscular routes. an additional pregnant cattle served as the unvaccinated control group. after calving, the number of animals in each group was increased by the newborn calves. in the dams, the humoral immune response was evaluated before calving and, subsequently, at different times until post-calving day (pcd ). in addition, the antibodies in colostrum, milk, and in serum samples from newborn calves were evaluated at different times until pcd . the results indicated that inactivated glycoprotein e (ge)-deleted marker vaccines are safe and produce a good humoral immune response in pregnant cattle until calving and pcd . moreover, results showed that, in calf serum, passive immunity persists until pcd . bovine alphaherpesvirus (bohv- ) causes various clinical syndromes among cattle populations, including respiratory disease, vaginitis, balanoposthitis, abortions, enteritis, and encephalitis, which may be observed after acute infection or subsequent to viral recrudescence, following periods of stress [ , ] . to date, finland, sweden, norway, denmark, austria, switzerland, as well as the province of bolzano in italy, have successfully employed a "test-and-slaughter" strategy. other european union states have implemented compulsory eradication programmes combining "test-and-removal" with "vaccination" using marker vaccines, conversely to vaccination strategies used in the usa, where non-marker vaccines are used [ ] . marker vaccines are derived from the deletion of one or more genes responsible for the synthesis of glycoproteins or enzymes [ , ] . in europe, glycoprotein e (ge) of bohv- is the gene marker that is most commonly deleted in modified live or inactivated bohv- diva (differentiating infected from vaccinated animals) vaccines [ , ] . this type of immunisation makes it possible to differentiate animals immunised with marker vaccines (ge negative) from those infected or inoculated with traditional non-deleted vaccines (ge positive) through diagnostic tests specific for bohv- ge [ , , ] . however, a disadvantage of using live marker vaccines is that the virus can remain in a latent state in the immunised animals such that it can be reactivated and excreted following exposure to an immunosuppressive stimulus [ ] . further, marker vaccines administered both intramuscularly and intranasally induce a marked humoral and cell-mediated immune responses [ ] , however, little information is available regarding the induction of passive immunity. regarding traditional vaccines, several reports have shown that cattle vaccinated with non-deleted modified-live vaccines transfer neutralising antibodies (na) to the newborn calves that can protect them from experimental infection [ , ] , and other studies have demonstrated that a poor titre of colostral antibodies increases the risk of contracting bohv- infection in the calf [ ] . it has also been reported that the concentration of maternal antibodies against bohv- in calves after birth and after colostrum ingestion is directly proportional to that present in the mother's serum [ , ] . though extensive studies have reported on marker vaccines, there is a need to assess their ability to induce passive immunity. here, we have made a concerted effort to evaluate passive immunity transmitted from cattle to newborn calves following immunisation with different inactivated marker vaccines. two commercial vaccines were selected for the study ( table ) . the products were identified as follows: vaccine a, a ge-negative, inactivated strain of bohv- containing light paraffin oil; and vaccine b, a ge-negative, inactivated strain of bohv- containing aluminium hydroxide, saponin, and thiomersal. the vaccines were administered as follows: doses of ml each were given to each animal (pregnant females) days apart. vaccine a was injected to the animals intranasally (i.n.), whereas vaccine b was injected intramuscularly (i.m.) into the neck muscle. the cattle received their first dose of vaccine injection in the th month of pregnancy. all animals in the study were from a single dairy herd, located in central italy and owned and run by a local veterinarian. according to the farm records, no vaccine against bohv- had been used before and no recent history of respiratory disease was registered. eighteen pregnant friesian cattle of approximately - years of age and devoid of na and elisa antibodies against glycoprotein e (ge-elisa) of bohv- were selected. the animals had previously had two pregnancies. the status of the animals was confirmed throughout the experimental period based on the absence of clinical symptoms and serological and virological investigation against ibr. the animals were fed twice per day with unifeed mixtures and water ad libitum. moreover, after calving, milking was performed three times per day. the maintenance and experimental protocols were established according to the european legislation on the protection of animals used for scientific purpose [ ] . the experiments were carried out with the approval of the italian ministry of health under the authorisation number / -pr. the number of animals in each group was determined through the sampling procedure envisaged for an experimental clinical study that was used to compare the proportions in terms of superiority. a percentage of was considered for the proportion of the appearance of the study event in the control group and a percentage of was used as the proportion of the appearance of the study event in the experimental group, with an alpha error of % and a study power of %. the cattle were divided into three groups with six pregnant animals each. the first and second group were injected with vaccine a and b, respectively (table ) , whereas the third group served as an unvaccinated control group. the animals in each group were housed in separate pens on the farm. after calving, the number of animals in each cattle group increased by six newborn calves, and these were fed with l of colostrum or milk administered twice per day throughout the experimental period. subsequently, days post-birth, the newborn calves were fed with concentrated feed and hay in addition to milk from approximately days after birth. data on the following possible adverse reactions after vaccination were obtained from the local veterinarian: (i) localised swelling at the injection site; (ii) slight transient slight increase in body temperature; (iii) anaphylactic shock; (iv) abortion; (v) stillbirth; or (vi) birth of weak, unthrifty calves. in the case of abortion, the foetus and placenta with serum samples were submitted for laboratory investigations. clinical conditions were monitored throughout the experiment. on the day of vaccination (time ), day , and day post-vaccination days (pvds), serum samples were collected from the cattle and tested for the presence of bohv- ge-elisa antibodies and nas. in addition, serum samples were taken from all animals on , , , , , , , , , , and post-calving days (pcds) to assess bohv- antibodies by gb-elisa, ge-elisa, and virus neutralisation test (vnt). simultaneously, colostrum or milk samples were collected from lactating cattle to test for gb-elisa, ge-elisa, or indirect elisa of bohv- . blood samples of approximately ml were collected from the coccygeal or jugular vein of pregnant cattle and calves, respectively. for each animal, disposable needles and vacutainer tubes were used. subsequently, the samples were centrifuged at × g for min at • c to extract the serum. in addition, colostrum and milk samples were uniformly collected from the teats of each animal to obtain ml of each using conical tubes. later, they were centrifuged at × g for min at • c to obtain skimmed samples. all samples were transported with refrigeration to the laboratory within h of collection before testing. afterwards, all samples were stored at − • c for further serological studies. two commercial elisa tests (idexx ibr ge ab test, maine, usa; idexx ibr gb x ab, maine, usa) were used in parallel to examine the collected sera, colostrum, or milk samples. in addition, indirect elisa (idexx bhv bulk milk ab, maine, usa) was used only for colostrum and milk samples. the protocols described by the kit manufacturer were followed and the results were also expressed according to the instructions of the manufacturer. the microplates were read using an automated plate reader and the data were analysed using the magellan software (tecan ag, switzerland). the serum samples were tested using the protocol described by the oie manual of diagnostic tests and vaccines for terrestrial animals [ ] . briefly, µl of undiluted serum samples and two-fold dilutions of each were mixed with µl of tcid of bohv- (los angeles strain / ) in -well microtitre plates. the samples were incubated at • c for h and then , madin-darby bovine kidney cells in µl were added to each well. the cells were provided by biobanking of veterinary resources (bvr, brescia, italy) and identified with the code bs cl . after days of incubation at • c, the plates were read using the inverted tissue culture microscope to determine cytopathic effects. neutralisation titres were expressed as the highest dilution inhibiting cytopathology. overall, animals were used in this study, which included the pregnant cattle and their newborn calves. the titres of antibodies were measured on a logarithmic scale with base . means of the titres were calculated for each animal group and for all sampling times. the nonparametric wilcoxon mann-whitney test was used to evaluate the presence of any statistically significant differences in immunity induced by vaccination between the two ge-deleted marker vaccines and the unvaccinated controls. the differences between group a and group b with respect to the control group at each sampling time were studied considering a significance level at p-value ≤ . . all statistical analyses were performed using stata software v. . (statacorp lcc, texas, usa). after immunisation, no clinical signs or adverse reactions were observed in any of the pregnant cattle or animals immunised i.n. with vaccine a or i.m. with vaccine b. moreover, throughout the experimental period, no clinical signs of ibr infection were seen in the calves born from cattle immunised with vaccine a or b, except for two calves. these two animals, born to vaccine a-immunised cattle showed mono-lateral discharge at two months of age, and, following a nasal swab for virologic and bacteriologic investigations, were found to be infected with only one staphylococcus spp. consequently, they were treated with antibiotics (ceftiofur hydrochloride). after days post-vaccination, all pregnant cattle had nas to bohv- at a mean na titre of . log . titres in cattle vaccinated with both vaccine a and vaccine b showed a significant difference compared to the control (p = . ); the mean titre was . log . this value was increased to . log (p = . ; vaccine a) and . log (p = . ; vaccine b) at pvd . no seroconversion was detected in the unvaccinated controls (table ) . after days post-calving, the mean na titre of cattle immunised with vaccine a did not decrease compared to that on the day of calving ( . log ; p = . ). however, pcd onwards, the na titres started declining and continued to do so till pcd (antibody titres reached . log ; p = . ), after which it remained constant until pcd (table ) . conversely, after days post-calving, the mean na titres of cattle immunised with vaccine b decreased to . log (p = . ) and, then, remained constant up to pcd . subsequently, they decreased up to pcd to an antibody titre of . log (p = . ). in addition, after vaccination, all pregnant cattle remained seropositive for gb and seronegative for ge. conversely, no seroconversion was detected in the unvaccinated controls (table ) . on the day of calving (approximately h after birth), calves born to vaccine a-immunised cattle had nas with a mean titre of . log (p = . ), which remained constant up to pcd . this titre decreased to . log (p = . ) on pcd . subsequently, it declined up to pcd , reaching . log (p = . ). conversely, on the day of calving, the calves of cattle immunised with vaccine b had nas titre of . log (p = . ), which persisted till pcd . it, then, started declining up to pcd , when antibody titres reached . log (p = . ). after calving, all calves remained seropositive for bohv- gb and seronegative for bohv- ge. in contrast, no seroconversion was detected in the control group (table ). the colostrum and milk samples collected from cattle immunised with vaccine a or b were seronegative for ge and seropositive for gb or indirect elisa antibodies throughout the experimental period. in contrast, the colostrum or milk samples collected from unvaccinated cattle were seronegative for gb, ge, and indirect elisa antibodies (table ). currently, in european countries, the diva strategy is considered to be one of the first-line interventions in bohv- eradication programs in areas with a high prevalence of the disease [ , ] . in italy, a national surveillance plan for ibr is only active for autochthonous cattle breeds and recommends the use of marker vaccines to decrease ibr seroprevalence [ ] . it is widely known that marker vaccines induce a marked humoral and cell-mediated immune response [ , ] ; however, only little information is available regarding passive immunity induced by these vaccines. previous studies have suggested that passive immunity would be induced in the calves after maternal vaccination. however, this study is important to provide this. we performed several experiments using two ge-deleted marker vaccines, administered via an intranasal (vaccine a) or intramuscular (vaccine b) route. both inactivated ge-deleted marker vaccines did not induce any clinical signs or adverse reactions. such effects, especially abortion and anaphylactic shock, have been reported when modified-live attenuated vaccines are administered to pregnant cattle in the pre-partum period [ , ] . the results of this study, in accordance with those reported previously [ , ] , suggested no risk of adverse reactions following the administration of either inactivated ge-deleted marker vaccine. in addition, the outcomes of the present study are in accordance with those of other studies [ ] , showing that the vaccination of pregnant cattle can prevent ibr-induced abortion. it is currently known [ , ] that other herpesviruses are involved in cattle abortion, but little information is available on the ability of vaccines prepared against bohv- to protect against abortion induced by bovine gammaherpesvirus (bohv- ) and bovine alphaherpesvirus (bohv- ). the response to vaccination was determined by the assessment of na titres against bohv- or by ge-elisa of serum bohv- in pregnant cattle. results indicated that na titres against bohv- were increased at pvd , compared to control levels. significantly (p = . ; p = . ) elevated titres were also observed at pvd . these findings are in agreement with those of other studies in which an increased colostral antibody production against bohv- and various pathogens were detected after the vaccination of pregnant cattle during the pre-partum period [ , , , ] . in contrast, in a study conducted by lemaire et al., a low level of antibodies against bohv- was observed in two cows after two vaccinations with inactivated ge-deleted marker vaccines [ ] . moreover, in calves, a decline in maternal antibodies against bohv- and bovine viral diarrhea virus was shown at or days of age, respectively [ , ] . similar results were also seen in another study, in which a vaccine containing rotavirus, coronavirus, and escherichia coli was used to immunise pregnant cattle and led to high titres of specific antibodies in their colostrum and milk [ ] . further, agianniotaki et al. demonstrated the presence of nas against lumpy skin disease virus until days post-calving [ ] . in the present study, the pregnant cattle showed negative results for ge-elisa during the vaccination period. these results are in agreement with those of reports demonstrating that serum samples collected from animals immunised with ge-deleted marker vaccines were also negative for ge-elisa [ , , ] . moreover, this result showed that, during the experimental period, the field virus did not circulate in the experimental groups. cattle naturally or experimentally exposed to bohv- can be positive for ge-elisa antibodies [ ] . the experiments performed herein showed that pregnant cattle immunised with the two ge-deleted marker vaccines could transfer passive immunity to calves. in fact, on the day of calving, both groups injected with vaccine a or b showed high na serum levels against bohv- . after birth, calves are born in an agammaglobulinemia state and need colostrum with a high level of immunoglobulins (igs). in the first h, these igs can penetrate the bloodstream by binding to the fc receptors located in the intestinal brush border [ , ] . produced by b cells or plasma cells in the blood of lactating cows [ ] , these igs are responsible for the early protection of newborns against different infections. furthermore, colostrum contains leukocytes and several antimicrobial proteins, such as complement c , lactoferrin, lactoperoxidase, and lysozyme. these leukocytes, together with maternal igs, are involved in conferring orogastric protection [ ] ; specifically, they can enter the circulation through intestinal adsorption to promote neonatal cellular immunity [ ] . in our research, on the day of calving, the cattle immunised with vaccines showed a good level of nas, which decreased progressively up to pcd . in addition, calves in both the groups showed na titres similar to those of their mothers approximatively h after birth. subsequently, the antibody titres decreased until the end of the experiment, when they reached negative values. in both of the vaccinated groups, the difference in antibody titres between cattle and newborn calves could be due to the passage of igs from mothers to newborns through the colostrum. these results are similar to those obtained by cervenak et al., showing that when the time of calving approaches, igg massively decreases in maternal serum samples as it binds to mammary epithelial cells. then, igg is released with colostrum and milk during lactation. calves undergo so-called gut closure by approximately - h after birth and the adsorbed colostral igg (igg , igg ) levels fall below % [ ] . after pcd , the antibody titres in milk gradually decreased up to pcd . the data obtained in this study supported the results of mechor et al., who reported a correlation in serum na titres between cattle and newborn calves [ ] . in addition, the antibody titres observed in both the groups of newborn calves, born to the cattle immunised with vaccine a or b ( . log and . log , respectively), on pcd were lower than those required to protect them against infection by bohv- . several studies have shown that nas higher than a value of . log can protect calves against experimental infection [ , , ] . moreover, it has been shown that these antibodies appear in nasal secretions from calves as early as the first day after the ingestion of colostrum [ ] . the nas secreted in the respiratory tract mucosa, primarily of the igg class, persist for to days after birth and serum antibodies can be detected until calves reach several months of age [ ] . these results are similar to those of other studies [ ] showing the persistence of nas up to pcd when using non-marker vaccines. this study was carried out based on current field conditions with different variables, including the geographical position of the farm, weather, nutrition, and health status of the herd. generally, dairy cattle are subjected to stress that can negatively affect their serological responses to vaccination. in this study, there was no evidence of stressors as both intramuscularly-and intranasally-vaccinated animals produced humoral immunity with high antibody titres. conversely, other authors, using modified-live vaccines, found that the aforementioned factors can negatively affect the antibody response after vaccination [ ] . in this study, no challenge with virulent bohv- virus was given because this research aimed to evaluate the ability of the two ge-deleted marker vaccines to induce passive immunity. moreover, this study was intended to form the basis of additional research evaluating the effect of different vaccines against ibr. thus, future studies will be conducted to assess whether passive immunity in calves can protect them against experimental infection using virulent bohv- virus. overall, the two inactivated ge-deleted marker vaccines against bohv- were shown to (i) be innocuous for pregnant cattle, (ii) effectively transfer passive immunity from dams to calves up to pcd , and (iii) be suitable for immunisation in ibr eradication programs. further studies will be conducted to evaluate if passive immunity induced by these vaccines can protect calves from challenge with a virulent (wt) strain of bohv- . author contributions: conceptualization, s.p., c.r., and c.i.; stata software v. . , e.s.; methodology and investigations, e.r., c.p., p.g. and g.v.; data curation, c.r. and c.p.; manuscript writing, review, and editing, s.p. and g.m.d.m. all authors have read and agreed to the published version of the manuscript. bhv- infection in cattle: an update bovine herpes virus infections in cattle bovine herpesvirus- : evaluation of genetic diversity of subtypes derived from fields strains of varied clinical syndromes and their relationship to vaccine strains the use of marker vaccines in eradication of herpesviruses vaccination of calves against bovine herpesvirus- : assessment of the protective value of eight vaccines epidemiology and control of bovine herpesvirus infection in europe cell-mediated immune responses induced by bhv- : rational vaccine design surveillance of infectious bovine rhinotracheitis in marker-vaccinated dairy herds: application of a recombinant ge elisa on bulk milk samples antibody response to bovine alphaherpesvirus (bohv- ) in passively immunized calves a study on latency in calves by five vaccines against bovine herpes virus- infection enhancement of the immune response and virological protection of calves against bovine herpesvirus type with an inactivated ge-deleted vaccine effect of maternally antibody upon vaccination with infectious bovine rhinotracheitis and bovine virus diarrhea vaccines protection of newborn calves against fatal multisystemic infectious bovine rhinotracheitis by feeding colostrums from vaccinated cows maternally derived humoral immunity to bovine viral diarrhea virus (bvdv) a, bvdv b, bvdv , bovine herpes virus- , parainfluenza- virus bovine respiratory syncyntial virus, mannheimia haemolytica and pasteurella multocida in beef calves, antibody decline by half-life studies and effect on response to vaccination predicted ages of dairy calves when colostrum-derived bovine viral diarrhea virus antibodies would no longer offer protection against diseases or interfere with vaccination effect of age at time of vaccination on antibody titres and feedlot performance in beef calves on the protection of the animals used for scientific purposes manual of diagnostic tests and vaccines for terrestial animals national surveillance plan for infectious bovine rhinotracheitis (ibr) in autochthonous italian cattle breeds: results of the first year of activity inactivated bovine herpesvirus marker vaccines are more efficacious in reducing virus excretion after reactivation than a live marker vaccine anti-bovine herpesvirus and anti-bovine viral diarrhea virus antibody responses in pregnant holstein dairy cattle following administration of a multivalent killed virus vaccine safety of a modified-live combination vaccine against respiratory and reproductive diseases in pregnant cows bovine herpesvirus modified live virus vaccines for cattle reproduction: balancing protection with undesired effects prevention of abortion in cattle following vaccination against bovine herpesvirus : a meta-analysis partial protection induced by a bhv- recombinant vaccine against challenge with bhv- a recombinant bovine herpesvirus defective in thymidine kinase and glycoprotein e is immunogenic for calves and confers protection upon homologous challenge and bohv- challenge stimulation and analysis of the immune response in calves from vaccinated pregnant cows antibody response to glycoprotein e after bovine herpesvirus type infection in passively immunized, glycoprotein e-negative calves colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and escherichia coli f (k ) colostrum transfer of neutralizing antibodies against lumpy skin disease virus from vaccinated cows to their calves passive immunization, an old idea revisited: basic principles and application to modern animal production system the neonatal fc receptor plays a crucial role in the metabolism of igg in livestock animals passive immunity in newborn calves cytotoxicity of human peripheral blood and colostral leukocytes against shigella species humoral and cellular factors of maternal immunity in swine evaluation of safety and efficacy of dna vaccines against bovine herpesvirus- (bohv- ) in calves demonstration of colostral antibodies in the nasal secretion of calves and their protective effect against infection evaluation of safety and efficacy of an intranasal vaccine containing a temperature-sensitive strain of infectious bovine rhinotracheitis virus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to professor fernando a. osorio, school of veterinary medicine & biomedical sciences, university of nebraska-lincoln (usa) for providing a critical review of this manuscript and to professor gigliola canepa, university of milan (i), for revising the language of the manuscript. the authors also thank dr. roberto sabato for kindly taking part in sample collection. the authors declare no conflict of interest. key: cord- -xqkre z authors: muller, janine d.; wilkins, michelle; foord, adam j.; dolezal, olan; yu, meng; heine, hans g.; wang, lin-fa title: improvement of a recombinant antibody-based serological assay for foot-and-mouth disease virus date: - - journal: journal of immunological methods doi: . /j.jim. . . sha: doc_id: cord_uid: xqkre z abstract differentiating foot-and-mouth disease virus (fmdv) antibodies generated during a natural infection from those due to vaccination (diva) is crucial for proving freedom from disease after an outbreak and allowing resumption of trade in livestock products. the world organisation for animal health (oie) recommends that fmdv vaccines are composed of inactivated virus that has been purified to remove non-structural viral proteins. such purified vaccines primarily induce antibodies to viral structural proteins, whereas replicating virus stimulates host antibodies specific for both structural and non-structural proteins. the current preferred fmdv diva test is a competitive elisa (c-elisa) designed to detect antibodies to the non-structural protein abc. previously, we described the development of an fmdv diva test based entirely on recombinant proteins (the recombinant detecting antibody and the abc coating antigen) produced in escherichia coli. in this study, we have determined the precise binding site of the recombinant detecting antibody to a conserved sequence within the b region of the abc protein, replaced the original e-tag of the detecting antibody with two in-house tags and engineered a direct antibody–reporting enzyme (alkaline phosphatase) fusion protein. these modifications have further improved the diva test, providing great potential for large scale production and uptake due to its simplicity, reproducibility and low cost. foot-and-mouth disease (fmd) remains the greatest threat to livestock industries worldwide. for a country to be considered free from the fmd virus (fmdv), different diagnostic approaches are required according to whether vaccine has been used and if so whether it was used in response to an outbreak of disease or as part of routine prophylaxis. the ability to differentiate between these different immune states has been an area of much research. the current preferred test to differentiate infected from vaccinated (diva) animals is a competition elisa (c-elisa) designed to detect antibodies to the non-structural protein abc as an indicator of infection (sorensen et al., ; clavijo et al., ; foord et al., ) . most approved fmdv vaccines are composed of mainly viral structural proteins and hence primarily induce antibodies to these proteins, whereas replicating virus stimulates host antibodies against both structural and non-structural proteins. although non-structural proteins can in theory contaminate the vaccine preparation, it is expected that fmdv vaccines produced by most current commercial manufacturers will not induce significant antibody responses to non-structural proteins. provided these diva tests are used under controlled circumstances and knowledge of vaccine quality understood, they can be of tremendous benefit for fmdv diagnosis and surveillance. previous fmdv diva tests relied on polyclonal or hybridoma-derived monoclonal antibody reagents which are expensive, difficult to produce and maintain. to improve the diva c-elisa, we produced the two critical reagents, the detecting antibody and coating antigen, in e. coli, making them safe and more economical to produce without the requirement for infectious virus or animals (foord et al., ) . however, the recombinant antibody-based test still relies on two additional commercial antibodies (the epitope-tag-specific antibody and an enzyme conjugated antibody), which makes it expensive and difficult for quality assurance due to the requirement for multiple antibodies. in the current study, we have further improved this recombinant protein-based diva test by: ) determination of the precise binding site and binding affinity of the detecting antibodies which can facilitate fine tuning of assay sensitivity and specificity if required; ) replacement of the commercial etag with two in-house epitope tags allowing further reduction of production costs; ) development of an antibody-reporter fusion protein for a simple assay requiring fewer steps. the non-structural protein abc previously described by foord et al. ( ) was used as the coating antigen for the c-elisa. production of the two detecting antibodies crab-fm and fm with different epitope tags is described in section . . monoclonal antibody f g , specific to the sars coronavirus spike protein (berry et al., ) was kindly provided by dr. j. berry and mab - d against the e protein of classical swine fever virus was produced in-house . the following panels of experimental sera were used in the evaluation of the c-elisa. the sera from fmdv-infected and fmdv-vaccinated cattle representing o, a, asia- and c serotypes and the associated pre-treatment sera were kindly provided by dr. alan r. samuel, iah, pirbright, uk. infected pig sera were generated against serotype a and were kindly provided by dr. j. lubroth, plum island animal disease center, new york, usa. naïve pig sera were obtained in-house at aahl. the vaccinated pig sera were serotype o and were kindly provided by dr. dong manh hoa, regional animal health center ho chi minh city, vietnam. two sera from sheep infected with o-ukg were kindly provided by dr. bob armstrong, iah, pirbright, uk. serum from a sheep that had been experimentally infected sequentially with multiple serotypes; o -tunisia, a , a , o -bfs and c -detmold was kindly provided by dr. aldo dekker, central institute for animal disease control, lelystad, the netherlands. naïve sheep sera and sera from sheep vaccinated with serotypes o, a and asia- were kindly provided by dr. jef hammond, aahl. primers were designed to amplify the three fragments of the fmdv b coding region denoted b- , b- and b- (table ) and engineered to include asc i and not i unique restriction sites. small gene fragments were amplified by pcr ( °c min, cycles [ °c s; °c s; °c min], °c min, °c) using the original pgd - b plasmid (foord et al., ) as the template. purified pcr products were digested with the restriction enzymes asc i and not i and cloned in-frame with the gst-tag in the pgd vector . protein expression was performed as previously described for abc with e. coli mc cells (foord et al., ) . pcr fragments containing coding regions for the sars (yu et al., ) and yyep epitopes were created by pcr assembly of overlapping primers (see table ) under the following conditions: °c min, cycles [ °c s; °c s; °c s], °c min, °c. pcr products were purified and digested with the restriction enzymes not i and eco ri to facilitate cloning in-frame with the antibody gene sequence in the modified pcantab-link vector (sapats et al., ) . the ph.d.- phage display peptide library (new england biolabs) was utilized for epitope mapping of the fm and fm crabs, following the manufacturer's instructions. briefly, µl (∼ . × phage particles) of phage peptide library was added to µl of fm or fm scfv solution and incubated at room temperature for min. for affinity binding of fm or fm to protein g, µl of anti-e-tag monoclonal antibody (pharmacia biotech) was added to the phage/scfv solution and incubation continued for a further h. protein g-dynabeads (invitrogen) were prepared by washing four times in pbst and blocking in . m nahco (ph . ), mg/ml bsa for h. the phage/scfv/monoclonal antibody complex was added to the blocked dynabeads and incubated for h at room temperature. the bead complex was washed ten times in pbst and bound phage eluted using ml . m glycine-hcl (ph . ), mg/ml bsa, followed by neutralization with m tris-hcl (ph . ). phage titrations were performed to give inputs of to × plaque forming units for the following three rounds of binding and recovery. single plaques from the third round of bio-panning were isolated, ssdna purified and the -mer peptide sequence determined by dna sequencing. . . affinity determination using biosensor . . . cloning, expression and purification of crab scfvs large scale protein expression of crab-fm and fm was performed using a pgc e. coli expression vector (coia et al., ) . specifically, the pcantab-fm and fm clones were digested with nco i and not i and the resulting scfv gene fragments cloned into a likewise digested pgc vector that contained c-terminal flag and his tags (robert et al., ). the resulting pgc-fm / -flaghis constructs were expressed according to previously described methodology (dolezal et al., ) . proteins were isolated from the periplasmic space (skerra and pluckthun, ) . the subsequent supernatant was filtered through a . µm filter and the fm and fm scfv proteins purified by a two-step automated procedure using an aktaxpress purification system (ge healthcare). in brief, the scfv proteins were first purified using a ni + -nta histrap ff ( ml) affinity column followed by size exclusion chromatography on a hiload / superdex column equilibrated in pbs. purified proteins were quantified by their absorbance at nm using the theoretical extinction coefficient calculated from the sequence (gill and von hippel, ). a biacore t biosensor instrument (papalia et al., ) was used to measure the kinetic binding interactions of abc antigen with immobilized fm and fm . the scfv proteins were immobilized at °c in running buffer (hbs-ep + buffer [ mm hepes, mm nacl, mm edta and . % surfactant p , ph . ]) in separate flow-cells on a cm (s series) sensor chip using standard amine-coupling (johnsson et al., ) . the scfv proteins were diluted to μg/ml in mm sodium acetate ph . and injected for min ( μl/ min) over the activated surface. final immobilized protein levels achieved were ru ( ru = pg protein/mm ) for fm in flow-cell and ru for fm in flow-cell . for all subsequent binding experiments flow-cell was used as a "mock surface" for referencing purposes. the abc antigen in . % sds was subjected to size exclusion chromatography on a superdex hr / column (ge healthcare) in pbs (ph . ) calibrated with bio-rad gel filtration standard proteins. protein fractions corresponding to monomeric species of abc were collected and concentrated to . mg/ml ( . µm). binding experiments were performed in triplicate in hbs-ep + running buffer at °c with a series of antigen concentrations, diluted two-fold from nm to nm, serially injected at a constant flow rate of µl/min over the four flow-cells within the biacore t instrument. antigen association and dissociation phases were each monitored for s and s, respectively. at least one buffer blank injection was included for the purpose of doublereferencing (myszka, ) . the fm and fm scfv protein surfaces were regenerated with a s injection of mm phosphoric acid at µl/min. scrubber software (version . c; biologic software, campbell, australia) was utilized to process all biacore t generated sensorgrams and to determine association rate (k a ) and dissociation rate (k d ) constants by globally fitting to the processed data sets to a : langmuir binding model. the equilibrium dissociation constant (k d ) was calculated from the quotient of k d /k a . plasmids containing a gene encoding a his tag and an e. coli ap gene, designated pdap and pdap /s, were sourced from dr. randolf kerschbaumer (kerschbaumer et al., (kerschbaumer et al., , . both pdap and pdap /s were compatible with the pcantab-series of vectors, designed for the simple construction and production of crabs. pdap , pdap /s and the plasmid encoding crab-fm were digested with the restriction enzymes sfi i and not i, to enable the antibodyencoding gene to be cloned in-frame with both the his tag and the ap gene. all constructs were transformed into electrocompetent mc e. coli cells. positive clones were confirmed by pcr ( °c min, cycles [ °c s; °c s; °c s], °c min, °c) using the chicken heavychain primer and a primer specific for the ap gene in the vector (table , sequencing primers) and direct sequencing of the pcr product. the resulting plasmids, pdap - and pdap /s- , were then transformed into electrocompetent e. coli tg cells for protein expression. a single transformant was inoculated into lb broth containing µg/ml of ampicillin and % glucose and incubated at °c overnight with shaking. the overnight culture was diluted : in m zb/ampicillin broth (kerschbaumer et al., ) and grown at °c with shaking, until the optical density of the culture was . as measured using an lkb biochrom ultrospec (λ = nm). cells were harvested by centrifugation at ×g for min at °c, the supernatant was discarded and the bacterial pellet resuspended in fresh m zb-gy/ampicillin media (kerschbaumer et al., ) , and further incubated at °c for h to achieve maximal expression of crab proteins. the cultures were centrifuged at , ×g for min at °c, the pellet was discarded and the supernatant was stored at °c until further analysis. equal amounts of each b protein, approximately μg/ lane, were loaded onto a % sds-page and electrophoresed. the proteins were transferred to a nitrocellulose membrane, blocked and probed with either goat anti-gst-hrp ( : ); crab-fm -e-tag ( : ), crab-fm -e-tag ( : ) or the control crab- -t-tag ( : ) (sapats et al., ) . each membrane was washed three times for min in tbst and all bound antibodies, with the exception of the anti-gst-hrp control, had the addition of a secondary mouse anti-e-tag antibody ( : ), followed by goat anti-mouse-hrp ( : ). the bound antibodies were detected using a hrp substrate ( ml pbst containing tablet [ mg] -chloro- napthol [sigma] dissolved in ml methanol and µl h o % [w/v] just before use). color development was stopped after min by washing the membrane in water. crabs with different epitope tags were compared in a c-elisa format. the c-elisa was performed as previously described in foord et al. ( ) with the exception of % sterile filtered bsa (sigma, usa) in pbst used as blocking buffer. the % bsa/pbst was used in place of the skim milk powder/pbst block because it had been shown in previous studies to interfere with binding of the yyep-mab (m. yu, unpublished results). the optimal working concentration of each antibody was determined by serial titration across the coating antigen abc ( ng/well) and were as follows; crab-fm -e-tag ( : final : ), crab-fm -sars ( : final : ), crab-fm -yyep ( : final : ); crab-fm -e-tag ( : final : ), crab-fm -sars ( : final : ), crab-fm -yyep ( : final : ). each crab was detected using either the e-tag-specific antibody (pharmacia; : ), the sars-specific antibody f g ( : ) or the yyep-specific antibody - d ( : ). all of these tag-specific mabs were detected using goat antimouse-hrp (jackson immunoresearch laboratories inc.; : ). for rapid and simple determination of enzyme activity of the scfv-ap fusion proteins, to a microtitre plate well was added µl of water, µl of crab-fm -ap supernatant and µl of ap substrate ( mm tris ph containing mm nacl, mm mgcl and mg/ml p-nitrophenyl phosphate [sigma]). the plate was placed at room temperature in the dark for h and the enzyme activity was determined by measuring the change in optical density of the solution at a wavelength of nm on an automated elisa plate reader (thermo multiskan ascent, finland). the c-elisa was performed essentially as outlined above with the following modifications: the incubation of test serum with the recombinant crab-fm -ap/s produced in . above ( : : final : ) was performed at room temperature for min and then developed by incubation with ap substrate for h at room temperature in the dark as described above. the fmdv b region contains three repetitive sequences as shown in fig. a . to determine the exact binding site(s) utilized by the crabs, each b peptide was cloned, expressed and tested for reactivity with fm and fm and the nonrelated crab- (specific to ibdv) in a western blot. the results shown in fig. b indicated that both fm and fm bound the full-length b, the b- and b- peptides, but not the b- peptide. the non-related crab- exhibited no reactivity to any of these fusion proteins (data not shown). the relative mobility and quantity of each fusion protein were determined using the goat anti-gst hrp antibody. amino acid sequence alignment of the three repeated regions within the b protein indicates a high degree of similarity between b- and b- . in comparison, the b- region is less similar. this observation is consistent with the western blot results obtained (fig. b) . taken together, the data suggests that there are two independent binding sites in the abc protein, which are recognised by both fm and fm . after three rounds of affinity selection as detailed in the materials and methods, specific reactivity of the selected phage clones with the crabs was confirmed by elisa (data not shown). of those showing a positive elisa reactivity, twelve clones were randomly selected and sequenced to determine their peptide insert sequences. the results obtained (fig. ) revealed several important findings: a) a consensus sequence motif was identified from the peptide sequences selected by both crabs; b) the motifs recognised by both crabs share a high level of similarity all contained a Φ-dple(d) sequence (Φ = aromatic amino acid residue); c) the Φ-ple(d) motif was found in the b- and b- peptides; d) there is a subtle difference between the two motifs recognised by both crabs where the p residue is more conserved in the motif recognised by fm than that of fm ; and e) the le(d) residues are highly conserved in both motifs, but missing from the b- peptide, which may explain the failure of both crabs to react with this peptide. kinetic parameters were determined for abc antigen binding to immobilized crab-fm and fm (table ) . differences were detected mainly in the association rate constant (k a ) whereby abc associated approximately -fold faster with fm than with fm . dissociation rate constants (k d ) were comparable for both crabs although fm dissociated approximately . -fold slower. it is therefore the association rate that contributes most to the overall difference in affinity (k d ) whereby fm exhibits a four times higher affinity for abc in comparison to fm . to enhance the diagnostic application of the crabs, the commercial e-tag was replaced with in-house epitope tags to decrease associated costs with secondary reagents. a c-elisa was used to compare the relative performance of each crab with different epitope tags. results obtained with paired sera of pre-treatment and fmdv-infected cattle, sheep and pigs indicated that the performance of the crabs with in-house tags was comparable to those with the e-tag (fig. ) . performance of the yyep tag was shown to be slightly better than the original e-tag. to further simplify the assay, an antibody-reporter fusion protein using ap as the reporting enzyme was generated. the plasmids pdap and pdap /s contain the wild type ap gene and a mutant gene (ap/s), respectively (kerschbaumer et al., ) . the ap/s has been shown to enhance the specific activity of the ap up to -fold (kerschbaumer et al., ) . because the ap gene is derived from e. coli, fm -yyep was included as a control alongside the fm -ap and fm -ap/s constructs for all studies. western blot analysis demonstrated the presence of the crab-ap fusion protein with the predicted molecular mass of approximately kda (fig. a ). the functionality of the fusion protein was demonstrated by directly measuring ap activity in the crude supernatant as shown in fig. b . background ap activity was negligible as indicated by the control fm -yyep. as expected, the fm -ap/s clone exhibited higher enzyme activity than the fm -ap clone. furthermore, the specific reactivity of the crab component in the fusion was confirmed by elisa, which indicated that the crab-ap proteins reacted with abc, but not with a non-related sars virus antigen containing the same his tag (data not shown). since the ap/s fusion showed higher specific activity, subsequent studies were conducted using fm -ap/s only. after optimization of the one-step c-elisa using different dilutions of the fm -ap/s protein, a diva test was performed with a panel of sera representing naïve, fmdv-infected and fmdv-vaccinated cattle, sheep and pigs (fig. ) . the results demonstrated that the fm -ap/s protein retained the ability to function in the c-elisa format and differentiate between naïve, fmdv-infected and fmdv-vaccinated animals despite the introduction of an enzyme molecule fused at its c-terminus. mapping of epitopes using phage display random peptide library. random -mer aa sequences selected by fm (on the left) or fm (on the right) are aligned for identification of consensus motifs, which are in turn aligned with the predicted matching residues in the b repetitive sequences shown underneath. residues in red indicate conserved random peptide sequences present in the b region whereas residues in blue are those conserved in the peptide sequences, but absent in the b region. of the cattle sera, the six naïve and six fmdv-vaccinated sera demonstrated less than % inhibition. the six sera from fmdvinfected cattle, representing o -manisa, c-oberbayern, asia- india and three different strains of serotype a (a , a iraq and a cruzeiro), all had an inhibition of greater than % with the exception of c-oberbayern with % inhibition. the six sera from naïve and fmdv-vaccinated pigs (serotype o) showed an inhibition of less than % and % respectively, with the sera from fmdv-infected pigs (serotype a) demonstrating a range of inhibition from % to %. for the sheep sera tested, the six naïve and six fmdv-vaccinated sera showed an inhibition of less than % and % respectively and the sera from fmdvinfected sheep showed inhibition of , and %. recombinant antibodies are being used as an alternative to mabs for a range of research applications. chicken recombinant antibodies are not only simple to produce but they are also less cross-reactive when used in combination with mammalian antibodies in immunoassays. our previous fmdv diva c-elisa (foord et al., ) utilized a commercial e-tag which can only be detected using the commercial anti-e-tag antibodies (the e-tag mab is no longer commercially available). in this current study, we determined the epitope sequences recognised by the two closely related crabs and the binding affinities by biosensor. to further improve their application, the crabs were modified to include non-commercial tags and an antibody-enzyme fusion protein. using two independent methods, i.e., expression of truncated peptide sequences and selection of mimotopes from phage display random peptide libraries, the key binding sites/ residues of crab-fm and fm were determined. it was interesting to note that both crabs have two binding sites within the b region, designated b- and b- in this study. although both crabs bound the same site, the data obtained from the phage library suggests a subtle difference in the key residual contacts between the two antibodies. in the Φ-dple (d) motif, crab-fm has a more stringent requirement for the y residue at the Φ position and the presence of the p residue than that of crab-fm . this difference in binding between fm and fm was further demonstrated by absolute affinity measurements using a biosensor. this was most notable with the association rate constant (k a ), which showed an approximate -fold difference between the antibodies, with fm having a faster on-rate. since the variable heavy (v h ) chain complementary determining regions of the two antibody molecules is identical (foord et al., ) , one could conclude that the observed difference in binding affinity and mimotope specificity is entirely dependent on the variable light (v l ) chain. these data would suggest that further v l -randomization may be used as a strategy to improve the assay sensitivity in the future. to improve the viability of the recombinant antibody as a potential commercial reagent, alternative epitope tags were explored. for this application we chose two epitope tags derived from the sars coronavirus spike protein and the classical swine fever virus e protein, which have been developed in our group (m. yu, unpublished results) . although both tags maintained the functionality of the crabs, the yyep tag was more effective in the c-elisa than the sars-tag. for this reason, the in-house produced yyep tag will be used in all future studies. a potential future improvement will be a direct conjugation of hrp with the - d antibody for detection of the yyep epitope tag. although the introduction of the yyep epitope tag had improved the diva c-elisa, the assay strategy remained reliant upon a mab and a conjugate for detection. we moved to a direct antibody-enzyme fusion system to simplify the assay to a single antibody in a one-step assay format. to do this, we employed the ap-fusion vectors developed by kershbaumer's group (kerschbaumer et al., (kerschbaumer et al., , , that contained both the wild type and a mutant ap gene. the mutant gene denoted ap/s contained a serine residue (s) at position instead of the wildtype aspartate residue (d), which has the potential to increase the specific ap activity by fold (kerschbaumer et al., ) . the crab-ap fusion proteins generated in this study table apparent kinetic rate constants and equilibrium binding constants for the interaction of recombinant abc with immobilized fm and fm scfv proteins (± standard deviation; n = ). scfv k a × (m − s − ) k d × − (s − ) k d (nm) crab-fm . ± . . ± . ± crab-fm . ± . . ± . ± fig. . performance comparison of three different epitope tags in c-elisa. results shown are c-elisa readings conducted under identical conditions using fm with the e-tag (green), the yyep-tag (blue) and the sars-tag (red). error bars represent the standard deviation of the mean. maintained both the ap activity and the antibody specificity of the two fusion partner proteins. however, the fm -ap/s was only fold more reactive than the fm -ap. nevertheless, the fm -ap/s represents a very promising reagent for wider application of the one-step fmdv diva test. in conclusion, the current study has made significant progress in our understanding of the two fmdv-specific crabs produced in our previous study. the introduction of the antibody-enzyme fusion protein into the diva test has realized the potential of a one-antibody one-step assay format which will greatly facilitate its production and application in both developed and developing countries. the advantages of the current test platform over other existing tests include simple operation, less assay time, low production cost and high reproducibility. development and characterisation of neutralising monoclonal antibody to the sarscoronavirus development and use of a biotinylated abc recombinant protein in a solid-phase competitive elisa for the detection of antibodies against foot-and-mouth disease virus use of mutator cells as a means for increasing production levels of a recombinant antibody directed against hepatitis b scfv multimers of the anti-neuraminidase antibody nc : shortening of the linker in single-chain fv fragment assembled in vl to vh orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers production and application of recombinant antibodies to foot-and-mouth disease virus non-structural protein abc calculation of protein extinction coefficients from amino acid sequence data immobilization of proteins to a carboxymethyldextran modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors pdap : a vector for construction of alkaline phosphatase fusion-proteins single-chain fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay high-resolution characterization of antibody fragment/antigen interactions using biacore t engineered antibody intervention strategies for alzheimer's disease and related dementias by targeting amyloid and toxic oligomers generation of chicken single chain antibody variable fragments (scfv) that differentiate and neutralize infectious bursal disease virus (ibdv) assembly of a functional immunoglobulin fv fragment in escherichia coli differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking elisa based on baculovirus expressed fmdv abc antigen and a abc monoclonal antibody btag: a novel six-residue epitope tag for surveillance and purification of recombinant proteins fine mapping of c-terminal linear epitope highly conserved among the major envelope glycoprotein e (gp to gp ) of different pestiviruses determination and application of immunodominant regions of sars coronavirus spike and nucleocapsid proteins recognized by sera from different animal species this research was supported through funds from the australian biosecurity cooperative research centre for emerging infectious diseases (project . re) and meat livestock australia. j.d. muller was a recipient of phd scholarships from mla and ab-crc. key: cord- -eiobmxp authors: zhao, shan; li, wentao; schuurman, nancy; van kuppeveld, frank; bosch, berend-jan; egberink, herman title: serological screening for coronavirus infections in cats date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eiobmxp coronaviruses (covs) are widespread among mammals and birds and known for their potential for cross-species transmission. in cats, infections with feline coronaviruses (fcovs) are common. several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. however, whether cats might become naturally infected with covs of other species is unknown. we analyzed coronavirus infections in cats by serological monitoring. in total cat serum samples and fcov type or type -specific antisera were screened for the presence of antibodies against the s receptor binding subunit of the cov spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. seventy-eight sera were positive for antibodies that recognized one or more coronavirus s s whereas serum exclusively reacted with human coronavirus e (hcov- e) and two sera exclusively reacted with porcine delta coronavirus (pdcov). we observed antigenic cross-reactivity between s s of type and type fcovs, and between fcov type and porcine epidemic diarrhea virus (pedv). domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the cd domains of s . the cross-reactivity of fcov type and pedv was also observed at the level of virus neutralization. to conclude, we provide the first evidence of antigenic cross-reactivity among s proteins of coronaviruses, which should be considered in the development of serological diagnoses. in addition, the potential role of cats in cross-species transmission of coronaviruses cannot be excluded. coronaviruses (covs) are enveloped viruses with a positive-stranded rna genome and classified into four genera (alpha-, beta-, gamma-and deltacoronavirus) within the subfamily orthocoronavirinae in the family coronaviridae of the order nidovirales. covs are found in a variety of mammals and birds, in which they can cause respiratory, enteric and systemic infections [ ] [ ] [ ] . additionally, covs have proven ability for cross-species transmission, exemplified by the emergence of severe acute respiratory syndrome (sars) coronavirus in / , and of the middle-east respiratory syndrome (mers) coronavirus in [ ] . both viruses belong to the betacoronavirus genus and have an animal origin. sars coronavirus crossed over from bats via intermediate hosts to humans, became human-adapted and quickly spread worldwide before its containment. mers coronavirus recurrently enters the human population via its dromedary camel reservoir host, with limited, non-sustained human-to-human transmission particularly in healthcare settings [ ] [ ] [ ] . apart from sars-and mers-cov, all four globally endemic human covs (hcov-oc , hcov-nl , hcov- e and hcov-hku ) originate viruses , , of from animals [ ] [ ] [ ] [ ] . in addition, cross-species transmission potential of covs is also illustrated by the occurrence of chimeric coronaviruses that resulted from recombination events between feline covs (fcov) and canine covs (ccov) [ , ] . in order to get insight into the frequency of interspecies transmission of coronaviruses within and between animal and human populations and the risk of subsequent development of a pandemic, it is useful to screen for coronavirus infections in animal species; especially those that are in close contact with humans. serological assays that can detect virus-specific antibody responses against infection play an important role in these epidemiological studies [ ] . cats live in close contact with humans and often roam around freely in the environment. hence cats are an interesting species to study for infections with coronaviruses. infections with feline coronaviruses (fcovs) are recognized and widespread [ , ] . fcovs are classified into two types, type and type , based on the genetic and antigenic difference of their spike (s) protein [ ] . in the field, the majority of fcov infections are caused by fcov type , while fcov type , derived from recombination events of type fcovs and ccovs obtaining the s gene and some flanking regions of ccovs, is less prevalent [ , ] . depending on the virulence of the fcov strain and the immune response of the cat, the clinical presentation can range from apparently asymptomatic, through diarrhea, to full-blown feline infectious peritonitis [ ] . fcovs are members of the genus alphacoronavirus, to which also hcov- e, porcine transmissible gastroenteritis virus (tgev), and ccov belong. the latter three viruses and fcov type have been proven to use feline aminopeptidase n (fapn) as a functional receptor in vitro [ ] . the receptor for type fcov has still not been identified [ ] . notably, previous studies have shown that hcov- e and ccov could infect cats after experimental inoculation, causing an asymptomatic infection [ , ] . thus, cats might potentially become naturally infected with covs of other species which may lead to virus-host adaptation e.g., mutation or recombination, resulting in emergence of novel coronaviruses and potentially new diseases [ , ] . the extent to which infections with covs of other species occur in the field, has not been explored in previous epidemiological studies of cov infections in cats [ , [ ] [ ] [ ] . being the main envelope protein of coronaviruses, the spike (s) protein mediates cell attachment and membrane fusion to allow viral entry. s functions as the main determinant of cell-, organ-and host-tropism. additionally, it is also the major target of neutralizing antibodies. spike comprises two functionally interdependent subunits, s and s , with s responsible for receptor binding and s for membrane fusion [ , ] . the s subunit is the least conserved and the most variable immunogenic antigen between coronavirus species [ ] . therefore, the s subunit is well suited as an antigen to screen for coronavirus type specific antibodies [ ] . in this study, covs infection in cats were detected through profiling antibody presence in serum samples from cats. recombinant cov spike s subunits of different animal and human covs were expressed in a mammalian expression system and used for screening of cat sera for the presence of antibodies against the respective proteins. positive samples were also tested by virus neutralization assays to support the specificity of the reaction [ ] [ ] [ ] . this investigation intends to extend our knowledge of cov epidemiology, potential reservoirs, and cross-species transmission. specific fcov type and fcov type sera were obtained from specific pathogen free (spf) cats previously infected with strain uu or rm and fipv- respectively [ , ] . in addition, for the serological survey, feline sera were retrieved from the serum bank in our lab. these had all been collected from cats in the netherlands. most of the samples (> %) were from a study on antibody titer testing for feline panleukopenia virus. the other samples were send to our lab for fip or felv-fiv diagnostics. sera of uninfected spf cats were included as negative controls. all samples were stored at − • c until analysis. african green monkey kidney cells (vero-ccl ), human hepatoma cells (huh ), pig kidney epithelial cells (llc-pk ), human embryonic kidney cells stably expressing the sv large t antigen (hek- t) were maintained in dulbecco modified eagle medium (dmem, lonza, basel, switzerland) supplemented with % fetal bovine serum (fbs, bodinco, alkmaar, the netherlands). virus strains used in this study have been described previously [ ] [ ] [ ] . briefly, recombinant porcine epidemic diarrhea virus (pedv) (rpedv-s dr -gfp) was propagated and titrated in vero cells, and hcov- e in huh cells. pdcov was propagated and titrated in llc-pk cells, but supplemented with µg/ml tpck-treated trypsin (sigma-aldrich, inc., st louis, mo, usa) in dmem. synthetic sequences of coronavirus spike s subunits (hcov-hku (gb: yp_ . ), mers-cov (gb:yp_ . ), sars-cov (gb: aax . ), hcov-oc (gb: aar . ), hcov- e (gb: np_ . ), hcov-nl (gb: yp_ . ), tgev (gb: abg . ), pedv (gb: aog . ), bcov (gb: p . ), pdcov (gb: aml . ), fcov type (gb: fj . ), fcov type (gb: ay . )) and different domains of pedv s subunit (s and s a-d , as identified and described in [ ] ) were cloned into pcaggs expression plasmids as described previously [ ] . similarly, the expression constructs encoding chimeric proteins in which s s were fused to the fc domain of mouse igg a. for protein production, hek- t cells were transfected with plasmid dna conjugated to polyethyleneimine (polysciences, inc., warrington, pa, usa). at - h post transfection, inoculum was removed and the transfection mixture was replaced by sfm ii expression medium (gibco®, life technologies inc., grand island, ny, usa). at - days post transfection, cell supernatants were harvested and proteins were collected by protein a sepharose beads (ge healthcare bio-sciences ab, uppsala, sweden). proteins were then eluted with . m citric acid, ph . and neutralized with m tris-hcl, ph . . concentrations of proteins were assessed by nanodrop spectrophotometry (thermofisher scientific inc., waltham, ma, usa) and confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) with bovine serum albumin (bsa, bioivt, west sussex, uk) as standard. typical yields for proteins were . - . mg/ml. for long term storage, proteins were stored at − • c upon usage. to study the potential cross-reaction between fcov type and in more detail, models of fcov type (strain: uu ; genbank accession no.: fj . ) and fcov type (strain: - ; genbank accession no.: ay . ) s proteins were generated via the automated protein structure swiss-model homology modelling server (https://swissmodel.expasy.org/) [ ] using the elucidated hcov-nl cryo-em structure (pdb code: szs) as the input model. figures were made with pymol (the pymol molecular graphics system, version . schrödinger, llc.). fcov s domains of both type and , namely s -cd , were expressed as murine fc fusion proteins in hek- t cells as described above. high binding microtiter plates (greiner bio-one bv, alphen aan den rijn, the netherlands) were coated overnight at • c with equal molar amount of protein ( . pmol per well, diluted in phosphate buffered saline (pbs, ph . )). after three washes with washing buffer (pbs containing . % tween- ), the plates were blocked for h at • c with blocking buffer (pbs containing % milk powder (protifar, nutricia, zoetermeer, the netherlands), . % tween- ). protein coating efficiency was assessed by binding of anti-mouse igg antibodies in a direct elisa, and confirmed the equimolar coatings of all proteins. to detect antigenic reaction with serum samples, sera were tested in duplicate at a : dilution in blocking buffer, and then incubated in the plates at • c for h. after washing, plates were incubated with a : diluted horseradish peroxidase (hrp)-conjugated goat anti cat igg (rockland immunochemicals, inc., pottstown, pa, usa) at • c for h. the peroxidase reaction was then visualized via adding tmb super slow one component hrp microwell substrate (biofx®, surmodics ivd, inc., eden prairie, mn, usa) for min. reaction was stopped with . % sulfuric acid and optical densities (od) were measured at nm. negative sera (from uninfected spf cats) were included to determine the elisa cut-off values; sera with od values higher than -fold the od of negative sera were considered positive. all s proteins were coated on the same elisa plates making it easy to screen and compare the od values of individual sera in one assay. hereby we excluded the sera that give high background od values against all proteins being considered false positive. neutralization assays were performed with some of the covs to support the specificity of elisa results. cat sera were serially diluted -fold in dmem and mixed : with rpedv-s dr -gfp, hcov- e or pdcov ( % tissue culture infective doses [tcid ]/ml). these mixtures were then incubated at • c for h, and µl of each mixture was used for inoculation with vero, huh and llc-pk cell monolayers in -well plates, respectively. for pdcov infection, tpck-treated trypsin (sigma-aldrich, inc., st louis, mo, usa) was supplied to llc-pk tissue culture medium at a final concentration of µg/ml. at - days post infection, cytopathic effect (cpe) could be observed via microscopy. virus neutralization titers (vnt) were expressed as the highest serum dilution resulting in % reduction of cytopathic effect (hcov- e and pdcov) or virus-induced fluorescent cells (pedv). before virus neutralization, sera were inactivated through incubation at • c for min. experiments were performed in triplicate. feline sera (n = ) were screened by indirect elisa for antibody reactivity against cov s antigens. the od values against these antigens are shown in figure . in total, of the sera ( . %) contained anti-cov antibodies, while sera showed reactivity against more than one cov s antigen. none of the samples had to be discarded because of reactivity against all of the proteins indicating a potential false positive result. the frequency of different combinations of cov-s reactive samples is summarized in table . reactivity against eight out of cov s antigens could be observed, whereas none of the sera recognized the s protein of hcov-hku , mers-cov, sars-cov and hcov-oc . different s antigens were grouped by amino acid sequence phylogeny (left panel) using mega . each column of the heat map represents an individual sample, and columns were arranged in a descending order based on elisa-reactivity against feline coronavirus (fcov) type . table . numbers of positive cat samples and different combinations of reactivity found. the number of positive sera against each individual s is shown in the bottom row. positive elisa reactions are colored in orange. cut-off value was determined as the -fold over the od of negative sera. fcov type -s fcov type -s pdcov-s tgev-s e-s bcov-s different s antigens were grouped by amino acid sequence phylogeny (left panel) using mega . each column of the heat map represents an individual sample, and columns were arranged in a descending order based on elisa-reactivity against feline coronavirus (fcov) type . table . numbers of positive cat samples and different combinations of reactivity found. the number of positive sera against each individual s is shown in the bottom row. positive elisa reactions are colored in orange. cut-off value was determined as the -fold over the od of negative sera. number of cats (total = ) fcov type -s fcov type -s pedv-s pdcov-s tgev-s e-s nl -s bcov-s as expected, many sera were positive for fcov s , with sera ( . %) positive for fcov type and sera ( . %) for fcov type s ( figure ). all of the fcov type s positive sera also tested positive for fcov type s , while of fcov type s positive sera also reacted with tgev s . the fcov type s and tgev s elisa reactivities showed a strong nonparametric spearman correlation (spearman r = . , p < . ). with respect to this, we suggest that the tgev s positivity was due to cross-reactivity of fcov type s , as fcov type shows close antigenic and genetic relationship with tgev (s shares . % amino acid sequence identity). the remaining fcov type s positive but tgev s negative sera do react with fcov type s . an explanation might be the cross-reactivity between fcov type and type . remarkably, feline sera were reactive with s proteins from human, porcine and bovine covs (table ) , including hcov- e ( / ), hcov-nl ( / ), pedv ( / ), pdcov ( / ) and bcov ( / ). od values of feline sera positive for hcov-nl s and bcov s were relatively low ( figure a ). elisa reactivity towards non-feline cov s proteins might be explained by infection with the respective or related covs or by the presence of cross-reacting antibodies, although there was low sequence identity (< . %) between s proteins of fcov type and related non-feline coronaviruses (for the complete comparison of s sequence identities, see table ). yet, all of pedv-s positive sera were also positive for fcov type s ( figure b , table ). the elisa results of fcov type s and pedv s showed a strong nonparametric spearman correlation (spearman r = . , p < . ). thus, this might indicate the occurrence of antibody cross-reactivity against fcov type and pedv s antigens. many of the hcov- e and pdcov s positive sera also reacted with fcov type s , but no strong nonparametric spearman correlation was observed (hcov- e, r = . ; pdcov, r = . ). one feline serum only reacted with hcov- e viruses , , of s , and two feline sera only recognized pdcov s . ( figure b , table ). this observation led us to hypothesize that cross-reactivity may not play a role in elisa reactivity of these three sera, but that the three cats had been infected with these viruses or related viruses. fcov type -s (fj . ) fcov type -s (ay . ) . pedv-s (aog . ) . . pdcov-s (aml . in our screening, samples were shown to be positive for two or more s proteins including fcov type . the data prompted us to test different hypotheses which may explain this phenomenon: specific reaction through natural virus infection or reaction due to cross-reactivity with fcov-s antigens. to explore this further, we employed fcov type specific sera derived from specific pathogen free (spf) cats that had been experimentally infected with fcov type i strain rm (n = ) or strain uu (n = ). these sera were tested for their elisa reactivity against seven cov s proteins (excluding tgev s ) that showed positive reactivity in the previous serological screening. as expected, all sera were positive for fcov type s in our elisa; interestingly, four samples also reacted with fcov type s , and five samples with pedv s . no positive elisa-reactivity was detected with s of hcov- e, pdcov, hcov-nl or bcov (table s ). thus, fcov type infection could lead to the generation of antibodies that cross-react in the s -elisa with fcov type and pedv s proteins. the elisa cross-reactivity of fcov type specific sera with fcov type s antigens prompted us to map the domains responsible for cross-reaction within the s subunit. hence, to identify domain borders within s , we built homology-based models of both fcov type and type spike using the related elucidated hcov-nl cryo-em structure as the template model. as shown in figure a , continuous structural domains can be identified for the s subunit of both spikes, namely s , and s a through s d . amino acid sequence identities of these domains between fcov type and type differ, ranging from . % to . % ( figure b ). several s proteins for both type and type fcov-s comprising one or two domains were expressed and purified ( figure c ). fcov type specific sera (n = for strain rm and n = for strain uu ) and type (n = , strain - ) specific sera were then tested against these proteins in elisa format. the four fcov type specific sera that cross-reacted with s of fcov type again showed binding to fcov type s . but the fcov type specific sera showed little to no reactivity against fcov type s . as shown in figure , the type specific antisera reacted with all of the homologous s domains, with s and s b of both type and type displaying the strongest reaction. interestingly, the cd domain showed the highest level of cross-reactivity between fcov type and , in agreement with its highest sequence identity among s domains (figure ) . the other three domains showed little to no cross-reactivity. antisera reacted with all of the homologous s domains, with s and s b of both type and type displaying the strongest reaction. interestingly, the cd domain showed the highest level of crossreactivity between fcov type and , in agreement with its highest sequence identity among s domains ( figure ). the other three domains showed little to no cross-reactivity. the s subunit of one protomer are colored, with s shown in cyan, s a in blue, s b in green, and the domains s cd in red. the s part of the protomer is marked in light gray. (b) schematic presentation of the fcov type (strain uu ) and type (stain - ) s protein with the signal peptide (sp), the s subunit (the domains are colored as described in the legend of figure a ) and the s subunit (the c-terminal transmembrane domain is indicated by a black box). amino acid sequence identities between fcov type and type s domains are indicated. (c) diagram of the different s subdomains sequence. all s subdomains were c-terminally tagged with the fc part of mouse igg a (not shown in the figure) and expressed as fc fusion proteins. the s subunit of one protomer are colored, with s shown in cyan, s a in blue, s b in green, and the domains s cd in red. the s part of the protomer is marked in light gray. (b) schematic presentation of the fcov type (strain uu ) and type (stain - ) s protein with the signal peptide (sp), the s subunit (the domains are colored as described in the legend of figure a ) and the s subunit (the c-terminal transmembrane domain is indicated by a black box). amino acid sequence identities between fcov type and type s domains are indicated. (c) diagram of the different s subdomains sequence. all s subdomains were c-terminally tagged with the fc part of mouse igg a (not shown in the figure) and expressed as fc fusion proteins. viruses , , x for peer review of figure . elisa-reactivity of fcov specific antisera against different s subdomains of fcov type and . equimolar amount of purified s proteins and the four s subdomains were coated onto well plates and antibody binding was determined by elisa. the fcov type and specific antisera used in the screening were derived from experimentally infected specific pathogen free (spf) cats and are indicated at the right side of each panel, absorbance values and antigens in use are shown on the y-and x-axis, respectively. graphs represent the mean values from three independently performed experiments. standard deviations are indicated as error bars. because the fcov type specific cat sera also showed elisa reactivity with pedv-s (table s ), we analyzed the reaction of the five pedv-s positive cats in more detail. samples were analyzed via elisa using antigens comprising different pedv-s domains, as described in our previous study [ ] . cat sera taken pre-and post-fcov infection were collected and tested. as indicated in figure , all five cats had developed pedv-s reactivity to different extent after fcov type inoculation. noticeably, all sera showed the highest od values with the cd domain, while the other domains, including the s b containing the presumed receptor binding domain (rbd) [ ] , were non-reactive ( figure ). on the other hand, the swine pedv positive control serum exhibits strong reactivity against all pedv-s domains. the next question we asked was whether fcov type specific sera could neutralize pedv infection in tissue culture, as they showed no reactivity with the s b of pedv spike. as shown in figure , pedv neutralizing antibodies were detected in three out of five fcov type i specific cat sera. figure . elisa-reactivity of fcov specific antisera against different s subdomains of fcov type and . equimolar amount of purified s proteins and the four s subdomains were coated onto -well plates and antibody binding was determined by elisa. the fcov type and specific antisera used in the screening were derived from experimentally infected specific pathogen free (spf) cats and are indicated at the right side of each panel, absorbance values and antigens in use are shown on the y-and x-axis, respectively. graphs represent the mean values from three independently performed experiments. standard deviations are indicated as error bars. because the fcov type specific cat sera also showed elisa reactivity with pedv-s (table s ) , we analyzed the reaction of the five pedv-s positive cats in more detail. samples were analyzed via elisa using antigens comprising different pedv-s domains, as described in our previous study [ ] . cat sera taken pre-and post-fcov infection were collected and tested. as indicated in figure , all five cats had developed pedv-s reactivity to different extent after fcov type inoculation. noticeably, all sera showed the highest od values with the cd domain, while the other domains, including the s b containing the presumed receptor binding domain (rbd) [ ] , were non-reactive (figure ). on the other hand, the swine pedv positive control serum exhibits strong reactivity against all pedv-s domains. the next question we asked was whether fcov type specific sera could neutralize pedv infection in tissue culture, as they showed no reactivity with the s b of pedv spike. as shown in figure , pedv neutralizing antibodies were detected in three out of five fcov type i specific cat sera. the experiment was carried out in duplicate and repeated three times. error bars indicate standard deviations. sera were collected from spf cats prior (cat - p) and after (cat - ) experimentally inoculated with fcov type . positive serum: pedv positive swine serum collected from the field; negative serum: serum from fcov negative spf cat. several serum samples from field cats, but not virus-specific serum samples from fcov inoculated spf cats, were found to be elisa positive for hcov- e (n = ) and pdcov s (n = ) ( figure a) . also, a few feline sera displayed unique elisa positivity for s of hcov- e (n = ) or pdcov (n = ) ( figure b, table ). this could indicate that these antibodies were induced upon infection with these specific viruses. to corroborate the possibility of a natural infection in these cats with hcov- e or hcov- e-like viruses, we tested sera neutralization antibody titers. the results showed that one of the hcov- e s reactive feline sera was able to neutralize hcov- e infection (vnt = ); no neutralization of pdcov was detected for the all pdcov-s positive sera. coronavirus infections are endemic and ubiquitous in feline populations. two viral types, type and , are distinguished and both of them could well sustain themselves in the cat reservoir [ , ] . both have been shown to have worldwide distribution, with the seropositivity rate up to % among animal shelter populations and in multi-cat households [ , ] . the majority of natural infections are caused by type fcovs, while in the field type fcovs are less common and mainly occur in asia [ , , [ ] [ ] [ ] . covs are generally considered to be host-specific; however, cross-species transmission does occur which may lead to incidental infections like the spillover of mers-cov from dromedary camel to humans, where humans function as an incidental and ultimately dead-end host [ ] . but covs might also adapt to the new host exemplified by the animal origin of all four endemic human covs (hcov-oc , hcov-nl , hcov- e and hcov-hku ) [ ] [ ] [ ] [ ] . whereas in cats infections with fcov are well recognized, studies regarding possible natural infections with other animal and human coronaviruses are lacking to the best of our knowledge. knowing the genetic variability of coronaviruses and the use of orthologous receptors by non-feline covs, studies on cross-species transmission are desirable. this may provide insight regarding whether cross-species transmission does occur. in the present study we used the highly immunogenic s antigens to screen cat sera for the presence of antibodies against feline and non-feline coronaviruses, as a first indication of possible infections with these viruses. in our study, of the cat sera were shown to be seropositive for coronaviruses. the seropositive rate ( . %) against s of fcov type is consistent with previous studies [ , ] . all of the fcov type s positive sera of naturally infected cats were also positive for fcov type s , which might be the result of cross reaction between the two proteins, despite their low amino acid identity. elisa with specific antisera from experimentally fcov type and type infected cats showed that sera of several fcov type infected cats could cross-react with fcov type s . domain mapping elisa results showed that fcov type specific sera react to different levels with the s domains of fcov type s protein, and also reacts with fcov type s cd . vice versa, fcov type specific sera also reacted with s cd of fcov type . these observations pose a potential two-way cross-reactivity between s cd domains. interestingly, in parallel with our findings on feline coronaviruses, we identified a number of samples that were seropositive against the s of pedv, a viral pathogen that mainly replicates in the porcine intestinal epithelium. to study the possibility of cross-reaction, samples derived from preand post-fcov infected cats were screened against pedv s in elisa. the reactivity found against pedv s with fcov specific sera shows that cross-reaction can occur at the level of domain s cd ; the other pedv s domains showed no reaction with the fcov positive sera. judging from these observations, it seems that s cd plays an important role in cross-reaction between fcov type and , and also fcov and pedv. as s cd is the most conserved domain among fcov and also between fcov and other alphacoronaviruses (for a systematic assessment of sequence identities, see table ), it is reasonable to hypothesize that antibodies can develop against conserved epitopes within this region and subsequently cause cross-reaction. this should be taken into account when developing and interpreting serological assays. table . identities of amino acid sequences of fcov type (strain: uu ) s and s domains compared with the amino acid sequences of other alphacoronaviruses. (identities are shown in %; na: not available) the genbank accession numbers of these viruses are as follows: fcov type (uu ), fj . ; fcov type (rm), fj . ; fcov type , ay . ; tgev, abg . ; pedv, aog . ; hcov- e, np_ . ; hcov-nl , yp_ . . amino acid % identity to fcov type (uu ) s s s a s b s cd noticeably, elisa reactivity among cat sera towards the n-terminal fcov s domains and a was less consistent and generally lower compared to whole s , which seems to correlate with the higher antigenic variation in those domains found among fcov type strains [ ] (figure ). especially the sera from fcov-rm infected cats (cat , , and ) showed lower od values against s a . this phenomenon could be explained as the samples displaying higher reactivity were from cats inoculated with fcov-uu (cat , and ), the particular strain from which the s region was used as an antigen in the elisa studies. in the meantime, the possibility of the variable elisa reactivity might be due to the difference in individual antibody levels. in principle, the distinct antigenic reactivity of s and s a between the two fcov types might facilitate the development of a specific elisa method which allows the serological discrimination of fcov type and type infections in cats. in order to provide further insight regarding cross-reactivity between fcov type and pedv, we performed virus neutralization assays. cross-neutralization of pedv infection could be observed for some of the feline fcov type post-infection sera, in contrast to the pre-infection serum counterparts. since fcov specific pedv neutralizing sera did not react with pedv s , s a or s b , it is likely that the cross-neutralizing antibodies are targeting conserved epitopes in the s cd domain or the s subunit of the pedv spike protein [ ] . given the unknown tgev infection background of the pedv positive pigs, the cross-reaction of pedv specific sera against fcov type could not be explored in our study, as tgev positive pig samples would certainly influence the outcome [ , , ] . of note, our findings cannot exclude the possibility that field cats might incidentally get naturally infected with pedv or pedv-like viruses, as there had been one report showing the detection of pedv in one stray cat via pcr assay [ ] . it would be interesting to include more sera of cats from pig farms in future studies. considering the fact that cats play an important role in human society and have constant interaction with humans, it is of interest to conduct serological surveys for possible reverse zoonosis of human pathogens. in our study s antigens of several human coronaviruses were included and this led us to identify hcov- e seropositive feline samples in our elisa survey (table ) ; one serum in particular reacted solely with hcov- e s but not with any other coronavirus. of the hcov- e s reactive feline sera one showed low neutralizing activity against hcov- e infection. this might suggest that positive cats were indeed exposed to hcov- e or related viruses. mers seropositivity is also seen in other species besides the dromedary host [ ] . rare cases of seropositivity might be considered as spill-over infections from the dromedary camel reservoir. similar (perhaps dead-end) spill-over infections of e from the human reservoir to cats might also occur. a similar principle could also apply for pdcov, a porcine pathogen that emerged rather recently. both hcov- e and pdcov use apn as their receptor and have been reported to also be able to use feline apn for cellular entry [ , ] . although reports are lacking regarding the natural infection of these two viruses in cats, hcov- e was shown to cause a priming effect of fcov antibody in experimentally fcov infected cats suggesting that infection occurred [ ] . therefore, the detection of antibodies against s of hcov- e in a portion of the cats might be specific and due to the exposure to hcov- e through daily interaction with humans. eight cats were seropositive for pdcov of which two cats were seropositive only for pdcov and not for any other covs. this could be caused by infection with pdcov or pdcov-related viruses through avian sources, considering the fact that cats are natural avian predators and the presumed avian origin of pdcov [ , ] . our findings emphasize the potential role of cats as incidental hosts for non-feline coronaviruses and the need of in-depth study of naturally infected pathogens in cats. besides serological studies, efforts should also focus on isolation and identification of these viruses in cats. in conclusion, we presented a thorough serological survey in cats using s proteins of different animal and human coronaviruses. we demonstrated, despite the low amino acid identity, cross-reactivity between s proteins of fcov type and , and between that of fcov type and pedv. this should be considered when developing fcov serological assays as well as interpreting the results. our observation that some feline sera displayed antibody reactivity exclusively against non-feline cov s proteins warrant further research into the epidemiology and cross-species transmission of coronaviruses in cats and other animals that are in close contact with humans. further large scale serological studies regarding coronaviruses infection across animal species using arrays of cov s antigens can shed light into the hitherto unresolved host promiscuity of coronaviruses and the risk of cross-species transmission. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / / s , table s : elisa reactivity (od values) of fcov type specific antisera against s antigens of different coronaviruses. the authors declare no conflict of interest. pre-fusion structure of a human coronavirus spike protein discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer sars and mers: recent insights into emerging coronaviruses sars-cov and emergent coronaviruses: viral determinants of interspecies transmission middle east respiratory syndrome middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution link of a ubiquitous human coronavirus to dromedary camels evidence for an ancestral association of human coronavirus e with bats evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc surveillance of bat coronaviruses in kenya identifies relatives of human coronaviruses nl and e and their recombination history coronaviruses post-sars: update on replication and pathogenesis feline and canine coronaviruses: common genetic and pathobiological features serologic assays for influenza surveillance, diagnosis and vaccine evaluation the prevalence of types i and ii feline coronavirus infections in cats feline infectious peritonitis. abcd guidelines on prevention and management differentiation of feline coronavirus type i and ii infections by virus neutralization test feline coronavirus type ii strains - and - originate from a double recombination between feline coronavirus type i and canine coronavirus emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses an update on feline infectious peritonitis: virology and immunopathogenesis feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i type i feline coronavirus spike glycoprotein fails to recognize aminopeptidase n as a functional receptor on feline cell lines experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus experimental inoculation of cats with human coronavirus e and subsequent challenge with feline infectious peritonitis virus feline aminopeptidase n is a receptor for all group i coronaviruses an eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type i and ii feline coronavirus infections a retrospective clinical and epidemiological study on feline coronavirus (fcov) in cats in istanbul feline coronavirus serotypes and : seroprevalence and association with disease in switzerland coronavirus spike protein and tropism changes mechanisms of coronavirus cell entry mediated by the viral spike protein specific serology for emerging human coronaviruses by protein microarray sars-cov antibody prevalence in all hong kong patient contacts middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study porcine epidemic diarrhea virus (pedv) introduction into a naive dutch pig population in live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis pathogenic characteristics of persistent feline enteric coronavirus infection in cats broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein swiss-model: modelling protein tertiary and quaternary structure using evolutionary information characterization of monoclonal-antibodies against feline infectious peritonitis virus type-ii and antigenic relationship between feline, porcine, and canine coronaviruses a review of feline infectious peritonitis virus infection: - seroprevalence study of feline coronavirus in owned and feral cats in sydney prevalence of korean cats with natural feline coronavirus infections persistence and transmission of natural type i feline coronavirus infection spike protein fusion peptide and feline coronavirus virulence the s glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs prevalence of swine viral and bacterial pathogens in rodents and stray cats captured around pig farms in korea middle east respiratory syndrome coronavirus infection in non-camelid domestic mammals this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - wn ivq authors: berry, jody d; jones, steven; drebot, michael a; andonov, anton; sabara, marta; yuan, xin y; weingartl, hana; fernando, lisa; marszal, peter; gren, jason; nicolas, brigitte; andonova, maya; ranada, francesca; gubbins, michael j; ball, t.blake; kitching, paul; li, yan; kabani, amin; plummer, frank title: development and characterisation of neutralising monoclonal antibody to the sars-coronavirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: wn ivq there is a global need to elucidate protective antigens expressed by the sars-coronavirus (sars-cov). monoclonal antibody reagents that recognise specific antigens on sars-cov are needed urgently. in this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mabs) against the sars-cov is presented, based upon their specificity, binding requirements, and biological activity. initial screening by elisa, using highly purified virus as the coating antigen, resulted in the selection of mabs to the sars virus. subsequent screening steps reduced this panel to seventeen igg mabs. a single mab, f g , is specific for the nucleoprotein as seen in western immunoblot while five other mabs react with the spike protein. two of these spike-specific mabs demonstrate the ability to neutralise sars-cov in vitro while another four western immunoblot-negative mabs also neutralise the virus. the utility of these mabs for diagnostic development is demonstrated. antibody from convalescent sars patients, but not normal human serum, is also shown to specifically compete off binding of mabs to whole sars-cov. these studies highlight the importance of using standardised assays and reagents. these mabs will be useful for the development of diagnostic tests, studies of sars-cov pathogenesis and vaccine development. the sars-coronavirus (sars-cov) is recognised as the causal agent of severe acute respiratory syndrome (sars) in humans. this virus caused nearly deaths and infected more than people in various affected countries throughout the world (stadler et al., ) . the sars-coronavirus spike protein has only - % pairwise identity at the amino acid level to the spike proteins of other previously characterised coronaviruses. recently, the genomes of sars-cov isolates, implicated in the toronto outbreak, were sequenced in their entirety (marra et al., ; rota et al., ) . the production of mabs to the sars-cov virus is critical for diagnostic development, vaccine research and studies of viral pathogenesis. assays that detect the presence of virally encoded proteins or nucleic acids may be preferable for diagnosis of sars infections as the development of serum antibodies in infected individuals is quite protracted (li et al., a) . coronaviruses are enveloped, single-stranded rna viruses that replicate in the host cell cytoplasm (fields et al., ) . the coronaviruses form a single genus of the family coronaviridae and the virions are large ( - nm in diameter) pleomorphic, but generally spherical, particles. virions of most coronaviruses contain three major proteins: the phosphorylated nucleocapsid protein (n); a small membrane-embedded glycoprotein (m); and a large club-shaped peplomer glycoprotein (s) which appears in em micrographs as protruding spikes nm in length. the m protein is synthesised on ribosomes bound to the endoplasmic reticulum and accumulates in the golgi apparatus. the subcellular localisation of m protein to the golgi is believed to influence the site of virus budding in the infected cell. the s-protein mediates many of the biological properties of the virus, including attachment to cell receptors, penetration, and cell-fusion, and it is the major target for virus-neutralising antibodies (collins et al., ; talbot et al., ; wege et al., ; jimenez et al., ; laude et al., ; godet et al., ) . a portion of the s glycoprotein that is not incorporated into budding virions is transported to the plasma membrane of the cell where it remains bound to the cell surface (gerna et al., ) . coronaviruses infect a wide range of mammalian hosts to produce a variety of disease outcomes including respiratory disease, enteritis and encephalitis. antigenic similarities between various coronaviruses have been demonstrated to reside in the s-protein and have been used to study the evolution of this virus family (brian et al., ) . for most coronaviruses causing enteric and respiratory diseases the pathophysiological events leading to clinical symptoms are due to the acute cytocidal infection of the target cells. these infections can be limited by the local immune response resulting in the production of secretory antibodies specific for the s-protein (enjuanes et al., ) . in contrast, many coronaviruses are maintained and spread in the population as inapparent and subclinical infections. the sequence of events leading to chronic versus acute disease is unknown but likely depends on the expression of viral genes, the functional impairment of host cells, and the interaction with the host immune response. there is a critical need to elucidate the immunologic basis for protection against sars-cov infection. recently the sars s-protein was shown to be a functional fusogen and is about - kda in size (xiao et al., ) . a host cell receptor, angiotensin-converting enzyme (ace- ) was recently identified as a functional receptor for the sars-cov, and mediated infection of t cells in vitro (li et al., b) . therefore, antibody responses to the s-protein may neutralise the infectivity of the sars-cov. the immunogenetics of antibody responses to protective epitopes is of particular importance and will lead to a clearer understanding of the nature of protective antibody responses to sars. lastly, the production of protective monoclonal antibodies may lead to the development of new recombinant therapeutic antibodies in order to provide rapid protection in sars patients. in the present work, a description of the development of murine mabs against the sars-cov involved in the toronto sars outbreak is presented. the mabs were analysed for pertinent immunochemical properties and for their ability to neutralise sars-cov in vitro. for preparation of partially purified whole-virus antigen, sars-cov was expanded after plaque purification in vero- cell monolayers and partially purified through a sucrose cushion. highly purified sars-cov (tor- strain, isolated from a patient infected in the toronto sars outbreak; krokhin et al., ) was prepared in the same way, except the viral particles were further purified using gradient centrifugation. briefly, ml of supernatant from sars-cov infected vero- cells was concentrated first on top of a cushion of iodixanol in a beckman sw rotor (mississauga, on). the virus was subsequently mixed to form a suspension of % iodixanol and subjected to centrifugation in a beckman nvt rotor (mississauga, on) for . h at , × g. fractions were collected from the bottom of the self-generated gradient, tested by western immunoblot with sars-cov-infected, convalescent human patient serum, and the sars-cov positive fractions were pooled and dialysed against phosphate buffered saline (pbs). the dialysed virus preparation was further concentrated by ultracentifugation for . h at , × g. immunisation of mice was performed according to ncfad standard operating procedures under iso . five-to six-week-old female balb/c mice (charles river, wilmington, ma) were injected subcutaneously (sc) with g of beta-propiolactone-inactivated, partially purified sars-cov (tor- strain) with an equal part of complete freund's adjuvant (h -ra, cfa) from difco (bd, oakville, on) on day . on day the mice received g of partially purified sars-cov s.c. in incomplete freund's adjuvant (ifa) in a total volume of l. on days and , the mice received g of the same antigen in a total volume of l sc with ifa. the mice received a final booster injection with g of highly purified sars-cov in l pbs to the intra-peritoneal cavity days prior to hybridoma fusion. mice were euthanised by anaesthesia overdose and exsanguinated by cardiac puncture. the spleens were subsequently excised under aseptic conditions. infected vero cells were scraped off of cm corning tissue culture flasks (corning, ny) and clarified by centrifugation. a borate saline mixture ( . m boric acid, . m, nacl, . m naoh) was used to wash the cell pellet twice and the pellet was resuspended in ml borate saline + % triton x- for each t flask. the pellet was kept at • c using a water bath and sonicated for ten minutes at % power. the debris was pelleted via centrifugation at , × g for min and the supernatant was collected and stored at − • c in aliquots for later use. removal of mouse spleens, preparation of spleen and myeloma cells, and the fusion for hybridoma production were performed according to ncfad standard operating procedures under iso . ampoules of the myeloma cell line p x ag . (atcc, rockville, md) were thawed week prior to fusion and grown in bd cell mab quantum yield medium in the presence of -azaguanine (sigma, oakville, on). cells were in log-phase growth at the time of fusion. hybridoma fusion was performed essentially as originally described (kohler and milstein, ) with the following modifications. briefly, spleens were harvested days after a final boost with a given antigen and the splenocytes were prepared by splenic perfusion as follows. under aseptic conditions, the spleens were perforated with a cm syringe with a gauge sterile disposable needle. the spleen cells were perfused out of the spleen with injections of serum free bd cell mab quantum yield medium (bd-pharmingen, oakville, on). two identically immunised mouse spleens were used to produce these hybridoma clones. the fusion was performed using the p x ag . myeloma line in log-phase growth. peg ( ml; roche, basel, sw) was added drop-wise over min while gently tapping the tube containing the thoroughly washed myeloma-splenocyte pellet. the peg was slowly diluted out over three minutes with serum free bd-cell mab quantum yield medium. the cells were resuspended and mixed into ml of stemcell clonacell medium d (hat) (vancouver, bc) containing ml origen hybridoma cloning factor (hcf) (igen, gaithersburg, md) and plated out according to the manufacturer's instructions. the plates were incubated at • c under a % co overlay for - days in humidified chambers. visible colonies were picked from the plates after approximately weeks growth and placed into -well plates containing - l of complete hybridoma medium supplemented with × hypoxanthine thymidine (sigma, oakville, on), % hcf and % fbs (wisent). supernatants were screened days later via elisa using purified virus as antigen. isotyping was performed using a commercial murine isotyping dipstick test (roche, basel, sw) according to the manufacturer's instructions. hybridoma culture supernatants were concentrated - fold using amicon stirred cell nitrogen concentrators with kda cutoff millipore (ym- ) membranes (both from millipore, billerica, ma). hybridoma culture supernatants were assayed for binding to highly purified sars-cov in an elisa assay when the cultured cells were confluent in the culture plates. the costar -well / well elisa plates (corning, ny) were coated with either bovine serum albumin (bsa) or highly purified sars-cov ( - ng/well) in pbs overnight at • c and then blocked with . % bsa in pbs, for h at • c. the supernatant ( l/well) was incubated neat for h at • c. the elisa plates were washed times with distilled water and patted dry on a paper towel. a pan-goat anti-mouse igg-hrp antibody (southern biotechnology associates, birmingham, alabama) was diluted to : in . % bsa in pbs, applied to the elisa plates for min at • c, and then washed as described above. positive binding was detected with commercial abts used according to the manufacturer's instructions (roche, basel, sw). the od was read at nm at and min intervals after addition of the developing reagent. mouse immune and preimmune sera were diluted : with %-bsa in pbs for use as positive and negative controls, respectively, and for the establishment of the hybridoma screening assay. competition elisa (c-elisa) measured the binding of murine mabs to highly purified sars-cov in the presence of human serum. infected serum was from confirmed infected patients with well-established sars. these samples were from the initial set of patients from which the virus was isolated. plates were coated with highly purified sars-cov at ng per well, and normal human serum or serum from convalescent sars patient s , serially diluted in % bsa-pbs, was allowed to react on the pre-blocked plates for min. mab f g (spike specific) or f g (nucleoprotein specific) were pre-diluted to a concentration that gives approximately half-maximum optical density after h development in the presence of no competing serum. the diluted mab preparations were then applied to the wells, and the incubation and development of the c-elisa was performed as described above. whole virions and sars-cov-infected vero cell lysates, at a final total protein concentration of g per lane, were boiled in sds-loading buffer for min. the samples were loaded in criterion pre-cast gels (biorad, mississauga, on) and electrophoresed at v for min. the proteins were transferred to immobilon nylon membranes (millipore, billerica, ma) for h at room temperature at v, or overnight at v at • c. blots were blocked with % bsa in tbs, rinsed three times with tbs, and reacted with monoclonal antibody overnight at • c. the antibody supernatants were reacted neat and the concentrated supernatants were diluted : in . % bsa in pbs. blots were washed three times with tbs-tween- ( . %) for min before being incubated with secondary antibody (same as above) at : in tbs, . % bsa for h. the blots were washed as above and developed using dab insoluble substrate (pierce, rockford, il). monolayers of sars-infected vero cells were stained as follows. glass slides were coated with infected vero cell monolayers and fixed with acetone. the slides were irradiated with kilogreys from a cobalt gamma irradiator, removed from biocontainment, and then stored at − • c. dilutions of antibodies and test sera were made initially in -well plates (bd-falcon, oakville, on) in sterile phosphate-buffered saline (ph . ). samples were allowed to incubate for min in a • c incubator, and were washed with distilled water. fluorescein labelled goat anti-mouse secondary antibodies (sigma, oakville, on) diluted in pbs were added to the slides and incubated for min at • c, washed as above, and air dried. slides were coated with mounting medium and stored at • c until examined. immuno-dotblot analysis was performed using immobilon nylon membranes (millipore, billerica, ma). a total of l of sars-cov antigen (infected vero cell lysate) or g of highly purified virus is coated (per spot) for h at • c. in an attempt to further characterise the immunochemical properties of the individual mabs, antigen was pre-treated in several different conditions as follows: untreated native antigen; denatured by heat treatment at • c for min; denatured by sodium dodecyl sulphate treatment (sds) % prior to coating on the membrane; denatured by both heat treatment and sds as above together; reduced with beta-mercaptoethanol ( %) prior to coating on the membrane; denatured by temperature as above and reduced as above, together; denatured by both heat and sds in the presence of beta-mercaptoethanol. antigen-coated blots were blocked with % bsa in tbs for h at • c, and rinsed three times with tbs, prior to incubation with mabs. the concentrated mab supernatants were diluted : in . % bsa in tbs (tris-buffered saline, ph . ) and reacted overnight at • c on a rotary platform shaker with gentle agitation. blots were washed three times with tbs-tween- ( . %) for min before being incubated with secondary antibody (goat anti-mouse igg-hrp, southern biotech, same as above) at : in tbs, . % bsa for h. the blots were washed as above and developed using dab insoluble substrate (pierce, rockford, il). the elisa positive monoclonal antibodies were screened for neutralisation of sars-cov using two independent formats. the first was a standard plaque reduction assay and the second measured reduction of cytopathic effect (cpe) in a microtiter format. a standard plaque reduction neutralisation test was performed as previously described (beaty et al., ) using highly purified sars-cov. briefly, mixtures of pre-titred ( pfus) sars-cov and serial two-fold dilutions of hybridoma supernatant were incubated at • c for h and added to six well plates containing vero cell monolayers. after incubation at • c for h, a nutrient-agar overlay was added and the plates were placed in a co incubator at • c for approximately days. a second overlay was then added which contained neutral red as a vital stain. plates were then checked periodically over the next few days for plaque formation. the highest dilution tested that produced a plaque reduction of at least % was defined as the titration end point. a microtiter format cpe reduction assay was used to test the sars-cov reactive mabs for neutralisation of sars-cov and transmissible gastroenteritis virus diamond strain (kindly provided by dr. susy carman, lsd, university of guelph). briefly, concentrated hybridoma supernatants were diluted : in cell culture medium and incubated with tcid of either highly purified sars-cov (tor- strain), or tgev, for h at • c, for a final dilution of : . the virus-antibody mix was then transferred onto cell monolayers in -well plates (costar, corning, ny). vero v- cells were used for the sars-cov, st cells for the tgev. the plates were incubated until cpe developed in virus back titration controls. elisa screening on highly purified sars-cov identified a panel of mabs reactive to the sars-cov antigen preparation. negative screening on bsa reduced this number to a panel of igg/k type mabs ( fig. ; table ). in general, binding reactivity of these mabs is greatly affected by both the conformation and purity of the antigen as illustrated by the decreased binding of these mabs, when tested in elisa, to heat denatured pure sars-cov as antigen compared to native virus (fig. ) . this clearly shows the importance of selecting suitable antigen for screening assays. western immunoblot analysis identified mabs to the sars-cov spike and nucleoprotein. five mabs (f g , g , g , g and g ) react specifically with the sars-cov spike protein in western immunoblot. these mabs recognise spike in western immunoblot on both highly purified virus and infected cell lysates (fig. ) , but do not react with mock-infected cell lysates (data not shown). this result suggests that these mabs target linear epitopes within the spike protein. another mab, f g , bound fig. . elisa reactivity of mabs with whole, inactivated sars-cov. hybridoma supernatants were tested at a : dilution in pbs + . % bsa on pre-blocked plates, coated with ng per well of inactivated, highly purified sars-cov. positive clones were identified as having positive binding (color) in wells that were at least four-fold higher than the background level reactivity on bsa. antigen legend: black bars-native, highly purified sars-cov; white bars-heat denatured, highly purified sars-cov; grey bars-bsa control. a virus neutralisation tests were performed independently in separate containment laboratories (nml, national microbiology laboratory; ncfad, national centre for foreign animal disease). the last six rows denote neutralising clones. b only a single dilution of / was tested in microwell format. c protein specificity tests shown here were determined by western immunoblot with purified virus and infected cell lysate under denaturing conditions (fig. ) . u, unknown target antigen. d immuno-dotblot was performed using antigen treated under various conditions described in section . n, native; h, heat denatured, • c for min; d, sds treated ( %); h+d, heated in the presence of sds ( %); r, treated with reducing agent, beta-mercaptoethanol ( %); h + r, heated in the presence of reducing agent, beta-mercaptoethanol ( %); a, treated with heat, sds ( %) and reducing agent beta-mercaptoethanol ( %). e immune-fluorescence on whole cell slides infected with sars-cov (see fig. ); ++, strong positive reaction; +, positive reaction; ±, weak positive reaction; −, negative reaction. f epitope properties described as follows: l, linear or continuous; e, surface exposed; c, conformational; p, protective in vitro; nd, not determined. to the nucleoprotein in western immunoblot assays. interestingly, the majority of the identified mabs bound to the spike protein and not the nucleoprotein, despite the strong sero-reactivity of the polyclonal serum of the immunised mice for the nucleoprotein (fig. ) . the identity of the target antigen of eleven other mabs could not be determined by western immunoblot analysis. this observation suggests that these mabs likely target conformational epitopes that are sensitive to the conditions employed in such analyses. work is planned to identify the specific targets of these mabs. immuno-dotblot analysis reveals a spectrum of conformational requirements for binding (summarised in table ). the effects of different denaturing treatments on the binding activity of a subset of neutralising and some non-neutralising mabs were examined using immuno-dotblot assays on infected lysates compared to uninfected lysates. the series of conditions tested include exposure to heat, detergent, a reducing agent, and combinations thereof. interestingly, mab f g , which binds to the nucleoprotein in western immunoblot, does not bind to the sars-cov antigen in immuno-dotblot under any of the conditions tested. the inherent charge of the highly phosphorylated nucleoprotein, or of the immobilon membrane used in the assays, may provide an explanation for these results. immuno-dot blots were not performed with mabs f g , f g , f g , f g , or f g , however the binding of these mabs is considered conformational as they do not bind to sars-cov proteins in western immunoblot (fig. ) . the binding of spike protein specific mabs f g , f g , and f g is inhibited in immuno-dotblot assays when antigens are treated with heat plus detergent, or heat plus detergent plus reducing agent. when the immuno-dotblot assay was instead performed using purified virus particles, the result was identical for nucleoprotein specific mab f g , and spike specific mabs f g , f g , and f g (data not shown). antigen pre-treatment for western immunoblot, which depends on the application of an electrical current, differs when compared to passive adsorption in elisa and immuno-dotblot assays, and clearly do not always correlate with one another. these studies illustrate the need to use multiple assays for epitope characterisation and reveal limitations of current epitope classification schemes. the spike protein is an immunodominant antigen when inactivated sars-cov is used as an antigen. murine antibody responses to inactivated whole sars-cov are focused upon the sars spike and np protein as shown by the specificity of the recovered mabs. while multiple sars-cov proteins appear to be the target of mouse serum antibody as shown in western blot (fig. , immune sera) , the majority of the mabs recognise the spike protein in western immunoblot. in a sars-cov infection, the host immune system is exposed to a large load of nucleoprotein antigen, due to the presence of replicating virus in infected tissues. this would suggest that a competitive elisa format based upon the np as the target antigen might provide fig. . competition elisa measuring the binding of murine mabs to highly purified sars-cov in the presence of human patient serum. dilutions (as indicated) of a normal human serum control (white bar), or serum from convalescent sars patient s were applied to wells coated with highly purified whole sars-cov. mab f g (spike specific; black bars) or f g (nucleoprotein specific; grey bars) were then added to the reactions. the results depicted for the pooled normal human serum (nhs) represent the mean of three replicate tests performed in the presence of mab f g combined with three replicate tests performed in the presence of mab f g . results are representative of identical assays performed in duplicate with gamma-irradiated patient sera ( mrad) (*p = . , **p = . , student's t-test). increased sensitivity for early detection of infection. towards this goal, the ability of sars convalescent serum to compete for binding of mabs to the nucleoprotein or to spike on whole sars-cov was tested via elisa. the serum samples were gamma-irrradiated prior to use. antibodies in the serum of patient s clearly inhibit binding of both the nucleoprotein mab f g and the spike protein specific mab f g (fig. ) . in contrast, antibody in pooled normal human sera does not compete for binding by either mab. while not a statistical analysis these data show that these mabs are useful for the development of serological competition assays which can be subjected to validation tests. the sera from several other sars-cov infected patients demonstrate a similar ability to inhibit binding by the f g (nucleoprotein specific mab) and f g (spike specific mab). however, in some samples there is inhibition of only the binding to nucleoprotein without inhibition of the mab to the spike protein suggesting that spike antibody responses take longer to develop in infected humans (data not shown). the predominance of mabs to the spike protein in mice immunised with intact viral particles led us to test for biological activity in virus neutralisation assays. the sars-cov spike protein is the target of neutralising antibodies. neutralisation positive mabs bind both to linear epitopes in the spike protein and to unknown conformational epitope(s) either in the spike protein or in other proteins. a total of six mabs were identified that could neutralise sars-cov infectivity: f g , g , g , g , g , and g (table ) . significantly, two of these mabs, f g and , were positively identified as being specific for spike, as determined by western immunoblotting. the specific targets of the remaining neutralising mabs remain to be elucidated. no cross-neutralisation was observed for the animal coronavirus tgev. this shows that these mabs are specific for sars-cov epitopes and do not cross-neutralise via tgev epitopes. the remaining mabs were unable to prevent viral growth when they were applied to the neutralisation assays. these data suggest that vaccines capable of engendering neutralising antibody responses to the spike protein may be effective in blocking infection with sars-cov. the four western immunoblot negative, virus-neutralising mabs were tested for their ability to bind native sars-cov in infected cells by immunofluorescence assay. nonneutralising mab f g was used as a positive control, since this mab recognises spike protein in immunohistochemical staining of infected vero cells (data not shown). immunofluorescence analysis reveals that the neutralising mabs f g , g , g , and g specifically recognise sars-cov infected but not uninfected vero cells (fig. ) . irrelevant, isotype matched mabs, produced in an identical fashion, do not react with sars-cov infected vero cells. the sars-neutralising mabs bind epitopes with higher conformational requirements than the non-neutralising mabs as they are less tolerant to denaturation of the epitopes. the method of antigen preparation and quality of the antigen greatly affect the interpretation of mab binding results obtained from immuno-dotblot assays. importantly, none of the mabs react with mock-infected lysates as assayed in immuno-dotblots (data not shown). this observation suggests that the majority of the neutralising mabs likely target surface exposed, protein epitopes on the native viral particle. one such putative protective antigen has been identified as the spike protein by western immunoblot analysis with neutralising mabs f g and f g . antigen quality and conformation affects the binding of the anti-sars-cov mabs in elisa. while purified virus is clearly the optimal antigen tested in this series of experiments, the lower quality sars-cov-infected vero cell lysates are, however, much easier to prepare for diagnostic assays. therefore, the limits of mab binding to sars-cov infected vero cell lysates were further examined via elisa. the majority of these mabs exhibit decreased binding when the antigen is heat denatured ( table ) . heat denaturation has very little effect on the binding of non-neutralising mab f g , which also maintains a high level of binding in elisa using infected vero cell lysates. f g does, however, show higher background on the irrelevant antigen, bsa, and has inconsistent reactivity in western immunoblots with heat denatured viral lysate (fig. , table ). the combination of lower quality antigen in infected vero cell lysates, along with heat denaturation of the antigens, has a stronger negative effect on binding by the neutralising mabs compared to non-neutralising mabs (table ) . indeed, regardless of western immunoblot reactivity, the non-neutralising clones retain a greater ability to bind heat denatured antigen compared to the neutralising mabs (lower mean percent reduction in od per group p < . , student's t-test, table ). this supports the earlier observation in elisa on purified sars-cov (fig. ) that shows that the neutralising mabs have higher requirements for native epitope conformation compared to non-neutralising mabs. the higher conformational requirement by neutralising mabs may help to explain some discrepancies observed in the immuno-dotblot methods. the immuno-dotblot is, overall, a less sensitive method, and the results are more difficult to quantify, compared to elisa. for example, mab f g binds to the sars-cov spike protein in western immunoblot and neutralises sars-cov infection in vitro. while binding in western immunoblot generally suggests the epitope is linear in nature, nonetheless, heat treatments clearly affect binding of some mabs to the whole virion. the immuno-dotblot data reveal that with the lower quality antigen of the infected vero cell lysate, under the conditions of heat-plus detergent, or heat-plus detergent and reducing agent, mab f g cannot bind (table ) . this paper describes the development of murine mabs which recognise sars-cov antigens in elisa, immuno-dotblot, western immunoblot, on the surface of infected cells, and in neutralisation assays. these data are consistent with the appearance of coronavirus antigens on the surface of the infected cell during replication (talbot et al., ) , although the fixation process may allow for reactivity of these mabs with internal antigens as well. the conformational sensitivity of most of the sars-cov neutralising mabs is consistent with properties of neutralising mabs raised against other enveloped viruses, which generally require more native conformation for binding (wilson et al., ; zwick et al., ) . the immuno-dotblot assays contradict the classification of several putative epitopes as being linear as is suggested by positive western immunoblot reaction (for example with neutralising mab f g ). indeed, it appears that the strict traditional classification of epitopes as being linear or conformational must account for a broader spectrum of conformational requirements, especially when dealing with antigens of variable quality in different immunological tests. however, in general the neutralising mabs can be considered to have a higher requirement for correct epitope conformation compared to non-neutralising mabs in elisa. it will be important to verify, under optimised conditions (opstelten et al., ) the use of viral lysates designed for maximal recovery of coronavirus proteins, and to this end the production of high quality recombinant protein antigens will provide useful insights. unfortunately, preparation of highly purified viral antigen requires enormous efforts under bio-containment conditions, which emphasises the need for a quality recombinant antigen assay. collectively, these data demonstrate that development of mab-based diagnostic tools for the detection of sars-cov infection is well within reach. the spike protein of the sars-cov is a target of neutralising mabs. epitopes on the spike protein provide neutralising targets on sars-cov in vitro, and this is consistent with the spike being the target of neutralising antibodies for other coronavirus strains (godet et al., ) . moreover, these mabs may be useful for the identification of protective epitopes for vaccine formulations (enjuanes et al., ) . studies are underway to determine the identity of the antigen(s) recognised by the neutralising, western immunnoblot negative mabs. preliminary analysis of the nucleotide and predicted amino acid sequences of the cloned v h and v l coding regions of these mabs suggests that the mabs are distinct. this observation implies that the hybridoma clones expressing the anti-sars-cov neutralising mabs were derived from individually rearranged and clonally selected b cells in vivo. it also reveals that there is no apparent consensus sequence within the complementarity determining regions that is required for the mabs to exhibit virus neutralisation activity. this finding makes it feasible to engineer multiple mabs with high specificity and avidity for various sars-cov epitopes for preparations of defined cocktails of therapeutic mabs. a detailed description of the immunogenetic properties of these mabs is in preparation (berry et al., manuscript in preparation) . the np specific mab, f g , is useful in competitive elisa with patient sera. this is important as early detection of sars infections is key to risk management of this disease. this is the first description of neutralising mabs from a host immunised with whole sars-cov and these antibodies should prove useful for the development of new diagnostic tests, studies of antigenic variation, and vaccine development in the global fight against sars. arboviruses proceedings of the fourth international symposium on neonatal diarrhea. s.d. acres monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion tropism and immunoprotection in transmissible gastroenteritis coronaviruses fields virology reactivity of human coronavirus oc and neonatal calf diarrhoea coronavirus membrane-associated antigens major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein critical epitopes in transmissible gastroenteritis virus neutralization continuous cultures of fused cells secreting antibody of predefined specificity mass spectrometric characterization of proteins from the sars virus: a preliminary report antigenic structure of transmissible gastroenteritis virus. i. properties of monoclonal antibodies directed against virion proteins profile of specific antibodies to the sars-associated coronavirus angiotensin converting enzyme is a functional receptor for the sars coronavirus envelope glycoprotein interactions in coronavirus assembly sars-beginning to understand a new virus topographical mapping of epitopes on the glycoproteins of murine hepatitis virus- (strain jhm): correlation with biological activities hybridoma antibodies to the murine coronavirus jhm: characterization of epitopes on the peplomer protein (e ) epitopes involved in antibody-mediated protection from ebola-virus the sars-cov s glycoprotein: expression and functional characterization identification and characterization of a peptide that specifically binds the human broadly neutralizing anti-human immunodeficiency virus type antibody b the authors would like to thank ms. nicole beausoleil, mr. daryl dick, mr. darrell johnstone, ms. kathy frost, ms. hilary holland, and mr. richard nickel for expert technical assistance. mg is supported by a health canada ocs postdoctoral fellowship. thanks to dr. john copps (nc-fad) for expert veterinarian services and for assisting in the design of the emergency animal use document. thanks also to dr. susy carman (university of guelph, canada) and dr. lorne babiuk (vido, canada) for providing animal coronavirus strains. funding for this work was provided by health canada and the canadian food inspection agency. this work is dedicated to the memory of lloyd d. berry. key: cord- -sj ngpk authors: he, qigai; du, qingyun; lau, suelyn; manopo, ivanus; lu, liqun; chan, shzu-wei; fenner, beau j.; kwang, jimmy title: characterization of monoclonal antibody against sars coronavirus nucleocapsid antigen and development of an antigen capture elisa date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: sj ngpk this report describes the production of several mabs against n protein, a major immunodomain of sars cov nucleocapsid protein [he, q., chong, k.h., chang, h.h., leung, b., ling, a.e., wei, t., chan, s.w., ooi, e.e., kwang, j., . development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. clin. diagn. lab. immunol. ( ) – .]. one representative igg monoclonal antibody (mab), s-a d , was selected and characterized. s-a d reacted specifically react with both recombinant and native nucleocapsid protein of sars cov. the reactivity of s-a d with purified n protein and utilization of the mab as a detector antibody to develop an antigen capture elisa was assessed. as little as . pg of purified n protein and tcid( ) of sars cov could be detected by the antigen capture elisa. specific binding of the mab s-a d to both purified n and sars cov nucleocapsid antigen was effectively inhibited by human sars positive serum and guinea pig anti-n serum. the n protein in n -spike recombinant baculovirus-infected sf- cells could also be identified. n protein was detected in ifa igm-positive serum samples collected from sars confirmed patients, but not in nine samples collected from sars recovery patient. no false positive results were given when samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (ibv), chicken coronavirus, was tested. this monoclonal antibody-based antigen capture elisa is thus a powerful tool for early diagnosis of sars cov infection. severe acute respiratory syndrome coronavirus (sars cov) is the causative agent of a new and emerging disease worldwide marra et al., ) . the disease was widely prevalent in more than countries with reported cases and causing deaths in (who, . thus, the development of diagnostic tests for specific and early detection of sars cov will contribute to the risk management of the disease. at present, sars cov infection is confirmed by the detection of viral rna via pcr or rt-pcr (drosten et al., ; poon et al., ) , however, this is a technically demanding technique and this is susceptible to cross contamination. the determination of infectious virus in samples can be carried out by inoculating cell cultures, such as vero cells, with a patient specimen, though this is relatively time consuming. most serological assays developed so far are based on the detection of specific circulating antibodies he et al., ) . these assays are highly sensitive and specific for detecting antibodies against sars cov. however, a lack of detectable antibodies in sars cov infected patients at early stage or throughout the whole disease course has been reported. these cases oc- - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.jviromet. . . curred in sars patients who were immuno-compromised or who had chronic conditions, e.g., diabetes mellitus or chronic renal insufficiency and may remain afebrile when acutely ill or possess symptoms attributable to underlying diseases, thus delaying sars diagnosis (houng et al., ) . therefore, more effort should be directed towards developing a simple and inexpensive assay for the detection of sars cov proteins. such tests could be used for early detection and follow-up of patients during treatment and thus reducing the workload of laboratory personnel. antibodies against the nucleocapsid protein are longer lived and occur in greater abundance in sars patients than antibodies against other viral components such as the spike, membrane and envelope proteins chen et al., ; huang et al., ; kim et al., ; tan et al., ; timani et al., ; zhu et al., ) . this might be due to the higher expression of nucleocapsid as compared with other viral proteins after sars cov infection (rota et al., ) . these data indicated that nucleocapsid could play a crucial role in antibody response during infection. in our previous work, a major immunodomain of recombinant sars cov nucleocapsid (n ) was identified and used to develop a western blot for the detection of antibodies against sars cov infection, the sensitivity and specificity were . and %, respectively . from these results, we hypothesize that monoclonal antibodies against n would specifically recognize sars cov in immunoassays. in this study, we produced monoclonal antibodies against n protein. the monoclonal antibodies were characterized by sars cov-infected vero cells and nucleocapsid-spike fusion protein-based ifa, western blot, and n proteinbased elisa. the isotype of the promising monoclonal antibody, designated as s-a d , was determined and was further applied to develop a specific and sensitive antigen capture elisa for the detection of sars cov. the sensitivity and specificity of this antigen capture elisa was also assessed. rpmi medium and fetal bovine serum (fbs) were obtained from invitrogen (carlsbad, ca). hypoxanthineaminopeterin-thymidine (hat) supplement used for the propagation of hybridoma was purchased from sigma (st. louis, mo). mice myeloma cell s/p . and spodoptera frugiperda (sf- ) cells were available from our laboratory. human cells (thp- , a ) and vero cells were obtained from the institute of molecular cell biology (imcb), singapore. inactivated sars cov (sin strain) was provided by singapore general hospital. twenty-seven sera that were available from patients presenting symptoms satisfying the world health organization (who) definition of sars and sera were obtained from healthy individuals. sera were provided by tock seng hospital and were heat inactivated at • c for h before use. the recombinant baculovirus bearing the fusion gene encoding n and sc fragment of the spike protein was constructed as described previously (he et al., ) . a commercial sars ifa diagnostic kit (euroimmun, germany), in which inactivated sars cov infected vero cells was used as antigen, was purchased from medizinische labordiagnostika ag, germany. three serum samples from infectious bronchitis virus (ibv)infected chickens and ibv m strain were also tested. the experiments involving the use of the inactivated sars cov or human sars inactivated sera were performed in a bsl laboratory. sars cov n and the n-terminal amino acids of nucleocapsid (n ) protein were expressed and purified as described previously . n was used for animal immunization as well as the antigen in antibodydetection elisa and western blot assay while n acted as a heterologous antigen for optimization of antigen capture elisa. four - -week-old balb/c mice were injected subcutaneously with g of purified n protein emulsified with an equal volume of adjuvant (seppic, france) for three times with a -week interval. mice received a final booster injection with g of antigen in l pbs to the intraperitoneal cavity days prior to hybridoma fusion. mice were sacrificed and their spleen cells harvested. mice s/p . myeloma cells were in log-phase growth prior to fusion with spleen cells (yokoyama, ) . hybridoma culture supernatants were screened using elisa, ifa and western blot as described previously . when the desired clones were identified, they were expanded in cm flasks. one week later, the hybridoma suspension was harvested and cell debris pelleted via centrifugation at g for min, followed by collection of the supernatant and storage at − • c. fifty microliters of hybridoma supernatant was incubated in -well microplates (nunc, denmark) coated with purified nucleocapsid protein, and the bound antibody detected with a : dilution of horseradish peroxidase (hrp) labeled rabbit anti-mouse immunoglobulin (dako, denmark). after extensive washing, the plates were incubated with ophenylenediamine dihydrochloride (opd) (sigma, usa) for min and the reaction was stopped by addition of . l m h so . the absorbances at nm were read using a tecan microplate reader. hybridoma supernatants were diluted : in sample buffer and mixed thoroughly by vortexing, followed by analysis with a commercial kit (euroimmuno, germany), in which sars cov infected vero cells were used as the fluorescence antigen. the two techniques were used to further confirm the reactivity to n protein in accordance with our previous descriptions (he et al., , . the reactivity of mab was evaluated with antigen capture elisa in which purified n was employed as the standard antigen and two-fold serially diluted mab as the detector antibody. hybridoma supernatant that did not produce mab served as a negative control. isotyping was performed using a mouse mab isotyping kit (amersham bioscience, england). guinea pigs were immunized with g of purified n protein. booster injections were administered at week intervals. ten days later the animals were euthanized for serum preparation. samples were evaluated for antibody against the n by n -based western blot. subsequently, igg was extracted from the antiserum using protein a affinity chromatography (sigma, usa) and the concentration determined using a bca measurement kit (sigma, usa). the reactivity of the igg with n protein was assessed by an antigen capture elisa, as described below. the -well flat bottom microtiter plates (nunc, demark) were coated with ng of purified igg in l carbonate buffer ( mm sodium bicarbonate and mm sodium carbonate) per well, incubated at • c for h or at • c overnight. plates were washed three times with pbs-t and blocked with l of blocking solution ( % nonfat milk in pbs-t) and incubated at • c for h. after the plates were rinsed with pbs-t for three times, l of purified n protein in pbst containing % nonfat milk was added. the plate was washed three times with pbs-t, and l of mab was added to each well. the plate was washed again and l of rabbit anti-human immunoglobulin hrp-conjugated antibodies was added at : dilution. after another extensive wash with pbs-t, l of opd was added to each well. plates were incubated at room temperature for min and the absorbance was read at nm. for elisa optimization, monoclonal antibody and purified igg were serially diluted two-fold and were used as detector and capture antibody, respectively, in the new assay. optimization conditions were determined by comparing the homologous (n ) and heterologous (n ) reaction to achieve the highest specificity and the signal-to-noise ratio for this assay. the signal-to noise ratio was calculated by dividing the absorbance of homologous antigen by that of heterologous antigen. purified n ( ng/l) was -fold serially diluted with % nonfat milk in pbs-t as diluent and used as tested antigen in the capture elisa format as described above. the diluent was employed as a blank control. prior to being tested, inactivated sars cov culture ( . pfu/ml) was treated with sample lysis solution ( . % tween- , % triton, and % nonfat milk in pbs) and incubated at • c for h. after centrifugation at g for min, the supernatant was assayed. vero, thp- and a cells were used as negative controls. the detection limits of both standard protein and sars cov were determined according to the cut-off value, which was calculated by the formula (x + s.d.). chicken infectious bronchitis virus (ibv), one of the animal coronaviruses, was propagated in spf embryos. debris was removed from the harvested virus supernatant by centrifugation. the titer of virus was determined to be . eid / . ml. ibv was treated using the same sample lysis buffer and methods as those used for sars cov, and assayed in the same way. twenty five microliters of purified n , at concentration of and ng/l, respectively, were mixed with guinea pig anti-n serum and a serum from a sars convalescent patient. additionally, × and × tcid of inactivated sars cov were incubated with guinea pig anti-n serum. normal guinea pig and human normal sera were included as a negative serum controls. these contents were subsequently incubated at • c for h, followed by incubation at • c for h before sandwich elisa analysis, as described above. the percent inhibition of antibody binding was calculated by the following formula: % inhibition = [ . − (a of n + positive serum)/(a of n + normal serum)] × . the specificity of the blocking was confirmed if the percentage of inhibition was greater than . detection of sars cov protein from human infected cells was mimicked using sf- cells expressing recombinant nucleocapsid, protein according to our previous report (he et al., ) . pellets were resuspended in the sample lysis solution, incubated at • c for h, -fold serially diluted with % nonfat milk in pbs-t before being tested. non-infected sf- cells were used as a negative control. the detection limit of recombinant n protein in the cell lysates was determined according to the cut-off value (x + s.d.). thirty serum samples obtained from healthy individuals were used in the test to determine cut-off value for the new capture elisa. subsequently, serum samples, which were collected from patients infected with sars cov during the sars outbreak in singapore in and were shown to be igm-positive using inactivated sars cov-based ifa, and serum samples available from sars recovery patients and blood donors, respectively, were tested by the antigen capture elisa. fusion of spleen cells from immunized balb/c mice with s/p . myeloma cells produced several hybridoma clones secreting mabs against n proteins (data not shown). positive clones were determined through indirect elisa as having at least a three-fold higher absorbance than that of the background. the positive clones showed great variation in their ability to secret mab. the hybridoma cell lines yielding the highest antibody titer, s-a d , was selected to produce monoclonal antibody for further analyses and experiments. after incubation with mab and fitc-conjugated antibody and subsequent examination by fluorescence microscopy, positive cytoplasmic immunofluorescent stainings of the authentic virus antigen in infected vero cells and recombinant n expressed in sf- cells were shown (figs. a and a), identical to those obtained with guinea pig anti-n monospecific antibody (figs. b and b) and with human sars patient serum (figs. c and c), while fluorescent staining was not observed in the noninfected vero cells and sf- cells ( fig. d and e, fig. d and e). the specific reactivity of the mab s-a d with purified n protein (fig. a ) was identical to that of the human sars positive serum (fig. b) , while no reaction was observed when non-antibody secreting hybridoma was tested (fig. c) . the titrations of mab by elisa, n -based western blot, n -spike fusion protein based ifa and sars cov infected cell-based ifa were : , : , : and : , respectively. finally, the isotypes of the representative mab, s-a d , was determined as igg class; elisa reactivity with purified n is shown in fig. . with the increase in the dilution factors of mab, absorbance gradually decreased, while absorbances of mab at : and : dilution were nearly equivalent to those of the blank controls. thus, the reactivity was dose-dependent. to standardize the sandwich elisa, igg was isolated and purified from pooled guinea pig anti-n sera whose titer was determined to be : by western blot. the concentration of extracted igg was determined to be . g/l. sds-page analysis showed two bands of appropriately . and . kda, representing the heavy and light chain regions (data not shown). as expected, a dose-dependent reactivity of purified igg occurred (fig. ) . the absorbances were far greater than those obtained with the same concentration of nonimmunized guinea pig serum. the absorbances decreased dramatically when the igg concentration was . g/well, and the reactivity of . ng of igg was equal to that of the blank igg control. as a first step in the development of our elisa, mab and purified igg were diluted in coating buffer and was used as capture antibodies to coat the microtiter plates, respectively. however, igg binding to the hydrophobic and hydrophilic microtiter plates was more efficient than the mab. therefore, the elisa plates were coated with purified igg and incubated at • c overnight or at • c for h in the following experiments. when the hrp-conjugated antibody was used at the recommended concentration ( : ) in the test, : dilution of capture antibody ( . g/well) and : dilution of mab were determined as the optimal working conditions based on the signal-to-noise ratio ( . ) ( table ). the antigen capture elisa was standardized using the optimal conditions. the absorbance values of normal vero, thp- and a cells were . , . and . , respectively. therefore, the cut-off value for detection of viruses in cell culture was determined as . (x + s.d.). according to the cut-off threshold ( . ), a − dilution of n protein and a − dilution of sars cov suspension were considered positive (fig. ) . so, it was deduced that as little as . pg of purified n protein, tcid of sars cov could be detected. sf- cells ( × cells/ml) had a % infection rate with recombinant baculovirus at h post-infection based on fluorescence assays using mab against n as the primary antibody. a l aliquot of each dilution of sf- cell lysate supernatant was subjected to elisa. absorbance values of . - . were obtained at dilutions of + − to − . according to the calculated cut-off value ( . + [ × . ] = . ), the test was able to detect a − dilution of infected sf- cells ( × cells/ml) (fig. ) . thus, as few as infected sf- cells could be detected by this elisa. according to the cut-off value based on the detection of samples from healthy individuals, sars cov n protein could be detected in all of the ifa igm-positive sera, but none of the nine samples from sars recovery patients using the new elisa. no false positive results were produced in sera from healthy blood donors. the recognition site of the mab on n protein was blocked by guinea pig anti-n serum and human sars positive serum, leading to the lower absorbance (fig. ) . sars cov could not be detected after incubation with anti-n serum. human and guinea pig normal serum could not inhibit the binding of mab to n or native viral nucleocap- in this test, : and : dilution of igg and mab were used in the standardization of the capture elisa. a this number represents the signal-to-noise ratio of the sample measured at different combinations of the capture and detector antibody in the antigen capture elisa. fig. . inhibition effects of guinea pig anti-n serum and human sars positive serum on the recognition of nucleocapsid antigen by detector antibody. groups and : . g of n ; groups and : . g of n ; groups and : × and × tcid of sars cov. the different concentrations of n protein or sars cov were incubated with guinea pig anti-n serum, human sars positive serum (long bars), guinea pig and human normal serum (short bar), respectively. the contents were subjected to capture elisa. sid antigen (longer bars). the calculated inhibition rates were more than % (shorter bars). in addition, no cross-reaction was observed when ibv was tested. this result demonstrates that the antigen capture elisa is highly specific for detecting the nucleocapsid antigen of sars cov. sars cov is an etiological agent causing severe acute respiratory syndrome, a newly emergent disease. there is a global need to develop a sensitive and specific immunoassay to detect sars cov. in our previous work, a major immunodomain of n protein (n ) was identified and n -based western blot assay was developed to detect antibodies against sars cov. therefore, it is reasonable to assume that a mab against this n protein will improve the test specificity. this paper describes the production of monoclonal antibody against n protein as well as the development of an antigen capture elisa, offering a safe, cost-effective tool for detection of sars coronavirus. in this test, the mab reacted with both n and authentic nucleocapsid in sars cov, indicating the potential of this mab to detect sars cov by immunoassays. moreover, the mab at : dilution was detectable by sf- cell-based ifa whereas mab at : dilution could be detected by sars cov infected vero cells-based ifa. this is partially explained by the fact that the nucleocapsid gene, fused with truncated spike gene, was placed under the transcriptional control of the strong polyhedrin promoter of autographa californica nuclear polyhedrosis virus (acnpv) in the recombinant baculovirus, leading to an abundance of n in infected sf- cells. the recognition of the authentic nucleocapsid antigen indicated that the mab produced in this work can be used to develop immunoassays, for instance, ifa, for direct detection of sars cov from nasopharyngeal aspirates obtained from the patients that are diagnosed as probable or suspected cases under clinical criteria. compared to the mab, igg displayed stronger binding to microtiter plates and was therefore used as capture antibody with the mab as detector antibody in this antigen capture elisa. the detection limit of the test is . pg of standard protein and tcid of sars cov. this sensitivity is consistent with the previous descriptions of sensitivity in other antigen capture elisas. in an attempt to further improve the sensitivity of the assay, mab against the immunogenic sc fragment of spike protein was produced in this experiment and used as a detector antibody to detect spike protein in infected sf- cell lysates and inactivated sars cov. however, this method did not improve sensitivity. in the specificity test, the specific binding of both recombinant and native nucleocapsid by mab was uniformly inhibited by guinea pig monospecific antiserum to n and human sars positive serum, but not by human and guinea pig normal sera. due to a lack of coronavirus infected human samples, ibv was used to assess the specificity of the mab for other coronaviruses. in this experiment, ibv could not be recognized by the mab, indicating the mab's specificity. these observations suggest that the mab-based assay is specific for detection of sars cov infection. moreover, the n protein was consistently detected in our limited igm-positive samples but not in eight igg-positive sera from convalescent patients, indicating the potential of early detection of sars cov infection although it needs clinical trials, consistent with new finding (di et al., ) . theoretically, antigen should be released from infected cells or viruses and appear in the host blood system followed by the presence of relative specific antibody. additionally, due to the abundant expression of n protein compared to other proteins during viral replication in infected tissues, the host immune system is exposed to a larger load of n antigen. therefore, this antigen capture elisa, based on mab to n protein, might provide a more sensitive method for early detection of sars cov infection. antibody detection of sars-cov spike and nucleocapsid protein antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay reveals high sensitivity of the nucleocapsid protein in acutephage sea of severe acute respiratory syndrome patients identification of a novel coronavirus in patients with severe acute respiratory syndrome evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome novel immunofluorescence assay using recombinant nucleocapsidspike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus evaluation of antibody responses against sars coronaviral nucleocapsid or spike proteins by immunoblotting or elisa development and evaluation of an efficient -noncoding region based sars coronavirus (sars-cov) rt-pcr assayfor detection of sars-cov infections generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus a one step quantitative rt-pcr for detection of sars coronavirus with an internal control for pcr inhibitors profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers cloning, sequencing, expression, and purification of sars-associated coronavirus nucleocapsid protein for serodiagnosis of sars cumulative number of reported probable cases of severe acute respiratory syndrome (sars) production of monoclonal antibodies induction of sars-nucleoprotein-specific immune response by use of dna vaccine we would like to thank dr. ooi eng eong, dr. ling ai ee, dr. chang hiok hee and dr. bernard leung for providing technical support. this project was funded by agri-food and veterinary authority, singapore. key: cord- - wuttqw authors: pereira, e.p.v.; van tilburg, m.f.; florean, e.o.p.t.; guedes, m.i.f. title: egg yolk antibodies (igy) and their applications in human and veterinary health: a review date: - - journal: int immunopharmacol doi: . /j.intimp. . . sha: doc_id: cord_uid: wuttqw egg yolk constitutes a relevant alternative source of antibodies. it presents some advantages over mammalian serum immunoglobulins regarding productivity, animal welfare and specificity. the main immunoglobulin present in avian blood (igy) is transmitted to their offspring and accumulates in egg yolks, which enables the non-invasive harvesting of high amounts of antibodies. moreover, due to structural differences and phylogenetic distance, igy is more suitable for diagnostic purposes than mammalian antibodies, since it does not react with certain components of the human immune system and displays greater avidity for mammalian conserved proteins. igy has been extensively used in health researches, as both therapeutic and diagnostic tool. this article aims to review its applications in both human and veterinary health. antibodies are protein molecules produced in response to an antigen. due to their ability to bind to specific targets, they are widely used in research, diagnosis and therapy. most of the currently available antibodies are produced in mammals, especially in small rodents [ ] . however, the production of antibodies in mammals can be challenging due to the fact that some antigens elicit weak immune responses or are even completely non-immunogenic. moreover, the production of antibodies in mammals involves procedures that cause pain and distress to animals; such as immunization, blood sample collection and sacrifice [ ] . the search for more efficient and economical techniques, as well as for the reduction and refinement of the use of animals, has led to growing interest for egg yolk antibodies (igy). obtaining antibodies from egg yolk is a non-invasive method that eliminates the need for blood collection. hens produce a greater amount of antibodies compared to other animals, like rodents for example, considerably reducing the number of animals required for the production of antibodies [ , ] . this method presents several economical advantages over the use of other animals. for example, the cost of maintaining a hen is less expensive than maintaining animals like mice and rabbits; the amount of antibodies produced by chickens also corresponds to that of larger animals, such as goats and sheep. for instance, in one year, a hen lays about eggs and produces an average of , g of igy [ ] . moreover, the specific igy produced in chickens are about - % of the total amount of antibodies [ ] . nonetheless, the amount of antibodies produced is correlated to the quantity of antigen applied to the hen, its immunogenicity and molecular weight [ , ] . being a productive technique, as well as more refined from the point of view of animal welfare, igy technology has been used for several purposes in human and veterinary health, such as in immunodiagnostics [ ] , immunotherapy [ ] , neutralization of toxins from venomous animals [ ] and bacteria [ ] , and as functional food [ ] . considering the fast development of igy technology, this work aims to review its applications in human and animal health, in addition to increasing the potential use of these antibodies among researchers and consequently promoting the reduced use of non-invasive procedures on animals. the specific protective effect of egg yolk extracts from immunized hens, attributed to the transfer of serum chicken antibodies to eggs, was first described in [ ] . however, this knowledge remained without applications until it attracted the interest of the scientific community due to the search for animal welfare, which was driven by the works of russel & burch and the publication of the "principles of humane experimental technique" in . the use of igy increased in the s, possibly due to the development of commercially available secondary reagents, such as igy purification kits and anti-igy specific antibodies conjugated to alkaline phosphatase, fluorescein isothiocyanate and peroxidase markers [ ] . in , the european center for the validation of alternative methods (ecvam) workshop recommended the use of igy rather than mammalian igg, with the purpose of minimizing the pain generated by invasive collection of serum antibodies [ ] . igy is present in birds, reptiles, amphibians and lungfish and is the evolutionary precursor of igg and ige, present only in mammals [ ] . over time, igy was called igg due to the supposed similarity between the two. however, leslie & clem emphasized the distinct differences between igy and igg, such as antigenic differences and the major size of igy heavy chain, suggesting the use of this term instead of igg [ ] . the igy molecule has a structure similar to that of igg, with two heavy chains (h), each one with a molecular weight of to kda, and two light chains (l), with kda. the light chains have one constant region (cl) and one variable region (vl), similar to igg. the major difference between igy and igg is found at the heavy chains. igg has three constant regions at the heavy chains (ch -ch ), while igy has four constant regions (ch -ch ) (fig. ) . a further constant domain, with the corresponding carbohydrate chains, gives igy a higher molecular weight ( kda) in comparison to igg ( kda) [ ] . igy is less flexible than igg due to the absence of the hinge region between ch and ch [ ] , similar, in this aspect, to ige [ , ] . the presence of glycine and proline residues at ch -ch and ch -ch regions also limits igy flexibility [ ] . one advantage of this higher chain stiffness is that it may be associated with increased igy resistance to proteolytic degradation and fragmentation [ ] . also, igy is more hydrophobic than igg and has an isoelectric point between . and . . the half-life of purified igy is of months, retaining activity for up to six months at room temperature and one month at °c. moreover, affinity-purified and biotinylated igy retained high activity after five years of storage at °c [ ] . the structural and phylogenetic differences between igy and igg are manifested in different molecular and biochemical interactions. the fc portion of igy is unable to activate the human complement [ ] and to bind to rheumatoid factor [ ] and to protein g [ ] , it also does not react with human anti-mouse antibodies (hama) [ ] nor with the erythrocyte agglutinogens a and b [ ] . due to phylogenetic distance, chickens produce a stronger immune response against conserved mammalian proteins [ ] . although igy contains the variable (v), junction (j) and diversity (d) regions, the contribution of the v (d) j rearrangement to its immunogenetic diversity is small, due to the fact that only a single locus undergoes rearrangement [ ] . thus, the genetic diversity of igy is guaranteed by gene hyperconversion, a process in which pseudogenes donate homologous sequences to the functional gene (v) after v (d) j rearrangement [ ] . even though it is a protein molecule, igy is resistant to heat and ph, being stable between °and °c and active between ph . and . however, the affinity of igy to its antigen decreases with increasing temperature. nonetheless, the addition of sucrose, maltose and glycine protects igy from heat denaturation [ ] , while sorbitol stabilizes it at acid ph [ ] . igy is resistant to inactivation by the proteolytic enzymes trypsin and chymotrypsin, but is degraded by pepsin [ ] . the digestion of igy with papain leads to a fc fragment plus two monovalent fab fragments, like igg. nonetheless, igy digestion with pepsin leads to a pfc′ fragment and two monovalent fab fragments, unlike mammalian antibodies, which forms one bivalent fab fragment when cleaved by pepsin [ ] . the characteristics of igy, in comparison to igg, are summarized in table . several methods have been developed to protect igy from degradation by ph and pepsin present in the stomach, allowing it to arrive unscathed at the small intestine, which is especially important for the use of igy against enteric pathogens. among these treatments, chitosan and alginate microcapsules [ ] , β-cyclodextrin microcapsules [ , ] , copolymers of methacrylic acid [ ] , liposomes [ , ] , multiple emulsification [ ] , hydrogel [ ] and carbon nanotubes with hydrogel [ ] have been tested with different degrees of success. nonetheless, unprotected igy consumed as egg yolk in liquid [ ] and powder forms [ ] showed resistance to the gastrointestinal tract conditions in calves. the antibody titers are influenced by several factors, such as the antigen type and dose, the used adjuvant, the route of application, the inoculation frequency, age and stage of development in birds [ , ] . several protocols for obtaining igy, observing these different variables, have been applied [ , ] . basically, multiple immunization protocols were tested for different antigens and animals, in order to obtain the highest antibody titer for each case [ ] . several antigen types have been used to produce specific igy in birds, such as complex antigens (virus, bacteria and parasites) and single antigens (proteins, polysaccharides, peptides and nucleic acids) [ ] . different antigen concentrations can also be combined with adjuvant. however, it is common to use to μg of antigen per ml injected in two or three sites, with the age of hen ranging between seven and eight weeks [ ] . the induction of high antibodies titers depends on the use of adjuvants. though freund's complete adjuvant (fca) is the most potent to induce antibodies in laboratory animals, it can lead to severe inflammation at the injection site [ ] . although some studies have shown that the immunization with fca is more tolerated by birds than by mammals and seems to not generate tissue damage in chickens [ , ] , other studies using fca for bird immunization contradict these results [ , ] . since there is no consensus, one of the best substitutes for fca is freund's incomplete adjuvant (fia), which, unlike fca, does not contain mycobacteria. shade et al. recommended the use of fia, which can be used since the first immunization without any prejudice to the antibody titer [ ] . in spite of this, the first inoculation is generally performed using fca, while the subsequent inoculations are performed using fia, without the occurrence of inflammation at the injection site [ , ] . the most common route of antigen administration for igy production in chickens is the intramuscular route, where the inoculation is usually into the breast muscle. this technique is most suited for young hens, because subcutaneous injection into the neck is more difficult to perform and may cause more distress to the animal. the intramuscular injection into the leg must be avoided as it can lead to lameness [ ] . nonetheless, for a non-invasive method, the administration of antigen can also be performed orally [ ] . the number of inoculations required depends on the type and dose of the antigen, as well as the adjuvant used. at least two inoculations must be performed before the laying period, with an interval of four to six weeks. the titer of igy must be assessed days after the last immunization. if the antibody titer decreases, further immunizations must be done during the entire laying period to increase antibody titers year round [ ] . an increase in titer of egg yolk antibodies can be observed from the second week [ , ] or sometimes from the fifth week post antigen inoculation [ ] . after a steady increase, stabilization of the antibody titer occurs, reaching a plateau, and from there on it decreases progressively [ ] . through booster inoculations, it is possible to keep high titers of egg yolk antibodies for more than days [ ] . the igy extraction consists of the removal of lipids to form a water soluble fraction (wsf), followed by the precipitation of the antibodies contained therein. several extraction methods are available for obtaining igy from egg yolk and the choice of the suitable method depends on the purpose, which can require different purification degrees, as well as the extraction scale, cost and available technology [ ] . a frequently used method for the extraction of igy is the precipitation with polyethylene glycol (peg) . . the method consists on the delipidation by centrifuging egg yolk diluted in phosphate buffered saline (pbs) with . % of peg . next, the supernatant is twice centrifuged with % of peg to precipitate the antibodies [ ] . akita & nakai obtained highly purified igy by removing the lipids from the yolks by means of six-fold dilution in water at ph . , with an incubation time of h at °c, followed by igy precipitation with % of ammonium sulfate (nh ) so , which was supplemented by the addition of sodium chloride (nacl) or by ultrafiltration prior to gel filtration or ion exchange chromatography. the authors also achieved highly purified igy by means of ethanol precipitation at lower temperatures [ ] . nonetheless, a study conducted by araújo et al. showed that the most suited concentration of (nh ) so for igy's precipitation is %. in this study, higher concentrations of (nh ) so also caused the precipitation of non-specific proteins, while a lower concentration led to a greater loss of antibodies in the supernatant [ ] . since the fc region of igy does not bind to proteins a and g, which are commonly used in the affinity purification of mammalian igg, affinity chromatographic methods for igy purification requires other types of ligands. igy can be purified through the adsorption of the immunizing antigen to the solid phase of the affinity column, which generates monospecific antibodies that can be eluted under acid [ ] or alkaline conditions [ ] . the purification of igy can also be performed through thiophilic adsorption chromatography [ ] . in contrast to other affinity purification methods, it can be performed at mild conditions [ ] . hens are able to produce between and mg of igy antibodies by yolk, of which between and % are specific [ ] . in one year, a single laying hen produces between and g of total igy [ ] . although the production of igy for research is mainly done in chicken, other birds are also used for the same purpose, such as goose [ ] and quail [ ] , using an immunization protocol similar to that used for chickens. the production of igy and its possible applications in both human and veterinary health are summarized in fig. . the use of polyclonal igy against infectious diseases minimizes the risk of microbial resistance, since the antibody is directed to various antigens of the same microorganism, which requires multiple genes for its synthesis [ ] . therefore, specific igy antibodies are a relevant alternative to use as antimicrobials in human and veterinary health in the face of the emergence of resistant bacteria. igy antibacterial activity can be assessed by the inhibition of bacterial growth and biofilm formation in vitro, as well as adhesion ability, since they are prior conditions for successful colonization of a higher animal by bacteria and viruses [ ] . igy antibacterial activity against gastrointestinal pathogens has been widely investigated. nasiri et al. extracted igy from hens immunized with the recombinant protein fanc, from enterotoxigenic escherichia coli (etec) and these antibodies bound specifically to fanc in elisa, western blot and dot-blotting [ ] , demanding, thus, more investigations to evaluate its viability as a potential immunotherapeutic compound. li et al. evaluated the effect of specific and non-specific igy against salmonella typhimurium on the immune response of mice infected by the bacterium [ ] . specific igy reduced the expression of the pro-inflammatory cytokines tnf-α and inf-γ and elevated that of the antiinflammatory cytokine il- ; while non-specific igy also reduced the level of tnf-α, but did not alter the expression level of inf-γ and il- . although both specific and non-specific antibodies reduced the number of t lymphocytes in the lamina propria and of cd + , cd + and γδ t lymphocytes in the jejunum of mice, the animals treated with anti-s. typhimurium igy had a longer survival rate than those treated with nonspecific igy. igy activity against bacterial spores was also investigated. oral administration of igy against clostridium difficile spores both delayed the onset of diarrhea in rats treated prior to the infection and reduced its recurrence in infected rats. thus, specific igy could be used in the prophylaxis of c. difficile infection, as well as an immunotherapeutic to eliminate the remaining spores on the intestines of people who have already had the infection, which would reduce the recurrence of diarrhea [ ] . the therapeutic action of igy against helicobacter pylori has been largely studied as well, through antibodies directed to both the whole cell and to specific proteins. oral administration of anti-hpuc (h. pylori urease) igy inhibited the growth of h. pylori in vitro and reduced the inflammation of stomach in mice, with a significant reduction of neutrophils and lymphocytes infiltration on the tissue. a decrease in the serum level of antibodies against h. pylori was also observed in treated animals [ ] . similar results, as the reduction of serum levels of anti-h. pylori igg and the absence of inflammation signs on the stomach were observed in mice that received oral igy against the h. pylori vacuolating cytokine a (vaca) [ ] . specific igy against hp-nap protein, the main virulence factor of h. pylori, significantly inhibited the adhesion of the bacterium to an ags cells culture [ ] . solhi et al. evaluated the occurrence of cross reaction between strain-specific igy and four different h. pylori strains. the antibodies inhibited the cell growth and the urease enzyme not only of the strains for which they were specific, but also of other strains [ ] . the therapeutic potential of igy against bacterial respiratory tract infections has also been considered. igy against mycobacterium tuberculosis increased the proliferation of pbmc (peripheral blood mononuclear cells) in rats in a dose-dependent manner. an increased expression of cytokines that stimulate the cellular immune response was observed and an increase of mrna level of il- and ifn-γ was detected by rt-pcr, which indicates that anti-m. tuberculosis igy is a potential immunotherapeutic that stimulates t-helper immune response [ ] . shi et al. produced specific igy against multi-drug resistant strains of acinetobacter baumannii. the antibodies inhibited bacterial growth in vitro, significantly reduced the mortality of mice infected with the bacterium and attenuated the lung inflammation [ ] . igy has also been tested as a potential therapeutic resource in oral infections against prevotella intermedia. specific igy inhibited bacterial growth in liquid medium and showed therapeutic activity in rats with p. intermedia induced gingivitis. the reduction of gingival inflammation, bacterial plaque and bleeding, as well as the normalization of wbc (white blood cells) levels in the blood, were observed in treated animals. histopathological examination showed no signs of inflammation in the gums of rats treated with anti-p. intermedia igy [ ] . additionally, studies have shown inhibitory growth effect of therapeutic igy produced against fusobacterium nucleatum in liquid medium and in the formation of biofilm in polyestyrene plates. anti-f. nucleatum igy also reduced bone loss in mice with periodontal disease when applied after the infection, thus presenting therapeutic function [ ] . the therapeutic effect of igy against acne was also evaluated. revathy et al. produced and assessed the efficiency of specific igy against the bacterium propionibacterium acnes. the antibody inhibited bacterial growth and biofilm formation in vitro, proving to be a possible alternative to antibiotics in the treatment of acne [ ] . more recently, igy was tested for antibacterial effect in aquatic animals. gao et al. produced igy against vibrio spp., the major cause of death in white shrimp (litopenaeus vannamei), and demonstrated that powder egg yolk containing anti-vibrio igy significantly reduced the mortality of white shrimp infected with v. harveyi and v. parahaemolyticus [ ] , which indicates that therapeutic igy can also be applied in aquafarming. the prophylactic potential of igy against dental caries has been widely studied. an interesting study carried by bachtiar et al. evaluated the effect of soybean milk containing igy against the cariogenic bacterium streptococcus mutans in rats. the animals fed with this milk presented a decrease in the number of s. mutans in dental biofilm and fifteen days after milk intake igy could still be detected in the saliva [ ] . in other research, the effect of specific igy against the cell-associated glucosyltransferase enzyme (ca-gtf), of s. mutans, was evaluated. as a result, anti-ca-gtf igy inhibited bacterial adhesion in vitro and suppressed the oral cavity colonization [ ] . in addition, specific igy action against the comd protein, a quorumsensing signals receptor of s. mutans, was also explored. it inhibited, in vitro, the development of biofilm of s. mutans from the oral cavity of people with and without caries, and changes in the protein expression pattern were found in the bacteria treated with anti-comd igy [ ] . the protective effect of igy against opportunistic infections was also investigated by thomsen et al., where, by means of chemiluminescence, the production of reactive oxygen species (ros) in polymorphonuclear neutrophils (pmn) was evaluated as an indicator of the intensity of pmn mediated immune response against pseudomonas aeruginosas in vitro, as well as the influence of anti-p. aeruginosas igy in this process. the specific igy opsonized the bacterium and intensified the cellular immune response, probably due to the recognition of igy by receptors resembling avian igy receptors or physical-chemical changes in the bacterium, since igy does not activate mammalian fc receptors. anti-p. aeruginosas igy could be used in the prophylaxis of p. aeruginosas infection in patients with cystic fibrosis by increasing the cellular immune response [ ] . prophylaxis of bacterial infections in animals for consumption, using igy, was also considered. specific igy incorporated into hydrogel added to carbon nanotubes and chitosan (h-cnt) showed protective activity against enterotoxigenic escherichia coli (etec) in piglets [ ] . thus, anti-etec igy incorporated into h-cnt and orally applied could be used to prevent the intestinal infection in these animals. in order to generate antibodies against the bacterium campylobacter jejuni to be used as food additive aiming to control c. jejuni colonization in chickens, thibodeau et al. produced anti-c. jejuni igy in three different ways: oral administration of a pool of living bacteria from four c. jejuni strains, subcutaneous injection of extracts from the outer membrane of the bacterium or of formalin inactivated bacterium. the hens submitted to subcutaneous inoculation produced igy more intensely, although the antibodies obtained through oral inoculation also showed good activity. anti-c. jejuni igy, obtained from the three ways, presented bactericidal activity in vitro [ ] . igy against the bacterium aeromonas salmonicida, the causative agent of ulcers on fish skin, was added, in powder form, to the water in which koi carp (cyprinus carpio koi) ornamental fish were raised. anti-a. salmonicida antibodies showed good prophylactic activity against the infection, and a small amount of specific igy was enough to completely prevent the appearance of skin ulcers in healthy fish that cohabited with sick fish previously infected with a. salmonicida [ ] . the potential of igy technology to prevent bacterial infections in animals could be useful in the needful strategies to reduce the use of antibiotics in food animal husbandry, which has been linked to the selection and spread of resistant bacterial strains that threaten antibiotics as a therapeutic resource [ , ] . the therapeutic potential of igy against viral infections of the gastrointestinal tract in animals has been extensively investigated. oral administration of egg yolk powder rich in igy against bovine group a rotavirus (rva) was performed on calves infected by the virus. as a result, disease attenuation occurred in treated animals, with a reduction of the period of diarrhea and hyperthermia and the absence of other symptoms observed in untreated calves, such as anorexia, dehydration and depression. the egg yolk powder enriched with anti-rva igy retained the activity after two years, when kept at °c [ ] . in another study, igy against the s protein of porcine epidemical diarrhea virus (pedv) was produced and orally administered in piglets previously infected with pedv. anti-s igy reduced the severity of diarrhea and intestinal lesions caused by pedv infection and nullified the mortality of piglets due to the disease [ ] . another possible therapeutic use of specific igy is against dengue fever, as demonstrated by fink et al. anti-denv igy produced in goose was able to neutralize the virus in vitro and in vivo without binding to fcγ receptors on myeloid cells and generating ade (antibody dependent enhancement) in mice [ ] . the protective effect of igy against influenza has been widely studied. igy against the avian influenza a virus (h n ) were extracted from eggs available in supermarkets of vietnam, where the vaccination of chickens against h n virus is required. anti-h n igy was administrated intranasally in mice before and after their infection with h n and h n and completely prevented the disease onset [ ] . these results reveal that commercially available eggs that are produced in countries where anti-h n vaccination is mandatory constitute a considerable source of igy that could be used to prevent a potential pandemic of h n virus. following this rationale, wallach et al. produced igy against h n , h n and h n strains of influenza virus, which were tested for their ability to neutralize homologous and heterologous strains in vitro and to prevent the infection in vivo. the antibodies inhibited only homologous strains in the seroneutralization and in the haemagglutination inhibition assay, except anti-h n igy, which also inhibited h n . anti-h n and anti-h n igy were applied intranasally in mice h before the infection by influenza virus and showed prophylactic activity. thus, igy against influenza virus strains could be used in nasal, oral or spray applications to protect individuals and environments [ ] . igy against influenza b virus (ibv) was also tested and neutralized the activity of haemagglutinins and neuraminidase present in the virus and inhibited viral replication in vitro. when applied intranasally, anti-ibv igy prevented influenza development in mice treated prior to virus exposure and attenuated the disease in those treated post-infection [ ] . the prophylactic effect of igy against other viral infections of the respiratory tract was also evaluated. igy against andes virus (andv), the causative agent of the hantavirus pulmonary syndrome (hps), was obtained from geese eggs by inoculating these animals with the dna encoding the virus envelope glycoprotein. anti-andv igy was able to prevent hps development in recently infected mice but failed when applied after the onset of viremia, thus presenting prophylactic and non-therapeutic activity [ ] . the protective effects of igy were also considered for viral infections in poultry. aizenshtein et al. produced, in the same eggs, efficient igy against the newcastle disease virus (ndv), infectious bursal disease virus (ibdv), influenza and reovirus, which are pathogenic for birds [ ] , demonstrating that passive immunization with igy against several viruses is possible; nonetheless, it is a technology that must be further explored. a gel preparation for oral use containing igy against candida albicans was tested by tekeuchi et al. and caused a reduction in the number of colony-forming units (cfu) on the oral cavity of elderly people, showing promise for prophylactic use against c. albicans oral infection [ ] . in other research, specific igy inhibited the adhesion of candida albicans and candida glabrata to denture base material. anti-c. albicans igy was more effective against c. albicans than anti-c. glabrata igy, while both antibodies were equally effective in preventing the adhesion of c. glabrata [ ] . sampaio et al. produced a high avidity igy against the protozoan trypanosoma evansi. there was neither prophylactic action nor infection control in the treated rats, but anti-t. evansi igy increased their survival rate when used concomitantly to anti-hematozoa drugs [ ] . in another study, grando et al. inoculated trypanosoma cruzi trypomastigots in hens and extracted a high amount of anti-t. cruzi igy that cross-reacted with t. evansi antigens in western blot, showing that the anti-t. cruzi igy is not reliable as a diagnostic tool, but deserves more investigations as a possible therapeutic resource for trypanosomes infection [ ] . the phylogenetic distance between birds and mammals ensures a stronger immune response against mammalian antigens by birds [ ] . such a feature may be advantageous to produce igy against human tumor antigens. following this rationale, amirijavidv et al. produced highly specific igy against a sequence of amino acids present on the ectodomain of the trail (tnf-related apoptosis-inducing ligand) receptor trail-r (dr ). the antibodies bound to the amino acid sequence and activated the dr receptors in human breast cancer cells mcf , acting as a trail agonist and inducing apoptosis [ ] . igy against other receptors, such as the her receptor, was tested coupled to single walled carbon nanotubes (swnts) and specifically detected the her receptors on the surface of sk-br- cells. the binding of the complex to the receptors was measured by raman signals emitted by the nanotubes. swnt has a near infrared absorption (nir), which can be used for tumor ablation, and, coupled to anti-her igy, was able to kill sk-br- cells without needing internalization of the complex by the cell [ ] . these findings show that igy produced against tumor antigens is an attractive alternative for a more selective treatment of cancers and its use could, therefore, minimize the side effects of traditional chemotherapy. igy raised against porcine pancreatic lipase was used against the enzyme in vitro and in vivo. later, mice with obesity induced by high fat diet were orally treated with the antibody, which was given concomitantly with food, and a reduction of adipose tissue and liver fat level was observed, as well as an increase of fecal excretion of triglycerides and their decrease in blood plasma. anti-lipase igy inhibited the hydrolysis of diet fat and reduced its intestinal absorption, showing anti-obesity activity [ ] . wei-xu et al. evaluated the antiallergic effect of specific igy against the pro-inflammatory cytokines il-β and tnf-α in guinea pigs with induced allergic rhinitis. a reduction of the eosinophils number in the blood and in the nasal and bronchial lavages was found, as well as a decrease of eosinophils, neutrophils and lymphocytes infiltration into the nasal mucosa and the lungs of animals treated with anti-il-β and anti-tnf-α igy, alone or jointly [ ] . one of the side effects that occur in individuals receiving antivenom serum produced in goats, sheep and horses is due to the presence of serum proteins on the anti-venom serum derived from these animals, in which igg is not sufficiently purified [ , ] . one advantage of using igy in anti-venom serotherapy is that it is easily purified, which would minimize the occurrence of side effects due to nonspecific proteins. araujo et al. demonstrated this property when specific igy was produced as anti-venom of the snake genus bothrops sp. these antibodies neutralized a pool of venoms from five bothrops species, with an ed of μl/ ld , showing little to no side effects in mice [ ] . mendonza et al. also produced igy capable of neutralizing the venom of the peruvian snake bothrops atox. the anti-venom igy showed considerable cross reaction with the venom of bothrops brazili and could be used not only as b. atox anti-venom, but also as a tool for the research of cross reaction with venoms from different species [ ] . in another elegant work, andrade et al. produced igy against a pool of venoms from snakes of the genus bothrops and against the venom from the species crotalus durissus terrificus. anti-venom igy extracted from eggs was compared to the horse anti-venom igg in western blot. the results showed that specific egg's igy recognized the same antigens as the equine anti-venom [ ] . igy against coral snake venom was first produced in response to a pool of venoms from different species of micrurus. these antibodies recognized, by western blot, venom proteins from several snakes: m. isozonus, m. surinamensis, m. f. fulvius, naja kaouthia, n. pallida, bothrops colombiensis, crotalus durissus cumanensis and c. vegrandis and could, therefore, be used as a broad-spectrum snake anti-venom [ ] . zolfagharian and dounighi produced igy by inoculating the vipera lebetina snake venom, inactivated by γ radiation, in hens [ ] . these antibodies were effective in neutralizing the crude venom of vipera lebetina in mice. anti-venom igy were also obtained from eggs of hens immunized with the venom of the snake trimeresurus albolabris. these igy recognized, by western blot, most of the proteins present in the t. albolabris venom and neutralized it in mice in a dose-dependent manner [ ] . more recently, liu et al. extracted and purified igy from eggs of hens inoculated with the venom of the deinagkistrodon actus snake. these antibodies were able to neutralize the lethal effects of the venom, such as bleeding, edema formation and myotoxicity in a dose-dependent manner [ ] . da rocha et al. produced igy against ophidian toxins of crotalus durissus terrificus, bothrops jararaca and bitis arietans. the antibodies were able to bind to specific components of the venoms in western blot and protected % of the intoxicated mice when obtained after the ninth inoculation. the authors recommended the use of a small antigen dose ( μl) applied in successive inoculations for igy production, since this dose was enough to genetically alter the v(d)j segments on the naïve cells and to generate immunological memory [ ] . however, igy raised against the venom of the snake oxyuranus scutellatus was less effective than equine igg, being unable to neutralize the neurotoxic and coagulant effects of the venom [ ] . nonetheless, this result cannot be extended to igy produced against other venoms. igy against the tityus caripitensis scorpion venom, produced by alvarez et al., neutralized not only the venom of t. caripitensis, but also that of other tityus species (t. quirogae, t. discrepans and t. gonzalespongai), and inactivated the hyaluronidase, an enzyme that facilitates the toxin spread in the tissues, present in the t. serrulatus venom [ ] . thus, igy raised against t. caripitensis venom could be used as a broadspectrum anti-scorpionic serum. another application of igy technology, the prophylaxis of celiac disease, was demonstrated by gujral et al., who developed powdered egg yolk formula with protective sugars containing anti-gliadin igy, among which, the formula with mannitol (eyp-m) retained its activity after being submitted, in vitro, to chemical conditions analogous to those of the stomach and small intestine. the formula igy-epy-m neutralized in vitro both the isolated gliadin and that present in food matrix and inhibited its intestinal absorption in mice, showing promising for the prevention of celiac disease [ ] . bobeck et al. used igy against the human intestinal alkaline phosphatase (hiap) to assess the influence of iap on increased bioavailability of phytate phosphate in the presence of α-dihydroxycholecalciferol (vitamin d ) in chickens. anti-hiap igy was ingested by chickens and reduced the absorption of phytate phosphate, which suggests that although it performed less adequately than sevelamer chorhydrate, already used for the same purpose, anti-hiap igy can be optimized for the prevention of phytate phosphate toxicity induced by the consumption of the active form of vitamin d [ ] . the ability of igy to detect viral pathogens of the gastrointestinal tract in humans and animals has been widely studied. igy raised against canine parvovirus viral like particles (cpv-vlps) were used in elisa and immunochromatography, showing sensitivity and specificity in the detection of canine parvovirus in dog fecal samples [ ] . specific igy against the e protein of bovine viral diarrhea virus (bvdv) were used in elisa to detect pathogens that cause diarrhea in cattle. anti-e igy showed a high specificity, recognizing only bvdv. elisa and immunochromatography tests using these antibodies were efficient in detecting bvdv in serum samples of cows with diarrhea, showing a concordance of , % and % with rt-pcr, respectively [ ] . da silva et al. used igy against hepatitis a virus (hav) in a competitive immunoenzymatic assay to detect anti-hav igg in serum samples, showing satisfactory sensitivity and specificity [ ] . more recently, igy against hav was used to detect the virus in hepatic sections of infected rhesus monkeys by means of indirect immunofluorescence (iif). anti-hva igy was more efficient than the commercially available igg for the detection of the same antigen [ ] . igy was also used to detect viral pathogens in aquatic animals. specific igy against the soft-shelled turtle systemic septicemia spherical virus (stsssv) was used to compose a lateral flow assay to detect the virus in turtles. this assay was sensitive and specific, detecting stsssv in all infected turtles in serum and feces samples [ ] . a potential for diagnosis was presented by igy raised against the nucleocapsid protein (np) of coronavirus (cov). anti-np igy, used as capture antibody in elisa for detecting np, lowered its detection limit to a picogram level, which indicates that the antibody is promising for use in the diagnosis of acute respiratory syndrome associated to coronavirus (sars-cov) and is sensitive enough to detect small amounts of np [ ] . moreover, the ability of igy in diagnosing dengue fever was also evaluated. figueiredo et al. produced igy against the non-structural protein (ns ) of dengue virus (denv ). these antibodies were used to compose an immunosensor that was effective in electrically detecting the ns protein of denv in standard samples and could be used for dengue diagnosis in biological samples [ ] . one of the most studied diagnostic potential of igy is against staphylococcus aureus. the fact that the fc portion of igg reacts with the staphylococcal protein a makes igy a relevant resource for more specific detection of different s. aureus strains and their toxins, since, due to structural differences, the fc portion of igy does not react with protein a [ ] . following this rationale, walczak et al. produced igy against the fibrinogen binding protein (efb) of staphylococcus aureus and against a peptide epitope that encompasses the residues - of efb protein. anti-efb and anti-efb - antibodies presented high titers in elisa and strong avidity in western blot, showing promising use in the diagnosis of s. aureus infection [ ] . another elisa test using igy against the staphylococcal enterotoxin b (seb) of s. aureus as capture antibody and specific ssdna aptamers coupled to biotin as revealing was developed by mulidi et al. this assay was efficient in detecting seb, but also reacted with others staphylococcal toxins, such as staphylococcal endotoxins a (sea) and c (sec), toxic shock syndrome toxin (tsst) and α-hemolysin [ ] . igy raised against α-hemolysin was applied as capture antibody in elisa for detecting the toxin in the supernatant of s. aureus cultures. anti-αha igy showed high specificity against the toxin, without reacting with protein a, which is present in all s. aureus strains [ ] . the ability of igy to diagnose resistant s. aureus was also investigated. yamada et al. produced igy against the penicillin binding protein (pbp) a, which is present in methicillin resistant s. aureus (mrsa) strains. anti-pbp a igy was used in elisa, lateral flow and latex to detect mrsa and other s. aureus strains sensitive to methicillin and beta-lactams. the antibody was mrsa specific and did not react with the sensitive strains that express significant amounts of protein a [ ] . among the parasitic infections tested, cakir-koc evaluated the potential of igy in detecting the protozoan toxoplasma gondii by producing igy against its surface protein sag- , which reacted with the target antigen in elisa and western blot [ ] . therefore, this study revealed a potential diagnostic test for toxoplasmosis. more recently, anti-sag igy conjugated to fluorescein isothiocyanate detected t. gondii tachyzoites in a colony, by means of immunofluorescence, and may be used to detect the protozoan in other types of samples [ ] . in other interesting research, an indirect elisa using igy to detect cathepsin f (cf), a cysteine protease present in the helminth opisthorchirs viverrini, was developed. this assay showed good sensitivity in the detection of o. viverrini in fecal samples from humans living in endemics places; however, a cross reaction with taenia spp., echinostoma spp. and minute intestinal fluke (mif) was observed [ ] . therefore, improvements still have to be made before this technology is available. miura et al. produced igy against the gp protein, from the cryptosporidium hominis protozoan. these igy bound to gp in western blot and to c. parvum sporozoites in fecal samples by indirect immunofluorescence, suggesting that anti-gp igy could be used in the diagnosis of cryptosporidiosis caused by both c. hominis and c. parvum [ ] . several authors have been investigating the potential of igy in the detection of tumor markers. igy against the peptide antigen ca - , a commonly used breast cancer marker, was used as secondary antibody in a sandwich elisa aiming to detect ca - , showing potential for clinical use [ ] . sun et al. produced igy against two portions of the transmembrane glycoprotein her : her -a, proximal region, and her -b, distal region. anti-her -a and anti-her -b igy effectively detected the her glycoprotein in cultured breast cancer sk-br- cells by immunofluorescence and in sections of breast tumors over expressing her -b, using immunohistochemistry. in addition, the antibodies bound to the glycoprotein in elisa and western blot, which indicates that igy against her is promising for use in breast cancer diagnosis [ ] . in another study, Łupicka-słowik et al. developed a direct elisa test using a lysate of human malignant tumor cells and igy against bovine adenosine deaminase (c-ada). the assay was efficient in detecting human adenosine deaminase (h-ada) present in the tumor cells lysate, which was possible due to the high homology between c-ada and h-ada. the elisa test using anti-c-ada igy could be used to diagnose several types of malignant tumors in humans, as well as be an alternative to currently employed enzymatic methods for the detection and quantification of ada for pleural tuberculosis diagnosis [ ] . another igy developed to detect prostatic specific antigen (psa) and two peptide fragments of this protein demonstrated specificity and marked the antigen more strongly than the igg counterpart using western blot analysis. however, anti-psa igy had an unsatisfactory sensitivity when applied as secondary antibody in indirect elisa [ ] , thus deserving more investigation. in general, these findings suggest that the phylogenetic distance between birds and mammals, that ensures a stronger immune response against mammalian antigens by birds than by other mammals [ ] , makes the production of igy against tumor antigens advantageous not only for therapeutic purposes, as described above, but also for usage in several types of immunoassays for tumor detection in humans. hens immunized with umbilical cord sera produced specific igy against igg and the complement fractions c b and c d. these antibodies did not react with the c b fraction -which configures a higher specificity, since anti-c b antibodies often cross-reacts with the antigens of chido/rodgers rbc group -nor with erythrocyte antigens from abo group. these antibodies are, therefore, promising as a reagent for coombs test [ ] . igy immunoassays were used to detect the hepatic expression of cytochrome p e (cyp e ) in mice treated with medicinal herbs and products derived from plants rich in flavonoids. cyp e metabolizes a wide variety of chemicals with different structures, in particular small and hydrophobic compounds, including potential cytotoxic and carcinogenic agents. anti-cyp e igy was specific, without reacting with other cytochromes, and was able to detect the reduction of hepatic cype e expression due to the ingestion of natural products with hepatoprotective effects [ ] . the ability of igy in identifying harmful substances in consumer products has been evaluated. an elisa test was developed to detect the staphylococcal enterotoxin g (seg), using specific igy, and showed satisfactory sensitivity and specificity, reducing the interference of protein a that occurs in igg tests. this test was successfully used to detect seg in milk and dairy products samples and could therefore be used to identify the toxin in food [ ] . bittner et al. used igy in elisa to detect potentially allergenic proteins in commercially available latex gloves. this assay presented similar results to that of the gold standard test, which uses mammalian igg [ ] . igy can also be used in assays to identify antibiotic residues in food products of animal origin, as demonstrated in a study by he et al., in which produced anti-gentamycin igy specifically detected the target antibiotic present in animal origin products [ ] . following this rationale, li et al. used specific igy to detect kanamycin and gentamycin residues in milk and meat samples by means of competition elisa and fpia (fluorescence polarization immunoassay) [ ] . the potential of igy in identifying substances has also been used to evaluate the toxicity of a natural product employed in the alternative medicine. igy labeled with quantum dot were successfully used in a lateral flow assay for the detection of rhein; a toxic substance found in the plant rheum officinale, widely used in chinese traditional medicine; in plant samples and serum from users [ ] . igy raised against the bacterium listeria monocytogenes showed a significant inhibitory effect of bacterial growth in liquid medium and in fish samples stored between and °c in a dose-dependent manner, which indicates that anti-l. monocytogenes igy is a potential antimicrobial for use in the food industry [ ] . taking into consideration the versatility and the range of igy already tested against several bacteria, this result could be easily apply to other food poisoning bacteria and viruses. among the importance of this technology, leclair et al. demonstrated that igy produced against the staphylococcal enterotoxin b (seb), a potential biological weapon, can save individuals exposed to this material. the results with rhesus monkeys showed that animals that received anti-seb igy min before or h after exposure to a lethal seb aerosol survived [ ] , which indicates that anti-seb igy could be used to protect populations in a hypothetical context of bioterrorism involving seb [ ] . the latest findings using igy have clearly demonstrated the versatility of this technology. obtaining igy from birds presents several technical and economical advantages over mammalian igg, and as described in this review, igy technology has a broad spectrum of applications in human and veterinary health. it can be used in multiple types of therapies; it is useful in the prevention of various types of diseases and detects, by means of different techniques, several classes of antigens, such as microorganisms, tumor markers and substances. among the advantages of this technology, the replacement of invasive antibody collection by its extraction from eggs is one of the most interesting, considering the animal welfare benefits, with this technology it is possible to achieve great quantities of antibodies with a lower cost of production and less damage to animal welfare. due to its structural differences and phylogenetic distance, igy is more specific for diagnostic use and displays greater avidity for mammalian conserved proteins than igg, being, thus, an important alternative in the search for more effective diagnostics and therapies. in addition, in view of its proven ability to neutralize microorganisms, igy represents an important therapeutic resource in times of increasing resistance to antibiotics and emergence of viral diseases for which there is no treatment. there are no funding or competing interests to report. chicken egg yolk antibodies (igy) as an alternative to mammalian antibodies production of antibodies in chickens the production of avian (egg yolk) antibodies: igy. the report and recommendations of ecvam workshop igy technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (peg) precipitation avian antibodies (igy) against trypanosoma cruzi: purification and characterization studies antibodies to proteins from yolk of immunized hens chicken egg yolk antibodies (igy) for detecting circulating antigens of schistosoma japonicum oral passive igybased immunotherapeutics: a novel solution for prevention and treatment of alimentary tract diseases eficacia experimental de anticuerpos igy producidos en huevos, contra el veneno de la serpiente peruana bothrops atrox protection against bacterial superantigen staphylococcal enterotoxin b by passive vaccination suppressive effect of functional drinking yogurt containing specific egg yolk immunoglobulin on helicobacter pylori in humans ueber natürliche immunität und ihre verwerthung für die immunisirungstherapie igy-technology: application and trends, epc - th european poultry conference igy: clues to the origins of modern antibodies phylogeny of immunoglobulin structure and function chicken immunoglobulin gamma-heavy chains: limited vh gene repertoire, combinatorial diversification by d gene segments and evolution of the heavy chain lócus avian igy antibodies and their recombinant equivalents in research, diagnostics and therapy chicken antibodies: a tool to avoid interference by complement activation in elisa use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factors protein g: a powerful tool for binding and detection of monoclonal and polyclonal antibodies chicken antibodies: a clinical chemistry perspective, ups extraction of a monospecific coombs-reagent from chicken eggs efficient production of chicken egg yolk antibodies against a conserved mammalian protein rearrangement of immunoglobulin genes in chicken b cell development avian b-cell development: generation of an immunoglobulin repertoire by gene conversion acid stability of anti-helicobacter pyroli igy in aqueous polyol solution oral passive immunization effect of anti-human rotavirus igy and its behavior against proteolytic enzymes production and purification of fab′ fragments from chicken egg yolk immunoglobulin y (igy) chitosan-alginate microcapsules for oral delivery of egg yolk immunoglobulin (igy) passive immune-protection of litopenaeus vannamei against vibrio harveyi e vibrio parahaemolyticus infections with anti-vibrio egg yolk (igy)-encapsulated feed microencapsulation protects immunoglobulin in yolk (igy) specific against helicobacter pylori urease microencapsulation for the gastric passage and controlled intestinal release of immunoglobulin y encapsulation of chicken egg yolk immunoglobulin g (igy) by liposomes protective effect of microencapsulation consisting of multiple emulsification and heat gelation processes on immunoglobulin in yolk ph-responsive hydrogels to protect igy from gastric conditions: in vitro evaluation in vitro toxicity evaluation of hydrogel-carbon nanotubes composites on intestinal cells egg yolk igy: protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves egg yolk igy antibodies: a therapeutic intervention against group a rotavirus in calves chicken egg yolk antibodies (igytechnology): a review of progress in production and use in research and human and veterinary medicine hen egg yolk antibodies (igy), production and use for passive immunization against bacterial enteric infections in chicken: a review antibody production in rabbits and chickens immunized with human igg. a comparison of titre and avidity development in rabbit serum, chicken serum and egg yolk using three different adjuvants freund's complete adjuvant in the chicken: efficient immunostimulation with severe local inflammatory reaction stable biocompatible adjuvants-a new type of adjuvant based on solid lipid nanoparticles: a study on cytotoxicity, compatibility and efficacy in chicken evaluation of igy capture elisa for sensitive detection of alphahemolysin of staphylococcus aureus without staphylococcal protein a interference generation and application of polyclonal igy antibodies specific for full-length and nicked prostate-specific antigen production and characterization of anti-campylobacter jejuni igy derived from egg yolks antibacterial activity of egg yolk antibody (igy) against listeria monocytogenes and preliminary evaluation of its potential for food preservation preparation and characterization of egg yolk immunoglobulin y specific to influenza b vírus highly sensitive detection of cancer antigen - using novel avian igy antibodies development of chicken egg yolk antibodies against streptococcus mitis -purification and neutralizing efficacy lmmunoglobulins from egg yolk: isolation and purification brazilian igy-bothrops antivenom: studies on the development of a process in chicken egg yolk isolation of immunoglobulin in yolk (igy) and rabbit serum immunoglobulin g (igg) specific against bovine lactoferrin by immunoaffinity chromatograph hen egg yolk antibodies purified by antigen affinity under highly alkaline conditions provide new tools for diagnostics. human intact parathyrin as a model antigen production and characterization of igy against canine igg: prospect of a new tool for the immunodiagnostic of canine diseases thiophilic adsorption chromatography dengue virus specific igy provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement development of anti-helicobacter pylori immunoglobulins y (igys) in quail production and characterization of egg yolk antibody (igy) against recombinant vp -s antigen chicken egg yolk antibodies (igy) modulate the intestinal mucosal immune response in a mouse model of salmonella typhimurium infection characterization of chicken igy specific to clostridium difficile r spores and the effect of oral administration in mouse models of initiation and recurrent disease treatment of helicobacter pylori infection in mice with oral administration of egg yolk-driven anti-urec immunoglobulin preventive effect of anti-vaca egg yolk immunoglobulin (igy) on helicobacter pylori-infected mice inhibitory effects of rhp-nap igy against h. pylori attachment to ags cell line in vitro evaluation of cross-strain inhibitory effects of igy polyclonal antibody against h. pylori, microbial pathog koerniasari, the activity of immunoglobulin y anti-mycobacterium tuberculosis on proliferation and cytokine expression of rat peripheral blood mononuclear cells effects of specific egg yolk immunoglobulin on pan-drug-resistant acinetobacter baumannii protective effect of an egg yolk-derived immunoglobulin (igy) against prevotella intermedia-mediated gingivitis effectiveness of egg yolk immunoglobulin (igy) against periodontal diseasecausing fusobacterium nucleatum in vitro evaluation of the efficacy of chicken egg yolk antibodies (igy) generated against propionibacterium acnes effects of soybean milk, chitosan, and anti-streptococcus mutans igy in malnourished rats' dental biofilm and the igy persistencyin saliva anticell-associated glucosyltransferase immunoglobulin y suppression of salivary mutans streptococci in healthy young adults anti-pseudomonas aeruginosa igy antibodies promote bacterial opsonization and augment the phagocytic activity of polymorphonuclear neutrophils igy against enterotoxigenic escherichia coli administered by hidrogel-carbon nanotubes composites to prevent neonatal diarrhoea in experimentally challenged piglets ulcer disease prophylaxis in koi carp by bath immersion with chicken egg yolk containing anti-aeromonas salmonicida igy medical consequences of antibiotic use in agriculture industrial food animal production, antimicrobial resistance, and human health immunoprophylactic effect of chicken egg yolk antibody (igy) against a recombinant s domain of the porcine epidemic diarrhea vírus spike protein in piglets prophylactic and therapeutic efficacy of avian antibodies against influenza virus h n and h n in mice cross-protection of chicken immunoglobulin y antibodies against h n and h n viruses passively administered in mice antiviral biologic produced in dna vaccine/goose platform protects hamsters against hantavirus pulmonary syndrome when administered post-exposure practical aspects in the use of passive immunization as an alternative to attenuated viral vacines effects of oral moisturising gel containing egg yolk antibodies against candida albicans in older people use of candida-specific chicken egg yolk antibodies to inhibit the adhering of candida to denture base materials: prevention of denture stomatitis production, purification and therapeutic potential of egg yolk antibodies for treating trypanosoma evansi infection apoptotic killing of breast cancer cells by igys produced against a small aminoacid epitope of the human trail- receptor, asian pac anti-her igy antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells antiobesity activity of hen egg anti-lipase immunoglobulin yolk, a novel pancreatic lipase inhibitor anti-interleukin- beta/tumor necrosis factor-alpha igy antibodies reduce pathological allergic responses in guinea pigs with allergic rhinitis a comparison of ovine and equine antivenoms the production and characterization of antibothropic and anti-crotalic igy antibodies in laying hens: a long term experiment coral snake antivenom produced in chickens (gallus domesticus) study on development of vipera lebetina snake anti-venom in chicken egg yolk for passive immunization anti-trimeresurus albolabris venom igy antibodies: preparation, purification and neutralization efficacy preparation and neutralization efficacy of igy antibodies raised against deinagkistrodon acutus venom development of igy antibodies against anti-snake toxins endowed with highly lethal neutralizing activity development of a chicken-derived antivenom against the taipan snake (oxyuranus scutellatus) venom and comparison with an equine antivenom antibodies anti-tityus caripitensis venom: purification and neutralization efficacy in-vitro and in-vivo binding activity of chicken egg yolk immunoglobulin y (igy) against gliadin in food matrix oral antibodies to human intestinal alkaline phosphatase reduce dietary phytate phosphate bioavailability in the presence of dietary α-hydroxycholecalciferol evaluation of chicken igy generated against canine parvovirus viral-like particles and development of enzyme-linked immunosorbent assay and immunochromatographic assay for canine parvovirus detection preparation of chicken igy against recombinant e protein of bovine viral diarrhea virus (bvdv) and development of elisa and ica for bvdv detection an immunoenzymatic assay for the diagnosis of hepatitis a utilising immunoglobulin y using immunoglobulin y as an alternative antibody for the detection of hepatitis a virus in frozen liver sections development of a colloidal gold immunochromatographic strip for the rapid detection of soft-shelled turtle systemic septicemia spherical vírus diagnostics of severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid antigen using chicken immunoglobulin y electrical detection of dengue biomarker using egg yolk immunoglobulin as the biological recognition element the binding of staphylococcal protein a by the sera of different animal species method for generation of peptide-specific igy antibodies directed to staphylococcus aureus extracellular fibrinogen binding protein epitope a novel igy-aptamer hybrid system for cost-effective detection of seb and its evaluation on food and clinical samples detection of methicillin-resistant staphylococcus aureus using a specific anti-pbp a chicken igy antibody production of anti-sag- igy antibody against toxoplasma gondii parasites and evaluation of antibody activity by elisa method novel fitc-labeled igy antibody: fluorescence imaging toxoplasma gondii in vitro chicken igy-based coproantigen capture elisa for diagnosis of human opisthorchiasis evaluation of recombinant cryptosporidium hominis gp protein and anti-gp chicken polyclonal igy for research and diagnostic purposes highly sensitive detection of cancer antigen human epidermal growth factor receptor using novel chicken egg yolk immunoglobulin development of adenosine deaminase-specific igy antibodies: diagnostic and inhibitory application evaluation of igy antibody as a polyspecific coombs-reagent development of an igy antibody-based immunoassay for the screening of the cyp e inhibitor/enhancer from herbal medicines development of igy based sandwich elisa for the detection of staphylococcal enterotoxin g (seg), an egc toxin content of asthmagen natural rubber latex allergens in commercial disposable gloves development of indirect competitive elisa using egg yolk-derived immunoglobulin (igy) for the detection of gentamicin residues detection of kanamycin and gentamicin residues in animal-derived food using igy antibody based ic-elisa and fpia quantum dot-based lateral-flow immunoassay for rapid detection of rhein using specific egg yolk antibodies key: cord- - vm k authors: okba, nisreen m.a.; muller, marcel a; li, wentao; wang, chunyan; geurtsvankessel, corine h.; corman, victor m.; lamers, mart m.; sikkema, reina s.; de bruin, erwin; chandler, felicity d.; yazdanpanah, yazdan; le hingrat, quentin; descamps, diane; houhou-fidouh, nadhira; reusken, chantal b. e. m.; bosch, berend-jan; drosten, christian; koopmans, marion p.g.; haagmans, bart l. title: sars-cov- specific antibody responses in covid- patients date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: vm k a new coronavirus, sars-cov- , has recently emerged to cause a human pandemic. whereas molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. validated serologic assays are important for contact tracing, identifying the viral reservoir and epidemiological studies. here, we developed serological assays for the detection of sars-cov- neutralizing, spike- and nucleocapsid-specific antibodies. using serum samples from patients with pcr-confirmed infections of sars-cov- , other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial elisas. we demonstrate that most pcr-confirmed sars-cov- infected individuals seroconverted, as revealed by sensitive and specific in-house elisas. we found that commercial s igg or iga elisas were of lower specificity while sensitivity varied between the two, with iga showing higher sensitivity. overall, the validated assays described here can be instrumental for the detection of sars-cov- -specific antibodies for diagnostic, seroepidemiological and vaccine evaluation studies. in december , a new coronavirus (cov) emerged in china to cause an acute respiratory disease known as coronavirus disease (covid- ) ( ) . the virus was identified to be a betacoronavirus related to severe acute respiratory syndrome coronavirus (sars-cov) and thus, was named sars-cov- ( ) . in less than two decades, this virus is the third known coronavirus to cross the species barrier and cause severe respiratory infections in humans following sars-cov in and middle east respiratory syndrome in , yet with unprecedented spread compared to the earlier two. due to the rapid rise in number of cases and uncontrolled and vast worldwide spread, the who has declared sars-cov- a pandemic. as of march th , the virus has infected over , individuals in countries, . % of which had a fatal outcome ( ) . the rapid identification of the etiology and the sharing of the genetic sequence of the virus, followed by international collaborative efforts initiated due to the emergence of sars-cov- have led to the rapid availability of real-time pcr diagnostic assays that support the case ascertainment and tracking of the outbreak ( ) . the availability of these has helped in patient detection and efforts to contain the virus. however, specific and validated serologic assays are still lacking at the moment and are urgently needed to understand the epidemiology of sars-cov- . validated serologic assays are crucial for patient contact tracing, identifying the viral reservoir hosts and for epidemiological studies. epidemiological studies are urgently needed to help uncover the burden of disease, in particular, the rate of asymptomatic infections, and to get better estimates on morbidity and mortality. additionally, these epidemiological studies can help reveal the extent of virus spread in households, communities and specific settings; which could help guide control measures serological assays are also needed for evaluation of the results of vaccine trials and development of therapeutic antibodies. among the four coronavirus structural proteins, the spike (s) and the nucleocapsid (n) are the main immunogens ( ) . here, we describe development of serological assays for the detection of virus neutralizing antibodies and antibodies to the nucleocapsid (n) protein and various spike (s) domains including the s subunit, and receptor binding domain (rbd) of sars-cov- in elisa format. using a wellcharacterized cohort of serum samples from pcr-confirmed sars-cov- and patients pcr-confirmed to be infected with seasonal coronaviruses and other respiratory pathogens, we validated and tested various antigens in different platforms developed in-house as well as a commercial platform. serum samples were collected from pcr-confirmed mild and severe covid- patients ( table ) from france in accordance with the local ethical approvals. samples used for assay validation were from persons pcr-diagnosed infections with of human coronaviruses (hcov- e, nl or oc ), sars, mers, or with a range of other respiratory viruses ( table ) as published previously ( ) . samples from patients with recent cmv, ebv or mycoplasma pneumoniae infection were included as these have a higher likelihood of causing false positive results. we used serum samples from healthy blood donors (cohort a) as negative controls; sanquin blood bank (rotterdam, the netherlands) obtained written informed consent for research use. all samples were stored at - °c until use. the use of serum samples from the netherlands was approved by the local medical ethical committee (mec approval: - ). serum samples from sars patients ( ) were kindly provided by professor malik peiris, hong kong university. all serum samples from covid- pcr-confirmed cases ( n = ) were previously analyzed by recombinant sars-cov- spike protein-based immunofluorescence test and plaque reduction neutralization (wölfel et al submitted manuscript, doi:https://doi.org/ . / . . . ). sera were tested as part of an extended diagnostic regimen upon informed written consent of patients. all non-sars-cov- sera (n= ) stemmed from the serum collection of the national consiliary laboratory for coronavirus detection at charité, berlin, germany and were provided for diagnostic proposes upon informed written consent. the collection contained follow-up sera from pcr-confirmed human cases: n= hcov- e, n= hcov-hku , n= hcov-oc , n= mers-cov, n= hcov-nl , n= sars-cov and n= common cold cov antibody positive sera. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the spike ectodomains of sars-cov- (residues - , ; strain wuhan-hu- ; genbank: qhd . ), sars-cov (residues - , ; strain cuhk-w ; genbank: aap . ) and mers-cov (residues - ; strain emc; genbank: yp_ . ) were expressed in hek- t cells with a c-terminal trimerization motif and strep-tag using the pcaggs expression plasmid. likewise, the sars-cov- s subunit or its subdomains (s;s , residues - ; s a , residues - ; rbd, residues - ; genbank: qhd . ) were expressed in t cells as prnt was used as a reference for this study, because neutralization assays are the standard for cov serology. we tested serum samples for their neutralization capacity against sars-cov- (german isolate; gisaid id epi_isl ; european virus archive global # v- ) by plaque-reduction neutralization test (prnt) as previously described for with some modifications ( ) . heat-inactivated samples were -fold serially diluted in dmem medium supplemented with nahco , hepes buffer, penicillin, streptomycin, and % fetal bovine serum, starting at a dilution of : in μl. fifty μl of the virus suspension ( spot forming units) was added to each well and incubated at °c for h. following incubation, the mixtures were added on vero-e cells and incubated at °c for more hour. the cells were then washed and further incubated in medium for h. after the incubation, the cells were fixed and stained with polyclonal rabbit anti-sars-cov antibody (sino biological). the cells were then fixed and stained using a rabbit anti-sars-cov serum and a secondary peroxidase-labelled goat antirabbit igg (dako). the signal was developed using a precipitate forming tmb substrate (true blue, kpl) and the number of infected cells per well were counted using the immunospot® all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint image analyzer (ctl europe gmbh). the serum neutralization titer is the reciprocal of the highest dilution resulting in an infection reduction of > % (prnt ). a titer of > was considered to be positive. the prnt for the german sera was done as described previously using vero e cells after hour at °c the supernatants were discarded, the cells were washed once with pbs and supplemented with . % avicel solution in dmem. after days at plates were fixed and inactivated using a % formaldehyde/pbs solution and stained with crystal violet. we performed anti-sars-cov- igg and iga elisa using beta-versions of two commercial kits (euroimmun medizinische labordiagnostika ag, https://www.euroimmun.com) and performed the assay according to manufacturer's protocol. reagent wells of both assays are coated with recombinant structural protein (s domain) of sars-cov- . the optical density (od) was detected at nm, and a ratio of the reading of each sample to the reading of the calibrator, included in the kit, was calculated for each sample (od ratio). as the beta-version of the kit awaits ce validation, we determined an in-house cut-off value based on the mean background reactivity of all sars-cov- -negative sera in the study multiplied by . in case of iga this was od ratio = . and for igg od ratio = . . we performed the inhouse elisas by coating -well microtiter elisa plates with in-house produced spike antigens (s or s of sars-cov- , sars-cov or mers-cov; or sars-cov- s a , or rbd proteins) or sars-n (sinobiological) in pbs overnight at °c. following blocking, diluted serum ( : or -fold serially diluted for titers) was added and incubated at °c for h. antigen-specific antibodies were detected using peroxidase-labeled rabbit antihuman igg (dako, https://www.agilent.com) and tmb as a substrate. the absorbance of each all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint sample was measured at nm. we set cutoffs at standard deviations above the mean value of the negative cohort. serum samples were previously tested for antibodies against the s of different coronaviruses as described earlier ( ). the correlations between antibody responses detected by different elisas were and those detected by prnt, as the gold standard for cov serology, were analyzed using graphpad prism version (https://www.graphpad.com). we evaluated sars-cov- specific antibody responses in severe and mild cases using serum samples collected at different times post-disease onset from three french pcr-confirmed covid- patients. we tested sera for sars-cov- specific antibodies using different elisas. following infections, all three patients seroconverted between days and post onset of disease (figure ) , and antibodies were elicited against the sars-cov- s and s subunit including the n-terminal (s a ) domain and the receptor binding domain (rbd). since the n protein of sars-cov- is % similar to that of sars-cov ( table ) , we used sars-cov n as an antigen to test for sars-cov- n-directed antibodies in an elisa format. we found that, following infection, antibodies were elicited against the n protein and when tested in a prnt assay these sera were able to neutralize sars-cov- . we observed cross-reactivity with the sars-cov s and s proteins, and to a lower extent with mers-cov s protein, but not with the mers-cov s protein (figure g-h) . this was evident from analyzing the degree of similarity of the different cov s protein domains to their corresponding sars-cov- proteins ( table ) , where sars-cov showed high similarities in all different s domains. the analysis showed that the spike s subunit is more conserved among covs and thus plays a role in the cross-reactivity seen when the whole s was used as an antigen. thus, s is a more specific than s as an antigen for sars-cov- serological diagnosis. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. we further assessed the specificity of the s assay using cohorts a-e (table ) comprising of sera from healthy blood donors (a), pcr-confirmed acute respiratory non-cov infections (b), acute to convalescent pcr-confirmed alpha-and beta-hcov infections (c), and pcr-confirmed mers-cov (d) and sars-cov (e) infections. none of the sera from specificity cohorts a-d were reactive in our in-house s elisa at the set cut-off indicating % specificity, whereas sera from sars-cov patients cross-reacted (figure a) . additionally, the specificity of s as an antigen for sars-cov- serology was further supported by the fact that - % of the cohort a-c sera included in this study were seropositive for the endemic hcovs (hcov-hku , hcov-oc , hcov-nl , and - e) as determined by the s protein microarray (figure b) . nonetheless, all were seronegative for sars-cov and mers-cov. using the same cohort, we also validated the specificity of the anti-nucleocapsid and anti-rbd igg elisas for detecting sars-cov- specific antibodies. at the set cut-off, except of sars-cov patient sera, none of the control sera tested positive for anti-rbd nor antinucleocapsid antibodies (figure c,d) , whereas we detected seroconversion among the three covid- patients. these validated elisas for different antigens can be useful for epidemiological studies as well as for evaluation of vaccine-induced immune responses. next, we validated the sensitivity and specificity of commercial elisa kits for detecting s -specific igg and iga antibodies using the same cohort (table , figure ). all three covid- patients had reactive antibodies by both the igg ( / serum samples) and iga ( / serum samples) elisas (figure ) . while sars-cov patient's sera were reactive as noted earlier, we also detected reactivity of serum samples from the validation cohorts a-d; / for iga and / for igg elisas. two hcov-oc (a β-cov) patients' sera were reactive in both igg and iga elisa kits. we confirmed the cross-reactivity of the two sera by testing twelve serum samples from both patients collected at different time points, pre-and post-oc infection. while all pre-infection sera were negative, all post-infection sera were reactive in both igg and iga based elisas. we have earlier reported cross-reactivity of these sera in a mers-cov s igg elisa kit ( ) . further validation was also done in a different laboratory (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint nl , x hcov-oc ) as well as mers-cov (n= ) and sars-cov (n= ) infected persons collected - days post disease onset (figure ) . all covid- patients were previously confirmed to seroconvert at days - post onset of disease using recombinant immunofluorescence test and prnt. / seroconverted patients showed reactivity above the implemented cut-off values in the igg and iga elisa. one patient (figure , black line) maintained slightly below the cut-off which might be explained by an overall reduced antibody response of this patient (prnt = ). overall, the iga-based elisa kit was more sensitive but less specific than the igg based elisa kit. finally, we compared the performance of the different elisas for the detection of antibodies among pcr-confirmed covid- patients to that of prnt, as the gold standard for cov serology ( table ) . prnt correlated strongly with the different elisas, with the commercial iga showing the strongest correlation followed by the s and n elisas indicating their capacity to detect sars-cov- specific antibodies. however, a larger patient cohort is needed to assess the sensitivity of these platforms. validated sars-cov- serological assays are currently lacking, yet urgently needed for contact tracing, epidemiological and vaccine evaluation studies. since the n and the s proteins are the main immunogenic cov proteins, we developed elisa-based assays, which were able to detect antibodies to these two proteins along with the two spike domains, s a and rbd. those correlated strongly with virus neutralizing antibodies detected by a prnt assay. since the majority of the human population has antibodies against the four endemic human coronaviruses, it was crucial to verify the specificity of these assays to avoid false positive results. additionally, the two zoonotic coronaviruses, sars-cov and mers-cov, are also beta-coronaviruses, raising potential for cross-reactivity. among the spike protein antigens tested, the s was more specific than s in detecting sars-cov- antibodies, as mers-cov-s cross-reactive antibodies were detected in the serum of one of the covid- patients which was not seen when mers-cov s was used for testing. this could be explained by the high degree of conservation in the cov s subunit relative to the s ( table ) . therefore, consistent with our earlier findings for mers-cov serology ( ), s is a specific antigen for sars-cov- diagnostics. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. when testing the specificity s or its rbd for detecting sars-cov- antibodies, none of the sera form the validation cohorts (a-e) showed any reactivity; except for sars-cov patients sera. this -not-unexpected -cross-reactivity resulted from the high degree of similarity between the s and rbd of the sars-cov and sars-cov- ( table ) . however, sars-cov has not circulated in the human population since , i.e. years ago, and an earlier study reported waning of sars-cov-specific antibodies which made them undetectable in serum samples of % ( / ) of samples tested years following infection ( ) . it is therefore unlikely that antibodies to this virus are present in the population and thus there could hardly be a chance that false positives result from sars-cov-antibodies reactivity. meanwhile, we made use of the high degree of similarity between the sars-cov and sars-cov- proteins for the development of our inhouse n elisa, where we used sars-cov n ( % similar to sars-cov- ) as an antigen. the n-elisa could detect sars-cov- -specific antibodies with high specificity and sensitivity. using the three different validated elisas, we found that antibody levels were higher following the severe infection compared to the mild ones; similar findings has been reported earlier for mers-cov ( , ) . however, this needs to be confirmed with a larger cohort of patients with varying degrees of severity, while it still highlights the potential need of a sensitive assay to avoid missing those with milder infections in epidemiological studies. among the inhouse elisas tested, the rbd and n elisas were more sensitive than s elisa in detecting antibodies in mildly infected patients and showed stronger correlation with prnt titers. therefore, detecting antibodies against two different antigens might be needed to confirm the findings and avoid false negatives in surveillance studies. however, the sensitivities of the assays need to be further validated with a larger cohort. we further validated beta-versions of an iga and an igg s commercial elisa in two different labs. while the iga-based elisa showed higher sensitivity than the igg-based elisa, the opposite was true for the specificity where the igg elisa was more specific than the iga elisa. yet, we noted some cross reactivity in both elisas with serum samples from the same two hcov-oc patients that cross reacted in a mers-cov s igg elisa ( ) despite the different antigen coated. this indicates a response to another protein which could be in the blocking or coating matrix, apart from the specific antigen coated, resulting in this consistent false positive result. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. overall, the assays developed and validated in this study could be instrumental for patient contact tracing, serosurveillance studies, as well as vaccine evaluation studies. however, since various studies will be carried out in different labs, it is crucial to calibrate and standardize assays developed by different labs using well defined standard references as a part of diagnostic assay validation. this is not only needed to reduce interassay variability, but to also harmonize the results obtained from different labs using various assays ( ) . this is crucial for better comparison and interpertaion of results from different studies as well as evaluation of vaccine trials, allowing for uniform assessment of immunogenicity, efficacy and better understanding of correlates of immune protection ( ) . thus, setting up reference panels is a vital element in our preparedness approaches to emerging viruses. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. protein sequences were aligned using clustalw. n, nucleocapsid; s, spike; s ,the n-terminal subunit of the spike protein; s , the c-terminal subunit of the spike protein; s a, domain a of the spike s subunit; rbd, receptor binding domain; nd, not done. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. p value summary *** **** ns, non-significant p > . ; * reflects significance level: **, p ≤ . ; ***, p ≤ . ; ****, p ≤ . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint figure : kinetics of antibody responses against sars-cov- following infection. we tested one severe (red) and two mild (green and black) sars-cov- patients for antibody responses against the (a) spike (s), (b) spike s subunit, (c) spike n-terminal (s a ) domain, (d) receptor binding domain, (e) nucleocapsid proteins using elisas. (f) virus neutralizing antibodies were tested using the plaque reduction neutralization assay (prnt). reactivities of sera form the three patients at different time points against whole spikes (g) and s (h) of sars-cov- , sars-cov and mers-cov were tested by elisa. sars-cov, severe acute respiratory syndrome coronavirus; mers-cov, middle east respiratory syndrome coronavirus; all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. kinetics of antibody responses in three covid- patients (c,d). cross-reactivity of some hcov-oc sera in the commercial platforms (e,f). correlation between antibody responses detected by the elisas and the plaque reduction neutralization assay (prnt ; g,h). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . correlation between antibody responses detected by the elisas and the plaque reduction neutralization assay (prnt ; c,d). (e,f) the kits were tested for specificity using serum samples from hcov ( x hcov- e, x hcov-hku , x hcov-nl , x hcov-oc ) as well as mers-cov (n= ) and sars-cov (n= ) infected persons. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint a pneumonia outbreak associated with a new coronavirus of probable bat origin the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- . nature microbiology coronavirus disease (covid- ) situation reports detection of novel coronavirus ( -ncov) by real-time rt-pcr. euro surveillance : bulletin europeen sur les maladies transmissibles = european communicable disease bulletin serological assays for emerging coronaviruses: challenges and pitfalls. virus research sensitive and specific detection of low-level antibody responses in mild middle east respiratory syndrome coronavirus infections. emerging infectious diseases newly discovered coronavirus as the primary cause of severe acute respiratory syndrome dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc blocking transmission of middle east respiratory syndrome coronavirus (mers-cov) in llamas by vaccination with a recombinant spike protein. emerging microbes & infections transmission of merscoronavirus in household contacts. the new england journal of medicine lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study mers-cov antibody responses year after symptom onset, south korea antibody response and disease severity in healthcare worker mers survivors. emerging infectious diseases comparison of serologic assays for middle east respiratory syndrome coronavirus. emerg infect dis international biological reference preparations for epidemic infectious diseases. emerg infect dis this work was supported by the zoonoses anticipation and preparedness initiative (zapi project; imi grant agreement no. ), with the assistance and financial support of imi and the european commission, in-kind contributions from efpia partners. key: cord- -flhfagp authors: nicol, thomas; lefeuvre, caroline; serri, orianne; pivert, adeline; joubaud, françoise; ducancelle, alexandra; lunel-fabiani, françoise; le guillou-guillemette, hélène title: assessment of sars-cov- serological tests for the diagnosis of covid- through the evaluation of three immunoassays: two automated immunoassays (euroimmun and abbott) and one rapid lateral flow immunoassay (ng biotech) date: - - journal: j clin virol doi: . /j.jcv. . sha: doc_id: cord_uid: flhfagp background: the emergence of new sars-cov- has promoted the development of new serological tests that could be complementary to rt-pcr. nevertheless, the assessment of clinical performances of available tests is urgently required as their use has just been initiated for diagnose. objectives: the aim of this study was to assess the performance of three immunoassays for the detection of sars-cov- antibodies. methods: two automated immunoassays (abbott sars-cov- clia igg and euroimmun anti-sars-cov- elisa igg/iga assays) and one lateral flow immunoassay (lfia ng-test® igg-igm covid- ) were tested. specimens were analyzed from patients with a positive rt-pcr response, from patients with symptoms consistent with covid- but exhibiting a negative response to the rt-pcr detection test, and from control group specimens. days since symptoms onset were collected from clinical information sheet associated with respiratory tract samples. results: overall sensitivity for igg was equivalent (around %) for clia, elisa and lfia. sensitivity for igg detection, > days after onset of symptoms, was . % for all assays. overall specificity for igg was greater for clia and lfia (more than %) compared to elisa ( . %). specificity was significantly different between iga elisa ( . %) and igm lfia ( . %) (p < . ). the best agreement was observed between clia and lfia assays ( %; k = . ). conclusion: excellent sensitivity for igg detection was obtained > days after onset of symptoms for all immunoassays. specificity was also excellent for igg clia and igg lfia. our study shows that ng-test® is reliable and accurate for routine use in clinical laboratories. a new acute respiratory syndrome named coronavirus disease has emerged from the region of wuhan in china in december . this infection, widespread all over the world, is caused by a novel sarbecovirus designated severe acute respiratory syndrome coronavirus (sars-cov- ), associated with severe morbidity and mortality [ ] [ ] [ ] . the detection of viral rna by real time reverse transcriptase-polymerase chain reaction (rt-pcr) in respiratory tract samples is considered as the gold standard method for screening and diagnosis in the early phase of infection. however, sensitivity is variable depending on sample types, suitable sampling technique, the anatomic site, time of infection and viral load [ ] [ ] [ ] . chest computed tomography (ct) may be helpful for the diagnosis, complementary to rt-pcr, but it remains unspecific [ ] . development of new serological tests [ , ] , readily available and easier to perform compared to requirements of molecular assays in laboratories [ ] , could be helpful as a complementary diagnostic tool and to increase the sensitivity of tests especially in patients with late complications i.e. severe pneumonia. different assays have recently been commercialized: automated tests (enzyme-linked immunosorbent assays [elisa] or chemiluminescence enzyme immunoassays [clia]) or rapid detection test (lateral flow immunoassays, lfia). lfia seems to be very attractive for large seroprevalence studies because these tests can be used easily as point of care tests or in the laboratory, with a result in less than minutes. serological tests can be used for symptomatic individuals for which rt-pcr testing was either not performed at the time of acute illness or for which nasopharyngeal swab result was found to be negative, and also for epidemiological studies (close contacts screening, screening of health care workers …) [ , ] . however, the relevance of serological tests is highly related to their clinical performance, hence antibody (ab) assays with good sensitivity and specificity are needed. despite a growing number of available assays, related j o u r n a l p r e -p r o o f clinical performances are still scarce [ ] [ ] [ ] [ ] [ ] or unknown and individual studies are usually inconclusive. moreover, the quality and diagnostic performance of rapid tests have already been questioned in spain and united kingdom [ , ] . the aim of the study was to assess the clinical performance of ce marked assays available in europe to detect sars-cov- antibodies: two automated immunoassays (euroimmun and abbott assays) targeting two different proteins and also one lateral flow immunoassay (ng biotech). then, serum samples with a potential cross-reaction to the sars-cov- immunoassays were investigated (table ) . samples from pregnant women and sera from patients with positive rheumatoid factor (rf) were also tested. the study was approved by the institutional board of the angers university hospital. j o u r n a l p r e -p r o o f the euroimmun anti-sars-cov- elisa igg and iga assays (euroimmun, lüebeck, germany) were performed according to the manufacturer's guidelines on the ds ® system, an automated microplate technology (dynex technologies gmbh, denkendorf, germany). the microplate wells are coated with recombinant s structural protein and the assay detects anti-sars-cov- igg and iga against the viral spike protein (sp). the abbott sars-cov- igg (abbott, ireland) was performed according to the manufacturer's instructions on the automated abbott architect i sr instrument the assay is a clia for qualitative detection of igg antibodies against the sars-cov- nucleoprotein (np) in serum or plasma. ng-test® igg-igm covid- (ng biotech laboratoires, guipry-messac, france) is an immune colloidal technique intended for the qualitative detection of igg and igm antibodies against the sars-cov- nucleoprotein in serum or plasma. μl of specimen, were added onto the sample loading area followed by drops of sample dilution solution. the results were read and interpreted min after testing. all statistical analyses were performed using ibm® spss® . statistics software (statistical package for social sciences, ibm corp., chicago, il). to assess the sensitivity and specificity, j o u r n a l p r e -p r o o f we choose the rt-pcr method as gold standard. time from onset symptoms was used to determine sensitivity and specificity. grey zone was considered positive for the statistical analyses. a p value < . was considered statistically significant. the cohen's kappa value was determined for agreement between assays. sensitivities and specificities obtained with three immunoassays are summarized in table table summarized overall agreement and agreement relative to the time of symptoms onset between three immunoassays. overall, excellent agreement was observed between the three assays. the best agreement was observed between clia and lfia ( %; cohen kappa index of . ). even for the first week of symptoms onset, an excellent agreement was observed between elisa and lfia assays ( %; k= . ). however, poor agreement was observed between elisa and clia ( %; k= . ). overall agreement between igg/iga elisa and igg/igm lfia was excellent ( %; k= . ). the iga, igm and igg ab kinetics were studied using specimens from seven patients (positive rt-pcr) with serial results and interesting kinetics ( figure ) . then, five patients presented an earlier igg seroconversion using clia compared to elisa, the first week of symptom onset. among these patients, we observed in three patients an igm line with lfia and iga elisa was positive for four patients. using lfia, results were more easily interpretable for igg line than for igm line. igm line was difficult for reading, notably for seven sera. j o u r n a l p r e -p r o o f discussion a strong clinical performance of assays in diagnosis and management of covid- is essential to quickly contain the covid outbreak worldwide. therefore, the development of serological assays, routinely used in clinical laboratories to determine recent infection or previous contact with viruses, is a good option complementary to rt-pcr method [ ] . on may, , the french health authority (haute autorité de santé) and infectious diseases society of america recommended that patients with symptoms consistent with covid- but having a positive result for sars-cov- by rt-pcr may be diagnosed by serological tests [ , ] . various immunoassays are available on the european market [ , ] and subjected to european regulations with the mandatory ce marked for sales. nevertheless, the european commission, in its april recommendations, allowed exceptionally the marketing of tests that do not have the ce marked, in the interest of public health [ ] . here, we evaluated three different ce marked commercial immunoassays for detection of sars-cov- antibodies in human serum and plasma. elisa assay was performed on a semiautomated microplate technology requiring high handling and with a limited capacity of tests per day ( tests per h). in contrast, clia assay is a fully automated random-access test and that can perform over , tests per h. these two assays are used in clinical laboratories, unlike lfia, which can be used as a point of care test or in clinical laboratories and provides a result within min. performance of euroimmun assay has been evaluated in some studies [ , , [ ] [ ] [ ] [ ] , showing sensitivity for igg between % and % > days after symptoms onset and specificity between and %. few studies reported clinical performance of abbott assay [ , , , ] . sensitivity for igg was between % and % more than days post symptom onset and specificity between and %. in our study, we showed a sensitivity for igg of j o u r n a l p r e -p r o o f % for clia abbott and elisa euroimmun assays > days after symptoms onset and an overall specificity for igg of . % and . % with elisa and clia respectively. we carried out a large cross-reactivity study and more false positives results were observed using elisa than clia as previously described [ ] . recently, many ce marked lfia became available. two studies showed that sensitivity and specificity were similar to those of euroimmun assay [ , ] .however, to our knowledge, only one study compared clinical performance between clia abbott and lfia [ ] and no study described diagnostic performance of ng-test®. here, we observed an excellent agreement for igg between clia and lfia days after onset symptoms (k= . ), and an excellent sensitivity and specificity for both assays. lfia advantages are the ability to reach larger population groups, when used in point-of-care, and to evaluate the herd immunity without saturating the capacity of laboratories. however, these devices must be used with caution. trained staff or automated reader devices are needed for good interpretation of result. traceability of results may be at fault in case of use at the point-of-care and results may not be reported to the health authorities for seroprevalence studies. to evaluate sensitivity, some manufacturers or authors used the time from positive rt-pcr rather than the time from symptom onset. however, there is a risk of misestimating sensitivity as some patients presented late after the onset of symptoms with disease progression at time of the first pcr testing. then, sensitivity and specificity must be interpreted with caution. the use of rt-pcr as gold standard may decrease the real number of patients infected by sars-cov- due to false negative results. in our study, we observed false positive results with iga elisa and few with igm lfia. no false positive with igm lfia were observed with for rf specimens whereas interferences were described with some other immunoassays [ ] . elslande et al pointed out that the elisa iga should not be used for the screening of asymptomatic persons. it might be better not to measure igm or iga since it may result in a significant number of false-positive results without improving diagnostic performance. [ ] . it would appear here that igm detection with the lfia provides a gain in diagnostic performance. developed immunoassays target either the sp or the np of sars-cov- [ ] , involving different immune ab responses. however, related studies are controversial. some studies described that early antibody response was targeted against np and then sp inducing an earlier positivity of the tests targeting np [ , ] . by contrast, another study revealed that the spbased elisa was more sensitive than the np-based one in the detection of igm [ ] . here, we did not observe any significant difference between sensitivity of iga elisa and igm lfia in conclusion, our study showed equivalent clinical performance for igg of three immunoassays (elisa, clia and lfia) > days after symptoms onset. the three assays had, as expected, a poor sensitivity during first days of symptom onset. therefore, serological tests can be useful to confirm past covid- , to do epidemiologic studies days after symptoms j o u r n a l p r e -p r o o f onset [ ] or to identify people who could return to the workplace, even if its use is still widely discussed [ ] . for asymptomatic patients with rt-pcr negative, a higher threshold must be used [ ] . a lower threshold ( - days) should be used for symptomatic patients > days with negative rt-pcr and clinical presentation consistent with covid- . currently, it is not clear whether igg antibodies are protective against reinfection [ ] . finally, even if the lfia is reliable on serum or plasma, studies should be conducted to evaluate the performance on fingerstick; a process commonly used for seroprevalence studies. this research did not receive any specific grant from funding agencies. ng-test® igg-igm covid- rapid test cassettes (ng biotech laboratoires) were kindly provided by the manufacturer. the authors declare that they have no conflict of interest. world health organization china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study vitro diagnostic assays for covid- : recent advances and emerging trends laboratory diagnosis of emerging human coronavirus infections -the state of the art evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of -ncov infections, medrxiv chest ct for typical -ncov pneumonia: relationship to negative rt-pcr testing developing antibody tests for sars-cov- severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease patients world health organization significance of serology testing to assist timely diagnosis of sars-cov- infections: implication from a family cluster the laboratory diagnosis of covid- infection: current issues and challenges evaluation of two j o u r n a l p r e -p r o o f automated and three rapid lateral flow immunoassays for the detection of anti-sars-cov- antibodies clinical performance of two sars-cov- serologic assays serological immunochromatographic approach in diagnosis with sars-cov- infected covid- patients performance characteristics of the abbott architect sars-cov- igg assay and seroprevalence in diagnostic performance of covid- serology assays covid- in spain: unreliability of new tests delays effort to slow coronavirus spread in spain | society | el paÍs in english says millions of coronavirus test kits bought from china are unreliable for most patients clinical and virological data of the first cases of covid- in europe: a case series different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid- patients place des tests sérologiques dans la stratégie de prise en charge de la maladie covid- idsa covid- antibody primer sars-cov- diagnostic pipeline brief clinical evaluation of six high-throughput sars-cov- igg antibody assays clinical performance of different sars-cov- igg antibody tests performance characteristics of four high-throughput immunoassays for detection of igg antibodies against sars-cov- diagnostic performance of rapid igg/igm antibody tests and the euroimmun iga/igg elisa in covid- patients evaluation of covid- igg/igm rapid test from orient gene biotech a method to prevent sars-cov- igm false positives in gold immunochromatography and enzyme-linked immunosorbent assays interpreting diagnostic tests for sars-cov- the trinity of covid- : immunity, inflammation and intervention evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov- clinical performance of sars-cov- igg antibody tests and potential protective immunity, microbiology the important role of serology for covid- control waiting for certainty on covid- antibody tests -at what cost? protective immunity after covid- has been questioned: what can we do without sars-cov- -igg detection? %) se: . ( . - . %) se: . ( . - . %) the authors thank the laboratory technicians who helped us.the authors thank thomas le guillou for proofreading the english manuscript.j o u r n a l p r e -p r o o f key: cord- -vkzya uq authors: ijaz, m. k.; sabara, m. i.; frenchick, p. j.; babiuk, l. a. title: effect of different routes of immunization with bovine rotavirus on lactogenic antibody response in mice date: - - journal: antiviral research doi: . /s - ( ) - sha: doc_id: cord_uid: vkzya uq summary the effect of different routes of immunization with either live or killed bovine rotavirus (brv) on the production of lactogenic antibody response in mice was evaluated. the routes of immunization were intramuscular (im), oral (o) or intradermal in the mammary region (imam). following immunization, serum antibody responses were monitored by an enzyme linked immunosorbent assay (elisa). following whelping, the mice were allowed to stay with their mother until sacrificed on alternate days post-parturition from day – . milk from their stomach was collected for antibody titration by elisa and virus neutralization test. regardless of the routes of immunization, a rapid increase in serum anti-rotavirus antibody titers was observed for the first wk after immunization followed by a gradual decline. after parturition, the mean antibody titer of lacteal secretions, as determined by elisa, increased gradually for days with the greatest increase on day , followed by a decrease in anti-rotavirus antibody. these titers also correlated with antibody titers in milk as measured by virus neutralization test. the best lactogenic antibody response was observed when imam × im × route of immunization was used with live brv as the antigen. interestingly, immunization via the oral route with killed brv also resulted in good antibody responses. in contrast, in the group where killed brv was used, animals receiving × orally had the highest antibody titer. the distribution of different antibody subtypes in milk samples revealed igg to be the predominant antibody followed by igm and iga. irrespective of the route of administration, there was an increase in iga on day as compared to day in most of the groups. the significant role played by mucosal immunity in passive protection and the possible ways to modulate subtype specific lactogenic immune response are discussed. animals models; lactogenic immunity; rotaviruses rotavirus has been identified as the single most important causative agent of acute neonatal enteritis in a variety of domestic and laboratory animals and humans. consequently, a world-wide effort is being made to develop an appropriate vaccine against this pathogen (babiuk et al., ; flewett and babiuk, ; hanson et al., ) . although previous studies have indicated no correlation between protection from rotavirus infection and the titers of passively acquired antibodies in the serum of newborns (saif and smith, ; m.k. ijaz et al., unpublished data) , it has been shown that the continuous presence of anti-rotavirus antibodies in the intestine of neonates is important in preventing clinical disease associated with rotavirus infections (offit and clark, . ; saif and smith, ; snodgrass et al., ; m.k. ijaz et al., unpublished data) . in several animal species, including humans, immunization during pregnancy has been shown to result in the presence of antibodies in colostrum and milk (goldblum et al., ; saif and smith, ) . in addition to the humoral immune response, the role played by the cellular component of immunity in the production of lactogenic immune response has also been documented (parmerly and beer, ; ripenhoff-talty et al., ) . cell transfer experiments in mice have revealed the migration of lymphoblasts from mesenteric lymph nodes into the intestine and mammary gland and other secretory organs and peripheral lymph nodes (mcdermott et al., ; mcdowell and lascells, ) . most cells migrating into secretory organs and mesenteric lymph nodes synthesized iga, but most of those migrating to peripheral lymph nodes produced igg or igm (bohl et al., ; mcdermott et al., ; roux et al.. ) . experiments carried out in porcine and bovine models have revealed the predominance of iga and igg subtypes in porcine and bovine milk, respectively, with the decrease of these subtypes occurring during the post-parturition period (saif and bohl, ; saif and smith, ) . thus it becomes imperative to direct efforts in order to stimulate the lactogenic anti-rotavirus antibody response to higher titers for longer periods after parturition to ensure protection against rotavirus infection in neonates receiving these antibodies in mother's milk. earlier experiments conducted to determine the kinetics of brv replication in a mouse model revealed that the neonates are most susceptible to infection at the age of seven days (ijaz et al., in preparation) . in our protection studies the neonates are challenged on day seven. taking this into consideration, we have utilized the murine model to study the influence of different routes of immunization with either live or killed brv on the rate of decline of antibody titers and different subtypes in the lacteal secretions of the da-m following parturition up to eleven days. this report forms the basis of our protection studies which are currently under way. rotavirus-free mice were obtained from charles river breeding laboratories (wilmington, ma). in order to ensure that they were free of anti-rotavirus antibodies, they were bled on arrival from the coccigial vein. the sera were tested for anti-rotavirus antibodies using an elisa assay (bidwell et al., ) and all were found to be seronegative. to maintain them rotavirus free, the animals were housed in isolation units throughout the experiment. bovine rotavirus (brv) isolate c- was grown in ma- cells in the presence of pg of trypsin/ml (difco laboratories, detroit, mi) (babiuk et al., ) . after h of incubation at °c. supernatant containing the virus was harvested and cell debris removed by centrifugation at rpm for min. the virus was concentrated from the clarified supernatant fluids by pelleting through a % sucrose cushion at rpm in an sw rotor (beckman model l - ) for l/ h at °c. the virus pellet was resuspended in double distilled water and the amount of virus protein measured spectophotometrically by using the formula: (alj,,) - . (a)zho) x = kg/ml where a ,, and a,,,, represents adsorbance at nm and nm, respectively. when live brv was used as the antigen. each dam was given l.r.g ( x lo' plaque forming units) of purified virus contained in adjuvant (freund's incomplete adjuvant [fia] ) or double distilled water, according to the immunization protocol (table ) . killed brv was prepared as described above. in order to inactivate the virus, it was subjected to ultraviolet light ( ergs) for h and the extent of inactivation was tested in vitro. in no instance was active bovine rotavirus found after this inactivation procedure. each dam was given kg of purified virus contained in adjuvant or double distilled water, according to the immunization protocol (table ) . for oral immunization, mice were inoculated with kg of brv followed and wk later with the same viral dose inoculated orally. one week before the final booster inoculation, mice were bred in a female-to-male ratio of :l. all the dams gave birth naturally and the pups remained with their dams throughout the experiment. sampling and sample processing all mice were bled prior to immunization and their sera were tested for the presence of rotavirus antibody by both an elisa and virus neutralization test. all of the mice were seronegative for rotavirus on initiation of the study. thereafter, blood samples were taken weekly during the course of immunization to monitor antibody responses as indicated in the immunization protocol (table ). the immunization intervals were chosen arbitrarily at three weeks, since in most instances. maximal immunity occurs three weeks post-vaccination. the newborn mice were allowed to stay with their mother throughout the period of the experiment. in order to collect samples of colostrum and milk, three neonates from each group were sacrificed every second day post parturition starting on days through i. milk from their stomach was collected and diluted twofold in mem. samples were spun in an eppendorf centrifuge and supernatants were aliquotted into eppendorf tubes and stored at - °c. enzyme-litlked ittmut~osorhet~r rrssup (elisa) for detectiotl o.f an(i-rotavirus antibodies antibody levels in serum and milk samples against brv were determined by the method described previously (bidwell et al., ) . briefly. -well microtiter plates (immunolon : dynatech laboratories inc., alexandria. va) were incubated at °c overnight with fg of brv (isolate c- ) antigen diluted in x carbonate-bicarbonate (ph . ). the volume of brv per well was ~ . after incubation of plates with antigen. unadsorbed protein was removed by extensive washing in distilled water. the antigen was then overlaid with ~ of mouse antiserum per well in an undiluted form or diluted in . m phosphatebuffered saline containing . % fetal bovine serum (fbs). incubation of antigen was carried out for h at room temperature, after which time unbound antibody was removed by washing in distilled water. a : dilution of rabbit antimouse horseradish peroxidase conjugate (boehringer mannheim biochemical, calgary, alberta) was then added per well, and incubation of the conjugate proceeded at room temperature for an additional hour. after extensive washing to remove excess conjugate. the bound conjugate was reacted with ~ of chromagen and enzyme substrate (recrystallized -amino salicyclic acid. mg/ml in . phosphate buffer, ph . . , aldrich chemical co., milwaukee, wn) per well. to which . c/c hydrogen peroxide was added immediately before use. the reaction was allowed to proceed for min at room temperature before the absorbance ( nm) of each well was determined with a micro-elisa reader (dynatech instruments, inc., ca) and titers were expressed as the reciprocal of the highest dilution with an od of > sd over mean background levels. for the quantitation of different classes of antibody in mouse milk, mouse monoclonal subtyping kit -k was used following the procedure recommended by the manufacturer (hyclone laboratories. logan, ut). micro-tieiwalizutiotl test neutralization of brv c- by milk samples taken from hyperimmunized dams was determined by a micro-neutralization test using -well plates containing confluent monolayers of bsc- cells grown in eagle's minimal essential media (mem) with % fbs (gibco laboratories. grand island, ny). milk samples were se-rially diluted and incubated for min at °c. virus dilutions representing tcid ,, were added :l with various dilutions of milk and the virus-milk mixture was transferred to the plates containing confluent monolayers of bsc- cells in each well in duplicate. the plates containing virus-milk mixture were incubated for h at °c. the cell monolayers were washed with mem and replaced with ~ of mem. the plates were incubated at °c for two days. the dilution sample giving % cytopathic effect (cpe), as observed under the microscope. was taken as end point. since passive protection is important for preventing rotavirus infection in neonates, it is important that neonates receive sufficient quantities of neutralizing antibodies in milk from their mothers. in this study we were interested in determining the best regimen of immunizations against brv which would result in the highest neutralizing antibody levels in milk. to achieve this goal. mice were immunized with either live or killed brv via different routes and the levels of serum antibody against rotavirus were monitored throughout the immunization schedule up to parturition. after parturition, anti-rotavirus antibodies were determined in lacteal secretions for days. using an elisa and virus neutralization test. antibody titers in serum, colostrum and milk are shown in figs. - . milk samples were also examined for determination of different immunoglobulin (ig) classes. data showing ratios of various subtypes in milk collected from dams immunized with either live or killed brv are summarized in tables and . the distribution of different subtypes in milk samples revealed igg to be the most predominant antibody followed by igm and iga. it is interesting to note that regardless of the route of immunization, a gradual increase in the antibody titer in the lacteal secretions was observed up to days post-partum, with the greatest increase on day . followed by a decrease in titer on day ; a unique observation compared with other species. ltntnunizatiotz with killed bovirle rotavirm to determine whether killed brv can produce lactogenic anti-rotavirus antibody response, experiments were performed using this antigen orally and in combinations of other routes. animals were immunized by the following route using killed brv: x , x im x , im x and imam x . freund's complete adjuvant (fca) and freund's incomplete adjuvant (fia) were used only with the im route of immunization (table ) . immunization with killed brv significantly increased anti-rotavirus antibodies in serum in all groups regardless of the route of immunization (fig. ) . the rapid immune response observed a week after the first immunization as compared to an almost negligible titer on day , does not reflect that animals were primed before immunization because mice used in this study were pre-tested for anti-rotavirus antibodies and all were found to be seronegative and control mice remained seronegative throughout the study. however, it does show that rotavirus is highly immunogenic. among all the groups immunized with killed brv, the group immunized times via the imam route showed the best anti-rotavirus serum antibody response. an interesting but unexplainable decrease in antibody occured days post-immunization in animals immunized via the imam route (fig. ). however. a second immunization at days increased the antibody titer above that of any other group. although the second immunization boosted the antibody titers in all groups, the third immunization at days did not show any effect on the level of antibody production in any of the groups under study (fig. ) . antibody titers against rotavirus in lacteal secretions as determined by elisa. varied from - log,,, between groups one day post-parturition ( fig. ) . animals immunized x had the highest titers whereas the im x had the lowest antibody titers. a gradual increase in antibody was observed in all groups with the maximum antibody titer in lacteal secretions being achieved days after parturition. unlike serum antibodies, the highest lacteal antibody titer occurred in the group immunized x and x im x . the virus neutralization antibody titers in milk showed a similar pattern throughout lactation as did the elisa titers. however, anti-rotavirus antibody titers, as determined by elisa, were consistently higher than virus neutralization titers, with the exception of the group immunized x which showed slightly higher neutralization titers on day and after parturition (fig. ) . immunization with live bovine rotavirus since killed antigen produced an excellent response following oral immunization, attempts were made to do similar studies with live virus. animals were immunized by the following routes using live brv: x , x im x , im x , imam x and imam x im x with or without adjuvant as indicated in the immunization schedule (table ) . immunization with live brv induced the production of high levels of anti-rotavirus antibodies in serum of dams in all groups. the pattern of immune response was rapid, similar to the groups immunized with killed brv. one exception was the group immunized imam x im x , which revealed significantly higher antia rotavirus antibodies days after primary immunization ( wk after the second immunization). there was a rapid decrease in antibody titer in this particular group days after the first immunization, but the second immunization at days induced a higher level of antibody than in the rest of the groups. the third immunization at days did not increase antibody production, and antibody levels in serum started declining towards the time of parturition (fig. ) . titers of anti-rotavirus antibodies in lacteal secretions, as determined by elisa, also revealed a similar pattern as seen in the groups immunized with killed brv. the colostrum and milk samples collected up to a week after parturition showed a gradual increase in antibody titers as determined by elisa. the highest antirotavirus antibody titer in all groups was observed on day post-parturition. the maximum titer of antibody at this point was observed in the group immunized imam x im x . the titers started declining thereafter (fig. ) . virus neutralizing antibody titers in the lacteal secretions exhibited a similar pattern throughout lactation. the anti-rotavirus antibody titers, as determined by elisa, were consistently higher than virus neutralizing antibody titers in all the groups (fig. ) . the distribution of antibodies associated with various immunoglobulin (ig) subtypes in mammary secretions are presented in tables and . throughout the duration of the experiment, igg antibodies predominated, followed by igm and iga in each group. irrespective of the route of immunization and type of antigen used, there was a gradual reduction in the distribution of igg and igm antibodies until the end of the experiment. in contrast, iga antibodies, after a gradual decrease up to day post-parturition, showed an increase on day before decreasing again. since neonates are often exposed to enteric viruses before they have sufficient time to develop active immunity, they are highly susceptible to infection unless passively immunized before exposure to these pathogens. lactogenic immunity has been shown to play a major role in providing protection to neonates from a variety of mammalian species against enteric infections (hanson et al., ; knight and peaker, ; offit and clark, ; opdebeeck, ) . this further underlines the significance of passively required antibodies from colostrum and milk in the neonatal gut for preventing these infections (hanson et al., ) . therefore, considerable efforts are underway in order to develop vaccines which will stimulate lactogenic immunity against enteric infections in order to improve the immune status of neonates and thereby, prevent early infection (babiuk et al., ) . the role of maternal antibodies in the protection of neonates against diarrhea, caused by a number of viral agents, has been shown to depend on the continual presence of antibody in the intestinal lumen (saif and smith, ) . in the case of rotavirus induced neonatal diarrhea in calves and lambs, it has been experimen-tally shown that by feeding colostrum, milk, or serum containing suffcient anti-rotavirus antibodies, infection of the intestine can be prevented (snodgrass et al., ) . the degree of protection not only correlated with the quantity of anti-rotavirus antibodies present but also with the subtype of the ig involved (snodgrass et al., ) . local immunity against rotavirus can perform two functions. first. the presence of antibody in the intestine should protect the neonate from disease. secondly, it should help in the reduction of rotavirus in feces and hence reduce environmental contamination (saif and smith, ) . since the mouse model being used in our protective-challenge experiments against rotavirus infection, utilizes neonates at days of age, the time when mouse enterocytes have the highest number of rotavirus specific receptors, thereby making them more susceptible to infection (riepenhoff-talty et al., ) . we studied lacteal antibody responses spanning this time frame, plus a few days beyond. to achieve this, different routes of immunization were evaluated. although administration of live or killed brv at mucosal sites (intestine and mammary regions) not only induced a significant elevation of iga, igm and igg antibodies in lacteal secretions there was also a marked increase in serum anti-rotavirus antibodies as determined by elisa. this might be due to rapid transmission of the viral antigen from mucosal sites to other body sites. therefore, the sites of inoculation of antigen might only serve as a "portal of entry". it could also be due to spillover of mucosally produced igs into the circulation (chang et al., ) . the most prominent feature of this study and other studies, employing the murine model (halsey et al., ) , is the increase of milk antibody production approximately days following parturition. the increase in milk igs on day could occur as a result of the production of antibodies by sensitized cells residing within the mammary glands, which are turned on by hormones along with the influx of antibodies from the serum (halsey et al., ) . it has been shown by halsey et al. ( ) that such a transfer does take place in the mouse with as much as % of the milk iga on the fourth day of lactation coming from serum as compared to % on the eighth day. the rest of the iga is likely produced locally by sensitized cells, in the mouse model. in contrast, there is no evidence for local production of iga in the mammary gland in early or mid-lactation in sheep (a.j. husband et al., ; th international congress of immunology, toronto, canada, . . ). hanson et al. ( ) could not observe any transfer of dimeric iga from the circulatory system to milk in rats. bohl and saif ( ) vaccinated pregnant cows via the imam route with live attenuated transmissible gastroenteritis (tge) virus and found that antibodies in milk from these animals were primarily of the igg class. in contrast, after natural infection with tge virus via the oral route, they observed that the antibodies in lacteal secretions are mainly of the iga class (bohl et al., ) . thus, it suggests that the route of infection or vaccinations with virus may influence the ig class in secretions. although in the present study the majority of antibody in milk was of the igg class, in natural infection with rotavirus it is the secretory iga which is closely related to protection in mice and humans because of its resistance to degradation by trypsin, chymotrypsins, and pepsin (offit and clark, ) . the reason for relatively higher igm titers than iga in lacteal secretions is not clear. it could, however, reflect the primary nature of the immune response, as rotavirus-free mice were used in this study. also in the case of imam route of immunization, antigen was administered intradermally in the mammary regions. even though the igm and igg antibodies were predominant in lacteal secretions, their effectiveness in preventing enteric infection in mice has been questioned (newby, ) . since iga is not absorbed from the intestine, it will remain locally and therefore should be more effective in preventing infection as compared to igg and igm which are absorbed (hanson et al., ) . in rats and mice, milk antibody is probably locally produced since a marked number of ig-containing cells are present in the mammary gland from late pregnancy through parturition and lactation to involution (lee et al., ) . thus, inoculation of antigen(s) into the mammary gland of the rat during pregnancy results in an increase of specific ig-containing cells of iga and igg subtype (lee et al., ) . in contrast, the majority of milk igs in ruminants, are essentially serumderived, and the lactating gland contains few ig-containing cells (saif and smith, ) . thus the physiology of the mammary gland of the specific species being immunized must be taken into consideration when designing immunization protocols for controlling enteric infections in neonates. the results of our study do not correlate with the work of oflit and clark ( ) particularly regarding the antibody titer in serum and milk. following oral immunization they reported that anti-rotavirus antibody response in serum and milk, even with homotypic virus. was approximately fold and go-fold lower, than that found after parenteral inoculation. whereas in our study, different routes of immunization did not seem to make any difference as far as antibody titer in serum and milk were concerned. the plaque reduction neutralization (prn) titer of milk in particular was much lower ( log,,,) after oral immunization compared to parenteral immunization (offit and clark, ) . even though one would expect high antibody titer in milk when immunizations are carried out using the mucosal versus the parenteral route. no explanation for this obvious difference was given by the authors. whether the differences in our study and their work is related to different quantities and quality of antigen remains to be determined. further studies are required to explore possible factors influencing the regulation of lactogenic antibody response in mice and other animals against rotavirus if effective control measures are to be implemented. these studies must address the effect of hormones and neuropeptides on the development of immunity and secretion of igs (diamond, ; halsey et al., ; stanisz et al., ; weisz-carrington, ) . recent studies clearly indicate that neuropeptides can modulate in vitro immune response either in a positive or a negative manner. furthermore, different antibody subtypes may be altered more than others (stanisz et al., ; th international congress of immunology, toronto, ontario, canada, . . ). these studies clearly indicate the potential of regulating specific lactogenic immune responses by neuroimmunoregulation. the role of neuropeptides and hormones in the regulation of anti-rotavirus lactogenic immunity is presently under investigation. finally, selective production of iga by an antisuppressor (as) mechanism may selectively enhance iga production and thereby contribute to intestinal and lactogenic immune regulation (ernst et al. ; th international congress of immunology, toronto, canada, . . ). an understanding of these mechanisms and appropriate exploitation should greatly aid in the enhancement of rotavirus immunity in the neonate and newborn and thus have a tremendous impact on the morbidity and mortality due to rotavirus infections in animals and humans. rotavirus isolation and cultivation i n the presence of trypsin rotavirus and coronavirus infections i n animals enzyme immunoassays for viral diseases we would like to acknowledge the excellent technical assistance of donna dent and barry carroll, glen gifford for preparation of the figures and irene kosokowsky for typing the manuscripts.financial support was provided by the medical research council, agriculturalcanada and the natural sciences and engineering research council of canada. key: cord- - q oopaz authors: dobaño, carlota; vidal, marta; santano, rebeca; jiménez, alfons; chi, jordi; barrios, diana; ruiz-olalla, gemma; melero, natalia rodrigo; carolis, carlo; parras, daniel; serra, pau; de aguirre, paula martínez; carmona-torre, francisco; reina, gabriel; santamaria, pere; mayor, alfredo; garcía-basteiro, alberto; izquierdo, luis; aguilar, ruth; moncunill, gemma title: highly sensitive and specific multiplex antibody assays to quantify immunoglobulins m, a and g against sars-cov- antigens date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: q oopaz reliable serological tests are required to determine the prevalence of antibodies against sars-cov- antigens and to characterise immunity to the disease in order to address key knowledge gaps in the context of the covid- pandemic. quantitative suspension array technology (qsat) assays based on the xmap luminex platform overcome the limitations of rapid diagnostic tests and elisa with their higher precision, dynamic range, throughput, miniaturization, cost-efficacy and multiplexing capacity. we developed three qsat assays to detect igm, iga and igg to a panel of eight sars-cov- antigens including spike (s), nucleoprotein (n) and membrane (m) protein constructs. the assays were optimized to minimize processing time and maximize signal to noise ratio. we evaluated the performance of the assays using plasmas obtained before the covid- pandemic (negative controls) and plasmas from individuals with sars-cov- diagnosis (positive controls), of whom were asymptomatic, had mild symptoms and were hospitalized. pre-existing igg antibodies recognizing n, m and s proteins were detected in negative controls suggestive of cross-reactive to common cold coronaviruses. the best performing antibody isotype/antigen signatures had specificities of % and sensitivities of . % at ≥ days since the onset of symptoms and . % at ≥ days since the onset of symptoms, with auc of . and . , respectively. combining multiple antibody markers as assessed by qsat assays has the highest efficiency, breadth and versatility to accurately detect low-level antibody responses for obtaining reliable data on prevalence of exposure to novel pathogens in a population. our assays will allow gaining insights into antibody correlates of immunity required for vaccine development to combat pandemics like the covid- . in a globalized world where emerging infectious diseases of broad distribution can put at stake the health and economy of millions of people, there is a need for versatile and reliable serological tools that can be readily applicable (i) to determine the seroprevalence of antibodies against any new pathogen and, more importantly, (ii) to characterise immunity to the disease at the individual and community levels. in the case of the covid- pandemic caused by sars-cov- , one of the main priorities since the beginning of the epidemics in china by the end of ( ) was to ascertain the percentage of the population that had been exposed to the virus, considering that a considerable number of people could have been asymptomatic ( ) ( ) . the lack of sensitive and specific serological tests early in the covid- pandemic delayed the precise estimation of the burden of infection for the rational implementation of public health measures to control viral spread ( ) . furthermore, immunological assays that can measure a high breadth of antibody types and specificities are needed to dissect which are the naturally acquired protective responses and identify correlates of immunity ( ) . additionally, when a vaccine becomes available, such assays would be valuable to evaluate immunogenicity of candidate vaccines and monitor duration of immunity at the population level ( ) . common tools for antibody studies are (i) rapid diagnostic tests (rdt) as point of care (poc) devices that usually measure either total immunoglobulins or igg and igm, qualitatively ( ), (ii) traditional enzyme-linked immunosorbent assays (elisa) ( ) that can quantify different isotypes and subclasses of antibodies against single antigens at a time, and that require certain previous expertise, personnel and equipment, and (iii) chemiluminescent assays (clia), widely used in clinical practice, faster and with higher throughput than elisa ( ) . the performance characteristics of the commercial kits available in the early months of the covid- pandemic were questionable ( ) , while external evaluations validating their reliability and accuracy were not published. a number of in-house elisa assays have also been developed in hospital and research laboratories ( ) , but they have the limitations that (i) a relatively large amount of sample is required, (ii) the large surface area of the individual microplate wells and the hydrophobic binding of capture antibody can lead to non-specific binding and increased background, and (iii) most elisas rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity ( ). an alternative technique that offers the benefits of elisa but also a larger dynamic range of antibody quantification and higher sensitivity ( ) ( ) is based on the xmap luminex® platform (www.luminexcorp.com/bibliography). secondary antibodies are labelled with fluorescent phycoerythrin (pe) directly or with biotin that mediates binding to streptavidin-rphycoerythrin (sape), which does not depend on an additional reaction. the technique has the added value of higher throughput (up to -well plate format), increased flexibility, and lower cost with the same workflow as elisa, particularly if using magnetic magplex® microspheres. paramagnetic beads allow for automation of workflow and better reproducibility compared to the previous generation of microplex® microspheres. since the beads have the capture antigen immobilized on their much smaller surface area compared to a -well microplate well, reduced sample volumes are required and non-specific binding is diminished ( ) . furthermore, a chief advantage over elisa is the multiplex nature of the assay that allows measuring antibodies to different antigens simultaneously. this increases the probabilities to detect a positive antibody response due to the heterogeneity of the human response and therefore it has a higher sensitivity relevant for identifying seropositive individuals. the luminex technology, capable of measuring simultaneously antibodies against (magpix®), (luminex / ®) and up to different antigens (flexmap d®), makes it an invaluable tool for antigen and epitope screening. finally, its versatility to set up adapted antigen panels makes luminex an excellent platform to ensure better preparedness for faster response to future emerging diseases and pandemics. here we report on the establishment and validation of three quantitative suspension array technology (qsat) assays to measure igm, iga and igg antibodies against eight sars-cov- antigens, based on the adaptation of previous in-house protocols that measured antibodies to other infectious diseases, including malaria ( )( )( ) ( ) . due to the need to process a large amount of samples with the minimal time and cost during a pandemic like the covid- , we optimized several conditions to reduce the duration of the assays and report them here for the three main isotypes that have proved useful for seroprevalence studies ( ) . positive samples were plasmas from individuals with a confirmed past/current diagnosis of covid- . one hundred and eleven had sars-cov- infection confirmed by real time reverse-transcriptase polymerase chain reaction (rrt-pcr). fifty-five were recruited in a study of health care workers in hospital clínic in barcelona, most of them with mild symptoms, of them hospitalized and without symptoms, all rrt-pcr positive ( ) . fiftyseven were covid- patients recruited at the clínica universidad de navarra in pamplona (spain), of which had severe symptoms and were hospitalized and had mild symptoms (one clinically diagnosed with positive radiology and serology, and negative rrt-pcr); were asymptomatic health workers with positive diagnosis confirmed by four serological tests but no rrt-pcr data. time since onset of symptoms ranged from to days. positive samples were used individually or as pools of up to samples depending on the tests. for optimization tests, only a subset of samples were used. negative controls were plasmas from healthy european donors collected before the covid- pandemic, and were used individually. numbers of positive and negative samples were in line with protocol recommendations from the foundation for innovative new diagnostics (find). ethics. samples analyzed in this study received ethical clearance for immunological evaluation and/or inclusion as controls in immunoassays, and the protocols and informed consent forms were approved by the institutional review board (irb) at hcb (refs. ceic- and hcb/ / ) or universidad de navarra (ref. un/ / ) prior to study implementation. the receptor-binding domain (rbd) of the spike (s) glycoprotein of sars-cov- , the leading vaccine candidate target, was selected as the primary antigen to develop the initial qsat assay because (i) s is one of the most immunogenic surface proteins together with the nucleocapsid protein (n) ( ) (ii) rbd is the fragment of the virus that mediates binding to the host receptor ace in the lung cells ( ) (iii) antibodies to rbd correlate with neutralizing antibodies ( )( ) that could be associated with protection based on studies of other coronaviruses and animal models ( ) ( ) ( ) ( ) , and (iv) an elisa based on this same protein has received fda approval for covid- serology ( ) . the rbd was from the krammer lab different test concentrations of protein antigens were coupled to magnetic magplex . µm cooh-microspheres from luminex corporation (austin, tx) in reactions of a maximum of , beads, at , beads/µl ( ) . first, beads were washed twice with . µl of distilled water using a magnetic separator (life technologies, d), and resuspended in µl of activation buffer, mm monobasic sodium phosphate (sigma, s ), ph . we compared the performance of the assays when a subset of positive and negative plasma samples were incubated at different dilutions with the antigen-beads for h or h at rt in relation to our previous protocol on at ºc. antigen-coupled beads, initially including rbd singleplex, were added to a -well µclear® flat bottom plate (greiner bio-one, ) at beads/well in a volume of µl/well pbs-bn. next, individual positive plasma samples (range of dilutions tested from / to / ) and individual negative controls (at the same dilutions as the positive samples), were added per plate in a final volume of µl per well. two blank control wells with beads in pbs-bn were set up in each plate to control for background signal. plates were incubated on a microplate shaker at rpm and protected from light, and then washed three times with µl/well of pbs-tween . %, using a magnetic manual washer (millipore, . for more accurate igm measurements, we tested whether diluting samples : with gullsorb™ igg inactivation reagent (meridian bioscience™) prior to testing for igm levels could reduce high responses observed in some negative samples ( ) . additionally, we tested the levels of rbd and s antibodies obtained at different plasma dilutions when incubated in a multiplex panel with additional antigenbeads including s , s , m and n constructs, compared to those obtained in singleplex, to check for potential interferences. finally, since viral proteins have diverse immunogenicity, definitive plasma dilutions were established with titration experiments in individual positive and negative samples once the final multiplex antigen panel and all assay conditions had been selected. we compared the performance of the assays when using biotinylated secondary antibodies days (top markers), ≥ days (top markers) and ≥ days (top markers). for each model, we calculated the auc and selected three seropositivity cutoffs aiming at specificities of i) %, ii) ≥ % and < %, and iii) ≥ % and < %, and obtained the corresponding sensitivity. models with % specificity and the highest sensitivities were selected for roc curve representations. the analysis was carried out using the statistical software r studio version r- . . ( ) (packages used: randomforest ( ) and proc ( )). the characteristics of sars-cov- infected participants whose plasma samples have been used in the study, with regards to age, sex, days since rrt-pcr diagnosis and days since onset of symptoms, are included in table s . the optimal amount of protein to be coupled to beads depended on the antigen and needs to be tested with each new lot. among the concentrations tested ( , or µg/ml protein), titration curves did not usually change substantially, in which case the lower concentration was chosen for the subsequent experiments. an illustrative example is shown for which the medium concentration was slightly superior when tested for igg and igm and thus selected ( figure s ). plasmodium falciparum antigens were based on on incubations at ºc ( ) . for the covid-incubations at ºc versus shorter times at rt. we tested that the range of dilutions was still adequate when reducing the incubation time (figure a ) and compared antibody levels and number of seropositive samples incubating on at ºc versus h rt at / ( figure b) . although the mfi readings in positive samples generally diminished with shorter times, the mfi readings in the negative samples also reduced, i.e. the signal to noise ratio was the same or sometimes better, maintaining or increasing the overall proportion of seropositive among the positive samples and thus the sensitivity. based on these data, we adopted the h incubation time for an initial covid- seroprevalence study ( ) . we subsequently tested shorter incubations more extensively and found that h was non-inferior to h incubation ( figure c ) and thus h was selected for the optimized sop. reduction of background in igm assay. treatment with gullsorb™ reduced or did not change the mfi signal, depending on the sample, antigen and dilution ( figure s a ). this additional incubation generally increased the signal to noise ratio and thus sensitivity and number of seropositive igm responses among the positive controls, particularly at the lower dilutions, therefore the gullsorb™ incubation was adopted for this assay ( figure s b ). igm reactivity in negative controls was lower against s-based antigens than against m-or nbased antigens and thus gullsorb™ treatment benefited the signal to noise ratio the most in these later proteins. singleplex versus multiplex antigen testing. multiplexing the antigens ( -plex panel) did not significantly decrease the mfi antibody levels to rbd or s compared to singleplex testing ( figure a ) neither for any of the other antigens ( figure s a) . interestingly, there was no evidence of any interference between rbd, s, s or s antigens despite sharing epitopes within the same multiplex panel. a number of negative pre-pandemic samples had preexisting antibodies recognizing sars-cov- proteins for certain isotypes and dilutions ( figure s a ): igg to s , s , m and n constructs, and iga to s and n-term & c-term of n. furthermore, testing plasmas against multiple antigens increased the sensitivity of the assay since some individuals who were seronegative or low responders to rbd, responded with higher antibodies to s (figure b) . once the multiplex antigen panel was established, a set of positive and negative samples were tested at different dilution(s) covering the diverse immunogenicity of the proteins, and / and / were selected for the assay performance evaluation (figures c and s b) . secondary antibodies conjugated to pe performed as well as a two-step secondary antibody conjugated to biotin followed by sape incubation (figure ) . the pe-antibody reagent that resulted in a shorter assay was selected as the preferred option. finally, min incubation was non-inferior to min incubation ( figure s ). we sought for the combination of ig and antigen responses that yielded the highest specificity (primarily), sensitivity and auc to detect seropositive responses. for rbd and s, igg and iga at / dilution, and igm responses at / , gave higher percentages of seropositive responses among the positive controls and thus were selected for the calculations; for n constructs, igg and iga performed better at / except for n c-term in which igg was better at / . antibodies to m, s (igg & iga) and n n-term (igm) did not discern well positive from negative responses and were not included in the rf models. the contribution of each antibody/antigen variable was ranked according to an rf algorithm at different periods since onset of symptoms ( figure s ) and the top - variables were selected. we performed rf for all the combination of variables and assessed the sensitivity of each combination at three different seropositivity thresholds aiming at specificities of %, % and %. the specificity of the qsat assays in samples from participants with sars-cov- positive diagnosis with ≥ days since the onset of symptoms (n= ) was up to % with sensitivity up to . %, and auc up to . , for the best combinations of ig isotypes/antigens. the top performing antibody signatures for three different seropositivity thresholds targeting specificities of %, % and % are shown in table , and their roc are shown in figure . in samples from participants with ≥ days since the onset of symptoms (n= ), the specificity was up to % and the sensitivity up to . %, with auc up to . for the best combinations of ig isotypes/antigens (table , figure ). in samples from all participants regardless of time since symptoms onset (n= ), the specificity was up to % and the sensitivity up to . %, depending on the combinations of ig isotypes/antigens, with auc up to . for the best combinations (table s , figure ). the performance of the qsat assays to predict positivity was clearly superior using combinations of multiple ig isotypes/antigens to using single isotype/antigen markers ( figure ). higher sensitivities were obtained when specificities were set to % or % ( tables , & s ), reaching % for samples ≥ or ≥ days since the onset of symptoms. we developed three novel multiplex immunoassays for quantifying igm, iga and igg to eight sars-cov- protein constructs and evaluated by machine learning classification algorithms the performance of several isotype/antigen combinations to detect any positive antibody response to infection, obtaining specificities of % and sensitivities of . % (≥ days since symptoms onset) or . % (≥ days since symptoms onset), and very high predictability (auc ≥ . ). our qsat assays, based on the xmap technology, provide the best precision, accuracy and widest range of detection compared to classical qualitative (rdt) or quantitative (elisa) assays. for any given test, there is usually a trade-off between sensitivity and specificity. to evaluate the performance of the assays here, we prioritized specificity over sensitivity for the implications that false positives may pose at a personal level and the impact that specificity has in seroprevalence studies. particularly when prevalence of infection is low, the positive predictive value of a test strongly relies on a high specificity. for example, in a scenario of % prevalence and % sensitivity, the positive predictive value of the test decreases from % to %, with a reduction in specificity from % to %. however, other seropositivity thresholds can be used to have a balanced specificity/sensitivity or to maximise sensitivity. a time period after the onset of symptoms is usually established for these analyses, because antibodies take an average - days since infection to be produced and detected depending on the isotype and test (igm - days, iga - days, igg - days) ( )( )( )( )( ) ( ) . thus, it is not necessarily expected to detect antibodies in individuals who are acutely infected and diagnosed around the time of plasma collection. accordingly, when considering all samples, which included and individuals with less than and days since onset of symptoms, respectively, sensitivity was lower (up to . %) at specificities of %. however, we detected igm or iga as early as days, and igg as early as day, from onset of symptoms. in fact, since samples were collected in the early days of the covid- pandemic, it is expected that igm and iga, which are induced upon primary infection earlier than igg, could contribute to a higher sensitivity of detection. most of the best signatures identified included igm and iga besides igg, regardless of the time period since onset of symptoms, also beyond days. however, over time, the only antibodies that would be expected to remain in blood are igg due to the decay of igm and iga, e.g. igm levels may become undetectable by the fifth week after symptoms onset ( ) . therefore, with longer days since infection, the serological assays to detect maintenance of antibodies could focus on igg detection. the superior performance of the qsat assays is partly based on direct fluorescence detection as opposed to colorimetric detection mediated by an enzyme. also, antigens are covalently coupled to beads as opposed to passive coating of the elisa plates, leading to a higher density of antigen per surface area and less antigen wash off during the assay. the higher background of elisa microplates is related to the fact that they have a much larger surface area than the combined area of microspheres, which is more prone to the binding of non-specific antibodies if blocking is not performed correctly ( ) . the sensitivities and specificities of other sars-cov- serological assays externally validated with > positive and > negative samples (as recommended by find protocols), some of them approved by the usa fda, are summarized in table s ( ) ( ) . while luminex assays generally have high correlation to elisas in singleplex (r ~ . ) ( ), it is important that the assays perform equally well in multiplex format, with no interference noted between antigens, even if they had overlapping epitopes. a key value of multiplexing is that it allows to capture a wider breadth of responses and this is needed because some individuals may not respond to one antigen (e.g. rbd) but may do so to other antigens (e.g. s or n proteins) ( )( ) ( ) . here, we substantially increased the sensitivity of the assay when combining isotypes/antigens compared to using only one isotype/antigen. the addition of n was more beneficial to detect seropositive responses when the onset of symptoms was recent, as this antigen is the most abundant and immunogenic and specific antibodies appear to be elicited earlier ( ) . in contrast, combinations of s antigens seemed to be sufficient to detect seropositive responses with longer periods since the onset of symptoms. an added advantage of multiplexing is the reduced usage of sample volume, resources and time, if antibodies to several antigens are to be evaluated. the possibility to perform miniaturized assays in small amounts of blood is very attractive in paediatric studies, in large field surveys where fingerpick may be more logistically feasible, and to test special tissues of interest including mucosal fluids. those combined advantages have a direct impact on the cost-efficacy of the qsat assay, that is overall cheaper than rdt or elisa assays. the cost of the xmap assay can be less than one-fifth of the least expensive commercial elisa and less than one-sixteenth of the most expensive commercial kit. cost is reduced because there is less protein used due to the smaller surface area and less amounts of other materials and reagents. we reduced the dilutions of plasma and titrated the secondary antibody to use the minimal amounts of samples and reagents, without compromising sensitivity. the economy of scale will improve further when the assays are adapted to high throughput flexmap d -well plate format but they are also easily adaptable to the bench top magpix -well format that is more affordable and easy to maintain even in remote laboratory settings. interestingly, positive antibody responses to m, s , s and n antigen constructs were detected in samples collected before the covid- pandemic. the presence of such antibodies has been interpreted as cross-reactivity with antigens of coronaviruses causing the common cold ( )( ) ( ) . indeed, higher sequence homology at the protein level between sars-cov- and coronaviruses has been reported for n (particularly n-terminal and central regions), m and s ( )( ) ( ) . pre-existing sars-cov- -specific t cells have been recently reported and also attributed to cross-reactivity to human coronaviruses previously encountered ( ) ( ) . the multiplex nature of the assay will allow to test this hypothesis in the future with the addition of antigens to related coronaviruses e, hku , nl and oc in the same assay panel, by comparing the patterns of antibody reactivity, in order to address the significance of this in immunity to here, antibody responses to m were very marginal and did not contribute to higher assay sensitivity and this could partly be because the purity of the protein was not high. however, this antigen may be valuable in studies establishing the antibody correlates of protection since at present the targets of immunity have not been elucidated. it is possible that, in addition to neutralizing antibodies directed to the rbd region of s, antibodies of other specificities with non-neutralizing functions, for example fc-mediated opsonisation and phagocytosis, could be relevant in protection. in fact, t cell responses to epitopes located on m have been detected at high frequencies ( ) , and it is possible that antibodies to this or other less immunogenic antigens may also have a role in protection in some individuals. in our study, the addition of s from a commercial supplier did not have any added value but for future versions of the assay we will test s from different sources, as this subunit is expected to not cross-react with other beta-coronaviruses and be specific for sars-cov- diagnostics ( ) ( ) . the assays performances were excellent but further testing needs to be performed with longer periods of time since onset of symptoms, although we do expect to maintain high specificity and sensitivity albeit antibody signatures would be different and based on igg only. future studies will include additional positive samples of asymptomatic individuals, who probably have lower antibody levels than mild or severe cases and are rarely included in the validation of commercial kits. in addition, it will be interesting to include negative controls reacting with other coronaviruses or other infections (e.g. malaria) and pathologies known to induce polyclonal responses or rheumatoid factor, which may increase background responses. in conclusion, we developed % specific and fast assays with possibly one of the best diagnostic characteristics reported in the published literature to assess seroprevalence of covid- . considering their high sensitivity, these qsat assays would be suited to identify individuals with levels of antibodies below the lower limit of detection of rdt or the lower limit of quantification of elisa, such as asymptomatic children or immunosuppressed individuals, or long-term decaying antibodies ( ) . in addition this approach would be particularly suited to identify hyper immune donors with very high levels of antibodies and the largest antigenic breadth for immunotherapy. the assays are highly versatile, being easily adaptable to quantify other antibody igg and iga subclasses and avidity with the use of chaotropic agents, and even functional activity like binding inhibition to the virus receptor ace . the multiplex capabilities make them also ideal for sizeable peptide screenings to accelerate epitope mapping and selection for identifying fine-specificity of immune correlates of protection for vaccine development, and would also be applicable in vaccine evaluation when the first candidates reach larger-scale phase and clinical trials. the assays development and sample collection were performed with internal funds from the investigators groups and institutions, and the performance analysis received support from a novel coronavirus from patients with pneumonia in china covid- : four fifths of cases are asymptomatic, china figures indicate asymptomatic sars coronavirus infection among healthcare workers, singapore. emerg infect dis the important role of serology for covid- control what policy makers need to know about covid- protective immunity immune surveillance for vaccine-preventable diseases lateral flow assays enzyme-linked immunosorbent assay (elisa) chemiluminescent immunoassay technology: what does it change in autoantibody detection? auto-immun highlights. / / serological assays for emerging coronaviruses: challenges and pitfalls luminex corporation. overcoming the cost and performance limitations of elisa with xmap(r) technology. tech note characterization and development of a luminex(®)-based assay for the detection of human il- simultaneous quantitation of cytokines using a multiplexed flow cytometric assay development of quantitative suspension array assays for six immunoglobulin isotypes and subclasses to multiple plasmodium falciparum antigens optimization of incubation conditions of plasmodium falciparum antibody multiplex assays to measure igg, igg( - ), igm and ige using standard and customized reference pools for sero-epidemiological and vaccine studies development of a high-throughput flexible quantitative suspension array assay for igg against multiple plasmodium falciparum antigens analysis of factors affecting the variability of a quantitative suspension bead array assay measuring igg to multiple plasmodium antigens seroprevalence of antibodies against sars-cov- among health care workers in a large spanish reference hospital neutralizing antibodies against sars-cov- and other human coronaviruses structural and functional basis of sars-cov- entry by using human ace neutralizing epitopes of the sars-cov s-protein cluster independent of repertoire, antigen structure or mab technology neutralizing antibodies against sars-cov- and other human coronaviruses the time course of the immune response to experimental coronavirus infection of man reinfection could not occur in sars-cov- infected rhesus macaques two year prospective study of the humoral immune response of patients with severe acute respiratory syndrome immunoassay for serodiagnosis of zika virus infection based on time-resolved förster resonance energy transfer r: a language and environment for statistical computing. r foundation for statistical computing classification and regression by randomforest. r news proc: an open-source package for r and s+ to analyze and compare roc curves antibody responses to sars-cov- in covid- patients: the perspective application of serological tests in clinical practice antibody responses to sars-cov- in patients of novel coronavirus disease temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study antibody detection and dynamic characteristics in patients with covid- profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus (sars-cov- ) profiling early humoral response to diagnose novel coronavirus disease (covid- ) profile of specific antibodies to sars-cov- : the first report antibody testing for covid- diagnostic testing for severe acute respiratory syndrome-related coronavirus- : a narrative review antibody responses to individual proteins of sars coronavirus and their neutralization activities profiles of igg antibodies to nucleocapsid and spike proteins of the sars-associated coronavirus in sars patients development of an enzyme-linked immunosorbent assay-based test with a cocktail of nucleocapsid and spike proteins for detection of severe acute respiratory syndrome-associated coronavirus-specific antibody detection of nucleocapsid antibody to sars-cov- is more sensitive than antibody to spike protein in covid- patients. medrxiv analysis of serologic cross-reactivity between common human coronaviruses and sars-cov- using coronavirus antigen microarray. biorxiv antigenic crossreactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses e and oc sars-cov- specific antibody responses in covid- patients beyond the spike: identification of viral targets of the antibody response to sars-cov- in covid- patients. medrxiv targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals presence of sars-cov- reactive t cells in covid- patients and healthy donors. medrxiv serological signatures of sars-cov- infection: implications for antibody-based diagnostics. medrxiv we thank the volunteers who donated blood for covid- studies and the clinical and laboratory staff who participated in the sample collection and processing. special thanks to key: cord- - q vwoct authors: grzelak, ludivine; temmam, sarah; planchais, cyril; demeret, caroline; huon, christele; guivel, florence; staropoli, isabelle; chazal, maxime; dufloo, jeremy; planas, delphine; buchrieser, julian; rajah, maaran michael; robinot, remy; porrot, francoise; albert, melanie; chen, kuang-yu; crescenzo, bernadette; donati, flora; anna, francois; souque, philippe; gransagne, marion; bellalou, jacques; nowakowski, mireille; backovic, marija; bouadma, lila; le fevre, lucie; le hingrat, quentin; descamps, diane; pourbaix, anabelle; yazdanpanah, yazdan; tondeur, laura; besombes, camille; ungeheuer, marie-noelle; mellon, guillaume; morel, pascal; rolland, simon; rey, felix; behillil, sylvie; enouf, vincent; lemaitre, audrey; creach, marie-aude; petres, stephane; escriou, nicolas; charneau, pierre; fontanet, arnaud; hoen, bruno; bruel, timothee; eloit, marc; mouquet, hugo; schwartz, olivier; van der werf, sylvie title: sars-cov- serological analysis of covid- hospitalized patients, pauci-symptomatic individuals and blood donors. date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: q vwoct it is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of sars-cov- infection and their antibody response profile. here, we performed a pilot study to assess the levels of anti-sars-cov- antibodies in samples taken from pre- epidemic individuals, patients from hopital bichat (paris), pauci-symptomatic individuals in the french oise region and contemporary oise blood donors. two in-house elisa assays, that recognize the full-length nucleoprotein (n) or trimeric spike (s) ectodomain were implemented. we also developed two novel assays: the s-flow assay, which is based on the recognition of s at the cell surface by flow-cytometry, and the lips assay that recognizes diverse antigens (including s or n c- terminal domain) by immunoprecipitation. overall, the results obtained with the four assays were similar, with differences in sensitivity that can be attributed to the technique and the antigen in use. high antibody titers were associated with neutralisation activity, assessed using infectious sars-cov- or lentiviral-s pseudotypes. in hospitalized patients, seroconversion and neutralisation occurred on - days post symptom onset, confirming previous studies. seropositivity was detected in % of pauci-symptomatic individuals within days post-symptoms and % of blood of healthy donors collected in the area of a cluster of covid cases. altogether, our assays allow for a broad evaluation of sars-cov seroprevalence and antibody profiling in different population subsets. about four months after the initial description of atypical pneumonia cases in wuhan in december , covid- has become a major pandemic threat. as of april , , about half of the human population is under confinement, million infections have been officially diagnosed, with , fatalities and . million recovered cases. covid- is caused by sars-cov- , a betacoronavirus displaying % nucleotide homology with severe acute respiratory syndrome virus (now termed sars-cov- ), that was responsible for an outbreak of , estimated cases in . pcr-based tests are widely used for covid- diagnosis and for detection and quantification of sars-cov rna . these virological assays are instrumental to monitor individuals with active infections. the average virus rna load is copies per nasal or oropharyngeal swab at day post symptom onsets and may reach copies . a decline occurs after days - , but viral rna can be detected up to day post-onset in recovered patients at a time when antibodies (abs) are most often readily detectable . disease severity correlates with viral loads, and elderly patients, who are particularly sensitive to infection, display higher viral loads . serological assays are also being implemented. anti-spike (s) and nucleoprotein (n) humoral responses in covid- patients are assessed, because the two proteins are highly immunogenic. the viral spike (s) protein allows viral binding and entry into target cells. s binding to a cellular receptor, angiotensin-converting enzyme (ace ) for sars-cov- and -cov , is followed by s cleavage and priming by the cellular protease tmprss or other endosomal proteases . s genes from sars-cov and -cov share % amino-acid similarity . one noticeable difference between the two viruses is the presence of a furin cleavage site in sars-cov , which is suspected to enhance viral infectivity . the structures of s from sars-cov- and co-v- in complex with ace have been elucidated [ ] [ ] [ ] . s consists of three s -s dimers, displaying different conformational changes upon virus entry leading to fusion , , . some anti-s antibodies, including those targeting the receptor binding domain (rbd), display a neutralizing activity, but their relative frequency among the generated anti-sars-cov- antibodies during infection remains poorly characterized. the nucleoprotein n is highly conserved between sars-cov and -cov ( % amino-acid homology). n plays a crucial role in subgenomic viral rna transcription and viral replication and assembly. serological assays are currently being performed using in-house, pre-commercial versions or commercially available elisa-based diagnostics tests , , [ ] [ ] [ ] . other techniques, including point-of-care and auto-tests are also becoming available. in hospitalized patients, seroconversion is typically detected between - days post symptom onset, with a median time of - days for anti-s igm and days for igg and iga , , [ ] [ ] [ ] [ ] . the kinetics of anti-n response was described to be similar to that of anti-s, although n responses might appear earlier [ ] [ ] [ ] . anti-sars-cov- antibody titers correlate with . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint disease severity, likely reflecting higher viral replication rates and/or immune activation in patients with severe outcome. besides n and s, antibody responses to other viral proteins (mainly orf b and nsp ) were also identified by antibody microarray . cov, are considered to be relatively low , . with sars-cov- , neutralizing antibodies (nabs) have been detected in symptomatic individuals , , , , and their potency seems to be associated with high levels of antibodies. neutralisation is assessed using plaque neutralisation assays, microneutralisation assays, or inhibition of infection with viral pseudotypes carrying the s protein , , [ ] [ ] [ ] . of note, potent monoclonal nabs that target rbd have been cloned from infected individuals . whether asymptomatic infections, which are currently often undocumented , and most likely represent the majority of sars-cov- cases lead to protective immunity, and whether this immunity is mediated by nabs, remain outstanding questions. commercial tests are not yet widely distributed. thus, we have designed anti-n and anti-s elisa, as well as novel assays for anti-sars-cov- antibody detection and neutralisation. we compared their performance and carried out anti-sars-cov- antibody profiling in different population subsets. we first designed four tests to assess the levels of anti-sars-cov- antibodies in human sera. their characteristics are summarized table . elisas. the two in-house elisas are classical tests, using as target antigens the full-length n protein (elisa n) or the extracellular domain of s in a trimerized form (elisa tri-s). the two recombinant antigens were produced in e. coli (n) or in human cells (s). the elisa n assay is a classical indirect test for the detection of total immunoglobulins, using plates coated with a purified his-tagged sars-cov n protein. titration curves of sera from covid- patients and pre-pandemic sera initially led to the determination that a dilution of : was of optimal sensitivity and specificity, and was later used for testing of large cohorts. the elisa tri-s, for trimeric s, allows for the detection of igg antibodies directed against the sars-cov- spike. we developed an elisa using as antigen a purified, recombinant and tagged form of the s glycoprotein ectodomain, which was stabilized and trimerized using a foldon motif. serum igg from pre-epidemic (n= ), pauci-symptomatic (n= ), and hospitalized individuals (n= ) were titrated using serum dilutions ranging from : to : , , (fig. s ). receiving-operating characteristic analyses using either total area under the curve or single optical density measurements indicated that the : dilution provides the best sensitivity and specificity values and was therefore . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint used in subsequent analyses (fig. s ). of note, the tri-s elisa also permitted the titration of anti-s igm and iga antibodies in human sera (fig. s ). s-flow. the third assay, termed s-flow, is based on the recognition of the s protein expressed at the surface of t cells ( t-s cells). we reasoned that in-situ expression of s will allow detection of antibodies binding to various conformations and domains of the viral glycoprotein. we verified that s was functionally active, by mixing t-s cells with target cells expressing ace . large and numerous syncytia were detected, indicating that s binds to its receptor and performs fusion (not shown). the principle of the s-flow assay is depicted fig. s a . t-s cells are incubated with dilutions of sera to be tested. antibody binding is detected by adding a fluorescent secondary antibody (anti-igg or anti-igm). the signal is measured by flow-cytometry using an automated -well plate holder. the background signal is measured in t cells lacking s and subtracted in order to define a specific signal and a cut-off for positivity. to establish the specificity of the assay, we first analyzed a series of sera collected before , from the institut pasteur biobank (icareb). all sera were negative (fig. s ), strongly suggesting that antibodies against other coronaviruses circulating in france were not detected. we then measured the sensitivity of the assay, by assessing the reactivity of sera from covid- patients hospitalized at hôpital bichat (table s ). an example of binding with two patients' sera (b and b ) is depicted fig. s b . serial dilutions allowed for the determination of a titer, which reached a value of , and , for b and b , respectively (fig. s b ). of note, the median fluorescence intensity (mfi) of the signal decreased with the dilution, indicating that mfi, in addition to the % of positive cells, provides a quantitative measurement of the levels of specific antibodies. we thus selected a single dilution ( : ) to analyze large numbers of samples. we then analyzed samples from patients (b -b ) ( fig. s c and table s ). we observed an increase of the igg response over time, with positivity appearing days after symptoms onset. serial dilutions indicated that antibody titers raised over time (not shown). we observed similar patterns with the igm and igg responses (fig. s d ). the absence of an earlier igm response may be due to the lower sensitivity of the secondary anti-igm antibodies tested or because of a short delay between the two responses, which has been already observed in covid- patients. addressing this question will require the analysis of a higher number of individuals. we also tested a secondary anti-whole ig antibody, but it did not prove more sensitive than the anti-igg. we thus tested the different cohorts with the secondary anti-igg. the fourth assay, termed lips (luciferase immunoprecipitation assay) is based on the use of antigens made of viral proteins (or domains) fused to nanoluciferase (nanoluc) (fig. s ). the objective was to develop an assay that is able to test large diverse cohorts and evaluate the range of antibody responses against a set of viral proteins or domains. this opens the possibility to select the . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint best antigens for high throughput binding assays. each antigen is used at the same molar concentration, based on a standardisation by luciferase activity of the amount of ag engaged in each reaction. this allows for easy direct comparison of the ab responses (amplitude and kinetic) against each antigen. a panel of different s and n-derived antigens were first evaluated with a set of pre-epidemic human sera were along with those of with covid hospitalized patients (fig. s ) . two patients were sampled at different time points. the strongest signals in covid patients' sera compared to background of pre-epidemic sera were identified with s , s and n (c-term part) antigens ( fig. s ) . additional investigations on a limited panel of sera sampled in pauci-symptomatic patients showed that s responses were, regarding the diagnostic sensitivity and quantitative responses, similar to full s responses evaluated by s-flow (fig. s ). to avoid redundancy, we focused lips analysis to n, selecting it for its sensitivity regarding an intracellular viral protein not targeted by nabs and s as it is described as a target of most nabs. to establish the specificity of the assay, we first analyzed the same series of sera we used for s-flow and found all of the sera to be negative (fig. s ). we also measured the kinetic of apparition of antibodies in the same longitudinal samples from patients ( fig. and table ). we observed an increase of response overtime, with positivity appearing - days after symptoms onset. of note, the protein a/g beads used for precipitation of the immune complexes do not bind efficiently to igm or iga. protein l, which has a higher affinity binding to iga, has not yet been tested. we screened different cohorts to evaluate the performance of the four assays and corresponding antigens ( table ) . we first used sera from up to pre-epidemic individuals, collected before , to assess the specificity of the tests. we then measured antibody levels in hospitalized covid- patients from hôpital bichat (paris), to determine the sensitivity of the tests and analyze the kinetics of seroconversion. the clinical and virological characteristics of four of these patients have been recently described . we next studied the prevalence of sars-cov- positive individuals in a cohort of pauci-symptomatic individuals in crepy-en-valois, a city of , inhabitants in oise. on february , a staff member from a high school in crepy-en-valois was admitted to an hospital in paris with confirmed sars-cov- infection. on march - , students from the high school, parents of the students, teachers and staff were invited to participate to an epidemiological investigation around this case. blood samples were collected from individuals reporting mild signs compatible with covid- (fever, cough or dyspnea). finally, we tested sera from blood donors from the etablissement français du sang (efs) in lille (france). the blood samples were donated in two cities, clermont ( , inhabitants) on march and noyon ( , inhabitants) on march , each located at about kilometers from crepy-en-valois. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . results obtained with sera from each category of individuals are presented fig. . the preepidemic samples served as negative controls. with the four assays, signals were consistently negative (s-flow and lips s ) or low (elisas and lips n). this strongly suggested that a prior exposure to human seasonal coronaviruses associated to the "common cold" (such as hcov-oc , hcov- e, hcov-hku- or hcov-nl ) does not induce an obvious cross-reaction with our assays. this was expected, since these prevalent viruses are distantly related to sars-cov- at the protein level. with each assay, we established cut-off thresholds. for elisa n, the cut-off was set at % percentile of preepidemic sera. for elisa tri-s, the cut-off was established as the mean + standard deviations (sd) of the pre-epidemic samples analyzed, which corresponds to % specificity. for the s-flow, we established a cut-off that corresponded to a signal > % of cells positive by flow cytometry. for the lips assays, the cut-off was based on internal controls. s-flow and lips s cut-offs eliminated all preepidemic samples analyzed. having established these cut-off levels, we analyzed samples from patients from hôpital bichat. some of the patients were analyzed at different time points, representing a total of up to samples. the percentage of positive samples varied between and %, with a mean of %. the fact that not all patients were seropositive reflected the various sampling times from each individual. to study more precisely the kinetics of seroconversion, we selected patients with more than five longitudinal samples and known dates of symptom onsets (fig. ). in these patients, seroconversion was detected between - days post symptom onsets with elisa-n, lips-n, elisa tri-s and s-flow. the lips s assay became positive with a slower kinetic, and one of the patients remained just below the cut-off. for some patients, the lips n and elisa n signals appeared before the lips s and elisa tri-s, which suggest different kinetics of n-and s/s responses independently of the sensitivity of the test. we then tested the sera obtained from pauci-symptomatic individuals in oise. positivity rates varied from % to % between the assays, with a mean of % ( fig. and table ). this range of variation was more marked than with hospitalized patients, likely because pauci-symptomatic covid- individuals display lower viral loads than those requiring hospitalization and may generate lower levels and different patterns of antibodies. to our knowledge, these figures represent one of the first evaluations of sars-cov- prevalence in pauci-symptomatic individuals within a cluster of severe cases. the fact that only one third of the individuals were tested positive suggests that some of them may not have seroconverted at the time of sampling, and/or that other viruses or environmental causes were responsible for the reported symptoms. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint we next examined sars-cov- seroprevalence in samples collected from blood donors on march - , . eligibility criteria for blood donation included an absence of recent signs of infection or antibiotic treatment. the donors can thus be considered as asymptomatic individuals with stringent criteria. the donors were negative with elisa-n and lips assays. with s-flow, donors were positive, including two with a strong signal. these positive and negative donors were then tested with elisa tri-s, and only the two strong responders scored positive. therefore, the positivity rate in this cohort was low ( - % with the two most sensitive assays). this suggests that the virus had not circulated to a large extent in a radius of kilometers around the initial clusters. it is also likely that asymptomatic infection induces low and delayed seroconversion. further studies are warranted to evaluate sars-cov- prevalence in denser population environments. we performed a side-by-side comparison of the assays using the three cohorts. for a given assay, we first scored the number of positive samples measured with the other assays ( fig. ). with hospitalized patients, roughly similar numbers of positive cases were obtained with the four assays, with the exception of lips s , confirming that this assay is less sensitive, probably because it does not catch antibodies targeting other s domains. however, combining the lips s and n results gave similar detection rates than any of the three other tests. with the cohort of pauci-symptomatic individuals, the s-flow and elisa tri-s yielded very close results and higher detection rates than the other tests. in blood donors, positive cases were only detected with these two tests. we then mixed results obtained with the three cohorts and calculated correlation rates between each assay (fig. ) . the dot plots indicate that sera with high antibody levels are generally caught by the four assays. important differences are however observed with samples with a low antibody concentration, reflecting both the choice of the antigens and the intrinsic different sensitivities of the assays. we then thought to evaluate the presence of nabs in the sera of infected individuals. various tests have already been established , , , . we focused on two tests. the first is a microneutralisation (mnt) assay using infectious sars-cov- . this reference method is based on virus incubation with serial dilutions of the sera, and evaluation of titers on vero-e cells. we also developed a lentiviralbased pseudotype assay, as outlined fig. s a . lentiviral particles coated with s and encoding for a reporter gene (gfp) are pre-treated with dilutions of the sera to be tested, incubated with target cells ( t cells transiently expressing ace and the tmprss protease) and the signal is measured after . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint h. a pilot experiment with sera from hospitalized patients demonstrated a strong neutralizing activity with some of the samples (fig. s b, c) . as a control, we used lentiviral particles coated with an irrelevant viral protein (vsv-g), and they were insensitive to the same sera (fig. s c ). we also tested as a proof of concept the neutralisation activity of the first sera of the cohort of paucisymptomatic individuals (fig. s d) . a strong correlation was observed between mnt and neutralisation of pseudoviruses (fig. s e) . of note, with the pseudovirus assay, similar neutralisation results were obtained when target cells transiently transfected with ace and tmprss were replaced by stable t-ace cells, or when luciferase was used as a readout instead of gfp. the reference mnt assay is labour-intensive and requires access to a bsl facility. we thus performed a pilot correlative analysis between the four serological tests and the pseudovirus assay ( fig. a ). this analysis was performed with samples from hospitalized patients and paucisymptomatic individuals. a strong correlation was observed with the elisa n, elisa tri-s, s-flow and lips-n, with a similar but less marked trend with the lips-s assay. we also determined by linear regression the association between the intensity of antibody binding and pseudovirus neutralisation. a neutralisation activity > % was associated with the following signals: elisa n (> . ), elisa tri-s (> . ) s-flow (> % of positive cells) and lips-n (> . ). with this level of neutralisation, lips s mainly gave positive responses and a few responses below the cut-off. in hospitalized patients, the neutralisation activity increased over time, being detectable at day five and reaching % and - % at days - and - , respectively (fig. b) . these pilot experiments were so far performed with a limited number of samples originating from individuals with mild, severe or critical symptoms. it will be important to increase the number of pauci-symptomatic individuals tested, and to evaluate whether asymptomatic seropositive individuals exhibit a neutralisation activity. we have designed four serological assays to detect anti-sars-cov antibodies. the first two assays are elisa detecting anti-n and anti-s responses. the s-flow assay allows to identify and score the levels of antibodies binding to all domains and conformations of s expressed at the cell surface. the lips assays target different domains of s and n, and allow for the detailed profiling of the humoral responses. we have evaluated their performance and compared their results with two neutralisation assays, a reference mnt assay and a pseudovirus neutralisation assay. each assay presents advantages and drawbacks. elisas are widely used, either as in-house or commercial tests, and can be easily performed in routine diagnostic laboratories in large quantities. they can be performed at a high scale. the s-flow assay captures all anti-s antibodies and provides excellent sensitivity but requires access to a cell culture system and flow cytometry equipment. thus, . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . it would be less adapted to high-throughput screenings. the lips assay allows the testing of different target antigens in a liquid phase assay, also preserving as much as possible conformational epitopes and it appears to be as sensitive as elisa and s-flow for some of the antigens tested. it requires access to a bioluminescence detection instrument. the two neutralisation assays require cell culture systems, with mnt using infectious virus and necessitating access to a bsl facility, whereas pseudovirus neutralisation is adaptable to high-throughput screenings. serological diagnostic tests are complementary to viral detection by rt-pcr for diagnostic purposes in patients. results from our study and others indicate that in severe and critical cases (hospitalized patients), seroconversion is detectable as soon as - days post symptom onset , , [ ] [ ] [ ] . in such cases, antibody titers can reach high levels, and the different assays gave similar results. detection of anti-n and anti-full s responses demonstrated similar rates of seroconversion, whereas the s response was delayed. the anti-n response appeared slightly more rapidly than s/s responses for a given type of test, which could be of interest to develop routine diagnostics tests, if confirmed. at the population level, serological tests are used in surveys to identify persons who have been infected. regarding the identification of pauci-symptomatic or asymptomatic individuals, we consistently observed similar levels of seroprevalence, again with different sensitivities depending on the assay. elisa tri-s, s-flow and the combined lips s +n gave slightly higher detection rates than elisa-n. combining elisa n and s assays may also increase the sensitivity of detection. in our cohort of pauci-symptomatic individuals, only a minor fraction of individuals was tested by rt-pcr (not shown). it will be useful to perform a similar analysis on individuals that have been fully characterized virologically, to further assess the serological parameters of patients with diagnosed sars-cov infection. it has been reported that in convalescent patients with mild symptoms, nabs are detected from day - after disease onset in a large fraction of patients . the titers of nab correlated with the titers of anti-s antibodies (targeting s, rbd, and s regions) . a critical question is the detection of antibodies, their neutralisation potential in asymptomatic individuals, and, more generally, the correlates of protection. in our pilot study with healthy blood donors, the elisa n and lip s +n assays were negative, whereas six individuals scored positive with s-flow. when reanalyzed with the elisa tri-s, two of the six individuals were positive. these results indicate that the most sensitive assays are required for identification of asymptomatic sars-cov- infected individuals, who will likely mount a weaker response than patients experiencing a mild or severe infection. indeed, this should not be at the expense of specificity, as this could considerably impact the predictive value of positive results in low prevalence areas. we are currently exploring the levels of antibody responses in other contemporary cohorts to address this question. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . beyond the simple detection of individuals that have been in contact with the virus, the knowledge of immune protection (or on the contrary facilitation in case of re-infection in individuals with low antibody responses) detected with sensitive tests is key to avoid misuse of serological tests. neutralizing antibodies have a major role in preventing reinfections for many viral diseases. a major point is the relationship between in vivo protection and the levels of antibody binding to the virus or neutralizing it. we compared our serological assays to mnt and pseudovirus neutralisation assay, in a limited number of individuals. we observed a strong correlation between the extent of anti-full s and even anti-n response and the neutralisation capacity of the sera. we are currently examining whether antibody levels and which viral protein best correlate with neutralisation in paucisymptomatic or asymptomatic seropositive individuals. answering this question will help determining whether a serological high throughput assay may serve as a surrogate to estimate the level of protection at the individual or population level. this is an important parameter to understand and model the dynamics and evolution of the epidemics and define serological tools for population control. non-neutralizing antibodies, or neutralizing antibodies at sub-optimal doses can also lead to antibody-dependent enhancement of infection (ade). ade exacerbates diseases caused by feline coronavirus, mers-cov and sars-cov- [ ] [ ] [ ] [ ] . ade might thus also play a deleterious role in covid- . covid- cases were from included at hôpital bichat-claude-bernard in the french covid- cohort. some of the patients have been previously described . each participant provided written consent to participate to the study, which was approved by the regional investigational review board (irb; comité de protection des personnes ile-de-france vii, paris, france) and performed according to the european guidelines and the declaration of helsinki. pauci-symptomatic individuals: on feb , , a patient from crepy-en-valois (oise region, northern france) was admitted to a hospital in paris with confirmed sars-cov- infection. as part of . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . an epidemiological investigation around this case, a cluster of covid- cases, based around a high school with an enrolment of pupils, was identified. on march - , students at the high school, their parents, teachers and staff (administrative staff, cleaners, catering staff) were invited to participate to the investigation. a ml blood sample was taken from individuals who reported fever or mild respiratory symptoms (cough or dyspnea) since mid-january . the median age was years (interquartile range: - ), and % were female. this study was registered with clinicaltrials.gov (nct ) and received ethical approval by the comité de protection des personnes ile de france iii. informed consent was obtained from all participants. samples from blood donors were collected in accordance with local ethical guidelines by using polyethylenimine (pei)-precipitation method as previously described . recombinant tri-s proteins were purified by affinity chromatography using the ni sepharose® excel resin according to manufacturer's instructions (thermofisher scientific). protein purity was evaluated by in-gel protein silver-staining using pierce® silver stain kit (thermofisher scientific) following sds-page in reducing and non-reducing conditions using nupage™ - % tris-acetate gels (life technologies). high-binding . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . lips assay. ten recombinant antigens were designed based on the viral genome sequence of the sars-cov- strain france/idf / (accession no epi_isl_ ) obtained from gisaid database . five targeted different domains of s: full s sub-unit (residues - ), n-terminal domain of s (s -ntd, residues - ), domain connecting the s -ntd to the rbd (s -cd, residues - and - connected by a gggsgg linker), full s sub-unit (residues - ), and s - . for constructs that did not contain an endogenous signal peptide (residues - ) i.e. s -cd and s . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint neutralisation was calculated using the formula: x ((mean of replicates -mean of negative controls)/(mean positive controls -mean of negative controls)). s-pseudotypes incubated without serum and medium alone were used as positive and negative controls, respectively. t-cells stably expressing ace were also used in this assay and yielded similar results. for luciferase-expressing pseudotypes, samples were analyzed with the enspire instrument (perkinelmer). data processing and analysis. flow cytometry data were analyzed with flowjo v software (tristar). calculations were performed using excel (microsoft). figures were drawn on prism (graphpad software). statistical analysis were calculated using prism . sera from pre-epidemic individuals sampled between and (first row), hospitalized cases with confirmed covid- (second row), paucisymptomatics individual from the crépy-en-vallois epidemic cluster (third row) and healthy blood donors (last row) were surveyed for anti-sars-cov- antibodies using four serological assays. elisa n and elisa tri-s are conventional elisa, using either n or the trimeric ectodomain of s protein as antigens. s-flow is an assay detecting antibodies bound to cells expressing s by flow cytometry. lips s and n detect either s or n fused to luciferase by immunoprecipitation. pre-epidemic samples were used to determine the cut-off of each assay, which is indicated by a dashed line and a green area. elisas were set to % specificity. the number of positive samples is indicated. each dot represents a sample. neutralizing activity (dilution : ) of sera from the pauci-symptomatic cohort (c - ) and sera from hospitalized patients (b -b ) was determined by the pseudovirus neutralisation assay and compared to serology data obtain with the assays. numbers indicate the coefficient of correlation (spearman r). all correlations are significant (p> . ). b. neutralisation data from hospitalized patients were plotted against the day postsymptom onset. the red line corresponds to a non-linear fit of the data. table comparing the % of seroreactivity between groups according to auc value categories (see methods). (c) roc graphs comparing tri-s elisa igg seroreactivity between sars-cov- -exposed or infected individuals and pre-epidemic controls. table on the right indicates for each roc analysis the sensitivity and specificity values. (d) representative elisa graphs showing the igg, igm and iga reactivity against purified tri-s proteins of selected sera from sars-cov- -infected subjects. mean values ± sd from intra-assay duplicates are presented. sd, serum dilution. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin detection of novel coronavirus ( -ncov) by real-time rt-pcr molecular diagnosis of a novel coronavirus ( -ncov) causing an outbreak of pneumonia evaluation of a quantitative rt-pcr assay for the detection of the emerging coronavirus sars-cov- using a high throughput system virological assessment of hospitalized patients with covid- temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study the novel coronavirus ( -ncov) uses the sars-coronavirus receptor ace and the cellular protease tmprss for entry into target cells structure of sars coronavirus spike receptor-binding domain complexed with receptor cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace structural basis for the recognition of sars-cov- by full-length human ace unexpected receptor functional mimicry elucidates activation of coronavirus fusion a serological assay to detect sars-cov- seroconversion in humans sars-cov- specific antibody responses in covid- patients antibody responses to sars-cov- in patients of novel coronavirus disease profiling early humoral response to diagnose novel coronavirus disease (covid- ) global profiling of sars-cov- specific igg/ igm responses of convalescents using a proteome microarray presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov potent neutralizing antibodies in the sera of convalescent covid- patients are directed against conserved linear epitopes on the sars-cov- spike protein potent human neutralizing antibodies elicited by sars-cov- infection substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) clinical and virological data of the first cases of covid- in europe: a case series antibody-dependent enhancement of feline infectious peritonitis virus infection in feline alveolar macrophages and human monocyte cell line u by serum of cats experimentally or naturally infected with feline coronavirus antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcγrii-dependent entry into b cells in vitro molecular mechanism for antibody-dependent enhancement of coronavirus entry antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins the icareb platform: a human biobank for the institut pasteur and beyond efficient generation of human iga monoclonal antibodies disease and diplomacy: gisaid's innovative contribution to global health monitoring silent spillovers before emergence: a pilot study at the tick/human interface in thailand lentiviral vectors encoding hiv- polyepitopes induce broad ctl responses in vivo we thank the patients and individuals who donated their blood, nicoletta key: cord- -kbndie e authors: braesch-andersen, sten; beckman, lena; paulie, staffan; kumagai-braesch, makiko title: apod mediates binding of hdl to ldl and to growing t carcinoma date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: kbndie e apolipoprotein (apo) d is an important protein produced in many parts of the body. it is necessary for the development and repair of the brain and protection from oxidative stress. the purpose of this study was to investigate the extent to which apod interacts with lipoproteins in human plasma. by using detergent-free elisa, we show that immobilized monoclonal antibodies against apod very efficiently bind to low density lipoprotein (ldl) from plasma; this binding is as equally efficient as binding to an anti-apob monoclonal antibody. adding detergent to the plasma inhibited the binding, suggesting that the binding is dependent on the presence of intact lipoprotein particles. reversing the system by using immobilized anti-apob revealed that the affinity of apod for ldl is rather low, suggesting that multiple bindings are needed for a durable connection. biosensor experiments using purified lipoproteins also showed that purified apod and high density lipoprotein (hdl ), a lipoprotein fraction rich in apod, were both able to bind ldl very efficiently, indicating that the hdl -ldl interaction may be a physiological consequence of the affinity of apod for ldl. furthermore, we found that apod increases the binding of hdl to actively growing t bladder carcinoma cells but not to quiescent, contact-inhibited, confluent t cells. this result is especially intriguing given that the t supernatant only contained detectable levels of apod after growth inhibition, raising the possibility that alternating the expression of apod and a putative apod-receptor could give direction to the flow of lipids. in the current paper, we conclude that apod mediates binding of hdl to ldl and to growing t carcinomas, thereby highlighting the importance of apod in lipid metabolism. introduction upon growth arrest (quiescence) in cell lines [ , , , , ] . addition of purified apod to vascular smooth muscle cells inhibits pdgf-bb-induced proliferation [ ] and induces apoptosis in colorectal carcinoma cells [ ] . an important function of hdl particles is to deliver lipids to ldl and vldl particles, and it is reasonable to assume that this interaction is facilitated/regulated in some way. miller et al. [ ] showed that hdl and ldl mutually inhibited each other when binding to fibroblasts. their results indicate either that ldl and hdl compete for the same receptors on the fibroblasts or that the interaction between hdl and ldl in solution interfered with the binding to the fibroblasts. in the present study we have investigated the potential role of apod in the interaction between lipoproteins and between lipoproteins and cells. our results show that apod may provide an important link in the interaction between hdl and ldl particles and between hdl particles and cells, thus, apod may serve as an important regulator of lipid trafficking. monoclonal antibodies (mab) to apod were generated by immunizing mice with human recombinant apod. antibody specificity was confirmed by immunoprecipitation followed by mass spectrometry, and three mabs (d , d and d ) were selected and used in the study. other mabs used were the ldl and ldl mabs against human apob, the e mab against apoe, the h and h mabs against apoh, the j and j mabs against apoj and two control mabs, the -b - mab against human ifn-c and the il -i mab against il- (all from mabtech, nacka strand, sweden). a positive control antibody against human thioredoxin reductase (trxr ), which was used for immunostaining the t cells, was a kind gift from anders rosén, university of linköping. recombinant apod was obtained from biovendor (modrice, czech republic). to increase the solubility, this recombinant apod was modified at the following positions: trp -his, cys -ser, ile -ser. other modifications include leu -pro, pro -val and asn -ala. for more information, see the biovendor homepage. purified apoa , ldl, vldl, hdl, hdl and hdl were obtained from the academy bio-medical company inc. (houston, tx, usa). native apod was purified from plasma by affinity chromatography. in short, . ml edta/ aprotinin plasma was centrifuged first at g for minutes to remove cells, followed by centrifugation at g for minutes to remove platelets and particles. irrelevant mouse antibodies were added to the plasma to a final concentration of mg/ml in order to block hama-activity (human anti-mouse antibodies). the plasma was then diluted x with pbs tween for a final concentration of . % tween and passed at a flow rate of . ml/min over a ml affinity-column coupled with the anti-apod mab d . the column was washed with pbs, and apod was eluted with . % hac and immediately flashfrozen (s figure) . the bladder carcinoma cell line t (atcc, manassas, usa) was cultured in either dmem or rpmi medium (gibco, brl, life technology ltd. paisley, scotland) with % fbs (hyclone, thermo scientific). finger blood was obtained from consenting healthy volunteers (approved by regionala etikprövningsnämnden stockholm, / - / ) by piercing the top of the finger with a lancet and recovering ml blood with an adjustable pipette and a sterile tip. the blood was immediately transferred to eppendorf tubes containing ml dilution buffer (pbs, . mm edta, . % bsa and mg/ml irrelevant mouse igg), and cells were removed by spinning for minutes at g. finally, the supernatant was transferred to a new tube and centrifuged at g for minutes to remove platelets. for the purpose of this publication, we arbitrarily calculated the volumes of the finger blood plasma based on the observation that the finger blood samples were approximately half blood cells and half plasma. except when stated differently in the figure legends the finger blood plasma came from the same donor, although repetitions of the finger blood experiments were partly conducted using different donors. elisa assays to measure apod, apoa and apob (mabtech) were performed according to the manufacturer's instructions. dual-specific elisa, capable of detecting direct or indirect interactions, was performed using antibodies with different target specificities for capture and detection. to not disturb the integrity of the complexes, the incubation and washing buffers did not contain detergent. in short, the coating antibodies were diluted to mg/ml in pbs with . % azide, except for mab ldl , which was used at mg/ml, and ml per well were added to -well elisa plates (costar, corning, ny, usa). after a two hour incubation at room temperature, the plates were blocked with . % bsa in pbs azide (incubation buffer) for a further two hours at room temperature or in the refrigerator overnight. the plates were then washed in an automatic elisa washer (elx , biotek instruments, winooski, vt, usa), using pbs as the wash buffer. purified apolipoproteins or finger blood plasma was diluted in the incubation buffer, and ml/well were added to the elisa plates for a one hour incubation. after washing with pbs, the biotinylated antibodies were diluted in the incubation buffer to mg/ml, except for ldl -biotin, which was used at . mg/ml, and antibodies were added to the plates. following a one hour incubation, plates were again washed, streptavidin-alp (mabtech), diluted in the incubation buffer, was added, and the plates were incubated for hour. after further washing, the plates were developed by adding the substrate pnpp (para-nitrophenylphosphate, s , sigma-aldrich, st. louis, mo, usa) in a mm, ph . , tris-hcl buffer supplemented with mm mgcl . the readings were performed at nm in a thermo max microplate reader (molecular devices, ca. usa). t bladder carcinoma cells were seeded onto -well plates (costar) at varying concentrations to generate both confluent and non-confluent cell layers. to obtain approximately % confluent cultures, cells were seeded hours before the experiment, whereas confluent cells were seeded days before to obtain achieve full contact (confluency) and a cobblestone-like pattern. the cells were washed three times with serum-free rpmi buffered with mm hepes. next, ml of mg/ml hdl were added to each well, with or without ng/ml of apod or apoa , in an incubation buffer consisting of rpmi with hepes and . % bsa. after a hour incubation at room temperature, the cells were washed three times with ml of rpmi and ml of pbs with . % bsa and . % tween was added. after minutes, the supernatants were transferred to a lowbinding -well v-shaped polypropylene plate (no. , greiner bio one, germany) and centrifuged at g for minutes to remove non-solubilized material. finally, ml from each well were collected, and the concentration of apoa was determined by elisa. the binding between apod and lipoprotein particles was assessed using a blitz biosensor (fortebio, pall life science, menlo park, ca, usa). this biosensor measures mass by how it changes optical layer thickness and thus the interference patterns of reflected light. for more details, see the pall life science homepage. in brief, biotinylated mab d (anti-apod) and control mabs e (anti-apoe) and -b - (anti-ifnc isotype control for d ) were added to streptavidincoated biosensor surfaces, followed by rinsing and addition of purified apod and/ or lipoprotein particles. pbs with . % bsa and . % sodium azide was used as a buffer, and both the rinsing and loading steps were performed using a volume of ml. antibodies were loaded at a concentration of mg/ml, apod at mg/ml and hdl , hdl and ldl at mg/ml. the loading time was minutes for antibodies and min for lipoproteins, and the rinsing time was seconds. t cells were grown in dmem supplemented with % fbs in cm cell culture flasks (costar). supernatants from the confluent cultures, corresponding to approximately . million cells per flask, and non-confluent cultures, corresponding to - . million per flask ( - % confluent), were collected hours after changing the media. for each condition, the supernatants from three flasks were probed four times and tested for apod content via apod elisas (mabtech), with a detection limit of approximately . ng/ml. the same cells were also harvested and used for flow cytometry (see below). for intracellular staining, t cells were removed from cm tissue culture flasks by washing once with ml pbs containing mm edta, followed by a minute incubation at ˚c with ml of the same buffer. detached cells were transferred to a ml centrifuge tube and resuspended with a pipette to obtain a single cell suspension. the cells were washed once in pbs and, after sedimentation at g for minutes, the cell pellet was re-suspended in ml pbs and ml of a freshly prepared buffer of pbs with . % tritonx and % paraformaldehyde. after minutes fixation at room temperature, the cells were resuspended by adding ml pbs and then pelleted again by centrifugation at g for minutes. the supernatant was removed, and the remaining reactive groups were inactivated by addition of % glycine in pbs, azide, and . % bsa. after another minutes, the cells were pelleted again at g for minutes and then re-suspended in ml of pbs, azide, . % bsa. cell staining was performed in v-shaped -well plates (type , greiner bio one, frickenhausen, germany) using k cells/well, and primary antibodies were added to a final concentration of mg/ml. after a minute incubation at room temperature, the cells were washed twice with pbs before adding a secondary fitc-conjugated rabbit f(ab) anti-mouse igg antibody (f , dako, glostrup, denmark) diluted / in pbs, azide, . % bsa. after incubation for min, the cells were again washed, followed by analysis in in a guava easycyte flow cytometer (millipore, billerica, ma, usa). cells were seeded onto glass microscope slides placed in petri dishes to maintain humidity. after incubation for days (non-confluent) or days (confluent) in a ˚c incubator, the cells were washed in pbs and fixed for min in pbs containing . % paraformaldehyde and . % tritonx . the cells were then washed with pbs and blocked with pbs containing . % bsa and % glycine for min, followed by washing and incubation with primary antibodies at room temperature. in addition to the anti-apod mab d , a negative isotype-control mab (il - ) and a positive control mab (trxr ) were used (all at mg/ml). after h, the cells were washed and incubated with a secondary rabbit anti-mouse-igg fitc-conjugated f(ab) (dako) antibody ( : ) for another hour. after washing, cell nuclei were stained for min with dapi ( mg/ml) (pierce) and, after a final wash, were covered with a cover glass. cells were analyzed and photographed using a leica dmrb fluorescent microscope equipped with a nikon coolpix camera (shutter speed: fitc, sec; dapi, / sec). results are presented as means with standard deviation. statistical analysis was performed with the graphpad prism program using the mann-whitney-u-test. differences were considered statistically significant if p-values were less than . . apod in plasma is found primarily in hdl particles and to a lesser extent in ldl and vldl particles. to investigate the presence of particles containing both apod and apob, we performed a dual-specific elisa using the anti-apod mab d as a capture antibody and a biotinylated anti-apob mab, ldl , for detection. to avoid disturbing the integrity of the lipoprotein particles, the elisa was performed in the absence of detergents. using this approach, we could detect not only lipoprotein particles carrying both apod and apob but also potential complexes between hdl and ldl or vldl particles, as depicted in fig. . the dual-specific apod/b elisa analysis of plasma from finger blood yielded a surprisingly strong signal that was higher than that seen when immobilized apod was probed with the anti-apod antibody, d -biotin ( fig. a) . similar results were obtained when performing the dual-specific elisa with two other anti-apod capture antibodies, d and d (data not shown). similarly, the detection mab ldl -biotin in fig. a could be replaced by a different anti-apob antibody, ldl -biotin, with comparable results (s figure) . furthermore, diluting the plasma yielded very similar titration curves from the dual-specific elisa as the same plasma from an apob-specific elisa (fig. b) . as apod has been reported to be scarce in ldl and vldl particles, much of this signal is likely attributable to the recognition of complexes between apod-containing hdl and apob-containing ldl (or vldl) particles. using the same type of dual-specific elisa with antibodies against apoe (fig. c) , apoh (fig. d ) or apoj (fig. e ) for capture resulted in weak or absent signals. to demonstrate the presence of these putative lipoprotein complexes in the general population, finger blood from ten healthy volunteers was tested using the apod/b elisa, and as seen in fig. a , all samples displayed elisa values comparable to those seen in fig. . for reference, the apob and apod levels in the same samples were measured separately ( fig. b and c ). to determine the importance of having intact lipoprotein particles, we repeated the dual-specific apod/b elisa in the presence of detergent, using anti-apod-d as the capture mab and anti-apob-ldl as the detecting mab. as seen in fig. a , only small amounts of apob bound to d when detergent was present, indicating that intact lipoprotein particles are needed for the apod-ldl interaction. more surprisingly, when we reversed the system, using ldl as the capture antibody and biotinylated anti-apod d as the detecting antibody, no complexes could be detected, independent of the presence of detergent (fig. b ). to further investigate the potential interaction between apod-containing particles and ldl and the role of apod in this interaction, we used both purified apod and isolated hdl preparations in combination with purified ldl in the same dualspecific elisa. apod was purified from plasma by affinity chromatography in the presence of detergent, and a single band corresponding to apod was observed on sds-page (s figure) . we also used hdl and hdl particles, which differ both in size (hdl is significantly bigger) and apod content (hdl contains more apod). the apod and apob content in the different preparations was determined by elisa, confirming the differences in apod content (table i) . hdl contained some apob, which often happens when hdl is contaminated with small dense ldl. in fig. , purified ldl alone and ldl co-incubated with hdl , hdl or purified apod were analyzed via the dual-specific elisa. as expected, ldl itself gave a weak to moderate positive signal due to the concomitant presence of apod and apob in some of these particles. this signal was roughly doubled by the addition of either hdl or hdl , while addition of purified native apod led to a -fold binding enhancement. interestingly, recombinant apod, in which amino acids had been removed to reduce the hydrophobicity, did not promote ldl binding. purified vldl also binds to anti-apod in a detergent-sensitive manner and the binding is also enhanced by the addition of extra apod (fig. ) . however, the binding gives lower values and these values are not raised as much by addition of purified apod. fig. . anti-apod antibodies capture apob in a detergent-free dual specific elisa. the following capture antibodies functional under detergent-free conditions were used: a) anti-apod (d ), b) anti-apob (ldl ), c) anti-apoe (e ), d) anti-apoh (h ), e) anti-apob (ldl ) and f) anti-apoj (j ). human edta finger blood plasma, prepared as described in the methods, was diluted with pbs containing . % bsa. detecting antibodies in a-d were ldl -biotin (anti-apob), d -biotin (anti-apod), h -biotin (anti-apoh), b -biotin (anti-ifnc) and e (anti-apoe), and in e and f were d -biotin (anti-apod), d -biotin (anti-apod), ldl -biotin (anti-apob) and j -biotin (anti-apod). note that e was also used for capture in c). the means ¡ sd of four replicates are shown. results within each panel are from the same donor. experiments a-d were repeated three times using the same donor, and experiments e and f were repeated four times using four different donors. doi: . /journal.pone. .g to measure binding in a more direct manner, we also used biosensor technology. we were able to not only register binding but also obtain information about the relative association affinities of the interactions. in agreement with the previous elisa results, the anti-apod mab d bound purified ldl due to the presence fig. . the interaction between apod and apob is a general mechanism. finger blood from ten donors was assayed for a) apod/apob interaction in a detergent free dual-specific elisa using d for capture and biotinylated ldl for detection b) elisa for apob levels and c) elisa for apod levels. note that finger blood plasma, in figures b and c, was arbitrary calculated as half the finger blood volume, with the remaining volume assumed to be cells. of the ten donors, no. - were women, and no. - were men, all between - years old. means ¡ sd of four replicates are shown. apod mediates binding of hdl to ldl of apod in some of the ldl particles. (fig. a) . however, ldl binding was considerably faster and stronger when the d mab was first loaded with purified apod. the mab d yielded similar results (s figure) . as expected, the antibody also bound hdl and hdl to an extent reflecting the amount of apod in the respective particle. similar to the enhanced ldl binding observed in the presence apod, preloading the d mab with hdl , and to some extent with hdl , also increased the initial speed of ldl binding, though this effect was less strong compared to using purified apod (fig. b ). as apod is expressed by many cell types, we also investigated the interaction between apod and lipoprotein particles in a cellular setting. for this we used the fig. . binding between apob and apod is disturbed by detergents and is dependent on the immobilized antigen. finger blood plasma was treated with or without detergent before being added to elisa plates. a) anti-apod (d ) was used as the coating antibody, allowing for multiple apod bonds to each ldl particle, as detected using anti-apob (ldl -biotin). b) ldl was used as the coating antibody, and biotinylated d was used as the detection antibody. the means ¡ sd of four replicates are shown. experiments were repeated three times with same donor. doi: . /journal.pone. .g urinary bladder carcinoma cell line t , which produces apod and displays contact inhibition when cultured in vitro, which has previously been shown to affect apod production [ , , , , ] . in agreement with this latter finding, apod production was high in confluent, growth-arrested t cell cultures ( , million cells in ml), but was not detectable in non-confluent proliferating anti-apod (d ) was used as the capture antibody and anti-apob (ldl -biotin) was used as the detection antibody in a detergent free dualspecific elisa. ldl ( ng/ml) was added either alone or in combination with apoa ( ng/ml), hdl ( ng/ml), hdl ( ng/ml), apod (native) ( ng/ml) or recombinant apod ( ng/ml). as a the control, ldl was omitted, but the same amounts of apoa , hdl , hdl , apod or recombinant apod were added. the control without ldl is shown in grey bars. relative absorbance was calculated by dividing the absorbance value by the absorbance of ldl alone. the experiment was repeated times, each time using four replicates, and mean of the experiments ¡ sd is shown. ** p, . compared with ldl alone; p-values were calculated using the mann-whitney test with graphpad prism . the effect of adding extra apod ( ng/ml) to vldl ( ng/ml) was analyzed using a dual-specific elisa with or without detergent present. the capture antibody was anti-apod (d ), and the detection antibody was anti-apob (ldl -biotin). the means ¡ sd of four replicates are shown. experiments were repeated three times. * p, . ; p-values were calculated using the mann-whitney test with graphpad prism . cultures ( , million cells in ml) (fig. a) . however, despite the absence of apod in the supernatant of proliferating cells, apod was abundant intracellularly, as shown by flow cytometry (fig. b ) and by immunocytochemistry (fig. ) . to measure potential interactions between cells and lipoprotein particles, cells were incubated with hdl in the presence or absence of apod. after removing unbound material by washing, the bound particles were solubilized, and the apoa (hdl) content in the soluble fraction was analyzed by elisa. as fig. . biosensor monitoring of apod-mediated binding of ldl. in the blitz biosensor analysis, the signal corresponds to the mass collected on the biosensor surface. here we used streptavidin-coated sensor surfaces that were loaded with biotinylated antibody ( mg/ml for seconds) followed by apod ( mg/ml for seconds) or buffer-control ( seconds). the surfaces were subsequently loaded with the ldl, hdl or hdl lipoprotein particles ( mg/ml for seconds). between loading reagents, the sensors were rinsed for seconds. the buffer used for all steps and dilutions was pbs, azide, . % bsa. a) the green, black, grey and red lines represent experiments preloaded with anti-apod d . the blue line indicates the control preloaded with isotype control antibody b (anti-ifnc). the green and blue lines indicate that apod was preloaded, while the black, grey, and red lines indicate the buffer controls. the panel shows the final step in which the lipoprotein particles bind to the antibody loaded with or without apod. the green, black and blue lines show binding of ldl, the grey line shows binding of hdl and the red line shows binding of hdl . b) the binding step of the biotinylated antibody was omitted. green, black, red and grey lines indicate experiments that used d -biotin, and the blue line indicates that e -biotin (anti-apoe) was used. subsequent reactions show the addition of hdl in red, hdl in grey, apod in green and blue, or buffer in black to the preloaded antibodies. in the last step, ldl was loaded in all the conditions. doi: . /journal.pone. .g demonstrated in fig. , significant amounts of hdl were bound to both the confluent and non-confluent cultures. however, addition of purified apod to the non-confluent, proliferating cells significantly promoted the binding of hdl, whereas the same addition to confluent cells only had a marginal positive effect. similar experiments with ldl showed that apod did not help ldl bind to the cells (data not shown). fig. . apod production in t cells was assayed by elisa for culture supernatant and by facs for intracellular staining. a) t cells were grown in dmem with % fbs in cm flasks. three flasks each of confluent cells ( . million in ml) and non-confluent cells ( . million in ml) were sampled times. apod content was assayed by apod elisa; detection limit . ng/ml. b) d (anti apod) was used to stain confluent (black) and non-confluent (grey) t carcinoma cells. the isotype control is depicted with a dotted black line for confluent t cells and with a dashed grey line for non-confluent t cells. experiments were repeated three times. doi: . /journal.pone. .g we have here shown that apod, immobilized by specific monoclonal antibodies on a solid surface, mediates the binding of ldl to the surface. we used a dualspecific elisa in which antibodies to apod bound intact apob-containing lipoproteins equally efficient as antibodies specific for apob. this property was unique for apod and was not seen with other exchangeable apolipoproteins such apoe, apoh and apoj, all of which are present in hdl and ldl/vldl lipoprotein particles [ , , ] . adding detergent inhibited the apod-mediated binding of apob, demonstrating that changes in the ldl structure affect the formation of the complex. surprisingly, ldl/vldl particles immobilized by antibodies to apob could only marginally capture apod, suggesting that the affinity of a single apod is not sufficient to withstand the repeated washings and incubations of the elisa protocol. therefore, several apod molecules will likely be needed to achieve sufficient avidity to immobilize ldl to a surface via apod-binding. this moderate affinity between apod and ldl is likely suitable for the rapid encounters between fig. . non-confluent t cells stain positive for apod. cells were seeded on objective glasses in petri dishes. after either days (non-confluent) or days (confluent), the cells were washed and incubated with a negative isotype-control mab (anti-il ), a positive control anti-thioredoxin reductase mab (anti-trxred), or anti-apod d mab. bound monoclonal antibodies were stained with polyclonal anti-mouse fitcconjugated fab fragments. nuclei were visualized with dapi. cells were photographed with a leica dmrb fluorescent microscope (shutter speed fitc sec, and dapi / sek). staining was repeated three times. hdl-containing apod and ldl particles in the plasma, as a high affinity interaction could yield more stable complexes and aggregation. notably, adding purified apod helped d to bind more ldl, indicating that the apod contact with ldl is a very rapid event. hydrophilized recombinant apod, on the other hand, was not capable of binding ldl, probably because some of the hydrophobic amino acids required for binding have been removed in the recombinant apod. these results emphasize the need to use native apod for functional studies of this lipocalin. biosensor analysis is a nice complement to elisa as it measures binding in a direct way. mass binding to the sensor-tip results indiscriminately in a stronger signal, and the speed of binding can be monitored and is proportional to the fig. . apod facilitates binding of hdl to growing t cells. t cells grown in -well plates were washed with protein-free rpmi two times, and ml/well rpmi supplemented with . %bsa was added with or without hdl (total hdl, ng/well) and with or without apod ( ng/well) or apoa ( ng/well). after incubation for hour at ˚c, the plate was washed three times with protein-free rpmi, and bound apoa was released using detergent and assayed by apoa elisa. in a) the cells were non-confluent and in b) the cells were confluent. means ¡ sd of four experiments are shown. ** p, . , ****p, . ; p-values were calculated using two-way anova with holm-sidal multiple comparison test, graphpad prism . doi: . /journal.pone. .g affinity/avidity of the reaction. we demonstrate that although ldl contains some apod and will bind to anti-apod (d ), the binding was considerably faster if the d antibodies were preloaded with apod. in addition, hdl bound both more quickly and with more mass to d -coated surfaces than did hdl . as mentioned above, hdl has a higher density than hdl but is also considerably smaller and carries more apod. preloading the surface with purified apod is the most efficient way to tie down ldl, but hdl also yields a high initial speed of ldl binding compared to d alone or loaded with hdl . one explanation for the difference between the quick binding of ldl to apod-or hdl -coated surfaces and the slower linear binding of ldl to d alone is that, in the latter case, apod needs to reposition to allow multiple bonds between the surface and ldl. in animal experiments, apod was shown to be important in protecting against oxidative stress [ , , ] . interestingly, hdl has been reported to be more important than hdl for protection against ldl-copper-catalyzed oxidation [ , ] . the biosensor results obtained here show that hdl bound ldl more avidly, supplying a potential explanation for the protection of ldl by hdl . t carcinoma cells display contact inhibition if the cells are washed with fresh medium after reaching confluency. washing removes growth factors and ensures that the cells are kept below the threshold needed to overcome contact inhibition [ ] . interestingly, purified apod may aid growing t cells in binding hdl, but not contact-inhibited, growth-arrested cells, indicating a possible growthregulated receptor for apod. additionally, only supernatants from growthinhibited t cells contained detectable amounts of apod. these results agree with several reports showing that apod production increases upon growth arrest . however, intracellular staining with d revealed that non-confluent t cells contained substantial amounts of apod, albeit less than confluent cells. the reason for these unexpected results may be that non-confluent cells produce, but do not export, apod. another possibility is that non-confluent cells secrete apod but then internalize it efficiently via receptors not expressed by quiescent cells. one might speculate that apod plays a role in the lipid efflux to and from t cells. a putative mechanism is that apod helps to supply lipids to growing cells, presumably via an apod-recognizing receptor. once contact inhibition is induced, the cells downregulate this receptor and secrete surplus lipids via apod-containing lipoproteins. however, verifying such a mechanism would require extensive studies to find an apod binding receptor. large amounts of apod accumulate at sites of nerve injury [ ] . human genetic studies and studies in apod knockout mice have revealed that apod is important not only for brain and nerve functions but also for controlling inflammation and macrophage homeostasis and to maintain a normal lipid profile [ , ] . here we propose that apod acts a mediator/cofactor in the interaction of hdl particles with ldl particle and with cells. apod would presumably anchor hdl to ldl or cells with an affinity/avidity that result in a suitable on-off rate for lipid trafficking. this mechanism could partly explain several of the physiological roles of apod. for example, apod has been reported to help regulate lcat activity [ , , ] . if apod is an important mediator of lipid trafficking, one might speculate that the intense up-regulation of apod at the site of nerve injury is necessary to attract and increase the local concentration of hdl particles for lipid transport between glial cells or macrophages and nerve cells and schwann cells. apod is an enigmatic protein, but the results of the current study reveal an important function of apod as a mediator of the interaction between hdl particles to ldl particles and to growing cells. however, further studies are needed to fully elucidate the physiological function of apod. several important questions remain, such as determining whether other cofactors are necessary for these lipoprotein interactions and how the lipid hormone-carrying role of apod affects these interactions. will the lipid load of this lipocalin change its function? identifying and characterizing the potential cellular receptor for apod and how it is regulated during cell growth will also be necessary. supporting information s figure. affinity purified apod. the mabtech apod standard was purified by affinity purification from edta aprotinin plasma in the presence of a non-ionic detergent. approximately . mg was analyzed by sds-page in the presence of a reducing agent and stained with simplyblue, invitrogen. doi: . /journal.pone. .s (tif) s figure. ldl/vldl captured by anti-apod can be detected by either ldl biotin or ldl -biotin. d was used to capture lipoproteins from human edta finger blood plasma, prepared as described in the methods. biotinylated detection antibodies were ldl (anti-apob), and ldl (anti-apob). d biotin was used as a positive control. means ¡ sd of three replicates are shown. doi: . /journal.pone. .s (tif) s figure. biosensor monitoring of apod-mediated binding of ldl. in the blitz biosensor analysis, the signal corresponds to the mass collected on the biosensor surface. here we used streptavidin-coated sensor surfaces that were loaded with either d -biotin or the isotype control b -biotin ( mg/ml for seconds). sensors were rinsed ( seconds) and loaded with apod ( mg/ml) or buffer-control ( seconds), and then rinsed again and loaded with mg/ml ldl ( seconds). doi: . /journal.pone. .s (tif) author contributions preparative isotachophoresis of human plasma high density lipoproteins hdl and hdl apolipoprotein d apolipoprotein d in lipid metabolism and its functional implication in atherosclerosis and aging structural insight into the dual ligand specificity and mode of high density lipoprotein association of apolipoprotein d apolipoprotein d is the major protein component in cyst fluid from women with human breast gross cystic disease human apod, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in drosophila apolipoprotein d is involved in the mechanisms regulating protection from oxidative stress accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. identification of apolipoprotein d, apolipoprotein a-iv, apolipoprotein e, and apolipoprotein a-i apod, a glia-derived apolipoprotein, is required for peripheral nerve functional integrity and a timely response to injury regeneration-associated high level expression of apolipoprotein d mrna in endoneurial fibroblasts of peripheral nerve apolipoprotein d: an overview of its role in aging and age-related diseases apolipoprotein d takes center stage in the stress response of the aging and degenerative brain identification of apolipoprotein d as a cardioprotective gene using a mouse model of lethal atherosclerotic coronary artery disease neuroprotective effect of apolipoprotein d against human coronavirus oc -induced encephalitis in mice apolipoproteins in the brain: implications for neurological and psychiatric disorders genetic variation in apolipoprotein d affects the risk of alzheimer disease in african-americans genetic variation in apolipoprotein d and alzheimer's disease association between polymorphisms in the apolipoprotein d gene and sporadic alzheimer's disease a role of apolipoprotein d in triglyceride metabolism genetic deficiency of apolipoprotein d in the mouse is associated with nonfasting hypertriglyceridemia and hyperinsulinemia separation of free and apolipoprotein d-associated human plasma lecithin: cholesterol acyltransferase activation of lecithin-cholesterol acyltransferase by apolipoprotein d: comparison of proteoliposomes containing apolipoprotein d, a-i or c-i a cholesteryl ester transfer complex in human plasma selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein d: implications for lipid antioxidant activity and alzheimer's disease apolipoprotein d transcription occurs specifically in nonproliferating quiescent and senescent fibroblast cultures inverse relationships between cell proliferation and basal or androgen-stimulated apolipoprotein d secretion in lncap human prostate cancer cells growth inhibition of human breast cancer cells by , -dihydroxyvitamin d is accompanied by induction of apolipoprotein d expression modulation of apolipoprotein d and apolipoprotein e mrna expression by growth arrest and identification of key elements in the promoter apolipoprotein d inhibits platelet-derived growth factor-bb-induced vascular smooth muscle cell proliferated by preventing translocation of phosphorylated extracellular signal regulated kinase / to the nucleus expression and potential role of apolipoprotein d on the death-survival balance of human colorectal cancer cells under oxidative stress conditions interaction between high density and low density lipoproteins uptake and degradation by cultured human fibroblasts proteomic analysis of electronegative low-density lipoprotein a -kda apolipoprotein designated apoj is a marker for subclasses of human plasma high density lipoproteins genetic variation in the apolipoprotein h (beta -glycoprotein i) gene affects plasma apolipoprotein h concentrations hdl exerts more powerful antioxidative, protective effects against copper-catalyzed ldl oxidation than hdl hdl, lipid peroxidation, and atherosclerosis apod mediates binding of hdl to ldl matrix stiffening sensitizes epithelial cells to egf and enables the loss of contact inhibition of proliferation key: cord- -l f gp authors: nan title: oral and poster manuscripts date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: l f gp nan pandemic influenza h n (h n pdm) virus of swine-origin causes mild disease, but occasionally is associated with acute respiratory distress syndrome and death. , it is important to understand the pathogenesis of this new disease. previously we showed a comparable virus tropism and host innate immune responses between h n pdm and seasonal h n influenza virus in the human respiratory tract, however h n pdm virus differed from seasonal h n influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection. we now compare the tropism and host responses elicited by pandemic h n with that of related swine influenza viruses and a pandemic-swine reassortant virus in ex vivo and in vitro cultures of the human respiratory tract and conjunctiva. we have used recombinant virus to investigate the role of the hemagglutinin (ha) and neuraminidase (na) of h n pdm virus in its conjunctival tropism. these findings are relevant for understanding transmission and therapy. fragments of human conjunctiva, bronchi, and lung tissues were cut into - mm fragments within h of collection and infected with influenza a viruses at a titer of tcid ⁄ ml. viruses investigated included h n pdm (a ⁄ hk ⁄ ⁄ ), swine h n virus (a ⁄ swine ⁄ hk ⁄ ⁄ ), which shares a common derivation for seven genes with h n pdm, a natural swine reassortant h n (a ⁄ swine ⁄ hk ⁄ ⁄ ), which has acquired the na gene from h n pdm and other swine influenza h n viruses. reverse genetics derived recombinant viruses with ha and na gene segments of seasonal h n and pandemic h n swapped were also studied. lung fragments were cultured at °c in culture plates; conjunctival and bronchial biopsies were cultured in air-liquid interface at and °c respectively. tissue fragments were infected for h and incubated for , , and h post infection. infectious viral yield was assessed by titration in mdck cells. the infected tissues were fixed with formalin and analyzed by immunohistochemistry for influenza antigen. cytokines profiles induced by influenza virus infected respiratory epithelial cells in vitro were measured by quantitative rt-pcr and elisa. we found comparable replication in seasonal and pandemic h n viruses in human respiratory tract, while the swine influenza a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) virus and a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) virus failed to infect and replicate in human lung ex vivo culture, but it replicated productively in human bronchus ex vivo. interestingly, the swine reassortant influenza h n (a ⁄ swine ⁄ hk ⁄ ⁄ ) virus (with the na from h n pdm) infected and productivity replicated in lung ex vivo and in vitro. pandemic h n pdm virus, but not seasonal h n virus, was able to infect ex vivo cultures of human conjunctiva, suggesting subtle differences in receptor binding profile in h n pdm, seasonal viruses, and the swine related h n viruses. using reverse genetics derived recombinant viruses, we were able to demonstrate that the ha and na segments of h n pdm, but not the polymerase genes, were required for the conjunctival tropism of h n pdm ( figure ). in contrast with highly pathogenic influenza h n virus, which induced high cytokine and chemokine decretion, the related swine viruses, a ⁄ swine ⁄ hk ⁄ ⁄ (h n ), as well as the swine pandemic reassortant virus, a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) we studied were similar to h n pdm and seasonal influenza viruses in their intrinsic capacity for cytokine dysregulation. collectively, our results suggest that pandemic h n pdm virus differs in modest but subtle ways from seasonal h n virus in its intrinsic virulence for humans, findings that are in accord with the epidemiology of the pandemic to date. the ha and na gene segments are key to the conjunctival tropism manifested by the h n pdm virus. the pandemic reassortant influenza h n (a ⁄ swine ⁄ hk ⁄ ⁄ ) virus isolated from swine with the na from h n pdm shares with h n pdm the capacity for productive replication in lung ex vivo and in vitro. these findings are relevant for understanding transmission and therapy. isolation of influenza viruses from specimens is traditionally performed in two classical systems: embryonated chicken eggs and mdck cell culture. nevertheless, several publications are dedicated to the theme of alternative cell culture systems, which may be used for influenza virus isolation and cultivation. [ ] [ ] [ ] this is in part because mdck cells are of animal origin, which means that they cannot be used as a proper model for estimating interactions between a human virus and a human cell culture as a host. a variety of human monolayer and suspension cell cultures have been tested on their capability to support influenza virus replication. among them, some support influenza a virus growth as well as mdck cells do, others support replication of a virus, but do not enable the formation of mature viral particles, whereas others show only a weak level of replication or are not permissive at all. caco- cells, for example, represent a good substitute for mdck cells, because it has been shown that the rate of viral isolation in caco- cells is as effective as in mdck, and sometimes is even better. the success of viral replication is determined not only by the cell culture type, but also by the virus itself. despite the accepted view that it is the type of receptor that defines the interaction between the virus and the host cell, there is evidence that it is not the only factor that predetermines the fate of the cell. the fate of the infected cell can also differ. a series of articles show that apoptosis is the most probable mechanism of cell killing by influenza viruses. , influenza a viruses of different subtypes induce apoptosis to a different extent (e.g. h viruses provoke more strong apoptotic response than h viruses do ). nevertheless, it has been demonstrated that caco- cells do not follow the apoptotic pathway and die through necrosis. the sjpl cell line also dies through necrotic pathway and not apoptosis. the aim of our work was to compare growth characteristics of different flu viruses (e.g. avian, swine, and human) in various human and animal cell cultures and to evaluate their influence on cell culture growth. the parameters measured in the study were as follows: cytopathic changes of cell cultures following virus infection, hemagglutinin production, np synthesis, the dose-dependent effect of infection on cell proliferation, and the ability of viruses to induce apoptosis. influenza viruses used included: highly pathogenic avian h n a ⁄ kurgan ⁄ ⁄ , low pathogenic avian h n a ⁄ gull ⁄ kostanai ⁄ ⁄ , swine h n a ⁄ swine ⁄ ⁄ , human h n v a ⁄ california ⁄ ⁄ , human h n v a ⁄ saint-petersburg ⁄ ⁄ , human h n a ⁄ brisbane ⁄ ⁄ , and human h n a ⁄ brisbane ⁄ ⁄ . the viruses were propagated in -days embryonated chicken eggs, the allantoic fluid was collected, the aliquots were made and stored at ) °c for further use. to evaluate tcid for each virus on all cell cultures, -well plates were used. the cells were seeded ae ml per well (concentration of - ae · cells ⁄ ml). the confluent -h old monolayer was used for viral inoculation. the cells were washed twice with serum-free medium, then ae ml of tenfold viral dilutions from viral aliquots were added and left for min for contact at °c. the cells were then washed to remove the non-attached particles, and the wells were filled with tpck-trypsin ( lg ⁄ ml)-containing medium without bovine fetal serum. the plates were observed daily for cytopathic effect, and the results were evaluated at h after infection for cytopathic effect and by reaction of hemagglutination with suspension of chicken erythrocytes ( ae %). infection of suspension cell cultures was done in centrifuge tubes. cells (concentration - · ) were inoculated with viral dilutions (moi = - ). after min of contact, cells were washed, resuspended in rpmi with trypsin and fetal serum, and seeded in -well plates ( ml in each well). the results were fixed after h, calculating the number of cells grown and estimating the rate of apoptosis by hoechst- staining. cells were grown in -well plates with seeding concentration · cells ⁄ ml. one millilitre of cell suspension was placed in each well, inoculated with viral dilution (moi = - ) and left for h. after, the cells were detached from plastic with versene and calculated in fuks-rosental camera to evaluate the number of cells. the monoclonal antibodies obtained in research institute of influenza towards viral nucleoprotein np were used following the standard protocol described in. for all viruses tested, mdck turned out to be more permissive than sp cell culture. avian viruses, independently of their pathogenicity, replicated efficiently on both animal cultures tested. human h n and h n viruses demonstrated weaker replication in sp cells. the most significant differences were seen for swine influenza and pandemic h n v viruses which replicated in mdck cells at the rates comparable with other viruses, but showed poorer growth in sp cell line (see table ). human cell lines displayed clear differences in their susceptibility to viruses of various origins. avian influenza viruses replicated in all cell lines except girardi heart, and the most intense replication rate was observed for ecv- , l- , and rd lines. a- and a- were poorly infected, as well as all suspension cell lines tested. seasonal human h n , as well as h n viruses, replicated in all cell cultures tested, but the rate of infectivity was rather low in practically all cultures tested with the exception of rd and t- g cell lines. strikingly, swine influenza virus and human pandemic h n v viruses didn't replicate well in any of human lines tested. a weak replication rate was observed in ecv- , rd, and t- g, but in general, human cell lines were the titers produced by swine and pandemic influenza viruses are shaded in grey. *low-pathogenic avian influenza virus; **highly-pathogenic avian influenza virus poorly susceptible to pandemic h n v. swine influenza virus differed because it infected weakly a- and girardi heart cell cultures, which was not the case for h n v viruses. our study has shown that all influenza viruses were able to induce apoptosis in the cell cultures tested. the degradation of chromatin found in the nucleus with hoechst- staining was seen before the first symptoms of cytopathic effect (cpe) in monolayer of cells. in cell cultures where the cpe was not visible, high doses of virus still induced apoptotic response. the process of apoptosis is rather well studied in mdck cells and some other cell types, so we've focused on three human monolayer cell cultures that are relatively poorly studied: a- , ecv- , and flech. these cell cultures are less susceptible to viral infection, and besides, it was interesting to find out whether the viruses that do not cause any cpe do infect these cultures. a- turned out to be most sensitive to apoptotic response, while flech turned out to demonstrate weak reaction. time needed for apoptosis induction by different flu viruses also varied. the earliest apoptosis was noted for h n and h n viruses and h n viruses induced apoptosis at about h postinfection. it is well-known that apoptosis can be induced only by a reproducing virus, and that uv-kills viruses that are not capable of it. we tested whether swine and pandemic h n v viruses (that do not show cpe in these cultures) do replicate in them and induce apoptosis with the help of monoclonal antibodies against viral np. the obtained data show that they indeed do replicate in these cell cultures, as we observed np fluorescence, and that they also induce apoptosis (see table ). we've shown earlier thus, we've tested the ability of swine and pandemic h n v viruses in this aspect. it was shown that these viruses were comparable with the effect seen for seasonal h n virus. moreover, swine influenza virus induced stronger apoptotic response in hemablastoid cell lines in comparison with pandemic h n v viruses, which also have a swine origin. we also checked the ability of flu viruses to influence monolayer cell cultures growth. the data clearly indicated that only ecv- endothelial line and t- g glioblastoma line displayed cell proliferation in response to low moi. apoptosis wasn't registered in these stimulated cultures, apparently because the moi was very low. all the other monolayer cultures didn't respond to low moi by stimulation of their proliferation. interaction between an influenza virus particle and a host cell can follow several scenarios. cpe seen in infected cells is accompanied with high rates of viral particles production and leads to cell death. the death itself may be through apoptotic or necrotic pathways. , also, infection process in low doses can stimulate cell proliferation -the effect seen for hemablastoid lines, histiocytes, peripheral blood cell lines, , and in glioblastoma and endothelial cell lines as it was described here. considering the origin of ecv line, these cells bear all the antigenic, biochemical, and physiological traits of umbilical cord and are actively used in pharmacological tests as well as glioblastoma cells; they also are of special interest for oncogenesis studies. table . replication, apoptosis induction, and np synthesis of influenza viruses in a- , ecv- and flech cell cultures. the numbers represent the log tcid ⁄ ae ml calculated by reed-muench method as described in. the ()) symbol means that no cpe could be observed in any dilution and no hemagglutination could be registered. the (+) symbol means that apoptosis was observed with hoechst- staining though the productive replication and production of progeny viruses in human cell lines was generally low, it is evident that viral infection does occur in these cells, even for swine and h n v viruses. it can be demonstrated by the presence of np de novo synthesis and by stimulation of virus-induced apoptosis. in fact, we observe a contradiction: avian influenza viruses actively reproduce in human cell lines, but we do not see their vast spreading in human population, while h n v viruses that hardly replicate in all human cultures tested have caused the latest pandemic. influenza viruses continue to cause problems globally in humans and their livestock, particularly poultry and pigs, as a consequence of antigenic drift and shift, resulting frequently and unpredictably in novel mutant and reassortant strains, some of which acquire the ability to cross species barriers and become pathogenic in their new hosts. long-term surveillance of influenza in migratory waterfowl in north america and europe have established the importance of anseriformes (waterfowl) and charadriiformes (gull and shorebird) in the perpetuation of all known subtypes of influenza a viruses. the available evidence suggests that each of the hemagglutinin (ha) and nine neuraminidase (na) subtype combinations exist in harmony with their natural hosts, cause no overt disease, and are shed predominantly in the feces. , in this study we determined the subtypes and prevalence of low-pathogenic influenza a viruses present on the territory of kazakhstan in - and further analysed the ha and na genes of these isolates in order to obtain a more detailed knowledge about the genetic variation of influenza a virus in their natural hosts. (institute for biological safety problems, gvardeiskiy, zhambyl oblast, kazakhstan)). samples that were identified as influenza a virus positive by matrix rrt-pcr were thawed, mixed with an equal volume of phosphate buffered saline containing antibiotics (penicillin u ⁄ ml, streptomycin mg ⁄ ml, and gentamicin lg ⁄ ml), incubated for minutes at room temperature, and centrifuged at g for minutes. the supernatant ( ae ml ⁄ egg) was inoculated into the allantoic cavity of four -day old embryonated hens' eggs as described in european union council directive ⁄ ⁄ eec. embryonic death within the first hour of incubation was considered as non-specific, and these eggs were discarded. after incubation at °c for days the allantoic fluid was harvested and tested by haemagglutination (ha) assay as describe in european union council directive ⁄ ⁄ eec. in the cases where no influenza a virus was detected on the initial virus isolation attempt, the allantoic fluid was passaged twice in embryonated hens eggs. the number of virus passages in embryonated eggs was limited to the maximum two to limit laboratory manipulation. a sample was considered negative when the second passage ha test was negative. the subtypes of the virus isolates were determined by conventional haemagglutination inhibition (hi) test and neuraminidase inhibition (ni) test, as describe in european union council directive ⁄ ⁄ eec. rna extraction and pcr with specific primers rna was extracted from infective allantoic fluid using rneasy mini kit (qiagene, gmbh, germany) according to the manufacturer's instructions. the rna was converted to full-length cdna using reverse transcriptase. the rt mix comprised ae ll of dmpc water, ll of · first strand buffer (invitrogen), ae ll of mm dntp mix (amersham biosciences), ll of mm uni primer, u of rnaguard (amersham biosciences), u of mmlv reverse transcriptase (invitrogen) and ll rna solution in total volume of ll. the reactions were incubated at °c for minutes followed by inactivation of the enzyme at °c for min. pcr amplification with ha and na gene specific primers was performed to amplify the product containing the full length ns gene. twenty-five microliter pcr-mix contained · platinum taq buffer (invitrogen), lm dntp, ae mm mgcl , nm each of fw primer and rw primer, u platinum taq dna polymerase (invitrogen) and ll cdna. reactions were placed in a thermal cycler at °c for min, then cycled times between °c seconds, annealing at °c for seconds, and elongation at °c for seconds and were finally kept at °c until later use. sequences of the purified pcr products were determined using gene specific primers and bigdye terminator version ae chemistry (applied biosystems, foster city, ca), according to the manufacturer's instructions. reactions were run on a abi tm dna analyzer (applied biosystems). sequencing was performed at least twice in each direction. after sequencing, assembly of sequences, removal of low quality sequence data, nucleotide sequence translation into protein sequence, additional multiple sequence alignments, and processing were performed with the bioedit software version ae ae ae with an engine based on the custal w algorithm. the phylogenetic analysis, based on complete gene nucleotide sequences were conducted using molecular evolutionary genetics analysis (mega, version ae ) software using neighbor joining tree inference analysis with the tamura-nei c-model, with bootstrap replications to assign confidence levels to branches. [ ] [ ] [ ] [ ] ha and na sequences obtained from genbank the ha and na gene was analyzed both with selected number of influenza isolates and in comparison with virus genes obtained from genbank were used in phylogenetic studies [ ] . the nucleotide sequence data obtained in this study has been submitted to the genbank database and is available under accession numbers fj , fj ae , fj , fj , gu -gu for ha and fj , fj ae , fj , fj , gu -gu for na. avian influenza prevalence in our study h , h , and h influenza a virus subtypes were found to circulate at the same time, in the same geographic region in the kazakhstan. this finding most likely indicates the existence of a large reservoir of different influenza a viruses in kazakhstan. we analyzed the ha and na gene sequences of the eight influenza a viruses isolated in kazakhstan together with selected number of isolates, reported between year to , and previously published in the genbank. phylogenetic analysis of the h ha gene showed that all viruses separated into the american and eurasian lineages ( figure ). an evolutionary tree suggests that north american isolates have diverged extensively from those circulating in other parts of the world. geographic barriers which determine flyway outlay may prevent the gene pools from extensive mixing. the lack of correlation between date of isolation and evolutionary distance suggests that different h ha genes co circulate in a fashion similar to avian h ha genes and influenza c genes, implying the absence of selective pressure by antibody that would give a significant advantage to antigenic variants. analysis of phylogenetic relationships among the ha ha genes reported in this study clearly shows that viruses belong to the western pacific flyway, one of the major migratory flyways in this region that have subsequently spread throughout eurasia. these findings provide further evidence of the dynamic influenza virus gene pool in this region. along the western pacific migratory flyway, the influenza virus gene pool in the domestic waterfowl of southern china has 'mixed' longitudinally with viruses isolated from japan, mongolia, and siberia. however, it appears that there has also been 'mixing' latitudinally through overlapping migratory flyways, thereby facilitating interaction between the influenza virus gene pool in domestic waterfowl in the eastern and western extremities of the eurasian continent. this helps to explain the latitudinal spread of the qinghai-like (clade ae ) h n virus in the last years, while h n outbreaks in korea and japan may represent the longitudinally transmitting pathway. ha of subtype h so far has been found exclusively in shorebirds, such as gulls, and in a pilot whale (potentially a spillover from shorebirds), but not in other avian species that are natural hosts of influenza a virus, such as ducks and geese; therefore the study of the evolution of these viruses is very interesting. phylogenetic analysis h ha gene revealed three significantly different evolutionary lines: an american line, a european line, and a line comprising the isolates from america and eurasia. further we analyzed na genes of influenza viruses (figure ) . the na gene is important both because of its functional role in promoting the dissemination of the virus during infection, and because, like ha, it is a principal target of the immune system. it was shown that phylogeny of na genes of influenza have the same properties as hemagglutinin. na genes of kazakhstanian viruses belong to eurasian lineage of virus evolution. obtained data are important for surveillance and diagnostics because some of the lpai viruses examined in this study can infect and be shed by chickens and turkeys and may have epidemiology potential during further recombination with other influenza viruses. influenza virus is divided into different subtypes based on hemagglutinin (ha) and neuraminidase (na) on the virus surface. within each subtype, ha continues to mutate and produce immunologically distinct strains, as antigenic drift. the continuous mutation of influenza virus (iv) is important for annual epidemics and occasional pandemics of disease in humans. antigenic drift requires vaccines to be updated to correspond with the dominant epidemic strains. in humans, ivs show both antigenic drift frequently. in contrast, ivs from birds are in evolutionary stasis, and they show little amino acid changes. , the reason is that ivs in bird intestine are not subjected to strong immune selection. hemagglutinin (ha) gene of influenza a virus encodes the major surface antigen, which is the target for the protective neutralizing antibody response that is generated by infection or vaccination. in humans, influenza a viruses show antigenic drift with amino acid changes in the globular head of the ha so as to evade herd immunity of the population. on the contrary, avian influenza a viruses show evolutionary stasis in wild birds. h aivs have occurred frequently in chicken farms in the world. although vaccination is not permitted, h n aivs have circulated in taiwan for a time. the seroprevalence in chicken flocks reaches about % in the field. h n aivs invades internal organs, such as kidney and lung. thus, viruses in chicken flocks are pressured into antibody selection. here, we report that h n aivs in the field have showed evolutional changes instead of evolutional stasis. in response to requests from poultry farmers for diagnostic investigations of illness in poultry flocks, the authors did necropsy at the pen-site. after careful examination, tracheae were taken and kept in cold for virus isolation in the laboratory. for avian influenza virus isolation, trachea was homogenized : in tpb with antibiotics. the homogenate was frozen and thawed three times and then centrifuged at g for minutes. the supernatant was passed through a ae lm filter. the homogenate was examined for the presence of virus by inoculation into five -to -day-old specific-pathogen-free (spf) chicken eggs for two passages. thirteen h n aivs were isolated in this laboratory during and from different parts of taiwan. besides the viruses isolated in this laboratory, the ha sequences of chicken h n aivs were from the genbank. the accession numbers of hemagglutinin of aiv reference strains included in this study were as the following: g ⁄ , dq ; g ⁄ , dq ; ⁄ , dq ; na ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ns ⁄ , dq ; sp ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; pf ⁄ , dq ; pf ⁄ , dq ; pf ⁄ , dq ; a ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ch ⁄ , dq ; ⁄ , dq ; a ⁄ , dq and ⁄ , dq . the viruses isolated were propagated in the allantoic cavities of -day-old embryonated spf eggs for hour. the virus rna was extracted using qiaamp viral rna miniprep kit (qiagen) . six-week-old balb ⁄ c mice were injected emulsion intraperitoneally with lg of purified and concentrated a ⁄ chicken ⁄ taiwan ⁄ v ⁄ (h n ) virion with complete freund's adjuvant. every two weeks, the mice were boosted supplementary five times with lg of virion in incomplete freund's adjuvant. when the mice were boosted, blood was collected from tail vein and tested by the western blot assay to check the antibody titers. the mice were then injected intraperitoneally with lg of virion at week . five days after the last injection, the splenocytes in the mice were fused with myeloma cells (sp ⁄ -ag ). one week before fusion, the myeloma cell line was expended in dmem medium (hyclone laboratories, logan, ut) with % fetal bovine serum at °c to ensure they were in the exponential growth phase. the spleen cells from immunized mice were washed, harvested, and mixed with the previously prepared myeloma cells and fused by gradually adding % polyethylene glycol- . the resulting pellet was plated into well tissue culture plates. only the fused cells grew in medium with hypoxanthine-aminopterin-thymidine (hat). with fresh medium replacement over weeks, the hybridomas were ready for screening. hightiter monoclonal antibody (mab) preparations were obtained from the ascetic fluid of mice injected with the selected hybridoma clones. the antibody from mouse ascetic fluids was purified by precipitation with ammonium sulfate, then aliquoted and frozen at ) °c, avoiding repeated freezing and thawing. eventually, six mabs were obtained and named ch -d , eb -b , eb -e , eb -f , ff -f , and ff -f , respectively. the hi test was performed following a standard method. all the viruses were diluted twofold and reacted with % chicken erythrocytes in the v-bottomed microtiter plate by the hemagglutination test. after agglutination, four hemagglutinating units of a ⁄ chicken ⁄ taiwan ⁄ v ⁄ (h n ) and ascetic fluids from the immunized mice of the six mabs were prepared for hi test. hi titers of or more were regarded as positive. the cases submitted for diagnosis from chicken farms had respiratory signs, increase in mortality, or drop in egg production (e.g. egg production dropped from % to %). the extent of drop in egg production depended on the chicken ages. for example, the age of case was weeks, a stage of increasing egg production. however, after h n aiv infection, the egg production decreased % instead of increasing and then stayed at % for a week. the infected chickens showed signs of decreasing activity, anorexia from g per bird to g per bird, and respiratory signs. case showed infection in the second floor first and then transmitted to third and fourth floor, indicating that the virus transmitted by air or human movement. however, most cases showed air borne transmission from one flock to another in spite of enforcing restrictions of persons entering the poultry pens and changing clothes and booths. in most cases, males' mortality was higher than that of female pen mates. by comparing the sequences of ha of those h n viruses, we found that amino acid changes in ha were higher than those in ha , showing that antigenic changes on the globular head of ha molecule rather than randomly on the whole ha protein, indicating that h n viruses in taiwan had been selected in the presence of antibody pressure. the aa residues and changes that showed yearly trends were the followings: a- s, i s, v i, n s, e k, l m, e d, q k, a v, or t, s n, s r, k n, y d, n t, s i, g d, l v, i v, g e, t n, g s, a v, k e, d n, i m, and m i. however, their significance on antigenic variation was previously unknown. by hemagglutinination inhibition (hi) assays, except mab ch -d , all other monoclonal antibodies elicited from v ⁄ showed different hi titers with the different h n viruses (table ). however, those mabs showed negative hi to and , the early h n strains. this indicated that the epitopes recognized by those mabs were undergoing antigenic drift. introduction aquatic birds are recognized as the natural reservoirs of the influenza a virus as all known subtypes (h -h , n -n ) have been found in them. phylogenetic analyses of influenza viruses found in other animals revealed that all were directly or indirectly derived from viruses resident in aquatic birds. however, the prevalence, movement, and evolutionary dynamics of influenza viruses in these avian hosts have not been well defined. southern china was hypothesized to be an 'epicenter' for the generation of human pandemic influenza viruses as all major influenza pandemic viruses in the th century emerged from this region. the ecological background that facilitates the occurrence of these pandemic influenza strains has not been fully explored. in the past two decades, four lineages, belonging to h n , h n , and h n viruses, have become established and long-term endemic in different types of poultry in this region. [ ] [ ] [ ] some of these viruses were disseminated to many countries in eurasia and africa and have continued to cause sporadic human infection, posing a persistent pandemic threat to the world. in the mean time, the endemic influenza lineages have undergone extensive genetic reassortment events giving rise to many variants, dramatically increasing the genetic diversity of the influenza virus in this region. questions remain as to how and where these viruses emerged, and what were the sources of the gene segments incorporated within the novel reassortant variants of the h n , h n , and h n virus lineages. to address these questions, surveillance of influenza in migratory and domestic (sentinel) ducks has been conducted since at poyang lake, the biggest fresh-water lake and the major migratory bird aggregation site in southern china. the aim of this study is to identify the prevalence, seasonality, and movement of virus between migratory and domestic ducks. migratory ducks were captured during over-wintering, from november to march. cloacal swabs and blood samples were collected from each individual bird. all birds were released after sampling. to observe the interaction between migratory ducks and domestic birds, we also sampled domestic ducks from two duck farms (designated as sentinel ducks) surrounded by rice fields and inaccessible to other types of poultry, but accessible to migratory birds. that is, the sentinel ducks share the same water body with migratory ducks and have the chance to spread viruses to each other. for sentinel ducks, sampling was conducted fortnightly, all year-round, on the two farms from august onwards. cloacal swabs and fresh fecal droppings were taken. about birds were randomly sampled fortnightly from these farmed ducks. all swabs were soaked in vials containing ae ml transport medium with antibiotics and kept on ice-packs during sampling and immediately stored in ) °c freezers for further use. blood samples from migratory ducks were treated according to methods previously described. serological survey and virus subtyping in migratory and sentinel ducks used hemagglutination inhibition (hi) and neuraminidase inhibition (ni) tests as previously described. for isolates that were not identified by reference antisera, subtypes were determined by rt-pcr using subtype specific ha and na diagnostic primers. prevalence and seasonal patterns of influenza virus in migratory and sentinel ducks during during - a total of cloacal swabs from migratory ducks and cloacal or fecal swabs from sentinel ducks were collected at poyang lake. from these specimens, influenza isolates were obtained from migra- tory ducks and from sentinel ducks; isolation rates of ae % and ae %, respectively (table ) . it was noted in sentinel ducks that virus occurrence formed a seasonal peak from november to february, which completely overlapped the over-wintering months of migratory ducks. this suggests that virus movement or transmission between migratory and sentinel ducks occurred during this period at poyang lake. thirty positive samples (hi titer ‡ ) were identified from blood samples collected during november and december in . among these, samples were positive to h , were positive to h , were positive to h , and were positive to h . one serum sample was positive to both h and h , which suggested co-infection of influenza virus in migratory ducks might occur in natural conditions. poyang lake, which is located in the northeastern part of jiangxi province, is the largest freshwater lake in china and is part of the eastern asia-australia migration route. every year, hundreds of thousands of migratory ducks congregate at poyang lake during the migration season. recent farming practice involves raising domestic waterfowl in dense populations in the poyang lake region. farmraised domestic waterfowl are allowed to feed in and share the same water body with migratory birds, thereby facilitating direct interactions between domestic waterfowl and freeranging migratory birds. this makes poyang lake an ideal site to observe the dynamics of influenza virus interactions between migratory and sentinel ducks in southern china. in our longitudinal surveillance during [ ] [ ] [ ] [ ] [ ] [ ] , the overall virus isolation rate from migratory ducks was less than %, which suggests a low prevalence of viral infection during the birds' southern migration. similar results have been observed in taiwan, which is also an important stopover site for migratory birds along the eastern asia-australia migration route during years of surveillance. the overlap in seasonal patterns of virus infection between migratory and sentinel ducks found in our study suggests that virus movement or transmission between migratory and sentinel ducks occurred during the period of time migratory birds were at poyang lake. the ha subtypes harbored in migratory and sentinel ducks were similar in our study. for migratory ducks, h , h , h were the predominant subtypes, while h , h , and h were the major subtypes in sentinel ducks. hpai h n was only detected from migratory ducks in early on two sampling occasions. from phylogenetic analyses the h n viruses isolated from migratory ducks were closely related to the viruses endemic in domestic poultry in southern china. therefore, it appears that h n viruses endemic in domestic poultry could be transmitted to migratory ducks via close contact in southern china. only lp h viruses were detected from sentinel ducks at poyang lake during this period. whether h n virus infection was absent from sentinel ducks at poyang lake needs further investigation. serological surveys provided further evidence for the prevalence of aiv in migratory ducks at poyang lake. the serological results in did not match well with the epidemiological results during [ ] [ ] [ ] [ ] [ ] [ ] , which suggests that influenza virus infection in migratory birds could be influenced by multiple factors, such as host immune status, population size, spatial and temporal variations, and migration routes. southern china has the biggest domestic duck population in the world. our study demonstrates that dynamic interactions between migratory ducks and sentinel ducks occurred frequently throughout the surveillance period. thus, sentinel ducks could be treated as intermediate hosts between the ''real gene pool'' from migratory ducks and domestic poultry in the whole influenza virus ecosystem. a sentinel duck sampling system may be a feasible method to represent the viruses in the natural gene pool and a baseline for virus or gene interactions between migratory and domestic ducks. further investigations and surveillance are required to better understand the role of the domestic duck population in facilitating virus interactions and the generation of genetic diversity. two distinct lineages of h n influenza viruses represented by a ⁄ chicken ⁄ beijing ⁄ ⁄ (ck ⁄ bei-like) and a ⁄ quail ⁄ hong kong ⁄ g ⁄ (g -like) have become established and endemic in poultry in southern china. these established h n lineages continue evolving to generate many different reassortant variants (or genotypes) , and are causing sporadic cases of human infection. , studies of h n viruses isolated from pigs in hong kong and shandong province have also raised the possibility of reassortment with human-like viruses from pigs. , in addition, h n viruses isolated beyond the late s had preferential binding with a- , -neuacgal human-like receptors. these observations suggest that the h n influenza viruses still have pandemic potential. unlike highly pathogenic h n influenza viruses that have been rarely detected in the live-poultry markets in hong kong since , h n viruses are still frequently isolated in our surveillance program. therefore, we try to understand the continuing evolution of h n viruses through genetic characterization and phylogenetic analyses of the viruses isolated in hong kong live-poultry markets from to . a total of terrestrial poultry were sampled at different live-poultry markets in the hong kong sar between january and december . of those samples, were from chickens and the others were from minor poultry species including chukar, pheasant, guinea fowl, silky chicken, and pigeon. fecal droppings, cloacal and tracheal swabs, drinking water, and environmental samples from cages were collected into transport medium. viruses were isolated in -to -day old embryonated eggs as described previously. virus isolates from positive sampling occasions were selected for sequence analysis. rna extraction, cdna synthesis, and pcr were carried out as described previously. dna sequencing was performed using bigdye terminator v ae cycle sequencing kit on an abi dna analyzer (applied biosystems) following manufacturer's instructions. all sequences were assembled and edited with lasergene ae (dnastar, madison, wi) software. sequence alignment and residue analysis were performed with the bioedit sequence alignment editor, version ae . all eight gene segments of sequenced viruses were characterized and analyzed phylogenetically together with virus sequence data available in public databases. maximum-likelihood trees were constructed using garli ae . estimates of the phylogenies were calculated by performing neighbor-joining bootstrap replicates using paup* ae . systematic surveillance of live-poultry in hong kong from to resulted in h n isolates from samples (overall isolation rate, ae %) ( table ). there were strains isolated from chicken samples (isolation rate, ae %). of these viruses, four were isolated from tracheal swabs (isolation rate, ae %), while isolates were isolated from cloacal or fecal swabs (isolation rate, ae %). an additional isolates were collected from drinking water samples (isolation rate, ae %). there were strains of h n viruses isolated from minor poultry samples (isolation rate, ae %) ( table ) . of these viruses, only one was isolated from tracheal swabs (isolation rate, ae %), whereas strains of viruses were isolated from cloacal or fecal swabs (isolation rate, ae %). the isolation rate in drinking water in minor poultry was again higher when compared with other sampling methods with strains isolated from drinking water samples (isolation rate, %). taken together, these findings suggest that the h n viruses mainly replicated in the intestinal tract of chickens and minor poultry species. also, the high isolation rate in drinking water samples could be a sensitive indicator for monitoring the prevalence of h n viruses in the field. to better understand the evolutionary pathway of h n viruses in southern china, representative viruses, isolated from hong kong live-poultry markets from to , were sequenced and genetically characterized. phylogenetic analysis of the h ha gene revealed that ck ⁄ bei-like viruses were predominant and one chicken isolate had a g -like ha gene ( figure ). this is the first time the g like h ha gene has been detected in chickens from livepoultry markets in hong kong. the ck ⁄ bei-like lineage is further divided into two subgroups as previously described. subgroup is represented by qa ⁄ st ⁄ ⁄ and subgroup is represented by dk ⁄ hk ⁄ y ⁄ . all h n viruses in this study belonged to subgroup of the ck ⁄ bei-like lineage except for the virus with the g -like ha gene. phylogenetic analysis of the na gene also showed a similar evolutionary pattern to the ha gene with all viruses clustered within the ck ⁄ bei-like lineage. these results revealed that ck ⁄ bei-like viruses are predominant in both chickens and minor poultry. all of the pb , pa, np, ns and m genes clustered with those of h n lineage viruses previously prevailing in ter- restrial poultry in southern china. phylogenetic analysis of the pb gene revealed three different lineages; g -like (n = ), ck ⁄ sh ⁄ f ⁄ -like (n = ), and unknown avian (n = ). the sh ⁄ f ⁄ -like lineage (or f ⁄ -like) was previously reported in eastern china and was used previously for vaccine production in an intensive vaccination program. this pb gene lineage was also distinguishable from the ck ⁄ bei-like lineage and its presence in the viral genome may be due to reassortment between the vaccine strain and field isolates, followed by selective establishment in terrestrial poultry. gene constellation analyses of the viruses revealed six genotypes. thirty-four of the viruses analyzed belonged to two genotypes, b and b , which were also the prevailing reassortants found in other provinces in southern china since . the remaining sixteen viruses belonged to four novel genotypes that have not been identified before in this region. characterization of h n influenza viruses isolated from live poultry in hong kong markets from a year surveillance program revealed that ck ⁄ bei-like viruses were predominant in southern china and were continuing to evolve. two recognized and four novel genotypes were identified in this study. one characterized virus, ck ⁄ hk ⁄ nt ⁄ , had a g like ha gene (the first time this has been detected in hong kong poultry markets) that showed a close relationship with two human h n strains isolated in . g -like viruses were usually detected and caused outbreaks in chickens of middle eastern and european countries, [ ] [ ] [ ] and minor poultry, mainly quail, in southern china. whether the g -like virus was transmitted from china to middle eastern and european countries, as the highly pathogenic h n virus did in the last five years, or vice versa, is still unknown. since the ck ⁄ hk ⁄ nt ⁄ strain clustered with other g -like strains isolated previously in minor poultry in southern china, the g -like viruses in chicken may be due to interspecies transmission from minor poultry species. genetic studies demonstrated that reassortants with genotypes b and b persistently occurred in either chickens or other minor poultry species from to . other genotypes that were prevalent in southern china might be being gradually replaced and four novel genotypes were identified in this study. these novel genotypes were generated through reassortment of viruses with different lineages. a newly emerged f ⁄ -like lineage originating from eastern china is responsible for generation of some of the novel genotypes found in this study. the ck ⁄ bei-like lineage is gradually being replaced by f ⁄ -like lineages which are becoming dominant in northern and eastern china. , animal experiments have also demonstrated that f ⁄ -like viruses are more effective in replication and transmission in chickens compared with ck ⁄ bei-like viruses. since the f ⁄ -like lineage of the pb gene has been introduced into southern china, this newly emerged lineage may have a higher tendency to replace the rnp genes in the circulating ck ⁄ bei-like viruses and subsequently become the endemic virus in terrestrial poultry. in vietnam, the modelling of the pandemic h n progression estimates that ( - ) pigs might be exposed to the virus on the basis of cases among swine owners ( - ). a poor level of biosecurity, high animal densities, and a mix of species could increase the risk of influenza virus flow, persistence, and emergence on swine and poultry farms. this study was set up in the red river delta, where a third of the national pig husbandry is produced. the aims are to give preliminary information of the epidemiological state of swine influenza and in order to further assess the risk of infection of swiv, through cross-species transmissions from poultry to pigs. this paper will present the preliminary results on swiv and the risk factors of pig seropositivity in vietnam. a cross-sectional study was conducted in two provinces of the red river delta in april . pig farms were randomly selected from nine communes representative of at risk area of avian h n . in each farm, pig and poultry were sampled and collected to virological and serological analyses. interviews were conducted in all farms by trained interviewees. questionnaires included closed and open questions on ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - livestock husbandry ⁄ management and household characteristics, such as herd size and structure, health history and vaccination, pig housing, watering and feeding system, reproduction, purchasing of animals, biosecurity measures, pig contact with poultry, and environmental factors. the virological detection assay was performed on pools of nasal swab specimens from pigs. we investigated whether real-time rt-pcr assay could detect gene m on pools of nasal swab specimens before attempting virus isolation from individual nasal swab specimens. the poultry and pig sera were tested against influenza type a with an enzyme-like immunosorbant assay (elisa) competition test idvetª. this commercial kit is designed to specifically detect antibodies directed against the np protein antigen of influenza type a viruses. the positive serum samples were examined in hemagglutination inhibition (hi) to determine antibody titers and subtypes. the hi test was tailored for h , h , and h subtypes in pigs and h and h subtypes in poultry. seroneutralization tests by pseudo particles were used to test the presence of antibodies directed against h subtype. we analysed the data for relationships between influenza a serological status (the outcome variable) and possible risk factors using r version ae ae (r development core team). the statistical unit was the individual. initially, the quantitative variables were encoded into categorical variables according to the quartiles or median. descriptive statistics (e.g., means or medians, proportions, standard deviations) were calculated for all herd-level and commune level predictors to assist in the subsequent modeling process. we also performed the independence test among all variables to determine if variables were dependant. then, univariate analysis of potential risk factors for the pigs being positive for swiv and estimation of odds ratios were performed using generalised linear mixed models with binary outcome and logit link function for each herd-level and commune-level variable to determine which variables were individually associated with influenza a seropositivity at a significance level of p < ae . herd and commune of residence were included as a random effect to account for the correlation of observations at the herd level. the third stage of the analyses included the four herdlevel variables found to be significantly (p < ae ) associated with influenza a seropositivity. an automatic process using all possible associations between the selected variables was computed into a mixed logistic regression models, with random effects. when two variables were collinear, as determined before, only one variable was likely to enter the multivariable model, and therefore, the selection of which collinear variable to enter the model was guided by biological plausibility and statistical significance. all of the pools of nasal swabs were rt-pcr negative. the maximal possible prevalence considering perfect diagnostic tests would be of ae % at a confidence level of %, in an infinite population within these regions (win-episcope ae ). six hundred-and-nine pig sera were tested in nonvaccinating farms. the herd seroprevalence of swine influenza in the commune previously infected by the avian h n in the red river delta raised by ae % [ ae ; ae ] in april . but among seropositive farms, only four had at least two seropositive pigs. the within-herd seroprevalence is very low, and no seropositivity was detected in the majority of farms. estimates had large confidence intervals due to small sample sizes. the individual seroprevalence raised ae % [ ae ; ae ]. the subtyping of seropositive sera is still in process. descriptive statistical analyses on five major risk factors of swiv: farm size, breeding vs. fattening, purchasing, percentage of family income, and poultry production, were conducted. based on this analysis, three types of farming systems were identified and included in mixed models ( table ) . percentage of family income by pig production and poultry production were not differentiating factors for this typology. whereas types and seem to be specialized in fattening, the type produces and might sell piglets on the farm site. the exploration of the different variance components indicated that the random effect variances were mainly associated with the herd, while the commune did not seem to have any effect. therefore we included in all models only the herd as a random effect. the random effect term for herd was modelled, assuming a normal distribution with a table . typology of farming system type : large fattening farms largest scale production, with more than pigs per year specialized in fattening, and purchase more than pigs per year type : small fattening farms small scale of production, with less than pigs per year specialized in fattening, and purchase less than pigs per year type : medium breeding-fattening farms medium scale of production, with less than pigs per year breeding and fattening piglets, with rare purchase common variance [$n( ,r herd)]. the univariate analyses were conducted on variables and typology variables, with herd as random effect. some coefficient or confidence intervals were inconsistent because of small effectives, especially for the percentage of self-product culture or the pig freegrazing because of the lack of positive results in the dataset. the only one significant (p value < ae ) parameter was the percentage of pig sales in the familial annual income. surprisingly, common risk factors of swine influenza infection, such as farm size, animal movements, and sanitary parameters got low odds ratio individually (without being significant); the typology provides the hypothesis of complex interactions effects that increase the risk of infection. as shown in table , the farming system type got a higher seroprevalence of ae % [ ae - ae ] and a higher risk indicator, with or = ae (p-value = ae ) in comparison with type . this finding was not significant. in the multivariate mixed model, the percentage of familial income provided by pig production was the only one significant variable, with or = ae [ ae - ae ]. the focus on diseased animals in the winter-time is usually required in order to increase the likelihood to isolate the virus, although the isolation rate on healthy or clinical samples never exceed %. the season and the lack of disease reports might explain the difficulties to detect influenza viruses. additionally, the pooling method tends to decrease the isolation rate because of a dilution effect, potential presence of pcr assay inhibitors, or uneven distribution of virus in the sample. our seroprevalence results must be confirmed and the subtypes identified, especially because we found only one positive animal in a few farms that could be attributed to false positive results of the elisa test (performances are not known). these preliminary results are in favor of a virus circulation at low level in the spring, but must be completed by further surveys in the winter and before the new year (têt celebration) when pig production, trade, and movement increase at their maximum. no clear prior information on the expected prevalence of swine influenza in vietnam, tests sensitivity, and speci-ficity could be obtained from literature or reliable sources. bayesian methods will be carried out in the future in order to compute prevalence and ⁄ or to estimate the probabilities of freedom. the risk factors analysis was limited by the lack of positive results. further studies are necessary to identify the at-risk season and type of farming systems at risk of swine influenza infection. however, this investigation of risk factors leads to the hypothesis that medium size breeding-fattening farms had a higher risk than large or small size fattening farms. further investigation are needed to precise this typology. the risk of swiv infection increases with a combination of three major factors. poultry production does not seem to play any role on swine infection. the generalized linear mixed model afforded to take into account all the non investigated parameters at the herd level. although we investigated the most common risk factors of swine influenza infection covering different kind of fields, the herd random effect might explain risk variations. mixed models have become a frequently used tool in epidemiology. due to software limitations, random effects are often assumed to be normally distributed. since random effects are not observed, the accuracy of this assumption is difficult to check. further studies, such as case-control or cohort studies could help to identify more precisely risk factors of swine influenza seropositivity, as these study designs are more adapted than cross-sectional studies. the concept that swine are a mixing-vessel for the reassortment of influenza viruses and for the emergence of pandemic influenza viruses has been re-enforced by the emergence of the recent pandemic. the pandemic h n virus of (h n pdm) is believed to have emerged through the reassortment of north american triple reassortant and eurasian avian-like swine influenza viruses. since the immediate precursor of this pandemic virus has not yet been identified, it is not possible to be definite whether the reassortment leading to the pandemic occurred in swine, but swine influenza viruses are the nearest known ancestors of each gene segment of h n pdm. , the mechanisms of pandemic emergence are not clear. it is believed that the pandemics of and arose through reassortment of the pre-existing human seasonal influenza virus with avian influenza viruses, and swine have been proposed to be a possible intermediate host where such reassortment between human and avian viruses may take place. the pandemic was the first to arise for over years and the first to occur after the understanding that pandemics arise from animal influenza viruses. systematic studies of influenza virus ecology and evolution in swine are, therefore, important in order to understand the dynamics of pandemic emergence. furthermore, since swine are the likely host within which h n pdm virus originated, it was predicted that this virus would readily infect swine and may reassort with endemic swine influenza viruses. these predictions have now been confirmed with reports of h n pdm being detected in pigs in many countries and reassortment with endemic swine influenza virus being confirmed. while h n pdm has been genetically and antigenically stable in humans, reassortment between h n pdm, which is well adapted to transmission in humans, and other avian or swine viruses may lead to the origin of novel viruses posing a threat to public health. in addition to endemic swine virus lineages, avian influenza viruses such as h n and highly pathogenic avian influenza (hpai) h n have also been occasionally identified in pigs in parts of asia. , it has been shown that h n pdm readily reassorts with h n to generate viable progeny in vitro. it is therefore essential to monitor the ecology, evolution, and biological characteristics of swine influenza viruses so that their continued evolution and zoonotic and pandemic potential can be monitored. there is however, a paucity of surveillance data on swine influenza viruses worldwide. this is in part related to the negative commercial consequences that may arise from detection of influenza in a swine herd leading to a major economic loss to the producer. here we outline a surveillance system that has been in place in hong kong for the last decade, based on sampling animals arriving at an abattoir in hong kong. we demonstrate the feasibility of such surveillance in an abattoir setting and compare methods used for detection influenza viruses in swine. virus isolation was carried out by inoculation into mdck cells and by allantoic inoculation in embryonated eggs as previously described. virus isolates were subtyped by haemagglutination inhibition tests using specific antisera and genetically characterized by sequencing and phylogenetic analysis of the haemagglutin gene. , virus detection by rt-pcr a subset of recent specimens was tested in parallel by real time pcr using the biorobot universal system (qiagen) that enables fully-automated viral nucleic acid extraction and downstream reaction setup in a -well plate format. total viral nucleic acids were extracted in a -well plate format with the qiaamp virus biorobot mdx kit (qiagen) on the biorobot universal system (qiagen) according to the manufacturer's instructions. briefly, ll of sample was lysed in ll buffer al, supplemented with ae lg carrier rna in a s block (qiagen), which placed the samples into a well plate format. after protease digestion, samples were transferred to silica based membrane in well plate format for binding. following two washing steps, rna was eluted in ll of elution buffer (buffer ave) into a well elution microplate cl (qiagen) . for the synthesis of cdna, ll of purified rna was used in a ll reaction containing ll of · buffer, ae nm of each deoxynucleotide triphosphate (dntp), mm dithiothreitol, lg random primer, u of rnaseout recombinant ribonuclease inhibitor, and u of superscript iii reverse transcriptase (all from invitrogen). reactions were performed in the geneamp thermocycler (applied biosystems) with the following parameters: minutes at °c, minutes at °c, and soak at °c. subsequent to the reactions, ll of cdna was diluted ⁄ by adding ll of ae buffer (qiagen) . real-time pcr was performed using the power sybrÒ green pcr master mix (applied biosystems) according to the manufacturer's instructions. briefly, ll of ⁄ diluted cdna was amplified in a ll reaction containing ae ll of · power sybr green pcr master mix, nm of forward primer m c ( ¢-ctt cta acc gag gtc gaa acg- ¢) and nm of reverse primer m r ( ¢-agg gca ttt tgg aca aag ⁄ t cgt cta- ¢). the primers have been designed to amplify the sequences in the conserved region of influenza a virus matrix gene, thereby detecting viruses from different species including swine influenza viruses. real-time pcr was performed in the abi fast system (applied biosystems) with the following cycling conditions: minutes at °c once, seconds at °c, and minutes at °c for cycles, followed by melting curve analysis with seconds at °c, minutes at °c, and seconds at °c. in each assay, serially diluted plasmids containing the full length m gene cloned from a ⁄ vietnam ⁄ ⁄ (h n ) were included as standards to perform absolute quantification. a manual baseline was set from cycles - and a manual cycle threshold (ct) was set at ae . samples that were positive or unequivocal results from the real-time pcr were confirmed by performing gel electrophoresis on the pcr products. positive visual identification was made in the presence of the target pcr product at bp in length. a total of tracheal and nasal swabs were processed during the years january -april and yielded influenza virus isolates, an overall virus isolation rate of ae %. of these, were subtype h (classical swine, eurasian avian-like swine, and triple-reassortant), were human-like h viruses, and were eurasian avianlike swine h n viruses. culture in mdck cells yielded % of h subtype viruses, % of the human seasonal-like h n viruses, and ae % of the avian-like eurasian swine h n viruses. culture in embryonated eggs yielded ae % of the h subtype viruses, % of the human seasonal-like h n viruses, and ae % of eurasian avian-like swine h n viruses ( figure ). tracheal and nasal swabs each gave comparable overall virus isolation rates ( ae %). however, isolation rates for human-like h n viruses were ae fold higher in nasal swabs ( ae % versus ae % respectively; p = ae ) ( figure ) . a parallel evaluation of rt-pcr and culture was carried out in specimens. rt-pcr detected ⁄ ( %) of the culture positive specimens. rt-pcr was also positive in ⁄ ( ae %) culture negative specimens, but all these specimens had very low virus load in the rt pcr tests. virus could not be cultured from these culture negative specimens even by attempts at virus re-isolation from the frozen specimen. surveillance in an abattoir setting provides an acceptable yield of influenza viruses and is a feasible method of swine influenza surveillance. sampling in a large abattoir setting allows surveillance to be carried out anonymously with no negative consequences to the supplier. the supply-chain of pigs to the hong kong abattoir involves pigs being trucked in over long distances and may provide opportunity for virus amplification during transport. thus, virus isolation rates may be lower in more vertically integrated and homogenous production and slaughter systems where less mixing of pigs occurs. our results indicate that mdck cell culture is essential for optimizing virus isolation during swine influenza surveillance. allantoic inoculation of embryonated eggs by itself is sub-optimal for isolation of swine influenza viruses. it is however possible that inoculation of embryonated eggs by the amniotic route may lead to better isolation rates than allantoic inoculation. rt-pcr detection is an alternative method for virus detection. but the additional specimens detected by rt-pcr did not yield culturable virus, even following attempts at re-isolation and sequential passage. the rt-pcr positive ⁄ virus isolation negative specimens had very low virus load, and this may be the explanation for the inability to isolate such viruses. in addition, rt-pcr did not detect all viruses isolated by culture. tracheal and nasal swabs gave comparable isolation rates with the exception of human-like h n viruses which were more frequently isolated from nasal swabs. this may suggest that, in contrast to endemic swine influenza virus lineages, these human-like h n viruses are less adapted to replication in the lower respiratory tract. in summary, collection of nasal or tracheal swabs in an abattoir setting together with virus isolation in mdck cells provides a feasible approach to surveillance of swine influenza viruses. kong, kong, - introduction wild waterfowl are the natural reservoir of influenza a viruses (aiv), and they play an important role in the genesis of pandemic influenza. it is suggested that the pandemic virus was purely derived from avian virus, which adapted to humans and caused efficient human-to-human transmission, while the pandemics of and had acquired the viral haemagglutinin, pb polymerase, and in , the neuraminidase gene segments from the avian gene pool. the major regional outbreaks of highly pathogenic avian influenza (hpai) h n in asia, europe, and africa highlight the potential role played by migratory waterfowl in disseminating highly pathogenic influenza viruses. therefore defining the influenza virus gene-pool in wild birds is of vital importance. surveillance was carried out - times weekly from to during the winter months of october to april in the hong kong mai po nature reserve and lok ma chau, hong kong. the hong kong mai po nature reserve and lok ma chau are along the east asia-australian flyway where a peak of more than ducks and grebes congregate every winter. fecal droppings were collected and transported in vials containing ae ml of vtm, which was prepared from m ( ae g ⁄ l), penicillin g ( · u ⁄ l), polymyxin b ( · u ⁄ l), gentamicin ( mg ⁄ l), nystatin ( ae · u ⁄ l), ofloxacin hcl ( mg ⁄ l), and sulfamethoxazole ( g ⁄ l). an aliquot of ll from each swab sample was inoculated into the allantoic cavity of a -to -day-old chicken embryonated egg, and incubated for days at °c. positive ha isolates were subtyped using standard antisera , and rt-pcr was performed with the used of one-step rt-pcr assay (invitrogen) described earlier, followed by sequencing on abi prism xl dna analyzer. the determination of species of origin was performed by dna barcoding of the mitochondrial cyto-chrome oxidase i gene from dna extracted from the fecal droppings. during the -year surveillance period, a total of influenza viruses were isolated from samples collected, an overall isolation rate of ae %. a total of isolates were obtained from specimens collected during the winter period coinciding with the southern migration of waterfowl along the east asian flyway and one isolate obtained from samples collected in spring during the period when northern migration of waterfowl took place along the east asian-australasian flyway. the isolation in hong kong was slightly lower than a similar study conducted in south korea in which the isolation rate of migratory birds was ae % in - . this suggested a slightly lower prevalence of influenza virus present in hong kong as the birds migrated southwards. the viruses isolated in hong kong, representing hemagglutinin (ha) subtypes of h -h and neuramidinase (na) subtypes of n -n , were all from wild waterfowl ( table ) . out of the twelve ha subtypes isolated, h and h were the two subtypes that were isolated frequently every year for h and in six out of seven years for h , respectively. h and h viruses accounted for ae % and ae % of all virus isolated, respectively. on the other hand, h , h , and h were the least prevalence ( ae %) and were only isolated once in years. of the na subtypes, n and n were isolated most often ( ae % and ae % of all isolates, respectively) and n was the least ( ae %). november was the month that had the highest prevalence of influenza virus ( ae % of samples being positive) compared to only ae % in march. the subtype's variation was the most diverse in december during our years of surveillance. this suggested that more of these wild migratory birds may be carrying influenza virus when they arrive in hong kong. however the continued isolation of viruses suggests continued circulation of these viruses in the vicinity of mai po. the study of dna barcoding for the mitochondrial cytochrome oxidase i gene retrieved from fecal droppings revealed that the isolates originated mainly ( ae %) from birds of the order anseriform, family anatidae including eurasian wigeon, northern shoveler, northern pintail, common teal, and garganey. non-anseriformes which were found to have shed aiv viruses were cormorant, grey heron, and stint. none of the water samples collected from the ponds where these birds congregate were found to be positive for the virus. phylogenetic analyses of the ha gene of the lpai h viruses isolated in this study clustered with that of the other lpai h viruses isolated from hokkaido, mongolia, and siberia and were not closely related to the hpai h n . satellite tracking of eurasian wigeons and northern pintails in dec and revealed their flyway from hong kong to as far north as eastern russia, eastern mongolia, and northern china. no hpai h n viruses were isolated in this study from apparently healthy birds. however, as part of the surveillance of dead wild birds carried out by the department of agricultural, fisheries and conservation of the government of hong kong during this same period, over dead wild birds were tested positive for hpai h n and has been reported elsewhere. our influenza surveillance in hong kong has revealed a diversity of influenza virus subtypes the migratory waterfowl infected within the region. the result of the phylogenetic analysis correlated with the findings from satellite tracking that viruses isolated in hong kong were closely related to those isolated in areas along the migratory route. no healthy bird was isolated with hpai h n, although dead wild birds have been regularly found to have hpai h n virus, suggesting that infected birds might not live for a long period. introduction a novel swine-origin h n influenza virus emerged in mexico in april and rapidly spread worldwide, causing the first influenza pandemic of the st century. most confirmed human cases of h n ⁄ influenza have been uncomplicated and mild, but the increasing number of cases and affected persons worldwide warrant optimal prevention and treatment measures. today, almost all of the pandemic h n ⁄ viruses tested are resistant to m blockers. therefore, only the neuraminidase (na) inhibitors are currently recommended for treatment of this pandemic influenza. for the control of influenza infection, the clinical use of oseltamivir has increased substantially during the pandemic. to date, the majority of tested clinical isolates have remained susceptible to na inhibitors, oseltamivir and zanamivir, but oseltamivir-resistant variants with h y na mutation (n numbering) have been isolated from individuals taking prophylaxis, from immunocompromised patients, and from a few community clusters. , in view of the high prevalence of oseltamivirresistant seasonal h n influenza viruses in - , the isolation of resistant h n ⁄ viruses without known oseltamivir exposure raised great concern about the transmissibility and fitness of these resistant viruses. here we studied the transmissibility of a closely matched pair of pandemic h n ⁄ clinical isolates, one oseltamivir-sensitive and one resistant, in both direct contact and respiratory droplets routes among ferrets. viral fitness was evaluated by co-infecting a ferret with both the oseltamivir-sensitive and -resistant viruses. the viruses were also characterized by full genome sequencing, susceptibility to na inhibitors, and growth in mdck and mdck-siat cells. oseltamivir-resistant influenza a ⁄ denmark ⁄ ⁄ (h n ) virus (a ⁄ dm ⁄ ⁄ ) was isolated from the throat swab of a patient who had influenza-like symptoms and received post-exposure oseltamivir prophylaxis ( mg once daily). wild-type influenza a ⁄ denmark ⁄ ⁄ (h n ) virus (a ⁄ dm ⁄ ⁄ ) was isolated from a patient in the same cluster of infection as the a ⁄ dm ⁄ ⁄ virus. to assess growth kinetics of viruses, confluent mdck or mdck siat cell monolayers were infected with viruses at a multiplicity of infection (moi) of approximately ae pfu ⁄ cell (single-step) or ae pfu ⁄ cell (multi-step). supernatants were collected every h or h p.i. for time points. a modified fluorometric assay using the fluorogenic substrate ¢-( -methylumbelliferyl)a-d-n-acetylneuraminic acid (munana) was used to determine viral na activity. the drug concentration required to inhibit % of the na enzymatic activity (ic ) was determined by plotting the percent inhibition of na activity as a function of compound concentration calculated in the graphpad prism (la jolla, ca) software from the inhibitor-response curve. na enzyme kinetics were determined by measuring na activity every seconds for minutes under the same conditions as above, when all viruses were standardized to an equivalent dose of ae pfu ⁄ ml. the k m and v max were calculated by fitting the data to the appropriate michaelis-menten equations using nonlinear regression in the graphpad prism software. young adult ferrets ( - months of age) were obtained from the ferret breeding program at st. jude children's research hospital. all ferrets were seronegative for influenza a h n and h n viruses and for influenza b viruses. for transmission studies, the donor ferrets were lightly anesthetized with isoflurane and inoculated intranasally with tcid virus in ae ml sterile pbs . after the donor ferrets were confirmed to shed virus on day p.i., each donor was then housed in the same cage with two naïve direct-contact ferrets. two additional recipient ferrets were placed in an adjacent cage isolated from the donor's cage by a two layers of wire mesh (approximately cm apart) that prevented physical contact but allowed the passage of respiratory droplets. ferret weight and temperature were recorded daily for days. nasal washes were collected from donors and recipients on day , , , , , , , and p.i. by flushing both nostrils with ae ml pbs, and tcid titers were determined in mdck cells. serum samples were collected weeks after virus inoculation, and were tested for seroconvention by hi assay. full genome sequencing revealed that the pair of h n ⁄ viruses differed only at na amino acid position , where the pandemic a ⁄ dm ⁄ ⁄ virus had an h y amino acid mutation caused by a single t-to-c nucleotide substitution at codon . the wild-type a ⁄ dm ⁄ ⁄ was susceptible to oseltamivir carboxylate (mean ic : ae nm), but the a ⁄ dm ⁄ ⁄ carrying the h y na mutation had ic values approximately - times of the wild-type viruses (mean ic : nm). the ic of zanamivir was comparable for both viruses and were uniformly low (mean ic £ ae nm). the h y na mutation confers resistance to oseltamivir carboxylate but did not alter susceptibility to zanamivir. to understand the impact of the h y mutation on the na enzymatic properties, na enzyme kinetics was determined. the na of the oseltamivir-resistant virus had a slightly higher k m (mean = lm) and lower v max (mean = u ⁄ sec) than na of the sensitive virus (k m , mean = lm; vmax, mean = u ⁄ sec). the results suggested that the h y na mutation reduced na affinity for substrate and na catalytic activity, although the function of na was not severely impaired. to further evaluate the impact of the h y na mutation on virus growth in vitro, single-and multi-cycle growth studies of both viruses were performed in mdck and mdck-siat cells. in the both single-and multiple-cycle growth curves, the two viruses reached comparable levels eventually, but the initial growth of the resistant virus was significantly delayed by at least - logs in comparison to that of wild-type virus (p < ae ). the donor ferrets inoculated with wild-type a ⁄ dm ⁄ ⁄ or oseltamivir-resistant virus shed virus productively until day or day p.i., with a peak virus titer comparable to that of a ⁄ dm ⁄ ⁄ virus (table ). in a ⁄ dm ⁄ ⁄ virus group, two of direct-contact ferrets the weight loss in ferrets is the maximum percentage loss compared with the initial weight. virus shedding is indicated as number of virus-shedding animals ⁄ total number; mean peak virus titer (log tcid ⁄ ml) in nasal wash samples is indicated in parentheses. serum hemagglutination inhibition (hi) titer to homologous virus in ferret serum was determined on day p.i. duan et al. and of respiratory droplet-contact ferrets were infected through virus transmission, as indicated by the virus titers and inflammatory cell counts in their nasal washes and also by sero-conversion. under identical conditions, in a ⁄ dm ⁄ ⁄ group, only of direct-contact ferrets were infected through virus transmission, but neither respiratory droplet-contact ferrets was infected, as confirmed by the absence of sero-conversion (table ) . virus shedding in the direct-contact ferrets was lower and peaked after a longer interval in this group than in the oseltamivir-sensitive a ⁄ dm ⁄ ⁄ group (table ) , but the resistant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. these results showed that an oseltamivir-resistant h y mutant of pandemic h n virus, a ⁄ dm ⁄ ⁄ virus could be only transmitted efficiently by direct contact. to compare the relative fitness, growth capability, and transmissibility of the sensitive and resistant h n ⁄ viruses within host, a donor ferret was co-inoculated with a : ratio of the sensitive and resistant viruses, and another two naive ferrets were housed with the donor to test direct contact. during co-infection, the pattern of virus shedding and the clinical signs were similar to those in ferrets inoculated with either a ⁄ dm ⁄ ⁄ or a ⁄ dm ⁄ ⁄ virus (table ). in the inoculated donor ferret, the virus population in the nasal washes remained mixed but wild-type viruses outgrew the resistant virus progressively ( figure ). two of direct-contact ferrets were infected through virus transmission, but only wild-type virus was detected in both direct-contact ferrets ( figure ). in summary, oseltamivir-sensitive a ⁄ dm ⁄ ⁄ virus possessed better growth capability in the upper respiratory tract than did resistant a ⁄ dm ⁄ ⁄ virus, and thus had an advantage in directcontact transmission. our study determined the comparative transmissibility of two naturally circulating oseltamivir-sensitive and -resistant pandemic h n ⁄ viruses; we demonstrated inefficient respiratory-droplet transmission of an oseltamivir-resistant h y mutant of pandemic h n virus among ferrets, although it retained efficient direct-contact transmission. we suggest that the lower fitness of resistant virus within the host along with its reduced na function and delayed growth in vitro may in part explain its less efficient transmission. notably, the h y mutant of h n ⁄ used in this study was the first oseltamivir-resistant h n ⁄ isolate from a patient on oseltamivir prophylaxis to be characterized for transmissibility. our observation in the animal model is consistent with the epidemiological data collected from humans, which showed no evidence of predominant or continued circulation of oseltamivir-resistant viruses. as this study was undertaken, additional h y mutants of h n ⁄ viruses have emerged in the absence of oseltamivir use. , the emergence of these viruses should raise concerns as to whether resistant h n ⁄ viruses will acquire greater fitness and spread worldwide as the naturally resistant h n viruses did during the - season. two independent studies have evaluated the pathogenecity and transmission of other oseltamivir-resistant pandemic h n ⁄ clinical isolates in the animal models. , one of the studies, which also used an oseltamivir-resistant virus isolated from a patient under oseltamivir prophylaxis, observed similar results as ours: although the respiratory-droplet route of transmission was not investigated, it was shown that the resistant isolate was transmitted though direct-contact route and was as virulent as wild-type virus in ferrets. in another study, two oseltamivir-resistant isolates were transmitted through the respiratory-droplet route in ferrets, and the dynamics of transmission were different between the two isolates. apparently, these two oseltamivir-resistant isolates were still unequal in their transmissibility and were disparate from the resistant isolate in our study. the isolation history of the two resistant isolates was unclear in this study, and this would be an important factor to understand the fitness of drug-resistant viruses. further studies with more clinical isolates of diverse isolation background are warranted to identify how these novel h y mutants of pandemic h n ⁄ virus have changed to retain their full transmissibility. taken together, all these related studies underline the necessity of continuous monitoring of drug resistance and characterization of potential evolving viral proteins. this study was supported by contract hhsn c from the national institute of allergy and infectious diseases, national institutes of pigs have been considered as hypothetical ''mixing vessels'' facilitating the genesis of pandemic influenza viruses. , the pandemic h n ⁄ virus (ph n ⁄ ) contained a very unique genetic combination and was thought to be of swine origin, as each of its eight gene segments had been found to be circulating in pig populations for more than a decade. however, such a gene constellation had not been found previously in pig herds all around the world. only after its initial emergence in humans has this virus been repeatedly detected in pigs, and found to further reassort with other swine influenza virus. [ ] [ ] [ ] a primary question remaining to be answered is whether the ph n ⁄ -like and their genetically related viruses could become established in pig populations, thereby posing novel threats to public health. despite the fact that ph n ⁄ first appeared in mexico and the united states, and six of its eight gene segments were derived from the established north american triple reassortant swine influenza virus (trig), its neuraminidase (na) and matrix protein (m) genes belonged to the eurasian avian-like swine lineage (ea), which had never been detected in north america previously. , likewise, the trig-like viruses were never reported in europe. in contrast, both lineages of virus were frequently detected in asia, and reassortants between them have also been documented in recent years. , this has given rise to a complicated ecological situation, i.e. the simultaneous prevalence of multiple genotypes of h n and h n viruses in pigs. , among them, two representative reassortants showed the most similar genotypic characterization to the ph n ⁄ virus, the sw ⁄ hk ⁄ ⁄ (h n ) and sw ⁄ hk ⁄ ⁄ (h n ), which respectively harbor seven and six gene segments closely related to the pandemic strains. , to understand their in vivo characteristics and zoonotic potential, these two viruses, together with a human prototype strain and a swine ph n ⁄ -like isolate, were chosen for a study of their pathogenicity and transmissibility in domestic pigs, ferrets, and mice. the prototype ph n ⁄ virus, a ⁄ california ⁄ ⁄ (ca ), was provided by the world health organization collaborating centers for reference and research on influenza (atlanta, ga, usa). three ph n ⁄ -related swine influenza viruses were isolated through our surveillance program in south china as previously described. , the a ⁄ swine ⁄ guangdong ⁄ ⁄ (h n , gd ) virus was a ph n ⁄ -like swine isolate. a ⁄ swine ⁄ hong kong ⁄ ⁄ (h n , hk ), the closest pandemic ancestor known to date, possesses an m gene derived from the ea lineage, with the other gene segments from trig viruses. a ⁄ swine ⁄ hong kong ⁄ ⁄ (h n , hk ), a recent pandemic reassortant progeny, had a ph n ⁄ like na gene (also belonging to the ea lineage), an ea-like hemagglutinin (ha) gene, and six trig-like internal genes. all viruses were propagated in madin-darby canine kidney (mdck) cells for three passages, and their titers were determined by plaque assays. all experiments with live viruses were conducted in biosafety level (bsl- ) containment laboratories. pigs ( - week old, n = - ) and ferrets ( month old, male, n = ) were intranasally infected with pfu of each virus, and mice ( ) ( ) week old, female balb ⁄ c, n = ) with a dose of pfu. naïve uninfected pigs (n = ) were co-housed in the same cage with the inoculated ones from each group. body weights and clinical signs were recorded daily. virus replication was determined by titration of the virus in nasal and rectal swabs (pigs), nasal washes (ferrets), as well as from lungs and other organs (pigs and mice). seroconversion was tested by hemagglutination inhibition (hi) assays. histopathological and immunohistochemical analysis were performed as previously described. statistical analysis was performed by mean analysis with pasw statistics (spss inc., chicago, il, usa). the probability of a significant difference was computed using anova (analysis of variance). results were considered significant at p < ae . the pathogenicity of the four viruses tested differed significantly in inoculated mice. animals infected with pfu of hk experienced the most severe body weight loss ( ae ± ae %) but started to recover after days post-infection (dpi). hk caused similar peak body weight loss ( ae ± ae % on dpi) in mice as did ca ( ae ± ae %, on dpi), but the onset of clinical signs and weight loss (on dpi) was day later than those caused by the other three viruses. the gd -infected group suffered the least body weight loss ( ae ± ae %, dpi) and was the earliest to recover. although all four viruses were detected in the lungs with comparable virus titers on dpi (p > ae ), mice inoculated with gd consistently showed the lowest lung index (lung weight ⁄ body weight, %) on , , and dpi (p < ae ), suggesting the slightest injury and consolidation of the lungs. in concordance with the body weight change, the lung index from the hk group was higher than that from any other groups on and dpi, indicating the marked virulence of hk in mice. notably, virus titer of hk in the nasal turbinate was lower than the other groups both on and dpi (p < ae ), but virus replication in the lower respiratory tract was either higher (in the trachea) or similar (in the lungs). observations of the body weight changes caused by infection of ph n ⁄ or its genetically related swine viruses in ferrets have come to a similar conclusion as that for the mouse experiment. after nasal inoculation with pfu of each virus, all groups of ferrets experienced transient body weight loss for - days, except for those infected with gd , which showed no significant weight loss (p > ae ). although ferrets from the ca -infected group reached their peak weight loss ( ae ± ae %, dpi) one day earlier than those from the hk and hk groups, they began to regain body weight quickly thereafter. hk -infected ferrets also recovered rapidly and their body weights reached the same level as those of the gd -infected group at dpi. comparatively, ferrets inoculated with hk had the most retarded body weight recovery, which did not get back to the baseline level until dpi. hk was only detectable in the nasal wash on dpi, whereas the duration of virus shedding for gd , hk , and ca was - days. by combining the data obtained from the virus titration in the mouse turbinate and ferret nasal washes, a possible conclusion can be made that hk may have lower transmissibility than the other three viruses. after inoculation or exposure by direct contact (physical contact) with the ph n ⁄ virus and its close relatives, most pigs experience no or mild symptoms, such as slight loss of appetite and inactivity. body weight loss was only recorded in pigs inoculated with hk during the second week post-inoculation, but not in their contact pigs or in the other groups. diarrhea was observed intermittently in each of the inoculated or contact groups throughout the experiment, and viruses could be recovered in the rectal swabs, saliva, drinking water, and environmental swabs (inner cage walls accessible to the pigs) at various time points. however, virus titers in the positive rectal swabs were just slightly above the detection limit, while those from the environment sometimes could be higher. whether these viruses can replicate in the digestive tract or were just carried-over by contaminated foods and water requires further investigation. although virus could be detected in the nasal swabs of all infected or contact animals, the lowest peak titer was from pigs inoculated or in contact with hk ( ae - ae log tcid ⁄ ml lower than the other groups), suggesting unfavorable replication in the nasal cavity for this virus. postmortem examination on and dpi revealed that pigs infected with hk had the most extensive gross lesions in the lungs, and histochemical staining of viral nucleoprotein (np) in lung tissues on dpi also suggested the best replication for hk in the lower respiratory tract. on days post-contact (dpc), all pigs exposed to the inoculated animals developed sero-conversions (hi = - ) except for one from the gd contact group. however, on dpc, its hi titer reached , indicating slower seroconversion. this study revealed that both the pandemic h n and its genetically related swine viruses could readily infect mice, ferrets, and pigs causing mild to moderate clinical symptoms. they could also transmit efficiently between pigs. when compared with the pandemic stains and its reassortant progeny (hk ), the hk (h n ) virus containing the ea-like m gene in the genetic context of the trig virus showed consistently higher virulence in all three mammalian models tested, but it is still unknown what might happen if such a virus further reassorts to obtain the pandemic-like or ea-like na gene. however, our findings suggest that pigs could likely maintain the prevalence of different genotypes of pandemic-related influenza viruses, and highlight the zoonotic potential of multiple strains of swine influenza virus. pandemic influenza viruses emerge from the animal reservoirs. among the three pandemics that occurred in the last century, we learned that the h n and the h n pandemic viruses emerged by reassortment between circulating human virus and avian-origin influenza virus(es). studies on the emergence of the catastrophic spanish h n virus suggest that the virus may have obtained all of its eight gene segments from the avian reservoir, , or alternatively is a reassortant between mammalian and a previously circulating human influenza virus. over years since the last pandemic, the first pandemic in the st century arose in and was caused by a swine-origin influenza virus containing a unique gene combination, with gene segments derived from the circulating north america ''triple reassortant'' (pb , pb , pa, ha, np, and ns) and the ''eurasian'' (na and m) swine influenza viruses. , analysis of the pandemic h n viruses failed to identify known molecular markers predictive of adaptation to humans. the ''triple reassortant'' swine influenza viruses emerged in late s in north america is a reassortant between classical swine (descendent of the virus after adaptation in swine population), avian, and human influenza viruses. the eurasian influenza virus was originally an avian influenza virus that was introduced into the european swine population in the late s. , while incidents of zoonotic infection with triple reassortant or eurasian influenza in humans have been reported, , sustained human-to-human transmission has never been established. these results suggest that the unique gene combination seen with the pandemic h n viruses may confer its transmissibility among humans. we have carried out systematic prospective surveillance of swine influenza in southern china over that last years through samples routinely collected at an abattoir in hong kong. during this time, the surveillance results suggest co-circulation of classical swine h n , triple reassortant h n , eurasian swine h n , and a range of reassortants between these three virus lineages. , ferrets have been reported as a suitable model for the study of influenza transmission as they are naturally susceptible to influenza infection, exhibit similar clinical signs (including sneezing), and possess receptor distribution in the airway similar to that of humans. [ ] [ ] [ ] to identify molecular determinants that enable sustained human-to-human transmission, we compared the pandemic virus with genetically related swine influenza viruses obtained from this surveillance program for their ability to transmit from ferret to ferret by direct contact or aerosol transmission. viruses human h n influenza virus [a ⁄ wuhan ⁄ ⁄ (wuhan )] and pandemic h n influenza viruses [a ⁄ california ⁄ ⁄ (ca )] were included for the study. swine influenza viruses that are genetically related with the pandemic h n virus were selected from our surveillance system, including classical swine-like influenza virus a ⁄ sw ⁄ hk ⁄ ⁄ (h n ) (swhk ), triple reassortant-like a ⁄ sw ⁄ arkansas ⁄ ⁄ (h n ) (swar ), and one reassortant between triple reassortant and eurasia swine influenza viruses [a ⁄ sw ⁄ hk ⁄ ⁄ (h n ) (swhk )]. swhk contains seven gene segments (pb ,pb ,pa,ha,np,m,ns) closely related to the pandemic h n viruses. transmissibility was tested in -to -month-old male ferrets obtained from triple f farm (sayre, pa); all ferrets were tested to have hi titer £ against human seasonal influenza h n (a ⁄ tennessee ⁄ ⁄ ), h n (a ⁄ brisbane ⁄ ⁄ ), and influenza b (b ⁄ florida ⁄ ⁄ ) prior the experiments. in each virus group, three ferrets were inoculated with tcid of the virus. at day postinoculation (dpi), we introduced one naïve direct contact ferret to share the cage with inoculated ferret, and one naïve aerosol contact ferret into the adjacent compartment of the cage separated by a double-layered perforated divider. nasal washes were collected every other day and tested for influenza virus antigen and to determine viral titers (tcid ). weight changes, temperature, and clinical signs were monitored daily. transmission is defined by detection of virus from nasal washes and ⁄ or by seroconversion (> fold rise in the post-sera collected after - days post contact). experiments were performed in the p + laboratory at st. jude children's research hospital. all studies were conducted under applicable laws and guidelines and after approval from the st. jude children's research hospital animal care and use committee. at tcid inoculation dose, all viruses replicated efficiently in the ferret upper respiratory tract with peak titers detected from inoculated ferrets at dpi. lower peak titers were detected from swhk and swhk inoculated ferrets, however, the differences were not statistically significant (table ) . tissues collected from inoculated ferrets at dpi showed that pandemic h n and swine influenza viruses replicated both in the upper and lower respiratory tract of the ferrets, while the replication of human seasonal influenza wuhan was restricted in the upper respiratory tract. direct contact transmission from inoculated donor ferrets to their cage-mates was observed for all viruses studied, albeit at different efficiency. human seasonal influenza (wuhan ) and pandemic h n viruses (ca ) transmitted most efficiently via direct contact route as the virus can be detected on dpi from direct contact ferrets, and the peak titers were detected on dpi from direct contacts. moderate direct contact transmission efficiency was detected from swar and swhk viruses as the virus can be detected from direct contact ferrets at dpi, with peak titers detected at dpi or dpi. classical swine-like swhk showed least efficient contact transmission as virus could be detected from all direct contacts only at dpi, and the peak titer detected on dpi. aerosol transmission was detected in groups of human seasonal influenza virus wuhan ( ⁄ ), pandemic h n influenza virus ca ( ⁄ ), as well as swine precursor virus swhk ( ⁄ ). transmission of wuhan and ca to aerosol contacts was detected at dpi or dpi, while transmission of swhk was detected later at dpi, suggesting that the swhk virus possessed aerosol transmission potential, but may require further adaptation to acquire efficient aerosol transmissibility. in addition to viral detection from nasal washes, we also detected viruses from the rectal swabs of ferrets inoculated or infected with pandemic h n viruses (ca ) or classical swine-like virus (swhk ), which share the common origin for the ha, np, and ns gene segments. while many of the swine influenza viruses studied were able to transmit via the direct contact route, swhk , which shares a common genetic derivation for seven genes with h n pdm, possessed capacity for aerosol transmission, albeit of moderate efficiency. swhk differed from swine triple reassortant viruses in the origins of its m gene. it is possible that the m gene derived from eurasian avian- like swine viruses also contributes to the transmissibility of h n pdm influenza viruses. outbreaks of highly pathogenic avian influenza (hpai) of the h n subtype are of extreme concern to global health organisations as human infection can result in severe acute respiratory distress syndrome, multi-organ failure, and coma. hpai viruses of either h or h subtypes contain a characteristic multi-basic cleavage site in the hemagglutinin glycoprotein as well as other virulence factors that expand the viral tropism beyond the respiratory tract of poultry. there is also emerging evidence of viral rna or antigen in multiple organs and the cns of humans infected with h n that is consistent with systemic infection , and raises the question of the role of the cleavage site in dissemination of the virus in this species. the majority of human cases with h n have involved contact with sick or contaminated poultry and exposure to respiratory secretions of birds that can be inhaled and ingested. particular risk factors for h n infection include bathing with sick birds, improper hand washing after handling sick birds, or slaughtering poultry. viral inoculum may also be consumed directly during a variety of religious and cultural practices, such as drinking contaminated duck blood and kissing of merit release birds. h n infection is lethal in % of human cases, and the pathogenetic mechanisms leading to this level of mortality are unclear. to date cases have been reported to the who, although many more people have potentially been exposed to h n through contact with infected bird populations. some studies have suggested that genetic factors may predispose an individual to severe h n disease, but little is known about the influence of route of virus exposure on morbidity and mortality. in ferrets, an animal model frequently used to study influenza because of its similar disease profile to humans, swayne et al. observed that exposure to a virulent h n strain a ⁄ vietnam ⁄ ⁄ by intra-gastric gavage did not lead to disease and did not generate an antibody response, whereas ferrets that experienced a more natural exposure by being fed contaminated meat developed severe signs of infection. in this study we further assessed the disease profile of h n following a natural oral exposure in the ferret model. to achieve this inoculation condition, conscious ferrets voluntarily consumed a liquid inoculum of h n hpai strain a ⁄ vietnam ⁄ ⁄ . as a comparison anesthetised ferrets were exposed by intranasal administration of inoculum and the ensuing disease profiles of the different routes of infection were compared. eight ferrets per group were inoculated with egg infectious dose of a ⁄ vietnam ⁄ ⁄ in a volume of ll that was given to the nares of anaesthetized ferrets to establish a total respiratory tract (trt) infection or voluntarily consumed by conscious ferrets to establish an oral infection. ferrets were culled at a predetermined humane endpoint that was defined as either a > % weight loss and ⁄ or evidence of neurological signs, discussed in ; animals that did not reach the humane endpoint were euthanased on day after challenge. nasal washes and oral swabs collected during the course of infection and organ homogenates were assessed for the presence of replicating virus by growth in embryonated-chicken eggs; viral loads were determined by titration on vero cells and expressed as tcid . tissue samples were fixed with formalin and embedded in paraffin for sectioning. viral lesions were identified by hematoxylin and eosin staining of the sections and the presence of viral antigen in the sections was determined by staining with antibody to influenza a nucleoprotein. pre-and post-exposure antibody responses were assessed by hemagglutination-inhibition assays using irradiated a ⁄ vietnam ⁄ ⁄ virus. the majority ( %) of ferrets infected by the trt route rapidly became inactive, developed severe disease, and were euthanased at the humane endpoint following infection ( figure ). ferrets infected orally had an improved chance of survival, as only % of animals developed severe disease (figure ), and the surviving ferrets were more active than ferrets infected by the trt throughout the stage of acute infection (data not shown). the improved survival rate and wellbeing of ferrets infected orally was not a result of poor infection rates by this route, as of surviving ferrets developed h specific antibodies by day post-infection, and they did not have pre-existing antibodies to h n (data not shown). the two ferrets that developed severe disease after oral infection had similar disease profiles to ferrets infected by the trt route; they both progressed to a > % weight loss and exhibited neurological signs (data not shown). viral loads in organs of these two ferrets confirmed dissemination to extra-pulmonary sites (table ) : replicating virus was detected at high titres in the spleen, pancreas, liver, and brain. similar findings were recorded in ferrets with trt infections in this study (not shown) and elsewhere. viral load in nasal washes and oral swabs taken at days , , and post-infection by the oral route did not correlate with the development of severe disease, and virus was isolated only sporadically and at low titre from the nasopharynx of these animals (data not shown). interestingly, the two ferrets with severe disease after being infected orally had no detectable viral antigen or lesions in the olfactory epithelium and bulb (table ) , whereas of ferrets culled after infection by the trt route had lesions and viral antigen in both the olfactory epithelium and bulb (data not shown). trt oral figure . percentage of ferrets that survived infection after oral or trt infection. ferrets were exposed to a ⁄ vietnam ⁄ ⁄ by the total respiratory tract (trt) route (circles) or the oral route (triangles). the percentages of ferrets that survived infection are indicated at each day following challenge. ferrets exposed orally were more likely to survive h n infection than ferrets exposed to the same dose of virus by the trt. the improved survival rates that were observed after an oral infection could be a consequence of low-level viral replication in the upper respiratory tract in combination with delivery of a substantial portion of the inoculum directly to the stomach where it may have been inactivated by the harsh environment of the gastro-intestinal tract. most ferrets infected orally developed an h -specific antibody response which differs from the studies of swayne et al. in which ferrets gavaged with a liquid inoculum neither developed signs of disease nor an antibody response. however swayne et al. administered virus to anaesthetized ferrets by gastric gavage that would have bypassed the oropharynx. in our study virus was administered to the oral cavity directly and would have had access to the oropharynx. low level of replication at this site may have been sufficient to trigger an antibody response. the two ferrets that developed severe disease following oral infection had a similar profile of viral dissemination as ferrets infected by the trt route. differences were seen in the olfactory epithelium and bulb as lesions, and viral antigen did not occur in these sites following oral infection, although cerebral involvement was identified. one route of dissemination of h n into the cns may be by transport within nerves through the olfactory bulb into the cerebrum. due to the absence of lesions and antigen in these sites following oral infection the spread of virus into the brain in these two animals may be occurring through involvement of other cranial nerves or the hematagenous routes. nasal turbinates ) ae ) ) ) ) pharyngeal lymph node interactions of oseltamivir-sensitive and -resistant highly pathogenic h n influenza viruses in a ferret model < ae b ) + + + + olfactory epithelium nd a nd ) ) ) ) olfactory bulb nd nd ) ) ) ) trachea < ae ) ) nd ) nd lung ) < ae + + ) + spleen ae ) + + ) + small intestine ) ) ) ) ) + pancreas ) ae + ) + + the pandemic potential of highly pathogenic h n influenza viruses remains a serious public health concern. while the neuraminidase (na) inhibitors are currently our first treatment option, the possibility of the emergence of virulent and transmissible drug-resistant h n variants has important implications. clinically derived drug-resistant viruses have carried mutations that are na subtype-specific and differ with the na inhibitor used. the most commonly observed mutations are h y and n s in the influenza a n na subtype (n numbering here and throughout the text); e a ⁄ g ⁄ d ⁄ v and r k in the n na subtype; and r k and d n in influenza b viruses. h n influenza viruses isolated from untreated patients are susceptible to the na inhibitors oseltamivir and zanamivir, although oseltamivir-resistant variants with the h y na mutation have been reported in five patients after , or before drug treatment; and the isolation of two oseltamivir-resistant h n viruses with n s na mutation from an egyptian girl and her uncle after oseltamivir treatment were described. the impact of drug resistance would depend on the fitness (i.e., infectivity in vitro, virulence, and transmissibility in vivo) of the drug-resistant virus. if the resistance mutation only modestly reduces the virus' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. the recombinant wild-type h n influenza a ⁄ vietnam ⁄ ⁄ (vn-wt), a ⁄ turkey ⁄ ⁄ (tk-wt) viruses, and oseltamivir-resistant viruses with h y na mutation (vn-h y and tk-h y) were generated by using the -plasmid reverse genetics system. susceptibility to na inhibitors was tested by using a fluorescence-based na enzyme inhibition assay with munana substrate at a final concentration of lm. viral fitness was studied in vivo in a ferret model: groups of three ferrets were lightly anesthetized with isoflurane and inoculated intranasally with vn-wt, vn-h y, or mixtures of the two at a different ratios at a dose of pfu in ae ml pbs; they were inoculated with tk-wt, tk-h y, or mixtures of the two at a different ratios at a dose of pfu in ae ml pbs. respiratory signs (labored breezing, sneezing, wheezing, and nasal discharge), neurologic signs (hind-limb paresis, ataxia, torticollis, and tremor), relative inactivity index, weight, and body temperature were recorded daily. virus replication in the upper respiratory tract (urt) was determined on days , , and p.i. the competitive fitness (i.e., co-inoculation of ferrets with different ratios of oseltamivir-resistant and -sensitive h n viruses) was evaluated by the proportion of clones in day- nasal washes that contained the h y na mutation. na mutations were analyzed by sequence analysis of individual clones ($ clones ⁄ sample) created by ligation of purified pcr products extracted from nasal wash samples into a topo vector. introduction of the h y na mutation conferred high resistance to oseltamivir carboxylate in vitro; the mean ic of the vn-h y and tk-h y viruses was and times, respectively, that of the corresponding wildtype viruses. the oseltamivir ic of the tk-wt virus was $ times that of the vn-wt virus. all four recombinant h n viruses were susceptible to zanamivir. introduction of the h y na mutation reduced $ % and % of the na activity of vn-h y and tk-h y viruses, respectively, as compared to the wild-type virus activity (p < ae ; two-tailed t-test). all ferrets inoculated with either vn-wt or vn-h y virus exhibited acute disease signs (high fever, marked weight loss, anorexia, extreme lethargy), rapid progression, and death by day - p.i., and no differences in clinical signs and replication in the urt of ferrets were observed between wild-type and oseltamivir-resistant viruses ( table ) . both of the tk viruses caused milder illness than did the vn viruses, despite a much higher dose ( pfu ⁄ ferret), and the tk-h y virus caused less weight loss and fever than the tk-wt virus (table ) . however, competitive fitness experiments revealed a disparity in the growth capacity of vn-h y and tk-h y viruses as compared to their wild-type counterparts: clonal analysis established the uncompromised fitness of vn-h y virus and the impaired fitness of tk-h y virus (table ) . although, the trend towards an increase ⁄decrease in the frequency of the h y na mutation relative to the wild-type was statistically significant (p > ae ) for two studied groups only. mutations within the na catalytic (r k) and framework (e a ⁄ k, i l, h l, n s) sites or near the na active enzyme site (v i, i t ⁄ v, q h, k n, a t) emerged spontaneously (without drug pressure) in both pairs of viruses (results not shown). the na substitutions i v and e a could exert compensatory effect on the fitness of vn-h y and tk-h y viruses. the lethality and continuing circulation of h n influenza viruses warrants an urgent search for an optimal therapy. our results showed that the h y na mutation affects the fitness of two h n influenza viruses differently: the oseltamivir-resistant a ⁄ vietnam ⁄ ⁄ -like virus outgrew its wild-type counterpart, while the oseltamivir-resistant a ⁄ turkey ⁄ ⁄ -like virus showed less fitness than its wild-type counterpart. we used a novel approach to compare the fitness of oseltamivir-sensitive and -resistant influenza viruses that included analysis of virus-virus interactions within the host (competitive fitness) during co-infection with these viruses. although mixed populations were present in the urt of ferrets on day p.i., the fitness of vn-h y virus was uncompromised as compared to that of its drug-sensitive counterpart, while that of tk-h y virus was impaired. a minor population of na inhibitor-resistant variants may gain a replication advantage under suboptimal therapy in two ways: (i) preexisting variants less sensitive to the drug are selected from the quasispecies population, leading to an increase of the number of resistant clones, and (ii) outgrowing variants may acquire additional compensatory mutations that enhance their fitness. it is possible that use of antiviral drugs (particularly at suboptimal concentration) against mixtures of oseltamivir-resistant and sensitive viruses will promote the spread of drug-resistant variants * ferrets in all groups inoculated with a ⁄ vietnam ⁄ ⁄ virus died by day - p.i. and were observed once daily for days. ** results obtained from one ferret. *** by inhibiting drug-sensitive variants that are competing with them for the dominance in the infected host. the influence of multiple genes on the fitness of viruses carrying h na mutation cannot be excluded. in our study we focused on additional na mutations, and sequence analysis of individual na clones was done to identify potential host-dependent and compensatory na mutations. we found that the na mutations e a and n s, which confer cross-resistance to oseltamivir and zanamivir, , can emerge spontaneously in clade . h n influenza virus in ferrets. further, we observed that mutations at na catalytic (r k) and framework (i l and n s) sites and in close proximity to the na enzyme active site (v i, i t ⁄ v, q h, k n, a t) emerged without drug pressure in both pairs of h n viruses. compensatory mutations in na or other genes may mitigate any fitness cost imposed by resistance mutations. our study identified six potential compensatory na changes (d v, f s, i v, e a, h l, and f s) that may affect the fitness of viruses with the h y na mutation. we suggest that na mutations at residues i v and e a are of importance. interestingly, we observed differences in predominance of i v and e a na mutations in different genetic backgrounds: i v mutation was identified in a ⁄ vietnam ⁄ ⁄ (h n )-like and e a in a ⁄ turkey ⁄ ⁄ (h n )-like genetic background. moreover, i v na mutation was identified only when ferrets were inoculated with the mixtures of vn-wt and vn-h y viruses, but not in ferrets inoculated with vn-h y virus. none of the potential compensatory na mutations was identified in the original inoculum used to infect ferrets. the h y na mutation causes a large shift in the position of the side chain of the neighboring e residue, which must form a salt bridge with r to accommodate the large hydrophobic pentyl ether group of oseltamivir. residue i is located near the na active site, and although it does not alter polarity, it results in a shorter side-chain and, thus, may indirectly affect the residues in the na active site. we suggest that antigenic and genetic diversity, virulence, the degree of na functional loss, and differences in host immune response and genetic background can contribute to the observed differences in the fitness of h n influenza viruses. therefore, the risk of emergence of drugresistant influenza viruses with uncompromised fitness should be monitored closely and considered in pandemic planning. this study was supported by contract hhsn c from the national institute of allergy and infectious diseases, national institutes of health, and by the american lebanese syrian associated charities (alsac). the data presented in the manuscript have been published at: govorkova ea, ilyushina na, marathe bm, mcclaren laninamivir (r- ) is a strong na inhibitor against various influenza viruses, including oseltamivir-resistant viruses. [ ] [ ] [ ] [ ] [ ] [ ] we discovered a single intranasal administration of laninamivir octanoate (cs- ), a prodrug of laninamivir, showed a superior anti-virus efficacy in mouse and ferret infection models compared to repeated administra-tion of oseltamivir and zanamivir. [ ] [ ] [ ] this suggested that cs- works as a novel long-acting na inhibitor of influenza virus in vivo. a single inhalation of cs- proved noninferiority in adult patients and significantly superior in child patients, compared to an approved dosage regimen of oseltamivir for treatment. cs- has been commercially available as an inhaled drug, inavir Ò , for the treatment of influenza in japan since october . the long-acting characteristics of cs- are explained by several reasons. first, cs- was quickly hydrolyzed to an active metabolite, laninamivir, after an intranasal administration to mice, and was retained for a long time as laninamivir in target organs, such as lung and trachea. however, with an intranasal administration of laninamivir, it disappeared quickly and did not demonstrate its longlasting characteristics. another reason is a strong binding of laninamivir to nas of seasonal influenza viruses compared to other three na inhibitors, oseltamivir carboxylate, zanamivir, and peramivir. in the following, the tight-binding ability of laninamivir to pandemic (h n ) na, as well as to the seasonal influenza virus nas, was demonstrated. in addition, we present a hypothesis of the mechanism of the long-lasting property of cs- in mouse based on a localization of an enzyme that hydrolyzes cs- to laninamivir. the influenza viruses, pandemic(h n ) (inf ), a ⁄ new caledonia ⁄ ⁄ (h n ), a ⁄ panama ⁄ ⁄ (h n ), and b ⁄ mie ⁄ ⁄ were treated with excess na inhibitors, such as oseltamivir carboxylate, zanamivir, peramivir, and laninamivir, and then unbound na inhibitors were removed from the mixtures with a bio-spin column bio-gel p- (bio-rad laboratories, hercules, ca, usa). the na substrate, -methylumbelliferyl-n-acetyl-a-d-neuraminic acid (nacalai tesque, japan) was added to the virus-na inhibitor complex, and the na activities were followed for hours at room temperature by measuring the fluorescence at an excitation wavelength of nm and an emission wavelength of nm. the enzyme which hydrolyzes cs- to laninamivir was partially purified from rat lungs using ion exchange column chromatography, and almost all bands separated by an sdspolyacrylamide gel electrophoresis were identified by mass spectrometry. the gene expression profiles of the enzyme were investigated by the bioexpress database (genelogic inc., gaithersburg, md, usa). the enzyme gene cloned from mouse lung mrna was transiently expressed in cos cells. antiserum to the esterase was prepared by immunizing rabbits, and immunostaining was done using histomouse-tm-max kit (invitorgen corp., carlsbad, ca, usa) according to the manufacturer's manual. binding stability of na inhibitors to the four viruses are shown in figure the enzyme that hydrolyzes cs- to laninamivir in rat lungs was identified as carboxyesterase. this esterase was shown to be expressed in epithelial cells of rat lung by in situ hybridization. the mouse homolog of the rat esterase was carboxylesterase (ces ). the mrna of the mouse ces was shown to be highly expressed in lung and liver by the gene expression profile, and ces was also found to contain signal sequences for retention in endoplasmic reticulum (er) and golgi at the c-terminus. the cloned ces gene and the ces gene lacking the signal sequence were exogenously expressed in the cos cells. the cs- -hydrolyzing activity associated with the cos cells expressing ces was recovered from the culture sup of the cos cells expressing ces lacking the retention signal sequence. localization of ces was immunohistologically confirmed inside the airway epithelium cells of mice, which are the target cells for influenza virus infection. the long acting property of intranasal administration of cs- in mice can be explained both by the long retention of laninamivir in the respiratory tract and by the stable binding of laninamivir to influenza virus na. again, stable binding of laninamivir to na of pandemic (h n ) virus was also observed similar to that of seasonal h n virus. the following are speculated as the mechanisms for the long-lasting characteristics of cs- in mice. we explain the mechanism by clarifying a cs- hydrolyzing enzyme and its localization inside cells. the hypothesis of the mechanism is presented in figure . briefly, hydrophilic laninamivir may not enter easily inside cells, whereas hydrophobic cs- may enter inside cells. ces with er ⁄ golgi retention signal hydrolyzes octanoate of cs- figure . difference of binding stabilities of various na inhibitors to influenza virus neuraminidases. the na substrate was added to the influenza virus-na inhibitor complex (oseltamivir carboxylate, n; zanamivir, h; peramivir, s; laninamivir, •; distilled water, ¤), and the na reaction was followed for minutes. the background (only the na substrate [d] ) is also shown. a part of data from. to generate the hydrophilic drug, laninamivir, and then it is trapped inside er ⁄ golgi because of its high hydrophilicity. the glycoprotein, na, which matures in er ⁄ golgi, meets laninamivir there and efficiently makes a stable complex with it. there are some questions that remain. how does cs- move from the cell membrane to er ⁄ golgi? is laninamivir indeed trapped inside er ⁄ golgi, and does it make a complex with na in mice? we are now making an attempt to clarify these concerns. in our study, we have explored the antiviral potential of two newly synthesized compounds to provide protection against the novel pandemic influenza virus h n ( ) strain. the compounds were reconstituted in dimethylsulphoxide (dmso), and so the initial studies began with cytotoxicity determination of solvent on uninfected and untreated madin-darby canine kidney (mdck) cells. on obtaining an upper limit for dmso, the compounds were tested for estimation of their maximum non-toxic dose to the mdck cells. thereafter, the effective dose of the compounds was evaluated and validated by a number of assays and gene expression profiling at both nucleic acid and protein level. we found that these newly synthesized compounds possess potent inhibitory activity towards the novel pandemic influenza h n ( ) virus. these findings are being evaluated in vivo for a better understanding of their inhibitory capabilities and also their effect on the host metabolism. this will be required in the course of development of new drugs for use in the prophylaxis and treatment against the influenza virus. the mdck cell line (from nccs, pune) was maintained in · dmem media (sigma, st. louis, mo, usa) supplemented with % fetal calf serum and antibiotics viz. unit ⁄ ml penicillin and lg ⁄ ml streptomycin at °c ⁄ % co . the synthesized compounds used in this study were kindly provided by the department of chemistry, university of delhi, delhi, india. the pandemic influenza h n ( ) virus was isolated and propagated in the allantoic cavities of embryonated chicken eggs during the pandemic period. the virus stocks were prepared and stored at ) °c. plaque assay was performed as previously described by hui et al., . briefly, ae · mdck cells ⁄ ml were seeded in six-well plates and maintained in dmem for hours at °c ⁄ %co . the monolayer of the cells was inoculated with serially diluted virus samples for minutes at °c ⁄ %co . subsequently, a mixture of agar overlay was added, and the plates were incubated at °c for days or until formation of plaques. the plaques were visualized after removal of the agar plug and staining with ae % crystal violet or neutral red solution. the virus titre was expressed as plaque forming unit (pfu) per milliliter. the in vitro cytotoxicity analysis was performed to determine the % cytotoxic concentration (cc ) of the compounds on mdck cells. the compounds were dissolved in dimethylsulfoxide (dmso), and so a prior cytotoxicity analysis was performed to determine the toxic concentration of dmso on the cells. various concentrations of compounds were mixed with dmem containing % fcs before addition to the preformed monolayer of mdck cells in -well plates. a series of suitable controls for in vitro cc determination was included in every plate, and the plates were incubated in the optimum environment for mdck cell culture. the cc of test compounds was analyzed by estimation of percentage cell viability of the compound-and mocktreated mdck cells by performing a colorimetric assay using tetrazolium salt -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide (mtt) at end-point of hours post-incubation. the assay was performed as described by mosman . briefly, mtt stock at a concentration of ae mg ⁄ ml was prepared in · pbs. the media was aspirated from the wells and ll of mtt dye from the stock was added to each well. following incubation at °c ⁄ % co for - hours, the dye was very carefully removed from the wells, and the cells were incubated with ll of stop solution (dmso) per well at °c ⁄ % co for hour. the absorbance of the supernatants from each well was measured at nm, and the percentage cell viability was calculated. madin-darby canine kidney cells were maintained overnight in a -well tissue culture plate at °c ⁄ % co . the cells were inoculated with various virus dilutions at °c ⁄ % co for minutes and observed for cytopathic effect (cpe). the media from the experimental wells were aspirated after - hours of infection and were subjected to plaque assay. the percentage cell viability was determined by performing mtt assay. the results of both these tests were used to assess tcid of the virus. the pre-formed monolayer of mdck cells was inoculated with the -fold dilution corresponding to tcid of the virus for hour at °c ⁄ co . the experimental setup included control wells for the cells, virus, and compound. meanwhile, the concentrated stocks of the synthesized compounds were diluted with dmem (with % fcs) to various concentrations within their respective cc ranges. one hour post-infection, the cells were incubated with these diluted solutions. the cells were observed at various time intervals post-inoculation for cpe, and ll media was collected from each experimental well for performing hemagglutination test. after h, the media was collected for plaque assay and the cells were subjected to mtt cell viability assay. preformed monolayers of mdck cells were infected with virus and treated with the respective inhibitory concentration of the compounds. forty-eight to hours post-incubation, total cellular rna was isolated using ribozol (amresco, solon, oh, usa) and treated with lg ⁄ ml of dnase (promega, madison, usa). the concentration and quality of the rna from each well were determined by measuring their absorbance at and nm. one microgram of the cdna synthesized from each rna sample was used for sybr green-based real-time pcr detection of the ha gene of pandemic influenza h n ( ) virus. as a control, human glyceraldehyde- -phosphate dehydrogenase (hgapdh) was also amplified using gene specific primers. , immunoblotting immunoblotting was performed to further validate the antiviral potential of the compounds. the experimental protocol was the same as for real time rt-pcr analysis. the cells were harvested hours post-treatment with the compounds to prepare whole cell lysates in mammalian cell lysis buffer [ ae m nacl, ae m tris cl (ph ae ), ae m edta (ph ae ), m m protease inhibitor cocktail, lg ⁄ ml pmsf]. the protein concentration was determined by bca protein assay. the cell lysates were fractionated on % polyacrylamide for western blotting. the blot was developed using sheep monoclonal antibody (santa cruz biotechnology, ca, usa) against ha protein of influenza virus and horseradish peroxide conjugated rabbit-anti sheep igg ( : dilutions) as secondary antibody. the median cytotoxic concentration for compound meuh came out to be lm, and that for flh was lm. compounds showing potent antiviral effect on the pandemic influenza h n ( ) virus propagation in madindarby canine kidney cells ( figure ). the viral titres remained constant in cells treated with the compounds, while they increased in the untreated virus infected cells. ed for the compounds meuh and flh were and lm, respectively. fifty-two percent (meuh) and % (flh) inhibition against the pandemic influenza h n ( ) virus was achieved using ed of the test compounds. both the compounds were able to reduce the rna levels of the ha gene by approximately - %, whereas approximately % inhibition was seen when both the compounds were used in combination. similar results were obtained by the immunoblotting analysis ( figure ). antiviral therapy has shown to be a promising tool in the management of various respiratory diseases, including those caused by influenza viruses. we have already shown inhibition of influenza virus replication in our earlier studies using catalytic nucleic acids, which can be used as an approach in the development of new therapeutic strategy. these therapies are very useful as the influenza virus vaccines need annual renewals due to frequent genetic drifts in the viral surface proteins. in pandemic situations the existing vaccines do not provide complete protection against the novel virus as the population generally remains naïve for the newly mutated surface antigens. the antiviral drugs play an important role in the control of novel viral strains for which there are no vaccines available. however, the key obstruction in the extensive use of antiviral drugs is their cost and relative therapeutic efficacy provided. two classes of drugs were being used for treatment and control of the influenza virus infection in humans, the m ionchannel blockers , (amantadine and rimantadine), which prevent viral uncoating, and the neuraminidase inhibitors , (zanamivir and oseltmivir), which prevent the release of influenza virions from the cytoplasmic membrane. but widespread resistance to these antiviral drugs , has limited their use. thus, novel drugs are required for the effective therapy against the emerging strains of influenza virus. the novel chemical compounds used in our study were tested for their antiviral efficacy against the pandemic influenza h n ( ) virus. a reduction in the cpe in compound treated virus infected mdck cells indicated presence of antiviral activity in chemical compounds. the persistence of constant viral titers in the compound treated cells provided evidence for the interference posed by the compounds in the replication of influenza virus. inhibition in the ha gene expression further validated our hypothesis for the antiviral effect of compounds. the efficacy of these compounds in animal models is currently being validated in our laboratory. further, molecular studies are required to ameliorate the awareness regarding the mode of action of these chemical compounds against the viruses. and is now licensed in japan, while another, laninamivir, is being developed as an inhaled prodrug. resistance to nais among circulating influenza viruses was previously low (< % worldwide). [ ] [ ] [ ] however, the - influenza season was marked by a worldwide emergence of oseltamivir-resistant seasonal influenza a (h n ) viruses with the h y (h y in n numbering) in the na. [ ] [ ] [ ] [ ] [ ] [ ] the prevalence of oseltamivir resistance was even higher in the subsequent - influenza season with many countries reporting up to % oseltamivir resistance, seasonal and pandemic influenza viruses collected globally between october , and september , were submitted to the who collaborating center for surveillance, epidemiology and control of influenza at the centers for disease control and prevention (cdc) in atlanta, ga, usa, and propagated in madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa). reference viruses representative of oseltamivir-sensitive and -resistant seasonal and pandemic viruses were also propagated in mdck cells. susceptibilities of virus isolates to the nais oseltamivir carboxylate (hoffman-la roche, basel, switzerland) and zanamivir (glaxosmithkline, uxbridge, uk) were assessed in the chemiluminescent ni assay using the na-star tm kit (applied biosystems, foster city, ca, usa) as previously described. additionally, subsets of virus isolates were tested for susceptibility to peramivir (biocryst pharmaceuticals, birmingham, al, usa). fifty percent inhibitory concentration (ic ) values were calculated using jaspr curve fitting software, an in-house program developed at cdc. curve fitting in jaspr was done using the equation: v = vmax · ( ) ([i] ⁄ (ki + [i]))), where vmax is the maximum rate of metabolism, [i] is the inhibitor concentration, v is the response being inhibited, and ki is the ic for the inhibition curve. box-and-whisker plot analyses of log-transformed ic s were performed for each virus type ⁄ subtype and nai using sas . software (sas institute, cary, nc, usa) to identify viruses with extreme ic values (outliers). outliers were characterized based on a statistical cutoff of ic greater than three interquartile ranges from the th percentile. outliers were subjected to genetic analysis by pyrosequencing and ⁄ or conventional sequencing to detect known or novel markers of nai resistance. those harboring previously characterized mutations in the na associated with nai resistance were considered drug-resistant; their descriptive statistics were determined separately from naisusceptible viruses. descriptive statistics to compute the mean, median, and standard deviation (sd), and a one-way analysis of variance were performed on original scale ic data, using sas . software (sas institute) for each nai and virus among seasonal influenza a (h n ) viruses tested for oseltamivir susceptibility (n = ), ( ae %) were outliers for the drug (table ) and harbored the oseltamivir-resistance conferring h y mutation in the na. by contrast, only a small proportion ( ae %) of tested h n pdm viruses (n = ) were resistant to oseltamivir. all influenza a (h n ) viruses (n = ) were sensitive to oseltamivir except for one outlier, a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, whose ic of ae nm was beyond the statistical cut-value off and > -fold the mean ic for the drug ( ae nm). all influenza b viruses (n = ) were sensitive to oseltamivir with exception of an outlier b ⁄ texas ⁄ ⁄ , with d e (d e in n numbering) mutation in the na, whose ic was beyond the cut-off, but only fourfold greater than the mean ic for the drug. all virus types ⁄ subtypes tested for zanamivir were sensitive to the drug (table ) , except for some outliers among seasonal influenza a (h n ) and a (h n ) outliers. the seasonal influenza a (h n ) outliers included a ⁄ thailand ⁄ ⁄ (h n ) and a ⁄ hawaii ⁄ ⁄ (h n ), both with combined h y and d d ⁄ g mutations in their na. the presence of concurrent mutations at na residues h and d in seasonal influenza a (h n ) virus isolates substantially enhances resistance to oseltamivir and peramivir and ⁄ or zanamivir, however, the changes at d are typically cell-derived and not present in clinical specimens. influenza a (h n ) outliers for zanamivir included a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, as well as a ⁄ maryland ⁄ ⁄ and a ⁄ vladivostok ⁄ ⁄ with d g and mixed d d ⁄ g mutations, respectively. some mild outliers for zanamivir among a (h n ) viruses with ic beyond the statistical cutoff but < -fold mean ic for the drug were also identified; their genetic analysis revealed presence of wildtype and mutant sequences at residue namely, d d ⁄ g, d d ⁄ n, or d d ⁄ a. mutations at residue d of the na are associated with reduced susceptibility to zanamivir in a (h n ) viruses, but were reported to be cell-culture derived in recent h n viruses. all virus isolates tested for peramivir (n = ) were sensitive to the drug, except for h y variants among seasonal influenza a (h n ) and h n pdm viruses, which exhibited reduced susceptibility to the drug. in addition, one influenza a (h n ) isolate, a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, showed reduced susceptibility to peramivir. the ic values determined in functional ni assays provide valuable information for detection of resistant viruses, but should not be used to draw direct correlations with drug concentrations needed to inhibit virus replication in the infected human host, as clinical data to support such inferences are inadequate. nevertheless, combining elevated ic values with the presence of established molecular markers of resistance in the na of virus isolates and their matching clinical specimens provides a reliable and reasonably comprehensive approach of identifying nai-resistant isolates for surveillance purposes. in this study, outliers with elevated ic values for oseltamivir among seasonal influenza a (h n ) and h n pdm viruses were confirmed to be oseltamivir-resistant based on the presence of the h y mutation in the na. outliers for oseltamivir and ⁄ or zanamivir among influenza a (h n ) viruses in this study were shown to harbor mutations at d , which were earlier associated with reduced susceptibility to zanamivir, and were cell-culture derived. the effects of d mutations on nai susceptibility appear to be strain-specific; however, there are no conclusive supporting data and further investigations are required. outliers among the influenza a viruses in this study exhibited changes in the na, derived naturally or through cell-culture, which altered their susceptibility to nais. however, mild outliers for oseltamivir and ⁄ or zanamivir among influenza a viruses with slightly elevated ic s, but without apparent changes in the na are sometimes identified. in such instances it is imperative to exclude the potential presence of influenza b among such outliers, using conclusive genetic tests such as real time pcr, since influenza b viruses exhibit higher ic values for oseltamivir and zanamivir than influenza a viruses. viruses exhibiting such mixes are typically excluded from statistical analyses of ic s for respective drugs and virus type ⁄ subtype. establishment of a clinically relevant ic cutoff value which could be used to differentiate statistical outliers from truly resistant viruses is imperative. global surveillance for nai susceptibility of influenza viruses circulating globally should be sustained to reflect the impact of seasonal and pandemic of influenza, given the limited pharmaceutical options available for control of influenza infections. nasopharyngeal swab specimens from patients with acute respiratory infection were collected at influenza sentinel surveillance units (outpatient and hospital-based) all over mongolia. specimens were transported to the virology laboratory, nccd, ulaanbaatar, and rt-rt pcr positive samples were grown in a mdck cell culture according to the protocol developed by cdc. and influenza virus gene segment (m genes) sequencing ( strains-genbank accession numbers: cy , cy , cy , cy , cy , cy , and cy ) and influenza virus gene segment (na gene) sequencing ( strains genbank accession numbers: cy and cy ) by the standard methods with applied biosystems xl genetic analyzer using primers supplied by who collaboration centers. a chemiluminescent na inhibition assay was performed with veritas microplate luminometer using the commercially available kit, na-star (applied biosystems, foster city, ca, usa), according to the manufacturers protocol. the na inhibitor susceptibility of influenza virus isolates was expressed at the concentration of na inhibitor needed to reduce na enzyme activity by % (ic ). oseltamivir carboxylate, was provided by f. hoffman-la roche ltd (basel, switzerland). na inhibition assay data were analyzed using robosage software comparing test data with the data produced by the reference na inhibitor sensitive and resistance strains, which were provided by the who influenza collaboration center, melbourne, australia. all viruses tested were sensitive to oseltamivir with two exceptions: a seasonal influenza virus a ⁄ ulaanbaatar ⁄ ⁄ (h n ) with ae nm ic value and a pandemic influenza virus a ⁄ dundgovi ⁄ ⁄ (h n ) with ae nm ic value ( figure ). there was oseltamivir resistance detected in ae % ( ⁄ ) of seasonal a (h n ) and in ae % ( ⁄ ) of a (h n ) pdm viruses. the oseltamivirresistant viruses were collected from untreated patients. in total, influenza b viruses were analyzed by na inhibition assay and all were sensitive to oseltamivir. the na of both oseltamivir-resistant strains contained h y mutation based on the sequencing analysis. the difference in the na amino-acid sequences between the mongolian oseltamivir-resistant viruses and the respective oseltamivir-sensitive reference viruses is shown in table all a(h n ) viruses analyzed for m channel inhibitor resistance by pyrosequencing contained the s n mutation and, thus, were resistant to this class of anti-influenza drugs. the segment sequencing revealed that seasonal a(h n ) viruses possess the common s n mutation. of note, a single strain a ⁄ zavkhan ⁄ ⁄ (h n ) contained an unusual s d change in the m protein. our study shows that the same prevalence [ ae % ( ⁄ )] of seasonal a(h n ) viruses with h y mutation in ⁄ season in mongolia with the published data for ⁄ season from japan. , however the prevalence of oseltamivir resistance in japan has dramatically increased in ⁄ season to % ( ⁄ ). the observed double mutations: h y and d g in a ⁄ ulaanbaatar ⁄ ⁄ (h n ) strain, which have been also found in japan in ⁄ season. the patient from whom the oseltamivir resistant seasonal influenza h n virus has been isolated was a -year-old boy, living in ulaanbaatar, the capital city, without history of using oseltamivir. the patient from whom the oseltamivir resistant a(h n )pdm virus was isolated was a year-old man, residing in the dundgovi, the southern province, also without history of antiviral treatment. according to the who data, isolation of the pandemic viruses carrying h y change from untreated patients has been uncommon. circulation of amantadine-resistant seasonal a (h n ) viruses has been increasing in mongolia since ⁄ influenza season. all pandemic influenza a(h n ) strains ( ) tested were resistant to m channel inhibitors due to the presence of the s n mutation in the m protein. among seasonal a(h n ) viruses, one contained a s d change whereas the others had s n, the well established marker of resistance to both amantadine and rimantadine. this is the first report of detecting the s d change in the seasonal a(h n ) viruses. according to the cdc data (unpublished), the s d change conferred the drug resistance in the a(h n ) viruses according to the virus yield reduction assay. it is essential to continue the antiviral resistance surveillance of influenza virus strains circulating in mongolia to ensure the efficiency of a proper clinical management of influenza patients. (conferred by the s n mutation). of note, the genotype and genotype dual resistant viruses from asia appear to be genetically similar to those previously reported dual resistant viruses from hong kong, sar. , the genotype virus was the only dual resistant virus with a nearly complete c genome. oseltamivir-resistance for this virus appears to be the result of a reassortment as demonstrated by the presence of the oseltamivir-resistant clade b na gene. although the detection of dual resistant seasonal influenza a (h n ) viruses is still rare, there has been an increased prevalence of dual resistance viruses during the last three seasons: . % ( of tested in - ), . % ( of in - ) , and % ( of in - ) (v p < . ). while the continued circulation or co-circulation of seasonal a (h n ) viruses is uncertain, the emergence of dual resistant influenza viruses in five countries does present a public health concern, especially since dual resistant viruses would limit the options for antiviral treatment to a single licensed antiviral drug: zanamivir. moreover, the markers of resistance seen in seasonal a (h n ) viruses also confer resistance in the more widely circulating pandemic a (h n ) virus. and, since the acquisition of mutations in influenza a viruses typically occur through drug selection, spontaneous mutation, or genetic reassortment with another drug resistant influenza a viruses, the detection of influenza a (h n ) viruses that are resistant to both adamantanes and oseltamivir warrants close monitoring, even if only detected at low frequency. new antiviral agents and strategies for antiviral therapy are likely to be necessary in the future. heightening concern that drug resistance will likewise become prominent in pandemic viral strains and highlighting the need for antiviral drug resistance surveillance. the h y mutation in h n neuraminidase is the most common mutation conferring resistance. however, due to the high mutation rates of viruses, new mutations can be expected that will also render viral neuraminidase less sensitive to antiviral drugs. pcr methods can be used to detect previously identified mutations; however, functional neuraminidase enzyme activity inhibition testing is necessary for detecting drug resistance that results from novel mutations. the two neuraminidase enzyme inhibition assays using either the fluorescent munana or chemiluminescent na-star Ò substrate are robust tools for ni susceptibility testing. the munan-a-based assay is broadly used by many groups, including many regional health organizations for ni susceptibility testing, yet no standardized protocol or dedicated kit has been in place for this assay, making comparison of data generated between different laboratories difficult. borrowing from multiple neuraminidase inhibitor susceptibility network (nisn)-published munana-based neuraminidase assay protocols, we have developed a kit-based fluorescent neuraminidase assay that offers both standardization and off-the-shelf quality-controlled reagents for ni susceptibility testing and other neuraminidase assay applications. the na-fluor tm influenza neuraminidase assay reagents and protocols were optimized in comparison to published nisn protocols according to the criteria of assay performance, ease-of-use, consideration of historically used assay conditions, reagent storage stability, and environmental impact. our optimized assay conditions consists of lm munana, ae mm mes, mm cacl , and ph ae in a ll assay volume, and performing the assay for minutes at °c following a minutes preincubation of drug with the virus. these conditions are consistent with the majority of published influenza ni screening data in publication. the standard na-fluor tm assay workflow for screening viral isolates for sensitivity to nis includes first titering the viral sample by neuraminidase activity to determine optimal virus concentration to be used in subsequent ic determination assays. the na-fluor tm assay is an ideal tool for titering virus based on neuraminidase activity in the viral coat. titering of viral samples prior to running the ic determination assays insured that assays would be performed within the fluorescence detection dynamic range of both the assay and the fluorometric instrument being used. viral titers giving rfus in the range of - were used for subsequent assays. comparison to traditional munana assays a primary goal of developing a standardized munana assay was to provide a standardized protocol and set of reagents that would allow for comparison of ni surveillance data between laboratories and over time. in addition, the assay should provide data comparable to historical data sets based on traditional munana-based protocols. to insure that our newly developed na-fluor tm assay met these criteria we performed side-by-side comparisons of the na-fluor tm assay to munana-based nisn protocols, as well as our na-xtd tm and na-star Ò chemiluminescent neuraminidase assays to compare assay sensitivity and dynamic range and for ni ic determination with multiple viral isolates. for all assay comparisons, assays were performed according to respective published protocols. for direct comparison of results, an equivalent amount of virus (and concomitant neuraminidase activity) was used for each assay. the na-fluor tm assay provides low-end sensitivity (by signal to noise ratio) and dynamic range similar to nisnpublished, munana-based protocols (data not shown). these assays all show a low-end detection of approximately ae u ⁄ well and dynamic range of - orders of magnitude when performed simultaneously side-by-side using serial dilutions of bacterial (clostridium perfringes) neuraminidase. these assays show approximately onefold less dynamic range and approximately fivefold less low-end sensitivity than chemiluminescent assays under these conditions. given the large amount of archived ni inhibition data for viral isolates over the past decade, it is very important for a standardized assay to generate data similar to established protocols so that data can be compared in relative terms. when run side-by-side, na-fluor tm assay provided oseltamivir carboxylate and zanamivir ic values similar to nisn-published, munana-based protocols. ic values vary somewhat for munana assays versus chemiluminescent assays depending on the viral isolate, as previously described. the na-fluor tm assay also exhibited similar sensitivity for detecting ni sensitive virus compared to nisn-published fluorescent assays as shown in figure . the large shift in ic values between oseltamivir-sensitive and resistant virus using the na-fluor tm assay enables detection of mutant virus in mixed viral samples ( figure ). this capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during ni susceptibility surveillance. several characteristics of the na-fluor tm assay make it an ideal assay for processing large numbers of viral isolates for ni sensitivity surveillance or for using the assay for high throughput screening for lead discovery of new antiviral reagents. the na-fluor tm assay signal was found to remain stable for up to hours after stop solution addition when stored at room temperature and for several days when stored at °c (data not shown). ic values did not change over these times, indicating that the assay is compatible with processing many samples in a short time frame. the na-fluor tm assay was also found to be highly reproducible giving a z' of ae or above indicating that the assay can be used confidently to identify nis in high throughput screening mode. the assay can tolerate up to % dmso, a common compound delivery reagent used in high throughput screens (data not shown). we have developed a standardized na-fluor tm assay suggested protocol that gives data similar to established mun-ana protocols. however, we have also found that several protocol adaptations can be made that generate comparable data while allowing the user more flexibility in assay mode, use of additional reagents, and to meet user-specified assay time requirements. the na-fluor tm assay can be run in either the standard minutes ⁄ °c endpoint mode described above or as real-time kinetic assay with repeated reads taken over time without the addition of stop solution, which both serves to terminate neuraminidase activity and to enhance the fluorescence of the product. for typical ni-sensitive viral strains, the rate of munana substrate turnover at °c is linear for at least hours (data not shown). as would be expected, rates of substrate turnover decrease in the presence of nis reflected in a decreased slope exhibited by real-time kinetic reads. real-time acquired rfus are typically - fold lower than rfus acquired after addition of stop solution at the same time point. ic values obtained using slope analysis for real-time assays are similar to values obtained by endpoint analysis. whether run in real-time or end-point mode, the linear rate of substrate turnover allows the user to run the assay for shorter or longer assay times than the standard protocol without compromise to assay performance. the na-fluor tm assay is also compatible with standard methods used in many laboratories to inactivate virus. we have shown that ni ic values for multiple viral strains remain unchanged when the assay is performed in the presence of ae % np- or % triton x- (data not shown). similar results are also obtained by adjusting the na-fluor tm stop solution to % ethanol prior to addition for assay termination. the assay is unaffected by phenol red concentrations present in cell culture media. we have developed a standardized munana-based fluorescent neuraminidase assay, the na-fluor tm influenza neuraminidase assay kit, which has been optimized for ni susceptibility screening. the assay provides data that can be compared to data generated using traditional munanabased protocols. the assay is economical, highly reproducible, easy to use, and environmentally friendly. the assay is flexible and amendable to user-specific adaptations including assay mode, assay timing, and reagent compatibility. trademarks ⁄ licensing ª life technologies corporation. all rights reserved. relenza is a registered trademark of glaxo- to test the prophylactic potency of h -vhhb, mice were treated intranasally with pbs, lg of h -vhhb, or negative control rsv-vhhb at , , or hours before infection with one ld of nibrg- ma virus. body weight loss was monitored daily, and on day mice were sacrificed to determine the viral load in the lungs. all mice that received h -vhhb retained their original body weight, whereas those receiving pbs or rsv-vhhb gradually lost weight (data not shown). intranasal administration of h -vhhb at or hours before challenge resulted in undetectable lung virus titers. when animals were treated with h -vhhb hours before challenge, virus titers were fold lower compared to pbs and rsv-vhhb treated mice, and three out of seven animals still had undetectable virus titers ( figure ). we next determined if h -vhhb nanobody Ò could be also be used therapeutically. we administered lg of this nanobody Ò intranasally to mice up to hours after chal-lenge with ld of nibrg- ma virus. four days after challenge, animals that received h -vhhb , , or hours after challenge had significantly higher body weight (data not shown) and lower lung virus loads than control mice. although mice treated with h -vhhb nanobody Ò hours after challenge were not clinically protected compared to control mice, they had significantly lower lung virus titers (figure ). to identify the ha amino acid residues that are potentially involved in h -vhh binding, escape viruses were selected by growth and plaque purification of nibrg- ma virus in the presence of h -vhhm or h -vhhb nanobodies Ò . the ha sequences of six independently isolated h -vhhm escape viruses revealed substitution of a lysine by a glutamic acid residue at position in ha (h numbering). in addition, two h -vhhm escape mutants carried an n d and four carried an n s substitution. the three-dimensional structure of nibrg- ha shows that n d ⁄ s and k e are close to each other as part of the corresponding antigenic site b in h ha. , interestingly, the n d ⁄ s mutations remove an n-glycosylation site, which is surmised to have evolved in h n ha as a strategy to mask an antigenic site. escape viruses selected in the presence of h -vhhb carried k n (n = ) or k e (n = ) substitutions. these results indicate that residues in antigenic site b, at the top of ha and very close to the receptor binding domain (rbd), are essential for neutralization of the virus by h -vhhm ⁄ b nanobodies Ò (figure ). the virus titer was measured in lung homogenates prepared on day after challenge. the x axis refers to the time points in hours relative to the challenge (time = hours) when ha-specific nanobodies (h -vhhb), control nanobodies (rsv-vhhb) or pbs was administered to the mice. # below detection limit, n not determined [n = - mice per condition: p values < ae (*)]. here we demonstrated that prophylactic and therapeutic treatment with llama-derived immunoglobulin single variable domain fragments is effective to control infection with h n influenza virus in a mouse model. we demonstrate that pulmonary delivery is a highly effective route of administration to treat or prevent influenza virus infection. in addition, we demonstrate that a homobivalent h -vhhb has powerful h n -neutralizing activity in vivo. it is important to note that we used a mouse-adapted derivative of the non-highly pathogenic nibrg- virus in our challenge model. nevertheless, this virus induces severe morbidity and lethality in mice. compared to conventional neutralizing monoclonal antibodies, vhhs offer the advantage that they are easy to produce in escherichia coli, typically with high yield. in addition, their small size ( kda for a monovalent vhh) and high folding capacity allow the generation of oligovalent vhh derivatives. in vitro escape selection revealed that a k e substitution in ha abolished the neutralizing effect of h -vhhm ⁄ b. a lys or arg residue at this position is conserved in all human h n virus isolates. of note, all selected escape mutants contained a glutamic acid or serine residue at position , which suggests that the conserved positively charged amino acid is important for neutralization by h -vhh nanobodies Ò . interestingly, escape mutants selected with h -vhhm also carried an n d ⁄ s co-mutation that removes an n-glycosylation site in this antigenic site of ha. the predicted n-glycosylation site at n in a ⁄ hong kong ⁄ ⁄ ha was shown to be glycosylated and may have evolved to mask an antigenic site near the rbd. , the selected amino acid changes are located near the receptor binding site of ha. therefore, it is possible that enhanced receptor binding properties of these escape viruses contribute to or are responsible for the loss of neutralizing activity of h -vhh nanobodies Ò . , we conclude that influenza virus neutralizing nanobodies Ò have considerable potential for the treatment of h n virus infections. although we focused on vhhs that presumably recognizes an epitope near the rbd, it is possible to select vhh molecules that bind to other epitopes in ha, including more conserved domains. more, a novel na (i m) substitution was discovered in a series of specimens from a patient. for the amantadine resistance, samples were tested, and all of them were confirmed to be resistant. we collected respiratory specimens from patients who had been clinically refractory to antiviral treatment since october upon ethical approval from the relevant institutions. to investigate the resistant pattern, sequence analysis to the na and matrix (m ) genes were conducted by reverse transcription (rt)-pcr and sequencing reaction. the obtained sequences were analyzed by the influenza sequences and epitopes database, which was developed in korea. eleven patients were found to be having oseltamivir-resistant pandemic (h n ) viruses with the h y substitution in the viral na genes (tables and ). some cases were associated with oseltamivir treatment on the basis of h y change from the oseltamivir-sensitive genotypes to oseltamivir-resistant genotypes in consecutive samples from the same patient. furthermore, a novel na (i m) substitution that may be associated with oseltamivir resistance was detected in specimens from one patient (patient g) who had myelodysplasia and received oseltamivir and peramivir (tables and ). in addition, we obtained viruses from clinical specimens (patients a and c) and evaluated antiviral susceptibility by measuring the dose of oseltamivir and zanamivir required for % inhibition (ic ) of na activity. these viruses (from patients a and c) were resistant only to oseltamivir (ic ae and ae nmol ⁄ l, respectively). susceptibility to zanamivir was not altered whether na contained y or h (ic ae and ae nmol ⁄ l, respectively). one isolate of pandemic (h n ) virus with an oseltamivir-sensitive genotype (h in its na) was susceptible to oseltamivir (ic ae nmol ⁄ l) and zanamivir (ic ae nmol ⁄ l). patients with oseltamivir-resistant pandemic (h n ) were treated during hospitalization with oseltamivir alone or with a combination of other antiviral drugs ( we found patients of oseltamivir resistance with h y mutation in the na gene of pandemic (h n ) virus through the surveillance of patient refractory to antiviral treatment. in addition, novel amino acid change (i to m) at position in the na gene, which might influence oseltamivir susceptibility, was detected in sequential specimens of a patient. these data showed that generation of oseltamivir resistance could be associated with oseltamivir treatment. therefore, it needs to strengthen the antiviral monitoring by supplementation of the clinical data including antiviral treatment. during the pandemic, oral oseltamivir was the primary antiviral medication used for treatment of hospitalized patients with ph n infection. many physicians worried that clinical deterioration or failure to respond to treatment with oseltamivir was due to either oseltamivir resistance or oseltamivir failure. in the united states, two investigational intravenous (iv) nais were available during - : peramivir through emergency use authorization and zanamivir by investigational new drug application. peramivir would be an option for patients with oseltamivir failure, but would not be appropriate for patients infected with h y oseltamivir resistant mutants. iv zanamivir was available in limited supply, but would be appropriate for severely ill patients infected with an oseltamivir-resistant ph n virus. during the pandemic, clinicians had few options for antiviral resistance testing in the united states. to respond to this need, the us centers for disease control and prevention (cdc) offered antiviral resistance testing for patients suspected to have clinical failure due to oseltamivir resistance. we describe the methods that cdc used to prioritize patients for testing during the pandemic and to detect markers for oseltamivir resistance, as well as the results from this testing. to facilitate decisions on which patients to test, we developed testing algorithms that were shared with state labora-tories, epidemiologists, and the emergency operation center at cdc. we prioritized patients who might benefit the most from antiviral testing given the inherent delay in providing antiviral results, e.g. patients who might have prolonged ph n shedding. patients that were critically ill [intensive care unit (icu) admission] or patients with severe immunocompromising conditions with clinical evidence for oseltamivir treatment failure (persistent detection of virus and clinical unresponsiveness to the drug) were prioritized. in addition, we tested specimens from patients that failed oseltamivir chemoprophylaxis. standard forms with information regarding specimen and minimal clinical information were collected on all patients. all protocols were validated and approved by clinical laboratory improvement amendments, e.g. quality standards to ensure accuracy, reliability, and timeliness of patient test results. information collected on patients was deemed public health response, not research, at cdc. clinical specimens, confirmed as pandemic influenza a (h n ), were tested for the h y mutation in the na using pyrosequencing. results were returned to sender within - hours of specimen receipt. from october until july , a total of specimens from patients were submitted for testing. viruses from ( %) of patients had h y mutation in the na in at least one submitted specimen. clinical information was available for patients (table ) . most patients had received oseltamivir for treatment prior to obtaining the specimen sent for antiviral testing. four patients received oseltamivir for chemoprophylaxis, all were immunosuppressed, and all had the h y mutant; duration of chemoprophylaxis until ph n infection was detected varied ( - days). among the patients with an h y mutant who were treated with oseltamivir, the median time on oseltamivir prior to collection of specimen with h y mutation was days (range - days). three patients were part of a hospital cluster of oseltamivir-resistant virus infections and were infected with h y mutants prior to oseltamivir treatment. patients with immunocompromising conditions accounted for almost half of all patient specimens tested, but they accounted for the majority of oseltamivir-resistant ph n virus infections (table ) ; among individuals with severe immunocompromising conditions and clinical failure while on oseltamivir therapy, ( %) had the h y mutant detected. among the immunosuppressed patients with an oseltamivir-resistant virus, ( %) had hematologic malignancies reported. in contrast, among the subset of icu patients without immunocompromising conditions and clinical failure while on oseltamivir therapy, we found little resistance: ( ae %) of icu patients had oseltamivir resistance detected. during the pandemic, we were able to provide timely and useful information to clinicians regarding suspected cases of oseltamivir resistance. our testing algorithm limited the number of specimens to specimens from the highest risk patients that would benefit the most from antiviral treatment. such an approach allowed us to offer this service without compromising our public health duties. in addition, the information we collected on patients from this service complimented our data on the national surveillance for antiviral resistance. we also performed national antiviral resistance surveillance from april to july . overall, resistant ph n viruses were identified from april to july in the united states among tested samples, including specimens described above, surveillance specimens, and resistant viruses reported in the literature. further studies to understand risk factors for oseltamivir-resistant ph n infection in patients with severe immunocompromising conditions are needed. while efforts to provide antiviral testing technology and materials to state laboratories are ongoing, clinicians still have limited options for such testing. rapid and inexpensive assays that could be performed by clinical laboratories, especially those caring for immunosuppressed patients, would be useful to inform patient care. the applied biosystems Ò na-xtd tm influenza neuraminidase assay kit provides the next-generation na-xtd tm , -dioxetane chemiluminescent neuraminidase (na) substrate, together with all necessary assay reagents and microplates, to quantitate sensitivity of influenza virus isolates to neuraminidase inhibitors. like the na-star Ò influenza neuraminidase inhibitor resistance detection kit, the na-xtd tm influenza neuraminidase assay provides highly sensitive detection of influenza neuraminidase activity. in addition, the na-xtd tm assay provides extended-glow light emission that eliminates the need for reagent injection and enables signal measurement either immediately or up to several hours after assay completion. the na-xtd tm assay is also used to quantitate influenza na activity directly in cellbased virus cultures to monitor viral growth or inhibition. global monitoring of influenza strains for resistance to neuraminidase inhibitors (nis) is essential for understanding their efficacy for seasonal, pandemic, or avian influenza, and studying the epidemiology of viral strains and resistance mutations. functional neuraminidase inhibition assays enable detection of any resistance mutation, making them extremely important for global monitoring of virus sensitivity to nis. the first-generation chemiluminescent na-star Ò influenza neuraminidase inhibitor resistance detection kit has been widely used for virus ni sensitivity assays, - including identification of a ⁄ h n pandemic virus resistant to oseltamivir. , in addition, this assay has been used for identification of new ni compounds, ni characterization, studies of virus transmission, drug delivery, na quantitation of virus-like particles, and cell-based virus quantitation. neuraminidase assays performed with chemiluminescent , -dioxetane substrates, including na-star Ò and na-xtd tm substrates, typically provide -to- -fold higher sensitivity by signal-to-noise ratio than assays performed with the fluorescent munana substrate. in addition, chemiluminescent assays provide linear results over - order of magnitude of neuraminidase concentration compared to - orders of magnitude with the fluorescent assay. the high assay sensitivity achieved with chemiluminescent assays enables use of lower concentrations of viral stocks, and the wide assay range minimizes the need to pre-titer virus stocks prior to ic determination. chemiluminescent reactions result in conversion of chemical energy to light energy, as light emission. the na-xtd tm substrate is a , -dioxetane structure bearing a sialic acid cleavable group. to perform the na-xtd assay, virus dilutions (from cell culture supernatant) are pre-incubated in the presence of neuraminidase inhibitor. then na-xtd substrate is added and incubated for minutes for substrate cleavage to proceed. finally, light emission is triggered upon addition of na-xtd accelerator, which provides a ph shift and a proprietary polymeric enhancer, both required for efficient light emission. chemiluminescent assays are performed in solid white microplates, and light emission is measured in a luminometer. the na-xtd tm substrate has a single structural difference from the na-star Ò substrate that provides a much longer-lasting chemiluminescent signal, with a signal half-life of approximately hours (not shown), compared to $ minutes with the na-star assay, eliminating the need for luminometer instruments equipped with reagent injectors and enabling more convenient batch-mode processing of assay plates. the na-xtd tm assay kit also provides a new accelerator solution, containing a next-generation polymer enhancer, and a triton Ò x- -containing sample prep buffer providing enhanced na activity. read-time flexibility is demonstrated by determination of oseltamivir ic values using data collected over hours after addition of na-xtd tm accelerator. although signal intensity slowly decreases over time, the ic curves and values are identical at each time point, shown using influenza b ⁄ lee ⁄ ( figure ) . triton x- detergent at % has been shown to inactivate flu virus while increasing neuraminidase activity. the addition of na sample prep buffer (containing % triton x- ) to virus stocks (at ⁄ volume, achieving a final concentration of %) provides increase in na activity up to fourfold, but is not consistently observed, and seems to be most effective with more concentrated virus stocks. ic values are unaffected by the addition of triton x- to the virus stock prior to virus dilution (not shown), so the assay is compatible with known virus inactivation reagents. assay sensitivity and ic values determined with the na-xtd assay have been compared to those obtained with both the chemiluminescent na-star assay and the fluorescent na-fluor assay (not shown). the chemiluminescent assays provide -to -fold higher sensitivity by signal-to-noise ratio, depending on the virus strain, wider assay dynamic range, and better low-end detection limit than the fluorescent assay. the wide assay range with the chemiluminescent assays enables determination of ic values over a range of virus concentrations, eliminating the need to titer virus prior to performing ic determination assays. ic values obtained with the na-xtd assay are nearly identical to those obtained with the na-star assay, with both oseltamivir and zanamivir neuraminidase inhibitors, and tend to be slightly lower than ic values obtained with the fluorescent assay. viral na quantitation provides a convenient read-out to measure viral growth or inhibition, including inhibition in the presence of inhibitory compounds or antibodies, described as accelerated viral inhibition with na as readout assay (avina). bation in the presence of varying concentrations of oseltamivir carboxylate. samples of culture media were assayed hours later. quantitation of na activity with the na-xtd tm assay demonstrates inhibition of viral growth by oseltamivir carboxylate in cell culture ( figure ). different volumes of culture media were assayed with the na-xtd assay, either in the culture plate or in a separate assay plate (not shown). performing the assay using the entire well contents ( ll) reduces assay sensitivity due to the high concentration of phenol red. assaying a smaller volume of culture medium (either in culture plate or a separate assay plate) provides higher sensitivity, and enables temporal monitoring or use of remaining culture medium for other assays. the applied biosystems Ò na-xtd tm influenza neuraminidase assay kit is a next-generation chemiluminescent neuraminidase assay providing high assay sensitivity and ''glow'' light emission kinetics for improved ease-ofuse. the applied biosystems Ò na-fluor tm influenza neuraminidase assay kit, based on the fluorescent mun-ana substrate, has also been developed to complement the na-xtd tm and na-star Ò chemiluminescence assays, for users lacking luminometer instrumentation or choosing to use fluorescence assay detection. together these kits offer: • standardized reagents and protocols • choice of detection technology • simple instrumentation requirements • high sensitivity for use with low virus concentrations • compatibility with batch-mode processing and largescale assay throughput • broad specificity of influenza detection • flexibility in assay format • additional na assay applications -cell-based viral assays, screening for new nis, detection of na from other organisms functional neuraminidase inhibition assays enable detection of any resistance mutation and are extremely important in conjunction with sequence-based screening assays for global monitoring of virus isolates for ni resistance mutations, including known and new mutations. together, these assays provide highly sensitive, convenient and versatile assay systems with standardized assay reagents, and simple assay protocols for influenza researchers. over hospitalizations and deaths in the us annually are attributable to seasonal influenza, primarily in chronically ill persons and the elderly. - following the emergence of pandemic h n influenza, severe illnesses have also been observed in children and young healthy adults. the occurrences of staphylococcal and pneumococcal pneumonia complicating influenza pandemics are well described. [ ] [ ] [ ] although temporal associations of bacterial pneumonia and influenza circulation have been reported, there is little precise data on rates of bacterial complications of seasonal or pandemic influenza. the study of bacterial lung infection has been hampered by insensitive tests for invasive disease and the difficulty of interpreting routinely obtained sputum culture results. , procalcitonin (proct), the prohormone of calcitonin, can discriminate viral and bacterial infections. this -aminoacid precursor protein normally produced by neuroendocrine cells of the lungs and thyroid gland was first shown to be elevated in bacterial infections in patients with pulmonary injury and pneumonitis. stimuli of proct include tnf-a, endotoxin, and other bacterial products. several studies indicate that bacterial infections commonly induce hyperprocalcitonemia, but that viral infections, including h n , are associated with only minimal increases. , , of note, proct induction is attenuated by viral-induced interferon-c. a meta-analysis of studies comparing proct and crp as markers for bacterial infection found that proct was more sensitive and specific than crp for differentiating bacterial from other causes of inflammation. , therefore, we measured proct levels in patients with seasonal and pandemic influenza and compared results with conventional methods for bacterial diagnosis. adults ‡ years of age admitted to rochester general hospital (rgh) from november st to june th for two winter seasons ( - ) with an admitting diagnosis compatible with acute respiratory tract infection were recruited for the study. patients were screened within hours of admission, and those with prior antibiotic use, immunosupression, or pregnancy were excluded. subjects or their legal guardian provided written informed consent. the study was approved by the university of rochester and rgh research subjects review board. at enrollment demographic, clinical and laboratory information was collected. influenza testing included nosethroat swabs (nts) for rapid antigen, viral culture, and reverse transcription-polymerase chain reaction (rt-pcr) and serology. testing for bacterial pathogens included blood cultures, sputum for culture and gram stain, nts for mycoplasma pneumoniae and chlamydophila pneumoniae pcr, s. pneumoniae antigen testing, and pneumococcal serology. if patients were unable to expectorate, sputum was induced with normal saline and bronchodilators. specimens were considered adequate by the standard criteria of > neutrophils (pmns) and < epithelial cells per high power field. serum was collected at admission and hospital day for proct measurements. influenza infection was defined a positive result for any of the following tests: . cloned proteins were coated on eia plates at ug ⁄ ml in bicarbonate buffer. after overnight incubation, plates were washed and two-fold dilutions of serum were incubated overnight at room temperature. plates were washed and incubated with alkaline phosphatase conjugate for hours, followed by substrate. a greater than or equal to fourfold rise in titer was considered evidence of infection with s. pneumoniae. urinary antigen for s. pneumoniae samples were assayed for antigen using the binax now kit. (binax inc, scarborough, me, usa). the proct was measured using time resolved amplified cryptate emission technology (kryptor pct; brahms, henningsdorf, germany). functional sensitivity is ae ng ⁄ ml (normal levels are ae ± ae ng ⁄ ml). mycoplasma and chlamydia pcr real-time pcr targeting the p adhesion gene for m. pneumoniae and the ompa gene for c. pneumoniae was used to detect atypical bacteria. results fifty-one of ( ae %) illnesses evaluated tested positive for influenza virus. of these, were due to ''seasonal influenza'' ( influenza a ⁄ h n and influenza b), and were identified as ''pandemic influenza'' ( h n ). demographics of both groups were similar: mean ages ± and ± years, respectively, and equivalent sex and racial characteristics. other than a higher incidence of underlying lung disease in the seasonal group ( % versus %, p = ae ), pre-existing medical conditions including obesity were similar. symptoms, physical findings, and discharge diagnoses did not differ, and chest radiographs (cxr) showed infiltrates in % and % of seasonal and pandemic subjects, respectively. two pandemic and one seasonal influenza patient developed respiratory failure, and none died. overall, bacterial infections were diagnosed in ( %) subjects ( -seasonal and -pandemic), and none were bacteremic. bacterial infections included: -s. pneumoniae, -m. pneumoniae, -s. aureus, and -h. influenzae. all seasonal patients were diagnosed with asthma or bronchitis, whereas three pandemic patients had pneumonia. mean serum proct (ng ⁄ ml) levels in seasonal versus pandemic patients on admission and day were: ae ± ae versus ae ± ae and ae ± ae versus ae ± ae , respectively, and were not significantly different (table ) . several patients in the pandemic group had high proct levels, and there was a trend toward more pandemic patients having admission proct values ‡ ae ng ⁄ ml than seasonal subjects [ ( %) versus ( %), p = ae ] ( figure a , b). of the four patients with proc-t > ae ng ⁄ ml, two had dense infiltrates on cxr, one had a peripheral wbc of ⁄ ml with a threefold increase in s. pneumoniae antibody, and one developed respiratory failure associated with copd exacerbation. reliable sputum samples (within hours of antibiotics) were collected in only ( %) subjects. of these, proct was ‡ ae ng ⁄ ml in two with influenza alone and three associated with bacterial infection, and < ae ng ⁄ ml in with influenza alone and five associated with bacterial infection. in the with reliable sputa and accepting the conventional bacterial diagnosis, sensitivity of a proc-t ‡ ae ng ⁄ ml for bacterial infection was %, specificity %, positive predictive value %, and negative predictive value %. notably, one patient considered to have influenza alone (proct - ae ng ⁄ ml) had group a streptococcus and s. aureus in a contaminated sputum and bilateral infiltrates on cxr. three of five patients with bacterial infections and proct < ae ng ⁄ ml had a clinical diagnosis of bronchitis. mean proct values were significantly higher in patients with infiltrates versus those with atelectasis or no acute disease on cxr ( ae ± ae ng ⁄ ml versus ae ± ae ng ⁄ ml, p = ae ). combining patients with proct values ‡ ae ng ⁄ ml with those having positive bacterial tests, rates of bacterial infection associated with seasonal and pandemic influenza were % and %, respectively. notably, antibiotics were administered to % of subjects despite % having no acute disease on cxr. in our study, bacterial infections were diagnosed in approximately % of adults hospitalized with influenza with no significant difference in rates noted between seasonal and pandemic influenza infected subjects. previous reports of bacterial infection rates of - % with seasonal influenza are difficult to compare with recent studies of pandemic influenza, because the latter tended to focus on more severely ill patients. [ ] [ ] [ ] bacterial pneumonia has been suspected or diagnosed in - % of patients in intensive care associated with h n infection and up to % of patients who died. , despite aggressive pursuit of specimens for bacterial testing, diagnoses could be confirmed in only ( ae %) of patients using conventional methodology. given the difficulty in establishing a diagnosis of bacterial infection, elevated proct values may be helpful to identify patients at high risk for invasive disease. in a study of patients with severe h n or bacterial infection necessitating intensive care, a threshold proct level of ae ng ⁄ ml, demonstrated % sensitivity and % specificity for bacterial infection. among patients with h n associated pneumonia, many of whom had respiratory failure, a threshold proct value of ae ng ⁄ ml provided a sensitivity of % and specificity of % for bacterial infection. access to samples from lower airways in ventilated patients in these studies may have improved recovery of bacteria and account for the different results we observed. it should be noted that none of our patients were bacteremic, which is a very strong stimulus for proct release. proct levels have been used successfully to guide therapy in community acquired pneumonia, and our data showing high proct levels in patients with infiltrates on cxr suggests proct may be most useful for excluding invasive disease. , elevated proct levels were not observed in patients with purulent sputum and clear cxr. it is notable that a proct level of < ae ng ⁄ ml did not exclude patients with bacterial bronchitis since proct has been used to guide antibiotic therapy in copd exacerbations. while it could be argued that healthy patients with bacterial bronchitis do not require antibiotic treatment, physician behavior in our study indicates antibiotics are frequently prescribed. combining patients with proct values ‡ ae ng ⁄ ml and those with a positive bacterial test, approximately % in patients in our study had bacterial complications associated with influenza infection. efforts should be made to curtail antibiotic use in hemodynamically stable patients with clear cxrs. given physician discomfort regarding discontinuing antibiotics, proct measurements in combination with routine bacterial cultures should be useful tools to guide therapy. influenza, mrsa, cytokines: diagnosis, treatment, prevention -a possible strategy for outpatient care we started the antiviral treatment of influenza in humans using neuraminidase inhibitors on january , in a successful attempt to cure a -year-old patient. since then, we have used the inhalant antiviral drug zanamivir, and later (october , ) changed to the use of oseltamivir with systemic bioavailability for treating patients with influenza. after years of experience with antiviral treatment of outpatients, we highlight the importance of early diagnosis and early treatment. the necessity of an earliest possible diagnosis was confirmed in the pandemic of . large hospitals reported that patients with an h n ⁄ infection had to be treated with extracorporeal membrane oxygenation. we are convinced this is due to delayed recognition of infection in most cases. valuable time is lost when the patient with a sudden onset has to be brought to a hospital for emergency treatment. the point at which the patient goes to the doctor is decisive, and this problem of timing and the delivery of early treatment is not specific to germany. in our medical office, we assessed patients with suspected influenza (to date seasonal infections, and in , h n ⁄ ) through clinical diagnosis, and then proven by point of care rapid test (quickvue; quidel, san diego, ca, usa) followed by pcr. all of the patients undergo concomitant lab tests: leukopenia, serum iron level, and the humoral inflammation status [sum of the c-reactive protein (crp) and fibrinogen levels]. because of the constant threat of a bacterial superinfection, a bacterial swab and antibiogram is carried out on every patient. in all cases positive for influenza, oseltamivir was given immediately. nowadays it is important that a double infection with influenza and mrsa must be recognized immediately and treatment started at once with antivirals and, when appropriate, with a suitable antibiotic. we pay particular attention to an extremely low iron level (signum mali ominis). in addition we monitor oxygen saturation and the course of the humoral inflammation status every - hours for every of our outpatients. among our patients with seasonal influenza, we saw within hours, within hours, and within hours after disease onset. for pandemic influenza, it was patients within hours, within hours, and two within hours. for all patients, we measured crp < ae mg and fibrinogen < mg ⁄ dl ( hours), crp < mg and fibrinogen < mg ⁄ dl ( hours), and crp > mg and fibrinogen < mg ⁄ dl ( hours, only seasonal cases). antibiotics were necessary in cases, heparin and oxygen administration in cases. one hundred forty-eight patients had a superinfection following influenza. the most common strains were haemophilus parainfluenzae and staphylococcus aureus. the subsequent use of a suitable antibiotic was only necessary in % of the patients. in all cases diagnosed, treatment (including heparin and oxygen administration) and monitoring were conducted in our medical office. none of our patients (seasonal and pandemic) had to be admitted to hospital. the early decision of whether or not antiviral and antibacterial treatment is taking effect is the only way the threat of a cytokine storm can be averted. not only does the primary care physician have to be aware of the pathophysiology involved, but also the necessary diagnostic and therapeutic options have to be made available to him. the result will lead to a saving of both lives and healthcare costs. this applies both in epidemic as well as in pandemic times. today we know that influenza leaves behind a defenceless immune system, and that the proteases of s. aureus contribute to influenza associated pneumonia. mark von itzstein, who discovered neuraminidase inhibitors, emphasized the synergistic cooperation of viruses and bacteria (personal communication, ). mrsa and influenza viruses are posing problems worldwide. the case of a -year-old boy with h n ⁄ infection demonstrates how fatal developments can be prevented. due to his constantly recurring colds, we had already detected the mrsa colonization years earlier and had always worked on boosting his general health and resistance. both the patient and his family were included in dealing with the problem. the patient was, and is, always vaccinated early with a virosomal vaccine (baxter). during the oktoberfest in munich in september , when h n infections were increasingly occurring, we learned that our patient had come down with an extremely acute feverish illness. with the help of the rapid test, we diagnosed an h n ⁄ virus infection and started treatment with oseltamivir immediately. the humoral inflammation status, which had increased very rapidly to more than ae mg ⁄ dl within hours, was treated with the effective cotrimoxazol from the antibiogram. at the same time, the patient was heparinized. the following day the patient had no fever and was symptom-free. it was only through our early knowledge of what could develop pathophysiologically that we were in a position to make the right decision at the right time. every doctor treating outpatients can follow this procedure if he is familiar with the pathophysiology of the disease and has the available tests on hand: virus rapid test, additional laboratory parameters (leukopenia, iron), and the humoral inflammation status. the decisive factor, however, is the constant clinical alertness towards the course of every acute feverish cold with acute onset. the patient has to remain in the care of the attending physician, and the chosen treatment has to be administered and monitored. this means constant spo measurements and checking the humoral inflammation status every hours. if a clinical worsening occurs during monitoring, the treatment regime has to be changed immediately, which means the administration of an appropriate antibiotic. this outpatient care on the part of the doctor has to be available days a week so that no time will be lost. reports from the netherlands and denmark show that, with the help of this preventive strategy under the motto 'search and destroy,' the dangerous, fatal course of infections reported in germany with at least four deaths a day, can be avoided. however, the doctor has to be adequately remunerated for the elaborate amount of time this intensive outpatient care requires. with our strategy, we have moved from divergence to convergence in the care of our patients. we reported on our years of clinical experience with this approach at the antivirals congress in peking. our main message was early diagnosis and early treatment. we were able to demonstrate this in outpatients with seasonal influenza and h n ⁄ outpatients. our creed is: as much outpatient care as possible and as little hospitalization as possible. virological and autopsy findings in suspected and confirmed fatal cases of h n pandemic influenza in the czech republic -preliminary results influenza viruses cause substantial morbidity and mortality. pandemic influenza may have a serious impact on certain (mainly younger) age groups in comparison with seasonal flu. influenza is one of few viral infections capable of causing a pneumonia that is difficult to cure and ⁄ or leads to sudden death. the aim of this study was to analyze and compare virological and autopsy findings in patients who died with suspected or confirmed h n pandemic influenza virus infection. there were virologically confirmed cases of pandemic influenza and deaths in the czech republic during pandemic wave. more than influenza strains belonging to the new pandemic variant were isolated in the national influenza reference laboratory. postmortem biological samples were collected from any patient who died with suspected influenza infection to test for respiratory viruses. the samples were screened for h n pandemic influenza virus by real-time pcr (rt pcr), and when rt pcr positive, by virus isolation assay. no immunohistochemical staining for influenza antigen was done on the rna pcr positive cases. other important respiratory viruses such as respiratory syncytial virus, parainfluenza viruses, and adenoviruses were detected by virus isolation assay in a suitable cell culture. epidemiological analysis of postmortem histopathologic findings in the airway tissue was carried out in of fatal cases. virological findings were subsequently correlated with histological changes and available demographic and clinical data. statistical analysis was performed by t-test using spss software. sixty-one deaths ( males, females) were analyzed. the rna of the h n pandemic influenza virus was detected by pcr in cases, while cases remained negative. five respiratory syncytial viruses and two adenoviruses were detected in the influenza negative group. the mean age of confirmed h n pandemic influenza victims was ae years, age range - years and median ae years. the mean age of influenza negative victims was ae years, age range - years and median ae years. the % ci for the difference in the age between the two groups is ) ae ; ae . the test is statistically significant at the % level. the obtained significance (p = ae ) can be explained by the relatively small size of the study group. the most common postmortem histopathologic finding in the lung tissue of the h n pandemic influenza virus-positive victims was diffuse alveolar damage (often bilateral) and ⁄ or hyaline membrane formation, possibly with signs of respiratory distress syndrome (in , i.e., ae %, of autopsied patients). in the h n pandemic influenza virus negatives, the most common finding was pneumonia or bronchopneumonia with the detection of various bacterial species (in , i.e., ae % of autopsied patients). the cause might be either primary bacterial infection or superinfection following primary infection with influenza virus that remained undetected. the h n pandemic influenza victims were younger than the patients who died with suspected but undetected h n pandemic influenza. the majority of deaths were primarily linked to rapidly developing respiratory failure. this result supports the previous reports of severe respiratory outcomes in younger age groups that are typically linked to the spread of a pandemic strain of influenza. due to limited amount of pandemic vaccine, especially at the beginning of pandemic, it is advisable to assess experiences with antiviral treatment, mainly dosing, and way of antiviral administration. primers specific for each of the eight genes of pandemic h n ⁄ were adopted from assays as described previously to discriminate against seasonal human h n and h n viral segments (table ) . the primers were allowed to cross-react specifically with the sister clade viral segments of pandemic h n ⁄ . the method we employed in this study was a -step singleplex sybr green-based real-time rt-pcr. this approach helped lower the running cost of the assays and facilitated downstream molecular analyses (e.g., sequencing) by using screened cdna samples. viral rna was extracted from viral cultures or clinical samples as described , and was converted to cdna in a universal rt-pcr. each ll rt reaction containing ae ll of purified rna, ll of · firststrand buffer (invitrogen), u of superscript ii reverse transcriptase (invitrogen), ae lg of uni ( ¢-ag-caaaagcagg- ¢), ae mm of deoxynucleoside triphosphates and mm of dithiothreitol was incubated at °c for minutes, followed by °c for minutes for heat inactivation. for each segment-specific real-time pcr, the ll reaction contained ll of a -fold diluted cdna samples, ll of fast sybr green master mix (applied biosystems), and ae lm of the corresponding primer pair. the thermocycling conditions of all eight segment-specific pcrs were optimized as °c for seconds, followed by cycles of °c for seconds and °c for seconds, and all eight assays were performed simultaneously in a sequence detection system (applied biosystems). at the end of the amplification step, pcr products went through a melting curve analysis to determine the specificity of the assay ( - °c; temperature increment: ae °c ⁄ seconds). cdna of a ⁄ california ⁄ ⁄ virus was used as a positive control. robust and specific amplification was achieved in all eight segment-specific real-time rt-pcr reactions. pcr product for each segment of pandemic h n ⁄ yielded unique melting curve pattern with distinctive melting temperature (tm), which was not observed in negative and water controls ( figure ). reactions with tm value within sds of the mean tm were determined as positive. we evaluated the assays with a number of serologically confirmed human clinical samples. all pandemic h n ⁄ samples (n = ) were positive in all eight assays, while all seasonal samples (h n = ; h n = ) were negative in all assays, as expected ( figure and data not shown). these results showed that no reassortant of pandemic h n and seasonal viruses was present in the tested human isolates. we applied these assays to our on-going influenza virus surveillance program in swine. nasal and tracheal swab samples were collected at an abattoir in hong kong and cultured in madin darby canine kidney cells or embryonated eggs as described. positive viral cultures in hemagglutination assays were tested with the established segmentspecific real-time rt-pcr assays. among swine viral isolates collected from to september , of them were recognized as pandemic h n ⁄ in all eight segments. they were confirmed to be of pandemic h n ⁄ origin by subsequent full genome sequencing analyses, showing that there were interspecies transmissions of the virus from humans to pigs. , the remaining viruses had one to seven gene segments positive in the segment-specific real-time rt-pcrs. thirty of them were selected as representative samples for full genome sequencing analyses based on the genotyping data generated in our assays. they were swine h n or h n viruses with their gene segments derived from tr or eurasian avian-like swine lineages. it should be highlighted that all of their positive gene segments in our assays belonged to the sister groups of pandemic h n ⁄ . their melting curve patterns were very similar to those derived from segments of pandemic h n ⁄ , except for ha of tr lineage. our results successfully demonstrated the use of these segment-specific real-time rt-pcrs to recognize gene segments of contemporary tr (pb , pb , pa, ha, np, and ns) and ea (na and m) swine viruses. the ha-specific assay was able to discriminate pandemic h n ⁄ from other contemporary swine viruses in the same lineage. nevertheless, to confirm the identity and to examine all the genetic variations in the viruses of interest, full genome sequencing analyses were necessary. in this study, the biggest obstacles in primer design were sequence similarity and diversity of influenza viruses. we attempted to use degenerated primers, but they were highly non-specific. the finalized non-degenerated primers crossreacted with genes from pandemic h n ⁄ and its sister clade tr (pb , pb , pa, ha, np, and ns) and ea (na and m) swine viruses with some minor sequence mismatches. three avian (h n , h n , and h n ) and classical swine (h n ) were also tested with our assays. all of these animal viruses were negative, except for ns gene of the classical swine virus. our segment-specific real-time rt-pcr assays might be used in high throughput genotyping. they detected pandemic h n ⁄ viruses and acted as a preliminary screen-ing tool to select virus reassortants of interesting genotypes for further sequencing analyses. in fact, we identified a novel reassortant in january during the course of this study. this sw ⁄ hk ⁄ ⁄ has a previously unidentified viral gene combination as shown in figure . it was confirmed to be a reassortant between pandemic h n ⁄ and other swine viruses in full genome sequencing characterization. it has a pandemic h n -like n gene, an ea-like h , and the other six internal genes derived from tr swine viruses. , the eight established real-time rt-pcrs can rapidly reveal the gene-origins of influenza viruses. we are currently using these assays in influenza surveillance in humans and other animals. it is believed that similar strategy might be applied to detect and genotype other influenza viruses and possible reassortants in the future. pandemic influenza a ⁄ h n ⁄ infects millions of people around the world. a significant fraction of the world's population may also already have been exposed to the virus and, although asymptomatic, may be at least partially immune to the disease. a precise assessment of the number of people exposed to the influenza a ⁄ h n ⁄ virus is epidemiologically relevant. however, assays typically used to estimate antibody titers against a particular influenza strain, namely hi and neutralization, require use of the actual virus. this seriously limits broad implementation, particularly in regions where high biosafety facilities are unavailable. we developed an elisa method for the evaluation of presence of specific h n influenza virus-antibodies in serum samples. mouse anti-histidine tagged antibodies ( ll; lg ⁄ ml; abd serotec Ò , uk) in pbs (ph ae ) were dispensed into standard -well plates and incubated for - hour at room temperature. excess antibody was removed by at least two successive alternate washings with pbs-tween ae % and pbs. commercial blocking solution ( ll, superblock Ò t ; pierce Ò , usa) was added and incubated for at least hour at room temperature. after successive washing steps with pbs-tween ae %, non-glycosylated histidine-tagged recombinant protein ( ll; lg ⁄ ml) was added to each well. this protein consisted of the receptor-binding domain of the hemagglutinin of the influenza a ⁄ h n virus. , after hour incubation, wells were washed for at least two alternating minutes cycles with pbs-tween and pbs. a : dilution of the serum or plasma sample to be assayed ( ll) was added to each well and incubated at room temperature for hour. after repeated alternating minutes pbs-tween ae % and pbs washes, anti-human igg antibody solution ( ll ⁄ well; : dilution in pbs-tween ae %) marked with horse radish peroxidase (pierce Ò , usa) was added and incubated for hour at room temperature. after repeated alternate washes with pbs-tween ae % and pbs), substrate solution ( ll; -step ultra tmb-elisa; pierce Ò ) was added to each well. after incubation for minutes at room temperature in darkness, the enzymatic reaction was stopped by addition of m h so ( ll ⁄ well). yellow color produced by the enzymatic reaction was evaluated by absorbance at nm in a biotek Ò microplate reader (usa). blank assays using albumin in place of human sera established the elisa background signal, which was subtracted from sample absorbance signals: abs serum sample ¼ abs serum sample before correction À abs albumin sample : absorbance values were normalized based on the average signal of non-exposed subjects (uninfected subjects), and expressed as normalized absorbance (abs norm ): where abs serum ample is the sample absorbance signal, abs albumin sample is the albumin control absorbance signal, abs non exposed subjects is the average absorbance signal of non-exposed subject samples. for ferret serum samples, the same basic protocol was followed, with minor modifications. an anti-igg anti-ferret polyclonal antibody preparation was used at a dilution of : in pbs-tween ae %. a recombinant receptor-binding domain of the ha of the influenza a ⁄ h n ⁄ virus, expressed in escherichia coli strains, was used as the elisa antigen. this kda protein, designated here as ha - -rbd, contained amino acids - of the influenza a ⁄ mexico ⁄ indre ⁄ (h n ) hemagglutinin. a sequence coding for a series of six histidines at the n-terminus of the protein was included in the genetic construct to allow purification using immobilized metal affinity chromatography (imac) and attachment to assay surfaces treated with anti-histidine antibodies (or alternatively co + or ni + ). a panel of four samples (kindly provided by st. jude from ferrets exposed to different influenza strains, namely h n , h n swine, and h n , was also tested by the elisa method using : dilutions. protein ha - -rbd specifically and selectively recognizes antibodies from serum samples from convalescent h n ⁄ influenza subjects. dubois et al. demonstrated that this protein, produced in e. coli, folds properly into a -d structure practically indistinguishable from the analogous region in the ha of the influenza a ⁄ h n ⁄ virus. ha - -rbd preserves three of the conformational immunogenic epitopes (sa, sb, and cb) described for influenza a ⁄ h n hemagglutinins. the recombinant protein was used as the antigen, attached through histidine tags to microplate surfaces treated with anti-histidine antibodies to discriminate between serum samples from subjects exposed and non-exposed to influenza a ⁄ h n ⁄ . samples collected before the pandemic onset, and therefore presumed to exhibit low specific antibody titers against influenza a ⁄ h n ⁄ , were analyzed by elisa using the antigen ha - -rbd. the histogram of normalized absorbance values from this sample set displayed a normal behavior with a standard deviation of ae units. only ae , ae , and ae % of these samples exhibited normalized absorbance values higher than ae , ae , and ae , respectively. no sample from non-exposed individuals presented an absorbance value higher than ae . variability among samples from non-exposed subjects was much lower than in samples with high specific serum antibody titers from convalescent h n ⁄ patients. exposure to the h n ⁄ influenza virus with this elisa method can be predicted by absorbance values normalized to those of abs norm ¼ ðabs serum ample À abs albumin sample Þ=ðabs non exposed subjects À abs albumin sample Þ ð Þ serum from uninfected subjects. consequently, for reliable results, inclusion of samples from non-exposed subjects on every assay microplate is necessary. figure shows the analysis of human serum samples, including samples from convalescent patients with positive diagnosis by rt-pcr. three positive (dark gray bars) and two negative controls (light gray bars) were included in the same microplate. all serum samples corresponding to convalescent subjects exhibited absorbance values ae - ae times higher than negative samples ( figure ). normalized absorbance values above ae suggested exposure to the virus, although, a more conservative threshold value of ae units is proposed for discrimination between exposed and non-exposed subjects. the elisa method described here yields adequate reproducibility and a high signal ⁄ noise ratio within determinations in the same microplate and among different microplates. using a normalized absorbance value of ae , the method was able to discriminate samples from convalescent patients, preferably after the third week of infection, and at least up to the twentyfourth week of exposure. assay sensibility was further validated against results from hi assays. a previously reported study showed that all members in a pool of fourteen samples diagnosed as positive by hi exhibited normalized absorbance values higher than ae , and % of them exhibited normalized absorbance values higher than ae . in general, high hi titers (> ) were correlated with normalized absorbance values higher than ae . figure a shows results using the ha-rbd elisa method and the hi assay on a pool of seventeen known positive serum samples corresponding to convalescent h n ⁄ patients. all samples determined as positive by hi ( samples) were also positive by elisa. while sensitivity of the hi assay was ⁄ = ae %, the elisa method recognized all samples correctly as positive ( % sensitivity) when a threshold of ae or ae was used. figure b shows that sera from ferrets infected with other influenza strains (h n , h n swine, and h n ) showed no cross-reactivity when analyzed by elisa. in summary, the ha-rbd elisa method presented here consistently distinguished influenza a ⁄ h n ⁄ infected and non-infected individuals, particularly after the third week of infection ⁄ exposure. since no actual viral particles are required, this assay can be readily implemented in any basic laboratory. in addition, should sufficient vaccine be unavailable, this elisa could determine the level of specific antibodies against the virus and presumably the extent of partial protection in a subject. therefore, the elisa protocol might allow better administration of vaccination programs during pandemic or seasonal influenza outbreaks. in april , a novel h n influenza virus emerged in north america and caused the first influenza pandemic of the st century. [ ] [ ] [ ] [ ] the pandemic h n (pdmh n ) has a unique gene constellation that was not previously identified in any species or elsewhere. it is genetically related to the triple reassortant swine h n influenza viruses currently circulating in north america, with the exception of the neuraminidase (na) and matrix (m) genes, which are derived from a eurasian swine influenza virus. swine h n influenza viruses were first isolated in and continued to circulate in north america with very little antigenic changes (classical swine h n ) until . since , however, the antigenic make up of swine h viruses has shown increased diversity due to multiple reassortment events and the introduction of h n genes from human influenza viruses. currently, four swine h clusters (a, b, c, d) are found endemic in the north american swine population. , these swine h viruses show substantial antigenic drift compared to the classical swine h viruses. cluster d swine h is derived from current human h viruses, and there is a substantial antigenic divergence between classical swine h and human seasonal h viruses. epidemiological evidence shows a two-way transmission of influenza viruses between swine and humans, and such events lead to the emergence of the pdmh n virus. , , phylogenetic analysis have suggested that possible ancestors of the eight genes of pdmh n were circulating in the swine population for at least years prior to the emergence of the pdmh n virus in humans, although the pdmh n virus itself was not isolated from pigs until after the pandemic. interestingly, pdmh n infections have been reported not only in humans and pigs, but also in other animal species such as turkeys, cats, ferrets, cheetahs, and dogs. [ ] [ ] [ ] after the first report of pdmh n infection in swine in canada, other countries, including argentina, australia, singapore, northern ireland, finland, iceland, england, united states, japan, and china reported outbreaks of pdmh n in swine as well. , [ ] [ ] [ ] the ample geographic range of pdmh n outbreaks in swine, its apparent broad host range, and the possibility of two-way transmission between swine and humans poses a tremendous challenge for controlling the virus. therefore, to differentiate pdmh n from other h strains, particularly in swine and human populations, is an important issue to ascertain the magnitude of the disease caused by the pdmh n . in this study, we developed an elisa assay to discriminate pdmh n strains from other swine and human h viruses. madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa) were maintained in modified eagle's medium (mem) containing % fbs. a ⁄ california ⁄ ⁄ ⁄ h n virus (ca ⁄ ) was kindly provided by the centers for disease control and prevention (cdc), atlanta, georgia. other viruses are listed in table . viruses were propagated in mdck cells and stored at ) °c until use. viruses were titrated by the reed and muench method to determine the median tissue culture infectious dose (tcid ). three monoclonal antibodies ( b , h , and f ) against ha of pandemic h n were prepared in our laboratory following previously described methods (shao and perez et al., unpublished). purification and labeling of mabs mab b , h and f were purified on a protein g-sepharose affinity column (upstate biotechnology, lake placid, ny, usa). biotinylation of the detection antibody in the elisa was performed using sulfo-nhs-lc-biotin (sulfosuccinimidyl- -(biotinamido)hexanoate; pierce, rockford, il, usa) according to the manufacturer's instructions. purified h and f were selected as the capture antibody, and biotin-conjugated b was selected as the detection antibody, and hrp-conjugated streptavidin (abcom, cambridge, ma, usa) was developed using the tmb substrate system (kpl, gaithersburg, md, usa). in brief, the mixture of the purified h and f ( ae and ae lg ⁄ ml respectively, in carbonate ⁄ bicarbonate buffer, ph ae ) was coated to -well plates (test well, t) for h at °c. at the same time, a control antibody was coated to -well plates (control well, c). after blocking the plates with % (w ⁄ v) non-fat milk in pbs for hour at °c, the samples were diluted in extract buffer ( %tween- , ae %bsa in pbs) and added to the wells ( ll ⁄ well, each sample was table . specificity assay of the sandwich elisa result (t ⁄ c) added to four wells-two for t wells and two for c wellsand the mixture was incubated at °c for hour. after four washes, ll biotin-conjugated b ( ae lg ⁄ ml) in dilution buffer ( ae % bsa in pbs) was added to the wells and the mixture was incubated for h at °c. following three washes, ll diluted hrp-conjugated streptavidin ( ae ng ⁄ ml) in dilution buffer was added to the plates. after incubation for h at °c, the plates were washed five times, and the binding developed using the tmb substrate system for minutes. the ratio of the average od value of the t wells to that of the c wells (t ⁄ c) of individual samples was calculated. t ⁄ c values > ae were considered positive in the sandwich elisa. we developed three monoclonal antibodies, b , h , and f , against a prototypical pdmh n strain, a ⁄ california ⁄ ⁄ (h n ) (ca ⁄ ). these monoclonals were used to develop a rapid sandwich elisa for specific diagnosis of pdmh n strains. purified h and f were used as capture antibodies, whereas the biotin-conjugated b was used as detection antibody. the sandwich elisa showed strong reaction with different pdmh n strains as described in in order to evaluate if the sandwich elisa could distinguish the pdmh n from other swine h clusters (a, b, c, d), swine influenza strains spanning these clusters were tested. these viruses were first diluted : in extract buffer, and then added to the coated plates. as shown in table , the t ⁄ c ratios of these viruses were < ae , and therefore showed negative elisa result. likewise, testing of human seasonal virus strains a ⁄ brisbane ⁄ ⁄ (h n ), a ⁄ malaya ⁄ ⁄ (h n ), a ⁄ wsn ⁄ (h n ), and a ⁄ brisbane ⁄ ⁄ (h n ) also showed negative elisa results. furthermore, the sandwich elisa showed no cross reaction with avian influenza viruses, including strains of the h , h , h , h , h , h , h , h , h , h , and h subtypes. more recently, the mutation d g in the ha of some pdmh n strains has been associated with exacerbated disease and altered receptor binding. [ ] [ ] [ ] [ ] [ ] to evaluate if such mutant could be detected in our sandwich elisa, we tested a mutant of a ⁄ netherland ⁄ ⁄ (h n ) carrying the d g mutation (engineered by reverse genetics). as described in table , our elisa could still capture the d g mutant virus and showed a positive reaction, which highlights the specificity of our assay for pdmh n strains, even those with mutations. to evaluate the sensitivity of the elisa, we used the serially diluted pdmh n viruses to determine the limit of detection (lod). as shown in table , in our elisa the highest positive dilutions of nl ⁄ and ca ⁄ were : and : , respectively. the lod of the sandwich elisa by tcid was ae · and ae · tcid ⁄ ml, for nl ⁄ and ca ⁄ , respectively. it is important to note that the t ⁄ c ratio from nl ⁄ and ca ⁄ viruses showed clearly a dose dependent effect, while the t ⁄ c ratio of a ⁄ swine ⁄ iowa ⁄ (h n ) did not show the same dependence and was always < ae , corroborating the high specificity of the sandwich elisa for pdmh n strains. although we did not compare our elisa with other current commercial rapid influenza detection kits, the lod of our elisa assay is similar to other commercial kits that detect human seasonal influenza virus. comparison of the sandwich elisa with the ''gold standard'' -virus isolation in order to further evaluate the feasibility of the application of the elisa to clinical samples, nasal wash samples ae · ^ )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) -from ferrets, of those previously infected with ca ⁄ and shown positive by virus isolation, were tested. the samples were diluted : in extract buffer and then tested using the sandwich elisa. result showed out of positive samples by virus isolation were positive also by the sandwich elisa (sensitivity ae %). the samples tested that were negative by virus isolation were also negative in the elisa, indicating % specificity for our assay. these results show not only that our elisa has high compatibility with the virus culture method, but also indicates this application can be used for clinical samples. although real time rt-pcr targeting the ha gene has been used for specific diagnosis of pdmh n with high sensitivity, [ ] [ ] [ ] [ ] [ ] [ ] it is a method that requires manipulation of the sample to extract viral rna, and it is prone to crosscontamination during the pcr steps. in this study, we described a convenient sandwich elisa based on three mabs developed against the pdmh n strain. the elisa not only shows high specificity for pdmh n strain, but also shows great sensitivity. the elisa could distinguish pdmh n strains from human seasonal h and h viruses and, more importantly, from other swine h viruses. we must note that current rapid diagnostic tests cannot be used to differentiate pdmh n from swine or human h viruses. it is also worth noting that the sensitivity of commercial rapid antigen-based diagnostic tests for detecting pdmh n is lower than that for human seasonal influenza viruses. , a study by kok et al. showed that sensitivity of the current rapid antigenic tests for pdmh n is only ae %, whereas that for seasonal influenza a is ae %. chen et al. developed a dot-elisa and increased the sensitivity for influenza rapid antigen detection. however, the dot-elisa developed by chen cannot distinguish among subtypes. the lod of our elisa is between ae · to ae · tcid ⁄ ml, comparable to the lod of rapid diagnostic tests for human seasonal influenza viruses. compared to the ''gold standard''-virus isolation-our sandwich elisa showed ae % sensitivity using ferret nasal washes. our results highlight the potential application of our sandwich elisa for the specific diagnosis of pdmh n viruses. the timely and reliable laboratory evidences are vital factors for field epidemiologists trying to control outbreaks of infectious diseases and for the practicing clinicians to properly manage disease cases. therefore, analysis of new detection methods in comparison to the routine ''classical'' methods is essential to select new methods to be introduced into health service practices, especially in developing countries. in this study we have compared rt-rt-pcr detection of influenza viruses and direct fluorescent-antibody assay using r-mix hybrid cells (a &mv lu) with the ''classical'' cell culture methods in developing country settings. in this study, we analyzed nasopharyngeal swabs col- the detection of influenza h , h , b, and pandemic influenza (h )pdm virus-specific nucleic acids was performed by rt-rt-pcr in abi fast real time pcr system using primers recommended by cdc, usa, and super-scriptÔ iii one-step rt-pcr and platinum Ò taq dna polymerase kits (invitrogen). the cycling protocol was: minutes at °c, minutes at °c, and cycles of seconds at °c, seconds at °c. rapid detection of influenza infected cells has been performed by dfa using the infected hybrid cells of r-mix within hours after inoculation, according to the manufacturers instruction (diagnostic hybrids, inc., usa). the isolation of influenza viruses was performed on mdck cell culture by the protocol recommended by cdc, usa. we detected ( ae %) influenza virus-specific nucleic acid fragments from all tested samples by rt-rt-pcr. among the positive samples, there were ae % a(h n ), ae % a(h n ), ae % influenza b, and ae % a(h n )pdm with different distributions by time series in different age-groups. inoculation of the cell lines by rt-rt-pcr positive samples selected randomly has detected influenza virus in ae % ( ⁄ ) on mdck cell culture and % ( ⁄ ) on r-mix hybrid cell culture with varying distribution for different strains. in other words, mdck cell culture technique was better for isolation for pandemic influenza viruses and dfa using r-mix hybrid cell culture technique for detection of seasonal influenza viruses (table ) . average times needed for the final results for different methods were: hours for rt-rt-pcr, hours for dfa on r-mix and days for mdck cell culture with two passages at least. the peak of the seasonal influenza a virus detection occurred in the - th weeks of , however the pandemic influenza detection peak was observed in the - th weeks of ( figure ). the outbreaks by seasonal influenza viruses was observed mostly among the children of - years of age, and pandemic influenza virus outbreak was observed mostly in the adults of - years of age. the results of this study indicate that rt-rt-pcr is the most suitable method for decision makers in epidemiological and clinical settings by sensitivity and timeliness. the final results show that r-mix dfa requires times longer, and by mdck cell culturing, times longer periods, than by rtrt-pcr. mdck cell culture technique has a higher isolation of pandemic influenza viruses, and r-mix dfa has a greater detection rate of seasonal influenza viruses by our results. according to our study, with rtrt-pcr, the isolation of positive samples by tissue culture of influenza a viruses was % and influenza b viruses was ae %, which is lower than in similar spanish study. however our study illustrates similar results with a canadian study where the sensitivity of dfa method and tissue culture technique was shown to be lower than rtrt-pcr sensitivity. as recorded by a study of american researchers, r-mix hybrid and conventional cell culture techniques have had similar sensitivity, which does not match the results of our study. however, the results of our study match with the results of italian and american scientists , where the r-mix hybrid method for seasonal influenza viruses is higher than mdck cell culture technique. background: viral kinetics is increasingly used to study influenza infectiousness. the choice of the study design, i.e. when and how many times nasal samples are to be collected in individuals depending on the sample size, is crucial to efficiently estimate the viral kinetics (vk) parameters. material and methods: we performed a model based optimal design analysis in order to determine the minimal number of nasal samples needed to be collected per subject and when to collect them in order to correctly estimate the vk parameters. the model used was a non linear mixed effect model developed with data collected from patients sampled nine times in days (initial design - samples collected), and we used d-optimization for design identification. we also computed the minimal number of participants necessary. results: considering that % of the influenza-like illness cases are not due to volunteer challenge studies have been used since the 's to provide data on virus shedding from the respiratory tract during influenza infection. recently, vk was studied in naturally acquired influenza infection. , these data are invaluable to describe the natural history of influenza-infection and to compute natural history parameters such as the latent period, generation time, or the duration of infectiousness. [ ] [ ] [ ] [ ] however, among the studies used in a meta-analysis about viral shedding kinetics, the designs varied greatly from one to another. these differences led to variable amount of available information concerning the vk. the lack of adequate sampling leads to imprecise estimates. on the other hand, intensive sampling or over-sampling, while associated with highly informative data, may lead to unnecessary discomfort for the patient and cost to the investigator. optimal design is increasingly used to conceive studies and provides cost-efficient designs. here we propose an optimised design to model vk in the case of influenza infection. we defined the number of participants, the number of samples to collect and their allocations. this design allows, at a minimum cost and discomfort, accurate vk curves and allows the natural history parameters to be well described. model a vk population model was proposed for influenza infection. this model describes with eight parameters the relations between free virus, uninfected target epithelial cells, infected epithelial cells, and early immune response. this model was built on a dataset of volunteers from which nasal samples were collected once a day over days. we call this dataset the ''original dataset''. three parameters, the induction of the early immune response, the virus production rate, and the virus clearance, did not show inter-individual variability and were precisely estimated (relative standard error below %). we considered them as fixed in this research work. five parameters were hence considered here: b the infection rate, d the infected cell mortality rate, w the effect of early immune response on virus production rate and v init the initial value of virus titre. in order to correctly estimate these parameters it is crucial to determine a design to collect informative data. optimal designs maximise the amount of information provided by the study. it involves the determination of the number and allocation of sample times per subject as well as the number of participants. d-optimization is based on the maximization of the determinant of the fisher information matrix and thus minimizes the variance of the parameters. we used the fedorov-wynn algorithm implemented in pfim . to maximize this determinant, which implies to pre-define a set of possible sample times. with the hypothesis that the inoculation occurred at : am, we chose three possible hours ( : , : , and : ) for each day with respect of the sleep-time. to validate the design, we simulated datasets of volunteers with the optimised design obtained. we then estimated the population parameters using monolix . for each of the datasets. we compared the estimated parameters obtained with the simulated datasets to the parameters used to build the optimal design. we computed the relative bias as: with n: number of successful estimations among the simulated datasets. h i : parameter value obtained with the ith dataset. h: parameter value obtained with the original dataset. we also compared the observed rse from these simulations with the rse predicted by pfim and the rse obtained with the original dataset. the rse is proportional to ffiffiffi n p , where n is the population size. we can hence deduce the smallest number of participants necessary to obtain rse below %. where rse predicted is the highest predicted rse (here rse for w) with participants and n predicted = and rse min is equal to ae . considering that % of the influenza-like illness cases are not due to influenza virus, the total number of participants should be multiplied by ae . we found that the best design was when all the participants are sampled five times: three times during the second day post-inoculation at : , : , and : hours and twice on the third day post-inoculation at : and : ( figure ). the comparison of the relative bias and rse predicted by pfim and those obtained after simulation and re-estimation of the parameters are shown in figure . v init and d in a lesser extent present bias. fixed effect parameters are precisely estimated and accordingly to pfim except for v init . we found that participants shedding virus or participants with ili symptoms are necessary if % of them are not infected with influenza virus. we propose an optimised design to accurately study the vk of influenza virus with the minimal number of samples. this design is well balanced between the amount of necessary information and the precision of estimation. we found that samples are necessary to precisely fit the vk curves, which is five times less than the number of samples collected in the original study. ??? the samples should be collected during the second and third days after inoculation. yet we showed in a previous work that the incubation period lasted ae days. ??? hence, the optimised sample times correspond to the two-first days of symptoms and this design could be applied to naturally acquired infections studies in which the inoculation time is unknown. an advantage of this design is its practicality and convenience. all samples are collected during the daytime and after the onset of symptoms. it can thus be used for studies with naturally acquired infections. the design was validated with several criteria concerning the accuracy of the estimation with the optimised design. the parameters estimates were generally satisfactory. the parameter describing the effect of the early immune response on the virus production rate was, however, less precisely estimated (predicted rse = %), and the initial value of the viral titre was very different of the one obtained with the original dataset (bias v init on figure ). this is probably due to the fact that it was measured at day post inoculation, and that the inter-individual variability is much higher than at day . furthermore, d (the infected cell mortality rate) seems also to be biased. this may be due to the fact that three parameters were fixed. the model used was developed from experimentally inoculated healthy volunteers with low serum haemagglutinin antibody titre and with virus inoculation time at : am. the applicability of the design to naturally acquired infection would depend on the pathogenicity of the virus as well as pre-existing immunity and the relevance of challenge method to natural influenza acquisition. our design could be directly used to accurately study vk during influenza infections and would reduce the discomfort of patients and the cost of the experimentation. usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test introduction influenza rapid antigen tests (irat) have become very popular and are widely used for confirming suspected clinical diagnosis of influenza in japan. most of the currently used irat that are based on immunochromatography (ic), nasopharyngeal swab, nasopharyngeal aspiration, and throat swab have been approved as specimens for japanese national health insurance purposes. but the specimen collection by these methods gives patients considerable discomfort, and sometimes appropriate specimens cannot be obtained due to patient resistance, especially by children. in the present studies, self-blown nasal discharge was used as the specimen for an irat, and the results were compared with the results of viral isolation and an identical kit primed with nasopharyngeal swab specimens for seasonal influenza viruses and pandemic (h n ) virus. patients who visited any of the clinics that belong to the influenza study group of the japan physicians association in the - and the - influenza seasons with influenza-like illnesses exhibiting findings were registered after providing informed consent. a square plastic sheet of · cm was handed to the patient. nasal discharge was collected by blowing the nose into the plastic sheet as a specimen for irat, i.e. self-blown specimen. two nasopharyngeal swab specimens were also obtained at the same time for irat and virus isolation. self-blown specimens were obtained successfully by ( ae %) of consecutive outpatients in the - season, as seen in table the sensitivity and specificity of various influenza rapid antigen tests have been reported in various settings. [ ] [ ] [ ] [ ] direct comparison of the results is difficult because of differences in patient or influenza virus, characteristics such as age, study designs, and other features. in this study of the - influenza season, the sensitivity, specificity, and accuracy of the ic kit primed with nasopharyngeal swab specimens were ae %, ae %, and ae %, respectively. these results were quite comparable to our results of the - season, in which the overall results of other ic kits were ae %, ae %, and ae %, respectively, indicating that the ic kit used is quite reliable. the sensitivity, specificity, and accuracy of an ic kit will vary by the method of specimen collection. in general, virus titer is considered to be highest with nasopharyngeal aspiration, lower with nasopharyngeal swabs, and lowest with throat swabs. practically, nasopharyngeal swab is the most popular. the sensitivity, specificity, and accuracy of the ic kit with self-blown discharge specimens compared well with those of an identical ic kit primed with nasopharyngeal swab specimens. for self-blown specimens, sensitivity and specificity were ae % and ae % for influenza a, ae % and ae % for influenza b, % and ae % for pandemic (h n ) . self-blown specimens display sensitivity, specificity, and accuracy comparable to that of conventional nasopharyngeal swab specimens. there was no significant difference in sensitivity, specificity, or accuracy between self-blown specimens and nasopharyngeal swab for influenza a, influenza b, and pandemic (h n ) . these results suggest that selfblown specimens are as useful as nasal cavity swab specimens for the diagnosis of influenza in the clinical settings. nasal discharge, obviously, cannot be collected from infants incapable of blowing their own nose or patients who do not develop a nasal discharge. in this study, self-blown specimens were obtained from ae % of the patients. the rate of successful collection was over % in the age groups of - and - years. these rates would seem to be sufficient for clinical use. the procedure of self-blown specimen collection using a plastic sheet is easy and causes no pain or discomfort. it seems to be more acceptable and safe than the other methods, especially for children. furthermore, this procedure reduces the risk of influenza transmission from patients to the medical staff members involved in sample collection. self-blown sample collection may be superior to other sample collection methods in these respects. we previously reported an inverse correlation between the amount of virus in a specimen and the time to a positive reaction. in this study, there was no significant difference in the mean time to a positive between self-blown self-blown specimens enough to be examined were obtained from consecutive outpatients, and specimens showed a tendency to be obtained large amount from children rather than the aged. there were no statistically significant differences between the ic kit results primed with self-blown discharge and nasopharyngeal swab specimens for influenza a, influenza b and pandemic (h n ) . and nasal swab specimens, suggesting that the self-blown specimens contained sufficient viral antigen for the ic kits. the influence of the presence or absence of nasal congestion on the results of the kit was assessed. the sensitivity of selfblown specimens from patients with nasal congestion was significantly lower than that from patients without nasal congestion. it is possible that insufficient capability to blow the nose due to nasal congestion might tend to lead to false negatives. the observation that the time to positive is longer for patients with nasal congestion than for patients without nasal congestion is concordant. application of self-blown specimen collection only to appropriate patients would increase the sensitivity, which would be important in a clinical setting. we tested only two commercial antigen detection kit, the quick vue rapid sp influ kit and quicknaviÔ-flu (denka-seiken co., ltd). the resulting sensitivity, specificity, and accuracy of the ic kit primed with self-blown specimens were considered adequate for clinical use. to confirm the usefulness of self-blown nasal discharge specimens, further investigation is necessary using other kits and in different settings. the usefulness of a self-blown nasal discharge specimen for an influenza rapid antigen test based on immunochroma-tography was evaluated in the - and - influenza season. results suggest that self-blown nasal discharge specimens are useful as specimens for influenza rapid antigen tests based on immunochromatography for not only seasonal influenza viruses, but also pandemic (h n ) virus. the specimen collection by the patients themselves will reduce the burden of other collection methods and the risk of infection to the medical staff. in april , a mixed-origin h n influenza virus was recognized as a new causative agent of influenza-like illnesses (ili) in humans. since its emergence, the virus has spread rapidly throughout the world and caused a pandemic. most commercial rapid antigen tests (rat) can detect influenza a or b viruses, but cannot specifically distinguish pandemic (h n ) virus with seasonal influenza. recent studies have indicated that the poor performance of the rat approach and nonspecific detec-tion of the pandemic (h n ) virus was the main obstacle to their widespread use in private clinics. , with the need for a new rapid kit with reasonable sensitivity and specificity for pandemic (h n ) virus, we developed a new rat kit in collaboration with company, standard diagnostics, inc., (yongin-si, gyonggi, korea). monoclonal antibody (mab) against haemagglutinin (ha) of the pandemic (h n ) virus was developed using korean isolate and applied to the new kit with the mab to seasonal influenza virus. we examined the detection limit of the kit using the serial dilution of korean pandemic virus isolate (a ⁄ korea ⁄ ⁄ ). during december , clinical specimens from patients with ili were collected at sentinel clinics of six provinces in korea. the specimens were tested by the new rat, and the results were compared with those of real-time reverse transcription polymerase chain reaction (rrt-pcr) by us cdc and virus isolation in mdck cell culture to determine the sensitivity and specificity for the diagnosis of pandemic (h n ) . the detection limit of the new kit against ha of a ⁄ korea ⁄ ⁄ virus was confirmed to be pfu ⁄ ml. by contrast, the detection limit against the np protein was pfu. however, when the kit was applied to clinical specimens, no difference between the two targets was found. using rrt-pcr and viral culture as the references, the performance of the ridt is shown in table . among specimens, were tested positive by rrt-pcr and were tested positive by viral culture. among the rrt-pcr confirmed cases, were positive, and among the viral culture confirmed cases, were positive with the new rat. using rrt-pcr as the reference standard, the overall sensitivity of rat was ae % ( % confidence interval (ci): ae - ae %) and specificity was ae % (ci: ae - ae %). with viral culture as the reference, the rat sensitivity and specificity was ae % (ci: ae - ae %) and ae % (ci: ae - ae %), respectively. when analyzed by the regions tested, the sensitivity ranged between ae % and ae % for rrt-pcr and between ae % and ae % for viral culture as a reference. among patients who had a record of their symptom onset and sample collection date, ( ae %) visited the clinic on the day of symptom onset, and ( ae %) visited day later. when the rat performance was evaluated by day of onset, the sensitivity was lower at three or more days after the onset of symptoms; however, the sensitivity was highest at days after onset and reasonable on the day of onset or at day after ( table ). we found that this new rat had reasonable sensitivity and high specificity compared with rrt-pcr and viral culture for detecting the pandemic (h n ) virus. in one recent study, the sensitivity and specificity of the new rat kit was % and %, respectively, and the ha protein for pandemic (h n ) was detected more sensitively than the np protein for influenza a virus. the sensitivity and specificity of our new rat were lower than those of that study. we found that the test performance varied depending on the clinics in which the tests were performed, and this might be attributable to the persons who collected the specimens. although the clinicians were trained well for *ci, confidence interval. **ppv, positive predictive value. ***npv, negative predictive value. collecting specimens, there might be some differences in performance. the new rat kit could detect pandemic (h n ) virus specifically. although the sensitivity was lower than those of rrt-pcr and virus culture, and negative rat results should be confirmed with more sensitive methods, this kit could be useful in sentinel clinics if used with caution. determination of infectious virus titres is central to many experiments designed to study the biology of influenza virus. assays based on the measurements of viral components, whether viral protein or nucleic acid, does not differentiate infectious virus from non-infectious or defective viral particles, which may have no infectivity or biological *three hundred and forty samples with a known date of onset and sample collection were analyzed. ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - activity. therefore the ''gold standard'' of virus measurement requires bioassays that examine the ability of viral particles to replicate and further infect other cells. titration on madin-darby canine kidney (mdck) cells in a well plate format is commonly used to measure influenza virus titre. this method is labour intensive, subjective in their read out of cytopathic effect, and takes several days to obtain a result. microneutralization tests that quantitate neutralizing antibody titres and assays of drugs for antiviral activity also require well based assays of residual virus infectivity. therefore, technologies that improve on the titration of infectious virus will be of great benefit. this study utilized the xcelligence system (roche applied science), which adopts microelectronic biosensor technology to monitor dynamic, real-time label free and non-invasive analysis of cellular events. the system measures electronic impedance using an array of microelectrodes located at the bottom of each culture well (e-plate ). adherent cells are attached to the sensor surface of electrode arrays, and changes in impedance can be detected and recorded. the xcelligence system can monitor cell events induced by viral infection, such as changes in cell number, adhesion, viability, morphology, and motility. measured electrode impedance is expressed as dimensionless cell index and is graphically represented using software to show the phenotypic changes of a cell population over time. the aim of this study is to demonstrate that using this platform to measure real-time cell index has potential to circumvent many of the limitations of the currently established procedures of end point titration of virus infectivity and for microneutralization assays. madin-darby canine kidney cells were propagated in growth medium consisting of minimum eagle's medium (invitrogen) supplemented with % fetal bovine serum (invitrogen), ae mg ⁄ l penicillin (invitrogen), and mg ⁄ l streptomycin (invitrogen), with incubation at °c in a % co humidified atmosphere. influenza a ⁄ hong kong ⁄ ⁄ (h n ), a seasonal influenza virus from a patient who suffered from a mild febrile illness, was propagated in mdck cells maintained in virus medium consisting of minimum eagle's medium (invitrogen) supplemented with ae mg ⁄ l penicillin (invitrogen), mg ⁄ l streptomycin (invitrogen), and mg ⁄ l np-tosyl-l-phenylalaninechloromethyl ketone-treated trypsin (sigma, st louis, mo, usa), with incubation at °c in a % co humidified atmosphere. virus stocks were aliquoted and stored at °c until use, and the % tissue culture infectious dose (tcid ) of the virus stock was determined by titration in mdck cells according to standard procedures, and the tcid of the stock virus was calculated by the method of reed and muench. to perform a microneutralization assay, mdck cells seeded at a density of cells ⁄ well in an e-plate was removed from the xcelligence system after approximately hour; growth medium was then removed, cells washed, and replaced with ll virus-medium. a human serum, which is known to contain high titre antibody against the h n virus was heat inactivated for min at °c, and twofold serial dilutions were performed in virus medium. the diluted serum was mixed with an equal volume of virus medium containing influenza virus at tcid ⁄ ll. after incubation for h at °c in a % co humidified atmosphere, ll of virus-antibody mixture was added to the mdck cells to give each well an equivalent virus dose of tcid . a back titration of the virus challenge dose was performed, and a cell control (free of virus) was performed in quadruplicates. after incubation at room temperature for minutes, the e-plate was then placed back onto the xcelligence system in the incubator and maintain at °c with % co , and the cell index values were measured every minutes for at least a further hour. the same procedures were performed with cells seeded in conventional well cell culture plates for parallel comparison with the currently used standard method. in this case, cells were examined for cytopathic effect under an inverted microscope after days of infection and the lowest virus dilution, which protected the cells from viral induced cytopathic effect taken as the neutralizing end point. after hour of seeding mdck cells at cells ⁄ well, standard microneutralization assay for influenza virus was performed. integral to this assay, a serial titration of the input virus at ae log increments was carried out. wells infected with the undiluted virus ( tcid ⁄ well), the cell index commenced dropping at a steeper gradient than the no-virus cell control after approximately hour of infection ( figure ). this drop in cell index continues at a consistent slope until it flattened out when approaching zero cell index. this steep decrease in cell index with constant gradient was also observed for virus dilutions up to and including log ( -folds), and the profile shifted with increased time in proportion to the dilution made to the virus. virus dilutions beyond log have cell index profiles similar to the no virus input control, and this corresponds to the absence of cytopathic effect as determined by microscopic observation at hour after infection. hence, there was a correlation between the amount of virus used for infection, the onset of the influenza virus-mediated cytopathic effect, and the steep decline in cell index. a human serum with known microneutralization antibody titre to h n virus was used in this study to investigate the real time cell index changes that occur during the assay ( figure ). using influenza virus treated with serum dilutions up to and including a dilution of : , the cell index profile remained essentially the same as the no virus cell control, which correlates with the lack of cytopathic effects under microscopic observation at hour of infection. at a serum dilution of : , the steep decrease in cell index, which is characteristic of cellular cytopathic effect induced by the virus, became evident at around hour post infection, and this was reduced to hour when serum dilution of : was used. in contrast, for the virus -no antibody control, the onset time for this steep decrease in cell index occurs at approximately hour. for both serum dilutions of : and : , full cytopathic effect was observed microscopically at hour of infection. from microscopic observation of cytopathic effect, according to the current standard procedures, the neutralizing titre of the human serum used in this study is at : as it is the last dilution of the serum that prevented cytopathic effect from being detected. an essential part of the microneutralization assay is to confirm the titre of the input virus (normally tcid ⁄well) by performing a titration assay with decreasing serial dilutions of the virus. under normal procedures, cells are examined microscopically after hour of infection for sign of cytopathic effects. in the case of mdck cells, the cytopathic effect is cell death, which is indicative of the presence of live influenza virus infecting and replicating in the cells. therefore, the titre of the virus is taken as the last dilution in which cytopathic effect is present. parallel realtime cell index measurements demonstrated that for wells with cytopathic effects, the profile exhibits a steep gradient linear decrease in cell index after infection with the virus, which can be termed the ''cpe plunge.'' the time in which the cpe plunge became evident appears to be inversely proportional to the amount of virus, therefore the opportunity exists to utilize this aspect to calculate or compare quantitatively different virus concentrations. for unequivocal assignment of cytopathic effect, it normally requires - days after infecting the cells, with days after infection being the standard time to read virus titration and microneutralization assays. using the real-time cell index monitoring, it is found that apparent cytopathic effect can only be observed microscopically when the cell index has dropped to near zero. as the time of onset of the ''cpe plunge'' becomes evident many hours prior to observable cytopathic effect, it is possible that the time to results can be drastically reduced after some formulation of the method. we compared the current standard method in perfoming a microneutralization assay with one utilizing the real-time cell index measurement to investigate whether this approach is able to offer better performance over the existing one. the current standard neutralization assay is the microscopic observation of antibody mediated protection from virus cytopathic effect in mdck cells. this study showed that this may also be achieved by examining the profile generated from the real-time measurements of the cell index. using real-time cell index monitoring, it is possible to detect inhibitory activity at higher dilutions of the anti-serum than can be detected by the standard microscopic observation of cytopathic effect. therefore, the realtime cell index monitoring could potentially be developed to be a more sensitive method for measuring anti-viral activity. as drug resistant strains of influenza a viruses including the pandemic h n are being reported, the real-time cell based monitoring system may also have the potential to be developed for use as a diagnostic platform for drug resistance assays. this study suggests that real-time cell index monitoring has the potential to substantially reduce human resources in reading results, as well as reducing time-to-result of these assays from days to two. the saving could be substantial for work involving bio-hazard level ⁄ pathogens such as h n viruses as personnel working with these organisms are require to be highly trained and experienced. in addition, the reduction in transferring plates to and from the microscope in reading cytopathic effect will substantially reduce the possibility of accidents from occurring. furthermore, the system provides objective digital data to an otherwise subjective assay method, which can improve standardization, data exchange, and hence collaboration between different laboratories. with more detailed validation and development, real-time cell index monitoring could transform the way we study and diagnose infection with pathogens such as influenza viruses. the emergence of a novel h n influenza a virus of swine origin, the pandemic a(h n ) , with transmissibility from human to human in april posed pandemic con-cern and required modifications to laboratory testing protocols. a new protocol for universal detection of influenza a and b viruses and simultaneous subtyping of influenza a (h n ) virus, composed of two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h n v (relab, italy), was evaluated and compared to the reference protocol recommended by who. fast set infa ⁄ infb was able to detect influenza a and b viruses circulating between and belonging to different subtypes and lineages, and no cross reactions were observed by either fast set infa ⁄ infb or fast set h n v. the who assay was found to have a slightly lower end-point detection limit ( ) dilution) in comparison to the new protocol ( ) ). specificity of the assays was % as assessed on a panel of stored clinical samples including adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, s. pneumoniae, n. meningitidis, h. influenza, and human influenza viruses. the new assay panel allows the detection, typing, and subtyping of influenza viruses as requested for diagnostic and surveillance purposes. the high sensitivity of the protocol is coupled with capacity to detect viruses presenting significant heterogeneity by fast set infa ⁄ infb and with high discriminatory ability by fast set h n v. a rapid and sensitive assay for the detection of influenza virus in clinical samples from subjects with ili or low respiratory tract infections is a fundamental tool for epidemiological and virological surveillance, management of hospitalized patients, and control of virus nosocomial transmission. the emergence, in april , of a novel h n influenza a virus of swine origin, the pandemic (a(h n ) ), with transmissibility from human to human poses pandemic concern and required modifications to the laboratory testing protocols. molecular diagnosis of influenza is generally achieved through a twophase process: a screening phase for the detection of virus, and the subsequent strain characterization performed by either sub-type-specific rt-pcr or entire ⁄ partial genome sequencing. during a pandemic, simultaneous implementation of both the detection of influenza a and b influenza viruses and identification of the new subtype is useful for clinical and epidemiological reasons. here, we describe a new protocol including two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h n v (relab, italy) that allows universal detection of all influenza a viruses and, simultaneously, all subtypes that are influenza a(h n ) . specificity and clinical sensitivity of the two-one-step rt-pcrs (fast set infa ⁄ infb and fast set h n v; relab, italy) were evaluated by testing selected specimens, including: • fifty samples collected from nasopharyngeal swabs representative of influenza viruses, belonging to differ-ent subtypes and lineages, and other respiratory viruses and bacteria circulating in italy between and . • six purified a(h n ), a(h n ), and a(h n ) strains, kindly supplied by alan hay, who influenza centre, london, uk. • two hundred-fifty influenza positive samples selected according to type, subtype, clade and viral concentration from > specimens received by the liguria influenza reference laboratory between january st and december st, . since , nasopharyngeal swabs sampled from patients suspected of having contracted the influenza virus have been collected in viral transport medium, and upon arrival into the laboratory, the samples were divided in ‡ aliquots. those not immediately processed were stored frozen at ) °c. stored samples were used for this evaluation, and all specimens were re-extracted for the study. samples collected between and included specimens positive for: no seasonal a(h n ) have been detected since january st, . furthermore, weak positive sample using fast set infa ⁄ infb, but negative at block pcr and typing ⁄ subtyping assays was tested. the analytical sensitivity of the test under investigation was determined testing ten-fold serial dilutions of seasonal influenza a(h n ), seasonal influenza a(h n ), new pandemic influenza a(h n ) , and b cell culture-grown viruses. the intra-assay reproducibility was measured by testing the same a(h n ) positive sample times in the same experiment, while the inter-assay reproducibility was confirmed by testing the same samples in independent experiments. to evaluate the performance of the protocol, all samples were tested using a block pcr confirmation test (seeplex Ò rv ace detection), and all specimens collected between january st and december st, and dilutions were also assayed using the recommended who ⁄ cdc protocol of real-time rtpcr for influenza a(h n ). typing and sub typing were performed using the who protocol and ⁄ or sequencing. viral rna was extracted from swabs using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's protocol. fast set infa ⁄ infb and fast set h n v are two multiplex one-step real time pcr assays developed and evaluated by the liguria regional reference centre for diagnosis and surveillance of influenza in collaboration with relab diagnostics. both assays contain primers and a dual-labelled hydrolysis probe that targets two regions of the matrix gene (table ) . amplification conditions were as follows: reverse-transcription °c for minutes, denaturation °c for minutes, then cycles of °c for seconds, °c for seconds. the entire amplification process extended for minutes. an internal control real-time assay was also incorporated in order to detect pcr inhibition, failed extraction ⁄ pcr and technical error. the cdc realtime rtpcr (rrtpcr) protocol for detection and characterization of swine influenza includes a panel of oligonucleotide primers and dual-labelled hydrolysis (taqman Ò ) probes to be used in real-time rtpcr assays for the in vitro qualitative detection and characterization of swine influenza viruses in respiratory specimens and viral cultures. this protocol recommends three primer-and-probe sets: infa, amplifying a conserved region of the matrix gene from all influenza a viruses; sw infa, designed to specifically detect the nucleoprotein (np) gene segment from all swine influenza viruses and sw h , designed to specifically detect the hemagglutinin gene segment from a(h n ) . the seeplex Ò rv ace detection for auto-capil-lary electrophoresis is a multiplex block rt-pcr that applies dpoÔ (dual priming oligonucleotide) technology and is designed to detect major respiratory viruses, respiratory rna (influenza a and b virus, parainfluenza virus type , and , respiratory syncytial virus a and b, rhinovirus a ⁄ b, coronavirus oc and e ⁄ nl ) viruses and dna (adenovirus) virus, from patients' samples including nasopharyngeal aspirates, nasopharyngeal swabs and bronchoalveolar lavage. conventional viral culture was performed inoculating ae ml of each specimen into mdck-siat seeded into -well plates for influenza isolation. virus detection was performed by the hemagglutination test using ae % guinea pig red blood cells (rbc). specificity and clinical sensitivity results of the new protocol are reported in table . fast set infa ⁄ infb was able to detect influenza a and b virus circulating between and belonging to different subtypes and lineages, and no cross-reactions were observed by either fast set infa ⁄ infb or fast set h n v. among specimens collected between january st and december st, , all fast set infa ⁄ infb and fast set h n v high titre positive samples resulted positive using the who ⁄ cdc assay and showing reactivity using infa and sw infa primer-andprobe sets. among low titre a(h n ) positive samples at fast set infa ⁄ infb, ( ae %) were not detected by the who ⁄ cdc assay, but were positive using seeplex Ò rv . the who ⁄ cdc sw h primer-and-probe set works in ae % ( ⁄ ) and ae % ( ⁄ ) of high and low titre a(h n ) positive samples, respectively. all a(h n ) strains collected during and initially detected by fast set infa ⁄ infb were confirmed after rna re-extraction by seeplex Ò rv and who ⁄ cdc assay showing reactivity using the infa primer-and-probe set. all infa ⁄ infb were confirmed after rna re-extraction by seeplex Ò rv . one influenza a case identified by the who ⁄ cdc kit (infa primer-and-probe set, ct values: ae , sw infa primerand-probe set: negative) and new protocol (a primer-andprobe set, ct values: ae , a(h n ) primer-and-probe set, ct values: ae ) was not detected by either seeplex Ò rv or by who subtyping protocol and ⁄ or sequencing, suggesting a very low viral load or unspecific results by real time assays. the analysis of serial dilutions of cell culturegrown a(h n ) showed that the detection limit of fast set infa ⁄ infb, fast set h n v, and seeplex Ò rv was identical ( ) ) and log lower than that using the who ⁄ cdc protocol ( ) ). a similar analysis with respect to a(h n ) and a(h n ) strains indicated that fast set infa ⁄ infb sensitivity ( ) and ) , respectively) was log lower than that showed by seeplex Ò rv ( ) and ) , respectively). in comparison with the new protocol, the who ⁄ cdc assays, considering infa primer-and-probe set, was found to have a slightly lower end-point detection, detecting the ) a(h n ) and a(h n ) dilution. also in detecting influenza b virus, fast set infa ⁄ infb sensitivity ( ) and ) , respectively) was log lower than that showed by seeplex Ò rv and the who ⁄ cdc protocol. data on intra-assay and inter-assay precision, measured as cv% of ct showed that the dispersion indices observed had values of less than %. since samples were detected using the new protocol that resulted negative using the who ⁄ cdc assays. the unfortunately low quantity of low titre a(h n ) samples collected during did not allow us to highlight differences between assays fast set infa ⁄ infb, and fast set h n v positivity was always confirmed by seeplex Ò rv , which demonstrated high sensitivity, showing a detection limit comparable or lower when compared with those observed using the who ⁄ cdc assays. the high analytical sensitivity of seeplex Ò rv is reported by kim who observed a detection limit of copies per reaction for each type ⁄ subtype of influenza viruses. the high sensitivity of the new protocol is coupled with its capacity to detect viruses presenting a significant heterogeneity by fast set infa ⁄ infb and high discriminatory ability by fast set h n v. fast set infa ⁄ infb was able to identify representative influenza viruses of circulating strains during the last decade belonging to different subtypes, lineages, and clusters, and fast set h n v primerand-probe set reacted selectively with a(h n ) target. a recent report demonstrated that the sw infa assay is not specific to a(h n ) and is able to detect both human and avian (h n ) influenza a viruses and so there is the potential for misidentification. high titre (ct ae and ae at fast set infa ⁄ infb) a(h n ) viruses did not react with fast set h n v primer-and-probe set (data not shown). available human a(h n ) sequences are similar within the h n v primer-and-probe regions, but having - mismatches in the forward primer and, more notably, two of the mismatches occurred within nucleotides of the end, an important determinant for primer specificity. in conclusion, this protocol can be a powerful tool in the diagnostic laboratory setting for specific simultaneous analysis of several samples in minimal time, showing enhanced sensitivity in detecting influenza viruses, and high discriminatory ability in identifying the new pandemic a(h n ) . a university-corporate partnership to enhance vaccination rates among the elderly: an example of a corporate public health care delivery public health campaigns usually rely on governmental infrastructure and finance for vaccine implementation programs. however, there are many financial and physical barriers which preclude widespread and effective vaccine administration, especially among the elderly. on an international scale, both government agencies and citizen groups have a vested interest in searching for more resourceful methods of attaining significant immunization levels (> % of the population). in fact, it seems to have become both a grassroots civic and governmental goal, especially among developing countries. we implemented the unique strategy of enlisting the assistance of a privately-owned food market chain to address the public health issue of mass vaccination for the elderly. in this context, publix pharmacy and the university of south florida (usf) recently developed both a handbook and a training program to facilitate the administration of vaccinations. between and , the publix-usf partnership resulted in administration of over thirty thousand influenza a (h n ) vaccinations, % of which were given to adults over years of age. consequently, vaccine administration costs were decreased by using corporate resources and bypassing overly strained municipal resources. this unique university-corporate partnership successfully delivered h n vaccine to a vulnerable cross-section of society at a lower cost and with minimal side effects and morbidity. it may be safely projected that university-corporate partnerships could result in an effective method for rendering a vital service to an aging and especially vulnerable segment of the population. government policy and funding are the foundation of immunization programs on an international scale. for example, in the united states, governmental programs account for over % of the monetary outlay used for immunization. until , the global alliance for vaccines and immunizations (gavi) acted as a catalyst for implementing vaccine and immunization programs in each targeted country. under the auspices of gavi-collaborations between governments, charitable organizations, and multinational health agencies (such as uncief and the who)-many countries have increased their spending for vaccination programs. however, development of financially sustainable immunization programs geared toward reaching the majority of the population are still at a nascent level of evolution. the development of more innovative and costeffective approaches has become imperative in order to reach a greater number of vaccination candidates. administering the influenza vaccine only to the subpopulation of over year olds would save an estimated quality-adjusted life years in a cohort of approximately half the world's population. widespread public vaccination programs are made more complex by the continuing development of newer vaccines, concomitant specialized administration costs, and the logistical challenge of conveying recipients to vaccination points of service. , in spite of the increasing complexity of mass vaccination, cost-benefit analyses clearly favor annual influenza vaccination in the elderly population on an international scale. , recently, in , influenza vaccine administration was reported to reach between % and % of the elderly population, which denotes varying degrees of success within each particular country. , however, there was also a report of a uniform plateau effect at around % of the population, beyond which additional vaccination coverage was difficult to achieve. physical limitations to vaccination seem to be more insurmountable for the elderly. unfortunately, this is the population segment which could experience the most significant vaccination-associated mortality reduction. we employed the unique strategy of involving the resources of publix supermarkets, a corporate food market chain, to address the public health issue of widespread vaccination for the elderly. we took advantage of recent changes in the florida statutes, which expanded the scope of pharmacists' practice to include administration of vaccines. subsequently, publix pharmacy and the university of south florida (usf) developed a handbook and training program to facilitate and enhance vaccine administration by publix pharmacists. by using proprietary pharmacists and more practical supply storage, we were able to decrease the costs of vaccine administration. the consumer was charged $ for administration costs plus the cost of the injection itself, regardless of insurance or eligibility for governmental subsidy. although patients were initially self-selected, they were ultimately excluded if they had demonstrated prior adverse effects to influenza vaccinations or to any of the components of such vaccinations. between and , the publix-usf partnership vaccinated people against influenza a (h n ), of which were florida residents. the age range was - years old with a median age of years old. seventysix percent of the participants were over years old (see figure ). within the population surveyed, the reported side effects of the vaccine in this study were not serious, but included: vertigo, cold sweats, chills, vomiting, syncope, rash, nausea, stomach pain, elevated blood pressure, injection site reaction, inflamed bursa, and bilateral thigh discomfort. participants from all socioeconomic classes were vaccinated. an income-by-zip code analysis revealed % of those vaccinated resided in zip code areas where the average household income was <$ per year. of those remaining, % had an average income of $ -$ per year, and % had an income of >$ per year. each person vaccinated was charged ten dollars for administration costs. this represents a decrease in the administration costs ranging from one dollar to ten dollars saved per vaccine. , conclusion this unique university-corporate partnership successfully delivered h n vaccine to a high-risk population with decreased vaccine administration costs. the influenza vaccine is well-tolerated, with minimal side effects when patients who have a history of adverse reactions are excluded. we can postulate that university-corporate partnerships may indeed be effective at reaching the aging population which is a challenge in most communities. this delivery model may prove to be another tool for improving the efficiency of mass immunization by facilitating accessibility, which results in wider coverage. this model also enhances delivery of healthcare by decreasing costs of immunization regardless of whether the payer is a government, insurance company, or self-pay consumer. the gavi initiative stressed three goals for accomplishing sustainability and independence in immunization programs. the goals were to: (i) mobilize additional resources from governmental and non-governmental sources; (ii) improve program efficiency to minimize additional administration resources needed; and (iii) increase the reliability of funding. empowering privately owned corporations within the community, such as food markets or pharmacies, to administer vaccines mobilizes additional resources to readily achieve the first goal of gavi. mobilizing resources of non-healthcare, corporate vaccination locations enhances accessibility due to travel convenience. in our study, participants came from all socioeconomic classes, suggesting that ease of access is independently hindering mass vaccination, and that people of all incomes are more likely engaged when access issues are eliminated. the second and third goals were also accomplished by recruiting a corporation's resources for vaccine administration (refrigeration, storage, and employees). this minimizes the money spent from vaccine program funds to support the infrastructure of immunizations, thus improving financial efficiency and sustainability. financial efficiency implies that money is spent to safely reach as large a portion of the population as possible. by using corporate storage facilities instead of paying for independent facilities, money can be spent elsewhere. more vaccines can be purchased and more money can be spent on media communications to encourage vaccination. sustainability requires the ability to fund annual vaccination programs which reach % of the population or greater. key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. in addition, they must minimise false alarms during a normal influenza season. we develop a method that uses historical syndromic influenza data from the existing surveillance system 'servis' monitoring seasonal ili activities in scotland. we develop an algorithm based on wcr of reported ili cases to generate an alarm for pandemic influenza. wcr is defined as the ratio of the number of reported cases in a week to the number of cases reported in the previous week. from the seasonal influenza data from scottish health boards, we estimate the joint probability distribution ( figure ) we compare our method, based on our simulation study, to the mov-avg cusum and ili rate threshold methods and find it to be more sensitive and rapid. the wcr method detects pandemics in larger fraction of total runs within the same early weeks of pandemic starting than does any of the other two methods ( figure ). as shown in the table, for % pandemic case reporting rate and detection specificity of %, our method is % sensitive and has mdt of weeks, while the mov-avg cusum and ili rate threshold methods are, respectively, % and % sensitive with mdt of weeks. at % specificity, our method remains % sensitive with mdt of weeks. although the threshold method maintains its sensitivity of % with mdt of weeks, sensitivity of mov-avg cusum declines to % with increased mdt of weeks. for a two-fold decrease in the case reporting rate ( ae %) and % specificity, the wcr and threshold methods, respectively, have mdt of and weeks with both having sensitivity close to %, while the mov-avg cusum method can only manage sensitivity of % with mdt of weeks. the first cases of the pandemic were reported in scotland in the th week of the season. the wcr algorithm as well as the mov-avg cusum method detects the pandemic weeks later in week . the ili threshold method detects it week later in week . both the wcr and mov-avg cusum methods therefore outperform the ili threshold method by week in the retrospective detection of the pandemic in scotland. while computationally and statistically very simple to implement, the wcr method is capable of raising alarms rapidly and sensitively for influenza pandemics against a background of seasonal influenza. although the algorithm has been developed using the servis data, it has the capacity to be used at large scale and for different disease systems where buying some early extra time is critical. more generally, we suggest that a combination of different statistical methods should be employed in generating alarms for infectious disease outbreaks. different detection methods would provide cross-checks on one another, boosting confidence in the outputs of the surveillance system as a whole. real-time evidence being created worldwide will greatly contribute to the full understanding of influenza pandemics. here we report the real-time epidemiology and virology findings of the influenza a(h n ) pandemics in mongolia. the epidemiological and virological data collected through isss of nic, nccd, mongolia (real-time information on registered ili cases and virological laboratory results are available from the weekly updates in the nic, mongolia website: http://www.flu.mn/eng/index.php?option=com_ content&task=category&sectionid= &id= &itemid= ) were used for analysis in relation to the previous seasonal influenza activities in the country. influenza viruses were detected in naso-pharyngeal samples from ili patients by rt-rt-pcr with applied biosystems fast real time pcr system , using primers and instructions supplied by cdc, usa. influenza viruses were isolated by inoculation of rt-rt-pcr-positive samples of mdck cell culture according to the standard protocol. ten representative strains of a(h n )pdm viruses were selected for sequencing of different gene segments, namely: a ⁄ ula- , and a ⁄ dundgovi ⁄ ⁄ . sequencing of influenza virus gene segments was performed in applied biosystems xl genetic analyzer using primers and instructions supplied by cdc, usa, and bioinformatic analysis was performed with abi ⁄ seqscape v. . and mega programs. the pandemic alert in mongolia was announced by the government on april , , just after the who announcement of the pandemic alert phase, and planned containment measures were intensified. despite intensive surveillance, no a(h n )pdm virus was detected in mongolia until the beginning of october . around suspected cases, mostly arriving from the a(h n )pdm epidemic countries, tested zero by rt-rt-pcr for a(h n )pdm virus. the first a(h n )pdm case detected by the routine surveillance system in ulaanbaatar city, the capital of mongolia, was confirmed by rt-rt-pcr on october , ( st week of ). the reported ili cases escalated rapidly, reached the peak in the - th week of , and gradually decreased thereafter ( figure ). week of . however, the registered ili cases increased again from the th week of , and peaked at the - th weeks of . the viruses isolated during this nd peak were influenza b strains ( figure and table ). for the genetic characterization of the mongolian pandemic isolates, gene segments i (pb ), gene segments ii (pb ), gene segments iii (pa), gene segments iv (ha), gene segments v (np), gene segments vi (na), gene segments vii (m), and gene segments viii (ns) of the representative a(h n )pdm mongolian strains were sequenced, and all sequences have been deposited in the genbank (accession numbers: cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy ). all genes of mongolian strains were possessing ae - ae % similarity with the genbank deposited gene sequences of the original pandemic strain a ⁄ california ⁄ (h n ). the who declared the pandemic alert phase (phase iv) on april , , and was prompted to announce the pandemic phase (phase v) two days later. after days, the who declared the beginning of the pandemic peak period (phase vi) on june , . however, in mongolia, the pandemic alert period continued for days. mongolia was free of the pandemic virus during the whole first wave of the pandemics in the northern hemisphere. with the confirmation of the st influenza a(h n )pdm case on october , in ulaanbaatar, mongolia entered into the pandemic phase (phase v), and after just weeks, the registered ili cases peaked, confirming mongolia shifted into the pandemic peak period (phase vi), which i i i v i i v i v v v i i i i iii iv v vi vii viii worldwide by who in mongolia coincided with the nd wave of pandemics in many countries of the northern hemisphere (see, picture and table ). despite the relatively milder clinical manifestations, the disease burden for the health service was enormous, while the morbidity per population at the peak period was - times higher above the upper tolerant limit, and - times higher above the seasonal influenza outbreaks. in contrast to the seasonal influenza outbreaks where over % of the registered ili cases have been in the age group under , it has been observed that over % of the registered cases in this pandemic peak period were in the age group of - . on january , , we regarded the pandemic had entered into the post-peak period (phase vii) when the registered ili cases became lower than the upper tolerant limit, during which time mongolia experienced an influenza b outbreak. on may , we determined that mongolia entered the post-pandemic period (phase viii) as the influenza virus isolations were almost stopped, and after no pandemic virus detected for months. who announced pandemic vii and viii phases much later. , this first ever real-time laboratory confirmed influenza pandemics in mongolia and confirmed some variations of pandemic spread in different parts of the world. the comparison of deduced amino-acid sequence changes have shown that the mongolian strains belong to the clade , according to the classification of a(h n )pdm influenza strains suggested by m. nelson, which has circulated worldwide since july . this is also evidence that the st wave of the pandemics did not hit mongolia. the who public health research agenda for influenza is aimed to support the development of evidence needed to strengthen public health guidance and actions essential for limiting the impact of influenza on individuals and populations. each stream-specific group reviewed and discussed the proposed organization, content, rationale, and global health importance of their designated research stream. specific research recommendations were made for topics within each stream: background: a syndromic surveillance system using nonclassical data sources for detection and monitoring evolution of flu and flu-like illness (ili) in djibouti is reported here as part of the preliminary report of djibouti who-copanflu international study (wcis)**. methodology: clinical reports, over-the-counter drug sales, lab diagnosis report, and health communication trends were obtained for an integrated statistical analysis. results: transition to winter is concomitant with upsurge of ili cases and ili drug sales. in addition, more rural folks manage ili infections on self medicament than through clinical consultancy. inefficient and vague data collections were observed. a successful implementation of wcis will create a platform upon which challenges faced in djibouti health department in routine surveillance will be addressed to achieve a near-real time surveillance of flu pandemic. conclusion: innovations, prompt reporting, and instituting open source syndromic surveillance system software's in resource limited environment like djibouti will enhance early detection and evolution monitoring of pandemic flu. the spanish flu in ⁄ infected and killed millions of people, and threatened to wipe humanity off the face of planet. however, the recent scenarios of influenza h n ( ) pandemics' worldwide occurrence fell short of most scientific prediction on its magnitude and intensity. this dampened their confidence; they cannot state precisely as to when, how, where, and which of the spanish flu-like pandemic will occur in the future. in support of scientific community and governments, the who hasn't gone to slumber, but is reminding its member states to up their post pandemic surveillance and monitoring of influenza virus in circulation for advance preparedness in case of an outbreak. despite all uncertainty around the pandemic flu h n , there remains a common knowledge and understanding that this flu has shown a great potential to evolve and cause huge morbidity and mortality. although its future magnitude may be unpredictable, its recurring events have severe consequences on human health and the economic well being of everyone. and therefore, advance planning and preparedness is critical in protecting any population in the future, especially those located in resource limited environment without universal health cover and generous disaster emergency funds. . two collapsed sets of a weekly and monthly mean data (of four years period) were clustered in five categories of ili cases, drug sales, lab results, vaccine consumption, and health promotion. this was followed by a descriptive statistics analysis of cumulative weekly and monthly data to establish presence or absence of trend. time series analysis was not done due to data limitation. copanflu program: as at the time of going to press, the cohort study is at the household recruitment and inclusion phase and the study covers the djibouti city. it is in our intention to use the cohort study findings to validate or improve the niph ministry of health djibouti ili surveillance effort for better preparedness. clinical service: % of all health facilities are in djibouti city. of the ae % ( ) of the population that seeks medical care on influenza and influenza like illness each year, ae % ( ) and ae % ( ) of them are attended to at the city's public and private clinics, respectively ( figure ). the rest are attended from the regional health centers. the majority of ili incidence sharply rise with the onset of the winter season (october to april), affecting mostly the middle age group ( - years). pharmaco-surveillance: % ( ) of total prescriptions were antipyretic and antiflu drugs, ae % ( ) of which were consumed by peripheral regions, the non dji- lab diagnostics: the annual ili lab diagnosis was negligible ae % ( ), which can be attributed to less equipped virology laboratories to warrant routine service utility. documented cases were from previous bouts of avian influenza that had a human incidence from and . with support of egypt-based naval army medical research unit three (namru ), clinicians were motivated to sample all ili patients and submit to collaborating international reference influenza lab in cairo, egypt. vaccination: influenza vaccinations were undocumented, but at least ae % ( ) of population sought the service (for yellow fever and meningitis) as mandatory travel advisory or as childhood immunization need. at the time of going to the press, there were at least vaccine doses of h n ( ) virus donations yet to be administered. health promotion and hygiene: print and audiovisual risk communication remained favorite means of reaching out to urban dwellers ( ae %). while to the rural and nomadic population, person to person communications was the preferable means. to increasing public awareness that will encourage reporting of ili cases and entrench risk aversion health behavior that limits flu spread, who-copanflu international study djibouti has incorporated basic training on ili infection and personal hygiene by interviewers during household inclusion. improving national epidemic surveillance capacity and response under new international health regulation is important for any nation, including djibouti. our finding indicates the winter season predisposes one to ili infections; they therefore opt for medical services or self medication depending on their capability and ⁄ or understanding. in djibouti, almost no city dwellers favors self medication over clinical consultation, suggesting the presence of inhibitory factors like distance from the health centers and the cost of accessing consultancy. common in the absence of universal primary health care setting, it therefore calls for active innovativeness in outbreak detection, disease reporting, and preventive medicine on the part of health authority so as to achieve good population health. in respond to these, niph has turned resource limitation to a motivation instead and is working towards institutionalizing a near-real-time syndromic surveillance system as a core functional unit. it capitalizes on three major aspects within its reach: prompt accurate data generation for analysis, ehesp wcic-study input, and information technology use. prompt accurate data generation for analysis: data used in our analysis suffered from un-timeliness (weekly instead of daily basis), incompleteness (vague over-counter drug sales records), entry errors (incidence case reports), and poor collection format (most of data collection forms). use of satellite handset phones for regional health centers and mobile phones for city sentinel clinics will reduce unnecessary data delivery delays. in addition, creating awareness to data entry personnel on the importance of careful and completeness of entries is important, as is the need to reformat data collection forms to capture exact aspects of surveillance needs for relevant executable analysis. besides alerting for immediate impending epidemics, these data can also be adopted for projective predictive modeling of annual epidemics, including that for influenza. ehesp wcic-study input: djibouti wcic-study is complementary to the existing syndromic surveillance system, but with emphasis on flu and flu-like illness. various innovations as suggested above are used in seeking to overcome the prevailing challenges. while every attempt is made to realize its (wcic-study) objective and for global comparison, lessons learned from successful implementation will form a platform for future refined syndromic surveillance protocol as equally reported elsewhere in asian countries. , information technology: national institute of public health djibouti has an informatics department with sufficient working pcs and personnel to execute efficient data collection and management for epidemiological analysis. however, licensing cost of near-real time syndromic surveillance software is prohibitive, but the open access software with capacity to generate custom graphs, maps, plots, and temporal-spatial analysis output for specific syndromes should make implementation a lot easier. such output for conditions like flu (or gastroenteritis) will be essential to cause prompt response of the local public health office and international partners in saving lives and suffering of djibouti people. pandemic flu surveillance and preparedness requires multifaceted, interdisciplinary, and international approach whose efficiency and efficacy can only be refined over time. building on the health care system's swot for preparedness, the ehesp wcic-study promises to refine surveillance system operation and knowledge on individual's risk determinants to swine flu (h n ) virus infection at the household level in djibouti. these efforts are ultimately creating available control options at the time of need (pandemic occurrence), and at the same time exploring investment in quality data profiling and information technology, which will include syndrome surveillance software systems like essence, ewors, or other open sourced ones. the antibody efficacy -which compares the illness frequency between those with and those without a protective level of pre-epidemic hi antibodies ( ‡ : ) -has been proposed ; however, this index has rarely been used due to practical difficulties in confirming the strain-spe-cific disease corresponding to each of the vaccine-induced antibodies. we followed elderly individuals residing in a nursing home, whose serum specimens were obtained before and after undergoing trivalent influenza vaccination, in ⁄ influenza season (medium-scale mixed [a ⁄ h n and b] epidemic in study area, and a ⁄ h n was circulating at the nursing home). the serum antibody titre to each strain of influenza virus was measured by the hi method, using the same antigens as those in the vaccine. all participants' body temperatures, respiratory symptoms, other general symptoms, hospitalization, discharge, and death were recorded daily from november to april in a prospective manner. when the participants suffered any influenzalike symptoms, such as sudden fever ‡ ae °c, throat swabs were collected and tested using a rapid diagnosis kit for influenza, which utilizes an immunochromatographic method. the adjusted odds ratios (or adj ) for febrile illness and kit diagnosed influenza were evaluated using multiple logistic regression models adjusting for possible confounders (i.e., age, sex, coexisting conditions, and vaccine strains). after vaccination, the proportion of subjects achieving an hi antibody titre ‡ : (seroprotection level) were ae % ( ae - ae %) for a ⁄ h n , ae % ( ae - ae %) for a ⁄ h n , and ae % ( ae - ae %) for b. during the follow-up period, the a ⁄ h n strain was isolated therein, and subjects experienced sudden-onset fever ( ‡ ae °c), and eight subjects were positive for rapid diagnosis kit. patients with a seroprotection level of the hi antibody titre ( ‡ : ) had lower incidences of febrile illness (or adj , ae ; % ci, ae - ae ) and rapid kit diagnosed influenza (or adj , ae ; % ci, ae - ae ) than those with a lower titre. thus antibody efficacy ( ) or adj ) against fever related to a ⁄ h n and kit diagnosed influenza were both estimated to be %. although statistical significance was not detected due to limited sample size, these results lend support for the usefulness of antibody efficacy. some data presented within this manuscript was also published in hara et al. asia via a regional network from which epidemics in the temperate regions were seeded. the virus isolates obtained from nasopharyngeal swab specimens from outpatients were typed and subtyped by the hemagglutination (ha) inhibition assay. the emergence of a ⁄ fujian ⁄ ⁄ coincided with higher levels of influenza-like illness in korea than what is typically seen at the peak of a normal season. most of the intermediates and fujian-like strains were isolated from asian countries, and the mutational events associated with the fujian strains took place in asia. closely dated phylogeny from december , to august , showed that the antigenic evolution of the h n fujian strains had periods of rapid antigenic changes, equivalent to amino acid changes per year ( figure ). the fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the ha domain. the antigenic evolution of the fujian strains was initiated by exceptionally rapid antigenic change that occurred in asia, which was then followed by relatively modest changes. some of the data presented in this manuscript was previously published in kang et al. we compared reactivity to the novel virus strain using haemagglutination inhibition (hi) assays performed on discarded plasma specimens left over from routine testing. samples were taken from healthy adult blood donors (> years) before and after the ph n influenza epidemic that occurred during the southern hemisphere winter of , and again prior to onset of the southern hemisphere influenza season. reactivity to the novel h n strain of influenza was relatively uncommon among the healthy adult population during the first australian winter wave, rising from a baseline of % to %. a further increase in the seropositive proportion from % to % was observed over the summer months, most likely attributable to immunisation. this level of immunity appears to have been sufficient to constrain the winter epidemic. together with a final serum collection, planned for late , these data will aid evaluation of the extent and severity of disease in this 'second wave' of ph n . assessment of the extent of disease due to novel influenza a(h n ) virus (ph n ) during the winter outbreaks in australia was made difficult by the generally mild nature of disease. the epidemic was experienced in a staggered fashion around the country, reflecting the considerable geographical distances between state and territory capital cities ( figure ). differences in the intensity of case-finding during the evolving pandemic response and between jurisdictions hindered comparisons of disease burden in distinct geographical regions. rates of reported hospitalisations and deaths appeared fairly similar across states but, without a consistent exposure denominator, assessment of relative severity was difficult. we conducted a national serosurvey of antibody to ph n using residual plasma from healthy blood donors collected before and after the epidemic to estimate ph n exposure. here we report the findings of that first collection, together with new data on seroprevalence of ph n antibody in specimens gathered in march-april . these latter samples were collected prior to onset of seasonal influenza activity to assess the impact of a national ph n vaccine program conducted in spring ⁄ summer ⁄ on the proportion of individuals with antibody titres deemed protective. findings informed estimates of population susceptibility to ph n prior to the influenza season and provided a baseline for a subsequent serosurvey that will be collected at the end of to assess the extent of exposure during the 'second wave.' tralian red cross blood service (the blood service) for dengue fever surveillance studies. these samples were used to provide a baseline estimate of prevalence of cross-reactive antibody to ph n in the australian population. discarded plasma specimens, taken for virologic testing from healthy adult blood service donors, were prospectively collected at two additional timepoints for measurement of antibody to ph n . collection periods were as follows: approximately plasma samples were randomly selected from donors in each of brisbane, hobart, melbourne, newcastle, perth, sydney, and townsville on each occasion. up to specimens were identified in each of the following age strata: - , - , - , - , - , and > years. at the last collection timepoint, there was deliberate over-sampling of the oldest and youngest age strata in which approximately specimens were collected (i.e., up to specimens per site). in accordance with the provisions of the national health and medical research council's national statement on ethical conduct in human research, individual consent was not required for use of these specimens, given the granting of institutional approval by the blood service human research ethics committee. reactivity of plasma against ph n was measured in haemagglutination inhibition (hi) assays using turkey red blood cells (rbc). egg-grown a ⁄ california ⁄ ⁄ virus was purified by sucrose gradient, concentrated and inactivated with b-propiolactone, to create an influenza zonal pool preparation (a gift from csl limited). plasma samples were pretreated with receptor destroying enzyme ii (denka seiken co. ltd), : (volume ⁄ volume) and tested as previously described. following hour incubation, ll % (volume ⁄ volume) of rbc was added to each well. hi was read after minutes. any samples that bound to the rbc in the absence of virus were adsorbed with rbc for hour and reassayed. samples in which background activity could not be eliminated by these means were excluded from the analysis. titres were expressed as the reciprocal of the highest dilution of plasma where haemagglutination was prevented. a panel of control sera and plasma samples was included in all assays. it comprised paired ferret sera pre-and postinfection with the pandemic virus or seasonal influenza a(h n ), a(h n ), or influenza b viruses and paired human plasma and sera collected from donors before april or after known infection with the pandemic virus or after immunisation with the australian monovalent pandemic vaccine. all assays were performed by the who collaborating centre for reference and research on influenza. for each of the three study timepoints and within each age group, the proportion of seropositive individuals (hi titres ‡ ) was calculated, with exact (clopper-pearson) confidence intervals. the contribution of individual variables (age, gender) and location to seropositive status was assessed in separate multivariate logistic regression models developed to assess the post-pandemic and pre-influenza season collections. all statistical analyses were conducted in stata . locations of specimen collection are shown in figure , together with the number of samples tested from each centre. samples with high background hi titres or discrepancies between assays were excluded at each timepoint as follows: at baseline, from the post-pandemic collection, and in early . pared with baseline was % overall, rising from % to % (table ). the only jurisdictions in which seropositive proportions were higher in october ⁄ november than in the baseline collection were hobart [ % ( % ci ae , ae )], perth [ % ( ae , ae )], and sydney [ % ( ae , ae )]. in the multivariate regression model, the only jurisdiction in which exposure appeared somewhat higher than the reference population of brisbane was hobart [or ae ( % ci ae , ae ), p = ae ]. a marked age effect on antibody status was observed at this timepoint, with an increase in the proportion of seropositive individuals in relation to the baseline collection only noted for those aged between and years (table ) . according to the multivariate model, the youngest and oldest cohorts had similar titres, with all other groups showing significantly lower seropositive proportions than the reference population of - years [e.g. - years or ae ( % ci ae , ae , p < ae )]. an overall increase in the seropositive proportion from % to % was observed between october and april , distributed throughout all jurisdictions ( ( , ) ]. antibody titres prior to the influenza season rose in all age groups, but remained significantly lower among [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] year olds than in the youngest age cohort (table ) . adjusted ors for the seropositive proportion in the multivariate model in these age groups were: - years [or ae ( % ci ae , ae )]; - years [or ae ( ae , ae )]. the relatively low titres observed in these groups reflected small incremental increases in the seropositive proportion across each of the time points studied, suggestive of both low rates of infection and vaccination. the rise in immunity observed across the population was most likely attributable to immunisation in the majority, given the absence of observed outbreaks and very few notified cases of ph n during the period between the two plasma collections. this study suggests that, while adult exposure to ph n during the southern hemisphere winter was uncommon at around %, vaccine uptake in the australian population over the period november -may was in the order of %. this latter estimate is in keeping with recently published figures for adult ph n vaccine coverage from a national immunisation survey conducted by the australian institute of health and welfare. in that survey, vaccine coverage was significantly higher in tasmania than in other states, but mostly in those over years of age, possibly in a subgroup whose health status may have differed from that of the donor population. no allowance has been made in this analysis for likely waning of natural or vaccine induced immunity, possibly resulting in lower estimates of natural and ⁄ or vaccine exposure than may have occurred over the period. regardless of such intervening processes, the seropositive proportion among australian adults at the start of the winter season appeared likely to be sufficient to constrain transmission of infection in the age groups tested. this assertion has been borne out in practice, with only modest levels of influenza reported during the late and protracted season. a final serum collection is planned for the end of the influenza season in australia from which to assess the level of exposure in relation to the baseline observed here. the need for epidemiologic studies such as this has been highlighted by groups such as the european centre for disease control to aid evaluation of the extent and severity of the 'second wave,' known to be variable from historical reports of past pandemics in disparate populations. in - , the first wave of the swine-origin novel h n flu (h n ) pandemic swept across the world, including japan. to examine the epidemiological nature of this novel infectious disease among school children within and among small regional communities, we have carried out a complete survey on the incidence of h n among school children using absentee reports provided by school health teachers in two small administrative districts (population: about in total) in japan. we then examined the epidemiological diversity on the inci-dence of h n within and among small regional communities. we investigated seventeen elementary and ten junior high schools in moroyama-town and sakado-city located in the central part of saitama prefecture. populations are: all ages, and ; elementary schools, and ; junior high schools, and , respectively. the number of school children in each school ranges from to . the surveillance system was built on an apache-and mysql-based web server using html, php, and java-script. school health teachers enter information on children absenteeism due to school infectious diseases via web browsers at each school infirmary on a daily basis. in addition to the trend graphs shown on the web browser, detailed analyses were reported to the schools and local educational boards weekly. the basic reproduction number (r ) of h n was estimated according to becker. agentbased modeling and simulations were also performed using a multi-paradigm simulator anylogic version . (xj technologies, st. petersburg, russia). by the end of march , cumulative incidence (ci) of h n among school children in moroyama and sakado reached % and %, respectively. the overall r among school children in this area was ae . vaccination rate of children in this area during the surveillance period was reported to be very low (< %). there was no considerable difference between the epidemic curves in this neighboring town and city. on the other hand, in the individual schools, the cis as of the end of march scattered from % to % ( figure ) even though the schools are closely located. to examine the cause of this diversity, we built an agent-based community model consisted of the same numbers of agents as those of children in the actual schools and people in moroyama and sakado to simulate the infection. the ratio of probability of infection in schools and the remaining places were assumed to be : or : . using a heuristic optimization scheme, we estimated the parameters for the simulations to give the overall ci of % (the ci as of the end of march ). we then performed simulations repeatedly. the cis obtained with the repetitive simulations with the assumption of higher probability of infection in schools scattered from % to %, indicating that the cis of the small population communities may vary considerably, even though all the agents were assumed to have the same susceptibility to infection at the beginning, and the other conditions were the same. the policies for surveillance ⁄ analyses ⁄ prevention of communicable diseases in local communities have generally been decided on governmental-and ⁄ or each local administrative district-basis (populations: several hundred thousands to several millions) in japan. we found the considerable variations in the cis of h n for children among much smaller areas, i.e., the school districts (populations: all ages, several thousands; school children, several hundreds). we thus conclude that the granularity of surveillance ⁄ analyses ⁄ prevention should be finer than in the past to achieve the most effective policies against influenza and similar communicable diseases in the local communities. the cause of this diversity can be explained in part by the stochastic nature of infection transmission processes in the small populations shown by the agent-based simulations. we have already conducted a complete questionnaire survey for the school children and their parents to clarify the relevance of the other issues including differences in environmental factors, preventive policies (e.g., vaccination, school closures), etc., in each school. the detailed analyses will be reported elsewhere. a www-based surveillance system for transmission of infectious diseases among school children within and among small regional communities. j epidemiol ; (s ):s . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. the first cases of the a ⁄ h n v pandemic influenza were reported in mexico and the united states in april . given the context of this new influenza virus and considering the likelihood of its pandemic spread, the cohorts for pandemic influenza (copanflu) international consortium was created in order to study individual and collective determinants of pandemic a ⁄ h n v influenza across different countries by setting up prospective cohorts of households, followed during years. this study relies on the first available data from the copanflu-france project, which is part of the copanflu international consortium. we studied factors associated with elevated haemagglutination antibody titers against a/h n v at entry in the copanflu-france cohort. we focused in this primary analysis on the association between the titers and influenza vaccination (seasonal or pandemic) across age groups. the copanflu-france cohort was set up in fall . inclusions began on december , and ended on july , . households were sampled using a random telephonic design (mitofsky-waksberg method) in a stratified geographical sampling scheme, aimed at including a sample of subjects representative of french general population. all household members were eligible to the cohort, without any age limit. the inclusion of a household required the participation of all members: the refusal of one or more member(s) prevented the inclusion of other members. the protocol was approved by a research ethics committee and written informed consent was obtained for all subjects. this study requires several visits to the households by nurses who collect written data with questionnaires and biological samples. during the inclusion visits, nurses collected from all subjects detailed data regarding medical history, including vaccination and preventive measures against influenza. blood samples were collected at entry and centralized. a standard hai technique was adapted to the detection and quantification of antibodies to the a ⁄ h n v virus. the titration endpoint was the highest dilution that exhibited complete inhibition of haemagglutination in two independent readings. the lowest read dilution was ⁄ . geometric mean titers were calculated for hai assays with the use of generalized estimating equations for interval-censored data, , taking into account a within-household correlation. multivariate models were derived from this method to identify factors associated with elevated gmts. we defined the ''gmt ratio'' (gmtr) as the multiplicative factor applied to the gmt in presence of an explanatory variable. for qualitative explanatory variables, a gmtr of n means a predicted n-fold higher gmt for subjects exposed to the considered factor compared to others. for continuous explanatory variable, the same interpretation applies to a unit difference. the following variables were included in the multivariate models: age, history of pandemic or seasonal influenza vaccination, and history of ili. age was categorized in three groups: - years (reference group), - years and over years. the definition of ili was that used by the cdc : fever ‡ ae °c and cough and ⁄ or sore throat without another known cause. history of ili was defined as an ili reported by the subject between september , (beginning of the influenza epidemic in france) and the date of inclusion. this preliminary analysis included subjects belonging to households. results reported hereafter do not account for missing data. participating households were sized - subjects, mean size = ae . in comparison, the mean size of french households is ae according to the latest national census. the median age of subjects at entry was ae years [iqr: ae ; ae ] versus ae [ ae ; ae ] for french population. the proportion of subjects reporting a history of ili since the beginning of the epidemic varied from ae % for subjects over years to ae % for subjects below years (table ) . vaccination with the pandemic strain was the highest in subjects below ( %) whereas vaccination with the seasonal strain was the highest in subjects over ( ae %). detailed data regarding vaccination is given in table . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. [ ] [ ] [ ] [ ] [ ] [ ] among non-vaccinated subjects, elevated gmt in the elderly may be the result of exposure to similar viruses in early life, whereas children and young adults with elevated gmt are likely to have been infected by the a ⁄ h n v virus. [ ] [ ] [ ] [ ] interestingly, a significant drop in the hai titer is observed during the months following vaccination with the pandemic strain. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. the reason for this association is not obvious: although we cannot discard the hypothesis of a higher incidence of a ⁄ h n v infections in seasonal vaccine recipients, as described by several other studies, - the main explanation may be a cross-reaction between pandemic and seasonal strains. , , further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. in april , the cdc alerted about the appearance of a new strain of ia h n with unknown virulence. infants under years old had higher risks of hospitalization, complications, and rate of death for sari. materials and methods: a cross-sectional study was executed from may to december in . the sources were: mandatory reporting form of the province surveillance system, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports. inclusion criteria: children under years old with diagnostic of ili or sari and confirmed cases with epidemiological nexus or laboratory confirmation (rrt-pcr, ifi). the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. results: the ili rate in infants under years old was ae ⁄ people ( % ci - ) being higher in infants of years old ( ⁄ people of years ( % ci - ) ( table ) . infants had less risk of getting sick in relation to the rest of the population (rr ae [ % ci ae - ae ]) (p < ae ). the chance of sari in infants was ae ( % ci ae - ae ) compared to the rest of the population. the lethality rate was higher in infants under year old ( ⁄ people [ ⁄ ]). discussion: the evidence suggests that the infants under years old had lower risk of getting sick than the rest of the population, but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under year old. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. in april , the cdc alerted about the appearance of a new strain of ia h n with unknown dissemination and virulence. in june, the world health organization declared the pandemic. , the ili often presents an unspecific clinical picture in infants under years old, from mild symptoms to sari, especially in the newborn babies. infants under years old have higher risks of hospitalization, complications, and rate of death for sari. , on may th, argentina declared the first imported case of ia h n , and by the end of the month, it announced the viral circulation in the country. the epidemiological surveillance system of the province arranged that all the patients with influenza diagnosis made by a doctor must be reported. from april th to november th, suspected cases of ili in the province of tucumán were reported. the ili rate was ⁄ people, and ia h n comprised ⁄ people. the lethal rate of sari ia h n was ae ⁄ people ( ⁄ ). the objective of this research was to determine the epidemiological characteristics of the pandemic ia h n in infants under years old in the province of tucumán between may and december in . the province of tucumán is placed in the center of the northwest of the republic of argentina. it has a population of inhabitants of which are infants under years old. the crude birth rate for was ae &. the infant mortality rate was ae &. respiratory pathologies in infants under years old were the third cause of death in the province ( %). the public health system of the province is composed by three sectors: public, private, and welfare. with health facilities as a total, the average of available beds is & per inhabitants and & per neonates. a cross-sectional study was executed from may to december in in the province of tucumán, argentina. the following sources were used: mandatory reporting form of the surveillance system of the province filled by a doctor, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports (patients with sari). inclusion criteria: • suspected case of ili: sudden appearance of fever higher than °c, cough, or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nausea or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea. ) were used for the analysis. the odds rations, risk ratio and % confidence interval were calculated to compare ambulatory with hospitalized patients, confirmed and dismissed, < years old and the rest of the population. it was considered significant a rate of p < ae . the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. the epidemiological surveillance system of the province received ili reports, ae % ( ⁄ ) were infants under years old. twenty seven percent were dismissed ( ⁄ ), and % ( ⁄ ) of suspected cases were confirmed. the first ia h n case was a child of years from the province of buenos aires, in th epidemiological week, and the last suspected case was reported in october , ( figure ). the ili rate in infants under years old was ae ⁄ people ( % ci - ), being higher in infants of years old ( ⁄ people of years, [ %ci - ]). the higher ili rates in confirmed the pandemic of ia h n ( ) was detected for the first time in the province of tucumán. the evidence suggests that infants under years old had lower risk of getting sick than the rest of the population (protective factor), but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under year old. towns with the highest demographic density had superior proportion of cases. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. outbreak of h n influenza - : behavior of influenza h n in school children in the province of tucumá n, argentina criteria: patients treated with antiviral medication for prophylaxis, respiratory pathologies which did not justify specific medication, and incomplete forms. results: from all notifications, were cases of ili in the group aged - years old; % were males. the incidence rate in this group was ae per thousands of inhabitants. the % of laboratory samples were influenza a h n , % were confirmed as unspecific influenza, and % were dismissed. the school aged children group had a high risks of getting sick (r.r. ae [ % c.i. ae - ae ]), especially males. it appeared that school aged children had a protective factor for presenting sari (or ae [ % c.i. ae - ae ], p < ae ). the lethality rate in this group was ae ⁄ thousands. headaches, myalgia, coryza, and sore throat were very common and significantly different (p < ae ) than the rest of the population. it was reported a decrease in the ew coinciding with winter holidays (ew ). the epidemic curve was different in males compared to females during the winter holidays. discussion: school aged children got sick more than the rest of the population, although they presented less proportions of sari. however, comorbidities were decisive in order to present sari or death. the epidemic curve was different in males compared to females. through its analysis, the beneficial effect of school closure was observed, as long as children meet the recommendation to stay home. in april , different countries reported cases of influenza a h n ; mexico reported a high mortality rate associates with this disease. the world health organization (who) declared the phase influenza pandemic alert on june . several reports from different countries describe the behavior of the pandemic in school aged children. this group plays an important role in the transmission of influenza. in germany, during the summer peak, pandemic hardly spread within this group. this might be explained by the timing of the summer school holidays, which started between ew and . since mid october, after the autumn holidays, the school-aged children began to be more affected, and the proportion increased from % in the initiation period to ae % in the acceleration period. in australia, % of h n cases were school aged children ( - years), with a median age of years ( % of cases were aged - years and, and % between - years). in canada, the infection rate was highest in this group. in chile, the incidence rate was ⁄ inhabitants, although in general they had mild desease. school closure can operate as a proactive measure, aimed at reducing transmission in the school and spread into the wider community, or reactive, when the high levels of absenteeism among students and staff make it impractical to continue classes. the main health benefit of proactive school closure comes from slowing down the spread of an outbreak within a given area and, thus, flattening the peak of infections. this benefit becomes especially important when the number of people requiring medical care threatens to saturate health care capacity. it has its greatest benefits when schools are closed very early in an outbreak, before % of the population falls ill. school closure can reduce the demand for health care by an estimated - % at the peak of the pandemic under ideal conditions, but too late in the course of a community-wide outbreak, the resulting reduction in transmission is likely to be very limited. policies for school closure need to include measures that limit contact among students when they are not in school. tucumán is placed in northwest argentina and has a total area of km . the population ( census, projection ) was inhabitants; of wich were - years old. the health system of the province is composed of sectors: public, private, and welfare. it has a total of health facilities with internement available and an average of & inhabitants. influenza-like illness (ili) has seasonal and endemic behavior in this province, as evidenced by past records from the national health surveillance system and influenza sentinel surveillance unit of the province. an increase of ili was reported in , with a peak in the ew . the objectives were: general objective to describe the behavior of the influenza a h n epidemic in school aged children from the province of tucumán, argentina. specific objectives • to explore the response to preventive measures by school aged population. • to assess the effect of the suspension of classes in this group. • to estimate the magnitude and severity of the disease. • to observe the effect of co-morbidities in this group. a cross-sectional study was executed from may to december . data were gathered through mandatory reporting forms, wich were collected from all public and private health centers. inclusion criteria: patients with compatible symptoms with influenza a; school aged children - years old. exclusion criteria: patients treated with antiv- iral medication as prophylaxis, respiratory pathologies which did not justify specific antiviral medication, and incomplete forms. • suspected case of ili: cases considered by clinical criteria (fever higher than °c, cough or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nauseas or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea). • confirmed case: person with positive laboratory results for influenza a h n or unspecificed influenza a (by laboratory results through rrt-pcr or immunofluorescence techniques). • dismissed case: by negative or different laboratory results, or different clinical evolution. • comorbidities: chronic illnesses like arterial hypertension, diabetes, asthma, recurrent obstructive bronchial syndrome (robs), smoking, chronic obstructive pulmonary disease (copd), immunosuppression, hiv ⁄ aids, cancer, nephropathy, obesity; pregnancy was also considered. data were analyzed using epi software (epi infoÔ cdc, atlanta, eeuu). rates were calculated and rr was estimated with their respective confidence interval (ci). population data were taken from national census projections. an estimation based on the same census was used for the group between and years old. to observe the effects of other co-variables, the or and their ci were calculated. logistic regression was used to evaluate the influence of the comorbidities. x was used to compare proportions. respiratory samples (nasopharyngeal and faryngeal swabs) were obtained. they were analyzed at influenza sentinel surveillance unit of tucumán, and ⁄ or sent to national reference laboratory dr. c. malbrán (rt-pcr). from all notifications ( ), were cases of ili in the group aged between and years old, % ( ⁄ ) of which were males. the incidence rate was ae , and it differed according to the sexes: ae males and ae females per thousands of inhabitants (p < ae ). of all laboratory samples ( ) % were confirmed as influenza h n , % were confirmed as unspecificied influenza, and % were dismissed. the remaining percentage corresponded to the isolation of other viruses (parainfluenza, respiratory syncytial virus, and adenovirus). the school aged group had higher risk of getting sick, in relation to the rest of the population (rr ae [ % ci ae - ae ]), especially males (rr ae ) compared with females (rr ae ). the highest attack rate was observed in the capital of tucumán ( ⁄ inhabitants). according to the rest of the population, it looked like being school aged children meant a protective factor for presenting sari (severe acute respiratory infection) (or ae [ % ci ae - ae ], p < ae ). the lethality rate was ae ⁄ thousand. the risk of dying was low compared to other ages. persons with comorbidities had significantly higher risk of presenting sari (or ae [ % ci ae - ae ], p < ae ) and of dying (or ae [ % ci ae - ae ], p < ae ). respiratory comorbidities were the most fre- quent: asthma ae % ( ⁄ ) and % rors ( ⁄ ). the symptoms headaches, myalgia, coryza, and sore throat were very common and significantly different (p < ae ) than the rest of the population. if we compared the group aged - years with - years old, the epidemic curve of the first group showed a decrease in the ew , coinciding with winter holidays (ew ) (figure ). there was a slight increase in the tendency when classes began, but it showed a clear declination afterwards. the analysis of rates in school aged children by ew showed a reduction of ae % in males and ae % in females (p < ae ) at ew . however, after the first week of winter holidays, the curve in males had a significant increased to ae % compared to ew , reaching the highest weekly rate of the epidemic ( ⁄ inhabitants). the reopening of classes coincided with a significant decrease of the rate ( ae %), from to ae ⁄ inhabitants in ew (p < ae ). in females, the school closure coincided with a plateau-shaped curve, and the reopening with a significant decrease of ae % of the rate, from ae to ae in ew ( figure ). the school children got sick a lot more than the rest of the population, although they presented less proportions of sari. however, comorbidities were determined in order to present sari or death. symptoms like headache, myalgia, coryza, and sore throat were considered more conducting for the definition of cases in this population in tucumán. the epidemic curve was different in males compared to females during the winter holidays. the beneficial effect of school closure was observed as long as persons met the recommendations. the difference between males compared to females during winter holidays could mean that women would have carried out social distance recommendations much better, for example, remained at home. the significant reduction after the opening of classes is a factor to be considered as an effective intervention in the declining stage of the curve. here, we report pdmh n infection attack rate (iar) during the first wave of the pandemic. we used our iar estimates to infer the severity of the pandemic strain, including the age-specific proportion of infections that led to laboratory confirmation, hospitalization, intensive care unit (icu) admission, and death. [ ] [ ] [ ] [ ] part of these results are now available in ref. subjects of a community study, - years old between november and october , we conducted a cohort study of pediatric seasonal influenza vaccination and household transmission of influenza. one hundred fifty-one children aged - were recruited and provided baseline sera in november and december . between september and december a further children aged - were recruited and provided baseline sera for the second phase of the study. for this serologic survey, we tested the sera collected before the first wave and the sera collected after the first pandemic wave. written informed consent was obtained from all participants. parental consent was obtained for participants aged or younger, and children between the ages of and gave written assent. all study protocols were approved by the institutional review board of the university of hong kong ⁄ hospital authority hong kong west cluster. age-stratified data on virologically confirmed outpatient consultations, hospitalizations, icu admissions, and deaths associated with pdmh n from april to november were provided by the hong kong hospital authority (the e-flu database). since may , patients admitted with acute respiratory illnesses routinely underwent laboratory testing for pdmh n virus by molecular methods. sera were tested for antibody responses to a ⁄ california ⁄ ⁄ by viral microneutralization (mn). most individuals infected with influenza develop antibody titers ‡ : by viral microneutralization after recovery. we defined the pdmh n seroprevalence rate as the proportion of individuals who had antibody titers ‡ : . while mn antibody titers of ‡ are not by themselves conclusive evidence for pdmh n infection, we have assumed that the increase in cross-sectional seroprevalence between the pre-and post-first wave time periods are evidence of recent pdmhn infection. the iar was defined as the proportion of individuals infected by pdmh n during the first wave. the case-confirmation rate (ccr), case-hospitalization rate (chr), case-icu-admission rate (cir), and case-fatality rate (cfr) were defined as the proportion of pdmh n infections that led to laboratory-confirmation, hospitalization, icu admission, and death. due to containment efforts until june , all laboratory-confirmed cases were required to be hospitalized for isolation regardless of disease severity. as such, only surveillance data from june onwards were used to estimate severity measures. we estimated the iar as the difference between the prefirst-wave and post-first-wave seroprevalence rate. we used the estimated iar as the denominator for calculating the ccr, chr, cir, and cfr. we used an age-structured sir model with age classes ( - , - , - , - , and ‡ ) to describe the transmission dynamics of pdmh n in hong kong between june and november . we assumed that the mean generation time was ae days. using the age-structured transmission model, we estimated the following transmission parameters from the serial cross-sectional serologic and hospitalization data: (i) r o , the basic reproductive number; (ii) p and p , the reduction in within-age-group transmission for - and - years old during summer vacation (compared to school days during september-december ); (iii) d r , the average time for neutralization antibodies titer to reach ‡ : after recovering from infection; (iv) h a , the age-specific relative susceptibility with - years old adults as the reference group. we assumed non-informative priors for all parameters and used monte carlo markov chain methods to obtain posterior distributions of the parameters. sources of specimens: [ ] pediatric cohort study ( - april virological surveillance data suggested that the first wave of pdmh n in hong kong occurred from august to october . most of the laboratory-confirmed infections in this first wave occurred in individuals aged below years old accounting for > % of the lab-confirmed cases and hospitalizations, % of icu admissions, and % of deaths. taking into account a delay of - weeks for antibody titers to appear during convalescence, we found that these virological surveillance data were consistent with our serial cross-sectional seroprevalence data, which indicated a sharp rise in seroprevalence among the - years old from september to november and a plateau thereafter (data not shown). among individuals aged - years, the seroprevalence rates were similar across time between pediatric outpatient subjects and pediatric cohort study subjects (data not shown). similarly, for older age groups, the seroprevalence rates were largely similar between blood donor subjects and hospital outpatient subjects (except for the - years old in november-december). this provided some evidence that despite biases in our convenience sampling scheme, the resulting serologic data provided a reasonably representative description of seroprevalence in the community. the estimated pre-and post-first-wave seroprevalence rates and the corresponding iar estimates are shown in table . the severity estimates (ccr, chr, cir, and cfr) are shown in table . in summary, we estimated the iar was ae % among - years old, ae % among - years old, ae % among - years old, ae % among - years old, ae % among - years old, and ae % among - years old. overall, we estimated a population-weighted iar of ae % ( - %) among individuals aged - years through the first wave in hong kong. ccr were around ae - ae % among the - years old. chr were around ae - ae % among the - years old. cir increased from ae ( ae - ae ) per infections in - years old to ( ae - ) per infections in - years old. cfr followed a similar trend with ae ( ae - ae ) death per infections in - years old to ae ( ae - ) deaths per infections in - years old. compared to children aged - , adults aged - were ae and times more likely to be admitted to icu and die if infected. the best-fit age-structured transmission model gave the following parameter estimates: . the basic reproductive number was ae ( %ci, ae - ae ). . it took an average of ( - ) days for recovered individuals to develop neutralization antibody titer ‡ : . table . estimated age-specific proportions of individuals with pdmh n infections that were laboratory-confirmed, were hospitalized, were admitted to icu, and died. case-icu and case-fatality rates are expressed as number of episodes per infections . compared to - years old, - years old children and - teenagers were ae ( ae - ae ) and ae ( ae - ) times more susceptible to pdmh n infection, respectively. . compared to - years old, - years old older adults and - years old elderly were only ae ( ae - ae ) and ae ( ae - ae ) times as susceptible as the - years old, respectively. . compared to the school period during september-december , summer vacation reduced within-agegroup transmission by % ( - %) among - years old, but only % ( - %) among - years old. using computer simulations, we estimated that if preexisting seroprevalence is zero, real-time serologic monitoring with about specimens per week would allow accurate estimates of iar and severity as soon as the true iar has reached % (data not shown). we estimated that during the first wave in hong kong, ae % of school-age children and ae % of individuals aged - were infected by pdmh n . a serologic survey in england found similar iars in london and the west midlands. both studies highlight the importance of including serologic surveys in pandemic surveillance. the geographically compact and well-mixed population in the urban environment of hong kong permits some degree of confidence in the validity of our iar and severity estimates. the completeness of the pdmh n surveillance system, welldefined population denominator, and our large-scale serologic survey provide accurate numerators and denominators for the severity measures. we based severity estimates for pdmh n on the iar as the denominator. in most previous studies of pdmh n severity, the denominator was clinical illness attack rate, which depends on the probability of symptoms as well as medical care seeking behavior of the population. , our estimated cirs and cfrs are broadly consistent with presanis et al.'s 'approach ' severity estimates, but around - times lower than their 'approach ' estimates. our estimates of chr are - times higher than their approach estimates of symptomatic chr. however, the hospitalization-death ratio was ⁄ = as of november in hong kong, but ⁄ = as of june in new york, suggesting that the clinical threshold for admission in terms of disease severity at presentation may have been lower in hong kong. our study has a number of limitations. first, we have used antibody titers of ‡ : by viral microneutralization as an indicator of recent infection, correcting for pre-existing seroprevalence levels, but this may lead to underestima-tion of the iar if some infections led to antibody titers < : , or if some individuals with baseline titers ‡ : were infected. second, our estimates of the iar would be biased upwards if infection with other circulating influenza viruses led to cross-reactive antibody responses resulting in antibody titers ‡ : . however between august and october , % of influenza a viruses detected in hong kong were pdmh n , and only % of isolated viruses were seasonal h n viruses. third, a minority of severe illnesses associated with pdmh n infection might not be identified by molecular detection methods, for example if admission occurred after viral shedding from the primary infection has ceased, in which case we may have underestimated the disease burden of pdmh n . finally, our analyses are primarily based on seroprevalence among blood donors to the hong kong red cross, who may not be representative of the whole population. we do not have detailed data on donors to compare their risk of infection with the general population, but we did observe very similar seroprevalence rates across the three groups of subjects in our study, i.e., blood donors, hospital outpatients and participants in a community cohort (data not shown). in conclusion, around ae % of the population aged - and half of all school-age children in hong kong were infected during the first wave of pandemic h n . compared to school-children aged - , older adults aged - , though less likely to acquire infection, had ae and times higher risk of icu-admission and death if infected. thus, although the iar of pdmh n is similar to that of a seasonal epidemic, the apparently low morbidity and mortality of pandemic influenza (h n ) appears to be due to low infection rates in older adults who had a much greater risk of severe illness if infected. the reasons why older adults appear relatively resistant to pdmh n infection even though they appear to lack neutralizing antibody remains unclear. if antigenic drift or other adaptation of the pdmh n virus allows these older age groups to be infected more efficiently, the morbidity and mortality of subsequent waves of the pandemic could yet become substantial. and the national institute of allergy and infectious diseases, national institutes of health (contract no. hhsn c; adb no. n -ai- ). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. bjc reports receiving research funding from medimmune inc., a manufacturer of influenza vaccines. the authors report no other conflicts of interest. some data presented in this manuscript were previously published in wu et al. it is well known that a primary goal of vaccination is to generate immunological memory against the targeted antigen to prevent disease in a vaccinated person. this ensures an accelerated immune response in the event of future contact with the pathogenic agent, such as a virus. therefore, it is very important to develop criteria for the assessment of vaccine immunogenicity by measuring both t and b memory cell levels from the vaccinated host. in contrast to inactivated influenza vaccines, live attenuated influenza vaccines (laivs) have been shown to provide primarily cellular and local immune responses. - to date, however, the hemagglutination-inhibition (hai) test (i.e. detection of serum antibodies) remains the method widely accepted for evaluation of an influenza vaccine's immunogenicity. improved understanding of the role of cellular and mucosal immunity and their contribution to protecting against severe illness caused by influenza infection has emphasized the need to reconsider methodologies used to evaluate the immunogenic impact of various influenza vaccines. such new assays need to include methods to measure local antibodies and virus-specific lymphocytes, especially in the case of live attenuated influenza vaccines, because of their potential to induce such broad-based immune responses. the aim of this study was to assess the ability of new russian pandemic laivs a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) ('ultragrivak,' registered ae ae ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) ('influvir,' registered ae ae ) to induce memory t-cells in naïve human subjects and to compare results to levels of hai antibodies from each subject. a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv was generated by : genetic reassortment of low-pathogenic avian influenza virus a ⁄ duck ⁄ potsdam ⁄ - (h n ) and master donor strain a ⁄ leningrad ⁄ ⁄ ⁄ (h n ). , the vaccine strain contains ha gene from avian virus, as well as na and internal genes from the master donor virus. a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv was generated by classical ( : ) reassortment of a ⁄ california ⁄ ⁄ (h n ) with the master donor virus. the vaccine strain contains ha and na genes from a 'wild-type' h n strain and internal genes from the master donor virus. participants were aged to years and were without contra-indication of laiv vaccination. immunogenicity of a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv was assessed in ten vaccinated persons and ten volunteers inoculated with a placebo (sterile physiological saline solution). immunogenicity of a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv was estimated in vaccinated volunteers and nine volunteers inoculated with placebo. viruses or placebo were administered intranasally twice with an interval period of days at a dosage of ae ml per nostril for each vaccination. physical examination, venous blood and nasal swab samples were collected at four time points during the study: (i) before vaccination (day ); (ii) days after first vaccination (day ); (iii) days after the second vaccination (day ); and (iv) weeks after the second vaccination (day ). serum hai antibodies were measured by standard hai assay using % human red blood cells. test antigens for the assay were a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) or a ⁄ ⁄ california ⁄ ⁄ (h n ) to match the appropriate vaccine antigen. local iga antibodies in nasal swabs were evaluated by elisa using whole purified a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) or a ⁄ ⁄ california ⁄ ⁄ (h n ) viruses at hau per ae ml for absorption to elisa plates. endpoint elisa titers were expressed as the highest dilution of sera that gave an optical density (od) greater than twice the mean od of six negative controls in the same assay. percentages of virus-specific cd + cd + ifn-c + and cd + cd + ifn-c + peripheral blood memory cells were determined using a flow cytometry iccs assay performed by the published method. pbmcs were prepared with standard histopaque- gradient centrifugation from heparinized whole blood. wilcoxon matched pair test, mann-whitney u test and the students t-test were used for statistical data analysis. prior to the first vaccination (day ), gmts of hai antibodies to a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) laivs were ⁄ ae and ⁄ ae , respectively. in addition, gmts of siga against these specific antigens from nasal swabs were ⁄ ae and ⁄ ae , respectively. no hai antibody titers greater than : were observed prior to vaccination. background levels of virusspecific t-cells varied significantly within groups. mean levels of virus-specific cd + ifnc + cells were ae % to a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) and ae % to a ⁄ ⁄ california ⁄ ⁄ (h n ). for cd + ifnc + cells, initial levels were ae % and ae %, respectively. thus, background levels of virus-specific antibodies were low, but prior vaccination or virus exposure in some volunteers produced some pre-existing levels of t cells, thus they were not absolutely immunologically naïve in this sense. preexistence of h n -crossreactive antibodies and t-cells has been observed previously. [ ] [ ] [ ] effect of vaccination antibody immune responses both influenza a (h n ) and influenza a (h n ) laivs stimulated production of serum hai antibodies and local iga antibodies in nasal swabs. following the first vaccination with influenza a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv, % percent of volunteers exhibited seroconversion of hai antibodies; after the second vaccination, % of volunteers exhibited seroconversion. after the first vaccination, a % conversion rate of siga was observed; after the second vaccination, % showed conversions in levels of siga. the first vaccination with a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv showed ae % of hai antibodies seroconversions vaccination, and % seroconversion after second vaccination. for local siga, those results were ae % and ae % following the first and second inoculation, respectively. figure summarizes cellular immune responses observed in the vaccinated versus the placebo group. after the influenza a (h n ) laiv inoculation, significant differences in both cd and cd ifnc-producing t-cells were observed at day after the second vaccination (d ). these data indicate that healthy young people who never received such avian influenza vaccines and were not exposed to h n wild-type viruses were able to respond to the live attenuated h n influenza vaccine. after the first influenza a (h n ) laiv vaccination, reliable increases were observed in cd + cells only. after the second vaccination, increases in both cd + and cd + fold changes were significantly higher in vaccinated volunteers compared to the placebo group. it is noteworthy that cellular immune responses (cd + and cd + cells) were more marked in the a ⁄ ⁄ california ⁄ ⁄ (h n ). considering the long-term circulation of h -subtype viruses among humans in contrast to the novelty of h viruses, such a result would be expected. similar data were also observed following vaccination with the h n laiv. after first vaccination, the percent of people with notable increases in virus-specific cd + and cd + t-cells was % and % to h n and % and % to h n , respectively. after the second vaccination, these results were % and % to h n and % and % to h n , respectively. importantly, a significant number of vaccinated volunteers without remarkable increases ( ‡ -fold) in hai antibodies had notable increases in cd + and ⁄ or cd + memory cells. the percent of people with notable increases in virus-specific t cells after the second vaccination among hai()) volunteers was % and % to h n and h n , respectively. these results indicate that laivs were able to induce broadly responsive, key antiviral immune responses that would not have been detected by the hai assay alone. thus, it can be deduced that hai data alone fails to reveal important broad and specific immune responses to laiv. consequently, the hai test alone is not suitable for assessment of laiv immunogenicity. furthermore, vaccination with h n laiv was able to induce cross-reactive memory t-cells to a seasonal vaccine strain, a ⁄ ⁄ solomon islands ⁄ ⁄ (h n ) ( table ) . reliable increases to a (h n ) were observed in up to % of volunteers. there was an inverse dependence between levels of memory t cells before and after vaccination. authors are thankful to path for the financial support of these studies. we are also thankful to jessica d'amico and dr. rick bright for their editorial review. options for the control of influenza vii background: increased susceptibility of older populations to secondary bacterial pneumonia-like infections following influenza infection has been well documented. recent evidence in mouse models suggests that this increased risk from secondary bacterial infection occurs through a desensitization of the innate immune response. this recent finding, however, does not account for potential differences in immune responsiveness due to age. materials and methods: to address this parameter, we used three age groups (aged, adult, and young mice) to evaluate the role of age in influenza-mediated vulnerability to secondary bacterial challenge with pseudomonas aeruginosa. all mice were evaluated for multiple parameters including: (i) survival; (ii) lung bacterial load; (iii) total lung protein content; (iv) immune cell infiltration; (v) cytokine ⁄ chemokine expression; and (vi) toll-like receptor (tlr) rna expression profiles. results: prior challenge with influenza contributed to aberrant cytokine ⁄ chemokine profiles and increased lung cellular infiltrate in response to secondary bacterial infection across all age groups, supporting a critical role for influenza infection in the alteration of immune responses to other pathogens. also similar to human influenza, these changes were exacerbated by age in mice as demonstrated by increased bacterial load, mortality, and total lung protein content (an indicator of lung damage) after p. aeruginosa challenge. conclusions: these data support a potential role for virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and the increased susceptibility of influenza virus infected mice to secondary bacterial infection. the understanding of the complex interaction of host and pathogen -and the role of age -in human influenza is critical in the development of novel therapeutics and improved vaccine approaches for influenza. our results support further examination of influenza-mediated alterations in innate immune responses in aged and non-aged animals to allow elucidation of the molecular mechanisms of influenza pathogenesis in humans. there is considerable evidence in the clinical literature to support the role of influenza infections with an enhanced risk for secondary bacterial pneumonias. [ ] [ ] [ ] given the increased pneumonia-related morbidity and mortality in both the young and elderly populations, there is rationale for gaining a deeper understanding as to the systemic changes in the pulmonary microenvironment. although there are some recent reports that account for some of the molecular mechanisms at work in this disease process, there is a paucity of experimental evidence that considers the potential effects of age. developmental changes in the immune system that occur in the aged environment have been well documented with regard to senescence of the adaptive immunity, global changes in myeloid cell function, and the establishment of a general pro-inflammatory state. , the aim of this work was to provide evidence for the contribution of the aged immune environment to the pathology of influenza mediated secondary bacterial infections. animals used in this study were housed under conditions approved by tulane university's institutional animal use and care committee. female balb ⁄ c mice used in these studies were divided into three age groups: aged ( months old), adult ( months old), and young ( months old). each age group was subdivided into two groups: influenza infected and naïve (control). mice were infected by the intranasal route with · pfu of mouse-adapted influenza a ⁄ pr ⁄ ⁄ . clinical disease was measured by body weight changes over a week period post influenza challenge, and recovery was determined as return to pre-infection weight. all mice were subsequently challenged intransally with · cfu pseudomonas aeruginosa strain pao . twenty-four hours post-pseudomonas challenge, bal with sterile pbs was performed on all mice in all groups. total rna from the cellular fraction was pooled from three experimental animals from each group. tlr mrna was detected by qrt-pcr, where expression levels were determined as relative to b-actin mrna levels. cdna was synthesized from total cellular rna from bal samples using iscript cdna synthesis kit (biorad). pcr reactions were composed of ae lg cdna forward and reverse primers according to optimized conditions and ae ll of · syber green icycler supermix (biorad), in a total vol-ume of ll and were run using a biorad icycler utilizing melting point determination. primers and concentrations used in this study included: mus_tlr f: tgctttcct-gctggagattt- nm, mus_tlr r: tgtaacgcaac agcttcagg- nm, mus_tlr f: atatgcgcttcaa tccgttc- nm, mus_tlr r: caggagcatactggt gctga- nm, mus_tlr f: ggcagcaggtggaattg tat- nm, mus_tlr r: aggccccagagttttgttc t- nm, mus_tlr f: ctggggacccagtatgctaa- nm, mus_tlr r: acagccgaagttccaagaga- nm, mus_tlr f: ggagctctgtccttgagtgg- nm, mus_tlr r: caaggcatgtcctaggtggt- nm, mus_ b-actinf: agccatgtacgtagccatcc- nm, mus_b-actinr: ctctcagctgtggtggtgaa- nm. as a measure of protein leakage into the alveolar space, total protein content in each bal was measured by bca assay of each supernatant fraction according to manufacturer's instructions (pierce). cytokine and chemokines levels were measured by multiplexed bead array (bioplex, biorad). immune cell characterization of bal was estimated by flow cytometry. lymphocyte populations were gated by forward versus side scatter and characterized as b cells (f ⁄ ) , cd + ) or t cells (cd b ) , cd + ). the myeloid population that is composed of macrophages, neutrophils, dendritic cells, and natural killer cells was enumerated by gating all but those found in the lymphocyte gate using forward versus side scatter plots. flow cytometry data was analyzed using flojo software (treestar). statistical analysis, where appropriate, was performed using a two-way analysis of variance (age versus influenza infection status) supported by bonferonni's correction for multiple comparisons. a recent finding by didierlaurent, et al., described an influenza mediated desensitization of tlr function as a primary contributor to an increase in bacterial burden when challenged after resolution of the primary influenza infection. this finding, however, was obtained using animals that were - weeks of age, where our study included two cohorts of older mice ( months and months). using whole protein content of the bal as an estimate of protein leakage into the lumen of the lung, we found elevated protein content in aged mice as compared to young and adult mice. in aged mice, a slightly lower total lung protein when comparing influenza infected to protein in the bal from influenza naïve mice challenged with p. aeruginosa (table ) . supporting previously published studies showing a generalized pro-inflammatory cytokine environment in the aged immune system, we provide evidence for significantly (p = ae ) and an increase in ifnc (p = ae ) was detected. the decrease in gm-csf correlates well with a previous report that gm-csf is less prevalent in influenza resolved animals (table ). we also report a noticeable change in the immune cell populations with respect to b-cells, cd + t-cells, and the myeloid cell populations. there is a trend of increased prevalence in cd t-cells in the post-influenza environment across all ages. b-cell numbers also trend toward increase in influenza treated animals in young and adult animals; however, there is a noticeable decrease in the bcells in aged animals. across all age groups, there is a general decrease in frequency of cells that would normally make up the myeloid cellular fraction of the bal (macrophages, neutrophils, dendritic cells, and natural killer cells) ( table ). our study also shows, as cited by others, that toll-like receptor (tlr) gene expression in the post-influenza environment is decreased in cells found in the bal after both influenza and pseudomonas infection. our data support the previous finding of a reduced expression of tlr mrna in influenza-cleared mice when we measured tlr , , , and . only tlr showed differences with respect to age with young mice showing little or no detectable change in tlr mrna expression. our results show an increase in the expression across all tlrs examined in the aged mice group (table ) irrespective of influenza infection status. these data support earlier studies performed with adult mice that showed reduced tlr mrna expression in the post-influenza environment. this study also expands the current understanding of the potential role of age in influenza mediated bacterial infection-induced mortality. the impact of these alterations in the immune microenvironment across age groups and infection status is highlighted by the ability of bacterially challenged animals to clear infection. assessment of bacterial load in the lungs of p. aeruginosa challenged mice indicated a difference in young and adult mice if previously infected with influenza virus. in aged mice, both influenza challenged and influenza-naïve mice had higher bacterial loads and less variability when comparing within the age group, supporting the risk of age alone in susceptibility to bacterial pneumonia (table , figure ). taken together, these data support the potential role for both virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and increased the susceptibility to secondary bacterial infection that results from influenza infection in mice. these findings highlight distinct differences in the immune environment between age groups and thus reveal necessity for further examination as to the mechanisms of immunity across age with respect to current infection status. garnering a clearer understanding as to the complex interaction of host and pathogen with respect to age in influenza infections is central to the development of increased efficacy in vaccine and therapeutic strategies. prospective estimation of the effective reproduction background pandemic influenza a (h n ) virus (ph n ) emerged in early and rapidly spread to every continent. an urgent priority for international and national public health authorities was to estimate the transmissibility of the pandemic strain for situational awareness and to permit calibration of mitigation strategies. the basic reproductive number, r , is defined as the average number of secondary cases that index case generates in a completely susceptible population, and is a common measure of transmissibility. however, it is difficult to estimate r without an understanding of the degree of any pre-existing immunity in the population. the effective reproductive number, r, is defined as the average number of secondary cases that index case generates, and can be estimated over time (i.e. r t ). wallinga and teunis described a method to estimate r t based on illness onset dates of the cases while assuming that all secondary cases would have been detected, and cauchemez et al. extended the method to permit prospective estimation by adjusting for secondary cases that have not yet experienced illness onset at the time of analysis. we describe how the method can further be extended to account for reporting delays, allowing true real-time estimation of r t during an epidemic, and we illustrate the methodology on notifications of ph n and associated hospitalizations in hong kong. we obtained data on all laboratory-confirmed ph n infections ('cases') reported between may and november , to the hospital authority and center for health protection in hong kong collated in the eflu database. a subset of the cases was hospitalised. the database also included information on age, sex, illness onset date, laboratory confirmation date, and contact history (for the early cases). laboratory-confirmed ph n infection was a notifiable condition throughout our study period. we extended existing methods for estimating r t over time to allow for reporting delays between illness onset and notification, and between illness onset, notification, and hospitalisation for those cases that were hospitalised, where the reporting delay distribution were estimated empirically from the data. we further extended the methodology to allow for imported cases (infected outside hong kong) contributing to the estimation of r t as infectors but not infectees. we used multiple imputation to allow for missing data on some symptom onset dates to make best use of all available data. we used a serial interval with mean (standard deviation) of ae ( ae ) days, and in sensitivity analyses, we used serial intervals with mean ae days and ae days. statistical analyses were performed in r version . . (r development core team, vienna, austria). in late april following the who global alert, hong kong initiated containment protocols to attempt to delay local transmission of ph n for as long as possible. these measures included screening at ports, airports, and border crossings, and enhanced surveillance for people with influenza-like illness, particularly for those who had recently returned from abroad. laboratory testing capacity was substantial due to heavy investment in local infrastructure following previous experiences with avian influenza a ⁄ h n in and severe acute respiratory syndrome in . laboratory-confirmed ph n cases were isolated until recovery, and their close contacts were placed under quarantine for days. imported cases were identified sporadically through may and early june . the first case of ph n not traceable to importation (i.e. a local case) was identified on june and triggered a change to mitigation phase measures. some containment measures, including isolation of cases, were continued until the end of june to allow a soft transition between containment and mitigation phases. as an immediate measure to try to reduce community transmission of ph n , all childcare centres, kindergartens, and primary schools were proactively closed for days (subsequently extended for another - days to summer vacation in early july). any secondary schools in which one or more confirmed ph n case was identified were reactively closed for days. on june the government opened eight designated flu clinics across the territory to provide free medical consultation for outpatients with influenza-like illness and free laboratory testing for ph n . these clinics resumed regular chronic disease services in mid-august, and laboratory testing and antiviral treatment was restricted to high risk groups in september. the various interventions are highlighted in figure (a), superimposed on the epidemic curve of laboratory-confirmed ph n cases and ph n -associated hospitalizations. around % of the cases were hospitalised, and this proportion increased somewhat towards the end of the epidemic. figure (b) shows the estimates of r t based on laboratory-confirmed ph n cases. the estimated r t peaked at ae on june , and fell below between june and july (which was within the school closure period). r t fluctuated between ae and ae through the school summer vacations in july and august, it subsequently increased to around ae - ae after schools reopened in september until the epidemic peaked in late september, and then fluctuated below as the epidemic declined. the trends in r t based on h n -associated hospitalizations were similar, although with wider confidence intervals due to the smaller number of events ( figure c ). the extension of the methods to allow for reporting delays avoided substantial bias in realtime estimates of r during the epidemic for the most recent days, and closely tracked the final estimates of r t . our results suggest that ph n may have had slightly lower transmissibility in hong kong than elsewhere. for example, estimates of r t were around ae - ae in new zealand and australia. lower transmissibility in hong kong has been associated with school closures in june and july followed by summer vacations from july through august. furthermore, in hong kong the influenza virus usually does not circulate after august, and therefore seasonality could also be a cause for the lower r t . on the other hand, the interventions applied during the mitigation phase, such as the widespread use of antiviral treatment in hong kong and the pre-existing immunity in the ageing population in hong kong, may also be associated with lower transmissibility. there are some limitations to our work. first, we only used aggregated data, and we did not consider the heterogeneity among the cases in terms of sex and age or other factors. therefore our estimates can only provide a snapshot of the overall trend, but limited information for any specific subset of population. secondly, we did not consider the possibility that cases might be infected in hong kong and exported to other countries, which could lead to slight underestimation of the transmissibility. one has to be careful in translating the estimated r t to the effectiveness of any specific interventions, as interventions may not be the only factor influencing the transmissibility; for example, a depletion of the susceptible population during an epidemic can also be a factor for the decline in r t . in conclusion, real-time monitoring of the effective reproduction number is feasible and can provide useful information to public health authorities for situational awareness and planning. in affected regions, laboratory capacity was typically focused on more severe cases, and changes in laboratory testing and notification rates meant that that case counts may not necessarily reflect the underlying epidemic. a useful alternative to case-based surveillance is surveillance of the subset of severe infections, for example hospital admissions, or icu admissions, and our results show that it was feasible to monitor ph n -associated admissions in real-time to estimate transmissibility. influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. a robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. however, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incom-pleteness, high noises, and low reactors. to overcome these challenges, we developed a computational method, temporal matrix completion-multidimensional scaling (mc-mds), by adapting the low rank mc concept from the movie recommendation system in netflix and the mds method from geographic cartography construction. the application on h n and pandemic h n influenza a viruses demonstrates that temporal mc-mds is effective and efficient in constructing influenza antigenic cartography. the web sever is available at http://sysbio.cvm. msstate.edu/antigenmap. as a segmented, negative stranded rna virus, influenza virus is notorious for rapid mutations and reassortments. the mutations on the surface glycoproteins (ha and na) of influenza viruses are called antigenic drifts, and these antigenic drift events allow the virus to evade the accumulating immunity from previous infection or vaccination and lead to seasonal influenza epidemics. a reassortment event with a novel influenza antigen may result in antigenic shift and cause influenza pandemic. for instance, the h n pandemic virus is a reassortant with a swine origin ha antigen. vaccination is the primary option for reducing the effect of influenza, and identification of the right vaccine strains is the key to development of an effective vaccination program. the antigenicity of an optimal vaccine strain should match that of the epidemic strain. in influenza surveillance program, the influenza antigenic variants are generally identified by the immunological tests, such as hemagglutination inhibition (hi) assay, microneutralization (mn) assay, or elisa. these immunological assays measure the antigenic diversity between influenza viruses by comparing the reaction titers among the test antigens and reference antisera. however, data interpretation of the data from these assays is not trivial due to the embedded challenges such as data incompleteness, high noises, and low reactors. by mimicking geographic cartography, influenza antigenic cartography projects influenza antigens into a two or three dimensional map using immunological datasets. antigenic cartography can simplify the data interpretation, and thus, facilitate influenza antigenic variant identification. recently, we developed a novel computational method, temporal matrix completion-multidimensional scaling (mc-mds), in antigenic cartography construction. in this paper, we described the details of temporal mc-mds, especially the original concepts introduced in this method, and how they can achieve the robustness in antigenic cartography construction. our method included two integrative steps: it first reconstructs the hi matrices using low rank mc method, and then generates antigenic cartography using mds with a temporal regularization. the mc concept was adapted from the movie recommendation system in netflix and the cartography concept from geographic cartography. in , netflix, an online dvd and blu-ray disc rentalby-mail and video streaming company, held a -year netflix prize contest (http://www.netflixprize.com/) on computational methods for improving its recommendation system. in its recommendation system, netflix collected the rating data from the individuals. based on his or her renting history and the ratings in the systems (e.g., from evaluators and other renters), netflix recommendation system suggests certain movies to a renter. apparently, no individuals would be feasible to provide ratings for all of the movies, as it will take hundreds of years for a single person to rate over movies available from netflix. thus, the resulting rating data is an incomplete matrix, and it can be as sparse as less as %. the challenge in netflix recommendation system is a classic mc problem. [ ] [ ] [ ] [ ] [ ] as the inspiration of netflix prize contest, many efficient low rank mc algorithms were developed, for instance, opt-space, svt, cf, bellkor, pf, and fwls. eventually, the team bellkor's pragmatic chaos won this contest. their methods combines nonlinear probe blending and linear quiz blending to come up with a predictor bigchaos. matrix completion estimates the unobserved values based on the observed values. the users can refill the missing data without repeating the experiments. furthermore, mc will help reduce the noises in the data, for instance, those biases by different individuals performing experiments. in influenza antigenic characterization, hi assay is a commonly used assay for antigenic analysis, since hi assay is relatively economic and easy to perform. however, hi is labor intensive, and it is almost impossible for any individual lab to complete the hi assays for all pairs of antigens and antisera during influenza surveillance. in addition, both testing antigens and the reference antisera are dynamic. for instance, in seasonal influenza surveillance, generally only contemporary antisera are used in experiments. thus, we will have to integrate multiple hi tables in order to evaluate the overall antigenic changes for influenza vaccine strain selection. the resulting hi tables will be incomplete, and the observed entries in the integrated hi data can be as less as %. the completion of this matrix can be formulated as a typical mc. briefly, given the combination of hi matrix with m antigens and n antisera, the hi matrix can be represented as m m·n = (m ij ) m·n , where m ij denotes the hi values from the reaction between testing antigen i and antiserum j. the low rank mc assumes that both antigen and antiserum can be embedded into a low rank space. to be specific, the low rank mc method is to seek matrix u m·r , v n·r and a diagonal matrix r r·r , where m = u m·r r r·r (v n·r ) t . in order to achieve this goal, the optimization formulation has been employed, which can be represent as following, where e denotes the observed entries in hi matrix and g(x) is a regularization function. the eqn ( ) is the standard format of a low rank mc formulation. the geographic cartography is a common technique to display the cities and their geographic distances in a map. this cartography can be generated using mds based on a geographic distance matrix. figure (a) shows the antigenic cartography generated using a distance matrix with seven cities, and figure (b) is a map for comparison. as an analog of geographic cartography, the influenza antigenic cartography maps the influenza antigens into a two or three dimensional map based on the distance matrix generated using immunological data. this incomplete matrix can be filled through mc algorithm discussed in section mc and netflix. low reactors, non-random date incompleteness, and temporal model generally, three types of data are present in a combined hi matrix: high reactor, low reactor, and missing values. among these three data types, high reactors are the most reliable data points. the low reactors are those values present in the hi matrix as ''equal to or less than a threshold h'', where h can be , , , or . low reactors have similar values in the affinity dataset but could be from different binding settings. these low reactors are present due to the detection limits of biotechnology, and they are not reliable. both these missing values and low reactors make it very difficult to analyze and interpret antigenic correlations amongst tested antigens and reference antigens. to our best knowledge, none of the existing mc method can handle the threshold values. in addition, the non-random incompleteness of influenza immunological datasets generates an additional challenge in traditional mc methods, which are based on the assumption that the observed values are randomly distributed among the matrix. in a typical combined antigenic hi data, most of the off-diagonal entries are missing values or low reactor values. in order to overcome the above issues, we incorporated a regularization function into the eqn ( ), where this indicator function is only valid for those entries with low reactor values. an alternating gradient decent method is applied to solve the optimization problem in eqn ( ) . in addition, a temporal mds method is proposed to project the antigens into a or dimensional map. x where d ij is the average distance between virus i and virus j, t i is the isolation year of virus i, d ij is the distance between virus i and virus j in cartography, d ac i is the distance between virus a and center of group i, and d c i c j is the distance between the centers of group i and group j. all the parameters are tuned by cross validation. we named this method as temporal mc-mds. by applying temporal mc-mds method in an h n dataset, low reactors. figure (a) is a three-dimensional influenza antigenic map based on this data by using mc-mds method. the reported clusters (hk , en , vi , tx , bk , si , be , be , wu , sy , and fu ) were displayed in the core of a spiral s-shape, and bk and be are located at the turning point of this s-shape. however, the antigenic distances between some viruses are incorrect. for example, the distance between hk and fu in the projection is ae units, which is close to the distance between hk and bk ( ae units). the main reason leading to those inaccurate distances is the unique distribution of hi datasets described in section . . in comparison, with the temporal model, not only the viruses in clusters have been clearly separated, but also the antigenic distances between each cluster are proportional to their isolation time interval. in this updated cartography ( figure b ), the antigenic distance between hk and fu is ae units, where the distance between hk and fu is ae units. this result suggested that the temporal information is critical for antigenic cartography construction for immunological datasets spanning a long time period. the hi data from seasonal influenza surveillance belong to this category. for seasonal influenza virus ⁄ pandemic influenza viruses within a short time span, the temporal model is probably not necessary, as there is lack of long-term immunological pressure present in the population. figure (c) is an antigenic cartography generated using a hi dataset with h n influenza viruses spanning from april of to june of . this map demonstrates that there is lack of antigenic drifts during the first wave of this pandemic influenza as all of these viruses are mixed altogether. our limited studies on h and h avian influenza viruses suggested the temporal model is not needed for avian influenza viruses. however, extensive studies are required to investigate whether there is any special data structure present in this type of data. in this study, we described in details the concepts and applications of new computational method, temporal mc-mds for influenza antigenic cartography construction. we formulate the influenza cartography as two integrative steps: low rank mc problem from the concept of netflix movie recommendation system and mds from geographic cartography construction. in order to handle two additional challenges, including low reactor and non random distribution of antigenic data, a temporal model is incorporated into mc-mds as temporal mc-mds. our applications demonstrated that temporal mc-mds is effective in constructing influenza antigenic cartography. the three dimensional antigenic cartography for a ⁄ h n seasonal influenza virus without temporal model, and the antigenic clusters were defined in ref. [ ] ; (b) the three dimensional antigenic cartography for a ⁄ h n seasonal influenza virus with temporal model; (c) the two dimensional antigenic cartography for a ⁄ h n pandemic influenza without temporal model, and these viruses were labeled in shape by the corresponding month for them to be detected. one grid is corresponding to a twofold change in hemagglutination inhibition experiment. the mechanisms driving the three waves of infection and mortality in the uk in - are uncertain. although the circulation of three distinct viruses could have generated three waves of infection, the virological evidence required to prove or disprove this hypothesis is lacking. social distancing, an alternate mechanism for generating fluctuations in the effective susceptible pool and therefore explaining multiple waves of infection, , was not generally imposed in the uk as it was in the us and australia. we are therefore motivated to explore the possible role of continual population-level changes in the average protective response against the circulating virus in generating a multi-wave pandemic, within a biologically motivated deterministic model for influenza transmission. the nature and duration of protection against further infection following recovery from influenza is uncertain and depends on the mode and tempo of viral evolution, as well as the response of the cellular and humoral arms of the adaptive immune system. for a given seasonal ⁄ pandemic strain, memory b-cells may generate a specific antibody response in a portion of the adult ⁄ elderly population, depending on the exposure to related antigenic sub-types. however neutralising antibodies are unlikely to be a widespread immunological response to a novel (pandemic) strain. memory t-cells which recognise conserved internal viral proteins may be a more common mechanism for protection; the generation of very high levels of cytotoxic cd + t-cells potentially facilitates rapid viral clearance, , and lower levels of cd + t-cells perhaps provide partial protection. in this work we explore key drivers of multi-wave pandemics within phenomenological models that incorporate different immune response mechanisms building on existing models , incorporating the role of evolving population-level protection in multi-wave pandemics. we use weekly reports of influenza mortality rates for five administrative units in the uk (blackburn, leicester, newcastle, manchester and wigan) where records from block censuses instigated by local medical officers to record the cumulative incidence of reported symptoms in each wave in a sample of or more households are also available. the symptom reporting data allows us to estimate the case fatality rate and thus use the mortality time series to constrain our transmission model. furthermore, the incidence of individuals reporting symptoms in multiple waves provides information about the acquisition and loss of immunity. we extract the death rate and symptomatic (re)infection rates predicted by our model prevalence for a given set of parameters and estimate a likelihood-based on a comparison to all the death and cumulative reported incidence data assuming a negative binomial error distribution. we utilise monte carlo markov chain (mcmc) methods with parallel tempering algorithms to maximise this likelihood and obtain parameter estimates. parallel tempering -which concurrently searches for maximal likelihood parameter solutions on a set of scaled likelihood surfaces -allows for relatively rapid exploration of the parameter space. we use bayesian information criteria (combined with qualitative assessment of biological plausibility) to aid model selection. we have implemented a deterministic compartmental transmission model, which allows for a variety of phenomenological modes of protection against the pandemic virus. to facilitate this, we stratify the population into two groups; the 'experienced' population (stratum ) who have had been exposed to an influenza virus and the 'naive' population (stratum ) who have not. in each stratum, i hosts may be classified as either susceptible s i , exposed e i and e i , having (recovered from) a symptomatic i i (r i ), or asymptomatic a i (ra i ) infection. note that the states tq i , tq i , e i , t i , and t i are included so that the hosts move between the key epidemiological states with a peaked (rather than exponential) distribution of waiting times. hosts in the experienced stratum may exhibit reduced susceptibility, infectiousness, and symptomatic proportion compared to naive hosts, parameterised by e i , e s , and e a , respectively; however note that depending on the model parameters, there may be fully susceptible hosts within the experienced stratum. in addition, we assume homogeneous population mixing and a constant basic reproduction number r with the force of infection: modulated by a sinusoidal seasonal term with amplitude b with phase chosen to maximise transmission in the winter season. here n is the total population size, and x e is the initial fraction in the experienced strata. the proportion of symptomatic cases a and the case fatality rate l are permitted to vary from wave to wave (and given indices , or accordingly). the transmission dynamics is described by the following set of coupled ordinary differential equations. where s in, = p utq i and s in, = in order to divert recovered infectious hosts from the naive stratum into the experienced stratum. the probabilities of gaining permanent protection are q = q and q = . the latent exposed period is fixed to be c = ⁄ ae days, and the rate of recovery is parameterised by m = ⁄ t inf , where t inf is the infectious period. hosts with prior sterilising protection begin in q and move into s at rate u q = ⁄ t wq . recovered hosts (r i ) migrate back to s at a rate u = ⁄ t w . the state p contains hosts with permanent protection. the modes of protection captured in this model are: i. permanent prior protection (beginning in state p ), ii. waning prior protection (beginning in state q ), iii. permanent acquired protection with probability q (moving into state p ), iv. waning acquired protection with probability ) q, and, v. partial prior protection (beginning in state s ) resulting in reduced infectiousness (e i ), susceptibility (e s ), and symptomatic proportion (e a ). in the context of this model, 'permanent' protection refers to protection which lasts for the duration of the epidemic. here we explore the results of parameter fitting to two models which differ in the nature of the assumed pre-existing protection in the community at the beginning of the pandemic. protection hypothesis assumes that the prior protection is sterilising but temporary, whilst protection hypothesis assumes that the prior protection is partial but permanent and may act on susceptibility, infectiousness, and ⁄ or asymptomatic proportion. each model allows waning acquired protection and for a proportion q of the experienced population to gain permanent protection following infection. fitted parameters common to each model are t inf , b , q, t w , a, l and the proportion beginning in p x i . prior protection hypothesis : sterilising, waning prior protection we fix x e = and fit for q (t = ) ⁄ n and t wq so that protective modes i, ii, iii, and iv are enabled ( figure ). it is important to note that due to the slow convergence of the mcmc chains, we cannot guarantee that our parameter estimates correspond to the global minimum. furthermore, parameter estimates can only be meaningfully interpreted for good fits to the data. due to the prediction of a fourth (unobserved) wave for the model fit to blackburn, we do not report these parameter estimates here. the fits to the leicester data are generated with the parameter set r = ae , a = ae , a = ae , a = ae , t w = ae years, t wq = ae years, we fix q (t = ) ⁄ n = and fit for x e , e a , e i , and e s so that protective modes i, iii, iv, and v are enabled (figure ) . the parameters corresponding to the fit in figure for leicester are r = ae , a = ae , a = ae , a = ae , t w = ae years, p (t = ) ⁄ n = ae , s (t = ) ⁄ n = ae , b = ae , t inf = ae days, q = ae , e a = ae , e i = ae , and e s = ae . our model with protection hypothesis -which, similarly to the model discussed in ref. [ ] , assumes that a sub-population has waning sterilising prior protection -is able to generate multiple waves of infection via the continual replenishment of s from an initially large proportion (over %) of hosts with prior protection in q combined with the waning of acquired immunity in around % of cases on a time-scale of months. disease severity as measured by symptomatic proportion increases from % in the first wave to above % for the second and third waves. over a quarter of the population are initially permanently immune, and a large r value of ae drives transmission in the remaining population. protection hypothesis -which assumes that prior protection offers partial susceptibility and ⁄ or reduced infectiousness or symptomatic disease -performs slightly more poorly; the fit to the leicester data has an inferior likelihood (although the mortality data only likelihood is a little larger), despite the higher dimensionality of the model. nevertheless, the model fit still mirrors many characteristics of the data, particularly for leicester. we note that for this model, a is very near the lower limit, corresponding to ubiquitous exposure in the first wave. in this scenario, refuelling of the susceptible pool to generate secondary and tertiary waves is still possible due to a shorter waning time of acquired protection (well within months) and a lower probability of gaining permanent protection following infection, when compared with the parameter estimate for hypothesis . the parameter estimates suggest that approximately % of the population initially experiences reduced disease severity (e a $ ae ), but similar susceptibility and infectiousness. a larger value for r $ ae is required to drive transmission despite low numbers beginning in p , due to the large number of hosts who acquire temporary or permanent immunity early on in the pandemic. it is clear that, at least mathematically and perhaps biologically, there are multiple possibilities for the structure of population-level protection which are compatible with the generation of multiple pandemic waves. however, whilst the models considered here are able to explain the observed mortality and reinfection data for some patterns of infection and mortality (e.g. leicester), they are not consistently able to reproduce a pandemic which dies out after three waves across the connected populations we are studying (e.g. for blackburn). it is challenging to construct a deterministic model for the spread of disease within multiple locations in the uk in , which assumes homogeneous mixing without modulation of the transmission rate by social distancing. an improved model working with these assumptions likely requires a richer structure for the host protection response than the structures we have explored thus far. we are currently seeking improved fits to the data by implementing a number of biologically defensible exten-sions to our model, including incremental immunity whereby t w increases by a factor v after each exposure to the pandemic flu, and incremental loss of prior protection whereby a increases as hosts lose their sterilising prior protection. it is important to note that the mechanism(s) generating differences in the pandemic experience recorded in geographically connected locations is an open question; true differences in demography, varying degrees of reactive social distancing, inhomogeneities in the circulation (or circulation history, i.e. prior immunity) of viral strains, stochastic variations, and ⁄ or unique socio-cultural ⁄ behavioural conditions may all contribute to this effect. the h n experience in australia and elsewhere highlighted the difficulties faced by public health authorities in diagnosing infections and delivering antiviral agents (e.g. oseltamivir) as treatment for cases and prophylaxis for contacts in a timely manner. consequently, forecasts from mathematical models of the possible benefits of widespread antiviral interventions were largely unmet. we summarise results from a recently developed model that includes realworld constraints, such as finite diagnostic and antiviral distribution capacities. we find that use of antiviral agents might be capable of containing or substantially mitigating an epidemic in only a small proportion of epidemic scenarios given australia's existing public health capacities. we then introduce a statistical model that, based on just three characteristics of a hypothetical outbreak [(i) the basic reproduction number, (ii) the reduction in infectiousness of cases governments and public health agencies worldwide, spurred by outbreaks of sars and h n , have developed preparedness strategies to mitigate the impact of emerging infectious diseases, including pandemic influenza. pandemic response plans are presently being revised in light of the h n experience. [ ] [ ] [ ] many developed countries amassed large stockpiles of neuraminidase inhibitors (nais) with the expectation that they could be used to not only treat the most severely ill, but curb transmission in the community. without relevant field experience indicating how nais should be distributed, mathematical and computational modelling has been used to inform optimal deployment policy in a pandemic scenario. - models of population transmission were used to infer likely effects on epidemic dynamics, using data from human and animal studies of experimental infection and nai efficacy trials. in the australian (and wider) context, models indicated the potential for substantial benefit at the population level if nais were distributed in a liberal manner, targeting close contacts of indentified cases. furthermore, results indicated that use of limited nai resources in this way may improve the impact of case treatment due to the effects on epidemic dynamics. however, these models did not take into account logistic and other real-world constraints, such as finite diagnostic and antiviral distribution capacities, which were identified as limiting factors during the australian h n pandemic response. [ ] [ ] [ ] in particular, if using positive pcr diagnosis as a 'decision to treat' test, delays to confirmation of diagnosis, particularly once total laboratory capacity was exceeded, prevented timely delivery of nais to both cases and contacts of cases. in previous work, we have extended our existing models to examine how diagnostic strategies [e.g. using pcr confirmation versus syndromic influenza-like illness (ili) presentation as a decision to treat], diagnostic-capacity, and nai distribution capacity each impact on the ability to deliver an effective intervention. the model uses case severity (the proportion of infections deemed severe) to determine the overall presentation proportion, and so the ability to identify individuals eligible for nai treatment and contact prophylaxis. figure (a) shows a key result from the model. for each curve shown, we simulated thousands of epidemics, sam-pling across plausible ranges of parameters describing virus, population, and intervention characteristics using a latin hypercube sampling (lhs) approach. without intervention, the proportion of the population infected either symptomatically or subclinically by the end of the epidemic is around %. if a syndromic strategy (ili presentation) is used to determine provision of nais as treatment and prophylaxis, excessive distribution of drug to individuals who are not infected with influenza occurs early in the epidemic. early stockpile expiry accounts for a marginal impact of the antiviral intervention on the final outbreak size, in the order of a few percent. the second strategy modelled (pcr ⁄ syndromic) is one where pcr confirmation of diagnosis is required early in the epidemic to make treatment decisions until such time as laboratory capacity is exceeded. from this point, individuals are treated on the basis of symptoms alone -during an epidemic phase in which a substantial proportion of ili presentations will be attributable to influenza. under this strategy, the intervention is able to control the outbreak in approximately % of the simulated epidemics given the 'base case' constraints on diagnosis and delivery assumed in the model. the results highlight that a successful antiviral intervention requires a highly sensitive diagnostic strategy in the initial stages of the epidemic and comprehensive distribution of post-exposure prophylaxis. a pcr ⁄ syndromic strategy for decision to treat and provide contacts with prophylaxis is thus optimal. the surface in figure (b) shows the percentage of simulation runs for the pcr ⁄ syndromic strategy that have a final population attack rate of < % (a substantial reduction from the no intervention case of approximately %) as a function of pcr capacity and nai daily distribution capacity. as indicated by the arrow, the estimated australian pcr laboratory capacity appears to be sufficient, while significant benefits for the public health outcome may be achieved if logistical delivery constraints for nai distribution can be ameliorated. however, the probability that such an interventioneven with substantial increases in pcr and nai distribution capacity -would successfully mitigate an epidemic is low ( - %), and consequently it is difficult to universally recommend an antiviral intervention. in this study, we introduce a statistical model that predicts whether or not an nai distribution strategy based on a pcr ⁄ syndromic antiviral distribution policy will be successful in mitigating an epidemic. we thereby provide proof-of-principle for the design of a decision support tool that may be used by public health policy makers during an epidemic when faced with formulation of context specific nai distribution policy. synthetic data of hypothetical outbreaks and interventions were generated using the lhs simulations developed in ref. [ ] . we selected a random sample of outbreaks from a total of simulated epidemics ( % of model simulations). using these data, we identified independent model parameters that were most highly rank-correlated with the final attack rate. these parameters were included in a logistic regression model to assess their ability to predict whether an influenza epidemic would be successfully mitigated by an antiviral intervention (ar < %). model predictions were then validated against the full simulated dataset. full details of the simulation model, its structure, parameterisation and parameter distributions are available in ref. [ ] . use of the lhs simulation approach, and the method of model analysis and evaluation was similar to that previously described. matlab a (mathworks, natick, ma, usa) was used for the analysis and statistical model fitting. table shows results from our logistic regression model. key parameters sufficient to predict whether or not an outbreak may be controlled by the deployment of av agents are: . r , the basic reproductive number of the outbreak (assigned values between ae and ae for this example). as the value of r increases, the epidemic progresses more rapidly and is more difficult to control, explaining the negative correlation coefficient. . e t , the relative infectiousness of treated individuals (assigned values between ae and ae ). higher values for this parameter indicate only modest drug effects on transmission, explaining the negative correlation coefficient. . g, the proportion of infections that are severe (assigned values between ae and ae ), and which in turn determines the presenting proportion (derived values between ae and ae ). as the presenting proportion increases, the ability to identify and treat cases and deliver prophylaxis to contacts also rises, increasing the impact of the antiviral intervention. the roc curve ( -specificity versus sensitivity, not shown) for the logistic regression model specified in table has an area under the curve of ae , demonstrating that the model predicts the success of an antiviral intervention extremely well. for example, with a sensitivity of % we still have a specificity of approximately %. evaluation of the pandemic response has emphasised the need for early informed decision-making to implement proportionate disease control measures. our model identifies a low probability of successful epidemic mitigation using targeted antivirals alone (figure and ref. ), in distinction to results from models that fail to account for the diagnosis and delivery constraints inherent in any public health response. the decision support tool (table ) highlights key epidemic characteristics that are predictive of a high likelihood of effective mitigation. the reproduction number was one of the earliest parameters estimated from early outbreak data during the h n outbreak. , our findings reinforce the importance of characterising epidemic severity as early and as accurately as possible, in order to inform a proportionate pandemic response. critically, a typically mild pandemic (low g), such as that experienced in , is predictably difficult to contain using a targeted antiviral strategy due to the low proportion of infectious cases that present to health authorities. the relative infectiousness of treated individuals, e t , is strongly negatively correlated with successful mitigation, perhaps a surprising result given the model's underlying assumption (based on available epidemiological and human clinical trials data) that e t lies in the range [ ae , ]. that is, nais provided as treatment have a maximum impact of just a % reduction in infectiousness. however, our previous results show a strong synergistic effect of treatment when overlayed on a contact prophylaxis strategy, explaining the observation here that e t is critical in determining likely success of an intervention. despite the limited impact of treatment at the individual-level, the model outcomes are highly sensitive to the value of the relative infectiousness of treated cases. it follows that determination of e t is important for predicting the population-level outcome of a control effort. a 'small' reduction (of the order approximately %) may be extremely valuable in terms of success of a public health control strategy, and so should not be discounted. using a mathematical model which takes into account some of the key logistic constraints that are inherent to healthcare responses, we have derived a logistic regression model for estimating the probability that an antiviral intervention based on liberal distribution of nais as treatment and prophylaxis could successfully mitigate an influenza epidemic. the model demonstrates an excellent degree of accuracy when applied to synthetic data. the choice of parameters for the regression model was restricted to those that were both highly correlated with the success of the intervention and hopefully feasible to measure during the early stages of an emerging epidemic. the model could therefore be a useful near real-time decision support tool for public health policy in the face of an influenza epidemic, although further validation on a range of synthetic data (and real-world data where available) is required. influenza to seasonal flu status to avoid overstretching the demands on healthcare services. a great deal of information has emerged as the result of the pandemic response exercises conducted by affected countries. however, uncertainties remain regarding the effectiveness of intervention measures, as well as the feasibility and the timing of their implementation. mathematical and computational models [ ] [ ] [ ] have been used to project the outcomes of influenza outbreaks under various scenarios and epidemiological hypotheses. motivated by the events of and public health measures adopted by the taiwan cdc, we use a stochastic, individual-based simulation model to study the spatio-temporal transmission characteristics of the h n virus, so as to quantitatively assess the effects of early intervention strategies. our stochastic disease simulation model builds upon a highly connected network of individuals interacting with each other via social contact groups. to represent the daily interactions of approximately million people living in taiwan, we constructed a computer-generated mock population based on national demographic and employment statistics (to derive daily commute patterns) from the taiwan census (http://www.stat.gov.tw/). each individual is created with a set of attributes, including age, sex, residence, family structure, and social standing (employment status, etc.). based on their attributes and the time of day, each individual is assigned to miscellaneous contact groups, where the potential of interactions between any two individuals resulting in flu virus transmission occurs. such epidemiological properties are defined by empirically parameterized attributes such as basic reproduction number r , transmission probability, contact probability and associated probability distributions outlining the disease's natural history. additionally, intervention measures are implemented as scheduled events that could alter control parameters during the course of a simulation run. the targeted basic reproduction number (r ) in all our simulations is ae , following the suggested range by who of ae - ae . as the latent ⁄ incubation and infectious periods for h n have not yet been reliably ascertained, we adopt the natural history of the and pandemic influenza viruses. , here, the latent period ranged from to days, with a median value of ae days. the infectious periods begin day prior to symptom onset and can continue for - days, with a median value of ae days. twothirds of the infected individuals will develop clinical symptoms, and the asymptomatic cases will have half the infectious strength. the efficacy of antiviral drugs (oseltamivir) and vaccines are based on these studies. , for the source region of the infected cases, we use the north american continent (canada, mexico and united states) with an estimated total population of and an average hours of flight time to taiwan. the average daily passenger number is based on the annual statistical report on tourism, tourism bureau, taiwan (http://admin.taiwan.net.tw/english/statistics/year.asp? relno= ). each simulation lasts days and starts with a baseline simulation of r % ae h n pdm outbreak at the source region. the outbreak was adjusted to approximate clinical attack rate (car) in the united states, april -march , . we estimate the daily number of imported cases according to average daily passenger numbers and their probability of holding a disease status. we then apply airport exit ⁄ entry screening per corresponding success rates, by subtracting the number of identified symptomatic cases. we also consider latently infected passengers with inflight disease progression, by fitting a gamma distribution to the cumulative distribution of time to onset data with hours average flight-time, as presented by pitman et al. the daily imported cases are seeded according to the traveling patterns of foreign tourists and residents returning home. from the disease's natural history, we derive that roughly % of the infected travelers present no symptoms; the percentage increases if most symptomatic individuals elect not to travel in their condition, or are stopped by airport screening. we use the official epidemic data provided by the taiwan cdc to calibrate the simulation model and perform regression analysis on scenario parameters. this data is a close estimation of the weekly new clinical cases of h n pdm patients. it consists of weekly opd (outpatient department) icd- code (influenza) tallies collected by the bureau of national health insurance, taiwanadjusted to exclude seasonal flu patients and to account for uninsured patients. we formulate our scenario settings according to events in taiwan, and establish settings to approximate the actual events. with domestic events and intervention schedules fixed in time, the start date determines the simulation outcomes and the data range for selected indicators, such as the mean car, the epidemic peak, and several significant dates for the incoming index case events. we plot the taiwan weekly h n opd cases alongside the weekly new clinical cases from our simulation results in figure . our simulations not only capture the epidemic trend, but also pick out the most likely date, may , for identifying the first symptomatic case at airport screening based on practical assumptions. we further analyze the effectiveness of various mitigation measures with february , as the empirical start date for h n pdm in north america. the simulation result confirms that by the time we identified the first symptomatic case at the border screening, infected cases had already made their way to the public. by our calculation, roughly four such cases had passed in each of our scenario settings, with the first case happening as early as weeks before detection. figure also highlights the importance of the timing for the implementation of mitigation measures; for example, a -day-delay of the identical intervention plan results in nearly an additional % of the population being infected. therefore, the rule of thumb for healthcare officials is to implement intervention measures as early as possible. in our study, we have ignored the possibility of inflight transmission and any false positive results by airport screening procedures. to assess the effectiveness of each mitigation strategy of interest and their combinations, we take the calibrated simulation model and perform simulation realizations for groups of scenarios containing only those intended mitigation measures, and analyze the averaged results. for example, in the airport exit screening policy only scenario, the first imported symptomatic case can be delayed up to months, and the epidemic peak can be delayed up to days. as the data suggests, the exit screening policy alone has very little impact on car. combining various screening success rates for both exit and entry screening allows us to quantitatively assess their beneficial ramifications on the epidemic. for example, there is very little additional benefit between % and % suc-cess rates for entry screening policies when exit screening policies are adequate, as the enhanced border screening only delayed the epidemic peak by day, and reduced car by < ae %. base on this result, the government should not attempt to exhaust all its resources in securing the border during a pandemic event, because the return of such a policy will be disappointing. instead, a response plan with a shifting focus on health resource allocation and the capacity of adjusting intervention strategies in line with the developing epidemic will be most effective. based on the same principle, we perform experiments with assorted scenarios, including relaxing entry screening policies after identifying the first imported symptomatic case, mass vaccination based on the actual vaccination schedule of h n pdm in taiwan, and altering the start dates of the vaccination schedule. our results show that with a reasonable reduction in the airport entry screening success rate, we conserve valuable healthcare resources, but loose a few days for the strategic planning and preparation of subsequent response measures. in other simulation scenarios, a national vaccination campaign has very little impact on the outcome, due to the late start of the vaccination schedule. we then explore the effect of a national vaccination campaign with various starting dates. the simulation results are illustrated in figure , where the benefit of an early start date for mass vaccination is clearly demonstrated. considering a scenario with an % airport exit screening success rate, % airport entry screening success rate and % symptomatic case tracing success rate, the combined intervention strategy results in: a % reduction in car if the vaccination campaign starts in mid-november; % reduction if the campaign starts in mid-october; % reduction if the campaign starts in mid-september; and % reduction if the campaign starts in mid-august. in retrospect, the taiwanese government's response to h n pdm proved to be effective. first and foremost, it initiated enhanced border monitoring and on-board quarantine inspection as soon as the threat of a flu pandemic became clear. at the same time, the domestic preparations towards h n pdm were escalated, such as antiviral drug stockpiling and distribution, and vaccine acquisition. as the h n cases increased worldwide, various revised plans were adopted and implemented; such as the shift from labor-extensive on-board quarantine inspection to the notifiable infectious disease reporting system and realtime outbreak and disease surveillance system in order to effectively track down symptomatic and exposed passengers, apply prophylaxis treatment and mandatory in-home quarantine. as a result, all h n pdm related statistics are well below the international average. in modern society, countries rely heavily on the global economy for their own prosperity. shutting down the border for any length of time is not only costly, but could have disastrous economic effects that linger long after the event is over. moreover, with nearly % of the infected passengers presenting no symptoms whatsoever, they are not detectable by any port authority's screening procedures, and the importation of the novel flu virus is therefore inevitable. many studies conclude that entry screening is unlikely to be effective in preventing or delaying the importation of influenza, and has negligible impact on the course of subsequent epidemic. however, these studies are based on the assumption that effective exit screening is in place. our study shows that as the exit screening success rate decreases, the sensitivity of the entry screening policy becomes more pronounced. with the same methodology, we can also study the effects of varying the length of flight time, or the disease's incubation time. lastly, the benefit of entry screening is even more crucial for a small island country such as taiwan, since all incoming traffic must go through the port authority where entry screening can be enforced. in england and wales, three waves of the pandemic struck in summer, autumn, and winter seasons of - . although the proportion of people reporting symptoms was often greater in the first wave, - a puzzling feature was the much higher mortality in the second wave, in which . % of the population died, compared with . % in the out-of-season first wave and . % in the third wave. an obvious hypothesis to explain the changes in mortality from wave to wave would be that the virus mutated to higher virulence after the (lower mortality) first wave. although pandemic virus reconstituted from the high mortality waves has proven to have high virulence in animals, it has not been possible to recover virus from the first wave in for comparative purposes. indeed it is questionable whether virulence mutation(s) occurring between wave and wave could have spread to so many different populations in the time-frames observed. furthermore, in all three pandemic waves, there was the same agedistribution of mortality, with more deaths occurring amongst younger adults than older adults. [ ] [ ] [ ] this 'pandemic signature', arguably due to immune protection of older adults who were exposed to a similar virus in the years before , , suggests that the - viruses were at least immunologically similar in all three waves. a second hypothesis would be that the higher case fatality in the later waves was due to higher rates of complicating bacterial pneumonia, to increased transmission of influenza virus in the cooler months of the year, or to other seasonal effects. we have considered a third (immunological) hypothesis to explain the greatly increased mortality in waves and . the underlying idea is that the mortality rate in the first wave was lower than in later waves because most persons were protected by prior immunity in the first wave, and that the mortality was higher in later waves because of waning of that short-lived immunity. this hypothesis builds on our earlier modelling papers suggesting that even before the first wave in , military, school, and urban populations in england and wales apparently had (short-lived) immune protection, presumably induced by recent prior exposure to seasonal influenza. [ ] [ ] [ ] we suggest that this short-lived strain-transcending protection was in addition to the longer-lasting immunity, presumably induced by exposures to a similar virus circulating prior to , that arguably reduced pandemic mortality for older adults in - . , cumulative mortality rates attributed to pandemic influenza were available for each of the three waves in - for populations in england and wales. we have built immunological models to potentially explain the variation in mortality rates across waves and populations. to show proof of principle, we have fitted these models to mortality data from a randomly selected sub-set of twenty populations. our key assumption was that the risk of a fatal infection would be limited to persons with inadequate immunity who were being exposed to the pandemic virus for the first time. persons who were exposed and who survived an earlier wave were assumed to be protected against death in a later wave. model a and assumptions (see figure ) before the first wave, we assumed that people could be fully susceptible (s ), or partially protected (q ), or fully protected (p) by prior immunity which was not necessarily specific for the new virus. we assumed that exposure to the new pandemic virus would be fatal (m) in a proportion h of fully susceptible persons who were actually exposed (e) in the relevant wave. for those surviving that first exposure, it was assumed that they would be permanently protected against death in later waves by an immune assumed that viral exposure and multiplication would induce an immune response specific for the pandemic virus that would protect them against death in that wave and in subsequent waves. in contrast, for persons with strong prior immune protection, p, the virus would not be able to multiply to induce pandemic-specific immune protection. between waves, it is assumed that due to the waning of non-specific prior immunity, persons in the p state can move to the q state, and persons in the q state can move to an s state before the next wave. the proportion (e) of susceptible persons exposed to productive infection in each population was estimated by applying the following version of the final size equation to the proportion susceptible (s & q) in each wave, for each population: note: in both figures and , we have omitted the flows out of the q and e states that removed persons from the risk of death. parameters: s = proportion fully susceptible to infection and death before wave ; q = proportion susceptible to immunising infection, but not to death from exposure in wave ; p = proportion temporarily protected against both immunising infection and death from exposure in wave ; n = proportion even more protected against both immunising infection and death from exposure in wave (model b only); r = basic reproduction number (the average number of secondary cases for each primary case) in a fully susceptible population; f = proportion moving from q to s between waves; g = proportion moving from p to q between waves; d = proportion moving from n to p between waves (model b); h = proportion of e that actually move to m and die. model a could provide a very good fit for the summer, autumn, and winter waves of the - pandemic (results not shown). however, because of the replenishment of the pool of susceptible persons over time, model a also predicted a fourth wave of influenza in the spring season of . as no such wave was seen, and as we could not find parameters values for model a that did not predict a fourth wave, we must regard model a as inadequate. model b was similar to model a, but with an additional stage of prior immunity (n), which could wane to p. model b allowed us to not only fit the three observed waves, but also to fit the imputed data (zero cases) corresponding to the absent fourth wave. following earlier work, , we used a bayesian approach with markov chain monte carlo (mcmc) procedures to estimate model parameters, and we used hyper-parameters to allow for parameter variation between populations. the initial conditions were specified by the parameters: p , q , s and n . from these and the other parameters, it was possible to simulate the behaviour of model a over three waves, and of model b over four waves, and to estimate the expected numbers dying in each wave in each population. we calculated the log likelihood of the observed numbers of deaths given the parameter estimates, and we used mcmc simulation to generate the posterior distributions of parameters. although we obtained an excellent fit between observed and expected numbers of deaths in each of the three waves for the populations for model a, we could not find parameter values for model a that would fit the three observed waves without giving rise to a fourth wave in the spring of . accordingly, in the modified model b, we allowed for an additional stage of prior immunity (figure ) , and we fitted the model to the same data, plus imputed data corresponding to 'the absent fourth wave'. we obtained a very good fit to the three observed waves and the absent fourth wave in each population. the % credibility intervals for parameter estimates, derived from the posterior distributions of the hyper-parameters were: h = . - . , s = . - . , q = . - . ; n = . (fixed); p = ) s ) q ) n ; f = . - . ; g = . - . ; d = . - . and r = . - . . this analysis had allowed all parameters to vary from population to population under the constraints of the hyper-parameters. however, several of the biologically determined parameters might be expected to be more constant from population to population, whereas those dependent on mixing history and other social characteristics which vary more widely from population to population. to test this possibility, we fixed the mean values for the more biological parameters (f = . ; d = . ; g = . ) and estimated the % credibility intervals for the others as: h = . - . ; s = . - . , q = . - . ; and as before n = . (fixed); in a subsequent paper we will be able to provide more details of the method, the robustness of the assumptions, and the results from fitting to many more populations. this short report suggests that the observed patterns of mortality in england and wales over the three waves of the - influenza pandemic , can be explained by an immunological model. in particular, the lower mortality in wave one can be explained by the assumption of protective immunity antedating the first wave, arguably induced by prior exposure to seasonal influenza. , the much greater mortality in wave two can be explained by the waning, between wave one and wave two, of that short-lived and less-specific immune protection. the somewhat lesser mortality in wave three and the 'absent fourth wave' can be explained in terms of the progressive acquisition of immunity specific to the pandemic virus. the credibility estimates for parameters are of potential interest. for example, r estimates of . - . across different populations are consistent with our earlier findings. , if all persons had been susceptible, such r values imply that the virus would have infected most people in all populations. however, even in the first wave, the proportion susceptible, s + q , was < % in all populations, so that a considerable number of persons escaped productive infection in that wave; as their immunity waned, they became susceptible to infection in the later waves. it is likely that the variation in r between populations is due to different rates of population mixing. estimates for h indicate that between % and % of infections in the most susceptible persons were fatal; the higher values of h could reflect higher rates of secondary bacterial infection in the most socially disadvantaged and overcrowded populations. although we have shown the plausibility of an immunological explanation for wave to wave changes in pandemic mortality, we cannot assume that our particular model is even approximately correct. nor can we exclude the possibility that the higher mortality in the later pandemic waves in - was because of genetic change in the virus in later waves, or because of changing rates of secondary bacterial infection or seasonal effects. nevertheless, there is growing evidence that the population spread of pandemic influenza, whether in - , or in , , can be constrained by significant prior immunity, even for viruses that are ostensibly novel. previous reports, reviewed in ref. [ , ] , support the idea of strain-transcending immune protection, which can wane over periods of a few months. this form of protection, probably induced by recent exposure to seasonal influenza, may not be mediated by hi or neutralizing antibody. in contrast, strain-specific immunity, most often mediated by hi or neutralizing antibodies can be so long-lasting that after several decades it will still provide significant protection against any closely-related virus that re-appears in the population. it has not escaped our notice that although attack-rates in the h n pandemic were low in many countries, with generally mild symptoms, the virus did cause lifethreatening illness in a small proportion of younger affected persons. it seems likely that those who were most severely affected in were doubly unlucky: they had missed out on seasonal influenza infection or vaccination in the preceding season(s), and they were born too late to have been protected by the closely-related viruses that are thought to have circulated before . during the early phases of the influenza pandemic in italy, real-time modeling analysis were conducted in order to estimate the impact of the pandemic. in order to evaluate the results obtained by the model we compared simulated epidemics to the estimated number of influenza-like illness (ili) collected by the italian sentinel surveillance system (influnet), showing a good agreement with the timing of the observed epidemic. by assuming in the model mitigation measures implemented in italy, the peak was expected on week ( % ci: , ). results were consistent with the influnet data showing that the peak in italy was reached in week . these predictions have proved to be a valuable support for public health policy makers for planning interventions for mitigating the spread of the pandemic. mathematical models have recently become a useful tool to analyse disease dynamics of pandemic influenza virus can-didates. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] as of april , after the pandemic threat emerged worldwide, it was crucial for policy makers to have early predictions on the possible spread of the pandemic influenza virus in order to support, with quantitative insight into epidemic, policy decisions. thus, after the first pandemic alert was announced by the world health organization (who) in late april , a national crisis management committee headed by the minister of health was established in italy in order to provide weekly advice to the italian ministry of health. real-time analyses using an individual based model were undertaken. the transmission model was previously used for evaluating the effectiveness of the control measures adopted in the national pandemic preparedness plan and for assessing the age-prioritized distribution of antiviral doses during an influenza pandemic. to parameterize the transmission model, we used data derived from the national surveillance system until june and estimates of key epidemiological parameters as available at that time. in order to provide a preliminary assessment of the model predictions performed during the early stages of the epidemic, we compare model predictions with surveillance data of influenza-like illness (ili) available since august . after the first pandemic alert was announced by the who in late april , a national active surveillance system for the pandemic influenza was set up from april to july . however, over the period from april to october , surveillance systems, laboratory testing, and diagnostic strategies have varied considerably in italy. since end of july , following who recommendations, the focus of surveillance activities has changed in reporting requirements, as active case-finding became unsustainable and unnecessary. for this reason, the ministry of health (ministry of health, available in italian at the website: http://www.normativasanitaria.it) requested regional health authorities to report the weekly aggregated ili cases according to a new case definition (sudden onset of acute respiratory symptoms and fever > °c plus at least one of the following systemic symptoms: headache, malaise, chills, sweats, fatigue; plus at least one of the following respiratory symptoms: cough, sore throat, nasal obstruction). by october , following the increasing number of cases, the sentinel influenza surveillance system (influ-net available at: http://www.flu.iss.it) became the official surveillance system for ili cases in italy (ministry of health, available in italian at the website: http://www. normativasanitaria.it). since , influnet is routinely based on a nation-wide, voluntary sentinel network of sentinel community based physicians in the regions and autonomous provinces of the country. incidence rates are, therefore, not based on consultations, but on the served population of each reporting physician each week. influ-net usually consists of an average of (range - ) general practitioners (including physicians and pediatricians) per year, covering about ae - % of the general population, (representative for age, geographic distribution, and urbanization level) reporting ili cases (according with a specific case definition). italian influnet surveillance system is part of the european influenza surveillance scheme (eiss). a stochastic, spatially explicit, individual-based simulation model was used. individuals are explicitly represented and can transmit the infection to household members, to school ⁄ work colleagues, and in the general population (where the force of infection is assumed to depend explicitly on the geographic distance). the national transmission model was coupled with a global homogeneous mixing susceptible-exposed-infectious-removed (seir) model accounting for the worldwide epidemic, which is used for determining the number of cases imported over time. regarding the epidemiological assumptions (e.g., length and shape of the infectivity period, which lead to an effective generation time of ae days), this study is consistent with refs [ , , , ] , but for the proportion of symptomatic individuals, which is assumed to be ae %. the basic reproductive number of the national transmission model was set to ae , according to the early estimates as obtained during the initial phase of the epidemic in mexico in a community setting. , we initialized our simulations through the global homogeneous mixing model in such a way that imported cases were generated until june . this gives a reliable way for fixing the time in the simulations and thus determining the timing of school closure and vaccination in the simulations. the model accounts for school closure for both summer and christmas holidays: we assumed that in these periods contacts among students decrease, while contacts in the general community increase, as in ref. [ ] . we also considered scenarios accounting for partial immunity in the population. in order to investigate the effects of recommendations of the ministry of health (confirmed cases coming from affected areas were isolated for - days, either in hospital or at home) established in the early phase of the pandemic (april-july ), we assumed that a fraction of the imported symptomatic cases were isolated on the first day after the symptoms onset. this recommendation was in place until july . we also assumed, according to the italian school calendar, that schools were closed from june to september for the summer holidays, and from december to january for christmas holidays. the effects of prolonged school closure were also investigated. when considering vaccination, we assumed weeks for the logistical distribution of doses of pandemic vaccine. since at the time of simulation specific recommendations regarding the administration of a single dose of pandemic vaccine from ema were not available yet, we considered the administration of vaccine doses month apart). the pandemic vaccine was considered effective after the administration of the second dose with a vaccine efficacy of %. we assumed the vaccine to be administered by priority, vaccinating first the target population accounting for essential services workers (including health care workers and blood donors), pregnant women at the second or third trimester, and at risk patients (with chronic underlying conditions) younger than years old. the vaccination coverage was assumed %. regarding antiviral treatment and prophylaxis, recommendations of the ministry of health in the initial phase of the epidemic were to administer antivirals to all confirmed cases and to their close contacts. we assumed that the surveillance system would be able to detect % of symptomatic cases. after july , recommendations changed and antiviral treatment was considered only for cases with severe complications and in case of local clusters. since it was difficult to establish the proportion of treated cases, we considered different scenarios: antiviral treatment from % to % of the symptomatic cases. consistently with ref. [ ] , both treatment and prophylaxis were assumed to start day after the clinical onset of symptoms in the index case. treatment was assumed to reduce infectiousness by %, whereas antiviral prophylaxis was assumed to reduce susceptibility to infection by %, infectiousness by %, and the occurrence of symptomatic disease by %. as of july , approximately confirmed cases have been reported to the italian surveillance system for pandemic influenza. during july, the sudden increase of ili confirmed cases suggests for sustained autochthonous transmission in italy. by analyzing the number of ili cases reported to the surveillance during the weeks from to , we found that the exponential growth rate was ae ⁄ week and thus we estimated the national reproductive number to be r = ae . this estimate of the basic reproductive number supports the choice of the value adopted in the model simulations (r = ae ). in the absence of intervention measures, the predicted cumulative attack rate was ae % ( % ci: ae , ae ), and the peak was expected on week ( % ci: , ) with a peak day incidence of ae % ( % ci: ae %, ae %). by assuming case isolation, antiviral treatment, and prophylaxis to % of symptomatic cases until july , the peak was expected on week ( % ci: , ). when considering ae % of natural immunity in the population aged more than years, the peak was expected week later than in the previous scenario, i.e., on week ( % ci: , ). to validate the model, we compared model predictions (which are based only on the available information on the early phases of the epidemic) with ili data (figure ). based on model predictions, we estimated the underreporting factor of influnet ranging from ae to ae , considering different scenarios. by aligning the simulations with the ili data adjusted by the underreporting factor, we can observe that almost all the points in the increasing phase of the epidemic lie within the % ci of the model results (both considering or not natural immunity). the decay phase of the simulated epidemics shows a small delay with respect to the ili data. when introducing single and combined mitigation measures, such as case isolation, antiviral treatment, prophylaxis, and vaccination in the model, results showed that even a low proportion of symptomatic cases treated with antiviral drugs could have led to a relevant reduction in the epidemic size (table ) . we simulated the planned italian vaccination strategy (begun on october ), obtaining a limited but not negligible reduction in the attack rate with respect to the scenarios accounting only for antiviral treatment. moreover, the effect of vaccination would be higher if coupled with antiviral treatment; vaccination would have no effect on delaying the peak incidence. model predictions produced in italy during the early phase of the pandemic influenza are in excellent agreement with italian surveillance data on the beginning of the epidemic (when case isolation, antiviral treatment of index cases, and antiviral prophylaxis to close contacts were implemented by the italian regional public health authorities) and are basically consistent with the influ- net data during the course of the epidemic. the model has been useful for predicting the timing of the epidemic, while it has overestimated the impact of the influenza pandemic for adult and elderly individuals. however, the disalignment is probably due to the model parameterization. based on literature values, , we assumed a similar fraction of cases in the different social contexts considered in the model (namely ⁄ in households, ⁄ in schools ⁄ workplaces, and ⁄ in the general community), since analysis on the relative transmissibility of the virus was not carried out for any country yet. we were also able to estimate an underreporting factor for the influnet data in the range ae - ae . if we focus our attention on the reporting factor computed by considering the total number of cases (instead of symptomatic cases), the resulting value lies in the range - ae %, which is in excellent agreement with the range estimated in ref. [ ] on previous a ⁄ h n influenza seasons, namely ae %- ae %. moreover, based on our results showing that vaccinating % of the italian population was more than adequate to mitigate the pandemic, the ministry of health decided to stockpile a limited number of vaccines. we have also shown that starting the vaccination program in october (or later) could have had only a limited effect on reducing the impact of the epidemic, although it may have been useful to prevent a possible second wave and to protect essential workers and at-risk patients. finally, our results have shown that antiviral treatment would have been the most efficient strategy to reduce the impact of the influenza pandemic, even with a limited antiviral stockpile. a population-wide passive immunotherapy program in this paper, we assume that convalescent plasma (cp) is efficacious in treating severe cases of pandemic influenza. under this premise, we test the hypothesis that a population-wide passive immunotherapy program that collects plasma from a small percentage of convalescent individuals can harvest sufficient cp to treat a substantial percentage of severe cases during the first wave of the pandemic. the proposed program involves recruiting adults (individuals age - years) to donate blood if they have experienced influenza-like symptoms more than weeks ago (to account for the time needed for neutralizing antibodies to build up). the blood samples would be screened for infectious diseases (including hiv, hbv, hcv, htlv, and syphilis, etc., as in routine blood donation screening) and neutralizing antibodies against the pandemic virus. donors whose blood samples are free of known infectious agents and contain a sufficiently high titer of neutralizing antibodies would then be invited to donate plasma by plasmapheresis or routine whole blood donation. qualified donors with higher titers may be given higher priority for plasma donation. in this paper, we use the demographic and logistical parameters of hong kong as a case study. see figure for a schematic of the proposed passive immunotherapy program. we examine the following questions regarding the logistical feasibility and potential benefits of the proposed passive immunotherapy program: (i) what percentage of convalescent individuals (donor percentage) is needed in order for the program to significantly reduce pandemic mortality? (ii) how many severe cases can be offered passive immunotherapy? (iii) what are the ratelimiting factors in the supply of passive immunotherapy? (iv) what are the epidemiologic and logistical factors that determine the demand-supply balance of passive immunotherapy? a more detailed presentation of our results is now available in ref. [ ] . transmission and natural history model for pandemic influenza we use an age-structured disease transmission model to simulate the spread of pandemic influenza. the natural history model is similar to that used by basta et al. , the most important parameter in characterizing the growth of an epidemic is the basic reproductive number r , which is defined as the average number of secondary cases generated by a typically infectious individual in a completely susceptible population. we consider values of r between ae and , which is consistent with recent estimates. , [ ] [ ] [ ] logistical model for the passive immunotherapy program we assume that q d (%) of to year-old individuals who have recovered from symptomatic infections of pandemic influenza donate their blood for screening t r = days after cessation of symptoms. follow-ups of convalescent individuals infected with h n pdm in an ongoing clinical trial of passive immunotherapy suggested that neutralizing antibodies level reaches maximal level around - days after recovery and stays at that level for months after. we assume that q s (%) of these donors are qualified for plasma donation of which q r (%) are recurrent donors who return to donate plasma every t w = days. screening involves both detection of infectious agents and neutralizing antibodies against the pandemic virus. the latter is the rate-limiting step because neutralization tests of pandemic viruses can only be done in a bsl setting. we assume that five bsl -trained technicians are available to test the blood specimens, each running viral neutralization tests in days. therefore, the capacity and turnaround time of blood screening are u s = and t s = days, respectively. hong kong currently has nine plasmapheresis machines which allow a maximal throughput of plasma donations per day (assuming -hour daily operation with each donation taking minutes). therefore, the capacity and turnaround time of plasmapheresis are u p = and t p = ⁄ days, respectively. collected cp are ready for use in transfusion after final quality check, which takes t q = days. we assume that r t plasma donations are required to treat one severe case on average. the expert panel of the abovementioned study of passive immunotherapy for h n pdm in hong kong suggested that r t < . we assume that p h (%) of symptomatic cases will be severe cases for whom passive immunotherapy is suitable. although p h will be smaller than the case-hospitalization rate (passive immunotherapy may not be suitable for some hospitalized cases), we assume that the two have similar ranges and consider p h ranging from ae % to %. because each severe case requires r t plasma donations on average, demand for cp is simply r t p h times the number of symptomatic cases. therefore, r t p h can be regarded as a single parameter, which we refer to as the lumped demand parameter. we define the outcome as the percentage of severe cases that can be offered passive immunotherapy by the proposed program during the first wave of the local epidemic. we refer to this outcome as treatment coverage and denote it by q. we consider the base case scenarios assuming q r = % and q s = %. in general, the treatment coverage q increases sharply as the basic reproductive number r and the lumped demand parameter r t p h decrease (figure a ). in particular, when r is large and r t p h is small, q is very sensitive to r t p h , but insensitive to r . similarly, when r and r t p h are small, q is very sensitive to both. with a donor percentage of q d = %, the proposed program can supply passive immunotherapy to more than % of severe cases (q > %) if r < ae and r t p h < ae %, but < % if r > ae and r t p h > ae %. in general, the treatment coverage q increases sharply as the donor percentage q d rises from %, but with rapidly decreasing marginal increase ( figure b ). when r < ae and r t p h < ae %, q > % even if q d is as low as %, which is comparable to the current average blood donation rate of ae donations per population in developed countries. when q d is > %, q becomes largely insensitive to further increase in q d in most scenarios. the treatment coverage q for q d = % is more than % that for q d = % across all values of r and r t p h considered in the base case. therefore, increasing the donor percentage q d beyond % has a relatively small impact on cp supply. this is because increasing q d can boost supply only when plasmapheresis is not yet the supply bottleneck. for the same reason, once the donor percentage q d has reached %, the treatment coverage q is insensitive to further increase in q d even when the plasmapheresis and screening capacity are doubled ( figure b , lower panel). we conduct an extensive multivariate sensitivity analysis to test the robustness of our base case observations against uncertainties in parameter values. we generate epidemic scenarios by randomly selecting parameter values from their plausible ranges using latin-hypercube sampling. although there are numerous model parameters, the treatment coverage q is mainly determined by three lumped parameters: (i) r t p h , which indicates the magnitude of demand; (ii) q s q d , which indicates the magnitude of supply; (iii) the initial growth rate of the epidemic r (results not shown). while the dependence of q on r t p h and q s q d is readily comprehensible, it is not obvious a priori that q depends on the natural history and transmission dynamics of the disease via only the initial epidemic growth rate. when the plasmapheresis and screening capacity are very large, the supply-demand dynamics is further simplified: the treatment coverage q depends on lumped demand parameter r t p h and the lumped supply parameter q s q d only via their ratio. finally, q becomes insensitive to q s q d when the latter increases beyond - %, which is consistent with our base case observations. our results suggest that with plasmapheresis capacity similar to that in hong kong, the proposed passive immunotherapy program can supply cp transfusion to treat - % of severe cases in a moderate pandemic (basic reproductive number r < ae , lumped demand parameter r t p h < ae %) when the donor percentage is - %. increasing the donor percentage beyond % has little additional benefit because cp supply is constrained by the capacity of plasmapheresis during most stages of the epidemic. increasing plasmapheresis capacity could significantly boost cp supply, especially when there is a substantial pool of recurrent donors to alleviate the dependence of cp supply on donor percentage. in an ongoing clinical trial of passive immunotherapy for h n pdm virus infection in hong kong, % of convales- cent individuals agreed to donate their plasma for the study. therefore, the donor percentage required by the proposed passive immunotherapy program ( - %) is likely to be feasible. in view of the logistical feasibility of such program, we recommend that further clinical studies are conducted to evaluate the safety and efficacy of passive immunotherapy as a treatment for severe cases of pandemic influenza virus infection. our study is based on the premise that cp will be efficacious in reducing morbidity and mortality associated with pandemic influenza. in theory, the polyclonal nature of neutralizing antibodies in cp would lower the probability of an escape mutant emerging in treated patients. further, besides providing neutralizing antibodies against the pandemic virus, cp also might carry antibodies to other bacterial pathogens, which might decrease the severity of coexisting bacterial infections. as such, cp not only might reduce the case fatality rate but might also increase the recovery rate and shorten duration of hospitalization of severe cases. the proposed passive immunotherapy program can thus significantly reduce the burden on the healthcare system, especially the intensive care unit, which will likely be stressed, if not overloaded, at the peak of an influenza pandemic wave, hence benefiting the general public and not only those receiving passive immunotherapy. although the hypothesized efficacy of cp has yet to be proven in clinical trials, our modeling results show that a public health system similar to that in hong kong has the capacity to support a population-wide passive immunotherapy program that can supply cp treatment to a substantial percentage of the severe cases in a moderately severe pandemic. we estimate that compared to other developed countries, hong kong has a relatively low plasmapheresis capacity. our conclusions regarding donor percentage needed and rate-limiting factors remain valid for plasmapheresis capacity ranging from % to % of what we have assumed in the base case (results not shown). our conclusions are robust against uncertainties in the natural history and transmission dynamics of pandemic influenza. our sensitivity analysis shows that the outcome depends on these epidemiological characteristics only via the initial growth rate of the epidemic. as such, our results are applicable not only to pandemic influenza, but also to other emerging infectious diseases for which the time-scales of disease transmission and antibody response are similar to that for influenza virus. the three determinants of treatment coverage (the initial epidemic growth rate, the lumped demand parameter r t p h , and the lumped supply parameter q d q s ) are all readily measurable in real-time during an epidemic. therefore, our methods and results can be used as a general reference for estimating the treatment coverage of the proposed passive immunotherapy program for a given plasmapheresis capacity. background highly pathogenic h n virus continues to pose a serious threat to human health and appears to have the capacity to cause severe disease in previously healthy young children and adults. at present, antiviral therapy by oseltamivir remains the mainstay for managing h n patients. while early treatment improves survival, approximately % of patients treated within days of illness still succumb to the disease. in addition to the role of viral replication, there is good evidence that the host proinflammatory responses contributes to h n pathogenesis. this suggests that both antiviral and immune-modulatory drugs may have a role in therapy. we previously demonstrated that cyclooxygenase (cox- ) plays a regulatory role in h n hyperinduced pro-inflammatory responses, and its inhibitor has potent effects at modulating this host response. now we demonstrate that, in addition to its immune-modulatory effect, a selective cox- inhibitor, ns- has a direct antiviral effect against h n infection. materials and methods human primary monocytederived macrophages or alveolar epithelial cells (a ) were pre-treated with ns- or drug-vehicle for hour before h n virus infection. h n viruses at multipicity of infection (moi) of was used to infect the cells. following virus adsorption for mins, the virus inoculum was removed, and the cells were washed and incubated in corresponding medium with ns- or drug-vehicle as controls for , , , , and hours post-infection. cells were harvested for rna isolation at hours post-infection to study viral matrix (m) gene expression. supernatants were collected for % tissue culture infection dose (tcid ) assay to determine the virus titers at , , , and hours after h n infection. results ns- was found to suppress virus gene transcription and infectious virus yield in h n -infected human cells. conclusion we demonstrate that a selective cox- inhibitor, ns- , shows an inhibitory effect on h n viral replication in addition to its immune-modulatory effect that could counter the detrimental effects of excessive proinflammatory cytokine production. the findings suggest that selective cox- inhibitors may be a therapeutic target for treating h n disease in combination with appropriate antiviral therapy. the emergence and spread of the highly pathogenic avain influenza viruses (h n ) in poultry and wild birds with repeated zoonotic transmission to humans has raised pandemic concern. at the time of writing, human cases have been reported with fatalities, an overall case fatality rate of around % (cumulative number of confirmed human cases of avian influenza a ⁄ (h n ) reported to world health organization updated to october ). our previous data demonstrated that cox- was markedly up-regulated in h n -infected primary human macrophages, and that it played a regulatory role in the h n hyperinduced host pro-inflammatory responses. such cytokine dysregulation is proposed to be a major contributor to the pathogenesis of h n disease in humans. with the use of selective cox- inhibitors, we found that the h n -hyperinduced cytokine response was significantly suppressed by the drug in a dose-dependent manner. selective cox- inhibitor is a form of a non-steroidal anti-inflammatory drug that selectively targets cox- , and it is an inducible enzyme responsible for inflammatory process and immune response. here, we report a novel finding of a direct antiviral effect of a selective cox- inhibitor, ns- , against h n infection in human primary macrophages and alveolar epithelial cells. taken together with our previous findings that suggest an immuno-modulatory effect that can modulate virus driven cytokine dysregula-tion, these findings highlight a role for cox- and its downstream signaling as potential novel targets for adjunctive therapy of severe viral pneumonia, such as that caused by h n . such therapy may be combined with conventional antiviral drugs. the h n virus used was a ⁄ vietnam ⁄ ⁄ ( ⁄ ) (h n ), a virus from a patient with h n disease in vietnam during . the viruses were grown and titrated in madin-darby canine kidney cells cells as described elsewhere. virus infectivity was expressed as tcid . all experiments were performed in a biosafety level facility. monocyte-derived macrophages: peripheral-blood leucocytes were separated from buffy coats of healthy blood donors (provided by the hong kong red cross blood transfusion service) by centrifugation on a ficoll-paque density gradient (pharmacia biotech) and purified by adherence as reported previously. the research protocol was approved by the ethics committee of the university of hong kong. macrophages were seeded onto tissue culture plates in rpmi medium supplemented with % heat-inactivated autologous plasma. the cells were allowed to differentiate for days in vitro before use in the infectious experiments. alveolar epithelial cells: a cells were obtained from atcc and maintained in culture using dulbecco's modified eagle medium supplemented with % fetal calf serum, . mg ⁄ l penicillin, and mg ⁄ l streptomycin. differentiated macrophages or a cells were pre-treated with a selective cox- inhibitor, ns- (cayman), at concentrations as indicated or drug-vehicle for hour before infection. cells were infected with h n viruses at moi of . following virus adsorption for min, the virus inoculum was removed, the cells were washed and incubated in corresponding medium with ns- or drug-vehicle as controls throughout the experiments. cells were harvested for rna isolation at hours post-infection to study viral m gene expression. supernatants were collected for tcid assay to determine the virus titers at , , , and hours after h n infection. total rna was isolated using the rneasy mini kit (qiagen) according to the manufacturer's instructions. the cdna was synthesized from mrna with poly(dt) primers and superscript iii reverse transcriptase (invitrogen). transcript expression was monitored by real-time pcr using power sybr Ò green pcr master mix kit (applied biosystems) with specific primers. the fluorescence signals were measured using the real-time pcr system (applied biosystems). the specificity of the sybr Ò green pcr signal was confirmed by melting curve analysis. the threshold cycle (ct) was defined as the fractional cycle number at which the fluorescence reached times the standard deviation of the base-line (from cycle to ). the ratio change in target gene relative to the b-actin control gene was determined by the )ddct method as described elsewhere. ns- reduced the viral m gene expression in h n infected human macrophages in a dose-dependent manner ( figure ) . similarly, production of infectious virus yield in h n infected macrophages was found to be suppressed in the presence of ns- at lm compared to vehicletreated cells (figure a) . a comparable effect of ns- was observed in h n -infected human alveolar epithelial cells ( figure b ). we have previously demonstrated that cox- expression was dramatically upregulated following h n infection in human macrophages in vitro and in epithelial cells of lung tissue samples obtained from autopsy of patients who died of h n disease. this suggests that cox- may be an important host factor involved in h n pathogenesis and also provide a possible explanation on why h n virus replication is susceptible to a selective cox- inhibitor. cox- was previously reported to play an important role in the pathogenesis of other influenza a viruses. an in vivo study has highlighted the importance of cox- in h n - infected mice. findings showed that infection induced less severe illness and reduced mortality in cox- knock-out mice than in wild-type mice. on the other hand, cox- knock-out mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the bal fluid, whereas the inflammatory and cytokine responses were dampened in cox- knock-out mice. these data suggests that cox- and cox- may lead to opposite totally contrasting effects on influenza h n infected mice. cox- deficiency is detrimental, whereas cox- deficiency is beneficial to the host during influenza viral infection. therefore in the present study, instead of blocking cox enzymes in general as reported by others, we have chosen ns- that selectively block cox- but preserve cox- activity and showed that this drug significantly reduced h n virus replication in a dose-dependent manner. taken together with our previous report suggesting its immuno-modulatory effects, we believe that selective cox- inhibitors and cox- signaling pathways deserve investigation as a promising approach for targeting therapy in h n diseases. however, a few reports have suggested the importance of cox- in the late stage of inflammation for the resulution of inflammation, [ ] [ ] [ ] and this raises concern whether inhibition of cox- may be harmful in treating diseases related to dysregulation of host inflammatory response such as acute lung injury, which is a leading cause of death in h n patients. we previously looked at the autopsy samples of lung tissues from h n patients and found that cox- expression was markedly up-regulated compared with that from persons who died of non-respiratory causes. moreover, data also demonstrated that pro-inflammatory cytokines, such as tnf-a, was markedly elevated in the h n infected lung autopsies. taken together, with the histo-pathological findings, which showed predominant features of exudative inflammatory phase in autopsy lung samples from h n patients, , we may therefore speculate that people who had fatal h n infection died during acute inflammation phase, and before the resolution could occur, especially for the cases with a short disease duration (< - days). in conclusion, the roles of cox- in both pro-inflammation and pro-resolution phases deserves detailed investi-gation. the timing of selective cox- inhibitor therapy in h n infected patients may be extremely critical. therefore a time-dependent study using selective cox- inhibitors on h n -infected animal models will be particularly important in order to address the effectiveness of this drug in treating h n disease. avian antibodies to combat potential h n pandemic and seasonal influenza highly pathogenic avian influenza a virus (hpaiv) strain a ⁄ h n with unprecedented spread through much of asia and parts of europe in poultry remains a serious threat to human health. passive immunization (transfer of protective immunoglobulins) offers an alternative and ⁄ or additional strategy to prevent and cure influenza. here, we report that virus-specific immunoglobulin y (igy) isolated from eggs of immunized hens provide protection in mice against lethal h n virus infection by neutralization of the viruses in the lungs upon intranasal administration. importantly, chicken eggs obtained from randomly selected supermarkets and farms in vietnam, where mass poultry vaccination against a ⁄ h n is mandatory, contain high levels of igy specific for a ⁄ h n virus. when administered before or after the infection, igy prevented and significantly reduced replication and spread of hpaiv h n and related h n strains. thus, the consumable eggs readily available in markets of countries that impose poultry vaccination against a ⁄ h n could offer an enormous source of valuable biological material that provides protection against a ⁄ h n virus with pandemic potential. the approach could be used to control seasonal influenza. since , hpaiv of the h n subtype has resulted in more than cases of laboratory-confirmed human infection in countries with a death rate of more than % (http://www.who.int/csr/disease/avian_influenza/). h n influenza virus remains a global threat because of its continued transmission among domestic poultry and wild birds. passive immunization (the transfer of antigen-specific antibodies (abs) to a previously non-immune recipient host) offers an alternative and ⁄ or additional countermeasure against influenza. development of human monoclonal antibodies (mabs) against h n influenza haemagglutinin (ha) using epstein-barr virus (ebv) immortalization of b cells isolated from patients infected with h n , phage display, humanized mabs, and human recombinant abs has been attempted. chickens produce a unique immunoglobulin molecule called igy that is functionally equivalent to mammalian igg. igy is found in the sera of chickens and is passed from hens to the embryo via the egg yolk. egg igy has been used to prevent bacterial and viral infections (see review ) of the gastrointestinal tract and recently for protection against pseudomonas aeruginosa infection of the respiratory tract of patients with cystic fibrosis (cf). the epidemic of hpaiv h n virus has resulted in serious economic losses to the poultry industry, mostly in southeast asia. therefore, many countries including china, indonesia, thailand, and vietnam have introduced mass vaccination of poultry with h n virus vaccines that controls the h n epidemic to some extent. chickens immunized with recombinant h and ⁄ or inactivated h n reassortant vaccines produced a high level of virus-specific serum antibodies (abs) and were protected from h n virus challenge. theoretically, these abs could be found in egg yolk and separated for use in humans to prevent and cure h n hpaiv infection and disease, respectively. here, we examined the possibility that igy isolated from consumable eggs available in supermarkets in vietnam, where mandatory h n vaccination has been implemented, provide prophylaxis and therapy of hpaiv h n infection in mice. six-to -week-old female balb ⁄ canncrl (h- d) mice (charles river and jackson laboratory) and hy-line . igy abs were extracted from egg yolks as previously described. the % egg infectious dose (eid ) was determined by serial titration of virus stock in eggs, and eid ⁄ ml values were calculated according to the method of reed and muench. human virus stocks were grown in mdck cells as described previously , with viral titers determined by standard plaque assay. the % tissue culture infectious dose (tcid ) of virus was determined by titration in mdck cells. the standard elisa was performed for detection of anti-igy in the sera of igy-immunized mice. fifty percent lethal dose (ld ) titers were determined by inoculating groups of eight mice i.n. with serial -fold dilutions of virus as previously described. for infection, ketamine-anesthetized mice were inoculated intranasally with a lethal dose with pfu ( · ld ) of a ⁄ pr ⁄ ⁄ (h n ) virus as previously described, · ld of vn ⁄ (h n ) or · ld a ⁄ aquatic bird ⁄ korea ⁄ w ⁄ (h n ) resuspended in ll pbs per animal. ketamine-anesthetized mice were treated intranasally with ll of igy before or after infection. mice were observed for weight loss and mortality. subsets of animals were scarified for virus titre. we found comparable hai titers in the sera and egg yolks obtained from a farm in vietnam that was participating in a national mass vaccination program. furthermore we found % of eggs purchased in randomly selected supermarkets in hanoi, vietnam containing h -specific igy. the hai and vn titers of pooled egg yolk igy are comparable with those of sera obtained from hens selected randomly from the farm that underwent supervised h n vaccination. in contrast, igy separated from eggs purchased in korean markets where poultry are not vaccinated against avian influenza h n has no detectable h -specific hai or vn activity. we first treated naïve mice intranasally with h n -specific igy before infection with hpaiv h n strain, a ⁄ vietnam ⁄ ⁄ , isolated from a fatal case. such treated mice displayed mild weight loss and recovered completely by the end of the first week after inoculation ( figure a ). when animals were treated once with h n specific igy after h n inoculation they exhibited minimal weight loss during the first week after inoculation, and virus titers in the lungs were substantial reduced at day after infection; however, % of treated mice succumbed to infection during the second week after inoculation ( figure b) . it is possible that not all the hpaiv a ⁄ h n viruses were neutralized upon the single treatment with igy, and escaping viruses can spread systemically to organs outside of the lungs. these viruses may reappear in lung tissue later when specific igy is absent. indeed, vn ⁄ virus injected intravenously or into the brain can spread to the lungs. to circumvent the virus escape, we administered multiple treatments with h n specific igy after the infection. as a result, all infected mice recovered completely by the second week post-infection ( figure c) , and virus titers in the lungs were substantially reduced to the level that seen in protected mice that received single prior-infection treatment ( figure d) . similarly, the protective efficacy of h n -specific igy was observed in mice infected with lethal dose of mouseadapted avian influenza virus strain a ⁄ aquatic bird ⁄ korea ⁄ w ⁄ (h n ). this virus shares . % nucleotide sequence homology with ha (h ) but has different na (n ) from the one used for mass immunization in vietnam (reassortant avian h n influenza virus a ⁄ goose ⁄ gd ⁄ -derived, strain re- ). the results indicate that h n -specific igy isolated from eggs purchased in markets have preventive and therapeutic effects against infection with hpaiv h n and the related strain h n . the findings suggest that while a single treatment with igy prior to lethal infection was sufficient to protect the animals from the infection, multiple treatment is required for complete therapeutic effect after infection with hpaiv such as vn ⁄ strain. we further examined the protective efficacy of igy isolated from eggs laid by hens immunized in the laboratory with heat-inactivated human influenza a ⁄ h n virus, a ⁄ pr ⁄ ⁄ . we found substantial levels of hai and vn abs in the sera and yolks derived from immunized hens. when naïve mice were administered intranasally with such anti-pr ⁄ igy at - hours before or after infection with lethal dose of pr ⁄ virus, they were protected from the infection or lethal disease, respectively. the virus titers in the lungs of a ⁄ pr specific igy-treated mice at day after infection were also significantly lower than those seen in untreated mice or mice receiving normal igy. intranasal administration is the most effective route as compared to oral or peritoneal or intravenous administration for protection against lethal challenge, and the presence of virus-specific igy in bronchoalveolar lavage (bal) is required for the protection. the results provide a proof-of-concept that intranasal administration of virus-specific igy prevents influenza virus infection and cures the disease. the concept could be applied to control influenza outbreaks including seasonal and pandemic influenza. the protection was correlated with hai and vn activities of the igy and reduced virus titers in the lungs after treatments, suggesting that the protection is mediated by vn. we asked if administration of igy in the respiratory tract induces anti-igy ab response in mice. if this is the case, the next question is whether pre-existing anti-igy abs block igy-mediated protection. indeed, significant levels of anti-igy were observed in animals that received single or multiple administration of igy. when igy-immune mice were treated with virus-specific igy before or after lethal challenge, the results were identical to those obtained from treated naive mice, indicating that pre-existing anti-igy abs do not interfere with the protection mediated by virus-specific igy. consistently, incubation with anti-igy serum did not interfere with hai and vn activity of the virus-specific igy, indicating that anti-igy abs do not block virus binding by virus-specific igy (figure ). the finding suggests that the igy treatment could be applied to persons who have developed anti-igy during the individuals' life, and such treatment strategy could be repeated if multiple treatment is required and ⁄ or necessary later on to protect infections with other pathogens. the approach using specific igy for prevention and therapy of hpaiv h n infection offers a practical alternative to immunotherapy using convalescent plasma and an additional therapeutic option to antiviral drugs since widespread drug resistance has been recently reported among influenza virus strains. igy is relatively stable. we found no change in protective activity after at least months storage at °c, and lyophilization does not affect the activity, making production of igy practical. the use of igy immunotherapy has many advantages, since igy does not activate the human complement system or human fc-receptors, which all are well-known cell activators and mediators of inflammation. we chose the water dilution method for preparation of igy. the method is simple, efficient and does not require any toxic compounds or any additives. such igy preparations by this method have been used in other human study. , eggs are normal dietary components, so there is minimal risk of toxic side effects, except for those with egg allergy. thus, our study demonstrated that influenza virus-specific igy can be used in passive immunization that provides great help for immunocompromised patients and elderly who have weaken immune response to influenza vaccines. importantly, the consumable eggs readily available in the markets of countries that impose mandatory h n vaccination offer an enormous source of valuable, affordable, and safe biological material for prevention and protection against potential h n pandemic influenza. parts of the information and data presented in this manuscript were previously published in http://www.plosone.org/ article/info:doi% f . % fjournal.pone. . the polyphenol rich plant extract cystus is highly introduction the ⁄ h n influenza a virus pandemic clearly demonstrates that influenza is still a major risk for the public health. although the pandemic swine origin influenza a virus (soiv) caused only mild symptoms, the control of the outbreak still remains difficult. even as vaccine is available against this virus, the possibility of reassortment between the pandemic and a seasonal or avian a ⁄ h n influenza virus strain is indeed a frightening, but a likely event. this reassortant strain might be able to transmit easily between humans causing fatal infections, and the current soiv vaccine might no longer be sufficient to protect against the reassorted virus. in such a case, we can only rely on effective antiviral drugs. today, neuraminidaseinhibitors, such as oseltamivir, represent the most common clinically approved medication against influenza a viruses. unfortunately, the frequency of reports describing the appearance of drug-resistant seasonal h n and also h n influenza a viruses dramatically increased in the recent past. [ ] [ ] [ ] [ ] drug resistance to the known antivirals highlights the urgent need for alternative antiviral compounds with novel defense mechanisms. recently, we have reported that a polyphenol rich plant extract, cystus , which showed antiviral activity against influenza a viruses in cell culture and in mice. , moreover, the antiviral activity of cy-stus against seasonal influenza virus and common colds was also demonstrated in humans. however, the efficiency of cystus against soiv and a ⁄ h n isolates was unknown so far. therefore, we investigated cy-stus effectiveness against the pandemic strain and seven natural influenza a ⁄ h n isolates detected in several avian species during ⁄ avian influenza outbreak. additionally, the potency of the most common neuraminidase inhibitor oseltamivir was also investigated against these isolates. here, we show that cystus treatment was effective in in vitro studies against soiv and a ⁄ h n influenza virus. viruses avian h n isolates were originally obtained from the bavarian health and food safety authority, oberschleissheim, germany. the soiv a ⁄ hamburg ⁄ ⁄ was obtained from the robert-koch-institut, berlin, germany. all h n viruses were further propagated in embryonated chicken eggs or mdck ii (h n v) cells at the friedrich-loeffler-institut, tübingen, germany. for the cytopathological effect (cpe) inhibition screening, in accordance with sidwell, mdck ii cells were infected with different viruses at moi of ae . virus-infected cells were then treated with antiviral compounds cystus from ae to lg ⁄ ml or oseltamivir from ae nm to mm. after incubation for hours at °c and % co , cells were fixed, and viable cells were stained with crystal violet. after extraction of crystal violet from viable cells with % methanol, the extinction was measured with an elisa reader. immediately before infection, mdck ii cells ( · cells ⁄ well) were washed with pbs and subsequently incubated with virus diluted in pbs ⁄ ba ( ae % ba) mm mgcl , ae mm cacl , penicillin and streptomycin to a multiplicity of infection (moi) of ae for minutes at °c. cystus was added in a concentration of lg ⁄ ml directly to the virus-stock and on the cell monolayer simultaneously with the infection. after minutes incubation period, the inoculums were aspirated and cells were incubated with either mem or mem containing lm oseltamivir. at indicated time points, supernatants were collected. infectious particles (plaque titers) in the supernatants were assessed by a plaque assay under avicel as described previously. in order to investigate the antiviral potential of cy-stus , ec values based on the inhibition of the cpe on mdck ii cells were determined for cystus and in addition for oseltamivir. the ec values for cystus ranged from ae to ae lg ⁄ ml. cystus demonstrated the highest sensitivity against the soiv, sn and mb isolates with ec values below lg ⁄ ml. compared to these virus strains, cystus showed a slightly increased ec value for gsb ( ae lg ⁄ ml). in contrast the ec values for bb and bb were notably elevated ( ae and ae lg ⁄ ml). thus, the weakest antiviral effect of cystus was observed against these two isolates. the ec values evaluated for oseltamivir ranged from ae to ae lm ( table ), indicating that bb ( ae ) and gsb ( ae lm) can be considered resistant against oseltamivir. to confirm these results we investigated the ability of cystus to block virus replication as published before. as a control, virus infected cells were treated with oseltamivir as described earlier. in the absence of the drugs all influenza strains showed similar growth properties (figure , black squares) . first progeny viruses were detectable between and hours post infection (figure , black squares) . treatment with cystus resulted in reduction of virus titers of all influenza virus strains (fig. a-h, open triangles) . surprisingly, oseltamivir failed to inhibit the replication of two h n influenza virus strains (gsb and bb ), supporting the data of ec values ( figure d+h , grey rhombes). we assessed the antiviral activity of cystus against the newly emerged soiv and seven avian h n influenza viruses. cystus showed efficient antiviral activity against the pandemic h n v strain and was effective to a wide range of h n viruses. furthermore, cystus demonstrated a broader and more efficient antiviral potential than oseltamivir. cystus treatment leads to a stronger reduction of progeny virus titers, and more importantly, cystus was effective against all tested viruses, while oseltamivir was unresponsive against two of seven a ⁄ h n viruses. even though the pandemic strain in general is still sensitive to oseltamivir treatment, there are increasing numbers of reports of emerging resistant variants. the treatment with cystus does not result in the emergence of viral drug resistance since the mode of action is an unspecific physical binding of the virus particle that is also beneficial to reduce opportunistic bacterial infections. , , cystus is an extract from a special variety of the plant cistus incanus, and it is very rich in polymeric polyphenols. it is well known that polyphenols exhibit protein-binding capacity. however, cystus exhibited no neuraminidase inhibiting activity. therefore, ingredients of cystus may act in a rather unspecific physical manner by interfering with the viral hemagglutinin at the surface of the virus particle as demonstrated before. while this prevents binding of the virion to cellular receptors, it does not block accessibility and action of the viral neuraminidase. since, infections with influenza a viruses are still a major health burden and the options for control and treatment of the disease are limited, plant extracts such as cystus should be considered as a new candidate drug for a save prophylactic and therapeutic use against influenza viruses. attenuation of respiratory immune responses by antiviral neuraminidase inhibitor treatment and boost of mucosal immunoglobulin a response by co-administration of immuno-modulator clarithromycin in paediatric influenza the antiviral neuraminidase inhibitor osv and zanamivir are widely used treatment options for influenza infection and are being stockpiled in many countries. although mucosal immunity is the frontline of defense against pathogens, the effects of neuraminidase inhibitor treatment on airway mucosal immunity have not been reported. the suppression of viral rna replication and viral antigenic production by these drugs may result in a limited immune response against influenza virus. macrolides, such as cam and azithromycin, have anti-inflammatory and immunomodulatory properties that are separate from their antibacterial effects. [ ] [ ] [ ] this study examined the impact of osv treatment on immune responses in the airway mucosa and plasma in mice infected with iav and pediatric influenza patients. we also assessed the immuno-modulatory effects of cam in influenza patients who were treated with or without osv. female ae -week-old weanling balb ⁄ c mice were nasally inoculated with pfu of iav ⁄ pr ⁄ h n at day . immediately after infection, mice were given lg of osv orally or vehicle at -hours intervals for days. the levels of virus-specific siga in nws and bronchoalveolar fluids (balf) and igg in plasma were measured by elisa as reported previously. a retrospective clinical study was conducted. for the study, children with acute influenza were recruited and grouped according to the treatment received: days treatment with osv (n = ), cam (n = ), osv + cam (n = ), and untreated (n = ). since parents in japan are well aware of the adverse effects of osv especially the neuropsychiatric complications, the decision on whether to administer osv or not and to prescribe cam was made by the parents and the attending paediatricians, based on their anti-viral and immuno-modulatory activity. , comparisons were made of the levels of siga against iav ⁄ h n and iav ⁄ h n , total siga, in nws and disease symptoms before and after treatment. anti-ha siga and total siga in nws of patients were determined from the standard regression curves with human iga of known concentration in a human iga quantitation kit (bethy laboratories). because an affinity purified human anti-ha-specific siga standard of each influenza a subtype is not available, the relative value of anti-haspecific siga amount was expressed as unit (u). one unit was defined as the amount of one lg of human iga detected in the assay system as reported previously. the concentrations of siga in individual nws were normalized by the levels of total siga (lg ⁄ ml). oseltamivir suppresses viral rna replication and viral antigenic protein production. to investigate the influence of daily treatment with osv on ha-specific mucosal and systemic immune responses, we analyzed ha-specific siga levels in nws and balf as well as igg levels in plasma at days and post-infection in mice treated orally with osv or methylcellulose (mc) as vehicle. the osv treated mice showed lower antibody responses in nws and balf than control mice treated with mc solution (table ) . significantly reduced ha-specific siga responses were particularly noted in the osv group at day , the period of maximal mucosal siga induction. the airway secretions and plasma from mice at day did not contain detectable levels of ha-specific antibodies. these findings were supported by other data whereby mice treated with osv displayed significantly lower numbers of ha-specific iga antibody-forming cells (afcs) in the nasal lamina propria, mediastinal lymph nodes, and lungs compared with mc-treated mice. these results clearly indicate that oral administration of osv downregulates ha-specific siga responses in mucosa. on the other hand, there were no significant differences in the elevated levels of ha-specific plasma iga and igg antibodies or the increased numbers of ha-specific iga and igg afcs in the spleen between osv-and mc-treated mice. taken together, these results implicated the oral administration of osv in a suppressed induction of haspecific siga responses in respiratory lymphoid tissues, although systemic ha-specific antibody responses were not significantly affected by osv. since cam up-regulates il- , a mucosal adjuvant cytokine in the airways, and promotes the induction of siga and igg in the airway fluids of mice infected with iav, , we assessed the impact of treatment with osv and ⁄ or cam on the levels of anti-influenza siga in nws and clinical status of influenza patients. the concentration ratio of table . anti-ha-specific siga to total siga in nws was expressed as titer: anti-ha-specific siga (u ⁄ mg) ⁄ total siga (lg ⁄ mg) · . figure shows changes in the anti-ha(h n ) siga ratio (titer) and fold of increase in siga titer in each patient during the -days' treatment for the four different treatment groups. it is noteworthy that, upon admission to the hospital, the siga titers were < in % of patients. during the days of treatment, rapid increases in the titers were observed in almost all patients in cam, osv + cam, and no treatment groups. in contrast, in the osv group, the anti-ha-specific siga titers remained unchanged or decreased in the majority of patients. the finding of significant low induction of anti-viral siga in the osv group was supported by the results of animal experiments. however, the addition of cam to osv augmented siga production and restored mucosal siga levels; % of patients treated with osv + cam showed > -fold increase in the titers during treatment. these observations suggest that cam stimulated the local mucosal immunoresponse in the nasopharyngeal region of patients treated with osv. the prevalence of disease manifestations was also analyzed. among the symptoms listed, a significant decrease in the prevalence of cough was recorded between the no treatment group and the osv + cam group and between the osv group and the osv + cam group (**p < ae ), despite the limited number of patients in each group. the duration of the febrile period was significantly shorter in the osv and osv + cam groups than the no treatment group. however, no significant difference was observed between the osv group and osv + cam group. it has been reported that osv does not affect the cellular immune responses, such as cytotoxic t lymphocytes and natural killer cells. however, the effects of osv on mucosal immunity have not been studied so far. the present study showed that osv treatment of mice infected with iav induced insufficient protective mucosal siga responses in the respiratory tract, although treated mice showed the similar levels of systemic igg and iga antibody responses in plasma to those in mice treated with vehicle (table ) . the observed effect of osv on mucosal immunity was probably due to a suppression of viral replication and viral antigen production in the mucosal layer. these observations in mice are further supported by our clinical reports of siga in nws and balf of osv treated influenza patients. the membered-and membered-ring macrolides have been found to possess a wide range of anti-inflammatory and immuno-modulatory properties, , and to be effective in the treatment of respiratory syncytia and iav infection. , the efficacy of low doses administered on the long term against pathogens that are insensitive to macrolides indicates a mode of action that is separate from their antibacterial activity. , , , in the present study, we evaluated the immunomodulatory effects of cam on mucosal immune responses in pediatric influenza. a decrease in the proportion of total siga that was anti-ha-specific siga during treatment was observed in . % of patients in the osv group (those represented by the dotted lines and closed diamonds in figure ), whereas an increase in the proportion was observed in most patients of the other groups (except for one patient of the untreated group). despite the low or unchanged induction of anti-ha-specific siga in the majority of osv-treated patients, the additional use of cam with osv boosted the mucosal immune response and restored local mucosal siga levels. we are currently engaged in detailed immunological studies of the effects of cam and osv on the levels of mediators controlling iga class switching in nws of influenza patients and airway secretion of mice infected with iav. further studies should clarify the boost mechanisms of cam and the suppression mechanisms of osv in iga class switching. our findings suggest the risk of re-infection in patients showing a low mucosal response following osv treatment and cam effectively boosts the siga production for protection of re-infection. to date there is an urgent need to develop new antivirals against influenza. most of the molecules reported target influenza proteins that acquire rapid mutations of resistance. the development of new molecules that have a broad antiviral activity and are not subjected to influenza mutation is of particular interest. our laboratory and others recently showed that proteases can participate to the innate immune response in the airways through the activation of a family of receptors called par. in particular, through the release of interferon, par agonists curbed viral replication significantly in infected cells. in this study, since erk activation is crucial for virus replication, we investigated whether par could inhibit virus replication through inhibition of the erk pathway. results showed that while influenza a infection alone or par stimulation alone induced erk activation, par stimulation does not inhibit erk activation in influenza infected cells. thus, par agonists may be a potential new drug against influenza viruses that could be used in combination with other anti flu therapy such as the inhibition of the erk pathway. respiratory tract-resident proteases are key players during influenza virus type a infection. , in addition to their direct activating effect on surface viral proteins, lung mucosal proteases can regulate cellular processes by their ability to signal through protease-activated receptors (pars). after cleavage of the receptor by proteases, the new aminoterminal sequence of par binds and activates the receptor internally. these receptors are highly expressed at epithelial surfaces, in particular in the lung, where human influenza virus replicate in vivo. pars are thus directly exposed to proteases present in the airways. among the four different pars, par acts as an antiviral through an interferondependent pathway. , thus, agonists of par are potential new drugs against a broad range of influenza viruses, which is in accordance with the broad antiviral action of interferon. however, the signalling pathway induced by par agonists in influenza a infected cells has still to be investigated. in this manuscript, we showed that influenza infection or activation of par induced erk activation, a crucial step for efficient virus replication. , however, par agonists do not impaired erk activation in influenza a virus infected cells. since the pathway of par protection is likely to be erk-independent, the use of anti erk molecules in combination with par agonists maybe of potential interest in future anti-influenza therapy. influenza viruses a ⁄ wsn ⁄ (h n ) (a kind gift from nadia naffakh) was used in the present study. mdck (madin-darby canine kidney) and the human alveolar type ii a cell were obtained from atcc and grown as previously described. for western blot analysis, the following antibodies were used: monoclonal antibody for phospho-erk ⁄ (t ⁄ y ) and for erk ⁄ antibodies from cell signaling technology (beverly, ma), horseradish peroxydase (hrp)-coupled rabbit polyclonal antibodies against mouse or rabbit igg from paris (compiègne, france). a cells were infected with iav at an moi of in emem medium, as previously described. , at various time points post infection, cells were collected and proteins were analysed as previously described. , par stimulation was performed at °c in emem medium as previously described. after infection and ⁄ or stimulation, cells were lysed in ice-cold lysis buffer. lysates were centrifuged at g for min, and total proteins of the supernatants were analyzed by western blot analysis as previously described. , results since activation of the erk pathway is essential for efficient influenza replication, we first investigated the kinetics of erk activation after influenza infection in human a alveolar epithelial cells. for this purpose, a cells were infected with influenza viruses at a moi of at different time point post-infection, and activation of erk ⁄ pathway was assessed by western blot analysis using an anti-erk antibody. results showed that erk was phosphorylated after influenza infection in a time course depen-dent manner when compared to uninfected cells. in contrast, erk phosphorylation was not observed with heatinactivated viruses, suggesting that productive infection is needed for erk activation ( figure a ). antibodies against erk ⁄ were used as controls. since erk is activated after influenza infection, we then tested whether activation of par in uninfected cells also leads to activation of this pathway. for this purpose, a cells were stimulated with the selective human (h) or mouse (m) par agonist or a control peptide for the indicated time ( figure b ). when exposed to the par agonists and compared to controltreated cells, erk phosphorylation increased over the time course of stimulation. thus, influenza infection or stimulation of par without infection in a cells induced activation of the erk pathway at different time point post-infection. since influenza infection and par stimulation induced erk activation, we then investigated whether par could inhibit erk activation in influenza infected a cells. results in figure showed that in influenza infected cells, par activation for ten minutes does not inhibit erk activation after influenza infection. thus, erk activation is not inhibited by par activation in influenza stimulated cells. in this manuscript, we studied the activation of the erk pathway after par stimulation and or influenza infection. particularly interesting is the fact that either influenza infection or par stimulation alone induce erk phosphorylation in a epithelial cells, while erk activation is not inhibited in a infected cells compared to uninfected ones after par stimulation. proteases are key factor in the pathogenicity of influenza viruses. in addition to the cleavage of ha, necessary for iav replication, extracellular proteases also play a role in the modulation of the immune system against influenza viruses through the activation of pars. particularly par , activated by extracellular trypsin-like proteases, could inhibit virus replication through the release of interferon, , thus, strengthening the immune system via agonist peptides and providing new therapeutic potential against a broad range of influenza strains. in addition, targeting the host instead of the virus could provide a way to escape from virus resistance. thus, a better understanding of how virus escapes from immune surveillance may provide new therapeutic strategies to block iav. in addition, combinations of drugs that block virus replication via different pathways are of interest. the non classical molecules hla-g maybe an interesting new target as we recently showed that it is upregulated after influenza infection, and it is a well known immunotolerant molecule. indeed, it inhibits the innate immune response as well as the adaptive immune response. , also, as previously suggested, the erk signal transduction cascade is also of potential interest since it is crucial for virus replication and particularly influenza replication. , as shown here, it is unlikely that par protection occurs through an erkdependent pathway. thus strengthening the immune response with par agonists and blocking nuclear retention of the viral ribonucleoprotein complexes with inhibitors of the mek ⁄ erk pathway may be alternative combinatory approaches for influenza therapy. in addition, since those potential drugs target the host instead of the virus, this could help in the design of new antivirals molecules more resilient to iav mutations and thus to virus resistance. the initial waves of the first influenza pandemic of the st century have passed. in june , vaccine companies estimated they could produce in months almost . billion doses of pandemic vaccine. instead, they actually produced only million doses, of which % were non adjuvanted preparations. had these doses been produced with adjuvants (i.e., . lg instead of lg ha per dose), an additional billion doses could have been made available. yet there was public opposition to adjuvants in many countries, especially by regulatory officials in the united states. misperceptions about the safety of both adjuvanted and nonadjuvanted vaccines were widespread. added to this, shortfalls in vaccine production, delays in vaccine delivery, and the ''mildness'' of the pandemic itself meant that only a few countries achieved reasonable levels of vaccine coverage. millions of doses went unused and had to be destroyed. supplies of antiviral agents were even more limited. thus, despite the best efforts of influenza scientists, health officials, and companies, more than % of the world's people did not have timely access to affordable supplies of vaccines and antiviral agents. instead, they had to rely on th century public health ''technologies.'' given current understanding of biology in the early st century, they should have had -and probably could have had -something better. this report reviews evidence for an alternative approach to serious and pandemic influenza that could be used in all countries with basic health care systems. instead of confronting the influenza virus with vaccines and antiviral agents, it suggests that we might be able to modify the host response to influenza virus infection by using anti-inflammatory and immunomodulatory agents. this idea was introduced several years ago and has been reviewed in several publications. [ ] [ ] [ ] [ ] [ ] [ ] the central importance of the host response in the pandemic, young adults had high mortality rates. ever since, influenza virologists have sought to answer the question ''why did young adults die?'' by defining the molecular characteristics of the virus that were responsible for its virulence. in doing so, they have overlooked a crucial piece of clinical evidence from the pandemic: compared with young adults, children were infected more frequently with the same virus, yet they seldom died. consequently, the more important question is ''why did children live?'' this can only be explained by recognizing that children must have had a different host response to the influenza virus than adults. physicians have long recognized that for several other medical conditions, both infectious (e.g., pneumococcal bacteremia) and non-infectious (e.g., multiple trauma), children have a more benign clinical course than adults. , a corollary of this observation is that secondary bacterial pneumonia, although commonly found in young adults in , could not have been the primary cause of death. children must have had the same or higher rates of nasopharyngeal colonization with the same bacteria that were associated with pneumonia deaths in adults, yet children seldom died of secondary bacterial pneumonia. if young adults died with secondary bacterial pneumonia, underlying host factors must have made them more susceptible. few people who die of influenza do so during the first few days of illness when pro-inflammatory cytokine levels are high. instead, like patients with sepsis, they usually die in the second week, when anti-inflammatory cytokines and immunosuppression dominate. , , influenza deaths occur more frequently in older persons with cardiopulmonary conditions, diabetes, and renal disease, but as seen in the h n pandemic, they also occur in younger adults with obesity, asthma, and in women who are pregnant. regardless of age, people with all of these conditions share one characteristic in common: they have chronic low-grade inflammation. in effect, their ''innate immune rheostats'' have been set at different, and perhaps more precarious, levels that make them more vulnerable to influenza-related complications. laboratory studies of influenza virus infection confirm the importance of the host response. in several studies in mice in which the host response has been modified (e.g., cytokine knockout), survival has been improved without increasing virus replication in the lung. in fact, severe disease can be induced without any influenza virus replication. for example, fatal acute lung injury has been induced in mice by inactivated (not live) h n virus. in this model, antiviral agents would be useless; only the host response could be responsible for disease. these observations raise the following question: could the host response be modified so patients with severe seasonal and pandemic influenza might have a better chance of surviving? influenza is associated with acute coronary syndromes, and influenza vaccination and statins reduce their occurrence. these associations led to the suggestion in that statins might be used to treat pandemic influenza. other agents that might also be effective include ppara and pparc agonists (fibrates and glitazones, respectively) and ampk agonists (e.g., metformin). , these agents have been studied in laboratory models of inflammation, sepsis, acute lung injury, ischemia ⁄ reperfusion injury, energy metabolism, mitochondrial function, and programmed cell death. the results of these studies cannot be reviewed in detail here, but the major findings for cell signaling are summarized in the table . unfortunately, the results of experimental studies are not always clear cut. for example, in one study of influenza virus infected mice, il- was necessary for containing infection, but in another study il- appeared to be harmful. nonetheless, overall understand-ing of cell signaling pathways in influenza virus infections and the actions of statins, glitazones, fibrates, and ampk agonists strongly suggest that these agents could benefit patients with severe influenza. laboratory studies in mice infected with pr (h n ) h n and pandemic h n viruses show that resveratrol, fibrates, glitazones, and ampk agonists reduce mortality by - %, often when treatment is started - days following infection. - (resveratrol is a polyphenol found in red wine. it shares with these other agents many of the same cell signaling effects.) in h n -infected mice, treatment with celecoxib and mesalazine, together with zanamivir, showed better protection than zanamivir alone. remarkably, these immunomodulatory agents have not increased virus replication. even more remarkable, in another model of a highly inflammatory and frequently fatal conditionhepatic ischemia ⁄ reperfusion injury -glitazone treatment ''rolled back'' the host response of ''young adult'' mice ( - weeks old) to that of ''children'' ( - weeks old). this unique study suggests that immunomodulatory treatment might roll back the damaging and sometimes fatal host response of young adults with influenza to the more benign and rarely fatal response of children. several, but not all, observational studies have shown that outpatient statins decrease hospital admissions and mortality due to community-acquired pneumonia. for influenza itself, preliminary evidence presented in october suggests that immunomodulatory treatment of influ- table . cell signaling targets that might be affected by immunomodulatory treatment of severe seasonal and pandemic influenza* down regulate pro-inflammatory cytokines (e.g., nf-kappab, tnfa, il- , il- ) up regulate anti-inflammatory cytokines (il- , tgfb) up regulate pro-resolution factors (lipoxin a , resolvin e ) up regulate ho- and decrease tlr signaling by pamps and damps up regulate enos, downregulate inos, restore inos ⁄ enos balance and stabilize cardiovascular function decrease formation of reactive oxygen species and decrease oxidative stress improve mitochondrial function and restore mitochondrial biogenesis decrease tissue factor and its associated pro-thrombotic state stabilize the actin cytoskeleton in endothelial cells and intracellular adherins junctions, and thereby increase pulmonary barrier integrity and decrease vascular leak differentially modify caspase activation and apoptosis in epithelial and endothelial cells, macrophages, neutrophils and lymphocytes in the lung and other organs increase the bcl- ⁄ bax ratio in influenza virus-infected cells and prevent the apoptosis necessary for virus replication. *see references , , , for details. nf-kappab, nuclear factor kappab; tnfa, tumor necrosis factor alpha; tgfb, transforming growth factor beta; ho- , heme oxygenase - ; tlr, toll-like receptor; pamp, pathogen-associated molecular pattern; damp, damage associated molecular pattern; enos, endothelial nitric oxide synthase; inos, inducible nitric oxide synthase. enza patients with severe illness could be beneficial. in a study of almost patients hospitalized with laboratoryconfirmed seasonal influenza, inpatient statin treatment reduced hospital mortality by %. in these patients, the cell signaling effects of statin treatment, summarized in the table , probably acted to reduce pulmonary infiltrates, maintain oxygenation, stabilize myocardial contractility and the peripheral circulation, reverse immunosuppression, restore mitochondrial biogenesis, and prevent multi-organ failure. achieving these clinical effects led to a decrease in mortality. because of the molecular cross-talk between statins, fibrates, glitazones, and ampk agonists, , similar clinical benefits might be expected from other members of this ''family'' of immunomodulatory agents. simvastatin, pioglitazone, and metformin are produced as inexpensive generics in developing countries. they are used throughout the world in the daily treatment of millions of patients with cardiovascular diseases and diabetes. global supplies are huge. because most people with influenza recover without specific treatment (this was true in ), not all patients would require immunomodulatory agents. instead, only those at risk of ards, multi-organ failure, and death would need to be treated. importantly, the cost of treatment for an individual patient would be less than $ . (d.s. fedson, unpublished observations). moreover, unlike vaccines they could be used on the first pandemic day. thus far, influenza scientists and the institutions that support their work (e.g., nih and cdc, national health agencies in many countries, the bill and melinda gates foundation, the welcome trust, and the world health organization) have shown little interest in immunomodulatory treatment. nonetheless, when more than % of the world's people have no access to influenza vaccines and antiviral agents, their physicians must have access to an effective ''option,'' especially one that might be lifesaving. research on immunomodulatory agents for influenza must involve investigators in many fields outside influenza science -those with expertise in the molecular and cell biology of inflammation, immunity, sepsis, cardiopulmonary diseases, endocrinology and metabolism, ischemia ⁄ reperfusion injury, mitochondrial function, and cell death. laboratory studies needed to identify promising treatment agents would probably cost $ - million (d.s. the results of these studies would inform clinical trials that critical care physicians are already eager to undertake. , this work will be especially important for people in developing countries where critical care capacity is extremely limited and not likely to improve. like critical care physicians, influenza scientists too must recognize that they cannot afford not to undertake research to determine whether generic immunomodulatory agents might be useful in managing severe seasonal and pandemic influenza. the nf-kappab-inhibitor sc efficiently blocks h n influenza virus propagation in vitro and in vivo without the tendency to induce resistant virus variants introduction influenza is still one of the major plagues worldwide. the appearance of highly pathogenic avian influenza (hpai) h n viruses in humans and the emergence of resistant h n variants against neuraminidase inhibitors highlight the need for new and amply available antiviral drugs. we and others have demonstrated that influenza virus misuses the cellular ikk ⁄ nf-kappab signalling pathway for efficient replication, suggesting that this module may be a suitable target for antiviral intervention. here, we show that the novel nf-kappab inhibitor sc efficiently blocks replication of influenza a viruses, including avian and human a ⁄ h n isolates in vitro in concentrations that do not affect cell viability or metabolism. in a mouse infection model with hpai a ⁄ h n and a ⁄ h n viruses, we were able to demonstrate reduced clinical symptoms and survival of sc treated mice. moreover, influenza virus was reduced in the lung of drug-treated animals. besides this direct antiviral effect, the drug also suppresses h n -induced overproduction of cytokines and chemokines in the lung, suggesting that it might prevent hypercytokinemia we hypothesise to be associated with pathogenesis after infections with highly pathogenic influenza viruses, such as the a ⁄ h n strains. thus, a sc -based drug may serve as a broadly active nontoxic anti-influenza agent. to assess the number of infectious particles (plaque titers) in organs a plaque assay using avicel Ò was performed in -well plates as described by mastrosovich and colleagues. virus-infected cells were immunostained by incubating for hour with a monoclonal antibody specific for the influenza a virus nucleoprotein (serotec) followed by minutes incubation with peroxidase-labeled anti-mouse antibody (dianova) and minutes incubation with true blueÔ peroxidase substrate (kpl). stained plates were scanned on a flat bed scanner and the data were acquired using microsoft Ò paint software. the virus titer is given as the logarithm to the basis of the mean value. the detection limit for this test was < ae log pfu ⁄ ml. organs of infected and control mice were homogenized and incubated over night in ml trizol Ò reagent (invitrogen) at °c. total rna isolation was performed as specified by the manufacturer (invitrogen). rna was solubilised in ll rnase free water and diluted to a working concentration of ng rna ⁄ ll. reverse transcription real-time pcr was performed using quantifastÔ sybr Ò green rt-pcr kit and quantitect primer assays (qiagen) . all samples were normalized to gapdh and fold expression analyzed relative to uninfected controls. ct values were obtained with the smartcycler Ò (cepheid). to answer the question whether the nf-kappab inhibitor sc shows antiviral properties against influenza virus, h n infected mdck cells were treated with different concentrations of the inhibitor (figure ). already treatment with nm of sc led to a reduction of viral cpe of more than %. almost % protection of cells was achieved when cells were treated with lm sc . the results indicated that sc has antiviral properties at concentrations ranging from to nm. we next tested whether sc would also be effective in the mouse model of influenza virus infection. when h n mice were treated i.v. once daily for days with mg ⁄ kg sc , survival rate of the animals increased significantly (p < ae ). the same results were found when h n influenza virus infected mice were treated i.p. with mg ⁄ kg sc (data not shown). moreover, sc treatment was not only effective when the inhibitor was given prior to h n influenza virus infection, but also in a therapeutic setup when sc was applied to the animals days after infection (data not shown). since influenza virus infected mice showed increased survival after lethal infection, we next questioned whether the amount of influenza virus was reduced in the lung. therefore, we performed quantitative real-time (qrt) pcr to detect viral mrna. mice were treated with either sc or the solvent, and hour later the lungs were prepared to perform qrt-pcr. as shown in figure a the amount of viral mrna was reduced by % in sc treated mice compared to solvent treated controls, indicating that sc leads to a reduced expression of h n specific mrna in the lung of infected mice. since infection of mice with h n leads to hypercytekinemia, we also investigated the expression of cytokines in sc treated mice. as shown in figure b the amount of il- specific mrna was drastically reduced in sc treated mice compared to solvent treated controls. moreover, also the expression of ip- was altered in sc treated h n influenza virus infected mice. here, roughly % reduction of specific mrna was detectable ( figure c ). thus, sc leads to a reduced transcription of il- and ip- in h n infected mice. there is an urgent need for new concepts to develop antiviral drugs against influenza virus. targeting cellular factors is a promising but challenging approach, and the concerns about side effects are obvious. however, it should be considered that drugs targeting viral factors, such as amantadine or oseltamivir, also exhibit a wide range of side effects in patients. thus, drug safety has to be rigorously tested in clinical trials regardless whether a drug targets a cellular or a viral factor. moreover, resistance against human h n influenza viruses and highly pathogenic avian h n virus strains to oseltamivir and amantadine have been reported. in that respect, the strategy to target cellular factors , might be one way to ensure that new drugs against influenza virus will be useful and effective for a long time without causing the development of resistant virus variants. we were able to demonstrate that the nfkappab inhibitor sc is able to reduce influenza virus activity in cell culture. moreover, the compound was also effective against highly pathogenic avian influenza viruses of the h n and h n subtypes in the mouse model. next to the reduction of virus sc was also able to reduce h n -induced overproduction of cytokines and chemokines in the lung in the lung of mice after infection with h n . most importantly, the drug did not show any tendency to induce resistant virus variants (data not shown). thus, a sc based drug may serve as a broadly active non-toxic antiinfluenza agent. [ ] [ ] [ ] [ ] [ ] in hong kong, the first confirmed case was a tourist from mexico reported on may , . the local government made its first attempt to contain the spread of h n in the local community by closing the metropark hotel where that tourist was staying, and quarantining guests and staff for days. following identification of the first local case around weeks later on june , , the government closed all kindergartens and primary schools from june until early july. fever clinics were also opened, the alarm levels in hospitals were raised to the highest, and a public education campaign was implemented. previous studies of the community responses to severe acute respiratory syndrome (sars) and human-to-human h n avian flu identified the importance of understanding the background perceptions of risk and psychological impact on the community. [ ] [ ] [ ] [ ] [ ] in this study we investigated the psychological and behavioral responses of the general local community throughout the first wave of ph n , and we also examined the factors associated with greater use of preventive measures. a total of surveys were conducted between april and november , covering the entire first wave of the ph n pandemic. computer generated random-household telephone numbers from all land-based local telephone numbers covering over % of hong kong households were used to recruit a total of local adults. one cantonese-speaking adult (age ‡ ) was invited for interview in each selected household on the basis of a kish grid. the survey instrument was based on previous experience in sars and avian influenza projects. information, including knowledge on modes of transmission, psychological responses to pandemic influenza, preventive behaviors, attitudes towards the new vaccines and socio-demographics, was collected. informed consent was obtained prior to the interview. ethics approval was obtained from the institutional review board of the university of hong kong. descriptive statistics were weighted by sex and age based on the reference population data provided by the hong kong government census and statistics department. multivariable logistic regression analyses were used to examine the association between the use of preventive measures and knowledge, perceptions and behaviors, sociodemographic characteristics, and psychological responses to pandemic influenza. multiple imputation was used to cope with a small proportion of missing data and make the best use of all available data. statistical analyses were conducted in r version . . (r development core team, vienna, austria). twelve thousand and nine hundred and sixty-five local adults were recruited throughout the study period, with a total of telephone calls being made; the response rate among eligible participants was . %. hong kong entered the containment phase after the world health organization (who) announced a global alert, and policies including border screening, tracing, and quarantine of doi: . /j. - . . .x www.influenzajournal.com suspected cases were implemented. hong kong transitioned to the mitigation phase on june , when the first local case was reported. the chronology of these and other events plus the epidemic curve of laboratory-confirmed ph n cases are shown in figure (a) . the anxiety scores and risk perception of the respondents are shown in figure (b,c) . anxiety, measured by the state trait anxiety inventory, remained steady throughout the study period. in response to the announcement made by who and the unknown nature of the new virus, a higher proportion of the respondents expressed worry (more, much more, or extremely more worried than normal) if developed ili and perceived ph n severity (same, more, or much more serious than sars) initially in early may . fewer respondents reported worry if they developed ili as the pandemic proceeded, with a slight perturbation around the first deaths in july and a steady decline to . %, while perceived severity of ph n declined more dramatically after an early high. perceived risks of infection of respondents (absolute susceptibility) and risk relative to others (relative susceptibility) were also investigated and found to remain relatively stable throughout the first wave, with no indication of an increase during the period of peak ph n activity in september (figure c) . as the first wave of ph n progressed, knowledge on modes of transmission did not improve. on the contrary, later in the epidemic increasing proportions of respondents reported oral-fecal and cold weather as modes of transmission of ph n . around - % of the respondents did not recognize direct and indirect contact or touching infected persons and contaminated objects as transmission routes for ph n throughout the first wave ( figure d ). higher proportions of respondents avoided crowded places and rescheduled travel plans in the second half of june when local kindergartens and primary schools were closed and the first ph n -associated deaths were announced. social distancing measures such as avoiding crowded places and rescheduling travel plans remained stable with slightly decreasing trends thereafter. the use of hygiene measures and other social distancing strategies was relatively stable with slightly decreasing trends during the study period ( figure ). female sex and older age were generally associated with greater reported use of hand hygiene measures, home disinfection, avoidance of crowded places, and rescheduling of travel plans. female sex was also positively correlated with use of face masks and cough etiquette. we found a negative correlation between anxiety and use of all hand hygiene measures and cough etiquette, but a positive correlation between anxiety and use of home disinfection and (c) proportion of the respondents reporting higher worry if developed flu-like symptoms (more, much more, or extremely worried), higher perceived seriousness of h n compared to sars (much more or more severe), higher probability to contract h n over the next month (certain, much more, or more likely), higher probability to contract h n over the next month compared to others outside family (certain, much more, or more likely). (d) proportion of the respondents identifying possible modes of transmission as the actual modes of transmission of h n . social distancing measures. other significant factors contributing to greater use of preventive measures were worry and knowledge. greater worry was associated with higher probability of home disinfection, social distancing measures, and use of face masks. knowledge that h n could be spread by indirect contact was associated all the investigated preventive measures, and knowledge that h n could be spread by droplets was associated with cough etiquette, but not face masks. there were no consistent trends between all the investigated preventive measures and absolute and relative susceptibility. community transmission emerged in hong kong in mid-june , and prior to emergence of community transmission, perceived risk and perceived severity were high. as ph n spread in hong kong, risk perception declined, even at the same time as incidence was increasing. anxiety was low throughout, at around . on the -point scale, compared to a maximum of . during sars on the same scale. anxiety has been showed to be positively correlated to personal hygiene measures and social distancing in previous studies; , however, we found a negative correlation between anxiety and use of all hand hygiene measures, cough etiquette, and face masks, and a positive correlation between anxiety and home disinfection. the differences in findings may be due to the fact that our anxiety measure was not specific to h n , and the score could be affected by other factors including economics. unlike hygiene measures, higher anxiety level, greater worry, and higher risk of perception were all associated with more social distancing. , , , social distancing is the most direct strategy in avoiding infection from other people, and it is commonly observed in an outbreak that the general public avoids crowded places, travelling to other countries, and social gatherings, , but the economic impact could be substantial. as community incidence of h n peaked, we did not observe any increase in use of preventive measures (figure ) . we found that face mask use peaked at the early stage of the pandemic, while hand hygiene remained fairly constant, and the knowledge on the modes of transmission of ph n did not improve over time. the lack of substantial change in preventive measures or knowledge about the modes of ph n transmission in the general population suggests that community mitigation measures played little role in mitigating the impact of ph n in hong kong. on the other hand, knowledge that ph n could be spread by indirect contact was associated with all of the preventive measures studied. consistent with reports during the sars period, , this study also showed that females and those of older age were more likely than others to use hygiene measures, avoid crowded places, and reschedule travel plans. this study has some limitations. first, this was a crosssectional study that was carried out at different time points, rather than a longitudinal study following the same individuals over time, and so the inferences on changes in behavior may need to be interpreted more cautiously. second, we recruited samples from all land-based local telephone numbers that cover % of hong kong households, but the response rate was not high enough to guarantee a representative sample, and this could be a source of selection bias. third, the responses were self-reported, and this may lead to social desirability bias in estimating knowledge, attitudes, and preventive behaviors. fourth, since the hong kong population has previously gone through unique experiences from sars in and avian flu in , our results may not be comparable to other countries or settings. in conclusion, this study revealed that the ph n pandemic failed to generate an increase use of preventive measures in the local community. there was no association between anxiety level and the events of the pandemic. with a relatively low mortality and morbidity rates compared to sars, ph n was not a matter of concern in the hong kong community. the lack of substantial change in the use of preventive measures and improvement in knowledge on the modes of transmission of ph n suggested that public health campaigns during the pandemic may not have had substantial effects on the general public. london is a major tourist destination, the seat of government and finance in the uk, and in will host much of the olympic and paralympic games. along with the rest of the global community, in and early london faced the challenges of responding to the first pandemic of the st century. at the time, nhs in london was composed of organisations, including the london ambulance service, acute hospitals, mental health and primary care trusts, and the strategic health authority. while london's nhs is well practiced at responding to large, big bang incidents, the influenza a ⁄ h n v pandemic was a rising tide event that lasted many months. significant preparatory work had been undertaken prior to april , which meant that the nhs in london was ready to respond. nhs london (the strategic health authority for london) led the response in partnership with local managers in all nhs organisations. the first uk cases of influenza a ⁄ h n v were reported in scotland on april, with the first in london on april. cases continued to increase, and the first wave peaked in london in july. cases reduced over the school summer holidays, but increased again when children returned to school at the start of september, and a second, smaller wave occurred. it is essential that the nhs learns from the ⁄ influenza a ⁄ h n v pandemic to ensure it is prepared for future challenges. nhs london provided a standardised debriefing pack to all nhs organisations in the region to identify, capture, and learn lessons. each debrief event involved health and inter-agency partners to ensure all viewpoints were considered and brought together in a single local report. all local reports were compiled in an over-arching document, which brings together common themes to inform ongoing preparedness in the region. the debrief process identified a number of common themes, such as the need for clear and appropriate communication, the importance of working with partners, and the benefits of strong and early leadership. however, differences between and within organisations were also highlighted; for example, some wanted more freedom for local decision making, whereas others would have preferred more stringently applied central direction. the following paragraphs considers individual areas assessed in the debrief process. command and control was in the main effective, with clear direction delivered from the national centre through nhs london to local nhs organisations. effective leadership is essential; the identification of senior local individuals to lead the response with teams of people to support them was critical. appropriate use of technology to communicate messages and coordinate command and control processes greatly aided the response. this included the development of the nhs london noon brief, a daily digest and associated web portal, and regular teleconferencing. key points are: • operational management at all levels must be considered in pandemic planning. • appointing an executive lead in each organisation was invaluable in the response. • pandemic flu planning for london must continue to be regionally led. communication is an essential component of the response to any incident. it must be clear, timely, and accurate. in the main, communication was excellent and met these criteria. one of the most challenging aspects was when messages from partner organisations differed, which occasionally led to confusion, unnecessary work, or frustration. the use of technology greatly aided communication across the region and supported the response; this included secure web sites, bluetooth, and text messaging etc. key points are: • regular internal communications and staff briefings are critical in the response to emergencies. • regular teleconferencing should be incorporated into future plans. • organisations should consider proactive and innovative methods for communicating during emergencies. robust partnership working was an essential component of pandemic preparedness work; however in the event, the a ⁄ h n v pandemic had little impact on sectors in london other than health. resilient communication networks between organisations, a common understanding, and the ability to make decisions were essential to the response at local level. ipcs proved an excellent mechanism to maintain local working relationships and resolve problems. clarity on the seniority of those attending these meetings and whether multi-site organisations such as mental health trusts should attend every ipc should be considered on a local and regional basis. key points are: • pandemic planning must remain part of inter-agency working. • social care resilience and planning must be embedded and integrated in health planning. 'vulnerable groups' is a universal term that covers a large and fluid group of individuals with different needs. ensuring access to healthcare during the pandemic for those who became vulnerable due to the situation, or those identified as such prior to the event, was the role of the pct in partnership with the local authorities. work continues to ensure that communication with vulnerable people is appropriate and timely in all incidents, and that organisations work together to achieve this. key points are: • planning to support the breadth of vulnerable people must continue. • pandemic preparedness for the prison sector should be further developed. • red ⁄ amber ⁄ green ratings for assessing vulnerabilities of mental health service users in an emergency should be further developed across the region. correct and appropriate usage of ppe is an essential component of reducing influenza spread, particularly in healthcare settings. london's nhs had been working towards developing local stockpiles of ppe when the pandemic commenced; however, there was little in place. the unanticipated national stockpile, while providing ppe to all organisations, was accompanied with some challenges in that it was often unfamiliar stock. key points are: • work around local stockpiling of non-standard consumables should continue. • regular training and fit testing of respirators should be embedded in all organisations. antiviral treatment was a core component of the response to influenza a ⁄ h n v, and was provided free of charge from a national stockpile. npfs reduced pressure on frontline nhs services once it was activated; however, there were concerns that patients could 'cheat' the system and obtain the drugs prior their clinical need. information about storage requirements of countermeasures must be clearly explained when they are delivered to frontline services, and the potential for recall into national stockpiles should be planned for. key points are: • regular exercising of local mass countermeasures centres and antiviral collection points (acps) should continue. • the use of community pharmacies as acps should be further considered in the capital. pandemic influenza vaccine uptake by healthcare workers was better than usual seasonal influenza uptake in the majority of nhs organisations, but could have been even better. this was largely due to the second pandemic wave not being as significant as expected, lack of clarity around when the vaccine would be delivered, and limited amounts being available initially. • gp-led and mass vaccination models for pandemic vaccination should be considered in local plans. • local lessons from the pandemic vaccination campaign should be applied to seasonal flu vaccination. the ability to maintain or increase capacity in response to a surge in demand, no matter what the cause, must be planned for. any of a number of situations could result in reduced staff or more patients, such as industrial action, transport disruption, disease outbreak, major incident, or poor weather. the work undertaken during planning for and responding to the pandemic will stand organisations in good stead for future disruptions. the importance of robust business continuity planning locally cannot be overlooked, as this is a key component of maintaining and increasing capacity. key points are: • local gp 'buddy schemes' should be encouraged for response to extreme pressure events. • organisations should regularly run staff skills audits so as to be aware of their overall capability for managing emergencies. • less emphasis should be placed on the use of retired staff when planning service continuity. reporting is a necessary but onerous task, and is often one of the most time-demanding parts of any incident response. it is also the aspect least likely to be tested through exercising. nhs london worked with organisations to endeavour to reduce reporting pressures, but much of this was dictated by central government. it is essential that future reporting requirements are proportional, informative, and realistic. while recognising it is not possible to predict the detail of information that may be requested, some broad assumptions can be made. key points are: • organisations should consider how they would collect and collate data from disparate parts of their organisation, rather than focussing on the detail of what that might be. • national and regional planning should consider the need for information and how this is balanced with the demand this places on organisations. • the introduction of the concept of a daily dashboard to identify areas of pressure should be incorporated into pandemic flu planning. the winter and pandemic influenza resilience assurance process undertaken in autumn was a useful process to inform planning for the first winter when the pandemic virus would be circulating in the uk. this consisted of a regional inter-agency exercise and a comprehensive review of the winter and pandemic plans of all nhs organisations in london. • regular assurance of pandemic flu preparedness should be maintained. • future resilience assurance processes should be undertaken in a timely and measured manner. • local organisations should continue to undertake regular pandemic flu exercises. the recovery period is as important as the response, but often receives minimal attention and has the potential to suffer as staff return to their normal jobs. one of the aspects that was not anticipated during the pandemic was the amount of stock (ppe, antivirals, and vaccine consumables) that would be recalled into national stockpiles. this proved particularly challenging for pcts who had to coordinate the process across their local areas. key points are: • the recovery period of an emergency must be given the same status and importance as the response. • future pandemic flu planning must include the recovery of national stockpiles of equipment and medicines. it is essential the lessons from the ⁄ influenza a ⁄ h n v pandemic are learnt and embedded into business-as-usual and emergency response processes in preparation for the next pandemic and other incidents. even though the a ⁄ h n v pandemic was generally milder than previous pandemics, it still presented challenges to the nhs in london. the biggest challenge that remains is to ensure that the public and nhs staff are aware that a more virulent virus could cause significantly more illness, death, and disruption, and that we must maintain our preparedness should this happen. the influenza a ⁄ h n v pandemic has been a major stimulus to business continuity planning and emergency preparedness across health in london, and many of the experiences during the pandemic proved invaluable in the unusually severe weather in early . it is important that this impetus and focus is maintained. changes to the nhs landscape in london will be considered in ongoing pandemic and emergency preparedness to ensure we remain as well prepared as possible for future events, particularly as london approaches the olympic and paralympic games. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. the way national health systems operate during inter-pandemic and the pandemic alert periods and the methods they use to address potential threats posed by zoonotic viruses with pandemic potential, as well as sea-sonal influenza epidemics, can clearly indicate whether the countries have enough capacities to respond adequately to unexpected influenza outbreaks. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during a pandemic scenario, the group prioritized topics and questions relating to rapid action and response. five to key public health needs associated with a pandemic scenario have been identified for each of the research agenda streams: five priority public health topics were identified for a pandemic scenario as follows: • examination of host range and transmission dynamics of animal influenza viruses to guide surveillance, control strategies, and risk communication. • enhanced surveillance in animals and humans to monitor virus evolution: o early detection of novel reassortants or changes in genotype and ⁄ or phenotype related to virulence. o development of epidemiological and laboratory diagnostic tools and capacity building to optimize case finding. o develop a framework for surveillance in animals that address ethical, legal, and social barriers to intra-pandemic surveillance and reporting. • deconstruct the origins of the pandemic virus to identify factors that permitted efficient human transmission. • develop strategies to limit economic, social, and cultural disincentives of animal-based interventions to reduce intra-and inter-species transmission. • operational research to optimize risk communication in the early phases of the pandemic linked to animal husbandry and food safety. stream : limiting the spread of pandemic, zoonotic and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: transmissibility of influenza across the progression of infection and spectrum of disease: • relative contributions of the different modes of transmission for influenza. five priority public health topics were identified for a pandemic scenario as follows: • identification of groups at higher risk of infection and severe disease outcome through enhanced surveillance. • understanding disease severity and identification of predictors of severe outcomes. • investigation of vaccine effectiveness, especially in high risk groups in diverse geographic areas. • establishment ⁄ enhancement of pharmacovigilance, particularly for adverse events among at-risk groups. • optimization of strategies for rapid and targeted vaccine deployment. • rapid assessment to optimize acceptance of pandemic vaccine. six priority public health topics were identified for a pandemic scenario as follows: • collaboration and coordinated sharing of data, protocols, regulatory, and other implementation strategies and databases from different countries on all aspects of patient management and outcome to accelerate improvements in patient care. • development of best practices in patient management in different settings, including checklists and algorithms for clinical care and treatment, prognostic parameters, and tests to predict potential for the development of severe disease. • rapid, reliable, simple, low-cost point-of-care diagnostic tools for influenza. • best use of current antiviral drugs and optimal formulations in different target populations, such as parenteral and other routes of administration for severe infections. • use of combination therapies, including use of adjunctive therapies (e.g., use of convalescent serum and immunomodulators). • role of ongoing viral replication, host responses, and the effect of co-infections in the pathogenesis of severe disease. modern tools for early detection and monitoring of disease the group on surveillance tools concluded that the agreed topics of interest were equally applicable during a pandemic or inter-pandemic period: • studies to appraise and adapt modern technologies for early detection of influenza outbreaks in surveillance at the human-animal interface. • develop, integrate, and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems. • study efficient mechanisms on sharing data, clinical specimens, and viruses with consideration for local, ethical, legal, and research perspectives. • examine the timeliness and quality of data required for early detection from local to national and global levels for the respective stakeholders. five priority public health topics were identified for a pandemic scenario as follows: • identify environmental determinants of seasonal variation in influenza transmissibility in tropical and temperate regions. • estimate the transmission risk associated with types of contacts by comparing measured contact patterns with outbreak data. • incorporation of validated models of behavioral responses to risk and control measures in virus transmission. • development and implementation of novel technology for real-time sero-surveillance during a pandemic. • develop experimental and theoretical framework to assess host adaptation to study host receptor, antigenicity, and virulence. modern tools for strategic communication three priority public health topics were identified for a pandemic scenario as follows: • evaluate tools to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups to guide future communication efforts; develop tools and methods to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups, and thereby, guide future communication efforts. for communicating in different cultural settings, which engage and empower individuals and communities to practice and promote appropriate risk reduction measures. implementation of the identified research priorities is expected to underpin public health decision making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs and economic loss, and mitigate potential social disruption. complemented by an analogous research roadmap for a pandemic influenza scenario, the research recommendations for an interpandemic period represent a framework to provide evidence to guide public health policies on influenza control. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting public health needs for inter-pandemic periods according to its five key research streams: • stream . reducing the risk of emergence of pandemic influenza. • stream . limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza. • stream . minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza. • stream . optimizing the treatment of patients. • stream . promoting the development and application of modern public health tools. stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza inter-pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during an inter-pandemic phase, a more comprehensive approach was applied to establish research topics and prioritizing a range of questions that will build a solid foundation to guide research activities to support public health decision making. five to ten key public health needs associated with an inter-pandemic scenario have been identified for each of the research agenda streams: stream : limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: . transmissibility of influenza across the progression of infection and spectrum of disease . relative contributions of the different modes of transmission for influenza . biological, behavioral, and social host factors that influence the risk of transmission and infection . patterns, drivers, and mechanisms affecting the seasonality of transmission . viral and population factors that influence transmission and spread of different influenza types, subtypes, and strains . strategies to reduce the transmission of influenza in community, household, and health care settings, especially in less-resourced areas . impact and cost effectiveness of social measures, such as school closures, and the role of surveillance in assessing timing of these interventions . impact, effectiveness, and cost effectiveness of individual measures, such as isolation and quarantine . role of vaccination in limiting the spread of influenza and strategies for its use . impact of antiviral treatment and prophylaxis in reducing transmission of influenza stream : minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza . identify higher risk groups and severe disease through surveillance; disease severity and identification of predictors of severe outcomes . evaluate vaccination preventable disease burden and the potential impact of immunization programs through vaccine demonstration projects . enhancement of the properties of existing vaccines, including duration and breadth of protection, safety, immunogenicity, and dosesparing . development of new vaccines and vaccine platforms, especially suitable for under-resourced country settings . study the effectiveness of vaccine strategies to reduce disease burden in children and other high risk groups in a wide range of settings . improved uptake and acceptability of vaccines for both seasonal and pandemic influenza seven priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: inter-pandemic seasonal influenza scenario . research on the burden of severe disease with a focus on regionalspecific factors, such as the burden of tb and hiv and optimization of pandemic and management . development of new antiviral strategies and validation of surrogate endpoints which may aid in advancing understanding of disease progression . further clinical evaluation of current antiviral drugs, particularly in populations at risk . integration of seasonal influenza with pandemic preparedness; strengthen surveillance, health care systems, capacity, and preparedness planning . improving diagnostics (e.g., multiplex assays for viruses and bacteria), including antiviral resistance testing at point-of-care . dissemination of best practices, situation analysis, preparation for next epidemic (e.g., establish protocols for rotating stockpiles of antiviral drugs) . increased attention to basic science research such as studying immunomodulatory drugs five priority public health topics were identified for an inter-pandemic zoonotic influenza scenario as follows: inter-pandemic zoonotic influenza . antiviral susceptibility of circulating zoonotic viruses (e.g., h , h , h influenza viruses) . reassortment between zoonotic and human influenza viruses and the potential for inter sub-type spread of antiviral resistance and virulence modern tools for early detection and monitoring of disease the group focusing on surveillance tools concluded that the agreed topics of interest were equally applicable during both pandemic and inter-pandemic period: . identify modern technologies for early detection of influenza outbreaks as well as their application in surveillance at the human-animal interface . develop and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems . studies to address challenges on data, clinical specimens, and viruses sharing with consideration for local, ethical, legal, and research perspectives . examine the timeliness and quality of data required for early detection from local to regional, national, and global levels role of modeling in public health decision making five priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: . integration of genetic and epidemiological data to understand spatiotemporal spread to forecasts evolution for vaccine strain selection and to anticipate likely burden of disease . quantifying the relative contributions of different modes of transmission of human influenza and developing mechanistic modeling of transmission processes . research using data-capture technologies to characterize human contact and mobility patterns at local, regional, and global scales, and their correlation with transmission risk . integration of genetic, antigenic, and epidemiological analyses to optimize surveillance for newly emerging pathogens at the animal ⁄ human interface . identifying and quantifying human and environmental ecological, behavioral, and demographic determinants of the risk of cross-species transmission and pandemic emergence modern tools for strategic communication four priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: . review of evidence and experience related to health crisis communication from fields to organize knowledge and support evidencebased practice in strategic communication . identify and develop tools to rapidly and accurately monitor knowledge, attitudes, and practices in different population groups and guide future communication efforts . identify and develop communication tools and approaches for cultural settings and communities to practice and promote appropriate risk reduction measures . understand the potential ethical, social, economic, and political communication in crisis and develop strategies to work within constraints while maximizing opportunities complemented by an analogous research roadmap for a pandemic influenza scenario, the research topic recommendations for an inter-pandemic period represent an important outcome of joint international efforts by who, academicians, and public health experts. implementation of the identified research priorities is expected to underpin public health decision-making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs, and economic loss and mitigate potential social disruption over a medium-tolong term period. the impacts of school resumption on the incidence of pandemic (h n ) in school students introduction school closure is one non-pharmaceutical intervention that is often suggested in pandemic preparedness plans, and it was widely implemented in pandemic (h n ) to reduce transmission amongst school students. however, from past epidemiological studies, the effect of school closure in reducing respiratory disease transmission was inconclusive. given this public health intervention causes major disruption to the education system and potentially raises childcare issues to working parents, evaluating its effect in the recent pandemic is necessary to improve future pandemic planning. in hong kong, since school closure was implemented early in the pandemic and closure was effectively continued with the commencement of summer holiday, the lack of incidence data in the absence of school closure makes it difficult to analyse its effect directly. this has prompted us to analyse the situation indirectly from the angle of school resumption after summer holiday. in hong kong, public health surveillance on pandemic (h n ) was effective from th april- th september : healthcare professionals were advised to report suspected cases of infection to centre for health protection, department of health, hksar, for further laboratorial confirmation. demographics of reported cases were subsequently recorded into a computerised system (the ''e-flu'' database). following institutional approval, a dataset of all confirmed cases diagnosed from may to september was obtained, which included the age, gender, confirmation date, and notification date of each report. all cases were classified into four defined socio-economic classes by age: pre-schoolers ( - ), school students ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , adults ( - ), and retirees ( ‡ ). assuming cases had contracted infection on the earlier date between confirmation and notification, daily incidence in each age class was counted for epidemic curve construction. upon observing an unusual rise in the epidemic curve of school students when school season resumed in september, interrupted time series analysis (also known as intervention analysis) was applied to obtain the statistical significance of this observation. the analysis was applied to the incidence in school students from th july to th september , which covered the period from the start of summer holiday to the end of the th week of new school season. incidence in school students before summer holiday was deliberately dropped since not all schools were closed when the school closure policy was effective: all primary schools were closed proactively, whereas secondary schools were individually closed on a reactive basis if students were identified to have contracted the infection. school activity was formulated as a step function, which takes value from st september onwards (st = : t < st september, st = otherwise). a range of times series models were fitted by the maximum likelihood method and aic (akakine information criterion) was used to select the one with best fit. all computations were performed in sas version . . a total of ( ae %) pre-schoolers, ( ae %) school students, ( ae %) adults, and ( ae %) retirees were diagnosed with the infection in the surveillance period. the epidemic curves of preschoolers, school students, and adults showed a steady rise from th june onwards when local transmission of pandemic influenza was identified. an upsurge in the epidemic curve of school students can be observed in early september, coinciding with the commencement of the new school year (figure ) . interrupted time series analysis on the epidemic curve of school students returned an arima( , , ) model with equations: where st, yt, yt denote school activity, predicted and actual incidence in school students on day t, respectively. standard error and significance for model constants were: ae (se = ae , p = ae ), ae (se = ae , p = ae ). in short, the model can be interpreted as: the number of infected school students rose by ae per day on average during the entire study period, with a sharp increase by ae coming into effect when the new school year began. time series analysis showed, at the marginally significance level, that daily incidence in school students had a major increase when school season resumed. on the assumption that the increase was not caused by any change in health seeking behaviour, this result suggests that school resumption had facilitated transmission amongst school students. on the basis that school activity significantly increases incidence of pandemic influenza in school students, this study suggests closures of schools in the early phase of pandemic (h n ) and subsequently in the summer holiday probably had a major effect in mitigating transmission amongst school students. youngsters were postulated to be major vector for transmission in pandemic (h n ) . if this were true, it would be reasonable to expect the epidemic curves of the other age classes to show a similar upsurge when one is observed in school students. the absence of such observation in the epidemic curve of hong kong suggests school students were mostly disseminating the virus amongst themselves, but not to the other age groups. in november , gip convened the first global consultation on a public health research agenda for influenza to identify key research topics in each of the five main streams of public health research. during this meeting, the scientific working group (swg) of the sub-stream in ''modern tools for risk communication'' identified the requirements in research during influenza pandemics and inter-pandemic periods to provide clear, credible, and appropriate messages which meet the needs of diverse communities. the swg suggested that who hold a follow-up workshop to assess the use of modern tools related to strategic and risk communication and to further promote research in these areas. communication'' in may . one of the main objectives of the meeting was to generate a roadmap of public health research priorities related to strategic and risk communication. the research roadmap was developed by the group of invited experts on the basis of an analysis of available evidence and experience on public health and health crisis communication from relevant disciplines across global regions, as well as critical assessment of existing communication methods related to influenza control in different cultural, social, and ethnic settings. the workshop consisted of a series of presentations by experts in relation to experiences and lessons learned about communication during the sars, h n epidemic, and h n pandemic. there were also a series of group discussions on identifying research needs for pandemic and interpandemic periods in order to strengthen the research agenda. the expert group identified important public health needs in relation to communication during pandemics as well as in the inter-pandemic times. the main topics of discussion centered on communicating issues of influenza virus transmission, the use of influenza vaccines safety and efficacy, and use of antivirals as well as definition of the severity of the pandemic and the phase changes. in this context a number of research areas were identified, which can be broadly classified into four areas: understanding of communication principles and mechanisms is associated with an array of research topics covering different subject areas. one of the key questions here relates to the link between communication and ''behaviour change'' models and their application and appropriateness for different settings. the expert group defined the term ''behaviour change'' in this context as the modification of behaviour towards better health practices that are supported by clinical and scientific evidence for personal protection against infectious diseases and other adverse health risks. research topics related to these models require understanding and differentiating information and ''behaviour change'' needs of different audience segments, such as stakeholder mapping, target audience analysis, research into behaviour motivation, social norms, and the cultural, religious, social, legal, and political barriers and enablers of particular behaviors that are beneficial in influenza control. this research area also includes the analysis of media consumption among different audiences, role models, including ways to analyse how rumours and misinformation are spread, and ways to provide evidence-based information correctly. other important areas of investigation embrace methods to communicate uncertainty, learning how to build trust while communicating about a pandemic, and understanding what needs to be done before, during, and after a pandemic in order to create the best environment for influenza pandemic communication. critical key audiences identified for more intensive analysis were health workers, religious, public health, and societal (political and community) leaders. • investigation of the role of different communication channels and communication formats for different target audiences in a pandemic, particularly for groups that are ''hard-to-reach.'' • determining effects of perceptions related to pandemic influenza (severity, susceptibility, response efficacy, self efficacy, perceived social norms) on protective behaviours in different groups. • understanding audience in terms of their knowledge, preventive activities, and reasons why engaged ⁄ not engaged. • developing mechanisms to synergies between risk communication and behavior oriented approaches in the pandemic and inter-pandemic phases. • determining social, economic, cultural, and religious factors which support behaviours to limit spread and minimize impact in different settings. • identification of the key predictors ⁄ factors that influence people's behavior among different groups and populations vis-à -vis pandemic flu behaviors. • identification of elements that contribute to trust among populations and in different settings (country, public, professional, community), particularly where trust was previously compromised. • understanding psychology of different groups regarding their response to uncertainty, and finding the best way to communicate uncertainty. the research questions in this section relate to the planning, development, and evaluation of tools that can be quickly accessed and used in a pandemic situation. these may include communication materials and channels; the setting up of key stakeholder and champion communication networks; research protocols that are ready for rapid assessment during a pandemic or new communication tools. the use and understanding of terminology and language by both lay and professional groups and communities in planning for and ⁄ or reacting to a pandemic are important areas of research. acute examples, such as the naming of the viruses or the use of the word ''pandemic,'' illustrate this need well. the research focus of this area is to look at lessons learned from the a(h n ) pandemic and to document and evaluate case studies, both looking at best practices, challenges, and barriers that were experienced. different communication strategies need to be evaluated and models to be built not only in terms of reach, but also in terms of impact on thinking, emotional response, and behavioural modification. a key question was how to prepare communication for a pandemic and how can the pandemic communication contribute to longer term ''behavioural change.'' mathematical modelling on gauging outcomes of such ''behaviour change'' would provide strategic approaches in risk communication. this section aims to answer the question whether the modeling, mapping, and scenario planning are actually useful in the pandemic situation. the expert group agreed that the research on the above issues should use a variety of methods and engage a number of disciplines. this would include literature reviews, case studies, trials, ethnographic studies, modelling, surveys, network analysis, as well as any other useful methodology. in an inter-pandemic situation for actual behaviour under pandemic conditions. • study the synergies and develop priority research topics on strategic ⁄ risk communication for influenza under inter-pandemic situations that includes zoonotic and seasonal infections. the who public health research agenda for influenza initiated and facilitated a multi-disciplinary discussion for communication during pandemic and inter-pandemic situations. it focused on both theoretical and practical issues to improve practice and ensure the health of the public for influenza. critical areas for research were identified to build evidence in this field. it was recognized that there are extensive bodies of knowledge in a number of disciplines, , such as health promotion, behavioural psychology, social sciences, social and behaviour change communication, social marketing, and communication for development relating to these questions, and that these should be explored. outcomes of these research activities are expected to widen the evidence base which will support developing communication strategies for influenza by countries, institutions, and individuals and will, consequently, help to improve public health world-wide. abstract background: cytokine dysregulation contributes to the unusual severity of h n (reviewed in ). previously, we demonstrated that interferon regulatory factor (irf ) and p map kinase (p ) signaling pathways separately contribute to the induction of pro-inflammatory cytokines and chemokines in h n -infected cells. here we investigate the role of innate sensing receptors in the induction of these cytokines and chemokines in response to h n and seasonal h n infection. materials and methods: human macrophages derived from peripheral blood monocytes were infected with h n ( ⁄ ) or seasonal h n ( ⁄ ) viruses. the role of innate sensing receptors in cytokine and chemokine induction by h n virus was investigated using transient knock-down of these receptors with sirnas. the expression of innate sensing receptors in infected cells, and as a result of paracrine activation (by virus free supernatants of infected cells) of adjacent uninfected cells were also monitored by real-time pcr and ⁄ or western blotting. the involvement of janus kinase (jak) signaling pathways in these autocrine ⁄ paracrine cascades was investigated using a jak inhibitor. results: we previously showed that tnf-alpha, ifn-beta, and ifn-lambda are the key mediators directly induced by the h n virus in primary human macrophages with other cytokines and chemokines being induced as part of a secondary autocrine and paracrine cascade. here we demonstrated that retinoicacid-inducible gene i (rig-i) rather than toll-like receptor (tlr ) plays the predominant role in h n -induced cytokines and chemokines in human macrophages via the regulation of irf and nf-kb nuclear translocation. in addition to the effects on virus infected cells, paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the upregulation of sensing receptors. conclusions: h n directly induced tnf-alpha and ifnbeta mainly via rig-i signaling, and the subsequent activa-tion and nuclear translocation of irf and nf-kb in human macrophages. in addition to the effects on cytokine signaling, the innate immune sensing regulators themselves were also up-regulated by h n infection, much more so than by seasonal influenza infection, via jak signaling. the up-regulation of innate sensing receptors was not limited to the infected cells, but was also found in adjacent uninfected cells through paracrine feedback mechanisms. this may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. a more precise understanding of the signaling pathways triggered by h n virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h n influenza and also for treating other causes of acute respiratory disease syndrome. human h n infection is associated with a mortality rate of more than %. the basis for the unusual severity of h n disease has not been fully explained. cytokine dysregulation has been suggested to contribute to the disease severity of h n (reviewed in ). however, signaling pathways involved in the cytokine induction by h n virus are not fully understood. previously, we demonstrated that irf and p map kinase (p ) are separate signaling pathways which contribute to the induction of pro-inflammatory cytokines and chemokines in h n -infected cells. rig-i and melanoma differentiation-associated gene (mda ) are important cytosolic sensors of nucleic acid of pathogens, while tlr and tlr also recognize nucleic acid species of pathogens, but they are localized at the endosomal membrane. rig-i was found to be responsible for the recognition of influenza a virus infection, and the transfection of vrnps induces ifn-beta expression. while many studies have shown the role of rig-i in the induction of ifn-beta by influenza virus infection, the majority of these studies used either immortalized cell lines or mouse embryonic fibroblasts. there is a lack of data on the role of these innate sensing receptors in highly pathogenic avian influenza h n infection in primary human cells in vitro, which are more physiologically relevant. furthermore, there is little data on the autocrine and paracrine up-regulation of these innate immune sensors following virus infection. human macrophages were obtained from peripheral blood monocytes by adhesion and differentiation in vitro for days in rpmi medium supplemented with % autologous plasma. the cells were infected with h n ( ⁄ ) or seasonal h n ( ⁄ ) viruses at a moi of ae . a cells were obtained from atcc and cultured in mem medium supplemented with % fcs and % penicillin and streptomycin. the role of innate sensing receptors in cytokine induction by h n and h n viruses was investigated using transient knock down of these receptors with sirnas in human macrophages as previously described using specific sirnas purchased from qiagen. immunofluorescence staining assay of irf and nf-jb was employed to detect the nuclear translocation of these transcription factors after h n infection. rabbit polyclonal antibodies against human irf and and nf-kb were obtained from santa cruz biotechnology. goat anti-rabbit igg antibody conjugated with alexa fluor was a product of molecular probes. for investigation of paracrine effects on rig-i and tlr expression, culture supernatants collected from mock, ⁄ or ⁄ infected human macrophages were used to treat uninfected cells. the supernatants were first passed through a filter with -kda cut-off. virus particles as well as molecules with a molecular weight higher than kda were retained and removed, while the filtrate was collected for treatment of uninfected cells. the expression of innate sensing receptors in infected cells and in adjacent uninfected cells following paracrine activation by virus free supernatants of infected cells was monitored by real-time pcr. the involvement of jak signaling pathways in these paracrine cascades was investigated using a jak inhibitor (calbiochem). we previously showed that tnf-alpha, ifn-beta, and ifnlambda are the key mediators directly induced by the h n virus in primary human macrophages with others being induced as part of a secondary autocrine and paracrine cascade. in this study, we demonstrate that knockdown of rig-i or tlr led to the reduction of ifn-beta and tnf-alpha in human macrophages by both ⁄ (h n ) and ⁄ (h n ) infection. as shown in figure a , ⁄ virus induced higher level of ifn-beta mrna expression than ⁄ infection. cells transfected with rig-i or tlr sirna significantly reduced the expression of ifn-beta after ⁄ infection, by % and %, respectively. rig-i silencing also significantly reduced the ifn-beta expression in ⁄ infected cells by %. in contrast, silencing of mda or tlr did not suppress the induction of ifn-beta by either ⁄ or ⁄ infection; in fact, there was a slight ( %) increase of ifn-beta in cells transfected with mda sirna. based on these results we conclude that while both rig-i and tlr contribute to h n -induced interferon-beta induction in human macrophages, rig-i plays the dominant role. in order to investigate the relationship between these innate sensing receptors and the activation of transcription factors irf and nf-jb, we next measured the nuclear translocation of irf and nf-jb in cells with rig-i or tlr silencing after h n infection. immunofluorescence staining assay on irf and nf-jb was performed and the number of cells with nuclear translocation was quantitated. the percentages of cells with nuclear translocation were plotted in figure b . we demonstrated that rig-i knockdown led to a significant reduction of irf nuclear translocation after ⁄ infection, whereas the nuclear translocation of nf-jb after ⁄ infection was significantly suppressed by rig-i or tlr silencing. these results suggest that the involvement of rig-i and tlr in the cytokine induction by ⁄ was via the regulation of irf and nf-jb nuclear translocation. since rig-i and tlr are important in influenza a virus-induced cytokine expression, we next explored the expression of these innate receptors in neighboring uninfected human macrophages by treating the uninfected macrophages with the filtered culture supernatants collected from mock, ⁄ , or ⁄ infected macrophages. as shown in figure a , ⁄ supernatant differentially induced the mrna expression of rig-i, mda , and tlr compared to ⁄ supernatant treated human macrophages. the induction of rig-i was higher than the induction of mda and tlr . in the presence of lm of jak inhibitor, the up-regulation of all three innate sensing receptors was significantly reduced showing their induction was dependent on jak activity. human lung epithelial a cells were also treated with the supernatants collected from macrophages infected with mock, ⁄ , or ⁄ virus. differential induction of rig-i, mda and tlr by ⁄ supernatant compared to ⁄ supernatant treated cells was observed (figure b) . ⁄ supernatant dramatically induced all three innate sensing receptors, while ⁄ supernatant only marginally induced rig-i and mda , but not tlr . as in human macrophages, treatment with lm of jak inhibitor caused a significant suppression of ⁄ supernatantinduced rig-i, mda , and tlr expression in a cells. these results, taken together with the direct effects on virus infected cells, suggest that paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the up-regulation of innate sensing receptors. h n directly induced ifn-beta ( figure ) and tnf-alpha (data not shown) mainly via rig-i signaling and the consequent activation and nuclear translocation of irf and nf-kb in human macrophages. these results were consistent with a previous study using beas- b cells showing the essential role of rig-i in ifn-beta reporter activity by h n influenza virus infection. while tlr also played a role in induction of ifn-beta and the activation of irf and nf-kb, it plays a less important role compared to rig-i. the reduction of irf and nf-kb activation was also confirmed with the study by le goffic showing differential regulation of irf and nf-kb by rig-i and nf-kb can also be regulated by tlr . in addition to the direct role of rig-i and tlr in sensing and signaling the presence of influenza virus, the innate immune sensing regulators were themselves also highly upregulated in both infected (data not shown) and adjacent uninfected cells by influenza virus infection. compared with seasonal h n virus, the h n viruses had a much more dramatic effect on inducing innate sensing receptors via jak signaling pathways activated by autocrine and paracrine mediators. the up-regulation of rig-i, mda , and tlr was markedly induced by virus free culture supernatants from h n -infected macrophages, while supernatant from ⁄ -infected cells induced the expression of these receptors only to a lesser degree. the soluble mediators in the virus infected cell supernatant caused paracrine upregulation of rig-i, mda , and tlr in uninfected macrophages as well as human lung epithelial cells. these effects may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. taken together these results provide, at least, part of the explanation on the hyper-induction of cytokines in h n infection. a more precise identification of the signaling pathways triggered by h n virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h n influenza and also for treating other causes of acute respiratory disease syndrome. we generated mutants of y (h n ) and a ⁄ duck ⁄ hokkaido ⁄ vac generation and characterization of mutant viruses rgy sub (h n ), rgvac sub (h n ), and rgvac ins (h n ), which have a serial basic amino acid residues at their ha cleavage sites were generated by site-directedmutagenesis and reverse genetics. rgy sub (h n ) and rgvac ins (h n ) required trypsin to replicate in mdck cells, and showed similar levels of growth to their parental viruses (table ) . chickens intravenously inoculated with rgy sub (h n ) or rgvac ins (h n ) did not show any signs of disease. rgvac sub (h n ) replicated in mdck cells without exogenous trypsin, and one of the eight chickens inoculated with the virus showed slight depression at day post-infection. the h and h mutant viruses were serially passaged in the air sacs of chicks to assess their ability to acquire pathogenicity. plaque formation in mdck cells and pathogenicity in -day-old chicks and -week-old chickens are shown in table . rgy sub (h n ) replicated in mdck cells in the absence of trypsin and killed all of the chicks after six consecutive passages. two of the eight-four-weekold chickens inoculated intravenously with rgy sub-p (h n ) died within days. eventually, over % of the chickens intravenously infected with rgy sub-p (h n ) died by days post inoculation, and its pathogenicity was comparable to that of hpaivs. rgvac sub-p (h n ) was pathogenic to both chicks and -week-old chickens, and mortality increased after one more passage. rgvac ins-p (h n ) replicated in mdck cells in the absence of trypsin, killed all of the chicks, and caused % mortality among -week-old chickens. the lethal effect of rgvac ins-p (h n ) on chickens increased with one additional passage in the air sacs of chicks, as in the case of rgvac sub (h n ). to examine whether the pathogenicity of each virus via the natural route of infection correlated with that by intravenous infection or not, three -week-old chickens were challenged intranasally with the viruses at an eid of ae and observed for clinical signs until day post-infection (data not shown). all chickens inoculated with rgy sub-p (h n ) or its parental viruses survived without showing any clinical signs, and serum antibody responses were detected in the hi test. on the other hand, rgvac sub-p (h n ) and rgvac ins-p (h n ) were pathogenic as in the intravenous experiment, killing two of three chickens by day post-inoculation. one of three chickens were not infected with rgvac sub-p (h n ) or rgvac ins-p (h n ) via intranasal route (data not shown), indicating these p viruses had not been completely adapted to the host. to investigate the possibility of these p viruses to acquire further pathogenicity for chicken, rgvac sub-p (h n ) and rgvac ins-p (h n ) were obtained from the brain homogenates of the chickens that died on days post intranasal inoculation with the p viruses. although mortality rate of chickens inoculated with the p viruses was equal to that with p viruses, enhancement of pathogenicity was observed in intranasal inoculation study; all of the chickens inoculated with rgvac sub-p (h n ) were infected, and time to death was shortened to - days post inoculation in chickens with rgvac ins-p (h n ) (data not shown). to investigate whether tissue tropism of the viruses was involved in their pathogenicity, we determined viral titers in the tissue and blood samples from -week-old chickens intranasally inoculated with each virus on days post infection ( table ) . rgy (h n ) and rgvac (h n ) were scarcely recovered from the samples, and the mutant strains before passage showed broader tissue tropism than the parental viruses. none of the chickens inoculated with rgy sub-p (h n ) showed any signs of disease, and viruses were recovered from each of the samples except the brain and the blood. one chicken inoculated with rgvac sub-p (h n ) showed clinical signs such as depression, and viruses were recovered from virtually all of its organs and blood samples. two of three chickens inoculated with rgvac ins-p (h n ) showed disease signs, and one died days post inoculation. the viruses were recovered from almost all samples of the two chickens showing signs of disease. p viruses were efficiently replicated in systemic organs of the chickens as compared with p viruses. throughout the study, the viruses were recovered from the brains of all of the chickens showing clinical signs. here, we demonstrated that the h influenza virus acquired intravenous pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the ha and passaged in chicks. rgy sub-p (h n ) killed % of chickens infected intravenously, and its pathogenicity was comparable to that of hpaivs (table ) . however, chickens intranasally inoculated with rgy sub-p (h n ) did not show any clinical signs of disease (data not shown). these results are consistent with a previous study in chickens that found some h influenza viruses did not show intranasal pathogenicity although their intravenous pathogenicity index was over ae , classified as hpaiv according to the definition by european union. ohuchi et al. reported that the insertion of additional basic amino acids into the h ha cleavage site resulted in intracellular proteolytic cleavage. other groups reported that h and h has tolerated amino acid mutations into their cleavage sites, and the viruses with the mutated has replicated in mdck and ⁄ or qt cells in the absence of trypsin. , the results in the present study is in agreement with these, namely, cleavage-based activation by a ubiquitous protease is not restricted to the h and h has. the intranasal pathogenicity of the h and h mutants were different (data not shown), although these viruses similarly replicated in mdck cells in the absence of trypsin and killed chickens by intravenous inoculation ( table ) . the viruses were recovered from the brain and the blood of some chickens infected with rgvac mutants (h n ), and morbidity was closely associated with viral titers in the brain (table ) . on the other hand, no viruses were recovered from the brain of chickens infected with rgy mutants (h n ), explaining why rgy sub-p (h n ) did not show intranasal pathogenicity. all the viruses passaged in the air sacs of chicks killed chicken embryos by hours post allantoic inoculation (data not shown). rgvac sub-p (h n ) and rgvac ins-p (h n ) were more pathogenic to chicken embryos than rgy sub-p (h n ); the allantoic fluid obtained from the embryonated eggs inoculated with the h viruses passaged in air sacs was turbid. it has been reported that infection of a highly pathogenic h virus were strictly confined to endotherial cells in chicken embryos or chickens. , therefore, it is suggested that endotheliotropism differed between the h and h viruses passaged in air sacs and affected their intranasal pathogenicity. taken together, it is assumed that rgvac sub-p (h n ) and rgvac ins-p (h n ) showed marked intranasal pathogenicity with high levels of viremia caused by replication in vascular endothelial cells, leading to invasion of the brain. in the intravenous experiment, rgy sub-p (h n ) easily reached systemic organs, including the brain hematogenously, replicated through the cleavage of ha by a ubiquitous protease, and then exerted its pathogenicity. further study including a pathological analysis is currently underway to test this hypothesis. for all hpai viruses of subtypes h and h known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k ⁄ r-r with r at position p and k-k ⁄ r-k ⁄ t-r with k at p , and leads to a systemic infection. early studies demonstrated that the ubiquitously expressed furin and pcs are activating proteases of hpai viruses. furin and pcs cleave the consensus multi-basic motif r-x-k ⁄ r ⁄ x-r with r at position p . however, replacement of p r by k and a nonbasic amino acid significantly suppresses the processing activities of furin and pcs. most of the type ii transmembrane serine protease identified so far recognize a single r at position p , but the newly isolated mspl and its transcript variant tmprss preferentially recognize paired basic residue, particularly r and k at position p , at the cleavage site. [ ] [ ] [ ] thus, mspl and tmprss can activate various bioactive polypeptides with multibasic residue motifs, including fusogenic viral envelope glycoproteins. the present study was designed to characterize the proteolytic processing of the hpai virus ha by mspl and tmprss in comparison with furin. hpai virus a ⁄ crow ⁄ kyoto ⁄ ⁄ (h n ) was isolated from embryonated eggs inoculated with tracheal homogenates from dead crows. then, the mutant ha sequence was constructed by changing r residue to k residue (n'-rkkr-c' to n'-kkkr-c') at the ha cleavage site by sitedirected mutagenic pcr as described. we used human cell line ecv , which expresses mspl and tmprss at levels below detection, and established the cells stably expressing mspl and tmprss , such as ecv -mspl and ecv -tmprss . to determine the cleavage specificities of mspl ⁄ tmprss and furin, peptides ( lg each) were incubated with ae mu mspl ⁄ tmprss for hour and furin for hours at °c, respectively. after incubation, the samples were separated by reverse-phasehigh-performance liquid chromatography (rp-hplc) with the use of a c column. the elution samples were then identified by amino acid sequence analysis and by maldi-tof-ms. we analyzed the cleavability of -residue synthetic peptides derived from ha cleavage sites of hpai strains, such as a ⁄ chick ⁄ penn ⁄ ⁄ (h n ) and a ⁄ fpv ⁄ rostock ⁄ (h n ), and low pathogenic strain a ⁄ aich ⁄ ⁄ (h n ). after incubation with human mspl or human furin, the digested samples were separated by rp-hplc, and peptide fragments were characterized by mass-spectrometry and protein sequencing. in contrast to the low cleavage efficiencies of the h ha peptide with a single r at the cleavage site ( figure a) , both the h ha peptide with the k-k-k-r motif ( figure b ) and the h ha peptide with the r-k-k-r motif ( figure c) were fully processed at the correct positions by mspl within hour. in the case of h ha peptide with multiple basic residues, mspl cleaved two carboxyl-terminal sides of r in the cleavage site sequence of n'-k-k-rfl-k-k-rfl-g-c', while furin cleaved only at a single site of r with r at position p , n'-k-k-r-k-k-rfl-g-c' in the presence of mm cacl . these cleavage site specificities of furin were consistent with that reported for the h ha peptide of hpai virus a ⁄ hong kong ⁄ ⁄ (h n ) with r-k-k-r motif. however, the h ha peptide with k at position p ( figure b) was hardly cleaved by furin under the same experimental conditions. tmprss showed similar results (data not shown). these findings suggest that mspl and tmprss cover diverse cleavage specificities, including non-susceptible specificity to furin. full length recombinant ha of hpai virus with kkkr cleavage motif was converted to mature ha subunits with membrane-fused giant cell formation in mspl or tmprss transfectant cells. in addition, this conversion was suppressed by bowman-birk trypsin inhibitor, a membrane non-permeable highmolecular mass inhibitor against mspl ⁄ tmprss . to test for the generation of infective virus, the conditioned media of -day culture of ecv -wt and ecv -mspl cells infected with wt and mutant hpai h n viruses were inoculated into newly prepared cells and cultured for hours. although spreading of wt virus infection with ha cleavage motif of r-k-k-r was detected from the conditioned medium of both ecv -wt and ecv -mspl cells, that of mutant virus with ha cleavage motif of k-k-k-r was only detected from the condition medium of ecv -mspl cells. these results strongly suggest that the expression of mspl, but not furin, potentiates multicycles of hpai virus with k-k-k-r ha cleavage motif. seasonal human influenza a virus has have consensus monobasic cleavage site sequence, n'-q ⁄ e-x-rfl-g-c', and all hpai virus has have two types of cleavage site sequences with multiple basic amino acids, n'-r-k ⁄ r-k ⁄ r ⁄ x-rfl-g-c' with r at position p in a large number of hpai viruses and n'-k-k ⁄ r-k ⁄ t-rfl-g-c' with k at position p in a small number of hpai viruses. figure shows furin efficiently cleaved synthetic hpai a ⁄ hong kong ⁄ ⁄ (h n ) ha cleavage site peptide with the r-k-k-r motif, but hardly cleaved the hpai virus a ⁄ chick ⁄ penn ⁄ ⁄ ha cleavage site peptide with the k-k-k-r motif. furthermore, cleavage of the full-length ha of hpai virus with r-k-k-r motif was detected, but cleavage of hpai virus ha with k-k-k-r motif was hardly detected in ecv -wt cells containing furin ( figure ). these substrate specificities of furin suggest that proteases other than furin and pc ⁄ play a role in the processing of has of hpai virus with k-k ⁄ r-k ⁄ t-r cleavage motif. mspl and tmprss show unique cleavage site specificities of the double basic residues at the cleavage site, and r or k at position p greatly enhanced the efficiency, which none of the other ttsps have shown similar substrate specificities so far. furthermore, infectious and multicycle viral replication along with ha processing was also noted in genetically modified mutant recombinant live hpai virus a ⁄ crow ⁄ kyoto ⁄ ⁄ (h n ) with k-k-k-r cleavage motif in ecv -mspl cells (figure ) . these results were supported by the data of two cleaved peptides by mspl in figure c . these findings suggest that mspl has diverse cleavage specificities and may cleave ha at least two sites, although multiplicity of the mutant hpai virus was observed under the conditions. these results also suggest that mspl and tmprss in the membrane might potently activate the ha membrane fusion activity of hpai viruses and promote their spread. highly pathogenic avian influenza viruses replicate in various organs in birds, and the ha processing proteases might be widely distributed in these organs. indeed, tmprss and mspl are ubiquitously expressed in almost all human organs tested and are highly expressed in lungs, leukocytes, pancreas, spleen, and placenta. , in addition, mspl and tmprss are strictly localized in the plasma membranes, suggesting that proteolytic activation of hpai virus ha occurs not only through the trans-golgi network by furin and pc ⁄ , but also on the cell surface by mspl and tmprss . the pb -f protein, which is translated from the + reading frame of the pb gene segment, has been linked to the pathogenesis of both primary viral and secondary bacterial infections in a mouse model. - a mitochondrial targeting sequence is located in the c-terminal portion of the pb -f open reading frame, and expression of full length pb -f has been associated with mitochondrial targeting and apoptosis in a monocyte dependent manner. , it has been theorized that enhanced virulence could result from mitochondrial disruption with subsequent cell death mediated by pb -f . , a suggested second function of the pb -f protein is that it enhances immunopathology by triggering the inflammatory response. , in earlier studies from our group, the pro-inflammatory phenotype was markedly upregulated when the pb -f from the pandemic strain was expressed, arguing that this protein may be an important virulence factor for highly pathogenic pandemic viruses. , in this report we analyze the pb -f protein's contribution to pathogenesis in a mouse model, examining both inflammation and cell death. pb -f proteins from a variety of epidemiologically important iav strains including all pandemic strains from the th century, a highly pathogenic avian influenza virus of the h n subtype, and representative seasonal strains were utilized to determine the relevance to pandemic disease. we demonstrate that macrophage mediated immunopathology, but not apoptosis, are relevant functions of pb -f proteins from past or potential pandemic influenza viruses. using the predicted amino acid sequences of the pb -f proteins from pr , a ⁄ brevig mission ⁄ ⁄ , ⁄ singapore ⁄ ⁄ , a ⁄ hong kong ⁄ ⁄ , a ⁄ wuhan ⁄ ⁄ , and a ⁄ vietnam ⁄ ⁄ , peptides from the c-terminal end were synthesized as described. an additional n-terminal peptide was synthesized from the pr sequence as a positive control (mgqeqdtpwilstghistqk) as described. a panel of viruses were reverse engineered as described , and included laboratory strain pr , a virus unable to express pb -f (dpb -f ⁄ pr ), or expressing the pb -f of the pandemic strain ( pb -f ⁄ pr ) or the truncated h n strain (beij pb -f ⁄ pr ). , in addition, : reassortants encoding pb gene segments from a *current address: department of immunology and microbiology, university of melbourne, melbourne, vic., australia. highly pathogenic avian influenza of the h n subtype (h n pb ⁄ pr ), or from a human h n strain (h n pb ⁄ pr ) were utilized along with their isogenic deletion mutants for pb -f (h n dpb -f ⁄ pr and h n dpb -f ⁄ pr ). cell lines and cell death assays raw . cells were grown under conditions as described. cells were infected with one multiplicity of infection (moi) of virus for - hours, or exposed to lm (final concentration) of peptides derived from the c-terminal portion of pb -f for hour. cells from the supernatant and monolayers were harvested, washed, and stained with annexin (apc) and propidium iodide (pi) (becton dickinson, san jose, ca, usa), then analysed for cell death as described. six-to eight week old female balb ⁄ cj mice (jackson laboratory, bar harbor, me, usa) were maintained in a biosafety level facility in the animal resource center and procedures approved by the animal care and use committee at sjcrh. infectious agents and peptides were diluted in sterile pbs and administered intranasally to anesthetized mice (n = - ) in a volume of ll ( ll per nare) and monitored for overt signs of illness and weight loss daily. following euthanasia by co inhalation, the trachea was exposed and cannulated with a gauge plastic catheter (bd insyte; becton dickinson, sandy, ut, usa). bronchoalveolar lavage fluid (balf) was collected, red blood cell depleted, and cellular content analyzed via flow cytometry as described. one way analysis of variance (anova) was used for multiple comparisons of cell death and cellularity of balf. a p-value of < ae was considered significant for these comparisons. graphpad prism version . for windows (graphpad software, san diego, ca, usa) was utilized for all statistical analyses. to assess the contribution of pb -f to inflammation, we utilized a panel of previously described reverse engineered viruses in the mouse infection model. , the effect of pb -f expression was observed clearly in the inflammatory infiltrate in response to infection in the lungs. deleting pb -f from pr or expression of the c-terminally truncated beij pb -f had a significantly reduced influx of macrophages ( figure a) . expression of the pb -f caused similar inflammatory effects as the pr virus. disruption of pb -f expression the virus containing the h n pb gene segment in a pr background also significantly decreased the inflammatory response compared to the virus maintaining the ability to express full length pb -f ( figure a) . however, no differences were seen that could be attributed to the h n derived pb -f . the lungs of mice infected with the panel of pb -f variant viruses were examined at hours. pathologic changes typical of pr viral infection were observed in all lungs. these typical findings included perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris (figure ). in the lungs of mice infected with pr or pb -f ⁄ pr , however, significantly more perivascular cuffing was noted, with a prominent increase in numbers of macrophages (figure a, c) . the overall number of inflammatory cells throughout the lungs, including both airways and alveoli, was quantitatively greater in these mice than in mice infected with dpb -f ⁄ pr or beij pb -f ⁄ pr ( figure b, d) . as the function and influence of pb -f protein on normal viral function is not currently understood, and given the abrogation of enhanced inflammation induced by the truncated pb -f beij ⁄ pr virus, we sought to elucidate whether the c-terminal domain of pb -f could alone induce this inflammatory response. mice were exposed to a panel of peptides and were euthanized hours later for collection of balf. significant influxes of macrophages into the balf were seen following exposure to c-terminal pb -f peptides derived from pr , the pandemic strains from (h n ), (h n ), and (h n ), and the h n virus compared to controls ( figure b) . similar effects were not seen with the peptide derived from a more recent h n strain, a ⁄ wuhan ⁄ ⁄ . when peptide exposed mice were followed for morbidity for days, peptides proven to induce a heightened inflammatory response correlated strongly with overt clinical signs of illness (data not shown). thus, the ability to cause lung inflammation appears to be a property of pb -f proteins of viruses containing pb gene segments reassorted directly from the avian reservoir. the pb -f protein may contribute to virulence by rendering the host cellular immune response ineffective through inducing apoptosis. we sought to determine whether this was an epidemiologically important function for combating the host immune response to infection by testing the ability of pb -f proteins from several different iav strains to cause cell death. we therefore infected raw . cells with the panel of recombinant viruses at an moi of for - hours. as has been demonstrated previously, , , pr virus induces significant cell death compared to uninfected controls ( figure c ). when raw . cells were infected with pr virus, necrotic death peaked hours after infection. viruses lacking the c-terminal portion of pb -f , including the dpb -f ⁄ pr and the beij pb -f ⁄ pr were unable to cause cell death ( figure c ). in addition, expression of the pb -f also did not cause significant increases in cell death over controls. expression of pb -f or deletion of pb -f in either an h n or h n pb gene segment background similarly did not alter the cell death phenotype. to examine additional strains for which we did not have isogenic virus pairs, we next exposed the balbcj mouse derived macrophage cell line raw . to the panel of pb -f peptides derived from pr , the pandemic strains from (h n ), (h n ) and (h n ), and the h n for hours. cell death in raw . cells was caused only by the peptides derived from the laboratory strain pr and the peptide derived from the pandemic strain ( figure d ). viability was not affected by exposure of raw . to peptides derived from other virus strains. we conclude from these data that the mechanism by which pb -f contributes to the pathogenicity of pandemic influenza is unlikely to be through its reported ability to cause cell death. these data presented here demonstrate that the lung inflammatory response is enhanced by the influenza a virus pb -f protein in a mouse model. this inflammatory response was characterized by increased cellular infiltration of macrophages into the interstitial and alveolar spaces of the lungs, as well as enhanced perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris. this augmentation was shown to be induced by pb -f proteins only from those strains contributing to the formation of all pandemic strains of the th century and from the currently circulating, highly virulent h n strains that constitute an imminent pandemic threat. the iav h n strains circulating in humans since around code for a truncated pb -f . these viruses may lack the cterminal residues responsible for the inflammatory effects demonstrated in this publication. additionally, recently circulating h n strains, in contrast to their pandemic forbear from , have lost the capacity to cause pb -f mediated inflammation through mutation of the c-terminus of this protein. in a novel h n iav emerged from an animal reservoir and caused a human pandemic. disease burden from this strain has been considered mild in contrast to the three pandemics of the th century. the reasons for this disparity in pathogenesis are unclear. an examination of the origins of the three th century pandemics shows that only the hemagglutinin (ha) and pb gene segments were reassorted directly from the avian reservoir in every case, suggesting gene products of one or both of these may be important. the ha surface glycoprotein provided the antigenic novelty required for the each virus to achieve pandemic status. however, the significance of inclusion of a novel pb gene segment in each of the th century pandemics is not yet understood. we show here that the pb -f of these pandemic strains contributes to virulence through induction of inflammatory responses. thus pb -f may serve as a marker of the pathogenicity of pandemic strains. since the h n strain codes a truncated pb -f of only predicted amino acids, the lack of pb -f mediated inflammation may account in part for its relatively lower virulence. , of the panel of pb -f proteins studied, only that from the laboratory strain pr was capable of rendering responding host-immune cells ineffective by induction of cell death. we therefore hypothesize that molecular signatures specific to induction of apoptosis may have been lost through genetic mutation of the pb -f gene throughout the evolution of the iavs. our findings suggest that this apoptotic function is unlikely to be important for the virulence of any of the known pandemics. rather, the inflammatory phenotype appears to be the dominant contribution of pb -f to pandemic disease. influenza virus-cytokine-protease cycles are principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches introduction influenza a virus is the most common infectious pathogen in humans, causing significant morbidity and mortality, particularly in infants and the elderly. mof with severe edema is observed in the advanced stage of influenza pneumonia. however, the relationships amongst factors that induce vascular hyper-permeability in severe influenza remain unclear. it is reported that significant increases in levels of pro-inflammatory cytokine levels, such as tnf-a, il- , and il- b, affect host survival both positively and negatively. the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and mof. in addition, iav infection up-regulates several cellular proteases including ectopic trypsin and mmp- . up-regulated ectopic trypsin mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin (ha), which is crucial for viral entry and replication and the subsequent tissue damage in various organs. the aim of the this study was to define the pathogenic impact of cytokine storm in iav infection and the molecular mechanisms by which pro-inflammatory cytokines and proteases cause vascular dysfunction in animal model. weanling female mice aged weeks (c bl ⁄ crslc) were infected with iav ⁄ wsn ⁄ ( pfu) with and without treatment of pdtc ( . mg ⁄ kg), nac ( mg ⁄ kg), and ndga ( mg ⁄ kg). these inhibitors were administrated once daily for days after infection. the levels of cytokines in tissue homogenates were measured by elisa kits. the effect of inhibitors on viral replications was determined by real-time pcr. gelatin zymography and western blotting were conducted as reported previously. host cellular responses in the airway after iav infection figure shows schematic view of typical biological responses in the airway of mice after iav infection. an initial response before viral proliferation is significant increases in pro-inflammatory cytokine levels. immediately after cytokine inductions, there is a marked up-regulation of ectopic trypsin along with an increase in virus titer in the airway, lung, and brain. ectopic trypsin mediates the post-translational proteolytic cleavage of iav ha, which is crucial for viral entry and replication and the subsequent tissue damage in various organs. we also found that iav infection markedly induces mmp- and matrix degradation. just after the peak of viral proliferation, the innate and adaptive immune responses of protective immunity are induced for defense and recovery, or oppositely on rare occasions, mof with vascular hyper-permeability is started into the advanced stage of influenza. the levels of tnf-a and il- in the lungs were increased persistently for days after iav wsn infection, and that of il- b peaked at days - post-infection (figure a ). since these cytokine responses are associated with activation of nf-jb and ap- , we treated mice once daily for days with anti-oxidant inhibitors: pdtc and nac against nf-jb activation, and ndga against ap- activation. pdtc and ndga significantly suppressed the up-regulation of tnf-a and il- b (p < . ), and nac suppressed tnf-a (p < . ), and il- (p < . ) at day post-infection. gelatin zymography showed up-regulation of ectopic trypsin and mmp- in mice lung, brain, and heart during infection for days ( figure b ). trypsin and mmp- induction was inhibited by treatment with pdtc, nac, and ndga, probably via blockade of nf-jb and ap- binding in the promoter region of the genes. viral rna replication in various organs at day post-infection was suppressed by more than one order of magnitude by pdtc, nac, and ndga ( figure c ). suppression of viral multiplication and induction of cellular factors by pdtc, nac, and ndga, significantly improved the survival of mice at day post-infection, the late stage of infection ( figure d ). to elucidate the mechanisms underlying brain vascular dysfunction of influenza-associated encephalopathy, changes in the levels of tight-junction proteins, intracellular zonula occludens- (zo- ) and transmembrane occludin, and the matrix protein laminin, were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day post-infection, which were partly rescued by pdtc, nac, or ndga (figure e ). no other tight-junction protein, claudin- or matrix fibronectin and type iv collagen, were affected. the present study reports several new observations: (i) proinflammatory cytokines, tnf-a, il- b, and il- , when up-regulated by iav infection, induce trypsin and mmp- expression in various organs in mice; (ii) inhibitors of nf-jb and ap- effectively suppress the up-regulation of proinflammatory cytokines, trypsin, and mmp- and improve survival rates of infected mice. based on these results, we propose the 'influenza virus-cytokine-protease cycle' hypothesis as one of the mechanisms of vascular dysfunction in mof with cytokine storm in severe influenza and influenza-associated encephalopathy. the significance of pro-inflammatory hyper-cytokinemia, or 'cytokine storm,' in the pathogenesis of iav infection remains unclear. on the positive effects, cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, on the negative effects of excess cytokines, the hyper inflammatory process evoked by viral infection may become harmful through intracellular activation of nf-jb, ap- , and the janus kinase-signal transducers and activators of transcription signaling pathways. , [ ] [ ] [ ] the in vivo experiments presented here showed that nf-jb and ap- inhibitors markedly suppress the expression of cytokines, trypsin, mmp- , and viral replication, resulting in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production ⁄ activation of vasodilatory mediators such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs. the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyper-permeability following the 'cytokine storm' remain unclear. tnfa up-regulation alters the cellular redox state, reduces the expression of four complex i subunits by increasing mitochondrial o ) production and depleting atp synthesis, decreases oxygen consumption thereby resulting in mitochondrial damage, , and increases [ca + ] i atp depletion dissociates zo- from the actin cytoskeleton and thereby increases junctional permeability. endothelial dysfunction induced by 'influenza virus-cytokine-protease cycle' in the early stage of severe influenza may further affect various circulating factors, coagulation factors and complement systems, and vascular interacting cells, such as neutrophils, macrophages and lymphocytes. mof is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h n and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and formation of oxidized phospholipids, which induce lung injury via toll-like receptor signaling pathway. in addition to these data, up-regulated trypsin and pro-inflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of iav infection. further studies are required on the role of the 'influenza virus-cytokine-protease cycle' in the pathogenesis of mof, particularly in the late stage of viral infection. though influenza a virus replication kinetics and host responses have been previously studied in umbilical vein endothelial cell or transformed endothelial cell lines, the tropism of influenza a virus including h n and pandemic h n pdm for primary human lung microvascular endothelial cell has not been well defined. in this study we employed primary human lung microvascular endothelial cells, which are more physiologically relevant for understanding pathogenesis of influenza in the lung as to obtain a better understanding of the links of endothelial cell infection to systematic virus dissemination and multiple organ involvement in severe human influenza. supernatants of cells infected at moi of two were collected for cytokine protein assays, and total rna was extracted for gene expression analysis using qpcr. we found that seasonal influenza h n and h n viruses initiated viral gene transcription and viral protein expres- sion, but did not produce infectious progeny, while the highly pathogenic avian influenza h n and the pandemic influenza h n pdm virus could replicate even with the absence of exogenous protease (figure ) . furthermore, when compared to seasonal h n and h n , the h n virus was a more potent inducer of cytokine and chemokine including ifn-b, mcp- , rantes, ip- (figure ) , and il- , in virus infected endothelial cells, whereas h n pdm induced intermediate levels of cytokine and chemokine. avian influenza h n and pandemic h n pdm virus (but not the seasonal h n and h n virus) can productively replicate in human lung microvascular endothelial cells. this is likely to be of relevant to pathogenesis and provides a possible explanation for the extra-pulmonary infection seen in animal infection models. this extra-pulmonary spread may support the previous speculation and anecdotal evidence that h n and h n pdm virus can infect the gastrointestinal tract through the virus dissemination from the infected respiratory tract as the first target cells for influenza infection. [ ] [ ] [ ] in addition, the release of proinflammatory cytokine and chemokine induced by influenza h n and h n pdm virus infection in lung microvascular endothelial cells may be important contributors to the pathogenesis of severe human influenza disease leading to endothelial cell dysfunction that contributes to severe pulmonary disease symptoms. during its replication, influenza virus utilizes the host cellular machinery for many aspects of its life cycle. characterization of such virus-host protein-protein interactions is a must to identify determinants of pathogenesis. the m ion channel protein plays a crucial role during the entry and late stages of the viral life cycle where its c-terminal domain, well conserved among influenza a viruses, is accessible to cellular machinery after fusion with endosomal membrane and during its trafficking along the secretory pathway prior to assembly and budding. the aim of the study is to identify cellular interactants of m that play important regulatory roles during influenza infection. to identify cellular partners of m we performed a genome-wide yeast-two-hybrid (y h) screening approach using the cytosolic domain of m as bait and a human placenta random primed cdna library as prey and tested more than million interactions. from the y h screening, an interesting interaction with the human annexin a (anxa ) protein, a member of annexin family proteins that binds to phospholipds in a ca + -dependent manner, was identified. co-immunopre-cipitation of myc-tagged anxa and viral m proteins coexpressed in hek t cells after transfection and infection confirmed the direct interaction between anxa and m . we further investigated whether this interaction had any functional significance with regards to influenza life cycle. using a rna interference strategy to silence the anxa gene in human lung epithelial a cells, we observed increased progeny virus titers either in a single or multiple viral growth kinetics study, suggesting a negative regulatory role for anax during viral infection (figure ). a novel interaction between m and anxa was identified. more functional studies are in progress to define precisely the potential negative regulatory role of this interaction during viral infection. a systematic dissection of the viral life cycle will be performed to identify the step(s) affected by the anxa cellular factor using specific assays such as real-time quantitative rt-pcr in a single or multiple viral growth kinetics study, cell transduction with ha-and m -pseudotyped lentiviral particles, virion attachment and internalization assay, immunofluorescence staining of np protein as a marker of viral ribonucleoproteins localization, viral polymerase activity measurement, and viral budding observation by electron microscopy. rna extraction was achieved by qiagen biorobot ez prior to respiratory multiplex pcr analysis. what remained of the extracted material of each specimen was stored by refrigeration at °c. electronic patient records were searched for parameters, such as c-reactive protein (crp), white cell count (wcc), length of admission in days, and patient co-morbidities. patients were divided into three groups according to clinical severity: mild, moderate, and severe. the 'mild' group comprised of those admitted for three days or fewer, or not admitted at all. the 'moderate' group comprised those who required admission to hospital for more than days as a result of swine flu, but who did not require admission to an intensive care unit (itu). the 'severe' group comprised those who had required itu admission. invitrogen ' · reaction mix': ae mm of each dntp + mm magnesium sulphate. primer ⁄ probe mix recipe applied biosystems fast real-time pcr system, 'respiratory multiplex' program. well content ll; thermocycler initial stage ae °c for minutes, then °c for minutes. subsequent cycles of ae °c for seconds followed by °c for seconds for cycles. sequence detection software version . (applied biosystems). of clinical isolates analyzed, all samples produced amplification of pdh material; produced amplification of both swine flu and pdh material. human male dna (lot no. at ng ⁄ l, applied biosystems) at concentration calculated at ae cells ⁄ ll was diluted from ) to ) , yielding mean average ct values of respectively ae , ae , ae , and ae . plotting log of cell number versus ct gave a y = mx + c line from which ct could be interpolated into cell numbers. for swine flu quantification, a sample of swine flu ct ae was diluted through ) to ) . it must be noted that due to variability in resultant swine flu ct values, repetitions at these dilutions were done using an rna carrier ( lg ⁄ l, qiagen; cat no. ) in place of rnasefree water. the ) concentration was positive in nine out of assays; this fraction was used in the calculation described by simmonds to obtain a copy number of targets per reaction by the equation copy value = )ln(f), where f is decimal fraction of failure rate. here, f = ⁄ = ae ; )ln ae = ae copies. a control curve was generated with ct values of ae , ae , ae , and ae giving copy values of , ae , ae , and ae , respectively. using excel (microsoft office, ), these control series were adapted into formulae to convert swine flu and pdh ct values into copy numbers of these elements per reaction. simple division derived a value for swine flu copy per pdh copy, but this was chosen to be expressed as swine flu copy number per human cells. this will be referred to as the 'c' value. forty-two patients had known clinical details; average age was ae , female to male ratio : , and average admission length of days. of the mild group (n = ), nine cases were not admitted to hospital. of the remainder, the mean average admission length was ae days. mean average c value for all samples was ae · , with a standard deviation of ae · ; geometric mean was ae , and median average was ae . log(mean average c value) is shown for each severity group and for identified risk factors in the 'mild' severity group (figures a, b respectively) . in each case variation was too great to yield statistical significance. figure shows the range of c values observed in the 'moderate' severity group. > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > · ; < · > · ; < · > · ; < · > · ; < · > · ; < · > · ; < · in a study by duchamp et al., no significant correlation was observed between viral ct value and presence or absence of cardiaorespiratory disease, myalgia, digestive symptoms, or upper or lower respiratory tract infection (although a trend was observed towards patients presenting with signs of upper respiratory tract infection). to our knowledge, no other study has used a dual pcr for analysis of respiratory virus concentrations, and no study has attempted to correlate biochemical markers with respiratory virus concentration. the data exhibited a spectrum of c values, from values < · ) to over · . the three severity group standard deviations all overlapped with each other, preventing statistical significance. analysis of co-morbidities showed a high mean average c value when asthma was present ( ae · ), but again this was associated with an excessive standard deviation. whereas the median average c value in the presence of asthma was higher than the overall average c value ( ae versus ae ), it was significantly lower than the median c value when no co-morbidity was documented ( ae ). there are multiple caveats that may be the cause of such variety of c values obtained. the duration between initial rna extraction and study pcr had a range of to days, with mean average delay of days. the degradation of viral rna is an important contributor to assay variance and failure; rna degradation in clinical samples has been studied. [ ] [ ] [ ] degradation of human dna in clinical samples may have occurred. several studies have chartered degradation of stored human dna. , with regards to sampling, the clinical collection of throat swabs is naturally variable according to the method of the collector. a small number of bronchoalveolar lavage samples were analyzed, yet did not amplify, presumably due to rna degradation. the upper respiratory tract may be only a physical stepping stone for the virus, and take no further role in pathogenesis of severe disease (although undoubtedly is crucial for transmission). interestingly, a ferret study of pathogenesis observed that swine flu yields from the upper respiratory tract were greater than those given by ordinary seasonal h n , with consequently increased shedding. the review by mansfield cites significant findings regarding influenza pathogenesis, including the predilection of h n strains for type ii pneumocyte cells and alveolar macrophages. it also highlights the limitation of knowledge through dearth of human autopsy studies; an exception is the recognition of haematophagocytic syndrome in severe cases. it is known that specific immunoglobulin is effective against establishment of infection in the upper respiratory tract, whereas specific cytotoxic t lymphocytes (ctls) are necessary for clearance of the virus from the lower respiratory tract. it is also suggestive that a gap of two whole days transpires between initial infection and instigation of a specific immune response. it is plausible that in the healthy individual, virus progression is confounded by efficient natural mucosal immunity, in part through good secretory immunoglobulin levels. airway inflammation associated with asthma exacerbation is known to increase both risk of respiratory viral infection and poorer outcome. it is unproven but likely that the local inflammatory processes give rise to increased virion burdens in the upper airways; however, the same effect is conceivable for epithelial cell turnover. there will likely be variance within each clinical category due to patient circumstances and clinicians' judgment of required admission. unfortunately, the duration of symptoms prior to swab collection was often omitted in the clinical notes. finally, stratification of patient group by receipt of antiviral treatment was not studied. no correlations were observed with c values and crp, wcc or admission length. trends were observed towards higher c values in 'mild' cases, but without statistical significance. the relative small study size, coupled with the intrinsic variability of the parameters studied, warrants larger, better controlled, prospective studies to elucidate clinical use of the c value for influenza illness prediction and management. in mid-april a novel variant of a(h n ) influenza virus began to spread rapidly throughout the world, causing the first pandemic of the st century. the majority of the cases associated with this new virus show to be mild, but severe and fatal cases have been reported. molecular markers associated with severity have already been identified, as is the case of the mutation d g. resistant viruses to antiviral drugs have also been identified, highlighting the importance of rapid determination of the antiviral drug profile. global a(h n ) genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. the objective of this ongoing research study was, primarily, to thoroughly characterize the genetic profile and evolution of the emergent influenza a(h n ) virus circulating in portugal and its phenotypic expression on antiviral drugs susceptibility. the cases considered in this study were obtained from the community and from two collaborating hospitals in lisbon -a reference hospital for adults (hospital de curry cabral) and a reference hospital for children (hospital dona estefânia). the cdc real-time pcr protocol, recommended by world health organization (who), was the method used to confirmed all influenza a(h n ) cases. from a total of a(h n ) positive cases diagnosed and confirmed, were selected for this study, taking in consideration that they should cover the period of epidemic activity in portugal and include cases from persons belonging to risk groups and cases associated with more severe clinical features. ninety-six a(h n ) strains were isolated in mdck-siat cells, from combined naso-oropharyngeal swabs. for the evaluation of the genetic profile of a(h n ) virus circulating in portugal, of the isolates were characterized by genetic analysis of the ha, na, and mp genes. the remaining five gene segments (pb , pb , pa, ns, and np) were also sequenced for six of this isolates. briefly, sequencing was performed according to the protocol developed by cdc and recommended by who, using bigdye terminator v. . technology. nucleotide sequences were determined in a dna automatic sequencer abi prism xl genetic analyzer. for each genomic segment, genetic analysis was performed with lasergene v. . software (dnastar inc, usa) using an average of - overlapping readings, including sense and antisense, for precise nucleotide and amino acid sequence determination. genetic mutation and phylogenetic analysis were performed by neighbor-joining method, using mega . software, against published sequences from the vaccine strain (a ⁄ california ⁄ ⁄ ) and from selected a(h n ) strains available on gisaid epiflu database. all mutations were identified with reference to the vaccine strain genome sequence. antiviral drug susceptibility profile of a(h n ) influenza virus circulating in portugal was evaluated both phenotypically and genotypically for nais and genotypically for amantadine. phenotypic evaluation to nais, oseltamivir and zanamivir, was performed for all isolates by ic determination through munana fluorescence assays. genotypic evaluation was performed by searching for mutations associated with resistance to nais in all na gene sequences. amantadine susceptibility profile was performed for all isolates by searching on m sequence for the molecular markers associated with resistance to this antiviral drug (l f ⁄ i; v a ⁄ d; a t; s n; g e). genetic characterisation of the ha subunit of ha reveals point mutations in different strains. all analysed strains present p s and i v mutations, which distinguish them from the vaccine strain ( figure a ). thirty-three of the sequenced strains group in the s t branch. this mutation is referred in the literature as being associated with the putative antigenic site ca. most of these strains ( ) further subgroup in the d e branch, this mutation being associated with one loop of the receptor-binding site. from the early to the late epidemic period, an increased circulation of virus carrying the mutation s t was observed. this is in agreement with the association between this mutation and an enhanced viral fitness that is described in the literature. additional mutations were also observed in a small number of virus, of which we highlight: regarding the genetic characterisation of na, the majority of strains analysed ( of ) presents the mutations n d and v i ( figure b) . as mutation s t in ha gene, these two na mutations are described in the literature as associated with enhanced viral fitness. the few strains not carrying these mutations have circulated in the beginning of the epidemic period. fifteen of the analysed strains further subgroup in y h branch. additionally, mutation i v was identified in two strains. for the remaining gene segments available for the six analysed strains, the observations include: (i) no previously described virulence markers in pb , pb -f , and ns were detected; (ii) pb -f protein is present in the truncated form of amino acids; (iii) the presence of mutations i v and l q in ns and v i in np; (iv) the described association of mutation i v in ns and v i in np genes with viral fitness. phenotypic evaluation of nais susceptibility revealed the existence of three minor and two major outliers to oseltamivir ( figure ). the two minor outliers exhibited a reduction of approximately twofold in the susceptibility to this antiviral drug, comparing to the baseline level, while the reduction exhibited by the two major outliers was of approximately three-and fourfold. regarding zanamivir, two minor outliers were identified with a reduction of approximately twofold in the susceptibility, compared to the baseline level. these two minor outliers (a ⁄ portugal ⁄ ⁄ and a ⁄ portugal ⁄ ⁄ ) correspond to the two major outliers identified for oseltamivir. genetic analysis revealed the presence of the mutation i v in the na sequence of these two strains. the contribution of this mutation for the profile of reduced susceptibility identified for both nais is not known, but a mutation in the same na position (i r) has been referred to as being associated with a reduction in nais susceptibility. full genome sequence analysis of these strains shows that both strains also present the v i mutation in pb gene. however, no association of this mutation with antiviral drug susceptibility is referred in the literature. concerning genetic evaluation of susceptibility to amantadine, all analysed strains present a serine in position , which is a molecular marker of resistance to m inhibitors. these preliminary results allow us to discuss several points. however, the additional data that is being obtained through this ongoing study will be essential for a more complete analysis. for example, more information is needed to determine if the mutations found alter the biology and the fitness of the virus or if there are associated with an increased prevalence of the virus. the majority of the mutations identified in ha subunit have been detected in a(h n ) strains distributed throughout the epidemic curve, not evidencing a specific evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h n ) virus. the occurrence of mutations in the position of the ha subunit of a(h n ) virus have been described. however, more studies are needed to clarify the outcome of these mutations, as for example in patients with severe complications. it could also be relevant to investigate the presence of single and mixed variants in viruses and in clinical specimens and the possibility of these mutations affecting the binding specificity. regarding the susceptibility of a(h n ) pandemic viruses to antiviral drugs, all analysed strains were found to be resistant to amantadine. this resistant profile was not unexpected since the mp gene from this new variant had originated in the eurasian swine lineage, which is characterised by being resistant to this antiviral drug. the majority of the a(h n ) strains analysed revealed to be susceptible to both nais, with only five strains exhibiting a profile of reduced susceptibility, three to oseltamivir and two to both nais. for these last two, the presence of the i v mutation in the na sequence could explain the reduction observed, but a more complete analysis is needed to confirm this. the french national pandemic plan includes an early containment phase followed by a limitation phase. the efficacy of such a plan depends on pre-existing surveillance and laboratory networks. the grog community surveillance network and the hospital lab networks organized by the two french nics carried out the virological monitor- the efficacy of such plan depends on pre-existing influenza surveillance and laboratory networks. in france, the community surveillance is carried through the grog surveillance network. in addition, surveillance is also carried out in hospitals by the renal network. this renal network is divided in two sub-networks: the so-called h -labs network, activated during the containment phase and the extended renal lab network activated in the limitation phase. the h -labs have bsl- facilities that can be used for diagnosis purposes. as part of the national influenza surveillance system led by the french institute for public health surveillance (invs), the grog community surveillance network and the lab networks linked to the two french nics carried out the virological monitoring of the a(h n ) pandemic from the early containment phase up until the end of the pandemic phase. during the containment phase, all suspected cases were hospitalized and declared to invs. each patient was tested on the same day by specific virological diagnosis. hospital admission was not mandatory during the limitation phase, (i) the clustered cases were monitored to study transmission chains, and (ii) the circulation of the virus in the community was monitored through grog swabs collected by practitioners. the nics organized the influenza surveillance to fulfill several objectives according to the epidemiological situation. first, rt-pcr tools (influenza a m gene rt-pcr and a(h n ) specific h and n genes rt-pcrs) were developped and distributed to the lab networks on the th of may . from the early phase, the nics and the h -lab network analyzed all the samples collected from hospitalized and community patients. during the early phase of the limitation phase, an increasing number of labs were performing the specific assays. when the pandemic wave started, all hospital labs could do the testing. results were centralised by nic and reported on a weekly basis. in addition, nics carried out the monitoring of antiviral resistance emergence (na pyrosequencing, specific h y rt-pcr, and phenotypic assays), and real-time surveillance of genetic changes involved in virus adaptation (pb ) virulence factors or antigenic variations (ha). this sequencing was carried out by the pf sequencing platform of the institut pasteur. the first imported a(h n ) influenza cases were observed from the th of april . a limited number of cases have been reported in may. local transmission could be detected end of may. clusters were observed in schools in june and in summer camps during summer. as opposed to the epidemiology of the a(h n ) virus in other european countries, no summer wave was observed in france. only a limited number of sporadic cases were reported up until october. early september, a significant number of cases presenting with influenza-like illness was reported (figure ). the virological investigation of these cases showed high prevalence of rhinovirus infection. this circulation of rhinovirus was a counfounding factor of the pandemic. the pandemic wave lasted weeks between mid-october and the end of december (week to week , figure ). the pandemic wave started week - in the ile-de-france area, and only week - in the rest of france. the peak was recorded week ( figure ). the impact of the pandemic was mainly observed in the - years group of age. overall, severe cases have been admitted to the hospital, and deaths have been recorded by the end of the pandemic wave. the major impact was observed in the - years group of age ( % of deaths recorded). amongst the severe cases and the deceased cases, % and % of cases had no risk factor, respectively. these specimens, were positives for h n , representing ae % of total influenza virus detections. only nine brisbane-like h n , brisbane-like h n , and eight b viruses have been detected in the same period of time. the weekly positive rate ranged from % to %. phylogenetic and antigenic analyses of the viruses collected during the pandemic wave did not show any emerging genetic or antigenic variants (figure a,b) . eight patients, all among cases presenting with severe illness, were infected by a virus harbouring the d g mutation in the ha. amongst the virus tested for antiviral susceptibility or screened for the h y mutation by or specific rt-pcr, only oseltamivir-resistant viruses related to the na h y mutation have been detected. one of these cases also had an i r mutation associated to a reduced sensitivity to zanamivir. all but one resistant virus were detected in treated immunocompromised patients. overall, eight patients presented a virus with the d g mutation in the ha. all these patients had a severe infection; one of these had also a h y mutation in the na asociated to oseltamivir resistance. the pandemic started by the end of april . although the first cases recorded were as early as the th of april, the epidemic wave associated with a widespread spread of the virus was only recorded in october. the french population did not have to face a summer wave, as observed in north america and in numerous european countries. , it is difficult to speculate the reasons for the lack of summer wave; the specimens collected were negative for influenza. moreover, during september, it was anticipated that school openings would be the trigger for the beginning of the pandemic wave. as a matter of fact, a significant increase of influenza-like syndromes were observed at that time, but the virological investigation carried out by the laboratories showed thta is was related to a very large epidemic of rhinovirus. the epidemic circulation of other respiratory viruses can be counfounding factors for the surveillance of the influenza epidemic clinical when the survellance is only based on collection of clinical information. the starting of the pandemic wave was heterogeneous in france. the ilede-france region (paris and its suburbean area), where the population is dense, experienced an early start as compared to the rest of france. however, once the pandemic started in the rest of the county, the epidemic curves were quite similar. the peak was reached at identical times, although it may have been delayed in some remote places in france. overall, we estimate that % of the french population consulted for an ili presentation. the impact was mainly observed in the - years groupe of. however, this age groupe represented only a limited number of severe cases and deaths. on the other hand, the - years groupe of age, where the prevalence was not high, was the age group where the majority of severe cases and deaths was recorded ( % and %, respectively). this data is consistent with the observational data reported by numerous other countries. according to the profile of hospitalized cases, a(h n ) was more aggressive than seasonal viruses. the number of admission to the hospital was ten-fold that observed during a normal influenza epidemic. even if the mortality was limited ( cases), the age distribution of the deceased patients was different as compared to seasonal influenza ( % mortality in < years of age). the lack of recordeable excess mortality has been interpreted to be the consequence of a very mild pandemic, milder than some seasonal epidemics. however, the median age of the fatal cases was much younger than those observed during the seasonal flu, leading to a mis-interpretation of the real impact of the pandemic. when the impact is measurered in loss of years of life, the impact of this pandemic is larger than seen with seasonal influenza, and is quite comparable to these of the two last pandemics. the pandemic preparadness of numerous countries, the develoment of new intensive care techniques and equipment, and the large use of antivirals have reduced the overall impact of this pandemic. these are new factors that should be taken into account when evaluating the real impact of the h n virus. the virological monitoring of the pandemic was achieved by the community-based and hospital-based sea- sonal influenza networks, reminding the importance of maintining such networks. the diagnosis of influenza in most of the patients was carried out by molecular techniques. it has been clearly stated from the beginning of the pandemic that near-patient tests were lacking of susceptibility and could not be used for patient management. the distribution of a set of validated and comprehensive techniques by the two nic was very helpfull for the monitoring of the pandemic and the patients. however, this diagnostic procedure change should not preclude maintaining virus isolation that is necessary for whole genome analysis, monitoring of antigenic changes, and phenotypic testing for antiviral testing. some of the mutants that have been recorded, including viruses with antiviral resistance phenotype or genotype, could be analysed from grown virus strains. it is striking that despite a large antiviral usage, only a limited number of isolates had mutations associated to resistance. however, the frequent isolation of such resistant virus was observed in immunocompromised patients that presented severe infections and long virus shedding. the impact of the pandemic is still under evaluation. sero-epidemiological analysis will be performed to asses for the real attack rate of the pandemic virus. as in other countries, it has been recorded that asymptomatic infections could be observed frequently. it is quite unlikely that the impact of the pandemic was reduced by the vaccination campaign, although this vaccination started on the th of november, just when the pandemic started in france. it is estimated that millions received the vaccination. pandemic strains of the influenza virus sporadically emerge, deviating from the regular endemic strains of seasonal influenza. in april , a novel pandemic influenza virus a ⁄ h n emerged, swiftly spreading across the world. immediately, domestic and international public health agencies were forced to develop containment and mitiga-tion strategies in response to the pandemic. however, the dynamics and transmission patterns of this novel virus are yet to be fully understood. simultaneously, seasonal strains of influenza (a ⁄ h n , a ⁄ h n , and b) continued to circulate in many nations. both pandemic and seasonal variants of influenza are responsible for significant morbidity and mortality. to characterize the dynamics of this disease and the variation within strains, a more detailed understanding of the patterns in viral shedding during natural infection is required. the majority of data on the patterns of viral shedding during influenza infection are a result of volunteer challenge studies. in these studies, volunteers are commonly screened for pre-existing immunity against the challenge strain and are of a certain demographic and age. information on the patterns of viral shedding in natural influenza infections, pandemic or seasonal, is limited but should provide greater generalizability. we describe the trends of viral shedding and clinical illness in community acquired cases of pandemic and seasonal strains of influenza. in , a community-based study was conducted to analyse the effectiveness of non-pharmaceutical interventions to prevent the spread of influenza in households. in , a similar community-based study was initiated to collect comparative data from individuals infected with seasonal and pandemic influenza. both studies were conducted with very similar protocols, involving households in total. the specimens and symptom data required for this study all arise from secondary infections ascertained in these two community-based studies. the recruitment process in both studies was essentially identical. index cases were first recruited from their healthcare provider if they presented with influenza-like illness (ili). this individual would be included in the follow-up if he ⁄ she tested positive for influenza virus infection by rapid antigen test (quickvue) and was the first person in his ⁄ her household that showed signs of ili in the previous weeks. follow-up consisted of three home visits that spanned approximately - days. at each home visit, nasal and throat swab (nts) specimens were collected from all household members, regardless of the presence or absence of symptoms. symptoms were recorded in daily symptom diaries provided for every household member, and digital thermometers were provided to record daily tympanic temperature. the symptoms recorded were fever ‡ ae °c, headache, myalgia, cough, sore throat, runny nose, and phlegm. influenza virus infection and subtype was identified by reverse transcription polymerase chain reaction (rt-pcr) on the nts specimens. viral shedding was quantified from the same specimens by rt-pcr to determine viral loads, as well as by quantitative viral dilutions to determine median tissue culture infectious dose (tcid ). the details concerning laboratory methods have been described in a previous study. all analyses in this study focus exclusively on secondary cases; these are household contacts of recruited index cases who acquire influenza virus infection following the initial home visit. index cases generally presented with a certain threshold of illness severity requiring medical attention, whereas infections among household contacts can vary from asymptomatic to severe representing naturally acquired influenza infections. these secondary cases must be negative for influenza for their first nts specimen, and subsequently tested positive. we analysed mean viral loads measured by rt-pcr and quantitative culture by plotting by day since acute respiratory illness (ari) onset according to strain of influenza (pandemic a ⁄ h n , seasonal a ⁄ h n , seasonal a ⁄ h n , and seasonal b). ari is the reference time point, because the day of infection is unknown and is defined as the presence of ‡ of the symptoms mentioned above. average symptom scores were also plotted according to ari onset and grouped into upper respiratory symptoms (sore throat and runny nose), lower respiratory symptoms (cough and phlegm), and systemic signs and symptoms (fever ‡ ae °c, headache, and myalgia). mean daily tympanic temperatures were also plotted since date of ari onset and according to strain of influenza virus. all analyses were conducted using r software (version . . ; r development core team). a total of households and individuals were followed-up in the two studies. of household con-tacts tested by rt-pcr, were found to be influenza positive. among these influenza infections, ( ae %) were asymptomatic (rt-pcr positive plus symptoms recorded), were subclinical (rt-pcr positive plus symptom recorded), and presented with an onset of ari during the follow-up period. from the cases with ari onset, seven pandemic a ⁄ h n , seasonal a ⁄ h n , seasonal a ⁄ h n , and seasonal b influenza virus infections were identified. the age distribution among secondary cases was observed to be largely comparable across the four strains of interest (table ). there were a lower proportion of males who acquired pandemic a ⁄ h n compared to the seasonal strains of the virus. cough was the most commonly reported symptoms during follow-up in cases of pandemic a ⁄ h n and seasonal b, whereas runny nose was most common in seasonal a ⁄ h n and a ⁄ h n cases. cumulatively, fever ( ‡ ae °c) was reported in approximately half ( %) of the secondary cases. patterns of viral shedding were analysed in a subset of influenza positive individuals who recorded an onset of ari in their symptoms diaries (figure ). household contacts that were asymptomatic, subclinical, or did not have an ari onset were excluded from the analysis. viral shedding in all three influenza a strains were recorded to occur on the day of ari onset or day post-ari onset. follow- ing the peak, measured levels of viral shedding declined steadily to undetectable levels over - days. the trend of viral shedding in influenza b infected individuals rose days before ari onset, fluctuated for around days before eventually resolving. the patterns of viral shedding over time measured by quantitative viral culture were generally similar to the patterns measured by rt-pcr. the patterns of symptoms and signs were comparable in the four strains of influenza included in this study, peaking on the day or day post-ari onset, and gradually declining over a period of - days. in all strains, systemic symptoms and signs were observed to resolved faster than upper and lower respiratory symptoms. the trend of tympanic temperature in each influenza strain was comparable to the respective symptom pattern. patterns of viral shedding observed in influenza a strain infections (pandemic a ⁄ h n , seasonal a ⁄ h n , and seasonal a ⁄ h n ) were broadly similar. the pattern differed from the observed pattern of viral shedding in seasonal influenza b infections. the majority of viral shedding in influenza a strains occurred at and near ari onset, whereas there were variable amounts of viral shedding preand post-ari onset for those with influenza b. the biological reason for this difference is yet to be clarified. these differences are consistently observed regardless of laboratory method used to quantify the viral loads. it was observed that viral shedding measured by tcid resolved more quickly than when measured by rt-pcr, suggesting that rt-pcr is more sensitive, but it could be detecting inactivated fragments of rna instead of active virus. the trends observed for the seasonal strains of influenza in this study were similar to those reported in literature. the patterns of symptoms and signs as well as tympanic temperature in the four different strains of interest in this study were found to be comparable. these patterns closely resemble the patterns of viral shedding observed in the influenza a virus strains, but not in the influenza b virus strain. the trends of viral shedding, symptom scores, and tympanic temperature for pandemic a ⁄ h n were similar to trends observed for seasonal a ⁄ h n and seasonal a ⁄ h n infections, suggesting that the dynamics of these viruses are largely the same. the clinical course of infection with pandemic a ⁄ h n influenza virus appeared to be similar to the seasonal b influenza virus, but the patterns of viral shedding over time diverges. in general, our results suggest that the dynamics of the pandemic a ⁄ h n virus were similar to the seasonal a ⁄ h n and a ⁄ h n viruses, and clinically similar to the seasonal b virus. this study faced sample size limitations; very few cases of pandemic a ⁄ h n were detected and the secondary attack rate in general was low, though a total of households were followed up. this lack of power led to the inability to analyse the differences between adult and children and other characteristics that could be correlated with amount of viral shedding. there are also biases that must be factored in during recruitment. the eligibility criteria of only healthy households could select for households with higher innate immunity. on the other hand, recruitment at health care providers can be biased towards index cases that had more severe illness that required medical attention. the strength of the study is the broad generalizability of the results due to the strict classification of secondary cases. the infections reported in this study were all community-based and should represent true natural infections. pandemic potency of the influenza virus is largely determined by its transmissibility. the first objective of this study was to model the transmission of influenza h n and h n viruses. at present, vaccination with laiv has been used as a widespread, effective public health measure for influenza prophylaxis. some unsubstantiated concerns have been raised about a potential possibility of reassortment of circulating influenza viruses with laiv viruses following vaccination with laiv. thus, another objective of this study was to assess the probability of pig-to-pig transmission of cold-adapted viruses and their potential reassortment with wt influenza strains. female albino guinea pigs weighing - g were inoculated intranasally with eid of virus without anaesthesia. transmission studies were then performed hours after inoculation. inoculated animals were housed at % relative humidity and °c in the same cage with noninfected guinea pigs or in cages placed m away from non-infected pigs. virus replication was determined by virus isolation in hen eggs and by pcr. sera were collected at and days post inoculation. seroconversions were assessed by routine hai test. genome composition of reassortants was monitored by rflp analysis. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated, and virus growth properties were observed following virus titration in hen eggs. when infected pigs were co-caged with non-infected (naïve) individuals, vn , indo ⁄ , a ⁄ california ⁄ ⁄ , and nibrg- were isolated in %, % ae %, and % of contact animals, respectively. serological confirmation of virus transmission was higher than virological data ( %, %, %, and %, respectively). in addition, it was shown that when pigs inoculated with a ⁄ california ⁄ ⁄ were co-caged with animals inoculated with nibrg- , they got infected with both viruses ( table ) . the ability of direct transmission of cold-adapted viruses was also investigated. data show that the a ⁄ ⁄ california ⁄ ⁄ laiv candidate was detected in the upper respiratory tract of ae % vaccinated pigs. the mdv was identified in % of infected animals. however, neither group of contact pigs, co-housed with the vaccinate pigs, had evidence of infection with cold-adapted viruses. in addition, none of the contact pigs had any evidence of seroconversion to the coldadapted viruses as determined by hai assay. it was also most interesting to note that pig-to-pig transmission of the highly transmittable nibrg- reassortant virus was not seen when pigs, vaccinated with mdv, were co-caged with animals infected with nibrg- virus (table ) . this strongly implies a form of interference or protection from transmissibility that was provided by the cold-adapted virus. the results show that nibrg- and indo ⁄ viruses were able to spread between cages over the m distance ( % and % naïve animals were successfully infected, respectively). a ⁄ california ⁄ ⁄ influenza and vn viruses did not transmit between infected and non-infected guinea pigs housed in separated cages (table ) . pigs with confirmed a ⁄ california ⁄ ⁄ virus replication were also infected with nibrg- virus if h n -and h n -infected animals were separated by a space. thus, influenza virus transmission from h n -to h n -infected pigs has been shown, but the reverse pattern did not occur. transmission of nibrg- or a ⁄ california ⁄ ⁄ viruses was not observed when contact pigs were first vaccinated with the mdv and housed at a m distance ( table ) . it was also shown that efficiency of transmission of nibrg- was much higher than of other studied h n viruses; it can be transmitted between naïve guinea pigs separated from infected animals at a distance of - m (data not shown). five reassortants were isolated from animals which were infected with a ⁄ california ⁄ ⁄ virus and co-caged with pigs inoculated with nibrg- . two reassortants possessed different combinations of pr , nibrg- , and a ⁄ california ⁄ ⁄ genes and demonstrated the non-ca ⁄ non-ts phenotype typical of wt viruses. unexpectedly, two other reassortants inherited ha gene from nibrg- , na gene from a ⁄ california ⁄ ⁄ , and other genes from pr became ca and ts. : non-ts reassortant inherited pa gene from pr and seven other genes from a ⁄ california ⁄ ⁄ , gained ca properties. in spite of aforesaid experimental data, we cannot exclude the theoretical possibility of simultaneous infection of human host with cold-adapted and wt influenza viruses. to better understand possible consequences of such a reassortment event, we co-infected guinea pigs with a mixture of mdv and nibrg- viruses. nasal washes were collected and cloned by limited dilutions in hen eggs in the presence or absence of immune serum to the mdv. cloning of nasal washes without antiserum led to isolation of over clones, which were all identical to the mdv (data not shown). when nasal washes were cloned in the presence of antiserum, only nine clones were isolated. genome composition analysis showed that all isolates were triple reassortants, which had inherited pb and na genes from mdv, pa gene from pr , and ha gene from nibrg- . the origin of the other gene segments (pb , np, m, ns) in the genome of guinea pig-derived reassortants varied. reassuringly, all reassortants generated in vivo had the phenotype typical of the mdv. the severity of influenza outbreaks is partly determined by efficient spreading of the causative virus strain between human hosts. however, little is known about mechanisms underlying influenza virus transmission in humans. guinea pigs have been shown to be a suitable model for influenza transmission studies. our in vivo study showed that influenza a viruses vary in their transmissibility. nib-rg- and indo ⁄ viruses were able to transmit to naïve animals caged distantly from infected animals. in contrast, cold-adapted viruses, the same as those used for licensed laivs, showed no signs of transmission from one guinea pig to another. our study also provided evidence of a lower level of transmissibility of the novel pandemic h n virus compared to the nibrg- and indo ⁄ h n strains evaluated. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. in our study, the mdv inoculated into guinea pigs appeared to interfere with and even offer protection from transmission of the highly transmissible nibrg- virus. the ability to immunize with the laiv and subsequently block the spread of a homologous h n subtype and a heterologous h n subtype influenza virus between guinea pigs has been shown. interference between cold-adapted and wildtype influenza virus infection was the most likely explanation for the data observed in our study. the mdv inoculated into guinea pigs might in some way interfere with transmission of highly transmissible influenza viruses. it is believed by some that widespread use of laiv could increase the potential risk of reassortment of the vaccine strain with circulating influenza viruses immediately following vaccination. however, it was shown that any such potential reassortments would most likely lead to yet attenuated viruses. our in vivo studies have shown that introduction of mdv genes into the genome of nib-rg- virus led to the generation of triple reassortants inherited pb and na genes of mdv and ha gene of h n virus. all isolates possessed phenotypical markers associated with attenuation of mdv. our data suggest that even if a reassortment event of such rare occurrence between a laiv strain and a circulating virus were to occur, it would most likely lead to a reassortant that would retain highly attenuated phenotypic properties of the vaccine strain. our data strongly support the safety of laivs, especially those developed against highly transmissible h n and h n pandemic influenza viruses. this information builds upon databases that have clearly shown the low likelihood of transmitting an laiv, as well as the high likelihood of any field reassortment of laiv with a circulating influenza virus to retain important properties of the cold-adapted, temperature-sensitive vaccine master composition. very interestingly, we also present data that show the potential of a laiv to prevent the transmission of highly infectious influenza viruses, perhaps identifying a broader role for laiv in the overall scheme of influenza virus prophylactic use. background: schlieren imaging is a non-invasive, real-time airflow visualization technique that relies on differences in air temperatures (and the resulting changes in the refractive index) to allow exhaled human airflows to be seen clearly against the background of more-stationary, ambient air. recently, this technique, well-known to engineers, has been applied to better understand and characterize airflow behaviors associated with everyday, as well as healthcarerelated, human respiratory activities. materials and methods: as a surrogate marker for the behavior of airborne infectious agents, schlieren imaging was used to visualize the airflow patterns produced by adult human volunteers of different ages while coughing with and without the wearing of standard surgical and n masks. results: the cough plumes were generally similar in shape and range for all the adult volunteers used in this study. although both the surgical and n masks decelerated and blocked some of the forward momentum of the coughed airflows, much of the cough plume was redirected and escaped around the top, bottom, and side edges of the masks to merge with the volunteer's natural, verticallymoving thermal plume. conclusions: schlieren imaging is a safe technique for visualizing exhaled airflows from human volunteers without the need for potentially-irritant or toxic particle tracers. findings from these schlieren imaging experiments will assist the development of more effective aerosol infection control guidelines in healthcare premises where patients infected with potentially airborne infectious agents (e.g., influenza and tuberculosis) are present. these infectious agents may be transmitted to healthcare workers, other patients, and their visitors by way of exhaled airflows. with the recent influenza pandemic , and the ongoing concerns about human cases of avian influenza h n infections, there is now a very real concern about the potential for the aerosol transmission of respiratory pathogens. such concerns amongst staff and patients in healthcare environments have led to a greater emphasis on the understanding and control of infectious airflows. , previous visualization techniques have used potentially-toxic or irritant gas or particulate tracers with hazardous laser light sources that have precluded the use of human volunteers as subjects. instead, various forms of lung models that simulate human respiratory patterns with such particulate tracers have been used. , schlieren imaging is a technique familiar to engineers and offers a non-invasive (i.e., no tracer required) airflow visualization method that depends only on differences in the refractive index of the warmer, human-exhaled air and the cooler ambient air. the use of a simple incandescent or light-emitting diode (i.e., non-laser) light source is safe and allows human volunteers to be used as experimental subjects, where their exhaled airflows are then observed using a large, precise spherical or parabolic telescopic mirror and a camera, and are recorded for later analysis and presentation. [ ] [ ] [ ] the analysis of these patterns of 'real-life' human airflows will be useful in optimizing aerosol infection control guidelines, which aim to reduce the transmission of airborne infectious agents to other healthcare personnel, patients, or their visitors. the images and analysis presented here have all been obtained from the large m diameter parabolic mirror (figure ) situated at the gas dynamics laboratory of penn state (directed by gary s. settles). this large schlie-ren imaging system has been in use for over years to obtain high quality schlieren images for various engineering applications. it has only recently been applied to clinically-relevant imaging. the objective of this paper is to augment and expand upon the details of the methods and results presented in an earlier study using this same schlieren imaging system. the aim of this series of studies is to visualize and capture a series of airflow images produced by coughing from adult human volunteers of different ages ( - years old). these included males (three of years, one of years of age) and females (one of years, one of - years, and one of - years of age). each volunteer was tested with and without wearing either a standard surgical mask or n mask. more specifically, the aim was to visualize the extent and direction of leakage around the mask whilst each subject was coughing. penn state institutional approval for experiments involving human subjects was also obtained. each volunteer was asked to stand approximately m in front of the schlieren mirror, facing across the surface of the mirror on one side, and to cough several times as the real-time, color image and video footage was recorded by the operator (using a nikon d camera; nikon inc. melville, ny, usa). this process was repeated whilst each volunteer was wearing a standard surgical mask then an n mask (supplied by mÔ, st paul, mn, usa). some of the schlieren images obtained from some of these volunteers have been published previously: for a -year old male, the year-old female and a -year old male, and the - year-old female. this article completes this series of schlieren images obtained from these experiments by including the images recorded for the older, year-old man. generally, it was found that the shape of the cough plumes (shown in the figure as darker shadows emanating from the subject's mouth) produced by adult humans of different ages was relatively similar. cough plumes are roughly conical in shape and very turbulent, usually passing beyond the extent of the m mirror (figure a) . a previous detailed study of one of these images measured a maximum airflow velocity of m ⁄ second for an adult cough. similarly, the effects of wearing surgical and n masks can be generalized across different ages. wearing a surgical mask allows leakage of the coughed air from the sides, top, and bottom of the mask ( figure b ). there is also some leakage through the mask, as indicated by the darker patches of air directly in front of the mask ( figure b, c) . the useful effect of the mask appears to be a deceleration and redirection of this coughed (and potentially infectious) air into the natural, upward-rising human thermal plume, which captures it and carries it upwards where it is diluted and less likely to transmit infection to others. the effects of the n mask are similar (i.e., deceleration and redirection), yet due to its tighter (mask-fitted) face seal, more of the coughed air appears to penetrate the front of the mask ( figure c ). this penetrating air is, however, also decelerated sufficiently to allow the wearer's natural thermal plume to carry it upwards. , discussion from these series of schlieren images presented in this and other related studies, [ ] [ ] [ ] it is clear that schlieren imaging offers a safe, non-invasive, real-time technique to visualize human exhaled airflows for all age groups. it is apparent that, at least where airflow patterns are an acceptable surrogate marker for airborne transmission risks, there are beneficial effects of wearing either type of mask, even when the mask fit is relatively poor. this is often the case when n -style masks are purchased and used by the general public -in contrast to the situation with healthcare workers, who are often accurately fit-tested for this type of mask. the immediate significance of this can be seen when masks are bought by parents for their children. often, these will not be of pediatric size and the mask-fit will be loose. children are well-known to be major sources of infection in the community because of their relatively poor immunity to many types of infectious agents due to their young age and, therefore, limited past-exposure history. these images allow infection control teams to literally see how far and how fast potentially-infectious human exhaled airflows can travel from an individual. this may have significant implications for guidance on the wearing of masks for infected staff and patients, on ward bed-spacing, as well as for the types of masks to be used in different situations. the important practical potential lies in the non-intrusive visualization of airflows associated with human volunteers, to assist in heightening the awareness amongst healthcare workers of the risks and potential for the airborne transmission of infectious agents, as well as the development of more effective aerosol infection control policies. schlieren images can be analysed more quantitatively, e.g., with the 'schlieren-piv' technique, , though this additional quantitative data is probably more of research interest than being of immediate practical use to everyday hospital infection control teams. these are the subtypes that we have studied. clearly, the question arises as to whether the changes in antigenicity are coupled with changes in germicide susceptibility. we have employed a modified log-reduction method in a cell culture system employing mdck cells in serum-free ex-cellÔ medium supplemented with trypsin. microscopic examination of cpe was the marker for infectivity together with plaque assay. we confirmed antiviral potency by using specific subtype influenza identification subtype technology, quidel quickvue Ò influenza a + b test. the log inactivation and percent inactivation by bac after a second contact time for the h , h , and h pandemic strains are as follows: a ⁄ swine ⁄ iowa ⁄ ⁄ h n , ae log ⁄ ae %; a ⁄ swine ⁄ cal ⁄ h n , ae logs ⁄ ae %; a ⁄ j ⁄ ⁄ h n , logs ⁄ ae %; and a ⁄ hong kong ⁄ h n , ae logs ⁄ ae % (table ). comparable results of antiviral efficacy are obtained with the tcid and plaque assays against all subtypes studied. when performing the plaque assay the sensitivity of virus recovery was better in the vessel with a larger surface area and overall recovery was in agreement with the potency determined by tcid assay. in our plaque assay, we inoculated a ⁄ hong kong ⁄ ⁄ virus dilutions into two different vessels with hours adsorption time: -well plate and t- flask, ml inoculum per replicate. virus titers obtained were: ae · pfu ⁄ ml from -well plate and ae · pfu ⁄ ml from t- flask ( table ). the discrepancy on virus potency can possibly be explained as: the binding of virus to host cell occurs only when virus gets a chance to interact with the cell on the monolayer during adsorption time. the percentage of virus population in the inoculum that has the opportunity to bind to the cell mainly depends on the surface area where this interaction takes place. therefore, in our experiment the plaque assay in the t- flask gave higher virus recovery ae versus ae · pfu ⁄ ml. the increased virus recovery can translate into better sensitivity of the test system for disinfectant and antiviral agents. the potency of the virus used in this study was determined by tcid was · tcid ⁄ ml. rapid diagnostic testing for influenza (quickvue Ò influenza a + b test, quidel) for aj versus bac was studied. the presence of influenza viral nucleoprotein a determined by quickvue kit correlated % with the viral infection based on by cpe in viral culture. interestingly, the inactivation of viral nucleoprotein was able to be revealed with diagnostic kit in the dilutions of virus ⁄ bac reaction mixture, which possessed prominent cytotoxic effect for the host cells in viral culture system. this type of molecular testing method is useful for interpreting antiviral efficacy against a background of cytotoxicity. these experiments are intended for the sponsor to substantiate to us fda that their antiviral substances are safe and effective. the data shows that the three hemagglutinin subtypes were highly susceptible to the quaternary ammonium compound in the short term in vitro experiment. the appearance of novel subtypes in the future can be met with the assurance that disinfectant and ⁄ or antiseptic resistance will be unlikely. certainly, from the above data, although genetic reassortment of human and swine viruses may modulate influenza pathogenesis and limit existing vaccine benefit, it is not likely be a factor in control of viruses on environmental surfaces by benzalkonium-type disinfectant ⁄ cleaning agents in community or health care environments. table . comparison of viral titer obtained in different vessels using quantal tcid and plaque assay methods plaque assay tcid assay t- ( cm ) -well plate ( cm ) tcid ⁄ ml tcid ⁄ ml ae · pfu ⁄ ml ae · pfu ⁄ ml · ae · options for the control of influenza vii outbreak influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. in this paper we share our experience of managing an outbreak of viral respiratory infection in an acf very early in the influenza pandemic and also describe some of the emerging issues relating to crossreacting antibodies to the pandemic (h n ) influenza virus in the very elderly. the outbreak investigation was conducted as part of an urgent public health intervention initiated by the new south wales (nsw) department of health during the early stages of the first southern hemisphere wave of the pandemic. nose and throat swabs for nucleic acid testing (nat) plus acute and convalescent serum samples ( weeks apart) were collected from all the residents of an acf where an influenza-like illness (ili) outbreak occurred. the investigation revealed dual outbreaks of pandemic (h n ) influenza and rhinovirus infection. out of residents, three had laboratory confirmed influenza [two with pandemic (h n ) ], and had rhinovirus infection on nat. testing of acute sera collected from every subject found elevated ( ‡ : ) pandemic (h n ) hai antibody in % ( ⁄ ) subjects aged years or more (born before and median age years; geometric mean titre-gmt ae ) compared with none of the residents aged under years (born after and median age years; gmt ae , p = ae ). the acf was closed to visi-tors for days. the symptomatic residents received treatment-dose oseltamivir, and all other residents were given oseltamivir prophylaxis. more than one virus may be circulating in an acf with an ili outbreak at any one time in winter. a significant proportion of elderly residents had pre-existing cross reacting antibody to the pandemic (h n ) , which may explain the minimal clinical impact of pandemic (h n ) in this elderly population. influenza is one of the leading causes of infectious death in elderly people, principally due to co-morbidities and declining immune competence with age. it is the most important agent in outbreaks of respiratory illness. influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. the clinical presentation of influenza in residents of acfs can be subtle, with a blunted febrile response and a non-specific decline in mental and functional status. residents commonly have underlying diseases that can be exacerbated by influenza infection, and in addition, they are at higher risk of serious influenza-related complications than community dwelling elderly people. people aged over years are also at higher risk of influenza-related death, and more than % of annual influenza-related mortality is usually confined to this high risk group. in australia, influenza and pneumonia have sub-stantial health impacts; recorded as being the underlying causes of death for persons in . since the world health organization declared an influenza pandemic in june , australia has suffered one of the highest rates of confirmed infection during the first southern hemisphere wave. by late october there were reported deaths due to pandemic influenza in australia, and to date there have been about deaths reported worldwide. although disproportionately far fewer elderly people developed clinical influenza during the current pandemic than occurs with seasonal influenza, their case-fatality rate remained substantial. early in the pandemic (june ), we investigated a suspected pandemic influenza outbreak in a rural acf in the state of nsw, australia. the epidemiology (including virulence and clinical outcome in the elderly) of the pandemic (h n ) virus was mostly unknown at the time of investigation, and as time passed, this investigation provided clarity on some important issues of the influenza epidemiology in the elderly population. in this paper we share our experience of managing a dual outbreak of viral respiratory infections early in the pandemic, and also describe some of the emerging issues relating to the cross-reacting antibodies to pandemic influenza in the very elderly. the outbreak investigation was conducted as part of urgent public health intervention initiated by the nsw department of heath in conjunction with the local public health unit, the national centre for immunisation research and surveillance (ncirs), and the institute of clinical pathology and medical research (a who national influenza centre). to determine the extent and cause of the outbreak, a public health research doctor (gk) was dispatched from sydney over a weekend to assist with outbreak investigation and control. on june th , the greater southern public health unit surveillance officer (bd) received a report of a possible pandemic (h n ) outbreak in a local acf. on investigation, it was discovered that days earlier a year old female resident had become generally unwell, but without specific symptoms of influenza like illness (ili). soon after, nine of the co-residents (but no staff) had developed symptoms suggestive of influenza. one other resident had returned from a melbourne (victoria) hospital (where pandemic (h n ) was known to be circulating) the previous week after surgery, but did not have ili symptoms. on june th, the symptomatic residents had nasal swabs taken by the local doctor for influenza [including pandemic (h n ) ] nucleic acid testing (nat). there was rising concern due to reports of widespread pandemic (h n ) influenza in a local army camp just over the border in nearby victoria, where pandemic (h n ) influenza was known to be circulating widely. on june th, the year old lady proved nat positive for pandemic (h n ) , but none of the other samples were pandemic (h n ) nat positive. concern arose that there might be an outbreak of pandemic (h n ) in the facility, and that some of the swabs from other residents might be false negatives. between and june, after consent was obtained, directly or through next of kin in demented residents, all submitted to venipuncture for serology, successfully, and the other as yet un-swabbed residents were swabbed. basic demographic data were collected from every resident with clinical information on co-morbidities and current medication use. convalescent blood samples were collected after weeks on th july from of the residents. swabs were sent to icpmr where nat for influenza a [including pandemic (h n ) ] and b was performed. the acute and convalescent serum samples were tested later (in december ), using haemagglutination inhibition assay (hai) to detect pandemic (h n ) antibody. , interventions the acf was closed to visitors from th until th june. treatment of the positive case and the nine symptomatic residents, with twice daily oseltamivir, was begun on saturday june th, and all other residents were started on once daily oseltamivir prophylaxis. the facility manager and local general practitioner (gp) monitored patient health on a daily basis, and none had to stop oseltamivir due to adverse events. one resident with ili who was known to have moderately impaired renal function was given once daily rather than twice daily oseltamivir treatment. the age range of the residents was - years with a median of years. all residents had underlying medical conditions, e.g., chronic cardiac and respiratory diseases ( table ) testing of acute sera collected from every subject found elevated ( ‡ : ) cross-reacting hai antibody to the pandemic (h n ) in % ( ⁄ ) of subjects aged years or more (born before and median age years; geometric mean titre-gmt ae ). however, the hai titre was consistently < : and significantly lower (gmt ae , p = ae ) in the residents aged under years (range - years, median years) (figure ). the index case (nat positive) did not show a significant raise in hai level in convalescence (going from to ). the pandemic (h n ) case that was determined by serology was pandemic (h n ) nat negative. to our surprise, seven of the other asymptomatic residents had rhinovirus detected on extended nat (reported on june th), despite being asymptomatic at time of swabbing and remaining so. the original nine influenza nat negative samples were then tested and three of these were also nat positive for rhinovirus; in total, ten proved nat positive for rhinovirus ( ae %). the serologically confirmed pandemic (h n ) case was also positive for rhinovirus infection. of interest was that only one resident had a documented fever. this investigation illustrates some of the difficulties in managing and investigating possible influenza outbreaks in real time in the context of an influenza pandemic. finding a nat positive case of pandemic (h n ) influenza among many other symptomatic cases raised the possibility (although not the probability) that pandemic (h n ) was the cause of the outbreak. rhinovirus infection, however, was confirmed by nat in ten residents. this outbreak illustrates that more than one virus (in this case and perhaps ) may be circulating in an acf at any one time in winter. in ili outbreaks in acfs, broad laboratory testing is recommended; nat is the most sensitive method of detecting influenza or other viruses in respiratory tract samples. studies have found that the pandemic (h n ) haemagglutinin (ha) gene is more closely related phylogenetically to the h n virus and classical swine influenza a ⁄ h n viruses than more recent seasonal human influenza a ⁄ h n viruses. it is antigenically similar to the h n pandemic virus in terms of the immunodominant antibody response to haemagglutinin. [ ] [ ] [ ] it is likely that individuals alive during the emergence and initial persistence of the pandemic virus would have higher levels of cross-reacting hai antibodies to the pandemic (h n ) , which would contribute towards better clinical protection. in our investigation, % of the residents born before (aged years or above in ) had pre-existing cross-reacting hai antibody to the pandemic (h n ) . in elderly populations, severe illness may be associated with organisms typically considered to be mild, such as rhinovirus. however, studies have shown that nursing home residents may be susceptible to outbreaks of rhinovirus that may cause mild to severe respiratory illness, particularly in those with a history of lung disease. one rhinovirus outbreak in a nursing home in the usa caused fatalities. another outbreak showed residents with underlying lung disease are more likely to have longer infection, require antibiotics, develop bronchospasm, and have difficulty breathing; two residents with underlying lung disease required emergency treatment and one died. a previous influenza outbreak in a nsw aged care facility in caused significant mortality and morbidity. that outbreak resulted in hospital admissions and six deaths. in our investigation we have found that % of the residents had chronic lung disease and % had chronic cardiac conditions both considered as high risk for severe complications of both rhinovirus and influenza infection. however, there were no hospitalisations or deaths in our outbreak investigation. indeed only one resident developed fever, indicating that non-specific signs of illness (such as in our index case) may be the only, or early, indication of an ili. our own experience with managing other ili outbreaks has also taught us that staff of acfs may not be vigilant enough to detect fevers. in this outbreak, the nursing home staff, local gp, public health unit and the outbreak investigation team and supporting laboratory staff acted quickly and in a coordinated way. pre-existing cross-reacting antibody in the very elderly (aged ‡ years) probably helped to limit the spread of the pandemic virus (compared to the circulation of rhinovirus) within the acf. exposure to the pandemic (or a close variant occurring before ) appears to be responsible for a high hai titre in the very elderly, which contributed towards better clinical protection. however, wider testing early on would have alerted us more quickly to the main cause of the outbreak. treatment and prophylactic use of oseltamivir may also have contributed to halting the spread of pandemic (h n ) and also to symptom relief. pandemic (h n ) influenza virus (ah pdm) has spread worldwide since march . in a paper of ah pdm, % of infected individuals have experienced gastrointestinal symptoms such as diarrhea and vomiting, which is higher than that of seasonal influenza. however, little is known whether viable virus shed from stool and replication of viruses are ongoing in the gastrointestinal tract. , viral load and isolation of ah pdm in cell culture in stool samples has been reported. stool specimens were collected from patients suspected to have pandemic (h n ) infection from november through may . virus isolation was conducted in cell culture by using madin-darby canine kidney (mdck) cells and taqman based rt-pcr from % (w ⁄ v) stool suspension in phosphate-buffered saline. taqman based rt-pcr was conducted by using primers, probes, and positive controls provided by niid (national institute of infectious diseases of japan). to confirm presence of ah pdm viral rna, lamp (loop-mediated isothermal amplification) was used as supplemental testing. of patients, one child (case ) submitted one nasal swab and four stool samples, another one nasal swab and two stool samples, and the other one stool sample. informed consent was obtained. strand specific rt-nested pcr was performed for only case by using only one primer at the rt reaction and also assayed neu aca - gal and neu aca - gal binding specificity about isolated strain derived from nasal swab and stool. receptor binding specificity was performed using a solid-phase binding assay with the sialylglycopolymers (poly a-l-glutamic acid backbones containing neu aca - galb - glcnacb-pap or neu aca - galb - glcnacb-pap bond as described. ) nucleotide sequences of the ha gene of ah pdm viruses isolated from stool sample and nasal swab were analysed. in order to exclude the possibility of contamination, the stool samples and nasal swabs were subjected to virus isolation separately. after getting the results on the nucleotide sequence, we also confirmed no strain harboring identical sequence was isolated in our laboratory before and after the day of sample collection. ah pdm viral rna was detected in nine ( %) of the subjects from stool samples. among nine subjects, one case (case no. ) was positive for viral isolation. case , a healthy -year-old girl, experienced fever and abdominal pain, and the others had gastrointestinal symptoms without upper respiratory symptoms. in case , influenza a virus was diagnosed by rapid antigen test on the day of symptom onset. viable ah pdm virus was isolated from the stool sample and nasal swab on the second day from onset using mdck cells (table ). viral load decreased gradually after symptom onset. however, viral shedding was still present days after symptom onset. positive stranded rna was detected days after symptom onset from the stool specimen ( figure ). above two ah pdm strains (isolated from nasal swab and stool specimen) bound exclusively to human type receptor, neu aca - gal. sequence analysis demonstrated that isolated virus from stool samples was identical with that from nasal swabs in comparison of ha gene ( bp). ah pdm influenza virus was isolated from the stool and nasal swab samples in the same patient simultaneously by using mdck cells. our results suggests the detection of viral rna and viable ah pdm influenza virus from stool samples may serve as a potential mode of transmission and has important implications in understanding the context of ah pdm influenza virus. strategies to prevent transmission of influenza include use of respirators. ffp and n respirators are certified to fil-ter at least % of particles ( ae lm in diameter), and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. [ ] [ ] [ ] we have developed a proprietary acid-polymer formulation to coat a standard ffp respirator with an antiviral layer. we aimed to test this coated respirator for antiviral efficacy against a range of influenza viruses. a series of tests compared the antiviral efficacy of coated and uncoated respirators in conditions designed to simulate real-life exposure to influenza by varying the route of inoculation, contact time, temperature, humidity, moisture, and contaminating substances. we also investigated whether infectious viruses could be transferred from contaminated respirator surfaces to gloves. we tested human, swine, and avian influenza viruses, including influenza a and b viruses. influenza a subtypes were the a ⁄ h n pandemic strain, seasonal h n , h n , h n , h n , and h n . in each test, suspensions of influenza viruses were prepared to - log tcid ⁄ ml in mem. in some tests, organic contaminants (yeast, bsa, and mucin) were added. one set of respirators was maintained at °c and % relative humidity for hours before the viral challenge, and repeatedly sprayed with he-pes buffer to simulate respiratory secretions. for each test, three coated (glaxosmithkline actiprotect) and three uncoated (sperian willson easy fit) ffp respirator samples were inoculated with ae ml of a viral suspension, which was applied with a pipette, sprayed, or aerosolised to create airborne droplets. after minute at room temperature (on a shaker), the respirator samples were assayed for the presence of infectious viruses using standard methods. in one test, after a minute contact time of the respirator with the virus, nitrile gloves were applied with light pressure to the outer surface of inoculated respirator samples and then assayed after minute. samples were put into test medium (mem, supplemented with antibiotics [penicillin, gentamycin, or streptomycin] and amphotericin b or l-glutamine). the supernatants were vortexed, extracted, and used to prepare serial -fold dilutions in mem. each dilution was used to inoculate four wells of rmk cells in a multi-well plate, and these cultures were incubated and scored over days for cytopathic effects, cytotoxicity, and viability. (some tests substituted mdck cells; others used inoculated embryonated chick eggs.) all tests included negative cell controls, cytotoxicity controls, and neutralisation controls. the spearman-karber formula was used to calculate viral loads as tcid or eid . antiviral efficacy was calculated from the difference between the geometric mean loads of influenza virus on the coated and uncoated respirators after minute of exposure. the viral loads applied to respirators in these experiments ranged from ae to ae log tcid , and were therefore high in comparison with respiratory secretions from infected patients at the peak of influenza symptoms (range - log tcid ). tables - show that the average viral loads detected on uncoated ffp respirator samples remained high in all conditions tested, ranging from ae to ae log tcid (or ae - ae log eid ). in contrast, the average viral load on coated respirators after minute of exposure ranged from below the limits of detection to £ ae log tcid ( ae log eid ). therefore, the relative antiviral efficacy of the coating ranged from ‡ ae to ae log . table shows that the relative antiviral efficacy of the coated mask remained high in simulated-use conditions such as organic contaminants and repeated saturation at high temperature and humidity. in the experiment to test transfer of viruses from respirators, the gloves applied to regular uncoated inoculated respirators had a viral load of ae log eid (table ) . by contrast, no viruses were detected on either the coated respirators or the gloves applied to them. the relative reduction in contamination was therefore ‡ ae log . ‡ ae log viral load with organic contaminants* ae ae ae log viral load after heat, moisture, and simulated secretions** ae ae ae log viral load transferred to glove** ae £ ae ‡ ae log eid *influenza subtype was a ⁄ h n , and strain was vnh n -pr ⁄ cdc-rg. **influenza subtype was a ⁄ h n , and the strain was hong kong ⁄ ⁄ . results are mean log tcid , unless specified otherwise. results are mean log tcid , unless specified otherwise, based on an infectivity assay in triplicate. limits of detection varied. * pandemic strains. **results are mean log eid , based on a haemagglutinin assay in duplicate. options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - strategies to prevent transmission of influenza include use of respirators, and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings for protection against pandemic influenza. - ffp respirators are certified in europe to filter at least % of nacl particles ( ae lm in diameter), and ffp and ffp respirators must filter at least % and % of these particles, respectively. influenza a viruses are typically ae lm, and can be carried in aerosolised droplets smaller than lm in diameter, which can disperse widely, remain airborne for hours, and be inhaled deeply into the respiratory tract. we have developed an acid-polymer formulation to coat the outer layer of a standard ffp respirator, in order to provide antiviral activity on the outer surface. we compared this coated respirator against standard ffp , ffp , and ffp respirators for filtration of aerosolised influenza viruses. the aim was to simulate protection against infectious viruses in droplets released when infected people cough and sneeze, and during aerosol-generating procedures in healthcare settings. the first assay compared three samples of coated ffp respirators (glaxosmithkline actiprotect) with three ffp controls (sperian willson easy fit). for each test, suspensions of influenza a (h n ) at ae log tcid ⁄ ml in ae · minimum essential medium (mem) were aerosolised with a nebulizer. the airborne droplets were introduced into a sterile chamber upstream of a respirator sample for minutes, at a flow rate of ae l ⁄ minute. constant airflow was maintained for another minutes after exposure to the virus. then the collection dish in the downstream sieve sampler (anderson) was assayed for infectious viruses using standard techniques. briefly, serial dilutions of the collection medium (mem with % fbs, % gelatine, and % hepes, supplemented with antibiotics and amphotericin b) in mem + trypsin were used to inoculate madin-darby canine kidney epithelial (mdck) cells in quadruplicate in a multi-well plate. these cultures were then incubated and scored over - days for cytopathic effects, cytotoxicity, and viability. negative cell controls and cytotoxicity and neutralisation controls were also performed. the spearman-karber formula was used to calculate tcid . the second assay compared five samples of coated respirators with five ffp controls ( m ) and five ffp controls ( m ). a suspension of influenza a (h n ), at ae tcid ⁄ ml, was nebulized for minute and seconds into the aerosol chamber, at a flow rate of ae l ⁄ minute, followed by constant airflow for minutes after exposure to the virus. then the collection medium in the downstream chamber (as before, with % nahco ) was assayed as described above. initial viral loads in the first and second assays were ae and ae log tcid , respectively, and were therefore high in comparison with respiratory secretions from infected patients at the peak of their influenza symptoms (range - log t-cid ). table shows that the average viral load that passed through the uncoated ffp respirators in the first assay was ae log tcid . the average viral load that passed through the coated respirators was ae log tcid . therefore, for active filtration of viruses, the relative efficacy of the respirator with antiviral coating was ae log greater than the uncoated respirator. for surface inactivation, the relative antiviral efficacy of the coated respirator was ae log . in the second study, table shows that the average viral load that passed through the uncoated ffp respirators was ae log tcid . in contrast, ae log tcid passed through the coated ffp respirators. by comparison with the viral load when no respirator was present ( ae log tcid ), the ffp respirators reduced the viral load by ae log , and the coated ffp by ae log . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load by ae log more than the ffp respirators. in this second study, the average viral load that passed through the uncoated ffp respirators was also ae log tcid . by comparison with the viral load when no respirator was present ( ae log tcid ), the ffp respirators reduced the viral load by ae log . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load passing through the mask by ae log more than the ffp respirators. table also shows that the coated respirators reduced the infectious viruses remaining on the mask surfaces by ae log more than the ffp respirators, and ae log more than the ffp respirators. even with a very high viral challenge, the coated respirators prevented passage of at least an additional ae log infectious viruses, compared with uncoated respirators. large numbers of infectious virions passed through all uncoated respirators tested. ffp respirators were no more effective than ffp respirators at blocking airborne influenza viruses. based on these in-vitro results, respirators with the antiviral coating could be expected to provide more protection than standard respirators from the risk of inhaling influenza viruses. strategies to prevent transmission of influenza include use of respiratory protection. ffp and n respirators are certified to filter at least % of nacl particles ( ae lm in diameter), and many guidelines have recommended that healthcare workers wear these respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. , we have developed a proprietary acid-polymer formulation, designed to coat a standard respirator and inactivate influenza viruses on contact. we tested this coated respirator for cytotoxicity, skin irritation, and sensitisation potential. the antiviral coating was also tested for stability and leaching under extreme environmental conditions, such as physical abrasion and simulated breathing at different temperatures, levels of humidity and co , and saturation with contaminants. eight coated respirators were tested at standard relative humidity ( % rh) for hours, and one at elevated humidity ( % rh) for hours. four coated masks were treated with synthetic blood or oral secretions, and then tested at % rh for hour. the sample respirators were sealed onto a mannequin head inside an airtight chamber, and air at °c and ppm co was pumped through the masks by a cyclic breathing machine at l ⁄ minute. a mm glass-fibre filter was placed behind the respirator, over the mannequin's mouth opening. at the end of all tests, these filters were eluted and analysed using high-performance liquid chromatography (hplc). standard in vitro methods were used to assess the cytotoxicity of the coated polyester and uncoated polypropylene layers of the respirator (glaxosmithkline actiprotect). samples were extracted in minimum essential medium (mem), supplemented with serum, penicillin, streptomycin, amphotericin b, and l-glutamine, at °c for hours. triplicate monolayers of mouse fibroblast cells (l- ) were dosed with each extract (including a reagent control and negative and positive controls), and incubated at °c in % co for hours. after hours of incubation with samples or controls, the monolayers of mouse fibroblast cells were examined microscopically for abnormal cell morphology or cellular degeneration. samples of the coated respirator (comprising four polypropylene layers bonded to the coated polyester outer layer) were applied under occlusive patch conditions to the skin of adults. controls, including individual layers, were applied in the same way. in a separate patch test, samples of the coated polyester outer layer and controls were applied under the same conditions to adults. after hours, test patches and controls were removed. sites were then scored for itching, erythema, oedema, epidermal damage, and papular response after and hours. the patches were applied three times a week for weeks. to evaluate sensitisation, test patches were applied - days later for hours at different sites to the original samples. after this challenge, skin was assessed and graded for sensitisation potential after and hours. table shows that no residues of the antiviral coating or degradation products were detected in the air that had passed through any of the eight respirators. cytotoxicity tests showed that the coated respirator material caused % cell lysis or toxicity, classified as slight reactivity (grade ), and that uncoated material caused no cell lysis or toxicity (grade ) ( table ) . results for positive and negative controls were severe reactivity and no reaction, respectively. from the results of the two human repeat-insult patch tests, neither the coated or uncoated layers nor the fullthickness respirator fabric caused irritation (including itching, erythema, edema, vesiculation, epidermal damage, papules, or reactions beyond the patch site) or sensitisation in any of the adult volunteers at any of the time points. based on these results, in conjunction with published data on acute and repeat-dose toxicity, mutagenicity, local irritation, dermal sensitisation, and inhalation safety for all components of the antiviral coating, the potential topical or inhalation exposure to the coated antiviral respirator does not pose a safety risk. the antiviral coating is durable and stable, and stays on the outer surface of the respirator, even in extreme environmental conditions. the coated respirator is non-irritating and non-sensitising. therefore, this respirator is considered to be well-tolerated and safe for its intended use. ies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. knowing how influenza virus is transmitted at home and in school is the key to preventing its spread. at the previous two meetings of this conference, , we introduced our study of household transmission of seasonal influenza and reported our conclusion that protracted survival of the virus even after treatment increases household transmission, and is a major factor in the transmission of the virus to infants. on the other hand, during the recent pandemic, many schoolchildren developed serious respiratory tract disorders, which again highlights the significance of schoolbased transmission of the disease. in this study, we compared transmission of a new influenza strain at home and in school with that of seasonal influenza and proposed countermeasures. the for the analysis of school-based transmission, the epidemic status of seasonal influenza in children at six elementary schools over the past two seasons ( - and - seasons) was compared with that of pdmh in children at two primary schools. using observational data of school-based transmission, we also constructed a model for influenza transmission , and evaluated the effects of factors that could affect influenza transmission (e.g., antibody prevalence, transmission rate, non-infectious latent period, infectious latent period, school closure) through the use of simulations. in this study, a diagnosis of influenza was confirmed by rapid influenza antigen detection kit. we previously reported the high sensitivity of the kits, - not only for seasonal influenza, but also for h n pandemic compared to virus isolation and pcr. serum antibody was not investigated. most of the index patients were treated with oseltamivir or zanamivir, and patients were treated with amatadine. no treatment was done for patients. no nai therapy was done as prophylaxis within the family. the incidence of households with an initial case patient who subsequently infected another member of the household was ae % ( of households) for seasonal influenza or ae % ( of households) for pdmh . thus, the household incidence of pdmh was lower than that of seasonal influenza. in addition, the percentage of family members in households who were infected by initial case patients (household transmission rate) was ae % ( of individuals) for seasonal influenza or ae % ( of individuals) for pdmh . thus, the household transmission rate was also lower for pdmh than that for seasonal influenza. effect of family size on household incidence and household transmission rate an analysis of the effect of family size on household incidence showed that, in families consisting of - individuals, the incidence of seasonal influenza in order of increasing family size was ae %, ae %, ae %, ae %, ae %, and ae %, respectively, and the incidence of pdmh was ae %, ae %, ae %, ae %, ae %, and ae %, respectively, indicating that household incidence tends to increase with increasing family size. in contrast, no definite relationship was noted between household transmission rate and family size. transmission rates for seasonal influenza in order of increasing family size were ae %, ae %, ae %, ae %, ae %, and ae %, respectively, or ae %, ae %, ae %, ae %, ae %, and ae %, respectively, for pdmh (shown in table ). effect of age cohort of initial case patient in household on household incidence and household transmission rate an analysis of the effect of the age cohort of the initial case patient in the household on household incidence and transmission rate showed that the household incidence of seasonal influenza in c , c , c , and c was ae % ( of households), ae % ( of households), ae % ( of households), ae % ( of households), and for m and f was ae % ( of households) and ae % ( of households), respectively. therefore, household incidence was the highest in c , followed by the parents. when the initial case patient was a child, the household incidence increased with decreasing patient age. in contrast, the household incidence of pdmh in c , c , c , and c was ae % ( of households), ae % ( of households), ae % ( of households), ae % ( of households), and for m and f was ae % ( of households) and ae % ( of households), respectively. therefore, household incidence was higher when the initial case patient was a parent, rather than a child. the household transmission rates for seasonal influenza from c to f were ae %, ae %, ae %, ae %, ae %, and ae %, respectively. therefore, as for household incidence, the highest rate ( ae %) was observed in c . the corresponding household transmission rates for pdmh were ae %, ae %, ae %, ae %, ae %, and ae %, respectively, with the highest transmission rates observed for infections from parents (shown in table ). if the rate of individuals with a secondary infection transmitted from the initial case patient in a household is presented as a percentage of the total number of affected individuals, the rates for seasonal influenza and pdmh were ae % ( of individuals) and ae % ( of individuals), respectively. therefore, the rate of individuals with a secondary infection was lower for pdmh than that for seasonal influenza. by age cohort, the corresponding rates of individuals for seasonal influenza in c , c , c , and c were ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), and for m and f was ae % ( of individuals) and ae % ( of individuals), respectively. for pdmh , the corresponding rates in c , c , c , and c were ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), and for m and f was ae % ( of individuals) and ae % ( of individuals), respectively. these findings indicate that, especially in the case of pdmh , most secondary infections in parents tend to be transmitted from another household member. the mean annual prevalence of seasonal influenza and the new influenza strain at the elementary schools for the two seasons was ae % and ae %, respectively, whereas the prevalence determined days after appearance of the first case in school was ae % and ae %, respectively. in the recent season at the same elementary schools, however, the prevalence was a high ae %. since the prevalence at days after the appearance of the first case in school was already ae %, these data show that the influenza virus spread quickly throughout the schools. at the schools with high transmission rates in the early period of the pandemic, new infections were confirmed even days after the school closure action was taken. these findings indicate that pdmh , the current influenza virus, has a long latent period during which it becomes infectious and spreads from infected individuals to numerous others in their vicinity. we constructed a model for influenza transmission in schools and estimated the time course of changes in the number of expected cases and the expected prevalence during the season. in this model, school children were divided into six groups depending on the stage of infection: uninfected period with no immunity, non-infectious latent period, infectious latent period, onset, post-onset infectious period, and immune period. it was assumed that schoolbased transmission occurred during the infectious latent period prior to onset and that no infections occurred during the post-onset infectious period because children were absent from school. due to the long latent period of pdmh , the distribution of the non-infectious latent period of pdmh was established as (day , day , day , day ) = ( %, %, %, %) and the distribution of the infectious period as (day , day , day ) = ( %, %, %). when simulations were performed under these conditions using the model for school-based transmission of influenza in which children from classes with an outbreak were kept at home for days, the time course of changes in the number of affected individuals actually observed and the time course of changes in the number of expected cases were determined. the expected prevalence under these conditions was %. to evaluate the effect of school closure, simulations were performed based on the assumption that children from affected classes were not kept at home for days. it was shown that there was an increase in the expected number of cases during the days corresponding to the period of actual school closure and that the expected prevalence increased to %. based on these findings, it was concluded that keeping children home from classes with an outbreak is an effective means of controlling the transmission of influenza in schools (shown in figure ). if the transmissibility of pdmh virus at home is estimated based on the speed of transmission and the degree to which pdmh is prevalent in schools, it would be expected that the household transmission of pdmh is also higher than that of seasonal influenza. in fact, the opposite is the case. this paradox can be explained in two ways. . the number of children aged or more and parents with pdmh influenza as a percentage of the total number of affected individuals is lower than those with seasonal influenza ( ae % versus ae %). further, although the number of parents with a secondary infection was high at home, the percentage of the total number of individuals with pdmh was a low ae % ( of individuals), compared to that for seasonal influenza ( ae % [ of individuals]). in other words, adults are less susceptible to pdmh infections and there was a correspondingly small number of affected individuals. therefore, it was considered that the transmission rate at home was lower than that at school for this reason. . the percentage of households with more than one affected individual within the same family was higher for pdmh at ae % ( of households) than for seasonal influenza at ae % ( of households). in the patients secondarily infected with pdmh , ae % of them showed symptoms of infection days or more after the onset in the first patient, suggesting that they were not infected at home, and the actual household transmission was ae % ( of households). therefore, although the prevalence was higher for pdmh , it seems that household transmission was lower because households with an affected individual implemented satisfactory control measures against infection. seasonal influenza differs greatly from pdmh influenza in its transmissibility at home and in school. in the household transmission of pdmh influenza, both the household incidence and household transmission rate of pdmh were low compared to those for seasonal influenza. although transmission of seasonal influenza from infants to parents was marked, in the case of pdmh , the reverse was true with transmission from parents to children being predomi-nant. it should be noted that household transmission in mothers was common in all eight seasons, suggesting the need to reconsider control measures against infection when nursing unwell family members. in the case of school-based transmission, pdmh was more prevalent than seasonal influenza, indicating that the virus spread quickly throughout the schools. this difference was attributed to the long infectious latent period when pdmh rapidly became rampant in the schools. an analysis of school-based transmission using a model for influenza transmission showed that, when % of the student population is infected, schools should be closed for five consecutive days in order to minimize the spread of the disease. the effectiveness of seasonal influenza vaccine in preventing pandemic and seasonal influenza infection: a randomized controlled trial introduction household transmission has been estimated to account for one-third of all influenza transmission, , and children are at high risk of spreading the disease. with reference to previous evidence, - some vaccine deployment strategies target children to prevent them from infection and transmitting influenza. nevertheless, few studies evaluated the effectiveness of vaccinating children in reducing household transmission. , during - , a pilot randomized controlled trial was conducted to investigate such effect by studying households with school age children randomized to receive trivalent inactivated seasonal influenza vaccine (tiv). the monovalent vaccine against pandemic influenza a (h n ) (ph n ) had yet been available until the end of the first wave. various conclusions have been made as to whether seasonal influenza vaccine might possibly protect against ph n . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] we report findings on the effectiveness of tiv against ph n observed in our cohort. households were screened if they expressed interest after receiving invitation letters distributed via their children's school or an existing pediatric cohort study. to be eligible, the household had to include at least one child aged - years who was not allergic or hypersensitive to any of the tiv components. children known to have immunosuppressive conditions or other contraindications against tiv were also excluded. written consent and assent were obtained from participants aged above years and those aged - years, respectively. proxy written consent was obtained from legal guardians or parents for participants younger than years. ethical approval was obtained from the institutional review board of the university of hong kong. consented households were allocated to the tiv and placebo group (in ratio : ) according a code generated by block randomization with random block sizes of , , and . an independent nurse prepared . one child (study subjects) from each household in the tiv group received a single dose of tiv with one child from each household in the placebo group receiving a single dose of saline placebo. parents and legal guardians were asked to report any adverse reactions days following vaccination. all participants, study nurses, and other research staff were blinded to the allocation and administration of vaccine or placebo. the vaccine allocation sequence was only disclosed to the investigators at completion of the study. serum specimens were collected from subjects shortly before (november-december ), one month after vaccination (december -january ), and after the winter (april ) and summer influenza seasons (august-october ). serum specimens were obtained from household contacts at baseline and after the winter and summer influenza seasons. all household members recorded any fever ‡ . °c, chills, headache, sore throat, cough, presence of phlegm, coryza, or myalgia daily on a symptom diary. they were also invited to report to the study hotline immediately if they experienced at least of the above signs or symptoms. as a response, the study nurse would visit the households with any sick members and collect nose and throat swab from all household members. the households were also telephoned monthly or increased to fortnightly during influenza seasons to monitor for signs and symptoms and remind them to report to the hotline. supermarket or book vouchers (for children) were given to the households including us$ for each serum specimen collected, us$ . for each home visit, and us$ for completion of the study. serologically-indicated influenza infection was the primary outcome of this study. it was define as a ‡ fold rise in antibody titer within each influenza season. other study outcomes included rt-pcr confirmed influenza virus infection, acute respiratory illness (ari) (two of any of the above listed signs or symptoms), and influenza-like illness (ili) (fever ‡ . °c with cough or sore throat). antibody titers against the vaccine strains were obtained by testing each serum specimens by haemagluttination inhibition (hai). viral microneutralization (vn) using standard methods was found to be more sensitive than hai in detecting antibody response against a ⁄ california ⁄ ⁄ (h n ) in another study conducted by our group and was, therefore, used in this study. the sera was initially diluted at ⁄ and further tested in serial doubling dilutions. nose and throat swabs were tested by reverse transcription polymerase chain reaction (rt-pcr) for influenza a and b viruses. technical details of the laboratory methods have been reported elsewhere. , fisher's exact test and chi-squared tests were used to compare count data including occurrence of side effects, laboratory confirmed, and clinically defined influenza infections. wilcoxon signed-rank test were used to compare the serum antibody titers between groups. exact binomial method or the wald approximation was used to estimate % confidence intervals where appropriate. all analyses were carried out in r version . . (r development core team, vienna, austria). twenty-five primary and secondary schools in the district of the study clinic were invited to participate. to parents of three schools that agreed to take part and another study cohort, invitation letters were sent and households were enrolled. personal referrals were made from these parents to enroll additional households. among enrolled households, subject with history of epileptic seizure was assessed to be contra-indicated against receiving the vaccine. blood taking failed in another subject, and both of them withdrew from the study. eleven households did not complete the study. table shows subject and household contacts of the tiv and placebo group were similar in demographics and prior influenza vaccination history. antibody titers before vaccination were comparable between groups (data not shown). most study subjects who received tiv showed antibody titer ‡ against the vaccine strains month after receiving tiv, and the proportion was significantly higher than those who received placebo (a ⁄ h n % in tiv versus % in placebo group, p < . ; a ⁄ h n % versus %, p < . ; b % versus %, p = . ). none of the study subjects had antibody titer ‡ against ph n following receipt of seasonal tiv. no serious adverse reactions were reported, and only pain at injection sites was slightly higher in tiv group (data not shown). subjects who received tiv had lower rates of serologically confirmed seasonal influenza a(h n ) ( % versus %, p = . ), a(h n ) ( % versus %, p = . ) and b infection ( % versus %, p = . , although the differences were not statistically significant (table ) . study subjects had higher rate of serologically confirmed ph n infection ( % versus %, p = . ), yet it was not statistically significant. after adjusting for potential cross reactive antibody response, % of subjects in tiv versus % in placebo groups showed ph n infection confirmed by either serology or rt-pcr (p = . ). little differences were observed for rt-pcr confirmed infection, ari, and ili in results combining the winter and summer influenza seasons. during winter season when seasonal influenza predominated, study subjects who had received tiv showed a lower tendency to develop ili ( % versus %, p = . ) or ari ( % versus %, p = . ). an opposite tendency was seen (ili % versus %, p = . ; ari % versus %, p = . ) during summer when ph n predominated. however, these differences were not statistically significant. rates of ili in subjects infected with ph n did not differ statistical significantly between subject who received tiv and placebo ( % versus %, p = . ). the study was not powered to detect indirect benefits to household contacts of vaccines resulting from reduced household transmission. attack rates were found to be similar between household contacts of subjects received tiv and placebo (data not shown). to examine potential factors that might affect risk of laboratory confirmed ph n infection, a multivariable logistic regression model was fitted to study all subjects and their household contacts. younger participants aged below years were found to have a higher risk (< years or = . , % ci . , . ; - years or = . , % ci . , . , > or = . ). after adjusting for age, sex, and date of study completion, receipt of tiv for the - influenza season was not found to affect risk of ph n infection. however, participants who had laboratory confirmed seasonal influenza infection during the study period had % lower risk of ph n infection (infected with seasonal influenza or = . , % ci . , . ; not infected with seasonal influenza or = . ). as (see table s for winter and summer results separately). influenza-like illness (ili) defined as temperature ‡ . °c plus cough or sore throat; acute respiratory illness (ari) defined at least any two of fever ‡ . °c, chills, headache, sore throat, cough, presence of phlegm, nasal congestion, runny nose, muscle or joint pain. limited by the sample size, we were not able to differentiate between the protective effect of seasonal a(h n ) and a(h n ) infection against ph n . other details of the results from the study were published elsewhere. discussion a non-significantly higher rate of ph n infection was observed in study subjects who received tiv compared to placebo. results from a multivariable logistic regression suggested that such a pattern might be explained by more common seasonal influenza infection in placebo group prior to the pandemic, protecting the placebo group against ph n . seasonal influenza infection within - months observed in our study might have conferred better cross protection than tiv against ph n . this resembles similar previous findings on cross protection between influenza infections in human and animal studies. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, the same phenomenon has not been observed in some studies on seasonal influenza vaccine against ph n . , , [ ] [ ] [ ] apart from differences in study design and vaccine used, we speculate that a short time interval between ph n and most recent seasonal influenza peak activities might be crucial for the phenomenon. hong kong is a subtropical area where the pandemic was preceded immediately by summer seasonal influenza circulation and a few months apart from the winter - influenza peak. if cross protection from seasonal influenza lasts for only a short period, it might have waned below partial cross protection from tiv over time from last seasonal influenza infection. the current study is limited by a small sample size, and further studies are required to confirm our hypothesis. while tiv is only effective against matching strains, a universal influenza vaccine could provide better protection against the ever evolving influenza viruses. introduction immunisation of healthy, as well as high risk, children has been the focus of much recent attention both in prevention of seasonal influenza and during the h n pandemic. detailed information on reactogenicity, particularly for newer vaccine formulations that include adjuvants, is limited. we recently reported results of a head-to-head comparison of two h n pandemic influenza vaccines in children in the uk. here we present new, detailed analyses of reactogenicity data from that study, which has important potential implications for future paediatric influenza vaccine development and use. we compared the safety, reactogenicity, and immunogenicity of two h n influenza vaccines, one as b (tocopherol based oil in water emulsion) adjuvanted egg culture derived split virion, the other non-adjuvanted cell culture derived whole virion, given as two dose schedules days apart, in a randomised, open label trial as previously reported. the study was age stratified ( months to under years & - years) to ensure adequate data in young children. age appropriate safety data (simplified for under year olds) were collected for days after each vaccine dose and serum was collected at enrolment & days after the second dose. nine hundred-thirty seven children received vaccines as per-protocol. when comparing the two vaccines, grade ( ‡ mm) local reactions were seen more frequently following the adjuvanted than the non-adjuvanted vaccine in both age groups, after both vaccine doses. in children over years old, ae % versus ae %, p < ae , after dose one; ae % versus ae %, p = ae , after dose two, in children under years old, ae % versus ae %, p = ae , after dose one (non significant, ns); ae % versus ae %, p < ae after dose two. fever ‡ °c (axillary measurement) was seen more frequently following the second dose of the adjuvanted vaccine compared to the non-adjuvanted vaccine in < year olds ( ae % versus ae %; p < ae ). looking specifically at the adjuvanted vaccine in under year olds, comparing the second dose with the first, there were significantly higher rates of fever ‡ °c (axillary measurement) ( ae % versus ae %, p < ae ), local grade ( ‡ mm) reactions ( ae % versus ae %, p = ae ), pain ( ae % versus ae , p = ae ), use of analgesia or antipyretic medication ( ae % versus ae %, p < ae ), and decreased activity ( ae % versus ae %, p < ae ). the adjuvanted vaccine was significantly more immunogenic, most notably in the younger children. in < year olds, haemagglutination inhibition (hi) seroconversion rates were ae % versus ae %, p < ae . among all general and local reactions measured, only the maximum temperature measured during the days after the second dose of the adjuvanted vaccine showed a significant (positive) association with post vaccination hi titres. for each °c rise in temperature there was a % increase in titre (p < ae ). these reactogenicity data demonstrate a step towards the future possibility of one-dose influenza immunisation programmes for young children associated with low rates of fever and other reactions. the occurrence of fever following adjuvanted vaccine, seen particularly after a second dose in younger children, was quantitatively associated with enhanced antibody titres. this association was not seen with unadjuvanted vaccine. this apparent difference between the relatedness of the pyrogenic and immunogenic effects of the two vaccines merits further investigation. novel adjuvants appear to have the potential to overcome the relatively poor immunogenicity previously experienced with inactivated influenza vaccines in infants and young children. however, careful adjustment may be needed to optimise the balance between high protection and acceptable reaction rates. tries causing sporadic human infections. vaccination has been used as an effective public health tool for influenza prophylaxis. the goal of this study was to evaluate live attenuated influenza vaccine (laiv) vaccine candidates for subtypes h and h . the attenuated phenotype of h and h laiv candidates has been proven in experiments in ovo and in vivo. in randomized clinical trials among adult volunteers, no significant adverse reactions attributable to the live vaccine occurred. our results indicate that pandemic laiv candidates were well tolerated and elicited serum, local, and cellular immune responses. the emergence and spread of highly pathogenic avian influenza h n viruses in avian populations and concurrent infections in humans since has prompted efforts to develop vaccines for use in the event of an influenza pandemic. in , the world faced a new h n pandemic. immunization with inactivated or live vaccines is the primary measure for preventing influenza. laivs appear to be safe and efficacious, and might possibly provide broader immune responses than inactivated vaccines. our study evaluated laiv pandemic candidates as part of the global influenza pandemic preparation project outlined by the who. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated by routine technique in embryonated hen eggs. laiv and placebo were supplied by microgen (irkutsk, russia). the monovalent laiv was produced from the pandemic vaccine candidates and formulated to contain and ae eid per dose ( ae ml) of a ⁄ ⁄ california ⁄ ⁄ and a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ , respectively. the vaccine or placebo was administered intranasally with a single-use dosing nasal sprayer. two doses were given at an interval of days. one hundred-ninety healthy adults aged - years were randomly divided into groups to receive either pandemic vaccine candidates ( ) or placebo ( ) . subjects were informed about purposes and methods of the study and potential risks associated with participation. all participants had an hai antibody titer of £ : to a ⁄ california ⁄ ⁄ (h n ) pandemic virus. in all there were and vaccines and and participants who received placebo, and were further tested for immune responses to h n or h n pandemic vaccine, respectively. another participants vaccinated with h n laiv were children between to years old. before the children were vaccinated, their parents were advised about study and their consent was required before any child was enrolled. on the advice of the national ethics committee, we did not include a placebo group in this study. individuals were not enrolled if they had an acute illness or fever at the beginning of the study or a history of egg allergy. immune responses of subjects were assessed by routine hai test (evaluation of serum igg antibodies), elisa (evaluation of iga antibodies eluted from the nasal swabs into steril pbs), and cytokine flow cytometry assay (evaluation of virus-specific cd + cd + ifnc + and cd + cd + ifnc + peripheral blood mononuclear cells). the results of phenotypic analysis in ovo showed that pandemic vaccine candidates retained the cold adapted-temperature sensitive (ca ⁄ ts) phenotype, typical of the coldadapted parental mdv. in contrast and as expected, a ⁄ california ⁄ ⁄ and a ⁄ duck ⁄ potsdam ⁄ - parental strains had the non-ts ⁄ non-ca phenotype typical of wt viruses. the h n pandemic vaccine candidate demonstrated an attenuated phenotype in mice and in java macaques and did not infect chickens. the vaccine attenuation study confirmed the attenuated phenotype of a a ⁄ ⁄ california ⁄ ⁄ pandemic laiv candidate in mouse, ferret, and guinea pig models. the phase i ⁄ ii randomized, controlled, double-blind clinical study safety evaluation of pandemic vaccine candidates in adults clinical examination of subjects who received two doses of pandemic vaccine candidates indicated that both vaccines were well tolerated. no fever reactions were observed after the first or second vaccination. after the first vaccination, ae % and ae % of reactogenicity events consisting of catarrhal symptoms, such as pharyngeal irritation or hyperemia, were observed for h n and h n vaccine candidates, respectively. after revaccination, subjects did not report local or systemic reactions. to determine whether a serological response occurred in the cohort of immunologically naïve subjects vaccinated with pandemic vaccine candidates, hai and elisa tests were used (table ) . post-vaccination geometrical mean titers (gmt) among subjects who received two doses of h n vaccine were significantly higher than pre-vaccination titers. the frequency of ‡ fold antibody rises was significantly higher ( ae %) after revaccination than after one dose ( ae %). the percentage of subjects with post-vaccination serum hai titers to h n ‡ : was ae % and for titers ‡ : , it was ae %. no seroconversions in the placebo group were detected. the virus-specific nasal iga antibody response to vaccination after two doses of the h n vaccine candidate demonstrated significant increases of ‡ fold rise iga antibodies ( %) compared to one dose. cumulative data of h n vaccination (all applied tests) showed % and % of conversions after the first and the second vaccination, respectively. increasing h n vaccine virus infectivity from ae to ae eid ⁄ dose lead to an enhancement of post-vaccination hai titers in vaccinees after the first vaccination to homologous h n antigen from ae % to ae % of ‡ fold antibody rises. values of post-vaccination serum hai antibody titers in subjects vaccinated with another pandemic vaccine candidate, a ⁄ ⁄ california ⁄ ⁄ , also proved to be rather low. after the primary vaccination, the percentage of subjects with hai protective antibody titers ‡ : were ae %. after revaccination, this parameter increased to ae %. four-fold increases in serum hai antibody titres were four-fold conversions after the first and the second vaccination was ae % and ae %, respectively. elisa antibodies in nasal swabs showed had an advantage in detecting induction of local iga as compared to serum hai antibodies. after revaccination four-fold serum hai antibody conversions were ae % vs. ae % of iga conversions in nasal swabs, respectively. taking into account cumulative data of h n vaccination (hai and elisa data), the obtained results were here and in the ae % and ae % of conversions after the first and the second vaccination, respectively. fourty-seven subjects were vaccinated with h n laiv, and who received a placebo were chosen for evaluation of cellular immune response by cytokine assays. after revaccination, the mean increases of both cd + and cd + memory cells were significantly higher in vaccinated subjects compared to the placebo group. interestingly, the same effect of vaccination was observed in vaccinees without detectable conversions of hai antibody titers. even after a single vaccination, the rate of subjects with significant increases of these cells in the blood was ae % (cd + ) and % (cd + ). after the revaccination, the percentage of subjects with significant increases in cd + and in cd + cells was ae %. immunogenicity of h n pandemic vaccine candidate in children hai antibody results among children aged to years proved to be significantly higher when compared to adult subjects: after the first vaccination, ae % of the children seroconverted; after revaccination, seroconversions reached ae % ( table ). the gmt rise to h n vaccine with primary vaccination was : ; after revaccination it increased to : . benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. many years of laiv seasonal trials have shown excellent tolerability and low reactogenicity. [ ] [ ] [ ] indeed, data showed that live influenza vaccines cause minimal systemic, local, and thermal reactions, generally from to %. a different situation was observed in the cohort of immunologically naïve volunteers vaccinated with pandemic vaccines. the rate of local reactions to a ⁄ ⁄ california ⁄ ⁄ and a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ vaccine candidates increased to ae % and ae %, respectively. after revaccination no significant local and systemic reactions were observed. this confirms, indirectly, the development of a sufficiently high level of protection after the first vaccination with pandemic laiv. the most important criterion for assessing the quality of vaccines is their estimated safety, epidemic effectiveness, and immunogenicity. however, current regulatory documentation mandates that induction of serum antibodies, measured by hai, as the only criterion for a laiv immunogenicity evaluation. in addition to the standard hai assay, we determined serum (igg) and local (iga) antibodies in adult subjects vaccinated with an h n pandemic vaccine candidate. evaluation of overall results obtained in these additional serological tests, as well as those from the hai assay, showed an immune response to the vaccine in the majority of subjects ( ae % of ab seroconversions after the single vaccination and ae % after revaccination, respectively). these data show that methods used to routinely measure laiv immunogenicity should be revised to include a number of additional immunological methods such as igg and iga elisa, and cytokine assays consistent with the recently updated who recommendations on laiv monitoring. these clinical studies clearly demonstrated that pandemic laiv candidates are effective at generating pandemic specific influenza immunity. a key finding from this study is that it may be practical to give the vaccine as a single dose to both children and adults. evaluation of our laiv pandemic vaccine candidates was performed as part of the global influenza pandemic preparation project outlined by the who. it was considered that laiv could be produced in greater quantities and more rapidly than inactivated vaccines. together with the generation of herd immunity by laiv, this suggests that laiv implementation during the first wave of a pandemic may provide significant social, economic, and health benefits to the community. authors are thankful to path for the financial support of h n pandemic vaccine study. we are grateful for the the main evolutionary mechanism of influenza viruses during inter-pandemic period is the antigenic drift, but the epidemiological picture of circulating viruses is complicated by a high level of heterogeneity of strains, even though drift does not occur, due to co-circulation of drifted and old strains or to co-circulation of viruses belonging to the same type ⁄ subtype but with different antigenic patterns. [ ] [ ] [ ] [ ] [ ] [ ] lack of data exists on the impact of the wide heterogeneity of circulating strains on the seroprotection and on-field effectiveness of influenza vaccine: in particular, little is known about the ability of influenza vaccine to elicit an effective immune response against isolates with few amino acid mutations with respect to vaccine strains that represent the majority of circulating viruses. mf -adjuvanted vaccines, which are currently used for the prevention of seasonal influenza epidemics in elderly, are showed to confer higher seroprotection against homologous and drifted a(h n ) strains than non-adjuvanted vaccines. [ ] [ ] [ ] the broader immune response showed by mf -adjuvanted vaccine was measured using hi and nt assays against egg-grown drifted strains representing vaccine composition changes during the following seasons, but its ability to elicit a broader immune response against circulating viruses belonging to vaccine cluster and presenting amino acid mutations onto antigenic sites or against on-field isolates not-antigenically distant from vaccine strains has not yet been investigated. showing amino acid changes onto antigenic sites in position (n k), (n k), and (p s) with respect to a ⁄ california ⁄ ⁄ . in particular, a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ presents n d amino acid mutation detected in clade a ⁄ wyoming ⁄ ⁄ -like viruses. the ha sequences of a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ fell within the clade represented by the ha of a ⁄ califor-nia ⁄ ⁄ ; among these isolates, a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ showed antigenic site sequences very close to that of the ⁄ vaccine strain, whereas ha sequences of a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ posses amino changes onto antigenic site a(r k), c(g e) and d(r k), respectively. the ha sequences of more recent isolates fell within the clade represented by the ha of a ⁄ brisbane ⁄ ⁄ and characterized by the amino acid changes, relative to the ha of a a ⁄ california ⁄ ⁄ , g e and k i, with the exception of a ⁄ genoa ⁄ ⁄ , showing r g and l s amino acid changes present in viruses belonging to a ⁄ nepal ⁄ ⁄ clade. measure of genetic distance between vaccine and circulating strains was calculated as previously described by gupta. two blood samples were collected from each subject, just before and ± day post-vaccination. all sera were stored at ) °c. all samples were tested at the laboratory of health sciences department, university of genoa, by haemagglutination-inhibition (hi) and neutralization (nt) assays, performed following the who criteria and standardised method in our laboratory, respectively. [ ] [ ] [ ] guinea pig red blood cells were used for hi assay. all samples were assayed twice for hi and for nt. the obtained antibody titre was expressed as the reciprocal of the last sera haemagglutinating or inhibiting virus dilution. immunogenicity was determined by: geometric mean titre (gmt); mean-fold increase (mfi; ratio of post-to pre-vaccination titre); seroprotection rate (the percentage of subjects achieving an hi and nt titre ‡ iu); and seroconversion rate (percentage of subjects with a fourfold increase in hi or nt antibody titers, providing a minimal post vaccination titer of : ). post-vaccination gmt was reported as ratio, with the corresponding % confidence interval, of gmts after vaccination with mf -adjuvanted vaccine and with non-adjuvanted subunit vaccine. seroprotection and seroconversion rate % confidence interval was calculated using modified wald method. comparisons of seroconversion and seroprotection rates between subunit and mf -adjuvanted vaccine groups have been analyzed by fischer's exact test. the results were evaluated against the committee for medicinal products for human use (chmp) criteria for approval of influenza vaccines in the elderly, which require that at least one of the following criteria be met: mfi > ; seroprotection rate > %, or seroconversion rate > %. furthermore, hi titres were also transformed into binary logarithms, corrected for pre-vaccination status, as described by beyer et al. and were expressed as median titres, with the corresponding °- °i nter-quantile range. comparisons of corrected post-vaccination titers between subunit and mf -adjuvanted vaccine groups were analyzed by wilcoxon test. difference in immunogenicity profile between vaccine groups, expressed by ratio of different parameters, was correlated with genetic and antigenic distance between vaccine and viruses used in the study using spearman test. pre-vaccination titres were not significantly different between vaccine groups, for all strains (data not shown). post-vaccination gmt ratios between mf -adjuvanted and non-adjuvanted vaccine groups determined using hi and nt assays, with the corresponding % confidence interval, according to viral strain are shown in figure . both vaccines met chmp requirements for mfi (> ), seroconversion (> %), and seroprotection rate (> %) against a ⁄ wyoming ⁄ ⁄ -like, with the exception of a ⁄ genoa ⁄ ⁄ and a ⁄ california ⁄ ⁄ -like circulating viruses and against egg-grown a ⁄ wyoming ⁄ ⁄ , a ⁄ california ⁄ ⁄ , and a ⁄ wisconsin ⁄ ⁄ strains; the immune response against a ⁄ genoa ⁄ ⁄ met the requirements for mfi and seroprotection rate only in mf -adjuvanted vaccine group. requirements for mfi, seroconversion, and seroprotection rate against the a ⁄ brisbane ⁄ ⁄ -like virus a ⁄ genoa ⁄ ⁄ and the a ⁄ nepal ⁄ ⁄ -like genoa ⁄ ⁄ viruses and against egg-grown a ⁄ brisbane ⁄ ⁄ strain were reached only in subjects vaccinated with the mf adjuvanted vaccine. a similar pattern emerged from the analysis of mfi, seroconversion and seroprotection rates using nt assays. subjects vaccinated with the mf -adjuvanted vaccine showed significantly higher post-vaccination hi gmts against a ⁄ wyoming ⁄ ⁄ -like, a ⁄ california ⁄ ⁄ -like, a ⁄ nepal ⁄ ⁄ -like and a ⁄ brisbane ⁄ ⁄ like viruses, with the exception of a ⁄ genoa ⁄ ⁄ , and against egg-grown a ⁄ california ⁄ ⁄ , a ⁄ wisconsin ⁄ ⁄ , and a ⁄ brisbane ⁄ ⁄ strains, compared with individuals immunized with the non-adjuvanted vaccine ( figure ). the mf -adjuvanted vaccine also induced significantly higher seroconversion and seroprotection rates against following correction for pre-vaccination status, hi titres were significantly higher for the mf -adjuvanted vaccine group when evaluated against a ⁄ wyoming ⁄ ⁄ -like viruses, a ⁄ brisbane ⁄ ⁄ -like a ⁄ genoa ⁄ ⁄ , and a ⁄ nepal ⁄ ⁄ -like a ⁄ genoa ⁄ ⁄ strain ( figure ). pre-vaccination titre corrected response was higher in subjects vaccinated with mf adjuvanted vaccine also against egg-grown a ⁄ wyoming ⁄ ⁄ , a ⁄ california ⁄ ⁄ ⁄ , a ⁄ wisconsin ⁄ ⁄ , and a ⁄ brisbane ⁄ ⁄ . among viruses more closely related to a ⁄ california ⁄ ⁄ , subjects immunized with mf -adjuvanted vaccine showed a significantly higher corrected titres against a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ strains compared with the non-adjuvanted vaccine ( figure ) . spearman test showed a clear correlation between the distances and the advantage offered by mf expressed by ratio between mfi, post-vaccination gmts, corrected post-vaccination median, seroconversion, and seroprotection rates calculated using hi test in the two vaccine groups. similarly, ratio between mfi, seroconversion, and seroprotection rates calculated with nt test correlated with the genetic and antigenic distance between vaccine and viruses used for the study. the ability of mf to enhance the immunogenicity and to elicit a broader immune response against drifted strains than non-adjuvanted vaccine is consistent with other findings reported during the last decade. [ ] [ ] [ ] in subjects vaccinated with the mf -adjuvanted vaccine containing a ⁄ california ⁄ ⁄ , the immune response, expressed by a number of parameters, such as crude and corrected postvaccination titers, seroconversion, and seroprotection rates calculated using hi and nt assays, is higher than that observed in individuals immunized with subunit vaccine when it is evaluated against a drifted strains, such as a ⁄ brisbane ⁄ ⁄ -like and a ⁄ nepal ⁄ ⁄ -like strains, and against egg-grown a ⁄ brisbane ⁄ ⁄ virus. for the first time in this study, the impact of heterogeneity of circulating strains antigenically close to the vaccine on the antibody response elicited by mf -and non-adiuvanted vaccines is evaluated. immune response against viruses isolated during the ⁄ season, that appear more phylogenetically close to ⁄ vaccine strain a ⁄ wyoming ⁄ ⁄ , was higher in subjects vaccinated with mf -adiuvanted vaccine as demonstrated by higher crude and corrected post-vaccination hi titres and higher postvaccination nt titres, with the exception of a ⁄ genoa ⁄ ⁄ , against whom the nt post-vaccination gmt is identical in mf and subunit vaccine groups. furthermore, hi seroconversion and seroprotection rates were higher in mf vaccine group when evaluated against a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ . as far as the immune response against a ⁄ california ⁄ ⁄ -like viruses, the small number of enrolled subjects did not allow appreciating differences using qualitative response indicators, but crude post-vaccination hi titres were higher in mf vaccine group for all the strains. interestingly, a ⁄ california ⁄ ⁄ -like viruses with at least one amino acid change onto antigenic sites, i.e. a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ , showed a more marked difference in terms of response between the two vaccine groups. individuals immunized with mf -adiuvanted vaccine showed higher corrected post-vaccination hi titres and post-vaccination nt titres in comparison with subjects vaccinated with plain vaccine. these response indicators were similar in the two vaccine groups when the response was evaluated against a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ , which present no amino acid changes onto antigenic sites and identical hi titers respect with a ⁄ california ⁄ ⁄ at molecular and antigenic characterization, respectively. thus, the advantage offered by mf in terms of higher immunogenicity expressed by higher post-vaccination hi titres is observable also against viruses showing antigenic and molecular pattern undistinguishable from vaccine strain, but it became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew. as emerged for a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ , one amino acid was a sufficient change in antigenic sites for -fold decrease of hi titre against homologous vaccine strain to observe -fold higher post-vaccination nt titers (mf ⁄ subunit postvaccination gmt ratio range between ae and ae , figure ) and one-dilution higher corrected post-vaccination hi titers in mf vaccine group ( figure ) . finally, the correlation between the distance and the improvement offered by mf in terms of higher immunogenicity clearly emerged by spearman correlation analysis: it remains wellfounded both using a number of different response parameters obtained from hi and nt assays and calculating the distance by serological and genetic methods. outbreaks of h n pdm in pigs in commercial swine operations have been reported in several countries. in all incidents, epidemiological investigations have linked humans as the possible source of the infection to pigs. experimentally, it was established that the virus is pathogenic and transmits readily in pigs. the natural outbreaks of h n pdm and laboratory studies underscore the threat that the virus poses to the swine industry and highlight the need for developing effective control strategies. in the united states, a trivalent live attenuated influenza vaccine (flumistÒ) has been licensed for use in humans since . in swine medicine, however, temperature-sensitive laivs are not available. currently, only inactivated vaccines are available for pigs, but they provide limited protection against antigenically diverse influenza viruses. additionally, the use of inactivated vaccines has been associated with enhanced pneumonia when immunized pigs were challenged with divergent viruses. thus, the development of laivs has the potential to circumvent the drawbacks associated with commercial vaccines. with the aim of developing laiv temperature-sensitive influenza vaccines against the h n pdm virus, we have used reverse genetics to introduce attenuation markers in the polymerase genes of a swine-like tr h n influenza virus, a ⁄ turkey ⁄ ohio ⁄ ⁄ (h n ) (ty ⁄ ). we chose this isolate because it grows well in both eggs and cell culturebased substrates, displays a broad host range, and has internal genes similar to the h n pdm virus. safety and efficacy studies of the ty ⁄ att vaccine candidates in pigs demonstrated that this vaccine backbone is attenuated in swine and conferred sterilizing immunity upon an aggressive intratracheal challenge of pigs with the h n pandemic virus. thus, introduction of genetic signatures for att in the backbone of a swine-like tr influenza virus resulted in highly attenuated and efficacious live influenza vaccines with promising applications veterinary medicine. -t cells and mdck cells were maintained as previously described. a ⁄ turkey ⁄ ohio ⁄ ⁄ (h n ) (ty ⁄ ) has options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - been previously described and it was kindly provided by yehia saif, ohio state university. a ⁄ california ⁄ ⁄ (h n ) (ca ⁄ ) was kindly provided by the centers for disease control and prevention (cdc). generation of recombinant viruses by reverse genetics (rg) was done using a previously described method. the genetic signatures for attenuation were introduced into the pb and pb genes of ty ⁄ . ny : ty ⁄ att is a : reassortant with the surface genes from the a ⁄ new york ⁄ ⁄ (h n ) virus and the ty ⁄ att internal genes. all viruses were amplified in mdck cells to produce viral stocks. twenty-five pigs were divided into five groups (n = ) and intranasally inoculated with tcid ⁄ animal of either h n : ty ⁄ att or with ny(h n ) : ty ⁄ att vaccines diluted in ml of mem. two other groups were similarly inoculated with h n : ty ⁄ wt and h n : ty ⁄ rg and served as controls, whereas a fifth group was mockvaccinated with pbs alone. clinical observations were performed as previously described. , efficacy of h n ty ⁄ att vaccine in pigs fourty pigs were divided in four groups (n = )( table ) . group was vaccinated with tcid ⁄ animal of ny(h n ) : ty ⁄ att through intranasal route, whereas group was vaccinated intramuscularly with ml of an adjuvanted uv-inactivated ca ⁄ vaccine (uvadj-ca ⁄ ). group , non-vaccinated and challenged (nv+ca ⁄ ), and group , non-vaccinated, mock-challenged (nv+mock), were also included. pigs were boosted two weeks later. fourteen days post boost (dpb), pigs from groups - were challenged intratracheally with ml of · tcid of ca ⁄ . following challenge, pigs were monitored using methods as previously described. all statistical analyses were performed using graphpad prism software version ae (graphpad software inc., san diego, ca). the differences were considered statistically significant at p < ae . the ty ⁄ att-based vaccines are attenuated in swine pigs inoculated with wt ty ⁄ viruses developed fever (> °c) that peaked hpi ( figure a) and shed large amounts of in nasal secretions ( figure b) . similarly, viral titers in bronchoalveolar lavage fluid (balf) collected at dpi ranged from to tcid ⁄ ml ( figure c ). at necropsy, the lungs from animals inoculated with these viruses had severe pneumonia ( figure d ). in contrast, none of the animals inoculated with h n or h n ty ⁄ att viruses developed clinical signs following vaccination, indicating that the ty ⁄ att viruses were safe for administration to pigs ( figure a) . correspondingly, there was - fold less virus shedding from the nose of pigs vaccinated with ty ⁄ att viruses as compared to unmodified ty ⁄ viruses. in general, ny(h n ) : ty ⁄ att -vaccinated pigs shed less virus than h n : ty ⁄ att inoculated pigs ( figure b ). in addition, viral titers in balf were significantly reduced (p < ae ) in ty ⁄ attvaccinated pigs as compared to ty ⁄ wt-infected pigs ( figure c ). although both vaccines caused mild gross and microscopic lesions in the lungs, the percentage of lung ae ± ae * ± * ± * ± * balf, bronchoalveolar lavage fluid, uvadj-ca ⁄ , uv-inactivated ca ⁄ vaccine; nv+ca ⁄ , non-vaccinated, challenged positive control group; nv+mock, non-vaccinated, non-challenged negative control group. *significantly different from nv+ca ⁄ control group at p < ae . geometric mean hi titer against ca ⁄ at the day of challenge. à percentage of macroscopic lung lesions given as mean score ± sem. § average viral titer (log ) measure as tcid per ml. -average viral titer (log ) in balf at dpc. involvement was not significantly different from mock-vaccinated pigs, corroborating the clinical findings that these vaccines are sufficiently attenuated in pigs ( figure d, e) . histopathologically, nasal turbinates and trachea obtained from pigs immunized with either vaccine were similar to control animals, as opposed to the wt-inoculated pigs ( figure e ). vaccination with h n ty ⁄ att-based vaccines provides sterilizing immunity against h n pdm in pigs the clinical performance in pigs of the h n vaccines is summarized in table . nv+ca ⁄ animals had macroscopic pneumonia, viral replication in balf and shedding in the nose. uvadj-ca ⁄ vaccine provided satisfactory protection, but this protection was not sterilizing. remarkably, animals vaccinated with ny(h n ) : ty ⁄ att had sterilizing immunity. in both vaccine groups there was a significant reduction (p < ae ) in the percentage of macroscopic lung pathology compared to the nv+ca ⁄ group. control pigs had neither significant macroscopic nor microscopic lesions in the lungs. hi antibody titers measured at the day of challenge in both vaccine groups were approximately the same (table ). in the present study, we developed for the first time, temperature-sensitive laiv for use in pigs. data from our safety studies showed that both the h n and h n ty ⁄ att vaccines were attenuated in pigs. although the ty ⁄ att vaccines were detected in balf samples, the level of viral replication was significantly reduced in comparison to unmodified virus and, more importantly, caused no overt clinical signs. a minimal amount of replication is likely beneficial for eliciting t-cell responses to internal genes that may provide heterologous cross-protection. one of the most challenging tasks in producing effective live attenuated vaccines is to achieve an adequate balance between safety and efficacy. by introducing the att modifications into the polymerase genes of a swine-like tr strain, this desirable balance was achieved. the vaccines were histopathologic scores of nasal turbinates, trachea and lungs at dpi. ny(h n ) : ty ⁄ att (a virus that carries the surface genes of a ⁄ new york ⁄ ⁄ (h n ) and ty ⁄ att internal genes). all h n viruses have their surface genes derived from ty ⁄ . values are shown as the mean ± sem. * p < ae ; **p < ae ; *** p < ae . options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - attenuated in pigs and, more importantly, provided sterilizing immunity upon an aggressive challenge with pandemic h n as opposed to an experimental ca ⁄ inactivated vaccine, which elicited protective but not sterilizing immunity in all animals. in the face of influenza pandemics that have the ability to overcome the species barriers such as the h n , the supply of vaccines for use in agriculture could be jeopardized. our cell culture-based live att h n vaccines could be an attractive alternative for this possible pandemic vaccine shortage. because the ty ⁄ att live vaccines developed here are efficacious in swine, are easier to manufacture than inactivated vaccines, and do not require adjuvants, our study represents a major advance in vaccine development for the h n pandemic. in conclusion, our second generation of live att influenza vaccines based on modifications of the pb and pb genes of ty ⁄ retains its safety properties in vivo and can induce excellent protection against aggressive h n challenges in the swine host. influenza virus is one of the most important respiratory pathogens worldwide. , type a influenza causes an acute disease of the upper airways, and affects - million persons yearly. moreover, the threat of human influenza epidemic and pandemic has dramatically increased in recent years. vaccination is one of the crucial interventions for reducing the spread and impact of influenza. the generally used parenteral inactivated influenza vaccines induce mainly systemic antibody responses and only weak cell-mediated immunity and low levels if any mucosal immunity. on the other hand, intranasal immunization with live virus can induces a broad spectrum of both systemic and mucosal antibodies, and the immune response localized in the mucosa blocks the virus even during the first phase of infection. unfortunately, the use of live vaccines is always associated with a certain risk. the development of a crossprotective vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. we induced intrasubtypic and intersubtypic cross-protection in balb ⁄ c mice by intratracheal (it) immunization with inactivated influenza viruses together with dead delipidated bacillus firmus (dbf) as an adjuvant. ten days after the nd immunization dose, the mice were infected with live influenza virus b ⁄ lee ⁄ lethal for mice (total infection dose corresponded to · ld ) or a⁄ pr ⁄ (total infection dose corresponded to ae - ld ). dbf adjuvant markedly increased both systemic and mucosal anti-viral antibody formation when applied together with inactivated influenza a or b viruses. protective significance was tested in vivo. mice were preimmunized with ) pbs (controls), ) dbf alone, ) virus alone, and ) vir-us+dbf. influenza b virus strains b ⁄ lee and b ⁄ yamanashi ⁄ ( years phylogenetically distant and antigenically substantially different, especially in terms of the main protective antigen -surface haemagglutinin) or two different influenza a subtypes -a ⁄ pr ⁄ (h n ) and a ⁄ california ⁄ (h n ) -were used (figures and ) . the mice were challenged with · ld of either b ⁄ lee ⁄ or a ⁄ pr ⁄ as appropriate. all controls died. the mice treated with dbf alone died with a delay or survived, which could be explained by stimulation of innate immunity. the animals immunized with virus alone were protected against homologous strains. adjuvant immunization was cross-protective: the mice immunized with a heterologous b strain (figure ) fell ill (pronounced body mass loss), but almost all survived and recovered. the mice immunized with a heterologous a subtype were excellently protected (negligible weight loss and zero mortality). intratracheal dbf ( lg per mouse) given to non-immunized mice hour before influenza infection eliminated the lethal effect in - % of infected animals depending on infection dose ( ae - ld ); in mice infected with lower than lethal doses ( ae ld ), weight loss was minimized or did not occur. the current mode of vaccination-induced immunity is mostly effective against a homologous strain of the virus used for vaccination. the attention is therefore focused on vaccines that are able to induce cross-protection and could be effective also in case of sudden appearance of a new virus variant. inactivated influenza viruses are known to be often insufficiently effective when used for mucosal immunization and for induction of cross-protection against drifted influenza viruses or novel subtypes. the drawback of vaccination with dead virus can be overcome by using a suitable adjuvant. mouse models were successfully immunized with vaccine containing inactivated virus in combination with cholera toxin or the escheria coli heat-labile toxin (lt). [ ] [ ] [ ] the use of cholera toxin in humans is precluded because of its high toxicity; a number of lt mutants that retain their adjuvant activity have been prepared; these mutants were likewise tested on the mouse model and should not cause any serious side effect in humans. for this reason, current studies aim at finding a suitable and safe mucosal and systemic immune response. dbf has been shown to be a very efficient adjuvant for mucosal immunization stimulating both innate and adaptive immunity. intratracheal immunization with inactivated influenza viruses and dbf as adjuvant induced efficient and even heterosubtypic cross-protection. dbf given hour before infection provided partial protection probably because of its strong stimulatory effect on the innate immunity. temperature-sensitive and cold-adapted candidates for live attenuated influenza vaccine with genomic composition of : based on highly pathogenic influenza a ⁄ h n viruses with pandemic potential were generated by the replacement of six internal genes from the influenza a ⁄ puerto rico ⁄ ⁄ (pr ) virus from pr -based rg-candidates for inactivated vaccine with appropriate internal genes of influenza a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) master donor virus (mdv) for russian laiv by methods of classical reassortment. all attempts to capture avian n neuraminidase into the genome of the mdv laiv production were ineffective. : reassortants were not generated. step by step co-infection of triple reassortants (h n -h n -h n ) with h n mdv in some cases was the only possibility to generate influenza a ⁄ h n cold-adapted vaccine reassortants. difficulties in generating : reassortants could be explained by a substantial gene constellation in the genome of pr based h n reassortant viruses. strong coupling of pb ⁄ pr and avian n genes in a ⁄ h n -pr -rg reassortants was revealed. annually updated laiv strains are generated by classical reassortment of circulating influenza viruses with well characterized, attenuated, ts ⁄ ca mdvs. resulting attenuated reassortants inherit the relevant ha and na of wild type parental virus and six internal genes of the mdv. candidates for inactivated influenza vaccines based upon avian influenza viruses with pandemic potential are generally generated by reverse genetics methods. in these cases, like with laiv, vaccine strains are : reassortants which possess the modified ha and na from potentially pandemic virus and six internal genes from the pr virus. the pr virus is considered to be of low virulence, i.e. attenuated, for humans, yet offers properties of high seed virus growth for influenza vaccine production. the ha of avian h influenza viruses with pandemic potential is engineered to remove four basic amino acid codons from the cleavage site of ha, resulting in a virus that is considered attenuated for natural hosts and safe for people. the objective of this study was to safely generate vaccine candidates for a laiv using highly pathogenic avian influenza viruses by the replacement of six internal pr genes in the genome of candidates for inactivated vaccine subtype h n (a ⁄ h n -pr -rg) with internal genes of the laiv mdv by methods of classical reassortment. len -mdv and a ⁄ h n -pr -rg virus were co-infected in embryonated chicken eggs. five rounds of selective propagation were performed, three of which were at low temperature ( °c). the production and selection of reassortants were carried out in the presence of rabbit antiserum to len -mdv. cloning by endpoint dilution was performed in each of the last three passages. a virus sample in an open petri dish was rocked gently for sec while being irradiated with a ge watt germicidal lamp at a distance of cm from the dish. the residual infection titer was measured by titration in embryonated chicken eggs. genome composition of reassortant viruses was monitored by rflp analysis. in addition, capacity of reassortant viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) for influenza viruses was determined by virus titration in chicken eggs. reassortment of the mdv with the vn-pr or indo-pr viruses either resulted in reassortants that contained six internal genes from len -mdv. however, all generated clones contained the na from the mdv. of ten such : reassortants based on vn-pr three reassortants had the pa gene from pr and one had ns gene from pr . : reassortants from the targeted h n composition were not generated. after repeated attempts, : temperature sensitive and cold adapted reassortants based on vn-pr and indo-pr viruses were obtained, but again, none had inherited the avian n neuraminidase (table ) . in contrast, nibrg- didn't reassort with the mdv at all. twelve unsucsessful attempts to develop : or : reassortants of nibrg- with mdv showed that the classical reassortment procedure (cloning by limited dilutions in the presence of anti-mdv serum, followed by co-infection of equal doses of two parental viruses in eggs and two selective passages at °c) did not work for this virus pair. to disharmonize the incredibly strong gene constellation of nibrg- , various modifications of the co-infection step were studied, such as: altering the nibrg- to mdv ratio (from : to : tions of anti-mdv serum alone or together with anti-pr serum. it was noted that even if the h n to mdv ratio was : , the clones obtained were presumably parental h n viruses without the transfer of any mdv-genes into genome of nibrg- . in all, clones were isolated, and of them were identical to nibrg- parental virus. in nine clones, only the pa gene from mdv was included, whereas in three clones only the 'cold' ns gene was included (data not shown). using uv inactivation of nibrg- prior co-infection was more encouraging. after the first round of co-infection of partially uv-inactivated nibrg- with mdv (at ratio : ), reassortants that inherited several internal genes of mdv were obtained in the context of the nibrg- background (b , c , c , d ) ( table ). some of them (c , c , d ) were chosen for the next round of co-infection. after the second round of co-infection, c , c , and d 'intermediate' reassortants with mdv (at ratio : or : ) : vaccine reassortants finally were obtained. live attenuated influenza vaccine is considered as one of the most promising pandemic vaccines. according to the who there is evidence that laiv might be more effective than inactivated vaccines. this study attempted the safe development of laiv for potential pandemic highly pathogenic avian a ⁄ h n viruses on the base of rg-reassortants for inactivated vaccine with modified h hemagglutinin and mdv for laiv. replacement of pr based internal genes into genome of vn-pr and indo-pr reassortants with appropriate genes of mdv was realized by the classical reassortment procedure. difficulties were encountered in obtaining : reassortants that contained both the ha and na from the wild type avian h n parental virus. in attempts to reassort the nibrg- with mdv, the classical reassortment procedure was unsuccessful. the challenge faced was to break an incredibly strong gene constellation of the nibrg- virus. partial uv-inactivation of nibrg- was encouraged in replacement of some pr internal genes with mdv genes in some cases avian-human reassortant viruses with gull h n and human influenza h n genes were difficult to generate, and reassortants with the desired genotype of six gull virus genes with human influenza a h and n genes were not isolated despite repeated attempts. the gull pb , np, and ns genes were not present in any of the gull-human h n reassortants generated. it is difficult to fully understand potential reasons for observed difficulties to reassort some avian viruses with human strains. unsuccessful attempts to develop : vaccine reassortants may be caused by an observed strong connection of pb and na genes in the genome of a ⁄ h n -pr -rg viruses. in our attempts, each reassortant that possessed avian n neuraminidase inheritied pb gene of pr as well. and vice versa, the 'cold' pb gene always appeared to be coupled with the n neuraminidase of the mdv. in some cases, step by step co-infection of triple reassortants (h n -h n -h n ) with h n mdv may be the only possibility to generate a cold-adapted vaccine reassortant. our studies demonstrate unique and significant challenges that are faced in the development of influenza vaccines for avian influenza viruses with pandemic potential. such challenges must be further studied to identify methodologies to allow for rapid development and response to emerging viruses in a crisis. it is imperative that these studies be continued and expanded to identify either mechanisms of such tight gene constellations in influenza viruses produced by rg-derived vaccine strains or inability some genes of human h n and avian h n viruses to cross. in addition, further studies to improve the efficiency of classical reassortment processes will be conducted. during the period from to , avian influenza outbreaks among humans have been registered in countries of asia, europe, and africa. morbidity and mortality of humans followed the global spread of avian influenza h n among wild and domestic birds, which caused great economic loss to the poultry industry in many regions including some highly developed countries. the global threat from avian influenza forced scientists to develop technologies for the production of a ⁄ h n human vaccine. the development of ai a ⁄ h n vaccines using strains isolated in kazakhstan and the organization of local production and creation of strategic stockpiles of effective vaccines is the an important issue for public health protection in the republic of kazakhstan. to address this, a scientific program 'influenza a ⁄ h n vaccine development for public health protection in kazakhstan' was approved and financed from to . in this article we give basic results of the development of a recombinant ai a ⁄ h n inactivated whole virion vaccine with aluminium hydroxide as adjuvant for public health protection in kazakhstan. [ ] [ ] [ ] the development of vaccine technology was conducted with the use of a ⁄ astanarg ⁄ : ⁄ (a ⁄ h n ) recombinant strain made of a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) and a ⁄ pr ⁄ ⁄ (h n ) strains by the reverse genetics. inactivation of virus containing allantoic fluid was carried out with the use of formalin in different concentrations. complete-ness of the virus inactivation was tested by -fold virus passaging in embryos. , purification and concentration of the inactivated viruscontaining allantoic fluid was conducted with the use of ultra filtration in tangential flow, which was followed by gel filtration. then we evaluated the content of total protein, hemagglutinin, and ovalbumin in purified and concentrated material. vaccine was composed of clarified and inactivated virus concentrate with the known ha dose containment, and ae % aluminum hydroxide was added in : proportions. composition components and quality control of finished vaccine was determined in the stages of semi-finished product and finished biopreparation. determination of quantitative ovalbumin content was conducted by elisa applying a strip test-system chicken egg ovalbumin elisa kit cat. n (alpha diagnostic international, usa). vaccine immunogenicity was evaluated by hai micro test in u-bottom -well plates produced by 'costar' (usa). vaccine apyrogenicity was evaluated after intravenous injection of the studied preparation to rabbits. , for confirmation of the results vaccine series were tested for bacterial endotoxins with the use of limulus amebocyte lysate produced by charles river laboratories, inc. usa. the vaccine toxicity was evaluated in white mice with body weight - gm and in rats with body weight - g both males and females according to glp principles. allergenic characteristics of the inactivated vaccine was determined in white outbred mice and guinea-pigs both males and females according to 'methodic guideline for evaluation of allergenic characteristics of pharmacological substances'. in the first series of experiments, we conducted work for obtaining influenza a(h n ) recombinant strain. bidirectional expression plasmid phw_b with full-length sequences of ha and na gene segments of the strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) isolated in kazakhstan were synthesized in geneart ag, (regensburg, germany). ha gene was modified by deleting the region encoding multiple basic amino acid rrrk motif in ha cleavage site. moreover, to prevent recovery of repeating basic amino acids motif due to polymerase slide, we inserted replacements g fi t and k fi t. thus the ha cleavage site consists of the following sequence ntpqgerrrkkrglfgai ntpqtetrglfgai. the basic amino acid motif of highly pathogenic strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) was replaced by the sequence tetr ⁄ glf, which is characteristic of low pathogenic strains of influenza h n . sequence of gene coding na in the strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) was cloned without modifications. the other segments pb , pb , pa, np, m and ns were obtained from influenza virus ivr- and synthesized and cloned in two-forked expression plasmid phw_b in geneart ag company, germany. the origin of genetic segments of vaccine strain a ⁄ astanarg ⁄ : ⁄ (h n ) is presented in table . vero cell culture ( passage) (who) was received from european cell culture collection (salisbury, wiltshire sp jg, great britain). the cell culture was grown in dmem ⁄ f medium with the addition of % of fetal bovine serum and mm l-glutamine. to obtain reassortant virus a ⁄ astana ⁄ ⁄ r- : , vero cells were infected with correlative plasmids by way of electroporation using nucleofector ii (amaxa) equipment. infected cells were placed in -well plates. after hour, dmem ⁄ f medium was changed into ml of opti-pro sfm (gibco) medium adding mm l-glutamine and lg ⁄ ml trypsin. two days after cytopathic effect appearance supernatant was collected and used for infection of spf-eggs. the virus a ⁄ astanarg ⁄ ⁄ - : was grown in chicken embryos, and then virus titer was determined in chicken embryos and madine-darby canine kidney (mdck) cell culture. the titer of two final a ⁄ astanarg ⁄ - : virus stocks was ae log eid ⁄ ml (chicken embryos); ae log tcid ⁄ ml (mdck cells); ha titer : . a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) virus contains motif of repeating basic amino acids in ha cleavage site. it is known that this sequence is the main determinant of ai virus pathogenicity. that is why this site was deleted in vaccine candidate strain. sequence results confirmed that influenza virus a ⁄ astanarg ⁄ ⁄ r- : strain ha gene sequence contains modified ha cleavage site and keeps mutations inserted for prevention of return to virus wild type. to confirm stability of modified ha gene sequence, five additional passages of recombinant strain a ⁄ astana rg ⁄ ⁄ - : were conducted in chicken embryos. sequencing and following phylogenetic analysis of the recombinant strain a ⁄ astana rg ⁄ ⁄ - : ha gene sequence proved the presence of modification in ha cleavage site. deletion of pathogenicity site of the obtained virus was confirmed by lethality test for chicken embryos, intravenous pathogenicity test in chicken, and in plaque-forming test with trypsin. pathogenicity test in chicken embryos showed that recombinant strain a ⁄ astanarg ⁄ - : is capable of growing up to high titers without causing embryos' death. a ⁄ astanarg ⁄ - : strain pathogenicity evaluation was conducted in - week-age white leghorns chicken, and this study proved that the strain a ⁄ astanarg ⁄ - : (h n ) is not virus pathogenicity inductor in chickens, which got intravenous injections of this virus (pathogenicity index is equal to ). h n strain ha cleavage site modification provides its cleavage capability only with tripsin-like proteases, which shows low level of pathogenicity. aiming at confirmation of ha cleavage site modification, we experimentally studied virus replication ability both with trypsin and without this enzyme. and we got the following results. in the plaque-forming test, a ⁄ astanarg ⁄ - : strain produced plaques in mdck cells only with trypsin, proving the trypsin-dependent phenotype characteristic of low pathogenic avian influenza viruses. to prove the ha subtype antigenic analyses of a ⁄ astana ⁄ ⁄ r- : strain was conducted by means of serological methods in hemagglutinin inghibition test with the use of postinfection antisera of rabbits and rats (influenza research institute swd rams), standard serum received from cdc, atlanta, usa. hai test proved that a ⁄ astana ⁄ ⁄ r- : strain belongs to h subtype. furthermore, toxicity of vaccine candidate strain was evaluated by way of subcutaneous injection of viral material to balb mice. the strain appeared to be non-toxic for white mice getting subcutaneous injection of ae ml of the preparation. the conducted research showed that according to all tested characteristics, a ⁄ astana ⁄ ⁄ r- : strain can be used for influenza a ⁄ h n inactivated vaccine production. according to its genetic characteristics, this strain belongs to the group of vaccine strains recommended by who for the development of influenza pre-pandemic inactivated vaccines. we determined basic cultivation parameters of the recombinant strain a ⁄ astanarg ⁄ - : in - day chicken embryos. the determined parameters are the following: infection dose, - eid ; cultivation period, hour; incubation temperature, °c. these cultivation parameters allow obtaining virus containing material with biological eid and hemagglutinating activity of ae - ae log eid ⁄ cm and : ha titre and even higher. in the next series of experiments, we conducted research on the determination of optimal sequence of technological stages of virus clarification, concentration, and inactivation in the order of vaccine production. samples of viral material were subjected to inactivation before and after clarification and concentration. the regimen of virus inactivation by formaldehyde with final concentration of ae %, period of inactivation of days, temperature of inactivation medium of - °c, ph of inactivation medium of - ae . on the basis of the conducted experiments we determined that the selected regimen of inactivation provides complete and irreversible inactivation of viral suspensions of the hpai strain irrespective of the kind of inactivated material. we did not observe reduction of ha activity in non-clarified viral suspensions. however, when we inactivated clarified and concentrated material, ha activity reduced by an order of magnitude. comparison of forms and sizes of virion structural elements in native (non-clarified) and formalin inactivated preparations did not reveal any significant differences. concentration of virus particles in the studied preparations was similar. the selected inactivation regimen provides obtaining completely avirulent viral suspension of the strain a ⁄ astanarg ⁄ - : , and it does not influence the structure of the virus. on the basis of the experiments results, we selected method of viral allantoic fluid inactivation without preliminary clarification. during further research, we tried to get highly clarified viral concentrate. this study resulted in the combined scheme, which includes clarification of inactivated viral allantoic fluid by low speed centrifugation at circulations per min for minutes, filtration through membrane filters with pore diameter of ae lm, ultrafilatration ⁄ diafiltration, gel filtration in b sepharose, and sterilization of viral suspension through membrane filters with pore diameter of ae lm. the experiments resulted in the development of production technology of embryonic inactivated vaccine based on recombinant strain a ⁄ astanarg ⁄ : ⁄ (h n ) contain-ing aluminium hydroxide as adjuvant. the developed influenza a ⁄ h n human vaccine has the trade name kazfluvacÒ. its composition components are presented in table . preclinical testing of the vaccine kazfluvacÒ was conducted according to the following parameters: general health condition of animals, change of body weight and temperature of immunised animals (for ferrets), presence of post vaccination antibodies response in sera, forming protective immune response against reassortant viruses of h subtype, study of acute and chronic toxicity of three experimental vaccine series in different doses and semi-finished vaccine product applying different ways of injection, study of allergic and immunotoxic characteristics of the vaccine, as well as study of pyrogenic reaction and analysis for bacterial endotoxins presence. [ ] [ ] [ ] [ ] preclinical tests of kazfluvacÒ vaccine safety showed that this vaccine does not have toxic effect on organisms of warm-blooded laboratory animals. double intramuscular injection of kazfluvacÒ vaccine in inoculative dose does not effect appearance, general health condition, behaviour of animals, their muscular strength and physical activity, does not have negative effect on biochemical parameters of blood and basic physical functions of animals organism, and does not cause pathomorphological changes. this shows the safety of the vaccine. local irritation action was not observed. the results of the vaccine allergic action study showed that the vaccine does not have allergic effect at the intravenous injection. the research also showed that the vaccine does not have negative effect on immune system of laboratory animals. research conducted on mice and ferrets showed high immunogenic activity of the vaccine at one-and two-dose regimen of injection. the research showed % of protective effect of kazfluvacÒ vaccine at two-dose injection regimen in ferrets infected by homological strain of influenza virus. the devised inactivated influenza a ⁄ h n vaccine kaz-fluvacÒ is a safe and immunogenic biopreparation that is not worse than the overseas analogues in its immunobiological characteristics. [ ] [ ] [ ] [ ] to date the whole-virion inactivated influenza a ⁄ h n vaccines of the producers such as omnivest (hungary), biken, denka seiken, kitasato institute, kaketsuken (japan), gsk biologicals (belgium), sinovac biotech (china) are registered. all of them are produced on the basis of chicken embryos and aluminum is used as an adjuvant. kazfluvacÒ differs from its analogues in the flowchart of the virus purification and concentration that makes possible to produce a safer preparation. , the results of the conducted research and preclinical testing allow starting work towards implementation of phase i preclinical tests on volunteers. it is planned to conduct a randomized blind placebo-controlled phase i study on double application of kazfluvacÒ vaccine in increasing doses. the preparation will be administered to volunteers aged - years for assessment of its safety and immunogenicity in doses of ae and ae lg of ha. when the world health organization (who) announced the sixth phase of a ⁄ h n v influenza pandemic, scientists all over the world started investigation to develop technology for production of prophylactic means against the disease. having taken into consideration the threat of a pandemic for kazakhstan, the ministry of education and science of the republic of kazakhstan launched the program ''monitoring, study, and development of diagnostic, prophylactic, and therapeutic means for influenza a ⁄ h n .'' this paper presents the experimental data obtained at the ribsp in the course of the studies towards the development of technology for production of an inactivated a ⁄ h n influenza vaccine, as well as the results of pre-clinical testing of the developed vaccine. the development of vaccine production technology was conducted with the use of who recommended vaccine strain nibrg- xp constructed by the method of reverse genetics in the national institute for biological standards and control (nibsc, great britain). the virus was inactivated with formalin at different final concentrations, and the extent of inactivation was evaluated via threefold virus passages in developing chicken embryos. the inactivated virus was purified and concentrated by the method of ultrafiltration in tangential flow followed by gel filtration. the purified and concentrated material was evaluated judging on the total protein, hemagglutinin (ha), and ovalbumin. the vaccine was prepared by pooling the purified and concentrated virus material with the certain weight content of ha and the work solution of aluminum hydroxide ( ae %) in the ratio : . the ovalbumin content was quantified in elisa with the use of the strip test system chicken egg ovalbumin elisa kit (cat. no. alpha diagnostic international, san antonio, texas, usa). weight content of the virus ha was determined according to sominina, burtseva. the content of the residual formaldehyde, aluminum (al + ) ions, and thiomersal in the vaccine was measured according to the operating instructions. the vaccine immunogenicity was assessed in the hemagglutination inhibition test, which was carried out as a microassay in -welled u-bottomed plates (''costar'', new york, usa). , apyrogenicity of the vaccine was assessed post intravenous administration of the tested preparation to rabbits. , to confirm the obtained results the vaccine batches were tested for bacterial endotoxins with use of the limulus amebocyte lysate (charles river laboratories, inc., wilmington, ma, usa). the toxicity of the vaccine was assayed in white mice weighing - g and in rats weighing - g (male and female) in compliance with the principles of good laboratory practice. allergenic properties of the inactivated vaccine were determined according to the ''operating instructions on assessment of allergenic properties of pharmaceutical substances'' in white outbred laboratory mice and guinea-pigs of both sexes. the first step in the course of developing technology for vaccine production was to determine the major conditions for influenza virus cultivation: usage of -days embryonated chicken eggs at the infectious dose within - eid , incubation temperature ( ± ae )°c, and duration of the incubation period hours. the established parameters for virus cultivation made it possible to produce virus-containing materials of infectious activity within ae - ae log eid ⁄ cm and hemagglutinating activity : and higher. in the subsequent experiments, an optimal method for virus inactivation was selected. on the basis of the experimental findings, the following conditions for inactivation of the native virus-containing material were elected: formalin of ae % final concentration as an inactivating agent; inactivation period of hours at temperature ( ± )°c. these conditions provide the complete inactivation of the virus (nibrg- xp strain) material, did not impact distinctly the structural organization of the virus, and did not reduce the antigenic activity. as it is well known, virus purification and concentration means very much in the development of technology for production of an inactivated whole-virion influenza vaccine. the investigation into optimization of the technological step of purification and concentration of the recombinant influenza virus nibrg- xp strain resulted in selection of an optimal pattern including such steps as clarification of the virus suspension by filtration through membranes with pore size ae lm, virus concentration by ultrafiltration in a tangential flow, dialysis filtration in a tangential flow, gel filtration on sepharose b, and sterilization of the viral suspension through membrane filters with pore size ae lm. the studies conducted by the ribsp specialists resulted in the development of technology for production of the first domestic whole-virion inactivated a ⁄ h n influenza vaccine with aluminum hydroxide as adjuvant and with the brand name refluvac Ò . the key processing characteristics of the whole-virion inactivated a ⁄ h n influenza vaccine vaccine refluvac Ò are shown in table . simultaneous with the performance of all process operations, the parameters such as sterility, inactivation extent, ph, vaccine specificity, total protein content, weight content of has, aluminum and formalin contents, content of thiomersal, and ovalbumin, pyrogenicity of the vaccine and its immunogenicity for mice, were optimized. the key qualitative characteristics of the designed influenza a ⁄ h n vaccine refluvac Ò are shown in table . before implementation of phase i clinical trials on volunteers, preclinical testing of three experimental batches of refluvac for immunogenic activity and safety was carried out. it was conducted in three laboratory bases of research institutions: the toxicology institute ⁄ federal medicobiological agency, russia (st petersburg), the research institute for biological safety problems (republic of kazakhstan), and the influenza research institute ⁄ north-western branch of the russian academy of medical sciences (st petersburg), with use of different animal models (mice, rats, chinchilla rabbits, guinea-pigs, ferrets). the results of the preclinical testing are as follows: • electron microscopy of the preparation has shown that the viral particles are well dispersed and do not aggregate. the portion of whole (intact) particles is over %, which is evidence of virion integrity; • assessment of polypeptide composition of the vaccine refluvac by electrophoresis in % polyacrylamide gel with sodium dodecyl sulfate has shown the vaccine to contain both surface antigens (ha, na) and highly purified inner virion proteins (np, m ) that are typespecific antigens, so the vaccine is a preparation of full immunological value; • judging on the parameters of acute and chronic toxicity for white mice and rats of both sexes, the vaccine is a non-toxic and safe preparation; • under conditions of a chronic experiment on white mice and rats, it was found that refluvac does not produce changes in behavior, somatic, or vegetative responses; • assay of hematological and biochemical blood characteristics of white mice and rats following vaccine administration did not reveal any significant differences as compared to the animals of the control group; • refluvac does not cause allergenic and immunotoxic impact; • the vaccine refluvac does not cause local irritative effect; • refluvac is apyrogenic for laboratory animals; • the pathomorphological and hystopathological analysis did not reveal any changes due to immunization in animal organs; • testing of immunogenic characteristics of the vaccine on mice and ferrets has shown formation of hemagglutinating antibodies in animals after single administration; • refluvac induces % protection in immunized ferrets at their challenge with the wild-type influenza virus a ⁄ california ⁄ ⁄ (h n v). the results of the performed preclinical testing have allowed concluding that refluvac, an inactivated whole-virion vaccine with aluminum hydroxide as adjuvant, is a safe and highly effective preparation against influenza a ⁄ h n v. the implemented study resulted in development of technology for production of the first domestic inactivated allantoic whole-virion influenza a ⁄ h n vaccine with aluminum hydroxide as an adjuvant under the brand name refluvac Ò based on the recombinant strain nibrg- xp. the devised pandemic vaccine meets who requirements as well as requirements concerning safety and immunogenicity of the national pharmacopeias of the republic of kazakhstan and russian federation. [ ] [ ] [ ] [ ] the devised technology for vaccine production differs from the previous technologies for production of allantoic whole-virion influenza a ⁄ h n vaccines in its processdependent parameters. presence of an adjuvant (aluminum hydroxide) increases significantly the vaccine immunogenicity and allows maximal reduction of the dose of the administered antigen that, in turn, results in diminished reactogenicity of the vaccine. aluminum hydroxide is an adjuvant that is most frequently used in clinical practice. to date the results of the double-centered randomized study of the europe-licensed vaccine fluval p [monovalent inactivated whole-virion influenza vaccine with aluminum phosphate based on strain a ⁄ california ⁄ ⁄ (h n ) nymc x- a (omninvest, pilisborosjeno, hungary)] that is similar to the refluvac preparation are published. the data of this research are an evidence of safety and high immunological effectiveness of the vaccine in dose lg ha at single administration both in adults and elderly persons. the results of the pre-clinical tests allow recommending carrying out phase clinical testing of the refluvac Ò vaccine for safety and immunogenicity. single immunization of volunteers with refluvac Ò in doses ae , ae , and ae lg of ha are planned. mid , respectively. the study results confirm that new h n laiv and h n laiv candidates are safe and immunogenic and confer protection from homologues influenza virus infection in mice. the recent emergence of a new pandemic h n virus and the threat of transmission of avian viruses to humans had stimulated research and development of live attenuated cold-adapted influenza vaccines against newly appeared influenza viruses. formulations of live attenuated influenza a vaccine (laiv) against pandemic influenza strains, including h n , h n , h n , and h n are currently being tested in preclinical and phase i clinical studies. the following paper describes the preclinical study of new h n and h n laiv candidates in mice. the study addressed the following three objectives: (i) to demonstrate that cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates are indistinguishable from the parental a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) master donor strain (mds) virus with regard to replication efficiency in upper and lower respiratory tract of mice; (ii) to demonstrate the immunogenicity of different doses of cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates in mice; and (iii) to demonstrate the protective efficacy of cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates in mice against a homologous wild-type virus challenge. the a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) reassortant containing the ha and na genes from a ⁄ mallard ⁄ netherlands ⁄ (h n ) and six other genes from mds, the a ⁄ ⁄ california ⁄ ⁄ (h n ) reassortant containing the ha and na genes from a ⁄ california ⁄ ⁄ (h n ) and six other genes from a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) were generated by classical genetic reassortment in embryonated chicken eggs (ec). viruses were propagated in days old eggs ( °c, hours). fifty percent egg infectious dose (eid ) titers were determined by serial titration of viruses in eggs. titers were calculated by the method of reed and muench. female balb ⁄ c mice, - weeks of age were used in all experiments. mice were lightly anesthetized with ether and then inoculated intranasally (i.n.) with ll of infectious virus diluted in phosphate-buffered saline (pbs). mice were inoculated with mid ( % mouse infectious dose) of a ⁄ ⁄ california ⁄ ⁄ (h n ), a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ), and a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) mds. viral loads were measured in respiratory and brain tissues collected at and days post-infection (dpi). tissue homogenates prepared using a disruptor and clarified supernatants were titrated on eggs at permissive temperature to determine infectious concentrations. groups of animals were inoculated with mid or mid of either h n laiv or h n laiv intranasally after collecting a pre-immunization blood sample. a second blood sample was collected at dpi. on the same day, the animals received a second intranasal inoculation with the same virus that was used for priming at dpi. to assess protection, all animals were infected dpi with either mid of a ⁄ california ⁄ ⁄ (h n ) or mid a ⁄ mallard ⁄ netherlands ⁄ (h n ) virus by the intranasal route. four animals from each group were euthanized at dpi, and the respiratory and systemic organs were harvested for virus titration. a forth blood sample was collected at dpi from the remaining animals. hi antibody titers were determined for individual serum samples collected on days , , , and . body weights were taken daily following challenge through day postchallenge. sera were tested for hi against homologous h n and h n viruses. the h n laiv, h n laiv and h n mds influenza viruses replicate in mice lungs at level ae - ae lgeid ⁄ ml at dpi (figure ). at dpi, replication of the viruses in the lungs decreased to ae - ae lgeid ⁄ ml (data not shown). in contrast, the wild-type virus a ⁄ mallard ⁄ netherlands ⁄ (h n ) demonstrated high level replication in lungs - ae lgeid ⁄ ml. the levels of replication of studied viruses in nasal turbinates were ae - ae lg eid ⁄ ml at dpi (figure ) , and ae - ae lgeid ⁄ ml at dpi (data not shown). there were no significant differences between the viruses in regard to replication in upper respiratory tract of mice. thus, it was shown that a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) vaccine candidates was indistinguishable from parental a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) in terms of replication in the lungs and noses of mice at and dpi. no virus was found in the brain tissue of immunized mice at and dpi (in undiluted samples tested). thus, it was shown that a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ), a ⁄ ⁄ cali-fornia ⁄ ⁄ (h n ) vaccine candidates are identical to a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) in lacking neuroivasive capacity, and all three viruses similarly fail to replicate in the brain. it was shown that all immunized animals survived after challenge with wild-type a ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) virus. the mice in vaccine groups showed no signs of morbidity. average weight changes were tracked from day to day in all study groups, but the changes did not exceed %. as shown in figure , the challenge virus actively replicated in respiratory tissue taken from mock immunized animals ( ae lgeid in the lung and ae lgeid in the nose), but failed to infect the brain and spleen. on the other hand, in both h n laiv vaccinated groups, all tested organs were free from presence of challenge virus. thus, immunization of mice with either mid or mid h n laiv protected the animals from the subsequent challenge infection with a homologous with wild-type h n virus. both h n and h n laiv candidates were found to be immunogenic. after one dose of mid of h n laiv, gmt of hi antibodies were ae . one dose of mid or mid h n laiv elicited hi antibody level with gmt of ae and ae , respectively. the second dose of h n laiv further stimulated serum hi antibody levels to gmt ae and ae , for mid or mid , respectively (data not shown). the mouse model is widely used to better understand the pathogenicity of avian influenza viruses for mammalian species, to be able to predict the pandemic potential of such viruses, and to develop improved methods for the prevention and control of the virus in a potential pandemic. a subset of the h viruses was evaluated for the ability to replicate and cause disease in balb ⁄ c mice following intranasal administration. h subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. there is limited preclinical information available for laiv. thus, live monovalent vaccine against pandemic influenza virus h n (influvir) was tested for acute toxicity and its effect on the systems and organs of laboratory animals. according to toxicology and necroscopy results, the live monovalent influenza vaccine influvir, when applied intranasally, was safe and was well tolerated. in our current study we demonstrate that a(h n ) and a(h n ) laiv are indistinguishable from the parental mds virus with regards to replication kinetics in the upper and lower respiratory tract of mice. both h n and h n laiv candidates were immunogenic and protect mice against subsequent a challenge with the wild-type virus. live attenuated cold-adapted (ca) influenza vaccines are an effective means for the control of influenza, most likely due to their ability to induce both humoral and cellular immune responses. in our study we confirm that new h n laiv and h n laiv candidates are safe, immunogenic, and confer protection from influenza infection in mice. health organization (who) declared a pandemic by raising the worldwide pandemic alert level to phase . therefore, h n inactivated monovalent vaccine formulated with our proprietary oil-in-water emulsion based adjuvant was evaluated in ferrets for its potential to induce with low antigen dose efficient, robust, and rapid protective immunity against a wild type challenge virus (a ⁄ netherlands ⁄ ⁄ ). this adjuvant was also tested in ferrets in a h n avian influenza model for its ability to induce a cross-clade immunity and cross-protection. two independent studies (a&b) were carried out with male and female outbred ferrets (musleta putorius furo) in compliance with ''guide for the care and use of laboratory animals,'' ilar recommendations and aaalac standards. ferrets used in both studies were influenza seronegative by anti-nucleoprotein elisa and by hi assay against the pandemic and seasonal strains. in study a, four groups of seven ferrets aged approximately of months received one or two im vaccinations weeks apart of either af -adjuvanted ( ae lg of ha with af ) or unadjuvanted ( body weight loss was monitored as an indicator of disease and a mean body weight loss of % was recorded in the control group at day of necropsy. body weight loss was reduced to £ % and £ % in animals that had received and doses of either unadjuvanted or af -adjuvanted vaccine, respectively. viral lung titration showed high levels of virus replication ( ‡ ae tcid ⁄ g tissue) in the lungs of all control ferrets days after challenge. one or two administrations of unadjuvanted vaccine reduced lung viral load by and log , respectively. interestingly, ferrets that received either one or two doses of af -adjuvanted h n vaccine, showed significantly greater reduction of lung viral loads (> log ). no virus was detected in the lungs of ⁄ ( %) animals immunized with a single injection of the af -adjuvanted vaccine and in % of ferrets vaccinated twice. assessment of viral shedding from the upper respiratory tract showed that the af -adjuvanted a ⁄ h n monovalent vaccine was able to reduce the viral load in the nose and in the throat by ae and ae log , respectively, as compared to the control group. conversely, viral loads were only slightly reduced in the nose and mostly unchanged in the throat in ferrets immunized with either one or two doses of unadjuvanted a ⁄ h n monovalent vaccine. gross pathology and histology examinations revealed lung lesions consistent with influenza a ⁄ h n virus infec- however, a second dose of af -adjuvanted vaccine strongly increased hi and mn titers, which persisted for months (table ). antibody responses cross-reactive to heterologous clade . strain were elicited ferrets vaccinated with the af -adjuvanted clade . vaccine. hi antibody titers ‡ crossreactive to clade . and persistent up to d were observed in vaccinated animals. an inter-clade low crossreactive hi response to a clade strain was only detected in a few ferrets that had been vaccinated with the af -adjuvanted clade . . all af -adjuvanted clade . antigen vaccinated animals survived challenge either with the homologous or heterologous virus until euthanized day . after challenge, mean body temperature and mean body weights were monitored as indicators of disease. in the control ferrets, mean body temperature increased by - °c (depending on the challenge virus strain) h post challenge, with an accompanying mean body weight loss ranging from ae % to ae %. ferrets vaccinated with the af -adjuvanted clade . vaccine showed a lower and delayed fever compared to control ferrets that received the same viral challenge, whereas no significant differences were observed between vaccinated animals and their respective controls upon challenge with clade . or clade viruses. body weight loss was reduced in all vaccinated animals when compared to controls after challenge with either the homologous clade . strain or with one of the heterologous strains. lung virus titration showed high levels of virus replication in all control animals days after homologous challenge with the clade . virus. lung viral loads of all ferrets immunized with the af -adjuvanted clade . vaccine were reduced more than log . vaccination resulted in complete viral clearance from the lungs of % of animals assessed days after challenge. as compared to controls, a reduction of the mean viral load of about log was observed in ferrets vaccinated with the af -adjuvanted clade . vaccine after heterologous challenge with either the clade or clade virus. conversely, vaccination with af -adjuvanted clade . vaccine did not result in reduction of lung viral loads after challenge with the clade . heterologous virus strain. titration of pharyngeal swabs showed high levels of viral shedding in all control ferrets after challenge with clade . strain, whereas virus was not detected in any vaccinated animal. similarly, log reduction of viral shedding was seen in vaccinated versus control ferrets following clade heterologous challenge. lower reductions in viral shedding were observed after clade . challenge ( ae log ) and clade challenge ( ae log ). gross pathology and histology revealed lung lesions consistent with influenza a ⁄ h n virus infection all control animals challenged with the clade . , clade . or clade strains. mild to moderate lung lesions were observed in control animals following challenge with clade virus. macroscopic evaluation (percentage of affected lung parenchyma) and histopathological analysis (extent and severity of alveolitis, alveolar oedema and hemorrhage) showed that lung lesions were significantly reduced in af -adjuvanted clade . vaccinated animals after challenge with the homologous clade . virus strain as compared to controls. similarly, a reduction of the macroscopic and microscopic lung lesions was observed in vaccinated animals upon heterologous challenge with clade . and clade virus strains, whereas no differences were observed between control and vaccinated animals after challenge with clade virus. the results of these ferret challenge studies demonstrated that low doses of pandemic influenza vaccines formulated with an oil-in-water emulsion adjuvant, af , elicited strong antibody responses specific to the immunizing strain. importantly, these vaccines provided protection after homologous challenge with complete virus clearance in ferret lungs and reduced viral shedding from the upper respiratory tract suggesting an ability to reduce virus transmission. moreover, af -adjuvanted h n vaccine can provide cross-protection upon challenge with different h n clades by preventing mortality and reducing the viral burden in the lower and the upper respiratory tract. in conclusion, the results of these studies highlighted the ability of af -adjuvanted influenza vaccines to induce potent immune responses and full protection in ferrets against homologous challenge and suggested that protection may be mediated, at least in part, by antigenspecific humoral immunity. since , outbreaks of h n influenza virus infection in poultry have occurred in eurasian countries. phylogenetic and antigenic analysis of h n isolates revealed that there are three sublineages, consisting of g , g , and korean, among ha genes of the eurasian h n viruses. h n viruses do not cause severe disease in poultry, but co-infection of h n viruses with bacteria such as staphylococcus aureus, haemophilus paragallinarum, or attenuated coronavirus vaccine may exacerbate the disease. , h n viruses were isolated from domestic pigs in china and korea and from humans with febrile respiratory illness in hong kong in kong in , kong in , and it is, thus, postulated that in the present study, h virus strains were analyzed antigenically and phylogenetically to select a proper h n vaccine strain. inactivated whole virus particle vaccine was prepared, and its potency against h virus challenge was assessed in mice. viral rnas were extracted from the allantoic fluid of chicken embryos infected with viruses by using a commercial kit (trizol ls reagent; invitrogen, california, usa) and reverse-transcribed with the uni primer and m-mlv reverse transcriptase (invitrogen). the primers used for the ha gene amplification were h - f and h - r. for phylogenetic analysis, sequence data of the genes together with those from public database were analyzed by the neighbor-joining method. h influenza viruses were analyzed by hemagglutinationinhibition (hi) test. chicken hyperimmunized antisera against seven h viruses were prepared according to previous report. virus replication and pathogenicity against embryonated chicken eggs viruses were inoculated into -day-old embryonated chicken eggs and incubated for hours at °c. ha titers and % egg infectious dose (eid ) were measured every hours post-inoculation. pathogenicity of dk ⁄ hok ⁄ ⁄ against embryonated chicken eggs was evaluated by mean death time (mdt) as described previously. dk ⁄ hok ⁄ ⁄ was injected into the allantoic cavities of -day-old embryonated chicken eggs and propagated at °c for hours. the virus in the allantoic fluids ( ha) was purified by differential centrifugation and sedimentation through a sucrose gradient according to previous report. the concentration of protein was measured by od using ultrospec pro (amersham biosciences, tokyo, japan). the purified virus was inactivated with ae % formalin at °c for days. immunization of mice and challenge of immunized mice with hk ⁄ ⁄ four-week-old female balb ⁄ c mice were purchased from japan slc, inc. (shizuoka, japan). the mice were injected subcutaneously with , , ae , or ae lg proteins of inactivated dk ⁄ hok ⁄ ⁄ whole virus vaccine. two weeks later, the mice were boosted by subcutaneous injection with the same dose of the vaccine. control mice were injected with pbs. serum samples were tested by enzyme-linked immunosorbent assay (elisa) according to previous report. one week after the second vaccination, mice in each group were challenged intranasally with ll of ae eid of hk ⁄ ⁄ under anesthesia. on days postinfection, five mice in each group were sacrificed, and the lungs were separately homogenized to make a % (w ⁄ v) suspension with minimal essential medium (nissui, tokyo, japan). the virus titers of the supernatants of lung tissue homogenates were calculated in -day-old embryonated chicken eggs and expressed as the eid ⁄ gram of tissue. the other five mice in each group were monitored for body weight for days after challenge. the ha genes of h viruses were sequenced and analyzed by the neighbor-joining method. all of the h viruses were classified into the eurasian lineage ( figure ) . eleven, seven, and four strains were classified in the korean, g , and g sublineages, respectively. the h viruses of the korean and g sublineages were isolated from waterfowl, poultry, pigs, and humans in the east asian countries, and those of the g sublineage were isolated from poultry in the west asian countries. the cross-reactivity between these antisera and h n viruses were analyzed by hi test. the antisera against h viruses belonging to the korean sublineage were broadly cross-reacted to h viruses belonging to the g and g sublineages. h viruses belonging to the korean lineage were reacted to the antisera against h viruses belonging to the g and g sublineage compared with h viruses belonging to the other sublineage (data not shown). thus, it was suggested that h vaccine strain should be selected from the viruses of korean sublineage to prepare for the vaccine strain of h viruses. dk ⁄ hok ⁄ ⁄ replicated efficiently in -day-old embryonated chicken eggs (data not shown). pathogenicity of dk ⁄ hok ⁄ ⁄ against embryonated chicken eggs was determined by mdt. dk ⁄ hok ⁄ ⁄ was low pathogenic against embryonated chicken eggs (data not shown) and was selected as an h vaccine strain. to assess the potency of the vaccine against h virus infection, mice vaccinated subcutaneously with inactivated dk ⁄ hok ⁄ ⁄ were challenged intra-nasally with hk ⁄ ⁄ . immunogenicity of the inactivated vaccine was assessed by measuring the igg antibodies in mouse sera by elisa. antibody was detected in the group of mice injected lg protein after the first immunization and detected in the group of mice injected lg protein after the second immunization. thus, potency of the present inactivated whole virus vaccine was demonstrated in mice. next, to assess the protective immunity of the inactivated vaccine in mice, viral titers in the lungs was determined. the virus titers in the lungs were ae - ae eid ⁄ g in the groups of mice injected , and lg protein, and ae - ae eid ⁄ g in the other vaccinated groups. body weight reduction of mice were observed in the group of mice injected ae , ae lg protein, and control groups from dpi, and reached to % body weight loss from -to -day post-infection ( figure ). this result correlates with antibody titer in mouse sera and viral titers in the lungs. these results suggest that the test h inactivated whole vaccine confers prevent of weight loss and reduction of virus replication against h influenza virus infection in mice. recently, h n viruses of all of three sublineage have been isolated from wild birds and poultry in worldwide. h n viruses were isolated from pigs and humans in china and korea, suggesting that h n virus would be a potential for a pandemic influenza virus in human population. h n viruses were isolated from pigs in china and korea and were classified into the g and korean sublineage. in human cases, all h n virus isolated from humans in china was classified into the g sublineage. it was suggested that h n viruses isolated from pigs and humans vary in antigenicity of isolates between the korean, g , and g sublineages. therefore, it is important for the preparedness of influenza pandemic to develop h influenza virus vaccine, which could broadly cross-react to antisera of all sublineage viruses. so, we selected the vaccine candidate strain, dk ⁄ hok ⁄ ⁄ , which could broadly cross-react to antisera of all sublineage viruses, and which could replicate in this study, it was suggested that the test vaccine has potency to protect against challenge with h virus using mice for mammalian model. the challenge virus, hk ⁄ ⁄ , was isolated from human, replicates efficiently in mice, and shows pathogenicity in mice. the test vaccine inhibited viral replication and body weight loss in mice. whole inactivated vaccine produced protective immunity, supporting our approach of using whole virus particles for vaccine development. furthermore, whole particle virus vaccine could induce igg and mucosal iga levels after intranasal vaccination with whole particle vaccine. the present results may facilitate the studies of the vaccine for future pandemic caused by h influenza virus in humans. tants to attempt to improve growth. to determine whether wild type h n pdm grew better in the novartis mdck suspension cell line (mdck pf) than in eggs, isolations from h n pdm positive clinical samples were attempted in both substrates. the isolation rate of h n pdm viruses was higher in mdck pf cells ( %) ( ⁄ ) compared to allantoically inoculated eggs ( %) ( ⁄ ) . however the yields were lower than observed with seasonal viruses. little improvement in virus yield was seen with extra passaging or dilutions of h n pdm viruses isolated in mdck pf cells. with the emergence of the swine-origin pandemic h n (h n pdm) influenza in april , the need for efficient production of a suitable vaccine was a high priority. virus isolates were distributed by the who for the urgent development of suitable vaccine strains early in the pandemic. vaccine viruses can be grown in embryonated chicken eggs or in certified mammalian cells. , unfortunately wildtype h n pdm virus strains distributed by the who grew poorly in cell lines and eggs, requiring the generation of a series of conventional and reverse genetics derived reassortants to attempt to improve growth. from these reassortants, only the conventional egg derived reassortants nymc-x- a and nymc-x- (both based on one of the earliest known viruses a ⁄ california ⁄ ⁄ ) showed high enough growth and yield in eggs and cell culture to make them suitable for vaccine manufacture. these reassortants, while acceptable, still only gave haemagglutinin (ha) yields of approximately % that of seasonal h n reassortants. to determine if more recent wild type h n pdm viruses grew better in the novartis mdck suspension cell line (mdck pf), h n pdm positive clinical samples were cultured in mdck pf cells and also in embryonated hen's eggs. in addition, to improve virus yields from mdck pf isolates, extended passaging of three wild type h n pdm influenza viruses was performed using various virus dilutions at each passage level. the results were assessed using various serological and molecular biology techniques and compared to viruses isolated in eggs and conventional mdck cells. h n pdm viruses were received at the centre from who national influenza centres, who influenza collaborating centres and other regional laboratories and hospitals in australia, new zealand, and the asia ⁄ pacific region. viruses were received as original clinical specimens consisting of nasal swabs, throat swabs, nasopharyngeal aspirates, or nasal washes that had previously been shown to be h n pdm positive by real time rt-pcr. these specimens were then cultured in mdck pf cells with serum free medium containing trypzean (optaflu) and also independently inoculated into the allantoic cavity of day-old embryonated hen's eggs. virus cultures in mdck pf cells were sampled at and hour and evaluated by various means including ha titres. at hour, virus cultures were further passaged at varying dilutions ranging from ) to ) up to a total of passages. embryonated hen's eggs were incubated at °c for days and allantoic fluid was harvested and ha titres performed to determine whether a further passage was required in order to improve growth. the conventional reassortants were produced by a mixed infection of eggs or mdck pf cells with the wild type virus and a donor virus carrying the internal genes of the a ⁄ puerto rico ⁄ ⁄ virus. the reassortants were obtained by sequential passages using immuno-selective antisera against the surface antigen of the donor virus to remove virus populations carrying the ha and na protein of the donor strain. the reverse genetics viruses were rescued in vero cells using the plasmid system. both types of reassortants were generated and supplied by who collaborating centres and essential regulatory laboratories except the nvd-c- strain, which was produced by novartis. in this small study with recent h n pdm viruses, the isolation rate was higher in mdck pf cells ( %) ( ⁄ ) compared to allantoically inoculated eggs ( %) ( ⁄ ) . assessment of ha titres, however, showed higher ha titres in egg-isolated viruses compared to viruses isolated in mdck pf cells after two passages. egg generated or cell generated reassortant viruses gave higher ha titres compared to the homologous wild type viruses (table ) . no amino acid changes were observed in mdck pf isolated influenza viruses compared to original specimens or viruses isolated in conventional atcc derived mdck cells, unlike egg isolated viruses which showed a number of amino acid changes, many consistent with egg adaptation mutations (table ) . viruses isolated in mdck pf cells grouped phylogenetically with viruses isolated in conventional atcc derived mdck cells or viruses sequenced from original clinical samples, while egg isolated viruses grouped slightly differently (data not shown). as a result of the poor growth of h n pdm viruses in mdck pf cells, serial dilutions were performed over a number of passages ( figure ). based on the results obtained from the virus isolates, a ⁄ victoria ⁄ ⁄ , a ⁄ wellington ⁄ ⁄ , and a ⁄ darwin ⁄ ⁄ , a supplemental protocol was developed and used in the isolation of a ⁄ brisbane ⁄ ⁄ (figure ). only small differences in ha titer were seen between different dilutions, and copy number showed a similar trend to ha titer at each passage ( figure ). following the supplemental protocol for the isolation of a ⁄ brisbane ⁄ ⁄ results showed slightly higher ha titres with little variation between passages. the egg derived reassortants nymc-x- a and nymc-x- were also assessed for growth in mdck pf cells and were found to be superior by ha titer to other conventional reassortants (egg or cell derived), reverse genetics derived reassortants, or wild type viruses (table ) . two methods were used to determine the ratio of ha to other viral proteins: densiometric analysis using sds-page and reversed-phase hplc using a subtype specific standard. ha content in different vaccine seeds of influenza a subtypes demonstrated that the ha content per total virus protein from the nymc h n pdm reassortants was significantly different to the seasonal influenza a subtypes. for the seasonal h n the ratio of ha to p p p p p p p p p p p p p p p p n and m was ‡ %, for the h n the ratio of ha to n and m was £ %, while for the pandemic a ⁄ h n , the ratio of ha to n and m was much lower at £ % (data not shown). the results of this study has observed the growth of a series of - h n pdm viruses in vaccine suitable mdck pf cells to be generally lower than what has been seen with other seasonal influenza viruses. little improvement in virus yield was seen with extra passaging of h n pdm viruses isolated and passaged in mdck pf cells. passaging up to times in mdck pf cells using dilutions ranging from ) to ) resulted in supernatants with viral ha titres ranging from ha ⁄ ll to ha ⁄ ll. the isolation rate of h n pdm viruses was higher in mdck pf cells ( %) compared to allantoically inoculated (and passaged) eggs ( %), a trend also seen in previous work with seasonal influenza viruses. in contrast a study by hussain and colleagues found similar rates of isolation and replication of seasonal influenza viruses in mdck cells and eggs. the virus load as determined by matrix gene copy number showed a similar trend to ha titers. two of the isolates exhibited small rises and falls in ha titer during passaging, while a third, a ⁄ victoria ⁄ ⁄ gave consistently higher titers. interestingly this virus was unable to be isolated in eggs. the ha sequences of all strains were assessed at p , p , p , p , p and when available compared to the original clinical sample ha sequence. mdck pf-isolated viruses had few if any changes in their ha amino acid sequence, while the majority of egg isolates showed - amino acid changes compared to the clinical sample, with an egg adaption change (l i) evident in a number of them. the ha sequence of one of the better growing viruses, a ⁄ victoria ⁄ ⁄ , was found to have a g e change compared to the a ⁄ california ⁄ ⁄ reference virus. this change was also seen in the virus isolated in conventional, adherent mdck cells. these viruses with g e change when tested by hai have shown reduced reactivity with ferret antisera to a ⁄ california ⁄ ⁄ -like viruses, but normal reactivity with ferret antisera to h n pdm a ⁄ bayern ⁄ ⁄ -like viruses. despite this mutation all mdck pf derived viruses appeared to be a ⁄ california ⁄ ⁄ -like by hai. the h n pdm egg-derived reassortants (nymc x- a and nymc x- ) when grown in mdck pf cells were superior to wild type h n pdm viruses, reverse genetics derived reassortants, and other egg-derived reassortants. the yields of haemagglutinin from the nymc h n pdm reassortants were still below those seen with sea-sonal h n reassortants as was also seen in eggs. this trend has also been noted in other studies. in summary, attempts to improve growth and yield of the h n pdm wild types for mdck pf cells by extended passaging were not successful, and reassortants did not perform as well as seasonal h n reassortants have in the past. however, using higher dilutions for the passaging of h n pdm viruses in mdck pf cells did result in higher ha titres (a ⁄ brisbane ⁄ ⁄ ). further work is therefore required to generate pandemic h n seed viruses that grow well in a variety of cell culture and egg based vaccine production systems. the aim of this study is to evaluate antibody response to influenza virus neuraminidase (na) following immunization with live attenuated influenza vaccine (laiv). we adjusted the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. ( ) to assay neuraminidase inhibition (ni) antibody in sera taken from immunized mice and from human subjects in a clinical trial. for the assay, we prepared the a(h n ) reassortant virus containing the na of a ⁄ california ⁄ ⁄ (h n ) and the hemagglutinin (ha) of a ⁄ equine ⁄ prague ⁄ ⁄ (h n ). in addition, we used an na-specific igg elisa assay to test sera from immunized mice and volunteers. in mice, one dose of laiv induced ni antibody of a geometric mean titer (gmt) of ae , compared to ae in the control group. gmt of ni from human subjects who received two doses of pandemic a(h n ) were significantly higher than pre-vaccination titers. in unvaccinated human subjects, na-specific cross-reactive antibodies to pandemic a(h n ) were detected more often than cross-reactive antibodies to ha. antibody response to influenza virus na contributes to the overall immune response to influenza and may provide partial protection against influenza infection and reduce severity of disease in the host. a number of preclinical studies using purified or recombinant na have shown that various two-dose vaccine regimens in mice may significantly reduce pulmonary virus titers following viral challenge. [ ] [ ] [ ] a plasmid dna-vaccine model demonstrated cross-reactive antibodies to human n in mice could provide partial protection against a lethal challenge against h n or recombinant pr bearing the avian n . immunogenicity of current influenza vaccines, including laivs, is measured primarily as a level of strain-specific hemagglutination inhibition (hi) antibodies. however, the who meeting on the role of na in inducing protective immunity against influenza infection ( ) specified a need to develop suitable assays for anti-na antibody detection to enhance influenza vaccine evaluation in preclinical and clinical studies. the aim of the current study was to evaluate anti-na antibodies to pandemic a(h n ) influenza virus following laiv immunization. the rn ⁄ -swine a(h n ) reassortant influenza virus containing the na of a ⁄ california ⁄ ⁄ (h n ) and the ha of a ⁄ equine ⁄ prague ⁄ ⁄ (h n ) generated by classical genetic reassortment in embryonated chicken eggs (ce). parental a ⁄ equine ⁄ prague ⁄ ⁄ (h n ) influenza virus was obtained from the center for disease control and prevention, atlanta, ga, usa. viruses were propagated in day old ce and purified by sedimentation out of the allantoic fluid, followed by ultracentrifugation on - % sucrose step gradient. for the mouse studies, week old cba mice were inoculated intranasally with one dose eid ⁄ ae ml of a ⁄ ⁄ california ⁄ ⁄ (h n ) vaccine strain or received ae ml pbs. blood samples were collected on day post inoculation. healthy young adults were immunized twice, or days apart in the fall with a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv manufactured by microgen, irkutsk, russia. for the human studies, peripheral blood specimens were collected from volunteers before vaccination, days after the first vaccination, and days after the second dose of vaccine. sera from five subjects diagnosed with influenza a(h n ) were collected in december , to weeks post infection and kindly provided by e. vo ıtsekhovskaia from biotechnology laboratory, institute of influenza, rams. also, sera obtained in from unvaccinated vol-unteers were tested for presence of cross-reactive antibodies to a ⁄ california ⁄ ⁄ (h n ). sera were treated with a receptor-destroying enzyme from vibrio cholera (denka-seiken, tokyo, japan) and then were tested in duplicates for hemagglutination-inhibition (hi) h specific antibodies by standard procedures using a ⁄ ⁄ california ⁄ ⁄ (h n ) test antigen. the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. was adjusted to assay ni antibody. briefly, -well plates (sarstedt, inc., nümbrecht, germany) were coated overnight with ll of lg ⁄ ml fetuin. the purified a(h n ) reassortant virus was diluted in pbs with % bsa and mm ca + to give a four times higher optical density at nm (od ) compared to control wells not containing virus. fifty-microliter volumes of serially diluted serum samples were incubated with an equal volume of prediluted virus for hour at °c. after incubation, the plates were washed and neuraminidase activity was measured by subsequently adding peroxidase-labeled lectin ( lg ⁄ ml; sigma, st. louis, mo, usa), incubating for hour at room temperature, washing the plates, and adding ll of peroxidase substrate (tmb). the reaction was stopped after minute by adding ll of n sulfuric acid. od values were measured at nm using the universal microplate reader (el x ; bio-tek instruments, inc., winooski, vt, usa). the ni titers were expressed as the reciprocal dilution that gave % od of positive control (virus, no serum control). in addition we used an igg elisa assay with ae lg ⁄ ml of purified na from a ⁄ california ⁄ ⁄ (h n ) to test sera from immunized mice and volunteers. data were analyzed with statistica software (version ae ) (statsoft, inc. tulsa, oklahoma, usa). geometric mean titers (gmt) were calculated and used to represent the antibody response. the comparisons were made within groups between pre-and postvaccinated titers (expressed as log ) after first and second vaccination using wilcoxon matched pairs test. to compare multiple independent groups we used a kruskal-wallis anova with subsequent multiple pairwise comparison based on kruskal-wallis' sums of ranks. a p-value of < ae was considered to be statistically significant. in mice, one dose of laiv induced antibody responses to both ha and na components of the a ⁄ california ⁄ ⁄ (h n ) influenza virus vaccine (table ) . geometric mean titers of ni antibody levels from vaccinated mice were ae and were significantly higher compared to those in unvaccinated control animals (p < ae ). elisa igg titers expressed as log were ae compared to ae in control group. there was good correlation between antibody rises obtained using ni or elisa tests (r = ae ). in a study during the fall of , % of examined unvaccinated subjects were negative to pandemic a(h n ) (hi titers £ : ). serum hi antibody titers to pandemic a(h n ) ‡ : were considered to be protective against *the postvaccination gmts of hi antibodies after revaccination were higher than respective prevaccination titers (p = ae ) **the postvaccination gmts of ni antibodies after revaccination were higher than respective prevaccination titers (p = ae ) serum hi and ni antibodies to a ⁄ california ⁄ ⁄ (h n ) after one or two doses of pandemic laiv were evaluated in subjects who had pre-vaccination hi titers £ : ( table ) . post-vaccination gmts of a(h n )-specific antibodies were significantly higher than pre-vaccination titers only among subjects who received two doses of laiv ( table ). the frequency of subjects with ‡ fourfold rises in hi antibody titers was higher after two doses ( ae %) compared to responses after one dose ( ae %) although the differences were not statistically significant ( table ). the highest antibody titers of hi and ni antibodies were achieved after natural infection (p < ae compared to all post-vaccination groups). all five subjects with confirmed influenza also had high levels of n -specific igg measured by elisa using purified na as the coating antigen (data not shown). influenza ha and na surface proteins are primary targets of neutralizing antibodies that provide protection against influenza infection. the correlation of strain-specific hi antibody titers ‡ : to protection of % of the subjects against influenza infection is based on a number of reports published in s. serum antibodies against viral na as result of influenza infection or vaccination also can neutralize the virus from infecting cells; however, little is known about protective levels of such antibodies. to evaluate ni antibodies directed against pandemic a(h n ) we used the reassortant a(h n ) influenza virus with mismatched ha to avoid non-specific inhibition. we demonstrated laiv immunization effectively increased levels of ni antibody, although in smaller amounts compared to influenza infection. our data suggest that an antibody to neuraminidase, resulting from an earlier infection of the circulating seasonal influenza a(h n ), evidently cross-reacted with the n of pandemic influenza virus, perhaps due to the previously reported % of conserved na epitopes in pandemic a(h n ). the peroxidase-linked lectin test using the reassortant a(h n ) influenza virus was shown to be a sensitive and time effective means of revealing homologous and cross-reactive anti-na antibodies after laiv immunization or influenza infection. this could be a useful method for influenza vaccine evaluation. significant levels of anti-na antibodies detected in peripheral serum from subjects infected with wildtype h n virus or with h n laiv. and the cross-antibody response to ph n . for calculation of geometric mean titer (gmt), a titer of < was assigned a value of . statistical significance was determined by paired t-test. cross-reactive antibody response to ph n in vaccinated populations of seasonal influenza virus table shows the antibody response to seasonal influenza viruses and ph n of participants. before vaccination, no or little antibody response to ph n had been detected in all age groups. vaccination with seasonal influenza vaccines resulted in seroresponse in over % of subjects, except children aged - years ( %) and subjects aged of - years ( %) vaccinated with - season influenza vaccine and adults aged ‡ years ( %) vaccinated with - season influenza vaccine. seroconversion was detected in over % of subjects of all ages. postvaccination to prevaccination gmt ratios for response to seasonal influenza viruses was more than ae -fold. in contrast, seroresponse to a ⁄ california ⁄ ⁄ after vaccination with - and - seasonal influenza vaccines were detected in only % and % of those aged - years, % of those aged - years, % and % of those aged - years, % and % of those aged ‡ years, respectively. seroconversion in all participants ranged from % to %, and postvaccination to prevaccination gmt ratios were < ae -fold. preexisting antibody response to ph n among subjects born before s in china according to a recent report, people who were born from to had a preexisting immunity to ph n . although only a very low level of cross-reactive antibody response to ph n had been observed among older subjects aged more than years old in china, we further analyzed these data by different age distribution of subjects, which can trace back to the previous infection that is genetically and antigenically more closely related to this new ph n influenza virus. the proportion of seroresponse to ph n with the titer of , , and (highest titer detected from participants of all ages in this study) and the value of gmt were analyzed according to the birth decade of subjects from . similarly, a peak of antibody response and the value of gmt occurred both in subjects born from to and sharply decreased afterward ( figure ). the seroresponse of subjects born in and before is significantly higher than subjects born afterward (p < ae ). similar to recent studies in some asia countries (guangxi province of china and singapore), limited antibody response to ph n had been detected in children and adults. , but, some other studies from european countries (finland, germany, the united kingdom) and the united states reported a high proportion of older individuals aged > years with pre-existing cross-reactive antibodies to ph n , which may possibly ba a result of previous exposure to antigenically related h n influenza viruses circulating in earlier decades or a lifetime of exposure to influenza a, which has resulted in broad heterosubtypic immunity among older individuals in those countries. previous infection and vaccination with a ⁄ new jersey ⁄ may also contribute to the high level of cross-reactive antibody response to ph n among adults older than years in the us. , the peak of the antibody response to ph n in subjects born between and , which is consistent with recent reports, may suggest the previous viral infections of spanish flu or closely related influenza viruses, which is before and little after the year of . recent antigenic report of new ph n viruses indicated that they are antigenically homogeneous among historical viruses, which are most similar to classical swine a(h n ) viruses. a number of reviews [ ] [ ] [ ] [ ] confirmed that the virus is the likely ancestor of all four of the human and swine h n and h n lineages, as well as the 'extinct' h n lineage. in , a(h n ) influenza viruses were first isolated from swine. they have been shown to be antigenically highly similar to the recently reconstructed human a(h n ) virus. the cellular responses may contribute to the sustaining and long term antibody response. probably, boosting by persisting antigenically related viruses in the early decades of the th century, may have contributed to the ability of these subjects to sustain memory b cells, and it is well established that a subset of plasma cells is long-lived, and these cells contribute to durable humoral immune responses, such as that observed after childhood smallpox vaccination. furthermore, t cells that recognize cross-reactive epitopes are preserved and might be enriched in the memory population; the course of each infection is influenced by the t-cell memory pool that has been laid down by a host's history of previous successive infections. our study indicated that wide transmission of this new virus or any antigenically close related influenza a(h n ) viruses may not have circulated among populations in china before the outbreak of ph n . our data also suggests the need for vaccination with ph n vaccine in all age groups. hypo-and agammaglobulinemia patients have an impaired immune system and are particularly susceptible to bacterial infections that are normally defended against by antibodies. therefore, patients routinely receive replacement therapy with immunoglobulins isolated from healthy blood donors. these patients are also prone to get viral infections, possibly due to defects in toll-like receptors and . because these patients lack an antigen specific humoral immune response, they are rarely vaccinated. the ability of hypogammaglobulinemic patients to produce a specific cell-mediated immune response upon vaccination has only been sparsely investigated. in contrast to local mucosal antibodies, vaccine-induced cell-mediated immunity is not believed to protect against pathogen entry per se, but may be sufficient to provide protection against severe disease and death following transmission of some microbes. , the aim of this pilot study was to investigate if influenza vaccination of hypogammaglobulinemic patients can induce an influenza-specific cell-mediated immune response. we therefore vaccinated hypogammaglobulinemic patients and healthy controls with pandemic h n virus vaccine and subsequently investigated the bcell and t-cell responses. the percentages of ifn-c, il- , and tnf-a cytokine producing cd + th -cells were determined, as these cytokines are important indicators of cell-mediated immunity. five a-or hypogammaglobulinemic patients were classified based on the freiburg classification : patient # is diagnosed with x-linked agammaglobulinemia, patient # and # are in group ia, patient # is in group ib and patient # is in group ii. the monovalent egg grown split virus vaccine adjuvanted with as was manufactured by glaxosmithkline (gsk), belgium. the vaccine strain was produced by reassortment between influenza a ⁄ california ⁄ ⁄ (h n ) and a ⁄ pr ⁄ ⁄ (h n ) to produce a ⁄ california ⁄ ⁄ -like virus (x a). the vaccine was mixed with adjuvant to contain ae lg haemagglutinin (ha) of a ⁄ california ⁄ ⁄ -like virus (h n ), squalene ( ae mg), dl-atocopherol ( ae mg), and polysorbate ( ae mg) per ml. healthy controls and hypogammaglobulinemia patients were vaccinated by intramuscular (im) injection. hypogammaglobulinemia patients received one or two vaccine doses days apart. the intention was to vaccinate the hypogammaglobulinemic patients with two doses of ae lg ha, but ae lg ha was inadvertently administered to the patients as the first dose. for patient # this was the second dose as he had received an initial dose of ae lg ha months prior to the study. patient # , # , and # received a second dose of ae lg ha. four healthy controls were immunised with one dose of ae lg ha according to norwegian national guidelines. peripheral blood mononuclear cells (pbmcs) were harvested and washed in pbs with % fbs. the pbmcs were resuspended in lymphocyte medium (rpmi with l-glutamine, ae mm non-essential amino acids, mm hepes ph ae , mm sodium pyruvate, iu ⁄ ml penicillin, lg ⁄ ml streptomycin, ae lg ⁄ ml fungizone and % fbs) prior to use in the enzyme-linked immunospot (elispot) and influenza-specific cd + t-cell assays. serum haemagglutination inhibition antibodies were tested by a standard method using ha units and ae % turkey erythrocytes. all samples were tested in duplicate and the test was repeated at least two times. titres < were assigned a value of for calculation purposes. for numeration of antibody-secreting cells (asc), an eli-spot assay was conducted as previously described with the following modifications. ninety-six well elispot plates were coated with lg ⁄ ml of a ⁄ california ⁄ ⁄ like (x a) h n virus diluted in pbs overnight at °c. after blocking with rpmi ( % fbs), pbmcs were added and incubated ( °c, % co ) for hour. secreted antibodies were detected with biotinylated goat anti-human igg, iga and igm specific antibody (southern biotech, birmingham, alabama, usa), incubated for hour at room temperature and developed with extravidin peroxidase and aec substrate. the numbers of spots were counted using an elispot reader (immunoscanÔ) and immunospot Ò software. the influenza-specific cd + th -cell response was measured by intracellular cytokine production of ifn-c, il- , and tnf-a. peripheral blood mononuclear cells ( per well) were incubated for hour ( °c, % co ) in ll lymphocyte medium containing lg ⁄ ml anti-cd , lg ⁄ ml anti-cd d, ae lg ⁄ ml monensin, lg ⁄ ml brefeldin a, (bd biosciences, franklin lakes, new jersey, usa), and the h n influenza split virus vaccine x a (either ae lg ⁄ ml or lg ⁄ ml ha). basal cytokine production was determined by incubating pbmcs in lymphocyte medium without influenza virus, and the percentage of cytokine positive cells without influenza stimulation were subtracted from influenza-stimulated cells. cells were stained for cd , cd , cd , ifn-c, il- , and tnf-a (bd biosciences) as previously described. finally, cells were resuspended in pbs containing % fbs and ae % sodium azide and analysed by bd facscanto flow cytometer ( - cells acquired). flowjo v ae ae (tree star, ashland, oregon, usa) was used for data analysis. five to six fold lower gmts were found in the patient group as compared to the healthy controls throughout the study ( figure a) . the lowest hi titres were obtained in patients # , # , and # , whilst patients # and # and all healthy controls fulfilled two of three european medicines agency committee for medicinal products for human use (chmp) seasonal influenza vaccine licensing criteria, by obtaining an hi titre > and a mean geometric increase of ae between pre-and post-vaccination. thus, the hi data indicate that two vaccine doses was sufficient to induce a protective hi antibody response in two out of five of the hypogammaglobulinemia patients tested in this study. the numbers of influenza-specific iga, igg, and igm asc were tested pre-vaccination and days post-vaccination with the h x a virus. few or no ascs were detected pre-vaccination (data not shown). at days post-vaccination the patient's iga, igg, and igm asc levels were significantly lower (p < ae ) compared to the healthy controls ( figure b) . but, the post-vaccination asc numbers in the patients were generally higher than at pre-vaccination stage ( - ascs). patient # had the highest iga and igg asc numbers, followed by patients # and # , whilst patient # and # had few or no asc's. these results confirm that the patients are indeed hypogammaglobulinemic and that some of the patients (# and # ) could be agammaglobulinemic in the context of producing influenza-specific antibodies. the asc levels of patients # , # , and # were lower than those of the healthy controls, but could possibly be adequate for reducing the severity of influenza disease. the influenza-specific th -cell response was evaluated by stimulating pbmcs with the influenza x a virus , , and days post-vaccination. stimulation of healthy control pbmcs with x a days after vaccination, induced ifn-c, il- , and tnf-a production by an average of ae %, ae %, and ae % cd + t-cells, respectively. patient # and # had higher responses than the healthy controls and stimulation with x a induced ae %, ae %, and ae % of t-cells from patient # to produce ifn-c, il- , and tnf-a, respectively (figure a) . the response of patient # was further boosted by a second vaccine dose, which resulted in ae %, ae %, and ae % cd t-cells producing ifn-c, il- , and tnf-a, respectively at day ( figure b ). these results show that the hypogammaglobulinemia patients studied here did not have a common impaired influenza-specific cd + th cytokine response. rather, there was a tendency towards increased responses, suggesting that the diminished antigen specific b-cell responses could induce a compensatory antigen specific th -cell response. the results from this pilot study suggest that some hypogammaglobulinemia patients may benefit from influenza vaccination. we found very different patient responses to influenza vaccination, but some of the patients (patient # and # ) did mount low influenza-specific asc responses. in addition, the vaccine-induced hi antibody titres above the protective level in patient # and # . these results are in accordance with previous publications, which described that polypeptide vaccines induce humoral responses in subgroups of common variable immunodeficiency patients. [ ] [ ] [ ] in this study, we also investigated cell-mediated immunity and found the percentages of homologous and cross-reactive influenza-specific cd + th -cells to be in the same range (for patient # , # , and # ) or higher (for patient # and # ) in the a-or hypogammaglobulinemic patients compared to the healthy controls. the higher response is probably due to the patients having received a vaccine dose of ae lg ha, whilst the controls received ae lg ha. in addition, the patients received a second booster dose, which influences the day and months responses. nonetheless, these results are the first to demonstrate that proliferation of pandemic influenza antigen specific th cells can be induced in hypogammaglobulinemic patients. in addition, vaccination induced influenza-specific asc's in some patients. the findings are promising and provide hope that hypogammaglobulineamic patients could be vaccinated against influenza and other diseases preventable by figure . peripheral blood mononuclear cells s from patients and healthy controls were isolated at day (a), (b), and day (c) and stimulated for hour with x a virus before staining and flow cytometric analysis. the figure shows the mean ± sd frequency of influenza-specific cd + cytokine producing cells (%) where the basal cytokine production from unstimulated cells has been subtracted. data for the hypogammaglobulinemia patients are additionally shown as a number for each patient. **significantly higher frequency of il- producing cd + t-cells in the patients compared to the healthy controls (students t-test p < ae ). titres are presented as the geometric mean titre ± % confidence interval. elispot data (b) are presented as the mean number of influenza-specific iga, igg, and igm ascs per peripheral blood mononuclear cells ± sem. data for the hypogammaglobulinemia patients are additionally presented by a number for each patient. *significantly higher numbers of ascs were detected in the healthy controls as compared with the hypogammaglobulinemia group (students t-test, p < ae ). vaccination. however, this hypothesis should be tested in larger clinical studies. the influenza virus undergoes antigenic evolution under intense immune selection pressure from herd immunity in humans through the process called antigenic drift and shift. , because of antigenic drift, yearly updating of vaccine strain is needed. a mismatch between the circulating strains and the vaccine strain in the subsequent season is often encountered, resulting in reduction of vaccine effectiveness and lack of protection from the circulating strain. in order to address this, a universal influenza vaccine based on a more conserved part of the influenza virus, which is not affected by antigenic change and that is conserved across all strains, remains the ultimate goal to afford cross-protection to drifted strains as well as to other subtypes of influenza which may arise from antigenic shift. , previous studies have investigated the potential of the m e. , m e has remained highly conserved since it was first isolated in . several studies have examined the use of m e as a vaccine component, using various approaches including proteins, peptides, dna vectors, and attenuated viral vectors. , [ ] [ ] [ ] [ ] [ ] [ ] although m e is a weak antigen, by linking the protein to a carrier hepatitis b virus core particle, protection against influenza has been achieved in mice particularly when administered with an adjuvant. some articles found that vaccination with m e coupled to hbc induces protective antibodies, whereas the contribution of t cells to protection was negligible. protection induced by vaccination with m e-hbc was weak overall and failed to prevent weight loss in vaccinated infected animals, and mice succumbed to high dose infection. we aimed to address the poor immunogenicity of m e-hbc by using igv as adjuvant. igv domain is common and conserved in the tim family. ligand binding sites of t cell immunoglobulin mucin (tim) located at igv domain. [ ] [ ] [ ] tim function is done by anti tim antibody which recognized the ligand binding sites of igv domain. tim family members share a common motif, including an igv domain. they are differentially expressed on th cells and th cells with the ability to regulate the immune system. , the igv domain of human b - is sufficient to co-stimulate t lymphocytes and induce cytokine secretion. soo hoo et al. vaccinated with tim- antibody and inactivated influenza and found enhanced vaccine-specific immune response. we report here for the first time the use of igv recombinant protein as adjuvant to immunize mice with influenza m e-hbc. results indicated that igv can induce the strong cellular immune response and cross reaction with different subtype influenza virus antigen. target igv may be used to develop the new method for vaccination strategies. expression and purification of recombinant igv protein rna was extracted from healthy human pbmc. one-step rt-pcr (qiagen, valencia, ca, usa) was done for the amplification igv gene. the pcr product was purified and cloned into pet a (novagen, madison, germany). the resultant construct pet a-igv has a histidine (his) tag ( his) at the n terminus. dna sequence of the insert was determined by sequencing. igv. recombinant protein was expressed in escherichia coli and was purified on a ni column (novagen). the purified protein was examined by sds-page and western blotting. six-eight weeks female balb ⁄ c mice (institute of zoology chinese academy of sciences, china) was used for the study. mice were immunized twice intradermally with ug m e-hbc (provided by cnic, china) combined with different doses of recombinant igv protein , , ug, respectively, or without igv as control. the area proximal to the tibialis anterior muscle was sterilized with % ethanol and different groups of mice were injected bilaterally with , , ug igv plus ug m e-hbc in ul phosphate buffer saline per mouse using a ml syringe with attached ⁄ ¢¢ g needle. the immunization was given at weeks intervals. four blood samples were obtained from every mouse: before immunization, after the first and second immunization, and after virus challenge by retro-orbital plexus puncture. after clotting and centrifugation, serum samples were collected and stored at ) °c prior to use for assays. mouse-adapted a ⁄ pr ⁄ ⁄ (h n ), a ⁄ brisbane ⁄ ⁄ (h n ), a ⁄ xinjiang ⁄ ⁄ (h n ), and a ⁄ guangzhou ⁄ ⁄ (h n ) were provided by chinese national influenza centre. nine to eleven days old embroynated specific pathogen free (spf) chicken eggs were inoculated with virus, and the eggs were incubated at °c for - days. the allantoic fluid was collected and purified by sucrose density gradient centrifugation, and the virus was inactivated by formaldehyde at °c overnight. to identify igg, igg , igg a against m e, elisa assays were used. in brief, -well (nunc, brunei, denmark) were coated with ul ⁄ well of m e recombinant protein (provided by gene lab of ivdc, xuanwu district, beijing, china) in carbonate buffer (ph ae ) overnight at °c. immediately before use, the coated plates were incubated with blocking solution ( % bsa in pbs) for h at °c and washed four times with pbs containing ae % tween (pbs-t). the serum samples were serially diluted and added in the plates. the detection color was developed by adding hrp-labeled goat anti-mouse igg, igg , or igg a ( figure ) . no cross-strain response was observed in the control group. the igv adjuvented groups show splenocytes stimulation with seasonal h n , h n , h n , and h n antigens. m e-hbc immunization without igv showed splenocyte stimulation, but the extent was lower than animals immunized in the presence of the igv adjuvent. these data suggested that igv had enhanced effect on priming against the conserved viral antigen matrix protein and generation cross-strain immune response. influenza is a respiratory disease causing epidemics every year. h n viruses and swine-origin h n have also infected humans in recent years. seasonal influenza vaccine cannot cope with significant antigenic drift or with the emergence of pandemic viruses of different subtypes not contained in the vaccine. the high extent of conservation of the m e makes it a promising immunogen. a vaccine based on coupling of the m e peptide to an appropriate carrier may provide a universal vaccine with effectiveness and safety. m e based vaccination induces protective antibodies not only in mice, but also in ferrets and monkeys. the carrier hepatitis b core as carrier with m e forms a virus like particle (vlp). vaccination with m e coupled to hbc induces protective antibody, whereas the contribution of t cell protection was negligible. protection induced by vaccination with m coupled to hbc was weak overall. in order to improve the vaccination effect of m e-hbc, new adjuvant igv was evaluated in combination with the m e-hbc. the tim molecules are a recently discovered class of proteins with the ability to regulate the immune system. crystal structures of the tim molecules has revealed a unique, conserved structure with ligand-binding sites in the igv domain. to determine the potential immunostimulatory molecular properties of igv, we have evaluated immune response of the igv in combination with m e-hbc vlp. previous papers reported that vlp immunized mice can induce the th and th immune response. different adjuvant combined vlp can produce biased immune response th ⁄ th mixed immune response, or th -preferred th ⁄ th profile. thus, the response following the use of igv as a new adjuvant combined with m e-hbc vlp needs to be evaluated. results indicated that igv combined groups showed th biased immune response and enhanced cross reactive t cell immune responses. this may show that igv immunized the mice and antiigv antibody can cross link the igv on t cells and enhance the cell figure . t cell proliferation assay. mice were immunized twice with , , , ug ⁄ ml igv plus m e-hbc, respectively, and naive group was immunized with pbs. three weeks after a boosting immunization, spleens were harvested from immunized and naive mice. different subtypes of inactivated virus antigen (a) h n , (b) h n , (c) h n , (d) h n were added and cocultured with different group splenocytes for h. quick cell proliferation assay kit was used to detect the cell proliferation. the - nm absorbance was read on a plate reader. data were showed were shown as mean values. the difference between naive group and different doses igv plus m e groups was determined using the student's t-test. all significance level is p < ae . response. we also evaluated the cross-protection produced by igv combined m e-hbc. we challenge with mouseadapted strain pr and prove the cross protection via reaction between the cells from the immunized animal and different subtypes of virus antigen. some subtypes of virus cannot infect the mice naturally, and therefore, virus challenge cannot be used to evaluate the effect. we co-cultured the t cells with inactivated antigen h , h , h , and h , and t cell proliferation was measured. results indicated that after immunization with igv plus m e-hbc, the t cells show cross-protection with other subtypes. this provides evidence that igv can enhance the cross protection across subtypes. the results of this study demonstrated that recombinant igv can be useful as an adjuvant and polarize the m e-hbc vlp immune response to a th profile. igv induced the m e-hbc vlp to induce t cell proliferation and cross-reactive responses to different influenza virus subtypes. this finding represents a new direction for the promotion of cell mediated immunity in m e based vaccine against influenza. a core european protocol, i-move, describing the methods to estimate influenza vaccine effectiveness (ive) was proposed by the european centre for disease prevention and control (ecdc) and epiconcept for the - season. it includes a case control method for pooled analysis based on a randomized ''systematic'' sample of swabs. , collection of swabs using a non randomized, i.e., ''ad hoc,'' sampling strategy, left at the appreciation of sentinel practitioners, provides a greater number of cases and con-trols for ive estimation more easily than using a systematic randomized sampling strategy. the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than specimens yearly from cases of acute respiratory illness (ari), using both sampling methods. , during the circulation of pandemic influenza viruses in france, it gave an opportunity to compare ive estimates using systematic randomized versus non systematic ''ad hoc'' sampling. influenza vaccine effectiveness was estimated by a casecontrol methodology according to ecdc i-move protocol, using on the one hand a systematic random sampling, on the other hand ''ad hoc'' non random sampling. the study was proposed to primary care practitioners of the grog network ( general practitioners and pediatricians) trained to collect data and swabs. the study population was patients from the community of all ages consulting a grog practitioner for an influenza like illness (ili) and having a nasal or throat swab taken within an interval of < days after symptom onset. ili was defined according to the european union (eu) case definition as sudden onset of symptoms with at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath. swabs were performed through usual surveillance. no ethical approval was needed, but an oral informed consent was requested. cases were excluded if they refused to participate in the study or if they were unable to give informed consent or to follow the interview in native language because of aphasia, reduced consciousness, or other reasons. an individual was considered as vaccinated against pandemic influenza if he or she reported having received a pandemic influenza vaccination during the current season, and if at least one vaccine dose occurred more than days before ili onset. the study period started with the initiation of active influenza surveillance by the grog network, i.e., days after the beginning of the influenza vaccination campaign, and finished at the end of the influenza period defined as the last week with at least one swab positive for influenza within the grog network. ''ad hoc'' sampling patients from which swabs were taken were selected by the grog practitioners during the study period. systematic random sampling during the same period, patients were selected at random as follows. an age-group - years (gps and pediatricians); - years (gps and pediatricians); - years (gps); years or more (gps) was assigned to each practitioner, who was requested to swab the first patient of the week presenting with an ili within the pre-assigned age-group. swabs were collected in appropriate transport medium (virocult Ò , viralpack Ò , utm copan Ò ) and sent by post to the laboratory in triple packaging following the international guidelines for the transport of infectious substances (category b, classification un ). laboratory confirmation of influenza was by rt-pcr to detect currently circulating influenza a (subtypes h , seasonal and pandemic h ) and b viruses. an influenza case was defined as an ili case with a respiratory sample positive for influenza during the study period. controls were cases of ili having a swab negative for influenza during the study period. the outcome of interest is laboratory confirmed influenza. confounding factors and effects modifiers identified during the i-move preliminary study were registered: risk factors, chronic diseases, severity of underlying conditions, smoking history, former vaccinations, and functional status. data on cases and controls were collected by the practitioners using a standardized questionnaire adapted from the i-move study. questionnaires were sent by the practitioners with the swab to the virology laboratory, and sent to the grog national coordination. data entry and validation were ensured by open rome through the vircases computing tool. validation steps included control of exhaustiveness of centralization of questionnaires, comparison of data entered by the labs and the national grog coordination, coherence control, and identification of missing data. analysis was done for the two sampling groups (systematic and ad hoc) on cases ⁄ controls following the european method proposed by epiconcept, using excel ª (microsoft corp. redmond, washington, usa) and stata ª . baseline characteristics of cases and controls in unmatched studies were compared using the chi-square test, fisher's exact test, or the mann-whitney test (depending on the nature of the variable and the sample size). the association between vaccination status and baseline characteristics was assessed for both case and control groups. the vaccine effectiveness was computed as ive = )or (odds ratio). an exact % confidence interval (ci) was computed around the point estimate. analysis was stratified according to age groups, time (month of onset), presence or absence of chronic disease, and previous influenza vaccination. effect modification was assessed comparing the or across the strata of the baseline characteristics. confounding factors were assessed by comparing crude and adjusted or for each baseline characteristic. a multivariable logistic regression analysis was conducted to control for negative and positive confounding factors using a complete case analysis (with records with missing data dropped) and using multiple imputation with chained equations. the complete model included age group, number of gp visits, onset week, seasonal vaccination, previous seasonal influenza vaccination, presence of chronic disease and associated hospitalizations in the previous months, gender, and smoking status. variables were tested for multi-colinearity. interactions were tested using the likelihood ratio test (or wald test) and included in the model if significant at % level. a model with fewer variables (age group, number of gp visit, onset week, and seasonal vaccination) was also tested. several models were applied to both the ''ad hoc'' and systematic sampling groups of cases and controls. as shown in table , whatever the analysis method used, the ''ad hoc'' sampling strategy led to a slightly lower estimate of ive. the ci were extended when data were missing and reduced when using multiple imputations with chained equations. however, from a statistical point of view, comparison of ''ad hoc'' versus systematic strategies is not straightforward, because ''ad hoc'' sampling is not randomized and does not allow comparisons with statistical tests using statistical distribution laws. there are more missing data with the ad hoc sampling method. this is mainly due to our validation procedure: in the case of missing data in the systematic sampling group, as required by the i-move study protocol, queries were sent to sentinel practitioners using mail and phone calls. this specific heavy workload is not usually performed during routine surveillance and has not been achieved for the ''ad hoc'' sampling group given the great number of cases and controls ( ). within the framework of the i-move study, several items were added to the grog's usual clinical form accompanying swabs (hospitalizations, number of gp visits, smoking status, help needed for bathing or walking). in - , gps explained that this added workload was not compatible with their daily additional workload due to the pandemic situation. therefore, many of them refused to fill these new items systematically and threatened to leave the network. we thus obtained that the ''i-move items'' would be filled in for the clinical forms linked to systematic sampling, but were not in a position to obtain that for ''ad hoc'' sampling. the weekly distribution of systematic swabbing is not similar to that of ad hoc swabbing. the percentage of ad hoc swabs was higher than systematic swabs during the pandemic wave (mid-november to end of december) during which time the percentage of swabs positive for influenza was also higher ( figure ). this could explain the higher rate of positive swabs within the ''ad hoc'' samples. the vaccination campaign was launched by the ministry of health on october , , and vaccination coverage increased during the surveillance period. in february, the vaccination coverage was ae % in patients swabbed in the systematic group ( ae % on imputed data) and ae % [ ae - ae ] in the ad hoc group ( ae % on imputed data). at the national level, vaccine coverage is estimated at ae %. due to the over-mediatisation of pandemic vaccination and to rumors about its poor effectiveness, overconsultation of vaccinated patients and over-swabbing of vaccinated patients in the ad hoc group are not surprising. age distribution is significantly different between our two samples (p < ae ): the rate of - years old is lower in the systematic sampling group ( ae %) than in the ad hoc sampling group ( ae %). this can be explained by the fact that for the systematic sampling procedure, each grog practitioner had to swab the first ili patient in his assigned age group, whereas for ''ad hoc'' sampling, every grog practitioner could swab any ili patient irrespective of age. given the emphasis by health authorities and media on the burden of pandemic influenza among children and teenagers, one can hypothesize that when they were able to, sentinel practitioners focused on these age groups. gps in the ad hoc sampling scheme seem to have been more likely to select cases and further, to select vaccinated cases. those patients may have consulted earlier with specific symptoms (strong headache being more prevalent among cases). over-swabbing of patients having these symptoms in the ad hoc group is likely. the - pandemic influenza season was markedly different from previous ones: vaccination rate increased during and mainly after the pandemic peak; behaviors were strongly modified by unusual media hype; clinical features and risk factors might be different. it will be necessary to see if similar results are observed during a regular influenza season during which the vaccination rate increases before the epidemic peak with usual messages about vaccination and usual clinical influenza features. influenza early warning networks can estimate ive, taking into account many covariates. from a stakeholders and patients point of view, during the - influenza pandemic wave, there were no major discrepancies between ive estimated with an ad hoc sampling strategy, based on sentinel practitioners instinct, and ive estimated with a systematic random sampling strategy whatever the multivariable analysis methodology. although from a statistical point of view, comparison of the two strategies is not readily feasible because of the non random nature of ad hoc sampling. this latter strategy seems to result in slightly lower ive estimates, which could potentially be attributed to sentinel practitioners swabbing behavior. the ability to avoid missing data is a key point to decide which sampling method must be adopted, because ci extent depends greatly on the proportion of missing data among covariates. to match ive evaluation to surveillance networks practicality, selection of only those data essential for the study endpoint and easily collected by sentinel practitioners is paramount. it will be necessary to determine if results similar to those observed during the - pandemic season are found during a regular influenza season. influenza a viruses are important pathogens which remain a major cause of morbidity and mortality worldwide, and large numbers of the human population are affected every year. the first influenza pandemic in this century broke out in humans in march , and it was declared to be pandemic by mid-june. as of august jul , the pandemic virus had caused more than deaths worldwide, according to the world health organization (http:// www.who.int/csr/don/ _ _ /en/index.html). the infection and spread of the pandemic influenza was reduced in part due to the use of vaccines. however, the lack of h n pdm vaccine early in the pandemic illustrates the need to improve vaccine production and to generate vaccines that induce stronger cross-protection. inactivated split vaccines or live attenuated influenza virus vaccines (laivs) against h n pdm viruses were approved for human use by the united states food and drug administration. both the inactivated vaccines and laivs are produced by creating reassortant viruses that generally contain six vrnas (pb , pb , pa, np, m, and ns) from a master donor strain, plus the two glycoprotein vrnas (ha and na) from a virus that antigenically matches the strain predicted to circulate in upcoming influenza season (e.g. a ⁄ ca ⁄ ⁄ ). the reference viruses containing inactivated split virus vaccines are produced in embryonated chicken eggs, and primarily result in the production of antibodies that recognize the viral glycoproteins. both of these vaccine approaches require significant lead time for vaccine production, and modern approaches to speed preparation of vaccines and improve their efficacy is a global priority. , the ns protein of influenza a virus is a multifunctional protein that plays important roles in virus replication and as potent type i ifn antagonist. , mutations and ⁄ or deletions in ns typically induce stronger ifn responses by the host; those in turn suppress the replication of influenza virus - and can enhance immune recognition. [ ] [ ] [ ] [ ] in this study, we created a panel of experimental h n pdm ns-laiv candidates that have different deletions in the ns vrna and analyzed the vaccine potential of each ns-laiv in mice and ferrets to identify the best candidate(s). wt h n pdm influenza a virus a ⁄ new york ⁄ ⁄ (ny ) was created by reverse-genetics directly from a human swab specimen collected in new york state in april . deletions were introduced into the ny ns plasmid to create three mutant ns segments: ns - , ns - , and nsd . nucleotides - (cdna of ns segment) and - were replaced by stop codons to generate ns - and ns - ; nucleotides - were deleted to generate nsd , whose open reading frames for ns and nep were maintained. recombinant viruses were generated by co-transfection of eight reverse-genetics plasmids carrying the cdna of each gene segment into t ⁄ mdck cocultured monolayer adapted from hoffmann et al. , mouse studies experiments were performed in a biosafety level laboratories approved by the u.s. centers for disease control and prevention and the u.s. department of agriculture, and were conducted under approved animal care and use protocols. groups of -week-old female balb ⁄ cj (jackson laboratory, bar harbor, me, usa) were anesthetized with isoflurane and inoculated intranasally with tcid of each recombinant virus in ll of pbs diluent, or pbs as controls. body weights and clinical symptoms of the mice were monitored daily for days. nine mice in each group were euthanized on , , and days post inoculation (dpi), and nasal washes and lungs were collected for virus titration by tcid assay in mdck cells. at dpi, mice per group were challenged intranasally with · tcid ( ld in -week-old mice) of a mouse-adapted variant of ny (a ⁄ ny ⁄ ⁄ -ma ) (accepted, journal of virology). disease symptoms and weights of the vaccinated mice were monitored for days, and four mice from each virus group were euthanized at and days post challenge. lungs were removed and homogenized for virus titration by tcid assay. the mice that became moribund or lost > % of their starting body weight were euthanized for humane reasons. male fitch ferrets (triple f farms, sayre, pa, usa), - months of age and serologically negative by hemagglutination inhibition (hi) assay for currently circulating influenza viruses were used in this study. groups of or ferrets were inoculated intranasally with ae tcid of one of the viruses: ny wt (n = ), ns - (n = ), ns - (n = ), or nsd (n = ). ferrets were monitored for clinical signs through dpi as previously described. nasal washes were collected on , , , and dpi and were titrated in mdck cells by tcid assay. serum was isolated from blood collected ae weeks after immunization and used for neutralization assays. the ferrets were challenged with pfu of a ⁄ mexico ⁄ ⁄ ae weeks postimmunization and monitored for clinical signs of disease through dpi. nasal washes were collected on , , , and dpi, and were titrated in mdck cells by plaque assay. using reverse genetics, we created three laiv candidates weight loss of wt virus inoculated mice became evident at dpi, and the mice did not recover until dpi (figure a) . in contrast, mice inoculated with any one of the vaccine candidates had no clinical signs of disease and continued to gain weight at the same rate as did the mock- inoculated mice ( figure a ). viral titers in the lungs of ns - , and ns - infected mice were $ -fold lower than titers from wt virus-infected mice at all the time points analyzed ( , , and dpi) ( figure b) . notably, the nsd laiv was cleared from the mouse lungs very rapidly, and the mean titers were $ -fold and -fold lower than the titers of the wt virus at and dpi, respectively ( figure b) . the vaccinated mice were challenged with a mouseadapted variant of ny (accepted, journal of virology) on dpi. no disease symptoms were observed in the mice immunized by any of the ns-laiv candidates or the wt control. in contrast, disease symptoms including ruffled fur, hunched posture, and weight loss were observed in the mock-immunized mice as early as days post challenge (dpc); the symptoms progressed to severe disease, and the animals showed dramatic weight loss, became moribund, and succumbed to infection by dpc (figure c ). high titers of virus ($ tcid ⁄ ml) were present in the mock-immunized mice at dpc and at dpc ( figure d ). in contrast, virus was not detected in the lungs of immunized mice ( figure d ). this challenge data demonstrates that all of the ns-laiv candidates, including the highly attenuated nsd , induced sterilizing immunity that protected mice from a lethal ny h n pdm variant. groups of ferrets were intranasally immunized with ae tcid of each vaccine candidate or the wt virus. the titer of viruses recovered from nasal washes ranged from ae to ae tcid ⁄ ml through day in the wt virusinfected group, while the ns-laivs showed various degrees of attenuation (figure a) . the viral titer of all of the ns-laivs is at least -fold lower than that of wt in the nasal wash collected at dpi. the ns - laiv was the least attenuated in ferrets, and its replication was similar to that observed in mice. relative to the wt virus, the ns - laiv showed -fold reduction in titer, and the nsd laiv was below the limit of detection (at least fold reduction) at dpi. sera from blood collected ae weeks after immunization was analyzed for the presence of neutralizing antibodies by micro-neutralization assays. the ns-laiv candidates all induced very strong neutralizing antibody responses ( - ) that were similar to the titer elicited by wt virus infection ( figure b ). the ferrets were challenged with pfu of a ⁄ mexico ⁄ ⁄ (h n pdm) ae weeks post immunization. little disease or weight loss were observed in the naïve ferrets, and the ferrets immunized by infection with wt virus or the ns-laiv candidates didn't show any disease symptoms or weight loss. in contrast to the high titer of virus detected in the naïve ferrets through dpc, the ns-laiv immunized ferrets had very low levels of a ⁄ mexico ⁄ ⁄ in their nasal washes at dpc ( figure c ). the ferrets immunized with the ny ns-laivs had $ -to -fold lower viral titers than did the naïve animals ( figure d ). in summary, the ns-laiv candidates dramatically inhibited initial replication of the h n pdm virus under stringent challenge conditions ( pfu), and that the vaccinated animals rapidly cleared the infection (to below the limit of detection, by dpc). our results demonstrate that all of the ns-laiv candidates are attenuated compared to the wt h n pdm virus, and the degree of attenuation is dependent on the specific ns mutation. ns - was the least attenuated and does not represent a good vaccine candidate; whereas, nsd and ns - were highly attenuated in both the mouse and ferret models. although they were markedly attenuated, they elicited strong neutralizing antibody responses and protected mice and ferrets from subsequent challenge. nsd has a subtle in-frame deletion ( nt) that affects both the ns (residues - ) and nep (residues - ), and is analogous to a naturally attenuated variant of a normally highly pathogenic h n virus (a ⁄ sw ⁄ fj ⁄ ). the analogous ns deletion in a ⁄ sw ⁄ fj ⁄ (residues - ) was shown to reduce binding to host cleavage and polyadenylation specificity factor (cpsf), reduce ns protein stability, and enhance the type i ifn response of this h n virus. our study indicates that deletion of these nt in the ns vrna of the h n pdm also stimulates the host ifn response, specifically, ifn-ß, ifn-k , ip , and mxa (data not shown). the role of the deletion of residues - from nep has not been elucidated, but the induction of ifn and isgs by nsd was similar to, or slightly lower than, their induction by ns- , suggesting that the nep mutation also has an attenuating effect that warrants future investigation. in summary, we have generated a panel of laivs directly from a swab specimen containing a new pandemic virus and analyzed their attenuation and immunogenicity in two animal models. our study demonstrates that nsd is a novel ns-laiv that could be used to create laivs for diverse influenza a viruses. this study also validates the use of ns-laiv candidates, which are not only highly attenuated, but they also elicit strong innate and adaptive immune responses, resulting in protection of mice from subsequent challenge with a lethal mouse-adapted variant of ny , and ferrets from challenge with a ⁄ mexico ⁄ ⁄ (h n pdm). currently, a total of approximately million doses of inactivated influenza vaccine are being produced worldwide each year. one of the limitations in vaccine production is poor growth of human isolates in embryonated chicken eggs. this is essential to develop high yield seed viruses for large scale production of influenza vaccines. influenza a vaccine production utilizes high yield reassortants carrying ha and na genes from a wild type (wt) strain with generally - internal genes from the a ⁄ pr ⁄ ⁄ (pr ) strain, an highly egg adapted high growth donor strain. influenza b vaccines, however, have been produced directly from wt strains, partly because no high yield donor analogous to pr has been identified. in recent years, reverse genetics has been used as an alternative means of developing high growth vaccine viruses. , since in this plasmid-based technology, a : reassortant (six internal genes from a donor strain and two surface antigen genes from wild type strain) can be directly rescued, reverse genetics-derived reassortant viruses were expected to grow as efficiently as those derived from classical reassortment. however, reverse genetics reassortants have not produced the expected high growth for several reasons: (i) the : configuration is not always the best for virus yield, (ii) there is no process included for positive selection of adaptive mutants from quasispecies, and (iii) cell-derived viruses are not readily adapted to grow efficiently in eggs. our laboratory at new york medical college has been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ as a donor. it has been possible to develop b reassortants, which produce higher virus yields than wt strains in eggs, and it was found that the np gene of b ⁄ lee ⁄ was important in producing high yield b reassortants. however, b ⁄ lee ⁄ is inconsistent in providing high yield properties to b reassortants. in this study, in an attempt to find an alternative donor, we investigated the usefulness of b ⁄ panama ⁄ ⁄ for developing high yield b reassortants. as a wt strain, b ⁄ brisbane ⁄ ⁄ was used, which is one of the recommended influenza b virus vaccine strains for the ⁄ and ⁄ seasons. we found that b ⁄ panama ⁄ ⁄ is a useful donor, and some of the resultant reassortants were considered as vaccine candidates. b reassortant viruses were prepared by the classical reassortment method described by kilbourne. the antiserum to b ⁄ panama ⁄ ⁄ hemagglutinin and neuraminidase (hana) was raised in this study by immunizing rabbits with hana isolated from b ⁄ panama ⁄ ⁄ ; purified igg was used for antibody selection. the yields of the reassortants and their corresponding parent viruses were assessed by hemagglutination assay. viral rna was extracted directly from the allantoic fluid and amplified by rt-pcr to produce cdna for analyzing the gene composition. restriction fragment length polymorphism (rflp) analyses were performed to determine the origin of each gene segment of the high yield reassortants. restriction enzyme sets for each gene segment are available upon request. in this study we investigated the usefulness of b ⁄ panama ⁄ ⁄ as a donor for transferring high yield phenotype. b ⁄ panama ⁄ is a yamagata lineage strain with high growth phenotype (ha titer: - ). b ⁄ panama ⁄ ⁄ itself was a recommended b virus vaccine strain for ⁄ - ⁄ seasons. as a wt virus, a victoria lineage strain, b ⁄ brisbane ⁄ ⁄ , was used, which is a recommended b virus vaccine strain for use in the ⁄ and ⁄ seasons. reassortants were prepared according to classical reassortment protocol. after co-infection of b ⁄ panama ⁄ ⁄ and b ⁄ brisbane ⁄ ⁄ , progeny viruses carrying surface antigens (ha and na) of the vaccine strain were negatively selected by anti-b ⁄ panama hana antibodies, followed by passages without antibodies for positive selection of eggadapted viruses and finally limited dilution cloning. nymc bx- , bx- b, bx- d, and r- a are representative of resultant reassortants, which have significantly higher ha titers than the wt strain. the complete gene compositions of these reassortants were determined by rt-pcr ⁄ rflp analyses. as shown in table , all of these reassortants contained the pb of b ⁄ panama ⁄ ⁄ . other genes of b ⁄ panama ⁄ ⁄ (np of bx- , m of bx- b, and pb of bx- d) may not be involved in the high virus yield, since no significant growth difference among these reassortants in eggs was found as assayed by hemagglutination test. accordingly, the pb of b ⁄ panama ⁄ ⁄ is considered to be the sole factor involved in the high yield phenotype donated to the vaccine strain. we previously found that the b ⁄ lee ⁄ np gene was important in producing high yield b reassortants. it was of interest to examine whether b ⁄ lee ⁄ np and b ⁄ panama pb could work together to produce even higher yields. to test this possibility, bx- b ( : reassortant: pb and m genes from b ⁄ panama and the rest of the genes from b ⁄ brisbane) was selected and further reassorted with b ⁄ lee ⁄ . despite some difficulty in removing the na gene of b ⁄ lee ⁄ (r- c, b, b in table ), by monitoring ha and na genes of resultant viruses after each antibody selection passage with anti b ⁄ lee ⁄ hana antibodies, we were able to isolate and clone a triple reassortant, nymc bx- , which contains the np gene from b ⁄ lee ⁄ and pb and m genes from b ⁄ panama; the remaining genes are from b ⁄ brisbane ⁄ ⁄ (table ). in comparison with bx- b, no significant growth enhancement (nor reduction) in eggs was found for bx- over that seen for bx- b. nevertheless, bx- stably produces high virus yield and has been utilized as a seed virus for influenza b vaccine production for the - season by one or more vaccine manufacturers. there are contradictory reports - about the usefulness of reassortment for high yield influenza b viruses. however, we have been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ as a donor, and have been able to generate higher virus yield than wt strains. in this study, we found that b ⁄ panama ⁄ ⁄ serves as an efficient donor in providing the high growth capacity to b ⁄ brisbane ⁄ ⁄ (a recommended vaccine virus of victoria lineage for ⁄ - ⁄ seasons), and that the pb of b ⁄ panama ⁄ ⁄ is associated with the high yield phenotype. this particular strain from yamagata lineage might be useful to prepare high yield reassortants for other victoria lineage vaccine viruses. we noticed in this study that there may be segment incompatibilities between b ⁄ panama ⁄ ⁄ and b ⁄ brisbane ⁄ ⁄ . as shown in table , the pa and ns genes of all the high yield reassortants examined are derived from wt, b ⁄ brisbane ⁄ ⁄ , not from the donor, b ⁄ panama ⁄ ⁄ . this indicates that in this reassortment, the pa and ns genes are not replaceable with that of the donor to obtain high yield viruses. this degree of incompatibility might be common in b reassortment, resulting in low donor ⁄ wt reassortants, such as : and even : reassortants that we obtained in this study. if this is the case, reverse genetics based on : configuration may not result in generating high yield b reassortants unless a variety of donor ⁄ wt combinations are designed. one can speculate that in influenza b viruses, the surface glycoproteins (ha and na) and some of the internal proteins are functionally more closely related than in influenza a virus, as was seen in that pa and ns genes of b ⁄ brisbane ⁄ ⁄ reassort together with the ha and na genes of the same parent (table ). in our recent study on a reassortment between b ⁄ lee ⁄ and b ⁄ panama ⁄ ⁄ , it appeared that ha shapes overall gene constellations of the resultant reassortants, namely the reassortants tend to have more internal genes from the same parent of ha, no matter which parent's ha is selected by antibodies against the surface antigens of the other parent (data not shown). because of success in influenza a virus reassortment with pr , it is generally believed that reassortant with : or : configuration is optimal for virus yield. this may be the case in most instances of influenza a reassortment, but is not necessarily so in b reassortment. as shown in this study, only a single donor gene is capable of improving the yield of vaccine strain by reassortment. influenza a ⁄ h n v has spread rapidly in all parts of the world in as a true pandemic. epidemic events in russia occurred during the last week of september starting from far east region (yuzhno-sakhalinsk). kaliningrad (the western most russian city) was the second starting point of the epidemic. during october the epidemic spread over the whole russian territory. in a short period the new virus started to change genetically as it began to adapt to human populations during this pandemic (http://www.who.int; http://www.euroflu.org). in the period from may to december , clinical samples (nasopharyngeal swabs and postmortem materials) of patients with influenza-like illness from different regions of russian federation were analyzed to confirm the diagnosis using real-time reverse transcription pcr (rrt-pcr). clinical nasopharyngeal swabs and bronchoalveolar lavage and post mortal fragments of trachea, lungs, bronchi, spleen from saint petersburg hospitals and basic laboratories of federal influenza center were included in this study. all specimens were taken from patients with influenza-like illness or viral pneumonia. specimens were tested by rrt-pcr according to cdc protocols, i.e. using superscript iii platinum one-step qrt-pcr system (invitrogen) with primers and probes for infa, h seasonal, and h sw (biosearch technologies). in addition, the test-systems 'amplisense influenza virus a ⁄ b-fl' and 'influenza virus a ⁄ h -swine-fl' for pcr-detection, typing and subtyping of influenza viruses were also used. these test-systems are produced by central institute of epidemiology, moscow, russia and recommended by russian ministry of health as tests for influenza diagnosis. sequencing was carried out on an abi prism -avant genetic analyzer (applied biosystems, usa) with bigdye terminator cycle sequencing kit. phylogenetic analysis was performed using programs vector nti . (invitrogen) and mega . (psu, usa) by maximum likelihood with the tim+i+g model for ha, and -hky+i+g model for na. evolutionary model was selected by akaike information criterion (aic) in model-test (posada, crandall, ). statistical reliability of tree branches was evaluated by bootstrap test ( replications). immunohistochemical study was performed using novalink antibodies to ha and np with novocastra visualization system. influenza virus a ⁄ h n v rna was detected in patients with severe form of influenza-like illness and fatal cases. out of pcr-confirmed flu recovered cases % were patients under years of age, % were aged - years, and % were older than years. mean age of recovered patients was ae years (from month to years). viral rna in postmortem materials was detected mostly in lung tissue ( % of specimens) and trachea fragments ( %), and less commonly in spleen ( %). mean age of the deceased with confirmed flu (h n v) infection was ae years with age ranging from months to years. in % of fatal cases, influenza was complicated by viral or secondary bacterial pneumonia. median time from the onset of illness until death was days. according to our data, % of patients died had diabetes, ae % were obese, and % were pregnant women in the nd or rd trimester. ha and np were detected by immunohistochemical assay in lung tissue of dead patients with confirmed influenza virus a ⁄ h n v infection. ha and np was revealed in the endothelium of different sized blood vessels (capillaries and arterioles). these influenza virus proteins were also detected in some tissue macrophages apart from epithelium and endothelium. the localization of the two proteins was different: ha is mostly localized in cell membrane and cytoplasm, and np -mostly in the nucleus. here we present data on molecular genetic characteristics of strains of pandemic virus, strains obtained from clinical specimens, and from post mortal ones isolated in the research institute of influenza. all the strains studied contain the s n substitution in m protein, which indicates resistance to the adamantane antivirals, and have no h y substitution in the neuraminidase, which indicates resistance to oseltamivir. the phylogenetic analysis showed that russian viruses were similar to influenza viruses a ⁄ texas ⁄ ⁄ and a ⁄ california ⁄ ⁄ (ha similarity ae %). all russian viruses could be divided in two clusters: the first one includes viruses similar to the reference strain a ⁄ california ⁄ ⁄ , and the second one, which is the majority of viruses analyzed includes strains with substitutions ha s t, na n d, v i, and ns i v (figure ). bootstrap support was . the isolates with ha s t substitution can be classified in one of the five minor genome variants of a ⁄ h n v viruses found in the united states and mexico in . several viruses had strain-specific substitutions in antigenic sites sb and ca and the mutation d g in ha receptor-binding site. the substitution of amino acid residue asp to gly at position of ha was found in eight of eleven isolates ( %) from postmortem lung and trachea samples and two of forty isolates ( %) from nasopharyngeal swabs of patients with severe course of the disease. appearance of amino acid substitutions in the ha receptor-binding site (d e and d g ⁄ e) could be associated with influenza virus passaging on eggs. five strains that contained g at position of ha were isolated from post mortal specimens on mdck cells in this study, thereby excluding the possibility of substitution appearance hence to virus adaptation on eggs. in order to reveal genome changes in a(h n )v, strains isolated on the territory of russian federation during the pandemic, full genome sequences from genbank, and research institute of influenza database were analyzed comparing two groups of viruses (isolated before and after sept ). nine amino acid changes observed predominantly in late pandemic strains were found. five of them (s p, s n, d g, v i, v i) reside in ha, two in na (i v, n k), two in pb (k n, t i), and one in pa (f l). towards the end of the epidemic the viral population had demonstrated statistically certain rise in number of strains containing mutations in four genes. difference between groups was statistically significant (chisquare test, p = ae ). if v > ae , than difference between early and late strains is statistically significant. additionally fisher's test determined whether 'early strains' and 'late strains' differ significantly in the proportion of 'no mutation event' and 'mutation event' attributed to them in each particular position. all calculations were performed in fisher_tk freeware by vladimir belyaev similar to calcfisher (haseeb, ) fully described here (http://www.jstatsoft.org/v /i /paper). we have selected positions with statistically significant amino acid changes in late strains (p-value ae ). according to full genome analysis of influenza virus a ⁄ h n v strains, seven clades were distinguished, but the divergence between representatives of different clades remained small. (figure ). besides the strain a ⁄ perth ⁄ ⁄ also contains substitution s f in the same ha antigenic site. according to data obtained, the epidemic in russia was caused only by influenza virus a ⁄ h n v. unlike the previous epidemic periods when most severe influenza cases were registered among the children under years and among elderly people aged over years, the first wave of pandemic due to influenza virus a (h n )v resulted in increased level of mortality mainly among the people aged - years. though all pandemic viruses showed comparative genetic homogeneity, some evolutionary trends could be outlined. for clarification of the exact pathogenic role of mutation d g in ha receptor binding site, further studies are necessary. full-genome analysis of influenza virus a ⁄ h n v strains circulating in the southern hemisphere in the new epidemic season revealed the phylogenetic subgroup distinguished by seven substitutions in inner proteins (pb , pb , np, ns ) and sa antigenic site of ha (n d). the changes revealed could be caused by adaptation of the virus to an immunized human population. nasal and throat swabs (placed in ml mem and frozen at ) °c until use for viral rna extraction and tissue culture inoculation) were collected from patients with febrile illness, i.e., > ae °c. samples were received from clinics in us embassies and us military laboratories located throughout the world since the initial who declaration of novel h n outbreaks as a global pandemic on june , . viral isolates were obtained from inoculating cultures of mdck cells with ae - ae ml viral suspensions collected in mem originated from patients after - days incubation. [ ] [ ] [ ] [ ] due to low viral titers in normal clinical samples, most of full viral genome sequences were derived from viral stocks obtained by tissue culturing passages (mdck, - times). viral rna was extracted from clarified supernatant fluid of nasal ⁄ throat swabs or mdck cultures using the 'charge-switch' rna extraction system based on the user manual protocol from the manufacturer (invitrogen inc., ca, usa). total rna was eluted into volume equal to original sample volume, i.e., ll starting viral supernatant used to yield final ll rna in molecular grade water (invotrogen inc.) and stored at ) °c until tested. generating ⁄ preparing overlapped cdnas for full genome coverage of novel h n viruses by multiple rt-pcr amplifications the first step in the high-throughput sequencing pipeline for full influenza genome sequences was to establish a robust rt-pcr amplification scheme consisting different rt-pcr primer pairs covering all rna segments to ensure % amplification coverage of full viral genomes of all the incoming targeted viruses (houng, hs. , submitted for publication). extracted viral rna ( ll), derived from mostly mdck culturing stock or clinical sample containing sufficient viral load (> infectious units per ml) was added to primer-free rt-pcr total master mixture ( ae ll) for each virus followed by adding primer pair ( ll, pmole ⁄ ll per primer). rt-pcr was then performed: rt reaction through two hold-steps ( °c, minutes and °c, minutes); cycling amplifications ( °c for seconds, °c for seconds, °c for ae minutes). specific cdna amplicons corresponding to each individual primer pair were routinely monitored and visualized by agarose gel electrophoresis. pooled cdna products ( - lg) from each viral rt-pcr amplification run were used as sequencing substrates according to the roche flx user manual and bulletins by incorporating adaptors containing individually multiplex identifier [mid]-key assigned to each individually pooled viral cdna. up to different mid-keyed viral cdna were further pooled together to be clonally amplified on capture beads in water-in-oil emulsion micro-reactors (em amplifications), and pyrosequenced using one of two regions of a · mm picotiterplate. for each individual viral genome containing multiple assemblies ( rna segments), we obtained sff file(s) containing raw sequencing reads from which nucleotide sequence data and phredlike quality scores were extracted. on average, ae - ae % of - million mid-key specific nucleotides were extracted and mapped for consensus genome sequences. roche gsmapper (v. . and . ) software was used to assemble all sequencing raw data and sff files into consensus sequences. new reference mapping projects were created to assemble each individually mid-keyed viral cdna into consensus viral sequences. one of the earliest h n genomes of california origin, a ⁄ california ⁄ ⁄ (h n ), deposited in genbank, was routinely employed as a reference genome sequence for most of gsmapper projects. the resultant consensus sequences obtained were further verified and validated through the ncbi annotation utility check and ultimately deposited to the ncbi influenza database, genbank. nucleotide sequences specific to each individual rna gene were aligned by the geneious pro . . software (http://www.geneious.com). trees were built based on the tamura-nei genetic distance model using the neighbor-joining method with no outgroup used via geneious pro . . . phylogenetic trees of the h n genomes were constructed by importing fasta files containing specific concatenated target sequences of pb , pb , pa, ha, np, na, mp, and ns from each individual virus into the geneous pro software and going through the sequence assembling and tree building steps. high-throughput pyrosequencing of pooled novel h n cdnas by roche flx system up to viral cdnas could be routinely sequenced to completion for different full viral genomes from a single roche flx picotiter plate by utilizing the combination of pico-titer plate's two distinct regions as well as different mid-keyed adaptors. the 'shotgun' sequencing approach employed in this study is a feasible method to viral isolates (n) sequence multiple pooled h n viral genomes. for each pyrosequencing experiment, approximately - passed key reads (single fragment per bead) were obtained that yielded readable nucleic acid sequences. among those close to a million passed key reads, only - passed key reads had an average sequencing read length of > bps, defined as 'long reads' ( bps · reads = total of million bases of nucleic acid sequences) that were used to assemble into influenza genome sequences. mathematically, - million bases of raw sequencing data from each single roche flx experiment would provide sufficient sequencing bases to cover full genome sequences with approximate - · of sequencing depth coverage of influenza a with average genome size of bps for the total of eight segmented rnas. so far, more than full h n genomes sampled worldwide have been successfully sequenced and deposited in the ncbi database by division of viral diseases, walter reed army institute of research (wrair). the bioinformatics derived from unique viral genome sequences generated from this study based on constant rt-pcr amplification scheme and identical roche pyrosequencing protocols provide a reliable data set in predicting the evolutionary patterns of pandemic viruses. wrair received clinical samples from us embassies and military personnel throughout the world since the initial who announcement of novel h n outbreaks. nearly equal distributions of sequenced viruses derived from three broadly categorized geographic regions, north america, central ⁄ south america, and asia ⁄ europe ⁄ africa (data not shown). besides the geographic distribution pattern of viral isolates, figure displays the viral isolation time lines of all the sequenced viruses reflecting two peaks that coincided with two waves of pandemic infections, early-mid summer and fall of . phylogenetic trees of the eight influenza a segments of all sequenced viruses were tentatively generated. it was found that the substitution frequencies per site for the ha, na, and ns genes are at much higher rate than the other five genes, pb , pb , pa, np, and mp genes (data not shown). the observed higher genetic variations for ha and na genes of h n are consistent with the historical genomic and epidemiological dynamics data of human influenza a revealing higher temporal fluctuations in ha and na genes. [ ] [ ] [ ] [ ] analysis of full influenza genomes containing concatenated eight complete rna segments revealed the existence of two distinctive genetic clades in circulation since the beginning of pandemic, as shown in fig-ure . it is noteworthy that all viruses of mexico and california origins (clade shown at the top of figure ) were isolated at the beginning of pandemic prior to the isolation of all other viruses belonging to the second genetic clade . , discussion during the past decade, the advance of dna sequencing technology, such as development of ngs, in making full viral genome sequences readily available have enabled study of far broader and more detailed aspects of evolutionary change for any new emergent infectious pathogen. the massive sequencing capacity of roche flx system allows simultaneously process and sequence millions of individual cdna molecules, in contrast to processing and sequencing individual cdna fragments by conventional sanger sequencing method. within a short period of few months since the beginning of the pandemics, wrair accomplished large number of representative h n full genomes of worldwide origins via roche flx system. sequencing data derived from this study illustrates a much higher genetic variation rate for ha and na genes of h n that is compatible to the higher temporal fluctuation rate for ha and na genes of seasonal influenza a derived from decades of intensive monitoring and comparison studies and analyses. [ ] [ ] [ ] [ ] following the mexican and us reported cases, confirmed outbreaks of swine h n rapidly proliferated and spread throughout europe, asia, africa, and south america, most probably via global airline travel. , it seemed that new cases in the us and most cases throughout the world had been clinically mild relative to the initial reported cases in mexico. [ ] [ ] [ ] [ ] here we demonstrate through the phylogenetic relationship of sequenced h n full genomes that the clinical isolates could be divided into two different clades of viruses, i.e., the clade genetic group contains only viruses isolated at the beginning (march ⁄ april , mexico and california) of pandemics and the rest of other viruses all belong to the nd genetic group, clade . thus, it's likely that the currently circulating h n of clade causing worldwide infections is genetically different from the initial h n isolates that caused the early infections in mexico and california. , introduction a pandemic influenza virus ( h n ) was recently introduced into the human population. the hemagglutinin (ha) gene of h n is derived from 'classical swine h n ' virus, which likely shares a common progenitor strain with the human h n virus that caused the pandemic in . since antigenic changes of influenza virus ha occur more slowly in swine than in humans, we hypothesized that h n might still retain an antigenic structure similar to that of h n or the early isolates of its descendants. in this study, we compared ha antigenic structures of h n and human h n viruses by a molecular modeling approach to demonstrate the existence of shared epi-topes for neutralizing antibodies. we found that has of h n and the h n virus shared a significant number of amino acid residues in known antigenic regions. from this observation, we hypothesize that the h n ha antigenic sites will be targeted by antibody-mediated selection pressure in humans in the near future. we further discuss possible directions of antigenic changes in the evolutionary process of h n . sequence data of ha genes modeller v was used for homology modeling of ha structures. after models of the ha trimer were generated, the model was chosen by a combination of the mod-eller objective function value and the discrete optimized protein energy statistical potential score. after addition of hydrogen atoms, the model was refined by energy minimization with the minimization protocols in the accelrys discovery studio . software package using a charmm force field. steepest descent followed by conjugate gradient minimizations was carried out until the root mean square gradient was less than or equal to ae kcal ⁄ mol ⁄ a. the generalized born implicit solvent model was used to model the effects of solvation. the ha model was finally evaluated by using procheck, whatcheck, and verify- d. custom-made programs were developed with the ruby language and used for investigating the numbers of potential n-glycosylation sites and candidate codons (cand ) in ha sequences. it is known that the h ha molecules have four distinct antigenic sites: sa, sb, ca, and cb. , as a result, these sites consist of the most variable amino acids in the ha molecule of the seasonal human h n viruses that have been subjected to antibody-mediated immune pressure since its emergence in , although it was absent in humans from to . to investigate the structures of these antigenic sites of h n , d structures of the ha molecules of sc , the recent seasonal human h n virus (br ), and h n (ca ) were constructed by a homology modeling approach, and compared by mapping all the amino acid residues that were distinct from those of sc ha (data not shown). we found that most of these antigenic sites of br ha predominantly contained altered amino acid residues if compared with sc . by contrast, amino acid residues at these positions were relatively conserved in ca ha when compared with sc ha. notably, the sa and sb sites, which contain many amino acids involved in neutralizing epitopes near the receptor binding pockets, remain almost intact ( table ), suggesting that antibodies raised by natural infection with sc or its antigenically related descendant viruses play a role in specific immunity against ca . these observations lead us to hypothesize that such antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in the human population. based on this hypothesis, we speculated that h n would undergo patterns of amino acid substitutions in ha similar to those seen in seasonal human h n viruses during its epidemic period (i.e. those that have been substituted since ) (figure ) . we then predicted possible amino acid substitutions of h n from the sequence similarity of the antigenic sites. for example, both sc and ca had an asn residue at position in the sa site. for sc , the residue at this position has altered from asn to lys since . combining these two facts, it seems reasonable to hypothesize that ca will also undergo an amino acid substitution from asn to lys at position in the future. interestingly, we found that some of the recent variants of the h n virus have indeed undergone substitutions identical to those predicted in figure . it is important to monitor whether such variants will be selected and survive in sustained circulation in humans. next, we analyzed the acquisition of potential n-glycosylation sites associated with antigenic changes. previously, we reported that cand sites, a set of three codons that require single nucleotide substitution to produce n-glycosylation sequons, were important motifs to rapidly acquire n-glycosylation sequons. therefore, we investigated the number and location of potential n-glycosylation sites and cand sites in h n ha. we found that ca also had a single n-glycosylation sequon at the same position in the globular head region of ha, and lacked the multiple n-glycosylations that have been observed in the antigenic changes of the human h n virus during the early epidemic of this virus. we also found that ca ha possessed three cand sites that were present at the same position in sc ha (positions of the first asn residue, , , and ). of these, the cand sites with positions at and had actually become potential n-glycosylation sites in human h n viruses. this result suggests the likelihood of additional n-glycosylation at these sites during future antigenic changes of h n ha. notably, some of the recent h n variants (as of march , ) have an additional n-glycosylation sequon at position , where the h n virus readily acquired an n-glycosylation site during its circulation. the present study suggests that the antigenic structure of h n ha is similar, at least in part, to that of the h n ha. the and h n has share unique three-codon motifs that are important to readily acquire n-glycosylation sequons in their globular head region. based on these similarities, we predicted possible amino acid substitutions that might be associated with future antigenic changes of h n , and confirmed that such substitutions occurred in some of the recent variants of this virus. the present study provides an insight into likely future antigenic changes in the evolutionary process of h n in the human population. influenza viruses are classified into three types, a, b, and c, based upon the antigenic properties of nucleoproteins and matrix proteins. influenza a virus infects a wide range of hosts, including human, bird, swine, equine, and marine mammal species, while influenza b and c are less pathogenic than influenza a and are mainly found in humans, although there is evidence that they can also infect other species. influenza a has evolved in association with its various hosts on different continents for extended periods of time. to survive as a successful pathogen, the influenza viruses have developed a number of mechanisms, including antigenic mutation and genome reassortment, to continuously evolve and evade the surveillance of the host immune systems. antigenic and genetic analyses have provided important insights into the molecular dynamics of influenza virus evolution. however, a comprehensive understanding of influenza viral genetic divergence and diversity remains lacking. neuraminidase (na) is a major surface glycoprotein of influenza a and b, but is absent in influenza c. it plays a key role in virus replication through removing sialic acids from the surface of the host cell and releasing newly formed virions. influenza a viral na genes are classified into nine subtypes (na -na ) based upon their antigenic properties, while na genes of influenza b are not classified into subtypes. furthermore, most na subtypes of influenza a have evolved into distinct lineages and sub-lineages, which correspond to specific hosts or geographical locations. in this study, we conducted large-scale analyses of influenza na sequences in order to infer their evolution and to identify lineages (or sub-lineages) of influenza a viruses. a total of na sequences that excluded laboratory recombinant sequences were downloaded from genbank. sequences were aligned with muscle and mafft. the alignments were adjusted manually using translatorx, based upon corresponding protein sequences. phylogenetic analyses were conducted using the maximum-likelihood (ml) method in raxml. a set of perl scripts were written by us to facilitate this computational analysis. lineages and sub-lineages were determined based on the topology of the ml trees. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, but with the following modifications: a single digit is used to represent one of the nine subtypes and a letter is used to represent a lineage; a sub-lineage is also represented using a digit; a dot is used to separate a lineage and a sublineage. for example, a. means na subtype, lineage a, and sub-lineage . the time of most recent common ancestor (tmrca) was estimated using the bayesian mcmc method in beast. in all cases, we employed the gtr + u nucleotide substitution model, in which the first and second codon positions are allowed different rates relative to the third codon position. all data sets were analyzed under a relaxed molecular clock and the bayesian skyline population coalescent prior. the maximum clade credibility (mcc) tree across all plausible trees was computed from the beast trees using the treeannotator program, with the first % trees removed as burn-in. phylogenetic analysis based upon na sequences revealed two large groups corresponding to influenza a and b, respectively ( figure a ). within influenza a, two subgroups were found, one consisting of na , na , na , and na and the other consisting of the remaining five subtypes. subtype na was found to be a sister subtype of na , na being a sister subtype of na , and na a sister subtype of na . finally, each na subtype forms a distinct cluster, indicating its genetic uniqueness. influenza a and b viral na were estimated to have diverged around years ago ( figure b ). however, it had large % hpd values which ranged from years to years ago. the na subtypes of influenza a diverged from more than to several hundred years ago. the time of most recent common ancestor (tmrca) of each subtype of influenza a virus was generally recent and ranged from the calendar years to (figure b ). in addition, the tmrca for influenza b viral na was dated back to . a total of lineages were identified in influenza a (table ) . three lineages, a, b, and c, were identified for na based upon the tree topology. linage a originated from avian viruses and was further divided into sub-lineages: a. , a. , a. , a. , and a. . linage b consists of north american swine influenza viruses whereas c is a human lineage. two large lineages, a and b, were identified in na . lineage a is a human-specific lineage. interestingly, five major swine clades were observed within this lineage. lineage b is an avian-specific lineage, and consists of sub-lineages, b. , b. , and b. . three lineages were found in na . lineage a was found in north american avian, b in eurasian ⁄ oceanian avian, and c also in avian, but it does not show any geographical pattern. for na , na , and na , each was classified into lineages, one found in north american avian ( a, a, a) and the other in eurasian ⁄ oceanian avian ( b, b, b). three lineages identified respectively in na and na are north american avian ( a, a), equine ( b, b), and eurasian avian ( c, c). na was also found to have lineages: north american avian ( a), eurasian ⁄ oceanian avian i ( b), and eurassian ⁄ oceanian avian ii ( c), respectively. in this study, we conducted large-scale phylogeny and evolutionary analyses using influenza viral na sequences. the results showed that divergence between influenza a and b viruses occurred earlier than between any influenza a subtypes. this observation was consistent with previous findings based upon phylogenetic analysis of the ha gene, one of the most important genes related to host infection. within influenza a, two sub-groups were found, one consisting of na , , , and and the other consisting of the rest of five subtypes (na , , , , ) . this observation does not agree with the result described by liu et al., where na subtypes , , , , and formed one group and the remaining four subtypes (na , , , and ) formed the other group. this difference is apparently caused by the fact that an outgroup was not used in their phylogenetic analyses. in the present study, both influenza a and b viral na sequences were included in the analysis. high bootstrap values were obtained for major groups, indicating that the inferred evolutionary relationship should be highly reliable. classification and designation of the lineages and sublineages within the influenza a virus are essential for studies of viral evolution, ecology, and epidemiology. a total of lineages were identified within nine influenza a viral na subtypes and with the majority of the identified lineages found to be host or geographic specific or both. our results demonstrated a comprehensive view for the evolution of na genes and provided a framework for the inference of evolutionary history of pandemic viruses and for further exploring of viral circulations in multiple hosts. for example, the global pandemics of human h n in , h n in , the pandemic of human h n virus in , the crisis of h n hpai in hong kong in , and swine-origin h n influenza in , all can be mapped onto the lineages and sub-lineages identified in this study. such information will facilitate not only identification of known genetic origins but also early detection of novel influenza a viruses. influenza viruses constantly evolve to avoid the human immune pressure in the process of antigenic drift. through sequencing of viral genomes, the rates and direction of virus evolution can be observed. moreover, comparison of protein sequences allows us to determine amino acid substitutions that are related to immune pressure and antigenic drift. the creation of global influenza genetic databases, along with concurrent development of analytical tools, allows the comparison of multiple influenza virus strains. the main aim of this study was to perform antigenic and genetic comparison of pandemic influenza viruses (h n ) isolated during the - pandemic in ukraine and in other countries. nasopharyngeal swabs and autopsy materials collected from infected patients were received from the areas of ukraine. in addition, field isolates of influenza viruses from the ⁄ season and strain specific serum were used for identification by hemagglutinin inhibition assay. influenza viruses were identified and subtyped using real-time rt-pcr analyses using cdc primers and adopted protocols. sequencing was performed in two world health organization (who) influenza collaboration centers (centers for disease control and prevention, atlanta and national institute for medical research, london). hemagglutinin inhibition assay was conducted using chicken and guinea pig red blood cells following standard who protocols. the all ukrainian isolates of influenza viruses, which were isolated in ukraine during august-november , were identified as a ⁄ california the phylogenetic analyses confirmed the evolutionary relationship between ukrainian isolates and viruses from other countries, which were isolated during the first wave of the pandemic. high genetic and antigenic conservation of pandemic influenza viruses from ukraine and other countries also were demonstrated. considering that the emergence of the novel pandemic influenza strain occurred in countries of northern hemisphere during summer, it was very interesting and significant tracking the dynamics of genetic changes in influenza viruses, which were isolated at the beginning of epidemic and those isolated during the rise of the epidemic in ukraine. influenza a virus causes moderate to severe epidemics annually and catastrophic pandemics sporadically. due to the evasiveness of the influenza virus and the nature of its genome (eight single-stranded and negative-sense rna segments), it is essential to understand the evolution of this important pathogen. influenza virus evolves by two major mechanisms: mutation and reassortment. antigenic and genetic analyses have revealed partially the molecular dynamics of influenza virus evolution. , however, important questions, such as how many genotypes in the influenza a virus, remain unanswered. one of the major issues pertaining to this genotyping problem is how many lineages or sub-lineages can be determined for a subtype and according to what criteria. because of the unique structure of the influenza a viral genome, the computational genotyping methods developed for other viruses cannot be applied to the influenza virus. constructing phylogenetic trees is a powerful technique for the identification of evolutionary groupings (i.e., lineages ⁄ clades). however, for large trees, it is hard to determine how many lineages and the boundaries for each lineage. in this regard, multivariate analysis methods, such as multidimensional scaling (mds) and model-based hierarchical clustering, both taking advantage of dimension reduction and visualization, can complement conventional phylogenetic methods. hemagglutinin (ha), the fastest evolving segment, is recognized as the most important gene in the influenza virus that plays a key role in viral pathogenesis. however, we have only limited knowledge of lineages and sub-lineages occurring in the hemagglutinin (ha) gene of influenza a virus, although much effort has been made in assigning clades or sub-clades in highly pathogenic avian influenza (hpai) virus ha. in this study, both model-based hierarchical clustering and phylogenetic methods were used for sequence analysis. one objective for this study is to explore and develop a more accurate lineage approach for further comprehensive influenza lineage and genotype analyses. a total of hemagglutinin (ha) sequences (approximately nucleotides long), excluding laboratory recombinant sequences, were downloaded from genbank as of march, . sequences were aligned with muscle and mafft. the genetic distance matrix of all pairwise sequences was computed using the k p model under mega . . we then used the distance matrix as input to the cmdscale module in r . . for the mds analysis. the principle coordinates resulting from mds were used for the model-based hierarchical clustering analysis, again in r . . (the r foundation. available at: http://www.r-project.org/). the bayesian information criterion (bic) values were computed based upon ten different statistical data models -eii, vii, eei, evi, vei, vvi, eee, eev, vev, and vvv. the highest bic value was used to determine the number of clusters in the given sequence data. phylogenetic analysis was conducted using maximumlikelihood (ml) in raxml. raxml uses rapid algorithms for bootstrap and maximum likelihood searches and is considered one of the fastest and most accurate phylogeny programs for large-scale sequence analysis. all the analyses were conducted on the supercomputer cluster (holland computing center, http://hcc.unl.edu/main/index.php). the trees were visualized in figtree (version . . ) . lineages and sub-lineages were determined based on both the topology of the ml trees and model-based clustering results. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, with the following modifications: lineage analysis was conducted for each ha subtype, which agrees with the convention of influenza virologists that ha subtypes were identified in influenza a virus; ha lineages are represented with digits and letters, where the digit(s) represent one of the subtypes and a letter represents a lineage; here, we present sub-lineages or sub-sub-lineages also in digits, with smaller numbers representing earlier lineages or sub-lineages within the same subtype (e.g., lineage occurs earlier than lineage ); the digit is used to indicate inclusion of ancestral viruses in a lineage (or sub-lineage); a dot is used to separate lineages, sub-lineages, and sub-sub-lineages. for example, a. ae means ha subtype, lineage a, and sub-lineage , and sub-sub-lineage . the sub-lineage level can be extended as necessary. the model-based clustering method corroborates commonly used phylogenetic methods in lineage and sub-lineage assignment. here we use the h subtype as an example to show the lineage and sub-lineage assignment. the bayesian information criterion (bic) reaches its maximum when the number of clusters for h equals , regardless of which mode we choose ( figure a ). therefore, based on bic, the optimal number of clusters for the h subtype is . as a result, a total of clusters based upon the vvv model were identified ( figure b) . a significant correlation was found in lineage assignments by the phylogenetic method and the model-based hierarchical clustering method ( figure b,c) . lineages a and b were identified for h , which correspond to north american avian and eurasian avian, respectively. lineage a was further divided into sub-lineages, a. , a. , where a. is the ancestral sub-lineage in a. based on both model-based hierarchical clustering and phylogenetic analyses, a total of distinct lineages were identified among subtypes, averaging out to be ae lineages per subtype ( table ). the majority of the identified lineages were found to be host or geographic specific or both. for example, three lineages, a, b, and c, were identified for ha . lineage a was further divided into two sub-lineages, a. and a. . the a. is swine-specific, whereas a. is a human pandemic h n sub-line- how to accurately identify an evolutionary lineage of influenza a viruses is challenging. one commonly used approach is molecular phylogeny, where phylogenetic trees are constructed, and the tree topology is used for lineage determination. here, we used a bayesian model-based clustering method, along with phylogenetic methods, to decide lineages and sub-lineages of influenza a viruses based upon sequence data. the results demonstrated that the modelbased clustering method corroborates phylogenetic methods and increases the accuracy of lineage assignment. one salient feature of this study is its large-scale analysis of all available influenza a hemagglutinin sequences. a total of distinct lineages and sub-lineages were classified; the majority of them were found to be host or geographic specific. this observation agrees largely with previous findings. we are conducting further analyses of other influenza a segments and expect to identify their lineages and create a comprehensive genotypes database for all influenza a viruses. such information will allow us to detect the genetic origin of newly found viruses, track their genetic changes, and identify potential genome reassortments. a hierarchical nomenclature system has been proposed and adopted for hpai ha clades and sub-clades by who influenza surveillance centers. wan et al. also proposed a hierarchical approach for influenza a viral genotypes system. the work presented here is one of the first steps towards the development of a nomenclature system for influenza a virus lineages (at the segment level) and genotypes (at the genome level). whether the naming system will be accepted and used by the influenza research community is more challenging than the lineage analysis itself. identification of the genetic origins of influenza a viruses will enhance our understanding the evolution and adaptation mechanisms of influenza viruses. the phylogenetic analysis is the traditional approach to identify the influenza progenitor. first, the nucleotide sequences are aligned using multiple sequence alignment methods, such as clustalw, muscle, and t-coffee. second, phylogenetic analysis is performed on these aligned sequences to infer their evolutionary relationship using neighbor-joining (nj), likelihood, or bayesian inference. bootstrap analyses or computation of posterior probability are usually applied to estimate the phylogenetic uncertainty. however, this phylogenetic analysis is time consuming due to intensive computations in multiple sequence alignments and phylogenetic inferences. it is difficult to perform an analysis using this method on a large dataset, for instance, with more than taxa, as is the common case for influenza studies. alternatively, blast is applied to identify the prototype genes in the database. blast determines a similarity by identifying initial short matches and starting local alignments. since influenza viral sequences have very high similarities, especially for most conserved regions, blast usually generates a large number of outputs, which will not be helpful for progenitor identification. since blast is a local sequence alignment, the results from blast may not reflect the global evolutionary information between the sequences. the blast scores cannot be used to define the evolutionary relations between viruses, especially in the context of the entire genetic pool. recently, we have developed a distance measurement method, complete composition vector (ccv), that can calculate genetic distance between influenza a viruses without performing multiple sequence alignments. , we also adapted the minimum spanning tree (mst) clustering algorithm for influenza reassortment identification. the application of this approach in the analyses of pb genes of influenza a virus showed that the integration of ccv and mst allows us to identify the potential progenitor genes rapidly and effectively. based on these results, here we develop a webserver called ipminer for influenza progenitor identification. ipminer can identify potential progenitors for a query sequence against all public influenza datasets within a few minutes. in order to improve the computing efficiency, distance matrices were pre-computed by ccv, and they include for ha (h to ), for na (n to n ), and one for each of the internal gene segments (pb , pb , pa, np, ns, and mp). these pre-computed matrices will be updated weekly. ipminer just needs to compute the query matrices for a query sequence and sequences in the database. the standalone ccv program is also available at http://sysbio.cvm.msstate.edu/ipminer. in order to identify the influenza progenitor genes, ipminer first integrates the query matrix and a corresponding pre-computed matrix into a full distance matrix, which is then clustered by mst clustering algorithm. we adapted the threshold we measured previously in mst, u + nr, where u is the average distance and r is the standard deviation of a cluster. as a result, mst will generate a hierarchical structure for the clusters. in each cluster, we will randomly select viruses or % of the cluster size if this cluster has more than viruses. ipminer will return the viruses with the smallest distances when the search reaches to the lowest level (the largest n) in this hierarchical structure. our analyses have shown that the level has generally yielded good results for influenza a viruses. to visualize the overall mst structure, ipminer applies multi-dimensional scaling (mds) method to project all the viruses in the genetic pool onto a two dimensional graph, and the precursor viruses are marked in different shapes ( figure ). the users can select other prototype viruses from the graph for further phylogenetic analyses. a single job with one query sequence takes < min. the genbank identifiers and associated genetic distances and sequence identities are displayed. the users can download the sequences for the identified precursor viruses as well as those from the prototypes viruses. in addition, for the users' convenience, ipminer generates a phylogenetic tree using nj method implemented in phylip to illustrate the phylogenetic relationship among the query sequence(s), the identified progenitors, and the selected prototypes viruses. the programs in this solution package are written in java. the shell scripts are written in korn shell script in order to achieve high performance. cascading style sheets (css) are used for a consistent look across the pages. this also enables to change the overall design just by replacing the css definition file. php has been used as server side scripting and is written in java. in order to achieve high performance for computing in a genomic scale, we apply hash function or a binary tree, which enables that the precursor identification has a time complexity of o(n). for single queries, the users can visualize the results online. for batch queries of multiple sequences, the results will be sent to the users by e-mail. ipminer has been tested on microsoft internet explorer, mozilla firefox, and safari. the users need javascript to obtain full function of ipminer server. the webserver is available at http://sysbio.cvm.msstate.edu/ipminer. in summary, ipminer webserver has three major computational features for influenza progenitor identification: (i) it calculates the genetic distances through ccv and identifies the viruses with the shortest ccv distances against the query virus to be the progenitor genes; (ii) it projects influenza viruses onto a two dimensional map, which illustrates the global relationship between the progenitor genes and other viruses in the genetic pool; and (iii) it performs phylogenetic analyses between the query virus, the identified progenitor genes, and other selected prototype viruses. ipminer provides a user friendly web service for influenza progenitor identification in real time. the gisaid initiative offers an alternative to current public-domain database models in response to growing needs of the global influenza community for the sharing of genetic sequence and associated epidemiological and clinical data of all influenza strains. gisaid's publicly accessible epifluÔ database is governed by a unique sharing mechanism that protects the rights of the submitter, while permitting ongoing research as well as the development of medical interventions, such as drugs and vaccines. for the gisaid initiative, the max planck institute for informatics (mpii) saarbrücken, germany, has developed a web portal that is accessible at http://www.gisaid.org featuring the gisaid epifluÔ database that offers a unique collection of nucleotide sequence and other relevant data on influenza viruses. the database is based on software by oracle and the dante Ò system by a systems gmbh, germany. extensive metadata are also collected for most isolates. the database provides features for searching, filtering specific datasets for download, and user friendly upload functionality. to uphold gisaid's unique sharing mechanism, all users must positively identify themselves. while access is free of charge, all users agree that they will not attach any restrictions on the data, but will acknowledge both the originator of the specimen and the submitter of the data, and seek to undertake to collaborate with the submitter. all uploaded sequence data are submitted to rigorous curation by the friedrich-loeffler-institute for animal health (fli), germany. the database has been live since september , . among its contributors are all five who collaborating centers for influenza who routinely contribute data in addition to using the epifluÔ database for their semiannual vaccine strain selection. to provide a complete picture of data, all data available in the public domain is routinely imported. as of october , , the rapidly growing gisaid dataset comprises nucleotide sequences (from isolates) with (from isolates) uniquely submitted to this database. software development is underway to continually extend the spectrum of available data analysis tools. the intergovernmental process of the nd world health assembly specifically mentions gisaid as a publicly available database for depositing virus sequence data. starting in , germany's federal ministry of food, agriculture and consumer protection will be the long-term host of the gisaid platform. the mpii will continue to develop the portal and database software and enable gisaid to act as a catalyst for the development of advanced bioinformatics software connected directly to the database. gisaid has become an indispensible resource for the international scientific community on influenza. the consortium will expand its activities and offers to catalyze research and development on a wide variety of issues pertaining to risk analysis, drug development, and therapy of influenza. options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - the pandemic h n virus emerged in and spread rapidly throughout the world, principally affecting children and young adults. as this virus is new to the human population, it is important to determine if these influenza infections are more commonly associated with other respiratory pathogens compared to previously circulating influenza strains. co-infecting respiratory viruses may cause increased morbidity in individuals with pandemic h n , and may also be unwanted contaminants in influenza vaccines if original clinical samples containing these adventitious viruses are used to directly inoculate certified cell lines for vaccine production. to examine this issue, stored rna from original clinical samples (nasal swabs, nasal aspirates, throat swabs) from australian and new zealand subjects that were collected in that were positive for pandemic h n and samples collected in that were positive for seasonal influenza by real time pcr assay (using the cdc, usa kits), were subjected to a resplex ii -panel version . (qiagen) pathogen screen. the resplex ii assay detects common respiratory viruses, such as respiratory syncytial viruses (rsv a, b), influenza a and b viruses, parainfluenza viruses (piv - ), human metapneumo-viruses (hmpv), coxsackieviruses ⁄ echovirus (cvev), rhinoviruses (rhv), adenoviruses (adv b, e), coronaviruses (nl , hku , e, oc ), and bocaviruses. resplex ii uses a combination of multiplex rt-pcr, hybridization of pcr onto target specific beads followed by detection using luminex-xmap technology. original clinical samples were received at the center from who national influenza centers, who influenza collaborating centers, and other regional laboratories and hospitals from australia, new zealand, and the asia ⁄ pacific region. most samples were from australia and new zealand. these samples consisted of nasal swabs, nasopharyngeal swabs, nasal washes, throat washes, and throat swabs. all samples were stored at ) °c until rna was extracted. rna was extracted from ll of clinical sample using either the magnapure extraction system (roche, australia) or the qiaxtractor system (qiagen, australia) according to the manufacturer's recommendations with an elution volume of ll and stored at ) °c until used. a ll aliquot of rna was used to amplify the selected influenza virus gene using specific primers and probes as supplied by cdc (atlanta, usa) along with super-script iii platinum one-step rt-pcr reagents (invitrogen, australia). real time pcr detection was performed on a fast system with sds software (applied biosystems, ca, usa). a cut off of a cycle threshold (c t ) of or below was considered positive. resplex ii panel ver . detection the qiagen molecular differential detection (mdd) system was used, which combines qiaplex amplification (multiplex rt-pcr) with detection on the liquichip workstation (luminex's xmap microsphere based multiplexing system) and qiaplex mdd software according to the manufacturer's instructions. a low level cutoff was used ( ) to obtain maximum sensitivity. from the clinical specimens that were positive for influenza from by real time pcr, there were ( %) a(h n ) seasonal influenza viruses, ( ae %) a(h n ) viruses, ( %) b viruses, and ( ae %) viruses which were influenza a positive, but could not be typed. clinical samples from selected to study were all influenza a(h n ) pandemic positive by real time pcr. detection of influenza virus in respiratory samples was much lower with the resplex ii assay (using a low cut off of units) for pandemic influenza a virus ( ⁄ ; sensitivity ae %) and to a lesser extent for seasonal influenza a ( ⁄ ; sensitivity of ae %) and b viruses ( ⁄ ; sensitivity of ae %) when compared to real time pcr. there were relatively few co-infecting respiratory viruses with either pandemic h infections in ( ae %) or seasonal influenza infections in ( ae %) ( table ). the most common dual infection seen with pandemic h n viruses and seasonal b viruses was with cvev ( ⁄ ; and ⁄ ; , respectively) while for a(h ) viruses there were no dominant co-infecting viruses ( table ). in one case was detected with three respira- tory pathogens in the same sample, a year old female who had pandemic h n , cvev, and rhv, and in a seasonal influenza sample, one case with a triple infection was detected (bocavirus, piv and influenza b). the median age of subjects with co-infections was younger for both pandemic h n with a median age of years (range: months to years), compared to the full sample set which had a median age of years (range: months to years), while for the patients from with seasonal influenza viruses with co-infections they had a median age of ae years (range: months to years) compared to all samples which had a median age of years (range: months to years). there was good concordance in detecting influenza a and b in respiratory samples collected in between real time rt-pcr and the resplex ii system ( % versus > ae % for seasonal influenza a and b respectively). this data compares well with other studies such as li et al. who found that resplex ii had ae % sensitivity and % specificity for seasonal influenza a viruses and ae % sensitivity and % specificity for influenza b viruses. in contrast, the present study found only ae % sensitivity for the resplex ii detection of influenza a with the samples that were positive for pandemic h n by real time rt-pcr. a recent study by rebbapragada et al. also showed lower sensitivity for pandemic h n viruses in nasopharyngeal samples with the resplex ii system ( % sensitivity and % specificity) compared to other commercial platforms seeplex rvp ( % sensitivity and % specificity) and luminex rvp ( % sensitivity and % specificity). interestingly the latest version of the resplex system offered by qiagen the resplex ii plus panel ruo now has a separate target for the pandemic h n virus (mexico ). in terms of detection of other respiratory viruses such as piv- , piv- , rsv and hmpv, high sensitivities ( ae %, ae %, ae %, and %, respectively) and specificities ( ae - %) compared to taqman rt-pcr have been reported from testing of nasal wash and nasopharyngeal clinical samples. in both the seasonal influenza positive and the pandemic h n positive (by real time rt-pcr) clinical specimens, few other respiratory viruses were detected. only of the samples had another virus detectable and one had two other viruses, while in out had another virus and one had two other viruses detected from a total of influenza virus positive samples collected in each year. enteroviruses, coronaviruses, and parainfluenza viruses were most often found with both seasonal and pandemic infections. younger age appeared to be associated with co-infections with those subjects in with dual infections having a median age of only years compared to the study groups years; and similarly for , the median age for subjects with dual infections was only ae years compared to the study groups' median age of years. a study by chong et al. on nasopharyngeal swabs collected during - using resplex ii and luminex xtag rvp fast, they found dual respiratory virus infections in ⁄ ( ae %) of samples and only ( ae %) with triple respiratory viral infections; however, these were from cases with any combination of multiple respiratory viruses not necessarily influenza, although influenza positive cases were the most common respiratory virus detected ( ae % of all positive samples). given the low level and variety of viral co-infections along with both seasonal and pandemic influenza seen in this study, it is unlikely that influenza infections predispose subjects to particular respiratory viruses, but may still allow bacterial colonization, such as has been seen with severe and fatal cases with pandemic h n with various bacteria including streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus, or haemophilus influenzae. , low levels of other respiratory viruses along with the finding that certain cell lines (like the mdck -cells used in this study) do not propagate a number of these viruses (e.g. rsv a and b, rhinoviruses, coronaviruses), but do propagate others (e.g. parainfluenza ) should make testing for unwanted viruses that might be co-isolated with influenza viruses more focused and hence easier to detect and eliminate this isolate for future vaccine production. global influenza surveillance is one of the most important approaches to combat spread of disease. current laboratory methods for characterizing influenza are time-consuming and labor-intensive, and few viral strains undergo full characterization. even fewer strains from domestic poultry and swine or from wild aquatic birds are wellcharacterized. these strains are important for global surveillance since they are thought to be the precursors to pandemic influenza strains. we have designed a highthroughput global bio laboratory to address these surveillance needs. the goal of this project was to develop highspeed and high-volume laboratory capabilities for extensive surveillance and rapid and accurate detection and analysis of influenza. the workflow consists of surveillance, sample transportation, laboratory testing, data management and analysis. five robotic systems have been designed for this laboratory: sample accessioning, biobanking, screening, viral culture, and sequencing. sample accessioning logs barcodes, centrifuges, and aliquots samples are then sent to biobanking. the robotic biobank stores samples at ) °c and reformats tubes for screening. the screening system extracts rna and confirms the presence and subtype of influenza. aliquots of positive samples are sent to the viral culturing system for scale-up. finally, cultured samples are extracted and sent to the sequencing system for full genome sequencing. the sample accessioning, sequencing, and biobanking systems have been built, delivered, and validation processes are currently being completed. robotic screening and culturing systems have been fully designed and are ready to be built. a biosafety level -enhanced containment laboratory was built to enable the flow of samples containing highly pathogenic avian influenza viruses. in full operation, this approach to surveillance is designed to enable the sequencing of up to full virus genomes per year, more than the total of all full influenza genomes sequenced to date. the design of a robotic laboratory for influenza surveillance presents unique challenges and opportunities. before a robotic system is built, each assay is worked out on the bench top, each movement of the plates and reagents is defined, and the laboratory information management system (lims) must be able to address each step of the process. alternate assays are conceived for processes that are not automation-friendly. waste streams, worker safety, and space constraints are considered. each possibility is taken to reduce processes that have the potential to aerosolize or cross-contaminate influenza samples. instruments must be found that fit the capabilities needed. detailed specifications for each of the robotic systems were written including all the parameters listed above. once the systems are built, a long validation process takes place where the processes and instruments in each system are adjusted to function together properly. finally, a validation study is performed to ensure that the system is able to produce useful data for influenza research. the entire process takes months from start to finish for each robotic system and requires complete cooperation from a diverse team of researchers. the accessioning system logs initial sample information with the lims system. samples arrive in barcoded cryotubes. the liquid handler brings all samples up to a common volume and clarifies samples by centrifugation. samples are then transferred from screw-cap sample vials into storage plates containing individually punchable storage tubes. each tube ( ae ml) is individually identifiable with a d barcode on the bottom. six archive aliquots are made, and tubes are individually weld-sealed for storage. tips for aspiration are fixed and undergo a high-pressure plasma process between each use to sterilize tips and destroy nucleic acids. samples are stored at ) °c. each module has a capacity of remp plates or $ samples. the automated freezer system can assemble requested samples as -well plates while samples remain frozen. the screening system uses magnetic bead extraction chemistry, real-time pcr, and a liquid handling system to extract samples, confirm and quantify the presence of influenza, and reformat extracted samples for input into the sequencing system. serotype of human influenza samples will be performed by real-time pcr. many samples will not have enough material for further analysis and will need to be scaled up. the culturing system combines incubators, a liquid handling platform, plate reader, and real-time pcr to culture, monitor growth, harvest, and quantify influenza. when the system is not being used for culture and scale-up, it can be used to assay previously cultured influenza samples for drug resistance. a challenge to sequencing large numbers of influenza samples is the manpower required for sample preparation. the sequencing system has the capacity to prepare up to samples for sequencing per year for sanger sequencing. sanger sequencing was chosen because it is well-established for influenza surveillance, and automation-friendly. the system is designed to work with multiple primer sets ( , , ) . robotic systems all report to the lims. each process completion, plate movement, and data point are entered and checked by an online, web-based lims. status updates, notification, reporting, and data analysis can be achieved without entering the bsl containment facility. routine data analysis such as determining whether a cultured sample is ready to be harvested will be performed by the lims. complex data analysis, while still requiring significant human input, will be made easier by the data-acquisition functions of the lims. the implementation of a high-throughput influenza surveillance laboratory will provide an influenza research and response capacity that far exceeds what is available today. with the addition of each new system, we add a new capability to the influenza community and new opportunities to foster partnerships and collaborations with government, foundations, businesses, and academic institutions. this laboratory will not only enable cutting edge research, but will also enable a more effective response of near real-time surveillance during a pandemic outbreak. pandemics of and were believed to arise from avian influenza viruses. the tropism of avian and human seasonal influenza viruses for the human lower respiratory tract deserves investigation. the target cell types that support replication of avian influenza a viruses in the human respiratory tract in the early stages of clinical infection have not well defined. in a previous autopsy studies of human h n disease, influenza a virus were found to infect alveolar epithelial cells and macrophages. in this study, viral infectivity and replication competence of human and high and low pathogenic avian influenza viruses were systematically investigated in the human conducting and lower respiratory tract using ex vivo organ cultures. we compared the replication kinetics of human seasonal influenza viruses (h n and h n ), low pathogenic avian influenza viruses (h n , h n ) with that of the highly pathogenic h n viruses isolated from human h n disease. a range of human seasonal influenza a viruses of subtypes h n and h n viruses were included in this study from to . two isolates of low pathogenic avian influenza a (lpai) (h n ) viruses from different virus lineages isolated from poultry in hong kong in , a low pathogenic influenza a (h n ) virus isolate from wild ducks in hong kong in , and two virus isolates of highly pathogenic avian influenza (hpai) a subtype h n were included. fragments of human bronchi and lung were cut into multiple - mm fragments within hours of collection and infected in parallel with influenza a viruses at a titer of tcid ⁄ ml and as control cultures were infected with ultraviolet light inactivated virus. these tissues fragments were infected for hours and washed twice with pbs and incubated for , , and h at °c. the bronchial tissue was cultured in an air-liquid interface using sponge. viral yield was assessed by titration in mdck cells. one part of the infected tissue were fixed in formalin and processed for immunohistochemistry for influenza antigen. other part of infected tissue was homogenized and underwent rna extraction, and the expression of influenza virus matrix gene was measured by quantitative rt-pcr. human bronchus ex vivo cultures supported human seasonal influenza virus to replicate efficiently. avian influenza h n virus replicated, although less efficiently than that of seasonal influenza viruses, whereas hpai h n did not productively replicate in ex vivo cultures of human bronchus. this is in agreement with our previous finding in the well-differentiated bronchial epithelial cells in vitro. on the other hand, human lung ex vivo cultures supported prominent productive replication of human seasonal influenza h n ( figure a ) and hpai h n ( figure f ) viruses. lpai, such as h n ( figure c -d) and h n ( figure e ), also replicated productively, but with a lower viral yield. surprisingly, the replication of human influenza h n viruses ( figure b ) across the last three decades was greatly inhibited. there are clear differences in viral tropism of human seasonal and avian influenza viruses for replication in the human bronchus and lung. hpai h n virus can infect and productively replicate in the lower lung, which may account for the severity of human h n disease, but not in the conducting airways. surprisingly, there are marked differences in the replication competence of seasonal influenza viruses in ex vivo lung tissues, with influenza h n viruses being able to replicate efficiently while h n viruses do not. this may be related to the more strict siaa - gal binding preference of h n viruses. on the other hand, the efficient replication of influenza h n viruses in the alveolar spaces indicates factors other than tissues tropism alone play a role in the differences in disease severity between human seasonal h n and avian h n virus infections. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells, suggesting that viral protein(s) other than m gene-translational products facilitates the splicing of viral mrnas. the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns (c ⁄ ns ) and ns (c ⁄ ns ), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns and c ⁄ ns proteins, drastically reduced the splicing of ns mrna, raising the possibility that c ⁄ ns or c ⁄ ns enhances the splicing of viral mrnas. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns , whereas it was reduced by co-expression of influenza a virus ns protein (a ⁄ ns ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns , whereas it was inhibited by that of a ⁄ ns . these results suggest that influenza c virus ns , but not a ⁄ ns , can up-regulate the splicing of viral mrnas. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. , the inefficient splicing of viral pre-mrnas can be understood partly by the fact that influenza a virus ns protein is associated with spliceosomes and inhibits pre-mrna splicing. , cis-acting sequences in the ns transcript also negatively regulate splicing. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. the yamagata ⁄ ⁄ strain of influenza c virus was grown in the amniotic cavity of -day-old embryonated hen's eggs. cos- and t cells were cultured in dulbecco's modified eagle's medium containing % fetal calf serum. subconfluent monolayers of cos- cells were transfected with pme s containing influenza c virus m gene cdna using the lipofectamine procedure and then incubated at °c. total rna was extracted from both the transfected cells and cells infected with c ⁄ yamagata ⁄ ⁄ virus using the rneasy mini kit (qiagen). ribonuclease protection assay was performed using a ribonuclease protection assay kit rpa iii (ambion). briefly, a [ p]-labeled influenza c virus rna -specific rna probe (vrna sense) was synthesized by in vitro transcription and hybridized with the total rna at °c overnight. hybrids were digested with rnase a ( ae u) and rnase t ( u) at °c for minutes and then analyzed on a % polyacrylamide gel containing m urea. hmv-ii cells infected with c ⁄ yamagata ⁄ ⁄ and cos- cells transfected with pme s expressing influenza c virus ns were fixed with carbon tetrachloride at various times after infection and transfection, respectively. the cells were then stained by an indirect method using anti-gst ⁄ ns serum as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit igg (seikagaku kogyo) as the secondary antibody. the splicing efficiency of influenza c virus m gene-specific mrna (m mrna) in infected cells was higher than that in m gene-transfected cells the ratio of m encoded by a spliced m mrna to cm encoded by an unspliced m mrna in influenza c virusinfected cells was about times larger than that in m gene-transfected cells. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells (figure ). these data suggest that viral protein(s) other than m gene-translational products facilitates viral mrna splicing. the influenza c virus ns gene translational product may up-regulate the splicing of viral mrnas the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns (c ⁄ ns ) and ns (c ⁄ ns ), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns and c ⁄ ns proteins, drastically reduced the splicing of ns mrna, suggesting that c ⁄ ns or c ⁄ ns enhances viral mrna splicing. immunofluorescent staining showed that ns localized in the nucleus in the early phase of infection, and was distributed in both the nucleus and cytoplasm in the late phase of infection, raising the possibility that influenza c virus ns protein plays a role in viral mrna splicing that occurs in the nucleus. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns , whereas it was reduced by co-expression of influenza a virus ns protein (a ⁄ ns ) (figure a ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns , though it was inhibited by that of a ⁄ ns ( figure b ). these results suggest that influenza c virus ns , but not a ⁄ ns , can up-regulate the splicing of viral mrnas. in influenza a virus-infected cells, splicing is controlled so that the steady-state amount of spliced mrnas is only - % of that of unspliced mrnas. , the mechanisms by which influenza a virus ns pre-mrnas are poorly spliced have been investigated and the following confirmed. influenza a virus ns protein associates with spliceosomes and inhibits pre-mrna splicing. , two cis-acting sequences in the ns transcript (positions - in the intron and positions - in the ¢ exon region) inhibit splicing. by contrast, influenza c virus m gene-specific mrna (m mrna) is efficiently spliced in influenza c virus-infected cells. in this study, we examined the mechanism by which influenza c virus m mrna is efficiently spliced and the regulatory mechanism of the splicing of ns gene-specific mrna (ns mrna). the introduction of a translational pre-mature termination into the influenza c virus ns gene, thereby blocking the synthesis of influenza c virus ns (c ⁄ ns ) and ns (c ⁄ ns ) proteins, drastically reduced the splicing rate of ns mrna. we further examined whether c ⁄ ns potentially facilitates viral mrna splicing. the splicing rate of m mrna of influenza c virus was increased by co-expression with c ⁄ ns , whereas it was reduced by co-expression with influenza a virus ns protein (a ⁄ ns ) (figure a ). the splicing of influenza a virus m gene-specific mrna was also increased by co-expression with c ⁄ ns , though it was inhibited by co-expression with a ⁄ ns ( figure b ). these results suggest that influenza c virus ns can facilitate viral mrna splicing, but in no way inhibit it, which is in striking contrast to the inhibitory effect of influenza a virus ns on pre-mrna splicing. , the mechanism for splicing enhancement by c ⁄ ns also remains to be determined. we speculate that c ⁄ ns may interact with some host proteins involved in splicing, thereby leading to an up-regulation in splicing, or that c ⁄ ns may bind to pre-mrna, increasing its accessibility to the spliceosome. the spliced mrna of the influenza c virus m gene encodes the m protein, which plays an important role in virus formation and determines virion morphology. , therefore, it is speculated that the mechanism for efficient splicing of m mrna, which provides the m protein necessary for virus assembly in a redundant amount, has been maintained in the influenza c virus. by contrast, unspliced mrna from the influenza c virus m gene encodes the cm ion channel, which is permeable to chloride ions, and also has ph-modulating activity. although the role of the influenza c virus cm ion channel in virus replication remains to be determined, it is conceivable that the over-expression of the cm protein has a deleterious effect on virus replication since the fact that a high level of influenza a virus m protein expression inhibits the rate of intracellular transport of the influenza a virus ha protein and other integral membrane glycoproteins has been demonstrated. if this is the case, efficient splicing of m mrna may control the amount of cm synthesized to optimize virus replication. therefore, we speculate that efficient splicing of m mrna leads to a high level of m expression and the reduced expression of cm , thereby creating conditions that are optimal for virus replication. in this study, we provided evidence that c ⁄ ns facilitates the splicing of m mrna. furthermore, c ⁄ ns may regulate the splicing efficiency of its own ns mrna during infection, controlling the amount of c ⁄ ns and c ⁄ ns proteins in infected cells. c ⁄ ns plays an important role in the nuclear export of vrnp, and is also associated with vrnp in the later stages of infection in virus-infected cells and is incorporated into virions, suggesting that c ⁄ ns is involved, not only in the sorting of vrnp into the assembly site, but also in virus assembly. therefore, it is likely that there is a mechanism by which an appropriate amount of c ⁄ ns is provided during infection to accomplish these functions. in conclusion, c ⁄ ns , which enhances the splicing of viral mrna, may regulate both the expression level of m gene-derived m and cm proteins, and that of ns gene-derived ns and ns proteins, thereby leading to optimal virus replication. propagation of the human influenza viruses in embryonated hen's eggs always results in a selection of variants with amino acid substitutions in the hemagglutinin (ha) that affect viral receptor-binding characteristics (reviewed ). brookes et al. recently studied infection in pigs using the egg-grown virus that contained a mixture of the original a ⁄ california ⁄ ⁄ (h n pdm) and its two egg-adaptation mutants with single amino acid substitutions d g and q r ( and in h numbering system). only the original virus and the variant with g were detected in the directly inoculated animals, indicating that the variant with r failed to infect. only the original virus was detected in nasal secretions of contact infected pigs, suggesting that the d g mutant failed to transmit. in contrast, there was an apparent selection of the d g mutant in the lower respiratory tract samples from directly inoculated pigs. the d g substitution is of a special interest as it can emerge during virus replication in humans and was associated with severe and fatal cases of pandemic influenza in - - and . here we compared phenotypic properties of the original clinical isolate of h n pdm virus a ⁄ hamburg ⁄ ⁄ and its d g and d r mutants to explain observed effects of these mutations on virus replication in swine and to predict their potential effects on virus replication in humans. a ⁄ hamburg ⁄ ⁄ (ham) was isolated from clinical material by two passages in mdck cells. the virus was passaged twice in -day-old embryonated hen's eggs and plaqued in mdck cells. the plaques were amplified in mdck cells and the sequences of the viral ha were determined. the variants with single mutation d g and q r were aliquoted and designated ham-e and ham-e , respectively. the receptor-binding specificity of the viruses was assessed by assaying their binding to desialylated-resialylated peroxidase-labeled fetuin containing either a - -linked sialic acid ( - -fet) or a - -linked sialic acid ( - -fet). in brief, viruses adsorbed in the wells of -well eia micro plates were incubated with serial dilutions of - -fet or - -fet, and the amount of bound fetuin probe was quantified by peroxidase activity. the binding data were converted to scatchard plots (a ⁄ c versus a ), and the association constants of the virus-fetuin complexes were determined from the slopes of these plots. viral cell tropism and replication efficiency in human airway epithelium were studied using fully differentiated cultures of human tracheo-bronchial epithelial cells (htbe). , to determine cell tropism, cultures were infected at a moi , fixed hours after infection, and double immuno-stained for virus antigen and cilia of ciliated cells. infected cells were counted under the microscope ( · objective with oil immersion) in the epithelial segment that included - consecutive microscopic fields containing between % and % ciliated cells relative to the total number of superficial cells. percentages of infected ciliated cells and infected non-ciliated cells relative to the total number of infected cells were calculated. ten segments per culture were analyzed and the results were averaged. to compare growth kinetics of ham and ham-e, replicate htbe cultures were infected with plaque-forming units of the viruses followed by incubation at °c under airliquid interface conditions. at , , and hours postinfection, we added dmem to the apical compartments of the cultures and incubated for minutes at °c. the apical washes were harvested, stored at ) °c, and analyzed simultaneously for the presence of infectious virus by titration in mdck cells as described previously. the non-egg-adapted h n pdm virus ham, similarly to the seasonal human virus a ⁄ memphis ⁄ ⁄ (h n ), bound to - -fet ( figure a ) and did not show any significant binding to - -fet. this result contrasted with the binding of h n pdm viruses to several - -specific probes in carbohydrate microarray analysis. reduced avidity of virus interactions with soluble glycoprotein in solution as compared to its binding to the probe clustered on the microarray surface could account for these differences in the assay results. the d g mutant ham-e differed from the parent virus by its ability to bind to -fet and by its reduced binding to -fet. the q r mutant only bound to - -fet, although less strongly than did the avian virus a ⁄ duck ⁄ alberta ⁄ ⁄ (h n ). the viral cell tropism in htbe cultures ( figure b ) correlated with receptor specificity. ham and mem ⁄ showed a typical human-virus-like tropism , with preferential infection of non-ciliated cells (< % of infected cells were ciliated). the mutant with r and control duck virus displayed a typical avian-virus-like tropism (preferential infection of ciliated cells). the d g mutant displayed a cell tropism that was intermediate between those of human and avian viruses; in particular, this mutant infected significantly higher proportion of ciliated cells than ham and mem ⁄ . observed alteration of receptor specificity and cell tropism ( figure ) suggested that egg-derived mutations can affect replication of the h n pdm virus in human airway epithelium. to test this, we first compared the capacity of the viruses to initiate infection in htbe cultures. replicate cultures were infected with identical doses of the viruses, fixed hours post-infection, and immuno-stained for viral antigen. under these conditions, ham and ham-e infected comparable numbers of cells, whereas ham-e infected at least times less cells (data not shown). this result indicated that the mutation q r markedly impaired the ability of ham-e to infect human airway epithelial cultures. we next compared two other viruses ham and ham-e for their multi-cycle replication in htbe cultures and found that the original virus reached threefold higher peak titers hours post infection than did the d g mutant ( figure ). the d g mutation in h n pdm virus facilitates virus binding to - -linked receptors and alters viral cell tropism in human airway epithelium. these changes could account for increased replication of the d g mutant in the lower respiratory tract in humans - and pigs and correlation of this mutation with severe pulmonary disease. [ ] [ ] [ ] [ ] [ ] the d g mutant replicates less efficiently in human airway cultures than the original virus. this finding correlates with an apparent lack of transmission of variants with g in humans and pigs. egg-derived mutation q r abolishes virus binding to - -linked receptors and strongly decreases infection in cultures of human airway epithelium. this result agrees with poor infectivity of the q r mutant in pigs and highlights potential pitfalls of using egg-adapted viruses with this mutation for the preparation of live influenza vaccines. nin-esterase-fusion (hef), nucleoprotein (np), matrix (m ) protein, cm , and the non-structural proteins ns and ns . , cm is the second membrane protein of the virus and is encoded by rna segment (m gene). [ ] [ ] [ ] [ ] [ ] [ ] it is composed of three distinct domains: a -residue n-terminal extracellular domain, a -residue transmembrane domain, and a -residue cytoplasmic domain. , , it is abundantly expressed at the plasma membranes of infected cells and is incorporated in a small amount into virions. , cm forms disulphide-linked dimers and tetramers, and is posttranslationally modified by n-glycosylation, palmitoylation, and phosphorylation. [ ] [ ] [ ] analyses of a number of cm mutants revealed the positions of the amino acids involved in the posttranslational modifications. , evidence was obtained that the n-glycosylation was not required for either the formation of disulfide-linked multimers or transport to the cell surface, and that none of dimer-or tetramer-formation, palmitoylation or phosphorylation was essential to the transport of cm to the cell surface. in the present study, in order to investigate the effect of cm palmitoylation on influenza c virus replication, we generated a cm palmitoylation-deficient influenza c virus, in which a cysteine at residue of cm was mutated to alanine, and examined the viral growth and viral protein synthesis in infected cells. t and hmv-ii cells were maintained as described previously. , llc-mk cells were maintained at °c in minimal essential medium with % foetal bovine serum and % calf serum. monoclonal antibodies (mabs) against the hef, np, and m proteins of c ⁄ ann arbor ⁄ ⁄ (aa ⁄ ), and antisera against the aa ⁄ virion and the cm protein were prepared as described previously. , [ ] [ ] [ ] the seven pol i plasmids for the expression of viral rnas of aa ⁄ , and the nine plasmid dnas for the expression of the influenza c viral proteins were reported previously. , plasmid dna, ppoli ⁄ cm -acy(-), in which -tgt- of the m gene was replaced with -gct- , was constructed based on ppoli ⁄ m. to generate a recombinant wild-type (rwt) virus, the above-mentioned plasmids were transfected into t cells as described previously. to rescue a mutant virus, rcm -c a, a recombinant influenza c virus lacking a cm palmitoylation site, the plasmid ppoli ⁄ cm -acy(-), instead of ppoli ⁄ m, was transfected together with the other plas-mids. at hours posttransfection (p.t.), the respective culture medium of the transfected- t cells was inoculated into the amniotic cavity of -day-old embryonated chicken eggs, and a stock of the recombinant virus was prepared. the infectious titres of the stocked recombinant viruses and the supernatants of recombinant-infected hmv-ii cells were determined according to the procedure reported previously. radioimmunoprecipitation hmv-ii cells infected with recombinants were labeled with [ s]methionine or [ h]palmitic acid. cells were then disrupted and subjected to immunoprecipitation with the indicated antibodies. the immunoprecipitates obtained were then analysed by sds-page on ae % gels containing m urea, and processed for fluorography. flotation analysis was performed according to the procedure described previously. to examine whether the cm protein without palmitoylation is synthesized in rcm -c a-infected cells, hmv-ii cells infected with the recombinants were subjected to , and the lysates of the cells were immunoprecipitated with anti-cm serum and analysed by sds-page. as shown in figure , the cm protein was synthesized both in the rwt-and rcm -c a-infected cells, but no incorporation of [ h]palmitic acid into the cm proteins synthesized in the rcm -c ainfected cells was observed, indicating that cm in the rcm -c a-infected cells was not palmitoylated. the rwt or rcm -c a viruses were infected to hmv-ii cells at an m.o.i. of and incubated at °c for up to hours. the infectious titres (p.f.u. ⁄ ml) of rwt were approximately -to -fold higher than those of rcm -c a at - hours p.i. (data not shown), indicating that rwt grew more efficiently than did rcm -c a. thus palmitoylation of cm appears to have some effect on the generation of infectious virions in cultured cells. to investigate the reason(s) for the difference in growth kinetics between the two recombinants, we analysed viral proteins synthesized in the infected hmv-ii cells. pulsechase experiments of hmv-ii cells revealed no significant differences in the synthesis and maturation of the hef, np, m , and cm proteins between the rwt-and rcm -c a-infected cells (data not shown). the infected cells pulse-labeled and chased were respectively immunoprecipitated with anti-cm serum in the presence of mm iodoacetamide and analysed by sds-page in non-reducing condition. in both populations of infected cells, several bands corresponding to cm a-monomer, -dimer, and -tetramer, as well as cm b-dimer and -tetramer were detected (data not shown). these results demonstrate an absence of any significant differences between palmitoylation-deficient cm and authentic cm in terms of conformational maturation and transport in infected cells. membrane flotation analysis revealed that no significant differences in the kinetics of the hef, m , and cm proteins were observed between rwt-and rcm -c ainfected cells (data not shown). in contrast, a slight difference in np kinetics was observed. the pulse-labeled np proteins were recovered in the bottom fractions in both rwt-and rcm -c a-infected cells. in the chase experiment, the amount of membrane-associated np proteins in fractions and was % of the total np in the rwt-infected cells, which was higher than that ( %) in the rcm -c a-infected cells (data not shown). this finding may suggest that the affinity of the np protein, presumably representing the viral ribonucleoprotein (vrnp) complex, to the plasma membrane in the rcm -c ainfected cells is lower than that in rwt-infected cells, leading to the less efficient generation of infectious virions. since cm is structurally similar to m , an influenza a virus membrane protein known to be involved in infectious virus production, [ ] [ ] [ ] [ ] [ ] it is possible that the cytoplasmic tail of cm participates in the genome packaging through interaction with vrnp. in the present study, we showed that the affinity of np to the plasma membrane of rcm -c a-infected cells was slightly lower than that to the plasma membrane of rwt-infected cells. this observation may suggest that palmitoylation of cm is involved in the viral ribonucleoprotein (vrnp) incorporation, leading to efficient infectious virion generation. we hypothesize that palmitoylation contributes to proper regional structure formation in the cm cytoplasmic tail, which is competent to recruit vrnp efficiently into virions. alternatively, the cm cytoplasmic tail without palmitoylation is not likely to reach the proper conformation, resulting in reduced interaction with vrnp and less efficient generation of infectious progeny virions. the questions of if and how the m protein is involved in the interaction between the cm cytoplasmic tail and np remains to be clarified. we showed that cm synthesized in rcm -c ainfected cells was oligomerized and transported to the cell surface. this finding is consistent with the previous observation that palmitoylation is not required for the transport of cm to the cell surface in cm -expressing cos- cells. however, the use of reverse-genetics system has enabled us to conclude that the palmitoylation of cm is required for efficient infectious virus production. this suggests that the significance of the other posttranslational modifications of cm during virus replication can be clarified using recombinant viruses lacking the respective modification sites. sialic acid (sia) linked glycoproteins are the classical influenza receptors for influenza virus haemagglutinin to bind. the distribution of sia on cell surfaces is one of the determinants of host tropism, and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. previous research has shown the differences in apical versus basolateral infection and release of different influenza virus from polarized epithelial cells and correlated this with sialic acid distribution in the human respiratory tract. moreover, mass spectrometric analysis was recently employed to elucidate the glycans present in the tissue in a higher resolution in human lung. the objective of this study was to examine in detail the distribution of these sia-linked glycans at the cellular level by the use of confocal microscopy. human primary type i-like and type ii pneumocytes were isolated from human non-tumor lung tissue by tissue fragmentation, percoll density gradient centrifugation, and magnetic cell sorting. the cells were seeded on coverslips and maintained in small airway growth medium. when confluence was reached, cell monolayers were fixed with % paraformaldehyde. we used the plant lectins, sambucus nigra glutinin (sna) from roche which binds to siaa - gal, maackia amurensis agglutinin (maa)i and maaii from vector lab, which bind the siaa - gal linked glycans using vector red as fluorescent chromogen. the cells were counter-stained with dapi or with fitc-conjugated antibody against endoplasmic recticulum (protein disulfideisomerase, pdi). the cells were imaged with multi-photon excitation laser scanning microscopy using zeiss lsm. the optical cross-section pictures were reconstructed by zeiss lsm meta. we found that there was more binding of maai and ma-aii to type ii pneumocytes than type i-like pneumocytes and more overall binding of these lectins than binding of sna ( figure ). in keeping with results from other polarized cells there was more binding to the apical than basolateral aspect, thus, explaining the previously published data on apical versus basolateral infection. as sialic acid has been implicated in the targeting of proteins to the surface, the relative lack of sialic acid on the basolateral aspect can explain why there is little seasonal influenza virus dissemination to the systemic circulation in human infections. furthermore, though there was little binding of sna to the figure . primary human type i-like and type ii pneumocytes stained with lecins (red), pdi (green), and dapi (blue) and imaged captured with confocal microscope. apical or basolateral aspects of the pneumocytes, the experimental findings of infection by influenza h n virus that has a strict siaa - gal tropism suggests that there are siaa - gal glycans present, which are not readily bound by the lectin sna. the in vitro model of primary human type i-like and type ii pneumocytes system formed a polarized epithelium that has a similar lectin distribution to human alveoli in vivo which demonstrated that it is a physiologically relevant model to study the tropism and pathogenesis of influenza a virus. human disease caused by highly pathogenic avian influenza (hpai) h n virus is associated with fulminant viral pneumonia and mortality rates in excess of %. cytokine dysregulation is thought to contribute to its pathogenesis. , we previously found delayed onset of apoptosis in h n infected human macrophages and, therefore, a longer survival time of the target cells for prolonged virus replication and cytokine and chemokine secretion, which may contribute to the pathogenesis of h n disease in humans. as bronchial and alveolar epithelial cells are target cells of influenza virus because of their proximal physiological location and interaction with macrophages, we further investigated if the differential onset of apoptosis could be found in influenza h n and seasonal influenza h n infected human respiratory epithelia. we dissected the apoptotic pathways triggered by influenza virus infection. seasonal influenza h n virus (a ⁄ hk ⁄ ⁄ ), a low pathogenic avian influenza h n lineage isolated from poultry (a ⁄ quail ⁄ hk ⁄ g ⁄ ), and two virus isolates of hpai a subtype (a ⁄ hk ⁄ ⁄ and a ⁄ vn ⁄ ⁄ ) were included. primary human bronchial and alveolar epithelial cells were infected with influenza viruses at moi of and the cell monolayer was collected at , , and hours post infection for tunel assay, and supernatant were collected for ldh assay. fragments of human lung tissues were cut into multiple - mm fragments within hours of collection and infected with influenza a viruses at a titer of tcid ⁄ ml. these tissues fragments were infected for hours and incubated for hours at °c. one part of the infected tissue was fixed in formalin and processed for immunohistochemistry for influenza antigen, and the other part was homogenized and underwent rna extraction. apoptosis cdna superarray platform (sabioscience) was employed to conduct apoptosis pathway analysis. in bronchial epithelial cells, seasonal influenza h n virus induced a high percentage of apoptotic cells by tunel assay at , , and hours post infection with a peak of (figure ) . a similar observation of delayed onset of apoptosis was found in influenza h n and h n infected alveolar epithelial cells. besides, cdna array data of ex vivo infected human lung showed that both influenza h n and h n virus induced trail expression compared with mock-infected tissue (approximately folds) at hours post infection, but influenza h n virus infected lung induced significantly more trail ( folds compared to mock infected cells), albeit with a limited viral replication ( figure ). influenza h n virus infected lung also elicited more tnf-alpha and fasr transcription than either h n or h n . these observations can account for the greater apoptotic response in influenza h n virus infected lung. as little impact on the expression of intrinsic pathway components was observed, it seems that the apoptotic response to influenza virus infection in lung was mainly through the extrinsic pathways. no significant changes in the expression of anti-apoptotic protein gene was found, except for a moderate induction of birc by influenza h n virus, which may act to modulate the apoptotic response. the delayed onset of apoptosis by hpai h n and low pathogenic avian influenza h n virus infected respiratory epithelial cells may be a mechanism for the influenza viruses to have more prolonged replication within the human respiratory tract, and this may contribute to the pathogenesis of human disease. hemagglutination (ha) assay % crbc suspension was treated by mu a , -specific sialidase at °c for minutes. complete elimination of a , -receptor on sialidase-treated crbcs was confirmed by receptor staining and flow cytometry. ha assay of live viruses with % crbc or % sialidase-treated crbc were performed in bsl- facility. synthetic ¢sln-paa-biotin(pa ), ¢sln-paa-biotin(pa ), ¢sln-ln-paa-biotin(pa ) was provided by the scripps research institute (tsri). as described elsewhere with some modifications, generally, serial dilutions of sialyglycopolymers were coated in -well-flat-bottom polystryrene plates, and hau live virus ⁄ well were added. alternatively, the plates were precoated with lg ⁄ ml sialyglycopolymers, and then , , , , hau live virus ⁄ well influenza viruses were added. rabbit antisera against a ⁄ ah ⁄ ⁄ diluted in pbs containing % bsa was added into the wells. bound antibody was detected by use of hrp-conjugated anti-rabbit igg antibody and tetramethylbenzidine substrate solution. each sample was determined in duplicates and the absorbance read at nm. a total of h n virus strains were obtained from to . the name and passage history of influenza viruses used in the study are listed in table . as the same sequences of eight rna segments were detected in a ⁄ js ⁄ ⁄ and a ⁄ js ⁄ ⁄ , only a ⁄ js ⁄ ⁄ was tested here. three amantadine-resistant variants with m mutation of screening of receptor-binding preference by ha assay representative results from three sets of independent experiments are shown in table . complete ha with sialidasetreated crbcs, which were only with a , -receptors, was detected in human influenza virus (a ⁄ brisbane ⁄ ⁄ , h n ) and two human h n virus strains, a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . high binding of a , oligosaccharides to h n viruses was detected ( figure a -c). and enhanced a , -binding preference was also detected in a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . the a , -binding was dose dependent for sialyglycopolymers and virus titer. notably, as compared with a ⁄ gd ⁄ ⁄ of both short-and long-a , recognition, a ⁄ gx ⁄ ⁄ prefers to bind to long-a , six oligosaccharides at low viral titer ( figure b,c) . however, both of them showed strong affinity to short-and long-a , oligosaccharides at high viral loads ( figure d ). sialoside-, galactoside-, mannoside-and sulfo-os-binding are the four types of carbohydrate-binding properties of influenza virus. binding of influenza virus to the a , -or a , -linked sialylated glycans on cell surface is important for host range restriction, and the preference to a , of h n virus limited its efficient infection in human. here, dual receptor-binding preferences were detected in a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ , which are of clade ae ae . although there is no direct evidence supporting the occurrence of human-to-human transmission in these infection events or the association between viral virulence and receptor-binding switching, viral systemic disseminations are found in the both fatal cases (data not shown). furthermore, with the introduction of clade ae ae into the adjacent countries of china, the finding of h n virus with - binding in human should be of concern. though h n virus with human-type receptor-binding was isolated from one patient treated by oseltamivir and those viruses were with ha and ⁄ or na substitutions, whether the substitutions responsible for receptor specificity switching is pre-existed or selected in human host remains unknown. our finding that three mutant viruses bearing m mutations of a s, a t, and s n cloned from one isolate a ⁄ hb ⁄ ⁄ suggested it is likely that the resistant viruses emerged in the host environment. no variation was found in their ha and na sequence, and all of them show high affinity to a - -binding. our data suggest that the binding-specificity was not affected by the mutations on viral envelope protein m . with the adaptation from wild aquatic birds to domestic poultry or even in human host environment, influenza virus may possess broader carbohydrate-binding spectrum or topology conformation. , we demonstrated differential a , -binding property of two human h n viruses, a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . though minor effect of short-a , -binding was detected in viruses a ⁄ gx ⁄ ⁄ at low virus titer, both were of high affinity to long-a , glycans, even at the low titer which are rich on apical side of human upper respiratory epithelia. notably, no evident binding preference switching was detected in the viruses isolated from the sporadic human infection cases at the early of in china (table ) . however, higher affinity to the long-a , glycans was observed in bj ⁄ ⁄ , gz ⁄ ⁄ , and xj ⁄ ⁄ (data not shown). the discrepancy from the findings obtained by sialidase-treated crbc maybe associated with a limited abundance of n-linked a - with long branches on crbc, as demonstrated in a recent study. therefore, glycan dose-dependent binding assay is valuable and should be applied in flu surveillance. the underlying cause of the tendency is unknown, and further research on receptor-binding specificity of h n viruses is required. influenza a viruses of migrating wild aquatic birds in north america towards improved influenza a virus surveillance in migrating birds european union council directive ⁄ ⁄ eec the neighbor-joining method: a new method for reconstructing phylogenetic trees confidence limits on phylogenies: an approach using the bootstrap prospects for inferring very large phylogenies by using the neighbor-joining method mega : molecular evolutionary genetics analysis (mega) software version . the influenza virus resource at the national center for biotechnology information characterization of low-pathogenic h subtype influenza viruses from eurasia: implications for the origin of highly pathogenic h n viruses h n virus outbreak in migratory waterfowl a ⁄ h and a ⁄ h influenza viruses: different lines of one precursor evolution and ecology of influenza a viruses evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis antigenic and genetic conservation of h influenza virus in wild ducks biologic characterization of chicken-derived h n low pathogenic avian influenza viruses in chickens and ducks genetic and pathogenic characterization of h n avian influenza viruses isolated in taiwan between and experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity h n avian influenza virus field isolate evolution and ecology of influenza a viruses is china an influenza epicenter genesis of a highly pathogenic and potentially pandemic h n influenza virus in eastern asia evolution and molecular epidemiology of h n influenza a viruses from quail in southern china establishment of influenza a virus (h n ) in minor poultry species in southern china h n influenza viruses: outbreaks and biological properties seroprevalance and identification of influenza a virus infection from migratory wild waterfowl in china avian flu: h n virus outbreak in migratory waterfowl migration of waterfowl in the east asian flyway and spatial relationship to hpai h n outbreaks avian influenza monitoring in migrating birds in taiwan during establishment of an h n influenza virus lineage in domestic ducks in southern china evolution and molecular epidemiology of h n influenza a viruses from quail in southern china the genesis and evolution of h n influenza viruses in poultry from southern china human infection with influenza h n human infection with an avian h n influenza a virus in hong kong in antigenic and genetic characterization of h n swine influenza in china cocirculation of avian h n and contemporary ''human'' h n influenza a viruses in pigs in southeastern china: potential for genetic reassortment? h n influenza a viruses from poultry in asia have human virus-like receptor specificity characterization of h subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? bioedit: a user-friendly biological sequence alignment editor and analysis program for window ⁄ ⁄ nt genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion phylogenetic analysis using parsimony (and other methods) . beta a novel genotype h n influenza virus possessing human h n internal genomes has been circulating in poultry in eastern china since characterization of h n influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation in eastern china during a year period characterization of avian h n influenza viruses from united arab emirates phylogenetic analysis of influenza a viruses of h haemagglutinin subtype h n subtype influenza a viruses in poultry in pakistan are closely related to the h n viruses responsible for human infection in hong kong diversified reassortants h n avian influenza viruses in chicken flocks in northern and eastern china genotypic evolution and antigenic drift of h n influenza viruses in china from the nucleoprotein as a possible major factor in determining host specificity of influenza h n viruses pigs as the ''mixing vessel'' for the creation of new pandemic influenza a viruses origins and evolutionary genomics of the swine-origin h n influenza a epidemic pandemic (h n ) outbreak on pig farm reassortment of pandemic h n ⁄ influenza a virus in swine from where did the 'swine-origin' influenza a virus (h n ) emerge? substitution of lysine at position in pb protein does not change virulence of the pandemic h n virus in mice evolution and ecology of influenza a viruses the origins of pandemic influenza -lessons from the virus characterization of the influenza virus polymerase genes origins and evolutionary genomics of the swine-origin h n influenza a epidemic emergence of a novel swine-origin influenza a (h n ) virus in humans antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans genetic reassortment of avian, swine, and human influenza a viruses in american pigs detection of two antigenic subpopulations of a(h n ) influenza viruses from pigs: antigenic drift or interspecies transmission genetic relatedness of hemagglutinins of the h subtype of influenza a viruses isolated from swine and birds cases of swine influenza in humans: a review of the literature triple-reassortant swine influenza a (h ) in humans in the united states reassortment of pandemic h n ⁄ influenza a virus in swine ferrets as a transmission model for influenza: sequence changes in ha of type a (h n ) virus the ferret: an animal model to study influenza virus human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals medical management of influenza infection incidence of adamantane resistance among influenza a (h n ) viruses isolated worldwide from to : a cause for concern surveillance of resistance to adamantanes among influenza a(h n ) and a(h n ) viruses isolated worldwide peramivir emergency use authorization peramivir and its use in h n influenza cs- , a prodrug of the new neuraminidase inhibitor r- , shows long-acting antiinfluenza virus activity detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the first years of their use surveillance of influenza isolates for susceptibility to neuraminidase inhibitors during the - influenza seasons surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from oseltamivir-resistant influenza viruses a (h n ), norway, - influenza activity -united states and worldwide, - season emergence of resistance to oseltamivir among influenza a(h n ) viruses in europe oseltamivir-resistant influenza virus a (h n ), europe, - season widespread oseltamivir resistance in influenza a viruses (h n ), south africa and composition of the - influenza vaccine emergence of h y oseltamivir-resistant a(h n ) influenza viruses in japan during the - season pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza a viruses neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in a(h n ) viruses neuraminidase receptor binding variants of human influenza a(h n ) viruses due to substitution of aspartic acid in the catalytic site -role in virus attachment? neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective update: drug susceptibility of swine-origin influenza a (h n ) viruses comprehensive assessment of pandemic influenza a (h n ) virus drug susceptibility in vitro detection of molecular markers of drug resistance in pandemic influenza a (h n ) viruses by pyrosequencing pandemic (h n ) and oseltamivir resistance in hematology/oncology patients fluview: a weekly influenza surveillance report prepared by the influenza division development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from different neuraminidase inhibitor susceptibilities of human h n , h n , and h n influenza viruses isolated in germany from oseltamivir-and amantadine-resistant influenza viruses a (h n ) infections with oseltamivirresistant influenza a (h n ) virus in the united states isolation of oseltamivirresistant influenza a ⁄ h n virus of different origins in yokohama city, japan, during the - influenza season a two-year survey of the oseltamivir-resistant influenza a(h n ) virus in yamagata, japan and clinical effectiveness of oseltamivir and zanamivir oseltamivir-resistant influenza a pandemic (h n ) virus detection of an oseltamivir-resistant pandemic influenza a ⁄ h n virus in hong kong a novel small-molecule inhibitor of the avian influenza h n virus determined through computational screening against the neuraminidase aurintricarboxylic acid is a potent inhibitor of influenza a and b virus neuraminidases oseltamivir-resistant influenza a viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol oseltamivir is adequately absorbed following nasogastric administration to adult patients with severe h n influenza prevention and control of influenza influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states severe respiratory disease concurrent with the circulation of h n influenza staphlococcus aureus pneumonia studies on influenza in the pandemic of - . ii. pulmonary complications of influenza bacterial pneumonia during the hong kong influenza epidemic of - streptococcus pneumonia diagnostic value of microscopic examination of gram-stained sputum and sputum cultures in patients with bacteremic pneumococcal pneumonia pneumonitis-associated hyperprocalcitoninemia clinical review : procalcitonin and the calcitonin gene family of peptides in inflammation, infection, and sepsis: a journey from calcitonin back to its precursors procalcitonin in sepsis and systemic inflammation: a harmful biomarker and a therapeutic target procalcitonin in children with suspected novel influenza a (h n ) infection in vitro and in vivo calcitonin i gene expression in parenchymal cells: a novel product of human adipose tissue serum procalcitonin and c-reactive protein levels as markers of bacterial infection: a systematic review and meta-analysis procalcitonin as a diagnostic test for sepsis in critically ill adults and after surgery or trauma: a systematic review and meta-analysis respiratory syncytial virus infection in elderly and high-risk adults procalcitonin assay in systemic inflammation, infection, and sepsis: clinical utility and limitations simultaneous detection of chlamydophila pneumoniae and mycoplasma pneumoniae by use of molecular beacons in a duplex real-time pcr years influenza therapy with antivirals (zanamivir and oseltamivir), nd world summit of antivirals the art of viewing and influenza -its importance for diagnosis, rd european influenza conference emergence of a novel swine origin influenza a (h n ) virus in humans origins and evolutionary genomics of the swine-origin h n influenza a epidemic molecular detection of a novel human influenza (h n ) of pandemic potential by conventional and real-time quantitative rt-pcr assays pandemic influenza a(h n )v: human to pig transmission in norway? an investigation into human pandemic influenza virus (h n ) on an alberta swine farm pandemic (h n ) infection in swine herds implications of the emergence of a novel h influenza virus emergence of a novel swine-origin influenza a virus (s-oiv) h n virus in humans rapid detection of reassortment of pandemic h n ⁄ influenza virus rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) universal primer set for the fulllength amplification of all influenza a viruses cocirculation of avian h n and contemporary ''human'' h n influenza a viruses in pigs in southeastern china: potential for genetic reassortment? hog in the limelight: swine flu's got new genes on reassortment of pandemic h n ⁄ influenza a virus in swine pediatric hospitalizations associated with pandemic influenza a (h n ) in argentina factors associated with death or hospitalization due to pandemic influenza a(h n ) infection in california mortality from pandemic a ⁄ h n influenza in england: public health surveillance study antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans characterization of a newly emerged genetic cluster of h n and h n swine influenza virus in the united states experimental inoculation of pigs with pandemic h n virus and hi cross-reactivity with contemporary swine influenza virus antisera triple-reassortant swine influenza a (h ) in humans in the united states identification of human h n and human-swine reassortant h n and h n influenza a viruses among pigs in ontario genetic and pathobiologic characterization of pandemic h n influenza viruses from a naturally infected swine herd archive number pandemic (h n ) outbreak on pig farm reassortment of pandemic h n ⁄ influenza a virus in swine a simple method for estimating percent endpoints occurrence of haemagglutinin mutation d g in pandemic influenza a(h n ) infected patients in the west of scotland rrt-pcr) protocol for detection and characterization of swine influenza (version diagnostic hybrids, inc. readycells respiratory training r-mixÔ&d Ô dfa respiratory virus screening & id kit handbook centers for disease and control and prevention. influenza laboratory course comparison study of a real-time reverse transcription polymerase chain reaction assay with an enzyme immunoassay and shell vial culture for influenza a and b virus detection in adult patients comparison of a commercial qualitative real-time rt-pcr kit with direct immunofluorescence assay (dfa) and cell culture for detection of influenza a and b in children prospective comparison of r-mix shell vial system with direct antigen tests and conventional cell culture for respiratory virus detection optimized detection of respiratory viruses in nasopharyngeal secretions evaluation of r-mix freshcells in shell vials for detection of respiratory viruses time lines of infection and disease in human influenza: a review of volunteer challenge studies comparative epidemiology of pandemic and seasonal influenza a in households viral shedding and clinical illness in naturally acquired influenza virus infections population modeling of influenza a/h n virus kinetics and symptom dynamics sensitivity of influenza rapid diagnostic tests to h n and pandemic h n viruses clinical evaluation of an immunochromatography test kit, capilia flua,b, for rapid diagnosis of influenza time to positive reaction of influenza rapid diagnosis kit and its clinical significance the speed and reliability of the antigen detection kit capilia flu a, b when used in a clinical setting options for the control of influenza vi role of rapid immunochromatographic antigen testing in diagnosis of influenza a virus h n infection poor clinical sensitivity of rapid antigen test for influenza a pandemic (h n ) virus evaluation of new rapid antigen test for the detection of pandemic influenza a ⁄ h n virus application of real-time cell electronic sensing (rt-ces) technology to cell-based assays world health organization. pandemic (h n ) role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics rapid detection and identification of respiratory viruses using a dual priming oligonucleotide systembased multiplex pcr assay world health organization. influenza world health organization. cdc protocol of realtime rtpcr for influenza a (h n ) development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (h n ) virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings one-step real-time rt-pcr for pandemic influenza a virus (h n ) matrix gene detection in swine samples detection of pandemic influenza a ⁄ h n ⁄ virus by real-time reverse transcription polymerase chain reaction detection of influenza a(h n )v virus by real-time rt-pcr development of a real-time rt-pcr for the detection of swine-lineage influenza a (h n ) virus infections evaluation of multiple test methods for the detection of the novel influenza a (h n ) during the new york city outbreak design and clinical application of a molecular method for detection and typing of the influenza a ⁄ h n pdm virus confirmation of the first hong kong case of human infection by novel swine origin influenza a (h n ) virus diagnosed using ultrarapid, real-time reverse transcriptase pcr evaluation of four real-time pcr assays for detection of influenza a(h n )v viruses diagnostic assay recommended by the world health organization for swine origin influenza a (h n ) virus cross-reacts with h n influenza virus increasing immunization coverage the cost-effectiveness of influenza vaccination for people aged to years: an international model national seasonal influenza vaccination survey in europe seasonal influenza and vaccination coverage influenza vaccination coverage rates in five european countries during season ⁄ and trends over six consecutive seasons influenza in the elderly -a mini-review cost of vaccine administration among pediatric practices cost of universal influenza vaccination of children in pediatric practices surveillance report: respiratory infections strategies for mitigating an influenza pandemic methods for monitoring influenza surveillance data approaches to syndromic surveillance when data consist of small regional counts rrt-pcr) protocol for detection and characterization of swine influenza (version influenza virus strains circulating in mongolia in - swine influenza who ( ) influenza a(h n ) world now at the start of influenza pandemic general statement following the ninth meeting of the emergency committee who ( ) influenza updates us dept of defense global emerging infections surveillance è me recensement general de la population et l'habitat (rgph ), ministè re de l'economie des finances et de la planification ministry of health djibouti a survey of human cases of h n avian influenza report by who before international health regulation (ihr) electronic public health surveillance in developing settings statistical analyses in disease surveillance systems potential for a global dynamic of influenza a (h n ) infectious disease informatics: syndromic surveillance for public health and biodefense visualization techniques and graphical user interfaces in syndromic surveillance systems statistical inference for infectious diseases. risk-specific household and community transmission parameters evaluation of seasonal influenza vaccination effectiveness based on antibody efficacy among the institutionalized elderly in japan australian influenza surveillance summary report. canberra: australian government department of health and ageing australia's winter with the pandemic influenza a (h n ) virus seroprevalence of pandemic influenza a(h n ) virus in australian blood donors pandemic vaccination survey: summary results. cat. no. phe concepts and procedures for laboratory-based influenza surveillance. who collaborating centres for reference and research on influenza influenza a(h n ) seroconversion rates and risk factors among distinct adult cohorts in singapore ecdc. forward look risk assessment: likely scenarios for influenza in and the ⁄ influenza season in europe and the consequent work priorities. stockholm: european centre for disease prevention and control epidemiology of influenza -summary of influenza workshop iv sampling of populations: methods and applications comparison of influenza serological techniques by international collaborative study eliminating bias in the estimation of the geometric mean of hi titres reply to ''nauta jjp, eliminating bias in the estimation of the geometric mean of hi titers novel influenza a virus infections. case definition institut national de la statistique et des é tudes é conomiques. taille des mé nages dans l'union europé enne estimation de la population au er janvier par ré gion, dé partement, sexe et â ge sero-immunity and serologic response to pandemic influenza a (h n ) virus in hong kong pandemic influenza a(h n ) virus in scotland: geographically variable immunity in spring , following the winter outbreak high prevalence of antibodies to the pandemic influenza a(h n ) virus in the norwegian population following a major epidemic and a large vaccination campaign in autumn incidence of pandemic influenza a h n infection in england: a cross-sectional serological study prevalence of seroprotection against the pandemic (h n ) virus after the pandemic antibodies in residents of new south wales, australia, after the first pandemic wave in the southern hemisphere winter older age and a reduced likelihood of h n virus infection pre-existing antibody response against pandemic influenza h n viruses in taiwanese population cross-reactive antibody responses to the pandemic h n influenza virus seasonal influenza vaccine and increased risk of pandemic a ⁄ h n -related illness: first detection of the association in british columbia association between the - seasonal influenza vaccine and pandemic h n illness during spring-summer : four observational studies from canada does seasonal influenza vaccination increase the risk of illness with the a ⁄ h n pandemic virus seasonal h n influenza virus infection is associated with elevated pre-exposure antibody titers to the serum cross-reactive antibody response to a novel influenza a (h n ) virus after vaccination with seasonal influenza vaccine world now at the start of influenza pandemic rki working group pandemic. the first wave of pandemic influenza (h n ) in germany: from initiation to acceleration department of health and ageing. australian influenza surveillance canadian surveillance studies help to understand h n patterns influenza pandé mica a un añ o de la primera ola. ¿qué podemos decir ahora? revista chilena de infectología pandemic (h n ) briefing note options for the control of influenza vii managing and reducing uncertainty in an emerging influenza pandemic the severity of pandemic h n influenza in the united states assessing the severity of the novel influenza a ⁄ h n pandemic tecumseh study of illness. xiii. influenza infection and disease, - the infection attack rate and severity of pandemic influenza (h n ) in hong kong school closure and mitigation of pandemic (h n ) , hong kong comparative epidemiology of pandemic and seasonal influenza a in households incidence of pandemic influenza a h n infection in england: a cross-sectional serological study mortality from pandemic a ⁄ h n influenza in england: public health surveillance study monthly summary tables of influenza virus isolation different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures estimating in real time the efficacy of measures to control emerging communicable diseases the effective reproduction number of pandemic influenza in hong kong: prospective estimation multiple imputation: a primer comparative epidemiology of pandemic and seasonal influenza a in households strategies for containing an emerging influenza pandemic in southeast asia estimation of the serial interval of influenza school closure and mitigation of pandemic (h n ) , hong kong estimating the reproduction number of the novel influenza a virus (h n ) in a southern hemisphere setting: preliminary estimate in new zealand early transmission characteristics of influenza a(h n )v in australia: victorian state methods for monitoring influenza surveillance data comparative estimation of the reproduction number for pandemic influenza from daily case notification data how to maintain surveillance for novel influenza a h n when there are too many cases to count a computational framework for influenza antigenic cartography mapping the antigenic and genetic evolution of influenza virus proc of kdd cup and workshop matrix factorization techniques for recommender systems exact matrix completion via convex optimization a singular value thresholding algorithm for matrix completion matrix completion from a few entries matrices with prescribed entries and eigenvalues combining predictions for accurate recommender systems the bellkor solution to the netflix grand. prize the pragmatic theory solution to the netix grand prize feature-weighted linear stacking technical report. of health and ageing (australia). the australian health management plan for pandemic influenza influenza pandemic contingency plan, version . world health organisation. pandemic influenza preparedness and response prophylaxis or treatment? optimal use of an antiviral stockpile during an influenza pandemic the response to novel influenza a (h n ) epidemic in taiwan and analysis of the initial confirmed cases mitigation strategies for pandemic influenza in the united states strategies for mitigating an influenza pandemic containing pandemic influenza at the source efficient simulation of the spatial transmission dynamics of influenza world health organization. mathematical modelling of the pandemic h n a mathematical model for the global spread of influenza pandemic potential of a strain of influenza a (h n ): early findings effectiveness of the - influenza vaccine among children months to years of age, with vs doses cdc estimates of h n influenza cases, hospitalizations and deaths in the united states accessed entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation pandemic of influenza - america's forgotten pandemic. the influenza of influenza: a survey of the last years in the light of modern work on the virus of epidemic influenza understanding mortality in the - influenza pandemic in england and wales genomic analysis of increased host immune and cell death responses induced by influenza virus the age pattern of mortality in the - influenza pandemic: an attempted explanation based on data for england and wales protective immunity and susceptibility to infectious diseases: lessons from the influenza pandemic deaths from bacterial pneumonia during - influenza pandemic understanding influenza transmission, immunity and pandemic threats a biological model for influenza transmission: pandemic planning implications of asymptomatic infection and immunity prior immunity helps to explain wave-like behaviour of pandemic influenza in - influenza: accounting for prior immunity - influenza pandemic mortality in england and wales. colchester, essex: uk data archive [distributor] cross-reactive antibody responses to the pandemic h n influenza virus australia's winter with the pandemic influenza a (h n ) virus model predictions and evaluation of possible control strategies for the a ⁄ h n v influenza pandemic in italy the role of population heterogeneity and human mobility in the spread of pandemic influenza age-prioritized use of antivirals during an influenza pandemic mitigation measures for pandemic influenza in italy: an individual based model considering different scenarios strategies for containing an emerging influenza pandemic in southeast asia modeling targeted layered containment of an influenza pandemic in the united states reducing the impact of the next influenza pandemic using household-based public health interventions mitigation strategies for pandemic influenza in the united states effective, robust design of community mitigation for pandemic influenza: a sytematic examination of proposed us guidance containing pandemic influenza at the source strategies for mitigating an influenza pandemic new influenza a (h n ) virus: global epidemiological situation velluci l. enhanced epidemiological surveillance of influenza a(h n )v in italy changes in reporting requirements for pandemic (h n ) virus infection use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza guidance document: pandemic influenza preparedness and response situation updates -pandemic (h n ) pandemic potential of a strain of influenza a (h n ): early findings estimating the impact of school closure on influenza transmission from sentinel data preexisting immunity to pandemic (h n ) understanding the dynamics of seasonal influenza in italy: an analysis of disease incidence and population susceptibility what is the optimal therapy for patients with h n influenza meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h n treatment? treatment with convalescent plasma for influenza a (h n ) infection prophylactic and therapeutic efficacy of human monoclonal antibodies against h n influenza logistical feasibility and potential benefits of a population-wide passive-immunotherapy program during an influenza pandemic strategies for pandemic and seasonal influenza vaccination of schoolchildren in the united states mitigation strategies for pandemic influenza in the united states pandemic potential of a strain of influenza a (h n ): early findings transmission potential of the new influenza a(h n ) virus and its age-specificity in japan the transmissibility and control of pandemic influenza a (h n ) virus effect of clinical and virological parameters on the level of neutralizing antibody against pandemic influenza a virus h n world health organization. facts on blood transfusion hyperinduction of cyclooxygenase- -mediated proinflammatory cascade: a mechanism for the pathogenesis of avian influenza h n infection the role of influenza virus gene constellation and viral morphology on cytokine induction, pathogenesis, and viral virulence induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? analysis of relative gene expression data using real-time quantitative pcr and the acetylsalicylic acid (asa) blocks influenza virus propagation via its nf-kappab-inhibiting activity cyclooxygenase plays a pivotal role in the resolution of acute lung injury role of -deoxy delta( , ) prostaglandin j and nrf pathways in protection against acute lung injury resolution of inflammation in murine autoimmune arthritis is disrupted by cyclooxygenase- inhibition and restored by prostaglandin e -mediated lipoxin a production inhibition of cox- aggravates neutrophil migration and pneumocyte apoptosis in surfactantdepleted rat lungs re-emergence of fatal human influenza a subtype h n disease apoptosis and pathogenesis of avian influenza a (h n ) virus in humans h n infection of the respiratory tract and beyond: a molecular pathology study molecular biology, and pathogenesis of avian influenza a (h n ) infection in humans passive immunity in prevention and treatment of infectious diseases prophylactic and therapeutic efficacy of human monoclonal antibodies against h n influenza neutralizing human monoclonal antibody against h n influenza ha selected from a fab-phage display library passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza a h hemagglutinin in mice generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza a h n viruses igy: clues to the origins of modern antibodies chicken antibodies: a clinical chemistry perspective oral immunotherapy with yolk antibodies to prevent infections in humans and animals good effect of igy against pseudomonas aeruginosa infections in cystic fibrosis patients avian influenza vaccines: a practical review in relation to their application in the field with a focus on the asian experience vaccination: a tool for the control of avian influenza immunoglobulins from egg yolk: isolation and purification a simple method of estimating fifty per cent endpoints characterization of the reconstructed spanish influenza pandemic virus detection of antibody to avian influenza a (h n ) virus in human serum by using a combination of serologic assays avian influenza (h n ) viruses isolated from humans in asia in exhibit increased virulence in mammals heterosubtypic immunity to influenza a virus infection requires a properly diversified antibody repertoire early control of h n influenza virus replication by the type i interferon response in mice successful treatment of avian influenza with convalescent plasma chicken antibodies: taking advantage of evolution -a review oral administration of specific yolk antibodies (igy) may prevent pseudomonas aeruginosa infections in patients with cystic fibrosis: a phase i feasibility study avian flu: isolation of drug-resistant h n virus reduced sensitivity of influenza a (h n ) to oseltamivir enzymatic properties of the neuraminidase of seasonal h n influenza viruses provide insights for the emergence of natural resistance to oseltamivir surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from a polyphenol rich plant extract, cystus , exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance cystus , a polyphenol-rich plant extract, exerts anti-influenza virus activity in mice cistus incanus (cystus ) for treating patients with infection of the upper respiratory tract. a prospective, randomised, placebocontrolled clinical study in vitro and in vivo assay systems for study of influenza virus inhibitors new low-viscosity overlay medium for viral plaque assays antibacterial and antifungal activities of cistus incanus and c. monspeliensis leaf extracts flavan- -ols and proanthocyanidins from cistus incanus inhibition of the infectivity of influenza virus by tea polyphenols attenuation of inducible respiratory immune responses by oseltamivir treatment in mice infected with influenza a virus anti-inflammatory activity of macrolides: a new therapeutic potential? macrolide antibiotics as immunomodulatory medications: proposed mechanisms of action proteases essential for human influenza virus entry into cells and their inhibitors as potential therapeutic agents modified pulmonary surfactant is a potent adjuvant that stimulates the mucosal iga production in response to the influenza virus antigen boost of mucosal sectretory immunoglobulin a response by clarithromycin in paediatric influenza intranasal interleukin- is a powerful adjuvant for protective mucosal immunity immunological effects of the orally administered neuraminidase inhibitor oseltamivir in influenza virus-infected and uninfected mice clarithromycin in the treatment of rsv bronchitis: a double-blind, randomized placebo-controlled trial bird flu''), inflammation and anti-inflammatory ⁄ analgesic drugs clinical effects of low-dose long-term erythromycin chemotherapy on diffuse panbronchiolitis proteolytic activation of influenza viruses by serine proteases tmprss and hat from human airway epithelium proteolytic activation of the influenza virus hemagglutinin protease-activated receptors: new concepts in regulation of g protein-coupled receptor signaling and trafficking protective role for protease-activated receptor- against influenza virus pathogenesis via an ifn-gamma-dependent pathway agonists of proteinaseactivated receptor- enhance ifn-gamma-inducible effects on human monocytes: role in influenza a infection plasmacytoid dendritic cells migrate in afferent skin lymph annexin ii incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin plasminogen promotes influenza a virus replication through an annexin ii-dependent pathway in absence of neuraminidase costimulatory receptors in a teleost fish: typical cd , elusive ctla trypsin increases pseudorabies virus production through activation of the erk signalling pathway characterization of hla-g , -g , -g , and -g isoforms transfected in a human melanoma cell line immunosuppressive hla-g molecule is upregulated in alveolar epithelial cells after influenza a virus infection influenza virus propagation is impaired by inhibition of the raf ⁄ mek ⁄ erk signalling cascade influenza viruses and the nf-kappab signaling pathway -towards a novel concept of antiviral therapy the immunotolerance role of hla-g hla-g co-expression boosts the hla class i-mediated nk lysis inhibition hla-g inhibits the allogeneic proliferative response hla-g-mediated inhibition of antigen-specific cytotoxic t lymphocytes the world health organization. h n influenza vaccine task force pandemic influenza: a potential role for statins in treatment and prophylaxis commentary: from scarcity to abundance: pandemic vaccines and other agents for ''have not'' countries new technologies for meeting the global demand for pandemic vaccines confronting the next influenza pandemic with inexpensive generic agents: can it be done? was bacterial pneumonia the predominant cause of death in the - influenza pandemic? meeting the challenge of influenza pandemic preparedness in developing countries confronting the next influenza pandemic with antiinflammatory and immunomodulatory agents: why they are needed and how they might work molecular and cellular aspects of sepsis-induced immunosuppression immune homeostasis in the respiratory tract and its impact on heterologous infection identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury effector t cells control lung inflammation during acute influenza virus infection by producing il- a detrimental effect of interleukin- on protective pulmonary humoral immunity during primary influenza a virus infection inhibition of influenza a virus replication by resveratrol increased survival after gemfibrozil treatment of severe mouse influenza tnf ⁄ inos-producing dendritic cells are the necessary evil of lethal influenza virus infection peroxisome proliferator-activated receptor and amp-activated protein kinase agonists protect against lethal influenza virus challenge in mice acetylsalicylic acid (asa) blocks influenza virus propagation via its nf-kappab-inhibiting activity new low-viscosity overlay medium for viral plaque assays cyproquant-pcr: a real time rt-pcr technique for profiling human cytokines, based on external rna standards, readily automatable for clinical use role of hypercytokinemia in nf-kap-pab p deficient mice after h n influenza a virus infection developing new antiviral agents for influenza treatment: what does the future hold? influenza viruses and the nf-kappab signaling pathway -towards a novel concept of antiviral therapy rna viruses and the mitogenic raf ⁄ mek ⁄ erk signal transduction cascade world health organization writing group. non-pharmaceutical interventions for pandemic influenza, international measures effects of school closures, winter influenza season, hong kong entry screening to delay local transmission of pandemic influenza a (h n ) simple physical interventions such as hand washing and wearing masks can reduce spread of respiratory viruses hand hygiene and virus transmission demographic and attitudinal determinants of protective behaviours during a pandemic: a review perceptions related to human avian influenza and their associations with anticipated psychological and behavioral responses at the onset of outbreak in the hong kong chinese general population perceptions about status and modes of h n transmission and associations with immediate behavioral responses in the hong kong general population longitudinal assessment of community psychobehavioral responses during and after the outbreak of severe acute respiratory syndrome in hong kong public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey community psychological and behavioral responses through the first wave of the influenza a(h n ) pandemic in hong kong main tables of the population census. hong kong: government of the statistical analysis with missing data the impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong economic impact of sars: the case of hong kong sars-related perceptions in hong kong group members: stream -ilaria oie -world organisation for animal health, france; malik peiris us department of health and human services, usa; masato tashiro, national institute of infectious diseases, japan; marie-paule kieny , david wood . stream -tawee chotpitayasunondh world influenza centre at national institute for medical research philippe veltsos , cathy roth , gregory härtl options for the control of influenza vii who public health research agenda for influenza outbreak communication best practices for communicating with the public during an outbreak, doc who ⁄ cds ⁄ . . geneva: world health organization who outbreak communications guidelines, geneva: world health organization characterization of h subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? genetic conservation of hemagglutinin gene of h influenza virus in chicken population in mainland china continuing evolution of h influenza viruses in korean poultry evolution and molecular epidemiology of h n influenza a viruses from quail in southern china characterization of the pathogenicity of members of the newly established h n influenza virus lineages in asia sequence analysis of the hemagglutinin gene of h n korean avian influenza viruses and assessment of the pathogenic potential of isolate ms molecular characterization of h n influenza viruses: were they the donors of the ''internal'' genes of h n viruses in hong kong? h n influenza viruses possessing h n -like internal genomes continue to circulate in poultry in southeastern china role of infectious bronchitis live vaccine on pathogenicity of h n avian influenza virus manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees) an avian influenza virus of h subtype that is highly pathogenic for chickens, but lacks multiple basic amino acids at the haemagglutinin cleavage site human influenza virus hemagglutinin with high sensitivity to proteolytic activation insertion of a multibasic cleavage motif into the hemagglutinin of a low-pathogenic avian influenza h n virus induces a highly pathogenic phenotype acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain pathology and virus distribution in chickens naturally infected with highly pathogenic avian influenza a virus (h n ) during the outbreak in the netherlands implication of the proprotein convertases furin, pc and pc in the cleavage of surface glycoproteins of hong kong, ebola and respiratory syncytial viruses: a comparative analysis with fluorogenic peptides substrate cleavage analysis of furin and related proprotein convertases. a comparative study cloning and expression of novel mosaic serine proteases with and without a transmembrane domain from human lung host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses world health organization ⁄ oie ⁄ fao h n evolution working group. toward a unified nomenclature system for highly pathogenic avian influenza virus (h n ) novel type ii transmembrane serine proteases, mspl and tmprss , proteolytically activate membrane fusion activity of hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication is virulence of h n influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? targeted infection of endothelial cells by avian influenza virus a ⁄ fpv ⁄ rostock ⁄ (h n ) in chicken embryos detection of circulating asian h n viruses by a newly established monoclonal antibody a novel influenza a virus mitochondrial protein that induces cell death influenza a virus pb -f protein contributes to viral pathogenesis in mice expression of the influenza a virus pb -f enhances the pathogenesis of viral and secondary bacterial pneumonia the influenza a virus pb -f protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function influenza virus pb -f protein induces cell death through mitochondrial ant and vdac a single mutation in the pb -f of h n (hk ⁄ ) and influenza a viruses contributes to increased virulence the effects of influenza a virus pb -f protein on polymerase activity are strain specific and do not impact pathogenesis eight-plasmid system for rapid generation of influenza virus vaccines pb -f proteins from h n and century pandemic influenza viruses cause immunopathology pneumonia and respiratory failure from swine-origin influenza a (h n ) in mexico emergence of a novel swine-origin influenza a (h n ) virus in humans dating the emergence of pandemic influenza viruses in vitro and in vivo characterization of new swine-origin h n influenza viruses emergence of a novel swine-origin influenza a virus (s-oiv) h n virus in humans influenza: emergence and control systemic cytokine responses in patients with influenza-associated encephalopathy inflammatory cytokines in vascular dysfunction and vascular disease identification of trypsin i as a candidate for influenza a virus and sendai virus envelope glycoprotein processing protease in rat brain mechanisms of matrix metalloprotease- upregulation and tissue destruction in various organs in influenza a virus infection activation of influenza a viruses by trypsin treatment host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression active nf-jb signaling is a prerequisite for influenza virus infection nf-jb and virus infection: who controls whom tnf-induced mitochondrial damage: a link between mitochondrial complex i activity and left ventricular dysfunction acute encephalopathy associated with influenza and other viral infections molecular structure and assembly of the tight junction identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury human influenza virus infection and apoptosis induction in human vascular endothelial cells fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia h n infection of the respiratory tract and beyond: a molecular pathology study fatal avian influenza a (h n ) in a child presenting with diarrhea followed by coma influenza virus m protein mediates escrt-independent membrane scission the protein-protein interaction map of helicobacter pylori development of a real-time rt-pcr for the detection of swine-lineage influenza a (h n ) virus infections long-term storage and recovery of buccal cell dna from treated cards pandemic a(h n ) influenza virus detection by real time rt-pcr: is viral quantification useful? the effect of storage at different temperatures on the stability of hepatitis c virus rna in plasma samples impact of various handling and storage conditions on quantitative detection of hepatitis c virus rna blood storage at degrees c-factors involved in dna yield and quality effects of prolonged storage of whole plasma or isolated plasma dna on the results of circulating dna quantification assays pathogenesis and transmission of swine-origin a(h n ) influenza virus in ferrets viral tropism and the pathogenesis of influenza in the mammalian host influenza: pathogenesis and host defense cutting edge: stealth influenza virus replication precedes the initiation of adaptive immunity genetic and antiviral drug susceptibility profiles of pandemic a(h n )v influenza virus circulating in portugal observed association between the ha mutation d g in the pandemic influenza a(h n ) virus and severe clinical outcome world health organization. sequencing primers and protocol antiviral drug profile of seasonal influenza viruses circulating in portugal from predicting the antigenic structure of the pandemic (h n ) influenza virus hemagglutinin genomic signature and mutation trend analysis of pandemic (h n ) influenza a virus weekly update on oseltamivir resistance to influenza a (h n ) viruses pandemic a(h n ) influenza virus detection by real-time rt-pcr: is viral quantification useful? virulence associated substitution d g in hemagglutinin of pandemic influenza a(h n ) virus affects receptor binding the first wave of pandemic influenza (h n ) in germany: from initiation to acceleration new york city swine flu investigation team, et al. the severity of pandemic h n influenza in the united states from rhinovirus delayed the circulation of the pandemic influenza a(h n ) virus in france dynamics and impact of the a(h n ) epidemic in metropolitan france clinical aspects of pandemic influenza a(h n ) virus infection preliminary estimates of mortality and years of life lost associated with the a ⁄ h n pandemic in the us and comparison with past influenza seasons oseltamivir resistance in adult oncology and hematology patients infected with pandemic (h n ) virus household transmission of the pandemic a ⁄ h n influenza virus: elevated laboratoryconfirmed secondary attack nrates and evidence of assymptomatic infections time lines of infection and disease in human influenza: a review of volunteer challenge studies facemasks and hand hygiene to prevent influenza transmission in households comparative epidemiology of pandemic and seasonal influenza a in households viral shedding and clinical illness in naturally acquired influenza virus infections r: a language and environment for statistical computing the guinea pig as a transmission model for human influenza viruses in the control of epidemic and pandemic influenza - blocking interhost transmission of influenza virus by vaccination in the guinea pig model phenotypic properties resulting from directed gene segment reassortment between wild-type a ⁄ sydney ⁄ ⁄ influenza virus and the live attenuated vaccine strain emergence of a novel swine-origin influenza a (h n ) virus in humans pneumonia and respiratory failure from swine-origin influenza a (h n ) in mexico cumulative number of confirmed human cases of avian influenza a ⁄ (h n ) reported to who factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises airborne transmission of disease in hospitals noninvasive positive-pressure ventilation: an experimental model to assess air and particle dispersion airflow and droplet spreading around oxygen masks: a simulation model for infection control research coughing and aerosols. images in clinical medicine antimicrobial products registered for use against influenza a virus on hard surfaces. office of pesticide programs u.s. environmental protection agency antimicrobials division the genesis of a pandemic influenza virus influenza in the elderly -a mini review control of influenza in the long term care facility: a review of established approaches and newer options a study of the impact of influenza on the functional status of frail older people influenza in long-term care facilities: preventable, detectable, treatable mortality associated with influenza and respiratory syncytial virus in the united states causes of death australia : leading causes of death australian government department of heath and ageing. pandemic (h n ) update bulletins for pandemic (h n ) update bulletins for world health organization. pandemic (h n ) -update factors associated with death or hospitalization due to pandemic influenza a (h n ) infection in california influenza a (h n ) antibodies in residents of new south wales, australia, after the first pandemic wave in the southern hemisphere winter qualification of the hemagglutination inhibition assay in support of pandemic influenza vaccine licensure managing outbreaks of viral respiratory infection in aged care facilities -challenges and difficulties during the first pandemic wave laboratory diagnosis of influenza virus infection antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans incidence of pandemic influenza a h n infection in england: a cross-sectional serological study cross-reactive antibody responses to the pandemic h n influenza virus in vitro and in vivo characterization of new swine-origin h n influenza viruses cross-reacting antibodies against pandemic influenza a (h n ) virus in elderly australians rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality a rhinovirus outbreak among residents of a long term care facility lessons from a respiratory illness outbreak in an aged-care facility detection and isolation of pandemic (h n ) influenza virus infection among pediatric patients reporting diarrhea and influenza-like illness significance of seasonal influenza viruses in the stool of pediatric patients viral load in patients infected with pandemic h n influenza a virus haemagglutinin mutations responsible for the binding of h n influenza a viruses to human-type receptors biedermann e references world health organization writing group. nonpharmaceutical interventions for pandemic influenza, international measures avian influenza, including influenza a (h n ), in humans: who interim infection control guideline for health care facilities inactivation of influenza a viruses in the environment and modes of transmission: a critical review quantitative method for evaluating virucidal activity of biocides used on hard surfaces nonpharmaceutical interventions for pandemic influenza, international measures avian influenza, including influenza a (h n ), in humans: who interim infection control guideline for health care facilities inactivation of influenza a viruses in the environment and modes of transmission: a critical review distribution of airborne influenza virus and respiratory syncytial virus in an urgent care medical clinic characterisation of stability, cytotoxicity, and skin sensitisation and irritation for respirators with antiviral coating nonpharmaceutical interventions for pandemic influenza, international measures avian influenza, including influenza a (h n ), in humans: who interim infection control guideline for health care facilities c cf/ /infectioncontrol.pdf international organization for standardization. : biological evaluation of medical devices, part : tests for cytotoxicity: in vitro methods effects of antiviral drugs on viral detection in influenza patients and on the sequential infection to their family members. serial examination by quick diagnosis (capiria) and virus culture intrafamilial transmission of influenza a and b analysis of infectious diseases data stochastic models for analysis of household transmission data: examining human-made transmission experiments factors influencing the effectiveness of oseltamivir and amantadine for the treatment of influenza. a japanese study of the multi-center study of the ⁄ influenza season clinical evaluation of an immunochromatography test kit, capilia flua, b for rapid diagnosis of influenza usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test. options for control of influenza vii ; abstract book. options for the control of influenza vii strategies for containing an emerging influenza pandemic in southeast asia flute, a publicly available stochastic influenza epidemic simulation model the japanese experience with vaccinating schoolchildren against influenza mass vaccination of schoolchildren against influenza and its impact on the influenza-associated mortality rate among children in japan effect of vaccination of a school-age population upon the course of an a -hong kong influenza epidemic herd immunity in adults against influenza-related illnesses with use of the trivalent-live attenuated influenza vaccine (caiv-t) in children the effect of mass influenza immunization in children on the morbidity of the unvaccinated elderly effect of influenza vaccination of children on infection rates in hutterite communities: a randomized trial efficacy of live attenuated and inactivated influenza vaccines in schoolchildren and their unvaccinated contacts in novgorod, russia effectiveness of influenza vaccination of day care children in reducing influenza-related morbidity among household contacts effectiveness of schoolbased influenza vaccination strategies for pandemic and seasonal influenza vaccination of schoolchildren in the united states strategy for distribution of influenza vaccine to high-risk groups and children estimating the impact of childhood influenza vaccination programmes in england and wales population-wide benefits of routine vaccination of children against influenza prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) protective efficacy of seasonal influenza vaccination against seasonal and pandemic influenza virus infection during in hong kong partial protection of seasonal trivalent inactivated vaccine against novel pandemic influenza a ⁄ h n : case-control study in mexico city association between the - seasonal influenza vaccine and pandemic h n illness during spring-summer : four observational studies from canada cross-reactive antibody responses to the pandemic h n influenza virus effectiveness of - trivalent influenza vaccine against pandemic influenza a (h n ) -united states seasonal influenza vaccine and protection against pandemic (h n ) -associated illness among us military personnel interim analysis of pandemic influenza (h n ) in australia: surveillance trends, age of infection and effectiveness of seasonal vaccination pandemic (h n ) risk for nurses after trivalent vaccination infection and death from influenza a h n virus in mexico: a retrospective analysis birth weight, infant growth, and childhood body mass index: hong kong's children of birth cohort comparative epidemiology of pandemic and seasonal influenza a in households facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial partial protection against challenge with the highly pathogenic h n influenza virus isolated in japan in chickens infected with the h n influenza virus prior h n influenza infection and susceptibility of cleveland family study participants during the h n pandemic of : an experiment of nature transmission of pandemic h n influenza virus and impact of prior exposure to seasonal strains or interferon treatment prior infection with an h n swine influenza virus partially protects pigs against a low pathogenic h n avian influenza virus protection against a european h n swine influenza virus in pigs previously infected with h n and ⁄ or h n subtypes infection of mice with a human influenza a ⁄ h n virus induces protective immunity against lethal infection with influenza a ⁄ h n virus evidence of a cross-protective immune response to influenza a in the cotton rat model heterosubtypic immunity to influenza a virus: where do we stand? safety and immunogenicity of as b adjuvanted split virion versus non-adjuvanted whole virion h n influenza vaccine in uk children aged months- years: open label, randomised, parallel group, multicentre study options for live attenuated influenza vaccines (laiv) in the control of epidemic and pandemic influenza - pilot studies on recombinant cold-adapted live type a and b influenza virus vaccines development of cold-adapted recombinant live, attenuated influenza vaccines in usa and ussr clinical and epidemiological evaluation of a live, cold-adapted influenza vaccine for - -year-olds revised requirements for influenza vaccine (live) global pandemic influenza action plan to increase vaccine supply characteristics of human influenza a ⁄ h n , a ⁄ h n and b viruses isolated characteristics of human influenza a ⁄ h n , a ⁄ h n and b viruses isolated characteristics of human influenza a ⁄ h n , a ⁄ h n and b viruses isolated characteristics of human influenza a ⁄ h n , a ⁄ h n and b viruses isolated dynamics of antigenic and genetic changes in the hemagglutinins of influenza a ⁄ h n viruses of three consecutive seasons molecular epidemiology of a ⁄ h n and a ⁄ h n influenza virus during a single epidemic season in the united states an mf -adjuvanted inactivated influenza vaccine containing a ⁄ panama ⁄ (h n ) induced broader serological protection against heterovariant influenza virus strain a ⁄ fujian ⁄ than a subunit and a split influenza vaccine response of influenza vaccines against heterovariant influenza virus strains in adults with chronic diseases cross-protection by mf tmadjuvanted influenza vaccine: neutralizing and hemagglutinationinhibiting antibody activity against a (h n ) drifted influenza viruses quantifying influenza vaccine efficacy and antigenic distance detection of antibody to avian influenza a (h n ) virus in human serum by using a combination of serologic assays world health organization global influenza programme. who manual on animal influenza diagnosis and surveillance antigenic characterization of influenza b virus with a new microneutralisation assay: comparison to haemagglutination and sequence analysis seroprotection rate, meanfold increase, seroconversion rate: which parameter adequately expresses seroresponse to influenza vaccination? the adjuvanted influenza vaccines with novel adjuvants: experience with the mf -adjuvanted vaccine experimental inoculation of pigs with pandemic h n virus and hi cross-reactivity with contemporary swine influenza virus antisera current status of live attenuated influenza vaccine in the united states for seasonal and pandemic influenza failure of protection and enhanced pneumonia with a us h n swine influenza virus in pigs vaccinated with an inactivated classical swine h n vaccine isolation and characterization of h n influenza a virus from turkeys a new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates a dna transfection system for generation of influenza a virus from eight plasmids efficacy of inactivated swine influenza virus vaccines against the a ⁄ h n influenza virus in pigs mortality associated with influenza and respiratory syncytial virus on the united states realities and enigma of human viral influenza: pathogenesis, epidemiology and control immune response after adjuvant mucosal immunization of mice with inactivated influenza virus stimulation of protective and cross-protective imunity against influenza b virus after adjuvant mucosal immunization of mice protective and cross-protective mucosal immunization of mice by influenza virus type a with bacterial adjuvant synergistic action of cholera toxin b subunit (and escherichia coli heat-labile toxin b subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine effects of intanasal administration of chlera toxine (or escherichia coli heat-labila enterotoxin b subunits)supplemented with a trace amount of the holotoxin on the brain the adjuvants mf and lt-k enhance the mucosal and systemic immunogenicity of subunit influenza vaccine administrated intranasaly in mice generation of influenza vaccine strains by genetic reassortment a description of the process of seasonal and h n influenza vaccine virus selection and development pcr restriction analysis if genome composition and stability of cold-adapted reassortants live influenza vaccines options for live attenuated influenza vaccines (laiv) in the control of epidemic and pandemic influenza - characterization of an influenza a h n reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic h n viruses with pandemic potential are there alternative avian influenza viruses for generation of stable attenuated avian-human influenza a reassortant viruses? organization of production, quality control and provision of the republic of kazakhstan with the a ⁄ h n vaccine development of diagnostics and vaccines against influenza a ⁄ h n and a ⁄ h n is the basis for biological safety and protection of population in kazakhstan maior results of developing a technology for production of a pandemic influenza a ⁄ h n vaccine» abstract book ''options for the control of influenza vii abstract book international scientific conference of students, graduates and young scientists ''lomonosov isolation of influenza viruses in cell cultures and chicken embryos and their identification development of quality and safety control methods of the inactivated vaccine based on the strain a ⁄ astanarg ⁄ : ⁄ (h n control methods of medical immunobiological preparations injected to humans. guidelines, muk . / . . - (adopted by goskomsanepidnadzor rf ob scientific center for expertise of medical products, moscow. russian national pharmacopeia. ist edn the guidance for pre-clinical testing of pharmaceutical substances for safety and effectiveness (regulations of good laboratory practice in the russian federation -glp). manual on experimental (pre-clinical) assay of new pharmaceutical substances. moscow: ministry of public health ⁄ department of quality, effectiveness and safety control of medicines ⁄ scientific center of expert examination and national control of medicines ⁄ pharmacological national committee manual on experimental (pre-clinical) assay of new pharmaceutical substances. moscow: medline status of the development of influenza a ⁄ h n vaccines in the world and in russia approval of instruction for preclinical testing of pharmacological and medical substances in the repubilic of kazakhstan preclinical testing of new medical immunobiological substances. basic principles. moscow: ussr ministry of health order and methods of control of vaccine immunobiological safety. general methodic principles. moscow: ussr ministry of health planning and performance of clinical study of medicines federal pharmaceutical substances law. # -fz russian federation national standard. good clinical practice. # -st order of the minister of health of russian federation # signed . . . rules for clinical practice in russian federation a single-dose influenza a (h n ) vaccine safe and immunogenic in adult and elderly patients: an approach to pandemic vaccine development cross-reactive immunity to clade strains of influenza virus a subtype h n induced in adults and elderly patients by fluval, a prototype pandemic influenza virus vaccine derived by reverse genetics, formulated with a phosphate adjuvant, and directed to clade strains develop of domestic vaccinal and diagnostic preparations for influenza a ⁄ h n and a ⁄ h n as a basic principle of biological safety and security for the population of kazakhstan guidelines: ''isolation of influenza viruses in cell cultures and chicken embryos and their identification''. moscow: a-print validation of control methods for chemical and physicochemical characteristics of mibp quality: organization the guidance for pre-clinical testing of pharmaceutical substances for safety and effectiveness (regulations of good laboratory practice in the russian federation -glp). manual on experimental (pre-clinical) assay of new pharmaceutical substances. moscow: ministry of public health ⁄ department of quality, effectiveness and safety control of guidance on assessment of allergenic characteristics of pharmaceutical substances on adoption of the instructions for performance of pre-clinical assays and tests of pharmaceutical substances and medicines in the republic of kazakhstan national pharmacopoeia of the republic of kazakhstan planning and performance of clinical study of medicines federal law on medicines no. -aii dated national standard of the russian federation ''proper clinical practice'' no. -st guidance on clinical practice in the russian federation'' (in russian) aluminum hydroxide adjuvants activate caspase- and induce il- beta and il- release safety and immunogenicity of a pandemic influenza a h n vaccine when administered alone or simultaneously with the seasonal influenza vaccine for the - influenza season: a multicentre, randomised controlled trial current status of live attenuated influenza vaccine in the united states for seasonal and pandemic influenza a simple method of estimation fifty per cent endpoints use of animal models to understand the pandemic potential of highly pathogenic avian influenza viruses evaluation of replication and pathogenicity of avian influenza a h subtype viruses in a mouse model results of preclinical studies of live monovalent influenza vaccine influvir swine influenza a (h n ) infection in two children -southern california comparison of rna hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro concepts and procedures for laboratory-based influenza surveillance. us department of health and human services and pan-american health organization detection of antibody to avian influenza a (h n ) virus in human serum by using a combination of serologic assays fda ⁄ nih ⁄ who public workshop on immune correlates of protection against influenza a viruses in support of pandemic vaccine development coinfection of avian influenza virus (h n subtype) with infectious bronchitis live vaccine co-infection of staphylococcus aureus or haemophilus paragallinarum exacerbates h n influenza a virus infection in chickens human infection with an avian h n influenza a virus in hong kong in avian-to-human transmission of h n subtype influenza a viruses: relationship between h n and h n human isolates human infection with influenza h n universal primer set for the full-length amplification of all influenza a viruses phylogenetic analysis of hemagglutinin and neuraminidase genes of h n viruses isolated from migratory ducks the neighbor-joining method: a new method for reconstructing phylogenetic trees application of a microtechnique to viral serological investigations biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of a ⁄ seal ⁄ massachusetts ⁄ ⁄ (h n ) influenza virus development of vaccine strains of h and h influenza viruses isolation of ortho-and paramyxoviruses from feral birds in hokkaido head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of comparison, spatial organization and immunization route on the immunogenicity in a murine challenge model evaluation of growth, genetic, and antigenic characteristics of pandemic h n viruses isolated and passaged in qualified mdck suspension cells a (h n ) influenza virus pandemic: a review influenza vaccine: from surveillance through production to protection trivalent mdck cell culture-derived influenza vaccine optaflu (novartis vaccines) validation of the safety of mdck cells as a substrate for the production of a cell-derived influenza vaccine genetic composition of a high-yielding influenza a virus recombinant: a vaccine strain against ''swine'' influenza rescue of influenza a virus from recombinant dna of health, public health service. concepts and procedures for laboratory based influenza surveillance a rapid method for immunotitration of influenza viruses using flow cytometry generation of live attenuated novel influenza a ⁄ california ⁄ ⁄ (h n ) vaccines with high yield in embryonated chicken eggs haemagglutinin quantification and identification of influenza a&b strains propagated in per.c cells: a novel rp-hplc method comparison of egg and high yielding mdck cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells effect of neuraminidase antibody on hong kong influenza infection-permissive immunization with influenza virus neuraminidase prevents weight loss in infected mice protection of mice with recombinant influenza virus neuraminidase cross-reactive neuraminidase antibodies afford partial protection against h n in mice and are present in unexposed humans note for guidance on harmonization of requirements for influenza vaccines who meeting on the role of neuraminidase in inducing protective immunity against influenza infection who manual on animal influenza diagnosis and surveillance [electronic resource] ⁄ world health organization, department of communicable disease surveillance and response measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates detection of antibody to avian influenza a(h n ) virus in human serum by using a combination of serologic assays large trials confirm immunogenicity of h n vaccines pre-existing immunity against swine-origin h n influenza viruses in the general human population yuelong shu references novel swine-origin influenza a (h n ) virus investigation team. emergence of a novel swine-origin influenza a (h n ) virus in humans serum crossreactive antibody response to a novel influenza a (h n ) virus after vaccination with seasonal influenza vaccine detection of antibody to avian influenza a (h n ) virus in human serum by using a combination of serologic assays concepts and procedures for laboratory-based influenza surveillance manual on animal influenza diagnosis note for guidance on harmonization of requirements for influenza vaccines (cpmp ⁄ bwp ⁄ ⁄ ). european agency for the evaluation of medicinal products cross-reactive antibody responses to the pandemic h n influenza virus serologic survey of pandemic (h n ) virus influenza a(h n ) seroconversion rates and risk factors among distinct adult cohorts in singapore swine influenza a outbreak reflections on the swine flu vaccination program the flu and other influenza pandemics: ''over there'' and back again evidence of an absence: the genetic origins of the pandemic influenza virus the influenza virus: a killer comes into view antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans pathogenesis and immunogenicity of influenza viruses with genes from the pandemic virus lifetime of plasma cells in the bone marrow humoral immunity due to long-lived plasma cells no one is naive: the significance of heterologous t-cell immunity infection outcomes in patients with common variable immunodeficiency disorders: relationship to immunoglobulin therapy over years signaling by toll-like receptors and requires bruton's tyrosine kinase antiviral cytotoxic t-cell memory by vaccination with recombinant listeria monocytogenes cell-mediated immune response of human lymphocytes to influenza a ⁄ ussr (h n ) virus infection severe deficiency of switched memory b cells (cd (+)igm(-)igd(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease an early humoral immune response in peripheral blood following parenteral inactivated influenza vaccination multifunctional th cells define a correlate of vaccine-mediated protection against leishmania major note for guidance on harmonization of requirements for influenza vaccines (cpmp ⁄ bwp ⁄ ⁄ ) active vaccination in patients with common variable immunodeficiency (cvid) serum bactericidal antibody response to serogroup c polysaccharide meningococcal vaccination in children with primary antibody deficiencies specific anti-influenza virus antibody production in vitro by lymphocytes from a subset of patients with hypogammaglobulinemia shu a a state key laboratory for infectious disease control and prevention, national institute for viral disease control and prevention, chinese center for disease control and prevention, beijing, china. b state key laboratory for molecular virology and genetic engineering, national institute for viral disease control and prevention roles of adjuvant igv combined with influenza m e based virus like particle in the immune response and cross protection usa) ae h at °c. tetramethylbenzidine (tmb) was applied, and the optical den epidemiology and pathogenesis of influenza epidemiology and virology of influenza illness influenza vaccine: the challenge of antigenic drift evolving strategies for the prevention of influenza infection: potential for multistrains targeting a ''universal human influenza'' a vaccine universal influenza a vaccine: optimization of m -based constructs the epitope recognized by a monoclonal antibody in influenza a virus m protein is immunogenic and confers immune protection high-epitope density in a single recombinant protein molecule of the extracellular domain of influenza a virus m protein significantly enhances protective immunity pre-clinical study of influenza virus a m peptide conjugate vaccines in mice, ferrets, and rhesus monkeys induction of influenza type a virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein a universal influenza a vaccine based on the extracellular domain of the m protein protective immunity against influenza a virus induced by immunization with dna plasmid containing influenza m gene protection against influenza virus challenged by topical application of influenza dna vaccine influenza a vaccine based on the extracellular domain of m : weak protection mediated via antibody-dependent nk cell activity t cell immunoglobulin mucin- crystal structure reveals a novel ligand binding surface structures of t cell immunoglobulin mucin protein show a metal-ion-dependent ligand binding site where phosphatidylserine binds structures of t cell immunoglobulin mucin receptors and reveal mechanisms for regulation of immune responses by the tim receptor family the tim gene family: emerging roles in immunity and disease th -specific cell surface protein tim- regulates macrophage activation and severity of and autoimmune disease identification of a surface glycoprotein on african green monkey kidney cells as a receptor for hepatitis a virus the igv domain of human b - (cd ) is sufficient to co-stimulate t lymphocytes and induce cytokine secretion vaccination with cell immunoglobulin mucin- antibodies and inactivated influenza enhances vaccine-specific lymphocyte proliferation, interferon-r production and cross-strain reactivity incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune response virus-like particle induces protective immunity against homologous and heterologous strains of influenza virus site accessed on st study designs for timely estimation of influenza vaccine effectiveness using european sentinel practitioner networks a new influenza surveillance system in france: the ile-de-france ''grog''. . principles and methodology site accessed on st i-move'' towards monitoring seasonal and pandemic influenza vaccine effectiveness: lessons learnt from a pilot multi-centric case-control study in europe population and risk group uptake of h n influenza vaccine in mainland france - : results of a national vaccination campaign of global health and emerging pathogens ns deletions convert the -h n pandemic virus into a live attenuated vaccine. influenza and other respiratory viruses the influenza virus enigma vaccine production capacity for seasonal and pandemic (h n ) influenza interferon-induced isg conjugation inhibits influenza a virus gene expression and replication in human cells the multifunctional ns protein of influenza a viruses influenza a and b viruses expressing altered ns proteins: a vaccine approach multiple anti-interferon actions of the influenza a virus ns protein the ns protein of a human influenza virus inhibits type i interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells immunization with live attenuated influenza viruses that express altered ns proteins results in potent and protective memory cd + t-cell responses plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes regulation of adaptive immunity by the innate immune system the influenza virus ns protein: inhibitor of innate and adaptive immunity single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses unidirectional rna polymerase i-polymerase ii transcription system for the generation of influenza a virus from eight plasmids avian influenza (h n ) viruses isolated from humans in asia in exhibit increased virulence in mammals transmission and pathogenesis of swine-origin a(h n ) influenza viruses in ferrets and mice a naturally occurring deletion in its ns gene contributes to the attenuation of an h n swine influenza virus in chickens future influenza vaccines and use of genetic recombinants eight-plasmid system for rapid generation of influenza virus vaccines rescue of influenza b virus from eight plasmid high yield reassortants of influenza type b viruses np gene obtained from high yield donor. options for control of influenza vi. abstract # summary of status of development and availability of b ⁄ brisbane ⁄ ⁄ -like candidate vaccine viruses and potency testing reagent reassortants of influenza b viruses for use in vaccines: an evaluation preparation of influenza b virus recombinant strains genetic and phenotypic analysis of reassortants of high growth and low growth strains of influenza b virues pandemic influenza in russia: detection and molecular references evolutionary pattern of pandemic influenza (h n ) virus in the late phases of the pandemic antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans effects of egg adaptation on the receptor-binding properties of human influenza a and b viruses the early diversification of influenza a ⁄ h n pdm update on influenza a (h n ) monovalent vaccines clinical and epidemiologic characteristics of early cases of influenza a pandemic options for the control of influenza vii virus infection, people's republic of china genome evolution of novel influenza a (h n ) viruses in humans introduction and transmission of pandemic influenza a (h n ) virus -kenya emergence of a novel swine-origin influenza a (h n ) virus in humans first season of h n influenza origins and evolutionary genomics of the swine-origin h n influenza a epidemic modeling gene sequences over time in h n influenza a virus populations differentiation of two distinct clusters among currently circulating influenza a(h n )v viruses conversion of mdck cell line to suspension culture by transfecting with human siat e gene and its application for influenza virus production trivalent mdck cell culture-derived influenza vaccine optaflu (novartis vaccines) use of mdck cells for production of live attenuated influenza vaccine flow cytometric monitoring of influenza a virus infection in mdck cells during vaccine production cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences seroprevalence following the second wave of pandemic molecular evolution of human influenza a viruses in a local area during eight influenza epidemics from evaluation of hemagglutinin subtype swine influenza viruses from the united states the early molecular epidemiology of the swine-origin a ⁄ h n human influenza pandemic the genomic and epidemiological dynamics of human influenza a virus reconstructing the initial global spread of a human influenza pandemic: a bayesian spatial-temporal model for the global spread of h n pdm mild form of h n influenza infection detected by active surveillance: implications for infection control the pandemic (h n ) swine influenza virus is mild compared to the pandemic (h n ) virus because of a prolineto-serine substitution in the receptor-binding site of its hemagglutinin -a hypothesis pathogenesis and transmission of swine-origin a(h n ) influenza virus in ferrets investigation of the first cases of human-to-human infection with the new swine-origin influenza a (h n ) virus in canada antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans molecular evolution of hemagglutinin genes of h n swine and human influenza a viruses antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies the predicted antigenicity of the haemagglutinin of the spanish influenza pandemic suggests an avian origin genetically destined potentials for nlinked glycosylation of influenza virus hemagglutinin global patterns of influenza a virus in wild birds antigenic and genetic characteristics of swine-origin a (h n ) influenza viruses circulating in humans translatorx: multiple alignment of nucleotide sequences guided by amino acid translations raxml-iii: a fast program for maximum likelihood-based inference of large phylogenetic trees flugenome: a web tool for genotyping influenza a virus beast: bayesian evolutionary analysis by sampling trees origin and evolution of influenza virus hemagglutinin genes panorama phylogenetic diversity and distribution of type a influenza virus early alterations of the receptor-binding properties of h , h , and h avian influenza virus hemagglutinins after their introduction into mammals human influenza a h n virus related to a highly pathogenic avian influenza virus origin and evolutionary genomics of the swine-origin h n influenza a epidemic keywords novel influenza viruses, oseltamivir resistance phylogenetic analyses of pandemic influenza viruses textbook of influenza mapping the antigenic and genetic evolution of influenza virus cdc protocol of realtime rt-pcr for swine influenza a(h n ) revision the hemagglutination and hemagglutination ingibitions test. who collaborating center for surveillance molecular evolutionary genetics analysis (mega) software version . pandemic influenza: an inconvenient mutation large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution origin and evolutionary genomics of the swine-origin h n influenza a epidemic antigenic and genetic characteristics of swine-origin a (h n ) influenza viruses circulating in humans genetic subtyping using cluster analysis panorama phylogenetic diversity and distribution of type a influenza virus continuing progress towards a unified nomenclature for the highly pathogenic h n avian influenza viruses: divergence of clade . viruses the influenza virus resource at the national center for biotechnology information muscle: multiple sequence alignment with high accuracy and high throughput multiple alignment of dna sequences with mafft raxml-iii: a fast program for maximum likelihood-based inference of large phylogenetic trees flugenome: a web tool for genotyping influenza a virus evolution of influenza viral neuraminidase (na) genes revealed by large-scale sequence analysis. proceedings of the options for the control of influenza vii a quantitative genotype algorithm reflecting h n avian influenza niches xiu-feng wan a a department of basic sciences complete composition vector, influenza a virus, minimum spanning tree, phylogeny, progenitor ipminer: a progenitor gene identifier for influenza a virus avian-to-human transmission of the pb gene of influenza a viruses in the and pandemics triple-reassortant swine influenza a (h ) in humans in the united states clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, positionspecific gap penalties and weight matrix choice muscle: multiple sequence alignment with high accuracy and high throughput coffee: an objective function for multiple sequence alignments the neighbour-joining method: a new method for reconstructing phylogenetic trees mrbayes: bayesian inference of phylogenetic trees basic local alignment search tool a quantitative genotype algorithm reflecting h n avian influenza niches computational identification of reassortments in avian influenza viruses ubiquitous reassortments in influenza a viruses phylip -phylogeny inference package (version . ) pandemic influenza in russia: isolation, antigenic produced in our laboratory. normal equine serum was purchased from sigma (usa) comparative data on the development of influenza pandemic in russia and in the other european countries (in russian). materials of the ii annual all-russian congress on infectious diseases isolation on . . and deposition in the state virus collection of the first strain a ⁄ moscow ⁄ ⁄ (h n )swl, similar to the swine virus a(h n ) from the first patient diagnosed in moscow isolation of influenza viruses in cell cultures and in chicken embryos and their identification. methods guidance (in russian) molecular genetic characterization of the strains of pandemic influenza a(h n )v isolated on the territory of russian federation in (in russian). materials of the ii annual all-russian congress on infectious diseases genetic and antigenic analyses of influenza a (h n ) viruses, - sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses pandemic influenza a(h n ) virus mutations reported to be associated with severe disease antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans barr b,c a novartis vaccines and diagnostics gmbh, marburg, germany. b who collaborating centre for reference and research on influenza co-infecting viruses in pandemic h n and seasonal influenza positive human respiratory samples emergence of a novel swine-origin influenza a (h n ) virus in humans simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections comparison of multiple commercial assays for the detection of swine-origin influenza a (h n ) virus (soiv) evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections performance comparison of res-plex ii and xtag rvp fast for detecting respiratory viruses clinical virology symposium communityacquired respiratory co-infection (carc) in critically ill patients infected with pandemic influenza a (h n ) virus infection bacterial co-infections in lung tissue specimens from fatal cases of pandemic influenza a (h n ) -united states a quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine john's hopkins bloomberg school of public health design of an automated laboratory for high-throughput influenza surveillance human influenza surveillance: the demand to expand influenza: an emerging disease avian-to-human transmission of the pb gene of influenza a viruses in the and pandemics proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? influenza h n and h n virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation mapping of the two overlapping genes for polypepetides ns and ns on rna segment of influenza virus genome sequences of mrnas derived from genome rna segment of influenza virus: collinear and interrupted mrnas code for overlapping proteins influenza virus ns protein inhibits pre-mrna splicing and blocks mrna nucleocytoplasmic transport the influenza virus ns protein: a novel inhibitor of pre-mrna splicing identification of cis-acting intron and exon regions in influenza virus ns mrna that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes identification of a second protein encoded by influenza c virus rna segment influenza c virus ns protein upregulates the splicing of viral mrnas identification of an amino acid residue on influenza c virus m protein responsible for formation of the cord-like structures of the virus a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein detection of ion channel activity in xenopus laevis oocytes expressing influenza c virus cm protein evidence that the cm protein of influenza c virus can modify the ph of the exocytic pathway of transfected cells the ion channel activity of the influenza virus m protein affects transport through the golgi apparatus intracellular localization of influenza c virus ns protein (nep) in infected cells and its incorporation into virions receptor specificity, host range and pathogenicity of influenza viruses replication, pathogenesis and transmission of pandemic (h n ) virus in non-immune pigs world health organization. preliminary review of d g amino acid substitution in the haemagglutinin of pandemic influenza a (h n ) viruses observed association between the ha mutation d g in the pandemic influenza a(h n ) virus and severe clinical outcome quasispecies of the d g substitution in the hemagglutinin of pandemic influenza a(h n ) virus from patients with severe disease in hong kong a possible association of fatal pneumonia with mutations of pandemic influenza a ⁄ h n sw virus in the receptor-binding site of the ha subunit severe outcome of influenza a ⁄ h n ⁄ v infection associated with g ⁄ n polymorphisms in the haemagglutinin: a multicenter study influenza pandemic caused by highly conserved viruses with two receptor-binding variants overexpression of the alpha- , -sialyltransferase in mdck cells increases influenza virus sensitivity to neuraminidase inhibitors human and avian influenza viruses target different cell types in cultures of human airway epithelium avian-virus-like receptor specificity of the hemagglutinin impedes influenza virus replication in cultures of human airway epithelium receptor-binding specificity of pandemic influenza a (h n ) virus determined by carbohydrate microarray fields virology. philadelphia, pa: lippincott williams & wilkins the molecular virology and reverse genetics of influenza c virus identification of a second protein encoded by influenza c virus rna segment identification of a amino acid protein encoded by rna segment of influenza c virus influenza c virus cm protein is produced from a amino acid protein (p ) by signal peptidase cleavage a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein influenza c virus cm integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence evidence that the matrix protein of influenza c virus is coded for by a spliced mrna functional properties of the virus ion channels the cm protein of influenza c virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza a virus m and influenza b virus nb proteins characterization of a second protein (cm ) encoded by rna segment of influenza c virus phosphorylation of influenza c virus cm protein the sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza c virus cm protein identification of an amino acid residue on influenza c virus m protein responsible for formation of the cord-like structures of the virus a human melanoma cell line highly susceptible to influenza c virus antigenic characterization of the nucleoprotein and matrix protein of influenza c virus with monoclonal antibodies construction of an antigenic map of the haemagglutinin-esterase protein of influenza c virus the synthesis of polypeptides in influenza c virus-infected cells new low-viscosity overlay medium for viral plaque assays the influenza virus m protein cytoplasmic tail interacts with the m protein and influences virus assembly at the site of virus budding the cytoplasmic tail of the influenza a virus m protein plays a role in viral assembly the influenza a virus m cytoplasmic tail is required for infectious virus production and efficient genome packaging distinct domains of the influenza a virus m protein cytoplasmic tail mediate binding to the m protein and facilitate infectious virus production influenza virus m ion channel protein is necessary for filamentous virion formation influenza h n virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells das inhibits h n influenza virus infection of human lung tissues receptor binding specificity of recent human h n influenza viruses differential onset of apoptosis in avian influenza h n and seasonal h n virus infected human bronchial and alveolar epithelial cells: an in vitro and ex vivo study human influenza virus a ⁄ hongkong ⁄ ⁄ (h n ) infection induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells differential onset of apoptosis in influenza a virus h n -and h n -infected human blood macrophages avian flu: influenza virus receptors in the human airway haemagglutinin mutations responsible for the binding of h n influenza a viruses to humantype receptors an avian influenza h n virus that binds to a human-type receptor evolution of highly pathogenic h n avian influenza viruses in vietnam between evolutionary dynamics and emergence of panzootic h n influenza viruses writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h n ) virus recent avian h n viruses exhibit increased propensity for acquiring human receptor specificity a simple screening assay for receptor switching of avian influenza viruses glycan topology determines human adaptation of avian h n virus hemagglutinin h n chicken influenza viruses display a high binding affinity for neu acalpha - galbeta - ( -hso )glcnac-containing receptors a strain of human influenza a virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay search for additional influenza virus to cell interactions avian flu: isolation of drug-resistant h n virus the surface glycoproteins of h influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties this study was supported by the li ka shing foundation, the national institutes of health (niaid contract hhsn c), and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn c, the li ka shing foundation, and we thank all french and vietnamese field staff involved in the data collection in viet nam for their enthusiasm and support and we are grateful to the pig farmers participating in the study for their cooperation and patience. this study was a part of the gripavi project and was funded by the french ministry of foreign affairs. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn c and the area of excellence scheme of the university grants commission (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. we acknowledge the food and environmental hygiene department of hong kong for facilitating the study. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn c, the li ka shing foundation, and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. we gratefully acknowledge our colleagues from iiii, shantou university and skleid, hku for their excellent technical assistance. the study was supported by the rfcid commissioned study (lab# ) from research fund secretariat, food and health bureau, hong kong sar; area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ), hong kong sar; and by niaid contract (sjceirs, hhsn c), nih, usa.ferrets in all groups inoculated with a ⁄ turkey ⁄ ⁄ virus survived the infection and were observed once daily for days. below lower limit of detection (< ae log eid ⁄ ml).statistical cutoff of ic values for nai susceptibility, determined by x ae + iqr. outliers with ic above this cutoff and > times the mean ic for each drug were characterized as extreme outliers; those with known drug-resistance mutations such as h y were classified as resistant and analyzed separately. h wildtype, oseltamivir-susceptible isolates. h y variants, oseltamivir-resistant virus isolates. iqr, interquartile ranges; nai, neuraminidase inhibitors. we wish to thank our collaborators in the who global influenza surveillance network and united states public health laboratories for the submission of virus isolates and clinical specimens. we also thank our colleagues from the virus reference team and the influenza sequence activity, influenza division, cdc, for their valuable technical assis-the findings and conclusions of this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention (cdc). we are indebted to yonas araya, theresa wolter, and ivan gomez-osorio for their excellent laboratory techniques and animal handling assistance. we would like to thank andrea ferrero for her laboratory managerial skills. this research was possible through funding by the cdc-hhs grant ( u ci ), niaid-nih grant, (r ai ), csrees-usda grant ( - ), and niaid-nih contract (hhsn c). we thank c bazzoli for advice. this work was supported by a grant from the european union fp project flu-modcont (no. ). we thank staff at seoul, incheon, daejeon, gwangju, gangwon, and jeonbuk provincial research institute of health and environments for their laboratory testing. additionally, we would like to acknowledge the contributions of participating sentinel doctors for evaluating the new rat kit. this study was supported by a grant from the korea cdc. we thank roche applied science for providing the materials and equipment for this evaluation. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn c and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. the authors would like express their sincere thanks to cdc, usa for supporting the routine surveillance of ili in we would like to acknowledge the australian red cross blood service (the blood service) and the australian government, which fully fund the blood service for the provision of blood products and services to the australian community. we also wish to thank the donors and staff of the blood service, who have assisted in provision of specimens for testing in this protocol, as well as the staff at the who we are grateful to liping long for her assistance in map generation. this project was supported by nih niaid rc ai . cz is supported partially by canadian nserc postdoc fellowship. the authors thank the national investigation team based at the national institute of health (istituto superiore di sanita'), italy (in particular antonino bella, maria cristina rota, stefania salmaso) for providing their support in data collection, and the european union this study was supported in part by a grant-in-aid ( ) and the special coordination funds for promoting science and technology of ministry of education, science, sports and culture of japan. this study was supported in part by a grant-in-aid from the ministry of education, science, and culture of japan ( ) and the special coordination funds for promoting science and technology of mext of japan. the work described here was supported by phs grant ai- (jam) and alsac. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. py, po, dw and kb are employees of glaxosmithkline. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the stud- authors are thankful to path for the financial support of this research. we would like to acknowledge jessica d'amico and dr. rick bright of path for their editorial review. this study was supported by path. the authors would like to thank rick bright, jessica d'amico, and vadim tsvetnitsky for editing assistance. the we thank dr. m. enami (kanazawa university) for generously providing plasmids containing cdnas to influenza a virus m and ns genes. we also gratefully thank dr. r. sho (department of public health, yamagata university faculty of medicine) for statistical analysis. some data shown in this study have also been presented in the reference paper. this work was supported in part by a grant-in-aid for scientific research from the ministry of education, culture, sports, science, and technology, japan, takeda science foundation, terumo life science foundation, and a grant-in-aid from the global coe program of the japan society for the promotion of science. we thank markus eickmann for his help in isolation and initial characterization of a ⁄ hamburg ⁄ ⁄ and for providing antisera against h n pdm. this study was supported by the european union fp global a(h n ) genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. in this study we analysed the genetic and antiviral drug susceptibility profiles of pandemic a(h n ) influenza virus circulating in portugal. genetic profile analysis was performed in isolates to the hemagglutinin (ha), neuraminidase (na) and mp genes, and in six of these isolates the pb , pb , pa, np and ns genes were also analysed. antiviral drug susceptibility profile was analysed for isolates, phenotypically and genotypically to neuraminidase inhibitors (nai) and genotypically to amantadine. the point mutations identified in ha, na, and mp genes of different strains do not seem to evidence an evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h n ) virus. all analysed strains were found to be resistant to amantadine, and five of these strains exhibited a reduced susceptibility profile to nai, three only for oseltamivir and two for both inhibitors. introduction: the dynamics of pandemic influenza a ⁄ h n compared to seasonal strains of influenza is not clearly understood. it is important to understand the patterns of viral shedding and symptoms over time in community-based infections.materials and methods: household infections were followed-up in two large community-based studies. patterns of viral shedding, symptoms and signs, and tympanic temperature were plotted over time and grouped according to strain for analysis.results: the patterns of viral shedding, symptoms and signs, and tympanic temperature in three influenza a strains (pandemic a ⁄ h n , seasonal a ⁄ h n , and seasonal a ⁄ h n ) were comparable. peak viral shedding occurred close to the onset of symptoms and resolved after - days. patterns of viral shedding in influenza b virus infections differed.discussion: the patterns of viral shedding and clinical course of pandemic influenza a ⁄ h n infections were broadly similar to seasonal influenza a ⁄ h n and a ⁄ h n . only the clinical course of seasonal influenza b infections was similar to pandemic influenza a ⁄ h n . the dynamics of pandemic influenza a ⁄ h n were observed to be largely alike to the dynamics of seasonal influenza a ⁄ h n and a ⁄ h n . the coated respirators inactivated a broad range of influenza strains within minute, including the pandemic strain and human, swine, and avian influenza viruses. antiviral effectiveness was not reduced by hot, humid conditions or repeated saturation, which might occur during prolonged use of respirators. in contrast, infectious virions were detected on the surfaces of all uncoated ffp respirators, and could be transferred to glove surfaces during handling of contaminated masks. growth of the viruses was monitored by ha titer using turkey red blood cells, by quantitative real time rt-pcr (qrt-pcr) to detect the influenza a matrix gene, and also by flow cytometry to detect virus positive cells using monoclonal antibodies (imagen influenza virus a and b). , matrix gene copy number was determined using qrt-pcr and analysed using the sequence detection software on a fast system sds (applied biosystems, california, usa). further characterisation was performed through sequence analysis and the ha inhibition (hai) assay. sequence analysis was performed using dnastar and all sequences obtained were compared with the sequence of either the original clinical specimen if available or the conventional atcc derived mdck cell isolate. the hai assay was used to characterize the viruses against a panel of known standard reference viruses and their homologous ferret antiserum.options for the control of influenza vii abstract background: we measured the cross-reactive antibody response to pandemic h n in children and adults before and after vaccination with [ ] [ ] [ ] [ ] influenza season vaccines as part of the rapid public health response to the emergence of ph n and to provide evidence for ph n vaccination policy development in mainland china. materials and methods: archived serum specimens from previous vaccine studies were detected by hemagglutination inhibition assay. results: limited crossreactive antibody response to ph n had been detected among participants of all age groups before and after they had been vaccinated with - , - influenza seasonal vaccines. vaccination with seasonal influenza viruses resulted in limited seroconversion to ph n in all age groups, compared with - % of seroconversion to seasonal influenza viruses. but similar to recent studies, a peak of cross-reactive antibody response to ph n was observed in % and % of participants born from to before and after vaccination. conclusions: in order to protect our populations in china, our study strongly suggests vaccination with ph n is required in all age groups and that older populations born before may be associated with a lower infection rate of ph n . on april and april , , cases of ph n were identified in specimens obtained from two epidemiologically unlinked patients in the united states and soon thereafter in texas and mexico. since that time, the virus has spread across the globe. assessment of cross-reactive antibody response to the ph n after vaccination with sea-sonal influenza vaccine was first reported from us centers of disease control and prevention (us cdc). according to their results, the seasonal influenza vaccines provided little or no protection against the ph n , but some degree of preexisting immunity to the virus existed, especially among adults aged ‡ years. in this study, using archived serum samples from previous vaccine studies, we measure the level of cross-reactive antibody response to ph n in children and adults vaccinated intramuscularly with trivalent inactivated vaccine developed for the northern hemi- serum specimens were collected and provided by provincial centers for disease control and prevention of china as a public health response to the emergence of ph n exempt from human-subjects review. a total of serum samples were collected from xinjiang uygur autonomous region, yunnan, and shandong provinces. all the serum specimens were grouped by the age of subjects ( - , - , - , ‡ years) and by different influenza seasons.hemagglutination inhibition assay was performed according to standard procedures in this study. [ ] [ ] [ ] as with h n components of the vaccine, the seasonal influenza viruses used in this study were a ⁄ solomon islands ⁄ ⁄ and a ⁄ brisbane ⁄ ⁄ . the ph n influenza virus used in this study was a ⁄ california ⁄ ⁄ provided by us cdc. all the viruses were propagated in specific pathogenfree embryonated chicken eggs and inactivated by & paraformaldehyde. the criteria recommended by the european agency for the evaluation of medical product was applied for the assessment of seasonal influenza vaccine gmt, geometric mean titer; hi, hemagglutination inhibition. three weeks after boosting immunization, spleens were harvested from immunized and control mice. splenocytes were prepared by lymphocyte separation media (ez-sepÔ, shen zhen, china). the cells were washed and resuspended in complete rpmi- containing fetal bovine serum (hyclone, logan, ut, usa), glutamax, um b-me. splenocytes were cultured in vitro in the presence of inactivated h n , h n , h n , and h n influenza virus antigen for h. quick cell proliferation assay kit (biovision, san francisco, ca, usa) was used to detect the cell proliferation. the - nm absorbance was read on a plate reader. all experiments have been repeated at least three times.results are presented as mean standard error of the mean (sem). comparison of the data was performed using the student's t-test. significance was defined as a p value of < ae . to evaluate the adjuvant effect of recombinant igv, the anti m e antibody subclasses was measured. igg and igg a were detected after the first and second immunization ( table ). the ratio of igg a ⁄ igg was calculated. immunization with only m e-hbc showed a lower igg a ⁄ igg ratio < ae . igv combined with m e-hbc led to a high igg a ⁄ igg ratio of up to - after first and second immunization. these igg subclass distributions indicated that igv can induce a th immune response. to determine whether the splenocytes were stimulated in vitro with different subtypes of inactivated influenza antigen after the igv plus m e-hbc antigen immunization, h n , h n , h n , h n inactivated antigen was used table . the serum igg, igg , igg a, and igg a ⁄ igg ratio were measured by elisa after first and second immunization. m e were coated on the wells plate overnight, and serial dilution sera of day , , after first and second immunization were added , ae , , , , ug ⁄ ml of igg, igg , igg a purified antibody were also added for obtaining the standard curve. hrp-labeled goat anti-mouse igg, igg , or igg a was then added, washed, and the optical density was read at nm. the results were showed at mean ± sem. day after first immunization days after second immunizationoptions for the control of influenza vii the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than specimens yearly from cases of acute respiratory illness (ari), using two sampling methods: systematic randomized and non systematic ''ad hoc'' sampling. although vaccines against influenza a virus are the most effective method by which to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. a panel of recombinant live attenuated human influenza a vaccines (laivs), including ns - , ns - , nsd , were generated by rationally engineering mutations directly into the genome of a pandemic-h n virus. the vaccine potential of each laiv was determined through analysis of attenuation, immunogenicity, and their ability to protect mice and ferrets. the data indicate that the novel nsd -laiv was ideally attenuated and elicited strong protective immunity. this study also shows that attenuating mutations can be rapidly engineered into the genomes of emerging ⁄ circulating influenza a viruses in order to produce laivs. the influenza virus exhibits complicated evolutionary dynamics due to multiple reasons, such as diverse hosts, high mutation rates, and rapid replications. in this study, large-scale analyses of influenza neuraminidase (na) sequences revealed influenza a and b na genes diverged first around years ago, and subsequently the na subtypes of influenza a emerged around years ago. all nine na subtypes of influenza a were genetically distinct from each other, with a total of lineages identified. in addition, five and three sub-lineages were further identified in lineage a of na and lineage b of na , respectively. the majority of lineages and sub-lineages were found to be host or geographic specific. this study provides not only a better understanding of influenza na evolution, but also a database of lineages and sub-lineages that can be used for early detection of novel genetic changes for improved influenza surveillance. although phylogenetic approaches are commonly used and often found to be powerful, how to accurately identify lineages or sub-lineages of a gene segment of the influenza a virus remains a challenging issue. in this study, we address this issue by analyzing hemagglutinin (ha) sequences using a combination of statistical and phylogenetic methods. following a hierarchical nomenclature system that uses a letter to represent a lineage and a digit for a sub-lineage, we identified distinct lineages and sub-lineages in all ha subtypes through large-scale analyses of influenza a hemagglutinin sequences. the majority of the lineages or sub-lineages were host or geographic specific or both. further analysis of other segments will allow us to construct a comprehensive database for influenza a lineages and genotypes, facilitating early detection of new viral strains and genome reassortments and hence improve influenza surveillance. identification of the genetic origin of influenza a viruses will facilitate understanding of the genomic dynamics, evolutionary pathway, and viral fitness of influenza a viruses. the exponential increases of influenza sequences have expanded the coverage of influenza genetic pool, thus potentially reducing the biases for influenza progenitor identification. however, these large amounts of data generate a great challenge in progenitor identification. clinical (nasopharyngeal swabs) and post-mortem materials (fragments of trachea, bronchi, lungs, spleen) were obtained from clinics and ⁄ or out-patients from st. petersburg and from base virological laboratories (bvls) of the research institute of influenza in different regions of the country, which cover approximately ⁄ of the territory of russia. the informed consent for the bio-materials collection and studies was obtained from research subjects or from their relatives in cases of post-mortem materials. isolation of viruses was carried out in the mdck cell culture (cdc, atlanta, ga, usa) and in -day-old chicken embryos (e). isolation was done according the standard internationally accepted methods. the reaction of hemagglutination (ha) and the inhibition of hemagglutination (hai) were performed according the who recommended standard method. for the identification of epidemic isolates, we used the hyperimmune diagnostic bovine or ovine antisera annually obtained from the who reference center (cdc). for a detailed antigenic analysis we used the hyperimmune rat antisera against epidemic and reference influenza strains during the period from july , up to april , , we have obtained swabs from clinics and out-patients in st. petersburg and swabs from the bvls. in this period, rather high incidence of lethality from pneumonia was observed, which developed on the background of the pandemic flu h n v. thus, we received from bvls postmortem materials from deceased patients which manifested pcr+ influenza h n v-specific rna. all materials were tested for a possibility of isolation of influenza virus h n v both in eggs and in mdck cells. pcr-negative materials were discarded. we isolated strains of pandemic influenza from the materials collected in st. petersburg and region, which comprised ae % of the total number of analyzed samples. at the same time, we did not isolate any other sub-types of influenza in the season - except the pandemic flu. from the swabs purchased from bvls, strains were isolated, which compose ae % of the pcr+ samples, and strains from the post-mortem materials ( ae % of the pcr+ samples).altogether in the season - , we isolated, retrieved, and analyzed in hai influenza strains. ae % of them were pandemic strains a(h n )v, and only ae % influenza b viruses. these data together with the epidemiologic data and the results of pcr-diagnostic provide evidence in favor of nearly mono-etiological character of epidemic season - in russia for pandemic influenza a(h n )v.though the isolation of pandemic viruses was fulfilled in two traditional model systems, in the case of pandemic virus, we could observe the tendency of preferential multiplication in embryos compared to mdck, especially in cases of post-mortem material for which chicken embryos are the preferential system of isolation.h n v viruses, which were isolated and passaged in mdck, even with significant ha titers, quickly lost their ha activity provided they were kept at + °c. moreover, some other tested cell lines proved to be practically nonsensitive to the pandemic viruses h n v. we used hai reaction for the typing and antigenic characterization of isolated viruses. in the course of isolation of viruses in the reported period, we produced rat polyclonal antisera to the strains a ⁄ california ⁄ ⁄ and a ⁄ st. petersburg ⁄ ⁄ (h n )v and the antisera to the strains a ⁄ new jersey ⁄ ⁄ -the virus isolated during the epidemic in the united states and also of the swine origin -and to the 'swine' strains a ⁄ sw ⁄ ⁄ and a ⁄ iowa ⁄ ⁄ . the hai results of representative strains are given in figure . table shows that the isolated strains were homogenous in their antigenic properties and interacted with the diagnostic antiserum cdc for a(h n )v and also with the antisera to the strains a ⁄ california ⁄ ⁄ and a ⁄ st. petersburg ⁄ ⁄ up to - ⁄ homologous titer. viruses that were isolated from post-mortem materials did not differ by their antigenic characteristics from those isolated from swabs of live patients. only two strains could be attributed to the drift-variants of the strain a ⁄ california ⁄ ⁄ because they reacted with the appropriate antiserum up to ⁄ homologous titer; these strains were a ⁄ pskov ⁄ ⁄ and a ⁄ belgorod ⁄ ⁄ . it is interesting that the isolated strains reacted with the antisera to the strains a ⁄ new jersey ⁄ ⁄ and a ⁄ sw ⁄ ⁄ to ⁄ - ⁄ , and some particular strains even to ⁄ homologous titer. it is even more interesting that some pandemic isolates reacted with the antiserum to the strain iowa isolated in up to ⁄ - ⁄ homologous titer. despite of the fact that since the outbreak of 'swine flu' in the usa in new jersey years had gone (and for the strain iowa this period is nearly years) the ha of these viruses and of the pandemic influenza share some common antigenic determinants as was shown in hai.one more interesting feature of a considerable part of isolated strains is their capability to react with high titers with normal equine serum heated to and to °c, while all the strains of swine origin isolated earlier were inhibitor-resistant ( figure ). russian isolates of divided, in this respect, in two clear and approximately equal in number groups: one of them is similar to the reference strain amino acid substitutions, among them more than were disclosed in antigenic sites, so the degree of similarity to this strain is %. a new site of glycosylation was also discovered in the position of ha. essential distinctions of the aminoacid sequence of ha and antigenic properties of the h n v strains as compared with actual circulating and vaccine strains is one of the factors that determine the pandemic potential of this new influenza virus.according to the literature, the mutation in the ha gene d g could cause a broadening of the spectrum of receptor specificity of influenza virus by the acquisition of the capacity to bind both the residues a( fi ) and a( fi ) of the sialic acid of cellular receptors. both types of receptors are present at the human respiratory tract, but in different parts of it, and they exist in different proportions. according to the data of the european center of disease control and prevention (ecdc), the varieties g of the h n v virus were isolated in countries from subjects deceased of influenza or who suffered a severe form of illness, as well as from those who sustained only a light course of influenza. concerning the strains isolated in rii, this mutation was discovered in nine cases: four were isolated from live patients and five from post-mortem materials. thus, there are no convincing data at present that could prove a causal relationship of the given substitution and the aggravation of a disease course. this is in accordance with previous observations. concerning the resistance of studied strains to the widely used antiviral preparations, it was shown that all tested strains possessed the substitution s n in the m protein that determine the resistance to adamantanes. there was no substitution in the position of neuraminidase (na), which determines the resistance to oseltamivir (h y). these substitutions are the characteristic indices of the eurasian lineage of swine influenza viruses. thus all studied russian h n v isolates were resistant to adamantanes (rimantadine) and sensitive to oseltamivir. respiratory clinical samples taken in and that tested positive by real time reverse transcription (rt)-pcr for seasonal influenza viruses (a and b) and pandemic h n respectively were assessed for other respiratory viruses using the resplex ii panel ver . system distributed by qiagen. results showed that co-infections with another respiratory viruses were relatively rare, with a small number of samples having another co-infecting virus present, very few samples having two other viruses detectable in their samples, and none with further viruses. this low number of co-infecting viruses and the ability of certain cell lines not to support infection with particular viruses may make primary isolation of influenza viruses in cell lines easier than might have been thought previously. cm is the second membrane protein of influenza c virus and is posttranslationally modified by phosphorylation, palmitoylation, n-glycosylation, and dimer ⁄ tetramer formation. in the present study, we generated rcm -c a, a recombinant influenza c virus lacking cm palmitoylation site, and examined viral growth and viral protein synthesis in the recombinant-infected cells. the rcm -c a virus grew less efficiently than did the wild-type virus. membrane flotation analysis of the infected cells revealed that less np was recovered in the plasma membrane fractions of the rcm -c a-infected cells than that in the wild-type virus-infected cells, suggesting that palmitoylation of cm is involved in the affinity of the ribonucleoprotein complex to the plasma membrane, leading to the efficient generation of infectious viruses. influenza c virus has seven single-stranded rna segments of negative polarity, encoding pb , pb , p , haemaggluti- both the a , linkage and its topology on target cells were critical for human adaptation of influenza a viruses. the binding preference of avian flu virus h n ha to the a , -linked sialylated glycans is considered the major factor that limited its efficient infection in human. currently, the switch in binding-specificity of human h n viruses from a , to a , -glycans did naturally occur, and limited humanto-human transmission was found. to monitor their potential adaptation in the human population, receptor-binding specificity surveillance was made in china. here, the binding specificity of human h n virus strains isolated from to was demonstrated. dual binding preference to a , and a , -glycans were found in a ⁄ guangdong ⁄ ⁄ and a ⁄ guangxi ⁄ ⁄ . furthermore, both of them showed a high affinity to the long-branched a , -glycans, which predominate on the upper respiratory epithelial in human. our data suggests that the existence of h n virus with binding specificity to humans should be of concern.introduction via envelope glycoprotein hemagglutinin (ha), influenza viruses bind to cell-surface glycosylated oligosaccharides terminated by sialic acids (sa) where their linkage is celland species-specific. differential receptor binding preference is a host barrier for influenza virus transmission. although most h n viruses have low affinity to neu aca , gal (human-type) receptor, recent findings suggested that the adaptation of h n virus to human by mutations in the receptor-binding site (rbs) do indeed happen and resulted in enhanced affinity to human-type receptor. [ ] [ ] [ ] in contrast to its putative precursor, a ⁄ gs ⁄ gd ⁄ ⁄ , diverse genotypes were presented in currently circulating h n virus, accelerating evolution and widespread occurrence. , to date, distinct phylogenetic clades ( - ) were identified based on h n ha, and the confirmed human infections were caused by clade , , ae , ae , ae , and . in china, human h n disease was mostly caused by clade ae ae , which was identified in isolates from confirmed patients from provinces since . clade and clade ae are responsible for the case in and , respectively. two current cases of and were due to clade ae ae . now information on receptor property has been documented in some h n viruses of clade , ae , and ae . [ ] [ ] [ ] little is known about h n virus of clade ae ae , particularly from human.recently, a , -specific sialidase-treated red blood cell (rbc) agglutination assay was developed and used for receptor specificity screening of h n virus. , the a , or a , -binding preference can be distinguished by the change of hemagglutination titer reacted with rbcs and enzymatic rbcs. since fine receptor specificity existed in h n viruses, , the glycan array including sulfated-, fucosylated-, linear sialosides, di-sialosides, or direct binding assay with synthetic polyacrylamide (paa)-based sialylglycopolymers was also recommended for the receptor-specificity surveillance on h n viruses. furthermore, the long-branched a , sialylated glycans were currently identified to predominate on the upper respiratory epithelial in human and the recognition of this topology, ¢sln-ln is the key determinant for the human-adaptation of influenza a virus. here, we analyzed the receptor-binding specificity of human h n viruses isolated in china from to . since , a total of h n infection cases were confirmed in china from provinces. the pharyngeal swabs and lower airway aspirations from the patients were collected within days after disease onset, maintained in viral-transport medium, and tested within hours.options for the control of influenza vii key: cord- - nxhvvm authors: lei, xi-mei; yang, yong-le; he, yong-qiang; peng, lei; zhao, pengwei; xu, shu-ya; cao, hongwei; fang, pengfei; qiu, wenying; qin, pan; wang, bin; huang, yao-wei title: specific recombinant proteins of porcine epidemic diarrhea virus are immunogenic, revealing their potential use as diagnostic markers date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: nxhvvm given the highly contagious and acute nature of porcine epidemic diarrhea (ped), especially in piglets, there is an urgent need for the development of rapid and sensitive diagnostic assays. the diagnostic potentials of specific porcine epidemic diarrhea virus (pedv) accessory and nonstructural proteins, if any, have not yet been investigated. in order to determine and compare which of the viral proteins may be useful as diagnostic antigens, whole virus (wv) particles and a panel of structural and nonstructural pedv proteins [spike subunit (s ), the c-terminal part of orf (orf c), envelope (e), nonstructural protein (nsp ), nsp , ac (acidic domain of nsp ), and adrp (adp-ribose- -monophosphatase domain of nsp ), expressed individually in bacterial and/or mammalian cells] were tested for reactivity with sera from pedv-infected pigs by elisa and/or western blot analysis. according to western blots, serum antibody interactions with the s protein were relatively more sensitive and specific than orf c, e and ac. furthermore, a total of serum samples from diarrheal pigs of different ages were analyzed by elisa, with most showing immune-reactivity towards the wv, s , orf c, and e proteins. the earliest igg antibody response was observed in the one-week-old piglets, with similar antibody ontogeny and patterns of seroconversion for s , orf c, e, and wv antigens. in addition, the pattern of neutralizing antibody was more similar to that of iga in weaning piglets after pedv infection. collectively, these data provide more reliable information on the host immune response to different viral proteins, which will be useful for development of novel serological assays and for design of vaccines that better stimulate protective immunity. porcine epidemic diarrhea (ped) is characterized by severe diarrhea, vomiting, and dehydration followed by high mortality in suckling piglets . the causative agent, porcine epidemic diarrhea virus (pedv), was initially identified in europe in , and its genome (˜ kb in size) consists of seven open reading frames (orfs) (kocherhans et al., ) . the ' two-thirds of pedv genome encodes orf (consisting of overlapping orf a and orf b), and the ' onethird harbors orfs encoding four structural proteins, the spike (s), envelope (e), membrane (m) and nucleocapsid (n), and an accessory orf between s and e kocherhans et al., ; tian et al., ) . rna synthesis in pedv is carried out by a replicase-transcriptase composed of nonstructural proteins (nsp - ) encoded by orf a and orf b. among them, nsp comprises multiple structural domains, including a highly acidic domain at the amino terminus (ac), and a highly conserved adp-ribose- -phosphatase (adrp) macrodomain. the ac domain of nsp is essential for virion assembly and plays a critical role in interaction with the viral nucleocapsid during early infection, whereas the adrp provides activities necessary for synthesis of genomic and subgenomic rnas (hurst-hess et al., ) . as pigs of all ages are susceptible to pedv (alvarez et al., ; annamalai et al., ) , there is an urgent need for the development of highly sensitive and specific diagnostic assays for use in the field (diel et al., ) . since the identification of pedv, several diagnostic tests based on pcr detection of viral rna have been described in the literature (diel et al., ) . another common diagnostic method is serological testing for the presence of specific antibodies against viral proteins, which is also fast and convenient for epidemiologic investigations. many tools have been developed for the detection of anti-pedv antibodies based upon the major structural proteins (such as s, m or n proteins) in serum, colostrum, milk, feces and oral fluid, including indirect immunofluorescence assays (ifa), virus neutralization assays (sn), enzyme-linked immunosorbent assays (elisa), and fluorescent microsphere immunoassays (fmia) (diel et al., ; gerber et al., ; gerber and opriessnig, ; gimenez-lirola et al., ; okda et al., ) . but comparative studies of the above assays using different pedv structural and nonstructural proteins as antigens are rarely conducted. meanwhile, the diagnostic potentials of specific pedv accessory and nonstructural proteins, if any, have not yet been investigated in details. in this study, a panel of recombinant pedv orfs encoding structural and nonstructural proteins were expressed in mammalian and/or bacterial cells and screened for reactivity with porcine sera from seven provinces of china by elisa and/or western blot analysis, in order to determine which antigen is most suitable as a diagnostic marker for pedv infection. several rabbit polyclonal antibodies against these recombinant proteins were also generated and validated for use as diagnostic tools upon pedv infection in vitro. vero cells (atcc® ccl- ™) were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs). the pedv virulent strain zju/g / (genbank accession no. ku ) was used in the study qin et al., ) . a total of serum samples were collected in - from diarrheic pigs at commercial farms in the shandong, henan, jiangxi, hunan, jiangsu, heilongjiang and zhejiang provinces of eastern and northern china. there were samples from sows ( primiparous sows, and multiparous), from -week-old piglets, from week-olds, from week-olds, from week-olds, from week-olds, from week-olds, and from week-olds. another serum and fecal samples were obtained from weaning piglets experimentally challenged with pedv-zju/g / in three pedv challenged experiments. the procedure of the pedv challenge has been described previously . the briefly, the -dayold conventional piglets were inoculated with about ml infectious titer (tcid ) pedv in × pbs (ph . ). blood and fecal samples were collected prior to inoculation and at , , , , , , days postinoculation (dpi). samples from a total of survival piglets but not from dead pigs at each time point were collected. after centrifugation at ×g for min, serum was harvested, aliquoted and stored at − °c until use. fecal swab samples were individually mixed with ml of × pbs (ph . ) immediately after collection, placed in a ml cryogenic tube (bd falcon™), and stored at − °c until use. the animal experiments were approved by the experimental animal ethics committee of zhejiang university (approval no. zju ). the pedv-zju/g / strain was propagated in vero cells in dmem supplemented with μg/ml trypsin according to the standard method . briefly, a confluent cell monolayer was washed with minimum essential media (mem) twice before infecting with the virus [moi (multiplicity of infection) = ] for h at °c, after which additional culture medium was added without removing the inoculum. observed cytopathic effects (cpe) reached approximately % in - days, and the virus culture supernatant was collected after three freezethaw cycles, then clarified by high speed centrifugation ( ×g for min) and further purified by ultracentrifugation through a % (wt/ vol) sucrose cushion ( , ×g for h). the purified virus was further exposed by a negative staining technique, examined using electron microscopy, then stored at − °c until use. concentration of viral proteins was measured by bca protein assay kit (beyotime, shanghai, china). full-length pedv cdna was used as a template for amplification and cloning of various pedv genes, including those encoding the c terminus of pedv orf (orf c) and the complete sequences of e, nsp , nsp , ac, and adrp. the constructs used for this study are listed in table . for transient expression in mammalian cells, the pcdna- . vector was used, and the expression plasmid pcdna-pedv-s -fc has been described in the previous study (gerber et al., ) . for expression of his-tag fusion proteins in bacteria, pedv target genes were cloned in-frame with n-terminal ×his tags in the pet- a (novagen; for ac, adrp, nsp and nsp ) or the pet- a-derived psmart-i vector with an n-terminal sumo (small ubiquitin-related modifier) tag (smart-lifesciences, changzhou, china; for orf c and e). the oligonucleotide primer sequences and approaches used are available upon request. the sequences of all constructs were confirmed by dna sequencing (huada gene technology co., ltd). the above recombinant plasmids were transformed into bl (de ) competent cells, respectively, and grown in l of luria-bertani (lb) medium (invitrogen) containing μg/ml kanamycin at °c with shaking at rpm. when an od of . was reached, m isopropyl β-d- -thiogalactopyranoside (iptg) was added to the final concentration of . mm in the l of lb medium, and bacteria were grown for an additional h at °c. cells were chilled at °c and harvested by centrifugation at ×g for min, resuspended in ml lysis buffer ( mm tris−hcl, ph . ), and disrupted by ultrasonication. crude extracts were centrifuged at , ×g for min at °c, and soluble expression of the his-tagged fusion peptides was confirmed by sds-page analysis prior to purification by the ni-nta his•bind® resin system (transgen tech, dp , beijing, china) according to the manufacturer's instructions. for the polypeptides that were expressed in inclusion bodies, they were first solubilized in a denaturing buffer ( mm tris−hcl, with m urea, ph . ), and purified by ni-chelating chromatography (ge healthcare). elutions were pooled, dialyzed at °c against mm pbs (ph . ) with mm nacl and m urea, and analyzed by sds-page and western blot. five purified, recombinant pedv peptides (orf c, ac, adrp, nsp , and nsp ) were selected and separately used to immunize two new zealand white rabbits, using a custom antibody production service at hangzhou belta-biotechnology co., ltd. (hangzhou, china). rabbits were not immunized with recombinant e protein. the purified recombinant pedv s , orf c, e, adrp, nsp , nsp , and ac peptides were resolved on a - % bis-tris polyacrylamide gel (invitrogen) by electrophoresis and subsequently transferred onto a polyvinylidene difluoride (pvdf) membrane. proteins were then detected using an anti- ×his mab ( : dilution; protein-tech, wuhan, china) or rabbit polyclonal antisera at °c, followed by incubation with hrp-conjugated goat anti-mouse or anti-rabbit igg ( : , dilution; thermo fisher scientific, usa), as appropriate, at room temperature. after four washing steps using tris-buffered saline with . % tween (tbst), membranes were analyzed using an enhanced chemiluminescence kit (beyotime ecl plus kit, china). for serum western blot analysis, purified s , orf c, e, and ac peptides were incubated after sds-page and membrane transfer with individual porcine sera diluted : , or with anti-his mab or polyclonal rabbit serum as positive controls. hrp-conjugated rabbit antiswine igg and goat anti-mouse igg ( : , dilution; abcam, united states) were used, as appropriate, as secondary antibodies. antigen concentration and dilutions of sera and hrp-conjugated antibodies were optimized by checkerboard titrations. the optimal amount of pedv wv, s , orf c or e antigen used for coating was . , . , . or . ng/well/ μl. microtiter plates were blocked with μl/well blocking buffer (thermo fisher scientific, usa) for . h at °c. after coating, μl of serum samples ( : dilution) were transferred in triplicate and incubated at °c for h. afterwards, μl diluted hrp-conjugated goat anti-swine igg or iga ( : , dilution; thermo fisher scientific, usa) was added to each well and incubated at °c for h. wells were washed between incubation steps three times with μl pbs ( mm, ph . ) with . % tween- (pbs-t washing buffer). finally, μl tmb color liquid (solarbio, beijing, china) was added to each well and incubated for min at room temperature, after which the reaction was stopped by addition of μl/well of m sulfuric acid, and the plates were read at nm using a spectrophotometer. initial pedv-negative sera were obtained from the united states (a gift from dr. tanja opriessnig) that was subsequently used for screening negative porcine sera in china as reported previously (gerber et al., ) . the elisa positive cutoff values were calculated as the mean od of negative controls (n = ) plus three standard deviations. the positive and negative sera from experimentally-infected piglets were also confirmed by western blot on purified pedv wv and s protein antigens as previously described (huang et al., ) . positive and negative controls were run in duplicate on each elisa plate. antibody response in all tested samples was represented as a corrected sample-to-positive (s/p) ratio, calculated as follows: s/p ratio = (sample odmean od, negative controls) / (mean od, positive controlsmean od, negative controls). vero cells were seeded in confluent monolayers in -well plates (co star™, corning) for h, then infected with μl/well of pedv ( tcid /ml) for h at °c, followed by removal of the inoculum. mem supplemented with . % (w/v) trypsin (mmt) was added to each well and incubated at °c for h, then cells were fixed with % paraformaldehyde. for specific detection of pedv proteins, different rabbit anti-pedv polyclonal antibodies were used as appropriate, with a mouse anti-pedv s mab (cat no: , jbt, korea) used as a positive control. secondary alexa fluor -conjugated goat anti-mouse igg or goat anti-rabbit igg (invitrogen) were used at a : dilution, incubated for h at room temperature. plates were washed three times between antibody incubations with μl/well of pbs-t. nuclei were stained with ', -diamidino- -phenylindole (dapi; kpl, inc.) at a : dilution, and visualized under a fluorescence microscope. sera from challenged piglets were tested for neutralizing antibodies (na), according to the protocol with slight modification (kusannagi et al., ) . briefly, serum samples were inactivated at °c for min and then -fold serial dilutions ( : ˜ : ) were prepared in well plates. after mixing μl of each dilution with μl pedv ( tcid /ml), samples were incubated for h at °c and used to infect monolayers of vero cells in -well plates. after adsorption for h at °c, the inoculum was discarded, plates were washed three times with mem, and maintenance medium (containing μg/ml trypsin) was added to each well. after incubation at °c for h, cells were observed on an inverted microscope for cpe such as cell fusion and nuclear atrophy. sn titers were calculated using the reed and muench method and expressed as the reciprocal of the highest serum dilution resulting in % inhibition of pedv infection, relative to controls. all data were processed using spss (version . ) software and the graphpad prism program as described previously (gimenez-lirola et al., ; huang et al., ) . the cutoff value and diagnostic performance of each pedv antigen was determined by receiver operating characteristic (roc) analysis (sas version . , sas institute, inc., cary, nc, usa) based upon the elisa results. pedv-zju/g / strain was propagated in vero cells and purified by ultracentrifugation on a sucrose-gradient. electron microscopy revealed that the purified virus was comprised of a great number of vesicles with morphological heterogeneity and envelope fragments carrying spikes (fig. a) . previously, the virions of several coronaviruses such as transmissible gastroenteritis virus (tgev), turkey and bovine enteric coronaviruses have been observed with a diameter ranging between - nm (dea and garzon, ) . to our knowledge, the pleomorphic property of pedv virions containing not only the small or defective particles, but also the giant spherical particles ranging up to nm in diameter, is reported here for the first time. although a few of the pedv particles had lost partial spikes, they were relatively intact. as spike glycoproteins are known to be the most immunogenic proteins of coronaviruses , purification by ultracentrifugation through sucrose cushions in this study proved to be reliable. also, the level of pedv protein was relatively high as detected by bca protein kits, thus confirming that the quality, integrity and quantity of the purified virions were sufficient for use as the antigen in subsequent elisa assays. initially, we failed to detect the expression of the complete orf using the pet- a, the psmart or the other bacterial expression vectors under different conditions by western blot analysis (data not shown). therefore, the -aa of the c terminal part of orf (orf c) showing the predicted hydrophilicity profile was chosen as the target antigen for orf . the ac, e and orf c recombinant peptides were expressed in the inclusion bodies, whereas the nsp , adrp and nsp proteins displayed soluble expression in the cultured supernatants. expression yields of ac, e, and orf c were very low, hardly visible, when examined by coomassie blue staining after sds-page (data not shown); but confirmation of these three peptides with predicted sizes (ac:˜ kda; e fused with a sumo tag:˜ kda; orf c fused with a sumo tag:˜ kda) could be done using an anti-his-tag antibody by western blot (fig. b) . on the other hand, sds-page and western blot analyses of purified nsp , adrp and nsp soluble proteins showed bands that were consistent with the predicted sizes of , and kda, respectively (fig. c) . the purified s protein expressed in mammalian cells was also identified as a single band by sds-page (fig. d ) and by western blot using an anti-s monoclonal antibody as described previously (gerber et al., ) . due to glycosylation of the s protein, the size of the band in the gel was larger than its predicted size (˜ kda). . . antibodies generated against recombinant pedv ac, orf c, and nsp proteins resulted in specific fluorescence in vitro purified recombinant pedv peptides (orf c, ac, adrp, nsp , and nsp ) were used to immunize rabbits, generating polyclonal sera that were used to detect viral proteins on pedv-infected vero cells by ifa. the e protein was not used to immunize rabbits in this study. staining of anti-ac polyclonal serum resulted in specific fluorescence at h post-infection ( fig. a, b ) similar to the signal observed using the anti-s mab as the positive control (fig. g, h) . specific fluorescence was also detected with the anti-orf c (fig. c, d) and the anti-nsp polyclonal antibodies (fig. e, f) . in contrast, no specific fluorescence was observed when using the anti-adrp and anti-nsp polyclonal antibodies. ifa with pre-immune rabbit sera displayed no fluorescent signal in vero cells infected with pedv (fig. i, j) . the viral antigens were all detected in the cytoplasm of the infected cells. the anti-s mab reacted more strongly than the three positive rabbit antisera based on a comparison of the positive cell numbers and fluorescence intensities. infection of vero cells or vero cells expressing the entry receptor porcine apn with the other swine enteric coronaviruses , such as swine enteric alphacoronavirus , porcine deltacoronavirus (pdcov) and tgev, had no detectable fluorescence after ifa with the anti-pedv polyclonal antibodies described above (data not shown). therefore, anti-pedv-ac, anti-pedv-orf c and anti-pedv-nsp are pedv-specific and do not cross-react with these known porcine coronaviruses. previously, we have developed and validated indirect elisa based on the s protein to monitor serum anti-pedv igg and serum and fecal anti-pedv iga antibodies in postweaning pigs (gerber et al., ; gerber and opriessnig, ) . in this study, in order to determine the pattern of antibody response of weaning piglets in a -day weaning period after pedv infection, serum or fecal samples from experimentally-infected -day-old piglets were examined by elisa based on the pedv wv or the s protein, and by serum neutralization test (fig. ) . the results indicated that igg and iga responses against both antigens were detected in serum at different time points after pedv infection ( fig. a and b ). despite challenge with pedv in these piglets, levels of serum igg and iga decreased from dpi, reaching a minimum after dpi as detected by both wv and s antigens (fig. a, b) , and the pattern or trend of neutralizing antibody (na) was more similar to that of iga (fig. c) . a good linear relationship between the s -based iga elisa titers and na titers was observed (spearman's rank correlation coefficient of . ; p < . ), demonstrating the correlation between them (fig. d ). there were some differences in the sensitivity of the antigens to detect antibodies, as levels of serum iga were slightly higher when the s protein was used as the detection antigen. levels of fecal iga were also highest before challenge ( dpi), and continuously declined after challenge (fig. e) . the specificity and sensitivity of detection of s and wv antigens were similar for serum iga. the high igg and iga antibodies and na detected at the early stages of the weaning piglets are presumably maternal antibodies received from sows that were not pedv negative. in addition, piglets during weaning have not developed their own immunity to the virus. these results also demonstrated that the s -based elisa is an alternative (to wv-based) and ideal serological assay for detection of anti-pedv antibodies. . . antibodies against specific pedv peptides were detectable in sera from naturally infected pigs recombinant pedv s , orf c, e, ac and nsp peptides were used as antigens in western blots to detect antibodies in porcine sera from commercial farms since they produced antibodies in rabbits reacted to pedv antigens in infected cells except for the e protein that was not tested (fig. ) . the criterion for determining the seropositivity to a particular antigen of a sample was whether the expected protein band a mouse anti-s monoclonal antibody (g, h) and pre-immunization rabbit serum (i, j) were used as positive and negative controls, respectively. alexafluor -conjugated goat anti-rabbit igg and goat anti-mouse igg (green) were used as secondary antibodies, as appropriate. antibody staining merged with dapi nuclear staining (blue) is shown; magnification = × (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). was presented on the membrane. according to this, serum western blot analysis of the anti-nsp antibody showed high-background results and thus further investigation of nsp was not performed. a main specific band appeared for s , ac, e or orf c protein antigen tested (arrows indicated in representative samples in fig. ) , though additional "fuzzy" bands were present at low or high molecular weights that were likely from nonspecific reactivity of serum components. compared with the other proteins, the s band appeared to be more specific, with a cleaner background (fig. a) . pedv-positive sera were screened and verified viathis method, which were used to compare with the results of the elisa (fig. ; see below) , whereas negative sera from s -based elisa were also analyzed as negative controls, and none of them showed reactivity (fig. , the second lanes in all panels) . however, in the case of ac, the specific -kda bands also appeared in some sera that were negative by elisa (data not shown). furthermore, the specific igg antibody responses in sera from a total of diarrheic pigs from farms in china were analyzed using elisa assays based on pedv wv, s , orf c or e proteins (fig. a-d) . the results indicated good correlations among these antigens. overall, the levels of antibodies against pedv wv and the three proteins antigens were generally higher in primiparous and multiparous sows than those in - weeks-old pigs (p < . ). the trend of serum igg response to each pedv antigen from the -week-old to the -week-old pigs was similar to that of the experimentally-infected weaning piglets (fig. a) . antibodies were highest in the week-old piglets (p < . ) except for wv antigen, and then declined to a minimum by weeks-of-age significantly (p < . ) before increasing again to a relatively stable level from to weeks-of-age (fig. b ,c; p < . ). this pattern was in agreement with a recent report about the prevalence of pedv antibodies in swine farms (bertasio et al., ) , which might reflect that newborn piglets receive maternal igg antibodies from sows, though the level of the antibodies decline quickly, and the weaned piglets must begin to develop their own immunity to pedv. however, there were some differences in reactivity to the antigens used in the elisas. igg detected by wv-based elisa had a similar pattern to that detected by the other antigens, as the antibodies were in a relatively dispersed distribution, although its cutoff value ( . ) was closest to the orf c elisa ( . ). antibody levels against the pedv e protein were higher with a relatively concentrated distribution, especially in pigs older than weeks. however, the e protein-based elisa had the highest cutoff value ( . ), and the distribution of antibodies from to weeks-of-age was obviously different from the s -based elisa, which had the lowest cutoff ( . ). collectively, the data indicated that the pedv s protein was more specific and accurate as a detection antigen in elisa tests. with the introduction of pedv into the north american herd in - tian et al., ) , the need for a suitable diagnostic marker for the accurate, rapid, and early diagnose of pedv infection has become much more urgent. pedv herd-infection status is very important for biosecurity and the control of ped. compared to rt-pcr and other nuclei acid detection assays, serological tests are advantageous to study immune responses related to vaccination, wild-type virus infection, and to determine whether sow immunity is adequate in individual litters after pedv exposure (diel et al., ) . pedv infection is not always obvious in finishing pigs, which increases the risk of widespread disease in pigs of all ages (bertasio et al., ) . thus, sensitive serological tests would allow detection of recent infections, to avoid the introduction of these animals into naïve herds. however, there are many different structural and non-structural proteins to choose from when selecting an antigen to use in novel diagnostic assays. previously, we have developed and validated indirect elisa based on the s protein to detect anti-pedv igg and iga antibodies in postweaning pigs (gerber et al., ) . the present study was set up to investigate diagnostic potentials of specific pedv accessory and nonstructural proteins, which have not yet been reported systematically. fig. . detection of antibodies against pedv peptides in porcine sera by western blot. representative serum samples from diarrheic pigs were used in western blots to detect pedv s (a), ac (b), orf c (c), and e proteins (d); μg of recombinant peptides were loaded in each lane. hrp-conjugated goat anti-porcine igg was used as the secondary antibody, and pedv-negative serum was used as negative control. fig. . distribution of cumulative igg elisa sample-to-positive (s/p) ratios in serum samples collected from diarrheic pigs at commercial pig farms. various indirect elisa assays based on pedv whole virus (wv) (a), s (b), orf c (c), and e proteins (d) were used to test serum samples from commercial sows and pigs with diarrhea at different ages with unknown pedv infectious status. sera from naïve piglets were used as negative controls; samples above the determined s/p cutoff (dashed line) were considered positive. one-way analysis of variance (anova) was used for multiple comparisons between different ages among the individual antigens with an alpha value of . . the "ns" denoted no statistical differences. the sera and feces from the experimental infected piglets at weaning were first used for detection of antibodies using the pedv wv and s protein-based indirect elisas. the pattern of change in anti-pedv igg/iga from serum samples and in anti-pedv iga fecal samples (immediate decline post-infection) indicates that the piglets had obtained maternal antibodies at birth, and did not produced antibodies even after pedv challenge until their immune system matured. additionally, levels of na were more closely correlated with iga than igg (fig. b-d) , as seen in other studies (paudel et al., ) . abundant anti-pedv na have been demonstrated in colostrum on the day of birth, decreasing rapidly in milk by day , and gradually declining further from days - post-farrowing, which may contribute to variable protective capacity . the current study showed a similar decline, further confirming the reliability of the s -based elisa assays applicable to weaning pigs in addition to postweaning pigs (gerber et al., ; gerber and opriessnig, ) , and also highlighting the importance of accurate diagnosis in a short window for proper immunization of sows and piglets. compared to the pedv wv, s -based assays showed good reactivity and high sensitivity/specificity (figs. , a and b) . it is worth noting that the recombinant s protein was expressed in a eukaryotic expression system and should display a natural conformation with high glycosylation, as shown in fig. d , which may be one reason for its higher detection sensitivity. on the other hand, wv was mainly purified by sucrose density gradient centrifugation, differential centrifugation or polyethylene glycol (peg) precipitation (hoffmann and robert, ) . these methods would damage the integrity of the virus, especially the surface spike glycoprotein. therefore, the eukaryotic-expressed s protein is likely more advantageous than the wv as the antigen for pedv serological assays. for another two major structural proteins m and n, due to common epitopes shared by pedv, tgev and pdcov, several studies have previously demonstrated that pedv m or n presented some cross-reactivity to tgev or pdcov (gimenez-lirola et al., ; lin et al., ; ma et al., ) . in contrast, recombinant pedv-s had no cross-reactivity with sera from these porcine coronaviruses, showing the best diagnostic sensitivity (gimenez-lirola et al., ) . therefore, we did not pursue the development of serological assays based on m or n proteins in this study. the accessory orf protein is thought to have high potassium channel activity and may be associated with the virulence of pedv (wang et al., ) . the small structural e protein has important roles in the assembly of coronavirus virions, virus egress and in the host stress response (ruch and machamer, ) . besides the structural proteins, pedv has several non-structural proteins (nsp , nsp , nsp , among others) that express in the early stage of virus infection and have important functions in the viral replication cycle. the coronavirus nsp is a conserved component of the viral protein processing machinery, and may be incorporated in the virion viaits intimate association with viral rna (neuman et al., ) . the nsp is known as the largest replicase subunit, consisting of numerous distinct structural domains separated by disordered linkers. some of these, such as the ac and adrp (macrodomain), are well conserved across all genera of coronaviruses, though there have been no reports about serological assays based on pedv ac and adrp domains. considering their potential use in the study of host immune response, these proteins were specifically included in the current study. recombinant orf c, e, ac, adrp, nsp and nsp peptides were expressed and purified (fig. ) ; however, only orf c, ac and nsp produced functional rabbit antibodies recognizing pedv antigens in infected cells (fig. ; the anti-e was not generated and tested). subsequently, they were used individually as detection antigens in western blot and/or indirect elisas to detect anti-pedv igg antibodies in sera from diarrheic pigs (fig. ) . the pedv wv and the recombinant s expressed from mammalian cells was used for comparison. the s , orf c, and e peptides each reacted strongly with the sera, reflecting expected distributions of pedv-specific antibodies. the reactivity of ac, adrp, nsp and nsp peptides was less pronounced (data not shown), thus they were discarded from consideration as novel diagnostic antigens. the generally high level of igg against s , orf c, and e proteins in old-age pigs was consistent with recent reports that anti-pedv igg in infected pigs persisted for over than weeks after the onset of diarrhea symptoms (lin et al., ) . all antibody levels declined to their lowest levels at weeks-of-age, which was consistent with the result from experimentally-infected piglets at weaning (fig. ) . western blot was implemented to confirm the elisas, with additional "fuzzy" bands appeared when the orf c, ac and e were used as detection antigens, indicating non-specific recognition (fig. ) . this may be related to differences in antigens used and/or the sensitivity of the assays for detecting anti-pedv antibodies. the orf c antigen has moderate immune reactivity as evidenced by staining of anti-orf c in pedv-infected cells displaying specific fluorescence in ifa ( fig. c and d) , by serum western blot (fig. d) , and by indirect elisa (fig. c) . in future studies, we plan to employ the pedv mutants with the orf deletion generated by reverse genetics zhao et al., ) in comparison with the wild type pedv for more detailed evaluation of the protein for use as a marker in diagnostic assays. the accessory protein e showed intermediate sensitivity in western blot and extremely high sensitivity in elisa assays compared with the other antigens ( fig. c and d) . the elisa sensitivity was too strong, as nearly all of the sera from sows and - weekold piglets were strongly positive, thus it could not properly reflect the trend of pedv-specific antibodies. to our knowledge, this is the first report about the use of an orf -or e-based elisa on such a large scale. of the non-structural proteins, ac displayed strong immune reactivity ( figs. a and b) , suggesting that it may be released into circulation or is picked up by antigen-presenting cells (hurst-hess et al., ) . therefore, the study of anti-ac antibodies may contribute to a better understanding of the detailed function of nsp . our results also complement previous mass spec identification of nsp within purified virions (neuman et al., ) . in summary, this study is the first to dissect the range of antibody responses against pedv during infection, using different assays (elisa, western blot, sn) to comprehensively analyze pedv antibodies in porcine sera from china. the results confirmed high pedv prevalence in china (sun et al., ) . the antibody profiles provided by the study offer more reliable information on the host immune response to different viral proteins, and will be useful for design of vaccines that better stimulate protective immunity. above all, our data identified that besides s , the recombinant orf c and e proteins can also be used as diagnostic markers; but s represents greater sensitivity for a wide range of pedv-specific antibodies. the authors declare no conflict of interest. impact of porcine epidemic diarrhea on performance of growing pigs age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study evaluation of serological cross-reactivity and cross-neutralization between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains identification of coronaviruses by the use of indirect protein agold immunoelectron microscopy porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa detection of immunoglobulin (ig) a antibodies against porcine epidemic diarrhea virus (pedv) in fecal and serum samples reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states expression of the putative orf capsid protein of torque teno sus virus (ttsuv ) and development of western blot and elisa serodiagnostic assays: correlation between ttsuv viral load and igg antibody level in pigs serological profile of torque teno sus virus species (ttsuv ) in pigs and antigenic relationships between two ttsuv genotypes ( a and b), between two species (ttsuv and - ), and between porcine and human anelloviruses dissection of amino-terminal functional domains of murine coronavirus nonstructural protein aminopeptidase-n-independent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate.pdf antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein development of an indirect elisa, blocking elisa, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to north american strains of porcine epidemic diarrhea virus discovery of a novel swine enteric alphacoronavirus (seacov) in southern china comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus (mrv ) from a diarrheic piglet and seroepidemiological survey of mrv in diarrheic pigs from east china the coronavirus e protein: assembly and beyond characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review evidence of recombinant strains of porcine epidemic diarrhea virus porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry pedv orf encodes an ion channel protein and regulates virus production identification of a peptide derived from the heptad repeat region of the porcine epidemic diarrhea virus (pedv) spike glycoprotein that is capable of suppressing pedv entry and inducing neutralizing antibodies this work was supported by the national key research and development program of china ( yfd ), the key research and development program of zhejiang province ( c ), and the national natural science foundation of china ( ). we thank the professional editing service nb revisions for technical preparation of the text prior to submission. key: cord- -h o yqv authors: nan title: oral communications and posters date: - - journal: inflamm res doi: . /bf sha: doc_id: cord_uid: h o yqv nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : ( ) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? ( ),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; ( ) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd + tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp( - ) peptide in complex with hla-dr b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr b gene and a human t-cell receptor (tcr) that recognises the mbp - peptide in the context of hla-dr b either spontaneously or after immunization with mbp - develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far genes in this concert have been identified; the mhc class ii ab gene in the mouse ( ) and the ncf gene in the rat ( ) and in the mouse ( ) . the ncf protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf deficiency linking the ncf pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats ( ). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to % of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry- (azd ) is an inhibitor of mek / currently in development for cancer. phase determined the msd ( mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (- %) and proliferative index (- %). the trough plasma concentration ( ng/ml) corresponded to~ % inhibition of perk. about % of pts had stable disease after months. these results demonstrate that arry- (azd ) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase studies, in melanoma, colorectal, lung and pancreatic cancer. arry- is another potent, selective mek / inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry- inhibited tnfa, il- b and il- (ic s of , and nm, respectively). % inhibition of perk required nm. arry- was highly efficacious in cia and aia rat models, with ed s of and~ mg/ kg, respectively. in normal volunteers arry- was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry- monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry- in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock- isoform.these rock- inhibitors, typified by slx- and slx- , are potent (ic < nm), selective for rock- (> fold selectivity for rock- over rock- ) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx- inhibits cell proliferation and migration in several tumor cell lines including ht- , panc- and mda-mb- . moreover in xenograft studies using nude mice, slx- significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx- prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx- is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock- selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx- and slx- will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox- is constitutively expressed.the cox- gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox- are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox- can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox- plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox- and tumorigenesis come from genetic studies.overexpression of cox- has been observed to drive tumor formation whereas cox- deficiency protects against several tumor types.selective cox- inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox- inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox- -derived pge are found in both inflamed and neoplastic tissues.the fact that pge can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox- contributes to both wound healing and tumor growth.taken together, it seems likely that cox- induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. ( ), k hagihara( ), t nishikawa( ), j song( ), a matsumura ( ) ( ) health care center, osaka university, japan ( ) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il- in the inflammatory status. to know the pathogenic role of il- and the efficacy of il- blockade in inflammation, a humanized anti-il- receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il- blocking therapy improved the clinical symptoms and the laboratory findings, the il- function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il- , il- and tnf-alpha. in result, il- was an essential cytokine in induction of saamrna through the activation of stat which constructed the complex with nf-kappab p and a cofactor p . although there was no stat consensus region on saa promoter, stat bound at the site of nf-kappab re. the above research proved that il- signal is essential on the synergistic induction of saa via newly discovered stat transcriptional mechanism, suggesting the presence of this stat mechanism in inflammation, and confirming the normalization of serum saa level by il- blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada( ), k matsushima( ), s kaneko ( ) ( ) kanazawa university, japan ( ) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)- / monocyte chemotactic and activating factor (mcaf)/ ccl , a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. ( ), p lacamera ( ), b shea ( ), g campanella ( ), b karimi-shah ( ) , n kim ( ), z zhao( ), v polosukhin ( ), y xu( ), t blackwell ( ) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa , are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa -deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r receptor. we have shown that tnf-r is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor- (par- ) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par- is implicated in innate defense responses associated with lung inflammation. we showed that par- is expressed by human alveolar (a ) and bronchial ( hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh . in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par- , but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh . preincubation of a par- transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par- , generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par- in the respiratory tract, thereby altering the host innate defense mechanisms. caspase- -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor (par ) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars , and , suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (> %), which is not seen in dizygotic twins (< %) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before . incidence has increased since now leading to a lifetime prevalence of up to . % in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome being most consistent between different populations. in three coding variations in the card gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card gene. variations in the card gene do not fully explain the linkage finding in the pericentromeric region of chromosome . after stratification for card variants, the broad linkage peak is reduced to two more defined peaks on p and q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes and , respectively). dlg- is the example of a low-risk susceptibility gene with a modest associated odds ratio ( . - . ) . interestingly, the associa-tion signal appears to be confined to young males. slc a / which encode the kation-transporters octn and have been suggested to represent the disease gene in the + kb haplotype block on chromosome q . mdr has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a k illumina scan has been published identifying il- r as a further disease gene. we used a genome wide candidate gene approach (with appr. . csnps) to identify atg l as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr , teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr +/ cd + t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx -b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr .the compound proved to be highly efficacious in the tnf-aare and mdr a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx -b was well-tolerated, and no drug-related saes were reported.a -day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx -b was shown to be both safe and to have encouraging clinical results: % of patients on ccx -b (cdai ! , crp > . mg/l) exhibited a -point drop in cdai compared to % on placebo. furthermore, a crp decrease of mg/l was seen in the ccx -b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il- p , ifn-gamma, and the chemokine rantes was shown in the ccx -b group while levels remained stable in the placebo group. ccx -b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg d in a model of inflammatory bowel disease, where cd +cd -t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg d antibody therapy. the expression of nkg d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg d on the cell surface as well as increased levels of nkg d mrna in cd + t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg d antibody (cx ) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx treatment decreased the cellsurface expression of nkg d and prophylactic administration of cx attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx administration to mildly colitic animals, whereas cx did not attenuate severe colitis. thus, nkg d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg d in cd + t cells increased markedly during the development of disease and since administration of cx early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than have gained approval. the pipeline contains many more potential biopharmaceuticals and at present in new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled % human biologicals are potentially immunogenic ( ) ( ) . i shall discuss two examples: ) recombinant human cytokines (ifn-beta- a and - b), and ) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used ( - ). it took more than years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized ( , ). ) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, - % of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnß antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnß in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnß antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit , was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, : - , ) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par and par also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par activation in joint inflammation and pain. male c bl/ mice received an intra-articular injection of either the par activating peptide aypgkf-nh or the inactive peptide yapgkf-nh ( mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par activating peptide caused knee joint blood flow to gradually increase by up to % over the succeeding hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par antagonist pepducin p pal ( mg i.p.). the control peptide yapgkf-nh had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par receptors in mice knees causes joint inflammation and pain. vincent lagente ( ), e boichot ( ) ( ) air liquide, centre de recherche claude-delorme, jouy en josas, france ( ) inserm u , universitØ de rennes matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp- ) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp- /timp- ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that members of the par family (par , par and par ) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par and par in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par and par -dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par and par dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par -dependent mechanism.in contrast, activation of par on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled consecutive dogs (group b). results: dogs were excluded from group a where / dogs ( %) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in , a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including consecutive dogs with a final diagnosis known to predispose for dic ( % were finally diagnosed with dic). outcome was -day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim : ; - ) . relative risk according to bpw (rr [ % confidence interval]) for (a) a dic diagnosis and (b) -day mortality, were assessed. -day mortality in the study population was %. % were bpw positive. bpw was not a significant diagnostic factor for dic (rr= . [ . ; . ] ), but strongly so for outcome (rr= . [ . ; . ] ) with a % ( / ) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. ( ), b hideo ( ) ( ) department of molecular pathology, kumamoto university, japan ( ) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of f¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of f¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing f¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin- expression (graÅa-souza et al., ) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., ) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. ( ) ( ) university of melbourne, victoria, australia ( ) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx ), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx -/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx -/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx -/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx -/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase- (mmp ) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx -/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp . chris bolton( ), c paul( ), s barker ( ), r mongru ( ) ( ) william harvey research institute, london, uk ( ) university west of england, bristol ( ) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a subunit (glyr a ) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a , the reduction of glyr a was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a , while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a may be a target molecule for pkg to induce allodynia. ( ), r leite( ), ys bakhle ( ) ( ) federal university of minas gerais, belo horizonte, brazil ( ) medical college of georgia, augusta, usa ( ) imperial college, london, uk selective cyclooxygenase inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats ( - g; - animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and min later given cx ( mg/kg, s.c.). after a further min, rats were injected (ipl) with the inflammatory stimulus, carrageenan ( mg/paw). mechanical pain threshold was hourly measured over the next h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin % reversal = . nanomoles), higher doses of microtubule inhibitors (taxol % reversal = . nanomoles) with no effect of acrylamide ( up to nanomoles).we conclude that ) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and ) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox- in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox- inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for hrs daily for a period of seven days. nimesulide ( . mg/kg, i.p.) was administered daily for days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in . ul. ngf ( . , and ug) injected in the tmj challenged h prior by cg ( ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k a ( and ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production and h after the tmj cg ( ug) injection. the tmj injection of ngf ( ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf ( ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd +/f / -ve/cd c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam- , although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl /mcp- , and its receptor ccr , and in response to anti-cxcr .this was supported by in vitro experiments showing that mif induced ccl /mcp- release from cultured murine endothelial cells.finally, mice lacking cd , the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd .this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf- ) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap- activities. gm-bmm preferentially produced tnfa, il- , il- and il- while, conversely, bmm generated more il- and ccl ; strikingly the latter population could not produce detectable il- and il- . following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap- activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il- following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type diabetes mellitus (t dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad , ia- and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd and cd t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th , tc ) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b: - cause diabetes . knockouts of the insulin gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin gene (islet expression) prevents % of diabetes . dual insulin knockout with transgenic insulin with altered peptide (b :a) prevents all diabetes . islets with native b: - sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells .anti-b - t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain . alpha chain as transgene sufficient to engender anti-insulin autoantibodies . kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as % of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a -step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam- and lfa- , have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa- antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuíon molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, düsseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il- and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin- , tnf-µ, p mapk, msk / , mk , nf-kappab and ap- . histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h receptor (h r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h r deficient mice and mice treated with h r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th responses. ex vivo restimulation of primed t cells showed decreases in th cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h r was responsible for the the t cell effects. the influence of h r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h r deficient mice. the h r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h r was minor. surprisingly the h r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h r antagonists. ( ), bsp reddy ( ) ( ) nizams institute of medical sciences, hyderabad; india ( ) genix pharma, india rupatidine, carries the majority of the histamine h receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either mg rupatidineformulation after overnight fast. washout was days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc - mmsq/hr and auc - %/hr) both for untransformed and log transformed data were found to be within - % of % ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet ( ), h yoshimura( ), k ohara ( ), y mastui ( ), h hara ( ), h inoue ( ), h kitasato ( ), c yokoyama( ), s narumiya ( ), m majima ( ) ( ) kitasato university school of medicine, kanagawa, japan ( ) tokyo dental medical colledge, tokyo, japan ( ) kyoto university school of medicine, kyoto, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitorsconstitute revascularization. we hypothesized that txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggrega- inflamm. res., supplement ( ) tion.to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out(ipko) and thromboxane (tx) a receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf- and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. purpose: the s calcium-binding proteins, a , a and a are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a /a is anti-microbial; a has oxidant-scavenging functions. a is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys to ala a mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s s in asthmatic lung. the s s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a . a , a or the a /a complex had relatively low ability to induce il , tnf, il , and chemokines mrna from pbmc compared to a . only a induced significant levels of il ; none induced il or gm-csf mrna compared to lps. in contrast to a which is activating, a significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys . conclusions: the cytokine profile generated by a in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a in oxidant defence, particularly to hypobromite generated by activated eosinophils. ( ), d mankuta( ), g gleich ( ), f levi-schaffer ( ) ( ) hebrew university of jerusalem, israel ( ) hadassah university hospital, israel ( ) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson ( ), d kashanin ( ), f odowd( ), v williams( ), g walsh ( ) ( ) cellix ltd, institute of molecular medicine, dublin, ireland ( ) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam- and vcam- , is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. - dynes/cm ). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam- , icam- , and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam- are high ( - %, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam- , with pma-stimulated cells showing a higher affinity for icam- than nonstimulated cells (approx. % and %, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph . ). male c bl/ were instilled with ml/kg of gj. after or h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. . ae . , sg h: . ae . ; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il- ratio in balf at the nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il- ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c bl/ mice were subjected to min of intestinal ischemia and challenged with . mg/kg of intranasal lps at the th hour of reperfusion (two-hit). balf and culture of lung explants were performed h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r ( . ae . vs . ae . , mg/mg). lung mpo was increased ( . ae . vs . ae . ; od nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group ( . ae . vs . ae . ; x e cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf ( . ae . vs . ae . ; mm) and in lung explants ( . ae . vs . ae . ; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. ( ) ( ) novartis institutes for biomedical research, horsham, uk ( ) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over % (p< x - ) for exercise capacity, measured by running on a treadmill.after months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p< . ); however there was a - -fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p< . ).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p< . ). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. ( ), ap ligeiro-oliveira( ), jm ferreira-jr( ), sr almeida( ), w tavares de lima( ), shp farsky ( ) ( ) university of s¼o paulo, brazil ( ) regional integrated university of alto uruguai and missðes, brazil methods: male wistar rats were exposed to vehicle or hq ( mg/kg; ip.;daily, days, two-day interval every five days). on day , animals were ip sensitized with ovalbumin (oa). assays were performed on day . results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound / . in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd and cd on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missðes / university of s¼o p, department of clinical and toxicological analyses, s¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br ( ) ( ) radboud university nijmegen, medical centre, nijmegen, the netherlands ( ) university hospital, zürich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr and tlr was associated with the expression of ifna, tnfa, il- b, il- , il- , and il- and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr / tlr expression in synovial tissue was associated with the presence of ifna, il- b and il- , but not tnfa, il- and il- . to investigate whether ifna, il- b and il- could induce tlr / tlr upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr / mrna expression. ifna incubation resulted in significant tlr /tlr upregulation, whereas il- b and il- did not. pre-incubation with ifna and subsequent stimulation of tlr /tlr significantly enhanced il- , tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il- b were produced upon stimulation with atp, but not upon tlr / tlr stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il- b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il- b / il- in synovial tissue. first, involvement in tlr /tlr regulation and second, involvement in atp-induced production of biologically active il- b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod (nucleotide/ binding oligomerization domain) and nod in model of acute joint inflammation induced by intraarticular injection of tlr (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod and nod occurs also in humans, we exposed pbmcs carrying either nod frameshift or nod frameshift mutation with scw fragments in vitro. both tnfa and il- b production was clearly impaired in pbmcs carrying the nod fs compared to pbmcs isolated from healthy controls. in line with the nod gene deficient mice, human pbmcs bearing the nod mutation produced enhanced levels of proinflammatory cytokines after h stimulation with scw fragments. these data indicated that the nlr family members nod and nod have a different function in controlling tlr -mediated pathways. we hypothesize that intracellular nod -nod interactions determine the cellular response to tlr triggers. whether lack of controlling tlr -driven pathways by nod signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp- cells with vitamin-d . lilra expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra expression (p= . ). treatment of macrophages with lps, tnf-a, il- b and ifn-g but not il- caused significant down-regulation of lilra (p< . ). function of lilra was assessed by cross-linking with plate-immobilised lilra -specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at h. correlation between lilra expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra could potentially provide therapeutic benefit. ( ) ( ) university of valencia, spain ( ) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm- ) on an animal model of human rheumatoid arthritis. dba- /j mice were treated with corm- ( , or mg/kg/day, i.p.) from day to after collagen-induced arthritis (cia) and sacrificed on day . administration of corm- resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm- treatment with the most pronounced protective effect observed at mg/kg. the levels of pro-inflammatory mediators (pge , il- beta, tnfalpha, il- and il- ) in the hind paw homogenates were significantly inhibited by corm- . in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm- alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm- appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without ng/ml il- a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a - were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < . ). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il- a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr and tlr activation in the expression of arthritis was studied using interleukin- receptor antagonist deficient (il- ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il- ra-/-tlr -/-mice developed more severe arthritis compared to il-ra-/-tlr +/+ littermates; whereas, tlr -/-il- ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr and tlr differentially regulated the disease expression, we studied the role of these tlrs in il- production and th development, both important in il- ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il- upon stimulation with tlr and tlr ligands; however, il- ra-/-bmdcs produced less il- than wild type dcs upon tlr stimulation and more il- than wild type dcs upon tlr stimulation. furthermore, il- ra-/-t cells produced lower amounts of il- when cultured with tlr -activated apcs and higher amounts of il- when cultured with tlr -stimulated apcs, both in combination with cd stimulation. facs analysis of th (cd +/il- +) cells from both spleen and draining lymph nodes revealed % reduction in il- ra-/-tlr -/-mice compared to il- ra-/-tlr +/+ littermates. specific cd /cd stimulation of non-adherent splenocytes confirmed lower il- production in il- ra-/-tlr -/-. these findings suggest important roles for tlr and tlr in regulation of th development and expression of arthritis. prostaglandine (pge ) stimulates the transactivational activity of p through p map kinase-dependent ser phosphorylation (jbc ) .p controls cell-cycle progression, in part, by differential regulation of ap- proto-oncogenes (jun/fos).currently we studied pge control of cyclin d promoter activity with particular attention to the role of ap- oncogenes.pge induced a . fold increase in junb mrna expression (northern blot), a . -fold increase in junb promoter activity (luciferase assay), and increased ser junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra / , and fosb expression were upregulated by pge .in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g v) resulted in a . fold increase in cyclin-d promoter activity, cyclin-d synthesis, thr /tyr phosphorylation of erk / ( . fold) and ap- (c-jun)-dependent transactivity ( . fold); cyclin d /cdk - inhibitor p ink a synthesis was suppressed. addition of excess rass n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d promoter activity/protein synthesis, blocked p ink a synthesis; the latter effects were reversed by the addition of excess junb.pge exerted temporal and bi-phasic dose-dependent control of the cyclin d promoter activity, largely through differential ap- activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla / cox/pges biosynthetic axis and highlight the complexity of pge action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., , inflammation res. (s ) s ) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider ( , pnas. : - ) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha ( mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c bl/ mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day . hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic < . mm). diga ( ml, mm) i.a. prevented hour aia (- . +/- . mm).diga at mg/kg reduced aia when administered i.v. (- . +/- . mm, p< . ) and i.p. (- . +/- . mm, p< . ), but is hydrolysed p.o. ( . +/- . mm ns). polysulphated diga (diga s) was unstable, but stabilised by n-acetylation (nacdi-ga s).tnf synthesis was potently inhibited by both nacdiga and nacdiga s (ic < . mm).nacdiga s ( mg/kg p.o.) inhibited aia (- . +/- . mm), and nacdiga s with lower degrees of sulphation (mw and kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on persons, divided into groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: ) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. ) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs expression following exposure of murine macrophages to lps implicated socs in the control of lps-mediated signaling. socs regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs in regulating the production tnf by lps and pam csk -activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs (adv-socs ), control vector (adv-gfp) or left untreated. adv-socs monocytes were exposed to tlr and tlr ligands, lps ( ng/ml) or pam csk ( ng/ml). facs analysis demonstrated infection efficiencies of ae % and ae % (n= , mean ae sem) of monocytes expressing adv-gfp or adv-socs at moi . adv-socs blocked lps and pam csk induced tnf mrna and protein production in a dose-dependent manner. in contrast, il- and il- production by adv-socs -infected monocytes was not blocked. adv-socs also blocked lps and pam csk induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of ae % or ae % were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p activation. furthermore, the kinetics of lps and pam csk induced ikba phosphorylation and degradation in adv-socs monocytes remained unaffected (n= and donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p or erk mapk pathways. these data suggest that socs regulation of lps and pam csk -induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels ( - mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: ) measuring cytosolic free calcium ([ca +] i) in fura- loaded hdsmc using spectrofluorometry and ) force measurements in - mg detrusor strips. hdsmc responded to beta-nad+ ( - mm) with an immediate and transient increase in [ca +] i. the ca + transient was followed by one or two much slower and transient increases in [ca +] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca + responses persisted in the absence of extracellular calcium. the ca + responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p x purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp -induced neurotoxicity. we recently showed that tat induces the overexpression of il- via cellular kinase pkr and activation of transcription factor ets- . in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat tyrosine phosphorylation, but had no effects on the serine residue of stat and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat was abrogated by tat. the inhibition of phospho-stat led to the deformation of stat homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and , oligoadenylate synthetase ( , oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of , oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase- belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin- (il- ), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase- in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase- and il- . interestingly, increased caspase- activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p mapk dependent increased secretion of procaspase- and active caspase- . furthermore, anisomycin increased the mrna expression of il- through a p mapk dependent but caspase- independent mechanism, reaching a maximum level after hours of stimulation. finally, anisomycin caused a rapid ( hours) increase in the secretion of proil- and active il- . secretion of active il- was mediated through a p mapk/caspase- dependent mechanism, whereas secretion of proil- was mediated by a p mapk dependent but capsase- independent mechanism. these data demonstrate that the activity of caspase- is increased in psoriatic skin and that il- secretion is regulated by a p mapk/caspase- dependent mechanism, making caspase- a potential target in the treatment of psoriasis. prostaglandin e (pge ) regulates the stability of cyclooxygenase- (cox- ) mrna through adenylate/uridylate-rich elements (ares) in the untranslated region ( utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge dependent stabilization of cox- in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox- utr] reporter fusion mrna ( ae . % decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il- b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~ kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox- gene transcription, thus enabling ttp to regulate cox- gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox- mrna stability and transcription and whose localization and interaction with other factors is regulated by pge . these data can provide important insight into deciphering the role of pge in fine-tuning physiological and pathophysiological gene regulation. ( ) ( ) chinese academy of sciences, shanghai, pr china ( ) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)- is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp- has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp- is regulated is still not fully understood. here, we show that inhibition of p map kinase by specific inhibitor sb or rna interference (rnai) markedly reduced the expression of mkp- in lps or pgn-treated macrophages, which is correlated with prolonged activation of p and jnk. depletion of mapkap kinase (mk ), a downstream substrate of p , by rnai also inhibited the expression of mkp- . the mrna level of mkp- is not affected by inhibition of p , but the expression of mkp- is inhibited by treatment of cycloheximide. thus, p mapk plays a critical role in mediating expression of mkp- at a posttranscriptional level. furthermore, inhibition of p by sb prevented the expression of mkp- in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p -mk -dependent induction of mkp- in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il- and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd /cd mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th cytokine il- is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar -/-and ifnar -/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p x receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin- beta (il- b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin- , a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin- is required for processing of caspase- and release of mature il- b induced by p x receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il- b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin- -independent phase induced by p x receptor activation, and this was unaltered by pannexin- inhibition.nigericin did not induce dye uptake.inhibition of pannexin- blocked caspase- and il- b processing and release in response to this two stimuli.thus, while pannexin- is required for il- b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin- hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il- . here we verify the effect of saa on the mrna expression and release of mip- alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip- alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa ( ug/ml) induced the expression of mip- alpha mrna at , and hours. mip- alpha protein was found in the suppernatant of and hours cultures (p< , ) and the addition of sb (p inhibitor) and pd (erk / inhibitor) completely abolished the release of mip- alpha. conclusions: saa is an inducer of mip- alpha expression in pbmc and p and erk / are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p decrease mediator production and therefore can produce profound anti-inflammatory effects. arry- is a potent inhibitor of p enzyme (ic < nm) with a novel pharmacophore and physiochemical properties distinct from those of other p inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic < nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed ~ mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of , , , or mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry- was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il , pge and tnffz with > % inhibition observed at the mg dose level. the plasma concentrations of drug peaked at~ ng/ml at the mg dose and cytokine inhibition was sustained for > hours, showing that low doses of arry- produced profound effects on clinical biomarkers. further evaluation of arry- in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, mg/kg; oral route; days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam- on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il- b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name ( mg/kg; oral route; days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained hours after i.p. injection of oyster glycogen ( %; ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of x cultured cells before and hours after lps stimulation ( m;g/ml). results: levels of no, tnf and il- b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il- b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin- (il- )-induced mmp- expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il- beta alone or in combination. mmp- mrna was analyzed by rt-pcr. mmp- protein, phospho-stat and p / mapk levels were measured by western blotting. mmp- promoter-luciferase, cmv-cbp/p plasmids and stat sirna were transfected by calcium phosphate method. ap- activity was monitored by elisa. stat -cbp/p interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il- -induced expression of mmp- and promoter activity. blockade with neutralizing ifn-gr antibody revealed that mmp- inhibition by ifn-¼ was mediated by the ifn-¼ receptor. ifn-beta-stimulated activation of stat was directly correlated with mmp- suppression. knockdown of stat gene by specific sirna or its inhibition with fludarabine partially restored the il- induction of mmp- expression and promoter activity. ifn-g did not alter activator protein (ap- ) binding ability but promoted physical interaction of stat and cbp/p co-activator. p overexpression reversed ifn-g inhibition of endogenous mmp- mrna expression and exogenous mmp- promoter activity. conclusions: ifn-g through its receptor activates stat , which binds with cbp/p co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp- gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for ( ), p asmawidjaja ( ), r hendriks( ), erik lubberts ( ) ( ) erasmus medical center, department of rheumatology, rotterdam, the netherlands ( ) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il- in th polarization in the prone autoimmune dba- mice with and without collagen-induced arthritis and to evaluate th specific cytokine and transcription factor expression. il- induced th cells in vitro from spleen cells of naïve and collagen-type ii (cii) immunized dba- mice. the percentage of th cells is markedly higher in cii-immunized versus naïve dba- mice. adding il- to tgf-beta/il- stimulated cd + t cells did not significantly increase the percentage of th cells. tgfbeta/il- in contrast to il- induced a relatively high percentage of il- +/ifn-gamma-cells and low il- -ifn-gamma+ cells. tgf-beta/il- did not increase il- receptor expression, which may explain why adding il- directly or two days after tgf-beta/il- did not result in an increase in the percentage of th cells. elevated expression of il- a and il- f as well as the th specific transcription factor rorgammat was found under il- as well as tgfbeta/il- conditions. interestingly, il- but not tgf-beta/il- is critical in the th cytokine il- expression in t cells from ciiimmunized dba- mice. these data show that il- was more pronounced in inducing il- +/ifn-gamma-(th ) cells under cii-immunized conditions. furthermore, il- did not markedly increase the percentage of th cells induced by tgf-beta/il- . however, il- is critical for the induction of il- expression, suggesting a unique role for il- in the induction of specific th cytokines ebi was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p of il- . ebi protein has been shown to form heterodimers with p . p /ebi termed il- , can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi and p is very limited. these data lead to the hypothesis that ebi may play a role independently from its association with p . thus, to define the function of ebi , we generated ebi transgenic (tg) mice expressing in multiple tissues. ebi tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd +, cd + t cells, b cells, nk cells and nkt cells. cd +t cells isolated from spleens of ebi tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd and anti-cd antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi tg mice were recovered with anti-ebi polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi antibody. in contrast, anti-p antibody suppressed chs responses in wt mice. these data suggest that ebi acts in different from il- , and reduces th responses. ( ), o thaunat( ), x houard ( ), o meilhac ( ), g caligiuri( ), a nicoletti ( ) ( ) inserm u and university denis diderot-paris , chu xavier bichat, paris, france ( ) inserm umr s , universitØ pierre et marie curie-paris , centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep -i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk / m and vascular inflammation leads to formation of leukotrienes through the -lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b (ltb ) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb -induced atherogenesis. targeting ltb -induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox- ). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox- ) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox- and the inducible enzyme responsible for pge synthesis (mpges- ) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi is the major biological active prostanoid produced in the normal physiological conditions. however, when cox- is induced, pgi and pge are equally produced. the role of cox- , cox- and mpges- activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep , ep , ep or ep ) preferentially stimulated by pge in the vascular wall. for these reasons, we have characterized the receptors activated by pge in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin- â) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox- inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn (a gene that regulates treshold of lumphocyte activation) the allele c t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in dutch ra cases and unrelated dutch controls. carrier analysis of the snp (rs ) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p = . and or = . ). in a meta-analysis of all studies comparing individuals, the or for the cc genotype to develop ra was . and the p-value < . . in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd / q , card /nod and il r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card /nod is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card appear to show opposite effects to other models and human systems. in humans, card mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp /cryopyrin/ cias gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp protein associates with asc and caspase- (thereby forming a molecular machine termed inflammasome that displays high proil- beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il- beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp -dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. ( ) ( ) erasmus mc, rotterdam, the netherlands ( ) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd c-diphtheria toxin (dt) receptor (cd cdtr) transgenic mice by airway administration of dt h before ova aerosol ( x) challenge or during an ongoing inflammation (depletion after x ova aerosols continued with additional ova aerosols). numbers of balf eosinophils, th cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th -type cytokines (e.g. il- and il- ), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th -type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi -kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd r, cd r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a (spla ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla , hdl and ldl and of their hydrolysis products on mitogenesis and pge and ltb release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla . spla hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge from vsmc was enhanced by group x spla s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla .native ldl and its spla hydrolysis products enhanced the release of pge in order x>v>iia.the release of ltb from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla .migration of vsmc was significantly enhanced by spla iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca ( ) ( ) ipmc-cnrs umr , valbonne, france ( ) university of washington, seattle, usa ( ) inserm umrs , paris, france ( ) university of naples, italy the superfamily of phospholipase a comprises at least intracellular enzymes and up to secreted pla s (spla s). elucidating the biological roles of each pla member is currently the most challenging issue in the pla field. the different spla s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla can trigger secretion of tnf alpha, il and il in a non-enzymatic manner. on colorectal cancer cells, hgx spla stimulates cell proliferation, produces potent eicosanoids including pge , and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla s, we have examined fatty acid release by spla siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla was observed when the residual sm/ pdcho molar ratio had reached . compared to a starting ratio of . .group v spla showed preferential release of linoleate at residual sm/ptdcho molar ratio . , while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla , possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla . the fatty acid specificity of pla is significantly affected by sm. thus spla x exhibited enhanced specificity for the release of arachidonic acid ( : ) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of : by spla v. ceramide selectively stimulated the release of : by both enzymes. only the long chain ceramides (> carbons) were effective, while ceramide phosphate did not stimulate spla activity. sm-deficient cells released more : in response to spla -treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a (spla ) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a- , an inhibitor of spla enzymes, on atherosclerosis and cholesterol levels over weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study ( % fat; . % cholesterol, . % casein) and were treated with vehicle or a- bid at mg/kg or mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a- significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for weeks, oral dosing of a- ( mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a- is a potential novel therapeutic agent for the treatment of atherosclerosis. ( ), s doty( ), c antonescu ( ), c staniloae ( ) ( ) saint vincents hospital manhattan, new york, usa ( ) hospital for special surgery, new york, usa ( ) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene (tsg- ) is induced by tnf-a during inflammation and its secreted product tsg- glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox- and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg- is expressed in atherosclerotic plaques and investigated tsg- protein expression and cellular distribution on superficial femoral artery endarterectomy specimens from diabetic and non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg- antibody. tsg- expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg- expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg- stained the foamy macrophages. tsg- expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg- gene (more that fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg- in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg- is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. ( ), r krohn ( ), h lue ( ), jl gregory( ), a zernecke ( ), rr koenen ( ), t kooistra ( ), p ghezzi( ), r kleemann ( ), r bucala( ), mj hickey ( ), c weber ( ) ( ) university hospital of the rwth aachen, germany ( ) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr , inducing rapid integrin activation. mif directly bound to cxcr with high affinity (kd of . nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr as well as cd , a recently identified membrane receptor moiety for mif. accordingly, cxcr and cd were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr , serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. ( ), r toes ( ), h van bockel( ), paul quax ( , ) ( ) tno bioscienses, leiden, the netherlands ( ) department of vascular surgery, leiden university medical center, the netherlands ( ) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c bl/ mice depleted for natural killer (nk)-cells by anti-nk . antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jµ knockout mice that lack nk . + natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c bl/ mice depleted for cd + tlymphocytes by anti-cd antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd + t-lymphocytes. this impairment was even more profound in anti-nk . treated mhc-class-ii-deficient mice that lack both nkand cd + t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c bl/ mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd -positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd + t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf: u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by % (p= . ), when compared to saline treated controls (n= ).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c convertases). crry-ig ( mg every other day) led to % decrease in vein graft thickening (p= . ) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. ( ), m-c koutsing tine( ), p borgeat( ), h ong ( ), sylvie marleau ( ) ( ) universite de montreal, quebec, canada ( ) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep , a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd , elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from weeks of age. at weeks old, mice were treated daily with a s.c. injection of ep ( mg/kg) for days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for minutes, followed by minutes reperfusion. our results show that ep significantly reduced leukocyte accumulation by % in the lungs, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice (n = - per group), as assessed by myeloperoxidase assay. this was associated with a % reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd -/-deficient mice, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice. we conclude that ep protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd -mediated pathway. glycogen synthase kinase beta (gsk- beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk- beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk- beta, tdzd- and sb , on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to mmhg for min) and subsequently resuscitated with shed blood for h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd- ( mg/kg, i.v.) or sb ( . mg/kg, i.v.) min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd- , but not sb , attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin . neither of the gsk- beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk- beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. ( ), y ito ( ), h yoshimura ( ), h inoue ( ), n kurouzu ( ), h hara ( ), y mastui ( ), h kitasato ( ), s narumiya( ), c yokoyama ( ), m majima ( ) ( ) kitasato university school of medicine, japan ( ) kyoto university school of medicine, japan ( ) tokyo medical and dental university, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out mice (ipko) and thromboxane (tx) a receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf- and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from patients with synovitis of "t months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from patients undergoing vitrectomy for vitreoretinal disorders. one dimensional h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il- , ccl ), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. ( ), am artoli( ), a sequeira( ), c saldanha ( ) ( ) instituto de medicina molecular,faculdade de medicina de lisboa, portugal ( ) cemat, instituto superior tØcnico, universidade de tØcnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn- , slfn- , and slfn- were maximally induced after hours. the maximal induction of slfn- and slfn- was observed after hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn- , slfn- , slfn- and slfn- were elevated in joints affected by cia. to investigate the role of slfn- , we have generated a transgenic mouse line, which over expresses slfn- specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal . further characterisation of the slfn- over expressing mouse line will be used to assess the function of slfn- in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin , lcn- , is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn- determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn- is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn- . ( ), y cao ( ), s adhikari ( ), m wallig ( ) ( ) national university of singapore, department of pharmacology, singapore ( ) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein ( mg/kg) for , and hours respectively. neutralizing monoclonal anti-il- antibody ( . mg/kg) was administered either with or without crambene ( mg/kg) hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp- , tnf-a and il- â and up-regulating anti-inflammatory mediators like il- . phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd .the anti-inflammatory mediator il- plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il- is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. ( , ) ( ) northern ontario school of medicine, thunder bay, ontario, canada ( ) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, , : l -l ). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn delivery system.using several fluorescence-based detection systems, we established that the natural beta integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a a receptor (a ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b formation while potentiating that of prostaglandin e through the up-regulation of the cyclooxygenase (cox)- pathway. moreover, our laboratory determined that a ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a ar, migrated neutrophils expressed less cox- than their wild type counterpart while displaying higher mrna levels of tnf-and mip- . mononuclear cells from a ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p and pi- k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a amino acid protein that contains a s domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr (tandem repeat unit b), rhrnr (tandem repeat unit a) and rhrnr (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. ( ), n lu( ), r jonsson( ), d gullberg ( ) ( ) department of biomedicine, university of bergen, norway ( ) the gade institute, university of bergen, norway a ß is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a ß expression. specifically, we have analyzed the influence of cell passage, growth factors and the -d microenvironment. using sv immortalized as well as primary fibroblasts, we show that a ß integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a ß is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a ß that need to be to taken into consideration when evaluating the role of a ß in wound healing and fibrotic disorders. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par in the process of inflammation in the gut.colonic inflammation in c bl wildtype and par -/-mice was induced by treatment with . % dss (in drinking water) or tnbs ( mg or mg in ul of % ethanol, single intracolonic injection) or pre-sensitizing mice with % oxazolone (in olive oil) applied to the skin of the abdomen, and days later, a single intracolonic injection of % oxazolone (dissolved in % ethanol).intravital microscopy was performed, days (tnbs/dss) or days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par -/-.our evidences indicate that deficiency in par attenuates inflammatory responses in the experimental models of colitis associated with either th (tnbs/dss) or th (oxazolone) cytokine profile.therefore, par deficiency in the gut exerts antiinflammatory properties that are independent of th or th cytokine profile.the present study further highlights par as a potential target for inflammatory bowel diseases. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par (par -/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par deficiency. aim of this study was to assess the effects of par deficiency (via par -/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp ( mm) or paf ( nm) or by intraperitoneal injection of tnf-a; ( . mg -given hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par -/-versus par +/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par -/-mice compared to wild types as well. no difference was observed between par -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par activation in mouse colon leukocyte trafficking highlights par as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. ( ), kk hansen( ), k chapman( ), n vergnolle ( ), ep diamandis ( ), md hollenberg ( ) ( ) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada ( ) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks and to activate pars , and in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk is able to activate both of pars and and to prevent thrombin from activating par . on the other hand, klk was a specific activator of par . kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars and and by inactivating par , kallikreins, like klks and , may play a role in regulating the inflammatory response and perception of pain. ( ), d park ( ), b short( ), n brouard( ), p simmons( ), s graves ( ), j hamilton ( ) ( ) melbourne university, melbourne, victoria, ( ) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p subunit and nrf into hepg or other cells, as well as sirna technique to knockdown endogenous p in cells, we found that nf-kb p subunit repressed the anti-inflammatory and anticarcinogenetic nrf -are pathway at transcriptional level. in p -overexpressed cells, the are-dependent expression of heme oxygenase- was strongly repressed. in the cells where nf-kb and nrf were simultaneously activated, p unidirectionally antagonized nrf transcriptional activity. the p -mediated are inhibition was independent of the transcriptional and dna-binding activities of p . co-transfection and rna interference experiments revealed two mechanisms which coordinate the p -mediated repression of are: ( ) p selectively deprives creb binding protein (cbp) from nrf , but not mafk, by competitive interaction with the ch -kix domain of cbp, resulting in inactivation of nrf transactivation domain and concomitant abrogation of the nrf -stimulated coactivator activity of cbp; ( ) p promotes recruitment of histone deacetylases (hdac ) to are by enhancing the interaction of hdac with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p subunit. since various inflammatory and tumor tissues constitutively overexpresses p in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd l, only lps-matured dc expressed par and par on their surface. stimulation of dc with thrombin, par -or par -activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk / and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain (mlc ). the erk / -and rock-i-mediated phosphorylation of mlc was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine- ( f) by either nh -reactive n-succinimidyl- -[ f]fluorobenzoate or sh-reactive n-[ -( -[ f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, - %; specific radioactivity, - gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of f-nldl and f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of f-nldl and f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-Þb (nf-Þb), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p mapk and the down stream kinases msk and in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il- b before kinase activation was determined by western blotting. a significant inhibition of both msk and activations was seen after pre-incubation with dmf and stimulation with il- b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p (ser ), which is known to be transactivated by msk . furthermore, incubation with dmf before stimulation with il- b resulted in a significant decrease in nf-kb binding to the il- kb and the il- kb binding sites as well as a subsequent decrease in il- and il- mrna expression. our results suggest that dmf specifically inhibits msk and activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki- and ck- . epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol ; , - ) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer ( ), e hasselager( ), j clausen( ), l iversen ( ), k kragballe ( ) ( ) aarhus university hospital, denmark ( ) novo nordisk a/s, denmark interleukin- (il- ) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il- are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il- is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il- expression in non-lesional psoriatic skin ex vivo. mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il- beta ( ng/ml) for , , , , , and hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il- beta rapidly induced il- mrna expression in the biopsies. the highest level of il- mrna was detected after hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il- mrna. increased levels of il- protein were detected in the supernatant of the il- beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il- protein in the keratinocytes, whereas the il- protein was present in epidermal cd a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il- expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il- protein in epidermal dendritic cells whereas no il- was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il- . one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp- was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp- activity and mrna levels induced by tnf was inhibited by~ % following h treatment with calcitriol. using specific inhibitors we established that the induction of mmp- was dependent upon the erk pathway, while p -mapk and pkc inhibited, and jun-kinase, pi- -kinase and src did not affect it. levels of c-fos, a component of ap- transcription complex known to mediate mmp- induction, were elevated by tnf and further increased by calcitriol. the induction of mmp- by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp- by egf. we conclude that calcitriol inhibits the induction of mmp- gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. ( ), p verzaal ( ), t lagerweij ( ), c persoon-deen ( ), l havekes ( ), a oranje ( ) ( ) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands ( ) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd + t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th -mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. ( ), y sawanobori ( ), u bang-olsen ( ), c vestergaard( ), c grønhøj-larsen ( ) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il- mrna has been shown. also, transgenic mice over-expressing il- exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il- antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il- ratanti-mouse mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il- as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il- antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il- antibody treatment in the time interval day - . our results suggest an anti pruritic role for il- antibody in an atopic dermatitis-like animal model. anti il- antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. ( ), p ferro ( ), hm asnagli ( ), v ardissone ( ), t ruckle ( ), f altruda ( ), ch ladel ( ) ( ) rbm merck serono/university of torino, italy ( ) merck serono pharmaceutical research institute, geneva, switzerland ( ) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide -kinases (pi ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi k-g inhibitor as on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose , mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi k-g are demonstrable after oral administration of selective inhibitors like as in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box (hmgb ) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb absorber. the purpose of this study was to test the hypothesis that hmgb removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb absorber group (group i),hmgb nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at hours after lps injection. the rats were sacrificed hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb with anova. results: survival rate was % in the group i at hours after lps injection, as compared with % in the group ii and % in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb therapeutically reverses lethality of established sepsis indicating that hmgb inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp ( ) ( ) department of pharmacology, national university of singapore, singapore ( ) dso national laboratories, singapore hydrogen sulfide (h s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, mg/kg i.p.), an inhibitor of h s formation or sodium hydrogen sulfide (nahs) ( mg/kg, i.p.), a h s donor. pag was administered either hour before or hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam- , p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs ( mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-x×b inhibitor, bay - . h s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay - . moreover, expression of cxcr in neutrophils obtained from h s treated mice was significantly upregulated leading to an obvious elevation in mip- directed migration of neutrophils. therefore, h s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid (trpv ) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv antagonists are not available for use in the mouse in vivo, thus established trpv knockout (-/-) mice were used. c bl wt and trpv -/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv -/-mice, with trpv -/-mice showing an enhanced drop at h. in a separate group temperature, a proposed pre-mortality marker was also reduced by h, again with a significantly increased drop in trpv ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv -/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv -/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv plays a protective role in sepsis. trpv is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. ( ), da souza-junior( ), l de paula ( ), mc jamur ( ), c oliver ( ), sg ramos ( ), cl silva ( ), lucia helena faccioli ( ) ( h rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il- , kc, mcp- and mip- were detected in the lungs by day . in vivo degranulation of mast cells by c / led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c / . histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c / . of interest, the number of mycobacterial bacilli recovered from the lungs was log higher after treatment of infected mice with c / . these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. ( ), ms chadfield ( ), db sørensen ( ), h offenberg ( ), m bisgaard ( ), he jensen ( ) ( ) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark ( ) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: female balb/ c-j mice (app. g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp ) and porcine (p ) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp- and metalloproteinase inhibitor timp- . results: all mice infected with the avian strain were euthanized after hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after , and h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp- and timp- between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp- and timp- . ( ), r molinaro ( ), a franÅa ( ), m bozza ( ), f cunha( ), s kunkel ( ) ( ) universidade do rio de janeiro, brazil ( ) universidade de s¼o paulo, brazil ( ) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr knockout mice (ccr -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr -/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c /bl mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since h after and until days. ccr -/-mice subjected to clp presented an increase in survival rate ( %) compared with wildtype mice ( %), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr -/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr -/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr -/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -µ and il- â) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa ( ), commiphora mukul ( ) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il- , cxcl and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl , which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr , the receptor for cxcl . while plasmablasts migrate in response to cxcl , plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr , the receptor for the interferon-gamma-induced chemokines cxcl , and , which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, ( ), h lee ( ) c a is a potent inflammatory mediator produced during complement activation. unregulated c a signalling through its receptor (c ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c ar antagonists for human therapy. we took neutrophils from genetically modified human c ar knock-in mice in which the mouse c ar coding region was replaced with human c ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c ar. these mabs inhibit c a-induced neutrophil migration and calcium-flux, and bind to a region of the nd extracellular loop of c ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c ar knock-in mice were given - mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single mg/kg dose of anti-c ar mab given days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g arrest in macrophage cell line raw . , their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba- mice by immunisation with bovine type ii collagen/adjuvant on day and . cor , synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day . etanercept ( mg/kg ip q d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry- is a potent (ic = nm), selective, atp-uncompetitive mek / inhibitor.arry- is highly efficacious in cia and aia rat models, with ed s of and mg/kg, respectively, equal to or better than standard agents. addition of arry- to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il- and il- production (ic s of , and nm, respectively). in contrast, inhibition of perk required nm to achieve a % reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of , , , or mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry- was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il and tnffz with > % inhibition observed at plasma concentrations of and ng/ml, respectively. similar inhibition of perk required ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry- and helped define tolerability. clinical evaluation of arry- in combination with methotrexate in patients with rheumatoid arthritis is on-going. p (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il- a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p activity is expected to regulate the levels of tnf-a and il- b thereby alleviating the effects of inflammation in ra. a new class of p inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. ( ), h aaes ( ), w-h boehncke( ), j pfeffer( ), t skak-nielsen( ), i teige ( ), k abell ( ), ph kvist ( ), e ottosen ( ), tk petersen ( ), lars svensson ( ) ( ) discovery, leo pharma, industriparken, ballerup, denmark ( ) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il- b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately % of psoriasis patients develop psoriatic arthritis. leo is a member of newly developed class of selective p map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice ( % reduction in mice and % in rats at mg/kg p.o.). in the humanised psoriasis model, leo at a dose of mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by %) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx- is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock- . its ic for rock- and rock- inhibition is nm and > mm respectively. the ability of slx- to inhibit septic liver injury was investigated in c bl/ j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps ( mg/mouse) and d-gal ( mg/ mouse) i.p and . hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a > fold increase in serum alt and ast levels. this increase was reduced by > % in mice pretreated with slx- either orally ( mg/kg, and hrs prechallenge) or i.p. ( - mg/kg, min pre-challenge). slx- inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by > %. to assess the kinetics of slx- s benefit, slx- ( mg/kg i.p.) was given min prior to, or or min after the lps/ d-gal. slx- was effective at inhibiting the rise in alt and ast levels at all time points suggesting that inhibiting rock- even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, out of mice given lps/d-gal were dead by hrs whereas in mice given slx- ( mg/kg orally) animal died at hours and the remaining mice were alive hrs later. these results show that specific inhibitors of rock- may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il- secretion in human purified t-cells stimulated with anti-cd and anti-cd or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. ( ), j zhang ( ), k henley ( ), m white ( ), d hilton ( ), b kile ( ) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c bl/ control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments . f(ab) or iv. f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il- beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt ( ), u hüffmeier ( ), i kçnig ( ), j lacorz ( ), a reis ( ), k reich ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*- a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*- t was detected (or= . , % ci . - . ; pcorr= . ) also in patients negative for the psors risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*- a and psoriasis seems to primarily reflect ld with psors , tnf*- t may represent a risk factor for psa independent of psors . ( ), n modi ( ), m stanford ( ), e kondeatis ( ), r vaughan ( ), f fortune ( ), w madanat ( ), c kanawati( ), p murray ( ) methods: dna was obtained from patients with bd, from the uk and from the middle east (me), and controls individuals, from the uk and from me. dna was prepared by proteinase k digestion, and salt extraction and - and snp were detected by a pcr-ssp. results: there was no significant difference in expression of - c/t or a/g when all bd patients were compared to all control individuals, (p= . and . , respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p= . , p= . ) or me bd patients (p= . , p= . ) when compared to the appropriate controls. ( ), da brown ( ), h johnen ( ), mr qiu ( ), t kuffner ( ), pgm curmi ( ), l brown ( ), m mazzanti ( ), sn breit ( ) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il- gene is single copy gene located on chromosome q . - in humans. interleukin- is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin- concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il - snp in the children with the cp. methods: dnas obtained from peripheral blood of cp patients and unrelated healthy volunteers were genotyped for the il - snp pcr-rflp method. results and conclusions: il - genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il fngene polymorphisms and cerebral palsy has to be investigated in the future studies. ( ), d delbro ( ), e hansson ( ) ( ) kalmar university, sweden ( ) gçteborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: . the expression of alpha nachrs and alpha nachrs by immunofluorescence and western blot. . intracellular ca +-transients spread within the astroglial networks after stimulation with nicotine. . pro-inflammatory cytokines, il- b, il- , and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca + transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca + responses were blocked by the alpha nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database ). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at mg/kg dose, and above. in the th test weak % effect was seen at side effect free dose ( mg/kg). in the arthritis model b.i.d. mg/kg dose of tramadol given on days - after cfa caused a maximum % effect on weight bearing incapacitance (day ). however, its effect seemed to diminish ( % on day ) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at but not at mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge and pgd in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk- .biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge and pgd levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk- was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo( ), lm yshii ( ) ( ), sk costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college, london, uk ( ) butantan institute, s¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr , a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin ( %, h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il- b and il- , but not tnfa) and anti-inflammatory (il- ) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr ( nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. ( ) ( ) university of s¼o paulo, s¼o paulo, brazil ( ) butantan institute, s¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or , -naphthoquinone ( , -nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and , -nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep ( mg/kg) and , -nq ( nmol/kg). after min, dep and , -nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following h treatment with pollutants. this treatment contributes to augment the ach ( - - - m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: , -nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and , -nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv ( , , mm) dose-dependently inhibited ng/ml lps induced tnfa, il- b, il- , mcp- and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk / and jnk but not p . nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk / , jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. ( ), cs patil( ), sv padi ( ), vp singh ( ) ( ) university institute of pharmaceutical sciences, panjab university, chandigarh, india ( ) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia days after the nerve ligation. sildenafil ( and fÝ g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) and glyt are expressed in glia and neurons, respectively. glyt make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt (sarcosine and org ) and glyt (alx and org ) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt and glyt inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp ff mouse to assess the effect of the gp ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il- family cytokine receptor gp preventing shp /ras/erk signaling and leading to constitutive stat transcriptional activation. tumour development is inflammation dependent, there is a requirement for il- , and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s inflamm. res., supplement ( ) posters dispensable for tumorigenesis. in gastric antrum the gp ff mutation results, decreased erk / activation and constitutive phosphorylation of stat , abnormal signaling is replicated in macrophages. antral stat activation is unaffected by depletion of il- . however in macrophages an absence of il- results in higher stat activation demonstrating the anti-stimulatory role of il- on macrophages. the gp ff mutation results in macrophages with decreased il- and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp ff mice display aberrant gp signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. ( ), y riffo-vasquez( ), s brain( ), s costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and , -naphthoquinone ( , -nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv knockout (ko) mice exposed to dep and , -nq. dep ( mg/kg) and , -nq ( nmol/kg)-induced airway inflammation was assessed via i-labelled albumin h post pollutants injection. mpo assay and histopathology were performed h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and , -nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv ko, suggesting these pollutants act via a trpv -mediated mechanism. in contrast, mpo activity in the airways of trpv ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv ko, indicating that the lack of trpv receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv receptors. acknowledgements: capes, cnpq, fapesp. ( ), n sato( , ), y endo ( ), s sugawara ( ) ( ) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan ( ) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j . . female balb/c mice ( weeks old) received a basal diet or a biotin-deficient diet for weeks.ten days after sensitization with intraperitoneal injection of lps and nicl , the mice were challenged with intradermal injection of nicl into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j . cells were cultured in biotin-sufficient or deficient medium for weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il- beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j . cells produced il- beta significantly higher than biotinsufficient j . cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il- beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il- , icam- and e-selectin were studied in patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in patients and severe -in patients. the highest levels of il- were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il- and adhesion molecules and severity of pancreatitis. during first week the levels of il- gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam- levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il- and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il- and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for â-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd and dnam- on mc, and b and nectin- on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells ( - weeks with scf, interleukin- and prostaglandin e , cbmc). cbmc were activated, after their culture for days with myeloma ige ( mg/ml), by rabbit anti-human ige antibodies ( mg/ml), for , and h at c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl- , bcl- , bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl- that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl- and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl- family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. ( ), c armishaw( ), z yang ( ), h cai ( ), p alewood( ), c geczy ( ) ( ) university of new south wales, faculty of medicne, sydney, australia ( ) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s a , s a and s a are closely related proteins associated with inflammation. s a is expressed in the human genome, but not in the mouse. s a is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s a and human (h) s a share % structural identity, but the hinge domains are more divergent. the ms a hinge (ms a - ) is a potent chemoattractant for leukocytes whereas this sequence in hs a (hs a - ) is inactive. methods: s a hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp cells or murine mc in vitro and mouse footpad injection. results: s a hinge domain (s a - ) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s a , the hinge domain only weakly induced cytokine production (il , il ) by macrophages. residues essential for oedema were hydrophobic in nature (leu , isoleu , isoleu and isoleu ). n and i were essential for responses provoked by - m s a - whereas mutation of k , l , n , i , d , k and i significantly reduced migration with - m s a - . conclusions: s a and ms a may be functional chemattractant equivalents; s a may have arisen by duplication of human s a . isoleucine residues in s a hinge domain are essential for its proinflammatory properties. ( ), g kiriakopoulou ( ), e tsimara( ), a voultsou( ), k zarkadis ( ) ( ) general hospital of zakynthos, greece ( ) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes . eo . %, hb . g/dl, ht . %, mcv , fl, mch . pg, thrombocytes . , blood sedimentation mm, fe mg/ml, ferritin . ng/ml. normal biochemical examinations. u/s: livers size lightly increased . mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules ( - mm) with a thin wall and quills on the side, as well as three imago worms (> mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites . , eo . %, hb , g/dl, ht . %. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement ( ) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average . mg/ l; p < . ) and with attacks (average . mg/l; p < . ) were significantly higher than those of normal controls (average . mg/l). serum hs-crp levels were inversely correlated with fev . % in asthmatic patients (r = - . , p < . ). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was % hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. ( ), sk bk( ), v sharma( ), rp bhandari ( ) ( ) nepal medical college teaching hospital, nepal ( ) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s inflamm. res., supplement ( ) posters from january to december . demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was , , giving an incidence of . per deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over % were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were . and . per deliveries (p <. );the corresponding cases of deepvenous-thrombosis diagnosed were . and . per deliveries, respectively (p <. ). the majority ( %) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of < weeks, a bmi of , or more and maternal-age of or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. ( ), ch ladel ( ), t ruckle( ), c rommel( ), r cirillo ( ) ( ) rbm-merck serono, colleretto giacosa, italy ( ) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi k signalling inhibition resulted in clinical amelioration of disease symptoms. as , a novel isoform non-selective, yet specific class-i-pi k inhibitor, administered at mg/kg twice-a-day for days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as was able to decrease its levels down to values comparable with naïve animals. controls and as treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi kg) and immunosuppressive (via pi kd/a/b) activity. ( ) ( ) institute of biomedical science, university of sao paulo, brazil ( ) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol ( ìg/kg) or progesterone ( ìg/kg) h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement ( ) s the release of il- and reduced that of il- and tnf. the release of il- by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il- and increased il- production in bal and increased that of il- and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam- and mac- expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after (ovx- ) or days (ovx- ) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, %; aerosol) and sacrificed h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam- , mac- and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day , e-selectin expression increased(ovx- = , ae , ) and at day , decreased (ovx- = , ae , ) as compared to respective controls (sh- = , ae , ; sh- = , ae , ). estrogen treatment reverted this profile in both groups (ovx- +e= , ae , ; ovx- +e= , ae , ). mean fluorescence intensity of bal cells showed increase of mac- expression (ovx- = ae , vs sh- = , ae , ), icam- (ovx- = , ae , vs sh- = , ae , ) and l-selectin (ovx- = , ae , vs sh- = , ae , ) at day i.e., ovx- group. on the other hand, we observed a decrease in icam- (ovx- = , ae , vs sh- = ae , ) and mac- expression (ovx- = , ae , vs sh- = , ae , ) was seen in ovx- group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx- ) and decreased (ovx- ) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell ( ), s lim ( ), y lin ( ), bp leung ( ), c thiemermann ( ), wsf wong ( ) ( ) national university of singapore ( ) the william harvey research institute, london, uk glycogen synthase kinase b (gsk- b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk- b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd- , a non-atp competitive gsk- b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd- significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il- , il- and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd- also suppressed the mrna levels of icam- , vcam- and chitinase proteins in the lung. histological studies revealed that tdzd- substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd- also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd- . our findings suggest that inhibition of gsk- b may have therapeutic potential for the treatment of allergic airway inflammation. ( ), a yildirim ( ), f ercan ( ), n gedik ( ), m yuksel( ), inci alican ( ) ( materials and methods: sprague-dawley rats ( - g) were exposed to oc (burn group) or oc water bath (control group) for s under ether anesthesia. adm ( ng/kg; s.c) was administered min before burn and all rats were decapitated h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p< . - . ) and mpo activity showed a marked increase in all tested tissues (p< . ) of the burn group. adm reversed these parameters effectively (p< . - . ). luminol chemiluminescence levels showed increases in both ileum and lung (p< . - . ) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p< . ) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p< . ). adm reduced plasma tnf-alpha level (p< . ) which showed a significant increase in burned animals compared to controls (p< . ). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday ( ), sd joshi ( ), kr reddy ( ) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg +, and an inhibitor, hql- , with anti-inflammatory activities in vivo, at . resolution. hql- was found to reside within the catalytic cleft between trp and gsh in the quaternary complex. hql- inhibited h-pgds competitively against the substrate pgh as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql- bound to h-pgds with an affinity that was -fold higher in the presence of gsh and mg + than in their absence. hql- inhibited selectively pgd production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql- inhibited antigen-induced production of pgd , and airway inflammation in mice without affecting the production of pge and pgf f¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats ( - g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for h ( breath/min). one lung was subjected to hyper-ventilation ( ml/kg/lung) and the other was ventilated with a normal volume ( ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip- was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip- expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip- between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. ( ), j-y gillon( ), v lagente ( ), e boichot ( ) ( ) university of rennes , rennes, france ( ) serono international s.a., geneva, switzerland macrophage elastase (mmp- ) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp- in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp- (rhmmp ) on il- /cxcl release from alveolar epithelial cell line a and we explored the underlying mechanisms. a cells were stimulated with rhmmp- ( . - - . - u/ml) during hours and il- /cxcl level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p kit. we observed that mmp- elicited il- /cxcl release in a dose-dependent manner. this production could be prevented by a pretreatment for hour with a selective mmp- inhibitor (as - mm) or with the nonselective inhibitor batimastat ( - mm) . the il- /cxcl production was also inhibited by actinomycin d ( mg/ml), erk / inhibitors (u mm and pd mm) and nfkb inhibitors including bay - ( mm) and nfkb activation inhibitor ( nm), whereas p kinase inhibitor (sb ) had no effect. stimulation with mmp- was rapidly followed by a phosphorylation of erk / ( min) and an nfkb nuclear translocation and activation ( h). the nfkb activation was not inhibited by a treatment with u . these data suggest that alveolar epithelium is a target of mmp- since it upregulates gene expres-s inflamm. res., supplement ( ) posters sion and release of il- /cxcl , via erk / and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)- , in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h cells were exposed to serial dilutions of test agents in serum-free medium for hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il- , matrix-metalloprotease- (mmp- ), the gel-forming mucin muc ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p s cyp a and cyp b were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il- alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th ) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase- (cox- ) and cox- dependent phases of prostaglandin d (pgd ) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic values of . mm and . mm, respectively. this compound inhibited cox- and -dependent conversion of the exogenous arachidonic acid to pgd in a dose-dependent manner with an ic values of . mm and . mm, respectively. however, dpt did not inhibit cox- protein expression up to a concentration of mm in the bmmc, indicating that dpt directly inhibits cox- activity. furthermore, this compound consistently inhibited the production of leukotriene c (ltc ) in a dose dependent manner, with an ic value of . mm. these posters inflamm. res., supplement ( ) s results clearly demonstrate that dpt has a dual cox- / -lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf- , could block the inflammation induced by an acute exposure to cigarette smoke in strains of mice, balb/c and c /bl .animals were administered the compound ( - mg/kg) either per os (p.o.) or intranasally (i.n.) hour before and hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf- dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~ % at mg/kg; p < . )or i.n. (~ % at mg/kg; p < . ).however, the compound had no clear effect on bal neutrophil infiltration in c /bl mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < . ). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il- beta, kc, and mip- , pkf- had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table ) cytokine analysis showed significant increases in tnfalpha, il- , kc, gm-csf, mcp- , il- beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in ( ) tnfa + gm-csf ( ) gm-csf ( ) il- + gm-csf ( ) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst- assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd a low cd b + cd c + cd + cd + cd low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd + t cell proliferation, il- production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after weeks. the hmscs were cultured and in passage ,the cell surface antigens of both groups were analyzed by flow cytometory.in passage , in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after weeks, alp staining and activity were measured in each group.after weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for weeks and weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal høgh ( ), hm lindegaard ( ), gl sorensen ( ), a høj ( ), c bendixen ( ), p junker ( ), u holmskov ( ) ( cytosolic phospholipase a (cpla ) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla -deficient mice are resistant to cia. thus we aimed to study whether cpla is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla antibodies revealed low positive cpla protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla were detected. in addition, robust cpla protein expression in the skeletal muscles surrounding the joints and strong cpla positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla protein, due to an inflammatory infiltrate and increased cpla expression, suggests an important role of cpla in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b .q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome a disease protective locus (cia ) and another on chromosome a disease-promoting locus (cia ). nod.q allele at cia promotes arthritis whereas cia , has a protective effect in contrast to the b .q allele on chromosome . a promising candidate gene in cia locus is complement factor (c ) as the nod.q allele produced defective c protein. cia locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf , fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf mutated and wildtype mice. we could show that ncf mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf wildtype dc. in addition, ncf mutated dc induced higher levels of il- production by hybridoma t cells. to analyze the role of ncf in dc on arthritis, mice were developed expressing functional ncf restricted to dc (b .qdcn). these mice are characterized for burst, ncf expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf deficient mice. this suggests that ncf plays a role in the uptake and processing of posters inflamm. res., supplement ( ) s antigens, probably by dc. this will be further investigated with the b .qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days and in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day and % incidence was observed on day . mif -/-mice exhibited delayed arthritis, with onset on day and % incidence on day . mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo ( ), a denadai-souza ( ), lm yshii ( ), a schenka ( ), ma barreto ( ), d boletini-santos ( ), c lima ( ), v rioli ( ), mn muscar ( ), e ferro ( ), sk costa ( ) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin ( or ìg/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day . results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day , there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag , we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo ( ), r saldanha-gama ( ), ja moraes ( ), r zingali ( ), c marcienkewicz ( ) ( ) universidade do estado do rio de janeiro, rio de janeiro, brazil ( ) universidade federal do rio de janeiro, rio de janeiro, brazil ( ) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb , a b and/or avb ) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a /a b integrins, vlo , induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo interaction with a b integrin, activating the focal adhesion cascade. vlo effects on the delay of neutrophil is modulated by pi k, erk- mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl- family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a b integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) ( ), h serezani ( ), m peters-golden( ), sonia jancar ( ) '( ) university of s¼o paulo, brazil ( ) university of michigan, usa it is been shown that leukotriene (lt) b and cysteinyl lt (ltc , ltd and lte ) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin - um), pkc-a (ro- - - nm), pi k (ly - um and wortmannin- nm), erk / (pd - um), cpla (aacocf - um), p mapk (sb - um) and ca++ (bapta/am- um), before stimulation with ltb or ltd and addition of igg-opsonized red blood cells for min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb -enhanced phagocytosis is dependent on pkc-a, while ltd effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin- mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin- is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin- as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin- at gene and protein level in monocytic thp- cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin- on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin- and - expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin- and- expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc was incubated with pap ( ng/ml) and/or tnfa ( ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p . results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u macrophages. methods: ha was added to u macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule- (icam- ) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)- b, and il- were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il- b, and il- . in contrast to kda ha, kda ha at mg/ ml inhibited lps-induced cytokine production. anti-icam- antibody blocked the effects of ha on the lps actions on u cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam- antibody reversed the inhibitory effects of ha on phosphorylation of p nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam- through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox- is prostacyclin (pgi ), which is a potent vascular patency factor. induction of endothelial cox- under hypoxic conditions is well documented. this response, along with associated pgi release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox- expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox- promoter region in huvec. cox- induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox- expression than those for nfkb. furthermore, expression of cox- was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi but not vaso-occlusive thromboxane a was seen. thus the putative protective induction of cox- in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th -predominant immune responses remains to be investigated. therefore, the effects of a th selective inhibitor tak- were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak- was administered orally, starting at the time of induction of glomerulonephritis. in group , the drug was administered daily for the initial days. tak- was administered on day only in group , and from day to in group . in each group, nephritic rats were killed on days and . results: in group , glomerular damage, including crescent formation, was improved on day , with reductions in the numbers of cd , cd and ed- positive cells, as well as in urinary protein excretion. protein and transcript levels of th cytokines in the diseased kidneys were markedly decreased by tak- treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group , in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group , partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s inflamm. res., supplement ( ) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd- mice were injected intraperitoneally with mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline ( mg/kg), lisofylline ( mg/kg) or prednisolone ( mg/kg). the tissues were harvested , , , , and h following lps administration and stored at - c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin- beta (il- beta), interleukin- (il- ), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at or h after lps administration, whereas the expression of tnf-alpha and il- beta in lungs and il- in liver reached the peak values at h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately %), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz( ), m losada ( ), c company ( ), c lope-gines( ), l piqueras( ), j cortijo ( ) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx cl (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii microm or with tnfalpha ( ng/ml) for , and h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after h and h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for and h. these effects were caused by the interaction of aii with its at receptor since they were abolished by losartan (at receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for and h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th -type cytokine profiles, i.e. m. globosa induced il- , il- , and il- secretion from the keratinocytes, whereas m. restricta induced il- secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il- and il- , respectively. the m. globosainduced increase in il- secretion was inhibited by mabs against cd and cd . in case of m. restricta, mabs against toll-like receptor (tlr ) and cd suppressed significantly il- secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl ) and tarc (thymus and activation-regulated chemokine/ccl ) are th type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e sub-fraction showed inhibitory activity on the stat protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il- family now consists of members, most of which have assigned functions.there are members of the il- receptor family (including the decoy receptor type ii il- r).many of the il- family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il- family members il- f , f and f signal through a complex of the il- r family member rp in association with il- r acp to activate common inflammatory pathways.the specific activity is is low, on the order of - ug/ml ec .we have found that removal of a few n-terminal amino acids from il- f , f , f and f can increase their bioactivity approximately -fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il- f is capable of antagonizing signaling via il- rrp , but full-length f is inactive. ( ) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr (hvem/tnfrsf ), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf ), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr pathway. c bl/ tr knockout mice and their wild-type control were fed a high-fat diet for weeks and the obesity phenotypes were determined in the obese tr knockout mice and the control. the obese tr mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, mg/kg, i.v.) and controls were sensitized with ova ( ìg) and al(oh) ( mg, s.c.) days after alloxan or saline injection. the animals were challenged days later by the intratracheal instillation of ova ( mg/ . ml). the following analyses were performed h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il- beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils ( %) and mononuclear cells ( %); reduced levels of tnf-alpha ( %) and il- beta ( %), and reduced p-selectin expression ( %) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin ( iu, s.c.), h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il- beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il- and the adhesion molecule icam- by real-time pcr. icam and il- were induced rapidly peaking after h, their mrna levels increased again from h to reach a plateau between h to h after exposure to tnf, whereas tnf mrna levels increased steadily between h and h. h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il- but did not affect that of icam- or tnf h following exposure while calcitriol markedly inhibited the induction of all pro-inflammatory genes h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p by tnf. this action was mimicked by the posters inflamm. res., supplement ( ) s jnk inhibitor sp and the p inhibitor sb .the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p cascades. ( ), cm lotufo ( ), p borelli ( ), zs ferreira ( ), rp markus ( ), shp farsky ( ) ( ) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of s¼o paulo, brazil ( ) department of physiology, bioscience institute,university of s¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru -treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru -treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam- , vcam- and pecam- . participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru -treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. ( ), y naito( ), t okuda ( ), k mizushima ( ), t okayama ( ), i hirata ( ), h tsuboi ( ), t suzuki ( ), o handa ( ) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co( ppm- ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc- mrna/protein and p-p mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc- mrna/protein and p-p mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero ( ), m maróstica ( ), m saad( ), j pedrazzoli jr ( ) ( ) s¼o francisco university medical school, brazil ( ) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass ( . +- . and . +- . % of body weight for colitis and control, respectively; p< . ) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter ( . +- . and . +- . um for colitis and control, respectively; p< . ), higher basal lipolysis ( . +- . and . +- . ug.mg- for colitis and control, respectively; p< . ) and tnf-alpha production ( . +- . and . +- . ng.mg- for colitis and control, respectively; p< . ). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis ( . +- . and . . +- . ug.mg- for colitis and control, respectively; p< . ), increased tnfalpha ( . +- . and . +- . ng.ml- for colitis and control, respectively; p< . ), leptin ( . +- . and . +- . pg.ml- for colitis and control, respectively; p< . ) and adiponectin production ( +- and +- ng.ml- for colitis and control, respectively; p< . ). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il- â, il- , il- , kc, cox- , igg heavy chain, and phospho-stat in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p mapk, the activation of the p mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p mapk and of mk and hsp , two down-stream targets, were not increased in dss treated animals compared to controls. leo , a potent inhibitor of p activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr + cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/- ng/ml lps. for mlr, balb/c tcells were added for days. cells were incubated with sb ( -( -fluorophenyl)- -( -ethylsulfinylphenyl)- -( -pyridyl) h-imidazole, sb) or ml ((rs)-{ -[ -( -fluorophenyl)- -methylsulfanyl- h-imidazol- -yl]pyridine- -yl}-( -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il- production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic spdc . mm, bmdc . mm) and ml (ic spdc . mm, bmdc . mm). preincubation with dcs had no effect, despite reduced lps stimulated il- and tnfa synthesis by sb (ic il- . mm, tnf . mm) and ml (ic il- . mm, tnf . mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p mapk in the mlr is under investigation. p inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. ( ), s luik( ), s laufer( ), m seed ( ), v holan( ), s fiorucci ( ) ( ) synovo gmbh, tübingen, germany ( ) university of tübingen, germany in vivo anti-inflammatory activity of certain p kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically ( d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at mmolkg- d- . we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il- , il- ) and adhesion molecules (e-selectin and icam- ) were measured in patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the , , and day. all patients were divided on two groups: first group compiled patients, in which dexamethasone ( mg/day during - days) was applied in the complex management of acute pancreatitis, and control group - patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il- , il- , il- , icam- , and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th transcription factor gata- can inhibit th /th cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day . at day , t cell specific gata- tg mice did not show any difference in arthritis score compared to wild type mice. however, at day , wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata- tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata- protects against development of severe joint inflammation. facs analysis revealed low levels of il- +/ifn-gammacells in wild type as well as in gata- tg mice at day . as expected, il- positive cells were higher in gata- tg mice compared with wild type mice. interestingly, at day , the percentage of il- +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata- tg mice, suggesting prevention of th expansion under gata- overexpression in vivo. these data revealed that t cell specific overexpression of the th transcription factor gata- protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il- +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata- overexpression in t cells prevents expansion of the il- +/ifn-gamma-t cell subset. pingping jiang ( ), pt sangild( ), t thymann ( ), hh-y ngai ( ), w-h sit ( ), k-l chan ( ), jm-f wan ( ) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis ( d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to days of parenteral feeding followed by day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = ) were compared with corresponding pigs remained healthy (n = ). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase and chain a, medium-chain acyl-coa dehydrogenase with -thiaoctanoyl-coa etc.), inflammation (peptide-binding protein (pbp ) and snail homolog ), signal transduction proteins (thyroid hormone binding protein precursor, park protein and chain b, structure of the rho family gtp-binding protein cdc in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli :b ) by inhalation. the lungs were lavaged hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of mg ( ml/animal) of lps (p.aeruginosa) to c bl/ j mice. control animals received a single dose of sterile ml . % saline/animal. the mice were terminated , , , and h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by %). treatment with daivonex / dovobet also showed an anti psoriatic effect with a % reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res ( ): ) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or % protein diet (c-group) during the first days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o -, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin (k ) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k /vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with -o-tetradecanoylphorbol- -acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone- -valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd -positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone- -valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type diabetes (t d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t d. we aim to detect how gimap affects the lifespan of t cells. results: overexpression in c cells of both wt gimap and gimap with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap causes apoptosis in jurkat cells and primary naïve t cells but not in activated t cells. finally, gimap -mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap causes rapid death of the cells. this suggests that gimap is pro-apoptotic. the results with human wt gimap support this conclusion: recently, much focus has been on the cellular cd / cadpr signaling system during inflammatory processes. the cd /cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il- . to our knowledge, the expression and function of the cd /cadpr signaling system in the human detrusor muscle have not been described. cd protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca + concentration ([ca +] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd which could be upregulated by il- ( ng/ml). in hdsmc briefly activated with il- ( ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca +]i. cyclic adpr-mediated ca + increase was greatly reduced in ca + free medium suggesting ca + entry as well as ca + release. cyclic adpr -elicited ca + increase was mimicked by -deaza-cadpr, and blocked by -bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il- , cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that ) hdsmc express cell surface cd , ) the expression and function of cd are augmented by il- , ) externally added cadpr elicited a rapid, il- -dependent, and -bromo-cadpr-inhibitable ca + mobilization, ) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd /cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for days before induction of arthritis and days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin ( mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration ( mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t ), and compared with the original formula (t ) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t cream (n= ) or no cream (n= ) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment ( . ml ; stirred continuously at c). min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ,) or not in the presence of % t , % t , or no treatment. hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < . ). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t with zinc only was more effective than t (treat:f , = . , p < . ; lps:f , = . , p < . ; treat by lps:f , = . , p < . ). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. ( ) ( ) osprey pharmaceuticals limited, canada ( ) probetex, inc., texas, usa the ccl /ccr chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl chemokine fused to a truncated form of the enzymatically active a domain of shiga toxin has been developed. the ccl portion binds specifically to ccr -bearing leukocytes and enters the cells, where the sa portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day and treated intravenously with vehicle, or mg/kg of the recombinant protein q d from day until day . urine and blood collections were made prior to ats injection and on days and . animals were sacrificed on day . no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of , , , and % for glomerular lesions, m/m count, fibronectin and µ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement ( ) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning ( h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd b expression on resting monocytes harvested from h blister was lower in patients as compared to controls (p= . ). lower expression of cd b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p= . and p= . , respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip- µ was higher among patients (p= . ) and similar levels were seen in blister fluids. concentration of mcp- was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru inhibited no production, and inos protein and mrna expression in murine j macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac selective mc . hdac inhibitors reversed the effects of dexamethasone and ru on inos expression or no production. stably transfected a / cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv -dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd mice ( - g) were given intraplantar injections (i.pl.) of tnfa ( pmol/ microl) and tyrode (as vehicle, contralateral paw; microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and h after injection. indomethacin ( mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle ( % nahco ). another group of mice were injected with tnfa i.pl. plus % nahco with the contralateral paw injected with tyrode plus indomethacin ( mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa ( pmol) leads to significantly reduced (p< . compared to baseline values) paw withdrawal latency in both paws h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin ( mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox- inhibitor, nimesulide. in conclusion, cox- derived prostaglandins are important in the development of hyperalgesia. local cox- inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats ( - g) were administered dexamathasone ( mg/kg, i.p.) either h before or h after lps ( mg/kg, i.p.) injection. animals were killed h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il- b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h s concentration and liver and kidney h s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h s and tissue h s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps ( ng/ml, h, oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone ( nm) significantly (p< . ) reduced expression of both cse and inos. these data provide further evidence that h s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h s production. ( ), u jalonen ( ), h kankaanranta ( ), r tuominen( ), e moilanen ( ) ( ) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland ( ) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup , tis , g s and zfp , is a factor that binds to utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s inflamm. res., supplement ( ) posters concentration of pma for h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro (inhibits pkcb, & and e), gÖ (inhibits pkca, b and &) and cgp (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp did not affect ttp mrna half-life and therefore we measured the effects of cgp on the activation of transcription factors involved in ttp expression. cgp had no effect on the activation of nf-kb, egr or sp . in contrast, cgp reduced the activation of transcription factor ap- , which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap- . the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il- , il- and no) in raw . cells. among them, -oxooctadec- -enoic acid and -oxooctadec- -enoic acid inhibited the production of tnf-a, il- , il- and no at the concentration of mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il- , il- and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen( ), p hjarnaa ( ), j fensholdt ( ), p-p elena( ), k abell ( ), tk petersen ( ) ( ) discovery, leo pharma, ballerup, denmark ( ) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr- . additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd induced il- response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to mg/kg i.p. or mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi kg gene depletion and pi kd kinase-inactive mutation, pik cgd koi, exhibited thymus atrophy, similar to previously reported pi kg and d double knockout (p g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg / igg a ratio and ige level were elevated in pik cgd koi mice corresponding to a skewed th profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi k d or together with pi k g selective inhibitors, while th cytokines were suppressed th cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th reaction but attenuate th medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, h before i.d. injection of . ml pbs containing des-arg -bradykinin (dapk, nmol right hind paw) and . ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) - minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk / . dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp and erk / pd inhibitors. western blot analysis shows that phosphorylation of jnk and erk / is important in the up-regulation of b receptors. our results clearly show that the phosphorylation of both erk / and jnk mapkinases is an important step for the in vivo up-regulation of b receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b receptor require further investigation. continued interest in development of small molecule inhibitors of p mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p leads to increase production of pro-inflammatory cytokines such as tnf-a and il- b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified -( -chlorophenyl)- -(( -methoxyphenoxy)methyl)- [ , , ] triazolo [ , -b] [ , , ] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the -substituted- -amino- -mercapto- h- , , -triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position and . synthesis and sar will be presented. cytokines like il- b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il- b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il- b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p mapk inhibitor sb and a highly active compound developed in our lab. a data set was generated by determining whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il- and tnf a. inhibition of p map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p map kinase. scope of this work was to investigate -thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised -alkylsulfanyl, -( -fluorophenyl), -( -aminopyridin- -yl) substituted imidazoles as p map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the -aminopyridin- -yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the -thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met , for inhibitory activity. less clear is the importance of the hydrogen bond between n of the imidazole ring and lys of p map kinase.to investigate the role of lys in interacting with the scaffold we prepared two sets of , diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys , contrary to the observation on the compound vk- and p map kinase, that a nitrogen atom bearing a lone pair in position of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys rather than to form an attractive interaction with p map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of -substituted and unsubstituted , -diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p . these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw . cells. treatment of c bl/ mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il- b in the serum. the serum level of the anti-inflammatory cytokine il- was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk / and p mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw . cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide mg for days followed by mg daily for weeks. at week all patients started infliximab mg/kg, and received a further four infusions at weeks , , and . results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a , the active metabolite of leflunomide, and adverse events. the mean disease activity score (das ) fell from . at week to . (p< . ) at week and remained between . and . up to week . in those patients remaining on treatment, more than % achieved an acr response from week to week , and up to % achieved an acr response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il- those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of anticollagentype ii mabs on day , followed by i.p. injection of lps on day to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide ( mg/kg/day) starting on the day of first immunization (day ) in prophylactic treatment group and after the onset of arthritis (day ) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il- b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group ( . ae . ) and therapeutic treatment group ( . ae . ) compared to untreated group ( . ae . , p = . and p = . respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p< . ). steady state mrna levels of il- b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p< . ). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine ( ìm) to detect oxygen free radical production. the cells ( , x cell/ml) were then incubated with a range of concentrations of fibrinogen ( - mg/dl) for minutes. our results show that posters inflamm. res., supplement ( ) s fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations ( - mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox- and cox- ) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox- is mainly involved in homeostatic processes, while the inducible isoform (cox- ) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox- and cox- of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox- and anti-cox- effects by prostaglandin e (pge ) measurement after a (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing reference nsaids, selective or not for cox- or cox- . fifty % inhibitory concentration (ic ) values against cox- and cox- and cox- :cox- ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= . ). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox- and cox- . for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day . the inflammatory response was measured after , and hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at / hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp- were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd b, were cd lo, ssclo and rp- + (granulocyte marker). in humans, transmigrated granulocytes expressed cd and cd b. these cells had a significantly higher cd b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated consecutive patients with mean age ae . years who were admitted within h after ischemic stroke. a control group of patients with mean age ae . years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: . ae . and ae . nmol/l. correlation analysis revealed p< . . discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p< . ) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from ffae in the control to ffae at the concentration of ffmg/ml (p< . ). in the ischemic phase, cynodon dactylon ( ffmg/ml) also decreased the incidence of vt from % (control) to %. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p< . for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during min followed by h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox- (hypothalamus) associated to a marked reduction of ca +-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca +-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson ( ), h qui ( ), m wang ( ), i vedin ( ), jz haeggstrçm ( ), j palmblad ( ) ( ( ), r carnuccio( ), p romagnoli ( ), f rossi ( ) ( ) second university of naples, italy ( ) university of naples, italy ( ) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury ( ) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox- , nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone ( mg/kg/ die, by gavage) for days before carotid injury and days after injury. control rats received physiological solution. days after injury cox- expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p< . ). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by % in treated versus control carotids (p< . ). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h o production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h o production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( ( ), b sehnert ( ), h lanig( ), s päßler( ), r holmdahl ( ), h burkhardt ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander university of erlangen-nuremberg, germany ( ) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic that is highly somatically mutated and its epitope on type ii collagen (cii, aa - ). methods: the establishment of a dynamic simulation modelling of a ciic single-chain fragment (scfv) in complex with the triple helical ciic epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c scfvs and the respective ciic epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr and cdr regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c scfv sequence into the respective germline does not affect cii binding affinity (kd x - ) could be confirmed experimentally by the mutagenesis of (!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd + t-cell proliferation and active brain lesions, on the one hand, and il- , il- and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il- production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il- , but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il- responses, and disease activity is associated with mbp-induced proliferation of cd + t cells. ( ), k goula ( ), p georgakopoulos ( ) ( ) renal unit, st. anrdew hospital, patras, greece ( ) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men ( %). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of maintenance haemodialysis patients at our center over the past years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture. patients ( . %) from the study group were diabetic. results: cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli ( cases), enterobacter aerogenes ( case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone ( mg/kg/day). thereafter the amount of steroid was reduced to . mg/kg/day and a low dose of paquinimod ( . mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd +, cd + and on cd -cd -t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at : . mp were separated on -d gels and tested in wb ( : ) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around , , , kda ( total positive/ tested patients). the dominant antigen was at kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m muscarinic receptor was overcome. proteins were separated on d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for h, exposed to uvb and further cultured for another h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase (msk ) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin- â (il- ß) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase- (mmp- ) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp- activity and mmp- expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp- with the ic s of . - . ìm, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp- induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin ( . - . ìm) strongly inhibited mmp- induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap- , whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p mapk activation, and kaempferol inhibited the p mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp- inhibition and mmp- down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. ( ) ( ) university of valencia, spain ( ) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol- -thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-Þb activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta ( mg/ml) produced a % inhibition of oedema and a strong reduction of pge ( %), ltb ( %) and mpo activity ( %) in skin homogenates. the inhibitory effect of avarol at the same dose was % for oedema, % for pge , and % for ltb and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-Þb nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-Þb activation and can be mediated by the downregulation of intracellular signal-transduction ( ), ams silva( ), cmm santos( ), dcga pinto( ), jas cavaleiro( ), jlfc lima ( ) ( ) requimte, departamento de química-física, faculdade de farmµcia da universidade do porto, porto, portugal ( ) departamento de química, universidade de aveiro, aveiro, portugal -styrylchromones are a novel class of chromones, vinylogues of flavones ( -phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of -styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic -styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several -styrylchromones on the proinflammatory enzymes cyclooxygenase (cox- ), cyclooxygenase (cox- ) and -lipoxygenase ( -lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied -styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested -styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of -styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers f-fluorodeoxyglucose (fdg), -o-methyl- - f-fluoro-l-dopa (omfd), and f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp- was compared with that by squamous cell carcinoma (fadu), mamma (mcf- ) and colorectal adenocarcinoma (ht ) cell lines (without or in the presence of specific inhibitiors). several thp- phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp- cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp- cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp- phenotypes and between thp- phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a -end sage ( sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by sage in colon cancer cell lines treated with epigenetically affecting agents, -aza- deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin- (il- ) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il- is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il- contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il- . serum levels of il- are elevated in patients with colorectal cancer, however serum levels of il- were found to be independent of il- mrna expression in tumor tissue. in this study we analyzed il- mrna expression by real-time pcr in sporadic colon cancer tissue as well as corresponding normal mucous tissue. il- mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p= , ). there was no correlation betweenil- mrna expression and tumor grade or stage. thus we can conclude that il- produced at the tumor site is not involved in sporadic colon cancer progression. ( ), t aiamsa-ard ( ), v chinswangwatanakul ( ), ki techatraisak ( ), s chotewuttakorn ( ), a thaworn ( ) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with % of serum and incubated for hours. cells were analyzed by using [ h] thymidine and immunoblotting assay for cell proliferation and cox- /nos- protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox- but not nos- in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox- . conclusions: this result suggested that some factors in serum of cancer patients could induce cox- protein expression in huvec. the increasing of cox- might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok- @on neutrophil-derived superoxide production and tumor growth. methods: ah a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok- , a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok- on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok- . ( ), m jokic ( ), v zjacic-rotkvic( ), s kapitanovic ( ) ( ) university hospital sestre milosrdnice, bucharest, romania ( ) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il- is a pleiotropic cytokine mapped to chromosome p - . its promoter snp - g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from patients diagnosed with gep-net and age and sex-matched volunteers were analyzed for - g/c snp of the il- gene. to analyze il - c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis Ä test and fishers exact test were used. the level of significance was . . results: there were no differences observed in the frequencies of the - high expression (gc and gg) genotypes between the patients and healthy volunteers (p= . ), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p= . ). - g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p= . ). conclusions: high expression genotypes of il- - snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type receptor (nts ). here we show that hmc- human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc- cell rna yielded a band with % sequence identity to pront and a band corresponding to the pront processing enzyme, pc a.immunocytochemistry on hmc- cells showed specific staining for pront. stimulation of hmc- cells with a + pma, pge , c / or mastoparan released immunoreactive nt.rt-pcr on hmc- cell rna yielded a band with % sequence identity to human nts . western blotting gave bands corresponding to unglycosylated ( kda) and glycosylated ( kda) nts .immunocytotochemistry on hmc- cells showed specific staining for nts . these finding have significance for the role of mast cells in tumor growth. ( ), j buddenkotte ( ), mp schçn( ), m steinhoff ( ) ( ) university hospital münster, germany ( ) university hospital würzburg, germany the proteinase-activated receptor par- has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par- in cutaneous cancerogenesis is still unknown. here we could show a protective role of par- in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par- -deficient and wild-type mice were painted once with dmba ( , -dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate ( -o-tetradecanoylphorbol- -acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after weeks, par- -deficient mice showed a significantly increased number of skin tumors ( per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par- stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase / (erk / ) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta (tgf-â ). thus, our results provide the first experimental evidence for a tumor-protective role of par- . ( ), ma arbós ( ), a fraga ( ), i de torres( ), j reventós ( ), j morote ( ) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf /cxcr and mcp /ccr chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf /cxcr and mcp /ccr expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b f (sc). paf-r antagonist, web ( mg/kg, ip) was given daily for days. we found that eat growth was significantly reduced by pretreatment with web , and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web pretreatment. eat growth was accompanied by increased production of prostaglandin e , vegf and no which was reduced significantly by web treatment. in b f melanoma, web , alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web- reducedactive caspase- expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. ( ), mt quiles ( ), a figueras( ), r mangues( ), f vinals( ), jr germa( ), g capella ( ) ( ) institut de recerca vall de hebron, barcelona, spain ( ) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl (sdf- )/ cxcr chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys and asp nih t transfectants. cys tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with µ-smooth muscle actin positives cells surrounded by f / macrophages were only observed in asp tumors associated with a shorter growth period. asp tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp- protein levels were similarly diminished in both transfectants. higher mmp and mmp activities and expression were observed in asp tumors probably produced by macrophages or stromal cells. total and active mmp levels were higher in cys tumors. the expression of sdf- and cxcr remained unchanged while sdf- g isoform was selectively induced in cys tumors, suggesting sdf- a or b are induced in asp tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf- expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase- and substrates; tnf-alpha; il- ). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher ( -fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ and gencat_agaur_ xt_ ). ( ), m spinola( ), c pignatiello( ), w cabrera ( ), og ribeiro ( ), n starobinas( ), t dragani ( ) ( ) butantan institute, sao paulo, brazil ( ) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c bl/ and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing , mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin (vnn ) and major histocompatibility antigen e alpha (h -ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome ( . cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr- and - , pdgfr, kit, csf- r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability ( %) has translated into superior in vivo anti- inflamm. res., supplement ( ) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk . thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a and du- ) or low passage patient derived tumors (cxf ; colon cancer, rxf ; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. ( ), p bobrowski( ), m shukla ( ), t haqqi ( ) ( ) albany medical college, usa ( ) rainforest nutritionals, inc, usa ( ) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol # . proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il- b ( ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado ( - mg/ml) was tested for its ability to maintain optimal igf- transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity ( mg/kg po) leading to gsh safety rating of /unclassifiable. oligomeric proanthocyanidin content was high ( mg/kg) with the majority of oligomers > mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il- b induced glycosaminoglycan release from human cartilage explants. progrado prevented il- b induced suppression of igf- production from human cartilage explants as well as stimulating basal igf- production (p< . ). comparable changes in igf- gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf- . this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h o -induced cytotoxicity and oxidative dna damage in the nih/ t fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of , -diphenyl- picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h o -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h o , without cytotoxicity except for the hexane extract at high concentrations (> mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk project from korea research foundation). sirt is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt activity, comparing heart (h) and adipose (a) tissue of sedentary young (n ), sedentary old (n ) and trained old (n ) rats. the trained old rats performed a -weeks moderate training on treadmill. on h and a tissue of all rats sirt activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts -hydroxynonenal ( -hne), mnsod, catalase and foxo a by western blot, and gadd a, cyclin d and foxo a mrna by rt-pcr. aging reduced sirt activity in h (p< . ) without effects in a, producing an increase of mda (h, p< . ; a, p< . ) and -hne (h, p< . ; a, p< . ), and a decrease of mn-sod (p< . ) and catalase (p< . ) expression in both h and a. aging did not affect foxo a protein expression in h, and foxo a mrna in a. exercise produced an increase in h foxo a protein expression (p< . ) and in a foxo a mrna, associated to higher mn-sod (h, p< . ; a, p< . ) and catalase (h, p< . ; a, p= . ) levels in both h and a of aged rats. in heart exercise-induced higher sirt activity bring on decrease in cyclin d and increase in gadd a mrna expression. in a we found a similar decrease in cyclin d , without changes in gadd a mrna expression. these findings suggest that exercise is able to increase sirt activity in aged rats. ( ), t horiguchi( ), k abe( ), h inoue( ), t noma ( ) ( ) institute of health biosciences, the university of tokushima graduate school, tokushima, japan ( ) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl -induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with f×l/ kg bw of ccl w/wo mg/ kg bw of gl. after hours and hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between hour-treated-livers (ccl or ccl and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at hours compared to that in ccl treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a , hsp , and procollagen were still very high in gl-treated liver. after hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl -treated liver. then, we screened , genes by microarray and found that genes were up-regulated and genes were down regulated in ccl +gl compared to ccl treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn ). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp- monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp- cells. we also demonstrate that such modulation involves both erk / and p mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. ( ), b arnold( ), g opdenakker ( ) ( ) jagiellonian university, department of evolutionary immunobiology, krakow, poland ( ) german cancer research center, department of molecular immunology, heidelberg, germany ( ) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase (mmp- -/-) at least one compensatory mechanism operates as there are elevated levels of pge of cox- origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp- -/-mice as at hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp- -/-mice. for this numbers of apoptotic (annexin v) and necrotic ( -aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase were significantly lowered in mmp- -/-mice while levels of caspase , and were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp- -/mice during zymosan peritonitis and it is due to the decreased levels of active caspase . the increased activity of other examined caspases is most probably independent of apoptosis. ( ), h james ( ) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), and mg/kg produced a dose-dependent inhibition of paw edema ( % and % at h; % and % at h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), mg/kg( % at h; % at h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of mg/ kg( % at h; % at h) but not with a dose of mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin mg and mg/kg ( % and % at h; % and % at h respectively) and selective cox- inhibitor rofecoxib, mg/kg ( % and % respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. ( ), p hart( ), j edwards ( ), c quirk ( ) ( ) molecular pharmacology limited (usa), australian division, perth, western australia ( ) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il- â production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen ( %) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at min, or at hrs after injury. a third injury site was not treated. photographs were taken before, hrs, and weeks after injury. cytokines: human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of % or % active ingredient. hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at hrs after oxsoralen-uv, the min treatment site showed slight erythema, the hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. weeks after injury, the min site was normal, the hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il â secretion by . % and . %, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. ( ), p hart( ), j snowden ( ), maud eijkenboom ( ) ( ) molecular pharmacology limited (usa), australian division,perth, western australia ( ) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of %, . %, %, . %, %, % or % bov-zn (two pooled experiments). hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for %, %, %, % fcs against %, . %, %, % bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter ) in treated and untreated cell cultures over - hrs at intervals. the ic for tnf-a suppression was reached at . % bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by % bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-£\ and il- fÒ. these cytokines are known to be regulated by the stress-activated p fnfnmap kinase pathway. because of this, inhibition of p map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last years, numerous groups have reported on the development of p map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p vs other kinases, enabling the development of p selective molecules. inflamm. res., supplement ( ) posters anti-inflammatory activities. a series of labdane-type diterpenoids ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, and were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of and % were noted in the -o-tetradecanoyl-phorbol- -acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e , and tumour necrosis factor-alpha in bacterial endotoxin-activated raw . macrophage cells with ic in the range - mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase- , as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbß, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of mg/kg and substance ffw- Ñ (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect ( %) at a dosage of mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first min and hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw- Ñ that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th -response with increased levels of proinflammatory cytokines like tnfa and il , and mouse models of crohns disease show that the balance of il / is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il , il and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d , which were able to suppress the cocktail induced il secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p - inflammation and human incisional hernia pathophysiology maria antonia arbos via( ) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney ( ae vs. ae ae ; p< . ), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of s¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge mimetic combination therapy (dp- ) that inhibits basal and lps/tlr induced tnf-?, il- ß, and mmp- , , , in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp dramatically inhibited il- ß, tnf-?, mip , mcp- and il- expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il- ß, mmp- and to a lesser extent mmp- expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data ( , anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb from the -lipoxygenase pathway, pge through the inducible cyclooxygenase (cox- ) pathway and cytokines/chemokines as tnf-alpha, il- beta, il- , mip- alpha, mip- beta, mip- alpha and mip- alpha. engagement of the adenosine a a receptor (a ar) blocks the in vitro synthesis of ltb while it potentiates the cox- pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip- alpha, mip- beta, mip- alpha and mip- alpha in toll-like receptor- -stimulated human neutrophils. little effect was observed on il- beta and il- . using the murine air pouch model of inflammation with a ar-knockout mice, we observed that the activation of a ar positively impacts the expression of cox- in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a a receptor, neutrophils that migrated into the air pouch h following lps injection expressed higher mrna levels of tnf-alpha, mip- alpha and mip- beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a ar-knockout mice and in view of the potent inhibitory actions of pge on inflammatory cells, an increased cox- expression and a prevented release of tnf-alpha, mip- alpha and mip- beta caused by a ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis ( d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw . after challenged with lps (escherichia coli :b ) for , and hours. we identified proteins from approximately detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein , capg protein, poly(rc) binding protein , isocitrate dehydrogenase (nad+) alpha, lactate dehydrogenase , a chain, guanine nucleotide binding protein (g protein), beta polypeptide like , triosephosphate isomerase and proteasome alpha subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p , malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified -amino- -benzyl- -( -bromophenyl)- -methyl- , -dihydropyrano[ , c] pyrazole- -c arbonitrile as a potential novel lead structure as p map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol- ( h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha -acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from . to . ìg/ml in male, and from . to . ìg/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at . - . ìg/ml or days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from to ìg/ml in male, and from to ìg/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at - , ìg/ml. despite a high value of , - , ìg/ml being observed for serum aag levels in pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid ( ìg/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi- , a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi- .the ability of pbi to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi- treated animals, n= ) were used to assess leukocyte infiltration and to measure by elisa tnf-µ, mcp- and pge production.in vitro, pbi- is able to significantly decrease by . % ae . % (p< . ), tnf-a production by human lpsactivated pmn.in vivo, pbi- significantly decreased the production of tnf-a ( . % ae . %; p< . ), mcp- ( . % ae . %; p< . ) and pge ( . % ae . %; p< . ) induced by lps injection.however, pbi- did not significantly inhibit leukocyte infiltration.these results show that pbi- is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. ( ), lj lowenstine,( ), aj norris( ), t spangler( ), lm woods ( ) ( ) zoological society of san diego, usa ( ) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n= each) were selected for parasitology and em (group ), and gross and histopathology (group ). all outbreak cases and group controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in % ( / ) of outbreak cases and in % ( / ) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in archived crows submitted to the vmth from - , suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk / by mek / is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as , a selective mek inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for days (twice daily), by oral route at the doses of , and mg/kg. as at and mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o -.), hydrogen peroxide (h o ), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. ( ), h spalteholz ( ), u reibetanz ( ), p salavei ( ), m fischlechner ( ), h-j glander( ), j arnhold ( ) ( ) university of leipzig, medical faculty, institute for medical physics and biophysics, germany ( ) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects - % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h o ) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor- (par ) in inflammation and innate immunity. par is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par . par is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation ( hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. ( ), k bendtzen ( ), f sellebjerg ( ), ch nielsen ( ) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from ms patients and healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd + monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il- , tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione( ), g tarantino( ), a spanò( ), p ladogana ( ), fl perrone( ), s padula( ), a riccio ( ) ( ) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy ( ) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: patients with arthritis were selected in a population of subjects affected by hcv infection. each patients was evaluated by clinical examination ( denoted poliarticular and mono-oligoarticualr involvement), by x-graphic aspects of joint involvement ( patients presented join erosions), by ana, rf and anti-ccp positiveness.results: , % of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in out of the patients with joint erosions, and only in out of the patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg d on cd + t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type diabetes. the monoclonal antibody cx recognizes murine nkg d and has been shown to block ligandbinding and mediate internalization of nkg d. furthermore, cx can inhibit and/or ameliorate disease in animal models of type diabetes and inflammatory bowel disease. thus, it is very likely that nkg d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb .scid mice. different doses of cx antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx elisa in serum) and flow cytometry (down-regulation of nkg d on cd b+ nk cells) for up to two weeks after administration. we found that cx very efficiently down-regulate nkg d on cd b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx antibody in vivo. ( ), k mehta( ), n deo( ), j chaudhary( ), p bobrowski ( ) ( ) albany medical college, usa ( ) vedic lifesciences, usa ( ) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n= ) were screened and randomized to receive glucosamine sulfate (n= , mg/day) or reparagen (n= , mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a % improvement in womac pain, with assessments at , , , and weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p< . ), with a similar, progressive improvement over the course of the week treatment protocol ( - % reduction in total womac or vas scores). the response rate was substantial for both glucosamine ( %) and reparagen ( %), which exceeded placebo responses ( %, p < . ) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p < . ), and serum igf- levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin- (il- ) and monocyte chemoattracting protein- , two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by % and %, respectively. corresponding reduction in ibuprofen group was % and %. protective mechanisms involved were assessed in human cultured u macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha ( % and % reduction by supplementation with nm and ibuprofen, respectively) and il- ( % and % in corresponding reduction). nm supplementation dramatically reduced prostaglandin e secretion by stimulated macrophages along with cyclooxigenase- (cox ) cellular protein expression. mrna levels forcox and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. ( ), hp kim ( ), kh son ( ) ( ) college of pharmacy, kangwon national university, south korea ( ) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw . cells. among the tested compounds, -methoxy- , -dichlorochalcone (ch ), -hydroxy- -methoxychalcone (ch ), -hydroxy- -bromo- -methoxychalcone (ch ) and -hydroxy- , -dimethoxychalcone (ch ) potently inhibited no production (ic s, . - . mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position ( or ) to carbonyl moiety with/without -(or -)hydroxyl group and -halogen substitution in b-ring. when the cellular action mechanisms of ch , ch and ch were further examined, it was revealed that ch and ch clearly down-regulated inos expression while ch did not. moreover, ch and ch were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)- activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox- expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox- activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, cyclohexylimino- -methyl- , -dihydro- h-benzo [ , ] oxathiol- -one (lyr- ) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw . . the lyr- reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr- could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr- inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys- to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser- and to glu). therefore, lyr- could inhibit lps-induced nf-kb activating pathway by targeting ser- and/or residues on the activation domain of ikkbeta. as pharmacological actions, lyr- prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox- , or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw . . furthermore, lyr- protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline ( mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, nm, , and mm, increased guinea pig plasma dao activity by , , and %, respectively, while sertraline increased it at mm (by %). at higher concentrations ( and mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at and nm amitriptyline the activity of hnmt increased by and %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider( , ), c macarthur ( ), d trune ( ), s mccoy ( ) ( ) oregon health and science university, portland, oregon, usa ( ) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p , for treatment of inflammation induced by either lps or bacteria.peptide p was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p inhibited lps-induced production of serum inflammatory mediators.moreover, p treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p .simultaneous injection of bacteria and peptide p resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p treatment.-subcutaneous and oral administrations of p , but not intravenous administration, were also efficacious in reducing inflammation. administration of p after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. ( ), c zhou( ), y zhang( ), m sun( ), x wan ( ), h yu( ), x yang( ), rd ye ( ), j-k shen ( ) formyl peptide receptor-like (fprl ) is a structural homologue of fpr, which binds chemotactic peptides of as small as amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl ligands include peptides of - amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl -transfected cell line. although substitution of d-met resulted in loss of activity, removal of val together with d-met produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl ligand. further substitution of lys with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl through aromatic interactions involving the side chains of trp and tyr , hydrophobic interaction of nle , and the thio-based hydrogen bonding of met , with the respective residues in fprl which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl -related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il- , tnf-a and il- levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects ( , and mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses ( . and mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin ( and mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin . mg/ mouse/day in combination with glycine or mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. ( ), p sambrook( ), k fukudome( ), m xue ( ) ( ) university of sydney, st leonards, nsw, australia ( ) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr . cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein- -induced chemotaxis of monocytes by approximately %. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than % in ra monocytes. the inhibitory effects of apc were reversed by rcr , indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord- - ipj z authors: fung, joshua; lau, susanna k. p.; woo, patrick c. y. title: antigen capture enzyme-linked immunosorbent assay for detecting middle east respiratory syndrome coronavirus in humans date: - - journal: mers coronavirus doi: . / - - - - _ sha: doc_id: cord_uid: ipj z the middle east respiratory syndrome (mers) is the second novel zoonotic disease infecting humans caused by coronavirus (cov) in this century. to date, more than laboratory-confirmed human cases have been identified in countries, and more than mers-cov associated deaths have been reported since its outbreak in . rapid laboratory diagnosis of mers-cov is the key to successful containment and prevention of the spread of infection. though the gold standard for diagnosing mers-cov infection in humans is still nucleic acid amplification test (naat) of the up-e region, an antigen capture enzyme-linked immunosorbent assay (elisa) could also be of use for early diagnosis in less developed locations. in the present method, a step-by-step guide to perform a mers-cov nucleocapsid protein (np) capture elisa using two np-specific monoclonal antibodies is provided for readers to develop their in-house workflow or diagnostic kit for clinical use and for mass-screening project of animals (e.g., dromedaries and bats) to better understand the spread and evolution of the virus. the middle east respiratory syndrome (mers) is the second novel zoonotic disease infecting humans caused by coronavirus (cov) in this century. to date, more than laboratory-confirmed human cases have been identified in countries, and more than mers-cov associated deaths have been reported since its outbreak in [ ] . rapid laboratory diagnosis of mers-cov is the key to successful containment and prevention of the spread. nucleic acid amplification test (naat, e.g., real-time reverse transcription quantitative polymerase chain reaction [real-time rt-qpcr]), virus isolation, transmission electron microscopy, immunohistochemistry, and serological methods (e.g., antigen capture enzyme-linked immunosorbent assay [elisa] and immunofluorescence assay [ifa] ) have been developed and used for mers-cov diagnosis [ ] [ ] [ ] [ ] [ ] [ ] . while the "gold standard" for mers-cov diagnosis is naat of the upper region of the envelope gene (up-e) or the nucleocapsid (n) gene as suggested by the world health organization (who), antigen capture elisa assay for mers-cov can also be informative when naat is not available or when the serological assay is used to confirm the findings and aid treatment decision [ , ] . further to diagnosing possible human infection of mers-cov, this method is also useful for screening the virus in the wildlife or agricultural applications. government agencies and research groups may find serological tests like antigen capture elisa to be more economical than naats for routine screening of mers-cov in farm-held or city-dwelling animals. the antigen capture elisa described in this method offers four significant advantages over traditional naats. firstly, serological screening requires less space in facilities and can be performed in point-of-care locations to minimize sample transporting and reduce turnover time. to avoid crosscontamination from amplicons in naats, the workflow usually requires four separate physical locations: ( ) sample preparation (lysis, extraction of nucleic acids, and reverse transcription), ( ) naat master mix preparation, ( ) template addition, and ( ) amplification and analysis. though technologies like real-time rt-qpcr simplify the workflow, such requirements limit the assay to be performed in regional laboratories designed or designated for this application. antigen capture elisa, on the other hand, can be performed on open bench in a single location after virus inactivation, allowing it to be performed in even the most minimally designed facility. secondly, antigen capture elisa can be performed with simple equipment and can be established with limited initial investment. for performing naats at a modern standard, uv cabinets or workstations for master mix preparation and sample addition, thermal cyclers, agarose gel running, and visualization equipment are the least requirement. for more stringent testing and faster turnaround, it calls for a real-time pcr thermal cycler (e.g., roche's lightcycler systems or bio-rad touch detection systems) which requires a fair amount of initial investment and limits the assay from being performed in remote or less developed locations. in contrast, antigen capture elisa and other serological methods can be performed with much simpler equipment. multichannel pipettes, automatic plate washer, and plate reader are the only specialized tools needed for this application and can be purchased with ease if those are not already available. thirdly, much less training is required for technicians to handle serological testing than naats. though naats and elisa are some of the most basic assays performed in a medical laboratory and minimal training is needed for an experienced worker to perform such task, to allow quicker and broader surveillance of mers-cov in human and animal population, it would be beneficial to set up more surveillance facilities in the less developed parts of the world. the time and resources needed to train a novice laboratory worker to perform elisa are much less, as only dilution and pipetting skills are required. fourthly, common nucleic staining chemicals used in naats for amplicon visualization are a possible mutagen and post potential health risk to workers and the surrounding environment; while chemicals and solutions used in elisa are relatively safer. to visualize the amplicons after agarose gel electrophoresis or during the qpcr thermal cycles, dyes like ethidium bromide (etbr), sybr green, or gel red are used; while etbr is a known mutagen, others are a relatively new addition to the market and extensive safety data is not widely available [ ] . in comparison, the chemicals and solutions used in elisa are commonly found in clinical and research laboratories and are generally safe when used properly. finally, and most importantly, antigen capture elisa can offer high sensitivity and specificity for mers-cov diagnosis in even early infection and animal samples. we have previously demonstrated that by using two mers-cov nucleocapsid protein (np) specific monoclonal antibodies (mabs) in performing capture elisa, the test can accurately detect mers-cov virus down to tcid / . ml and has a specificity of % [ ] . as the nasopharyngeal aspirate viral load from patients during acute infection are around copies/ml and nasal samples in dromedaries are usually around - copies/ml, this test offers sufficient sensitivity for mer-cov diagnosis and screening [ ] [ ] [ ] . other forms of mers-cov serological diagnostic test have also been developed based on different principles and are designed to fulfill different purposes, one should also review those options and evaluate their needs. to detect seroconversion from previous infection of mers-cov, the who suggests laboratories to perform ifa or elisa together with neutralization assay, the result alone can be used to determine if it is a confirmed case, regardless of the results from naat assay [ ] . for rapid on-site diagnosis of mers-cov, we have previously reported the adaptation of the antigen capture elisa in the format of lateral flow immunoassay (lfia). this assay can yield results in under half an hour, requires minimal equipment, training, and can be stored at room temperature, thus allowing it to be performed in the field [ ] . this lfia is also able to detect mers-cov-like viruses (e.g., tylonycteris bat cov hku and pipistrellus bat cov hku ) and is useful for the research to understand the evolutional history of mers-cov [ , ] . in the current manuscript, the method for performing np capture elisa using two mers-cov-np-specific monoclonal antibodies (mabs) will be introduced. the general workflow of the assay is summarized in a figure for quick referencing [ , ] (fig. ). the antigen capture elisa is also known as sandwich elisa and makes use of a "capture" antibody and a "detection" antibody. the capture antibody is coated onto the wells of a microtiter plate before the assay. then following sample processing, the lysate is incubated in the wells of the microtiter plate. if the sample contains peptides from mers-cov (specifically nucleocapsid protein), they will bind with the coated antibody and be "captured" onto the microtiter plate. even minute amount of viral peptide can be retained in the well if the capture antibody has a high affinity to the peptide and was coated at high concentration. unbonded proteins are then washed away before the addition of the second, "detection" antibody. the secondary mab also recognizes the mers-cov np, presumably binds to a distinct epitope, and is conjugated with horseradish peroxidase for detection. the combination of two mabs in an elisa assay offers increased sensitivity for mers-cov np. on the other hand, this "sandwich" approach also allows improved specificity for the mers-cov nucleocapsid protein by combining the specificities of the two mabs, allowing it to differentiate and identify mers-cov spiked sample from other samples from healthy and patients who contracted various respiratory tract infections, as previously demonstrated [ ] . in this assay the nucleocapsid protein was selected as the target for generating antibodies to detect mers-cov. according to previous experience when working with sars-cov, we observed that the np is a highly immunogenic and abundantly expressed structural protein, and a more preferable target than the spike (s) protein [ , ] . working with the hypothesis that the np protein of mers-cov might also be a desirable target when developing an antigen capture elisa for it, we have shown that the assay offers high specificity and sensitivity, as mentioned above. the steps related to the cloning and purification of (his) -tagged recombinant np (rnp) of mers-cov for the generation of anti-mers-cov-rnp mabs will not be described, as there are commercially available antibodies readily available for purchase. the horseradish peroxidase (hrp) system was used for the colorimetric visualization at the final stage of the assay. commercial elisa kits may utilize other detection methods; optimization may be needed. for readers who would like to generate their own hrp conjugated detection antibody, there are also kits available. when preparing solutions and buffers for the assay, investigators should be aware that "old" buffers may be more likely to be contaminated. the accuracy and reproducibility of the assay can be affected, as the peptides from fungus or other microorganisms may compete with the target antigen. prepare fresh solutions periodically (~ month); autoclave or filter sterilize the buffers if available. if contaminations are a common occurrence, the addition of . % sodium azide (nan ) as a preservative is an option. microtiter plates with antibody . dilute the mers-cov-rnp mab f in blocking buffer. (see note ). . coat the microtiter plates by adding μl of the solution prepared per well. . cover the plate with an adhesive plastic cover and incubate at c overnight (see note ). . discard the adhesive plastic cover and remove the solution. . wash the plate with μl of washing buffer per well for five times using an automatic microplate washer. . dry the plate by patting the plate on a paper towel. . allow the plate to air-dry. proceed to the next step or cover the plate with adhesive plastic cover and store at c until use. all processes with potentially infectious mers-cov materials should be handled according to institutional, local, and international regulations, guidelines, and standard operating procedures (sop) to avoid spreading and contamination of the facility. all work with infectious mers-cov was performed inside a biosafety level- cabinet with sop in approved biosafety level- facilities during development and evaluation of the assay [ , , ]. . aliquot μl of viral lysis buffer to new . ml conical screw cap tubes according to the number of samples and controls (see note ). . pipette μl of specimen from the sample collection tube to the . ml conical screw cap tubes with viral lysis buffer, mix well. allow sufficient time for inactivation. . transfer the inactivated sample out of the biosafety cabinet to the general laboratory area, according to established sop. . there are many viral lysis buffers available for purchase from bio-reagents vendors, e.g., buffer al from qiagen. readers could request samples and perform their own testing on the conditions required to efficiently inactivate mers-cov. . tmb solutions are normally purchased from bio-reagents vendors at ready-to-use dilations, follow manufacturer's instructions. world health organization: world health organization, avenue appia laboratory testing for middle east respiratory syndrome coronavirus a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt are other fluorescent tags used instead of ethidium bromide safer clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia a highly specific rapid antigen detection assay for on-site diagnosis of mers rapid detection of mers coronavirus-like viruses in bats: pote n-tial for tracking mers coronavirus transmission and animal origin genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus differential diagnosis of pandemic (h n ) infection by detection of haemagglutinin with an enzyme-linked immunoassay sars coronavirus detection methods detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay differential cell line susceptibility to the emerging novel human betacoronavirus c emc/ : implications for disease pathogenesis and clinical manifestation cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc ( ) by both immunofluorescent and neutralizing antibody tests . serially dilute the inactivated sample in sample dilution buffer, add μl of the mixture into the wells in duplicates. . gently shake the plate for min to mix well, and then incubate at c for min while being covered with an adhesive plastic cover. . discard the adhesive plastic cover and remove the solution. . wash the plate with μl of washing buffer per well for five times using an automatic microtiter plate washer. . dilute the secondary detection antibody (mab c conjugated with hrp) in enzyme dilution buffer immediately before use. . add μl of the diluted detection antibody to each well using an -channel pipette. . cover the plate with an adhesive plastic cover and incubate at c for min. . discard the adhesive plastic cover and remove the solution. . wash the plate with μl of washing buffer per well for five times using an automatic microtiter plate washer. . add μl of , , , -tetramethylbenzidine (tmb) substrate solution to each well (see note ). . cover the plate with aluminum foil to protect from light, incubate for min at room temperature. . add μl of stop solution to each well to stop the reaction. . read the plate using an automatic plate reader at wavelength nm. . analyze the data by using the predetermined cutoff value. . an automated microtiter plate washer-dispenser would be a good addition to the workflow as the washing steps can be performed in a shorter amount of time and with greater consistency. but multichannel or even single-channel pipettes can be used instead. . the actual dilution of antibodies depends on the batch and quality of the mabs used. evaluations and characterization to determine the specificity and sensitivity have to be performed to establish the optimal dilution for the highest signal-to-noise ratio. . prevent the microtiter plates to dry up by placing the plates in a box with some moist tissue paper laying under while storing in an incubator or on a rack in a warm water bath. key: cord- -nrodyagi authors: schutzer, steven e. title: the use of host factors in microbial forensics date: - - journal: microbial forensics doi: . /b - - - - . - sha: doc_id: cord_uid: nrodyagi advances have been made in the forensic analysis of microbes and toxins. an underdeveloped and underutilized area in microbial forensics is how the host interacts with microorganisms in a way that provides unique signatures for forensic use. for forensic purposes, an immediate goal is to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection. principal methods that are sufficiently developed are characterization of the humoral immune response to microbial antigens including vaccine-induced immunity and detection of antibiotics that may be present in a possible perpetrator. this chapter presents central elements of the host response in a simplified fashion and describes a representative example, which, in the appropriate context, has a high potential of providing evidence that may aid an investigation to distinguish a perpetrator from a victim. this chapter also presents information about the immune system so that the interested reader can have a fuller understanding of the immune response in general. considerable advances have been made in the forensic analysis of microbes and toxins. these advances include sequencing, genomics, and microscopy. an underdeveloped and underutilized area in microbial forensics is how the host interacts with microorganisms in a way that provides unique signatures for forensic use. for forensic purposes, an immediate goal is to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection. two principal methods that are sufficiently developed are characterization of the humoral immune response and identification of vaccine-induced immunity or antibiotics that may be present in a possible perpetrator. this chapter presents central elements of the host response in a simplified fashion and describes a few representative examples, which, in the appropriate context, have a high potential of providing evidence that may aid an investigation to distinguish a perpetrator from a victim who has been exposed to a particular microbe or by-product, such as a toxin. this chapter also presents nonmicrobial forensicedirected information about the immune system so that the interested reader can have a fuller understanding of the immune response in general. the primary aims of a microbial forensics are to identify the biological agent, its source, and the individuals responsible for the event (budowle et al., ) . analytic approaches differ when the suspected biothreat agent is encountered in a container or the environment, as opposed to in vivo in a human, animal, or plant. analyses of trace elements, pollens, growth media, latent fingerprints, and microbial and nonmicrobial nucleic acids are all applicable to the container and environmental sample (states et al., ) . however, once the microorganism or its toxin is in the living host, it is no longer possible to analyze the preceding items except the microbial nucleic acid. however, the host's response to the biological agent may be available for analysis for clues. this is akin to other forensic studies where physical traces of bite marks, scratches, wound trajectories, and sizes of wounds are often surrogate evidence of the teeth, fingernails, and bullets (averill and odontology, ) . while the forensic pathologist is familiar with evidence related to determining the manner of death including the host response, those involved with healthcare alone are more familiar with the host response. in the context of microbial forensics, it is important to integrate all of these with intelligence information so that they may be included in the analytical data and attribution picture. the physician and other healthcare providers may be among the first to realize that a patient is a victim of a biocrime. in the case of a covert attack, it may be the physician or medical examiner who first recognizes the index case. these healthcare workers are in key positions to preserve critical evidence and, thereby, contribute to the investigation (schutzer et al., ) . there are a number of steps that should be followed when the possibility of a biological attack arises, either with the consent of the patient or because individuals are compelled by law to interact with public health and law enforcement. a joint statement by the fbi, the cdc, and the dhs advises calling the fbi and public health authorities if a suspicious situation arises (investigation et al.) . some guidelines on the procedure(s) to report of suspicions of biocrimes are provided by the centers for disease control and prevention (cdc; http://www.cdc.gov), the federal bureau of investigation (fbi; http://www.fbi.gov), and the department of homeland security (dhs; http://www.dhs. gov) and detailed in previous article (schutzer et al., ) . the host response to a microorganism or other foreign substance is often a wellorchestrated series of events, which may protect the individual from harm (zabriskie, ). at the same time, these host responses may provide clues as to identity of the offending microorganism or toxin as well as a rough chronology of when it occurred and for how long it has been persisting. emerging technologies such as transcriptional arrays and bioinformatic analysis will eventually be refined and methods validated to provide even greater help in delineating more of the pathways and components of the host response to an infectious agent (sala et al., ; popper et al., ; ko et al., ) . other technologies are sufficiently mature to be of use today. the immune system and its components are a mainstay of our protection against infections and malignancies (zabriskie, ; paul, ; murphy and weaver, ) . inflammation is often a side effect as the immune system contains and eradicates a microorganism or eliminates foreign tissue. specific arms of the immune system can be used as markers in support of or against the presence of an infection. the humoral or antibody response to an invading microorganism is one example of a specific arm that can have forensic value. some of the antibodies produced may have a protective role together with other parts of the immune system by eradicating the pathogen or neutralizing a toxin. other antibodies may not be as effective in this role. however, in their ability to recognize unique and specific microbial antigens, they can serve as indicators that a specific microorganism was recently present or was present in the past. in the case of a vaccine, specific antibodies may recognize highly specific epitopes of one microbe versus those of a related microbe (e.g., influenza virus). this is especially so with different recombinant vaccines and could have forensic importance. substances such as antibiotics, which can rapidly kill a pathogen, may modify the immune response by removing or reducing the infectious driving force for a full-scale response. as noted above, in clinical and veterinary medicine, measurement of the immune response helps the diagnostician decide what infection was present and how recently. in these situations, the intent is to provide treatment. the forensic scientist may exploit parts of the immune response to discover who is likely a victim of an attack and who might be responsible. this chapter will discuss the basics of the host immune response in a simplified manner that can have utility in a forensic sense. examples will provide a sense of what information is achievable and what is . the use of host factors in microbial forensics not likely to provide clues with a high degree of certainty. in response to a new exposure to a microbe, the innate immune system may be the first line of defense. then the immune system starts to activate the antibody system. typically, a cell known as a macrophage ingests and degrades some of the invading pathogens. it then presents part (antigens) of the microorganism to a helper t cell (a lymphocyte), which then directs other lymphocytes known as b cells to produce antibodies to those antigens of that particular microbe that were presented. it usually takes at least days before any microbe-specific antibody can be detected (parslow, ) . antibodies are a specific form of the proteins known as immunoglobulins (igs). igm, igg, iga, and secretory iga are the principal classes of immunoglobulins with prime relevance to this chapter and will be discussed in more detail. in an infection, immunoglobulins usually appear in the order of igm, igg, and iga. b cells first begin to produce igm, and then some b cells undergo an irreversible switch to those that produce igg. later, some of these b cells undergo a switch to become iga-secreting b cells. immunoglobulins persist for varying times; for example, the half-life of particular igm antibodies is approximately days, while that of igg can be as long as e days (table . ) (paul, ; murphy and weaver, ) . in certain circumstances of ruling in or ruling out a suspect, the specific ige may be of value. those individuals unfortunate to have allergies have problems due to ige against allergens (such as ragweed, peanut, or cat dander). in this case, the ige molecules sit on the surface of mast cells and basophils. these cells can release histamine and other allergic mediators when the offending allergen bridges two ige molecules. similar to the effect from an infection with a live microbe, vaccines are often designed to provoke an antibody response. the vaccine can be composed of a live or attenuated microbe, a whole nonproliferating microbe, or an antigenic (recombinant) part of the microbe or a toxoid. regardless, the intent of immunization with a vaccine is to engender protection, often by the generation of protective neutralizing antibodies. although the half-life of an individual igg molecule is less than a month, a population of antibodies of the igg isotype form may persist for life. memory b cells can sustain these antibodies and retain the ability to quickly generate the appropriate antibodies when challenged. when the immune system encounters another infection or is subjected to a revaccination (booster), the result is an accelerated production of the particular antibody and increase in the levels of antibodies that circulate in the blood ( fig. . ) . perhaps, the pattern of antibody response which has the most forensic value, by providing a timeframe, is the appearance of igm first, followed by a b-cell switch to the longer-lasting igg. during the early phase of exposure, igm predominates, as time goes on, igg may wax and wane and igm is no longer found ( fig. . ) . the antibody response to a particular agent may be directed to different antigens at different times, that is, early or later after the initial exposure. that response often involves igm at the early stage and igg later. late in the course of (bennett et al., ) , a virus known to cause mononucleosis. during acute early disease, it is common to find high levels of antibody of the igm isotype to the viral early antigen (ea) and viral capsid antigen (vca). it is rare to find igg antibody to the vca or to epsteinebarr nuclear acid (ebna) in anything but low titers (levels). as the patient recovers from their first infection with ebv, it is rare to find anything but low levels of igm to ea or vca, but igg to vca in higher or increasing levels is common. antibodies to ebna are often very low during this stage. several months after clinical recovery, igm to ea and vca remain at low levels, whereas igg to vca and ebna are present at high levels, often for years. a controlled experiment or normal clinical event illustrates what happens when the immune system responds to the infectious agent or a vaccine again. the controlled experiment may be in a laboratory animal or a patient illustrative concepts receiving a booster vaccine. the uncontrolled but normal clinical event occurs when the patient is reexposed to the infectious agent. consider a generic antigen exposure. the first time the immune system encounters antigen x (agx), it responds as shown in figs. . and . . initially, antibodies to agx are barely discernible; then levels rise and later fall to a plateau. if a simultaneous exposure were to occur with agx and a new agy from another microorganism, the immune system would quickly mount a brisk response with high levels of ab to agx, while the course of ab to agy would be slow and delayed, just as it was in the response to the first exposure to agx. this phenomenon is termed immunological memory or an amnestic response. this can be useful when the symptoms and signs of exposure to either x or y are similar. this is the case with the early flu-like symptoms of pulmonary anthrax (raymond et al., ; waterer and robertson, ; bush et al., ) and with the influenza virus itself (meltzer et al., ; cao et al., ; lessler et al., ) . another example common to all of us is repetitive exposure to different strains of flu viruses (meltzer et al., ; janeway, ) . as illustrated in table . , a person infected for the first time with one strain of the influenza virus has a response to most of its antigens (as a theoretical example, ag , , , , , ). three years later, the same individual exposed to a partially similar influenza virus responds preferentially to those antigens that were also present on the original influenza virus. the person also makes a smaller initial antibody response to new antigens, that is, those not shared with the first virus. ten or years later, during a new flu season and exposure to a third strain of influenza, the most brisk responses would be to antigens previously recognized by the immune system. this is the scientific basis for giving the flu vaccine, which contains a variety of possible antigens common to multiple strains of the flu virus so that a rapid and protective antibody response will occur. utility of serologic analysis of people exposed to anthrax: strengths and limitations our knowledge of the humoral response to infection with biothreat microbes is limited compared with our knowledge of the kinetic response to common human infections. nevertheless, in the appropriate context and with sufficient background information, detection of antibodies to a particular microbe and its antigens can have important value for a microbial forensic investigation. this information may have critical probative value or it can guide investigative leads. the absence of a specific antibody response may also have value in a particular investigation. certainly, its importance is increased in the context of information of what organism could be involved, when the exposure was likely to have occurred, the route of exposure, what symptoms and signs are manifesting in the host, and other data such as presence of antigens and microbial nucleic acids (jackson et al., ) . other information such as how many hosts (people or animals) have had this infection in the geographic region, what is the incidence, and background prevalence of antibody titer to the organism in question or a related organism, in the population being studied, is also important. vaccination responses can have forensic value. the current protective antigen (pa) vaccine has small amounts of ef and lethal factor (lf), which are responsible for some of the side effects, so one might expect to see antibodies against these antigens as well as to pa. the recombinant pa is just pa so anti-lf and anti-ef would be absent in an immunized individual. the anthrax-letter attacks raised multiple questions for every person infected, possibly exposed, vaccinated, or treated. some of these questions included how these persons were infected by spores, if at all; that is, through breaks in the skin (cutaneous anthrax); by inhalation of spores (pulmonary anthrax (bennett et al., ) ), or by ingestion (gastrointestinal anthrax (bravata et al., ; tutrone et al., ) ). or, were they among the "worried well"? consider the situation where a close associate comes down with symptoms compatible with inhalational anthrax after receiving a letter containing powder and that material is no longer available. until this is shown not to be anthrax, great worry will ensue. in several cases of documented exposure, there was not enough time for the patient to develop antibody to a specific anthrax antigen, at least as probed for igg. serial serum samples obtained on november , , , and of were tested for igg antibody to the pa component of the anthrax toxins by enzymelinked immunosorbent assay (elisa); all samples were nonreactive. serial tests for serum igg antibody to the pa toxin of anthrax were performed on workplace-exposed persons. all but one test was negative. most of the specimens were collected on october and (traeger et al., ) . it is instructive to look at the positive antibody case in the context of the nature and utility of serologic analysis of people exposed to anthrax: strengths and limitations duration of that individual's symptoms when he developed a positive test. ernesto blanco, a -year-old mailroom clerk (case ), experienced fatigue on september . he worked in the mailroom of the ami building and delivered mail to the index case. on september , he developed a nonproductive cough, intermittent fever, runny nose, and conjunctivitis. these signs worsened through october when he was hospitalized. in addition, he had shortness of breath with exertion, sweats, mild abdominal pain and vomiting, and episodes of confusion. his temperature was elevated to . c ( . f), heart rate was rapid at /min, respiratory rate was slightly fast at /min, and blood pressure was / mm hg. he had bilateral conjunctival injection and bilateral pulmonary rhonchi. at the time of admission, his neurologic exam was normal. no skin lesions were observed. the only laboratory abnormalities were low albumin, elevated liver transaminases, borderline low serum sodium, increased creatinine, and low oxygen content in the blood. blood cultures were negative on hospital day , after antibiotics had been started. the chest x-ray showed a leftsided pneumonia and a small left pleural effusion but no "classical" mediastinal widening (dewan et al., ) . the patient was initially given intravenous azithromycin; cefotaxime and ciprofloxacin were subsequently added. a nasal swab obtained on october grew bacillus anthracis on culture. computed tomography (ct) of the chest showed bilateral effusions and multilobar pulmonary consolidation but still no significant mediastinal lymphadenopathy. pleural fluid aspiration was positive for b. anthracis dna by pcr. bacterial cultures of bronchial washings and pleural fluid were negative. immunohistochemical staining of a transbronchial biopsy demonstrated the presence of b. anthracis capsule and cell wall antigens. during hospitalization, his white blood count rose to , /mm , and fluid from a second thoracentesis was positive for b. anthracis dna by pcr. immunohistochemical staining of both pleural fluid cells and pleural biopsy tissue demonstrated the presence of b. anthracis capsule and cell wall antigens. serial serum samples demonstrated > fourfold rise in serum igg antibody to the pa component of the anthrax toxins by an elisa assay. the patient was able to leave the hospital on october on oral ciprofloxacin. table . illustrates both the clinical and microbial forensic approach and context in which to analyze such a patient. it is likely to be common to most situations where a biocrime ( ). these questions include was the infection acquired naturally or was it an intentional action that led to the infection; how did the particular individual acquire it if it was not a natural infectiondwas he the target or a bystander. an alternative possibility in the right circumstances is a laboratory-acquired infection. this case also demonstrates that cultures may be negative at different times from different fluids and tissues because of early administration of antibiotics. however, the remnants of the infection, even dead organisms, can be found by probing for antigens and dna. this patient's response demonstrated a classic principle of infectious disease, a rising antibody titer over time. in this case, it was igg to a particular antigenic component of the anthrax toxins (friedlander and little, ; cunningham et al., ) . the subject's antibody response may have been detected earlier if igm to this component or to other antigens of anthrax had been sought. the case also points out the utility of integrating the presence of antibody with other indications of an anthrax infection such as culture, pcr, and antigen detection. these take on their greatest significance during clinical illness in someone who was possibly exposed. early administration of antibiotics can prevent or interfere with the isolation of a pathogen by culture (kaeberlein et al., ) . of the first pulmonary anthrax cases associated with the letter attacks, three patients had no isolate of b. anthracis from any clinical samples; however, culture was attempted after initiation of antibiotic therapy. history of exposure in conjunction with compatible symptoms and signs of disease and objective laboratory findings were the basis for the diagnosis. b. anthracis was identified in pleural fluid, pleural biopsy, or transbronchial biopsy specimens by reactivity with b. anthracis-specific cell wall and capsular antibodies or by the detection of dna in pleural fluid or blood by pcr (jernigan et al., ) . it is important to understand the limitations of any assay used in medicine or forensics (budowle et al., ; schutzer et al., ). an igg-based elisa for anti-pa illustrates the importance of understanding the limitations of an assay. the elisa was developed at the us army medical research institute of infectious disease (usamriid) and put into operation after optimization and internal validation at the cdc for functional sensitivity and specificity in detecting an antibody response to b. anthracis infection. its major limitation was that only one antigen was used and only igg was measured. therefore, a negative result shortly after exposure may, in effect, be a false-negative result. a gap such as this may be filled by development of an assay for antigenspecific igm, and by probing for other b. anthracis antigens or epitopes yet to be characterized. the assay may be very useful in its present form to screen asymptomatic people with possible exposure. the study by dewan et al. (dewan et al., ) provide a contemporary background database on a group of postal workers who may have been exposed to b. anthracis. beginning on october , , postal employees and others who had been to the washington d.c. postal facility went to the d.c. general hospital for antibiotics in addition to those people whose treatment began on october , . serum samples were also obtained from the individuals who had been to the washington d.c. postal facility during the precious weeks. all were negative for specific anti-pa igg, including three individuals who reported a remote history of anthrax vaccination. the consistent negative findings may be explained by the fact that antibiotic therapy was initiated before serum testing and that there were no baseline serum samples available for testing. in addition, the time period from exposure to sampling was very short. among individuals in the capitol region with culture-positive nasal swabs who received prophylactic antibiotics immediately, none had a positive culture from a nasal swab repeated days later, and none developed igg to pa antigen days after exposure. this again emphasizes the limitation and interpretation of a test in someone who had early antibiotic treatment. it does raise forensic utility considerations. even with these easily disseminated spores, an antibody response may be aborted or modified with antibiotics by early eradication. furthermore, antibiotics taken before exposure would likely be effective in preventing laboratory and clinical signs of an infection. detection of microbial dna, antigen, or the organism itself on a person's body, clothing, or possessions should raise a red flag for exposure. the route of infection is important in interpreting results and the limitations of the assay used. the example of cutaneous anthrax in paraguay illustrates this notion, as well as the need to search for other antigens as markers of exposure (harrison et al., ) . in an analysis of an outbreak of cases of cutaneous anthrax that followed contact with raw meat from a sick cow, sera from cases and colony and noncolony controls were examined by western blot for antibodies to pa and lf weeks after the outbreak. an elisa was used to probe for antibodies to the poly-d-glutamic acid capsule. of the cases, had antibody to pa, for a sensitivity of . %; none of the controls was positive. only of cases had antibody to lf; all controls were negative. anticapsule antibodies were positive in of but were also positive in of controls. the results of this study demonstrate the need to consider other antigens. some of the principles discussed above are highlighted by a report on severe acute respiratory syndrome (sars). the appearance alone of this coronavirus responsible for this disease evoked concern of a possible terrorist origin at the onset. a report in the morbidity and mortality weekly report (mmwr (cfdcap, ) ) on the "prevalence of igg antibody to sars-associated coronavirus in animal traders" discussed the need to validate and interpret tests in appropriate populations. also discussed was the inability to date the time of infection by the igg assay, and the possibility of assay crossreactivity to a near neighbor that might be unknown. in a promed bulletin, dr. steve berger looked at the same data from a different perspective and reported "this week's study in mmwr indicates that animal contact may indeed promote infection; however, the most striking finding seems to have eluded the authors: . percent to . percent of individuals in a healthy control group of adults were also found to be seropositive! the population of guangdong province is . million ( ), of whom . million are adults over age . if we assume that the seropositivity rates among controls is representative of the province as a whole, , to , , adults in guangdong have at some time been infected by the sars virus. these figures are -to -fold the total number ( ) of sars patients reported worldwide to date!" this comparison is a good illustration of the advantage of open dissemination and discussion of information as well as the need to question the methodology of acquisition of data before accepting their application in formulas or for analyses for forensics and epidemiology. it is also of value to remember that many infections include many with sars coronavirus have been asymptomatic or mildly symptomatic. plague, is a zoonotic infection caused by yersinia pestis, which occurs in the western united states with regularity and has an animal reservoir (bennett et al., ) . the situation with the naturally occurring yersinia is in contrast to the appearance of a case of smallpox which would raise an immediate red flag for a bioterrorist event. cases need to be approached from an epidemiologic standpoint first to determine whether it is a naturally acquired case or whether the facts point to a deliberate introduction of the organism. analytic techniques could include genomic analysis of an isolated organism and immunological response of the host. in the new era of rapid and deep sequencing, our capacity to investigate the genomics is growing (mardis, ; stavnsbjerg et al., ) . in consideration of animal reservoirs, elisa assays were compared with other tests for detection of plague antibody and antigen in multimammate mice (mastomys coucha and. mastomys natalensis) (shepherd et al., ) , which were experimentally infected and then sacrificed at daily intervals. igg elisa was equivalent in sensitivity to passive hemagglutination and more sensitive than the igm elisa and complement fixation. antibody was detectable by day after infection using all four tests. igm elisa titers fell to undetectable levels after weeks. plague fraction antigen was detected in of bacteremic sera from m. coucha and m. natalensis. this antibody pattern comparison shows that the principle of igm versus igg to this pathogen works to temporally situate the infection as an early versus late or past event. it also shows that when the information is combined with antigen detection, it engenders more confidence in the results. it should be noted that conclusions from this older reference has been substantiated with more defined antigens and assay technologies. melioidosis is caused by burkholderia pseudomallei (ashdown, ) . key clinical signs and laboratory results may raise the possibility of an infection with this pathogen. whether it is an acute, persistent, or past infection can be determined by assessing several host responses. often a simple indicator such as erythrocyte sedimentation rate or c-reactive protein (crp) can raise a clinical suspicion of an infection. in a study of patients with clinical melioidosis, ( culture-positive and culturenegative) had relatively uneventful disease courses. initially, they had elevated serum crp that decreased with antibiotic therapy and returned to normal as the disease resolved. in another series of patients, igm and igg were measured by elisa in sera from septicemic cases and sera from cases with localized melioidosis (chenthamarakshan et al., ) . sixty-five sera from culture-negative cases seronegative for other endemic infections but suspected of melioidosis were also examined. other controls included serum from nonmelioidosis cases, high-exposure risk cases, and healthy individuals. the igg-elisa was % sensitive and % specific. all sera from cases with septicemic and localized infections and of sera from clinically suspected melioidosis cases were positive for igg antibody. the sensitivity and specificity of the igm elisa were % and %, respectively. a geometric antibody index for igm antibody in the sera of the melioidosis cases was significantly higher in cases compared with that of the noncase controls. in another study by some of the same authors, a rapid test for igg and igm was shown to have clinical utility (cuzzubbo et al., ) . a study with the intent of evaluating the utility of an igg assay compared with other assays illustrates how the clinical and temporal context must be integrated for interpretation (dharakul et al., ) . it also illustrates how there is room for technical improvement in tests but the best setting is often the endemic area itself or at least using samples from that area in which the infections are occurring. these tests were evaluated in the actual clinical setting in an area endemic for melioidosis. specificity of igg ( . %) and igm ( . %) assays was significantly better than that of an indirect hemagglutination test (iha) ( . %). the sensitivity of the igg assay ( . %) was higher than that of the iha test ( . %) and the igm test ( . %). specific igg was found in septicemic cases ( . %) and localized infections ( . %). the igg test was also better than the igm test and the iha test in identifying acute melioidosis cases in the first days after admission. igg antibody to a b. pseudomallei antigen remained high for longer than years in recovered, disease-free patients. because this is a disease that may have an incubation of days to years, an acute case may very well be detected by a rise in specific igm if it were a matter of days from infection. although endemic for southeast asia, if b. pseudomallei was used as a biothreat agent in a different environment, its course and manifestations may not be recognized due to unfamiliarity with the disease. the above example also points out how the context in which a test is used determine is valuable. the concept of predictive value is instructive in determining how useful a test may be. in terms of disease detection, a high positive predictive value indicates the test is useful in determining that the disease is present. a high negativity predictive value would indicate that the test is useful in excluding the presence of the disease. another zoonotic agent is rift valley fever virus (rvfv), which can be transmitted via aerosols (clark et al., ) . one study with the intent at looking for improved tests showed the utility of igm to determine an early exposure to rvfv (niklasson et al., ) . two elisa igm tests detected specific igm antibodies to rvfv during the first weeks after vaccination. three inactivated vaccine doses were given on days , to , and to . elisa serum igm on days e were negative or in the lower range of detection; on days e the serum antibody values were strongly positive; on days e , they were waning and in later collected samples were negative. the plaque reduction neutralization test was negative on days e and became positive in later samples. similar to the examples shown above, these data suggest that three doses of rvfv vaccine induced a prolonged primary antibody response. the authors of that study concluded that the elisa igm may be useful for early diagnosis of acute human infection. good correlation of a neutralization test and elisa igg would indicate a later infection. taken together, these examples illustrate that an ideal test or analysis for both clinical and forensic use would incorporate endemic and incident area controls, historical contextual information, knowledge of the route of exposure, background incidence, and kinetics of transmission. each of these scenarios must take into account multiple factors and the limitations of any analytic process to be applied. on one extreme is the situation that occurred with the onset of acquired immunodeficiency syndrome (aids) from the human immune deficiency virus (hiv) in the united states. initially, there were no cases, and therefore a precise highly sensitive and specific test with excellent positive and negative predictive values (such as exists now when a combination of tests are used) would not likely yield a positive result in an area where there was little hiv infection and disease at the onset such as kansas. a positive test by today's methodologies from a serum sample from kansas would be considered a probable falsepositive and warrant further investigation. today, several viral and nucleic acid assays are available that would provide a definitive diagnosis in a short period of time (bennett et al., ) . however the same sample tested at the beginning of hiv testing could have been positive if the person had adult t-cell leukemia, which is caused by human t-cell leukemia virus- (htlv- ) because the original tests for what became known as aids involved whole viral lysates in which up to % of the htlv- sera cross-reacted. questions regarding the interpretation of the test results could be raised by knowledge of different presentations of the infection. for example, htlv- can actually be used in the laboratory to immortalize cells. in the patient, it actually increases the t-cell count, as is the nature with leukemia, instead of decreasing them, as with hiv infections. other laboratory indicators such as hypercalcemia would now raise the leukemia as a consideration. interpretation of a positive laboratory test must also take into account the health status of the person being tested. this is important for the practice of medicine and can have relevance when extended to forensic analysis (schutzer et al., ) . the following examples illustrate this concept. individuals who have syphilis, a treponemal bacterial infection, can typically have a positive fluorescent treponemal antibody test result for years, even after successful treatment. however, while infected they would have a positive venereal disease research laboratory (vdrl) test, which reverts to negative following successful antibiotic therapy. the vdrl test detects nonspecific anticardiolipid antibodies and can produce false-positive results with other conditions (e.g., pregnancy). there are some notable exceptions related to crossreactive epitopes or autoimmune diseases. these are readily distinguishable by history and clinical information. similarly, individuals infected with active tuberculosis will likely have a positive skin test (mantoux) or a positive interferon-gamma release assay (dewan et al., ; ota and kato, ) , whereas the uninfected healthy person will be negative. in certain instances, a sick person with a cell-mediated immune deficiency will be anergic, that is, he/she will be negative to multiple skin tests including common antigens such as candida. the key difference here is that there is a great difference between the healthy person being tested and an ill or immunocompromised individual being subjected to the same test. tests may also discriminate between the length of the infection (i.e., acute or chronic); limitations of these tests may lead to different interpretations unless one is familiar with those limitations. an example of this occurred with the bacterial infection of borrelia burgdorferi, which causes lyme disease. antibiotics can abrogate the antibody response because elisa results were negative in % of patients with known disease who were treated early (dattwyler et al., ) . in early cases, reactivity to a unique antigen, ospa, was also negative in serological assays despite a demonstrable t-cell possible scenarios of bioterrorism attacks: distinguishing victims from perpetrators response (krause et al., ) . analysis of these same sera found that there was antibody to b. burgdorferi, but it was below the threshold of detection by conventional assays. it was detectable in its bound form, in immune complexes (schutzer et al., ; schutzer and coyle, ) . anthrax can be used as an example where investigatory leads can be generated by considering a scenario in toto. the elderly woman who died in connecticut from inhalation of anthrax clearly had no occupational exposure nor was she known to have had contact with anyone who had anthrax. it was possible that she had contact with cross-contaminated mail. however, if this case had occurred as the index case or out of context of the mail attacks, it would have been reasonable to question her travel history, what her work if any was, or if she received or used spore-contaminated products from an anthrax endemic area. similarly, the vietnamese woman who died of inhalation anthrax in new york city would also have had these questions investigated. it would have been useful to search for direct or indirect evidence of anthrax by physical examinations of her contacts or close neighbors. inspection and cultures from her workplace, apartment, and apartment complex (especially contiguous neighbors) are important for detecting the presence of b. anthracis. coworkers, friends, neighbors, and other contacts could have had their serum analyzed for antibody to antigens of b. anthracis. these samples could have been frozen so that if one were positive it would be available for a comparison study in the future. at a minimum, these types of studies could serve as future control data for the geographic region. with molecular methods, even trace amounts might be detectable (lasken and egholm, ) although parallel investigation as to background control would be necessary. although hypothetical, several results could have occurred, and each will be considered separately. first example, a close contact is positive for igm to one of the b. anthracis antigens, such as pa. this finding would suggest that this person had recent exposure and, if nothing else, should be treated. this individual could conceivably be the one who knowingly or unknowingly passed the spores to the patient. given the october onset of illness, which is late in the mailing sequence, it would be less likely that this individual was a perpetrator but rather a recent victim. however, if this person were igg-positive, then there are several other possibilities. perhaps, this person had past exposure in an endemic region and was treated (e.g., haiti, where anthrax is known as "charcoal disease"). or this person could have been vaccinated for bona fide reasons such as a researcher who received it for occupational exposure. or this person could have obtained the vaccine originally for legitimate or illegal purposes but was nevertheless vaccinated. the vaccine usage may have been for a clinical trial or animal experimentation. animal vaccines may be more obtainable without strict record keeping. this person could have loaded the mail with relative impunity if there was protective antibody generated from the vaccination. situations similar to this one will require intelligence information regarding access, ability, and motive. in an area where recombinant vaccines are being developed or used antibody response would be different between someone using one type of recombinant vaccine as compared with someone using another type of vaccine. nevertheless, finding igg to one or more antigens of b. anthracis could point investigators toward such a seropositive individual, whereas an igm finding could justify critical therapy. where information points to a particular individual, investigation could be extended to search for ingestion or injection of antibiotics as illustrated below in the ciprofloxacin example. questions would be raised regarding access to antibiotics, recent ingestion/injection of them, half-life of the antibiotic, half-life of the metabolites of the antibiotics, and in which body fluids or tissues can the residual be found. as illustrated from . the use of host factors in microbial forensics the data in the earlier sections, someone with antibiotics in their system may be protected following exposure to a potential pathogen. this person would be antibody-negative and likely antigen-and microbial dna/rnanegative, because the infection would have been eradicated before the organism can proliferate in any significant quantity. the widespread prophylactic use of ciprofloxacin during the period following the anthrax mailing attacks is illustrative of an understudied area. ciprofloxacin has been increasingly associated with tendonitis and ruptured achilles tendons (akali and niranjan, ; palin and gough, ; godoy-santos et al., ) . in the future, better methodology to follow the pharmacokinetics of an antiinfective compound may have forensic implications. in the last example, someone who takes an antibiotic prophylactically while manipulating a lethal microbe may exhibit side effects that in the proper context of an investigation may add to the picture of possible culpability. this area is far from established at this point in time. strategies can be employed to examine suspicious but possible accidental transmission of infections. this approach is illustrated by a recent study of avian influenza using a multitude of assays. tools to determine person-to-person spread as the mode of transmission included viral culture, serologic analysis, immunohistochemical assay, reverse transcriptasee polymerase chain reaction (rt-pcr) analysis, and genetic sequencing (ungchusak et al., ; meinel et al., ) . it is likely that future understanding of the immune system and evolving technologies will bring new analytic power to the field, but in the interim we can make good use of proven principles for forensic purposes. serial serum c-reactive protein levels as an aid to the management of melioidosis management of bilateral achilles tendon rupture associated with ciprofloxacin: a review and case presentation manual of forensic odontology mandell, douglas, and bennett's principles and practice of infectious diseases inhalational, gastrointestinal, and cutaneous anthrax in children: a systematic review of cases index case of fatal inhalational anthrax due to bioterrorism in the united states clinical features of the initial cases of pandemic influenza a (h n ) virus infection in china detection of immunoglobulins m and g using culture filtrate antigen of burkholderia pseudomallei systematic literature review of rift valley fever virus seroprevalence in livestock, wildlife and humans in africa from mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen evaluation of a new commercially available immunoglobulin m and immunoglobulin g immunochromatographic test for diagnosis of melioidosis infection seronegative lyme disease. dissociation of specific t-and b-lymphocyte responses to borrelia burgdorferi inhalational anthrax outbreak among postal workers diagnostic value of an antibody enzyme-linked immunosorbent assay using affinitypurified antigen in an area endemic for melioidosis advances in the development of next-generation anthrax vaccines fluoroquinolones and the risk of achilles tendon disorders: update on a neglected complication evaluation of serologic tests for diagnosis of anthrax after an outbreak of cutaneous anthrax in paraguay pcr analysis of tissue samples from the sverdlovsk anthrax victims: the presence of multiple bacillus anthracis strains in different victims immunobiology the immune system in health and disease bioterrorism-related inhalational anthrax: the first cases reported in the united states isolating "uncultivable" microorganisms in pure culture in a simulated natural environment what was old is new again: using the host response to diagnose infectious disease cellular immune reactivity to recombinant ospa and flagellin from borrelia burgdorferi in patients with lyme borreliosis. complexity of humoral and cellular immune responses whole genome amplification: abundant supplies of dna from precious samples or clinical specimens outbreak of pandemic influenza a (h n ) at a new york city school next-generation dna sequencing methods whole genome sequencing identifies influenza a h n transmission and offers superior resolution to classical typing methods laboratory surge capacity and pandemic influenza janeway's immunobiology detection of human immunoglobulins g and m antibodies to rift valley fever virus by enzymelinked immunosorbent assay risk of tuberculosis among air passengers estimated by interferon gamma release assay: survey of contact investigations rupture of the achilles tendon associated with ciprofloxacin medical immunology, tenth ed. lange medical books/mcgraw-hill medical pub. division fundamental immunology, sixth ed. wolters kluwer health gene transcript abundance profiles distinguish kawasaki disease from adenovirus infection specific, sensitive, and quantitative enzyme-linked immunosorbent assay for anthrax lethal toxin impairs il- expression in epithelial cells through inhibition of histone h modification dissecting regulatory networks in host-pathogen interaction using chip-onchip technology sequestration of antibody to borrelia burgdorferi in immune complexes in seronegative lyme disease biocrimes, microbial forensics, and the physician use of forensic methods under exigent circumstances without full validation comparative tests for detection of plague antigen and antibody in experimentally infected wild rodents handbook of forensic services comparison of two commercial broad-range pcr and sequencing assays for identification of bacteria in culture-negative clinical samples first case of bioterrorism-related inhalational anthrax in the united states cutaneous anthrax: a concise review probable person-to-person transmission of avian influenza a (h n ) bioterrorism for the respiratory physician essential clinical immunology key: cord- - kwny authors: mariani, stefano; minunni, maria title: surface plasmon resonance applications in clinical analysis date: - - journal: anal bioanal chem doi: . /s - - - sha: doc_id: cord_uid: kwny in the last years, surface plasmon resonance (spr) and its advancement with imaging (spri) emerged as a suitable and reliable platform in clinical analysis for label-free, sensitive, and real-time monitoring of biomolecular interactions. thus, we report in this review the state of the art of clinical target detection with spr-based biosensors in complex matrices (e.g., serum, saliva, blood, and urine) as well as in standard solution when innovative approaches or advanced instrumentations were employed for improved detection. the principles of spr-based biosensors are summarized first, focusing on the physical properties of the transducer, on the assays design, on the immobilization chemistry, and on new trends for implementing system analytical performances (e.g., coupling with nanoparticles (nps). then we critically review the detection of analytes of interest in molecular diagnostics, such as hormones (relevant also for anti-doping control) and biomarkers of interest in inflammatory, cancer, and heart failure diseases. antibody detection is reported in relation to immune disorder diagnostics. subsequently, nucleic acid targets are considered for revealing genetic diseases (e.g., point mutation and single nucleotides polymorphism, snps) as well as new emerging clinical markers (microrna) and for pathogen detection. finally, examples of pathogen detection by immunosensing were also analyzed. a parallel comparison with the reference methods was duly made, indicating the progress brought about by spr technologies in clinical routine analysis. surface plasmon resonance (spr) appeared as revolutionary technology almost years ago, when the first commercial instrumentation was launched on the market by pharmacia biosensors ab, a swedish company, derived from pharmacia ab. the company developed an innovative technology by the joint effort of physicists, chemists, biologist, engineers, and computer scientists. since then, many scientists joined the spr to test new applications in various analytical fields, such as food safety [ ] (e.g., mycotoxins [ ] , genetically modified organism (gmo) [ ] ), microbial contamination such as escherichia coli [ ] , doping analysis [ ] , laboratory medicine [ , ] , proteomics [ , ] , bacteria detection [ ] , and also environmental monitoring [ ] . among them, assuredly, clinical analysis has also been explored as a fruitful application field. the advantages brought about by current spr technology include real-time monitoring of the analyte/molecular markers, label free and parallel analysis (with spri), minimal sample pretreatment, quantitative response, and very good sensitivity and reproducibility, (reported detection limits are in atto-or femtomolar ranges and coefficient of variations below %). these features, coupled to miniaturization, make spr suitable for point of care (poc) diagnostics [ ] , where fast analysis and multi-analyte detection are mandatory. in this review, we focused on the analysis of target of interest in molecular diagnostics in complex and real matrices (e.g., serum, saliva, blood, and urine) but also in standard solution when the detection strategy is innovative and involves the improvement of analytical performances. so the panel of revised analytes includes hormones (steroids and peptides), protein clinical markers, antibodies involved in immune disorders, nucleic acids for genetic disease and as clinical markers (i.e., mirna), bacterial cells, and viruses for pathogens detection. so far, most of the research articles come from academic exercises but we are more and more confident that the application of spr to the clinical and medical analysis will gain momentum in the next future. the physical principles and the state of the art of surface plasmon resonance [ ] [ ] [ ] [ ] [ ] and surface plasmon resonance imaging-based [ ] [ ] [ ] [ ] biosensors were reviewed in many excellent works. at the beginning of the th century ( ), wood was the first scientist who described the inhomogeneous distribution of light in a diffraction grating spectrum caused by surface plasmon wave (spw) [ ] . sixty-six years later ( ), kretschmann [ ] and otto [ ] rigorously demonstrated the optical excitation of surface plasmons (sps) with the method of attenuated total reflection (atr). spr is defined as a charge-density oscillation at the interface between two media, with dielectric constants of opposite signs (e.g., metal and a dielectric), which generate a surface plasmon wave (spw) with a propagation constant β, expressed by the following equation [ ] : where ω is the angular frequency, c is speed of light in vacuum, and ε d ε m are the dielectric constants of dielectric and metal, respectively. at the metal-dielectric interface the electromagnetic field of the spw has as a maximum intensity that exponentially decreases (evanescent wave, ew) into both media with a variable penetration from to nm (for vis and nir wavelengths) [ ] . prism couplers, grating couplers, or metal-dielectric waveguides are the configuration used for the excitation of surface plasmons but the first one represents the most common approach for the plasmon excitation via the attenuated total reflection method (atr) with kretschmann geometry becoming also the most suitable for sensing and biosensing applications. in the kretschmann geometry, the light wave is totally reflected at the boundary between a thin metal layer (typically gold or silver with nm of thickness) and a high refractive prism coupler (typically in glass). the reflected light excites the surface plasmons of the metal film generating an ew (or spw) penetrating the metal layer (fig. ) . spr based biosensor belongs to refractometric devices, since the propagation of the spw is sensitive to changes in the refractive index of the dielectric. the binding between analytes and bioreceptors immobilized on the sensing surface causes a local change in refractive index (fig. ) and the variation of the propagation constant β, generating a real time signal in label-free way, since no labels have to be added in solution for the development of the analytical signal. classic spr spectroscopy is based on the monitoring of coupling angle, coupling wavelength, intensity, phase [ ] , whereas spr imaging (spri) is based on the measurement of reflectivity of monochromatic incident light at a fixed angle by means of a ccd camera. compared with traditional spr, spri offers main breakthroughs such as multiplexed detection (by designing -d patterned microarrays of molecular probes) and the real-time visualization of the whole biochip surface to monitor simultaneously multiple bioreceptor/analyte bindings with the parallel support of a digital image, representing the intensity of binding in a color scale [ ] . on the other side, spri suffers from order of magnitude worse resolution than classical spr ( − versus − riu) [ ] . biosensor selectivity depends on the bioreceptors immobilized on the sensing surface. here we will present and discuss works that appeared in the last years aiming at developing robust and reliable spr assays in clinical diagnostics. among bioreceptors, we can enumerate antibodies, nucleic acid probes or synthetic receptors (molecular imprinted polymers (mips), aptamers, or artificial dna (xna). accordingly, it is customary to talk about immunosensors, nucleic acid sensor, aptasensor, and so on. an immunosensor uses the affinity interaction between an antigen and an antibody [ , ] . nucleic acid (na) sensors are based on oligonucleotidic probes and na hybridization reaction [ , ] . in na sensors, artificial nucleosides (xna) oligomers have also been employed as bioreceptor for clinical applications. xnas increase sensor stability by avoiding biodegradation or exploiting improved affinity of xna versus the target sequence compared with conventional dna probes. d'agata et al. reviewed works dealing with the great potential of artificial dna (pna, lna, hna, and morf) in nucleic acid spr-based sensing, exhibiting higher selectivity and sensitivity in detecting complementary or mismatching nucleic acid targets [ ] . behind that, aptasensors emerged in the last years as an interesting alternative to immunosensing. aptamers are na molecules that can bind to predetermined targets (small molecules as well as proteins and peptides) with high affinity and specificity [ , ] . these nucleic acid aptamers are engineered through repeated rounds of in vitro selection or equivalently with systematic evolution of ligands by exponential enrichment (selex) [ , ] . finally, mip sensors take advantage of totally synthetic molecular recognition elements, constituted by highly crosslinked polymers engineered to bind one target compound or fig. in a spr biosensor based on a prism coupler and working in kretschmann geometry with the atr method, light is totally reflected by a thin gold layer ( nm) that excites the surface plasmons (sps) and generates a spw (a). the affinity binding between bioreceptors and analytes causes a change of refractive index vs. time (b) entailing a shift of the reflectivity curve vs wavelength of source light or angle of reflected light (c) a class of structurally related target compounds with high selectivity [ , ] . interesting applications to clinical diagnostics have recently been reported for synthetic receptor such as xna, but still very few are using aptamers and mips instead. bioreceptors have first to be immobilized on the sensing surface and for this purpose, different approaches were reported. furthermore, the suitable assay format has to be applied depending on the clinical application. both aspects will be discussed in the next paragraphs. in spr-based biosensor development, the immobilization of the biomolecule on the sensing surface is a key step essential for the success of the analysis. in particular the features to be considered for the optimal bioreceptors immobilization procedure deal with the retentions of biological activity for the analyte after immobilization, the compatibility between binding activity and range of analyte detection, amount of immobilized bioreceptor (to avoid steric hindrance), reproducibility of immobilization (to assure reproducibility of results), and finally the regenerability of the biosensing surface (receptor-analyte dissociation without compromising the biological activity of the receptor). for the sensor applicability to real matrices, the immobilization chemistry should be optimized to also prevent unspecific interaction on the surface arising from matrix components (i.e., proteins in blood, serum, etc.). the binding of biomolecules to gold-sensing surface can be achieved through many suitable immobilization approaches, such as physical absorption, hydrophobic (via lipid layer), electrostatic interactions, covalent coupling (coupling of nucleophiles to carboxylic groups, to thiol groups or to aldehyde groups), and coupling of native and tagged molecules (avidin/ biotin) [ ] . among them, the last two approaches are surely the most exploited in clinical diagnosis application for cheapness, ease of realization, stability, and robustness. in particular, nucleotides are efficiently immobilized after modification with the thiol group, exploiting the high affinity between sulphur and gold surface or, alternatively, with biotin labeling via affinity interaction with avidin or streptavidin coated chip surface. in both strategies, the formation of a self-assembled monolayer (sam) increases the degrees of freedom of the bioreceptor and, consequently, the bindings with analytes. proteins (e.g., antibodies) and other peptides are widely immobilized by covalent amine coupling between amino groups of proteins and activated (with nhs/edc solutions) carboxylic linkers of alkanethiols (or disulfide) in sam format or carboxymethylated dextran ( d matrix immobilization). these approaches are rapid, simple, and inexpensive but bring about random orientation of bioreceptors (attributable to multiple functional groups on the protein), which could decrease the conformational flexibility, hindering the bioreceptors/analytes interaction and thus lowering the sensor sensitivity. an alternative strategy for a controlled orientation is, instead, based on the affinity reaction between biotin-conjugated protein and (strept)avidin-coated chip. in addition, the interaction between fc and protein g or a could be also exploited for the abs immobilization. preventing nonspecific adsorption of biomolecules (e.g., protein) on the spr sensing surface is another key-step for the development of specific biosensor, with real application to clinical diagnostics where complex matrices (such as serum, blood, and urine) are analyzed. a strategy frequently adopted is the fuctionalization of surface with non-fouling materials resistant to nonspecific interaction such as glycol-derivate oligo/polymers: oligo(ethylene glycol) (oeg), poly(ethylene glycol)(peg), eg -cooh, eg -oh but also poly(l-lysine) grafted with poly(ethylene glycol) (pll-g-peg) [ ] . the success of protection from nonspecific interaction is due to steric-entropic barrier properties coupled with a high degree of hydration of peg molecules as reported by blättler et al. [ ] . two assay formats, direct and indirect, were mostly adopted on spr biosensor for the detection of analyte in clinical diagnosis (schemes in fig. ). the assay selection is based on the mw analyte, on the bioreceptors/analyte affinity, and on the matrices under investigation. in the direct assay, bioreceptors are immobilized on the sensing surface and analytes are added in solution. the affinity interaction causes a refractive index variation, providing analytical signals proportional to analyte concentration. this strategy is usually adopted for analyte with mw greater than da, sufficient to develop a refractive index variation and resulting in detectable signal. moreover with a multi-step direct assay (fig. ) dls can be further lowered using a secondary bioreceptor (specific for analyte) in a sandwich format. labeled secondary bioreceptors (with biomolecules or nanoparticles) are often employed to increase the assay sensitivity. indirect assays are preferred for low mw analytes ( x da). here, purified analytes (or conjugates) are immobilized on the spr surface, while specific bioreceptors are incubated with the samples to be analyzed and injected to sensing surface; when the solution reaches the surface, only remaining free bioreceptors bind to immobilized analyte, providing a signal inversely proportional to its concentration in the sample. besides, secondary bioreceptors (labeled or not) can be added in solution to further improve sensitivity and lowering dls with multi-step assays. recently, many developments in spr technology were performed for the improvement of sensor analytical performances in terms of sensitivity, specificity and detection in real matrixes [ ] . a strategy was offered by nanoparticles coupled to plasmonic sensors in miniaturized and low cost systems [ ] . in , gao et al. examined recent works in engineering hybrid spr interfaces such as oxide-based hybrid spr interfaces and nanocomposite films, analyzing the optical properties and the suitable applications in sensing [ ] . more recently ( ), bedford et al. have specifically reviewed the recent trend of incorporation of gnps within the sensing surface itself or as tagging molecules in spr biosensor. significant sensitivity increases and dls decreases were described for a variety of analytes [ ] . as reported by minunni and spoto, the increase of sensitivity is explainable by both the mass enhancement of the nps in the ew as well as by the optical coupling between surface plasmons (sps) and electric fields of localized surface plasmons (lsps) of nps when they are close to the metallic surface [ ] . lsps are defined as collective electron oscillations confined in the mnp surface and promoted by interaction with the light. if the mnp dimension is much smaller than the wavelength of the excitation light, the local electromagnetic fields are strongly enhanced. a further contribution to the increase of sensitivity is also expected if nps clustered since it was proven that clusters of spherical metal and dielectric colloids show strong electric, magnetic, and fano-like resonances from the electromagnetic coupling between closely spaced particles [ ] . zanoli et al. reviewed the employment of functionalized gold nanoparticles for ultrasensitive dna detection on different transduction systems (including spr), useful to bypass pcr amplification steps [ ] . since we have previously defined the spr-based biosensor as a label-free system, for the sake of clarity we specify that the addition of bioreceptors labeled with nanoparticles is not employed for signal development itself but for the improvement of system analytical performance (sensitivity increases and dls decreases), really essential in clinical diagnostics where analytes are in complex matrices at very low concentrations. some drawbacks of nps uses could be found in the nps synthetic step, in tuning dimensions, and absorption wavelengths (for coupling between plasmons), since these steps are time-and reagent-consuming. furthermore, it seems constrictive to employ nps in reusable sensors since surface poisoning could occur, compromising serial measurements on the same chip: no data of regeneration are available in literature. very recently, graphene-based surface plasmon resonance interfaces were also studied for suitable application in spr sensor. the functionalized surfaces showed several advantages such as high surface-to-volume ratio, adsorption of organic or biological molecules (pi stacking), passivation against oxidation, and controlling the number of graphene layers [ ] . other strategies, different from nps-based approach, have been also adopted in this last decade for the improvement of spr based sensor. in , abbas et al. collected the last forefront tendencies in spr instrumentation with advanced performances identified in: new excitation schemes and optical configurations, liquid handling by microfluidics, and microsystems for integrated spr chips generally based on glass or poly(dimethylsiloxane) (pdms). new material such conducting metal oxides zno and zno/au in the adhesion layer to replace cr or ti were also discussed as well as forefront spr-related optoelectronic components (light source and detector) and finally the spr coupling with other analytical techniques (hyphenation approach with scanning probe microscopy, electrochemistry, fluorescence and raman spectroscopy) were also evaluated for the achievement of higher sensitivity and resolution [ ] . after focusing on different features of the spr technology (physical principle and instrumental details with the most recent trends) on bioreceptors immobilization approaches and on assay designs (indirect and direct, simple, multistep), we move on to review the applications of these strategies to real cases of interest in clinical diagnostics. hormones and small peptides analysis is a key area in diagnostic and, more recently, also in anti-doping controls. endocrine diseases diagnosis may be difficult, and it requires to directly assaying hormone levels in blood or eventually making indirect measurements. for example, diabetes mellitus is diagnosed via measurements of blood glucose rather than direct assays of plasma insulin. some interesting examples of hormones detection are provided by different authors, demonstrating the ability of spr to provide interesting solutions also in this clinical area. frasconi et al. developed a spr immunosensor for the detection of cortisol and cortisone levels in urine and saliva samples with an eco chemie autolab spr system. the measurement of free cortisol level in these matrices was an indicator for adrenal or pituitary gland disorder, and in doping analysis a marker of glucocorticoids illicit use. urine samples ( ml) were hydrolyzed by an incubation for h at °c with ml of pbs (ph . ), μl of α-methyltestosterone (internal standard), and μl of β-glucuronidase from escherichia coli. saliva samples were analyzed without pretreatment. specific antibodies were immobilized on polycarboxylate-hydrogel-based coatings, and the proposed method resulted to be simple, inexpensive, reproducible (rsd% < %), and sensitive, with detection limits less than μg /l (∼ . nm). a linear detection range from to μg/l for cortisol and - μg/l for cortisone was found and a very good correlation with the reference lc/ ms-ms method was proven, confirming the potential utility of spr for clinical, pharmacological and anti-doping application [ ] . estriol- -glucuronide, a hormone for the monitoring of ovarian function, was detected by jiang et al. with biacore x- system in urine samples from nonpregnant and pregnant subjects. the authors developed an inhibition (indirect) immunoassay by the steroid immobilization on the sensor surface, through estriol- -glucuronide-ovalbumin conjugate with an oligoethylene glycol (oeg) as linker. au nanoparticle-ab conjugate (au-ab) was used to enhance the sensitivity of the spr assay. a detection limit of . μg/l (∼ pm) in urine diluted with tm buffer ( : , : and : ) was rapidly achieved in min without complicated sample pretreatment and with a good reproducibility (cv% < . %). the assay was fast and inexpensive compared with traditional method based on ria, hplc-ms, or hplc with uv and fluorescence detection. it required less clean-up steps, resulting an efficient way to monitor e - g production rates in urinary samples of a normal menstrual cycle [ ] . besides the analysis of very low mw hormones, spr sensing has been also applied to the detection of peptide hormones such as insulin (mw da), which regulates carbohydrate metabolism. insulin sensing in serum is greatly important for clinical diagnostics and to follow-up patients affected by various types of diabetes but also for doping control. recently ( ) frasconi et al. detected insulin with indirect immunoassay in human serum samples from healthy and diabetic patients using eco chemie autolab spr system. hydroxyl/lc-pdp-functionalized g -pamam dendrimerencapsulating gnp was covalently bound by amino coupling to an alkanethiolates (carboxylated) sam-modified gold surface. then insulin was covalently immobilized on the functionalized surface exploiting the amino-coupling chemistry for the immunoassay (fig. ) . the innovative surface chemistry reduced nonspecific surface interactions and the effect of coupling between localized plasmon of the nps and surface plasmon lowered the dl down to . pm (∼ . ng/l). ten-fold diluted sera were analyzed in the - pm linear concentration range and radioimmunoassay reference method confirmed the reliability of the analytical device; indeed the biological level for insulin in the diabetic patients is - pm, corresponding to a range . - pm in a -fold diluted sample. moreover, the sensor provided reproducible results (cvs ranged between . % and . %) and it was reusable up to times [ ] . other high mw peptide hormones, such as human pituitary hormones, were recently detected with spr. in particular, lechuga et al. developed binding inhibition (indirect) immunoassays on a spr from sensia sl, where hormones were immobilized on the sensor surface by amino-coupling and working conditions (assay buffer and regeneration solution) were optimized. they detected first hgh (growth hormone) in human serum samples (mixed : in pbst), essential for development and normal growth [ ] . then they also performed, using the same strategy, the single and multi-analyte determination of two other gonadotropic hormones in urine and serum sample (both mixed : in pbst): follicle stimulating hormone (hfsh) and luteinizing hormone (hlh) involved in the development and function of the reproductive system [ ] . finally they detected hgh, hlh, hfsh, and htsh (thyroid gland stimulation for thyroxine production) in urine and serum sample (again both mixed : in pbst) using an indirect immunoassay coupled to multi-analyte sensor. all biosensors resulted sensitive (about μg/l (∼ pm) as dl for each analyte), reusable from to consecutive assay cycles and specific for each analyte. the spr based immunoassays resulted reproducible with mean intra-and inter-day cvs about %, appearing as a highly reliable tool for endocrine real-time and daily monitoring in clinical laboratory compared to official methods as ria, ima or elisa [ ] . as reported by segura in , there are many areas of common interest and overlaps between clinical analysis and anti-doping monitoring such as methodological similarities, analysis of human biological matrices, and detection of the same analytes (e.g., hormone). the author was confident that the mutual interaction between the fields would be able to bring mutual improvements in terms of technological solutions and innovative strategy [ ] . in the anti-doping code, the list of prohibited substances, under the category peptide hormones, growth factors and related substances, a variety of hormones and their releasing factors can be found: ( ) erythropoiesis-stimulating agents [e.g., erythropoietin (epo), darbepoetin (depo), hypoxiainducible factor (hif) stabilizers, methoxy polyethylene glycol-epoetin beta (cera), peginesatide (hematide)]; ( ) chorionic gonadotrophin (cg) and luteinizing hormone (lh) and their releasing factors, in males; ( ) corticotrophins and their releasing factors; ( ) growth hormone (gh) and its releasing factors and insulin-like growth factor- (igf- ). in addition, several growth factors are also banned [ ] . an excellent review on the potential use of spr in anti-doping analysis is provided by the segura group where it is highlighted how conventional analytical methods for detecting human biotics as doping agents (i.e., recombinant products), may hardly distinguish between endogenous and exogenous origin. for larger molecules (i.e., protein hormones) the innate structural complexity, the heterogeneous nature, and the extremely low levels in biological fluids made the analytical procedures heavily dependent from the immunological approaches [ ] . a protein biomarker is an indicator of a specific biological stage used in clinical diagnosis to monitor the stage of related diseases, to guide molecularly targeted therapy, and to evaluate a therapeutic response [ ] . the increasing number of clinically relevant protein biomarkers and the advances of proteomics techniques indicate the need of reliable methods for their detection in complex matrices. at this purpose, spr and spri provide a suitable platform for daily routine clinical analysis thanks to real-time and label-free detection [ ] . in this section, we focused on the most recent assays based on spr biosensor for the detection of protein biomarkers for multiple diseases first and then specific for cancer and cardiac diseases. the c-reactive protein (crp, kda) is an important blood serum marker for inflammatory processes (e.g., cardiovascular disease (cvd), inflammatory bowel diseases), currently detected by latex-enhanced turbidimetric immunoassay. jung et al. developed a label-free array system using amidelinked (al) nhs-dextran biacore cm chip for crp detection in human sera. after afm studies, they found this surface [ ] more suitable for proteins immobilization than a normal epoxide-linked carboxymethyl-dextran layer. monoclonal anti-crp antibodies were immobilized onto the surface and rapid analysis of crp was performed down to μg/l (∼ . nm) concentration in human sera diluted ( : in pbs, ph . ); results showed a good correlation with the turbidimetry reference immunoassay [ ] . bini et al. immobilized a biotinylated mer rna aptamer for crp on a carboxylated dextran-streptavidin-modified cm chip of a biacore x. in the assay optimization they evaluated the effect of different buffers, the effect of ca + ion on the interaction between the aptamer and crp, and finally the specificity of the aptasensor against putative interfering biomolecules. the highest spr signals and the lowest detection limit ( μg/l, ∼ . nm, suitable for clinical applications) were recorded with the following experimental condition: serum diluted : with hepes buffer at ph . and mm ca + concentration. cv% was estimated to be about % and a liner correlation was found within - μg/l range of concentration. experiments in serum solution showed an interfering effect caused by igg, suggesting the need of sample pretreatment [ ] . the two assays described above were suitable for clinical monitoring of crp because the protein is about . mg/l in adult serum (above the achieved dls) [ ] . circulating annexin a (anxa , kda), a biomarker related to various diseases (acute myocardial infarction, trauma, thrombosis, inflammation, and cancer) was rapidly and inexpensively detected in human blood samples (diluted : in hbs-ep) by trouvé et al. using a biacore . they immobilized anxa antibody on a carboxymethylated dextran cm sensor chip by amino coupling, and they found for the first time that the level of circulating anxa was higher in a male . (± . ) μg/l than in a female . (± . ) μg/l, indicating a difference by gender. a good linearity was observed between . and . μg/l with a good correlation (r = . ) and cv% below %. these results proved that the label-free spr biosensor was a very good alternative to the label-based conventional elisa immunoassay with advantages such as very good sensitivity, high reproducibility, and fast responses (in few min) [ ] . the long-term outcome of cancer therapy depends on early diagnosis and the response to therapy. an important aspect of cancer management includes careful monitoring of cancer biomarkers in physiological fluids [ ] . vaisocherová et al. used a home-built spr instrument for the immunodetection in human serum of alcam [ ] (activated leukocyte cell adhesion molecule/cd ), a - kd transmembrane glycoprotein biomarker of pancreatic cancer, typically with a concentration of μg/l in human serum [ ] . anti-alcam was immobilized via physical adsorption on positively charged amine-terminated alkanethiol sam surface. despite nonspecific binding recorded, a sufficiently low dl was achieved ( μg/l, ∼ pm in serum diluted : in pbs) for the discrimination of alcam levels in cancer cases from control sera. a good reproducibility was also confirmed by the cv% less than %. the direct detection (without signal amplification) showed also an excellent correlation with elisa reference method [ ] . in the same year, ladd et al. developed an immunosensor for the simultaneous and specific detection of alcam and transgelin- (tagln ), a -kda protein that has been a biomarker of breast and colorectal carcinoma). specific antibodies were immobilized by amino-coupling on cooh-oeg-coated gold sensor chip of a home-built spri platform. dls for alcam and tagln in pbs buffer were μg/l (∼ pm) and μg/l (∼ . nm), respectively. no crossreactivity was recorded but high levels of nonspecific adsorption were found with serum solution ( : in pbs), indicating the need of highly non-fouling surfaces in sensor applications [ ] . in , piliarik et al. combined high-resolution homebuilt spri sensor and high-density protein array with lowfouling background for the parallel detection of protein cancer biomarkers alcam and hcg (human chorionic gonadotropin) in diluted blood plasma samples ( % in te buffer). specific antibodies were immobilized by amino coupling on a low-fouling chip surface (bsa immobilized on carboxylterminated thiols). the biosensor showed a very low nonspecific protein adsorption (less than ng/cm ), and a linear dependence between response and analyte concentration was found (below μg/l for hcg and below μg/l for alcam). the following dls were reached: μg/l ( . nm) for alcam and μg/l ( . nm) for hcg and a very good reproducibility was confirmed by the cvs% less than % [ ] . in , kazuno et al. developed a novel spr biosensor based method for multi-sequential detection of sna- , aal, and pha-l lectins to estimate the glycosylation of haptoglobin (a complementary marker to ca in ovarian cancer) in sera of patients with prostate cancer disease. the method involved anti-haptoglobin immobilization by amino-coupling, sera dilution : in hbs-p buffer, filtration, and finally detection of the sugar chain by lectin solution. a calibration curve for haptoglobin was obtained in - mg/ml concentration range. multi-sequential analysis of sna- and haptoglobin represented an accurate diagnosistic approach for prostate cancer. the label-free spr biosensor was less complex and time-consuming than conventional elisa, where labeling step with enzymes, radioactive isotopes, or fluorescein are required [ ] . gill et al. quantified p αmap kinase, a prognostic marker in head and neck squamous cell carcinoma (hnscc), by an immunosensor (biacore ). they investigated the correlation between p α and hnscc in diluted serum ( : in hbs-ep) of patients undergoing radiation therapy (rt). they immobilized anti-p α abs by amino-coupling on a cm sensor chip and used western blot and elisa as reference methods. patients with hnscc showed -fold increase in p α levels ( . mg/l∼ nm) compared with control sample while values during-rt and post-rt treatments decreased to . mg/l (∼ . nm) and . mg/l (∼ . nm), respectively, showing a reduction attributable to a clinical tumor regression by rt. these results confirmed the suitability of p α as a serum marker in hnscc, and that if coupled to spr sensing offers higher sensitivity than traditional antibody-based methods such as elisa and western blotting [ ] . pimková et al. developed spr biosensor with dispersionless microfluidics (home-built) for the detection of svegfr- (soluble vascular endothelial growth factor receptor), a biomarker abnormally produced from cancer cells in myelodysplastic syndromes (mds). vegf-a (homodimeric glycoprotein) was covalently immobilized on the sensor surface by amino coupling, allowing the detection of svegfr- in human blood ( % in pbs) down to a dl of μg/l ( . nm) and with a good reproducibility between % and %. the approach suggested a model for future protein multi-array for mds diagnosis based on protein-protein interactions. the assay resulted in being less sensitive than conventional detection of svegfr- based on elisa, capable of detecting . μg/l and up to . μg/l, respectively, for healthy individuals and mds patients [ ] . an innovative approach for the same biomarker detection was presented by liu et al. where vegf was released in solution by living skov- ovarian cells (i.e., cancer cell) (fig. ) . anti-vegf abs were immobilized on a protein gcoated chip surface, and the analyte produced in the flow chamber (in pdms) was directly monitored. the linearity of the response in the . - . mg/l vegf concentration range and reproducibility (inter-assay rsds% less than . %) were assessed in pbs buffer. further, the cell viability was demonstrated and then the in vivo microenvironment of the vegf signaling pathways was mimicked. from the quantification of vegf released by cells, the carcinoma cell number was accurately predicted, showing the suitability of an innovative strategy for the real-time monitoring of biomarker expressed from living cells [ ] . we underline here vegf is also a target molecule in anti-doping analysis, present in the wada list of prohibited substances [ ]. battaglia et al. developed a fiberoptic spr biosensor for the simultaneous detection of three cytokines related to chronic wound healing, interleukin- (il- ), interleukin- (il- ), and tumor necrosis factor-r (tnf-r), reaching a detection limit below μg/l in hbs buffer and in spiked cell culture medium (ccm). specific antibodies were immobilized on the spr fiberoptics by aminocoupling, and no nonspecific bindings from the cell culture medium proteins were observed on the biosensor surface. the sensor emerged as a reliable device for multiple and specific biomarker detection since the il- and tnf-r concentrations are ∼ μg/l in normally healing wounds and and μg/l, respectively, in chronic wounds. moreover, the il- levels are∼ . μg/l (not detectable) in a normally healing wound and∼ ng/ml (detectable) in not healing wounds [ ] . cardiac markers are proteins released by injured myocardial cells into the bloodstream during cardiovascular diseases (cvds) and so are useful to better diagnose cvd conditions promptly and efficiently [ ] . in particular, brain natriuretic peptide (bnp, . kda) is a hormone secreted mainly from the cardiac ventricle into the blood, frequently monitored as marker in cardiac failure [ ] . teramura et al. developed a sandwich-type immunosensor for bnp detection in donor plasma (not pretreated) using nanobeads for signal amplification (fig. ) . primary anti-bnp abs were immobilized on cooh-sam chip surface (amino coupling) of a home-built spr instrument, and a sandwich assay was performed with a secondary biotinylated antibody after immunoaffinity interaction with bnp. finally, streptavidin-conjugated nanobeads ( nm in diameter) were added for signal amplification, enhancing sensitivity, and lowering dl from μg/l ( . nm) to ng/l ( . pm). linearity was observed in the - ng/l bnp concentration range, confirming the suitability of this immunosensor for clinical necessities since the normal level of bnp in plasma is about ng/l but it increases approximately to μg/l during acute or chronic cardiac failure. sensitivity was comparable to commercially available reference assays for bnp (ria and chemiluminescent reactions ( - ng/l) [ ] . reproduced by permission of the royal society of chemistry [ ] in addition, an indirect immunoassay coupled to portable spr instrumentation (with developed microfluidic) was also reported for bnp detection in human serum ( : in . m pbs). samples were incubated with acetylcholine esteraselabeled antibodies and then introduced into the microchannel of the device so that only free-bnp conjugate bound to bnp previously immobilized on the surface (by amino-coupling). then acetylthiocholine was added as substrate and hydrolyzed by acetylcholine esterase. the thiol compound (thiocholine) produced covalently bound to a thin gold layer located downstream in the microchannel, generating a spr angle shift monitored as analytical signal. a concentration range from to ng/l was examined and trace levels of analyte ( fg) were detected in about min. cv% for five measurements of ng/l bnp was . % and although this value was high compared with a commercial immunosensing system, the authors considered the rsd% acceptable for clinical diagnosis since bnp concentration significantly increases in heart failure patients [ ] , as mentioned above. two other proteic myocardial infarction biomarkers, myoglobin (mg) and cardiac troponin i (ctni), were quantified at biological levels in undiluted serum and without any sample pretreatment, using a fiberoptic spr immunosensor (jobin-yvon spex m spectrometer). specific antibodies were immobilized via amino coupling on a -mercaptohexadecanoic sam, minimizing the nonspecific signal the serum proteins. dls were . μg/l ( pm) for mg and . μg/l ( pm) for ctni, well below threshold limits to detect myocardial infarction disease (approximately - μg/l for mg and - μg/l for ctni). a good reproducibly (cv% < %) and linear correspondences between signals and concentrations up to μg/l were also assessed for both analytes [ ] . liu et al. designed a spr biosensor with high anti-fouling ability for the detection of the cardiac marker troponin t in d-pbs buffer. anti-troponin t antibodies were immobilized by amino-coupling on the chip surface (navi spr) coated with a homogeneous sam of oligo(ethylene glycol) (oeg)terminated alkanethiolate and mercaptohexadecanoic acid (mhda). the system showed interesting high anti-fouling properties (high resistance to nonspecific adsorption of hsa) and high affinity and specificity for the detection of troponin t. a good linear correlation (r= . ) below μg/l and a dl of μg/l ( . nm) were also confirmed [ ] . biosensing plays a crucial role in the analysis of autoantibodies in human serum for the real-time monitoring of autoimmune diseases. an increasing number of researches were available in literature. schlichtiger et al. recently reviewed different biosensor-based approaches in this field. authors also emphasized the main advantage of spr for monitoring bindings in real-time and for multiplexed analyses of autoantibodies by use of microarrays (e.g., spri) [ ] . the detection of pathogenic anti-dsdna abs in sera of patients affected by lupus erythematosus (sle) was reported buhl et al. an antigenic construct was formed as follow: a synthetic oligonucleotide ′-aldehyde-modified (cho) was coupled to biotinylated human transferrin using -hydrazinonicotinate acetone hydrazone (sanh) as covalent linker; then it was hybridized with the complementary antistrand and ligated with a human recombinant dsdna fragment bp in length. the conjugate was finally immobilized on a gold surface coated with carboxymethyldextran functionalized with streptavidin, and diluted sera ( : in diluents from sle patients and healthy donors) were analyzed with the spr biosensor system (biacore x). as a result, authors confirmed sle in patients with . % specificity at a sensitivity fig. schematic illustration of spr-based sandwich-type immunoassay for bnp and amplification of spr signal by accumulation of streptavidin nanobeads. reprinted with permission from elsevier [ ] of . % compared with positive results in the farr ria assay with . % specificity at a sensitivity of . %. moreover, a very good reproducibility was confirmed by intra-assay imprecision ( . %- . %) and day-to-day variation ( . %), making the spr biosensor really suited for anti-dsdna abs detection in sle disease. in addition, the device provided information about binding kinetics and affinities of the specific autoantibodies [ ] . the same group also studied interactions between dsdna and anti-dsdna autoantibodies from sle sera of patients, again with biacore x and with the same dsdna immobilization strategy previously described. they characterized kinetics, off-rates, and functional affinities (avidities) for three anti-dsdna mabs, confirming the importance of kinetic properties for the explanation of behavior of mabs in traditional methods such as the farr ria and elisa [ ] . metzger et al. developed a biosensor for the diagnosis of antiphospholipid syndrome (aps) through the discrimination of disease-relevant autoantibodies (anti-β -gpi) from crossreactive antibodies developed in other infections. human β -gpi (β -glycoprotein i) was covalently linked to a cm chip surface coated with n-alkanethiol sam (biacore x). sera ( : in hbs) from aps patients or patients with sle, syphilis, parvovirus b , and healthy donors were analyzed and the results were compared with those from elisa test. no significant antibody bindings (signal < ru) were recorded in samples from healthy individuals or patients with other infections, whereas response levels in the range of - ru were recorded from positive aps patient sera, with a correlation coefficient of . with reference elisa test. the spr-based assay was also reproducible since no significant loss of activity (< %) was recorded after measurement cycles of patient sera [ ] . in a later study, müller et al. used the same instrumental setup to prove the correlation between the affinity of anti-β -gpi in patient sera ( : in hbs) and the antigen β -gpi preparations (manufacturer, origin, and purification method). the study explained the inter-assay differences of anti-β -gpi elisas, confirming that only one common β -gpi preparation would have improved the inter-assay comparability [ ] . in the same year, the authors also demonstrated that the employment of β -gpi-derived domain-specific peptides would have offered diagnostic advantages in primary autoantibody screening for aps and in discrimination of sera of aps patients from sera of healthy patients [ ] . schlichtiger et al. detected antiphospholipid antibodies (apl, a serological indicators of the disease) with an immunosensor developed on biacore x platform. in particular, they detected cardiolipin antibodies (acl) in sera ( : in hepes) from healthy donors and aps patients. cardiolipin was immobilized by amino coupling (after activation with edc/nhs) on -mercaptoundecanoic acid sam-coated chip surface. the binding ag/ab was monitored and the assay confirmed aps with % diagnostic specificity, equal to the standard elisa method used in routine diagnostics but showing more sensitivity than elisa ( % versus . %). moreover, the chip was regenerable (up to measurements) with excellent intra-chip reproducibility (rsd%= . %) and chip-to-chip reproducibility (rsd%= . %) [ ] . rutgers et al. reported kinetic analysis of anti-gbm autoantibodies, from sera of nephritic patients, performed with biacore instrumentation. purified bovine collagen α (iv)nc (control) and α (iv)nc (ag) were immobilized onto a carboxymethylated dextran hydrogel surface after edc/nhs activation. autoantibodies from patient sera bound to α (iv)nc whereas no binding was recorded to α (iv)nc (control). from the estimation of the dissociation and association constants, they demonstrated the high affinity ab/ag, the high velocity of association and the slowness of dissociation, explaining the rapid course of the disease as well as the resistance to therapy (persistent ab glomerular aggregation could generate potentially incessant inflammation) [ ] . alaedini et al. detected autoantibodies against the monosialotetrahexosyl ganglioside gm (anti-gm ) related to acute and chronic motor neuropathies. gm (ag) and gm (control) gangliosides were absorbed on a methyl dextran layer of a chip and the discrimination between patients sera and healthy controls was possible with sensitivity and specificity comparable to classic elisa tests [ ] . antiglutamic acid decarboxylase (gad) autoantibody detection, an indicator of type i diabetes mellitus, was developed by lee et al. with biacore . the ratio between -mpa and -mua ( : ) thiols was first optimized for covalent binding of biotinylated gad via streptavidin immobilization (after nhs/edc activation). anti-gad abs were analyzed in hbs buffer within . - . μm concentration range and the interference with other biomolecules (bsa as control) was found to be negligible [ ] . carlsson et al. detected insulin autoantibodies (iaa) in serum samples from individuals at high risk of developing type diabetes (t d). they designed an indirect competitive immunoassay to bypass nonspecific adsorption of matrix proteins that could mask the analytical signal. insulin was immobilized on cm chip of a biacore x after activation with nhs/edc and the sensor was calibrated with iaa ( - u/ml) in sera derived from pooled nondiabetic serum spiked with pooled high iaa-positive serum to obtain identical matrix composition. excellent assay performances were reported since analysis time was -fold reduced from days to min, and the sensitivity was comparable to that offered by ria; the cut-off for positivity was . u/ml (in healthy swedish children) [ ] . a parallelized immunoassays on spr microarray imaging (ibis technologies) was used by lokate et al. to detect anti-citrullinated protein antibodies (acpa) in sera of patients ( : in pbst) affected by rheumatoid arthritis (ra). carboxylated xantec hc nm sensor chips were activated with nhs/edc and then two different linear citrullinated peptides (cita and citb) were immobilized on the surface; arga and argb (with arginine instead of citrulline) were spotted as corresponding control peptides. interactions between citrullinated peptides and serum autoantibodies of ra patients and controls were monitored with an experimental dl of . pm. although the sensitivity was slightly lower than that of the reference elisa test, the spr system presented the advantage of automation and surface regeneration by repetitive incubations with mm glycine•hcl for s, making the test really suitable for daily routine clinical control [ ] . three years later, a similar approach was applied by van beers et al. with ibis-ispr (imaging) for the analysis of the autoantibodies against peptide fractions in ra patients diluted sera ( -fold in pbst). peptides obtained from citrullinated human fibrinogen were immobilized on a dextran hydrogel surface (after activation with nhs/edc) and three major citrullinated epitope were identified. the multi-array enabled the simultaneous detection of autoantibodies/peptides interactions with a significant reduction of analysis time, resulting in less time-consuming and more accurate than reference elisa screening methods [ ] . spr imaging-based sensing was also used by scarano et al. for anti-bovine igg detection in untreated human serum and milk since high levels of these antibodies are related to type diabetes in serum of children. bovine iggs were immobilized in microarray-mode (spri lab + , horiba) by amino coupling on mua-coated gold chip surface. the nonspecific adsorption and the matrix effect were evaluated by a dedicate software [ ] aimed to optimize a guided automated selection of best-reacting spot (fig. ). anti-bovine igg was detected in a range of concentrations from . to . mg/l in real diluted matrices ( / for serum and / for milk in hepes buffer) with the standard addition method. a good linear response (r = . ), a good reproducibility among experiments (cv%= . %), and a dl of . mg/l ( nm) were achieved in serum while in milk the equally good performances were evaluated as r = . , cv%= . %, and dl= mg/l ( nm). the assay opened new horizons for the detection of protein in complex real matrices with spri-based biosensing, since clinical data (e.g., in serum) displayed anti-bovine igg values up to mg/l with median value of mg/l [ ] . carcinoembryonic antigen (cea) autoantibodies (specific for cea, a biomarker associated to colorectal, gastric, and pancreatic cancer) were monitored in cancerous human serum samples by ladd et al. with a home-built spr instrument. cea was immobilized on a carboxylated gold surface by aminocoupling and autoantibodies were detected by a sandwich assay adding a secondary antibody (goat anti-human igg-hrp conjugate). sera were diluted in pbsa buffer ( : , : , and : ) and tested; cea autoantibody concentrations were around mg/l, resulting∼ . μg/l after dilution ( : ). autoantibodies were distinguishable in cancerous sera samples from the healthy sample, using elisa as reference assay. this finding could be used as a diagnostic criterion for early cancer detection as well as for the diagnosis of disease progression [ ] . recently, two excellent reviews summarized the progresses made by spr-and spri-based nucleic acid (na) sensing [ , ] and their employment in clinical analysis. many oligonucleotidic analytes of interest in medical diagnostics have been detected by na spr based sensing. here we reviewed the na assays in two main classes: assay for the detection of chromosome anomalies (i.e., point mutations and snps) and assay for the detection of genetic material as biomarkers (i.e., micrornas and biomarkers of pathogens). as for the other analytes, we focused only on na assays applied to real matrices with the exception of those developed in standard solution that in our opinion improved on the analytical approach or the system analytical performance. the study of chromosome anomalies by spr in real samples deals with the detection of point mutation and single nucleotide polymorphisms (snps), which are point mutations that occur with frequencies > %. both mutations may have remarkable involvements in clinical diagnosis if they occur in a specific coding region of chromosome-related to common diseases [ , ] . in particular, spr was used as a tool for the detection of point mutation related to tumor suppressor genes (e.g., tp [ ] [ ] [ ] [ ] and k-ras [ ] ), cancer disease [ , ] , and hereditary disease [ ] [ ] [ ] [ ] [ ] [ ] . the tumor suppressor gene tp is the mostly mutated gene in presence of human cancers [ ] and, for this reason, it is one of the most studied genes in cancer research also with assays-based on spr transduction systems. in , jiang et al. detected tp point mutation with a dna spr-based sensing using portable instrumentation (a texas instruments spreeta). synthetic thiolated oligonucleotides ( mer) were immobilized on the gold chip surface as capturing probe for the snp analysis of real dna samples (extracted and amplified at a . μm concentration) from both normal wild-type (jurkat) and mutated (molt ) cell lines. the biosensor distinguished between sequences differing by only one base in the snp position (signal lowered by % for the molt ) in only min with a very low nonspecific response and with the ability to regenerate the sensor up to times. these findings coupled to the good reproducibility (cv%= %) proved that the method has potential for routine clinical analysis [ ] . in the same year, wilson et al. reported the use of the muts, a natural protein able to recognize selectively only mismatching sequences, for the detection of snp in μm synthetic oligonucleotides (tp analogue) with the same instrument. the prior injection of ssb (single strand binding) protein prevented the nonspecific interaction between muts and thiolated ssdna immobilized on the sensor. the sensor was regenerable for times (at least) making the whole approach really inexpensive for the detection of clinically relevant mutations [ ] . sipova et al. developed a sandwich-like assay for the detection of snp (codon ) in a short ( mer) tp synthetic analogue with a four-channel spr biosensor developed at the institute of photonics and electronics (prague). immobilized thiolated probes hybridized the target while streptavidin-oligonucleotide (son) complexes investigated the polymorphic site with a secondary hybridization (enhancement). a good sensitivity down to pm and a good specificity were achieved in the detection [ ] . with the beginning of the nanotech era, metal noble nanoparticles assumed a central role in the development of more sensitive assays for point mutation discrimination. yao et al. developed a method based on sandwich-like assay coupled to nanoparticles, for the detection of snps again in tp gene (excised from the pc -sn plasmid and bp) with a home-built spr. thiolated oligonucleotides were linked to gnps (signal enhancer) for the snp discrimination down to . f. target concentration in μl of sample. the method was adequately reproducible (rsd% < %) and specific thanks to the carboxylated dextran film that prevented the nonspecific adsorption of nucleotide-capped gnps. therefore, the spr-based test was attractive when analyte concentrations were very low and the sample availability was very limited [ ] . another original approach, based again on nps coupled to spr imaging dna sensor (spr imager, gwc technologies), was developed by the corn group for the high sensitive detection of snps of clinical interest. in particular, picomolar detection of snp in brca gene, associated with breast cancer, was reached in non-amplified human dna samples by measurements of surface enzymatic ligation reactions enhanced by gold nanoparticles. the coupling of spri and thiolated dna microarrays for snp genotyping was very attractive because it could be multiplexed for the simultaneous analysis of multiple snps [ ] . the same instrument was used by spoto and coworkers to further enhance snp detection with the employment of streptavin-coated gnps linked to biotinylated oligonucleotide and pna probes immobilized on a gold surface previously functionalized with dithiobis(n-succinimidylpropionate) (dtps). femtomolar sensitivity was reached with synthetic dna target in pbs buffer, making the strategy suitable for detecting dna samples without pcr amplification [ ] . indeed, years later the same approach (fig. ) was applied for the snp discrimination in the human β globin gene involved in several type of β thalassemia, directly in nonamplified genetic material (isolated from leucocytes in peripheral blood) and down to attomolar concentration bypassing the pcr step [ ] . as mentioned in the introduction, the labeling with nps was demonstrated to be useful for the improvements of analytical performances such as increase of sensitivity and lowering of dls. with this strategy, genomic dna was directly detected without any pcr amplification step but, on the other side, synthesis of nps was time-and reagent-consuming. interesting examples of the combined use of artificial dna and spr, able to enhance sensitivity of diagnostic snp investigations, were eventually reported in literature [ ] . in , feriotto et al. reported the first application of pnas and spr to human hereditary mutations. in particular, these authors detected the w x snp on cf gene, associated to cystic fibrosis, with biacore spr. pcr-amplified samples were biotinylated and immobilized on the sensor surface with streptavidin-biotin chemistry while pna probes were hybridized on the immobilized pcr products for the recognition of polymorphism. the reported results suggested that spr was an easy, fast (few min), and automatable platform for detecting mutations with real-time monitoring of hybridization, unlike other methods based on gel electrophoresis and/or dot-spot analysis [ ] . three years later, corradini et al. used biacore to discriminate the same snp in cf gene ( μm) with chiral chains modified pna (based on d-lisine), improving the recognition ability compared to simple pna [ ] . in other approaches reviewed below, the snp discrimination was performed without the employment of enhancing factor (i.e., protein or nanoparticles) or artificial dna, speeding the assays and lowering costs. recently ( ), ermini et al. have developed a very sensitive ( am) and label-free dna sensor for the specific detection of non-amplified abcb gene fragment (extracted from human lymphocytes) on spri lab + platform (horiba scientific) [ ] . abcb is a key gene in pharmacogenomics, with many snps implicated in a common set of problems such as altered drug levels and susceptibility to diseases (e.g., inflammatory bowel disease, parkinson's disease, refractory seizures, and cd cell recovery during human immunodeficiency virus therapy) [ ] . the sandwich assay was based on the optimization of sample pretreatment (fragmentation and denaturation) coupled to a rational design of very selective and performing probes. in particular, thiolated capturing probes, immobilized on the sensor surface, hybridized the target (abcb gene fragment) while a secondary probe hybridization confirmed the selectivity of the capturing [ ] . in the same year, the assay described before was optimized (secondary probes labeling and length) and successfully applied for the detection of rs snp (in the abcb gene) related to susceptibility of many diseases such as colorectal [ ] , breast, and renal cancer [ ] . snp discrimination was performed in genomic dna extracted from human lymphocytes and randomly enriched at mg/l (∼ . fm) by whole genome amplification (wga). the measures were reproducible (cv% < %) and the sensor was reusable for up to times [ ] . micrornas (mirnas) are important gene regulatory nucleic acids (about mer) in humans, affecting transcriptional and post-transcriptional regulation of gene expression [ ] . mirna also has a key role as marker of serious disease (i.e., cancer [ , ] , heart disease [ , ] , diabetes [ ] , nervous system diseases [ ] , and liver disease [ ] , included from high femtomolar to low nanomolar range of concentration in human blood [ , ] . however, mirnas are not yet mentioned in literature coupled to spr for clinical routine analysis; below, we reviewed some academic researches for the detection of mir- , the most abundant mirna in [ ] hepatocytes with an achievable future key role in clinical diagnosis as a predictive marker for viral, alcohol, and chemical-induced liver disease [ ] . a fast and simple assay was recently ( ) described by zhang et al. for the detection of mir- extracted from human breast tumor cells. the detection was based on a simple and hybrid sandwich-like assay: mir- was hybridized from a fully complementary thiolated dna probe immobilized on the sensor surface using a biacore x. a secondary biotinylated dna probe (linked to streptavidin) was hybridized to the target, enhancing sensitivity and lowering the detection limit down to pm [ ] . in addition, the assay was pcr-free and showed a good reproducibility (cv%= . %). corn and coworkers described an innovative approach for the detection of mirna, extracted from mouse liver tissue, based on thiolated lna microarray immobilized on the gold chip surface (spr imager, gwc technologies). simultaneous detection of mir- , mir- b, and mir- b from mouse liver tissue, was achieved down to fm, enhancing spri transduction with poly-(a)enzyme chemistry and t -coated au nanoparticles (fig. ) . despite the high sensitivity and specificity, the method required an elaborate multistep protocol [ ] . a portable na sensor based on sprcd (coupler and dispenser) with a special diffraction grating was developed by sipova et al. nonamplified mir- extracted from mouse liver tissue was captured by thiolated dna immobilized on the sensor surface. a specific antibody recognized dna/ rna-formed hybrid, enhancing the response and allowing the detection in less than min and down to pm (results that were in good agreement with those obtained using qpcr) [ ] . although the two described assays were developed for the detection of mirna from mouse liver, they represented excellent model systems for the improvement of analytical performance (e.g., dls lowering) suitable for next applications to detection of human mirna samples. since current methods for the detection of pathogens are expensive, time consuming, and involve culture-based techniques [ ] [ ] [ ] , spr platforms were also employed for the survey of bacteria (e.g., escherichia coli) in environmental monitoring and in clinical diagnostics, through the detection of bacterial genetic material as biomarker [ ] . kai et al. developed a method for the rapid detection of verotoxin-producing escherichia coli o :h isolated from stools, amplified by pcr, and using te as sample buffer. biotinylated pnas (bioreceptors) were immobilized on a streptavidin-coated sensorchip sa of biacore . a good correlation was found with positive results for pcr ( cfu per . g of stool) samples from patients and healthy carriers. the sensor was reusable for times after regeneration with a washing solution ( mm naoh) [ ] . wang et al. detected simultaneously four pathogenic microorganisms (pseudomonas aeruginosa, staphylococcus aureus, clostridium tetani, and clostridium perfringens). dna was extracted from the four pathogens, amplified simultaneously by universal primers ( s rna) and then hybridized on a spr-based multichannel sensor with specific dna probes immobilized by thiol chemistry. the assay was specific, fast, and with dls down to pm (for c. tetani). in addition, a good linear dependence (r > . ) was recorded between signals and pcr products concentrations ( . - nm). although the dna was extracted from commercial bacterial strains and not from human matrices, the study clearly showed potential of multi array for high analytical productivity in clinical diagnostics ( min for four specific interactions) [ ] . finally, in zhang et al. developed a label-free and sensitive method for the detection of inva gene, extracted from bacterial culture of salmonella and amplified by pcr. biotinylated ssdna probes were immobilized on streptavidin-coated dextran chip surface of a biacore x. the sensor was previously calibrated with synthetic target dna and a good linearity from o nm and a detection limit of . nm were assessed. detection of salmonella was possible as low as cfu/ml within . h and the excellent regeneration of sensor surface (up to cycles binding/ regeneration) allowed sensor reuse with cost reduction [ ] . beyond the identification of pathogens via na detection, other approaches based on the developing of different spr immunosensors were reported. in particular, we reviewed the pathogens detection in real matrices, and examples in buffer solution were reported only when these approaches were found very promising. viruses spr-based detection of antibodies against viral pathogens in medical diagnostics was previously reviewed by homola in . in particular, the review focused on antibodies detection for hepatitis g and c, hepatitis b, herpes simplex virus type (hsv- ) and type (hsv- ), epstein-barr virus, varicella-zolter virus, human respiratory syncytial virus, and adenovirus [ ] . antibodies for herpes simplex virus type (hsv- ) and type (hsv- ) were detected with biacore x in diluted human serum ( : in hbs buffer). biotinylated peptides, from corresponding segments of the n-terminus of hsv- and hsv- glycoprotein b (gb), were immobilized as antigen on chips coated with streptavidin. spr biosensor discriminated specific ab/ag binding better than conventional reference method (elisa), evaluating low-avidity background reactivity recorded frequently in human sera [ ] . abad et al. detected anti-adenoviral antibodies (rad/p ) in diluted sera of patients ( : in hepes buffer) after dosing with an adenoviral-based gene therapy vector. dextran and amino-coupling chemistry were used for the immobilization of intact virus on chip surface of a biacore . the assay was useful for designing more efficient dosing schedules automated and requiring less analysis set-up time than reference elisa method [ ] . antibodies detection against hepatitis g in patient serum was performed by rojo et al. with synthetic peptide (antigens) immobilized on biacore via dextran and aminocoupling chemistry, finding a good correlation with the elisa reference test [ ] . the same chemistry of immobilization was used by mcgill et al. for the detection of human respiratory syncytial virus (rsv) in diluted serum ( : in hbs buffer) with biacore . antibodies against virus glycoproteins (f and g) were covalently immobilized on dextran for the discrimination of the antigenic differences between the two virus genotypes (f-and g-glycoproteins) [ ] . an indirect assay was described by chung et al. for the detection of antibodies against human hepatitis b virus (hhbv) in diluted patient serum ( % in pbs). hhbvantigens were immobilized (via aminocoupling) on the spreeta surface platform and dls of . nm or . nm (after amplification with avidin-biotinylated secondary antibodies) were achieved, with very similar results of the reference elisa kit for the diagnosis of hepatitis [ ] . regnault et al. developed an assay for the detection of antiprotein s antibodies, expressed after varicella-zolter viral diseases in diluted plasma ( % in hbs buffer) of infected patients. a dextran layer was employed for the immobilization of protein s via amine coupling on the surface (with biacore x). high binding ( ru) was observed in serum of patients whereas low signals ranging from . to ru were recorded in plasma from healthy donors [ ] . vaisocherová et al. described the detection of antibodies for epstein-barr virus (anti-ena) in diluted human serum ( % in pbs buffer) with a home-built spr sensor platform based on the wavelength division multiplexing (wdm). synthetic peptides (bioreceptors) were immobilized on the surface via hydrophobic and electrostatic interaction and anti-ebna antibodies were detected down to . μg/l (∼ pm). inter-chips rsds% of the sensor response was estimated to be within the %- % range whereas intra-chip rsd% was found to be lower (∼ %), showing a good reproducibility in both situations. in summary, the analytical performances were comparable with that of a conventional peptide-based immunoassay (elisa) [ ] . kumbhat et al. developed an immunosensor for the detection of dengue virus specific igm antibodies in infected patients serum (different dilutions in pbs), using an autolab model springle. dengue antigen-bsa conjugates were immobilized as bioreceptor via aminocoupling on gold sensing surface functionalized with a mercaptoundecanoic sam. in addition, the sensor was regenerable with pepsin solution in glycin-hcl buffer (ph . ), and the results were comparable with those obtained by mac-elisa [ ] . nilsson et al. designed an inhibition assay (indirect) for the quantification of hemagglutinin (ha), a glycoprotein on the capsid of the seasonal influenza viruses. recombinant ha proteic ags (a/h n , a/h n , and b) were immobilized via aminocoupling on the dextran matrix of a biacore t chip while specific antibodies were mixed in serum solution of virus antigen (fig. ). the detection limit was . mg/l and the method resulted in more precise (intra-cv= %) and faster ( h) than single-radial immune-diffusion (srid) reference method (intra-cv= %; analysis time= h) [ ] . park et al. reported a preliminary study in pbs buffer for the detection of sars coronaviral envelope protein (scvme) in the diagnosis of severe acute respiratory syndrome (sars). scvme was anchored to gold chip surface (spri, k-mac) by gold binding protein (gbp) and selective and sensitive detections of anti-scvme antibodies were performed down to μg/l (dl) in min. afm and spr imaging analyses confirmed the anti-scvme-specific capturing by antigen domain with an appropriate orientation [ ] . wang et al. employed high-resolution spr microscopy (sprm) for the label-free and imaging detection of single viruses particles (h n influenza a/pr/ / and hcmv) in pbs buffer down to ∼ . fg/mm (mass dl). anti-influenza a antibodies were covalently immobilized on peg/peg-cooh coated gold chip surface via aminocoupling. this study demonstrated the suitability of the method to assess masses and binding activity of individual viral particles [ ] . in this section detections of pathogenic bacteria or other microorganisms were reviewed focusing on immunological assays for direct pathogen or anti-pathogen antibodies detection. salmonella, a frequent gastrointestinal pathogen involved in many diseases such as diarrhea, gastrointestinal inflammation, and life-threatening typhoid fever [ ] , has been widely reported in literature coupled to spr-based sensing. a spr-based sandwich immunoassay was developed in pbs buffer by muzumdar et al. for serotyping of salmonella b, c, d. polyclonal antibody for salmonella, immobilized on the spr chip (plasmonic biosensoren ag), captured the salmonella bacterial cells further probed with antigenspecific antibodies for serotyping. the method was rapid and quantitative with a detection limit of about cell/ml, and the spr immunosensor provided more comprehensive and quantitative information than conventional slide agglutination test (sat) [ ] . more recently ( ), gupta et al. designed an immunosensor for salmonella typhi antibody detection in pbs buffer and then in patients sera. salmonella typhi antigen was immobilized on a -mercaptobenzoic acid ( -mba) modified gold surface chip of an electrochemical surface plasmon resonance system (autolab esprit). the discrimination between positive (widal positive) and negative sera (widal negative) was achieved in less than min. experimental dls were, respectively, : dilution for mab and : dilution for salmonella typhi antibodies, and a good reproducibility ( measures) was recorded (cv%= . %) [ ] . boer et al. detected glycan-specific serum antibodies, biomarkers of exogenous pathogens, in human sera infected by the parasite schistosoma mansoni. sera were diluted ( : in pbst) while glycans samples were printed on an epoxide-activated chip (biacore flexchip). different serum antibody profiles between infected sera and control sera were revealed in real-time and without fluorescent labeling [ ] . up to now, isi web of knowledge reported almost , publications related to spr sensing and, if we analyze the distribution of the topics, the technology impacts significantly on clinical chemistry. however, studies demonstrate proofs of principle and only very recently, reports on applications to real matrices have appeared. in this review, we considered a panel of analytes of interest for clinical diagnostics, also focusing on emerging technological innovations such as coupling between spr and nanoparticles, microfluidics, or new chip design, for improving the analytical performance. the detection of hormones related to endocrine disorders, protein biomarkers in relation to various conditions (inflammation states or traumas) or, more specifically, for cancer and overlay plot showing sensorgrams of injected serum mixed with a concentration series of virus standard. report points (marked as x) are taken before and after injection and response levels are measured between those (arrow). after each injection, the surface is regenerated in preparation for a new injection. reprinted with permission from elsevier [ ] cardiac diseases were discussed as case studies. antibody detection was also reported in relation to diagnosis of infections and immune system diseases. in addition, we reviewed nucleic acid sensing for the detection of genetic disorders, but also for recognizing specific sequences for pathogens (virus, bacteria) detection, or for mirna as emerging biomarkers. finally, we discussed immunosensing approaches for pathogens detection. in our selection, we privileged studies conducted on real matrices (blood, serum, and urine) with a few exceptions, when the analytical strategies appeared especially original and innovative. the development of innovative chip chemistry and antifouling strategies guarantees reduced nonspecific binding, which is essential for complex biological fluids analysis. amine coupling allows for covalent immobilization of receptors, ensuring repeated measurement on the same chip, after a simple dissociation of the receptor-ligand complex with chaotropic reagents. interestingly, sample pretreatment is reduced to a minimum: dilution, sometimes a short heating or filtration. in summary, spr-based biosensors offer analytical performances (sensitivity, dls, and reproducibility) comparable to conventional methods employed in clinical analysis (electrophoresis, chromatography, elisa, ria, etc.); in addition, they ensure real-time monitoring, label-free solutions, parallel analysis (spri), high throughput, little sample pretreatment, fast responses, and cheapness. for all these reasons, we believe that in the near future, spr will emerge as an efficient, powerful, and alternative tool for daily routine clinical analysis, opening also new horizons for future developments in personalized medicine and in point of care diagnostics. advances in surface plasmon resonance biosensor technology towards high-throughput, food-safety analysis recent developments and applications of surface plasmon resonance biosensors for the detection of mycotoxins in foodstuffs surface plasmon resonance for detection of genetically modified organisms in the food supply direct detection of e. coli o :h in selected food systems by a surface plasmon resonance biosensor surface plasmon resonance in doping analysis realtime and label-free bio-sensing of molecular interactions by surface plasmon resonance: a laboratory medicine perspective cancer biomarker detection by surface plasmon resonance biosensors proteomic applications of surface plasmon resonance biosensors: analysis of protein arrays surface plasmon resonance mass spectrometry in proteomics rapid and label-free bacteria detection by surface plasmon resonance (spr) biosensors recent advancements in surface plasmon resonance immunosensors for detection of small molecules of biomedical, food and environmental interest emerging optofluidic technologies for point-of-care genetic analysis systems: a review surface plasmon resonance sensors: review advances in surface plasmon resonance biosensor analysis present and future of surface plasmon resonance biosensors recent advances in surface plasmon resonance based techniques for bioanalysis surface plasmon resonance sensors for detection of chemical and biological species surface plasmon resonance imaging surface plasmon resonance imaging for affinity-based biosensors surface plasmon resonance imaging measurements of ultrathin organic films surface plasmon resonance imaging as a tool to monitor biomolecular interactions in an array based format on a remarkable case of uneven distribution of light in a diffraction grating spectrum radiative decay of non-radiative surface plasmons excited by light excitation of nonradiative surface plasma waves in silver by the method of frustrated total reflection surface plasmon resonance sensing of nucleic acids: a review immunosensors-principles and applications to clinical chemistry surface plasmon resonancebased immunoassays surface plasmon resonance imaging for nucleic acid detection artificial dna and surface plasmon resonance new trends in affinity sensing: aptamers for ligand binding aptamer-based molecular recognition for biosensor development from oligonucleotide shapes to genomic selex: novel biological regulatory loops methods developed for selex advances in the manufacture of mip nanoparticles molecularly imprinted polymers and their use in biomimetic sensors the art of immobilization for spr sensors. in: homola j (ed) surface plasmon resonance-based sensors se- high salt stability and protein resistance of poly(l-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates surface plasmon resonance imaging: what next? nanostructured plasmonic sensors recent developments and applications of hybrid surface plasmon resonance interfaces in optical sensing surface plasmon resonance biosensors incorporating gold nanoparticles functionalized gold nanoparticles for ultrasensitive dna detection recent advances in the development of graphene-based surface plasmon resonance (spr) interfaces new trends in instrumental design for surface plasmon resonance-based biosensors surface plasmon resonance immunosensor for cortisol and cortisone determination determination of estriol -glucuronide in human urine with surface plasmon resonance and lateral flow immunoassays multifunctional au nanoparticle dendrimer-based surface plasmon resonance biosensor and its application for improved insulin detection determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay single-and multi-analyte determination of gonadotropic hormones in urine by surface plasmon resonance immunoassay surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples is anti-doping analysis so far from clinical, legal or forensic targets? the added value of close relationships between related disciplines protein biomarker discovery and validation: the long and uncertain path to clinical utility multiplexed surface plasmon resonance imaging for protein biomarker analysis analysis of c-reactive protein on amide-linked n-hydroxysuccinimide-dextran arrays with a spectral surface plasmon resonance biosensor for serodiagnosis development of an optical rna-based aptasensor for c-reactive protein surface plasmon resonance shows a gender difference in circulating annexin a in human multi-sequential surface plasmon resonance analysis of haptoglobin-lectin complex in sera of patients with malignant and benign prostate diseases comparative study of spr and elisa methods based on analysis of cd /alcam levels in cancer and control human sera label-free detection of cancer biomarker candidates using surface plasmon resonance imaging surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma quantification of p α map kinase: a prognostic marker in hnscc with respect to radiation therapy surface plasmon resonance biosensor for the detection of vegfr- -a protein marker of myelodysplastic syndromes real-time monitoring biomarker expression of carcinoma cells by surface plasmon resonance biosensors quantification of cytokines involved in wound healing using surface plasmon resonance brain natriuretic peptide as a novel cardiac hormone in humans: evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide, and brain natriuretic peptide surface plasmon resonancebased highly sensitive immunosensing for brain natriuretic peptide using nanobeads for signal amplification on-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system quantitative measurement of cardiac markers in undiluted serum surface plasmon resonance biosensor with high anti-fouling ability for the detection of cardiac marker troponin t biosensor approaches for the detection of autoantibodies in human serum novel biosensor-based analytic device for the detection of anti-double-stranded dna antibodies optical biosensor-based characterization of antidouble-stranded dna monoclonal antibodies as possible new standards for laboratory tests biosensor analysis of beta -glycoprotein ireactive autoantibodies: evidence for isotype-specific binding and differentiation of pathogenic from infection-induced antibodies standardized antigen preparation to achieve comparability of anti-β -glycoprotein i assays -glycoprotein i-derived peptides as antigenic structures for the detection of antiphospholipid antibodies covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum high affinity of anti-gbm antibodies from goodpasture and transplanted alport patients to alpha (iv)nc collagen a surface plasmon resonance biosensor assay for measurement of anti-gm( ) antibodies in neuropathy characterization of a selfassembled monolayer of thiol on a gold surface and the fabrication of a biosensor chip based on surface plasmon resonance for detecting anti-gad antibody an indirect competitive immunoassay for insulin autoantibodies based on surface plasmon resonance biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging mapping of citrullinated fibrinogen b-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance surface plasmon resonance imaging (spri)-based sensing: a new approach in signal sampling and management surface plasmon resonance imaging-based sensing for anti-bovine immunoglobulins detection in human milk and serum direct detection of carcinoembryonic antigen autoantibodies in clinical human serum samples using a surface plasmon resonance sensor large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome characterization of single-nucleotide polymorphisms in coding regions of human genes detection of tp mutation using a portable surface plasmon resonance dna-based biosensor a novel optical biosensor format for the detection of clinically relevant tp mutations streptavidin-enhanced assay for sensitive and specific detection of single nucleotide polymorphism in tp sub-attomole oligonucleotide and p cdna determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification detection of a k-ras point mutation employing peptide nucleic acid at the surface of a spr biosensor single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions single nucleotide polymorphism detection by optical dnabased sensing coupled with whole genomic amplification direct detection of point mutations in nonamplified human genomic dna biosensor technology for real-time detection of the cystic fibrosis w x mutation in cftr enhanced recognition of cystic fibrosis w x dna point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor point mutation detection by surface plasmon resonance imaging coupled with a temperature scan method in a model system surface plasmon resonance imaging as a multidimensional surface characterization instrument-application to biochip genotyping surface plasmon resonance imaging (spri) system and real-time monitoring of dna biochip for human genetic mutation diagnosis of dna amplified samples the iarc tp database: new online mutation analysis and recommendations to users ultrasensitive detection of dna by pna and nanoparticleenhanced surface plasmon resonance imaging direct detection of genomic dna by surface plasmon resonance imaging: an optimized approach polymorphisms in human mdr (p-glycoprotein): recent advances and clinical relevance a comprehensive investigation on common polymorphisms in the mdr /abcb transporter gene and susceptibility to colorectal cancer mdr gene c t polymorphism and cancer risk: a meta-analysis of case-control studies micrornas: genomics, biogenesis, mechanism, and function oncomirs: micrornas with a role in cancer microrna expression profiles classify human cancers micrornas: novel regulators in cardiac development and disease microrna regulatory networks in cardiovascular development plasma microrna profiling reveals loss of endothelial mir- and other micrornas in type diabetes a microrna feedback circuit in midbrain dopamine neurons plasma microrna- as a biomarker for viral-, alcohol-, and chemical-related hepatic diseases multiplexed detection methods for profiling microrna expression in biological samples streptavidin-enhanced surface plasmon resonance biosensor for highly sensitive and specific detection of microrna attomole microarray detection of micrornas by nanoparticle-amplified spr imaging measurements of surface polyadenylation reactions surface plasmon resonance biosensor for rapid label-free detection of microribonucleic acid at subfemtomole level a review on viral biosensors to detect human pathogens biosensors as innovative tools for the detection of food borne pathogens review of biosensors for foodborne pathogens and toxins label-free detection of s ribosomal rna hybridization on reusable dna arrays using surface plasmon resonance imaging detection of pcr products of escherichia coli o : h in human stool samples using surface plasmon resonance (spr) rapid labelfree identification of mixed bacterial infections by surface plasmon resonance label-free and high-sensitive detection of salmonella using a surface plasmon resonance dna-based biosensor detection of human serum antibodies against type-specifically reactive peptides from the n-terminus of glycoprotein b of herpes simplex virus type and type by surface plasmon resonance development of a biosensor-based method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors gb virus c (gbv-c)/hepatitis g virus (hgv): towards the design of synthetic peptides-based biosensors for immunodiagnosis of gbv-c/hgv infection detection of human respiratory syncytial virus genotype specific antibody responses in infants application of spr biosensor for medical diagnostics of human hepatitis b virus ( hhbv ) anti-protein s antibodies following a varicella infection: detection, characterization and influence on thrombin generation surface plasmon resonance biosensor for direct detection of antibody against epstein-barr virus surface plasmon resonance based immunosensor for serological diagnosis of dengue virus infection a novel assay for influenza virus quantification using surface plasmon resonance protein nanopatterns and biosensors using gold binding polypeptide as a fusion partner labelfree imaging, detection, and mass measurement of single viruses by surface plasmon resonance cellular aspects of immunity to intracellular salmonella enterica surface plasmon resonance (spr) as a rapid tool for serotyping of salmonella surface plasmon resonance immunosensor for the detection of salmonella typhi antibodies in buffer and patient serum serum antibody screening by surface plasmon resonance using a natural glycan microarray acknowledgments the authors acknowledge fondazione arpa progetto dolore and miur -prin biosensori realizzati con nanomateriali per una rapida identificazione di biomarcatori tumorali for financial support. key: cord- - chpmgry authors: leung, danny t. m.; lim, pak-leong; cheung, tak-hong; wong, raymond r. y.; yim, so-fan; ng, margaret h. l.; tam, frankie c. h.; chung, tony k. h.; wong, yick-fu title: osteopontin fragments with intact thrombin-sensitive site circulate in cervical cancer patients date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: chpmgry we investigated whether circulating osteopontin (opn) could be used as a biomarker for cervical cancer. we employed a monoclonal antibody (mab ) specific for the unique and intact thrombin-sensitive site in opn using an inhibition elisa. we found significantly higher levels of opn in cervical cancer patients in both the plasma (mean +/- sd, +/- ng/ml) and serum ( +/- ng/ml) compared to healthy subjects [ +/- ng/ml, from plasma samples (p < . ), and +/- ng/ml, from serum samples (p = . ), respectively]. similar results were obtained when the plasma from a bigger group ( individuals) of cervical cancer patients ( +/- ng/ml) were compared with the same plasma samples of the healthy individuals (p = . ). more significantly, the opn level was highest in stage iii-iv disease ( +/- ng/ml, from individuals; p = . ) and least and non-discriminatory in stage i ( +/- ng/ml, from individuals; p = . ). no such discrimination was found when a mab of a different specificity (mab ) was used in a similar inhibition elisa to compare the two groups in the first study; a commercial capture elisa also failed. the possibility that the target epitope recognized by the antibody probe in these assays was absent from the circulating opn due to protein truncation was supported by gel fractionation of the opn found in patients’ plasma: – kda fragments were found instead of the presumably full-length opn ( kda) seen in healthy people. how these fragments are generated and what possible role they play in cancer biology remain interesting questions. osteopontin (opn), known previously as spp (secreted phosphoprotein ) or eta- (early t-lymphocyte activation protein ), was initially identified as an extracellular matrix protein produced by osteoclasts [ ] . it is now considered to be a pleiotropic, pro-inflammatory purchased from r & d systems (minneapolis, mn) (cat. no.: -op) and from sigma (st. louis, md) (cat. no.: o ). thrombin and glutathione-coupled sepharose b beads were obtained from amersham biosciences (piscataway, nj). protein g-coupled sepharose b beads, protease inhibitor cocktail, pmsf, tmb, pma, fitc-conjugated goat anti-mouse ig antibodies, and n-hydroxy-sucinimidobiotin were obtained from sigma. bio-gels of various retention limit [p ( kda), p ( kda), p ( kda), p ( kda)] were obtained from bio-rad laboratories (berkeley, ca). human opn assay kit was obtained from immuno-biological laboratories (ibl) co. ltd., gunma, japan. pre-operative paired plasma and serum samples were obtained with consent from a first cohort of patients diagnosed with cervical cancer [ patients with squamous cell carcinoma, patients with adenocarcinoma; ages ranging from - years (mean +/-sd = . +/- . years)], and from a second cohort of cervical cancer patients [ patients with disease stage i, ages ranging from - years (mean +/-sd = . +/- . years); patients with disease stage ii, ages ranging from - years (mean +/-sd = . +/- . years); patients with disease stage iii-iv, ages ranging from - years (mean +/-sd = . +/- . years)], from the department of obstetrics and gynecology, prince of wales hospital, hong kong. as controls, plasma samples from healthy women (ages ranging from - years (mean +/-sd = . +/- . years) and serum samples (ages ranging from - years (mean +/-sd = . +/- . years) sent for routine laboratory test were used. all samples were stored at - °c before use. all the data were analyzed anonymously. this study was approved by the the joint chinese university of hong kong-new territories east cluster clinical research ethics committee. bacterially derived recombinant human opn of n-terminal and c-terminal half were generated as gst fusion proteins, opn-o-gst and opn-p-gst, respectively, in escherichia coli bl using the pgex- t expression vector (amersham biosciences) as previously described [ ] . cdna obtained from hep- cells was pcr-amplified to generate dna fragments using the following forward (f) primers and reverse (r) primers: opn-o [(aa - ), f, '-cgtgg atccatgagaattgcagtgatttgc- '(containing a bamh site, underlined); r, '-c gatgaattccgcgaaacttcttagattttga- ' (containing an ecor site, underlined)], and opn-p [(aa - ), f, '-cgtggatcctcaaaatctaagaagtttcgc- ' (containing a bamh site, underlined); r, '-aattcccggggattaattgacctcagaa gatgc- ' (containing a smai site, underlined)] and subcloned into the pgex- t vector. the fusion proteins derived from the above constructs were induced with isopropyl-β-d-thiogalactopyranoside, purified by affinity chromatography, and examined on sds-page by commassie blue staining and wb. insect-cell derived recombinant human opn were generated as v peptide-tagged (v ) fusion proteins (opn-o-v or opn-p-v ) in sf using the baculodirect baculovirus expression system according to manufacturer's instruction (invitrogen). cdna obtained from hep- cells was pcr-amplified to generate dna fragments using the following forward (f) primers (containing a kozak sequence, underlined) and reverse (r) primers: opn-o (f, '-ccaccat gggaatgagaattgcagtgatttgc- '; r, '-gcgaaacttcttagattttga- '), and opn-p (f, '-ccaccatgggatcaaaatctaagaagtttcgc- '; r, '-attgac ctcagaagatgcact- ') and subcloned into the entry vector pcr /gw/topo (invitrogen). purified plasmid with insert of right orientation was recombined with the baculodirect linear dna by incubation with lr clonase overnight at room temperature. sf cells growing at log-phase were transfected with the recombined dna mixture by cellfectin (invitrogen) and selected with mm ganciclovir. low titer of virus preparation (p viral stock) bearing the opn-o or opn-p gene constructs were harvested from the spent culture supernatant at day - post-transfection under ganciclovir selection, where less than % of sf cells was infected as revealed by anti-v antibody staining in ifa. high titer virus preparation (p viral stock) was harvested from sf cells inoculated with an aliquot of p viral stock for - days under ganciclovir selection where - % of cells were infected. fusion proteins were obtained from lysates of sf cells incubated with p viral stock without selection ( hr at °c) where almost % of cells were infected at the time of harvest as revealed by anti-v antibody staining. cytosolic extracts were prepared by incubating the infected sf cells with lysis buffer ( % np- , mm nacl, mm tris, ph . , x protease inhibitor, mm pmsf) at °c for min, followed by centrifugation ( , rpm, min, °c) the culture supernatant of hl ( x /ml) cells with or without treatment with pma (ng/ml) for hr was harvested and concentrated : the original volume by centrifugation at , rpm for min at °c (centricon ym- , kda cutoff; millipore, billerica, ma). samples were stored at - °c before analysis. balb/c mice were hyperimmunized with purified bacterially-derived opn-o-gst or opn-p-gst protein, and spleen cells obtained from these animals were fused with ns myeloma cells as described [ ] . hybridomas obtained were screened for reactivity against insect cell-derived recombinant proteins (opn-o-v or opn-p-v ) by direct-binding elisa; positive hybridomas were further characterized by ifa and wb. monoclonal antibodies from selected clones (mab , mab , mab ) were obtained from the spent-culture supernatant or ascites fluid and purified by ammonia sulfate precipitation. in some cases, the antibodies were protein-g selected and biotinylated using n-hydroxy-sucinimidobiotin. opn was detected by determining the ability of the test sample (human serum or plasma, or hl -derived culture supernatant) to block the binding of the opn-specific indicator antibody (mab , mab or mab ) to the insolubilized antigen [bacterially-derived opn-o-gst (for mabs and ) or opn-p-gst (for mab ); ns -derived fulllength opn]. the concentration of indicator antibody used was determined previously by titration of the antibody against the respective antigen, based on % od max . thus, wells of -well elisa plates (immunlon ii; dynatech, chantilly, va) were coated with the different antigens ( μg/ml) at °c using bicarbonate buffer (ph . ). fifty μl of diluted sample (for plasma and serum, dilution ratio = : . ) were mixed with μl of diluted indicator antibody, and incubated with the immobilized antigen for hr at °c. after washing, bound indicator antibody was detected using hrp-conjugated goat anti-mouse ig ( : ) ( . hr at °c) and tmb, and the results read at od nm in an elisa reader. a calibration curve was constructed for each experiment using serial dilutions of full-length opn ( to ng/ml). this was employed to determine whether mab and mab shared a common binding site in opn. thus, μl of serially diluted mab (competitor) were mixed with μl of biotin-coupled mab of a pre-determined concentration and incubated with immobilized opn-o-gst at °c overnight. cold (unbiotinylated) mab was used as positive control. following washing, bound indicator antibody was detected by incubation with μl hrpconjugated streptavidin ( : ) for . hr at °c, and the reaction developed using tmb; the results were read at od nm. binding specificity was further confirmed by a reverse assay, using mab as competitor, and biotin-coupled mab as indicator. cytospin preparations of sf cells expressing opn-o-or opn-p-v protein were methanolfixed, blocked with . % bsa and stained with the reagent mab for min at room temperature. following incubation with fitc-conjugated goat anti-mouse ig antibodies ( : ) for min at room temperature, the cells were examined by uv microscopy [ ] . anti-v antibody (invitrogen) was used as a positive control. recombinant proteins or sf cell lysates were treated in reducing sample buffer at °c for min, resolved on - . % sds-page gel, and then transferred to pvdf membranes. the membranes were blocked with . % bsa and later incubated with the detecting mab (mab or mab ) at °c overnight; following this, the membrane was incubated with hrp-conjugated goat anti-mouse ig antibodies for hr at room temperature. membranes were then thoroughly washed and used for film development by ecl chemoluminescence (amersham biosciences). full-length opn ( ng) was incubated with graded doses of thrombin ( , . , . , . , . , and . u/ml) in tris-buffer, ph . at °c for hr. after heating with reducing sample buffer at °c for min, the samples were resolved on - . % sds-page gel, transferred to pvdf membranes, and examined by wb. in the mab-protection study, opn ( ng) was preincubated with graded doses of mab in tris-buffer, ph . , at °c overnight, followed by incubation with u/ml thrombin at °c for hr. samples were then treated with reducing sample buffer at °c for min, resolved on - . % sds-page gel, transferred to pvdf membranes, and examined by wb. mini-columns ( ml bed-size) were prepared using bio-gel (bio-rad laboratories, berkeley, ca) of different retention limits: p ( kda), p ( kda), p ( kda), p ( kda). these were thoroughly equilibrated with pbs ( μl) by repeated ( x) loading and subsequent centrifugation ( x g, min). each column was first calibrated using blue dextran and dnplysine. in the experiment, μl human plasma was loaded to each column and centrifuged ( x g, min). the eluate ( μl) was collected and the column was loaded with pbs ( μl) again, centrifuged and the eluate collected. the procedure of pbs-loading was repeated, so that, for each sample, such eluates (fractions) were obtained. the opn concentration in each fraction was determined using the mab inhibition elisa. as a control, bovine serum albumin ( mg/ml) was also run through each column using the same procedure, but the protein content in the fractions was determined using the bicinchoninic acid (bca) protein assay kit (pierce, rockford). microdissection of the tumor tissue, rna extraction from the processed tissue, as well as reverse transcription and real-time pcr of the extracted rna, were all performed as described [ ] . comparison of difference between two groups was performed using the unpaired t-test. correlation between groups of data was performed using regression analysis, and a p-value of < . was considered statistically significant. variable data were expressed by scatterplot analysis (graphpad prism , graphpad software, inc., san diego, ca). recombinant opn proteins were produced in e. coli from dna cloned from hep- cells. thus, two gst-linked opn fragments were obtained, one representing roughly the n-terminal half (opn-o) and the other, the c-terminal half (opn-p) of the protein (fig a and b) . corresponding proteins of these fragments (opn-o-v , opn-p-v ) were also expressed in insect cells ( fig c) . ten hybridomas were produced from mice immunized against opn-o or opn-p. of the clones that bound well to bacterial opn-o, mab and mab are igg antibodies which recognized linear epitopes (elisa + , ifa + , and wb + ), while mab is also an igg but it recognized a conformational epitope (elisa + , ifa + , wb -). all the opn-p-specific antibodies (mab , mab , mab , mab ) are igms which bound only to linear epitopes. mab , mab and mab bound specifically in an elisa to the recombinant opn antigen derived from both bacteria and insect cells (fig a and b) ; the binding was comparable to that for the full-length opn obtained commercially ( fig b) . mab and mab bound to different sites in opn-o since they did not cross-inhibit each other ( fig c) . with all three mabs, specificity of binding was also demonstrated by wb analysis ( fig d) and immunofluorescence-staining of insect cells transfected with the respective opn genes ( fig e) . we discovered, by chance, that mab recognized the evolutionary-conserved and unique thrombin-sensitive site (arg -ser ) in opn (see fig a) . first, it is known that digestion of opn by thrombin produces two fragments of roughly equal size. accordingly, we incubated intact opn (obtained commercially) with increasing amounts of thrombin and monitored the result using mab in an elisa to detect how much intact opn remained: increasing degradation was observed with increasing enzyme (fig a) . in contrast, this effect was not observable when probed with mab (fig a) . secondly, using mab as probe, full-length opn could be observed as a kda band in western blots; this disappeared completely when the protein was pre-treated with u/ml thrombin (fig b, upper panel) , while smaller amounts of thrombin had little or no effect. when mab was used to probe the same blot where u/ ml thrombin had been used, there was also significant reduction in the amount of full-length opn but for reasons unclear, this did not disappeared completely; more importantly, an additional antigen of kda appeared (fig b, lower panel) . thirdly, mab was able to protect opn from thrombin digestion in a dose-dependent fashion (fig c) . concentration of detecting antibody used was pre-determined from the titration curves of these mabs (data not shown), chosen at % maximal binding (fig b) . full-length opn obtained commercially or produced from mouse ns cells as histidine -tagged proteins, was used as the standard. assay sensitivity was determined as the concentration of full-length opn (ng/ml) required to inhibit the maximal antibody binding by %. the sensitivities obtained for the different assays are shown in fig a. of particular note is the excellent detection by mab ( ng/ml) when opn-o-gst was used as substrate, compared to the poor detection by mab ( ng/ml); both antibodies detected the commercially-obtained full-length opn very poorly ( ng/ml). detection by mab was extremely poor with all opn substrates. inhibition elisas were developed using either mab or mab as the reagent antibody and bacterially-produced opn-o-gst as the substrate. both assays efficiently and specifically detected the appropriate opn produced from insect cells or mouse ns cells (fig b) . interestingly, however, mab was . fold more sensitive than mab in detecting the opn produced by hl cells (fig c) . we next used the mab opn-o-gst inhibition elisa to detect opn from the plasma of cervical cancer patients [ squamous cell carcinoma, adenocarcinoma, cervical intraepithelial neoplasia (cin) (grade iii)]. we found highly significant (p < . ) levels of opn (mean +/-sd, +/- ng/ml) in these patients compared to healthy subjects ( +/- ng/ml) (fig a) . based on sample size, this discrimination is highly significant (minimum no. of patients and control required for % confidence at . power is and , respectively). the assay sensitivity was %, and the specificity, %. similar results were found when the serum of these individuals was examined: the cancer patients ( +/- ng/ ml) had significantly (p = . ) higher levels of opn than healthy subjects ( +/- ng/ ml) (fig b) , the assay sensitivity and specificity being % and %, respectively. there is, in fact, good correlation (r = . , p < . ) between the plasma and serum opn levels in the cervical cancer patients (fig c) , even though the serum levels were significantly lower than the corresponding plasma levels. however, based on sample size, the discrimination between patients and control is not robust (minimum no. of patients and control required for % confidence and . power is and , respectively). for comparison, when mab was used in the inhibition based on plasma, there was no discrimination between the cancer and healthy groups (fig d) . this was also the case using a correlation between the small number of patients found positive by this kit or the mab inhibition elisa and the corresponding patients in the mab inhibition elisa (fig d and e) . we extended the mab inhibition elisa study using more cervical cancer patients and plasma only, this time identifying the stage of the disease as well in order to make a more precise correlation between opn level and disease. thus, again, there was excellent discrimination (p = . ) between the new cohort of patients ( +/- ng/ml) and the healthy subjects (mean +/- ng/ml) (fig f) . more interestingly, if the patients are subdivided according to the stage of the disease (fig f) , discrimination was seen in stage ii ( +/- ng/ml; p = . ) and, more pronouncedly, in stage iii-iv ( +/- ng/ml; p = . ), but not stage i ( +/- ng/ml; p = . ). tumor tissues were available from patients ( in stage i, in stage ii, in stage i) in this cohort which were used to determine the opn gene expression. as shown in fig g, there was good concordance (r = . , p = . ) between the plasma opn results and the opn gene expression results, thus validating the immunological detection. the finding that the mab inhibition elisa and the commercial opn kit could not detect elevated opn levels in the cancer patients suggested the possibility that the opn present could be fragmented i.e. the target sites for the antibodies used in these assays could be missing, whereas, by virtue of the design of the mab -based assay, the thrombin-sensitive site must be present. thus, we fractionated the plasma of cancer patients by gel chromatography using a small but long, thin column made of bio-gel of different pore sizes (p , p , p , p ). serial . ml fractions were collected and assayed for opn activity using the mab inhibition elisa. each column was pre-calibrated with blue dextran, dnp-lysine and bsa [a protein of similar molecular size ( . kda) to intact opn (around kda)]. as shown in fig a, bsa was totally excluded in p (exclusion limit, kda) and p ( kda); in both cases, the bulk ( %) of the protein appeared in fraction and the rest in fraction . in the p ( kda) and p ( kda) elution, however, bsa was not excluded but instead appeared mainly ( %) in fraction and in smaller amounts in fraction , but none thereafter. when the plasma pooled from four cervical cancer patients (selected on the basis of high opn activity) was similarly fractionated, only in p was opn totally excluded, with the bulk of the activity ( %) appearing in fraction ( fig a) . significantly, in p , the main opn activity ( %) shifted to fraction . in the p and p elution, however, very little activity was recovered from this fraction (fraction ); instead, there was a spread of activity from fraction to fraction , peaking at fraction (p ) or fraction (p ). collectively, the findings suggest that, in the cancer patients, opn exists not as an intact protein but rather as fragments of between kda and kda in size. interestingly, when the plasma pooled from healthy individuals was fractionated as above, the overall fractionation profiles obtained (fig a) were slightly different from those of the cancer patients. thus, total exclusion of opn activity was observed with both p and p , and the spread of activity in the included fractions of p and p was more limited (confined to fractions - ). collectively, this suggests a protein size very similar to that of bsa ( - kda). we performed western blotting of the p fractions obtained from both patients and control subjects (fig b) using mab as probe. the results are consistent with those of the elisa detection. significantly, in the patients, no activity was detected from fractions and , but fraction revealed an antigen of about kda in size that was present in small amounts. fraction contained a marginally-smaller antigen ( kda) in significantly greater abundance, while the antigen found in modest amounts in fraction was in turn marginally smaller in size ( kda). fraction had no activity. in contrast, by western blotting, in healthy subjects, the antigenic activity appeared earlier, in fraction (close to the void volume of the column), which forms the bulk of the activity from the sample, while the molecular size of this antigen (roughly kda) is bigger than those of the fragments found in patients. a smaller antigen of about kda was found in smaller quantities from the subsequent fraction (fraction ) while no activity was observed thereafter. we sought to answer an important question in cancer diagnosis: is opn present at diagnostic levels in the blood of cervical cancer patients? we found convincing evidence proving this. however, this was only true because we had used a very unique mab in a highly-sensitive but not-so-common detection system. this mouse antibody (mab ) is highly specific for the unique thrombin-sensitive site in opn, and opn is efficiently detected from a patient by its ability to block the binding between this antibody and the target antigen. thus, although healthy people were found to have nominal amounts of opn in their blood in the assay, cancer patients showed significantly higher levels depending on several factors. this discrimination was found in two separate cohorts of cervical cancer patients. plasma samples gave better results than serum in this assay, the reason being the fact that, as the blood clots to form serum, thrombin is activated which can digest the opn in the sample. presumably the conditions were not met for complete degradation of the protein. validity of the plasma mab inhibition elisa results is shown not only by the good correlation between these results and those of the corresponding serum samples, but also between these and the opn gene expression results. further validation is seen when the patients are re-grouped according to the stage of the disease: the opn levels found by the elisa increased with the severity of the disease. thus, the most severe of these, stage iii-iv, showed the highest level of antigen, while at the other extreme, the least severe, stage i, could not be distinguished from healthy individuals. indeed, the ability to detect stage i patients possibly poses the greatest challenge to all of immunological assays. the most important revelation of the study which sets it apart from past publications is the finding that opn circulates in our patients not as an intact protein but rather, as fragments of about - kda in size. these opn fragments, which by inference must bear the intact thrombin-sensitive site, are probably truncated somewhere at the n-terminal end of the protein based on the observation that two immunoassays used in parallel with the mab inhibition elisa had failed to detect similar opn increases in the cancer patients: ( ) an inhibition elisa which uses mab as the detecting reagent known to bind to the n-terminal half of opn (actual location unknown). ( ) a commercially-available capture elisa (human opn detection kit, ibl) which employed polyclonal antibodies directed at two sites in opn separated by amino-acids, one of which ( ipvkqadsgsseekq ) is situated at the n-terminal end of the protein (fig ) . since this capture assay detects ns -derived full-length opn as efficiently as the mab inhibition elisa, a likely reason for its failure is the absence of the n-terminal site in the patient's opn. the fact that this commercial kit was successfully used with other types of cancer previously [ ] suggests that opn might be cleaved differently (by different enzymes) in different types of cancer or in different individuals. there are many opn detection kits in the market but they all invariably use capture (sandwich) elisa. they can be different, however, in the pair of antibodies utilized not only in terms of fine specificity but also whether these are polyclonal or monoclonal in nature; most polyclonal antibodies target a very small peptide-segment of the protein. differences in the antibody pairs used can affect the type of opn fragment detected and hence the vast discordances among test kits for the same set of plasma samples [ ] . the inhibition elisa we developed is the first of its kind for opn detection. a distinct advantage of this format is the fact that the target site needs only be present in fragments or peptides as small as the site itself. it is instructive that at the start of our study, we had actually experimented with pairs of mabs (e.g. biotinylated-mab and -mab ) in capture eli-sas to detect plasma opn from patients, but none had succeeded. in general, inhibition assays are not common, but one which has proven efficacious in detecting typhoid fever is based on a rapid dual-particle system ('tubex') to detect a single specificity of antibody [ ] . the opn fragments were indirectly identified from pooled patient plasma by gel filtration. although the results are preliminary and need to be verified more robustly in terms of sample size and methodology, they nevertheless merit some discussion. to compensate for the imprecision of the fractionation, the bio-gel columns were carefully calibrated using known markers and the results were based on a comparison of the plasma samples between the cancer and healthy groups run under identical conditions. against this background, a notable difference in the p elution profiles between the two groups was indeed found: whereas in healthy people the bulk of the opn antigenic activity appeared in the void fraction (fraction ), in the cancer group, this appeared later (fraction )-suggesting an antigen smaller in size i.e. cleaved. support for this is seen when the p or p elution profiles between these groups are compared. thus, in the case of p , whereas in healthy people the main antigenic activity was found in fraction , this appeared much later in fraction in the cancer group, and there was a greater spread of activity. western blot analysis performed on the p fractions using the same antibody probe (mab ) confirmed the elisa findings. thus, the main antigenic activity was found in fractions and from the healthy and cancer groups, respectively. more instructively, the antigen found in patients was significantly smaller (about kda) than that observed in healthy people (about kda). it seems likely the latter is the intact, full-length opn even though it is slightly smaller than the intact opn ( kda) produced by hl cells used early in our study-this difference could be due to post-translational modification (see later). thus, the kda antigen found in patients is not intact but a major fragment. interestingly, this fragment was also observed in healthy people as a minor component. in the patients too, other fragments ( kda in fraction and kda in fraction ) were also found in smaller quantities. it is possible that these three antigens are actually the same fragment but have varying degrees of posttranslational modification. such modifications can increase the molecular weight of the protein considerably. this is highly possible with opn because although the protein is only roughly half as long (amino-acid) as bsa, it nevertheless has a very similar molecular weight. indeed, opn is known to be extensively phosphorylated because of which it is highly active biologically [ , ] . there are indeed many potential sites in the protein both for phosphorylation (about sites) and for glycosylation ( for o-glycosylation, for n-glycosylation) [ ] [ ] [ ] . in addition, the protein also undergoes sulfation and transglutamination. a possible candidate for the antigen found in the patients is the major fragment cleaved from whole opn by caspase- . this enzyme cleaves opn at two sites, asp and asp (fig ) , yielding two major peptides of and amino-acid long, respectively, but both could yield molecular weights greater than kda [ ] depending on the extent of post-translational modification. while these glycan or phosphate appendages mattered importantly to the linearized antigen in western blotting by way of increasing the molecular weight, they do not seem to affect the molecular size of the free-form antigen in solution. thus, in the p gel chromatography, the opn antigen appeared more like a globular protein with a molecular weight of less than - kda (i.e. a peptide with fewer than amino-acids) than a kda antigen as deduced by western blotting. the possibility that this antigen could be retarded in its passage through the gel due to interaction between the appended phosphate or carbohydrate groups and the bio-gel resin is belittled by the observation that the opn antigen from healthy people seemed to be unaffected. the surprising finding is the absence of intact opn in our patients. the argument that this could be present but is bound at the thrombin-sensitive site by a co-factor such as syndecan- [ ] or factor h [ ] and becomes masked as such, is ruled unlikely by the western blot results. this is because any factor bound to opn would have dissociated from it under the denaturing conditions used. the question arises whether the opn fragments found in our patients play a role in the cancer biology of the patient. on the one hand, this seems unlikely because the same fragment found in patients was also present in healthy people. indeed, the greater abundance of this fragment in patients may simply reflect the general heightened activity of tumor cells-not only more opn is produced, but also very quickly it becomes cleaved by the caspases and other enzymes in the tissue with the end result that vast amounts of opn fragments are generated and released to the circulation. it is not clear, however, how intact opn produced by other (non-cancerous) tissues becomes fragmented in our patients. on the other hand, there are numerous reports which described the potent biological activities of opn fragments. this includes the opn fragments generated by caspase- , which bear the rgd domain (fig ) that was recently shown to promote tumor growth and metastasis [ ] . another opn fragment which could remotely fit as the candidate antigen in our patients is the kda c-terminal peptide generated by mmp- , mmp- or mmp- (see fig ) [ ] ; this, again, could be bigger in molecular weight if it becomes glycosylated or phosphorylated. the tumorigenic potential of this fragment is not known but recently, takafuji et al. [ ] found a small opn fragment (residues - ) generated by mmp- which seemed able to induce tumor cell invasion via cd receptors in hepatocellular carcinoma [ ] . thus, the opn fragments which circulate in our cancer patients and which bear both the rgd domain and an intact thrombin-sensitive site, may be more important than just a diagnostic (or prognostic) biomarker-they could very well determine the biology of the cancer. cloning and sequence analysis of rat bone sialoprotein (osteopontin) cdna reveals an arg-gly-asp cell-binding sequence osteopontin: role in immune regulation and stress responses small integrin-binding ligand n-linked glycoproteins (siblings): multifunctional proteins in cancer autocrine activation of an osteopontin-cd -rac pathway enhances invasion and transformation by h-rasv osteopontin: regulation in tumor metastasis osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival the influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease osteopontin deficiency protects joints against destruction in anti-type ii collagen antibody-induced arthritis in mice the bridge between dendritic cells and asthma systemic endocrine instigation of indolent tumor growth requires osteopontin osteopontin is a marker for cancer aggressiveness and patient survival lentiviral-mediated mirna against osteopontin suppresses tumor growth and metastasis of human hepatocellular carcinoma osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells intracellular cleavage of osteopontin by caspase- modulates hypoxia/reoxygenation cell death through p an osteopontin splice variant induces anchorage independence in human breast cancer cells an osteopontin fragment is essential for tumor cell invasion in hepatocellular carcinoma osteopontin-c is a selective marker of breast cancer role of the integrin-binding protein osteopontin in lymphatic metastasis of breast cancer osteopontin overexpression in breast cancer: knowledge gained and possible implications for clinical management osteopontin expression in lung cancer correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies osteopontin expression in ovarian carcinomas and tumors of low malignant potential (lmp) genome-wide gene expression profiling of cervical cancer in hong kong women by oligonucleotide microarray osteopontin expression correlates with invasiveness in cervical cancer clinical significance of osteopontin expression in cervical cancer clinical implications of osteopontin in metastatic lesions of uterine cervical cancers plasma osteopontin as a biomarker of prostate cancer aggression: relationship to risk category and treatment response post-operative plasma osteopontin predicts distant metastasis in human colorectal cancer identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas prognostic significance of plasma osteopontin in patients with locoregionally advanced head and neck squamous cell carcinoma treated on trog . phase iii trial new dual monoclonal elisa for measuring plasma osteopontin as a biomarker associated with survival in prostate cancer: clinical validation and comparison of multiple elisas antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid molecular mimicry: anti-dna antibodies may arise inadvertently as a response to antibodies generated to microorganisms extremely low exposure of a community to severe acute respiratory syndrome coronavirus: false seropositivity due to use of bacterially derived antigens combined rapid (tubex) test for typhoid-paratyphoid a fever based on strong anti-o response: design and critical assessment of sensitivity regulation of vascular calcification: roles of phosphate and osteopontin control of osteopontin signaling and function by posttranslational phosphorylation and protein folding post-translationally modified residues of native human osteopontin are located in clusters: identification of phosphorylation and five o-glycosylation sites and their biological implications cell type-specific post-translational modifications of mouse osteopontin are associated with different adhesive properties post-translational modification and proteolytic processing of urinary osteopontin syndecan- protects against osteopontin-mediated acute hepatic injury by masking functional domains of osteopontin factor h binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack the rgd domain of human osteopontin promotes tumor growth and metastasis through activation of survival pathways osteopontin, a novel substrate for matrix metalloproteinase- (stromelysin- ) and matrix metalloproteinase- (matrilysin) activation of the osteopontin/matrix metalloproteinase- pathway correlates with prostate cancer progression we thank mr. man-hin leung for technical assistance. this work was supported in part by a direct grant from the chinese university of hong kong to yfw. designed assays/experiments: pll dtml. key: cord- - alsigxk authors: okda, faten; liu, xiaodong; singrey, aaron; clement, travis; nelson, julie; christopher-hennings, jane; nelson, eric a.; lawson, steven title: development of an indirect elisa, blocking elisa, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to north american strains of porcine epidemic diarrhea virus date: - - journal: bmc vet res doi: . /s - - -z sha: doc_id: cord_uid: alsigxk background: recent, severe outbreaks of porcine epidemic diarrhea virus (pedv) in asia and north america highlight the need for well-validated diagnostic tests for the identification of pedv infected animals and evaluation of their immune status to this virus. pedv was first detected in the u.s. in may and spread rapidly across the country. some serological assays for pedv have been previously described, but few were readily available in the u.s. several u.s. laboratories quickly developed indirect fluorescent antibody (ifa) assays for the detection of antibodies to pedv in swine serum, indicating prior exposure. however, the ifa has several disadvantages, including low throughput and relatively subjective interpretation. different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. therefore, the objective of this study was to develop and validate multiple improved serological assays for pedv, including an indirect elisa (ielisa); a highly specific monoclonal antibody-based blocking elisa (belisa); fluorescent microsphere immunoassays (fmia) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (ffn) to measure functional virus neutralizing antibodies. results: a recombinant north american nucleoprotein (np) based ielisa was developed and validated along with a belisa using newly developed pedv-np specific biotinylated monoclonal antibodies (mabs) and an fmia using magnetic beads coupled with expressed na pedv-np. receiver operating characteristic (roc) analysis was performed using swine serum samples (ielisa n = , belisa n = , fmia n = ). the roc analysis for the fmia showed estimated sensitivity and specificity of . and . %, respectively. the ielisa and belisa showed a sensitivity and specificity of . and . %; and . and . %, respectively. inter-rater (kappa) agreement was calculated to be . between ielisa and ifa, . between belisa and ifa and . between fmia and ifa. similar comparative kappa values were observed between the ielisa, belisa and fmia, which demonstrated a significant level of testing agreement among the three assays. no cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (tgev) or porcine respiratory coronavirus (prcv) was noted with these assays. all three assays detected seroconversion of naïve animals within – days post exposure. the ffn assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples. conclusion: well-validated ielisa, belisa and fmia assays for the detection of pedv antibodies were developed and showed good correlation with ifa and each other. each assay format has advantages that dictate how they will be used in the field. newly developed mabs to the pedv-np were used in the belisa and for expediting ffn testing in the detection and quantitation of neutralizing antibodies. in addition, these pedv mabs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests. measurement of neutralizing antibody responses using the ffn assay may provide a valuable tool for assessment of vaccine candidates or protective immunity. porcine epidemic diarrhea virus (pedv) was first described in europe in the s with more recent and severe outbreaks in asia [ , ] . the virus was identified in the united states in may , causing severe diarrhea and vomiting in pigs across age groups, with high mortality of up to − % in suckling pigs [ ] . pedv is an enveloped, single stranded rna virus belonging to the coronaviridae family. the coronaviruses taxonomically form a subfamily (coronavirinae) within the order nidovirales. recently, the international committee on taxonomy of viruses (ictv) recognized four genera within the coronavirinae subfamily: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [ ] . pedv belongs to the genus alphacoronavirus along with other swine viruses including transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv). the genome is composed of a large~ kb molecule consisting of a ′ untranslated region (utr), a ′ utr, and at least seven open reading frames (orfs) encoding three nonstructural proteins: orf ab (pp a and pp ab) and orf , an accessory protein. the four major structural proteins of the mature virion include the spike (s) glycoprotein (mr - kda), the nucleoprotein (np) (mr - kda) that is associated with the positive stranded rna providing integral support for its helical structure, the glycosylated membrane protein (m) (mr - kda), and the glycosylated envelope protein (e) (mr kda) [ ] [ ] [ ] . coronaviruses are taxonomically assigned to different genera based on their rooted phylogeny and calculated evolutionary distance for seven highly conserved genomic domains within orf ab [ ] . the genetic diversity of coronaviruses may be due to their high frequency of recombination [ ] . the heterogeneity among coronavirus subfamilies is well documented [ ] , and the factors that contribute to pedv's ability to gain or lose parts of its transcriptome are believed to have contributed to quasispecies with novel traits that are able to adapt to new hosts, ecological niches and zoonotic events. the exact origin of pedv in north america is not entirely clear, but there is evidence of genetic similarities to chinese pedv strains [ ] . recently, a novel na pedv recombinant strain was identified (s indel) containing both insertions and deletions within the n-terminal domain of the orf and s genes. specifically, sequence alignment indicated spike gene nucleotide deletions at positions - that correspond to amino acid deletions at positions and in addition to substitutions at positions (i), (h), (k), and (e) as compared to the cv strain [ , ] . the relatedness of several pedv strains circulating in china was evaluated by li et al. [ ] using phylogenic analysis of the np gene and no insertions or deletions were noted. sequence comparison with other european and korean pedv strains obtained from genbank indicated that the np genes were highly conserved ( . − . %) even though these strains originated from different geographic regions [ ] . in addition to being highly conserved among pedv variants, the np is the most abundant viral protein expressed in pedv infected cells [ , ] . in contrast, the spike protein is presented on the viral surface and subject to various host immune pressures, which predisposes it to a greater range of genetic heterogeneity including insertions and deletions. because the np protein is highly abundant in virus infected cells, it provides an attractive target for the development of antigen-based serological assays. taken together, this evidence provided rationale for using it as our antigen of choice for the ielisa, belisa and fmia. in response to the recent outbreaks of highly virulent pedv in north america (na), pcr assays were quickly developed to detect the presence of pedv rna in intestine or fecal material. these assays provide an important tool in control of the virus; however, well-validated, high-throughput assays to detect antibodies following infection would provide additional valuable diagnostic tools for the swine industry. the ability to detect and evaluate antibody responses using serologic tests is important in efforts to answer basic production related questions. these questions may include whether a production site is naïve or has historically experienced a pedv exposure, even though a producer has not seen obvious clinical signs; the level of immune response sows may have in relation to vaccination, initial wildtype virus infection or intentional feedback exposure; and whether sow immunity is inadequate when clinical infection occurs in individual litters after initial pedv exposure in a herd. one of the most pressing issues of pedv disease is maintaining herd site biosecurity through exclusion measures to prevent viral entrance into swine units. however, pedv infection may not always be obvious in finishing pigs so the widespread transport of these animals may represent additional risks. thus, sensitive serological tests provide a valuable tool in the detection of recent infection to avoid the introduction of these animals into naïve herds. since pedv was widespread in europe in the s and s and more recently in asia, various serologic tests have been developed and subjected to varying degrees of validation [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, few assays have been developed using antigens associated with contemporary strains currently circulating across na. the need to develop more sensitive serological assays has become paramount in order to address questions regarding pedv infections and epidemiological transmission patterns, as well as to analyze disease progression. currently, serum virus neutralization (svn) tests are the most widely employed serological assays used to detect pedv antibodies. it is a test that is highly specific and useful for screening of antibody titer post vaccination [ , ] . however, the test is expensive and labor intensive, requiring manual reading and interpretation of virus induced cytopathic effect (cpe) endpoints. moreover, serum cytotoxicity can be mistaken for viral cpe, giving rise to false interpretations at lower serum dilutions. several laboratories have generated in-house indirect elisas using either virus derived antigen or recombinant structural proteins. early indirect elisas were developed using vero cell derived, whole virus preparations [ , ] or vero cell expressed viral proteins [ ] . these methods may be problematic because serum from animals vaccinated with cell culture derived pedv may cross-react with cellular components of elisa antigen, causing low specificity and high background. other groups have used recombinantly expressed, purified, structural s and np proteins for ielisa serodiagnosis, but because low numbers of experimentally derived samples were used to evaluate the performance of the assay, full validation of the diagnostic sensitivities and specificities could not be assessed [ , ] . both the ielisa and belisa formats have proven useful for the serodiagnosis of experimental and natural infections. blocking or competitive elisas have been shown to be especially useful where a higher level of specificity is required. the increased specificity has been shown to be dependent on both the isotype and on the target specificity of the monoclonal or polyclonal antibodies [ , , ] . various laboratories have developed sensitive blocking elisas, and carvajal et al. [ ] demonstrated their belisa was able to detect an antibody response to days earlier than ifa, which suggested higher sensitivity of the belisa. in addition, the belisa is valuable as a confirmatory test where unexpected positive results appear in presumably negative herds. the fluorescent microsphere immunoassay is based on fluidic, particle array technology (luminex corp., austin, tx) and has become increasingly standardized and accepted in applications involving the serologic diagnosis of autoimmune and animal infectious diseases [ , ] . there are distinct advantages of the fmia over the elisa, which include higher sensitivity, higher sample throughput analysis, and the ability to multiplex and monitor exposure to multiple pathogens simultaneously in a single sample. in addition, multiple bead sets in the fmia could be added to a standardized assay against newer virus subtypes that continue to emerge in the field or to assess antigenic/phylogenetic differences between genera of coronaviruses. in this study, we report the adaptation of a recombinant, highly purified, na pedv-np antigen to the development of ielisa, belisa and fmia platforms for the detection of pedv antibodies in serum. these assays provide high throughput serological tests designed to address pedv disease diagnostics. they were fully validated using a large number of serum samples of known status, and validation of the tests was detailed using methods for the validation of serological assays for the diagnosis of infectious diseases previously described by jacobson for the office international des epizooties [ ] . in addition, a fluorescent focus neutralization (ffn) assay was developed for the rapid evaluation of neutralizing antibody responses. procedures involving animals were approved by the south dakota state university institutional animal care and use committee (iacuc) under approval numbers - a and -a . time course swine serum samples provided by kansas state university were part of a separate pedv challenge study conducted at the biosecurity research institute approved by the kansas state university institutional animal care and use committee. all other serum samples were obtained as routine diagnostic sample submissions at the south dakota adrdl. for time course studies, serum samples from experimentally infected animals were obtained courtesy of dr. richard hesse (kansas state university veterinary diagnostic laboratory, national pork board grant # - ). thirty-three pedv naïve -week-old feeder pigs were obtained from a private, high-health status swine production farm. . of the pigs, were inoculated with pedv at weeks of age via intranasal and oral routes with a pool of gut derived intestinal contents that had been used as "feedback" inocula for controlled exposure of a sow herd. serum was collected prior to challenge and at days , , , , , , and days post-infection (dpi). multiple aliquots of all samples collected were shared with requesting laboratories to expand diagnostic testing and vaccine development capabilities. to accurately assess the diagnostic sensitivity and specificity of the assays, samples of known serostatus for pedv were used. this included sera from multiple animal populations including experimentally infected animals and serum samples from animals with known historical exposure to pedv that were submitted to the south dakota animal disease research and diagnostic laboratory (adrdl). pedv negative sample sets included samples from pedv negative control pigs used in experimental studies and selected high biosecurity herds with no history of pedv. in addition, archived serum samples collected prior to the emergence of pedv in the u.s., including samples testing positive for the related swine coronaviruses tgev and prcv (n= > ), were used. the exact number of positive and negative sera used for sensitivity and specificity calculations per assay with statistical testing agreement calculations based on serum numbers is listed in table . the majority of these sera were identical among assays, but limited serum volume did not allow for use of all sera samples among all assays. the development and validation of the ielisa and belisa made use of a recombinantly expressed full length na pedv-np. the np open reading frame (orf) of pedv was amplified from rna extracted directly from intestinal contents by rt-pcr from a case submitted to the south dakota adrdl. it was subsequently directionally cloned into the e. coli, pet a(+), plasmid expression vector (novagen, madison, wi), then transformed into bl -codon plus (de )-rp competent cells (stratagene, la jolla, ca) for protein expression. primers used for the amplification of the full length ( bp) nucleoprotein were: pedv-np-fwd ( ′-cg cggatccatggcttctgtcagttttcag- ′); ped v-np-rev ( ′-cacactcgagatttcctgtgtcgaa gatctc- ′). next, μl of transformed cells were plated onto luria-bertani agar plates containing μg of kanamycin/ml and incubated overnight. the following morning, colonies from the agar plates were added to l of pre-warmed x yeast extract tryptone (yt) culture medium containing μg kanamycin/ml and allowed to grow to an od of . at °c. pedv-np expression was induced using isopropyl β-d- -thiogalactopyranoside (iptg) at a final concentration of . mm to induce transcription of the lac operon, and the e. coli was allowed to incubate for an additional h at °c with shaking at rpm. the agar was strained out and bacteria pelleted by centrifugation at , g for min at °c. the pellet was resuspended in ml of lysis buffer solution (b-per, pierce, rockford, il), incubated for min at - °c, then centrifuged at , g to separate the soluble from insoluble proteins. the pedv-np recombinant protein was expressed as insoluble periplasmic inclusion bodies. the resulting amino acid recombinant protein was denatured using m urea, subsequently purified three times using nickel-nta affinity column chromatography and refolded back to its native conformational state. individual affinity column elutions were collected, pooled and confirmed by sds-page, then aliquoted/frozen at − °c. the correct nucleotide sequence was confirmed by sequence and restriction endonuclease analysis. the average protein yield produced by the pet a-pedv-np plasmid construct was calculated to be mg pedv-np per liter of xyt under the aforementioned conditions. the recombinant protein was detected and a predicted molecular weight of kda was confirmed via western blotting using convalescent sera, a x histidinespecific mab (novagen, madison, wi) and a pedv-np specific mab (figs. and ). two separate mabs were developed in our laboratory (sd - and sd - ) that recognize both the native conformation of the pedv-np and the full length, linear, recombinant protein used in all antibody capture assays. hybridomas were produced as previously described [ , ] . immunoglobulin isotyping of the resulting mabs was performed using a commercial lateral flow assay (serotec, raleigh, nc). subsequently, mouse ascites fluid was produced in pristane-primed mice, and the antibodies were purified and biotinylated for use as the detection moiety for the belisa [ ] . the conjugated antibody solution was quantified via the lowry protein method, and carrier bsa was added to a final concentration of mg/ml, then aliquoted and stored at − °c. after h of incubation at °c, plates were rinsed x with pbs and examined using fluorescent microscopy. for each individual test, each pedv infected well was compared to its respective uninfected partner well, and a positive sample was indicated if a pedv specific fluorescent signal was observed at a serum dilution of : or greater. all samples were tested in duplicate, and the antibody titer was expressed as the mean of all replicates. ielisa the serological pedv-np indirect elisa was performed by coating alternate wells of immulon b, -well, microtiter plates (thermo labsystems, franklin, ma) with ng/well of purified, recombinantly expressed pedv-np antigen. the optimal dilution of the recombinant protein and secondary detection antibody was determined by a checkerboard titration that gave the highest signal to noise ratio. in addition, a single lot of pooled convalescent serum from pedv infected pigs was used to generate quality control standards that gave high and low optical density (high od = . to . ; low od = . to . ; and negative od < . ). pedv-np recombinant protein was diluted to . μg/ml in mm sodium carbonate- mm sodium bicarbonate-antigen coating buffer (acb) ph . . odd-numbered columns were coated with μl of acb plus antigen, while the evennumbered columns were coated with acb without antigen, serving as background control. the plates were incubated for one hour at °c, then washed x with pbs plus . % tween (pbst). each well was then blocked with μl of sample milk diluent (pbst plus % nonfat dry milk, (smd)) and allowed to incubate overnight at °c. the following day, the plates were washed x with μl of pbst. test and control sera were diluted / in smd, mixed, and μl of the solution was added to each well. the plates were incubated for h at - °c. next, μl of biotinylated, goat anti-swine detection antibody (bethyl laboratories, tx) was added at a concentration of ng/ml of pbst and allowed to incubate at - °c for h. the plate was washed x with μl of pbst, then μl of streptavidin-hrp conjugate (pierce, rockford, il) was added and incubated for another hour at - °c, then washed and developed with , ′, , ′-tetramethylbenzidine, peroxidase substrate (tmb) (surmodics, eden prairie, mn). color development progressed until the positive control attained a standard od and was stopped using n h so . colorimetric development was quantified spectrophotometrically at nm with a elx microplate reader (biotek instruments inc., winooski, vt) controlled by xcheck software (idexx laboratories, westbrook, me). the raw data was normalized and transformed into an excel spreadsheet. sample to positive (s/p) ratios were calculated using the following formula: s/p = optical density (od) of sample -od of buffer/od of positive control -od of buffer. belisa the serological belisa was performed using immulon b, -well microtiter plates (thermo labsystems, franklin, ma). alternate wells of each plate were coated with ng per well of expressed pedv-np antigen. the optimal dilution of the recombinant protein and mab antibody was determined by a checkerboard titration that gave the highest signal to noise ratio with an od reading of approximately . , in the absence of swine serum/competitor antibody. first, pedv-np recombinant protein was diluted to . μg/ml of acb. odd-numbered columns were coated with μl of acb plus antigen, while the even-numbered columns were coated with acb without antigen serving as background control. the plates were incubated for h at °c, washed times with pbst, then placed at °c overnight. the following day, each well was blocked with μl of smd and incubated one hour at °c. plates were washed times with pbst, and μl of test and control sera were diluted / with pbst + . % nonfat dry milk and added to each of the duplicate wells. plates were incubated h at °c. during sample incubation, pedv-np specific biotinylated, mabs (sd - and sd - ) were adjusted to equal titers and mixed together in a : ratio. next, μl of a : , dilution of the antibody detection mixture was added to the microtiter plate containing the competitive swine antibody, then swirled and incubated for an additional min at °c. the plates were washed times, and μl of high sensitivity, streptavidin-horseradish peroxidase conjugate (pierce, rockford, il) was added to all wells of the microtiter plate for h at °c. plates were washed times with pbst, and μl of tmb was added to all wells and gently swirled. after ap-proximately min, color development progressed until the negative control attained a standard od of approximately . and was subsequently stopped using n h so . colorimetric development was quantified spectrophotometrically at nm with an elx microplate reader (biotek instruments inc., winooski, vt) controlled by xcheck software (idexx laboratories, westbrook, me). the raw data was normalized and transformed into a excel spreadsheet, and the percent inhibition (pi) ratio was calculated using the following formula: pi = -{(od of sample -od of buffer)/(od of negative control -od of buffer)} x . a two-step carbodiimide coupling procedure was used to couple na pedv-np protein to luminex™ microspheres. briefly, the coupling of fluorescent microsphere was performed by washing . × microspheres twice with μl activation buffer ( . mnah po , ph . ) and sonicating them for s after each wash. microspheres were activated for min at - °c in μl activation buffer containing . mg n-hydroxysulfosuccinimide (sulfo-nhs) and . mg n-( dimethylaminopropyl)-n-ethylcarbodiimide (edc) (pierce chemical, rockford, il). activated microspheres were washed twice with pbs and sonicated. coupling was initiated by the addition of . μg of recombinant na pedv-np protein, brought to a final volume of μl with pbs and incubated in the dark for h at - °c with rotation. coupled microspheres were washed once with ml of pbs plus . % nan and . % bovine serum albumin (pbs-nb). next, the microspheres were blocked with ml of pbs-nb for min to reduce nonspecific binding. microspheres were then washed twice with pbs-nb and resuspended in pbs-nb to a final concentration of . × antigen-coupled microspheres/ml. a -well hydrophilic membrane filter plate was blocked for min with μl of pbs-nb, and then the liquid was aspirated via vacuum manifold. the plates were wetted with μl of pbs-nb buffer to prevent drying. next, μl of serum (diluted : in pbs-nb) was added to duplicate wells of the filter plate along with μl of pbs-nb containing . × antigen-coupled microspheres. since the microspheres and reporter moieties are light sensitive, all incubations were performed in the dark by sealing the plate with foil. subsequently, the fmia plate was incubated at - °c for h on a plate shaker rotating at rpm. the plate was washed times with μl of pbst. next, μl of anti-swine, biotinylated iga (heavy & light chain, diluted in pbs-nb; bethyl laboratories) or igg-fc specific polyclonal antibodies (diluted : , dilution in pbs-nb; bethyl laboratories) were added to the filter plate and incubated at - °c for h. np igm and igg isotypespecific antibody levels were detected using pedv-np coated microspheres, but speciated by means of individual and separate igm and igg-specific secondary antibodies. since validation was performed using serum, and because iga is present in very low amounts, iga specific secondary antibodies were not used at this step. after incubation with the secondary antibodies, μl of streptavidin phycoerythrin ( . μg/ml in pbs-nb, molecular probes) was added to each well and incubated for min at - °c with shaking. the supernatant was aspirated, and the plate was washed times with pbst. finally, the microspheres were resuspended in μl of pbst per well and transferred to a clear -well polystyrene optical plate. coupled microspheres were analyzed through a dual-laser bio-rad bio-plex instrument. the median fluorescent intensity (mfi) for microspheres corresponding to each individual bead analyte was recorded for each well. all reported mfi measurements were normalized via f -f , where f was the background signal determined from the fluorescence measurement of a test sample in uncoated beads and f was the mfi for a serological test sample using antigencoated beads. four serological reference serum sets were constructed as standards termed high, medium, low and negative to serve as internal quality control standards and to mathematically normalize individual samples for objective comparisons between testing platforms. the high-labeled standard was designed to generate an od above . for the ielisa and belisa and an mfi of approximately , for the fmia. the high standard was used exclusively for the mathematical determination of the serological response (s/p ratio) of samples used for test validation. the medium standard generated a response of between . and . od for the two elisas and approximately , mfi for the fmia. the low standard was designed to deliver a signal slightly above threshold level for all tests, and the negative serum generated an od or mfi to a background level of less than . od for the elisas and mfi for the fmia. validation methods for the determination of diagnostic sensitivity, specificity, repeatability and threshold cutoff level to accurately assess the diagnostic sensitivity and specificity of the assays, samples of known serostatus for pedv were used. this included sera from multiple animal populations including experimentally infected animals and serum samples submitted to the south dakota adrdl. pedv negative sample sets included samples from selected high biosecurity herds with no history of pedv and archived serum samples collected prior to the emergence of pedv in the u.s., including samples testing positive for the related swine coronaviruses tgev and prcv. known positive samples were collected from pigs that were naturally infected at least weeks prior to collection and were previously positive by pcr. the negative-testing sample population (uninfected animals) consisted of maximally pedv negative serum samples, while the positive-testing (infected) population was composed of serum samples. receiver operating characteristic (roc) analysis was calculated for each assay to assess diagnostic performance, which included determination of sensitivity, specificity and threshold cutoff using medcalc version . . . (medcalc software, mariakerke, belgium). the repeatability of each assay was assessed by running the same internal quality control serum standards in multiple replicates within the same run or between runs. for the ielisa and the belisa, the intra-assay repeatability was calculated for replicates on separate plates, then repeated over a -day period for inter-assay repeatability assessment. the values for each assay were expressed as a mean, standard deviation and percent coefficient of variation (cv%) for repeated measure. multiple comparison, inter-rater agreement (kappa measure of association) was calculated among all four tests (belisa, ielisa, fmia and ifa) using ibm, spss version software (spss inc., chicago, il). the sample cohort used was a well-characterized set of serum samples collected from "positive testing" experimentally infected pigs over time courtesy of dr. richard hesse (n = ) and from archived experimental control uninfected pedv "negative testing" animals. the interpretation of kappa can be rated as follows: kappa less than . , "poor" agreement; between . and . , "slight" agreement; between . and . , "fair" agreement; between . and . , "moderate" agreement; between . and . , "substantial" agreement; and between . and . , "almost perfect" agreement [ , ] . a pedv virus neutralization assay using a ffn format was developed for rapid detection of neutralizing antibodies produced in response to pedv infection. the ffn was evaluated using serum samples or rennet treated milk and colostrum samples. heat-inactivated samples were diluted in a -fold dilution series starting at : in mem plus . μg/ml tpck-treated trypsin in -well plates. an equal amount of cell culture adapted pedv stock at a concentration of foci forming units/ μl was added to each well and plates incubated for h at °c. the virus/sample mixture was then added to washed confluent monolayers of vero- cells and incubated for h at °c. plates were washed again with mem/tpck-trypsin medium and incubated - h to allow for replication of non-neutralized virus. plates were then fixed with % acetone and stained with fitc conjugated mab sd - to allow visualization of infected cells. endpoint neutralization titers were determined as the highest serum, milk or colostrum dilution resulting in a % or greater reduction in fluorescent foci relative to controls. as shown in fig. , the purity of the recombinant protein was assessed via sds-page and gave a band that migrated corresponding to the expected molecular mass of kda upon staining with coomassie brilliant blue r . the protein yield of the iptg induced e. coli culture was calculated to be approximately mg pedv-np/liter of xyt medium with a purity of greater than %. the identity of the protein was further characterized by western blot using convalescent swine serum, an anti-his mab and an anti-pedv-np mab (fig. ) . to optimize the serologic assays, various antigen and serum dilutions were used to determine optimum concentrations. all tests were optimized in a checkerboard fashion to maximize signal-to-noise ratios. it was determined by antigen titration that the optimal coating of luminex™/fmia microspheres was achieved at a concentration of . μg protein per . × microspheres. similarly, the optimum coating of both the ielisa and belisa plates was achieved at a concentration of ng/well. in addition, to determine the optimum serum dilution for each of the testing platforms, a well-characterized pedv "high" positive serum standard was serially diluted in a log titration against antigen coated microspheres (fmia) or antigen coated elisa wells at a fixed concentration. figure shows concentration-dependent od or mfi signals of various serum standards. overall, sample absorbance increased inversely proportional to the serum dilution. however, based upon the highest signalto-noise ratio, it was determined that the optimal serum dilution for the belisa was / , while the ielisa and fmia each demonstrated an optimum dilution of / as indicated by arrows (fig. ) . roc analysis to determine sensitivity, specificity and threshold cut-off levels was performed using large numbers of swine serum samples and demonstrated excellent agreement (> . kappa scores) between assays with good intra and inter assay repeatability ( table ) . none of the known positive tgev or prcv samples tested was shown to cross-react. the optimal cutoff values and corresponding sensitivity and specificity of each individual test are presented in fig. . specifically, roc analysis for the ielisa and belisa showed similar sensitivity and specificity of . and . %; and . and . %, respectively. the roc analysis for the fmia showed estimated sensitivity and specificity of . and . %, respectively. although the fmia showed an identical sensitivity as the belisa, it demonstrated the highest degree of specificity of all three assays at . %. this observation was not surprising given that fmia technology inherently imparts greater sensitivity and a larger dynamic range than the elisa platform [ ] . in addition to determining cutoff values, sensitivities and specificities, multiple comparison tests were performed to calculate the degree of agreement among the elisa, fmia and ifa tests. specifically, the kappa test demonstrated all diagnostic platforms had kappa values greater than . , which demonstrates that all tests are in "almost perfect" agreement with each other. the ielisa and belisa demonstrated slightly lower %cvs than the fmia with . %, . %, . % intra-assay variability for belisa, ielisa and fmia respectively. inter-assay %cvs were . , . and . % for the belisa, ielisa and fmia respectively. nonetheless, all the cvs were . % or less, which demonstrated that the tests were highly repeatable in a diagnostic application. as shown in fig. , a mean antibody response to pedv-np could be detected as early as dpi for both the ielisa and belisa. the fmia detected pedv-np antibodies slightly earlier at dpi. all tests detected the duration of antibody out to the dpi time-point in this study but demonstrated a decline in detectable antibody after dpi. high levels of pedv-np specific igm antibodies were observed at dpi compared to igg (fig. ) . however, the igm antibodies decreased to barely detectable levels by dpi. igg continued to increase linearly to dpi. there is a concomitant appearance of neutralizing antibodies by dpi. the ffn assay was initially evaluated using sequential serum samples from experimentally inoculated piglets. additional evaluation was conducted using serum samples from known pedv naïve herds and samples from herds with documented pedv exposure, collected at least weeks after initial pcr diagnosis and whole herd feedback. experimentally inoculated piglets demonstrated detectable seroconversion by dpi (fig. ). essentially all samples from pedv naïve animals had serum ffn endpoint titers of < : while most samples from the pedv positive set had endpoint titers ranging from : to : (data not shown). further evaluation of the ffn included serum, milk and colostrum samples from sows from a herd that had experienced an acute pedv outbreak to weeks prior to farrowing. all animals were exposed to live virus twice within the first week of the outbreak, followed by one dose of harrisvaccines porcine epidemic diarrhea vaccine, rna (harrisvaccines, inc., ames, ia) at week pre-farrow. serum and colostrum samples were tested at the time of farrowing, followed by serum and milk samples at week and weeks later. as shown in fig. , mean colostrum titers were approximately -fold higher than serum titers at the time of farrowing. at later time-points, serum and milk titers were similar in magnitude, although substantial animal to animal variation was apparent. overall, this repertoire of assays is useful for initial identification and efficient, high throughput quantitation of pedv antibodies. we evaluated all three diagnostic platforms against a well characterized ifa and compared the individual serum igm and igg kinetic antibody responses in an fmia to the appearance of neutralizing antibody as detected by the ffn assay. each of the antibody-capture assays was validated using a large number of serum samples (n > ) based upon the assay validation methods of jacobson, which is supported by the office international des epizooties [ ] . since pedv was first identified in the u.s. in may , it has spread rapidly to at least states (www.aasv.org) and has been reported in mexico and canada [ ] . the virus causes severe gastroenteritis, destroying villus enterocytes in pigs of all ages, and is characterized by vomiting and diarrhea, leading to subsequent dehydration, high mortality rates and economic losses, particularly in nursery piglets [ , ] . a variety of serological tests have been developed against pedv, but they vary by antigen used and in the degree of validation. in addition, few have used north american np based antigens or compared the array of serologic assays described here. in the current study, four tests (ifa, belisa, ielisa, fmia) showed strong correlation. each has advantages, which dictate how they will be used in the field. in addition, newly developed np mabs were used in the belisa and for expediting ffn testing in the detection of neutralizing antibodies. in the development of the elisas and fmia, the full length na pedv-np gene was amplified directly from rna extracted from pedv-infected ileal tissue. multiple sequence alignment analysis showed that the amplified np gene shared a % nucleotide homology with that of the us colorado strain isolated in (genbank accession no. - ). several authors confirm that the np carries multiple antigenic determinants that are conserved among the coronaviridae [ , ] . however, we performed one-way cross-reactivity testing using serum from tgev and prcv, and no antibody crossreactivity was detected within any of our assays. in addition to being highly conserved among various pedv variants, the np is the most abundant viral protein expressed in pedv infected cells, making it an attractive target antigen [ , ] . using western blotting experiments, we confirmed the finding of hou et al. [ ] , in which they observed the level of expression of np protein to be significantly higher than the level of the spike protein. our study demonstrated that it is possible to achieve a protein yield of over mg per liter of culture with a purity of greater than %. the recombinant np has previously been identified as a useful antigen in other elisas developed to detect antibodies in pigs located in china and korea [ ] . in a study by hou et al. [ ] , the authors showed similar sensitivities and specificities of their ielisa compared to the ielisa described in this study. however, smaller numbers of known positive and negative samples were evaluated than in the current study. since no test has % specificity, a belisa was developed that is useful for confirmatory testing due to its higher inherent specificity than the ielisa [ ] . blocking or competitive elisas have been constructed using monoclonal antibodies in pedv serodiagnosis, and the specific methodology can affect the overall specificity and performance of the assay. our method was based upon coating plates with highly purified na pedv-np, then using a combination of two separate na, anti-pedv-np specific, biotinylated, monoclonal antibodies as the blocking/competitive detection step. this allows the capture of anti-np antibodies at higher quantities and those with a greater range of antigen specificities. the analytical specificity of the np-based belisa is also dependent on the affinity of the mabs used. the antibodies used in this study are directed against conserved epitopes on the nucleocapsid protein without any evidence of cross-reactivity to any other genera of alphacoronavirus tested. a previous assessment of antigenic cross-reactivity was performed using these same mabs against different strains of pedv and tgev [ ] . in that study, the authors reported that both mabs used in the belisa reacted with all pedv strains tested, namely the homologous us isolate pc a and the heterologous strains s indel iowa , s del pc and cv , at similar titers. neither of the pedv-np mabs cross-reacted with either the tgev miller or purdue strains. not only were the belisa mabs tested for heterologous cross-reactivity, but all three diagnostic platforms were evaluated in their ability to capture antibody against tgev and prcv, and there was no cross-reactivity to either heterologous virus. serology testing with ifa, ielisa, belisa or fmia is useful in determining whether pigs were previously infected with pedv, or if piglets have acquired antibodies through colostrum (eg. passive antibody transfer). however, tests that evaluate the functionality of the antibodies such as the ffn are needed to determine if the detected immune response could be helpful in providing protection to nursing piglets. neutralizing antibodies may be protective through actions including blocking uptake of the virus into cells, preventing virus binding to receptors on cells, preventing uncoating of the virus genomes in endosomes and/or causing aggregation of virus particles. for enveloped viruses, such as pedv, lysis of the virus may also occur when antiviral antibodies and serum complement disrupt the viral membrane. for these reasons, an ffn-based virus neutralization assay was developed to assess levels of pedv neutralizing antibodies in serum, milk or colostrum samples. the ffn provides a more rapid determination of neutralizing antibody levels than is possible with traditional virus neutralization assays that rely on visualization of virusinduced cpe after three or more days incubation to allow for full development of pedv cpe. the direct observation of fluorescent stained infected cells, or lack of stained infected cells in the case of virus neutralization, allows for simple endpoint determination. this feature is particularly valuable when dealing with a fastidious, trypsin-dependent virus such as pedv where cpe-based endpoints may not be obvious or may be confused with trypsin-induced cpe in the cell monolayer. although neutralizing antibodies present in the serum would not be expected to provide direct protection from a strictly enteric infection such as pedv, our data suggest a correlation between detectable neutralizing antibody levels in the serum and those present in milk and colostrum of previously exposed or vaccinated sows. some correlation between pedv neutralization results and elisa results exists as described in the literature. one study performed a comparative analysis between a whole-virus antigen elisa and a serum neutralization test for the serodiagnosis of pedv [ ] . the presence of antibodies was confirmed by each test, and an overall testing agreement of . % was demonstrated using field serum samples. furthermore, a pairwise correlation was performed that showed corrected cutoff values between the elisa od and sn titers having an r value of . , indicating that the cpe-based neutralization test had roughly the same reliability as the elisa test [ ] . newer technologies such as the fmia are useful for the detection of antibodies against multiple antigens simultaneously for surveillance purposes. fmia are bead based assays for simultaneous high throughput detection of antibodies to multiple antigens. the fmia differs from the elisa since it involves a fluid incubation step with "beads suspended in solution, which allows for higher surface area exposure in dimensions" [ ] . therefore, there is a shorter diffusion path to antibody binding sites on the antigen coated beads resulting in rapid reaction times. instead of a method using an enzymatic reaction such as with the elisa, the fmia detection is with laser technology, which results in a shorter detection time. this pedv antigen specific bead set can be "mixed" with additional coated beads to other antigens, such as siv, pcv , prrsv or other pathogens, for simultaneous detection of antibodies to these antigens. in addition, an fmia could be developed for differentiation of wild-type infected vs vaccinated animals (diva) if proteins used in the vaccine were different from those produced in a wild-type infection. individual kinetic serum igg and igm levels were measured by fmia in experimentally infected animals over time. the appearance of the igm subclass is considered an immunological parameter of early infection and generally appears prior to the appearance of igg, and this was confirmed in our study. this was in contrast to the data of woo et al. [ ] , which was unable to detect igm antibodies using their np-based indirect elisa. igg antibodies may be more easily detected as they are characterized by higher antigen affinity but lower avidity than igm [ ] . further understanding of various antibody profiles will provide important information on the ability of vaccines to stimulate a protective immune response. these well-validated na pedv ielisa, belisa, fmia and ffn assays are useful for a range of serological investigations. they can serve as a complement to nucleic acid detection and determine the pedv status of asymptomatic individuals for cost-effective tools in management strategies and monitoring virus exposure within the herd. the fmia will be useful for isotyping the antibody responses and in multiplexing for determining exposure to multiple pathogens simultaneously. in addition, the ffn is useful for determining whether the antibodies measured are providing a biological function of blocking virus infectivity. work is ongoing to further validate these assays on other sample matrices such as milk and colostrum for measuring passive transfer of antibodies and oral fluids for pen-based surveillance. porcine epidemic diarrhea virus; ifa: immunofluorescent assay; ielisa: indirect enzyme linked immunosorbent assay; belisa: blocking enzyme linked immunosorbent assay; fmia: fluorescent microsphere immunoassay; ffn: fluorescent focus neutralization; orf: open reading frame; utr: untranslated region tgev: transmissible gastroenteritis virus; prcv: porcine respiratory coronavirus; na: north american; cpe: cytopathic effect; dpi: days post-infection; adrdl: animal disease research and diagnostic laboratory sodium dodecyl sulfate-polyacrylamide gel electrophoresis iacuc: institutional animal care and use committee; hat medium: hypoxanthine-aminopterin-thymidine medium; dmso: dimethyl sulfoxide; bsa: bovine serum albumin fbs: fetal bovine serum; nvsl: national veterinary services laboratories moi: multiplicity of infection; tpck: l- -tosylamide- -phenylethyl chloromethyl ketone; fitc: fluorescein isothiocyanate; acb: antigen coating buffer; smd: sample milk diluent; hrp: horseradish peroxidase ′-tetramethylbenzidine; pi: percent inhibition; mfi: median fluorescent intensity; siv: swine influenza virus; pcv- : porcine circovirus type ; prrsv: porcine reproductive and respiratory syndrome virus evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study experimental infection of pigs with a new porcine enteric coronavirus, cv emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences ratification vote on taxonomic proposals to the international committee on taxonomy of viruses sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china an elisa optimized for porcine epidemic diarrhoea virus detection in faeces antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans establishing a genetic recombination map for murine coronavirus strain a complementation groups origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states third strain of porcine epidemic diarrhea virus, united states coronavirus immunogens the molecular biology of coronaviruses a new coronavirus-like particle associated with diarrhea in swine enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera an elisa for detection of antibodies against porcine epidemic diarrhoea virus (pedv) based on the specific solubility of the viral surface glycoprotein evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies development of a porcine epidemic diarrhea virus m protein-based elisa for virus detection development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (pedv) antibodies detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection porcine epidemic diarrhea: kinetics of actively and passively acquired serum antibodies and the effect of reinfection development of an -plex luminex assay to detect swine cytokines for vaccine development: assessment of immunity after porcine reproductive and respiratory syndrome virus (prrsv) vaccination development of a fluorescent microsphere immunoassay for detection of antibodies against prrsv using oral fluid samples as an alternative to serum-based assays validation of serological assays for diagnosis of infectious diseases differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies antibodies to major histocompatibility antigens produced by hybrid cell lines the measurement of observer agreement for categorical data development and laboratory evaluation of a lateral flow device (lfd) for the serodiagnosis of theileria annulata infection opportunities for bead-based multiplex assays in veterinary diagnostic laboratories distinct characteristics and complex evolution of pedv strains pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs antigenic relationships among homologous structural nucleotide sequences of porcine, feline and canine coronaviruses porcine epidemic diarrhea virus (cv ) and feline infectious peritonitis virus (fipv) are antigenically related critical factors affecting the diagnostic reliability of enzyme-linked immunosorbent assay formats longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus the distribution and functions of immunoglobulin classes the authors declare that they have no competing interests.authors' contributions fo: conducted elisa and fmia development/validation, statistical analysis and co-wrote paper. xl: conducted elisa and fmia development/validation. as: assisted with sample acquisition, assay development and study design. tc: assisted with sample acquisition and study design. jn: conducted virus neutralization assay development and testing. jch: assisted with study design and co-wrote paper. ean: developed study concept and design, edited paper. sl: directed assay development and validation, co-wrote paper. all authors read and approved the final manuscript. key: cord- - lswjro authors: fan, jing-hui; zuo, yu-zhu; shen, xiao-qiang; gu, wen-yuan; di, jing-mei title: development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: lswjro the recent dramatic increase in reported cases of porcine epidemic diarrhea (ped) in pig farms is a potential threat to the global swine industry. therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (pedv) infection or vaccination would be essential in helping to control the spread of ped. we developed and validated an indirect enzyme-linked immunosorbent assay (elisa) based on the recombinant membrane (m) protein of pedv. to detect pedv antibodies in eight herds, serum samples were collected from sows that had been immunized with a ped vaccine, and screened using the developed elisa in parallel with a serum neutralization (sn) assay. of the tested samples, were positive for the presence of pedv antibodies according to both assays, while were negative. an excellent agreement between the elisa and the sn assay was observed (kappa = . ; % confidence interval = . – . ; mcnemar's test, p = . ). no cross-reaction was detected for the developed elisa with other coronaviruses or other common pig pathogens. the developed elisa could be used for serological evaluation and indirect diagnosis of ped infection. porcine epidemic diarrhea (ped) is a devastating swine disease and its etiological agent is ped virus (pedv). porcine epidemic diarrhea is characterized by watery diarrhea, dehydration, and a high death rate among suckling pigs (pensaert and debouck, ) . the disease was first documented in the united kingdom in as a swine disease that resembled transmissible gastroenteritis (oldham, ) . outbreaks of ped have since been reported in europe, asia, and north america (nagy et al., ; takahashi et al., ; sun et al., ; fan et al., ; stevenson et al., ) . porcine epidemic diarrhea virus has spread rapidly across the united states, resulting in the high mortality of piglets, and substantial economic losses (cima, ) . its recent emergence in north america suggests that this virus is a threat to the swine industry worldwide. accurate diagnosis and serological detection of specific antibodies in pigs as a result of pedv infection or vaccination are required to control the spread of ped. porcine epidemic diarrhea virus is a coronavirus within the coronaviridae family. the genome of pedv contains genes encoding spike (s), membrane (m), small membrane (sm), open reading frame , and nucleocapsid (n) proteins (park et al., ) . the m protein is a structural membrane glycoprotein, and the most abundant of all the envelope proteins. it has a short amino-terminal domain that exists outside of the virion, with the long carboxyterminal domain of the protein present inside the virion (utiger et al., ) . immune reactions to the m protein of coronaviruses play an important role in the induction of protection, and in mediating the course of the disease (fleming et al., ; vennema et al., ) . the nucleotide sequence of the pedv m gene exhibits low homology (around %) with the m gene of other coronaviruses; however, its sequence is highly conserved among different pedv strains arndt et al., ) . therefore, the m protein could be a suitable candidate for the detection of pedvspecific antibodies and in diagnosing pedv infections. many enzyme-linked immunosorbent assays (elisas) have been developed for the detection of antibodies against pedv. however, the preparation of an appropriate antigen for most of these methods requires cultivation of pedv, which is time consuming and expensive. in the current study, we expressed and purified the m protein of pedv. was used as a coating antigen in an indirect elisa that we developed. we then screened serum samples from pigs to determine the presence of antibodies specific for the pedv m protein. we collected blood samples from healthy unvaccinated pedv-free pigs of various ages. the sera derived from these samples were negative for the presence of antibodies against pedv according to serum neutralization (sn) assays (sn titers < : ). the sera from blood samples, taken from pedv-infected pigs, were used as reference positive sera (sn titers ≥ : ). the pedv infection status of pigs was confirmed by reverse transcription polymerase chain reaction (rt-pcr) assays targeting the pedv m gene, with viral rna extracted from fecal samples. porcine sera containing antibodies against transmissible gastroenteritis virus (tgev; sn titer : ), porcine rotavirus (prv; : ), porcine reproductive and respiratory syndrome virus (prrsv; : ), porcine circovirus (pcv- ; : ), and classical swine fever virus (csfv; : ) were obtained from the hebei center for disease prevention and control (shijiazhuang, people's republic of china). a total of serum samples were collected from eight pig herds, in which the animals had been administered a pedv vaccine. all samples were tested by the developed indirect elisa. porcine epidemic diarrhea virus strain hb/bd (genbank accession no. jf . ) was isolated from the feces of a pig from hebei province, china, and adapted to cell culture for passages in vero cells. viral rna was extracted from pedv using trizol reagent (invitrogen, carlsbad, ca, usa), following the manufacturer's instructions. the sequence corresponding to the m gene was amplified from viral rna by rt-pcr using oligonucleotide primers mp ( -gga tcc atg tct aac ggt- ) and mp ( -aag ctt tct gtt tag act aaa t- ). the resulting pcr products were separated by agarose gel electrophoresis and purified using a gel extraction mini kit (tiangen biotech co. ltd., beijing, china) according to the manufacturer's recommendations. purified amplicons were cloned into the pmd -t vector (takara biotech co. ltd., dalian, china) to yield the recombinant plasmid pmd-m, which was then transformed into competent escherichia coli jm . positive clones were selected according to their lacz phenotype, and verified by restriction enzyme digestion, pcr screening, and dna sequencing. the m gene contained within pmd-m was subcloned into the prokaryotic expression vector pgex- p- (sunbiotech inc., beijing, china) to yield pgex- p-m. the pgex- p- vector also contained a sequence for a gst tag, which was designed so as to be added at the amino terminal of the expressed protein. the pgex- p-m vector was verified by dna sequencing. the recombinant m protein was transformed into e. coli bl (tiangen biotech co. ltd., beijing, china). e. coli bl cells were cultured and transformed with pgex- p-m, in luria-bertani broth supplemented with g/ml ampicillin until the optical density at nm (od ) was . - . , at which point protein expression was induced via the addition of . mm isopropyl-␤-d-thio-galactopyranoside. at h post-induction, bacterial cells were collected by centrifugation. the recombinant protein was purified from the bacterial lysate using a gst fusion protein purification kit (transgen biotech co. ltd., beijing, china), according to the manufacturer's recommendations. fractions of purified recombinant m protein were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), with polyacrylamide gels stained with coomassie brilliant blue r- . expression of the recombinant m protein was confirmed by immunoblotting, using porcine pedv antisera. the prokaryotic expression vector pgex- p- was also transformed into e. coli bl as a negative control. fractions of purified recombinant proteins were separated by sds-page and electrophoretically transferred to nitrocellulose membranes (gibco brl, gaithersburg, md, usa). to prevent nonspecific reactions, membranes were blocked with % (w/v) non-fat milk powder in tris-buffered saline containing . % (v/v) tween (tbst) at • c for h. membranes were incubated with pig pedv antisera (diluted : ) at • c for h. after three washes with tbst, membranes were incubated with an anti-pig igg conjugated to horseradish peroxidase (hrp; sigma-aldrich, st louis, mo, usa) at • c for h. we used , -diaminobenzidine (sigma-aldrich) for the development of color reactions to visualize protein bands. a checkerboard titration involving each combination of antigen (purified recombinant pedv m protein) and sera was used to determine optimal dilutions for use in the indirect elisa we developed. the antigen and serum were diluted from to . g/ml and : to : (two-fold dilution), respectively. ninety six well plates (nunc maxisorp, denmark) were coated with the recombinant m protein, which was diluted in phosphate-buffered saline (pbs), and allowed the plates to incubate at • c overnight. pbs was used as a blank control. wells were blocked with % fetal bovine serum in pbs (ph . ) for h at • c. wells were then washed three times with pbs containing . % tween (pbst) and diluted serum samples were added, in duplicate, to the antigen-coated wells. following incubation at • c for h, plates were washed three times with pbst and l of diluted goat anti-pig igg conjugated to hrp (kpl inc., gaithersburg, md, usa) was added to each well. after incubation at • c for min, wells were washed with pbst, and l of , , , ,-tetramethyl-benzidine (tmb) was added to each well and the plates were incubated for min at room temperature. color development reactions were stopped with l of m h so . the od was determined for each well using a microplate reader. dilutions that resulted in the highest od ratio between the positive and negative serum samples (p/n value), along with an od value for positive serum samples close to . , were considered optimal working conditions. to establish negative and positive cutoff values for this assay, pedv-negative serum samples, as determined by sn assays, were tested in triplicate using the elisa we developed. a cutoff value was defined as the mean absorbance value plus three standard deviations (sd). repeatability assays were conducted by comparing the ratios of od values for triplicate results from each serum sample tested on the same plate (intra-assay repeatability) or on different plates (inter-assay repeatability). the coefficient of variation (cv) was calculated according to the following formula: where x was the average od for the field serum samples. a cv value less than % was considered an acceptable level of variation. to determine the specificity of the developed elisa, crossreactivity was assessed by determining the reactivity of the purified antigen with serum samples containing antibodies against tgev, prv, prrsv, pcv , csfv and pedv. reference serum samples were diluted : , with three replicates of each dilution tested using the indirect elisa. serum samples from pedv-infected pigs (n = ) were selected at random to determine the sensitivity of the indirect elisa. samples were serially diluted two-fold from : to : . eight replicates of each diluted serum sample were tested to determine the titers of antibodies in samples. the titers were also determined by sn. the indirect elisa that we developed was used to evaluate the presence of pedv antibodies in serum samples from sows with varying immune status. these serum samples were also subjected to sn assays. the level of agreement between the sn and elisa assay was determined using a cohen's kappa, and a mcnemar's test was performed to investigate the differences between both assays. a p-value < . was considered significant. all analyses were performed in spss version . . the m gene (approximately bp) of pedv was successfully amplified and cloned it into a ta vector. positive clones were identified by sequencing. sequence analysis indicated that the pedv m gene was bp, and encoded amino acids. the complete nucleotide sequence of the m gene has been deposited in genbank (accession number jf ). recombinant pedv m gene was expressed in e. coli bl . analysis of the bacterial cell lysate by sds-page and western blotting revealed a prominent band of around kda. the recombinant protein we identified was approximately kda heavier than the predicted molecular weight of kda, which was due to the gst tag (fig. ) . the recombinant protein was predominantly in the insoluble fraction of the bacterial cell extract, and purified using a gst fusion protein purification kit. western blotting analysis showed that the purified protein was recognized by pedv antiserum (fig. ) . the optimum antigen concentration and serum sample dilution were determined to be . g/ml and : , respectively. the optimum dilution of the anti-pig igg conjugated to hrp was found to be : . the incubation conditions for primary and secondary antibodies were h at • c and min at • c, respectively. the optimum blocking buffer was found to be % fetal bovine serum in pbs, while pbst was the most appropriate washing buffer. the detection threshold of our indirect elisa was determined using serum samples that were negative for the presence of pedv antibodies. the cutoff point was specific for each individual assay and was based on the od of negative samples included in each assay. the mean od value for the elisa was . , with an sd of . . the cutoff value was determined to be . (mean + sd); therefore the negative-positive threshold was set at . . serum samples returning an elisa od value below table cross-reaction analysis of the m-based i-elisa with anti-sera against other pig viruses: od value (mean ± sd). pedv, tgev, prv, pcv- , csfv, prrsv represent pig sera that were positive for pedv, tgev, prv, pcv- , csfv, prrsv, respectively. 'noninfected' represents the serum from a pig that was negative for pedv. . were regarded as negative; samples that returned an od value higher than this were considered to be seropositive for pedv antibodies. reproducibility within and between elisa plates was evaluated by testing ten sn-negative serum samples and ten sn-positive serum samples in triplicate. the inter-assay cv of the indirect elisa ranged from . % to . % with a median value of . %. the intraassay cv ranged from . % to . %, with a median value of . %. these results indicate that the indirect elisa we developed to detect antibodies against the pedv m protein was repeatable, with low and acceptable levels of variation. the specificity of our elisa was evaluated by testing the reactivity of the pedv m protein antigen with antibodies against pedv, tgev, prv, prrsv, pcv- , and csfv. we found that pedv antisera significantly reacted with the pedv m antigen, as expected. in contrast, the od values for all other serum sample containing antibodies against other porcine viruses were significantly lower than the established negative cutoff value (table ) . our results suggest that little or no cross-reactivity occurs between the pedv m protein and antibodies against other porcine viruses, and that the m protein antigen was specific for antibodies against pedv. we evaluated the sensitivity of our indirect elisa using ten pedv-positive serum samples, and found that we could detect pedv antibodies in a serum sample that had been diluted out to : , while only : . in sn. this result indicated that our indirect elisa was more sensitive than the sn assay for pedv antibody detection (table ) . of the serum samples collected from animals with a known immunization history across eight pig farms, ( . %) were positive for pedv antibodies according to our elisa, while ( . %) were pedv antibody-negative. in comparison, according to the sn assay we used in parallel with the indirect elisa, % ( / ) of samples were pedv antibody-positive and . % ( / ) of samples were antibody-negative (table ) . according to both assays, and of the screened serum samples were pedv antibody-positive and -negative, respectively. three of the serum samples that were positive according to the sn assay, were porcine epidemic diarrhea virus is a member of the coronaviridae, and is known to cause fatal diarrhea in newborn piglets. the virus was first identified in (pensaert and debouck, ; chasey and cartwright, ) and has since been found to be prevalent in many countries. infection with pedv has resulted in significant economic losses, mainly in europe and asia, and recently the usa (fan et al., ; chae et al., ; martelli et al., ; stevenson et al., ) . although there are commercial vaccines available to prevent and control ped in china, the damage caused by pedv is significant and the threat continuous. to effectively control and prevent this disease, detection methods that can assess the current epidemic situation in herds are required. in addition, for subsequent immunoprophylaxis, assays that can monitor serum antibody levels of immunized or infected pigs need to be developed. before local immunity is actively established, the piglet intestine is protected against pedv infection by maternal antibodies. although the presence of antibodies in serum is not directly related to the protection for sows or for piglets, serological examination facilitates the assessment of humoral response to pedv elicited either through vaccination or natural infection. porcine epidemic diarrhea virus was isolated for the first time in using the vero cell line and trypsin-supplemented medium (hofmann and wyler, ) . various diagnostic methods have been described for the detection of pedv antigen and pedv antibodies (carvajal et al., ; knuchel et al., ; kweon et al., ; song et al., ) . at present, serological diagnostic methods for the diagnosis of pedv infection are more common. given that elisas are simple, sensitive, and convenient serological detection methods, a number of commercial and in-house elisas have been developed to detect pedv antigens, or antibodies against pedv (song and park, ) . the majority of in-house assays for the detection of antibodies against pedv are based on using the whole virus as an antigen (carvajal et al., ; oh et al., ) , or preparations of viral antigen (knuchel et al., ) . the cultivation of pedv is laborious and time consuming, especially considering the bio-security measures that must be taken in handling this virus; therefore it is difficult to upscale the production of these serological assays. the use of a recombinant viral protein as an antigen would allow researchers to avoid some of the problems associated with the large-scale preparation of native pedv antigen. recently, an elisa based on a recombinant n protein was validated in china (hou et al., ) . however, these researchers did not assess the cross-reactivity of the elisa with other coronaviruses. the m protein of pedv protrudes from the viral envelope, and is considered a superior diagnostic antigen compared with other pedv proteins (shenyang et al., ) . in the current study, we chose the pedv m protein for use as an elisa antigen because it is highly conserved among pedv strains, and because it can elicit the formation of protective antibodies (zhang et al., ) . we expressed the m protein in e. coli and confirmed its antigenicity using pedv-specific antibodies. our results indicated that the expressed recombinant m protein was indeed antigenic, and could feasibly be used as a coating antigen in an indirect elisa. the indirect elisa that we developed exhibited low levels of variability among replicates, according to intra-and inter-assay tests. these minor variations in results indicated that the indirect elisa was reproducible. furthermore, this elisa was able to detect pedv antibodies in serum samples that had been diluted out to : , and exhibited no cross-reactivity to antibodies against other common pig pathogens. these findings indicated that the developed indirect elisa could be used widely in the future. vaccination is one of the most effective techniques in controlling ped in china. however, since late , ped has been reemerging in immunized swine herds with devastating impact. to confirm the effects of targeted immunoprophylactic measures, we used our indirect elisa to screen serum samples from vaccinated pigs. we found that . % and % of samples were seropositive according to elisa and sn assay, respectively. this indicated that vaccination elicited neutralizing antibodies against pedv in sows to some degree. however, . % and % of samples from vaccinated pigs were seronegative by the elisa and sn assays, respectively. although these antibodies could not be used to assess protection against pedv, these vaccinated, but seronegative sows are not able to vertically transmit effective antibodies to their neonates. it indicating that the vaccine used requires some improvement in antigenicity. a comparison of the elisa and sn assay results revealed eight differences between positive and negative samples. there were five elisa-positive samples that were negative according to the sn assay we used, which could be explained by the higher sensitivity of the elisa. the reason for the elisa-negative results for the three sn-positive serum samples might be due to errors inherent in the tests that we applied. in conclusion, this is the first report of an indirect elisa using the recombinant pedv m protein as a coating antigen for the detection of antibodies against pedv in china. the developed assay is quick, convenient, and not labor-intensive, and could facilitate the development of a reliable tool or kit for the large-scale detection of antibodies against pedv. the authors have no conflicts of interest concerning the work reported in this paper. a conserved domain in the coronavirus membrane protein tail is important for virus assembly evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies prevalence of porcine epidemic diarrhea virus and transmissible gastroenteritis virus infection in korean pigs virus-like particles associated with porcine epidemic diarrhoea viral disease affects us pigs: porcine epidemic diarrhea found in at least states heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in china monoclonal antibodies to the matrix (el) glycoprotein of mouse hepatitis virus protect mice from encephalitis propagation of the virus of porcine epidemic diarrhea in cell culture development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (pedv) antibodies in situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets an elisa for the detection of antibodies against porcine epidemic diarrhea virus (pedv) based on the specific solubility of viral surface glycoprotein rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy enterotoxigenic escherichia coli, rotavirus, porcine epidemic diarrhoea virus, adenovirus and calici-like virus in porcine postweaning diarrhoea in hungary comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection letter to the editor. pig farm molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea a new coronavirus-like particles associated with diarrhea in swine high-level prokaryotic expression of envelope exterior of membrane protein of porcine epidemic diarrhea virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan identification of the membrane protein of porcine epidemic diarrhea virus primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens identification of a conserved linear b-cell epitope in the m protein of porcine epidemic diarrhea virus this work was supported by the research foundation of the education bureau of hebei province, china (no. ). key: cord- -i tuhqk authors: yu, fuxun; du, yanhua; huang, xueyong; ma, hong; xu, bianli; adungo, ferdinard; hayasaka, daisuke; buerano, corazon c.; morita, kouichi title: application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of sftsv-specific human igg and igm antibodies by indirect elisa date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: i tuhqk background: severe fever with thrombocytopenia syndrome (sfts) is an emerging disease that was first reported in china in . it is caused by sfts virus (sftsv) which is a member of the phlebovirus genus in the bunyaviridae family. sftsv has been classified as a bsl pathogen. there is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. methods: the full length nucleocapsid (n) gene of sftsv yamaguchi strain was amplified by rt-pcr and cloned to an expression vector pqe . the recombinant (r) sftsv-n protein was expressed by using escherichia coli (e. coli) expression system and purified under native conditions. rsftsv-n protein based indirect igg and igm enzyme linked immunosorbent assay (elisa) systems were established to detect specific human igg and igm antibodies, respectively. one hundred fifteen serum samples from clinically suspected-sfts patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich elisa system. results: the native form of recombinant (r) sftsv-n protein was expressed and purified. application of the rsftsv-n protein based indirect igg elisa to the serum samples showed results that perfectly matched those of the total antibody sandwich elisa with a sensitivity and specificity of %. the rsftsv-n protein based indirect igm elisa missed positive samples that were detected by the total antibody sandwich elisa. the sensitivity and specificity of rsftsv-n-igm capture elisa were . and %, respectively. conclusions: the rsftsv-n protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. its preparation is simpler in comparison with that used for the total antibody sandwich system. our rsftsv-n protein-based igg and igm elisa systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. they are safe to use for diagnosis of sfts virus infection and especially fit in large-scale epidemiological investigations. severe fever with thrombocytopenia syndrome virus (sftsv), also named as fever, thrombocytopenia and leukopenia syndrome virus (ftlsv) or huaiyangshan virus, is an emergent virus that was first reported in [ ] [ ] [ ] . the sources of serum samples where the virus was identified were from patients infected in and in china. severe fever with thrombocytopenia syndrome (sfts), the disease caused by the virus has a major clinical presentations that include fever, thrombocytopenia, leukocytopenia, gastrointestinal symptoms, neurological symptoms, bleeding tendency, as well as less specific clinical manifestations [ , ] . this disease has a case-fatality rate ranging from . to % in different areas of endemicity [ ] . human-to-human transmission of sftsv was reported to occur through close contact with the blood and/or body secretions of infected patients [ ] [ ] [ ] [ ] [ ] . after the first identification of sfts, sfts cases have been reported in provinces of china [ ] . recently, the existence of this disease has also been confirmed in japan and south korea [ ] [ ] [ ] [ ] [ ] . in japan, the case-fatality rate of % ( / ) was apparently higher than that in china, where an average of % of cases was fatal [ ] . data on the high fatality rate due to sftsv indicate that sftsv is a threat to human health. another tick-borne phlebovirus, the heartland virus, which was detected in missouri, is phylogenetically associated with sftsv. it causes severe febrile illness with thrombocytopenia, leukopenia in the total blood cell count, and elevated levels of liver enzymes [ ] . for the diagnosis of sfts, laboratory confirmation is essential because the clinical manifestations of sfts are non-specific. virus isolation from the blood of viremic patients is the direct evidence of sftsv infection, however, it is time-consuming and needs high security biocontainment facility [ , ] . detection of sftsv genome could be achieved by different nucleic acid detection techniques such as reverse transcription-pcr (rt-pcr) [ , ] , real-time rt-pcr [ , ] , reverse transcriptionloop-mediated isothermal amplification assay (rt-lamp) [ ] [ ] [ ] , reverse transcription-cross-priming amplification coupled (rt-cpa) with vertical flow (vf) visualization [ ] . although these techniques have high sensitivity and specificity in early diagnosis, the duration of viraemia in sftsv infection is very short, generally - days after the disease onset [ ] . hence, the nucleic acid detecting techniques are applicable only during the acute phase of the disease which is within week after its onset. the final confirmation of infection in many cases may rely on the detection of the specific antibodies to sftsv. sftsv is a member of the phlebovirus genus in the bunyaviridae family. like other bunyaviruses, the l segment encodes the rna-dependent rna polymerase; the m segment has an open reading frame (orf) coding for a gngc precursor in the order gn-gc; whereas the s segment uses ambisense coding to express two proteins: one is a nucleocapsid (n) protein encoded by the ′ half of viral complementary sense s rna, and the other is a nonstructural (ns) protein encoded by viral sense s rna [ , , ] . nucleocapsid (n) protein is one of the most immunodominant viral proteins among members of the bunyaviridae family. recombinant n protein of rift valley fever (rvf) virus, another member of the phlebovirus genus, was reported to be used in a detection system for the laboratory diagnosis of rfv infection in humans and animals [ ] [ ] [ ] . in sftsv, jiao et al. developed a recombinant n protein based sandwich enzyme linked immunosorbent assay (elisa) for detecting the total antibodies against this virus in humans and animals [ ] . in our present report, recombinant sftsv-n (rsftsv-n) protein was expressed by using escherichia coli (e. coli) expression system and then purified. rsftsv-n protein based igg elisa and igm elisa systems were established. serum samples from clinically-suspected sfts patients were used to evaluate the newly established systems and results were compared with those obtained by using the total antibody detecting sandwich elisa system. the sftsv nucleocapsid gene encoding amino acid residues - of the full length nucleocapsid protein was successfully amplified by rt-pcr and cloned into an expression vector in frame and downstream of the sixhistidine tag (fig. a) . the sequence and reading frame of the n gene were confirmed by dna sequencing of the recombinant plasmid. the recombinant protein was successfully expressed in e. coli. most of the expressed protein was soluble ( fig. b lane ) . the rsftsv-n protein was purified from the supernatant under native conditions. analysis of purified recombinant protein by sds-page and coomassie blue staining revealed a single protein band of kd as predicted by the amino acid sequence of the nucleocapsid protein ( fig. b lane ) . the identity of the rsftsv-n protein was further confirmed by western blot assay with mouse monoclonal antibody against histidine and the use of sfts patient serum sample (fig. ) . to determine the appropriate dilutions of serum samples for the indirect igg and igm elisas using the rsftsv-n protein mentioned above, samples from healthy volunteers and sfts confirmed patients were diluted two-fold from : up to : . after the application of igg elisa to serum samples from healthy volunteers, gave an optical density (od) value of . at : and lower dilutions of the samples. at higher dilutions, : and : , the od values of all the samples were between . - . . the od values of two serum samples for sfts-confirmed patients did not change much; the value was . at : dilution and was reduced only to . at : dilution. based on these results, the dilution of serum samples for igg elisa was set at : for convenience. in igm elisa, of the healthy volunteer samples gave an od value of . in : and : dilutions but all samples gave reduced values between . - . at : dilution. the od value of two samples from sfts-confirmed patients was reduced from . at : dilution to . at : dilution. thus, the dilution for igm elisa was set at : . at the set serum dilutions, : for igg and : for igm, all the healthy volunteer serum samples gave an od values between . and . , hence, the cut-off for giving a negative result was set at . . among the serum samples from sfts-suspected patients, samples were positive by rsftsv-n proteinbased indirect igg elisa and were negative. the od value for negative samples ranged from . to . , and from . - . for positive samples. these results perfectly matched the results obtained by using the total antibody table ) . among the serum samples from sfts-suspected patients, samples were positive and were negative by rsftsv-n protein-based indirect igm elisa. the od values for negative samples ranged from . to . , and from . - . for positive samples. our indirect igm elisa failed to detect the eight samples that were positive by the total antibody sandwich elisa kit. compared with the kit, the sensitivity and specificity of the rsftsv-n-igm capture elisa were . and %, respectively ( table ). in the present study, we expressed rsftsv nucleocapsid protein in e. coli and purified the recombinant protein to near homogeneity by the his-tag based affinity chromatography under native conditions and used it as an assay antigen in the indirect igg and igm elisa. nucleocapsid protein is the most abundant protein in many viruses and recombinant nucleocapsid protein has been used for the sero-diagnosis of many viruses like, sars and nipah viruses [ , ] . in the phlebovirus family, rvf virus n protein is a good target for igg and igm elisa systems [ ] [ ] [ ] . for sfts virus, there has been only one report on the development of an elisa system (double-antigen sandwich assay system) which makes use of a recombinant n protein for detecting total antibodies and this system was validated by neutralization test [ ] . the total antibody elisa kit used in the present study was based on this system. in contrast to the preparation of the recombinant n protein used in this antigen sandwich assay system, our recombinant protein was mostly soluble and was purified under native condition without using any detergent thereby skipping the arduous work of refolding the denatured protein. the expression and purification procedures described in this study provide a simple and efficient way to obtain pure sftsv n protein in large quantity. using this rsftsv-n protein, we developed indirect igg and igm elisa for human serum, and compared with a commercial total antibody detection sandwitch elisa kit. evaluation of serum samples from sftsvsuspected patients showed a concordance rate of and . % for the igg and igm elisa in comparison with the total antibody detection sandwitch elisa kit, respectively. the sensitivity and specificity of the rsftsv-n protein based indirect igg elisa system were both % with regard to total antibody detection sandwitch elisa kit ( table ) . the sensitivity and specificity of the rsftsv-n protein based indirect igm elisa system were . % and % respectively, with regard to total antibody detection sandwitch elisa kit ( table ). the indirect igm elisa system failed to catch eight samples that were positive by the total antibody kit. this may be caused by the lower sensitivity of indirect igm elisa method or the igm antibody against sftsv did not exist in these samples. all the serum samples were collected from sfts suspected patients who recovered after their illness and all the positive samples have the specific igg (table ) . it is well known that the igg competes with the igm in binding to antigen thereby reducing the sensitivity of indirect igm elisa [ ] . still our indirect igm elisa has a sensitivity of . %; it is an acceptable level for clinical diagnosis, especially for use in developing countries. for more sensitive diagnosis method, we are currently developing an igm capture elisa system. the total antibody sandwich-elisa system which was developed by jiao et al. was widely used in china because it is simple to perform, could be used to human and all kinds of animals. but the disadvantages of this method are that it cannot distinguish igg from igm and that more volume of the serum (serum used without dilution) is required for the assay thus, limiting its application for clinical diagnosis and large scale epidemiological studies. our sftsv-n protein based systems detect igg and igm separately, so it can distinguish previous or recent infection, respectively. the serum dilution is : for igg and : for igm; this greatly save the serum used and will be quite beneficial for precious samples and large scale epidemiological studies. the rsftsv-n protein based indirect igg and igm elisa systems presented here eliminate the use of infectious virus in the antigen production, which requires high level of microbiological security facilities. hence they are safer methods for diagnosis. the expression and purification procedure for recombinant sftsv-n protein is simple and easy allowing an easy standardization of the antigen production. the advantages of using a prokaryotic host to produce recombinant sftsv-n protein would be considerable due to the ease of scale-up, and the low costs involved in growing bacteria. it would be especially useful in cases of large-scale epidemiological investigation and for application in developing countries. in conclusion, the rsftsv-n protein is highly immunoreactive in human infection and it is a good target for laboratory diagnosis. our rsftsv-n protein-based igg and igm elisa systems are safe, specific and sensitive tools for serological diagnosis of sfts virus infections and especially fit to for use in large-scale epidemiological investigations. two serum samples from sfts-confirmed patients collected in and serum samples from healthy volunteers collected in -several years before the earliest identified sfts patient was reported-were used as positive and negative controls, respectively, in determining the serum dilution for the igg and igm indirect elisa we developed in the present study. to evaluate these two assays, serum samples collected in in henan province, china were used in this study. these samples were collected from patients who recovered from an illness that was suspected to be sfts. the yamaguchi strain of sftsv (genbank accession no. ab , ab , and ab ) that was isolated in from a patient in yamaguchi prefecture, japan was inoculated to confluent monolayer of vero-e cells. these cells were then maintained at °c in eagle's minimum essential medium supplemented with % fetal calf serum and . mm of each non-essential amino acids for days. the infected culture fluid (icf) was harvested and from a μl of this icf, viral rna was extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. the extracted rna was eluted in μl of elution buffer and then used as template for rt-pcr. rt-pcr was performed by using the primers '-ggag catgcatgtcggagtggtccagg- ' and '-aataa gcttttacaggtttctgtaagca- ' to generate the full length n gene of sftsv. sense and reverse primers contained sphi and hind iii restriction sites (underlined), respectively. the pcr amplified dna fragments were digested with sphi and hind iii, purified by a qiaex ii gel extraction kit (qiagen, hilden, germany), and subsequently cloned into the corresponding restriction site of the pqe vector (qiagen, hilden, germany). the insert of recombinant plasmid was confirmed to be in frame by dna sequencing. the expression construct encompassing amino acid (aa) - , the full length of sftsv n protein with a vector derived his-tag (histidine hexmer) at the n-terminus, was obtained. the resultant recombinant protein was designated as rsftsv-n protein. the rsftsv-n protein was expressed by inserting the recombinant plasmid containing the sftsv-n sequence into e. coli strain xl- blue and cultured at °c in luria-bertani (lb) medium containing μg/ml of ampicillin. when the optical density (od nm) of the culture reached . , the expression of recombinant protein was induced for h by the addition of . mm isopropyl β-d-thiogalactoside (iptg). after harvest by centrifugation, the e. coli pellet was washed in phosphate buffered saline solution (pbs), then resuspended in mm pbs ph . with mm nacl and frozen at − °c. after freezing and thawing three times, the cell suspension was sonicated for min with an interval of s between pulses and centrifuged at , g for min at °c. the supernatant was then applied to a talon™ imac resin column (clontech, usa). after being washed with a binding buffer ( mm pbs with mm nacl containing mm imidazole, ph . ), the purified protein was eluted with an elution buffer ( mm pbs with mm nacl containing mm imidazole, ph . ). the protein solution was aliquoted and stored in a final concentration of % glycerol at − °c until use. protein concentrations were determined by the bradford method using a bio-rad protein assay reagent kit (bio-rad, usa), and the purity of the protein was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). western blot analysis was performed as described before [ ] . briefly, protein marker (precision plus protein standards, bio-rad), sftsv infected vero-e cell lysates and the purified recombinant protein were separated in a - % gradient polyacrylamide gel (atto corporation, japan) before being electrotransferred onto a pvdf membrane (immobilon, millipore, usa) by using a semidry electroblotter (sartorius, germany). the membrane was blocked with blockace (yokijirushi, sapporo, japan) overnight at °c to prevent nonspecific staining, then subjected to reaction with mouse anti-histidine (ge healthcare life sciences, : dilution), or patient serum sample ( : dilution) for h at °c before incubation with rabbit anti-mouse igg, or goat antihuman peroxidase conjugate (american qualex, califonia, usa, : dilution) for h at °c. finally, the reaction was visualized by dimethyl aminobenzidine (dab) staining. serum samples from two sfts-confirmed patients and from healthy volunteers were diluted at : , : , : , : and : dilutions. the diluted sera were checked by rsftsv-n based indirect igg and igm elisa separately as described below. the appropriate dilution of the serum samples for these two assays was determined based on od values at nm. to evaluate the usefulness for diagnosis of the rsftsv-n protein we developed, we established an indirect igg and igm elisa for the laboratory diagnosis of sftsv infection in humans with the serum samples as clinical specimens. these assays have common procedures which will be described here and the procedures specific for each assay will be described under each assay. the plates used were -well nunc immunoplates (thermo scientific, denmark), and all the reagents were in μl volumes. the optimal concentration of rsftsv-n protein used to coat the microplates was determined by checkerboard titration with reference serum samples ( from sftsconfirmed patients and from healthy volunteers). the coating buffer was . m pbs, ph . , and plate coating was conducted at °c overnight. wash buffer was . m pbs with . % (vol/vol) tween (pbs-t). plates were washed three times with pbs-t after exposure to a specific reagent at each step of the procedure except at the last step as described below. dilution of all serum samples and reagents were in % nonfat milk (difco, detroit, usa) in pbs-t. incubations, except for substrate, were done for h at °c. plates with μl h o -abts substrate (kirkegaatrd & perry, gaithersburg, md) in each well were incubated for min at °c. the plates were read spectrophotometrically and od values at nm were recorded. in the indirect igg elisa, -well plates were subjected to the following steps: coating of each well with ng rsftsv-n protein per well followed by human serum samples that were diluted : , in % nonfat milk in pbs-t, then detection of bound igg with : , diluted horseradish-peroxidase-conjugated goat anti-human igg (american qualex, califonia, usa) which was made visible after adding h o -abts substrate, the last reagent in this series of procedure. od values at nm were recorded on a microplate spectrophotometer. each serum sample was tested in duplicate, and the mean od for each sample was calculated. reference serum samples were run in every assay. the mean od of a sample more than twice the mean od of the negative control serum was considered positive. the procedure for indirect igm elisa was similar to igg elisa. the changes were that the serum samples were diluted at : and the detection of bound igm was donewith : , diluted horseradish peroxidaseconjugated goat anti-human igm (american qualex, califonia, usa). serum samples from suspected sfts patients were subjected to a commercial elisa kit (xinlianxin biomedical technology co., ltd, wuxi, jiangsu, china) following the manufacturer's protocol. the kit is a double-antigen sandwich enzyme-linked immunosorbent assay kit that detects total antibodies including igg and igm against sftsv [ ] . results obtained by using this total antibody elisa kit was compared to the results obtained by applying the igg and igm indirect elisa described above. this research was approved by the institutional review board at the center for disease control and prevention of henan province. all participants gave written informed consent for the use of their serum samples for research purposes. metagenomic analysis of fever fever with thrombocytopenia associated with a novel bunyavirus in china hemorrhagic fever caused by a novel tick-borne bunyavirus in huaiyangshan, china case-fatality ratio and effectiveness of ribavirin therapy among hospitalized patients in china who had severe fever with thrombocytopenia syndrome a family cluster of infections by a newly recognized bunyavirus in eastern china, : further evidence of person-to-person transmission a cluster of cases of human-to-human transmission caused by severe fever with thrombocytopenia syndrome bunyavirus person-to-person transmission of severe fever with thrombocytopenia syndrome bunyavirus through blood contact person-to-person transmission of severe fever with thrombocytopenia syndrome virus. vector borne zoonotic dis human-to-human transmission of severe fever with thrombocytopenia syndrome bunyavirus through contact with infectious blood a highly pathogenic new bunyavirus emerged in china severe fever with thrombocytopenia syndrome: tickmediated viral disease severe fever with thrombocytopenia syndrome the first identification and retrospective study of severe fever with thrombocytopenia syndrome in japan sensitive and specific pcr systems for detection of both chinese and japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load severe fever with thrombocytopenia syndrome virus in ticks collected from humans, south korea a new phlebovirus associated with severe febrile illness in missouri early diagnosis of novel sfts bunyavirus infection by quantitative real-time rt-pcr assay development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for rapid detection of a new sfts bunyavirus establishment of a novel one-step reverse transcription loop-mediated isothermal amplification assay for rapid identification of rna from the severe fever with thrombocytopenia syndrome virus detection of new bunyavirus rna by reverse transcription-loop-mediated isothermal amplification detection of severe fever with thrombocytopenia syndrome virus by reverse transcription-cross-priming amplification coupled with vertical flow visualization severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis the ecology, genetic diversity, and phylogeny of huaiyangshan virus in china cloning and expression of rift valley fever virus nucleocapsid (n) protein and evaluation of a n-protein based indirect elisa for the detection of specific igg and igm antibodies in domestic ruminants preparation and evaluation of a recombinant rift valley fever virus n protein for the detection of igg and igm antibodies in humans and animals by indirect elisa validation of an igm antibody capture elisa based on a recombinant nucleoprotein for identification of domestic ruminants infected with rift valley fever virus preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by doubleantigen sandwich enzyme-linked immunosorbent assay evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay serodiagnosis using recombinant nipah virus nucleocapsid protein expressed in escherichia coli immunoglobulin m antibody capture enzyme-linked immunosorbent assay for diagnosis of st. louis encephalitis this study was supported by a grant-in-aid for scientific research from the ministry of education, culture, sports, science and technology (mext), japan, the japan initiative for global network on infectious diseases (j-grid), mext, the authors declared they have no competing interests. key: cord- -srenbxa authors: zhao, jincun; wang, wei; wang, guang-fa; li, yonghua; zhuang, hui; xu, xiaoyuan; ren, furong; zhao, zhendong; gao, xiao-ming title: development and evaluation of an enzyme-linked immunosorbent assay for detection of antibodies against the spike protein of sars-coronavirus date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: srenbxa background: severe acute respiratory syndrome (sars) is caused by infection with sars-associated coronavirus (cov). amino acid residues – of the spike (s) glycoprotein of sars-cov (s - ) contains dominant epitopes for anti-viral antibodies (abs) in patient sera. objectives: to develop and evaluate an elisa system for detection of anti-s abs in patient sera. study design: express recombinant s - in e. coli and evaluate the sensitivity and specificity of an elisa system based on the s - polypeptide. results: the s - -based elisa detected igg abs in out of serum samples from hospitalized patients with probable sars, a result closely correlated with that obtained with a virus-based elisa (r = . , k = . ). differential anti-s igg responses were observed amongst sars patients. some of them produced anti-s abs early during their infection, while others failed to make igg abs against the s - polypeptide. none of the serum samples from healthy blood donors was positive in the s - -based assay. conclusion: the s - -based elisa can detect anti-s igg abs with high sensitivity and specificity. severe acute respiratory syndrome (sars) is caused by sars-associated coronavirus (sars-cov), an enveloped, positive-stranded rna virus of the coronaviridae family (rota et al., ; peiris et al., ) . the genome of sars-cov encodes several structural proteins including the spike (s) glycoprotein, nucleocapsid protein, membrane protein and envelope protein (marra et al., ) . membrane fusion between sars-cov and the host cell is mediated by its s glycoprotein xiao et al., ; dimitrov, ; wong et al., ; wang et al., ; sui et al., ) , which consists of amino acid residues with approximately % homology to that of the other human covs (spiga et al., ; ho et al., ) . the s subunit (amino acids - ) of the s glycoprotein contains a receptor-binding domain and is apparently the main target for neutralizing abs in patient sera (xiao et al., ; sui et al., ; wong et al., ; wang et al., ; babcock et al., ) . western blot (wb) and indirect immunofluorescence assays have been developed for detection of abs against the s protein chang et al., ; wu et al., ; woo et al., ; ho et al., ; he et al., ) . however, so far there is no enzyme-linked immunosorbent assay (elisa) available for easier and more sensitive detection of anti-s abs. our computer-assisted analysis suggested that amino acid residues - of the s glycoprotein (s - ) of sars-cov is largely solvent accessible and likely to contain dominant b cell epitopes. this coincided with recently published results by lu et al. ( ) showing that residues - of the s protein of sars-cov contained dominant epitope(s) for anti-s abs in patient sera, as determined in wb assays. sequences outside the - region were relatively poorly recognized by patient sera . it is also supported by findings of zhou et al. ( ) that residues - of the s protein of sars-cov elicited neutralizing abs against the virus. in this study, the s - fragment was expressed in e. coli and used as ag in an elisa system for detection of anti-s abs. restriction enzymes and t ligase were from invitrogen (usa). a kit for dna extraction and purification was from qiagen (hilden, germany). e. coli strains of bl (oe ) and dh ␣ were obtained from novagen (germany) and invitrogen (usa), respectively. complementary dna encoding full length s of sars-cov was a gift from china cdc. from march , , a major outbreak of sars had taken place in beijing, china. we collected sequential blood samples ( samples in total) from patients (both sexes, - years of age, average . years), admitted to the first affiliated hospital of peking university, beijing, china between th april and th june, . all patients fulfilled the who definition of probable sars (fever • c or higher, cough, new pulmonary infiltrates on chest radiography in the absence of an alternative diagnosis to explain the clinical presentation). the blood samples were processed within h of collection. all patient sera were tested for anti-sars-cov igg abs using an elisa kit produced by huada institute (see below). a total of serum samples from patients, randomly selected from the above patient group, were tested using our s - -based elisa kit. in addition, sera from healthy blood donors (both sexes, - years of age, collected between may and july ) were provided by beijing red cross blood center. a small outbreak of sars took place in april involving nine patients in anhui and beijing, china. sequential serum samples from four of the confirmed patients (second or third generation cases), admitted to ditan hospital between th april and th june , were also included in this study. dna coding for s - of sars cov s protein was pcr amplified using high fidelity taq dna polymerase (takara biotech co. ltd., japan). the sequences of the primers employed in the pcr reaction were s - : cgc gga tcc atg ccc ttt gag aga gac ata tct (forward primer, carrying a bamhi restriction site) and s - : ccc gaa ttc tta aat gtc gca ctc ata aga agt g (reverse primer, carrying an ecori site). the pcr product was gel-purified and cloned into expression vector pet- a (novagen, germany). the resultant construct, pet a-s / , encodes for the s - fragment with a histidine (his) tag ( his) at the n terminus. dna sequence of the insert was determined and compared with the s gene sequence of sars-cov strain urbani (accession number ay ) using dnastar software. pet a-s / was transformed into e. coli bl cells for expansion. a bacterial colony harboring the plasmid was inoculated into yt medium containing kanamycin ( g/ml) and cultured, with continuous shaking, at • c to appropriate density. isopropyl-␤-d-thiogalactopyranoside (iptg) was then added to a final concentration of . mm to induce the expression of the recombinant protein (for further . h). after centrifugation at × g for min, the pellet was resuspended in pbs and subjected to sonication in an ice bath for min. the inclusion bodies were solubilized with m urea, after centrifugation at × g at • c for min, the supernatant was applied to an equilibrated ni column (novagen, germany). after on-column refolding procedure, proteins bound to the column were eluted with mm imidazole in tris-buffered saline (ph . ). the eluted fractions were examined by sds-page and the proteins were either stained with coomassie blue or transferred to nitro-cellulose membrane for wb assays. the nitro-cellulose membranes, on which protein bands had been transferred, were blocked at room temperature for h with block solution ( % non-fat dry milk) and then incubated for h at room temperature with serum sample. after washing in tris-buffered saline (tbs, ph . ) containing . % tween , the membranes were incubated with hrp-labeled goat anti-human igg ab (zhongshan biotech co., china). , -diaminobenzidine tetrahydrochloride (dab, sigma) was used to visualize the reaction. elisa plates (nunc, demark) were coated at • c overnight with recombinant protein ( . pmol/well) in carbonate buffer (ph . ). immediately before use, the coated plates were incubated with blocking solution ( % bsa in pbs) for h at • c and then washed times with pbs containing . % tween (pbs-t). serum samples ( / diluted, l/well) were added in triplicate and incubated for min at • c. after washes with pbs-t, horseradish peroxidase (hrp)-labeled goat anti-human igg ab was added and incubated for h at • c. ortho-phenylenediamine (opd) substrate was used ( l/well) as substrate with m h so solution as stop buffer. the optical density (od) was immediately read at nm. for elisas using the kit produced by huada institute (beijing, china), the manufacturers' instruction was followed. briefly, dilution buffer ( l/well) was added to pre-coated wells followed by l serum and incubated for min at • c. after washes, hrp-labeled detection ab ( / diluted) was dispensed ( l/well) and incubated for min at • c before further washes. substrate buffer containing abts [ , -azino-di-( -ethylbenzo-thiazoline sulfonate)] was then added and allowed to develop for min. after stop buffer was added and the plates were read at nm. all experiments describe here have been repeated at least three times. results obtained using s - recombinant protein-based elisa and sars-cov-specific kit were compared using the correl module of microsoft excel software. the cohen kappa test was performed to analyze the agreement between the results of the two elisa kits using spss software. complementary dna encoding s - of sars-cov was cloned into expression vector pet- a for expression in e. coli bl . the his-tag-containing recombinant protein was purified to more than % homogeneity using a ni column. sds-page analysis of the affinity purified product revealed protein band of expected molecular weight (fig. ) . in wb assays, convalescent serum from a sars patient (pt-lx) and anti-his-tag mab specifically recognized the recombinant s - (fig. ) . in the subsequent study, recombinant s - was employed as coat- ing ag for an elisa kit with hrp-labeled goat-anti-human igg abs as secondary ab. checker-board titration experiments were carried out to optimize the concentration of the coating ag (s - ) and secondary ab (data not shown). a sars-cov-specific elisa kit, developed by huada institute, beijing, china, has been licensed by the ministry for public health of china. it employs the lysate of sars-covinfected vero-e cells as coating ags and has been widely used in china for sars-cov-specific ab testing with reliable results. by using the huada kit, we analyzed sequential serum samples (total sera) from hospitalized patients with probable sars and the results are summarized in table . half of the patients seroconverted for igg abs against sars-cov by day after the onset of illness. nearly % of them produced virus-specific serum abs by days - . sera from three convalescent sars patients and two healthy individuals were serial diluted and tested in the s - -based elisas, which detected anti-s igg abs in a specific and sensitive manner, with the reactivity end point from : to : diluted patient sera (fig. ) . serum samples from healthy blood donors were subsequently screened and none of them was positive (fig. a) , suggesting a high specificity for the s - -based system. when sequential serum samples ( total, / diluted) from patients (randomly selected from the abovementioned patient set) with probable sars were analyzed using the s - -baesd assays, were positive (fig. b) . the huada kit detected anti-viral igg abs in out of the samples (fig. c) , of which were positive in both assays. when the s - -based and virus-based elisa results were plotted against each other, a linear correlation (first degree regression, r = . ) was observed (fig. ) , fig. . sensitivity of the s - -based elisa. elisa plates were coated with recombinant s - . serum samples from convalescent sars patients ( , ♦, ) and healthy individuals ( , ) were serial diluted and dispensed, in triplicates, into the wells. hrp-labeled goat anti-human igg was used as secondary ab with opd as substrate. the results are expressed as absorbance reading at nm wavelength. fig. . comparison of s - -based and virus-based elisa results. the s - -based elisa kit was used to screen serum samples from (a) healthy blood donors and (b) patients (pt to pt , collected between and days from the onset of illness) with probable sars. the serum samples were folds diluted, results are expressed as absorbance reading at nm wavelength. cutoff value was . . elisa results obtained with the huada kit are shown in (c) for comparison. in this case, serum samples were folds diluted, results are expressed as absorbance reading at wavelength of nm. cutoff value was . . each bar represents one serum sample, samples from each patient were group together in the order of collection time (days) after onset of illness. the cohen kappa test also confirmed an agreement between them (κ = . ). time courses of igg responses against the s protein and the whole virus in out of the patients are compared in fig. a and b. sera from patients pt , pt and pt contained high titer anti-s but low titer anti-virus igg abs. in contrast, patient pt was a high responder against the whole virus but low responder against s - . patient pt seroconverted days after the onset of symptoms, suggesting a possible case of sars-cov infection being acquired in the hospital ward. patient pt remained negative in both tests months after the onset of illness and was eventually excluded as a sars patient. both the s - -based and the virus-based elisa kits were employed to screen sera from an additional sars patients infected in april . three of them were high responders against the s protein, while the other one (pt ) responded poorly to s - but strongly to the whole virus ( fig. c and d) . in patient pt , anti-s serum abs appeared earlier than that specific for other viral proteins. specific and sensitive detection methods for anti-viral abs in patient sera are of great value in diagnosis as well as research. virus-based assays have the advantage of being able to detect abs specific for all structural components of the virus. on the other hand, recombinant protein-based tests would allow analysis of anti-viral humoral immunity in much detail. the s protein of sars-cov is heavily glycosylated and contains many disulfide bonds (spiga et al., ; krokhin et al., ; tripet et al., ; ying et al., ) . it is difficult, however, to obtain s glycoprotein expressed in eukaryotic systems, which would otherwise be ideal for detection of anti-s abs in vitro. full-length s protein expressed in prokaryotic systems is often insoluble and therefore unsuitable as coating ag in elisa systems. our on-column refolding procedure allowed correct formation of some of the disulfide bonds in the s - fragment, producing soluble recombinant protein at a reasonably high concentration (data not shown). results reported herein indicate that the s - -based elisa can detect anti-s abs in patient sera with high sensitivity and specificity. it has been suggested that the n-glycans of the s glycoprotein could have a significant effect on its antigenicity, as the presence of n-glycans contributes to the correct folding and biological function of glycoproteins. however, recently published results indicate that bacterial expressed fragment of the s protein (non-glycosylated) could maintain its antigenicity (zhou et al., ; lu et al., ) . in addition, neutralizing abs were successfully raised in mice after immunization using prokaryotically expressed s - of sars-cov (zhou et al., ) . in this study we wound that most sars patients developed strong igg responses against the s glycoprotein of sars-cov. some of them had anti-s abs in significantly higher titer than that against other structural proteins of the virus (e.g. pt and pt ). in this situation, our s protein-based elisa would be more sensitive than the virus-based assays in detecting antiviral abs. there were also cases where the s - was poorly recognized by abs in patient sera (e.g. pt in fig. c and d). it should be emphasized, however, that the s - polypeptide covers less than / of the s protein sequence. a negative result such as this does not rule out possible presence of anti-s abs against epitopes outside the - region of the s protein. moreover, xu et al. ( ) have reported relatively high frequency of variations in s gene of sars-cov. natural variability in the s protein might also affect the validity of the s - -based elisa, although antigenic variation in s - has not yet been fully characterized. amino acids to of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor antibody detection of sars-cov spike and nucleocapsid protein the secret life of ace as a receptor for the sars virus development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome antigenicity and receptor-binding ability of recombinant sars coronavirus spike protein mass spectrometric characterization of proteins from the sars virus: a preliminary report angiotensin-converting enzyme is a functional receptor for the sars coronavirus immunological characterization of the spike protein of sars-coronavirus the genome sequence of the sars-associated coronavirus coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome molecular modeling of s and s subunits of sars coronavirus spike glycoprotein potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers structural characterization of the sars-coronavirus spike s fusion protein core expression cloning of functional receptor used by sars coronavirus a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensinconverting enzyme relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia early detection of antibodies against various structural proteins of the sars-associated coronavirus in sars patients the sars-cov s glycoprotein: expression and functional characterization sars-associated coronavirus quasispecies in individual patients proteomic analysis on structural proteins of severe acute respiratory syndrome coronavirus an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies the first two authors contributed equally to this work. key: cord- -a b hyyr authors: nan title: th annual meeting of the gth (gesellschaft für thrombose- und hämostaseforschung) date: journal: ann hematol doi: . /bf sha: doc_id: cord_uid: a b hyyr nan the variable molecular weight (mw) of vwf is due to differences in the number of subunits comprising the protein. it is assumed that endothelial cells secrete large polymeric forms of vwf and that smaller species arise from proteolytic cleavage. vwf has two main properties: it stabilizes factor viii protecting it from inactivation by activated protein c or factor xa, and it mediates platelet adhesion to subendothelium of the damaged blood vessel wall. each vwf subunit contains binding sites for collagen and for platelet giycoproteins gp ib and gp iib/i~a. multiple interactions of the multivalent vwf lead to extremely strong binding of platelets to subendothelial surface, that is capable of resisting high wall shear rate in the circulating blood. only the largest multimers are hemostatically active. lack of the largest vwf multimers was observed in patients with yon wiuebrand disease type a. unusually large molecular forms of vwf were found in patients with thrombotic thrombocytopenlc purpura. proteolytic enzyme(s) may be involved in the physiologic regulation of the polymeric size of vwf and thus play an important role in the pathogenesis of vwf abnormalities in some patients with congenital or acquired disorders of hemostasis. we have purified (- , -fold) from human plasma a vwf degrading proteas¢ using affinity chromatography and gel filtration. the proteolytic activity was associated with a high mw protein (mr - kd). vwf was resistant against the protease in a physiologic buffer but became degraded at low salt concentration or in the presence of m urea. proteolytic activity had a ph optimum at g- and was not inhibited by serine protease inhibitors or sulfl~ydryl reagents. inhibition by chelating agents was best reversed by barium ions, the observed properties of the vwf degrading enzyme differ from those of all hitherto described pretenses. analysis of cleaved vwf showed that the peptide bond tyr-g met had been cleaved -the same bond that has been proposed to be cleaved in rive. the endothelium releases the vasodilator nitric oxide (no) and the vasoconstrictor endothelin (et)-i. no is formed from l-arginine via the activity of constitutive nitric oxide synthase (cnos or enos). an inducible form of nos (inos) is activated by cytokines. no activates guanylyl cyclase in vascular smooth muscle and platelets, leading to the formation of cgmp which induces relaxation or platelet inhibition, respectively. in vessels, no is responsible for endothelium-dependent relaxations; in vivo it exerts a vasodilator tone which can be enhanced by shear forces and receptor-operated agonists such as acetylcholine, bradykinin, thrombin, atp and adp. infusion of no-inhibitors in vivo leads to vasoconstriction and increases in blood pressure and oral administration to hypertension in the rat. within the endothelium, no inhibits et gene expression and release of the peptide via cgmp. hence, no-induced hypertension is associated with increased plasma et levels. et, a -amino acid peptide, has potent vasoconstrictor properties via eta-and in part etb-raceptors on vascular smooth muscle. in endothelial cells, et activates etb-receptors linked to no and prostacyclin formation. under basal conditions, little et is formed, but is increased by thrombin, angiotensin ii, arginine vasopressin, cytokines and ox-ldl. et antagonists have been developed and allow to study the effects of et in vivo. et and no most likely play an important role in disease states such as hypertension, atherosclerosis, coronary artery disease, heart failure, pulmonary hypertension and subarachnoid hemorrhage. clinical trials to further define their role in these disease states are now under way. in summary, the endothelium is an important regulator of vascular tone and structure in vitro and in vivo. in disease states, their interaction is imbalanced leading to enhanced vasoconstriction, thrombus formation and structural changes of the blood vessel wall. pharmacological tools aiming to inhibit those changes are now being developed. j.m. harlan, r.k. w/nn, s. sharar, and n. vedder universit , of washington, seattle, washington ischemia-reperfusion injury has been implicated in the pathogenesis of a wide variety of efinical disorders. [n preclinical models, tissue damage .clearly occurs during ischemia, but, parado.,dcally, may be exacerbated during reperfusion. this reperfusion injury appears to involve activation of the intlammato~, cascade with generation of complement 'components, lipoxygenase products, and chemokines as proximal mediators and neutrophils as final effectors of vascular and tissue damage. we have examined the role of leukocyte adhesion in reperfusion injury in two models -the rabbit ear as a model of isolated organ injury and hemorrhagic shock and resuscitation in the rabbit and primate as a model of traumatic shock and multiple organ failure. data regarding the efficacy, timing, and safety of leukocyte adhesion blockade using selectin-or integrindirected reagents in these models w/ll be presented. the current status of anti-adhesion therapy in other pre-clinical models and early clinical trials will be re~ ewed. an amidolytic assay for the determination of activated protein c (apc)-resistant factor va (fva) has been developed. this assay measures the cofactor activity of fva in diluted plasma samples via the rate of thrombin formation. the apc response is calculated from two fv determinations: one performed in the presence (apc-fv) and one in the absence of recombinant apc. the apc-fv activity is expressed as percentage of the initial fv activity and indicates the sensitivity of fva to apc. normal ranges were established by analysing plasma samples of healthy individuals and an apc-fv activity above % was found to be indicative for apc-resistance (apc-r). in a control group of patients the apc-r assay gave abnormal results in patients. dna analysis confirmed heterozygous fv r q mutation in all patients and confirmed the non-carrier status in all of the patients yielding normal results. an aptt-based apc-r assay performed on the same group of patients showed abnormal results in two of the non-carrier patients. one of these patients was diagnosed as positive for lupus anticoagulant, whereas the reason for the wrong positive result in the second patient remains unclear. eleven patients were analyzed before start of oral anticoagulation and during oral anticoagulant treatment. comparison of the assay results demonstrate a correlation of % indicating that the assay is independent of the activities of vitamin k-dependent clotting factors. the apc-r amidolytic assays allows specific and sensitive detection of fva-resistant to apc. the assay is performable in plasma samples of all persons in whom the diagnosis of apc-r may be indicated. in patients treated with oral anticoagulants or showing other clotting abnormalities affecting the aptt the apc-r amidolytic assay is helpful to establish the diagnosis of apc-r. dept of pediatrics, university hospitals kiel and mtinster, germany resistance to activated protein c (apcr), in the majority of cases associated with the arg gin point mutation in the factor v gene is present in more than % of patients < years of age with unexplained thrombophilia. to determine to what exteut this relativdy common gene mutation affects the risk of thromboembolie events in infants and children, its occurrence was investigated in a population of children with unexplained venous or arterial thromboernbotism: thrombosis of the central nervous system (cns, n= ), vena portae (n= ), deep vein thrombosis (n= ), vena caval occlusion (n= ), neonatal renal venous thrombosis (rvi'; n-- ), neonatal stroke (n= ), stroke (n= ), arteria femoralis ocdusion (n= ). four ont of these patients showed a positive history of unexplained familial thrombophilia. apcr was measured in an activated thromboplastin time (afit) according to dahlbtick. the results were expressed as apc-ratios: clotting time obtained using the apc/caci -solution divided by dotting time obtained with cac . concerning the special properties of the childhood hemostatic system, infants and children with apcr < were considered to be apc-resistent only when the results were confirmed in a : l dilution with factor v deficient plasma (instrumentation laboratory munich, germany). plasma of healthy children served as controls. the arg gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mul i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. consistent with the ap'it based method out of children with venous (v) thrombosis and eight out of patients with arterial (a) vascular insults showed the common factor v mutation. additional coagulation defects (autithrombin, protein c type i, enhanced antiphospbolipid igg, enhanced lipoprotein (a)) were found in % (v) and % (a). furthermore, we diagnosed exogenlc reasons (septicemia, postpastal asphyxia, fetopathia diabetica, central line and steroid/asparaginase administration) in six out of (v) and three out of (a) children with thrombosis and apcr. all four patients with a positive family history of thrombophilia (mothers only !) showed the common factor v mutation arg gin. in the control group the prevalence of apcr was . %. the high incidence of additional exengenic factors in children with apcr confirm literature data of previously described inherited coagulation disorders during infancy and childhood: an acquired risk of thromboembolic disorders masks the coagulation deficiency in the majority of patients with an inherited prethrombotic state. furthermore, the incidence of % apc resistant children with arterial insults in this study challenge the view that apcr is associated with venous but not with arterial thrombo-st . activated protein c resistance and plasminogen deficiency in a family with thrombophilia m, zttger , f. demarmels biasiutti , ch. mannhalter , m. furlan , b.~e httmatologisches zentrallabor der universit~t, inselspital, ch- bern klinisches institut fur medizinische und ehemische labordiagnostik, universit~t wien, a- wien several hereditary defects of the proteins regulating blood coagulation have been associated with familial thrombophilia. since the recent discovery of activated protein c (apc) resistance due to the factor v rf q mutation as a highly prevalent hereditary risk factor for venous thromboembolism (tel, evidence is accumulating that familial thrombophilia may be due to a combination of genetic defects. thus, protein c-or protein s-deficient patients having suffered from te seem to be more likely to carry the factor v r q mutation than expected from its allelic frequency in the population. we report a family (see figure) in which plasminogen deficiency ( . u/ml) had been found in the propositus having had twice postoperative deep vein thrombosis (dvt) at ages of and yrs, respectively, as well as in family members ( . - . u/ml). out of these plasminogen deficient individuals, only the propositus' daughter had suffered from recurrent dvt at age < yrs. reinvestigation of this family in showed factor v r q mutation in the propositus, his daughter, an asymptomatic sister and a brother with postoperative pulmonary embolism (pe). his father had had postoperative pe; he is deceased and could not be examined. ~, [] plasminogen deficiency ~ ,rll factor v rf q mutation /~ propositus ~¢ bistory of dvt and/or pe e superficial phlebitis j ~,,~ not investigated " " deceased even though this family is small for establishing unequivocal association of te with known defects, the two most severely affected individuals with recurrent te at ages < yrs had combined plasminogen deficiency and apc resistance whereas those with isolated plasminogen deficiency were asymptomatic. these data support the concept of multigenie interactions leading to familial thrombophilia. resistance to activated protein c (apc resistance) is the most common dsk factor for venous thrombosis (vt). in most cases apc resistance is caused by a single point mutation at position arg in the factor v gene (factor v leiden). while ample data in hetarozygous patients have been published, reports in homozygous patients are limited. we studied patients ( males [m] , females it]) in whom a homozygous mutation had been verified by dna analysis. the median age at the time of the study was . years (y) (range - y). twenty-five patients had experienced vt ( m, f). four patients were discovered dunng family studies and were asymptomatic, three were children (between and y) and one patient was a y old man. in males the first thrombosis occurred at a median age of y (range - y), in females this was at a significantly younger median age of y (range - y). twelve of the symptomatic females had taken oral contraceptives (oc, estregen content . - .ling) for to months (median m) pdor to thrombosis. in women vt occurred during pregnancy, in female it was precipitated by hormone replacement therapy. in contrast, in ,< males the thrombosis happened spontaneously, in males it followed surgery. the sites of thrombosis were dvt in males and females, dvt and pulmonary embolism (pe) in females and male, dvt and caval vein thrombosis in female and superficial thrombophlebitis in males and female. eight females had at least one pregnancy, in total children and abortions. two had thrombotic events during pregnancy and after delivery. all homozygous patients showed apc ratios between . and . (mean . + . ). conclusion: patients with homozygous fv leiden have similar clinical symptoms as patients with deficiencies of antithrombin-, protein c or protein s deficiency. however, in contrast to these defects a very high dsk dudng oral contraceptive medication leading to an ealier manifestation in females can be observed. several synthetic (efegatran, argatroban, inogatran and napsagatran) and recombinant (hirudin, peg-hirudin and hirulog) antithrombin agents are in different stages of nlinical development for cardiovascular and thrombotic indications. while the specificity of these agents for thrombin is a concern, little has been done to study the effects of these agents on other serine proteases involved in coagulation and fibrinolytic processes. fihrinolytic compromise by site directed thrombin inhibitors has been reported recently (thromb res ( ): - , ) . while these agents have been shown to inhibit plasmin and related enzymes, little or no informatienentheir effects onthe generation and functionality of apc is available. sinceapc plays an increasingly important role both as an antienagulant enzyme, by inhibiting factors v and viii, end a pro-fibrinelytic enzyme, by stimulating the release of t-pa fi'om endothelial sites, an inhibition of apc may result in both pro-coagulant state and fibrinolytic deficit. representative thrombin inhibitors (dup -a prototype boronic acid peptide derivative, efega~an, argatroban, hirulog, hirudin and feg-hirudin) have been compared for their ability to inhibit apc (american red cross). these bionhemically defined studies in which the remaining activity of apc after incubation with a thrombin inhibitor was determined speclrophotometrically with a ehromogenic substrate (s- , pharmacia, franklin, oh) , demonstrated that dup and efegatran inhibit apc in a coneentrafinn dependent manner (ic~ = . and , gm resp~tively), hirnlog inhibits apc weakly ( p ¢i produced only % inhibition), while argatroban and ~ have no anti-apc activities, while hirulog, hirudin and argatroban produced no direct enti-apc activities, it is conceivable that they may inhibit thrombomodnlin-bound thrombin and thus prevent activation of protein c, resulting in a functional apc deficit and failure to improve clinical outcomes despite higher dosage. while initially it was thought that sole targeting of thrombin will provide monospacifin anticoagulant agents devoid of some of the adverse effects observed with heparin, the recent clinical trials clearly suggest that thrombin is not the only determinant of thrombogenesis. furthermore, potent antithrombin agents such as hirudin, hirulog and peptides, indirectly inhibit the generation of apc, by compromising thrombomodulin-bound thrornhin and such agents as efegalran and dup also produce direct apc inhibition. endogenous inhibition of formed ape by thrombin inhibitors may therefore compromise the feedback regulatory funetiens of apc and may lead to thrombotic amplification in fully enticoagulated patients. these studies warrant prcelinlcal assessment of thrombin inhibitors to evaluate their relative inhibitory effects on apc. poor anticoagulant response to activated protein c (apc resistance) causes a significant portion of deep vein thrombosis (dvt) whereas its association with coronary artery disease (cad) and myocardial infarction (md is still controversial. therefore, we investigated recently hospitalised patients suffering from cad with or without previous mi. the cad was proven by coronary angiography. apc resistance was analysed by using the ap'l-i'-based assay coatest apc resistance (chromogenix). eleven patients showed an apc sensitivity index below . viewed as apc resistance. using pcr technology, the factor v mutation causing apc resistance g "-) a) has been shown in nine of these eleven patients. this represents . % ( / ), compared to , % found in healthy blood donors ( / ). one homozygous carrier (male, age ) was identified (apc sensitivity index . ) who suffered from dvt at age . recent angiography demonstrated diffuse cad, no thrombotic events were reported in his family. in contrast, multiple thrombotic manifestations (dvt, mi, stroke) occurred in the relatives of four heterozygous patients. we conclude that the prevalence of apc resistance is rather low in patients with cad. nevertheless, the natural history of coronary manifestation of apc resistance seems to vary, probably depending on the presence and severity of cardiovascular risk factors. resitanee to activated protein c (apc resistance) is the most cormnon hereditary cause of thrombophilia and significantly linked to factor v leiden pcr based methods are used to identify the crucial point mutation in the factor v g(me. we designed primers in order to identify factor v leiden by allele-specific pcr amplification. amplification specificity for factor v was ensured by the 'primer fv , located at the introng/intronl border of the g~ae. one sense and two antisense primers were used in ~vo separate primer mixes specific for factor v arts (wildtypo) or factor v otn (factor v leiden) yielding bp products each. in each pcr reaction a pair of primers amplifying a fragment of the human growth hormone gene was included, fimctioning as an internal positive amplification control ( bp pcrfragment). after an initial denaturation step /.tl samples ( rig genomic dna) were subjected to two-temperature cycles fouowed by threetemperature cycles. for visualisation p of the amplification product were run on a % agarose gel presmined with ethidittm bromide. the presence or absence of specific pcr amplification allowed defiu/te allele assignment without the need for any postamplifieation specificity step. the in~ernal positive control primers it~cate a sucessf-u/pcr amplification, allowing the assignment of homozygosity. in a prospective study p~e.ients with tlaromboembolic events were analysed using this technique and enmpared with pcr -rflp according to bertina et al. the concordance between these techniques was %. in patients a heterozygous factor v ohas mutation was detected, whereas one pa ent with recurrent thrombcembolism was homoz-ygous. no false-positive or false-negative results worn observed in the homozygous as well as hcterozygous samples. in addition in samples identified to carry the point mutation by al/ele-specifin pcr anxplification automatic :~equencing confirmed the heterozygous or homozygous point mutation. due to its time-and cost-saving features allele-specific amplification should be considered for screening of factor v leiden. background: an initial intravenous course of tmfractionated heparin ~ljasted on the basis of the activated partial thromboplastin time is the currmt standard treatment for most patients with venous thrombosis. low-molecularweight hqmrin pre~a~tious can be administered subcutaneously, once or twice daily, without laboratory monitoring. we compared the relative effic.~y and safety of low-molecular weight heparin versus anfractionated heparin for the initial treatment of deep venous thrombosis. methods: english-language reports of randomized trials vtta'e identified th~ a medline search ( through ) and a complementary extensive manual search. reasons for exclusion from the analysis were no hepada dosage adjustments, the lack of um of obj~tive tests for deep venous thrombosis, dos~ranging studies that used higher doses of low-molecularweight heparin than are ctareatly in use, and the failure to provide blind endpoint ~sossmeat. we assessed the incidence of symptomatic recurrent vinous thromboembolic disease, the incidence of clinically ii~t bleeding and mortality. results: twelve of the identified trials satisfied the predetermined criteria. the relative risk reductions for symptomatic thromboembolic complicatious, clinically ~t bleeding, and mortality varied firom . % aad were all statistically significantly in favor of low-molecular-wedght hqtmrin. coadusions: low-mol~ular--weight hoparim administered subcutaneously in fixed doses adjusted for body weight aml without laboratory nmaitori~ =re more effective and safea" tlum adjn~_-dose standard h~. sauce low molec~dar weight hqmrim vary in o~apositiou =ad pharma~ogical im)fil~ the benefits of each ~ shodd ~tabllsbcd separately. unfraetionated hcparin (uh) and low molecular weight heparin (lmwh) are widely used for the prevention and treatment of thrombotic disorders. uh and lmwh induce platelet aggregation in vitro. rgd peptides compete with fibrinogm for the binding to the glycoprotein receptor (gp lib-ilia) of platelets and inhibit platelet aggregation. to inhibit the heparin-indueed platelet ~tion and prolong the half-life in blood of rgd peptides, we linked ac-rgdv-ssggs-ahx-yk eovalently to lmwh in a ratio : . the peptide is composed of three regions: a. rgd-gives the specificity for the receptor gp lib-ilia; b..ssggs-ahx-is the spacer between carrier and ligand, which should facilitate the intnraetion between the conjugate and the gp lib-ills receptor; c. -yk arc functional antino acids for iodination (y) and for covalent attachment (k) to the cattier lmwh. the aggregation achieved with different concentrations of lmwh, lmwh-eonjugate and lmwh/rgd-peptide mixture in a ratio : was mea.~ared after rain.; maximum aggregation after platelet activation with pm adp was set equal to %. platelet aggregation in normal human plateletrich eitrated plasma (prp; /p.l) was induced by lmwh in a dose ~ndent manner. heparin can induce antbodies which interact with platelets and endothelial cells. this causes thrombocytopenia and thromboembolic complications. hitpatients do need effective parenteral anticoagulation. we treated patients ( m, fm), median age years ( - ) with laboratory prooven hit (hipa-test) with recombinant (r-)hirudin. as these patients had been preseleeted by their immunological response during heparin treatment and the treatment duration of the study was longer than in any other study using thlrudin, all patient samples were investigated for anti-r-hirudin antibodies himdin antibodies were screened by a sandwich elisa using r-hirudin fixed to the solid phase as antigen. all plasma samples were screened for anti-hirudin antibodies of the igg class, but solar only a suset of samples for lge anti-r-himdin antibodies. of patients ( . %) developed anti-hirudin antibodies of the igg class. anti-hirudin antibodies were not detectable not before days of r-hirudin administration solar no ige anti-hirudin antibodies were found. none of the patients devdoped thromboeytopenia or allergic symptoms. however, in a subset of patients the anti-hirudin antibodies enhanced the anticoagulatory effect of r-hirudin. in patients the hirudin dosage had to be decreased by - fold to maintain a stable aptt level, in patients, despite stable r-hirudin maintenance dose the aptt increased to values > see.. during the study patients with anti-hirudin antibodies had to be reexposed to a second course of r-hirudin for parenteral anticoaguhtion none of these patients developed any allergic reaction. in conclusion we found a high proportion of anti-hirudin antibodies in hatpatients treated with r-hirudin for more than days. these antibodies seem to have minor clinical relevance in regard to allergic reactions. however, one has to consider that these antibodies may influence the pharmacokinetics of rhimdin and thereby enhance its antieoagulatory potency. therefore, aptt must be monitored closely in patients receiying r-hirudin for more than days a major concern in the use of hirndin, the most potent and specific thrombin inhibitor, is the risk of bleeding associated with the potential effect of this drug on hemostasis, particularly when the antithrombotic therapy is combined with invasivo procedures, fibrinolytic treatment, or patient's predisposition to abnormal bleeding. thus, availability of an antagonist to hirudin would be essential for instant neutralization of the antithrombotie action. however, thueh a hirudin antagonist is unknown in nature. to prepare an antagonist to hirudin, a mutant derivative of human prothrombin, in which active site aspartate at position is replaced by an asparagine, has been designed, expressed in recombinant chinese hamster ovary cells, and purified to homogeneity. d n-prothrombin was converted to the related molecules d n-meizothrombin and d n-thrombin by limited proteolysis by e. carinatue venom and o. scvutellatus venom, respectively. both d n-thrombin and d nmeizothrombin exhibited no thrombin activity and titration resulted no detection of the active site. however, binding to solid phase immobilized hirudin and fluorescence studies confirmed that the binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins, hi vitro examinations showed that d n-thrombin and d nmeizothrombin bind to immobilized hirudin, neutralize hirudin as well as in the purified system and in human blood plasma and re-activate the thrombin-hirudin complex. animal model studies confirmed that d nthrombin and d n-meizothromi.,in act as hirudin antagonist in blood cireulatlon without detectable effects on the coagulation system. while i.v. injections of hirudin in mice resulted in an increase in partial thromboplastin time, thrombin time and anti-thrombin potential, additional injections of d n-thrombin and d n-meizothrombin resulted in a normalization of these coagulation parameters. elevation of plasma homocysteine is a hereditary disorder of methionine metabolism associated with a high risk of arterial vascular disease. however, as yet relatively little attention has been directed towards the association between hyperhomocysteinemia and juvenile venous thromboembolism (vte). consequently the aim of our study was to evaluate the prevalence of hyperhomocysteinemia (hyper-hcys) and juvenile vte. patients: patients ( men, median age ys; women, median age ys) who had at least one verified episode of vte before the age of ys were investigated in regard to their total plasma hcys levels. none of the patients had renal or liver dysfunction or evidence of any autoimmune or neoplastic disease. methods: plasma total homocysteine levels were determined by hplc with fluorescence detection. hyperhomocysteinemia was defined as hcys levels exceeding the upper limit of the normal range obtained in our laboratory from healthy control subjects ( males, median age ys, hcys % ci: . - . pmol/l; females, median age . ys, hcys % ci: . - . ,gruel/l). resuits: out of patients had hyper-hcys, giving a prevalence of . %. of these patients, were male and female, indicating that the relation between elevated plasma hcys levels and vte may not be as strong in woman as in men. discussion: according to previous reports, our study shows that there is a high prevalence of hyper-hcys in patients with juvenile vte. however, the mechanisms by which hyper-hcys can provoke vte and whether hcys is an exclusive risk factor or if it contributes to other existing predispositions, possibly working as a trigger factor is unknown yet. some authors suggest hcys-iaduced effect on factor v activation or inhibition of thrombomodalin-dependent protein c activation. in addition an influence on thrombocyte aggregation has been postulated. conclusion: measurement of hcys levels may be useful in the evaluation of patients with a history of juvenile venous thromboembolism and could be clinically important as hyper-hcys is easily corrected by vitamin supplementation. detailed determination of the pathogenesis of vte in patients with hyper-hcys should be the aim of further investigations. a deficiency of one of the coagulation inhibitors antithrombin (at), protein c (pc) or protein s (ps) and resistance to activated protein c (apc resistance) are established risk factors for venous thromboembolism (vte). in the majority of patients with apc resistance, the .tug gin mutation (factor v leiden) is present. whereas deficiencies of one of the coagulation inhibitors are rare in the normal population, the allele frequency of factor v leiden is - % in western europe. heterozygous individuals have a - fold, homozygous an fold increased risk for vte the typical clinical features of all abnormalities are deep vein thrombosis, pulmonary embolism, superficial vein thrombosis and thrombosis at unusual sites, like mesenteric vein thrombosis or cerebral vein thrombosis. the thrombotic risk is low during childhood, but increases considerably after the th year of age. a retrospective study in adult patients out of families with a symptomatic deficiency of at, pc or ps revealed that around % of surgical interventions and traumas of the lower extremities were complicated by vte. therefore, these patients should receive thrombosis prophylaxis al~er surgery and trauma if their age is higher than years. pregnancy is associated with a very high risk for vte in individuals with at deficiency and prophylaxis should be initiated already in the first trimester. after delivery, thrombosis prophylaxis is adviced for all females known to have an abnormality. oc increase the risk, especially in at deficient and in homozygous factor v leiden females and are therefore contraindicated in these individuals. oc do also increase the risk for vte in patients heterozygous for factor v leiden and females known to have this abnormality should be discouraged from taking oc or should at least be informed on their increased risk. university hospital-', jerusalem, israel, hospital bcan.iou ~. paris, france, increased frequency of thrombocmbolie events have been observed iu patients with b-thalassemia. our findings of shortened platelet survival and enhanced urinary excretion ofthmmboxanc a: metabolitcs (blood : (blood : , suggested an increased platclet activation in tbese patients. we also fouud that isolated thalassemie rbc enhance prothronlbin activation, suggesting an increased membrane exposure of procoagulant phospholipids i.e, phasphatidylserine (am j. hematol. : , ) . we now show that annoxin v, which has a high specificity and affinity for anionic phospholipids inhibits pmthrombm activation by factor xa, by binding to thalassemic rbc (ic~, = . nm). kerckhoff-klinik, bad nauheim ~, medizinische poliklinik bonn , institut for immunologie und transfusionsmedizin universit~ll greifswald a antibody-mediated intravascular platelet activation is believed to be the basis for both arterial and venous thrombosis in patients with hat. while the development of arterial thrombosis can explained sufficiently by intravascular platelet activation, it is a matter of discussion whether additional risk factors are involved in the pathogenesis of hat-related venous thrombosis. since resistance to activated protein c (apc) is the most common inherited risk factor for venous thrombosis described the frequency of apc resistance among a population of hat patients has been studied. hat was diagnosed using the heparin-induced platelet aggregation assay and confirmed by the ~ c-serotoninrelease test. the diagnosis of apc resistance was established by two functional assays and genetic analysis. at time of diagnosis of hat, patients showed venous thromboembolic complications. among these, were found positive for apc-resistance. pulmonary embolism was diagnosed in hat patients, of them were apc resistance positive. none of the hat patients who showed exclusively thrombocytopenia were apc resistance positive. early oral anticoagulation (oa) was initiated in patients after the diagnosis of hat has been established. six of these patients developed serious thrombotic complications including skin necrosis. these results demonstrate that apc resistance is an additional and common risk factor for the development of hat-related venous thrombosis. early initiation of oa during an acute episode of hat dramatically increases the risk of thrombosis. therefore, oa in hat patients should be initiated only after platelet counts have been returned to baseline levels and effective parenteral anticoagulation is achieved. controlled trials for primary and secondary prevention of stroke g. de ( aetano, c. cerletti and v. bertel~ consorzio mario negri sud, santa maria imbaro, italy this presentation will review the antithrombotic treatments to prevent ischemic stroke that have been evaluated in controlled clinical trials. in two studies of aspirin therapy for pdmary prevention in male physicians there was no reduction in the incidence of stroke, while that of first myocardial infarction was significantly lowered. similar results were obtained in a prospective study in a large cohort of women taking aspirin daily. the incidence of vascular death was not modified by aspirin in any of these trials. this is possibly due .to an excess of strokes associated to aspirin treatment: indeed the four vascular events avoided in us physicians under aspirin prevention for five years would result from five myocardial infarction and one vascular death avoided and two additional strokes occurred. oral anticoagulant therapy decreases the relative risk of stroke in patients with non valvular atrial fibrillation. warfarin appears to be superior to aspirin, but the latter drug is a useful alternative when long-term anticoagulant therapy cannot be administered. a metanalysis of about trials and over , patients with different vascular diseases treated with aspirin (at different doses) and/or other platelet inhibitors showed % overall reduction of vascular events including stroke. the optimal dose of aspirin for secondary stroke prevention could not be established. in patients with previous minor strokes or tia there was % reduction of vascular events and % of non fatal strokes. the avoidance of nine strokes of any cause among the expected in patients at risk would result from the sum of ischemic events avoided and a haemorrhagic one occurred in excess. ticlopidine was reported to reduce the risk of stroke in two large tdals (one in patients with major stroke), but there is no evidence that it is better or safer than aspirin. we compared the effect of the direct specific thrombin inhibitors, napsagatran (na) and rec. hirudin (rh) with unfractionated heparin (uh) on the further growth of preformed thrombi. as a model of thrombogenesls, an annular perfusion chamber exposing rabbit aortic subendothelium was perfused with native rabbit blood at an arterial wall shear rate ( /s). fibrin and platelet thrombi were allowed to form during a min perfusion period after which the test agents were given iv as a bolus and a continuous infusion ( and pg/kg/min, n= ) and the perfusion continued for min. the control groups were perfused for or rain (n= ). fibrin deposited and platelet thrombi formed on subendothelium were evaluated by microscopic morphometry. the % surface coverage with fibrin was not reduced in the drug-treated groups since fibrin deposition was similar in the and min control groups ( + % and : %, respectively, mean:l:sem). platelet thrombus area (ta) in the control groups increased from + pm /pm after min to + pm /lim after rain perfusion. na at g/kg/min reduced ta by % to values ( +_ ptm / ~m) lower than those of the min control group whereas rh at this dose reduced ta by % ( -j: .tm /i.tm). uh at both doses was ineffective. these findings show that in contrast to uh the direct thrombin inhibitors na and rh inhibit the growth of preexisting thrombi. these results could be explained by the higher potency of na and rh as compared to uh for inhibiting clotbound thrombin (gast et al., blood coagul fibrinol , .' - ) and suggest that thrombus-bound thrombin is an important modulator of platelet thrombus growth and/or stability in this thrombosis model. platelet adhesion -the initial event of thrombosis -is believed to be completely prevented by intact endothelium. we challenged this theory by superfusing intact human umbilical vein endothelial monolayers with activated human platelet rich plasma utilizing the stagnation point flow adhesio-aggregometer (spaa). the spaa provides flow mediated contact of platelets with the superfused surface. heparinized ( . - . u/ml) platelet rich plasma (prp) was obtained from healthy volunteers and activated by addition of adenosine diphosphate (adp " - m). platelet deposition was recorded on-line by video as well as by measuring scattered light. fixed samples were examined by phase contrast and electron microscopy, inhibition experiments were performed with either the tetrapeptide rgds, the non-peptide gpiib/llla-inhibitor ro- - or a monoclonal antibody directed against the gpilb/llla complex. stimulation with adp prompted platelets to adhere to intact endothelium single or as microaggregates of a diameter of up to micrometer. adhesion was dependent upon convective transport resulting in platelet collision with the endothelial monolayer. infusion of rgds or ro- - into the flowing, adp-stimulated prp completely prevented platelet adhesion to the endothelium as well as subsequent aggregation. when the inhibitor inflow was stopped while adp stimulation persisted, adhesion and aggregation occurred immediately. re-establishing the inflow of the inhibitors -with still continued adp stimulation -led to disintegration of the adhering aggregates. when prp preincubated with the monoclonal antibody against gpllb/llla was superfused, platelet adhesion to the endothelium and aggregation were irreversibly blocked. our results suggest that convective transport and stimulation of platelets are prerequisites to overcome endothelial thromboresistance and that subsequent platelet adhesion to the endothelium is mediated via the platelet gpilb/llla receptor complex. prevent thrombus formation affer ptca i.p. tepanova t, g.v. bashkov , l.p.kapralova, s.p. domogatsky ~ cardiology research center t and national haematology scientific cettter russian academy of medical sciences, moscow, russia percutaneous transluminal coronary angioplasty (ptca) results in atheroselerotie plague rapture, vascular wall damage and thrombogenic collagen exposure. subendothelial collagen type i-lll is a very ~rong agonist of platelet-dependent thrombus formation in arteries. the anlithrombotic action of rabbit polyclonal antibodies to rat collagen type i-ill and their chemically synthetized conjugate with monoclonals to human recombinant two-chain/one-chain urokinase type plasminogen activator (rtcu-pa/rseu-pa), cross reacting with rat tcu-pa/scu-pa was studied both an in vifro and in vivo. anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like ~ructures induced by rat collagen immobilized with the polystiroi surface in a condition mimics the high shear rate in the large elastic-type arteries. the short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by _+ % (p< . ) as well as by the bispecific conjugate ( _+ %, p< . ). the treatment of collagen-adsorbed conjugate by rtcu-pa did not increase the autithrombotic effect of bifunctional antibodies. the present date suggest, that the local administration of the anticollagen antibodies at the site of atherosclerotic plague rapture may tm the efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries after ptca. increased levels of certain hemostatic factors have been shown to be related to an increased risk of cardiovascular events. hypercoagulability is suggested to predispose to arterial thrombosis and thereby to participate in atherogcncsis. we therefore assessed fibrinogen, prothrombin fragment + (fi+ ) and yon willebrand factor (vwf) antigen in consecutive patients (aged + years) with known coronary artery disease (cad) who all underwent coronary angiography. the extent of coronary artery disease was quantified according to modified criteria of the american heart association (total, proximal and distal "score"). furthermore the intima-media thickness (imt) was determined in the carotid and femoral arteries by standardized ultrasonographie measurement, vwf antigen was found to correlate positively with the total and proximal coronary score (r= , p< . and r=o. , p< . ). while fi+ showed no correlation with the coronary scores, it was significantly correlated with the imt values in the carotid arteries (r= . . p< . ). after differentiating tertiles of the parameters patients belonging to the upper tertile of fi+ concentrations had significantly higher imt values of the carotid and femoral arteries ( . _+ . mm vs. . +_ . mm in the lower tertile, p< . : . _+ . mm vs. i. -+ . ram, p= . ) whereas in patients belonging to the upper tertile of vwf antigen concentrations the proximal coronary artery score was significantly higher (!. -+ . vs. . + . in the lower fertile, p< . ). fro correlation of fibrinogen concentrations and extent of cad or imt values of the carotid and femoral arteries could be demonstrated. in conclusion procoagnlatory mechanisms as indicated by elevated concentrations of yon willehrand factor antigen and fi+ may be contributing factors in atherogenesis. we have previously shown that pgei is a potent inhibitor of pdgf-ioducod proliferation of vascniar smooth muscle cells (vscm) and inhibits dna replication by a camp-related mechanism (grol~er et al, ) . the present study investigates of whether or not this aatimitogeni¢ activity of pget can be amplified by trapidil, a compound that has been shown recently to inhibit the incidence of restenosis of hmnan coronary arteries subsequenmt to ptca (maresta et al. ) , vsmc were prepared from coronary arteries of adult bovine hearts, passagod and kept under standard tissue culture conditions. cells of passage - wore incubated in serumfree medium for h in the presence of indomethacin ( p.m). addition of pdgf-bb ( ng/ml) under these conditions stimulated dna-replication as assessed from 'hthymidin lncm'poration, by .- laid above control level. trapidil at idvl caused a minor reduction of pdgf-induced mitogenesis whereas t) of the compound resulted in a marked reduction of dna replication by % (p < . , n = ). pgei at . nm diminished the incorporation rate by t % while the simultaneous administration of both pged and trapidil ( idyll caused a significantly stronger response as seen from n reduction of ~h-thymidine incorporation rate by % (p < . , n = ). as a possible mechanism of action, trapidil might have inhibited phosphodiesterases. to establish this, we measured the camp-depcudont proteinkiaasc (pk) a activity in cell homogenates. trapfdil increased the basal fka-activity from % to % of the maximum response while the response to pget ( am) amounted to %. coincubation of pgei with trapidil caused a % stimulation of pka activity, sugesting a small though detectable inhibition of vscm phosphodiesterases by trapidil at anttmitogenic concentrations. essentially similar results wore obtained when thrombin was used as the mitogenic agent. the data demonstrate a significant antimitogenic effect of trapidil at p.molar concentrations that are in the range of plasma levels after therapeutic administration of the compound in rive. at these concentratrations, pget induced inhibition of mitogenesis is markedly enhanced by trapidil. inc. i~enna, and ~cenlral itematnlogy laboroto~. , university hospital of bern pibrinogen (fg), yon willebrand factor antigen (vwf) and tissue-type plasminogeu activator antigen (l-pal have recently been shown in be independent risk factors for subsequent coronary events in patients with angina pectoris (nejm ; : ) although paul antigen has also been proposed as a risk factor, conclusive dam showing its predictive value is still lacking. furthermore, we have recently shown in a study investigating survivors of myocardial infarction that not only are fg, t-pa and pai-i significantly increased in these patients when compared to a heahhy conlro[ group, but pci activity is also elevated ( hrornb. tfaemost. ; : abst.) , hi order to obtain cut.off points for the individual parameters, frequency histogram plotl; were transformed into straight line cumulative frequency (probit) plots (thromb i/aemost. ; : ) . the cut-off valu~ for the four parameters were determined as follows: fg at . g/l, t-pa at . ng/ml, pal-i at ng/ml and pc[ at % of a normal pooled plasma. utilising there cut.off points it was then possible to determine the accumulative discriminatow effectiveness of the parameters. when fg w;qs employed alone as the discriminatow factor, it was observed that % ( ) of the coronary heart dir, ease (chd) group eilher had the cul..off value or were below it aud % ( ) of the normal group were above the cut-off value, thus, resulting in % false ne$atives and % false positives. when a second additional risk factor, t-pa wa_~ introduced, the number of false negatives dropped to % [i.e. % ( / ) had two, risk factors elevated] and the number of false positives to % to investigate whether a third parameter could discriminate further, pai-i antigen was used to analyse the rcnudning false positives and negatives. an additional % could be detected, resuhing in % of the chd group having three risk factors elevated. similarly, the number of normal aubjecta with three parameters elevated dropped by % to % furthermore. when a fourth parameter was introduced, namely pci, it was round to discriminate a further % in the chd group, thereby increasing tile di~riminalion to %. the number of false positives dropped to %, additionally, determination of pci increased the discrimination of patienta having had multiple infarctions from °/= when thrce parameters were mcasured to %. from these results it can be concloded that determination of fibrinogen levels alone is not sumcicnt to separate patients from controls as t-pa adds significant discrimination. pai-i antigen which correlated strongly with t-pa did not significantly increase the discriminatory potential of both fg and i-pa. however, by employing pci as a fourth paramctcr, virtually complete separation between the chd and normal groups as well as rurthcr recoguitiou of' patients having had multiple infarctions could be obtained. to test the hypothesis that oral contraceptives (oc) enhance exercise-induced activation of blood coagulation we examined women ( + (sd) years, bmi . + . kg/m , vozm.. + ml/kg/min) without oc between day and of the menstrual cycle and women ( + (sd) years, bmi , ± , kg/m', vo max + ml/kg/min) taking oc ( mg desogestrel and mg ethinylestradiol) between day and of drug intake. prothrombin fragment + (ptf + ) and fibrtnopeptide a (fpa) were measured before and after running for one hour on a treadmill at a speed corresponding to the anaerobic threshold. mean heart rate [ ± vs. ± min ) and mean plasma lactate ( . ± . vs. . + . mmot/i) wera comparable during exercise between control and oc group, respectively. results for markers of thrombin and fibrin formation were: ptf + (nmol/i) fpa (ng/ml) control before , ± . . + . after . + . . + . " oc before . + . . + . after . + . * + . -+ . * + * p < . vs. baseline, + p < . between groups. we conclude that oral contraception with mg desogestrel and mg ethinylestradiol enhances exercise-induced thrombin and fibrin formation, our data suggest that exercise testing might be useful for evaluating the risk of thrombosis associated with different compositions of oc. a. haushofer +, wm. halbmayer +, j. radek +, m. dittel *, r. spiel *, h prachar *, j. mtczoch *, m. fischer + + zentraltaboratorium mit thrombose-und c~rinnungsambulanz -krankenhaus lai~: * . medizinische ab[eilung mtt ka~liolo$i¢ -krankenhaus lainz und ludwig bottzmann-lnstitut ftlr herzinfarktforsohung, wien fifty-one patients (age . ± . a; m / ) implanted with coronary stems palm~-schatz, gianturco-roubin, micro stcnts) received a now antithrombotic treatment using a combination of ti¢lopidine (tic) × mg/d for days and acetyl salicylic acid (asa) zoo mg/d for long-term treatment. patients (pat) only received tu standard hepartn as i.v. bolus immediately before stent implantation (day l ). side effects and changes in hematological (day i to , . and [= without t[cil liver and kidney parameters (day , , , ) were monitored. thirty-eight pat ( %) came for the controls to our del~rtment and were additionally monitored by thromboelastograpy (teg) and bleeding time (bt) (day g and ). the other pat were monitored externally, side effects were reported. thrombin geucration after stenting was monitored from day i to by prothrombin fragment + (f + ) and thrombin-antithrombin-lll-comptex (tat). "k" of the "leg decreased (day vs ; p< . l ). bt prolongation was negatively correlated with the bodysurf ace area (tic+asa: p< . , asa: p< . l) and showed a reduction after withdrawal of tic ( l sec, / so: [median, quartiles] vs. sec, sec; p< .ix) ). f + and tat of day i (blood collection: , , , h after intervention, f + : . nmol/i, . /i. nmol/l: tat: . pg/ , . / . ilg/ ) were lower compared to day to (f i + : . nmol/l, ]. /i . nmol/l; tat: . pg/i, . / . ijg/ ; p< . ). tic scorns not to be a strong thrombin generation inhibitor. during stenting one pat (i. %) sustained a non penetrating mci and one ( . %) an ischaemic stroke. tic+asa were very effective, only with one pat ( . %) stent thrombosis (acute) occurred. side effects: / . % gastrointestinal (one lead to hospitalization), / . % hematomas at the needle site in the groin (one surgical intervention), / . % leucopcnias (one agranulozytosis with hospitalization), / . % allergic skin reactions and / . % increased liver enzymes (got, gpt, "pgt, alkaline phosphatase; > × of the j. ). with one pat with gastrointestinal disturbances and skin reactions tic had to be withdrawn and treatment was changed to oral anticoagulatlon + asa. one pat showed a combination of skin reactions, gastrointestinal distufl~aneas and on day a heavy reaction of the liver enzymes ( j. after weeks). a decrease of the white blood count (day : . gh, . / . g/l, day : . g/l, . / . g/l; p< . i) could be observed. the safety of the therapy with tic+asa should be elucidated and extensively discussed. the serpins c esterase inhibitor (cllnh), antithrombin iii (atiii), alantitrypsin (slat), and a -antiplasmin (azap) are known inhibitors of coagulation factor xla (fxla). although initial studies suggested al at to be the main inhibitor of fxla, we recently demonstrated cllnh to be a predominant inhibitor of fxla in vitro in human plasma (wuillemin et el., blood ; : ) . the present study was performed to investigate the plasma elimination kinetics of human fxla-fxla inhibitor complexes injected in rats. the amounts of complexes remaining in circulation were measured using elisas. the plasma tl/ of clearance was min for fxla-alat complexes, whereas it was , , and min for fxla-cllnh, fxla-a ap, and fxla-atiii complexes, respectively. thus, due to this different plasma tl/ , preferentially fxla-alat complexes may be detected in clinical samples. this was indeed shown in plasma samples from thirteen children with meningococcal septic shock (mss), a clinical syndrome which is complicated by activation of coagulation, fibrinolytic, and complement systems. fxla-fxla inhibitor complexes were assessed upon admittance to the intensive care unit. fxla-a at complexes were elevated in all patients, fxla-c nh complexes in nine, fxla-atiii complexes in one patient, and no elevated fxla-a ap complexes were found. we conclude from this study that, ( ) although c inh is the predominant fxla inhibitor, fxla-alat complexes may be the best parameter to assess activation of fxi in clinical samples, ( ) measuring fxla-fxla inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of fxla in rive, and ( ) fxl is activated in patients with meningococcal septic shock. dudng the coagulation of plasma about % of the (x ap present is covalently crosslinked to fibrin by factor xiila (aoki und sakata , thomb. res. : - ) . we investigated the binding of azap by factor xiila to soluble fibrin (desaabb-fibdno) whose polymerization was inhibited by an isolated fibrin ddomain named d=,,, (haverkate and tiemann , thromb. res. : - ) . d==. is known to have an intact fibrin-polymerization site and is able to block the prolongation of the fibrin protofibrils at an early stage depending on its concentration. lateral association to fibrin fibers does not take place, since the inhibited protofibnls formed at the conditions used here do not reach a sufficient length (williams et el. , biochem. j. : - ; hantgan et al. , ann. n. y. acad sci. : - ) . material and method: soluble desaabb-fibrino was prepared by incubation of (lztl)-fibrinogen ( . mg/ml), d= o ( . mg/ml; molar ratio d==o to fibrin : ) and . u/ml thmmbin for min. then q sl)-c~ ap ( p.g/ml), faktor ×ill ( ulml) and ca ) ( mmol/i) were added. the crosslinking reaction was stopped at different times of factor xiila-incubation by adding of urea/edtasolution. the suspension was analysed by ultracentrifugation on gradients containing saccharose, urea and sos. re~ultl: the elution profiles of the ultrecentifugation-gradients show the formation of cmsslinked fibrin oligomers of increasing size depending on the time of factor xiila-action. the crosslinked fibrin polymers contained about % of the fibrin initially added. although factor xiila acted well, crosslinking of azap in the fibrin oligomers could not be observed. conclusl n: as we already demonstrated (kelach et el. , ann, hematol. (suppll) : a ) the crosslinking of azap to fibnn clots depends on the structure of the fibdn network, especially on the degree of lateral association of the fibrin pmtofibdla. in desaabb-fibrino no lateral association of fibrin protofibnls takes place under the conditions chosen here. thus it is consistent with our theory that we did not observe any binding of aiap to the fibrin oligomers of desaabb-fibrino. human pci is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as tissue kallikrein and the sperm protease acrosin. it is synthesized in many organs including liver, pancreas, and testis. the physiological role of pci has not been defined yet. recently, we have cloned and sequenced the mouse pci gene (zechmeister-machhart etal., manuscript in prep.) . this enabled us to study pci gone expression in murino tissues using mouse pci edna and crna probes. by northern blot analysis, mouse pci tar.ha was exclusively found in the reproductive tract (testis, seminal vesicle, ovary), all other organs analyzed -including the liver were negative for pci mkna, indicating that in the mouse pci is not a plasma protein. to determine which cells of the reproductive tract synthesize pci, cellular localization was assessed by in situ hybridization of mouse testis and ovary sections. in testis, pci mrna was present in the spermstogonia layer and in leydig cells, while sertoli cells and peritubular myoid cells were negative. these results are consistent with the immunohistological localization of human pc (laurell et al,, ) . in the mouse ovary, stroma cells of the medulla and around the follicles were positive for pci mrna. no pci expression was detected in theca or granulosa cells. we also studied the regulation of mouse pci gone expression by steroid hormones in vivo. [n mature male mice castration caused an increase in pci mrna in seminal vesicles, which was reversible upon the administration of testosterone. in tissues of intact adult male and female mice, pci mrna levels decreased after injection of human chorionic gonedotropin (hcg), while in castrated male mice, hco had no effect on seminal vesicle pci mrna. progesterone and -b estradiol decreased ovarian pci mrna levels in immature female mice. these data suggest direct down-regulation of mouse pci by sex steroids. the different tissue specific pci-geoe expression in men and mice furthermore indicates a different biological role of this serpin in the two species. ctr. transgene technology, leuven "[' -tissue factor ('it) is a kda glycoprotein mainly known a the primary cellular initiator of blood coagulation. whether tf expression may also play a role in development is unknown, but the lack of spontaneous viable mutations of the tf gene in rive leads to the speculation that its absence may not be compatible with normal embryonic development. to determine the significance of "if in ontogenesis, the pattern of tf expression in mouse development was examined and compared to the 'if distribution in human postlmplantation embryos and fetuses of corresponding gestational age. at early embryonic period of both murine ( . and . pc) and human (stage ) development there is a strong tf expression in both ectodermal and entodermal cells. "if decoration was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express "if in the adult organism. surprisingly, during renal development and in adult organism tf expression differs between men and mice. in humans maturing stage glomerali were "if positive whereas in mice glomeruli were negative and instead epithelia of tubular segments were tf positive. in ncuroepithelial cells there was a striking 'if expression indicating a possible role of'if in neumlation. moreover, there was a robust tf expression in tissues such as skeletal muscle, and pancreas, which do not express in adult. in contrast to tf, its physiologic ligand factor vii was not expressed in early stages of human embryogenesis, but was detectable in fetal liver, the temporal and spatial pattern of tf expression during murine and human development support the hypothesis, that 'if serves as an important mo~hojzenic factor darinz embrvozenesis. to serve as an anticoagulant, protein c (pc) must be activated by a complex formed between the enzyme thrombin (t) and its cofactor thrombomodulin (tm). therefore, downregulation of endothelial cell surface expressed tm, for example, triggered by an inflammatory stimulus, could become a critical factor in effective pc activation. in order to develop a recombinant (r) pc mutant which can be activated independently of the tkm-complex, a peptide sequence including p - in the activation peptide of pc was modified to be identical to the factor xa (fxa)-cleavage site in prothrombin. the mutant was expressed in hu cells, purified and its anticoagulant properties characterized. using purified fxa the mutant showed activation rates between . and . nmlmin at pc concentrations between and nm, while the rpc wild type was insensitive for fxa activation. the activation reaction is calcium-dependent reaching maximal activation rates at a calcium concentration of mm and was enhanced to . -fold by addition of anionic phospholipids (pl). in contrast to the wild type pc the rpc mutant was insensitive for activation by the t/i-m complex. addition of the mutant to normal human plasma induces a prolongation of tissue-factor and p-it-based clotting assays. using normal human plasma as a source for fxa the the activation rates of the mutant were found -fold higher than in the purified system if tissue factor was used to generate fxa. in conclusion, our data demonstrate that the rpc mutant is effectively activated by fxa in a purified as well as in a plasma system. interestingly, the activation rates are enhanced in the presence of pl and normal human plasma. fudher studies should clarify the potential use of this mutant as a novel anticoagulant. thrombln plays a pivotal role in thrombotic events. the time course of thrombln concentration in blood or plasma after activation is of special interest to answer a variety of questions. with a chromogenic assay developed by hemker et el. [thromb. haemostas, , , ] it became possible to measure the generation of thrombin in activated plasma continuously. inhibitors of clotting enzymes which are to be developed as anticoagulants should be able to inhibit thrombin generation or to immediately block generated thrombin. we have used a test based on hemker's thrombin generation assay to elucidate which potency and specificity an inhibitor of factor xa needs to efficiently block thrombin generation in human plasma. thrombin generation after extrinsic (tissue factor) or intrinsic (ellagic acid) activation was followed using the chromogenic substrate h-~ala-gly-arg-pna (pentapharm ltd.). a series of synthetic low molecular weight inhibitors as well as naturally occurring inhibitors of factor xa with different potency were investigated. because of the inhibition of activated factor x the generation of thrombin in plasma is delayed and the amount of the generated thrombin is reduced. the concentrations which cause a % inhibition of thrombin generation (icso) correlate with the k~ values of the inhibitors. low molecular weight inhibitors with k~ values of about nmol/i inhibit the generation of thrombin after extrinsic activation with icso in micromolar range. after activation of the intrinsic pathway tenfold lower concentrations are effective. the strongest inhibitory activity after extrinsic as well as intrinsic activation is shown by recombinant tick anticoagulant peptide (r-tap) with ic~o of . pmol/i (axtdqsic) and . pmo/i (intdnsic). in the compadson of synthetic low molecular weight inhibitors of thrombin end factor xa which have similar k= values for the inhibition of the respective enzyme (lowest i< nmol/i), factor xa inhibitors are less effective tn the thrombin generation assay. in contrast, the highly potent xa inhibitor r-tap shows a stronger inhibition of thrombin generation than the tight binding thrombin inhibitor hirudin. background: resistance to degradation of coagulation factor v by activated protein c is associated with a point mutation in which adenine is substituted for guanine at nucleotide in the gene coding for factor v. to date this specifc mutation appears to be the most common inherited abnormality which predisposes patients to venous thrombosis. for this reason a reliable, fast and automatable system for the diagnosis of the described point mutation is required. the conventional methods used to identify the mutation are based on allele-specific restriction enzyme site analysis or direct sequencing. these methods have disadvantages for a large scale dna diagnosis, which include the need for electrophoresis or a high cost and time consumption. methods: an alternative strategy of dna diagnosis, the allele-specific oligonucleotide ligation assay, was adapted for the diagnosis of tile point mutation of factor v. following pcr amplification of the target dna, tile procedure was performed completely automatically on a robotic workstation with an integrated elisa reader using a -well microtiter plate. allelespecific restriction enzyme site analysis was performed to confirm the genotypes. results: in patients with the mutation and in individuals without the mutation the genotypes determined with the conventional allele-specific restriction enzyme site analysis were in % concordance with the elisabased oligonucleotide ligation assay. discussion: the pck-oligonucleotide ligation assay applied as automated detection system for the identification of the coagul;mon factor v point mutation allows tile rapid, reliable, and large scale analysis of patients at risk for thrombosis. resistance to the asticoa=m~lant activity of activated protein c (apc resistance) has emerged as the most con'anon inherited thrombophilic state. patients lreterozygous for factor v leiden are more likely to suffer from thromboembolie events than controls. this risk is even more pronounced in homozygotes. due to the low sensitivity and speeifity of most coagulation tests some investigators suggest to examine patients for the presence of factor v leiden mutation by pcr-based methods. re e~tly we presented an aptt-based functional test (acceleria inactivation test ait): : diluted plasma ( bi) is mixed with factor v deficient plasma ( ~tl) and aptt reagent ( .d), incubated at °c and then coagulation is induced by caci and a.pc ( ~d). using a standard curve, the clotting time (see) is transferred in per cent accelerin inactivation (%ai). using this test, the widely used apc-ratio as well as pcr-based factor v leiden detection (confirmed by direct sequencing) we prospectively studied consecutive patients with thromboembolic events. patients without the factor v mntation eonsitently showed more flazm % al with the exception of one patient with severe factor deficiencies (including factor v) due to hepatic failure and heterozygous for factor v-leiden resulting in */. ai, there was a complete concordance between the pcrbased method and dysaseelerinemia detected by ait. due to these result a specifity and sensitivity of ait above % was calculated. furthermore, a clear discrimination could be obsoved beween heterozygotes ( %<ai< %) and (incl. samples obtained from jeaa) homozygotes (ai< %). in contrast a.pc-ratio resulted in a great overlap between patients homozygous for the wildtype , hcterozgous or homozygous for factor v leiden. in addition we confirmed thc di.~eulties nsing the apc-rauo for patients trader anticoagulation, or with different types of other d~rombophilie defects such as lupus anticoagulant or protein s deficiency. due to the time and cost-saving featttres the/kit should be considered for the rapid detection of apc resistance without the use of pcr-based techniques. in summary we found, well in accordance with previous reports, that apcr due to the fv-leiden mutation is • highly prevalent defect in vte pts. pts with the fv-leiden mutation were eharacterised by a higher incidence of vte's without recognised prothrombotic risk factors and more frequent positive family history; no higher incidence of recurrent vte's or other prothrombotie coagulation defects was found; the age at initial manifestation of thrombophitia was comparable. acquired a recently identified mechanism for famdial thrombophilia is characterized by a poor anticoagulant response to actavated protein c (apc), it is now welt recoginzed to be due to a genetic defect in the factor v gene, which results in the syn-,thesis of an abnormal factor v molecule and to be a frequent risk factor for thromb.osis, however apc resistenee can also be found in certain acquired conditions. since pregnancy ~s known to be associated with a hypercongulable state resulting in a prevalance of thrombosis up to % and thromboembolic events, we determined the prevalence of apc resistance during the course of pregnancy at the city hoslfital ruesselsbeim. germany. blood samples from pregnant woman were drawn at mmester , , and at term. besides the routine coagulatton screening, protein c and viii:c, the apc resistance were evaluated. a modified aptt method utilizing human plasma fracnonated apc (amencan red cross. bethesda, maryland, ~usa) was used to assay apc resistance. this method relies on the determination of two aptt's in the patient plasma, one in the absence of apc and one in the presence of a fixed amount of apc. clotting tune was determined after mcubation using the aptt reagent ( we investigated the sensitivity of various species towards activated, human protein c (apc) and found an impaired response in the order monkey < horse < pig < dog < rabbit. we assume that this effect is mainly caused by differences in the apc cleavage region of factor v, as recently described for a factor v mutation in bumat~s: factor v-leiden. this was corroborated by the finding that addition of human factor v similar to the factor v added with the animal plasma had only minor influence on test outcome. coagulation assays dependent on apc, such as for protein c and protein s determination, in the standard apc assay, the more factor v-leiden specific apc assay and in the pcat, a new screening assay for the whole protein c system, are based on the inactivation of factor va via apc. thus, in all these assays, % of rabbit plasma added to human plasma mimicked a apc response as observed for heterozygous carriers of factor v-leiden and % as for homozygous carriers. we exploited this factor v-leiden like behaviour to prepare plasma standards for calibration of apc dependent assays by mixing rabbit plasma with human plasma~ as reference for these standards the % value was assigned to the clotting time in the absence of apc, while the % value was assigned to the clotting time in presence of apc as obtained with a human, normal plasma pool. then these calibrated standards were used for establishing reference curves. on automated coagulation analyzers, the results obtained were automatically reported in '% of normal' or '% sensitivity'• this not only eases evaluation, more importantly, this procedure also considerably improves the comparability of results obtained with different reagents and instruments. in order to establish the methods for activated protein c resistance and the faktor v leiden-mutation we investigated healthy individuals ( women, men, median age ys) using the assay "contest ® apc-resistance" (chromogenix, sweden). the mean apc-ratio was . , the cut-off level for low response to apc was calculated as . , / women were oral-contraceptive users. they showed a slight decreased apc-ratio (mean . ). of the individuals ( , %) had a poor response to apc (mean ratio . ) as a apc-resistance. for detecting the factor v gene mutation we a used non commercial own modified easy to handle pcr-based test. in of the individuals ( . %) the factor v-leiden mutation could be found as heterozygotes. in this group, the apc-ratio was significant lower (mean . ) as in the group with apc-resistanee and without deteetiun of the factor v mutation (mean . ). nevertheless in one individual with beterozygote factor v-leiden, the apc-ratio was found in the normal range ( . ) , also in a few individuals with very iow apc-ratio the factor v mutation could not he detected. finally this data suggests a high prevalence of apc-resistance and factor v-leiden mutation in the population of the southwest of germany. resistance to activated protein c (apc) has been reported to interfere in clotting assays for protein c (pc) and protein s (ps) from various manufacturers, resulting in decreased values or even apparent deficiency (type ii') of pc. or is. in our laboratory, when using functional, avit-based assay kits for pc., is, and apc sensitivity from behring (marburg, germany) on an automatic turbidimctric coagulation analyzer (fibrintiraer a, behring), the interference in the is assay has been observed in a very pronounced manner soon after the introduction of apc sensitivity testing into the standard laboratory procedure for patients with thrumbophilia. yet we were not aware of an interferenca caused by apc resistance in the pc test. we therefore evaluated our results for pc eed ps with respnet to apc sensitivity status in a retrospective analysis. exclusion criteria were: oral anticoagulant therapy with vitamin k antagonists, impaired liver function, borderline result in the apc sensitivity test, known analytical intedercnc* other than apc resistance (e.g. elevated factor viii:c). resu/ts: apparant pc activity was slightly ( %) lower and apparent ps activity was grossly ( %) lower in ape resistent patients than in patients with normal apc sensitivity: there was no case with a presumed du~l defect of both at~ rnsistaac¢ and pc deficiency. in addition, none of the few patients diagnosed with pc deficiency before the introduction of the apc sensitivity assay turned out to ix: apc resistent. much more of a problem is the known sensitivity of the pc test to an elevated factor viii concentration which is quite a common finding among hospital patients but rarely known in an individual case. by aeccelerating the clotting process, elevated factor viii can cause apparent pc deficiency that can not be confirmed in a chromogunie pc assay. ,clearly, when combined with apc resistance, this problem is exspacted to be more severe. on the other hand, the comhination of both apc resistance and apparent is deficiency was found on a regular basis with many decreased and some bordeflinc values for is but only few in the lower normal range and virtually none in the upper non~al range. also, several #eats previously diagnosed with is deficiency turned out to be pc resistent, and the diagnosis had to be corrected, but dual defects cannot be ruled out. conclusion: for, the interpretation of pc and/or :" values from clotting assays the apc sensitivity status must be considered. when apc resistance and an apparent deficiency of pc or is is found, a type i deficiency may be confirmed or ruled out using antigen assays, but at present, there seems to be no reliable way other than genetic analysis to address the question of dual defects with presumed type ii deficiency, especially in the case of ps. therefore, there is a clear need for analytical improvements and the development of functional assays that are less sensitive to interference by apc resistance. the most common inherited risk factor for venous thrombosis is the resistance to activated protein c (apc). the main reason for this resistance is a point mutation in the gene of dotting factor v (f v). a nueleotide exchange leads to a different amino acid sequence in the protein and thus to the loss of one of the cleavage sides which is important for the inactivation of the procoagulatory form off v. the imbalance in the coagalation cascade leads to a strong risk for venous thrombosis, which is thought to be inoreaced by factor to for heterocygotes and about for homocygote individuals. the prevalence in patients with a history of thrombosis is about %, among apcresistant patients as high as %. prevalence-off v ,leiden" is reported to be % of the normal population in leiden/netherlands and as high as % in a certain city in the south of sweden. due to the obviously different geographic distribution and the highly increased risk of venous thrombosis in carriers of the mutation investigated the distribution of this mutation in the normal population of different regions in germany. first attempts to assess the prevalence off v mutation by molecular methods (polymerase chain reaction followed by restriction fi'agment length polymorphism analysis) revealed that in the area of wiirzburg . % of blood donors carry the mutation, which is significantly different (fischer t-test, level of significance . %) from published data observed in freiburg ( . %). homburg/saar and harmover revealed . % and . %, respectively, and were not significantly different from wfwzborg. further investigations of blood somples fi:om other regions should give an overview of the geographic distribution of this mutation in germany and might finally help to draw a map era regional risk of venous thrombosis. the regular apc resistance test is not applicable to plasma from orally anticoagulated (oac) or heparinized patients due to decreased levels of vitamin k-dependent clotting factors and to thrombin inhibition by antithrombin, respectively. the former limitation is overcome by prediluting samples - - with fv-defident plasma. for this purpose a special quality of fv-deficient plasma has been prepared, which also allows analysis of heparinized plasmas. this fv-deficient plasma has now been evaluated with the coatest apc resistance assay. a complete ape ratio discrimination for the fv q mutation is obtained for plasmas from oac and heparin patients as well as from non-treated individuals. ratios above . are obtained in normal genotype plasmas, whereas heterozygotes for the mutation yield ratios in the range . - . . the basal apt times for oacplasmas with widely spread inr-values were reestablished into the normal range. .the stability of the reconstituted fv-deficient plasma was found to be' at least hours at room tempetature, indicating its suitability for .routin.e u.~. f .u~ .e~mv~., the influence of preanalytical procedures is rmm~ single or double cenuifugation as well as freezing of samples do not alter the apc ratios to any significant extent. our results prove the usefulness of the modified coatest apc resistance kit method as a reliable, simple and rapid tool when screening for the presence of the fv q mutation. hereditary resistance against the anticoagulatory action of activated protein c (apc-resistance, apcr) which was first described by dahlb~ick et al. (pnas, : , was identified as a possible new thrombophilic factor in a high percentage ( - %) of young adults with thrombotic events. a single missense mutation (r q) due to a g/a transition (g a) in exon of the factor v gene (bertina et al., nature, : , ) is tightly linked with apcr and is regarded as the causative molecular defect. identification of this mutation by pcr-based methods is easy to perform and avoids preanalytie and analytic errors in the eoagulometric assay for apcr. since the impact of this mutation in children with thromboembolic disease has not been determined to date, we initiated a multi-center prevalence study on two pediatric populations, with and without thromboembolie events. we compared pediatric patients with thrombosis, divided into different age groups ( to < , years; > , to < years; > to < years) with a normal population of children. the mutation g a was found with an unexpected high prevalenee of % in our normal controls. however, the prevalence was significantly higher in the age groups: to< , years ( %) and > to < years ( %). in patients between > , to < years the overall prevalence was similar to the control ( %). however in patients of this age with spontaneous thrombosis apcr was also a significant risk factor ( %). our results emphasize the impact of apcr for thrombogenesis in children. however, the significance is agedependent and does possibly reflect the different physiology of haemostasis in our three age groups. activated protein c (apc)-resistanec is a newly reeognised risk factor for thrombosis. in at least % of the cases it is caused by a single point mutation in the factor v gene (g->a at nucleotide ), which predicts replacement of arginin with ghitamin. one of the apc cleavage sites in factor va is located c-terminal of arginin , and mutated factor va (factor v leiden) is resistant to apc-mediated inactivation. from epidemiologic studies it is known, that this abnormality can be found in about one third of patients with thrombosis. apc-resistance is a major basis for venous thromboembolism and is prevalent in about . % of the general caucasian population. recurrent spontaneous abortion (rsa) affects - % of couples and represents a major concern for reproductive medicine. in spite of extensive endocrine, genetic, serologic and anatomic evaluation some - % of rsa women remain unexplained. a frequent morphologic finding in placentae of aborted pregnancies is an increase of fibrin deposition within the intervitlous space. because of these findings we studied the prevalence of apc-resistance in women with rsa (more than miscarriages) of unknown origin. in of cases we found a pathologic apc-resistance, both patients had a history of recurrent thrombosis and were heterozygous for factor v leiden. the prevalance of apc-resistance is , % and thus equals the prevalence in the general population. our data do not support the hypothesis that apc-resistanee is a risk factor for recurrent spontaneous abortion. h~matologisches zentrallabor der universit~t, inselspital, bern resistance to activated protein c (apc) due to the mutation arg --~ (]in of factor v (factor v leiden mutation) is the most frequent hereditary thrombophilic defect known today, with a prevalence of - % in patients with idiopathic venous thromboembolism and of about - % in the general population. with an allele frequency of % the expected number of homozygous individuals is about in . homozygous and heterozygons individuals differ considerably with respect to the relative risk of thrombosis ( -fold versus -fold) as well as to the age of the first thrombotic event ( versus years). deficiency of the vitamin k dependent protein s (p$), an important cofactor of apc, is another hereditary thrombophilia which is, however, much rarer than apc resistance with a prevalence of to % in patients with venous thromboembolism. factor v leiden mutation as well as ps deficiency are associated with impaired anticoagulatory activity of apc, which is most pronounced in case of the combination of the two defects. the combination of ps deficiency (with an assumed prevalence similar to that of pc deficiency) with heterozygous or homozygous apc resistance can be expected with a probability of : ~ or : ~ , respectively. it is well known that ps levels decrease towards the low normal or even subnormal range during pregnancy. moreovar, there is increasing evidence that the sensitivity of plasma to the antieoagulatory effect of apc decreases during pregnancy resulting in an acquired apc resistance. these pregnancy associated effects art obviously much more relevant in case of preexisting ps deficiency or apc resistance and should contribute to the elevated thrombotic risk during pregnancy in a subject with either of the two defects, and even more so for a woman who suffers from both defects. we describe a young woman with a combination of homozygens apc resistance ( apc ratio . , normal range: . - . ), pronounced ps deficiency (free ps .ll u/i, total ps . u/i, normal range: . - . u/ and . -lag u/i, respectively) and, moreover, impaired fibrinolysis (no change of euglobulin -lysis time after rain venous occlusion) who developed deep vein thrombosis after cesarean section in her first pregnancy. examination of her familiy showed heterozygous apc resistance in her asymptomatie father (apc -ratio . ) , combination of heterozygous apc resistance (apc -ratio . ) and ps deficiency (free ps . u/i, total ps . u/i) in her nsymptomatic mother and no defect in her sister. considering the fact that the mother was still thrombosis free at the age of one may assume that the thrombosis risk in the proposita was mainly influenced by the homozygnsity for apc resistance. s. ehrenforth, m. adam, b. zwinge, i. scharrer university hospital, dept. of angiology, frankfurt a.m., germany introduction: apc resistance has been shown to be the most commonly inherited defect which constitutes a risk factor for venous thrombosis (vt). however, most of the present epidemiological studies concerning apc-r prevalence in thrombophilia were derived from results of tests conducted onplasmas collected under various conditions. this may influence the great differences reported on the prevalence of apc-r among these patients. for example, it has been shown that freezing of plasma specimens prior to analysis of apc-r causes a significant decrease in the assay results.the aim of our study was to evaluate the influence of eentrifugation conditions on the results obtained with the chromogenic apc-r assay. patients and methods: blood was collected from patients (t women, men; fv gent.type: r/r , r/q , q/q ) through veinpuncture into trisodmm ciwat ( : ). platelet-rieh and platelet-poor plasma was obtained by immediately centrifugation at "c for , , , , , rain at , , , , and rpm. additional, pnp obtained from healthy individuals ( male, female without hormonal trealraent) was prepared equally. apc-response was determined within one hour after centrifugation using the coatest apc resistance kit from chromogenix. results: for both, pnp and sin/gle plasma samples, we observed continuous higher af'c-ratios after increasing cenwifugation intensity. for example, an increase from to rpm resulted m an increased apcratio from . to . ( min), from . to . ( rain) respectively. even though less distinctive, similar results were observed concerning the duration ol eentrifugation: when the duration was increased from to minutes we observed a continuous increase in apc-ratio, for example from . to . when using rpm and from . to . when using rpm. the decrease of the ratio after low eentrifugation is the eonse- nence of the shortening of affft in the presence of apc, without a signhcant influence of basal al:rl~ without apc. conclusion: our results demonstrate that centrifugation conditions are important to consider for the interpretation of apc-r results. supporting our observations, recent studies from sidelmann et al. have shown that an increase in plasma platelet concentration, low eentrifugation respectively, causes a signficant decrease in the apc-response. however, so far the mechanism responsible for the significant effect of both on apc-r assay results is unknown. although technically simple, the biochemical cemplexitiy inherent in the chromogenic apc-r assay necessitates a standardized plasma handling procedure to secure a reproducible determination of apc-il compapjson of different assays for determination of apc-resistance with the geno'fyping factor v (arg -> glu) g. siegert*, s. gehrisch*, e. runge**. r. naumann**, r. kn fler*** *institute of clinical chemistry, **clinic of internal medicine, *** childrens hospital resistance to apc diagnosed on the basis of prolongated clotting time in the aptt assay" is now considered a major cause of thrombophilia. in the majority apc resistance is ~ted with a point mutation in factor v molecule (arg glu), but both are not synonym. protongated baseline aptt is a limitation of the assay. following the determination is not possible in risk groups of patients (factor)ill deficiency, lupus anticoagulan and in patients under anticoagulant therapy. in these causes a dilution of plasma in factor v deficient plasma is recommended. the immunochrom assay is based on the inactivation of factor villa by apc. the aim of the study was to compare different functional apc response assays with the result of the dna analysis. apc response was tested in healthy probands, thro~ patients and family members using the lmmtmochrem assay, the contest (chromogeaix) and the contest with + dilution of the plasma in native factor v deficient plasma (immune). the dna analysis was performed as described by bertina. one patiem was homozygoas for factor v mutstion~ a hetemzygous result was obtained in members of the control group, in patients and in family members. in all cases with factor v mutation the ratio of the immunochrom assay was lower than the laboratory own value, independent on anticoagulant therapy. pathological ratios in this assay were also obtained in one member of a family" with high thrombotic incidence (dna arg/arg) and in patients under anticoagulant therapy ( two of this patients are one cloned twins). in the contest a ai~ response was diagnosed in all cases with factor v mutation without anticoagulant therapy and in % of heterozygous patients under anticoaglant therapy. results of the test using the dilution in factor v deficient plasma showed a good agreement vath the results of the dna analysis but the method is obviously only sensitive for the factor v mutation. the reason for pathogical ratios in the lrnmunechrem assay in wildtype patients is unclear. the majority of this patients is treated with anticoagulants, a comparison with the contest is not possible. interestingly in one patient under heparin and low ratio in the immunochrom assay' after reduction of hepann the ratio of the coatest was also low. it seems necessary to investigate in which distance to the thrombotic events the apc resistance should be tested. following pathological ratios in ftmctional apc assays must be discussed: high levels of factor viii and or v wiuebrand antigen (acute phase reactien), other mutations in factor v and viii. the factor v dilution assay should be replaced by the dna analysis. due to their differing compositions, the "sensitivities" of various aptf reagealts differ not only with respect to factor depletions, heparin and fibrin-fibrinogen degradation products, but also with regard to pathological inhibitors. for lupus anticoagulants this means that "lupus-sensitive" reagents can be delineated from "lupus-insensitive" reagents. with a "lupus-insensitive" ai~ reagent there is no or only slight prolongation of the aptt in the plasma under investigation, whereas with a "lupus-sensitive" reagent marked prolongation is observed. for the meaninof~l use of aptr reagents it is necessary to know the extent to which they are influenced by lupus anticoagulants. the following apti' reagents were tested: • ptt-reageaz, p'rta, ptta liquid, ptt-la, pti'-lt (boehringer/stago) • pathromtin, pathromfin sl, necthroratin (behring) • platelin s, piatehn excel ls (organon tekinka) • actin-fs, actin-fsl (dade) • aptt silica lye, aptt silica liquid (instntmentation laboratory) the material for investigation consisted of plasmas from patients with lupus anticoagulants. a confirmatory test (lupus anticoagulant test, immune) was positive for all of the patients. measurements were made using the sta coagulation analyser (boehringer/stago). it can be seen from the results that in some instances very different prolongations were obtained in identical plasmas by using differing aptt reagents. low susceptibility to lupus anticoagulants was shown by actirt fs (dade), ptt-reagenz (bcehrlnger) and neothromfin (behring). high susceptibility was shown by platetin excel ls (organon teknika), ptt-la and pti'-lt (boehringer/stago). lupus anticoaguhant screening with the aptt reaction is promising when two aptr reagents differing as greatly as possible in their lupus anticoagulant sensitivity are used. the resistance to the anticoagulant response of activated protein c (apc) is a major cause of venous thrombosis. apcresistance is due to a single mutation in factor v gene, which predicts replacement of arg in the apc-cleavage site with gln (factor v leiden mutation). in contrast to other known genetic risk factors for thrombosis, this factor v g-a mutation has a high prevalence in the common population of western europe (average - %). we have determined the prevalence of the factor v g-a mutation in a population of probands of north-eastern part of germany. the mutation was found in %. (heterozygoty were found in subjects person was homozygous.) the results are compared with our studies of populations from argentine and poland. me analysed the factor v g-a mutation in patients with thrombosis from germany and hungary. this mutation has been found in about % of these patients. in contrast, the frequency of this mutation was strongly reduced in a group of patients with thrombosis and pulmonary embolism of argentine ( heterozygotes in patients; %). the results of these different populations will be described and discussed. past medical history: venous thromboembolic events (re) at , and i years; intermittent oral anticoagulation (oac) without te's. diagnosis of autoimmune disorder with elevated antinuelear-antibody-fiters and positive lupus-anticoagulant test. no other relevant illnesses; family history uneventful. two weeks prior to the referral to us -acute febrile illness with nausea, diarrhea, abdominal pain; hospitalisation, treatment with iv antibiotics and anticoagulation with fraetionated heparin; development of extensive deep vein thrombosis (dvt) of the right leg; initiation of full-dose unfractionated heparin; decline of platelet count from to a nadir of g/l; referral to our department. on admission an extensive coagulation screen yielded the following results (n/normal, t/elevated, i/reduced, +/positive, -/negative): pt t, aptt t, tr n, factor ii, v, viii n, factor vii, ix, xi, xii /,, fibrinogan t, atiii n, protein c, s *, activated protein c sensitivity ratio . ($), fv-leidenmutation pcr -, fibrinolytic system n, tat t, ft÷ t, lupus anticoagulant +, heparin induced platelet antibodies +; no diagnosis of a specific autoimmuna disorder could be made. an immunosuppressive therapy with corticosteroids and anticoagulation with recombinant hirudin were init'~at~; no p~ogr~zsion of the dvt oeeured and normalisation of the platelet count was observed. during follow-up under oac ) and low-dose corticosteroids, the patient was well, the pathologic coagulat;.on results, including lupusanticoagulant and activated protein c resistance, have returned to normal; no further te's have been observed. in summary we present a case of a complex coagulation disorder as part of an autoimmune process, resulting in a clinically manifest prothrombotie dysbalance including lupus anticoagulant, acquired resistance against activated protein c and heparin induced thrombocytopenia (type ii), entering complete remission under combined immunosuppressive and anticoagulant therapy. in the last years, a vast number of simplified analytical procedures have been developed for the diagnosis of haemostatic disorders. today the detection method have evolved from the mechanical hooking method or ball coagulometry to optical systems, which additionally can utilise chromogenic substrates or immunological methods. in these systems the clotting time is derived from algorithms (e.g. threshold or maximum of the first or second integral). we studied healthy subjects, aged to years and patients, aged to years using a new aptt reagent (pathromtin $l). the results were compared with those obtained with a routinely used reagent (pathromtin). the reference range, factor-, heparin-and lupus anticoagulant sensitivity were determined. analysis was performed using the behring fibrintimer a (bfa) with optomechanical clot detection, the behring coagulation timer (bct) with op-"dcal clot detection by threshold and the dw test and dw confirm for lupus anticoagulant diagnostic. our results showed that the new pathromtin sl reagent met the demands for a higher factor and lupus anticoagulant sensitivity. it is highly suitable for monitoring heparin therapy and gave comparable results with the optical and the optomechanical analyser systems, hence reagent c~n be used for both systems. restenosis following percutaneous transluminal angioplasty (pta) continous to be a major clinical problem. neoinfimal hyperplasia, being the major undedying cause, can not be sufficiently avoided. vadous plasmatic coagulation and fibrinolytic factors, have been associated with artedal restenosis. anticardiolipin antibodies (act_) have been established as dsk factors for venous or arterial thrombosis. methods: in a cohort of patients ( men and women, age ± years) undergoing pta of a peripheral artery we prospectively evaluated whether acl could influence months restenosis rate. patients were clinically examined before, and months after pta. noninvasive grading of artedal stenosis was done by duplex scanning of jet peak velocities. restenosis was arbitrarily defined as more than % occlusion of the lumen at the site of dilatation months after successful intervention. laboratory investigation at the same time included acl and other known atherosklerosis risk markers, such as fibdnogen (fbg), yon willebrand factor (vwf), homocystein (hcy), c-reactive protein (crp). thrombin generation markers, such as thrombin-antithrombin iii complexes and prothrombin fragments + , as well as thrombomodulin (fm) as an endothelial activation marker, were also measured. results: / ( . %) patients were considered to have developed restenosis after months. / ( %) patients were found to have positive igg-( - gpl) and/or igm-acl ( - mpl) at all three measurements. / was negative before but seroconverted (igm) months after pta. / ( %) acl-positive and ( . %) acl-negative developed restenesis at months (chi-square p-value= . ). all above mentioned coagulation parameters did not differ between acl-positive and -negative patients, measured before or months after pta. some of them are shown below (values before pta): fbg ( basilar artery stenosis is a rare event in young children. risk factors are head or neck trauma with consecutive dissection of the vertebral artery, cardiac diseases or hypercoagulability. elevated lipoprotein (a) (lp(a)) serum levels in adults can mediate atherosclerosis. in addition, lp(a) might interfere with fibrinolysis. here we report on a year old boy , who presented with acute brain stem symptoms. history revealed neither trauma nor infectious disease. conventional and mr angiography showed stenosis of basilar artery without ischemic lesions. laboratory findings were normal in routine blood and csf tests. global coagulation parameters as well as procoagulant and anticoagulant factors were normal. cardiac and autoimmune disease could be ruled out. lp(a) serum levels were significantly elevated to mg/dl (normal range < mg/dl). analysis of other family members revealed a hereditary hyperlipoproteinemia (a) which might explain family history of an increased incidence of myocardial infarction and cve in elderly family members. clinically the patient recovered completely from brain stem symptoms after heparinization and subsequent oral anticoagulation with phenprocoumon. however, radiological signs of basilar artery stenosis were progredient. in a recently developed specific test, an elevated anti-phosphatidylserin antibody titer was detected one year after primary diagnosis. in conclusion, this is the first report on a child with stenosis of the basilar artery and elevated levels of lp (a). it is unclear, whether apa contributed to the onset of basilar artery stenosis or developed secondary due to endothelial defects after thrombosis and anticoagulation. apa, however, might increase the risk of further thrombotic events in this patient. in patients with thrombotic events respectively patients with systemic lupus erythematodes antioardiolipin antibodies (aca) aund lupus anticoagulant (la) were ~ea~ured. for aca detecting we use the assays from elias for igg-and ig~}-antibodies. we use as sensitive methods for detecting la in our laboratory the testkits from diagnostlca stago (staclot la with hexagonal array of phospholipids, ,ptt-la a very sensitive pttmethod and staclot p~p-a platelet neutralization procedure) and the ptt from organon teknik~ (platelin excel ls with two incubation times, and minutes). i"~e results of this tests were compared with three new or~e on german market: specktin apot (aktlvated plasma clotting time), specktin aptt (aptt wlth purified soy extract) and pecktin la (phospholipid preparation in concentrations between and ~g/ml); all wak chemie. traditional aptt reagents were developed for the sensitive detection of factor vib an ix as a cause of hemorhage. high sensitivity against lupus anticoagulants, which also prolong aptt, was not required for this purpose, with increasing recognition of the importance of antiphospholipid antibodies as a risk factor for thrombembolism, more sensitive reagents were designed, which now reliable detect this condition. using such reagents as a screening test in a general hospital makes it necessary to distinguish both conditions quickly. we here report an algorhythm, by which we use an inhibitor (lupus anticoagulant) sensitive (sta aptt, boehringer) and an inhibitor insensitive reagent (actin fs, dade) to distinguish anticoagulants and factor deficiencies as a cause of prolonged aptt. citrate plasma from patients with various diseases showed an unexpectedly abnormal inhibitor sensitive aptt (> s). plasmas with factor deficiencies remained abnormal with the insensitive aptt reagent. a regular correction of their defect occured on mixing with normal plasma. by measurement of single coagulation factors five patients with contact factor xii deficiency were found. this condition is associated with thrombosis and very rarly with bleeding. three patients with factor xi deficiency and two patient with factor ix deficiency were also identified. antiplatelets, of any kind, permits a secondary prevention of myocard ischemic lesions. there is no general consensus regarding secondary prevention of cerebral ischemic lesions. aspirin remains the most common substance, ticlopidlne also brings about prevention, but with important secondary effects. european stroke prevention study i has demonstrated that the combination of antiplatelets, in particular aspirin/dipyridamole (persantln), is also very active. to collect more information, esps was organized and patients receiving either a placebo,either mg aspirin,either mg sustained release form of dipyridamole (persantin (r)), or the combination aspirin/dipyrldamole, were recruited. it ended march st with the following conclusions: i-aspirin, mg a day, brings about a significant secondary reduction of stroke ( .z %), after a two year follow-up. notwithstanding the low dose of aspirin, haemorrhages remain important. -dipyridamole, at mg a day, brings about a significant reduction of stroke (i . ~), similar to that of aspirin. one could thus substitute mg aspirin by mg dipyridamole. -the combination of mg aspirin and mg dipyridamole brings about a significantly greater reduction of stroke ( . ~). esps revealed that a low dosage of aspirin is active, that dipyridamole alone is also active, but that the combination of both gives far better results. the study of the primary end-points,the study of the survival curves, the factorial statistical analysis and the pairwise comparison analysis, led to these conclusions. the conclusions drawn from esp£ underline that the combination aspirin/dipyridamole is a privileged choice for cerebral ischemia, the state of activation of circulating platelets in acute cerebral ischemia is controversial. activation of platelets on single cell level can be assessed by determining the shape change or the expression of antigens such as p-selectin (cd ). shape change is an early and rapidly reversible event in platelet activation whereas p-selectin is irreversibly expressed on the platelet surface upon stimulation. methods: we investigated untreated patients within one day after cerebral ischemia, patients months after stroke treated with warfarin, and age and sex matched control subjects without vascular risk factors. venous blood was collected into a fixation solution blocking the metabolic processes in platelets within milliseconds. we determined the fraction of resting discoid platelets by phase contrast microscopy. the expression of p-selectin was measured by flowcytometry. results: the fraction of platelets expressing p-selectin was higher in patients with acute cerebral ischemia ( . _+ . %) than in control subjects ( . _+ . %; p< . , u-test). patients with stroke (n= , . + . %) and patients with transient ischemic attack (tia; n= , . -+ . %) had similar values. patients months after stroke still had higher values ( . + , %, p< . ) than control subjects. the rate of discoid platelets was not different between patients with acute ischemia (n= , . -+ . %), patients months after stroke (n= , . -+ . %) and control subjects (n= , . _+ . %). platelet count was not significantly different between groups. conclusion: the elevated proportion of platelets expressing pselectin indicates strong platelet activation in acute cerebral ischemia and in a majority of patients months after stroke. assessment of pselectin revealed a higher sensitivity for platelet activation after stroke or tia than analysing the reversible shape change. further studies have to clarify if monitoring of platelet activation by flowcytometry is helpful as a prognostic tool and to evaluate therapeutic strategies after stroke. vascular smooth muscle cell (smc) proliferation and migration into neointima are the hallmarks of atherogenesis. the complexity of these processes and their concerted action and interaction of molecules are yet to be fully elucidated, one crucial molecule seems to be the urokinase-type plasminogen activator receptor (upar) recently also assigned as cd antigen, upar serves a dual function: ( ) it directs upa proteolytic activity to a special location on the cell surface and ( ) induces cellular signals leading to various phenotypic changes. we have investigated the signal-transducing capacity of upar in human smcs and provide here a molecular explanation for uparrelated cellular events. activation of these cells with upa (even with inactivated catalytic center) results in the induction of tyrosine phosphorylation, suggesting modulation of upar-associated protein tyrosine kinases (ptks) upon ligand binding. we obtained patterns of tyrosine-phosphorylated proteins with molecular masses of ~ - and - kd. using antibodies against different types of ptks as well as immunoprecipitation-and immunoblotting techniques the ptks involved in the upar-signalling complex were identified to be members of the src-ptk family. the cotocalization of upar and ptks at the cell surface of smcs was further confirmed by confocal microscopy studies. we conclude that the upar-ptk complex is most likely involved in this signal transduction pathway that provides the coordinated action of extracellular proteolysis, adhesion, and cell activation, which is required for cell migration. this mechanism may be crucial for the progression of atherosclerotic plaques. activation markers of haemostasis have been found elevated in relation to diabetic vascular lesions. simultaneous pancreas-and kidney transplantation (pkt) in type i diabetes has been shown to improve diabetic complications and long term survival. we measured haemostatic vascular risk factors and activation markers in plasma of patients after successful pki', patients after pkt and rejection of the pancreas graft and patients after pkt and rejection of the renal graft. blood samples were taken during routine ambulatory visits, patients were free of any ongoing acute disorder or transplant rejection and under continuous immunosuppressive medication. despite individually adjusted insulin therapy hba plasma levels increased after pancreas rejection ( , vs ,i , p< . ). platelet counts and plasma levels of fibrinogen, f + fragment, tat-, app-complex and-fibrin monomer were found significantly elevated as compared to diabetic controls but not significantly different with respect to complete or partial successful pkt. one major reason of the increased activation state of haemostasis may be cyclosporin treatment given to all patients, t-pa and pal i plasma levels were within the normal range and significantly correlated to plasma triglyceddes (r. . ; p< . ). d-dimer plasma levels were significantly lowered after pancreas rejection ( ( ) vs ( ) nglml; mean(sem) p< . ), which might reflect impaired fibrin degradation related to increased glycosylation of fibrinolytic factors. in conclusion, despite the marked improvement of glucose and lipid metabolism, plasma markers of activation of coagulation and flbrinolysis are not decreased to normal after simultaneous pancreas and kidney transplantation. according to the investigations of fowler et al. and pepe et al. the probability of an ards occurring with one risk factor is - %, and in the presence of several risk factors, %. goris et al. and johnson et al. determined the level of severity with the aid of a fixed scale: the injury severity score. all these investigations are however not to be interpreted as typical following coronary surgery. these investigations demonstrated that the kallikrein and factor xii systems are of great importance as intraoperative risk factors. here the factor xii system plays a major role with direct or indirect activation of the kauikrein-kinin system with the splitting products alpha-factor xiia and bfactor xha respectively. all ards scores take the pmn-elastase into account. if the pmn-elastase values ( pg/l) are constantly high postoperatively then lung complications are to be expected. patients developing an ards displayed significantly lower alpha -macroglobulin values. patients who developed a highly significantly raised kallikrein-like acdvity (> u/i) after the beginning of bypass and showed constantly high values during ecc are difficult to keep under control due to the blood pressure behaviour. the platelet pal also shows a significant rise and intraoperatively runs analogous to platelet factor , only antiparailel, since it attacks the endothelium. we were able to show that pai- is suitable as an indirect marker for a possibly developing restenosis. % of the patients investigated with lowered pai- values in the postoperative phase did not develop a restenosis. however, with patients showing significantly rising pa[- values from the st. to rd. postoperative day % of all the cases had a restenosis. a further risk factor in this respect are significantly raised fibrinogen levels which lay over % at the end of surgery. if these fibrinogen values do not fall from the st. postoperative day onwards a raised risk of thrombosis must be reckoned with in the absence of therapeutic intervention. the following parameters represented haemostaseological risk parameters with significant behaviour within the framework of this study: ) regards the blood pressure behaviour, the kallikrein-like activity (> u/i); ) with regards to the lung complications, aipha -macroglobulin and pmn-elastase (> g/i); ) and final/y as a possible marker for a developing restenosis pai- and fibrinogen (> %). resulting from numerous clinical studies homocysteinemia is found to be an almost independent risk factor of atherosclerosis including thrombotic complications as well as of venous thromboembolism. experimental investigations on the underlying mechanisms suggest endothelial cell damage accompartied by the development of an atherogenic and thrombogenic potential, increased platelet reactivity, oxidative modification of ldl, and enhanced affinity of lp(a) for fibrin. to our knowledge no results are published on the influence of homoeysteine on leukoeytes although these cells are deeply involved in pathological events within the vasculature. therefore, as a first approach different functional parameters of human polymorphonuclear leukozytes (pmnl) were followed under incubation with , , and i.tm (final concentration) dl-homocysteine (hc) in isolated fractions or whole blood, respectively: l) spontaneous mobility of pmnl, measured as migration distance into micropore filters in a modified boyden-chamber, is found to be significantly enhanced by the two smaller hc concentrations. ) chemotaxis induced by . i.tm formylmethionylleueylphenylaianine (tmlp) shows no significant differences. )monitoring of chemiluminescence signals (autolumat lb , berthold) is complicated as hc influences the luminol-mediated indicator reaction. adjusting appropriate conditions the following results are obtained: spontaneous chemiluminescence and that induced by zymosane, tmlp, and the ca +-ionophore a are entranced by the two higher hc concentrations. there are, however, differences between the blood donors as a minority does not respond to hc in repeated measurements. with phorbol -myristate acetate the signal is diminished by hc in all cases and with all concentrations. ) phagoeytosis induced by zymosane (microscopic evaluation) as well as by opsonized e. coil (cytoflowmetric evaluation) is significantly increased by the two higher hc concentrations. conclusion: the activation of human pmnl is enhanced with respect to the majority of investigated stimuli by hc in concentrations reached under pathophysiological condititions. the effect of pysical exercise on hemostatic parameters was studied in patients (male, mean age: [range - ] yrs) with angiographically documented coronary artery disease (cad) and in controls (male, [ - ] yrs) both participating in an hour group exercise session for cardiac rehabilitation. in each group relevant arteriosclerotic lesions in carotid, abdominal and leg arteries were excluded by doppler ultrasound examinations. patients were all under -blocking agents and aspirin. plasma levels of prothrombin fragment + (ptfi+ ) and fibrinopeptide a (fpa) reflecting formation of thrombin and fibrin, respectively, were measured at rest and immediately after hour of exercise consisting of jogging, light gymnastics and ball games. training intensity in both groups was comparable as indicated by the mean heart rate during exercise corresponding in patients to + % (mean-+sd) and in controls to -+ % of the maximal heart rate previously determined on a bicycle ergometer. baseline values for ptf + were significantly lower in oatients ( . -+ . nmol/i; mean-+sd) than in controls ( . -+ . ; p< . i. after exercise we found an increase of ptf + in controls to . -+ . nmol/i (p< . ) while in patients ptfi+ remained unchanged ( . -+ . after). accordingly, exercise induced r se of fpa was more pronounced in controls (from . -+ . to . -+ . ng/mt; p< . ) than in patients (from . -+ . to . + . ng/ml; p< . t). we conclude that in terms of thrombin and fibrin generation exercise training does not exert detrimental effects on hemostasis in patients with cad. lower baseline values and lack of exercise induced increases of ptf + in patients with cad might be attributed to medication with aspirin and/or b-blocking agents. periodontitis marginalis (pm) is an inflammatory oral disease that is caused by gram-negative bacteria and that has a high incidence in the second half of the life. clinical signs of pm are gingival bleeding, periodontal pockets, alveolar bone destruction and loss of teeth. recent epidemiologlcal studies have provided some evidence for an association between pm and atherosclerosis. in the present paper we will summarise some of the results that we have obtained in studies on patients with pm as well as on patients with hypercholesterolaemia (hc) and atherosclerosis. pm was frequently found to be associated with hc ( % in rapidly progressive pm) and increased reactivity of peripheral blood neutrophils and platelets (e.g. generation of oxygen radicals and paf-induced aggregation). patients with hc and atherosclerosis had a higher frequency of severe pm when compared with data on the community periodontal health. the severity of pm was higher in patients with plasma cholesterol levels _> . mm when compared to those with plasma cholesterol < . mm. in patients with coronary atherosclerosis the severity of pm was significantly correlated with plasma cholesterol level, systolic blood pressure and the number of diseased coronary arteries. these results provide further evidence for an association between pm, hc and atherosclerosis. it can be speculated that hc is not only a risk factor for atheroscterosis but also a risk factor for pm and acts by increasing the reactivity of neutrophils and platelets. on the other hand, pm as a mild chronic inflammation could promote the development of atherosclerosis due to effects of endotoxins on vessel wall, blood cells and haemostatic factors. it has been also speculated that phagocyting leukocytes in the inflamed periodontal tissues could contribute to oxidative modification of ldl. so far, there is no evidence that atherosclerosis may contribute to the pathogenesis of pro protein z (pz) is a vitamin k dependant plasma protein synthesized in the liver. it promotes the association of thrombin with phosphorlipid surfaces. recently it has been shown that a deficiency of pz may lead to a bleeding tendency. in patients undergoing chronic hemodialysis, disorders of hemostasis are common. to examine if plasma levels of pz are altered in patients with end stage renal disease we determined pz in plasma of patients at the beginning of hemodialysis treatment. the results were compared with a group of healthy controls. the difference of pz levels in plasma of patients with end stage renal disease with the control group was not significant. control group was + ng/ml and in patient group was + - ng/ml. one patient with marked bleeding tendency after hemodialysis pz was ng/ml. we concluded that patients with bleeding disorders pz determination may be helpful. the normal range of actin fs was reinvestigated in a multicentric approach. a protocol was developed which requests from each center to assess the aptt with one common and one variable lot of actin fs in samples of suspected normals. inclusion and exclusion criteria based upon the results of clotting assays, liver enzymes and clinical data were defined. results: a total of results was obtained. the majority of centers in this study used the electra or c (mla). results for the electra group (n = ) showed a precision for the common lot of actin fs with a common lot of a three level control from . % (level ) to . % (level ) with an excellent accuracy between the centers. clotting times with the variable lots of actin fs were very similar. the results from normals, however, showed a somewhat higher dispersion using the common lot of actin fs. of centers had almost identical mean values (range . to . sue) whereas one reported shorter and one longer clotting times ( . and . sec). results with the variable lots gave almost identical results as the common one. a total of results of all lots gave a normal range of . to . ( - % percentiles) on electra. mean values on acl (n = ) were . , on bct, . sec, on amga coagulometric, . sec, on amga turbidimetric, . sec (n = each). all centers used sarstedt monovettes with . sodium citrate. discussion: the results of this study demonstrate the lot to lot consistency of all lots of reagents included in this study since the common and variable lots showed very consistent results. interestingly in the large group of electra users the normal ranges showed some differences, though the controls in all centers were almost identical. this confirms the recommendation that a normal range as stated from the manufacturer should be used for orientation only and that each laboratory should assess its own range. direct acting anticoagulant agents such as hirudin (r-h), argatroban (arg), efegatran (efe) and peg-hirudin (ph), represent specific and potent inhibitors of thrombin. blood samples collected in r-h ( ~g/ml), arg ( ~tg/ml), efe ( ~tg/ml) and ph ( ~tg/ml) do not clot for extended periods (> hours), thus allowing for the collection of plasma for analytical purposes. unlike heparin, these agents do not require any plasma cofactor for their anticoagulant effect. in contrast to citrate, oxalate, edta and heparin, these antithrombin agents do not alter the electrolyte or protein composition of blood. thus, blood collected in these agents may provide a physiologically intact (native) sample for clinical laboratory profiling. we have used all of these agents to prepare whole blood and plasma samples for various diuical laboratory measuroments. plasma samples collected with these agents are obviously not suitable for global clotting tests (pt, aptt, thrombin time, fibrinogen); however, these agents are optimal anticoagulants for the collection of samples for the molecular markers of hemostatie activation, such as fibrinogen/fibrin related degradation products, prothrombin fragment, protease cleavage products, tfpi, tnf and other protein mediators. electrolytes, blood gases, enzymes and protein profiling can also be satisfactorily measured on blood samples collected with these agents. antithrombin anticoagelatad blood used fur hematologic analysis showed equivalent blood count and differential results as that obtained with edta blood. unlike other anticoagulants, these agents do not interfere in the cell staining process. washed blood cells can also be prepared using antithrombin aents supplemented buffers for morphologic and fuuctional studies. thrombin inhibitors such as hirudin have also been used for flow cytometry and image analysis of blood cells and tissue exudates. our observations suggest that these anticoagulants can be used as suitable anticoagulants for clinical laboratory blood sampling. these agents can also be used as a flush anticoagnlant fur most automated instruments as these exhibit superior anticoagulant properties to heparin. furthermore, the hematologic parameters obtained in antithrombin anticeagnlated blood may be physiologically more relevant than those determined on blood collected in edta, citrate or heparin. antithrombin ul determination is one of the most popular method for in vitro diagnostic of number of different disorders. human fhrombin a~nity purified on heparin-rnodified silica-based sorbents was used for level of antithrombine lu determination by abilgaard method in blood of patients with pregnancy pathology, acute leukemia, thrombocytopenia and anemia. it was founded, that antithrombin level is decreased to - % of normal values in case of pregnancy pathology, to < % -in case of acute leukemia and thrombocytopenia, to s % -in case of anemia. obtained results show the strong relationships between named disorders and patient antifhrombin iii level. therefore anfifhrombine iii estimation may be used as simple and quick method for preliminary diagnosis of above named disorders. bm coasys is a complete automated analyzer system for coagulation tests. it is well suited for routine coagulation testing in random access in a medium throughput laboratory environment. analytical performance and practicability were tested by a common evaluation program in five hospital laboratories. within run and day to day cv's were below % in different samples (controls, patients) . comparison in different therapeutic ranges confirms the declaration of the isi-value for calculating inr-values. normal values for coagulation tests with results in pdmary units were checked in samples and confirmed. due to the optical measuring principle of the bm coasys there was a little tendency to shorter times with the thrombin reagent. in conclusion the performance of coagulation tests with the bm coasys was rated as well or better compared to existing systems in the laboratories with advantages due to short timed familiarizing and easy handling. flexibility and stability of the system permit optimal integration and innovation into the w rkflow of the routine laboratory. the purified thrombin and antithrombin iii (at iii) have a great interest in the clinical diagnostic and treatment practice, so their isolation methods are very important. molecules of these proteins have some fragments replying for interaction with native glycosaminoglycan, heparin. this interaction is used for isolation and purification of thrombin and at ul from native materials, blood plasma or its fractional products. we have done comparative studying these proteins purification on heparin sorbenfs, which contain heparin immobilized on sificagel, modified by glycidooxipropyl, gamma-aminopropyl and tosyl chloride groups, or on cellulose: heparin-epoxy-silica ( ), heparin-gammapropyl-silica ( ), heparjn-tozylsilica ( ), and heparin-cellulose ( ). we founded that thrombin binds with all sorbents, while at iii doesn't binds with sorbents and . there wasn't any difference between silica and cellulose sorbents in thrombin desorbfion by t m naci. at iii binds more stronger with heparin-ceuulose t[~,~n with silica sorbents but specific activity and purity degree were approximately the same on both kinds of sorbents. thrombin specific activity and purity degree were approximately twice higher on sorbents and in comparison with sorbents ! and ( - nih units/mg versus -t nih unlts/mg). therefore, sorbents and can be used for isolation and purification of thrombin and sorbents t and can be used for isolation and puriiication of at ii . we used these sorbents for large scale purification of named proteins. purified thrombin was used for production of diagnostic kits for anfithrombine iii, fibrinogen, fibrin/fibrinogen degradation products and thrombin time determination. after an aerobic or anaerobic physical exercise various alterations of the hemostatic system were detected. numerous investigations of the hemostatic system exist of running and of bicycle ergometer exercise but not of swimming. young volunteers (n= ; median age years) were investigeted~ there was an aerobic exercise (achieved heart rate -- /min, lactate < mmol; n= ) and an anaerobic exercise (achieved heart rate ~ lo/min, lactate > mmol/ ; n= ). in both groups there was a significant shortening of the ptt. under anaerobic conditions hematocrit and quick significantly increased. factor viii activity rose significantly in both groups. indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f + only under anaerobic conditions (tat from , to , pg/ ; f + from , to , nmol/ ). indicating activation of fibrinolysis t-pa activity increased significantly in the anaerobic group (from , to , iu/ml) but not in the other group. this findings indicate that there is e balance in the hemostatic system by activation of clotting as well as of fibrinolytic system in young volunteers during exercise by swimming dependend on the degree of exercise load. membranes as well as compact, porous disks are successfully used for fast analytical separations of biopolymers. as far as capacity, speed and performance of separation are concerned, the supports are as effective as other recently developed fast media for the separation of biopolymers °). so far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations. in this report, the use of a compact tube made of poly(glycidyl methacrylate) for fast preparative separations of proteins is shown as a possible solution of these problems. the units have yielded excellent results, regarding performance and speed of separation as well as capacity. the application of compact tubes made of poly(glycidyl methacrylate) for the preparative isolation of the coagulation factors viii and ix from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. in terms of yield and purity of the isolated proteins, this method was comparable to preparative column chromatography. the period of time required for separation was five times shorter than with corresponding column chromatographical methods. our measurements showed an excellent correlation of the two systems (r= , ). the maximum amplitudes on the roteg were on average . % higher than on the hteg, corresponding to a slightly lower reverse momentum of the measuring system in comparison to the hteg. we report first results out of the evaluation of sta compact (boehringer mannheim/diagnostica stack)). sta compact is designed for automated analyses of routine and special coagulation (chronometfic, photometric [ nm] and turbidimetric [ run]) tests. in addition, it does measure ,,derived" fihrinogen. tests as follows were evaluated: prothrombin time (pt), partial thromboplastin time (aptt), fibrinogen (clauss method), thrombin time, at iii (chromogen), hepato quick, as well as the factors ii, v, vii, x, and viii. results: within run cvs of the clotting tests were below % (calculated on the basis of seconds) in most cases, day to day cvs below % (not measured for factors, yet). at iii yielded within run cvs below % in the decision range. measuring ranges: at iii: - %; fibrinogen: . - . g/l (plasma -dilution / ), after rerun with other dilutions: from . g/ (dilution: / ) to g[l (dilution: / ). method comparisons, using sta as reference, yielded slopes close to . and negligible intercepts. throughput: with routine clotting tests about tests/h, in a sample selective access mode. we conclude, that sta compact allows precise measurement of routine and special coagulation tests. it is also a reliable system for photometric tests and well suited for intermediate workloads as well as stat analyses. we did evaluate ptt lt, a new liquid, silica based ptt reagent. special attention was given to reference interval and heparin sensitivity. the new reagent is well suited for the measurement of intrinsic clotting factors and is reported to have high sensitivity for lupus anticoagulants (higher sensitivity than sta aptt [boehringer mannheim = bm]). it is stable for days in the cooled compartment of the sta analyzer. methods: all experiments were made on sta. for comparison, we used three other ptt reagents (a lab. routine, silica based aptt, as well as sta aptt and sta ptt kaolin from bm). in addition, thrombin time ( u/ml thrombin, sta thrombin reagent) and heparin (chromogenic xa test, rotachrom heparin) were measured. results: within mn imprecision (n= ) was below . % cv in the normal range and in two controls (mean values: s, s), and . % in a heparin plasma (mean: s). between day imprecision (d= ) was below % in two controls ( mean values: s and s). the upper limit of the reference range is s ( . th perc., median: s; patients with normal coagulation status [routine aptt, fib., pt], median age: years); almost identical reference ranges were obtained with sta aptt and the routine ptt reagent, while sta ptt kaolin showed significantly lower values ( . th perc.: s, median s). method comparison study: good agreement using plasmas from patients without heparin: (y= a + . x, n= , range of(x) from to s, r = . ; x = sta aptt). the median values from patients under high dose heparin were: routine ptt: s, sta aptt: s, ptt -lt: s sta ptt kaolin s, thrombin time s and heparin . iu/ml in conclusion, results of the new reagent compare well to our routine ptt and to sta aptt system reagent. it allows sensitive monitoring of high dose heparin therapy and is well suited for detecting abnormalities of the intrinsic clotting factor pathway. is a standard technique since many years. the interpretation of the thrombelastograms has been widely based on phenomenologic observations, while there is a lack of exact information concerning the coagulation mechanisms leading to the teg amplitude (a're~). the aa'ec is a measure for the mechanical stiffness of the clot and depends on: a) fibrin formation and adequate polymerisatiun of a -dlmensional network: measurements with nonrecalcified citrated blood activated with adp or epinephrine (both n= ) did not show any clot formation in the teg this relies on the need for a mechanical coupling between the teg pin and cup over a distance of mm, which is accomplished by the fibrin network therefore, teg can only be performed under thrombin formation and thus under thrombin-activation of the platelets in the sample. factors, which inhibit platelet aggregation but don't limit thrombin-activation of platelets, cannot be monitored by teg. b) the attachment of the dot on the surface of the teg pin and cup. according to recent literature we suggest that the attachment of the clot in the teg relies exclusively on fibrinogen/fibrin adsorption to the surfaces of the pin and cup. interruption of this attachment can result in lower amplitudes or the so-called ,,stairway" phenomenon. we could show a complete interruption of the clot attachment by dipping the pin for one second in % albumine solution (n= ). c) the fibrinogen concentration (fg) and platelet count (pc) of the sample. in volunteers we found only a poor correlation of the maximum amplitude (ma) with fg alone (r= . ) or pc respectively (r= . ), while there was a very good nonlinear correlation to the product of fg and pc. we suggest that the fibrin network forms the main structure of the clot while the thrombocytes enhance its stiffness in a concentration-dependent manner. this effect of the ptatelets can be completely reversed by gplibfllla antagonists. d) adequate coagulation activation: in nonactivated teg even small amounts of inhibitors can lead to a significant reduction of the ateg. conclusion: alterations in teg measurements can be judged more properly when the underlying mechanisms are understood. the consideration of the limitations of the method allows a more specific interpretation of the results. as a response on a customer request we did investigate the sample stability of blood samples for the aptt. the study was set up in a way that simulated the conditions of a large private laboratory in which the samples arrive several hours after blood collection. blood was drawn from donors into . % sodium citrate and mixed well before it was divided into several aliquots which were kept at room temperature. the aliquots were centrifuged after ~ , , , and h after venopuncture and the plasma was analyzed immediately with different reagents on electra . results: there was a clear difference in the change of the apttover time with these reagents. also f viii (determined with a chromogenic assay with complete and standardized activation) change considerably. reagent a: ellagic acid, plant phospholipid, reagent b: sulfatide/kaolin, phopholipids, reagent c: ¢llagi¢ acid, plant and rabbit brain phospholipids the increase of aptt was apparently not a function of the decrease of fviii because the in vitro f viii sensitivity of reagent b. was inferior to reagent a though reagent b showed more prolongation of the aptt than reagent a. reagent c, however, showed only minor changes in the aptt. discussion: these data show that the sample stabifity of the aptt is reagent dependent and that it is not simply a function off viii sensitivity. other factors such as the buffer system but also the sensitivitiy towards other factors than f viii seem to contribute. a comparison of the technical principle of the roteg coagulation analyser and conventional thrombelastographic systems an. calatzis, p. fritzsche. al calatzis, +m. kling, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology yechnische universit~t m nchen thrombelastography (teg) was introduced by hartert in as a method for continous registration of the coagulation process. in we presented the roteg coagulation analyser, using a newly developed technical method. in teg systems according to i/artert the sample (blood or plasma) is placed in a cup which is alternately rotated to the right and left by , °. a cylindrical pin, which is suspended freely on a torsion wire, is lowered into the blood. when coagulation starts, the clot begins to transfer the rotation of the cup to the pin against the reverse momentum of the torsion wire. the angle of the pin is electromagnetically detected, transformed to the teg amplitude and continously recorded. in the roteg the pin is attached to a short axis, which is guided by a ball beating. thus all possible movement is limited to rpotation (r_oteg). the cup is stationary, and the pin is rotated alternately by ° to the right and left by a feather system. when a clot is formed, it attaches to the surfaces of the pin and cup and starts preventing their relative movements against the reverse momentum of the feather. here the reduction of the rotation of the pin, which is detected optically, is tranformed to the teg amplitude. as can be shown by theoretical analysis and by control measurements, the roteg provides the same measuring capabilities as conventional teg systems. the main advantage is the solid guiding of the measuring system, which makes the roteg easily transportable and less susceptible to shock or vibration during measurement. yhrombelastography (teg) is a standard monitoring procedure for evaluation of coagulation. usually only nonactivated native blood teg measurements (nateg) are performed, which leads to a) a long time interval until coagulation and fibrinolysis parameters are available b) very high susceptibility of the measurement to inhibitors like heparin, which disturbes the judgement of other components of coagulation, c) unspecific results. our aim was to develop a coagulation monitoring system based on teg providing fast and specific information on the different components of coagulation. methods: the following measurements are performed in paralel using disposable pins/cups (haemoscope): a) extrinsic activated teg (exteg): al whole blood (wb) + ~tl innovin (recombinant thromboplastin reagent, dade). b) intrinsic activated teg (integ): al wb + ~tl kaolin (suspension g/l, behring). c) aprotinin teg (apteg): exteg + kie aprotinin (trasylol, bayer). d) heparinase teg (hepteg) as decribed in ( ). results: exteg and integ provide information on the extrinsic/intrinsic system within - min and information on the platelet/fibrinogen status within - min. because of the addition of potent activators integ and exteg can be performed when inhibitors like heparin are present in the circulation. fibrinolysis effects can be seen on exteg and integ and by comparison of exteg and apteg (apteg: invitro-fibrinolysis inhibition by aprotinin). if fibrinolysis is detected by exteg or integ and aprotinin-susceptibility is verified by apteg, aprotinin therapy will be initiated. heparin effects are revealed by hepteg. discussion: by the comparison of parallel teg measurements which have been differently activated, specific and fast information on the different aspects of the clinical coagulation status is provided. the presented tests can be easily performed bedside and only a small specimen of whole blood is needed ( , - , ml). introduction: a severely prolonged aptt ( s; normal: ~os) was observed during preoperative screening for a planned splenectomy in a -year-old man with an year history of osteomyelofibrosis. fellewing neer-normal~atien ( s) of the ap'ci" after rain preincubation in a kaolin based aptt assay, pk deficiency was suspected and studies were performed to further investigate the nature of the pk deficiency as well as the mechanism underlying the normalization of the prolonged aptt by increasing the preincubation time. methods: the apl-r assay was peal'armed using kaolin/inesithin. high molecular weight kininogen clotting activitiy (hk:c), fxii:c end pk:c were measured by an aptt based assay using neothromtin ® (behnng) and rain (pk:c) or min (hk:c, fxli:c) preincubation. pk amidolytic activity (pk:am) was assayed using cosset pk ~ (chromogenix) and pk antigen (pk:ag) by quantitative immunoblotting. fxll and hk proteolysis dunng activation of plasma by kaolin ( mg/ml at =c) or ds ( . ~tglml at =c) was demonstrated by immunotilotting assays of fxii and hk following sds-page. assay pk:c pk:am pk:a~i fxfi: the propositus had pk:c< %, pk:am= % and pi~ag< . % as compared to normal pooled human p(asma (nhp). his son and two daughters had pk:c- % and normal aptt values, incubation of the propositus' plasma with ds did not result in fxii or hk cleavage within rain, whereas jn nhp detectable f×ii and hk proteolysis occurred after rain and complete proteolysis was observed after - rain. in contrast, kaolin activation of propositus' plasma led to slow activation of fxii after rain, presumably by autoactivation, and to fxlla-induced hk proteolysis. near-normalization of the propositus' aptt by prolongation of the preincubation time paralleled fxii autoactivation as evidenced by immunobletting. we describe a propositus with severely prolonged aptt due to hereditary, crm negative pk deficiency suffering from omf. activation with a particulate suspension of kaolin led to slow fxii autoactivation and hk proteolysis, whereas ds in solution did not induce fxii or hk cleavage. fxii autoactivation seems to be responsible for the normalization of the prolonged aptt in pk deficiency after prolonged preincubation times. in our study we compared a conventional bag with silicone tubing (a) for blood donation with new ones (] from biotrans and c from baxter) with a newly developed y-shaped adapter. this adapter is integrated into the tubing and therefore provides the advantage for drawing blood samples in a closed system. the systems were identical in amount and content of anticoagulant, i. e. ml of cpd per bag resulting in approximately % of the final whole blood volume. the purpose of the study was to determine whether the different tubings can influence the quality of plasma products conceming the blood coagulation system. in plasma samples we measured several factors of the procoagnlatory and fibrinolytic systems. intralndividual control eitrated (. m) blood samples were initially drawn from the contralateral cubital vein from the same male donor ( in each group). in all bag samples we found small but significantly higher levels of the global test parameters ap'it and ti" compared to controls, indicating a higher amount of anticoagulant. pt, however, revealed no differences, thus suggesting that factor activities were not altered (statistics according to mann-whimey). increase of procoagulatory activity measured as tat complexes showed elevated levels in bags a and c whereas prothrombin fragments fl+ decreased only in a. conceming the fibrinolytic system, plasminogen a~tivators and pai- values were diminished in all three systems < a < c) compared to controls. d-dimers were lowest in a followed by slightly higher values in c, controls and b. fibrin monomers did not reveal any significant differences: a < c < controls < b. in summary, the quality, of the different blood sampling devices was comparable to the intraindividual controls as to factor activities measured by global tests. the activation of the procoagulatory and fibrinotytic systems was slightly but in most cases significantly higher in the two new devices than in the conventional one. all values, however, obtained from the plasma samples did not exceed the normal range of healthy blood donors. therefore we concluded that the two new closed blood drawing systems are favorable for blood donating procedures. in patients with acute myocardial infarction (ami) and thromholytie therapy ( patients with rt-pa, patients with streptokinase and one with heparln) with ck, myoglobin and ekg criterions the patients were divided in two groups (reperfusion/no fellow two hours after starting the thrombolytic therapy) . blood samples were taken before, rain, i h, h, h, h, h after lysis and than every day till day . because of the central role of factor xii in activation of coagulation, fibrinolysis, kallikreln-kinin-system and complement cascade we investlgate the role of factor xila initiated by ami and the relation of factor xiia to the thrombolytie agent and reoeclusion rate. for the investigatlens we take the kits from shield diagnostics (xiia), behring diagnostica (c~-inactivator, pl~nogen, ~-antip]~n~n, pap), chromogefiilx ab (prekallikrein) and di~nostica stage (vile). the results: there is an increase of factor xiia immediately after starting the fihrinolysis (max. rain after starting); the increase /i independently of the thrombolytie agent. parallel to factor xiia raises factor viia without significant changes of c - naotor and prekalllkrein. that means: activation of xiia and fibrinolytic pathway leads to relatively mild c.hanges in kallikrein system, hut to significant activation of extrinsic system by vila-tissue factor. in some patients is an additional rise in the system xiia -viia, when the fibrinolytic system is already in the normal range. there will need further investigations to define the risk of reocclusion as a result of activation of faktor viia by faktor >li ia. autoimmune thrombocytopenic purpura (aitp) is a frequent complication of chronic lymphocytic leukemia (cll] which developes on different stages of the disease and needs special treatment measure. mechanism of autoimmune disorders in cll remains uncleared. we investigated immunologic phenotype of blood lymphoid cells in patients suffering from cll with aitp. in these patients we did not observe disorders in expression of b-lineage markers as compared with cll patients without immune complications ( patients). but in the st group of the patients the greater number of b-celts expressed markers of activation. according to ig heavy chain expression, the lymphocytes in most cases of cll complicated by aitp had more mature phenotype. in all patients with k phenofype of cll lymphocytes we found immune disorders. the development of aitp was accompanied by lowered level of t cells and changed dis'flibution of their immunoreguiatory subsets: diminished number of cdz~cells and increased one of cd~'÷lymphocytes. the results of our investigations undirectly proved that malignant b-cells in cll are involved in production of autoantibodies against blood cells. dysbalance in t-cell system with functional disturbances of immunoregulation are significant in development of autoimmune complications in cll a in women with severe fvii deficency (< %) hypermenorrhagia may cause life threatening blood loss. therefore, hysterectomy at a young age is reported frequently in the literature. a year old girl without history for a bleeding disorder was transfered with hypermenorrhagia. the initial laboratory data revealed an abnormal quick-test of % due to fvll of , %, normal platelet count and hemoglobin level of , g/dl. antifibrinolytic therapy (tranexamic acid x mg/kg bw/d) and lynestrenol substitution were started to reduce the hemorrrhage. despite treatment the daily blood loss increased to a maximum of ml. therefore, substitution therapy with recombinant fvila (rfvila) (novonordisk) was started at a dose of ilg/kg bw q h. subsequently blood loss decreased to ml/d, but even with an increasing dose of rfvlla up to i~g/kg bwq h (fvil activity max. % min after injection) and additional hormonal support with a lh-fsh-anatgonist some hemorrhage remained. a short .course of methergin was stopped due to severe pain. ultrasound of the uterus revealed a hypertrophic endometrium causing the persistent bleeding. it decreased slowly over several weeks and hemorrhage stopped completely after d. the total rfvlla dose administered was rag. no side effects were observed. no transfusions of blood products were necessary. currently, menstrual cycle is suppressed by estriosuccinate. conclusion: due to close cooperation with a specialised gynecologist, hypermenorrhagia was controlled and in this woman with severe fvll deficiency hysterectomy was avoided. in three male members aged between and years of a family suffering from inherited bleeding disorders the diagnosis of protein z deficiency was established. plasma protein z evaluated by elisa (asserachrom protein z, diagnostica stago, france) ranged between and ng/ml. the patients mostly suffered from moderate bleeding complications like prolonged bleeding secondary to trauma or invasive measures and also spontaneous hematuria. previous laboratory investigations revealed variable platelet function deficiencies and transitory boderline decrease of von-willebrand factor. spontaneous bleedings were rarely recognized, however, they occured more frequently when analgetics were taken. bleeding complications showed good response to hemostyptic measures and antifibrinolytic therapy. the use of pcc containing a high level of protein z in these patients is restrained to severe bleeding disorders or major surgery. defibrotide is a mammalian polydeoxyribonucleotide derived anti-ischemic and antithrombotic drug (crinos s.p.a., v"flla guardia, italy). while the drug is known to produce polytherapeutic effects owing to its multicomponent nature, the exact mechanisms of its anti-ischemic effects remain unknown at this time. since defibrotide is found to be effective in ischemic disorders such as paod, vod related occlusive disorders and related rnicroangiopathic conditions, we studied the effect of this drug on the contraction of dog and pig arterial strip/rings obtained from various sites. in vitro supplementation ofdefibrotide to the organ bath containing control dog and pig arterial rings did not modulate the serotonin and thromboxane (generated) contraction, however, tissues obtained from dogs treated with mg/kg defibrotide iv exhibited a profound desensitization to the agonist induced contractile process. the time course of these effects was found to be much larger than the plasma half-life of defibrotide. this presentation will provide additional data on the effect of defibrotide on the contraction of vascular smooth muscles as a possible explanation for the anti-ischemic effects of defibrotide. a. wehmeier, a. popescu, w. schneider klinik for h,~matologie, onkologie und klinische immunologie der heinrich-heine-universit&t d sseldorf in chronic myeloid leukemia (cml), evolution of blast crisis is the limiting factor of survival. however, as in other chronic myeloproliferative disorders, bleeding and thrombotic complications are a major source of morbidity but their incidence has rarely been analysed in larger patient groups. we retrospectively evaluated patients with cml during chronic phase ( cases), accelerated disease ( cases), and blast cdsis ( cases), and determined the incidence of thrombohemorrhagic complications in relation to the stage of the disease. in chronic phase, patients had bleeding complications ( . %/patient year) and patients thrombotic episodes ( %/patient year). the incidence of bleeding increased significantly in accelerated disease ( patients, . %/patient year) and blast crisis ( patients, %/patient year), and many patients had repeated complications. contrary to our expectations, the incidence of thrombotic complications also increased to . %/patient year in accelerated phase and . % /patient year in blast crisis, tn chronic phase, patients died because of bleeding events. in accelerated phase, patients died due to bleeding and patient due to thrombotic complications. in blast crisis, bleeding was associated with deaths, and pulmonary embolism with deaths. analysis of the cause of thrombohemorrhagic complications revealed that in chronic phase, bleeding was often associated with uncontrolled busutfan therapy, whereas in blast crisis, severe bleeding occurred mainly when platelet counts were low and peripheral blasts increased. however, there was no obvious explanation for thrombotic complications. we conclude that bleeding and thrombotic complications are a major source of morbidity and mortality also in cml, and that the incidence of such complications increase in advanced stages of the disease. klinik for innere medizin °, klinikum schwerin patients suffering from primary or secondary amyloidosis may occasionally acquire a coagulation disorder characterised by isolated factor x deficiency. we report on a -years-old man who presented with lower gastrointestinal bleeding and prolonged prothrombin time (quick %). amyloidosis was suspected and proven using biopsy of the rectum and histological analysis. in addition, a monoclonal gammopathy of undetermined significance was diagnosed by immunofixation (light chain, type x). detailed investigation of the prolonged prothrombin time led to the discovery of a pronounced factor x deficiency (residual activity %). inhibitors of coagulation factors could not be demonstrated. the treatment of the patient consisted of red blood cell transfusion and infusion of prothrombin complex concentrates. due to the extremely rapid clearance of infused factor x, no increase of its activity was observed. chemotherapy of the monoclonal gammopathy was initiated (melphalan/ prednisone). over the following six months the frequency of major bleeding episodes gradually decreased. however, subclinical occult bleeding continued. the factor x activity was repeatedly found between and %. we support the suggestion from literature data that clinically relevant bleeding episodes are likely to occur in patients with amyloidosis-associated factor x deficiency if the residual activity is below %. sepsis and septic shock is a disease entity which is characterized by inflammatory reactions (sirs), coagulation abnormalities (dic), organ failure (mof) and severe hemodynamic alteration frequently leading to death in a shock. the aim of our studies was to investigate the efficacy of antithrombin iii (kybernin ®) on ~he outcome of septic shock in a pig endotoxemic model. pigs, in this model respond to lps with elevated tnflevels, decreased leukocytes and platelets counts, increased tat and fibrin monomer levels, hypotension and in increase of the pulmonary arterial pressure (pap), indicating impaired lung function. a total number of male castrated juvenile domestic pigs ( - kg) were anaesthetized, ventilated mechanically and infused with saimonella abortus equi lipopolysaccharide (s. equ-lps) over three hours ( . ~g/kg * h). a swan-ganz-catheter was inserted into the pulmonary artery to measure the pap. animals were allocated to two groups,, the treatment group (n = ) received antithrombin iii (at iii) according to the following regimen: u/kg (t = - , i. v. infusion), u/kg (i. v. bolus, t = ) and u/kg (t = - rain, i. v. infusion). the placebo group ( n = ) received the appropriate amount of human serum albumin: - - mg/kg (same schedule as with at iii). main objective was defined as the mortality rate at six hours a_~er s. equ-lps infusion. whereas in the placebo group out of animal died (mortality rate: %) all at iii-treated pigs survived the observation period of hours (p < . , x -test). the at iii group was shown to have a lower pap than the control group, especially the second peak of hypertension was abolished by at iii. it is therefore concluded that at iii should be a useful tool for the treatment of severe sepsis and septic shock. in a nationwide monthly survey all childrens hospitals in germany (esped) were asked to clinical and therapeutical informations about children suffering from pmi. during july till june children were registered. from these, had either ecchymoses and/or necroses related to an increased mordibity and mortality ( %), whereas showed no bleeding signs except for petechiae. of these children one died. the therapeutic interventions concerning hemostasis are listed according to the defined two risk ~oups. from the patients with ecchymoses or necroses, / received combination therapy (compared to / with petechiae or no bleeding sign) of at iii, heparin and/or plasma. only t child received protein c concentrate. the data show that children with low risk did in part receive higher doses of heparin and/or at iii concentrate than did high risk patients, whereas plasma therapy was adjusted to severity of eoagnlopathy. furthermore, the wide range of given therapeutics allows no information about the different medications. therefore, controlled studies with respect to the different therapeutic interventions in children with high risk pmi is desirable. a fully automated procedure for the reptilase time assay y. schmitt ( ) and h.j. kolde ( ) ( ) institute for laboratory medicine, st~dtisches klinikum, darmstadt, frg, ( ) dade diagnostics, unterschlei heim the reptilase time assay is a relatively simple technique for the detection of fibrinogen degradation products and fibrinogen deficiency or abnormality. the procedure is performed with citrated plasma and batroxobin reagent, a snake venom enzyme from bothrops atrox. this enzyme cleaves fibrinogen by releasing fibdno peptide a only but not fibdno peptide b. in contrast to the physiological enzyme thrombin that is readily neutralized by antithrombin iii and hepadn batroxobin is not inactivated by physiological inhibitors. at present this assay is mainly performed manually or on mechanical instruments. we have adapted this assay to the electra fully automated coagulation analyzer (medical laboratory automation, pleasantville, n.y.) using the thrombin clotting time procedure in the instrument software with batroxobin reagent (dade dia- the clot formation is registrated turbidimetrically and the dotting time is pdnted. the within run precision (n= ) of this procedure was tested with two plasmas from the daily routine and was between . and . %. in normal samples we found clotting times from . to . sec. in samples with liver disease (confirmed by pseudochlinesterase < u/ml) or on thrombolysis therapy with streptokinase or urokinase the fully automated assay on the electra was compared to the semiautomatic method using a kc coagulometer (amelung, lemgo, germany) based on a rolling metal ball pdnciple and magnetic endpoint detection. the two assays agreed very well with a correlation coefficient of r = , and a regression line according to passing and bablok of y = . x + . . these data show that the reptilase time can be performed with good precision and with good correlation to the manual technique on mechanical instruments on the electra . introduction: disseminated intravasal coagulation (dic), due to a massive activation of the coagulation system, is frequently observed in intensive care patients suffering from severe underlying diseases. laboratory diagnosis of dic is based on different coagulation tests, but unfortunately the routine haemostaseological parameters react with latency in the course of acute dic objective: in four cases from a cohort of patients with severe sepsis and dic we analysed special haemostaseological parameters (tat, f -t , d-dimers, human-leucocyte-elastese (file), catepsin g and heparin cofactor ii (hc ii)) and correlated them with a mof-score in order to test their predictability on the prognosis of these patients. results: all patients were substituted with at iii concentrate. l, the investigated patients median time of treatment with at iii concentrate was ( - ) days and median time of dic-duration was ( - ) days. none of the presented patients died during observation period. all analysed parameters, except d-dimers, showed a sufficient correlation with the evaluated mof-score (tat: r= , ; f -f : r= , ; hle: r= , ; catepsin g: r=- , ; hc ii: "=- , ). the d-dimers did not correlate with the mof-score, which is probably due to the delayed reactive hyperfibrinolysis in the course of dic. furthermore, the decrease of the tat-complexes, f -f , hle and catepsin g levels were followed by an increase of at hi and hc ii activity. conclusion: in general the analysed activation markers and coagulation parameters are sufficiently to describe the ongoing process of the dic. the hyperfibrinolytic activity of dic is sufficiently represented by the d-dimer test, but is of defered reactivity in the course of dic. unfortunately these parameters are not established in the routine monitoring of dic on intensive care units and therefore further studies are needed to investigate the practicability and reliability in the daily routine monitoring. we have previously reported that notoginsenoside r (ng-r ) has an effect on counteracting lipopolysaccharide (lps) induced upregulation of plasminogen activator inhibitor- and tissue factor expression in cultured human umbilical vein endothelial ceils in vitro and in mice in vivo [fibrinolysis ; :(suppl ) ]. in this study we investigated the effect of ng-r on prevention of lps induced lethal toxicity in mice. because mice are relatively resistant to lps when applied as a single agent, we sensitized them by simultaneous treatment with d-galactosamlne. the % lethality induced by lps ( . mg/mouse) plus d-galactosamine ( mg/mouse) in c hs-ie mice was reduced to % by simultaneous administration of ng-r ( . mg/mouse) with lps/galactosamine (p< . by x test). ng-r also significantly delayed lps/galactosamine induced lethal toxicity from hours to hours with all animals surviving beyond hours. because lethality induced by lps involves the synergistic effect of multiple effector molecules such as tumor necrosis factor (tnf)-ct, interleukin (il)-i, interferon ' etc., we also investigated the effect of ng-r on lps induced tnf-ct production from leukocytes in cultured human whole blood cells (hwbcs) ex vivo. the production of tnf--ct induced by lps ( ng/ml for hours) in the supernatant of hwbcs was inhibited by % and % respectively, when the cells were incubated ng/ml or ng/ml lps together with i~g/ml ng-r , respectively (tnf-ct concentration, ng/ml lps treated cells: + pg/ml, i ng/ml lps plus l.tg/ml ng-ri treated cells: + pg/ml, p< . ; ng/ml lps treated cells: _+ pg/ml, ng/ml lps plus pg/ml ng-r treated cells + pg/ml, /'=- . ). the present results suggest that ng-r can prevent the onset of lps toxicity as well as the lps induction of cytokines. therefor ng-ri may be effective in preventing the effects of septic shock in gram-negative infections. to elucidate the mechanisms by which coagulation is initiated in septic patients in vivo, coagulation measurements were prospectively evaluated in patients with severe chemotherapyinduced neutropenia. this group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing prior to and during evolving sepsis or septic shock. patients with febrile infectious events were accrued to the study. of these, patients progressed to severe sepsis and an additional patients to septic shock. at onset of fever, factor (f) vlla activity, f vii antigen and antithrombin iii (at iii) activity decreased from normal baseline revels and were significantly lower in the group of patients who progressed to septic shock compared to those that developed severe sepsis (medians: . versus . ng/ml, versus u/dl and versus %; p < . ). the decrease of these variables in septic shock was accompanied by an increase in a marker of thrombin generation like prothrombin fragment + (medians: . versus . rim; p=o. ). these differences were sustained throughout the septic episode (p < . ). f vlla and at ill levels of < . ng/ml and < %, respectively, at onset of fever predicted a lethal outcome with a sensitivity of and %, and a specificity of and %, respectively. in contrast, fxila-alpha antigen levels were not different between both groups at onset of fever and were only marginally higher further during the course of septic shock (p=o. ). thus, septic shock in neutropenia is associated with significant coagulation activation, presumably driven by the tissue factor pathway rather than the contact system. furthermore, in septicemia both f vlla and at iii measurements are sensitive markers of an unfavourable prognosis. hemostatic parameters in sepsis patients treated with anti-tnfct monoclonal antibodies c. salat , p. boekstegers , e. holler , , b. reinhardt i, r. pihusch , k. werdan , m. kaul , t. beinert , e. hiller med. klinik iii und i , klinikum grosshadern der ludwig-maximilians-universitat monchen, h~imatologikum der gsf , knoll ag ludwigshafen tumor necrosis factor et (tnfc~) is a central mediator in the pathogenesis of sepsis and septic shock. as administration of anti-tnfct monoclonal antibodies was able to protect animals from an otherwise lethal endotoxin challenge clinical studies were initiated in patients with sepis. tnfct exerts a procoagulant effect, e.g. by enhancing pai-i and activating thrombin as indicated by an increase in tat and pf / levels. therefore it may be involved in disseminated intravascular coagulation in sepsis. we determined tat, pf / , d-dimers, tpa, upa, pai-i and vwf levels in patients with sepsis or septic shock. patients received the anti-tnfa monoclonal antibody mak f (knoll ag, ludwigshafen), whereas patients served as controls. we found a significantly lower level ofupa in anti-tnfc~ treated patients. since the difference existed before onset of treatment it can not be attributed to tnfot antagonisation. all other parameters investigated did not differ significantly between the two groups throughout the study period. failure to detect modulation of hemostasis by anti-tnf~ might be explained by delayed initiation of treatment in clinical sepsis. in animal experiments it has been observed that the antibody prevented lethal endotoxin effects when given prophylactically or minutes after endotoxin challenge, but not when it was administered . hours later. in addition, beneficial clinical and hemostatic effects of tnfet antagonisation might be observed only in subgroups of patients with hyperinflammatory sepsis. larger studies addressing this point are under way. protease receptors for thrombin and trypsin have been described for different cell lines. we investigated the ability of trypsin to activate human umbilical vein endothelial cells (huvec). cell activation was measured by the increase of intracellular free ca * (caff) with help of microscope fiuorometry (fura- ) and by the von willebrand factor release measured by a sandwich elisa. incubation of huvec with thrombin ( u/ml) or trypsin ( nm) showed a - fold increase of c~ff. a subsequent homologous stimulation after s lead to a - fold lower concentration of ca~ ÷ compared to the first stimulation. therefore cells have been desensitised by the first stimulation. inhibition of the proteolytical activity of trypsin by soybean trypsin inhibitor was followed by failure of trypsin inducing an increase of ca~ ÷ concentration. in cross stimulation experiments with thrombin and trypsin, we could demonstrate, that cells first stimulated with thrombin showed a second maximal response by subsequent stimulation with trypsin. the same effect was measured with first stimulus trypsin and second stimulus thrombin. trypsin and thrombin induced a release of von willebrand factor ( - fold in comparison to unstimulated cells). we found a vwf release dependent on the concentration of trypsin similar to thrombin. an electrophoretic analysis of the released von willebrand factor showed a different multimeric composition of vwf between trypsin and thrombin stimulation. these results indicate, that there might be a protease receptor on huvec for trypsin being different from the thrombin receptor. clinical and laboratory findings of coagulopathy were investigated by an -year-survey to children's hospitals. meningococcal infections were evaluable. severe disease (characterized by need for mechanical ventilation, dialysis and/or catecholamines) was seen in of these children; of those survived and died. clinical signs of severe coagulopathy were seen in children: ecchymoses (n = ) and skin necrosis (n = ) were associated with increased mortality ( % and %, resp., compared to . % overall mortality). five of surviving children with skin necroses required surgical interventions (skin transplantation and/or amputations). petechiae were frequent (n = ) and as isolated finding not related to severe disease or fatal outcome ( % mortaliy). platelet counts at admission were lower in non-survivors ( th- th percentile: - . /gl, median: . /i.tl) than in survivors ( th- th percentile: - . /i.tl, median: . /gl). at iii values showed no difference between survivors and non-survivors. protein c was available in few patients (n = ): in this subgroup, protein c was lowered in patients with limited disease ( th- th percentile: - %, median: %) as well as severe disease ( th- th percentile: - %, median: %). in conclusion, the findings "ecehymoses" and "skin necroses" were related to fatal outcome and therefore included in a prognostic score for severity of meningncoccal disease. the influence of irradiation on pai-i and vwf levels in human umbilical vein endothelial cell cultures k. fragiadaki, c. salat, r. pihusch, b. reinhardt, m penovici, e. hiller med. klinik iii, klinikum grosshadern der ludwig-maximilians-universitat monchen an elevation of pai- in bone marrow transplant recipients developing veno-occlusive disease (vod) of the liver has been described earlier. endothelial cell damage due to the preparative myeloablative radioehemotherapy is supposed to be an important step in the pathogenesis of the disease, which is characterized by an obstruction of small intrahepatic venules. in order to investigate a possible role of irradiation we studied the influence of several doses ( , , , gy) on pai- and vwf levels in the supematant of human umbilical vein endothelial cell cultures (huvec). pai- antigen and vwf were determined by enzyme immunoassays. whereas pai- and vwf levels remained unchanged alter irradiation with gy and in control cultures, a rise was observed one day after irradiation with gy (mean day "-)day + ) in pai- ( , % --) , %) and vwf ( %--) , %) levels. the increase was more pronounced and reached levels of statistical significance after a dose of cry (pai- %--) , % and vwf %--) %). both pai- and vwf levels decreased on day after irradiation with and gy. our results indicate that irradiation induces an increase of pal- and vwf in endothelial cells. nevertheless, this effect was observed only in doses above those ones used during conditioning when patients receive x gy. additional factors seem to be of significance. cytokines like tnfo~ enhance pai- and vwf in endothelial cell cultures and are known to be elevated in bmt-associated complications. it can be speculated that irradiation in concert with these factors may contribute to the development of veno-occlusive disease. disseminated intravascular coagulation is characterized by high consumption of coagulation factors, systemic elevation of fibrinolysis by tpa and concomitant elevation of pai-i secreted from inflamed endothelial cells. in an attempt to investigate the contribution of inflammatory cytokines, endothelial cells lines of microvascular origin were stimulated in vitro and pal- antigen was measured h, h and h after stimulation. in contrast to results published from experiments performed with macrovascular human umbilical vein cells (huve), our results obtained with different microvascular endothelia isolated from skin, solid tumor tissue and bone marrow revealed that inflammatory cytokines reduced pal- antigen levels. in addition to tnf-a ( ng/ml) and lps ( pg/ml), we found that il- ( u/ml) and gm-csf ( u/rot) also reduced pai-i levels within the first h of incubation (from ng/ml to - ng/mll and the effect was even more pronounced after h and h (from ng/ml to ng/ml). il- ( u/ml) and lps ( pg/l) also reduced constitutive levels of pal- but the effect occured later than h after addition of the stimulator. the strongest synergistic effect was demonstrated with gm-csf plus il- resulting in pal- suppression of % after h and % after h. in contrast, g-csf ( u/ml) induced the immediate ( to ng/ml after h and to ng/ml after h) upregulation of pal- antigen. stimulation of pat- levels was also observed with tgf-i~ ( pg/ml), however not earlier than h of incubation. interestingly, both stimulatory cytokines, ie. g-csf and tgf- , alone were able to counteract the decrease of pat- antigen by tnf-a but only a combination of g-csf plus tgf-g neutralized the effect by il- . results indicate that inflammatory cytokines regulate pal- fibrinolysis in a synergistic and antagonistic fashion. we established the culture of human brain microvascular endothelial cells (hbmec) in order to investigate the pathophysiology of hu~man cerebral malada, which is still associated with a high mortality rate. it is widely accepted that among the reasons for the fatal outcome of cerebral malaria, the interaction of endothelial cells with cytokines and paras lites with subsequent changes in haemostaseological parameters is involved. the human microvascular endothelium may therefore play a deci §ive role in the pathophysiology of cerebral malaria. ery throcytes containing later stages of p. falciparum specifically bind to capillary ec in vivo (sequestration). tnf-cq il- and il- are considerably elevated in severe malaria. coagulation factors such as tissue factor and von willebrand factor are affected by malada suggesting the involvement of the hbmec in cerebral malada. so far, research on the involvement of the hbmec has been performed on ec cultured from human umblilical veins (huvec). the relevance of this model may be questioned on t, ,he grounds that the capillary endothelium probably plays a greater role than the endothelium of the large vessels. besides, some propertie.$ of the endothelioum seem to vary, upon the organ of origi/n. for the~ reasons, our laboratory has established the hbmec as a model to study the pathophysiology of human cerebral malaria. to demonstrate the relevance of this model in the context of malaria, hbmec were challenged with sera from different patients with severe p. falciparum malaria and with serum from a healthy donor. we can demonstrate that in cells challenged with malaria patient sera icam- and substance p were upregulated. on the other hand cells challenged with serum from a healthy donor expressed neither icam- nor substance p. these results strongly suggest the relevance of this model for vessel involvement in malaria. both, histamine and serotonin have been described as potent stimulators of yon willebrand factor (vwf) release from human umbilical vein endothelial cells (huvec). we performed experiments to differentiate the receptors for histamine and serotonin induced vwf release. absolutely unexpected we don't found any significant vwf release after the addition of serotonin to huvec or human artery endothelial cells (huaec) in concentrations from . ijm to pm. in the case of histamine ( . pm - pm) we measured a vwf release - fold compared to unstimulated cells. this release was in the same order of magnitude as the release induced with u thrombin. to verify these results we measured the effect of histamine and serotonin on the intracellular ca ÷ concentration (ca~ ÷) in huvec and huaec. cells were labelled with fura- and the change in fluorescence after agonist addition was measured with a microscope fluorometer. using the same agonist concentrations as above we found an - fold increase of caj . with histamine or thrombin but no effect by addition of serotonin. this results indicate a similar activation of human endothelial cells by histamine and thrombin and that serotonin don't stimulate endothelial vwf release or increase of cay. activation and/or dysfunction of the endothelium can be triggered by cytokines (e.g. interleukin- , tumor necrosis factor-alpha) or bacterial substances (e.g. endotoxins) and may contribute to shock and multi organ failure. pal-l and tm were assessed as parameters of activated endothelium following bsct in three to four days intervals from start of conditioning therapy through day + . data were compared to the occurrence of sepsis, veno-occlusive disease (vod), capillary leakage syndrome (cls) and graftversus-host-disease (gvhd). patients with neither complication served as controls. no *days after stem cell tranplantation pai- and tm were increased in all patients with sepsis, cls~ vod and/or gvhd. pai- peaked at days to and the increase was highest in sepsis and lowest in cls. the increase in tm values was somewhat delayed (day + ) and was highest in vod and cls and lowest in gvhd. pai- and tm are sensitive markers of endothelial activation in sepsis, vod, cls, and/or gvhd, but they do not allow a differention between these complications. endothelin (et) is the most potent vasoconstrictor. it is known that et plasma concentration is correlated with a poor prognosis in patients with non ischemic cardiomyopathy (cm). the contribution of the heart to the production of et is still unknown. to investigate the pathogenetic mechanism in patients without coronary artery disease (cad), we examined patients with hypertension ( . pulmonary capillary wedge pressure (pcwp) was measured in all patients. et and its precursor big-endothelin (bet) were determined at rest and after pharmacological stimulation with dipyridamole ( . mg/kg body weight), that increases coronary blood flow by factor - on a non endothelial pathway. cardiac coronary et and bet concentrations were determined from the arterial blood samples, obtained from the aorta, and simultaneously from the coronary sinus (venous blood). blood samples were collected into ice chilled vacutainer tubes and stored after centrifugation at - *c. et and bet were analysed after extraction by a sepal< c cartridge by radio immuno assay technique (immundiagnostik). it is concluded that et is increased with elevated filling pressures of the heart in patients with cm. it is not produced in considerable quantity by the heart neither at rest nor at increased blood flow. there ore the lung has to be considered as the major organ for the production of et and bet in patients without cad. to characterize the incompatibility of blood with foreign surfaces valide in vitro methods especially in testing of platelet function are neceessary. it seems to be effective to use test systems which can also be helpful lateron in the clinic when foreign surfaces (e.g. venous catheters) are used and evaluated in so called phase- -studies. we studied the influence of reference polymers under standardized and controlled flow conditions on platelets in citrated blood specimen of healthy blood donors.the following tests were performed pre and post platelet-pol)aner contact: decrease of platelet count, platelet aggregation (wu-gmtemeyer index), analysis of platelet spreading capacity on standardized plastic surfaces by using a visual microscopic evaluation according to breddin and bfirck ( ) and an interactive computer-aided system (ibas, kontron gmbh, manchen, frg) by digitalizing the morphological picture of the platelet slides and area detection with a resolution of x pixels. results: platelet counts showed significant differences pre and post polymer contact, the wu-grotemeyer index demonstrated platelet activation only by blood contact with large volumes of polymeric material whereas both visual and computer-assisted evaluation of platelet spreading ability revealed a marked shift in the different classes of platelets: platelet activation results in a decrease of large structural elements and an increase of elements with spider threads. (pre contact (n= ): :~- large forms of platelets, ~- small forms and :l- spider forms; post contact (n= ): -+- large forms, a: small forms and ± platelets with spider threads). in some series there were significant differences between visual and computer-aided evaluation in the detection of small and spider forms. however, the relative increase of these nonspread spider forms could be stated with beth methods (wilcoxon test). we therefore conclude, that platelet morphometry with both methods is a sensitive and reliable ex vivo method to evaluate platelet interactions with artificial surfaces and can also be used lateron in phase- -studies in patients. however, the ibas-system requires further maprovement in hard-and so,ware to reduce the high expenditure of this method. despite for the most part standardised methods such as hypothermia, cardioplegia the perioperative myocardial infartion rate is still high at approx. %. in cardiovascular surgery it is well known that various cardioplegic solutions are employed for myocardial protection during the ischemic phase. in order to evaluate the possible influence of these solutions we selected two of the most commonly used cardioplegic solutions for investigation in a randomised double-blind study: htk (group ) and st. thomas (group ). after randomisation each group consisted of patients who had to undergo aortocoronary bypass surgery. aim of the investigation was to establish possible varying cellular changes during the reperfusion phase or in the early operative phase in order to be better able to apply reinforcing clinical measures. in the context of this study the classical enzyme-diagnostical methods ck,ck-mb and ldh as most useful, however not as convincing. still, we have in the meanwhile been able to show that the cardiac muscle troponin t proves a particularly sensitive parameter regards differentiated ischemic damage to the myocardium. ~his we were able to conflrm in extensive preliminary trials. cardiac troponin t was registered with a one-step lmmunoassay using two highly specific monoclonal antibodies directly via two different epitopes of cardiac troponin t. simultaneously the corresponding pre-and postoperative ecg was registered. further, within this context we investigated parameters that indicate cellular damage, such as platelet factor (pf ), t-pa, interleukin- and pmn-elastase. in the reperfusion phase in group there is a significant rise in tmponin t while in group these values remain practically unchanged up to the st. postoperative day. of special importance is interleucin since according to most recent studies the release of this substance leads to platelet activation via the arachidonic acid metabolism. this pathway must, further, be regarded within the context of free radical formation. on the st. postoperative day the values in group are significantly higher. the effects of membrane damage is also observed via pf and the pmn-elastase to be different in both groups. on the basis of this study we arrive at the conclusion that the htkcardioplegia is essentially less damaging than that of the st. thomas solution. ( ) r. hetzer ( ) ( ) department of hematology and oncology, vimhow klinikum, humboldt university, berlin, germany ( ) we investigated the influence of two different vad systems on these hemostatic changes. vads were implanted in patients [ bi-vad (berlin heart), left vad (novacor n )] with end-stage heart disease who were awaiting heart transplantation. the following hemostatic parameters were measured during the first days of bddging or until heart transplantation: thrombin-antithrembin iii (tat) complexes, prekallikrein, factor (f) xll, plasminogen, or -antiplasmin, and i?,thremboglobulin. results: during the first week of bridging, significantly higher tat levels were observed in novacor patients compared to berlin heart patients. prekallikrein activity levels were significantly lower in the berlin heart patients in the early bridging period. all other parameters were comparable in both groups throughout the entire observation period. differences in hemostatic parameters became apparent only in the early bridging period with more enhanced pmthrombin activation in the novacor group and more prominent contact activation in the berlin heart group. avoidance of the transmission of viral infections and saving in the use of blood products encouraged the use of apparatwe intraoperative autetransfusion techniques. patients and methods: arer randomization apparative intraoperative autotransfusion was performed in x patients during elective hip surgery using i-iaemonetics cell saver ill, haemonetics cell saver v, electromedics elmd, haemolite and fresenius continuous autotransfnsion system (cats). at defined tmaes we detenmned a lab panel (clinical chemistry, lipids, proteolytic capacity, hemolysis, coagulation panel) at determination points in the reservoir, the retransfused blood and in the patient. results: no significant differences concerning proteolytic capacity, prothrombin time, platelets, lipids, electrolytes. increased hemolysis (p< . ) in the hcs iii group vs. the other groups (lo rain. after application of the retransfnsed blood). low heparin concentrations of retransfused blood in the hcs iii group( . +- . u/ml) vs. high concentrations in the cats group ( . +- . ;p-- . ). parameters of thrombin generation were elevated in the hcs iii group vs. the other groups (p= . ). conclusions: the use of different apparative autotransfnsion systems dunng elective hip surgery results in dysturbances of hemocompatibility. the activation of the coagulation system during the collection and filtering is partly influenced by the elimination kinetics and the dose regime of heparin. however intraoperative autotransfusion must be roan~ged very carefully and possibly adverse effects of perioperadve heparin peak levels have to be considered. little information is available on the management of patients with factor viii deficiency who require cardiac surgery. we report the case of a year old man with factor viii deficiency and combined severe aortic stenosis and incompetence and mitral incompetence who underwent a double valve replacement at our institution. he had a history of several bleeding episodes following minor surgery. previous factor viii levels were between and %. using standard cardiopulmonary bypass, a double valve replacement with a and mm bileaflet prosthesis in aortic and mitral position, respectively, was performed. a high dose aprotinin regime was used ( . x a iu). three doses of factor viii concentrate were given in the perioperative period, totalung u until the st postoperative day. repeated measurements of the factor viii level were performed. the postoperative chest tube drainage was rot. until the th postoperative day an additional dose of iu of factor viii was given to maintain a level of at least %. the obligatory anticoagulation was achieved initially with heparin i.v. in therapeutic dosage. due to a persistent rd degree av block a permanent pacemaker was inserted with additional iu of factor viii. on the th postoperative day warfarin was commenced aiming for an inr of . - . . the patient was discharged home therearer. he was trained to monitor his inr with a coagu chek device. no bleeding episode occurred during the first months follow up. open heart surgery can be performed safely in patients with factor viii deficiency with the use of factor viii concentrates and monitoring of factor viii levels. coating of biomaterials was developed using synthetic polymers with incorporated anticoagulants. stents were coated with a thin layer consisting of a polylactide polymer containing peg-hirudin and a stable prostacyclin analogue. these materials were tested with a ,,human shunt model" using nonant/coagulated blood of healthy volunteers. within minutes uncoated stents were covered by fibrin and aggregated platelets, which could be seen macroscopically and by scanning electron microscopy; coated stents were free from coaguiation plugs. this observations were supported by analysis of coagulatiuon activation markers. unlike coated stents, uncoated stents revealed high levels (>detection limit) of tat complexes and prothrombin fragments (f - ). in a series of experiments stents were tested in sheep. in sheep stents (coated/uncoated patmaz-schatz stents) were ptaced by conventional techniques in the left anterior descending artery. anticoagulant therapy consisted of a heparin bolus and intravenously given aspirin before stent implantation. no ant/coagulation was given thereafter. existing data show hyperplasia in the area of uncoated stents which was reduced around coated stents (this study will be finished in january ). this coating technique with incorporated anticoagulants reduces thrombogenicity during the early and late phase of biomaterial implantation. studies concerning catheters, vascular prosthesis and oxygenators are in progress. the mechanical circulatory support (mcs) is a therapy for patients (pts) with endstage cardiac insufficiency. during mcs thrombeembolic events, due to the surface thrombogenicity of the implanted device, are feared complications. activated blood platelcts play a major role in this context. therefore, patient's platelet morphology was investigated. during the period of mcs, using the novacor left ventricular assist system n , blood samples of pts were observed by means of scanning electron microscopy (sem). blood was collected preoperatively and after implantation daily during the first week as well as weekly for the first months. samples were drawn via an gauge cannula into caeodylic-acid buffered glutaraldehyde and platelets were prepared for morphological investigations. platelet alterations were classified as non activated, activated and aggregated, based on "shape change" morphology. additionally, the common blood coagulation parameters were evaluated. preoperatively, . + . % of activated platelets were found. within the first postoperative week, the mean level of activated platelets raised to . + . % (p< . ). comparing short-(< days) vs. long-term (> days) mcs, a significant difference of activated ptatelets (overall mean values) could be seen ( . +_ . % vs. . _+ . %, p= . ). during mcs a correlation between hemolysis and platelet aggregates, as well as the values of activated dotting time and activated platelets were observed. also, specific platelet deformations and damages appeared during mcs, which could not be found preoperatively. all pts with mcs showed alterations of their platelet morphology induced by the activation of the implanted synthetic material. with regard to the postoperative antithrombotic therapy, these observations should be taken into consideration. during extracorporeal circulation (ecc) the blood and its compenents are exposed to artificial surfaces and inflammatory respenses are activated, especially the complement, coagulation, fibrinolytic and kallikrein systems. furthermore leukocyte activation occurs and platelet function is impaired. these humoral and cellular systemic responses are known as the "pustperfusion syndrome" with clinical symptomes like lenkocytosis, increased capillary perraeability, accumulation of interstitial fluid and organ dysfunction. the impertance and even perhaps the existence of the damaging effects of cpb have been widely debated in the literature over the past years. many efforts have been made to reduce traumatizing factors, e.g. the use of membrane instead of bubble oxygenators. recently, heparin-coated equipmen~ and tubings have been proposed to avoid excessive contact activation during cpb, the here presented study was designed to assess changes in coagulation and flbrinolytie activity in patients undergoing cpb. in this regard we investigated coagulation parameters like fibrinogen, antithrombin, pmthrombin-fragments fl+ , thrombin-anthhmmhin complex, tissue-factor, fibrin-monomeres and parameters of the fibrinolytic system like tissue-plasminogen-activator, plasminantiplasmin-complex, d-dimers and plasminogen-activator inhibitor before, during and after cpb. the activation of the complement cascade was followed by measuring the concentration of c a, c and c c. the results demonstrate distinct alterations in above mentioned parameters. in spite of a high dose hepariulzation (act> s) combined with an antifibrinolytic tw, atment an activation of the coagulation system was observed immediately after the onset of cpb followed by an activation of the fibrinolytic system. therefore further efforts should be done to develop new anticoagulatory regiments and improve the biocompatibility of materials used for cpb. during cardiopulmonary bypass blood is exposed to nonphysiologic conditions. the contact with artificial surfaces and mechanical stress result in a periopemtive response which includes activation of the complement, coagulation, fibrinolytic and kallikrein system, activation of nentrophils with degranulation and pmtease enzyme release, oxygen radical production and the synthesis of various proinflammatory cytokines. this so-called "pest-pump intlammatory response" has been linked to respiratory distress syndrome, renal failure and neurologic injmy. our goal was to investigate the time course of eytokine levels and the activation of leukozytes and platelets and to quantitate leucocyte subpepulatioas in patients undergoing cpb. at different time points, pre, during and pest cpb, we determined the levels of interleukin (il) , il- , il- , il- , il- , il- , tumor necrosis factor ¢z (tnf-a) and interferon " ' (ifn'--/) using elisa-techulques. lymphozyte subpepulations were characterized by flow cytometry and specific monoclonal antibodies against cd (pan t-cell marker), cd (surface antigen on t-helper cells), cd (surface antigen on b-cells), monocytes were determined by cd and platelets by cd (act. gpilb/llla) and cd b (gp ib). single cell activation was analyzed using markers against cd (il- receptor), cd (il- receptor), hla-dr (mhc class ii), cd (transferrin receptor) and cd (activation inducer molecule), platelet activation was monitored with an antibody against cd (gmp- ). preliminary results revealed distinct increases in r,- , il- , and il-io following cpb whereas tnf-a and ifn--/levels were not significantly influenced. fttnhermore, activation of particular cell populations was observed. finally, our investigations should contribute to a better understanding of the complex humeral and cellular respenses induced by cpb and thus might help to develop new strategies to circumvent the negative impacts of cpb. optimal adjustment of anticoagulation in machine plasmapheresis is important for the quality of the prepared fresh frozen plasma (ffp) as well as for the safety of the donation. in the present study the suitability of prothrombin fragment ( ft+ ) in the assessment of anticoagulation during plasmapheresis was investigated. matarlal and methods: plasmapheresis procedures were performed on donors ( ~, o" ) using different plasmapheresis machines (a , baxter; mcs p, haemonetics; pph , electromedics/medtronic). acid citrate dextrose formula a (acd-a) in a ratio to whole blood of : was used for anticoagulation. the concentration of fi+ in the donor's blood was measured before and after plasmapheresis and in the prepared ffp. the actual acd-a volume used was also registered. results: there was a significant rise of the ft+ -concentration in the donors blood after plasmapheresis with each of the three automatons: a : . vs . , p < . ; mcs p: . vs . , p < . ; pph : . vs . , p < . . the ffp prepared with each machine showed the following f~+ concentrations: . ± . , . : ± . and . ± . respectively. the difference between the groups was not significant. the elevation of the ft+ -concentration in the donor's blood showed a negative correlation with the volume of the acd-a used. during of the procedures technical problems occurred (inadequate venous acces, occlusion of the citrate tube, reduced whole blood flow). after these procedures there was a marked elevation of f~+ in the donors blood ( . ± . ), accompanied by an elevated f~+ -concentration in the prepared ffp's. conclusion: these data show that ft+ is a suitable parameter for the assessment of anticoagulation during plasmapheresis. several epidemiologic studies demonstrated that fibrinogen is an independent cardiovascular risk factor and should be considered for screening programs. prothrombin time derived fibrinogen (df) measurement combines the advantage of an established highly reproducible automated method with no additional reagents, except for calibration. several studies showed that the df values correspond well with the clanss method except in cases such as thrombolytic therapy in which the df results are higher. however, no results exist whether in patients with coronary heart disease with fibrinogen as a risk factor the df values are also comparable to the established clausss method. the aim of our study was to compare df values to clauss method results in cardiac patients, especially in patients before and after coronary bypass grafting (cab(]). measurements of df were performed on an acl (il) using the pt-fibrinogen-hs reagent. fibrinogen clanss method was done on the acl using fibrinogen c reagent (il) and on a kc (amelung) with fibrinogen a reagent (boehringer maanheim). for calibration we used the calibration plasma half volume (it.) with the fihrinogen concentration proposed by the manufacturer. plasma samples were obtained from patients at admission before cabg and postoperatively up to week, and from healthy persons (staff). within assay imprecisious using normal and abnormal controls (il) were comparable with both methods showing cvs between . and . %. in normal healthy persons the medians of the df and the clanss method run on the acl were very similar ( vs rag/all), whereas kc values were about % lower ( md/dl). in cabg patients at admission we found the same differences as in normals with the clanss method (acl: vs kc : rag/all), however the df values were siginficantly higher (median mg/dl). if we took a cutoff value of mg/dl, as suggested by the results from the northwick park heart study, we would categorize into the high risk group out of patients using the df method, with the clanss-acl method and with the clanss-kc method, i.e. nearly % more patients were classified in the high risk group using the df method. postoperative samples showed the expected increases due to the acute phase response with the same magnitude of differences. because of its rapidity and reproducibility the df method is well suited for routine measurements, however, standardization remains an urgent task in order to avoid misinterpretation of results. for fibdnogen measurements in clinical laboratories, the two most widely used methods are the clotting time method according to clauss (cfib) and the sn called "derived" fibrinogen method (dfib) implemented in optical coagulometera with the fibrinogen concentration being derived flora the optical density of the fibrin clot in a standard prothrnmbin time (pt) assay. it is well known that under certain circumstances, e.g. in the presence of fibrin(ogen) degradation products (fdp), there is a discrepancy between the two methods with higher values for dfib than for cfib. yet the opposite discrepancy, i.e. fibrinogen values derived from the optical density of the clot grossly lower than values from dotting time assays, seems to be very rare and is poorly understood so far. the patient (male, years) had ingested the esterase inhibitor parathion (e ) in an attempt o f suizide and was treated with high doses of atmpin. he had no clinical signs or history or family history of bleeding or thrombotic disorders. except for a very low pseudocholinesterase activity, all laboratory results were normal ineinding pt, afft, thrombin time, and factor xiii. pt and aptt did nnt differ between an optical coagulometer (electra c, mla) and a mechanical one (kc.. , amelung). there was no evidence of disorders known to interfere with hemostasis like paraproteinemia or dyslipldemia. however, in all blood samples received for dotting tests during a period of days the macroscopic appearance of the fibrin clot was quite unusual (only slightly turbid/almost transparent) and there was a striking discrepancy between a very low or low dfib on the electra (pt reagent: thromboplastin is, dade) and a normal or high cfib (kc ; thrombin reagent, dade). on admission, values were mgml (derived) vs. mgldl (clauss). cfib rose to s mg]dl with dfib at mg/dl in the last sample on day . ~ al! samples dfib was about % (ls- ) of cf[b. when the patient's plasma was added m normal pooled plasma it caused, in a dose-dependent manner, values lower than predicted for dfib and values slightly higher than predicted for cfib. in the absence of data from additional (e.g. immunologic) methods the following principal possibilities (and combinations) have to be considered: ) normal fibrinogen concentration and clot formation rate, but abnormal optical properties of the clot (cfib correct, dfib falsely tow); ) normal optical properties of the clot, but accelerated clot formation and very low fibrinogen concentration (dfib correct, cfib falsely high). in either case, the molecular basis could be: a) a genetic or acquired molecular abnnrmality of fibrin/fibfinogen; b) an interfering substance. direct effects of the loxic agent parathion and/or the antidot drug atropin are not likely to be the cause since other patients, often with more severe parathion inmxicatian requiring higher doses of atmpin, showed normal optical density of the clot. we hope to perform a more in depth investigation of this abnormality in the future, including various methods, reagents, and instruments for fibrinogen measurement, a survey of the patient "s family, and studies of the molecular nature of the phenomenon. increased fibrinogen is known to be an independent predictor of subseqtmnt acut~ coronary syndromes. however. a multitude of methods for fibrinogen determination is available. there is a lack of standardisation among fibrinogen assays. in a family cohort study (patients'with combined hyperlipidaemia and f or hypemricaemia) fibrinogen was determined in plasma samples from family members using a functional and an immunochemical assay. the fimctional assay according to clauss was performed on the analyser ca using the test fibrinogen a from boehringer. the immmmephelometric assay was performed on ~e behring nephelometer system using the reagent and standard from behring. a good similarity between both assays was obtained at low and high flbrinogen levels as well as in samples with increased c-reactive protein (crp). values obtained by both assays correlated similar with total cholesterol, ldl--cbelesterol and apolipeprntein b. the ratio functional fibrinagen / immlmochemial fibrinogen showed no dependence on cholesterol, t-pa, v wiuebrand factor and crp. release of two fibrinopeptides a from fibrinogen generates desaa-fibrin monomer, which rapidly aggregates, forming fibrin complexes. fibrin monomers can be detected in plasma samples after chemical desaggregation of fibrin complexes using thiocyanate by monoclonal antibody binding to the alpha-chain neo-n-termini generated by fibrinopeptide release. although postulated, an intermediate of fibrin formation, carrying one fibrinopeptide a and one fibrin alpha-chain neo-n-terminus has so far escaped analytical procedures. we have employed a monoctonal antibody specific for fibrin alpha-chain neo-n-terminus, mab b , attached to magnetic microparticles, for isolation of fibrin-related material from plasma samples of patients with elevated soluble fibrin. the material was desorbed by sds-urea buffer and subjected to sds-page and immunoblotting. immunostaining with panspecific anti-fibrinogen and anti-fdp-e antisera showed a range of bands corresponding to fibrin monomers, and fibrin derivatives containing the fibrin e-domain. lmmunostaining with monoclonal anti-fibrinopeptide a antibody resulted in a doublet band corresponding in size to fibrin monomer. similar results were obtained with polyclonal antisera against fibrinopeptide a. for a more quantitative approach, desa-fibrin monomer was detected by an elisa procedure using mab b as capture and monoclonal anti-fibrinopeptide a antibody as tag. a sample with extremely high level of desaa-fibrin monomer, determined by elisa (enzymun®-test fm) was used for calibration, since reference material is not available. a correlation of r=o.g was found between desaa-fibrin monomer and relative desa-fibrin monomer levels. detection of desa-fibrin monomer required sample pretreatment with thiocyanate for desaggregadon of fibrin complexes. from these preliminary data it appears that desa-fibrin monomer accounts for a fairly constant proportion of soluble fibrin and is a polymerizing species. fibrinogen has been shown to be a major cardiovascular risk factor. especially for epidemiological studies, exact quantitation of fibrinogen in clinical plasma samples is of great imporance. fibrinogen levels are generally measured by clotting assay according to clauss, or by determination of derived fibrinogen values upon photometric measurement of prothrombin time (derfbg). the clotting assay has been shown to be influenced by high levels of soluble fibrin derivatives. the pt-derived fibrinogen levels appear rather convenient in clinical routine, since no additional reagents are needed. we have compared the clauss assay and derfbg with a turbidimetric fibrinogen assay using snake venom protease for fibrinopeptide release, performed in photometric autoanalyzers. d-direct antigen was measured in parallel using tinaqaant d-dimer lpia. results were correlated with total fibrinopeptide a release by thrombin, measured by elisa. a total of samples were included, of which samples ( %) were recorded as above measurung range by derfbg. these samples encompassed a range of . - . g/l and . - . g/l in clauss, and turbidimetric assay, respectively. the range of values measured by derfbg assay was . - . g/i, corresponding to . - . gll and . - . g/ in the clauss and turbidimetric assay, respectively. the correlation of derfbg with the clauss assay was re . , correlation with turbidimetric assay was r= . for the values actually detected. the correlation between clauss and turbidimetric assay was r= . for all values. there was no dependency of test results or inter-test variation upon d-direct. correlation graphs displayed a decreased test response of clauss assay in the high concentration range, resulting in an underestimation of fibrinogen concentration. the derfbg assay, in contrast, showed normal range values in samples from patients with fibrinotytic treatment and low fibrinogen levels in the other assays. correlation with fibrinopeptide a release was r= . for clauss assay, r= . for turbidimetric assay, and r= . for derfbg. for clinical routine, derfbg appears to be applicable for all samples between . and . g/l with exclusion of samples from patients with fibrinolytic treatment or endogeneous hyperfibrinolysis. other samples may be analyzed by clotting assay or turbidimetric assay, although the latter appears to be more suited for measurement of high range samples. for inhibition of pk is . pmol/l the antifibrinolytic activity of the inhibitors was determined by measuring the lysis of radiolabelled human plasma clots• the compounds which inhibit plasmin and pk influence remarkably the streptokinase-induced clot lysis but not lysis induced by uk and tpa. surprisingly, inhibitors of uk and tpa do not influence clot lysis induced by uk or tpa. the structure-activity relationships for inhibition of ptasmin, uk, tpa and pk could help in the design of more potent inhibitors of fibrinotytic enzymes. uk inhibitors are of interest for the development of anti-invasiveness drugs, while plasmin/pk inhibitors could be prototypes of a "synthetic aprotinin". in the ecat angina pectoris study t-pa antigen was an indepcndem risk factor of subsequent acute coronary syndromes. pat indicates the risk bat depends on other known risk factors. it should be tested in members of a family cohort study (patients with combined hyperlipidaemia and / or hyperuricaemia), if the active pal antigen or the whole pai antigen showed a stronger relation to t-pa and metabolic variables. the active pall antigen was determined using elisa actibind pat- (technoclone / lmmuno) , the whole pai-i antigen was measured using the f_lisa pat- (technoclone i immuno). t-pa activity was determined with the coaset t-pa from chromogenix, the tintelize tpa from biopool was the used test for determination of t-pa antigen. the active pat antigen showed a stronger correlation to t-pa activity and t-pa antigen than the whole pal antigen. circulating t-pa activity was influenced predominantly by the active pal antigen. both pat antigens were correlated in similar manner with metabolic variables, lipoproteins and b/vii. table: correlations of active and whole pal antigen (** p < , ) active pal antigen whole pal antigen active pat antigen , , ** whole pal antigen , ** , t-pa activity - , ** - , ** bpa antigen , ** , ** body mass index , ** , ** triglycerides , ** , ** total cholesterol , ** , ** ldl-cholesterol , ** , ** hdl-cholesterol - , ** - , ** apolipoprotein b , ** , ** apolipoprotein a i - , ** - , ** the lower relationship of the whole pat antigen to t-pa is obviously caused by patient samples with high levels of whole pat antigen in contrast to normal values of active pat as well'as of t-pa. possibly, a high ratio of whole pai antigen / active pat antigen is caused by a raise of latent pal the main form of pat in the platelets. the clinical importance of an increased ratio whole pal antigen / active pal antigen remains under investigation. the cyclic antibiotics-polypeptides bacifracin a, bacilliquin from boci/lu~, licheniformis and gramicidin s from bocil/us brevis, var. ( . b., were used for investigation. we studied their influence on the fibrinoly~c and coagulation activity in vitro• me~hods. to solution of human plasmin (thrombin). containing . mg of protein ( nih unff)/ml, the analyses' solution of antibiotics ( . - , mg) was added. then we defined the tlbrinolytlc activity of the mixes using azofibrin lysis, and fhrombin activity was determined according to the speed of fibrin clots formation from fibrinogen solution. results. in following table are submifled the results received in our laboratory {we also offer results of antibiotics influence on urokinase activity): ki, mm ki --the constant of inhibition. n. d. ~ in studied lirnils the inhibitor's activity was not observed. ---the inhibitor's activity was not define. i. --the inhibitor% activity was observed but ki not determined. +, +% +++ --effect of inhibffion (in rela*iive indexes). conc/us/on.~ the results received by us testify to the necessity of cautious approach to the use of antibiofics-polypeptides for various sorts of therapy in view of their possible influence on fibrinolytic and coagulation actlvlfy, of the organism. these results were used for preparation in our laboratory of biospeciflc sorbents containing c-ramicidin a, bacil}iquirt and gramicidin s.as ligands, they can reversibly bind thrombin, plasmin {plosminogen) and urokinase directly from crude exkacts. the enzymes are selectively eluted without substantial losses of specific activity in e yield of - %. there is a great body of rather contradictory informations dealing with fibrinolysis in liver.. cirrhosis, which can be accelerated, normal or reduced, depending on the type of cirrhosis and investigation techniques (clot-lysis, fibrinolytic component measurements). our previous finding was, that in vitro plasma-clot lysis, induced by exogeneously added tpa or streptokinase proved to be reduced, and this had a good correlation with severity of the disease and the elevation of plasmatic yon willebrand factor levels. in vitro clo~/-[ lysis tests, induced by tpa were performed in patients with alcoholic liver cirrhosis, utilising a microplate light-scattering assessment method. the tests were repeated using the same plasma samples in each patients with a microplate which was covered by cultured endothelial-cell monolayer (umbilical vein, huvec}. clot lysis speed proved to be . - times slower with huvec milieu in the control group, while in the cirrhotic patients this inhibition was stronger and resulted in -fold reduction of lysis speed. our results suggest, that cirrhotic plasma is able to accelerate the release of fibrinolytic inhibitors from cultured endothelial cells, which phenomenon may also contribute to the complex alterations of in vivo fibrinolysis in cirrhotic patients. deep vein thrombosis (dvt) is a systemic disease with prolonged clinical manifectation. anticoagulation therapy in dvt is not completely effective. thrombolytic therapy may give rise to a systemic lytic state, the fibrinospesific agents (scu-pa and t-pa) have short half-lives in the circulation. we investigated the potency of the acylated plasminogen streptokinase activator complex (gbpg-sk) to deep vein clot dissolution as compared to well known sk and apsac both in v~tro and in vivo in the model of venous thrombosis in artherio-venous shunt in rats. it was shown in in vitro study that fibrinolytic activity of plasminogen activators mainly depends on their stability in plasma. stability studies carried out by incubating sk and pg-sk activator complexes in plasma with euglobulin precipitation . total fibrinolytic activity was measured by the fibrin plate method. gbpg-sk possessed the greatest stability in human plasma than apsac or sk because of its prolonged inactivation period (the deacylation half-life for gbpg-sk was :e rain in contrast with -~ min for apsac). the stability degree of two acylated thrombolytics (gbpg-sk and apsac) was in order to inverse proportion of their first order rate deacylation constants ( . • - and . • -s sec- respectively). the fibrinolytic potency of sk, apsac and gbpg-sk was measured by -labeled fibrin clot lysis in plasma and in vivo by lysis of the preliminary formed -labeled fibrin clot inserted into the jugular vein. fibrinolytjc activity of acylated plasminogen activators gradually increased in time. under sk administration, the clot lysis came to the end by hours while apsac and gbpg-sk haven't lost their activity for - hours. gbpg-sk possessed significantly more prolonged fibrinolytic activity than apsac, the acyl-enzymes did not significantly influence on plasminogen,,.~ -anfiplasmin and fibrinogen levels in plasma according to their activity specific to fibrin-bound plasminogen. in opposite, sk produced a significant depletion of plasminogen, ~- antiplasmin and fibdnogen levels in plasma. it seems, on the basis on in vitro and an animal experimentation, than apsac with its moderately fast deacylation rate is more suitable for rapid thrombolytic effect, but gbpg-sk with its slow deacylation rate is suitable for deep vein thrombosis, when the rapid thrombolysis is less critical. it's well known that the complete lysis of thrombi usually isn't observed at the thrombolytic therapy. at present study we have attempted to quantify the possible mechanism of fibrinolysis inhibition during the thrombolysis. i-labelled partially cross-linked fibrin clots of different volume ( . - . ml) were immersed in tris-hcl buffer ( ml) containing plasmin ( - nm) at °c. the lysis rate was detected by counting of soluble fibrin degradation products (fdp). at all the eases lysis slowed down and stopped in hs though clots dissolved up to only - %. no irreversible inlaibition of plasmin caused by denaturation occur as was judged by the measurement of fibrinolytic activity at the diluted samples. however the increase of fdp concentration in surrounding buffer led to the reversible inhibition of fibrinolytic activity of plasmin up to % of baseline. the sds-page analysis under non-reduced conditions shown the acoumulation of high-molecular weight fdp at the surrounding buffer. the inhibition phenomenon could be connected with the specific binding of plasrnin with soluble fdp having exposed lysine residues and the subsequent removal of enzyme from fibrin surface. unexpectedly since the heterogeneous character of occurred reactions tile change of the clots surface area during lysis didn't affect the fibrinolysis kinetics in all the concentration intervals. to estimate the kinetic parameters the kinetic curves were linear in the coordinates [p /t (l/t*ln(isl°{(lslo.lpi)). the obtained parameters were following: keat=l. min-l,km=l. ixm,kp= . ~tm. the clinical trials have shown that fdp concentrations at the thrombolytic therapy of deep venous thrombosis and acute myocardial infarction usually was approximately in the range . - . ~tm. therefore the described phenomenon of fibrinolysis inhibition by formed fdp may take place during thrombolytic therapy. al. calatzis, an. calatzis, +m. klmg, +l. mielke, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology technische universit~.t monchen thrombelastography (teg) is an established method for the detection of fibrinolysis. fibfinolysis is usually determined when the teg amplitude decreases by more than % atter the maximum amplitude is reached. this takes a considerable amount of time (more than minutes). our approach bases on the understanding of fibrinolysis as a process which runs in paraue[ to coagulation and is not exclusively subsidiary to it. the effect of fibrinolysis on the growing clot in the teg is shown by the comparison of two parallely performed teg measurements: exteg: teg measurement with standardised activation of the extrinsic system. apteg: exteg with in-vitro-fibrinolysis inhibition via aprotinin. exteg-reagent (ex): : dilution of innovin (recombinant thromboplastin reagent, dade) with aqua dest. apteg-reagent (ap): parts innovin, parts trasy[ol (aprotinin, bayer, i . kie/ml), parts aqua dest. test procedure: l p ex or ap + ~l citrated blood (cb) + lal cacl -solution , m. the only difference of the two reagents is the addition of kie aprotinin in the apteg, leading to an in-vitro fibrinolysis inhibition. the usage of disposable pins and cups (haemoscope, illinois, usa/e.m.s., vienna) is recommended for ensuring standardised conditions for both measurements. results and discussion: when there is a better clot formation in the apteg (corresponding to a lower so-cafled k-value) than in the exteg, fibdnolysis can be suspected. this technique requires only commercially available reagents and is easy to perform on conventional teg systems. due to the standardised coagulation activation with a thromboplastin reagent, fibrinolysis can be detected also when inhibitors like heparin are present in the circulation. according to our experience using this technique during liver transplantation, clinical relevant fibrinolysis can be detected as described in less than l minutes. many thromboembolic (massive pulmonary embolism, proximal deepvein thrombosis, etc.) and coronary diseases (infarction, acute phase, etc.) require fibrinolytic therapy to early recanalizafion. the application of the well-known or new thrombolytic agents needs the use of specific, simple and reproducible methods for the determination of fibdnolyfic activity. we suggest new methods for measuring the blood plasma concentrations of plasmin, plasminogen, antiplasmins, and urine urokinase activity. these methods involve the employment of chromogenic substrafe azofibrin (human fibrin, covalently labeled with p-diazobenzenesulfonic acid). method~. . ml of studied solution was added to , ml of azofibrin suspension in certain buffer ( - mg/ml) and the mixture incubated at oc for - rain. after the end of incubation the mixture was filtered, the volume of solution brought up to ml by . m naoh and the optical density was determined at nm. resuffs. azofibrin can be used for quantitative determination of proteinases activity in search of new fibrinolytic means. for comparison the results of our studies fibrinolytic activity of some proteinases with the use of azofibrin are presented: activity. with an increase of pal and ldl-and a decrease of hdl-cholesterol concentrations k is concluded that the increased cardiovascular risk in diabetes meilitus was partly caused by a down regulation of the fibrinolytic system, increase of erythrocyte aggregation and plasma viscosity. also disturbances of lipid metabolism an abnormal whr seems to be of an additional atherogenous factor in dm. plasma concentrations of thrombin-anfithrombin-iii (tat), alpha- antiplasmin-plasmin (app) complexes and ddimer were investigated in patients treated with thrombolytic therapy for acute myocardial infarction (ami) either with streptokinase (n= ), urokinase (n= ) or recombinant t-pa (rt-pa, n= ). all patients received an intravenous heparin bolus of , iu on admission, which was followed at once by an infusion of , iu/hr for the next three days titrated to maintain the partial thromboplastin time at twice control value. tat, pap and ddimer were measured by enzyme immunoassay on admission, , , , , , , hours and on day and after admission. groups did not differ significantly in regard to age, sex, delay and infarct location. on admission, no marker differed significantly between groups. thereafter, tat levels increased significantly exclusively in rtpa treated group. from to hours after admission, tat were significantly higher in rtpa treated patients than in streptokinase and urokinase treated group (p< . ). however, during continous heparin infusion, which was started immediately after stop of thrombolytic therapy, in each group tat concentrations decreased below admission values. app were significantly higher only hour after admission in the rt-pa group (p= . ). ddimer did not differ signifieanfly between groups. our results demonstrate, that rtpa induces a hypercoagulable state, which may contribute to reocclusion after successful reopening of the infarctrelated coronary artery. the significant tat decrease during continous heparin infusion supports the concomitant use of thrombin inhibitors as adjunctive therapy with thrombolytlc treatment for ami. thus, in acute myocardial infarction patients, thrombin generation is markedly influenced by the thrombolytic agent used and concomitant heparin therapy. endothelium derived relaxing factor-no (edrf-no) plays a major role in regulation of vascular tonicity and also exerts platelet inhibitory action~ however, due to the chemical nature of edrf-no few is known about its production and activity as a general index or marker of vascular function in human diseases. one way to achieve this can be measurement of nitrate/nitrite excretion in the urine, which seems to reflect vascular edrf-no production. in this report a self-developed elisa method is described, which was used for this perpose. nitrate/nitrite urinary exretion proved to significantly decreased in insulin dependent and in non-insulin dependent diabetes mellitus as well after a comparison of the excretion values to other markers of angiopathy (yon willebrand factod soluble thrombomodulin, beta -thromboglobulin) it seems to be acceptable, that urinary nitrate/nitrite excretion can be a useful indicate of diabetic vascular disorders. two major concerns still accompany the application of prothrombin complex concentrates (pcc). viral safety has to be guaranteed and therefore several measures for virus inactivation or elimination are taken during the manufacturing process. the inherent risk of thrombo-embolic side effects has to be considered. to minimize these risks and to achieve good clinical efficiency the quality criteria for pcc's are under pending discussion. it is generally accepted that a modem pcc-preparation should contain all of the four coagulation factors in a well balanced proportion and that it should also contain protein c and protein s. additionally, the concentration of activated coagulation factors should be kept at a minimum. a present pcc-produedon process mainly consists of a qae-sephadex extraction of cryopeer plasma followed by a solvent/detergent virus inactivation step. further purification is achieved by subsequent chromatography on deae-sephamse. the aim of this study was to improve product quality by avoiding f viiactivation without implementing major changes to the production process. at the same time, a second virus eliminating step was added to the production process. it could be shown that speeding up the chromatographical process by switching the deae-sepharose-chromatography from a classical axial column to a radial chromatography resulted in a significant reduction of f viia-genemtion. mainly the reduction of contact time, resulting from the highest possible flow rates, leads to the wanted effect. the relation between f vii/f viia was : or more. in order to investigate the feasibility of virus filtration the eluate of the deae-sepharose column was filtered through a virus removing ultipor vffilter. the analysis of the solution before and after fillration showed that the filtration had no influence on coagulation factors activity, protein content, proteolytic activity etc. preliminary studies showed significant virus reduction values. in the past few years the problem of expediency of the treatment aimed at developing immunological tolerance in hemophil;a patients by way of complete removal of inhibitor with high doses of factor viii has been discussed in literature. we observed patients with hemophilia. inhibitors to factor viii:c were revealed in . % of patients with hemophilia a and fo factor ix --in . % of patients with hemophilia b. the level of an inhibitor was not higher than befhesda u/ml, that is those patients were not regarded as "high responders". a high incidence of inhibifors in young patients [from to years of age, . %) compared with older patients (from to years of age, . %) testifies to the probability of inhibitors development during treatment with modern concentrated preparation of factor viii, ix. inhibitor development in patients ( . %] in the course of antihemophilic concentrates transfusions is an evidence of alloimmunization of patients with proteins. the investigations show that in the course of transfusion therapy patients develop secondary immunodeficiency due to chronic antigenic stimulation of immune system with high doses of allogenic proteins. against the background of immunodeficiency patients with hemophilia develop complications of immune character: infections complications -- . %, aufoimmune processes -- . %, secondary tumours -- . %. plasmapheresis is the most rational method of removing inhibitor in patients with low level of inhibitor ("low responders", < bu/ ml) and in patients with mean response. thus it should be noted that the treatment of patients aimed at developing immunological tolerance is not only expensive and economically unprofitable but also not indifferent fo the organism. in a recent multicenter study previously untreated patientens (pups) with severe hemophilia a were treated with a recombinant factor viii concentrate (rfviii, recombinate©). during fviii treatment ( %) developed inhibitors, high titer (> bethesda units (bu)/ml), low titer (< bu/ml) and transient inhibitors. plasma samples from before treatment and during treatment but before inhibitor occurrence were available in inhibitor patients. these plasma samples were analyzed by a highly sensitive immunoprecipitation (ip) assay for the presence of anti-tviii antibodies. in ( %) a significant increase of anti-fv]]i antibodies was seen indicating the development of a clinical relevant inhibitor titer. this immune response occurred after to (median ) exposure days (ed). in the same period only out of inhibitor patients showed a decreased in vivo recovery. in pups who developed no inhibitors plasma samples from the entire treatment period were available. an immune response to rfviii treatment was seen in pups after to ed (median ed). the immune response was later and less pronounced in comparison to inhibitor pups before inhibitor occurrence. with the ip method the detection of an early immune response is possible which might be predictive for a later inhibitor development. the inclusion of the lip method should be considered for future multicenter pup studies. in the past anaphylactie reactions to plasma and plasma components have been a common complication of replacement therapy in patients with hemophilia a and b. we report on severe bleeding episodes in patients with hemophilia a and b, respectively. both patients had a history of life threatening anaphylactic reactions after exposure to different plasma derived clotting factor concentrations including intermediate purity factor viii-and factor ix-concentrate, respectively. high purity factor concentrates were tolerated well without any allergic side effects. a years old patient with a moderate form of hemophilia a (f viii %) had a history of severe immediate reactions with skin manifestations and bronchospasm after exposure to fresh frozen plasma, ctyoprecipitate and different plasma derived factor viii-concentrates of intermediate purity. in all episodes pretreatment with corticosteroids and antihistamines was unsuccessfull in avoiding severe bronchospasm. replacement therapy with two different recombinant factor viii concentrates was tolerated well without any side effects. a years old haemophiita b patient developed hypersensitivity reactions to prophylactic factor ix substitution, which could be overcome by using a factor ix .concentrate with improved purity. a recent recurrence of hypersensitmty under this treatment was finally overcome by the use of highly purified (monoclonal antibodies) factor ix concentrate. we conclude from these findings that high purity of factor concentrates, possibly due to the absence of soluble hla-antigens, are advantageous in patients disposed to allergic reactions. introduction: antibody formation against factor (f) viii remains one of the most severe complications of repeatedly transfused patients with haemophilia a. as reported previously in our study about the incidence of fviii inhibitors, we have observed a high incidence of fviii inhibitors among our haemophilia a patients. it is still not clear why certain haemophiliacs develop antibodies and others do not. a number of previous studies suggest that there is a genetic predisposition for the fviii inhibitor development. thus, the purpose of our study was to examine, if there is a correlation between fviii antibody-formation and genetically determined histoeompatibility antigen (hla) patterns in our haemophiliacs. patients and methods: hla-class i (a, b, c) and hla-class ii (dr, dq) typing was carried out for respectively multi-transfused paediatric haemophilia a patients (fviii:c activity < %), including who had developed an antibody to fviii: were high responders (> bu), were low responders (< bu). hla-typing has been performed by a standurcl two-stage microlymphoc~.ftotoxicity procedure (drk frankfurt) using antisera with defiend hla-specifity (biotest diagnostica). results: we found an under-representation of hla-a in fviii inhibitor patients when compared with the subgroup without inhibitor. in regard to the hla-b and hla-c antigen frequencies there are no apparent differences between the groups. among the class ii antigens there were higher frequencies of dr , drw and dqwl in the non-inhibitor group. however, the reduction in hla-a , hla-cw , hla-dqw respectively hla-dr frequency for inhibitor patients as reported previously could not be confirmed in our study. conclusion: so far it remains unclear if there is a significant association of a certain hla allels with the development of fviii antibodies. recombinant factor sq (r-viii sq, pharmacia) is a b-domain-deleted recombinant factor viii. it is formulated without albumin (hsa). the product has been shown to have in vitro and in vivo biochemical characteristics similar to a plasma derived full-length protein (p-viii). the international clinical trial programme was initiated in march . pharmacokinetic studies have shown that the b-deleted r-viii sq should be given according to the same dosage principles as a full length p-viii. at present, the product is being tested in previously treated patients (ptps) and untreated patients (pups) with severe haemophilia a (viii:c < %), both during long-term treatment (on demand therapy or prophylaxis) as well as during surgery. the long-term study in previously treated patients in germany was started in january . thirteen patients have been included in centers. all patients are still on treatment with r-viii sq, most of them receiving prophylactic treatment. global treatment efficacy has in general been considered excellent or good. no serious clinical adverse events related to the study product have been reported, nor have any inhibiting antibodies to factor viii or antibodies to mouse-lgg or cho-cell components developed in the patients. further results such as data on efficacy, half-life, recovery and safety will be presented in detail at the meeting. nowadays it is not sufficient to regard hemophilia only as hemorrhagic diafhesis of coagulation genesis, caused by deficiency or molecular anomalies of coagulation factor, without taking into account the immunity state. on examination of patients (pts) (hemophilia a -- pts, hemophilia b -- pts, willebrandt's disease u pfs) the development of immune complications was revealed in . %. chronic persistent hepatitis ( . %), chronic active hepatitis ( . %), herpes simplex ( . %), chlamidiosis ( . %), bacterial infection ( . %} were regarded as infectious complications. bacterial infections have a routine course due to preserved phagocytic function of neufrophils. and viral infections, whose ability to resistance is connected with t -cell link immunity, take on a chronic persistent course, mechanism of the development of autoimmune processes (autoimmune thrombocytopenic purpura -- . % of pts, immunocomplex disease -- . % of pts, the appearance of immune inhibifors -- . % of pts} is connected with the impairment of immunological surveillance over b -cells aufoimmune clones as a result of dysbalance in the system of t -lymphocyfes immunoregulatory subpopulations. lymphadenopathy and splenomegaly ( . %) develop due fo benign proliferation of lymphoid tissue as a result of impairment of regulatory function of t -lymphocytes system, or they may be an evidence of virus infection. we observed one episode of acute leukemia. immune complications in hemophilia patients develop against the background of secondary immunodeficiency caused by chronic antigenic stimulation of patients' immune system with high doses of allogenic proteins, which plasma preparations contain. in immune complications hemophilia patients develop hemorrhages, whose pathogenesis is quite different from that caused by coagulation factor, so it should be taken into account in the course of treatment. control of hemophilia therapy classically was based on four parameters: life span expectancy of patients, orthopedic status (normal zero), pettersson score and social integration. oren, however, these parameters described an irreversible status with permanent damage particularly of the joints, especially when patients were grown-up. in order to establish risk-adapted therapy protocols to prevent hemophllic osteoarthropathies, quality control programs have to he set-up that allow for early adjustment of dosage and substitution frequency. here bleeding frequency is one the main parameters, being a clear hint for the possible development of a target joint. since we have established a computer database (haemopat) that contains data from all patients treated in our center. tables and graphs allow for early detection of increased bleeding tendency in a given joint, and accordingly for adjustment of therapy. the results of years of measuring reasons of joint damage and not documenting the orthopathies as such will be demonstrated. parallelly a new program (haemopat win . ) will he introduced allowing for easier handling of data and their evaluation. this program will be used as of december . in combination with a substitution calender to be filled in by all patients, in which factor concentrates, lot numbers, dosage, and date of administration will he constantly recorded, this program will extend our existing database in order to follow closely clinical and orthopedic parameters of each patient, and consequently acts as strict control of therapy quality. additionally, it provides sufficient data to fulfil any documentation needs, requested by medical authorities. the program will be available for all those interested free of charge. ) kinderklinik der westf. wilhelms univ. mttuster - ) biotest pharma gmbh, dreieich haemoctin® sdh; the fviii sdh (sdh = solvent detergent and dry heat = °c, rain) from biotest pharma is a high purity (specific activity ~ ) fviii concentrate manufactured from large human plasma pools. virus validation studies have shown virus inactivation/reduction (log ) during the manufacturing process for lipid coated vints~ such as: h]v- > . ; psr > . ; vsv > , ; bvdv > . ; hcv > . * and non enveloped vimsas such as: parvo** = . ; reo > . *** and hav > . . more than hemophilia a patients (ptps = previously treated patients), baseline fviii activity < %, were included in an international drug monitoring study to follow their fviii inhibitur status. the hemophilia centers included were three centers from hungaria (helm pal children hospital and the national inst. of haematology, budapest and regional blood transfusion center, debrecen) and four centers from germany (two from berlin, one fraukfurffmain and one monster). patients were enrolled in the drug monitoring beginning aug. . at the entry none of the patients had a detectable inhibitor. at the end of sept. there were no side effects or adverse events in connection with the use of haemuetin®. before the haemoctin drug monitoring study, the patients were treated with cryoprecipitate, or purified fviii products. inhibitor testing was done on patients plasma samples using the bethesda method. repeated fviii recovery determination at one time (between to hrs) after haemoctin® application demonstrated the expected recovery and normal half life time. none of the hemophilia a patients, treated with haemuetin® sdh developed a clinical relevant inhibitor. at the beginning of the stud)', the clinical efficacy of haemuetin® was studied in hemophilia a patients and shown to give an in vivo recovery of + % by one stage assay and + % by a chromogenic assay. t ½ values were + . and . + . hrs respectively. the study for the clinical efficacy of haemoctin® sdh was repeated in a group of patients approximately two years later. although cd lymphocyte counts are known as reasonable predictors of prognosis in hiv infection, the cd count is not in all cases an infallible indicator of prognosis. therefore several serological markers are used to predict disease outcome, including beta- microglobulin ( m), immunoglobulin a (iga), lymphocyte counts (lymph) and others. in this study we followed a cohort of haemophiliacs ( with haemophilia a, with haemophilia b) and patients with severe von willebrands disease over a period of months (mean, range: - ). testing for l~ m, igg, iga, igm, cd and cd cell counts (abs. and relat.), cd /cd ratio, and absolute resp. relative leucocyte and lymphocyte counts was performed at least times a year. at the same time clinical examinations and review of history were undertaken. mean of laboratory tests for every quarter of a year and significant changes during time of observation were calculated and correlated with clinical data. - - - - - - cd + + + .- : .+. + cd ~ + + + + ± + ~ m z . + . . + . . + . . + . . ± . . ± . lymph ~ . + . . + . . + . . ± . . ± . . ± . means/pl ± standard deviation means mg/l ± standard deviation during time ef observation we found significant changes of cd (abs. and relat.), abs. cd counts, cd /cd ratio, f~ m, leucocytes and lymphocytes. the abs. cd and cd counts correlated clearly with lymphocytes und leucocytes counts but not with ~ m. the prognostic value of the tested parameters is discussed by calculation of correlations with clinical data, anti-retroviral treatment and treatment of haemophilia. the availability of high purity factor concentrates has recently encouraged clinicians to use perioperative continuous infusion of fviii or fix to prevent or reduce bleeding in patients with haemophilia. in conliast to repeated highdose bolus injections, the continuous infusion trealment regime maintains constant coagulation factor activity at a level necessary for hemostasis, reducing the total cost of treatment by about % and preventing possible side effects of bolus doses. the new application mode, however, requires stable products which tolerate slow passage through an infusion device. our objective was to test in vitro the fviii concentrate immunate (stim plus) and the fix concentrate immi.ynine (stim plus) at room temperature, under conditions of long-term contact with polypropylene tubing in an infusion pump. infusion rates were chosen to mimic clinical situation. the control samples were not infused through the pump but were otherwise treated identically. test samples were drawn before and at , , , and hours after the onset of each infusion run. fviii (one-stage, two-stage and chromogenic assay) and fix (one-stage) activity were measured using immuno reagents. presence of activated factors were measured by napt'i', while flla, fxa, plasmin and pre-kallikrein activator were detected with specific chromogenic substrates. the data showed equivalent results between test and control samples with no loss of fviii or fix activity. the potencies of both immunate (stim plus) and immunine (stim plus) remained within + % of labeued values within hours after onset of infusion. in conclusion, immunate (stim plus) and immunine (st m plus) are suitable for contiuous infusion when using automatic infusion device within applied test criteria. in htanans, circulating half-lives of asparaginase enzymes from e. coli and erwinia chrysanthemi vary within a wide range. moreover, half-lives differ not only among different e. coli strains but also among commercial e. coli preparations. to investigate the possible influence of two different sources of e. coil asparagmase (asn) preparations on the fibfinolytic system of leukemic children a prospective randomized study was performed correlating asn pharmacokiuetics (asn activity, asparagine depletion) with fibrinolytic parameters (plasminogen (plas), o. -antiphismin (ct ap), tissue-type plasminogen activator (t-pa), tissue type plasminogen activator inhibitor (pal ), d -i)imer (i)-d)). together with prednisono, vincristine and an anthracycline children received i iu-/m asn medae r (originally purchased: kyowa hakko, kyogo japan) and children iu/m crasintin r (bayer, leverkusen, germany). blood samples for pharmacokinetic and coagulation analysis were drawn before the first asn administration and every third day whilst on medication. the results are shown in the . asn activity shows a negative correlation (spearman: rho/p) to plas (-, / . ) and ct ap (-, / . ). a positive correlation was found between asn activity and d -dimer formation ( . / . ). t-pa and pal showed no relationship to asn activity. all children showed complete aspamgiue depletion at a detection limit of . um during the course of asn admiatstration. two thrombotic events occurred in the kyowa group, one of the distinctions between the two e. coli asn preparations administered ill this stndy is the absence of cystine in the kyowa asn, which also has a lower isoelectric point and a longer half-life than the bayer type a asn. with respect to this observations this may lead to longer inhibition of protein synthesis, which then may be the cause of a bigher rate of side effects. along with studies on asn pharmacokinoties dose recommendations need to be tailored to the specific asn preparation employed to ensure optimal antineoplastic efficacy while minimizing the hazard of complications. different types of coagulopathy in hepatic veno-occlusive disease (vod) and capillary leakage syn-drome (cls) after bone marrow transplantation w. ntimberger, s. eckhof-donovan, st. burdaeh and u. g bel department for pediatric hematology and oncology, heinrich heine university medical center, diisseldoff, germany it is generally accepted, that cls, coagulation activation and refractoriness to platelet transfusions are part of the syndrome of hepatic vod. we assessed patients with either vod or cls or both vod and cls, in order to analyze the influence of either syndrome on different aspects of hemostasis. vod was diagnosed according to jones et al. [transplantation ( ) ]. diagnosis of cls was >_ % increase of body weight in the past hours and non-responsiveness to furosemide [niirnberger et al., ann hematol ( ) ] . patients with vod, cls or both were compared to control patients without either diagnosis. eight patients suffered from both vod and cls, patients only from vod, and only from cls. patients had neither syndrome and served as control population. activation of the coagulation system was assessed by increase of tat-complexes and/or increased consumption of at iil the hemostasis patterns were as follows: no. introduction: lung cancer goes along with coagulation activation and increased thromboembolic risk. acute phase reaction in cancer patients leads to elevated levels of c b-binding protein (c b-bp) followed by a shift from free to c b-bp-bound protein s. we tried to find out whether there is a correlation between alterations of c b-bp, protein c protein s system and interleukin (il- ), which is one of the most potent inducers of hepatic acute phase reaction. patients: i. patients with lung cancer; . control group: patients in complete remission after lung cancer. methods: clotting methods: protein c and s activity; elisa tests: protein c antigen, tat-complexes, prothrombin fragments f i+ , il- . electroimmuno-diffusion (laurell): free and total protein s, c b-bp. results: tat-complexes and f i+ were elevated in cancer patients. c b-bp levels were slighthly increased ( ± % of n.), protein s activity was ± % of n. (control group: ± % of n.). il- in lung cancer patients was . . pg/l (control: . ± . pg/l). conclusion: one source of the hypercoagulable state in lung cancer patients is decreased protein s activity due to elevated c b-bp levels. this is probably caused by hepatic acute phase reaction which is triggered by increased il- levels. these plasma levels correlate with levels of the tumor marker ca and with the stage of the disease but correlations with patient outcome (disease recurrence and overall survival) have not previously been shown. plasma levels of d -dimer and ca (determined by sandwich elisa assays} were measured prior to treatment in women with figo stage t to iii ovarian cancer and correlated with tumor stage, relapse and overall survival over a mean follow -up period of months (range to months). levels in healthy women and patients with benign ovarian disease served as controls. the occurrence of deep vein thrombosis in the cancer patients was also determined by impedance plethysmography that, when positive , was confirmed by contrast venography. preoperative d -dimer and ca i levels in ovarian cancer patients were statistically signfficantly higher than in controls. preoperative cut off values were calculated for the prediction of cancer relapse and survival for both measurements. d -dimer levels above a cut off level of ng/ml were statistically significantly associated with the rate of relapse but ca levels were not. deep venous thrombosis occurred in % of cases but there was no difference between properafive levels of d -dimer in patients who subsequently did versus did not develop deep vein thrombosis. high levels of d -dimer are associated with more advanced disease and with poor prognosis in patients with ovarian cancer. the high levels of d -dimer are a biologic feature of the malignancy itself that may be attributable, at least in part, to increased conversion of fibrinogen to fibrin in the tumor bed with subsequent degradation of fibrin by the fibrinolytic mechanism. thus d -dimer levels may serve as a marker for overall tumor burden as well as "disease activity". a high incidence of deep vein thrombosis exists in the course of the disease in ovarian cancer patients but preoperative levels of d -dimer are not predictive of this occurence. yon tempelhoff georg -friedrich, michael dietrich, dirk schneider, lothar heilmann. dept. obstet. gynecol. city hospital of ruesselsheim. -germany. an increase of plasminogen activator inhibitor activity (pai act.) in the plasma of cancer patients has been recently discribed. we have longitudinally investigated pai act. in patients with primary breast cancer and compared the results with the outcome of malignancy. patients with untreated primary breast cancer and without proof of metastasis (t - n - m ) were eligible for this study. in all patients coagulation tests including fibrinogen {method according to clauss), d -dimer (elisa} and pal act. (upa dependent inhibition test) were performed prior to primary operation, months thereafter and at the time of cancer relapse. seventy -two healthy women and patients with benign breast disease served as controls. during a mean follow -up of + months patients ( %) developed cancer recurrence and ( . %) patients died. in all cancer patients preoperative levels of fibrinogen and pai act. were significantly higher compared to healthy women and to patients with benign breast disease. preoperatively only pal act. was significantly higher in patients with vs. without cancer recurrence ( . _+ . u/ml vs. . + . u/ml; p = . ). in patients with later recurrence pai a~t. significantly dropped months after operation (p = . ) and was again significantly increased at the time of cancer recurrence ( . _+ . ; p = . ). a preoperative cut off value (calculated via cox model) of pai act. above . u/ml was significantly associated with the rate of relapse (tog rank: p = . ) and in % of patients who died of cancer preoperative pai act. were also above this cut off. impaired fibrinolysis in patients with breast cancer is significantly associated with the outcome of cancer. a monoclonal heparin antibody (mab) has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. for further analyses tyramine, which was covalently bound to low molecular mass heparin by endp int attachment (malsch r et al: anal biochem ; : - ) , was labeled with -iodine at the aryl residue. the tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline igg. the mab recognized specifically intact heparin and heparin fractions. the lower detection limit of heparin preparations was ng/ml. no cross reactivity of the mab occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate a and c. oversulfated heparin showed lower affinity to the antibody hl. than - -and - -desulfated beparin. the method established for the purification of the mab was ammonium sulfate precipitation with followed dialysis. sds-page and high pressure capillary electrophoresis prooved the high purity of the received antibody. the biological activity of mab was tested by the chromogenic assay $ and remained stabile while purified. in conclusion, the present abstract describes an purified igg monoclonal antibody directed against heparin and heparin fractions, which can be used for biological measurements. the concentration of heparin and dermatan sulfate in biological fluids is usually measured using radiolabeling. for this purpose aromatic compounds are usually used to insert radioactive iodine labeling at the saccharide backbone of the glycosaminoglycan. we developed methods for the specific labeling of hepann and dermatan sulfate at the terminal residue. tyramine was bound by reductive amination to the , anhydromannitosyl end of heparin, produced by nitrous acid degradation and confirmed by c.nm r spectroscopy. (anal biochem : - , ) this method was also used to produce a low molecular mass dermatan sulfate (lmmd)derivative after partial deacatylation. in order to choose the proper method for evaluating the specific anticoagulant activity in the row of chitosan polysulphate (cp) samples with different degrees of pol ~merization and sulphation we applied to pharmaeapea article (a~) when assessing the ability of direct anticoagulants to depress the coagulability of recalcificated sheep blood (using the rd international heparin standard), and to measuring such acti¢ity as per pharmacokinetic model (a ). the model admits the "kinetics of cp elimination be linear in ease of intravenous injection to rabbits, as it is observed in heparin: ct=co exp(-i~ x t), where ct is cp concentration at the time moment t; co is cp concentration at the moment of injection; i~ is the elimination constant. besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific actidty a : t=kt ct + tin, where t is the time of clot formation at different tlme intervals after of cp injection; t~, is the time of clot formation prior to cp injection. t value was assessed in two tests: in blood coagulation time (bct) and in activated partial thromboplastin time (aptt). no correlation was observed between a and a . at the same time the values of ifm and the period of semieliminatinn (tvz) with the use of the original method that were obtained with the help of the quantitative determination of cp in rabbit's blood taken at different time intervals after injection, showed a close correlation ( "= , p< , ) between the same parameters, obtained with the help of the of the pharmacokinetic model in bct test. thus, experimentally it was proved that the assumption of the linear elimination and the effect-dose dependence was true, which is necessary for a calculation. we recommend to use intravenous injection of the samples to animals with further assessment of the results according to the pliarmacokinetic model to calculate the specific anticoagulant activity in the row of chemically related potential direct anticoagulants. in this investigation we compared the biological activity of a low-molecular-heparm (lmw-heparin, mono embolcx®) after intravenous, subcutaneous and oral application in rats. sprague-dawly rats were anaesthetized by ketamine/diazepam and the blood samples were taken from the retro orbital sinuus. axa u/kg body weight of the lmw-heparin were injected intravenously and subcutaneously to rats each. between minutes and hours after injection serial blood samples were taken. mg/kg ( . axa u/kg) body weight of the lmw-heparin were applicated orally using a stomach tube. blood samples were taken between and hours after oral application. the antifactor xa and antithrombin activities of the plasma samples were measured, using ehromogenic assays and the substances s and s (kabi vitmm). after i.v. injection the maximum axa and alia activities were . axa u/ml and . aiia u/nil respectively. after s.c. application the antifactor xa activity of the lmw-heparin showed a maximum of . axa u/ml atter minutes. the antithrombin activity exhibited an eatiier maximum activity of . alia u/nil minutes after injection. after the oral application no increase of the axa or alia activities was measured. the lmw-heparin has a high antifaetor xa and antithrombin activity after i.v. and s.c. injection. after oral application no activity of the lmw-heparin was measurable. these results implicate that fractionated heparin is not absorbed after oral application or is inactivated in the gastrointestinal tract. to improve the activity after oral application modified hepatins have to be synthesized. in an in vitro study the effect of various heparin derivatives (calciparin, fraxiparin, cy , cy , astenose, hexasaccharide, ssh ) on thrombin-and adp-induced platelet aggregation as well as on adpmediated platelet activation in whole blood was investigated. all heparin derivatives caused a concentration-dependent inhibition of thrombin-induced aggregation of washed platelets. calciparin and astenose were found to be the most effective compounds with ic o values of . and . p, mol/l, resp.; higher concentrations ( - times) were required for the other compounds. furthermore, the heparin derivatives were studied with regard to their potentiating effect on adp-induced platelet aggregation. in a concenwation range from to u/nil calciparin, fraxiparin, cy and astenose led to a potentiation of the adpinduced aggregation whereas cy , hexasaccharide and ssh did not show this effect. the increase in aggregation was associated with an increase in thromboxane a lbrmation. in addition, the effect of calciparin, fraxiparin, cy and astenose on adp-induced platelet activation in whole blood was investigated by flow cytometric analysis using monoelonal antibodies to platelet surface receptors opiiia (cd- ) and p-selectin (cd- ). at concentrations that caused a maximum potentiation of adp-induced platelet aggregation these substances led to a strong increase of adp-mediated activation of platelets in whole blood. the effect was most pronounced when the blood was anticoagulated with calciparin and astenose, resp. in conclusion, the results suggest that the aggregation-promoting effect of heparin derivatives included in this study is dependent on the molecular weight and the degree of sulfation and is in part due to the generation of thromboxane. heparins are negatively charged polysaccharides and bind protamine forming a stable complex. here we report on the properties of microbeads ( . pro) coated by protamine. protamine chloride ( . ijm) was covalently bound to . mg paramagnetic tosyl..activated microbeads m- (dynal). the covalent binding of protamine was from . to , mg/g beads. protamine-dynabeads were produced in a phosphate buffer at different ph ( , ; , ; , and , ). the protamine-dynabeads produced ph . showed the best properties for flow cytometry analysis. in saline solution they bound lmm-heparin-tyramine-fitc (lmmh-tyr-fitc) dose dependently from . to u/ml, whereas in plasma and blood they bound lmmh-tyr-fitc from . to u/ml. dependent on the binding protocol, the microbeads also bind proteins unspecifically, i.e bovine serum albumine and protamine to a lower extent.the adsorbed proteins, however do not bind lmmh-tyr-fitc dose dependently. the saturation of the proteins on the beads was determined as their relative fluorescence intensity (rfi). in saline solution the saturation was measured at rfi, in human plasma at rfi and in whole blood at rfi. using flow cytometry erythrocyctes, lymphocytes, monocytes and granulocytes were not bound to protamine dynabeads. these data demonstrate that protamine-dynabeads can be used to measure the concentration of lmmh-tyr-fitc in saline solution, plasma and blood because they do not bind to human blood cells. the present study was designed to investigate the anticoagulant action of inhaled low molecular weight (lmw)-heparin in healthy volunteers. , iu (group t), , iu (group ), , iu (group ) or , iu (group ) lmw-heparin were given to healthy volunteers each at weeks intervals. in group tissue iactor pathway inhibitor (tfpi) antigen and activity, chromogenic factor xa assay, heptest, aptt and thrombin clotting time (tot) remained unchanged during the days observation period. in group tfpi antigen and activity, aptt, tct and the $ method remained uneffected. heptest coagulation times were . + . before, . + . sec. hrs and to . + . sec. hrs after inhalation. in group tfpi antigen increased from . + . to . + . ng/ml hrs after inhalation. tfpi activity remained unchanged. $ method increased from . to . + . iu/ml hrs after inhalation. heptest coagulation values were prolonged up to _+ . s ec after hrs and returned to normal within hrs after inhalation. aptt and tct remained unchanged. after inhalation of , iu lmw-heparin, the following changes were observed: tfpi antigen increased to +_. . ng/ml and normalized within hrs. -i'fpi activity increased to . _+ . u hrs after inhalation and was normal after hrs. antifactor xa activity, as measured by s method, increased to . + . u/ml after hrs and was normal after hrs. heptest coagulation values increased to . + . sec hrs after inhalation and normalized after hrs. aptt and tct did not change throughout the observation period. the data demonstrate a resorption of lmw-heparin by intrapulmonary route in man. no side effects were observed. recently we developed a tritium-labelled arachidonic acid ([ h]aa) release test with high sensitivity to membrane-toxic agents. the assay performed in u cells is intended to evaluate ehemicals, drugs and biomatefials with regard to their eytomembrane toxicity [kloeking et at. ( ) , toxicology in vitro , - ]. local irritation reactions are described in patients receiving therapeurieat dosages of lmw heparin. this fact prompted us to examine the following lmw hepafins and heparinoids for their membrane toxicity in u cells: reviparin-sodium, enoxaparine-sodium, mueopolysccharide polysulphate (mps), pentosan polysulfate sodium (pps), polysulfated bis-lactobionic acid amide derivatives lw (aprosulate) and lw . for this purpose, [ -- ]aa labelled u ceils were incubated with different concentrations of lmw heparins and heparinoids at °c for hour. compared with untreated cells, the [~h]aa release of cells treated with mg of the drugs was two times higher with reviparin sodium, three rimes higher with bis-lactobionic acid amide lw , five times higher with pentosan polysulfate, times higher with ertoxaparine-sodlum, but it was equal to the control with mucopolysaccharide polysulphate. the rate of araehidonic acid release in response to a test chemical may therefore be used to assess the membrane-toxic effect of this substance and to predict its the inflammatory potential in the skin. semi-synthetic glyensaminoglycans (gags) with antithrombotic properties can be prepared from the e. coli k polysaecharide by coupled chemical and enzymatic methods. the molecular weight of these semi-synthetic gags can be adjusted to obtain products mimicking the molecular profile of a low molecular weight hepatm. in order to compare the biochemical and pharmacologic properties of a semi-synthetic gag (sr a, sanofi/choay) with a commereiany available low molecular weight heparin, fraxiparine (sanofi, paris, france), valid biooheanical and pharmacologic methods were used. the molecular profile of this agent as determined by hplc exhibited a comparable distribution profile (mr= . kda) in comparison to fraxiparine (ma= . kda) . the anticoagulant properties of sr a were comparable to fraxiparine in the aptt and heptest. however, in the usp assay, this agent showed slightly weaker activity. sr a also exhibi~d comparable affinity to atffl and hcii. in comparison to fraxiparine, it produced a much weaker response in the hit screening system. in~ viv studies, sr a preduecd strong dose-dependent antithrombotic actions in both the iv and sc studies in the rabbit jugular vein stasis thrombosis model (ed =i - gg/kg). additionally, it also produced antithrombotic aefiorts in a rat jugular vein clamping model. the hemorrhagic effects of this agent were comparable to those of fraxipafine as measured in a rabbit ear blood loss model. intravenous administration of sr a also revealed a comparable pharmaeokinetie behavior to fraxiparine. no abnomaiitias of the clinical chemistry (change in liver enzymes) and hematology profile (thrombocytopenia and lencecytosis, etc.) were noted in primates. at a dosage of i and . mg/kg iv, this agent also caused a release of functional tfpi which was comparable to the observed responses of other low molecular weight heparins. these studies suggest that sr a is capable of producing similar pharmacologic effects as other low molecular weight heparms, however, additional optimization studies are required for demonslrating product equivalence. limited information on the comparative pharmacoldnetics of low molecular weight heparin (lmwh) is available on the data obtained from aptt, heptest, anti-xa and antmia assays. since these drugs are currently used for therapeutic indications using relatively high dosages and intravenous administration. aptt, heptest and antmia test may be valuable in the assessment of their effects. in order to investigate the relative pharmacokinetics of lmwh using apt'i', heptest, anti-xa and anti-iia methods, certoparin (sandoz, basel, switzerland) was administered to individual groups of healthy male volunteers ( - kg) via intravenous ( mg) and subcutaneous ( nag) routes in a crossover study. blood samples were drawn at , , , , , , , , and minutes. using a baseline pool plasma obtained from the same volunteers, calibration curves for each of the individual tests were constructed to extrapolate circulating levels of certoparin. a non-compartmental model using trapezoidal technique was used to obtain pharmacokinetic parameters such as t / , vd, and clsys. in the intravenous studies, the t / was found to be dosedependent for aptt, heptest, anti-xa and antm]a. the auc, however, was significantly different for each test and was dose-dependent following the order: aptt<heptest<anti-xa<anti-iia. in the subcutaneous studies, the t / was both test and dose-dependent. the auc was also dose-dependent and followed the order: anti-xa>heptest>aptt>antmia. the clsys of the antma was much faster in comparison to the other tests. the clsys of the aptt and heptest was independent of dose. however, anti-xa clsys by this route was lower than other tests. the apparent vd followed the order aptt>antmia>heptest>anti-xa. the bioavailability of the certoparin as measured by various tests ranged from - %. these studies suggest that beside providing pharmacokinetic data, aptt, heptest and anti-iia assays may provide useful data on thier safety and efficacy at high dosages. the immunological type of heparin associated thrombocytopenla (hat ii) is a severe complication of heparin treatment and is associated with arterial and venous thrombosis. only patients with absolute thrombocytopenia have prompted suspicion of hat in clinical practice. we report on a year old male, who developed thromboembolic episodes after coronary angiography like reinfarction and thrombotic episodes of a. brachialis. fibrinolytic therapy combined with i.v. unffactionated heparin treatment was the therapy of choice and was followed by severe fua~er thromboembolic adverse effects. besides an impaired fibrinolytic response and elevated antiphospholipid anitbodies, we diagnosed hat type ii in hipa and elisa (stago-boehringer, marmheim). this special patient had platelet counts within a normal range, when developing the thromboembolic episodes. it appears that the normal platelet count during the thromboembolic episodes reflect a relative thrombocytopenia. from a clinical point of view we recommend the use of a lab panel to exclude hat type ii in patients with thromboembolic episodes under therapy with fractionated or unfractionated hepafin. platelet counts within a normal range are no absolute exclusion criterion for hat ii. low molecular weight heparins (lmwhs) are now commonly used for the prophylaxis of post-surgical thromboembolic complications. in this indication, lmwhs are administered as a single or twice a day subcutaneous regimen. usually these agents are administered at - mg total dose which is equal to - anti-xa (axa) iu. newer methods such as ehromogenic substrate based axa methods and the heptest clotting time can be used to determine the effects of lmwhs during the initial phases of prophylactic therapy. this may be useful in the elderly and weight compromised patients where a fixed dosage may not be optimal and may produce bleeding effects. similarly in the overweight patients, a fixed dose may not be efficacious. thus, monitoring of lmwhs in these patients may be useful in the optimization of their therapy. lmwhs are also used in the treatment of deep vein thrombosis using both intravenous and subcutaneous protocols. high dosages of up to mg sc/day and infusions of up to axa iu/kg/hr have been administered. in these conditions, the monitoring of the circulating lmwh levels may be useful in optimizing the dosage. we have modified the aca heparin (do_pont merck, wilmington, de) method to measure the lmwh levels in the plasma of patients treated with both the prophylactic and therapeutic dosage. owing to the required turnaround time, simple operation and reliable results, this method was found to be of value in the monitoring of these agents. this presentation provides an overview of the clinical application of various lmwhs with particular reference to the need of monitoring for their effects to optimize the clinical outcome. a double-blind, multicentric, controlled trial was performed in order to compare the antithrombotic efficacy and safety of single daily doses of ie anti-xa of low molecular weight heparin (lmwh) sandoz (certoparin) and ie unfractionated heparin (ufh) tid. in patients undergoing elective total hip replacement blood samples were drawn before the first subcutaneous injection of lmwh or ufh resp., two hours after administration on the first and th postop, day and on the last day of prophylaxis (day - ), anti-xaactivity was measured by chromogenic substrate assay, heptest and aptt by clotting assays and tissue factor pathway inhibitor (tfpi) and heparin-pf -antibodies by elisa techiques. as expected, the anti-xa-activity and the heptest values were significantly higher in the lmwh-group at all time points after administration of the drugs; the mean values of heptest were sec in the ufh-and sec in the lmwh-group respectively, the aptt was not different in both groups. at the end of prophylaxis positive antibodies to heparin-pf complexes were detected ~n both groups; this however was not correlated with clinical thrombocytopenia. a detailed correlation between patients with deep vein thrombosis (dvt) and positive antibodies has still to be done (all patients were screened for asymptomatic dvt between day - by bilateral phlebography. tfpi was markedly increased in the lmwh-and only slightly elevated in the ufh-group; the differences are statistically significant. summarizing it can be concluded that antibodies to heparin-pf complexes may occur without clinical symptoms of hepafin-induced thrombocytopenia type ii and that tfpi may play a sigificant role for the antithrombotic efficacy of ufh and lmwh. unfractienated heparin represents one of the most severe and frequent causes of drug-induced thrombooytopenia. heparin-indueed thrombocytopeala (hit) occurring early in therapy is often mild and serf-limited, appearing to be caused by a direct aggregant effect of heparin on platelets (hit type i). hit type ii, however, is immune-related an may result in absolute thrombocytopenia (platelet count <i /~l) or a relative decrease in platelet count (?_ % reduction from baseline values). unlike most other immune thrombecytopenias in which bleeding complications often predominate, hit type ii may results in severe arterial and/or venous thromboses. the management of patients with hit type ii and thrombosis is often difficult. heparin must be stopped immediately. antithrombotic, fibfinolytic, or antiplatelet agents have been used in these situations, as have anticoagulants other than unfraetionated hepafirl plasmapheresis, used in other immune-related disorders, has also been occasionally reported. at our institution this intervention appears to provide significant elininal benefit to hit patients. several recent reports have noted that the heparin-dependent antibodies in hit type ii are directed against a complex of heparin bound to platelet factor (pf ) and an elisa-based test system for hepafin-pf eomplexinduced antibodies (hpia; stago) has been develuped~ we report our experience in patients with hit type ii in whom plasmapheresis was instituted and the antibody levels in blood were measured pre-and post-treatment to identify wkether circulating antibody titers decreased thus providing some explanation for the clinical benefit. the patients developed profound absolute thrombocytopenia (range, - k/~tl) and positive heparin-induced platetet aggregation studies following treamlent with uofractionated heparin. thromh cytopenia induced by hepariu (hat-syndrome) typically appears several days after initiation of heparin therapy. this phenomenon is caused by antibodies of the lgg type which are induced by a heparin-pf -complex and which also activate platelets. in a recent study (n engl j med ; : - ) the hat-syndrom was diagnosed in . % ( of ) patients who had received unfractionated heparin (ufh) and in none of those patients who had been treated with low-molecular-weight heparin (lmwh). out of the patients treated with ufh presented with at least one thrombotic event ( times a venous, once an arterial event). the incidence of heparin-dependent igg antibodies ( . %) was higher in the ufh treated group than in patients treated with lmwh ( . %). our patient was a male caucasian white, years of age, otherwise in good clinical condition with no severe diseases in his pmh. both parents, however, had suffered several thrombotic episodes. the patient was admitted to the hospital in september " with clinical signs of a deep vein thrombosis (dvt) of the right leg. the diagnosis was phiebographically confirmed. after having given informed consent the patient was treated with cycles of r-tpa (loco-regional application) and cycles of uhsk as part of a clinical trial (uhsk vs. rtpa, boehringer/m). lysis was successful with complete recanalisation of the veins. after completion of lysis the patient was further anticoagulated with coumarin overlapped by heparin (ufh). as a complication the patient suffered from a retroperitoneal and an upper gi ract bleeding from esophagitis grade ii to hi while being on coumarin prophylaxis. therefore the patient was put on lmwh and then discharged with this prophylaxis. after less than two weeks the patient presented again to the hospital with a relapse of dvt in the same leg. phiebographically nearly all deep veins were occluded to a higher degree than before, i. e. also with concomitant pelvic vein affection and with hea~ , swelling of the whole leg. blood count showed a marked decrease of platelets to /nl. the platelet aggregation test demonstrated a hat-syndrome which was confirmed by the hipa-assay. the heparinoid orgaran® was not reactive in the assay. having seen a previous bleeding episode in this patient, we did not see an indication for another b'sis therapy. therefore we decided to put the patient on an intermediate dose of coumarin overlapped by hiradin (hat study, behring) instead of orgaran®, since hirudin allows a much more precise dose adjustment. after days the patient was discharged with no further complications. risk factors for dvt have not been detected until now. to our knowledge this patient is one of the first cases of hat-syndrome induced by lmveh. under routine conditions aptt is the most frequently used test method for the surveillance of patients undergoing hepann therapy. in literature a , fold to , fold prolongation of the initial value is recommended for individual therapy a~ixatian. in contrast to the control of the enmarin therapy by means of prothrombin time measurement the individual hupafin sensitivity of the ~ reagents of this standard value hardly receives notice. commercially marketed ~ reagents differ from one another method and concentration of the activator as well as their phosphelipids. when choosing a reagent the user must only take into consideration the sensitivity against its factors, heparin and lupus but also the adalxability of the reagent towards the given measurement system ( software variability towards incubation time, sodimentalion problems in reagents with high kaolin concentration). reagents offering a high heparin sensitivity already are showing a prolongation of the coagulation time at low heparin dosage ( . - . u/ml). in the therapeutic range ( . - . u/ml) very often no linear relationship between prolongation of the aptr and heparin concentration level is found_ therefore very often coagulation times above the routine measuring range ( s) can be observed in this range. moreover, various medical measuring devices differ in their ability to recogniso the retarded entry of coagulation. coagulation limes with different apq't reagents in correlation with the heparin concentration level can be found in the adjoined table. significantiy prolonged coagulation ti.~es of aptt lead to a reduction of the hel~ dose in clinical practice. due to the discrepancy between heparin concentmon level and the prolongation of the afrt in reagents with a non-linear heparin sensitivity a reduction of heparin dosage leads to an inmt~cient anticeagulalion and the risk of further thrombosis. therefore the producers should be asked to include precise directions on the heparin sensitivity of the aptt reagent in the therapeutic range in order to enable an optimal surveillance of the heparin therapy. a coordination between clinic and laboratory should be compulsory for the choosing of the aptt reagent. introduction: hat is a rare but sometimes fatal complication of therapy with unfractionated (ufh) and low molecular weight beparins (lmwh). we report on our experience with the clinical coume and management in patients (pts). in all cases a severe decline of platelet count and in cases a positive hipa test (= heparin-induced platelet antibodies) were documented. patients, clinical course and outcome: female and male patients (median age years, range - ) were analysed in this retrospective study. pts were referred from outside hospitals. all pts had been treated with ufh ( pts additionally with lmwh) for prophylaxis (n = . in pts pedoperatively) or treatment (n = ) of venous thromboembolism (v'te). in pts at least one vte occured during heparin therapy, in all cases prior to hat-diagnosis (vte: n = ; putmonary embolism: n = ; artenal embolism n = ; thrombosis of sinus transversus n = ). hat was suspected . days (median; range - ) after first heparin application. hepadn therapy was discontinued immediately, at that time the median plateiet count was g/i (range - ). pts were treated with orgaran r, followed by recombinant hirudin (r-hirudin, behdngwerke ag: clinical study) in cases; pts were treated exclusively with r-hirudin. plat~let count normalized in pts within days (median; range - ). one patient in the orgaran" group died on recurrent pulmonary embolism. pts recovered without any further thromboembolic complication. conclusion: in our small series of ts hat was complicated with vte in / cases. in many pts hat was diagnosed strikingly late. for early diagnosis careful monitonng of platelet counts are mandatory dunng hepadn therapy, both orgaran ~ and r-hirudin seem to be safe and effective in preventing further thromboembolism, p to prospectively evaluate coagulation or fibrinolytic activation in children long-term anticoagulation with lmwh was administered a longitudinal study was perfomed. within a months period children and adolescents ( - years) suffering from malignant bone tumors (ewing's sarcoma or osteosarcoma) received enoxaparin (clexane r/rhone-poulenc rorer, france) for primary propylams. the majority of patients received . mg/kg bw enoxaparin hours before major surgery (limb salavage). in addition, ten neonates and children received enoxaparin in the same dosage for secondary prophylaxis. in both groups therapy was adjusted to . - . iu/ml anti xa activity. median (range) duration of therapy was performed for l ( - ) weeks. before (' " ), one (t ) and weeks (t ) within the context of the present study an investigation on the physiology of clotting was conducted in three groups each consisting of patients, who had to undergo a total end-prosthetic hip implantation (teph) for the first time. criterion for allocation to a particular group was type of intraoperative transfusion the patient received : infusion of whole blood, erythrocyte concentrate or fresh frozen plasma. at eight fixed times the activity or concentration of the components of the kallikrein-kinin and inhibitory systems was determined. the statistic evaluation ensued with the spss software package. for all three patient collectives an activation of both systems was to be observed with the teph-implantation whereby there were no statistically striking differences between the three groups. thus in the case of the kallikrein-like activity an intraoperative rise of over % was registered, while at the same point in time there was only a % reduction in the prekallikrein activity. postoperatively a % rise in the prekallikrein activity was to be observed. as a reserve inhibitor alpha -macroglobalin showed no significant change in activity during the period of observation whereas the acute phase proteins alpha -proteinase inhibitor and cl-esterase-inhibitor yielded a postoperative rise of % and up to %. the beta-factor xllainhibition showed a rise of % during the reconvalescent phase. the year old female caucasian patient presented here, was admitted to a nearby district hospital in december ' with the acute symptoms of pulmonary embolism. the source of the embolus could not be detected. high dose heparin was administered. pmi-i revealed a coronary artery disease and a peripheral artery occlusive disease of the left lower leg, yet blood pressure values of the lower ex~emities were only slightly below brachial pressures. the first episode of pulmonary embolism was followed by a second one two weeks later for which the patient received heparin again. following this treatment the platelet count decreased significantly to /ni. with an acute thrombosis of the lower caval vein and with leriche's syndrome, i. e. occlusion of the abdominal antra from the bifurcation down to the ileofemoral arteries bilaterally, the patient was transferred to our hospital. on initial examination we saw a patient in no acute distress, no dyspnea, blood pressure / mmhg, pulse /rain, no peripheral edema or disturbances of sensitivity. neither femoral, popliteal, nor pedic pulses were palpable on either side. a heparin associated thrombopenia was detected with the platelet aggregation assay and confirmed by the heparin induced platelet aggregation assay (hipaa). the autoantibodies targeting the underlying pf -heparin complex were determined later by an elisa method in serum samples of the patient (asserachrom hia, diagnostica st,ago). a temporary anth&~r filter system was inserted transbmchially into the lower eaval vein and placed jnst above the thrombus. in the following days, cycles of lysis therapy with mil u streptokinase were administered taking six hours for each cycle. heparin was given in the mean time via the filter system in therapeutic dosages in order to prolong values . to times the individual base line level. at the end of the second cycle, pulses were palpable inguinally, two hours later both popliteal and the right pedic pulse were present. the left leg showed better microperfusion than before. computer tumoglaphy did not reveal an)-thrombotic material in the descending aorta and the iliac arteries. for continuous anticoagulation after lysis we used the heparinoid orgaran® to overlap the period until full anticoagulation was achieved by coumarin. the heparinoid was tested before us non-reactlve in the hipaa. with this prophylaxis the platelet count increased to normal values. hat ii related thrombosis is the most severe complication of heparin therapy, especially in patients with pre-existing thromoembolic disorders (dvt, dic). we present to our knowledge the first case of hat ii during thrombolytic therapy (it) with simultaneous hepatin administration. at admission a year old girl showed complete occlusion of the pelvic veins of the right leg (r common and external iliac v., common and proximal superficial femoral v.). as in children no prospective studies on the thrornbolysis of dvt with rt-pa are available, we started tt according to the frankfurt/m. regimen: rt-pa (actilyse) bolus injection . nag/kg bw followed by continuous infusion of mg/kg bw/d; simultaneous low dose heparin - u/kg bw/d. doppler ultrasonographie control on day nine after the beginning of tr revealed a further distal growth of the thrombus into the popliteal vein. at the same time at ir was decreased to % and there was a dramatic drop in platelet count to < % of the baseline value ( to gpt/ ). the diagnosis hat ]i was confirmed by positive hipa-test. the only heparin preparation not cross-reacting with the antibody was the lmw heparinoid orgaran. heparin therefore was replaced by orgaran: bolus injection , u/h followed by infusion of u/h. rt-pa-therapy was not discontinued. at ri substitution was performed at levels < %. under orgaran administration platelet count rose from to gpt/ within days. on day complete reperfusion was achieved and rt-pa was discontinued ,on day . the patient was discharged from the hospital on prophylaxis with phenprocomnon in good general condition at admission thrombophilia parameters (protein c and s, apc) were within normal range. heparin induced thrombocgopenia (hit) is a serious but rare side effect of treatment with unfractionated heparin. patients with hit present with a rapid decrease in thrombocyte count and occurence of venous or arterial thrombosis under heparin treatment. patients with arterial occlusive disease who need surgical intervention are of high risk to develop rethrombosis especially during the first few postoperative days while they are under i.v. heparin-treatment. in our study we investigated prospectively all patients who were admitted to a surgical unit because of arterial occlusive disease and received surgical intervention. patients were screened on the rd and th postoperative day for heparin-antiplatelet -antibodies, decrease in platelet count and thromboembolic complications. patients developed antibodies against heparin-platelet factor . one of the patients developed a venous thrombosis in the operated leg in combination with a decrease in thrombocyte count. we conclude that heparin-platelet factor -antibodies are often produced in patients under i.v. heparin treatment. only few of these patients actually suffer from clinically s anptomatic hit. medizinische universit~itsklinik, institut far klinische biochemie und pathobiochemie, d- wtirzburg, germany the important physiological and pathophysiological role of platetets in health and disease is well established. platelets are also the primary target of many vasoactive hormones, factors and drugs. in addition, platelets provide a very important model system for studying agonist-and antagonist-mediated signal transduction events ( , ). platelet activation by agonists is associated with shape change, extension of filopodia, generation of intracellular messengers, secondary agonists and activation of adhesion receptors and integrins including the fibrinogen receptor gp iib/iiia. activation of protein tyrosine ldnases and calcium-dependent protein kinases appear to be of critical importance for the mechanism of platelet activation. in contrast, platelet inhibition by vasoactive factors and drugs is often mediated by the camp and/or cgmp signal transduction cascades. recent data form our laboratory suggest that the focal adhesion protein vasp, a major target of the intracellular no/cgmp and prostacyclin/camp effector systems, is an important link between signal transduction pathways and elements controlling cell motility ( - ). the implications of the recent advances in platelet signal transduction research for understanding the physiology, pathophysiology and pharmacology of human platelets will be discussed. domain. however, in vivo truncated "i'po is much less potent than full length tpo due to its rapid clearance. this suggest that the c-terminal glycosylated domain acts to stabilize circulating tpo. tpo stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet producing megakaryocytes in vitro. in vivo tpo induces dramatic increa~ses in megakaryocyte number and platelet production in animals, indicating that tpo regulates both thrombopoiesis and megakaryocytopoiesis and is the primary regulator of physiological platelet production. this later notion is supported by the phenotype of ti~ and cmpl knockout mice in which platelet levels are only - % of normal while other blood lineages are unaffected. in animal models of myetosuppresion ,and marrow transplant, tpo reduces the platelet nadir seen in the~se models and significantly accelerates platelet recovery, tpo also has profound effects on red cell recovery in these models. these results suggest that tpo will be clinically u~ful for the treatment of thrombocytopenia in caucer patients who undergo chemotherapy or bone marrow transplant. the haparg-study ( .~a.emostatic parameters as risk factors in healthy volunteers) was sponsored by the german ministry of research and started in . a previous study ( pard, intern. angiology , - , ) had shown that spontaneously enhanced platelet aggregation as measured with the pat-ill-test and a high fibrinogen level were significant risk factors for new vascular occlusions in diabetic patients. it was the aim of the haparg study to establish if spontaneous plateletsggregation and other hemostatic variables are independent risk factors for vascular occlusions also in healthy volunteers. employees of hschst ag, frankfurt aged - years and personnel of the university of mainz, aged - y. entered this prospective study. besides anamnestic data on smoking, hypertension and diabetes blood pressure, the ankle/arm doppler -index and an ecg were recorded and serum cholesterol, hdl, ldl, triglycerides, uric acid and glucose were measured. men and women entered the study and were followed for - years. during this period vascular occlusions ocoured ( coronary and cerebral events and peripheral vascular occlusions). only three of these events occured in women. besides age and gender as expected risk factors the multivariate logistic stepwise regression analysis revealed as significant risk factors slightly elevated levels of blood glucose (p= . ), smoking (p= . ), an elevated diastolic blood pressure (p= , ),followed by spontaneous aggregation (p = . ) and higher hdl-levels (p= . ) as significant risk factors for men. higher fibrinogen levels just missed significance in this analysis (p= . ). none of the other hemostatic parameters showed any significant correlation with the vascular events.to our knowledge this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. spontaneously enhanced platelet aggregation seems to be an independent risk factor and may be an indicator of an ongoing active atherosclerotic process. mild bleeding symptoms -petechia, prolonged bleeding after tooth extraction -were observed in a patient with normal coagulation tests but slightly disturbed phtelet function: decreased aggregation in response to adp, pal: and thrombin receptor-derived peptide (trap- ). platelet count, agglutination with both ristocetin and botrocetin and thrombininduced aggregation were in the normal range. an abnormal membrane glycoprotein detected in electrophoretic studies of the patient's platelet proteins was shown to be related to gp llxx by immunoblotting. the variant gp differed even from the largest normal polymorphic gp iba form a by an increase in molecular mass, platelets of the patient contain the variant and normal c form in equal amounts. the quantity of gp ib as detected by flow cytometry is normal. lmmunoblotting studies of proteolytic fragments of the abnormal gp indicated that the defect is located in the c-terminal part of the glycocalicin fragment. measurement of the length of glycocalicin molecules after glycerol-spraying rotary shadowing electron microscopy demonstrated two populations of molecules in the patient sample: a normal on with an average size of angstr m and a fraction with an average size f angstr n~ preliminary results of molecular analysis of the gp iba gene in the patient confirm the localization of the genetic defect by protein studies. in summary, the patient is heterozygous for a hitherto undescdbed molecular variant ofgp ~a. barnes and co-workers have shown that simple triple-helical collagenlike peptides, comprising basically a repeat gly-pro-hyp structure, are highly platelet reactive in polymeric form. they additionally have shown that the activity does not involve gpla/lla (integdn c~z~l). platelets adhere under static conditions to these fibrillar peptides mainly in a bivalent-cation independent manner; the bivalent cation dependent adhesion can be inhibited by an antibody against gpflb/llla (integfin m~d ). however, platelets of a patient with type i glanzmann's thrombasthenia secreted their ¢z-granule-and dense body content just like normal controls while activated with the triple-helical gly-pro-hyp peptide. the lack of an essential involvement of cd was shown with cd deficient platelets, which respond to the peptides as readily as normal platelets when fibdnogen binding and cd and cd expression were measured. to study the involvement of the yon willebrand factor (vwf) platetets from a patient with severe type vwd (vwf not detectable in platelets and plasma) were investigated. the vwf deficient ptatelets responded quite normally to the fibritlar collagen-like peptide. addition of pudfied vwf to the vwf-deficient platelets before adding the paptide raised the platelet respond slightly we conclude that neither ~ ~ integrin, nor m~ integdn, nor cd , nor vwf are the pdmary platelet receptor for the collagen-like peptide. there is evidence that native low density lipoprotein (ldl) can be oxidatively modified in vivo and the oxidized ldl may contribute to thrombogenesis and atherogenesis by damaging endothelial cells, eliciting vasocontractiola and stimulating blood platelets. by means of biochemical methods, electron microscopy, reflection contrast microscopy and computer image analysis, we find that oxidized ldl, compared to native ldl, affects platelets at least in the following five aspects. ) oxidized ldl alters platelet functional morphology in a time and dose dependent manner. ) oxidized ldl induces secretion of serotonin from both morphological resting platelets and shape changed platelets. ) oxidized ldl advances, accelerates and enlarges platelet adhesion. ) oxidized ldl causes cytodamage in platelets and cultured endothelial cells. ) oxidized ldl stimulates platelets and endothelium, which leads to a formation of microthrombi on the monolayer of human umbilical vein endothelial cells. further experiments show that oxidized ldl inhibits the activity of ca +-atpase in purified plasma membranes of platelets, and therefore increases the cytosolic calcium level in platelets. these results clearly indicate that calcium signaling pathway is intimately involved in the mechanism of platelet activation induced by oxidized ldl. since oxidized ldl, a relevant platelet agonist, has been confirmed to be present in vivo, further studies on its role in platelet activation and plateletendothelium-interaction can help for better understanding thrombogenesis and atherogenesis. recantly physical parameters such as sheer stress, hypo-or byperoxic conditions and hyperthermia have been shown to modulate endothelial cell dependent fibrinolysis. in this study we investigated whether yet another physie~ stimulus namely hypothermia might have an impact on the expression of components of the fibdnolytic system of human endothelial cells in culture. confluent cultures of human umbilical vein endothelial cells (hlivecs) were exposed to gc ° for , , , , or rain, respectively. control cultures were exposed to °c for the same time periods. thereafter medium was removed, fresh medium was added and the cells were incubated for hours at °c conditioned media (cm) were harvested and plasminogen activator inhibitor-i (pal-i) antigen was determined by a specific elisa. our data showed that huvecs responded to exposure to °c with a time-dependent increase of pai-i in cm. maximum induction of pal-! antigen was seen after minutes exposure to °c (pal-! antigen: _+. ng/ cells for treated cells; _ ng/ cells for control cells). the inereese in pal- antigen was also reflected on the level of specific mrna synthesis as evidenced by northern blotting which showed a twofold increase in pai-i specific mrna in huvecs exposed to °c as compared to control cells. in conclusion, our data gives evidence that hypothermic stress like several other forms of physiological or pathological stress, results in activation of endothelial ceils as evidenced by an increased expression of pai-i. our findings provide ~rther evidence that such activation of endothelial cells can be induced not only by biochemical mediators but also by physical stimuli. nitric oxide (no) is formed from intracellular l-arginine by two no synthase (nos) isoforms: a ca+ +-dependent constitutive form (cnos) and a ca+÷-independent inducible form (inos) which is expressed after challenge of cells with immunologic or inflammatory stimuli. endothelial cell derived no is an important signaling molecule inducing smooth muscle cell relaxation and platelet inhibition, however its role in cell proliferation is poorly understood. the present study investigates the pattern of nos isoforms and no production in actively proliferating low density human umbilical vein endothelial cells (ld-huvec) and in high density monolayers of quiescent cells (hd-huvec). ld-huvec but not hd-huvec expressed inos (measured as ca ++independent citrulline formation from l-arginine in cell lysates) with a maximal activity of - pmoles/min/mg protein, cnos (ca +÷dependent citrulline formation) was demonstrated in hd-huvec ( . - . pmoles/min/mg) but not measurable in ld-huvec lysates. lmmunoprecipitation of cell lysate with a mab against murine macrophage inos decreased inos activity of supernatant by % in ld-huvec. immunoprecipitation with an anti-ecnos mab did not modify inos activity in ld-huvec but abolished cnos activity in hd-huvec. no release (measured as nitrite + nitrate production in an h incubation in the presence of mm l-arginine) was . nmol/mg cell protein in the supernatant of hd-huvec whereas it was t fold higher with ld-huvec. measurement of intracellular cgmp after stimulation of cells with i- #m ionomycin (a sensitive index of enos activity) showed a - % increase of basal levels in hd-huvec but only - % increase in ld-huvec. on the contrary, sodium nitroprusside-induced cgmp increase was similar in non-confluent and confluent cells. the expression of inos and the high no production in ld-huvec suggest a role of endogenous no in mediating cell proliferation. indeed, the nos inhibitors l-nmma and l-canavanine inhibited [~h]thymidine incorporation in ld-huvec by %. on the ++ nt no r uctlon ar x r d m other hand enos and ca -depende p od " e e p esse " differentiated huvec and correspond to the ability of the cells to regulate vascular tone and platelet activation. the processes of replication, maturation and motility of progenitor cells of different lineages in the bone marrow are tightly regulated, particulady by various cellular contacts and their modulation involving pericellular proteolysis. while mononuclear phagocytes and related cell lines are well characterized for surface localized plasminogen activation by receptor bound urokinase, megakaryocytes are poorly described in this respect. in the present study the expression of mrna of urokinase receptor in megakaryoblastic cell lines chrf- , dami and meg was found at a low basal level and increased - fold after pma-treatment. urokinase receptor mrna levels remained differentiation dependent elevated for up to days. functional analysis of upar expression on the cell surface was documented immunologically by facs-anatysis and on the functional level by direct binding of radio-labeled amine-terminal fragment of urokjnase and mirrawed the increase in urakinase recptar mrna level. as expected, antibody and ligand binding was sensitive towards treatment with phosphatidyl-inositol-specific phospholipase c, since the urokinase receptor belongs to the group of gpi-anchored receptors. in cultered megakaryocytes derived from bone marrow urokinase receptor mrna was demonstrated by rt-pcr, as well. these results indicate a differentiation-dependent increase of urokinase receptor expression on cells of megakaryoblastic cell lineage suggesting the participation of urokinase dependent events during maturation process and intravasation of these platelet precurors. regulation of am gene expression may be coupled to oxidative stress through redox sensitive transcriptional regulatory factors. the aim of this study was to investigate the influence of selected antioxidants on tnfa ( u/ml, h)-induced surface expression of elam-i, icam-i and vcam-i in human umbilical vein endothelial cells. am expression was determined by quantitative flow cytometry and immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase method). the following antioxidants were tested: pyrrolidine dithiocarbamate (pdtc), n-acetylcysteine (nac), glutathionemonoethyl ester (gshmee), probucol and the estradiol-derived scavestrogens j , j , j . additionally, fi-estradiol was examined. the used concentrations were - mm for nac and gshmee and - ~m for all other substances. according to their influence on tnf-induced am expression, the tested antioxidants can be divided into three groups: ) pdtc, nac and gshmee inhibit the expression of all three am, ) j and j reduce vcam-i and icam-i and increase elam-i, ) j and probucol show no effect. fl-estradiol elicits a potentiation of tnfa-induced expression of all three am. tnfa-induced vcam-i expression was most effectively inhibited by ptdc and j ( em: % and % inhibition, respectively). the best inhibitors of icam-i induction were pdtc ( ~m: % inhibition) and j ( t~m: % inhibition). j and j most effectively increased the elam-t induction ( /~m: % and %, respectively). the results confirm that antioxidant-sensitive mechanisms play a role in tnfct-induced am upregulation. it is suggested that icam- -and vcam-l-expression is regulated differently from elam-i. the diversity of antioxidant effects might be due to distinct levels of redox control of transcription with an antioxidant-inhibited and an antioxidant-promoted step. heamophiliacs who continue to produce tilers of f.v i inhibitors no higher than to bethesda units (bu) when given continuous f,viii replacement therapy are classified as low responders. studies carried out on low responders to assess the effect of immune tole~lcc therapy flit) in these patients. it was found that elimination ofiahibitors in low responders could be achieved using f.vlii at to too u/kg given every to days over a period of weeks. we consider that tt is justified in this group of patients. prevents rebooster effects and reduces elimimfion time of f.viii inhibitors. it also reduces the enhanced mortality in patients with inhibitors and has been shown to be cost-effective. in view of the improved prognosis, we recontmend starting itt in low respor, ders soon after the diagnosis has been made in order to minimize f.vill exposure before isfi.tiating el'. itr in low responders has a better success rate an.,d requires shorter u'eatment than itt in high responders. w.schramm, med.klin~, innenstadt, lmu, mfinchen following replacement therapie~ in hemphilm t_be developmem of inhibimrs to the p*ficat's deficient factor is the most serious sequelae. the incidence of inhibitors varies between and % of persons with hemophilia a. inhibitor develop, mostly in paticm~ with sovere bemoph/lia a, early in life and afmr few exposure days ( - days). using the bethesdamethod the inh~itom can be derided in low (< bu) and high msponder (> bu) hemopb~iacs with high fitcrs have ~ually serious ~ problems. they are resistent to mg,,flary replacement therapy, the ~ goal in the treawnent is to control severn acum bleedin~ and to eradicate the inlu'bitor perrmnanfly and to induce tolea'ance. in the tream'tmt of acute blcedings in patients with hlhibitors factor viii inhibitor bypassing ag~ts like activated prothro~ complex concenuxtes (feiba) or prothrombin complex concentrates (pcc) arc mostly used. the meehani~n of aefiou of theses concentrates is net fully investigated. their effect is usually related to the high coment of activated clotting factors ~d phosphoupids. since some years acdwated recombinant factor vii (f vii a) is used to treat patients with inl'dbitocs successfully in several clinical situations including surgery. in addition porcine factor vii is widely used in particular in the uk for the treatment of factor v].ii inhibitor patients and could demonstrade good clir cal results, in case of life threatening bleedings a temporary reducfic~ of inhihitors could be. ~hieved by using extem*,ivc plasma exchange (protein a adsorption) and immune suppression with cyclophosphamid (~alm protocol). follow~g the first description by h. bmc~'~mn some modifications for tlm induction of irmnune tolerance in hemophilia a patients have ~en propet'ed. these schedule, can be derided into high, intemxxfime and low dosage roglrmms di:ffea'jng in the dosage of factor viii infused. successful rates about to go % can be obtained with ~ and high dose regimens. but is has to be co~sidered that the~ expensive trea.t~nt regimens have a great physical and p .syc.hosocial impact to the benx~-li~s and thch" farm~e& the different immu~ mler-a.~ze mg~-'~ predominantly used in high rcsponder inhibitor. most of the patients with low concentrations of inhibitors cm be managed with factor viii in increased dosage. this is in agreement with the consensus recorrnr~rdadons for u'eatlncnt hemophiliacs in germany fi'orn . before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was : both idiopathic and secondary types. since , nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early . however, in a small number of eases, vk deficiency oceured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacental transport of vk in the perinatal period,following studies were carried out. t)hepaplastintest(normotest) were performed on women in the last stage of pregnancy and each coagulation factor was estimated as well. )correlations were made between mothers'and babies'hepaplastin test values. )transplacental transport of vk was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastin test value of less than % of the normal adult value, the value of the hepaplastintest was less than % of normal adult value in the cord venous blood° we also demonstrated that vk passed through the placenta but only in small qualities. hiv-negative patients (median age ]yrs, - }, formerly treated with non-virusinactivated coagulation products, underwent hepatologic examination, including afp screening and sonography. .suffer from severe, from moderate or mild haemophilia a or b, from other severe coagulation factor deficiencies. had been treated with products of the swiss red cross (src) only ( with small pool cryoprecipitate}, with foreign products only, with both src and foreign products. treatment intensity was variable with> ' iu/yr in ,< ' in ] , < treatment episode/yr in ] , a total of only - treatment courses in patients. afibrinogenemic patients had prophylactic replacement therapy. hcv serology was positive in / ] patients ( %), in with detectable hcv rna ( %). the persons who escaped hcv infection, with normal alt-levels and without sonographic alterations, had low intensity treatment with small pool src preparations only. alt-levels were elevated in / anti-hcv positive patients ( %). / had abnormal sonographic findings ( %). there was a clear correlation between elevated alt-levels and abnormal sonographies: of patients with elevated alt had abnormal sonography, of with normal alt had abnormal sonography. patients had liver cirrhosis ( with clinically overt hepatopathy), ( / = %!) with hepatocellu]ar carcinoma (hcc) with elevated afp-leveis. of these patients had intraarterial embolization with ]ipiodol-epirubicin; in patients hcc diagnosis was made in a late stage. i patient with advanced liver cirrhosis underwent successful liver transplantation. of the patients with hepatopathy had severe haemophilia with temporary high alcohol intake, had mild coagulatlon disorder with few treatment episodes. possible precipitating factors were coinfection with hbv, high alcohol consumation and first exposure to hcv contaminated blood products in an advanced age, but not intensive replacement therapy. very similar results for f vlll and vwf. since the factor viii level is kept steady above the level where there is an increased risk of haemorrhage, continuous infusion is haemostatically safer and more efficacious than bolus injections, another advantage is a progressive decrease of clearence during the first days after surgery which leads to a substantial reduction of factor concentrate consumption by avoiding the innecessary peaks of bolus injections. children with severe form of haemophilia a undergoing elective surgery received continuous infusions with different plasma-derlved and recombinant f viii concentrates. before surgery, patients got bolus injections to raise the factor viii levels to more than %. during continuous infusion factor viii levels were measured two to three times a day and the infusion rate of to iu/kg/h could be reduced on the second or third day to - iu/kg/h. the clinical efficacy was excellent with no bleeding events. in children with vwd also undergoing elective surgery continuous infusions with humate pr were performed in the same way. no bleeding events were observed in these patients. none of the patients developed postoperative wound infections. the overall doses of f vtll concentrate 'were about - % lower than those required during replacement therapy with bolus doses. lg factor x frankfurt i : molecular and functional characterisation of a hereditary factor x defect (gla + lys) huhmann i., holler b., krinninger b., turecek p.l, richter g., scharrer i., forberg e., watzke h. univ. klinik f r inhere med.i, abteilung for h~tmatologie und h~mostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrombin to thrombin. the congenital fx-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a year old patient presenting a mild bleeding tendency. his p'fi ( sec) is within the normal range, the pt ( % of normal) is slightly reduced. the factor x antigen level is reduced to % of normal. molecular charactedsation of the genetic defect was performed by amplification of the eight exerts and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of + gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboll site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exert ii. fx was isolated from plasma of the propositus by monoq ion exchange chromatography. performing clotting assays with purified fx frankfurt i we determined an activity of % of normal fx upon activation with rw, % upon intdnsic activation (aptt) and % upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt %, ptt % and rw % of normal) when the reduced fx-ag-level of the plasma ( %) is taken into account. we therefore conclude that the substitution of gla + to lys results in a fx molecule which is severely defective in both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardiothoracic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardiopulmonary bypass surgery. blood samples were drawn preoperatively as well as , , hours and , , , , , , days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, thrombin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= patients were examined (age: __+ y). they lost +__ ml blood (mean+sd) into the drains within the first hours after end of surgery. a severe bleeding was defined to exist, if the blood loss exceeded this range (> ml within h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding mg/i at end of surgery (n = ) had a negative predictive value of %, positive predictive value of %, specificity of % and a diagnostic efficacy of %. in contrast, soluble fibrin which correlated well with fibrinopeptidea (r> . , n= ) did not correlate neither with degradation products nor with bleeding complications (n = ). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near % and a diagnostic efficacy of > % (pat. without antifibrinolytic drugs), which complies to findings from bredbacka ( ). other parameters were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibrin for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrin will provide further insights into fibrin(ogen) metabolism. heparin induced thrombocytopenia represents a multicomponent syndrome associated with the use of heparin and related drugs resulting in not only thrombocytopenia, but also arterial thrombosis of varying magnitude. the initial diagnosis ofthis syndrome is usually made by clinical observation and a drop in platelet count. conventional diagnostic methods include platelet aggregation responses to patient's serum and ~ c serotonin release in response to patient's serum, aggregation/agglutination of patient's platdets in response to heparins and the detection of patients anti-heparin platelet factor (hpf -ab) ned-antibodies by using elisa methodology. several other individualized methods are also used to demonstrate platelet activation. to test the diagnostic validity of the platelet aggregation (pa) c serotonin release (sr) and the relevance of hpf~-ab serum samples collected from patients with clinically eunfwmed eases of lilt syndrome were compared in parallel in various assay systems. the diagnostic efficacy of these tests varied from - % with the pa test providing better results than others. when the pa test was compared with serotonin release, a poor correlation was noted (r= . ). in contrast, the correlation between the pa and hp -ab was somewhat better (r= . ). in another study, blood samples collected from patients treated with ahigh dose low molecular weight beparin for two weeks ( mg o.d.) were tested. of these patients showed a high titre of hpf .ab without any decrease of platelet count. none of these patients were found to be positive in the c serotonin release assay. a third study included blood samples from dvt patients administered with iv heparin infusion, high dose sc lmw heparin (certoparin) and iv lmw heparin for the management of dvt. none of these patient groups ( - ) exhibited any hit responses, hmvever, the incidence of high hpf -ab titre was found to be % in heparin, % in patients with lmw heparin iv and % in lmw heparin sc groups. pa and sr studies revealed % and % false positive ~ respeetively. these studies clearty suggest that the currently available ~ for laboratory diagnosis of hit syndrome are of limited value, and caution should be exercised in the interpretation of the results obtained with these tests. heparin-induced thrombocytopenia (hit) is one of the major severe side effects during treatment with heparin. in postoperative medicine clinical studies demonstrated the prevalence of hit with unfractionated over fractionated heparins. few data are available from the non-ope "ative medicine and from patients without thmmboembolism before heparinization. in a controlled prospective randomized study the safety and efficacy of low-dose heparin was compared with a lowmolecular-weight (lmw) heparin over days in bedridden medical inpatients (haemostasis, in press). patients were randomized and controlled for the development of thrombocytopenia. thrombocytopenia was defined as a platelet count below . lid at day . patients developed thrombocytopenia in the heparin group and no patient in the lmwheparin group (p< . ). none of the patients with thrombocytopenia developed a thromboembolic complication. in a second prospective case control study patients with side effects on anticoagulants were treated with lmw-heparin once daily subcutaneously for a period of month to years. platelet count was performed every to months. none of these patients developed thrombocytopenia during heparinization with lmw-heparin. it is concluded that hit is a very rare complication in nonoperated bedridden medical patients. a decrease of platetet count may occur in about . % of patients receiving low-dose heparin. the incidence of hit with thrombosis during low-dose heparin and of hit during lmw-heparin in non-operated patients is manyfold lower and remains to be determined. terminology: instead of the term "hemorrhagic disease of the newborn (hdn)" the term vkdb should be used, since neonatal bleeding is often not due to vkdeficieacy and vkdb may occur after the neonatal period (i.e. after weeks). definition: vkdb is a bleeding disorder caused by reduced activity of vkdependent coagulation factors which responds to vk. diagnnsis: in a bleeding infant a prolonged pt (inr > . ) together with normal fibrinogen and platelet count is almost diagnostic of vkdb. the diagnosis is proven, if vk shortens the pt (after only - minutes) and/or stops bleeding. classification: classification by age of onset into early (< h~. classic fdav - ) and lale form (> i week < months), and by etiology into idionathic and ~ec nd~'y. in secondary vkdb in addition to breast feeding other factors can be demonstrated, such as poor intake or absorption of vk and increased consumption of vk. vk-prophylaxis: benefits: oral and intramuscular (i.m) vk (one dose of i nag) prevents equally well the classic form of vkdb. lm. vk appears to be more effective in preventing the late form (times -> ). the protection achieved by single oral prophylaxis (times - ) is improved by triple oral vk (times - ). risks: because of poten[ial ri~l~ associated with extremely high levels of vk and the possibility of injection injury, i.m. vk has been questioned as the prophylaxis of choice for normal neonates. since vk is involved not only in coagulation but 'also in carboxviation with multiple effects, excessive deviations from the low physiologic concentrations, which prevail in the fully breast-fed healthy mature infant should be avoided. proposal: repeated (daily or weekly) small oral doses of vk are closer to physiologic conditions than single i.m. bolus doses, which expose neonates to excessively high vk levels. the incidence of intracranial vkdb can be reduced if the grave significance of warning signs is recognized (i.e, icterns, failure to thrive, feeding problems, minor bleeding, disease with cholostasis). whether or not the more reliable absorption of the new mixed mieellar (mm~ nrenaral~i n of vk can reduce the protective oral dose of vk-.prophylaxis has to be evaluated. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was : both idiopathic and secondary types. since , nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early . however, in a small number of cases, vk deficiency occured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacentel transport of vk in the perlnatal period,followlng studies were carried out. )hepaplastlntest(normotest) were performed on women in the last stage of pregnancy and each coagulation factor was estimated as well. )correlatlons were made between mothers'and babies'hepaplastin test values. )transplacental transport of vk was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastln test value of less than % of the normal adult value, the value of the bepaplastlntest was less than % of normal adult value in the cord venous blood. we also demonstrated that vk passed through the placenta but only in small qualities. the point mutation g to a at nt in exon v of the factor x gene (gin to lys) has previously been found in two independent kindreds with fx deficiency. it occured in both families in an heterozygote state and was associated with two other genetic defect in the fx gene. we have identified another familiy in which this mutation occurs in a homozygote state. in this family the mutation is associated with the previously reported mutation gla to lys which also occurs in a homozygote state. the pt and ptt of the proposita and her siter are markedly prolonged. the fx activity is reduced to < % in the extrinsic system, to % in the intrinsic system and to % after activation with rvv. the fx antigen is reduced to %. the coagulation profile of this family thus is identical with that of fx vorarlberg despite the fact that the fx vorarlberg kindred is only heterozygous for the mutation glal to lys. haplotype analysis could not rule out consanquinity with the fx vorarlberg kindred. these data suggest that the mutation at nt which leads to a fairly dramatic amino acid change from glu to lys would indeed represent a polymorphism. to further address this question we cloned the fx gene in an expression vector (pcep ) for transient expression in the human embryonic kidney cell line and introduced the mutation at nt by site directed mutagenesis. hereditary deficiency of factor ixa, a key enzyme in blood coagulation, causes hemophilia b, a severe x-chromosomelinked bleeding disorder; clinical studies have identified nearly deleterious variants. the x-ray structure of porcine factor ixa shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for fx activation by phospholipid-bound flxa and cofactor villa. the . a resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (egf)-like modules; the n-terminal gla module is partially disordered. the catalytic module, with covalent inhibitor d-phe-pro-arg chloromethyl ketone, most closely resembles fxa but differs significantly at several positions. particularly noteworthy is the strained conformation of glu- , a residue strictly conserved in known fixa sequences but conserved as gly among other trypsin-like serine proteinase. flexibility apparent in electron density together with modelling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fixa. most hemophilic mutation sites of surface fix residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary flxa-fvilla-fx complex structure: the stabilizing fvilla interactions force the catalytic modules together, completing flxa active site formation and catalytic enhancement. factor x frankfurt i molecular and functional characterisation of a hereditary factor x defect (gla + ---, lys) huhmann i., holler b., krinninger b., turecek pi., richter g., scharrer i., forberg e., watzke h.. univ. klinik ftlr innere medi, abteilung for h~matoiogie und hamostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrembin to thrombin. the congenital f×-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a year old patient presenting a mild bleeding tendency. his ptt ( sec) is within the normal range, the pt ( % of normal) is slightly reduced. the factor x antigen level is reduced to % of normal. molecular characterisauon of the genetic defect was performed by amplification of the eight exons and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of + gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboti site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exon i . fx was isolated from plasma of the propositus by monoq ion exchange chromatogrephy. performing clotting assays with purified fx frankfurt i we determined an activity of % of normal fx upon activation with rw, % upon intrinsic activation (aptt) and % upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt %, ptt % and rw % of normal) when the reduced fx-ag-level of the plasma ( %) is taken into account_ we therefore conclude that the substitution of gla + to lys results in a fx molecule which is severely defective ip both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardioth~)racic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardlopulmonary bypass surgery. blood samples were drawn preoperatively as well as , , hours and , , , , , , days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, throm. bin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= patients were examined (age: + y). they lost +__ ml blood (mean_+sd) into the drains within the first hours after end of sur. gory. a severe bleeding was defined to exist, if the blood loss exceeded this range (> ml within h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding mg/i at end of surgery (n = ) had a negative predictive value of %, positive predictive value of %, specificily of % and a diagnostic efficacy of %. in contrast, soluble fibrin which correlated well with fibrinopeptide a (r> . , n= ) did not correlate neither with degradation products nor with bleeding complications (n= ). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near % and a diagnostic efficacy of > % (pat. without antifibrinolytic drugs), which complies to findings from bredbacka ( ). other paramelers were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibdn for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrln will provide further insighls into fibrin(ogen) metabolism. this study was conducted as a randomized parallel -group clinical trial comparing the safety and efficacy of a low molecular weight heparin {lmwh} -monoembolex sandoz and unfractionated standard heparin glfh) for the perioperative prevention of venous thromboembolie disease (dvt) following major surgms' in patients with gynecologic malignancy.. three hundred and twenty women (six drop outsl werr randomized and received either times daily [l" s.c. ul.'i-i (sandoz nuemberg germany] (n = ) or once a day t i~'v'. units s.c. monoembolex (n = ) plus two placebo injections. heparin therapy was started the morning before opcrati(m and continued until the th postoperative day. up to the th poatop, day the incidence of dvt was . % (n = ; incl. pulmona~ embolisms pe) in the lmwh group and . % (n = ; incl. pe} in the ufh group. the overall incidence of clinically hemorrhagic wound complications was significantly decreased in the lmwh group . % (n = hi compared to the ufh group . % {n = ; p < . . the incidence of major hemorrhagic episodes was . % in = in the lmwh group and . %/n = ) in the ufh group. this difference was not statisticauy significant. one case of fatal pe was observed in the lmwh -treated group. five women deaths in the lmwh group were observed during the study and in the ufh group. this study demonstrates that the perioperative treaunent of low molecular weight heparins is more safety than standard heparins in gynecologic -oncologic patients undergoing major surge .ry. however, the incidence of thromboembohc complications is simmilar in both treatment regimes. to explore the effect of targeting an antithrombin to the surface of a thrombus, recombinant hirudin (hir) was covalently linked to the fab' fragment of fibrin-specific monoclonal antibody d (fab) resulting in a stable conjugate (hir-fab). in vitro, hir-fab was times more efficient than hir alone in inhibiting fibrin deposition on experimental clot surfaces in human or baboon plasma (p< . ). to validate these results in vivo, hir-fab was compared to hir in a baboon model. the deposition of ill-in-labeled platelets onto a segment of dacron vascular graft present in an extracorporeal arteriovenous shunt was measured. blood flow rate was ml/min. one hour local infusions of atu of either hir-fab or hir resulted in deposition of . x and . x plate!ets, respectively. equieffective dosages were atu hir-fab and atu hir resulting in deposition of . x and . x platelets, respectively. based on full dose response curves (n = ), hir-fab was found to be > . -fold more potent (based on activity) than hir. because of the small total amounts of antithrombins used and the short duration of these experiments, no significant systemic effects were observed. thus, fibrin-targeted recombinant hirudin prevents platelet deposition and thrombus formation more effectively than uncoupled hirudin in vitro and in an in vivo primate model. triabin, a kda protein from the saliva of the assassin bug triatoma pallidipennis, is a new specific thrombin inhibitor ( ). tt does not block the catalytic center but interferes with the anionbinding exosite of thrombin. the recombinant protein was produced with the baculovirus/insect cell system and used to study the inhibitory effect of triabin on thrombin-induced responses of human blood platelets and blood vessels. aggregation of platelets in tyrode's solution was measured turbidimetrically at °c. for the studies on blood vessels rings ( - mm) from small porcine pulmonary arteries were placed in organ baths for isometric tension recording. the integrity of the endothelium was assessed by the relaxant response to bradykinin. like hirudin, triabin inhibited the thrombin ( . u/ml)-induced aggregation of washed human platelets at nanomolar concentrations (ec = . nmol/l); whereas the adp-and collagen-induced aggregation were not suppressed. in pgf c~-precontracted porcine pulmonary arteries, the thrombin ( . u/ml)-induced endothelium-dependent relaxation was inhibited by triabin in the same concentration range as found for inhibition of platelet aggregation. higher concentrations of triabin were required fo affect the contractile response of endothelium-denuded porcine pulmonary arteries to thrombin ( u/ml). in all these assays, the inhibitory potency of triabin was dependent on the thrombin concentration used. these studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of the thrombin-mediated cellular effects. dept. of medicine, university hospital benjamin franklin, free university of berlin, dept. of medicine and dept. of surgery, heinrich-heine:university dusseldod after standardized training in home prothrombine estimation using the coaguchek system, consecutive patients (p) who had st. jude medical aortic or mitral valve implantation were allocated to two random arms; p were asked to control the inr themselves every third day. in the remaining p anticoagulation was managed by the home physician without recommending an interval for these controls. all p were monitored during the education period to a target therapeutic range of inr . - . . p were asked to contact their home physician immediately if the inr was measured . below or above the target range (inr-corrider . - . ). all p had out-patient re-examinations every three months. thrombotic, thromboembelic and hemorrhagic complications were documented by the p using special documentation cards. the following findings were documented during the follow-up period: . . the results of this randomized study demonstrate a significant improvement in the management of oral anticeagulation by home prothrombine estimation. significant (p< . ) more inr measurements were found inside the target therapeutic range. moreover. bleeding and thromboembolic complications could be reduced (p = . ) in the study group with home prothrombine estimation. life-threatening thromboembolic and hemorrhagic complications were not observed in p who were on home prothrembine estimation, while three such events ( . %/year) were documented in group a. local vascular injury following ptca exposes circulating platelets to prodmmbogenic stimuli. by binding to platelet gp iiblliia fibrinogen crosslinks platelets, which represents the final common pathway of platelet aggregation. fradafiban (bibu zw) is a non-peptide compound with effective, reversible inhibitory effects on fibrinogen binding to gp iib/ii/a on human platelets. in the first double-blinded, prospective phase ii study three escalating doses of bibu zw as a continuous h-i.v, infusion were tested in comparison to placebo in patients with stable angina pectoris undergoing elective ptca. the mean receptor occupancy with rag, ms and ms per hour were . , . % and . % at hours, respectively. as compared to placebo breeding time was significantly prolonged ( vs rain) during fi-adaiiban infusion with a weak dose-dependency. platelet aggregation in platetet rich plasma ex vivo with collagen ( . and . gg/ml), adp ( . and . gmol/ml) or ca-ionophor a ( . and . gg/ml) was significantly and dose-dependently inhibited as compared to placebo. using the two upper doses of fradafiban, we observed major bleeding complications in patients requiring blood transfusions or vascular surreal repair. in these patients, too, maximal antiplatelet effects could be documented. these data sugest that bibu zw is an effective fibrinogen receptor antagonist in patients. the requirement of ad hoc receptor occupancy determination or platelet function monitoring for safe and effective clinical use should be evaluated. in a placebo controlled interaction study healthy volunteers were randomized to receive either a hour infusion of peg-hirudin ( . mg/kg/h) after an i.v, bolus of . mg/kg + placebo, or mg/day acetylsalicylic acid (asa) for three days followed by a placebo infusion or the peg-hirudin infusion + asa. each volunteer received all three treaments. there was a washout period of at least days between the infusions. at short intervals aptt, activated clotting time (act), ecadntime (ect), alia-activity using the chromogenic substrate , collagen-induced aggregation, platelet adhesion and platelet induced thrombin gene,ration time (pitt) were measured, bleeding time (simplate) was studied before drug administration, on day three before the infusion and hours after start of the infusion.the infusion of peg-hirudin after and hours led to a mean hirudin plasma level of . pg/ml. asa markedly inhibited collagen induced aggregation as expected. the mean bleeding time was prolonged under the influence of peg-hirudin from . to . min, after asa from . - . min and after the combination of peg-hirudin + asa from . - . min. in each volunteer the bleeding time was longer under the combination than after asa alone. in two volunteers receiving peg-hirudin + asa the bleeding time measurement was stopped after rain. none of the coagulation parameters or platelet function tests correlated with the prolongation of the bleeding time. however the bleeding time was excessively prolonged in those volunteers who had a marked prolongation under asa alone.the combination of hirudin at a higher dosage with asa probably is associated with a relative high risk of bleeding. either the hirudin dosage should be reduced if the combination seems feasabie or asa should be given after the end of hirudin treatment. fibrinogen with the sta/stago and the mla/dade systems correlated well, but neither system correlated well with the acl/il system. at iii, protein c, protein s, and anti-xa heparin assays using stago reagents performed as expected for normals and low abnormals on the sta. factor levels on the sta/stago system were less sensitive than factor levels obtained with the dade reagents on the mla or fibrometer. using the sta/stago system, thrombin time results correlated well with the aptt and heparin levels. the thrombin time was not associated with additional manipulation for assay preparation, nor any cross-contamination of reagent or sample, since on the sta reagents do not come into contact with tubing. the sta was not sensitive to hemolytic, icteric or lipernic samples for clotting assays artd showed the same sensitivity as the mla for chromogenic assays. the overall data comparisons, high throughput, minimal operator intervention for reagent/assay change and ease of operation warrant further evaluation of the sta hemostasis analyzer. a. wehmeier, d. s hngen, c. rieth klinik for h#,matologie, onkologie und klinische immunologie der heinrich-heine-universit~it d sseldorf hirudin selectively inhibits thrombin by direct interaction. because the effect of hirudin is independent of antithrombin iii and other factors, it seems an attractive alternative to current anticoagulants. however, it is uncertain whether hirudin influences plateletassociated thrombotic disorders and how it compares with conventional and lmw heparin. we investigated the effect of recombinant hirudin preparations (rhein biotech, dt sseldorf) on platelet function tests: in vitro bleeding time, adhesion to glass beads, aggregation in platelet-rich plasma and whole blood. hirudin was used in concentrations of . - i.tg/mi, and was compared to trisodium citrate ( . %), conventional heparin ( iu/ml) or lmw heparin (fraxiparin, iu/ml). both recombinant hirudins showed normal activity in thrombin neutralization tests, and prolongation of thrombin time and aptt. however, in vitro bleeding time was not prolonged by hirudin, but was more than doubled by addition of conventional and lmw heparins. platelet retention to glass bead columns was reduced by hirudin in a dose-dependent manner to about % but was more effectively reduced by both heparin preparations and citrate. hirudin had an inhibitory effect on p!atelet aggregation in prp induced by thrombin, collagen, and predominantly epinephrine but not adp and ristocetin. in whole blood, a small effect could only be observed with hirudin concentrations of > ~g/ml as compared to citrateanticoagulated blood. in summary, thrombin inhibition by recombinant hirudin has little effect on in vitro platelet function tests in comparison to heparins and calcium depletion. the role of endothelin (et), prostaglandins and the coagulation system in the pathogenesis of acute renal failure is still to be defined. in anaesthesized pigs the effects of i.v. infusion of et ( /~g/kg) alone (group , n= ) and after pretreatment with the potent thrombin-inhibitor hirudin ( , mg/kg)(group , n= ) on haemodynamics, coagulation parameters (factor viii, antithrombin iii, precallicrein, fibrin monomers, aptt) and prostaglandins were investigated. plasma renin activity (pra)-, creatinine clearance-, urine volume-measurement and blood gas analysis were performed hourly. et-infusion caused an initial bp-reduction and marked hr-reduction followed by a transient bp-elevation and hr-reduction. activation of platelets can be directly measured by flow cytometry using monoclunal antibodies. in an in vitro study the effect of the thrombin inkibitors argatroban, efegatran, dup , recombinant hirudin and peghirudin on platelet activation induced by various agonists was studied in whole blood. blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregatiun of platelets. for anticoagulation of blood the thrombin inhibitors mentioned above were used at a final concentration of ~tg/ml each. blood samples were then incubated at °c either with saline, r-tissue factor (rtf), arachidonic acid (aa), adenosine diphosphate (adp) or collagen. at definite times ( , . , , rain) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured by means of fluorescent monoclonal antibodies to platelet surface receptors gpiiia (cd- ) and p-selectin (cd- ). the agunists used induced a platelet activation of . + . % (rtf), . + . % (aa), . + . % (adp) and . + . % (collagen). flow cytometric analysis showed that all thrombin inlaibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. in contrast, after induction of platelet activation with the other agonists an increased percent cd- expression was found showing a strong platelet activation with a maximum at the same times as in non-anticoagulated blood. in conclusion, the results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. the formation of active serine proteases including thrombin may be effectively inldbked by these agents. the observations further suggest that, while thrombin inkibitors may control serine proteases, these agents do not inhibit the activation ofplatelets mediated by other agonists. this work was supported by the grant bmft nbl . animal experimental studies on the pharmacokinetics of peg-hirudin e. bucha, a. kossmehl, g. nowak max-pianck-gesellschaft e. v., arbeitsgruppe "pharmakologische h~imostaseologie", jena hirudin, when complexed with polyethylene glycol (peg), increases its molecular weight from to kda, thereby preventing extravasation of this drug. peg-hirudin is distributed almost only in the intravascular blood space. in addition, its increased molecular weight retards the renal elimination. the elimination half-life of hirudin in rats ( + min, as determined) is increased five-fold ( ± min). with the same hirudin dose applied, the blood level of hirudin is increased -fold, measured in the -elimination phase. in the urine of rats, - % of the hirudin activity were recovered following hirudin administration, but % could be detected after peg-hirudin had been applied. after subcutaneous administration of peg-hirudin, the trnaxwalue is reached at rain (r-hirudin: min); the cmax-value is increased -fold, compared to that of r-hirudin ( . pg/ml). hours later, still one fifth of the maximum concentration (cma,) is present in the blood, and the renal elimination is still retarded. in the urine of rats, % of the hirudin activity applied were recovered in the -h urine sample. with intact renal function, following subcutaneous administration, peghirudin is abte to produce a constant blood level of hirudin over a long pedod. thrombin inkibitors such as r-hirudin (rh), argatroban (a), efegatran (e), and peghiradin (ph) are currently undergoing extensive clinical trials in such cardiovascular indications as ptca, ami, and treatment of unstable angina. a rapid assessment of the anticoagulant actions of these agents is, therefore, crucial to assure their efficacy and safety. currently, act and aptt are used to measure the anticoagulant effect of these agents. we have utilized a dry reagent technology based on the motion of paramagnetic iron oxide particles (plop) to measure the antithrombin effects of various thrombin inhibitors (cv diagnostics, raleight, nc). the heparin monitoring card has been modified to measure antithrombin agents in various anticoagulant ranges for (a) (e), (rh), and (ph). blood samples drawn from patients treated with (a) and (rh) have been evaluated and concentrations of these agents have been calculated using an external calibration curve. in the in vitro setting, citrated whole blood or citrated frozen plasma can be used to evaluate the anticoagulant effects of these agents. the results obtained are comparable to the act which is conventionally used for the monitoring of these agents. both (rh) and ( period. we would like to present a case of heparin-induced-thrombocytopenia (hit) in a years old woman who underwend open heart surgery. she suffered from a combined aortic valve disease and leading stenosis. laboratory analysis showed constant low platelet counts ( /nl) without heparin application, so that an idiopathic thrombocytopenlc purpura was suspected. but platelets also decreased after heparin application. heparin-antibodies were found in the heparin induced platelet activation assay (hipaa). treatment with corticosteroids and immunoglobulines, respectively, showed no improvement but the patient unfortunately developed a pneumonia with legionetla pneumophila. therefore, the only suitable anticoagulant for the necessary aortic valve replacement was hirudin: a bolus injection of r-hirudin of , mg/kg b.w. was administered min. bevore start of the extracorporal circulation (ecc), the heart-lung machine (hlm) was primed with mg r-hirudin and another bolus of mg of r-hirudin was administered. additionally mg of r-hirudin was applicated to the cell-saver-reservoir. during the period of ecc ecarin clotting time and aptt values were taken every ten minutes for monitoring r-hirudin concentration. the postoperative anticoagulation was performed with a constant infusion of r-hirudin starting eight hours after the end of ecc and monitored by aptt. due to mechanical aortic valve the further anticoagulation was performed with phenprocoumon, starting days postop. the therapy with hirudin showed no side-effects. hirudin, threrefore seems to be a suitable anticoagulant in patients with high risk for bleeding complications like this. doses fi:om - mg/kg gave similar post-op blood loss measurements without s dnseresponse ( - oc/kg) (less blood oozing than a historical heparin control but equivalent post-op blood loss; q- ec/kg). doses > mg/kg showed more intra-op blood loss than the lowe~ doses, but equal post-.op blood loss. the bleeding time test was less elevated than for heparin. platelet counts and hematoerit did not vary except for hemodihition on pump. liver enzymes did not vary significantly pre-op to post. act values showed arg was eliminated (dose-dependently) by hour post-op. dogs were hemodymamieally stable during the peri-operative period, and overall gave predictable responses to arg (as opposed to variable responses to heparin). in a substudy it was demonstrated that hypothermia did not affect the activity of arg, nor did varioos formnlations. this dose finding study strongly suggests that arg may be a safe and effective alternative to heparin for patients undergoing cpb. this is particularly important for the growing population of patients with hit who require cardiac surgery, for which no anticoagulant alternative is presently available. three recent clinical tdals with r-hirudin (timi , gusto and hit) have shown that the risk of severe haemorrhagic side effects was strongly associated with high aptt-levels. the large intedndividual variability of the aptt and the lack of a linear dose-effect ratio, however, limits its value for reliable monitoring of the anticoagulant effect of hirudin since even severe overdosage due to impaired renal elimination may not be detected with this assay. we have therefore evaluated the ecadn clotting time (ect) as descdbed by nowak and bucha (thromb. haemost. ; : ) under conditions which allow conclusions on its reliability in the clinical situation.for this, citrated venous blood obtained from healthy volunteers, patients with unstable angina pectoris, and patients treated with marcumar was supplemented with different concentrations of peghirudin. measurements of aptt and ect were made in duplicate. in contrast to the aptt, the ect showed a close, linear relationship with peg-hirudin plasma concentrations in the range of and ng/ml. the lineadty of this relationship was not affected by the presence of unfractionated or low molecular weight hepadns in concentrations of up to pg/ml. the ect was not affected by fibdnogen concentrations % below normal. a somewhat higher slope but no change in linearity was found in plasma from marcumar-patients with quick-values between and %. no significant differences were found between values measured in citrated blood or plasma or using different coagulation timers. the most potent thrombin inhibitor containing a benzamidine moiety is napap (k i = nmol/i). unfortunately, the pharmacokinetic properties (fast elimination by hepatic uptake and biliary excretion, poor enteral absorption) are unsuitable for the use of napap as an oral anticoagulant. the application of choice of a synthetic thrombin inhibitor would be the oral one, therefore, we looked for other lead structures. with the nc~-arylsulfonylated piperazides of -amidinophenylalanine we found a new group of derivatives which inhibit thrombin with ki-values in the nanomolar range. the piperazides exert anticoagulant activities with high selectivity, leaving activated protein c and components of the fibdnolytic system unaffected. in rats, the piperazides are rapidly eliminated from the circulation (tl/ ~ min) upon i.v. administration, too. after oral administration, the systemic bioavailability is low. upon intraduodenal administration of high doses widely varying blood levels were seen, depending on the mode of administration. to cladfy the importance of a possible hepatic first pass effect we studied in more detail the pharmacokinetics of the n~-( naphthylsulfonyl)- -amidinophenylalanine n'-acetylpiperazide in rats using hplc-analysis. like other benzamidines the piperazide is excreted via the bile to a high extent. enteral absorption rates of about % are found after blocking the hepatic uptake and biliary excretion. hence, a hepatic first pass effect appears to be the main reason for low systemic bioavailability after orallenteral administration. at the same time, fast elimination from the circulation by hepatic uptake is the main problem for maintaining effective blood levels with benzamidines. therefore, the elucidation of the structural elements influencing the absorption and elimination processes of these types of inhibitors is necessary. the piperazides of -amidinophenylalanine bear the possibility to easily introduce a wide variety of substituents on the second nitrogen of the piperazine moiety. a -year-old female patient with diabetic nephropathy increasingly developed signs of allergisation combined with dyspnea, erythema, pruritus, and circulatory insufficiency two months after start of heparin-anticoagulated haemodialysis und initial surgical application of a double lumen venous catheter. in addition, growing thrombocytopenia was observed involving a drop in platelets by %, compared to the initial values. the haemodialytic efficiency was reduced by massive thrombosis of the dialyzer and subsequent repeated interruption of treatment. at the end of may heparin antibodies were detected and the hat diagnosis was confirmed. immediately afterwards, haemodialysis treatment was continued, applying hirudin as anticoagulant. using steam-stedlised haemophan dialyzers and . mg/kg r-hirudin (iketon, italy), the minimum therapeutic blood level of hirudin ( . pg/ml whole blood) was reached. this provided therapeutically relevant blood level conditions during a . h haemodialysis. more than regular haemodialyses were run without problems. in all hirudin-anticoagulated haemodialysis treatments the ecarin clotting time was used as the method of choice for bedside blood level and dosage control. after the th haemodialysis, the frequency was reduced from ( ) to haemodialyses a week. accordingly, the hirudin dose was increased to . mg/kg. the creatinine clearance increased continuously from initially . to . ml/min after the th week of hirudin-anticoagulated haemodialysis. platelet count and haemodialytic efficiency normalized. we could demonstrate that the regular use of hirudin as anticoagulant along with dialyzers impermeable to hirudin enables very good results in haemodialysis treatment in heparin-associated thrombocytopenia, hirudin is suited for use as anticoagulant in problem patients with hepadn-induced allergy when combined with a drug monitoring method fit for bedside use. capillary electrophoresis methods provide a fast measurement of proteins. thus we developed for pharmacokinetic measurements of r-hirudin and peg-hirudin capillary electrophoresis methods. for the measurement of r-hirudin we used fused silica capillary and a borate buffer. this buffer was used to detect r-hirudin, but could not be used to measure peg-hirudin. for simultaneous measurement we used a neutral capillary to prevent protein absorption to the capillary wall. the buffer was a mm tricine buffer (ph = . field strength v/cm). it resolved r-hirudin from peg-hirudin at nm using reverse polarity. a linear correlation between the peak area and the concentration was found between pgtml and mg/ml for hirudin (r = . ) and between , and mg/ml for peg-hirudin (r = . ) was found by coinspiking of human plasma and urine with r-hirudin and peghirudin the two proteins were completely resolved. a linear correlation between the peak area and the concentration was found. the method separates r-hirudin from peg-hirudin and may be applied to biological systems to measure the concentration of r-hirudin. triabin is a thrombin inhibitor from the saliva oft. pallidipennis structurally unrelated to any protease inhibitor known and which probably functions by an interaction with the anionbinding exosite of thrombin. we used sf insect cells infected with recombinant baculovirus to produce sufficient triabin for a detailed biochemical characterization. the activity of the protein purified from cell lysates was assessed in a fibrinogen clotting assay and was found to be similar to that of the natural protein. a -fold prolongation of thrombin-clotting time and aptt was achieved with nm and nm triabin, respectively. a kinetic analysis of the thrombin-catalyzed fibrinopeptide a release from fibrinogen showed that triabin is a tight-binding inhibitor. using the graphical method of dixon, the ki was determined to be pm. introduction: thrombocytopenia is a common adverse effect of heparin therapy, in type ii hit platelet decrease induces severe complications. we here present two special cases of type ii hit. case report i: a year old male patient with dvt of the left leg was treated with therapeutic doses of heparin. from the first to the th day of therapy, platelet count decreased from to /ui. hit was confirmed by hipa-test, heparin therapy was s~opped and treatment with the heparinoid orgaran n was started. during the following days, arterial thromboses in the right a. femoralis occurred. several thrombe~tomies were not successful and although orgaran ~" was stopped because of suspected crossreactivity, amputation of the right leg could not be avoided. during the following days under hirudin-treatment platelet count normalized and no further complications occurred. case report : a year old female patients suffering from hip fracture was treated by surgery with tep-operation and received prophylactic heparin treatment. after days, platelet count decreased from initially to /ul and dvt of the right leg was diagnosed. on the same day, severe bleeding into the left leg was observed and hemoglobin concentration was diminished to . g% (before surgery . g%). hit was confirmed by hipa te~t, heparin was stopped and treatment with orgaran started. thrombocyte count normalized and no further complications occured. conclusion: hit type ii can cause severe bleeding as well as thromboembolic complications. because of possible cross-reactivity between heparin and orgaran~,, hirudin should be given in hit patients. currently thrombin time (ti), aptt, activated clotting time (act) or anti ila -activity (alia), measured by a chromogenic substrate test are used to monitor hirudin treatment or prophylaxis. the " " responds very sensitive to hirudin plasma levels end thus requires variable thrombin concentrations. aptt appears to be more adequate, however, it shows large interindividual variations and does not respond sensitive enough to higher hirudin concentrations. act is a simple whole blood clotting assay, but it is strongly influenced by the blood collection technique. the ecadn clotting time (ect) is a new clotting assay, recently described by nowak and bucha (thromb.haemost , , ) . it measures the clotting time of citrated blood or plasma after prothrombin activation by ecarin, a snake venom of echis carinatus. ec.t shows a linear dependence on different hirudin concentrations over a wide concentration range ( e.g. . - pg/ml). in a clinical interaction study healthy volunteers were administered hirudin, asa or both. male volunteers received an i.v. infusion of peg-hirudin ( . mg/kg/h) for hours after an initial i.v. bolus of . mg/kg to compare the sensitivity and reliability of ect with aptt, l-r end act. the act was measured on the hemochron , usa, ect on a fibrin timer, aptf using the aptt lyophylized silica reagent by il and alia on an acl (il-milan) with the chromogenic substrate . all tests were performed in duplicate. ect was more sensitive to different hirudin concentrations than aptt, or act. the ect results were better correlated with the alia-activity than ap'l"r and act. the lower detection range for ect is . pg/ml hirudin. ect is a very sensitive, simple and reliable test for the monitoring of hirudin treatment and prophylaxis. recombinant and synthetic inhibitors of thrombin such as hirudin, efegatran and argatroban are currently in various phases of clinical trials in several surgical and medical indications. the therapeutic effects of these agents are usually monitored by aptt whereas in cardiovascular indications, cefite act and hemotech® act are used. the reliability of both aptt and the act tests in predieting the safety of various thrombin inhibitors has been heavily debated. furthermore, some of these inkibiturs are administered simultaneously to heperinized or coumadinized patients and the obtained aptt and act results do not lady refleot the effects of these agents. fcafin is a snake venom derived fi'om echis carinatus which converts prothrombin into mesothrombin, targeting the arg~ -ile tm bond between the a and b chains of prothrombm. while thrombin inhibitors are capable of inhibiting mesothrombin, atiii/beparin complex does not have any effect. using purified ecarin, nowak and bucha ( thromb haemost : ) proposed to assay hirudin. since thrombin inhibitors exhibit similar mechanisms of thrombin inhibition, ecarin clotting time (ect) was evaluated to test its diagnostic efficacy in various experimental and clinical settings. lyphilized eoarin was obtained from knoll ag, ludwigshafen, germany). concentration dependent clotting times for himdin, efegatran and argatroban were obtained in a range of - p.g/ml. all of the antithrombin agents produced a concentration dependent prolongation of ect and showed va~angpotendies inthe order ofefegatran> argatreban> hirudin, on a gravimetriebasis. on a molar basis, the anticoagulant order of potency was found to be hirudin> afegatran> argatroban. utilizing the ect, the effect of these inhibitors on patients undergoing bolus or infusion therapy, resulting in a concentration level of ~ gt g/rnt, have been measured. unlike such global tests as pt and aptt, patients receiving simultaneous heparin or oral anticeagulants can be monitored for antithrombin specific prolongation ofthe ect. plasma samples from heparinized (aptt - sec) or coumadinized ('pt - see) patients, supplemented with argatroban or hiredin did not show any differences m the ect. a medified ecarm act comparable to the celite act has also been developed. initial results demonstrate that this test is not affected by aprotinin, heparin and reduction of the prothrorabin complexes in the inr range of . - . . these results indicate that ecarin based clotting times provide slx~etlie ~lts of circulating levels of thrombin inhibitors, which can provide reliable information to optimize their safe(y and efficacy. r-hirudin is a highly potent and selective inhibitor of the serine proteinase thrombin. after intravenous administration, r-hirudin is eliminated exclusively with the urine. its plasma half-life is very short, - h. peg-hirudin is a derivative produced by coupling polyethylene glycol (peg) to a specially designed recombinant hirudin mutein. peg-coupling results in a considerable prolongation of the plasma half-life of peg-hirudin, compared to r-hirudin. after intravenous administration of r-hirudin into rats, a very small amount of ,,hirudin-like" activity ( - % of applied activity) was recovered in the urine. in contrast, after peg-hirudin had been administered, more than % of the applied activity could be recovered in rat urine. these results suggest differences in the renal metabolism of peg-hirudin and r-hirudin. within the scope of pharmacokinetie studies in rats we investigated the appearance of biologically active metabolites of peg-hirudin after kidney passage in urine. affinity chromatography on immobilised thrombin was used as a quick and gentle method in searching for biologically active hirudin metabolites in rat urine. but it had to be completed by anion-exchange and/or reversed-phase chromatography to ensure that all active metabolites were detected. the isolated biologically active metabolites were purified by reversed-phase hplc and were biochemieally characterized. in previously reported studies we found a hlrud n derivative consisting of the amino acids - as the main metabolite in rat urine following intravenous administration of r-hirudin. this metabolite was not detected in the urine after administration of peg-hirudin, confirming the suggestion of a different renal metabolism. carrageenans are high molecular weight sulfated polygalactans of plant origin (derived from red algae) with anticoagulant properties. in previous studies we investigated the anticoagulant activity of lambda-carrageenan, a highly sulfated type of carrageonans. unlike heparin, lambda-earrageenan exerts its anticoagulant activity primarily through direct inhibition of the serine proteinase thrombin. only a part of its antithrombin activity is indirectly mediated through antithrombin iii. to investigate relations between molecular weight and biological activities, tambda-carrageenan has been hydrolysed and fractionated. the molecular weight has been determined with the aid of size exclusion hplc using dextrans as molecular weight standards. the degree of sulfation has been determined by anion-exchange hplc. we have obtained low molecular weight lambdaearrageenans ranging from , dalton to , dalton with degrees of sulfation of - % and - %. the anticoagulant and antithrombin activity of low molecular weight carrageenans have been determined using coagulation assays and purified systems, and we have compared their activities with those of heparin and other sulfated polysaecharides. further, we have investigated the ability of lambda-carrageenan and its low molecular weight derivatives to inhibit the activity of human blood phagocytes. the activity has been determined by measuring the cellular chemiluminescence in a mieroplate himinometer using a himinol-dependent assay and zymosan as phagocytosis activating agent. we have used an assay in human whole blood and assays with isolated human mononuclear and polymorphnuclear cells. the anticoagulant activity and also the ability of carrageenans to inhibit the activity of human macrophages decrease with decreasing molecular weight and decreasing degree of sulfation. the natural ocouring, yellow pigment curcumin is the major component of tumeric and is commonly used as a spice and food-coloring agent. since curcumin has been reported to have anti-tumorpromoting, antithrombotic and anti-inflammatory properties, we studied, whether curcumin acts on the transcription factors ap-l(jun/fos) and nf-~:b in cultured endothelial cells (ec). when ec were cultured in the presence of curcumin, electrophoretic mobility shift assays (emsa) demonstrated, that binding of endogenous ap- to its dna recognition motif was suppressed. inhibition was due to direct interactions of curcumin with the dna-binding motif for ap-i. enhanced ap- binding, induced after tnfa stimulation of ec, was decreased in cells pretreated with curcumin. this resulted in reduced transcription and expression of tissue factor, known to be controlled by ap-f and nf-~b. nuclear run on assays proofed, that curcumin directly reduced the tnfa mediated transcription of genes, regulated by ap- , as tf, endothelin- and c-jun. thus, curcumin did not only suppress apl(jun/fos)-binding, but also inhibited tnfa induced jun transcription, transient transfections with tissue factor promotor plasmids confirmed, that inhibition by curcumin was dependent on intact ap-i sites. beside its effect on ap-l-binding, curcumin reduced the radical dependent activation of nf-kb due to its antioxidant properties, however, this inhibition was indirect and less prominent. the relevance of the in vitro data was confirmed in vivo in mice bearing meth-a-sarcoma. when mice received curcumin before tnfa was injected, tumors showed reduced ap- activation. simultanously fibrin/fibrinogen deposition decreased, most probably due to reduced tissue factor expression. thus, curcumin inhibits ap-t activation and expression of endothelial genes controlled by ap-t in vitro and in vivo. (jung, ) . additionally, haemorhenlogical parameters (plasma viscosity, erythrocyte aggregation) were measured. in all patients aptt, bleeding time, platelet adhesiveness, von wiuebrand f~ctor and factor viii concentration and activity were determined. the patients with von willebrand disease showed characteristic morphological changes of capillary geometry. tortuosity of nailfold capillaries was markedly increased as well as the diameter of capillariez on the arterial and venous side. plasma viscosity was significantly low. multiple parameter analysis concerning to galen and gambino ( ) and using the parameters ,,plasma viscosity below . mpas", ,,torquation index higher than ", ,,erythrocyte column diameter bigger than , gin" showed a positive predictive value of %. capillary diameter and capillary tortuosity have a positive predictive value of , %. additionally, a reduction of the vasomotorie reserve and/or a decreased erythrocyte velocity in the capillaries below the reference range was found in most of the yon willebrand patients. it was quite remarkable, that of of the yon willebrand patients showed significant capillary bleedings. these findings confirm some former observations (e.g. o'brian ) and preliminary reports of our group (koscielny ). polymerase chain reaction (pcr)-based quantitation of mrna transcripts is an important tool in the investigation of the underlying molecular defects in inherited platelet disorders, such as the bernard-soulier syndrome. however, for the exact quantitation of mrna a number of methological requirements has to be met. first, a standard (s) mrna must be synthesized which is able to undergo the same processing as the target wild type (wt) mrna. secondly, the quantitation step following the pcr must differentially recognize standard and target dna, and thirdly, the assay must be precise with respect to both inter-and intraassay variability. in order to satisfy these requirements we constructed a s-gpib mrna which is identical to the wt-gpib mrna except a bp long primer recognition site at its " end allowing differentiation between the pcr amplified wt-or s-gpib cdna through incorporation of a fluorescein or biotin labelled " primer. both standard and w[ gpib mrna showed identical amplification kinetics in the pcr reaction. the amplified dna was quantified using an dna binding assay. in this assay binding of amplified dna to gcn fusionprotein-coated microtiterplates is measured. since the gcn binding motif is incorporated into the wt-and s-gpib cdna through an identical " primer, competition between s-and wt-cdna during amplification has been analyzed. at a given concentration of nm of gcn . primer no competition between the sdna and wt-dna for the primer was observed during pcr cycles. the sensitivity limit of the assay performed in this way was amol wt-gpib~, dna, and intraassay variability reached from . % to . % calculated for fmol and fmol dna, respectively. to sum up, combination of rt-pcr with the amplified dna binding assay and usage of an internal standard mrna allows sensitive and accurate quantitation of gpiba mrna in human platelets. since upa and thrombin are main conrtibutors to the process of proliferation and migration of vascular smooth muscle cells (vsmc), which is part of the pathogenesis of atherosclerosis. we are currently assessing the role of spatial expression of upa and thrombin receptor (tr) on cells with human carotid artery plaques (n= ). we have used a double immunolabeling approach, combining anti-upa and anfi-tr antibodies. to identify the different cell types, we used the following antibodies: anti a-smooth muscle actin (a-sma) for smooth muscle cells, ulex europaeus agglutinin i (uea i) for endothelial cells, inflammation cell cocktail (cd +cd ) for monocyte/macrophage and lymphocytes and an anti-proliferation cell nuclear antigen antibody (pcna) to stain proliferating cells. in the carotid atherosclerotic plaques, upa immunostaining was distributed focally, preferentially in the fibrous cap and some cells of the foam cell rich region (fcrr). it was present in distinct patterns: cytoplasmic staining. tr staining was distributed similar to upa staining. with double staining combining anti upa antibodies with anti-tr antibodies, cellular co-localisation of both upa and tr was demonstrated. these cells were identified as smooth muscle cells by -sma. inflammatory cells were mainly localized within the fcrr, they only stained for upa. in conclusion: our data demonstrates that upa and tr are coexpressed in vsmcs in human carotid artery atherosclerotic plaque tissue. we therefore conclude, that the mitogenic activity of upa is associated with the thrombin signalling pathway. in the proficiency test of the ,,deutsche gesellschaft flir klinische chemie" (dgkc) / , lyophilised plasma samples (immuno ag) were sent to participants: a normal plasma and plasmas from persons under oral anticoagulation (oac-plasmas. inr . to . ). the participants (n= ) returned the pt times obtained and in most cases (n= ) also the isi value for the thromboplastin used (isi of pack insert). the inr was calculated using the pt of normal plasma and the isi of pack insert (method i). two additional methods for inr calculation were compared with method i. according to the concept of calibrated plasmas (houbouyan et al., t ), a calibration curve was constructed using the normal plasma and the ac-plasmas. the inr calculated using the pt •fn•rma¿ plasma and the laboratory-specific isi value given as /slope of the calibration curve (method ii) or was read off directly (method hi). for inr values, calculated by the methods from the participants data (n= ), outlier elimination ( sd, iterative) was performed. the inr mean values for all calculation models remain in a narrow range. using calibrated plasmas (method i and m), less outlier were eliminated and cv's obtained were smaller than using the conventional procedure ( i ). obviously, the inr inherited problems, such as accurate isi value, pt value of normal plasma and instrument/laboratory influences on isi, can be reduced using calibrated oac-plasmas. practical approach and educational considera-tions of home prothrombin time estimation a. bernardo, a. bernardo, c. halhuber herz-kreislauf-klinik, bad berleburg, germany specific training is necessary for the patient to achieve reliable and reproducible results in prolhrombin time measurement. the training scheme is based in many respects on experience with similar training courses for home control and management of diabetes and asthma. the education program is divided into a theoretical and a practical part. the theory part has group sessions of twenty patients of a time. the practical course is reduced to a maximum of five patients. the sessions are conducted by a medical doctor and by specialized medicaf/technical assistants. on average eight hours of theoretical education and two hours of practical training are sufficient. the contents of the theoretical lessons are: • need for anticoagulation after heart valve replacement, • potential interaction between anticoagulants and other medication, • accurate recording of the measured prothrombin time results, • techniques of prospective determination of the necessary amount of anticoagulant, • calculation of the individual doses, • potential pitfalls and mistakes, • corrections in case of over-and under-dosage, • early recognition of thromboembelic and/or bleeding complications. an alternative is a full-day intensive course which can be held during the weekend. our recently reported ( ) observation that oral anticoagulant treatment causes an increase of heparin cofactor ii (hc ii) activity in plasma is now confirmed by a more extensive study. in thrombophilic patients who were on vitamin k antagonist therapy (marcumar r) we found a median hc ii level of % as compared to % for thrombophilic patients without any therapy (p < . " ) and % for healthy controls (p < . " ). moreover we observed that the increase of hc ii level was significantly correlated with increasing inr-values (r = . , p < . ). follow-up observations on some patients showed, however, clear differences in the levels of hc ii activity after onset of vitamin k antagonist therapy. thus, some patients responded rapidly with a significant increase in activity ("strong responders") while others showed only slight changes ("weak responders"). in conclusion, the determination of hc ii activity may result in an improved estimation of the risk of bleeding, especially in high intensity treated patients (inr > . ). after intracoronary stent implantation an aggressive oral anticoagulation (oac) therapy is mandatory. to find out whether coagulation activation occurs after coronary stent implantation during high dose oac therapy markers of plasmatic coagulation and d-dimer were measured. patients male patients (average age years) were examined. blood samples were taken before and right after stent implantation and during the following week. patients got mg phenprocoumon during the first three days and additionally heparin and acetylsalicylic acid (asa) were given. methods ptz, aptt, tz, protein c, tat-complexes, fi+ and d-dimer were measured. results d-dimer levels increased steadily between day and day . tatcomplexes showed a slight increase from day ( . bg/i) to day ( . ~tg/i). on day tat levels were down again ( . p,g/l). fl+ (day : . ng/ml) also showed a slight increase on day ( . ng/ml). protein c decreased steadily from day ( %) to day ( %). conclusion during the initial phase of oac therapy a coagulation activation is reported but no significant elevation of tat or fl+ was found. this result shows that additional heparin and asa therapy was sufficient to avoid systemic coagulation activation. the increase of d-direct should be interpreted as a si~=m of local fibrinolytic reaction due to stent implantation. three methods for the determination of prothrombin time from capillary blood in patients under oral anticoagulation have been investigated. two methods were run on coaguchek® monitors (boehringer mannheim) from capillary whole blood. after fingerpuncture the first drop of blood was applied to the well of a coaguchek® test strip directly from the finger-tip, whereas the second drop was sucked into a non-anticoagulated plastic capillary (hirschmann) and immediately applied to the test strip -and vice versa to eliminate any influence of first and second drop of blood. the third method was hepato quick (boehringer mannheim) which was determined out of citrated capillary blood from an earlappuncture. specimen of patients under oral anticoagulation were investigated. the method comparisons between each of the coaguchek® methods and the laboratory method show good results and the correlation between the coaguchek® methods is excellent. mean differences to the lab methods are - . inr in both cases. no mean deviation was detectable between the coaguchek® methods. scattering of coaguchek® versus hepato quick was +/- . inr in the range to inr except for three outliers and one patient with fluctuating results in the lab method which could not be resolved. introduction: haemorrhagic coumarin skin necrosis is a severe complication during initial phase of oral anticoagulant therapy. histological examination shows thrombotic occlusion of small vessels, but little is known concerning the pathophysiologic background of the bleeding component. recently, we described protein z deficiency in patients with bleeding complications of otherwise unknown origin. thus, we were prompted to measure protein z in patients with coumarin skin necrosis. patients: patients (i man, women; age: ± years) suffering from haemorrhagic coumarin skin necrosis were examined. all patients had normal liver protein synthesis function, none was under oral anticoagulant treatment during this study. method: protein z antigen test, diagnostika stago, france. results: out of the patients examined had diminished protein z levels ( , , , ug/l) in comparison to normals ( ug/l). in one of our patients, protein z was normal ( ug/l). conclusion: low protein z levels are additional risk factors for haemorrhagic coumarin skin necrosis. oral anticoagulant therapy is the treatment of choice in patients with need for long-term anticoagulation. since oral anticoagulants interfere with the function of vitamin k, it is not clear whether stable oral anticoagulation can be achieved in patients with need for continous substitution of fat-soluble vitamins including vitamin k. we report about a -year-old man who had experienced progressive hypertrophic obstructive cardiomyopathy over the preceeding years. atrial fibrillation has been first diagnosed years ago. latter on, recurrent ischemic attacks and embolism of the right arteria iliaca occurred. in the patient received extirpation of the ileum and subtotal amputation of the jejunum because of mesenteric infarction. the resulting short bowel syndrome requires continous substitution of fat-soluble vitamins. since vitamin k free preparations of fat-soluble vitamins for parenteral use are not available, prophylaxis of thrombosis has been performed with unfractionated hepadn. as a consequence of the longterm treatment with hepadn the patient developed severe osteoporosis. therefore, the decission :o discontinuate heparin therapy and initiate oral anticoagulation has been made. because of its shorter halflife warfarin (coumadin) was used instead of dicoumarol. over a weeks lasting induction phase inr values were controlled daily. a dosage regime starting with ' mg warfarin at the day of vitamin application (day ) followed by . mg on day and . mg on days , , and , respectively, was found to be optimal to maintain inr values within the target range (inr: . - . ). in order to minimize the risk of hemorrhage the vitamin administration was changed to the subcutaneous route. during an observation period of months neither any bleeding or thrombotic complications nor a vitamin deficiency occurred. these data indicate that stable oral anticoagulation can be achieved despite extreme variation of vitamin k plasma levels. portable monitors for home monitoring of inr are well established for adults on oral anticoagulants. patient's compliance is improved as well as long term outcome. experience concerning accuracy of the procedure in children is limited. inr determinations were performed in parallel from venous and capillaryblood samples of an infant on phenprocoumon, starting at the age of months. the coaguchek® monitor from boehringer mannheim was used. choosing an arbitrary range of agreement of ,qnr . for both determinations, % of the measurements were within the defined range. / outliers were due to low inr resulting from difficulties in capillary blood sampling. the degree of agreement increased when the procedure was performed at least once a week. in conclusion: inr determination with a portable monitor may be helpful in home monitoring oral an.ticoagulant therapy in young children. a dose adjustment should be done only on the base of inr determination of venous blood -if it is considered the gold standard -to avoid over-anticoagulation. a stable anticoagulation is one of the most difficult tasks in attending patients with heart-valve-prosthesis. if prothrombin times are out of the therapeutic range, the risk of bleeding or thromboembolism increases disproportionately. for this reason any improvement in anticoagulant control and/or management can have far reaching consequences in decreasing complications, in extending longevity and in improving quality of life. for the first time a clinical trial was started in and continues until today at the cardiac rehabilitation center bad berleburg, germany with patients mainly after heart valve replacement. the patients were trained to measure their own prothrombin time and to adjust their own dosage of the oral anticoagulant. within six years patients were trained: patients could be followed up with regard to their selfdetermined prothrombin times. the results were within the therapeutic range in . % of the measurements (n= . ) taken by the patients themselves. on average, the patients who determine their prothrombin time themselves did so at a weekly interval. neither major bleeding nor thromboembolic complications could be observed in the patient-years of home prothrombin estimation. it is to be hoped that the usual rate of complications can be reduced when patients determine their prothrombin time themselves at a close interval, resulting in more constant values in the therapeutic range and slight corrections of the anticoagulant dose. home prothrombin estimation promises better quality of life and has a considerable potential to achieve this goal. circulating plasma thrombomodulin (tm) is a novel endothelial cell marker, which may reflect endothelial injury. tm acts as thrombin receptor which neutralises the fibrin-forming effect of thrombin, and also accelerates the formation of the anticoagulant protein c/s pathway. tm therefore belongs to the anticoagulant defence system against thrombosis. increased tm levels have been described in various diseases such as ards, thromboemboembolic diseases, ttp, diabetes, le and cml reflecting alterations of the vascular system at the endothelial level. to find out to what extent cardiac catheterisation imtates vascular endothelium, tm concentrations (stago, asnieres, france: x iu/ml) were investigated prospectively in infants and children (three days - years). blood samples were drawn before the intervention, immediately at the end and h later, snap frozen (- °c) and investigated serially in dublicate six weeks - months later. the results (median and range values) are shown in the enhanced tm concentrations immedately after the operative intervention, followed by normalisation within h, indicates that cardiac catheterisation in pediatric patients rather leads to a short lasting irritation of the vascalar endothelium than to severe irreversible endothelial damage. recently in an al=wl" based method dahlb~ick et al described in vitro resistance to the anticoagulant effect of activated protein c (apc) in thrombophilic adult patients. apcr is in the majority of cases associated with the arg gin point mutation in the factor v gene. concerning the special properties of the neonatal hemostatic system (low vitamin k dependent coagulation factors, physiological prolongation of the pt and aptf) we adjusted this ap'it based method (chromogenix, m~,lndal, sweden) to neonatal requirements: apcr was measured in healthy infants according to dahlb~ck. the results were expressed as apc-ratios: clotting time obtained in a : , : and : dilution with factor v deficient plasma (instrumentation laboratory munich. germany) using the apc/caci solution divided by clotting time obtained with cac in the same i: , : and : dilution. in addition, plasma of neonates with septicaemia were investigated and data of infants aged birth -three months with arg gin +/-were shown. the arg gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mnl i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. results are shown in the . ( . - . ) neonates and infants were considered to be apcr when the aptt ratio was < or = . concerning the special properties of the neonatal hemostatic system, our data show concordance with the pcr method in neonates and infants only, when the aptt based method was performed in the i: plasma dilution. case report: we report on an -year old boy with severe hemophilia b and frequent screaming at night. eeg showed spike wave activity, starting from the temporal lobe, but generalizing within seconds. complex partial seizures were diagnosed and therapy with carbamazepine was initiated. as no improvement was seen nmr was performed. this revealed lesions within the right frontal cortex. higher doses of carbamazepine were not succcssfull as was therapy with phenytoin and pfimidone respectevely. the patient is now treated with carbamazepine and valproate. he still suffers from one short seizure per day. because of his seizures we started prophylactic replacement therapy with i.e. factor ix twice per week. discussion: in wilson et al. first detected brain abnormalities in of children and adolescents with hemophilia a or b who were negative for immunodeficieney virus ( ). the most common findings ( / patients) were small, focal, nonhemorrhagic white matter lesions of high signal intensity on t weighed images. similar lesions have been reported in children with sickle cell cerebral infarction ( ) . only three of these patients had seizures, all of those having a documented history of intracranial hemorrhage. our patient has similar lesions as those described by wilson et al. but no history of intracranial hemorrhage is documented. even if tuberous sclerosis might be a differential diagnosis, we think that the abnormalities are related to hemophili a or its treatment, because the patient has no further signs of this disorder. conclusions: . in patients with hemophilia and seizures nmr might be useful as a high sensitive method for the detection of gray and white matter changes. . further studies should be initiated to determine the prevalence of pathological conditions in the brain of hemophiliac patients. disseminated intravascular coagulation (dic) is a rare, but foudroyant disease occuring in gram-negative sepsis like meningococcal septicemia. despite the avallibility of potent antibiotics, mortality in mertingococcal disease remains high ( about % ), rising to % in patients presenting with severe shock and consecutive dic. as the clinical course and the severity of manifestations of systemic meningococcal infections varies there is a need for early diagnosis of the infection and stage of coagulopathy in order to reduce the high mortality rate. few and rapidly available parameters are needed to classify the wide spectrum of clinical and laboratory findings in patients with dic. the parameters include partial thmmboplastin time, pmthmmbin time, plasma levels of fibrinogen, fibrin monomers and dimers, fibrin degradation products and the thrombocyte count. monitoring the course of hemostaseologicai findings in pediatric patients with systemic meningococeal infections we observed a change of coagulation parameters as early as in the first stages of the infection: a prolongation of partial thromboplastin time to an average of . sec (range - sec, norreal - sec), a decrease of prothrombin time to . % (range - %, normal - %) and of antithrombin iii to an average level of . u/ml (normal - u/ml ) was found to (- ) hours after admission. the consecutive development of hemostaseological parameters mentioned above permitted to define the stage of coagulopathy and thus to induce a stage related therapy. primary treatment consisted in control of shock by liquid substitution, compensation of metabolic acidosis, correction of clotting disorders ( at iii and heparin in stage of pre-dic ; at iii and fresh frozen plasma in case of advanced dic ) and treatment with g-lactam antibiotics ( e. g. cefotaxime or ceftriaxone ). an early assessment of the coagulation disorders in meningococcal disease can be based on few coagulation parameters, thus an appropriate treatment may be arranged to prevenl the patient from a fatal outcome of meningococcai septicemia and protect him from the development of a waterhouse-friderichsen-syndrome. this study was designed to prospectivdy evaluate coagulation and flbrinolyfie activation in children (neonate - years) during cardiac catheterisation with low dose flush heparin ( iu/ml saline). aptt (instrumentation laboratory: see), anti xa activity (xa; chromogenix: iu/ml), prothrombin fragment ft. (f . ; behring werkc marburg: nmol/l) and d -dimer formation (d-d; bnhring werke/vhrburg: ug/l) were investigated before (t ), at the end (t ) and h after cardiac catheterisation (t ). in addition, to evaluate the influence of inherited thrombophilia in all patients resistance to activated protein c (apcr), protein c, protein s and antithrombin were investigated. during catheterisation median (range) hepadn was administered in a total dose of ( - ) iu/kg bw. in addition infants < months of age (arterial catheterisatiun only) or patients with known thrombophilia received - iu/kg hepafin for fmther hours. the results (median and range) are shown in the ft. was sigificanfly elevated above the pediatric boundary immediately after the intervcation and nearly reached baseline values h later. in contrast no cfinically relevant fibrinolytic activation was seen: d -dimer formation increased within the pediatric boundary immediately after the catheter and returned to basdine levels h later. three children showed resitance to apc. tn one child stroke occurred before. not knowing the result of apcr in the remaining two patients only one neonate received further prophylactic heparin. the third neonate without heparin prophylaxis suffered from venous occlusion within two days after the intervenfon~ in addition, no protein c, protein s or antithrombin deficiencies were found. although administration of low dose flush heparinisation during cardiac cathetefisation could not prevent short -term coagulation activation, no thrombotic events occurred in children without inherited thrombophilia. if fnrther prophylactic hepariuisation in children with a~r, protein c, protein s or antithrombin deficiencies may prevent vascular occlusion requires a more intensive study. a.sandvoss, w.eberl, m.b rchert introduction: capillary leakage, edema and hypovolemia are common complications in preterm infants especially if birth weigth is below . g. septicemia, asphyxia and immaturity seem to be most important risk factors. to determine the influence of c -esterase inhibitor (cilna) in preventing contact phase and complement activation we investigated c na concentrations in normal and symptomatic preterm infants. methods: activity of cilna were measured by chromogenic substrate method (behringwerke), cilna concentration with radial immunodiffusion (behringwerke,germany). results: cllna-activity in asymptomatic preterm infants (n= ) was +/- % of normal at birth. healthy newborns showed activities of +/- %. cilna reached normal adult values - days after birth. preterm infants with respiratory distress syndrome(n = ) showed lower activity on day - , patients with additional septicemia (n= ) had decreasing c ina-activities in the first three days of life. individual course of cllna-activity and thrombocyte count correlated in the group with irds with and without septicemia. in children with capillary leakage onset of diuresis went parallel with raising cllna-activity. markers of contact phase (f xlla) and complement activation (c al were investigated in single cases and evidence for involvement of both systems was found. conclusion: contact activation and complement system play an important role in capillary leakage in preterm infants. cilna regulates both systems. activity of cilna correlates with clinical course, substitution therapy is possible and may improve outcome of these critical ill patients. antiphospholipid antibodies (apa) interfere with hemostasis probably by inhibition of protein c or prothrombinase complex. thereby, apa might lead to thrombosis or increased bleeding. however, incidence and clinical importance of apa has not yet been investigated in children. therefore, we assayed plasma samples of children, aged , to years (mean years) by elisa detecting igg-and lgm-antibodies directed against eardiolipin, phosphatidyl serine and phosphatidic acid. in patients with increased bleeding, thrombophilia or prolonged clotting tests a detailed coagulation analysis was performed. according to their diagnosis children were devided into groups: i. autoimmune diseases, ii. infections, iii. metabolic diseases, iv. other diseases, v. healthy children. results: apa were found in / patients. in the respective groups we demonstrated apa in the following proportions: . lgg-isotype: activitiy of c esterase inhibitor (c na) is reduced in preterm infants especially if birth weigth is below . g and respiratory distress syndrome and/or septicemia is present. capillary leakage with generalized edema, hypovolemia and hypotension is resulting in imbalance between inhibition and activation of contact phase and complement system. iln four patients we investigated seven courses of substitution ;with commercial c esterase inhibitor preparation (berinertr,behringwerke), case reports are given. all patients had clinical symptoms of capillary leakage, all had septicemia accompanied by either respiratory distress, disiseminated intravascular coagulation or mutiple organ failure. jefficiacy of substitution therapy is dose related, supranormal iactivities of cilna are necessary, reflecting raised consumption of inhibitor in ongoing disease. clinical effects on diuresis, catecholamine need and especially on thrombocyte counts are demonstrated. or arterial thromboembolic event in children e. lenz, c. heller, w. schr ter*, w. kreuz johann w. goethe-universit~itskinderklinlk, frankfurt a. main, germany * georg-augast-universit/itskinderidinik, g/ ttingen, germany venous thrombosis as well as arterial thrombo-occlusive events are rarely observed in childhood, but can lead to life-threatening situations and longterm sequelae in these patients. after the initial stage of treatment (thrembolysis or thrombectomy) the pediatrician has to decide how to efficiently prevent re-thrombosis in the individual patient. anticoagulation after venous thrombosis is generauy recommended for months after the event; if an underlying thrombophilic condition has been detected in the patient anticoagulation has to be considered lifelong. when evaluating antithrombotic therapies for children it is of importance to consider whether the anticoagulatory effect is mainly necessary in the venous or arterial vessel system. the hemorrhagic risk and side effects of the different anticoagulatory preparations have to be taken into account, especially when treating small children. only limited experiences exist concerning the suitability of the preparations for long-term anticoagulation in children and general recommendations on the ideal dosage in pediatric patients are still missing. we want to disscuss different types of anticoagulants (such as coumarins, unfractionated heparin, low molecular weight heparin (lmwh) and inhibitors of platelet aggregation) their mode of action, their suitability for pediatric patients and their side effects and relevance of these side effects especially in children. from the experience in our own pediatric patients, we would like to report on the indications, which can be given to administer these different preparations, the dosage regimen we recommend and the laboratory tests to monitor save and efficient re-occlusion prophylaxis in our patients. in this context we would like to present our data on patients with either thrombosis or arterial infarction due to a thrombophilic condition, who had all contraindicatioas to oral anticoagulation by coumarins. because prophylaxis for re-thrombosis was mandatory in these patients, lmwh was given for long-term anticoagulation in a dally subcutaneous dosage of - anti-xa u/kgbw. monitoring was done by anti-xa-test ( , - , anti-xa u/ml). under this regimen none of the patients developed re-thrombosis or bleeding complications. alopecia was seen as a side-effect. this study was designed to prospectively evaluate coagulation and fihrinolytic activation after cardiopulmonary bypass with aprotinin ( x u/kg bw) in infants and children aged . - years, and to correlate these findings to the clinical outcome. prothrombin fragment f . (f . ; behring werke marburg: nmol/l), antithrombin-serinesterase -complex (atm; stago: ng/ml), d -dimer formation (d-d; behring werke marburg: ug/l), tissue-type-plasminogen activator ag (t-pa; chromogenix: ng/ml), plasminogen activator inhibitor antigen (pai; chromogenix: ng/ml) and cl-inhibitor (c ; behring werke marburg: x - g/l) were investigated before the operation (t ), at the end of the operation (t ), and on postoperative days (t ), - (t ) and - (t ), respectively. the results are shown in the table (median and median absolut deviation): t t t t " " nv fi. . +/- . . +/- . . +/- . +/- . . +/- . the platelet (pl) function defect induced by thrombolytic agents has been attributed either to the degradation of pl surface receptors or to the anti-aggregatory effect of fgdps. in contrast to other plasminogen activators scu-pa is intimately inked with pl: they can rapidly incorporate exogenous seu-pa, release it upon stimulation and bind the proenzyme. recently we have reported that exposure of prp to recombinant scu-pa ( . -t um) in timed interval - min resulted in dose-dependent inhibition of pl aggregation. timecourse changes of the process were followed by the biexpotential kinetics: a rapid initial inhibition during the first - rain with the moderate suppression of pl aggregation in the min period. when tcu-pa ( - nm) was exposed to prp in the same conditions dose-and time-dependent inhibition of pl aggregation was also observed. since the effect was obtained no earlier than t min after exposure of tcu-pa to prp, and the threshold dose was higher. comparable inhibition of pl aggregation was obtained with nm of scu-pa versus nm of tcu-pa and the llbrinogen depletion by the end of the min period was % and % respectively. it's likely that tcu-pa and its precursor have different mechanisms of action on the pl aggregatory function. in a recent study we have shown that recombinant rscu-pa inhibits platelet (pl) aggregation in prp. to exclude the possible influence of rscu-pa/plasma interfere on this process the aggregation of washed pls was under the investigation. pls were washed according to modified mustard's method, suspended in buffer and adjusted to , / . the resuspended pls were exposed to - nm of rscu-pa for min at (;. at time points , , and min the aggregation with . iu/ml of thrombin was measured. it was found that the exposure of pls to rscu-pa ( - nm) for man resulted in marked inhibition of their aggregation. since after - man of incubation with - nm of rscu-pa the inhibitory effect on pl aggregation became less pronounce or even disappeared. when nm of rseu-pa was used the inhibition of pl aggregation became significant only by rain of exposure period and didn't change for man of investigation. the observed results may be cormeeted with uptake of rscu-pa by pls from surrounding buffer as well as with individual variations of pl response to the same concentration of rscu-pa. loss of glycosylation may result in a reduced platelet (p) survival and perhaps altered function. we analyzed the structural and functional effect of specific deglycosylation (combinations of n/o-glycosidase and neuraminidase treatment) of p and isolated p gpib. washed and formaldehyde-fixed p were digested as follows: ) with neuraminidase ( . u/ml) + o-glycosidase ( . mu/ml) + n-glycosidase ( . u/ml), ) with neuraminidase alone ( . u/ml), ) with n-glycosidase ( u/rnl) and ) with neuraminldase ( . u/ml) + o-glycosidase ( mu/ml). all reactions were performed in the presence of protease inhibitors (pmsf, leupeptin, sbti), after washing x the p and identically treated controls were analyzed by flowcytometry with the antibodies di (mab: a-gpib), i-l vlab: a-gpiiia), and the lectins wheat germ agglutinatinln (wga, for neunac) and peanut agglutinin (pna, for [ dgal( - )-galnac) which confirmed effective and specific deglycosylation by the respective enzymes (but gave only minor differences with di and h ). the botrocetin ( ) and ristocetin (r)induced agglutinations showed arer treatment ) (all enzymes) a full inhibition of r-induced agglutination but only a mildly reduced b-induced agglutination ( % of normal). treatment and (neuraminidase alone, and n-glycosidase alone) affected both agglutinations only mildly ( - % of normal).treatrnent ) (o-deglycosylation) however showed a major inhibition of r-agglutination down to %, while b-agglutination interestingly was almost fully retained. the results of the rotary shadowing electron microscopy of purified gpib suggested a collapse of the normally stretched, glycosylated, gplb, not only after the treatment with all three glycosidases, but also .after o-deglycosylation alone. we conclude that oglycosylation is most important for ristocetin-induced platelet-von willebrand factor-interaction and responsible for the typical stretched shape. the phenomenon of in vitro platelet aggregation and consequent pseudothrombocytopenia (ptcp) in the presence of calciumchelatization by na-edta and sodium-citrate was studied in blood samples of a patient. initial platelet counts electronically measured were /ul blood anticoagulated with na-edta and sodium-citrate. normal platelet counts were found in heparin-anticoagulated blood and in capillary blood. immunoglobulines of the igg and igm subclass were identified in the patients plasma. by incubation of the patient's serum with platelets of healthy individuals, platelet-clumping occurred in the presence of na-edta and sodium-citrate but not in the presence of heparin. the platelet membrane glycoproteins (gp) hb/llia, ix and iiia/vnr g-chain were involved in the antigen antibody reaction as demonstrated by specific antibodies and flow-cytometry. on platelet surface permanent calcium-exchange and -replacement is dependent on external calcium concentration. calcium depletion induced by calcium chelators as na-edta and sodium-citrate might conformationally change platelet surfaces and induce formation of neoantigens. the decrease of gp llb/illa platelet surface antigen to % (normal > %) indicated the important role of the gp iib/iiia receptor at ptcp. the saliva of tdatoma pallidipennis, a triatomine bug, was found to contain a protein called "pallidipin", that specifically inhibits collageninduced platelet aggregation but not adhesion or shape change. to investigate the mechanism of action of recombinant pallidipin the influence on platelet fibdnogen binding after activation by collagen type i in different concentrations was measured by flow cytometry. the same concentrations of pallidipin that inhibited the couagen-induced platelet aggregation completely did not cause any inhibitory effect on fibdnogen-binding in the prp from the same donor measured contemporaryly. collagen type i-induced platelet aggregation of cd -deficient platelets from two different unrelated blood donors was inhibited by the same concentration of pallidipin that inhibited aggregation of control platelets. there was no inhibition of collagen-induced fibdnogen-binding in the cd -deficient platelets as well. pallidipin did not cause inhibition of collagen-induced membrane expression of cd and cd of control and cd -deflcient platetets as measured by flow cytometry. however eadier studies had shown an inhibition of collagen-induced atp and { tg secretion by pallidipin. therefore we compared the effect of pallidipin in unstirred and stirred prp samples. while pallidipin had no effect in unstirred samples it showed strong inhibition of ptg secretion in stirred samples. we therefore conclude that pallidipin does not act on collagen-induced aggregation through cd and that the inhibition is a post fibdnogenbinding event. pallidipin does not influence the first steps in secretion, which are independent from cytoskeleton and platelet-platelet contact, but inhibits the following steps. -hydroxy-wortmannin does not inhibit the transport of nm-gold labelled fibrinogen in resting platelets. e. morgenstem, b. kehrel and k.j. clemetson medical biology, saarland univ., homburg, germany, haemostasis research, univ. muenster, germany and theedor-kocher-lnstitut, univ. bern, switzerland. wortmannin, an inhibitor of phosphoinositide -kinase and of myosin light chain kinase blocks reactions of the activated platelet. to obtain informations about the role of the contractile cytoskeleton in receptor-mediated transport of resting platelets, the effect of -hydroxy-wodmannin (hw) on the endocytosis of fibrinogen from the surface of resting platelets was studied. gel filtered platelets (gfp) were incubated for min at °c with hw ( x - m) or with iloprost. controls and gfp preincubated with hw or ilopmst were incubated with . nm-gold labelled fibrinogen molecules (fg-au; final concentration p.g/ml) at °c. the experiments were stopped after or min by rapid freezing. after freeze substitution in acetone with % osmiumtetroxide, sedal sections were prepared. the sections were examined after incubation with ascorbic acid ( % in h ) for rain at °c (to reduce metallic osmium) and silver-enhancement using danscher's ( ) method (to visualize the fg-au). examination of adp stimulated platelets in the presence of fg/ml fg-au shows that the ligand is able to mediate aggregation. the examination reveals, that fg-au was present in a low density on the platelet surface, in higher density in the surface connected system (scs), in coated pits and vesicles and separated smooth vesicles (representing endosomes?) as well as in the matrix of alpha-granules. after rain, the number of labeled granules was increasing. labels on the surface and on the mentioned cytoplasmic membranes were observed during the whole period of incubation. hw or iloprost did not alter the resting gfp and the mentioned qualitative ultrastructural findings in both preparations did not show differences to the controls. we conclude from the results with hwthat the regular contractile function of the cytoskeleton is not necessary to transport the fg-au in resting platelets. methods: edta anticoagulated whole blood was incubated with thiazole orange and analyzed with a flow cytometer. young platelets were defined by having a high fluorescence from thiazole orange (normalized to platelet size). platelets were also incubated with fluorescent antibodies to gpib, gp lib/ilia and gmp- (two colour method). results: surface expression of gpib was the same in young and older platelets. results for gp lib/ilia and gmp- (in resting and activated platelets) will be presented. conclusion: young platelets can easily be detected using thiazole orange and flow cytometry. there is no differential expression for gpib. further results will be presented. the influence of erythrocyte and thrombocyte content on the release of atp by different agents in whole blood specimens was tested. the measurement had been performed in the lumi-aggregometer using the principle of the luciferin-luciferase reaction. altogether blood samples were diluted gradually before induction of the release reaction by arachidonic acid ( , mmol/i final concentration), adp ( ijmol/i) and collagen ( , and , tjg/ml). the peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (= peak in relation) and into atp concentrations (= absolute peak) after testing the atp standard in parallel for each dilution step separately. the peak in relation increases by increasing dilution with all inducers. it was identic with the atp standard and with collagen, somewhat lower with arachidonic acid and much higher by adp. a luminescence-optical effect may influence all these results. the absolute peak decreases by dilution under arachidonic acid and collagen as it was expected by the decreasing thrombocyte content of the samples. under induction by adp no decrease of the absolute peaks by increasing dilution of the samples was abserved. this can be explained only by liberation of atp from the erythrocytes. the atp standard is essential for the quantification of the release reaction. adp doesn't suit for it. collagen with a final concentration of pg/ml was proven as the best inducer. platelet aggregation induced by several agents has been photometrically investigated in disc shaped rotating cuvettes coated with vessel wall tissues obtained from human umbilical cord, either endothelium or smooth muscle cells or extracellular matrix or combinations of them. in addition, effects of endothelium incubated with several cytokines on platelet aggregation have been studied. endothelial cells strongly inhibited aggregation depending on their cell count and the concentration of the inducer. smooth muscle cells showed the same effect but very less marked. in presence of extracellnlar matrix spontaneous aggregation occured. endothelium could inhibit this spontaneous aggregation when present in the same cuvette, smooth muscle cell could not. incubation of endothelium with several cytokines increased its anti-thombotic properties. for example, at a platelet count of x /id in the prp, - m adp led to maximal aggregation in uncoated cuvettes, in presence of , x endothelial cells aggregation was completely abolished, in presence of , x " cells aggregation was decreased to %. smooth muscle cells diminished the aggregation effect of , nih thrombin to % when only one side of the cuvette was coated and to % when both sides were coated. endothelium could not inhibit aggregation induced by , x - m adp but endothelium incubated with u/ml tnf-a or u/ml intedeukin-lfl or lmm l-nitro-arginin for h did completely inhibit aggregation. platelets become sticky and adhere to surfaces or to another without contracting and secreting. during maturation of megakaryocytes finally platelets lost their genomic nuclear message. only mitochondrial dna of platelets can be identified. we focused our attention on the impact of mitochondrial dna and the mitochondrial transscriptive mechausisms during platelet activation in normals. materials and methods: leucocyte free (nagentte chamber, flow cytometric analysis) platelet rich plasma or platelet concentrates a_~er hemapheresis were filtered by pall leucocyte filters. the influence of different anticoagulants (commercially available sarstedt tubes containing citrate, heparim edta and atu/ml hirudin wacker) was examined. activation was due to a nun. hemapheresis procedure ( - fold increase of cd , cd ) and ex rive stmaulation due to niy u/ml thrombin, . m cac or combmatious. the guanidiurn method for total rna preparation were used according to t. brown: current protocols in molecular biology . - . . , . different primers of mitochondrial genome (e.g. cytochrome b and atpase) were prepared using pcr and mitochondrial transscription was examined using northern-blot-technique. results: ., there is less activation of mitochondrias using hirudin anticoagnlation, but a fold increase of mitochoindrial rna content in heparinized samples. ., stimulation with thrombin leas to an increase to . e-l rna btg/platelet, compared to . - . e- rna ~tg/platelet under unstimulated conditions.. conclusion: there is evidence for the importance of platelets mitochondrial dna and mitochondrinl transsefiption in regulation of cytosceleton and platelet activation. thrombospondin- (tsp- ) is a large homotrimeric glycoprotein originally identified as a platelet alpha-granule component. the investigation of its putative role in a variety of pathophysiologies like haemostatic disturbance, malignancy and wound healing requires specific laboratory reagents. monoclonal antibodies are one of the most powerful of these reagents. therefore, we purified human tsp- from thrombin-stimulated platelets using affinity chromatography to generate monoclonal antibodies in mice. a subclass igg monoclonal antibody designated . was purified from ascitic fluid and further characterised. western blot experiments demonstrated that this antibody reacted only with the unreduced molecule whereas the tsp- subunit chain was not recognised. no cross-reactivities with human fibrinogen, fibronectin, vitronectin and von willebrand factor were found. preliminary results indicate that the monoclonal antibody . can be used to investigate tsp- function in several assays including immunocytochemistry and cell adhesion as has been demonstrated for hl- cells. in addition, a sandwich enzyme immunoassay was developed using goat-antihuman tsp- igg and derivatised monoclonal antibody . (peroxidase, biotin) as a sensitive method for detection of tsp- in human body fluids. in the following study the expression of the platelet antigen (cd p) and the leukocyte antigen (cdllb) were measured in whole blood, in addition to platelet-leukoeyte adhesion (rosette formation) by means of multicolour fluorescent labelling (cd , cd , cd a). the measurements were carded out both in freshly drawn whole blood which had been antieoagulated with different agents, and in stirred samples of whole blood under controlled conditions ( °c, rpm, different stirring times). the results are presented as the percent positive events in each gate (platelets, leukocytes -pmnl, monocytes, lymphocytes and rosettes -plateletpositive events in the pmnl, monocyte and lymphocyte gates), whose mean fluorescence is given in addition to an index comprising the product of the percent positive events and their mean fluorescence. stirring (max rain) induced an increase of cd p on the platelet surface of ca. %, without any change in the mean fluorescence. under these conditions increased cdllb on pmnl and monoeytes could be detected. an increase in the rosette formation could also be measured (greater index), in that the percent of monocytes which were platelet-positive increased with no change in the mean fluorescence of the positive events, whereas pmnl showed an increased mean fluorescence, but not an increased number, of platelet-positive events. the time-dependent changes in rosette formation on stirring could be further increased by addition of adp. these results show that it is possible to measure rosette formation, and also the influence of effector agents (inhibitors or activators of platelets or leukocytes) on rosette formation, in whole blood using flow eytometry. itp patients undergoing splenectomy were observed after - years following operation and divided into groups. first group consisted of patients with normal platelets count and absence of haemorrhagic syndrome. second group was formed of itp-patienfs with episodes of thrombocytopenia recovery following certain time period after splenectomy. in the aim to study the cellular immunity there were carried out immunophenotypical investigations of blood samples using immunofluorescence method with monoclonal antibodies application. the increase of b-cells, expressing cd , cd , hla-dr-antigen has been revealed in the nd group. quantity of srfc, cd +, cd + cells in the blood of recovered patients was lower than in patients of the first group. this group was also characterized by statistically significantly increased level of cd + cells while the cd /cd ratio was equal to . :i: . % ( , + , % in patients of the second group, respectively, p>o, }. also the relatively high expression of activating antigens in patients with thrombocytopenia recovery after splenectomy was stated. among infectious complications in all patients observed were predominantly found various types of throat infection, mainly with unsatisfactory treatment possibilities. we have observed the opsi-syndrome in patients, being featured with marked tiredness, breath loss, intolerance of hard physical working, diminished ability to maintain physical activity. extracellular matrix (ecm) produced by human endothelial cells closely resembles the vascular subendothellal basal lamina in its organization and chemical composition. thus it contains collagens, fibroneetin, von witlebrand factor, thrombospondin, fibrinogen, vitronectin, laminin and heparin-sulphate. platelets carry different receptors on their membrane surface with specific binding capacities for one or more of these extracellular matrix proteins, such as glycoprotein (gp) iibiiia, gp ib/ix and gpiiib. incubation of platelets with ecm results in platelet adhesion, degranulation, prostaglandin synthesis and aggregation. we studied patients whose platelets showed either a receptor defect in gpiibiiia or gpiiib or a storage pool disease. adhesion experiments were performed using siliconised glass, collagen coated surfaces, immobilized fibrinogen as well as human subendothelial matrix. platelet adhesion of patients with thrombasthenia glanzmann (receptor defect of gpiibiiia) resulted in a total lack of binding to silieonised glass and immobilized fibfinogen. adhesion to collagen was almost normal in spite of the fact that only single platelets sticked to the surface and no microaggregates were observed. the adhesion to ecm was diminished and also no aggregates were detected. patients with a receptor defect in gpiiib showed normal platelet adhesion to siliconised glass and immobilized fibrinogen but binding to collagen and ecm was markedly reduced, while platelets with a storage pool defect sticked to siliconised glass but failed to adhere to ecm. by centrifugation of citrate blood ( x g, min) erythrocytes and leucocytes go to the bottom, whereas plasma and thrombocytes stream in the upper part of the probe. so the thrombocyte count doubbles in the platelet rich plasma in contrast to the platelet count in the whole blood volume. if the thrombocytes are more or less activated, they adhaere on erythrocytes, leucocytes or aggregate end are not able to stream upwards. the quotient between thrombocyte counts in prp and whole blood is a measure for thrombocyte activation. we chequed the value of this screening in different groups of patients with arterial occlusions disease (aod), chronical venous disease (cvd), diabetes mellitus (dm] and in healthy control persons (control). variation coefficient of the method is . (prp) and . (tc) respectively (coulter counter). differences to the control group are significant. changes in the patient groups in dispensaires follow up years are also significant. nicardipin -induced immunthrombocytopenia p. eichler , c. hinrichs , g greinacher l i.institut fur immunologic und transfusionsmedizin, ernst-moritz-arndt-universitat greifswald, . deister-s ntel-klinik, bad m nder drug-dependent immune-thrombocytopenias are a rare but clinically important variant of immune-thrombocytopenias. patients are at risk to suffer from severe bleeding complications. especially in patients receiving multiple drugs, diagnosis of drug-dependent immune-thromboeytopenia is often difficult. we report the case of a year old male patient who received allopurinol, captopril, digitoxin, furosemid, and nieardipin. the patient presented with hematomas (pit. count < g/l) and later developed bone marrow dysplasia. in an elisa using whole platelets and patient serum, a weak reactivity in the presence of furosemid, but a stronger reactivity in the presence of nicardipin (antagonil, ciba-geigy) could be demonstrated. the reaction pattern is given in the the enzyme-immunological determination of soluble fibrin (sf) proved to be highly sensitive and specific. this sf-elisa detected fibrin hacking fibrinopeptide a (fpa) via the monoclonal antibody t specific for the neoepitope generated on the aa-chain after the split of fpa. lill et al. recently introduced a new assay modification which utilizes the same antibody as the old one but takes advantage of a pretreatment of plasma specimens with kscn. this strong chaotropic ion is used to dissociate the various fibrin complexes possibly hiding fibrin epitopes. it was the aim of this study, therefore, to compare the two sf-elisa modifications (with and without kscn-pretreatment of specimens) . in order to examine the dynamics of thrombin-induced fibrin(ogen) metabolism we made course observations in patients with a certain form of septicemia. both assay modifications detected fibrin(ogen) derivatives which differed considerably in kinetics (n= samples from courses). the former sf-elisa (no kscn) correlated well with prothrombin fragments, thrombin-antithrombin ! -complexes and with the release of fibrinopeptide a ( r > . , n= ). results of the new sf-elisa with kscn pretreatment of patients' plasma, however, correlated conspiciously well with d-dimer levels (r > . ) but distinctly less with the markers of thrombin generation (- . < r < . ). this good correlation with d-dimer levels was unaccountable since the d-dimer maximum occured significantly later than the peak of markers of thrombin generation (p < . ). therefore, kscnpretreatment of fibrin specimens seems to lead to a change in the specificity of the fibrin assay despite usage of the same catching antibody. different half-iifes of differently composed fibrin complexes should be considered in trying to explain the findings. nevertheless, the results of the former assay without kscn-treatment correlated much better with the well-known dynamics of thrombin-induced fibrin generation during hemostasis activation than the data from the new assay modification. consequently, further examinations are necessary to specify the effect of kscn on soluble fibrin complexes and the resulting assay specificity. a rapid assay for the determination of the primary hemostasis potential (php) of whole blood has been developed (kundu et al, ) from the original method of kratzer and born. the new system employs a disposable test cartridge which holds the sample (citrated whole blood) and all components for the tests at the same time. the test procedure is very simple. the cartridge is loaded with - p.l citrated whole blood and is inserted into the platelet function analyzer (pfa aaw). the test is started automatically after a preincubation phase of . rain. the reaction starts with the contact of the whole blood and the capillary which is connected with a collagerdephinephrin coated membrane with a small aperture inside the test cartridge. under constant negative pressure the sample is aspirated and through the contact ofplatelets and vwf with collagen adherence and aggregation begins. the adhesion and aggregation process leads to the formation of a platelet plug which obstructs the flow through the aperture. the result of the php is reported as closure time (ct). additional parameters such as bleeding volumes are possible as well. first results show good reproducibility, normal values in the range of up to sec. and a good discrimination of healthy donors from patients with congenital or acquired platelet dysfunctions. the system detects aspirin induced thrombocyte function defects and von willebrand disease. in ease of an abnormal result in the collagerdepinephrin system a second type of cartridge with a collagerdadp coating can be employed. in the majority of cases aspirin induced dysfunctions are normalized and could thus detect aspirin use. the proposed system may be a valuable tool for routine assessment of the primary hemostasis potential in a routine citrate blood sample laboratory. inducing mental stress in young healthy male volunteers aged to ),ears with no previous history of thmmbophilia or a hemorrhagic diathesis was performed by a first time parachute descent from an altitude of meters. the purpose of this investigation was to find out whether there are any changes in the corpuscular and plasmatic fractions of peripheral blood. we were especially interested in elucidating changes in the procoagulatory and/or fibrinolysis systems. venous blood samples were obtained directly before and directly after the jump. flight time from the departure of the airplane to the landing of the parachutists was approximately minutes. the maximum time that elapsed between the two blood withdrawals were minutes. in a preliminary study with different voinnteem, certain fluid imbalances had been observed. absolute numbers of leukoeytes ( . vs. . l/n , erythrocytes ( . vs. . /pl), and platelets ( vs. /nl) significantly increased (p < . ), as well as the hemoglobin concentration from to g/l (p < . ). even though fluid imbalances before and after the jump had practically been excluded by measuring nearly identical hematoerit values (. vs.. ), we noticed a marked drop in aptr ( vs. sec) and a significant increase in factor viii ~tivity. as a direct stress response, we found a rise in fibrinogen concentration ( . vs. . g/l) which is one of the shortest acting acute phase proteins. concerning reactive fibrinolysis, d-dimers showed an increase in concentration from lag/l to still normal values of lag/l, which was not significant due to low numbers of values (p = . ). we observed similar changes in fibrin monomers and prothrombin fragments fl+ . from other investigations on the kinetics of the activation of the procoagulatory system we know that maximum activil is not reached until hours after initiation of activation.these investigations studied perioperative changes in different kind of operations which served as a control group concerning the degrees of tissue damage and resulting coagulation disturbances. to better understand these phenomena we plan to induce mental stress in a laboratoq' environment to further exclude unknow~a influences on the mechanisms which can activate the procoagulatory and fibrinolytic systems. triodena (t) / / ug ee, / / ug gestodene) were tested for their effect on hemostatic parameters. three groups (n= ) of healthy female volunteers were treated for months with one of these oc. blood was taken before treatment (day - of pretreatment cycle, ) and on days - of the ~ (i) and (ii) treatment cycle. indications of an activation of blood coagulation and fibrinolysis were detected as the plasma levels of prothrombin fragment f i+ and of fibrin split product d-dimer and plasmin antiplasmin complexes were found elevated during treatment. the following main regulatory components of blood coagulation, activators and inhibitors, were investigated: factor vii antigen fviiag, fvii clotting activity fviie, circulating activated factor vii cfviia and antithrombin at activity, total protein s antigen tps-ag, free protein s antigen fps-ag, protein s activity psact, circulating thrombomodulin etm fviiag, fviie and cfviia significantly increased during treatment; cfviia: : c . mu/ml a prethrombotic condition characterized by elevated levels of circulating soluble fibrin has been claimed to be a predisposing factor for accumulation of coronary thrombotic material in acute myocardial infarction. the present study includes patients with clinical suspicion of myocardial infarction. blood samples were drawn by the primary care physician, upon arrival in the hospital, and after , , , and hours of hospital stay. patients with myocardial infarction were identified by typical course in lead ecg, and upon sequential determination of troponine t, myoglobin, ck, and ck-mb. patients with primary cpr were excluded from evaluation. soluble fibrin was measured by enzymun®-test fm (boehringer mannheim). patients with acute myocardial infarction display soluble fibrin levels within the normal range (< ~tg/ml) during the initial two hours after onset of symptoms. there was no significant difference between patients with myocardial infarction and patients with coronary heart disease without myocardial infarction. slightly elevated levels were found in patients with atrial fibrillation, reflecting intracardiac fibrin formation. in patients without fibrinolytie treatment, a slight increase of soluble fibrin levels with a maximum after approximately hours is observed. most patients with fibrinolytic treatment display a considerable increase in soluble fibrin, with maximum levels immediately after infusion of the fibrinolytic agent. four patients with pulmonary embolism showed soluble fibrin levels in the range of - [.tg/ml, which remained in the same range during the entire observation period. in conclusion, circulating soluble fibrin is not increased in patients with acute myocardial infarction and does not appear to be a predictor of acute coronary events. high levels of soluble fibrin in patients with fibrinolytic therapy may reflect release of fibrin from thrombotic material, but also de novo generation of fibrin due to release of active thrombin from thrombi not necessarily located in the coronary vessels. detection of elevated levels of soluble fibrin in patients with acute chest pain should result in careful examination for signs of pulmonary embolism or aortic aneurysm. the possibility to determine activated coagulation factors opens the question if data provide evidence of an activated coagulation or fibrinolysis and if this has a prospective value. we investigated patients with confirmed thrombosis, postsurgical septieaemia and also after liver transplantation. in all patients factor viia, xii, xiia and also the fibrinolytic parameters t-pa, pai- , pap, plasminogen and a -ap were determined. in addition, f + and apc-resistance with heterocygote factor v-leiden-mutation and confirmed thrombosis. we found increased factor viia which showed partly also an increased fl+ . patients with other pathological results such as a reduced t-pa and/or increased pai- showed a low incidence of elevations in factor vii or f + . the activation of factor xii seems to be of minor importance in patients with thrombosis. a different picture is found in septic and transplanted patients. obviously factor xii-activation is of major importance in this group. a deterioration of the clinical symptoms is correlated with an increased factor xiia which is paralleled by a decrease of factor xiiactivity. the investigation of fibrinolysis parameters such as pai- and pap demonstrate a fibrinolytic disturbance of the balance. statistically significant are differences in septicaemic patients both in the surgical and in the internistical group in contrast to polytrauma patients. in patients with liver transplantations significant changes are apparently related to rejection of the transplanted organ together with a deterioration of the clinical picture. the possibility to detect activated coagulation factors may be a tool to detect changes in the hemostasis system at an early stage and to use this for an improved therapy. control of long-term oral anticoagulation is usually performed by serial determinations of the prothrombin time. however, the assessment of effective anticoagulation versus the potential risk of bleeding complication is difficult to achieve. molecular markers of blood coagulation activation might add valuable information in individual cases. we investigated patients with thromboembolic manifestations (deep vein thrombosis n= , pulmonary embolism n= , myocardial infarction n= ) for one year beginning with admission to the hospital. tat, prothrombin fragments f + , d-dirner and fibrin monomer concentrations were analysed. all markers were significantly increased at the time of initiation of anticoagulant therapy thus reflecting a prethrombotic situation. patients suffering from venous thromboembolism demonstrated higher concentrations of tat and f + in comparison to myocardial infarction ( . vs . pg/ , p=o. ; . vs . nmol/i, p= . ). f + , tat and d-dimer concentrations decreased gradually over the first days of anticoagulant therapy reaching values within the established normal ranges in all cases. f + and tat concentrations reflect the activity of the coagulation system during long-term anticoagulation whereas analysis of fibrin monomer yielded partly controversial results. we conclude that f + and tat appear to be superior to fibrin monomer for the individual control of oral anticoagulant therapy. the influence of thyroid failure on haemostasis is controversial. mainly hypoceagulable states have been described in clinically overt hypothyroidism. since hypothyroidism has been associated with an increased risk of atherosclerosis, we studied a wide range of haemostatic factors in untreated female patients with subclinical (b, n= , age + ) or overt (c, n= , age -zcj) hypothyroidism, as well as in hypothyroid women under " treatment (d, n= , age + ) and euthyroid controls (a, n= , age + ). simple screening tests (prothrombin time, activated partial thromboplastin time, fibdnogen), procoagulant factors (fvii, fviii, von willebrand factor), coagulation inhibitors (antithrombin ill, hepadn cofactor ii, protein c, protein s) and fibdnolytic factors (plasminogen, antiplasmin, plasminogen activator inhibitor, tissue plasminogen activator) were measured. results factor vii activity (vii:c), factor vii antigen (vii:ag) and their ratio were found increased in hypothyroid patients. factor viii activity showed the same tendency, whereas von willebrand factor ramained unchanged, as did all other parameters with exception of free protein s, which declined in overt hypothyroidism and in t treated subjects. these differences tended to diminish after exclusion of women with estrogen replacement therapy for menopause, but the ratio vii:cnii:ag, as well as fvii:c still remained significantly higher in hypothyroid patients. conclusions: subclinical and overt hypothyroidism are associated with significantly higher levels of factor vii:c and vii:ag. the disproportionate increase in vii:c compared to vll:ag, as shown by their ratio, might reflect the presence of activated factor vii (vila), which in turn indicates a hypercoagulable state. this pattern becomes more pronounced with the concomitant estrogen replacement after menopause. exocytosis following platelet activation leads to translocation of cd p (p-selectin), cd , and thrombospondin, from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. we here report detectability of these molecules preformedprior to platelet activation -inside the cytoplasm of resting platelets. two different methods are compared, i. e. using either methanol or the fix&perm kit (an der grub) for cell membrane permeabilization. in addition, interleukin(il)-ice is shown to be present in platelet cytoplasm after methanol treatment, but not after permeabilization using fix&perm. whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining. our data demonstrate the feasibility of the methods presented for the detection of intracellular platelet molecules. this technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilization procedure does not lead to antigen leakage or destruction. physical exercise activates the clotting as well as the fibrinolytic system as indicated in numerous investigations of exercise by running and by bicycle ergometer but not by swimming. the positive effect of an endurance training in coronary sport groups is induced also by influences on the hemostatic system. the influences are suppression of the clotting activation by the acute exercise and by an increased fibrinolysis response. different hemostatic parameters, therefore, were analyzed before and after swimming of male coronary patients (n= ; median ag~ years, achieved heart rate: /min). indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f + among the coronary patients (tat from , to , pg/ ; fi+ from , to , nmol/ ). the degree of clotting activation among the coronary patients was less than that observed in a group of young volunteers in a former investigation. this must be explained by existence of the coronary heart disease or by the higher age in the patient group. indicating an activation of fibrinolysis t-pa activity increased significantly in coronary patients (from , to , iu/ml) resulting in an unchanged balance between coagulation and fibrinolysis. from this findings of the hemostatic systems no increased risk of the coronary patients by swimming can be derived. a prerequisite, however, are precautions l±ke to devoid exercise in the anaerobic range, exclusion of major heart failure and of cardiac arrhythmias before begirming of the swim training. the principle of the fontan operation consists in anastomosing the right atrium to the pulmonary arteria, thus bypassing the right ventricle and using the only functional single ventricle as a pump for the systemic circulation. there are only few data about the influence of the changes in hemodynamics on coagulation and fibrinolysis. we investigated the coagulation system in children and young adults aged to years in a general examination to months after fontan procedure. besides other abnormalities of the coagulation system, there were significantly increased values for the thrombin-antithrombin-iii-complex (tat) in patients ( %). as a marker for an activation of the fibdnolytic system we found elevated plasmin-alpha -antiplasmin-(pap-) levels in patients ( %). less frequently, the concentrations for the prothrombin-fragments and (f and ) ( patients, %) or the d-dimer ( patients, %) were increased. we didn't find significant differences in a clot-lysis-assay between fontanoperated patients and an age-matched control group. there was no significant correlation between activation of coagulation and clinical situation or diameter of the pulmonary arteria. whether the present data can help to estimate the risk for a thrombo-embolic complication following fontan procedure, still has to be investigated. the results of the clot-lysis-assay suggest, that for lysis of thrombi the same dose of rt-pa should be used as for other patients. a nd generation functional protein s assay p. van dreden* and e. adema** * serbio, gennevilliers france, ** boehringer mannheim, tutzing germany a second generation protein s test was developed with improved sensitivity to protein s and better reagent stability. the test result was found to be unaffected by apc-resistence ( patients, heterozygote for the mutation with a apti' + apc ratio between . and . ), heparin up to iu/ml and f viii activity between and %. in the test, diluted sample is mixed with protein s deficient plasma, activated factor v, activated protein c, phospholipids and an intrinsic pathway activator. this mixture is incubated for minutes. during this time, the activated protein c inactivates part of the f va. the extend of f va inactivation depends on the protein s concentration. after minutes caci is added and the time untill clot formation is measured. the clotting time is a linear function of the protein s concentration between and % protein s. for the three preproduction lots the difference in dotting time between and % protein s was - seconds. this compares to - seconds typically obtained with the old test. within run precision (n= i on sta) is cv= - % on the basis of protein s. day to day precision (n= on sta) was found to be cv= - %, again calculated on the basis of protein s concentration. the cv of % was obtained for an avk plasma with % protein s; it corresponds to a standard deviation of only . % in protein s. the insensitivity to interferences, in particular apc-resistence and better precision and stability are expected to improve the quality/reliability of a protein s determination. in this study we evaluated the use of hormonal contraception on the parameters protein c, protein s and pal. samples from women with, without hormonal contraception and in menopause were assayed by coagulometric (protein s clotting test (behdngwerke, marburg, frg) or chromogenic methods (protein c activity test and pal reagent from behringwerke, marburg, frg) in double determination and were compared with the reference ranges. in addition thromboplastin time (thromborel s reagent) and fibrinogen (multifibrin) from behringwerke, marburg, frg, and aptt (actin fs reagent from dade corp., unterschlei heim, frg) were determined. in women using hormonal contraceptives (p< , ) and in menopause (p< , ) protein s activity was significantly reduced compared to other women (< years) while protein c acitivity did not change. in menopausal women a higher susceptibility to thrombosis was supported by an increase of aptt (p< , ) and fibronogen (p< , ). while there was no change for pal, plasminogen was significantly lower in women using hormonal contraceptives and in menopause (p< , ). we could not observe a higher turnover of coagulation and fibdnolytjc system with hormonal contraception. noteworthy was the occurence of low (< mg/dl) and borderline fibrinogen (max. mg/dl) in , % of women res. in , % of women (together with borderline aptt) who had an individuell risk for arterial disease. protein s protein c fibdno~en aptt plasminog~ without hcc , -+ , , -+ , , -+ , , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] with hcc , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ± , , .+ , , [ ] [ ] [ ] , menopause , + , , ± , , : , , [ ] [ ] [ ] , hcc= hormonal contraception hemostatic parameters in a patient undergoing bone marrow and subsequent liver transplantation due to veno-occlusive disease c. salat , , e. holler t, , hi. kolbl, , b. reinhardt l, r. pihusch , p. g hring , s. poley , e. hiller l=med. klinik iii, = institut flit klin. chemie, klinikum grosshadern der ludwig-maximilians-universit~tt mfinchen, =h~tmatologikum der gsf a year old patient suffering from all received allogeneic bone marrow transplantation (bmt). after an uncomplicated early posttransplant period the patient was dismissed after weeks. a bilirubin rise with subsequent liver failure was observed during the following weeks. according to biopsy proven hepatic veno-occlusive disease (vod) liver transplantation was performed on day . unfortunately the patient died on day due to aspergillosis. we monitored levels of protein c (pc) and s (ps) as well as pall during the pre-and posttranspiant period. pal level was normal (< ng/ml) during the first weeks after bmt but increased with the manifestation of vod ( . ng/ml on day ). it reached its peak immediately before liver transplantation ( . ng/ml) and returned to normal levels within the next few days. pc levels which were normal before bmt decreased prior to clinical diagnosis of vod and were normal after liver transplantation. ps levels lay within the normal range at all timepoints. vwf was elevated before bmt ( %) and remained relatively stable during the whole investigatonal period ranging from to %. it is assumed that vod is initiated by an endothelial cell injury -possibly due to radiochemotherapy -and subsequent hypercoagulability. our results indicate that the "endothelial cell marker" vwf is not helpful in predicting vod. the kinetics of the investigated parameters underline the significance of pc and pai- as described by others and our group earlier, whereas ps does not seem to play a role in the pathogenesis of vod. the budd-chiari syndrome (bcs) is characterized by hepatic venous outflow obstruction that may be caused by the precipitation of a thrombus. it frequently coseggregates with other major diseases like myoloproliferative diseases or defects in the haemostatic system (antiprotein c and protein s deficiencies e.g.). only recently, the factor v leiden mutation (fvlm) has also been associated with bcs. we hypothesized that defects in the thrombo-modelling associated anticoagulant pathways (tmaap) are a major risk factor for the precipitation of bcs. we screened our cohort of patients (pts) with bcs for the presence of defects in the tmaap and identified pts with protein s deficiency (psd). these pts were screened for the three point mutations in exon (codon- ; ins t), exon (codon ; a-->t) and in intron (g-->a + ) of the ps alpha-gene that have been demonstrated by bertina et al to coseggregate with psd. restriction enzyme analysis and confirmation-sensitive gel electrophoresis for the detection of single-base differences in doublestranded pcr-products were employed. all living family members of the indicator pts were also screened for heterogeneties in the three point mutation as described. no single abnormality in these genes despite presence of pbd in those family members was found. in addition, pts and family members were also screened for fvlm. one pt and two of his family members, in addition to psd, were subject to fvlm. the other two lots and their family members were not subject to fvlm. in contrast to the first family, despite psd, those two pts suffered from morbus crohn and acute myeloid leukaemia as risk factors for bcs. we conclude: psd is one major risk factor for the precipitation of bcs. to precipitate this disease, one additional risk factor is required. psd may be caused by genomic defects in the protein s gene other than those described by bertina. only a few publications describe a thromboembolic disease due to dramatically reduced protein s levels being associated with viral or bacterial infections, autoimmune mechanisms are suspected but the aetiopathogenesis is still under discussion. we report on a year old boy who developed purpura fulminans of the left leg during varicella infection. on the fourth day of infection the disease started with pain and haemorrhagic efflorescence localized at the left taft. on admission the boy suffered from a purpura fulminans with central necrosis measuring x era. suspecting a hereditary thrombophilic disease we started therapy with protein c concentrate and recombinant tissue type plasminogen activator. the fellowing coagulation investigation showed a severe deficiency of protein s (total protein s-antigen < u/ml, free antigen not measurable) in combination with factor v leiden mutation. other thrombophilie and coagulation parameters did not show deviation from normal range. after weeks we saw a slight improvement of the total protein s antigen up to u/ml. the free protein s antigen was still undetectable. during the following weeks the patient recovered slowly and the protein s activity and antigen normalized. because of skin necrosis thromboembolie prophylaxis was initiated with low molecular weight heparin (fragmin®, ie/kgbw/die) and continued for months. under this therapy there were no further thromboembolic events. these results suggested an autoimmune protein s deficiency in a patient suffering from chickenpo×. an analyses of autoantibodies at the time of diagnosis showed a slight increase of the antieardiolipin antibodies (igg , iu/ml, igm , iu/ml) which normalized during hospitalisation. we suspect an antibody to protein s probably caused by similar presented viral antigens. we suppose that autoimmune mechanism during different infections in combination with a heterzygous apc-resistance may be a potential risk factor for developing thrombotic disease. in the central nervous system mrna encoding for prothrombin and thrombin receptor is present and astroglial cells in culture process and secrete thrombin. moreover, effects of thrombin on brain cells including change of neudte outgrowth and astrocyte shape are described, but the molecular mechanisms are unclear. we investigated the effects of human <z-thrombin and a six amino acid thrombin receptor activating peptide (trap- , sfllrn) on [ca +]i, phosphoinositide hydrolysis and protein kinase c activation in the rat glioma c cell line which is a widely used in vitro model for studying glial functions. stimulation of c cells with both c{-thrombin and trap- resulted in transient [ca +]i mobilization, [ h]inositol phosphate response and activation of the pkc isoenzymes c{,i], , , and s. these results suggest that ~-thrombin and trap- activate at least partially the same intraceltular signaling pathways in rat glioma c cells which is an evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma c cells. further investigations in this field are in progress including both receptor binding studies and more detailed analysis of thrombin receptor mediated signaling network and the cellular response of thrombin induced transmembranal signal transduction pathways in c glioma. thrombin is a potent mitogen in vascular smooth muscle cells (smc). the mitogenic effect of bovine thrombin and human thrombin receptor-activating peptide (hutrap- ) was investigated in bovine coronary artery smooth muscle cells. confluent cells (passage - ) were incubated for h in serum-free medium with thrombin ( nm) or trap ( - pm). the mitogenic response was assessed using [sh]thymidine incorporation. results demonstrated that thrombin induced a three-to fourfold increase in [ h]thymidine incorporation compared to the control, thus being as active as the potent mitogen pdgf-bb ( ng/ml). the mitogenic effect of thrombin was dependent on its proteolytic activity. when the active site of thrombin was irreversibly inhibited by d-phe-pro-arg-chloromethylketone (ppack) no increase in [°h]thymidine incorporation was observed even at the high concentration of nm ppackthrombin. thrombin-induced [ h]thymidine incorporation was significantly reduced by the reversible direct thrombin inhibitor nc£-( naphthylsulfonylglycyl)- -amidinophenylalanine piperidide (napap, lpm) . in contrast to thrombin trap- at a concentration up to pm did not increase [ h]thymidine incorporation in the absence or presence of the aminopeptidase inhibitor amastatin ( ijm). in order to demonstrate whether hutrap caused a stimulation of the receptor and signal transduction in bovine smc the activation of protein kinase c (pkc) was measured as translocation of the cytosolic to the membrane bound fraction. it was found that both thrombin ( nm) and trap ( pm) induced a translocation of pkc after rain. in conclusion, thrembin and trap activated pkc in coronary smooth muscle cells but only thrombin acted as a mitogen. endothelial cells once stimulated with thrombin are resistant to subsequent stimulation directly after the first. but after a recovery period of about min the cells are sensitised again for activation by thrombin. in our experiments this resensitisation couldn't be inhibited by actinomycin d or cycloheximide nor by the phosphatase inhibitor ocadaic acid. therefore an intracellularly pool of thrombin receptors has been proposed possibly co-localised with golgi vesicles. brefeldin a (bfa) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organisation of the golgi apparatus. because of this we examined the effects of bfa on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. the vwf release from weibei-patade vesicles was used as physiological parameter of activation of thrombin receptors by thrombin. the addition of bfa ( pg/ml - ng/ml) to endothelial cells blocked the receptor response regeneration, whereas the first stimulation was not influenced by bfa at the concentrations used. these results give further evidence that the proposed storage pool of thrombin receptors is iocalised in vesicles of the golgi apparatus. randomized comparison of double bolus reteplase (r-pa) and front-loaded alteplase (rt-pa) in patients with acute myocardial infarction (rapid ii) c. bode, rw. smalling, j. kalbfleisch, g. berg, dj. odenheimer, e. bshm, wd weaver and the rapid ii investigators. med. klinik iii, university of heidelberg, germany the study was undertaken to assess the efficacy of a double bolus regimen of + megaunits of reteplase, a recently developed deletion mutant of tissue-type plasminogen activator, relative to front-loaded (gusto-regimen) alteplase. there was no age limit and patients were recruited up to hrs after onset of symptoms. the primary endpoint "patency at minutes" was centrally assessed in a blinded fashion. infarct related coronary artery patency (timi grade or ) and complete patency (timi grade ) at min for reteplase vs. alteplase were . % vs. . % (p= . ) and . % vs. . % (p= . ), respectively. at minutes, the incidence of both, patency and complete patency was also significantly higher in retep ase vs. aiteplase treated patients: . % vs. . % (p= . ). and . % vs. . % (p= . ). reteplase treated patients required fewer additional coronary interventions within the first hrs of treatment ( . % vs. . %, p= . ). there were no significant differences between reteplase vs. alteplase regimens in day mortality ( . % vs. . %), severe bleedings ( . % vs . %) • or hemorrhagic stroke ( . % vs. . %). reteplase, when given as a double bolus of + mu to patients with acute myocardial infarction, achieves significantly higher rates of early patency and requires fewer acute coronary interventions than front-loaded alteplase without an apparent increased risk of complications. lysis therapy with uhsk of deep vein thrombosis (dvt) of the lower extremity commoniy results in a relatively high patency rate. in our patients, patency was achieved in approx. - %. these results were supported by several studies. in dvt of the pelvic veins, however, this kind of aggressive therapy is excluded due to the risk of iatrogenically induced pulmonary embolism. on the other hand, the need for an effective therapy is evident because of the usually occtnring complete occlusion of the pelvic veins and the following severe clinical symptoms phlegnmsia cerulca dolens etc.). until september " , consecutive patients ( males, mean age ranging from to years) with dvt of pelvic veins were included in this study. after clinical and phiebographic/computer tomographic (ct) diagnosis, a temporary filter system was placed intravenously either by a wambmchial ( pat.) access route, lransfemorally ( pat_) or wansjngularly ( pat.) under mdiological control. the open filter was positioned above the thrembus in the lower caval vein: times an antbeor system, times an anglocor and times a cook filter were used. in the following three days up to cycles of lysis therapy with mli u of streptokinase were administered, each cycle lasted six hours. heparin was given simultanously via the filter system in therapeutic dosages in order to prolong alrit values . to times the individual base line level. effective lysis was controlled by a second phlebography/ct, and the filter system removed through the introducer sheath valve. after lysis therapy all patients were put on coumarin with overlapping heparinization. successful lysis (complete recaanlisation without remaining thrombi and lysis of more than per cent of the original thrombus) could be achieved in % ( patients). in two cases a fatal pulmonary embolism occurred. times great parts of a thrombus were captured by the filter and could be removed with the system. due to an insufficient boparin therapy we detected a newly developed thrombosis of the lower cavul vein twice after lysis. this complication could be resolved by additional administration of streptokinase. in nearly all cases we found extensive hematomas on the catheter insertion sites. patients with transfemoral applications had to be transfitsed with red packed cells due to retroperitoneal hematoma. one filter system was broken after use. lysis of dvt of the pelvic veins with uhsk and with the protection of a removable temporary filter system situated in the lower caval vein enables us to carry out an effective therapy. the filter systems, however, have to be ameliorated in order to better prevent fatal pulmonary embolism. heparin should be administered at least in the remainder of the day of lysis. the insertion site should be the enbital vein because compression for bleeding complication is easily feasible at this location. we report about seven of our patients (pts.) with bcs treated by fibrinolytic therapy ( female, male, age , years + ). only one case was of unknown origin while in four pts. myeloproliferative syndromes, in one protein c deficiency and in another one morbus behcet was detected in five pts. chronical and in two acute bcs was diagnosed. as complications were found: in three pts• thrombosis of vena iv.) cava, in one ofv. porta and v. lienalis, in one more of v• porta and v• cava. we treated just one patient with urokinase ( x . iu/d for days). four pts. were treated with rt-pa in concentrations of to mg/d (for a period of up to days) and continuous heparin infusion. high doses of rt-pa ( to mg/d for one or four days) were given in one case of acute and one of chronic bcs. partial (after high-dose rt-pa nearly complete) recanalisation of thrombotic hepaticel veins was only achieved in the two acute cases of bcs. no significant recanalisation was proven in hepatical veins of chronic bcs. three times complete fibrinolysis of v. cava thrombosis (two after highdose therapy) and one partial lysis could be attained. in one patient partial lysis of v. porta and in another one of v. lienalis was found. furthermore complete recanatisation of v. porta followed local rt-pa lysis during surgical intervention. in our two pts. with acute bcs significant improvement of hepatical venous flow could be measured after fibrinolytic therapy• in chronic bcs hepatical venous flow did not improve while the complication of v. cava thrombosis could eventually be treated successfully. probably due to fibrinolytic therapy one case of esophageal varix bleeding occurred. the benefit of flbrinolytic therapy of bcs seems to be influenced by the dur~ion of the disease• according to our results fibrinolytic therapy should be considered in cases of acute bcs as well as in the above mentioned thrombotic complications• recent studies suggest that transport of thrombolytic agents through the clot can play a major role in determining the reperfusion time needed for thrombolysis. as the mechanisms of this phenomena are rather unclear we studied fibrin ( . - . ~tm) clot lysis by plasmin ( nm) presented outside or inside the clot in purified system (tris-hc buffer, °c). the clots was formed by addition of cac ( mm), thromboplastin ( u/ml) and human thrombin ( nih/ml)t the lysis degree was determined either by counting of radiolabelled fibrin degradation products or by time of total lysis. obtained kinetic parameters of this reaction: kest= . rain - , km=i. ~tm (plasmin inside clot); kcat=l. rain - , km=i. lzm (plasmin outside clot). this data indicate that eatalytie efficiency of plasmin from inside the clot is -fold higher than from outside as measured from kcat/km. furthermore we studied plasma clot ( . ml) lysis in original plasma ( ml) containing sk ( - nm) and seu-pa ( - rim) in the final concentrations from inside or outside the clot. the results are presented in the the similar results have been got when the fibrin clots were formed with batroxobin instead of thrombin. it may be concluded that fibfinolysis is a surface-dependent process. it's likely that the higher level of plasminogen activators in blood would prevent thrombus formation, while physiological clots would stay resistant. for the improvement of the thrombolytic therapy in children more clinical data are needed. therefore we report on our experiences with rt-pa presenting the clinical course in patients (age range: days to years) with venous thrombosis (n= ) and purpura fulminans by meningococcosis (n= ). the patients were treated with rt-pa (actilyse r) for , hours to days. the venous thromboses were localized in lilac/femoral veins (n= ), brachiocephalic/jugular/subclavian veins (n= ) and the cava superior vein (n=l). central venous catheters were causative in cases. the dosage range was between . to . mg/kg for the initial dose and . to . mg/kg/d for the subsequent continous infusion. ten patients received simultaneously heparin. complete clot dissolution occured in cases, partial clot dissolution in patient. in patients with a history of the first clinical signs of thrombosis of weeks or more the lysis was not successful. concerning the side effects only small bleeding episodes on former venipuncture sites were observed in patients. systemic effects such as the decrease of the plasma flbrinogen concentration were rare too. in conclusion, the thrombelysis with rt-pa presents an effective and safe therapy in children at: the rt-pa dosage used. in case of a history of the thrombotic event of more than weeks a thrombotytic therapy should not be carried out. d.dimer is widely used in clinical situations associated with thrombotic diseases. the most popular application is the exclusion diagnosis of deep-veinthrombosis and pulmonary-embolism and the assay is performed in emergency. d.dimer is also frequently measured for evaluating the thrombotic risk in the post-operative period. quantitative, flexible and sensitive assays are required. we have developed a new automatic method which works on the coagulation walk-away instrument, sta. this assay was developed with two monocloual antibodies specific for d.dimer coated on microlatex particles and it uses an immuno-turbidimetric technology. absorbance is measured at nm and is a direct relationship of d.dimer concentrations. the assay works with ~tl undiluted plasma and is performed in less than minutes. the dynamic range is from . to [~g/ml and no prozone effect is observed up to concentrations higher than ~tg/ml. the detection threshold is of . [lg/ml. the intra-assay reproducibility is below % and the inter-assay reproducibility is below %. recovery studies of purified d.dimer added to normal plasma were between and %. no interference of heparin, bflirubine, lipids or of rheumatoid factor up to concentrations of u/ml is observed. there is no effect of high fibrinogen concentrations (> g/l). when compared with conventional elisa techniques (asserachrom ddi), the assay demonstrated a correlation coefficient of . on samples from normal individuals and hospitalised patients with elevated d.dimer concentrations. slope was of . and intercept was of - . . this new assay offers a full flexibility for individual testing as the calibration curve is stable for at least one week on the instrument. it is then well adapted for all the applications of d.dimer measurements in coagulation laboratories. children between an age of days and months ( median weeks ) with thrombotic or embolic occlusion of major vessels were treated with rt-pa for thrombolysis. the affected vessels were both sided renal veins or one sided renal vein and v. cava inf. in cases, the v. cava superior in , the v. cava inf. plus renal veins plus aorta in , the left ventdcle in , the aorta in , the a. femoralis in and the v. portae in case. out of occlusions were associated with an indwelling catheter. underlying dieseases were sepsis ( ), prematudty ( ), vitiurn ( ), asphyxia ( ), short bowel syndrome ( ), hus ( ), diabetes ( ), cmv ( ), exsiccosis ( ) and m. hirschsprung ( ). thrombolysis was performed with an bolus of rt-pa ( . - . mg/kg) followed by continuous infusion ( . - . (- ) mg/kg/ h, median . mg/kg/ h). low dose hepadn ( ie/kg/ h) was given dudng full dose hepadn (aptt , - times normal) after the thrombolysis. in pts. rt-pa was administered locally through the catheter and in cases systemically. in patients the vessels could be recanalised completely, in partially, in patient the therapy had to be discontinued. in vessels a reocclusion occurred. bleedings were noted in three patients, all from recent venous puncture sites. the results encouraged us to start a multi-canter trial which has been approved by the ethical committee and is open for recrural. the aim is to compare efficacy and safety of rt-pa with urokinase, the only recommended standard in the management of critical major vessel obstruction in newborns and infants. the design is a randomised, notblinded trial with a cross-over option after three days in cases without success. study end points are recanalisations, major bleedings and number of cross-overs. inclusion criteria are age under year, lifethreatening vessel obstruction, age of thrombus up to days, no precaeding fibdnolytic therapy. exclusion cdteda are cerebral hemorrage, pedventricular leukomalacia, surgery dudng the last days and cns injuries during the last months. although our knowledge on inherited thrombotic coagulation disorders has greatly expanded within the last years, there are still man}, patients with recurrent venous thrombosis in whom no obvious predasposition can be identified.thus we decided to include also so-called rare defects associated with thrombosis in our routine thrombophilia screening programme, such as fxii deficiency. fxii is an important element m the intrinsic pathway of fibrinolysis and there is evidence for an insufficient fibrinolytic activity in fxii deficient pts..up to date only few and controversial data exist about the frequency of fxii deficiency in pts. with thrombophilia. cons~uently the aim of our study was to evaluate the association between fxii deficiency and juvenile venous thrombosis in a great population. patients and methods: pts. ( female, male, aged i to ys, median age . ys) with venous thromboembolism before the age of ys were studied. one-stage clotting activity assay of fxii (fxii:c) was performed on acl using fxii deficient plasma from instrumentation laborato~. fxii antigen concentration (fxii:ag) was measured by electroimmundiffusion using reagents from behfingwerke, enzym research respectively. the normal ranges are tl~. routine reference values obtained m our labratory from healthy subjects ( males, females, median age . ys); % range: fxii:c - %, fxii:ag - %). results: / pts.were classified as fxi deficient (f , m ), giving a prevalence of . %. severe fxii deficiencies with fxii:c below % were observed in pts..ll pts= proved to have moderate fxii deficiency with fxihc ranging lrom to % and fxii:ag ranging from to %. in none of them inherited deficiencies of other well established thrombophila risk factors could be detected. none of the fxii deficient pts. had positive lupus anticoagulant tests. familial fxii deficiency was found m cases. discussion and conclusion: the precedences of fxii deficiency amongpts, with venous thromboembolism was previously described to be . - %. supporting these data, we have shown a praevalence of fxii deficiency of . %. in comparison to the frequency of other well established thrombophila risk factors we consequently have observed a relatively high prevalence of fxii deficiency m our study group.these data, from the largest such study reported, strongly indicate that fxii deficiency may not be a rare deficiency and may be more frequently associated with thrombosis than currently suspected. we describe a family with an exceptionally rare, i.e. plasminogen, deficiency, combined with subnormal activities of coagulation factor xii (hageman factor). the first thromboembolic event, pulmonary embolism in the proposita was diagnosed at age . since that time, 'spontaneous' venous thromboembolic events verified by phlebography and perfusion/ventilation lung scan recurred once every year despite oral coumarin therapy, whose intensity varied over an exceptionally wide range despite tight control the patient was repeatedly given succesful thrombolytic therapy with streptokinase or recombinant tissue plasminogen activator. her plasma plasminogen chromogenic activity was - % compared to a normal plasma pool (reference range - %), plasminogen antigen was diminished to the same extent. the patient's factor xii exhibited only - % activity in a factor-deficient plasma assay as compared to a normal plasma pool. other known risk factors for recurrent venous thromboembolism were not present : no evidence of malignancy, no obvious precipitating events, normal values of antithrombin iii, protein c, protein s, fihrinogen, thrombin time, platelets, lupus-like anticoagulant, aptt prolongation after addition of activated protein c. the proposita's mother had died at age from pulmonary embolisnt no coagulation studies are available. the proposita's sister was first diagnosed deep leg vein thrombosis at age , since that time recurrent episodes of venous thromboembolism have been diagnosed also in an other hospital. this sister's plasminogen activity was %, but factor xii activity was reduced to %. three brothers of the proposita were examined, too, all in their rd decade of life. none of them recalled symptoms of or treatment for thromboembolic disease. in one brother, factor xii activity was normal ( - %), but plasminogen only about %. in the nd brother, factor xii was very variable ( , and %), plasminogen was in the lower normal range, in the rd brother, factor xii was about % (repeatedly), plasminogen was normal. current knowledge about the risk of thromboembolism with both enzymes is limited, the optimal management remains controversial. msrgit serbsn,maria cucuruz,dan madras,carmen petrescu, natalie rosiu,rodica costa iii rd psediatric clintc,universtt v of medicine, the unsatisfactory efficiency of entihepetitis b vaccination in our haemsphiliscs suggested the control of the immune status in hiv negative patients,by establishing through flowcitomstrie with monoclonsl antibodies the lymphocyte subsets (cd ,cd ,cds,cd&/cd ratio and cd ) and by seric tmmunoglobulins levels; the immunological parameters have been correlated with the serological markers of hepatitis infections (hay, hbv,hcv ebd hdv) as well as on dependence with the treatment (blood,plasma,crysprecipitate,fector viii/ix concentrate) and the quantity of their consumption (ui/k weight/yesr).the interpretation of the results pointed out • significant lower level of cd ,cd& (p<o,o ) st the group of hsemophiliscs treated with blood,plasma and cryopraclpitate; sdditionsly,in this group of patients it was found s significant drop of the cd counts (p< , ) without an immunoglobulin-deficiency. the same behaviour was found at the group of haemophiliacs carriers of hb~ and hcv markers.the superpositisn of hepatitis infections on the treatment with blood and native blood products in most of the cases has let unanswered the question related to the mechanism of the immunodeficiency: the hepatitis b or c infections or the negative impact of the itarative important alloantigenlc load ? illumination with l~tm methylenblue (mb) is used for inactivation of enveloped viruses in human plasma. via a photooxidative mechanism viral structures as well as plasmaproteins especially fibrinogen can be damaged. fibrinogen is an important mediator in the cell-cell interaction of platelets. the four rgd sequences (arg-gly-asp) of fibrinogen can be recognized by the activated fibrinogenreceptor gphb/iiia . after binding to gpiib/iiia one molecule fibdnogen can link two platelets. the aim of this study was to investigate the influence of mb treatment on the fibrinogen activity in human plasma. furthermore we were interested to demonstrate if altered fibrinogen inhibit platelet aggregation by blocking the gpiib/]iia receptor. human plasma was treated with - ~m mb. after illumination fibdnogen was isolated by fl-alanin precipitation. purified fibdnogen was analysed by one-and twodimensional gelelectrophoresis. influence of modified fibrinogen on platetet aggregation was measured with gelfiltrated platelets. possible receptorblockade were examined in an j-fibrinogen competition assay. densitometric scanning of reduced sds-pages showed a decrease of the a-subunit as well as an increase of high molecular aggregates with increased mb concentrations. this effect was not detectable for fibrinogen isolated from plasma with bm mb. this results were confirmed by two-dimensional gelelectrophoresis. ibm mb treatment showed no receptor blockade in aggrement with normal platelet aggregation after stimulation with adp and fibrinogen. proposed validation of a quantitive quality-assured pcr method f. dorner, j. eibl, g. zerlauth immuno ag we have developed a highly sensitive and reliable quantitative method, based on the polymerase chain reaction (pcr) and on the reverse transcriptase polymerase chain reaction (rt-pcr), to screen for the nucleic acids of human immunodeficiency virus type (hiv-i), hepatitis b virus (hbv) and hepatitis c virus (hcv) in plasma and plasma products. the detection of pcr products is carried out by laser induced fluorescence, the procedure is therefore termed "laser induced fluorescence pcr" (lif-pcr). the procedure allows the precise quantitation of genome equivalents due to the use of two calibrators that are co-amplified by the same set of primers as the target template in one and the same test tube. a high degree of reliability is achieved by co-precipitation of the extract, co-amplification and co-determination of the calibrators together with the genome equivalents to be determined. in this report we present validation data for the lif-pcr and discuss them in the light of the recently published ich-guidelines on the validation of analytical procedures. taking into account the pecularitles of the replacement therapy of haemcphiliacs in romania the study aimed at evaluating the dimension and profile of blood-born infections in these patients; hsemophlllacs,elasslfled in groups: a-ulth no exposure to blood or bloodproducts, b-treated ~ith factor-concentrates and c-ulth plasma,cryoprecipitate + factor-concentrates in their therapy have been investigated (elisa method) for serological markers of hepatitis a,b,c end d, cvtomegalovirus (cmv) and hiv i and hiu infection. the infections profile of our haemophiliacs~hes some peculsrities. the hepatitis infection is the most extended,lnvolving more than % of the patients. the frequency is for hepatitis -bz,z~ , u -"/~,~ , g -bb, % end o - % : the multiple infection was diagnosed in more than % of cases. the prevalence uas significantly hlgher in the group c. we studied blood count and relevant coagulation system parameters of autologous blood donors. indications for autologous blood donation were divided in categories: a group of diverse (a, n= ), reduction mammoplasty (b, n: ), prostatectomy (c, n=io) and total hip replacement (d, n= ). we performed bleod count (bc), thromboplastin time (tt), partial thromboplastin time (aptt), fibrinogen (fib), f vhi act, vwf rco, vwf ag, f xiii, at iii, d-dimer-fragments (dd) and fibrinmonomers (fm). all parameters were analyzed as initial value, bc, tt, aptt, fib, f xiii and at iii were measured additionally at each donation. the percentage of initial pathologic data was ( ) were included in the study. the inclusion criterittm was: an endegen(y.~ red cell reserve (ercr) of less than ml in men and less than ml in women. ercr is the volume of blood that can be withdrawn up to tim hematocrit of . . the selected dosages of rhepo ( , and iu/kg body weight biweekly over weeks) were based on the calculated blood volume and the ercr. a unit of whole blood, separated into an autologens red cell concentrate and one unit of fresh frozen plasma, was collected when ]mnoglobin concentration was at least . g/ ml (or hematocrit was at least . ) up to an autologons predeposit of ml red cell volume (rcv). additionaly, platelet concentrations and serum ferritin levels were measured~ results: at the initial clinical examination the ercr was ml in women and in men. this resulted in a weekly dosage of x . iu rlaepo in women and x . iu rhepo in men. patients donated red cell each ( ml rcv on an average) within . days. the targeted rcv could not be reached in patients because the operation was reschednled. the collection from one patient had to be aborted because of angina pcetoris complains. tic following side-cffczts occurred: episodic hypcrtejlsion ( tim~), chest pain ( ), fafigne ( ), inflnenza-lihe syndrome ( ). platelet count increased from initially - x '//d (i %) during therapy to a maximum level of + v~ ( %) without any clinical signs of thrombeembolism. in the control group (autologous blood donation weekly or times without erythrupoiedn stimulation, n= ) platelets increased from initially __+ '/fi ( %) to + v#i ( %) i ]aximlzm colldttsions: the adlninist/'ation of lhepo in the above described manner to autologous donors undergoing heart surgery is well tolerated. it allows to collect a sufficient number of autologous blood units and the preparation of a predeposit at short notice. essential thrombocythemia is a chronic myeloproliferative disease with a benign clinical course that is mainly complicated by microcirculatory or thrombotic vascular disorders. it is at present uncertain which patients need (lifelong) therapy and whether cytoreduction is necessary. we retrospectively evaluated vascular complications in patients ( men, women) with a median age of years in relation to therapy. three major treatment groups were analysed: patients treated with acetylsalicylic acid (ass) alone, patients given alkylating agents, and patients treated with interferon alpha (ifn). there was no significant number of patients treated with hydroxyurea. ifn-treated patients were younger (median age years) than patients in the other groups, but platelet counts before therapy were similar (median . - . x /i). before therapy, % of patients treated with ass, % treated with alkylating drugs, and % treated with ifn had bleeding events, microcirculatory or thrombotic complications. dudng therapy, median platelet count was . x /i in patients on ass, . x /i in patients treated with alkylants, and . x /i in ifn-teated patients. the rate of patients with complications during therapy was % ( . %/patient year) for ass, % ( . %/patient year) for patients on alkylating drugs, and % ( . °/dpatient year) for ifn-treated patients. we conclude that all three treatment regimes seemed to have a beneficial effect on the incidence of vascular complications in essential thrombocythemia. the results give a rough estimate of complication rates to be expected and may form the basis for randomized clinical trials. adhesion of activated platelets to leukocyte* has been shown to be mediated by at least two molecules on the platelet surface: p-selectin (cd ) and fibrinogen (fg) bound to the gp iib/iiia complex. interaction of platelets with teukocytes via cd is believed to stimulate the production of reactive oxygen species (ros) in leukocyte*. the role of platetet-bound fg as a signalling molecule in platelet-leukocyte interaction is controversially discussed: stimulatory as well as inhibitory effects on leukocyte ros production has been described. in the present study we measured ros production in undiluted samples of citrated whole blood (wb) as well as in suspensions of erythrocytes and leukocytes in platelet-rich plasma (platelet-rich wb) or platelet-poor plasma (ptatelet-poor wb). ros production was determined by measuring luminol-enhanced chemiluminescence (cl). stirring wb samples at , rpm for min resulted in an activation of leukocyte* (increase of cl, upregulation of cd lb) and platelets (platelet aggregation, upregulation of cd ). stirring-induced cl was significantly higher in platelet-poor wb when compared to platelet-rich wb. higher cl in plateletpoor wb than in platelet-rich wb was also found when adp (moderate stimulation of platelets and leukocyte*) or fmlp (strong stimulation of leukocyte*) but not when pma (strong platelet and leukocyte stimulation) was added. dextran sulphate that is known to block cd -mediated interaction ofplatelets with leukocyte* caused a dose-dependent inhibition of stirring-induced cl in wb samples. in contrast, when binding of fg to the platelet surface was blocked by rgds or the fg receptor antagonist gr , stirring-induced cl, but not cl in unstirred samples, was significantly enhanced. gr also reduced adhesion of platelets to neutrophils, but did not affect cd exposure. the results indicate that interaction of platelets with leukocyte* in whole blood via cd and fg may have opposite effects on leukocyte ros production: stimulation by cd and inhibition by platelet-bound fibrinogen. late reocclusive processes represent one of the most commonly seen serious adverse events after coronary angioplasty. the pathophysiology of both of these processes is multifunctional and represens complex time dependent pathophysiologic events involving both the humeral and cellular processes. to test the effects of low molecular weight heparin (certoparin, sandoz ag, numberg germany) against late occlusive processes, four groups (groups a-d) of rats ( - ) were anesthetized and the external common carotid arteries were exposed. all groups received a u/kg of unfraetionated heparin through the femoral vein. a f balloon emboleetomy catheter was inserted into the left carotid artery and passed throug the left common carotid artery leading to the aortic arck vascdar injury was induced by repeatedly (x ) inflating the catheter balloon and withdrawing it to the bifurcation. following this procedure, two hours after, equlgravimetric amounts of various agents were administered via sc route to each of the individual groups. group a was administered with certoparin od for days. group b received a lower low molecular weight heprin (mr . kda). group c received unfraetionated heparin and group d served as a control. on day , perfusion fixation was performed on all animals. the injured and contralateral carotid arteries were excised for histelogic examinatior~ in comparison to the saline control, varying degrees of the inhibition of myointimal proliferation following arterial injury was noted in all groups. however, the degree of inhibition followed the order: heparin<certopann<llmwh_ in contrast to heparin treated group (c), the other groups a, b, and d did not exhibit any hemorrhagic lesions. however, degree of perivascular fibrosis, hemorrhage and inflammation were observed at the infusion site. in comparison to heparin, certoparin and its lower low molecular weight derivatives exhibit better efficacy in reducing balloon angioplasty induced restenosis. the observed efficacy of these agents may be due to better bioavailability and sustained duration of action. in the present paper the binding of heparin to granulocytes of human and animal species was analyzed and compared with the binding to monocytes and lymphocytes. a low-molecularmass heparin (lmmh) was covalently bound to tyramine (tyr) and subsequently tagged with fluorescein- -isothiocyanate (fitc) by endpoint attachmenc. binding to leukocytes was quantified using flow cytometry analysis. the leukocyte populations were identified by their light scatter properties in man by cd , cd , and cd antibodies, in mice by cd and gr antibodies, and in rat by cd , ed , and rp antibodies. granulocytes, monocytes and lymphocytes of all species bound dose-dependently lmm-heparin. the binding of lmmh-tyr-fitc ~to granulocytes was higher in human, rat, rabbit, dog, and pig as compared to monocytes and lymphocytes. mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. binding of lmmh-tyr-fitc was reversible in the presence of unlabeled heparin or lmmh. more than % of human, rat, rabbit, dog and sheep granulocyte populations could be'distinguished from monocytes and lymphocytes by their fluorescence intensity after incubation with lmmh-tyr-fitc. this separation was not obtained for mouse and pig granulocytes. the data present evidence of a specific heparin binding to granulocytes of many species and indicate the significance of fluorescent labeled lmm-heparin for biological investigations. background and purpose: ptca is an effective treatment for restoring coronary artery pat~cy; however, acute thrombotic rencclusion is a drawback of the procedure. currem prevention of rencclusion relies on antiplatelet agmts (aspirin) and antithrombin agents aeparin). in the present study we examined the time course of changes in blood coagulation and fibrinolytic function following ptca in patients with ischaemic heart disease. patients were randomly assigned to receive either standard therapy ( nag aspirin p.o.; . iu hepafin i.e. bolus, and - . iu heparin intra-proeadurally)(n= ), or adjunctive therapy (aspirin, hepann; mg sodium pmtosan polysulphate, spp intra-cortmarily and subcutaneously) (n= ). the sensitive markers of thrombin activation (thrombin-amithrombin iii complex ('rat), prothrombin fragmem f + (pl+ ), d-dimer) and of fibrinolytic activation (plasmin-antiplasmin complex (pap), tissue type plasminogan activator (t-pa) and plasminogen activator inhibitor (pal)-i activities) were measured before, immediately after, and hs after ptca. results: we found elevated mean levels of tat and fl+ in both groups during the procedure, while plasma coneentrafiuns of d-dimer and pap remained in normal range. activity of pai-i was not elevated compared to normal comrol. the t-pa activity obtained immediately post-ptca showed significant increase in patients given spp. primary angiologic success was achieved in % of both groups. one patient in each group had recurrent ischaemia and emergency interventions (angioplasty, stent, bypass surgery) were performed. one patient receiving adjunctive therapy died in cardiogenic shock within hs arer ptca. major bleeding was observed in one patient given spp. conclusion: no significant intracoronary haemostatic activation occurred in the majority of patients during and up to hs after ptca. treatment with spp was associated with enhanced fibrinolytic activity. the benefits and risks of adding spp to heparm and aspirin should be evaluated by further studies, in july ,¢ the paul ehdich institute became responsible for quality and safety of bt~d and blood products. since ~his time, batch m~se of ppsb and factor ix prapamtions were carded out regularly, whereas ihe obligatop i testlng of all other products derived frem pooled plasma will be starling in janualy . the european pharmacopoeia does .or contain directions for the quality control of ppsb preparations. therefore, ifua ~ coneenning factor ix clotting concentrates are applied, ircludmg the non activated ptt and the classical tkrombln test these m~ods are not su'rlable for reliable exclusion of ituombogenk~b/ ,of pp,sfl, because vlla actlvry is not d~. a hllhedo unsolved problem is the high sensitivity of file classical thrombin test often not correlating with ~ data of thrombogenlci~. in ordm td es~abl~ an effective quality conlrd protocol, different methods for the qual~ative detennlna~on of ila, vlla. ixa, and xa (chrornogenic, fluorege~c clolfing reactions) wera instaued. in agreement with the [deralure, considerable differences were found among the ppsb pr~ons registered in germany. cedain amounts of activated do~ng racers may be well tole~ted in redp/ents. ~no are in a steady state condition. however, in severely ill patienls, eg. post operatively, tissue factor may be expressed which pofenllatesthe dsk of ~aclor vlta. in the case of a certain ppsb preparation hlgh ¢onceatratio~ of acthrated cto~ng factors, especially ~vated factor vii, cotmlaled with fatal comlffcal~ns, evaluating ~ expe~ concenlralbn limits for adjvated prothrombin co~np~.x factors as prerequisite for ~e batch r~ease have to be applied. aprotinin is used as a hemostatic agent in cardiopulmonary bypass surgery and other indications where hemostatic compromise results in hemorrhagic manifestations. the mechanism by which this agent produces hemostatic effects is not known, however, inhibition of serine proteasas such as plasmin, kallikrein and dastase and modulataion of platelet receptors have been considered as possible target sites. to determine the effect of aprotinin on tissue factor mediated activation of human platelets, whole blood samples were drawn at baseline and hours aider aspirin ( mg po) ingestion (n= ). blood samples were supplemented with saline and aprotinin ( ug/ml) and tissue factor activation was studied at pg/ral. platelet activation was investigated using gtycoprotein ffla and gmp specific antibodies on a flow cytometer (epic coulter). tissue factor was found to produce the activation of both the baseline and asp' "mnized platelets. aprotinirt augmented the tissue factor activation of platelets in both the control and aspirin supplemented groups. however, the relative increase in the aspifinized group was much monger ( % of the baseline). these results suggest that aprotinin augments the tissue factor mediated activation of platelets in both the normal and aspirinized individuals. while these effects may result in hemostatic restoration in aspirin compromised patients, in normal individuals the clinical implications of this effect remain to be clarified. , - (group ) or > years (group ) duration. anticoagulated whole blood was incubated with fluorescent antibodies to gpib and gmp- (two colour method) and analyzed with a flow cytometer. thrombomodulin, f + , protein s, -thromboglobulin were measured according to standard procedures. results: surface expression of gmp- was not different in groups to , however, there was a tendency to higher acitvation in group (< years iddm). results for thrombomodulin, f + , protein s, -thromboglobulin will also be presented. conclusion: though it did not reach statistical significance, platelet acitvation seems to be more important during early diabetes. this wilt be correlated with endothelial and plasmatic activation markers. in our clinic four patients with hiv-related thrombocytopenia were treated with a lot of gammagard ( f abllf), which later turned out to be hcv contaminated. before infusion all patients were negative for hcv antibodies and hcv rna. to months after infusion / patients, who suffered from arc at the time of hcv infection with cd counts > /pl, seroconverted, whereas in the two other patients, who suffered from aids with cd counts below /pl, there was no seroconversion. in all cases hcv rna was found. genotyping with inno-lipa (innogenetice) showed hcv genotype l(b) in all patients. liver enzymes and hcv rna copies were measured repeatedly over a period of one year after infection. the patients with arc showed a strong increase of hcv rna titre during the first to months after infection, followed by a rapid decrease within the next months. in the patients with aids hcv rna copies increased moderately within the first to months, followed by a slow decrease. elevation of liver enzymes was mild in the aids patients and seems to be independent from the hcv rna titre. in the arc patients liver enzymes changed parallel to hcv rna titers with a delay of to months. the course of hiv infection was only slightly influenced by the acute hepatitis c as measured by cd counts, i% microglobulin and hiv rna copies. introduction:mechanisms underlying ischemia/reperfusion injury have been thoumughly investigated in experimental models. leucocytes appear to play a main role through production of cytokines and overexpresssion of adhesion molecules. in experimental animals, administration of monocional antibodies (mab) recognizing cd can reduce organ injury following ischemia/repedusion. no data, however, have been reported concerning clinical ischemia situations. patients and methods:we investigated expression of cdt , cd la, cdf l b and cd lc in granulocytes, monocytes and lymphocytes from peripheral blood of five patients undergoing elective hand surgery. the tourniquet was applied on the upper arm and heparinized samples from cubital veins were obtained before and at the end of ischemia. control samples were drawn from the nonischemic contralataral arm with the same timing, duration ot ischemia ranged between sixty and one hundred minutes ( ~ ). whole blood samples were incubated with specific, fluorochmme labelled antibodies and analyzed by fluorocytometry (facscan, becton dickinson, san jose, ca). mean fluorescence intensity (mfi), quantitatively reflecting surface expression of the indicated markers was evaluated for the individual cell populations. data were compared by the paired student's t-test, p< , was evaluated as significant. results:mfi for all markers was comparable in all cell populations in samples obtained before ischemia from both arms. in contrast, expression of cd was significantly enhanced in granulocytes ( _+ vs. _+ ), monocytes ( -+ vs. + ) and lymphocytes ( _+ vs. -+ ) from samples derived from the ischamic arm, as compared with the nonischemic arm, as measured at end of ischemia. at the same time, an increase of cdf lb on granulocytes ( ~_ vs. + ) and monocytes ( + vs. -+ ) but not on lymphocytes was found, no modifications of cdlta and cdttc expression could be observed. there was no correlation between duration of ischemia and quantitative expression of these markers, conclusions:our data indicate that relatively short ischemia periods induce an increased expression of ~ " integrins adhesion molecules on leucocytes. these results suggest, at close similarity with findings from expodmental models, that overexpression of adhesion molecules might play an important role in the induction of ischemia/reperfusion injury, in humans. in patients suffering from chronic inflammatory bowel diseases, such as morbus crohn and colitis ulcerosa, we observe massive, sometimes barely staunchable bleedings. hereby, the deficiency of coagulation factors, especially of factor xiii in plasma is established. ttowever the influence of factor xiii on the pathomechanism of the underlying disease is still under discussion. therefore we studied the f xiii content in the intestinal mucosa. an immunohistochemicat method was developed using commercially available antibodies against f xiii subunit-a, the detection of mucosal factor xiii depends on the amount of chromogen bound to the antibody-horseradish-peroxidase complex. with this method, it is possible to locate but not to quantify f xili in the intestinal tissue. therefore we developed an elisa-metbod in homogenized intestinal tissue, using commercially available antibodies. its precision was validated using a standard curve with commercially available factor xiii preparations (fibrogemmin®). the detection limit of this method is > . i.u. f xiii/ml of tissue solution. freezed dried intestinal tissue (lmg) was homogenized in ml buffer using a potter. specimens of the large bowel revealed f xiii values of , + , i.u. (x __+ sd), tissue solution. with this method it is possible to quantify tissue-bound faxtor xiii. studies are in progress to elucidate the content of f xiii in the intestine of patient's suffering from infammatory bowel diseases in order to contribute data to the pathomechanisms of f xiii deficiency. in a previous double-blind, controlled trial we were able to show that aprotinin administration has significantly contributed to reduce periand postoperative bleeding complications without increasing the risk of thromboembotic complications. the question arises whether this beneficial effect may be associated with its effects on intraoperative fibrinolysis. therefore, patients were treated with or without aprotinin ( million kiu loading dose over minutes followed by , kiu per hour), and citrated blood samples were obtained at the following time points: before operation, after induction of the anesthesia, at the beginning of operation, intraoperatively when the femur shaft was implanted, and hours postoperatively. the determinations of plasmin/antiplasmin-complexes, d-dimers, thrombin/antithrombin iii-complexes, and prothrombinfragments + were performed by means of test kits from behring, germany (enzygnostrpap micro, enzygnost r d-dimer testkit, enzygnost r tat micro and enzygnost r f + respectively). -all markers of activated fibrinolysis and blood coagulation were significantly increased in the groups with and without aprotinin treatment, the highest activities to be seen when the femur shaft was implanted. however, the values of pap and d-directs of the aprotinin group were below the values of the control group until the end of operation. the markers of activated coagulation showed the opposite effect, however the differences between the two groups were not significant. as expected, the aptt was significantly prolonged in the aprotiningroup. the aprotinin treatment was also associated with a significantly lower blood loss in these patients. -concluding it can be said it is not clear whether the blood saving effect of aprotinin may be exclusively attributed to its antiplasmin activity since the differences of the fibrinolysis parameters were not statistically significant. further blood samples should be analysed between the implantation of the femur shaft and the end of operation. in our laboratory large amounts of human prothrombin are required ( - mg/week). as we try to produce meizothrombin and meizothrombin-des-fragment- from human prothrombin and to apply it as an antidote for hirudin, the classical adsorption to barium sulphate or aluminum hydroxide from human plasma cannot be used. commercially available human prothrombin is expensive and of an unacceptable quality for our applications. in most of these batches we found small amounts of factor x and prothrombin activation products. we now developed a procedure to isolate prothrombin from "prothrombin complex concentrates" (ppsb- -bulk, drk-blutspendedienst nds.). the concentrate also contains fac-tor vii, factor ix, and factor x. the prothrombin had to be separated from these factors. the concentrate we used contained amounts of other proteins and activation products of prothrombin (e.g. prethrombin- ) as well. for the preparation of prothrombin from ppsb we used anion exchangechromatography (resource-q ®) on an fplc ®. we applied dissolved ppsb directly or after buffer exchange on sephadex g- onto the column at room temperature. the prothrombin was eluted with an naci-gradient in trisodium citrate buffer, ph . . the buffer conditions are similar to the conditions used in the preparation of ppsb. the quality of the prothrombin so obtained was sufficient for most of our experiments. a second purification step on ion-exchange resulted in a % pure product devoid of contaminating factor activities and activation intermediates as examined with coomassie and silver stained sds-page electrophoresis and assays for factor x. this prothrombin contained full enzymatic activity and its activation by specific snake venom prothrombin activators showed the known activation products. we are now able to isolate the amounts of pure prothrombin required for preclinical investigations. most of the commercially available lmwhs such as enoxaparin, fraxiparin, and fragrnin are prepared by chemical methods which can result in desulfation and other chemical modifications of the internal structure leading to differences in the pharmacologic effects. on the other hand, tiactionated lmwhs retain their native characteristics and are structurally similar to heparin. in addition, the oligosaocharide sequence responsible for atiii binding is not modified. physical methods such as gamma irradiation (~co) have been used to fi'agment sulfated glycosaminoglyeans yielding fragraents without chemical modifications (deambrosi et at. in : biomedical and biotechnological advances in industrial polysaccharides, pp. - ). utilizing this technique, depolymerized heparius exhibiting different molecular weights can be obtained. this communication reports on the biochemical and pharmacologic effects of several such depolymerized heparins to demonstrate the molecular weight dependence on biologic activity. fragments exhibiting molecular weights of , , , and kda were prepared by exposing concentrated heparin solutions to a rectilinear gamma ray beam at intermittent doses of . to mrad under controlled temperatures. unlike the chemically depolymerized heparins, these fractions did not exhibit any decrease in charge density or atiii affinity. in routine assays for heparin, a clear cut molecular weight dependance on the anticoagulant and antiprotease actions was observed. on a gravimetric basis, these agents produce superior antithrombotic actions in comparison to chemically depolymerized derivatives. these studies suggest that gamma irradiation can be used to prepare lmwhs which retain their molecular integrity and therefore may prove to exhibit a more comparable biologic profile to hepari~ futthermore, lmwhs produced by gamma irradiation lack the usual double bond fommtion which requires the use of additives which can alter the product profile. university hospital, dept. of angiology, frankfurt a.m., germany introduction: thromboembolic disease constitutes a major clinical problem and among others a defective fibrinolytic system has been suggested as a predisposing factor for the development of thrombosis. the plasma fibrinolytic system can be impaired by inherited deficiencies of plasminogen defective release from the wessel wall tissue plasminogen activator (t-p'a) or by high ptusma levels of regulatory proteins, such as plasmino- en. activator inhibilors (pal). the aim ....... of the present study w~s to eshmate the prevalence of decreased fibnnolyl~c actwlty m young pls. with thrombophilia. patients: a great population of pts. (fenmle , male ; age - ys median . ys) with venous thromtx~emolism before the age of years were investigated in regard to their plasma fibrinolytie system. in none of them well established thrombophilia risk factors could be identified previously. methods: plasminogen ~behdngwerke), pai- activity (ehromogenic assay, biopool), pal-i anugen coneentration (elisa, biopool), t-pa activity (chromogenic assay, biopool) and antigen concentration (elisa, biopool) were measured before and after venous oeclusion.vo was performed z month after the last thromboembolic epi~xle. healthy subjects (median age . ys) served as controls. results." pts.( . %) were classified as plasminogen deficiencies (activity and antigen). pts.( %) had significantly elevated levels of pal activity (up to u/ml) and pal antigen (up to ng/ml). none of the pts. with high pal levels had laboratory signs of acute phase reaction. low t-pa activity could be demonstrated and confirmed in pts., aecordingto a prevalence of . % (range: - . u/ml; reference limils: . - . u/ml). however, there was a significant negative correlation between t-pa activity and pal values. in pts. ( . %) the low t-pa activity was associated with increased pal levels whereas the t-pa antigen concentration was normal. a parallel reduction of t-pa activity and t-pa antigen (range: . - . ng/ml; reference limits: . - . ng/ml) were determined repeatedly in pts. (f , m , median age ys). thus, the prevalence of a defective t-pa release was . % in our study group. conclusion." in comparison to the frequency of inherited deficiencies of other well established thrombophila risk factors we have observed a relativel~ high prevalence of diminished t-pa activity, elevation of pal respectively in our study group. our data strongly indicate that besides t-pa and pal acuvity, antigen concentration for both parameters should be determined in pts. with thrombophilia. the antithrombotic and anticoagulant effect of the supersulfated low molecular weight heparin ssh was studied after i.v. and s.c. administration in rats. thrombus formation in the jugular vein was induced by i.v. injection of activated human serum and following stasis for rain and was assessed by a thrombus score ranging from (no thrombus formation) until (complete thrombus formation). ssh t injected either min (i.v.) or rain (s.c.) before thrombus induction caused a dose-dependent antithrombotic effect in a range from . to mg/kg i.v. and to mg/kg s.c. there were clear differences in the antithromboric effectiveness between female and male animals, i.e, in female rats antithrombotically effective doses were lower than in male rats (edh after i.v. injection in females . mg/kg, in males . mg/kg). the sex differences were confirmed in studies on the time course of the antithrombotic effect. after i.v. injection of fully effective doses ( mg/kg i.v. and mg/kg s.c., resp.) the antithrombotic effect disappeared after h in female or after h in male rats. for studies on the anticoagulant action blood was drawn from the femoral artery and after centrifugation global clotting assays were performed in plasma. similar to its antithrombotic action ssh also caused doseand sex-dependent anticoagulant effects. the most sensitive assays were the aptt and the heptest; thrombin time and prothrombin time were less or not influenced by ssh . in conclusion, ssh was found to be an effective anticoagulant and antithrombotic agent in experimental studies in rats. at present there is no explanation for the clear sex differences found in this species. venous thromboembolic disease is the most frequent complication in patients undergoing total knee replacement therapy. patients and methods: after informed consent x patients were included in an open randomized clinical study and the incidence of venous thromboembolisrn was examined using different regimes for heparin prophylaxis ( patients received fraxiparin rag once daily, patients clexane once daily and patients u calciparin twice daily). there were no differences between the groups concerning age, sex, body weight, risk factors, surgeons, decrease in hemoglobin~ and requirements for blood products. pre surgery, day , day - phiebograms were performed and also tat, dimers, fl+ prothrombin fragments were examined. results: ., dvt in patients ( . %). dvt in / patients under calciparm prophylaxis, / patients under fraxiparin and / patients under clexane treatment. ., low speciflty ( . %) of dimers and tat ( %) for detecting a dvt in these special patients undergoing knee replacement therapy, elevated fi+ fragments in the dvt group at ti and t vs the patients without dvt (t dvt: . +- . vs. . +- . -p= . ). , only / patients ( %) with dvt had clinical signs of thrombosis. conclusions: ., there is an increase of thrombin gneeration measured by tat and dimers after knee replacement therapy. there are further studies with more patients necessary to confirm that fl+ prothrombin fragments can discriminate between patients with and without dvt from a clinician's point of view. ., phlebographicauy confimled dvt in almost % of our patients demonstrate the high thromboembolic risk in these patients. von willebrand's disease (vwd) type is characterized by absence of high molecular weight muitimers. qualitative changes in the structure of the molecule might be associated with enhanced binding of von willebrand factor (vwf) to platelet glycoprotein lb. therefore in some patients vwd type is associated with severe thrombocytopenia. here, we report on a year old boy who presented with severe purpura and platelet counts about /gl at the age of years. thrombocytopenia did not respond to corticosteroids. a normalized platelet count of short duration was observed after high-dose immunoglobulins. in addition, increase of platelets was seen after anti-d treatment. thus, although platelet associated antibodies were not detected, thrombocytopenia seemed to be caused by an autoimmune mechanism. despite platelet counts above /gl, the patient experienced severe bleedings with a significant decrease of hemoglobin levels. therefore, he needed several transfusions. coagulation analysis revealed vwd. application of ddavp lead to a normalization of partial thromboplastin time (ptt) and an increase of factor viii with subsequent cessation of bleeding symptoms. recently, vwd was typed by lack of high molecular weight multimers. in conclusion, we report a case with vwd type responding to ddavp. however it is unclear, whether thrombocytopenia is part of the vwd type or of autoimmune origin. since autoimmune antibodies have not been detected, the effect of immunoglobulin treatment might be explained by blockade of enhanced binding of vwf to glycoprotein lb. von willebrad disease (vwd) with a prevalence of , % (ruggeri , rodeignere ) seems to be the most frequent inherited hemostatic disorder. • the diagnostic criteria for vwd are clinical picture, family hostory, laboratory findings: bleeding time, partial tromboplastine time (ptt), level of factor viii:e, vwf, vwf:ag, ristocetin induced platelets aggregation (ripa) and multim~-analysis.the diagnosis ofvwd is occasionally difficult, especially in early childhood because the laboratory data may vary due to time of investigation, as well as abnormalities may not be present in all sub-types the aim of this study was the evaluation of diagnostic approach to vwd in childhood and diagnostic reliability of all available laboratory tests. all previously mentioned laboratory tests have been done on our own material ( child who satisfied all criteria for vwd, boys and girls, - years old) except mulfimer analysis which was unavailable in some cases. majority of laboratory tests proved to be highly specific and necessary for diagnosis. however, the diagnostic reliability of fviii:c and adhesion of platelets is much lower in mild cases in comparison to total sample, while ptt is an unvaiied test. the most specific screening test for vwd is vwf which diagnostic reliability is almost , . the optimal strategy to establish general diagnosis of mild forms ofvwd is use of vwf and vwf:ag plus ripa if necessary and multimer analysis to classify variant types. we report on a new multimeric structural defect of vwf detected in a german family (two sisters and their three children): all members of the family who presented to our outpatient clinic had an increased spontanous bleeding tendency (moderate or strong hematoma, epistaxis, menorrhagia). prolonged bleeding could be observed after surgical procedures (adenotomia, tooth extraction) and after trauma (laceration). wound heeling was impaired in two cases. clotting assays showed slightly prolonged apti" and a mild decrease of f viii:c, vwf:ag and vwf:rcof levels. collagen binding activity was within normal ranges. bleeding time (simplate i) was slightly prolonged. the analysis of the multimeric structure in plasma showed quantitative and qualitative abnormalities: all multimers were detectable; the structure of vwf was reproducably abnormal in all family members so that the defect must be caused genetically. the thmmbocytic vwf showed neither qualitative nor quantitative alterations. minirin@ (ddavp) was administered as a test dose of , ~tg/kg bw in ml , % nacl-solution i.v. to evaluate efficacy and tolerance: clotting assays showed normalization of a_vrt, f viii:c, vwf:ag, vwf:rcof in plasma and shortening of bleeding time in three cases. an insufficient rise of vwf:ag and vwf:rcof levels could be observed in one case. one patient had no rise of f vm:c but a corrected bleeding time. multimeric analysis showed no structural change. the administration of ddavp was well tolcrated in all cases. the existance of all multimers in plasma and the normal collagen binding activity suggest that the structural abnormalities of vwf in this family does not cause functional defects so that the defect could be classified as a type i vwd. the response to ddavp was only partially effective. mild von willebrand disease (vwd) is far the most frequent congenital bleeding tendency. its diagnosis is very helpful in pre-operative check-up in order to avoid bleeding complications during surgery. following post-operative periods or monitoring the management of haemorrhagic episodes in vwd patients is also strongly recommended. current methods involve complex technologies, are time consuming and require large series. these assays lack the expected flexibility for rapid individual testing in patients. a new and flexible assay which works on the fully automatic walk-away coagulation instrument, sta, has been developed for these applications (liatest vwf). the technology is an immuno-turbidimetric method using mierolatex particles coated with rabbit polyelonal antibodies specific for vwf. the assay has a dynamic range from to % yon willebrand factor (vwf) concentration, it works with a fold dilution of tested plasma ( td) and it offers a calibration established with the nibsc international standard. the total assay time is of less than minutes and the detection threshold is of % there is no prozone effect up to concentrations higher than , % vwf. intra-assay reproducibility is < % and inter-assay one < %. in dilution studies a mean recovery of % was obtained. in a study on plasma samples from norma~ individuals, patients with high vwf concentrations, and vwd, comparison with the elisa technique demonstrated a correlation coefficient of . with a slope of . and an intercept of . . in the low assay range too, a good agreement was obtained with the elisa. we conclude that liatest vwf is a reliable, flexible, sensitive, and rapid automated assay which fits well the vw'f assay applications in coagulation laboratories. fibrinolysis, the process during which the active enzyme plasmin is generated in a regulated and localised way, is -in a classical understanding -responsible for the dissolution of blood clots formed in a vessel. for this activity, t-pa is generally assumed to be the most important plasminogen activator and its activity, is regulated by enzyme kinetic mechanisms dependent on the presence of fibrin. with this background t-pa is used for thrembolytic therapy with great success. however, data from t-pa knockout mice indicate that t-pa might not be responsible for inhibiting the spontaneous development of intravsacular thrombi but only for dissolution of fibrin formed upon a coagulation challenge. in contrast, u-pa, generally assumed to be important for extravascular proteolytie activity on activated or tumour cells, seems to lead to the development of spontaneous fibrin formation in a mouse knockout model. on the other hand, the major plasminogea activator inhibitor pal-i seems not only to regulate intravascular fibrinolysis but seems to also be important for the progression of vascular diseases (neointima formation is e.g. increased in a pai- knockout model, but increased levels of pai- seem to predict reocclusion after angioplasty). in addition to their functioning as enzymes and inhibitors, components of the fibrinolytic system seem also to be involved in signalling processes in tumour and other cells. the u-pa/u-pa-receptor system could be shown to function as a chemotactic system and to elicit a migratory and mitogenle response in monoeytes and tumour ceils as well as in vascular cells. for such a response activation of tyrosine kinases of the sre-family might be responsible in some cell lines, but other signal transduction pathways e.g. involving caveolae and the starprotein can not be excluded. there seems to be a further important role of components of the fibrinolytic system which involves serine protease inhibitors (serpins): serpins have homologies to hormone binding proteins and cleavage of serpins by their target enzymes not only leads to inactivation of the enzyme but also to a possible release of bound hormones from the serpins. from these data clearly the relevance of any regulation of the fibrinolytie, system depends on the specific function of the system to be dealt with. in addition to "fibrin binding", "receptor mediated" and "genetic control" (e.g. g vs. g in the pai-i promotor) also "signal transduction" and "hormone delivery" are distinct functions of the system with specific regulation. plasmatic for both, healthy persons as well as for patients with angina pectoris it could be shown that increased values of plasma fibrinogen, factor viic and vwf:ag are significantly associated with the risk to suffer an acute myocardial infarction or cardiac sudden death. the same holds for tpa:ag. however, a group analysis in quintiles reveals that particularly low tpa:ag values are connected with a particularly low coronary risk. unexpectedly also the acute phase protein crp is positively associated with increased coronary risk. for clinical purposes these factors have already been included into coronary risk scores in order to improve the individual risk prediction in combination with lipids and other risk factors. the assessment of the pathophysiological significance of these observations remains at dispute. pathways are discussed: . the assumption that increased plasma values of those factors indicate increased coagulation activity could so far not be established in prospective studies. . both vwf:ag and tpa:ag are produced in endothelial cells. an increase of their plasma level could therefore indicate increased endothelial cell functions which accompanies progressive atheromatosis. the risk association of the two acute phase proteins crp and fibrinogen could be interpreted analogously. . first prospective studies favour the assumption of a genetic determination to an increased production of coagulation proteins in persons at particular coronary risk. it could also be shown that there is a certain dependance of the gene-polymorphism for co-and -fibrinogen chains from the coronary risk. . even slightly elevated concentrations of fibrinogen and/or vwf:ag may influence the quality of a coronary thrombus both by increased physical stability and by reduced fibrinolytic lysibility. this could mean that an early coronary clot under these conditions could more readily develop to a stable, occlusive thrombus. a newborn with pronounced bleeding tendency had a prothrombin (prth) deficiency below . % in a clotting assay. both parents had activities of % and %, respectively. however, the immunological determination ofprth by elisa revealed normal concentrations in all family members ( %- %). furthermore, thrombin generation as investigated by a chromogenic assay using ecarin for activation of prth was normal as well. activation of prth by fxa was investigated by reealcificafion of the plasma samples and further analyzed for prth and its derivatives produced. although clotting times still were different, finally, normal levels of fl+ and tat were generated as determined by elisa. western blot analysis using polyclonal (rabbit) antibodies to prth and a monclonal antibody specific to human thrombin, revealed different patterns of prth degradation products. tat was only weakly visible in the serum of the mother and nearly absent in the child.the mobility of prothrombin and thrombin was different compared to normals indicating a lower molecular weight. after reduction of disulfide bridges a higher molecular weight of thrombin was observed compared to normals indicating an insufficient cleavage ofprth and formation ofprethrombin . these observations let suggest that prothrombin marburg is a deletion mutant lacking the cleavage region arg -ile . upon cleavage by factor xa only prethrombin is formed under liberation of fl+ . this prethrombin is able to cleave chromogenic substrates in the ecarin assay. probably, prethrombin forms a complex with atiii which is detected by elisa, but unstable under denaturing conditions as in the western blot. as a major complication of haemophilia a treatment, up to % of the severely affected patients develop antibodies to substituted factor viii. investigating patients and considering the data of further patients of the haemophilia database, we could show, that risk of inhibitor developement depends on the patient's mutation type. patients with more severe gene defects, like intron inversions, stop mutations or large deletions had a risk of about % for inhibitor developement, which was about times higher than for missense mutations or small deletions. besides an influence of mutation type, we investigated other parameters e. g. immune response genes (i-ila-genotype) and clinical aspects (treatment onset and frequency, type of concentrate) that might also affect inhibitor formation. to exclude any effect of mutation type, we focussed on patients with an intron inversion. hla-typing showed that some t-ila-alleles (dqb , bt) occurred more otten and others (dqa , dqb , dr , c ) less frequent in inhibitor patients. treatment onset, frequency and type of concentrate apparently do not affect inhibitor incidence. the results presented here, prove that inhibitor development is considerably influenced by the mutation type. this supports the hypothesis that patients with severe molecular defects have no endogenous factor viii protein and that substituted factor viii represents a foreign protein, leading to an immune response, e. g. the production of alloantibodies. in addition, the immune response seems to be modified by the hla-genotype. however oar findings (in terms of genotype and treatment parameters) can only explain part of the inhibitor pathogenesis. it is still unsolved why substituted factor viii does not lead to a recognizable immune response in / of the patients with severe molecular factor viii gene defects. consequently other factors, probably concerning the antenatal phase, must be involved. viia in the treatment of patients with inhibitors against factor viii or ix: a german/swiss/austrian multi~center trial d. ellbriiek*, i. scharrer**, j. dethling***, and the rfviia study group *section h~mostaseology, university ulm **dept. of angiology, jwg-university hospital frankfurt a.m. ***novo nordisk, mainz administration of activated recombinant factor vii (rfviia) can by-pass the fvnlwlx pathway and offers an alternative treatment for patients with antibodies (inhibitors) against these factors. from november to october , a total of bleeding episodes and surgical interventions in patients were treated with rfviia in a phase iiib multicenter trial. diagnosis was hemophilia a (n = ) or b (n=l) with inhibitor, and acquired inhibitor against factor viii (n= ). various serious bleeds, from complicated joint and gingival bleeds to lifethreatening psoas bleeds, have been treated. operations have been tooth extractions, radiosynovectomy, implantation and explantadon of porth-acaths and one adenotomy. dose regimen was - /zg/kg bw every two to three hours until clinical improvement, with subsequent dose reduction. results: for bleeding episodes, response to rfviia after hours was effective in %, partially effective in " , ineffective in "o and not evaluable in ( %) of the patients. two of the three treatment failures were associated with very long dosage intervals of rfviia. the third patient was in a critical situation with artificial high pressure respiration and polytransfusion because of a hematothorax, and suffered a terminal intracerebral bleed. the efficacy of rfviia for surgery was very good. response to treatment was independent of antibody titer. no signs of dic or activation of coagulation were noted. conduslon: in our experience, rfviia is an efficient and safe treatment for inhibitor patients with acute bleeding episodes. it should be investigated, whether rfviia can be an alternative treatment also for the hometreatment situation. successful immunetolerance therapy of f vih-inhibitor in children after changing from high to intermediate purity f vih concentrate w. kreuz, j. joseph-$teiner, d. mentzer, g. auerswald*, t. beeg, s. becker zentrum der kinderheilkunde, j. w. goethe-universit~itj frankfurt am main *professor hess kinderklinik bremen introduction: inhibitor to f viii is the most severe complication in treatment of patients with haemophilia a. the incidence of f viii inhibitors is estimated to range between - %. several authors reported that the immunetolerance therapy (itr) of f viii-inhibitors can be induced with high dose f viii concentrate. objective: this presentation will show data of four children with haemophilia a and f viii inhibitor (high responder), who had an unsuccessful lit with high dose f viii concentrate (high purity) in the first step. f viii concentrate was changed to an intermediate purity product (haemate hs®) in the subsequent course of h't. all patients received bleeding prophylaxis with an activated-prothrombin-complex-concentrate (feiba®). results: median age was ( - ) months, when the inhibitor was first detected. in all four patients the f viii inhibitor titre increased under immunetolerance treatment with f viii concentrate (high purity) in the first step of therapy. after changing the f viii concentrate (intermediate purity) the inhibitor titres decreased continuously after a rebooster effect to be within months. median duration of f viii inhibitor elimination time (until first testing of be) was ( - ) months. in all patients the f viii inhibitor was successfully eliminated. until now all patients are under prophylactic treatment with f viii concentrate and had no positive inhibitor testing since. median observation time since the first testing of be is ( - ) months. conclusion: different studies concerning immunetolerance treatment have been successful with f viii concentrates of different purity. according to our experience in these four presented patients, we assume that probably not the purity of the f viii concentrate is important for the induction of immunetoleranee, rather than the type of f viii presentation in the used concentrate. the used preparation (haemate hs®) is a f viii concentrate with high concentration of vwf, which is known to be important for the protection of f viii against degradation by proteases. this may be a mechanism for a prolonged antigen presentation to the immunesystem and thus may have a positive impact on the outcome ot itr. long scale trials are needed to prove the above assumptions. thrombasthenia glanzmann is a disease affecting platelet function because of a partial or total lack of glycoprotein (gp) ilbllla expression or a modification of this complex. since the receptor dysfunction goes along with reduced or absent platelet aggregation and adhesion, it causes bleeding complications in case of injury. here we report about a years old women, who suffered since early childhood from a severe bleeding disorder. life threating bleeding complications occured after tooth extraction and after abdominal surgery. analysis of the patients platelets revealed normal values for the platelet count, whereas their volume showed to be increased ( fl). clot retraction was diminished to %. platelet adhesion to siliconised glass and human subendothelial matrix was reduced, as was the spreading of the platelets. adp (i#m) induced platelet aggregation was inhibited, while collagen-, ristocetin-and thrombin-induced aggregation showed to be normal. cross immunelectrophoresis resulted in an atypical peak of gpiibllla with reduced electrophoretic mobility. in the electroimmunoassay according to laurell % of gpiibllla was detected. moreover we observed a markedly diminished j-fibrinogen binding. sequence analysis of the gpiib and gpiila cdna after pcr amplification unraveled a g --, a transition in gpiib, substituting gly --* glu. the structure/function relationship of this mutation has still to be investigated. we report two new abnormal fibrinogen variants, denoted as bem iv and milano xi, both having an exchange of arginine to histidine in position of the ac~-chain. routine coagulation studies revealed prolonged thrombin and reptilase clotting times, low plasma fibrinogen concentrations determined by a functional assay but normal fibrinogen levels measured by the immunological assay. the onset of turbidity increase following addition ofthrombin to purified fibrinogen was markedly delayed in both variants. release of fibrinopeptide b by thrombin, measured by reversed phase hplc, was normal whereas only one half amount of normal fibrinopeptide a was released. in addition to normal fibrinopeptide a, an abnormal fibrinopeptide a* was cleaved from both dysfunctional fibrinogens. the structural defect was determined by asymmetric pcr and direct sequencing of a gene fragment coding for the nh -terminus of the aachain. both variants were found to be heterozygous for the transition g to a at nucleotide position , leading to the substitution actl arg-->his, resulting in a delayed fibrin polymerization. the simple assay permits detection of the most common amino acid substitutions occuring in the nh -terminus of the ac~-chain of the functionally abnormal fibrinogen variants. protein c inhibitor (pci) a member of the serpin family is also known as plasminogen activator- (pal- ). pci was first described as a component of human plasma, regulating the activity of activated protein c and other sedne proteases of the human coagulation and fibdnolysis system. since then pci was found to be present in extra-plasmatic systems also. high concentrations of pci were detected in human seminal plasma suggesting a role for pci in human fertility. significant concentrations of pci mrna and antigen were located in lysosomes of proximal tubular kidney cells suggesting an intracellular function for pci in this environment. in this study we present evidence that pci is also present in human pancreas. rna from human pancreas was reverse transcribed and pcr amplified. the resulting pci cdna was identical with pci cdna from human liver. ~p labeled antisense rna probes used in in situ hybridization experiments with human pancreas tissue sections showed that pci rna was located in the acinar ceils. pancreatic fluid was analyzed by sds-page and immunoblotting. using monospecific antibodies directed against human plasma pci, a mw protein band was observed which comigrated with purified human plasma pci. our results show that pancreas cells contain a significant concentration of pci mrna. this message is localized in the secretory acinar cells. therefore we conclude that pci antigen found in pancreatic fluid is likely to originate in the pancreas. the role of pancreatic pci is unknown at present. however, since thrombosis and systemic hypercoagulable states are known complications of pancreatic diseases our results and in vitro experiments by others showing that pci can inhibit pancreatic enzymes such as chymotrypsin and trypsin indicate that pci may be part of the inhibitor potential which protects pancreatic tissue from auto degradation. these inhibitors normally prevent the release of active pancreatic proteases into the vasculature or microcirculation where destabilization of the coagulation balance and subsequent thrombus formation could occur. institute for clinical chemistry and laboratory diagnostics and *clinic for cardiology, universi w of duesseldorf p-selectin (cd p, the former granule membrane protein or gmp ) is an integrated membrane protein of platelets and endothelial cells. under inactivated conditions it is stored in the alpha granules of platelets and in the weibei-palade bodies of endothelial cells. endothelial cells covering atherosclerotic plaques show an increased expression of p-selectin. -thromboglobulin ( -tg), which is also expressed from the alpha granules of platelets during adhesion or aggregation, is regarded as a marker of platelet activation in vivo. coronary thrombosis plays a central role in the pathogenesis of acute coronary syndromes. we therefore analysed cd p and -tg in acute coronary syndromes, healthy subjects (hs, n=l i), patients with stable angina pectoris (sap, n= ), unstable angina pectoris (uap, n=l ) and acute myocardial infarction (ami, n= ). plasma samples were obtained by using ctad vacutainer tubes ( . m na~-citrate, theophylline, adenosine dipyridamole). patients with cad showed significantly increased plasma concentrations of cd p (hs: + versus sap: + ng/ml, p< . ; versus uap: + ng/ml, p< . ; versus ami: + ng/ml, p< . ) independent of the severity of clinical symptoms. in comparison only patients with ami showed significant higher -tg concentrations compared with hs (hs: + versus ami: + ng/ml, p< . ). although the cd p plasma concentrations showed no relationship to the clinical severity, hence there was a positive correlation between cd p (r= . ; p< . ; n= ) to the severity of cad classified as i, , vessel disease. it is concluded that elevated cd p concentrations are correlated with the severity of cardiovascular disease. cd p is not suitable for differential diagnosis of acute coronary syndromes, because it is elevated independently of the clinical status of the patients. the involvement of platelets in the pathogenesis of acute myocardial infarction may be indicated by the increased -tg concentrations. iklinik nr herz-, thorax-und herznahe gef&schirurgie und institut x~tr klinische chemie und laberatodumsmedizin der universint regensburg an increased blood loss following surgery with extracorporeal circulation (ecc) contributes to the morbidity and mortality. postoperative haemorrhage following ecc has been related to a platelet function defect and the activation of the blood dotting and fibrinulytic system. we investigated platelet surface antigen expression and parameters indicating activation of the clotting and fibrinolytic cascade to assess the predictive potential of these variables for increased blood loss after ecc. g patients referred for coronary bypass gra~ing with no history of a bleeding disorder and normal routine clotting tests were included. on the day prior to surge~ and immediately upon arrival on the intensive care unit blood samples were drawn. the surface expression of glycoprotein (gp) lib-ilia, gp lb, and p-selectin was meamred with and without in vitro stimulation with adenosine diphosphate (adp) using whole blood flow cytomet~y. platelet counts and platelet factor (pf ), as well as, routine clotting tests were performed. activation of the clotting and fibrinolytic system were judged from thrombin-antithrombin-iii complex fiat), fibrinogen fig), d-dimers (dd), cc -antiplasmin (tz a), prothrombin fragment + (fl+ ),and tissue plasm~ activator (t-pa). blood loss fxom chest tubes was measured hourly until removal of drains. following ecc the levels of pf , tat, dd, o~ a, fl+ , and dd were sigulticnatly increased (p< . ) compared to baseline values. gp iib-iila, gp ib, p-selectin, platelet count, and fg were significantly reduced (p< . ). analysis of variance (anova) revealed that postoperative values of gp ib (p< . ), dd (p<o. ), fg (p< . ),and platelet count ( < . ) had a significant influence on blood loss. none of the preoperative data was significantly associated with increased blood loss. a subgroup of patients who bled more than the mean + sd ( cases) was formed_ in logistic regression analysis gp ib expression (p= . ) and dd (p= . ) were of value in identifying bleeders. the sensitivity of the equation was %, the stx~ificity % and the positive predictive value %. although the reasons for an increased blood loss following ecc are multifactorial, we were able to identify a small number parameters with a significant infl~nco. the combination of the expression of yon willebrand receptor on the platelet su_rfaco and d-dimer levels in plasma was successful in predicling an increased blood loss following heart surgery. this may be helpful to define indications for platelet transfusion and flesh frozen plasma. the most widely distributed receptor for the fc-region of igg is the fcreceptor iia (fcyriia). for this receptor a functionally relevant polymorphism of amino acids argmhis is known. we previously showed hit-antibodies to bind to platelets via feyriia and now whether clinical manifestation of hit is associated with the fe'/riia polymorphism. therefore, we developed a rapid two step allelle-specific pcr (asp) method for genotyping the foyriia. with control samples the asp was compared with the time consuming and complicated allele-specific oligonucleofide hybridization technique, the phenotype expression of fctriia (using the moabs iv. and h ) and the monocyte stimulation assay. complete concordance between the assays was found. we used the asp to determine the allelic distribution of the inst. physiol. chem., marburg; *)univ. of virginia, charlottesville, the functional heterogeneity of blood platelets is clinically significant and may contribute to the pathogenesis of cardiovascular and atherosclerotic diseases l). in order to understand the biochemical basis of the functional heterogeneity of platelets, we have investigated the signal transducing pathway of platelet activation a) in terms of guanylate cyclase and phosphodiesterase activities and b) at the level of protein phosphorylation and the receptor-effector coupling via g i-and gs-proteins in functional heterogeneous blood platelet subpopulations separated according to their densities. furthermore, we analyzed the adp-ribosylation of rhoa, which is a lowmolecular weight gtp-binding protein and is thought to be involved in the platelet "shape change" reaction and aggregation. aggregation was enhanced in low-density platelets (sp i) and less inhibited when cgmp was increased by no, compared to high-density platelets (spiii). sp i possessed a lower basal level of cgmp and exhibited a smaller increase in cgmp after stimulation with no. cyclic amp-dependent phosphodiesterase activity was higher in sp i compared to spiii, whereas cgmp-dependent phosphodiesterase activity was similar in sp i and sp iii. sp i, when compared to sp iii platelets, showed a higher degree of prephosphorylation of proteins in the range of , - , and - kda. after thrombin stimulation, the phosphorylation of these proteins was similar in the subpopulations. pertussis toxin,induced adp-ribosylation of the - kda gi-protein was highest in sp i, whereas cholera toxin induced adpribosylation of the gs-protein and botulinum c exoenzyme-induced adpfibosylation of rhoa were highest in sp iii. the findings suggest that a) the higher aggregability in sp i compared to sp iii may be caused by a poorer ability of guanylate cyclase to form cgmp and a higher activitiy of campdependent phosphodiesterase and that b) the adp-ribosylation of g-protein subunits and rhea may contribute to the functional heterogeneity in the subpopulation leading to the greater ability of sp i for aggregation. ) opper et al., atherosclerosis, , - , . causes of accelerated atherogenesis in non-insulin-dependent diabetes mellitus (niddm), known to be associated with increased plasma activity of plasminogen activator inhibitor type (pal-l) as well as with increased plasma concentrations of proinsulin and insulin, remain enigmatic. this study was designed to determine whether proinsulin and insulin increase pal- in vivo thereby potentially accelerating atherogenesis. to simulate hyper(pro)insulinemia seen with niddm, we infused equimolar concentrations of insulin ( iu/kg), proinsulin, c-peptide, or placebo, all endotoxin-free, i.v. over h in conscious rabbits maintained euglycemic. plasma pal- did not increase with placebo (n= ) or c-peptide (n= ), but did with proinsulin and insulin peaking in h at . -and . -fold (n= and n= , p< .o for each) in parallel with pal- protein quantified by reverse fibrin autography. the increases were specific as reflected by unchanged activities of o~-antiplasmin and t-pa. pat- gene expression (pal- mrna) increased with proinsulin and insulin by . -and . -fold in h in aorta, . -and . -fold in liver (p< . for each), but not in heart, lung, spleen, or kidney and returned to baseline in h (n= each). in situ hybridization localized the increased pal- mrna in aorta to endothelium (n= each). thus, both proinsulin and insulin directly augment gene expression of pal- in arterial endothelium in vivo, thereby increasing plasma pal- activity, attenuating endogenous fibrinolysis, and potentially accelerating atherogenesis. the sarco-endoplasmic-retikulum ca+÷atpases (serca) that sequester calcium into intracellular stores, represent the most important mechanism to maintain cytosolic calcium levels low and to return to a resting level after activation. recent studies in platelets and rat endothelial cells have identified two forms belonging to the serca b and serca types. the monoclonal antibody pl/im raised to human platelet intracellular membranes has been shown to recognise the serca ÷+ isoform and to inhibit ca sequestration in human platelets. the aim of this study was to identify ca atpase distribution in human umbilical vein endothelial cells (huvec) and human polymorphonuclear leukocytes (pmnl). polyclonal antibodies with known specificity towards serca b (r / ) and serca ( / ) in addition to pl/im were used in histochemical studies of permeabilised cells and in western blotting experiments using cell lysates and mixed membranes. using the alkaline phosphatase anti-alkaline phosphatase (apaap) method r / and / reacted strongly with proteins in permeabilised huvec and pmnl, however pl/im showed good staining only with pmnl. in western blotting experiments r / recognised proteins with molecular sizes of kda (known to be the parent ca++atpase) and kda (which may represent antibody recognised degradation product), in both huvec and pmnl. / ÷+ recognised bands at kda (parent ca atpase), kda and kda in both huvec and pmnl membranes. in agreement with histochemical results pl/im recognised proteins ( , and + + kda) only in pmnl membranes. in ca transport studies pl/im ÷+ inhibited ca uptake in membranes prepared from pmnl but not in huvec. the distinct reaction of pl/im with pmnl and huvec suggests the possible presence of distinct sub-isoforms of serca with the pmnl and platelet proteins being similar and different to the serca isoform present in huvec. microvascular endothelial cell (ec) lines with a limited life span of - months in vitro were established from human bone marrow, skin or solid tumor tissue. homogeneity of the lines was verified by flow cytometric analysis and cytokine expression patterns. the cultured ec constitutively expressed hla-class i antigens, cd , procollagen type i, cd e, cd , and receptors for granulocyte colony stimulating factor (g-csf). endothelial cells constitutively produced il- and il- as well as tpa and pai-i as determined by elisa. in the presence of iu/ml of exogeneous il-la, g-csf was secreted at high amounts (lng/ml) and gm-csf at low amounts ( pg/ml) in confluent monolayers and during over night incubation. these ec potently bound to isolated neutrophilic granulocytes. when granulocytes were stimulated by either ~ or . m formyl-methionyl-leucyl-phenylanaline (fmlp) to produce oxygen radicals, the preincubation with a single cell suspension of ec caused oxygen radical scavenging. a ratio of : ec to neutrophilic granulocytes resulted in a __+ % inhibition of fmlp-induced and luminol-enhanced chemiluminescence in vitro. this effect was probably due to the constitutive nitrogen oxide (no) production by ec. unexpectedly, preincubation of ec with iu/ml of interferon-y (ifn-y) to increase no-secretion did not alter the extent of the oxygen scavenging effect measured at different ratios of ec to neutrophilic granulocytes. according to the surface antigen expression pattern, identical ifn-f treatment upregulated adhesion receptors on ec. concomitantly, binding of neutrophils to ec was significantly increased. thus, the net oxygen-scavenging effect by ec appears to be stable under the condition of ifn-y stimulation which indicates maintenance of ec function and effective neutralizition of non-specific oxygen radical damage. moreover, the established test system can be useful to investigate the selective contribution of other inflammatory cytokines, adhesion molecules, macrophages and platelets on ec function in vitro. atherosclerosis is characterized by intimal migration and proliferation of smooth muscle cells (smcs), by macrophage infiltration and extracellular matrix remodeling. circumstantial evidence suggests that urokinasetype plasminogen activator (upa) may be relevant in these processes. this study examines upa expression and upa antigen localization in human coronary arteries with ,carious degrees of atherosclerotic lesions. representative segments of macroscopically normal areas (mnas), early lesions (els) and fibrous plaques (fps) were processed in parallel for in situ hybddization and immunohistochemical analysis. in mnas, upa was detected both in the luminal endothelium and in celocalization with ~-actin-positive smcs throughout the tunica media. in els, upa mrna and antigen were substantially reduced in the luminal endothelium. however, cellular upa expression was detected in the subendothelial area in colocalization with tissue-infiltrating cd positive macrophages. in the deeper intimal and in the medial layers strong upa expression was observed in association with ~-actinpositive smcs, with most of the upa-positive cells being located in areas with a high proliferative activity as detected by pcna staining. in fps, upa mrna was widely expressed in macrophage-containing areas within the fibrous cap and at the periphery of the necrotic core of the lesions. in addition, upa antigen was identified in acellular areas of the necrotic core regions. compared to the diseased intima of these lesions, the adjacent media stained only weakly for upa. in conclusion, this study demonstrates an enhanced expression and synthesis of upa by intimal smcs and macrophages in advanced atherosclerotic lesions of human coronary artedes these findings suggest a role for upa in the progression of the coronary atherosclerosis. adenosine produced by vascular cells, platelets and other tissues has been proposed to be a vasodilator, inhibits platelet aggregation, stimulates proliferation and migration of endothelial cells as well as exhibits antiinflammatory effects. adenosine of endothelial origin intra-and extracellularly formed and highly concentrated at the luminal surface may constitute an important antiaggregatory mechanism, which is part of the well-known antithrombogenicity of an intact endothelial lining. it was the aim of this study to investigate whether adenosine could influence the fibfinolytic potential of endothelial cells. when confluent human umbilical vein endothelial cells were incubated with adenosine ( . - mm) a significant dose dependent decrease in plasminogen activator inhibitor type- (pai-i) antigen production by this cells was seen as compared to control. this effect by adenosine was also time dependent and seen after hours of incubation with adenosine. as determined by elisa, pai-i antigen in conditioned media of endothelial cells incubated with mm adenosine decreased to % as compared to control. pai-i antigen in extracelullar matrix of these cells was also reduced by incubation with adenosine. these results were also reflected on the level of specific mrna as determined by northern blotting. pai-i mrna decreased in endothelial cells after incubation for hours with adenosine ( mm) to % ( . kb) and % ( . kb) of control. in conclusion our data demonstrate that adenosine can downregulate the expression of pai- in cultured endothelial cells thereby increasing the profibrinolytic capacity of these cells. this effect of adenosine might contribute to its antithrombotic potential in addtion to its known antiaggregatory effect. sowohl die effizienz als such die komplikationen einer antikoagulantien-dauerbehandlung sind in erster linie yon der gore und engmaschigkeit der gednnungskontrellen abh~ngig seit wird die schulung zur gerinnungs-selbstkontrolte in der herz-kreistauf-klinik bad bedebarg in gruppen eingeobt. die theoretische und praktische anleitung erfolgt in stunden durch eine in der patientenschuiung erfahrene arzthelfedn (oder krankenschwested-pfleger) und einen arzt. die praktische schulung wird durch einen patientennahen theoretischen tell erg~nzt. verglichen mit konventionell ~berwachten oral antikoagulierten patienten (ca. % der tpz-werte liegen im theapeutischen bereich) erreichten quickwert-selbstbestimmer, daf$ , % yon tpz-werten im therapeutischen bereich lagen (tabelle ). schwerwiegende thromboernbolische ereignisse oder blutungskomplikatienen wurden nicht beobachtet. durchschnittlich kontrolliert der quickwert-selbstbestimmer seine gerineungswerte w chentlich, itmgstens im zweiwochen-abstand. ob sich blutungs-und thremboembolierisiko langfristig senken lassen bei gleichze~iger verbesserung yon prognose und lebensqualit~it, bedarf des nachweises im rahmen einer randomisierten, kontrollierten und prospektiven studie. tabelle i verteilun~l der selbstbestimrnten gednnur ~swerte zeitraum zeitraum - zeitraum zeitraum - zeitraum zeitraum - to evaluate the potential benefit of an optimized oral anticoagulation management with inr measurements performed by the patients themselves, two prospective randomized studies have been completed. the first study (n= ) was performed to evaluate the accuracy of inr self-testing (inr-st) compared with standard laboratory controls as well as the improvement of anticoagutation monitoring by inr-st, e.g. the percentage of measurements found inside the target therapeutic inr-range. the parallel measurements revealed a high accuracy of inr-st with a . % median difference between parallel controls of single measurements. in addition, inr-st resulted in a significant increase of measurements inside the target therapeutic range (from to %) in the entire group. however, inr-st performed every weeks did not result in a significant improvement, while selfcontrols every week (from to %) and every days (from to %) did. in a second study ( patients) the incidence of bleeding and thromboembolic complications were evaluated in patients who either had standard anticoagulation measurements o[ performed inr-st. during a median follow-up of . years in the group with standard oral anticoagulation management an incidence for bleedings of . % per year and for thromboembolic complications of . % per year was found, while in the group of patients with inr-st bteedings occured with an incidence of . % per year and thromboembolisms with . % per year. it can be concluded that inr-st can be performed with high accuracy by the patients themselves. inr-st results in more frequent controls of the inr and consequently in significantly longer time periods where the patients are inside the optimal target therapeutic inr range. the more accurate anticoagulation management resulted in our study group in a significantly lower incidence of thromboembolic and hemorrhagic complications. the cell biology of tissue factor (thromboplastin) h. prydz, the biotechnology centre of oslo, norway tissue factor (tf,thromboplastin) is the most potent trigger of blood clotting knuwn.lts presence in tissue extracts has been known since before the turn of the century.erwin chargaff (of dna fame) first described that tf consisted of two parts (a protein and a lipid moiety), a fact that was independently rediscovered in the early sixties by deutsch and lechner in vienna and by myself in oslo. in tf was first purified and shown to be an integral membrane protein of mr about kda (this was a slight overestimate,recent studies indicate - kda). in four groupes independently cloned tf edna, later also genomic dna. the structure of tf suggested that it was a membrane spanning receptor, with an extracellular part of about amino acids folding into two domains remotely like the domains of the ig-superfamiliy, and more specifically the eytokine receptors. tf is synthesized to a varying degree in many tissues, but not normally in tissues in contact with blood. however, during acute inflammatory situations and under the influence of a number of agents (immune cnmplexcs, lectins, cytuklnes,bacterial components, complement components etc) monucytes and vascular endothelial cells are induced to synthesize tf. another possible way tu get tf into contact with blood is thruugh trauma, surgical ur otherwise. the fact that tf is inducible has created a lot uf interest in its cell biology. we reported recently that binding of factor viia tu tf un the cell surface elicited a marked increase in intracenular ca spikes, thus rendering the final proof that tf not only luoks like a receptor but really is one, since all the three essential criteria are fulfilled: tf has a transmembrane segment and a cytoplasmic tail tf has a high specificity/high affinity ligaud (factor vii and factor x) tf induces an intracellular signal upon ligand binding recent results regarding tf as a receptor and regarding its intracellular traffic will be reported. both f.ix and f.x are activated by the tf/f.viia complex. we have used a tf-initiated model system to examine the roles played by f.ixa and f.xa in initiating coagulation. the in vitro model contained a cellular tf source; plasma concentrations of f.ii, v, viii, ix and x, tissue factor pathway inhibitor and antithrombin iii. we tested the ability of exogenously added f.ixa or f.xa to substitute for tf/viia activity in initiating platelet activation and thrombin generation. f.xa was times more potent than f.ixa in initiating platelet activation, but f.ixa was more potent in promoting iia generation. in the presence of tf/viia, zymogen f.x was required both for platelet activation and iia generation, while f.ix was only required for iia generation. thus, tf/viia-activated f.ixa and f.xa have distinct physiological roles. the main role of f.xa is to activate platelets by producing an initial small amount of iia. the process of platelet activation is much more efficient if this initial iia is produced on the tf-bearing surface. f.ixa, by contrast, enhances iia generation on platelets by providing f.xa for platelet surface prothrombinase assembly. we conclude that activation of both f.ix and f.x by tf/viia is essential for efficient initiation of coagulation. furthermore, f.x activated on the surface of a tf-bearing cell isnot functionally equi.valent to f.x activated on the platelet surface. therefore, f.x activation by tf/viia does not compensate for a lack of f.ix or viii in hemophilia because the f.xa activated by tf/viia is located on the wrong surface for efficient thrombin generation. our results emphasize that the location, of an activated coagulation factor is a critical determinant of its role in hemostasis. the x-ray crystallographic structure of the fviia-tf complex located the areas within fviia in contact with tf to the first egflike and the protease domains. high-affinity binding of fviia to tf depends on ca + and previous studies suggest that ca + binding to a site in the protease domain is required. however, using surface plasrnon resonance we have shown that removal of the n-terminal gla domain and hydrophobic stack (hs) from fviia results in a decreased affinity for tf. in search for the mechanism by which regions in fvua not in direct contact with tf affect the affinity, various techniques have been employed. a plausible model is one in which the gla domain and hs optimize the interaction with tf by affecting regions that mediate the association with tf. using gel filtration we demonstrated that the hydrodynamic radii of des( - )-fviia and, especially, fviia decreased upon ca + binding, whereas des( - )-fviia was virtually unaffected. this suggested that the gla domain and the hs interact in a ca +-dependent manner with other regions in fviia, presumably the protease domain, making fviia more globular. using cd spectroscopy, we could detect ca +-induced changes in the secondary structure of fviia, all assignable to an increased helical content of the gla domain. the amidolytic activity of fviia in the absence of tf has a ca + dependency that suggests involvement of the gla domain in obtaining full activity. this activity was very sensitive to guanidine hydrochloride (c < . m) and its quenching corresponded to unfolding of the ca +-loaded gla domain as measured by changes in the trp fluorescence of fviia. based on these results it is conceivable that the gla domain and perhaps the hs of fviia interacts with the protease domain and that this globular form mediates the initial contact with "it. several studies have indicated a profound role for fvii(a) in arterial thrombogenesis. in the present study we quantified the inhibitory effect of dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone recombinant fviia (degr-rfviia) on acute thrombus formation at medium-sized artery blood flow conditions characterized by a wall shear rate of s - . native human blood was drawn directly from an antecubital vein by a pump into a heparin coated mixing device where degr-rfviia ( . - nmol/l final plasma concentration) or buffer were mixed homogeneously with the flowing blood. subsequently, the blood entered a parallel-plate perfusion chamber in which a thrombogenic surface consisting of immobilized tf and phospholipids was positioned. fibrin deposition, platelet-fibrin adhesion and platetet thrombus volume triggered by this surface were measured by morphometry. degr-rfviia inhibited these thrombus parameters in a dose dependent manner with ic values between . and . nmol/l the plasma levels of thrombin-antithrombin m complexes were inhibited at an ic of . nmot/l since the concentration of fvii and fviia in normal plasma is about and . nmol/l, respectively, theic values indicate that degr-rfviia is a very efficient inhibitor of thrombus formation in this model. thus, inhibition of the extrinsic pathway of coagulation may represent and entirely new and effective arterial anti-thrombotic approach. the anticoagulant agent from medicinal leeches, hirudin, is presently gaining popularity as an antithrombotic drug. the design of recombinant forms of the minipretein and their availability led to the clinical use of this naturally occurring thrombin inhibitor. extensive studies in experimental animals have shown that due to its pronounced and specific antithrombin effect hirudin is an antithrombotic agent of high quality. apart from its anticoagulant effect, hirudin is pharmacodynamically inert and is well tolerated in vivo. brain abnormalities in male children and adoleescents with hemophilia: detection with mr imaging mri of sickle cell cerebral infarction in addition, portions of the preparations were incubated with . nm-gold labelled fibrinegen molecules (fg-au; final concentration gp_g/ml) for see at °c. the expenments were stopped al~er or rain by glutaraldehyde fixation. serial sections were prepared and examined after silver-enhancement using danscher's ( ) method to visualize the fg-au. hwdid not alter the resting gfp. after . ,rain, activated hw-platelets changed their shape moderately but in contrast to the controls did not aggregate. moreover, the conatdcting cytoskeleton that centralizes platelet organelles was not formed. fg-au was found on the surface of the control platelets, in high density in the contact spaces between the aggregated platelets and internalized in the surface connected system. only a very small number of hwtreated cells had fg-au on their surface and showed internalization. rain after adp most of the platelets in both preparations had retumod to a discospherecytic shape it is confirmed that hw inhibits the platetet aggregation and in addition, ) the formation of the contractile cytnskeleton as well as ) the binding and interalization of the used ligand fibrinogen santoso l.institute for immunology and transfusion medicine, emst-moritz-arndt-university greifswald; . institute for transplant vasculopathy in cardiac allografts is the leading limitation to long-term survival of cardiac transplant recipients. activation of the coagulation mechanism by adherent monocytes expressing tissue factor (tf) is expected to be involved in the development of transplant atherosclerosis. in this in vitro study the effect of the immunosuppressant cyclosporine a (csa) on monocyte tf expression and the regulation of the tf gene transcription was analysed. csa was shown to inhibit the induction of monocyte procoagulant activity (pca) by decreasing lipopolysaccharide (lps)-induced tissue factor (tf) expression in monocytes. csa exerted a dosedependent inhibitory effect on monocyte tf expression at concentrations ( , txmol/l csa: % inhibition) not decreasing the cellular protein synthetic rate. addition of csa during lpsstimulation prevented activation of the nuclear factor (nf) ~b as demonstrated by electrophoretic mobility shift assay. western blot analysis confirmed reduced nuclear translocation of nf-~b in the presence of csa. inhibition of the lps-induced nf-~b activation by csa resulted in a reduced tf gene transcription as demonstrated by rt-pcr-analysis. thus, cyclosporine a prevents tf expression by inhibiting the induction of tf gene transcription in activated monocytes. csa may therefore provide a therapeutical tool acting not only as a direct immunomodulating agent, but also by influencing local coagulation activation. the n-glyean pattern of recombinant human coagulation factors ii (rf-i~ and ix (rf-ix), derived from both transfected chinese hamster ovary (cho) cells and african green monkey (vero) cells, and produced at industrial scale were analyzed by binding to carbohydrate specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. human plasma-derived coagulation factors i (hpf-i ) and ix (hpf-ix) exhibited complex-type glycan structures with carbohydrate chains capped with c~( - )sialic acid. terminal galactose-b( - )-n-acetylglucosamine units were detected in hpf-ix. both cho cell-derived rf-]i and rf-ix exhibited complot-type glycosylafion and contained ~ - )-sialio acid in addition to terminal galactose-b( - )-n-acetylglucosamine. vero cell-derived if-ix exhibited a complex-type glycan structure sinailar to that of cho cell derived rf-ix. in contrast, rf-li produced by vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "highmarmose" structures and complex-type glycosylation containing et( - )-sialie acid. galaetose-b( - )-n-acetylglucosamine structures and a low concentration of ct( - )-sialic acid were detected in both micro-heterogeneity fractions of veto cell-derived rf-li. although different in the'tr carbohydrate structures, coagulation factors ii and ix obtained recombinantly from both transformed cho-cells and vero-cells exhibited coagulation activities comparable to the plasma-derived proteins. exposure of tissue factor (tf) to flowing blood, as a consequence of vascular injury, leads to tf/f.viia-initiated coagulation which may play a key role in triggering intravascular thrombosis. in order to evaluate the potential advantages of tf pathway blockade over existing therapy, the antithrombotic and the anticoagulant .effects of a monoclonal anti-rabbit tf antibody (ap- ) and heparin were compared in a rabbit arterial thrombosis model. thrombosis was induced by a local injury to the carotid artery and the recurrence of thrombus formation was followed by monitoring the cyclic flow variations (cfv) of the arterial blood flow. rabbits received intravenously either ap- , heparin or vehicle, and cfv were monitored on line for min. activated partial thromboplastin time (aptt), and activated clotting time (act) were measured at the end of the experiment control ap-i heparin <lmg/kg* . mg/kg/hr . mg/kg/hr intraabdominallretroperitoneal, cns) on a named-patient basis. later also mild/moderate bleeds, predominantly joint bleeds were included.in - % of the bleeds novoseven was found to be hemostatically efficacious. also in major surgery (hip-and knee-replacement including a bilateral total condylar arthroplasty, herniotomy) hemostatic efficacy has been found to be % since rfvlla is proteotytically active only in complex with tf the risk for systemic activation of the coagulation system should be minimal. also very few side-effects have been seen.doses between - tjg/kg have been used, which initially have to be given at hours intervals. to achieve satisfactory hemostasis both an initial dose high enough to induce immediate hemostasis and an appropriate dose interval ensuring a plasma level of fvli:c well above the hemostatic lower level for a period of time, the length of which is dependent on the type of bleeding, are important. accordingly, the highel the initial dose the more robust with regard to dose interval will the treatment be. acute myocardial ischemia is usually triggered by coronary thrombosis. heparin (hep), along with aspirin, is commonly used for the treatment of unstable angina (ua) and myocardial infarction (mi). hirudin (hiq is a potent antitbrombin agent which may offer therapeutic advantages over standard hep for the treatment ua and mi. the oasis phase i pilot study conducted in canadian centres, has evaluated the safety, feasibility and efficacy of hir (hbw , behringwerke ag, marburg) compared to hep in patients with mi or suspected non q-wave mi. patients ( % received aspirin) were randomized to a hr infusion of either hep ( u bolus followed by u/hr; n= ), or low dose hir ( . mg/kg bolus then . mg/kg/hr; n= ) or medium dose lair ( . ms/ks bolus then . mg/kg/hr; n= ). at seven days of follow-up the incidence of major bleeds was . % in the heparin group, . % low dose hit, . % medium dose lair (p=ns), minor bleeds were . %, . % and . % respectively (p--- . hep vs med dose); there were no hemorrhagic strokes. the incidence of the primary outcome of cardiovascular death, new mi and severe recurrent ischemia (refractory or severe angina) was . %, . % and . % respectively (p--- . hep vs reed dose). there was also a trend towards lower rates of coronary artery bypass surgery in reed dose hir compared to hep. in a patient substudy, levels of thrombin antithrombin complex, fibfinopeptide a and d-dimer were all suppressed to a greater extent by mr compared to hep. these results indicate that hirudin may be a more effective agent for the treatment of acute coronary syndromes, but more information on safety and efficacy is needed from larger randomized trials. full clinical and coagulation resuks will be presented. hat is caused by an immunologic response to heparin. affected patients need effective parenteral anticoagulation. we treated patients ( m, fm), median age years ( - ) with laboratory confirmed hat (hipa-test) with r-hirudin (behringwcrke ag, marburg). treatment regimens were: ai acute hat: . mg/kg b.w., continuous infusion a mg/kg b.w/h ( patients); a acute hat with thrombolysis: bolus . mg/kg b.w., continuous infusion . mg/kg b.w./h ( patients); b: proph, treatment: continuous infusion . i mg/kg b.w.pa ( patients); c: during cardio-pulmunary bypass. primary objective was: increase in platelet counts or maintenance of normal baseline values during effective anticoagulation. secondary objectives were the incidence of new thromboembolic complications (tecs), major hemorrhage, limb amputations and deaths. results were compared with a historical control of ix-at patients, all confirmed by the hipa test, but treated before rhirudin was available (danaparoid, marcumar, no anticoagulation). primary efficacy analysis: respunders ai: %o, a : %, b: %, c: %, total: %. all adverse events beside bleeding complications had been judged to be caused by the underlying disease and not directly related to r-hirndin. proportion of patients with event in the obse~wation period; death: a . %, a %, b . , c %, total: . %, during rhirudin treatment: . %; new tec: ai . %, a %, b . %, c %, total . %, during rhirudin: . %; limb amputation: ai . %, a %, b . °/ , c %, total: . %, during rbirudin: . % eaapoiat we conclude, that r-hirndin is an effective anticoagulant in hat typa ii patients allowing rapid re, coved" of platelet counts. compared to a historical control the r-hirudin group had a strongly reduced incidence of combined endpuint of mortality, limb amputation and new tecs. major bleedings occurred slightly more frequent than in the historical control. however, the dosage used in regimen b might have been too low, as flds group revealed an effective anticoagulatiun and increase in platelet counts in only % and concomitantly had the higl)est incidence of new tecs. key: cord- -u cufg authors: finger, paula fonseca; pepe, michele soares; dummer, luana alves; magalhães, carolina georg; de castro, clarissa caetano; de oliveira hübner, silvia; leite, fábio pereira leivas; ritterbusch, giseli aparecida; esteves, paulo augusto; conceição, fabricio rochedo title: combined use of elisa and western blot with recombinant n protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: u cufg background: the avian infectious bronchitis virus (ibv) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. this study examined the combined use of an elisa and western blot (wb) to detect antibodies against the nucleocapsid protein (n) of ibv. the coding sequence for n was amplified by rt-pcr and expressed in escherichia coli. a soluble recombinant n protein (rn) of approximately kda was obtained. a total of sera were tested against the rn in elisa and the results were compared with those of the commercial idexx ibv ab test. elisa-rn achieved a . % sensitivity and . % specificity. wb confirmed all false negative sera in elisa-rn or idexx test as truly positive. the current study indicate that the combined use of rn in elisa and wb is a powerful tool for the immunodiagnosis of avian infectious bronchitis. methods: constructed recombinant pae/n expression vectors were used to transform e. coli bl (de ) star competent cells (invitrogen). the rn of infectious bronchitis virus was purified by affinity chromatography using histrap hp ml columns pre-packed with pre-charged ni sepharose in the Äktaprime automated liquid chromatography system (ge healthcare). a total of serum samples from chickens were used to develop and evaluate the elisa-rn test. to standardize the indirect elisa development, serum dilutions ( : , : and : ) and different concentrations of purified rn antigen ( , and ng/well) were tested. positive and negative sera for ibv were used as controls. the results were compared with those obtained from a commercial kit. serum samples scored as negative with the commercial kit but as positive with the elisa-rn were further analysed by western blot analyses using the rn protein as an antigen. the results of the elisa-rn were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software graphpad prism version . for windows (graphpad software, usa). results: the expected cdna fragment of approximately bp was successfully amplified by pcr using primers designed to select for the coding region of the n protein. the rn was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately and kda. out of sera that scored positive in the commercial elisa idexx ibv ab test, were also positive in the elisa-rn, yielding an elisa-rn test sensitivity of . %. out of sera that scored negative in the idexx ibv ab test, also scored negative in the elisa-rn, indicating a specificity of . %. sera that tested negative in the elisa-rn and positive in the commercial test also reacted with the rn protein in western blot. conclusions: the association between the elisa and western blot techniques developed in this study with a subunit of ibv (rn) were able to detect antibodies that the commercial elisa did not detect suggesting that the elisa-rn has greater sensitivity. methods: constructed recombinant pae/n expression vectors were used to transform e. coli bl (de ) star competent cells (invitrogen). the rn of infectious bronchitis virus was purified by affinity chromatography using histrap hp ml columns pre-packed with pre-charged ni sepharose in the Äktaprime automated liquid chromatography system (ge healthcare). a total of serum samples from chickens were used to develop and evaluate the elisa-rn test. to standardize the indirect elisa development, serum dilutions ( : , : and : ) and different concentrations of purified rn antigen ( , and ng/well) were tested. positive and negative sera for ibv were used as controls. the results were compared with those obtained from a commercial kit. serum samples scored as negative with the commercial kit but as positive with the elisa-rn were further analysed by western blot analyses using the rn protein as an antigen. the results of the elisa-rn were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software graphpad prism version . for windows (graphpad software, usa). (continued on next page) (continued from previous page) results: the expected cdna fragment of approximately bp was successfully amplified by pcr using primers designed to select for the coding region of the n protein. the rn was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately and kda. out of sera that scored positive in the commercial elisa idexx ibv ab test, were also positive in the elisa-rn, yielding an elisa-rn test sensitivity of . %. out of sera that scored negative in the idexx ibv ab test, also scored negative in the elisa-rn, indicating a specificity of . %. sera that tested negative in the elisa-rn and positive in the commercial test also reacted with the rn protein in western blot. conclusions: the association between the elisa and western blot techniques developed in this study with a subunit of ibv (rn) were able to detect antibodies that the commercial elisa did not detect suggesting that the elisa-rn has greater sensitivity. keywords: ibv, nucleoprotein, recombinant antigen, diagnosis, indirect elisa background avian infectious bronchitis (ib) is caused by a virus in the coronaviridae family, genera gammacoronavirus. it is a highly contagious disease with a short incubation period [ ] . the avian coronavirus was previously classified, and is most commonly referred to, as avian infectious bronchitis virus (ibv). the ibv is responsible for respiratory disease, which manifests in clinical symptoms such as sneezing and tracheal-bronchial rales that can lead to the development of more severe symptoms [ , ] . infected birds exhibit reduced performance, consequently leading to a reduction in weight gain and deterioration in egg quality and quantity. secondary bacterial infections will also contribute to economic losses. carcass condemnation due to the development of airsacculitis [ , ] negatively impacts commercial sales of bird meat and eggs. brazil was once the world's largest exporter of poultry and currently the world's third largest producer of bird meat [ ] . the consequences of ibv are a significant threat to brazil's poultry industry. the ibv genome consists of a non-segmented positivesense single-stranded rna that is approximately . kb in length. it encodes non-structural (accessory proteins) and four structural proteins: the nucleocapsid protein (n), the spike protein (s), the envelope protein (e), and the matrix protein (m). the nucleocapsid protein, or n protein, consists of amino acids. it has a molecular mass of approximately kda and directly binds with the viral genome to form the virion nucleocapsid [ , ] . its structure is highly conserved, with different strains of ibv sharing a high degree of identity ( - %) [ ] . the n protein is also known for its immunogenicity, inducing specific antibody and cytotoxic t-cells mediated responses [ , ] . there is significant interest in the use of the ibv n protein as an important target for diagnosis since it possesses the antigenic characteristics required for the development of serological assays that can be applied to detect or quantify antibodies against the ibv [ ] . the laboratory diagnosis of ib is dependent on direct and indirect techniques. the direct techniques are employed for viral isolation and genomic or phenotypic identification of the virus, while the indirect methods are used to detect specific antibodies [ ] . in addition to being applied for serodiagnosis, serological techniques can also be employed to evaluate the immune responses stimulated by vaccines. commercial elisa kits are typically used to indirectly diagnose ibv. these kits, however, are expensive when large number of samples require screening and they are not acessible for applications with the scale of the brazilian poultry industry [ ] [ ] [ ] . elisa techniques currently available are designed to detect polyclonal antibodies that target the whole virion. the use of nucleoprotein as the antigen for diagnosis and evaluation of vaccine immune responses is an interesting target to explore since this protein plays a important role in ibv virus replication and the induction of a specific immune response in infected birds [ , ] . the use of recombinant antigens in the design of a specific diagnostic technique facilitates the development of highly sensitive and specific assays that display a high antigen concentration and, thereby, reduce or eliminate background reactions. the use of recombinant antigens also represents a viable method of reducing immunoassay development costs. easy production of antigens in expression systems leads to simple and efficient antigen development which can reduce the production costs associated with diagnosis [ ] . the aim of the current study was to evaluate the combined use of an elisa and western blot (wb) to detect antibodies against the nucleocapsid protein of ibv. a previously characterized brazilian viral sample of ibv strain massachusetts (m -cnpsa -embrapa -concórdia, sc, brazil) was propagated after days of incubation in the chorioallantoic cavity of specific pathogen free (spf) embryonated chicken eggs. the allantoic fluid was then collected and stored at − °c. viral rna extraction was carried out with trizol® ls reagent (invitrogen™, eua), according to the manufacturer's instructions. extracted rna from ibv strain m was used for cdna synthesis with random oligonucleotides. reverse transcription (rt) was carried out using superscript® one-step rt-pcr system (invitrogen, usa). the resulting cdna samples were used to for pcr amplification of the whole orf of the n protein gene. primers based on the ibv m n protein gene sequence available at gen-bank (accession number m ) were designed to align between and and - bp of the gene and include cleavage sites for restriction enzymes. there was a restriction site for xhoi in the forward primer ( ′ -ccgctcgagatggcaagcggtaaggcaa - ′) and a restriction site for kpni in the reverse primer ( ′ -ggggtacctcaaagttcattctctccta - ′). the pcr reaction was performed with approximately ng of the extracted cdna, . mm mgcl, . mm dntps, units of taq dna polymerase, x reaction buffer, pmol of each primer, and m n,n,n-trimethylglycine (betaine) under the following conditions: cycle of °c for min, cycle of °c for min, then cycles of °c for min, °c for min, and °c for min, and a final extension of °c for min. the pcr amplification product was confirmed on a % agarose gel and purified using gfx pcr dna and gel band purification kit (ge healthcare, chicago, usa), according to the manufacturer's instructions. the pcr product was cloned into pae vectors by a t dna ligase (invitrogen) binding reaction after cleavage with restriction enzymes kpni and xhoi. the constructed recombinant pae/n expression vector was used to transform e. coli bl (de ) star competent cells (invitrogen). the resulting recombinant clones were cultivated in ml of lb broth medium with μg/ml of ampicillin ( °c, h, rpm). the whole culture volume was transferred to flasks containing ml of lb and incubated at °c with agitation ( rpm) until the optical density (o.d.) at nm reached . . the expression of the recombinant n protein (rn) was induced by adding isopropyl-β-d-thiogalactopyranoside (iptg, . mm final concentration) to the culture and incubating for h at °c with agitation ( rpm). the cells were harvested at , x g for min at °c and the culture pellet containing the rn protein was subjected to a solubilization procedure in Äkta wash buffer ( . % nah po , . % of nacl, . % imidazole, ph . , supplemented with μg/ml lysozyme). the resuspended cells were submitted to seven sonication cycles of s at hz and centrifuged again. the rn was purified by affinity chromatography using histrap hp ml columns pre-packed with pre-charged ni sepharose in the Äktaprime automated liquid chromatography system (ge healthcare). the protein concentration was determined with qubit™ protein assay kits (thermo-fisher scientific, usa) according to the manufacture's instructions. the sds-page was carried out on % polyacrylamide gel. the gels were either stained with coomassie blue r- (bio-rad, california, usa) or electroblotted onto hybond-ecl . μm nitrocellulose membranes (ge healthcare) using the bio-rad mini trans-blot cell (bio-rad) for western blot. briefly, the membrane was blocked with % non-fat milk in phosphate buffer saline containing . % tween- (pbs-t) ( mm nacl, . mm kcl, mm na hpo , mm kh po , ph . ), incubated with mouse monoclonal antibody (mab) anti- xhis (sigma, usa) diluted to : in pbs-t, and incubated with polyclonal antibody anti-mouse igg conjugated to hrp (sigma). all incubation steps were performed at °c for h under slight agitation followed by three washes with pbs-t. the immunoblot was developed using , ′-diaminobenzidine (sigma). a total of chicken serum samples (n = ) were used to develop and evaluate the elisa-rn. the serum samples were kindly provided by mercolab laboratories (garibaldi, brazil), a laboratory accredited by the mapa (ministry of agriculture, livestock and food supply) that uses the commercial elisa kit ibv ab test (idexx) for characterization. serum samples from chickens with a positive diagnosis for newcastle disease were used for the evaluation of the elisa-rn specificity. the samples used in this study originated from birds with a known history of vaccination, including vaccination against the newcastle disease virus, and were categorized into three groups: commercial egg-layers, broiler breeders, and meat-producing chickens. this information was further used to improve the evaluation of the elisa-rn test. were tested. positive and negative sera for ibv were used as controls. the -well microtiter plates (nunc max-isorp®, thermo fisher, usa) were coated with ng/ well of rn diluted in . m carbonate-bicarbonate buffer (ph . ) and incubated overnight at °c. the plates were incubated at °c for h with a blocking solution ( % non fat dry milk in pbs-t). serum samples diluted : in pbs-t were added in duplicate and the plates were again incubated for h at °c. after this period, the secondary antibody horseradish peroxidase (hrp) -rabbit anti-chicken igy peroxidase conjugate (sigma) diluted : , in pbs-t was added and plates were once more incubated at °c for . h. after each incubation, the plates were washed three times with pbs-t. in the final step, plates were washed five times and μl/well of peroxidase substrate o-phenylenediamine dihydrochloride (sigma aldrich) was added. the reaction was interrupted with n h so and the results were read as o.d. using a spectrophotometer at nm. to determine the cutoff value, negative sera were used, totalizing sera, considering the mean of sera added of two standard deviation. the roc analysis was used to simulate the influence of different cutoff values on the sensitivity and specificity of the test. the results were compared with those obtained with the commercial kit. serum that scored as negative in the commercial kit but as positive in the elisa-rn were submitted to western blot analyses using the rn protein as antigen. in order to evaluate the repeatability of the elisa-rn, three separate batches of recombinant n protein were produced and purified following the methodology described above with distinct purification times, to demonstrate the reproducibility of the recombinant protein expression procedure. serum samples were selected for testing against each batch of antigen and the averages, and standard deviations, of the o.d. at nm were calculated. of the samples used for this test, one was strongly reactive positive, four were moderately reactive positive, and four were negative serum samples. to evaluate the specificity of the test, negative serum samples to ibv and positive serum samples to newcastle disease were tested in the elisa-rn. the western blot was performed almost as described above. the rn was deposited in all wells of the sds-page gel and, after transfer to the nitrocellulose membrane, it was cut to obtain strips. each strip was incubated with a : serum dilution after blocking with % non-fat milk. the antibody horseradish peroxidase (hrp) -rabbit anti-chicken igy peroxidase conjugate (sigma) ( : ) was added to all membranes and the reaction was revealed using , ′-diaminobenzidine (sigma). all incubation steps were performed at °c for h under slight agitation and were followed by three washes with pbs-t. the results of the elisa-rn compared to the commercial kit results, the receiver-operating characteristics (roc) curves, the area under the curve (auc) and the confidence intervals were obtained through the software graphpad prism version . for windows (graphpad software). the expected cdna fragment of approximately bp was successfully amplified by pcr using primers designed to obtain the coding region of the n protein. successful recombinant pae/n vector construction was confirmed through cleavage with the same restriction enzymes used for the plasmid construction (date not shown). the recombinant n protein (rn) was expressed as a soluble protein, dispensing refolding steps, and, after purification, a yield of mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately kda and kda (fig. a) . the same bands were observed in the western blot and confirmed the presence of bands that corresponded to the protein of interest (fig. b) . other studies have also described the cloning of the bp corresponding to the n protein gene from ibv [ , ] . they reported that the recombinant protein presented as two distinct molecular masses, one kda and other kda. the kda mass is the truncated form of the n protein [ , ] . this corroborates with the results observed through sds-page and western blot in the current study (fig. ) . the results of the elisa-rn were compared with the results obtained with the ibv ab test (idexx). receiver-operating characteristics (roc) with % confidence intervals were used to analyse these results (fig. ) . out of sera that scored positive in the ibv ab test, also scored positive in the elisa-rn test. the sensitivity of the elisa-rn test was . %. out of the sera that scored negative in the idexx test, also scored negative in the elisa-rn, indicating a specificity of . %. the area under the curve (auc) was . (p < . and cutoff . ). three different protein batches were used as coating antigens for testing the elisa-rn and the results from all three were similar (p > . ) and, thus, indicate antigen stability (fig. ) . all newcastle disease positive sera tested negative in the developed elisa-rn, providing evidence of test specificity (data not shown). all sera that tested negative in the commercial test and positive in the elisa-rn were submitted for western blot analyses using the rn protein as an antigen. all serum samples reacted with the two bands of rn, confirming the results obtained in the developed elisa (fig. ) . sera that tested negative in the elisa-rn and previous studies that focused on the development of more efficient diagnostic techniques were not limited to avian infectious bronchitis detection, but also aimed to control and monitor chickens vaccination [ , ] . the nucleoprotein from ibv is widely considered the choice protein for the development of immunoassays for antibody detection since this protein plays an important role in inducing an antibody response in ibv infected or vaccinated animals [ , ] . other studies have demonstrated that the nucleocapsid protein is highly conserved among ibv isolates ( - . % similarity), immunogenic, and abundantly expressed during infection [ , , ] . these features make this protein an interesting candidate for use in diagnostic techniques, such as elisa and wb [ ] . the elisa is a powerful tool because it provides a safe and an easy way to evaluate the ibv vaccine efficiency, and perform serological diagnosis as well as epidemiological surveillance [ ] . in contrast to the elisa presented in this study, the elisa developed by lugovskaya et al. [ ] used two fragments of the recombinant n protein expressed in e. coli as an antigen, achieved a specificity of . %, and a sensitivity of . %. these results are comparable with the commercial test that is currently used for routine ibv diagnosis that has a specificity of . % and a sensitivity of . % [ ] . the elisa-rn developed in this study presented similar specificity and sensitivity results ( . and . %, respectively), and thus highlights that our study uses a western blot technique to complement the results obtained through elisa and further support the ibv diagnosis. the western blot technique may prove useful for epidemiological studies, for monitoring specific pathogen free farms, and in vaccine potency tests. the association of elisa and western blot can be seen as a tool to be considered to increase sensitivity for serodiagnosis purposes [ ] . since positive sera react with two bands in the western blot, the technique is even more specific. the recombinant n protein produced in this study was expressed in the soluble fraction. our rn was easily recoverable from the culture without using denaturing agents. in contrast to previous studies where the n protein was expressed in its insoluble form [ , ] , our procedure avoids the necessity to refold the recombinant protein. the production of rn was repeatable since the same elisa-rn test results, and about the same yield, were obtained when different production and purification times were used. besides the use of the n protein for the diagnosis of ibv, an elisa that was developed using the s protein was also described and compared to a commercial test [ ] , and achieved a specificity and sensitivity of . and . %, respectively. however, the n protein offers specific advantages when used for the purpose of diagnosis during viral infection since it is produced in larger quantities than the s protein (the n proteins is produced at a ratio of : relative to the s protein [ ] and plays an important role in the virus's replications and assembly process [ ] . additionally, the s protein has hypervariable regions that cause mutations in its sequence and therefore offers low efficiency as a protein for diagnosis [ ] . the results from the elisa test developed using the rn protein indicate that the test could be effectively applied for ibv diagnosis [ ] . the yield of rn per litre of lb broth culture would coat approximately -well microtiter plates allowing for the diagnostic analysis of approximately , serum samples in duplicate. also noteworthy is the production cost reduction from using rn in a soluble form, since this eliminates costs associated with the refolding step while it also preserves important conformational epitopes. the currently available commercial test on the other hand, employs the whole ibv as an antigen which requires viral propagation and the implementation of robust laboratory biosafety standards [ ] . it is worth mentioning that, by comparing the developed elisa-rn and wb with the idexx ibv ab test, the analysis conducted in the current study indicated that it was possible that some serum identified as negative for anti-ibv antibodies on the commercial test be positive when tested using the elisa-rn and by wb. this could be of concern as false negative results are undesirable, especially if a lot of birds are misdiagnosed. thus, the elisa-rn could be applied together with the wb developed in this study for the routine detection of ibv in diagnostic laboratories. false negative results that contribute to the spread of the disease can be avoided, or at least decreased, when the two tests are applied together. the indirect elisa developed here with rn as an antigen allowed for the detection of anti-ibv antibodies in chicken serum at high specificity and sensitivity. the association between elisa and western blot techniques developed with a subunit of ibv (rn) were able to detect antibodies that were not detected with the commercial elisa test suggesting greater sensitivity in the developed elisa-rn. in addition, the elisa-rn with the advantages of easy preparation and improved safety could be a promising alternative to the whole live virus elisa. relationship between sequence variation in the s spike protein of infectious bronchitis virus and the extent of crossprotection in vivo infectious bronchitis virus: immunopathogenesis of infection in the chicken efficacy of infectious bronchitis virus vaccines against heterologous challenge coronavirus avian infectious bronchitis virus bronquite infecciosa das galinhas: conhecimentos atuais, cepas e vacinas no brasil polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses comparisons of the structural proteins of avian infectious bronchitis virus as determined by western blot analysis specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus characterization of infectious bronchitis viruses isolated from outbreaks of disease in commercial flocks in brazil recombinant nucleocapsid protein based single serum dilution elisa for the detection of antibodies to infectious bronchitis virus in poultry high-level protein expression following single and dual gene cloning of infectious bronchitis virus n and s genes using baculovirus systems detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay the s glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens selective replication of coronavirus genomes that express nucleocapsid protein elisa for antibodies to infectious bronchitis virus based on nucleocapsid protein produced in escherichia coli evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus diversidade genética de amostras brasileiras do vírus da bronquite infecciosa determinada pelo sequenciamento de nucleotídeos dos genes n e s development of a recombinant nucleoprotein-based enzyme-linked immunosorbent assay for quantification of antibodies against porcine reproductive and respiratory syndrome virus combined use of western blot / elisa to improve the serological diagnosis of human tuberculosis development of a multiepitope antigen of s protein-based elisa for antibodies detection against infectious bronchitis virus the amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with ′ genomic rna coronavirus ibv: further evidence that the surface projections are associated with two glycopolypeptides we thank mercolab laboratory (garibaldi, rs, brazil) for giving the sera that were analyzed in this study. not applicable. all data generated or analyzed during this study are included in this article. all authors read and approved the final manuscript. this article does not contain any studies with human participants or animals performed by the author. the authors authorize the publication. no known competing interest. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - b erfj authors: nguyen, t. d.; bernard, s.; bottreau, e.; lantier, i.; aynaud, j. m. title: Étude comparée de trois souches du coronavirus de la gastroentérite transmissible: conditions de la réplicationvirale et de la synthèse des antigènes structuraux date: - - journal: annales de l'institut pasteur / virologie doi: . /s - ( ) - sha: doc_id: cord_uid: b erfj summary purdue- and d- strains of tgev were compared with the -sg strain, which was obtained by means of a survivor selection process in gastric juice of adult pig. the -sg strain was characterized by (a) low infectivity, (b) delayed and restricted growth associated with low and delayed rna synthesis, and (c) a high content of structural antigens. in contrast, purdue- and d- strains were characterized by (a) high infectivity, and (b) a normal pattern of virus replication and rna and structural antigen synthesis. tunicamycin induced the inhibition of synthesis of el and e glycoproteins (detected by the elisa test using monoclonal and polyclonal antibodies) as well as a significant reduction in the np protein. the inhibitory effect of tunicamycin was influenced by the cell type and virus strain. la gastroenterite transmissible (get) est une enterite neonatale mortelle, hautement contagieuse, causee par un coronavirus specifique de l'espece porcine; ia get est responsable de lourdes pertes economiques en production porcine. la relation entre ie pouvoir pathogene du virus et d'eventuels marqueurs in vitro lies par exemple aux conditions de sa replication, fait encore l'objet de controverse [ , , , ] . apres un certain nombre de passages en serie sur culture cellulaire, le virus oet perd une grande partie de son pouvoir pathogene. des resultats recents suggerent qu'il n'est pas possible de distinguer la souche attenuee -s (utilisable comme vaccin vivant oral chez la truie en vue de l'induction de l'immunite lactogene) des souches d- (dont la souche -s tire son origine) et purdue- (souche americaine de reference), par l'etude des conditions d'attachement in vitro des particu-ies virales (ann. rech. veterin., , sous presse). c'est pourquoi nous avons entrepris l'etude comparee de la replication de ces trois souches de virus get dans deux systemes cellulaires differents : les cellules st (testicule de pore) et les cellules rpd (rein de pore). nous avons con centre nos efforts d'une part sur l'etude de la cinetique des syntheses vir ales et, d'autre part, sur l'analyse des antigenes structuraux du virus produit a la fin du cycle, al'aide d'une batterie d'anticorps monoclonaux specifiques du virus oet. la resistance de la souche -s al'acidite et ai'action des enzymes proteolytiques [ ] suggere l'existence probable de changements structuraux ou conformationneis, qui pourraient etre lies aux conditions de glycosylation des proteines virales. c'est pourquoi nous nous sommes aussi interesses ai'influence d'une substance connue pour son activite a ce niveau: la tunicamycine. cellutes. la lignee de cellules de rein de pore rpd a ete decrite precedemrnent [ ] . la lignee de cellules de testicules de pore st [ ] a ete fournie par le dr e.h. bohl (wooster, ohio, usa). ces cellules sont cultivees en milieu mem (milieu essentiel minimal) additionne de serum de fcetus de veau ( %). la souche purdue-us, qui a subi passages sur cultures cellulaires, a fait i'objet de nombreuses publications [ , , , ] cinetlque de la synthase de l'arn, des proteines et de la formation virale en cycle unique. decrite precedemment [ ] , la technique pour i'etude du developpement du virus en cycle unique a he reprise pour examiner la synthese de l' arn et des proteines et la formation des particules infectieuses virales. les modifications suivantes ont ete apportees : apres l'attachement du virus, esinoculums ont ete elimines par deux lavages. le milieu mem additionne de ug/rnl d'actinomycine d et de , fici/ml d 'uridine au de leucine tritiees, a ete utilise pour la culture des cellules infectees, qui ant ete ensuite mises aincuber a °c. preleves en fonction du temps, les echantil-ions ( , ml chacun) ant ete deposes sur des papiers filtre (titertek cell harvester filter, flow laboratories). apres trempage pendant h it °c dans de l'acide trichloroacetique , ces papiers sont rinces trois fois avec de l'alcool a °et seches. la radioactivite de l'uridine au de la leucine tritiee incorporee dans les macromole- les antigenes viraux neoforrnes sont titres al'aide d'un test elisa. l'effet inhibiteur de la tunicamycine sur ie rendement de la multiplication virale est exprime par la baisse du titre infectieux et de la densite optique pour le test elisa. titrage des antlgenes structuraux du coronavirus get it i'aide d'une technique elisa mixte. anticorps monoclonaux anti-coronavirus get. -une batterie d'anticorps monoclonaux de souris specifiques du virus oet a ete preparee recemment [ ] . un certain nombre d'entre eux ant ete fournis par h. laude et utilises dans cette etude (tableau i). anticorps polyclonal anti-coronavlrus get_ -le serum de la truie n° , qui a ete utilisee dans une etude anterieure [ ] , a servi a la preparation de l'anticorps polyclonal. cette truie a ete vaccinee par voie orale avec la souche -s ( semaines avant la mise-bas) et la portee de porcelets a ete eprouvee (au e jour d'age); le serum recolte lors de i' abattage (deux semaines apres la mise-bas) presente un titre neutralisant de . . preparation du conjugue, -le marquage des anticorps al'aide de la peroxidase a ete realise selon la technique de nakane et kawaoi [ ] . detection des antlgenes structuraux du coronavirus de la get. -l' application de la technique elisa-sandwich [ ] aux etudes virologiques et immunologiques de la get a ete developpee recemment [ ] . la particularite de cette technique est la suivante: ) la sensibilisation des plaques plastiques (nunc, immunoplate ii, inter. ) cinetique d'apparition des particules infectieuses. -quel que soit ie systeme cellulaire, la souche -s se distingue des deux autres souches par un allongement de la phase de latence ( fig. c et if). en particulier dans les cellules rpd, on observe un retard d'environ h. de plus, la production de la souche -s ne represente que de celle des deux autres souches. cependant, la phase exponentielle presente une pente similaire pour les trois souches. par rapport aux cellules rpd, les cellules st offrent au virus oet des meilleures conditions pour sa replication puisque celle-ci est plus precoce et plus productive (titres infectieux plus eleves), ) cinetique de la synthese de i'arn et des proteines virales. -presentes par la figure (a, b, d, e), les resultats sont les suivants. par rapport aux deux autres souches, la souche -s est caracterisee par une incorporation plus faible d'uridine tritiee. en revanche, la souche -s presente une incorporation plus importante de la leucine tritiee, en particulier dans les cellules st, ce qui suggere un niveau de synthese de proteines plus elevee, enfin, ie systerne cellulaire a une influence importante: on constate en effet que la synthese de i'arn viral dans les cellules st est presque le double de celle qui est observee dans les cellules rpd. notons par ailleurs que la synthese de l'arn cellulaire est pratiquement negligeable en presence d' [j.g/ml d'actinomycine d. analyse des antlgenes structuraux du virus get produit a la fin du cycle. .. . . .: :- souche d- presente de facon generale un comportement identique a celui de la souche purdue- (resultats non presentes). en resume, ces resultats mettent en evidence l'abondance particuliere d'antigenes structuraux synthetises par la souche -s en culture cellulaire. influence de la tunicamycine sur ie rendement en particules infectieuses. l'addition de la tunicamycine dans le milieu a pour effet de diminuer le rendement en virus infectieux ( fig. ). l'effet inhibiteur de la tunicamycine est plus intense a la concentration de ug/ml pour la souche -s . par contre, pour les autres souches, on observe un effet inhibiteur proportionnel a la concentration de tunicamycine. de plus, on constate les faits suivants: dans les cellules st, la multiplication virale de la souche -s est plus affectee par la tunicamycine que celie des autres souches ; inversement, dans les cellules rpd, la sensibilite ala tunicamycine est differente ; la souche -s est mains affectee que les deux autres souches virales. influence de la tunicamycine sur la synthese des antigenes structuraux. la determination quantitative, par elisa, des antigenes viraux contenus dans les suspensions virales demontre qu'en presence de la tunicamycine la synthese globale des proteines virales diminue parallelement ala formation des particules infectieuses ( fig. ). les resultats de la reaction elisa mettant en oeuvre les anticorps monodonaux, demontrent que la synthese non seulement des glycoproteines mais aussi de la nucleoproteine est affectee par l'addition de la tunicamycine ( fig. ). influence du traitement du virus seml-purlfle par le metaperiodate sur son aetlvlte antlgenlque. cette experience a ete menee atitre de controle pour permettre de savoir si i'alteration des parties glycosidiques des glycoproteines virales avait, ou non, une influence significativesur l'activite antigenique du materiel viral etudie dans ces conditions experimentales (elisa). pour cela nous avons choisi le metaperiodate dont les interactions sur les sucres sont connues [ ] . le traitement d'une suspension virale (souche purdue- ) semi-purifiee par ultracentrifugation n'a pas d'influence significative sur l'expression de l'activite antigenique, en elisa, des glycoproteinesel et e jusqu'a la dose de ug/rnl. les antigenes structuraux sont titres par un test elisa utilisant, pour la sensibilisation des plaques, un melange des differents anticorps monoclonaux specifiques de chacune des trois proteines virales, et, pour la revelation, l'anticorps polyclonal . la difference de la synthese des proteines entre les deux types de cellules, est determinee par celie propreaux cellules (synthese endogene) plutot que par l'influence de la multiplication virale. quelle que soit la souche, il est interessant de noter que les conditions de synthese des proteines sont differentes dẽ celle de i' arn. dans le cas de la souche -s , le faible niveau observe dans la synthese d'arn et dans l'apparition de particules infectieuses mais non dans la synthese de proteines, n'a pas encore d'explication. pour la souche -sa, it existe un nombre eleve de particules virales physiquement completes qui ne sont pas toutes infectieuses quel que soit le systeme cellulaire. en effet, malgre une quantite plus faible de particules infectieuses, dans les cellules st, la tunicamycine a un effet inhibiteur sur la formation du virus infectieux plus prononce pour la souche -s que pour les autres souches. en revanche dans lescellules rpd, un effet inverse est observe. ainsi, il semble que la souche -s , resistante al'acidite et aux proteases digestives, est plus vulnerable a l'inhibition de la glycosylation. les conditions de glycosylation des proteines sont dependantes de la machinerie cellulaire [ , ] . la difference d'activite de la tunicamycine observee entre les deux types de cellules pour une souche virale confirme cette observation. nos resultats experimentaux montrent egalernent que la synthese non seulement des glycoproteines mais aussi des proteines non glycosylees (np) est diminuee en presence de tunicamycine. nos resultats confirment done que les proteines el et e du coronavirus oet sont toutes les deux n-glycosyiees [ ] . par ailleurs, il est interessant de noter que l'inhibition de la synthese des proteines el et e entraine parallelement une diminution de la synthese de la proteine np. dans le cas du virus de i'hepatite murine, la tunicamycine n'inhibe que la synthese de proteine e [ ] . ainsi, dans le cas du virus oet la synthese de el et e pourrait intervenir dans la regulation de la synthese des autres composants du virus de la get, dont la proteine np. cependant, nos resultats ainsi que ceux de rottier et coil. [ ] apartir du virus de i'hepatite murine montrent que, en presence de la tunicamycine, des particules virales infectieuses continuent aetre formees, bien que moins nombreuses « / ). selon holmes et coli. [ ] , en presence de la tunicamycine, des particules virales «nues » (depourvues de spicules)sont formees et depourvues de pouvoir infectant. on sait en outre que la tunicamycine inhibe completernent la production de virus infectieux de l'herpesvirus bovin [ ] . les limites de la technique elisa (reactions positives detectables apartir de particules infectieuses) ne nous permet pas de determiner si ces particules infectieuses produites en presence de tunicamycine sont « nues » ou pas. le probleme majeur qui s'est pose lors de la mise en oeuvre de la technique elisa etait l' antigenicite des proteines virales synthetisees en presence et en absence de tunicamycine. les arguments suivants sont en faveur d'une reactivite identique de ces proteines avec les anticorps monoclonaux: ( ) les apoproteines, el et e , reagissent avec les anticorps induits par les virus complets [ , ]; ( ) nous n'avons pas constate de difference dans le pouvoir neutralisant des anticorps sur le virus cultive sur cellules st ou sur cellules rpd, malgre la difference de la sensibilite atunicamycine; et ( ) le traitement du virus purifie par le metaperiodate, qui modifie profondement les parties glycosydiques des glycoproteines, n'a pas d'influence significative sur l'antigenicite des proteines virales evaluee al'aide de la technique elisa, sauf pour les concentrations de metaperiodate qui affectent egalement la nucleoproteine (np) non glycosylee; toutefois cet argument n'est valable que pour les epitopes . -enfin, quelle que soit la souche de virus, l'inhibition de la synthese des proteines el et e -par le biais de l'inhibition de la n-glycosylation par la tunicamycine -entraine une diminution sensible de la formation de la proteine np non glycosylee. transmissible gastroenteritis (toe) of swine: survivor selection of tge virus mutants in stomach juice of adult pigs detection of transmissible gastroenteritis coronavirus by a sandwich elisa technique antibody response in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus the periodate oxydation of amino acides with references to studies of glycoproteins sindbis virus glycoproteins are abnormally glycosylated in chinese hamster ovary cells deprived of glucose alteration in the hemagglutinin associated with adaptation of influenza b virus to growth in eggs enzyme-linked immunosorbent assay. -iii. quantitation of specific antibodies by enzyme labeled antiimmunoglobulin in antigen coated tubes local and systemic cell-mediated immunity against transmissible gastroenteritis, an intestinal infection of swine comparison of properties between virulent and attenuated transmissible gastroenteritis virus defective replication of porcine transmissible gastroenteritis virus in a continious cell line in vitro differenciation and ph sensitivity of field and cell culture attenuated strains of transmissible gastroenteritis virus analysis of the fonctions of coronavirus glycoproteins by dfferential inhibition of synthesis with tunicamycin cloning and expression of the surface glycoprotein gp of porcine transmissible gastroenteritis virus in vitro properties of low and high passaged strains of transmissible gastroenteritis coronavirus of swine antigenic structure of transmissible gastroenteritis virus. -i. properties of monoclonal antibodies directed against virion proteins studies on transmissible gastroenteritis of swine. -ii. selected characteristics of a cytopathogenic virus common to five isolants from transmissible gastroenteritis physicochemical properties of field and cell culture-attenuated strains of swine transmissible gastroenteritis (tge) coronavirus peroxidase-labelled antibody a new method of conjugation neutralizing secretory a and igg do not inhibit attachment of transmissible gastroenteritis virus viral protein synthesis in mouse hepatitis virus strain a -infected cells: effects of tunicamycin the structure and behaviour of coronavirus effects of tunicarnycin and monensin on biosynthesis, transport and maturation of bovine herpes virus type glycoproteins key: cord- - tdfvlqd authors: tan, xiaotian; lin, cory; zhang, jie; khaing oo, maung kyaw; fan, xudong title: rapid and quantitative detection of covid- markers in micro-liter sized samples date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: tdfvlqd covid- pandemic has caused tens of thousands of deaths and is now a severe threat to global health. clinical practice has demonstrated that the sars-cov- s specific antibodies and viral antigens can be used as diagnostic and prognostic markers of covid- . however, the popular point-of-care biomarker detection technologies, such as the lateral-flow test strips, provide only yes/no information and have very limited sensitivities. thus, it has a high false negative rate and cannot be used for the quantitative evaluation of patient’s immune response. conventional elisa (enzyme-linked immunosorbent assay), on the other hand, can provide quantitative, accurate, and sensitive results, but it involves complicated and expensive instruments and long assay time. in addition, samples need to be sent to centralized labs, which significantly increases the turn-around time. here, we present a microfluidic elisa technology for rapid ( - minutes), quantitative, sensitive detection of sars-cov- biomarkers using sars-cov- specific igg and viral antigen – s protein in serum. we also characterized various humanized monoclonal igg, and identified a candidate with a high binding affinity towards sars-cov- s protein that can serve as the calibration standard of anti-sars-cov- s igg in serological analyses. furthermore, we demonstrated that our microfluidic elisa platform can be used for rapid affinity evaluation of monoclonal anti-s antibodies. the microfluidic elisa device is highly portable and requires less than μl of samples for each channel. therefore, our technology will greatly facilitate rapid and quantitative analysis of covid- patients and vaccine recipients at point-of-care. the disease (covid- ) related to novel coronavirus (sars-cov- ) has caused tens of thousands of deaths and is becoming a severe threat to global health. however, diagnosis and prognosis of covid- are still facing serious challenges. the current "gold standard" method to diagnose sars-cov- infection is based on the detection of viral rna (n gene) in nasal swab samples with rt-pcr. however, pcr-based diagnosis is prone to false negatives due to the uncertainties in sample collection and the molecular mechanism of the test and is unable to track the immune response to the viral infection . earlier sars-cov related research , and most recent studies on sars-cov- , have shown that sars-specific antibodies, such as igg, igm, and iga, can be detected in serum as early as seven days after viral infection ( - days after the symptoms appear) and can last for several years after recovery . similar immune response should also occur after a successful vaccination (> days after vaccination) , , . therefore, virus-specific antibodies in serum and secretory mucus (e.g., saliva and sputum) can be used as diagnostic markers for viral infection and for the evaluation of patient's adaptive immune responses (either from virus infection or from vaccination). as such, sars-cov- -specific antibodies are currently listed among the diagnostic markers in the "covid- diagnosis and treatment plan (provisional th edition)" published in china. in addition to sars-cov- specific antibodies, the viral antigen (such as the spike protein or s protein) in circulating blood can be used for the prognosis of covid- -related viremia , . as one of the most commonly used targets for vaccine development, the serum concentration of the s protein is also a potential marker for early-stage vaccine responses, especially for sub-unit vaccines . unfortunately, the existing antibody detection methods are still far from adequate. the gold nanoparticle based lateral flow assay (e.g., paper-based test strips) is very popular in rapid detection of igg/igm antibodies (especially for point-of-care diagnostics) - . although fast ( - minutes), it provides only yes/no information and has very limited sensitivities, making this method very easy to generate false positives/negative and impossible to track the patients' immune response to infection, treatment, and vaccination. conventional elisa (enzyme-linked immunosorbent assay), on the other hand, can provide quantitative, accurate, and sensitive results, but it involves complicated and expensive instruments and long assay time ( - hours) , . in addition, samples need to be sent to centralized labs, which significantly increases the turn-around time. in this work, we present a microfluidic elisa technology for rapid ( - minutes), quantitative, and sensitive detection of sars-cov- biomarkers using sars-cov- specific igg and viral antigen -s protein, both of which are spiked in serum, as a model system. we also characterized various humanized monoclonal igg and identified one with a high binding affinity towards sars-cov- s protein that can serve as the calibration standard of anti-sars-cov- s igg in serological analyses. furthermore, we demonstrated that our microfluidic elisa platform can be used for rapid affinity evaluation of monoclonal anti-s antibodies. the microfluidic elisa device is highly portable and requires only l of samples for each channel, which can be easily collected from a drop of fingertip blood. therefore, our technology will greatly facilitate rapid and quantitative analysis of covid- patients and vaccine recipients at point-ofcare. a detailed description of the automated elisa system and corresponding capillary sensor arrays can be found in our previous publications , . a photo of the capillary sensor array can be found in figure (a). it is made of polystyrene using the injection molding method. the sensor array has channels, each of which has an inner diameter of . mm and approximately l volume, and acts as an elisa reactor. the chemiluminescent substrate (supersignal™ elisa femto substrate, ), the ultrapure™ dnase/rnase-free distilled water ( ), and the superblock™ (pbs) blocking buffer ( ) were purchased from thermo fisher. the elisa coating buffer ( × pbs, dy ), concentrated wash buffer (wa ), and concentrated reagent diluent ( % bsa in × pbs, dy ) were purchased from r&d systems. the human serum was purchased from millipore sigma (h - ml). human-cell-expressed sars-cov- spike s -his recombinant protein was provided by sino biological ( -v h). the human-cell-expressed sars-cov spike s -his recombinant protein was purchased from creative biolabs (vang-wyb ). the recombinant cr therapeutic antibody was purchased from creative biolabs (mro- lc). the humanized chimeric antibodies d , d , and d were developed and provided by sino biological (catalog number: -d , -d , and -d ). the anti-polyhistidine antibody that was used in polyhistidine-mediated s protein immobilization (see figure s ) was purchased from thermo fisher (ma - ). the horseradish peroxide (hrp) conjugated secondary antibody used in the igg detection experiment was from the detection antibody in thermo fisher's human total igg elisa kit ( - - ). the hrp conjugation of cr , d , d , and d antibodies were carried out with abcam's hrp conjugation kit (ab ) with a molar ratio of antibody : hrp = : . the illustrations for the reactor preparation protocol and the elisa protocols can be found in figure s . for all steps, the working solution of the wash buffer and reagent diluent were diluted with ultrapure™ dnase/rnase-free distilled water to achieve × working concentration. in the first step of the reactor preparation process ( figure s (a)), the working solution of the capture antibody (i.e., d in s detection experiments) or the anti-his antibody (in igg detection and antibody affinity experiments) were prepared by diluting the stock solution with the elisa coating buffer ( × pbs, ph = . ) to achieve final concentrations of μg/ml. note that for the anti-s igg detection and antibody affinity experiments, the second incubation step was used for blocking plus s protein immobilization (with μg/ml of s -his protein dissolved in % bsa). for the s protein detection, this step is used for blocking only (with % bsa in × pbs). for the anti-s igg detection experiments (see figure s (b) for the detailed protocol), various concentrations of monoclonal antibodies were prepared by diluting the stock solutions with times diluted human serum (the serum was diluted with × reagent diluent, which correlates to % bsa). the working solution of the detection antibody (in this case, the hrp-conjugated secondary antibody) was prepared by diluting the stock solution times in × reagent diluent. for the s detection experiments (see figure s (c) for the detailed protocol), various concentrations of the s proteins were prepared by diluting the stock solution with times diluted human serum. the working solution of the detection antibody (i.e., hrp-conjugated d ) was prepared by diluting the stock solution times in × reagent diluent. the final concentration of the detection antibody was µg/ml. for the antibody affinity experiments, various concentrations of hrp-conjugated monoclonal antibodies were prepared by diluting the stock solution in × reagent diluent. the signal intensities of the microfluidic elisa were measured with the chemiluminescent imaging method, using a cmos camera (see figure (b) for an example of the chemiluminescent signals). to enhance the dynamic ranges of the elisa, multiple exposures with adjustable exposure time were applied. all chemiluminescent intensities (cl intensity) were normalized to three seconds of exposure time. detailed explanations about the chemiluminescent imaging and the multiple exposure approaches can be found in our previous publications . the receptor binding domain (rbd) in the spike protein (located in the s subunit) on a coronavirus is responsible for binding to the membrane receptors on the target cell. it plays a critical role in the coronavirus cell entry process. for a patient infected by a coronavirus, his/her immune system will develop antibodies to bind and block the rbd of that specific coronavirus. out of all types of neutralization antibodies, igg has the longest lifetime in a person's circulating blood. it is used as a biomarker for the evaluation of patient's adaptive immune responses and the degree of recovery). in addition, as the major active ingredient, the concentration of sars-cov- s specific igg can be used as the indicator for the strength of the convalescent plasma [ ] [ ] [ ] [ ] [ ] . for these reasons, igg that binds specifically to sars-cov- s protein (especially the rbd) is the first biomarker that we aim to detect. the mechanism of igg detection is illustrated in figure (a). first, s protein is immobilized on the capillary inner surface through a poly-histidine mediation approach (see figures s and s (a) for details). then, s specific igg in the sample (such as serum) is attracted to the surface through immunosorbent reaction. finally, the hrpconjugated detection antibody is used to visualize the binding of the immobilized igg. to ensure detection specificity, a monoclonal detection antibody that binds specifically to the fc domain on human igg was used. according to the protocol in figure s (b), the entire assay was completed in minutes. to validate the feasibility of our assay, we selected three recombinant and humanized monoclonal anti-sars-cov- s antibodies as the positive control antibodies. the first antibody, cr , is a therapeutic human igg originally developed against the s protein of sars-cov. it was recently reported to have cross-reactivity towards the s protein of sars-cov- [ ] [ ] [ ] . the second and the third antibodies, d and d , are humanized chimeric iggs (the precursors of d and d were originally raised in mouse and rabbit, respectively) that are specific to the s protein of sars-cov. based on the preliminary results conducted at sino biological internally, they were also believed to have high binding affinities towards the s protein of sars-cov- . in order to mimic the actual clinical situations, we decided to use times diluted human serum as the solvent of the igg antibodies ( - are the typical dilution factors of serum in actual serological analyses). to evaluate the differences in antibody's affinity towards sars-cov- s and sars-cov s , we performed a side-by-side study with these two types of s proteins for all three clones of antibodies. the corresponding results are shown in fig. (b)-(d) . in general, the chemiluminescent intensities are proportional to the concentration of the spiked-in monoclonal antibodies. all these three antibodies are still detectable at . ng/ml with both types of s proteins. for d and d , the signal for both types of s proteins is very similar, indicating that the antibodies' binding affinity towards sars-cov- s and sars-cov s should be very similar (d may have a slightly higher affinity towards sars-cov- s than sars-cov s ). however, for cr , the signal for sars-cov- s is systematically lower than that for sars-cov s , indicative of a weaker affinity of cr towards sars-cov- s than sars-cov s , which agrees with recently published findings about cr 's binding ability , . the entire dynamic ranges of these three antibodies against sars-cov- s can be found in fig. s . the linear dynamic range in the log-log scale for cr , d , and d are - ng/ml, - ng/ml, and - ng/ml. the corresponding slope in the linear range is . , . , and . , respectively. as a negative control, a human igg isotype does not generate any detectable signal within the entire range of detection ( . - ng/ml). due to the narrow linear dynamic range and relatively low binding affinity, cr should not be used as the calibration standard of anti-sars-cov- s igg. the remaining two antibodies, d and d , are nearly the same in terms of the dynamic range and affinity towards sars-cov- s . however, d has a better specificity for sars-cov- s compared to sars-cov s . therefore, d may be a better candidate as a calibration antibody. in addition to anti-s igg, s protein itself may also be a marker in the prognosis of covid- . it may appear in blood for the patients who develop coronavirus viremia and the people who receive certain types of coronavirus vaccines (especially subunit vaccines). recent evidence indicates that the viral proteins may also exist in mucus samples such as the saliva. to detect the s protein, we employed a standard sandwich elisa format, as illustrated in fig. (a) , in which a monoclonal sars-cov- /sars-cov s rbd-specific antibody (d ) was used as the capture antibody and another s rbd-specific antibody (d ) was used as the detection antibody sars-cov- /sars-cov. to reduce the number of steps as well as the total assay time, we directly conjugated hrp molecules on the detection antibody. according to the protocol in figure s (c), the entire assay was completed in minutes. same as in the igg detection experiment, we performed a side-by-side study with the s proteins from sars-cov- and sars-cov. to mimic actual clinical settings, we used times diluted human serum as the solvent of the s protein, as we do not expect to see a high concentration of viral s protein in serum (or saliva). the entire dynamic range of the s detection assay is presented in fig. (b) . the linear dynamic range for sars-cov- s and sars-cov s is . - ng/ml and . - ng/ml with a slope of . and . (in the log-log scale), respectively. according to figures (b) and (c), the detection of sars-cov s appears to have a higher sensitivity than sars-cov- s . this is because both of antibodies (d and d ) used in this assay were originally raised against the rbd of sars-cov s . a higher sensitivity in detecting sars-cov- s protein may be achieved in the future with the antibodies specifically developed against the s protein (or s rbd) of sars-cov- . based on the studies in the previous sections, it is obvious that a good calibration standard (monoclonal human or humanized igg towards sars-cov- s protein) is essential for performing quantitative evaluation of the patient's immune response. high affinity antibodies are also essential for building a sensitive sars-cov- s elisa kit. however, due to nascence of sars-cov- there is no "gold standard" antibody that can be used in igg calibration or s antigen detection yet. consequently, it is urgent to find humanized antibodies with high binding affinities. unfortunately, traditional antibody evaluation approaches, such as plate-based elisa, bio-layer interferometry (bli), and surface plasmonic resonance (spr), suffer from long assay time ( - hours for plate-based elisa), small dynamic range ( orders of magnitude for plate-based elisa), and large sample concentration and consumption ( - µg/ml for bli or spr) [ ] [ ] [ ] . to address these problems, here we present a simple and rapid approach for the affinity assessment of monoclonal antibodies. the assay mechanism and the corresponding protocol are illustrated in figures (a) and s (d), respectively. our assay follows a single-step elisa format. same as in the igg detection experiment, recombinant s proteins were first immobilized on the supporting surface ( µg/ml), followed by the binding of igg. to reduce potential sources of errors, we directly conjugated hrp molecule with purified igg molecules (molar ratio igg : hrp = : , with the hrp conjugation kit from abcam). the antibodies were then diluted to six different concentrations ( - ng/ml with × serial dilution) and then applied to the s protein-coated elisa reactors. the immobilized igg can be quantified after a short minutes of incubation and times of rinsing. although the antibodies may not reach equilibrium by the end of the incubation, the quantity of the immobilized antibody (also the signal intensity) should still have a positive correlation with the affinity of the antibody. to compare the antibody's affinity towards the s protein of sars-cov- and sars-cov, we performed a side-by-side experiment for all antibodies under test . the antibodies were cr , d , d , and d . the first three antibodies were used as calibration standards in the igg detection experiments and d was used as the detection antibody in the s detection experiments. thanks to the simple protocol, our assay exhibits excellent intra-assay consistencies (see fig. s as an example), thus ensuring highly reliable measurements. as shown in figure (b), these four antibodies have very different affinities towards the s protein of sars-cov- . note that the data for ng/ml are not presented because the signal is not detectable for the three antibodies except d (the signal for d is very weak, see figure s ). for the points within the linear dynamic ranges, the signal intensities with the strongest antibody (d ) are - times higher than the weakest antibody (cr ). for example, the signal intensities for these two antibodies at ng/ml are and . , respectively. on the other hand, the lowest detectable concentration for d is ng/ml and the lowest detectable concentration for cr is ng/ml. this can be another evidence for the difference in antibody's affinity. these observations agree with several recently published experiment results and our own preliminary results [ ] [ ] [ ] and our measurements at sino biological (the equilibrium dissociation constant, kd, for cr and d is - nm and < nm, respectively, based on bli measurements. in contrast, as shown in figure (c) the antibodies' affinity towards sars-cov s can be very different from sars-cov- s . for example, cr 's binding affinity towards sars-cov s is stronger than its affinity towards sars-cov- s . conversely, d 's binding affinity towards sars-cov s is weaker than sars-cov- s . in addition, the pattern of calibration curves with sars-cov- s is significantly different from that with sars-cov s . while d , d , and cr 's affinities towards sars-cov- s vary significantly, they appear to be very similar to each other towards sars-cov s . although our current method allows us to rapidly evaluate the relatively affinity among all antibodies, it is unable to extract the exact value of kd. this problem can be resolved by introducing multiple calibration antibodies with known affinities. we have demonstrated a portable chemiluminescent microfluidic elisa system that is able to conduct sensitive detection and quantification of sars-cov- -related biomarkers in only - minutes using micro-liter sized sample volumes. the llod of ng/ml for igg in serum was achieved using the humanized chimeric antibodies as the model system. we also successfully characterized different antibodies and identified an antibody candidate, d , which can be used as the calibration antibody for quantitative evaluation of anti-sars-cov- s igg. this approach can also be extended to evaluation of therapeutic convalescent plasma. furthermore, we demonstrated sensitive detection of sars-cov- s protein (antigen) with the llod of . ng/ml. finally, we showed that our technology can be used as an alternative approach for rapid ( . minutes) screening and validation of monoclonal anti-s antibodies. our method requires only tens of nanograms, which is several orders of magnitude smaller than used traditional label-free methods (such as bli and spr), and will be useful in screening and selection of high affinity therapeutic neutralization antibodies and research-use antibodies , [ ] [ ] [ ] . we will continue to optimize our assays in multiple aspects. for igg detection, we will investigate more humanized or patient-derived sars-cov- s antibodies to identify optimal calibrators with a high binding affinity and a large linear dynamic range. the differences in the antibodies' affinity against monomeric s (most commonly used in antibody and vaccine development) and trimeric s (the natural conformation of s on sars-cov- virus) can also be explored . for s protein detection, we will improve the detection sensitivity, since the abundance of sars-cov- s may be very low in actual patient samples. based on our previous publications , , the llod can be greatly enhanced when we replace the hrp-conjugated detection antibody with a biotinylated detection antibody once it become available. for antibody affinity evaluation, we aim to further optimize the assay's protocol (such as adjusting the incubation time and rinsing time) and employ it to evaluate more therapeutic and research use antibodies. our approach also opens a door for other covid- related clinical or laboratory researches. for example, the diagnostic value of the covid- related biomarkers in serum and saliva (especially s specific iga, igm, and viral antigens such as the s and nucleocapsid (n) proteins) is currently under intensive evaluation , , . the igg detection method described in this work can be easily adapted to detect and quantify other types of antibodies such as igm and iga , , . the concept for sars-cov- s protein detection can also be adapted to detect other types of viral antigens (such as the sars-cov- n protein), as described in figure s . direct detection of viral antigens in patient samples such as serum and saliva may facilitate the rapid and cost-effective diagnosis of covid- , . finally, the microfluidic elisa platform can be used to study the neutralization efficacy of therapeutic antibodies (see figure s ), as well as for the recognition, evaluation, and phenotyping of natural and recombinant (fake) viral particles . and sars-cov s protein (black circles) in times diluted human serum. the averaged background is subtracted from all data points. the solid lines are the linear fit of the data in the loglog scale. the grey shaded area marks ×standard deviation of the background. the lower limit of detection (llod) for sars-cov- s protein and sars-cov s is . ng/ml and . ng/ml, respectively. (c) calibration curves for s proteins between . and ng/ml. the error bars are generated from duplicate measurements. different monoclonal humanized s specific igg against the s protein from sars-cov- (b) and sars-cov (c). the solid lines are the linear fit of the data in the log-log scale. the sample-to-answer time of this assay is minutes. (b)-(d) detection of s specific igg in times diluted serum, against the s protein from sars-cov- (red squares) and sars-cov (black circles). the calibration curves are generated with three different monoclonal humanized antibodies (cr in (b) error bars are generated from duplicate measurements. see also figure s for the entire dynamic range of cr , d , and d , and their respective lower limits of detection the authors thank the financial support from the department of biomedical engineering. the authors declare the following competing financial interest): m. k. k. o. and x. f. are cofounders of and have an equity interest in optofluidic bioassay, llc. key: cord- - lxc rj authors: soltan, mohamed a.; tsai, yun-long; lee, pei-yu a.; tsai, chuan-fu; chang, hsiao-fen g.; wang, hwa-tang t.; wilkes, rebecca p. title: comparison of electron microscopy, elisa, real time rt-pcr and insulated isothermal rt-pcr for the detection of rotavirus group a (rva) in feces of different animal species date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: lxc rj there is no gold standard for detection of rotavirus group a (rva), one of the main causes of diarrhea in neonatal animals. sensitive and specific real-time rt-pcr (rtrt-pcr) assays are available for rva but require submission of the clinical samples to diagnostic laboratories. patient-side immunoassays for rva protein detection have shown variable results, particularly with samples from unintended species. a sensitive and specific test for detection of rva on the farm would facilitate rapid management decisions. the insulated isothermal rt-pcr (rt-iipcr) assay works in a portable machine to allow sensitive and specific on-site testing. the aim of this investigation was to evaluate a commercially available rt-iipcr assay for rva detection in feces from different animal species. this assay was compared to an in-house rtrt-pcr assay and a commercially available rtrt-pcr kit, as well as an elisa and em for rva detection. all three pcr assays targeted the well-conserved nsp gene. clinical fecal samples from diarrheic animals (mainly cattle and horses) were tested. the percentage of positive samples by elisa, em, in-house rtrt-pcr, commercial rtrt-pcr, and rt-iipcr was . %, %, . %, . %, . %, respectively. the agreement between different assays was high ( . – %) in samples containing high viral loads. the sensitivity of the rt-iipcr assay appeared to be higher than the commercially available rtrt-pcr assay, with a limit of detection ( % confidence index) of – copies of in vitro transcribed dsrna. in conclusion, the user-friendly, field-deployable rt-iipcr system holds substantial promise for on-site detection of rva. rotavirus is classified as a member of family reoviridae, genus rotavirus. it is non-enveloped, - nm in diameter, and the genome length is approximately . kb. the genome is composed of segments of double-stranded rna and encodes six structural proteins (vp - , and ) and six non-structural proteins (nsp - ) (desselberger, ; zhou et al., ) . the virus capsid displays icosahedral symmetry and contains three layers, including an outer layer composed of the vp protein, with vp protein spikes, an inner or middle vp glycoprotein layer, and the core shell formed by vp (desselberger, ) . antigenic epitope analysis of the vp glycoprotein classifies the genus rotavirus into groups (a-h) (chandler-bostock et al., ; matthijnssens et al., ) . rotaviruses are characterized by relatively high antigenic and genetic diversity, as a result of accumulation of point mutations (genetic drift), and/or reassortment of genomic segments (genetic shift) (matthijnssens et al., ) . although host species barriers and host range restriction exist in rotavirus, reassortment can result in interspecies transmission, which also contributes to the diversity and evolution of rotavirus (martella et al., ; zhou et al., ) . rva is one of the main causative agents of diarrhea in young humans and animals (cho et al., ; zhou et al., ) . it is ubiq- uitous in the environment and relatively resistance to disinfectants. the adult animals are the main source of infection for newborn animals, and serological surveys revealed that - % of adult animals have an immune response against rva (schlafer and scott, ) . clinically healthy newborn animals may also shed rva, but the prevalence is lower compared to animals exhibiting diarrhea (kaminjolo and adesiyun, ) . studies have shown that detection of rotavirus in the presence of diarrhea is a significant finding in calf diarrhea (cho et al., ) . additionally, rotavirus has been shown to be significantly associated with liquid diarrhea in -to -day-old dairy calves (al mawly et al., ) and also in beef calves (cho et al., ) . there are many assays used for diagnosis of rva. historically, diagnostic laboratories routinely used electron microscopy (em) for detection of the virus. however, due to the costs of the microscope and its maintenance, as well as the required technical expertise, this method has lost favor. additionally, em lacks sensitivity, requiring ∼ viral particles/ml for virus detection (maes et al., ) . immunoassays (such as elisas) have replaced em as the diagnostic test of choice for detection of viral antigen because of their ease of use and speed of obtaining a result (desselberger, ) . most of the commercially available elisa kits are based on monoclonal antibodies against vp glycoprotein, which is expressed at a high level during infection. the vp glycoprotein is conserved among rva of different animal species, allowing cross-reactivity and detection of the virus from different hosts. therefore, the commercially available human rva elisa assays have been used for detection of rva in animals (bailey et al., ) . however, as a result of antigenic drift, there are several variants or genotypes of rva vp that have been described (mino et al., ) . differences among these vp genotypes influence the performance of various commercially available vp based immunoassays. therefore, diagnostic kit validation for each species is necessary. not all the assays have been validated for use in all animal species, and this is particularly true for horses (mino et al., ) . in fact, one human rva rapid immunoassay developed for patient-side use was shown not to work in detection of rva in horses (slovis et al., ) . recently, molecular techniques such as rt-pcr and real-time rtrt-pcr have replaced other diagnostic tests with the advantage of higher analytical sensitivity and specificity (slovis et al., ) . however, these types of assays are run in commercial or diagnostic laboratories and require expensive equipment and advanced technical skills, resulting in increasing costs to the producer, which leads to a reduction in the use of laboratory assays to support field disease investigations (izzo et al., ) . therefore, the aim of our investigation was to evaluate a recently available insulated isothermal rt-pcr (rt-iipcr) reagent set (pockit tm rotavirus a reagent set, genereach usa, lexington, ma, usa) with use of a portable pcr machine, which could potentially be used for point-of-need detection for rva in the feces of different animal species. the assay was compared to an in-house rtrt-pcr assay, a commercially available rtrt-pcr kit, a commercially available elisa, and em. a total of fecal samples from clinically affected animals, submitted to the university of tennessee, college of veterinary medicine, clinical virology laboratory, were used for comparison between different diagnostic assays. nucleic acids were extracted with an automated nucleic acid extraction system according to the manufacturer's instructions (taco tm mini, genereach usa) from all samples for molecular testing (in-house real-time rt-pcr, the commercially available real-time rt-pcr, and the rt-iipcr reagent set). briefly, mg of fecal sample was added to ml pbs and l of the supernatant was used for extraction. nucleic acids were tested immediately following extraction or were stored at − • c until tested. approximately five grams of fecal material were suspended in ml distilled water and centrifuged at , g for min. the fecal pellet was re-suspended in ml of distilled water and l of the suspension was mixed with l of % phosphotungstic acid (ph . ) in ml of distilled water. the mixture was then nebulized onto a carbon type-b, -mesh copper grid (ted pella, redding, ca, usa). the grids were examined by an electron microscope (zeiss auriga, university of tennessee, advanced microscopy and imaging center, knoxville, tn, usa). eighty four of the fecal samples were available for testing by em. a commercially available sandwich elisa, targeting the vp of human rva (premier tm rotaclone elisa kit, meridian bioscience, cincinnati, oh, usa), was used for testing the fecal samples, according to the manufacturer's instructions. eighty-five of the fecal samples were available for testing by elisa. the published nucleotide sequences of rva non-structural protein (nsp ), from different animal species ( bovine, equine, caprine, ovine and canine), were retrieved from genbank and aligned using mafft software. primers and a probe were designed, using the genscript online software, to amplify an area of bp. primer and probe sequences are shown in table . onestep real-time rt-pcr assay was performed using the superscript ® iii platinum ® one-step qrt-pcr kit (invitrogen, thermo fisher scientific, carlsbad, ca, usa) in a stepone tm real-time pcr system (applied biosystems, thermo fisher scientific, foster city, ca, usa). the proper concentrations of primers, probe and magnesium were optimized. the test was performed in -l total reaction volume containing l of the extracted nucleic acid, nm of each primer, nm of probe, mm magnesium and nm rox reference dye. following optimization, the cycle parameters were: • c for min, • c for min, followed by cycles at • c for s and • c for min. the assay sensitivity was determined and optimized by using standard rna, which was produced by cloning the pcr product from a positive clinical sample with the ta cloning ® kit with pcr tm . vector and one shot ® inv␣f' chemically competent escherichia coli (invitrogen, thermo fisher scientific, usa) according to manufacturer's instructions. the purified plasmids were sequenced to confirm correct orientation, linearized, and used as a template for synthesis of in vitro transcribed rna using the megascript t transcription kit (invitrogen, thermo fisher scientific, usa), according to manufacturer's instructions. rna copy numbers were calculated and a standard curve was generated from ten-fold serial dilutions of the rna standard at a range from × − to × − . the reproducibility of the assay was evaluated by calculation of intra-and inter-assay coefficient of variation (cv). the assay specificity was evaluated by testing dna and rna of different pathogens that are known to cause diarrhea in various animal species, including neorickettsia risticii (potomac horse the rt-iipcr test (pockit tm rotavirus a reagent set) targets the nsp gene. all the components of the rt-iipcr reaction were lyophilized in one tube. the lyophilized premix was rehydrated before reaction in l premix buffer b and l of sample rna were added to the mixture. the mixture was transferred to an rtube, centrifuged and tested by the pockit tm nucleic acid analyzer (genereach usa) as previously described (wilkes et al., ) . the assay specificity was evaluated as described for the in-house rtrt-pcr assay. double-stranded rotavirus ns rna was synthesized and used to determine sensitivity of the rt-iipcr reagent set. briefly, a plasmid containing a partial sequence of the nsp gene of the rva/horse-wt/zaf/eqrv-sa / /g p[ ] strain (genbank accession jq ) and the bovine rotavirus strain kj strain (genbank accession dq ) were used to generate positive and negative strand rna by in vitro transcription using the maxiscript ® t kit and megascript ® sp kit (life technologies, darmstadt, germany), respectively. residual dna was removed using the ambion ® turbo dna-free tm kit (life technologies). the two rna products were annealed to form double-stranded rna. residual single strand rna was removed by rnase a treatment. after phenol-chloroform extraction, integrity of the double-stranded rna preparation was confirmed by polyacrylamide gel electrophoresis analysis. concentration of rna was determined in a nanodrop spectrophotometer (nanodrop technologies, houston, tx, usa). serial dilutions of double-stranded rna were made in ng/l yeast trna. single use aliquots were stored at − • c. additionally, the sensitivity of the rotavirus rt-iipcr reagent set was evaluated by comparison with the commercially available rtrt-pcr assay using -fold serial dilutions of nucleic acid extracted from a positive bovine clinical sample. the nucleic acid samples were tested by the lsi vetmax tm triplex ruminant rotavirus & coronavirus real-time pcr kit (thermo fisher scientific, usa), according to manufacturer's instructions. the percentage of agreement between the different diagnostic assays and cohen's kappa coefficient were calculated using spss statistics software. the standard curve for the in-house rtrt-pcr assay generated from serially diluted standard rna was linear (slope = − . ) http://www- .ibm.com/software/analytics/spss/products/statistics/. the assay was linear over orders of magnitude and was able to detect as few as genomic equivalents per reaction. ( fig. ) and the coefficient of linear regression (r ) was . . the assay efficiency was estimated to be . %. the developed assay was sensitive and able to detect genomic equivalents of the target nsp gene per reaction. furthermore, it was specific, with no amplification detected from dna or rna from other pathogens known to cause diarrhea in animal species. the assay was reproducible with intra-and inter-assay cvs ranging from . to . and . to . , respectively. the limit of detection ( % confidence interval) for the rt-iipcr reagent set was and copies of dsrna, based on using log dilutions of in vitro transcribed dsrna containing target bovine and equine rotavirus sequences, respectively. the comparison between sensitivity of the rt-iipcr assay and commercially available rtrt-pcr assay using -fold serial dilutions of nucleic acid from a positive sample showed that the rt-iipcr reagent set had a fold increase in sensitivity. like the in-house assay, this assay was also specific, with no amplification in any of the samples used for specificity testing. there was variation in the percentage of positive samples detected by each assay. the rt-iipcr assay detected the most, while em detected the least. the overall percentages of positive samples by each diagnostic assay are shown in table . there was a significant difference in the number of positive samples detected with the in-house rtrt-pcr assay versus the other two molecular tests. these differences are evident when comparing the kappa coefficients (table ) . the em assay results showed substantial agreement in comparison to elisa results. this agreement becomes fair when compared to the rt-iipcr reagent set and the commercial rtrt-pcr kit, and moderate in comparison to the in-house rtrt-pcr assay. the elisa showed moderate agreement with the rt-iipcr reagent set and commercial rtrt-pcr assay but had substantial agreement with the in-house rtrt-pcr assay. a perfect agreement was found between the rt-iipcr reagent set and the commercial rtrt-pcr assay (table ) . comparison between the different assays showed a strong correlation for samples containing high viral loads (ct values ≤ ) (table ) . rva is one of the most prevalent causative agents of diarrhea in farm animals (athanassious et al., ; izzo et al., ; slovis et al., ) . it causes significant economic losses as a result of decreased weight gain, treatment costs, and high mortalities. therefore, development of a highly sensitive and specific test for point-of-need diagnosis would be beneficial to the veterinary practitioner and producer because delays associated with shipping samples to a diagnostic laboratory could be avoided, aiding in rapid control and prevention decisions (izzo et al., ) . there are many commercially available lateral flow immunochromatography assays (lat) that can be used for on-site detection of rva. one brand of lat showed favorable results in comparison to virus isolation and elisa (maes et al., ) . however, when compared to qrt-pcr, the results have been variable. the limitation of antibody-based tests for the detection of enteric pathogens is the requirement of high concentration of free antigen to generate a positive reaction, the free antigen is decreased significantly during the course of disease. therefore, these tests have lower sensitivity and could miss positive samples collected late in the course of clinical disease, when compared to rt-pcr (izzo et al., ; maes et al., ) . depending on the test used and species tested, these tests can also have low specificity (izzo et al., ) or may not work at all (slovis et al., ) . commercial elisa assays and other immunoassays, while easy to use and rapid for detection, are mostly designed for human use and care must be taken when applying these tests for animal use. the elisa used in this study was able to detect rva from three different animal species. the agreement between em and elisa was . %, which was similar to previously published reports (athanassious et al., ; benfield et al., ; reynolds et al., ) , and both of these methods lacked the sensitivity achieved with molecular assays, which has also been previously reported (izzo et al., ) . interestingly, three positive samples by elisa in this study were negative by em and all three molecular assays. the elisa assay absorbance values for these samples ranged from . to . , which indicates low viral load according to the manufacturer. these samples were considered false positives. false positive results are not uncommon for elisa assays as a result of the complex sample matrix (i.e. gut microbiota), which can increase the probability of cross-reactions. the type of antibodies (polyclonal versus monoclonal) used strongly affects their detection efficiency. the use of monoclonal antibodies is usually associated with increased specificity of the assay, but this also creates potential problems with regard to amino acid variability among the rotaviruses from different species another complication with detection of rotavirus is the fact that it can be detected in both healthy and diseased animals. establishing a causal relationship may be difficult without demonstration of classic histopathological changes (izzo et al., ) , particularly when using highly sensitive molecular detection methods. considering animals shed up to - virions/ml of feces during the acute phase of infection (izzo et al., ) , positive results obtained with less sensitive methods (em and elisa) are more likely to be associated with causality. in human medicine, elisa diagnosis is highly correlated with disease in rva infection (phillips et al., ). related to this concept is the idea that magnitude of viral shedding (based on ct value) can help determine disease etiology (phillips et al., ) . evidence of high viral load in samples tested gives the clinician more confidence that the virus is the cause of the disease process (izzo et al., ) . a high correlation between ct values ≤ and clinical disease was found in one human study that compared rva shedding in clinically healthy subjects versus those with diarrhea (phillips et al., ). we found a higher correlation between test methods when we evaluated samples with ct values ≤ . however, ct values from different protocols must be interpreted with care because the values may not equate to the same viral load per gram of feces (phillips et al., ) . this was actually seen in this study when comparing between the commercial rtrt-pcr assay and the in-house rtrt-pcr assay, particularly with the positive equine samples. the ct values obtained by the in-house rtrt-pcr assay were and . , compared to . and . , respectively, by the commercial rtrt-pcr kit. while we did not have additional equine samples to further examine this, these high ct values from equine samples are consistent with a previous report (matthijnssens et al., ) in which the same commercial molecular assay was also used for diarrheic samples from equine. ct values for positive samples from that study ranged from . - . . the authors attributed the high ct values to degradation of the viral rna in the samples or mild infection. while these are certainly possibilities, these results do raise questions about the sensitivity of this commercial rtrt-pcr assay in the diagnosis of equine rva. this disparity may potentially be attributed to mismatches in primer and probe binding areas. the designed primers and probe of the in-house rtrt-pcr assay contain several degenerate bases, in an attempt to avoid problems with base mismatches. however, as seen with the sensitivity testing and comparison between the assays, the in-house molecular test was less sensitive that the other molecular assays. based on the findings in this study when comparing ct values, it is important to note that the possibility of the significance of lower concentrations of rna may not be excluded. ct values can be affected by many factors, not just mismatches in primer/probe binding regions or inappropriate handling and storage of the sample, but also stage of the disease and the quality of the sample collected (izzo et al., ) . the commercial rt-iipcr assay performed in a portable pcr machine was shown to have higher sensitivity than the other molecular methods tested in this study. while it is possible some of the positive samples could have been false positives, we believe it is more likely associated with the increased sensitivity seen with this test, which was demonstrated with side-by-side testing, versus the commercial rtrt-pcr assay, of serial dilutions of nucleic acid from a clinical sample. high sensitivity of rt-iipcr assays has been consistently demonstrated in previous reports (ambagala et al., ; balasuriya et al., ; wilkes et al., ) . the higher sensitivity may be attributed to the performance of the reaction in gradient temperature that results from the thermal convective phenomena associated with this type of pcr (krishnan et al., ) . this allows primers and probes to anneal to sequences with mismatches (ambagala et al., ) . the commercial rt-iipcr assay incorporates a fluorescent hydrolysis probe, which increases the specificity of the assay, functioning more like a real-time pcr than a conventional pcr. however, rather than obtaining a ct value that requires interpretation, the portable machine detects the fluorescent signal before and after the reaction and automatically converts it into a positive or negative result. the numerical value for these fluorescent signals can be obtained from the machine for some determination about amount of virus present in the sample, but the sensitivity of the signal does not correlate as well as a threshold cycle value does for real-time pcr. the automated interpretation of the iipcr machine does however make the method easier to use without the need for advanced training. the portable machine is small and light weight and can be operated with a car battery. the three rt-pcr assays evaluated in the study were shown in general to have comparable performance for rva detection in fecal samples. the real-time pcr assays are excellent tools for diagnostic laboratories, and the rt-iipcr assay working in a portable pcr machine is highly sensitive and specific and shows promise for on-farm molecular detection of rva. m.a. soltan and r. p. wilkes declares no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. p.y. lee, y.l. tsai, c.f. tsai, h.f. chang, and h.t. wang are affiliated with genereach usa. however, this work does not alter our adherence to all the veterinary journal's policies on sharing data and materials. risk factors for neonatal calf diarrhoea and enteropathogen shedding in new zealand dairy farms a rapid field-deployable reverse transcription-insulated isothermal polymerase chain reaction assay for sensitive and specific detection of bluetongue virus detection of bovine coronavirus and type a rotavirus in neonatal calf diarrhea and winter dysentery of cattle in quebec: evaluation of three diagnostic methods equine rotaviruses-current understanding and continuing challenges rapid detection of equine influenza virus h n subtype by insulated isothermal rt-pcr (iirt-pcr) assay using the pockit tm nucleic acid analyzer comparison of a commercial enzyme-linked immunosorbent assay with electron microscopy fluorescent antibody, and virus isolation for the detection of bovine and porcine rotavirus diversity of group a rotavirus on a uk pig farm case-control study of microbiological etiology associated with calf diarrhea prevalence of major enteric pathogens in australian dairy calves with diarrhoea comparison of three diagnostic techniques for detection of rotavirus and coronavirus in calf faeces in australia rotavirus infection in calves piglets, lambs and goat kids in trinidad pcr in a rayleigh-benard convection cell evaluation of a human group a rotavirus assay for on-site detection of bovine rotavirus zoonotic aspects of rotaviruses vp -sequence-based cutoff values as a criterion for rotavirus species demarcation molecular characterization of equine rotaviruses isolated in europe in : implications for vaccination comparison of two commercial kits and an in-house elisa for the detection of equine rotavirus in foal feces diagnosing rotavirus a associated iid: using elisa to identify a cut-off for real time rt-pcr evaluation of elisa and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces prevalence of neutralizing antibody to the calf rotavirus in new york cattle infectious agents associated with diarrhoea in neonatal foals in central kentucky: a comprehensive molecular study understanding interobserver agreement: the kappa statistic rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction evidence for interspecies transmission and reassortment events we wish to thank all the veterinarians who submitted samples and made this work possible. key: cord- -qa uph authors: nan title: poster discussion session pds date: - - journal: allergy doi: . /all. sha: doc_id: cord_uid: qa uph nan objectives: since bradykinin is a short-lived, low-abundance mediator in the systemic circulation, the discovery of additional biochemical biomarkers correlating hae disease activity with contact system dysregulation may be useful for further elucidation of hae pathophysiology and pharmacodynamic therapeutic modulation of the contact system. results: activated pkal cleaves single chain high molecular weight kininogen to generate bradykinin and cleaved chain hmwk. cleaved -chain hmwk was measured in human plasma using both a semi-quantitative western blot assay with fluorescent detection (licor) and a novel elisa with a capture antibody that specifically binds -chain hmwk. the western blot assay was previously used to monitor pharmacodynamic activity in hae patients treated with lanadelumab, a fully human antibody inhibitor of pkal that is in clinical development for hae attack prophylaxis. lanadelumab inhibited -chain hmwk generation following contact system activation in vitro, confirming that -chain hmwk is a product of pkal activity. plasma -chain hmwk levels from hae patients during or between attacks were compared to that from healthy volunteers using both the western blot assay and elisa. roc curve analyses with both methods suggest that -chain hmwk is a trait-specific biomarker capable of differentiating hae patients from healthy volunteers. the elisa was able to differentiate samples from hae patients collected during an attack from those collected between attacks with moderate sensitivity and specificity (c-statis-tic= . ). a comparison of -chain hmwk levels in citrated plasma versus plasma that contains protease inhibitors (scat plasma) provides estimates of endogenous versus ex vivo activation. the measurement of -chain hmwk using the specific assays described may find use in further dissecting the role of the contact system in disease pathology, to identify additional indications for modulators of this pathway, and to investigate therapeutics targeting the contact system. | g protein coupled receptor kinase (grk ) regulates endothelial permeability induced by bradykinin | pharmacokinetics (pk) and pharmacodynamics (pd) of c esterase inhibitor of chronic urticaria challenges most commonly identified were the following: time of onset of disease; frequency/duration of and provoking factors for wheals; diurnal variation; occurrence in relation to weekends, holidays, and foreign travel; shape, size, and distribution of wheals; associated angioedema; associated subjective symptoms of lesions; family and personal history regarding urticaria, atopy; previous or current allergies, infections, internal diseases, or other possible causes; psychosomatic and psychiatric diseases; surgical implantations and events during surgery; gastric/ intestinal problems; induction by physical agents or exercise; use of drugs; food allergies; relationship to the menstrual cycle; smoking habits; type of work, hobbies; stress; quality of life and emotional impact; previous therapy and response to therapy, and previous diagnostic procedures/results. we included all of these aspects in our guide and as a result we developed a chronic urticaria check list. conclusions: our guide of clinical history for chronic urticaria (gur) contributes to have an easy tool in order to achieve a better diagnosis and evaluation of chronic urticaria. | clinical and diagnostic features in acquired cold urticaria patients in a coruña sanitary area, spain physicians and dermatologists/allergists; c/sa patients were more likely to visit dermatologists/allergists ( % vs. %) and less likely to visit general physicians ( % vs. %) than european patients. emergency room visits due to cu were more common in c/sa ( %, mean [sd] number= . [ . ] ) than europe ( %, mean [sd] number= . [ . ] ). conversely, hospital admissions due to cu were more likely to occur in europe ( %) than c/sa ( %), but the average (sd) number of admissions among those hospitalised was greater in c/sa ( . [ . ] vs. . [ . ]). variations were seen in subregion comparisons. mean (sd) overall wpai scores were . ( . ), . ( . ), . ( . ), and . ( . ) for absenteeism, presenteeism, work productivity loss, and activity impairment, respectively; patients in c/sa reported a higher rate of impairment (range, %- %) on all domains compared with patients in europe. conclusions: cu is associated with substantial hru and work and activity impairment in both europe and c/sa. general physicians should be considered key members of the treatment team in the care of patients with cu in these regions. objectives: cu patients (n= ) received monthly subcutaneous injections of omalizumab for up to six months. urticaria-related symptoms were assessed by both the urticaria control test (uct) and the chronic urticaria quality of life score (cu-q ol). peripheral blood was drawn prior to each injection for determining the concentration-dependent reactivity of patients' basophils to specific anti-fceri and unspecific fmlp stimulation by basophil activation test. furthermore, the impact of anti-ige treatment on ige-bearing cell populations was characterized by flow cytometry analyzing the surface expression of both fceri (e.g. on monocytes, dendritic cells, basophils) and the low-affinity receptor for ige, fcerii (e.g. on b cells, eosinophils). results: anti-ige treatment of cu patients significantly improved clinical symptoms of cu already after the first injection as evaluated by cu-q ol and uct, the latter of which correlated with an increase of basophil numbers and a decrease of basophil surface bound ige. of note, cell-bound ige on fcerii-expressing cells was not altered. furthermore, while clinical amelioration also was accompanied by reduced fceri expression on basophils, the basophil responsiveness to anti-fceri stimulation increased in % ( / ) of treated patients. in contrast, ige-independent activation of basophils by fmlp was unchanged. conclusions: clinical improvement of cu patients treated with omalizumab is associated with characteristic immune alterations in basophils, like rapid, cell-specific reduction of surface bound ige and fceri-expression as well as normalization of basophil responsiveness to fceri-stimulation. while our findings might help to better understand the mode of action of anti-ige therapy in cu, they also can shed new light on the pathomechanisms underlying cu. | omalizumab in patients with severe active chronic spontaneous urticaria (csu) heavily treated with corticosteroids and cyclosporine introduction: increased levels of blood d-dimers (d-d), the byproducts of fibrin degradation, is linked to the severity of chronic spontaneous urticaria (csu) and to poor response to antihistamines h (ah ). omalizumab (oma) is a human monoclonal anti-immunoglobulin-e antibody registered as an add-on treatment of csu in adults and adolescent (≥ years old) and with insufficient response to ah . the sunrise study assessed the efficacy of omalizumab on csu symptoms and the correlation between d-d levels and response over time to treatment with oma to explore its potential predictive value. objectives: sunrise was a french prospective non comparative phase study. included patients had to have been diagnosed with csu for at least months, be resistant to ah treatment, and have a uct score (assessed by patient over the last weeks, values from (maximal disease)to (full control)) < , indicative of a poorly controlled disease. the widely used uas score (assessed by patients over week which captures intensity of pruritus and number of hives, values ranging from (no disease) to (severe disease)) was further used to evaluate the proportion of patients achieving a well controlled disease(uas≤ ). all patients received mg oma by sub-cutaneous injections at day , weeks (w) and . blood levels of d-d were assessed (turbidimetric immunoassay) at d , w and w . response to treatment was evaluated at w by means of the uas . results: median level of d-d assessed at d in patients was increased at baseline ( ng/ml, extremes - ) and normalized as early as w reaching ng/ml ( - ) at w . correlation between d-d concentration and uas score at w was weakly positive (spearman coefficient . ). among the patients with a very high baseline de d-d level (> ng/ml) were responder (uas ≤ ) at w . conclusions: baseline d-d levels were increased in more than half of patients of this study in line with relevant literature. a fast normalization was observed with oma as early as w of treatment. d-d levels at w were weakly correlated with uas . subgroup analyses may help to better understand the link between d-d and clinical response, as these preliminary results do not yet allow the predictive use as a biomarker. the sunrise study explored for the first time in a prospective way the impact of oma on dd and found weak correlations were measured between dd level and response to oma. further studies will be needed to evaluate its predictive value for response. (crp, esr, il- , il- , il- , ccl /mcp- ), and the disease severity in patients with chronic spontaneous urticaria introduction: pru p is the primary sensitizer of some fruits and responsible for severe reactions in the mediterranean area. sublingual immunotherapy (slit) using peach extract enriched in pru p (prup -enriched-slit) brings a new perspective to treat patients with reactions to peach considering that currently the treatment of the allergy to peach is based on avoidable ingestion of fruit. we performed a pilot study to examine the immune modulation by slit in patients with peach allergy over a -year treatment period. objectives: we aimed to evaluate the effect of the slit during one year in patients with peach allergy. we analysed the capacity pru p enriched-slit to modulate immune response, from a th to th response with increases of treg cells. we studied three groups of subjects: peach allergic patients who received prup -enriched-slit for year, peach allergic patients non treated, and healthy controls who tolerated peach. monocyte-derived dendritic cells (dcs) maturation, peripheral blood mononuclear cell phenotype and lymphocyte proliferation after prup stimulation were assessed by flow cytometry from samples obtained before, and , and months during slit. results: we found statistically significant differences in dcs activation and maturation between allergic patients and controls at the basal state. when we analyzed the effect of prup -enriched-slit over time, we found a significant reduction of activation and maturation markers (ccr , cd , cd , cd and cd ) at the first month of treatment that was maintained after year. concerning lymphocytes, we observed a significant decrease of effector cells immune cells. recent studies showed that fructo-oligosaccharides (fos) increase the efficacy of oral immunotherapy (oit) in a mouse model for cow's milk allergy, however, the mechanism is unknown. objectives: investigating the effect of oit+fos on the effector response and the process of tolerance induction. methods: female c h/heouj mice ( - weeks old, n= /group) were sensitized to the cow's milk protein whey ( mg in pbs, intragastrically (i.g.)) with cholera toxin ( lg) once a week for weeks (d -d ). the mice received a diet with % fos or a control diet from d -d . oit ( mg in pbs or pbs) was provided days a week for weeks (d -d ). intradermal (i.d.) and i.g. challenges were performed to measure the acute allergic response. serum, bone marrow, caecum content, small intestines and mesenteric lymph nodes (mln) were collected at d , d and d . spleen-derived t cell fractions (whole spleen, cd +cd -and cd +cd +, using macs) were transferred to na€ ıve recipient mice at d . the recipients were sensitized and challenged as described for the donor mice. conclusions: this study shows that oit+fos results in an early induction of functional tregs and a reduction of mast cell degranulation upon challenge. the latter may be caused by inhibition of mast cell activation by galectin- and/or butyric acid. moreover, the effect of fos on bone marrow suggests possible epigenetic changes reducing development of mast cells. further research is needed to investigate if this approach may be of potential value to treat food allergies. | safety and feasibility of slow low-dose oral immunotherapy sugiura s; kitamura k; tajima n; takasato y; kato t; tajima i; ono m; tagami k; sakai k; nakagawa t; ito k aichi children's health and medical center, obu, japan introduction: slow low-dose oral immunotherapy (sloit) is an ongoing clinical trial conducted in our institute (umin registry number ). this is a type of oral immunotherapy with a low dose antigen increased very slowly. objectives: to evaluate the safety and feasibility of the protocol. results: sloit enrolled the patients who were diagnosed as severe egg, milk or wheat allergies, with a threshold dose of g (or ml) or less in the oral food challenge (ofc, -minutes boiled egg white, whole milk, udon noodle). subjects were divided into two groups, intervention (sloit) group or elimination (control) group, based on their preference. sloit group began to ingest / to / amount of the final dose of the ofc based on the severity of provoked symptoms. intake was continued everyday with an increasing dose less than . times/ month, expecting a times increase from the starting dose after months. feasibility was evaluated based on the proportion of patients who complied the protocol. safety was evaluated based on the frequency of immediate allergic reactions observed in the programmed intakes. fifty-nine patients were enrolled from april to december , and of them (egg: , milk: , wheat: ) preferred the sloit group. among them, patients ( . %) dropped out from the study protocol because of provoked allergic symptoms (n= ) or the other personal reasons (n= ). among a total of ingestions by patients who continued the protocol over months, mild symptoms were observed times ( . %), to which rescue medicine such as oral antihistamines was used in cases ( . %). no one needed an emergency visit or an adrenalin auto-injector. low threshold dose at the initial ofc ( . g or less, n= ) was associated with higher proportion of provoked allergic symptoms in the protocol (low threshold: . %, non-low threshold: . %, p=. ). the level of specific ige titer was not associated with the safety. conclusions: sloit protocol has sufficient safety and feasibility, but low threshold patients should be monitored closely. the efficacy of this protocol is being evaluated by an ofc after - months of the treatment, and the overall data will be presented after march . objectives: the aim of this phase i, randomized, non-controlled, multicenter, opened, with parallel groups clinical trial, is to evaluate the safety and tolerability of subcutaneous immunotherapy (scit), in a polymerized mixture ( / ) depot presentation. patients with rhino-conjunctivitis polysensitized to olea europaea/ phleum pratense received a schedule consisting of two weeks of initiation with three weekly injections; or a program comprising two administrations in the same day separated by minutes. both treatments continued with a maintenance period of three months with a monthly administration. the primary outcomes are the number, percentage, and severity of adverse reactions. secondary endpoint included subrogate efficacy parameters evaluation: changes in immunoglobulin titers (specific ige, igg and igg ) and changes in cutaneous reactivity at different concentrations. systemic reactions were registered, representing . % of the included patients: one grade , described as general discomfort plus dizziness and one grade i, such as rhinoconjunctivitis. there were no local reactions. all were classified as of mild intensity and took place with the cluster schedule. symptomatic treatment was not required. conclusions: both schedules with polymerized mixture of phleum pratense/olea europaea, ( / ), presented a good safety and tolerability. a statistically significant decrease in cutaneous reactivity to olive and grass allergens was observed after immunotherapy. a | design of the pivotal phase iii study to assess the efficacy and safety of subcutaneous hdm allergoid immunotherapy in patients with hdm induced allergic rhinitis/ rhinoconjunctivitis introduction: in order to comply with ema guidelines on development of allergen immunotherapy products, a clinical development program was started to obtain full marketing authorization for a allergoid subcutaneous immunotherapy (scit) product for the treatment of house dust mite (hdm) allergy. previously, the safety and tolerability of increasing doses of this allergoid scit product was evaluated in patients with hdm-induced allergic rhinitis/rhinoconjunctivitis (arc) [eudract - - ] . no safety or tolerability issues were identified for doses up to aueq. subsequently, a dose-finding study to identify the optimal, i.e. effective and safe dose in hdm arc with or without concomitant asthma was performed [eudract - - ; pfaar, allergy ] . this study demonstrated a dose response relationship with doses of aueq ( . ml of aueq/ml) up to aueq ( . ml of aueq/ml) showing significant improvements compared to placebo. the current pivotal phase iii study [eudract - - ] aims to confirm safety and efficacy of this hdm allergoid scit product at a dose level of aueq/ml ( . ml) compared to placebo after one year of treatment in patients with hdm-induced arc. objectives: the current study is a multi-center ( clinical study centers in european countries) randomized, double-blind, placebocontrolled, parallel-group study in adult patients, with moderate to severe hdm induced arc with or without mild to moderate persistent asthma. the primary outcome of the study is the difference in mean combined symptom and medication score (nasal symptoms only) (csms (n)) between aueq/ml allergoid scit and placebo treatment, assessed during the last weeks of the approximately year treatment period. results: patients from centers, (mean age . years) have been included and analyzed. the . % are men, . % presented associated asthma. a large majority of patients have received subcutaneous sit ( . %), and . % of them containing a single allergenic source. . % in polymerized formulation and . % in depot. accelerated schedule has been the most prescribed ( . %), followed by clustered one ( . %). from the patients who have completed the study, . % of them improved from persistent to intermittent rhinoconjunctivitis (p<. ) and . % from moderate/severe to mild intensity (aria) (p<. ). moreover, % of asthmatic patients at baseline, did not have any bronchial symptoms after -year treatment. the improvement in quality of life was possible to be analyzed in patients. mean values in rqlq questionnaire (total score) decreased from . to . points ( . % of score reduction) in final visit, reflecting a statistically significant improvement (p<. ). mean value of treatment satisfaction was . (sd= . ) and . (sd= . ) for physician and patients respectively. for safety assessment, out of analyzed patients, only systemic reactions were reported in patients ( . %). seven of them were classified as grade i, and one as grade ii according to eaaci grading system. strasbourg is a m chamber, located into the university hospital of strasbourg at less than to minutes to the intensive care unit. one of the characteristics of this unit is that the maximum parameters are controlled: temperature, relative humidity, ventilation rate, particles number and particles size, concentration of airborne of der p and airborne voc. mite allergens extract were nebulized through a nebulizer. airborne der p concentrations were sampled using glass fiber filters and measured with an elisa assay. particles number and particles size were monitored continuously during nebulization, using particles counters distributed inside the exposure room. the cleaning process was also controlled and validated. objectives: to validate the technical parameters of the environmental exposure chamber (eec) of strasbourg with mite allergens. results: the reproducibility was excellent for the indoor temperature, relative humidity and airflow ( cv interassay < %). three concentrations of der p were measured: , , ng/m (n= ). for all concentrations, the cv intra-assay of airborne der p was ae . %, the interassay was less than %. for the particle size . - and - lm, the cv interassay was and %, respectively (n= ). the cv of the mmad was . % (n= ). no measurable airborne der p was detected in the toilets and the medical supervision room (n= ) neither in the exposure room minutes after the end of the allergen exposure (n= ). no airborne voc was measured in the chamber and the other rooms of the clinical unit (n= ). there was no significant change in particles size and number when to persons entered the room (n= ). introduction: pathogenesis of systemic sclerosis (ssc) includes vasculopathy with endothelial dysfunction which is considered to be one of the earliest changes in the pathogenesis of ssc. several biological molecules, including e-selectin (e-sel), inter-cellular adhesion molecule (icam- ), endothelin (et- ), von willebrand factor (vwf) and interleukin (il- ) have been associated with endothelial activation. objectives: we aimed to determine if these vascular biomarkers are associated with distinct capillaroscopic ssc patterns and/or more severe disease in ssc patients. results: correlations between serum levels of all vascular biomarkers were good to moderate and statistically significant, with r indices varying between . and . , the only exception being et- which did not correlate with e-sel. good correlations (r . to . ) were also found between all biomarkers and crp. patients with severe vasculopathy, as reflected by the nfc "late" pattern, had higher levels of il- (median . vs . pg/ml, p=. ), et- (median . vs . pg/ml, p=. ), vwf (median vs iu/ml, p=. ) and e-sel (median . vs . ng/ml, p>. ), compared to patients with nfc "early" or "active" patterns. there was a significant, negative correlation between lung transfer for carbon monoxide (dlco) and e-sel, icam- (both p<. ) and vwf (p=. ). et- was higher in patients with more severe disease (dcssc, patients positive for anti-topoisomerase antibodies and patients with a history of digital ulcers-all p<. ). conclusions: serum endothelial activation biomarkers are elevated in patients with more severe ssc-associated vasculopathy and correlate with serum crp. together with nfc data they may be used for assessing vasculopathy severity in ssc. objectives: here we present the imagination findings of eye involvement in a family whose members have mws. method: clinical data was collected during the course of ongoing patient care. results: we evaluated the clinical features of patients who were referred to our center. the median age of the patients was years (range: - years). the ratio of females /males was . ( / ). all patients had arthritis with exacerbation on exposure to cold and ocular involvement, mostly in the form of conjunctivitis and far less other forms. the median age of onset of ocular involvement was years ( - y). chronic eye damage were detected in three patients. corneal involvement and clouding was detected in four patient. two conclusions: in this study, it has been shown that eye findings related to mws can vary from conjunctivitis to severe uveitis. we want to emphasize that ocular involvement in mws should be carefully assessed, since it can lead to visual impairment. | antiretroviral activity of the conjugates '-azido- 'deoxythymidine and derivatives of , -diacylglycerides case report: aids epidemics remain the critical problem due to both their emergent and long development. treatment of aids with azt reduces p antigenemia, increases cd + lymphocyte counts, reduces the frequency and severity of opportunistic infections and prolongs life. however, zidovudine and other dideoxynucleotides do not decrease the ability to isolate hiv from pbmc, and in addition, these drugs are very toxic. this phenomenon is caused by insufficient inhibition of hiv due to low levels of nucleoside kinases in ccr -positive cells, including in macrophages, which are a major reservoir of hiv. potential advantages of these liponucleotide prodrugs include: greater in vivo efficacy and lower toxicity due to a greater delivery to monocytes/macrophages, ability to bypass the initial anabolic phosphorylation due to the presence of the phosphorous center in the structure, and the prospect of improved pharmacokinetics and prolonged intracellular persistence. the aim of this work is the study of cytotoxicity and anti-hiv activity of glycerolipids derivatives of azt. evaluation of the cytotoxicity of azt and test compounds was per- | bone mineralisation defect in patients with hax- deficiency and osteopenia (z score <À ) in patients. bone mineralisation defect was found in all female patients while only one male had osteopenia (table ) . conclusions: in this study, a significant decrease ( . %) of bone mineralization was observed in patients with hax- deficiency. female patients were found more prone to bone mineralisation defect. to conclude on this subject, more studies are needed with large number of patients having not only hax- deficiency but also ela- mutations. gene mutation age ( introduction: bronchopulmonary diseases are kept as one of the actual problems of the pediatricians. nevertheless fulfilment of several scientific works on study of these diseases, presently we meet the complication of the respiratory diseases, recurrency, changing to the chronical type. objectives: the main purpose of our work to study the cytokine status and substance p, mutual connection they in frequently ill chil- results: in fic with respiratory diseases in the acute period of the disease increase of levels proinflammatory cytokines il- beta, il- , il- , tnf-alpha and substance p,decrease of levels il- and ifngamma was marked. clinical remission in these children is not accompanied by normalization of cytokine status and substance p. the high level of proinflammatory cytokines and substance p testifies to proceeding of inflammatory process that is possible connected with persistence of the infections agent. acute decrease in level of cytokines il- and ifn-gamma in these children,most likely, is caused by the presence of a immunodeficiency of cellular type. conclusions: in this connection it is necessary to carry out an adequate therapy of fic with arvi. introduction: atopic diseases are known to be characterized by a t helper (th) -skewed immunity. th -skewed immunity at birth, th -associated cc chemokine ligand (ccl)- , appears to be associated with high total ige levels but not of allergic outcomes later in life. the prevalence of asthma increases with a rapid upward trend after age ; however, there are few studies addressing the changes of ccl chemokine levels during infancy related to the development of atopic diseases in early childhood. objectives: we investigated children followed up regularly at the clinic for years in a birth cohort study. the levels of th related chemokine ccl were quantified in cord blood and age . by multiplex luminex kits. specific immunoglobulin e antibodies against food (egg white, milk, and wheat) and inhalant allergens (d. pteronyssinus, d. farina, and c. herbarum) were measured at months as well as . , , and years of age. results: a total of pairs of ccl chemokine levels from birth to age . were recruited in this study. k-means clustering was performed using r software and package mfuzz and this resulted in groups of ccl chemokine levels that declined from around to pg/ml (cluster a, n= ), from around to pg/ml (cluster b, n= ) , and raised from around to pg/ml (cluster c, n= ) . in children with raised ccl chemokine levels appeared to be associated with a higher prevalence of house dust mite sensitization at age . . furthermore, raised ccl levels during infancy were significantly associated with higher risk of asthma at age (p=. ). conclusions: raised ccl chemokine levels during infancy appear not only to be associated with an increase in the prevalence of house dust mite sensitization but also the risk of asthma in early childhood. | serum periostin is "not" a biomarker for pediatric asthma suzuki n ; hirayama j ; nagao m ; kameda k ; kuwabara y ; kainuma k ; ono j ; ohta s ; izuhara k ; fujisawa t mie national hospital, tsu, japan; shino-test corporation, kanagawa, japan; saga medical school, saga, japan introduction: periostin is a matricellular protein induced by type helper t-cell cytokines, expressed by airway structural cells and is thought to contribute to airway remodeling and progressive lung function decline in severe asthma in adults. we sought a possible clinical utility of serum periostin in children with asthma. objectives: we recruited volunteer children and adolescents (age range, to years) who were otherwise healthy except for allergic diseases including bronchial asthma. allergic diseases were classified with isaac questionnaire and serum levels of periostin were measured with elisa. abstracts | results: a total of volunteers were enrolled. among them had no allergic diseases (na), had only allergic rhinitis (ar) and the rest of had bronchial asthma (ba) and/or atopic dermatitis (ad) . serum levels of periostin in na younger than were significantly higher than the older counterpart and the levels in children < years old were similar across each age. data distribution of serum periostin in ar were very similar with that in na and we defined na and ar groups as reference population. reference values, geometric mean (+ geometric sd range), for serum periostin were ( ) and ( ) ng/ml in children/adolescents < and ≥ , respectively. there were no gender differences in serum periostin in the reference population. we then compared the serum levels of periostin in ad and/or ba with the reference group and found that serum periostin in ad and ad+ba, not ba, in < years old were significantly higher than the reference group. in addition, severity of asthma had no association with serum periostin levels in age group of < . on the other hand, the levels in ba aged ≥ were slightly higher than non-ba (statistically significant). conclusions: serum periostin is physiologically high in children and adolescents, possibly in growing age, and the levels are elevated in those with ad, not ba. serum periostin is "not" a useful biomarker for pediatric asthma. | clinical, biochemical and radiological factors for response to aspirin desensitization in patients with aspirin exacerbated respiratory disease-pilot study introduction: aspirin desensitization is regarded as an effective and well-tolerated therapy for patients with aspirin exacerbated respiratory disease (aerd). despite many studies investigating the pathophysiology of aerd, the underlying mechanism responsible for the beneficial effects of aspirin therapy has not yet been clarified. the aim of the study was to evaluate the influence of aspirin desensitization on clinical, biochemical and radiological changes in aerd individuals. objectives: this is a prospective study of twenty-one aerd patients subjected to one-year aspirin desensitization. all participants were hospitalized three times over the period of one year. at baseline and during each follow-up visit ( nd and th month) blood, urine, induced sputum (is) and nasal lavage (nl) were collected from all participants. the acquired material was processed in order to evaluate ) is and nl cell count ) concentrations of is and nl eicosanoids ) leukotriene e (lte ) in urine and ) periostin in blood. additionally, participants underwent a ct scan of the paranasal sinuses at baseline and after months of aspirin therapy. the lund-mackay score values were compared. for statistical analysis, summary statistics and repeated measures anova with post-hoc test were applied. results: twenty participants completed a one-year aspirin therapy. there was a statistically significant decrease in the number (p=. ) and percentage (p=. ) of eosinophils in is between baseline and after aspirin desensitisation. significant increase of urine lte in the course of aspirin therapy was observed (p=. ). the levels of prostaglandins and leukotrienes in is and nl as well as blood periostin level and the differential cell count in nl did not change during aspirin desensitization. in ct scan images the regression of the lesions in paranasal sinuses was observed in %, the worsening in % and in % of patients no changes were noted. in is count of eosinophils in aspirin-sensitive individuals, which may potentially be used in disease monitoring and tailoring asthma therapy. aspirin desensitization did not lead to significant changes in local eicosanoid levels in is and nl. blood periostin level is not a good marker for patient's response to aspirin desensitization. introduction: one of the main severe asthma phenotype is the "severe eosinophilic" or "eosinophilic refractory" asthma, for which novel biologic agents are emerging as therapeutical options. in this context, blood eosinophils count are one of the most reliable biomarker. objectives: the aim of our study is to evaluate the performance of a point-of-care peripheral blood counter in a clinical setting of severe asthmatics. seventy-six patients with severe asthma were evaluated, for blood cells count, by both a point-of-care and a standard analyser. results: the inter-and intra-assay variation was acceptable for leukocytes, neutrophils, lymphocytes and eosinophils. this was not the case of monocytes and basophils. a significant correlation between blood eosinophils assessed by the two devices was found (r = . , p<. ); similar correlations were found also for white blood cells, neutrophils and lymphocytes. the point-of-care device showed ability to predict blood eosinophils cutoffs used to select patients for biologic treatments for severe eosinophilic asthma, and the elen index, a composite score useful to predict sputum eosinophilia. introduction: over years ago there was revealed periostin should play an important role in pathogenesis of allergic inflammation including asthma and processes of tissue remodeling and fibrosis. its expression has been observed in the thickened basement membrane as well as in serum of asthmatic patients. thus, measuring of periostin serum levels may shed some light on these elusive asthma features. periostin has already demonstrated a convenient value in clinical studies as a companion diagnostics for lebrikizumab, tralokinumab or omalizumab treatment. however, to date, the changes of periostin serum levels following asthma therapy except for inhaled corticosteroids remain unclear. objectives: to emphasize this issue we have collected clinical and laboratory data including serum periostin of asthma patients ( males/ females) in a cross-sectional study. all patients were treated either by conventional therapy comprising inhaled corticosteroids (n= ) or by inhaled corticosteroids with additional biological therapy (omalizumab) (n= ). asthma phenotype, control, complications, comorbidities and other available biomarkers have been assessed and statistically analysed. results: we have observed a weak but statistically significant correlation between serum periostin and total ige levels (p=. ) and absolute eosinophil count (p=. ). association between periostin and total ige had nonlinear character (p=. ), and was expressed particularly in non-severe asthma patients without omalizumab treatment (p=. ). despite mutual correlation between serum periostin a total ige levels, both biomarkers showed different reaction on asthma treatment. multivariate analysis demonstrated, that only periostin levels, in contrast to all other assessed biomarkers, were significantly decreased in severe asthma patients treated by omalizumab (p=. ). this relationship was independent of asthma control (assessed by asthma control test -act), exacerbation rate, hrct or spirometry measurement results or comorbidities, except chronic rhinosinusitis with nasal polyps (crswnp) (p<. ). conclusions: we demonstrate, serum periostin levels are dependent on therapy, thus it may contribute not only to asthma phenotype stratification, prediction of treatment responsiveness, complications such as remodeling but probably more precise monitoring of therapy effect too. | usefulness of serum pteridines as a biomarker for childhood asthma kasuga s ; hamazaki t ; fujitani h ; fujikawa s ; niihira s ; shintaku h department of pediatrics, osaka city university graduate school of medicine, osaka, japan; the national institute of special education, tokyo, japan introduction: reliable and stable biomarker of airway inflammation is essential to determine intensity of asthma treatment. exhaled nitric oxide (feno) has been introduced to assess useful marker of airway inflammation but it fluctuates depending on steroid inhalation. nitric oxide is produced by nitric oxide synthase which requires tetrahydrobiopterin (one of pteridines) as a cofactor. objectives: to assess pteridines as a biomarker of childhood asthma control. results: asthmatic children were recruited for periodical asthma checkup program in japan to assess asthma control status by using objective questionnaire, respiratory function tests, airway resistance, feno, and serum pteridine levels. serum pteridine levels were measured by high performance liquid chromatography. total japanese children ( - years) were participated in this program. children who have no asthma attack over three years were divided as remission group to evaluate the long-term asthma control. the other children were divided as asthma group. furthermore, we divided asthma group for three groups by childhood asthma control test (c-act) scores to evaluate the short-term asthma control. and we had age matched children as control group. pteridine levels tended to decrease in patients who showed higher feno in asthma group. asthmatic children showed lower pteridine levels than other groups. there are significant differences between control group and remission groups and asthma groups (p<. , tukey's honestly significant difference test). but there are no significant differences between the three groups in asthma group (well-controlled group, partly-controlled groups and uncontrolled groups) which were divided by c-act scores. the low concentration of serum pteridines in children with stable asthma may indicate poor long-term control of asthma. but that not indicate poor short-term control of asthma. since pteridine biosynthesis pathways were suppressed by th mediated immune response, these results suggest that th mediated immune response was dominating the th response in asthmatic children. therefore, serum pteridines could be a novel biomarker of stable asthmatic children. introduction: asthma is a syndrome with chronic airway inflammation. the goal in the treatment of asthma is to control the inflammation. however, there has been a scarcity of useful noninvasive tests for monitoring the airway inflammation in clinical setting. metabolites of the eicosanoids pathways in induced sputum of the patients with asthma could be valuable biomarkers that can reflect the airway inflammation. objectives: to investigate eicosanoid metabolites and to find out their phenotypic differences, induced sputum supernatants from patients with refractory asthma, patients with controlled asthma, and normal control subjects were analyzed by using liquid chromatography tandem mass spectrometry. in addition, we evaluated the relationship between asthma exacerbation and eicosanoid metabolites. results: among metabolites which were measured, metabolites were detected in the induced sputum. we found that normal control subjects had higher concentrations of prostaglandin (pg) d (normal subjects vs controlled asthma vs refractory asthma, median conclusions: eicosanoid profiles in induced sputum supernatant were different between patients with refractory asthma, those with controlled asthma, and normal control subjects. our findings suggest that they could be biomarkers to differentiate refractory asthma from controllable asthma or non-asthma. this work was supported by the research program funded by the korea centers for disease control and prevention ( -er - ) . | serum periostin in asthma is related to disease severity, eosinophilia and il- introduction: introduction: asthma is a chronic inflammatory disease where more than powerful inflammatory mediators are associated with airway hyperresponsiveness, mucus hypersecretion, activation of fibroblasts and hyperplasia and hypertrophy of smooth muscles of the airways and if they do not use preventive antiasthma treatment can cause irreversible airway remodeling. objectives: the aim of this study was to determine the effect of adding montelukast to combined therapy icss/labas in patients with uncontrolled asthma by analyzing of serum level of il- , il- , eosinophils and symptom score. methods: in study we included patients, they were treated with icss/labas ( / mcg-twice daily) plus montelukast ( mgdaily). in each of them were measured serum levels of il- and il- by the elisa method, value of eosinophils were obtained with visual examination of peripheral blood smear, and assessing symptom scores with -point likert scale of breathlessness at the beginning and after months of therapy. objectives: this study has been focussed on a detailed comparison of two samplers-cyclone and chemvol-and on the parameters that could influence their efficiency. results: airborne concentrations of two key olive and grass allergens, ole e and phl p , respectively, were monitored over two years with different weather patterns, and , in c ordoba, located in south-western spain. allergenic particles were quantified by elisa assay and results were compared with pollen concentrations monitored using a hirst-type volumetric spore trap over the same study periods. the influence of weather-related parameters on local airborne pollen and allergen concentrations was also analysed. inter-year differences were observed with regard to pollen season timing and intensity. for both olive and grass, the pollen season was longer and the pollen index (pi) higher in ; that year the peak value was higher and it was recorded earlier. although a positive correlation was detected between results obtained using the two samplers during the pollen season, results for the cumulative annual allergen index varied considerably. the two samplers revealed a positive correlation between pollen concentrations and both minimum temperature during the warmer year ( ) and maximum temperature during the cooler year ( ); a negative significant correlation was observed in both cases with rainfall and relative humidity. conclusions: in summary, although differences were observed between the two samplers studied, both samplers may be suitable for allergen detection. objectives: the scientific council of rnsa was asked to update the allergy potency (ap) of plant species that can be established in urban areas. to update the allergy potency of plant species, the rnsa used scientific work on the subject, and also the opinions of allergists and botanists. the pollen grains of anemophilous species are transported by wind; they produce very large quantities of pollen grains so that the fertilization of female flowers has a greater chance of being effective. the majority of allergenic species are anemophilous. results: the pollen allergy potency of a plant species is the ability of its pollen to cause an allergy to a significant part of the population. the allergenic potential can be: low or negligible: no problem to plant them in urban garden moderate: only a few species can be planted in the same garden high: this species cannot be planted in urban places. the table presented on the poster will permit to know the level of allergy potency of more than species. conclusions: species or genus with a strong ap should be labeled as "not to be planted in habitation or residence area ", those with moderate ap should be labeled as "not to be planted in big quantities in habitation or residence area". other species with low or negligible ap may not be affected by public information. gadermaier g ; metz-favre c ; stemeseder t ; de blay f ; pauli g universit e de salzbourg, salzbourg, austria; chru strasbourg-allergologie, strasbourg, france introduction: the purpose is to study the profile of cutaneous and molecular sensitization of patients sensitized to plantain pollen living in the north-east of france. objectives: the sera of patients with seasonal pollinosis and cutaneous polysensitization including a positive test to plantain, are investigated through immunoblot (ib), elisa and immunocap. the plantain immunoblot is inhibited with plantain, grass, ash and birch pollen extracts as well as with pla l . the specific iges against pla l , phl p - and profilin are measured. results: pla l , the major plantain allergen is detected by ib in cases out of , either in glycosylated or non-glycosylated form. an allergen of kda is detected in out of cases, always and exclusively inhibited by the grass extract. the intensity of the spot is proportional to the anti-phl p / phl p ige levels, and corresponds to an allergen which is cross-reacting with grass. other cross-reacting allergens with grasses are detected at kda ( / ), and at - kda ( / ). at the molecular weights of to kda, we detected in addition to pla l , cross-reacting allergens with grass, ash and birch pollen which predominantly corresponded to profilin which was confirmed by specific ige measurements in cases. conclusions: among the patients included in this study, only % had genuine sensitization to plantago, which never corresponded to a monosensitization in our cohort. most often cutaneous sensitizations to plantain pollen were based on ige cross-reactivity with grass pollen allergens mainly through a kda allergen and/or profilin. the strong environmental grass pollen pressure, compared to the low plantain pollen exposure, seems to be at the origin of this profile of molecular sensitization. these results reinforce the superiority of molecular diagnosis in patients with pollinosis, who are polysensitized. | sensitisation to peach tree pollen in a highly exposed population results: the % of subjects were sensitized to pp, which was the most prevalent after olive tree, grass and cypress pollens, respectively. sensitization to peach fruit was %. pru p spt was tested in / pp positive cases, being % positive ( / ). specific ige to pru p was detected in / pp sensitized individuals. immunoblot showed specific ige to different components in the pp extract, being the most frequent band recognised a - kda protein. conclusions: pp is a prevalent inhalant allergen in highly exposure areas. in our population pru p was not identified as the major allergen. other pp molecular components need to be identified and their clinical relevance should be further investigated. introduction: allergic respiratory diseases increase after an exposure to airborne pollen, as asthma and allergic rhinitis, are deeply increasing and nowadays, they represent one of the major public health problems. olive pollen is one of the main causes of allergic disease in the mediterranean area, especially in northwest and west of the turkey. olive pollen has been characterized proteins with allergic activity and ole e is the major allergen of olea pollen. objectives: the aim of this study was to estimate the correlation between daily airborne olive pollen and ole e in the atmosphere. aeroallergen load of ole e detected by cascade impactor (chemvol) using prewashed polyurethane foam and pollen counts recorded by hirst trap. chemvol sampler collects particles at l/minutes and it contains impaction stages pm> micron and >pm> . lm. this sampler is being tested in the frame of the project hialine. results: generally, similar behaviour of pollen count and total allergenic load of ole e was observed during the main pollen season. nevertheless, in some occasions, before and later of main period, airborne ole e activity was recorded and some differences between pollen grain/m and allergen concentration/m were detected. pollen from different days released -fold different amounts of ole e per pollen. average allergen release from pollen was much higher in ( . pg ole e /pollen, r =. ) than in ( . pg ole e /pollen, r =. ). indeed, yearly olive pollen counts in were . times higher than in , but ole e concentrations were . times higher. these results have shown that ole e is mostly associated with olive pollen grains but aeroallergen load was not always directly proportional to airborne pollen counts. this suggests that ole e quantification is a better marker for olive allergen exposure. in conclusion, aeroallergen monitoring may contribute to a better understanding of the ole e exposure from airborne pollen. objectives: to describe the clinical profile of the patients sensitized to alt at and to assess the sensitivity of the prick test with the alternaria extract in comparison to the patients sensitized to both allergens alt a and alt a . out of the patients, ( . %) were sensitized to alternaria with specific ige of ≥ . isu to alt at or alt a . twenty patients ( females and males, age - years old) who were sensitized to alt a were selected. we analyzed the clinical profile, total ige values, other co sensitizations as well as the clinical relevance of alternaria sensitization in these patients. : of the patients sensitized to alternaria, ( . %) were sensitized to alt a , of which ( . %) were monosensitized to alt at . the mean age was lower in monosensitized (ẋ . vs . ). among the patients monosensitized to alt a , ( %) had prick test negative with the alternaria extract. eight out of patients were polysensitized to more than three different aeroallergens and were asthmatics ( persistent). the median total ige was higher in monosensitized ( ku/l vs ku/l). of these patients, alternaria sensitization has clinical relevance in ( %), of which have positive prick test to alternaria. all the patients ( / ) sensitized to both allergens have positive prick test to alternaria. ten of them were polysensitized, were asthmatic ( persistent) and sensitized patients showed clinical relevance. introduction: the role of vitamin d as a potential immune-modulator has been recently elucidated. dendritic cell, a key regulator driving towards th immune response in allergic diseases is known to be affected by vitamin d. however, the role of vitamin d in the pathogenesis of allergic rhinitis is unclear and its anti-allergic effect has not been established yet, especially in the mouse model. objectives: the aims of this study are to evaluate ) the anti-allergic effect of topically applied vitamin d in the allergic rhinitis mouse model, and ) the effect of vitamin d on dendritic cell activation. results: balb/c mice were intraperitoneally sensitized with ovalbumin (ova) and alum, and they were intranasally challenged with ova. intranasal , -dihydroxyvitamin d was given to treatment group and solvent was given intranasally to sham treatment group. allergic symptom scores, eosinophil infiltration, cytokine mrna levels (il- , il- , il- , il- , ifn-c) in the nasal mucosa, serum total and ova-specific ige, igg , and igg a were analyzed and compared with negative and positive controls. cervical lymph nodes were harvested for flow cytometry analysis. in the treatment group, allergic symptom scores, eosinophil infiltration, and the mrna levels of il- and il- were significantly reduced compared to positive control. il- mrna level, serum total ige, and ova-specific ige and igg levels showed a tendency to decrease in the treatment group, but did not reach to a significant level. il- did not show a significant difference between groups. cd c + , mhcii hi , cd + activated dendritic cells were significantly reduced in the treatment group. cd + , cd + , foxp + treg cells tended to increase in the treatment group, however it was not significant. the intranasal instillation of , -dihydroxyvitamin d has an anti-allergic effect in the allergic rhinitis mouse model. we believe that the anti-allergic effect of vitamin d is mediated by the inhibition of dendritic cell activation, and therefore decreased objectives: we wanted to assess whether treatment with cyclosporine and tacrolimus allows children with vkc to improve the level of vitamin d due to the higher summer sun exposure for good control of the ocular symptoms. objectives: our objective was to assess the expression of smad and smad proteins in patients with asthma in correlation with clinical parameters and the expression's changes in response to allergen and methacholine challenge test. the study included patients with asthma and healthy volunteers. spirometry, skin prick tests (spt), allergen and methacholine provocation tests were performed in compliance with standards. personalized clinic surveys including act tm were collected. venous blood was collected before and after provocation. the expression levels of il- and il- and smads were evaluated by qrt-pcr using isoform-specific primers. results: we showed correlation between the mrna expression of smad and the asthma control according to act tm (p<. ). the expression of smad is higher in the group of uncontrolled ( -Δct = . , p<. ) and in the group of partially controlled patients ( -Δct = . , p<. ) in comparison to the group of patients with controlled asthma ( -Δct = . ). we proved that expression of mrna of smad correlates significantly with expression of il- and il- in patients with asthma (il- r= . ; il- r= . ) and in the control group (il- r= . ; il- r= . ) (p<. ). expression of smad elevates more after methacholine provocation test (median -Δct = . ) rather than after allergen provocation test (median -Δct = . ; p=. ). we showed also that skin prick test results correlate positively with smad level after the provocation (p<. ). conclusions: the loss of asthma control is connected with expression of smad , which is part of tgf-ßrii related pathway. il- and il- correlates with smad expression, which can indicate the participation of these cytokines in the regulation of this pathway. the expression of smad elevates after methacholine and allergen provocation as well as it correlates positively with skin prick test results, which can be useful clinically. to sum up, tgf-ß-tgfßrii-smad proteins are an important mediators of inflammation in asthma. | deletion of nfatc in t lymphocytes affects th and th cell differentiation as well as il- -mediated mast cell activation in allergic asthma objectives: analysing the role of nfatc in the allergic trait of human asthma. additionally, we investigated the influence of nfatc on t cell differentiation and immunoglobulin class switch and explored its impact on mast cell differentiation in experimental asthma. with the children s hospital in erlangen, we studied nfatc mrna expression in freshly isolated pbmcs from pre-school children with and without asthma. in asthmatic children, we found increased nfatc mrna expression, especially in those with a positive skin test. these results were confirmed in the asthma bio-repository for integrative genomic exploration (asthma bridge) study, where the isoform a and d of nfatc were found significantly increased in asthmatic adults with a positive skin test. moreover, il- was also found increased in the supernatants of pbmcs of asthmatic children with a positive skin test. furthermore, mice with a deficiency of nfatc in cd + t cells display significantly lower levels of il- . additionally, these mice show reduced numbers of lung th and th cells. moreover, basic leucine zipper atf like (batf), which is known as an important transcription factor for t cell differentiation as well as immunoglobulin class switch, was found decreased in these mice. consequently, ova-specific ige and total igg levels were found significantly decreased after allergen exposure and in the absence of nfatc . furthermore, nfatc deletion also resulted in decreased mast cell numbers. we then analyzed the effect of il- on mast cell differentiation and histamine release. we observed that bone marrow differentiated mast cells incubated with ova and il- had an induced histamine release. conclusions: thus, nfatc deficiency in t cells results in defective ige production affecting ige-orchestrated mast cell activation mediated through il- . therefore, nfatc emerges as a novel target for anti-allergy intervention. | efficiency of the use of nitric oxide donors for the treatment of bronchial asthma bazarova s; djambekova g center of therapy, tashkent, uzbekistan introduction: to study the influence of nitric oxide donor-l-arginine-on indicators of endothelial system in patients with bronchial asthma. objectives: patients aged - years ( ae . years) with moderate persistent bronchial asthma were examined. ratio of men to women was / . two age-and sex-matched groups were randomly selected. main group ( patients) received nitric oxide donor -l-arginine in addition to standard background therapy (gina, ) . the medication ( ml of . % solution, tivortin, "yuria-farm", ukraine) was administered intravenously once daily for days. the control group ( patients) only received the background therapy. the efficiency was assessed with the use of conventional methods of study (clinical laboratory methods, instrumental methods: spirography, peak flow monitoring, bronchomotor tests). concentration of nitric oxide stable metabolites in exhaled breath condensate (ebc) and in blood was studied. their ratio was also assessed. results: baseline data in patients of both groups demonstrated the increase of level of nitric oxide stable metabolites in blood ( . ae . mmol/l) and in ebc ( . ae . mmol/l). after the treatment the positive clinical efficiency of inclusion of l-arginine was much higher than that in the control group (p<. ). in the main group, reduction of requirement for beta -agonists, decrease of frequency of night-time symptoms, decrease of frequency of lowest pef rate in the morning and improvement of respiratory function were observed on average on the rd/ th day compared to the control group were such improvements were evident on the th/ th day. in the main group the concentration of nitric oxide stable metabolites significantly increased in blood ( . ae . mmol /l, p<. ) and in ebc ( . ae . mmol/l, p<. ). in the control group the concentration of nitric oxide stable metabolites changed in blood ( . ae . mmol/l) and in ebc ( . ae . mmol/l), but not significantly. introduction: abpa is currently believed to be an exaggerated form of aspergillus sensitization, and is probably the first step in its development. objectives: the aim of this study was to investigate the clinical and immunologic characteristics of fungal-sensitive asthma (fa), nonfungal-sensitive asthma(nfa) and abpa. conclusions: there were different clinical and immunological features among nfa, fa and abpa. the abpa had worse function as well as higher percentage of bronchiectasis, and higher dose of oral corticosteroid. besides, the sensitivity to aspergillus was more severe in abpa. the level of sige-a.f was associated with the damage of lung function. | self-reported allergic rhinitis and/or allergic conjunctivitis associate with il rs genotypes in finnish adult asthma patients introduction: the increased prevalence of asthma and allergic diseases is a major public health problem worldwide. atopy, family history, inhaled irritants, and upper airway inflammation are known risk factors of asthma. a population-based sample of finnish adult asthma patients (n= ) and matched controls (n= ) filled a questionnaire. asthma was diagnosed based on a typical history of asthma symptoms and lung function tests. skin prick tests (spt) with aeroallergens and blood tests including analysis of interleukin (il ) rs (g/a) genotypes were performed for a subsample (n= ). objectives: our aim was to observe in adult asthmatics with and without allergic co-morbidities e.g. subject-reported allergic rhinitis and/or allergic conjunctivitis (ar/ac) association with il rs genotypes and other factors. results: the proportion of asthmatics reporting ar was . % and ac was . %. after adjustments, the presence of il rs aallele (or= . ci= . - . , p=. ) or multi-sensitization (adjusted or= . , ci= . - . , p=. ) associated with ar/ac-asthma. nasal polyps and aspirin-exacerbated respiratory disease associated also with ar/ac-asthma. conclusions: adult ar/ac-asthma could putatively be a phenotype, characterized by the presence of atopic and/or eosinophilic factors and a high prevalence of the il rs a-allele. studies on the mechanisms behind this and in other populations are needed. | immunoregulatory role of nfatinteracting protein (nip) in adaptive and innate immune responses in allergic asthma introduction: nfat-interacting protein (nip) is a th associated transcription factor. after t cell receptor stimulation, the arginine methylation domain of nip supports the interaction with nfat, thereby enhancing the production of the th cytokine il- . moreover, nip deficient mice have been shown to be deficient in il- and ifn-gamma production indicating that nip controls both th and th cytokine production and might therefore play a protective role in allergic asthma . objectives: we wanted to analyze the importance of nip in allergic asthma in pre-school children as well as adults. furthermore, we investigated the role of nip in a murine model of allergic asthma to find out more about its contribution to adaptive as well as innate immune responses in the disease. results: in the european study predicta, in collaboration with the children s hospital in erlangen, we analyzed nip mrna expression by using quantitative real time pcr in rna extracted from pbmcs isolated from pre-school children with and without asthma. in the pbmcs of the asthmatic children, nip mrna expression was found significantly increased compared to healthy control children. furthermore, nip mrna expression was also found induced in cd + t cells in adult asthmatics from the asthma bio-repository for integrative genomic exploration (asthma bridge) study. we further analyzed the importance of nip in a murine model of allergic asthma. after allergen sensitization and challenge nip (-/-) mice showed decreased airway hyperresponsiveness, inflammation and mucus production, three of the main patho-physiological features of asthma. additionally, we discovered that nip (-/-) mice released decreased th type cytokines and also expressed less st , which is the receptor for il- , after allergen challenge. furthermore, a defect in innate lymphoid cell type (ilc ) differentiation was observed in the absence of nip in allergic asthma and in bone marrow differentiated ilc s indicating a crucial role for nip in mediating asthma via ilc s. conclusions: in summary, we found that the lack of nip influences not only immune responses of the adaptive immune system but also influences components of innate immunity resulting in a abstracts | protective phenotype to allergic diseases such as asthma. objectives: in the study were included ap and healthy controls (hc). fraction of exhaled no (feno), standard lung function parameters, complete blood count and absolute count of cells, serum ige, crp, il- , il- a and periostin were measured. results: four clusters were identified by cluster analysis: cluster (c )(n= )-late-onset, non-atopic, eosinophilic asthma with impaired lung function, cluster (c )(n= )-late-onset, atopic asthma, cluster (c )(n= )-late-onset, aspirin sensitivity, eosinophilic asthma and cluster (c )(n= )-early-onset, atopic asthma. we have found higher levels of il- in all clusters ap as compared to hc (c : p=. , c : p=. , c : p=. and c : p=. ). tendency for higher levels of serum il- in c compared with c or c (p=. or p=. , respectively) was observed. periostin levels were significantly higher in c (p<. ), c (p<. ) and c (p<. ) as compared to hc. there were no differences in periostin levels between all clusters (anova, p=. ). il- a levels were significantly higher only in c as compared to hc or to c and c (p<. , for each comparison). we have found correlation between il- and crp (r=. ; p=. ) in c , il- a and periostin in c (r=. ; p=. ) and in c (r=. ; p=. ). interestingly, we have observed negative correlation between the duration of asthma and il- a (r=À. ; p=. ), but positive between the duration of asthma and crp (r=. , p=. ) in c . our results have shown higher levels of il- in all clusters as compared to hc that are associated with marker for systemic inflammation. periostin levels were significantly elevated in c , c and c as compared to hc with no differences between the clusters. a positive correlation between periostin and il- a in c and c was observed, that rising the question about their interrelationship in the pathogenesis of late-onset asthma. serum il- a was significantly higher in c in comparison with hc or c and c suggesting that th mediated immunity may be involved in early-onset, atopic asthma. these data support the concept of heterogeneity of the bronchial asthma. | eosinophil activation with autophagy and extracellular dna traps is involved in severe asthma results: il- +lps treatment significantly increased autophagy and eet levels from pbes of the study subjects (p<. for all), which were in a positive correlation (r=. , p<. ). compared to nsa patients, both untreated and il- +lps-treated pbes from sa patients had significantly higher autophagy levels (p=. and . , respectively), while only il- +lps-treated pbes from sa patients had higher eet level (p=. ). eet level from untreated pbes was correlated with serum eosinophil cationic protein level (r= . , p= . ) and fev % predicted value (r=À. , p=. ). co-culture of aec with pbes slightly increased il- production, which was significantly enhanced by il- +lps treatment (p<. ). the il- production in co-culture system was reduced by pretreatments with dexamethasone ( mm, p=. ), antibodies against major basic protein ( ng/ml), il- receptor ( mm) and il- receptor ( mm, p=. for all), but increased by cotreatment with micrococcal nuclease ( iu/ml, p=. ). conclusions: pbes from sa patients are highly susceptible to be activated to produce high levels of autophagy and eet, which could enhance and maintain airway inflammation. we suggest that steroid and anti-il- /il- receptor antibodies may be beneficial to control airway inflammation in severe eosinophilic asthma via inhibition of eet production, while dna digesting reagents may increase airway inflammation. introduction: it has been demonstrated that exposure to stress induce hyporeactivity of the hpa system by modifying the secretion of cortisol and also that psychosocial stressors such as poor social status are associated with an increased risk of childhood asthma, decreased serum cortisol and high ige response. thus, from this concept it could be postulated that a blunted hpa axis response may increase the risk for allergic inflammatory reaction. objectives: aim of this study was to evaluate the association of serum cortisol in pediatric allergic bronchial asthma and its influence on the ige immune response in a poor children community with psychosocial chronic stress. results: this was a pilot analytical case control study( ipa positive subjects and healthy control paired by age and gender, both from poor areas of barranquilla objectives: in order to characterize bp an immunoproteomics analysis was conducted, i.e. electrophoretic separation of cypress pollen extracted proteins, ige western blotting using cypress pollen allergic patient's sera and mass spectrometry (lc/ms/ms) for identification of ige-binding proteins. results: ms analysis using chymotrypsin identified in bp a peptide also found in the family of protein snakin/gibberellin-regulated protein (grp). the snakin- , an anti-microbial peptide of potato, produced as a amino-acid recombinant protein (homologous to the c-terminal part of grp), is recognized by ige from cypress pollen allergic patients with ige to bp . this ige reactivity is abolished after reduction of disulfide bridges and is inhibited by a cypress pollen extract. ige reactivity to bp is however barely inhibited by the recombinant potato snakin- . conclusions: bp exhibits a molecular mass closer to grp than to snakin and the absence or very low inhibition of the ige reactivity to bp with snakin may be explained by peptidic ige epitopes on the n-terminal part of bp . the potato snakin- share % sequence identity with peamaclein, the peach allergen pru p , also shown in citrus. these results might explain the peach/cypress and citrus/cypress syndromes described and point out bp as the cross reactive allergen. the proteins of snakin/grp family present in many fruits and vegetables might include allergens involved in pollen/food associated syndromes. introduction: exposure to high levels of grass pollen may lead to a high degree of allergic inflammation, sensitization to minor allergens, such as profilin, and development of severe profilin mediated food reactions, similar to those described due to sublingual immunotherapy (slit). objectives: our objective here was to identify genetic biomarkers in order to generate a model that can improve the classification and treatment of patients with a higher probability of developing severe adverse reactions. results: healthy subjects (group , control) and patients with mild (group ) or severe (group ) profilin mediated food reactions were studied. rna extraction was performed on ficoll-isolated pbmcs using the rneasy ® mini kit (qiagen) and its integrity was analyzed with experion rna stdsens analysis kit (bio-rad). the gene expression profile of all the samples was analyzed using the gene-chip ® wt plus reagent kit (affymetrix) and two specific software: affymetrix ® expression console ™ and affymetrix ® transcriptome analysis console (tac). finally, the microarray data was validated by quantitative real-time pcr (qpcr). the hierarchical clustering of the samples shows the separation of the patients into three clusters coincident with the three established clinical groups (control, mild and severe). genetic profile of patients with mild reactions is similar to healthy patients while severe subjects were significantly different from the other two groups. genes regulated in the severe group were related to histone modification pathways, human complement system, cell adhesion and tgf-b and its receptor. these changes may be associated with the different degree of inflammation between patients in each experimental group. in the course of our study we found out that severe patients had different rna expression patterns compared to mild and non-allergic patients. this lead to the identification of genetic biomarkers useful for the generation of a model able to predict severe reactions and/or adverse reaction during immunotherapy, thus improving the diagnosis and treatment of this type of allergic results: his-tagged recombinant allergens, namely parvalbumin, aldolase, enolase and tropomyosin from c. idella and l. crocea, as well as cod parvalbumin, were synthesized in e. coli and purified using immobilized metal-chelate chromatography. children with history of immediate-type fish allergy were included in this study and their serological ige reactivity to the fish allergen components were measured by elisa. children were positive to at least one allergen component, with nine of them being positive to parvalbumin. despite the high similarity between l. crocea, c. idella and cod parvalbumins ( . - . %), three children only reacted to l. crocea and c. idella parvalbumin but not to cod parvalbumin, while the other six children were positive to all three parvalbumins. competitive inhibition elisa revealed that c. idella parvalbumin inhibited > % of the binding of specific ige to both l. crocea and cod parvalbumin, while reciprocally only inhibition of % and % could be achieved respectively. two children were reactive to aldolase and enolase but not parvalbumin. one of them had positive sige to both enolase and aldolase from l. crocea, while the other reacted to aldolase from both species. these two children exhibited relatively mild subjective allergy symptoms (itchy skin and throat). notably, three children were non-reactive to all components tested, and two of them were outgrowing fish allergy clinically. introduction: anti-a-gal antibodies are naturally produced in response to the gastrointestinal flora. in red meat allergy, patients develop ige antibodies towards the a-gal epitope, which itself are structurally closely related to the blood group b antigen. objectives: this study aimed to explore the relationship between a-galand b-antigen-specific antibodies in red meat allergic patients compared to healthy individuals with blood group b or a/o. sera from red meat allergic patients and healthy blood donors of whom belonged to blood group b and to blood group a or o were included. ige reactivity against a-gal and the b-antigen were determined using immunocap. allergen-specific igg, igg , igg , igg , igg and ige were assessed by indirect elisa assay. statistical analysis was performed using spearman rank correlation and unpaired t-tests. results: all red meat allergic patients, of the healthy a/o and of the healthy b donors were ige positive to a-gal. however, the ige levels to a-gal were significantly higher in the allergic group compared to the healthy a/o and b individuals. the majority of the meat allergic patients, but none of the healthy individuals had ige antibodies against the b-antigen. a moderate correlation between a-gal and b-antigen specific ige was noted (r =. ). the red meat allergic patients had significantly higher igg levels against a-gal with igg and igg antibodies as the predominant difference compared to the healthy individuals. the healthy a/o ige positive individuals had significantly higher igg , igg and igg compared to the ige negative individuals. the igg response to the b-antigen followed the same pattern as to a-gal. there was a low correlation between the igg levels against a-gal and the b-antigen in both meat allergic patients and healthy a/o individuals (r =. and . ). introduction: fx , a food mixture of milk, egg white, fish, peanut, wheat and soybean, is largely used for food allergy detection. besides the fact that it is questionable if this is the better approach to identify a food allergy, the information that the test provides may represent a pitfall because a positive or negative result does not mean food allergy presence or absence, respectively. objectives: the goal of our study was to access the reason for fx request in a pediatric hospital and to analyze its suitability. methods: the fx requests, performed in a pediatric population of d. estefânia hospital over a five months' period, were analyzed, concerning demographic data, reason for request and attitude taken due to the result. this test was requested due to respiratory symptoms in patients, gastrointestinal symptoms in patients, cutaneous symptoms in patients and nonspecific complaints in patients. all of these symptoms were not directly related with food intake. a positive result was obtained in patients; of those, only were referenced to our immunoallergology department. in all of them a detailed clinical history was obtained and diagnostic tests (skin prick tests and specific ige) were performed in the ones considered suitable. food allergy was diagnosed in only one patient. conclusions: in the vast majority of patients, fx was asked for nonspecific complaints and often without a clinical history suggestive of food allergy. moreover, a positive fx test does not mean clinical reactivity or food allergy. on the other hand, the clinical history allows us to identify a suspect trigger in most of the children with food allergy. in such cases, it is preferable to request the specific ige towards the allergen, which gives a more precise and accurate result, instead of the fx . as our results showed, in the majority of the cases, the fx request was often made without complying with a reasonable criterion, implying unnecessary costs. the high number of requests verified may be explained because it is an easily accessible analysis but this attitude should be discouraged. tuppo l ; alessandri c ; pasquariello s ; petriccione m ; giangrieco i ; tamburrini m ; rafaiani c ; ciancamerla m ; mari a ; ciardiello ma istituto di bioscienze e biorisorse -ibbr-cnr, naples, italy; caam -centri associati di allergologia molecolare, rome, italy; cra, research unit on fruit trees, caserta, italy introduction: pomegranate, punica granatum l., is one of the oldest cultivated fruit trees. the fruit contains the arils, which are seeds covered by a red pulp, that is a juice sac. the arils are surrounded by the white and fleshy mesocarp. pomegranate can trigger allergic reactions, but the allergenic pattern of this fruit is still poorly characterized and only one allergen, the ltp pun g , was reported. objectives: the aim of this study was the investigation of the allergen pattern in pomegranate tissues and cultivars. reported symptoms were food impaction ( %), dysphagia ( %), heartburn ( %) and vomiting ( %). the first symptoms were more frequent in adolescents and adults ( %). children's main complaint was vomiting ( %) and cases presented failure to thrive. most patients had associated allergic diseases ( %), % had previous food allergy and % were sensitized to aeroallergens. endoscopic evaluation revealed esophageal stricture in patients. at least half of diagnostic esophageal biopsies had > eosinophils per highpower field and % showed microabscesses. food sensitization was found in % of the patients, mainly to cow's milk ( %), nuts and peanut ( %), cereals ( %) and egg ( %). considering therapeutic approach, % were treated with swallowed fluticasone and dietary elimination was recommended in %. oral corticosteroids were prescribed in patients. at present time patients had done endoscopic reevaluation, ( %) showed histologic resolution. five patients had clinical and histologic relapse during follow-up. conclusions: eoe has a balanced distribution but a distinctly clinical presentation accordingly to age group: children may present unspecific symptoms like vomiting, whereas adolescents and adults complain of food impaction and dysphagia. other atopic diseases and food sensitization is very common. introduction: air pollution, particularly ambient air particulate matter (pm), is considered as one of the most important environmental risks for human health. pm could potentially disrupt immune regulatory mechanisms and predispose exposed individuals to asthma. in contrast, children exposed to traditional farm environment seem to have a natural resistance to asthma. this phenomenon links with exposure to stable dust and subsequent immune regulatory mechanisms initiated in the airways. the underlying exposure agents and definitive pathways determining the risk of asthma are still to be identified. objectives: our aim was to investigate the effect of urban air pm (high risk environment) and farm dust (protective environment) on immune responses in finnish children. briefly, peripheral blood mononuclear cells (pbmcs) of -year-old children (n= ) were stimulated with farm dust extract ( lg/ml, stable in northern savonia, finland) and pm samples ( lg/ml, pm . - , pm - . or pm < . , nanjing, china) for hours. expression of immunogenic cd and tolerogenic ilt in circulating myeloid dendritic cells (mdcs) and plasmacytoid dcs (pdcs) and monocytes were analyzed by flow cytometry. pm samples were analyzed for polycyclic aromatic hydrocarbons (pahs), inorganic ions and elements. farm dust sample will be analyzed for microbial content. results: pm stimulation decreased the percentage of cd + monocytes and dcs among children's pbmcs, whereas farm dust stimulation increased the percentage of cells positive for this marker. the percentage of tolerogenic ilt + was decreased in all cell types after stimulation with pm. farm dust also decreased the percentage of ilt + monocytes, but not dcs. specific metals and pahs in pm associated with the studied immunological parameters. conclusions: samples from high risk and protective environments have differing capacities to influence immunogenic and tolerogenic properties in children's immune cells. the importance of these findings in relation to the risk of asthma in exposed populations will be studied further. introduction: alterations in cell surface glycosylation pattern is a common feature of tumor cells that might be related to immune evasion and malignancy. objectives: to study the capacity of the carbohydrate a (ca ) located in the surface of murine ehrlich tumor (et) cells and also in certain human adenocarcinomas to condition the phenotype and function of human dendritic cells (dcs) and the capacity to polarize t cell responses. results: nf-jb/ap- are not activated in thp cells by ca , however, this carbohydrate partially impairs the activation of these transcription factors induced by the tlr ligand pam csk. ca induces the expression of the tolerogenic marker pdl in human monocyte-derived dcs (hmodcs) as well as the production of il- and il- , analyzed by flow cytometry and elisa assays respectively. ca -activated hmodcs generate functional il- -producing cd + cd high cd -foxp + regulatory t (treg) cells that were able to inhibit the proliferation of cd + t cells from pbmcs in a dose-dependent manner. supporting the role of ca in the induction of treg cells, our in vivo data showed that the ca is present in the sera of tumor bearing mice and the percentage of foxp + treg cells is increased in the regional (inguinal) lymph nodes from tumor bearers. our results showed that ca -activated hmodcs induce the generation of foxp + treg cells both in vitro and in vivo, which might well condition the immune response against the tumor and promote the tumor escape. | assessment of changes in expression of immune system biomarkers to assist the differential diagnosis of acute bacterial infections introduction: biomarkers for acute infections include c-reactive protein, mmp- , sicam- , procalcitonin, and neutrophil band counts for bacterial infection. a rapid means of assessment of acute bacterial infections via biomarker assessment was sought. objectives: the expression of toll-like receptors (cd and cd ), complement receptors (cd and cd ), integrins (cd b and cd c), fc-receptors (cd and cd ) and l-selectin (cd l) on the surface of blood neutrophils and monocytes stimulated with inactivated e. coli, l. acidophilus, e. coli derived bacterial ghosts, e. coli lps and l. acidophilus cell walls was assayed using flow cytometry. both the percent of expression and mean fluorescence intensity (mfi) were analyzed for each molecule. results: all the bacterial components used exerted similar activation capabilities even in low concentrations. while the expression of cd b, cd c, cd , cd and cd was enhanced by both neutrophils and monocytes under activation, the expression of cd significantly increased only in neutrophils. the expression of tlr and tlr was slightly increased by neutrophils and monocytes. the expression of cd l by monocytes and neutrophils (the percent of activated cells as well as the mfi) was decreased during activation. there was a negative correlation between cd l expression and integrins (cd b and cd b). the activation index was calculated for each molecule as a ratio of expression of molecule by activated cells vs cells used as a negative control (resting). the highest values for the activation index was seen with cd b, cd c, cd , cd , cd l and cd mfi by neutrophils and monocytes, and the percent of cd expression by neutrophils. conclusions: e. coli and bacterial ghosts significantly increased the expression of cd b, cd c, cd , cd , cd l and cd by neutrophils and monocytes even in very low concentrations, suggesting use as potential biomarkers in the differential diagnosis of the etiology of acute infections. objectives: the aim of this study was to evaluate changes in peripheral blood monocyte expression of cd , cd , cd , cd , hla-dr, and cd in kidney allograft recipients. in total, patients who underwent renal transplantation from a deceased donor were enrolled in the study. the phenotype was evaluated by a multicolor flow cytometry in defined time points and in the case of complications requiring fine needle aspiration biopsy procedure. the results confirmed our pilot data, proportions of peripheral cd +cd + monocytes were downregulated during the first week after the kidney transplantation while the percentage of cd +cd + monocytes dramatically increased early after the kidney transplantation and remained high for at least four months in most patients. the expression of cd (marker of m macrophages) was limited only to a small population of monocytes (less than % in most patients) but the receiver operating characteristic (roc) curve analysis showed its potential importance by significant correlation with acute rejection with a sensitivity of % and specificity of . % (area under the roc curve . , p-value: . ). no correlation between two different m markers cd and cd has been found. the expression of cd (dc-sign) was low and did not show any changes in time or association with acute rejection. hla-dr (mhc ii) and cd (integrin associated protein) were constitutively expressed without any significant changes in patients with acute rejection of the allograft. we assume from our data that kidney allograft transplantation is associated with early reciprocal modulation of monocyte subpopulations (cd +cd + and cd +cd +). a decreased proportion of cd positive blood monocytes seems to be associated with an increased risk of acute rejection of kidney allograft. introduction: thioredoxin (trx), a -kda oxidoreductase enzyme, is well known to be a redox-active protein that regulates reactive oxidative metabolism. in addition to its anti-oxidative activ- | progranulin-dependent regulation of th airway inflammation by house dust mite allergen introduction: progranulin is a growth factor that consists of amino acids including / repeats of cysteine-rich motifs, and produced by variety kinds of cell. progranulin is involved in the regulation and maintenance of inflammatory response, and its role is wellstudied in neuronal and metabolic diseases such as neurodegeneration and type diabetes. however, the role of progranulin during the development of airway inflammation induced by inhaled allergen is still obscure. objectives: in this study, we evaluated the role of progranulin in the development of th airway inflammation induced by house dust mite allergen. results: to find the main source of progranulin, we stimulated each cell line with various doses of house dust mite allergens. the production of each cytokine, including progranulin, was estimated in culture supernatant by elisa. to investigate the role of progranulin in airway inflammation, we intranasally administrated house dust mite allergens to -week-old female progranulin knock-out mice (macrophage-specific) or littermate mice. lung inflammation and immunological parameters were evaluated at h after first sensitization with allergen or h after final allergen challenge. the production of progranulin was significantly elevated by house dust mite allergen stimulation in innate immune cells, especially in alveolar macrophages over other cells. in the house dust mite allergeninduced airway inflammation model, we found that the level of progranulin increased earlier than other pro-inflammatory cytokines. in addition, in macrophage-specific progranulin knock-out mice, airway inflammation was down-regulated in the earlier phase after exposure to house dust mite allergen. moreover, we stimulated mice with house dust mite allergen for a longer period to observe the changes in the adaptive immune response of th airway inflammation, which was found to be decreased in conditional knock-out mice. conclusions: these findings indicate that th airway inflammation induced by house dust mite allergen is dependent on progranulin. objectives: the aim of the present study was therefore to investigate the immunomodulatory effect of epinephrine on m a macrophages and its consequence on cross talk to mast cells in a human model of allergic inflammation. results: primary monocytes from healthy pbmcs were first differentiated into m a macrophages using m-csf in the presence of il- and il- cytokines. after overnight incubation with epinephrine, supernatants were collected and analyzed by elisa for il- , tnf, il- , ccl , il- and ifn-c, whereas cell surface markers including cd , cd and cd were evaluated using flow cytometry. subsequently, both m a and epinephrine-treated m a supernatants were transferred onto cord blood-derived mast cells (cbmcs) for further overnight incubation, after which ige-mediated degranulation was assessed by the ß-hexosaminidase release assay. after overnight epinephrine treatment, m a macrophages showed an increase in il- , ccl , tnf and il- production, but no ifn-c and il- expression was observed. epinephrine treatment also downregulated surface markers cd and cd and upregulated cd . when supernatants from epinephrine-treated m a macrophages were added to cbmc cultures, ige-mediated degranulation was impaired compared to cbmcs treated with supernatants of unstimulated m a macrophages. conclusions: taken together, epinephrine promoted a phenotypic shift of m a polarized human macrophages toward an m b-like regulatory phenotype that was able to reduce the ige-mediated degranulation of cbmcs. we conclude that prolonged acute stress exposure in allergic patients may attenuate symptoms of acute allergy by directing macrophages towards an immunosuppressive phenotype, which can further dampen mast cell degranulation. objectives: a murine local lymph node assay was used to investigate the effect of oa on the immune response to the known skin sensitizer hexyl cinnamic aldehyde (hca, % w/v). the ear lobes tape stripped prior to immunization. test solutions ( ll) were applied on the dorsal side of each ear on three consecutive days. female balb/c mice ( groups a mice), were exposed to the vehicle acetone:olive oil ( : ) alone, or in combination with hca, with or without oa in the concentrations , and %, or oa alone ( , and %). on day , the animals were weighed and exsanguinated by cardiac puncture. the auricular lymph nodes were harvested for single cell preparation, stimulation with cona and cytokine release of il- and il- . the earlobes were excised and fixed for immunohistochemistry. results: no group differences were found for bodyweights or bodyweight change, number of lymph node cells or il- secretion. il- showed a tendency of dose-related increase, but a significant difference were only found between hca and hca+ % oa (p=. ) in one out of the two experiments. in he stained sections, the epidermal thickness was significantly increased in groups given hca + and % oa (p≤ . ). sections immunostained with anti-ly g showed significant increase in neutrophil influx for the same groups as above (p≤ . ). oa alone showed no effects or effects significantly lower than hca + oa. objectives: we hypothesized that plasma s p levels in cf patients might be associated with cftr mutations and could influence disease presentation. results: plasma was collected with a defined standard operation procedure to impede unspecific s p release from blood cells from double lung transplanted adult cf patients as well as sex-and age-matched, non-allergic healthy controls all being fasted overnight. total plasma s p was measured by mass spectrometry and unbound plasma s p by elisa. levels were correlated with cftr mutation status, routine laboratory parameters and clinical symptoms. we observed higher total and unbound plasma s p levels in healthy controls compared to cf patients with the latter value reaching statistical significance (p=. ) after exclusion of two statistical outliers. unbound plasma s p levels were significantly higher in df homozygous cf patients compared to patients with df heterozygosity (p=. ). patients with other mutations were excluded. levels of unbound s p were positively correlated with hemoglobin and negatively correlated with triglyceride levels. additionally, we observed a positive correlation of total plasma s p levels in cf patients with hba c. gastrointestinal symptoms were more common in df heterozygous ( / ) compared to df homozygous cf patients ( / ). fecal calprotectin levels were found to be significantly higher in df homozygous compared to heterozygous cf patients (p=. ). differences in unbound s p levels were not correlated with immunosuppressive treatment after transplantation. conclusions: to the best of our knowledge this is the first clinical study directly correlating plasma s p levels with cf genotypes and clinical presentation in cf patients. we emphasize to evaluate s p as a potential novel disease biomarker as well as a therapeutic target for cf in future studies. supported by the austria science fund grant kli (to eu). ochoa-grull on j ; tejera-alhambra m ; guevara k ; guzm an-fulgencio m ; benavente cm ; mart ınez r ; p erez c ; peña a ; rodr ıguez de la peña a ; llano hern andez k ; rodr ıguez-fr ıas e ; s anchez-ram on s objectives: we show preliminary data of one study aimed to evaluate the use of this strategy in the prevention of rrti in infants and preschool children. results: patients: children ( male and female, age range - months) were included in a randomized double blind and placebo-controlled study (eudract - - ) . all of them showed negative in vivo and in vitro allergy tests. active treatment consisted in a suspension of a mixture of selected strains, grown and inactivated in optimal conditions, of s. aureus ( %), s. epidermidis ( %), s. pneumoniae ( %), k. pneumoniae ( %), m. catarrhalis ( %) and h. influenzae ( %) in physiologic saline solution with % glycerol at a concentration of formazin turbidity units (ftu)/ml (equivalent to bacteria/ml). placebo did not contain any bacteria. patient were treated for a period of months, receiving daily sublingual puffs of active or placebo and with a follow-up of other months (total period of evaluation was year). symptom (cough, dyspnea, wheeze, mucus, fever, discomfort) and medication (inhaled corticosteroids, beta adrenergics, montelukast, antibiotics) scores were evaluated since the first day of treatment to the end of the study ( year) . any adverse event was recorded to assess safety. for the comparison between both groups, t test was used. patients who received active treatment experienced an improvement of % over placebo in overall symptoms and % in medications scores (p<. ). in the combination of symptoms and medication scores the improvement was % (p<. ). no adverse events related to treatment were recorded. conclusions: immunostimulation with these selected strains of bacteria is safe and can be successfully used in infants and preschool children in order to prevent rrti. introduction: to reduce the duration and the risk of the allergen specific immunotherapy using commonly used allergen extracts, new highly immunogenic and non reactogenic vaccines are needed. objectives: the goal of the present study was to employ the ap spytag/catcher system to develop a virus-like particle (vlp) vaccine based on the major house dust mite (hdm) allergen der p and to evaluate its reactogenicity in vitro. spycatcher-ap vlps and recombinant der p , fused at the c-terminus to the amino acid spytag binding-partner, were expressed in e. coli. purified spytagged der p was mixed with spycatcher-vlps, which resulted in covalent conjugation of der p to the surface of spycatcher-vlps. excess unbound der p was removed by dialysis. dynamic lightscattering (dls) was used to analyse the size and aggregation state of vlp-der p . the ige reactivity of vlp-der p was assayed by direct elisa and by rat basophil degranulation assays. conclusions: our results demonstrate that coupling of spy-tagged der p to ap spycatcher-vlps dramatically reduces the reactogenicity of the allergen, suggesting that vaccination with ap vlp-der p may be a safe and effective treatment for hdm allergy. objectives: the qm s hybrid protein is a qm variant where cysteine amino acids have been replaced by serine. the expression of qm s hybrid protein was performed in e. coli bl (de ) after iptg induction. the purification of qm s protein was performed from inclusion bodies by a three-step chromatography process. the stability of qm s was analyzed by sds-page and total protein assay. qm s ige-binding capacity was compared with natural der p and der p by elisa-inhibition and allergenicity was studied by mediator release from rbl cells. immunogenicity was evaluated in mice by analysis of the specific igg response to der p and der p . results: qm s was expressed in complex media as inclusion bodies that were solubilized in urea. soluble protein was purified by anionic ion exchange, hydrophobic interaction, and size exclusion chromatography in the presence of a detergent. qm s purification process was shorter and more efficient than that of qm . the purity obtained was > %. elisa inhibition assay showed that qm s hybrid protein was almost unable to inhibit ige-binding to the hdm extract, less than % in all the range of concentrations tested ( . - ng/ml) and representing a -fold reduction as compared to qm . qm s showed a great reduction of the b-hexosaminidase release in rbl cells, compared to der p and der p . qm s was able to induce der p -and der p -specific lgg antibodies responses comparable with those induced by the mixture of wildtype allergens. mouse igg antibodies induced by the hybrid proteins qm s and qm showed similar ige-blocking antibodies properties to mixture of der p and der p . the stability of qm s was studied in solution at °c, °c, and - °c and lyophilized at °c, being the frozen and lyophilized forms the best conservation conditions. the qm s hybrid exhibited less ige-binding activity than qm and the natural der p and der p while retained the immunogenicity. these properties together with the improved manufacturability made qm s a good candidate for sit to hdm allergy. objectives: slit tablets of cockroach, slit tablets of parthenium, slit tablets containing both allergens together (mix) and slit bilayer tablets containing one layer with parthenium allergen and other layer with cockroach allergen, compress to single tablet were prepared. punches and dies of mm were used for compression. slit drops containing cockroach, slit drops of parthenium and slit drops containing both allergens together, were prepared and filled in ml amber colored dropper vials. results: tablet formulations were evaluated for thickness ( . - . mm), weight variation ( - mg), hardness ( . - . kg/cm ), disintegration time (not more than min.), and biologically active content ( %- % of the stated label claim). in-vitro dissolution test was performed as per usp using distilled water as the medium and the release was shown between to % in minutes. the liquid formulations were analyzed for ph ( . - . ), the biological content ( % - % of the label claim), specific gravity ( introduction: virtually all patients suffering from the common birch pollen allergy exhibit ige against the bet v relevant allergen. as such, an elisa method for the quantification of bet v was selected and validated as part of the bsp project, aiming to establish reference methods for the european pharmacopoeia. herein, we report the mapping of the epitopes recognized on recombinant bet v allergen by the two specific murine monoclonal antibodies (mabs) used for the accurate and precise quantification of bet v within birch pollen extracts. objectives: in order to investigate the ability of mabs b and h to recognize various bet v isoallergens, we first carried out immunochromatography combined with electrophoresis. epitope mapping was performed by hydrogen/deuterium exchange (hdx) coupled with mass spectrometry (ms) analysis, using a gmp-grade purified rbet v molecule. results: immunochromatography unveiled that both mab b and mab h capture most of bet v isoallergens present within birch pollen natural extracts. those two mabs cross-react with the aln g allergen from alder pollen but do not react with the cas s chestnut pollen allergen. hdx-ms experiments combined with site-directed mutagenesis evidenced that mabs b and h target two distinct epitopes. the hdx-defined b epitope is discontinuous and contains a dominating sequential element (i.e., loop ile -lys ). the hdx-defined h epitope is also discontinuous and mainly composed of regions ile -lys and arg -phe . conclusions: overall, this study provides a precise molecular characterization of epitopes within bet v recognized by mabs b and h , confirming that these two antibodies target distinct non-overlapping epitopes and recognize the vast majority of currently introduction: short or common ragweed (ambrosia artemisiifolia), belonging to the aster plant family, sheds enormous amounts of highly allergenic pollen late in the summer. due to its high allergenic potential ambrosia artemisiifolia is becoming a health threat in north america and europe. hal allergy is developing a subcutaneous immunotherapy for patients suffering from ragweed pollen allergy. a standardized ragweed pollen extract, chemically modified and adsorbed to aluminium hydroxide (al(oh) ), is being investigated for its potential use in immunotherapy. objectives: ragweed extract (re) was modified by glutaraldehyde followed by adsorption to al(oh ). in vitro, a mediator release assay (mra) using hurbl (humanized rat basophil leukemia) cells was performed. hurbl cells were pre-sensitized using individual sera of ragweed-allergic patients and challenged with serial dilutions of re and modified re starting at lg/ml followed by eight / dilutions ( - . ng/ml). antigen-specific release of ß-hexosaminidase was measured and calculated in relation to % release values. in vivo, the immunogenic potency of modified re was evaluated by measuring the induction of re-specific igg in mice. female balb/c mice (n= per group) were subcutaneously (s.c.) injected with . aueq/ml re or modified re adsorbed to al(oh) ( . mg/ml) per mouse on days , , and . control mice (n= ) were injected with matrix only. specific igg titres were determined in serum obtained at days - , and . results: the potency of modified re in mra was drastically reduced in all patients, with a mean reduction of fold or more. chemical modification resulted in a later onset of activation as well as a lowering of the maximum release of ß-hexosaminidase. in vivo, both re and modified re show comparable levels of re-specific igg antibodies in mice at day of the immunogenicity model. shown that chemical modification impairs the capacity of re to activate basophils while retaining its capability to be immunogenic. therefore, chemical modification of re may be a promising approach for the development of a safe and effective immunotherapy for ragweed allergy. objectives: the children who had taken subcutaneous conventional venom immunotherapy in our pediatric allergy outpatient clinic between and were evaluated with respect to the side effects. in addition, each child was called to ask if the patient was exposed to bee sting and the result of a sting during immunotherapy. introduction: the major unmet needs for allergen immunotherapy (ait) are improved efficacy with good tolerability, and high adherence. to achieve these, allergoids, peptides and recombinant proteins are potentially the answer but their low rate of systemic aes make selection of the optimal dose difficult. to select the dose for an ultra-short course subcutaneous birch ait, the company has adopted the use of a conjunctival provocation test (cpt), a wide range of doses and the multiple comparison procedure -modelling (mcp-mod) statistical analysis to test for a dose response and to determine the shape of the dose response curve. objectives: a range of dose regimens of su, , and su were compared with placebo with respect to reduction of total symptom score elicited by cpt after treatment. patients were administered weekly injections outside the pollen season. cpt was performed at screening, at baseline and weeks after completion of treatment. the study was undertaken in sites in germany and austria with patients. the primary efficacy analysis was performed on a modified full analysis set (fas). the mcp-mod methodology was used to test for a dose-response using the placebo and above doses to describe the shape of the dose response curve. three candidate models were pre-specified: a maximum possible effect for the agonist (emax) model, a logistic model, and a linear in log-dose model. a statistically significant dose-response (p<. ) was shown for the range of cumulative doses, which approached a plateau with the su dose. the median effective dose (ed- ) was su. only minor differences were observed between the six active treatment groups in prevalence of treatment-emergent adverse events (between . % and . % of patients with overlapping % two-sided confidence intervals), majority of which were local reactions, short-lived and mild. teaes classified as systemic reactions were seen in . % ( su group) and in up to . % ( su group) of patients in the active treatment groups, and in . % of patients in the placebo group. no treatment related saes were observed. adherence was > % in all treatment arms. the ed- was su, demonstrating that the currently marketed dose ( su) is effective. the highest su dose will be further investigated in a pivotal phase iii trial having achieved an increase in efficacy by % without differences in the onset of aes between the treatment arms. introduction: in order for allergen immunotherapy (ait) to induce long-term immunological and clinical effects prolonged administration is required. therefore adherence to treatment is crucial for its efficacy. there is currently limited data available on ait adherence beyond clinical trials i.e. in real-life clinical practice. objectives: this eaaci immunotherapy interest group endorsed survey aimed to prospectively evaluate adherence to sublingual and subcutaneous immunotherapy in adults with allergic respiratory diseases and hymenoptera venom allergy in real life practice across different european countries. in addition, the reasons for lack of adherence and discontinuation of treatment were explored. this was a prospective, multi-centre, observational survey which took place in eight countries: czech republic, georgia, germany, greece, italy, poland, portugal and spain. data collection involved an online survey that followed participants four-monthly for a period of months from the start date of ait. results: a total of participants were included in the analysis. introduction: allergic rhinitis is a multiple gene-regulated disease involved in many immune cells such as mast cells and eosinophils, and various inflammatory mediators, and mirna probably plays a critical regulatory role in its pathogenesis. therefore, studies on the functions of critical mirna and its regulatory mechanisms in activated mast cells will lay an important theoretical foundation for our understanding of ar pathogenesis and the development of therapeutic strategies. objectives: to investigate the effect of mir- a- p on mast cell activation in an ar mouse model. the number of sneezes and the frequency of nasal rubbing in ar+mir- a- p group were significantly reduced compared to those for ar+mir-nc group (p<. ). histological examination showed that inflammation in the nasal mucosa from ar+mir- a- p group was slighter than that in ar+mir-nc group. the number of mast cells in ar+mir- a- p group was increased compared to ar+mir-nc group (p<. ). the levels of histamine and il- in nasal lavage fluid supernatants, histamine in plasmas and il- in sera were significantly decreased in ar+mir- a- p group compared to ar+mir-nc group (p<. ). conclusions: upregulation of mir- a- p can reduce allergic inflammation in the nasal mucosa of ar and alleviate ar symptoms through inhibiting mast cell activation in vivo. mir- a- p probably becomes a new target for gene therapy of ar. | correlation between chronic cough and chronic rhinosinusitis in adults: nationwide, population-based, and cross-sectional study the second hospital of shandong university, ji nan, china; national university of singapore, singapore, singapore introduction: nasal polyp implies a refractory clinical course in case of chronic rhinosinusitis (crs). although hypoxia is believed to be associated with nasal polyposis, little is known about the mechanism underlying polypogenesis. objectives: the aim of this study was to assess mrna and protein introduction: nasal polyp is a multi-factorial disease commonly associated with inactive ciliary beating frequency (cbf), a condition partly attributed to the mis-localization of dnah , a component of the outer dynein arm in ciliary axoneme. so far however, there have yet to be a systematic histopathological investigation directly linking dnah localization pattern in nasal polyps. therefore, we sought to examine the localization of dnah in cilia structure of both nasal polyps and inferior turbinates from healthy individuals, and assess whether there are any localization changes that can account for the extensive inactive cbf observed in nasal polyps. objectives: the focus of this work is to compare the localization of dnah from the nasal polyps biopsies (n= ) and normal inferior turbinates (n= ) by immunofluorescence. the characterization of each sample was obtained from an average of fields at magnification. results: from the samples, we observed three distinct localization patterns of dnah in the nasal cilia. the three patterns were as follows: ) the localization of dnah in normal cilia is present throughout the entire axoneme (pattern a); ) the localization of dnah is within the axoneme except at their proximal regions (pattern b); ) the localization of dnah is restricted exclusively at the ciliary base and not present in the entire axoneme (pattern c). approximately % of the samples exhibited more than one distinct localization patterns for dnah within the observed fields. the percentage of pattern a, pattern b and pattern c were observed in . %, . %, and . % fields for samples from nasal polyps. correspondingly, . %, . %, and . % were observed for samples from healthy controls. the results indicated that the predominance of "pattern c" in nasal polyps countered by "pattern a" in inferior turbinates from healthy individuals. conclusions: our study indicated that there is a significant increase in the mis-localization of dnah among the cilia in nasal polyps as compared to controls. this mis-localization may account for the inactive cbf, a hallmark characteristic, observed in nasal polyps. zhao l ; zhi l ; jin p ; zi x ; tu y ; li a ; li t ; shi l ( . , . - . , p<. ) and +gc np patients ( . , . - . , p<. the results showed that the number of th + cells were correlated with eosinophil cells and macrophage (r=. , p<. , r=. , p <. ), but no correlation was found between th + cells and neutrophil cells. the significantly correlation were found between il- objectives: this study aimed to reveal if some features of the sinus wall and content (as homogeneity and density of the sinus content, or the continuity, thickness and density of the sinus wall), differ between the afrs and other forms of crs. we tried also to establish early diagnostic parameters for recognition of fungal rhinosinusitis on ct. results: the study included adult patients (mean age: . ae . years, m:f ratio= . : ) with clinically diagnosed crs. out of all maxillary sinuses (n= ) from study patients, ( . %) were opacified, and only these sinuses were included in further analyses. we found out that: ( ) positive fungal finding had % ( / ) crs patients and % ( / ) of these patients had severe forms of crs, ( ) patients with positive fungal finding had more often positive specific ige ab than those without fungi in sinuses ( . % vs. . %, p=. ), ( ) foci of non-homogeneity, mean and maximum densities and wall density were more common found in maxillary sinuses with present fungi than those without fungi (p=. , p=. , p=. ; respectively) and ( ) patients with crs lasting more than years had more often foci of non-homogeneity and presence of hyperattenuation centres, than patients with shorter length of crs. results: neutrophil-related gene mpo and eosinophil recruitment genes ccl and ccl showed higher expression level in acp than in controls, which were in line with the significantly elevated neutrophils and eosinophils infiltration in acp compared to control. increased cd + t cells, macrophages and cd + t cells infiltration in acp were also observed. the expression level of t-reg transcription factor foxp was significantly higher in patients with acp than in controls, but the expression of th /th /th transcription factor tbet, gata and rorc were significantly decreased in acp vs controls. we further investigated the relationships between these t-cellassociated genes in acp. the expression of foxp was positively correlated with t-bet, gata , il r and il a, while no significant correlation with rorc was evident. il was observed positively correlated with t-bet, gata , foxp , and il r. il had significant correlation with t-bet and il a. objectives: the aim of this study was to find the olfactory change pattern of crs after ess in short-term and the differences between crswnps and crssnps, secondary aim were to identify the relationship among olfactory dysfunction, ct scores and quality of life(qol). in this study, crs patients who underwent ess were evaluated preoperative by t&t recognition threshold tests, snot- score and lund-mackay ct score. patient outcomes were re-evaluated at clinical follow-up month, months and months postoperative. analysis of variance was performed and correlation was calculated, with results analysed separately for crswnp and crssnp subgroups. . subgroups of crs differed in the degree of olfactory dysfunction reported before and after the ess. a significant difference in the changes of olfactory dysfunction between the two groups was found at month postoperative. . the mean t&t and snot- scores showed significant improvement within months after ess in both crswnp and crssnp subgroups, however, no significantly recovery of olfactory dysfunction was observed at months compared to month postoperative. there is a plateau of olfactory recovery at months postoperative. . in crswnps, the mean t&t scores preoperative were correlated with lund-mackay ct score significantly(r=. , p<. ; r=. , p<. ). however, no relations were found in crssnps and the changes of olfactory dysfunction at the months postoperative with lund-mackay ct score. . olfactory scores, before and after the ess, and their changes did not correlate with sont- scores. conclusions: olfactory dysfunction was more severe in crswnps. olfaction and qol of crs patients were significantly improved after ess in both groups, but there was a plateau of olfactory recovery at months postoperative. ct scan may predict olfactory disorder, but the olfactory scores were not related with the qol. objectives: in this study, we investigated the effect of hgf, tgf-b , and pge as effective components for allergic rhinitis treatment using in vitro and in vivo mouse model studies. results: pge decreased infiltration of eosinophil in nasal mucosa. tgf-b decreased the infiltration of eosinophil in nasal tissue and increased the number of treg in spleen. however, there was no antiallergic effect of hgf in this experiment condition. in case of the combination treatment group (tgf-b +pge +hgf), eosinophil infiltrations and the expression of eotaxin- were reduced in the nasal tissue, and treg was increased in the spleen. in all treatment group, serum ige and systemic cytokine levels were not decreased due to intranasal administration rather than systemic administration. in vitro study showed that phosphorylation of map kinases such as erk, jnk, and p and translocation of p were inhibited after treatment of hgf, tgf-b and pge , suggesting their anti-allergic mechanism. conclusions: we found that tgf-b , and pge decreased allergic inflammation and these effects might be derived from changes in the frequency of treg and the activation of map kinase and p in the t cell receptor signaling pathway. furthermore, we hypothesized that tgf-b , and pge would be effective components for allergic rhinitis therapy. introduction: interleukin (il)- is implicated in suppression of allergic inflammation. the role of il- in the early-phase reaction in type hypersensitivity has been unclear, however. we investigated the contribution of il- in a mouse model of the ige-mediated early-phase reaction in allergic conjunctivitis. objectives: ige-mediated allergic conjunctivitis was induced in c bl/ -kit(+/+) wild-type mice, kit(+/+) il- -deficient mice, and kit(w-sh/w-sh) mast cell-deficient mice by means of passive conjunctival anaphylaxis. the mice were thus subjected to subconjunctival injection with anti-dinitrophenol ige (dnp-ige) followed after h by intravenous injection with dnp antigen. kit(w-sh/w-sh) mice that had received a subconjunctival graft of cultured bone marrowderived mast cells from kit(+/+) wild-type mice or kit(+/+) il- deficient mice were similarly treated. vascular permeability of the conjunctiva was examined min after antigen injection by colorimetric evaluation of the extravasation of evans blue dye. results: passive transfer of dnp-ige followed by intravenous antigen injection increased vascular permeability in the conjunctiva of kit(+/+) wild-type mice but not in that of kit(w-sh/w-sh) mice, suggesting that this effect was dependent on mast cells. vascular permeability was increased to a significantly greater extent in kit(+/+) il- -deficient mice than in kit(+/+) wild-type mice. reconstitution of kit(w-sh/w-sh) mice with kit(+/+) wild-type or kit(+/+) il- deficient mast cells restored the dnp-ige-and dnp-induced increase in vascular permeability to similar extents. our results suggest that il- produced by cells other that mast cells suppresses the mast cell-mediated early-phase reaction in ocular allergy. objectives: our aim was to evaluate the effectiveness and the safety of ccl treatment for keratoconus in children with vkc. forty-two boys (mean age . ae . years) and girls (mean age . ae years) with vkc were included in the study. tarsal, limbal and mixed vkc were detected in . %, . % and . % of the subjects, respectively. evaporative dry eye was detected in children out of ( . %), schirmer test results were < mm/ minutes in . % and < mm (severe dry eye) in out of children ( . %) and % of the subjects (n= ) had confirmed keratoconus/forme fruste keratoconus with corneal topography (sirius, cso, italy). allergic symptoms were controlled with topical steroids, cyclosporine, dual action antihistaminic/mast cell stabilizers and lubricant agents before the procedure. ccl surgery was performed under topical anesthesia. the children were followed-up at least year and preoperative and postoperative corneal topographic parameters were compared using paired sample t test. results: the visual acuity was between . and . (moderate visual loss) in . % of the subjects and less than . (severe visual loss) in . % of the children. ccl procedure was performed to eyes of children. at the end of one year, the disease was stable in all children with no differences in k and k corneal parameters before and after cxl (p>. ). there was a statistically significant improvement in maximum keratometry value after the procedure (before . ae d, after . ae . d, p=. ). in one subject, a corneal infiltrate was detected days after ccl, which was treated successfully with topical moxifloxacin. otherwise, no complications were observed in the postoperative period. conclusions: as keratoconus is common in vkc, these children should be referred to ophthalmologists for an eye examination and corneal topography. ccl seems to be a safe and effective option to halt the progression of keratoconus, which might be very aggressive in children with vkc. results: surprisingly, we found among them cases of celiac disease, cases of thyroid dysfunction (thyroiditis), cases of crohn's disease and case of ulcerative colitis, case of anterior uveitis and case of pemphigus respectively. we realized that an average percentage of % of the total of vkc cases are affected by an "autoimmune" systemic disease. limbal form of vkc was prevalent and more than % of children showed it. we can suppose that a racial and genetic predisposition to systemic diseases can coexist with vkc or that there is a group of vkc affected subjects in which the immune disorder is predominant on the allergic disease. introduction: nasal polyposis (np) is a heterogeneous inflammatory disease of nasal mucosa affecting - % of the population with a high rate of recidivism. polyps arise from nasal sinuses to nasal cavity and are often associated with a strong local eosinophilia. the pathophysiology of np remains controversial, as it seems to be a phenotypic manifestation of multiple possible immunologic processes, such as respiratory allergy, despite a lack of correlated systemic response. here we propose a multiparametric assessment of np patients, in order to shed light on the underlying mechanisms of the disease in allergic and non-allergic patients and aiming to find new predictive biomarkers. objectives: our main aim was to unravel the link between systemic and local allergic inflammation and polyp development, as well as the nasal epithelium condition in polyps and surrounding healthy tissue. methods: four groups of patients were included in the study: healthy donors with or without allergy and np patients with or without allergy. in this regard, several different approaches were followed: a metabolomics serum study, a polyp and nonpolypous nasal epithelium histology and transcriptomic study. results: as for the histological study, luna staining revealed differences in eosinophilia between allergic and non-allergic patients, especially when patients were polysensitized, including perennial allergens; and between nonpolypus tissue and polyps being higher in polyps. pas staining showed differences in epithelium integrity and submucous and goblet cell (pas positive) distribution. immunohistochemistry for cd + and cd c+ reveal a significant inflammatory infiltration in polyps. this inflammatory response was also asses by abstracts | gene expression quantitation. no differences were seen in the metabolic profile in patient sera between groups. for the first time, nasal epithelium from polyps and neighboring tissue were studied. histology techniques and image analysis revealed differences in eosinophil concentration in both mucosa and submucosa areas, as well as different features in epithelium and submucous tissue structure. some of these findings were confirmed by gene expression quantitation. conclusions: our data show an increased eosinophilia and inflammatory infiltration in polyp tissue suggesting a role for allergic inflammation in the progression of np. additionally, we provide clues for the role of inflammation in the damage on nasal mucosa and the following progression of the disease. | is specific immunotherapy effective in subjects suffering from vkc? a tertiary referral center ten years experience. objectives: our work shows the results of sit additional to usual treatments, in children suffering from vkc and followed in our tertiary referral center (lavagna hospital, genova, italy). we retrospectively analyzed the clinical data of subjects ( males and females); their mean age at the beginning of treatment was ae . years. the patients were treated both with scit (sub-cutaneous immune-therapy- . %) and slit (sub-lingual immune-therapy - . %) depending on patient's wishes. they had to be mono-sensitized to one of the usually more frequent allergens (dust-mites, grass pollen, and pellitory) which was detected by means of recombinant rast, prick test and conjunctival provocation test (cpt); these tests were performed after a complete ophthalmological and allergological history and examination. children selected for sit needed to be positive to all performed allergy tests. systemic involvement included cases of asthma, cases of atopic dermatitis and case of rhinitis; the remaining cases were asymptomatic. local involvement included only vkc cases, the % of which were of the limbal type and only subjects were suffering from the tarsal papillary type. mean follow-up was . years. all the patients included into the study completed their treatment and followed the therapeutic protocol. after one year of sit, no variation in clinical course and treatment was recorded. after the third year of sit, an average improvement in symptoms and signs score ( %) and an average decreased need for allergy systemic medications ( %.) (i.e. antihistamines and corticosteroids) was registered. also topical therapy (including steroid and cyclosporine eye-drops) was discontinued in % of children, in this group, short courses of steroid drops were necessary in less than % of children (as rescue treatment in the acute phases of the disease). these positive results after sit treatment were stable for the following years. few (only local sub-cutaneous) side effects were recorded and the treatment was generally well-tolerated. conclusions: our experience shows positive results with sit in vkc which can have sensitive-to sit-treatment subtypes. results: deaths occurred in children ( boys, %). median age at death was years (iqr - ). pamr of any cause was . ( %ci, . - . ) per children per year, with a decreasing rate over time (annual change: - . %; %ci, - . to . ). triggers were iatrogenic causes (n= , %), insect venom (n= , %) and food (n= , %). unspecified causes were frequently reported (n= , %). there was no difference in overall pamr between boys and girls (p=. ). there was no age group related differences in fatalities: preschool children (< years) (n= , %), school children ( - years) (n= , %), adolescent and toddlers (> years) (n= , %). the number of fatal cases was similar comparing the southern (n= , %) and the northern regions of france (n= , %) (p=. ). the first episode of anaphylaxis for each patient was captured to calculate incidence. we estimated incidence rate ratios using poisson regression models. results: between and there were anaphylaxis episodes in patients younger than years in hong kong. the incidence of admission for anaphylaxis increased markedly from . to . per person-years during the study period (p<. ). the incidence of food-related anaphylaxis increased significantly from . to . (p<. ). increases in anaphylaxis and food-related anaphylaxis were seen in all age groups, with the largest increase in those aged to years. at the beginning of the study period ( ), medication was a more common trigger for anaphylaxis than food ( . vs . per person-years). by , food had become the predominant trigger ( . vs . per personyears for medication). the incidence of medication-related anaphylaxis decreased significantly (p<. ). the incidence rate of anaphylaxis was significantly higher in boys than girls in the - and - year age groups, while there was no significant gender difference in the - year age group. the most common food triggers of anaphylaxis were peanuts, seafood, eggs, milk products, tree nuts & seeds (in descending order). conclusions: even though the incidence of anaphylaxis among children in hong kong is lower compared with other western countries, it has recently increased significantly, with food-related anaphylaxis predominant. | prevalence of anaphylaxis and prescription rates of epinephrine self-injector in korea based on national health insurance data results: the prevalence of anaphylaxis over the years were . %. the annual prevalence of anaphylaxis increased over the years. anaphylaxis was more prevalent in male than female ( % vs. %) and in population aged - years old. for the regional prevalence of anaphylaxis in korea, gangwon province showed the highest prevalence of anaphylaxis ( . per individuals) and relatively low prescription rates ( . %) of epinephrine self-injector for the patients with anaphylaxis. on the contrary, seoul showed relatively low prevalence of anaphylaxis ( . per individuals) and the highest prescription rates ( . %) of epinephrine self-injector for patients with anaphylaxis conclusions: the prevalence of anaphylaxis has increased annually in korea. the prevalence of anaphylaxis and prescription rates of epinephrine self-injector showed regional difference in korea. objectives: the aim of the study was to analyze the prevalence of allergic symptoms and anaphylaxis in mastocytosis patients analyzed in the registry of the ecnm. results: methods: a total of patient with mastocytosis were enrolled. in these patients, the prevalence of allergy, anaphylaxis, triggers of allergic reaction, and disease subtypes were analyzed. results: symptoms of allergy were observed in % of all patients. the most affected group were patients with bone marrow mastocytosis (bmm: . %) and indolent systemic mastocytosis (ism: . %). insect venom allergy (iva) was reported in . % of all subjects. in ism/bmm patients iva affected . % of the cases, while in other patient groups, only . % of the cases were affected (p<. ). most patients ( %) had wasp allergy, % had bee allergy, % polistes allergy, and . % allergy to more than one venom. in . % the culprit insect was not identified. food allergy was reported in . %, drug intolerance/allergy in . %, inhalant allergy in . %, and physical triggers in . % of patients. in mastocytosis patients iva is the most prevalent cause of anaphylactic reactions exceeding the prevalence of iva in the general population by far. iva affects mainly bmm and ism patients ( . % of cases). but ( . %) subjects didn't know whether adrenaline was administered. only within patients who had adrenaline autoinjectors used their autoinjectors during an anaphylaxis attack. most common symptoms were skin (n= , . %) and respiratory symptoms (n= , . %). syncope, hypotension or hypoxemia were present in cases( . %), at least three organ dysfunctions in ( . %) cases; patients ( . %) had to be hospitalized (f: , m: ).nearly a third ( . %) of the patients had stage - anaphylaxis and patients( . %) had stage - reactions. in cases ( . %), basal tryptase levels were examined and the average value was correlated with the severity. concomitant drugs being used by the patients were antihypertensives ( . %),oral antidiabetics ( . %); angiotensin converting enzyme inhibitors or angiotensin receptor blockers( . %), beta blockers( . %), diuretics( . %) and nsaid's ( . %). conclusions: male sex was noted as a risk factor for severe reactions and recurrent anaphylaxis. anaphylaxis requiring hospitalization was more frequent in the patients using oral antidiabetics or diuretics. baseline tryptase levels were higher in patients with neurological and gastrointestinal symptoms. cardiovascular symptoms were found to be higher if a cofactor was present. skin symptoms were seen more frequently and higher rate of hospitalization occurred in anaphylaxis in the presence of infections or nsaid use. this study is important to elucidate the factors affecting anaphylaxis severity. | serum levels of a, ß-pgf in combination with apolipoprotein a or cysleukotrienes are reliable biomarkers of anaphylaxis objectives: we analyzed mast cell mediators in sera derived from patients with acute anaphylactic symptoms (n= ) versus patients with acute cardiovascular or febrile reactions (n= ) and patients with a history of anaphylaxis but without displaying any symptoms when sera were taken (n= ). in addition, we identified proteins with substantial changes during anaphylaxis. matched serum samples were used to compare basal mediator levels with corresponding levels during acute anaphylaxis in the same patient (n= ). roc curve analysis was performed to determine the sensitivity/specificity of each mediator. results: serum levels of histamine and tryptase were not increased upon anaphylaxis and showed no relation to anaphylaxis severity. however, serum a, ß-pgf , a metabolite of pgd , was significantly increased in acute anaphylactic patients (~ -fold) and abstracts | correlated well with anaphylaxis severity. a, ß-pgf distinguished anaphylaxis from cardiovascular or febrile reactions and showed the highest diagnostic power observed by roc curve analysis. cys-leukotrienes (cys-lts=ltc , ltd , lte ) were increased upon anaphylaxis while apolipoprotein (apo) a was significantly decreased. the highest diagnostic power was observed with the combination of a, ß-pgf and apoa . conclusions: in conclusion, histamine might only be used to detect anaphylaxis when assessed shortly after onset of an anaphylactic response because of its short half-life, whereas tryptase is a useful biomarker if the baseline level of the same patient is known. a, ß-pgf seems to be the most reliable marker as demonstrated by the distinct increase upon anaphylaxis and could be supported by apoa or cys-lts. further investigations are needed to prove the suitability of these markers. objectives: objective of this study was to estimate the long-term bv of tryptase using certain chronic disease models and to compare it with those in food and drug allergy. results: serial determinations of tryptase concentrations (n≥ data points per patient) obtained from patients diagnosed with mastocytosis (n= ) or chronic urticaria (n= ) during a period of sometimes several years were measured by the immunocap assay and evaluated using sigmaplot software. polynomial curve fitting was performed and data points outside the % confidence interval of the curve were appointed as outliers and excluded. because the data points were not normally distributed due to long-term fluctuations in homeostatic set-point, a non-linear fitting was applied and used to compute the standard error of the estimate. these standard errors of the fit divided by the estimates themselves were used to calculate the total coefficient of variation within a subject (cv t ). the analytical cv (cv a ) was calculated based on quality control samples ( levels, n= data points) in a conventional way, while the within-subject bv (cv i ) was defined as cv l =(cv t À cv a ) ½ . eleven patients with a chronic disease were selected, of which patient was treated as a potential outlier and patients had to be excluded because of cv t <cv a . the between-subject bv (cv g ) was without the outlier . ae . % (n= data points during a period . ae . years) and with the outlier . ae . % (n= data points during a period of . ae . years). as a food and drug allergy control group additional patients were selected with an uncomprehended food (n= ) or drug allergy (n= ). this cv g was calculated under the same conditions and proved to be . ae . % (n= data points during a period of . ae . years). the long-term between-subject biological variation of tryptase concentrations of patients in chronic disease models was smaller than that of patients with an uncomprehended food or drug allergy. chronic disease models may be of interest to determine the long-term "physiological" biological variation of tryptase. vilà-nadal g; rodr ıguez-sanz a; s anchez-villanueva r; fiandor-roman a; dom ınguez-ortega j; bell on-heredia t introduction: hypersensitivity reactions during hemodialysis sessions have been reported. our aim was to study the mechanisms of the allergic or "pseudoallergic" reactions to synthetic polysulfone (ps) dialysis membranes in "ps-allergic" patients who tolerated cellulose triacetate (cta) membranes. objectives: ten patients with adverse reactions to ps and control non-allergic patients in hemodialysis were studied. ml of blood were collected and an ex-vivo hd were performed on experimental external circuits with low or high priming volumes and pediatric membranes (fx-paed helixone . m and sureflux). serum tryptase levels, basophil degranulation (hla-dr À cd + cd + leukocytes), and t cell activation (cd expression on cd + and cd + subpopulations) were analysed in pre-and post-dialysis samples. objectives: the aim was to investigate the influence of asa and alcohol on egg allergy. donor sera from egg allergic patients (i-iv) with s-ige to ovalbumin at (i) . , (ii) . , (iii) . , and (iv) ku/l were injected intracutaneously to the forearm of healthy/nonallergic volunteers. they were then orally challenged separately with: ) egg white ) egg white + asa and ) egg white + alcohol. 'time to wheal' and 'wheal size' were compared between the three experiments. results: two sera (i-ii) with the lowest s-ige to ovalbumin did not react sufficiently and where excluded from further analysis. the remaining sera (iii-iv) significantly decreased 'time to wheal' with both asa (p=. ) and alcohol (p=. ) added as co-factor compared to baseline. increase in 'wheal size' was only found with co-factors added for the less reactive serum (iii) and not for the serum with the highest s-ige (iv). introduction: eaaci have published numerous guidance documents providing evidence-based recommendations for the recognition, risk factor assessment and management of anaphylaxis. these guidelines clearly advocate intra-muscular adrenaline firstline for the treatment of anaphylaxis, and list absolute indications when an adrenaline auto-injector (aai) must always be prescribed; eg for those who have previously experienced an anaphylactic event, those who have a mast cell disorder and for those with co-existing unstable or moderate/severe asthma and a food allergy. furthermore, eaaci recommends that an aai should be prescribed to those at risk of anaphylaxis (termed relative indications), and that aais should be prescribed and carried by patients at all times. despite widespread dissemination of these guidelines, anaphylaxis hospitalizations continue to rise in many european countries. objectives: in this environment eaaci developed the anaphylaxis survey (axis) as part of its allergy awareness initiative to gain insight into guideline awareness. this survey was sent by email to eaaci members. members from countries responded. data were anonymous and analysed descriptively. results: . % (n= ) reported familiarly with the eaaci anaphylaxis management guidelines, and of these . % (n= ) reported following them. . % (n= ) responded that they would use adrenaline firstline for the treatment of anaphylaxis (although . % (n= ) would still administer it subcutaneously). however, when confronted with a list of indications, only . % (n= ) of eaaci members would prescribe an aai for all absolute indications. furthermore, although . % (n= ) of respondents considered they would prescribe an aai for those at risk, when provided with a list of relative indications, % would prescribe an aai for all of them. there was also a lack of consensus about when an aai should actually be used, with . % of respondents (n= ) considering aai use when individuals experienced breathing difficulties and felt that they were going to collapse. conclusions: these results reflect the latest findings that aais are under-prescribed in daily practice. there appears to be a mismatch between perception of guideline compliance and aai prescription behaviour. continued training, further simplification and wider dissemination of guideline messages are required. objectives: the aim of this study was to analyse the clinical practice of emergency physicians concerning the management of anaphylaxis in alsace and lorraine, a region of . million inhabitants, in france via an electronic survey. results: the response rate was . % ( ). fifty percent of physicians were female. . % ( ) ( ). in case of grade reactions, intramuscular adrenaline injection was performed by . % ( ) of physicians as first-line treatment, intravenous adrenaline injection by . % ( ), antihistamines (oral route or iv) by % ( ) and corticosteroids (oral route or iv) by . % ( ). in case of grade reactions, intramuscular adrenaline injection was performed by . % ( ) of physicians as first-line treatment, intravenous adrenaline injection by . % ( ), iv antihistamines by . % ( ) and corticosteroids (oral route or iv) by % ( ). following an acute anaphylaxis episode, only . % ( ) of physicians systematically recommended an allergy assessment, . % ( ) sometimes, and . % ( ) never. conclusions: this study highlights the discrepancies between current guidelines for the management of anaphylaxis in emergency departments and common clinical practice. our results emphasised the need for specific programs to improve clinical management of anaphylaxis in ed. conclusions: a significant number of individuals among those surveyed reported a severe allergic reaction requiring an eai, yet less than a quarter used an eai. the study also identifies most common challenges faced by at-risk individuals including eai availability, cost, and concerns over short expiry dates. objectives: to evaluate the implementation of a stock epinephrine (stock epi) program in a mall setting. stock epinephrine devices are not prescribed for a particular person and can be used in anaphylactic emergencies. methods: a mixed methods approach was used to evaluate the implementation of the stock epinephrine program regarding its acceptability and adoption by diners, food service, mall administration and security personnel; and the appropriateness, costs (included costs for personnel, epinephrine auto-injector devices, and training materials), fidelity, reach, and sustainability (measured by the national health service (nhs) sustainability model, united kingdom) of the program in this setting (hamilton, ontario, canada). the study revealed that individuals with food allergy and food service establishments were in favour of a stock epi program. a total of individuals with food allergy completed our national online survey. approximately % dine out once or twice per month, and one-third had experienced an allergic reaction. we also completed in-person surveys with diners at the mall food court. of these, % had a food allergy and % reported carrying their epinephrine auto-injector when dining out. the mean nhs sustainability score was among survey participants, well above the mean considered a sustainable program (> ). total cost of the stock epi program over a one-year period varied by ontarian stakeholders: $ for the mall, $ for fast-food restaurants, and $ for sitdown restaurants. conclusions: this is the first study to evaluate the implementation of a stock epi program. the stock epi program was well received abstracts | and sustainable. implementing a stock epi program provides enhanced access to emergency medication, however it does not replace the responsibility of individuals with food allergy to self-manage. objectives: to identify the optimal needle length for epinephrine prefilled syringe. results: three hundred seventy-two children aged month to years were enrolled. skin to muscle depth (stmd) and skin to bone depth (stbd), which can represent the minimum and maximum needle length respectively, were measured using an ultrasonography at the mid-anterolateral thigh. number of children who had stbd less than needle length (too long needle) and stmd greater than needle length (too short needle) were calculated. one hundred thirty-seven children weight < kg, children weight > - kg, and children weight > kg were enrolled: ( . %) children were male. one inch needle was too long in ( . %) children weight < kg, ( . %) children weight > - kg. it was too short in ( . %) children weight > kg. age≥ months, weight≥ kg, height≥ cm, bmi≥ kg/m and thigh circumfer-ence≥ cm, provided the sensitivity of - % in predicting the appropriateness for using inch needle for children weight < kg. in children weight > kg, thigh circumference≥ . cm provided the sensitivity of % and specificity of % for predicting the inappropriateness for using inch needle. objectives: we present a patient with a probable mdh syndrome to unusual drugs, including ah and cct. results: we report a case of a -years-old female, with history of moderate-persistent asthma and chronic urticaria, who experienced angioedema and exacerbation of urticaria hours after the administration of multiple ah (desloratadine, loratadine, cetirizine), systemic cct (hydrocortisone, methylprednisolone, deflazacort) and nonsteroidal anti-inflammatories (nsaids) (paracetamol, ibuprofen, flurbiprofen). patch tests (pt) with excipients (bial arestegui ® ) and drug provocation test (dpt) with placebo were negative. skin prick tests (spt) and intradermal tests (id) with hydroxyzine, hydrocortisone, methylprednisolone and prednisolone were positive to hydroxyzine ( mg/ml) and pt with corticosteroids (bial arestegui ® ) and hydroxyzine were negative. dpts with desloratadine and an alternative ah, dimetindene, were positive with facial angioedema and generalized urticaria within hours. lymphocytic transformation test (ltt) was positive to desloratadine, ebastine and clemastine. dpt with dexamethasone was negative, however, when administered as treatment, a reproducible reaction occurred. since dpt with montelukast was also positive, omalizumab mg was initiated to control angioedema and chronic urticaria. after year of treatment, dpt with nimesulide was negative. omalizumab dose was reduced to half after the patient found out she was pregnant. there were no further episodes after anti-ige therapy introduction and pregnancy went uneventfully. conclusions: mdh syndrome is rare, more so when the drugs reported are ah and cct. hr to ah was confirmed, but diagnostic workup remains incomplete, postponed due to the patient's pregnancy. this case is as challenging in terms of diagnosis as it is in terms of therapeutic, so much so that omalizumab was initiated as an off label therapy, maintained during pregnancy based on the premise that risk was lower than benefit. objectives: in this study, we aimed to present our patients who were admitted with oral iron hypersensitivity. conclusions: according to our clinical experience, we think that oral iron salts with different conjugates are safe and acceptable option in patients with suspected oral iron hypersensitivity. introduction: antineoplastic agents are consider nowadays an essential treatment for many kinds of cancer. the increased use of these drugs in recent years is in parallel with a high rise of hypersensitivity reactions to them. these reactions range from mild to severe and as other allergic reactions, are not predictable. a nursing protocol in the desensitization schedules with these drugs is essential in allergy daily hospitals. objectives: the aim of this study are to describe a nursing protocol during desensitization schedules with antineoplastic agents carried out in our allergy unit in order to detect symptoms suggestive an allergic reaction during drug administration and to assess a correct intervention in case of reaction. conclusions: an appropriate nursing protocol in desensitization schedules with antineoplastic agents is essential in order to achieve the correct administration of the treatment in safety conditions. | long term clinical effects of aspirin desensitization in patients with nerd: comparison of maintenance doses of mg vs mg aspirin Çelik ge ; karakaya g ; erkekol f € o ; dursun ba ; gelincik a ; celebioglu e ; y€ ucel t ; yorulmaz i ; dursun e ; tezcaner Ç ; s€ ozener zÇ ; b€ uy€ uk€ ozt€ urk s ; kalyoncu f ; aydin Ö introduction: aspirin desensitization (ad) treatment has been shown to be effective in relieving the respiratory symptoms as well in reducing recurrency of nasal polyps in patients with nsaids exacerbated respiratory diseases (nerd). however, a conflict occurs about effective maintenance doses of aspirin on clinical parameters. objectives: in this study, our aim was to compare the effects of two different maintenance doses of aspirin on clinical outcomes for years of ad. this was a multicenter study which involved tertiary centers. patients who completed at least one year of ad treatment were included to analysis. study outcomes were number of nasal surgery, sinus infections, asthma morbidity (number of severe asthma attacks, hospitalization) as well as medication uses for both clinical conditions. the study included subjects, of whom were under mg aspirin daily as maintenance treatment whereas remaining on mg aspirin for a mean of . ae months of ad duration. regardless of maintenance doses, number of nasal polyp surgery gradually decreased at ( . ae . /year) and years ( . ae . /year) compared to that of before ad ( . ae . /year) (p<. ) in all subjects and were comparable in and mg. considering asthma outcomes, decrease in asthma attacks were observed only in mg aspirin group (p<. ) at and years whereas hospitalization due to asthma and systemic corticosteroid use decreased in both groups at and years. conclusions: ad has a reducing effect on nasal polyp recurrence for at least years in patients with nerd. this effect was similar for both and mg maintenance doses of aspirin. considering both treatment arms provided decreased hospitalization due to asthma and systemic corticosteroid use, we think that at years evaluation both and mg/day aspirin has comparable effects on asthma as well. however, reducing effect of ad on asthma attacks was only existed in patients taking mg. aspirin. objectives: pregnant women with syphilis and history of immediate hypersensitivity reaction (hsr) to penicillin were enrolled. according to the risk stratification, which was based on the initial hsr, serum specific ige and skin tests, patients were re-exposed to penicillin either through desensitization or provocation. patients with a clinical history suggestive of penicillin-anaphylaxis and/or positive serum specific ige to penicillin and/or positive immediate skin test were considered at high risk and were desensitized. the remaining patients underwent penicillin provocation test. results: we evaluated pregnant women with latent syphilis and history of penicillin allergy. clinical history was suggestive of immediate hsr in out of these ( %) patients, who were desensitized. all of them had negative serum specific ige to penicillin. intradermal tests were positive in / ( %). three out of those four were desensitized with an oral protocol and reacted during the procedure. one patient had a severe breakthrough reaction with uterine contraction and did not finish the procedure. the only patient with positive intradermal test that didn't react during the rdd underwent an intravenous protocol. the remaining / ( %) patients had negative skin tests and an uneventfully rdd. there was a statistically significant association between positive intradermal tests and breakthrough reactions during the rdd (p=. ). the other / ( %) patients with inconclusive history and negative skin test were submitted to penicillin provocation, which were negative in all of them. conclusions: risk stratification based on the initial clinical reaction and skin testing to guide penicillin re-introduction was safe and effective, as well as rdd. skin testing identified allergic patients to penicillin with increased risk of reactions during rdd. | utility of basophil activation test for monitoring the acquisition of clinical tolerance after subcutaneous desensitization to brentuximab-vedotin in two patients many hypersensitivity reactions (hsrs) produced by biologic agents have been seen and their true incidence is unknown. desensitization is a method to counter hsrs from monoclonal antibodies in patients with no other adequate alternative options. objectives: we describe a successful rapid desensitization to bv in two patients with scleronodular type hl, refractory to several lines of chemotherapy and asct. this clinical tolerance to bv is easily observed through the basophil activation test (bat) as a decrease in activated basophils after desensitization was done as a treatment of hsrs. because there was no therapeutic alternative in the two patients, we planned to pursue bv administration using a rapid desensitization -step protocol. a total dose of mg of bv was given through increasing rate and concentration. the patients completed their infusion without difficulty. after desensitization to bv, bat was done in both patients. the percentage of activated stimulated basophils with bv descended in both patients. both values are similar to their corresponding negative controls. conclusions: the bat continues to be a useful in vitro tool for the study of drug allergic disease. also, the bat in flow cytometry is able to monitor an acquired tolerance induced by a desensitization treatment in hsrs to bv. however, studies involving a larger number of patients will be required to assess the safety and efficacy of this approach to bat as a method to validate rapid desensitization in patients with hsr to bv. objectives: we retrospectively reviewed desensitizations in patients with a history of ihsrs to chemotherapy agents performed in our center from january to december . the protocol consists of increases in infusion rate every to minutes, in a to steps depending on the drug. in all cases the protocol was performed without premedication (only using regular medication according to instructions for every drug). results: a total of patients with a history of (ihsrs) received desensitization protocol without premedication to chemotherapy agents. the most common involved drug was carboplatin in ( . %) patients (of these % presented positive skin test (st)), followed by paclitaxel in ( . %) patients (of these . % presented a positive st) and oxaliplatin in ( . %) patients (of these . % o the st were positive). other chemotherapy agents involved were cetuximab, rituximab, irinotecan, epirrubicin, etoposide, cisplatin and cyclophosphamide. all patients were able to successfully complete the desensitization protocol without premedication and none of them need to withdraw the drug. conclusions: this protocol for rapid desensitization to chemotherapy without premedication is safe and effective. in addition, minimizes secondary effects of the premedication in these patients that are polymedicated. | rapid desensitization for the management of hypersensitivity reaction to biologicals-infliximab and adalimumab in inflammatory bowel disease patients objectives: to identify barriers to best practice with regards to drug allergy history taking and documentation, and to elaborate the potential strategies to overcome them. results: a total prescribers responded to the survey: doctors in training . % consultants . % non-medical prescribers . % most respondents . % ( %ci . - . %) were not aware of the availability of penicillin allergy testing in our trust. among those that were aware of it, . % ( %ci . - %) had not referred any penicillin-allergic patient to immunology during the past year. barriers to accurate allergy history collection: . % ( %ci - . %) concurred that often it is not possible to draw a firm conclusion based on history alone. . % ( %ci . - . %) agreed with the statement saying that, regardless of the details of the allergy history, it is always better to "play it safe" and not to use alternative beta-lactams in patients labelled as being penicillin-allergic (figure will be attached in the poster). among the interventions proposed; practical educational sessions, an interactive questionnaire to guide allergy history taking and classification and a modified antibiotic policy to guide prescribing based on the allergy classification, were all rated as useful (average score > on a scale from -not useful at all-to -very useful). | the regulatory role of germinal center maturation during the early b cell response to inhalant allergens investigated in the piama cohort using the medall allergen microarray introduction: in contrast to common belief, igg to airborne allergens is higher in allergic subjects, even before immunotherapy. one of the confusing aspects of the allergic immune response is that not only the igg response, but also the ige response can follow more than a single trajectory (with or without gc maturation). for ige we assume that the direct isotype-switching pathway (igm to ige) is the most relevant for the initial, mature-gc independent phase of sensitization to low-dose airborne allergens (such a pollen, mite). in later phases and for higher exposure situations as well as for other immunization routes, indirect switching is assumed to be the more relevant pathway. methods: ige, igg and igg antibodies were measured using the medall allergen microarray in children from the piama cohort at age and . these results were analyzed in relation to the ige levels at age and . objectives: to find support for the hypothesis that the ige/igg ratio reflects not only exposure, but also details on immunological processes during sensitization, such as germinal center maturation. results: sigg and sigg levels to the major inhalant allergens were low at age and remained in general low at age . however, children who at that time were positive for ige an allergen had a significant increased sigg to the allergen in question. sigg also appeared, but this response was low. the sigg level at age in sigenegative children was not consistently predictive for sige at age . conclusions: the initial igg response to inhalant allergens is synchronized with the ige response. this result fits with the hypothesis abstracts | that in the initial phase of sensitization to inhalant allergens the allergen initiates a weak and incomplete germinal center response that allows parallel ige-and igg production. one of the consequences of the multiplicity of b cell developmental pathways is that the igg/ige ratio is potentially diagnostic: if a subject has sigg levels in the microgram range, and thus a high sigg/sige ratio, this indicates involvement of mature gcs and the indirect class-switching pathway for some or all of the sige in this subject. | synthetic allergoid consisted of plga nanoparticles covered with synthetic peptides from bet v objectives: the aim of this study was to test a hypothesis that the ahr signaling is critical in controlling sl homeostasis through the regulation of key sphingolipid enzymes involved in the s p synthesis. results: we found that an ahr ligand and a tryptophan photoproduct, -formylindolo ( , -b) carbazole (ficz; nm), induced a increase in s p level in a ros and ca + -dependent manner, leading to the degranulation as well as il- secretion in mast cells, when compared to those seen in vehicle-treated cells. this was concomitant with an increased level of sphk phosphorylation and with a reduction in the enzymatic activity of s p lyase, which could be reversed by the addition of an anti-oxidant, nac. moreover, s pl was found to be directly oxidized by ros in vitro and in vivo. conclusions: our findings suggested that ahr-mediated ros and ca + signals are critical for controlling sl homeostasis through regulating sphk and s pl metabolic pathways, providing a new regulatory pathway in mast cells. methods: peptide cytokine mimetics were selected by phage display technology. flow cytometry, elisa, elispot, t cell proliferation, reporter gene, mediator release, intravital microscopy and peritonitis assays were conducted to evaluate the capacity of the mimetic peptides to modulate the immune response. results: the synthetic tgf-b -like peptide was able to down-regulate the production of tnf-a, il- , ifn-c and il- , up-regulate il- , decrease basophil degranulation and induce t reg cell differentiation. furthermore, this peptide was able to decrease leukocyte rolling and neutrophil migration during an inflammatory condition in vivo. the synthetic il- -like peptide was able to decrease basophil degranulation and to inhibit the proliferation of allergen-specific t cell lines established from the peripheral blood of birch pollen-allergic patients. conclusions: the peptide cytokine mimetics tested herein, were able to modulate the immune response in the tested conditions. they, thus, represent promising novel candidates for therapeutic approaches. nonetheless, most studies focus on changes occurring early in life and there are rare data on differences in responses between allergic and non allergic subjects. objectives: we aimed to evaluate i) the maturation trajectory of the tlr antiviral pathway ii) if this trajectory varies between atopics and non atopics. peripheral blood mononuclear cells (pbmcs) were isolated from otherwise healthy atopic and non atopic subjects. atopy was assessed by medical history and skin prick testing to common aeroallergens and egg white. selected cytokines involved in the antiviral response were measured by luminex multiplexing technology in hour culture supernatants of poly:ic-stimulated pbmcs. data were analyzed by estimating the non-parametric correlation between age and cytokine expression in atopics and non atopics and comparison of regression curves for each cytokine between the groups was performed. results: the analysis comprised data from atopic and non atopic patients (mean age . years, age range - and mean . years, range - . , respectively). significant age-related increases in the production of ifn-a , ifn-c, il- b, il- a, tnf-a, and mip- b were found only in non atopics and of il- and il- in both groups. significant differences in the trajectories (slopes) of cytokine responses over time between atopics and non atopics were observed for ifn-a , ifn-c, il- , il- b, tnf-a and mip- b, with suboptimal production in atopics. conclusions: age-related increases in cytokines implicated in innate antiviral responses were observed mostly for non atopics. atopy was associated with suboptimal trl- induced cytokine responses. differences in the developmental pattern of those cytokines between atopics and non atopics may account for the reported increased susceptibility of atopics to infections. | a systems-immunology approach identifies a set of micrornas in shaping the th phenotype in allergic airway inflammation introduction: mouse allergy is a common disease in inner city households, affecting up to % of children who are exposed as determined by house dust analysis. it is associated with allergic rhinitis, atopic dermatitis and asthma and it has been reported that exposure and sensitization to mouse allergens is a strong predictor for asthmatic disease. despite a strong link between mouse exposure and asthmatic disease, the allergic immune response to mouse has been significantly understudied. to date, only one major allergen in mouse, mus m , has been identified and very little is known about the targets of the allergic immune response against mouse. objectives: using a proteomic/transcriptomic approach, we sought to identify t cell targets in mouse allergic and asthmatic patients. results: mouse urine and epithelial extracts were analyzed by d-ige/igg immunoblots using pooled sera from mouse-sensitized donors. mass spectrometry of selected protein spots identified novel antibody reactive proteins. predicted mhc binding peptides from these novel proteins and mouse homologs to mammalian allergens were screened for t cell reactivity in pbmcs from mouse allergic patients. overlapping peptides from the major mouse allergen mus m and its major urinary protein isoforms were screened in parallel. our screen for t cell responses in pbmc from mouse allergic donors demonstrated that major urinary proteins account for > % of the total t cell response but they are not the only target of mouse-specific t cell responses. reactivity to mouse peptides homologous to other mammalian allergens, specifically guinea pig, was also detected. conclusions: in summary, our data demonstrates that the cellular and serological targets of the allergic response overlap, with mus m being the major target for both t cells and antibodies. to the best of our knowledge, this is one of the first comprehensive studies of t cell epitope targets in mouse allergy, which provides important insights into cellular and serological targets. this data may form the basis for the development of a mouse-specific immunotherapeutic approach. introduction: food allergy has a complex etiology with many potential underlying factors proposed to contribute to and modify its development and progression. the use of a databases to collect and analyse all relevant data related to incidents of food allergy is essential to fully understand causal factors and improve treatment. objectives: we developed a database using sql, hibernate and java server pages (jsp) that was designed to allow allergy professionals to easily add and modify patient data, including medical history, reaction details and in vitro/in vivo test results. we then filled this database with clinical data relating to reactions to plant-based foods for patients who visited the allergy service of the regional university hospital of malaga between - . these data were then analysed in various ways. cluster analysis of skin test results was used to search for relationships between different allergens based on similarities between patient sensitisation profiles; descriptive statistics and graphical analysis were performed to search for relationships between food type, age of first reaction, number of reactions and reaction severity. results: cluster analysis placed the different skin test allergens in distinct groups, which generally correlated with the type of allergen. for example, nuts, rosacea plant-food, mites, trees and weeds formed distinct clusters. analysis of patient history data showed that the first reaction occurred most frequently between ages - , with a right skewed distribution. a relationship was also found between age at first reaction and reaction severity, being urticaria and angioedema more common when the initial reaction occurs at a younger age, and anaphylaxis when the initial reaction occurs later in life. oas remained relatively prevalent at all ranges (around a quarter of all reactions in all age groups). we found fruits to be the most frequent triggers, followed by nuts; within fruits peach and banana were the most frequent. conclusions: this preliminary study show the importance and utility of recording patient allergy information in a well-structured and easily managed database. future work is currently underway to collect a new set of patients from the same geographical area and to analyse similar data from a different area in order to identify what results are replicable within our population and which results are generalizable to other areas. introduction: cow's milk allergy is very common in children and its correct diagnosis is important to prevent possible dangerous allergic reactions. the aim of the present work was to evaluate the prevalence of allergic sensitization to both cow's milk and to its main proteins. with medcalc ® . normality distribution of data was evaluated through the kolmogorov-smirnov test. patients were divided into three age groups ( - years, - years- - years). chi-squared test was performed to verify a statistical difference between sensitization to whole milk and to its main proteins and patients' age. introduction: the order fagales represents an important cause of tree pollen allergy, which is ubiquitous in the northern hemisphere. a high degree of allergenic cross-reactivity has been observed among allergens from these plants, mainly represented by pathogenesis-related protein class (pr- ) pr- s, including inhalant and food allergens. conclusions: testing ige reactivity to a panel of pr- s unveils important associations between sensitization profiles and clinical presentation, and allows the identification of novel cluster patterns potentially useful to predict disease severity in patients with pr- allergy. results: patients were included, seven boys and four girls, with a median age at diagnosis of six months. the most common offending foods were cow's milk protein (cmp, n= ) and rice (n= ). other foods were fish, egg, chicken, wheat, carrot and potato. average time of symptom onset was . hours. the most frequent symptoms were vomiting (n= ) and diarrhea (n= ). six patients had a history of hospital admission related to this problem. seven patients had concomitant atopic diseases, being the most frequent allergic comobility atopic eczema (n= ). skin prick tests and/or specific ige to culprit foods were negative at diagnosis, except for one patient with low specific ige to cmp. another patient become sensitized to cmp during follow-up. open food challenges were performed in patients starting from six months of age. resolution was achieved in patients, at a medium age of months. results: a total of cases of immediate-type fa among children were reported, with . % involving patients younger than years of age. the major causative foods were hen's egg ( . %), cow's milk ( . %), walnut ( . %), wheat ( . %), peanut ( . %), soybean ( . %), and shrimp ( . %). the most common causative food in each age group was cow's milk ( - years), walnut ( - years), walnut and hen's egg ( - years), and buckwheat ( - years), respectively. the symptom onset time was less than minutes in %. food-induced anaphylaxis was reported in ( . %) out of cases, and the major causes were cow's milk ( . %), hen's egg ( . %), walnut ( . %), wheat ( . %), peanut ( . %), buckwheat ( . %), and shrimp ( . %). the proportion of anaphylaxis was highest in buckwheat ( . %), followed by walnut and pine nut ( . % each). korean children were hen's egg, cow's milk, walnut, wheat, and peanut, with distinctive distributions according to different age groups. anaphylaxis was reported in . % among immediate-type fa. results: a total of children with a median (inter-quartile) age of . years ( . - . ) were enrolled to the study; (male . %). their ages at diagnosis were . years ( . - . ); follow-up times were . years ( . - . ) and milk specific ige levels at diagnosis were . ku/l ( . - . ). in . % of the children there was only cma; the other children were polyallergic to different foods having most frequently egg white allergy. concomitant diseases were . % atopic dermatitis, . % were asthma, . % were allergic rhinitis. during the follow-up milk tolerance was developed in . %, . % and . % at the ages of , and years respectively. the specific ige level at the beginning of the disease was found to be a risk factor for the persistence of the disease (p<. ). conclusions: cma is frequently present with other food allergies. nearly half of the patients develop tolerance to cm up to the age of years; whereas / becomes tolerant when they are at the age of years. most frequent concomitant diseases are atopic dermatitis and asthma. objectives: two survey tools were used; a questionnaire based on similar surveys done overseas, and the validated food allergy quality of life questionnaire (faqlq). this was distributed throughout paediatric allergy clinics at two metropolitan centres. children and adolescents aged - completed the questionnaire independently, whilst parents assisted with children aged - years. results: surveys have been collected at the time of writing of which were answered by parents for young children. overall, / ( %) reported bullying, with a higher portion in older children and adolescents ( / ; %). of this group, / ( %) reported being bullied or teased because of their food allergies. from parental reports, / ( %) stated that their child had experienced bullying or teasing because of food allergies. for those not bullied, parents mentioned that this may be due to their child having friends at school, being too young for bullying or because other children at school had a good understanding of the severity of allergies and were educated by teachers. the most common location for bullying was "in the playground or sportsground" ( / ). the most common form of bullying involved being "teased, called names or someone has said mean things to me" ( / ). whilst food allergens were involved in bullying in many cases ( / ), there were no reports of children being forced to eat food to which they are allergic. of concern however, two adolescents reported experiencing an allergic reaction as a result of the bullying. the majority reported experiences of sadness from bullying ( / ) while seven stated that it had no effect. conclusions: our current research shows that % of children and adolescents with food allergies experience bullying, and that % ( / ) experience bullying specifically because of their food allergies. this indicates a significant social problem that requires addressing to positively assist those children living with food allergies. introduction: recently we demonstrated that intake of specific foods, types of fat and micronutrients was associated with inflammation and mucosal integrity in adults with eosinophilic oesophagitis (eoe). the current study aimed to compare dietary intake of these patients with dietary guidelines and intakes of the general dutch population to further investigate our hypotheses on the protective or allergy-provoking role of specific nutrients in eoe. results: the total faqlq score was low when assessed by teenagers and children ( . and . , respectively) and moderately low when assessed by parents ( . ). experience of anaphylaxis and having multiple food allergies impaired hrql according to faqlq parent form (p<. ). gender, having prescribed an adrenaline-auto injector, experience of food provocation test, peanut allergy and faim did not contribute to different hrql. hrql in kindergarten and schools were moderately diminished (sum score . in schools and . in kindergartens) (p>. ). perceived disease severity was moderately present with total faim scores being . , . and . , when assessed by children, teenagers and parents, respectively (p>. ). % of participants' reported at least some possibility of dying if child/teenagers would accidently eat a food allergen. after fulfilling faqlq and faim, all participants expressed content, ten children/teenagers decided to approach food provocation tests de novo, employees of children's schools/kindergartens were encouraged in written invitations to assess anaphylaxis training programs, and four families accepted additional psychological support. conclusions: food allergies impair hrql in children and teenagers. allergies to multiple foods and experience of anaphylaxis were associated with more severe impairment of hrql. hrqlq and faim are useful, additional tools to assess and discuss child's/teenager's/parent's fears and obstacles because of food allergy and identify further needs of support. introduction: fruit allergy is the most common cause of food allergy in children older than years and adults. regional variations have been observed in europe but there are few studies in pediatric population. objectives: in the context of a prospective and multicentric study on pollen and vegetable food allergy in spain, we enrolled patients (median age , range - , female %), who had suffered at least two episodes of immediate symptoms after ingestion of fruits and had positive skin test to the implicated fruits. immunocap isac was analyzed in all patients. our aim was to describe the clinical characteristics with the fruits involved and the usefulness of the allergens included in the immunocap isac to improve its characterization. symptoms were categorized into oral allergy syndrome (oas), systemic symptoms (ss) and anaphylaxis. results: a total of patients were included. all of them had symptoms with more than one fruit. patients had pollen sensitization. the main offending food associated with allergic reactions were peach ( %), kiwi ( %), melon ( %), apple ( %) and banana ( %). allergic patients to peach had oas, ss and anaphylaxis, were recognized prup in all patients with anaphylaxis, in patients witch oas and in with ss, also prup associated with abstracts | prup in patients with ss. for allergy to kiwi, patients had oas and ss, were recognized actd in patient with sao and patients with ss. actd in patient with ss. conclusions: in our population, the most prevalent fruit allergy was the peach, as in spanish adults. patients allergic to peach were the ones that presented the most ss and anaphylaxis, followed by allergic to apple. as previously have been reported most of them had sige to its components in isac; being prup the most prevalent ( % had prup ). only patients with ss with kiwi were sensitized to kiwi allergens .the majority of patients allergic to apple, melon and banana were not diagnosed by immunocap isac. introduction: studies have shown that asthma and allergy are prevalent among production workers processing seafood, particularly in workers processing crustaceans. a major ige-reactive proteins is the muscle protein tropomyosin. specific ige to tropomyosin is suggested as a central marker for crustacean allergy, however it is not the only protein characterised as an allergen in crab processing. objectives: the aim of our study was to characterise tropomyosin exposure and prevalence of sensitisation to allergens in workers processing king crab (paralithodes camtschaticus) and edible crab (cancer pagurus) in land based processing plants in norway. results: personal air samplers collected air from the workers' breathing zone during crab processing. workers' blood was collected for ige testing. extracts of both king crab and edible crab raw meat, cooked meat, intestines and shell were made in our lab and used for skin prick testing and immunoblotting. while processing cooked crab yielded highest tropomyosin levels in the edible crab plant, processing raw crab yielded highest levels in king crab plants. ten ( . %) edible crab and ( . %) king crab workers had positive ige test (> . ku/l blood, immunocap systems) to crab. four ( %) of skin prick tested king crab workers and ( %) of skin prick tested edible crab workers had one or more positive reactions to edible crab extracts. more workers reacted to cooked crabmeat extracts than to raw crabmeat extracts. immunoblotting showed ige binding to a large number of proteins in all four extracts of both king and edible crab. binding was found to high molecular weight proteins in all four extracts of the crab tested, and the ige-reactive proteins differed between king crab and edible crab. conclusions: workers are exposed to tropomyosin in their breathing zone during crab processing. both king crab and edible crab workers are sensitised to crab, shown with immunocap specific ige test to crab, as well as positive skin prick tests and immunoblots to four different crab extracts made in our lab. workers processing crab in norwegian processing plants have an increased risk for developing sensitisation to crab. objectives: the aim of the study was to assess frequency of skin symptoms in surgery clinic employees, to evaluate burnout as a predictor of the frequency of skin symptoms, and to determine latexspecific ige in surgery nurses with skin symptoms. results: skin symptoms were significantly more frequent in surgery nurses ( %) than in surgeons ( . %), other physicians ( ), and other nurses ( . %) (v = . ; p=. ). skin symptoms were also significantly more frequent in workers with high/medium than in workers with low emotional exhaustion ( . % vs . %; v = . ; p=. ), as well as in participants with burnout than in subjects without burnout introduction: baker's asthma sensitization pattern is changing due to the introduction of different types of grains and seeds. objectives: a year-old ecuadorian man showing ocular, nasal and pulmonary symptoms when handling grain flours (with or without seeds), while baking for the last years. he tolerated grain flours oral intake, but had oropharyngeal symptoms, rhinoconjunctivitis and dyspnea when eating sunflower and sesame seeds, mustard, and beer with alcohol. he tolerated alcohol-free beer. we performed an allergy study: prick-test with commercial extracts of pollens, dust and storage mite, fungus, animal danders, cereals, yeasts and mustard. prick by prick with patient's products: wheat, multicereals and seeded flour, sunflower seeds, regular and alcoholfree beer spirometry and niox. serial peak flow measurement at and away from work, handling tests with wheat flour and sunflower seeds. laboratory studies: specific ig e to cereals and seeds (cap-isac-microarrays). immunoblot with regular beer (at room temperature and boiled), wheat flour and sunflower seeds, and sequential chromatography. results: skin tests were positive with commercial mustard and all provided products except for the alcohol-free beer. spirometry was normal. niox: ppb. peak-flow monitoring showed a % variability during working period, remaining stable during holidays. handling tests with wheat flour and sunflower seeds were positive. specific ig e was positive for grains, malt, gluten, mustard and sunflower seed. the specific determinants were positive to s-viciline, -s globulin, several prolamins ( s-albumine, alfa-amylase inhibitors and gliadin) and ltp. immunoblot detected a band lower than kda in both regular beer extracts (not detected in alcohol-free beer) identified as barley's ltp, a band of kda in the sunflower seed extract ( salbumine), and two bands lower than kda in wheat flour extract (two kinds of alfa-amylase inhibitors). we present a non-atopic baker with occupational rhinoconjunctivitis and asthma due to prolamins (alfa-amylase inhibitor and gliadin of wheat flour together with s-albumin sunflower seed), and anaphylaxis when eating seeds ( s-albumins, s-vicilin and s-globulin) or drinking beer (sensitization to barley's ltp). it is interesting that the manufacturing process of non-alcoholic beer (high temperature and high pressure) seems to degrade barley's ltp, as suggested by both tolerance to its ingestion and loss of immunoblot band. objectives: the main objective of this study is to evaluate longitudinal change of lung function in workers employed in food preparation and distribution potentially exposed to food allergens. spirometries performed between and as part of medical surveillance of food-handlers workers were evaluated. data about occupational task, work years, smoking habits and diagnosis of atopy, asthma and copd were collected from a clinical database. differences in prevalence were calculated by chi-square test, differences in means were calculated using spirola software referred to european predicted values. results: the majority of workers were females (n= ; . %) and caucasians (n= ; . %). ( . %) subjects were current smokers, ( . %) were ex smokers, ( . %) were atopic and no one reported a diagnosis of asthma or chronic obstructive pulmonary disease. % workers were canteen service employees and % were cookery employees. mean yearly values of the pair-wise within person variation of fev and fvc were respectively À ml and À ml. . % of last observations had a fev below lln and . % of last observations had a fvc below lln. conclusions: this study may help in planning preventive programs and in facilitating early recognition and diagnosis of work-related respiratory diseases. wheat, foods and latex allergens may determine decline in fev and fvc; furthermore, in our study, a significant proportion of workers reported exposure to tobacco smoke. excessive loss in fev over time should be evaluated using a percentage decline ( % plus loss expected due to aging) that we will make afterwards adding more years of follow up spirometries. intervention of smoke avoidance are needed. | prevalence of wood dust sensitization in occupationally exposed workers in germany-what can be tested? objectives: in serum samples from patients with suspiciously allergic symptoms to wood dust overall specific (s)ige-tests with standardized wood-dust extracts coupled to streptavidin-immu-nocaps were conducted. additionally, ccd as known source of non-protein ige-target was evaluated. sensitization rates were calculated for different wood species. most frequently requested wood dust allergens were obeche (n= ), beech (n= ), oak (n= ), spruce (n= ) and pine wood (n= ) followed by - requested sige-tests to mahogany, ash, larch and maple wood. results: overall wood dust sensitization rate was about % (range: - %) with obeche, box wood and kambala as most prominent sensitization sources obtaining each more than % sensitization. no sensitizations were detectable to red cedar and meranti wood in more than requested tests, respectively. in patients at least one sige-sensitization to any wood was measured. there from were additionally tested with ccd resulting in % positive and % negative ige responses to ccd. some wood species were exclusively recognized by ccd-positive subjects: ash, maple, alder, mahogany, teak, mansonia and palisander. whereas other woods were recognized by sige of subjects with / or without sige to ccd: obeche, box wood, spruce, oak, beech, limba, pine and kambala. relevance of wood sensitization next to ccd was investigated in eight double positive subjects (wood + / ccd +). specific ige-binding to wood allergens was completely inhibited by ccd in three samples. these subjects were supposed to have no clinically relevant wood sensitization. whereas in five samples sige-binding to selected wood species was not significantly reduced by ccd. in four of these patients skin prick tests (spt) and challenge tests (bronchial and/or nasal) with corresponding wood allergens were performed. three of four challenge tests were positive with the respective wood extract and all spts with wood extracts whose sige-binding was not affected by ccd inhibition showed positive reactions. here clinical relevance of sige-mediated wood sensitization could be demonstrated. conclusions: in summary, our data demonstrate that standardized wood extracts and ccd tools are necessary for valid in-vitro diagnosis of wood dust sensitization. introduction: respiratory symptoms have been reported frequently among seafood processing workers. since seafood processing workers handle the raw material and participate in processing activities during work, they are exposed to inhalable bioaerosols. this put them at risk of developing respiratory symptoms, asthma and allergy. there is little knowledge about the respiratory health status among fish production workers on board fishing trawlers. objectives: the aim of this study was to assess the respiratory health status among norwegian fish production workers, processing fish on board fishing trawlers. the study population consisted of fish production workers, machinists, support crew members and non exposed controls, all were males. written informed consent was the fish production workers had a significantly decreased fev % predicted compared to the non-exposed control group, b=À . , % ci [À . , À . ], when controlling for age, pack-years and family history of asthma/allergy/eczema. the effect did not change when controlling for doctor diagnosed asthma or after dividing fish production workers by doctor diagnosed asthma. machinists and support crew members showed a similar decrease in fev % predicted, but the difference from the non-exposed control group was not statistically significant. furthermore fish production workers, reported a non-significantly increased prevalence of wheezing and daily morning cough compared to the non-exposed control group. conclusions: fish production workers, processing fish on board fishing trawlers, showed reduced lung function values compared to a non-exposed control group, and this finding is in accordance with previous findings in seafood industry worker populations from our research group. results: study group comprised patients with work-related respiratory symptoms suggesting wra. the research completion with sic allowed to recognise wra in persons ( oa and wea) and to exclude asthma in cases qualified to reference group (gr). workers with wea occupationally exposed do lmw-a manifested the highest level of baseline non-specific bronchial hyperresponsiveness (nsbhr) in comparison to the other groups (table ) . patients with oa exposed to lmw-a more frequently than exposed to hmw-a revealed nsbhr before sic (p=. ) with lower level of median (me) provocative methacholine concentration value causing % fall in fev (pc induced sputum (is) was obtained before and h after sic from patients ( gr and wra). in all gr samples and samples possessed before sic from wea subjects exposed to hmw-a, intermediate profile of is (neutrophils (ne)< % and eosinophils (eo)< %) dominated ( results: eg: in "granulation" exposure is relatively high and the number of different enzymes handled is low; here the risk of sensitization is highest. in contrast in the pilot plants the exposure compared to granulation in general is lower but the number of enzymes handled concurrently is higher. still the sensitization risk is lower than the range for granulation. conclusions: even though this approach may seem crude and not free from bias and potential misclassification, data does not support the hypothesis that the number of enzymes increases risk of sensitization, whereas increasing exposure level seems to be a risk factor. this suggests that each enzyme exposure acts as a risk in its own right and that the "cocktail effect" seems to be of minor relevance. phospholipids. bioinformatic studies of sequence homology conducted prior to this study showed no similarity between the mpla s and known allergens including ves v . however, the common enzymatic activity in ves v and the mpla s might still lead to crossreactivity. the goal of this project was therefore to test for possible cross-reactions between sige towards ves v and three different mpla s. methods: serum from known wasp allergic persons with sige towards ves v spanning from . ku/l to ku/l were used for inhibition studies. from each, ll serum was incubated with ll of either saline solution (negative control), lg/ml alk soluprick solution (positive control) or one of three mpla s, each in three concentrations (either . lg/ml, lg/ml and lg/ml (n= ) or lg/ml, lg/ml and lg/ml (n= )). the level of sige towards ves v was measured using the i immunocap, and a decrease in this level was calculated as %inhibition compared to the sige measured from serum incubated with the negative control. inhibition by mpla s would indicate cross-reactivity. results: the positive control caused . ae . % (n= ) inhibition of sige towards ves v . this was lower than expected but was found to be caused by a few sera where the fraction of sige towards ves v was < % of all sige towards wasp venom. in the remaining sera, %inhibition was . ae . % (n= ). for all three mpla s, no inhibition was found for any serum tested (n= ) at the highest concentration tested with %inhibition being . ae . %, . ae . % and . ae . % respectively. conclusions: no inhibition of sige to ves v was found to any of the three microbial phospholipases tested. this indicates that no cross-reaction is found between the phospholipase a in wasp, ves v , and phospholipase a from microorganisms despite the common enzymatic activity. objectives: the aim of study was to evaluate the prevalence and the impact of polyvalent ige-mediated allergy on the course of ad and the occurrence of allergic symptoms from other organs and systems in infants and young children. conclusions: polyvalent ige-mediated allergy is common in young children with ad and seems to be a risk factor for the severe course of the disease. introduction: non-steroid anti-inflammatory drugs (nsaid) are the second most common cause of drug allergies in childhood. objectives: the aim of the study is to determine the frequency of nsaid hypersensitivity in asthmatic children. results: patients who were being followed up for asthma were included in this study. the mean age of the patients was . ae . years, while . % ( ) were male. % (n= ) of the patients had a reaction history to nsaid (ibuprofen in , flurbiprofen in , diclofenac potassium in , metamizole+acetylsalicylic acid in , paracetamol+acetylsalicylic acid in and ibuprofen+acetylsalicylic acid in ). nsaid sensitivity was confirmed in ( . %; / ) patients who were tested with suspected drugs, while the provocation test was found negative in one patient who described reaction with ibuprofen. of the children who were assessed as a control group, only had a reaction history to acetylsalicylic acid and no reaction developed in the provocation test. conclusions: nsaid hypersensitivity is more common in patients with asthma. thus, these patients should be evaluated for nsaid hypersensitivity. results: from jan to dec , pediatric cases were received by kaers. of pediatric patients, . % were male, . % were female and . % were unknown. these pediatric cases included a total of adr events with an approximate average of . adr events per report. of those cases, . % were in infants (age - years), . % were in young children (age - years), . % were in old children (age - years), . % were in adolescent.(age - years) and unknown were . %. male to female ratio was : . and the mean age was . ae . years. regarding categorical ranking of reported adr agent groups, the most common group were antibiotics ( . %) followed by antineoplastic agents ( . %), vaccine ( . %), antipyretics ( . %), opioids ( . %), sedatives ( . %), antiepileptic drugs ( . %), contrast media ( . %), steroids ( . %). the most common adr symptoms were gastrointestinal system disorder in . %, skin-appendage disorder in . %, abstracts | body as a whole-general disorder in . %, central-peripheral nervous system disorder in . %. regarding seriousness of adrs, events ( . %) had episodes requiring hospitalization and were considered life threatening. of these, cases had anaphylaxis or anaphylactoid reactions. introduction: multiple drug allergy is an adverse reaction to two or more structurally unrelated drugs that appears to occur by immune mediated mechanism. patients with a history of reaction to two or more drugs often apply to allergy clinics. objectives: the aim of this study is to evaluate the test results of the patients who have a history of multiple drug allergies and underwent drug provocation tests. results: during the study period, drug provocation tests were performed in patients ( drug provocation tests). of these patients, ( . %) had a history of drug reactions to or more drugs. the mean age of the patients who had a history of reactions to two or more drugs was . ae . years, and . % (n= ) toms, and autoimmune manifestation in comparison to igm/iga responders (respectively, pneumonia: %, % and %; chronic diarrhea: %, % and %; autoimmunity %, % and %; autoimmune cytopenias: %, % and %). malignancies were found more frequently in the non-responders and igm-only responders groups in comparison to igm/iga responders (respectively, %, % and %). eleven ( %) patients died during the study time. survival analysis according to the igm/iga responder status showed that the -years estimated survival for non-responders vs igm-only vs igm/iga responders was respectively after one year %, % and %; after two year: %, % and %; after three years: %, % and %%; after years: %, % and %; after years: %, % and %; after years: %, % and %. interesting, in our series only two deaths were due to infective complications: five were consequent to malignancies, one to autoimmune cytopenias and three to not-cvid related conditions. between-infusions intervals ( - days) than pid patients ( - days). finally, a small number of patients with anti-cd -related sid was able to discontinue scig replacement therapy after recovery of spontaneous igg production. conclusions: this is, to our knowledge, the biggest single center cohort of scig-treated patients ever described. results suggest that safety and effectiveness of scig is similar in pid and sid, irrespective of the mechanisms underlying igg depletion. moreover, in sid a lower igg dosage is required and ig replacement does not always need to be lifelong, with obvious pharmacoeconomic implications. a | should we screen children with bronchial asthma for primary immune deficiencies? miteva d ; perenovska p ; papochieva v ; georgieva b ; lazova s ; naumova e ; petrova g the patient became febrile and cultures were repeated, being positive to campylobacter jejuni, resistant only to ciprofloxacin (blood) and to campylobacter coli, susceptible only to gentamicin and amoxicillin/ clavulanic acid (stools). treatment was thus switched to amoxicillin/ clavulanic acid ( / mg / h). the patient became apiretic at day and improvement of local inflammatory signs was noticed, treatment was prolonged for six weeks. one week after cessation, skin lesion worsened again in the same location, in association with fever. blood and stool cultures were repeated and gentamicin ( mg/day; iv) and cefixime ( mg / h) were started, in agreement with previous cultures results. curiously, there was no history of diarrhea, but the patient referred a period of recurrent abdominal colicly pain, before cutaneous lesions appear. conclusion: bacteremia with campylobacter species requires specific laboratory workup. diagnosis of campylobacter jejuni bacteremia should be considered in hypogammaglobulinemic patients with recurrent fever, particularly when typical copper color erysipela-like skin lesions occur. campylobacter eradication can only be achieved with prolonged antibiotic therapy guided by antibiogram in cultures. conclusions: in this study, genetic defects of five higm patients have been identified, for other patients further genetic investigation such as next generation sequence (ngs) is required. the study results can help diagnose of the disease more definitively and also can provide valuable information for genetic counseling especially for those who have a history of immunodeficiency in their families and also for prenatal testing. conclusions: mutation analysis of unc d gene can help the families with hlh patients give genetics counseling for carrier detection and prenatal diagnosis. woelke s ; valesky e ; bakhtiar s ; bader p ; schubert r ; zielen s department for children and adolescents, division of allergology, pulmonology and cystic fibrosis, goethe university hospital, frankfurt, germany; department of dermatology, venereology and allergology, goethe university hospital, frankfurt, germany; department for children and adolescents, division for stem cell transplantation and immunology, goethe university hospital, frankfurt, germany introduction: ataxia telangiectasia (a-t) is a devastating multi-system disorder characterized by progressive cerebellar ataxia, growth retardation, immunodeficiency, chronic pulmonary disease and genetic instability with an increased risk for malignoma. as described in other primary immunodeficiencies cutaneous granulomas are a known phenomenon also in a-t. still treatment indication and strategies remain controversial. objectives: from our cohort of classical a-t patients, eight patients in the aged to years presented with granulomas. histopathology of the lesions confirmed the presence of granulomatous inflammation without detection of any microbiological agent in all patients. five patients suffered from cutaneous manifestation, in two patients we detected a bone and in one a joint involvement. both patients with bone involvement (patients and ) as well as one patient with massive skin manifestation (patient ) were treated with tnf inhibitors (infliximab). the patient with granulomas in his finger joint (patient ) was bone marrow transplant (bmt) for other reasons. year led to a total remission for three years now. in patients and treatment with tnf inhibitors led to a partial regression of granulomas. treatment interruptions caused deterioration again. in the course of treatment the effects of infliximab diminished most likely caused by drug antibodies. after changing treatment to subcutaneous adalimumab a further regression could be detected. in patient granulomas totally disappeared with immune reconstitution after successful bmt. partially successful in treatment of granulomas. due to the known immunodeficiency in a-t patients, indication for immunosuppressive therapies as tnfa inhibitors should be held strictly. woelke s ; hess u ; knop v ; krausskopf d ; kieslich m ; schubert r ; zielen s of to years regarding c-reactive protein (crp), liver enzymes, abdominal ultrasound and neurological status (ataxia score). we divided the patients into two age groups of a-t patients aged to years and a-t patients aged years to years. ataxia score (r= . ), although the underlying pathomechanism is unclear. ultrasound revealed nonalcoholic fatty liver disease in only one young patient ( . %) compared to older patients ( . %). one female patient aged years died due to a hcc. conclusions: liver disease is present in almost all older a-t patients. structural changes, nonalcoholic fatty liver disease and fibrosis are frequent findings. there is a considerable risk for hcc. prospective studies are necessary using noninvasive techniques for the assessment of liver fibrosis (eg transient elastography) and to establish the risk of hcc in a-t patients. objectives: in this study, it was aimed to determine the frequency of pollen-food syndrome in children who have sensitization to pollens. results: pollen-sensitized patients were included in this study. the mean age of the patients was . ae . years, while . % (n= ) were male. in . % (n= ) of the patients, allergic rhinitis was concomitant with asthma. . % (n= ) of the patients described symptoms related to pfs. % ( / ) of them had a history of anaphylaxis with suspected food. the mean age of the patients describing pfs was . ae . years and % (n= ) of them were female. in ( . %) of these patients, skin tests performed with suspected food was positive, but in one patient the skin test was negative while specific ige was positive. suspected food was fruit/ vegetables in patients. in patients with pollen allergy, oropharyngeal symptoms related to fresh fruit, vegetables, dried fruits and nuts should be enquired. it should be noted that these patients might experience serious systemic reactions including anaphylaxis. patel nb ; vazquez-ortiz m ; lindsley s ; abrantes g ; bartra j ; dunn galvin a ; turner pj conclusions: there is no evidence that the occurrence of anaphylaxis at fc, and self-treatment with an adrenaline auto-injector device, result in adverse impact on hrql measures. the impact of a reaction at food challenge appears to confer greater benefit on the parent than the child. the relationship between confidence in management and hrql needs further assessment, since it is likely that these outcomes will be affected in different ways following therapeutic interventions. results: fifty-one patients ( females, males) (median age: . years, range - ) with cma were studied. spt to cm was . ae . mm as mean diameter. forty-eight out of fifty-one ( . %) patients underwent dm-opt ( patients refused to underwent opt due to positive spt or s-ige to dm introduction: bovine milk is the most common food allergen in children under years of age. milk allergy is treated by eliminating milk from the diet. the milk elimination diet endangers the child's energy intake and also exposes the child to shortage of multiple nutrients. this study was needed because there are no previous systematic reviews about this subject. objectives: the aim of the present study was to examine if the milk allergy or the other factors associated with milk elimination diet have an influence on child's growth. the present study was conducted according to international guidelines for systematic reviews. results: a total of studies were initially identified, of which three fulfilled our criteria. these three studies included children with cow's milk allergy and control children. in all these three studies, children with cow's milk allergy were lighter than controls. in addition, in two studies, the growth of the children with cow's milk allergy was stunted. in two studies the milk elimination was substituted with special infant formula. also in one study where both the growth and the weight were stunted, no major differences in energy or nutrient intake between cow's milk allergic cases and controls were reported. conclusions: current evidence suggests that milk allergy is associated with stunted growth in childhood, but reasons for this are unclear. in order to clarify the effect of cow's milk elimination diet on growth in childhood and the underpinning mechanisms, more studies need to be conducted. in addition, special attention to the diet and growth of children with cow's milk allergy is needed. results: a total of children were surveyed in this -year period (annual average: ). in , children ( . %) were diagnosed with food allergy, including cases of pfas, cases of egg allergy, and cases of dairy-product allergy. in , children ( . %) were diagnosed with food allergy, including cases of pfas, cases of egg allergy, and cases of dairy-product allergy. over this -year period, the prevalence of pfas increased . fold (from . % to . %). among the pfas cases, the prevalence of rosacea and apple allergy increased . -and . -fold, respectively. the most prevalent was apple allergy ( . %), followed by peach ( . %), loquat ( . %), plum ( . %), pear ( . %), and strawberry ( . %). the prevalence increased significantly for pfas but not for other types of food allergy (egg and dairy-product allergy). in the future, nationwide studies are needed to further elucidate the relationship between pfas and allergic rhinitis. | immune profile after oit in children with cow s milk allergy patients with elimination diet, group : patients with natural tolerance, group : healthy control). in all groups specified laboratory tests were performed at onset and also at month to treatment groups. in desensitization group at month of treatment we evaluated increase in total ige level, sp iga and igg antibody responses, decrease in cow's milk spige levels and an increase in il- , il- , tgf-b cytokine responses without a difference in cd +cd +fox-p % levels. il- levels were similar with pre-treatment levels whereas foxp mrna expression was similar with tolerance group. in elimination group at month treatment, there was a decrease in cow's milk spige whereas there was no change in sp iga levels and a minimal increase in igg levels. there was no change in il- and il- cytokine levels. and an increase in tgf-b cytokine response less than group , decreased il response, a foxp mrna expression differed from tolerance group was identified. objectives: although hyperuricemia has a significant prevalence in the general population, and has been related to exercise-induced asthma, could be related to bronchial asthma and nasal polyposis. hitherto, its possible association with hypersensitivity to nsaids and its convenience as biomarker have not been acquainted. conclusions: in our population, hyperuricemia has not demonstrated to be a reliable biomarker related to nsaids allergy and could not be used as a risk factor for assessing the triad nsaids allergy, asthma and nasal polyps. to study the vas of the total score was seen significant variance in: nsaid-cu ( . pt) and nsaid-pa ( . pt) (p=. ), nsaid-cu conclusions: data support that cutaneous manifestations have a common response with aerd to cox inhibitors. conclusions: this study showed that in a positive drug oral provocation test, respiratory symptoms were accompanied with nitric oxide changes in both nasal and exhaled way. barrionuevo e ; doña i ; salas m ; bogas g ; guerrero ma ; sanchez mi ; cornejo-garcia ja ; torres mj allergy unit, regional university hospital of malaga-ibima, malaga, spain; research laboratory, ibima-regional university hospital of malaga-uma, malaga, spain introduction: non-steroidal anti-inflammatory drugs (nsaids) are the most frequent triggers of drug hypersensitivity reactions, being cross-hypersensitivity reactions (chr) the most frequent. the categories included in chr are nsaids-exacerbated respiratory disease (nerd); nsaids-exacerbated cutaneous disease (necd) and nsaids induced urticaria/angioedema (niua). however, it has been reported patients with chr to nsaids who developed a combination of skin and respiratory symptomatology (blended reactions). objectives: our aim was to analyse the characteristics of patients with blended reactions and compare with those developing symptoms exclusively respiratory (nerd) or cutaneous (niua). episodes of cutaneous and/or respiratory symptoms after the intake abstracts | of ≥ different nsaids included strong cox- inhibitor (acetylsalicylic acid (asa) and/or indomethacin); ii) if they had < episodes of cutaneous and/or respiratory symptoms induced by < different nsaids, a positive drug provocation test (dpt) with asa was required; iii) if patients had respiratory symptoms accompanied or not by cutaneous involvement, a positive nasal provocation test with lysine aspirin (npt-lasa) was required. atopy was assessed by skin prick test using a panel of inhalant and food allergens. objectives: subjects with nsaids hypersensitivity were divided into two groups: a) those from to years and b) those from - years. diagnosis was established by a clinical history and controlled challenge with asa. atopic status was verified with a detailed allergological study including skin testing to inhalant allergens. clinical entities were classified in three categories: urticaria/angioedema, anaphylaxis and respiratory (asthma and/or rhinitis). cases. no differences were observed in the atopic status between both groups. there were significant differences between males/ females (p<. ). when we compared the clinical entities there were more cutaneous manifestations, mainly angioedema, in the group a) and more anaphylaxis in group b) although there were no significant differences due to sample size. conclusions: significant sex differences between hypersensitivity reactions to nsaid occurs with predominance of males in the first group (a). although there are also a predominance of clinical entities, an increase number of cases is needed to establish significance. studies on this direction are in progress. show an increase in all age groups. etiologic analysis was limited as the study was carried out using the icd- code (nhic records) and database of self reporting systems (kaers). so, further study was needed. introduction: genetic variants from the q locus are the strongest known genetic determinant for early-onset childhood asthma, and have also been associated with uncontrolled asthma despite asthma treatment. objectives: the aim of the study was to assess whether there is an association between a single nucleotide polymorphism (snp) in the q locus (rs ) and asthma exacerbations despite the objectives: our hypothesis is that the arg allele is associated with increased use of prescribed asthma medication, over a -year period. to explore this hypothesis, we have undertaken a secondary analysis of breathe, a study of gene-environment associations with asthma severity. breathe data were collected on participants with asthma, aged - years, between and , in tayside conclusions: in children and adults, the homozygous arg/arg status is associated with long-term increased prescribing for asthma medication compared to those carrying at least one gly allele. defining subgroups of individuals requiring more medicines could help predict treatment costs and develop targeted management strategies. objectives: considering the role of ptgdr in allergy, the goal of this study was to analyze the effect of ptgdr expression on cytokine levels in the a cell line. analyze ptgdr expression in a cell cultures. cytokine production assays in the culture supernatant were measured by cytometric bead assay using the bioplexpro tm human cytokine standard -plex, group i. the assays were conducted with bioplex high-throughput fluidics system, powered by the luminex technology. every sample was run at least in triplicate. the ptgdr expression in the transfected cells with the haplotypic variants differed by orders of magnitude relative to control cells (p<. ). we found significant differences in ptgdr expression between ctct and cccc haplotypic variants. the cccc (À c, À c, À c, and À c) introduction: c t polymorphism in the cd gene has been suggested in susceptibility to asthma. cd is a multifunctional receptor endotoxin, which is expressed on the surface of macrophages, monocytes and neutrophils. it is likely to play a role in the inflammation pathway. though data is available regarding association of cd gene with asthma but independent studies are in conflict. objectives: the present study was conducted to examine the association of promoter c t single nucleotide polymorphism (snp) in the cd gene for indian children with atopic asthma. we characterize the c t polymorphism in children with asthma ( ), cohort group ( ) and healthy control group ( ) by pcr rflp. association analysis was performed using v tests. we also analyzed the association of cd (c t) with total ige levels by elisa and foxp expression using flow cytometry. in this snp a base pair (bp) pcr product was generated using the standard primers. after restriction products showed that homozygous c allele was appeared as a single bp band, the homozygous t allele as bands of bp and bp, and heterozygous exhibits all three bands ( , and bp). the or of cc genotype frequency abstracts | was . in study group and . in cohort group and the or of c allele frequency was . in study group and . in cohort group. total ige level were found to be significantly higher in cc genotype compared to ct and tt genotype. foxp level is significantly higher in control group in all genotypes compared to cohort and study group. conclusions: the present study concludes that in cd gene polymorphism cc genotype was not significantly associated with asthma but other factors ie total ige and foxp showed significant association of cc genotype with asthma. on the other hand, there was significant association of c allele with asthma. | the disbalance of tlr , tlr gene expression and cytokines production in children with bronchial asthma svitich oa ; gankovskaya lv ; namazova-baranova ls ; bragvadze bg ; alekseeva aa ; gankovskii va objectives: examined were patients with bronchial asthma aged from to years and healthy children of the same age. cytokines were determined by elisa. determination of mrna expression in scrapings from the mucous membranes of the respiratory tract and in peripheral leukocytes was carried out polymerase chain reaction in real time. results: in scrapings from the mucous in patients with moderate to severe asthma found a significant increase in the gene expression of tlr , times, the gene tlr in times in comparison with the control group. in children with severe asthma also found a significant increase in the gene expression of tlr . times compared to healthy children, p≤. ). indicators of tlr gene expression in this group of patients have a tendency to increase, but not statistically significant. when comparing the indicators of innate immunity in samples with a leukocyte mass in the group of children with severe asthma showed a decreasing expression of tlr compared with the index in healthy children. also decreased the expression of tlr . in children with moderate ba similarly, a significant decrease of tlr gene expression in times. the trend towards reduced expression of tlr remains in this group, but is not reliable. in washings from the nasopharynx shows that patients with bronchial asthma il- is increased . times compared to the norm, to pcg/ml, tnf increased in times and pcg/mg in severe asthma, and . pkg/ mg-for mild, il- increased in times ( pcg/mg), pkg of . / mild blood, il- also increased . -fold and equal to . pkg/mgwith heavier with easy- . pkg/mg, normal À pkg/mg. ie is an increase in proinflammatory and anti-inflammatory cytokines. conclusions: the overexpression of tlr , tlr accompanied by increasing, the production of cytokines. this is a violation of mechanisms of innate immunity at the level of the mucous membrane of the nasal cavity on the role of inflammation in the pathogenesis of tlr. | sustained reduction in risk of experiencing asthma symptoms and using asthma medication in years following grass slittablet treatment-results from the paediatric gap trial were: wheezing, cough (for more than consecutive days), shortness of breath, chest tightness. the odds ratio for having asthma symptoms, using asthma medication, or having asthma symptoms and using asthma medication was significantly lower in the grass slit-tablet group during the followup years. for asthma symptoms, the results were: or= . , p=. days with csms< were defined as "no or minimal symptoms" and days with csms > were defined as severe symptoms. csms score was significant lower in the ilit group than in the placebo group for , and . in mean csms was . roth-walter f ; schmutz r ; mothes-luksch n ; zieglmayreer p ; zieglmayer r ; jensen-jarolim e objectives: here, we investigated whether the immune molecule lipocalin (lcn ) may discriminate between allergic and sensitized individuals, and between responding and non-responding patients. results: lcn -concentrations were assessed in sera of healthy and allergic subjects (n= ) as well as of house dust mite (hdm) allergies that underwent hdm-sublingual immunotherapy (slit) in a randomized, double-blind, placebo controlled trial for weeks. sera pre -, post-slit and at least months after slit were assessed for lcn and correlated with total nasal symptom score (tnss) obtained during chamber challenge at week in patients receiving hdm-(n= ) or placebo-slit (n= ). allergic individuals had significant lower lcn -levels than healthy controls, with women having lower lcn -levels than men in the patient cohort. hdm-allergic patients who received hdm-slit had a significant increase in hlcn months after termination of hdm-slit, whereas in subjects receiving placebo no increase in hlcn was observed. within the hdm-slit treated group, lcn -levels were significantly higher in patients whose symptoms improved during slit in contrast to those in which symptoms became more severe. hence, time-course of lcn in an allergic individual was predictive to assess clinical reactivity to hdm. objectives: here, we present the benefits of treatment in terms of nnt to prevent one additional child from having asthma symptoms and asthma medication use. children treated with grass slit-tablet had a reduced risk of asthma symptoms and asthma medication use during the -year follow-up period compared with placebo (or= . [ . , . ] for sq grass slit-tablet (n= ) vs placebo (n= ), p<. , relative risk reduc-tion= %). the risk reduction was independent of age at treatmentstart. younger children had a higher predicted probability of developing asthma symptoms and asthma medication use than older children. thus, the younger the children were at treatment-start, the greater the percentage was prevented from having asthma symptoms and asthma medication use during follow-up off treatment. for children aged at treatment-start, the risk was reduced from % to % and for those aged it was reduced from % to %. consequently, the nnt to prevent one additional child from having asthma symptoms and asthma medication use during the -year follow-up increased with age, with nnt= for children aged and nnt= for children aged . conclusions: the grass slit-tablet reduced the risk of asthma symptoms and asthma medication use during the -year follow-up period; the risk reduction was independent of age. however, the nnt increased with age as younger children had a higher risk of developing asthma symptoms and asthma medication use, emphasising the importance of treatment-start early in life. results: participants were screened for birch and grass allergy, of whom ultimately met randomization criteria and were treated with either slit-t or placebo for months. treatment was preceded by a successful baseline birch pollen challenge in the eeu where a minimum tnss of was achieved in the first of hours of pollen exposure. participants attended the post treatment challenge in the eeu, also hours in duration. no significant differences were noted in the reduction of birch-induced tnss compared to baseline between the slit-t and the placebo treated participants (the primary outcome measure). adverse events with a minimum % frequency occurred in % of participants in the placebo arm and % of participants in the active arm, with upper respiratory tract infection ( % in active arm and % in placebo arm) being the most common. oropharyngeal itch was the most common adverse event with causality at least possibly related to study medication ( % in active arm and % in placebo). no serious adverse events occurred including no anaphylaxis. objectives: here, we present a pooled subgroup analysis in adolescents from phase ii/iii-iii trials with the hdm slit-tablet ( sq-hdm dose), p in north america and to- - - in japan. results: to- - - was a randomised, dbpc phase ii/iii trial investigating the efficacy and safety of the hdm slit-tablet in japanese adolescents and adults ( to years) with moderate-tosevere hdm ar (n= , of which were adolescents). subjects were randomised to treatment with the hdm slit-tablet in doses of sq-hdm, sq-hdm or placebo for year. p was a randomised dbpc phase iii trial investigating the efficacy and safety of the hdm slit-tablet in north american adolescents and adults (≥ years) with moderate-severe hdm ar with or without asthma (n= , of which were adolescents). subjects were randomised to treatment with sq-hdm or placebo for up to year. in both trials, the primary endpoint was the average total combined rhinitis score (tcrs) during the last weeks of treatment. the pooled analysis was performed on mean values using a linear mixedeffects model. treatment with sq-hdm of the hdm slit-tablet resulted in a statistically significant reduction in the average tcrs of . ( %, p=. ) compared to placebo in the last weeks of treatment. statistically significant differences were seen for both components of the tcrs, the rhinitis medication score (difference= . , p=. ) and rhinitis symptom score (difference= . , p=. ). furthermore, treatment with sq-hdm resulted in a statistically significant reduction of the conjunctivitis symptom score compared to placebo (differ-ence= . , p=. ). treatment was well tolerated. the most frequent adverse events were mild-to-moderate local allergic reactions. the pooled subgroup analysis showed that the hdm slit-tablet ( sq-hdm) was effective in treating hdm allergic rhinitis in adolescents ( - years old). the reduction in the primary endpoint tcrs was statistically significant and comparable to what has been observed in adults. results: at the time of the data-cut for this analysis, a total of european patients were screened ( % male). % of the subjects were aged - , % were aged - , and % were adults. the mean age of the sample was . [sd . ] years, with a minimum of and a maximum of years. in europe were children. many were allergic to foods other than peanut, and most had at least one other atopic condition. even if allergic to multiple foods, patients and their families were nonetheless keen to participate in the trial. the majority of the screened patients were highly sensitive to peanut, reacting to mg (cumulative) or less of peanut protein but a significant proportion of screening failures were due to lack of reactivity to this dose. | efficacy of ir -grass pollen sublingual tablet: improvement in subjects with grass pollen-induced allergic rhinoconjunctivitis based on well days and severe days evaluation objectives: four phase ii/iii dbpc studies, in adults and one in children/adolescents, were conducted worldwide. participants were abstracts | randomised to receive the ir tablet or placebo starting months ( m) prior to the pollen season and continuing for its duration. data from the first season of the trials in adults were pooled. the well days were defined as those with not more than one moderate symptom or mild symptoms of the evaluated symptoms (sneezing, rhinorrhoea, nasal pruritus, nasal congestion, ocular pruritus, watery eyes) and no use of rescue medication. the severe days were defined as either at least one moderate symptom in subjects using rescue medication, or at least moderate symptoms or one severe symptom whether rescue medication was taken or not. for each subject, the proportions of well days and those of severe days were evaluated during the pollen period while on treatment. treatment groups were compared using a wilcoxon -sample test. introduction: the safety of subcutaneous immunotherapy (scit) has been proven in several studies, but the occurrence of side effects (se) remains a concern in daily clinical practice and understanding of their risk factors and avoidance strategies remains limited. objectives: the aim of presented study was to assess the incidence and risk factors of early and late side effects in patients undergoing scit. we conducted a year-long observation of over patients undergoing scit in our outpatient clinic. we recorded detailed information for each administration and screened subjects for both immediate and late reactions. we compiled the records with medical histories to build a database, which was analyzed using multiple logistic regression. casein is a major cow s milk allergen and very resistant to high temperatures. higher levels of ige towards caseins have been reported to associate with persistent cow s milk allergy and higher risk of adverse reactions before and during the milk oit. a follow-up study on oit to milk started in with a six-month built-up phase. seventy-six children (mean age . years) with challenge-verified cma participated the milk oit and their immunological changes were analyzed employing crd. during the year , long-term follow-up report was collected by questionnaires ( %, n= ) and blood samples ( %, n= ). specific antibody responses were characterized before oit and on long term follow up and compared to long-term questionnaire results: milk consumption (yes/no) and side-effects from milk from past months (yes/no). objectives: to define the utility of crd in long-term follow-up after milk oit. results: mean follow-up time was ( - ) years. ten patients ( %) avoided milk completely and patients ( %) reported routine consumption of dairy products. side effects after milk consumption from last months were reported by % of the patients. increased specific ige levels towards caseins were seen among patients who did not consume milk at the long-term (p=. *). there was no significant association between the long-term milk consumption and ige levels to whole milk (p=. ), caseins (p=. ), alpha-lactalbumin (p=. ) and beta-lactoglobulin (p=. ) measured before the oit in this small sample. patients who consumed milk at the longterm follow-up and reported side effects had higher specific ige levels towards caseins before oit (p=. *). conclusions: there was a high incidence of side effects even after eight years of milk consumption, which may indicate desensitization instead of true tolerance. component-resolved diagnostics may corroborate in predicting prognosis, as suggested by higher incidence of side effects among patients with high levels of ige towards caseins also in the long-term follow-up. objectives: retrospective study of patients who underwent subcutaneous ait for allergic rhinitis ( % had concomitant asthma). patients with less than months of treatment were excluded. large local reactions (wheal≥ mm) and systemic reactions were defined according to who grading system. patient's satisfaction was defined by vas score. results: the mean age of patients ( % males) was ae (range - ) years ( %< years).the most prevalent immunizing allergens were house dust mite ( %), olive ( %) mix of grasses ( %) and pets ( %). % of patients were immunized against a single allergen whereas % were immunized with≥ allergens. patients were followed for ae months. following ait the vas score decreased from . ae . to . ae . (p<. ). % of the patients declared that they will recommend immunotherapy to their relatives. systemic adverse reactions ( % class i, % class ii) were observed in introduction: metabolomics, one of the core disciplines of system biology, is a high-dimensional biology method that may allow hypothesis-free profiling of biomarkers, rather than a traditional hypothesis-driven approach. objectives: this study aimed to apply the metabolomic approach to serum to longitudinal alterations during allergy immunotherapy (ait) in dermatophagoides pteronyssinus (der p) sensitized asthmatic children. results: a robust hydroxyeicosatetraenoic acids (hetes) of inflammatory responses was found for discriminating during ait, including -hete, -hete and -hete. -hete and -hete were significantly decreased continuously from through months of ait. moreover, compared with baseline of ait -hete was detected in very low level during and months. the metabolomic profiling could clearly longitudinal alterations biochemical-metabolic profiles in der p-sensitized children during ait. these markers might be involved in asthma pathophysiology but also represent the therapeutic target for ait. background: metabolomics, one of the core disciplines of system biology, is a high-dimensional biology method that may allow hypothesis-free profiling of biomarkers, rather than a traditional hypothesis-driven approach. this study aimed to apply the metabolomic approach to serum to longitudinal alterations during allergy immunotherapy (ait) in dermatophagoides pteronyssinus (der p) sensitized asthmatic children. methods: in this longitudinal study, we recruited der p-sensitized asthmatic children with ait for months. serum samples were analyzed using a metabolomic approach based on mass spectrometry. results: a robust hydroxyeicosatetraenoic acids (hetes) of inflammatory responses was found for discriminating during ait, including -hete, -hete and -hete. -hete and -hete were significantly decreased continuously from through months of ait. moreover, compared with baseline of ait -hete was detected in very low level during and months. the metabolomic profiling could clearly longitudinal alterations biochemical-metabolic profiles in der p-sensitized children during ait. these markers might be involved in asthma pathophysiology but also represent the therapeutic target for ait. we performed desensitization with asa according to the wong protocol (doses of . , . , , , , , , , , mg of asa at intervals of - min). we pre-medicate with cetirizine mg. our patient successfully reached the necessary dose: mg of asa ( . , . , , , , , , and mg) conclusions: approximately % of patients with asthma undergo respiratory symptoms due to exposure to aspirin, meanwhile . % and . % of the population shall undergo hives when exposed to it. desensitization with aspirin is an efficient and safe treatment in patients with hypersensitivity type i, iii and iv. due to the benefits and low toxicity of the wong protocol, we recommend this protocol in order to desensitize patients with allergy to aspirin who undergo rheumatologic or cardiovascular disorders and need antiplatelet treatment. we finally recommend our patients to take mg of asa premedicated with mg of cetirizine every day. we warn that if you interrupt the administration of asa for more than hours, it shall have to be administered again under medical supervision. semedo fm ; cruz c ; reis r ; tomaz e ; in acio f horiuchi t ; takazawa t ; saito s department of anesthesiology, gunma university hospital, maebashi, japan; intensive care unit, gunma university hospital, maebashi, japan introduction: sugammadex is a synthetic c-dextrin derivative that is designed to selectively bind to steroidal neuromuscular blocking agent molecules. although sugammadex has been used in many cases of general anesthesia, there are several reports of anaphylaxis following its use. skin testing is the gold standard for detecting the causative agent of anaphylaxis. however, the test itself sometimes precipitates serious complications, including recurrence of anaphylaxis. hence, development of a novel test that can be performed in vitro without causing such complications is desired. recently, the basophil activation test (bat) has been established as a tool to detect the causative agent of anaphylaxis with high sensitivity and specificity. yet, there are few studies examining the utility of the bat in diagnosing sugammadex-induced anaphylaxis, besides our previous report. although both cd and cd c are currently used as the major markers for activated basophils, which one of them is more suitable for the bat depends on the targeted drugs. objectives: the aim of this study was to investigate whether bat could be utilized to diagnose sugammadex-induced anaphylaxis. in addition, we compared the capability of cd c and cd as markers for activated basophils. seven patients with perioperative anaphylaxis demonstrating a positive skin test for sugammadex were included. furthermore, individuals who tolerated sugammadex and had a negative skin test for allergy to this drug were enrolled as controls. results: the ratios of activated basophils in the patients were much higher than those in controls (mann-whitney u test, p<. ). cd c up-regulation. this was also true for cd . in the case of cd c, the sensitivity of bat for sugammadex was % and specificity was %, while sensitivity and specificity for cd were % and %, respectively. there were no significant differences between cd c and cd in the areas under the roc curve. conclusions: this study showed that bat is a reliable instrument to diagnose sugammadex-induced anaphylaxis. we did not find any difference between cd c and cd as markers for activated basophils in the bat for sugammadex. objectives: we report a retrospective study of patients who were referred to our allergology departments between and with a suggestive history of immediate hypersensitivity to ppis. our purpose was the analysis of the clinical presentations and the allergological investigation performed in order to confirm the diagnosis of drug hypersensitivity to ppis and to study the cross-reactivity among ppis. results: the culprit drugs were pantoprazole (n= ), omeprazole (n= ) and lansoprazole (n= ). the allergological investigation confirmed the diagnosis of hypersensitivity to ppis in patients ( by skin tests and oral challenge). we observed cross-reactions between omeprazole, pantoprazole and esomeprazole (n= ), omeprazole and pantoprazole (n= ), respectively omeprazole and esomeprazole (n= ). in patients the severity and the recurrence of the reaction did not allowed the provocation test with the culprit drug. two oral challenges allowed the use of an alternative ppi (based on the pattern of less cross-reactivity depending on the chemical structure: pantoprazole for a patient who had a grade iii anaphylaxis to lansoprazole and lansoprazole for a patient who had a grade ii anaphylaxis to pantoprazole). conclusions: a complete allergological investigation (skin tests and cautiously, oral challenge) is needed in order to confirm the diagnosis of drug hypersensitivity to ppis and to take a therapeutic decision. the diagnostic approach is limited by the low sensitivity of skin tests and the patient's background (comorbidities and severity of the reaction) which do not always allow the oral provocation test. an ige dependent mechanism may be involved in hypersensitivity reactions to ppis or their metabolites. herrero-lifona l ; muñoz-rom an c hospital quironsalud campo de gibraltar, los barrios, spain; hospital regional universitario de m alaga, m alaga, spain introduction: hypersensitivity reactions to beta-lactams are an important problem to study, especially in children, given that these antibiotics are the gold standard for the treatment of many infectious diseases in the infancy. most hypersensitivity reactions to betalactams in children are due to non-immediate response and to diagnose them is essential to perform a drug challenge test. although hypersensitivity is usually ruled out in children by drug challenge test, it is positive test up to %- %. objectives: a year-old boy is suspected to have experienced two drug reactions after the intake of amoxicillin with and without clavulanic acid for pharyngitis treatment. in the first one, he presented a maculopapular eruption in the back after one day treatment with amoxicillin-clavulanic acid. in the second reaction, two hours after the intake of amoxicillin, it appeared an itching maculopapular eruption in the back that was resolved with symptomatic treatment. in both cases, the patient tolerated treatment with cefuroxime afterwards. results: intradermal test with amoxicillin was negative in immediate and delayed reading. oral drug challenge test with amoxicillin ( mg/kg/day, total cumu- oral drug challenge test with penicillin v (total cumulative dose mg): it was well tolerated and the patient continued taking it for epicutaneous patch testing with amoxicillin in the lesion area and in an unaffected area: in the lesion area, it appeared mild erythema, but it was considered a negative result in both areas. conclusions: we report a case of an amoxicillin-induced multiple fixed exanthema: an unusual non-immediate reaction in childhood. most cases of non-immediate hypersensibility need to be confirmed by drug challenge test, given that skin testing lacks sensitivity both intradermal and patch tests. the patient presents a selective hypersensitivity to amoxicillin, being tolerant to penicillin and cephalosporins. jimenez-rodriguez t ; soriano-gomis v ; gonzalez-delgado p ; cueva-oliver b ; venegas-diaz ij ; fernandez j results: data from patients were analyzed, of which . % ( ) were women. the overall mean age was . ae . years, with no statistical difference between sexes. the . % ( ) of the patients presented symptoms with a single group of nsaids, and . % ( ) with or more different groups. the % ( ) of patients presented a history of atopy. in patients with rhinitis % had symptoms with only one nsaid and % with two or more groups of nsaid, while patients with asthma, % reacted to one nsaid and % to two or more groups. the most frequent clinical manifestations were: urticaria/angioedema in % ( ), pruritus in . % ( ), bronchospasm in . % ( ), other respiratory symptoms in % ( ) and anaphylaxis in % ( ) patients. the most frequently involved nsaids were: metamizole ( . %) and ibuprofen ( . %). skin tests were performed in only patients, of whom ( %) were positive to metamizol. dpt was performed in . % ( ) objectives: fifty patients diagnosed with "dress" ( - ), in a tertiary center were retrospectively analyzed, with cases meeting the regiscar criteria. we collected demographic, clinical, laboratory and therapeutic data from the electronic medical records. results: all cases occurred during hospitalization and in several hospital areas. the mean age was . years ( - ) with . % women and % of caucasian origin. reported clinical manifestations were skin rash ( %), fever ( . %), digestive symptoms ( . %), respiratory ( . %), neurological ( . %), head and neck ( . %), urological ( . %), ophthalmologic ( . %) and musculoskeletal ( . %). the most relevant laboratory findings were eosinophilia ( %), elevated transaminase levels ( %), lymphocytosis ( %), altered coagulation ( %) and altered renal function ( %). virus serology was positive in cases ( . %) involving hhv- , ebv, cmv and hiv. antinuclear antibodies were positive in / cases ( . %). skin biopsy, performed in cases ( . %) revealed findings suggestive of drug induced skin reaction. suspected culprit drugs were antibiotics ( %), nsaids ( %), antiepileptic drugs ( %), and antiparasitic agents ( %). one case was related to the administration of heparins and other to a monoclonal antibody. in % of cases, the episode was not related to a particular drug. treatment was accomplished by the administration of corticosteroids ( %), antihistamines ( %), and immunosuppressant drugs in cases. fluids and other medications were administered attending to symptoms. death occurred in patients, although only in case it was related to dress syndrome. the average time from reaction to death was . months. allergy evaluation, performed in cases addressed the potential role of drugs. skin tests were positive in / patients ( . %) and basophil activation test was negative ( / cases). results: from patients admitted with possible diagnosis of scar, only met the inclusion criteria ( f/ m, age - , average . years-old). they were all admitted to a medicine ward. five patients had diagnosis criteria of dress, of sjs and of ten. as for the drugs related with the reaction, antiepileptic drugs were the most frequent ( patients); allopurinol ( ), betahistine ( ), ciprofloxacin+nimesulide ( ) were the causative drugs in the remaining patients. average time to the beginning of symptoms was . days ( patients unknown). in patient the cause was unknown (he had criteria of ten and also diagnosis of malaria and was transferred to another hospital). there were no deaths. objectives: the objectives of the present study were to determine the efficacy of nfeno to: . establish the diagnosis of rhinitis; and . discriminate allergic rhinitis (ar) from non-allergic rhinitis (nar). material and methods: prospective and controlled study with healthy subjects, which included patients with rhinitis. ar and nar were phenotypically defined according to positive or negative prick test results, respectively; the control group was collected from people without upper or lower respiratory tract disease and the prick test was negative. in all sample included the following measurements were performed: feno and nfeno (novario analyzer) were performed; spirometry; eosinophil counts in blood and nose (by nasal brushing); bilateral nasal endoscopy; and acoustic rhinometry. results: we included cases (ar= , nar= and controls= ), with a mean age of (sd . ) years, % men. the table below shows the results of the variables analyzed by group. patients with rhinitis compared to controls had significantly greater feno production, as well as a higher proportion of eosinophils in blood and nose, but not in nfeno production. however, when the two subgroups of rhinitis were compared, ar patients had significantly higher feno and nfeno than nar, in addition to blood and nose eosinophils. there were no differences in acoustic rhinometry values between groups. conclusions: although nfeno does not appear to identify patients with rhinitis, it may be useful in discriminating ar from nar. in routine clinical practice, it could help guide to identify an ar in cases with inconclusive results of the complementary tests commonly used in its diagnosis. introduction: fractionated exhaled nitric oxide (feno) is used as a marker of eosinophilic airway inflammation in asthma, whereas clinical presentation of nasal no (nno) is unknown. objectives: the objective of this study was to evaluate the factors influencing nno levels. in patients with chronic nasal symptoms, total-nasal-symptom-scores (tnss) were calculated; skin-prick-test conclusions: in conclusion nasal no is useful in allergic inflammation in the absence of sinus obstruction in nasal cavity. however, its value is limited with paranasal sinus ostium occlusion. were recruited across australia and the uk via a patient panel. the aim was to assess which ar treatment attribute(s) drove patient preference. results: table shows the willingness to pay (wtp) for each attribute. all attributes were significant predictors of treatment choice, except administration method. however, patients in both countries showed a considerable preference for treatments that were more efficacious, fast acting and affordable. conclusions: although treatment relief remains of primary concern, time to treatment benefit and cost could dictate treatment preference. these data may be of value in optimizing the acceptability of future ar treatments and informing trial endpoint selection. australia ( izquierdo l; chiriac am; molinari n; demoly p introduction: allergic rhinitis is a frequent disease with an important impact on quality of life. self-administrated control assessment tests can help achieve a better management of this disease. however, none of these tools has been validated in teenagers. test in its original version, following the same protocol as the original study in adults. we designed a multicentre, observational, cross- conclusions: while the number of included patients is low, the first results are promising. indeed, a significant improvement of the rhinitis control score was found between d and d . this led us to the hypothesis that this questionnaire could be used as is to estimate the control of the allergic rhinitis in teenagers. analyses concerning the validation of the questionnaire itself do not reach at the moment the threshold of significance, the recruitment is on-going, to increase its power. objectives: the objective of this study was to evaluate the role of serum vitamin d in patients with symptomatic allergic rhinitis and active asthma during the allergy season and observe the effect of montelukast mg daily as treatment. results: this study included asthmatic and seasonal allergic rhinitis patients following a single-blind, placebo run-in period of - days, patients were randomized to oral montelukast mg (n= ) or placebo (n= ) daily during the -week, double-blind, active-treatment period. the serum vitamin d was also evaluated in both the groups. the serum vitamin d levels were found to be higher in patients taking montelukast compared to placebo after weeks (p<. ). montelukast reduced the daily rhinitis symptoms score: difference between montelukast and placebo (p<. ). similar improvements were seen in daytime nasal symptoms (p<. ) and nighttime symptoms (p<. ). conclusions: montelukast provides significant relief from symptoms of seasonal allergic rhinitis, while also conferring a benefit for asthma, in patients with both allergic rhinitis and asthma. further, it has a beneficial role in improving vitamin d levels. venegas-diaz ij ; jimenez-rodriguez t ; canto-reig vj ; lindo-gutarra m ; soriano-gomis v ; gonzalez-delgado p ; cueva-oliver b ; fernández j allergy section. general hospital alicante. isabial, alicante, spain; allergy section. general hospital alicante. isabial-umh, alicante, spain introduction: local allergic rhinitis is an accepted entity, which only can be recognized by nasal provocation test (npt) in patients with clinical rhinitis with negative skin tests or specific ige (sige). our aim was study this entity in our patients in alicante, spain. objectives: patients with symptoms of rhinitis with skin tests, and sige negative for common aeroallergens were selected, since . half of them ( ) most were young adults, % belonging to the range of age between - years; more than % had family history of atopy; the mean age of onset of symptoms was years and up to . % had had symptoms before the age of years; . % were nonsmokers. regarding comorbidity, . % of the patients presented with concomitant symptoms of conjunctivitis and . % of asthma. symptoms in . % of the patients were persistent and most of moderate ( %) to severe ( . %) intensity. the majority of patients ( . %) presented perennial symptoms and a tpn positive to mites in . %, to salsola pollen in % and to cypress pollen in . %. but patients ( . %) were simultaneously positive to allergens. conclusions: local allergic rhinitis represents an important proportion of patients with clinical rhinitis with negative skin tests and sige. we have to pay attention to identify clinical and demographic factors of ral and to use npt to demonstrate it. objectives: we report the extent of sinus involvement at baseline (bl) in pts with bilateral np refractory to intranasal corticosteroids from a dupilumab phase a study (nct ). ct scans from enrolled pts, adults < years with nasal polyp score of ≥ / , were pooled and analyzed at bl. for opacification, standard lund-mackay (lmk) scoring ( =normal, =partial opacification, =total opacification) in maxillary, anterior/posterior ethmoid, sphenoid, frontal sinuses was used. ten sinus scores plus bilateral ostiomeatal complex (omc) score ( =not occluded, =occluded) were summed to a bilateral total lmk ( - ). zinreich modified lmk score (zlmk), providing more granularity on opacification degree, was evaluated post-hoc; each sinus was given a - score based on opacification % from mucosal thickening ( = %, = %- %, = %- %, = %- %, = %- %, = %); omc results: the prevalence of asthma at the age of years was . %. % of them had an act tm value below , ie uncontrolled asthma, median . (range - ). independent risk factors for uncontrolled asthma at the age of were current doctor diagnosed rhinitis (adjusted or, aor . ; % ci . - . ), wheeze triggered by exercise (aor . ; . - . ) and cat at home (aor . ; . - . ). if at least one of the parents had higher education the risk of uncontrolled asthma was decreased (aor . ; % ci . - . ). six children reported hospitalisation due to asthma during the last months (ie . %) at years of age. hospitalisation was more common in individuals with uncontrolled asthma (or . ; . - . ) or when mites (or . ; . - . ) or pollen (or . ; . - . ) was reported as trigger factors. also, oral corticosteroids (betamethasone), in the last months increased the risk for hospitalisation (or . ; . - . ). to have a parent with asthma ( % compared to %, or . ; . - . ) or higher education ( % compared to %, or . ; . - . ) was numerically less common for children who were hospitalised for asthma but did not reach statistical significance. conclusions: uncontrolled asthma was associated with current allergic rhinitis, having a cat and exercise as trigger factor. at least one parent with higher education reduced the risk. hospitalisation due to asthma was more common in individuals with uncontrolled asthma. abstracts | adolescent asthma in relation to severity and gender-data from a prospective population based cohort study introduction: asthma often debuts early in life, but recent studies show that the development of asthma is a dynamic process with disease turnover throughout child-and young adulthood. objectives: to describe adolescents' asthma with focus on severity and gender. the study population consisted of adolescents participating in the cohort, followed since birth up to years of age with questionnaires and clinical investigations. blood samples from adolescents ( . %), sera analyzed for specific ige against common inhalant allergens. asthma at years was defined as fulfilling at least of the following criteria: symptoms of wheeze and/or breathing difficulties in the last months, ever doctor's diagnosis of asthma and/or asthma medicine occasionally or regularly last months. uncontrolled asthma was based on parental information on symptoms of asthma in the last months prior the -year follow-up, including or features: nocturnal asthma, activity limitation, wheeze times and hospitalization due to acute asthma. we looked into phenotypes; late-onset, defined as asthma at , or , but not at , and years of age and persistent, defined as asthma at , or and , or years of age. severe asthma, defined as asthma as above combined with prescribed and dispensed corticosteroids used within the last months plus uncontrolled asthma and/or lung function (fev < % of predicted). results: at years of age, . % (n= ) fulfilled the study definition of asthma, . % late onset and . % persistent asthma. persistent asthma was more frequent among boys than girls . % vs . % (p=. ). overall ige sensitization was common among adolescents with asthma, . % were sensitised to common inhalant allergens, and equally common in late-onset and persistent asthma ( . % vs . %, p=. ). however, more boys than girls were sensitised ( . % vs . %, p<. ). in total . % (n= ) adolescents had severe asthma, . % (n= ) boys and . % (n= ) girls (p=. ). the overall prevalence of severe asthma in the cohort was estimated to . %. severe asthma did not significantly differ among adolescents with late-onset compared to persistent asthma ( / , / , p=. ). conclusions: among adolescents with asthma, late-onset (age ≥ years) and persistent asthma were equally common. persistent asthma were more common among boys. there were no difference in asthma severity, or proportion of ige sensitization among adolescents with late-onset or persistent asthma. however, data investigating associations with wheeze and asthma in later childhood are scarce. objectives: our aim was to explore the association of maternal milk fatty acid composition with childhood wheezing phenotypes and asthma up to age years. breast milk was collected weeks and months post-delivery in the ulm birth cohort study (n= and n= , respectively). concentrations of - carbon atom chain length fatty acids were measured by high-resolution capillary gas-liquid chromatography. to control for constant sum constraint, concentration data were transformed using the centered log ratio method. compositional biplots and correlation matrices were used to group fatty acids based on within sample correlation. adjusted risk ratios with parent-reported wheezing phenotypes and doctor-diagnosed asthma were computed using a modified poisson regression. results: we observed no straightforward evidence of associations between overall breast milk fatty acid composition and specific wheeze phenotypes or doctor-diagnosed asthma. conclusions: despite our use of sophisticated statistical methodology, our results may have been biased toward the null by several cohort-specific factors associated with breast milk collection and fatty acid composition. to overcome potential selection bias by maternal lifestyle, further research should investigate fatty acid intake during the first year of life including sources other than breast milk among children who were never or not exclusively breastfed. introduction: fall is the most common season for asthma exacerbation. previous studies found that exposure and sensitization to house dust mites (hdms) can exacerbate asthma. the seasonal variation in indoor hdm concentration is highest in the fall, correlating with the incidence of asthma exacerbation. most of the studies conducted to date have focused on the effect of hdm exposure on the incidence of acute asthma exacerbation, but reports about the effect of hdm sensitization are few. thus, we aimed to determine whether sensitization to hdms acts as a risk factor of asthma exacerbation in the fall. in addition, we investigated whether asthma exacerbation in the fall had any distinctive features by comparing levels of various cytokines and chemokines with those in other seasons. objectives: we enrolled children aged - who visited the emergency department because of acute asthma exacerbation from january to december ( . %, males; mean age, . ae . years). they were treated in accordance with the standardized treatment protocol. blood samples were collected from the children during the course of treatment. by using residual sera from the blood samples, we measured the levels of total immunoglobulin e (ige), hdm-specific ige (sige), eosinophil cationic protein (ecp), and various cytokines and chemokines, and classified them according to season. we compared the date divided into fall group (from september to november, n= ) and other season group (from december to august, n= ). ci, confidence interval; or, odds ratio; der f-sige, dermatophagoides farinae -specific immunoglobulin e; der p-sige, dermatophagoides pteronyssinus-specific immunoglobulin e; ecp, eosinophil cationic protein; ige, immunoglobulin e. data are presented as n (%) or as meansaesems and median (range). p value refers to the difference between the fall and "other season" groups and was calculate by the chi-square statistics, fisher's exact test, student's t-test, or wilcoxon rank-sum test. a adjusted for total ige. results: this retrospective study obtained archived nasopharyngeal aspirate (npa) samples from patients aged below years who were hospitalized for acute respiratory illnesses in a university-affiliated hospital during the periods september-november and january-april . their clinical information was retrieved from computerised record. hrv was detected by rt-pcr, and isolates were sequenced to determine the genogroups and serotypes. ninety patients whose npa was positive for hrv and patients being negative for an extended panel of respiratory viruses by multiplex pcr method were identified. mean age of these groups was . years and . years respectively. hrv infection was significantly associated with asthma exacerbation (or . , results: a total of children were included: . % were boys; . % aged months to years, . % aged - years, . % aged - years and . % aged - years; . % were hospitalized, results: the long-term remission (≥ years without treatment) rate years after initiating early anti-inflammatory therapy was . % (intermittent asthma, %; mild persistent asthma, . %; moderate persistent asthma, . %; severe persistent asthma, . %). longterm remission rates improved compared with past asthmatic conva- objectives: the aim of this study is to explore the potential value of feno level for diagnosing chronic cough in children. objectives: in this study, we aimed to compare the efficacy of classical spirometry and impulse oscillometry (ios) in evaluation of late reversibility in children who received treatment with the diagnosis of atopic asthma. we enrolled patients aged - years who were diagnosed with atopic asthma. exclusion criteria were having received asthma treatment during the previous two months, having acute asthma exacerbation findings, having any other respiratory disease or cardiac disease that may affects the lung function test results. allergic sensitization was determined by skin prick test performed according to eeaci guidelines. lung function test measurements were performed at enrollment and after two months of inhaled steroid treatment. conclusions: classical spirometry is more valuable compared to ios in evaluation of late airway hyperreactivity in children with atopic asthma in children older than seven years age. | computer bronchophonographyfrequency analysis of the respiratory cycle. objectives: we performed mct in children with symptoms suggestive of asthma and without respiratory symptoms. after each inhalation step oscillometry and spirometry was performed. parameters analysed for ios were z , r , r , x , x and ax (the integrated impedance reactance at r and above) and fev for spirometry. a fall of % in fev from baseline after mtc was considered as a positive challenge. pc -fev and pc r , x and ax were calculated. results: a total of patients, female, with a mean age of . (ae . ) years were enrolled. had a ≥ % fall in fev after mtc. the mean variation in fev was % (- . %/ + . %), the mean variation in z , r , r , x , x , ax and fres were . % objectives: the objective of our study was to synthesize cationic peptides and study their antiviral activity (aa) in vitro. results: peptides were synthesized by solid phase method with different structures (linear, helical and dendrimeric). cytotoxicity of the peptides was studied by mtt assay using hela cells. objectives: the aim of the study is to evaluate the changes of il- results: subjects aged - years were enrolled in this study, which were divided into groups: patients with diagnosed moderate to severe ba ( st group), ba patients with rvi ( nd group), subjects with rvi only ( rd group) and healthy volunteers ( th group). all patients with ba from group and received inhaled corticosteroids. clinical blood test revealed increase of eosinophils in patients with ba and ba accompanied with rvi up to % and %, respectively. fev was decreased in st and nd groups to % and % compared % and % for rd and th groups, respectively. conclusions: this study is alarming for asthmatic nosocomial hazards in this govt. hospital. identification of ige specific reactive components of predominant fungal allergens and cross-reactivity among each other, delined in this study could minimize the hazard of therapeutic and diagnostic use of these cross-reactive components, in fungal allergen-specific immunotherapy. objectives: we conducted a longitudinal prospective study to examine the development, composition and diversity of the gut microbiota in healthy and allergic children. we followed children from months to years of age with clinical evaluation; specific ige levels and skin prick testing. fecal samples were collected at , , months and years. s rrna sequencing was used to profile the gut microbiome using illumina miseq. the composition and diversity of the gut microbiome were assessed using quantitative insights into microbial ecology (qiime). comparisons between groups were made using the lefse pipeline; non-parametric factorial kruskal-wallis test, unpaired wilcoxon rank test and linear discriminant analyses (lda) with score > . . to assess the interaction effect, the likelihood ratio test (lrt) was applied using r statistical program. p<. was considered significant after correction for multiple testing. results: to achieve our aim we divided balb/c mice into groups: mice with viba ( st group), mice with viba treated with nonspecific sirna against gfp (sigfp) ( nd group) and against il- (siil- ) ( d group). th group was intact mice. groups - were i.p. sensitized on days , , with ovalbumin (ova) mixed with aluminum hydroxide and i.n. challenged with ova on days - . the same mice were i.n. infected with tcid /mouse rsv strain a on day . mice from group and were i.n. treated by sirnas on days - in dose lg/mouse. on day hyperresponsiveness (ahr) to methacholine was measured. on day and lungs were removed for histological analysis. viral rna (vrna) load and il- gene expression were evaluated by qpcr in lungs. bronchoalveolar lavage (bal) was collected for differential cell count by light microscopy. so i.n. administration of siil- suppressed il- gene expression in lungs by % compared to sigfp treated mice. there were no significant changes in body weight and lung vrna amounts between mice received sigfp and siil- . mice treated with siil- demonstrated the tendency to improve lung function compared to mice of group - , that expressed in % reduction of specific resistance of airways and % increase of peak expiratory flow. bal cell count revealed decrease of total cell number, eosinophils and lymphocytes in mice received siil- by %, % and % compared to sigfp treated mice, that indicate reduction of inflammation, that was confirmed by histopathological studies showed % leukocyte reduction. downregulation of il- resulted in -and . -fold decrease of bronchial epithelium metaplasia and hyperplasia. and femur length measurements were collected from routine antenatal screenings. these and derived head to abdominal circumference ratio and estimated fetal weight were converted into z-scores adjusted for gestational age and gender and categorized as "low" (≤ sd below mean), "normal," or "high" (≥ sd above mean). ad cases were children with parent-or pediatrician-report of physician ad diagnosis assessed yearly up to age years and supplemented by clinical diagnoses during dermatological exams at . , , and years. modified poisson regression models were used to compute risk ratios (rr) adjusted for potential confounders. conclusions: these results provide further evidence for a role of fetal growth as an influence on atopic disease outcomes. unlike previous studies, our data suggests several patterns of fetal growth beginning as early as the st trimester may influence ad outcomes. objectives: we aimed to examine the role of eosinophil cationic protein (ecp), eosinophil derived neurotoxin (edn) and total immunoglobulin (ig) e as a bio-marker of disease severity. we examined the difference in level of total ige, ecp and edn between the two groups and whether any correlation existed between disease severity and ecp or edn. objectives: we aimed to identify the subgroup of ad patients with a good clinical response to probiotic treatment. we recruited children who suffered from moderate to severe ad with the scoring ad (scorad) index of or higher. after weeks of washout period, all patients were given lactobacillus plantarum cjlp at a dosage of colony-forming units once a day for weeks. we measured eosinophil counts in the peripheral blood, the proportion of cd + cd + foxp + regulatory t (treg) cells in cd + t cells, serum total ige levels, and specific ige to common allergens before the start of the treatment (t ) and at discontinuation (t ). logistic regression models were used for the statistical analysis. seventy-six patients ( boys and girls) with a mean age of . ae . years completed the study. there were responders and non-responders after probiotic treatment. the median scorad was reduced from . (range . - . ) at t to . (range . - . ) at t in the responder group (p<. ). in multivariable logistic regression analysis, a good clinical response was significantly associated with high total ige levels (aor . , % ci . - . ), increased expression of , and high proportion of cd + cd + foxp + cells in cd + t cells (aor . , % ci . - . ). in responder group, the proportion of cd + cd + foxp + cells of cd + t cells were significantly increased after weeks of treatment (p=. ), while the levels of tgf-b mrna expression were decreased (p=. ). there were no differences in total ige levels between t and t (p=. ) conclusions: the therapeutic effect of l. plantarum cjlp on ad is more pronounced in children with high total ige levels, objectives: the objective of the study was to examine the effect of a specific synbiotic mixture of short-chain galacto-, long-chain fructo-oligosaccharides (scgos/lcfos, ratio : ) and bifidobacterium breve m- v on the severity of ad and correlation to serum chemokines in infants with moderate to severe ad and elevated ige. in an exploratory randomized, double-blind, placebo-controlled trial the effect of extensively-hydrolyzed whey-based formula without intervention. serum obtained prior to start and at the end of intervention were analysed using luminex. six chemokines and nine ratio's thereof were correlated to ad severity (sample size= ). introduction: dyshidrotic eczema is one of the most common skin conditions. contact allergy is often associated with dyshidrotic eczema although the exact impact and the influence of contact allergens in different forms of dyshidrotic eczema remain unknown. hypersensitization to nickel is one of the most common contact allergies associated with pompholyx. the standard of care protocol is to use a medical treatment with topical corticosteroids and calcineurin inhibitors to treat the symptoms, together with occlusive barrier creams to avoid skin exposure to the allergens. after the symptoms have been cleared with the topical treatment, the recommendation is to use occlusive barrier creams to prevent recurrence of the symptoms. objectives: a new emollient with specific metal-scavenging agents and no occlusive ingredients has recently been developed and made commercially available. the aim of this study was to evaluate the effect of such cream to provide relief for patients with dyshidrotic eczema associated with nickel allergy. results: thirty-two subjects with dyshidrotic eczema and a positive patch test ppt (contact sensitized) reaction to nickel were selected. these were divided into two randomized groups, group-a was given nickel-scavenging cream (skintifique creamtm, paris) after medical treatment, (n= ) and group-b followed the standard protocol for pompholyx, (n= ). hand eczema was scored according to the dyshidrotic eczema area and severity index (dasi). dasi scores were evaluated at the beginning of the study (day- ), after the medical treatment (day- ) and two months after the end of medical treatment (day- ). results show a significant difference in the efficacy of treatment between the two groups at day- . a higher percentage of at least % reduction of initial dasi score ( . %) and a higher percentage of total clearance ( %) in patients using nickel-scavenging nonocclusive moisturizing cream was observed as compared to standard-of-care occlusive creams ( . % and %, respectively). objectives: the goal of this study was to investigate whether long-term emollient therapy is associated with alterations of skin barrier function and shifts of the skin microbiome in infants at high risk for developing ad. we prospectively enrolled newborns with a family history of ad to be randomized to either emollient treatment group or control group. at months of age, we tested the skin barrier (transepidermal water loss/tewl, water capacitance/cap, ph) and skin microbiome ( s rdna sequencing of skin swabs from cheek, dorsal and volar forearm). results: the emollient group (n= ) had significantly lower skin ph compared to controls (n= ) (p=. ), but without a statistically significant difference in tewl or cap. the emollient group had higher numbers of different bacterial taxa (chao richness) at cheeks (p=. ), dorsal forearms (p=. ), and volar forearms (p=. ) as compared to controls. both streptococcus pneumoniae and s. salivarius statistically significantly contributed to the observed skin microbiome differences between patient groups. s. salivarius was significantly more abundant in emollient subjects at all sampling sites (p=. ). we then analyzed our previous larger cohort of older children with ad and also observed higher s. salivarius proportions in ad patients with treated and less severe disease (p=. ). objectives: to evaluate the effect of overnight treatment with a temperature-controlled laminar airflow (tla) device in children/adolescents with severe eczema over a -month period. in an open-label study, subjects aged - years (median years) with longstanding severe eczema attended visits during the run-in period lasting - weeks (median . weeks) to optimize eczema management. the run-in was followed by a month treatment period using overnight tla device (airsonett ® , sweden), which included study visits. we used scorad-index results: the median duration of eczema was . months (interquartile range - . ). all subjects were sensitised to ≥ perennial allergen, and had multiple comorbidities ( / rhino-conjunctivitis, / food allergy, / asthma). there were no significant changes during the run-in period in any of the outcome measures. we observed a significant improvement in scorad after the -month tla-treatment period, from . [ . - . ] to . [ . - . ] , p=. . iga improved significantly from a median of [ - ] to [ ] [ ] [ ] , p=. . improvement in symptoms was paralleled by a significant reduction in medication usage. by months, there was a significant improvement in . ] to [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , p=. ), and an improvement in cdqli (marginal, p=. ). however, we observed no changes in poem (p=. ) . post-hoc cluster analysis of the patterns of changes in scorad over the month treatment period identified two clusters, with participants classified as responders and as non-responders. introduction: intestinal degradation has been shown to determine allergenicity of food allergens. however, it is unclear how allergens are degraded inside pivotal immune cells such as dendritic cells (dc), and how this affects the subsequent immune response to these allergens. in our studies, we determined whether we are able to measure uptake and degradation of allergens inside dc and whether differences in above mentioned factors exist between allergens. objectives: using mouse bone marrow-derived dc and fluorescent-labeled proteins, we studied the cellular uptake of the peanut proteins ara h , , , and . results: first, we observed that dc uptake of ara h was much higher than ara h , and . using blocking reagents and receptor binding assays for various uptake routes in dc, we observed that one of the principal routes of uptake for ara h was the mannose receptor. other uptake routes, and the routes of uptake for ara h , and are currently under investigation. second, using proteins coupled to beads we observed that intracellular protein degradation was higher for ara h and ara h than for ara h and . finally, cd +t cells from ara h , , or sensitized mice were added to matching allergen-pulsed dc. we observed that while ara h , and to lesser extend ara h elicited strong th type responses, ara h did not elicit any t cell responses. conclusions: together, we show that allergenicity of (peanut) proteins may be, at least partly, determined at the level of allergen uptake and breakdown inside the antigen presenting cell. these findings may be relevant to risk evaluation of existing allergens but also of novel or modified proteins. this also illustrates the usefulness of in vitro, cell based assays to examine initial processes of allergenic sensitization to proteins. | sensitising capacity of unmodified and acid hydrolysed gluten through the skin-a comparative study in na€ ıve vs tolerant brown norway rats ballegaard ar; madsen cb; bøgh kl national food institute, technical university of denmark, søborg, denmark introduction: allergic sensitisation to foods may occur in infancy without prior oral exposure to the offending food. this has led to the assumption that food allergy sensitisation may occur through alternative routes, such as the skin, supported by the observed correlation between skin barrier disruption and food allergy. recently, concerns have been raised regarding the safety of use of cosmetic and personal care products containing hydrolysed wheat proteins, since these products have been shown to induce allergy towards acid hydrolysed wheat through the skin, and even to cause an abrogation of the already established oral tolerance against unmodified wheat. objectives: the aim of the study was to compare the sensitising capacity of an unmodified and an acid hydrolysed wheat product via slightly damaged skin, in order to evaluate differences in conditions necessary for skin sensitisation in na€ ıve vs tolerant individuals. brown norway rats were raised and bred on either ( ) a diet free from wheat, resembling individuals with a na€ ıve immune system, or ( ) a conventional wheat containing rat chow, resembling individuals tolerant to wheat. results: in the na€ ıve rats both products were able to induce a statistically significant specific antibody response after application of the products on the slightly damaged skin, whereas in the wheat tolerant rats, only the acid hydrolysed wheat product was able to induce a statistically significant antibody response. for both the na€ ıve and the wheat tolerant rats the response was dose-dependent. in the na€ ıve rats both products were able to sensitise through the skin, inducing a specific ige response, whereas in the tolerant rats only the acid hydrolysed product were able to induce a specific ige response, though this ige response was much lower than in the na€ ıve rats. results from competitive elisas demonstrated that new epitopes had developed as a result of acid hydrolysis, though original epitopes were maintained at the same time. this may explain why only the acid hydrolysed wheat could induce specific antibody responses in the tolerant animals. conclusions: this study showed that the sensitising capacity through the skin of two different wheat products is heavily influenced by the tolerance status of the immune system and the degree of modification of the wheat products. results: using a germ-free c h/hen mouse model of food allergy, we examined the presence of a major peanut allergen, ara h , in the blood after intraperitoneal injection in previously sensitized and control (non-sensitized) mice using an untargeted, quantitative proteomic approach. previously sensitized mice underwent physiological (core temperature decrease, clinical symptoms) and biochemical (mast cell protease increase, ara h specific ige positivity) changes associated with severe allergic reactions. we were able to confidently detect multiple peptides derived from the ara h protein after intraperitoneal injection in both control and ara h sensitized mice. however, the ara h protein was present at - fold higher levels if mice were previously sensitized, suggesting increased transit across the peritoneal mesothelium with sensitization. an untargeted proteomic approach also allowed changes in the blood proteome of mice undergoing a severe allergic response to be examined. we identified proteins with significantly altered quantity in serum between control and sensitized mice and were therefore apparently associated with the allergic response. we demonstrate the applicability of untargeted proteomics to the study of the allergen transport and proteomic changes which co-occur with a resultant allergic reaction. the transit of allergens into the bloodstream is heavily dependent upon previous sensitization. we are continuing this work to examine the specificity of observed increases in trans-mesothelial transport and to address transport of allergens after intragastric challenge. | iga to cow's milk differs between breast milk and serum for its epitope specificity objectives: we sought to assess whether the profile of epitopespecific iga differs between mother's serum and bm. we also examined how infants' food epitope-specific iga develops in early infancy and the relationship of iga epitope recognition with development of cow's milk allergy (cma). results: . we measured iga specific to an array of overlapping peptides in major cow's milk allergens (alpha s -, alpha s -, betaand kappa-caseins and beta-lactoglobulin). diversity of peptide-specific iga (ie epitope diversity) was determined in paired maternal and infant sera as well as breast milk samples in mother-infant dyads within the first postpartum months utilizing peptide microarray. microarray data was converted to z-scores and filtered for noise. peptide epitopes were determined based on jaccard distance between neighboring peptides, and intra individual correlation between sample types was estimated using phi coefficient. comparisons between groups and individuals was done using non-parametric tests. we noted marked discordance in epitope recognition in paired breast milk and maternal serum samples. at least one shared epitope was recognized by both milk and serum samples ranging from % of mothers for alpha-s casein to % for kappa-casein. epitopespecific iga was detectable in infants' sera starting at less than months of age. sera of mothers with a cma infant had increased binding of epitope-specific iga to cow's milk proteins compared to those with a non-cma infant (p<. for all five proteins). conclusions: these findings support the concept that mothers' milk represents a product of the mucosal immune system that has an antibody repertoire distinct from that of peripheral blood. results: multiple ige-binding proteins were detected in the different wn preparations and many of which were dissemblance in dblotting. profiles of detectable proteins (peptides) of raw, roasted and boiled wn extracts were profoundly different in emi analysis. introduction: consumption of tree nuts is on the rise due to their beneficial health effects. however, tree nuts can led to severe allergic reactions in sensitized patients. thermal processing can modify the structure and function of food proteins and may alter (increase or decrease) their allergenic properties. knowledge about the effects of thermal processing on tree nuts such as cashew or pistachio is rare and based on traditional in vitro immunoassays. objectives: to elucidate the influence of thermal treatments (boiling and heat / pressure) on the ige reactivity of cashew and pistachio proteins, by means of traditional in vitro immunoassays and mediator release assays (mra). results: the allergenicity of untreated and treated cashew and pistachio nuts was evaluated by ige-elisa, ige-immunoblot and elisa inhibition assays using sera from spanish patients with clinical allergy to cashew and pistachio. rat basophilic leukaemia cell line transfected with the a-chain from the human high-affinity ige receptor fceri, was sensitized with a pooled sera and used for mra. sensitized cells were stimulated with untreated and treated protein extracts, in order to investigate the capability of untreated and treated cashew and pistachio proteins to cross-link ige on effector cells. the results showed that heat and pressure treatment at the harshest conditions considered in this study produced a higher decrease of the ige-binding capacity of cashew and pistachio proteins than boiling without pressure or soft conditions of heat and pressure. interestingly, although the treatments of heat and pressure seemed to affect cashew allergens to a greater extent than pistachio allergens (evaluated by ige-elisa and elisa inhibition), the results of mra using the cell line rbl- indicated that cashew proteins treated with heat and pressure, still retained some capacity to cross-link ige. introduction: previous research has indicated an important role for dietary non-digestible oligosaccharides in decreasing the incidence of atopic dermatitis in children at risk of allergy. it is assumed that these prebiotics promote colonization of beneficial bacteria in the gut. children with atopic dermatitis receiving a diet of galacto-and long chain fructo-oligosaccharides (scgos/lcfos) with bifidobacterium breve m- v had enhanced serum galectin- levels. galectin- has ige-binding capacities and can hereby suppress degranulation of mast cells and basophils. next to their function in the gut, oligosaccharides may also affect other immune cells directly, since they were found in plasma and urine. objectives: we investigated whether non-digestible oligosaccharides or galectin- can have a direct effect on immune cells, by determining the effect on basophil degranulation in peanut-allergic patients. whole heparinized blood samples were collected from peanut-allergic adult patients and incubated for hours with either a mixture of . % : scgos/lcfos or scfos/lcfos, or galectin- ( or lg/ml) at °c in the presence of il- ( . ng/ml). after hours, a basophil activation test (bat) was performed. basophils were stimulated for minutes at °c with increasing concentrations of whole roasted peanut extract or human anti-ige. degranulating basophils were determined as cd + cells and calculated as percentage positive cells. results: in each patient, the concentration of anti-ige or peanut extract that induced maximal degranulation in the untreated control sample was used as reference value to compare degranulation of the samples pretreated with scgos/lcfos, scfos/lcfos, or galectin- . pre-treatment of whole blood with scgos/lcfos resulted in an average decrease in degranulation of approximately %, while a significant reduction of % was observed after pre-treatment with scfos/lcfos (p<. ). pre-treatment with lg/ml galectin- decreased basophil degranulation with %, whereas lg/ml galectin- caused a significant decrease of % (p<. ). no differences were observed in the ec , indicating that the basophils are not becoming less sensitive to the peanut extract or anti-ige. the prebiotic mixture scfos/lcfos and galectin- can contribute to decreased degranulation of basophils in a igemediated bat assay using whole blood. further analysis is warranted to define the exact working mechanism of these oligosaccharides. introduction: the intestinal mucosa plays a key role in the development of food allergies. we studied the interaction between intestinal epithelial cells (iecs) and pbmcs of peanut-allergic patients in a transwell co-culture model. exposure of iecs to a mixture of galacto-and/or long chain fructo-oligosaccharides (scgos/lcfos) in combination with synthetic cpg dna (tlr ligand),modulated the cytokine response of activated pbmcs, driving away from the allergic phenotype. objectives: our aim was to compare the efficacy of scgos/lcfos with a scfos/lcfos mixture and to evaluate these effects in an allergen-specific co-culture model. iecs (ht- , human colon adenocarcinoma) were grown on transwell filters until confluence. iecs were apically exposed to . % : scgos/lcfos or scfos/lcfos either or not in combination with . lmol l À cpg dna to mimic presence of dna of beneficial bacterial in the gut. these iecs were co-cultured with basolateral pbmcs from peanut-allergic patients, either activated with anti-cd /cd ( hours) or peanut-extract (pe, days).cytokines ifn-c, il- , il- and tnf-a were measured in the basolateral supernatant and t-cell polarization of the pbmcs was determined (th , th , treg and tfh). results: apical exposure of iecs to cpg dna increased basolateral ifn-c and il- production by anti-cd /cd activated pbmcs (p<. ), and was enhanced by both oligosaccharide mixtures (p<. ). cpg exposure in transwells with pe stimulated pbmcs also increased ifn-c and il- production, which was further enhanced by scfos/lcfos (p<. ). in this pe-specific model, percentages of th and treg cells increased upon cpg exposure of iecs, and th frequency was increased by scfos/lcfos (p<. ). cpg dna exposed iecs suppressed il- and tnf-a production by anti-cd / cd activated pbmcs. both scgos/lcfos and scfos/lcfos reduced tnf-a production in combination with cpg dna (p<. ). tfh cells can produce il- , inhibiting class-switching to ige . upon cpg exposure of iecs, we observed an increase in tfh cells (p<. ) and similar tendency for cd + il- + cell frequency in anti-cd / cd activated pbmcs. conclusions: epithelial exposure to both scgos/lcfos and scfos/lcfos enhances the cpg dna induced th and regulatory il- response in an anti-cd /cd co-culture model, whereas only scfos/lcfos was effective in an peanut-specific co-culture model. introduction: in areas of spain with high level of grass pollen exposure, % of grass allergic patients were sensitized to profilin, and most of them developed severe profilin mediated food reactions. this specific population of patients constitute an ideal model to study the relation among respiratory and food allergy objectives: our aim here was to analyze the links between epithelial barrier integrity and inflammation in oral mucosa. methods: allergic patients and controls were included in the study. allergic patients were stratified into mild or severe according to their clinical history and response to profilin oral challenge test. immunohistochemistry (ihc) for cd c, cd , cd , claudin- ; and dapi nuclear staining were performed in formalin-fixed, paraffin embedded sections of oral mucosa biopsies. rt-pcr was performed to analyze il b and il gene expression and the number of circulating cd + cells in pbmc was measured by flow cytometry. additionally, basophil activation test was carried out in whole blood samples upon profilin stimulation and the ec was calculated. results: regarding epithelial barrier integrity, claudin- expression resulted inversely proportional to pollen-associated food allergy severity. furthermore, by dapi staining, we noticed a lower number of epithelial cells in allergic patients than in non-allergic, and the gene expression of il b and il resulted significantly increased in oral mucosa from severe group. as for immune response in oral mucosa, the number of cd c and cd + cells resulted significantly higher in severe group. in allergic patients, double ihc for cd c-cd showed an increase colocalization of t cells and apcs in the interface between epithelium and connective tissue. a decrease in blood circulating cd + cells was detected in allergic patients compared to non-allergic. as for the basophil sensitivity, ec in severe patients was -times lower than in mild. our results show that damage in oral mucosa epithelium, probably induced by high grass pollen exposure, might allow profilin to penetrate inside oral mucosa and induce inflammation with local recruitment of immune cells. furthermore, analyzing the immune response developed by effector cells, basophils from severe group result more sensitive to profilin as they react to a lower allergen concentration. these data explain the differences in food allergy severity between mild and severe group and propose oral mucosa as a new sensitization route. introduction: th cells producing the hallmark cytokines il- , il- and il- have been found to constitute the majority of the allergen-specific th cell responses in allergic diseases. subpopulations of the th responses have been described with an early primed th subtype characterized by production of il- and il- , and a highly differentiated th subtype, which in addition produce at least il- . objectives: we aimed to investigate the polarity of tree nut and peanut allergen-specific th cells in subjects with confirmed tolerance or allergy to multiple nuts, and hereby detect differences in allergenspecific th cell responses within the same study population. we also wanted to stress the question whether a th phenotype dominates in asymptomatic sensitization. methods: pbmcs from donors all assessed for clinical reactivity to hazelnut, walnut, cashew nut, pistachio nut and peanut was stained with cfse and stimulated ( cells/ml) with and without whole nut extracts ( - lg/ml) for days. allergen-specific cd + t cell phenotypes and cytokine release was analyzed with flow cytometry and luminex, respectively. the allergen-specific th cells of the allergic donors showed a trend of more highly differentiated il- +il- + th cells and higher abstracts | release of il- than in the tolerant donors. unexpected, except in the cashew nut stimulated cells, no difference was found in the relative percentage of the less differentiated th cells (single il- + allergen-specific cd + t cells) when comparing allergic with tolerant donors. when subdividing the tolerant donors into asymptomatically sensitized or ige-negative (< . kua/l), increased frequency of highly differentiated il- +il- + allergen-specific th cells were found in asymptomatically sensitized compared to tolerant-ige-negative donors. interestingly, a positive association of the allergen-specific ige level and the frequencies of allergen-specific il- +il- + and il- + th cells were found in hazelnut, pistachio nut and cashew nut but not in walnut and peanut allergic subjects. conclusions: an overrepresentation of allergen-specific highly differentiated il- +il- + th cells and an elevated il- production were observed in allergic subjects compared to subjects that tolerated the nuts. we furthermore found a trend that subjects asymptomatically sensitized to nuts differed from tolerant-ige-negative subjects by having relatively more highly differentiated il- +il- + allergen-specific th cells. introduction: it remains largely unknown which features of food proteins that render them allergenic vs tolerogenic. however, it has been suggested that the protein-chemical features affects protein uptake in the intestine, and that protein uptake route may impact on the risk of sensitisation. objectives: the aim of this study was to investigate the interplay between protein-chemical characteristics, the allergenic vs tolerogenic properties and the intestinal uptake of two protein products. the allergenic vs tolerogenic capacity of a heat-treated whey product, consisting of partly denatured and aggregated proteins, was compared to an unmodified whey product in: ( ) results: though this study showed that both unmodified and heattreated whey had immunogenic, sensitising and eliciting capacities as well as tolerance inducing capacity, significant differences between the two products were observed. the heat-treated product was found to have a lower allergenicity combined with high tolerogenicity compared to the unmodified product. competitive igg elisas indicated that heat-treatment of whey induced de novo epitopes while the original epitopes were maintained. newly established methods to study in vivo intestinal uptake were successfully applied to compare the uptake kinetics of the two products in different small intestinal tissues and serum. collectively the in vivo and in vitro uptake experiments suggested that uptake kinetics and the major intestinal uptake route differed between the heattreated and unmodified product. conclusions: this study showed that heat-treatment, which induces partly protein denaturation and aggregation, changes the immunological properties and intestinal uptake of a whey protein product. the heat-treated product was found to have a lower allergenicity combined with high tolerogenicity compared to the unmodified product, which highlights this products promising potential for induction of cow's milk tolerance. objectives: in this study, we enumerated tregs in esophageal tissue of patient with eoe, gerd and normal controls. ten patients with eoe, patients with gastroesophageal reflux disease (gerd), and patients with normal endoscopy and normal esophageal tissue were included. tregs were enumerated in paraffin embedded esophageal biopsy blocks, using immunohistochemistry assay. tregs were identified as foxp +, cd + cells. results: tregs were counted in high power fields (hpf, ) for patients and the average of hpf was recorded. the number of tregs in esophageal tissue of patient with eoe (mean . cells/ hpf) was significantly more than gerd(mean . cells/hpf) and control groups(mean . cells/hpf) (p value <. ). conclusions: there is an increase in number of regulatory t cells in esophagus of patients with eoe in comparison to gerd and control groups. the presence of these cells might be due to eosinophilic inflammation and help controlling the inflammation. objectives: to evaluate whether anti-cd (daratumumab) treatment results in a reduction of total and specific ige levels. results: samples from patients with relapsed/refractory multiple myeloma, treated with daratumumab monotherapy or daratumumab plus lenalidomide-dexamethasone in the umc utrecht between april and august , were tested for total ige levels as well as presence of specific ige against common inhalant allergens. in patients with detectable ige at baseline, the total and specific ige levels were evaluated during treatment up to weeks. of eight patients receiving treatment, four had detectable ige levels at baseline. one patient demonstrated sensitization to common inhalant allergens. for this patient, levels of total ige gradually decreased during weeks of treatment (from to ku/l; %), as well as specific ige against timothy grass ( . - . ku/l; %) and house dust mite ( . - . ku/l; %). a second patient, not sensitized to common inhalant allergens but with ige levels of ku/l at baseline, also demonstrated a decrease in ige levels, to ku/l ( %). the last two patients had total ige levels < ku/l at baseline, which dropped below detection limit after eight weeks of treatment. conclusions: this proof of concept demonstrates that (specific) ige depletion occurred during treatment with anti-cd (daratumumab). anti-cd could potentially play a role in the management of severe ige-mediated diseases. objectives: following individual and assessment, patients established and stable on omalizumab have been commenced on home therapy. training and education in self-administration has been provided. a service assessment has been carried out to review the ongoing safety, quality and patient experience of the service. results: to date, over doses of omalizumab have been self administered in the community with no adverse events or incidents related to home therapy. conclusions: self-administration of omalizumab at home by patients offers the potential to improve quality of life while also providing efficiency savings. introduction: chronic idiopathic/spontaneous urticaria (csu) is a chronic urticarial subtype, defined as itchy hives that last for at least weeks, with or without angioedema, and that have no apparent external trigger. although csu is more frequent in adult populationup to . %- %-, it can affect children and generally has a prolonged duration and has a detrimental effect on patients' quality of life. before the fda and ema approval of the anti-ige monoclonal therapy omalizumab for adults and children years and above, nonsedating h -antihistamines were the only agents licensed for use in patients with csu. however, a majority of patients did not respond to these drugs, even when they were administered at three to four times their licensed dose. objectives: we present pediatric patients (≥ years old) with csu non-responding to antihistamines, treated with omalizumab. results: at the diagnosis of csu, the patients were , , years old, respectively. a trial with non-sedating h -antihistamines, administered up to three to four times their licensed dose, were performed for all patients, without clinical efficacy. omalizumab was started at , , years, respectively. before anti-ige therapy, the urticaria activity score (uas) was , , respectively, indicating poor symptom control. omalizumab therapy was administered every weeks for months at the dosage of mg s.c. after month of therapy, all patients were symptom free with uas ; the patients remained asymptomatic for all the months of duration of monoclonal therapy and antihistamines were discontinued. by now, after , , months respectively from discontinuation of omalizumab, all patients are asymptomatic with uas . introduction: mepolizumab is a humanized monoclonal antibody directed against il- , and is licensed for the treatment of severe asthma in patients aged > years (eu only adults) with an eosinophilic phenotype as an add-on treatment. we were interested to know whether the substance has an antiallergic effect, too. objectives: here, we report the case of a year old man (non- results: in march he was switched from omalizumab to mepolizumab ( mg/month) weeks after the last omalizumab because of two asthma exacerbations under omalizumab in the last months and eosinophils/ll blood under mg prednisolone/day. after two injections of mepolizumab his fev increased from % to % pred., the act from to points, feno decreased from to ppb and the amount of prednisolone from mg to mg/day. but -the patient reported new nasal symptoms after the nd injection of mepolizumab, mainly blocked nose which has not been observed under omalizumab. he agreed to be challenged in the mobile gae²len exposure chamber* with house dust mite allergen for min. the total symptom score increased from to points after min exposure time remaining stable till the end, the positive nasal inspiratory flow decreased from to l after min and l after min, the fev decreased from % to % pred. following the inhalation of salbutamol the fev reversed. conclusions: mepolizumab has an anti-asthmatic effect but the anti-allergic efficacy seems to be small or absent. methods: all patients treated with omalizumab for severe allergic asthma or chronic spontaneous urticaria at the department of respiratory medicine and allergy at aarhus university hospital (n= ) were grouped after their home municipality. the number of omalizumab treated patients/inhabitant in these municipalities was correlated to the distance to the treatment centre at aarhus university hospital. results: mean distance to the treatment centre was . km for all inhabitants in the central region, while patients treated with omalizumab lived in average . km away. we found a negative linear correlation between the number of patients treated with omalizumab/inhabitant and the distance from the municipality to the treatment centre (slope: À . %ci: À . to À . ), p=. , n= ). conclusions: patients living at long distance from the treatment centre are less likely to be offered omalizumab treatment for severe allergic asthma or chronic spontaneous urticaria. objectives: demographics, clinical characteristics, and response to mepolizumab, were evaluated for atopic and non-atopic subgroups. sensitization to any one of the following; house dust mite, dog dander, cat dander, alternaria, or cockroach was considered as atopic, as assessed by specific serum ige of ≥ . ku/l. mensa (nct ) was a gsk sponsored study. results: of the severe asthma patients, ( %) were considered atopic, ( %) were considered non-atopic and atopic status was missing for ( %) patients. the majority of atopic patients (n= ) were sensitized to ≥ antigens. compared to the non-atopic sub-group the atopic sub-group was younger with a mean age of vs years of age, had a longer mean duration of asthma ( . vs . years), and a higher total baseline ige level ( u/ml vs u/ml). mean number of exacerbations in the months prior to the study was similar in the atopic and non-atopic subgroups ( . vs . ) as was the mean baseline peripheral blood eosinophil level ( cells/ll vs cells/ll). with mepolizumab an % reduction in eosinophils was achieved by week irrespective of atopic status and maintained throughout the treatment period. mepolizumab reduced the rate of exacerbations relative to placebo by % in the atopic subgroup and by % in the non-atopic subgroup. conclusions: the mensa study recruited an equivalent portion of atopic and non-atopic severe asthma patients. the atopic population was younger and had a longer duration of asthma than the non-atopic subgroup. while the baseline total ige level was > times greater in the atopic subgroup, the pharmacodynamic and efficacy response to mepolizumab treatment was similar. introduction: a treatment goal in the management of oral corticosteroid (ocs) dependent severe asthma is to reduce daily ocs use due to the side effects associated with long term use. anti ige treatment is used in atopic patients to reduce daily ocs. this analysis characterizes ocs reduction and asthma control in the sub-set of atopic and non-atopic ocs-dependent severe eosinophilic asthma (sea) patients from the -week sirius ocs reduction study. objectives: sirius study participants (n= ) were sub-grouped by atopic status in a post-hoc analysis; demographics, clinical characteristics, and response to mepolizumab were evaluated. sensitization to any one of the following; house dust mite, dog dander, cat dander, alternaria, or cockroach was considered as atopic, as assessed by specific serum ige of ≥ . ku/l. sirius (nct ) was a gsk sponsored study. results: of the ocs-dependent sea patients, ( %) were considered atopic ( % female), ( %) were considered non-atopic ( % female), atopic status was missing for patients ( %). compared to the non-atopic sub-group, the atopic sub-group was younger; mean age of vs years of age, while the mean duration of asthma was the same irrespective of atopic status ( years). the mean ocs daily dose and acq- score at baseline were similar between subgroups ( . mg vs . mg and . vs . , in the atopic and non-atopic subgroups respectively). the mean baseline peripheral blood eosinophil level was comparable in both subgroups ( cells/ll vs cells/ll) while the total ige level was higher in the atopic subgroup ( u/ml vs u/ml). mepolizumab lead to a ≥ % reduction in eosinophils by week irrespective of atopic status and eosinophil reduction was maintained throughout the study. mepolizumab reduced the ocs dose in the atopic group and non-atopic subgroups (odds ratio of a greater ocs reduction category of . ( % ci . , . ) and . ( % ci . , . ), respectively) and led to improvement in asthma control with a change in acq- of À . ( % ci À . , . ) in the atopic subgroup and À . ( % ci À . , . ) in the non-atopic subgroup. in an ocs dependent sea population mepolizumab reduced eosinophils independent of ige level and atopic status. in this limited post-hoc analysis, mepolizumab was an effective ocs sparing agent while improving asthma control in both atopic and non-atopic patients. patients who responded to retreatment had a similar mean time to response between the st dosing period ( . weeks) and nd dosing period ( . weeks). of all patients who were retreated (n= ), symptom control (uas ≤ ) after two doses was achieved in % ( st period) and % ( nd period) of patients; complete response (uas = ) occurred in % ( st period) and % ( nd period) of these patients. omalizumab was well-tolerated during both dosing periods. conclusions: omalizumab retreatment is safe and effective in patients with ciu/csu who respond to initial treatment and relapse after withdrawal, with most patients regaining symptom control after a nd course of omalizumab. : total ige reactivity of the grass allergoid formulation was diminished compared with native unmodified extracts. a difference in igg profiles was observed and indicated enhancement of accessible reactive igg epitopes across size distribution profiles of the grass allergoid formulation. detailed analysis of the epitope specificity showed retention of six lol p igg binding epitopes in the grass modified extract. all epitopes were mapped on the solvent exposed area of lol p homology model accessible for igg binding. one of the epitopes was identified as an "immunodominant" lol p igg binding epitope ( -ifkdgrgcgscfeik- ) and classified as a novel epitope. lastly, lol p igg antibodies showed functional capacity to block up to % of ige binding sites which provides evidence in protective function of immunotherapy induced antibodies against native allergens. the structural and immunological changes which take place following the grass allergen modification process were further unravelled revealing distinct igg immunological profiles. the results from this study support the concept that modification not only enhances the safety profile of scit but allows shorter-course objectives: design targeted sirna delivery system into liver cells. results: liposome surface was modified by lactose derivatives with different structures: lac(c ) and (lac) lac(c ) . the basic physicochemical and biological properties of the carriers have been examined. it is shown that glycoconjugate introduction has no effect on the physico-chemical characteristics. however the carbohydrate modification of the liposomal surface leads to increasing in transfection efficiency on a specific cell line hepg . moreover, the introduction of mono-carbohydrate derivative increases the transfection efficiency better than using a branched derivative of lactose. in the pharmacokinetic study target effect also was shown. unmodified liposomes start to be detected in the liver at minutes after administration, whereas modified liposomes were detected at minutes after administration. similarly, the excretion of modified liposomes from the liver was more slowly. also worth noting the high concentration of modified liposomes in the liver. furthermore liposomes modified by carbohydrates reduces the intensity of its accumulation in the lung. conclusions: it was shown that increasing attraction of drugs to the liver cells can be achieved by using liposome modification with lactose derivative. | sialylated fetuin-a is a candidate predictive biomarker for successful grass pollen allergen immunotherapy objectives: to identify new biomarkers of ait efficacy, pre-treatment sera obtained from grass pollen allergic patients responding or not to sublingual ait were differentially assessed by d-dige or label-free mass spectrometry. the role of fetuin-a in allergy physiopathology was studied by using gene silencing in a mouse asthma model, human dendritic cell stimulation assays and surface plasmon resonance. results: using comparative proteomics, we provide evidence for an increased o-linked sialylation of fetuin-a in sera collected before treatment from patients exhibiting clinical responses, when compared with low responders. whereas feta may either inhibit or promote chronic inflammation, no specific role in allergy had been ascribed to it until now. in ovalbumin-sensitized mice, silencing of the fetuin-a gene increased airway hyperresponsiveness and th responses. fetuin-a, but not its non sialytated counterpart, synergizes with lps and grass pollen or mite allergens in a tlr -dependent pathway to enhance the proallergic profile of human monocyte-derived dendritic cells. conclusions: quantification of sialylated fetuin-a glycoforms in the blood before treatment allows to identify patients more likely to benefit from grass pollen immunotherapy. validation of the hypothesis that this marker associated with "an inflammation status" can be used to predict ait efficacy is ongoing in larger cohorts of patients. introduction: kawasaki disease (kd) is a vasculitis that mainly affects small to medium-sized vessels, particularly the coronary arteries, and is a leading cause of acquired heart disease in children. previously, we found that the development of kd is associated with an elevation of both th and th immunity. gene hypomethylation is abstracts | an important form of epigenetic regulation, which results in increased gene expression. because m macrophages are associated with inflammation and th immunity while m macrophages are associated with immune regulation and th immunity, we hypothesize that kd is associated with hypomethylation of both m and m macrophage related genes. objectives: our objective was to investigate the methylation profile of m and m related genes in patients with kawasaki disease. twenty-four patients with kd and age-matched healthy controls (hc) were included in this study. in patients with kd, blood was sampled hours before ivig therapy (kd ) and days after ivig therapy (kd ). after dna extraction, samples were analyzed using human methylation bead chip (illumina) to examine the methylation ratios of genes related to m macrophages ( genes, cpg sites) and m macrophages ( genes, cpg sites). cpg sites with more than % difference in methylation levels and a pvalue less than . between groups were considered significant. objectives: primary aim of the study is the identification of differentially expressed genes (deg) associated with allergic rhinitis (ar) by using genome-wide transcriptomics data from blood immune cells. this set of candidate genes will then be used to define gene networks relevant for onset, severity and potential treatment outcome of house dust mite associated ar. with different ethnicity or populations exposed to a different environment is currently in preparation. introduction: allergic patients display abnormal immune responses to harmless antigens, leading to various symptoms from hay fever to life-threatening conditions. these responses include abnormal type immunity polarization and induction of allergen-specific memory t and b cells, resulting in development of allergy instead of immune tolerance. allergen-specific immunotherapy (ait) is currently the only causative treatment of allergic disorders. yet, in depth understanding of the underlying differences in allergen-specific cd + t cell and treg responses between allergy and tolerance and their changes during immunotherapy is lacking. objectives: we investigated whole-genome transcriptomics of circulating birch (bet v ) and grass (phl p a)-specific cd + t cells in allergic patients before and at , , and months of ait, as well in non-allergic healthy controls in corresponding seasonal time points. detailed immunophenotyping with flow cytometry and cd + mhc class ii tetramer staining with low rna/single cell next generation sequencing were performed. results: at baseline, out of the pollen season, there were more allergen-specific cd + t cells in allergic patients than in controls. at this time, over genes were significantly changed in allergenspecific as compared to total cd +t cells in patients, yet we found substantial differences in signal transduction and inflammatory response gene expression when compared to healthy controls. during ait we noted significant increase of allergen-specific cd + cells in patients with subsequent gene expression changes in the immune tolerance pathways. finally, we found increase in allergen-specific treg cells in patients upon ait, but not in tolerant controls in corresponding seasons. of interest yet, at early ait time points, allergenspecific treg cells displayed gene profiles suggesting their insufficient suppressive functions. conclusions: in summary, in vivo allergen exposure causes profound changes in the transcriptomic profiles of allergen-specific t cells. these gene profiles seem to be deficient at baseline in allergic patients, but ait is skewing them into the tolerant controls levels. introduction: the variability of the pharmacological response to beta (b )-agonists may be due to the polymorphism of the gene of results: singled out statistically significant differences of the genotype distribution. the gly/gly homozygous allele was discovered twice as often in the group with an insufficient response to b -agonists than in the group with a good response ( vs %, p<. ), while the distribution of heterozygous allele was detected the opposite pattern ( vs %, p<. ). in the arg/arg genotype distribution, there were no considerable differences in both groups ( % in each group). in the subgroups of children, receiving the high doses of the inhaled glucocorticoids, there was a trend for the prevalence of gly/gly homozygous allele prevalence. we have discovered the association of the gly/ gly genotype of the adrb gene with an insufficient effect of broncholytic therapy by means of short-term b -agonists; we also revealed the participation of the gly allele in the phenotype formation with the severe run of bronchial asthma and tolerance towards the therapy both by b -agonists and inhaled glucocorticoids. mckenna oe ; posselt g ; lackner p ; schmitt a ; h€ ollbacher b ; briza p ; wessler s ; gadermaier g ; ferreira f universit€ at salzburg, salzburg, austria; georg august-universit€ at g€ ottingen, g€ ottingen, germany introduction: an excess of million people worldwide have a reported allergy to birch pollen. proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. until now the protease content of betula pendula, a species endemic to the northern hemisphere, has not been studied in detail. hence, we aim to identify and characterise pollen and bacterial derived proteases found within betula pendula pollen. objectives: birch pollen transcriptome was constructed via de novo transcriptome sequencing. reads were assembled using the trinity software suite.. analysis of the birch pollen proteome was achieved via mass spectrometry and use of zymographic gels with the embedded substrates gelatinase and casein, which enabled visualisation of proteolytic activity. further to this, protease activity was quantified using a fluorescently labelled casein substrate protease assay. results: by using mass spectrometry, we were able to identify up to proteases within birch pollen. we could cluster the proteases into specific families based on their distinct proteolytic activities. further comparative analysis of the proteome and transcriptome revealed the relationship between transcript levels and the proteins they encode. zymographic methods enabled distinct visualisation of proteolytic activity for both casein and gelatin substrates. using fluorescently labelled casein, the birch pollen protease activity was quantified as . lg/ml when compared to a trypsin standard curve. additionally, bacterial isolates of the birch pollen were identified, and the proteolytic activity analysed. we report successful discovery of pollen and bacterial derived proteases endogenous to betula pendula. whilst none of the known birch pollen allergens have been recognised as a protease, we aim to investigate the role of tight junction degradation within epithelial cells and further enhance understanding of proteolytic activity on immune-polarization. objectives: to investigate the frequency and reasons of shortand long-term reintroduction failure in adults after a negative fc. method:: these are preliminary results of an ongoing prospective study. after a negative fc, patients receive standardized aftercare consisting of an introduction scheme to introduce the food at home and consultation by phone for advice and support hours, - weeks and months after the fc. short-term data about the frequency that patients failed introduction, defined as not completing the introduction scheme or patient-reported allergic complaints repeatedly during introduction, was obtained using a telephone interview. long-term data ( - months after negative fc) about frequency and reasons of reintroduction failure in daily diet, defined as not eating the food or only eating products with traces of the food, was obtained using a patient-reported questionnaire. results: from until now patients were included (mean age: years, male: %), who underwent a total of fcs with: hazelnut ( %), other nuts ( %), fruit ( %), composite meals ( %), fish and crustaceans ( %), cow's milk ( %), hen's egg ( %) and other ( %). in ( %) of the negative fcs, introduction using an introduction scheme was advised. in %, patients did not receive standardized aftercare for different reasons, eg negative fc with a composite meal. in %, introduction with an introduction scheme failed. long-term information was available for fcs. introduction failed in fcs ( %), including patient that even avoided traces of the food. patient-reported reasons for introduction failure were (n= ): symptoms after ingestion of the food (n= ), fear for allergic reactions (n= ) and not like the taste of the food (n= ). conclusions: short term introduction with an introduction scheme failed in %. however on the long-term in almost one third of the negative fcs, patients failed to reintroduce the food in daily diet, despite careful aftercare. it is recommended to give these patient even more intensive and tailored support. introduction: in order to help allergic patients manage often severe symptoms, food manufacturers are required to list allergens on labels and take precautions to avoid inadvertent contamination of foods with allergens. existing tools using generic immunoassays do not provide precise identification or quantification of specific food allergens. furthermore, existing elisa immunoassays are not able to be run simultaneously and are often unreliable. objectives: our goal was to develop and validate an accurate, sensitive and high throughput immunoassay that would enable simultaneous quantification of multiple allergens in foods. fluorescent beads coupled to allergen specific monoclonal antibodies were used to develop a multiplex array for simultaneous quantification of major allergens from peanut (ara h ), milk (bos d ), egg (gal d ), and shrimp tropomyosin (pen a ). target allergens were detected using biotinylated antibodies and streptavidin conjugated fluorochrome. the array was quantified using highly purified natural allergens as standards. allergen content was measured in various samples including samples spiked with purified allergen and allergen incurred food matrices. the results were compared to elisa. a multi laboratory validation was performed to validate the performance characteristics of the assay. results: there was a high correlation between the multiplex array and allergen specific elisa. the limit of detection of the array was as low as pg/ml. no significant cross reactivity was observed between the various food allergen assays. the recovery of allergen from spiked samples was generally between and %. inter and intra assay variability was observed to be less than %. in conclusion, an accurate, sensitive and reliable multiplex array for major food allergens was developed and validated. the flexibility of the microsphere technology allows for further expansion to produce a comprehensive array for the most important food allergens. this quantitative multiplex array may help to reduce the risk of inadvertent contamination of food. objectives: our aim was to create a food allergy web-based educational program for both schools and restaurants professionals. results: an interactive platform that hosts a free learning program, conclusions: the program and the toolkit are currently available online and are being implemented in schools at the north of portugal. we expect that fac program will give us an important insight on the professionals' knowledge about food allergy. additionally, acting as free integrated service and awareness effort, this program could be an educational tool easily adapted and disseminated, which may improve professionals 'commitment and skills to deal with food allergy in the community. | development of parallel reaction monitoring (prm) methods for soy and milk detection: consideration of allergen-derived ingredients chen s ; krawitzky m ; yang ct ; downs m university of nebraska-lincoln, lincoln, united states; thermo fisher scientific, san jose, united states introduction: the presence of undeclared food allergens poses both regulatory and health risks. in order to assess the magnitude of these risks, accurate quantitative detection methods are required. for some allergenic foods, the food industry uses not only the allergenic source but also a number of source-derived ingredients. some detection methods (both immunoassays and mass spectrometry methods) may fail to accurately detect and quantify these allergen-derived ingredients if they are not taken into consideration during development. objectives: the objective of this work was to select peptide targets for parallel reaction monitoring (prm) mass spectrometry methods that are suitable for detecting a variety of soy-and milk-derived ingredients. six soy-derived and six milk-derived ingredients were obtained for this study. the ingredients were extracted ( mmol l À tris-hcl, ph . , with mol l À urea, mmol l À dtt, and % pvpp) and subjected to in solution trypsin digestion. discovery proteomics analysis was conducted by lc-ms/ms using a q exactive tm plus orbitrap tm running in top data-dependent acquisition mode. peptides were identified using proteome discoverer . and relative, labelfree quantification was conducted on high-confidence (fdr< . ) peptides. selected peptides were subsequently analyzed in a targeted prm mode. results: the relative abundance of identified peptides varied among both the soy-derived and milk-derived ingredients. in the case of soy ingredients, for example, there were particularly marked decreases in the abundances of numerous peptides, across different proteins, in a hydrolyzed soy protein isolate. in the milk ingredients, variation in peptide abundance could be more directly attributed to differences in product protein fractionation (eg a decrease in abundance of whey proteins in a sodium caseinate product), although some peptides and proteins maintained consistent abundance across ingredients. after applying specificity and performance criteria, peptides from soy proteins and peptides from milk proteins were selected for further analysis in a targeted prm method. conclusions: peptide abundances vary widely among ingredients derived from allergenic foods. consideration of these peptide differences during ms method development by incorporating discovery proteomics may lead to more versatile and widely-applicable quantitative detection methods for food allergens. introduction: milk and its derivates are usual ingredients in many food products, which must be excluded by cow's milk allergic (cma) patients. oit protocols in patients with cma appear to be safe and effective in inducing desensitization and milk introduction in the diet. objectives: we conducted a survey in cma patients after successful completion of an oit programme, to assess their dietetic profile after introduction of fresh milk and dairy products (unrestricted diet) we considered cma patients (pts), males and females, age - (median . ), who successfully completed the oit protocol. these patients were on an unrestricted diet for milk and derivates, and they had been recommended to assume at least ml of fresh milk or yogurt every day. patients were given an ad hoc questionnaire to assess the milk/dairy products intake at least months after oit completion. results: from our investigation all of the pts have been assuming milk protein everyday without significant reactions after oit. % of the interviewee ( pts) referred to be drinking fresh milk at least three times a week, in a quantity of ml or higher. of note, % have to add cocoa powder ( pts) or coffee ( pts) to mask milk taste. dislike of milk taste was the cause of the refuse to take fresh milk in pts (over patients that didn't take milk at all); pts who didn't drink milk had at least ml yogurt instead, almost every day. fresh cheese was eaten at least once a week by % of all the pts; hard cheese was eaten as main course almost once a week by %. % of patients consumed biscuits and sweet baked products containing milk/derivates at least once a week, % every day. pizza was also present in the diet of the majority of pts ( %), once a week/month. ice cream was appreciated by all the patients and regularly assumed especially during the summer time. conclusions: taste seems to be the main factor that directs the daily choice of milk derivates or milk containing products. all pts easily consume baked products containing milk/derivates, and the majority of them accept fresh milk as advised in order to maintain unresponsiveness. our survey confirms that oit is effective in expanding food choice, but for some patients the change of dietetic habits is hampered due to taste reasons. a more detailed, qualitative and quantitative analysis of the milkunrestricted diet will derive from the -days food diary given to these patients. | frozen-defrosted dried skimmed milk is a suitable product for sublingual immunotherapy for cow's milk allergy introduction: sublingual immunotherapy (slit) is a promising treatment for cow's milk allergy (cma) due to its favourable safety profile. however, its efficacy is limited-probably due to the small volumes and doses that can be delivered sublingually, especially in children. milk products with preserved allergenicity that allow higher protein concentrations in smaller volumes might potentially improve the efficacy of slit for cma. dried skimmed milk (dsm) could fulfill these characteristics. in addition, once dsm is reconstituted, freezing individual vials from the same batch for further administrations could increase dose-consistency throughout the treatment, which would help ensure safety. however, little is known about whether processing to produce dsm, and further freezing-defrosting, may alter its allergenicity. objectives: to evaluate the allergenic protein composition of dsm, including once frozen-defrosted, in comparison to fully-allergenic usually consumed fresh milk. methods: sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by silver staining was performed following the laemmli method to determine the soluble protein composition of the following products: fresh pasteurized milk (friesland campina, amersfoort, the netherlands), dsm (marvel, the premier foods group ltd, london, uk) and frozen-defrosted dsm to identify all protein bands present. western blot with anti-whey and anti-casein antibodies was performed to identify the specific allergenic proteins. results: no significant differences were found amongst the milk products tested, with sds-page displaying all the bands corresponding to the major allergenic proteins in milk (alpha-lactalbumin, beta-lactoglobulin and alpha-, beta-and kappa-caseins) and the western blot showing recognition to all proteins with similar intensity. the major allergenic proteins present in unprocessed milk are well-preserved in dsm, even after freezing-defrosting, making it a convenient product for sublingual immunotherapy. results: nineteen children were randomized into the low dose group (n= ) and the elimination group (n= ). there were no significant differences in background between these groups. after weeks of therapy, the rate of passing the oral food challenge test with / th of a whole egg was significantly higher in the low dose group ( / ( %) vs / ( %), p=. ). ovomucoid-specific ige levels in the low dose group after weeks were significantly lower than those at baseline. adverse events associated with both therapies did not occur during the study period. conclusions: these results show that low dose ingestion of egg is safe and effective for tolerance or desensitization induction in children with egg allergy, without the need for dose elevation. | another brick in the wall: toward a national food allergy strategy for canada introduction: the world is experiencing an apparent epidemic of food allergies. with rising prevalence and increasing global spread, basic scientists continue to search for causes while clinical and social scientists continue to study the consequences. these include life-long chronic illness, fear and anxiety, social exclusion and stigma, all of which are underscored by inequalities across vulnerable groups (eg, low income families, immigrants, indigenous peoples). concomitantly, consumer organizations and patient support groups attempt to influence policy in order to maximize choice and minimize risk for individuals and families affected by food allergy, both directly and indirectly. this includes food labeling legislation, food safety regulations, and policies and practices in public places such as schools, restaurants and transportation modes (eg, airplanes). objectives: in canada, with the support of + years of science undertaken by the allergy genes and environment (allergen) networks of centre of excellence in allergic disease, we are leading a national team that aims to establish the building blocks for a national food allergy strategy. conclusions: the greatest challenges to building a national food allergy strategy are not those related to the basic or clinical science, but rather the activities and commitment of individual stakeholders. while gaps remain in the science, the challenges of transdisciplinary integrated knowledge translation hinder progress. we present lesions learned regarding culture shifts that will facilitate the progress we need to maximize choice and minimize risk for canadians affected by food allergy. | component resolved diagnostics reduces diet restrictions by half among finnish school children savolainen j ; mascialino b ; pensamo e ; hermansson l ; silvan m ; borres mp ; korhonen k university of turku, turku, finland; department of allergology, uppsala, sweden; thermo fisher scientific-immunodiagnostics, turku, finland; university of turku, uppsala, sweden; thermo fisher scientific-immunodiagnostics, lieto, finland introduction: there are - special diets because of food allergy in finnish schools. the finnish allergy program - was launched to reduce problems and costs induced by allergies. one of the special aims was to reduce diets caused by food allergy will by %. the h€ ark€ atie social and health care service region schools have required doctor's certificates for diets for over abstracts | years. all diets are listed in a register and checked on a regular basis. objectives: the purposes of this study were to evaluate whether it is possible reduce the special diets when the special diet system appears to be working well and to assess the added value of immu-nocap ® isac allergen component diagnostics in the intervention. results: there were children attending the schools in the h€ ark€ atie region. there were children with diet due to food allergy. children were recruited to study from the diet register and contacted by a nurse's phone interview. there were children who refused the study leaving children for the assessment of the diet. children were not following any diet and in children the diet was not regarded necessary by the study nurse. children agreed to take part in the study and were referred to laboratory tests for food specific ige and isac. children dropped out leaving children in the study. after the food specific ige results were available, children were contacted by physician. based on the results and/or interview a free diet was allowed for children. children were eating small amounts of the food and were encouraged to increase the use of the foods. children were advised to avoid the foods only if they caused symptoms. only children were advised to continue diet. after the results of isac were available, children conclusions: the study confirmed that it is possible to have diets decreased over % in accordance to the finnish allergy program. additionally, the study showed that food allergic children can benefit from the use of component resolved diagnostics. | omalizumab treatment for severe food allergy caused by lipid transfer protein: a preliminary case series mari a; zennaro d; ferrara r; bernardi ml; alessandri c caam-centri associati di allergologia molecolare, rome, italy introduction: lipid transfer proteins (ltp) are allergens from plantderived foods causing symptoms ranging from oas to anaphylaxis. ltp are present in almost all kind of plant species used for human feeding. ltp allergic patients suffer of reactions to a broad variety of foods, with a very personal reactivity profile which can involve few or many foods. in addition, patients who start to record severe reaction to several foods begin to be afraid of eating any other related or non-related food, leading to a poor diet. omalizumab (ozm) has been documented to be effective in treating food allergies with sensitization to other allergens. objectives: to document the approach for recruiting, studying, monitoring ltp allergic patients, and do a preliminary evaluation of the clinical impact of ozm therapy. results: six adult patients with documented reactions to plantderived foods in the last months and diagnosed to be ige positive to multiple ltp by using multiplex testing, namely isac at the beginning and faber during the follow up, were recruited. ozm was offered as an off label treatment adopting an open label study design. a questionnaire was submitted to each patient before, during and after the treatment. foods listed as "no more eaten" where considered for reintroduction, starting from those excluded because of fear, going for those with an increasing risk of reactions. re-introduction was done at home after receiving detailed instructions, having all rescue medication at hand. the allergist was connected real time with the patients using one or more ict tools. one patient dropped out because was not complying with the scheduled reintroduction. three patients completed the treatment with a successful re-introduction of all or almost all excluded foods, in two cases peach was reintroduced. the last two patients reintroduced %- % of the excluded foods, including some of those previously causing reactions. the attempt of re-introducing the most risky foods failed causing local allergic reactions promptly controlled by the therapy. all foods re-introduced were tolerated once the treatment was stopped. conclusions: ozm seems to be a promising treatment for severe ltp allergics, improving their qol. starting from the recruitment phase to the re-introduction, the overall procedure looks quite complicated and deserve much resources. ige sensitization to ltp, carefully defined before treatment, can be monitored during the ozm course as the drug doesn't interfere with faber test ige detection. | oral immunotherapy with peach-juice in lipid transfer protein (ltp) allergy: is it possible to reach tolerance? introduction: food allergy to rosaceae fruits and nuts, due to sensitization to lipid-transfer protein (ltp), is frequent in the mediterranean area countries. based on milk allergy oral immunotherapy (oit) protocols, a study is proposed to obtain an oit regimen in patients allergic to ltp. objectives: we included patients with anaphylactic reactions due to ltp-food allergy, from january/ to october/ . skin prick test (spt) was performed with a food and inhalant battery, ltp bial-aristegui ® ( . mg/ml), prick by prick (pbp) with a specific commercial peach-juice (dilutions to endpoint), and determination of pru p ige levels (immunocap). the presence of pru p protein in the peach-juice, was confirmed by sds-page and elisa method, and quantified with anti-pru p . we elaborated an oit guideline with progressive administration of peach-juice, - - - drops ( drops= . ml), sublingually, starting concentration chosen according to the endpoint spt results, up to ml daily dose. the dose reached at the allergy service, was continued at home, daily, for - weeks. all the increasing doses were performed at the allergy service, after pbp with peach-juice and ltp control spt. after a time, an oral challenge up to ml peach-juice was performed. patients were instructed on the importance of maintaining regular intakes and avoiding cofactors. results: patients started this protocol ( male/ female), aged - (media . ). peach-juice analysis in agarose gel electrophoresis, showed a kda band quantified as . lg/ml of ltp. there was no difference between wheal diameter on spt for ltp bial-aristegui ® and pbp with commercial peach-juice along all the study. average concentration on pbp to endpoint: / ( ), / ( ), / ( ). pru p ige level average: . ku/l. none anaphylactic or serious reactions during the oit protocol appeared. only five patients ( %) reported mild occasional symptoms (oral pruritus and mild located urticaria). patients reached ml daily dose ( . %), and of them ( %) completed oral food challenge with peach-juice up to ml, with good tolerance. this pattern also allowed us to reintroduce withdrawn foods from the diet due to previous reactions, with good tolerance. results: children (age - years) sensitized to dog dander extract (median . ku a /l), with or without known dog induced allergic airway disease, were included. nasal provocation testing with dog dander extract was performed. measurement of ige to dog dander extract and to can f -can f was performed with immunocap. an ige level ≥ . ku a /l was considered positive. among all children sensitization to can f ( %) and can f ( %) was most frequent. corresponding numbers for can f , can f , can f and can f were %, %, % and % respectively. based on the results from the nasal challenges three groups were identified; no (n= ), mild (n= ) and strong reaction (n= ). the median ige levels to dog dander and to all allergen molecules were higher in the strong reaction group than in the mild and the no reaction group. among children with a strong reaction, ige to can f was found in % ( / ) compared to % ( / ) among children with no reaction (p=. ). ige to can f showed a similar pattern (p=. ). % ( / ) of the children with a strong reaction were sensitized to can f compared to . % ( / ) of the patients with no reaction (p=. ). no associations were found between the ige levels to can f , can f and can f and the no reaction or strong reaction groups. using multiple regression analysis, with all allergens in the model, sensitization to can f showed a statistically significant difference between the groups. conclusions: molecular allergology may improve diagnostics of dog allergy in children. sensitization to the lipocalin can f was significantly associated to a strong positive nasal reaction implying its usefulness as a marker of clinically relevant dog allergy. tsolakis n ; malinovschi a ; nordvall l ; janson c ; mattson l ; lidholm j ; borres m ; alving k uppsala university, women's and children's health, uppsala, sweden; uppsala university, medical sciences, uppsala, sweden; thermo fisher scientific, uppsala, sweden introduction: cat allergy is common worldwide and a major trigger of asthma in many countries. molecular patterns of cat sensitisation vary between individuals but their relationship with allergic inflammation has not been extensively studied. objectives: the aim was to investigate the prevalence of ige to different cat allergen components and their relationship to type- inflammation and bronchial responsiveness in young asthmatics. conclusions: ige sensitisation to cat serum albumin (fel d ) and cat lipocalins (fel d , fel d ) , but not to secretoglobin (fel d ) or cat dander extract, were independently associated with feno and b-eos count. we suggest that cat serum albumin and cat lipocalins can be used as markers for increased risk of type- inflammation in young asthmatics, as shown in multiple regression analyses. | complete sequence and recombinant production of horse dander allergen equ c lidholm j; lundgren t; larsson h; mattsson l thermo fisher scientific, uppsala, sweden introduction: horse dander is an increasingly important cause of respiratory allergy. equ c was one of the first horse allergens to be recognised but only a small part of its amino acid sequence has been reported. objectives: the aim of this study was to determine the complete sequence of equ c and express it as an immunoreactive recombinant protein. methods: equ c was purified by size exclusion, hydrophobic interaction, anion exchange and reversed phase chromatography. recombinant equ c was expressed as a hexahistidine tagged protein in e. coli and purified by immobilized metal ion affinity and ion exchange chromatography. ige antibody reactivity to natural and recombinant equ c and other horse dander allergens was determined in sera of subjects allergic to horse. results: a putative equ c sequence predicted from genomic data revealed a lipocalin protein of amino acids with a highest sequence identity among known lipocalin allergens of % to can f . n-terminal sequencing and mass spectrometric analysis of purified natural equ c confirmed . % ( / residues) of the predicted sequence. recombinant equ c displayed ige antibody binding activity comparable to that of purified natural equ c (r=. ). of the horse allergic subjects studied, ( %) showed ige antibody binding to equ c , ( %) to equ c , ( %) to equ c and ( %) to equ c . the complete sequence of equ c was established. as a fully immunoreactive recombinant protein, it represents an important addition to the panel of allergens useful in the diagnosis of allergy to horse. | component resolved diagnosis using guinea-pig allergens elucidates allergen sensitization profiles in allergy to furry animals objectives: to identify major guinea-pig allergens and to evaluate their potential as reliable markers for a specific ige-diagnosis in comparison to dander extracts. results: forty-three patients with a clinical history of allergy to guinea-pig and patients allergic to cat and/or dog were recruited for the study. major guinea-pig allergens were identified by ige-immunoblot and n-terminal sequencing of ige-reactive proteins. corresponding cdnas were cloned and allergens were expressed as recombinant proteins in e. coli. specific ige to animal dander, fel d and can f were determined, specific ige to fel d , can f and to guinea-pig allergens were quantified by elisa. two new guinea-pig lipocalin allergens, cav p and cav p , were identified in guineapig dander. the combination of guinea-pig allergens, the new allergens and the previously isolated lipocalins cav p and cav p , enabled the identification of out of patients sensitized to guinea-pig. the vast majority of the patients had specific ige to cav p ( %). cav p shares % sequence identity with fel d and can f and was found to be cross-reactive with these cat and dog allergens. in the group of cat and/or dog allergic patients, % had also specific ige to guinea-pig dander. nearly half of those ( %) had ige to cat serum albumin fel d or to fel d ( %) and to can f ( %), explaining the high degree of cross-reactivity to guinea-pig dander. only % of the cat/dog allergic patients with a positive isle test to guinea-pig dander had specific ige to any of the non crossreactive guinea-pig allergens cav p , cav p or cav p . however, none allergen has been characterized from mongolian oak. objectives: in this study, we tried to characterize a major allergen from mongolian oak. results: a molecule homologous to pathogenesis-related protein , a putative que m , was cloned by rt-pcr and its recombinant protein along with que a , an allergen from white oak (q. alba) was produced. cloned que m sequence shares . %- . % amino acid sequence identity ( . %) with pr- -like allergens from various plants. allergenicity and diagnostic value of recombinant que m abstracts | , que a and bet v proteins were compared by elisa using sera from oak sensitized subjects. specific ige to recombinant que m , que a , and bet v were detected in . %, . %, and . % of serum samples from korean tree pollinosis patients. recombinant que m was able to inhibit ige reactivity to que a and bet v , indicating its strong cross-reactivity. activation pattern of basophils from five patients was similar in terms of cd c expression and protein concentration of challenged bet v and que m . objectives: over the course of one year, all patients who were seen at our immunoallergology clinic for the first time underwent spt with our standard series, to which four olive tree cultivar extracts were added (arbequina, blanqueta, hojiblanca e picual). we then recorded wheal size diameters, considering a wheal > mm to correspond to a positive test. we recorded individual sessions of spts, in which presented at least one positive result. two hundred and thirtysix of these patients ( . %) had a positive spt for olive tree pollen, only one of them being monosensitized. when looking only at pollen-sensitized patients, twenty-two patients ( . %) tested positive solely for olive tree pollen. in all allergic patients, the most frequent cultivar sensitization was to the cultivars hojiblanca ( . %) and arbequina ( . %), followed by the cultivars picual ( . %) and blanqueta ( %). twenty-six patients ( % of all olive pollen sensitizations) had a positive spt for the conventional extract and negative spt for the cultivars; patients ( . %) had a positive spt for both. we noted that . % (n= ) of patients sensitized to olive tree pollen had a negative spt with the conventional extract and positive with one of the cultivar extracts. the cultivar hojiblanca was the most frequent in this group ( . %, n= ), followed by the arbequina cultivar ( . %, n= ). in the group as a whole, there was a positive correlation between the results of the spts with the conventional extract and each of the cultivar extracts. this correlation was weaker with the cultivar hojiblanca (r=. ). conclusions: some patients, sensitized only to the pollen of certain olive tree cultivars, are not identified with the conventional extract. spt with the hojiblanca cultivar could identify most of these patients in our country, and therefore should be considered in patients with a history of pollinosis and a negative spt for the common extract of olive tree pollen. | structural and immunological comparison of heat treated pru p and art v , the non-specific lipid transfer proteins of peach and mugwort pollen wildner s ; stock l ; regl c ; alessandri c ; mari a ; huber c ; stutz h ; gadermaier g objectives: recombinant art v . and pru p . were expressed in e. coli rosetta-gamib plyss and purified using cation exchange chromatography. proteins were analyzed in gel electrophoresis and mass spectrometry. proteins were incubated at °c in time intervals up to min using buffers at ph . and . . physico-chemical properties of native and heated allergens were analyzed by circular dichroism spectroscopy, dynamic light scattering and size exclusion chromatography (sec). using sera from italian patients sensitized to pru p and art v (n= ), ige binding to native and heat-denatured allergens was investigated in elisa. results: highly pure recombinant pru p and art v were obtained as non-tagged proteins from e. coli. identity and formation of disulfide bonds was verified by mass spectrometry. circular dichroism showed high thermal stability of both proteins at acidic ph. the alpha-helical fold of art v was lost upon heating for min at ph . while pru p was already altered after min. purified pru p and art v are monomeric molecules with a hydrodynamic radius of . and . nm, respectively. structural relaxation is observed upon heat treatment which is not attributed to protein aggregation as determined by sec. the ige reactivity to both allergens was largely unaffected upon heating at ph . . notably, ige reactivity to art v was already significantly decreased upon min heating and was completely abrogated to both proteins after min denaturation at neutral conditions. conclusions: even though the fold of pru p is more compact compared to art v , susceptibility to structural changes upon thermal treatment at neutral conditions are more pronounced which do however not directly translate to lower ige binding capacities. particularly the buffer environment needs to be considered when formulating ltp-containing products which undergo heat treatment. objectives: we sought to determine the ige binding capacity and potential diagnostic value of a recombinant hybrid molecule. results: the codon-optimized nucleotide sequences of a hybrid molecule comprising the full sequences of blo t and der p at the amino and carboxyl ends respectively, here named bp- , was cloned into a plasmid vector and expressed in escherichia coli as a xhis tag protein. two hundred and thirty three sera from colombian (n= ) and cuban (n= ) allergic patients were tested by elisa for ige reactivity. thirty seven sera from non-allergic subjects and negative skin test (spt) to mites were used as controls. all subjects provided written informed consent to their participation in this study. hdm allergy was diagnosed on the basis of clinical symptoms in combination with mite extract spt. potential diagnostic value of bp- specific ige was determined by receiver operating characteristic (roc) analysis and area under roc curve (auc) calculated. positive serum ige values to hybrid molecule were defined as optical density (od) higher than . (mean od plus standard deviations in nonallergic subjects). in respiratory allergic patients, the overall frequency of positive ige reactivity to bp- was . %, in non-allergic subjects the frequency was %. serum ige levels to bp- were positively correlated to spt to b. tropicalis (spearman r=. , p=. ), and to d. pteronyssinus (spearman r=. , p=. ). using spt to mite extracts as gold standard, the sensitivity and specificity of serum ige levels to bp- were . % and . % respectively, with an auc of . ( % confidence interval . - . ). conclusions: these data suggest that bp- could be a useful reagent for identifying allergic patients sensitized to b. tropicalis and/or d. pteronyssinus. | igg, ige and igg specific antibodies to molecular allergens of aspergillus fumigatus introduction: in clinical allergy, alongside with skin prick tests, in vitro determination of specific ige for a particular patient contributes to the diagnosis and helps to estimate the risk associated with different food allergens. however, with commercial methods of specific ige antibodies detection (component-resolved diagnosis, crd), the clinician is typically limited by the list of the available allergens. objectives: to overcome this limitation, we developed two component-resolved diagnostic tests for food allergy in which natural extracts can be used. in the first developed method, the crd is performed using immunoaffinity capillary electrophoresis (iace) coupled with matrixassisted laser desorption/ionization mass spectrometry. meanwhile, the second method is based on in-tube immunomagnetic separation (ims) with mass spectrometry identification (mass spectrometry or peptide mass fingerprinting). in both techniques, magnetic beads coated with antihuman ige antibodies are used to extract the ige antibodies from the blood serum of the allergic patient. then, the immunocomplex, obtained on the magnetic beads, is used to quantify the total ige level or to probe the ige binding with standard allergens or natural allergenic extracts. afterwards, the identification of the extracted proteins, ie potential allergens, is performed by mass spectrometry with or without ce separation. after optimisation, the proposed methods have been successfully applied to a commercial blood sample of a patient with a known allergy to cow's milk, with results confirmed by standard tests. as a proof-of-concept, the sensitization profile of a patient suffering from protein contact dermatitis to the cow's whey fraction has been determined. we confirmed the presence of circulating ige antibodies binding lactoferrin and bovine serum albumin. cross-reactivity tests were also performed using goat and sheep milk and revealed the patient sensitivity to serum albumins from these two milks. such approaches open the possibility for direct identification of ige-bound allergens molecular mass and structure. these methods allow the discovery of yet unknown allergens and could be useful for precise personalized allergy diagnosis, allergens epitope mapping, and cross-reactivity studies. objectives: the objective of this study was to investigate the validity of cord blood ige for predicting atopy at years of age. methods: a total of children born in participated in the longitudinal investigation of global health in taiwanese schoolchildren (lights) cohort. total ige was measured in umbilical cord blood at birth. perinatal history was collected from medical records in the chang gung memorial hospital, taiwan. total and specific serum ige and questionnaires were carried out at years of age. receiver-operating characteristic (roc) curves were used to determine the validity of cord blood ige for predicting atopy at years of age. logistic regression models were applied to assess the association between cord blood ige and atopy at years of age. results: cord blood ige levels was significantly associated with total serum ige level at years of age (r=. , p<. ). the cord blood ige levels in atopic children aged years (meanaesd, . ae . ku/l) were significantly higher than those in nonatopic children ( . ae . ku/l) (p<. ). the area under the receiveroperating characteristic (roc) curve of cord blood ige for predicting atopy at years of age was . . the sensitivity, specificity, and positive and negative predictive values of cord blood at the optimal cutoff of . ku/l on the roc curve for predicting atopy were . %, . %, . %, and . %, respectively. higher cord blood ige levels (≥ . ku/l) was associated with a higher likelihood of atopy at years of age (or= . ; % ci: . - . ; p<. ). our results indicate that cord blood ige is a potential predictor of atopy at school age, with an optimal cutoff of . ku/l. bogomolov a vinnitsa national pirogov memorial medical university, vinnitsa, ukraine introduction: allergen sensitization is being diagnosed by commonly available methods in clinical practice-skin prick tests (spts) and specific immunoglobulin e test (sige). recently, a new thermographic (th) method for the assessment of spt was developed, and it was demonstrated that the th measurements of forearm temperature distribution during spt, supported by a mathematical model, offer a new quantification method of allergen-induced skin reactions. objectives: the aim of this study is a comprehensive comparison of the th method with spt and sige techniques. the group of patients who were participated in this study consist of patients. among them were patients ( . %) with allergic rhinitis and patient ( . %) with asthma, . % of them were men and . % were women aged - years (mean age . ae . years). spt and sige testings were performed by the standard techniques. for th analyses, set of thermograms of both forearms were acquired after prick and analyzed with the use of developed software. all results were converted into categorized scale for comparison. after counting patients who were true positive (tp), true negative (tn), false positive (fp), and false negative (fn), the sensitivity, specificity, and accuracy were calculated according to the following formula using the results of spt as the standard; sensitivity=tp/(tp+fn), specificity=tn/(tn+fp), and efficacy=(tp+tn)/(tp+tn+fp+fn). the results showed high correlation coefficients between the methods equal to . - . . the sensitivity and accuracy of the th assessment in respect of both the classical methods is at a good level ( . - . ). the acceptable level of specificity . - . was also achieved for the majority of allergic reactions. the best accordance was observed between th and sige results (r=. ), while th-spt was the most divergent pair r=. . in case of particular allergens, the biggest correlation was . , while the smallest value amounted to . . the results of diagnostic indicators of thermographic measurement of skin reactivity in comparison with the classical methods of determining the sensitivity to allergens show the prospect of using the method in routine practice. the main advantages of this method are its higher measuring ability and objectivity, by which the possibility of making error in diagnosis is significantly reduced. introduction: chronic urticaria symptoms may be worsened by factors such as temperature, exercise, hormones and stress. a salicylate rich diet has been reported to worsen symptoms in these patients. the mechanism by which natural salicylates do this is unclear but, like aspirin, is thought to be due to their ability to interfere with the arachidonic pathway via cyclooxygenase inhibition. studies have shown that low dose aspirin increases serum il- levels in patients with antiphospholipid syndrome. il- is important in basophil and mast cell function, inducing mediator release and cd c upregulation in the absence of ige stimuli. objectives: the gold standard for diagnosing salicylate exacerbated chronic urticaria is by challenge testing. there is no in vitro laboratory test approved for routine diagnosis. this study investigated whether il- levels are raised in patients with chronic urticaria and if these levels were affected by salicylate intake. we aimed to find an optimum method of measuring il- levels by comparing levels in serum and salivary samples. the quantikine human il- enzyme linked immunosorbent assay (elisa) was validated and used on both saliva and serum of patients with chronic urticaria and normal controls. this was a case control study of medicated patients with chronic urticaria and controls at university hospital southampton, to see whether there were any correlations with il- , and degree of salicylate intake (based on a questionnaire). introduction: since the introduction of molecular components in allergy, a big challenge of allergy diagnostics is to connect clinical symptoms with optimal test use and correct interpretation of results. objectives: the aim of the study was to ( ) develop an algorithm that would meet that need, and ( ) to evaluate the effect of introduction of algorithm to clinical practice. the algorithm was developed which groups clinical symptoms into six categories: rhinoconjunctivitis/ asthma, oral allergy syndrome (oas), urticaria/angioedema, eczema, anaphylaxis, and a combination of symptoms and combines them with knowledge of possible allergen specificity. this information is combined with two basic allergen mixtures (panels), reflex testing of selected food molecular components and accompanied by interpretative comments. the introduction of our algorithm led to less inhalation screenings, more food screenings and an increase in the requested molecular components. the oas was seldom recognized or used as a symptom by specialists. the reduction in costs, by using the possibility that the disease presentation may be a consequence of an relatively not dangerous oas, was therefore not achieved. all pr- positive proteins in various allergen sources showed also positivity for birch antigen. the screening based on this algorithm has potential to enable clinicians/general practitioners with a tool to increase the pre-test probability of allergy for the most frequently occurring allergens. allergy diagnostics may be more efficient if pr- component of birch (r bet v ) is included in early screening and can help in early recognition of oas. helbling a department of otorhinolaryngology-head and neck surgery | clinical and immunological evolution of patients who failed milk-oral immunotherapy there is lack of evidence on evolution among failures. objectives: to analyze clinical and immunological evolution of patients who failed milk-oral immunotherapy. data were obtained from medical records of a cohort of patients who failed moit in the past years in hospital infantil universitario niño jesus ( %) patients failed during build-up phase [ ( %) due to adverse events (ae) and ( %) to family decision. failed during maintenance phase: ( %) due to eosinophilic esophagitis, ( %) to family decision, to psychiatric disorder and (range - ). the most frequent aes were cutaneous and gastrointestinal symptoms. / ( %) patients underwent a second moit and was successful in . the second moit was performed between and years after the first one. there was no statistical differences between specific ige(ku/l) levels at baseline and months after the moit end portugal introduction: fish allergy is common in countries where consumption is high. parvalbumins present in fish muscle are the major allergens. allergy to multiple fish species is caused by parvalbuminspecific cross-reactive ige. cross-reactivity with parvalbumin from baltic cod retrospective study of patients with fish allergy followed in our immunoallergology department. fish tolerance acquisition was evaluated by oral food challenge. statistical analysis was performed using spss version (descriptive statistics, student test results: pts were included ( male, female), children and adults (age ae years). ( %) had previous history of rhinitis, ( %) of asthma and ( %) of eczema age of first contact with fish averaged . ae . months (min , max ) and the possible types of contact were: oral in ( %), cutaneous in ( %) and inhalation of cooking fish vapours age of first clinical manifestation (excluding the pts who developed allergy in adulthood) was at ae months (min , max ) the clinical manifestation of the reaction was: angioedema/urticaria ( %), gastrointestinal symptoms ( %), eczema ( %), respiratory symptoms ( %), oral allergy syndrome ( %), cardiovascular symptoms ( %) age at the first immunoallergology out-patient clinic consult averaged ae years (min . , max ) and time from first symptom to first thermo-fisher) was evaluated before and after acquisition of tolerance to at least fish. before tolerance, sige (ku/l) averaged roc curve (area under curve . ) showed that, for gad c ku/l, pts had a sensitivity of . % and specificity of . % that they would have a negative oral food challenge to a fish an sige< . ku/l had sensitivity of % of a negative challenge ku/l had specificity of . % of a positive challenge. conclusions: fish allergy is a common allergy in early childhood however, acquisition of tolerance is possible. rgad c appears to be a good marker for fish tolerance and could help allergologists as to when start testing for fish tolerance key: cord- -sgm q i authors: walter, justin d.; hutter, cedric a.j.; zimmermann, iwan; wyss, marianne; earp, jennifer; egloff, pascal; sorgenfrei, michèle; hürlimann, lea m.; gonda, imre; meier, gianmarco; remm, sille; thavarasah, sujani; plattet, philippe; seeger, markus a. title: sybodies targeting the sars-cov- receptor-binding domain date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: sgm q i the covid- pandemic, caused by the novel coronavirus sars-cov- , has resulted in a global health and economic crisis of unprecedented scale. the high transmissibility of sars-cov- , combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (rbd) of sars-cov- . in an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. we obtained six strongly enriched sybody pools against the isolated rbd and identified unique anti-rbd sybodies which also interact in the context of the full-length sars-cov- spike ectodomain. among the selected sybodies, six were found to bind to the viral spike with double-digit nanomolar affinity, and five of these also showed substantial inhibition of rbd interaction with human angiotensin-converting enzyme (ace ). additionally, we identified a pair of anti-rbd sybodies that can simultaneously bind to the rbd. it is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against covid- . moreover, generation of polyvalent antivirals, via fusion of anti-rbd sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. we present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource. the ongoing pandemic arising from the emergence of the novel coronavirus, sars-cov- , demands urgent development of effective antiviral therapeutics. several factors contribute to the adverse nature of sars-cov- from a global health perspective, including the absence of herd immunity [ ] , high transmissibility [ , ] , the prospect of asymptomatic carriers [ ] , and a high rate of clinically severe outcomes [ ] . moreover, a vaccine against sars-cov- is unlikely to be available for at least - months [ ] , despite earnest development efforts [ , ] , making alternative intervention strategies paramount. in addition to offering relief for patients suffering from the resulting covid- disease, therapeutics may also reduce the viral transmission rate by being administered to asymptomatic individuals subsequent to probable exposure [ ] . finally, given that sars-cov- represents the third global coronavirus outbreak in the past years [ , ] , development of rapid therapeutic strategies during the current crises could offer greater preparedness for future pandemics. akin to all coronaviruses, the viral envelope of sars-cov- harbors protruding, club-like, multidomain spike proteins that provide the machinery enabling entry into human cells [ ] [ ] [ ] . the spike ectodomain is segregated into two regions, termed s and s . the outer s subunit of sars-cov- is responsible for host recognition via interaction between its c-terminal receptor-binding domain (rbd) and human angiotensin converting enzyme (ace ), present on the exterior surface of airway cells [ , ] . while there is no known host-recognition role for the s n-terminal domain (ntd) of sars-cov- , it is notable that s ntds of other coronaviruses have been shown to bind host surface glycans [ , ] . in contrast to spike region s , the s subunit contains the membrane fusion apparatus, and also mediates trimerization of the ectodomain [ ] [ ] [ ] . prior to host recognition, spike proteins exist in a metastable pre-fusion state wherein the s subunits lay atop the s region and the rbd oscillates between "up" and "down" conformations that are, respectively, accessible and inaccessible to receptor binding [ , , ] . upon processing at the s /s and s ' cleavage sites by host proteases as well as engagement to the receptor, the s subunit undergoes dramatic conformational changes from the pre-fusion to the post-fusion state. such structural rearrangements are associated with the merging of the viral envelope with host membranes, thereby allowing injection of the genetic information into the cytoplasm of the host cell [ , ] . coronavirus spike proteins are highly immunogenic [ ] , and several experimental approaches have sought to target this molecular feature for the purpose of viral neutralization [ ] . the high specificity, potency, and modular nature of antibody-based antiviral therapeutics has shown exceptional promise [ ] [ ] [ ] , and the isolated, purified rbd has been a popular target for the development of anti-spike antibodies against pathogenic coronaviruses [ ] [ ] [ ] [ ] . however, binders against the isolated rbd may not effectively engage the aforementioned pre-fusion conformation of the full spike, which could account for the poor neutralization ability of recently described single-domain antibodies that were raised against the rbd of sars-cov- [ ] . therefore, to better identify molecules with qualities befitting a drug-like candidate, it would be advantageous to validate rbd-specific binders in the context of the full, stabilized, pre-fusion spike assembly [ , ] . single domain antibodies based on the variable vhh domain of heavy-chain-only antibodies of camelids -generally known as nanobodies -have emerged as a broadly utilized and highly successful antibody fragment format [ ] . nanobodies are small ( ) ( ) ( ) ( ) , stable, and inexpensive to produce in bacteria and yeast [ ] , yet they bind targets in a similar affinity range as conventional antibodies. due to their minimal size, they are particularly suited to reach hidden epitopes such as crevices of target proteins [ ] . we recently designed three libraries of synthetic nanobodies, termed sybodies, based on elucidated structures of nanobody-target complexes (fig. a) [ , ] . sybodies can be selected against any target protein within twelve working days, which is considerably faster than natural nanobodies, which requires the repetitive immunization during a period of two months prior to binder selection by phage display [ ] (fig. c) . a considerable advantage of our platform is that sybody selections are carried out under defined conditions -in case of coronavirus spike proteins, this offers the opportunity to generate binders recognizing the metastable pre-fusion conformation [ , ] . finally, due to the feasibility of inhaled therapeutic nanobody formulations [ ], virusneutralizing sybodies could offer a convenient and direct means of prophylaxis. here, we report of in vitro selection and characterization of sybodies against the rbd of sars-cov- spike protein. two independently prepared rbd constructs were used for in vitro sybody selections, and resulting single clones that could bind the full spike ectodomain were sequenced, expressed, and purified. six unique sybodies show favorable binding affinity to the sars-cov- spike, and five of these were also found to substantially attenuate the interaction between the viral rbd and human ace . moreover, pairs of sybodies were identified that can simultaneously bind to the rbd. we present all sequences for these clones, along with detailed protocols to enable the community to freely produce and further characterize these sars-cov- binders. based on sequence alignments with isolated rbd variants from sars-cov- that were amenable to purification and crystallization [ , ] , a sars-cov- rbd construct was designed, consisting of residues pro -gly fused to venus yfp (rbd-vyfp). this construct was expressed and secreted from expi cells, and rbd-vyfp was extracted directly from culture medium supernatant using an immobilized anti-gfp nanobody [ ], affording a highly purified product with negligible background contamination. initial efforts to cleave the c-terminal vyfp fusion partner with c protease resulted in unstable rbd, so experiments were continued with full rbd-vyfp fusion protein. to account for the presence of the vyfp fusion partner, a second rbd construct, consisting of a fusion to murine igg fc domain (rbd-fc), was commercially acquired. to remove any trace amines, buffers were exchanged to pbs via extensive dialysis. proteins were chemically biotinylated, and the degree of biotinylation was assessed by a streptavidin gel-shift assay and found to be greater than % of the target proteins [ ] . we note that while both rbd fusion proteins were well-behaved, a commercially acquired purified full-length sars-cov- spike ectodomain construct (ecd) was found to be aggregation-prone. very recently, we also produced an engineered spike protein ectodomain containing two point mutations known to stabilize the pre-fusion state, an inactivated furin cleavage site, and a c-terminal trimerization motif [ , , ] . while this purified pre-fusion spike (pfs) had not yet been available for binder selections and characterization by grating-coupled interferometry, it was used to conduct elisas in order to identify selected sybodies which recognize the rbd in the pre-fusion context (see below). since both our rbd constructs bear additional fusion domains (fc of mouse igg and vyfp, respectively), sybody selections were carried out with a "target swap" approach (fig. b) . hence, selections with the three sybody libraries (concave, loop and convex) were started with the rbd-vyfp construct using ribosome display, and the rbd-fc construct was then used for the two phage display rounds (selection variant : rbd-vyfp/rbd-fc/rbd-fc) and vice versa (selection variant : rbd-fc/rbd-vyfp/rbd-vyfp). accordingly, there were a total of six selection reactions (table , fig. b) . to increase the average affinity of the isolated sybodies, we included an off-rate selection step using the preenriched purified sybody pool after phage display round as competitor. to this end, sybody pools of all three libraries of the same selection variant were sub-cloned from the phage display vectors into the sybody expression vector psb_init. subsequently, the two separate pools (all sybodies of selection variants and , respectively) were expressed and purified. the purified pools were then added to the panning reactions of the respective selection variant in the second phage display round. thereby, rebinding of sybody-phage complexes with fast off-rates was suppressed. enrichment of sybodies against the rbd was monitored by qpcr. already in the first phage display round, the concave and loop sybodies of selection variant showed enrichment factors of and , respectively (table ) . after the second phage display round (which included the off-rate selections step), strong enrichment factors in the range of - were determined. after sub-cloning the pools from the phage display vector pdx_init into the sybody expression vector psb_init, clones of each of the selections reactions ( table , fig. b ) were picked at random and expressed in small scale. our standard elisa was initially performed using rbd-vyfp (rbd), spike ectodomain containing s and s (ecd), and maltose binding protein (mbp) as unrelated dummy protein. as outlined in the materials and methods section, elisa analysis revealed very high hit rates for the rbd and the ecd ranging from % to % and % to %, respectively (fig. , table ). the majority of the sybodies giving an elisa signal to the rbd also gave a clear signal the full-length spike protein (fig. ). however, there was a total of hits that only gave an elisa signal for rbd-vyfp, but not for the ecd. this could be due to the presence of cryptic rbd epitopes that are not accessible in the context of the full-length spike protein, or the respective sybodies may recognize the vyfp portion of the rbd-vyfp construct, though the selection procedure clearly disfavors the latter explanation. importantly, background binding to the dummy protein mbp was not observed for any of the analyzed sybodies, clearly showing that the binders are highly specific. we then sequenced sybodies that were elisa-positive against rbd-vyfp as well as the full-length spike ( for each of the selection reactions numbered from sb# - , see also fig. b ). subsequent to sybody sequencing, we also performed the elisa using engineered pre-fusion-stabilized spike ectodomain (pfs) (fig. ) , which was not available at the onset of the project. overall, the elisa signals for the ecd and pfs are highly similar. however, there are around sybodies that bind to the ecd clearly stronger than to the pfs (yet the opposite scenario was never observed). this could be explained by the fact that the pfs forms a trimer, while the oligomeric state of the ecd is not clear. in addition, the ecd might adopt partially or completely a post-fusion state, whereas pfs is expected to predominantly adopt the pre-fusion state. trimer formation as well as pre-fusion stabilization might shield certain binding epitopes on the rbd in the context of the pfs, which might become accessible as the spike falls apart into monomers and/or transits to the post-fusion state. in light of our elisa data, the pfs construct will be a crucial element in any future sybody selection campaigns. sequencing results of out of sybody clones were unambiguous. out of these clones, were found to be unique and the respective clone names are indicated in the elisa figure (fig. , table ). of note, there were no duplicate binders identified in both selection variants, indicating that the two separate selection streams gave rise to completely different arrays of sybodies. as an additional note, one sybody identified from the supposed convex library turned out to belong to the concave library; spill-over of sybodies across libraries is occasionally observed. hence, there was a total of concave, loop and convex sybodies, which were then aligned according to their library origin . as a final analysis, all sybody sequences were aligned to generate a phylogenetic tree, which shows a clear segregation across the three libraries and indicates a large sequence variability of the identified sybodies ( fig. ). the unique sybodies were individually expressed in e. coli and purified via ni-nta affinity chromatography and gel filtration. ultimately, sybodies were sufficiently well-behaved, with respect to solubility, yield, and monodispersity, to proceed with further characterization. for a kinetic analysis of sybody interactions with the viral spike, we employed grating-coupled interferometry (gci) to probe sybody binding to immobilized rbd-vyfp or ecd. first, the purified sybodies were subjected to an off-rate screen, which revealed six sybodies (sb# , sb# , sb# , sb# , sb# , and sb# ) with strong binding signals and comparatively slow off-rates. binding constants were then determined by measuring on-and off-rates over a range of sybody concentrations, revealing affinities within a range of - nm to the sars-cov- spike (fig. , table ). of note, binding affinities were consistently equal or higher for the ecd as compared to the rbd-vyfp, in particular in case of sb# for which the off-rate differs by more than two-fold. this might indicate a binding avidity effect arising from binding epitopes clustering in the context of the spike trimer or differences with regards to the glycan structures (rbd-vyfp was produced in hek cells, whereas the ecd was produced in insect cells). to our surprise, the majority of purified and elisa-positive sybodies ( out of ) displayed binding affinities worse than nm. this may be attributed to the presence of complex heterogeneous asnlinked glycans within the rbd, which could hinder the isolation of specific high-affinity binders. alternatively, given that the final elisa step of the selection process resulted in a substantial number of positive clones, insufficiently stringent conditions may have favored the high positive hit rate of lowaffinity binders. since virulence of sars-cov- is dependent on the ability of the viral rbd to bind to human ace (hace ), we sought to determine which of the selected sybodies that were well-behaved upon purification could inhibit interaction between the isolated rbd and purified hace . for this assessment, elisa plates were coated with purified hace , and the binding of purified rbd to the immobilized hace was measured in the presence or absence of an excess of each purified sybody (fig. ). while the absence of any added sybody resulted in a strong elisa signal corresponding to rbd association with hace , the pre-incubation of nearly all sybodies with the rbd resulted in an attenuated signal, implying that these binders inhibit rbd-hace association. this signal decrease relative to unchallenged rbd was modest for most sybodies, with an average signal reduction of about %, but five sybodies demonstrated exceptionally high apparent inhibition of rbd-hace interaction (sb# , sb# , sb# , sb# , and sb# ), showing ≥ % signal reduction. notably, the aforementioned kinetic analysis had shown that these sybodies were also among the strongest rbd binders. taken together, this data suggests that sb# , sb# , sb# , sb# , and sb# recognize a surface region on the rbd that overlaps with the hace binding site. while kinetic analysis had revealed sb# to be among the stronger binders to the sars-cov- ectodomain (kd ≈ nm, fig. , table ), the hace competition elisa revealed that sb# does not inhibit hace -rbd interaction to the same extent as other sybodies with comparable affinities ( % inhibition for sb# , compared to > % for sb# , sb# , sb# , and sb# ). therefore, it was hypothesized that sb# may interact with a non-or partially-overlapping surface on the rbd, relative to the more strongly-inhibiting sybodies. using sb# as a representative of the hace -inhibiting sybodies, we analyzed the ability of sb# and sb# to simultaneously associate with the rbd. first, elisa experiments demonstrate that incubation of sb# with the pre-fusion spike only slightly prevents the spike from binding to immobilized sb# , whereas pre-incubation with sb# , sb# , sb# , sb# , or sb# completely prevents spike interaction with immobilized sb# (fig. ). in agreement with the elisa data, gci experiments revealed that co-injection of sb# and sb# results in a clear (but not fully additive) increase of the response signal, relative to sb# or sb# injected alone, implying simultaneous binding of sb# and sb# (fig. ). the control gci experiment involving the co-injection of sb# and sb# did not result in a similar signal increase ( fig. ). in sum, this data plausibly suggests that sb# and sb# can simultaneously bind to the rbd. for the design of therapeutics against sars-cov- , the fusion of such a pair of non-overlapping binders could provide benefits via increased overall avidity to the spike protein. we have demonstrated the ability of our rapid in vitro selection platform to generate sybodies against the sars-cov- rbd, within a two-week timeframe. characterization of these sybodies has identified a high-affinity subset of binders that also inhibit the rbd-ace interaction. we anticipate that the presented panel of anti-rbd sybodies could be of use in the design of urgently required therapeutics to mitigate the covid- pandemic, particularly in the development of inhalable prophylactic formulations [ ] . furthermore, our identification of a pair of sybodies that can simultaneously associate with the rbd may offer an attractive foundation for the construction of a polyvalent sybodybased therapeutic. we have attempted to provide a complete account of the generation of these molecules, including full sequences and detailed methods, such that other researchers may contribute to their ongoing analysis. future work may include virus neutralization assays using the identified sybodies, as well as further selection campaigns targeting additional spike epitopes. finally, our recently described flycode technology could be utilized for deeper interrogation of selection pools, in order to facilitate discovery of exceptional sybodies that possess very slow off-rates or recognize rare epitopes [ ] . a gene encoding sars-cov- residues pro -gly (rbd, genbank accession qhd . ), downstream from a modified n-terminal human serum albumin secretion signal [ ] , was chemically synthesized (geneuniversal). this gene was subcloned using fx technology [ ] into a custom mammalian expression vector [ ] , appending a c-terminal c protease cleavage site, myc tag, venus yfp [ ] , and streptavidin-binding peptide [ ] onto the open reading frame (rbd-vyfp). - ml of suspension-adapted expi cells (thermo) were transiently transfected using expifectamine according to the manufacturer protocol (thermo), and expression was continued for - days in a humidified environment at °c, % co . cells were pelleted ( g, min), and culture supernatant was filtered ( . µm mesh size) before being passed three times over a gravity column containing nhsagarose beads covalently coupled to the anti-gfp nanobody k k [ ], at a resin:culture ratio of ml resin per ml expression culture. resin was washed with column-volumes of rbd buffer (phosphate-buffered saline, ph . , supplemented with additional . m nacl), and rbd-vyfp was eluted with . m glycine, ph . , via sequential . ml fractions, without prolonged incubation of resin with the acidic elution buffer. fractionation tubes were pre-filled with / vol m tris, ph . ( µl), such that elution fractions were immediately ph-neutralized. fractions containing rbd-vyfp were pooled, concentrated, and stored at °c. purity was estimated to be > %, based on sds-page (not shown). yield of rbd-vyfp was approximately - μg per ml expression culture. a second purified rbd construct, consisting of sars-cov- residues arg -phe fused to a murine igg fc domain (rbd-fc) expressed in hek cells, was purchased from sino biological (catalogue number: -v h, µg were ordered). purified full-length spike ectodomain (ecd) comprising s and s (residues val -pro ) with a c-terminal his-tag and expressed in baculovirus-insect cells was purchased from sino biological (catalogue number: -v b , µg were ordered). the prefusion ectodomain of the sars-cov spike protein (residues - ) [ ] , was transiently transfected into x suspension-adapted expicho cells (thermo fisher) using mg plasmid dna and mg of pei max (polysciences) per l procho medium (lonza) in a l erlenmeyer flask (corning) in an incubator shaker (kühner). one hour post-transfection, dimethyl sulfoxide (dmso; applichem) was added to % (v/v). incubation with agitation was continued at °c for days. l of filtered ( . um) cell culture supernatant was clarified. then, a ml gravity flow strep-tactin®xt superflow® column (iba lifescience) was rinsed with ml buffer w ( mm tris, ph . , mm nacl, mm edta) using gravity flow. the supernatant was added to the column, which was then rinsed with ml of buffer w (all with gravity flow). finally, six elution steps were performed by adding each time . ml of buffer bxt ( mm biotin in buffer w) to the resin. all purification steps were performed at °c. to remove amines, all proteins were first extensively dialyzed against rbd buffer. proteins were concentrated to µm using amicon ultra concentrator units with a molecular weight cutoff of - kda. subsequently, the proteins were chemically biotinylated for min at °c using nhs-biotin (thermo fisher, # ) added at a -fold molar excess over target protein. immediately after, the three samples were dialyzed against tbs ph . . during these processes (first dialysis/ concentrating/ biotinylation/ second dialysis), %, %, % and % of the rbd-vyfp, rbd-fc, ecd and pfs respectively were lost due to sticking to the concentrator filter or due to aggregation. biotinylated rbd-vyfp, rbd-fc and ecd were diluted to µm in tbs ph . , % glycerol and stored in small aliquots at - °c. biotinylated pfs was stored at °c in tbs ph . . sybody selections with the three sybody libraries concave, loop and convex were carried out as described in detail before [ ] . in short, one round of ribosome display followed by two rounds of phage display were carried out. binders were selected against two different constructs of the sars-cov- rbd; an rbd-vyfp fusion and an rbd-fc fusion. mbp was used as background control to determine the enrichment score by qpcr [ ] . in order to avoid enrichment of binders against the fusion proteins (yfp and fc), we switched the two targets after ribosome display (fig. b) . for the offrate selections we did not use non-biotinylated target proteins as described in the standard protocol, because we did not have enough purified protein at hand to do so. instead we sub-cloned all three libraries for both selections after the first round of phage display into the psb_init vector ( clones) and expressed the six pools in e. coli mc cells. then the pools corresponding to the same selection were pooled for purification. the two final pools were purified by ni-nta resin using gravity flow columns, followed by buffer exchange of the main peak fraction using a desalting pd column in tbs ph . to remove imidazole. the pools were eluted with . ml instead of . ml tbs ph . in order to ensure complete buffer exchange. these two purified pools were used for the off-rate selection in the second round of phage display at concentrations of approximately µm for selection variant (rbp-fc) and µm for selection variant (rbp-yfp). the volume used for off-rate selection was µl. just before the pools were used for the off-rate selection, . % bsa and . % tween- was added to each sample. off-rate selections were performed for minutes. elisas were performed as described in detail before [ ] . single clones were analyzed for each library of each selection. since the rbd-fc construct was incompatible with our elisa format due to the inclusion of protein a to capture an α-myc antibody, elisa was performed only for the rbd-vyfp ( nm) and the ecd ( nm) and later on with the pfs ( nm). of note, the three targets were analyzed in three separate elisas. as negative control to assess background binding of sybodies, we used biotinylated mbp ( nm). positive elisa hits were sequenced (microsynth, switzerland). the unique sybodies were expressed and purified as described [ ] . in short, all sybodies were expressed overnight in e.coli mc cells in ml cultures. the next day the sybodies were extracted from the periplasm and purified by ni-nta affinity chromatography (batch binding) followed by sizeexclusion chromatography using a sepax srt- c sec size-exclusion chromatography (sec) column equilibrated in tbs, ph . , containing . % (v/v) tween- (detergent was added for subsequent kinetic measurements). six out of the binders (sb# , sb# , sb# , sb# , sb# , sb# ) were excluded from further analysis due to suboptimal behavior during sec analysis (i.e. aggregation or excessive column matrix interaction). kinetic characterization of sybodies binding onto sars-cov- spike proteins was performed using gci on the wavesystem (creoptix ag, switzerland), a label-free biosensor. biotinylated rbd-vyfp and ecd were captured onto a streptavidin pcp-sta wavechip (polycarboxylate quasi-planar surface; creoptix ag) to a density of - pg/mm . sybodies were first analyzed by an off-rate screen performed at a concentration of nm (data not shown) to identify binders with sufficiently high affinities. the six sybodies sb# , sb# , sb# , sb# , sb# , and sb# were then injected at increasing concentrations ranging from . nm to μm (three-fold serial dilution, concentrations) in tbs buffer supplemented with . % tween- . sybodies were injected for s at a flow rate of μl/min per channel and dissociation was set to s to allow the return to baseline. sensorgrams were recorded at °c and the data analyzed on the wavecontrol (creoptix ag). data were double-referenced by subtracting the signals from blank injections and from the reference channel. a langmuir : model was used for data fitting. purified recombinant hace protein (mybiosource, cat# mbs ) was diluted to nm in phosphate-buffered saline (pbs), ph . , and μl aliquots were incubated overnight on nunc maxisorp -well elisa plates (thermofisher # - - ) at °c. elisa plates were washed three times with μl tbs containing . % (v/v) tween- (tbst). plates were blocked with μl of . % (w/v) bsa in tbs for h at room temperature. μl samples of biotinylated rbd-vyfp ( nm) mixed with individual purified sybodies ( nm) were prepared in tbs containing . % (w/v) bsa and . % (v/v) tween- (tbs-bsa-t) and incubated for . h at room temperature. these μl rbd-sybody mixtures were transferred to the plate and incubated for minutes at room temperature. μl of streptavidin-peroxidase (merck, cat#s ) diluted : in tbs-bsa-t was incubated on the plate for h. finally, to detect bound biotinylated rbd-vyfp, μl of development reagent containing , ′, , ′-tetramethylbenzidine (tmb), prepared as previously described [ ] , was added, color development was quenched after - min via addition of μl . m sulfuric acid, and absorbance at nm was measured. background-subtracted absorbance values were normalized to the signal corresponding to rbd-vyfp in the absence of added sybodies. purified sybodies carrying a c-terminal myc-his tag (sb_init expression vector) were diluted to nm in µl pbs ph . and directly coated on nunc maxisorp -well plates (thermofisher # - - ) at °c overnight. the plates were washed once with µl tbs ph . per well followed by blocking with µl tbs ph . containing . % (w/v) bsa per well. in parallel, chemically biotinylated prefusion spike protein (pfs) at a concentration of nm was incubated with nm sybodies for h at room temperature in tbs-bsa-t. the plates were washed three times with µl tbs-t per well. then, µl of the pfs-sybody mixtures were added to the corresponding wells and incubated for min, followed by washing three times with µl tbs-t per well. µl streptavidin-peroxidase polymer (merck, cat#s ) diluted : in tbs-bsa-t was added to each well and incubated for min, followed by washing three times with µl tbs-t per well. finally, to detect pfs bound to the immobilized sybodies, µl elisa developing buffer (prepared as described previously [ ] ) was added to each well, incubated for h (due to low signal) and absorbance was measured at nm. as a negative control, tbs-bsa-t devoid of protein was added to the corresponding wells instead of a pfssybody mixture. ( ) ( ) ) sb# belongs to the concave library (spill-over). ) two sequencing reactions failed. sb# qvqlvesggglvqaggslrlscaasgfpvrkanmhwyrqapgkerewvaaimskgeqtvyadsve grftisrdnakntvylqmnslkpedtavyycrvfvgwhyfgqgtqvtvs sb# qvqlvesggglvqaggslrlscatsgfpvyqanmhwyrqapgkerewvaaiqsygdgthyadsvk grftisrdnakntvylqmnslkpedtavyycravyvgmhyfgqgtqvtvs sb# qvqlvesggglvqaggslrlscaasgfpvnyktmwwyrqapgkerewvaaiwsyghtthyadsvk grftisrdnakntvylqmnslkpedtavyycvvwvghnyegqgtqvtvs sb# qvqlvesggglvqaggslrlscaasgfpvyaqnmhwyrqapgkerewvaaiyshgywtlyadsvk grftisrdnakntvylqmnslkpedtavyycevqvgawytgqgtqvtvs sb# qvqlvesggglvqaggslrlscaasgfpvfsghmhwyrqapgkerewvaailsngdsthyadsvk grftisrdnakntvylqmnslkpedtavyycrvhvgahyfgqgtqvtvs sb# qvqlvesggglvqaggslrlscaasgfpveqgrmywyrqapgkerewvaaiishgtvtvyadsvk grftisrdnakntvylqmnslkpedtavyycyvyvgaqywgqgtqvtvs sb# qvqlvesggglvqaggslrlscaasgfpvlftymhwyrqapgkerewvaaiwssgnstwyadsvk grftisrdnakntvylqmnslkpedtavyycfvkvgnwyagqgtqvtvs sb# 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qvqlvesgggsvqaggslrlscaasgfiygitylgwfrqapgkeregvaalvtwngqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaadwgydwplwdewywywgqgtqvtvs sb# qvqlvesgggsvqaggslrlscaasgtiadikylgwfrqapgkeregvaalmtrwgstyyadsvk grftvsldnakntvylqmnslkpedtalyycaaanyganyplysqqysywgqgtqvtvs sb# qvqlvesgggsvqaggslrlscaasgsissikylgwfrqapgkeregvaalmtrwgmtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaanyganeplqythynywgqgtqvtvs sb# qvqlvesgggsvqaggslrlscaasgeiesifylgwfrqapgkeregvaalytyvgqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaasygaahplsimryyywgqgtqvtvs sb# qvqlvesgggsvqaggslrlscaasgtiahikylgwfrqapgkeregvaalmtkwgqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaasyganfplkasdysywgqgtqvtvs sb# qvqlvesgggsvqaggslrlscaasgsiqaitylgwfrqapgkeregvaalvtwngqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaadwgydwplwdewywywgqgtqvtvs sb# qvqlvesgggsvqaggslrlscaasgsissitylgwfrqapgkeregvaalvtysgntyyadsvk grftvsldnakntvylqmnslkpedtalyycaaatwghswplyndeywywgqgsqvtvs sb# qvqlvesgggsvqaggslrlscaasgsissitylgwfrqapgkeregvaalitvnghtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaaawgyawplhqddywywgqgtqvtvs sb# qvqlvesgggsvqaggslrlscaasgsissitylgwfrqapgkeregvaalntfngttyyadsvk grftvsldnakntvylqmnslkpedtalyycaaatwgyswpliaeynwywgqgtqvtvs sb# qvqlvesgggsvqaggslrlscaasgsissitylgwfrqapgkeregvaalktqagftyyadsvk grftvsldnakntvylqmnslkpedtalyycaaanwgyswplyeaddwywgqgtqvtvs the plasmids encoding for the six highest affinity binders will very soon be available through addgene (addgene # -# ). for each of the six independent selec on reac ons, clones were picked at random and analyzed by elisa. a non-randomized sybody was used as nega ve control (wells h and h , respec vely). sybodies that were sequenced are marked with the respec ve sybody name (sb# - ). please note that iden cal sybodies that were found - mes are marked with the same sybody name (e.g. sb# ) . elisa analyses shown in these graphs were performed on three different days: ( ) rbd and mbp, ( ) ecd, ( ) . . . . concave adsvkgrftisrdnakntvylqmnslkpedtavyycx-vxvgxxyxgqgtqvtvs phylogene c tree of rbd sybodies. a radial tree was generated in clc . . . sybodies inhibit rbd binding to ace . the effect of sybodies on rbd associa on with human ace was assessed with an elisa. individual sybodies ( nm, sybody number shown on x-axis) were incubated with bio nylated rbd-vyfp ( nm) and the mixtures were exposed to immobilized ace . bound rbd-vyfp was detected with streptavidin-peroxidase/tmb. each column indicates background-subtracted absorbance at nm, normalized to the signal corresponding to rbd-vyfp in the absence of sybody (dashed red line). simultaneous binding of sb# and sb# . (a) simultaneous binding of sybodies was analyzed using gra ng-coupled interferometry on the wave system (creop x ag, switzerland). bio nylated ecd was immobilized and the binders were injected alone and simultaneously at satura ng concentra ons (sb# : nm, sb# : nm, sb# : nm). superimposed sensorgrams are shown. (b) compe on elisa. title of the graphs indicate the sybody which was directly coated on the plate at a concentra on of nm. the labels on the x-axes depict the sybody used for compe on. to determine the background signal, buffer devoid of protein was added. herd immunity -estimating the level required to halt the covid- epidemics in affected countries the reproductive number of covid- is higher compared to sars coronavirus estimation of the reproductive number of novel coronavirus (covid- ) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis presumed asymptomatic carrier transmission of covid- estimating clinical severity of covid- from the transmission dynamics in wuhan, china predicting the future trajectory of covid- preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies. viruses the sars-cov- vaccine pipeline: an overview use of antiviral drugs to reduce covid- transmission a novel coronavirus outbreak of global health concern a sars-like cluster of circulating bat coronaviruses shows potential for human emergence structure, function, and evolution of coronavirus spike proteins cryo-em structure of the -ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov- spike glycoprotein sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor we thank rony nehmé and andré heuer (creoptix ag, wädeswil, switzerland) for the acquisition, fitting and interpretation of gci measurements using the wavesystem. we thank florence projer, david hacker and kelvin lau (protein production and structure core facility, epfl, switzerland) for the production of the pre-fusion spike protein. we are grateful to jason mclellan (the university of texas at austin, u.s.) for having provided the pre-fusion-stabilized soluble spike expression vector. kine c characteriza on of the top six sybodies. (a) binding kine cs were measured by gra ng-coupled interferometry on the wave system (creop x ag, switzerland). rbd-vyfp and ecd were immobilized and the sybodies were injected at increasing concentra ons ranging from . nm to μm. data were fi ed using a langmuir : model. key: cord- -xx u h authors: tripathi, siddhartha; agrawal, amit title: blood plasma microfluidic device: aiming for the detection of covid- antibodies using an on-chip elisa platform date: - - journal: trans indian natl doi: . /s - - - sha: doc_id: cord_uid: xx u h covid- is a public health emergency of international concern. detection of sars-cov- virus is an important step towards containing the virus spread. although viral detection using molecular diagnostic methods is quite common and efficient, these methods are prone to errors, laborious and time consuming. there is an urgent need for blood-based tests which are simple to use, accurate, less time consuming, portable and cost-effective. human blood plasma contains water, proteins, organic and in-organic substances including bacteria and viruses. blood plasma can be effectively used to detect covid- antibodies. the immune system generates antibodies (igm/igg proteins) in response to the virus and identification of these antibodies is related to the presence of the infection in the patient in the past. therefore, detecting and testing the presence of these antibodies will be extremely useful for monitoring and surveillance of the population (petherick, lancet : – , ). herein, we describe and propose a microfluidic elisa (enzyme-linked immunosorbent assay) system to detect covid- antibodies on a lab-on-chip platform. we propose to first separate plasma from whole human blood using a microfluidic device and subsequently perform the detection of antibodies in the separated plasma using a semi-automated on-chip elisa. the technology presented comprises a microfluidic blood plasma separation device which is capable of effectively separating plasma from whole human blood. human blood constitutes cells and plasma. blood plasma is considered an important source of information pertaining to human health condition; this is due to the presence of important bio-markers. human blood plasma contains water, proteins, organic and in-organic substances including bacteria and viruses. blood plasma is separated from other constituents on a routine basis. use of plasma is preferred over whole blood in several diagnostic tests; this is due to clogging, cell lysis and cell interference issues associated with whole blood testing. we have developed a passive microdevice to separate blood plasma; the device design and other features are shown in fig. . the device is simple, compact and efficient. the principle behind plasma separation revolves around harnessing the bio-physical and geometrical effects of blood flow within a microchannel. experimental results indicate that almost pure plasma (separation efficiency . %, purity) is obtained by injecting whole blood at a flow rate of . ml/ min. the yield (or amount of plasma obtained to amount of blood infused) of the device is % with whole human blood. the plasma separated was found to be hemolysis free and few biomarkers of interest, namely proteins, hcg (human chorionic gonadotropin) hormone, and glucose were successfully recovered from the separated plasma. the device was fabricated in pdms using photolithography and soft lithography techniques (other fabrication materials and techniques can also be employed). the major advantage of such microdevice is its accuracy, ease of operation, use of small sample amount, small size, portability and ease of its integration with a bio-sensing platform. the device has been extensively studied, patented and has been reported in various publications (tripathi et al. (tripathi et al. , a (tripathi et al. , b (tripathi et al. , (tripathi et al. , prabhakar et al. ) . recently, this microdevice has been successfully employed by a research group for measuring dopamine from whole blood. researchers have successfully integrated this plasma separation microdevice with an enzyme-free plasmonic neurotransmitter dopamine biosensor to measure dopamine concentration with high detection selectivity (vázquez-guardado et al. ) . the reported plasma separation microdevice is not only an alternate to the centrifuge, but it can also be easily integrated with a biosensing platform/detection technology (for example, elisa) and result in a point-of-care device. microdevice ensures separation of high-quality plasma with minimal cell interference enabling selection of an analyte with high specificity and sensitivity. the technology was developed to realize a microdevice to enable blood plasma separation in a lab-on-chip format in an effective way. the study was motivated from the current worldwide effort of developing point-of-care microdevices. there are numerous novel features of this microdevice. the developed microdevice is passive and does not rely on active techniques of separation, the device uses elevated dimensions, so maintaining tight tolerances is not essential; the design is, therefore, easy to fabricate and is cost-effective. the device can work efficiently over a wide range of hematocrit, both whole blood and diluted blood can be used. whole blood is preferred as it ensures sufficiency of bio-markers in the separated plasma. the device can separate plasma in a continuous manner without clogging the microdevice. approximately µl of plasma can be removed using ml of whole human blood in approximately min. the device can easily be integrated with a bio-sensor to enable on-chip detection of a target analyte. the most common tests to detect the sars-cov- virus are the rt-pcr test (swab based) and the antibody test (blood based). in addition, field effect transistor (fet)-based sensors for point-of-care testing have been reported. recently, seo et al. ( ) have reported successful detection of sars-cov- virus with high sensitivity in swab specimens fig. a blood plasma microdevice design and zoomed view at the junction. symbols: i-blood inlet, o-blood outlet, p-plasma outlet. b experimental photograph showing plasma separation in the microdevice using whole blood at a flow rate of . ml/min. c exter-nal view of the blood plasma separation taking place in the pdms microdevice. d comparison of the device size with a coin (tripathi et al. ) using a fet-based biosensor. the graphene-based device used sars-cov- spike antibody to detect the antigen protein. the swab-based tests directly detects the virus and are useful for early detection of the virus whereas blood-based tests are indirect and informs about the infection in the past. the technology of blood plasma separation in a microdevice can be employed for testing of antibodies present in the blood plasma of a covid- -infected subject, similar to a point-of-care serological test. the immune system generates antibodies (igg/igm proteins) in response to the virus. identification of these antibodies is related to the presence of the infection in the patient in the past. therefore, detecting/ testing the presence of these antibodies will be extremely useful for surveillance of the population and also for plasma transfusion to combat the active virus (petherick ). also, blood-based test can allow for the measurement of additional bio-markers of interest such as crp (c-reactive protein). this protein has been found to correlate with the severity of covid- infection (vashist ) . in the past, various researchers have reported detection of hiv, zika, hepatitis b, dengue, influenza, measles and rubella using microfluidic techniques (yeh et al. ). immunoassays can be used to measure small amounts of analytes effectively (vashist ; yeh et al. ; lee and lee ; hsu et al. ; liu et al. ) . herein, we propose the integration of sandwich elisa (enzyme-linked immunoabsorbent assay) with the blood plasma separation microdevice to detect covid- antibodies after minor modifications in the design. although the separated plasma from the chip can be analyzed in a conventional elisa, we prefer to propose the on-chip detection of analyte for simplicity and cost-effectiveness. the idea, procedure and steps have been presented in fig. . the whole setup will be similar to an elisa on a microdevice. first, the detection zone area (the plasma reservoir, fig. a) is coated with the sars-cov- antigen (spike protein) by surface immobilization techniques (direct entrapment of antibodies by spotting and drying methods/physical absorption methods or plasma treatment) on the glass/pdms (heyries et al. ; welch et al. ) . note that the glass surface is used for bonding purpose. next, the pdms-based microdevice is bonded onto the glass plate such that the plasma reservoir of the device aligns with the antigen-immobilized area. next, bsa (bovine serum albumin) is added to prevent non-specific binding of antibodies. the device is now ready for the injection of whole blood. blood is injected using a syringe pump delivering a constant flow rate of . ml/min; however, the use of expensive syringe pump can be avoided by devising a spring-loaded syringe capable of delivering the required flow rate. as blood flows into the microchannel, the plasma gets fig. a top: mask of the original blood plasma separation microdevice design with additional inlet for carrying out washing steps and injecting antibodies. bottom: side view of the microsystem. b experi-mental sandwich elisa: showing steps to identify the presence of sars-cov- (covid- ) antibodies present in blood plasma separated and flows towards the plasma outlet reservoir. the antibodies, or the target of interest binds to the sars-cov- antigens coated on the reservoir; this step involves reaction and incubation time. subsequently, washing step is carried out by injecting pbs (phosphate-buffered saline and . % tween ) from an additional reservoir connected through a channel near the plasma outlet. this step is essential to remove the unbound molecules. next, labelled secondary antibodies (hrp-horseradish peroxidase conjugated) are injected and added to the reservoir. finally, the substrate addition (tmb- , ′, , ′-tetramethylbenzidine + h o ) will result in colorimetric signals for image analysis and determining the concentration of covid- antibody (hsu et al. ) . though colorimetric methods are simple to employ, the electrochemical methods integrated with smartphone technology can also be employed for detection purposes (lee and lee ) . realizing a fully automated testing on a lab-on-chip format is quite challenging, it is expected that the proposed idea will be useful in reducing the sample processing and detection time as compared to conventional elisa technique. in addition, the arrangement of the microdevice is such that other bio-markers of interest can also be measured simultaneously, if desired. the development of the elisa-based microfluidic platform will involve modifications in the current design and fabrication of the microdevice using photolithography and soft lithography techniques, plasma separation and off-chip testing of separated plasma using conventional -well elisa, procurement of spike proteins, antibodies, buffers, bio-safety cabinet and spectrophotometer, immobilization of antigens and related experiments, quantification of antibodies (image analysis), and comparison of results obtained from the current microdevice with those obtained using a conventional elisa kit. overall, months will be a reasonable estimate to accomplish the whole task. conflict of interest the authors declare that they have no competing interests. ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. developing antibody tests for sars-cov- straightforward protein immobilization on sylgard pdms microarray surface based elisa for the detection of autoimmune antibodies in body fluid the case of bullous pemphigoid lab on a chip for in situ diagnosis: from blood to point of care a fully integrated distance readout elisa-chip for point-of-care testing with sample-in-answer-out capability a novel, compact and efficient microchannel arrangement with multiple hydrodynamic effects for blood plasma separation rapid detection of covid- causative virus (sars-cov- ) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor blood plasma separation in elevated dimension t-shaped microchannel performance study of microfluidic device for blood plasma separation-a designer's perspective passive blood plasma separation at the microscale: a review of design principles and microdevices microdevice for plasma separation from whole human blood using bio-physical and geometrical effects international patent on microdevice for separating plasma from human blood enzyme-free plasmonic biosensor for direct detection of neurotransmitter dopamine from whole blood in vitro diagnostic assays for covid- : recent advances and emerging trends point-of-care microdevices for blood plasma analysis in viral infectious diseases orientation and characterization of immobilized antibodies for improved immunoassays key: cord- -q zeoqlb authors: carrat, f.; de lamballerie, x.; rahib, d.; blanche, h.; lapidus, n.; artaud, f.; kab, s.; renuy, a.; szabo de edelenyi, f.; meyer, l.; lydie, n.; charles, m.-a.; ancel, p.-y.; jusot, f.; rouquette, a.; priet, s.; saba villaroel, p. m.; fourie, t.; lusivika-nzinga, c.; nicol, j.; legot, s.; druesne-pecollo, n.; essedik, y.; lai, c.; gagliolo, j.-m.; deleuze, j.-f.; bajos, n.; severi, g.; touvier, m.; zins, m. title: seroprevalence of sars-cov- among adults in three regions of france following the lockdown and associated risk factors: a multicohort study. date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: q zeoqlb aim to estimate the seroprevalence of sars-cov- infection in may-june after the lockdown in adults living in three regions in france and to identify the associated risk factors. methods participants in a survey on covid- from an existing consortium of three general adult population cohorts living in the ile-de-france (idf) or grand est (ge), two regions with high rate of covid- , or in the nouvelle-aquitaine (na), with a low rate, were asked to take a dried-blood spot (dbs) for anti-sars-cov- antibodies assessment. the primary outcome was a positive anti-sars-cov- elisa igg result against the spike protein of the virus (elisa-s). the secondary outcomes were a positive elisa igg against the nucleocapsid protein (elisa-np), anti-sars-cov- neutralizing antibodies titers >= (sn), and predicted positivity obtained from a multiple imputation model (mi). prevalence estimates were adjusted using sampling weights and post-stratification methods. findings between may , and june , , , participants were asked to provide dbs, and , were included in the analysis, with a positive elisa-s, with a positive elisa-np, with sn>= and (standard deviation= ) with a positive mi. adjusted estimates of seroprevalence (positive elisa-s) were . % ( %ci . %; . %) in idf, . % ( %ci . %; . %) in ge and . % ( %ci . %; . %), in na. the adjusted prevalence of positive elisa-np, sn and mi were . %, . % and . % in idf, . %, . % and . % in ge, and . %, . % and . % in na, respectively. a higher seroprevalence was observed in younger participants and when at least one child or adolescent lived in the same household. a lower seroprevalence was observed in smokers compared to non-smokers. interpretation at the end of the lockdown the prevalence of anti-sars-cov- igg or neutralizing antibodies remained low in the french adult population, even in regions with high reported rates of covid- . to estimate the seroprevalence of sars-cov- infection in may-june after the lockdown in adults living in three regions in france and to identify the associated risk factors. participants in a survey on covid- from an existing consortium of three general adult population cohorts living in the ile-de-france (idf) or grand est (ge) -two regions with high rate of covid- , or in the nouvelle-aquitaine (na) -with a low rate, were asked to take a dried-blood spot (dbs) for anti-sars-cov- antibodies assessment. in idf, . %, . % and . % in ge, and . %, . % and . % in na, respectively. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint serological surveys help determine the extent of infection by a viral agent in a population and identify associated risk factors. in addition, characterizing the distribution of antibodies against this agent can help evaluate the portion of the population that is immunized to quantify herd immunity. however, despite the ongoing covid- pandemic, there are still very few serologic surveys describing the factors associated with sars-cov- seroprevalence, and only one study explored the distribution of neutralizing antibodies against sars-cov- in a general adult population in a very low prevalence area. serologic surveys of sars-cov- have been performed between january and july in the general population in iceland, switzerland, spain, uk, , italy, belgium, germany, china, brazil, canada, and the us. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint the present report combined data collected from questionnaires in the sapris ("santé, perception, pratiques, relations et inégalités sociales en population générale pendant la crise covid- ") survey in france, with serological results from the sapris-sero study. the sapris survey has been described elsewhere. briefly, the survey was created in march to evaluate the main epidemiological, social and behavioral challenges of the sars-cov epidemic in france in relation to social inequalities in health and healthcare. it is based on a consortium of prospective cohort studies including three general population-based adult cohorts and two child-cohorts (not presented in this study). [ ] [ ] [ ] all participants from the original cohorts with regular access to electronic (internet) questionnaires were invited to participate in the sapris survey (supplementary figure ). two self-administered questionnaires covering the lockdown and the post-lockdown periods were sent as of april , and returned before may , . variables collected in the questionnaires included socio-demographics, household size and composition, covid- diagnosis, sars-cov- rt-pcr test, a detailed description of the subject's symptoms in the two weeks before each questionnaire, comorbidities, healthcare use and treatment, employment, daily life, child care, alcohol, tobacco and cannabis use, social and sexual life, preventive measures, risk perception and beliefs. the goal of the sapris-sero study (#nct ) including participants enrolled in the sapris survey, was to quantify and follow the cumulative incidence of sars-cov- infection in the french population using serological tests and to assess the determinants of infection. self-sampling dried-blood spot (dbs) kits were . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint eluates were processed with a commercial elisa test (euroimmun®, lübeck, germany) to detect anti-sars-cov- antibodies (igg) directed against the s domain of the spike protein of the virus (elisa-s). the volume of eluate used corresponded to the amount of serum and dilution recommended in the manufacturer's instructions. all samples with an elisa-s test optical density ratio ≥ . were also tested with an elisa test to detect igg antibodies against the sars-cov- nucleocapsid protein (euroimmun®, lübeck, germany, elisa-np) and with an in-house micro-neutralization assay to detect neutralizing anti-sars-cov- antibodies (sn), as described elsewhere. briefly, we used veroe cells cultured in -well microplates, tcid of the sars-cov- strain bavpat (courtesy of pr. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint we randomly selected , of the participants of the sapris survey for this study who agreed to be tested and who were residents from one of the three french administrative regions: ile-de-france (idf) or grand est (ge), i.e. the two regions with the highest reported cumulated rates of covid- at the end of the lockdown period, or nouvelle-aquitaine (na), a region with a low reported rate. ethical approval and written or electronic informed consent were obtained from each participant before enrolment in the original cohort. the sapris survey was approved by the inserm ethics committee (approval # - dated march , ). the sapris-sero study was approved by the sud-mediterranée iii ethics committee (approval # . . . ) and electronic informed consent was obtained from all participants for dbs testing. the main outcome was a positive elisa-s test. in accordance with the manufacturer's instructions a test was considered to be elisa-positive with an optical density ratio ≥ . , elisa-indeterminate between . and . , and elisanegative, < . . the secondary outcomes were a positive elisa-np (using the same thresholds) and positive sn defined as a titer ≥ . because test sensitivity and specificity was not %, we also used a multiple imputation (mi) method to estimate a participant's positivity in which the likelihood of positivity was based on observed test results and covariates. the association of seroprevalence was evaluated in relation to age, gender, sociodemographic characteristics, bmi, chronic conditions (according to a pre-specified is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint list), tobacco and alcohol use before the lockdown. age groups were categorized according to predefined limits (< ; - ; - ; - ; ≥ years old) and bmi according to standard cut-offs (< . ; . -< ; ≥ -< ; ≥ kg/m ). the association of seroprevalence was also studied in relation to symptoms. possible covid- was defined according to the european centre for disease prevention and control as at least one of the following: cough, fever, dyspnea, and sudden anosmia, ageusia or dysgeusia. participants who did not report any of these symptoms on either questionnaire, did not have a positive covid- diagnosis, or did not experience cough, fever or feverishness from the beginning of the year were considered to be asymptomatic. inverse probability weighting and generalized raking were used to estimate seroprevalence in the adult population. weights were estimated from each cohort source by logistic regression, with selection or participation as response variables and socio-demographic characteristics as covariates. an initial cohort-specific calibration was performed by generalized raking in relation to the marginal totals of the distribution of age class, gender and socio-professional category in the target population. the weights were then rescaled according to the relative sample size of each cohort, then recalibrated according to the same covariates to provide representative estimates of the adult population. this weighting procedure was performed for each region independently. confidence intervals for weighted estimates were computed by bootstrapping. to fit the mi model, participants with all three positive elisa-s, elisa-np and sn test results were classified as "true" positives while those with all three negative . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint results or elisa-s < . were "true" negatives. the markov chain monte carlo method was used to imputing mi using numerical values from the three serological tests (log-transformed), region, age and gender. the mi was built from imputed data sets and estimates combined with rubin's rules. chi-square test for trend was used on unweighted data to compare symptoms and health care use according to elisa-s results. logistic regression models were used on unweighted data with stratification in the source cohort to identify the determinants of a positive elisa-s (primary outcome). indeterminate elisa-s results were grouped with negative results in the primary analysis. multivariable analysis was performed including region, age, gender and all factors associated with seroprevalence in univariable analysis. a backward elimination procedure was used to identify independent covariates associated with a positive elisa-s. contact with a rt-pcr positive household member was not considered to prevent the risk of reverse causation. multivariable analyses were repeated using secondary outcomes then performed in each region to identify any potential regional effect-modification. weighting and multiple imputation used the survey and mice package from r software version . . (r foundation for statistical computing, vienna, austria). other analyses were performed with sas · software (sas institute inc., cary, north carolina, usa). p<. was considered to be statistically significant. the sponsor and funders facilitated data acquisition but did not participate in the study design, analysis, interpretation or drafting. fc had full access to all data in the study and fc, xl, nb, mt, gs, mz made the final decision to submit the study for publication. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . participants with a positive elisa-s had a higher rate of self-reported symptoms than those with negative tests except for skin lesions (table ) is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . on univariable analysis, the rate of positive elisa-s was higher in idf and ge than in na (table ) multivariable analysis showed an independent and positive association between positive elisa-s, idf and ge compared to na, for younger age, and at least one child or adolescent living in the same household (table ). a negative association was found with active smoking (vs no smoking). the observed associations were confirmed with mi and were overall consistent with elisa-np and sn, although they did not all reach statistical significance due to a smaller number of events (supplementary tables - ). when multivariable analysis was performed in each region separately, the associations did not differ between idf and ge but the pattern in na was different from that in idf or ge, with a higher odds-ratio (or) in young age groups in the former (or= . . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint a lower seroprevalence with increasing age was reported in several populationbased serological studies , and a higher rate of possible covid- with decreasing age was described in an earlier study. although men are known to be at a higher risk of severe covid- , hospitalization and deaths than women, we found an association between seroprevalence and female gender in na, which was also reported in a recent italian study. this association was only found in the region with a lower prevalence and may be related to the specific dynamics of transmission in this area. based on the estimated -day median covid- incubation time and the appearance of symptoms within days after infection, participants who developed a possible covid- before march and tested positive were potentially infected before lockdown, probably in the workplace or in the community. this could explain why we did not find any specific association with social health inequalities, while, conversely, univariable analyses showed associations between seroprevalence and working adults with higher incomes and educational levels. as in other studies, univariable analysis identified the size of the household and the number of rooms, but only living with at least one child remained associated with seroprevalence on multivariable analysis, indicating that children could play an important role in household-related transmission. finally, active smoking was associated with a lower rate of elisa-s or sn positive results. , smoking status was collected before the peak of the pandemic and thus could not have been affected by preventive behaviours in smokers. although smoking is a risk factor for severe covid- in infected patients, its role in the risk of infection remains unclear because certain components of the smoke (such as nicotine) regulate ace receptor expression which is involved in sars-cov- entry into cells. , smoking is also known to be associated with lower serum levels of igg, is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint iga or igm, but this probably does not explain why smoking was also negatively correlated with sars-cov- rt-pcr positive results in several studies. , our study has several limitations. first, the primary endpoint is based on a test that does not have a % sensitivity and specificity. thus, certain participants were probably misclassified. we used manufacturer-defined cutoff points for positivity, although the test performance can increased by using other positive and negative cut-off values. however, prevalence correction using these reported test performances or by the manufacturer are not applicable to our study, since the use of is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint formally associate the self-reported covid- symptoms with a positive serological result and once again, misclassification may have occurred. this study has several strengths. in particular, it is based on well-characterized general population cohorts with a very high participation rate. moreover, serological samples were collected within to months after the period of intense circulation of sars-cov- and all serological tests were centralized and performed blinded to participants' characteristics or clinical history. several serological methods were combined, including neutralization, to improve the interpretation of seroprevalence results. in conclusion, our study shows that the level of seroprevalence remains low in the is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . prof fabrice carrat reports personal fees from imaxio and sanofi, outside the submitted work. all other authors declare no competing interest. this study anr (agence nationale de la recherche, #anr- -covi- , #anr- -coho- ), fondation pour la recherche médicale (# rr - ), inserm (institut national de la santé et de la recherche médicale, #c - ). the constances cohort study is supported by the caisse nationale d'assurance is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . . ( . ; . ) . ( . ; . ) . ( . ; . ) . ( . ; . ) . ( . ; . ) . ( . ; . ) . ( . ; . ) . ( . ; . ) week (first day) is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint assessing the extent of sars-cov- circulation through serological studies seroprevalence and correlates of sars-cov- neutralizing antibodies: results from a population-based study in bonn humoral immune response to sars-cov- in iceland seroprevalence of anti-sars-cov- igg antibodies in geneva, switzerland (serocov-pop): a population-based study prevalence of sars-cov- in spain (ene-covid): a nationwide, population-based seroepidemiological study uk biobank sars-cov- serology study. weekly report - population point prevalence of sars-cov- infection based on a statewide random sample -indiana incidence and risk factors of illnesses presumably caused by a sars-cov- infection in the general population during the lockdown period: a multi-cohort study cohort profile: the french e n cohort study the nutrinet-sante study: a web-based prospective study on the relationship between nutrition and health and determinants of dietary patterns and nutritional status the french constances population-based cohort: design, inclusion and follow-up lower prevalence of antibodies neutralizing sars-cov- in group o french blood donors covid- : point épidémiologique du mai obesity: preventing and managing the global epidemic case definition for coronavirus disease (covid- ), as of generalized raking procedures in survey sampling multiple imputation for nonresponse in surveys estimating the burden of sars-cov- in france clinical and immunological assessment of asymptomatic sars-cov- infections factors associated with covid- -related death using opensafely the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application cluster of covid- in northern france: a retrospective closed cohort study clinical characteristics of coronavirus disease in china nicotine and the renin-angiotensin system cigarette smoke exposure and inflammatory signaling increase the expression of the sars-cov- receptor ace in the respiratory tract smoking and immunoglobulin levels covid- testing, hospital admission, and intensive care among , , united states veterans aged the association of smoking status with sars-cov- infection, hospitalisation and mortality from covid- : a living rapid evidence review with bayesian meta-analyses validation of a commercially available sars-cov- serological immunoassay alcohol use before lockdown (in g/dy) < [ , bmi (kg/m ) < . [ . chronic diseases (y vs n) asthma, copd, other respir. diseases the authors warmly thank all the volunteers of the constances, e n-e n, and nutrinet-santé cohorts.we thank the staff of the constances, e n-e n and nutrinet-santé cohorts that have worked with dedication and engagement to collect and manage the data used for this study and to ensure continuing communication with the cohort participants.we thank the ceph-biobank staff for their adaptability and the quality of their work.in the virology department, dr nadège brisbarre and the technical staff for impeccable management of samples and serological assays. key: cord- -c bwky e authors: pickering, s.; betancor, g.; pedro galao, r.; merrick, b.; signell, a. w.; wilson, h. d.; tan kia ik, m.; seow, j.; graham, c.; acors, s.; kouphou, n.; steel, k. j.; hemmings, o.; patel, a.; nebbia, g.; douthwaite, s.; o'connell, l.; luptak, j.; mccoy, l.; brouwer, p. j.; van gils, m. j.; sanders, r. w.; martinez nunez, r.; bisnauthsing, k.; o'hara, g.; macmahon, e.; batra, r.; malim, m. h.; neil, s. j.; doores, k.; edgeworth, j. d. title: comparative assessment of multiple covid- serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: c bwky e there is a clear requirement for an accurate sars-cov- antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. we therefore evaluated the performance of a variety of antibody testing technologies and their potential as diagnostic tools. a highly specific in-house elisa was developed for the detection of anti-spike (s), -receptor binding domain (rbd) and -nucleocapsid (n) antibodies and used for the cross-comparison of ten commercial serological assays - a chemiluminescence-based platform, two elisas and seven colloidal gold lateral flow immunoassays (lfias) - on an identical panel of sars-cov- -positive samples and pre-pandemic negatives. there was a wide variation in the performance of the different platforms, with specificity ranging from % to %, and overall sensitivity from . % to . %. however, the head to head comparison of multiple serodiagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over % seen in several tests when assessing samples from more than days post onset of symptoms. furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. rigorous comparison of antibody testing platforms will inform the deployment of point of care technologies in healthcare settings and their use in the monitoring of sars-cov- infections. as of the st of june , over million cases of sars-cov- have been confirmed worldwide, accounting for more than , deaths (https://covid .who.int/). lack of treatments or vaccines have forced governments to adopt strict quarantine strategies in an attempt to control the spread of the virus, causing major economical disturbances as well as adversely affecting quality of life and healthcare provision. there is, therefore, a clear requirement for accurate serology testing as a companion diagnostic to pcr-based testing. this is highlighted by the recent appearance of clusters of paediatric inflammatory multisystem syndrome (pims) and other hyperimmune reactions associated with sars-cov- infection [ , ] . presentation is delayed relative to active viral infection, with the detection of antibody responses being key to clinical diagnosis. in addition, monitoring population seroprevalence will be central to future public health planning based on disease susceptibility and herd immunity [ ] . for this to be meaningful, it is imperative that antibody detection methods are affordable, reliable, and readily accessible. however, with no 'gold standard' for antibody testing and an incomplete knowledge of the immunology of covid- , evaluating tests with the assumption that antibodies 'should' be there, and comparatively to rt-pcr, is problematic. head-to-head comparisons of multiple sero-diagnostic assays on identical samples therefore provides a robust assessment of individual assay performance. accordingly, we developed a highly specific semi-quantitative elisa for the detection of anti-spike (s), -s receptor binding domain (rbd) and -n antibodies, and used this to cross-evaluate ten commercial antibody tests (seven lateral flow immunoassays (lfias), one chemiluminescent assay and two elisas) on a collection of serum samples from confirmed rna positive patients, and pre-pandemic samples from march . our results demonstrate a wide variation in the performance of the different platforms, ranging from . % to . % sensitivity and from % to % specificity. as expected, performance is highly dependent on the time the sample was taken post onset of symptoms (pos) and disease severity. results obtained in this work have enabled the diagnostic-grade validation for one of the lfias evaluated for pilot clinical use for adult and paediatric patients with a range of clinically-suspected covid- inflammatory syndromes at guy's and st thomas' hospitals. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . table ) . an in-house elisa was developed to measure antibody responses against the full-length s, the rbd and n. recombinant s and rbd were expressed in hek f cells and purified by affinity and size exclusion chromatography. n was expressed in and purified from e. coli. prepandemic serum samples from several cohorts were used to determine the lower limit of the assay, and samples from hospital patients with confirmed sars-cov- infection (from the described serum samples in table ) were used as positive controls. a total of negative control samples included sera from individuals attending st thomas' hospital in march , sera from vaccination studies, healthy volunteers, and individuals with acute ebv infection. all serum samples from pcr positive icu patients taken at least days post symptoms showed strong igg binding to s, rbd and n ( figure a) . in contrast, although high igm reactivity was also observed to s and rbd in some individuals (figure a) , only of the negative control samples showed igg reactivity to s or rbd. high igm and igg reactivity was observed in the pre-pandemic samples against n ( figure a ) suggestive of potential crossreaction with seasonal coronaviruses. of note, all individuals with acute ebv infection had high igm reactivity to n and rbd. importantly, none of the pre-covid sera had detectable igg binding to n and s or n and rbd ( figure b) . taking into account the reactivity of negative control samples in this elisa, % specificity could be reached using a cut-off where igg against n or s both have od values at least -fold above the wells containing secondary antibody only ( figure b) . all serological assays were evaluated with the same set of serum samples from confirmed sars-cov- -positive individuals. each of the samples was tested: for anti-sars-cov- igm by in-house elisa and seven lfias; for iga by commercial elisa (figure ) ; and for igg by inhouse elisa, seven lfias, a commercial elisa and for total antibody (igg, igm and iga) using a chemiluminescent assay (figure ) . the commercial elisas detect anti-s antibodies; for the lfias this is undisclosed proprietary information, but for some of the tests is known to be s. with no existing gold standard for the assessment of the serological response to sars-cov- , we started by comparing commercial serological assays with the best performing configuration of the in-house elisa (detection of anti-s igm and igg antibodies), which was also most likely to represent antibodies detected by the commercial tests. for the purpose of illustration, the intensity of bands shown in a positive lfia test, or signal strength in commercial elisa or chemiluminescent assay, is reproduced as a heatmap. however, the visible detection of a band or result above a given manufacturer's threshold scored as a positive result regardless of classification. for samples giving a strong response by elisa (> fold), the majority of the commercial assays show a consensus positive result. weaker antibody responses yielded mixed results from the lfias, with a clear pattern of increasing detection of antibodies seen across all tests with increasing time pos. samples from days - gave an extremely mixed picture, indicative of an early evolving immune response. for the detection of igm, of the samples from days or more pos were negative by anti-s elisa. of these samples were positive, in some cases only weakly, for at least two lfias and/or iga. the remaining were negative in all lfia tests and for iga (indicated with a yellow circle in figure ). these same samples were negative for igg by in-house and commercial elisa, in all lfias and by chemiluminescent assay (indicated with a yellow circle in figure ) . importantly, all samples came from individuals with a disease severity score of . later time points were obtained for three of these individuals (both of the day samples and the sixth day sample) and the next available samples were found to be strongly positive for igg and igm by lfia (days , and pos, respectively). further investigation into the nature of the negative day sample revealed that it was an error in self-reporting . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . the time of symptom onset: this individual had covid- -compatible symptoms for days prior to sampling, yet tested negative for rna days pos. days pos the individual tested positive for rna, therefore the more likely time of sampling was between and days pos. samples were not re-classified, as they are representative of the real-time analyses being performed during the peak of a pandemic. however, the day sample was omitted from sensitivity calculations. sample-by-sample analyses of multiple serological assays showed a trend for increasing detection of antibodies with increasing days pos (up to ). we also observed that individuals with severe disease had a more readily detectable antibody response, particularly compared to those who had a brief hospital stay with minimal intervention. samples were grouped . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint according to days pos and clinical severity, and compared for anti-s igm and igg by in-house elisa. significant differences in antibody levels were observed with increases in both days and severity, although there was no association between days and severity themselves ( figure a) . to assess the development of the antibody response in sequential samples from the same individual, and to evaluate the ability of lfias to detect nuances in antibody response, longitudinal samples (from individuals with disease severity scores of ) were tested by one of the best performing lfias (surescreen) and in-house elisa (figure b and c). a similar pattern of detection was seen for both types of assay, with igm detectable earlier than igg, and both tests showing consensus for the strength and timing of the response. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . this study describes the development of six in-house elisa configurations for the detection of igm and igg against sars-cov- s, rbd and n. anti-s and -n igg attained specificities of up to % - % when results for both targets are combined -and sensitivities of up to %. we used this semi-quantitative platform to cross-evaluate seven lfias, a chemiluminescent test and two commercial elisas. importantly, the availability of sequential serum samples from patients admitted from the start of the outbreak under an existing ethics agreement for storage and analysis of surplus amounts of routinely collected clinical samples, enabled us to conduct this detailed study including examining assay sensitivity with respect to time pos. our analysis demonstrates a broad range of performance across the different platforms, with several commercial tests performing above % specificity. we found that all platforms showed highest sensitivity, with narrowest confidence limits, in samples taken days pos, with most tests reaching a value of over % ( figure b ). when all commercial tests were compared, accu-tell, surescreen and spring demonstrated highest sensitivity at earlier time points, while maintaining specificities of % or above. these tests also gave the best crossassay agreements with each other and with the in-house elisa. in the best-performing tests, we also observed that signal strength aligned with that seen by elisa; this was further supported by the sequential signal increase seen in longitudinal samples from five individuals. we approached this study with the intention that an unbiased and transparent comparison will be of broad value to the scientific and infection diagnostics communities, both in terms of naming and comparing the kits, and in the nature of the samples likely to be encountered in hospitals during a sars-cov- outbreak. few studies have been published to date in which multiple lfias and elisas have been evaluated side-by-side with named kits [ - ]; and in those that have, only two tests overlap with our study, deep blue and medomics [ ] . early reports stating that lfias have insufficient sensitivity may have been due to testing samples from mixed time points and disease severities, or tests that differ from those evaluated here [ ] . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint the strength of our study is the head-to-head evaluation of multiple tests on identical serum samples. the sample set was not compiled retrospectively for the purpose of evaluation, but was part of an ongoing process to deploy a serological assay to broaden diagnostic capability at guy's and st thomas' hospitals during the peak of the sars-cov- epidemic in london. these samples are therefore entirely representative of the type that will be encountered by hospital laboratories, and the challenges associated with variable time to seroconversion and errors in self-reporting onset of symptoms are real. the cross-evaluation of multiple tests enabled the identification of samples that were negative in every test performed, despite the fact that samples were taken at days pos or later. while this could highlight a characteristic of covid- where a subset of patients do not produce a detectable antibody response, we have evidence that in three cases (the two day samples and sixth day ) where later samples were available ( , and days pos, respectively; the only next available samples) both igm and igg antibodies were detected in several lfias. in another case (sample at day pos), we uncovered a likely error in self-reported symptom onset and the sample could actually range anywhere between and days pos. it is important to note that all of the six unexpectedly negative samples were from covid- cases classified as severity level . while there is no current agreement on the relationship between disease severity and antibody titres (some show an association, some do not) [ , , - ], we observed a significant increase in detection of antibodies with increased severity of symptoms ( figure a ). importantly, this correlation is not explained by a concomitant increase in the days pos of the sample. therefore, before deployment in situations where the pre-test prevalence is likely to be low, such as seroprevalence studies, out-patient assessment or pre-admission screening for operations, these assays will require further evaluation with known sars-cov- asymptomatic and ambulatory cases, alongside an extended set of pre-pandemic samples. this is a priority as countries navigate their way out of lockdown and move towards living with the ongoing threat of sars-cov- , and seroprevalence studies will be important in the implementation and management of safe public health policies. to maintain the high specificity of our in-house elisa in community cohorts where pcr status is unknown, we would recommend determining seropositivity based on igg to both n and s. sequential or alternate detection of igm, iga and igg may also provide information on the history of infection. in the only iga test that we evaluated (euroimmun), specificity was high while . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint also showing a strong signal in early samples and a good overall sensitivity. detection of iga in serum, and potentially even earlier by mucosal sampling, may be a useful diagnostic tool. next generation antibody tests may improve on those currently being trialled, but our results demonstrate that lfias may have utility in a hospital setting as of now, particularly if deployed where a rapid result could aide a clinical pathway or decision in real time, such as ward location or prioritisation of further diagnostics and follow up. the ease of use and affordability of the lfias weighs heavily in their favour, especially for potential in resource-poor settings or as point-of-care solutions in hospitals. choosing the tests with the highest specificity will translate to confidence in a positive test result in the clinic; negative or borderline cases may be tested serially or used together with elisa testing [ ] . combination testing, in parallel with rt-pcr, and serial or sequential testing, would provide diagnostic solutions to the delayed-onset syndromes such as pims that are increasingly being reported post-peak pandemic [ , ] . a further consideration that should be given for healthcare service deployment in the hospital or community setting is consistency of use. although the lfias are marketed as home testing kits, in our experience user assiduity is essential to their optimal performance, particularly when scoring borderline cases and considering need for two independent readers. there will also likely be need to evaluate alternative sources of blood collection particularly pin-prick collection, and even different samples such as saliva, all of which should be fully evaluated before deployment. in summary, our study compares the performance of commercially available platforms and several combinations of in-house methods for the detection of anti-sars-cov- antibodies in serum samples. although lfias lack the semi-quantitative information provided by elisa tests, they have a clear utility advantage over elisa or chemiluminescence-based technologies. shortlisted tests, combined with confirmatory reflex testing using our in-house elisa, are now being taken forward into extended validations as part of a pilot clinical service at guy's and st thomas' hospitals. these incremental steps keeping different technologies in scope, whilst multiple different use-cases are still being defined, will help determine the clinical utility and cost-effectiveness of covid- serological testing in healthcare settings both in the hospital and the community. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . patients diagnosed with covid- were classified as follows: -asymptomatic or no requirement for supplemental oxygen. -requirement for supplemental oxygen (fio < . ) for at least hrs. -requirement for supplemental oxygen (fio ≥ . ) for at least hrs. -requirement for non-invasive ventilation (niv)/ continuous positive airways pressure (cpap) or proning or supplemental oxygen (fio > . ) for at least hrs and not a candidate for escalation above level care. -requirement for intubation and mechanical ventilation or supplemental oxygen (fio > . ) and peripheral oxygen saturations < % (with no history of type respiratory failure (t rf)) or < % (with known t rf) for at least hrs. all sera/plasma was heat-inactivated at °c for mins before use in the in-house elisa. high-binding elisa plates (corning, ) were coated with antigen (n, s or rbd) at µg/ml ( µl per well) in pbs, either overnight at °c or hr at °c. wells were washed with pbs-. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint dna using pei-max ( mg/ml, polysciences) at a : ratio. supernatant was harvested after days and purified using ni-nta agarose beads. we tested seven point-of-care colloidal-gold-based lfias detecting igg and igm antibodies against sars-cov- . with the exception of medomics, all lfias were ce ivd marked. target antigens were undisclosed proprietary information, but for several of them were known to be s. lfias were run according to manufacturer's instructions. typically, - µl of serum was added to the lfia membrane start point, followed by - drops of supplied buffer. kits were run at room temperature for minutes and then immediately scored using a -point scale (negative, borderline, positive, strong positive) for both igm and igg. scoring was performed independently by two individuals. the sars-cov- ab diagnostic test kit (shenzen watmind medical co., ltd.) detecting total antibody against sars-cov- was run on the chemical luminescence immunity analyzer mf (shenzen watmind medical co., ltd). the platform was calibrated with a supplied control cartridge daily prior to testing. panel samples were analysed according to manufacturer's instructions. results equal to and below . au (arbitrary units) /ml were . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint negative, scores above . au/ml were deemed positive. for comparison to other immunoassays, scores between < au/ml were deemed positive, scores > au/ml were deemed a strong positive. two commercial elisas detecting iga (ei - a) or igg (ei - g) antibodies to the sars-cov- s protein were obtained from euroimmun medizinische labordiagnostika expected binomial exact % confidence intervals were calculated on prism . using wilson/brown statistical analysis. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint (a) > pre-pandemic serum/plasma samples were assayed for igm and igg against sars-cov- n, a stabilised and uncleaved s protein, and the rbd from s. sera/plasma came from healthy volunteers (ucl healthy), vaccine trials (hird cohort [ ]), cancer patients, and emergency admissions to st thomas' hospital in march (sth healthy). the igm and igg binding were compared to that of sars-cov- pcr positive icu patients collected in march and april . sera and plasma were diluted to : and : respectively. the values reported are the fold-change in od above background. (b) analysis of igg and igm binding of pre-covid- human sera/plasma compared to sera from sars-cov- infected patients revealed that igg against both n and s distinguished these two groups. the fold-change above background for igg against n was plotted against the foldchange above background for s igg binding and rbd igg binding for each individual. comparison of ten serological assays for the detection of anti-sars cov- igg. as for in figure , the same serum samples were assessed for the presence of anti-sars cov- igg. each sample was assayed using an in-house anti-s elisa (shown in the graph across the top of each panel, black bars), seven colloidal gold lateral flow tests (deep blue, accu-tell, genbody, surescreen, spring, biohit and medomics), and a commercial elisa (euroimmun). a chemiluminescent assay for total anti-sars cov- igm, igg and iga (watmind) was also included. the threshold for a positive result in the in-house elisa is set at -fold above background, as indicated by the red dashed line. comparative tables serological assays were compared and the percentage agreement between each of the samples in the assays is represented within each box. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint sensitivity and specificity comparison of serological assays (a) specificity was determined for each serological assay using a panel of pre-pandemic serum samples from march . overall sensitivity was determined for each serological assay, based on results for serum samples from known sars-cov- -positive individuals (shown in figures and ) . for the lateral flow assays, a positive result for igm, igg or both is considered positive. for the euroimmun and in-house elisas, sensitivities for iga, igm and igg are calculated separately. (b) sensitivity and % confidence (dashed line) intervals were determined for each serological assay at increasing days pos. results for each test were categorised according to whether the serum sample was from < , ³ , ³ , or ³ days pos. serological assays were compared for igm or igg (left and right panels, respectively), and the percentage agreement between each of the samples in the assays is represented within each box. overall sensitivity (a) and specificity (b) were determined for each serological assay as in figure . sensitivity was determined for each serological assay at increasing days pos (c), or severity of illness (d). results for each test were either categorised according to whether the serum sample was from < , ³ , ³ , or ³ days pos, or severity of illness, with indicating mild illness (requiring no respiratory support) and indicating critical (requiring ecmo) (see materials and methods for full classification). % confidence intervals are shown for each assay in all panels (wilson/brown expected binomial). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint the work was supported by gifts from peking university donors and anhui deep blue company. thank you to dr terry wong for his support in acquiring test kits. we also thank the following sources for donation of test kits: the manufacturers of spring, biohit, genbody, medomics and watmind. thank you to surescreen diagnostics for their engagement and technical assistance. thank you to bindi patel, nicola varatharajah, abayomi fatola and amelia moore for laboratory assistance. thank you to florian krammer for provision of the rbd expression plasmid, and leo james and leo kiss for the provision of purified n protein. we thank king's college london infectious diseases biobank for provision of pre-covid- vaccine samples, all patients and control volunteers who participated in this study and to all clinical staff who helped with recruitment, including those working with the tapb project at the royal free hospital. we thank the maini lab at the division of infection and immunity for providing pre-covid pandemic healthy control samples. we are extremely grateful to all patients and staff at st thomas' hospital who participated in this study. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . (https://www.rcpath.org/uploads/assets/ - aca- - . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . * copd, interstitial lung disease, bronchiectasis, asthma . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . guidance and standard operating procedure covid- virus antibody testing for covid- : a report from the national covid scientific advisory panel antibody responses to sars-cov- in patients of novel coronavirus disease viral kinetics and antibody responses in patients with neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications antibody responses to sars-cov- in patients with covid- kinetics of sars-cov- specific igm and igg responses in covid- patients antibody responses to sars-cov- in covid- patients: the perspective application of serological tests in clinical practice recommendations for verification and validation methodology and sample sets for evaluation of assays for sars-cov- (covid- ) adjuvented influenza-h n vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events key: cord- -ocisq e authors: pallett, scott j c; rayment, michael; patel, aatish; fitzgerald-smith, sophia a m; denny, sarah j; charani, esmita; mai, annabelle l; gilmour, kimberly c; hatcher, james; scott, christopher; randell, paul; mughal, nabeela; jones, rachael; moore, luke s p; davies, gary w title: point-of-care serological assays for delayed sars-cov- case identification among health-care workers in the uk: a prospective multicentre cohort study date: - - journal: lancet respir med doi: . /s - ( ) - sha: doc_id: cord_uid: ocisq e background: health-care workers constitute a high-risk population for acquisition of severe acute respiratory syndrome coronavirus (sars-cov- ) infection. capacity for acute diagnosis via pcr testing was limited for individuals with mild to moderate sars-cov- infection in the early phase of the covid- pandemic and a substantial proportion of health-care workers with suspected infection were not tested. we aimed to investigate the performance of point-of-care and laboratory serology assays and their utility in late case identification, and to estimate sars-cov- seroprevalence. methods: we did a prospective multicentre cohort study between april and june , , in two phases. symptomatic health-care workers with mild to moderate symptoms were eligible to participate days after onset of covid- symptoms, as per the public health england (phe) case definition. health-care workers were recruited to the asymptomatic cohort if they had not developed phe-defined covid- symptoms since dec , . in phase , two point-of-care lateral flow serological assays, the onsite ctk biotech covid- split igg/igm rapid test (ctk bitotech, poway, ca, usa) and the encode sars-cov- split igm/igg one step rapid test device (zhuhai encode medical engineering, zhuhai, china), were evaluated for performance against a laboratory immunoassay (edi novel coronavirus covid- igg elisa kit [epitope diagnostics, san diego, ca, usa]) in samples from health-care workers and pre-covid- negative control samples. in phase (n= ), serosurveillance was done among ( · %) of health-care workers reporting symptoms, and in a subset of asymptomatic health-care workers ( [ · %] of ). findings: there was variation in test performance between the lateral flow serological assays; however, the encode assay displayed reasonable igg sensitivity ( of ; · % [ % ci · – · ]) and specificity ( of ; · % [ · – · ]) among pcr-proven cases and good agreement ( of ; · % [ · – · ]) with the laboratory immunoassay. by contrast, the onsite assay had reduced sensitivity ( of ; · % [ % ci · – · ]) and specificity ( of ; · % [ · – · ]) and agreement ( of ; · % [ · – · ]). five ( %) of pcr-positive cases were negative across all assays. late changes in lateral flow serological assay bands were recorded in ( · %) of cassettes ( [ · %] of encode assays; [ · %] of onsite assays), but only seven (all onsite assays) of these changes were concordant with the laboratory immunoassay. in phase , seroprevalence among the workforce was estimated to be · % ( % ci · – · ) in asymptomatic health-care workers and · % ( · – · ) in symptomatic health-care workers. seroprevalence across the entire workforce was estimated at · % ( % ci · – · ). interpretation: although a good positive predictive value was observed with both lateral flow serological assays and elisa, this agreement only occurred if the pre-test probability was modified by a strict clinical case definition. late development of lateral flow serological assay bands would preclude postal strategies and potentially home testing. identification of false-negative results among health-care workers across all assays suggest caution in interpretation of igg results at this stage; for now, testing is perhaps best delivered in a clinical setting, supported by government advice about physical distancing. funding: none. severe acute respiratory syndrome coronavirus (sars-cov- ) spread extensively following its identification in december, , becoming a global pandemic by march, . more than cases have been reported and deaths attributed to worldwide, as of july , . substantial public health isolation measures have been adopted in an attempt to slow the spread of infection. case finding strategies have predominantly relied on pcr assays during the acute infection phase, through centralised specialist laboratories. in the uk, testing capacity in the early period of the covid- pandemic was mostly limited to patients who were admitted to hospital with covid- symptoms, and only later extended to include symptomatic health-care workers. health-care workers constitute a population that is at substantially greater risk of con tracting sars-cov- infection due to the rate and nature of exposure associated with clinical care of positive cases. personal protective equipment (ppe) and stringent infection prevention and control measures aim to mitigate this risk and minimise both nosocomial infection of health-care workers and onward trans mission. during the initial period, when the case rate was at its peak but pcr testing was not yet widely available, a large proportion of symptomatic health-care workers were not tested. thus, sars-cov- prevalence among uk health-care workers remains largely unknown. where the infection rate in asymptomatic health-care workers is similar to that seen in general community transmission, targeted testing of sympto matic individuals might be better placed to inform infection rates among health-care workers. a variety of pathways to enhance case finding have been considered. these include point-of-care molecular platforms for acute phase testing, and laboratory elisa or lateral flow serological assays for antibodies specific to sars-cov- for delayed case identification. [ ] [ ] [ ] although lateral flow serological assays potentially offer rapid results in either the point-of-care setting or home reading or postal testing, concern exists around test performance characteristics, particularly in the first weeks after onset of symptoms, as well as their poor positive predictive value when applied to a general population. by contrast, although laboratory-based elisa kits might offer improved test performance characteristics, they have undergone only limited clinical evaluation. we have previously evaluated the performance of lateral flow serological assays against pcr in moderate to severe sars-cov- infection. in the present study we aimed to explore the utility of these assays in a population of health-care workers with mild to moderate community-managed sars-cov- infection (all healthcare workers delivering direct clinical care to high-acuity sars-cov- -positive patients). we compared two lateral flow serological assays against laboratory-based elisa and pcr, where available, doing an interval analysis of test performance characteristics and their suitability for evidence before this study the evidence for the performance and limitations of various serological assays for severe acute respiratory syndrome coronavirus (sars-cov- ) is scarce. we searched pubmed for academic publications and online search engines for relevant grey literature on may , , and repeated the search on may , . a summary provided by wu and mcgoogan in february, , of characteristics and important lessons from china, highlighted the considerable risk to health-care workers of the potential for nosocomial infection. studies by hunter and colleagues and treiber and colleagues provide further insight, through pcr testing, into the burden of covid- in health-care workers in the uk. the capacity to test health-care workers during the early phase of the covid- pandemic in the uk was limited but can now be mitigated by delayed case identification through serology testing, providing insight into the overall burden of sars-cov- infection among health-care workers. to date, suitably sized studies have not yet been done in the uk. this prospective, multicentre cohort study demonstrates the comparative utility of sars-cov- serology tests, looking at both lateral flow assays and elisa approaches. our study also provides one of the first large-scale insights into seroprevalence data in a cohort of health-care workers with high covid- exposure in the uk. methodologically, demonstration of colorimetric band intensity on lateral flow assays and its correlation with optical density on elisa provides a degree of confidence in the interpretation of high-intensity bands but reinforces the limitations of interpretation of lateral flow assays (and risks identification of false positives) when the bands appear weak. additionally, our study is the first to demonstrate the substantial risks associated with delayed reading of lateral flow assays (in terms of both false positives and false negatives) and is informative when considering delayed reading (eg, postal testing) as part of a testing strategy if shared at this early stage in the planning process. variation in performance characteristics between assays highlights the urgent need for individual evaluation of the large number of commercial sars-cov- serology tests that have become rapidly available. practically, the observation of falsenegative serology results among health-care workers provides valuable information considering their messaging around interpretation of serology results at this early stage in the scale up of serological testing. false-negative results have a clear impact on the manner in which serological testing might be used to augment and support physical distancing policies, as well as implications for the development of large-scale testing pathways. further research is required into the full scope of serological testing for sars-cov- infection and factors associated with failing to mount a detectable immune response to sars-cov- infection in otherwise healthy individuals. this study also demonstrates the potential limitations of singletarget immunoassays for sars-cov- and should help inform future research studies, where further evaluation is required not just of alternative assays but also through the comparison of the various epitope targets that are currently available. delayed case identification. subsequent screening of the remaining symptomatic health-care workers was completed to assess seroprevalence. a prospective multicentre sars-cov- serological testing programme was implemented on april , , and data were collected until june , , across two hospitals in london, uk, comprising employees. health-care workers were eligible for serological testing if they had delivered direct clinical care to sars-cov- positive inpatients in cohort areas or isolation rooms involving aerosol-generating procedures; and had experienced mild to moderate symptoms matching the public health england (phe) case definition for sars-cov- infection, including fever or cough, or both, with breathlessness or anosmia, or both, with onset at least days before testing. specialist staff (redeployed from hiv and sexual health services) screened staff for clinical symptoms; collected demographic data (including age, sex, ethnicity, and job role); and carried out venepuncture for the serum sample facilitating inoculation of the point-ofcare lateral flow serological assay and matched laboratory elisa. the study was done in two phases. phase involved an evaluation of serology performance characteristics, comparing the matched lateral flow serological assays with elisa. phase comprised an estimate of seroprevalence in symptomatic and asymptomatic health-care workers through further lateral flow serological assay testing. an interval analysis was done once testing had occurred for at least % of the reported number of symptomatic health-care workers to human resources during the study period (reached on june , ). health-care workers were recruited to the asymptomatic cohort if they had satisfied inclusion criteria as above but had not experienced any phe-defined covid- symptoms since dec , . recruitment was consecutive. statistical analysis was done with ibm spss statistics (version ). a stard (standards for reporting of diagnostic accuracy studies) checklist is available in the appendix (pp - ). lateral flow serological assay testing was done with the onsite ctk biotech covid- split igg/igm rapid test (ctk biotech, poway, ca, usa) and the encode sars-cov- split igm/igg one step rapid test device (zhuhai encode medical engineering, zhuhai, china). lateral flow serological assays were completed as per the manufacturers' instruction leaflets. lateral flow serological assays were read at min by two clinical staff experienced in the use of point-of-care analysis (appendix p ). lateral flow serological assay readers, and subsequently elisa staff, were masked with respect to any previous pcr results. a visual scoring system for evaluating immunochromatography rapid diagnostic kits has previously been described for chikungunya virus. based on this system, lateral flow serological assays were recorded as positive or negative with readings visually scored as absent ( ), very weak positive ( ), weak positive ( ), medium positive ( ) or strong positive ( ) , and compared with elisa optical density readings. sds of values in each visual group were compared with overall results. the mean ranks were compared for significance by use of the kruskal-wallis test. dunn's post-hoc tests were then done for pairwise comparisons and results reported as p values, with a significance threshold of p< · . comparator elisa testing was done with the qualitative edi novel coronavirus covid- igg elisa kit (epitope diagnostics, san diego, ca, usa), targeting igg antibodies to the nucleocapsid protein of sars-cov- . post-marketing manufacturer information reported a sensitivity of · % and specificity of · %. elisa was completed as per the manufacturer instruction leaflet and results were recorded as an optical density reading. where equivocal results were found, confirmation testing was done with the abbott sars-cov- igg (antinucleocapsid) chemi luminescent microparticle immunoassay (abbott laboratories, lake bluff, il, usa) on the architect i sr immunoassay analyser and the final result included in the analysis. to derive a measure of sensitivity, the results of lateral flow serological assays and elisa were compared in health-care workers who had previously received pcr testing (ausdiagnostics, sydney, australia) at initial presentation with covid- symptoms. as per local hospital guidelines, pcr testing was done where possible between days to inclusive since onset of symptoms. a measure of specificity was derived through testing, with elisa and lateral flow serological assays, of historical negative serum samples ( samples from among patients with infectious or inflammatory presen tations from august, , and maternal antenatal screening samples from august, ). a further evalu ation was made on agree ment between lateral flow serological assay results and elisa through interpretation of lateral flow serological assay visual scores in relation to elisa optical density readings, particularly very weak and weak positive bands, to identify potential issues with variability in reading of lateral flow serological assays. lateral flow serological assay cassettes were observed at min postsampling, h post-sampling, and h post-sampling, and readings recorded. if a change was noted in the appearance of a band (negative to positive reading) or disappearance of a band (positive to negative reading) at h or h, a correlation was made against the elisa result to establish the risk of late readings (eg, if postal return methods are used in conjunction with self-testing). see online for appendix phase : determining sars-cov- seroprevalence in health-care workers phase of the study analysed elisa against both lateral flow serological assays. in phase , the remaining healthcare workers fulfilling the inclusion criteria were invited for serological testing with a lateral flow serological assay. to assess the significance of the sensitivity of the lateral flow serological assays (using an estimated sensitivity of %), we did a power calculation, setting α at · and β at · . we estimated symptomatic infection to be % among health-care workers. we calculated that the study would require pcr-positive health-care workers in phase . in order to achieve this sample size, based on self-reporting of pcr-positive cases to human resources, we estimated that testing coverage in phase would need to capture at least % of all reported symptomatic health-care workers. additionally, following an update in government advice in mid-may, , to offer testing to asymptomatic health-care workers, we tested a subset of asymptomatic health-care workers who were offered serology testing in order to more accurately estimate sars-cov- seroprevalence in the entire workforce. positive and negative predictive values based on test performance characteristics were then calculated if considered for use in three cohorts: in the uk general . historical serum samples were evaluated to assess specificity (tables , ). equivocal results were retested with the abbott sars-cov- igg (anti-nucleocapsid) chemi luminescent microparticle immuno assay (abbott laboratories, lake bluff, il, usa). phase : further analysis of lfa testing offered to all symptomatic health-care workers, including initial lfas tested in phase (n= ) and phase (n= ). further analysis of asymptomatic health-care workers with lfa testing (n= ). lfa=lateral flow serological assay. phe=public health england. population (based on estimates of uk prevalence during our cohort's period of majority reported symptom onset of · %); in the asymptomatic health-care worker population if general screening was to be applied, with a derived prevalence from a subset of asymptomatic health-care workers; and in health-care workers reporting phe-defined symptoms during the study period, with a derived prevalence from serological testing of symptomatic health-care workers. there was no funding source for this study. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. in phase we compared matched two-way lateral flow serological assays against elisa for serum samples from health-care workers with an additional historical pre-covid- -negative control samples (figure samples from all health-care workers were run against both lateral flow serological assays and laboratory immunoassays. (table ) . all pcrpositive cases reported as igg positive on elisa were also positive on the encode lateral flow serological assay. five ( %) of samples that were negative on laboratory elisa were also negative on both lateral flow serological assays. health-care workers had a negative pcr recorded. of these, ( · %) of were negative on appendix p ). kruskal-wallis testing of visual scores for encode and onsite lateral flow serological assays against optical density showed a significant difference between groups (p< · ). sds were calculated for each visual score and dunn's pairwise tests were done between paired groups (figure ). all lateral flow serological assay cassettes were read at min, h, and h after sampling. at h no change was noted from the initial readings. at h a change was noted in ( · %) of encode lateral flow serological assay tests. of tests changed from negative to positive, and six of changed from positive to negative. elisa results were concordant with initial encode lateral flow serological assay readings at min. ( · %) of onsite lateral flow serological assays also demonstrated a change in readings at h, with of cassettes changing from negative to positive (elisa confirmed seven as positive and eight as negative) and of becoming unreadable. in phase , health-care workers working in highacuity areas were tested (including phase participants) with a lateral flow serological assay (mean age · years (table ) . of the symptomatic health-care workers, ( · %) reported fever, reported cough ( · %) and ( · %) reported anosmia. χ² testing for independent association of symptoms with seropositivity was significant for fever (χ² · , p= · ) and anosmia (χ² · , p< · ). a significant association was not found for cough (χ² · , p= · seroprevalence was considered in three separate cohorts as described. in the uk general population ( · %), for onsite the positive predictive value was · % ( % ci · - · ) and negative predictive value was · % ( · - · ); and for encode the positive predictive value was · % ( · - · ) and negative predictive value · % ( · - · ). in the asymptomatic health-care worker cohort ( · %), for onsite the positive predictive value was · % ( % ci · - · ) and negative predictive value was · % ( · - · ); and for encode the positive predictive value was · % ( · - · ) and negative predictive value was · % ( · - · ). in health-care workers reporting symptoms ( · % of symptomatic health-care workers), for onsite the positive predictive value was · % ( % ci · - · ) and negative predictive value was · % ( · - · ); and for encode the positive predictive value was · % ( · - · ) and negative predictive value was · % ( · - · ). in this study we report good positive and negative predictive values for delayed serological testing in a carefully selected population with suspected sars-cov- infection. these findings have the potential to support case identification strategies, depending on the device used. testing could be particularly useful in improving our understanding of the true prevalence of community managed, mild to moderate infection among high-risk health-care workers while informing the analysis of infection prevention and control strategies for any future waves of the ongoing covid- pandemic. testing in this high-exposure cohort based in london, uk, has identified several key lessons that could substantially affect future plans for wider serological testing among health-care workers. although a wide range of rapid diagnostic point-of-care serological assays have emerged in a short period of time, individual assay analysis has been challenging in individuals with mild to moderate infection as numbers of so-called true-positive pcr-tested individuals available for comparison are limited. concerns about variability in sensitivity between lateral flow serological assays are borne out in our data, when these assays were compared against laboratory elisas. we demonstrate good agreement for detection of igg with the encode lateral flow serological assay when our pre-test criteria are applied ( · %); however, there was reduced agreement with the onsite lateral flow serological assay ( · %). this result would therefore limit the utility of the onsite assay even within the high-risk health-care worker population, where a much higher pre-test probability exists than in the general population. among the pcrpositive subgroup, results were concordant between individual assays, with the encode lateral flow serological assay identifying %, and the onsite %, of healthcare workers who were also positive on elisa. the failure of all three assays to identify pcr-positive health-care workers with phe-defined symptoms highlights the limitations of relying on any single diagnostic platform, or perhaps identifies a cohort of individuals who did not mount a timely serological response. where reduced sensitivity among serological assays in the early infection period has been previously described, , reference photographs selected from mean optical density value cassette for each score. sds of encode lateral flow serological assay values: negative ( · ), very weak ( · ), weak ( · ), medium ( · ), and strong ( · ) visual scores. dunn's pairwise tests showed no significant difference between visual scores, comparing very weak to medium (p= · ), medium to strong (p= · ), very weak to weak (p= · ), and weak to medium (p= · ) visual scores. all other pairwise comparisons had a significant relationship with optical density (p< · ). sds of onsite lateral flow serological assay values: negative ( · ), very weak ( · ), weak ( · ), medium ( · ), and strong ( · ) visual scores. dunn's pairwise tests showed no significant difference between visual scores comparing very weak to weak (p= · ), weak to medium (p= · ), and medium to strong (p= · ) visual scores. all other pairwise comparisons had a significant relationship with optical density (p< · ). it is clear that any use of such assays in supporting early return-to-work policies could carry considerable risk. such policies or recommendations should therefore be actively discouraged. additionally, the proportion of individuals who are seronegative on both elisa and lateral flow serological assays, despite being pcrpositive, is of concern. any use of serological testing should be accompanied with a reinforcement of ppe and infection, prevention and control advice, regardless of result, and therefore testing should be undertaken face to face in a clinical setting. immunity inferred by the presence of antibodies is yet to be determined and clinical counselling of result interpretation at this stage as purely case identification is an important part of the testing process, preventing misunderstanding and incorrect behaviour modification (eg, relaxing physical distancing rules or reducing ppe). this assessment could be done at established uk national health service (nhs) facilities, as we have done here, or potentially done via mobile health units as demonstrated by early identification of hiv infection through point-of-care cd testing in south africa. we found visual scoring of immunochromatography lateral flow serological assay bands to correlate with elisa optical density. more prominent bands were seen in positive cases and the weakest bands were seen in those initially reported as equivocal. we did, however, find laboratory elisa-negative samples with very weak lateral flow serological assay bands, casting doubt on the ability to interpret these results. the risk of reporting false positives is likely to be increased further by non-expert reading, such as with home testing seroprevalence studies. where bands have a strong or medium immuno chromatography reading, however, elisa was positive in almost all ( [ %] of ) cases for the encode lateral flow serological assay, reflecting greater reliability. in com parison, seven of onsite cassettes with medium to strong bands or strong bands were elisa negative. across both lateral flow serological assays, the finding of igm-only bands might represent greater capability among the lateral flow serological assays to identify positive cases, or false-positive or cross-reactive igm results. among the historical negative samples, reactivation of epstein-barr virus might have accounted for a false-positive result. although manufacturers have reported no observed cross-reactivity with seasonal coronaviruses, the sample sizes in the present study were not large enough and further evaluation is required in a larger cohort of health-care workers. an apparent failure to seroconvert to igg among those with confirmed sars-cov- infection has been documented in one study and this effect might account for some of the igm-only cases. although we observed igg-positive results in pcr-positive individuals as late as months after symptom onset, the limited pcr positive rate among health-care workers makes analysis of test performance as a function of time difficult to assess in this study. further analyses with alternative laboratory elisa platforms, including those with igm targets as well as igg spikeprotein receptor-binding-domain targets (in addition to the nucleocapsid protein targets), are urgently required to determine the reasons for these discordant findings. longitudinal serial testing will also be informative. when considering the utility of lateral flow serological assays, one of the key benefits is the availability of realtime results at the point of testing. where we investigated delayed reading of lateral flow serological assay cassettes, a considerable number changed at h, becoming either entirely unreadable or leading to the reporting of a different result. this effect has previously been reported with a high incidence of true non-reactive results changing to weak positive when undergoing delayed reading of rapid antibody tests for hiv. we therefore strongly advise against any use of lateral flow serological assays in home testing programmes that involve nonexpert reading of cassettes or return postage of samples for evaluation at extended time intervals. early work in pcr testing of health-care workers in the uk, through testing of symptomatic individuals but also through serial screening of asymptomatic individuals, suggests that infection rates in this cohort might reflect wider community transmission rather than primarily nosocomial infection. , this observation is in keeping with first reports of large-scale testing of healthcare workers in china. where pcr testing is not as widely available, serological testing offers an additional avenue for estimating infection rate among health-care workers. where fewer than half of those working in the highest risk areas and presenting with phe-defined symptoms had reactive results, estimation of total rates was similar to previously reported rates. however, we found some discordance in our asymptomatic cohort when compared to previously reported data. serological screening done among health-care workers in essen, germany, found a seropositive rate of · %, of which the majority of individuals did not report phe-defined symptoms; yet in our study we reported a seroprevalence of · %. recent data from a study in oxford, uk, however are supportive of our findings, where ( · %) of igg-positive results were reported in a large asymptomatic health-care worker cohort. although all three studies were done during the same time period, the incidence rate and population density in the uk were considerably higher and such marked variation in seroconversion reflects the impact of geographical variation in exposure intensity and reinforces the need for granular serological data. in stark contrast to seroprevalence among individuals with phedefined symptoms ( · %), the findings from our asymptomatic cohort are perhaps more in keeping with the observed peak among the general population in london ( · %), with a slight increase reflecting the increased exposure faced by health-care workers. there is growing evidence, however, that those with mild disease are less likely to produce a detectable antibody response than those with more severe disease, and specifically those with lower respiratory tract involvement. a relative difference in exposure might also partly explain the observed variability in seroprevalence, with minimal doses of virus perhaps insufficient to induce an adaptive immune response, but instead dealt with by the innate immune system. in this context, until the differential antibody response and its neutralising capacity in sars-cov- are better understood, generalised testing of asymptomatic healthcare workers, where positive predictive value is substantially reduced, should be done only with considerable caution alongside detailed advice about the current limitations for interpretation. when pre-test criteria are applied ( days after onset of phe-defined symptoms), the positive predictive value is substantially increased. further investigations among this cohort with antireceptor binding domain assays might provide additional information to the anti-nucleocapsid assay used here, where reduced sensitivity is a clear issue. although pcr is recognised as the gold standard in the diagnosis of acute sars-cov- infection, our study was limited to a subgroup analysis of pcr-positive health-care workers because of the reduced availability of pcr testing during the early phase of the pandemic. as pcr testing capacity was increased, we conversely noted a substantial decline in health-care workers presenting with acute phase symptoms. where pcr data were available, reduced sensitivity for all three assays was observed compared to information provided by the manufacturer. this finding might reflect limitations in the use of these assays within individuals with mild to moderate infection, with many assay development characteristics reported in testing of inpatients with severe disease, and supports the requirement of individual assay evaluation within intended populations before widespread use. that our data demonstrate general agreement between lateral flow serological assays and pcr, however, is encouraging, but the reduced agreement among lateral flow serological assays in general is concerning. this potential discrepancy could be explained by the utilisation of separate assay targets (nucleocapsid or spike protein) in lateral flow serological assays. we were unable to discern further target information from both manufacturers. where clear information remains unavailable for assay targets, as with many currently available commercial lateral flow serological assays, greater understanding of any neutralising effect of different antibodies will be even more important. although our study was done at multiple sites, its capacity to reflect esti mated prevalence in secondary care facilities in london alone and elsewhere was probably limited; appropriately powered studies are now required in locations with a lower estimated prevalence to understand the likely prevalence among health-care workers at a national level. this study is further limited by the use of an igg-target laboratory elisa to the nucleocapsid protein only. additional analysis with a laboratory elisa capable of detecting the spike protein, and in particular the receptor binding domain, would allow for further evaluation of the reliability of the observed results with lateral flow serological assays. in conclusion, serological testing, whether with laboratory-based elisa platforms or lateral flow serological assays designed for use at the point of care, offer the potential for late case identification among health-care workers with mild to moderate sars-cov- infection. we found variability in the performance of different lateral flow serological assays when compared against an igg-target laboratory elisa. building on our previously reported work of moderate to severe sars-cov- infection among inpatients with pcrconfirmed sars-cov- infection, we found suitable performance characteristics from the encode lateral flow serological assay device (and slightly less so with onsite) but only if utilised in patients with phe-defined symptoms at least days following symptom onset. although lateral flow serological assays might provide a clear mechanism to identify those health-care workers who have contracted sars-cov- infection, it is crucial to note that cassettes provide incorrect results if delayed reading is done and this must be a key consideration for any future plans for large-scale testing with lateral flow serological assays. situation report - covid- : infection prevention and control guidance covid- : pcr screening of asymptomatic health-care workers at london hospital the detection of sars-cov- using the cepheid xpert xpress sars-cov- and roche cobas sars-cov- assays early detection of sars-cov- antibodies in covid- patients as a serologic marker of infection point-of-care serological assays for sars-cov- in a uk hospital population: potential for enhanced case finding covid- : investigation and initial clinical management of possible cases evaluation of an immunochromatography rapid diagnosis kit for detection of chikungunya virus antigen in india, a dengue-endemic country edi novel coronavirus covid- igg elisa kit sars-cov- igg instructions for use. h r an introduction to power and sample size estimation government to offer antibody tests to health and social care staff and patients in england report : estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european countries. imperial college london test performance evaluation of sars-cov- serological assays rapid point-ofcare cd testing at mobile units and linkage to hiv care: an evaluation of community-based mobile hiv testing services in south africa symptoms and immunoglobulin development in hospital staff exposed to a sars-cov- outbreak re-reading of oraquick hiv- / rapid antibody test results: quality assurance implications for hiv self-testing programmes first experience of covid- screening of health-care workers in england characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention sars-cov- -specific antibody detection in healthcare workers in germany with direct contact to covid- patients differential occupational risks to healthcare workers from sars-cov- : a prospective observational study sars-cov- shedding and seroconversion among passesngers quarantined after disembarking a cruise ship: a case series immunity and immunopathology to viruses: what decides the outcome? sjcp, gwd, and lspm designed the study methodology. sjcp, ap, samf-s, mr, cs, rj, and gwd collected samples and carried out lateral flow serological assay testing at satellite sites. alm, kgg, and jh carried out elisa testing. pr carried out molecular diagnostics and elisa testing of equivocal results. sjcp, mr, ap, sjd, and ec analysed the data. all authors reviewed the results and contributed to data analysis, and provided comments. sjcp and lspm drafted the initial manuscript with all authors contributing substantially to revisions before submission. all authors agreed on the final version for submission. ( ). rj has received honoraria, speaker fees, and travel support or research grant funding, or both, from gilead, viiv healthcare, bristol myers squibb, abbvie, janssen, and merck. sjcp has received a research grant from the scientific exploration society. ec has received speaker fees from biomerieux ( ). all other authors declare no competing interests. the data analysed during the current study and further details about the assays are available from the corresponding author on reasonable request, as long as this meets local ethical and research governance criteria. key: cord- - gfk m authors: stadlbauer, daniel; amanat, fatima; chromikova, veronika; jiang, kaijun; strohmeier, shirin; arunkumar, guha asthagiri; tan, jessica; bhavsar, disha; capuano, christina; kirkpatrick, ericka; meade, philip; brito, ruhi nichalle; teo, catherine; mcmahon, meagan; simon, viviana; krammer, florian title: sars‐cov‐ seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup date: - - journal: curr protoc microbiol doi: . /cpmc. sha: doc_id: cord_uid: gfk m in late , cases of atypical pneumonia were detected in china. the etiological agent was quickly identified as a betacoronavirus (named sars‐cov‐ ), which has since caused a pandemic. several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re‐infection. here we describe a detailed protocol for expression of antigens derived from the spike protein of sars‐cov‐ that can serve as a substrate for immunological assays, as well as a two‐stage serological enzyme‐linked immunosorbent assay (elisa). these assays can be used for research studies and for testing in clinical laboratories. © the authors. current protocols in microbiology published by wiley periodicals llc. basic protocol : mammalian cell transfection and protein purification basic protocol : a two‐stage elisa for high‐throughput screening of human serum samples for antibodies binding to the spike protein of sars‐cov‐ severe acute respiratory syndrome coronavirus (sars-cov- ), the virus that causes coronavirus disease (covid ; often written , emerged in late in wuhan, china (wu et al., ; zhu et al., ) . rapid, global spread of the virus is presently causing a pandemic. currently, no drugs or antivirals are available and countermeasures are limited to non-pharmaceutical interventions (npis). nucleic acid−based tests for detection of the virus during acute disease are in use worldwide corman et al., ) . however, the development of serological assays is lagging due to lack of suitable reagents. serological assays are needed to perform serosurveys aimed at determining the real infection rate and infection fatality rate in a given population. furthermore, they are useful to characterize the immune response to the virus in a detailed qualitative and quantitative manner. serological assays are also of immediate practical use. they can be used to identify individuals who were infected (including severe, mild, and asymptomatic cases) and who are now potentially immune. a recent study in non-human primates showed that re-infection, at least in the small number of animals used in the study, does not occur (bao et al., ) once antibody responses have been mounted. infection with coronaviruses circulating in human populations, such as hku, nl , etc., also leads to immunity that protects from re-infection for months to years (callow, parry, sergeant, & tyrrell, ) . therefore, individuals who have mounted an immune response to sars-cov- are likely immune, which means that they are unlikely to transmit the virus to others. as an example, healthcare workers who are immune could potentially care for covid patients with minimal risk to themselves, their colleagues, and other patients. in addition, the use of convalescent plasma may serve as a valuable treatment option for patients with severe covid , especially in the absence of other options. a serological assay is critical for identifying potential plasma donors. the surface glycoprotein of the virus, termed the spike (s) protein, mediates attachment of the virus to human cells via its receptor-binding domain (rbd; wrapp et al., ) and mediates fusion of viral and cellular membranes. antibodies binding to the spike protein, and especially to the rbd domain, can neutralize sars-cov- . therefore, we used different recombinant spike protein preparations as the antigens for our elisa. we reported in our earlier work that individuals not exposed to sars-cov- are completely naïve to the spike protein, and their serum samples show little or no reactivity in an elisa (amanat et al., ) . it is, therefore, easy to distinguish between exposed/immune and naïve individuals. community in the future. not every aspect of these protocols has been optimized in detail, and we provide notes and comments whenever further optimizations and testing are recommended. mammalian expression plasmids for the generation of the recombinant proteins are available from the corresponding author and from bei resources. this protocol can be used for both expression vectors: the one expressing secreted rbd as well as the one expressing a soluble, trimeric version of the sars-cov- spike protein. expression levels of the rbd are very high in our hands (> mg/l culture), while expression levels for the full-length spike are lower (approximately to mg/l). therefore, we use the recombinant rbd for initial screening elisas and the full-length spike for confirmatory elisas (as described in basic protocol ). the expression vector constructs were described previously (amanat et al., ) . in brief, the sequences used for both proteins are based on the genomic sequence of the first isolate, wuhan-hu- , which was released on january , (genbank: mn . ). sequences were codon-optimized for mammalian cell expression. the full-length spike protein sequence was modified to remove the polybasic cleavage site, which is recognized by furin, and to add a pair of stabilizing mutations (figure ). these two modifications were included to enhance the stability of the protein based on published literature (amanat et al., ) . the plasmids are grown in e. coli at °c (or °c) at rpm in luria-bertani (lb) broth with ampicillin (lb-amp) in shaker flasks overnight. high-quality plasmid dna can be obtained using commercially available maxiprep kits (ideally with an endotoxin-removal step). importantly, other cell lines ( t, cho, etc.), other media, transfection reagents, and more sophisticated protein purification methods might be used as alternatives if available. rbd = receptor-binding domain of sars-cov- (nr- ) pbs = phosphate-buffered saline rt = room temperature ( °to °c) mem = minimum essential medium ) and seeded at a density of , cells/ml in expi expression medium. the viability of the cells must be greater than % at all times. . cells are passaged every to days and incubated in an orbital shaking incubator at °c with shaking at rpm and % co . see current protocols article phelan and may ( ) for basic cell culture techniques. a maximum cell density of − × cells/ml is recommended, at which point cells should be immediately passaged. . × cells are suspended in ml ( × cell/ml) of expi expression medium in a -l erlenmeyer flask. transfections are performed according to manufacturer's instructions. . ml of opti-mem is added to two -ml sterile polypropylene conical tubes: one tube receives μg ( μg/μl final dilution in the total volume of culture) of respective plasmid dna (for rbd or full-length spike), while the other tube receives μl of expifectamine transfection reagent from the kit. . the contents of both -ml tubes are mixed together and incubated at rt for min to prepare the transfection mixture, after which the transfection mixture is added dropwise to the cells slowly, over a course of a few seconds using a micropipette while swirling the cells in a circular pattern. . cells are then returned to the shaking incubator. . at hr post-transfection, . ml of expifectamine transfection enhancer and . ml of expifectamine transfection enhancer from the kit are added to the culture, and subsequently the culture is returned to the shaking incubator. . at days post-transfection, the cells are harvested and centrifuged min at × g, °c. . the supernatant is filtered using a . -μm stericup filter; the cell pellet can be discarded. alternatively, cells can be spun down at × g for min, supernatant can be collected, and the same cells can be resuspended in ml of fresh expi expression medium and returned to the shaking incubator for another days. this makes it possible to collect more protein in the fresh supernatant (the cells continue to express the protein) and can be used to increase protein yield. the protein integrity needs to be verified in the same way as for the initial protein harvest. this alternate strategy works well with the rbd, but is less suitable for the full-length spike (we have detected protein degradation in that case). . continue to process the supernatant and purify protein immediately. alternatively, if the supernatant is stored, it must be kept at °c (and for no longer than overnight/ hr) in order to prevent denaturation of the protein at room temperature. these steps can be replaced by more advanced purification methodologies, for example, if an Äkta purifier is available. the methods described below work even in labs not geared toward protein purification. see current protocols article petty ( ) for additional detail on metal-chelate affinity chromatography. . prior to use, ni-nta resin ( ml per ml culture) is washed once with fresh pbs (transfer resin into a -ml tube and fill up with pbs), then spun min at × g, °c. . once the centrifugation is complete, the pbs is discarded and resin is resuspended with the cell culture supernatant and inverted two or three times. . the resin is then incubated with the supernatant for hr on a shaker ( rpm) at rt. . two clean -ml polypropylene columns are loaded with the supernatant-resin mixture and then washed with one column volume of wash buffer four times. . columns are then eluted using the elution buffer. . four fractions are collected from each column by incubating the resin in the column with ml of elution buffer for each fraction. incubate resin with elution buffer for min after each addition of elution buffer. . eluate is collected directly in a -ml polypropylene conical tube placed on ice. the total volume of eluate should be ml from the two columns. more columns can be used to speed up the purification time, depending on the volume of the culture. . eluate is spun through -kda amicon ultra centrifugal filter units (for rbd) or -kda amicon ultra centrifugal filter units (for full-length spike) at × g for min (or longer if eluate takes longer to pass through the membrane) at °c or until only to μl remain in the unit. amicon filter units should be equilibrated with pbs before use. . pbs is added twice to the amicon ultra centrifugal filter unit from step and the unit is spun at × g for min at °c or until only to μl remain in the unit. stadlbauer et al. current protocols in microbiology . finally, the protein is collected from the amicon ultra centrifugal filter unit, its concentration is measured (e.g., using the bradford protein assay or similar methods; see current protocols article; lovrien & matulis, ) , and a denaturing sds-page ( % to % gradient; see current protocols article: manns, ) is run to check the integrity of the purified protein. . after the elution step, protein should always be kept on ice or stored at °c. for storage longer than hr, protein should be frozen at − °c to avoid degradation. a concentration of mg/ml and an aliquot size of to μl is recommended. the purpose of this protocol is to describe the procedure for measuring human antibody responses to the recombinant receptor-binding domain (rbd) of the spike protein or fulllength spike protein of sars-cov- and to ensure the reproducibility and consistency of the obtained results. we developed this as a two-stage elisa in which the first stage ('a' steps below) includes relatively high-throughput screening of samples in a single serum dilution against the rbd (which expresses very well and therefore can be produced in greater quantities). this is followed by a second stage ('b' steps below) in which positive samples from the first stage undergo a confirmatory elisa against the full-length spike protein (which is harder to express; therefore there is usually less available). for the second stage, a dilution curve is performed. typically, if only one operator is available, screening elisas can be run in the morning ( samples/ plates per run) and confirmatory elisas can be run in the afternoon ( samples/ plates per run). of note, we describe the assay here as it is set up in our laboratory. we use a plate washer and a plate reader, but no automated system. the protocol can be adapted to use with an automated liquid handler. in addition, one of the difficulties in setting up the assay is the availability of appropriate negative and positive controls. negative controls are easier to come by, and can be serum pools taken before . positive controls can be convalescent samples from covid patients or monoclonal antibodies (mabs) like cr (ter meulen et al., ; tian et al., ) . if no human sera or mabs are available, mouse mabs, mouse sera against sars-cov- , other animal sera against sars-cov- , or anti−his tag antibodies (the proteins are histagged) can be used. however, in this case, a different secondary antibody for the species from which the primary antibody is derived is needed for the positive control. also, we recommend generating large batches of positive controls, which can be used for many runs. the positive control should be selected to result in a strong signal (recommend od of about . ), and should be clearly distinguishable from the negative controls. elisas can be run with either serum or plasma. caution: before starting to work with covid samples, please consult with your local biosafety officer regarding which precautions, personal protective equipment and protective measures are required. definitions elisa = enzyme-linked immunosorbent assay pbs = phosphate-buffered saline current protocols in microbiology rbd screening elisa a. coating elisa plates (day ). i. thaw the required number of vials of antigen (sars-cov- rbd protein) to coat -well microtiter elisa plates at a concentration of μg/ml. once thawed, mix by gently vortexing vials before diluting in × pbs. prepare approximately ml for each plate to be coated. ii. coat plates with μl of diluted protein per well using a multichannel pipettor and a reservoir. lightly tap plates against surface to ensure protein is evenly coating the bottom of every well. iii. incubate at °c overnight. always keep a plate cover on top of coated plates during all steps of the protocol! plates can likely be stored at °c for up to week, but this needs to be validated locally to ascertain that it does not change the results. a. heat inactivation of samples (day , this is a general safety precaution for work with human serum). caution: we have not tested if this procedure inactivates sars-cov- ; please consult with your local biosafety officer to discuss proper safety precautions. i. set the water bath to °c. once temperature is reached, place the serum/plasma samples in the water bath and immediately start the timer for hr. ii. remove samples when the timer goes off. do not leave samples at °c for longer than hr. ii. using an automated plate washer, wash coated elisa plates three times with pbs-t. iii. add μl blocking solution to all wells of the plates, starting the timer for hr (do not exceed hr) after completing the first plate. place plates in a °c (rt) incubator until step a. a. pre-diluting samples (day ). i. in a biological safety cabinet, set up sterile . -ml microcentrifuge tubes to predilute serum samples at a : ratio. ii. add μl of sterile × pbs to all tubes. iii. gently vortex each serum sample to mix and add μl to the microcentrifuge tubes, vortexing once more. do this for all remaining samples including the positive and negative controls. the volume of serum not needed in these 'a'steps will be stored and used for the 'b'steps, below. a. setting up dilution plates (day ). i. calculate and prepare at least ml of pbs-t + % (w/v) milk powder. ii. prepare one dilution plate (separate flat-bottomed cell culture plate) per antigencoated plate prepared. iii. add μl of pbs-t containing % milk to all wells of the dilution plate (including blank wells). iv. leaving columns and as blanks, add μl of pre-diluted sample (or control) to the designated wells. this results in a final serum dilution of : . v. continue until all samples and controls have been added to the designated wells. see reference plate layout in figure . a. transferring serum dilution (day ). i. after the blocking incubation in step a, substep iii, remove elisa plates from the rt incubator and throw off the blocking solution. tap the plates dry on a kimwipe or other absorbent material. ii. using a multichannel pipettor, pipette up and down four to six times in the wells of the first row of the dilution plate to mix. iii. transfer μl from each well of the first row of the dilution plate to the corresponding wells in the elisa plate. change tips and continue to transfer the second row of the dilution plate to the elisa plate in the same manner. iv. start the timer for hr as soon as the contents of all of the rows have been transferred to the first elisa plate. v. place plates in a °c (rt) incubator. do not exceed hr of incubation at rt before proceeding to step a. a. incubating with secondary antibody (day ). i. after hr incubation at rt, wash the elisa plates from step a three times with pbs-t using an automated plate washer. ii. dilute anti-human igg (fab-specific) hrp-labeled secondary antibody : in pbs-t containing % milk. prepare at least ml per plate. iii. add μl of the secondary antibody solution to all wells of the plate using a multichannel pipettor. be sure to avoid touching the walls of the wells with the pipette tips, to avoid carry-over and high background signals. stadlbauer et al. current protocols in microbiology iv. start the timer for hr (stay in a range of to min) as soon as the secondary antibody has been added to the first plate. v. place plates in a °c (rt) incubator. a. developing and reading plates (day ). i. after hr, wash plates three times with pbs-t using an automated plate washer. ii. prepare sigmafast opd solution and calculate amount needed. one set of tablets ( gold + silver tablet) dissolved in ml water for injection (wfi) can be used for two -well plates. iii. fully dissolve one gold tablet in ml wfi. do not add silver tablet to solution until ready to start adding to the plates. the opd solution needs to be prepared immediately before use. iv. add μl opd solution to all wells of the plate. begin the timer for min as soon as opd has been added to the first row of the first plate. do not exceed min of developing before stopping the reaction. v. to stop the reaction after exactly min, add μl of m hcl to all wells. ideally, read plates immediately after adding hcl. vi. read elisa plates in a plate reader at an absorbance of nm (immediately after adding hcl) and record data. samples that exceed a certain od cutoff value (proposed cutoff: od = . to . , or mean of negative controls plus times the standard deviation of the negative controls) are assigned as presumptive positives and will be tested in the confirmatory elisa using full-length spike protein ('b' steps, below) . the od cutoff has to be experimentally determined and depends on assay background and noise. spike confirmatory elisa b. coating elisa plates (day ). i. thaw the required number of vials of antigen (sars-cov- spike protein) to coat -well microtiter elisa plates at a concentration of μg/ml. once thawed, mix by gently vortexing vials before diluting in × pbs. prepare approximately ml for each plate to be coated. ii. coat plates with μl of diluted protein per well using a multichannel pipettor and a reservoir. lightly tap plates against surface to ensure protein is evenly coating the bottom of every well. iii. incubate at °c overnight. always keep a plate cover on top of coated plates during all steps of the protocol! plates can likely be stored in °c for up to week but this needs to be validated locally to ascertain that it does not change the results. b. blocking elisa plates (day ). i. calculate to prepare at least ml of blocking solution per plate. ii. using an automated plate washer, wash coated elisa plates three times with pbs-t. iii. add μl blocking solution to all wells of the plates, starting the timer for hr (do not exceed hr) after completing the first plate. iv. place plates in a °c (rt) incubator until step b. b. pre-diluting samples (day ). retrieve : pre-diluted samples from step a, above, to be tested and confirmed (samples that are above certain threshold in rbd screening elisa based on a set od value; see annotations after step a, substep vi). confirmatory spike elisa reference plate layout. the sample layout on the elisa plate is shown, including the serial dilution steps that need to be performed. wells designated for positive (+) and negative (−) controls are indicated. b. performing serial dilutions (day ). i. calculate and prepare at least ml of pbs-t + % (w/v) milk powder per plate. ii. after the blocking incubation in step b, substep iii, remove plates from the rt incubator and throw off the blocking solution. tap the plates dry on a kimwipe or other absorbent material. iii. using a multichannel pipettor, add μl of pbs-t containing % milk to all wells of each plate. iv. leaving columns and as blanks, add an extra μl of pbs-t containing % milk to wells only in columns and . wells of column and will be the sample wells. v. add μl of : pre-diluted sample (final dilution : on the plate) to the first well in column and continue to add samples to all wells. vi. in column , add samples to wells a through f. vii. transfer positive and negative controls into wells g and h respectively. see reference plate layout in figure . viii. with the multichannel pipettor, pipette up and down four to six times in column to mix. discard these tips. with new tips, transfer μl ( -fold dilution) from column to column , and pipette up and down four to six times to mix. repeat this until column ; discard μl before column . ix. taking fresh tips, mix column by pipetting up and down four to six times. repeat the same process of transferring, mixing, and discarding tips from columns to . once column is reached, discard μl. x. start the timer for hr once the first elisa plate has been serially diluted. do not exceed hr of incubation at rt before proceeding to step b. xi. place plates in a °c (rt) incubator. current protocols in microbiology b. incubating with secondary antibody (day ). i. after hr of incubation at rt, wash the plates from step b with pbs-t using the automated plate washer. ii. dilute anti−human igg (fab-specific) hrp-labeled secondary antibody : in pbs-t containing % milk. prepare at least ml per plate. iii. add μl of the secondary antibody solution to all wells of the plate using a multichannel pipettor. be sure to avoid touching the tips of the pipette to the walls of the well. iv. start the timer for hr (stay in a range of to min) as soon as the secondary antibody has been added to the first plate. v. place plates in a °c (rt) incubator. b. developing and reading plates (day ). i. after hr, wash plates three times with pbs-t using an automated plate washer. ii. prepare sigmafast opd solution and calculate amount needed. one set of tablets ( gold + silver tablet) dissolved in ml water for injection (wfi) can be used for two plates. iii. fully dissolve one gold tablet in ml wfi. do not add silver tablet to solution until ready to start adding to the plates. the opd solution needs to be prepared immediately before use. iv. add μl to all wells of the plate. begin timer for min as soon as opd has been added to the first row of the first plate. do not exceed min of developing before stopping the reaction. v. to stop the reaction after exactly min, add μl of m hcl to all wells. vi. read elisa plates in plate reader at an absorbance of nm and record data. true positive samples will have a signal higher than the negative control plus standard deviations of the negative controls in at least two consecutive dilutions. . g nah po .· h o . g nacl . g imidazole (sigma-aldrich # i or equivalent; final concentration is mm) l distilled water store at room temperature up to months use distilled water filtered using a . -μm stericup vacuum filtration system. l distilled water l × pbs (corning tm # cm or equivalent)) ml tween (fisher bioreagents #bp - or equivalent) store at room temperature for to months wash buffer ( l) . g nah po · h o . g nacl . g imidazole (sigma-aldrich # i or equivalent; final concentration is mm) store at room temperature up to months use distilled water filtered using a . -μm stericup vacuum filtration system. the protein expression and purification methods (basic protocol ) described in this article are based on well-established techniques. the expression plasmids and protein sequences have been optimized to increase protein stability and yield (amanat et al., ) . plasmids can be requested from the krammer laboratory or can be found on bei resources. the elisa protocol (basic protocol ) has been designed to allow for highthroughput screening of many samples per day, followed by a confirmatory step to verify presumptive positive results. the elisa assay itself is based on well-established protocols and has been optimized for the use of sars-cov- antigens. the most common problem for the transfection (basic protocol ) is low cell viability before performing the transfection. the cells need to be % to % viable. the absence of antibiotics/antifungals requires good sterile technique to prevent contamination. sterile plasmid preparations are also recommended, and it is important to add the enhancer to the shaking flasks hr post-transfection. for the protein purification, we recommend always using fresh ni-nta resin to prevent cross-contamination with other proteins. harvested supernatant should be ideally processed immediately to ensure protein integrity. to make filtering of the supernatant easier, an additional centrifugation step (after pelleting the cells) is recommended to pellet residual cells and other particles. when performing buffer exchange using amicon ultra centrifugal filter units, make sure to use the right-size cutoff (use smaller cut-off for rbd). it is recommended that purified protein be diluted to a concentration of about mg/ml. storage at higher concentrations may result in aggregation of protein. for the elisa (basic protocol ), performing all of the washing steps and adhering to the incubation times are important to achieve low background reactivity. most critical are the incubation times for the secondary antibody and the substrate (opd and hcl for stopping the reaction). in addition, touching wells with tips when transferring secondary antibody and substrate can result in higher background and possibly false positive wells, and needs to be avoided. in preparing the opd, it is also important to dissolve the gold tablet fully and only add the silver tablet right before the substrate is added to the elisa plate. we expect expression levels of the rbd to be above mg per l of culture cells and expression of the full-length spike protein to be approximately mg per l of fs, using a gravity-flow protein-purification strategy. when running the sds-page to confirm protein integrity, clear single bands are expected for the rbd and full-length spike at around to kda and ∼ kda, respectively. additionally, elisas with positive and negative controls (e.g., monoclonal antibodies) are performed to confirm correct protein folding. we expect a good binding profile for the positive control and low-to-no background reactivity for the negative control. basic protocols and can be completed in about days. basic protocol takes about days. growing up a cryostock of f cells, bringing them to passage (recommended before transfection), and obtaining a sufficient cell number would take another few days; this is not taken into account in the protocol. basic protocol takes at least days (antigen coating on day and running the elisa on day ). the screening elisa could be performed in the morning and the confirmatory elisa in the afternoon, or the assays can be done on consecutive days. a serological assay to detect sars-cov- seroconversion in humans. medrxiv reinfection could not occur in sars-cov- infected rhesus macaques. biorxiv the time course of the immune response to experimental coronavirus infection of man molecular diagnosis of a novel coronavirus ( -ncov) causing an outbreak of pneumonia detection of novel coronavirus ( -ncov) by real-time rt-pcr assays for total protein sds-polyacrylamide gel electrophoresis (sds-page) of proteins. current protocols in microbiology basic techniques in mammalian cell culture human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody cryo-em structure of the -ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china a novel coronavirus from patients with pneumonia in china we thank dr. raffael nachbagauer (icahn school for medicine at mount sinai) and dr. aubree gordon (university of michigan) for critical reading and constructive comments. development of this protocol was partially supported by the niaid centers of excellence for influenza research and surveillance (ceirs) contract hhsn c.philanthropic donations in support of our work are much appreciated, since the reagents stadlbauer et al. current protocols in microbiology are shared free of charge with the scientific community. please contact vanesa saric (vanesa.saric@mountsinai.org) for further information. key: cord- -up q k q authors: dortmans, j.c.f.m.; li, w.; van der wolf, p.j.; buter, g.j.; franssen, p.j.m.; van schaik, g.; houben, m.; bosch, b.j. title: porcine epidemic diarrhea virus (pedv) introduction into a naive dutch pig population in date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: up q k q porcine epidemic diarrhea virus (pedv) is the highly contagious, causative agent of an economically important acute enteric disease in pigs of all ages. the disease is characterized by diarrhea and dehydration causing mortality and growth retardation. in the last few decades, only classical pedv was reported sporadically in europe, but in outbreaks of pedv were described in germany. phylogenetic analysis showed a very high nucleotide similarity with a variant of pedv that was isolated in the us in january . the epidemiological situation of pedv infections in the netherlands in was unknown and a seroprevalence study in swine was performed. in total, blood samples from sows from farms and samples from wild boars were collected from may till november and tested for antibodies against pedv by elisa. the apparent herd prevalence of . % suggests that pedv was not circulating on a large scale in the netherlands at this time. however, in november a clinical outbreak of pedv was diagnosed in a fattener farm by pcr testing. this was the first confirmed pedv outbreak since the early nineties. sequence analyses showed that the viruses isolated in and in the netherlands cluster with recently found european g b strains. this suggests a one event introduction of pedv g b strains in europe in , which made the netherlands and other european countries endemic for this type of strains since then. porcine epidemic diarrhea (ped) is an economically important acute enteric disease in pigs of all ages. the disease is characterized by diarrhea and dehydration causing mortality -particularly in neonatal pigletsand growth retardation. the causative agent is porcine epidemic diarrhea virus (pedv), which is an enveloped, positive singlestranded rna virus, belonging to the family of coronaviridae (pensaert and de bouck, ) . the genome of pedv is approximately kb long and about two-third encodes for non-structural proteins and one-third of structural proteins (kocherhans et al., ) . among these proteins, the main research interest is focused on the spike (s) gene and its glycoprotein product s, mediating receptor binding and membrane fusion (li et al., ) . although only one serotype has been described, phylogenetic studies of the s gene showed that pedv can be genetically separated into two groups: genogroup (g ) and genogroup (g ). each genogroup can be further divided into subgroups a and b, and a and b, respectively (lee, ) . classical ped, now grouped g a, was first recognized as a severe swine enteric disease separate from transmissible gastro enteritis (tge) in the united kingdom in and first described in belgium in (pensaert and de bouck, ) . in the eighties and nineties, the virus was detected in many countries in europe including the netherlands and from europe pedv spread to asia, where it caused large outbreaks with considerable losses in the pig industry (song and park, ) . until , north america was considered to be free of pedv infections (cima, ) , but in that same year highly virulent strains of pedv emerged in the united states of america (us), causing diarrhea, vomiting and loss of appetite in pigs of all age groups and up to % of mortality in suckling piglets (chen et al., ; huang et al., ; stevenson et al., ) . this strain, typed as g b, rapidly spread across the us, canada, mexico and several countries in south america . in the last few decades, only classical pedv (or g a) was reported sporadically in europe (alborali et al., ; martelli et al., ) . in , outbreaks of pedv were described in germany and phylogenetic t analysis showed a very high nucleotide similarity with a variant of pedv (oh ) containing nucleotide insertions and deletions in the s gene (s-indel) that was isolated in the us in january stadler et al., ) . this variant, typed as g b, caused mild clinical signs and lower mortality rates in suckling piglets compared to other circulating pedv g b strains in the us . since the reports of outbreaks in germany, more reports about outbreaks of this particular s-indel virus in several european countries have been published, among which france, belgium, spain, portugal and austria (efsa, ; grasland et al., ; mesquita et al., ; steinrigl et al., ) . this suggests that this mild pedv variant is circulating in europe since the beginning of . the aim of this study was to determine the status of pedv in the netherlands with a serological survey and to investigate the first pedv outbreak in the netherlands since the early nineties. the number of required blood samples from animals and farms to estimate the seroprevalence of pedv in dutch sow herds was calculated based on the following assumptions: pedv is highly contagious and no vaccination against this virus was carried out in the netherlands. as a result, it was expected that, if pedv was present in a sow herd, the within herd prevalence would be very high (bertasio et al., ; goede et al., ) . the required number of blood samples per herd was calculated using winepiscope . (thrusfield et al., ) . to detect infection in a herd with % probability, an estimated within herd prevalence of %, and an average herd size of sows per farm (wur, ), three blood samples per farm were required. in , there were approximately sow farms in the netherlands (wur, ) . in order to show with a high probability ( %), that less than % of the dutch farms (n = ) were infected, farms would need to be tested (thrusfield et al., ) . herds were randomly selected stratified by pig density per province to represent the total dutch sow herd population ( fig. a and b). statistical analyses were performed using stata/se version . software (stata corporation, ). for the serological survey, blood samples were selected from samples collected for the obligatory monitoring of aujeszky's disease (pseudorabies), swine vesicular disease (svd) and classical swine fever (csfv). additionally, blood samples were collected from sows at slaughter (vion, groenlo, the netherlands). herds were identified based on their unique herd number (ubn). samples were randomly selected given the availability of enough serum to perform all assays. samples were collected from may till up to and including november . commissioned by the dutch government, gd animal health was also monitoring wild boars for aujeszky's disease, svd, foot and mouth disease, csfv, trichinellosis and african swine fever. because it is suggested that wild boars may play an important role as a pedv reservoir (lee et al., ) , blood samples of wild boar collected as part of this monitor were also tested. these blood samples were collected from regions close to the borders with germany and belgium, from april till august . blood samples collected were stored at − °c, before dispatched to the virology division of the faculty of veterinary medicine, at utrecht university (uu). for the detection of pedv antibodies in serum samples an in-house indirect elisa based on the viral spike (s) protein s -part of the g b strain gdu (non-s-indel, genbank ku . ) was used, similar as the elisa previously described (gerber et al., ) . the s antigen used in this study is produced in hek t cells, a mammalian expression system. to facilitate the purification from the cell culture supernatant, the protein is associated with the fc part of murine igg. per well ng of protein was used to coat the plates. the in-house elisa was optimized by uu with sera from pedvinfected (g b) pigs from the us and with hyperimmune sera of animals vaccinated with the pedv s protein. sera were tested in duplicate at a : dilution, the absorbance was measured with an elisa plate reader at nm. the mean od value of pedv-negative sera was . . sera with od values of > . ( × od value pedv negative sera) were considered positive. the aforementioned pedv-positive sera from infected animals were high positive (> . od) in this elisa ( supplementary fig. ). the calculated sensitivity and specificity of the elisa was % (ci %: - %; n = ) and % (ci %: - %; n = ), respectively. for the detection of virus-specific antibodies in serum samples the virus neutralization test (vnt) was used as an alternative sero-diagnostic assay. a previously described vnt was used to test all samples positive by elisa. the validation of this test is shown in fecal swabs or eswabs (copan diagnostics incorporated) of individual animals were taken by the local practitioner and sent in the same day for pcr testing. also three pigs of the index farm with severe diarrhea were euthanized and submitted for post mortem examination. rna was isolated from fecal samples and the detection pcr was performed as previously described (kim et al., ; lowe et al., ) . in order to type the viruses the pedv s gene was sequenced with primers previously described (chen et al., ; oka et al., ) and designed in this study (table ) , and compared with sequences available in genbank. a phylogenetic tree was constructed using mega software by neighbor-joining method maximum likelihood method. branch lengths represent the predicted number of substitutions and are proportional to the differences between the isolates (tamura et al., ) . after confirmation of the first g b outbreak in the netherlands it was unquestionable to try to prevent the further spread of the outbreak. therefore, it was decided to monitoring and follow up of specific management advices to the farmers. five fattening herds, five sow herds and one nursery herd were selected after pedv infection was confirmed by pcr on feces. for control and eradication of pedv, a tailor made advice per farm, mainly based on biosecurity measures, was given. advise was mainly focused on the separation between pedvinfected and non-infected healthy animals. this separation was applied in stables, animal categories and compartments. it was made using the following measures: dress code (change between compartments), additional disinfection cleaning and disinfection of compartments and corridors and improving pest control. furthermore, all professional herd visitors were informed and required to take measures, in particular those aimed at cleaning and disinfection, which would prevent the transfer of the disease to another pig farm. regular testing of pooled fecal samples was done to monitor the effect of interventions. in sow herds, nursery piglets and replacement gilts were sampled; in fattening herds a random sampling in all age groups was performed. after introduction of pedv, the virus could be detected in a compartment for - weeks. at farm level the virus was detectable much longer due to transmission to new susceptible animals. farms were presumed negative for pedv if three sampling rounds with at least -weeks interval, of thirty randomly taken individual fecal samples each, proved to be pcr negative. in total, sera from sows originating from farms, and sera from wild boar were collected. the blood samples came from all provinces in the netherlands and are fairly representative for the distribution of farms across provinces (fig. b ). all sera were tested in the indirect pedv s -based elisa (group , table ). nine samples, originating from nine different farms located in four different provinces, tested seropositive. the od values were low in all seropositive samples (od: . - . ) relative to positive control sera (convalescent or hyperimmune sera) with values od: . - . . two of the nine elisa-positive sera from the serological study also tested positive in the vnt. with two confirmed positive samples of the samples the animal prevalence was . % (ci %: - . %). the herd prevalence with out of farms was . % (ci %: - . %). for all herds, three samples were tested. the additional samples of seven herds with a seropositive sample of which one vnt positive, were examined. these samples, in total, were all negative in the pedv elisa. the proportion of seropositive samples ( / ) falls within the expected proportion of false positives given the specificity of the elisa which is estimated at > %. so we either detected pedv outbreaks that had not spread yet or the seven original samples were false positives. for two herds no additional samples from the monitoring programs were available (including one sow farm with a vnt positive sample). all tested sera obtained from wild boar were negative for pedv antibodies in the used indirect elisa (group , table ). in the first week of november , gd animal health received a report of pigs showing lethargy and anorexia for up to h after which profuse diarrhea occurred in almost all pigs in nine compartments within the fattening barn. diarrhea was watery, light greyish, sometimes yellow or green colored. body temperature in the clinical phase reached . °c. after the first days of disease lethargy subsided and lack of appetite diminished, a profuse diarrhea became more prominent. in later stages of the infection the consistency of the diarrhea changed to slightly more solid. pigs did not seem to suffer much and no animals died, although some pigs did not grow for a week and within the group body weights started to differ. after an extra week of feeding, pigs did recover and had a normal weight at slaughter. the fattening barn, located in a pig dense area of the netherlands, consisted of compartments with pigs each, divided over pens. first symptoms occurred on october th in one compartment followed by symptoms in consecutive compartments in the following days. initial diagnostic tests for the presence of e. coli, salmonella, lawsonia intracellularis and brachyspira pilosicoli were, except for low numbers of pathogenic e. coli, negative. based on the low numbers of pathogenic e. coli and the age of the pigs involved, e. coli was ruled out as causative agent in this case. subsequently, it was decided to test for porcine deltacoronavirus (pdcov) and pedv. six fecal samples were found to be negative for pdcov, but positive for pedv rna on november th . on the same day, three pigs with severe diarrhea were euthanized and were submitted for post mortem examination. pathological examination showed severe villus atrophy in the small intestine, and pcr tested positive for pedv. during the outbreak, in total % of the animals ( compartments) were affected by pedv as confirmed by pcr on fecal samples. based on the results of the serological survey, over % of the dutch farms were pedv negative in ; it was decided to try to prevent the further spread of the outbreak. to control and eradicate pedv the biosecurity measures taken, as described in the materials and methods section, seemed to be of great importance. also pet control was applied since some farms suffered from mice and rats. furthermore, it became clear that in compartments in which the infection was present, after thorough cleaning and disinfection, pedv free piglets could be introduced and that those stayed free from pedv infection. three fattening and three sow herds were presumed free of pedv within months after the diagnosis of ped was confirmed. the nursery herd was depopulated, and, after double cleaning and disinfection of all compartments, repopulated. despite the immediate action of all parties in the dutch pig production industry to optimize their hygiene measures to ensure that infection by pedv would be avoided as much as possible, it could not be prevented that pedv spread to other farms. most infected farms were located on the east side of the netherlands at the border of germany (fig. c) . at the end of , in total farms were confirmed pedv positive by gd animal health. in fig. the number of new pedv pcr positive farms per month since the beginning of the outbreak is shown. in order to characterize the virus originating from outbreaks in and , the s gene of three isolates of different farms was sequenced and the sequences ned/gd / , ned/gd / and ned/ gd / were deposited in the genbank database and received accession numbers kr (index farm), kr and mf , respectively. together with sequences available in genbank, the isolates were compared in a phylogenetic tree (fig. ) . sequence analyses showed that the isolates had a . % homology with the usa/oh strain and a . % homology with the german g b strains. furthermore, the european strains available in genbank ( - ) cluster together with this oh strain, or the so called s-indel strain (fig. ) . this study showed that most likely pedv and particularly the genogroup b (s-indel) strains did not circulate in the netherlands on a large scale till the end of . only a very small part of the dutch sow farms tested positive on antibodies against pedv ( . % (ci %: - . %)) and no pedv antibodies were detected in wild boars. the used elisa based on the s protein of a g b strain showed a high sensitivity and specificity against antibodies raised against g a, g b en g b strains. however, the elisa was validated with a limited amount of samples ( supplementary fig. ) and field samples may react differently, just like in a similar elisa recently described (gerber et al., ) . the viral spike (s) protein is prominent on the virus surface and is very immunogenic. all animals that have been infected with pedv have antibodies against the s protein and particularly against the s part. the s -part of the spike protein is the most variable part between related coronaviruses and there is no cross reactivity with other coronaviruses such as transmissible gastroenteritis coronavirus (tgev), porcine respiratory coronavirus (prcov) and porcine delta coronavirus (pdcov) as previously described (gimenez-lirola et al., ; lin et al., b) . because results of immunoassays based on the s protein correlate well with viral neutralization (paudel et al., ) , as shown in supplementary table , we decided to use the vnt as an alternative sero-diagnostic assay. based on case reports from germany stadler et al., ) , austria (steinrigl et al., ) and the netherlands (gd animal health) a pedv infection with the european circulating strain (g b) will spread very rapidly within a herd and a single pedv positive animal on a farm is not likely to occur. the number of elisa positive animals ( %, table , group ) on the index farm after the clinical signs started, seemed to justify the assumption of the high within herd prevalence of % for sample size calculation for the serological survey. eventually, there were fewer herds sampled than planned ( instead of ) since it was decided to stop the serological survey, because the first case of pedv was diagnosed in the netherlands on november th . sequence analyses of the viruses isolated in - showed a % homology with oh , a less virulent pedv strain found in the us and in germany in . although the g b virus strain present in europe is less virulent (efsa, ; grasland et al., ; hanke et al., ; mesquita et al., ; steinrigl et al., ) compared to the strain that caused the us outbreaks in , this european strain showed to be very contagious and still could cause severe economic damage in pedv naive herds. after infection on sow farms loss off piglets could range up to % in the sucking piglets ( (lin et al., a) and individual case reports, gd animal health). in the foreseeable future, vaccination will not be possible. therefore, the authorities in the netherlands advised all parties in the pig production industry to optimize their hygiene measures to ensure that infection by this virus would be avoided as much as possible. the strict preventive biosecurity measures taken in these herds demonstrated that most herds that were monitored could prevent the transfer to pedv naive compartments within the herd and some were able to obtain a presumed pedv free status for the whole farm. additionally, a higher biosecurity level helps preventing the introduction of other pathogens and controls the spread of infection within that herd (fao, ) . however, an increasing number of herds became infected (fig. ) and pedv spread across the country after the initial outbreak (fig. c) . the transport trucks with positive animals between herds seemed to have the highest transmission risk (data not shown). the number of new pedv pcr positive farms per month since the beginning of the outbreak is most likely an underestimation (fig. ) . since ped is not a notifiable disease, nor have all veterinarians performed diagnostic testing in all cases, as the clinical signs of an outbreak are very typical. it seems that pedv g b became endemic in the netherlands since its initial outbreak in november , just like in most countries in europe. that g b viruses isolated in europe in - phylogenetically cluster together with a high similarity (fig. ) suggests a onetime introduction event. however, pedv is endemic and many other coronaviruses are circulating that may result in coronavirus variants through mutation or recombination (fehr and perlman, ) . therefore, it is of upmost importance that the presence of circulating pedv is being monitored and genetically analyzed, in order to update diagnostic tools where necessary. we declare no conflict of interest. surveillance and control of ped coronavirus in pigs in italy porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states viral disease affects u.s. pigs: porcine epidemic diarrhea found in at least states updated epidemiological data on ped good practices for biosecurity in the pig sector -issues and options in developing and transition countries coronaviruses: an overview of their replication and pathogenesis detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france comparison of porcine epidemic diarrhea viruses from germany and the united states origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states multiplex realtime rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus wild boars harboring porcine epidemic diarrhea virus (pedv) may play an important role as a pedv reservoir manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination cellular entry of the porcine epidemic diarrhea virus experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains role of transportation in spread of porcine epidemic diarrhea virus infection epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy outbreak of porcine epidemic diarrhea virus in portugal cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines emergence of porcine epidemic diarrhea virus in southern germany first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences mega : molecular evolutionary genetics analysis version . win episcope . : improved epidemiological software for veterinary medicine distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus pig farming the authors would like to thank d. oorburg en c. sima of vion, groenlo, the netherlands, for providing blood samples from slaughter sows, g. spierts and laboratory staff for collecting and archiving blood samples and n. schuurman and j. de jong for performing the elisa. furthermore, r. dijkman for implementing the pcr at the gd animal health lab, a. van lenthe for advice during the survey, m. meijerink for assisting in sample collection during herd visits and a. veldhuis and h. brouwer-middelesch for making the maps of the netherlands (fig. ) . the authors thank the owner of the index farm and the practitioner involved in this farm for their information and their cooperation in collecting the samples.the authors would like to thank the dutch ministry of economic affairs and the product board for livestock and meat for funding the monitoring system for pig health in the netherlands. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- - zgc xrb authors: zhao, shan; smits, constance; schuurman, nancy; barnum, samantha; pusterla, nicola; van kuppeveld, frank; bosch, berend-jan; van maanen, kees; egberink, herman title: development and validation of a s protein-based elisa for the specific detection of antibodies against equine coronavirus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zgc xrb equine coronavirus (ecov) is considered to be involved in enteric diseases in foals. recently, several outbreaks of ecov infection have also been reported in adult horses from the usa, france and japan. epidemiological studies of ecov infection are still limited, and the seroprevalence of ecov infection in europe is unknown. in this study, an indirect enzyme-linked immunosorbent assay (elisa) method utilizing ecov spike s protein was developed in two formats, and further validated by analyzing paired serum samples (acute and convalescent sera) from horses involved in an ecov outbreak and sera of horses with unknown ecov exposure. both formats showed high diagnostic accuracy compared to virus neutralization (vn) assay. receiver-operating characteristic (roc) analyses were performed to determine the best cut-off values for both elisa formats, assuming a test specificity of %. employing the developed elisa method, we detected seroconversion in . % of horses from an ecov outbreak. among the horse sera, seropositivity varied from . % (young horses) to . % (adult horses) in dutch horse populations. further, sera of icelandic horses were included in this study and a significant number of sera ( %) were found to be positive. overall, the results demonstrated that the ecov s -based elisa has reliable diagnostic performance compared to the vn assay and is a useful assay to support seroconversion in horses involved with ecov outbreaks and to estimate ecov seroprevalence in populations of horses. coronaviruses (covs) are enveloped, positive single-stranded rna viruses that belong to the subfamily orthocoronavirinae in the family coronaviridae of the order nidovirales. they are classified into four genera (alpha-, beta-, gamma-and deltacoronavirus) and infect both mammalian and avian hosts [ , ] . equine coronavirus (ecov) belongs to betacoronavirus species, within the embecovirus subgenus of the betacoronavirus genus, as does human coronavirus oc , hku and bovine coronavirus [ ] . ecov was isolated for the first time from a two-week-old diarrheic foal in north carolina (usa) in , suggesting the role of ecov in causing enteric disease [ ] . since , several cases of ecov infections have also been reported in adult horses from the united states, europe and japan [ ] [ ] [ ] [ ] [ ] . equine coronavirus has been detected in fecal samples from horses with clinical signs that included anorexia, lethargy, fever and, less frequently, diarrhea, colic and neurologic deficits [ , ] . the morbidity rate varies from % to % during outbreaks. mortality is low and has been related to endotoxemia, septicemia or hyperammonemia-associated encephalopathy [ , ] . the outbreaks in adult horses demand further studies on the pathogenesis and epidemiology of ecov infections. for this, diagnostic assays with high sensitivity and specificity are crucial. ecov is known to be associated with enteric infections but can also be detected in a small percentage of horses with respiratory signs. virus shedding can be observed in fecal samples or nasal swabs from sick horses as well as healthy horses, but with a strong association between clinical signs assumed to be related to ecov infection and virus detection in fecal samples suggesting a possible etiological role of ecov [ , ] . recently, real-time quantitative pcr (qpcr) methods have been established and were shown to be able to detect ecov in feces efficiently. however, ecov viral nucleic acid is generally only detectable by qpcr within a limited timeframe of - days post infection, as reported from both field and experimental studies [ , , , ] . on the other hand, serological assays can be used to support the diagnosis of a clinical ecov infection by showing seroconversion or a significant increase in antibody titer in paired serum samples. serological assays are also needed to gain more insight into the transmission rate of infection within animal populations [ ] . antibodies induced by betacoronaviruses persist in blood for a longer period after infection [ , ] . the virus neutralization (vn) assay has long been used as a gold standard to confirm serological responses to coronavirus infections [ ] [ ] [ ] . although the vn assay is highly specific for the detection of antibodies, it is also time-consuming and laborious to perform. alternative high-throughput serologic assays that correlate well with neutralizing antibodies are therefore needed. severe infections of ecov have been shown to be associated with high viral load, but mild or asymptomatic infections may occur with low levels of virus replication being negative in pcr and with variable immune responses [ ] . consequently, specific, sensitive and high-throughput serodiagnostic methods are necessary to avoid the underestimation of prevalence in surveillance studies. the spike protein (s) of coronaviruses is the key mediator in virus cell entry and therefore the major target for neutralizing antibodies. the s ectodomain consists of two functionally interdependent subunits, s and s . the n-terminal s subunit is responsible for receptor binding, while the c-terminal s subunit mediates membrane fusion [ , ] . the s subunit is the most variable immunogenic antigen among coronaviruses, and therefore it is an ideal candidate for the detection of cov species-specific antibodies [ , ] . the objective of the study was to develop and validate an elisa method for the detection of specific antibodies to ecov and provide a tool for the diagnosis and the future estimation of ecov prevalence and incidence in various equine (sub) populations. a total of equine serum samples were included in this study. the details of serum panels a-h (n = ) are shown in table . they were retrieved from the serum bank at gd animal health deventer, the netherlands. all of them were collected for the monitoring of other diseases independent to this study, and their ecov exposure status was unknown. with the exception of panel h (collected from iceland), all serum samples from panel a to g were collected from horses in the netherlands. additionally, panel i included paired (acute-and convalescent-phase) serum samples that were collected during an ecov outbreak in the usa ( ). all samples were stored at − • c until tested. ecov strain nc was propagated and titrated in human rectal adenocarcinoma (hrt- g) cells. hrt- g cells and human embryonic kidney cells stably expressing the sv large t antigen (hek- t) were maintained in dulbecco modified eagle medium (dmem, lonza, basel, switzerland) the sequence of the s subunit of the spike protein of the ecov nc strain (residue - of the amino acid sequence) was derived from genbank (genbank no.: ef . ). human codon-optimized sequences encoding the ecov s subunit were synthesized and fused to the fc domain of mouse igg a, which was subsequently cloned into the pcaggs mammalian expression vector as described before [ ] . for ecov s -fc protein production, expression plasmid was transfected into hek- t cells using polyethyleneimine (polysciences, inc., warrington, pa, usa) in a ratio of : . after h of incubation, the transfection medium was removed and replaced by sfm ii expression medium (gibco ® , life technologies inc., grand island, ny, usa). at six days post transfection, cell culture supernatants were harvested and the soluble s was purified from the culture medium using protein a sepharose beads (ge healthcare bio-sciences ab, uppsala, sweden). subsequently, the proteins were eluted using . m citric acid, ph . , and immediately neutralized with m tris-hcl, ph . . the purity and integrity of proteins were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) and stained with gelcodeblue stain reagent (thermofisher scientific inc., waltham, ma, usa). purified proteins were quantified by nanodrop spectrophotometry (thermofisher scientific inc., waltham, ma, usa) and by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) with bovine serum albumin (bsa) as standard, then stored at − • c until further usage. equine sera (n = ) were randomly selected from different serum panels (a-d) and tested for neutralizing antibody titers in an ecov vn assay. heat-inactivated equine sera ( • c for min) were serially diluted -fold in dmem supplemented with % fetal bovine serum and mixed with an equal volume of ecov nc strain ( % tissue culture infective doses (tcid )/well) in -well cell culture plates (corning inc., kennebunk, me, usa). virus-serum mixtures were incubated at • c for min. then µl of the virus-serum mixture was added in duplicate to hrt- g cells monolayers in -well cell culture plates. at six days post infection, a clear cytopathic effect (cpe) was observed and the virus neutralization titers (vnt) were determined. the vnt of sera were expressed as the reciprocals of the highest serum dilution that resulted in % neutralization of cpe. a titer of ≥ was considered to be positive. two different formats were developed employing ecov s protein, a so-called wet format elisa (welisa) and a dry format elisa (delisa). . . . ecov s wet format elisa (welisa) high-binding microtiter plates (greiner bio-one bv, alphen aan den rijn, the netherlands) were coated with ecov s protein ( µl per well) in phosphate buffered saline (pbs, ph . ) overnight at • c. the optimal protein amount and dilution of secondary antibody conjugate were determined by checkerboard titration. the protein concentration in use was . µg/ml. after three washes with pbs containing . % tween- (pbst), the plates were blocked with pbst containing % milk powder (protifar, nutricia, zoetermeer, the netherlands) for h at • c. following blocking, plates were incubated with serum samples diluted : in pbst containing % milk powder for h at • c. after a washing step, µl/well : , diluted horseradish peroxidase (hrp)-conjugated goat anti-horse igg (h&l) (abnova, taiwan, china) was added to detect bound antibodies and plates were incubated for h at • c. subsequently, the plates were washed, and the peroxidase reaction was then visualized via incubating plated with tmb super slow one component hrp microwell substrate (biofx ® , surmodics ivd, inc., eden prairie, mn, usa) for min at room temperature. the reaction was stopped by adding . % sulfuric acid (h so (vwr international bv, amsterdam, the netherlands)) and optical densities (od) were immediately measured at nm using an elisa microplate reader (biotek instruments, inc., winooski, vt, usa). all serum samples were tested in duplicate. . . . ecov s dry format elisa (delisa) high-binding microtiter plates (greiner bio-one bv, alphen aan den rijn, the netherlands) were coated with ecov s protein ( µl per well) in ammoniumcarbonate solution ( . g/l (vwr international bv, amsterdam, the netherlands)) overnight at • c. then µl/well blocking solution ( . g/l ammoniumcarbonate + g/l caseine (vwr international bv, amsterdam, the netherlands) + g/l sucrose (merck and co., inc., kenilworth, nj, usa)) was added and plates were incubated for one hour at room temperature. subsequently, the contents of the plates were discarded, and plates were dried for four hours at • c, vacuum sealed and stored at - • c. the optimal protein amount and dilution of secondary antibody conjugate were determined by checkerboard titration. the protein concentration in use was . µg/ml. plates were incubated with serum samples µl per well and diluted : in pbs + . % tween- + . % dry milk (bio-rad laboratories, inc., hercules, ca, usa) for h at • c. after a washing step (five times with pbst µl/well on a biotek automatic washing station), µl per well : , diluted horseradish peroxidase (hrp)-conjugated goat anti-horse igg (h&l) (abnova, taiwan, china) was added to detect bound antibodies and incubated for h at • c. subsequently, the plates were washed again using the same washing procedure and the peroxidase reaction was then visualized by incubating plates with tmb (idexx laboratories, westbrook, nj, usa) for min at room temperature. the reaction was stopped by adding µl/well sulfuric acid (h so . m (vwr international bv, amsterdam, the netherlands)) and optical densities (od) were immediately measured at nm using an elisa microplate reader (biotek instruments, inc., winooski, vt, usa). all serum samples were tested in duplicate. s/p values were calculated with the formula: s/p = (od sample-od negative control)/(od positive control-od negative control). the correlation between od values scored with two elisa formats was measured by the pearson correlation coefficient using graph pad prism, version . the discriminating power of the two different elisa formats was analyzed by performing receiver operator characteristic (roc) analysis with sera, which were negative (vnt < ) and were positive (vnt ≥ ). the cut-off value, diagnostic specificity and sensitivity were determined by roc analysis using sigmaplot. a minimum specificity of % was chosen for the selection of cut-off values. additionally, the reproducibility of assays was evaluated by testing three samples with different od values. inter-assay coefficients of variation (cv) and intra-assay cv were determined testing each sample in triplicate on three different plates in three different runs and within the same plate, respectively. to identify equine sera containing ecov-neutralizing antibodies, we screened a subset of equine sera, composed of randomly selected serum samples from panels a-c, and all samples from panel d were screened in the vn assay. of the sera, sera were tested as negative (titers < ) and positive samples (titers ranging from to ). additionally, paired samples from horses (n = , panel i) were tested in the vn assay. twenty out of sera collected from the first time point exhibit titers ranging from to . the convalescent serum samples were collected - days following the first round of sample collections, and all of them showed neutralization responses with titers ranging from to . within these horses, seven of them showed seroconversion and showed a significant ( -fold or greater: log ) increase in titer in the vn assay. to confirm the presence of ecov specific igg in icelandic horses, horse sera with positive ecov s elisa results (in panel h, s/p value > . ) were tested in ecov neutralization assays. all of them had neutralizing antibodies with titers varying between and . besides the conventional wet elisa format (welisa) for general laboratory usage, a dry standardized elisa format (delisa) was also developed and validated to facilitate implementation as routine diagnostic method in different laboratories and possibly wider application as an elisa kit. both elisa formats were developed for the detection of ecov-specific antibodies in horse serum samples. the diagnostic performance of both elisas was evaluated using a subset of horse sera with known vn results as described above. the pearson correlation coefficient was calculated to assess the correlation between the od values obtained with the two elisa formats (figure ) . results indicate that od values obtained with both elisas show a high degree of correlation, with correlation and regression coefficients close to (r = . , regression coefficient = . , p < . ). thus, the performance of both elisa formats is very similar. additionally, paired samples from horses (n = , panel i) were tested in the vn assay. twenty out of sera collected from the first time point exhibit titers ranging from to . the convalescent serum samples were collected - days following the first round of sample collections, and all of them showed neutralization responses with titers ranging from to . within these horses, seven of them showed seroconversion and showed a significant ( -fold or greater: log ) increase in titer in the vn assay. to confirm the presence of ecov specific igg in icelandic horses, horse sera with positive ecov s elisa results (in panel h, s/p value > . ) were tested in ecov neutralization assays. all of them had neutralizing antibodies with titers varying between and . besides the conventional wet elisa format (welisa) for general laboratory usage, a dry standardized elisa format (delisa) was also developed and validated to facilitate implementation as routine diagnostic method in different laboratories and possibly wider application as an elisa kit. both elisa formats were developed for the detection of ecov-specific antibodies in horse serum samples. the diagnostic performance of both elisas was evaluated using a subset of horse sera with known vn results as described above. the pearson correlation coefficient was calculated to assess the correlation between the od values obtained with the two elisa formats (figure ) . results indicate that od values obtained with both elisas show a high degree of correlation, with correlation and regression coefficients close to (r = . , regression coefficient = . , p < . ). thus, the performance of both elisa formats is very similar. subsequently, the discriminating power of the welisa and delisa was evaluated via receiver operator characteristic (roc) analysis. the roc curves were plotted based on the previous classification of sera into negative and positive by vn assays (figure a,b) . then the optimal cutoff values, diagnostic specificity and sensitivity of both elisa formats were determined by the established roc curves. the elisa results of the vnt-positive and negative samples are shown in figure c ,d. the diagnostic accuracy of both elisa formats was considered to be high as the same area under the curve (auc) values were observed (auc = . ), with a relative sensitivity and specificity approximately % according to the youden plot of welisa and delisa. therefore, the test characteristics of both elisa formats were assigned the same weight. in this study, a minimum specificity of % was chosen for the threshold of cut-off values for both elisas. accordingly, the optimal cut-off for welisa was an od value of . -for which, the sensitivity was % and the specificity was %. for delisa, the test results were expressed as s/p values. a cut-off at an s/p value of . yielded a sensitivity of % and specificity of %, respectively. subsequently, the discriminating power of the welisa and delisa was evaluated via receiver operator characteristic (roc) analysis. the roc curves were plotted based on the previous classification of sera into negative and positive by vn assays (figure a,b) . then the optimal cut-off values, diagnostic specificity and sensitivity of both elisa formats were determined by the established roc curves. the elisa results of the vnt-positive and negative samples are shown in figure c ,d. the diagnostic accuracy of both elisa formats was considered to be high as the same area under the curve (auc) values were observed (auc = . ), with a relative sensitivity and specificity approximately % according to the youden plot of welisa and delisa. therefore, the test characteristics of both elisa formats were assigned the same weight. in this study, a minimum specificity of % was chosen for the threshold of cut-off values for both elisas. accordingly, the optimal cut-off for welisa was an od value of . -for which, the sensitivity was % and the specificity was %. for delisa, the test results were expressed as s/p values. a cut-off at an s/p value of . yielded a sensitivity of % and specificity of %, respectively. furthermore, the inter-and intra-coefficient of variation (cv) of the three ecov positive sera tested with both elisa formats were lower than %. more specifically, the intra-assay cv of welisa and delisa ranged from . % to . % and from . % to . %, respectively, while the inter-assay cv of welisa and delisa varied from . % to . % and from . % to . %, respectively. overall, these results indicate that the performances of both elisa formats were very much equivalent and that the results of both elisas were strongly correlated to vn results. to determine the diagnostic performance of the ecov s elisa, paired serum samples (panel i) collected from an acute ecov outbreak were investigated by welisa. the horses presented similar clinical signs as described in [ ] , and virus shedding was confirmed by qpcr analysis [ ] . at the acute stage, out of horses were qpcr positive, while at the convalescent stage, this number had decreased to six. serum samples were further validated by vn assay ( figure b ; table s ). seven out of horses showed seroconversion, while another horses showed a significant ( -fold or greater) increase in vnt. performing the welisa (see figure a ; table s ), the same seven out of horses showed seroconversion; acute phase sera were negative (od value < . ) whereas the convalescent phase sera all had od values greater than . ( . - . ). thus, seroconversion rates calculated from welisa and vnt showed a % correlation (table s ). for the horses that showed a -fold or greater increase in vnt (n = ), nine of the acute phase sera had positive od values between . and . ( × background) and also a higher than (n = ) to (n = ) fold increase in the od value in the convalescent serum. five of the vnt positive paired serum samples had od values of > . (twice the background od value) in the acute phase serum. two of these samples with an od value of . and . respectively in welisa also showed a greater than -fold increase in od value. the three vnt positive samples with less than -fold increase in od values already had high furthermore, the inter-and intra-coefficient of variation (cv) of the three ecov positive sera tested with both elisa formats were lower than %. more specifically, the intra-assay cv of welisa and delisa ranged from . % to . % and from . % to . %, respectively, while the inter-assay cv of welisa and delisa varied from . % to . % and from . % to . %, respectively. overall, these results indicate that the performances of both elisa formats were very much equivalent and that the results of both elisas were strongly correlated to vn results. to determine the diagnostic performance of the ecov s elisa, paired serum samples (panel i) collected from an acute ecov outbreak were investigated by welisa. the horses presented similar clinical signs as described in [ ] , and virus shedding was confirmed by qpcr analysis [ ] . at the acute stage, out of horses were qpcr positive, while at the convalescent stage, this number had decreased to six. serum samples were further validated by vn assay ( figure b ; table s ). seven out of horses showed seroconversion, while another horses showed a significant ( -fold or greater) increase in vnt. performing the welisa (see figure a ; table s ), the same seven out of horses showed seroconversion; acute phase sera were negative (od value < . ) whereas the convalescent phase sera all had od values greater than . ( . - . ). thus, seroconversion rates calculated from welisa and vnt showed a % correlation (table s ). for the horses that showed a -fold or greater increase in vnt (n = ), nine of the acute phase sera had positive od values between . and . ( x background) and also a higher than (n = ) to (n = ) fold increase in the od value in the convalescent serum. five of the vnt positive paired serum samples had od values of > . (twice the background od value) in the acute phase serum. two of these samples with an od value of . and . respectively in welisa also showed a greater than -fold increase in od value. the three vnt positive samples with less than -fold increase in od values already had high od values in the acute phase serum as well as high vnt (mean od value = . , mean vnt = . ). for the six horses that did not show a significant rise in vnt, five serum samples collected at the acute stage already had high antibody levels as shown by elisa and neutralization assay (mean od value > . , mean vnt > , table s ). further, the pearson correlation coefficient was calculated to assess the overall correlation between the od values obtained with welisa and vnt (log titers) from acute and convalescent-phase sera of the horses ( figure s ). results indicate that od values and vnt show a good degree of correlation (r = . , p < . ). these data support the use of the welisa as a diagnostic tool in case of suspected ecov outbreaks. od values in the acute phase serum as well as high vnt (mean od value = . , mean vnt = . ). for the six horses that did not show a significant rise in vnt, five serum samples collected at the acute stage already had high antibody levels as shown by elisa and neutralization assay (mean od value > . , mean vnt > , table s ). further, the pearson correlation coefficient was calculated to assess the overall correlation between the od values obtained with welisa and vnt (log titers) from acute and convalescent-phase sera of the horses ( figure s ). results indicate that od values and vnt show a good degree of correlation (r = . , p < . ). these data support the use of the welisa as a diagnostic tool in case of suspected ecov outbreaks. we further set out to determine the seroprevalence in horses with unknown ecov exposure using the delisa format. a total of serum samples (table , panel a-h) were analyzed. with the exception of panel d, all sera were from adult horses (older than months). seroprevalence varied from . % (panel d) to . % (panel c) among these eight serum panels. the lowest number of positive samples was found in panel d which contained young horses ( - months old, average age: . months ( % ci . - . )). in the other four serum panels (panel a, b, e and f) from dutch horses, the historical serum samples (panel g) and samples from iceland (panel h) higher seroprevalences were found ( . - . %). table . prevalence of ecov s -reactive antibodies in equine sera used in this study. we further set out to determine the seroprevalence in horses with unknown ecov exposure using the delisa format. a total of serum samples (table , since the beginning of the st century, ecov infections have been reported in horses, causing fever and enteric diseases [ ] . more recently, infections in adult horses were reported with clinical signs of fever, anorexia, lethargy and, less commonly, specific signs of diarrhea and colic [ , ] . nevertheless, information regarding the circulation of ecov in the equine population, especially in europe, is still limited [ , ] . serological studies are useful tools to investigate ecov prevalence in horse populations. in the present study, our aim was to develop a simple and reliable method for antibody detection against ecov that can be used for diagnostics and sero-epidemiological studies. as compared to virus neutralization assays, the elisa method has the advantage of being reproducible, potentially high-throughput and much less laborious. in our study, we set up an ecov s -based elisa method in two complementary formats. the conventional welisa format is for general laboratory usage with simplified, easy to perform coating procedures. on the other hand, coated plates of the delisa format could be stored for a longer time period, making it ideal for transportation and kit development. we showed that both formats performed equally well, and their results correlated nicely. when comparing with the vn assay by roc analysis, our elisa method with both formats was shown to have high accuracy. in our current study we applied welisa for the analysis of the paired outbreak samples, while the delisa was further validated and used for the high-throughput screening of larger amount of serum samples. we utilize ecov s as the viral antigen for antibody detection in this study. the s chimeric protein was expressed in mammalian cells, and hence both the protein conformation and modification (e.g., glycosylation) are mimicking the s proteins on the surface of virus particles [ ] . as the most divergent and immunodominant component of coronaviruses, s has been widely used in the development of methods for specific coronavirus serological studies [ , , , ] . our findings validate that ecov s is a highly suitable antigen for the detection of antibodies against ecov showing very good agreement between the elisa and vn assays. recently, similar conclusions were also drawn for the role of mers s in mers serology [ ] . with our welisa method, we were able to analyze paired samples that were collected during an ecov outbreak. in the virus neutralization assay seroconversion or a -fold or greater increase in ecov antibody titers could be detected in sera of out of horses within weeks of the initial observation of clinical disease and detection of viral rna in feces. of these positive horses showed seroconversion or a -fold or higher increase in od values in the welisa. the three remaining vnt positive samples had high od values already in the acute phase serum. of the six ecov negative paired samples five had high vn antibody titers and od values already at the acute phase. this might be due to late sampling of these horses or previous exposure to ecov (table s ). this study confirms that the ecov s elisa is a useful diagnostic test for the demonstration of a potential ecov outbreak and should be considered as a useful adjunct to investigation of fecal samples by qpcr. we also determined the seroprevalence of serum samples collected from horses with unknown ecov exposure via our delisa. results showed that the overall seroprevalence in the different cohorts tested is . %- . %. these percentages are in agreement with the study performed by hemida et al. [ ] , in which they detected coronavirus infections in horses in saudi arabia and oman and they found that % of them had detectable neutralizing antibodies to ecov. a lower percentage ( . %) of positive animals was found in another ecov seroprevalence study conducted in the usa [ ] . several factors might contribute to these differences in results. there is only limited information regarding ecov prevalence in europe including the netherlands [ , ] , and it is possible that the overall ecov distribution differs between continents. moreover, our study employs eukaryotically expressed ecov s protein as coating antigens, while in the us study chimeric s protein expressed in escherichia coli was used. the expression in mammalian cells guarantees a more native configuration of the protein, in particular of glycosylated antigens such as the coronavirus spike protein. reports had shown that both coronavirus s and s subunit elicit antibody responses, but the level of immune responses triggered by them may differ [ , ] . furthermore, the criteria for determining the cut-off value are different for the two studies. in our study we defined positive and negative samples on the basis of a vn assay, whereas the us study used negative qrt-pcr and absence of clinical signs as criteria to define horses as ecov negative. in this way, seropositive horses may have contributed to higher cut-off values and potentially a lower sensitivity of the assay. in our study, we noticed differences in seroprevalence between young and adult horses. in the group of young horses (panel d, table ), the lowest seroprevalence was found. young horses may initially be protected against ecov infection by maternal antibodies and may become gradually more susceptible as maternal antibodies wane. the risk of becoming infected increases with age. this hypothesis is further supported by the age distribution of pcr-confirmed ecov infection cases: foals (age - months) have the lowest infection rates, and the infection rate increases with age [ ] . we also observed a significant percentage of seropositive horse serum samples collected back in (panel g, table ). ecov-like viruses were detected in the s and s by electron microscopy in feces of horses with enteric disease, but virus isolation and characterization was not reported [ ] [ ] [ ] [ ] . the history of ecov presence, especially in europe, is possibly much longer than currently understood [ ] . intriguingly, we noticed that icelandic horses also are seropositive against ecov (panel h, table ). twenty-four serum samples showed high ecov elisa reactivity (s/p value > . ) and also had neutralizing antibodies with vnt varying between and . the horse population of iceland has been geographically isolated for more than years and is free from most common equine contagious diseases such as equine influenza, equine herpesvirus , strangles and equine viral arteritis [ ] . to date, no prior studies of ecov prevalence in horses from iceland had been performed. this is the first evidence of the existence of ecov infection in iceland. in conclusion, we developed a high-throughput, reliable and specific elisa method to study humoral immune responses in horses against ecov. with this method, we are able to perform the serodiagnosis of ecov infection and assess the seroprevalence within horse populations in the future. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . table s : detection of antibodies to ecov in equine serum samples during an ecov outbreak by welisa in winter ; figure s . correlation between the od values obtained with welisa and virus neutralization titers (vnt) of horses from acute and convalescent-phase sera. origin and evolution of pathogenic coronaviruses genetic recombination, and pathogenesis of coronaviruses genomic characterization of equine coronavirus characterization of a coronavirus isolated from a diarrheic foal isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the nc strain first detection of equine coronavirus (ecov) in europe emerging outbreaks associated with equine coronavirus in adult horses epidemic of equine coronavirus at obihiro racecourse, hokkaido, japan in the first detection of equine coronavirus in adult horses and foals in ireland enteric coronavirus infection in adult horses frequency of molecular detection of equine coronavirus in faeces and nasal secretions in horses with acute onset of fever disease associated with equine coronavirus infection and high case fatality rate clinical presentation, diagnostic findings, and outcome of adult horses with equine coronavirus infection at a veterinary teaching hospital: cases ( - ) evaluation of equine coronavirus fecal shedding among hospitalized horses experimental inoculation of equine coronavirus into japanese draft horses seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt persistence of antibodies against middle east respiratory syndrome coronavirus experimental reproduction of winter dysentery in lactating cows using bcv-comparison with bcv infection in milk-fed calves porcine epidemic diarrhea virus (pedv) introduction into a naive dutch pig population in middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study sars-cov antibody prevalence in all hong kong patient contacts cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer coronavirus spike protein and tropism changes mechanisms of coronavirus cell entry mediated by the viral spike protein serological assays for emerging coronaviruses: challenges and pitfalls serological screening for coronavirus infections in cats development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses detection of equine coronavirus in horses in the united kingdom production of recombinant protein therapeutics in cultivated mammalian cells specific serology for emerging human coronaviruses by protein microarray sensitive and specific detection of low-level antibody responses in mild middle east respiratory syndrome coronavirus infections coronavirus infections in horses in saudi arabia and oman seroprevalence and risk factors for infection with equine coronavirus in healthy horses in the usa identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design coronavirus and gastroenteritis in foals rotavirus and coronavirus associated diarrhoea in domestic animals isolation of coronavirus-like agent from horses suffering from acute equine diarrhoea syndrome concurrent cryptosporidium and coronavirus infections in an arabian foal with combined immunodeficiency syndrome genomic dissection of an icelandic epidemic of respiratory disease in horses and associated zoonotic cases we are grateful to udeni b.r. balasuriya (louisiana animal disease diagnostic laboratory and department of pathobiological sciences, school of veterinary medicine, louisiana state university, baton rouge, louisiana, usa) for providing strain nc and hrt- g cells and to sigríður björnsdóttir (icelandic food and veterinary authority, selfoss, iceland) and vilhjálmur svansson (institute for experimental pathology, university of iceland, reykjavik, iceland) for providing sera from icelandic horses. we also thank heleen zweerus for her practical assistance. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - f ecg authors: kompanikova, j.; zumdick, a.; neuschlova, m.; sadlonova, v.; novakova, e. title: microbiologic methods in the diagnostics of upper respiratory tract pathogens date: - - journal: clinical research and practice doi: . / _ _ sha: doc_id: cord_uid: f ecg upper respiratory tract infection (uri) is a nonspecific term used to describe acute infections involving the nose, paranasal sinuses, pharynx, and larynx above the vocal cords. the aim of this study was to provide a summary of the most common pathogens of uri and to compare advantages and disadvantages of traditional and new rapid microbiological tests used to identify them. blood samples were simultaneously examined by the enzyme-linked immunosorbent assay (elisa) and by the filmarray respiratory panel for eight different pathogens in a total of tests performed in nasopharyngeal swabs. the elisa method is unable to identify the pathologic agent until the host’s immune system elicits a response. the method is readily available in many laboratories at a low cost, which puts less strain on economic resources. the filmarray(®) panel, on the other hand, is more expensive, but it is fast and exact in the identification of a broad spectrum etiologic agents. nonetheless, since most repiratory tract infections are viral in origin and there is no treatment available, the diagnosis provided by the filmarray panel does not provide any additional clinical benefit and thus should be used only whenever necessary on the individual basis. upper respiratory tract infections (uris) involve the moist surface of the eyes and eyelids, the nasolacrimal ducts, the middle ear, paranasal sinuses, mastoid air cells, and the main respiratory passage of the nose and throat as far as the epiglottis and vocal cords. acute uris include the common cold, pharyngitis, epiglottitis, and laryngotracheitis (nester et al. ) . a variety of viruses, bacteria, fungi, and parasites can infect the respiratory tract. transmission of organisms occurs by aerosol droplet or direct hand-to-hand contact with infected secretions, with subsequent passage to the nares or eyes. most uris are of viral etiology (dasaraju and liu ) . epiglottitis and laryngotracheitis are exceptions with severe cases likely caused by haemophilus influenzae type b. bacterial pharyngitis is often caused by streptococcus pyogenes. bacterial and viral upper respiratory infections produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. proper treatment depends on the correct identification of a pathogen involved as antibiotics provide little or no benefit with viral infections (nester et al. ) . the uris are in % of viral origin (mäkelä et al. ) . orthomyxoviruses (influenza a and b), paramyxoviruses (parainfluenza and respiratory syncytial viruses), coronaviruses, adenoviruses, and enteroviruses (coxsackie and echo viruses) cause common cold. however, most colds are caused by more than types of rhinoviruses (musher ; cooper et al. ). more than strains of adenoviruses cause pharyngitis resembling a strep throat. rhinoviruses are unresponsive to antibiotics and other medications that control bacterial infections. antibiotic treatment of adenovirus infections is of no value and sometimes can even be harmful, because it supresses normal bacterial flora and enables the resistant opportunistic pathogens to grow in an uncontrolled way (nester et al. (murray et al. ) . the most common uri of bacterial origin is pharyngitis caused by group a beta-hemolytic streptococci, with streptococcous pyogenes as a main representative, which accounts for - % of pharyngitides (poole and portugal the filmarray panel is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple respiratory pathogen nucleic acids in nasopharyngeal swabs. this new platform combines automated sample preparation, nucleic acid extraction, and polymerase chain reaction (pcr)-based detection from a single unprocessed sample in h (biofire diagnostics; salt lake city, ut). this method allows for identification of different respiratory pathogens from a nasopharyngeal swab, of viral etiology and three of bacterial origin (idaho technology ). in the present study we seek to determine the individual advantages and disadvantages of different diagnostic tools for pathogens underlying the uris, as well as under which circumstances a specific method would have an advantage over another one. table . these results corresponded with a number of respiratory infections, as diagnosed before hand, such as uris, pneumonia, acute inflammation of nasopharynx, acute bronchitis, hypothermia unrelated to external temperature, and undefined fever. for comparison, specimens obtained from two patients were tested simultaneously with traditional elisa and filmarray panel methods. a venous blood sample and a nasopharyngeal swab were taken from both patients for either method, respectively. in one of these patients, all results were negative and no etiologic agent could be identied by the elisa method. in total, days were was required to obtain the full panel of results (table ; patient ). in addition, culture of the specimen obtained from a nasopharyngeal swab from the same patient also was negative for any pathogens. however, nasal swab specimen, examined by the filmarray panel, yielded a positive result for human rhinovirus or human enterovirus (table ; patient ), which was negative when with elisa. elisa performed in a second patient showed an elevation of igg against parainfluenza virus (igg positive - . , cut off - . , index - . ). this result, however, is inconclusive regarding an acute current infection as it rather confirms a previous encounter with the virus. the result regarding influenza a infection was also negative (igm - . , cut off - . , index - . ; and igg - . , cut off - . , index - . ). the testing time for all eight pathogens amounted to days (table ; patient ). the examination with the filmarray panel in this patient identified the etiologic agent as influenza a/h - (table ; patient ). the identification had to be confirmed with a serum hemagglutination inhibition test (hit), which was done at the department of virology of the regional institute of public health, in banská bystrica, slovakia. the hit was performed twice weeks apart to assess a possible change in antibody titers. while in the first sample influenza a and b titers were negative, there was a significant rise in the antibody titer against table . health insurance gave points for each of the tests, which makes a total points, plus points for sample culture from upper respiratory tract and points for a culture form lower respiratory tract. each insurance point has a value of . €. the laboratory therefore received . € for running the elisa and additionally . € for the cultures, which sums up to a total of . € or % of the real material costs. on the other hand, health insurance gave points for each of the pathogen targets in the filmarray panel, which makes a total of , points and comes to . € or % of the real material costs amounting to . € per sample. the insurance reimbursed . € for influenza a and b testing each. each type of influenza was tested twice giving the cost of . € in total. the actual laboratory costs for all hit tests were . €, which is about times less than the insurance reimbursement. laboratory testing is generally not recommended in the evaluation of upper respiratory infections. tests for specific pathogens are helpful when therapy depends on the results. targeted therapy is not available for most viruses that cause uri. therefore, viral testing is rarely indicated for uncomplicated uris in the outpatient setting. however, confirmation of a viral condition such as influenza may reduce inappropriate use of antibiotics (balentine and siamak ) . considering the benfits for patient, the speed to identify the etiologic agent clearly favors the filmarray system. this method readily identified the etiologic agent in both patients in whom it was applied in the present study, whereas the hit identified the virus only in the repeat sample of the second patient. however, identification of the etiologic agent did not have any benefit for the first patient with mild symptomatology of coryza because no targeted treatment for human rhinovirus/enterovirus infection was needed. the symptomatology in the second patient was severe and the speed of pathogen identification is crucial for the commencement of appropriate treatment, especially in suspected cases of influenza where early administration of neuraminidase inhibitors significantly reduces mortality rates. in such cases, accuratelly targeted therapy has an enormous benefit for the patient. the laboratory costs to run one examination with different methods showed that the filmarray multiplex pcr respiratory panel is more expensive than the elisa, hit, and the cultivation. one examination with the filmarray panel brought a . € per patient profit for the laboratory, whereas the profit from running elisa together with cultivation was . €, and that from hit was . € per patient. therefore, filmarray respiratory panel is best in terms of profit margin for the laboratory and least favorable for health insurance. serologic diagnostic methods, such as elisa and hit, cannot identify the pathologic agent until the host's immune system elicits a response. however, advantages of those methods are that they are readily available in many laboratories and are least pricey for health insurance. the disadvantage is that the spectrum of pathogens detected is small. a clinical benefit of the filmarray respiratory panel is that it is quick and exact and may identify a broader spectrum of possible pathogens. since most uris are viral in origin and there is no treatment available, the diagnosis provided by the filmarray panel is not always necessary and the method should be used on an individual basis when clinically justified. from the economic standpoint, filmarray respiratory panel is the most profitable for the laboratory. in critically ill patients, a spectrum of diagnostic methods should be used to obtain diagnosis as fast as possible. in patients with minor respiratory tract infections, a more rational approach should be undertaken since the speed and accuracy of diagnosis are less crucial. the decision to choose a specific diagnostic method rests with the medical caregiving staff. upper respiratory tract infection principles of appropriate antibiotic use for acute pharyngitis in adults: background infections of the respiratory system lippincott's illustrated reviews: microbiology viruses and bacteria in the etiology of the common cold upper respiratory tract infection workup medical microbiology, th edn how contagious are common respiratory tract infections? microbiology: a human perspective treatment of rhinosinusitis in the outpatient setting characteristics of streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and staphylococcus aureus isolated from the nasopharynx of healthy children attending day-care centres in the czech republic acknowledgments this study was supported by department of microbiology and immunology. we are thankful to our colleagues who provided expertise that greatly assisted the research. the authors declare no conflicts of interest in relation to this article. key: cord- -g go u authors: kovac, marc; risch, lorenz; thiel, sarah; weber, myriam; grossmann, kirsten; wohlwend, nadja; lung, thomas; hillmann, dorothea; ritzler, michael; bigler, susanna; ferrara, francesca; bodmer, thomas; egli, konrad; imperiali, mauro; heer, sonja; salimi, yacir; renz, harald; kohler, philipp; vernazza, pietro; kahlert, christian r.; paprotny, matthias; risch, martin title: edta-anticoagulated whole blood for sars-cov- antibody testing by electrochemiluminescence immunoassay (eclia) and enzyme-linked immunosorbent assay (elisa) date: - - journal: diagnostics (basel) doi: . /diagnostics sha: doc_id: cord_uid: g go u while lateral flow test formats can be utilized with whole blood and low sample volumes, their diagnostic characteristics are inferior to immunoassays based on chemiluminescence immunoassay (clia) or enzyme-linked immunosorbent assay (elisa) technology. clias and elisas can be automated to a high degree but commonly require larger serum or plasma volumes for sample processing. we addressed the suitability of edta-anticoagulated whole blood as an alternative sample material for antibody testing against sars-cov- by electro-clia (eclia; roche, rotkreuz, switzerland) and elisa (igg and iga; euroimmun, germany). simultaneously drawn venous serum and edta-anticoagulated whole blood samples from individuals were included. correction of the whole blood results for hematocrit led to a good agreement with the serum results for weakly to moderately positive antibody signals. in receiver-operating characteristic curve analysis, all three assays displayed comparable diagnostic accuracy (area under the curve (auc)) using corrected whole blood and serum (aucs: . for eclia and igg elisa; . for iga elisa). in conclusion, our results suggest that the investigated assays can reliably detect antibodies against sars-cov- in hemolyzed whole blood anticoagulated with edta. correction of these results for hematocrit is suggested. this study demonstrates that the automated processing of whole blood for identification of sars-cov- antibodies with common eclia and elisa methods is accurate and feasible. the coronavirus disease (covid- ) constitutes a recent global pandemic caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) virus. whereas acute disease diagnosis by laboratory methods predominantly utilizes demonstration of virus replication by real-time reverse transcriptase polymerase chain reaction (rt-pcr), serologic tests are mainly employed for diagnosis of past covid- infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . demonstration of specific antibodies against the sars-cov- also allows for confirmation of clinically suspected covid- cases for which rt-pcr testing has been negative [ , ] . from a public health perspective, serologic tests are also employed for estimating the proportion of individuals already infected with the sars-cov- and facilitating contact tracing, as well as the surveillance or identification of individuals who are susceptible to covid- infection [ ] . antibodies against the sars-cov- develop within - days after covid- symptom onset [ ] . specific igm or iga can precede the development of specific igg [ , ] . however, specific igg can also develop along with or before the occurrence of specific igm or iga [ ] . the specificity of antibodies against the sars-cov- is directed against several virus-specific proteins, such as the nucleocapsid (n) or spike (s) proteins [ , ] . n or s (s and/or s ) are the target antigens in most of the commonly employed immunoassays in clinical diagnostics [ ] [ ] [ ] [ ] . it has been shown that the antibodies against the receptor-binding domain (rbd) of s , as well as the n-protein, correlate closely with the virus neutralization titer [ ] . rbd-binding enables the sars-cov- to infect the target cell via the angiotensin-converting enzyme (ace ) receptor [ ] . several assay types have been developed for detection of the covid- -specific immune response, including immune chromatographic lateral flow assays [ ] , enzyme-linked immunosorbent assays (elisas) [ , , , ] , and chemiluminescence assays (clias) [ , ] . lateral flow assays were marketed early by a large variety of manufacturers. the diagnostic accuracy and utility of some of these products has been questioned. however, since lateral flow formats can be used with minimal sample volume (as little as µl) and a variety of materials (e.g., whole blood, serum, and plasma), capillary blood sampling is frequently employed with this assay format [ ] . other assay formats (elisas and clias) have been reported to possess better diagnostic characteristics and more efficient and higher throughput. however, due to the dead volume needed for trouble-free automated sample processing, these assays need more sample volume (minimum of - µl) than lateral flow tests. further, these assays have only been validated for cell-free matrices, such as serum and plasma. since the frequency of requests for the covid- antibody test continues to increase, automated testing is becoming more and more important. as capillary samples are characterized by low sample volume, and separation of cells from plasma or serum further reduces the sample volume [ ] , capillary samples are rarely processed on high-throughput laboratory analyzers [ ] . the issue of low sample volume associated with capillary blood sampling for testing with clias and elisas may be circumvented by employing whole blood as a matrix. a total sample volume of µl whole blood would allow for automated and efficient processing of capillary blood samples for sars-cov- antibody testing with clias or elisas. thus, we investigated whether the results from sars-cov- antibody testing with edta-anticoagulated whole blood would be comparable to the results obtained in serum. in this study, we analyzed paired serum and edta-whole blood samples from individuals who were tested for sars-cov- antibodies. the study was conducted with anonymized blood samples from patients referred to the labormedizinisches zentrum dr. risch ostschweiz ag (buchs sg, switzerland). the study protocol was verified by the ethics board of eastern switzerland (ekos; basec nr. req- - ; approval date . . ), which waived informed consent for performing laboratory analysis on anonymized samples. samples from patients referred for covid- serology for whom an edta-whole blood sample was available from the same venipuncture were included. for each sample, the age and gender of the individual, the results from sars-cov- rt-pcr analysis (if performed), and the delay from rt-pcr analysis to antibody testing (days) were documented. serum and edta-whole blood samples referred for complete blood count testing were kept at • c to • c before analysis of sars-cov- antibodies, which was conducted daily on weekdays. before placing the serum samples on laboratory analyzers, the samples were brought to room temperature over h and homogenized by vortexing. before antibodies were identified in edta-whole blood, hemolysis was induced by freezing the sample to − • c for h after homogenization on a sample roller for min. after freezing, the hemolyzed edta-whole blood samples were brought to room temperature over h on a sample roller and then put on the analyzer. for antibody testing using eclia, the antibodies were tested on a cobas (roche diagnostics, rotkreuz, switzerland) using the elecsys ® anti-sars-cov- assay (roche diagnostics, rotkreuz, switzerland). this assay was ce-marked, as well as cleared by the u.s. food and drug administration (fda), and employs a recombinantly engineered nucleocapsid antigen for detection of total immunoglobulin. according to the manufacturer, serum and edta-plasma or heparin-plasma, but not edta-whole blood have been specified as acceptable specimens. additionally, the samples were analyzed by elisa (euroimmun anti-sars-cov- igg and iga elisas, euroimmun, luzern, switzerland) run on a dsx analyzer (dynex technologies, denkendorf, germany) . these ce-marked assays detect specific igg and iga directed against the s spike protein of the sars-cov- . serum, edta-plasma, heparin-plasma, and citrate-plasma, but not edta-whole blood have been specified by the manufacturer as acceptable specimens. hematocrit was assessed before inducing hemolysis in the edta-whole blood samples using a sysmex xs- i instrument (sysmex, horgen, switzerland), employing the cumulative pulse height method . the inter-series coefficients of variation (cvs) were . % for the elecsys ® anti-sars-cov- assay (at a mean cutoff index [coi] of . ; coi for positivity > ), . % for the euroimmun anti-sars-cov- igg elisa (mean ratio of the extinction of the control patient sample over the extinction of the calibrator, [s/c] of . ; s/c for positivity > . ), . % for the euroimmun anti-sars-cov- iga elisa (mean s/c of . ; s/c for positivity > . ), and . % for hematocrit (at a mean level of . %). continuous variables are presented as medians and interquartile ranges (iqrs), whereas proportions are given as percentages with % confidence intervals (cis). associations between variables were calculated with the spearman's rank correlation. proportions were compared using the chi-square tests. results obtained by the eclia assay, as well as the elisas measuring edta-whole blood were corrected for hematocrit by multiplying the result with the reciprocal of the hematocrit to render them comparable to the serum measurements. the bland-altman analysis was performed by comparing the results corrected for measured hematocrit to the results obtained in serum. this type of analysis assesses accuracy and precision of the antibody measurements by relating the difference between the hemolyzed edta-whole blood and serum measurements to the average of the two results in each patient. the limits of agreement are given by the mean + . standard deviations (sds) containing % of the values. the mean difference is a measure for accuracy, and the limits of agreement are a measure of precision. a method comparison was also performed using the passing-bablok regression analysis. the proportion of positive results in the edta-whole blood samples among the positive results obtained by the same method in the corresponding serum samples, as well as the proportion of negative results in the edta-whole blood samples among the negative results in serum were calculated. diagnostic sensitivities and specificities for the cutoffs provided by the manufacturers were calculated for serum, edta-whole blood samples without hematocrit correction, and edta-whole blood results corrected for hematocrit. further, diagnostic accuracy was assessed for the different sample materials and assays by means of receiver-operating characteristic (roc) curves, which were compared by the method of delong . as a reference standard for determination of disease status, a positive rt-pcr result or one n-along with one s-antigen-positive antibody assay was considered to be covid- -positive, whereas cases with no or negative pcr and negative results from one n-and one s-antigen assay were considered negative for covid- . finally, p-values < . were considered to be statistically significant. the medcalc software version . . (mariakerke, belgium) was used for all statistical calculations. paired serum and edta-whole blood samples were available from patients ( females, %; % ci: , ). the median age of the patients was years old (iqr: , ). covid- was diagnosed in patients ( %; % ci: , ) with either rt-pcr (n = ) or two positive antibody results in one assay against the n-antigen and one assay against the s-antigen (igg or iga; n = ). for all patients with rt-pcr results (n = ), serum samples were taken after a median of days (iqr: , ) following rt-pcr. serum antibody testing was positive with the eclia, igg elisa, and iga elisa in , , and samples, respectively. the median hematocrit was % (iqr: , ). we observed a close correlation between the results obtained in serum and hemolyzed edta-whole blood with or without correcting for hematocrit. the correlations between the results obtained in serum and hemolyzed edta-whole blood not corrected for hematocrit were r = . (p < . ) in the igg elisa, r = . (p < . ) in the iga elisa, and r = . (p < . ) in the eclia. the respective correlations between the serum and hemolyzed edta-whole blood results corrected for hematocrit were similar: r = . (p < . ) in the igg elisa, r = . (p < . ) in the iga elisa, and r = . (p < . ) in the eclia. the passing-bablok regression analysis with the serum results as the independent variable and the whole blood results not corrected for hematocrit as the dependent variable revealed a slope (s) of . and an intercept (i) of . for the igg elisa, . (s) and . (i) for the iga elisa, and . (s) and − . (i) for the eclia. the respective parameters for serum and whole blood corrected for hematocrit were . (s) and − . (i) for the igg elisa, . (s) and − . (i) for the iga elisa, and , (s) and − . (i) for the eclia. inspection of the bland-altman plots ( figure ) illustrated that the results between serum and whole blood corrected for hematocrit were comparable up to a s/c of in the igg elisa (cutoff for positivity s/c ≥ . ; figure a ) and in the iga elisa (cutoff for positivity s/c ≥ . ; figure b ), as well as up to a coi of in the eclia (cutoff for positivity coi ≥ ; figure c ). it can thus be concluded that the anti-sars-cov- antibody results in whole blood corrected for hematocrit with weakly and moderately positive findings are comparable to those obtained from serum. in the whole blood results not corrected for hematocrit, of the positive eclia serum results were found to be positive ( %; % ci: , ). the other respective proportions were out of ( %; % ci: , ) in the igg elisa and out of ( %; % ci: , ) in the iga elisa. in the whole blood results corrected for hematocrit, of the positive eclia serum results were found to be positive ( %; % ci: , . among the initially negative eclia results in serum, one sample showed a positive result in the whole blood results corrected for hematocrit (coi in serum/corrected whole blood: . / . ). this sample had positive results in the igg and iga elisas. thus, this sample can be considered as a false-negative in the eclia serum results. among the initially negative igg elisa results in serum, nine samples showed positive results in the whole blood results corrected for hematocrit (s/c in serum/corrected whole blood: / . ; / . ; . / . ; . / . ; / . ; . / . ; . / . ; . / . ; . / . ). interestingly, all but one sample had positive eclia results, and the remaining sample ( . / . ) had a positive iga elisa result (s/c: in the whole blood results not corrected for hematocrit, of the positive eclia serum results were found to be positive ( %; % ci: , among the initially negative eclia results in serum, one sample showed a positive result in the whole blood results corrected for hematocrit (coi in serum/corrected whole blood: . / . ). this sample had positive results in the igg and iga elisas. thus, this sample can be considered as a false-negative in the eclia serum results. among the initially negative igg elisa results in serum, nine samples showed positive results in the whole blood results corrected for hematocrit (s/c in serum/corrected whole blood: / . ; / . ; . / . ; . / . ; / . ; . / . ; . / . ; . / . ; . / . ). interestingly, all but one sample had positive eclia results, and the remaining sample ( . / . ) had a positive iga elisa result (s/c: . ). it could thus be concluded that all nine samples were likely false-negatives in the igg elisa serum results. therefore, the specificity of the igg elisa assay using whole blood corrected for hematocrit is unaffected compared to its serum performance. among the initially negative iga elisa results in serum, samples showed positive results in the whole blood results corrected for hematocrit (s/c in serum/corrected whole blood: . / . ; . / . ; . / . ; / . ; / . ; . / . ; . / . ; . / . ; . / . ; . / . ; . / . ; / . ; / . ; / . ). only six of these samples had positive eclia results. the other samples were also clearly negative in the igg elisa. it can thus be concluded that the specificity of the iga elisa using whole blood corrected for hematocrit decreases compared to its serum performance. in summary, correcting the whole blood results for hematocrit improved the sensitivity of the whole blood measurement, while leaving specificity relatively unchanged compared to the eclia serum results. correcting the whole blood results for hematocrit in the igg elisa increased the sensitivity of the method compared to serum assessments, while specificity remained unchanged. finally, correcting the whole blood results for hematocrit in the iga elisa improved sensitivity at the cost of a somewhat decreased specificity. given these findings, it can be concluded that for the eclia and igg elisa, the results are not inferior when using whole blood corrected for hematocrit instead of serum. the diagnostic sensitivities and specificities of the different assays are shown in table . using the predefined cutoffs provided by the manufacturers, the eclia in serum exhibited a significantly higher diagnostic sensitivity than both elisas (p < . for both). in whole blood corrected for hematocrit, the sensitivities of the eclia and igg elisa did not differ significantly. the igg elisa had a significantly higher diagnostic sensitivity than the iga elisa (p < . ). in serum and whole blood corrected for hematocrit, the diagnostic specificities were significantly better for the eclia and igg elisa than the iga elisa (p < . ), whereas there was no significant difference between the eclia and igg elisa. for all three of the investigated assays, changes in the diagnostic sensitivities and specificities were not significantly different when using whole blood corrected for hematocrit instead of serum. roc analysis revealed comparable aucs between the different materials for all of the investigated assays: . for serum, . for whole blood, and . for whole blood corrected for hematocrit in the eclia; . , . , and . in the igg elisa; and . , . , and . in the iga elisa. there were no statistical differences in the aucs between the eclia and igg elisa regardless of the sample material used. both assays for all materials were significantly better than the iga elisa using whole blood with or without correction for hematocrit (p < . ). the aucs of the different assays in serum and hemolyzed edta-whole blood with correction for hematocrit are shown in figure . the three assays had comparable diagnostic characteristics when using serum or hemolyzed edta-whole blood with or without correction for hematocrit for analysis. with conventional cutoffs, correction of the whole blood results for hematocrit appeared to preserve, or even increase sensitivity in all methods. together, our results support the use of whole blood as a valid material for anti-sars-cov- antibody testing with the three investigated assays. to the best of our knowledge, the only anti-sars-cov tests that have been approved for edtawhole blood use are immunochromatographic lateral flow rapid tests [ ] . even though some of these formats have acceptable diagnostic characteristics, especially for specific igg-antibodies, lateral flow test formats are considered inferior to commonly used automated assays employing clia or elisa techniques [ ] . automated immunoassays, however, have not been validated with whole blood. our data show that whole blood constitutes an acceptable sample material for automated testing in the investigated test formats. this finding is important because it allows for the use of capillary blood on automated analyzers. such analyzers have a dead volume of up to µl, and covid- tests need test volumes that are approximately µl. collection of capillary blood is a method that can be used outside of medical institutions, which may present an advantage when testing is performed on a population level. in our experience, capillary samples rarely exceed a volume of µl. centrifuging such samples in order to obtain plasma or serum is laborious and does not provide enough sample volume (< µl) for appropriate processing. utilizing the entire blood sample of - µl without centrifugation may allow for the use of capillary blood samples in highly automated clia or elisa analysis systems. until now, capillary blood samples were primarily utilized for lateral flow tests. in addition to a somewhat inferior performance, these tests are limited by the fact that they cannot be automatized and do not allow for the provision of numeric results to assess antibody response development. thus, the combination of capillary blood sampling and high-quality tests may be very important in terms of planning and conducting large epidemiological studies [ ] . our data show that correcting for hematocrit aids detection rates in all three investigated assays compared to the results of whole blood without correction. should it not be possible to obtain a hematocrit measurement, the whole blood results may be corrected for a mean hematocrit value (e.g., %) to be used with the conventional antibody cutoffs. the sensitivity and specificity of such an the three assays had comparable diagnostic characteristics when using serum or hemolyzed edta-whole blood with or without correction for hematocrit for analysis. with conventional cutoffs, correction of the whole blood results for hematocrit appeared to preserve, or even increase sensitivity in all methods. together, our results support the use of whole blood as a valid material for anti-sars-cov- antibody testing with the three investigated assays. to the best of our knowledge, the only anti-sars-cov tests that have been approved for edta-whole blood use are immunochromatographic lateral flow rapid tests [ ] . even though some of these formats have acceptable diagnostic characteristics, especially for specific igg-antibodies, lateral flow test formats are considered inferior to commonly used automated assays employing clia or elisa techniques [ ] . automated immunoassays, however, have not been validated with whole blood. our data show that whole blood constitutes an acceptable sample material for automated testing in the investigated test formats. this finding is important because it allows for the use of capillary blood on automated analyzers. such analyzers have a dead volume of up to µl, and covid- tests need test volumes that are approximately µl. collection of capillary blood is a method that can be used outside of medical institutions, which may present an advantage when testing is performed on a population level. in our experience, capillary samples rarely exceed a volume of µl. centrifuging such samples in order to obtain plasma or serum is laborious and does not provide enough sample volume (< µl) for appropriate processing. utilizing the entire blood sample of - µl without centrifugation may allow for the use of capillary blood samples in highly automated clia or elisa analysis systems. until now, capillary blood samples were primarily utilized for lateral flow tests. in addition to a somewhat inferior performance, these tests are limited by the fact that they cannot be automatized and do not allow for the provision of numeric results to assess antibody response development. thus, the combination of capillary blood sampling and high-quality tests may be very important in terms of planning and conducting large epidemiological studies [ ] . our data show that correcting for hematocrit aids detection rates in all three investigated assays compared to the results of whole blood without correction. should it not be possible to obtain a hematocrit measurement, the whole blood results may be corrected for a mean hematocrit value (e.g., %) to be used with the conventional antibody cutoffs. the sensitivity and specificity of such an approach reveal comparable results to those provided for whole blood corrected for measured hematocrit in table (data not shown). however, it would also run the risk of false-positive antibody results, especially in patients with anemia, whereas false-negative antibody results would be found in patients with polyglobulia. in order to prevent misclassification as much as possible, we recommend correcting for hematocrit. if this is not possible, a coi value of < . in the eclia or a s/c value < . in the igg and iga elisas regardless of the hematocrit result would, with a high probability, correspond to a negative result in serum, even in patients with anemia (not lower than a hematocrit of %, which corresponds to a hemoglobin level of g/l using the rule of three for converting hematocrit into hemoglobin levels [ ] ). we did not assess the effect of edta concentration on antibody results, such as in the case of inadequately filled tubes [ ] . increased edta concentration leads to increased binding of metallic ions (e.g., europium, zinc, and magnesium), which are either used as immunoassay reagents (europium) or as cofactors (zinc and magnesium) for the enzymes used in signal generation, such as alkaline phosphatase [ ] . further, it has been shown that reagent antibodies in immunoassays can recognize divalent cation complex binding sites on proteins [ ] . reduced availability of such cation complexes may induce conformational changes in proteins with altered immunoreactivity [ ] . we do not believe that such interference would affect the investigated assays because we do not expect conformational changes in the epitope-recognizing site in the analyte (sars-cov- antibodies). additionally, neither europium nor alkaline phosphatase is employed as a reporter system in the investigated assays. a further limitation of the present study is that only one chemiluminescence assay, one igg elisa, and one iga elisa format have been employed. it is therefore not possible to infer that our findings would be applicable to all other elisa and chemiluminescence assay formats. however, we do not believe that these limitations invalidate our findings. in conclusion, we demonstrated that edta-anticoagulated whole blood represented a sample material that could be employed with commonly used immunoassay formats that allowed for highly automated throughput of samples for sars-cov- antibody testing. in clinical practice, serum samples are not always available for analysis, and our investigation may help patients with a need for sars-cov- antibody testing who are in this situation. the fact that whole blood was successfully utilized in the investigated test formats suggests that capillary blood samples, if properly taken, might also be suitable for sars-cov- antibody testing-not only with lateral flow test formats, but also immunoassays of higher quality. capillary blood samples have already been shown to facilitate epidemiological studies of infectious disease antibodies by means of home sampling [ ] . thus, the present study is useful for validating the aforementioned conditions for epidemiological studies or clinical practice. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. should rt-pcr be considered a gold standard in the diagnosis of covid- ? antibody responses to sars-cov- in patients with covid- value of diagnostic testing for sars-cov- /covid- role of serology in the covid- pandemic the role of antibody testing for sars-cov- : is there one? temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study early detection of sars-cov- antibodies in covid- patients as a serologic marker of infection molecular and serological tests for covid- a comparative review of sars-cov- coronavirus laboratory and point-of-care diagnostics covid- diagnostics, tools, and prevention. diagnostics covid- serological tests: how well do they actually perform? diagnostics profiling early humoral response to diagnose novel coronavirus disease (covid- ) will antibody tests for the coronavirus really change everything? in vitro diagnostic assays for covid- : recent advances and emerging trends performance characteristics of the abbott architect sars-cov- igg assay and seroprevalence in severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease analytical performances of a chemiluminescence immunoassay for sars-cov- igm/igg and antibody kinetics structure of the sars-cov- spike receptor-binding domain bound to the ace receptor evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-sars-cov- antibodies evaluation of commercial and automated sars-cov- igg and iga elisas using coronavirus disease (covid- ) patient samples evaluation of a covid- igm and igg rapid test; an efficient tool for assessment of past exposure to sars-cov- hemoglobin/hematocrit and other erythrocyte parameters capillary blood for point-of-care testing chlamydia trachomatis antibody detection in home-collected blood samples for use in epidemiological studies comparison of three-fold converted hematocrit and micro-hematocrit in pregnant women interferences from blood collection tube components on clinical chemistry assays interferences in immunoassay key: cord- - f qc authors: toftaker, ingrid; toft, nils; stokstad, maria; sølverød, liv; harkiss, gordon; watt, neil; o’ brien, amanda; nødtvedt, ane title: evaluation of a multiplex immunoassay for bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk against two indirect elisas using latent class analysis date: - - journal: prev vet med doi: . /j.prevetmed. . . sha: doc_id: cord_uid: f qc bovine respiratory syncytial virus (brsv) and bovine coronavirus (bcv) are responsible for respiratory disease and diarrhea in cattle worldwide. the norwegian control program against these infections is based on herd-level diagnosis using a new multiplex immunoassay. the objective of this study was to estimate sensitivity and specificity across different cut-off values for the mvd-enferplex bcv/brsv multiplex, by comparing them to a commercially available elisa, the svanovir(®) bcv-ab and svanovir(®) brsv-ab, respectively. we analyzed bulk tank milk samples from herds in a low- and herds in a high-prevalence area. as none of the tests were considered perfect, estimation of test characteristics was performed using bayesian latent class models. at the manufacturers’ recommended cut-off values, the median sensitivity for the brsv multiplex and the brsv elisa was . [ . – . % posterior credibility interval (pci)] and . [ . – % pci], respectively. the median specificity for the brsv multiplex was . [ . – . % pci], but only . [ . – . % pci] for the brsv elisa. however, increasing the cut-off of the brsv elisa increased specificity without compromising sensitivity. for the bcv multiplex we found that by using only one of the three antigens included in the test, the specificity increased, without concurrent loss in sensitivity. at the recommended cut-off this resulted in a sensitivity of . [ . – % pci] and specificity of . [ . – . % pci] for the multiplex and a sensitivity of . [ . – % pci] and a specificity of . [ . – % pci] for the bcv elisa. bovine coronavirus (bcv) and bovine respiratory syncytial virus (brsv) are commonly occurring agents among cattle worldwide (valarcher and taylor, ; boileau and kapil, ) . they are endemic and prevalent also in the norwegian dairy herd (gulliksen et al., ; klem et al., a) . bcv causes respiratory disease, calf diarrhea and winter dysentery (contagious diarrhea in adult cattle) (boileau and kapil, ) . brsv causes respiratory disease mostly in young animals but can affect animals of all ages, and is a common cause of respiratory outbreaks in norway (larsen, ; klem et al., a) . consequences of these infections are herd health problems, reduced animal welfare and increased use of antibiotics due to secondary bacterial infections (larsen, ; valarcher and taylor, ; boileau and kapil, ) . therapy costs and reduced production entails considerable financial loss for the farmer, and contributes to a present focus in nordic countries on how to limit the spread of these viruses in the cattle population. in , a national control program against brsv and bcv infections was launched in norway as a joint initiative between the producer organizations. this prompted the need for an easy and cost-effective way to screen dairy herds for a herd level diagnosis for brsv and bcv. the initial screening in the control program was conducted using bulk tank milk samples (btm). there are already commercially available indirect enzyme-linked immunosorbent assays (elisas) widely used in routine diagnostics and research in the nordic countries (svanovir ® brsv-ab and svanovir ® bcv-ab) (tråvén et al., ; klem et al., t b; toftaker et al., ) . however, in order to optimize cost-effectiveness of the control program, the development of a new multiplex antibody elisa was initiated (mvd-enferplex bcv/brsv multiplex). the new test allowed screening for both viruses by the use of a single test. the performance of a diagnostic test is characterized by the test's sensitivity (se) and specificity (sp), where se is the proportion of true positives correctly classified as positive by the test, and the sp is the proportion of true negative subjects correctly classified as negative. the true antibody status of each test subject can be determined in two ways: by use of a perfect reference test, or based on populations with known status. however, a perfect reference test (often termed a "gold standard") is rarely available and for endemic diseases, which is the case for brsv and bcv in norway, no reference population with complete certainty regarding disease or disease freedom exists. consequently, the underlying true infection status for test subjects remains unknown. test validation studies (erroneously) assuming perfect reference tests are common, even though this has been shown to introduce bias in the estimation of accuracy parameters (valenstein, ; lijmer et al., ) . latent class analysis (lca) allows for the estimation of test parameters in populations where the underlying true infection status cannot be determined (hui and walter, ) . in lca the true infection status is treated as an existing, but unknown (latent), variable and test accuracy and prevalence are parameterized according to this latent variable. as the brsv/bcv multiplex is a new test, it needs to be validated. test characteristics are different when a test is used as a herd test, compared to when it is used on individual samples (christensen and gardner, ) and validation for the relevant application is therefore important. btm testing is a key component of the norwegian brsv/ bcv control-program, it is therefore of interest to estimate test accuracy, at different cut-off values, for this application. the aim of this study was to estimate the test sensitivity and specificity of the newly developed mvd-enferplex bcv/brsv multiplex across different cut-off values, for detection of antibodies in btm. the bcv part of the multiplex was compared to the commercially available svanovir ® bcv-ab, and the brsv part of the multiplex was compared to the svanovir ® brsv-ab. as neither test could be considered perfect, the evaluation was done using lca. a cross-sectional sampling design was used for the present study. herds were eligible for inclusion if they delivered milk to the largest dairy company in norway (tine sa), and provided a btm sample during the study period (march ). herds from two counties with an expected difference in true prevalence (tp) were selected in order to meet the model assumptions, described in the lca section. using a random numbers generator, samples were randomly chosen from herds in "oppland" (pop ) and from herds in "sogn og fjordane" (pop ) counties. "sogn og fjordane" is located in western norway, and was assumed to have a relatively low prevalence, based on results from a previous study (toftaker et al., ) . oppland county, located in eastern norway, was thought to have higher prevalence based on known patterns of animal movements and a history of previous outbreaks of disease (toftaker et al., ) . btm samples were collected from nearly all norwegian dairy herds delivering milk to the largest dairy cooperation (tine sa) during march . the samples were collected as part of the national control program against brsv and bcv. the milk truck driver collected samples at ordinary milk shipment using standard procedures for btm sampling. the milk was then stored at °c until received at the laboratory (tine mastitis laboratory, molde, norway) where samples were frozen and shipped over-night to the enfer laboratory in ireland (enfer scientific, naas, ireland). samples were kept frozen until the time of laboratory analysis. the svanovir ® brsv-ab, hereafter designated the brsv elisa, and svanovir ® bcv-ab, hereafter designated the bcv elisa, were used on all samples, following the manufacturer's instructions. the optical density (od) reading of nm was corrected by the subtraction of od for the negative control antigen, and percent positivity (ppvalue) was calculated as (corrected od/positive control corrected od) × . according to the test manuals, the recommended cut-off values of sample positive > pp for both tests were used as a starting point for these tests (svanova; svanova) . for the brsv elisa the se and sp provided by the manufacturer were % and %, respectively. these parameters are calculated from serum samples, and parameters specific for btm samples have not been reported (elvander et al., ) . for the bcv elisa the test parameters provided by the manufacturer were se of . % and sp of %, and as for brsv the calculations are based on serum samples (alenius et al., ) . all samples were analyzed using the mvd-enferplex bcv/brsv multiplex, hereafter referred to as the brsv/bcv multiplex (enfer scientific, naas, ireland). a panel of three bcv recombinant proteins (bcv a-c), along with a panel of two recombinant proteins and two synthetic peptides for brsv (brsv a-d) were used as antigens. briefly, the antigens were deposited in a multiplex planar array as individual spots into wells of well microtiter plates to produce arrays of antigens. samples were diluted : into sample dilution buffer and mixed before added to the well and incubated at °c for min with agitation. after washing procedures, the detection antibody diluted in conjugate buffer was added and plates were incubated ( °c for min with agitation) before new washing. finally, the chemiluminescent substrate was added. relative light units (rlu) were captured ( s exposure) immediately, using quansys biosciences imaging system, and data was extracted using quansys q view software (v . . . ). antigens were combined in a parallel reading, i.e. the test was considered positive when the rlu-value of at least one antigen was above the applied cut-off. laboratory personnel were not formally blinded to test results, but due to the large volume of samples they were considered blinded for any practical purposes. because the multiplex consisted of several antigens each giving a separate response, a separate cut-off value was needed for each antigen. we calculated the proportion of herds that had a positive response to each of the individual antigens within the test-positive group (at manufacturers recommended cut-off values), and defined the antigen with the highest proportion of positive responses as the most influential. this was done for both viruses. when later choosing which cut-off values to assess, changing the cut-off for the most influential antigen for each virus was prioritized. we used an explorative approach to selecting cut-off values, and several different cut-off values were tried for the most influential antigen (fig. ) . furthermore, we evaluated test performance when including only the single most important antigen. data preparation and descriptive analysis were performed in stata (stata se/ ; stata corp., college station, tx). in the present study, we used guidelines for reporting of diagnostic accuracy in studies that use bayesian lca (kostoulas et al., ) . the target condition was herds with one or more animals producing bcv/brsv-antibodies while contributing to the bulk tank. the underlying latent state could be considered as previous exposure, leading to antibodies in btm. the use of lca methodology for diagnostic test evaluation requires a set of assumptions of the tests and test populations to be fulfilled. ( ) two or more populations with different prevalence are included, ( ) the se and sp of the diagnostic tests are the same across the populations, and ( ) the tests are conditionally independent (cid) given disease status (hui and walter, ) . we ran the analyses assuming cid between tests; however, we also explored the consequences of relaxing this assumption as explained below. for the cid-models, parameters were estimated for several cut-off values (fig. ). models were fit using bayesian lca in the openbugs version . . rev software. we used non-informative priors in the shape of uniform distributions on the interval between zero and one, modelled using the beta ( , ) distribution for test properties and sub-population prevalence in all analyses. models were run with , iterations, of which , were used as burn in and discarded. convergence of the markov chain monte carlo (mcmc) chains were assessed by visual inspection of history plots, time-series plots and gelman-rubin diagnostic plots using three sample chains with different initial values, as suggested by toft et al. ( ) . posterior inference was done by calculating medians and % posterior credibility intervals (pci) for se, sp and true prevalence. the model description is included in appendix a. a correlation between tests, if present, is not possible to estimate in a two tests scenario without including informative priors. we did not have any reliable prior information on test performance or prevalences in the present study. however, the consequences of relaxing the assumption of conditional independence given disease status was first explored by vacek ( ) , who examined the impact of conditional dependence by assuming a fixed proportion of the maximum possible covariance between tests. following this approach we explored the consequences of conditional dependence between tests for the cut-off values with the preferred test characteristics. (fig. : alternative for brsv, alternative for bcv.) see appendix a for details. we compared the results of the conditional independence model to models allowing , , and % of the maximum possible positive covariance, as well as a negative covariance of − %. a combination of different cut-off values for the included antigens, (cut-off alternatives - ) are presented in fig. for the brsv-and bcv multiplex. for the brsv multiplex, the brsv-a antigen was responsible for detecting the majority of the positive samples. for the bcv multiplex the antigen detecting the majority of positive samples was the bcv-a. counts of test outcomes for the tests are presented in tables and for the brsv and bcv tests, respectively. (top) and bcv antigens (bottom) included in the bcv/brsv multiplex. to the right are spider plots of median se and sp for the different cut-off alternatives. the brsv elisa cut-off was fixed at sample positive > pp, except for alternative where sample positive > pp was used. for the bcv elisa the cut-off was fixed at sample positive > pp. test parameters are estimated from a bayesian lcm analysis. counts of paired test outcomes in the two sub-populations for the brsv-antibody tests (brsv multiplex/brsv elisa). for the brsv multiplex varying cut-off values for the included antigens were used (shown in fig. ). the brsv elisa cut-off was fixed at sample positive > pp, except for alternative where sample positive > pp was used. a . . latent class analysis . . . brsv estimates of median se and sp and true prevalence in the two subpopulations for the brsv-multiplex and brsv elisa when applying different cut-off values are presented in table . as a starting point the recommended cut-off values from the test manufacturers were applied (alternative in fig. ), resulting in median se of . and sp of . for the brsv multiplex, and se . and sp . for the elisa. the sp of the elisa increased to . (se . ) when a cut-off of sample positive > pp was used. for the multiplex, increasing the cut-off value for the brsv-a antigen generally resulted in lower se and higher sp estimates as could be expected. discarding all antigens except the brsv-a resulted in increased specificity, however, with the cost of significantly reduced sensitivity, as can be seen from comparing cut-off alternative and in table . point estimates (median) of true brsv antibody prevalence ranged from . to . for pop , and from . to . for pop . results from the coc-models with fixed covariance, showed that allowing for covariance altered specificity estimates for both the elisa and the multiplex. the change was small for a covariance of . or less of the maximum possible covariance. the se estimates were not noticeably affected by allowing for covariance. results from the sensitivity analysis are presented in table . estimates of test parameters and true prevalence in the two subpopulations across different cut-off values for the bcv multiplex and the bcv elisa are presented in table . when we applied the cut-off values currently recommended by the test manufacturers (alternative in fig. . similar to what we observed for brsv, increasing the cutoff for the most important antigen (bcv-a) resulted in a lower se and a higher sp for the bcv multiplex. when we used the bcv-a as the sole antigen (cut-off alternative , table ) the median sp increased to . while the median se remained unchanged ( . ). point estimates (median) of true bcv antibody prevalence ranged from . to . for pop , and from . to . for pop . results from the sensitivity analysis, i.e. allowing for covariance between tests, showed negligible effect on the estimated test-parameters; less than % change in parameters for covariance at % of maximum possible (results not shown). we estimated the sensitivity and specificity of a new multiplex and two commercial elisas for detection of brsv and bcv antibodies in btm using lca. this is the first study evaluating the mvd-enferplex brsv/bcv multiplex. the present study is also the first to present test parameters for the svanovir ® brsv-ab and svanovir ® bcv-ab on btm. the brsv multiplex showed a somewhat lower se, but a much higher sp than the brsv elisa at the recommended cut-off values. however, when we increased the cut-off of the brsv elisa to sample positive > pp, this resulted in a large increase in sp without a notable decrease in se, as shown in table . our results therefore suggest that a higher cut-off than recommended by the manufacturer might be appropriate when using the svanovir ® brsv-ab on btm. for bcv, the specificity of the multiplex was notably lower than the bcv elisa at the recommended cut-off when using all three antigens. however, when using the bcv-a antigen only, the sp improved without the cost of reduced se, and the test performance was then similar to the bcv elisa. this implies that the extra antigens are adding false positive samples, hence reducing sp. overall; the two tests in this study both showed good performance for detection of both brsv and bcv antibodies. a possible benefit of choosing the multiplex therefore lies in enabling screening for both agents simultaneously as this will reduce screening costs. as the multiplex evaluated in the present study is a new table counts of paired test outcomes in the two sub-populations for the bcv-antibody tests (bcv multiplex/bcv elisa). for the bcv multiplex varying cut-off values for the included antigens were used (shown in fig. ) . the bcv elisa cut-off was fixed at sample positive > pp. table test parameter estimates for the brsv multiplex and brsv elisa: sensitivity, specificity, and estimates of true prevalence (tp) in the two sub-populations. cut-off alternative - represents different cut-off alternatives for the brsv multiplex (presented in table ). the brsv elisa cut-off was fixed at sample positive > pp for all alternatives except for alternative , where the brsv elisa cut-off was increased to sample positive > pp. test, there were no relevant studies we could compare estimates to. however, the multiplex technology has been shown useful for bovine tuberculosis in cattle and goats (clegg et al., ; o'brien et al., ) . the parameter estimates provided by the manufacturer for the svan-ovir ® bcv-ab are based on data from a study in which serum samples were analyzed using both the elisa and a virus neutralization test (vnt) (alenius et al., ) . the estimates, se of . % and sp of %, were calculated using vnt as gold standard. for the svanovir ® brsv-ab, the se ( %) and sp ( %) were calculated in a study comparing the test results to another elisa in serum samples. thus, test estimates were relative to the other elisa (elvander et al., ) . results from the former studies are not comparable to the present study due to different sample material (serum vs. btm). even so, it is important to note that in studies assuming a perfect reference test the estimated se and sp of the index test will never exceed those of the gold standard, thus the higher se of both the brsv and bcv elisa found in our study was not unexpected. to explore the effect of different cut-off values on test characteristics we applied a range of cut-off values for the multiplex antigens. whenever the cut-off is changed this could entail a change in the definition of the latent condition and change the number of true positive and true negative herds. there was relatively little variation in the se and sp estimates of the brsv-and bcv elisa across the different cutoff values explored, and the change in estimates of true prevalence was minor. the tests generally agreed on the proportion of positive herds indicating that tests had good agreement on the underlying target condition. the explorative approach to choosing cut-offs is a potential weakness of the current study; however, the different scenarios provide examples of expected performance for different cut-offs and do not represent an optimization of the diagnostic tests. the chosen cut-off will table results from the sensitivity analysis (brsv): median estimates and % posterior credibility intervals (pci) of the sensitivity (se) and specificity (sp) of bulk tank milk brsv multiplex and brsv elisa at the manufacturers' recommended cut-off (alternative , fig. ), for the conditionally independent (cid) model and conditionally dependent (coc) models where the covariance is expressed as proportions of maximum possible value. test parameters for the bcv multiplex and bcv elisa: sensitivity, specificity, and estimates of true prevalence (tp) in the two sub-populations. cut-off alternative - represents different cut-off alternatives for the bcv multiplex (presented in table ). the bcv elisa cut-off was fixed at sample positive > pp for all alternatives. likely affect the number of antibody producing animals needed for a positive btm result, and a positive correlation between within-herd prevalence and od-value has been shown for other diseases (muskens et al., ; nekouei et al., ) . because the typical norwegian dairy herd is small (mean herd size . ) (anon., ) compared to most other developed countries, this might influence the generalizability of our results: in larger herds antibodies might be diluted in the bulk tank, and hence cause the test se to decrease. however, larger herds might also have more positive animals. careful evaluation of the model assumptions is crucial when performing lca, as violation of assumptions might lead to biased results. the assumption of different prevalence between populations is central to lca models, and toft et al. ( ) showed that the precision of the accuracy parameters improved with increasing difference in prevalence among the populations studied. in the present study, the difference in prevalence between the two sub-populations was relatively large, which in addition to a sufficient sample size, leads to narrow posterior credibility intervals for the se and sp estimates. the second assumption is that the test characteristics are constant in both populations. the norwegian dairy herd is relatively homogeneous, and the two sub-populations in this study are likely similar in terms of breeds and production systems. a potential source of variation in test characteristics between sub-populations could be antigenic diversity within the norwegian dairy herd. findings of antigenic diversity of bcv are summarized by saif ( ) who concludes that only a single serotype is known based on virus cross-neutralization tests, and that a high level of cross protection has been shown between respiratory and enteric isolates. for brsv, a norwegian study found that the current norwegian strains of brsv belonged to the same subgroup as other north european isolates, indicating that the within-country diversity is likely to be limited (klem et al., a) . additionally, cross-reaction is likely to be common, and has even been shown for isolates from different species (oberst et al., ) . even though it seems unlikely that spatial antigenic diversity plays an important role as source of bias it cannot be excluded with complete certainty. the final assumption to be met is conditional independence of tests given the disease status. several papers argue that if tests have similar biological basis, this assumption is likely not met branscum et al., ) . conditional independence between tests means that the probability of a positive (or negative) result from one test is the same regardless of the result of the other test, given the true disease state (enøe et al., ; toft et al., ) . conditional dependence would, in terms of false positives, mean that the second test is more likely to pick up a herd as a false positive if it already tested (false) positive on the first test, for instance due to cross-reactivity with other agents. to estimate covariance between tests (γ se and γ sp ) two extra degrees of freedom are needed. in a two tests, two populations scenario this results in an unidentifiable model i.e. it is not possible to estimate covariance without including prior information. no reliable prior information could be obtained for test parameters or prevalences in the present study. another approach potentially allowing for estimation of covariance would be to include a third test: either another antibody test, or a test detecting the virus itself (e.g. a qpcr). the first option would not necessarily allow for estimation of covariance unless the third test had some underlying properties substantially different from the two other tests. adding an antigen test might ensure conditional independence, however, it would change the underlying disease status to involve not only serological response, but also a coherent shedding of virus. we explored the consequences of conditional dependence (sensitivity analysis) by including fixed covariances as proportions of the maximum possible covariance between tests. for the bcv estimates, allowing for covariance in the latent class models had negligible effect on parameter estimates of both tests. as the se of the bcv multiplex and the sp of the bcv elisa is close to one, the small effect of covariance was expected. it can be shown mathematically that test se (sp) are conditionally independent whenever one test has se (sp) = , see appendix a for details. this was also the situation for brsv-se where the se of the elisa is close to . however, the coc-models with fixed covariances did yield changes in the estimated specificity for brsv of both tests. this became most notable when the covariance was assumed larger than % of maximum. in summary, the effect of covariance was small except for brsv-sp for high values of covariance. it is important to note that the sensitivity analysis gives an indication of the effect of covariance if present, but does not answer whether covariance exists. even though both tests in this study are antibody tests, they differ in the way they are designed. first, the elisas uses crude whole virus in the elisa well, whereas the brsv/bcv multiplex uses peptides and recombinant proteins. second, the tests use different techniques for detection. the elisas use a chromogenic substrate and results are based on a reading of optical density, whereas the brsv/bcv multiplex uses a chemiluminescent substrate where results are based on a reading of light emission. these differences make a violation of the conditional independence assumption less likely. in conclusion, the brsv/bcv multiplex and the brsv/bcv elisa showed similar performance when applied on btm samples. the sp of the bcv multiplex can be improved by using the bcv-a antigen only, and the low sp of the brsv elisa can be improved by increasing the cut-off when using this test on btm. t., n.t., m.s., l.s. and a.n. declare no conflict of interest. g.h. and n.w. are the founders of mv diagnostics and developers of the multiplex test. a.o'b. is also developer of the multiplex test, and an enfer employee. however, neither mv diagnostics nor enfer were involved in the data-analysis, and could not have inappropriately influenced this work. then γ se and γ sp are the conditional covariances (cocs) among infected and non-infected test subjects, respectively, and presence of coc between tests given disease status implies that γ se ≠ and/or γ sp ≠ . the latent class model assumes that for the ith subpopulation the counts (o i ) of the different combinations of test results, e.g. pos/pos, pos/ neg, etc. for the two tests follow a multinomial distribution o i | se j ,sp j ,p i ∼ multinominal(prob i , n i ) for i = , ,…,s and j = , . where s is the number of subpopulations; j is the index for the test; and prob i is a vector of probabilities of observing the individual combinations of test results for the ith subpopulation (with true prevalence,tp i ): the model with cid between tests can be obtained by letting γ se = γ sp = in the above expression. from the expression for prob i it is possible to derive upper and lower limits for γ se and γ sp , since each of the elements of the probability vector must be between zero and one, thus: if we let the se or sp of either test be equal to in the above equations, it follows that the associated conditional covariance is limited to zero from above and below. thus implying conditional independence (with respect to se and/or sp) between the two tests given disease status. in frequentist statistics, a % confidence interval not including zero is evidence for statistical significance. if a similar approach is adopted in a bayesian setting, then a % posterior credibility interval for the conditional dependence without zero indicates that the conditional dependence should be included in the model. this covariance can be expressed as either γ se (or γ sp ) or as the proportion of covariance relative to its maximum value. bovine coronavirus as the causative agent of winter dysentery: serological evidence key numbers from the norwegian dairy herd recording system bovine coronavirus associated syndromes estimation of diagnostic-test sensitivity and specificity through bayesian modeling herd-level interpretation of test results for epidemiologic studies of animal diseases using latent class analysis to estimate the test characteristics of the γinterferon test, the single intradermal comparative tuberculin test and a multiplex immunoassay under irish conditions evaluation and application of an indirect elisa for the detection of antibodies to bovine respiratory syncytial virus in milk, bulk milk, and serum estimation of sensitivity and specificity of diagnostic tests and disease prevalence when the true disease state is unknown conditional dependence between tests affects the diagnosis and surveillance of animal diseases respiratory infections in norwegian dairy calves estimating the error rates of diagnostic tests occurrence and phylogenetic analysis of bovine respiratory syncytial virus in outbreaks of respiratory disease in norway association between the level of antibodies in bulk tank milk and bovine respiratory syncytial virus exposure in the herd stard-blcm: standards for the reporting of diagnostic accuracy studies that use bayesian latent class models bovine respiratory syncytial virus (brsv): a review empirical evidence of design-related bias in studies of diagnostic tests prevalence of coxiella burnetii infection in dutch dairy herds based on testing bulk tank milk and individual samples by pcr and elisa predicting within-herd prevalence of infection with bovine leukemia virus using bulktank milk antibody levels serological analysis of tuberculosis in goats by use of the enferplex caprine tb multiplex test characteristic differences in reverse transcription-polymerase chain reaction products of ovine, bovine, and human respiratory syncytial viruses bovine respiratory coronavirus: the veterinary clinics of north america svanova manual bovine respiratory syncytial virus antibody test diagnosing diagnostic tests: evaluating the assumptions underlying the estimation of sensitivity and specificity in the absence of a gold standard assessing the convergence of markov chain monte carlo methods: an example from evaluation of diagnostic tests in absence of a gold standard bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk-risk factors and spatial analysis a cohort study of the effect of winter dysentery on herd-level milk production nationwide survey of antibodies to bovine coronavirus in bulk milk from swedish dairy herds the effect of conditional dependence on the evaluation of diagnostic tests bovine respiratory syncytial virus infection evaluating diagnostic tests with imperfect standards this project was funded by the research council of norway (nfrproject no /e ), the norwegian research funding for agriculture and food industry and tine sa. the assumption of conditional independence between tests given disease status implies that for the population with infection present (d + ), the probability of test and both being positive given the test subject is truly infected is: similarly, for the population of non-infected subjects (d − ), the probability of test and both being negative given the test subject is truly noninfected: if we define key: cord- -juw xt q authors: pedersen, niels c.; lowenstine, linda; marx, preston; higgins, joanne; baulu, jean; mcguire, michael; gardner, murray b. title: the causes of false-positives encountered during the screening of old-world primates for antibodies to human and simian retroviruses by elisa date: - - journal: journal of virological methods doi: . / - ( ) - sha: doc_id: cord_uid: juw xt q abstract sera from old-world primates representing different species were screened by elisa for antibodies to human t-lymphotropic viruses i and iii, and simian retrovirus type (srv- ). about onefourth of the sera were positive by elisa. there was a tendency, however, for the same sera to be positive for all three human and simian retroviruses. only about one in five of the elisa antibodypositive sera were confirmed to be positive by western blotting. false-positive elisa antibody tests were particularly common among sera from mandrills, crab-eating macaques, lion-tailed macaques, african green monkeys, and debrazza's and moustached guenons. sera that were falsely positive in elisa antibody tests to the three human and simian retroviruses were found to contain antibodies that reacted at comparable intensity with feline leukemia, infectious peritonitis and panleukopenia viruses. the false anti-viral activity of these sera was found to be due to antibodies that reacted with non-viral proteins that were copurified with all five virus preparations. these proteins were present in normal cat and human cells used to grow the various viruses and in gelatin. the implications of nonspecific cell-protein antibodies in primate sera were discussed in the light of this and previous seroepidemiologic studies of man and old-world monkeys. we have reported in a companion paper on the serologic screening of oldworld primates of different species for antibodies to human t-lymphotropic virus, types i and iii (htlv-i, iii) and simian retrovirus type (srv- ) (lowenstine et al., ) . the preliminary screening of these animals was conducted with an enzyme-linked immunosorbent assay (elisa). a large number of positive reacting monkeys were identified by elisa, most of which were negative when tested by more specific procedures, such as western blotting. false positive elisa antibody tests, while sporadically encountered among most of the species of oldworld primates, were especially prevalent in mandrillus sphinx (mandrills), macaca fasicularis (crab-eating macaques) , macaca sifensus (lion-tailed macaques), cercopithecus aethiops (african green monkeys), cercopithecus neglectus (de-brazza's guenons), cercopithecus cephus (moustached guenons) and miopithecus talapoin (talapoins) . talapoins and mandrills were somewhat unique, however, in that they also possessed a considerable amount of true seropositivity that was being masked by the high background of false-reacting antibodies (lowenstine et al., ) . with the world-wide search for animal reservoirs for human retroviruses, it is natural for investigators to concentrate on man's closest relatives, the old-world primates. serologic screenings of old-world primates for other virus infections in the past have also yielded confusing results, and several investigators have cryptically mentioned the problems of false positive-reacting antibodies (kalter et al., ; kalter, ; strickland-cholmley and malherbe, ) . the cause of false positive serologic tests in these other virus systems was not determined, however. considering the importance of old-world primates as potential reservoirs for human pathogens, and the number of investigators doing serologic studies with these animals, we felt that it was important for us to spend some time on the causes of the false positive serologic reactions that we had encountered. in this paper, therefore, we will present evidence that a small proportion of monkeys of some species, and a large percentage of monkeys in others, possess serum antibodies that cross-react with a common cellular protein or proteins that are copurified with most virus antigen preparations. sera from old-world primates comprising different genera and species were obtained from six different zoos located across the united states. simian retrovirus type (srv- ) isolated at the california primate research center was grown in human raji cells (daniel et al., ; marx et al., ) . human t-lymphotropic virus type iii (htlv-iii), provided by dr. r.c. gallo (nih), was grown in h cells. lav provided by dr. f. barre-sinoussi (pasteur institute) was grown in hut- cells. human t-cell leukemia virus (htlv- ) and permissive in mt- cells were provided by dr. r.c. gallo (nih). feline leukemia virus (felv) was grown in fl cells (theilen et al., ) . procedures used for the purification of retroviruses have been described elsewhere . briefly, virus was concentrated by ultracentrifugation from cell culture fluid, clarified by low-speed centrifugation and filtration. virus was further concentrated by sucrose gradient centrifugation. fractions with a refractive index corresponding to a density of . - . g/ml in sucrose and containing the peak mg*+-dependent reverse transcriptase (rt) activity were collected by tube puncture, diluted with tris-nacl buffer, and pelleted by ultracentrifugation. virus pellets were resuspended in the tris-nacl buffer and the protein concentration determined by the method of bradford ( ) . this antigenic preparation was used for elisas, western blots and viral adsorption studies. the ucdl strain of fipv was grown on fcwf- feline cell cultures (pedersen et al., ) . the purification of fipv was carried out using the procedure of boyle and coworkers ( ) . feline panleukopenia virus was grown on crandell feline kidney cells and the virus purified from culture supernatants by low-speed centrifugation to remove cell debris, ultracentrifugation to pellet virus, and continuous cscl gradient separation (mathys et al., ) . cellular debris was obtained from the culture supernatants of hut- human t-lymphoblastoid cell cultures. culture supernatants were clarified of cells by low-speed centrifugation. subcellular debris was pelleted by ultracentrifugation and the pellets washed once in tris-edta saline buffer, ph . . techniques for elisa have been previously described (lutz et al., ) . briefly, microelisa plates (dynatech, alexandria, va) were coated with ng of purified virus per well. after washing the plates, monkey sera diluted :loo were added to duplicate wells. after incubation and washing, rabbit anti-cynomologous monkey igg conjugated to horseradish peroxidase and diluted was added to each well. anti-cynomologous monkey igg reacts well with igg from all species of cercopithecine monkeys and pongid and hylobatid apes (damian and greene, ) . after incubation, all trays were washed and substrate added to each well. the reaction was stopped after h and colorometric reactions read in a dynatech elisa reader. positive and negative control sera were used on every plate. a positive reaction was considered to be any corrected optical density (od) reading times that was greater than . this was usually - % of the values obtained from positive control sera. purified virus was disrupted in . % sds and separated on % polyacrylamide gels according to the procedure of laemmli ( ) . one lane of each gel contained the molecular weight standards. the protein bands were electrophoretically transferred to nitrocellulose paper by the technique of tsang and coworkers ( ) . unbound protein-binding sites were blocked with % gelatin in elisa dilution buffer. the nitrocellulose sheet was then cut into strips; molecular weight standards were stained with amido black. strips were incubated with monkey sera diluted :loo in elisa dilution buffer for h at °c. strips were then washed several times in elisa washing buffer and reacted with rabbit anti-monkey igg conjugated to horseradish peroxidase for h at °c. the strips were then washed and incubated with substrate solution ( mg diaminobenzidine, . m tris, . %) h,oz, ph . ) for - min. strips were washed several times with distilled water and air dried. elisa screening of old-world primate sera demonstrated a high incidence of positive reactions to one or more of the three principal screening retroviruses, htlv-i, htlv-iii and srv- (table ) . one hundred twenty-four out of sera from species of monkeys were elisa-positive for htlv-i antibodies, / sera from species were positive for antibodies to htlv-iii, and / sera from species had antibodies to srv- . the high incidence of elisa-positive reactions to htlv-i, htlv-iii and srv- was surprising and led us to question the specificity of the elisa antibody procedure. therefore, the same sera were retested by western blotting. a presumptive positive reaction was only scored on sera reactive by this procedure. antibodies to htlv-i, confirmable by western blotting, were detected in l/ pan troglodytes (chimpanzees), pan pan&us (pygmy chimpanzees), m. sphinx (mandrills), l/ m. fasicularis (crab-eating macaques), l/ m. silensus (lion-tailed macaques), / m. maurus (moor macaques), m. niger (black macaques), / m. tonkeana (tonkeana macaques), l/ m. nemistrina (pigtail macaques), l/l m. radiata (bonnet macaques), cercocebus atys (sooty mangabys), / c. aethiops (african green monkeys), / cercopithecus albogularis kolbi (kolb's guenons), l/ c. cephus (moustached guenon), and m. talapoin (talapoins) . specific antibodies to htlv-iii were found in monkeys, / sooty mangabys, / talapoins, / false-positive elisa antibody reactions to one or more viruses were found in a low proportion of individuals from many different species of old-world primates (table ) . six species in particular, mandrills, crab-eating macaques, lion-tailed macaques, african green monkeys and debrazza's and moustached guenons, were often falsely positive for all three viruses (table ) . we were interested, therefore, in determining why sera from these six species of monkeys possessed such high levels of nonspecific reacting antibodies. one explanation for the high incidence of false-positive elisa antibody tests was that the positive to negative ratio (p:n) that we used as a cutoff value was too low. in these studies, we assumed that a p:n ratio of . or greater was positive. non-specifically positive elisa tests (elisa positive, western blot negative) in langurs (presbytis entellus and p. obscurus), silver-leaf monkeys (presbytis cristata), spotnose guenons (cercopithecus nictitans), colobus, baboons, siamangs (symphalangus syndactylus), and orangutans (pongo pygamaeus) were almost always in the range of . - . (data not shown). exceptions were found, however, in sera from the six problem species that were antibody-positive across the board and in a high proportion of animals in each group. table shows the p:n ratios of representative monkeys from two of these six species, african green monkeys and lion-tailed macaques. false positives in these species frequently had p:n ratios above . it was concluded, therefore, that raising the p:n ratio to . would eliminate false-positive reactions in some monkeys, but not in the six problem species. in a further attempt to identify the causes of the nonspecific elisa antibody reactions that we had observed, we decided to concentrate the remainder of our studies on sera from african green monkeys. the pattern of false-positive reactions was identical in all six of the problem species, so we felt that african green monkey sera contained similar false-reacting factors. african green monkey sera were also much easier to obtain in sufficient number and quantity. african green monkey sera that were positive for htlv-iii antibodies by elisa were usually positive for htlv-i and srv- as well. in fact, plots graphing the optical density (od) values of the htlv-iii antibody assay against the od values for htlv-i, felv and srv- antibodies were virtually linear, with correlation coefficients greater than or equal to . (fig. i) . this type of reactivity made us question whether we were even dealing with an antibody-mediated reaction. the anti-viral activity of the positive sera, however, could be isolated to the globulin component and comigrated with the igg fraction obtained by biogel . m column chromatography (fig. ) . the retrovirus specificity of elisa-positive and -negative african green monkey sera was next studied. sera were reacted against two nonretroviruses: ( ) feline infectious peritonitis virus (fipv), a coronavirus, and ( ) penia virus (fpv), a parvovirus. sera that were positive against htlv-iii also reacted strongly against each of these non-retroviruses. the values for the antibody reactions to the two viruses, when plotted against the od values for htlv-iii antibodies, were also linear, with correlation coefficients greater than . (fig. ) . at this point, it was doubtful whether the elisa antibody reactivity contained in african green monkey sera was even viral specific. in our final test, mock pur- ified subcellular debris from non-infected hut- cells was used as the test antigen. the same sera that reacted positively against htlv-i, htlv-iii, srv- , felv, fipv and fpv also reacted positively at the same proportionate intensity with the cell debris (fig. ) . in an attempt to identify the cellular antigen present in all of our virus preparations against which the african green monkey sera reacted, elisa-positive and -negative sera were reacted in western blots against gradient purified virus, cell lysates of virus-infected cells, and cell lysates of non-infected cells. none of the elisa-positive or -negative african green monkey sera reacted positively against specific viral bands of htlv-i, srv- , fipv or fpv in western blots (data not shown). however, antibody activity of elisa-positive sera was strongly directed against the envelope proteins of htlv-iii and felv in western blots (fig. ) . interestingly, whether identifiable bands were present or not in the blots, elisapositive sera tended to produce a much more intense overall background stain than negative sera (data not shown). this reaction could be eliminated by blocking the strips with bovine serum albumin rather than gelatin (data not shown). a group of elisa-positive and -negative african green moneky sera were also reacted in western blotting against infected and non-infected cell lysates. none of the positive or negative sera demonstrated strong reactions against any single protein found in lysates of infected cells used to propagate htlv-iii (hut- cells) or srv- (ragi cells) (data not shown). similar to western blots prepared from whole virus, there was a pronounced tendency for positive sera to produce a greater diffuse background staining than negative sera (data not shown). this background was proportional in intensity to the reactivity of the sera in elisa against whole virus. it also could be eliminated by blocking the strips with bovine serum ablumin rather than gelatin. the fact that elisa-positive african green monkey sera reacted equally with a number of different virus and cell protein preparations suggested that they recognized a common antigen(s). this was confirmed by doing competition elisas. htlv-iii antibody-positive african green monkey sera, when preabsorbed with felv, no longer reacted with htlv-iii, srv- or felv (table ) . antibody could not be absorbed out with fetal bovine serum, indicating that the antibodies present in the sera were specific for cellular proteins. similarly, htlv-iii positive serum preabsorbed with srv- reacted much more weakly with htlv-iii, srv- and felv than nonabsorbed serum (table ). in contrast, human or rhesus monkey sera that were specifically positive for htlv-iii still reacted at the same relative intensity after absorption with felv or srv- (table ) . studies with african green monkey sera established the cause of false-positive elisa antibody tests in this species; sera appeared to contain a specific antibody or antibodies that was directed against cell-associated protein(s) that were co-purified with the various virus preparations. this protein was present in several di- all sera were diluted to half-maximal binding capacity. maximal binding occurred at :loo dilution for each serum, while half-maximal binding occurred at : . correlation between elisa antibody seropositivity to felv or cell-debris and to false-positive reactions to htlv-i, htlv-iii, or srv- . positive to felv or cell-debris, or both felv-and cell debrispositive sera that were also false-positive to one or more of the test viruses, htlv-i, iii, srv- verse species of animal cells and appeared to be present at low levels in gelatin, a complex animal protein. the possibility that this antibody was directed to glycoproteins was suggested by western blot studies. as a final experiment, we retested most of the old-world primate sera for elisa antibodies to felv and to cell debris. both of these antigen preparations, but in particular felv, seemed to identify sera that contained the anti-cell protein antibody. we found that / old-world primate sera reacted with felv in elisa and / with hut- cell debris (table ) . there was a high correlation between seropositivity to felv or cell debris and false-positive elisa antibody re-actions to htlv-i, htlv-iii or srv- , especially in the six problem species that were identified earlier in the study (table ). the influence of nonspecific antibody reactivity on seroepidemiologic studies was apparent from this study. about one-fourth of the monkeys that we tested were positive for one or more of the human and simian retrovirus antigen preparations that we used. however, only about one in five of these elisa antibody-positive sera was confirmed to be positive by western blotting. the cause of these falsepositive elisa reactions was found to be a serum antibody that reacted with cellular proteins that were co-purified with our viral antigen preparations. such contaminating proteins are virtually impossible to remove from preparations of viruses made from cell-culture fluid or infected tissues. the propensity of this antibody to react with cellular and viral membrane proteins, and its seeming lack of host specificity, indicated that it was directed against glycoproteins or carbohydrate moieties of glycoproteins. antibodies to forssman's protein, blood group substances, and histocompatibility antigens occur naturally in many animal sera and might have been involved in the phenomenon that we observed. the fact that certain species of monkeys possessed this antibody to a much higher degree than others indicated that it was not due just to environmental exposure. it is common knowledge, however, that many sera from african humans and primates tend to be 'sticky' in antibody assay procedures and some researchers believe that this might be due to the high degree of exposure to infectious diseases (biggar et al., ) . the problem of nonspecific antibody binding is intrinsic to elisa antibody procedures. the appearance of color in the reaction mixture indicates only that antibody has been bound to the plastic well. whether or not this binding is specific cannot be accurately answered without appropriate controls. this is not as great of a problem with western blots, which measure antibody binding to characteristic bands of viral protein. complement fixation tests suffer from the same problems as elisa procedures. if the complement fixating antigen is impure, and antibodies bind to antigens other than the ones being tested, then a nonspecific reaction occurs. this study was not the first to indicate that nonspecific antibody-binding reactions might be a problem in primate seroepidemiologic studies. kalter and colleagues ( ) reported that a large proportion of simians gave strong complement fixation reactions to marburg virus-positive guinea pig antigen prepared by the communicable disease center (cdc). strickland-cholmley and malherbe ( ), however, indicated that such reactions might have been nonspecific. they found that positive cdc antigen reacting sera were usually negative when tested in parallel with a monkey liver antigen preparation. these investigators postulated that the cdc antigen contained a second and stronger non-marburg virus antigen, and that this antigen accounted for the high number of simian reactors tested with this antigen. strickland-cholmley and malherbe ( ) also observed that many ba-boon and african green monkey sera reacted strongly in complement fixation tests with uninfected guinea pig and monkey control liver antigen. they postulated that these 'autoantibodies' might have resulted from previous liver damage. a more likely explanation, however, was that these sera contained antibodies that reacted specifically or nonspecifically with normal cellular proteins present in their liver preparations. kalter ( ) , after an exhaustive seroepidemiologic search for antibody to marburg virus in african green monkeys, concluded that "it is obvious that a larger number of sera were either nonspecific, anticomplementary, or both. ..". kalter ( ) also found, after examination of japanese macaque sera for antibody to a number of agents, " . ..that antibody to an uncommonly large number of antigens are detected by cf" and hi procedures. at this time it is difficult to evaluate these findings. it would appear that these animals have a rather high incidence of 'antibody' as a result of ( ) numerous contacts with various agents other than marburg virus, ( ) a nonspecific inhibitor in the sera of this species of macaca resulting in a high incidence of 'seropositives'. . .". kalter went on to state that " . ..testing old-world african simians for antibody continued to indicate a large number of serologic positives, with the talapoin stil showing the greatest incidence . " it is noteworthy that in our study several species of macaques and talapoins also possessed high levels of nonspecific cell protein antibodies. the problem of nonspecific antibodies in sera, especially when assayed by elisa, is not unique to primates. elisa antibody screening of human sera for htlv-iii is most accurate when high-risk candidates are tested. nonspecific positives are a much bigger problem, however, when the test is used for screening of low-risk groups (carlson et al., a) . many researchers believe, therefore, that all htlv-iii elisa positives should be confirmed by other procedures, such as western blotting (carlson et al., b) . in fact, some investigators state that even western blotting may not be accurate with freeze-thawed and hypergammaglobulinemic sera, and that radioimmunoprecipitation assays should be the standard for specificity (brun-vezinet et al., ) . false-positive htlv-iii antibody reactions can also be a problem in populations considered to be at high risk. biggar and associates ( ) found that from to % of zairian people reacted positively in elisa to htlv-i, ii and iii. there was a strong correlation between the levels of these antibodies and the levels of antibodies to plasmodium falciparum. one of their possible explanations for this phenomenon was "false-positive reactivity in the elisa assay due to cross-reactive antibodies or other unknown factors." biggar ( ) has also eluded to false-positive reactions in sera from african children tested against htlv-iii (epstein et al., ; sainger et al., ) . false-positive reactions to human retroviruses have also been described by snyder and associates ( ) and snyder and fox ( ) . these studies bring in the question of using elisa antibody tests to screen large populations of animals or people for viral antibodies. in most species, however, elisa antibody tests have proven fairly accurate for screening purposes. we were able to concentrate almost all of our 'true' positives into a smaller number of sera, on which we could concentrate our more specific testing. the only notable exception was in the case of sooty mangabys and kolb's guenons; only were positive for htlv-iii antibodies by elisa, while that were tested showed characteristic htlv-iii core protein bands on western blots. a lentivirus was readily isolated from the blood of the sooty mangabys (lowenstine et al., ) . care should also be taken not to eliminate sera that appear to be reacting falsely. talapoins and mandrills in our study were found to have a very high incidence of nonspecific felv and cell debris antibodies, but this was masking a large number of htlv-i, iii and srv- specific reactors (lowenstine et al., ) . lancet , pathology of simian primates. part th interscience conference on antimicrobial agents and chemotherapy (minneapolis) marburg virus disease marburg virus disease key: cord- -s lvhknm authors: watanabe, shumpei; omatsu, tsutomu; miranda, mary e.g.; masangkay, joseph s.; ueda, naoya; endo, maiko; kato, kentaro; tohya, yukinobu; yoshikawa, yasuhiro; akashi, hiroomi title: epizootology and experimental infection of yokose virus in bats date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . . sha: doc_id: cord_uid: s lvhknm to reveal whether bats serve as an amplifying host for yokose virus (yokv), we conducted a serological survey and experimentally infected fruit bats with yokv isolated from microbats in japan. yokv belongs to the entebbe bat virus group of vector unknown group within the genus flavivirus and family flaviviridae. to detect antibodies against yokv, we developed an enzyme-linked immunosorbent assay (elisa) using biotinylated anti-bat igg rabbit sera. serological surveillance was conducted with samples collected in the philippines and the sera supplied from malaysia. one of the samples from the philippines ( . %) and of the samples from malaysia ( %) had detectable elisa antibodies. in the experimental infections, no clinical signs of disease were observed. moreover, no significant viral genome amplification was detected. these findings revealed that yokv replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for yokv. bats, the only mammals capable of flight, display large amounts of diversity and account for % of the mammalian species recorded in the world. during the past decade, bats have been associated with several emerging zoonotic agents including hendra, nipah, lyssa, ebola, and severe acute respiratory syndrome coronavirus-like viruses [ ] [ ] [ ] [ ] [ ] . therefore, bats are thought to be an important reservoir for many mammalian viruses. specifically, numerous viruses have been isolated from the genus flavivirus, family flaviviridae [ ] [ ] [ ] [ ] . while the epizootology of these viruses remains unknown, nucleotide sequences for some flaviviruses have been reported [ ] [ ] [ ] . yokose virus (yokv) is a flavivirus that has been isolated from a bat in japan in . yokv belongs to the entebbe bat virus group within the genus flavivirus and family flaviviridae. to investigate the possibility that bats serve as a reservoir for japanese encephalitis virus (jev) during the winter, oya et al. attempted to isolate arthropod-borne viruses from bats in oita prefecture, japan [ ] . during this investigation, yokv was isolated from the long-fingered bat miniopterus fuliginosus. recently, tajima et al. [ ] reported the complete nucleotide sequence of yokv. kuno and chang [ ] compared the complete nucleotides sequences with those of other flaviviruses. they concluded that yokv is genetically closer to yellow fever virus or sepik virus than jev, and it is most closely related to entebbe bat virus. previous phylogenetic analyses of the genus flavivirus have revealed that flaviviruses can be divided into three groups: mosquito-borne, tick-borne, and unknown vector groups [ , ] . although yokv is classified into the entebbe bat virus group of vector unknown group, conserved sequence element (cs ) in the -untranslated region of yokv is similar to that of mosquito-borne viruses, which are known to display highly conserved cs regions [ , ] . therefore, tajima et al. [ ] suggested that yokv belongs to the mosquito-borne virus group. moreover, previous reports indicated that entebbe bat virus can replicate in mosquito cells in vitro [ ] , and experimental infection of entebbe bat virus using frugivorous and insectivorous bats showed no viral growth in bats [ ] . since the initial isolation with yokv in , there have been no additional reports on the isolation or antibody detection of yokv from bats or mosquitoes. therefore, to determine whether bats serve as a natural or amplifying host for yokv, we conducted a serological survey and experimental infection studies in bats with yokv. to detect antibodies against yokv, we developed an elisa using biotinylated anti-bat igg rabbit sera. in this system, polyclonal anti-bat igg rabbit sera were used as described in a previous paper [ ] . the developed anti-bat igg reacts only with bat igg but not with igg of other mammalian species. therefore, this elisa detects bat-specific igg antibodies. using the conventional elisa, a serological survey was performed on bat serum samples collected from the philippines and malaysia. vero cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal calf serum (fcs), penicillin, and streptomycin. the oita- strain of yokv was kindly provided by dr. t. takasaki (national institute of infectious diseases). the virus was grown in vero cells on -cm roller bottles. infection was performed at a multiplicity of . tcid /cell with an inoculum containing ml of serum-free maintenance medium (-smm) in dmem, . g triptose phosphate broth, g l-glutamine-na, g glucose, . g yeast extract, . g l-glutamine/l, penicillin, and streptomycin. following virus adsorption at c for h, cells were washed with -smm and ml of -smm was added per bottle. four days after infection, ten -ml bottles of infectious fluid were harvested and cellular debris was removed by low-speed centrifugation ( Â g, min, c). the resulting supernatant was collected, and half was used for virus purification while the remainder was used for virus inactivation. virus purification was performed according to the procedures outlined for jev [ , ] . briefly, yokv was precipitated from the supernatant by incubation with peg ( mm) and nacl ( mm). after an overnight incubation at c, the mixture was centrifuged ( Â g, min, c) and the pellet containing the virus was resuspended in ml tris-saline-edta buffer (ten buffer). virions were further purified on ml of a continuous - % sucrose gradient. the gradients were centrifuged in an rps t rotor (hitachi, tokyo, japan) at , Â g, for h at c. fifteen fractions were collected from the bottom of each tube. each fraction was assayed by using both elisa and titration method. peak fractions that demonstrated the highest elisa reactivity were collected and used as elisa antigens. virus inactivation was performed using % formalin at a final concentration of . %, according to the procedures for jev [ , ] . the inactivated virus was purified as described above. the peak fractions were collected and dialyzed with pbs. the inactivated virus was filtered through a . mm low protein-binding (gv-type) filter unit, and stored at c until it was used as a bat immunogen. two leschenault's rousette bats (rousettus leschenaulti) were immunized with inactivated and purified virus. one milliliter of the inactivated virus was inoculated intraperitoneally twice at -week intervals to obtain positive sera containing anti-yokv antibodies. serum samples were collected at days and postinoculation. briefly, a small vein on the patagium was cut with a scalpel for blood sampling, and filter papers were soaked with approximately ml of blood. the filter papers were transferred into eppendorf tubes and eluted in ml pbs by centrifugation ( rpm, min, c). after discarding the filter papers, eluates were centrifuged again and the supernatants were stored as serum samples. these samples were estimated to be equal to a : sera dilution. on day after collecting sera, two bats were inoculated with a secondary injection. on day after the first inoculation, the two bats were anesthetized using a . mg intraperitoneal injection of ketamine hydrochloride and were euthanized via intracardiac exsanguination. bat serum samples were collected in the philippines and malaysia. insectivorous bats were collected from two sites in the philippines (fig. ). sites were chosen on the basis of a previous survey [ ] . all captured bats were anesthetized by administering a mg/kg intraperitoneal injection of ketamine hydrochloride. we measured the size of each bat (weight, forearm length, head and body length, ear length, and tail length) and examined additional morphological features (uropatagium, tragus, and nose-leaf). species were identified based on gross morphology according to the classification criteria for chiropterans [ ] (table ) . blood was obtained by cardiac puncture and stored at c until centrifugation. sera were frozen at À c during transport and later stored in a À c freezer upon arrival. fruit bat sera collected in malaysia were kindly supplied by dr. a. rayari (university of malaysia, sarawak) and dr. t. imada (jica project leader at the veterinary research institute). elisas were performed according to the procedures described for jev [ ] . to standardize the reagents used for the labeled avidin-biotin enzyme-linked immunosorbent assay (lab-elisa), antigen and antibody concentrations were determined by checkerboard titration. briefly, yokvantigen was diluted with coating buffer at a concentration of mg/ml and elisa plates were coated with ml/well of the antigen solution. after washing the plates, ml of serum from each sample was appropriately diluted with blocking buffer (pbs, ph . , containing . % tween and % chicken serum) and delivered to each well. the plates were then kept at c for h. after washing the plates, biotin-labeled anti-bat igg rabbit serum was diluted in blocking buffer at : and ml of the diluted solution was added to each well. anti-bat igg rabbit serum [ ] was biotinlabeled by the methods described by chang et al. [ ] . the plates were incubated at c for min. after washing the plates, horseradish peroxidase-labeled avidin (sigma, st. louis, mo, usa) was diluted in blocking buffer at : and ml of the diluted solution was added to each well. after incubation at c for min, the plates were washed and ml of prepared tmb substrate solution (tmb microwell peroxidase substrate; kappel, usa) was added to each well. following incubation for min at room temperature, ml of stop solution ( m h so ) was added to each well and od values were determined. minor nonspecific reactions were observed in the control wells without serum and antigen. in the control wells without antigen, some of the od values were slightly higher than those with the antigen; however, the values were all lower than . and neared . the cutoff point was estimated as the sum of the average od values of the control wells without serum and antigen and an additional factor of . in accordance with the procedures for jev [ ] . according to the above criteria, when antiserum was used at the highest dilution in a checkerboard titration ( : ), the end point titer of antigen was determined at : (a putative unit). we examined negative control samples at a dilution of : - : using : diluted antigen (as a putative units). a cutoff value of . was set according to the following calculation: mean each wells plus (s.d.) Â . according to these criteria, when the antiserum was used at a dilution of : in the checkerboard titration, the end point titer of antigen was determined at : . therefore, the yokv antigen was used at : ( units) for the lab-elisa assay. neutralization tests were performed using the microtiter method. one hundred tcid of virus was incubated with twofold serially diluted serum samples and added into vero cells seeded in a -well plate. the titer of neutralization antibody was determined based on the highest serum dilution, which completely suppressed a cytopathic effect. leschenault's rousette bats were obtained from zoos in japan. these fruit bats were housed in separate cages in an air-conditioned room. the animals were fed several kinds of fruit with free access to water. serum samples were collected from the orbital sinus under anesthesia by diethylether. nine of the seronegative bats were randomly selected for experimental infection and placed in a negative-pressure isolator. the bats were inoculated intraperitoneally with ml of solution containing tcid /ml of yokv. the bats were separated into three groups, each containing three bats. one group each was sacrificed by cardiac puncture on days , , and postinoculation following anesthesia by a . mg intraperitoneal injection of ketamine hydrochloride. the experiment was conducted according to the guidelines for the care and use of laboratory animals, graduate school of agriculture and life sciences, the university of tokyo. serum samples were obtained via whole blood by centrifugation at rpm for min at c. organs (liver, kidney, spleen, lung, and brain) were also collected. during the experimental infection, bats were examined daily for clinical symptoms of infections. urine and fecal specimens were collected using a clean translucent plastic sheet spread along the bottom of the cage. virus isolation was attempted from these samples (i.e., organs, serum, urine, and feces). each sample was homogenized in dmem as % suspensions and assayed for viral titers using tcid on vero cells. each sample was also tested using rt-pcr to detect yokv rna. finally, serum samples were tested using nt and elisa. viral rna extraction was performed on samples obtained from infected bats using an sv total rna isolation system kit (promega, madison, wi, usa) according to the manufacturer's instructions. superscript tm one-step rt-pcr with platinum taq (invitrogen, carlsbad, ca, usa) was used for rt-pcr. the primer set ( -ataagacagccaaccattgc- and -tatccggcaaatccaatcac- ) was targeted to a -bp fragment of the envelope gene and designed to be specific to yokv according to a prior report [ ] . to obtain sera positive for anti-yokv antibodies, two bats were immunized with inactivated and purified virus. antibodies were first detected in bat with a titer of : on day postinoculation. anti-yokv antibodies reached the peak titer ( : in bat and : in bat ) a week after the second inoculation on day (fig. ) . on day , the bats were killed by intracardiac exsanguination under anesthesia. neutralizing antibodies were also tested on days and . we could not obtain sufficient serum volumes to perform the neutralizing test for days and . neutralizing titers were found to be low on day ( : in bat and : in bat ). of the bat serum samples collected in the philippines, had sufficient volumes and quality for analysis. twenty-six additional samples were supplied by dr. a. rayari (university of malaysia, sarawak) and dr. t. imada (jica project leader at the veterinary research institute). sera were screened at a dilution of : and each sample was tested three times by elisa. of the serum samples tested, ( . %) were determined to be positive in the elisa assay; elisa titers of each serum sample were determined. neutralization tests (nts) were also conducted using collected bat sera at a concentration of : . of the samples, five were determined to be positive. although the titers of these five samples were also determined, they were low. yokose antibody titers obtained by elisa were compared with those obtained by nt (fig. ) . close correlations existed between elisa and nt titers (r = . ). serological tests used for flaviviruses, such as elisas or fluorescent antibody techniques, have been reported to show high cross-reactivity with other flaviviruses [ , ] . therefore, to check the specificity of the yokv elisa, an elisa with the jev antigen substituted for yokvantigen was conducted. elisa titers of the jevantigen were determined using positive sera from bat . the heterologous titer using the jev antigen was : whereas the homologous titer (using the yokv antigen) was : . therefore, we found that the homologous titer was times higher than the heterologous titer. elisa with the jev antigen was also conducted using the bat serum samples described in section . . titers of all the samples were less than : . furthermore, the serum samples collected from bats which were obtained from japan zoos were also screened by elisa using the jev antigen. titers of all samples were less than : . an elisa was used to exclude bats that were positive for antibodies against yokv. fourteen percent of the fruit bats (leschenault's rousette bats) collected from several zoos in japan had antibodies against yokv ( table ). all of the bats seropositive against yokv were obtained from zoos in the western portion of japan. nine of the seronegative bats were selected and experimentally infected with yokv. during experimental infection, none of the bats inoculated with yokv showed clinical signs of infection. viral particles were not recovered from any of the collected samples. viral genome amplification was not detected in any of the samples including sera, organs (brain, heart, kidney, liver, lung, and spleen), feces, and urine; however, viral rna was detected in the liver of one bat that was killed days after inoculation (table ) . sera collected from the infected bats were tested using a nt (table ). nt titers of six preinoculated serum samples were less than : . for the remaining three samples, nt was À À À À À À À À À À À À À À À À (+) positive; (À) negative. table neutralizing titers of the bats experimentally infected with yokv bat no. days after inoculation (-) not tested. not performed due to insufficient volumes of serum. no nt antibodies were detected in sera obtained days after inoculation. on days and postinoculation, nt antibodies were detected in all samples except for one bat killed on day , and the nt titers were less than . we developed an elisa system using biotin-labeled anti-bat-igg rabbit serum to detect antibodies against yokv in bat sera and conducted serological surveys using this system. one of the samples collected from the philippines and five of the samples from malaysia had detectable antibodies against yokv. these results suggest that yokv is distributed not only in japan, but also in other asian countries. the possibility exists that antibodies detected in this survey were against other flaviviruses, since antibodies against flaviviruses have been reported to show high cross-reactivity with other flaviviral antigens [ ] . thus, we conducted an elisa in which we substituted the jev antigen to confirm assay specificity. the elisa using the jev antigen was found to react with the positive serum against yokv from the immunized bat; however, the titer against jev was much lower than the homologous titer using yokvantigen. we also tested field samples using an elisa with the jev antigen, and all samples were negative (data not shown). moreover, elisa and nt antibody titers from both field samples and positive sera from immunized bats showed a close correlation. these data suggest that the antibody detected in this survey was specific to yokv. serological surveys of several viruses have been conducted using bat sera collected in the field; however, most of the surveys were performed using a nt or fluorescent antibody tests [ ] [ ] [ ] [ ] ] . obtaining sufficient volumes of blood necessary for these serological tests is difficult, particularly in smaller species including microbats. moreover, these assays are not suitable for testing large numbers of samples at one time. therefore, an elisa is a powerful tool that can be helpful in serological surveys of infected bats. however, no known conventional elisas are available, except assays using protein g or competitive techniques with monoclonal antibodies [ , ] . in this study, we demonstrated that a conventional elisa using biotin-labeled anti-bat igg rabbit sera was able to detect antibodies in bat sera obtained from field studies. from complete nucleotide sequence analyses of yokv, tajima et al. [ ] suggested that yokv belongs to the mosquito-borne group. thus, to reveal whether bats serve as an amplifying host for yokv, we conducted an experimental infection study of yokv in bats and examined viral growth and pathogenicity. since yokv was originally isolated from a species of microbat, m. fuliginosus, it would be preferable to conduct experiments on viral characteristics of yokv not only with fruit bats but also with m. fuliginosus or other microbats. however, maintaining and feeding microbats, especially insectivorous bats, is difficult. therefore, we conducted our experimental infection studies on the fruit bat r. leschenaultia. prior to experimental infection, we performed an elisa to exclude fruit bats that had been previously exposed to (i.e., were positive for) antibodies against yokv. fourteen percent of the fruit bats collected from several zoos in the western portion of japan were positive for yokv antibodies. these positive sera against yokv were also tested using an elisa with the jev antigen. all samples were negative. given that leschenault's rousette bats have been bred in each zoo, and reared separately in openair cages, it seems likely that these antibody positive bats had been exposed to yokv at the zoos themselves. although no accounts of viral isolation or antibody detection of yokv have been reported since the initial one in , yokv appears to be present in japan. no clinical signs of disease were observed in fruit bats following viral infection. moreover, significant viral genome amplification was not detected in any of the samples, except for one liver sample obtained from a virus-inoculated bat killed at day postinoculation. no viral particles were isolated from any of the samples and antibody responses were low. these results reveal that yokv replicates poorly in leschenault's rousette bats, and might suggest that fruit bats do not serve as an amplifying host for yokv. our results from the serological field survey demonstrate a low prevalence of yokv in bats from the philippines and malaysia, further supporting this suggestion. yokv may have additional amplifying hosts besides bats, such as mosquitoes. to confirm the viral pathogenicity in microbats and also the relationship between yokv and mosquitoes, further studies are needed. although no cases of yokv infection have been reported in other animals, a single human case of febrile illness, possibly caused by sepik virus, has been published [ ] . interestingly, sepik virus exhibits high nucleotide sequence similarities with yokv. further studies are necessary to more fully elucidate the pathogenicity of yokv. severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats fruit bats as reservoirs of ebola virus an apparently new virus (family paramyxoviridae) infectious for pigs, humans, and fruit bats isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus nipah virus outbreak in malaysia isolation at dakar of a strain of arborvirus from the salivary glands of the bat (preliminary note) isolation of rio bravo and a hitherto undescribed agent, tamana bat virus, from insectivorous bats in trinidad, with serological evidence of infection in bats and man complete genome sequence of montana myotis leukoencephalitis virus, phylogenetic analysis and comparative study of the untranslated region of flaviviruses with no known vector characterization of sepik and entebbe bat viruses closely related to yellow fever virus phylogeny of the genus flavivirus using complete coding sequences of arthropod-borne viruses and viruses with no known vector genetic characterization of yokose virus, a flavivirus isolated from the bat in japan phylogeny of the genus flavivirus phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the modoc virus, a flavivirus with no known vector antigenic relationships of flaviviruses with undetermined arthropod-borne status studies on arboviruses and bats (chiroptera) in east africa. i. experimental infection of bats and virus transition attempts in aedes (stegomyia) aegypti (linnaeus) molecular evolution inferred from immunological cross-reactivity of immunoglobulin g among chiroptera and closely related species large-scale purification of japanese encephalitis virus from infected mouse brain for preparation of vaccine a purified inactivated japanese encephalitis virus vaccine made in vero cells serologic evidence of lyssavirus infections among bats, the philippines a key to the bats of the philippine islands labeled avidin-biotin enzyme-linked immunosorbent assay (lab-elisa) for detection of japanese encephalitis antibody in swine sera biotin-labeled antigen sandwich enzyme-linked immunosorbent assay (bla-s-elisa) for the detection of japanese encephalitis antibody in human and a variety of animal sera antigenic structure of the flavivirus envelope protein e at the molecular level, using tick-borne encephalitis virus as a model characterization of neutralizing antibodies to west nile virus traditional and novel approaches to flavivirus vaccines public health surveillance for australian bat lyssavirus in queensland a solid-phase blocking elisa for detection of antibodies to nipah virus supplement to international catalogue of arboviruses including certain other viruses of vertebrates we thank dr. tomohiko takasaki of the national institute of infectious diseases of japan for providing the oita- strain of yokv, and the members of the veterinary research department of the research institute for tropical medicine in the philippines for their assistance in collecting and processing specimens. this study was supported by a grant from the ministry of education, culture, sports, science and technology, japan. key: cord- -i x knq authors: böse, reinhard; peymann, berit title: diagnosis of babesia caballi infections in horses by enzyme-linked immunosorbent assay (elisa) and western blot date: - - journal: international journal for parasitology doi: . / - ( ) - sha: doc_id: cord_uid: i x knq abstract from babesia caballi in vitro cultures a preparation of % infected erythrocytes was obtained. from this, b. caballi antigens were extracted with the detergent -[( -cholamidopropyl)-dimethylammonio]- -propane-sulfonate (chaps) and used as elisa antigens. a control antigen of normal erythrocytes from the same donor horse was prepared in an identical manner. the elisa and western blot were validated by testing of sera from horses experimentally infected with b. caballi or b. equi or not infected with babesia spp. elisa and western blot results were compared with those obtained by the immunofluorescence antibody test (ifat) and complement fixation test (cft). the sensitivity of the elisa of . % obtained for sera from day after infection was superior to the western blot ( . %), the ifat ( . %) and the cft ( . %). no positive results were obtained in the elisa and western blot with sera from horses not infected with babesia spp. resulting in a calculated specificity of % for both tests. cross reactions of b. equi-positive sera did occur to a larger extent in the elisa ( %) than in the ifat ( %). no cross reactions were observed with the western blot and the cft. the higher sensitivity of the elisa was also demonstrated by testing of field sera: more positive results were obtained by elisa ( ) as compared to ifat ( ) or cft ( ). the validity of these results was confirmed by testing of sera by western blot. the elisa as the most sensitive test provides the best method for the identification of carrier horses to prevent the introduction into non-endemic areas (export testing). positive elisa results can be confirmed by western blot, if a species-specific diagnosis is required. index key words: babesia cahelli. sero diagnosis; elisa; western blot; ifat; cft. bubesiu caballi and . equiare obligate intraerythrocytic parasites of equines. they are the causative agents of equine babesioses which are endemic in most tropical and subtropical areas of the world (friedhoff, ; friedhoff, tenter & miller, ) . whereas . cabalii only invades erythrocytes b. equi is also capable of infecting lymphocytes (schein, rehbein, voigt & zweygarth, ) . both parasites are transmitted by tick vectors with almost worldwide distribution (fricdhoff, ) . consequently, it is important to prevent the introduction of carrier animals into nonendemic areas, particuiariy where the diseases could be spread by vector ticks. horses to be exported into the u.s.a., japan, australia or other countries have to be tested negative for babesioses by the camplement fixation test (cft) or the immunofluores&en~ antibody test (ifat) . the cft yiefds a considerable number of false-negative results (weiland, ; tenter l friedhoff, ). the ifat is laborious and not amenable to standardization. thus, there is a need for improved serological tests for the diagnosis of equine babesioses. a first step was made by deveioping a western blot for the diagnosis of b. cab& infections. this test can be used in case of contradicting cft and ifat results and provides a speciesspecific diagnosis (base & daemen, ) . for routine diagnosis, however, the test is too laborious. in general, the advantages of the elisa in contrast to cft or ifat are its high sensitivity and the possibility of standardization and computer evaluation. we here report the development of a sensitive elisa and the application of the western blot for the diagnosis of b. caballi infections. rnrigen preparation. parasites were cultured using the m~~roaeroph~ious stationary phase culture technique [levy bt risk, t ) essentially as described previously ( &e l daemen, ). parasitized erythrocytes were enriched to about % infected cells with density gradient centrifugation on a two-step percoll gradient (bhushan, miiller & friedhoff, ) , washed three times in rpmi- ~ tissue culture medium and stored in aliquots at - °c. a control antigen of non-infected erythrocytes of the same doner horse used for the cultures was prepared in the same manner. for the elisa both antigens were extracted with urea of with detergents. urea (gibco/brl, eggenbeim, germany, no. @ uv) was used in a final concentration of . m-sodium dodecyl sulfate (sds) (gibco/brl, no. ua), triton x- (boehringer, mannheim, germany, no. , nonidet p- (boehringer, no. ) and -[ -cholamidopropyl)-dimethyiammonio]-l-propane-sulfonate (chaps) (boehringer, no. ) were used in final concns of i %. antigen and reagent were mixed and incubated for min at °c (urea, triton x- , nonidet p- , chaps) or for mm at °c (sds) and centrifuged ( , g, io min, °c). the supernatant was used as antigen in the elisa. prokxol. the elisa was performed as a heterogeneous, indirect, non-competitive test for the detection of antibodies. extracted antigens were diluted in . mcarbonate-bicarbonate buffer, ph . , ~ transferred to each well of a microtitre plate and incubated for h at °c. pool and a good discrimination between positive and negative sera. plates were washed times for min with pbs ( mt+phosphate buffer m&i-nacl, ph . ) containing . % tween r (pbs--t). assays were performed without a blocking step or plates were blocked for min at °c. gelatin (sigma, deisenhofen, germany, no. g- ) bsa (sigma, no. a- }, ovalbumin (sigma, no a- ), rabbit serum, a *blocking reagent' (boehringer, mal~nheim, germany, no. ) and low-fat milk (ueltena mil~hwerke, uefzen, germany) were used in concns of % a chemically modified bsa (bsac) (aurion, wageningen, netherlands) was tested in a concn of . %. sera were diluted l/ in pbs with % bsa and plates incubated for i h at °c followed by a set of washes and a min incubation for the conjugate (rabbit anti-horse igg (h+l) hrp, dianova gmbh, hamburg, germany, no. - - ) diluted l/ in pbs with % bsa. when assays were performed without a blocking step, different dilution buffers were tested. sera and conjugate were diluted in pbs with % bsa. pbs with . % tween of pbs with % bsa and . % tween . after a further set of washes substrate ( mg ml i of -aminosali~ylic acid (ellens & gielkens, ) in . m-sodiumphosphate buffer, ph . containing . m&i-edta and mm-h&f was dispensed and plates were read. iveszertt blot. the western blot was carried out as described previously (bose & daemen, ) , except that tbs with % gelatin was substituted for tbs with . % tween . sera reacting with antigens of mol. wts of and so kda and one or more of the , f i or kda antigens were regarded positive. sera reacting strongly only with the and kda anfligens were also considered positive. sera. sera tested were as follows: (i) sera from horses not infected with bubesia spp., i.e. horses from germany tested negative in preliminary studies by ifat at a serum dilution of l/ and by cft at a serum dilution of < l/ ; the results for these sera were used to calculate the diagnostic specificity of the elisa and western blot; ( ) sera from horses experimentally infected with b. cabal/i or with b. caba~l~ and b. equi; these results were used to calculate the diagnostic sensitivity of the elisa, western blot, ifat and cft; ( ) sera from horses experimentally infected with . egui; these results were used to evaluate the extent of cross reactions; ( ) i field sera, i.e. sera from corsica, sera from different european countries and sera from horses from brazil. for the elisa the following standards were used: (i) a high&red b. caballi serum pool; ( ) a low-titred b. cabah serum pool; ( ) a pool of sera negative for b. cabdi and b. eqeti. the cft was performed following the instructions of the united states department of agriculture ( ) and the ifat according to tenter & friedhoff ( ) . l)ntu generation and ~va~ua~~on. to allow a comparison between different microtitre plates and different runs at each microtitre plate the three standards were run on each plate in duptica&es. the absorbance values were measured approx. , and min after substrate addition with an elisa reader (titertek multiskan plus mk ii, flow laboratories, meckenheim, germany) interfaced with an ibm compatible personal computer. the computer based kinetics linked immunosorbent assay (kela) programme was used to calculate slope values as the relationship between the rate of substrate conversion by enzyme and time (jacobson, downing & lynch, ; barlough, jacobson, downing, marcella, lynch & scott, : bose, jacobson, gale, walt~sbuhl & wright, ) . further, the kela programme was used to calculate 'delta values' by subtraction of the kela slope values for control antigen from those for b. cuba& antigen. data generated by the kela programme provided a basis for maintaining quality control in the assays. the kela programme included assessment of the: ( ) linearity of the reaction rate (od vs time) in each well of the microtitre plate to ascertain whether the enzyme-substratechromogen reactants were performing properly, ( ) mean, s.u. and coefficient of variation for the slopes of sample replicates to determine within-run variation, ( ) degree of correlation of observed slopes for standard sera compared with their expected values by linear regression analysis and ( ) normalization of mean slopes for each sample to the expected values for controls thus allowing direct comparison of results on a day-to-day basis. all of these data were generated automatically at the end of each run by the kela programme and presented in printed reports for easy evafuation. the threshold was determined arbitrarily under antigen preparation. when extracted with urea, triton x- , nonidet p- or chaps, a high specific activity of the antigen was observed. in contrast the use of sds led to a low reaction between antigen and the high-titred b. cab& serum pool (fig. .) . the best discrimination between positive and negative sera was obtained with the antigen extracted with chaps that was selected for further studies. checkerboard titrations with b. cu~a~i~ antigen, the control antigen and serum pools revealed suitable dilutions of l/ to i/ for the b. caballi antigen. for the control antigen the reaction with the high-titre b. caballi serum pool and the negative serum pool remained at an almost constant level for dilutions from l/ to l/ , . thus, we used the same working dilution of l/ for both, the b. caballi antigen and the control antigen. selection of the microtitre plate. ellsas were performed using different microtitre plates. considerable differences between the performance of the plates tested were observed and the selection of a suitable plate was crucial to obtain a good discrimination in the elisa. best results were obtained with the plates no. , nunc gmbh and no. -l l- gmbh, and the latter was chosen for the further studies. hocking of plates and diluents for sera. several blocking reagents were investigated regarding their ability to reduce the non-specific binding of sera and thereby improve the discrimination. results were compared with those obtained without a blocking step. no improvement in the discrimination could be achieved by introducing a blocking step with any of the blocking reagents tried. when different dilution buffers for sera and conjugate were tested, best results were obtained with pbs-t with % bsa. upt~m~zed elisa protocol. b. caballi antigen and control antigen were extracted with chaps and used in a final dilution of l/ . the microtitre plate no. - - , flow laboratories, was used; a blocking step was omitted. sera were diluted l/l and conjugate l/ in pbs-t with % bsa. as the problem of non-specific binding of test sera could not be solved by introducing a blocking step the control antigen was used. delta values were calculated by subtraction of slope values of control antigen from slope values for b. caballi antigen. determination of the threshold. under consideration of the results in all four tests the threshold was set at . for the delta values ( x ). the average of all delta values ( x ) of sera from horses not infected with babesiaspp. was . (range- . to . )with a s.d. of . . validation of the assay. calculations of the sensitivity of the tests were carried out either with the results of sera from day or from day after experimental infection with b. ca~ai~i (table ). in both cases the sensitivity of the elisa was superior to all other tests; the cft revealed the lowest sensitivity (table ). all sera from horses not infected did not react in the elisa and in the western blot leading to a specificity of % (table ). the specificity was not calculated for the ifat and cft as a negative result was required for the sera to be considered as originating from horses not infected. cross reactions. in the elisa for b. caballi ( %) sera from horses experimentally infected with b. equi reacted, the highest delta value ( x ) being . . none of these sera was positive for b. cabal& in the western blot. of the sera cross reacting in the elisa /l had a titre of q : in the ifat for . ?-data are based on the results of sera from horses not infected with babesiu spp., i.e. horses originating from germany and tested negative for b. cuballi and b. equi by ifat and cft. consequently, the specificity was not calculated (n.c.) for the ifat and cft. equi. the ifat revealed / cross reactions ( %); with the western blot and the cft no cross reactions were observed. sera. tested were field sera, i.e. sera from corsica, sera from different european countries, and sera from brazil. whereas elisa, western blot and ifat showed similar results, the cft revealed significantly fewer reactions; the agreement between cft results and those of the other tests was not calculated. in / cases ( %) identical results were obtained with elisa, western blot and ifat. the results of elisa and western blot agreed to % ( / ), the results of elisa and ifat agreed to % ( / ) and the results of western blot and ifat agreed to % ( / ). all / sera positive by cft and ifat were also positive by elisa and western blot (table ) . / sera reacted in the cft and not in the ifat; of these sera were positive in the elisa and in the western blot suggesting false negative results in ifat, whereas the remaining serum tested probably false positive by cft. / sera reacted not in the cft but in the ifat. two of these sera were negative in the elisa and western blot suggesting false positive results of the ifat. from sera negative by cft and ifat, sera, all from brazilian horses, reacted in the elisa. seven of these sera tested positive and io negative with the western blot; of the sera negative in the western blot originated from foals younger than months born to sero positive mares. one of the problems developing elisas for babesial infections is the contamination of antigen preparations with host proteins, namely from erythrocytes. for b. bovis a simple method for the enrichment of infected erythrocytes by selective lysis of noninfected erythrocytes is available (mahoney, ) and preparations of % infected erythrocytes have been used for the development of sensitive and specific elisas (waltisbuhl, goodger, wright, commins & mahoney, ; b&e et al., ) . for b. caballi only small amounts of % infected erythrocytes can be obtained using percoll gradients (bhushan et al., ) . attempts to develop an elisa for b. cab&i with crude antigen made from blood with low parasitaemias have led to limited improvements in the sero diagnosis of b. cab& (weiland, ) . in preliminary expts we used antigen with a percentage of parasitized erythrocytes (ppe) of % from in vitro cultures and found this preparation not suitable as antigen in the elisa. reactions with positive sera were low and a poor discrimination was obtained (data not shown). only antigens extracted from a preparation of % infected erythrocytes were suitable as elisa antigens. a particular problem in preliminary studies was the high background staining caused by negative sera. background staining could not be reduced by the introduction of a blocking step after antigen coating without a loss of sensitivity. however, when different methods of antigen extraction (fig. l) , different microtitre plates and different dilution buffers were tried simultaneously, conditions could be defined which reduced the background staining and improved the discrimination significantly. the reason for the high background staining caused by equine sera is not known. for b. bovis there is evidence, that serum components other than igg are involved (bose et al., ) . these serum components, possibly igm by nature, are recognised by conventional conjugates and thus cause the background staining. for b. caballi we found that igg itself is probably causing most of the background staining in the elisa. background staining was not reduced when an igg fc-specific conjugate was used, but was almost completely absent after equine sera were passed over a protein g affinity column (data not shown). it appears that immunoglobulins from horse sera exhibit a high tendency to adhere to the surface of polystyrene microtitre plates. thus, to date the only practical solution for the problem is the use of an appropriate control antigen. apart from the high non-specific binding of horse sera a strong reaction of some negative sera with b. cab&i antigens or erythrocyte antigens extracted from the preparation of % infected erythrocytes was observed. we did not attempt to solve this problem by adsorption with normal erythrocytes as described for b. bovis (waltisbuhl et al., ) as this would have introduced another time-consuming step into the elisa protocol. with the use of the control antigen the reaction of negative sera with the antigen preparation and the background staining were not eliminated, but did no longer lead to false positive results after calculation of delta values. defined sera were used to validate the elisa and western blot (table ). the elisa proved to be the test with the highest sensitivity (table ) . western blot and ifat provided comparable results, while the cft must be regarded as obsolete due to a low sensitivity which does not meet the requirements for export testing or epidemiological studies. the consideration of sera taken from day of the infection for the calculation of the sensitivity of a diagnostic test seems to be sufficient for the requirements of the export testing as no serological test is capable to detect fresh infections. the main disadvantage of the elisa is the high percentage of cross reactions with sera from horses infected with b. equi. thus a species-specific diagnosis is not always possible by elisa, i.e. positive reactions up to delta values ( x -l) of approx. can be due to a b. equi infection. for export testing the cross reactions are of little significance as horses infected with b. equi are excluded from export as well. the disadvantage of cross reactions in the elisa can be overcome by testing of elisa-positive sera by western blot. sera were regarded positive, when a clearly visible reaction with the and kda antigens was present and one or more of the , , and kda antigens was also recognized. a number of b. caballi sera, however, particularly shortly after infection, recognized only the and kda antigens, albeit strongly. thus strong reactions with the and kda antigens were also regarded positive. with these criteria applied no false positive reactions were obtained with b. equi sera. most b. equi sera did not react with any of the diagnostic antigens of b. cab& some sera showed a faint reaction with the and kda antigens which was clearly distinguishable from the strong reactions of b. caballi sera. thus, the western blot can serve as a confirmative test which provides a species-specific diagnosis. evaluation of a computer-assisted, kinetic-based enzymelinked immunosorbent assay for detection of coronavirus antibodies in cats enrichment of babesia cabal/i-infected erythrocytes from microaerophilous stationary-phase cultures using percoll gradients an improved elisa for the detection of antibodies against babesiu bovis using either a native or a recombinant b. bovis antigen demonstration of the humoral immune response of horses to babesia caballi by western blotting a simple method for the purification of -aminosalicylic acid. application of the product as substrate in enzyme-linked immunosorbent assay (elisa) die piroplasmen der equiden-bedeutung fiir den internationalen pferdeverkehr haemoparasites of equines: impact on international trade of horses computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office bubesia bovis: continuous cultivation in a microaerophilous stationary phase culture bovine babesiosis: preparation and assessment of complement fixing antigens babesia equi (laveran ) . development in horses and in lymphocyte culture serodiagnosis of experimental and natural babesia equi and b. caballi infections titers, tests, and truisms: rational interpretation of diagnostic serologic testing united states department of agriculture . . equine piroplasmosis complement fixation test antigen production. . piroplasmosis complement fixation testmicro method. united states department of agriculture, animal and plant health inspection service an enzyme-linked immunosorbent assay to diagnose babesia bovis infection in cattle species-specific serodiagnosis of equine piroplasma infections by means of complement fixation test (cft), immunofluorescence (iif), and enzyme-linked immunosorbent assay (elisa) key: cord- -lbmbp ca authors: hansen, c. b.; jarlhelt, i.; perez-alos, l.; hummelshoj landsy, l.; loftager, m.; rosbjerg, a.; helgstrand, c.; bjelke, j. r.; egebjerg, t.; jardine, j. g.; svaerke jorgensen, c.; iversen, k.; bayarri-olmos, r.; garred, p.; skjoedt, m.-o. title: sars-cov- antibody responses determine disease severity in covid- infected individuals date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: lbmbp ca globally, the covid- pandemic has had extreme consequences for the healthcare system and calls for diagnostic tools to monitor and understand the transmission, pathogenesis and epidemiology, as well as to evaluate future vaccination strategies. here we have developed novel flexible elisa-based assays for specific detection of sars-cov- antibodies against the receptor-binding domain (rbd): an antigen sandwich-elisa relevant for large population screening and three isotype-specific assays for in-depth diagnostics. their performance was evaluated in a cohort of convalescent participants with previous covid- infection, ranging from asymptomatic to critical cases. we mapped the antibody responses to different areas on protein n and s and showed that the igm, a and g antibody responses against rbd are significantly correlated to the disease severity. these assays-and the data generated from them-are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term covid- immunity. coronaviruses (covs) are zoonotic pathogens primarily targeting the human respiratory system ( ) . while most human cov infections are mild, three coronaviruses have appeared in the past two decades that cause deadly pneumonia. severe acute respiratory syndrome coronavirus (sars-cov- ), first observed in china in , spread rapidly to countries infecting more than , people and causing deaths before being contained in ( ) . barely a decade later in , middle east respiratory syndrome coronavirus (mers-cov) was identified on the arabian peninsula and has caused more than , cases and deaths ( ) ( ) ( ) . at the end of , a novel sars-cov strain (sars-cov- ) emerged in wuhan (china) and has been spreading at an unprecedented speed around the world ever since. the disease, named coronavirus disease (covid- ) , accounts for more than million confirmed cases and , related deaths at the time of manuscript preparation ( ) . the clinical features of the covid- infection are diverse, ranging from asymptomatic carriers of the infection to acute respiratory distress syndrome and multiple organ dysfunction ( ) . sars-cov- can infect people of all ages; however, elderly and people suffering from co-morbidities such as diabetes, cardiovascular disease, chronic respiratory disease or cancer are more inclined to suffer from a severe disease progression ( ) . sars-cov- is an enveloped rna virus with a diameter of - nm to accommodate one of the largest genomes of all known rna viruses ( . - . kb) ( ) . one-third of its genome encodes four structural proteins: spike (s), membrane (m), envelope (e) and nucleocapsid (n) proteins. the protein s that extends as homotrimers on the outer viral membrane binds to the host angiotensin-converting enzyme (ace ) receptor and allows the virus to enter and infect the host target cell ( ) . host proteases process the protein s into the s and s subunits: s is responsible for receptor recognition and is comprised of an n-terminal and a c-terminal domain, the later containing the receptorbinding domain (rbd) ( ) ; while the s mediates the fusion of the viral envelope with the membrane of the host cell. the protein s of sars-cov- shares % homology at the amino acid level with the protein s of sars-cov- and while both interact with the ace receptor, sars-cov- does so with a - times higher affinity ( ) , which may explain the higher transmission rate of covid- . precise diagnosis of covid- with rt-pcr detection of sars-cov- nucleic acids remains crucial to identify symptomatic carriers to secure correct treatment and as a tool in quarantine strategies to limit the infection rate in asymptomatic carriers. serological detection of specific sars-cov- antibodies is a useful tool to identify . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint convalescent individuals that have developed immunity and thereby might be protected against reinfection, although this issue is still not resolved ( ) . moreover, serological testing is necessary to understand the transmission, pathogenesis and epidemiology of sars-cov- , providing critical data to inform public health authorities for controlling the spread of covid- and eventually re-opening societies. another question that has arisen is whether an overwhelming antibody response may even aggravate covid- infection in patients ( ) . due to urgency and demand, many serological tests have been developed rapidly and made commercially available with only limited validation on clinical samples ( ) . we have developed a flexible elisa-based platform for rapid and specific detection of sars-cov- antibodies. the platform includes an indirect rbd sandwich elisa (s-elisa) for pan immunoglobulin (ig) detection suitable for large scale antibody surveillance and direct elisas for in-depth analyses of the igm, iga and igg isotype antibody responses towards rbd and protein n. moreover, we set out to characterize the antibody response levels in relation to symptom characteristics and disease severity, to elucidate the immunological response in covid- convalescent individuals. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the assays were optimized with a specific focus on diminishing nonspecific binding and the final setup was chosen based on the optimal intensity (od) of the absorbance signal and signal-to-noise (s/n) ratio between the positive sample and the negative quality control. the developed elisa-based assays proved to be suitable for automatization and were used in high-throughput setups with both -well and -well formats, correlating significantly a total of convalescence plasma samples from previously infected individuals with sars-cov- (positive samples) and plasma samples from healthy individuals collected before the pandemic outbreak (negative controls) were subjected to antibody measurement in the rbd s-elisa ( figure a ) and in the direct elisa setups ( figure b -d). s/n ratio between the od of a positive sample and the od of the negative quality control and receiver operating characteristic (roc) were assessed to calculate the best fit cut off to estimate the performance of the assay. the rbd s-elisa performed with a . % sensitivity and . % specificity ( figure a ). the sensitivities and specificities of the direct elisas were . %, . % for igm ( figure b) , . %, . % for iga ( figure c ) and . %, . % for igg ( figure d ), respectively. the intra-and inter-assay variation were found to be acceptable (< %) for all four assays ( table ). the limit of detection was determined by interpolating the cut off od value and converting it into antibody concentrations. the resulting values indicate an estimated -fold higher sensitivity of the direct elisa setup ( . ng/ml) compared to the s-elisa ( . ng/ml) ( table ) . detection of igm, iga and igg antibodies against sars-cov- protein n was evaluated by analyzing positive samples and negative controls and roc curve analyses were assessed to estimate the assay performance . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint to provide a better insight into antibody seroconversion during sars-cov- infection and reactivity against different locations on protein s and protein n, we conducted igm, iga and igg detection in positive samples against protein fragments and short peptides located on the protein s and protein n structures, full-length rbd, protein s and protein n (figure a ). the heatmap shown in figure b indicates a different reactivity of igm, iga and igg towards the proteins/peptides analyzed. figure c represents a simplified overview of the % of individuals with antibodies recognizing each protein/fragment. a clear tendency towards a higher prevalence of igg responses against the s subunit part of protein s and the middle part of protein n was observed. parts of the s subunit and most of the n terminal part of rbd showed little immunogenicity. the cut off was calculated as the average of the negative controls plus three times the standard deviation. levels of antibodies against sars-cov- were measured from plasma samples of recovered individuals with covid- . the rbd s-elisa measures total anti-rbd igs present in the samples using a single dilution ( figure a figure b ). in comparison, responses of igm and iga isotypes were detected in and of the individuals, respectively ( figure c and figure d ). a dynamic range of antibody titers expressed as arbitrary units (a.u.)/ml was obtained when od values were interpolated by regression analysis using a four-parameter logistic curve fitting. the convalescent individuals were classified according to disease severity ( figure a -c) and symptom onset calculated as the time from the first selfperceived symptom related to covid- to the moment of blood sampling ( figure d -f). we observed a highly significant difference in the antibody titers between the disease severity groups ( figure a -c) (p < . ), with a clear association between increasing antibody levels and more severe disease symptomatology for all three antibody isotypes. igg shows the most significant increase between the severity groups ( figure c ). in contrast, asymptomatic . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint individuals appeared not to follow the same severity tendency. this could be explained by the low number of individuals in this group, thus not being representative. when we assessed the difference in antibody titers between groups based on the time of symptom onset ( figure d -f), we observed a significant increase in igg levels continuously over the time of sampling ( figure f ), while igm ( figure d ) and iga levels ( figure e ) did not change significantly. table depicts the correlation between self-perceived covid- symptomatology and the igm, iga and igg titers, as well as the disease severity, sex and age. symptoms such as fever, shortness of breath and lack of appetite were significantly and positively correlated with igm and igg levels. in contrast, iga levels were significantly negatively correlated with the loss of sense of smell/taste and headache. both age, sex (male) and disease severity were significantly positively correlated to the level of all three antibody isotypes. when adjusting the analysis between antibody levels and disease severity for age and sex, there was no longer a significant correlation between iga and severity. whereas it remained highly significantly correlated for igm and igg. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint we have developed an elisa-based platform for detection sars-cov- antibodies comprising an indirect rbd s-elisa for pan ig detection and direct elisas for in-depth analyses of the igm, iga and igg isotype responses towards rbd and protein n. rbd was chosen as the primary antigen for the screening and estimation of antibody titers for several reasons. it is regarded to be a sufficient representative part of protein s to induce an immunogenicity response ( ) and we did not detect additional sensitivity improvements by employing the full protein s as a ligand-target in the direct elisa (to be described in detail elsewhere). in a recent phase trial, antibody responses against a vaccine candidate (s- p antigen) and the rbd were assessed, finding similar ig responses in pattern and magnitude between both antigens ( ) . one of the advantages of using rbd instead of full-length protein s is the more efficient production and higher stability of recombinant rbd due to its reduced size and simple tertiary structure. the relative unique primary sequence of the sars-cov- rbd ( ) also reduces the risk of cross-reactive antibody signals derived from prior b cell responses against other types of coronaviruses. it is; however, important to annotate that this setup is highly flexible, making it possible to substitute the antigen upon a change in demand or in case of viral mutations. the use of protein n as a target antigen for serological screening has several theoretical pitfalls: the location of protein n makes it less accessible for b cell receptor interaction on the naïve b cell and probably requires a viral membrane degradation. furthermore, protein n shows higher sequence conservation ( . %) and thereby increase the risk of false-positive detection ( ) . nevertheless, large commercial providers have chosen to use protein n as the serological target in different types of sars-cov- antibody assays ( ) . in our study, the detection of igm antibodies towards protein n or its fragments was weaker than igg, suggesting a fast seroconversion of igm into igg. furthermore, around - % of the convalescent individuals did not mature any detectable antibody response, which is in good agreement with a previous study ( ) . the direct elisa setup allows the use of different sars-cov- proteins in their full-length, shortened variants or fragmented immunogens, offering a useful tool to study the different reactivity patterns of igm, iga and igg towards specific exposed areas on the viral antigens. we measured several different antigen areas on protein n and s to establish a heatmap of the antibody landscape. based on the results, we could demonstrate a tendency towards immune dominating areas in the s unit and the central part of protein n. however, it is important to highlight that the heatmap does not represent the full sequences of the cov antigens and that the dissection of antigens into shorter fragments could have a significant impact on the antibody reactivity. it could; however, in a more extensive setup, provide valuable information towards a targeted . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . we show that igg levels increases during the first weeks after symptom onset, while the igm and iga levels remain stable for the same period of time. this was surprising as we would have expected a more pronounced decrease in igm levels over time. this observation could indicate an importance of the two isotypes, reinforced by the fact that severity is correlated with high levels of antibodies of all three isotypes. it has previously been shown that both igg independently and total antibody levels correlate with disease severity in patients during hospitalization ( ) , but to our knowledge, the prolonged clear correlation between iga, igm and igg titers and disease severity have not been reported before. the data illustrates that individuals with mild symptoms during infection with sars-cov- , in general, will mount a lower antibody response compared to individuals with moderate and severe symptoms several months after recovering from a covid- infection. this observation gives an essential insight into the immunological response regarding clinical disease presentation, which further highlights the demand for more quantitative assays. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint in this study, all three antibody isotypes correlate positively with age and sex (male), which can be explained by the fact that severe covid- infection is more pronounced in the elderly male population ( ) . we show that the antibody titers, especially igg levels, are correlated with specific symptom characteristics, including fever, pharyngalgia, shortness of breath and nausea, which again shows the link between clinical manifestations and the immunological response. the iga level, on the other hand, was negatively correlated with loss of sense of taste/smell and surprisingly showed no other correlation to the symptomatology of the upper respiratory tract in this study. the role of iga, which is considered the predominant antibody involved in mucosal immunity, remains to be fully understood. iga is suggested to mediate anti-viral defense functions at different anatomic levels in relation to mucosal epithelium ( ) . however, the mechanisms behind this remain unknown and often gain limited attention during infectious studies. in this respect, it could be interesting to examine the iga levels in mucosal tissue during sars-cov- infection and determine whether the mucosa-associated iga plays a significant role during sars-cov- infection. our findings provide support to the notion that antibodies towards sars-cov- represent a double-edged sword. antibodies are important in viral neutralization, but also in fc receptor-mediated phagocytosis, antibody-dependent cellular cytotoxicity (adcc) and complement-dependent cellular cytotoxicity (cdcc) and subsequent elimination of pathogens. however, it is known that particularly adcc and cdcc can drive harmful and systemic pro-inflammatory responses that can have severe pathophysiological consequences. thus, based on our findings and others, it may be suggested that an unwanted immune response towards sars-cov- may be one of the mechanisms causing hyperactivation of macrophages and monocytes, leading to the deadly cytokine storm, which seems to be a hallmark of covid- ( ) . whether a previously infected individual can expect stable long-term protection against reinfection with sars-cov- remains unknown. a recent study found a correlation between the production of neutralizing antibodies against rbd and elevated igg titers in convalescence covid- individuals ( ), reinforcing the use of rbd as the candidate to analyze for neutralizing antibodies in these individuals. the durability of neutralizing antibodies (primarily igg) against sars-cov- has yet to be defined, but persistence for up to days from symptom onset has been described ( , ) . in comparison, when following infection with sars-cov- , concentrations of igg remained high for approximately to months before subsequently declining slowly during the next to years ( ) . it is uncertain whether an individual with low antibodies titers, mainly igg, has a higher risk of reinfection compared to an individual with high levels of . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint antibodies post disease. it has recently been suggested that in the absence of antibody levels, an individual could be protected against reinfections by the presence of memory t cells ( ) . furthermore, the major proportion of convalescent individuals, included in this study, indeed show a dominating igg response, suggesting that both affinity maturation, isotype class switching and b cell memory response has occurred. these b cell populations could, together with the memory cd + and cd + cells, secure a fast and efficient response to a secondary exposure of sars-cov- . this study is limited by relying on participants' retrospectively self-reported symptomatology and symptom debut, which allows for an unknown amount of misclassification. moreover, with this design, we could not monitor the antibody response concerning survival. however, because of the detailed analysis of the antibody responses, and the clear associations despite the retrospective design, we assume that the associations would be even stronger in a carefully conducted prospective designed study. in conclusion, we have established robust, flexible and specific elisa-based platforms for detection sars-cov- antibodies and presented novel insight into the link between antibody responses and covid- disease severity. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the following buffers were used: pbs ( . mm na hpo , . the coding sequence for protein s rbd (qic . , aa r -s ) was synthesized by genscript and cloned into a pcdna . expression vector with an n-terminal human vh - signal peptide and a c-terminal xhis tag followed by an avitag (-gsg-hhhhhhhhhh-gsg-glndifeaqkiewhe). the sequence of protein n (qld . ) was optimized as described elsewhere ( ) and synthesized by geneart (thermo fisher scientific, waltham, ma, usa) into a pcdna . vector with the human serum albumin signal peptide and a c-terminal xhis tag (-gs-hhhhhhhh). both constructs were expressed using a mammalian transient expression system. on day five after transfection, the supernatants were harvested by centrifugation and sterile filtered. the recombinant proteins were purified using a -step automated purification method setup on an Äkta express chromatography system with an immobilized metal affinity excel histrap column and a size exclusion superdex column (chromatography system and columns cytiva, marlborough, ma, usa). the purified proteins were stored in a buffer composed of mm hepes, mm nacl, ph . . a portion of the rbd was specifically biotinylated in the avitag sequence using a bira kit (avidity llc, co, usa). a total of recovered individuals previously tested rt-pcr positive for sars-cov- were included in the study. the department of cardiology at herlev university hospital in denmark recruited the participants. the rt-pcr positive participants are comprised of males and females aged from - and course of disease ranged from asymptomatic to critically ill. all participants were invited to complete an electronic self-report questionnaire providing additional information about symptom onset, characteristics and disease severity divided into the following groups: asymptomatic, mild, moderate, severe or critical. the mild disease was defined as having few symptoms and generally feeling well, moderate disease as being bedridden at home, severe disease as the need for hospitalization and critical disease as need of admission to the intensive care unit (icu) for mechanical ventilation. characteristics of the study participants are detailed in table . serum and edta plasma samples were stored in aliquots and kept frozen at − °c . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint usa). plates were washed four times with pbs-t between the steps mentioned above. all -well plates were handled by the biomek fx automated workstation (beckman coulter, brea, ca, usa). development and validation: specificity of the signal, limit of detection, variation, parallelism and clinical performance the rbd s-elisa and the direct elisas was subjected to optimization with regards to dilution range, the use of blocking buffers and variations in incubation times. the final conditions were chosen based on s/n ratios. specificity and sensitivity were calculated based on roc curve representation and selection of the most appropriate cut off by prioritizing the specificity. assay sensitivity regarding the limit of detection was determined by the concentration given by interpolating the od value of the cut off. the calibrator was prepared by spiking µg/ml of recombinant human monoclonal igg antibody against sars-cov- spike (a , genscript, piscataway, nj, usa) into normal human serum and diluting in serum into a -fold dilution. samples were treated as patient samples and further diluted : in the s-elisa and : in the direct elisa in pbs-t followed by incubated as stated above. intra-assay variation was evaluated by calculating the coefficient of variation (cv) of an individual cvs for all the duplicates in a total of samples. inter-assay variation was evaluated by calculating the cv of two samples run in duplicates in separate plates on three different days. the parallelism between serum and plasma samples was evaluated by comparing pairs of serum and plasma samples using spearman rank correlation tests. to evaluate whether the antibody levels correlated with the disease severity and/or the days after symptom onset, sample absorbances were logistically transformed and a four-parameter logistic curve fitting was applied to calibrate the antibody levels into a.u./ml. the appropriate dilution for each sample was chosen based on the best fit in the linear range of the calibrator. the interpolated value in a.u./ml was corrected by the dilution factor ( , or ). a sample od value below . was automatically given the value of a.u./ml. a total of different sars-cov- protein fragments on the protein s and s and protein n coupled via an nterminal cysteine and maleimide conjugation to recombinant-human serum albumin (rhsa) (albix, novozymes, bagsvaerd, denmark), short proteins from protein s and full-length protein s, protein n and rbd were analyzed for immunogenicity capacity on the direct elisa. proteins details are illustrated in figure a . nunc™ maxisorp flat-bottom plates nonsterile -well plates were coated with µg/ml of the proteins in pbs on at °c. a total of rt-. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint pcr positive samples and six negative controls were diluted : in dilution buffer and incubated as mention above. detection and development procedures were followed, as described in subsection . . statistical analyses were performed using graphpad prism version statistical differences between disease severity and symptoms onset groups were analyzed using one-way anova (kruskal-wallis test) with dunn's multiple comparison test. spearman rank correlation tests were used to determine the correlation between different experimental parameters. data are represented as the average of sample duplicate and the median. significance levels: * = p < . , ** = p < . , *** = p < . **** = p < . and p < . was considered statistically significant. all procedures involving the handling of human samples are in accordance with the principles described in the declaration of helsinki and ethically approved by the regional ethical committee of the capital region of denmark (h- ) . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint kruskal-wallis test was performed. p value < . was considered significant. * = p < . , ** = p < . , *** = p < . **** = p < . . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint tables table . intra-and inter-assay variation and limit of detection for the s-elisa setup and the direct elisa setup. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . spearman rank correlation analysis was performed. p value < . was considered significant. * = p < . , ** = p < . , *** = p < . **** = p < . . a n = participants. b n = participants. c partial correlation adjusted for age and sex. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint origin and evolution of pathogenic coronaviruses sars -beginning to understand a new virus identification of a novel coronavirus in patients with severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group sars and mers: recent insights into emerging coronaviruses coronavirus disease (covid- ) pandemic clinical features of patients infected with novel coronavirus in wuhan, china estimates of the severity of coronavirus disease : a model-based analysis covid- infection: origin, transmission, and characteristics of human coronaviruses the covid- pandemic: biological evolution, treatment options and consequences structural and functional basis of sars-cov- entry by using human ace cryo-em structure of the -ncov spike in the prefusion conformation. science ( -. ) antibody responses to sars-cov- in patients of novel coronavirus disease dissecting antibody-mediated protection against sars-cov- the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov- patients an mrna vaccine against sars-cov- -preliminary report the coronavirus nucleocapsid is a multifunctional protein biochemical characterization of sars-cov- nucleocapsid protein diagnostic accuracy of serological tests for covid- : systematic review and meta-analysis seroprevalence of antibodies against sars-cov- among health care workers in a large spanish reference hospital gender differences in patients with covid- : focus on severity and mortality multiple functions of immunoglobulin a in mucosal defense against viruses: an in vitro measles virus model dysregulation of immune response in patients with covid- in wuhan, china detection of sars-cov- -specific humoral and cellular immunity in covid- convalescent individuals immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid- duration of antibody responses after severe acute respiratory syndrome robust t cell immunity in convalescent individuals with asymptomatic or mild covid- chimeric proteins containing map- and functional domains of c b-binding protein reveal strong complement inhibitory capacities the authors thank jytte bryde clausen and bettina eide holm for excellent technical assistance and josé juan almagro armenteros (university of copenhagen, copenhagen, denmark) for his statistical advice. this work was financially supported by the carlsberg foundation (cf - ) and the novo nordisk foundation ( a ). the authors have declared that no conflict of interest exists. the assays were developed in a non-commercial collaboration between rigshospitalet and novo nordisk a/s. key: cord- -gonx taq authors: pignatelli, jaime; alonso-padilla, julio; rodríguez, dolores title: lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (ptov-he) protein date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: gonx taq hemagglutinin-esterases (he) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. he proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. the role of the he protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. the phylogenetic analysis of he coding sequences from porcine torovirus (ptov) field strains revealed the existence of two distinct he lineages. in a previous study, ptov virus strains with he proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. in this work, we report antigenic differences between the two he lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of ptov infection in the farms. toroviruses (tov) are enveloped, positive single-stranded rna viruses of cattle, horses, pigs and humans [ ] . they have been associated with enteric infections and diarrhea, especially in young animals and children [ ] , and are considered a potential zoonotic threat [ ] . the torovirinae subfamily of the coronaviridae family (order nidovirales) comprises four species: equine torovirus (etov), bovine torovirus (btov), porcine torovirus (ptov) and human torovirus (htov). the few epidemiological studies performed in different countries indicate high prevalences of these viruses [ ] [ ] [ ] . the torovirus's genome size (~ kb) and organization are very similar to those of other coronaviruses, with two huge overlapping open reading frames (orf) shaping the ′-end of the genome, where the replicase/transcriptase machinery is encoded, and a final third of the rna molecule hosting the coding sequences for the four structural proteins (from ′ to ′): spike (s), hemagglutinin-esterase (he), membrane (m), and nucleocapsid (n) [ ] . by analogy to other nidoviruses, the s protein is considered to be the putative receptor binding molecule. copies of this molecule form the large spikes protruding from the viral particles as shown by electron microscopy [ , ] . the he protein forms homodimers which make up the smaller spikes [ , ] . this he protein is a class i membrane glycoprotein of about kda that belongs to the receptor destroying enzyme (rde) protein family. as an rde protein he provides reversible binding to glycosylated surfaces [ ] due to its ability to bind sialic acids and catalyze the disruption of that binding by means of its acetyl-esterase activity. the d structure and sialic acid preference of ptov-(strain markelo) and btov-(strain breda) he proteins have been solved [ ] . two main domains have been defined in the he monomer of both proteins: the enzymatic acetyl-esterase region (e) and the receptor binding (r) or lectin domain. despite the great amount of knowledge acquired on this protein family, it is not yet clear what is the exact role of he during the torovirus infectious cycle. the etov, the only cell culture adapted torovirus, as well as different btov strains that could be isolated in cell culture, all lack a functional he protein [ ] [ ] [ ] [ ] due to deletions or mutations in the he gene, acquired during their adaptation to in vitro growth conditions. thus, he expression seems to be detrimental for in vitro culture of these viruses, as it occurs with the coronavirus murine hepatitis virus (mhv) [ ] . however, the maintenance of the he protein in a vast majority of new ptov and btov field isolated strains indicates that it has to play an important function during in vivo infections. several hypothetical functions have been postulated for the tov-he: (i) being a viral co-receptor, (ii) digestion of mucus layers to allow the virus to reach the target cells in the respiratory and/or enteric tracts, (iii) release of viral particles bound to decoy receptors, (iv) influencing host/cell tropism, or as recently proposed by de groot's group, (v) acting as a molecular timer in the virus pre-attachment step to the host-cell [ ] . two ptov-he lineages have been identified, with representative strains being markelo and p [ ] . they share an amino acid sequence homology of %. recently, during a longitudinal survey of ptov in a spanish pig herd, several ptov-he isolates representative of both lineages were identified [ ] , and similar findings were described from a survey performed in korea [ ] . in the first case, the two ptov-he lineages were detected even within the same animal at two sequential sampling time points, indicating that both ptov strains carrying different he proteins coexisted on the same farm infecting the same piglets, and suggesting that the immune response generated against one ptov strain did not protect the animals against the infection by the other strain. to further investigate this hypothesis ptov-he proteins corresponding to each he lineage were expressed, characterized, and used to track the anti-he response in the animals from the farm where the ptov strains were obtained. the analysis of serum samples by hemagglutination inhibition assay and elisa revealed antigenic differences between the two he lineages. cells, viruses and antisera bsc (african green monkey kidney cells) cells were grown in dulbecco's modified eagle's medium (dmem), supplemented with % heat-inactivated neonatal calf serum (ncs), non-essential amino acids ( %), gentamicin ( μg/ml), penicillin ( iu/ml), streptomycin ( μg/ml) and fungizone ( . μg/ml). vaccinia virus (vacv), strain western reserve (wr) [ ] , and the recombinant vacv (rvv) derivatives were propagated and titrated in bsc cells. to obtain a polyclonal antiserum against ptov-he (αhe), a new zealand rabbit was inoculated with two different peptides coupled to keyhole limpet hemocyanin (klh). peptides comprising residues - (ctnpstpn sldipqqlc) and - (ltppenipshc) of ptov-he-bres [genbank: fj ] [ ] were chemically synthesized at the proteomics facility of our institution after selection of target ptov-he antigenic regions by bioinformatic analysis with protean software (dnastar inc. madison, wi, usa). the serum sample collection and their treatment were previously described [ ] . the samples corresponded to animals from three different litters (a, b and c; four animals per litter) of the same farm, and were collected at -, -, -, -, and -weeks of age. serum samples from the corresponding sow of each litter (n = ) collected at the first day of sampling were also obtained. . the amino acid residues that are conserved in all sequences from one lineage but are different from those in the strains corresponding to the other lineage are depicted in blue. other residues that are conserved in all sequences or are non-lineage specific are depicted in red. pgemt plasmids with ptov-he coding sequences from isolates . and . [ ] were used to sub-clone the indicated ptov-he sequences into the pjr vacv transfer vector [ ] by asymmetric digestion with restriction endonucleases bamhi and ncoi. the resulting plasmids, pjr-he . and pjr-he . , carried the he genes under the control of the vacv synthetic early/late promoter (pe/l). insertion of the he genes into the vacv hemagglutinin locus (ha) was achieved by homologous recombination between the transfer vectors and the virus genome following standard procedures [ ] . the resulting rvv-he . and rvv-he . viruses were selected and grown following standard procedures to yield viral stocks that were titrated and stored at − °c until use. a control rvv harboring a disrupted ha locus (rvv-ha -) was generated in parallel under the same procedures using the empty pjr vector. rvv expressing soluble forms of he . and he . proteins fused to the c-myc tag were also generated to facilitate protein purification by affinity chromatography. for this purpose, the sequences coding for the fusion proteins were cloned into the pjr vector, and the corresponding rvv were obtained as described above. bsc cells infected with the corresponding rvv at a multiplicity of infection (moi) of plaque-forming units per cell (pfu/cell) were collected at hours postinfection (hpi) in laemmli sample buffer. expression of ptov-he proteins in infected cells was analyzed by polyacrylamide gel electrophoresis (sds-page) and western blot with the rabbit polyclonal αhe serum. after incubation with a horseradish peroxidase-conjugated secondary antibody (sigma-aldrich, saint-louis, mo, usa), the reactive bands were detected by chemoluminescence using the commercial ecl reagent (ge healthcare, uppsala, sweden). subconfluent bsc cell monolayers were grown in mm-diameter coverslips and infected with rvv-he . , rvv-he . , or the control virus rvv-haat an moi of pfu/cell. at hpi, cells were washed, fixed with % paraformaldehyde and permeabilized with . % triton x- in pbs. after a blocking step with % fetal calf serum (fcs) in pbs, cells were incubated for h with αhe polyclonal antiserum diluted ( : ) in pbs containing % fcs, washed three times and stained with alexa fluor goat anti-rabbit igg (molecular probes™, life technologies, carlsbad, ca, usa) at : dilution. dapi reagent ( ′, ′-diamidino- -phenylindole) (molecular probes™) was used to stain cell nuclei. after washing with pbs, coverslips were mounted on microscope slides using the prolong® gold anti-fade reagent from molecular probes™. images were captured with a confocal radiance system (bio-rad, münchen, germany) and processed using imagej [ ] and adobe photoshop cs (adobe system inc., san josé, ca, usa). confluent bsc cell monolayers seeded in mm plates were infected with rvv-he . and rvv-he . at a moi of . at hpi cells were rinsed with pbs and harvested. after a centrifugation step at × rpm for min, cell pellets were resuspended in tne buffer ( mm tris-cl, mm edta and mm nacl) and subjected to three successive freeze-thaw cycles and sonication. the soluble fractions were stored at − °c until needed. to obtain esterase inactivated ptov-he cell extracts, rvv infected bsc monolayers were treated with mm diisopropyl fluorophosphate (dfp; sigma-aldrich) for min at room temperature before scraping the cells. any remaining excess of dfp was removed after thoroughly washing the cells with ice-cold pbs. protein concentrations in the cell extracts were determined by bradford reaction (bio-rad protein assay) using known amounts of purified bovine serum albumin (bsa) as standards. specific acetyl-esterase activity of both recombinant he proteins (he . and he . ) was tested by an in situ staining assay as previously described [ ] . bsc cell monolayers were infected with the corresponding rvv-he and with rvv-haas the negative control. at hpi viral plaques expressing he were visualized after incubation with α-naphtyl acetate-fast blue bb solution (anae assay kit, sigma-aldrich) according to the manufacturer's instructions. cell monolayers were then stained with crystal violet solution ( . % in % methanol) to visualize all viral plaques. sialate-o-acetylesterase activity of rvv-infected bsc cell extracts in tne solution was determined with the synthetic substrate p-nitrophenyl acetate (pnpa; sigma-aldrich) as previously described [ ] . briefly, cell extracts were incubated with mm pnpa at °c, and the hydrolysis of pnpa was recorded by reading the absorbance at nm at different times. the enzymatic activity per μg of cell lysate, after subtracting the background activity present in the control cell extract infected with rvv-ha -, was calculated as previously described [ ] . these assays were performed in u-shaped -well plates (nunc, roskilde, denmark). mouse (mus musculus, strain swiss) blood obtained from the retro-orbital cavity of anesthetized animals was collected in two volumes of sterile alsever solution to prevent clotting. animals were handled following the guidelines of the animal experimentation committee of our institution, in strict accordance with the spanish law (rd - ). the red blood cells (rbc) obtained were washed with alsever solution, counted and diluted in pbs to obtain a % stock solution ( % solution corresponds to · rbcs per ml). hemmagglutination assays (ha) were set-up using two-fold dilutions of extracts from dfp-treated and untreated rvv-infected cells. fresh mouse rbc in pbs solution were added ( μl) to the he containing wells and incubated for two hours at °c. hemagglutination was scored and documented by photography, and plates were then placed at °c and a second image was taken after h incubation at this temperature. the hemagglutination units (hau) of he containing cell extracts were established as the reciprocal of the highest dilution causing hemagglutination. for the hemagglutination inhibition assays (hi), he cell extracts ( hau in μl pbs) were incubated ( h at °c) with two-fold serial dilutions of kaolin treated pig sera [ ] prior to adding μl per well of a mouse erythrocyte suspension (final concentration of . %). the hi titer was defined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination by the viral antigen. serum samples with hi titers higher than log were considered positive. extracts from bsc cells infected with rvv-he . -myc or rvv-he . -myc, were prepared as described above in tne buffer containing % np . the soluble fractions were transferred to new tubes containing anti-c-myc coated beads previously equilibrated in tne buffer (mbl, naka-ku nagoya, japan). after h incubation at °c the mixtures of bead suspension and cell lysates were centrifuged in spin columns, washed thrice, and proteins were eluted with a c-myc tag peptide. the reagents and the protocol for the protein purification procedure were provided by the supplier. the purified proteins were analyzed by sds-page and western blot with the αhe polyclonal serum and anti-c-myc monoclonal antibodies to confirm their identity (clontech, takara bio inc., otsu, shiga, japan), and by coomasie blue staining of the gel to determine their purity and concentration using different amounts of bsa as the reference. an indirect elisa was set up to detect antibodies against ptov-he proteins in pig serum samples. the optimal protein concentration was established by checkerboard titration with positive and negative pig control sera. paired rows of a -well microtiter plate (maxisorp, nunc) were coated overnight ( °c) with purified he . -myc and he . -myc proteins diluted at a . μg/ml concentration. plates were thoroughly washed after each step by rinsing the plates thrice with pbs containing . % tween (pbst). after coating, plates were blocked for h at °c with pbst- % bsa. then, μl of each serum sample diluted : in pbst- % bsa was added in paired wells and incubated h at °c. a commercial goat anti-pig igg secondary antibody conjugated to horseradish peroxidase (sigma-aldrich) was used. the enzymatic reaction was developed using o-phenylenediamine dihydrochloride (opd-fast™, sigma-aldrich), and stopped after min by adding μl/well of n sulphuric acid. optical densities at nm (o.d. ) were recorded with a multichannel spectrophotometer (titertek multiscan mcc/ ). as negative control serum, a pool of sera from caesarianderived, colostrum-deprived (cd/cd) pigs kept under germ free conditions (spf) was used, and the positive control serum was a commercial porcine serum (abd serotec, kidlington, uk) previously determined to contain antibodies against ptov [ ] . the elisa cut-off was established for each antigen as the mean of the o.d. of negative control serum plus three times the standard deviation (he . -myc cut off = . ; and he . -myc cut off = . ). a previously described indirect elisa was used to detect antibodies to the highly conserved ptov nucleocapsid protein [ ] . statistical analyses of hi and elisa sera reactivities obtained from animals grouped by ages against both ptov-he . and ptov-he . were performed using two tailed t student's. means ± standard error of the mean (sem) for each group are shown. the detection of ptov strains belonging to the two defined he lineages, represented by markelo and p strains, in piglet fecal swabs collected on a spanish farm in the course of a ptov longitudinal survey was previously described [ ] . interestingly, two ptov strains, one from each of the described lineages were isolated from the same animal (pig , litter b) at different time-points of the piglet's life, at -and -weeks of age. the he from the virus isolated at week , named ptov-he . , was . % homologous to markelo he at the amino acid level, whereas the protein from the virus found at week , he . , was more closely related to p he ( . % homology). the direct comparison between both ptov-he . and ptov-he . gave a . % identity, a homology that is similar to those obtained when comparing previous reported he sequences from both he lineages [ , ] . to study the amino acid differences between both he lineages, a sequence alignment from he proteins corresponding to viruses identified in three different geographical regions was performed. the he sequences from the spanish ptov strains he . , he . , he . and he . [ ] were compared with those from other european strains markelo, p , p [ ] and bres [ ] , and from korean strains - and - and - and - [ ] . the alignment shows that several amino acid positions were conserved in a lineage specific manner ( figure a , grey shade), even in the geographically distant korean strains. these lineage specific amino acids were located in the markelo tridimensional structural model [ ] , and, as shown in figure b , these differential amino acids (blue balls) were found mainly placed on the surface of the receptor binding domain, suggesting that they could mark potential antigenic differences between both he lineages. to study the he proteins from ptov-he isolates . and . as the model for the markelo and p he lineages, and search for potential antigenic differences between them, both proteins were expressed by the recombinant vacv methodology. rvv carrying the corresponding he . or he . coding sequences inserted into the ha locus of the vacv genome, were generated (rvv-he . and rvv-he . ). in addition, a vacv lacking a functional ha protein, rvv-ha -, was produced to serve as the negative control. ptov-he proteins expressed upon infection of bsc cells with the rvv were specifically detected by western blot and immunofluorescence microscopy using the αhe rabbit antiserum. western blot analysis under denaturing conditions detected both he . and he . proteins in rvv-infected cell monolayers with the expected kda molecular weight (figure a ) corresponding to the glycosylated form of the protein [ ] . both he proteins were expressed at a similar extent. regarding the subcellular localization of recombinant he proteins analyzed by immunofluorescence, the ptov-he specific signal was widely distributed in the cytoplasm as well as associated to the nuclear and plasma membranes of bsc cells infected with either rvv-he . or rvv-he . but not in rvv-ha --infected cells ( figure b ). the acetyl-esterase activity of the recombinant ptov-he . and he . proteins was analyzed by in situ staining of infected cells by the anae assay [ ] . infected cells surrounding viral plaques produced by both rvv-he . and rvv-he . show a brownish staining whereas the rvv-haderived plaques remained unstained ( figure a ). ptov-he . and rvv-he . were also able to hydrolyze the acetyl group from the synthetic substrate p-nitrophenol acetate in the pnpa assay [ ] performed with extracts from infected cells. the graph in figure b shows the specific pnpa hydrolysis obtained with the he proteins. both he . and he . cell extracts show similar activities ( . and . mu/μg lysate respectively). ptov-he acetyl-esterase activity of the infected cell extracts was completely and irreversibly inhibited when the rvv-infected bsc cell monolayers were treated with the serine-esterase inhibitor dfp (see materials and methods) as shown in figure b . no esterase activity could be detected in extracts from cells infected with rvv-ha -, either untreated or treated with dfp. the lectin activity of he . and he . proteins was tested by hemagglutination assay using mouse erythrocytes. the hemagglutination assay was set up with two fold serial dilutions of dfp treated (dfp + ) and dfp untreated (dfp -) cell extracts, starting from μg per well of total protein. the lysate from cells infected with rvv-haserved as the negative control. at °c, both dfp + and dfp -rvv-he cell extracts were able to induce hemagglutination of mouse rbc at a similar ha titer, whereas the hacell extract did not hemagglutinate the mouse rbc at any of the amounts tested ( figure c ). when plates were placed at °c, the hemagglutination net induced by dfpcell extracts was disrupted due to their acetyl-esterase activity at that temperature, however hemagglutination was maintained in dfp-treated he containing wells, indicating that the acetyl-esterase activity was completely inhibited ( figure d ). the esterase activity and receptor binding results show that both recombinant ptov-he proteins were biologically active, indicating the acquisition of proper conformational folding. the hemagglutination caused by he proteins is due to the binding of their lectin or receptor binding domain to specific sialic acid determinants present on the surface of rbc. hence, in order to examine the potential existence of antigenic differences between the two lineages of ptov-he proteins, piglet serum samples were analyzed with an hi assay to detect the presence of specific antibodies against the receptor binding domain that prevents the rbc hemagglutination. in the hi test, dfp inactivated he . and he . cell lysates were used to avoid artifacts in the assay derived from the he esterase activity. using serum samples from the longitudinal survey, different hi antibody response dynamics against he . and he . proteins were observed. anti-he . antibodies were detected at week in % of the piglets ( / ) ( figure g ), even though all piglets from litter b were negative against that particular lineage at that time-point ( figure c ). with respect to the anti-he . inhibitory reaction in week sera, none of the animals in litter c was reactive against it ( figure f ), and only one piglet from litter b had specific anti-he . hi reactivity ( figure d ). however all piglets from litter a had high hi titers towards this lineage, in agreement with the high hi titer of their sow ( figure b ). independently of their lineage specificity, the hi titers decreased at week (coinciding with weaning) in all but one anti-he positive piglets, however, the percentages of positive animals against both he proteins remained constant until week ( figure g ). up to that week, the reactivity to he . was circumscribed to piglets within litter a ( figure b ). at week , just % of the piglets ( / ) were reactive against he . , and a bare % presented anti-he . antibodies ( figure g ). by week , % of the piglets ( / ) were positive against he . ( figure g ). although anti-he . reactivity was also observed in % ( / ) of the piglets, all those animals had higher hi titers against he . . to compare the reactivity of each serum sample against both he . and he . proteins the hi titers obtained against the two he proteins were plotted in figure h . while few samples had the same hi titers with both proteins (serum samples found over the diagonal), most of them showed preferential reactivity with one versus the other he lineage (serum samples found at both sides of the diagonal) and even some serum samples showed specific reactivity against only one of the he proteins ( serum samples were positive only for the he . and were specific for the he . ). these data clearly indicate that he lineage specific amino acid differences within the receptor domain were enough to determine that antibodies developed against one lineage do not interfere with the receptor recognition by the other he lineage. to further investigate the reactivity of antibodies in piglets' serum samples against he . and he . proteins and the dynamics of antibodies against each of them, an elisa method using purified myc-tagged he proteins (he . -myc and he . -myc) as antigens was used. the analysis by sds-page confirmed the purity of both preparations and their reactivities with both αhe polyclonal serum and anti-c-myc monoclonal antibodies were confirmed by western blot (see additional file ). all pig serum samples tested were positive against both he proteins by elisa ( figure ). one-week-old piglets had o.d. values akin to those of their corresponding sow against each he protein. piglets from litter a were highly reactive against both he . and he . , although the elisa values were slightly higher against the second one ( figure a and b) . piglets from litter b, had a low reactivity against both proteins ( figure c and d ). in contrast, piglets from litter c showed a higher reactivity against he . than against he . ( figure e and f). as it happened with hi titers, anti-he reactivity by elisa gradually diminished with piglet's age and by weeks of age, weeks after weaning, it reached the lowest level in most of the animals. at the next age analyzed ( weeks), most animals showed an increase in their anti-he reactivities against both ptov-he proteins, and these kept rising until weeks of age at least. to obtain an overview of the immune response against both ptov-he proteins, the mean elisa reactivities and hi titers of the serum samples from each litter grouped by ages were compared side-by-side. as shown in figure , both assays revealed statistically significant figure analysis by hi assay of the reactivity of sera obtained from pigs at different ages against ptov-he . and ptov-he . . extracts from bsc cells infected (moi ) for h with rvv-he . (panels a, c and e) or rvv-he . (panels b, d and f) and treated with dfp were diluted to contain hau in μl pbs and incubated ( h at °c) with two-fold serial dilutions of pig sera prior to adding the mouse rbc. serum samples were obtained from animals belonging to three litters (litter a, panels a and b; litter b, panels c and d; litter c; panels e and f) at different ages ( , , , antigenic differences between he . and he . . in animals from litters a and b, higher reactivity against he . was observed in the first weeks of the piglets' life, although low titers were observed in animals from litter b, which were barely detectable by the hi assay. on the contrary, in the first weeks, animals from litter c had higher antibody titers against he . by both assays. also, from these results it is clear that sera from -week old animals from the three litters reacted preferentially against he . , indicating a switch in antibody reactivity in animals from litter c. figure reactivity of sera obtained from pigs at different ages against ptov-he . and ptov-he . determined by elisa. purified he . -myc-and he . -myc proteins were used as coating antigen ( . ng/well). the same serum samples analyzed in figure were used in the elisa at a : dilution. igg elisa reactivities against he . -myc-(panels a, c and e) and he . -myc (panels b, d and f) of serum samples from pigs and their sows are represented. figure paired comparisons of the results of pig sera reactivities against ptov-he . and ptov-he . determined by hi and elisa. graphic representation of the mean hi (a) and elisa (b) titers of sera grouped by pig ages against both ptov-he . (black bars) and ptov-he . (grey bars). means ± standard error for each sample are shown. asterisks indicate statistically significant differences of sera reactivity against the two proteins (**p < . , *p < . ; student's t test). the dotted lines indicate the respective cut off values determined for each assay. the reactivity of the piglet's sera against the highly conserved n protein using a previously standardized elisa [ ] has already been analyzed [ ] . for comparison with the anti-he immune response here we provide the results of the analysis of sera from the individual piglets over time (see additional file ). although there are differences in the magnitude of the elisa titers among the different piglets, what is clear from these results is that once maternally acquired antibodies had vanished, which for the n protein occurs around weaning time (week of age), all animals developed their own anti-n antibodies that can readily be detected in most animals by week . these findings were in agreement with those obtained by hi and elisa using the he proteins as antigen, and indicate that all animals become infected by ptov soon after weaning. despite the great advances recently made on he knowledge, which include the elucidation of both btov-he and ptov-he tridimensional structures [ ] , the exact role of the he protein in the viral life cycle and the potential relevance of the differences between he lineages in the immunological response to the virus remain unclear. without an in vitro culture system and given the great difficulties and costs of in vivo research, the use of heterologous expression systems represents a useful and relatively inexpensive approach to study the ptov-he protein. here we used the recombinant vacv methodology to express two full ptov-he sequences, he . and he . , and their two corresponding soluble fractions attached to a c-myc tag. those he coding sequences had been previously identified during a thorough longitudinal study of ptov in a spanish farm [ ] , and each of them were found to belong to one of the two defined ptov-he lineages [ ] . these he sequences were obtained from the same animal in sequential collection points. this finding indicated that both lineages co-existed on the farm [ ] although with apparently different prevalences according to the age of the host. this result could lead to new possibilities to approach the ptov-he behavior in the virus' natural environment. both recombinant proteins show similar features regarding their molecular weight, glycosylation degree, and subcellular localization as those reported previously for ptov and btov [ , ] , indicating that the recombinant he proteins follow a correct biosynthetic pathway. in addition, the heterologous expressed he proteins were fully functional as receptor bindingreceptor destroying enzymes since both ptov-he proteins were able to hemagglutinate mouse erythrocytes, but also to hydrolyze the acetyl-ester linkage of glycan chains, as well as from acetylated synthetic compounds like pnpa and anae. the analysis of the amino acid changes found between both ptov-he lineages shows that there are amino acid residues that are conserved in a lineage specific manner even among strains identified in very distant geographic areas (different european countries and korea). this analysis also indicates that potential antigenic differences would be mainly determined by residues located at the receptor binding domain, and exposed on the surface of the protein according to the proposed structural model [ ] . hence, to elucidate if he lineage specific changes could determine antigenic differences on the receptor binding domain, the he . and . proteins were used as model proteins in hi assays with field serum samples from the same farm where both lineages were detected. significantly, by this assay most serum samples show preferential reactivity to one of the he proteins, or even specific reactivity against only one of them (see figure h ), indicating the existence of antigenic differences between the two he lineages. antigenic differences between he proteins from the two known btov lineages have also been described [ ] . in addition, to study the antibody response against the whole protein by a different approach, soluble c-myc tagged he . and he . proteins were generated by rvv methodology to obtain highly purified coating antigens that were used in elisa to test the same field serum samples. using both approaches, a high prevalence of antibodies against ptov-he was observed in both sows and piglets. these results were in agreement with those obtained using an elisa assay with the very immunogenic and highly conserved n protein as antigen [ ] . in the present study, similar anti-he response profiles over time were observed by both he-elisa and the more restricted lectin-specific hi test. a general decrease of antibody levels was seen from the first weeks of age until piglets' reached week , related to the extinction of maternally derived initial immunoglobulins. at -weeks of age, immune levels recovered due to the development of the pigs' own response to infections, and they increased at least until week . at this last sampled point, a general increase of reactivity against he . (p -like) was found in animal sera from the three litters, indicative of the prevalence of this lineage upon time. overall, similar antibody patterns were observed by elisa against the he proteins and the n protein, although a delay in both the extinction of maternally derived antibodies and the development of self-acquired antibodies against the he was observed in all animals analyzed (see figure and additional file ). the rising of antibodies to ptov antigens after weaning can be explained as a consequence of the animal grouping in livestock facilities for fattening purposes that provides encyclopedia of virology. rd edition etiology of community-acquired pediatric viral diarrhea: a prospective longitudinal study in hospitals, emergency departments, pediatric practices and child care centers during the winter rotavirus outbreak, to . the pediatric rotavirus epidemiology study for immunization study group emerging and re-emerging swine viruses bovine torovirus (breda virus) revisited identification and characterization of a porcine torovirus seroprevalence of porcine torovirus (ptov) in spanish farms a new family of vertebrate viruses: toroviridae. intervirology purification and partial characterization of a new enveloped rna virus (berne virus) new insights on the structure and morphogenesis of berne virus hemagglutinin-esterase, a novel structural protein of torovirus structure, function and evolution of the hemagglutininesterase proteins of corona-and toroviruses structural basis for ligand and substrate recognition by torovirus hemagglutinin esterases characterization of epidemic diarrhea outbreaks associated with bovine torovirus in adult cows genetic and antigenic characterization of newly isolated bovine toroviruses from japanese cattle the pathogenesis of torovirus infections in animals and humans hemagglutination mediated by the spike protein of cell-adapted bovine torovirus nidovirus sialate-o-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes the murine coronavirus hemagglutinin-esterase receptor-binding site: a major shift in ligand specificity through modest changes in architecture phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events longitudinal serological and virological study on porcine torovirus (ptov) in piglets from spanish farms detection and molecular characterization of porcine toroviruses in korea vaccinia virus: a suitable vehicle for recombinant vaccines? molecular characterization of a new ptov strain. evolutionary implications il- delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against hiv- env in a dose-dependent manner e. coli beta-glucuronidase (gus) as a marker for recombinant vaccinia viruses image processing and analysis in java identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza c virus and bovine coronavirus the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities comparison of macro and micro methods in kaolin and rbc treatment of sera used in the measles hemagglutination-inhibition test lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (ptov-he) protein we thank joquim segalés for providing porcine field serum samples. we also want to thank susana plazuelo for her excellent technical assistance in the analysis of sera by elisa. this work was supported by grants agl - and consolider-porcivir csd - from the spanish ministry of science and innovation. jaime pignatelli and julio alonso-padilla were both recipients of contracts financed with founding from the consolider-porcivir research project. the conditions for piglet infection and/or re-infection and the mixture of ptov strains in the same animal population. our results indicate that the immune response developed against one of the ptov lineages could not protect against the infection by other ptov isolates carrying an he protein belonging to a different lineage. hence, the specificity of the piglet's current immune response, its own or maternal, towards one or the other he lineage at a given time could determine the ptov strain that could infect or prevail in the animal. our hypothesis is that this ptov lineage alternation could explain the sustainment of both strains on the farm. in fact, even though the p -like (he . ) strain was not found by molecular detection in piglets at the earliest ages [ ] , the high reactivity observed by elisa in sows and young piglets and, more remarkably, by the hi in sow a and in -, -and -week old piglets from litter a meant that such a strain was already circulating in the herd from the very beginning of the sampling. particularly, in piglet the increasing reactivity against both he proteins from week to week indicates a temporal coexistence of both types of virus in the piglet at around weaning time. the lack of anti-he maternal antibodies with an hi capacity against either he protein might have facilitated the establishment of both ptov strains early on in the piglet's life. although we described that the two ptov-he lineages were detected within the same animal at two sequential sampling time points, the lack of detection of ptov-he . at week could have been due to the low abundance of this virus at the beginning of life, while at later times it became predominant, and therefore easier to detect. although the shift from ptov-he . to ptov-he . in the analyzed animals (markelo-like to p -like strains) seems to derive from immune pressure on the latter, the contribution of a potential better viral fitness provided by the former on older pigs' tissue environments cannot be discarded.the tendency to recombine modules of their genomes observed in nidovirales, together with the extensive mutation rates found, like in other rna viruses, would facilitate the evasion of immune responses and the rapid adaptation to new hosts and the new hosts' environments. from this study, where two ptov he genotypes were found co-existing on the same farm, we can speculate that the specificity of the immune response towards one or the other he lineage in piglets at a given time could determine the ptov strain that prevailed and spread. however, the potential additional contribution of the immune response to other viral antigens in virus selection also has to be considered. future studies with higher numbers of animals from different farms will be required to further support the proposed hypothesis.though the ptov-he protein in vivo function/s are still to be undoubtedly defined, its persistence in field strains, the tendency to undergo recombination events and the different antigenic characteristics of both he lineages indicate that he protein from torovirus plays an important role in virus-host interactions with implications in immune protection that could explain the broad spread of this virus in the pig population, causing chronic infections/re-infections of the animals. additional file : analysis of purified ptov-he . -myc and ptov-he . -myc proteins. (a) cell extract from bsc cells infected (moi ) with rvv-he . -myc or rvv-he . -myc (lysate), and affinity purified he . -myc and he . -myc proteins (protein) were fractionated by % sds-page, and the gel was stained with coomassie blue. (b) affinity purified he . -myc and he . -myc proteins were reacted in western blot with the αmyc and αhe antibodies. molecular size markers are given in kda.additional file : reactivity of sera obtained from pigs at different ages against the n protein. the same serum samples analyzed in figure were used in elisa at a : dilution using the ptov-n protein as antigen as previously described [ ] . the authors declare that they have no competing interests.authors' contributions jp and jap have contributed equally to this work. jp and jap participated in the design of the study, generated recombinant viruses to express recombinant he proteins, contributed to characterize the recombinant he proteins, carried out hi assays, participated in the analysis of the results and draft of the manuscript. dr conceived the study, participated in its design, coordination and draft of the manuscript. all authors read and approved the final manuscript.submit your next manuscript to biomed central and take full advantage of: key: cord- - la tm authors: hsueh, po-ren; kao, chuan-liang; lee, chun-nan; chen, li-kuan; ho, mei-shang; sia, charles; de fang, xin; lynn, shugene; chang, tseng yuan; liu, shi kau; walfield, alan m.; wang, chang yi title: sars antibody test for serosurveillance date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: la tm a peptide-based enzyme-linked immunosorbent assay (elisa) can be used for retrospective serosurveillance of severe acute respiratory syndrome (sars) by helping identify undetected chains of disease transmission. the assay was developed by epitope mapping, using synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of sars-associated coronavirus. the new peptide elisa consistently detected seroconversion by week of onset of fever, and seropositivity remained through day . specificity was % on normal blood donor samples, on serum samples associated with infection by other pathogens, and on an interference panel. the peptide-based test has advantages of safety, standardization, and automation over previous immunoassays for sars. the assay was used for a retrospective survey of healthy healthcare workers in taiwan who treated sars patients. asymptomatic seroconversions were detected in two hospitals that had nosocomial disease. a peptide-based enzyme-linked immunosorbent assay (elisa) can be used for retrospective serosurveillance of severe acute respiratory syndrome (sars) by helping identify undetected chains of disease transmission. the assay was developed by epitope mapping, using synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of sars-associated coronavirus. the new peptide elisa consistently detected seroconversion by week of onset of fever, and seropositivity remained through day . specificity was % on normal blood donor samples, on serum samples associated with infection by other pathogens, and on an interference panel. the peptide-based test has advantages of safety, standardization, and automation over previous immunoassays for sars. the assay was used for a retrospective survey of healthy healthcare workers in taiwan who treated sars patients. asymptomatic seroconversions were detected in two hospitals that had nosocomial disease. r etrospective surveillance for infection is an important means to screen for and interrupt undetected chains of disease transmission. such surveillance may be key to tracking the severe acute respiratory syndrome-associated coronavirus (sars-cov) because mild and asymptomatic cases of sars-cov infection that do not meet the world health organization's case definition ( ) have been identified by immunoassays ( ) ( ) ( ) , and sars-cov-like viruses have been isolated from wild mammals ( ) . sars-cov may have persisted over the summer in previously affected areas in such difficult-to-recognize reservoirs ( ) . the reemergence of sars in the city of guangzhou of the guangdong province of china in december and january ( ) is evidence that an unknown reservoir exists and signals the need for continued surveillance with laboratory testing. the current laboratory methods for identifying sars are not ideal tools for use in retrospective mass screening. reverse transcription-polymerase chain reaction (rt-pcr) for detecting viral rna is the most sensitive method for early identification of sars. however, viral load rapidly declines beginning or days after disease onset ( ) ( ) ( ) . moreover, rt-pcr requires sophisticated equipment and high laboratory quality-assurance standards ( , ) . identifying seroconversion to sars-cov by immunoassay also is a definitive criterion for laboratory determination of sars ( ) , and seroconversion is the preferred standard for retrospectively detecting sars-cov infection ( ) . sars immunoassays include the enzyme-linked immunosorbent assay (elisa) or western blot with antigen from whole virus or various recombinant proteins, a cumbersome immunofluorescence assay (ifa) using whole virus fixed on glass, and methods to determine neutralizing antibodies ( , ) . immunoglobulin (ig) g to sars-cov, detected by these immunoassays, begins to rise sharply by day after onset of symptoms. virtually all sars patients show virus-specific antibody by week , and anti-sars-cov igg persists through day ( , , ) . although any of these immunoassays can provide a definitive laboratory finding, all but the recombinant tests require biosafety level to contain the virus or are time-consuming to perform, have not been well-standardized, are of unknown specificities, and would be difficult to adapt to large-scale manufacture. improving laboratory methods for the large-scale serologic surveillance of sars, particularly in the presence of other respiratory illnesses, and standardization of diagnostic assays are key priorities for controlling sars ( ) . in this report, a standardized and rapid peptide-based sars elisa is characterized for sensitivity and specificity. beginning in april , delay in recognizing sars cases and in implementing isolation procedures led to several nosocomial clusters of sars-cov transmission in healthcare facilities in taiwan ( , ) . the results from a retrospective serologic survey by the peptide elisa of healthcare workers from facilities affected by nosocomial outbreaks are presented as a working example. we developed an elisa for sars that has synthetic peptide antigens as the solid-phase immunosorbent. over overlapping peptides, deduced from the tor sars-cov genomic sequence ( ) , were synthesized as candidate antigens from the spike (s), membrane (m), and nucleocapsid (n) proteins. candidate immunodominant s, m, and n peptides were selected and refined on the basis of serologic reactivities to a panel of serum samples from patients clinically diagnosed with sars at national taiwan university hospital in taipei and the xiaotangshan sars emergency hospital in beijing ( ) . epitope mapping by serologic validation has been described for the development of peptide-based elisa tests for hiv, hepatitis c virus, and foot-and-mouth disease virus ( ) ( ) ( ) . for the peptide-based sars elisa, wells of microtiter plates were coated with µg/ml of a mixture of the s, m, and n protein-derived peptides, and serologic reactivities were determined by a standard elisa procedure as previously described ( ) , except that the detector was horseradish peroxidase-conjugated goat anti-human igg, and the chromaphore was , ′, , ′tetramethylbenzidine (tmb). in brief, serum samples, including two normal human samples provided as nonreactive controls, were diluted : in phosphate-buffered saline with carrier proteins and preservative. the diluted serum samples were reacted to the peptide-coated microtiter wells for l h at °c. plates were washed times, reacted to the antibody conjugate, again washed times, and reacted to tmb; reactivity was then determined by a . assay results were obtained within h. results were scored on the basis of the signal/cutoff (s/c) ratio, and cutoff absorbance was determined from the mean of the two controls plus standard deviations (sd) from the distribution of normal human samples ( figure ). seroconversion panels were collected as serial serum samples from sars patients at national taiwan university hospital in the course of treatment. these panels tested positive for seroconversion by ifa ( ) and are used here to evaluate analytical sensitivity. a panel of serum samples from convalescent sars patients were provided by the center for disease control, department of health, taiwan, to evaluate diagnostic sensitivity. these serum specimens were confirmed for sars-cov infection clinically by the world health organization diagnostic criteria ( ) and serologically by whole virus-based elisa and ifa; some specimens were also confirmed by rt-pcr ( ) . samples were drawn with appropriate timing for serologic reactivity ( - weeks after onset of symptoms). a panel of , plasma samples collected from random blood donors in florida before was obtained from the gulf coast regional blood center (houston, tx) for specificity evaluation. additional specificity studies were conducted with serum that had serologic reactivities for bloodborne pathogens (hiv- , hiv- , hepatitis c virus, htlv /ii, and syphilis) obtained by united biomedical inc. before from various u.s. sources, an interference panel supplied by boston biomedica inc. (boston, ma) of serum samples with interference substances commonly found in processed clinical samples (edta, acid citrate dextrose, and citrate phosphate dextrose with adenine), and serum supplied by national taiwan university hospital from patients associated with typical and atypical respiratory pathogens other than sars-cov (influenza, rubella, cytomegalovirus, epstein-barr virus, and mycoplasma pneumoniae). serum samples were collected from healthy healthcare workers after interviews to confirm lack of signs and symptoms of sars including fever, respiratory symptoms, and diarrhea. the peptide-based elisa was evaluated for sensitivity to seroconversion on eight seroconversion panels obtained from national taiwan university hospital (figure , table ). in patient , seroconversion was detected by day with an a of . . absorbance remained at > at day . in patient , from whom blood was drawn on days , , , , and (no samples were collected from day to day ), seroconversion was apparent on day after the onset of fever. on day , the a remained > . in five of the other six seroconversion panels, from acute to convalescent phases, seropositivity was observed by days to , and by day in patient , from whom serum had not been collected from days to ( figure ). the peptide-based elisa showed an analytical sensitivity to earliest time of detection by week and for duration of detection beyond day . the seroreactivities of patients to were also evaluated by a standard ifa method ( ) for comparison. seroconversion was detected in all six patients by the ifa method within days of the peptide elisa (data not shown). the diagnostic sensitivity of the peptide elisa was % on a panel of convalescent-phase serum samples from sars patients provided as a reference panel by the center for disease control, department of health, taiwan. these sera, confirmed for sars by diagnostic and serologic criteria, displayed a mean s/c ratio of . (figure ). the specificity of the peptide-based sars elisa was tested on plasma obtained before from , random florida blood donors (gulf coast regional blood center, houston, tx). these normal plasma samples with a presumed zero seroprevalence rate gave a mean a of . ± . (sd). subsequently, the cutoff value for the peptide-based assay was set as the mean a for duplicate nonreactive controls plus . (based on sds from the mean for these , normal plasma samples). the distribution of the s/c ratio for the blood bank samples is plotted in figure with the mean s/c ratio of . . none showed positive reactivity, for a specificity on the normal samples of % ( table ) . the peptide elisa was further evaluated for specificity with a pre- collection of serum samples from patients with seropositivities for bloodborne pathogens such as hiv- , hiv , hepatitis c virus, htlv /ii, and syphilis (various u.s. sources), and with normal serum samples spiked with interference substances heparin, edta, acd, and cpda- (boston biomedica). the samples with seroreactivities for the pathogens all tested negative by the peptide-based sars elisa, as did the sera interference panel ( table ). the peptide elisa was evaluated for specificity on serum samples drawn from patients associated with typical and atypical respiratory pathogens other than sars-cov (national taiwan university hospital). these included samples from ) patients naturally infected with influenza (two sequential bleeds per influenza patient), ) patients with rubella, bacterial agent for atypical pneumonia, and ) pre-and postvaccine blood samples from patients given influenza vaccine. all samples were tested in duplicate. the sitespecific antigens of the peptide sars-cov elisa were free of cross-reactivities to the other respiratory pathogens ( table ) . a prospective study was performed to determine asymptomatic infection among primary healthcare workers in hospitals that treated sars patients. we collected serum samples from healthcare workers without symptoms, not all of whom were in direct contact with sars patients, who agreed to be tested for antibody to sars-cov at ho ping, yang ming, en chu kong, and hsin tai hospitals, approximately weeks after the outbreaks were recognized. ho ping and yang ming hospitals had admitted sars patients before the recognition of sars and before healthcare workers had implemented control measures. subsequently, these facilities experienced transmission to healthcare workers. the en chu kong and hsin tai facilities admitted patients once control measures had been implemented. neither of these hospitals recorded transmission of sars to healthcare workers. elisa detected three cases out of samples from ho ping and one case in blood samples from nursing aides at yang ming. these four positive samples, indicative of asymptomatic infection, were confirmed for seropositivity by ifa. none of the serum samples from the two hospitals without nosocomial infection displayed seroconversion. a convenient elisa to detect igg to sars-cov, based on site-specific synthetic antigens taken from the s, m, and n proteins of the virus, has high specificity. no cross-reactivity was detected in samples associated with common non-coronavirus respiratory pathogens. in addition, the lack of detectable reactivities among the , u.s. blood donors supports a specificity for the assay to distinguish sars-cov infection from infection by other human coronaviruses. the presence of anti-coronavirus antibodies among a u.s. population of this size is strongly anticipated because an incidence as high as % for oc and e respiratory infections has been observed, even among healthy young adults ( ) . the new peptide-based elisa is equivalent in sensitivity to other immunoassays for sars and can be detected after day . the synthetic antigens provide the advantages of high standardization, freedom from biohazard, and ease of scale-up production. moreover, testing by the elisa format can be readily automated for large-scale screening. the highly specific peptide-based sars antibody test is a convenient means to carry out widespread retrospective surveillance, such as that now being proposed for china to trace hotspots of persons carrying antibodies to sars-cov and to track the origins of the disease ( ) . a preliminary survey with the peptide elisa detected asymptomatic clusters of seroconversion among exposed healthcare workers in two taiwan hospitals that also had nosocomial disease. in contrast, no seroconversion was found among the exposed healthcare workers from two hospitals that had no apparent disease transmission to healthcare workers. the finding of asymptomatic seropositive persons indicates that the test will be useful in larger retrospective surveillance studies, which are needed to fully define the epidemiology and spectrum of disease. the protocol and the informed consent documents were approved by the ethics committee of medical research of the biomedical sciences, academia sinica. all participation was voluntary and was documented with written informed consent. dr. hsueh is an associate professor in the departments of laboratory medicine and internal medicine, national taiwan university hospital, national taiwan university college of medicine. his research interests include mechanisms of microbial resistance and molecular epidemiology of emerging pathogens. he is actively involved in a national research program for antimicrobial drug resistance and is a member of the sars research group of national taiwan university college of medicine and national taiwan university hospital. world health organization. case definitions of surveillance of severe acute respiratory syndrome (sars) mild severe acute respiratory syndrome asymptomatic severe acute respiratory syndrome-associated coronavirus infection early indications point to lab infection in new sars case isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars-one year later update : announcement of suspected sars case in southern china; investigation of source of infection for confirmed case begins tomorrow clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study detection of sars coronavirus in plasma by real-time rt-pcr serologic and molecular biologic methods for sars-associated coronavirus infection communicable disease surveillance & response. use of laboratory methods for sars diagnosis evaluation of reverse transcriptase-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus surveillance case definition for severe acute respiratory syndrome (sars) and update on sars cases-united states and worldwide the severe acute respiratory syndrome profile of specific antibodies to the sars-associated coronavirus sars laboratory workshop. summary of the discussion and recommendations of the sars laboratory workshop use of quarantine to prevent transmission of severe acute respiratory syndrome-taiwan the genome sequence of the sars-associated coronavirus peptides as diagnostic reagents for sars. united states patient application no detection of antibodies to human t-lymphotropic virus type iii by using a synthetic peptide of amino acid residues corresponding to a highly antigenic segment of gp envelope protein improved serodiagnosis of hepatitis c virus infection with synthetic peptide antigen from capsid protein differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide elisa microbiologic characteristics, serologic responses, and clinical manifestations in severe acute respiratory syndrome evidence-based medicine for student health services antibodies to sars-like virus hint at repeated infections key: cord- - g spkc authors: minami, shohei; terada, yutaka; shimoda, hiroshi; takizawa, masaki; onuma, mamoru; ota, akihiko; ota, yuichi; akabane, yoshihito; tamukai, kenichi; watanabe, keiichiro; naganuma, yumiko; kanagawa, eiichi; nakamura, kaneichi; ohashi, masanari; takami, yoshinori; miwa, yasutsugu; tanoue, tomoaki; ohwaki, masao; ohta, jouji; une, yumi; maeda, ken title: establishment of serological test to detect antibody against ferret coronavirus date: - - journal: j vet med sci doi: . /jvms. - sha: doc_id: cord_uid: g spkc since there is no available serological methods to detect antibodies to ferret coronavirus (frcov), an enzyme-linked immunosorbent assay (elisa) using recombinant partial nucleocapsid (n) proteins of the ferret coronavirus (frcov) yamaguchi- strain was developed to establish a serological method for detection of frcov infection. many serum samples collected from ferrets recognized both a.a. – and a.a. – of the n protein, but two serum samples did not a.a. – of the n protein. this different reactivity was also confirmed by immunoblot analysis using the serum from the ferret.therefore, the a.a. – of the n protein was used as an elisa antigen. serological test was carried out using sera or plasma of ferrets in japan. surprisingly, % ferrets in japan had been infected with frcov. these results indicated that our established elisa using a.a. – of the n protein is useful for detection of antibody to frcov for diagnosis and seroepidemiology of frcov infection. epizootic catarrhal enteritis (ece), a new enteric disease of domestic ferrets (mustelo putorius furo), was first described in the united states in the early s [ ] . a novel alphacoronavirus, ferret coronavirus (frcov), was detected as the causative agent of ece in and designated as ferret enteric coronavirus (frecv) [ , ] . ferrets with ece show general clinical signs including lethargy, anorexia and vomiting, and characteristic signs with foul-smelling, green mucous-laden diarrhea [ ] . frcov was also reported as the causative agent of feline infectious peritonitis (fip)-like disease in , and the virus was designated as ferret systemic coronavirus (frscv) [ ] [ ] [ ] . ferrets with fip-like disease show characteristic clinical signs of large palpable intraabdominal masses like dry type of fip [ ] [ ] [ ] . frcovs were divided into two genotypes, i and ii, based on differences in the spike (s) gene, and it was suggested that genotype i was associated with fip-like disease and genotype ii was with ece [ ] . however, we previously showed that there was no significant relationship between the genotypes of frcov and disease in japan [ ] . in addition, genotype i frcov was also detected from many asymptomatic ferrets in the netherlands [ ] . the relationship between genotypes of frcov and clinical symptoms remains unclear. although frcov genes were detected in ferrets by reverse transcription-polymerase chain reaction (rt-pcr), there is no method to detect antibodies to frcov. we attempted to isolate frcov using feline cell lines and our newly established ferret cell line (manuscript in preparation), but the virus has not yet been isolated. because the nucleocapsid (n) is conserved between coronaviruses and used as an antigen to detect antibody [ , ] , the n protein of frcov was one of the most likely antigen candidates to detect antibody to frcov. in this study, an enzyme-linked immunosorbent assay (elisa) using recombinant n proteins was established and applied to investigate the seroprevalence of frcov infection in japan. samples from domestic ferrets: from animal hospitals in japan, serum and plasma samples were collected from domestic ferrets between aug st, and feb th, and used for elisa and immunoblot analysis. we analyzed and reported the results for of the feces samples in our previous study [ ] . one fecal sample from a ferret in our animal facility was used to amplify the n gene of the frcov yamaguchi- strain. amplification of n genes: rna of the yamaguchi- strain was extracted from feces using a qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. n genes of the yamaguchi- strain was amplified by rt-pcr using takara rna la pcr tm kit (amv) ver. . (takara, otsu, japan). rt was performed using random -mer oligonucleotide primers, and pcr was performed using primer pairs, nf ( ′-tta cat atg gta taa gaa cta aac- ′) and nr ( ′-cga tgt agg aac ctt caa aat a- ′). pcr products were electrophoresed on a . % gel and extracted using a qiaex ii gel extraction kit (qiagen). construction of expression plasmids: yamaguchi- strain fragments were amplified using primer pairs, n f ( ′-tgg gat cca tgg ctg gaa acg gac cac- ′) and n r ( ′-gac tcg agt tag tta ttg gat cta ttg ttg gac- ′) for nt - encoding a.a. - , and n f ( ′-tgg gat cca tta aca gta aca gtg gtg ata t- ′) and n r ( ′-gac tcg agt tag ttt agt tca tca ata att tca- ′) for nt - encoding a.a. - . these forward and reverse primers contained bamhi and xhoi sites at the ′-end, respectively. fragments were purified using a minelute pcr purification kit (qiagen) and digested with restriction enzymes, bamhi and xhoi. two fragments of the yamaguchi- strain were electrophoresed on a . % gel and extracted using a qiaex ii gel extraction kit (qiagen). fragments were then cloned into bamhi and xhoi sites of the expression plasmid pgex- p- vector (ge healthcare, piscataway, nj, u.s.a.) using a dna ligation kit ver. . (takara). plasmids were transformed into escherichia (e.) coli strain dh α (toyobo, osaka, japan). expression and purification of glutathione-s transferase (gst)-fusion proteins: two n protein fragments, n - and n - , were expressed as fusion proteins with gst, gst-n ( - ) and gst-n ( - ), respectively. e. coli containing recombinant or control plasmid was cultured in × yeast extract and tryptone (yt) medium ( . % tryptone, % yeast extract and . % nacl, ph . ) containing µg ampicillin ml − . expression of recombinant proteins was induced by the addition of mm isopropyl β-d- thiogalactopyranoside (wako, osaka, japan) for hr. the bacterial cells were suspended in sonication buffer ( mm tris-hcl, ph . , mm nacl, mm edta and mm dithiothreitol) and lysed using a multi-beads shocker (yasui kikai, osaka, japan). after centrifugation, supernatants were mixed with triton x- at a final concentration of % for min and then centrifuged at , × g at °c for min. the supernatants were collected, mixed with glutathione sepharose b beads (ge healthcare) and incubated at °c for min. after centrifugation, beads were washed four times with phosphate-buffered saline (pbs) containing . % triton x- and once with sonication buffer. the beads were mixed with µl of mm glutathione and incubated at °c for hr. after incubation, supernatants were harvested as purified recombinant proteins and used for elisa and immunoblot analysis. the purified proteins were confirmed to be single bands by coomassie-brilliant blue (cbb) staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) analysis. sds-page analysis of recombinant proteins: purified recombinant proteins were mixed in equal volumes of × sample buffer ( mm tris-hcl, ph . , % glycerol, % sds, . % bromophenol blue and % -mercaptoethanol) and boiled for min. samples were electrophoresed by sds-page and stained with cbb. quantification of recombinant proteins: concentration of purified proteins was measured using bio-rad protein assay dye reagent concentrate (bio-rad, hercules, ca, u.s.a.) according to the manufacturer's instructions. a standard curve was constructed using bovine serum albumin (sigma-aldrich, st. louis, mo, u.s.a.). the absorbance was measured using a spectrophotometer (bio-rad) at nm. elisa: the concentration of purified recombinant proteins was adjusted to µg ml − with adsorption buffer ( . m carbonate-bicarbonate buffer, ph . ). gst was used as a control at µg ml − . one hundred microliters of purified recombinant proteins and gst were added to well microplates (maxisorp; nunc, roskilde, denmark). after incubation at °c for hr, plates were placed at °c overnight. the wells were washed three times with pbs containing . % tween (pbs-t) and then incubated with µl of % block ace (dainippon pharmaceutical, osaka, japan) in pbs at °c for min. after washing three times with pbs-t, µl of diluted sera or plasma were added to duplicate wells and incubated at °c for min. sera or plasma was diluted to : or : with pbs-t containing . % block ace. subsequently, wells were washed three times with pbs-t before µl of peroxidase-conjugated anti-ferret immunoglobulin (rockland, limerick, pa, u.s.a.) diluted with pbs-t containing . % block ace was added and incubated at °c for min. following three washes with pbs-t, µl of horseradish peroxidase substrate (bio-rad) was added to each well. after incubation at room temperature for min, the enzymatic reaction was stopped by adding µl of % oxalic acid to each well. the absorbance was measured using a spectrophotometer (bio-rad) at nm. all results were subtracted from the value for gst, and the cut-off value was arbitrarily set at . . phylogenetic analysis: a phylogenetic tree was constructed using the program mrbayes ver. . . [ ] for mrmodeltest analysis with a wag substitution matrix [ ] . we referred to the following sequences to construct the phylogenetic tree of n protein sequences; frecv strain msu- (gu ), frecv strain msu- (dq ), frscv strain msu- (gu ), mink cov strain wd (hm ), mink cov strain wd (hm ), ccov type ii strain fc (ab ), fcov type ii strain m - (ab ), fcov type i strain c (ab ), sars-cov strain bj - (eu ) and frcov strain yamaguchi- (lc ). the tree was represented graphically using figtree ver. . . [ ] . statistical analysis: significant differences were statistically analyzed using chi-square and fisher's exact probability tests. p values of < . were considered to be statistically significant. nucleotide sequence of the yamaguchi- strain n gene ( , bp) was determined, and the deduced amino acid sequence of n protein ( amino acids) was phylogenetically analyzed (fig. ) . two recombinant n proteins, gst-n ( - ) and gst-n ( - ), based on the yamaguchi- strain were expressed as gst fusion proteins in e. coli and used as elisa antigens with sera and plasma samples from ferrets. although most samples reacted to both recombinant proteins, the plasma of ferret no. and serum of ferret no. only reacted to gst-n ( - ) and did not recognize gst-n ( - ) (fig. ) . these results indicated that gst-n ( - ) was suitable for detection of antibodies to frcovs. therefore, we de- cided to use gst-n ( - ) in the subsequent investigation. in addition, a cut-off value was arbitrarily set at od= . . the plasma of no. and serum of ferret no. showed differ-ent reactivities from the other samples in elisa (fig. ) . to confirm the different antigenicity, immunoblot analysis was carried out using serum of ferret no. . plasma of ferret no. was used to compare with serum of ferret no. . the purified proteins were confirmed to be single bands by cbb staining after sds-page analysis and used (fig. a) . plasma of ferret no. and serum of ferret no. reacted with recombinant protein gst-n ( - ), but only plasma of ferret no. also reacted with gst-n ( - ) ( fig. b and c) . the results of the immunoblot analysis were consistent with those of the elisa. seroprevalence of frcov infection in ferrets in japan: elisa using gst-n ( - ) was carried out with : dilutions of nine sera and plasma samples from domestic ferrets in animal hospitals in five prefectures in japan. the results showed that of the ( %) ferrets were seropositive for frcov infection. there was no significant difference between seropositivity and age or sex (table ) . in this study, we attempted to clarify the seroprevalence of frcov in japan and developed an elisa using two yamaguchi- strain recombinant n proteins, gst-n ( - ) and gst-n ( - ). more ferret serum samples recognized gst-n ( - ) than gst-n ( - ) (fig. ). in addition, identities of n ( - ) between yamaguchi- and the other frcovs ( . - . %) were higher than those of n ( - ) ( . - . %) (data not shown). therefore, we selected gst-n ( - ) as the elisa antigen for our serosurvey. surprisingly, we found that % ( / ) of domestic ferrets were seropositive to this antigen by elisa (table ). there are reports of frcov gene detection in %- % of ferrets in japan and the netherlands [ , ] . these data indicate that frcov has already spread within the ferret population and that many ferrets may be persistently infected with frcov. however, there was no significant difference between seropositivity and symptoms, age or sex. further studies are required to clarify the pathogenesis of frcov in ferrets. plasma from ferret no. and serum from ferret no. showed different reactivities from those of other ferret samples in elisa, reacting only with gst-n ( - ), but not with gst-n ( - ) (fig. ) . the different reactivity of ferret no. serum was also confirmed by immunoblot analysis using gst-n ( - ) (fig. c) . these results indicated that gst-n ( - ) is a better choice of antigen for elisa than gst-n ( - ). elisa using gst-n ( - ) will be useful for serological surveys for frcov. in future studies, this frcov infected with ferret no. should be analyzed closely. in conclusion, a new elisa system using the recombinant n protein of frcov, gst-n ( - ), was established. this elisa will be useful for diagnosis and epidemiological studies on frcov infection in ferrets. acknowledgments. this experiment was supported by grants from the ministry of health, labour and welfare of japan (h -shinko-ippan- ; h -shinko-ippan- ) and by jsps kakenhi grant number h . figtree v . computer program and documentation distributed by the author clinicopathologic features of a systemic coronavirus-associated disease resembling feline infectious peritonitis in the domestic ferret (mustela putorius) systemic coronavirus-associated disease resembling feline infectious peritonitis in ferrets in the uk detection of feline infectious peritonitis virus-like antigen in ferrets comparison of the amino acid sequence and phylogenetic analysis of the peplomer, integral membrane and nucleocapsid proteins of feline, canine and porcine coronaviruses enteric coronavirus in ferrets mrbayes : bayesian phylogenetic inference under mixed models recombinant nucleocapsid protein-based igg enzyme-linked immunosorbent assay for the serological diagnosis of sars genetic characterization of coronaviruses from domestic ferrets a general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach coronavirus-associated epizootic catarrhal enteritis in ferrets molecular characterization of a novel coronavirus associated with epizootic catarrhal enteritis (ece) in ferrets comparative sequence analysis of the distal onethird of the genomes of a systemic and an enteric ferret coronavirus key: cord- -zngp tb authors: kadkhoda, kamran title: covid‐ : are neutralizing antibodies neutralizing enough? date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: zngp tb nan s ince its inception, coronavirus disease (covid- ) has caused significant morbidity and mortality globally. for that reason, treatment and prophylaxis are two quintessential ways to reduce harm as much as possible. the use of convalescent-phase plasma (cp) in severely ill patients with covid- has been attempted on an individual basis and it is being used in the context of ongoing clinical trials; thus, it is pivotal to understand the potential risks and caveats of using cp particularly taking immunopathologic phenomena into account. in a recent study, shen and colleagues assessed the clinical outcomes of five critically ill patients with covid- treated with cp. the study had several limitations mainly including lack of a control group; however, interesting results were derived from the study that are worth discussing. they used recombinant receptor-binding domain (rbd) of the sars-cov- spike protein in their igg enzyme-linked immunosorbent assay (elisa). the first important result was the apparent lack of correlation between igg elisa titers with those of the neutralization assay. neutralization titers as low as had different igg elisa titers of and , . these results highly suggest that there must exist more epitopes on rbd that do not engage in receptor binding on the cultured cells used in neutralization assay that can still bind anti-rbd iggs present in the sera, not to mention that the elisa design, expression, and purification of rbd, and more importantly coating of rbd on elisa plates, may create or unmask neoepitopes leading to eventual lack of correlation with the neutralizing antibody titer. the second main speculation is the potential interference by the original antigenic sin (oas) phenomenon. oas, first proposed over years ago, has been shown in the context of infection with a variety of viruses including influenza, dengue, zika, and coronaviruses (covs). [ ] [ ] [ ] according to oas, prior exposure to an antigen influences subsequent immune responses to the antigenically related agents because existing antibodies reduce the epitope burden; thereby this favors using memory instead of naïve b cells. this leads to a brisk and strong immune response that may not be "adequately neutralizing" while viral load remains high and immunopathologic mechanisms proceed such as in covid- . this may delay the generation of bona fide high-titer and high-avidity neutralizing antibody repertoire. in this context, previous exposure to common coronaviruses would lead to an early and high-titer immune response to sars-cov- . a similar phenomenon was frequently observed in serologic testing for the zika virus and dengue virus. furthermore, in the above study, despite diluting sera : , they still obtained extraordinarily high elisa titers as high as , (mean titer, , ) and , (mean titer, , ) for igm and igg, respectively, in critically ill patients to weeks after onset of symptoms whereas serum igm and igg elisa titers in asymptomatic convalescent donors to weeks after onset of symptoms only ranged to , (mean, ). the authors did not perform neutralization assays in parallel to assess crossreactivity with common covs: e, oc , nl , and hku . the last and perhaps another important observation is while patients had neutralizing antibody geometric mean titer (gmt) of before transfusion, their gmt only increased to day after transfusion of ml of plasma. this negligible increase in titer is barely one dilution difference, which could very well be due to the known ae dilution subjectivity associated with all neutralization assays. the donorsʼ gmt of neutralizing antibody was only as early as days after the resolution of their symptoms. this begs the question whether the so-called neutralizing antibodies were indeed "neutralizing" or not. in a more recent publication by duan and coworkers, patients with severe covid- transfused with cp collected from covid- -resolved asymptomatic donors. the donors had neutralizing antibody titers of more than at the time of donation while severely ill patients had relatively similar titers before transfusion as high as (range, - ; gmt, ). it should be highlighted that these titers were measured in patients to days (median, . days) after onset of symptoms. this study was also not controlled and, in addition to intensive supportive care, patients were on a range of agents including arbidol, ribavirin, remdesivir, interferon-α, oseltamivir, peramivir, and methylprednisolone; therefore, the observed slight clinical outcomes could not be reliably attributed to the infused plasma. historically, it was established that cats immunized with feline cov recombinant spike protein experienced worse outcomes when they were subsequently exposed to wild-type feline cov. liu and colleagues had also shown that macaques that were immunized with sars-cov (close relative of sars-cov- ) spike protein mounted anti-spike igg response that triggered acute lung injury. the anti-spike igg also skewed alveolar macrophages toward an inflammatory phenotype to launch hypercytokinemia (producing high levels of interleukin [il]- , il- , tumor necrosis factorα, among others). in their study, they showed that these newly polarized macrophages expressed high levels of cd a (fcγriia). antibody-dependent enhancement (ade) has been shown to work through non-or subneutralizing levels of "neutralizing antibodies" and cd a. ade has also been demonstrated in other infectious diseases such as zika, dengue, ebola, and human immunodeficiency virus. ade can lead to more viral propagation and/or generation of cytokine storm. more interestingly, patients who deceased due to sars had significantly higher titers of neutralizing antibodies. the us food and drug administration currently recommends using donated plasma with neutralizing antibody titers of or at a minimum a titer of in severely ill patients. although these cutoffs seem arbitrarily chosen, based on the above discussions, there remains a theoretical possibility that plasma recipients experience adverse outcomes due to iatrogenic ade. the potential issue of ade interference, albeit in the context of vaccine development, was also raised by coalition for epidemic preparedness innovations published recently in the new england journal of medicine. it needs emphasizing that the risk of ade, despite being clear in the context of certain infectious diseases such as dengue, has not been shown for coronaviruses in humans but this certainly remains a hypothetical risk. it is no-brainer that prime vaccine candidates would elicit neutralizing antibodies against sarv-cov- . all in all, whether cp therapy or prophylaxis would yield significant clinical benefits or not can only be answered through large-scale multicenter randomized clinical trials. treatment of critically ill patients with covid- with convalescent plasma the doctrine of original antigenic sin: separating good from evil dengue and zika virus diagnostic testing for patients with a clinically compatible illness and risk for infection with both viruses effectiveness of convalescent plasma therapy in severe covid- patients molecular mechanism for antibody-dependent enhancement of coronavirus entry anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection antibody responses against sars coronavirus are correlated with disease outcome of infected individuals recommendations for investigational covid- convalescent plasma food and drug administration developing covid- vaccines at pandemic speed the authors have disclosed no conflicts of interest. key: cord- -fjsuo yy authors: hoste, alexis c.r.; venteo, angel; fresco-taboada, alba; tapia, istar; monedero, alejandro; lópez, lissette; jebbink, maarten f.; pérez-ramírez, elisa; jimenez-clavero, miguel angel; almonacid, mercedes; muñoz, patricia; guinea, jesus; vela, carmen; van der hoek, lia; rueda, paloma; sastre, patricia title: two serological approaches for detection of antibodies to sars-cov- in different scenarios: a screening tool and a point-of-care test date: - - journal: diagn microbiol infect dis doi: . /j.diagmicrobio. . sha: doc_id: cord_uid: fjsuo yy severe acute respiratory syndrome coronavirus (sars-cov- ), has infected more than million people worldwide, becoming a pandemic. detecting antibodies against sars-cov- is of utmost importance and a good indicator of exposure and circulation of the virus within the general population. two serological tools based on a double recognition assay (enzyme-linked immunosorbent assay, dr-elisa and lateral flow assay, dr-lfa) to detect total antibodies to sars-cov- , have been developed based on the recombinant nucleocapsid protein. a total of serum samples, including positive for covid- and negative samples from healthy donors or infected with other respiratory pathogens, were analyzed. the results showed values of sensitivity between . %– %, and specificity of %– . %, for dr-lfa and dr-elisa, respectively. no cross-reactivity against seasonal coronavirus (hcov-nl , hcov- e, hcov-hku , hcov-oc ) was found. these results demonstrate the importance of serology as a complementary tool to pcr, for follow up of recovered patients and identification of asymptomatic individuals. in december , a novel coronavirus of animal origin (the severe acute respiratory syndrome coronavirus , sars-cov- ) emerged in the city of wuhan, in china, with the ability for human-to-human transmission ( ) . the associated disease, now named covid- , spread rapidly all over the world, and was declared a pandemic by the world health organization (who) on march th, . infection due to sars-cov- induces high rates of morbidity and mortality as described by the who ( ) . a significant concern is how rapidly the virus spreads, due in major part to the high number of asymptomatically infected individuals which could be an important source of viral dissemination ( , , ) . since, there is currently no vaccine available, neither efficient treatment, hygiene measures together with keeping social distance, are the main preventive ways to avoid the spread of the virus. serological studies can be used to collect epidemiological information on the prevalence of sars-cov- . moreover, in cases of covid- not detected by rt-pcr, the serological assays should be considered as a supplementary diagnostic tool, especially from the second week of illness, when the sensitivity of the current molecular tests decreases ( , ) . therefore, the aim of the present work was the development of serological tools to determine the presence of antibodies against sars-cov- in the population, as an indicator of an ongoing or previous infection. as for many other coronaviruses, one of the main structural proteins of sars-cov- is the nucleocapsid (n) protein. the n protein shows high immunogenic activity and is abundantly expressed during infection ( , , ) . these features make the n protein a potential target for serodiagnosis of sars-cov- infection. to date, some diagnostic methods have been developed based on the n protein, although validated methods are still j o u r n a l p r e -p r o o f journal pre-proof lacking to better understand the epidemiology of sars-cov- . in the current study, a double recognition enzyme-linked immunosorbent assay (dr-elisa) was developed to determine the presence of immunoglobulins of different classes (igg, igm and iga) to sars-cov- in human serum, to support the diagnosis of covid- . in parallel with this screening tool, a point-of-care test, based also on a double recognition format (a double recognition lateral flow assay, dr-lfa), and using the n protein as the target antigen, was produced to be used immediately and on site when there is suspicion for infection. a double recognition assay is based on the use of the same protein (in this case the n protein) as the target antigen and detection molecule, using the principle that antibodies possess multiple antigen binding regions (two for igg, four for iga and ten for igm), allowing their binding to both the target and detection antigen. double recognition tests have the advantage that they screen for all sars-cov- antibodies, regardless if it is iga, igg or igm. to carry out this study, a total of samples were analyzed with samples from positive patients to covid- and negative samples collected before or from patients negative to covid- . finally, a cohort of samples from patients infected with common-cold-coronavirus or respiratory pathogens that could potentially cross-react with sars-cov- were included in the study. the results shown in this paper reinforce the potential utility of serological testing as a the the sars-cov- n protein was labeled with peroxidase according to the method described by nakane and kawaoi ( ) , to be used as detector molecule in the dr-elisa described below. a double recognition elisa was developed as previously described ( ) at mg/ml was used. both reagents were dispensed in two parallel lines on nitrocellulose membrane (hf , merck millipore). after drying for min at °c, the membranes were sealed and stored at room temperature. black latex beads (merck millipore) were activated with edc ( -ethyl- -( dimethylaminopropyl) carbodiimide hydrochloride) and nhs (n-hydroxysuccinimide) and then coupled to n protein at a surface concentration of mg/m and blue latex beads were conjugated with the control protein. to prepare the conjugate solution, the n-latex and control-latex particles were diluted at a concentration of . % each, in a mm tris-hcl ph . buffer. the mixture was dispensed onto the conjugate pad, dried for min at °c and stored at room temperature. to assemble the cm master card, nitrocellulose membrane, conjugate pad, sample pad (cytosep , ahlstrom-munksjö) and wicking pad were pasted on a plastic backing with adhesive and covered with a protector film. the master card was then cut into strips of . mm width. the test was designed to be used with serum, plasma and blood samples. journal pre-proof twenty microliters of blood or ten microliters of serum/plasma were applied to the sample pad followed by μl of running buffer (tris-hcl ph . , nacl, casein and nan ). results were interpreted minutes after running buffer addition. a scale of the intensity of the signal of the test line from to was used, in order to give a semi-quantitative value for statistical purposes. data were statistically analyzed by a receiver-operator characteristics (roc) curve analysis using the medcalc® software to establish the optimal cut-off value for each assay. prior to the analyses, the samples were classified into positive or negative by pcr or by other commercial serological assays. using the same software, fisher's exact test was performed to determine the statistical dependence between the two assays developed. the complete sars-cov- n protein was cloned in the pdest vector, expressed in e. coli and further purified by immobilized metal affinity chromatography. the highly purified n protein was analyzed by gel electrophoresis followed by coomassie staining. a band of the expected molecular mass of the n protein (around kda) was observed ( figure ). journal pre-proof table . table : comparison between the dr-lfa and the dr-elisa to fully validate the dr-elisa and dr-lfa, the potential cross-reactivity by antibodies table ). table : cross-reactivity with other respiratory pathogens. abbreviation: nd: not determined. for the new emerging virus sars-cov- , the routinely used technique for testing patients is the rt-pcr, which detects the rna of the virus at early stages of the infection ( ) . fully validated serological tests are still missing as many of the commercial serological tests j o u r n a l p r e -p r o o f currently available for sars-cov- are poorly validated or display low sensitivity or specificity ( ) . in order to determine the prevalence of antibodies in the population and to complement the nucleic acid detection assays, especially at later days after the onset of the symptoms, serological assays are required ( ) . detection of antibodies is the most valuable indicator of the immune status of a person, identifying patients that have had covid- infection, and providing more accurate data related to risk of infection. in the present study, we developed two serological assays using the recombinant n protein table , a group of serum samples from early days post infection, positive to covid- by respiratory-pcr yet still negative in the commercial serological assay (with seroconversion a few days later) were also tested in our assays. four of these sera were positive in the dr-elisa and three in the dr-lfa, indicating that the dr-assays we developed are highly sensitive. regarding the specificity of the newly developed assays, we only found one sample positive to mycoplasma pneumonia that gave a positive signal in the dr-elisa. interestingly, no cross-reactivity by antibodies directed to seasonal alpha-or betacoronavirus was observed in our dr-assays, in contrast with regular sars-cov- antibody tests that do sometimes detect antibodies induced by hcov-oc infection ( ) . we tested samples from individuals that were negative for the virus in respiratory material (pcr) and also negative in serological assays, yet they were collected in a high risk group (personal communications). eleven serum samples showed a positive signal in the dr-elisa. five had a very high s/p (> ) and the others were found with lower s/p (between and ). three of the positives with high s/p were tested also for confirmation purposes in the dr-lfa, and also in this test the samples showed positive signals. these results could indicate that our tests can give false positives, or that the patients had experienced a previous infection, yet this was not diagnosed by the commercial assays, maybe not fully validated so far. it could also be that the serum samples contained only iga recognizing sars-cov- , since commercial assays used for classification only detect igm and igg ( ) . the serological assays could be a great complementary tool to the nucleic acid detection assays, as in this study, out of samples with a negative pcr in respiratory material, serum samples could diagnose a sars-cov- infection via serology (see table ). in these j o u r n a l p r e -p r o o f patients, were positive only to igg, were positive only to igm and were positive to igg and igm. an % correspondence was found between the dr-elisa and the commercial serological assay for the samples positive to igg or positive to igg and igm, but only one of the igm positive was found positive in the dr-elisa. this could demonstrate the higher affinity of igg compared to igm ( ) which could lead to lower specificity of serological assays specifically targeting igm. double recognition assays are sensitive tests, yet they also offer two additional advantages. first, it is a multi-specie test, detecting antibodies in human serum, but also in serum samples from other animal species, since it uses the target antigen as the detector molecule, instead of anti-specie antibody, and secondly, it detects total antibodies in a given sample. unlike the antibody response usually observed in other infectious diseases (first igm followed by igg), during covid- infection, igm and igg antibody responses appear almost simultaneously ( , ) . similar results were described previously for sars, where the igm appeared at the same time as the iga and igg ( ) . this shows the importance of having a test that detects total antibodies in serum. using the n protein of the sars-cov- as the coating antigen and as the enzymeconjugated antigen instead of enzyme-conjugated secondary antibody provides a specific and sensitive serological assay for detection of total antibodies to sars-cov- . the two assays developed in this study have been fully validated and received the ce marking. a novel coronavirus from patients with pneumonia in china covid- situation reports clinical characteristics of asymptomatic infections with covid- screened among close contacts in nanjing presumed asymptomatic carrier transmission of covid- serial interval of covid- among publicly reported confirmed cases sars-cov- viral load in upper respiratory specimens of infected patients viral load of sars-cov- in clinical samples serological assays for emerging coronaviruses: challenges and pitfalls nucleocapsid-independent specific viral rna packaging via viral envelope protein and viral rna signal sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome serum antibody response to respiratory syncytial virus f and n proteins in two populations at high risk of infection: children and elderly diverging trends in incidence of hiv versus other sexually transmitted infections in hiv-negative msm in amsterdam peroxidase-labeled antibody a new method of conjugation a novel double recognition enzyme-linked immunosorbent assay based on the nucleocapsid protein for early detection of european porcine reproductive and respiratory syndrome virus infection comparison of four new commercial serologic assays for determination of sars development of a duplex lateral flow assay for j o u r n a l p r e -p r o o f journal pre-proof simultaneous detection of antibodies against african and classical swine fever viruses a lateral flow assay for the rapid diagnosis of mycobacterium bovis infection in wild boar enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease good iga bad igg in sars-cov- infection? the distribution and functions of immunoglobulin classes. janeway's immunobiology interpreting diagnostic tests for sars-cov we thank dr. belén rebollo for technical support at eurofins ingenasa and maría garcía and alejandro soler for technical assistance at inia-cisa. we also thank dr. john n. barr for critical reading of the manuscript and assistance with the english language. training network (itn) honours under grant no (to alexis c. r. hoste). the datasets generated and analyzed during this work are available from the corresponding author on reasonable request. key: cord- -ro x qa authors: ingram, george a.; al-yaman, fadwa title: a comparative assessment of four serological methods used in the detection and measurement of anti-parasite antibodies in the serum of the amphibian, bufo viridis date: - - journal: international journal for parasitology doi: . / - ( ) - sha: doc_id: cord_uid: ro x qa abstract antibodies against crithidia fasciculata choanomastigotes were detected in green toad (bufo viridis) sera by direct agglutination, indirect haemagglutination (iha), complement-fixation test (cft) and enzyme-linked immunosorbent assay (elisa), correlation coefficients (r) were calculated for comparisons between each of the techniques and regression formulae derived in order to convert antibody levels as determined by one immunological method to that of another. the highest mean titre obtained by elisa was approximately . – . times greater than those obtained by the other techniques whilst cft gave the lowest values. iha and elisa titres were affected by different preparations of the crithidial antigen extracts. highly significant r values were determined for control sera when iha was compared to elisa (r > . ), and to both cft and elisa with immune animals (r > . ). elisa would seem most applicable for screening other lower vertebrates for anti-parasite antibodies especially in areas of human disease prevalence. amphibians are frequently parasitized by various protozoans present in body fluids and tissues and in some cases the parasites cause serious and debilitating diseases (abrams, ; roudabush & coatney, ) . trypanosomatid flagellates, in particular trypanosomes, have been reported in and isolated from the blood of frogs, toads and newts (bardsley & harmsen, ; woo & bogart ) . throughout the investigations into trypanosomatid infection in humans and mammals, sera have been examined for both anti-parasite antibodies and parasite antigens using various immunodiagnostic techniques (strickland & hunter, ; tiru & hennessen, ) . however there is a dearth of information pertaining to the use of serodiagnostic methods to detect kinetoplastid infections and resultant antibody production in poikilothermic vertebrates with only reptiles having been studied (dollahon, hager & hua, ; ingram & molyneux, a , b. a . in this paper we present the results of acomparative assessment of four serological tests (direct agglutination, indirect haemagglutination, complement-fixation test and elisa) used to detect and to determine the levels of antibodies in the sera of green toads (b. viridis), used as experimental models. these amphibians had been injected with the choanomastigotes of c. fusciculutu, a trypanosomatid flagellate. the parasites were chosen because of their ease of culture under laboratory conditions and more importantly because of their presence in the blood and alimentary canal of ranid anurans under natural conditions (smyth & smyth, ) . to the authors' knowledge, this is the first report of the use of an immunoenzyme method to detect antibodies in amphibians. furthermore, there are no previous data concerning anti-parasite antibody detection in amphibian serum. materials and methods injecrion und serrr. before injection of the parasites. a small sample of blood was taken by cardiac puncture from each of the toads and inspected for any current infection (with naturally-occurring trypanosomatid flagellates) by smear, wet mount preparation and slope culture as described elsewhere (ingram & molyneux, a. b ). in addition. blood smears were also examined by an immunoenzyme method reported previously by ingram and molyneux ( y b, c) but using a rabbit anti-c., firscic~tkctcr serum/swine anti-rabbit immunoglobulins antiserum/ peroxidase-rabbit antiperoxidase/amino-ethylcarbazole substrate system. furthermore, the gut contents and pbs extracts of randomly selected insects and worms respectively were also examined microscopically and by culture for the presence of trypanosomatids. the choanomastigotes in culture overlay were centrifuged at x g for min. the overlay was removed and the parasites were washed three times in phosphate buffered saline-pbs. ph . (i mm-naci; . mm-na?hpo, and i mm-nah,po,. h,o in de-ionized water). the number of parasites was counted and the suspension adjusted with pbs to give the required dose for injection purposes.toads were given a single intraperitoneal (ip) injection of x io" choanomastigotes in pbs. control animals consisted of those given pbs ip and normal, uninjected toads. the toads were anaesthetized. bled, killed and their weights and lengths noted. the parasite-and pbs-injected animals were bled at -day intervals. the uninjected controls were sampled at random intervals throughout the duration of the experiment. in order to detect parasite infection. blood was examined in a similar manner to that obtained from prrinjected animals and the uninjected controls. the sera were isolated and stored at - °c. specificifv. the promastigotes and procyclics of lershmania herfigi her@ and trpanosoma brucei brucei respectively, both kinetoplastid flagellate species related to c'. ~uscic~ulrtu, were used in the antibody assays to examine for possible non-specific reactions in the toad sera. antisera preparation. rabbit anti-toad serum was produced by an immunization schedule as described previously (ingram & alexander. ) and the immunoglobulin tttres of the antisera obtained were estimated by either countercurrent electrophoresis or elisa (ingram & molyneux, a). antigen extract. parasite antigen extracts were produced in two ways for use in the immunological techniques. the choanomastigotes were centrifuged at x g for io min in cold pbs. ph . . the pellet formed was resuspended in pbs and then washed and centrifuged a further three times. prior to use, the cells were subjected to either freezing and thawing (f&t) or sonication (son) treatments. in the former case the pellet was resuspended in chilled pbs, broken up by mixing and the parasites f&t at -min periods for min. the material wsas then centrifuged to remove cell debris and the supernatant protein concentration measured by the lowry method. alternatively, after addition of cold buffer, the pellet was disrupted by ultrasonication for three -min intervals whilst the mixture was kept chilled. the sonicated material was then left overnight at "c to further remove any protein. it was then centrifuged at x g for i min and the amount of soluble protein in the supernatant determined. agglutination assay two-fold serial dilutions of toad sera. inactivated by heating at x"c for min to destroy naturally-occurring complement activity, were preparcd with pbs. ph . (containing x mr+naci). to each dilution wsas added an equal volume of choanomastigotes (i x io cells ml-') and the mixtures incubated at °c for min. the direct agglutination (da) end point titre was regarded as that dilution in which visible agglutination was observed when compared to the pbs/parasite controls. normal toad sera were examined for the presence of natural haemagglutinins against sheep crythrocytes (she) before commencing the iha test as described by weir (iy x). she were tanned with . x it)-' mg ml-' tannic acid and coated with either f&t or son ant&n extract containing .y mg ml-' protein. doubling dilutionr of inactivated sera were made and to each was added the same volume of % tanned and coated she.the test samples were incubated at °c for min followed by{ overnight at c. the samples were then examined and the degree of haemapglutination assessed. untanned, tanned and antigencoated she were used as controls. antigen. toad antisera and either bvs or gpc were mixed together and fixation allowed to occur overnight at "c. sensitized she were then added and the mixtures incubated at °c for min after which the end-points were scored. following further incubation for h at sc, the samples under test were re-examined. the complement-fixing antibody (cfa) titre was taken as that dilution which gave % hacmolysis. ellsa. a modification of the method of chandler. cox. premier & hurrell (i yx ) was used. the concentrations of antigen. rabbit anti-toad antiserum and enzyme-conjugate used for the elisa were determined by chequerboard titration. the f&t and son antigen extracts (containing i mg ml-' protein) were prepared in . m-carbonate/bicarbonate coating buffer ph y. : c'. fi~scicrtlartr was adjusted to . x " cells ml-' in coating buffer and the three antigen preparations were separately dried onto the plates by overnight incubation at °c. after washing with pbs (containing o.os% tween ) ph . , a range of two-fold serial dilutions of control and immune sera were added to the appropriate wells and the plates were reincubated at °c for min. they were washed again. rabbit anti-toad antiserum (elisa titre i : ) at a dilution of : s was added and the plates were incubated as before. after re-washing, sheep anti-rabbit i@ immunoglobulin comugated to urease. diluted i in . was dispensed into the wells and the plates were similarly incubated. after a final three washes with pbs/tween followed by four washes with distilled water, urease substrate (sera-i.ab, crawley. u.k.) was added to the wells. the plate\ were subjected to a final incubation at °c for ~ min and the reaction halted by the addition of i % thiomersal solution (v/v). the end point antibody titre was considered to be that dilution which was visually different from the appropriate reference controls included with each experimental run. the mean toad serum antibody titres, s.e.m. and ranges in the control and c. fasciculuta -injected groups, together with the number of sera-containing detectable antibodies, are given in table . the mean value, range in titres and number of animals in which antibodies were detected were lowest for the cft and highest by elisa. of the control and parasiteinjected toads, and %, respectively contained antibodies detectable by elisa. when all four immunological assays were applied to each individual serum, in all cases antibodies were detected by at least two or more of the methods used. however the background 'positive antibody' titres in the control animals ranged from to -j depending on the technique employed. therefore values higher than -' were considered to be positive for the parasiteinjected toads. furthermore comparisons between control and immune sera for each method revealed significant differences in antibody titres (p< . ; student's t-test). moreover, 'antibody' levels were not significantly different between the two control groups (p> . ). the titres against l. hertigi and t. brucei varied from to -j and from to - in the control and immune sera, respectively. the results for each technique were compared in turn with those for the other methods and the half matrix of the pearson product-moment correlation coefficients (r) calculated for all experimental animals ( table ). the significance of each r value was tested by the t-test. in the case of the control sera, r values ranging from to sy% (p< . ) were calculated whilst overall higher correlations were determined for sera from immune animals which varied from to % (all i'< . ). in order that the results of the present study can be used by other investigators for comparative purposes with different experimental models, regression formulae to convert the antibody titres as determined for each technique to those of another are given in table . the regression equations, based on the rectilinear relationship y = mx + c, were only calculated for the highest mean titre found for each of the four immunological methods. the mean antibody titres against c. fasciculata and the number of control and immune sera containing detectable antibodies were the lowest for da and cft, intermediate for iha and highest for the elisa method (table ) . these findings may reflect the different sensitivities of the immunological techniques used (voller & de savigny, y ). the classical agglutination test has often been used for antibody titration in amphibian immunobiological studies (cooper, ). significant correlations (p < . ) were obtained for both control (range y- %) and immune sera ( -y %) when the da titres were compared to the values found for the other methods. although da is simple to perform, preconditions of the test include antigen-type specificity, the non-immobilization and non-autoagglutination of parasites and usually the use of living cells. in the current study, loss of cell motility was observed in some instances and the possibility of inclusion of nonviable cells in others cannot be ruled out. nevertheless caution must be taken in the interpretation of natural 'antibody' levels in view of the detection of 'antibodies' in normal bvs against l. hertigi and t. brucei with titres within the range found for c. fuscicdata. positive results for normal bvs would suggest the presence of low amounts of specific immunoglobulins induced by a current infection with or previous exposure to c. fasciculata parasites. however, the low levels of naturally-occurring 'antibodies' in normal bvs may also have been stimulated by the environmental presence of micro-organisms or other trypanosomatid flagellates which non-specifically cross react with shared cell wall carbohydrate antigenic determinants (andrews, reilly, ferris & hanson, ; schnaidman, yoshida, gorin & travassos, ; sharabi & gilboa-garber, ). in the present study, the low levels of 'antibody' in sera from the control groups are not likely to be caused by a current infection because no increase in titres were found throughout the o-week duration of the experiment. the lack of detection of c. fasciculata in blood microscopically or by culture coupled with the finding of parasite antigen(s) in blood up to weeks postinjection by the immunoenzyme method suggests that . grids possesses an efficient immune system responsible for the rapid elimination of antigen. therefore it was not possible in the work reported here to correlate the level of parasitaemia and antibody titres. however the exposure of b. grids to c. fasciculata evoked a specific immune response and resulted in significantly increased levels of serum antibody. under normal environmental situations, it is feasible that naturally-occurring immunoglobulins in bvs restrict parasite numbers to below a potential infectious threshold. the finding of serum antibody titres above those of normal background levels in amphibians or other animal hosts in similar habitats or areas endemic for certain diseases would indicate current infection with parasites, other infectious agents or pathogens within a population. no haemolytic anti-complementary activities were detected in normal bvs against the antigen extracts unlike previous reports for amphibians (gigli & austen, ) . whereas the cft is frequently nonspecific and inconvenient for handling many samples, it can utilize crude, soluble parasite antigen extracts. as with da cross reactivity can often lead to false positive results. when the results obtained by cft were compared with each other and with iha and elisa, the lowest range of correlations, although significant and similar to those determined for the da comparisons, were found for control bvs ( - %; igm which is an efficient complement fixer and also a good agglutinin (atwell & marchalonis, ; yamaguchi. kurashige & mitsuhashi, ) . however, the 'antibodies' in normal bvs would be present in limited amounts, fix complement less effectively and hence result in low background cfa values. this could also account for the lowest correlations found when the cfa titres were compared to those of the other methods for the controls. the source of complement seems to be important for the efficient fixation of toad antibodies. the use of toad serum as homologous complement source gave a higher mean value and usually slightly higher individual endpoint titres in both control and immunized animals compared to the antibody levels obtained with heterologous gpc. homologous serum has proved a reliable source of complement for the fixation of immunoglobulins in other anurans (alexander & steiner, ; sekizawa, fujii & katagiri, ) . however the use of commercially available gpc is also known to initiate good fixation in amphibian species (lallone, chambers & horton, : romano, geczy & steiner, ). in the current study, % correlation (p < . ) and "/,, (p< . ) were found for control and immune sera. respectively when the different complement sources were compared. low (so- %, controls) and high ( - x, immunized) but significant (i'< . ) r values were determined in comparisons between iha and cft. iha is prone to lack specificity in some cases and, in contrast to the cft, usually requires highly purified antigen preparations but is easier to perform. elisa gave the highest percentage positive titres in all the samples examined and appears to be the most sensitive of the techniques used to detect anticrithidial antibodies in toad sera. elisa was easy to peform, specific, an important factor which affects the values of the antibody titrcs is the preparation of the antigen extract (crouch & raybould, ; pappas. hajkowski, cannon & hockmeyer. ) . son antigen preparations gave higher titres than f&t antigen extracts. however, the coating of elisa plates with whole cells produced the highest values. in the work reported here, similar batches of antigen were used thus reducing potential variations due to different preparations. it is of interest to note that a comparison between iha and elisa control titres revealed - % (p < . i ) significant correlations and similar numbers of positive animals. this implies that the above two techniques have similar sensitivities in the screening of normal bvs for anti-parasite antibodies. nevertheless the method of antigen preparation ma> not be a salient criterion for antibody estimation in immune sera because correlation values of over % (f'< . ) were found when iha titres were compared to those of elisa. elisa seems appropriate for use in serodiagnostic surveillance programmes. applied to different amphibian species or other aquatic and semi-aquatic lower vertebrates, to detect antibodies stimulated against diverse parasite environmental pathogens. furthermore this technique would be of value in screening lower vertebrates for potential carriers or reservoirs of infective kinetoplastid tlagellates or indeed other pathogenic micro-organisms. such information may reflect the health status of animals within a population and aid in the determination of specific epidemiological and aetiological features of zoonoses and epizootics prevalence. ac~k~f~~*,lef~~.lg~l?~e~~t,s-'i'his work was undertaken whilst one of us (gai) was on an eec funded project contracted to the british council. in part subcontracted to the university of salford, for the development of the faculty of science at yarmouk university. jordan. ga would like to thank dr s. k. abdul-hafez for the use of his laboratory facilities for part of the work, dr n. ishmail for assistance with the bleeding of the animals and dr ellen lee of the university of salford computing centre for advice on the use of the computer for statistical analysis of the results. diseax\ in an amphihian colony the first component of complement from the bullfrog runa cutrsheianu: functional properties of cl and isolation of subcompon-entclq leptospiral agglutinins in sera from southern illinois herpetofauna immune response in xenopua iuevis and immunochemical properties of the serum antibodies. immunology : -l x. key: cord- - xn v s authors: rodák, l.; Šmíd, b.; nevoránková, z.; smítalová, r.; valíček, l. title: verification of sensitivity and specificity of group a rotavirus detection in piglets faeces with monoclonal blocking elisa methods date: - - journal: j vet med b infect dis vet public health doi: . /j. - . . .x sha: doc_id: cord_uid: xn v s monoclonal antibodies to group a rotavirus vp protein were prepared and used for verification of three blocking enzyme‐linked immunosorbent assay (elisa) modifications to detect rotavirus a. selected competitive blocking elisa (cb‐elisa) and electron microscopy (em) were used for examination of field faecal samples of piglets affected with diarrhoea. rotavirus was detected in samples ( . %) by cb‐elisa method, whereas in ( . %) samples by em examination. however, of samples positive by em, rotavirus a was detected by cb‐elisa in ( . %) samples; indicating the share of group a rotavirus in all cases of gastroenteritis caused by rotavirus. the sensitivity and specificity of the cb‐elisa was verified both by inclusion of control samples containing transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhoea virus (pedv) in each analysis and by comparative examination of samples with the commercial elisa kit. the cb‐elisa sensitivity was positively affected by examination of samples in the presence of chelating agent. rotaviruses rank among significant causes of gastroenteritis in the majority of vertebrates. data on their prevalence in human populations attest to their significance. a total of - million cases of gastroenteritis caused by rotaviruses with high mortality in children, particularly in developing countries, are annually reported (bajolet and chippaux-hyppolite, ; parashar et al., ) . it follows that rotaviral infections play a significant role in farm animals kept under much worse hygienic conditions. besides rotaviruses, two coronaviruses -transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhoea virus (pedv) -rank among the economically most important viruses causing diarrhoea in pigs. rotaviruses, which are about nm, with doublestranded rna genome, are highly resistant to the conditions of external environment and to both chemical and physical agents (ramos et al., ; fischer et al., ) . they replicate mainly in enterocytes covering small intestinal villi and are spread via the faecal-oral route (benfield et al., ; debouck and pensaert, ; gelberg et al., ) . previously, all rotaviruses were considered antigenically related, containing common vp antigen (group a rotavirus) with mr k. later, rotaviruses without the common antigen were classified as so-called non-group a rotaviruses. seven antigenically distinct groups of rotaviruses (a-g) were described; four (a, b, c and e) of them are pathogenic for pigs. group a rotavirus which is the most often ( . - . %) causal agent of rotaviral gastroenteritis (sigolo de san et al., ; janke et al., ; pongsuwanna et al., ) comprises a series of subgroups and serotypes according to antigenic analysis of vp and vp proteins present in the outer capsid of virion. common group a rotavirus vp antigen is found in the inner capsid of virion, and rotaviral infections caused by that agent may be diagnosed by use of specific vp antibodies regardless of animal species affected. rotavirus detection gives more exact information on the cause of gastroenteritis than determination of antiviral antibodies, which are present almost in all blood serum samples. therefore, both in veterinary and in human medicine, a series of methods are used [latex agglutination, reverse transcription polymerase chain reaction (rt-pcr), enzyme-linked immunosorbent assay (elisa), electron and immune electron microscopy (em) etc.]; some of them are available as commercial diagnostic kits (benfield et al., ; gerna et al., ; al yousif et al., ; raboni et al., ) . the objective of the present study was to compare three modifications of blocking elisa method for the detection of rotavirus a. the results obtained with selected competitive blocking elisa (cb-elisa) method were compared with those obtained by em and by commercial elisa kit. the reference strain of rotavirus a (osu strain, vr- ; serotype g /vp and serotype p /vp ) and three of our own rotavirus a field isolates were propagated in ma- cells grown in minimum essential medium eagle in the presence of trypsin ( lg/ml, t- ; sigma, st louis, mo, usa). the virus was released from the cells - hpi by repeated freezing/thawing. after centrifugation ( · g for min) of the culture medium, the pellet was resuspended in phosphate-buffered saline (pbs) in / of the original volume as crude viral antigen (v-ag). control antigen (c-ag) was prepared similarly from uninfected cells. purified v-ag was prepared by ultracentrifugation of supernatant on a cushion of % sucrose in pbs (optima le- k ultracentrifuge; beckman, palo alto, ca, usa). crude v-ag and c-ag were similarly prepared by propagation of tgev (strain capm v- ; collection of animal pathogenic microorganisms, brno) and pedv (strain cv- ) in porcine kidney (pk- ) and vero cell lines, respectively. they were used for specificity check of all elisa methods used. two hysterectomy-derived, colostrum-deprived, -week-old piglets were kept in sterile conditions, and orally infected with . , tcid of group a rotavirus. seven weeks post infection, piglets were challenged with . . tcid of the virus and killed under total anaesthesia days later. the titre of rotavirus a antibodies in the blood serum obtained (swspos.) was determined by indirect elisa. rotavirusnegative porcine blood serum (swsneg.) was obtained by exsanguination of -week-old uninfected piglet. the immunoglobulin fraction (swigrota) was obtained from positive serum by ion-exchange chromatography and used as a binding antibody in the blocking elisa methods. rota-and coronaviruses were detected by electron microscopic examination of culture media and faecal samples of piglets after negative staining with % ammonium molybdate solution in water, ph . (sˇmı´d et al., ) . inbred mice of the line balb/c were repeatedly immunized with purified rotavirus a. hybridomas were prepared by a standard procedure (galfre`and milstein, ) by fusion of splenic lymphoid cells with cells of the myeloma line sp / . specificity of mab produced by hybridomas was checked by elisa, western blot (wb) and immunoperoxidase (ip) detection of rotavirus a in infected cells. selected hybridomas were used for preparation of ascitic fluids. monoclonal antibodies (mab) purified from ascitic fluids by ion-exchange chromatography were stored at ) °c in % glycerol or used for the preparation of peroxidase conjugates (hrpo-mab-rota). antibodies to swine and mouse immunoglobulins were purified from hyperimmune swine (swamoig) or rabbit (raswig, ramoig) sera by affinity chromatography. these antibodies and mabrota were conjugated with horseradish peroxidase (hrpo, type vi-a; sigma) using the periodate method (boorsma and streefkerk, ) . the stock conjugate solutions were adjusted to mg antibodies/ml. for the ip, wb and elisa they were diluted · to · in pbs containing . % tween and . % lactalbumin hydrolysate (pbst-lah). rotavirus a antibodies in swine and mouse sera, in culture media of hybridomas and in purified mabrota preparations were assayed using the indirect elisa method. the wells of microtitre plates (nunc-immunoplate, polysorp, denmark) pre-coated alternatingly with crude rota a v-ag and c-ag were incubated ( h at °c) with various dilutions of tested samples. after washing the second identical incubation with conjugates to swine or mouse immunoglobulins followed. the reactions were visualized by incubation with chromogen tmb ( , ¢, , ¢-tetramethyl-benzidine; sigma) solution. after min, the reaction was stopped and absorbances were measured spectrophotometrically at nm. control wells filled with the diluent (blank) only, or with control negative or positive sera at the first incubation, were included in each examination. highest samples dilution showing a difference in optical density of at least . (after subtraction of absorbances of blank wells) between the wells with bound v-ag and c-ag were classified as positive. sensitivity and specificity of three variants of a blocking elisa were determined by box titration using faeces of an experimentally infected piglet. the wells of microtitre plates (nunc-immunoplate, maxisorp, denmark) were pre-coated overnight with binding antibodies in carbonate-bicarbonate buffer, ph . ( lg ig/ml; ll/well). during the first incubation ( h at °c), pairs of pre-coated wells were filled with ll mixtures of faeces with swsneg. or swspos. pbst-lah containing . m nacl and mm ethylenediaminetetraacetic acid (ed-ta)aena salt was used for sample dilution at the first incubation. after the second incubation (always h at °c) with ll of detection antibodies (conjugates hrpo-mabrota or hrpo-swigamoig), visualization of the reaction and assessment of absorbances (a) followed, similarly to indirect elisa. between incubations, the wells were rinsed with pbst four times. pairs of wells filled with the diluent (blank) or the mixtures of crude v-ag (rotavirus a, tgev, pedv) and sws neg./pos. during the first incubation were included in each analysis. the samples were regarded as positive if the net absorbance (na), i.e. the difference of average absorbances in the wells incubated with swsneg./pos. was > . , and reactions were blocked by > % in the wells with swspos. blocking percentages were determined using the formula: %b ¼ ) [(a swspos. · ) : (a swsneg. )]. the following variants of the blocking elisa method were investigated: double antibody sandwich elisa (das-elisa): wells precoated with binding mabrota. the first incubation with antigen test samples was performed for h, the second incubation with the detection antibody (hrpo-mabrota). competitive blocking elisa (cb-elisa): wells pre-coated with binding swigrota. after h of incubation with the antigen test samples the wells were supplemented with ll diluent containing lg mabrota/ml, and the incubation continued for another hour. the second incubation was undergone using detection antibody hrpo-swiga-moig. the das-elisa: wells pre-coated with binding swigrota. the first incubation with the antigen test samples was verification of sensitivity and specificity of group a rotavirus detection performed for h and the second incubation was conducted with the detection antibody hrpo-mabrota. commercial kit (ingezim rota das . .rt.k. ; ingenasa; inmunologia y genetica aplicada, s.a.; spain, thereafter das-elisa kit) was used for the detection of rotavirus a in faeces. the kit utilizes biotin-conjugated mab anti-rota a and streptavidin-peroxidase conjugate. examinations were carried out according to the manufacturer's instructions. samples were evaluated as positive at the values of corrected absorbance (ca) > . , dubious at ca ¼ . - . and negative at ca < . (ca ¼ a of the sample tested ) a of negative control antigen). results of the commercial kit examinations were compared with those obtained by em and cb-elisa methods. in total, faecal samples from piglets with diarrhoea were examined. most samples were from piglets younger than days. after delivery to the laboratory, they were diluted in two to three volumes of earle's medium, centrifuged ( min, · g), and the supernatants immediately examined by em. before cb-elisa examination, the samples were kept at ) °c. after verification of the specificity of hybridomas producing mab to rotavirus a, two of them were selected for further use. both mab of the isotype specificity igg a (g /d ) and igg b (b /f ) reacted with the viral protein vp in wb analysis (results not shown). indirect elisa titres of both mab stock solutions containing mg ig/ml reached · . cross-reac-tivity with other viral antigens could not be detected using any of the methods mentioned ( table ) . sensitivity comparison of three variants of the blocking elisa method of rotavirus a detection were performed by box titrations in microtitre plate wells pre-coated with binding antibodies. mixtures containing the faecal sample of an experimentally infected piglet and swspos./swsneg. fivefold diluted - · and - ·, respectively, were examined. the sample of faeces in the entire range of dilutions was assessed as positive by all of the three blocking elisa methods. only slight differences in sensitivity were detected. results obtained with minimal and maximal dilutions of sws only are shown in table . competitive blocking elisa methods proved to be most effective for the determination of coronaviruses (tgev: l. roda´k, b. smid, z. nevorankova, l. valicek and r. smitalova, unpublished data; pedv: l. roda´k, l. valicek, b. smid and z. nevorankova, unpublished data). therefore, cb-elisa method (variant ) was also used for routine rotavirus a detection in field samples; mixtures of faecal samples and swsneg./pos. were examined in working dilutions : and : , respectively. using diluent supplemented with chelating agent in the first incubation, more satisfactory results were obtained in most of samples in comparison with standard diluent (pbst-lah). net absorbance values (na > . ) and %b > was necessary to obtain for positive evaluation of the samples. under these conditions, sample evaluation was highly specific; as established by examination of selected positive and negative samples in eight wells (n ¼ ), determination of mean absorbances (ma) and standard deviations (±sd). calculated coefficients of variation (cv%) were . - . %. only with negative samples giving low absorbance values, the cv% was occasionally higher. sensitivity of the cb-elisa method and das-elisa kit was compared by examination of faecal sample of experimentally infected piglet twofold diluted · to ·. the results document that the highest sample dilution · and · was regarded as positive by das-elisa kit and cb-elisa respectively. cb-elisa absorbances (a) of the sample diluted : were . / . (table ) . it follows that sample with cb-elisa a < . will be probably negative by das-elisa kit. comparative examinations of crude v-ag (rota a, tgev, pedv) proved high specificity of both the methods (table ) . also randomly selected field faecal samples, assessed positively and negatively by em and cb-elisa, were comparatively examined by das-elisa kit (table ) (table ) . polyclonal and mab to group a rotavirus were prepared and used as binding or detection antibodies for sensitivity testing of three elisa variants for rotavirus a demonstration. in similar experiments dealing with tgev and pedv detection (l. roda´k et al., unpublished data), lower sensitivity of das-elisa variants using conjugated mab was proven. therefore, more sensitive variants of cb-elisa, based on the use of unconjugated mab, were selected for routine demonstration of both coronavirus and rotavirus a infections. the use of a uniform methodical procedure thus allows detecting simultaneously all three most important causative agents of viral gastroenteritis. specificity of examination was confirmed by comparative assessment of v-ag (rotavirus a, tgev, pedv) by cb-elisa and by commercial das-elisa kit ( table ). absence of cross-reactivity of cb-elisa with bacterial antigens present in field faecal samples confirmed the results of rotavirus a-negative samples. higher sensitivity of rotavirus a detection by cb-elisa in comparison with commercial das-elisa kit was proven. by examination of positive faecal sample twofold diluted · to ·, at least times higher sensitivity of cb-elisa method was demonstrated (table ) . this was also confirmed by comparative examination of randomly selected field faecal samples. all cb-elisa-negative samples were evaluated negatively by das-elisa kit examination. however, of the remaining cb-elisa-positive samples, only one half of samples were evaluated as positive by commercial das-elisa kit ( table ) . the sensitivity of rotavirus a detection by cb-elisa in comparison with em is also higher. of field faecal samples rotavirus was detected in samples by cb-elisa whereas in samples by em. on the contrary, of em positive samples rotavirus a was detected in ( . %) samples by cb-elisa. the fact that all groups of rotavirus are detected by em, while group a rotavirus only is detected by cb-elisa, can explain this difference. the congruency of em and cb-elisa examinations ( . %) indicates the share of rotavirus a in cases of rotaviral gastroenteritis and correlates with other findings (sigolo de san et al., ; janke et al., ; pongsuwanna et al., ) . the assumption that the remaining seven ( . %) samples likely contain non-group a rotavirus was partly confirmed by demonstration of group c rotavirus electropherotype in one em highly positive sample. therefore, more sensitive rt-pcr techniques will be applied in following experiments. it is well-known that chelating agents destroy the outer capsid of rotavirus and its infectivity (ward and ashley, ; fang et al., ) . therefore, we presumed that their presence in elisa examinations might improve accessibility of antigens localized in the inner capsid of virion, particularly the vp antigen. this was confirmed in our study, and better results were obtained in most samples using solution with chelating agent in comparison with the standard diluent. highly positive values of na and %b were obtained in of cb-elisa-positive faecal samples; it confirms suitability of working dilutions of faeces ( ·) and swsneg./pos. ( ·) selected for routine use. the remaining samples assessed as negative because of the %b < were also positive at higher working dilution. diagnosis of the causal agents of viral gastroenteritis is a basic prerequisite both for introduction of immunoprophylactic measures (schaller et al., ; ebina, ; hodgins et al., ) and confirmation of their effectivity. the use of uniform methods allowing diagnosing simultaneously rotavirus and coronavirus infections could contribute to this goal, as well as introduction of pcr in following experiments. evaluation of a latex agglutination kit (virogen rotatest) for detection of bovine rotavirus in fecal samples rotavirus and other viruses of diarrhea shedding of rotavirus in feces of sows before and after farrowing comparison of a commercial enzyme-linked immunosorbent assay with electron microscopy, fluorescent antibody, and virus isolation for the detection of bovine and porcine rotavirus periodate or glutaraldehyde for preparing peroxidase conjugates? rotavirus excretion in suckling piglets followed under field circumstances prophylaxis of rotavirus gastroenteritis using immunoglobulin purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins rotavirus particles can survive storage in ambient tropical temperatures for more than month preparation of monoclonal antibodies: strategies and procedures the shedding of group a rotavirus antigen in a newly established closed specific pathogen-free swine herd comparative evaluation of a commercial enzyme-linked immunoassay and solid-phase immune electron microscopy for rotavirus detection in stool specimens effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs relative prevalence of typical and atypical strains among rotaviruses from diarrheic piglets in conventional swine herds global illness and death caused by rotavirus disease in children serological and genomic characterization of porcine rotaviruses in thailand: detection of a g porcine rotavirus comparison of latex agglutination with enzyme immunoassay for detection of rotavirus in fecal specimens the stability of porcine rotavirus in feces prevention of human rotavirus-induced diarrhea in gnotobiotic piglets using bovine antibody electron microscopic demonstration of porcine epidemic diarrhea virus in the czech republic incidence of group a and atypical rotaviruses in brazilian pig herds comparative study on the mechanisms of rotavirus inactivation by sodium dodecyl sulfate and ethylenediaminetetraacetate this work was supported by projects qf and mze from ministry of agriculture of the czech republic.the authors wish to thank mrs farnı´kova´, mrs lickova´, ms hoydenova´and ms bacˇinska´for their skilful technical assistance. key: cord- -r x yaqj authors: ohnishi, kazuo title: establishment and characterization of monoclonal antibodies against sars coronavirus date: - - journal: sars- and other coronaviruses doi: . / - - - - _ sha: doc_id: cord_uid: r x yaqj immunological detection of viruses and their components by monoclonal antibodies is a powerful method for studying the structure and function of viral molecules. here we describe detailed methods for establishing monoclonal antibodies against severe acute respiratory syndrome coronavirus (sars-cov). b cell hybridomas are generated from mice that are hyperimmunized with inactivated sars-cov virions. the hybridomas produce monoclonal antibodies that recognize viral component molecules, including the spike protein (s) and the nucleocapsid protein (n), enabling the immunological detection of sars-cov by immunofluorescence staining, immunoblot, or an antigen-capture elisa system. in addition, several s protein-specific antibodies are shown to have in vitro neutralization activity. thus the monoclonal antibody approach provides useful tools for rapid and specific diagnosis of sars, as well as for possible antibody-based treatment of the disease. the outbreak of severe acute respiratory syndrome (sars) in ultimately led to people becoming infected, of whom died. the causative agent was identified as sars coronavirus (sars- cov) ( , ) . even though the epidemic ended, the threat of reemergence persists, compounded by the absence of an established vaccine. one of the critical issues in controlling a pandemic is a system for early diagnosis that distinguishes sars from other ohnishi types of pulmonary infections. based on clinical experience, several options have been considered in the quest to develop the capacity to accurately diagnose sars-cov infection, including molecular biology techniques and serological tests such as antigen-capture elisa assay and immunofluorescence assay to detect virus-infected cells in respiratory swabs ( - ) . the preparation of monoclonal antibodies (mabs) is considered to be especially valuable for serological testing. here we describe a method for the successful establishment and characterization of mabs against sars-cov structural components. these mabs enable the general immunological detection of sars-cov by methods such as immunofluorescent staining, immunoblotting, and immunohistology, in addition to the construction of a highly sensitive antigen-capture sandwich elisa ( ). . sars-cov, strain hku- , is expanded, purified, and inactivated by the method described in chapter . . balb/c mice, -to -week-old females, (japan slc, shizuoka, japan). . freund's complete adjuvant. . freund's incomplete adjuvant. the viruses generally elicit strong humoral immune response in mice and hence are good antigens for obtaining the mabs. the sars-cov also raises a high titer of serum antibody in mice. however, the immunization protocol should be optimized to obtain the desired specificity and quality of mabs of interest. the choice of parameters for immunization, such as the type of adjuvant, pretreatment of viral antigens (inactivation procedures), antigen dose, route of immunization, and the strain of mice, profoundly affect the properties of resulting mabs. for example, we found that inactivation of sars-cov with uv or formaldehyde gave rise to different immunoglobulin isotypes in mice ( ). in addition, the choice of host animals for the immunization affects the mode of epitope recognition. with the common fusion-partner cell lines such as ns- , p u , or sp /o, a variety of mouse strains, even different species such as rat and hamsters, are usable for the donor of antigen-specific b cells. however, balb/c mice are the most commonly used immunization host because the most efficient fusion-partner myelomas are derived from balb/c strain. the protocol described here is a general procedure to obtain mabs against viral antigens, though the choice of parameters, i.e., the immunization protocol, hybridoma production and so on, should be optimized for each purpose (see note ). . balb/c mice are immunized subcutaneously with g of uv-inactivated purified sars-cov using freund's complete adjuvant (fca). . after weeks, the mice are boosted with subcutaneous injection of g of uvinactivated sars-cov using freund's incomplete adjuvant (fia). . on day after the boost, sera from the mice are tested by elisa (see section . ) for the antibody titer against sars-cov. . the two mice showing the highest antibody titer are further boosted intravenously with g of the inactivated virus days after the previous boost. . if the antibody titer is lower than expected, the booster injection can be repeated several times before the final boost. three days after the final boost, spleen cells from immunized mice are fused with sp /o-ag myeloma by the polyethylene glycol method of kosbor et al. ( ) (see section . , step . this protocol gave rise to more than candidate hybridomas, some of which react with s and n protein of sars-cov ( table ) . . for to h before cell-fusion, g of peg in a sterile glass tube is melted in a microwave oven and dissolved in . ml of rpmi (abbreviated as rpmi hereafter), prewarmed to • c, by repeated pipetting. the peg solution is kept at • c for at least h. . one or two spleens from mice are excised and the spleen cell suspension is prepared by passing through a sterile stainless mesh or by smashing with two slide glasses. the cells are washed by centrifugation ( × g for min) twice with % fcs/rpmi and once with rpmi. . the log-phase growing sp /o-ag myeloma cells are washed twice with % fcs/rpmi and once with rpmi. . count the cells and mix them to a ratio of splenocyte:sp /o-ag = : . spin the cells down and remove the supernatant thoroughly. . add . ml peg/rpmi solution to the cell pellet slowly (taking about sec), loosening of cell pellet using the tip of a pipette. stir the cell clumps gently with a pipette for additional sec. the cells should be seen as small aggregates in this step. . add ml of prewarmed ( • c) rpmi slowly, taking - min for the first ml drop by drop, and then taking about min for the remaining ml. . incubate the tube at • c for at least min. . wash the cells twice with prewarmed rpmi. . suspend the cells with hat-medium to a concentration of - × sp /o-ag cells/ml and plate to . ml/well of the -well plates. . feed the cells with hat-medium by changing two-thirds of the volume of the wells on the days , , and . . the hybridoma colonies should be seen by microscope on days - and some fast-growing colonies should be recognizable by eye from day . generally, if the immunization and cell fusion step was successful, more than % of the wells contain hybridoma colonies. when the sizes of the colonies becomes one-tenth to one-fifth of the -well bottom area, the first screening of positive clones should be taken by elisa (see section . ). . the hybridoma cells in the elisa-positive wells are recovered and cloned by a limiting dilution method as follows. cells are counted, diluted with hatmedium, and plated into -well plates so that one well contains three or ten cells; the left-half of the plate contains three cells/well and the right-half of the plate contains ten cells/well. . the hybridoma colonies will be discernible after - days. then the elisa test should be performed again and the elisa-positive wells containing single colonies are selected. . the cells from the selected wells are expanded in a -well plate and adapted to ht-medium for at least week. . the cells are further adapted to the hybridoma medium for week. if necessary, the cells are further adapted to a serum-free hybridoma medium. . the hybridomas are now ready for collecting the mabs. the aliquots of the cells are resuspended in freezing buffer and stored at - • c or in liquid nitrogen. the first screening is conducted by elisa using sars-cov-infected vero e cell lysate as the antigen. in this first screening, the uninfected vero e cell lysate is used as the antigen for a negative control. in the case of the assay for the test bleed of immunized mice, the serial dilution of the sera with pbs-tween (start from / dilution) is used in place of hybridoma culture supernatants. . hybridomas are grown in about liters of serum-free hybridoma medium to the late-log phase/early stationary phase. . the culture supernatants are harvested by centrifugation at × g for min, and / volume of m tris-hcl (ph . ) and / volume of % nan are added. the following steps are carried out at • c (in a cold room) or on ice. . the protein g-sepharose b column ( - ml bed volume) is preequilibrated with pbs and the supernatant is loaded at a flow rate of about drop/ sec. this step takes - days. . the column is washed with pbs and the bound antibody is eluted with glycine/hcl solution. in this step, -ml fractions are collected into an eppendorf tube that contains . ml of m tris-hcl (ph . ). . after measuring the od of the fractions, the protein-containing fractions are pooled. an equal volume of saturated (nh ) so is gradually added to the pooled fraction. the solution is kept on ice overnight. . precipitated proteins are collected by centrifugation at × g for min and the pellet is dissolved with a small volume ( - ml) of pbs, dialyzed against pbs (three changes of ml, more than h each), . the dialyzed sample is centrifuged at , × g for min, aliquoted, and stored at - • c. this procedure generally gives - mg of purified mab from a -liter culture of hybridomas. . the protein concentration of the purified antibody is determined by od using a molecular extinction coefficient equal to . (for immunoglobulins). . the protein concentration is adjusted to mg/ml with pbs, and ml is dialyzed against mm sodium bicarbonate buffer (ph . ) at • c. . dissolve mg of sulfo-nhs-lc-biotin in ml of distilled water, and quickly add l of this solution to the antibody solution. mix well and stand in ice for h. . dialyze the solution against pbs at • c overnight with three changes of ml pbs dialysis solution, each for more than h. . the sample is transferred to an eppendorf tube and centrifuged at , × g, • c for min. . the supernatant is recovered and the protein concentration is determined by od as above. the aliquots are stored at - • to - • c. avoid repeated freeze-thawing. the working solution can be stored at • c for months to years. preservatives such as sodium azide can be added to . % if necessary. . uv-inactivated purified sars-cov is electrophoresed and blotted to pvdf membrane by the standard procedure (see note ). . the blotted pvdf membrane is blocked with starting block tm solution for h at room temperature. . the membrane is reacted with the first mabs diluted to g/ml (see note ) with % starting block tm /pbs-tween for h at room temperature. this incubation is done by placing the pvdf membrane in the hybridization bag with ml of antibody solution. . the membrane is washed with excess volume (about - ml) of pbs-tween three times for min each time in an appropriate container. . the membrane is reacted with peroxidase-conjugated f(ab ) fragment anti-mouse igg diluted to : , by % starting block tm /pbs-tween (see note ). the incubation is done in the hybridization bag for h at room temperature. . the membrane is washed thoroughly with pbs-tween four times for min each time. . after washing, the membrane is placed on mm filter paper and the liquid is removed but not dried. the membrane is then soaked with west femto a:b = : mixture (in case of minigel size, i.e., × cm, . - . ml of a:b mixture is required). the membrane should be completely soaked with excess volume of a:b mixture. . the membrane is picked up by a flat-tip forceps and placed in between two transparent polyester sheets (see note ). . the membrane/transparent sheet sandwich is placed in an x-ray film cassette with film and exposed for an appropriate time to obtain the best signals. an example of the result is shown in fig. , in which the purified sars-cov proteins ( . g/lane) are electrophoresed with sds-page, blotted to pvdf membrane, and detected by anti-s and anti-n mabs. for the diagnosis of sars-cov infection, serological tests such as immunofluorescence assay (ifa, described above) and antigen-capture elisa assay are two good options for detecting virus in, e.g., respiratory swabs or in virus-infected cells. by utilizing established mabs against sars-cov, it is possible to construct a highly sensitive antigen-capture sandwich elisa test system for detection of sars-cov. the sandwich elisa consists of two mabs, an antigen-capturing antibody and a detecting antibody, which recognize different epitopes of the target antigen. the antigen-capturing antibody is immobilized on the elisa plate and captures the viral antigens in the test sample. the detecting antibody is normally labeled with a signal-producing enzyme such as a peroxidase or a phosphatase. the following method is the basic procedure for constructing a sandwich elisa (see note ). chamber slides: lab-tek -well glass chamber slides ifa-staining buffer: % bsa (sigma) in pbs paraformaldehyde solution: % paraformaldehyde is freshly dissolved in pbs by heating to • c pbs/tritonx- solution: . % triton x- in pbs dapi solution: for the stock solution, , -diamidino- -phenylindole hydrochloride (dapi, invitrogen) is dissolved with dw at mg/ml and stored at - • c mounting solution: fluoromount g (southernbiotech immunoblot with monoclonal antibodies . blot membrane: pvdf membrane blocking reagent: starting block tm detecting (second) antibody: peroxidase-conjugated f(ab ) fragment anti-mouse igg (h + l) (use with : , dilution chemiluminescent reagent: supersignal west femto antigen-capture sandwich elisa . high-binding immunoassay microplate: immulon- (dynatech labs, va, usa) or the equivalent blocking solution: % ova in pbs-tween detecting (second) reagent: ␤-d-galactosidase-labeled streptavidine (zymed galactosidase substrate: -methy-lumbelliferyl-␤-d-galactoside (sigma) reaction stop solution: . m glycine-naoh purified mab for the antigen-capture is immobilized on an immulon- microplate by incubating g/ml antibody in the elisa-coating buffer at • c overnight the microplate is blocked with % ova for h at room temperature or overnight at • c the plate is washed three times with pbs-tween the uv-inactivated purified sars-cov samples (see note ), which are serially diluted with % ova/pbs-tween, are added to the wells and incubated for h at room temperature after washing three times with pbs-tween, wells are reacted with biotinylated detecting (second) mab ( . g/ml) for h at room temperature after washing three times with pbs-tween, wells are reacted with ␤-dgalactosidase-labeled streptavidine for h at room temperature after washing four times with pbs-tween, fluorescent substrate -methylumbelliferyl-␤-d-galactoside is added and incubated for h at • c the fluorescence of the reaction product, -methyl-umbelliferone ( -mu), is measured using fluoroscan ii (flow laboratories inc., inglewood, ca) at excitation and emission wavelengths of and nm identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study detection of sars-associated coronavirus in throat wash and saliva in early diagnosis immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome formalin-treated uv-inactivated sars coronavirus vaccine retains its immunogenicity and promotes th -type immune responses in vitro stimulated lymphocytes as a source of human hybridomas using antibodies: a laboratory manual monoclonal antibodies: a practical approach cleavage of structural proteins during the assembly of the head of bacteriophage t the author would like to thank drs. fumihiro taguchi and shigeru morikawa for their advice and discussion, dr. koji ishii for providing recombinant sars-cov proteins, and ms. sayuri yamaguchi for her technical assistance. this work was supported by grant from the ministry of public health and labor of japan. figure shows an example of an antigen-capture elisa system for sars-cov, in which two anti-n mabs, skot- (coating mab) and biotinylated skot- (detecting mab) are used. this sandwich elisa detects sars-cov protein at a concentration as low as pg/ml ( ). . for further understanding and detailed explanations of the hybridoma methodology, good textbooks are available ( , ). . the validation for the complete inactivation of the virus is crucial. for a detailed account see chapter in this volume. . in this step the culture supernatants are diluted to one-half in order to reduce the background of the elisa. if the background is still high, dilution of the culture supernatants can be one-fifth to one-tenth or more with pbs-tween. . the titration of the mabs and the second reagent is very important for obtaining the best results. it often varies from / to / , , and it should be determined for each mab. . the sds-polyacrylamide gel electrophoresis (page) is carried out by the method of laemmli ( ). the detailed conditions, such as the concentration of the gel or reducing/nonreducing and so on, should be changed case by case. generally, loading . - g/lane of purified virus fraction would be enough for the detection of the viral antigen by the chemiluminescence method. . we conveniently utilize transparent sheets such as those used for an overhead projector (ohp-sheet). . in order to obtain good sensitivity and specificity of the test system, the optimization of the combination of two antibodies, i.e., coating mab and detecting mab, is required. this can be done by the elisa with a matrix of candidate coating-mabs and detecting-mabs ( ). key: cord- -lhhjax s authors: pickering, suzanne; betancor, gilberto; galão, rui pedro; merrick, blair; signell, adrian w.; wilson, harry d.; kia ik, mark tan; seow, jeffrey; graham, carl; acors, sam; kouphou, neophytos; steel, kathryn j. a.; hemmings, oliver; patel, amita; nebbia, gaia; douthwaite, sam; o’connell, lorcan; luptak, jakub; mccoy, laura e.; brouwer, philip; van gils, marit j.; sanders, rogier w.; martinez nunez, rocio; bisnauthsing, karen; o’hara, geraldine; macmahon, eithne; batra, rahul; malim, michael h.; neil, stuart j. d.; doores, katie j.; edgeworth, jonathan d. title: comparative assessment of multiple covid- serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: lhhjax s there is a clear requirement for an accurate sars-cov- antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. we therefore evaluated the performance of a variety of antibody testing technologies and their potential use as diagnostic tools. highly specific in-house elisas were developed for the detection of anti-spike (s), -receptor binding domain (rbd) and -nucleocapsid (n) antibodies and used for the cross-comparison of ten commercial serological assays—a chemiluminescence-based platform, two elisas and seven colloidal gold lateral flow immunoassays (lfias)—on an identical panel of sars-cov- -positive samples and pre-pandemic negatives. there was a wide variation in the performance of the different platforms, with specificity ranging from % to %, and overall sensitivity from . % to . %. however, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over % seen in several tests when assessing samples from more than days post onset of symptoms. furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of sars-cov- infections. a a a a a as of the st of june , over million cases of sars-cov- have been confirmed worldwide, accounting for more than , deaths (https://covid .who.int/). lack of treatments or vaccines have forced governments to adopt strict quarantine strategies in an attempt to control the spread of the virus, causing major economical disturbances as well as adversely affecting quality of life and healthcare provision. current guidelines by leading health bodies, including the centers for disease control and prevention (cdc) in the us and public health england (phe) in the uk, recommend sars-cov- diagnosis from upper or lower respiratory specimens (including nasopharyngeal swabs and bronchoalveolar lavage) using real-time rt-pcr, typically targeting the nucleocapsid (n) or rna-dependent rna polymerase (rdrp) genes [ , ] . these tests are highly sensitivecapable of detecting vestigial viral rna levels-and are optimal for the early detection of the virus. however, the performance of the test is dependent on the time the sample is collected, with viral load declining after the first week of symptoms [ , ] . there is, therefore, a clear requirement for accurate serology testing as a companion diagnostic to pcr-based testing. this is highlighted by the recent appearance of clusters of paediatric inflammatory multisystem syndrome (pims) and other hyperimmune reactions associated with sars-cov- infection [ ] [ ] [ ] . presentation is delayed relative to active viral infection, with the detection of antibody responses being key to clinical diagnosis. in addition, monitoring population seroprevalence will be central to future public health planning based on disease susceptibility and herd immunity [ ] . for this to be meaningful, it is imperative that antibody detection methods are affordable, reliable, and readily accessible. however, with an incomplete knowledge of the immunology of covid- , evaluating tests with the assumption that antibodies 'should' be there, and comparatively to rt-pcr, is problematic. head-to-head comparisons of multiple sero-diagnostic assays on identical samples therefore provides a robust assessment of individual assay performance. accordingly, we developed a highly specific semi-quantitative elisa for the detection of anti-spike (s), -s receptor binding domain (rbd) and -n antibodies, and used this to cross-evaluate ten commercial antibody tests (seven lateral flow immunoassays (lfias), one chemiluminescent assay and two elisas) on a collection of serum samples from confirmed rna positive patients, and pre-pandemic samples from march . our results demonstrate a wide variation in the performance of the different platforms, ranging from . % to . % sensitivity and from % to % specificity. as expected, performance is highly dependent on the time the sample was taken post onset of symptoms (pos) and disease severity. results obtained in this work have enabled the diagnostic-grade validation for one of the lfias evaluated for pilot clinical use for adult and paediatric patients with a range of clinically-suspected covid- inflammatory syndromes at guy's and st thomas' hospitals. serum samples collected from individuals between the th of march and st of april at st thomas' hospital were used to compare a panel of serological assays. at the time of study, uk government guidelines limited sars-cov- testing to individuals requiring hospitalisation, and all individuals had rt-pcr-confirmed sars-cov- infection. samples were representative of typical hospital admissions during the period, with a spectrum of clinical severities from mild (requiring no respiratory support) to critical (requiring extra-corporeal membrane oxygenation (ecmo)), and a range of time points after self-reported onset of symptoms ( to days) ( table ). in-house elisas were developed to measure antibody responses against the full-length s, the rbd and n. recombinant s and rbd were expressed in hek f cells and purified by affinity and size exclusion chromatography. n was expressed in and purified from e. coli. a total of pre-pandemic serum samples from several cohorts were used to determine the lower limit of the assay, including sera from individuals attending st thomas' hospital in march , sera from vaccination studies, cancer patients, healthy volunteers, and individuals with acute ebv infection (s table) . samples from hospital patients with confirmed sars-cov- infection (from the described serum samples in table ) were used as positive controls. all serum samples from pcr positive icu patients taken at least days post symptoms showed strong igg binding to s, rbd and n (fig , s table) . in contrast, although high igm reactivity was also observed to s and rbd in some individuals, only of the negative control samples showed igg reactivity to s or rbd (fig , s table) . high igm and igg reactivity was observed in the pre-pandemic samples against n (fig ) suggestive of potential cross-reaction with seasonal coronaviruses. of note, all individuals with acute ebv infection had high igm reactivity to n and rbd. importantly, none of the pre-covid sera had detectable igg binding to n and s or n and rbd. taking into account the reactivity of negative control samples in this elisa, % specificity could be reached using a cut-off where igg against n or s both have od values at least -fold above the wells containing secondary antibody only (fig b, s table) . all serological assays were evaluated with the same set of serum samples from confirmed sars-cov- -positive individuals. each of the samples was tested: for anti-sars-cov- igm by in-house elisa and seven lfias; for iga by commercial elisa (fig ) ; and for igg by inhouse elisa, seven lfias, a commercial elisa and for total antibody (igg, igm and iga) using a chemiluminescent assay (fig ) . the commercial elisas detect anti-s antibodies; for the lfias this is undisclosed proprietary information, but for some of the tests is known to be s. with no existing standardised diagnostic test for the assessment of the serological response to sars-cov- , we started by comparing commercial serological assays with the configuration of the in-house elisa most likely to represent antibodies detected by the commercial tests (detection of anti-s igm and igg antibodies), and that had high specificity and sensitivity (s table) . for the purpose of illustration, the intensity of bands shown in a positive lfia test, or signal strength in commercial elisa or chemiluminescent assay, is reproduced as a heatmap. however, the visible detection of a band or result above a given manufacturer's threshold scored as a positive result regardless of classification. for samples giving a strong response by comparison of serological assays for detection of sars-cov- antibodies comparison of serological assays for detection of sars-cov- antibodies elisa (> -fold), the majority of the commercial assays show a consensus positive result. weaker antibody responses yielded mixed results from the lfias, with a clear pattern of increasing detection of antibodies seen across all tests with increasing time pos. samples from days - gave an extremely mixed picture, indicative of an early evolving immune response. for the detection of igm, of the samples from days or more pos were negative by anti-s elisa. of these samples were positive, in some cases only weakly, for at least two lfias and/or iga. the remaining were negative in at least lfia tests and for iga (indicated with a yellow circle in fig ) . these same samples were negative for igg by in-house and commercial elisa, in all lfias and by chemiluminescent assay (indicated with a yellow circle in fig ) . importantly, all samples came from individuals with a disease severity score of . later time points were obtained for three of these individuals (both of the day samples and the sixth day sample) and the next available samples were found to be strongly positive for igg and igm by lfia (days , and pos, respectively). further investigation into the nature of the negative day sample revealed that it was an error in self-reporting the time of symptom onset: this individual had covid- -compatible symptoms for days prior to sampling, yet tested negative for rna days pos. days pos the individual tested positive for rna, therefore the more likely time of sampling was between and days pos. samples were not re-classified, as they are representative of the real-time analyses being performed during the peak of a pandemic. however, the day sample was omitted from sensitivity calculations. overall, with the exception of genbody and watmind, all tests gave cross-assay agreements between . and . % (fig and s fig) . the highest level of agreement was seen between the in-house elisa, surescreen, accu-tell, spring and euroimmun tests. interestingly, the in-house elisa igm and euroimmun iga results showed particularly good agreement ( . %), although the euroimmun detected iga more frequently in early samples compared with the igm detected by in-house elisa (fig ) . results from the sars-cov- -positive samples and an identical set of pre-pandemic negative samples were used to evaluate assay sensitivities and specificities, with extended specificity assessments using larger sample numbers performed on selected tests (fig a, s fig and s -s tables). cross comparison of overall specificities and sensitivities led to the shortlisting of six tests with the highest specificity and sensitivity (elisa igm and igg, surescreen, accu-tell, spring and euroimmun iga). these were the same tests that gave the best agreement in the cross-assay comparisons. notably, sensitivity increased for all tests with increasing days pos, with antibodies being variably detected at early times ( fig b) . deep blue, accu-tell, surescreen and spring also displayed the highest levels of sensitivity at less than days. in sum, deep blue, accu-tell, surescreen, spring, biohit, medomics, euroimmun (iga and igg) and in-house elisas (igm and igg) all had sensitivities above % for samples taken � days pos. sample-by-sample analyses of multiple serological assays showed a trend for increasing detection of antibodies with increasing days pos (up to ). we also observed that individuals with � . to < are positive, and � are strong positive. samples are grouped according to days post onset of covid- symptoms, and squares aligned in columns under each bar of the graph show results for a single serum sample. yellow circles indicate samples from days or more pos that were negative by elisa and in at least other tests, as detailed in the text. https://doi.org/ . /journal.ppat. .g comparison of serological assays for detection of sars-cov- antibodies comparison of serological assays for detection of sars-cov- antibodies severe disease had a more readily detectable antibody response, particularly compared to those who had a brief hospital stay with minimal intervention. samples were grouped according to days pos and clinical severity, and compared for anti-s igm and igg by in-house elisa. significant differences in antibody levels were observed with increases in both days and severity, although there was no association between days and severity themselves (fig a) . to assess the development of the antibody response in sequential samples from the same individual, and to evaluate the ability of lfias to detect nuances in antibody response, longitudinal samples (from individuals with disease severity scores of ) were tested by one of the best performing lfias (surescreen) and in-house elisa (fig b and c) . a similar pattern of detection was seen for both types of assay, with igm detectable earlier than igg, and both tests showing consensus for the strength and timing of the response. comparison of serological assays for detection of sars-cov- antibodies this study describes the development of six in-house elisa configurations for the detection of igm and igg against sars-cov- s, rbd and n. anti-s and -n igg attained specificities of up to %- % when results for both targets are combined-and sensitivities of up to %. we used this semi-quantitative platform to cross-evaluate seven lfias, a chemiluminescent test and two commercial elisas. importantly, the availability of sequential serum samples from patients admitted from the start of the outbreak under an existing ethics agreement for storage and analysis of surplus amounts of routinely collected clinical samples, enabled us to conduct this detailed study including examining assay sensitivity with respect to time pos. our analysis demonstrates a broad range of performance across the different platforms, with several commercial tests performing above % specificity. we found that all platforms showed highest sensitivity, with narrowest confidence limits, in samples taken days pos, with most tests reaching a value of over % (fig b) . when all commercial tests were compared, accu-tell, surescreen and spring demonstrated highest sensitivity at earlier time points, while maintaining specificities of % or above. these tests also gave the best comparison of serological assays for detection of sars-cov- antibodies comparison of serological assays for detection of sars-cov- antibodies cross-assay agreements with each other and with the in-house elisa. in the best-performing tests, we also observed that signal strength aligned with that seen by elisa; this was further supported by the sequential signal increase seen in longitudinal samples from five individuals. we approached this study with the intention that an unbiased and transparent comparison will be of broad value to the scientific and infection diagnostics communities, both in terms of naming and comparing the kits, and in the nature of the samples likely to be encountered in hospitals during a sars-cov- outbreak. few studies have been published to date in which multiple lfias and elisas have been evaluated side-by-side with named kits [ ] [ ] ; and in those that have, only one test overlaps with our study, deep blue [ ] . early reports stating that lfias have insufficient sensitivity may have been due to testing samples from mixed time points and disease severities, or tests that differ from those evaluated here [ ] . the strength of our study is the head-to-head evaluation of multiple tests on identical serum samples. the sample set was not compiled retrospectively for the purpose of evaluation, but was part of an ongoing process to deploy a serological assay to broaden diagnostic capability at guy's and st thomas' hospitals during the peak of the sars-cov- epidemic in london. these samples are therefore entirely representative of the type that will be encountered by hospital laboratories, and the challenges associated with variable time to seroconversion and errors in self-reporting onset of symptoms are real. the cross-evaluation of multiple tests enabled the identification of samples that were negative in every test performed, despite the fact that samples were taken at days pos or later. while this could highlight a characteristic of covid- where a subset of patients do not produce a detectable antibody response, we have evidence that in three cases (the two day samples and sixth day ) where later samples were available ( , and days pos, respectively; the only next available samples) both igm and igg antibodies were detected in several lfias. in another case (sample at day pos), we uncovered a likely error in self-reported symptom onset and the sample could actually range anywhere between and days pos. it is important to note that all of the six unexpectedly negative samples were from covid- cases classified as severity level . the fact that several post-day samples were negative in all serological tests has practical implications for the use of such assays in diagnostic settings, and thought should be given to the meaning of a negative result. it also implies that serological surveys are likely to underestimate the level of exposure to sars-cov- , and the wide variation in the detection of antibodies, both in terms of time and disease severity, casts into doubt the utility of "immunity passports". however, although it is unclear at present whether detection of antibodies to sars-cov- indicates protection against future infection, measurement of antibodies to s, rather than n, is likely to better predict neutralisation function. in agreement with previous studies demonstrating a relationship between disease severity and antibody titres [ ] [ ] [ ] , we observed a significant increase in detection of antibodies with increased severity of symptoms ( fig a) . importantly, this correlation is not explained by a concomitant increase in the days pos of the sample. therefore, before deployment in situations where the pre-test prevalence is likely to be low, such as seroprevalence studies, outpatient assessment or pre-admission screening for operations, these assays will require further evaluation with known sars-cov- asymptomatic and ambulatory cases, alongside an extended set of pre-pandemic samples. this is a priority as countries navigate their way out of lockdown and move towards living with the ongoing threat of sars-cov- , and seroprevalence studies will be important in the implementation and management of safe public health policies. to maintain the high specificity of our in-house elisa in community cohorts where pcr status is unknown, we would recommend determining seropositivity based on igg to both n and s. sequential or alternate detection of igm, iga and igg may also provide information on the history of infection. in the only iga test that we evaluated (euroimmun), comparison of serological assays for detection of sars-cov- antibodies specificity was high while also showing a strong signal in early samples and a good overall sensitivity. detection of iga in serum, and potentially even earlier by mucosal sampling, may be a useful diagnostic tool. next generation antibody tests may improve on those currently being trialled, but our results demonstrate that lfias may have utility in a hospital setting as of now, particularly if deployed where a rapid result could aide a clinical pathway or decision in real time, such as ward location or prioritisation of further diagnostics and follow up. the ease of use and affordability of the lfias weighs heavily in their favour, especially for potential in resourcepoor settings or as point-of-care solutions in hospitals. choosing the tests with the highest specificity will translate to confidence in a positive test result in the clinic; negative or borderline cases may be tested serially or used together with elisa testing [ ] . combination testing, in parallel with rt-pcr, and serial or sequential testing, would provide diagnostic solutions to the delayed-onset syndromes such as pims that are increasingly being reported post-peak pandemic [ ] [ ] [ ] . further evaluations of candidate tests should be performed prior to use in clinical settings, ideally tailored to the intended usage and likely pre-test prevalence. a limitation of our study was the restricted number of pre-pandemic negative and confounding samples used to cross-validate the commercial kits, and further specificity and sensitivity studies with a shortlisted group of commercial tests are currently in progress. it will be particularly important to evaluate samples from individuals infected with other human coronaviruses or respiratory viruses for potential cross-reacting antibodies. a further consideration that should be given for healthcare service deployment in the hospital or community setting is consistency of use. although the lfias are marketed as home testing kits, in our experience user assiduity is essential to their optimal performance, particularly when scoring borderline cases and considering need for two independent readers. there will also likely be need to evaluate alternative sources of blood collection, particularly pin-prick collection, and even different samples such as saliva, all of which should be fully evaluated before deployment. in summary, our study compares the performance of commercially available platforms and several combinations of in-house methods for the detection of anti-sars-cov- antibodies in serum samples. although lfias lack the semi-quantitative information provided by elisa tests, they have a clear utility advantage over elisa or chemiluminescence-based technologies. shortlisted tests, combined with confirmatory reflex testing using our in-house elisa, are now being taken forward into extended validations as part of a pilot clinical service at guy's and st thomas' hospitals. these incremental steps keeping different technologies in scope, whilst multiple different use-cases are still being defined, will help determine the clinical utility and cost-effectiveness of covid- serological testing in healthcare settings both in the hospital and the community. for the st thomas' hospital samples, surplus serum was retrieved from the routine biochemistry laboratory at point of discard, and then aliquoted, stored and linked with a limited clinical dataset by the direct care team, before anonymisation under an existing ethics framework (rec reference /nw/ ) and with expedited r&d approval. serum/plasma samples used as negative controls in the in-house elisa development were obtained from the kcl infec comparison of serological assays for detection of sars-cov- antibodies patient overview and sample origin individual venous serum samples collected at st thomas' hospital, london from patients diagnosed as sars-cov- positive via real-time rt-pcr, were obtained for serological analysis. samples ranged from to days after onset of self-reported symptoms. for the longitudinal study serum samples ( - days after symptoms onset) were obtained from patients ( - samples each) with confirmed covid- diagnosis. two patients overlapped between the longitudinal study and validation study meaning in total there are unique patients between both studies. patient information is given in table . patients diagnosed with covid- were classified as follows: -asymptomatic or no requirement for supplemental oxygen. -requirement for supplemental oxygen (fio < . ) for at least hrs. -requirement for supplemental oxygen (fio � . ) for at least hrs. -requirement for non-invasive ventilation (niv)/ continuous positive airways pressure (cpap) or proning or supplemental oxygen (fio > . ) for at least hrs and not a candidate for escalation above level care. -requirement for intubation and mechanical ventilation or supplemental oxygen (fio > . ) and peripheral oxygen saturations < % (with no history of type respiratory failure (t rf)) or < % (with known t rf) for at least hrs. -requirement for ecmo. all sera/plasma was heat-inactivated at ˚c for mins before use in the in-house elisa. high-binding elisa plates (corning, ) were coated with antigen (n, s or rbd) at μg/ ml ( μl per well) in pbs, either overnight at ˚c or hr at ˚c. wells were washed with pbs-t (pbs with . % tween- ) and then blocked with μl % milk in pbs-t for hr at room temperature. wells were emptied and sera and plasma diluted at : and : respectively in milk were added and incubated for hr at room temperature. control reagents included cr ( μg/ml), cr ( . μg/ml), negative control plasma ( : dilution), positive control plasma ( : ) and blank wells. wells were washed with pbs-t. secondary antibody was added and incubated for hr at room temperature. igm was detected using goatanti-human-igm-hrp ( : , ) (sigma: a ) and igg was detected using goat-anti- comparison of serological assays for detection of sars-cov- antibodies human-fc-ap ( : , ) (jackson: - - -jir). wells were washed with pbs-t and either ap substrate (sigma) was added and read at nm (ap) or -step tmb substrate (thermo scientific) was added and quenched with . m h s before reading at nm (hrp). n protein was obtained from the james lab at lmb, cambridge. the n protein used is a truncated construct of the sars-cov- n protein comprising residues - (both ordered domains with the native linker) with an n terminal uncleavable hexahistidine tag. n was expressed in e. coli using autoinducing media for h at ˚c and purified using immobilised metal affinity chromatography (imac), size exclusion and heparin chromatography. s protein consists of a pre-fusion s ectodomain residues - with proline substitutions at amino acid positions and , a gggg substitution at the furin cleavage site (amino acids - ) and an n terminal t trimerisation domain followed by a strep-tag ii [ ] . the protein was expressed in l hek- f cells (invitrogen) grown in suspension at a density of . million cells/ml. the culture was transfected with μg of dna using pei-max ( mg/ml, polysciences) at a : ratio. supernatant was harvested after days and purified using streptactinxt superflow high capacity % suspension according to the manufacturer's protocol by gravity flow (iba life sciences). the rbd plasmid was obtained from florian krammer at mount sinai university [ ] . here the natural n-terminal signal peptide of s is fused to the rbd sequence ( to ) and joined to a c-terminal hexahistidine tag. this protein was expressed in ml hek- f cells (invitrogen) at a density of . million cells/ml. the culture was transfected with μg of dna using pei-max ( mg/ml, polysciences) at a : ratio. supernatant was harvested after days and purified using ni-nta agarose beads. we tested seven point-of-care colloidal-gold-based lfias detecting igg and igm antibodies against sars-cov- , full details of which are shown in table . with the exception of medomics, all lfias were ce ivd marked. target antigens were undisclosed proprietary information, but for several of them were known to be s. lfias were run according to manufacturer's instructions. typically, - μl of serum was added to the lfia membrane start point, followed by - drops of supplied buffer. kits were run at room temperature for minutes and then immediately scored using a -point scale (negative, borderline, positive, strong positive) for both igm and igg. scoring was performed independently by two individuals. comparison of serological assays for detection of sars-cov- antibodies the sars-cov- ab diagnostic test kit (shenzen watmind medical co., ltd.) detecting total antibody against sars-cov- was run on the chemical luminescence immunity analyzer mf (shenzen watmind medical co., ltd). the platform was calibrated with a supplied control cartridge daily prior to testing. panel samples were analysed according to manufacturer's instructions. results equal to and below . au (arbitrary units) /ml were negative, scores above . au/ml were deemed positive. for comparison to other immunoassays, scores between < au/ml were deemed positive, scores > au/ml were deemed a strong positive. expected binomial exact % confidence intervals were calculated on prism . using wilson/ brown statistical analysis. results for each test were either categorised according to whether the serum sample was from < , � , � , or � days pos, or severity of illness, with indicating mild illness (requiring no respiratory support) and indicating critical (requiring ecmo) (see materials and methods for full classification). % confidence intervals are shown for each assay in all panels (wilson/brown expected binomial). (tif) s table. specificity and sensitivity of in-house elisas during development phase. specificity and sensitivity (%) were determined for each configuration of the in-house elisa (detection of igm and igg to n, s and rbd) during initial development. pre-pandemic samples from several cohorts were used as negative controls for specificity calculations, and rt-pcr-confirmed sars-cov- positive samples were used as positive controls for sensitivity calculations. % cis are shown for each calculation. (docx) nhs. guidance and standard operating procedure covid- virus testing in nhs laboratories centers for disease control and prevention. interim guidelines for collecting, handling, and testing clinical specimens for covid- temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study sars-cov- viral load in upper respiratory specimens of infected patients an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study european centre for dieases prevention and control. paediatric inflammatory multisystem syndrome and sars-cov- infection in children clinical characteristics of children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov- herd immunity: understanding covid- test performance evaluation of sars-cov- serological assays evaluation of nine commercial sars-cov- immunoassays antibody testing for covid- : a report from the national covid scientific advisory panel antibody responses to sars-cov- in patients of novel coronavirus disease viral kinetics and antibody responses in patients with antibody responses to sars-cov- in patients with covid- recommendations for verification and validation methodology and sample sets for evaluation of assays for sars-cov- (covid- adjuvented influenza-h n vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events potent neutralizing antibodies from covid- patients define multiple targets of vulnerability a serological assay to detect sars-cov- seroconversion in humans thank you to dr terry wong for his support in acquiring test kits. thank you to surescreen diagnostics for their engagement and technical assistance.thank you to bindi patel, nicola varatharajah, abayomi fatola and amelia moore for laboratory assistance.thank you to florian krammer for provision of the rbd expression plasmid, and leo james and leo kiss for the provision of purified n protein.we thank king's college london infectious diseases biobank for provision of pre-covid- vaccine samples, all patients and control volunteers who participated in this study and to all clinical staff who helped with recruitment, including those working with the tapb project at the royal free hospital. we thank the maini lab at the division of infection and immunity for providing pre-covid pandemic healthy control samples.we are extremely grateful to all patients and staff at st thomas' hospital who participated in this study. comparison of serological assays for detection of sars-cov- antibodies comparison of serological assays for detection of sars-cov- antibodies key: cord- -mryazbnq authors: okba, nisreen m.a.; müller, marcel a.; li, wentao; wang, chunyan; geurtsvankessel, corine h.; corman, victor m.; lamers, mart m.; sikkema, reina s.; de bruin, erwin; chandler, felicity d.; yazdanpanah, yazdan; le hingrat, quentin; descamps, diane; houhou-fidouh, nadhira; reusken, chantal b.e.m.; bosch, berend-jan; drosten, christian; koopmans, marion p.g.; haagmans, bart l. title: severe acute respiratory syndrome coronavirus −specific antibody responses in coronavirus disease patients date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: mryazbnq a new coronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ), has recently emerged to cause a human pandemic. although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. we developed serologic assays for detection of sars-cov- neutralizing, spike protein–specific, and nucleocapsid-specific antibodies. using serum samples from patients with pcr-confirmed sars-cov- infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial elisas. we demonstrated that most pcr-confirmed sars-cov- –infected persons seroconverted by weeks after disease onset. we found that commercial s igg or iga elisas were of lower specificity, and sensitivity varied between the assays; the iga elisa showed higher sensitivity. overall, the validated assays described can be instrumental for detection of sars-cov- –specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies. dition, these epidemiologic studies can help identify the extent of virus spread in households, communities, and specific settings, which could help guide control measures. serologic assays are also needed for evaluation of results of vaccine trials and development of therapeutic antibodies. among the coronavirus structural proteins, the spike (s) and nucleocapsid (n) proteins are the main immunogens ( ) . we describe development of serologic assays for detection of virus neutralizing antibodies and antibodies to the n protein and various s protein domains, including the s subunit, and the receptor-binding domain (rbd) of sars-cov- in an elisa format. using a well-characterized cohort of serum samples from pcr-confirmed sars-cov- and patients pcr-confirmed to be infected with seasonal coronaviruses and other respiratory pathogens, we validated and tested various antigens in different platforms developed in-house, as well as a commercial platform. we used serum samples (n = ) collected from pcrconfirmed patients: with mild covid- and with severe covid- (table ) from france in accordance with local ethics approvals (f.-x. lescure et al., unpub. data, https://doi.org/ . / . . . ). for assay validation, we used samples obtained from persons who had pcr-diagnosed infections with human coronaviruses (hcov- e, nl , or oc ), sars-cov, mers-cov, or other respiratory viruses (table ) as reported ( ) . we also included samples from patients who had recent infections with cytomegalovirus, epstein-barr virus, or mycoplasma pneumoniae because these pathogens have a higher likelihood of causing false-positive results. as negative controls, we used serum samples from healthy blood donors (sanquin blood bank, https://www. sanquin.nl) (cohort a). we also tested serum samples from sars patients ( ) . all samples were stored at - °c until use. the sanquin blood bank obtained written informed consent for research use of samples from blood donors. use of serum samples from the netherlands was approved by the local medical ethics committee (approval no. - ). all serum samples (n = ) from patients with pcrconfirmed cases of covid- cases were previously analyzed by a recombinant sars-cov- s protein-based immunofluorescence test and plaque reduction neutralization (r. wölfel et al., unpub. . we tested serum samples as part of an extended diagnostic regimen after we obtained informed written consent from patients. we obtained non-sars-cov- -infected serum samples (n = ) from the serum collection of the national consiliary laboratory for coronavirus detection at charité-universitätsmedizin berlin (berlin, germany). samples were collected after we obtained informed written consent. the collection contained followup antibody-positive serum samples from pcrconfirmed virus-infected cases: hcov- e (n = ), hcov-hku (n = ), hcov-oc (n = ), mers-cov (n = ), hcov-nl (n = ), sars-cov (n = ), and common cold cov (n = ). we expressed the s ectodomains of sars-cov- (residues - , , strain wuhan-hu- , genbank accession no. qhd . ), sars-cov (residues - , , strain cuhk-w , accession no. aap . ), and mers-cov (residues - , strain emc, accession no. yp_ . ) in hek- t cells by using a c-terminal trimerization motif, strep-tag, and the pcaggs expression plasmid. likewise, we expressed the sars-cov- s subunit or its subdomains (s;s , residues - ; s a , residues - ; rbd, residues - ; accession no. qhd . ) in t cells, as described (c. wang et al., unpub. data, https://doi. org/ . / . . . ). we produced s proteins of other hcovs: hku (residues - ), oc (residues - ), nl (residues - ), e (residues - ), sars-cov (residues - ), and mers-cov as described ( , ) . we affinity purified all recombinant proteins from culture supernatant by using protein-a sepharose beads (catalog no. - - ; ge healthcare, ge healthcare, https://www.gehealthcare.com) or strep-tactin beads (catalog no. - - ; iba lifesciences, https://www.iba-lifesciences.com). we checked purity and integrity of all purified recombinant proteins by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with coomassie blue. we used the plaque reduction neutralization test (prnt) as a reference for this study because neutralization assays are the standard for coronavirus serologic analysis. we tested serum samples for their neutralization capacity against sars-cov- (german isolate; gisaid id epi_isl ; european virus archive global # v- ) by using prnt as described with some modifications ( ). we -fold serially diluted heat-inactivated samples in dulbecco modified eagle medium supplemented with nahco , hepes buffer, penicillin, streptomycin, and % fetal bovine serum, starting at a dilution of : in µl. we then added µl of virus suspension ( plaque-forming units) to each well and incubated at °c for h before placing the mixtures on vero-e cells. after incubation for h, we washed, cells supplemented with medium, and incubated for h. after incubation, we fixed the cells with % formaldehyde/ phosphate-buffered saline (pbs) and stained the cells with polyclonal rabbit anti-sars-cov antibody (sino biological, https://www.sinobiological.com) and a secondary peroxidase-labeled goat anti-rabbit igg (dako, https://www.agilent.com). we developed signal by using a precipitate forming , ′, , ′-tetramethylbenzidine substrate (true blue; kirkegaard and perry laboratories, https://www.seracare.com) and counted the number of infected cells per well by using an immunospot image analyzer (ctl europe gmbh, https://www.immunospot.eu). the serum neutralization titer is the reciprocal of the highest dilution resulting in an infection reduction of > % (prnt ). we considered a titer > to be positive. we performed the prnt for serum samples from germany by using vero e cells, as described (r. wölfel et al., unpub. data, https://doi.org/ . / . . . ) ( ) and -well plates. before the prnt, we heat-inactivated patient serum samples at °c for min. for each dilution step (in duplicate), we diluted patient serum samples in µl of optipro serum-free medium (https://www. thermofisher.com) and mixed : with µl of virus solution containing pfus. we vortexed the -µl serum-virus solution gently, incubated at °c for h, and then incubated each -well plate with µl serum-virus solution. after incubation for h at °c, we discarded supernatants, washed cells once with pbs, and supplemented them with . % microcrystalline cellulose solution in dulbecco modified eagle medium. after days, we fixed and inactivated the plates by using a % formaldehyde/pbs solution and stained with crystal violet. we performed anti-sars-cov- s igg and iga elisas by using β-versions of commercial kits (euroimmun medizinische labordiagnostika ag, https://www.euroimmun.com) and performed the assay according to the manufacturer's protocol. we detected optical density (od) at nm and calculated a ratio of the reading of each sample to the reading of the calibrator, included in the kit, for each sample (od ratio). because the β-version of the kit awaits validation and marking, we determined an in-house cutoff value based on the mean background reactivity of all sars-cov- -negative serum samples in the study multiplied by . the od ratio was . for iga and . for igg. we performed the in-house elisas by coating -well microtiter elisa plates with in-house-produced s antigens (s or s of sars-cov- , sars-cov or mers-cov; sars-cov- s a ; or rbd proteins) or sars-cov n protein (sino biological) in pbs overnight at °c. after blocking, we added diluted serum (diluted : or -fold serially diluted for titers) and incubated at °c for h. antigen-specific antibodies were detected by using peroxidase-labeled rabbit anti-human igg (dako) and , ′, , ′-tetramethylbenzidine as a substrate. the absorbance of each sample was measured at nm, and we set the cutoff value at sd above the mean value for the negative cohort. serum samples were previously tested for antibodies against s of different coronaviruses. we used a protein microassay that has been described ( ). we analyzed the correlations between antibody responses detected by different elisas and those detected by prnt, which is the standard for coronavirus serologic analysis. we used graphpad prism version (https://www.graphpad.com) for this analysis. we evaluated sars-cov- -specific antibody responses in severe and mild cases by using serum samples collected at different times postonset of disease from pcr-confirmed covid- patients from france. we tested serum samples for sars-cov- specific antibodies by using different elisas. after infection, all patients seroconverted between days and after onset of disease (figure ) , and antibodies were elicited against the sars-cov- s, s subunit, and rbd, but only / patients had detectable antibodies to the n-terminal (s a ) domain. because the n protein of sars-cov- is % similar to that of sars-cov (table ) , we used sars-cov n protein as an antigen to test for sars-cov- n protein-directed antibodies in an elisa format. we found that antibodies were elicited against the n protein in all three patients. when tested in a prnt, serum samples from all three patients neutralized sars-cov- infection. antibody responses detected by different assays correlated strongly with neutralizing antibody responses ( figure ). we observed cross-reactivity with the sars-cov s and s proteins, and to a lower extent with mers-cov s protein, but not with the mers-cov s protein ( figure , panels g, h). this finding was evident from analyzing the degree of similarity of the different coronavirus s protein domains to their corresponding sars-cov- proteins ( table ). this analysis showed that the s subunit is more conserved and thus plays a role in the cross-reactivity seen when the whole s was used as antigen. thus, s is more specific than s as an antigen for sars-cov- serologic diagnosis. we further assessed the specificity of the s assay by using cohorts a-e (table ) , which were composed of serum samples from healthy blood donors (a), pcr-confirmed acute respiratory non-cov infections (b), acute-phase and convalescent-phase pcr-confirmed αand β-hcov infections (c), pcrconfirmed mers-cov infections (d), and pcr-confirmed sars-cov infections (e). none of the serum samples from specificity cohorts a-d were reactive in our in-house s elisa at the set cutoff value, indicating % specificity, whereas serum samples from sars-cov patients cross-reacted (figure , panel a) . the specificity of s as an antigen for sars-cov- serologic analysis was further supported by the fact that %- % of serum samples in cohorts a-c included in this study were seropositive for endemic hcovs (hcov-hku , hcov-oc , hcov-nl , and hcov- e), as determined by the s protein microarray (figure , panel b) . nonetheless, all serum samples were seronegative for sars-cov and mers-cov. using the same cohort, we also validated the specificity of the n protein igg and rbd igg elisas for detecting sars-cov- -specific antibodies. at the set cutoff, except for serum samples from sars-cov patients, none of the control serum samples was positive for rbd antibodies, and mers-cov-positive serum sample was weakly positive for n protein antibodies ( figure , panels c, d). we also detected seroconversion among the patients with covid- . because serum samples from the patients were collected at a limited number of time points, it was difficult to accurately assess time for seroconversion. to accurately assess time of seroconversion, a larger number of longitudinal samples is needed. overall, these validated elisas for different antigens can be useful for epidemiologic studies and for evaluation of vaccine-induced immune responses. next, we validated the sensitivity and specificity of commercial elisa kits for detecting s -specific igg and iga by using the same cohort (table ; (figure ) . we also detected reactivity of serum samples from the validation cohorts a-d; / for iga and / for igg elisas. serum samples from patients infected with hcov-oc (a betacoronavirus) were reactive in both igg and iga elisa kits. we have reported the cross-reactivity of these serum samples in a mers-cov s igg elisa kit ( ) . we confirmed the cross-reactivity of the serum samples by testing serum samples from both patients that were collected at different time points were collected - days after onset of disease onset ( figure ). all covid- patients were previously confirmed to seroconvert at days - after onset of disease by use of a recombinant immunofluorescence test and prnt. a total of / seroconverted patients showed reactivity above the implemented cutoff values in the igg and iga elisa. a serum sample from patient ( figure , panels a, b) had an antibody level slightly below the cutoff value, which might be explained by an overall reduced antibody response of this patient (prnt = ). overall, the iga-based elisa kit was more sensitive but less specific than the igg-based elisa kit. finally, we compared the performance of different elisas for detection of antibodies among pcrconfirmed covid- patients with that of prnt, which is the standard for coronavirus serologic analysis (tables , ). the prnt correlated strongly with different elisas; the commercial iga elisa showed the strongest correlation, followed by the s and n elisa, which indicated their capacity to detect sars-cov- -specific antibodies. however, a larger patient cohort is needed to assess the sensitivities of these platforms. validated sars-cov- serologic assays are urgently needed for contact tracing, epidemiologic and vaccine evaluation studies. because the n and s proteins are the main immunogenic coronavirus proteins, we developed elisa-based assays that were able to detect antibodies to these proteins, and to the s domains, s a , and rbd. results for these assays correlated strongly with results of the prnt . because most humans have antibodies against the endemic human coronaviruses, it was crucial to verify the specificity of these assays to avoid false-positive results. in addition, the zoonotic coronaviruses, sars-cov and mers-cov, are also betacoronaviruses, increasing the potential for cross-reactivity. among the s antigens tested, s was more specific than s in detecting sars-cov- antibodies, as mers-cov s cross-reactive antibodies were detected in serum of of the covid- patients, which was not seen when mers-cov s was used for testing. this finding could be explained by the high degree of conservation in the coronavirus s subunit relative to s (table ) . therefore, consistent with our earlier findings for serologic analysis of mers-cov ( ), s is a specific antigen for sars-cov- diagnostics. when testing the specificity of s or its rbd for detecting sars-cov- antibodies, none of the serum samples from the validation cohorts (a-e) showed any reactivity, except for serum samples from patients with sars-cov. this finding is not unexpected because cross-reactivity resulted from the high degree of similarity between s and rbd of sars-cov and sars-cov- (table ) . however, sars-cov has not circulated in the human population since (i.e., years ago), and an earlier study reported waning of sars-cov-specific antibodies, which made them undetectable in ( %) of serum samples tested years after infection ( ) . it is therefore unlikely that antibodies to this virus are present in the population, and thus it is unlikely that false-positives results are caused by reactivity of sars-cov antibodies. we used the high degree of similarity between the sars-cov and sars-cov- proteins to develop a new in-house n protein elisa, in which we used sars-cov n protein ( % similar to sars-cov- n protein) as antigen. the n protein elisa could detect sars-cov- -specific antibodies with high specificity and sensitivity. using the different validated elisas, we found that antibody levels were higher after severe infection than after mild infections; similar findings have been reported earlier for mers-cov ( , ) . however, this finding needs to be confirmed in a larger cohort of patients with various degrees of disease severity, and it highlights the potential need for a sensitive emerging infectious diseases • www.cdc.gov/eid • vol. , no. , july assay to avoid missing persons who have milder infections in epidemiologic studies. in addition, igg seroconversion can be reliably confirmed in the second week after disease onset. however, because of the limited number of longitudinal serum samples from covid- patients tested by the in-house assays, it was difficult to accurately assess time for seroconversion. for this assessment, a larger number of longitudinal samples is needed. in the in-house elisas tested, the rbd and n protein elisas were more sensitive than s elisa in detecting antibodies in mildly infected patients and showed stronger correlations with prnt titers. therefore, detecting antibodies against different antigens might be needed to confirm the findings and avoid false-negative results in surveillance studies. however, the sensitivities of the assays need to be further validated with a larger cohort. we validated β-versions of iga and s igg commercial elisas in different laboratories. the igabased elisa showed higher sensitivity than the igg-based elisa, whereas the igg elisa showed higher specificity than the iga elisa. the iga and igg assays can be used for serologic diagnosis, igg is longer lived ( ) and thus is preferred for serosurveillance studies. we observed some cross-reactivity in both elisas with serum samples from the same hcov-oc patients in which these samples showed cross-reactivity in a mers-cov s igg eli-sa ( ) despite the different antigen used. this finding indicates a response to another protein that could be in the blocking or coating matrix, apart from the specific antigen coated, resulting in this consistent false-positive result. overall, the assays developed and validated in this study could be instrumental for patient contact tracing, serosurveillance studies, and vaccine evaluation studies. however, because various studies will be conducted in different laboratories, it is crucial to calibrate and standardize assays developed by different laboratories by using well-defined standard references as part of diagnostic assay validation. this standardization is not only needed to reduce interassay variability, but to also correlate results obtained from different laboratories that use various assays ( ) . this correlation is crucial for better comparison and interpretation of results from different studies; evaluating vaccine trials; enabling uniform assessment of immunogenicity, efficacy; and better understanding of correlates of immune protection ( ) . thus, setting up reference panels is a vital element in our preparedness approaches to emerging viruses. a pneumonia outbreak associated with a new coronavirus of probable bat origin coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- . nat microbiol world health organization. coronavirus disease (covid- ) situation reports detection of novel coronavirus ( -ncov) by real-time rt-pcr serological assays for emerging coronaviruses: challenges and pitfalls sensitive and specific detection of low-level antibody responses in mild middle east respiratory syndrome coronavirus infections newly discovered coronavirus as the primary cause of severe acute respiratory syndrome dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc blocking transmission of middle east respiratory syndrome coronavirus (mers-cov) in llamas by vaccination with a recombinant spike protein transmission of mers-coronavirus in household contacts lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study mers-cov antibody responses year after symptom onset, south korea antibody response and disease severity in healthcare worker mers survivors chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus study participants. comparison of serologic assays for middle east respiratory syndrome coronavirus international biological reference preparations for epidemic infectious diseases we thank malik peiris for providing serum samples from sars patients. dr. okba is a postdoctoral researcher in the viroscience department, erasmus medical center, rotterdam, the netherlands. her primary research interest is development of diagnostic and intervention strategies for emerging viruses. key: cord- -kaku xd authors: espejo, andrea p; akgun, yamac; al mana, abdulaziz f; tjendra, youley; millan, nicolas c; gomez-fernandez, carmen; cray, carolyn title: review of current advances in serologic testing for covid- date: - - journal: am j clin pathol doi: . /ajcp/aqaa sha: doc_id: cord_uid: kaku xd objectives: to examine and summarize the current literature on serologic methods for the detection of antibodies to severe acute respiratory syndrome coronavirus (sars-cov- ). methods: a literature review was performed using searches in databases including pubmed, medrxiv, and biorxiv. thirty-two peer-reviewed papers and preprints were examined. results: the studies included lateral flow immunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, and neutralizing antibody assays. the use of all major sars-cov- antigens was demonstrated to have diagnostic value. assays measuring total antibody reactivity had the highest sensitivity. in addition, all the methods provided opportunities to characterize the humoral immune response by isotype. the combined use of igm and igg detection resulted in a higher sensitivity than that observed when detecting either isotype alone. although iga was rarely studied, it was also demonstrated to be a sensitive marker of infection, and levels correlated with disease severity and neutralizing activity. conclusions: the use of serologic testing, in conjunction with reverse transcription polymerase chain reaction testing, was demonstrated to significantly increase the sensitivity of detection of patients infected with sars-cov- . there was conflicting evidence regarding whether antibody titers correlated with clinical severity. however, preliminary investigations indicated some immunoassays may be a surrogate for the prediction of neutralizing antibody titers and the selection of recovered patients for convalescent serum donation. the first cases of coronavirus disease (covid- ) were reported in wuhan, china, in december . as of june , , , , confirmed cases and , covid- -related deaths have been reported worldwide. in the americas, , , cases and , related deaths were confirmed. the first case of covid- in the united states was reported on january , , in washington. five months later, the united states has become one of the most affected regions with , , confirmed cases and , deaths. the rate of transmissibility, environmental stability of severe acute respiratory syndrome coronavirus (sars-cov- ), and the severity of disease in high-risk populations have all contributed to pandemic levels that challenge many health systems. consequently, understanding and implementing effective evidence-based testing is the cornerstone to correctly identify cases, predict clinical outcomes, and develop treatment strategies. reverse transcription polymerase chain reaction (rt-pcr) assays used to detect the presence of viral genetic material have become the gold standard of diagnosis. however, rna extraction • current peer-reviewed and non-peer-reviewed studies of serologic methods have diverse information. understanding the strengths and limitations of this literature is critical in the evolution of clinical applications of serologic testing. • the use of total antibody or simultaneous igg/igm measurements (regardless of method) significantly adds sensitivity to reverse transcription polymerase chain reaction testing protocols early post onset of symptoms and becomes the most accurate diagnostic test at later time points. • additional studies are needed to determine if antibody titers correlate with disease severity and whether certain antigen-specific antibodies determined by routine serologic testing may be surrogate markers for the presence of neutralizing antibodies and long-term immunity. the sars-cov- virus is genetically related to the severe acute respiratory syndrome coronavirus (sars-cov), which emerged in to and the middle east respiratory syndrome coronavirus (mers-cov), which was identified in . the genomic sequence of sars-cov- has approximately % homology to sars-cov and % homology with bat sars-like-covzxc . sars-cov- is a large ( - nm) positive-sense single-stranded rna virus with major structural proteins: nucleocapsid protein (np) holding the viral rna and envelope structural proteins including the spike protein (sp), envelope protein (ep), and membrane protein (mp). the np is the most abundant viral protein made and shed during infection. the sp consists of subunits referred to as s and s . s contains the receptorbinding domain (rbd) needed for binding to the host angiotensin-converting enzyme (ace ) receptor. s contains elements needed for membrane fusion. mp is the most abundant protein on the virion, and ep is the smallest protein and involved in the assembly and release of virions. the sp, rbd, and np proteins appear to be the main targets of the humoral immune response in coronavirus infections including sars-cov- and were the antigens used in the majority of the serologic assays examined in this literature review. [ ] [ ] [ ] studies varied widely in the detection of the different isotypes of antibody: igm, iga, and igg. many immunoassays, referred to as total antibody assays, were constructed to detect levels of all isotypes simultaneously. while a few studies presented quantitative data, the large volume of current literature described mostly qualitative and semiquantitative immunoassays. in the analysis of these studies, it is essential to examine the details of sample size and the patient population(s). , the design of a comprehensive validation study to address assay specificity requires the assessment of groups of samples: confirmed sars-cov- infected patients, confirmed healthy negative controls, and secondary sets of controls from patients with other viral infections and diseases. the latter group should include samples from patients with other human coronavirus infections. for sensitivity data, consideration of the timing of sample acquisition related to the onset of symptoms is crucial. samples acquired at very early times may represent a period where antibody levels are not present or are below the level of detection. finally, the performance of the assays will be affected by the technical component of the assays themselves. for lfia, reactivity is determined by visual inspection of bands on the immunochromatography paper present in single use devices. in the case of the semiautomated elisa and automated clia methods, the quantitation is provided by spectrometry and luminescence detection, respectively. it is important to note that many non-peer-reviewed manuscripts were included in this review; the reader is advised to search for final refereed versions that may have updated data and discussion points. the variable sensitivity and specificity of lfia have been the focus of the media and numerous studies due to the growing number of commercially available devices. , several investigations reported high sensitivity and specificity of these assays although many lacked information regarding the target antigens and well-defined negative control groups (ie, human coronavirus controls). ❚table ❚ summarizes the review of studies using lfia methods. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the experimental design and data of studies were judged to provide insight into lfia method for igm detection and % and % for igg detection, respectively. interestingly, the orient lfia, also constructed with the same antigens, showed a sensitivity of % for igm detection and % for igg detection. the assay specificity for each isotype was %, but it should be noted that the latter study tested a very small sample set of negative controls. another study utilized samples from both negative patients and patients with other viral infections and examined lfia devices. although no target antigen information was presented, the patient sample sets were robust. while evaluation of of the lfia demonstrated a poor performance, the remaining devices demonstrated % to % sensitivity and % specificity values. lastly, traugott et al presented data regarding lfia with specificity ranging from % %, but sensitivity was reported as poor at less than days post onset of symptoms ( %- %), poor to moderate at days to ( %- %), and excellent at or after day ( %). one study examined the accuracy of different lfia and elisa immunoassays using samples obtained from covid- patients at different time points of disease. specificity was assessed using samples from healthy subjects and samples from patients with other respiratory illnesses (not specified). the highest detection rate observed using lfia devices was obtained with combined igm and igg detection. accuracy peaked at days to post onset of symptoms with sensitivity results greater than % and specificity results greater than %. notably, this study reported several false-positive results obtained from banked specimens collected before the pandemic. these false-positive results suggest nonspecific binding by plasma proteins or cross-reactivity with antibodies produced during other viral infections. in a separate study of lfia devices, van elslande et al also used a secondary set of control samples to assess assay performance and reported a range of % to % specificity with the combination of positive igm and igg results but an % to % range of specificity when only igm or igg positive results were used. in their study, sensitivity was also reported to vary with the timing of sample acquisition post onset of symptoms. lfia is an appealing platform for sars-cov- testing with a relatively low cost per test and the advantage of potential use for point-of-care testing. lfia described in the literature thus far provided results for both igm and igg. the majority of the reports demonstrated a higher sensitivity and specificity with the detection of both isotypes, and a higher specificity was observed with the detection of the igg isotype over the detection of the igm isotype alone. antigens frequently used in lfia include np, rbd, and combined use of np and sp. a superior test accuracy based on the use of specific target antigen(s) was not evident at this time. studies utilizing samples obtained at various time points demonstrated the accuracy of lfia was optimal at approximately weeks post onset of symptoms. overall, the understanding of the limitations or advantages for the implementation of lfia-based assays is severely impacted by the presence of only a few properly designed studies in the current literature (table ) . publications that described elisa method validation often sought to address which antigens and antibody isotypes provided the best sensitivity and specificity ❚table ❚. , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in many of these studies, these calculations were reported solely to compare the performance of different antigens or the detection of different isotypes and not necessarily to propose the use of the assay for clinical implementation. additionally, as noted in table , many investigations utilized the elisa method to study the timeline of antibody expression in sars-cov- patients. to date, few investigations have focused on immunoassays utilizing the full sp antigen. , the sp antigen includes the rbd antigen as well as other essential peptides that may be the target of the humoral immune response. amanat et al generated different versions of the sars-cov- spike protein. one construct expressed the full length of sp and a second construct presented only the rbd. evaluating a small number of samples from rt-pcr-positive patients, reactivity to both antigens was excellent as demonstrated by optical density values. however, sp reactivity was significantly higher than that observed for rbd. igg , igm, and iga were the dominant isotypes observed in these patient samples. the investigators further reported that reactivity to a sample bank of sera obtained from patients with other human coronavirus infections was negligible. okba et al produced an array of different elisa to examine antibody reactivity to sp, s , np, and rbd antigens. a large cohort of samples was obtained from patients with other viral respiratory infections and was inclusive of banked samples from patients infected with various human coronaviruses. only samples from some sars-cov and mers-cov patients cross-reacted with ❚table ❚ studies reporting the use of enzyme-linked immunosorbent assay methods in the detection of antibody to sars-cov- the sars-cov- antigens. the rbd and np elisa were the most effective in the detection of antibodies in patients with mild infection. these antigens were examined in a second study using samples obtained from a cohort of patients obtained days or longer after symptom onset. the rbd elisa showed higher sensitivity ( % for igg and % for igm) compared to the np elisa ( % for igg and % for igm). okba et al also examined commercial elisa using the s antigen for the detection of iga and igg antibodies. both elisa showed some cross-reactivity with samples obtained from other human coronavirus positive patients. overall, the iga elisa displayed a higher sensitivity and the igg elisa displayed a higher specificity. lassaunière et al evaluated elisa constructed with rbd and s antigens using a control group of samples from patients with other non-sars-cov- viral infections. the performance of a sandwich elisa for the detection of total antibody reactivity to rbd was superior to that observed for the commercial igg and iga elisa using the s antigen. the elisa for total antibody detection provided the highest sensitivity and specificity of % and %, respectively. the specificity of the igg and iga assays were similar, but the iga elisa had a superior sensitivity of % vs % for igg. the authors reported a similar cross-reactivity of the commercial s elisa to other human coronaviruses as observed by okba et al. zhao et al also utilized a sandwich elisa to measure total antibody reactivity to rbd antigen as well as a rbd targeted elisa to measure igm reactivity alone and a np targeted elisa to detect igg reactivity. examining a cohort of over patient samples, the total antibody assay had superior performance vs the other assays with a sensitivity and specificity of % and %, respectively. while the specificity of the igm and igg elisa was %, the sensitivity of the igg elisa (np) was % compared to the igm elisa (rbd) at %. whitman et al evaluated different elisa and different lfia. in their study, the sensitivity for the elisa assays using samples obtained after day post onset of symptoms was greater than % and the specificity was greater than %. the agreement of the elisa with different lfia ranged from to %. overall, elisa assays with rbd and np antigens were the most frequently used in these early studies. although the sensitivity of the assays was affected by the timing of sample acquisition, a higher overall sensitivity was consistently observed with the use of total antibody detection. the elisa technique is labor intensive and unsuitable for point-of-care testing; however, it may offer the advantage of determining antibody titers and selective isotype detection. the importance of these options as clinical applications is unknown at this time. overall, while several elisa-based studies were robust in experimental design, many reports did not include examination of important control samples to best evaluate method specificity (table ) . the use of clia in the detection of sars-cov- antibody is of particular interest, as this method has excellent sensitivity with a high signal-to-noise ratio in the detection of other viral infections. clia uses recombinant antigen coated magnetic beads and a lumigen substrate with analysis on automated platforms. in current reports, np, combined np and sp, and rbd antigens were most commonly used ❚table ❚. , , , , [ ] [ ] [ ] [ ] [ ] [ ] many studies utilized clia to address differences in patient populations and time of onset of symptoms vs serologic results. long et al reported the use of the np and sp antigen combination in a study of samples from confirmed and suspect covid- patients. in their report, igg-positive serostatus approached % at days after onset of symptoms, and the median seroconversion for igm and igg was days. using a similar np and sp assay but a smaller cohort of positive and negative patients, jin et al reported a % sensitivity and % specificity for igm detection. however, for igg, the reported sensitivity was % and specificity was %. accuracy was higher for combined igm and igg testing with a posterior probability of greater than %; this was followed by igg alone ( %) and igm alone ( %) and was corroborated in a separate study. five studies warranted a closer examination, as each included control samples from patients with non-sars-cov- infections. lin et al used np antigen to detect igm and igg reactivity. sensitivity for both igm and igg assays was reported as %, and the specificity was % for igm detection and % for igg detection. ma et al compared rbd and np antigen targeted clia and reported a higher sensitivity and specificity when using the rbd antigen. a superior sensitivity and specificity were also demonstrated with the detection of iga over igm or igg detection alone. in addition, the use of iga in tandem with the detection of either igm or igg increased assay sensitivity and specificity. geurtsvankessel et al examined a total antibody assay for s /s reactivity and reported a sensitivity of % and a specificity of %. bryan et al validated an igg antibody assay using np antigen with a specificity of % based on the analysis of banked samples originally submitted for hsv testing. this study also included samples from confirmed covid- patients. the assay sensitivity increased from % at day to % at day . lastly, zhang et al used a large cohort of samples and showed an area under the curve of . and . for igm and igg, respectively, using a combined np and sp clia. the clia assay is traditionally considered a very sensitive method with the capability to detect low levels of antibodies. clia assays are automated and allow for a high throughput of samples. in the current literature review, rbd and np were the most commonly targeted antigens, and the studies detected igm and igg isotypes as well as total antibody reactivity. the use of rbd antigen and igg detection produced the highest accuracy data among studies. it is important to consider that the variation in performance among different platforms may have been related to sample acquisition timing, which was not consistently reported in the studies. rt-pcr testing for viral detection is a frontline tool to detect patients with suspected sars-cov- infection. however, several studies showed that the sensitivity of rt-pcr testing decreased over time post onset of symptoms and that this change was observed to be concurrent with the increasing sensitivity of antibody detection methods. guo et al evaluated the use of a np targeted elisa using igm, iga, and igg detection in a sample set of rt-pcr confirmed cases and suspected cases with negative rt-pcr. early in disease ( days post onset of symptoms), igm detection was positive in % of suspected cases with negative rt-pcr results and % of the rt-pcr confirmed cases. overall, the sensitivity of rt-pcr testing alone was %; however, when using rt-pcr in combination with the elisa, the total sensitivity increased to %. similar results were observed in another study where combined detection of igg and igm identified over % of suspected cases that were negative by rt-pcr testing. as the course of the disease progresses, the utility of serology increases as well. zhao et al evaluated the sensitivity of a total antibody elisa to the rbd antigen. in the early ( - ) days post onset of symptoms, the sensitivity was % for rt-pcr; this increased to % when rt-pcr was combined with elisa testing. the sensitivity for rt-pcr decreased to % at days to while the elisa sensitivity increased to %. at this time point, the use of both methods resulted in a sensitivity of %. lastly, on days to , rt-pcr sensitivity was reported as %, and the numerous studies indicated that antibody responses may vary according to disease severity, and some reports proposed that monitoring titers may be applied in clinical practice to guide earlier aggressive treatment. traditionally, the hallmarks of a humoral immune response include the early expression of igm isotype, which then matures into igg isotype expression. notably, many reports of sars-cov- patients indicated that igm expression was observed concurrently with igg expression. long et al conducted a large multicenter study using an np and sp targeted clia. the median seroconversion of both isotypes was recorded at day . in addition, a different np and sp targeted clia studied by suhandynanata et al showed a median seroconversion on days to for igm and igg. the goal of several elisa studies was to define the period of seroconversion, and all demonstrated the higher sensitivity of the assays by the second week post onset of symptoms. , , , zhao et al reported that the median seroconversion time of igm and igg to rbd antigen was days and , respectively. guo et al reported the median appearance of igm and iga at day and igg at day post symptom onset. their study used serial samples obtained from the same patients and an elisa for antibody detection to the np antigen. xiang et al also used a np targeted elisa and reported the median appearance of antibody at day post onset of symptoms. overall, while these results are similar to that reported in a review of serologic testing for mers-cov and sars-cov, they may have been affected by choice of target antigens, the various immunoassay kits, and the level of detail of case history used to categorize the time of sample acquisition post onset of symptoms. few studies have examined the differences in titers by case severity classification, and there was no consensus among the findings. tan et al reported that the igg and igm detection occurred earlier for severe cases compared to nonsevere cases (p < . ). higher titers were also observed in the former group. wang et al evaluated the titers of igm and igg in confirmed cases of which of the cases had mild to moderate disease and patients died. levels of igm were significantly higher in deceased patients (p = . ). however, no significant correlation was observed between case outcome and igg titers. in contrast, a large study of confirmed covid- patients reported elevated igm titers but lower igg levels in critical cases. ma et al evaluated iga titers in a cohort of patients and observed a significant correlation with disease severity (p < . ) and peak levels of iga to days after symptom onset. sun et al reported significantly higher igg titers to sp antigen in non-icu patients, whereas igg titers to np antigen were elevated in icu patients. lastly, to et al, using np and rbd targeted elisa observed no correlation between titers and severity. previously, quantification performed by elisa and neutralizing antibody assays showed that individuals over years of age had higher antibody titers than young healthy adults with human coronavirus (non-sars-cov- ) infection. notably, in a study of patients recovered from covid- , samples obtained from elderly and middle-aged individuals had higher titers of sp reactive antibody compared to young adults. thus, while increased age is often associated with severe sars-cov- disease and poor outcomes, high levels of antibody production do not appear to be detrimental given these preliminary reports. there were limited reports of testing approaches for the detection of asymptomatic individuals. much of the current literature was weakened given the limitations in the experimental design of studies used to validate the serologic methods. for example, paradiso et al used an lfia for igm and igg detection to screen health care workers. previously, this group reported an overall % sensitivity and % specificity obtained during the validation of this lfia. it was acknowledged that the reduced sensitivity was related to the selection of validation samples from patients early after the onset of symptoms. in the health care worker screening study, only . % of the cases were seropositive; these cases tested negative by rt-pcr. all the cases were positive for igm, and case was positive for both igm and igg. another study from the same investigation group examined asymptomatic cases presenting to the emergency department. twenty nine percent of the cases were positive by rt-pcr testing and % of the cases were positive by lfia testing. as the lfia was not fully validated, it is problematic to apply these results to develop screening strategies for asymptomatic patients. bendavid et al used lfia in a study of , individuals from santa clara county, ca, and found an unadjusted antibody prevalence of . % ( % confidence interval [ci], . %- . %). the weighted population prevalence was . % ( % ci, . %- . %). the kit was validated using samples from confirmed negative and positive sars-cov- patients with a combined igm/igg sensitivity of % and specificity of %. no secondary controls with non-sars-cov- viral infections were included in the validation study. as a result, this study likely overestimated the specificity of this lfia. zhao et al used a lab-developed elisa for the detection of igg antibody to s antigen with reported excellent sensitivity and specificity, as determined by the use of samples from symptomatic hospitalized patients and healthy individuals. in a subsequent study, the same elisa was examined in asymptomatic health care workers. ten percent of health care workers had seropositive results. samples from a small cohort of close contact individuals were also examined. all subjects tested negative by rt-pcr, but individual was seropositive. as with the study by bendavid et al, the specificity of the elisa assay was not fully assessed; this complicates the interpretation of these results. a report of serologic testing of a family cluster of individuals provided interesting data regarding the complexity of test sensitivity. two family members were hospitalized with sars-cov- symptoms. one individual tested positive by rt-pcr on presentation, and the second patient tested negative twice before obtaining a positive result on day . both individuals were positive for igm reactivity by elisa. the remaining family members remained asymptomatic. however, of tested positive for igm reactivity, and of were positive by rt-pcr testing at later dates. the fourth individual remained clinically asymptomatic and was negative on all testing. implementation of serology testing to screen the general population and asymptomatic health care workers is currently of significant interest. nonetheless, the available evidence is limited to support its use in these scenarios. with unclear population prevalence and the use of immunoassays that are not fully validated, the limitations of test sensitivity and specificity in the evaluation of asymptomatic individuals may be difficult to overcome. many studies focused on the detection of neutralizing antibodies for the potential use as a predictor of clinical outcome and in the identification of convalescent serum for use in a treatment strategy. several of these studies evaluated the correlation of data from elisa using rbd, s , s , and np antigens to the presence of neutralizing antibodies. wu et al studied neutralizing antibodies using a pseudotyped-lentiviral-vector-based assay using plasmids for sars-cov and sars-cov- sp protein. titers of neutralizing antibodies were reported as id (highest dilution resulting in % reduction of luciferase luminescence). parallel elisa for sars-cov and sars-cov- rbd, s , and s were conducted. the study samples were obtained from patients with mild to moderate symptoms who recovered from covid- . levels of neutralizing antibodies were low (id , < ) before day and appeared at to days after the onset of symptoms. titers persisted at similar levels on repeat testing weeks later. the levels of neutralizing antibodies in the patients were categorized as follows: low to mid in % (id , - ), mid to high % (id , , - , ), and high in % (id , > , ). samples from % of patients had very low levels (id , < ) and included samples from patients who had reactivity below the level of detection (id , < ). results obtained from elisa using rbd, s , and s antigens correlated moderately with neutralizing antibody titers. correlation coefficients ranged from . to . (p < . ). ni et al examined rbd and np elisa in tandem with the pseudovirus neutralization test in cohorts of patients tested at the time of discharge and weeks later. significant levels of igm and igg were present at both time points as determined by elisa methods. notably, while neutralizing antibody was detected at the time of discharge, levels were lower in of patients weeks later. okba et al compared the results of iga and igg elisa for rbd, s , and np to data obtained from a plaque reduction neutralization assay (using vero e cells) and reported a significant correlation (r = . , p < . ) with all elisa. the strongest correlation was observed with iga reactivity (r = . , p < . ). it is important to note that various elisa in this study were validated with a large secondary sample set obtained from patients with other respiratory viral infections ( table ) . the identification of novel antigenic epitopes that may be important in the humoral immune response was the focus of a limited number of studies. poh et al evaluated the reactivity of convalescent serum samples. six samples showed significant neutralization activity (id , > ) and were selected for further analysis. additional experiments to characterize antigen targets showed that the s and s peptides (within the sp) provided the strongest reactivity by elisa methods, and these results correlated with neutralizing activity. jiang at al developed a microarray of sars-cov- proteins to profile igg/igm responses of convalescent sera. all patients had combined igm/igg responses to np and s antigens but not to s antigen. furthermore, a significant number of samples were positive for anti-orf b and anti-nsp antibodies. these peptides may represent good predictors for immunity and possible therapeutic targets. overall, understanding the utility of routine serologic methods (ie, elisa, clia) in the prediction of convalescence is numerous immunoassays for the detection of antibodies to sars-cov- are rapidly emerging. these immunoassays have the potential to improve the diagnosis and monitoring of infection in different scenarios. , [ ] [ ] [ ] published and non-peer-reviewed studies varied dramatically in the definition of patient groups, time of sample acquisition, sample size, and inclusion of relevant control sera from patients with non-sars-cov- respiratory infections. these are all critical variables that will affect the sensitivity and specificity of the different immunoassays and should be a fundamental part of the review of any validated assay. determining the final roles of lfia and elisa immunoassays in sars-cov- testing and research is difficult at this time, as this is limited by the pitfalls in the experimental design of many of these foundational studies. further well-validated assays determined through studies with rigorous experimental design are needed. in the current literature, the study methods were heterogeneous regarding the specific antigens used and the different isotypes of antibodies measured; few studies compared these variables simultaneously. the most common antigens used in the assays included rbd, s , and np. there was preliminary evidence that the use of particular target antigens may provide value to increase the sensitivity of antibody detection methods. however, additional comprehensive studies need to be conducted to reproduce this early reported data. in addition, antigenic epitopes within the sp appeared to be important in the immune response and thus, this region remains of interest for future detailed studies. there is compelling evidence that using total antibody or combined igg/igm detection offered the highest sensitivity of detection. data from preliminary studies indicated that additional investigations should examine the clinical correlation of different isotypes and titers to disease severity. it is also clear that the timing of sample acquisition is a crucial determinant of test accuracy, although this important information was not always clearly presented in the current literature. the earliest positive results were reported by day post onset of symptoms, and accuracy peaked by the second week of symptoms. early in the course of the disease, when rt-pcr sensitivity was reported as % to %, the concomitant use of serologic tests significantly added sensitivity with consistently reported values over %. moreover, after to days post onset of symptoms, the sensitivity of rt-pcr dropped significantly while serology testing reached its peak. as fully validated methods become commercially available, serology methods may be utilized as an adjunct tool to rt-pcr testing protocols in patients with suspected infection. nearly all of the current literature focused on the results obtained using serologic testing in symptomatic patients. it will be essential to define antibody responses in individuals with subclinical and mild disease before immunoassays can be used in screening and seroprevalence studies. at the time of this writing, while the potential importance of quantitative titers was raised in the literature, testing platforms available to provide this information are largely absent from clinical laboratories. lastly, defining convalescence and the presence of long-lasting immunity to sars-cov- after infection or future vaccination is important. additional studies are needed to determine if ease-of-use assays of antibody detection and quantitation will compare well with traditional neutralizing antibody assays. while serologic testing continues to hold promise for various applications, there are still knowledge gaps that must be clarified to give meaningful recommendations for its use in different clinical scenarios. genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan covid- ) situation report- first case of novel coronavirus in the united states serological approaches for covid- : epidemiologic perspective on surveillance and control. front immunol virological assessment of hospitalized patients with covid- test performance evaluation of sars-cov- serological assays development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis diagnostic value and dynamic variance of serum antibody in coronavirus disease potential rapid diagnostics, vaccine and therapeutics for novel coronavirus ( -ncov): a systematic review immunological assays for sars-cov- : an analysis of available commercial tests to measure antigen and antibodies diagnostic testing for severe acute respiratory syndrome-related coronavirus- in vitro diagnostic assays for covid- : recent advances and emerging trends potent neutralizing antibodies in the sera of convalescent covid- patients are directed against conserved linear epitopes on the sars-cov- spike protein serological assays for emerging coronaviruses: challenges and pitfalls lessons learned from a rapid systematic review of early sars-cov- serosurveys antibody tests in detecting sars-cov- infection: a meta-analysis fast, portable tests come online to curb coronavirus pandemic evaluation of nine commercial sars-cov- immunoassays evaluation of enzyme-linked immunoassay and colloidal gold-immunochromatographic assay kit for detection of novel coronavirus (sars-cov- ) causing an outbreak of pneumonia (covid- ) antibody detection and dyanmic characteristics in patients with covid- clinical meanings of rapid serological assay in patients tested for sars-co rt-pcr serology characteristics of sars-cov- infection since the exposure and post symptoms onset diagnostic indexes of a rapid igg/ igm combined antibody test for sars-cov- . medrxiv serological immunochromatographic approach in diagnosis with sars-cov- infected covid- patients evaluation of the auxiliary diagnostic value of antibody assays for the detection of novel coronavirus (sars-cov- ) brief communication towards the next phase: evaluation of serological assays for diagnostics and exposure assessment evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-sars-cov- antibodies performance of sars-cov- antibody assays in different stages of the infection: comparison of commercial elisa and rapid tests diagnostic performance of rapid igg/igm antibody tests and the euroimmun iga/igg elisa in covid- patients lateral flow assays a serological assay to detect sars-cov- seroconversion in humans profiling early humoral response to diagnose novel coronavirus disease (covid- ) antibody responses to sars-cov- in patients of novel coronavirus disease severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease patients. emerg infect dis evaluation of nucleocapsid and spike protein-based elisas for detecting antibodies against sars-cov- a preliminary study on serological assay for severe acute respiratory syndrome coronavirus (sars-cov- ) in admitted hospital patients characterization of anti-viral immunity in recovered individuals infected by sars-cov- . medrxiv temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study early detection of sars-cov- antibodies in covid- patients as a serologic marker of infection kinetics of sars-cov- specific igm and igg responses in covid- patients. emerg microbes infect serological detection of -ncov respond to the epidemic: a useful complement to nucleic acid testing antibody responses to sars-cov- in patients with covid- evaluations of serological test in the diagnosis of novel coronavirus (sars-cov- ) infections during the covid- outbreak covid- diagnosis and study of serum sars-cov- specific iga, igm and igg by a quantitative and sensitive immunoassay longitudinal monitoring of sars-cov- igm and igg seropositivity to detect covid- performance characteristics of the abbott architect sars-cov- igg assay and seroprevalence in optimize clinical laboratory diagnosis of covid- from suspect cases by likelihood ratio of sars-cov- igm and igg antibody clinical significance of igm and igg test for diagnosis of highly suspected covid- infection viral kinetics and antibody responses in patients with covid- elevated serum igm levels indicate poor outcome in patients with coronavirus disease pneumonia: a retrospective case-control study detection of igm and igg antibodies in patients with coronavirus disease antibodies to coronaviruses are higher in older compared with younger adults and binding antibodies are more sensitive than neutralizing antibodies in identifying coronavirus-associated illnesses neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications rapid serologic test have a role in asymptomatic health workers covid- screening covid- antibody seroprevalence in significance of serology testing to assist timely diagnosis of sars-cov- infections: implication from a family cluster antibody testing for covid- : can it be used as a screening tool in areas with low prevalence? global profiling of sars-cov- specific igg/ igm responses of convalescents using a proteome microarray the role of antibody testing for sars-cov- : is there one? double-edged spike: are sars-cov- serologic tests safe right now? serology assays to manage covid- key: cord- -u y ttw authors: chen, keyan; zhao, kui; song, deguang; he, wenqi; gao, wei; zhao, chuanbo; wang, chengli; gao, feng title: development and evaluation of an immunochromatographic strip for rapid detection of porcine hemagglutinating encephalomyelitis virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: u y ttw background: the incidence of phe among pigs in many countries is on the rise, and it has caused great economic losses to the pig industry. therefore, the development of a sensitive, specific, and easily-performed assay is crucial for the rapid detection and surveillance of phe-cov infection and transmission. results: an immunochromatographic strip was developed for the detection of phe-cov. the colloidal gold-labeled mab d was used as the detection reagent, and the mab e and goat anti-mouse igg coated the strip's test and control lines, respectively. the immunochromatographic strip was capable of specifically detecting phe-cov with a ha unit of within min. storage of the strips at room temperature for months or at °c for months did not change their sensitivity or specificity. using rt-pcr as a reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be % and . %, respectively. there was an excellent agreement between the results obtained by rt-pcr and the immunochromatographic strips (kappa = . ). additionally, there was a strong agreement between the sandwich enzyme-linked immunosorbent assay (elisa) and immunochromatographic strips (kappa = . ). when the immunochromatographic strips were used for diagnosing phe-cov infection in the jilin province, the phe-cov-positive rate ranged from . % in the jilin district to . % in the songyuan district. conclusions: based on its high specificity, sensitivity, and stability, the immunochromatographic strip would be suitable for on-site detection of phe-cov for surveillance and epidemiological purposes. porcine hemagglutinating encephalomyelitis (phe) is an infectious disease primarily affecting pigs under weeks of age, causing vomiting, exhaustion, and obvious neurological symptoms [ ] . the mortality rate varies between - % [ ] . the disease is caused by phe coronavirus (phe-cov), which comprises a single strain and is the only known neurotropic cov affecting pigs [ ] . phe-cov was isolated for the first time in vivo from breastfeeding pigs suffering from encephalomyelitis in canada [ ] . in , an antigenically identical virus was isolated in england from suckling pigs presenting with anorexia, depression, and vomiting, but without clear signs of encephalomyelitis [ ] . surviving animals remained stunted in growth, and the condition was therefore called 'vomiting and wasting disease' (vwd). in china, phe-cov was first reported in , and it was later described both on the mainland and in the taiwan province [ , ] . the infection has also been reported in the major pig raising countries of europe, asia and north america, where it seemed to be endemic with no clinical outbreaks [ ] [ ] [ ] . currently, the incidence of phe among pigs in many countries is on the rise, and it has caused great economic losses to the pig industry. in , phe-cov was isolated from newborn and early-weaned pigs with vomiting and posterior paralysis on a canadian farm [ ] . in , this disease occurred twice in pig farms in the jilin province, with incidence rates among -dayold piglets as high as % and reported mortality rates ranging from % and % [ ] . in august , some pig farms in argentina experienced outbreaks of this disease, leading to deaths, morbidity rates as high as . % and a mortality rate of . % [ ] . therefore, early detection and control of phe-cov infection would be significant both from an economic and health viewpoint. at present, various laboratory methods are available for the detection and surveillance of phe-cov, including virus isolation [ ] , hemagglutination/hemagglutination inhibition (ha/hi) tests [ ] , immunohistochemistry (ihc) assays [ ] , and molecular tools such as nestedpolymerase chain reaction (nested pcr) and reverse transcriptase-polymerase chain reaction (rt-pcr) that enable detection of specific cov rna sequences from infected tissues [ , ] . however, these detection methods are laborious, time-consuming, and require laboratory procedures or special equipment, making them unsuitable for on-site inspection. current detection strategies are also insufficient to meet the needs of emergent management after phe outbreaks, thus restricting their application to veterinary clinical diagnosis. therefore, the development of a sensitive, specific, and easilyperformed assay is crucial for the rapid detection and surveillance of phe-cov infection and transmission. an immunochromatographic assay is a unique immunoassay developed in in which a cellulose membrane is used as the carrier and a colloidal gold-labeled antigen or antibody is used as the tracer [ ] . it combines the immune response with chromatographic theory in a test that is simple and quick, providing specific, sensitive, and clear results with simple or no instrumentation. thus, it is suitable for testing clinical samples on site, in clinics and in locales where medical treatment and laboratory facilities are not available. an immunochromatographic assay has been widely used for animal quarantine and medical reasons [ , ] . ( ) ( ) ( ) in this study, an immunochromatographic strip with high sensitivity and specificity was developed for the detection of phe-cov, combining monoclonal antibody (mab) and colloidal gold immunochromatography (gica), and the resulting product is suitable for the surveillance of phe-cov. balb/c mice were purchased from the laboratory animal center of general hospital of shenyang military area command in china. animal immunization experiments were performed in accordance with the guidelines for animal experimentation of the general hospital of shenyang military area command. the animals were maintained under pathogen-free conditions. the field samples (brain tissue samples from deceased piglets and nasal cavity or throat swabs from ill piglets) were provided by jilin center for disease control and prevention in order to perform general surveillance on the phe-cov infection. the virus strain used in this study was phe-cov- n (genbank accession no. ay ). the viruses were propagated and passaged in porcine kidney epithelial (pk)- cells [ ] , and purified by sucrose density gradient centrifugation. the viruses were stored at − °c until needed. the procedure employed for the production of the monoclonal antibodies (mab) against the he and s proteins of phe-cov were based on the protocol of kohler and milstein [ ] ,and the recombinant s protein of phe-cov were produced as previously described [ ] . briefly, balb/c mice at weeks of age were immunized subcutaneously with . ml ( units of ha) of phe-cov virus purified by sucrose density gradient centrifugation and emulsified : with freund's complete adjuvant (sigma, st. louis, mo). the mice were boosted three times with the same amount of antigen in % freund's incomplete adjuvant (sigma, st. louis, mo) every weeks, followed by an intraperitoneal injection of . ml phe-cov. three days later, their splenic mononuclear cells were isolated and fused with murine myeloma cells (sp / ) using % polyethylene glycol (peg)- (sigma, st. louis, mo). the hybridomas were generated through the selection of hat (sigma, st. louis, mo) medium and screened using a recombinant s protein-based enzyme-linked immunosorbent assay (elisa) and hi assays. the positive hybridoma cells were cloned by a limiting dilution to obtain four strains of secretory positive antibody hybridoma cells. the stable hybridoma clones of the immunoglobulin g mab d and e were injected into balb/c nude mice and the mouse abdominal dropsy was purified by sequential precipitation with caprylic acid [ ] and ammonium sulfate and dialyzed against phosphate buffer ( . m, ph . ) at °c. their purities were used in western blot analysis. the sandwich enzyme-linked immunosorbent assay (elisa) the elisa assay was based on the mab of anti-phe-cov for the detection of phe-cov. the purified mab d of phe-cov, diluted to a final concentration of . μg/ml with carbonate buffer ( . m, ph . ), was used as the capture antibody in elisa for coating well microtiter plates (costar corning inc., corning, ny.) with μl per well. the plate was incubated overnight at °c. the plates were washed times with pbs-tween (pbst), and nonspecific binding sites were blocked with % (w/v) bovine serum albumin (bsa) (sigma, st. louis, mo) in pbst ( μl/well) for h at °c. after washing the plates, the samples of the homogenate grind suspension or the supernatant of the cell cultures were diluted : with pbst containing % (w/v) bsa, and μl was added into the coated well. the phe-cov and pbst standards were added as positive and negative controls, respectively, and the samples were incubated for . h at °c. after washing the plates, the mab e ( μg/ml) was added to each well at μl per well, the samples were incubated for . h at °c, and the plates were washed times with pbst. horseradish peroxidase-conjugated goat anti-mouse igg (sigma, st. louis, mo) was added into each well at a working concentration of : , incubated for h at °c, and the plates were washed times with pbst. one hundred microliters of substrate solution o-phenylenediamine (opd containing h o ) was added to each well, and the color reaction was developed in the dark for minutes at room temperature. the reaction was then stopped with m of h so μl/hole, and the absorbance was read at od with a universal microplate reader (bio-rad laboratories inc., richmond, ca). colloidal gold was prepared as previously reported [ ] , with minor modifications. briefly, ml of . % (wt/ vol) haucl in doubly distilled water in a -ml siliconized conical flask was heated to boiling in a microwave oven, and then . ml % trisodium citrate was added to the solution. after the colloidal gold solution was boiled for an additional min, it turned a cardinal red color and was allowed to cool gradually and was stored at °c in a dark-colored glass bottle. the ph of the colloidal gold was adjusted to . with % potassium carbonate (wt/vol), followed by sub-installing tubes with ml colloidal gold solution each, to which were added μg, μg, μg, μg, μg, μg, μg, μg, or μg mab d . tubes were shaken for min, and then . ml of % nacl solution was added to each tube and mixed. two hours later, results were recorded. the mab d antibody ( . μl, . mg/ml) was added dropwise into ml of colloidal gold solution on a magnetic stirring apparatus for min, stood at °c for min, and then ml % (wt/vol) bsa was added to block excess reactivity of the gold colloid. the mixture was then stirred on the magnetic stirring apparatus for an additional min and stored at °c for h. after the mixture was centrifuged at , × g at °c for min, the supernatant was centrifuged at , × g at °c for min, and the resulting conjugate pellet was suspended in mm borax buffer (ph . ) containing % (wt/vol) bsa and . % nan . the sizes and shapes of the unconjugated colloidal gold and colloidal gold conjugated to antibodies were characterized using transmission electron microscopy. the immunochromatographic test device consisted of a plastic support to which an immunochromatographic strip composed of a sample pad, a conjugate pad, a nitrocellulose (nc) membrane, and an absorbent pad were mounted. the colloidal gold-labeled mab d solution was dispensed onto glass fiber paper at a speed of μl per cm using an xyz dispense workstation (beckman, usa), and the conjugate pad was dried under vacuum. the mab e ( . mg/ml) or the goat anti-mouse antibody ( mg/ml) was dispensed at the test or the control line on the nc membrane, at a rate of . μl/cm and a speed of cm/s using xyz- , and the membrane was dried under vacuum and stored at °c. the sample pad, pretreated conjugate pad, nc membrane, and absorbent pads were glued together on a support board and assembled into a test strip plate. then the strip plate was cut into -mm-wide pieces using an ln- cutting machine (beckman, usa). in addition, a sample pad completed the assembly with . -to . -mm overlap sequentially by mounting on the conjugate plastic card (figure ). the strips were stored in dry conditions at °c until required. during testing, approximately μl supernatant of the antigen samples were added to the sample hole of the immunochromatographic strip, and this liquid rapidly diffused into the conjugate pad. for positive samples, phe-cov was captured by mab d through percolation in the nitrocellulose membrane, while in the test line, the formation of a colloidal gold mab d -phe-cov-mab e complex caused the appearance of a red line; for negative samples, the test line zone did not form a colloidal gold mab d -phe-cov-mab e complex, and therefore no red line was evident. as a control, both the negative and positive sample control lines turned red by forming a colloidal gold mab d -goat anti-mouse igg complex; otherwise, the test results were invalid. the immunochromatographic strip is illustrated in figure . to evaluate the specificity of the immunochromatographic strips, phe-cov and other viruses, including tgev, pedv, prv, hcv, bcv, mhv and hcv-oc were simultaneously tested using the test strips. the virus samples of phe-cov ( units of ha) was diluted to : , : , : , : , : , : , : , : , and : with mmol/l borate buffer solution (ph . ), and these samples were simultaneously tested using the immunochromatographic strips to evaluate the strips' sensitivity. the same procedure was repeated three times by different operators. to determine the reproducibility of the strip, the same batch and five different batches of the immunochromatographic strip were used to detect phe-cov. all samples were repeated times and the data were collected. the immunochromatographic strips were stored at room temperature or at °c and used for testing positive ( ha units) and negative samples every months, to determine the stability of the test. to evaluate the correlation between the immunochromatographic strip and reference methods, a total of brain tissue samples were collected from deceased piglets with suspected phe-cov infection from several pig farms in the jilin province. a mg of brain tissue was measured and homogenized, then suspended : with pbs, followed by centrifuging at × g for min. the supernatant was collected for testing by elisa, the immunochromatographic strips, rt-pcr. the detection assay of phe-cov by rt-pcr were established as previously described [ ] . the kappa statistic [ ] and < . represented excellent agreement, good to fair agreement, and poor agreement, respectively [ ] . the immunochromatographic strips were applied to the diagnosis of phe-cov infection in the field. a total of nasal cavity or throat swabs were collected from approximately -to -week-old piglets, with vomiting and neurological symptoms consistent with phe-cov infection, from herds in the changchun, jilin, songyuan, siping, baishan, and liaoyuan districts of the jilin province, china in . the mab of anti-phe-cov were used in western blot analysis to identify the d mab, which recognizes the he protein, and the e mab, which recognizes s protein ( figure ) and was stored at − °c until use. the sandwich enzyme-linked immunosorbent assay (elisa) thirty negative and seven positive virus samples were detected by elisa (figure -a) . the threshold value of . was identified using a roc curve (figure -b ). an od > . indicated a positive result and ≤ . a negative result. additionally, phe-cov was diluted to ng/ml, ng/ml, ng/ml, ng/ml, ng/ml, ng/ml, . ng/ml, . ng/ml, and . ng/ml with pbs, which was detected by elisa. the standard curve was drawn by curveexpert software with phe-cov concentration on the x-axis and the average od value on the y-axis (figure -c) . the linear regression constant r was . , and the linear detection range was . - ng/ml. good linearity was observed, as the lowest detection limit was . ng/ml. to determine the optimal concentration of monoclonal antibody with colloidal gold, different concentrations of d mab were added in tubes with ml colloidal gold solution each. as shown in figure . the color of the tubes without sufficient protein changed from red to blue, whereas the color of the tubes remained unchanged if the amount of protein exceeded the minimum needed. the amount of protein in the lowest colloidal gold concentration tube, which remained red, was increased by % when the optimal antibody concentration was reached. the appearance of the colloidal gold solution was deep red and translucent, with a bright color. a bright band was visible when the test card faced the sun. the colloidal gold particles were consistent in size and uniformly distributed, with a mean diameter of about nm (figure -a) when observed under a transmission electron microscope. the colloidal gold-labeled mab d was observed with the transmission electron microscope to be evenly distributed, and the particle size was consistent. the colloidal gold particles had a visible clear space around the halo, then the surface of proteins and other adsorbed particles (figure -b). to determine the specificity of the immunochromatographic strip, phe-cov was simultaneously tested with tgev, hcv, pedv, prv, bcv, mhv and hcv-oc using the immunochromatographic strips. clearly, while each of the other samples resulted in one strong band on the control line, phe-cov displayed an additional band on the test line of the immunochromatographic strips ( figure ) . phe-cov was detected at different dilution strengths and repeated in triplicate with the immunochromatographic strip. the results are shown in figure . when the antigen were diluted from : to : (from to ha units, respectively), the reaction on the test and control lines were observed. thus, the sensitivity of the phe-cov colloidal gold immunochromatographic test was determined to be units of ha antigens. this procedure was repeated five times to detect the phe-cov of ha units at by the same batch and then by five different batches to determine reproducibility. these results (table ) showed little variability within the same batch or in different batches, demonstrating that the detection of the virus is highly reproducible. in addition, the stability of the immunochromatographic strip under various storage conditions was determined. the results are shown in table , they were stored at room temperature for months with a reduction in sensitivity of % and at °c for more than months with no loss of sensitivity or specificity for the detection of phe-cov. to evaluate the agreement between the immunochromatographic strip and reference methods, the clinical samples from deceased piglets were detected by sandwich elisa, rt-pcr, and the immunochromatographic figure the virus samples were tested by enzyme-linked immunosorbent assay (elisa). sample numbers - were negative and - were positive. a, a histogram of elisa results from both positive and negative virus samples; b, spss . for windows was used to create a roc curve to evaluate the threshold value; c, the standard curve was drawn by curveexpert software with the phe-cov concentration on the x-axis and the average od value on the y-axis. figure the optimal antibody concentration. the colloidal gold solution was made with different concentration of mab d . the color of the tubes without sufficient protein changed from red to blue, and when the amount of protein added to the tube exceeded the minimum needed, the color remained unchanged. table . of the clinical samples, were positive and were negative using the immunochromatographic strip, while were positive and were negative using elisa and rt-pcr. thus, the specificity and sensitivity of the immunochromatographic strip, as compared with elisa and rt-pcr, were % and . %, respectively. there were excellent agreement (kappa = . ) between the immunochromatographic strip to elisa and rt-pcr. the immunochromatographic strip was next applied to the diagnosis of phe-cov infection in the jilin province. characterization of these samples revealed that out of swab samples were positive for phe-cov infection (table ) . notably, the positive rate ranged from . % in the jilin district to . % in the songyuan district. these findings suggest that phe-cov is commonly transmitted in the jilin province, and the immunochromatographic strip can be used for the detection and differentiation of phe-cov in clinical diagnosis. phe-cov belongs to group of the coronaviridae family, a group characterized by the presence of a gene encoding the he protein [ ] . nucleotide sequence analysis of the region covering the s probe revealed . % nucleotide sequence homology to bcv and . % figure the colloidal gold and gold-labeled proteins were observed by electron microscopy. the results of transmission electron microscope imaging of colloidal gold and gold-labeled proteins. a: colloidal gold (× , ), b: gold-labeled mab d (× , ). homology to hcv-oc [ ] . although phe-cov causes two distinct clinical syndromes in pigs, only one serotype of the virus is known to exist. outbreaks of phe-cov-associated disease are now on the rise in many countries, inflicting considerable economic damage to the pig industry. remarkably, these recent isolates showed a high degree of genetic and antigenic homology with the reference strain phe-cov- n [ ] . currently, immunohistochemistry (ihc) for phe-cov, or molecular tools such as rt-pcr, enable the detection of specific cov rna sequences from infected tissues [ , ] . however, these detection techniques usually require special primers, a working laboratory, skilled technicians, and specialized equipment, making rapid and on-site detection of viruses in the field difficult. therefore, the development of a sensitive, specific, and easily performed assay is crucial for the rapid detection and surveillance of phe-cov infection and transmission. colloidal gold immunochromatographic assay (gica) is convenient, is rapid, has high specificity and sensitivity, and can be performed either without instruments or with only a simple instrument, making it suitable for clinical diagnosis and drug testing purposes in almost any context [ ] [ ] [ ] . colloidal gold is a negatively charged hydrophobic rubber particle that maintains a stable colloidal system through electrostatic repulsion [ ] . the key to successful colloidal gold labeling lies in the preparation of a homogeneous mixture of monodispersed colloidal gold particles [ ] . the microwave oven method was chosen in this experiment because it provides even heating without manual shaking, creating colloidal gold particles that are consistent in size and uniformly distributed, with a mean diameter of about nm under transmission electron microscope. in the preparation of gold markers, proteins and gold glue particles should also be present in an appropriate ratio. if too many antibodies are added, the amount of free antibodies instead of gold-labeled antibodies will increase, which will result in a decreased titer of gold-labeled mab d , affecting the test's sensitivity. conversely, if too few antibodies are added, it is easy for the colloidal particles to aggregate and precipitate, especially during purification by ultracentrifugation. therefore, it is essential to add sufficient colloidal gold antibodies to maintain stability. the specificity and sensitivity of the immunochromatographic strip are largely dependent on the following factors. first, the quality of the mabs used in the strip test is crucial for the specificity and sensitivity of the strip. in this experiment, both of the mabs used recognize the he and s proteins of phe-cov and have a high affinity for their respective antigen epitopes. analysis of the specificity showed the strip to be specific for the detection of phe-cov, because it reacted with neither two coronaviruses (tgev and pedv) in pigs nor with other group viruses of the coronaviridae family (bcv, hcv-oc and mhv), the common virus hcv, or prv, to which the clinical symptoms of phe-cov are similar. in addition, careful selection of a membrane is critical for high specificity, sensitivity, and rapid detection as the wicking rate and speed of liquid diffusion on the membrane (millipore corp shf , a liner). the membrane used in this experiment was nitrocellulose membrane (millipore corp shf , a liner) with a membrane pore size of . - . μm, igg binding force constant of - μg/cm , and a chromatography rate of - s/cm. the membrane ensures antibody absorption and the leaching and binding of gold-labeled proteins. these advantages enable the test results to be sensitive and easy to read [ ] . phe-cov is able to replicate in the upper respiratory tract with or without producing clinical signs. phe-cov can be isolated from the nasal cavity, trachea, brain, and lungs of diseased or healthy pigs [ ] [ ] [ ] and the brain had the highest detection rate by nested pcr and rt-pcr [ ] . because of rt-pcr and elisa are also widely applied in clinical practice due to its high sensitivity and specificity [ , ] . thus, a lot of brain tissue samples were collected from deceased piglets with suspected phe-cov infection, and using rt-pcr and elisa as reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be % and . %, respectively. there was an excellent agreement among the immunochromatographic strip to elisa and rt-pcr (kappa = . ). the quantifying test result indicated the sensitivity of the strip test to be close to, or slightly less than, that of elisa and rt-pcr. however, the elisa and rt-pcr assay each step of the procedure can strongly influence the result if not performed well. the procedure must be strictly followed, with good quality control inside and outside the laboratory, and reagents that ensure the test quality must be selected. although elisa and rt-pcr was developed for the detection of phe-cov with high specific and sensitivity, it is not suitable for use outside of the research laboratory. in contrast, the procedure for the immunochromatographic strip described in this paper is less laborious and time-consuming than the elisa and rt-pcr method. taken together, the results suggested that an immunochromatographic strip was developed with high sensitivity and specificity for detecting phe-cov, which could be used for clinical applications. phe-cov is excreted for - days in oronasal secreions [ , ] , with transmission occurring through exposure to nasal secretions. thus, a total of nasal cavity or throat swabs were collected from approximately -to -week-old piglets, with vomiting and neurological symptoms consistent with phe-cov infection, and application of the immunochromatographic strips for diagnosis of phe-cov infection in the jilin province. the positive rate ranged from . % in the jilin district to . % in the songyuan district. notably, the phe-cov positive rate was . % in liaoyuan district in the current study. however, the serum antibody positive rate was only . % in the previous study [ ] . this may be associated with the samples collected from different herds. these findings suggest that phe-cov is commonly transmitted in these districts in the jilin province. notably, specific pathogen-free (spf) pigs, derived from germ-free pigs and given artificial milk, have been introduced extensively in many farms in this area [ , ] . because these animals lack antibodies to certain pathogenic agents, including phe-cov, their susceptibility to these agents may be greater. therefore, reliable testing methods are important for the diagnosis and prevention of phe-cov disease, which can cause fatal outbreaks in phe-cov seronegative farms. from the results of clinical signs, an immunochromatographic strip was developed for detecting phe-cov. the immunochromatographic strip is a simple and quick test that requires no special training to use, and the sensitivity and specificity of the strip were similar to the elisa and rt-pcr test. this test would be a valuable addition to clinical detection techniques for phe-cov, especially in areas where laboratory facilities are not available. hemagglutination/hemagglutination inhibition ihc: immunohistochemistry; nested pcr: nested-polymerase chain reaction reverse transcriptase-polymerase chain reaction; mab: monoclonal antibody; gica: colloidal gold immunochromatography enzyme-linked immunosorbent assay; bsa: bovine serum albumin; pbs: phosphate buffered saline; nc: nitrocellulose; tgev: transmissible gastro-enteritis virus; pedv: pig epidemic diarrhea virus; prv: pseudorabies virus; hcv: hog cholera virus bcv: bovine coronavirus; mhv: mouse hepatitis virus isolation and identification of hemagglutinating encephalomyelitis virus form pigs in taiwan sequence of ther ′-terminal end ( . kb) on the genome of porcine hemagglutinating encephalomyelitis virus: comparison with other hemagglutinating coronavirus serological comparison of hemagglutinating encephalomyelitis viruses isolated from different outbreaks a hemagglutinating virus producing encephalomyelitis in baby pigs vomiting and wasting disease of piglets encephalomyelitis of swine caused by haemagglutinating virus. vi. morphology of the virus a presumptive case of vomiting and wasting disease in a swine nucleus herd. s h a p vomiting and wasting disease in piglets pathogenicity of fieldisolants of hemagglutinating encephalomyelitis virus for neonatal pigs biological and molecular characteristics of an hev isolate associated with recent acute outbreaks of encephalomyelitis in quebec pig farms isolation and identification of a high pathogenicitiy hemagglutinating encephalom yelitis virus continuous cultures of fused cells secreting antibody of predefined specificity microadaption of hemadsorption inhibition for neutralization tests with pig hemagglutinating encephalomyelitis virus a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl in children hospitalized with respiratory tract infections in belgium development of reverse transcriptase pcr and nested pcr to detect porcine hemagglutinating encephalomyelitis virus preliminary evaluation of an immunochromatographic strip test for specific treponema pallidum antibodies analytical performance and clinical application of a new rapid bedside assay for the detection of serum cardiac troponin i rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of herpes simplex virus type -specific immunoglobulin g antibodies in serum and whole blood characteristics of coronavirus (strain n) of pigs identification of ncam that interacts with the phe-cov spike protein purification of igg monoclonal antibody by caprylic acid precipit ation preparation and characterization of au colloid monolayers vomiting and wasting disease associated with hemagglutinating encephalomyelitis viruses infection in piglets in jilin, china understanding interobserver agreement: the kappa statistic evaluation of the tuberculin gamma interferon assay: potential to replace the mantoux skin test a comparative sequence analysis to revise the current taxonomy of the family coronaviridae genonlic relationship of porcine hemagglutinating encephalomyelifts virus to bovine coronavirus and human coronavirus oc as studied by the use of bovine coronavlrus s gene-specific probes hemagglutinating encephalomyelitis coronavirus infection in pigs rapid, sensitive, and specific lateral-flow immunochromatographic device to measure anti-anthrax protective antigen immunoglobulin g in serum and whole blood development and evaluation of an immunochromatographic strip for the detection of serum antibodies against bluetongue virus development of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype o manufacturing high quality gold sol the place of gold in rapid tests experimental infection of pigs with porcine hemagglutinating encephalomyelitis virus buoyant density of hemagglutinating encephalomyelitis of swine: comparison with avian bronchitis virus a seroepizootiologic study of vomiting and wasting disease virus in pigs comparison of elisa for the detection of porcine serum antibodies to non-structural proteins of foot-and-mouth disease virus osterhaus ad: evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections characteristics of a coronavirus causing vomition and wasting in pigs. arch gesamte virus forsch development of an immunochromatographic strip for serological diagnosis of porcine hemagglutinating encephalomyelitis virus the produce technology of spf pig were application of modern raising pig in china submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank american journal experts for excellent grammar revisions of this paper. the authors are extremely grateful to the staff working in the veterinary stations in the jilin province for collecting porcine virus samples for this study. this study was supported by the national natural science the authors declare that they have no competing interests.authors' contribution kc and kz carried out most of the experiments and wrote the manuscript. ds and wh participated in the planning of the project. cz and gw collected the samples. fg and cw conceived of the study and participated in its design and coordination. all authors read and approved the final manuscript. key: cord- -phsr jp authors: nan title: abstracts tps date: - - journal: allergy doi: . /all. sha: doc_id: cord_uid: phsr jp nan (either in men or women) between metabolic syndrome and incident asthma. conclusion: this study confirmed the significance of obesity as a risk factor for incident asthma. moreover, obesity appeared to be a stronger risk factor than metabolic syndrome. | relationship between helminth infection, blood eosinophils and asthma symptoms in a rural community from the tropics peñaranda d; alvarez l; sierra n; lopez j; zakzuk j; caraballo l institute for immunological research. university of cartagena, cartagena, colombia background: immune response to helminths shares many features with the allergic response. in tropical regions where helminths are highly prevalent, asthma is still a major public health burden. large clinical cohorts suggest that high blood eosinophils (hbe=> cells/ mm ) are associated with asthma exacerbations. however, the association between hbe and asthma severity in rural communities with prevalent helminthic infections is unclear. method: patients with wheezing symptoms in the last year living in a rural tropical community (santa catalina, colombia) where helminths are highly prevalent, were recruited for this study. blood eosinophils were assessed by complete blood count. parasitic infection was evaluated with two serial coprological exams (kato-katz method) and skin prick tests were conducted to determine reactivity to ascaris. results: seventy-three patients (mean age: ; range: - years old) were recruited in this study. a. lumbricoides and t. trichuria active infection ( . % and . %, respectively) were not related to age or gender. a positive spt to ascaris extract, aba- and d. pteronyssinus was observed in %, . % and . %, respectively. mean eosinophil count was cells/mm ; . % had hbe. rate of patients with at least one emergency department visit was . % and hospitalization, . %. blood eosinophil counts (as a continuous variable) were inversely associated with age (p = . ) and higher in helminth infection (p = . ). in crude univariate analysis, exacerbations (er and/or hospitalization) were associated with age (or: . ; % ci: . - . , p < . ) and hbe (or: . ; %ci: . - . , p = . ), but not with helminth infection. for a better definition of asthma, multivariate analysis done in those > years old indicated that hbe, helminth infection and positive ascaris spt were not associated with asthma exacerbations. conclusion: uncontrolled asthma is common in rural places of the tropics. since helminth infection influences eosinophilia, the clinical value of hbe to predict exacerbations is limited in helminth-endemic populations. castro mc ; ferreira j ; sarmento d ; carvalho c ; matos a ; bicho m chln-immunoallergy; lisbon medical school-genetic department, lisboa, portugal; lisbon medical school-genetic department, lisboa, portugal background: the bioavailability of no and endothelial homeostasis depends on the functional polymorphism of -bp del/ins within intron- of dhfr (dihydrofolate reductase enzyme) (rs ) that could interfere in the regeneration of bh (tetrahydrobiopterin) from bh ( , -dihydrobiopterin) and contributes to endothelial dysfunction in asthma. method: asthmatics (n = ) compared with control group (n = ).the polymorphism was analyzed by pcr. control of asthma assessed by (acq and paqlq). statistical analysis with spss . establishing a significance level of p < . . results: there are women and males in asthmatics and women and males in controls (p = . ). in asthmatics: age ( x ± sd): . ± . ; and in control group: age ( x ± sd): . ± . . the genotype frequencies in asthmatics are: dd ( . %); id ( . %); ii ( . %); in control group: dd ( . %); id ( . %); ii ( . %); there is no statistical difference between groups (p = . ). the allelic frequencies in asthmatics are: allele d ( . %); allele i ( . %); in control group: allele d ( %); allele i ( %); there is no statistical difference between groups (p = . ). the genotype frequencies in the uncontrolled asthmatics are: dd ( . %); id ( . %); ii ( . %) ; in the controlled asthmatics are: dd ( . %); id ( . %); ii ( . %); there is statistical difference between groups (p = . ). genotypes id and ii are more frequent in the uncontrolled asthmatics. the allelic frequencies in the uncontrolled asthmatics are: allele d ( . %); allele i ( . %); in the controlled asthmatics are: allele d ( . %); allele i ( . %); there is a trend to have differences between groups (p = . ). allele i is more frequent among uncontrolled asthmatics. the uncontrolled asthmatics are older than the controlled asthmatics (p < . ). there is no differences in gender distribution (p = . ). the genotype ii confers a risk of being uncontrolled asthmatic of . times when compared with controlled asthmatics and adjusted for age: or b : . [ . - . ]; p = . . physiopathology and the emergence of evidence-based clinical guidelines. however, variation still exists among some diagnostic aspects of asthma in real life. it is unknown to what degree diagnosis is affected by the treating physician's medical specialty. results: a total of gps, pediatricians, allergists, pulmonologists and otolaryngologists (orls) replied. although for general application of diagnostic clinical criteria all physicians rated similarly, in general accordance with the mag suggestions, a third of non-pulmonologist practitioners don't recognize chest discomfort as one of the clue symptoms of asthma, but they erroneously believe crackles are (p = . ). we found agreement in almost half of all physicians to erroneously believe that viral illness' induced wheezing in non atopic children predisposes asthma. conversely, - % are aware that allergic sensitization predisposes to asthma. most specialists -except pulmonologists (p = . )-incorrectly listed fev as the best parameter to identify airflow obstruction (ao) and fev /fvc to assess ao severity. % of gps do not know peak expiratory flow (pef) measurements could be valuable, and % of all specialists are not aware that changes in pef can also be used to confirm ao reversibility. to classify asthma, only pulmonologists adequately considered the level of control in similar proportion than severity ( % and %, respectively), which is uniformly the preferred method by most other specialists. conclusion: although in general many clinical aspects of asthma diagnosis seem to be accurately assessed, there is a wide specialityspecific variation regarding some aspects of phenotyping and classification, diverging from mag's recommendations. as such, our results can help to detect knowledge-gaps and to guide the development of more focused specialty-specific learning tools to improve clinical impressions, process medical evidence, and apply it to patient care. | issues, continuous medical education on treatment of acute asthma, exercise induced asthma and asthma in pregnancy should include, per medical specialty background: to unify and improve the management of asthma, including asthma exacerbations, the mexican guideline on asthma ( . %) of employees who stated increased symptoms with flour exposure. among all workers ( . %) employees were diagnosed as asthma and ( . %) workers were diagnosed as ba. conclusion: wheat flour sensitivity is high among workers who are exposed to wheat flour, however the prevalence of ba is similar to the previous data in the literature. johnsen cr ; callesen kt ; jensen bm ; poulsen lk clinic of allergy, dept. of dermato-allergology, gentofte hospital, copenhagen, denmark; laboratory of allergy, gentofte hospital, copenhagen, denmark background: enzymes are well known as sensitizers and causes of occupational allergy primarily in the industries producing and using the products. we present a case of occupational contact urticaria, rhino-conjunctivitis and asthma in a year male chef who was using a transglutaminase enzyme powder obtained from fermentation of streptomyces mobaraense as meat glue in processing of fine culinary dishes. this transglutaminase has been used for protein food preparation in industrial settings since to improve the texture of protein rich foods such as surimi or ham. in this case it was used in small scale in a gastronomy restaurant kitchen spraying enzyme powder with a sieve over raw meat without any protective equipment in contrast to the producer's recommendation. the chef was also found allergic to dried, edible mushrooms also forming part of the meat dish prepared with the transglutaminase enzyme powder. in one occasion he experienced an oral reaction with itching and swelling of the mucosa in the mouth, stridor, angioedema of the face, and urticaria after ingestion of beef meat treated with transglutaminase and rolled in horn of plenty dried mushroom powder. no other symptoms of food allergy were reported but a known cat allergy was. background: formaldehyde and xylene are occupational skin and respiratory irritant and/or sensitizer, exposure to those may be associated with dermatitis, rhinitis and asthma. health care workers, as nurses, laboratory technicians, doctors could be exposed in different tasks in operating rooms, endoscopy and in pathology laboratory. we describe three cases of work-related rhinitis in technicians employed in the same unit of hospital pathology . first case: a woman of years old underwent medical examination in our occupational allergy unit because allergy respiratory symptoms. she has been working for years in pathology laboratory and was exposed to xylene and formaldehyde. she developed rhinitis, rhinosinusitis, hyposmia and cough with sputum after years started work. she had negative skin prick test for common aeroallergens. lung function was normal with a fev /fvc ratio of % of predict. blood cells count reveled % of eosinophils ( /mmc) with total leucocytes. second case: a woman of years old was affected by moderate persistent allergic rhinitis with positive skin prick tests to house dust mite, dog and cat. in the last year rhinitis symptoms worsened in relation to work and improved during vacation. when she was exposed mostly to formaldehyde during shift at the end of it she usually experienced face skin and conjunctival erythema. she developed work-related symptoms after years of exposure in the pathology unit. third case: a woman of years old, who has been working for years in the pathology unit and was exposed to formaldehyde and xylene, in the last year developed moderate-severe persistent rhinitis with hyposmia and chronic cough. she referred to otorhinolaryngologist and an irritant induced rhinitis was diagnosed. she had negative skin prick test for common allergens and normal lung function. results and conclusion: the workers experienced respiratory symptoms in relation to work exposure to formaldehyde and xylene. the suspected causal agents were monitored in the work environment and an exceeding of the recommended limit values was found. preventive measure were adopted with a reduction of exposure and symptoms improve only in the second and third case. challenge test with mannitol is considered to be more specific than test with methacholine. also, duration of procedure is shorter and safer. therefore the study aim was to compare the usefulness of these two tests in monitoring of sict. method: four bakery workers with suspicion of oa underwent single-blind, placebo-controlled sict with workplace allergens accompanied by evaluation of nsbhr with mannitol and methacholine before and after sict. clinical examination, spirometry, skin prick tests (spts) to common aeroallergens and occupational allergens, serum specific ige antibodies to occupational aeroallergens were also performed. results: positive spts results to occupational aeroallergens were found in all bakery workers, specific ige to flours were detected only in two subjects. three out of the four patients displayed positive sict reaction (in two cases early spirometric response). in all of these patients, airway response to methacholine increased significantly. in the first two patients also airway reaction to mannitol was significant, whereas in one subject with early reaction there was no increase in nsbr after mannitol inhalation. the patient with negative sict results did not reveal any changes in nsbr before and after the test, neither to methacholine nor mannitol. | rice-induced occupational anaphylaxis and socio-economic impact-case report method: in this prospective study, a total of students completed a self-administrated questionnaire that comprised different questions and gave information about the participants and their glove use, working habits, signs and symptoms related to these gloves, precautions taken to minimize it, etc. skin prick test is performed through commercial extract latex gloves (stallergenes), while patch test is prepared through latex gloves and adhesives. two types of gloves are used: gloves that contain latex and gloves without latex (vinyl gloves), which are used also as e negative control. results: questionnaire items and diagnostic tests revealed that one-fourth of subjects were suspicious for latex gloves hypersensitivity. their mean value for skin reactions like irritant or allergic dermatitis or contact urticaria was between % and %, while for other symptoms the mean value was under %. logistic regression analysis revealed an association between different questionnaire items and positive allergy tests among suspected cases and diagnosed cases of latex allergy. approximately % of people who work with laboratory animals experience some allergic symptoms and about % of animal technicians go on to develop serious symptoms of asthma. uk government guidelines state that employers must prevent or adequately control exposure of employees to animal allergens and should undertake monitoring to ensure that suitable controls remain effective. the most widely used monitoring method is personal iom filters. however, these need to be attached to a pump and carried by the technician which can be cumbersome and awkward. previous data has demonstrated that allergens from dust mite, cat, dog and pollen could be captured and quantified by a novel type of nasal filter. in this current study, we sought to assess the feasibility of using the nasal filters for the assessment of exposure to mouse allergen in a laboratory facility. method: technicians working in a laboratory animal facility were asked to wear the filters during normal routine work. for comparison, they were also asked to wear an iom filter for the same duration. allergen was extracted from nasal and iom filters by gentle rocking in pbs-tween for two hours. levels of the major mouse urinary protein (mus m ) were quantified using our multiplex array technology, which is highly sensitive and allows for quantification of mus m down to . ng/ml. results: significant levels of mus m were detected in the nasal filter extracts and these levels correlated with the type of activity that was being performed by the technician, as well as the housing environment of the mice. levels were compared to the suggested 'safe' limit of allergen exposure of ng/m . we also found that the technicians grew accustomed to the nasal filters quickly and found them far more practical for every day monitoring that wearing the iom filter and pump. conclusion: these data indicate that nasal filters may be considered a simple and easily wearable method for monitoring laboratory animal allergen exposure. future studies are planned to assess the feasibility of wearing the filters for analysing exposure to other laboratory animal allergens from rat and guinea pig. havana university lower co emission, water and feed consumption and limited waste production. insects are currently allowed both for human and animal feeding in some eu countries, including italy and the risk profile related to production and consumption of insects as food and feed, including risk of allergenicity, is currently under evaluation by efsa. both food and feed products derived from insects require multiple manipulations by the breeder and/or by the workers who transform the insect into the commercial products, thus the occupational exposure have to be considered too. the aim of this work is to evaluate the allergenicity of tenebrio molitor, one most used species for animal feeding. method: t. molitor proteins were extracted from intact dried larvae and from flour of dried larvae. the protein extracts were separated in one-dimensional electrophoresis conclusion: according to these results, the larva flour seems to be less immunoreactive than the intact counterpart, probably due to the processing that causes the degradation of protein bands over kda. working in gastronomy is associated with exposure to many factors with an irritating and allergic potential influencing respiratory system. food products and organic dust are the source of inhaled allergens which may cause sensitization during apprenticeship. the study aim is a prospective observation of incidence of sensitization to selected environmental and occupational allergens among culinary school apprentices and identification of work-related allergic diseases in this group. method: the cohort comprised apprentices. they were examined in the first and the second year of education. questionnaire and allergological tests [(skin prick test) spt to common and occupational allergens, ige level evaluation (total and specific for occupational allergens) and pulmonary tests] were performed]. results: the most frequent symptoms reported by examined apprentices were rhinitis ( . %), conjunctivitis ( %), skin symptoms ( . %), dyspnea ( . %) and cough ( . %). subjects developed nasal symptoms during the second year of education, while in cases the skin symptoms and in subjects conjunctivitis appeared. in cases the work-related symptoms were reported. the most frequent positive results of spts were obtained with dermatophagoides pteronyssinus . %, dermatophagoides farinae . %, grass pollens . %. positive spt to rye and barley flour were found in respectively . % and . % apprentices. . % of apprentices had specific ige to flours. the preliminary results indicate that work-related allergy symptoms and hypersensitivity to occupational allergens are rarely found among culinary school apprentices in the first years of education. the further observation will allow to evaluate the trends in incidence of allergy to occupational allergens, as well as the clinical presentation of allergy in that group. | how multifaceted the clinical presentation and etiology of allergic diseases could be? method: the study was done in children from west georgia randomly and on based of questionnaire of representative cohort. ( ) ( ) ( ) . the cohort was children, - years old, risk factors were studied by way of interviewing, clinical-laboratory dates. for assessing the risk factors, was used 'case control' method. the statistical processing of material was done with computer program sps/sv . inclusion criteria for enrolment were: collectors of dust, gender, existence of moisture and mold consuming of tabasco, atopic dermatitis and seasonality. results: the groups, which we have studied, prevalence of acute respiratory viral infection was %, bronchitis − . %, allergic rhinitis . %, atopic dermatitis . %, food allergy . %. the reliability was high (p < . ) in families with bronchial asthma compared with healthy population. bronchial asthma was detected in . % of population. the hereditary load of allergic diseases in patients with bronchial asthma was . % and in healthy cohort it was . % (p < . ). conclusion: based on the results, we can conclude that, ecological factors and genetic predisposition significantly influences on prevalence of sensibilisation of house dust mite, molds and formation of bronchial asthma. as the genetic and environmental factors that act on an immune system are better elucidated and their roles established, the implementation of more enduring preventive efforts will be developed. however, at present, the best approach to the child at high risk for the development of allergies is to institute dietary and environmental control measures early to decrease sensitization, and to recognize and appropriately treat the evolving signs and symptoms of allergic disease. background: plantation of road-side avenue trees has become a major part of the urbanization programme in kolkata metropolis of india for megacity beautification and environmental management. due to evergreen habits, gulmohor (delonix regia) and chhatim (alstonia scholaris) are frequently selected for plantation programme to generate green belts. however, an increasing incidence of seasonal pollinosis was observed among the inhabitants living in close vicinity to these trees suggesting a possible link between the airborne pollen load and the concomitant respiratory hazards. this prompted us to investigate the allergens in the pollen of these two dominant avenue trees. method: aerobiological surveys were conducted at multiple sites of kolkata for a period of two years using seven-day volumetric burkard sampler to record the pollen concentration in the outdoor ambient air. clinical data and residual blood of pollinosis patients were collected from a public hospital. allergens were detected in the pollen proteome fractionated in d gel by ige-serology. the major igereactive proteins were partially purified by ammonium sulphate fractionation followed by ion-exchange chromatography. the allergenic activity of the fractions was tested by histamine release assay. results: a clear correlation was observed between the pollinosis related morbidity and the aeropollen load especially during the peak flowering period of these two trees. about % and % of the patients displayed positive spt response and ige-reactivity using pollen extracts of gulmohor and chhatim respectively. immunoproteomic analyses revealed the presence of - ige-reactive components in the d pollen proteome of these species. hierarchical cluster analysis with patient immunoblot data identified a kda and a kda protein as major allergens of gulmohor and chhatim respectively. the purified fractions containing each of these two major allergens induced histamine release from granulocytes within a range between and %. method: immortalized human keratinocyte cell line (hacat) and primary normal human epidermal keratinocytes (nheks) were differentiated with calcium chloride for and days, respectively. following the differentiation, the cells were treated with il- ( ng/ml), il- ( ng/ml), and/or hcho ( × ^- %) for hours. the mrna expression of flg, ivl, lor, dsg , dsg , dsc , dsc , as well as tslp was analyzed using quantitative real-time pcr. results: hcho exposure decreased the mrna expressions of structural components (flg, ivl, and lor) and cell adhesion molecules (dsg , dsg , dsc , and dsc ) in a short-period time of exposure ( hours). we also found that hcho exposure significantly enhanced il- -and/or il- -induced tslp production in nheks as well as hacat. interestingly, exposure to hcho alone is enough to increase the tslp mrna expression in both cells. conclusion: our results suggest that hcho exposure might synergistically damage the skin barrier function with il- and il- by increasing tslp expression and decreasing structural components as well as cell adhesion molecules. | skin prick test reactivity to aeroallergens in adult allergy clinic in a tertiary hospital: a -year retrospective study results: five different human sera were screened for specific ige level against different allergen sources using test methods of three different suppliers. the sensitivity of the three different methods can be arranged in the ascending order manufacturer a < manufacturer c < manufacturer b. with the test of manufacturer a, % of the measurements were below the detection limit ( . ku/l), with the test of manufacturer c, % of the measurements were below the detection limit, whereas the test of manufacturer b leads to values below the detection limit in % of the cases. in terms of variation coefficient, the test system of manufacturer c had the best performance. test systems of manufacturers a and b exhibited comparable variation coefficients, which were considerably higher than that of manufacturer c. conclusion: based on these test results, only the test of supplier c is recommendable for determination of levels of specific ige for diagnostics of allergic patients. with the test of manufacturer a, elevated levels of specific ige antibodies for many allergens cannot be detected due to the poor sensitivity of the test system. the test system of supplier b exhibits a good sensitivity but the coefficient of variation is rather high for a diagnostic test. this drawback could be circumvented by multiple determination of one test parameter. although this is an advisable strategy in general, the routine in diagnostic laboratories is incompatible with this approach, since throughput would decrease while costs would increase. this study is another good example for the need of the implementation of a characterized standard material with known values of sige, as demanded by wojtalewicz et al. method: cd c and cd expression on basophils were monitored upon exposure of whole blood samples (< hours) to anti-ige and/or allergenic extracts. staining was conducted on exposed samples using dry room temperature stable antibody panels (dura innovations format) coated in well plates, eliminating all antibody pipetting steps from the workflow. red blood cells were lysed and data was acquired (without further wash steps) on a cytoflex flow cytometer (beckman coulter). staining and lysing were automated using a biomek (beckman coulter). results: the described no-wash preparation protocol, already established for manual preparation mode in tubes, could be trans- conclusion: the hr-test was significantly less likely to be positive, if a patient suffered from monosymptomatic ae than in ae patients with concomitant urticaria. this could signify a higher likelihood of treatment response to antihistamine and other anti-allergic medication in the latter group. background: pathogenetic mechanisms of allergy are polymorphic. they include ige-dependent and ige-independent, allergen-specific granulocyte-mediated and lymphocytic reactions, as well as nonspecific hypersensitivity, which are realized through a variety of mediators: histamine, tryptase, etc. allergen bucal challenge mimics the natural situation and is useful for understanding the mechanisms of allergic airway inflammation and airway hyperresponsiveness (ahr).saliva used as a non-invasive readily available bio-sample for diagnosis instead of blood. biomarkers in saliva are associated with the pathogenesis and clinical outcome of allergic diseases method: aim: to examine mediators for ahr with buccal(mucosal) challenge tests. we examined patients with allergic asthma(the history, positive skin prick test, serum specific ige) and healthy volunteers. saliva were collected. then, both groups were subjected to buccal(mucosal) allergen challenge by a water-salt solution of the mite allergen dermatophagoides pteronyssinus. saliva was recollected in minutes and hours after the provocation. the level of myeloperoxidase, elastase, tryptase in saliva were determined by the elisa. that provocative test did not cause clinical symptoms development or reduction in nasal bronchial patency in any patient. results: in patients with allergopathology, an initially increased level of myeloperoxidase and tryptase in minutes after the provocation, elastase increased in hours (table ) . tryptase in saliva after minutes increased till . ( . ; . ) (me, pg/ml (lq;uq)), p = . . increased tryptase is presence of increased cellular inflammation, e.g. mast cells. its ige-dependent hypersensitivity, because there was the correlation between the elevated level of tryptase and positive prick tests. elevated levels of myeloperoxidase and elastase in saliva may be the criteria for the neutrophil hypersensitivity and ige-independent reactions. in healthy volunteers this increase was not observed. the identification of tryptase, myeloperoxidase, elastase can be used for diagnosis of types of ahr. tryptase is a mediator of early (immediate) response to allergen. increased myeloperoxidase and elastase indicates the involvement of the eosinophils and neutrophils in the oral mucous membrane in the allergic process. these mediators have additional roles in the late phase response. elevated levels of myeloperoxidase and elastase in saliva may be the criteria for the neutrophil hypersensitivity. conclusion: safe and acute in vitro methods allow to conduct early etiological diagnosis of allergy, which contributes to the effectiveness of therapy; the detection of polyvalent sensitization dictates the need for molecular diagnostics to single allergens, which has a higher prognostic level and the clinical significance of predicting the appropriateness and effectiveness of allergen-specific therapy; laboratory diagnostics of the allergy allows to reveal sensitization at the ( , ) ( , ) ( , . ) *p = . ; **p = . ; ***p = . . subclinical level, which increases early diagnosis and identify persons with a predisposition to allergy; the establishment of causal aller- background: antibacterial chemicals like parabens and triclosan have been associated with allergic disease in children. parabens are also suspected to affect metabolic functions, possibly due to their weak endocrine disrupting properties. furthermore, a possible link has been suggested for eczema and adiposity, and thus, how body burden of chemical exposures affect both of these outcomes are of interest. we aimed to describe the association between exposure to parabens and eczema and body mass index (bmi) in an adult population in norway. method: urine biomarkers of butyl-, ethyl-, methyl-and propylparabens were quantified by mass-spectrometry in adult participants (median age= years) from the rhinessa study in bergen, norway. linear regression models adjusted for gender, age and bmi (for eczema outcomes) and with clustering for siblings, were applied to model possible association between specific gravity standardized urine biomarker concentrations of parabens with bmi and eczema. results: propyl-(ppb) and methyl-parabens (mpb) were detected in % of the urine samples; ethyl (epb) in % and butyl (bpb) in % of the samples. in women, epb and bpb were detectable in % and %, respectively. participants with current eczema ( %) had lower level of several parabens compared to those without eczema (bpb for both genders; epb in women only and sum of all parabens in men only). body burden of epb (geometric mean (gm)) was . μg/l in women with current eczema compared to . μg/l in women without eczema (p = . ). body burden of parabens (mpb and epb) were inversely associated with obesity (bmi> , ( . %) ), as compared to normal range bmi (bmi= . - ( . %)) in both men and women. the concentration of mpb for obese women was gm= μg/l compared to μg/l in women with normal range bmi. for men, the gm for mpb was . μg/l in obese compared to . μg/l in normal weight men (p = . ). conclusion: person with eczema or obesity had lower paraben levels in urine. we speculate that these chemicals might be stored in adipose tissue, and therefore excreted in urine in lower levels among the obese. eczema and obesity was not strongly associated in the current study. method: we retrospectively analysed medical records of patients who were patch-tested with our dental screening series of substances. adverse reactions to dental materials were suspected based on subjective complaints in the oral cavity and/or objective conditions of the oral mucosa. square plastic chambers on hypoallergenic tape were used. patch tests were applied to the upper back and removed by the patient after hours. readings were performed and days after application (d and d ). results were evaluated according to the international contact dermatitis research group guidelines. positive patch test reactions fulfilled the criteria of at least a one plus (+) reaction on d and/or d . the term »contact allergy« is usually used for such reactions. we prefer the term »contact sensitization«. clinical relevance of positive reactions to dental materials was not systematically assessed in this analysis. conclusion: we report a high frequency of positive reactions on d that were not seen on d . this finding demonstrates the importance of an additional late patch test reading in patients with suspected contact allergy to dental materials. background: psoriasis is a chronic inflammatory skin disease. its etiopathogenesis is not exactly known. it is believed that the disease occurs in people with genetic tendency with the effect of a triggering factor. in some studies it is observed that contact dermatitis in psoriasis is increased with respect to normal population. for this reason it is proposed that allergen materials could trigger psoriasis. in this study it is aimed to determine contact allergy frequency in psoriasis cases using patch test. results: of the cases were plaque, of them were guttate, of them were palmoplantar and of them were inverse type. more positivity rate is observed in psoriasis cases ( . %) than control ( %). the positively responsed materials with respect to decreasing number of patients are found as follows: nickel sulphate ( . %), thimerosal ( . %), peru balsam( %), p-phenylenediamine( . %), colophony( . %), n-isopropyl-n-fenil- -fenilendiamin( . %), mercaptobenzothiazole( . %), benzocaine( . %), most frequently plaque type and following guttate type positive responses are observed in evaluations with respect to clinical types. no statistical significance is found between patch test results and pasi values in psoriasis cases. conclusion: patients with psoriasis should be carefully evaluated. sometimes some materials may trigger psoriasis. the composition of the pigments that professional tattooists use are varied inorganic salts of metals or organic vegetable pigments. red tattoos, especially those that contain mercury, are the most common cause of late reactions. method: year-old male patient with no previous allergy history known, who gets a tattoo on his right leg and develops within months, cutaneous erythema and pruritus on the same location as the tattoo. true test ® for skin allergy patch epicutaneous testing is performed. results: and hours reading: showed positiveness for mercury ++, with no late positive reactions after that. conclusion: as allergists we should be familiar with the different types of tattoos available, and know the possible cutaneous complications that each of these decorative techniques can present. it is our responsibility to be able to diagnose any complications at an early stage, establish the most appropriate treatment and, if possible, prevent them by informing the possible users. background: within otorhinolaryngological pathology chronic eczematous otitis externa is one of the most common, usually treated with topical medication successfully; however, there are cases in which the poor response to treatment, or the recurrence thereof, may be due to causes secondary to the medication itself, as observed in cases of allergic contact dermatitis caused by these drugs. case report: we present a -year-old male patient without relevant pathological antecedents or known allergies, who consulted the otorhinolaryngology service of our center for otorrhea of days of evolution, bilateral external otitis is diagnosed and a topical otological combination is recommended (beclomethasone dipropionate . % and clioquinol %, excipient: macrogol) with improvement. however, during the following years the patient presents exacerbations and remissions of the condition, with negative or inconclusive microbiological studies. during all that time he was using the prior topical treatment and other combination treatments of topical antibiotics, corticosteroids and local antiseptics. more aggressive causes of external otitis such as malignant external otitis were ruled out. during the third year of follow-up, a clear relationship of exacerbations was observed with the use of the first combination of topical drugs, so it was decided to investigate allergic sensitization. material and methods: we perform patch tests using true test ® , standard spanish series (geidac -spanish group for investigation of contact allergy dermatitis), topical corticosteroid battery, antiseptic, as well as topical medications used by the patient. results: from the first reading on day two, positivity was observed for: mixture of quinolines ++ patient's otological combination ++ and chlorquinaldol ++; being confirmed in the reading at day four. eczematous external chronic otitis is diagnosed with allergic sensitization to quinolines (clioquinol, chlorquinaldol). we conclude that in the case of chronic external otitis, allergic contact dermatitis should also be investigated as a possible cause, and it is important to perform epicutaneous tests with the patient's own products to evaluate non-common or hidden allergens that may be relevant to their current pathology. most common causes of acd. it is important to distinguish local findings of infection from acd caused by topical antibiotic treatments. here we present a patient with acd with topical use of bacitracin and neomycin combination therapy due to recurrent blepharitis. an -year-old male patient presented with the complaints of itching, redness, swelling of the eyelids and facial edema. he had used various topical ophthalmic antibiotherapy and eye shampoo for years due to recurrent blepharitis. five days ago, due to the redness of the eyelids, burning sensation in the eye, itching of the eyes; he was examined by an ophthalmologist. the eyelids and eyelashes were scaly and dry. the patient was treated with warm water soaked cloth dressing, mechanical eyelash cleaning and topical antibiotherapy (neomycin-simple combination therapy). after the second day of treatment, the patient's topical ophthalmic antibiotherapy was discontinued due to an augmentation of the redness in the eye- | contact allergy after exposure to ivy (hedera helix l) potent steroid treatment associated with systemic antihistamines, but with no improvement. on dermatological examination a small, well delineated, eczema-like plaque was noticed on a digital finger, as a new finding striking with her old burn scars. she denied any symptoms and was in good health condition. a mm punch biopsy was performed and histological report established the diagnosis of squamous cell carcinoma. the patient was transferred to oncology department for further investigation and treatment. conclusion: early diagnosis and prompt surgical therapy are recommended to all patients with chronic wounds and scars who develop malignant transformation. *written informed consent for the publication of potentially identifiable personal details of patient (gender, age, illness, location) was obtained. **in relation to this presentation, i declare that there are no conflicts of interest. ertugrul a; hizli demirkale z; bostanci i dr sami ulus maternity and children training and research hospital, ankara, turkey introduction: the incidence of contact sensitization among adolescent has been increasing. nickel is one of the important causes of allergic contact dermatitis (acd) in this age group. increased exposure to nickel and deterioration of the skin barrier are among the important risk factors in children. the gold standard for diagnosis is skin patch test. we report here an adolescent patient who has allergic sensitization to nickel and cobalt. case: a -year-old female patient admitted in our clinic with a complaint of edema on her face. the patient had applied chickpea water to her face at least once a day for one week because of her acnes. her medical history revealed that she had experienced similar edema on her face after applications of clay mask one year ago. she was diagnosed with cellulitis and she had been treated with antibiotics for five days. on her physical examination, angioedema was observed on her face, especially on the glabellar region. eosinophilia was not found on her laboratory data. c-reactive protein (crp), c and c esterase inhibitor protein levels were also normal. the skin prick test was performed with aeroallergens, chickpea, lentil, bean and nuts, and no reaction had been observed. the patch test was performed with 'thin-layer-rapid-use-epicutaneous' (t.r.u.e) test and chickpea. the patient had positive reactions to nickel and cobalt. detailed questioning disclosed that the patient was preparing the chickpea water in a metal pot. result: chickpea water and clay mask contain varying amounts of nickel. it was thought that the edema of the patient is due to nickel allergic contact sensitization. an increased exposure to nickel and cobalt raises the frequency of sensitization. nickel allergy can cause different clinics ranging from localized lesions to systemic reactions. we want to emphasize that a detailed medical history and the patch test would enable clinicians to demonstrate hidden allergens and then make a correct diagnosis. case report: autoimmune progesterone dermatitis is a condition of hypersensitivity to progestogens. it is not an easy diagnosis given the variety of clinical presentations it may have, ranging from eczema, urticaria, erythema multiforme, folliculitis, to angioedema or even anaphylaxis. manifestations are cyclical, occurring when the levels of progesterone are higher, this is, at the luteal phase of the menstrual cycle, and disappear during menses, with the physiological decrease of the hormone. it can also be triggered by exposure to exogenous progestins. we report the case of a -year-old woman with a cyclical erythematous and violaceous rash related to the menstrual period. the symptoms typically began - days before the onset of menses and ended - days before. the diagnosis was based in the clinical history and intradermal skin tests: skin prick testing with levonorgestrel and medroxyprogesterone were negative, but the intradermal skin test with medroxyprogesterone was positive at a concentration of mg/ml. we performed intradermal testing with the same concentration in three other women with no symptoms to exclude an irritative reaction, which were negative. autoimmune progesterone dermatitis is, perhaps, not so rare, but rather poorly recognized and reported, and thus, underdiagnosed. clinicians should be aware and include always this condition in the differential diagnosis, especially in cases of atypical or intractable skin eruptions. case report: a year old male was referred to a community allergy clinic for assessment of chronic urticaria (cu). allergy assessments for foods, inhalant and inducible physical triggers revealed no association. an autoimmune workup followed, with treatment consisting of standard dose antihistamines (h and h ). blood work revealed a persistently low hemoglobin with low-normal ferritin. hematology consulted and followed attempted iron replacement to no avail. skin biopsy revealed neutrophilic rich urticaria with the presence of eosinophils. serum protein electrophoresis (spep) revealed a monoclonal gammopathy with elevated igm, felt to be of undetermined significance (mgus). c-reactive protein (crp) was consistently elevated ( , ) in conjunction with anemia. rheumatology consulted and cleared of any evidence of vasculitis. hematology considered the anemia to be of chronic disease linked to cu. the cu was resistant to treatment including high dose antihistami- background: isolated head and neck angioedema (ae) can be mediated by bradykinin (bk) or histamin (hi) . the objective of our study was to determine which etiology was most frequent in cases of death by asphyxiating ae in france. we sought all cases of death by isolated asphyxiating ae reported in france between and via death certificates and/or the national pharmacovigilance database. results: the overall mortality by asphyxiating ae for all causes was . / million inhabitants. the death rate of bkae per million inhabitants was . and lethality of . per thousand patients per year. the death rate of hiae per million inhabitants was . and lethality of . per thousand patients per year. we found a times higher risk of death in case of bkae than hiae. conclusion: consequently, particularly severe episodes must be initially considered as bradykinin mediated and quickly reassess any first-line treatment that is inappropriate. case report: we present the case of a -year-old man who suffered recurrent abdominal pain since age of eight, leading to unnecessary emergency surgical interventions and endoscopies before hereditary angioedema due to c inhibitor deficiency (c -inh-hae) was diagnosed at the age of . rare subcutaneous swellings were considered allergic reactions preventing proper diagnosis. family history, positive for recurrent abdominal pain and swellings was totally neglected until diagnosis of c -inh-hae type i was established through appearance of severe oro-facial symptoms in the propositus' grandson. the diagnosis was suggested by the boy's mother, directed by educational materials available in the international hae patients' association website (www.haei.org). this report highlights and emphasizes the importance of accurately evaluated personal and family history to suspect condition that are scarcely known to the majority of physicians. highlights: diagnostic delay in hae and iatrogenic procedures are an underestimated problem, hiding undefined consequences, possibly destructing an entire lifetime. correct, publically available information provided by patients' associations raise awareness about the disease and could put the milestone of establishing correct diagnosis. de luque v ; lara p ; guardia p ; jimenez ar virgen macarena hospital, seville, spain; hospital virgen macarena sevilla, seville, spain background: in the protocol for the study of patients who consult for recurrent acute angioedema with facial involvement, the contactant battery is included (epicutaneous test). we review the results in our patients with facial angioedema to evaluate the contactants to which these patients present sensitization, some of them coexisting with contact dermatitis clinic. we reviewed the patients referred to the clinic for recurrent acute angioedema with facial involvement and to whom a standard battery epicutaneous test was requested. in all these patients, other habitual triggers included in the diagnostic protocol (food, medications, autoimmune diseases, bradyinergic aea/complement deficit …) were ruled out. conclusion: it seems to be profitable to continue including in the diagnostic battery of patients who consult for aea with facial affectation, study of epicutaneous with standard battery. it is a small sample, but the data correlate with what has been published, being more frequent the sensitization to contactants in women and the contactant more frequently involved nickel sulphate. | ace inhibitor-related angioedema-the value of history taking background: angioedema is a well-recognized side effect of angiotensin-converting enzyme (ace) inhibitor therapy. although it occurs in < % of the patients who take these drugs, it seems to be responsible for % of the episodes of angioedema. this entity is underdiagnosed and failure to recognize it leads to recurrence of episodes, with an impact on morbidity and increased risk of serious reactions. our objective is to analyze the clinical, therapeutic and orientation approach of patients diagnosed with ace inhibitor-related angioedema, evaluated at the outpatient consultation (oa) of immunoallergology (ia). a -year retrospective study was performed by analyzing the clinical files of all patients diagnosed with angioedema observed in oa of ia. the following variables were analyzed: gender; age; clinical data; evaluation in emergency department (ed); therapy in the episode; evolution and orientation. the chi-square test was used to study the association between categorical variables: "established therapy"/"disease evolution" and "place of reference"/"withdrawal of ace inhibitor". results: review of cases of patients referred for angioedema. only in % the final diagnosis was "ace inhibitor-related angioedema". the mean age of the patients was . years and % were male. the location of angioedema occurred in the tongue in % and in the remaining sites (lip, hemiface, tongue and hemiface, tongue and lip) appeared in the same frequency, %. none of the patients had airway obstruction. during the episode of angioedema, % of patients were not referred to ed and the therapeutic approach was done with antihistamines in %. in patients who were referred to ed ( %), antihistamines and corticosteroids medications were administered in %. regarding the evolution, it was verified that the duration of the episode was independent of the established therapy (p > . ). regarding the place of reference, % of the patients were referred form hospital (ec or ed) and, in these, the ace inhibitor was suspended in %. in patients referred from general practitioners ( %), in none of them the ace inhibitor had been withdrawal. a causal association between the use of ace inhibitors and the episode of angioedema becomes crucial, since drug withdrawal is indicated. a reference for ai oa should be weighed. therapy with antihistamines and corticosteroids has no proven efficacy. hereditary angioedema (hae) seen by physicians belonging to the hae scientific committee of the aaaeic background: patients with c -inh-hae frequently suffer from anxiety and stress. the impact of prophylactic treatment on anxiety and stress in c -inh-hae patients is largely unknown. here, we analyzed data from the apex- study, a phase ii study that investigated the effects of the oral kallikrein inhibitor bcx . method: c -inh-hae patients with a history of at least hae attacks per month were randomized to receive four different doses ( , , , . mg) of bcx or placebo for days. the depression anxiety stress scale (dass) was administered at baseline and at day . the dass consists of three self-reported scales designed to measure the negative emotional states of anxiety and stress. subjects used a -point severity/frequency scale to rate the extent to which they have experienced each state. results: baseline dass total scores as well as anxiety and stress domain mean (sd) scores for the mg treatment arm (n = ) were . ( . ), . ( . ), and . ( . ) points respectively. placebo scores were generally similar or slightly lower at baseline than for the mg treatment arm. the dass questionnaire data showed statistically significant improvements in total score vs. placebo at day (− . method: a two-phase mixed methods approach was used to develop the hae-rt tool including: phase : delphi study [hae specialists (n = ) and national patient advocacy group members (n = )] was conducted to reach consensus ( % agreement) on predictor variables to include in the tool. phase : retrospective chart review was conducted to assess the predictive findings of the decided variables. a convenient patient sample presenting with angioedema (with and without hae) between january -january were included in the study. results: nine of invited experts ( %) participated in the delphi study. of hae-specific predictive variables, reached consensuses including: (i) recurrent angioedema; (ii) absence of urticaria; (iii) recurrent abdominal pain/swelling; (iv) lack of response to allergic therapy. the retrospective study included patients (n = with hae; n = non-hae; overall % female). hae patients were significantly more likely to have a family history of hae ( % vs %; p < . ); previous recurrent angioedema ( %; p < . ); present with no hives ( %; p < . ); previous recurrent abdominal pain ( %; p < . ); and % responded to allergy treatments (p < . ). a regression analysis categorized observed frequencies (actual patient outcomes from chart review) versus predicted (by model); plotted on a by table and calculated the sensitivity and specificity of the hae-rt which resulted in one hundred percent for both. conclusion: our study demonstrated that expert involvement led to the identification and prioritization of variables that when included an hae-rt tool, were associated with a high level of sensitivity and specificity when applied to known patients. the next step is to observe the effect of the hae-rt tool on patient care in the ed. method: evaluation of cardiovascular manifestation included morphology, serum level of troponin t, electrocardiography (ecg) and echocardiography. evaluation of pulmonary manifestation included spirometry, diffusing capacity of the lung for carbon monoxide (dlco) and evaluation for mastocytosis included bone marrow biopsy and serum total tryptase measurements. results: in the study there were patients - women and men between and years old (the average age was ). there were ( . %) patients with mpcm, ( . %) with bmm, ( . %) with ism and ( . %) with ssm. the average level of serum tryptase was . μg/l ( . - ) . troponin levels was within the normal range in all patients. one patient had lowered the ejection fraction (eflv= %). no one patient had restriction. the average value of a forced lung capacity was . l ( %) and a total here, we describe a case series of twelve mis patients seen at our department over a -year period and report how many of these patients have sm. common phenotypical manifestations of acute hae episodes in this region, to review therapeutic challenges in a rural setting in comparison with world standards, and lastly to evaluate the socio-economic burden inflicted by the disease. method: a sample of individuals from a total of . the exclusion criteria was the inability to attend booked appointments more than times in year ( ). the following methods were used: an interview to formulate a family tree identifying affected individuals in contiguous generations, and review of the acute presentations in the past year ( ) . a questionnaire to obtain the relevant hae associated socio-economic burdens. a chart review to identify the therapeutic strategies in this region. c inh levels, and complement c to confirm the diagnosis. results: polygamy as a local culture was found to be an important factor that perpetuated the genetic burden of the disease. c inh and c levels confirmed hae in all participants individually. clinical features during acute attacks included swelling of extremities ( %), facial swelling ( %), neck swelling ( %), and laryngeal swelling ( %). therapeutic strategies for acute attacks included fresh frozen plasma or fresh dried plasma. danazol was used for prophylaxis. hae has had a significant negative impact upon the socioeconomic status of the affected individuals. conclusion: hae is a newly identified disorder in the broad spectrum of allergy medicine in kwazulu-natal. the diagnosis is simple to confirm but requires an initial high index of suspicion, and therapeutic management still poses a challenge in this region due to lack of resources. genetic counselling is of paramount importance during intervention since polygamy forms part of most cultures in this region. a support strategy is highly recommended in order to help alleviate the socio-economic burden posed by the disease in this region. the socio-economic burden secondary to hae ( participants/ identical questions each) question ( - points) question ( - points) question ( - points) question ( - points) question ( - point) method: a total of medical faculty senior year students were included to study on a voluntary basis. students are divided into two groups. one group was given visual user guide that has not been modified, and a visual user guide on which we have modified to the other group ( figure ). then they were asked to show how to use the inhaler spacer. results: the mean age of the volunteers was . ± years and ( . %) were male. there were students in the group without modification of the visual user guide and students in the other group with modified the visual user guide. sixty-four per cent of the modified user guide group showed correct use of the inhaler spacer, while % of the unmodified group showed correct use (p = . ). the group that given modified visual user guide was more successful in all of the display steps of the inhaler spacer. conclusion: modification of the currently available visual user guide of inhaler spacer in our country will increase the correct usage rate. results: mean fev , fvc, fev /fvc z-score were . ( . - . ), . ( . - . ), . ( . - . ), respectively. restriction had ( . %) and obturation ( . %) patients. fev (p < . , r = . ) and fvc (p = . , r = . ) decreased with age (%pv and z-score). background: children who were treated for leukemia are known to have developed long term impairment of lung function. the reasons that complication are only partially known. the aim of this study was to asses pulmonary function in children treated the lower dlco is the most frequent abnormality in childhood leukemic survivors. hsct and pulmonary infection (in particular cmv pneumonia) is a strong risk factor for impairment of dlco in children. clinical manifestation of dlco impairment is poor exercise tolerance. a screening for respiratory abnormalities in survivors following treatment for childhood haematologic malignancies, seems to be of significant importance. | phenotyping allergic respiratory diseases: an unsupervised classification using latent class analysis allergen groups was significantly associated to uawi (aor[ % ci]: . [ . - . ] ), compared to uasi. results: . % of br and % of py were women, median age was years, % br and % py reported having more than four years of training. although they recognized the main symptoms of ar, % br and % py never asked whether the patient had a medical diagnosis of ar; . % br and . % py did not ask whether the symptoms occurred when close to animals or allergens; % br and % of py did not ask if the patient had a medical diagnosis of asthma; % br and % py did not ask if rhinitis worsens asthma symptoms and . % br and . % py did not ask whether symptoms of rhinitis interfere with their daily activities. results: there was a predominance of female (br: %, py . %, uy: %) median age years old, / worked in the community and / in the emergency departments, % of the br had more than years of education, % from py had between and years, and % from uy had been graduated for less than year. br/uy recognize the main symptoms of ar, however % of those from uy do not ask: if the patient has physician diagnosis of ar, % present shortness of breath, and % a medical diagnosis of asthma, % if rhinitis worsens asthma symptoms and % if symptoms of rhinitis interfere with the patient's daily activities. the prescribed treatment varied a lot, the intranasal corticosteroid use rate was: bd: %, pd: % and ud: %. % of doctors in py, . % in br and % of uy never refer the patient to the specialist. . % of pcd of br, % of py and . % of uy are aware of aria guideline. conclusion: although ar is largely attended by pcp, recognition of symptoms and their impact on asthma, as well as the knowledge about aria guide is low and treatment is not always prescribed according to best practice. allergy education programs, with an emphasis on ar and aria guide, need to be directed to pcp in la for the better assistance of ar patients. | assessing knowledge of allergic rhinitis among final year medical and pharmacy students in croatia-curriculum change necessity? the two factor structured questionnaire was formed by the authors regarding the topics mentioned. t-test was used for statistical analysis. the global results were formed as composites of ( ) ar general characteristics, ( ) ar treatment approach, and ( ) the participants' overall knowledge. of the respondents, ( . %) were female and ( . %) were male (p < . ). medical students had a median score of of correct answers on ( ), of on ( ), and of on ( ), whereas pharmacy students had median score of of correct answers on ( ), of on ( ), and of on ( ). there were no significant differences in knowledge between two student groups. the results indicate an inadequate level of knowledge of ar in both groups, especially regarding the therapy approach. since general practitioners and community pharmacists have a major role in providing treatment to patients suffering from ar, it is important to develop advanced knowledge on this topic during medical and pharmacy degree courses. despite a relatively small study population, it would be advisable to introduce change by improving the core curriculum regarding ar with more emphasis on treatment, but additional research on this topic is necessary. tan r ; cvetkovski b ; kritikos v ; price d ; yan k ; smith p ; bosnic-anticevich s woolcock institute of medical research; university of sydney, sydney, australia; observational pragmatic research institute pte ltd, singapore, singapore; royal prince alfred hospital, sydney, australia; clinical medicine, southport, australia; griffith university, sydney, australia background: people with allergic rhinitis symptoms frequently selfselect over-the-counter medications from community pharmacies without seeking advice from a health care professional. this increases the incidence of complications due to delayed diagnosis and suboptimal treatment. this study aims to (i) compare the demographics, clinical characteristics and medication selected, between pharmacy customers who choose to self-select and those who interacted with a pharmacist when purchasing medication for allergic rhinitis symptoms, and (ii) identify the key factors associated with allergic rhinitis patients' medication self-selection behaviour. a cross-sectional observational study was conducted in a convenience sample of community pharmacies from the sydney metropolitan area. data were collected using a researcher administered questionnaire that included: demographics, pattern of allergic rhinitis symptoms, their impact on quality of life, factors triggering allergic rhinitis symptoms and medication(s) selected. logistic regression was used to identify key factors associated with participants' medication self-selection behaviour. results: of the recruited participants, were identified with allergic rhinitis, of which . % were female, . % were aged more than years old, . % had a diagnosis of allergic rhinitis, and . % self-selected medication(s). significant differences were noted in allergic rhinitis symptoms, impact of allergic rhinitis on quality of life and medication(s) selected between participants who chose to self-select and those who interacted with a pharmacist. participants who experienced moderate-severe wheeze were times more likely to self-select allergic rhinitis medication(s), and those who had allergic rhinitis symptoms impacting on their quality of life were . times less likely to self-select allergic rhinitis medication(s). conclusion: there is a high incidence of self-selection of over-thecounter treatments for allergic rhinitis symptoms in community pharmacy, with the majority of allergic rhinitis sufferers failing to seek pharmacist advice. this research identified predictors of medication self-selection behaviour in community pharmacy among people with allergic rhinitis, which can inform the design of tools/strategies and targeted interventions, aimed at improving pharmacist engagement and future practice in optimising allergic rhinitis management. the weir family health clinic, cork, ireland; university college, cork, ireland background: allergic rhinitis is a common condition that is predominantly managed in primary care. the incidence of allergic rhinitis is increasing. it is frequently under diagnosed, misdiagnosed and mistreated. it has a significant impact on patients' health related quality of life and represents a huge cost both to healthcare systems and society. the aim of this study was to implement appropriate guidelines regarding the management of allergic rhinitis in primary care and evaluate the effect on patients' health related quality of life. method: patients with a history of allergic rhinitis were selected from three general practice bases in west cork, ireland and quality of life of patients was assessed initially in year one and followed up one year later in a general practice setting using the standardised rhinoconjunctivitis quality of life questionnaire (rqlq). allergic rhinitis and its impact on asthma (aria) guidelines and appropriate prescribing were implemented during this year and patient education and structured follow up was arranged in the intervention group. this was compared with the control group who received usual care. results: valid responses were received, from the control group and from the intervention group. the study demonstrated a statistically significant difference in quality of life in the intervention group. in the adult intervention group the quality of life score decreased between and representing an improvement in their quality of life, (t = . ; df= ; p < . ). the difference in the score between the control and intervention groups in was also statistically significant.(t = . ; df= ; p < . ). the numbers in the adolescent groups and paediatric group also demonstrated an improvement in quality of life but the sample size was too small to demonstrate a statistically significant difference. conclusion: as the majority of patients rely on their general practitioners for treatment and diagnosis of allergic rhinitis, primary care represents an important area to target in the management of allergic rhinitis to improve patients' quality of life. the implementation of guidelines has been shown to improve patients' quality of life. this study demonstrates this care can be delivered in a primary care setting with an improvement in patients quality of life but substantial investment in education and resources available to primary care physicians is needed. | the predictive value of allergy tests in the diagnosis of peanut allergy in adults rey-garcia h; gunawardana n; wheeler k; scadding g; durham s; skypala i royal brompton hospital, london, united kingdom background: adults presenting with either new-onset symptoms attributed to peanuts or with early-onset peanut allergy, often wish to know whether they should continue to avoid peanuts. clinical history and standard tests may be sufficient to provide an answer, but for many the tests are inconclusive and an oral food challenge is required. this review was undertaken to determine the most accurate tests. conclusion: these data suggests that peanut spt and ara h provide the most accurate prediction of the outcome of oral food challenge in adults. should components not be available, then spt would be the test of choice being more accurate in all aspects than sige. combining spt and sige improves the sensitivity and negative predictive value of spt alone. however, the best combination is spt and ara h , which increases the overall accuracy to %. further studies are needed before it can be determined whether peanut diagnostic tests can replace the oral food challenge in adult patients. method: retrospective chart review was carried out in a community allergy clinic. patients with rap, bloating and altered stools who underwent bt were characterized by age, gender and atopic status. a separate study to assess patients' outcome post-dietary counselling was carried out to determine impact on symptom management. results: thirty-four patients were assessed for fi from january to december . female gender predominated ( / , %) with an average age of years at presentation. results of fi were positive in / ( %), borderline in / ( %) and negative in / ( %). the average age of patients with a positive, borderline and negative tests were , and , respectively. of the patients who tested positive for fi, ( . %) had comorbid inhalant allergies alone, ( . %) had comorbid (unrelated) food allergies alone, ( . %) had inhalant and food (unrelated) allergies, and ( . %) were non-atopic. of the patients who tested negative for fi, ( . %) had comorbid inhalant allergies alone, ( %) had comorbid (unrelated) food allergies alone, ( . %) had inhalant and food (unrelated) allergies, and ( . %) were non-atopic. conclusion: patients investigated for carbohydrate intolerance with rap, bloating and altered stools were predominantly female ( %). fi was confirmed in half. atopic status did not help differentiate between the fi positive or negative groups. results of a fod-map elimination diet are separately reported. conclusion: post-bt, % of patients reported symptom improvement. patients who implemented fructose or fodmap avoidance reported symptom improvement. one patient who tested negative for fi reported symptom improvement with a low fodmap diet. patients suspected as being fructose intolerant may benefit from a fructose restriction or fodmap diet, while awaiting bt confirmation. this form of dietary intervention may assist and shorten the natural history of non-specific chronic gi symptoms. inappropriate referrals to a uk paediatric tertiary allergy clinic demonstrate lack of allergy education and knowledge in primary care marriage de bristol royal hospital for children, bristol, united kingdom background: up to % of children have a food allergy. allergy has become an explanation for all manner of nebulous symptoms and self-diagnosis is common. sham allergy tests are easily available giving incorrect results and resulting in unnecessary, potentially harmful abstracts | parentally-imposed dietary exclusions. there are million allergyrelated google searches per year. the rising prevalence of perceived allergic disease has led to an increase in health service utilisation, including increased referrals to secondary care. clinic waiting lists are long and children with severe food allergies have to wait longer than necessary to be seen method: uk paediatric tertiary allergy clinic referrals were prospectively reviewed over three months. five inappropriate referrals deemed most reflective of poor knowledge in primary care were selected as case summaries to highlight this gap in knowledge. results: : schoolchild referred for investigation of allergic cause for a red, watery eye after splashing juice in her eye whilst cutting a kiwi, despite having a co-existent dendritic ulcer. : schoolchild referred for peanut allergy testing after inhaling a peanut and developing wheeze, with all respiratory symptoms resolving following peanut removal. : young child referred for peanut allergy testing after developing a rash on leg following skin contact with faeces hours after ingestion of peanut butter. : teenage boy referred for investigation of likely peanut allergy despite eating peanut butter and tree nuts almost every day. the family were concerned he was allergic to peanut butter. : toddler referred for milk allergy investigation after developing urticaria lasting hours minutes after drinking a bottle of milk. the child had consumed cow's milk formula since birth, and continued to consume milk daily for a further five months following the episode of urticaria. conclusion: provision of allergy services in the uk is poor and lack of investment in allergy services has led to suboptimal recognition and management of food allergy in primary care. allergy education provision for primary care practitioners is inadequate and fails to empower healthcare professionals to discern between allergy requiring full investigation and management, parentally-diagnosed allergy or symptoms which clearly have no association with allergy. progress to improve primary care training for allergy needs to be optimised to prevent further unnecessary referrals and lengthening clinic waiting lists. background: in case of allergic reactions to food or insect venom, quick and adequate treatment, based on clear instruction for use of emergency medication and calling for help, is necessary. however, daily practice shows that patients do not use the prescribed emergency medication because they are afraid to use the epinephrine auto-injector or they do not know how to use it. information and instruction offered by a reliable app could be a useful aid. we aim to develop an app for adult patients and children older than years with allergy to food or insect venom, which offers a step-wise approach to support patients, their relatives or acquaintances in case of an allergic reaction. method: first, the content of the app, including a step-wise approach to treat the allergic reaction has been determined, based on literature, a survey about needs of patients and on consultations of healthcare professionals. subsequently, a web-based prototype has been developed with an adult profile and children profile. the content and flow of this prototype was tested by the project group, as well as by selected healthcare professionals and patients and improved according to the test results. next, the revised prototype was submitted to representatives of patient and professional organizations for final approval. currently the procedure for ce approval is ongoing. finally, the app will be built and offered to the market for ios and android. results: a web-based prototype of the allergy app is available with two profiles: adult and older children. the app is useful for patients with a doctor's diagnosed allergy to food or insect venom, who received emergency medication and instructions to use prescribed medication in case of an allergic reaction. based on severity of complaints, the user is informed about the steps to treat the allergic reaction. in case of a moderate to severe reaction, the patient is advised to use an epinephrine auto-injector, to call the emergency number and, if prescribed, to use medication such as antihistamines, prednisone or inhaler. besides that, the app provides links to websites of expertise centres and patient organisations and includes instructions how to use the epinephrine auto-injector. conclusion: the allergy app will help patients, their relatives or acquaintances to adequately treat an allergic reaction to food or insect venom. involving patients and professionals in the development of the app will contribute to its acceptability and usability. | electronic documentation of drug allergies in a tertiary hospital in singapore: are we relying too much on it? choo kjl; garuna murthee k; naing cs singapore general hospital, singapore, singapore background: singapore has hospitals shared between public and private healthcare system. its healthcare system, ranked # in the world by who in serves a multi-racial population of . million of which % are above years old. drug allergy alert cards (medik awas) were started in s by singapore medical association to improve patient safety. work on computerisation of drug allergy and medical alerts started in the s, a precursor to today's critical medical information system (cmis). cmis serves as a platform across all public hospitals in singapore. it promotes uniform reporting of drug allergy and notification of adverse drug events to the health science authority. yet, we found that there is a lack of awareness of one's own drug allergies. method: all patients admitted to ward (general medicine ward) at singapore general hospital from july to oct were screen for any previously documented drug allergies. consenting patients who had previously documented drug allergies on cmis were interviewed to document their demographics, education level, current medications, knowledge of their own drug allergies and possession of a drug medication alert card. the answers were then compared with their electronic documentation of drug allergies for accuracy. we interviewed patients aged - with documented drug allergies during the recruitment period. % had secondary school education or higher. the majority ( %) spoke english and ( . %) mandarin. almost half had medical problems and are on long term medications (mean . medications); hypertension and diabetes being the top two common diseases. % of the patients could accurately relay their drug allergies; antibiotics and analgesia being the most labelled. only % had a drug allergy alert card while the rest both rely on the hospital's electronic documentation and/or their caregivers to record and relay their allergies to future prescribers. about % received prescription from multiple healthcare sites in both the public and private healthcare system. we found patients' knowledge of their own drug allergies dismal. the cmis electronic documentation provided a false sense of security. unfortunately, the cmis platform is not available to all private hospitals, increasing the risk of mis-prescription due to the lack of information. unless this is made available nationally, patients with drug allergies should be given some written documentation, either a letter or medik awas. how frequent are they and how are they treated? method: for this cross-sectional study, participants were recruited in the waiting rooms of local doctors in the rural bavarian forest region of southern germany (q / ). a paper questionnaire was handed out to the participants, asking for allergies (pollen, animal hair, bee and wasp venom, drugs, food, house dust mites, contact allergies and other allergies) and how or rather by whom (e.g. general practitioner, specialist, self-treatment) these allergies are treated. results: participants with a mean age of . years (sd= . ) and % women were included in this study. . % indicated to have at least one allergy, including pollen allergy most frequently ( . %). women had significantly more often at least one allergy than men (rr= . ; ci [ . ; . ] ) and for almost all examined allergies a significant higher risk of disease. younger age groups indicated more often to have at least one allergy ( - [. ; . ]) seemed to be affected less. participants indicated most frequently that their allergy was treated by a general practitioner ( . %), except of the - -yearold young adults who indicated "no treatment" most frequently ( . %). conclusion: there is a high self-reported prevalence of allergies in the examined rural bavarian region, that increases with decreasing age and is significantly higher among women. moreover, the data on the absence of an appropriated treatment for allergies is alarming. therefore, medical care needs to be improved in rural regions to lower the burden of allergies. | the prevalence of the burnout syndrome among medical professionals involved in allergology education programmes astafieva n ; kobzev d ; gamova i ; perfilova i ; udovichenko e ; skuchaeva l ; michailova i ≥ ) and rpa (low ≥ - , subscales were calculated and analyzed. results: on average students demonstrated: moderate/high ee scores ( - ); moderate/high dp scores ( - ) and moderate rpa scores ( ); higher rpa scores were common ( . %) among junior students, which is also linked with their levels of engagement, and lower ( %) among senior students. junior specialist (starting specialization) had very low scores in all subscales and expressed a very high motivation in their course and new profession. clinical allergologists with significant experience demonstrated moderate / high ee scores ( - ); low dp ( ) ( ) ( ) ( ) ( ) and rpa (below ) scores. high ee scores associated with pressures of service were compensated by a substantial loyalty to their profession and positive assessment of the outcomes of their work. clinical academics demonstrated the highest level of ee scores ( + among + % of the group) with low to moderate dp and rpa scores, the latter being associated with a loyalty to their profession. it was also possible to identify a correlation between engagement in research activities and lower rpa scores. conclusion: burnout is a complex and multifaceted phenomena, which requires further investigation. however, this research identified that students and junior specialists involved in allergology and clinical immunology programmes with higher levels of engagement and motivation to acquire new specialist knowledge had lower levels of ee, while loyalty to the profession and positive assessment of the outcomes of clinical and research work allows to compensate high levels of ee among experienced practitioners and clinical academics and to reduce burnout effects overall. | appeal (allergy to peanuts impacting emotions and life): the first pan-european study to evaluate the psychosocial burden of living with peanut allergy deutscher allergie-und asthmabund (daab)/german allergy and asthma association, mönchengladbach, germany; food allergy italia, padua, italy; aepnaa asociación española de personas con alergia a alimentos y látex, madrid, spain; afpral association pour la prevention des allergies, paris, france; asthma-allergy denmark, roskilde, denmark; anaphylaxis campaign ireland, cork, ireland; brainsell ltd, london, united kingdom; aimmune therapeutics, london, united kingdom background: peanut allergy, one of the most common and rapidly growing food allergies, is most frequently a lifelong condition. current management is limited to avoidance and symptomatic treatment of allergic reactions when accidental exposures occur. peanut allergy can affect the quality of life (qol) of individuals and also that of parents/caregivers and family members. appeal was designed to assess the impact of peanut allergy on qol in peanut-allergic individuals and their parents/caregivers and families. method: the first, quantitative part of appeal is described here and consisted of a pan-european, cross-sectional online survey of approximately minutes in length. the study was conducted in the uk, republic of ireland, france, spain, germany, italy, the netherlands and denmark. over participants were recruited via patient advocacy groups or directly through a specialist survey recruitment panel. ethics committee approval was obtained and all participants provided their informed consent. eligible participants were: ( ) parents or caregivers of a child/adult with peanut allergy; ( ) parents or caregivers responding as a proxy for a child aged under ; ( ) adults. all allergic subjects had self-reported diagnosed peanut allergy. after several screening questions, eligible participants answered a set of clinical questions about their (or their child's) allergies and other conditions, details on the peanut allergy diagnosis, contact with healthcare professionals, worst allergic reaction to date and use of emergency medicine. depending on whether they were an allergic adult, a parent responding on behalf of the child, or a parent/caregiver recounting their own experience, they then answered specific questions on restrictions on life choices, coping strategies and the impact of peanut allergy on feelings and emotions of families, friends and other people. sociodemographic questions completed the questionnaire. the results of the survey are being summarized using descriptive statistics and the data are being analysed on a pan-european level, by country, and according to the participant's perspective (parent, caregiver or individual). conclusion: this comprehensive, pan-european online survey has been specifically designed to uncover the psychosocial burden and effect on qol of peanut allergy in terms of individuals' lives and those of their families. results: pediatric patients were included in the study. . % (n = ) were male. the median age of onset of symptoms (interquartile range) was months ( - ).the median age (interquartile range) of diagnosis was months ( - ). initial reactions associated with cmpa were observed in . % of patients before months old. patients' diagnoses were atopic dermatitis ( %), urticariaangioedema ( %), anaphylaxis ( . %), proctocolitis ( . %), atopic dermatitis and urticaria ( . %), food protein induced enterocolitis ( . %) and eosinophilic esophagitis ( . % method: the study involved patients, who are two years old (and above), diagnosed with cow milk allergy and are observed for at least six months in our hospital. socio-demographic features of the patients, their symptoms, symptom-start ages, age of diagnosis, clinical findings at diagnosis and during observation were recorded in data collection questionnaires. results: the samples were ( . %) boys. the average symptomstart age was observed to be months , average diagnosis age months ( - ) and average age of final check months . when symptoms at entry were observed, . % of the patients had dermal system, . % gastrointestinal system, respiratory system disorders, and % were detected to have developed anaphylaxis. among the patients diagnosed with cow milk allergy, . % showed food reaction to nutrients with lge agents, . % to mixed types, and % to nutrients with non-lge agents. it was also observed that, in the end of . ± . -month observations, sensitivity to cow milk was observed to continue in ( . %) of our patients. when tolerance improvement rates among the patients were compared, anaphylaxis (p < . ) during entry were observed to be influential in continued allergic state. ( . %) patients were able to consume yoghurt, ( . %) patients could consume dairy products and ( . %) patients could not consume dairy products. conclusion: in the end of our investigation, it was observed that ( . %) of the patients developed cow milk tolerance before the age of . when the factors enabling the continuation of sensitivity in cow milk allergy were investigated, anaphylaxis during entry, entry specific lge and pasteurized milk antigen as well as high skinprick test results were detected to be significant. | initial lower threshold was a risk factor of severe adverse reaction during oral immunotherapy for cow's milk anaphylaxis results: before oit, median age was . years old, median threshold to induce initial reaction was . ml, to induce anaphylaxis was . ml of cm and median milk specific ige was . ku/l. twentyseven subjects ( %) dropped out from the protocol, subjects | investigation of heat and matrix effect on milk proteins' allergenicity and the development of hypoallergenic food products reduce some milk proteins' allergenicity (ß-lactoglobulin) . in this project we aimed to investigate the effect of heat and matrix on different milk protein fractions through maillard reaction and eventually develop hypoallergenic food products that have milk protein with low reaction risk. method: milk cake matrix is prepared in different flour/sugar (f/s) ratio ( f/ s, f/ s, . f/ s) and baked minutes at °c. proteins that cake contains are separated using sds page and stained with coomassie blue to check total protein. in parallel specific proteins are detected by western blotting using pooled sera from patients with milk specific ige> ku/l for incubation results: in normal milk cake recipe ( f/ s) ß-lactoglobulin bands are disappeared but casein bands did not differ in size. in order to investigate the matrix effect f/s ratio is changed and it is found that when this ratio decreases, with the affect of heat and maillard reaction, milk casein bands' intensities also decrease in sds gel coomassie staining. in western blot experiments it is also shown that milk specific ige bound weakly to casein bands in low f/s ratio cake ( . f/ s) whereas in cakes that have high f/s ( f/ s) ratio it bound significantly higher. and ß-lactoglobulin proteins' structure and lower the milk specific ige bindings to milk proteins in low f/s ratio cake through maillard reaction. | extensively hydrolyzed formulas for the management of cow's milk protein allergy in infants: is extensive hydrolysis sufficient to guarantee success? method: to better understand the range of ehfs, we aimed to analyse samples of commercially available ehfs from countries and various manufacturers, with a focus on suitability for cmpa management. samples were de-identified and coded for the analyses. molecular weight (mw) distribution of hydrolysates and residual proteins and peptide profiling were assessed with sds-page gel and size exclusion-high-performance liquid chromatography (se-hplc), as they reflect both the design of the formula and the quality management applied during production. osmolarity, nitrogen fractions, lactose content, total and free amino acids, β-lactoglobulin, and casein content were quantified and β-lactoglobulin residual allergenicity was assessed. results: peptide mw distribution displayed significant variation, with the percentage of peptides with mw > . kda varying from % to %. mw distribution was shown to be positively correlated with β-lactoglobulin specific in vitro degranulation. twenty % of samples had non-measurable β-lactoglobulin content (smaller than or at the limit of quantification (loq): . mg/kg); however, % of samples had β-lactoglobulin content greater than the loq, with high variability from . to mg/kg. surprisingly, even in samples featuring a high degree of hydrolysis, significant levels of residual β-lactoglobulin were quantified. conclusion: lack of consensus over the definition of 'extensively hydrolysed' is reflected in the wide range of degree of hydrolysis in commercially available ehfs, and can result in products that are mislabelled as 'extensively hydrolysed' and may be high-risk or even unsuitable for the management of cmpa. results of these analyses also highlight that degree of hydrolysis alone is not sensitive enough to characterise ehfs, and that whilst a high degree of hydrolysis is desirable, further quality control measures are essential to ensure clinically safe and suitable products. actionable guidelines to better define hypoallergenic formulas based on extensively hydrolysed milk proteins are warranted. background: assessing the effect of baked milk products on accelerating unheated milk tolerance in patients with cow's milk allergy. method: a randomized clinical trial was done on patients ( months- years old) divided randomly to case and control groups matched for age and sex. baked milk in form of muffin for months followed by baked cheese in form of pizza for next months was given to the patients in case group. skin prick test and serum ige (sige) levels (immunocap) of milk, casein and betalactoglobulin were measured before and after the study. the ones having milk sige less than ku/l and being asymptomatic during the study underwent oral food challenge test for evaluating unheated milk tolerance. chualalongkorn university, bangkok, thailand; kk women's and children's hospital, singapore, singapore; university of the philippines, philippine general hospital, manila, philippines background: problems in recognising cow's milk allergy (cma) and lactose intolerance (li) in infancy may lead to a delayed or incorrect diagnosis, as well as inappropriate dietary interventions. method: between january and november , a survey was conducted online in china, india, singapore, thailand, mexico, kuwait, united kingdom, australia, and paper-based in the philippines. the survey consisted of multiple-choice questions on cma and li in infants aged under months, two case scenarios (non-ige cma and anaphylaxis) and questions on educational needs (likert scale [ ] [ ] [ ] [ ] [ ] . data on the type of medical practitioner and clinical setting were collected. responses were summarised as percentages and categorised by country. results: responses were received from general practitioners ( . %), paediatricians ( . %), paediatric allergists ( . %), paediatric gastroenterologists ( . %) and other specialities ( . %). there were significant misconceptions about the clinical importance of primary li in infancy. while primary li rarely manifests before years of age, . % of participants felt it was a significant clinical problem in the first year of life. regarding secondary li, % of respondents recommended lactose restriction for viral gastroenteritis, and % for cow's milk protein-induced enteropathy. while the management of ige cma was relatively well understood, there were greater knowledge gaps for non-ige cma. % of practitioners appropriately identified extensively hydrolysed formula (ehf) as first-line treatment of cma in formula-fed infants. however, the distinction between lactose-free and lactose-containing ehf appeared to be an area of uncertainty. in india, . % used soy-based formulas as first- results: patch tests were positive in ( . %) and negative in others. positivity to milk was seen in patients ( . %), to soy in ( . %), to egg white in / ( . %), to wheat in / ( %), to potatoes / ( . %), to corn (maize) in / ( . %), to rice in / , and to peanut in / ( . %). patients were requested to withdraw the suspected food(s) from their diets during a months period. preliminary follow-up data show the improvement of one or more symptom in / patients (gastroesophageal reflux in , appetite in , stool consistency in , respiratory symptoms in , pain in , eczema in ). conclusion: patch tests are informative, easy to use tools in order to identify potential causes of common lasting symptoms in children with negative or weak rast results and introduce beneficial changes in the daily diet. longer follow-up is necessary in order to refine and assess the benefit of such strategy. background: currently in the us, in children suffer from food allergies. at present, there is no cure and strict avoidance of the relevant foods is the only way to prevent allergic reactions. elimination diets put infants and children at risk for nutritional deficiencies and impaired growth. we examined the role of the registered dietitian (rd) in advising patients and families of food allergic children. method: a retrospective review of clinical notes was performed for the first consecutive children who required a dietetic consultation in a dedicated food allergy clinic. we examined common questions from parents that were addressed by the dietician during the consultation. results: patients were aged months - years (median: months) and were diagnosed with the following food allergies: cow's milk: . %, egg: %, tree nut: %, peanut: %, wheat: %, soy: %, fruit/vegetable: %, legume: %, fish: %, sesame: %. the most common questions for the dietitian included: ways to meet nutritional needs following a prescribed allergen-restricted diet ( %), meeting vitamin d and calcium requirements on a milk protein-free diet ( %), suitable oral supplements and recommended serving sizes ( %), appropriate order of solid foods introduction in food protein-induced enterocolitis syndrome (fpies) ( %), cautionary food ingredient statements ( %), baked milk protein introduction ( %), cross-reactivity risk of milk protein with soy ( %) and crossreactivity of nuts in retail bakeries ( %). conclusion: parents of children with food allergies have multiple questions with regards to nutrition. dietetic input in the food allergy clinic addresses important issues for children and families including successful avoidance of allergen-containing foods while ensuring optimal nutrition, decreased exposure to high-risk situations and avoidance of allergen cross-contamination. | multicenter prospective study of a stepwise single dose oral food challenge of egg background: oral food challenges (ofcs) are necessary for allergy management. we previously reported that a low-dose ofc can avoid complete elimination, even if patients react to higher doses of causative foods. nevertheless, this approach has only been validated in a retrospective single-center trial. we have previously reported that the median time for initial symptom onset is minutes for egg ofc using a single exposure. therefore, this study aimed to confirm the safety and effectiveness of a stepwise single-dose ofc in a multicenter, prospective study. who showed a positive reaction to low-dose ofc, only patient ( %) showed a severe reaction: barking cough immediately improved with adrenaline inhalation. among patients with a positive reaction to medium-dose ofc, none had a severe reaction. the median times to symptom onset were and minutes following low-dose and medium-dose ofc, respectively. patients in the three groups, divided according to threshold doses, differed significantly in sige levels against egg white and ovomucoid. conclusion: this multicenter prospective study confirmed that stepwise single-dose ofc to egg will help to clarify the severity of egg allergy, and will contribute to improved food allergy manage- method: the study design was a retrospective cohort study extracting data from the electronic chart of children older than years who visited our out-patient clinic for egg or milk allergy and who underwent an oral food challenge test (ofc) twice within months between november and december . the patients were divided into five groups according to their treatment schedule, which consisted of those who: a) started from / of the first ofc reaction threshold and maintained / till the end of oit; b) started from / of the threshold and maintained / ; c) started from / of the threshold and maintained / , d); conventional slow oit (started from just below the first ofc reaction and increased . - . times every few weeks); or e) continued elimination. we determined the presence or absence of an increase in threshold reacted to the allergen, any adverse events during oit, and food-specific ige reduction. results: the number of participants was and their median age was years. the number of patients in groups a, b, c, d, and e was , , , , and , respectively. the percentage of patients in groups a, b and c showing an increase in reaction threshold to the allergen was higher than that in group e (p < . ), and that in group b was higher than that in group d (p < . ). the number (percentage) for group a, b, c, d, and e was ( . %), ( . %), ( . %), ( . %), and ( . %), respectively. there was a significant difference in the frequency of adverse events during oit between group a-c and d, which was as follows: ( . %), ( . %), ( . %), and ( . %), for the respective groups (p < . ). there was no significant difference in the percentage of patients showing a decrease in food-specific ige in each group. conclusion: the regimen starting from / of the ofc reaction threshold and maintaining the dose at / was safer and more effective for increasing the threshold reacted to the allergen than the 'conventional slow oit' regimen. elimination continuation was not effective for increasing the threshold reacted to the allergen. legumes allergy was presented in different clinical features; urticaria and angioedema in ( %) patients, anaphylaxis in ( . %) patients, atopic dermatitis in ( . %) patients, eosinophilic esophagitis in ( . %) patients and as food-related enterocolitis in ( . %) patient. thirteen ( . %) of the patients had asthma, ( . %) had allergic rhinitis. fourteen ( . %) of the patients with single legume allergy showed improvement. the patients who developed tolerance, of these ( . %) had peanut allergy, ( . %) had lentil allergy and ( . %) had chickpea allergy. two of patients with multiple legumes allergies, it developed tolerance to all the legumes they are allergic. conclusion: peanut and lentils were the most frequent legumes that displayed allergic reactions in our study. in these patients the rate of allergy to non-legumes food is high. in patients who were allergic to single legumes, the symptoms were ameliorated in . %. conclusion: cashew nut is a potent allergen and can cause quite severe reactions. avoidance of pistachio nut and other related allergens should be advised to patients after allergologic investigation. in the majority of the patients, presence of atopic dermatitis with food allergy is noteworthy. therefore, it would be useful to investigate these patients for cashew and other tree nut allergy before they present with a serious clinical reaction. and jellyfish sting. serum allergen-specific ige test was negative; skin prick test was positive for natto and pork. we performed an oral food challenge with natto, pork, crustaceans, and wheat, and she developed a general itchy rash after hours of eating natto. h blocker was administered and she recovered soon. however, the general itchy rash relapsed after hours. hence, we intramuscularly injected epinephrine, h -blocker, and steroids; then, her symptoms did not relapse. based on these findings, we inferred that anaphylaxis caused by natto could be associated with a jellyfish sting. discussion: although association between japanese fermented soybeans (natto) allergy and jellyfish sting has been previously reported, its anaphylaxis is a rare event. in this case, we suggest that anaphylaxis was caused by natto allergy, which was perhaps related to jellyfish sting. hence, further investigation is essential to elucidate the association between fermented soybeans allergy and jellyfish sting. introduction: non-celiac gluten sensitivity (ncgs) is a syndrome characterized by intestinal and extra intestinal symptoms related to the ingestion of gluten-containing food, in subjects that are not affected by either celiac disease (cd) or wheat allergy (wa).once the gluten-containing foodstuff is removed from the diet, the patients will have relief of their symptoms. case: a -year-old girl was referred by his general practitioner with history of occasional constipation and abdominal pain (especially after main meals and defecation), short stature and low weight. the growth indices were proper for her age till she was . then after there was a stunting. she had short stature and low weight. despite different types of supplementation, there was no improvement in growth indices, so she was referred to a pediatric endocrinologist for gh therapy. primary investigations and anti-ttg, iga, anti-ema all were normal. after a consultation with a pediatric gastroenterologist, a genetic study of hladq and were done because of the highly suspicion of celiac disease. the results were also negative. at last she was referred to immunology-allergy clinic for evaluation of probable food allergy. ige level was checked and a prick test was performed which they were not indicative of any suggestive food allergy. because of the history of the abdominal pain and constipation which was more prominent after meals, negative results of genetic study, spt to wheat, and serologic markers, a gluten free diet was suggested for her with the suspicious of non celiac gluten sensitivity. a significant improvement in her symptoms was noticed within weeks of starting gluten free diet. she has kg of weight gain and height improved from cm to cm in months. she continued to improve on a gfd and when seen in the follow-up clinic months later reported complete resolution of symptoms and another cm and kg gain in her height and weight. conclusion: non-celiac gluten sensitivity syndrome is a diagnosis made by excluding celiac disease and wheat allergy. it should be taken into consideration especially in patients who have the suspicious symptoms of celiac without supporting lab data, and also negative spt to wheat. the young man in question along with his parents were keen to proceed, so with some hesitation we proceeded to a hazelnut oral provocation challenge, having very carefully explained the risks of undertaking such a challenge. he successfully completed the challenge and experienced no allergic symptoms and is now able to have hazelnuts in his everyday diet. discussion: this young man wanted to confirm if indeed he was allergic to hazelnuts. not being hazelnut allergic would mean that he would be no longer allergic to any nut and would not have to take precautions prior eating products. positive results to both cor a and cor a , hazelnut storage proteins are associated with the patient possibly experiencing systemic reactions, at a higher risk of experiencing anaphylaxis it they were to ingest hazelnut. these facts in conjunction with his specific ige to hazelnut would have prevented us from proceeding to challenge was it not for this young man's persistence that he wanted to proceed to challenge despite the risks. conclusion: appearances are deceptive, as this case demonstrates; allergen-specific ige and component testing can only predict the probability of an allergic reaction, the final test in the diagnostic process is the oral provocation challenge. the patient and his family were happy for me to share the above with other health care professionals. method: we present the case of a female of years old diagnosed of acu with poor control of the symptoms at maximum doses of antihistamines. we decided to associate omalizumab treatment. the patient had a good control of the symptoms with omalizumab at dose of mg/ weeks, but in the th month she presented an erythematous, raised and pruritic lesion in the area of injection together with localized abdominal edema at hours of the administration, with two weeks of evolution without symptomatic treatment. we decided to discontinue omalizumab alter a second episode with half doses. results: we performed a skin biopsy of the lesion and epicutaneous tests with the drug. immediate hypersensitivity tests were not taking due to the impossibility of stopping antihistamines. skin biopsy showed a perivascular lymphocytic inflammation of the superficial and deep dermis with frequent presence of perivascular and interstitial eosinophils, suggestive of a hypersensitivity reaction. results: nine months before presentation at our clinic, the patient had been hospitalized and treated with imipenem and tmp/smx for pulmonary nocardiosis. once discharged, she had been prescribed oral tmp/smx alone, according to antimicrobial susceptibility. at our first evaluation, the patient presented with fever, macular erythematous non-pruritic (vasculitic-like) skin lesions on the upper limbs, polyarthralgia and bilateral ankle arthritis. tmp/smx was transiently stopped. after four days, there was a dramatic improvement, with resolution of all signs and symptoms. she was tentatively diagnosed with a viral infection and thus tmp/smx was started again. however, after three days, the symptoms (fever, arthritis and skin lesions) recurred. laboratory investigations showed increased levels of inflammatory markers. complete blood count with differential, serum creatinine, urinary sediment, liver enzymes, rheumatoid factor, antinuclear antibody, c , c , immune complexes, serology for rickettsia, borrelia and coxiella were all negative. hence, tmp/smx was stopped again and cutaneous lesions, fever and arthritis resolved spontaneously in five days. conclusion: given the clinical course and the resolution after the withdrawal of tmp/smx, we diagnosed a sslr due to sulfonamides. to the best of our knowledge, this is the first case of sslr occurring after a nine-month therapy with tmp/smx and allergists/immunologists should be aware of the possibility of such a reaction even after months. case report: * we received written informed consent for publication of these clinical details and/or clinical images included in my abstract was obtained from the patient. drug rash with eosinophilia and systemic symptoms (dress) syndrome is a severe adverse cutaneous reaction that usually appears - weeks after treatment with the causative drug. this syndrome is characterized by severe dermal rash, fever, eosinophilia, and internal organ involvement, and clinically, diffuse maculopapular eruption, exfoliative dermatitis, and facial edema are often observed. we performed blood tests and laryngeal fiberscopy for the diagnosis of the patient. intradermal test with delayed reading and patch test were performed months after the end of treatment. a -year-old man had begun treatment with carbamazepine for epilepsy. after weeks of treatment, he observed skin rash with pruritus on both lower extremities, and after weeks, his skin lesions had begun to spread over his whole body, and he complained of several new symptoms, including hoarseness, dyspnea at rest, and dysphagia. an examination revealed maculopapular rash, facial edema, and bilateral cervical lymphadenopathy. laryngeal fiberscopy revealed both arytenoid and epiglottic swelling. laboratory studies revealed eosinophil counts of /μl and increase in alanine aminotransferase level to u/l. a diagnosis of dress syndrome was definite according to the regiscar group criteria. carbamazepine, the suspected culprit drug, was withdrawn, and systemic corticosteroid was initiated. the patient experienced rapid improvements in hoarseness, dyspnea, and dysphagia. after days of treatment, laryngeal fiberscopy revealed complete resolution of both arytenoid and epiglottic swelling. to the best of our knowledge, our case is the first reported case of dress syndrome to manifest with laryngeal edema. case report: bortezomib (velcade Ⓡ ), a targeted therapy works by blocking the action of proteasomes in side cells, is commonly used to treat newly diagnosed as well as relapsed/refractory myeloma. bortezomib has been reported to have gastrointestinal symptoms, peripheral neuropathy, neuropathic pain and thrombocytopenia as its most common side-effects. although several cases of skin lesion caused by bortezomib have been reported, severe cutaneous adverse reaction (scar) such as stevens-johnson syndrome (sjs) is very rare. we here report a case of bortezomib induced sjs. a -year-old female patient, who was diagnosed with multiple myeloma, received bortezomib and melphalan /dexamethasone therapy. after the th dose of bortezomib, she presented with fever and maculopapular skin rashes spreading from face to the trunk. erosive lesions in the oral mucosa and corneal ulceration with conjuntival injection were observed. she was diagnosed as sjs. the symptoms of sjs improved after bortezomib was discontinued and systemic steroids and intravenous immunoglobulin were administered. drug patch test was performed, the result was positive in bortezomib. this is the first case report of bortezomib induced sjs in this country, which was diagnosed by a patch test. although the scar by bortezomib is generally considered very rare, we suggest that clinicians be aware of potential adverse reactions, including sjs. case report: we report the case of a healthy -year-old woman with history of red erythematous macules in both hands, one hour after taking a fluconazole ( mg) tab for a vaginal candidiasis. it faded spontaneously. she didn't recall if she had ever taken that medicine, but denied known drug allergies. although fde is primarily a clinical diagnosis, we conducted an oral challenge test with fluconazole ( mg). two hours after intake of the drug the patient started complaints of pain and erythema in both hands and the challenge was stopped. two days after the challenge, she developed red painful erythematous macules on the same sites of the first episode. due to the specificity of the challenge, local patch testing was not performed. introduction: the classic form of a fixed drug eruption is one or more anular or oval erythematous patches as a result of systemic exposure to a drug. these skin lesions normally resolve with hyperpigmentation and may recur in the same location with re-exposure to the drug. other types of fixed drug eruptions have been described, being fixed drug urticaria a rare form of presentation. ( , ) case report: in the last years, a year old woman has developed more than episodes of a wheal in the right supraciliary region minutes after taking mg of oral ibuprofen. the symptoms resolved in less than hours without treatment and without leaving residual lesion. after the last episode, she refers good tolerance to g of oral paracetamol. she denies local traumas. she also refers mild spring rhinoconjunctivitis well controlled with antihistamine, and sneezing with house dust. a -year-old-m patient had psoriasis vulgaris for years, and had been using methotrexate at intervals of years. despite the addition of phototherapy, he underwent a new treatment with biological agent (antitumor-necrosis-factor; anti-tnf), since the disease control was insufficient. before anti-tnf, preventive treatment against latent tuberculosis (tb) activation was indicated with positivity in tuberculin skin test ( mm). he was given inh mg/day, and at the th day of treatment, desquamation, erythema, and subsequent exfoliation developed in his hands and foots dorsum. inh was withdrawn. in order to distinguish the lesions from psoriasis attack, skin biopsy was performed and reported as erythema multiforme-like dermatitis with no relation to psoriasis. the lesions were completely improved at weeks of topical steroids, and inh was re-initiated at the same dose. a week after the initiation of the drug, skin lesions similar to previous reoccurred with more severity and progression from distal to proximal extremities. cell counts, renal and hepatic function tests, and hepatitis markers in blood were in normal limits. skin lesions were retracted after weeks of topical steroids, and withdrawal of inh. there was positivity in skin patch test with inh at hours. finally, for tb prevention an alternative drug rifampicin ( mg/kg/day) was given, and the patient successfully completed with no adverse event. his psoriasis lesions were improved with anti-tnf which was started after month of tb prevention with rifampicin. in these days which the use of biologic agents is increasingly widespread, inh use will be more prevalent than the past. even tough, it is effective and safe in most of the patients, its adverse event dermatitis may be a reason to withdraw in patients with dermatological diseases. in this case, diagnostic drug allergy evaluation should be performed to optimize the second-line treatment of tb infection, in addition to early withdrawal of the culprit drugs. background: around % of cancer patients will receive radiotherapy (ionizing radiations) as a treatment, either as a single therapy or as an adjuvant to chemotherapy and surgery. several side effects have been described due to radiotherapy, of which we can mention erythema multiforme and stevens johnson syndrome, but in lower prevalence. erythema multiforme can be described as an acute skin condition and may be present within a wide spectrum of severity. erythema multiforme minor represents a localized eruption of the skin with minimal or no mucosal involvement. the papules evolve into pathognomonic target or iris lesions that appear within a -hour period and begin on the extremities (see the following image). lesions remain in a fixed location for at least days and then begin to heal. it is considered to be a type iv hypersensitivity reaction associated with certain infections, medications, and other various triggers precipitating factors and complex interactions may trigger the appearance of signs and symptoms. these include especially recurrent herpes simplex virus (hsv), epstein-barr virus (ebv), histoplasmosis, alcohol, systemic diseases and immunological factors. method: -year-old male diagnosed with prostate adenocarcinoma who underwent transurethral resection and was taking trinomia (ramipril, atorvastatin, acetyl-salicylic acid) after his th rte external radiotherapy session, he presented erythematous maculo-papular lesions in the suprapubic area with some vesicles. therefore, withdrawal of treatment was decided and the performance of a skin biopsy. days later, regarding the improvement of the lesions, rte was continued, presenting incipient exacerbations of the lesions but it allowed us to end the cycle of treatment. results: skin biopsy results (anatomical pathology): basal keratinocytes, which blur the dermoepidermal interface, with lymphocyte exocytosis at this level, associated with isolated images of spongiosis. the dermis shows a superficial perivascular lymphocytic inflammatory infiltrate of moderate intensity. compatible with erythema multiforme. conclusion: radiotherapy is a technique of increasing use, so it is important to recognize the associated cutaneous lesions that appear less frequently and are sometimes underdiagnosed. diagnosis is both clinic and pathological and is usually late in most cases so it is vital to take into account this skin disease complication in order to be properly managed. including chinese herbal medicine is usually considered to be without any allergic and adverse reaction. method: visits were made to pharmacies in hong kong and luoyang, china and a martial art monastery/temple in dengfeng, china. some cam were found to have ingredients with potential allergic and adverse reaction. results: three cam, one from hong kong ( ), one from shaolin martial art monastery/temple in dengfeng ( ) and one from luoyang ( ), china were found to contain chinese herbal medicine with potential allergic and adverse reaction. ( ) cordyceps ling-zhi complex ingredients: cordyceps sinensis "caterpillar fungus", tremella fuciformis "snow fungus", ganoderma lucidum (ling-zhi) "reishi mushroom" and others years old female patient who has ulcers in oral mucosa and purple, itchy lesions on her right hand palmar area, little finger, index finger, on her left hand palmar area, pollex finger. in her history, she has relapsing vaginal yeast and she hasn't any hypersensitivity reaction with fluconazole before month ago she started to take fluconazole because of vaginal candidiasis. after using fluconazole she started to itch from described areas and dark redpurple eruptions appeared. she was prescribed oral methylprednisolone and topical pomade which included corticosteroid for four days but she didn't aware of fluconazole related drug reaction. lastly four days ago she took fluconazole and metronidazole for severe vaginal yeast. hours later pruritus, same eruption appear on the same area, lip and tongue angioedema than she had dyspnea, dizziness, hypotension, arrhythmia and consciousness. she had admitted to the emergency department and performed adrenalin. after a day bullae and ulcers came into existence in her oral mucosa. in her blood analysis there was mild increase in white blood cell count ( . /mm ), eosinophil count was normal ( /mm ), biochemistry parameters were in normal limits, crp and sedimentation rate were in normal limits, total ige was iu/l. introduction: tuberculosis is a disease that most commonly affects the lungs, which is transmitted by the respiratory tract and drugs are the most important factor in the treatment. non-resistant tuberculosis infection is usually treated with hrze. in rare cases, a hypersensitivity reaction may develop against one or more of the drugs during treatment. case: a -year-old female patient was diagnosed with culturepositive pulmonary tuberculosis and hrez treatment was started by the related department. seven days later she referred to the policlinic with edema and itchy erythematous lesions which are common in her extremities, which developed after hours of taking her medication. liver and kidney function tests and eosinophil count were normal. drug eruption was considered with current physical examination findings. the treatment was interrupted, short time systemic corticosteroids and antihistamine treatment started. desensitization planned. there was no feature in the prick and patch tests with drugs. desensitization was performed with isoniazid, no reaction was observed during the procedure. six hours after the procedure, the patient applied to the emergency department with painful edema and pruritic erythematous lesions in the extremities. desensitization procedures with rifampicin, ethambutol, pyrazinamide were performed without any problems after the lesions were regressed. isoniazid was withdrawn from the treatment protocol. outcome: we would like to present on this case that drug eruption may develop in the form of maculopapular rash after desensitization. this study aims to compare ethmoid mucosa and nasal polyp regarding density of tissue eosinophil and its sensitivity, specificity, and correlation with clinical characteristics for diagnosing ecrs. method: patients with crs with polyps scheduled for endoscopic sinus surgery were enrolled. specimens were collected from polyp apex, polyp pedicle and ethmoid mucosa. tissue eosinophil from these three sites in the same patient were compared. using eosinophilic mucin as a reference, sensitivity, and specificity of each site for diagnosing ecrs was assessed. correlations between tissue eosinophilia (defined as greater than / hpf) and clinical characteristics of ecrs including asthma, serum eosinophilia, and eosinophilic mucin were analyzed using each site of specimens. results: thirty patients with crs with polyps were enrolled. polyp apex, polyp pedicle and ethmoid mucosa gave similar results regarding tissue eosinophilia in patients ( . %). eleven ( . %) patients were ecrs (having tissue eosinophilia at all sites) and five ( . %) were non ecrs (no tissue eosinophilia at any sites). median tissue eosinophil was significantly greater in polyp apex ( , and polyp pedicle ( , iqr: - ) than ethmoid mucosa ( , iqr: - ), p = . . sensitivity of polyp apex, polyp pedicle and ethmoid mucosa for diagnosing ecrs were %, % and % respectively. specificity were %, % and % respectively. correlations between tissue eosinophilia and asthma were significant when assessing ethmoid mucosa (p = . ), and polyp pedicle (p = . ) but not polyp apex (p = . ). correlations with serum eosinophilia, and eosinophilic mucin were not significant (p > . ) when assessing any specimens. gov/pubmed/) was performed using the following key words: "obstructive sleep apnea syndrome"; "allergy rhinitis"; "hypoxia"; "intermittent hypoxia"; "fluctuating hypoxia"; "cyclic hypoxia"; and "hif- α" results: osas may affect the prognosis of ar patients based on the following evidence: ) ar is thought to be a cause of osas. ) exposure to hypoxia could mediate immune activation in ar and affect the response to treatment. ) hif- α expression may be a risk factor for ar. ) intermittent hypoxia can induce robust expression of hif- α. conclusion: first, improvement of ventilation during sleep represents an efficient strategy for treating ar. therefore, continuous positive airway pressure or nasal surgery to resolve a nasal obstruction could be added to ar treatment. finally, medications that target hif- α, such as digoxin, can be tested as adjuvant therapy. method: forty patients diagnosed with allergic rhinitis and olfactory dysfunction were recruited in current study in the group and . patients of group were administered with no treatment and patients administered with the traditional chinese acupuncture therapy were incorporated into the group . before the treatment, all of them underwent t&t olfactory testing, nasal sinus computer tomography scanning and visual analog scale (vas; - ), and repeated the assessment after four-week treatment. results: improved total t&t olfactory testing scoring averages and vas scoring averages was observed in eleven patients treated with traditional chinese acupuncture compared with four patients in the observation group. no side effect was found. no significant differences in olfaction recovery were found according to age, gender, or duration of disease between the two groups. the observation group underwent nasal endoscopic sinus surgery and the control group underwent external approach surgery, and the therapeutic effect of the two groups were investigated. results: the total effective rate was % in observation group and % in control group, the total effective rate of observation group is significantly higher than control group (p < . ). the recurrence rate was % in observation group and % in control group, the recurrence rate of observation group is significantly lower than control group (p < . ). complication occurrence rate of observation group was % which is significantly lower than control group % (p < . ). the therapeutic effects of endoscopic sinus surgery on chronic sinusitis in geriatric patients are better than conventional external approach surgery which is worth clinical application. results: (descriptive). the equick app is user-friendly even for vkc patients with sub-optimal reading ability. home use between clinic appointments allows responsive temporal data gathering of qol, symptoms, medication scores and impact of medical interventions. equick may be used in future as a research tool in gathering outcome data following interventions for vkc. ectoine, a substance deriving from halophilic micro-organisms, is a strong water structure forming solute exerting cell protective antiinflammatory and antiallergic properties. method: purpose of our study was to assess the efficacy of the preventive administration of % ectoine eye-drops ( times a day for months) to shorten the duration of vkc relapses (which begin, in our country, very early in spring and usually end in october), or to mitigate the attacks, which are only controlled by topical corticosteroids or cyclosporine resulting in an important burden of side-effects. in this retrospective study, we included children of both sexes ( males and females), under the age of years (mean age . years), affected by vkc from more than years/seasons and treated for more than months during a year, with cyclosporine eye-drops. these patients underwent, from february to september , the additional-to-the-usual protocol treatment with % ectoine eyedrops. results: % of the included subjects astonishingly had no relapse of vkc, % needed topical cs or cyc treatment but it was started months later compared to previous years, % needed the topical drugs months later and % had a similar to previous years course (no ectoine efficacy). the treatment was well tolerated and only child had to stop it because of local allergy to the eye-drops. the preventive administration of % ectoine eyedrops was able to stabilize and to delay vkc attacks in more than % of the selected patients showing the importance of anti-inflammatory and anti-allergic properties of this product. following international criteria we considered normal levels of vitamin d the levels between nmol/l ( ng/ml) and nmol/l ( ng/ml), a potential deficiency between nmol/l and nmol/l and a severe deficiency less than nmol/l. results: . % ( children) of akc group patients presented vitamin d low levels, among them children showed a potential deficiency and a severe deficiency. . % ( subjects) of vkc patients suffered a deficiency in vitamin d which was mild in and severe in patients. . % ( children) of sac group showed a deficiency in vitamin d which was potential in and severe in subjects. conclusion: our study shows that in different forms of allergic conjunctivitis many children are suffering a vit. d deficiency and it can be supposed that a correlation between the severity of the allergic form and the level of vit. d deficiency exists. we recommend allergists and ophthalmologists to check vit. d levels in children suffering from allergic conjunctivitis because its deficiency is very common and many are unaware of it; in case of a vit. d insufficiency it is fundamental to give a vit. d suitable-to-the-case supplementation. method: children ( males and females, mean age . ± months) affected by vkc and allergic rhinitis from more than years were treated with mometasone furoate nasal spray spray bid × weeks in a month, for consecutive months as a co-seasonal treatment at the beginning of eye allergic symptoms. other systemic or topical treatments did not vary compared to the previous years. results: a quick questionnaire administered to children and their care-givers showed that nasal symptoms regressed after a mean period of . days from their beginning but, impressively, in more than % of them, these patients did not show a vkc typical relapse along the months of mometasone treatment, moreover the following summer period was milder in subjective ocular symptoms in more than % of the patients. our experience pointed out that incs adjunctive treatment was positively associated with a regression of eye and nose symptoms in children suffering from vkc, confirming previous literature data which concern milder forms (seasonal allergic conjunctivitis or allergic rhino-conjunctivitis) compared to the severe forms (like vkc) we analyzed in our work. one of the involved mechanisms of action can be the alleged effect on the reduction of substance p in tears; it is supposed to reflect the neuropeptides levels in ocular tissues. | patient response to mp-azeflu in an allergen exposure chamber onset of action (ooa) timing may impact treatment adherence. mp-azeflu, intranasal azelastine hydrochloride (aze) and fluticasone propionate (fp) in a single device, has proven to have greater efficacy and faster ooa than a combination of oral loratadine and intranasal fp (lora/infp), but the clinical relevance for patients is unclear. this single-center (ontario, canada), randomized, double-blind, double-dummy, three-period crossover trial examined by which extent mp-azeflu provides clinically relevant symptom improvements according to different efficacy parameters. method: ar symptoms were induced in asymptomatic, ragweedsensitive patients via ragweed pollen challenge in an environmental exposure chamber. patients received a single dose of mp-azeflu, lora/infp, or placebo and were monitored for hours. symptoms were assessed using total nasal symptom score (tnss) and total ocular symptom score. responder analyses included the number of patients to achieve relevant response (rr) to therapy ( % or % reduction in tnss), time to rr (ie, first time point at which rr was reached), and minimal clinically important difference (mcid) in ooa. background: nasal allergen provocation test (napt) is a standardized diagnostic tool indicated in the diagnosis of allergic rhinitis, to design and monitoring allergen immunotherapy, and to study the pathophysiology of airway allergy. unfortunately, until now very few studies have evaluated its reproducibility and safety. in this study we wanted to analyse the safety and reproducibility of napt in a large group of rhinitis patients and healthy controls. unit until december . a bilateral saline challenge followed by a bilateral napt were performed in symptoms-free individuals. the response was assessed by nasal-ocular symptoms and acoustic rhinometry. all subjects signed a written informed consent. the safety of napt was checked by the occurrence of extra-nasal/ ocular reactions (enor), severe adverse events (sae), and use of rescue medication (rm). enor was assessed by clinical symptoms, physical examination, cardiopulmonary auscultation, spirometry, and oxygen saturation. the reproducibility of napt was tested by comparison of the results in or more sessions with≥ -month interval. background: nasal hyperreactivity (nhr) is self-reported by a majority of patients with allergic rhinitis (ar) and is likely mediated by neural-immune interactions. the combination of fluticasone propionate (fp) and azelastine (aze) hydrochloride administered in a single spray (mp-azeflu) has been shown to be superior to fp or aze alone in patients with seasonal ar (sar). we hypothesize mp-aze-flu may reduce neuro-immune mediators in ar with nhr. in a post hoc analysis of three pivotal studies of mp-azeflu, we analyzed the efficacy of mp-azeflu, fp, and aze in patients with ar with and without nonallergic triggers. method: in three randomized, double-blind, controlled trials, patients with sar were randomized : : : to mp-azeflu, fp, aze, or placebo (pbo). patients self-reported sensitivity to nonallergic triggers. change from baseline in total nasal symptom score (tnss) and treatment differences between active agents and pbo were calculated. results: across patients in three studies, mean age was . years and mean age at ar symptom onset was . years. overall, % reported ≥ nonallergic trigger, which included sudden temperature/humidity change ( %), tobacco smoke ( %), perfumes/fragrances ( %), incense/candles ( %), and cleaning products ( %). change from baseline in tnss for patients with ar and nonallergic triggers was greater with mp-azeflu than with fp or aze (table) , and patients with nonallergic triggers improved slightly less than patients without nonallergic triggers in both the mp-azeflu and fp groups. background: in low-income countries (lics), assessment of phenotypes, prevalence and risk factors for allergy-related diseases (ards) using allergen-specific ige may be complicated by environmental exposures such as helminths. these exposures may also induce cross-reactive carbohydrate-specific ige profiles that could inhibit allergic effector responses. we sought to elucidate the molecular basis of ige sensitisation among individuals in uganda, using a component-resolved approach to ige measurement. we employed the isac ® allergen microarray to assess plasma ige reactivity to purified natural and recombinant allergen components in participants of three studies: a trial of intensive versus standard anthelminthic treatment in the rural helminth-endemic lake victoria islands (n = ), a parallel urban survey of allergy outcomes in a lower helminth exposure community (n = ) and a study on asthma risk factors in children from the urban setting and from nearby rural schools (n = ). data on sensitisation to crude allergen extracts were obtained by skin prick testing (spt) with cockroach and house dust mites (hdm), and by immunocap ige testing (cockroach, hdm, and peanut). results: the rural setting was characterised by high prevalence (≥ %) of sensitisation to crude extracts (immunocap ige> . ku/ l) but low sensitisation to the major, established, allergenic components on the microarray (≤ %, ige> . isu). however, sensitisation to cross-reactive carbohydrate determinant (ccd)-bearing components and venoms was more common in rural (up to %) versus urban (up to %) individuals, and was associated with helminth infection. urban individuals mounted higher responses to allergenic components of dust mites but responses to other components were similar between the two settings. sensitisation to allergenic components was higher among asthmatics and spt+ children but ccd sensitisation profiles were similar between asthmatics and nonasthmatics, and between spt+ and spt-school children. conclusion: we show that, in lics, ige to crude allergen extracts (detected in standard immunocap assays) reflects sensitisation to a myriad of environmental exposures (absent in more developed countries), such as ccds expressed by helminths, and may not accurately define ard phenotypes in this setting. however, our data does not seem to indicate that ccd-specific ige detected by isac ® microarray protects against ards. considered minor allergens. due to their sequence homology and conserved structure, they show a high cross-reactivity. the objectives were to study the ige/igg binding properties of polcalcin in relation to the calcium ions, and the ige cross-reactivity between purified polcalcin from olea europea (ole e ) and two recombinant polcalcins (rphl p and rbet v ). method: ole e was purified by immune-affinity chromatography using polyclonal antibodies anti-rche a . serum samples were obtained from patients allergic to grasses recruited at hospital de guadalajara (spain), all of them positive to phl p with sige values ranging from . to . ku/l. equal volumes of all sera were used to prepare a pool. calcium binding assay was performed either by addition or not, or depletion of ca + . ole e was incubated with . mm cacl or with mm egta ph . (ca + chelator agent) at the same time as the antibody in immunoblot or elisa assays with the pool of sera or with anti-che a polyclonal antibody. crossreactivity assay was performed by immunocap inhibition. aliquots of the pool of sera were previously incubated with amounts of ole e ranging from . to . ng. the same dilution of the pool of sera without ole e was used as a control. after hours of incubation, sige (ku/l) binding to rbet v or rphl p was determined. results: a kda protein was purified from the o. europea extract and identified by lc/ms-ms as ole e . in the calcium binding assay there were no differences between the samples with or without ca + . however, the addition of egta to the reaction completely inhibited the binding of the polyclonal antibody by immunoblot and also produced a . % reduction of ige binding by elisa. in the cross-reactivity assay, a % inhibition of ige binding was obtained with . ng of ole e for rbet v and . ng for rphl p . the maximum rate of achieved inhibition was . % for rbet v and . % for rphl p . conclusion: native purified ole e contains the ca + necessary to bind to the specific antibodies and the depletion of ca + inhibited this binding. high cross-reactivity of ole e with rphl p and rbet v was demonstrated. | effect of glutathione-s-transferase pi on the cysteine protease activity of the house dust mite allergen der p background: environmental proteases have been proposed to be involved in the pathogenesis of allergic disorders via different mechanisms, such as the disruption of epithelial tight junctions, the cleavage of surface proteins, the activation of damage and pathogen-associated molecular patterns receptors, and the alteration of redox status. der p from house dust mite is one of the most clinically relevant indoor allergens worldwide, which exhibits cysteine protease activity and has been linked to allergenic rhinitis and asthma. however, it is unknown whether the host microenvironment could regulate der p activity once it reaches the mucosal surface. glutathione-s-transferase pi (gstpi) is an anti-oxidant and detoxification enzyme. gstpi is the predominant gst in human lung epithelial cells, where it is expressed in high levels. polymorphic variants of gstpi have been associated to various inflammatory lung disorders such as allergic asthma. more recently, gstpi has been identified as a redox regulator through protein s-glutathionylation, a post-translational modification where glutathione (gsh) is conjugated to cysteine residues. method: this work aimed at determining if gstpi affects the cysteine-protease activity of der p , compared to gstmu -a different gst isoform-by using different in vitro approaches. results: we found that gstpi increased der p -activity, but not gstmu. our results suggested a potential role of gstpi in upregulating the protease activity of der p allergen. however, the clinical implications of these findings in allergic airway diseases needs for further investigations. | cari p , a novel polygalacturonase allergen from papaya acting as respiratory and food sensitizer biswas sarkar m; sircar g; ghosh n; das ak; jana k; dasgupta a; gupta bhattacharya s bose institute, kolkata, india background: papaya was globally reported to elicit ige-mediated hypersensitivity. certain papaya sensitive patients with food allergic symptoms were found to experience recurrent respiratory distresses at peak flowering period of papaya even after quitting the consumption of papaya fruits. the immunoreactive protein present both in pollen and fruit proteome was detected by ige-serology and identified by mass spectrometry. one such allergen, designated as cari p was cloned, and purified as recombinant protein. the ige-reactivity of rcari p was examined by immunoblot using patient sera. the allergenic activity of rcari p was evaluated by histamine release assay from ige-sensitized granulocytes. the aggregation and folding pattern of rcari p was assessed by size exclusion chromatography and circular dichroism spectroscopy respectively. the presence of cari p in papaya fruit was searched by igg-immunoblot using allergen-specific rabbit antisera. a mouse model of papaya allergy was established to study the role of rcari p in eliciting respiratory and food hypersensitivity. results: a kda ige reactive protein commonly present in pollen and fruit proteome of papaya was identified as endopolygalacturonase. recombinant cari p remained monomer and the cd-spectra revealed predominantly β-sheet characters. the melting curve of the allergen showed partial refolding from a fully denatured state indicating the possible presence of conformational ige-epitopes in addition to the linear ige-epitopes of food allergens. out of papaya allergic patients displayed ige reactivity to rcari p . rcari p at μg/ml, induced histamine release from challenged granulocytes within a range of % to % (i.e. ± . %; n = patients). expression of cari p was detected in the peel and pulp tissues of papaya fruits at two edible stages of fruit maturation. in mouse model, rcari p exhibited a comparable level of eosinophil infiltration and goblet cell hyperplasia in lung and duodenum histology. conclusion: cari p the first major allergen reported from papaya with a dual role in respiratory sensitization via pollen inhalation and sensitization of gut mucosa via fruit consumption. the recombinant allergen can be used as marker allergen for molecular diagnosis and immunotherapeutic management of papaya allergy. background: lipids can be potent stimulators of the immune system, and their role in allergy is highly investigated and debated. since many allergens bind lipids, one question that arises is the relative importance of the lipids versus the lipid-allergen complex in eliciting the immune response. also of interest is an evaluation of the importance of the allergen-lipid complex. in our characterization of the structure of the cockroach allergen bla g , we discovered that it could promiscuously bind a variety of lipids in a large central cavity. this suggested that bla g could be used as a prototypical allergen and lipid delivery vehicle to test in various models of sensitization. method: cd spectroscopy. nmr spectroscopy. molecular modeling. we have developed an hplc procedure to strip the phospholipids derived from the e. coli-based expression system, and reconstitute the allergen with a variety of lipids. using cd spectroscopy and nmr, we have verified that the protein conformation is highly similar in the presence and absence of lipids. temperature dependent cd spectroscopy revealed that unloaded bla g is the least stable, and the melting temperature increased with increasing fatty acid chain length up to c . similar cd melting experiments revealed that bla g could bind lipoteichoic acid (lta) from gram positive bacteria, but did not interact with lipopolysaccharide (lps) from gram negative bacteria. molecular modeling studies have suggested that the stoichiometry of phospholipid binding is likely phospholipids per bla g and give insight as to the different binding characteristics that would allow bla g to bind lta but exclude conclusion: these biophysical studies will allow the design of bla g -lipid systems to test a variety of sensitization models. | sal k , a new allergen from salsola kali sola jp; pedreño y; fernández j; cerezo a; peñalver m probelte pharma, murcia, spain background: the polcalcin from salsola kali was identified and sequenced (genbank kt ) and the recombinant protein was characterized as a minor allergen with a prevalence of % of patients with a spt positive to s. kali. the objective of this study was to purify the polcalcin from s. kali pollen and to include the allergen in the website for the systematic allergen nomenclature (www.allergen.org). method: the native polcalcin from s. kali (npsk) has been purified from pollen after a first step of protein extraction and then diverse chromatographic steps: a size exclusion chromatography to remove particles minor than kda, an ionic exchange chromatography, a hydrophobic interaction chromatography and a final step of size exclusion chromatography to obtain the purified sample of polcalcin. the purity of the npsk has been determined by sds-page and the binding capacity to a specific polcalcin antibody from rabbit serum was tested by immunoblot. the specific antibody had previously been obtained by immunization with the recombinant polcalcin from s. kali. the allergenicity of the npsk has been assayed by immunoblot with a pool of sera of patients sensitized to s. kali. the identity of the purified npsk has been analyzed by peptide footprint in hplc-ms/ms after digestion with trypsin. all the information about the polcalcin from s. kali was sent to who/iuis allergen nomenclature sub-committee. the npsk showed a high purity in sds-page with a molecular weight of approximately kda and this purified protein reacted with the specific polcalcin antibody from rabbit serum. the ige binding capacity of the npsk was confirmed by immunoblot using a pool of sera from patients sensitized to s. kali. the analysis of peptide footprint confirmed that the purified protein is a polcalcin. the who/iuis allergen nomenclature sub-committee included the polcalcin from s. kali in the website for the systematic allergen nomenclature as a new minor allergen named sal k . conclusion: the polcalcin from s. kali has been purified from pollen and tested for its ige binding. it is included in the website for the systematic allergen nomenclature as the new allergen sal k . background: alt a protein is the major allergen from the fungus alternaria alternata and responsible for chronic asthma, yet little is known about its physiological role and immunological activity. our main purpose was to investigate the mechanism through which alt a induces an allergic response in bronchial epithelium. method: although alt a has a unique topology, we studied the structural relationship by in silico procedures consisting of three distinct structural alignment methods in order to understand its nature. the immunological properties of the allergen were investigated by using monocyte cell line thp and human peripheral blood mononuclear cells. results: its crystal structure has been recently reported and claimed to be exclusively in fungi without equivalent in the protein data bank. data obtained in silico show that this allergen shows some structural relationships with a number of other β-barrel proteins such as human lipocalin (lcn ). besides, our experimental data demonstrate that alt a is also able to interact with lcn , human lipocalin. in this way, the results obtained from several immunological assays showed that alt a is able to produce a response of the immune system through different immune innate receptor pathway inducing the th cytokines. background: increasing evidence of cross reactivity syndromes between pollen grains and fruits, with immediate or delayed reactions, has been reported. while some syndromes such as the birch pollen/apple syndrome are well documented, some other such as the cypress pollen/peach syndrome remain to be understood. for the latter, significant progress has recently been made with the discovery of a new allergen family, the gibberellin regulated proteins (grps), which has been shown to be responsible for the observed cross reactivity i.e. pru p and bp ( , ) for the peach and the cypress pollen respectively. grps are small cationic proteins with anti-microbial properties and have been shown to be over produced in response to a stress. herein, the case of a patient, born and raised in the south of france but currently living in paris, has been studied. this patient has been suffering since childhood from allergic rhinoconjunctivitis to cypress pollen and from some oral symptoms to peach and other fruits (including pomegranate). method: in addition to the clinical exploration and cutaneous tests, a very thorough biological characterization of the patient samples has been performed through various specific ige quantitation techniques, western blotting after one and two-dimensional gel electrophoresis and flow cytometry based basophil activation testing (bat). results: specific iges to cypress pollen, birch pollen, peach, orange and apple have been found. pr allergenic proteins are recognized by iges but no ltps. the presence of specific iges to cypress pollen bp , peach peamaclein (pru p ) and a cationic kda protein from pomegranate has been shown through western blotting after gel electrophoresis separation of the protein extracts. the use of bat finally enabled to demonstrate that the basophils of this patient were, ex vivo, strongly activated with protein extracted from orange and cypress pollen and also with purified proteins such as bp and pru p . conclusion: these results unambiguously show that the cypress pollen grp, bp , is clinically relevant, similarly to its homologous protein in peach, pru p . it can be proposed that these two allergens are at the basis of the observed cross-reactivity syndrome. the search for new cross-reactive allergenic grps in pollen, fruits or vegetables may enable to better understand other pollen/food associated syndromes that still remain unexplained. background: nine allergens of phleum pratense have been described until now (iuis database) and classified into groups based on their function and cross-reactivity. group and allergens are considered the most immunodominant, due both to their greater ige-binding capacity and the number of patients ige-reactive to them. previously published studies have estimated that group is recognized by almost % of grass pollen-allergic patients, and group by %. however, until now a comparative of the ability of these allergens to provoke an immune response has not been performed. the objective was to study the immunogenicity of the major allergens phl p and phl p , by analyzing the ability of the recombinant forms (rphl p and rphl p a) to induce a humoral immune response. method: five mice were immunized with the same amount of each recombinant protein: rphl p and rphl p a (indoor biotechnologies) ( μg plus two boosters of μg). the specific igg antibodies produced by each mouse were tested against the recombinant proteins by direct elisa and the title of each of them was determined by optical density (o.d.). additionally, the recognition of both allergens in native and depigmented-polymerized (dpg-pol) extracts of p. pratense was studied by direct elisa using these generated antibodies. results: preimmune sera were negative. all mice produced antibodies against the corresponding recombinant protein. the immune response (sigg) was statistically significant higher in mice immunized with rphl p than in those immunized with rphl p ; it was needed times more rphl p serum than rphl p a serum to obtain the same o.d. values. the difference in responses was higher in the group of mice immunized with rphl p than with rphl p a. differences in the recognition of phl p and phl p in native and depigmented-polymerized extracts of phleum pratense was also observed. it was necessary times more rphl p serum to produce the same signal than rphl p a serum in native extract and it was necessary times more rphl p serum to produce the same signal than rphl p a serum in dpg-pol extract. conclusion: rphl p a is more immunogenic than rphl p , which was also probed with native and dpg-pol extracts. background: glioblastoma (gbm) is an incurable primary malignant brain tumour with a median life span of less than months despite multimodal treatments. therefore, there is a serious need for the development of innovative medications. several epidemiological studies underlined an inverse correlation between pre-existing igemediated allergy and gbm risk, where having such an allergy decreased the odds of developing gbm by to %. we aim to delineate the intrinsic immuno-biological and molecular mechanisms that can be responsible for these correlations, based on the hypothesis that allergies may promote a state of increased immuno-surveillance in the brain through the presence of immunological factors such as immunoglobulins, cytokines and cells involved in th -driven allergic reactions. we consider that as the major immune cell type of the brain, microglia should be implicated in this beneficial association and may favour the elimination of the nascent tumour in brain parenchyma in an allergic context. we implemented a long term allergic airway inflammation by repeated nasal instillation of house dust mite (hdm) extract in a syngeneic orthotropic mouse model of gbm. we followed animal survival and the tumour growth by mri. in addition, we purified microglia from allergic vs non-allergic mice in order to assess their cytotoxic function against the gbm cell line ex vivo and their secretory capacities. finally, we investigated immunoglobulin reactivity against gbm antigens in the context of allergic reactions by reverse phase protein array (rppa). we demonstrated an increase of the animal survival that was correlated with a delayed tumour engraftment and a reduced tumour growth. these phenotypes were associated with functional modification of microglia from sensitized mice. indeed, these microglia showed a rise in the production of il- and tnf-a as well as an increase in cytotoxic functions against a gbm cell line ex vivo. in parallel, we observed an increase in serum igg reactivity against gbm antigens in mice sensitized with hdm compared to control mice. results: in patients ( %) with cvid we recorded at least one temporary platelet count decrease below × /l compared to only patient ( %) with xla (p = . ). more importantly in patients ( %) with cvid this decrease was observed in a period longer than months compared to patient ( %) with xla (p = . ). in patients ( %) with cvid we recorded at least one temporary platelet count decrease below × /l and only in patients ( . %) with cvid this decrease was observed in a period longer than months. we did not record any platelet count decrease bellow × /l in patients with xla however the difference with cvid did not reach statistical significance. no thrombocyte count decrease bellow × /l was observed in either group. none of patients required immunosuppressive treatment for immune thrombocytopenia (itp). conclusion: although the statistical significance was documented only in temporary platelet count decrease below × /l it is obvious that numbers of thrombocytes commonly fluctuate in some patients with cvid. the mechanism leading to these temporary decreases is unclear. monitoring of complete blood count is a basic follow-up investigation in patients with cvid. introduction: wegener's granulomatosis (wg) is a systemic disease that may affect all organs, most frequently the ears, noses, throats, sinuses, lungs and kidneys. it is a rare autoimmune disease, also called granulomatosis with polyangiitis, and characterized by necrotizing granulomatous inflammation in small and medium sized blood vessels. anti-neutrophil cytoplasmic antibody against to proteinase (c-anca) is thought to be responsible for autoimmune inflammation. the coexistence of wg and common variable immunodeficiency (cvid) is extremely rare. in this report, we describe a patient with wg and cvid who was treated with immunosuppressive drugs and intravenous immunoglobin concomitantly. case report: a twenty-four-year-old male patient was referred to our clinic for immunological evaluation due to recurrent infections, fever of unknown origin and neutropenia. the patient had been diagnosed with wg and taking immunosuppressive therapy for three years. he had chronic renal failure due to wg and had also been on peritoneal dialysis for three years. serum igg, iga levels, peripheral blood cd + b cell percentage and absolute count of the patients were found to be low according to reference limits. he was diagnosed with cvid after excluding secondary reasons for hypogammaglobulinemia and he started to receive mg/kg intravenous immunoglobulin (ivig) therapy once in a month. also, the treatment that consists of mycophenolate mofetil (mmf) and glucocorticoids was continued to decrease c-anca levels in serum. he has been accepted as a candidate for kidney transplantation, and prepare for this purpose. discussion: the management of the patient with cvid and wg may be complicated. it is considerably difficult and needs competency and courage. moreover, the cases similar to ours, are extremely rare. therefore, the authors should share their own experiences on cvid and discuss them by comparing the data obtained from other cases. background: leukocyte adhesion deficiencies (lads) are a group of three genetic disorders leading to defective leukocyte adhesion to the endothelium and as a consequence decreased leukocyte recruitment and immune defense. lad-i is caused by mutations in the gene encoding the ß -integrin cd on chromosome .lad-iii is a rare primary immunodeficiency syndrome, characterized by homozygous mutations in the kindlin- gene (official symbol fermt ). we have aimed to evaluate our patients who were followed up with lad for the last years, retrospectively. method: all data of the cases were obtained from the file records of age at diagnosis. results: seven patients from separate families were included in the study. four patients were lad-iii and patients were lad-i. the female to male rate was / . the age of diagnosis is ranged from days to years. the median umbilical cord detachment was days ( - days groups: up to times ( people) and from to times ( people). healthy donors were examined as a control. flow cytofluorometry method was used to study peripheral blood and assess the parameters of innate and adaptive immunity results: it was found that at a frequency of edema up to times a year there are changes in the t-system of adaptive immunity, which are shown by a decrease in the expression of late activation markers (cd + hladr+ . ± . %, in control . ± . %), an increase in the number of cd + cd + cytotoxic lymphocytes ( . ± . x /l, in control . ± . x /l) and as an increase in their functional activity (cd + gr+ . ± . x /l, in control . ± . x /l). the nature of disorders of cellular factors of the innate immunity is manifested by decrease in the adaptive resources of neutrophils (kstnbt . ± . u.e., in control . ± . u.e.). patients with hae with a frequency of edema up to times a year, we observed the disorders of the humoral link of adaptive immunity, which consist in an increase in the number of circulating b lymphocytes ( . ± . x /l, in control . ± . x /l). in addition, with the strengthening of the hae clinic, changes in the system of innate immunity progressed very fast and consisted in increasing the amount (cd + . ± . x /l, in control . ± . x /l), and functional activity (cd + gr+ . ± . x /l, in control . ± . /l) of natural killer cells results: we included children, mostly males ( %), aged between month and years. . % of patients (n = / ) showed abnormal absolute results of lymphocyte count for age. we found more patients evaluated in the age group of to years ( . %), followed by - years ( . %), lymphopenia was found in . % of patients. b lymphocyte deficiency was the most common pattern ( %) followed, in decreasing order, by low cd , t cd , tcd and nk. many patients have more than one affected population ( . %) . some patients were affected in all three series ( . %). the cd / cd ratio decreased in . % of the patients. the majority of the children were males between the ages of month and years. . % of patients showed abnormal absolute lymphocyte count for age. b-cell deficiency was the most common pattern followed, in decreasing order, by low cd , t cd , tcd and nk. many patients have more than one affected population. | indicators of the humoral immunity in the mechanical jaundice of benign genesis the aim of the investigation was to study the indices of humoral immunity in patients with benign mj, depending on the level of bilirubin. method: patients with mj and practically healthy volunteers were examined. patients with a level of bilirubin less than μmol / l - , with a bilirubin level of - μmol / l - and with a bilirubin level of more than μmol / l - patients. the concentration of immunoglobulin classes a, m, e and g in serum was determined by enzyme immunoassay. the statistical significance of the differences was determined using the ranked mann-whitney test. the critical level of significance in checking statistical hypotheses was assumed to be p < . . results: of the contacted dermatologists, participated ( women, men; mean age . ± . ) which results in a response rate of . %. the guideline compliant prescription rate of biologicals in patients with csu was . %. the most prevalent barriers in the prescription were the high cost of the treatment ( . %), low reimbursement for doctors ( . %) and the fear of a recourse claim ( . %). however, a lack of evidence or an insufficient efficiency were not con- case report: eosinophil associated gastrointestinal disorders (egids) including eosinophilic colitis are commonly associated with atopy. aeroallergen sensitization may accompany food allergy in these patients. a case with eosinophilic colitis responsive to anti-ige monoclonal antibody (omalizumab) treatment is presented. an eleven-year-old boy had bloody diarrhea lasting nearly one month in autumn for last years. this year diarrhea lasted more than months. colonoscopic biopsy revealed lymphoplasmacytic inflammatory cells including eosinophils leading to a diagnosis of ulcerative colitis. corticosteroid and mesalazine treatment was started with a good clinical response. recurrence of diarrhea during corticosteroid dose reduction suggested corticosteroid dependent ulcerative colitis. eosinophilic/allergic colitis was an alternative diagnosis when seasonal recurrence, lack of weight loss, eosinophils in biopsy and high serum ige level were considered. colonoscopy done after cessation of therapy for one month, revealed exudative ulcerous lesions, lacerations, loss of haustration compatible with colitis (inflammatory/allergic?). presence of significant mucosa associated lymphoid tissue in biopsy supported any inflammatory, reactive process. he had recurrent bronchiolitis until age six and allergic rhinitis in spring for three years. total ige and mix aeroallergen specific ige were high ( iu/ml, . kua/l), absolute eosinophil count was normal ( /mm ). food skin prick and patch tests were negative. he had positive skin reactions with dermatophagoides, grass and olea pollens (induration diameter: , , mm, respectively). pulmonary function test was normal. he was considered as eosinophilic/allergic colitis and omalizumab was started according to manufacturer's dosing table ( mg/ weeks). rectal bleeding decreased after first dose and ceased after the second dose. early colonoscopy examination after rd month of therapy showed that exudations disappeared and haustrations became evident. microscopy revealed mild nonspecific colitis. few patients with eosinophilic colitis improved with omalizumab were reported before. ige-mediated processes are responsible from eosinophilic inflammation in egids, making anti-ige therapy as a promising treatment option. | design of liposomal carriers modified by glycoconjugates for liver cell delivery of nucleic acids used. the surface of liposomal nanoparticles can be modified to increase the selectivity of intracellular delivery. it is well known that asialoglycoprotein receptors of hepatocytes have a strong affinity to galactose carbohydrate. therefore, the aim of this study was to assess the effect of the modification of the liposome surface by glycoconjugates on the selectivity of intracellular transport of nucleic acids into the liver cells. method: liposomes based on ornornglu(c ) were chosen previously as the effective nucleic acid delivery system. we modified liposomes with novel lactose-based derivatives. every of four glycoconjugates was added to ornornglu(c ) in an amount of , and %. as a result, variants of modified liposomes were obtained. to determine the cytotoxicity, an mtt test was used. using luciferase test, the selectivity of penetration was evaluated on nonspecific t (human embryonic kidney) and specific hepg (human liver cells) cell lines. results: modified liposomal compositions ornornglu(c ) - + lacc ( %) and ornornglu(c ) - + lacggg ( %) had the lowest cytotoxicity similar to that for unmodified ornornglu(c ) . the ic , calculated based on the data of mtt test, was . and . , vs. . mg/ml, respectively. ornornglu(c ) - + lacggg ( %) showed a . -fold increase in transfection activity on the nonspecific t cells, compared to unmodified ornornglu(c ) , whereas the modification of ornornglu(c ) - + lacc ( %) resulted in a -fold decrease in transfection activity. however, the ability of these variants to penetrate the specific liver hepg cell was significantly higher by and times, respectively, than for unmodified ornornglu(c ) . results: the greatest inhibitory effect of sbfhd was observed in mdm infected with hiv- bal: % and % suppression of hiv replication was achieved at concentrations of . μg/ml and . μg/ ml, respectively. the activity in pbmc and dc was less pronounced (the respective ic values were . μg/ml and . μg/ml). studies in endometrial hec- a cells demonstrated that sbfhd suppressed cd -independent entry of hiv- ( tcid /ml) by %, %, and %, respectively, at , , and μg/ml. the effect was also observed after increasing the dose of the virus. at tcid /ml, sbfhd suppressed hiv infection by % ( μg/ml) and % ( μg/ml). the cytotoxicity of sbfhd in this system was low. similar results were obtained with colorectal caco- cells. sbfhd exhibited no spermicidal activity at concentrations of up to mg/ml. combining within a single microbicide two agents that target distinct steps of hiv life cycle will maximize its efficacy (via synergistic effects and/or interference with multiple stages of the transmission). we therefore explored the synergistic potential of combinations of sbfhd and azt, the classical nucleoside rt inhibitor. in these experiments, % suppression of hiv infection was reached at concentrations of sbfhd and azt, which were significantly lower than the respective ic values of each component (determined in parallel experiments). the synergistic effect was most pronounced for the combination of . μg/ml sbfhd (which is times less than the ic ) and . nm azt (which is times less than its ic ). cd expression was increased after the co-culture with reishi, shiitake and boletus mushrooms (c - . ( . - . )%; pma - . ( . - . )%; )%; shiitake - . ( . - . )%; boletus - . ( . - . )%). method: the study included men (mean age ± . years) before and immediately after staying in countries with a hot climate. results: the development of lymphopenia observed in the first week of observation. this was accompanied by a decrease in the number cd + lymphocytes expressing the markers of late activation (cd + hladr+ . ± . x /л и . ± . x /л). revealed significant decrease of cd + cd + foxp + regulatory cells in the first week after returning from the area of adverse climatic conditions, as well as a significant sustained decrease in the number cd + cd + hladr+(p < . ). change of the effector link of innate immunity was determined in significant reliable decrease in relative (cd + . ± % and . ± . %, respectively, p < . ) and absolute (cd + . ± . x /l and . ± . %, respectively, p < . ) in the number of a population of natural killer cells in the first week of observation. in the context of acute stress marked a significant increase in relative and absolute numbers of b lymphocytes ( ± . % ( . ± . × /l) before a trip to countries with a hot climate and ± . % ( . ± . × /l) in the first week after returning, p < . ). the activity is the production of antibodies was not changed. (ast) which is the intramuscular injection of patients own serum, is a promising therapy with a substantial efficiency on ciu patients. in this study we aim to assess the efficacy of ast on chronic urticaria patients by dlqi questionnaire. method: this was a single-blind randomized clinical trial which evaluated the efficacy of autologous serum therapy compared to oral antihistamines in patients with ciu. ciu patients received the ast. every session cc of each patient's blood was centrifuged at the speed of rpm for minutes and . cc of the serum was injected intramuscular into the patient's deltoid muscle weekly for weeks. the control group consisted of ciu patients took mg of cetirizine daily for weeks. patients answered the dlqi questionnaire at the first session of treatment as baseline and weeks after the last session(week ) as response to treatment. the mean baseline score of dlqi for ast group was conclusion: pharmacotherapeutic and inpatient costs for patients with prevalent ar and asthma were lower in those prescribed ait than in those not prescribed ait in all years, both with and without including the cost of ait itself. this indicates that treatment with ait is associated with lower cost burden for health services. background: immunotherapy with peptides rather than conventional whole allergens is being developed to improve the benefit/risk balance of subcutaneous immunotherapy (scit). lolium perenne peptides (lpp) demonstrated reduced allergenicity following ex-vivo analyses, allowing higher doses to be given over a shorter period to improve treatment adherence and compliance. such treatment resulted in significant reduction in symptoms and rescue medication intake during the grass pollen season. here we report the safety of lpp immunotherapy in adults. background: a new allergoid from alternaria alternata was characterized to determine its reduced allergenicity in vitro. the objective of this study was to determine the skin response to the allergoid and to evaluate the clinical tolerance of the immunotherapy with the allergoid product using a rush schedule. method: to assess the skin response (sr) two groups of patients were included: group with patients sensitized to a. alternata and with respiratory disease caused by this mold; group (control) with patients sensitized to others allergens and non-atopic patients. the sr was determined by spt using three concentrations of the allergoid: p (lowest concentration), p (four times higher than p ) and p (estimated to obtain a wheal area similar to histamine mg/ml). in spt was also used a native extract of a. alternata (n) and histamine mg/ml (h). all products were tested in duplicate in all patients and the sr was evaluated by comparing the median of the wheal area produced by different products. to evaluate the clinical tolerance to immunotherapy the patients of group were treated with the allergoid product using a rush schedule consisting in a dose of . + . ml the first day and . ml after one month (maintenance dose). the clinical tolerance was determined as the percentage of adverse reactions (ar) to the treatment and the classification of ar was established according to eaaci. the number of patients included to evaluate the sr was (group : ; group : , atopic and non-atopic) with an average age of . (range . the spt data from group were expressed as median and interquartile range of wheal area (mm ): h: . ( . - . ); n: . ( . - . ); p : . ( - . ); p : . ( - . ); p : . ( . - . ). it was determined that sr of allergoid was reduced in % respect to the native. the products n, p , p and p did not produce any response in patients of group . to evaluate clinical tolerance, patients of group were treated with the allergoid product with a rush schedule and only two ar were registered ( . % of doses). these were retarded local reactions with a wheal diameter higher than cm. no systemic reactions were registered and all patients continued the treatment. the allergoid from a. alternata produces a significant reduced response to spt due to its reduced allergenicity. the treatment with an allergoid product in a rush schedule is safety and clinically well tolerated. background: in our study we aim to determine the more effective, the total cost of years of patients using scit was tl per person whereas the total cost of years of patients using slit was tl per person. when we compare the total cost data of both groups, we found that they are close to each other. while the greatest portion of the cost data of patients with scit treatment was direct costs associated with the treatment itself ( %); the remaining part of the total cost was indirect ( %) with non-medical expenses such as transportation ( %). in the slit group, direct costs including drug expenditures have a larger percentage ( %) and it was significantly more costly compared to the direct costs of the scit group ( %). transportation costs were found to be more costly in the scit group ( %) when compared to the slit group ( %). similarly loss of parent work days in the scit group(% ) was found to be significantly more expensive compared with slit group ( %). our study results show that slit is a similar treatment for clinically and laboratorially and has a similar efficacy to scit to reduce the patients' complaints and to the need for medication. for cost-effectiveness however medicines for treatment of scit are less costly; when long term total treatment costs are calculated slit and scit treatment are economically close treatments. the protein content of the new acd was . μg/mg and the protein profile in sds-page and sec-hplc confirmed the presence of proteins with high molecular weight and the absence of smaller proteins. the content of free lysine in acd, involved in glutaraldehyde modification, was reduced in . % respect to ncd and it can be considered as the polymerization degree. regarding to the allergenic profile, through elisa inhibition was determined a reduction of times in the capacity to bind ige of the proteins in acd respect to ncd, whilst the igg binding capacity was maintained. in immunoblot there was no reaction of acd proteins to specific ige from sera. the analysis by peptide footprint determined the presence of fel d and others allergens in acd. the content of major allergen fel d in acd was determined as . μg/mg. the new developed and characterized allergoid from cat dander has an excellent safety profile and will allow a safer immunotherapy to treat the allergy to felis domesticus. results: the protein content of the new aaa was . μg/mg and the protein profile in sds-page and sec-hplc confirmed the presence of proteins with high molecular weight and the absence of smaller proteins. the content of free lysine in aaa, involved in glutaraldehyde modification, was reduced more than % respect to naa and it can be considered as the polymerization degree. regarding to the allergenic profile, in immunoblot there was no reaction of aaa proteins to specific ige from sera and by elisa inhibition was determined a reduction of % in the capacity to bind ige of the proteins in aaa respect to naa. the igg binding capacity in aaa was maintained. the analysis by peptide footprint determined the presence of alt a and others allergens in aaa. the content of major allergen alt a in aaa was determined as . μg/mg. a. alternata shows an excellent safety profile and allows a safer immunotherapy to treat the allergy to this mold. she was an otherwise healthy woman: she took no drugs and she did not have any remarkable concomitant diseases. the distribution and appearance of the remaining body hair was normal and the hormonal level profiles (lh, fsh, estrogens, progesterone and testosterone) did not show any significant alteration according to her age. a year old woman with allergic rhinitis underwent sq glutaraldehyde-modified ait to house dust mites (d pteronyssinus and g domesticus) without any incidences and complete tolerance to maintenance dose without local reactions during a year period. two years after ait discontinuation, patient first experienced a local urticarial reaction with multiple hives at previous sq ait injection sites minutes after mg of ibuprofen intake. these symptoms recurred at least in seven occasions when patient was exposed to ibuprofen (in five) and metamizol (in two). results: case : dermatologist diagnoses localized hypertrichosis. case : a single blind, placebo controlled oral challenge (sbpcoc) with ibuprofen mg was performed and elicited multiples hives in the circumscribed area in the arm where ait was conducted. subsequently, sbpcoc with aspirin was carried out showing the same reaction although a controlled challenge with celecoxib was negative. conclusion: local hypertrichosis is a very rare injection-disease associated with injected allergen vaccine treatment. we also firstly described a recall urticaria phenomenon after allergen immunotherapy which has been only elicited after different nsaids intake. results: there were included patients, in five spanish hospitals. following aria guidelines, . % of patients were diagnosed of persistent moderate/severe rhinitis. the mean age was . ± . years, being . % female. moreover, . % of the patients had concomitant mild/moderated asthma. the period between the diagnosis of rhino-conjunctivitis and the informed consent signing was . ± . years. according to international guidelines, eight systemic reactions were registered, representing . % of the administered doses: five reactions grade , (described as nonspecific ocular pruritus, nasal herpes, general discomfort, localized non-specific pruritus plus nausea and non-specific pruritus in throat), a grade i reaction described as rhinoconjunctivitis and two reactions grade ii, registered as generalized urticaria and asthma. all reactions were classified of mild or moderate intensity and only two required symptomatic treatment. there were five clinically significant delayed local reactions, which were higher than cm or involved modifications in next dose. regarding efficacy parameters, immunoglobulin titers between baseline and final visit according to specific igg and igg significantly increased. cutaneous reactivity also decreased significantly in the dose response skin prick test. results: patients were included, to accelerated and to polymerized cluster group schedules. according to aria criteria, . % of patients presented persistent moderate/severe rhinitis. the mean age was . ± . years, being . % male. moreover, . % had concomitant mild/moderated asthma. immunoglobulin titers method: the quantification of total proteins in the products was carried out by means of a colorimetric technique using the bradford reagent (sigma-aldrich™, us) in accordance with the manufacturer's instructions. the absorbances of each standard and samples were obtained in a scinco™ s- spectrophotometer (seoul, korea) at nm. all samples were analyzed in duplicate. the electrophoretic profile of the proteins in the tested allergens was obtained according to the procedure described by laemmli, under denaturing conditions in a polyacrylamide gel at . % concentration and stained in silver. in each lane approximately μg of total proteins were applied. commercial extracts of the main allergens marketed in mexico were obtained, rossel ® , alk ® , alerquin ® , alergomex ® , allerstan ® , ipi asac ® ; and they were assigned randomly with the numbers , , , , and . results: the following protein concentrations were found in the various extracts analyzed: see table conclusion: differences were found in the protein profiles ana- background: a new allergoid from cat dander was developed and characterized to determine its reduced allergenicity in a % and the maintenance of igg binding capacity. the objective of this study was to develop an immunogenicity assay in mice with the new allergoid and a native extract from cat dander. the study included female balb/c mice separated in three groups of mice each: group , immunized with a mold allergen extract (control); group , immunized with a native extract from cat dander with a fel d content of . μg per dose; group , immunized with the new allergoid from cat dander with a fel d content of . μg per dose. all mice were immunized four times by subcutaneous injections with a volume corresponding to / of the recommended human maintenance dose with an interval between injections of weeks. one week after the last injection the mice were sacrificed and the serum was obtained. to determine the specific antibody title indirect elisa were performed using a cat dander extract as antigen, sera from mice as primary antibody and antimouse igg or igg as secondary antibody. elisa assays were performed using serial dilutions of sera or a simple dilution by duplicate to determine the specific antibody title as arbitrary units/ml (au/ml). the data were analyzed by one-way anova and tukey hsd test to compare the averages of specific antibodies in each group. results: the immunization with both the native extract and the allergoid from cat dander produces specific igg and igg . regarding to igg, a higher title was observed in group respect to group in a curve obtained after elisa with serial dilutions of sera. the specific igg title obtained in terms of au/ml was . ± . in group , . ± . in group and . ± . in group . concerning to igg the au/ml obtained was . ± . in group , . ± . in group and . ± . in group . the increase of specific igg or igg in mice from group respect to mice from group and control group was statistically significant (p ˂ . ). the safety profile of the allergoid from cat dander allows a treatment with higher dose of allergens to produce a greater response to immunotherapy to induce formation of specific this was an open, multicenter clinical trial, in patients aged between to years with rhinoconjunctivitis with or without concomitant mild asthma sensitized to house dust mites (hdm). the aim was to evaluate the safety and tolerability of the vaccine. secondary endpoints included were: changes in immunoglobulin levels (specific ige, igg and igg ) versus d. pteronyssinus and d. farinae and changes in cutaneous reactivity. patients were under study treatment for weeks: five for the induction phase (weekly injections) and for the maintenance phase (monthly injections). results: patients were included. there were withdrawals from the trial; no one was related to treatment. the patients mean age was . years, being % female. . % were diagnosed of persistent moderate/severe rhinitis according to aria guidelines and . % presented concomitant mild asthma. regarding to safety results, systemic adverse reactions were registered which corresponded to . % from a total of administered doses. the most of systemic reactions were grade i, ( . %) described as rhinitis or urticaria, grade or nonspecific ( . %) and reaction ( . %), was grade ii. all of them were mild or moderate and only needed treatment. among local reactions, ( . %) were clinically relevant late local reactions, meaning a wheal at injection site > cm and /or requiring a dose readjustment in the next administration; ( . %) were clinically relevant immediate local reactions meaning a wheal > cm. concerning the efficacy parameters, cutaneous reactivity at the final visit versus baseline was, in average, significantly decreased, and specific titers of igg and igg against tested hdm increased significantly at final visit. patients completed the study. mean values in rqlq questionnaire (total score) decreased from . to . points ( . % score reduction) in final visit, reflecting a statistically significant improvement (p < . ). annual episodes of rhinoconjunctivitis decreased from . to . (p < . ). . % of patients improved from persistent to intermittent rhinoconjunctivitis (p < . ) and . % from moderate/severe to mild intensity (aria) (p < . ). moreover, . % of asthmatic patients at baseline, did not have any bronchial symptoms after -year treatment (p < . ). mean value of treatment satisfaction was . (sd= . ) and . (sd= . ) for patients and physicians respectively. | evaluation of safety and tolerability of "allergovac poliplus" in polysensitized patients with allergic rhinitis-rhinoconjunctivitis with or without asthma: an observational prospective study (apolo) background: the objetive of this study was the safety and tolerance assessment of "allergovac poliplus" scit treatment, with allergen combination-mixtures in polysensitized patients, as well as the evaluation of the clinical improvement and patients' satisfaction after treatment. method: this is a prospective observational clinical study. allergovac poliplus treatment is being administered in a " -day" or in an abbreviated schedule. polysensitized patients (to pollens or mites), with rhinitis or rhinoconjunctivitis, with or without asthma, and between - years have been included. all adverse events are being recorded. visual analog scales (vass) are being used to evaluate clinical improvement, tolerance and satisfaction after treatment ( months). results: a total of patients have been included, with an aver- results: in all groups prevailed severe forms of the disease and the phenotype of frequent exacerbations. groups were comparable in age composition and structure of severity. observations in the group of vaccinated pcv continue the dynamics of decreased dyspnea up to . ( . ; . ) results: of a total of pts under scait, were excluded due to data unavailability, and included (♀ ( %), mean age ± years (minutes: max md ), age range [ - ] being most prevalent ( %). the most frequent diagnosis was rhinitis/rhinosinusitis ( %), followed by asthma ( %), diagnosis coexisting in abstracts | pts ( %). other diagnosis such as conjunctivitis ( %), atopic eczema ( %) and food allergy ( %) were also found. mite sensitization occurred in patients ( %) of which ( %) were monosensitized. the pollen sensitization was verified in ( %) with monosensitized pts ( %). the double sensitization mitespollens was displayed in ( %). sensitization to epithelia and fungi occurred respectively in ( %) and pts ( %). it was found that pts ( %) presented sensitization to the groups of allergens (mites, pollens, fungi, dander). an average of ± pts started this treatment per year. prescription included laboratories with the following %: a- . ; b- . ; c- . ; d- . ; e- . ; f- . ; g- . ; h- . ; i- . ; j- . . option for extract of physical modification ( %), physical-chemical ( %) and chemical ( %). table shows the frequency of distribution of scait composition. conclusion: in this population sensitization to mites was predominant being the most prescribed scait followed thru sensitization to grasses with the respective scait. the majority of the population was polysensitized. however, in composition preference the choice of group of allergens prevailed and only % had more than one sort of pollen and % pollen+mites. polysensitization is a reality, nonetheless the choice of ait composition should be guided thru scientific criteria and not through the availability of mixtures encouraged by laboratories. background: allergen immunotherapy (ait) has been proven to be an effective treatment of allergic diseases in numerous studies. however, its use in seniors remains limited and questionable, due to common comorbidities and limited evidence of efficacy and safety of ait in aging population. the aim of presented study was to assess the safety of ait in patients over years of age undergoing subcutaneous immunotherapy (scit) and analyze the potential risk factors of adverse reactions in this population, compared to younger adults. we followed subcutaneous immunotherapy in a group of patients treated in the outpatient clinic of medical university of lodz, of whom were aged and older ( between the age of - , aged - and patients above the age of ). we recorded detailed information of each administration and corresponding adverse reactions over the period of years. we compiled results of our observations with patients' medical records to compile a database, which we then analyzed using statistical software. method: a total of cases with seasonal allergic rhinitis undergoing pre-seasonal immunotherapy and cases followed with conventional drug treatment were included in the study. immunotherapy and control groups were divided into monosensitized (only pollen) and polysensitized (at least additional allergen except pollens) patient groups according to skin prick test reactivity. all patients were followed between march-september with symptom and medication scores, and visual analogue scale (vas). the quality of life was assessed using the mini-rqlq questionnaire. phleum pratense (phl p) specific ige and specific igg (uni-cap , phadia) measurements were performed before and after weeks of immunotherapy in all patients. gramineae pollens were counted during the grass pollen seasons. results: mean age was . ± . and . ± years, female/ male ratio was / and / , the number of monosensitized/polysensitized patients were / and / in immunotherapy and control groups, respectively. in the immunotherapy group, june-july symptom scores, may-june-july-august vas scores and june combined symptom-medication scores were lower than the control group (p = . ). furthermore, improvements in activities-practical problems and other quality of life scores were significantly different between two groups (p < . ). in immunotherapy group, phl p specific ige and phl p specific igg levels measured after immunotherapy were significantly higher compared to those before immunotherapy (p < . , p < . , respectively). phl p specific igg levels measured after immunotherapy were also significantly higher in the immunotherapy group than in the control group (p < . ). there was no difference in terms of clinical and immunologic parameters in monosensitized and polysensitized patients (p > . ). conclusion: clinical improvement with pre-seasonal allergoid immunotherapy is accompanied by an important increase in specific igg blocking antibodies despite short-term injections. our findings show that pre-seasonal allergoid immunotherapy has similar clinical efficacy and b cell response in polysensitized subjects compared to monosensitized patients. | the safety trial of sequential sublingual immunotherapy with japanese cedar droplet and house dust mite tablet matsuoka t ; kuroda y ; igarashi s ; fukano c ; natsui k ; ohashi-doi k ; masuyama k university of yamanashi, yamanashi, yamanashi, japan; torii pharmaceutical co. ltd., tokyo, japan background: sublingual immunotherapy (slit) is recognized as the only treatment option with the potential to provide long-term posttreatment benefits. in japan, the prevalence of japanese cedar (jc) pollinosis is very high, about % of the population, of which the majority are co-sensitized to hdm. slit is now well established, safe and convenient treatment form for allergic disease, and recently, jc slit-droplet and hdm slit-tablet products were approved in japan for treatment of jc and hdm induced allergic rhinitis, respectively. however, the safety of sequential jc slit-droplet and hdm slittablet has not yet been investigated. therefore, we investigated the safety trial on slit combined with jc droplet and hdm tablet in allergic patients. method: eleven subjects with jc pollinosis and hdm rhinitis were enrolled. patients were treated once-daily with jc slit-drops for weeks, followed by weeks of sequential slit treatment where the jc slit-drops and the hdm slit-tablets were administered daily with a minute interval ( st: jc-slit drops, nd: hdm slit-tablet). the primary endpoint was the frequency and severity of adverse events (aes) during sequential slit by common terminology criteria for adverse events (ctcae) v . and slit grading system. serum antibodies were measured as the secondary endpoint. results: eleven patients were recruited. aes after jc slit-drops administration were found in patients out of cases ( %). aes after sequential slit were found in patients out of cases ( %). all aes were graded or . no severe aes were observed during the study period. the levels of jc-and hdm-specific ige and igg in serum were increased during treatment. conclusion: sequential-administration of jc slit-drops and hdm slit-tablets was well tolerated by patients suffering from both jc pollinosis and hdm rhinitis. background: according to the ema guideline on the clinical development of products for specific immunotherapy products should be tested in phase ii at different doses in several study-arms to establish a dose-response relationship for clinical efficacy before confirmatory trials can be initiated. allergen exposure in an aec may be used as primary endpoint. the study was a single-center, randomized, double blind, placebo-controlled, phase ii trial, treatment duration months. grass pollen allergic patients ( - years of age) with seasonal rhinitis/rhinoconjunctivitis (arc) with (mild, gina i) or without concomitant asthma were randomized to three different dosages of a liquid phase iii study is in preparation. as part of an effort to prepare the analysis plan using the csms as primary endpoint, the grass pollen data of the european aeroallergen network (ean) was used to identify the window within the grass pollen season (gps) with optimal correlation between the grass pollen counts and the csms. method: ean currently includes information from more than active and historical pollen-monitoring stations in europe including countries. the ean database used for analysis included grass pollen data collected during - . the daily allergy symptoms and medication were recorded spontaneously using an app questionnaire on the subject's smart phone. the csms was re-calculated using the ean database, using the recorded symptom scores with estimation of the medication score using similar methods as recently published. the correlation between the daily grass pollen count and the daily csms was analyzed with a mixed effects model accounting for patient-specific correlations and symptom levels. conclusion: these results confirm a statistically significant correlation between grass pollen counts and the csms. importantly, these findings suggest that the optimal window to observe treatment effects after immunotherapy may be a short interval after start of the gps and during the peak gps, due to generally higher csms values. this provides sufficient basis to consider additional sensitivity analyses to evaluate the treatment effect of grass mata mpl scit on the primary csms endpoint during a shortened window after the start of the gps and to consider excluding the overlapping period between the bps and gps from the primary analysis. | combo-vas as a tool to assess efficacy of allergen immunotherapy ciprandi g ; silvestri m ; olcese r ; tosca ma ospedale policlinico san martino, genoa, italy; istituto g. gaslini, genoa, italy background: allergen immunotherapy (ait) is at present the unique cure for respiratory and venom allergy. usually, ait lasts for some years, but its efficacy is longstanding. criteria for assessing ait efficacy are mainly based on symptom severity improvement and saving of symptomatic medications. in this regard, there are different score grading for both measuring symptom severity and drug use. visual analogue scale (vas) is a well-defined and validated method widely used in many diseases, including allergic disorders. vas is a psychometric tool measuring the patient's perception of symptoms, emotions, pain, drug use, etc. recently, it has been published an eaaci position paper concerning the recommendations for the standardization of clinical outcomes used in ait trials for allergic rhinoconjunctivitis, but it is complex. so we would propose a simpler way to measure ait efficacy by vas, in particular a combo-vas based on one vas for symptom and one for medications. results: globally patients were retrospectively evaluated. all of them were treated with a -year ait course: were defined as responders and as non-responders. in responders group the combo-vas mean value was (iqr - ) at baseline and (iqr - ) after ait treatment. in non-responders group combo-vas mean value was at baseline and (iqr - . ) at the end of ait. the difference was significant (p = . ). the d combo-vas was − . % in responder group and − % in non-responders group (p < . ). conclusion: combo-vas, i.e. the sum of vas for symptoms and medications, could be an easy and quick tool for assessing ait efficacy and reflects the patient's perception. therefore, it could be very fruitful in clinical practice. | rapid up-dosing in sublingual specific immunotherapy is safe, well-tolerated and effective in patients suffering from tree pollen allergic rhinitis background: an optimised up-dosing period of specific immunotherapy (sit) is desirable for better patient compliance because a long or complicated up-dosing scheme is sensitive to disruption. the aim of this study was to compare the safety, tolerability and effectiveness of an optimised up-dosing scheme with two preexisting schemes of sublingual sit (slit) in patients under standard medical care. method: this was a prospective, open, active controlled, multi-center non-interventional study in germany and austria to document the treatment of children and adults with allergic rhinoconjunctivitis and/or allergic asthma treated with a slit containing purified, aqueous extracts of birch, alder and hazel pollen. the investigators were free to select an up-dosing scheme for included patients: scheme a consisted of an up-dosing period of up to days at the patient's home using three different solution strengths to reach the maximum dose; ultra-rush scheme b performed only with the highest solution strength at the physician's office within hours, and the optimised scheme c which was initiated at the physician′s office and continued at home using exclusively the highest solution strength within (long-term) or (pre-seasonal) days. data on up-dosing and maintenance treatments were documented by physicians during patient visits and by patient diaries. the study was approved by ethic committees, and all patients or parents gave their informed consent. results: in total, patients aged - years were included into this study. scheme a was applied by patients, patients decided on regimen b, and patients on the optimised scheme c. conclusion: one-day ur-scit conducted in an outpatient clinic was safe and well-tolerated in patients with ad sensitized to hdm. ur-scit can be a safe and useful option to start a subcutaneous allergen immunotherapy for ad. | factors affecting on adherence to allergen specific immunotherapy results: among enrolled patients, ( . %) patients failed to complete at least years of ait, which were regarded to be nonadherent in this study. univariate analysis revealed that male, younger age group less than years, cluster and ultra-rush schedules, atopic dermatitis, the absence of associated diseases, and follow up of other department were found to be associated with nonadherence to ait. in multivariate analysis, younger age group less than years (or . , % ci . - . ), cluster ( . , . - . ) and ultra-rush schedules ( . , . - . ) , and absence of follow up of other department ( . , . - . ) were independently associated with non-adherence to ait. no association was found in gender, diagnosis of allergic diseases, kind of allergen extracts, and patients' distance from hospital. conclusion: various factors are related with ait non-adherence to interfere the effectiveness of immunotherapy. clinicians need to be aware of the factors associated with non-adherence to ait and consider them when choose to maximize ait adherence. | cost-effectiveness of allergen immunotherapy to grass in patients with allergic rhino-conjunctivitis and asthma background: allergen immunotherapy (ait) has been shown to reduce symptoms and medication use in subjects with rhino-conjunctivitis and asthma. however, long-term cost effectiveness of this therapy needs to be evaluated. our aim was to assess cost effective of ait, both subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit), vs. pharmacotherapy alone in subjects with rhino-conjunctivitis, with or without allergic asthma, to grass pollens. method: a markov cohort state-transition model with a time horizon of years was used to assess the costs and effects of -year ait in adults. relative efficacy of the treatments expressed as standardized mean difference was estimated using an indirect comparison on symptom and medication score extracted from available meta-analyses. the rhinitis symptom utility index was used as a proxy to estimate utility values for symptom score. the societal perspective, through the human capital technique, was used to estimate indirect costs, to represent the scenario of a country with nationalized medicine. data on drug and other medical costs were derived from published sources as well as ait duration and asthma occurrence. additional sensitivity analyses were performed to test the robustness of our results. results: in the base case analysis, using italy clinical practice patients with moderate-to severe allergic rhino-conjunctivitis (ss ranging from to points) and a mean age at entry of years, both scit and slit were associated with increased cost but superior efficacy compared to pharmacotherapy alone. the results were most sensitive to variation in efficacy estimates and ait persistence rates. conclusion: this analysis suggests that ait is cost effective relative to pharmacotherapy alone. scit, despite significantly higher indirect cost burden, seems to be the most cost effective option. the results should be interpreted in the context of the data input and modelling assumption used. | ielisa as a tool to measure ige binding towards single modified peanut allergens background: immunotherapy has shown to be a potential treatment for food allergies but needs further research to improve safety. modification of peanut allergens to reduce their allergenicity is a promising approach to develop a safe and effective immunotherapy as shown by the successful completion of a first-in-human safety and tolerability study using hal-mpe in adult patients with peanut allergy (eudract - - ) . in order to assess the impact of modification on individual peanut allergens and to assess its impact on ige binding by individual patient sera, we have developed peanut allergen-specific inhibition elisas. with this methodology we are able to identify patients with residual ige binding to modified peanut allergens. method: ige inhibition elisas (ielisas) were developed and performed to test ige binding towards purified ara h and ara h and their reduced and alkylated (modified) versions, using the individual responses of single patient sera. results: ara h -specific ielisas showed that modification of ara h results in > % reduction in ige-binding for all individual sera tested. ara h -specific ielisas showed that modification of ara h also results in > % reduced ige-binding for most of the sera, but some sera were identified which showed residual, %- % ige binding to mara h . in some of the latter sera, the presence of ige binding to a linear hydroxyproline-containing peptide could be confirmed as a possible source for the residual ige binding to mara h . we have developed a methodology to assess residual ige binding to modified peanut allergens. the sensitivity of the allergen-specific ielisas allowed us to discriminate between patient sera in which ige binding to mara h and to mara h was virtually completely absent and sera in which - % residual ige binding to ara h was observed. the clinical importance of these observations is yet unknown. future clinical studies will need to reveal whether the patient-specific ige binding profiles to individual modified peanut allergens do correlate with the adverse events profile of immunotherapy with modified peanut extract. | design of a phase ii allergen immunotherapy study to determine the optimally effective and safe dose of subcutaneously administered tyrosine adsorbed modified grass allergen+mpl (mpl) adjuvants for the treatment of allergic rhinoconjunctivitis (arc) due to grass pollen. there is increasing evidence that the effectiveness of allergy immunotherapy to control arc symptoms is related to the cumulative allergen (or allergoid) dose administered. previously, two clinical studies have been conducted using a conjunctival provocation test (cpt) as primary efficacy measure for a similar scit mata mpl product for birch allergy [eudract - - and - - ] . these studies showed a . fold increase in cumulative dose to achieve~ % increase in efficacy, with a relative reduction in total symptom score (tss) of . % compared to placebo and no safety signals of concern. the shape of the dose response curve was curvilinear, where this high dose almost reached plateau. method: this is a multi-center (~ clinical study centers across europe), randomized, double-blind, placebo-controlled, parallel-group study in~ adult patients with moderate to severe seasonal arc with or without mild asthma. a positive cpt is to be achieved at screening and verified prior to randomization. the primary outcome is the post-treatment tss following cpt. a wide range of cumulative dose regimens is used ( , , and su) applied over weekly injections to establish the shape of the dose response to support dose selection for phase iii. the design of the current phase ii grass allergoid scit study will be discussed, including the rational of using cumulative dose regimens and placebo and the pre-selected shapes of the dose response curves. in addition, the number of patients screened and randomized will be presented by country, gender and/or age category and screen failures will be categorized. conclusion: this phase ii study was initiated to establish the dose response of a grass mata mpl scit product, using cpt to measure the effect of a wide range of cumulative dose regimens. the achievement of its aim will be an important milestone in the development of an efficacious and safe state-of-the-art grass scit. conclusion: we observed that the specific nasal challenge with house dust mite generates an inflammatory response within the first hours, but we did not demonstrate any correlation with the response to immunotherapy after six months. | tolerability of a two week rush updosing with modified allergens in pollen allergic subjects in the day-to-day practice background: in two phase iv studies the tolerability of a subcutaneous rush up-dosing, using three injections in two weeks, has been tested and proven to be save in adults. in the course of a non-interventional study (nis) now the tolerability of this treatment scheme was tested in the day-to-day practice. conclusion: over % of the patients could reach the highest dose of . ml. the overall tolerability is very good. the data from daily practice confirm the data that were previously obtained in two phase iv studies. siges from patients, evaluated during the st semester of at an outpatient clinic. all patients presented persistent moderatesevere allergic rhinitis, in pollen season and had not been submitted to it. all patients had positive spt for grasses (grass) and olive (olea). sige-tot for phleum pratense and olea europaea and some sige-crd (rphl p , rphl p , rphl p , rphl p , role and nole ) were determined. physicians were divided into groups (group if < years of practice and group if≥ years of practice) and were asked to choose which it to prescribe for each patient (none, only grass, only olive or both grass and olive), according to spt and sige results. results: fifteen physicians ( % with ≥ years of practice) participated in the survey. considering only the sige-tot results, the it choice (group / ) was: no vaccine in %/ %; grass vaccine %/ %; olive vaccine %/ % and both grass and olive vaccines in %/ % of the patients (p = . ), the intergroup agreement was % (kappa . ). according to the sige-crd results the physicians chose (group / ): no vaccine at %/ %; grass vaccine in %/ %; olive vaccine in %/ % and both vaccines in %/ % of patients however, data on control of allergic rhinitis (ar) after discontinuation of therapy are insufficient. the aim of our study was to assess sustained control of ar in three consecutive years after grass-pollen slit discontinuation. method: a total number of patients [ ( . %) males; mean age years, age range - ] well-controlled after a three-year course of slit with grass pollen extract were prospectively evaluated in three consecutive years after discontinuation of therapy. conclusion: a three-year course of grass-pollen slit seemed to have a long-term effect on control of symptoms in patients with ar. the authors declare no conflict of interest. results: all patients showed a positive sensitization profile by skin prick test to either betula and/or alnus. in % of patients this profile was furtherly confirmed with serum specific ige levels to betv , (mean . ku/l). allergic symptoms in patients with birch/alnus pollen allergy after ingestion of certain food can result from crossreactivity of bet-v -specific ige to homologous pathogenesis-related proteins, particularly the pr- protein. conclusion: within the allergy history we emphasize on focusing on sao symptoms as many patients under-recognize them. among the sensitization profile of these patients it is quite important to highlight cross reactivity between bet v and alnus. the other patient suffered from anaphylaxis(grade ii) induced by minimum amount of lettuce consumption without co-factors. both patients suffered from oral allergy syndrome to peach and allergic rhinitis. spts to foods and pollens were performed with commercial extracts, prick-through-prick with fresh plant foods, while specific ige was determined accordingly. ltp syndrome was defined as a sensitization to pru p and symptoms elicited by at least unrelated plant foods. co-factors were also investigated. results: the first patient was sensitized to lettuce, peanut, hazelnut, sunflower's seed, peach and banana, and plane tree, olive tree, grasses, parietaria and mugwort. sige to lettuce was . kua/l, to pru p was . kua/l and total ige was . u/ml. co-factors, such as exercise, were involved. the second patient was sensitized to peanut, walnut, hazelnut, almond, sunflower's seed, cashew, lettuce and peach, and, plane tree, olive tree, grasses, parietaria, mugwort and willow. sige to lettuce was . kua/l, to pru p was and total ige was . ku/l. no co-factors were identified. background: garlic (allium sativum) is a vegetable that belongs to amaryllidaceae's family. hypersensitivity to garlic is not very common. it has been mainly reported in occupational allergy but it also may cause contact dermatitis, rhinoconjunctivitis, asthma, urticaria, gastrointestinal symptoms and anaphylaxis after its ingestion. some studies have identified alliin lyase, a kda protein, as a major garlic allergen and it seems to be a heat-sensitive allergen. we report on a -month-old infant who presented, minutes after an accidental ingestion of garlic sauce, generalized erythema and cough. she was still breastfeeding and she had never abstracts | eaten garlic before (although the mother usually consumed garlic). the patient had never tasted other vegetables belonging to amarylidaceae's family either but zucchini, with good tolerance. we performed skin tests and specific ige (sige) to different vegetables. a raw garlic extract was also carried out and analysed in the patient by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). results: prick by prick with garlic was positive ( mm) and negative to onion, leek, asparagus, zucchini and saffron. skin prick tests to commercial extracts of mugwort, grass pollen, peach ltp and profilin were negative as well. specific ige to garlic was . ku/l (out from a total ige ku/l) and ku/l to onion and asparagus. sds-page immunoblotting assay with patient′s serum revealed ige reactivity with proteins of kda and kda. conclusion: we report a garlic ige-mediated anaphylaxis case in an infant with proteins of and kda as the relevant allergens. the mechanism of sensitization in the present case remains unclear. the authors hypothesized that breastfeeding, cutaneous contact or inhalation might be possible mechanisms involved. chong kw ; saffari se ; chan n ; seah r ; tan ch ; goh sh ; goh a ; loh w allergy service, department of paediatric medicine, kk women's and children's hospital, singapore, singapore; centre for quantitative medicine, office of clinical sciences, duke-nus medical school, singapore, singapore; yong loo lin school of medicine, national university of singapore, singapore, singapore background: the predictive decision points for both peanut skin prick test (spt) wheal size and serum ige concentrations, in peanutsensitized children, have not been evaluated in singapore. we aim to assess these for purposes of risk stratification and prediction of oral food challenge (ofc)s' outcomes by means of a retrospective chart review. results: the number of patients evaluated was , of which had clinical diagnosis of peanut allergy based on recent immediate reaction to peanut (pa group) and were tolerating peanuts regularly (pt group). the mean age of both groups were similar, . ± . and . ± . years in pa and pt groups respectively. there was a high prevalence of atopic diseases in both groups, with atopic dermatitis ( . % in pa, . % in pt), and other food allergies ( . % in pa, . % in pt). presence of rhinitis was statistically higher in the pa group compared to the pt group, with odds ratio of . ( % ci: . - . ) . a wheal size of ≥ mm and a peanutspecific ige of ≥ ku/l provided for a % positive predictive value. the larger the wheal size on spt, the higher the probability of a clinical reaction to peanuts. the results will help us in deriving preliminary cut-off values when conducting future prospective studies with ofcs in our peanut-sensitized cohort (whom had no prior peanut exposure), and to eventually reduce the need for expensive and potentially risky food challenges. | ginger: flavory, spicy …allergenic? a report of four patients with allergy to ginger background: ginger (zingiber officinale) belongs to the family zingiberoidae, along with cardamom and turmeric. the edible portion is the horizontal rhizome, and it is very appreciated for its aroma and spicy flavor. it also presents great interest for its therapeutic and culinary use. hypersensitivity to ginger is rare and has been scarcely reported. we report cases (p , p , p , p ) of adverse reactions to ginger after its ingestion and with good tolerance to cardamom and turmeric. method: skin prick tests (spt) to environmental allergens and prick-by-prick with ginger were carried out. total ige, and specific ige to ginger were also determined. a raw ginger extract was prepared. this extract was analyzed in all the patients by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). background: food allergy is divided into groups according to pathophysiology: ige-mediated, ige-and non-ige (mixed type), and non-ige (cellular type). however, in clinical practice, patients who fall under more than one group may be observed. method: patients who were diagnosed with food allergy at our clinic from january to december were included in the study. the medical files of patients were retrospectively evaluated, their symptoms and findings after consumption of foods were recorded, and they were categorized into groups (ige-mediated type, non-ige type, and mixed type) according to their symptoms and findings. results: a total of patients ( . % male) with food allergies were included in the study. according to categorization via symptoms and findings, the distribution of patients was as follows: ( . %) ige-mediated type, ( . %) non-ige type, ( . %) mixed type. the remaining ( %) patients were found to show various combinations of symptoms and findings that fit more than one group. in this study, we observed that food allergy symptoms and findings were distributed in a broad range which caused difficulties in the categorization of more than one-third of our patients. background: fish allergic patients suffer a lifetime of strict dietary restrictions. crocodile meat is a nutritious alternative choice in many countries around the world; however, it has recently been reported to also trigger severe allergic reactions. in these two case reports from pediatric patients were sensitised to the major fish allergen parvalbumin (pv), a potential cross-allergen. bony fish contain predominantly pvs of the β-lineage, which are the most common trigger of allergic reactions in fish allergic patients. in most other vertebrates, pvs of the α-lineage are most abundant, which have been reported as a causal allergen in frog, cartilaginous fish, and chicken allergies. we aimed to evaluate the allergenicity of crocodile meat in fish allergic children, with focus on pv. method: over children with clinical history of ige-mediated fish allergy were identified, skin tested to commonly consumed fish species using commercial and in-house preparations, and serum samples were collected. a sub-cohort was skin tested to crocodile using heat-treated tail muscle tissue from saltwater crocodile (crocodylus porosus). extracted proteins and purified pvs were analysed by sds-page, immunoblotting, and mass spectrometry. serum from all fish allergic children was analysed for ige reactivity to the crocodile pv. this reactivity was compared to those of raw and heated crocodile protein extracts as well as protein extracts and purified pvs from frequently consumed fish species. results: more than fish allergic children were positive on skin testing to crocodile (wheal size> mm), demonstrating its clinical reactivity. in vitro analyses revealed ige reactivity to crocodile pv in serum from more than half of all patients. two βand one α-crocodile-pv isoforms were identified. pvs constituted approximately % of total proteins in heated crocodile extracts, with β-pv ( kda) being times less abundant, but up to times more ige-reactive than α-pv ( kda). αand β-pvs from crocodilians, including alligators and crocodiles, share more than % and % of their amino acid sequence, respectively. conclusion: crocodile pv is a new allergen as per the iuis guidelines. fish allergic patients may be at risk of severe allergic reactions upon ingestion of crocodile meat due to strong ige cross-reactivity of β-pvs. this study suggests that fish allergic individuals and health professionals need to be aware of potential allergic reactions to meat from crocodilians, termed 'fish-crocodile syndrome'. background: we present a -year-old nonatopic woman that in december after eating a seafood paella with green pepper presented asthenia, nasal obstruction, incoercible vomiting and diarrhea. later she ate a grilled loin sandwich in a bar and she had the same symptoms (she asked the waiter at the bar and the chef had cooked her sandwich in the same pan where he had cooked green pepper just before). after that, she suffered from abdominal pain, nausea, abdominal distension without diarrhea two hours after she ate an omelet sandwich with certain flavor of green pepper. at present even the casual smell of pepper causes her nausea. the woman eats everything including spices and just avoids pepper. method: skin prick tests were performed using extracts from food (nuts, fish, mollusk, fruits, vegetables, legumes), aeroallergens (mites, abstracts | pollens and epithelia) and purified proteins (pru p , profilin, polcalcin, alfalactoalbumin, betalactoglobuline, casein). we also performed prick by prick to raw and cooked green pepper. sds-page immunoblotting according to laemli under reducing conditions (with -mercaptoethanol) was performed to study the molecular mass of the ige-reactive proteins. extracts from green pepper and green pepper seed were used. the prick tests were all negative and the prick by prick test to raw and cooked green pepper was positive in both cases. blond was taken from all patients to specify the levels of allergenspecific ige against allergen components of immunocap isac test, the result≥ . isu-e was assumed as positive. results: in the study group, in patients ( %) specific antibodies against ltp were detected, the isu-e level range . - average . isu-e on average, in patients with detected ltp ige was detected for . components belonging to the ltp, however the highest number % patients were detected ige only for ltp. in subjects ( % of respondents with detected ltp), ige was detected against art v and this is the only ltp component whose occurrence was statistically significant (p = . ). in patients ige was detected against pru p , jug r , pla a ,; patients ige to ara h ; patients cor a ; patients ole e , patients tri a , and in person para j . background: the birch pollen-associated oral allergy syndrome (oas), an ige-mediated local allergic reaction, is the most common manifestation of pollen-associated food allergies. its origin is explained by cross-reactions between birch pollen-and food-allergens belonging to the pathogenesis-related protein subfamily (pr- ). [ ] so far, there is no marker available for its detection and no standardized test established to evaluate objectively the subjective feelings experienced by oas. the diagnosis is based on a characteristic history and on detecting the sensitization to triggering allergens in skin prick test (spt) and laboratory examination. method: the aim of this study was to evaluate whether the food skin prick test could be a helpful marker in the diagnosis of a birch pollen-associated oas. for this exploratory study, data from - was collected retrospectively at the dermatological outpatient department of the ordensklinikum linz elisabethinen. patients with positive spt results for birch pollen were included. the variables age, gender, tree pollen-(birch, alder, hazel) and food-spt, laboratory tests (ige, bet v , bet v , gly m ) and symptoms (oas, rhinoconjunctivitis allergica, atopic dermatitis, anaphylaxis) for statistical analysis. results: there was an association between food-spt and oas but also between the negative oas patients and food-spt (p = . - . ). all of the bet v sensitized patients with positive gly m results had also a positive food-spt result. conclusion: there was no evidence for a possible role of food-spt as helpful markers in the diagnosis of birch pollen associated oas. maybe gly m could be a helpful marker, but more data are needed. bet v seems to be the cause of birch-pollen associated oas, due to the dominant sensitization pattern to major allergens in austria. background: eggs are among the foods most frequently causing allergy. the most common one is hen egg, although we may consume other bird's eggs such as duck's, those of goose, quails and seagulls. clinical and serological crossreactivity between hen egg proteins and those of other birds eggs have been described. allergy to other species eggs are less frequent and are usually described in patients allergic to hen eggs. we report a case of food allergy after ingestion of duck egg in an adult patient without hen egg allergy. the patient was a year-old man who had symptoms of generalized itching, swelling uvula, erythema neck and deglutition difficulty immediately after he ate eggs from duck and hen. he claimed to have eaten hen eggs almost daily without clinical symptoms and he had not previously ingested duck egg. he denied allergic reactions to any other food but did complain of seasonal allergic rhinoconjunctivitis in the spring. we performed skin prick test with extracts of egg and feathers, prick by prick test with cooked and fresh yolk and white from duck and hen egg and oral challenge with hen eggs. specific serum ige was measured to hen proteins and we carried out a western blot with the proteins of allergenic extracts from different eggs (white and yolk of quail, chicken, goose and duck) and an inhibition of western blot with ovalbumin as inhibitor allergen. results: skin test with extracts of eggs and feathers, cooked and fresh hen and duck eggs were positive. total serum ige was . ku/l. specific ige to hen's egg was class two for hen egg white, ovoalbumin and class one for yolk and ovomucoid. oral challenge with heated egg yolk negative and with egg white was positive. the patient's serum recognized mainly and intensely several proteins of white and egg yolk of quail and duck with a molecular mass around to kda respectively. on the other hand a protein around and - kda was unique recognized in white eggs of hen and goose. the western blot inhibition revealed ovalbumin inhibited the protein recognized in the egg white but not egg yolk involved in. background: management of tree nut allergy is usually based on the avoidance of the suspected tree nut (tn), as well as peanuts and seeds, either because of the risk of cross-reactivity and/or contamination, or due to the clinical severity. objective: to assess the sensitization pattern and clinical reactivity patterns to different tn, peanut and sesame seeds (ss) in patients with a history of reaction to at least one of these foods. results: a total of patients with confirmed nut allergy were included; % female, median age [interquartile range] of years; % were atopic. the most frequently involved foods were walnuts ( %), hazelnuts ( %), almonds ( %) and peanuts ( %). anaphylaxis was the clinical presentation in % of the patients. in those with a history of reaction to only one nut ( ), the most prevalent was peanut ( %). in the patients that reacted to more than one nut, the most frequent combinations were walnut/hazelnut ( ), walnut/almond ( ) and almond/ hazelnut ( ). of these patients, tolerated other nuts. two had sesame seed allergy, one reacted only to ss. nine patients ( %) were sensitized to foods that they tolerated. fourteen ( %) patients were sensitized to ltp and of them reacted to more than one nut. conclusion: these data concur with the existence of different sensitization profiles (primary, concomitant or cross-reactive), which may predict different clinical reactivity patterns and therefore, influence dietary recommendations. background: buckwheat (fagopyrum esculentum) is a polygonaceae weed, not a cereal, which is increasingly been consumed and used as an alternative food in the diet of celiac patients. despite its wide use, allergy to buckwheat has unfrequently been reported in our setting. method: two female patients, aged and , suffered an immediate severe allergic reaction after eating a bread and a pancake containing buckwheat among its ingredients. the first patient presented generalized urticaria, palpebral angioedema and pharyngeal occupation. the second one showed those same symptoms, as well as abdominal pain, nausea, dyspnea, dizziness, hypotension and loss of consciousness. skin tests and specific ige determination to aeroallergens and food allergens were carried out, including prick-prick test with buckwheat and all other components contained in the food involved. buckwheat allergens were studied by sds-page and ige-immunoblotting of one of the patients. results: prick-prick tests yielded strongly positive results to the food itself and buckwheat, and negative to the remaining food components in both cases. cap was positive to buckwheat ( . ku/l and > ku/l, respectively). basal serum tryptase levels were normal. both patients were not sensitized to cereals, ltps, profilins or pr- proteins. for the first patient, the skin tests were negative for other foods, seeds and nuts. the cap was negative for ltps, profilins and storage proteins of peanut and other nuts. the second patient who had the most severe reaction, was also sensitized to hazelnut(cap . ku/l), pistachio ( . ku/l), almond( . ku/l), walnut( . ku/l) and sesame( . ku/l), which were not included in the pancake. the buckwheat immunoblot of the first case, under non-denaturing conditions, revealed a ige-binding protein of -kda. ige-immunoblotting under reducing condition showed protein bands of , , and kda in the buckwheat extract. conclusion: two cases of anaphylaxis by buckwheat flour contained in two frequently consumed foods are presented. the absence of sensitization to ltps, together with the pattern of specific ige binding in the immunoblot in one of the cases suggests that the responsible allergen could correspond to a storage protein of buckwheat, without cross-reactivity with other seed and nut allergens. buckwheat must be taken into account as an unsuspected food allergen capable of causing severe allergic reactions. background: allergy to linseed (linum usitatissimum) has infrequently been reported despite of its wide use in bread and in a range of "health food" products. linseed contains potent allergens which have not yet been characterized. we studied three patients who presented allergic reaction after eating different foods which contain linseed. two of them (patient p and patient p ) had anaphylaxis and the third one (patient p ) had oral syndrome, abdominal pain and diarrhoea. p was also allergic to mustard and p to sesame seed. all of them tolerated the remaining seeds, nuts and food. baseline tryptase levels were in normal range in all patients. skin tests and specific ige determination to inhalants and food allergens were carried out. linseed allergens were studied by sds-page and ige-immunoblotting. skin tests: patient : prick tests were positive to pollens and linseed, and negative to ltp, profilin, nuts, seed and the remaining food; patient : prick tests were positive to linseed and mustard and negative to other food and inhalants; patient : prick tests were positive to linseed, pollens, sesame and nuts (peanut, hazelnut, almond, pistachio). we present three cases of severe allergic reactions to linseed. the pattern of specific ige binding in the immunoblot seems to lead to storage proteins as the responsible allergens. | pr- sensitization-looking it up in food allergy background: pr- protein group sensitization is found in patients with respiratory allergy, mainly in areas inhabited by trees of the betulacea or fagacea families. its role in food allergy is, however, more frequently described in the context of cross reactivity. our aim was to characterize the pattern of molecular sensitization of food allergic patients sensitized to pr- protein group with immu-nocap isac ® (isac). the remaining patients, with more severe reactions, were all co-sensitized to either ltp and/or storage proteins (sp). only of the patients without pfa were co-sensitized to ltp or sp versus of with pfa (p < . ). conclusion: pr- sensitization is rare in our population. approximately half of the patients had allergy to plant foods, but the majority were co-sensitized to ltp and/or sp. the few patients only sensitized to pr- had minor reactions. among the patients without plant food allergy, co-sensitization to ltp and sp was significantly less common. according to these results, in our population, pr- seems to be less relevant to food allergy, when compared to reported results from other european countries. gür Çetinkaya p; uysal soyer Ö; esenboga s; sahiner Üm; sekerel be hacettepe medical school, ankara, turkey background: pistachio is a tree nut belonging to "anacardiacea" family, and constitutes % of tree nut allergies. this nut most often abstracts | cross-reacts up to % with cashew which is located in the same family. pistachio allergy is mostly seen in iran, turkey, the united states, and china where this tree nut is frequently consumed. in this study, we analyzed age and cut-off values for development of tolerance to pistachio. method: children who had reported allergic reactions with pistachio, and who have not consumed pistachio, but had positive spt and/or specific ige levels were enrolled into the study. spt and specific ige levels were measured in all patients. oral provocation(op) tests with pistachio were performed by patients, and of them had positive test result. the median age of tolerance development was months (iqr: . - . months). the most commonly involved systems during op tests were skin ( %, n = ), gastrointestinal system ( %, n = ), and lower respiratory tract ( %, n = ). concomitant allergic diseases were atopic dermatitis ( %), asthma ( %) and allergic rhinitis ( %). there was a positive correlation between skin prick test diameter (spt) and specific ige levels (spige) (r = . , p = . ). spt of≥ . mm to pistachio nut was found as highly predictive of clinical allergy (auc: . , %ci: . - . , p < . ). any relation was not determined between eosinophil, basophil counts, triptase levels, and op test positivity. conclusion: pistachio allergy is one of the frequently seen nut allergies in turkey which may cause serious allergic reactions including anaphylaxis. op test showed that tolerance was achieved by the median age of months, and a cut-off level of . mm was best predictor for positive reaction. in an oral challenge test, the patient responded with generalized urticaria, swelling of the lips and difficulty breathing at a cumulative dose of . g peanut protein, however, without blood pressure drop. (grade , moderate symptoms). the patient was treated with anti-ige for months (with mg every weeks sc). oral desensitization began with ingestion of μg peanut, escalating to mg, on the first day and escalating weekly doses of peanut from mg to mg ( peanuts). then anti-ige was discontinued while the patient ingested peanuts every day. we have followed the patient's sensitivity to peanuts from before anti-ige treatment to after anti-ige washout with basophil testing. results: the patient completed desensitization without side effects, and continues to ingest peanuts a day. basophil sensitivity was reduced -fold by anti-ige treatment, but returned to baseline levels after anti-ige washout. conclusion: anti-ige allows rapid desensitisation of peanut allergic subjects with peanut oral immunotherapy. in the majority of subjects, this desensitization is sustained after anti-ige is discontinued. additional studies will help clarify which patients would benefit most from this approach. the return of basophil response to pre-treatment levels suggests that the patient is desensitised, and depends on the daily ingestion of allergen. what do we (not) know? background: the use of biologic prosthesis is a well-established surgical procedure. acute and delayed complications may occur, but accurate epidemiologic data about allergic reaction to graft tissue is lacking. methods: a years old boy referred to our unit for urticaria and gastrointestinal symptoms which developed a few years before. he received a biologic porcine vascular duct during a cardiovascular surgery at days of life. at the age of urticarial episodes occurred, and a diagnosis of beef allergy was made. after the exclusion of beef meat from the diet, most symptoms resolved, but the child began to complain about occasional episodes of vomit and diarrhoea. results: skin prick tests confirmed the beef meat sensitization and prick-prick test resulted positive against raw pork meat but not against cooked pork meat. in vitro tests demonstrated the presence of pork meat specific iges ( . kua/l). component-resolved diagnostic tests revealed allergy sensitization toward bos d (bovine serum albumin, bsa; porcine serum albumin was not tested due to lack of a specific test). after accurate exclusion of pork meat from diet, a complete remission was achieved, and the diagnosis of pork meat allergy was confirmed. conclusion: bsa is a major beef allergen, responsible for the raw beef-cow milk cross-reactivity but with a scarce importance in milk allergy. it is highly homologous with human serum albumin and other mammalian serum albumins, including porcine albumin. porcine albumin is also highly homologous with cat serum albumin and therefore responsible for cross-reactivity in patients affected by cat-pork syndrome. we hypothesize that the implanted porcine tissue was the trigger for pork meat allergy in our patient, as this condition is exceptional in childhood and that our patient never owned a cat. few cases of pork allergy due to porcine tissue implantation have been reported so far. of interest, pig sensitization was recognized as a rare but possible cause of blood-culture negative endocarditis in patients with porcine bioprosthesis according with anamnesis, increased ige level against pork, tissue eosinophilia during autopsy. background: desensitization to foods is assuming a new paradigm in food allergy. this technique is becoming widespread especially for patients with egg and milk allergy, but the effect of desensitization on the consumption of similar but not identical foods is still uncertain. material and methods: we describe the case of a -year-old patient, with a history of chicken egg allergy, who had been successfully desensitized tolerating cooked and raw chicken eggs for a year. the patient came to the office after presenting an episode of anaphylaxis immediately after eating fried quail eggs. an immunological study was carried out. we performed skin prick test with chicken egg′s proteins (white, yolk, ovoalbumin and ovomucoid). an immunoblotting to detect specific ige to egg proteins was also performed. for this purpose, the following extracts were used: chicken egg white and yolk (commercial extract form alk) and quail egg white and yolk prepared following a similar procedure (extracted in % phosphate buffer (w/v)). conclusion: we present the case of a patient with specific allergy to quail egg white and yolk, probably through ovotransferin, but not to chicken egg. the desensitization against chicken eggs does not allow the consumption of eggs of other species of birds, such as quail eggs, and this indication must be made specifically to patients after a protocol of desensitization against chicken′s eggs. case report: seven years ago we presented the case of a year-old male, who suffered two acute episodes of oral pruritus, lip angioedema, epigastric pain, generalized urticaria and dizziness after ingesting lettuce. he previously tolerated salads (including lettuce). he was later diagnosed with non-ltp-dependent lettuce anaphylaxis and an aspartyl protease was identified as a new lettuce allergen with cross-reactivity with other members of the compositae family. since the diagnosis he has avoided lettuce and all the other compositae. we present the patient's curious outcome after years of follow up. methods: total ige, basal tryptase, specific ige to lettuce by immu-nocap, prick-prick and oral challenge tests with lettuce and other compositae. sds-page, immunoblotting and molecular characterization of ige binding bands by mass spectrometry. skin prick tests and specific ige to lettuce were repeated on several occasions in the following years. after years an oral challenge test with lettuce was carried out. results: prick-prick test was positive to fresh lettuce ( × mm) and to other compositae (raw endive, chicory, thistle, artichoke and chamomile). prick-prick test was negative with these boiled compositae. basal tryptase was . μg/l. total ige in serum was ul/ml. specific ige by immunocap was positive to lettuce ( . ku/l) and negative to profilin, ltps and thaumatin. oral challenge test with endive was positive and negative with cooked compositae (artichokes and thistle). sds-page and immunoblotting detected an intensely binding ige band about kda, common for endive and chicory, which was identified by mass spectrometry as an aspartyl protease. no bands were detected at ltp ( kda) and profilin ( kda) . in the first two years after the diagnosis, prick-prick test and specific ige to lettuce remained positive (cap . ku/l and . ku/l, respectively). after years, prick-prick test and specific ige to lettuce became negative (cap . ku/l). with this findings a new oral challenge test with lettuce was carried out which result turned out negative. we report the first case of spontaneous tolerance to lettuce in a patient who previously presented lettuce anaphylaxis and identify an aspartyl protease as the causative allergen. introduction: eosinophilic esophagitis (eoe) is an emergent allergic inflammatory disease that is triggered by food allergens and characterized by progressive esophageal dysfunction. recently, it has been seen that eoe develops in up to . % of patients with ige-mediated food allergy undergoing oral immunotherapy (oit). ingestion of baked milk and egg was associated with increased development of tolerance to regular milk and immunologic changes have been reported in subjects ingesting baked milk and egg, similar to those seen in food oral immunotherapy studies. case description: we present the case of a -year-old girl, with history of ige mediated cow′s milk allergy and rhino conjunctival and bronchial asthma symptoms. prick test against milk proteins showed: milk mm, alpha-lactalbumin mm, casein mm and to beta-lactoglobulin: negative. total ige: ku/l. specific ige to milk: , alpha-lactalbumin: , ku/l; casein: ku/l and beta-lactoglobulin: . ku/l. we performed an oral food challenge with baked milk which was well tolerated. then, we performed an oral food challenge with fresh milk and she presented facial urticaria and pharyngeal pruritus. after months eating baked milk every day, she had symptoms of dysphagia and esophageal food impaction. for this reason, we performed an esophagogastroduodenoscopy (egd) and biopsies which showed white exudates and vertical furrows. the histological study showed eosinophil count> per high-power field. eosinophilic esophagitis was diagnosed and she started treatment with esomeprazole mg. the egd was repeated weeks later with similar results in the biopsy. she was treated with a comprehensive diet free of cow′s milk proteins. after weeks she was asymptomatic and endoscopy and biopsy findings were normal. we report the case of a cow′s milk allergic patient who developed eoe after introduction baked cow′s milk which apparently was tolerated in her diet. the avoidance proved efficacy in inducing the remission of eoe. method: we examined bottle-feeding infants with food allergy aged from to months. serum vitamin levels were measured by immunoassay methods (retinol binding protein (rbp), vitamin b , hydroxy vitamin d), biochemistry methods (vitamin c, vitamin e) and microbiology methods (vitamin b , vitamin b ). as criteria for complete sufficiency standards adopted in the russian federation were used (the lower limit of normal levels: rbp - . μmol/l; b - ng/ml; -hydroxy vitamin d- ng/ml; vitamin c - . mg/dl, vitamin e - . mg/dl; vitamin b - μg/ml; vitamin b - ng/ ml). results: complete sufficiency were observed in infants ( . % cases), one vitamin deficiency in infants ( . %), two vitamins deficiency in infants ( . %), three and more vitamins deficiency in infants ( . %). should be used after vitamin status asses, mainly using monovitamin medication. results: patients were followed with the diagnoses of cma and asthma. age at the beginning of symptoms and start of oit were in the range of - months and - years, respectively. they had high total ige ( - iu/ml), milk spige ( . - kua/l), casein spige background: food allergies may affect up to % of school-aged children. it has been shown that approximately % of all anaphylactic reactions caused by food allergy are firstly presented at (pre) school. therefore, it is of high importance that (pre)schools have a policy on food allergy management and the use of an epinephrine auto-injector (eai). to our knowledge, limited is known on the policies of food allergy management at (pre)schools. the aim of this study was to investigate the policy on food allergy management in preschools and primary schools in the northern part of the netherlands. results: we included preschools and primary schools in this study. we showed that . % of the preschools and . % of the primary schools had a child(ren) with food allergy. only . % of the participating preschools and . % of the primary schools had a policy on allergen avoidance and only . % of the preschools and . % of the primary schools had a policy for the use of an eai. the majority of the pre-and primary schools in the northern part of the netherlands have children with food allergy. however, only a limited number of (pre)schools do have written guidelines for food allergy management in (pre)schools. additionally, there is limited experience how to use an eai at (pre)schools. therefore, an evidence-based policy on food allergy management in (pre) schools is needed. background: food allergies are the most common cause of anaphylaxis in childhood. here we present cases had anaphylaxis due to cow 's milk allergy, and treated with specific oral tolerance induction (sotİ) to cow' s milk. method: soti protocol was administered according to previously published by longo et al. skin prick test were performed according to standard methods with allergens. cow's milk specific ige was investigated with immunocap system. in all cases, the wheal size of the cow's milk in skin prick tests or the specific ige levels was higher than level of positive predictive value of %. results: case : a years old male patient who had anaphylaxis after milk consumption. soti treatment was started one year ago. there was no complications during the dose increasing phase. however, he had two episodes of anaphylaxis during the maintenance phase. in the final visit, we observed that he could drink ml milk and could consume dairy products. case : eight-year-old male patient has an intensive care-of-hospitalization story due to anaphylaxis three times after milk consumption. his accordance to strict diet was bad, and had frequent asthma attack. anaphylaxis developed times during the dose increasing phase in soti protocol. after soti treatment, he could consume ml cow's milk and dairy products without problems. case : a -year-old male patient followed-up for asthma and cow's milk allergy. it was learned that anaphylaxis developed times after milk consumption. he was fed in accordance with the milk-free diet. he has used fluticasone nebules, montelukast, mometasone nasal spray, and cetirizine. anaphylaxis developed times during the dose increase phase of soti administration. there were many mild to moderate anaphylactic episodes during the maintaining phase. after the soti treatment, he could consume ml milk and dairy products. background: cow's milk protein allergy (cmpa) is one of the most common food allergies in early childhood. small dietetic group sessions for parents of infant with non-ige mediated cmpa were held to meet increasing demands and reduce waiting times. parents were given information on cmpa, advice on weaning and milk reintroduction using a locally designed milk ladder. parents were also advised to contact the dietitians via telephone if they had further questions. we aim to evaluate the sustained effectiveness and patient satisfaction of the group sessions. method: parents and carers who attended the group dietetic sessions held between november and july were included in the survey. feedback were obtained via a self-designed questionnaire using a likert-type scale, rating several questions from (least satisfied) to (most satisfied). initial feedback was obtained directly after the session. we followed these patients up a year after the initial session via telephone and postal questionnaire. results: overall attendance rate of the group sessions held was % (n = ). during the initial survey, participants found the group session useful (mean score . out of ) and felt more confident in managing cmpa (mean score . ). we successfully obtained follow up feedback from participants. majority agreed that the group sessions have been informative (mean score . ). they also said they felt confident weaning their child on milk-free diet (mean score . ), and in reintroduction of cow's milk in diet (mean score . ). % (n = ) said that they would have preferred an individual session. % (n = ) have contacted the dieticians via telephone after the initial session, and % (n = ) had requested individual consultations. % (n = ) have attempted reintroduction of cow's milk in their child's diet using our local milk reintroduction guide. the mean age at first challenge was months (age range to months), with average of two attempts. % (n = ) have been successfully challenged and are managing well on a normal diet. we recognised the limitation in obtaining feedback via telephone and postal questionnaire, which resulted in the poor follow-up response rate. overall, parents felt more confident in managing cmpa and the positive responses were sustained a year on, highlighting the success of these group sessions. follow up opt-in sessions could be offered to provide additional support and allay parental anxiety in challenging their child with cow's milk at an appropriate age. results: goats and rabbits were immunized with specific allergoids, the allergoid-specific igg titer determined and sera pools produced. the allergoid reference material was comprehensively characterized. while ige reactivity of the allergoids was not detectable anymore, igg reactivity was maintained. allergoid-specific assay parameters as serum dilution, reference dilution and sample dilution factor to obtain at least six data points within the pseudo-linear range of the inhibition curve were determined. with these set parameters the evaluation of the analytical method was performed. the assay showed very good results in terms of linearity, accuracy, precision, reproducibility and robustness for all investigated allergoids as well as aluminum-adsorbed allergoid-preparations. conclusion: with the developed immunological inhibition assay, it is possible to determine the specific igg reactivity of allergoids in different preparations. the performance of the analytical method met all pre-defined acceptance criteria, which will be confirmed in the next step by a validation procedure according to ich-guidelines. case report: we present the case of a -year-old patient, diagnosed with rhinitis and asthma due to sensitization to pollens, as well as dyshidrosis and allergic contact dermatitis to cobalt. the patient presented a cutaneous pattern consisting of erythematous papules, some scaly, very pruritic, of initial appearance in the upper limbs - days after initiation of administration of specific immunotherapy extract (depigoid forte grasses, leti ® ). subsequently generalize lower limbs and neck. she presented them repeatedly and late after - days of the first doses administered. she referred partial control of pruritus with oral antihistamines, without total resolution of lesions for weeks. immunotherapy was suspended persisting the skin lesions for more weeks. according to the personal history of sensitization to metals, and the clinic presented in a temporal relationship with the use of an extract of immunotherapy with aluminum hydroxide, a study was requested with epicutaneous tests with aluminum hydroxide as well as with epicutaneous tests with immunotherapy extract. aluminium hydroxide and depìgoid forte grasses extract epicutaneous test were negative. in subsequent visits the patient reported that coinciding with the start of immunotherapy, presented at home and mainly in her bedroom a plague of cimex lectularius, popularly known as bedbugs, proving that they had been the cause of bites on their skin, and later skin reaction. patient reported that with the elimination of said pest the skin lesions disappeared. the administration of its immunotherapy extract was tolerated. cimex lectularius, commonly known as bed bugs, is a hemiptera insect of the family cimicidae. the clinical picture usually corresponds to multiple pruriginous lesions from the prurigo type, to multiple erythematous plaques, some infiltrated and others with a urticarial appearance, or even bullous. the lesions last for to weeks without treatment, and while the older ones heal, new ones may appear. in our patient it was not considered as an initial diagnosis, having considered immunotherapy as an etiological factor, but we must not forget that although in our country it is not a reason for frequent consultation, either due to underdiagnosis, because of the transitory nature of the pathology or because of scarce number of causative agents, it is important to consider insect bites in the differential diagnosis of dermatosis. di cara g; salvatori c; testa i; pacitto a; bizzarri i; isidori c; tarsia m; esposito s università degli studi di perugia-dipartimento di scienze chirurgiche e biomediche, perugia, italy background: house dust mites (hdm) are one of the most important allergens involved in childhood respiratory diseases, and the most frequently prescribed extract for sublingual immunotherapy in children (slit). despite the improvement of standardization methods for the production of slit, the differences in cultivation and purification processes used to produce raw materials for specific immunotherapy extracts may still impact on the final composition of mite allergen extracts. our study investigated the total protein and main allergen content of five commercial hdm sublingual immunotherapy extracts using sds-page and immunoblotting. recombinant allergens of group and group major allergens were used to test the immunogenicity of such extracts. method: hdm slit extracts were purchased from five italian suppliers (alk-abellò, allergy therapeutics, anallergo, lofarma, stallergenes). the protein composition of extracts was evaluated analysing equal volumes ( ml/lane) by sds-page ( % separating gel) and subsequent immunoblotting. for identification of allergens in the extracts, western blot analyses were performed with rabbit monoclonal antibodies (raybiotech) against der p and der p . the total protein content in the five tested commercial extracts showed a relevant variability. the protein contents ranged from . to . μg/mg for what concerns der p , while der p showed a greater variability, ranging from . to . μg/mg. sds-page showed a similar pattern of distribution in of the tested extracts, which showed protein bands of comparable intensity, while extracts showed a lower total protein count. extract showed a higher intensity band corresponding to the molecular weight of tropomyosin. western-blotting showed a similar concentration of der p in most extracts, while der p was more variable. conclusion: our analysis of five commercial extracts commonly used for sublingual specific immunotherapy against hdm showed important variations in term of total protein content. a less evident but still relevant difference was also evidenced when testing the major allergen content, with up to % variation in der p and up to a -fold variation in der p concentration. this differences, likely related to the different production and extraction methods, could still be responsible of a different immunological response in children who underwent slit. method: we evaluated children who had completed their immunotherapy treatment. along with demographic data we were able to record skin prick test (spt) results and mrqlq at start and end of treatment. we only included patients who had completed pre and post treatment questionnaires in the study to allow a comparison. the scores were evaluated using a student t test. results: patients' starting age ranged from to years (mean years). of the children had completed pre and post treatment questionnaires. all had grass pollen allergy confirmed on spt at the start of treatment. patients ( %) had isolated grass pollen allergy on spt and ( %) had multiple allergies. mean start treatment score for all patients was on mrqlq. mean score at end of treatment was , indicating a % reduction in total mrqlq score (p value < . ). for those with multiple allergies the mean total mrqlq scores were at start of treatment and at end of treatment, indicating a % reduction (p value . ). for those with isolated grass pollen allergy scores were at start of treatment and at end of treatment indicating a % reduction (p value < . ). conclusion: for children with uncontrolled symptoms of allergic rhinoconjunctivitis, grass pollen immunotherapy is associated with statistically significant improvement in quality of life. this improvement is most beneficial for patients with isolated grass-pollen sensitivity on spt. those with multiple aeroallergen sensitivities on spt did show an improvement (not statistically significant) post-treatment. grass-pollen immunotherapy is an effective treatment for rhinoconjunctivitis to offer patients in a rural dgh setting. background: allergic asthma is a common clinical refractory disease, most patients with asthma are accompanied by varying degrees of allergy. in clinical practice, treatment of this disease using specific immunotherapy has proven effective. in the current study, we examined the effectiveness of specific immunotherapy in a total of patients admitted to our hospital from to . method: to investigate the clinical efficacy of allergic asthma-specific immunotherapy. patients were selected, of which were males, aged to years, females, aged between to years old, all patients were clinically diagnosed only as allergic asthma. the patients were randomly divided into two groups, including the observation group containing cases, the control group of cases. all patients were first treated with conventional basic treatment. the observation group was subsequently treated with specific immunotherapy. both groups were followed-up and the treatment efficiency were analyzed. results: after treatment, both two groups of patients showed improvement, in the observation group, the effective rate was %, while for the control group, the effective rate was %. observation group showed significantly better outcomes than the control group. conclusion: in allergic asthma treatment, adding specific immunotherapy on the basis of routine treatment is beneficial and could be widely used in clinic. case report: atopic dermatitis(ad)is the most common itchy dermatosis that affects millions of children and adults. during recent years, diagnosis and treatment based on component resolved diagnostics (crd)is recommended. we report a -year-old boy with severe atopic dermatitis. he had positive family history of atopy. the atopic dermatitis was developed since infancy. he was referred to our clinic when he was years old. he had generalized xerosis with ulcerative eczematous lesions on his neck, popliteal and antecubital areas. he had mild eosinophilia and his serum total ige level was iu/ml. daily bleach bath, moisturizing agents, topical steroids and systemic antibiotics in addition to antihistamine were prescribed. he had multiple food and aeroallergen sensitization in skin prick test (spt). he started to eliminate some foods according to the spt results. he was suffering from recurrent relapse even after strict food avoidance; so treatment with cyclosporine was initiated for him, with partial response. crd showed sensitization to alternaria alternata (alt a specific ige: . ku/l). allergen immunotherapy by alternaria alternata was started. after accomplishment of buildup phase, he had significant improvement and we were successful to taper and finally discontinue cyclosporine. now he is on maintenance phase of immunotherapy, his skin is in optimal condition only by hydration and moisturization. result: a -year-old thai girl is a known case of severe asthma since one year old. her asthma was uncontrolled asthma even treatment with high dose combination of inhaled corticosteroid and long acting beta agonist (ics/laba), montelukast and omalizumab. spirometry revealed the force expiratory volume in one second (fev ): % predicted, fev /forced vital capacity (fvc): % predicted and % improvement of fev after salbutamol ug inhalation. allergic sensitization showed specific ige to cat: . kua/l. slit with cat allergen started at the dose of au per month and increased to au per month (scit dose is au per month) for three-year-and-six-month course. after slit, her asthma symptom improved significantly. she can exercise without exacerbation and plays sport at school. her last episode of asthma exacerbation was . year ago. her fev (% predicted) was improved from % predicted to % and the fev bronchodilator response decreased from % to %. conclusion: an improvement of pulmonary function and asthmatic symptoms of the presenting case would support the efficacy of slit of cat allergen in a patient with severe asthma. ra developed during pollen scit in this case might be related with immunomodulation effect of immunotherapy. background: we report a case of -year-old woman with allergic rhinoconjunctivitis and mild persistent asthma due to sensitisation to seasonal pollens and molds with bad clinical evolution and not response to conventional drug therapy. we decided to start subcutaneous allergen specific immunotherapy with alternaria extract in our immunotherapy unit in accordance with the guidelines of the european academy of allergology and clinical immunology (eaaci) and we used a cluster regimen. the immunotherapy was not well tolerated: the patient had two grade systemic reactions with the first dose in two attempts. method: sensitisation was diagnosed through skin prick test with aeroallergens standard panel and serum specific ige by elisa. due to the bad tolerance to immunotherapy, molds molecular diagnosis by immunocap, study of the molecular weight to specific ige binding proteins by sds-page ige immunoblotting, and cross-reactivity study by means of immunoblotting-inhibition assay were performed. a. fumigatus extract was able to produce a total ige binding inhibition on the kda band of a. alternata extract when ige immunoblotting assay was performed. conclusion: respiratory allergic disease due to alternaria is difficult to control, the use of subcutaneous specific immunotherapy could be of significant benefit. most of the allergic patients to a. alternata are sensitized to alt a , major allergen from a. alternata. however, our patient is sensitized to a kda alternaria protein due to cross-reactivity with a. fumigatus allergens, this sensitization could explain the bad tolerance to the alternaria immunotherapy. background: the association between natural pollen exposure, clinical symptoms as well as allergen-specific immune responses has not been investigated at a molecular level. our aim was to monitor the effect of seasonal birch and grass pollen exposure on clinical symptoms as well as specific b cell and t cell responses to defined allergen molecules in sensitized subjects during two consecutive years. method: grass pollen sensitized (n = ) and birch pollen sensitized (n = ) subjects were included in this study and were followed for two consecutive years ( ) ( ) . subjects were taking part in a clinical trial for the recombinant grass pollen vaccine (bm ) but did not receive immunotherapy for the allergen they were sensitized to. before, during and after the respective seasons ige and igg levels as well as t cell responses to the major birch pollen allergen bet v and the major grass pollen allergens phl p , , and were measured. pollen counts were recorded throughout the year and patients kept a daily diary including symptom medication score (sms) and visual analogue scale (vas). results: we noted that ige levels specific for bet v and the grass pollen allergens increased most in the seasons in which patients experienced the highest peak symptoms according to vas and sms but not depending on cumulative pollen counts. increases in allergen-specific t cell responses were observed in the pollen seasons as compared to shortly before the pollen seasons in the grass pollenallergic patients also in association with vas and sms but not in the birch pollen allergic subjects. no relevant changes of allergen-specific igg levels were observed during the two years observation in grass and birch pollen allergic patients. we found an association of increases of allergen-specific ige increases shortly after the pollen season with clinical symptoms in the pollen season as reflected by vas and sms which was not necessarily reflected by cumulative pollen counts in the season. these results may be important for the analysis of allergen-specific immunotherapy trials. background: the morbidity and mortality of severe asthma is much higher than that of mild to moderate asthma. this study was performed to understand the clinical characteristics of severe asthma in korea. results: data from the questionnaire showed that bronchial asthma was diagnosed before pregnancy only in women ( . %). patients ( . %) were diagnosed with chronic bronchitis at the pregestation stage. asthma attacks were experienced repeatedly during a lifetime in . % of patients, . % of patients noted long periods of dry cough at night, among them . % had wheezing. the cold did not precede the wheezing breathing in . % of patients. difficulty in breathing on waking was noted in . % of patients, at night- . %. after examination, the diagnosis of asthma was confirmed in . % of the respondents ( people). symptoms of rhinitis are noted in % of women surveyed, % of rhinitis was allergic. before examination, the diagnosis of ar was only in . % of patients. the incidence of symptoms of asthma and ar in pregnant women is significantly higher than the reported cases of these diseases, which leads to untimely initiation of treatment. method: a total of nonsmoker asthmatic patients without concomitant pulmonary pathology are recruited to our study. all patients underwent spirometry tests, measurement of fraction of exhaled nitric oxide and sputum induction to asses sputum cell counts, demographic features and current medications were recorded. using the variables of age at onset, bmi, allergy status, fev %, fev /fvc, asthma severity and induced sputum cytology cluster analysis is performed. results: clusters are identified. cluster : (n = ) early onset atopic asthma, consists of mild asthmatics with a good asthma control and lower bmi. cluster : (n = ) severe atopic asthma, consists of lowest spirometry measurements with a least act scores. induced sputum cytology shows a neutrophilic character, while having also the highest percentage of eosinophils. cluster (n = ) late onset obese asthma, nonatopic asthmatics having high spirometry measurements, with a lower act scores. cluster (n = ) nonatopic mild asthma, consists of patients with the best respiratory functions and least inflammation in means of lowest total ige, feno, sputum cell counts. conclusion: identification of asthma phenotypes in different countries will improve our understanding on the heterogeneity of the disease among the different geographies. results: results and discussion. in the course of analysis, obesity was more common in children with bronchial asthma − % than in the comparison group-in . %. obesity of the st degree was diagnosed in patients of the main group, ii degree-in , and iii degree- and iv degree-in patients e diagnosis of obesity, the sds indices of body mass index (bmi) were determined. obesitymore than + . (i degree: sds bmi . - . , ii degree: sds bmi . - . , iii degree: sds bmi . - . , iv degree: sds bmi ≥ . ). conclusion: thus, the results obtained indicate a high prevalence of constitutional-exogenous obesity in children with bronchial asthma and precedes the formation of the underlying disease e diagnosis of obesity, the sds indices of body mass index (bmi) were determined. obesity-more than + . (i degree: sds bmi . - . , ii degree: sds bmi . - . , iii degree: sds bmi . - . , iv degree: method: postal questionnaires were distributed to an unselected group of asthma patients (n = ). healthy non-asthmatic volunteers were recruited amongst university and hospital co-workers (n = ). the presence of self-reported nhr, the type of triggers evoking nasal symptoms, asthma phenotype, medication use and environmental factors were evaluated. results: patients and controls completed the questionnaire (responder rate of % and % respectively). nhr was reported in % of asthma patients and % in non-asthmatic controls (p < . ), with changes in temperature being the most important inducer of nasal symptoms ( % of asthmatics), followed by strong odours ( %) and cigarette smoke ( %). interestingly, nhr was more prevalent in patients with severe ( %) compared to mild ( %) asthma symptoms (p = . ), and more prevalent in atopic ( %) compared to non-atopic ( %) asthmatics (p = . ). most asthma patients reported more than one trigger evoking nasal symptoms, with % of patients reporting or more triggers evoking nasal symptoms. results: the mean score of cbcl questionnaire in case group with . ± . was significantly higher than in comparison with a control group with . ± . (p = . ). the mean scores of the subscales of social isolation (the case group: . vs control: . , p = . ), anxiety-depression ( . vs , p = . ), intellectual problems ( . vs . , p = . ), and aggressive behaviors ( . vs . , p = . ) were significantly higher in children with asthma than in healthy children. the study showed a significant correlation between the mean duration of asthma and a general score of cbcl (p = . , cc= . ). moreover, there was also a significant correlation between asthma severity and cbcl scoring (p = / , cc= . ). conclusion: behavioral disorders in children with asthma are significantly more than healthy children. the duration of asthma and the severity of asthma, are related to and can predict behavioral disorders in children with asthma. background: assessment of asthma control is an integral part of the management of asthma. whilst asthma control test (act) is a commonly used questionnaire to assess symptom control, its utility in predicting long term risk of exacerbation has not been well studied. aim: to analyze the factors associated with uncontrolled asthma symptoms using act and its impact on predicting future exacerbation. method: severe asthma patients on at least step background: most of the asthma-scoring tools detect the asthma severity from patients' symptoms but there is no scoring tool using parameters to define risk of asthma exacerbation. thus, this study use factor analysis to evaluate the relationship of parameters in childhood asthma. method: the descriptive study using factor analysis in asthmatic children aged less than years old, who attended thammasat university, the center of excellence for allergy, asthma and pulmonary diseases, thailand. the participants or caregivers were inter- the factors which have the major impact on asthma control are changing bed sheets less than once per month and using dust mite-proof bed sheets. this study is supporting non-pharmacological strategies but further studies are needed to create a more efficient asthmatic symptom checker. were not different between the controlled and uncontrolled group. the act score in the controlled group was significantly higher than the uncontrolled group (p < . ). the study showed that cigarette smoke is one of the significant factors that can trigger asthma exacerbation (p < . ) and mosquito repellent coil smoke is also significantly associated with asthma exacerbation (p < . background: experimental studies have demonstrated that tumor necrosis factor family member (tnfsf /light) plays an important role in airway remodeling. there is little data available concerning in vivo regulation of tnfsf /light expression in humans. the aim of this study was to evaluate serum concentration of tnfsf / light in different subsets of asthmatic patients. the study was performed on nonsmoking asthmatic patients (a), including mild-moderate-severe asthmatics controlled on inhaled corticosteroids (aics) and asthmatics evaluated twice during asthma exacerbation (aex) and during subsequent remission (arem). in addition age and sex matched nonsmoking healthy controls were included (hc). serum tnfsf /light concentration was evaluated using elisa method. in asthmatic patients lung function tests, exhaled nitric oxide concentration (feno), serum total ige concentration (t-ige), allergen-specific ige concentration (s-ige) and peripheral blood eosinophilia were evaluated. ( + /- pg/ml) was significantly greater than that in hc ( + / - pg/ml; p < . ). among all asthmatic patients studied the greatest tnfsf /light serum concentration was demonstrated in aex ( + /- pg/ml), which was significantly greater than that in aics ( + /- pg/ml p < . ). during resolution of asthma exacerbation a significant decrease in serum tnfsf /light concentration ( + /- pg/ml; p < . ) was demonstrated. in arem the mean serum tnfsf /light concentration was comparable to that seen in aics (p = . ) but was still significantly greater than in hc (p < . ). no significant correlation could be demonstrated between serum tnfsf /light concentration and baseline lung function parameters, exhaled nitric oxide concentration, serum t-ige or s-ige concentration or peripheral blood eosinophilia. conclusion: enhanced production of tnfsf /light seen in asthmatic patients, which is further upregulated during asthma exacerbations may play an important role in asthma pathogenesis. method: two models of aspergillus fumigatus-induced allergic airway inflammation were used in the study: long terms ( weeks) and short terms ( weeks background: chalcone is identified as an inhibitor of the interaction between cxcr or cxcr and their ligand cxcl . therefore it is called a neutraligand. the chemokine cxcl , interacting with the cxc-receptor (cxcr ) can play a role in the progression and development of bronchial asthma. asthma is defined as a chronic disease characterized by episodes of obstructive events which affects about million people over the world. the aim of this study is to approach the mechanism of the anti-inflammatory effect of the cxcl neutraligand chalcone and also to assess its impact on the migration of dendritic cells in a murine model of allergic airway inflammation. method: chalcone is administered intranasally to balb/c ovalbumin (ova) asthma mice and control groups as well. our results indicate that the cxcl neutraligand chalcone can modify the inflammatory reaction in an airway allergic hypereosinophilia model. furthermore, found out that cxcl neutraligand chalcone prevents dc migration to the airways and airway jnc ganglia during allergic airway inflammation. the detection of the cxcr -cxcl pathway and its role in the pathophysiological actions of asthma offers a promising target for allergic diseases treatments. method: four groups of balb/c mice were defined: control and asthmatic, with and without treatment. asthmatic groups were sensi- overexpression of ptgdr in pulmonary cells associated to a generalized increase of cytokine expression. conclusion: in a mouse model we confirmed the involvement of ptgdr in allergic asthma by the increase of its expression levels after ovalbumin sensitization. we also identified a reduction of ptgdr levels in response to dexamethasone treatment. the in vitro model suggests that ptgdr induces an inflammatory response, increasing the cytokines levels. | immune imbalance between transcription factor t-bet/gata and allergic asthma results: t-bet mrna expression of peripheral blood lymphocytes in patients with allergic asthma was lower than that of the normal control group, and the expression level of gata- mrna was higher than that of the normal control group (p < . ). the th percentage of peripheral blood lymphocyte subsets was lower than that of the normal control group (p < . ), the percentage of th cells was significantly higher than that of the normal control group (p < . ), and the changes in t-bet/gata expression and th /th ratio was highly correlated. our objectives were to assess the changes of bmp and bmp serum levels in the response to allergen and methacholine challenge tests and the correlation between bmp and bmp serum levels and fev before and after allergen and methacholine challenge tests. method: study group consisted of patients with asthma and healthy volunteers. spirometry, skin prick tests, allergen and methacholine challenge tests were performed in compliance with eaaci, ers and ats guidelines. personalized clinic surveys including act ™ were performed. venous blood was collected before and after hour, and hours afterwards the provocation to edta-ke-filled test tubes. evaluation of bmp and bmp serum protein levels was performed using specific elisa immunoassay kits according to the manufacturer's protocol. results: the increase in bmp and bmp serum level hours after provocation test correlates significantly with the concentration methacholine during provocation time (p < . ). bmp serum level before the provocation, hour and hours after provocation, correlates negatively with fev change (p < . ). the median bmp level hours after provocation was significantly lower in patients with negative methacholine challenge test compared to the control group (p = . ). the median bmp level hours after provocation was higher in patients with positive allergen provocation test than in patients with negative test results (p = . ). the bmp serum level hours after positive methacholine test is lower and correlates inversely with fev change in every time point, which could indicate that serum level of bmp is a predictive factor of fev change. the higher bmp serum level, the lower fev change was observed. this could suggest the protective influence of bmp in patients with obstructive pulmonary disease, i.e. asthma. the higher bmp serum level hours after positive allergen provocation test result shows that the bmp could be an indicator of the response to a specific trigger. background: inflammation and coagulation are closely linked events. thrombin is the key enzyme in coagulation system. besides its well-known functions in hemostasis, thrombin plays a role in inflammation. the aim of our study was to evaluate thrombin generation in children with mild asthma and demonstrate associations between thrombin levels and control of asthma. method: forty-two children with mild asthma and forty-nine healthy children included in the study. asthmatic children had no asthma exacerbation during the last months. patients (n = ) who had mild persistent asthma, were using either inhaled steroid or montelukast. all patients performed spirometry. thrombin levels were measured by thrombin generation test. thrombin peak levels, endogenous thrombin potential, thrombin lag time, time to thrombin peak and thrombin tail time were recorded. results: thrombin lag time was significantly longer in children with asthma ( . ± . ) compared to those in control group ( . ± . ) (p < . ). children with asthma also had longer thrombin tail time compared to control group ( . ± . vs . ± . , p = . ). thrombin peak was inversely correlated with fef - (- . , p < . ). thrombin lag time was inversely correlated with fef - (- . , p < . ). thrombin generation parameters did not show difference according to asthma control treatment, asthma control scores and having atopy. conclusion: coagulation/anticoagulation balance is disturbed in mild asthma but this disturbance may not be as strong as to increase thrombin levels. factors increasing inflammation may cause an increase in lag time, and increase in inflammation and excessive fibrin deposition may contribute to airway narrowing. background: cytokines represent key mediators in the onset and persistence of inflammatory process, in both asthma and copd. il- , which belongs to il- family, it might act in a similar way with il- at the beginning of the inflammatory process. its role in atopic skin diseases has already been demonstrated, but there are conflicting results related to its role in respiratory allergic diseases. the aim of the study was the evaluation of il- plasmatic level in patients with asthma and copd and the its correlation with clinical and lung function parameters. method: fifty consecutive patients with bronchoobstructive diseases were included in the study. thirty-two patients presented asthma and patients had copd. the evaluation included: number of exacerbation in the last year, disease's severity, spirometry. plasmatic levels of il- and il- were determined in all patients. results: the mean age was higher in patients with copd dermatophagoides pteronyssinus [house duste mite (hdm), ug/ mouse] were administered oro-tracheally on days , , , , , , and . at was performed in a treadmill during weeks in moderate intensity, from day until day . results: at inhibited hdm-induced total cells (p < . ), eosinophils (p < . ), neutrophils (p < . ) and lymphocytes (p < . ) in bronchoalveolar lavage (bal), and eosinophils (p < . ), neutrophils (p < . ) and lymphocytes (p < . ) in peribronchial space. at also reduced bal levels of il- (p < . ), il- (p < . ), il- (p < . ), cxcl (p < . ), il- (p < . ), il- (p < . ), il- (p < . ), while increased il- (p < . ). airway collagen fibers (p < . ), elastic fibers p < . ) and mucin (p < . ) were also reduced by at. at also inhibited hdm-induced airway hyperresponsiveness (ahr) to methacholine . mg/ml (p < . ), . mg/ml (p < . ), mg/ml (p < . ) and mg/ml (p < . ). mechanistically, at reduced the expression of stat (p < . ), stat (p < . ), stat (p < . ) and jak (p < . ), similarly by peribronchial leukocytes and by airway epithelial cells. socs expression (p < . ) was upregulated in leukocytes and in airway epithelial cells, socs (p < . ) was upregulated in leukocytes and socs down-regulated in leukocytes (p < . ) and in airway epithelial cells (p < . ). conclusion: at reduces asthma phenotype which is followed by positive modulation of socs-jak-stat signaling in peribronchial leukocytes and in airway epithelial cells. rodolfo a ; paciência i ; rama t ; leão l ; silva d ; rufo j ; mendes f ; padrão p ; oliveira fernandes e ; moreira p ; delgado l ; moreira a porto, portugal; potentially irritating chemicals that may have a cutaneous drying side effect. this study aimed to evaluate if skin barrier function, as measured by transepidermal water loss (tewl), is affected by a training session in swimmers compared with football players. environment impact on the human respiratory health (clinicaltrials.gov identifier: nct ) and football players were invited to participate. due to the lack of prior information no sample size calculation was possible and all athletes that provided informed consent were included in the analysis (n = , females, aged to years). tewl was measured using the tewameter ® tm before, immediately after, and minutes after a hours training session. the probe was held on the dorsum of hand, the volar forearm and the antecubital flexure for s. the average of two consecutive measurements was recorded. non-parametric statistic was used were appropriate. ethical approval was obtained from the university clinical research ethics committee and informed consent provided. results: mann-whitney u test showed significantly higher baseline median tewl level on football players hand's dorsum compared with swimmers, median (p -p ) respectively . ( . to . ) and . ( . to . ); p = . . friedman test revealed a significant effect of swimming on tewl on the hand's dorsum, volar forearm and antecubital flexure (p < . ) while football training affected only the hand's dorsum (p = . ). differences in changes after swimming and football training were significant only for tewl in volar forearm (p = . ). in conclusion, our exploratory findings do not provide support for a specific deleterious effect of swimming, compared with football training, on the training induced changes in tewl. background: exercise-induced bronchoconstriction (eib) is defined as transient, reversible airway narrowing occurring during or after exercise, is common among elite athletes and associated with epithelial damage. however, little is known about the existence of eib in young athletes. the goal of this study is to investigate the presence and to evaluate potential (bio)markers of eib in young high-school elite athletes in different sport disciplines versus age-matched control subjects. method: high-school selected elite athletes ( - years) from different sport disciplines: basketball (n = ), football (n = ) and swimming (n = ) performing at least hours of sport per week (median= h) and control subjects (performing less than hours of sport per week) were recruited. the eucapnic voluntary hyperventilation (evh) test was performed according to ats guidelines and adapted for this age group. lung function was measured before, immediately after and , , minutes after the evh test. the test was considered positive if a maximal fall in fev of % was measured on at least one time point and exhaustion was excluded. a blood sample was obtained at baseline. sputum induction and skin prick test for the most common allergens were performed after the evh test. results: fifteen swimmers had a positive evh test ( . %), which is higher than in basketball players ( . %), football players ( . %) and controls ( . %). . % of the swimmers were atopic which is also higher than in basketball players ( . %), football players ( . %) and controls ( . %). serum clara cell secretory protein (cc ) levels are significantly higher in swimmers ( . ± . ng/ml) compared to indoor athletes ( . ± . ng/ml) and controls ( . ± . ng/ml). a significant positive correlation was found between the magnitude of maximal fall in conclusion: young elite swimmers have a higher prevalence of eib compared to basketball and football players. atopy and/or chlorine is a risk factor for the development of eib in young elite athletes. cc levels and sputum uric acid levels are increased in athletes compared to control subjects suggesting the presence of epithelial damage already at young age. this is especially observed in young elite swimmers, pointing to a probable role of exposure to chlorineby-products in combination with intensive exercise. results: data from subjects ( females, . %) were analyzed. frast was positive in ( . %) patients ( females, median age of years (iqr - )). in this group, ( . %) had a previous diagnosis of asthma, ( . %) practiced federated sports, ( . %) had smoke cigarette exposition and ( . %) had a bmi > kg/m . . % of patients showed a Δfev % > % in the first minutes after finishing the challenge. median fev reduction was . % ) and ml . frast was more frequently positive in patients with previous diagnosis of asthma (p < . ). there were no differences related to conclusion: frast is an important tool to diagnose exerciseinduced bronchospasm without asthma (eib wa ), as well as to diagnose asthma. in our study, frast was fundamental to access eib wa in % of patients with rsee, and confirmed asthma diagnosis in % of cases with previous asthma diagnosis and negative sbt. there was no difference in the prevalence of atopy between patients with positive and negative frast. patients older than years-old presented higher Δfev % compared to younger patients (p = . ). higher levels of feno were observed in patients with positive frast (p = . , p = . ), both in patients with and without previous diagnosis of asthma. a positive correlation was observed between feno levels and Δfev % in the whole sample (r = , p = . ); when these data were analyzed considering a previous diagnosis of asthma, only patients with this condition showed a positive correlation of feno and Δfev % (r = . , p = . ). conclusion: our results evidenced that higher feno was associated with atopy and a positive frast, both in patients with and without previous diagnosis of asthma. higher feno seems to correlate with Δfev % in patients with previous diagnosis of asthma. background: specific immunotherapy is the casual treatment for allergic rhinitis. a year old professional footballer suffer from severe allergic rhinitis since two years. during may, june and july his level of playing, concentration and durability decreased about %. patient was complaining of runny nose, nasal blockage, each eyes, sneezing, tearing. method: we did skin prick tests -which showed greatest allergy to grass pollen. we confirmed the allergy by specific ige and nasal provocation tests. spirometry was done-fev %. the patient was qualified to undergo specific immunotherapy. however, because of his profession, it was hard to find a day without trainings to get the vaccine. after long discussion, patient decided to start specific immunotherapy-scit. results: the patient start the immunotherapy. he was attuning very irregularly, because of matches, injuries, trips, trainings, and lack of time. several times we had to call the patient to remind him about the immunotherapy. after one year of scit the patient felt big improvement. during grass pollen season he suffered from mild allergic symptoms, and just for few days. after next year of immunotherapy, the patient had no symptoms of allergic rhinitis during the grass pollen season. however, it was the reason for him, to stop sit, before rd year of immunotherapy. conclusion: such a treatment-specific immunotherapy-is a burdensome method for both, for professional athletes and doctors. such a patients need to be on special observation, and cooperation with trainers must be obtained, if we want to see results. to improve compliance we have to keep in touch with patients, to remind them about next visit. gherasim a ; choual i ; radu c ; khayath n ; beck n ; jacob a ; schoettel f ; domis n ; de blay f alyatec, strasbourg, france; hôpitaux universitaires de strasbourg, strasbourg, france background: late allergic response (lar) is a good asthma model. it has been shown, in individual challenge tests that mite allergen induces more frequently late allergic responses (lar) than cat allergen. the aim of this study is to compare the frequency of lar in asthmatic subjects allergic to mite with asthmatic subjects allergic to cat. method: asthmatic subjects allergic to mite were compared to subjects allergic to cat (gina or ). the subjects had prick tests≥ mm compared to the negative controls and specific ige ≥ . ku/l. the dose selected for the mite and cat allergen was the airborne allergen concentration inducing the most frequently early asthmatic response (ear) (a % drop in fev ) and lar (a % drop in fev ). results: the frequency of lar with mite allergens was . % and % with cat allergens (p = . ). the frequency of ear for mites was . %; of . % for ear or lar, and % for ear and lar. in contrast, with cat allergens, % of patients had an ear, % had ear or lar and % had an ear and lar. no significant differences was observed between cat and mite allergen regarding the severity and the time necessary to obtain an ear and lar. no significant differences was observed between cat and mite allergen regarding the severity and the time necessary to obtain an ear and lar. the frequency of lar in asthmatic subjects allergic to dust mite exposed in alyatec ® eec was higher than in asthmatics sensitized to cats. our results confirmed previous results with individual bronchial challenge. therefore, it appears that the mite model is more interesting in the study of asthma. exposure chamber in strasbourg (alyatec ® ) in asthmatic patients allergic to cat allergens gherasim a ; choual i ; radu c ; khayath n ; beck n ; jacob a ; schoettel f ; domis n ; de blay f alyatec, strasbourg, france; hôpitaux universitaires de strasbourg, strasbourg, france background: as recommended by the task force on environmental exposure chamber (eec), allergenic and non-allergenic exposure must be better controlled in eec. it is the aim of alyatec's eec. the aim of the study is to validate alyatec's eec by determining the concentration of fel d inducing % of early asthmatic response (ear) and/or late phase asthmatic response (lar) in subjects sensitized to cat. method: it was a randomized, double blind, cross-over study including group a: asthmatic subjects allergic to cat and group b: asthmatic subjects allergic to another allergen. all subjects were first exposed to placebo. group a was exposed to fel d concentrations. the number and size of particles were recorded online during the exposure. group b was exposed to the concentration of fel d which fulfills the objective of the study. the mean age of subjects was years (± ). for the concentrations of fel d , we obtained more than % ear and/or lar. the mean time necessary to obtain an ear was: . ± minutes and . ± minutes for the lar. the mean fall in fev during ear and lar was − . % and − . % respectively. we didn't observe any severe reaction. no subjects in group b experienced any symptoms during exposure. we have validated alyatec's eec in asthmatic subjects allergic to cat allergens. we also demonstrated its specificity. background: the best test and strategy for diagnosing asthma especially in those patients with negative bronchodilator reversibility tests still remains unclear. in this study we aimed to investigate the diagnostic yield of peak expiratory flow (pef) variability for the patients with symptoms suggesting asthma but negative bronchodilator reversibility tests. method: subjects referred to our outpatient clinic with suspicion of asthma were enrolled in this study. demographics and referral symptoms were recorded, asthma control test (act) scores and health related quality-of-life scores (aqlq, sf ) were calculated. monitoring of pef variability during -weeks and bronchial challenge test with methacholine (bpt) were analyzed. asthma was diagnosed by having pef variability ≥ % and/or positive bpt. results: thirty out of enrolled patients were diagnosed as having asthma. when we compare asthmatic patients with nonspecific respiratory symptomatic subjects there were statistically-significant differences regarding to wheezing (p = . ), activity limitation (p = . ), total symptom score (p = . ) and basal fef (p = . ) in the favor of asthma cases. multiple logistic regression analysis revealed that lower basal fef - was an independent predictive factor of asthma diagnosis (p = . ). when the bpt positivity was assessed as gold standard for the diagnosis of asthma, the sensitivity and specificity of pef variability for different cut-offvalues (≥ %, > % and >% ) were . - . %, . - . % ve - . %, respectively. conclusion: fef - is an important diagnostic parameter for asthma. although current guidelines recommend pef variability of % for the diagnosis of asthma in general, this cut off level may not be appropriate for this defined group of subjects. our results suggest to use a cutoff level of > % while excluding asthma and ≥ % while confirming the diagnosis of asthma for patients with asthma suspicion but without shown reversibility. de barayazarra s background: in recent years obesity has been considered as a factor that contributes to the development of asthma, increases exacerbations and leads to poor control of it due to resistance to drugs to control this pathology. it is known that obesity produces chronic systemic inflammation; one of the markers that are affected is the levels of c-reactive protein (crp), which are increased. objective: evaluate, lung function, the use of medications to control asthma and systemic inflammation, after bariatric surgery. results: obese asthmatic patients with surgery, non-asthmatic obese patients with surgery, obese asthmatic patients without surgery. a significant difference was found between the severity of obesity and forced expiratory volume in patients with asthma and without asthma of second (fev ) before surgery with an average of . % at the beginning of the study and . % at months (p: . ). in the non-operated group, fev at the beginning was % and . % at months (p: . ). the crp, before surgery in all operated patients had crp: , at months after surgery they became negative, crp: (p: . ). in obese asthmatics with surgery at the beginning, % used medication, and at months only % in obese asthmatic patients without surgery, . % used the medication at the beginning, at months . % (p: . ). method: patients with asthma aged to and a predetermined positive methacholine pc were recruited and underwent a single challenge to cause bronchoconstriction of~ % comparing the outcome of the device with spirometry. the subjects were monitored at baseline, after a~ % fall in fev and after bronchodilation back to baseline. the study protocol allowed for an interim analysis of the initial subjects at which point the sensor was calibrated to optimise sensitivity. a further subjects were studied using the optimised sensor. results: all subjects successfully completed the study. the device was found to be straightforward to use by both operator and subject with no concerns regarding safety. the initial sensitivity of the device was found to be suboptimal in the first eight patients to reliably detect changes in lung function. after adjustment to the device the tests results of the remaining subjects were analysed. . % of subjects were female. the mean age of all subjects was . years. an average baseline fev value of . (s.d. . ) was observed. changes in lung function were detected in % of subjects. a baseline value, drop in lung function and reversal were measured in % of subjects. the mean percentage drop observed in % of subjects using the investigational device was . %. the mean percentage increase observed using the investigational from drop to reversal was . %. the device (using ebc ) was able to detect changes in lung function tracked using fev . this provided proof of concept that the device could potentially be used to monitor lung function more effectively in the home than peak flow and supports further development to optimise the device and demonstrate functionality in clinical asthma. method: ninety four patients under years of age seen in the allergy department due to common asthma symptoms (wheezing, dyspnea, cough, chest tightness) with normal spirometry and negative bronchodilator response, underwent mct during and . the variables studied were: sex, age, body mass index (bmi), asthma symptoms, exercise symptoms, rhinoconjunctivitis, family history of atopy, sensitization to respiratory allergens, spirometric data and fractional exhaled nitric oxide (feno). results: of the total sample, half were women ( . %) and the other half were males ( . %). mean age was . years. bmi was normal in most of them (with an average of . kg/m ). the most common symptom among the patients with positive mct was cough ( . %), followed by dyspnea ( . %), wheezing ( %) and chest tightness ( . %). . % had symptoms of asthma with exercise and . % had rhinoconjunctivitis. . % had a family history of atopy. . % were sensitized to aeroallergens, mainly to pollens (grass and olive tree). . % of the mct′s were positives, with a mean pc of . mg/ml. . % had a moderate-severe result (pc ≤ mg/ml), . % mild (pc - mg/ml) and . % bordering (pc - mg/ml). the mean feno was . ppb. conclusion: in our series, the completion of a test of hrb was decisive to confirm the diagnosis of asthma in most patients of a pediatric population with symptoms of suspicion (cough, mainly), normal spirometry and negative bronchodilator response (with normal feno in most of them). therefore, we consider it important to include in the routine clinical practice hrb tests in the pediatric population with suggestive symptoms of asthma, despite normal functional and/or inflammation tests. | cut-points of the ′control of allergic rhinitis and asthma test′ (carat) asthma subscale based on an international survey patients with asthma) in kashan, iran. the data collection tool was a questionnaire with questions, designed to gather information on demographic asthma patients, the current use of mobile functionalities, and the willingness to use these functionalities to receive selfmanagement services, which was distributed among patients with informed consent. the collected data were analyzed by descriptive statistics method using spss software. results: the most use of patients from mobile phone functionalities was to receive information about asthma symptoms and allergens and irritants via mobile internet ( . %). patients were most likely to use social networking ( . %) in comparison with other mobile phone functionalities, to receive reminders about appointments and medication. the respondents were most likely to use social networks through mobile phone functionalities, to receive asthma self-management information ( . %), to communicate with other patients ( . %), to receive reminders about medication use, and to perform a peak flow meter test ( . %) and to get an alert when the asthma is not controlled ( . %). the findings show that asthma patients are currently using the internet search for educational information and they have a tendency to use social networks to receive asthma-related services. patients believe that mobile health is an appropriate intervention for providing educational information, reminders, and alerts and communication with other patients. | concordance between the determination of asthma control through the gina guidelines and the act questionnaire-results of the efimera study background: the exacerbation of asthma, progressive worsening of acute episodes, is one of the most frequent attending reasons at hospital emergency unit . several factors causing poor control of asthma, such as inadequate therapy, have been described. in the present study, estimations of asthma severity by researchers were assessed by comparing the concordance between the assessment of asthma control through the gina guidelines and the act questionnaire method: cross-sectional observational study on the evaluation of factors related to treatment that influence the poor control of asthma was assessed through the gina guidelines and the act questionnaire. patients referred to a pneumologist or allergist by a primary care for the first time were evaluated. two variables were collected for the assessment of asthma control: one derived from the gina guidelines and another derived from the act questionnaire. regarding the gina assessment, researchers' evaluations guidelines were compared with the scoring calculated from the variables registered in the crd. both measures were compared in terms of sensitivity-specificity to determine their ability to classify patients. the patients included in this study (n = ) had a mean age of ± years, with a % of women and an average disease evolution of . ± . . the control of asthma according to "gina results: pts were reasonably representative of those in sls asthma (at sls baseline: . % male; mean age . yrs; mean asthma control test [act] score . ) . the most frequently reported symptoms during sls asthma for these pts were cough/ breathlessness, followed by wheeze, phlegm and chest tightness; breathlessness and wheeze were perceived as the biggest impactors on pts' lives. the aspects of daily life most impacted by asthma were reported as walking at a hurried pace, strenuous physical activity, and asthma-related frustration. since sls began, % of pts in this subset reported improvements in overall asthma ( % no change; % worsening). perceived changes in symptoms are shown (table) . most pts ( . %) reported avoiding places with dust, smoke or fumes. most pts ( . %) perceived no change in overall qol; . % reported improvement. being an act responder during sls (total act score ≥ or ≥ change at end of sls) was associated with reported improvements in overall asthma symptoms, lower impact of asthma on qol, and higher perceived confidence/control in managing asthma. more pts ( . %) in the ff/vi arm reported an overall improvement in asthma vs uc ( . %); the most evident differences between treatment groups were for breathlessness, wheezing and chest tightness. improvements in confidence/control in managing asthma were reported by . %/ . % of pts (ff/vi) vs . %/ . % (uc). conclusion: breathlessness and wheezing were key symptoms in sls asthma and had the biggest impact on pts' daily lives. this patient-centred study enriches the findings of sls asthma. funding: gsk (study ) | asthma and copd treatment adherence and breach using tai questionnaire suarez-vergara m; fuentes-soltero f; garcia-nunez i; ignacio-garcia j background: adherence is defined as medication (inhalator) intake following the dosage and schedule prescribed. adherence mistakes are a public healthy problem according to the big morbi-mortality presented in patients with an incorrect intake. our aim is to evaluate the adherence level and fulfillment in patients with asthma or copd using tai (inhalators adherence test) questionnaire. method: patients with a diagnosis of persistent asthma or copd were selected. we used to size adherence and breach type the tai questionnaire. adherence is defined as good when patient′s test reaches points, medium ( - points) and bad (less than points). breach type is defined as erratic when points between questions to are less than , deliberate when questions to are less than , and unconscious when questions and are less than points. a correct fulfillment is defined when questionnaire reaches points plus points of conscious fulfillment. results: fifty-five patients more than years old (mean age . years and . % males) were selected. a . % of them were asthmatics, . % copd and . % a mix phenotype. a . % presented a correct fulfillment with conscious fulfillment, and the other . % presented good, medium or bad adherence with a breach type. according to adherence level, a medium adherence was defined in patients ( . %), with an erratic mistake in patients ( %). bad adherence was seen in patients ( . %) , with the three breach types in patients ( %). good adherence with unconscious breach type was defined in patients ( . %). conclusion: tai questionnaire confirms a good adherence and fulfillment in less than % patients. an erratic mistake is the most frequent breach type defined in our patients. educational protocols should be applied to improve adherence and fulfillment. | what is adhesion to treatment of asthmatic patients like in argentina according to the tai questionnaire? background: asthma is a chronic inflammatory disease of the airways, which requires an adequate treatment and control. the adhesion of a patient to an asthma treatment is a critical factor in order to achieve and maintain control. this adherence arises from a consensual agreement of the doctor-patient relationship; it is a complex multifactorial variable in which the variability in human behaviour in relation to its environment influences. background: the association between ambient pollen and asthma has been studied intensively with inconsistent results, attributed to differences in study population, geographic factors (geoclimatic features), data sources, measurement of pollen (different types of traps), and different outcome occurrence (hospitalizations or emergency department visits). we investigated the associations between daily sales of short-acting β -agonists (saba) and outdoor pollen concentrations in the central france area. the relationship between daily changes in pollen concentrations and daily saba sales obtained from the social security database was analysed with generalized additive models, taking into account confounding factors such as air pollution, weather conditions, and day of the week. results: the daily saba sales (mean, sd) rose from . ( . ) conclusion: this study indicates that outdoor pollens contribute to asthma morbidity in the general population. it confirms the highly allergenic role of fraxinus, betula and quercus pollens, but also shows a relatively unknown association between treated asthma and carpinus and platanus pollens, despite their counts being less than % of overall pollen concentration. results: of asthma patients (mean age . years, female . %), regular ocs use was identified for patients ( . %), periodic ocs use for patients ( . %), and no ocs use for patients ( . %) -year post-index. regular ocs users had a greater mean age, were more often male, and had greater eosinophil counts, lower lung function, and greater prevalence of comorbidities than did the periodic and no ocs users (p < . ). total yearly cost was greatest for the regular ocs users (€ ), followed by periodic ocs users (€ ) and no ocs users (€ ) (p < . ). among regular ocs users, hospital admissions were the main cost driver ( . % of total cost), while gp consultations were driving the total cost in periodic and no ocs users ( . % and . % of total cost, respectively). conclusion: in this sample of patients with asthma in sweden, the total yearly cost of health care resource utilization for a regular ocs user is twice as high as for a patient with no ocs use, demonstrating substantial economic and clinical burden in asthma patients on regular oral steroid treatment. method: children with physician-diagnosed asthma who attended to an outpatient pediatric allergy and asthma center were enrolled in the study along with control subjects. asthma severity and control status of the patients were evaluated according to recent gina guidelines. laboratory investigations including skin prick tests, complete blood counts with differential, total ige levels, serum periostin levels and pulmonary function tests were performed. results: a total of children ( with asthma and age and sex-matched control subjects) with a median age of . years (range . - . ) were enrolled. asthma severity was mild in ( . %), moderate in ( . %) and severe in ( . %) children. children with asthma had significantly higher periostin levels than controls ( . ± . vs . ± . ng/ml; p < . ). the mean serum periostin levels of children with severe asthma ( . ± . ) were significantly higher than in children with moderate asthma ( . ± . ) and mild asthma ( . ± . ) (p < . ). serum periostin levels were found to be significantly correlated with asthma severity (spearman's rho [r]=. , p < . ). analysis using roc curves identified the role of periostin levels in determining children with severe asthma (auc: . , % ci: . - . , p < . ]. conclusion: serum levels of periostin, a novel asthma biomarker, were higher in asthmatic children, and were associated with asthma severity. adam i ; selevestru r ; rogut v ; sciuca s background: nowadays, data from several epidemiological studies confirm the important role of fungi in respiratory disease in the indoor as well as in the outdoor environment. in general, exposure to fungi occurs via inhalation, skin contact, or ingestion. alternaria alternata is one of the most common fungi associated with presence asthma and persistence and severity of asthma. although exposure to a. alternata is also may represent a risk factor for development of asthma. in ukraine has been an increase in the number of the mold sensitized children for the last few years. at the same time we can see increasing frequency ba at the children of pre-school age. method: thirty five children aged - years with allergic rhinitis and high level of asthma predictive index (api) sensitized to a. alternata were included in a -year cohort study of the efficacy and safety of slit (diater laboratories, spain) using standardized sublingual extracts containing molds (alternaria alternata). treatment efficacy was analyzed using the score of symptoms such as difficulty in nasal breathing, rhinorrhea, sneezing, itching of the nasal mucosa (upper palate) and discharge from the nose and recurring wheezing. we also have analyzed the level api during the period investigation. symptoms were measured before starting treatment, and at , and months after starting immunotherapy. results: slit significantly reduced both symptoms and medication score: nasal symptoms ( % vs. control group) and the use of rescue medications ( % vs. control group), and improved fev (in children aged≥ years). in the slit group, api decreased by % for the first year, by % for the second year. no patient had a systemic reaction during therapy. our results have shown that slit is an effective treatment in pediatric patients suffering from allergic rhinitis and high api with significantly improved clinical outcomes (less symptoms and less medication intake) in comparison with children treated with symptomatic drugs only. in this study, large and statistically significant differences in symptom and medication scores were demonstrated in patients receiving slit compared to control group. sublingual immunotherapy is effective for allergic rhinitis in children especially early age and is generally advantageous because of the convenient administration and safety profile and ensure prevention of developed ba. bednarek a background: the classification of asthma based on the severity of its clinical course has been recommended by gina since . this division is useful for the patient's initial assessment when asthma is being diagnosed and essential decisions concerning an appropriate therapy are made. the objective of the work is to evaluate the influence of a clinical form of asthma on vaccine immunity in preschoolers following three years after the programme of mandatory vaccination has been realised. the study encompassed preschool children (mean age of . ± . years old) with asthma being newly diagnosed, including patients with mild asthma and ones with moderate asthma, whose vaccine immunity (igg specific antibody titer) was assessed after the mandatory early childhood vaccines had been administered. monovalent vaccines (hbv+ipv+hib) along with a three-component combined vaccine (dtwp) were given to children while a six-component vaccine (dtap+ipv+hib+hbv) was given to the remaining children. the vaccine doses were consistent with the polish immunisation programme and manufacturers' recommendations. the elisa immunoenzymatic method was applied to assess titer of specific antibodies to diphtheria, tetanus, pertussis, poliomyelitis and h. influenza type b. the level of hbv antibodies was measured chemiluminescently. the immunity class for particular vaccinations was assessed according to the test manufacturers' instructions. results: children suffering from mild asthma had considerably more frequently vaccinations on time (p < . ) and the type of vaccines (monovalent, highly-combined) administered to them did not have a significant influence on a clinical form of asthma in the children examined (p > . ). apart from the vaccines against hepatitis b and rubella where considerably more frequently a high antibody titer occurred in children with mild asthma, the titers of antibodies to other vaccines, namely diphtheria, tetanus, pertussis, hib and mumps, were not associated with a clinical form of asthma. the protective antibody titers in the children with asthma were found in % after vaccinating them against poliomyelitis (≥ u/ ml) and measles (≥ ml u/ml). significantly higher current weight was solely found in the children with mild asthma (m = . , sd= . ; p < . ). conclusion: there are some clinical and cultural differences among the four southern chinese cities within the canton province. this study identifies potentially modifiable environmental and treatment factors associated with poor asthma control and qol for healthcare interventions. having a smoker in the family is independently associated with poor asthma control and qol. were classified into two groups (levocetirizine group (l) and montelukast group (m)) and we treated each group for another week. to evaluate the therapeutic effectiveness, we used symptom score (ss) and ebc leukotriene e (lte ). ebc samples were collected with rtube. each parameter was checked at , , week therapeutic period. results: most ar patient showed clinically improvement with and week fluticasone therapy ( wk ss= . , wk ss= . , wk ss= . p < . in l group; wk ss= . , wk ss= . , wk ss= . p < . in m group). lte levels of ar were higher than control ( wk vs. pg/ml), and were reduced after week fluti- mic were: md allergic rhinitis, wheezing apart from colds, eosinophilia ≥ %. outcome was defined as md asthma and at least episode of asthma during the previous year or more than episodes of wheezing during the months regardless of asthma diagnosis. results: from a total of of parents approached, ( %) agreed to participate in a phone interview. ( %) children were diagnosed with asthma. the age at the time of admission (mean, abstracts | (sd)) was . ( . ), at the time of phone survey . ( . ) months, respectively. positive loose api at - years of age had sensitivity of . %, specificity %, positive predictive value (ppv) . %, negative predictive value (npv) . %. positive stringent api at - years of age had sensitivity of %, specificity %, ppv . %, npv %. background: asthma is the most common chronic airway disease in childhood, with a high unmet need for new treatments due to insufficient symptom control in a relevant percentage of patients. ethics and resource factors limit the feasibility of large, long pediatric trials required to assess outcomes such as exacerbations and symptoms. for diseases like asthma, where the disease process is largely similar in children and adults, with the same expected therapy outcome, the international council for harmonisation advise extrapolating adult data to those of a younger age, reducing unnecessary pediatric trials. here we assess the partial extrapolation used in the clinical development of tiotropium. phase trials in adults (aged - ), adolescents (aged - ) and children (aged - ) with symptomatic severe (primotina-/pensie-tina-/vivatina-asthma) or moderate asthma (mezzotina-/rubatina-/ canotina-asthma), respectively. trials lasted - weeks, all with tiotropium respimat μg add-on vs placebo as two puffs once daily. results: in adult trials, lung function, symptoms and exacerbation endpoints were evaluated in a confirmatory manner: tiotropium significantly improves lung function and asthma control, and reduces risk of exacerbation, vs placebo ( conclusion: based on similarities in disease profile and magnitude of treatment responses between age groups, it is reasonable to expect tiotropium add-on to produce clinically meaningful improvements in exacerbation and symptom endpoints in children and adolescents, as in adults. the robust tiotropium clinical program supports using a partial extrapolation to avoid overly long and large trials in pediatrics. | clinical state of treatment and examination during last years before remission about asthmatic children in long-term remission cases method: remission cases (no symptom and no therapy) for years of asthmatic children were studied. clinical background and treatment (drugs) was studied during last years before remission annually. acetylcholine inhalation test by standard method was performed, and respiratory threshold of acetylcholine (rt-ach) was obtained. fev %, and serum ige also examined. these data were compared before remission with years after remission. results: mean age of cases at year before remission was . years old. male to female ratio was . . severity of asthma was all mild type, and number of attack was to times in a year. there was no admitted case during this study. the long-term therapeutic drugs were leukotriene receptor antagonist (anti lt) in cases, and/or inhaled corticosteroids (ics) in cases, but cases had no treatment for the control. geometric mean of rt-ach (after then: years before and after remission) was μg/ml and μg/ml. the mean fev % was % and %. geometric mean of serum ige level was iu/l and iu/l. complicated cases of atopic dermatitis decreased after remission, but the incidence of allergic rhinitis increased slightly. conclusion: characteristics of asthmatic children during last years before remission were mild type, had several times of attack in a year, and the treatment was mainly anti lt and/or ics. fev % was within normal range, and serum ige level was not changed after remission. rt-ach had the tendency to improve during years before and after remission. these data is supposed that airway hyperresponsiveness is one of the indicators for quitting treatment. | clinical aspects of polyvalent mechanic bacterial lysate (pmbl) treatment in children with uncontrolled asthma our results indicate that long-term treatment with omalizumab in children can help to achieve better asthma control and reduce the amount doses of basic therapy. method: allergic rhinitis (ar) and allergic rhinoconjunctivitis (arc) diagnosed-patients' demographic information, accompanying-asthma, the allergic history of the family, the onset of symptoms, types of aeroallergens sensitivity were noted from patients' files in our hospital's pediatric allergy clinic. results: in this study, patients were evaluated. the mean age of the patients were . ± . years and % (n = ) were male. ( %) patients had ar and ( %) patients had arc. background: allergic rhinitis (ar) is a disease characterized by symptoms of nasal discharge/congestion, sneezing, and pruritus, and is caused by an ige-mediated immunological response to inhaled allergens. we aimed to evaluate pollen season and out of pollen season pulmonary function tests (sft) of patients with ar in our study. method: in our study, the demographic characteristics and aeroallergens were recorded from patients' files with ar diagnosed. in addition, pollen season and out of pollen season sfts were evaluated and compared. conclusion: in patients with ar, fev and fvc values are seen to be lower during the season even though there is no lower respiratory symptom. therefore, sfts of patients with ar should be evaluated during pollen season. results: among the clinical manifestations, the most common combination of allergic rhinitis (ar) and conjunctivitis (ac) is noted in . % of adults and . % of children, but in children aged - , the combination of ar and ac is observed only in . %, among - years old- . %, while in the remaining age groups it is encountered in more than %. higher percentage of isolated ar is also observed among young children- . %, and those of the results: allergic rhinitis was a main symptom in . % of children with pollen-food sensitization. in all of them concomitant allergic disorders were noticed: bronchial asthma ( . %), atopic dermatitis ( . %). only in . % temporal association between ingestion of pollen-related foods and nasal symptoms was observed (mainly apple and peanuts); occurring also outside the pollen period. the simultaneously sensitization to animal origin food allergens was stated in . % of children with sar, but only in two of them milk and white egg proteins were an additional exacerbation factor of nasal symptoms. in . % anaphylactic reactions to food allergens were registered. . % of children were asymptomatic despite pollen-food sensitization. the statistically significant differences were noticed in comparison to the control group. conclusion: . allergic rhinitis in children, similar to adults, is a common manifestation of pollen-food syndrome and this type of sensitization should be taken into account regardless to age. . children with pollen-related food allergy have the predisposition to multiorgan clinical manifestation. . the lack of association of symptoms with plant-origin foods in the majority of cases and the asymptomatic course of food sensitization in more than one third of patients indicate the need for follow-up. | clinical benefit of the screening of suspected food allergen using multiple allergen simultaneous test in the patient with pollen-food allergy syndrome (pfas) background: the quantitative fluoresce enzyme immunoassay immunocap (ic) system has been widely used for detection of allergen-specific ige for the diagnosis of allergy. however, the system can only detect ige against a single allergen, the multiple antigen simultaneous tests has been developed such as the fluorescence enzyme immunoassay view allergy (va) or chemiluminescent enzyme assay mast iv (ma) and both assay detect more than allergen-specific ige. in this study we examined the diagnostic capability of these two systems for screening test in the patient with pfas. method: total number of participants are (male/female: / ), aged . ± . (range ~ ) years old. all the patients showed oral allergy syndrome (oas) to rosaceae family plants (apple, peach) and/ or kiwi and/or banana, also showed tree pollen allergy. specific ige assay were performed using ic, ma or va. results of greater than class were to be regarded as positive, and the concordance rates between the assays were assessed. results: the correlation of sensitivity between pr- (rbet v , rmal d , rpru p , measured by ic) and specific ige to apple (measured by va), specific ige to peach (measured by ma) in oas patients to rosaceae family plants were assessed. rbet v , rmal d , rpru p were found to be . %, . %, . % positive measured by ic while the specific ige to apple (supposed to be including pr- ) were found to be % positive measured by va. on the other hand, the specific ige to peach (supposed to be including pr- ) were found to be only . % positive measured by ma, this detection rate was lower than that of va (p < . ). also, the correlation of sensitivity between pr- (ract d , measured by ic) and specific ige to kiwi in patients with oas to kiwi were assessed. ract d were found to be . % positive measured by ic while the specific ige to kiwi (supposed to be including pr- ) were found to be . % and . % measured by va and ma, respectively (p < . ). additionally, all the oas patients to banana found to be positive for the specific ige to banana measured by va, but only patient was detected as positive measured by ma. conclusion: in this study, we found that va showed better agreement of sensitivity and specificity with ic compared to ma in the oas patients to rosaceae family plants, kiwi, or banana. therefore, it may be clinically useful for screening of allergen specific background: the hygiene hypothesis for autoimmune and allergic diseases, which exists nowadays, shows that human immune system is dependent on various environment factors. we consider the effects of humic substances (hs) to be important in understanding the hygiene hypothesis. due to urbanization, the amount of human interaction with hs found in soil has significantly dropped. the goal of our work was to study allergenic potential and antimicrobial activ- conclusion: hs appear to be exogenous immunocorrectors, and also to have an ability of suppressing propagation of allergic reactions and sensibilization, which leads to conclusion that they seem to play a major role in hygiene hypothesis. moreover, hs selectively interact with bacterial cell wall, and this effect could be used in order to create antimicrobial drugs based on hs. background: peach tree pollen has been identified as having relevant allergens (the third most prevalent after olive tree and grass pollen) in areas of high cultivars (murcia, east-spain). when analyzing molecular components in sensitized patients, along with pru p , we have identified other relevant inhalant allergens one of which was named pru p x. because pollen of different species share allergens and with plantderived food, we have also studied peach tree pollen sensitization in a non-exposed population (madrid, central-spain). the aim was to study the association between peach tree pollen and several panallergens, as well as the relevance of pru p x in our area (madrid). method: a total of patients who came to our allergy unit in those patients with positive spt to at least one pollen we also performed peach tree pollen spt. if positive, we tested pru p , pho d , pho d and pru p x. to study the clinical relevance of these findings, we also performed nasal provocation test (npt) with peach tree pollen and pru p x. results: a total of patients were sensitized to peach tree pollen. from these, % had also positive spt to pru p and none of them to pru p x. positive spt to polcalcin were found in the % of the cases and to profilin in the %. in patients sensitized to peach tree pollen npt was performed being cases positive to peach tree pollen and none to pru p x. conclusion: peach tree pollen sensitization in non-exposed patients with allergy to other pollens is high although primary sensitization is unlikely. these patients present clinical response when exposed to that pollen that needs further evaluation. in our study, one third of the patients were also sensitized to polcalcin and pru p and none to pru p x. we have not found clinical response to this new inhalant allergen identified in highly exposed peach tree pollen population. results: the bet v elisa . -ep complete kit format (including pre-coated plates and all buffers and reagents) allowed for the consistent measurement of bet v in birch pollen extracts within the same lab (intralab cv= . %) and between different labs (interlab cv= . %). the average recovery from matrix spiked samples (crs in birch pollen extracts) ranged from - %, with an average recovery of % (n = ). assay time was reduced from several days to two hours compared to the original method. the performance of the bet v elisa . -ep kit was comparable to that of the stallergenes greer candidate standard method and has been successfully cross-validated. this will enable allergen manufacturers and regulatory authorities to adopt a standard method for bet v determination, which, ultimately, may be included in the european pharmacopoeia. the development of a certified elisa represents a major step forward in the standardization and quality control of allergen products. | an isoform of the ole e allergen assembled by proteomics could explain the cross-reactivity with pollen and food nsltps results: a total of peptides were obtained by de novo sequencing. ten of them allowed the completion of the full-length amino acid sequence of the allergen. after purification, role e was obtained with a yield of . mg/l of cell culture. immunological assays confirmed that the recombinant isoform of ole e shared most of the allergenic and antigenic properties of the natural allergen. moreover, we observed its implication in cross-reactivity with pollen extracts, and plant-derived food extracts. conclusion: these results suggest that the presence of this isoform in the olive pollen could explain the co-sensitization observed in some allergic patients between ole e and nsltps from foodderived extracts and might be used for a more effective clinical diagnosis of olive pollen sensitized patients. background: penicillium oxalicum, one of the prevalent airborne fungi in india, was selected to detect its spores as potential source of allergens and also to identify and characterise its major ige-reactive component. the airborne spores of penicillium oxalicum was detected by andersen -stage air sampler at different parts of west bengal. the allergenic potency of p.oxalicum was tested by spt, elisa and immunoblotting. total protein was resolved in -d and -d gel electrophoresis and allergens were identified by -d and -d immunoblots. identification of major ige-reactive protein spots was made by mass spectrometry based maldi-tof-tof. major allergen was partially purified by ion exchange chromatography. results: aerobiological investigation clearly indicated the predominance of p. oxalicum spores ( cfu m − ) in the air of west bengal, india. sensitivity of patients to spore antigens was highly correlated with rhinitis. in sds-page, bands were detected with molecular weight range of - kda. the allergenic potency of spores was confirmed by skin-prick test, elisa and dot-blotting. eleven ige-reactive proteins were detected as allergens by -d and -d immunoblots, of which % patients were sensitized to kda allergen. this kda protein was found to be the major allergen which was further characterized by mass spectrometry based maldi-tof-tof. this major allergen (pi . ) was partially purified by ion exchange chromatography. the eleven allergens were identified from spore of penicillium oxalicum fungi for the first time from india. immuno-proteomic identification of major ige-reactive protein ( kda background: airway epithelium (ae) is one of the largest cellular surfaces exposed to the environment. ae constitutes a physical barrier due to the presence of intercellular apical junctional complexes between neighboring cells. in the past years evidence indicates an association between epithelial airway dysfunctionality and allergic asthma. it is still unclear if an impaired epithelial barrier could be the cause of allergy development as opposed to the consequence. one of the most common comorbidities of asthma is house dust mite (hdm) allergy. it has been shown that hdm allergen der p can disrupt the epithelial airway due to its protease action against cellular apical junction complexes damaging the epithelial monolayer. in the last decade, metabolomics has been successfully employed as a new approach to describe metabolic changes in biological systems. metabolomics focuses on describing and identifying small molecules to explain complex biological processes. we theorized that metabolomics could be used as a new tool to detect damage of epithelial barrier in vitro after der p exposure. method: human cell line calu- cultured at air-liquid interphase (ali) was used as an in vitro model of bronchial epithelium. ali culture system allows establishing different compartments, mimicking the conditions found in the human airways: a basolateral compartment in which basolateral surface of the cells is in contact with the culture medium, and an apical compartment where the apical cellsurface is exposed to air. after days in ali, the cells were exposed to either der p or pbs as a control in the apical side for hours. then, apical and basolateral media were collected and processed for metabolomics analyses. results: metabolic profiles from samples were obtained, these were composed by and features for apical and basolateral media, respectively. of these, using mann-whitney unpaired test as statistical analysis, and features were found changed within the apical and basolateral compartments, respectively. specifically, in the apical compartment there were signals significantly increased and decreased after der p exposure; whereas for the basolateral compartment, signals were found to be significantly decreased and increased after exposure. background: mites are one of the major causes of allergies. it is known that allergen concentration varies depending on the species of mites and the degree of allergy induction is different, but the difference in microbiota according to mite species is not known. in addition to allergen, endotoxin or bacterial dna, adjuvants of allergen derived from the microbiota in the mites, are also present in the feces. bacterial endotoxin is found in gram-negative bacteria, acting on tlr and acting as an adjuvant to allergies. method: three species of mites (d. farinae, d. pteronyssinus, and t. putrescentiae), known to cause allergies, are cultured in same condition(autoclaved media, %rh, °c)and analyzed for microbiota of each species. using the next generation sequencing that complements the existing sanger sequencing, we analyze the difference of microbiome according to the dust mite species and measure the level of endotoxin. method: six hundred and thirty five patients ( . % males and . % females, mean age . years old, range to years old) were included. all of them referred respiratory symptoms (rhinitis, conjunctivitis or bronchial asthma) and had skin prick tests positive with any pollen. patients were skin prick tested with a battery of common pollens in our area, including three species of chenopodiaceae: chenopodium album, salsola kali and salsola oppositifolia. results: three hundred and forty tree ( %) patients were sensitised to pollen of any chenopidaceae species: ( . %) to chenopodium album, ( . %) to salsola kali and ( . %) to salsola oppositifolia. the prevalence of skin sensitisation to pollen of salsola oppositifolia was . % in the population studied and . % in patients sensi- results: in patients aged - years of age in . % of the cases ige reactivity was at least to one allergen tested. the majority of patients (more than / ) had a complex sensitization profile and reacted on average to more than allergens. the highest frequency of sensitization in ukraine among patients who turned to the clinic among adults was found phl p ( . %), amb a ( . %), fel d ( . %), bet v ( . %) and children ( . %, . %, . %, . %), respectively. when analyzing the results of tests for the source of the allergen, most often among house dust mites (hdm) allergens in adults and children is sensitization to fel d ( . %), as well as to hdm: in adults (der f - . % der p − . %, der f - . %, der p - . %) and in children ( . %, . %, . %, . %), respectively. among fungal allergens the most common is sensitization to alt a and varies from . % in adults to . % in children. among pollen allergens in adults is sensitization to phl p ( . %), amb a ( . %), bet v ( . %), cynd ( . %), art v ( . %), bet v ( . %) and in children ( . %, . %, . %, . %, . %, . %), respectively. tests for food allergens in adults and children are more common on pr- proteins. in children, sensitization to milk and egg proteins is more common than in adults. conclusion: most patients who came to the clinic have a complex ige reactivity profile in which pollen sensitization predominates. among hdm allergens, more than / of the examined have sensitization to the cat's proteins. sensitization to mold alternaria alternata in children occurs times more often than in adults. results: total children were examined, aged - years (median years). % children were sensible to two and more components . %to and more components. the frequency of sensitization to inhalation components was . %, to food abstracts | components- . %. among the most frequent inhalation components were feld - %, betv - %, amba - %, phlp - %, alta - %, the sensitization to house dust mites (hdm) was most often observed to der p - %. however, the analysis of these protein by the level of isu showed that the highest levels were for der f median (iqr . - . ), whereas for fel d - . (iqr . - . ). among food allergens, sensitization was most commonly observed to pr- proteins - %. children sensitized to pr- proteins were in most cases sensitized to -mal d ( %), cor a . ( %), pru p ( %).this co-sensitization was accompanied by a high correlation of isu levels among these components. sensitization to celery and kiwi was less common, the level of these proteins was also low. the frequency of sensitization to storage proteins was %, among which the highest level of isu was in ara h median . sensitization to ltp proteins was detected in % of children, among which the most commonly detected pru p protein was . %. the sensitization to profilins, which was evaluated at the level of bet v , was found in % of children, but the levels of these proteins were not high. among the food products of animal origin, the most frequent was sensitization to egg component gal d − . %, however, isu levels were the highest to milk component bos d − . (iqr . - . ). the most frequent causative inhalation allergens were epidermal allergens and weed pollen, however, the highest level of isu was to hdm and mould. among food allergens, the most commonly observed sensitization was to pr- proteins. hypereosinophilia of peripheral blood was observed in children under study, which was % ( . %). as a result of testing patients with a wide panel of allergens, % of the patients had diagnostic levels of antibodies to allergens siged , . %to allergens siged . in % of cases, a significant level of antibodies to plantain allergens sige w was detected, . % to dandelion allergens sige w , . % to evergreen trees sige t , to maple sige t to . %, to allergens of olive tree sige t - %, to the banana allergens sige f - . %, to the egg protein sige f in . %, in % to the milk allergens sige f , to the food mixture sige f x - . %, to allergens of mold fungi mx − . %. among the leading household allergens were registered in the st group and in the nd group of the investigated children -d pteronyssinus ( . %, . %), and d. farinae results: the prevalence results are expressed in the table . we have not observed any significant association in allergic rhinitis patients group with any ltp or pr- molecules. for atopic dermatitis only rara h (or with % ci - . ( . - . ) and njug r (or with % ci - . ( . - . )) were associated significantly. for asthma, the most important molecules were rbet v , raln g , rcor a . , rcor a . , rmal d , rpru p and rapi g (p-values for or less than . ). conclusion: future studies focusing on the evaluation the association of cross-reactive molecules with allergy phenotype should be done. background: the fuzzy/green kiwifruit (actinidia deliciosa), widely grown commercially, contains various pulp allergenic molecules, including the major allergen cysteine protease actinidin. methods. this case report is about a -year-old male patient with house dust mite allergic persistent rhinitis and intermittent asthma, presenting a convincing history of anaphylaxis immediately after eating a kiwifruit on empty stomach, followed, a few months later, by a severe oral allergy syndrome after licking a slice of raw kiwi. previously, the patient ate kiwi without any problems and had no manifestations of pollen or latex allergy. skin prick testing was done with commercial allergen extracts, while prick-prick testing was performed with raw kiwifruit, avocado and banana. molecular approach consisted in assessment of serum specific ige to native extracts and molecular allergen components using patient-friendly allergen nanobead array multiplex test and singleplex capsule-enclosed activated cellulose solid phase fluorescence enzyme immunoassay. results. regarding kiwifruit allergy, the patient presented positive prick-prick tests with raw edible kiwifruit components: outer pericarp and inner pericarp (each mm wheal) and columella/core ( mm wheal) and negative with kiwifruit whole seeds, avocado and banana, and pollen extracts. serum specific ige to kiwifruit were detected ( . ku/l), but specific ige values were negative (≤ . fiu/ml) for actinidin act d , thaumatin act d , kiwellin act d , nsltp type act d , bet v -like major latex/ripening-related protein act c , act c chitinase_iv, act d cross-reactive profilins bet v (birch pollen profilin) and hev b (latex profilin), and also negative (< . ku/ l) for pr- ract d . moreover, specific ige to avocado were nor found (≤ , fiu/ml). although ige against seed proteins cupin/ s globulin act d and s albumin act d were not determined, this was not considered of great importance since allergic symptoms were also induced by licking kiwi pulp, in which abundantly expressed actinidin enzymatically degrades seed storage proteins, and prick-prick test was negative to kiwifruit seeds. conclusion: in a patient with anaphylaxis to kiwifruit, positive skin tests to its pulp and detectable serum specific ige to actinidia deliciosa, a detailed molecular allergy diagnosis is necessary, including assessment for act d glycoallergen or other molecules, not performed in this patient. | is pr- sensitization a portuguese phenomenon as well? background: bet v , a major allergen found in birch pollen, belongs to the pr- protein group. in our practice, some bet v sensitized patients have been identified, residing in areas without this tree genus in its flora. our aim was to characterize a portuguese patient population with pr sensitization. method: a group of patients in whom immunocap isac ® (isac) study was performed, between january and june , were analyzed. all subjects with one or more pr- sensitizations were selected, and their clinical records reviewed. a sequential sample of the last subjects (n = ) who underwent isac study, was then used for comparison. results: out of isac studies performed, only were positive for pr- protein group. median age was . years, % (n = ) were male. pr- sensitized individuals were more likely to live in portalegre district compared to the control group ( / vs / ; p < . ). patients were positive for pr- family pollens ( . %), frequently bet v (n = ), followed by aln g (n = ) and cor a (n = ). out of the patients were sensitized to pr- foods, mostly cor a . (n = ) and mal d (n = ). skin prick tests revealed birch as the main sensitizing pollen as well ( / ). moreover, only four patients were skin prick tested for fagaceae trees which were positive for oak ( ), chestnut tree ( ) and cork tree ( ) . all patients were co-sensitized to other pollens, namely grass and all had respiratory allergy. nine patients were food allergic, although seven of them were co-sensitized to other cross reactive (ltp/profilin) or species specific proteins. conclusion: although pr- sensitization is known to be rare in our population, mostly alto alentejo inhabitants showed sensitization to this protein family in our sample, either by in vitro and/or in vivo methods. this phenomenon is consistent with the native plant species of this region, which should be taken into account when studying the allergic profile of these patients. in our sample, all pr- sensitized patients had respiratory allergy while this protein didn't seem to be relevant when it comes to food allergy. further studies are needed to characterize which plant species belonging to this protein family are more significant for our country's aerobiology context and to determine its clinical relevance. included. allergic asthma, rhinitis, conjunctivitis and eczema allergic symptoms were diagnosed. all patients were tested by immunocap with mugwort pollen extract and the natural components nart v , nart ar , nart v , and nart an . results: the positive frequency and sige levels of the four components in the artemisia allergic patients from southwestern china were significantly lower than that from the north. art v and art an were the highest recognized allergens, followed by art v and art ar . patients from northern china were more likely to have abstracts | asthma ( %) than patients from southwestern china ( %), and being sensitized to more than two allergens increased the risk of asthma. sensitization to art v , art v and art an played a significant role in the development of asthma. artemisia pollen allergic patients is helpful to assess the potential risk of asthma. conclusion: a small but significant part of the population react to ragweed pollen extract and are not identified as disease-positive by standard sige tests. there is a need for targeted tests towards a larger spectrum of allergen molecules. in ragweed allergic individuals, this allergy can be the main cause of overall sige levels and also of in vivo reactions (tested by spt). | molecular profile of pollen sensitization of tashkent residents with respiratory allergy background: in paediatric cohorts, a correlation between specific ige (sige) levels to house dust mite extract or allergen components and the occurrence of asthma has been shown. higher levels of sige to mite extract were associated with a higher risk of wheezing. moreover, asthmatic children recognized more allergens and had higher sige levels to nder p as well as rder p , and . we sought to investigate potential differences in sige levels or sensitization patterns between asthmatic and non-asthmatic patients in a mixed paediatric and adult house dust mite allergic cohort. method: total ige and specific ige against house dust mite extracts (dermatophagoides pteronyssinus and farinae) and allergen components (rder p , , , and ) were determined in house dust mite allergic patients. patients had diagnosed asthma ("asthmatic", % females, mean age ± years, % younger than years), whereas had rhinitis (and conjunctivitis) without respiratory symptoms ("non-asthmatic", % females, mean age ± years, % younger than years). results: total ige levels were markedly higher in asthmatic compared to non-asthmatic patients ( vs. ku/l, p = . ). positivity to rder p ( vs. %, p = . ) as well as rder p ( vs. %, p = . ) differed between both groups. specific ige levels to house dust mite extracts and allergen components (rder p , , , and ) and positivity to rder p and did not differ between both groups. conclusion: in contrast to previously published data, sige levels to house dust mite extracts or allergen components were not statistically different between asthmatic and non-asthmatic patients in our mixed paediatric and adult house dust mite allergic cohort. only higher total ige levels and a higher reactivity to rder p and were found in asthmatic patients. however, larger studies are needed to confirm clinical relevance of these findings. results: prior treatments reported at baseline (bsl) included: . % of pts were receiving or more second-generation h -ah at approved dose (recommended first-line), . % were receiving them at increased dose (second-line); . % were receiving omalizumab (third-line); . % had no treatment. the majority of pts ( . %) had uncontrolled csu (uct< ) at bsl (table) . treatment changes were most evident at the bsl visit, with an increase in pts receiving omalizumab ( . %) and a decrease in those receiving no treatment ( . %) vs. prior therapy. these changes were associated with improvements in rates of hives and/or angioedema, uct and qol scores at month , but only modest improvements thereafter (table) . a sub-analysis of pts with uct< and who were receiving the approved ( . %) or increased dose h -ah ( . %), revealed that few pts had recommended escalation from the approved to increased dose h -ah ( . - . %) or from increased dose h -ah to omalizumab ( . - . %) (table) . conclusion: poor physician adherence to guidelines was evident throughout aware. initial improvements in disease activity and qol plateaued after month , possibly owing to fewer changes to recommended therapies. greater physician adherence to guidelines is needed for better symptom control in pts with uncontrolled csu. results: we revealed that in russians urticaria is associated with rs *arg/gln genotype of the il gene (p = . ) and rs *cc genotype of tlr (p = . ) gene polymorphism. in tatars the association with disease development was shown for rs *tt genotype of tlr gene snp (p = . ). the rs *c allele of tlr gene polymorphism is associated with acute and chronic forms of urticaria (p = . and p = . , respectively) and rs *c allele of il gene polymorphismwith acute urticaria (p = . ). method: csu patients from the urtica cohort (clinicalttrials.gov number: nct ) participated in the study. a questionnaire was carried out evaluating the triggers identified by the patients, the comorbidities and the treatments received. patients with a self-report of skin exacerbation by foods, nonsteroidal antiinflammatory drug (nsaid) or physical triggers were subjected to a controlled provocation test with the suspect food, medication or physical stimuli report by the patient. the levels of anti-tpo ige were measured during a period of clinical control and during two exacerbations in all patients. results: % of the patients had at less one inducible urticaria demonstrated by provocation tests ( % dermographism, % cold, % pressure). self-reported exacerbation for a food ( %) or medication ( %) were high, but positive provocation tests were low ( % and % respectively). patients had (+) anti-tpo ige during the baseline period. among them, % presented a significant elevation of anti-tpo ige during at less one of the two exacerbations. . % of patients (n = ) with (−) anti-tpo ige, presented elevation of anti-tpo ige one of the two exacerbations. conclusion: foods, drugs and physical triggers must be verified by challenge tests to avoid unnecessary lifestyle restrictions in patients with csu, nevertheless self-report is usually greater than positive provocation tests. increase concentrations of anti-tpo ige seems to be implicated in urticaria exacerbations in some patients with csu. brzoza z ; adamczyk k ; wcislo-dziadecka d ; zbiciak-nylec m ; brzezinska-wcislo l adipokines. the aim of the study was to evaluate the possible contribution of leptin to chronic spontaneous urticaria pathophysiology. the study included chronic spontaneous urticaria patients and healthy subjects. the leptin level in both examined groups was measured. results: no statistically significant difference in leptin level was determined between the studied subgroups. we are among the first to present the effects of exploration aimed at assessment of the possible role of adipokines in chronic spontaneous urticaria pathogenesis. in this study we did not prove any difference in leptin level. in our opinion it is valuable to perform further studies in this area. the microorganisms were inactivated with phenol, and the concentration was adjusted to microbial cells/ml (labeled as a / ). dilutions / and / were made from the product labeled / . the dot blot technique was used to detect the presence of specific ige to the different microbial antigens and controls (anti ige / and fold dilution ½ and ¼). the dot blot images were processed with a documentation system (gel doc ez, bio-rad), and the different microbial antigens in different dilutions were compared with the positive anti-ige controls. results: all patients have specific anti ige to microbial antigens (see table below). the presence of microorganism-specific ige could explain, the relationship between the infections and / or microorganisms in ciu, as well, the urticaria control by omalizumab, even when it has not been detected ige sensitizations to common allergens. finally, these findings, showed that the bacterial allergy could be one line of research to understand the unresolved etiology of urticaria. background: dermographism is the most common form of inducible urticaria. it shows itself as hives made by scratching or rubbing on the surface where it has been produced and with the same morphology. the pathogenesis has not been clarified nor has it been associated until now with the sensitization to allergens. we have studied the relationship between the presence of dermographism and domestic mites sensitization. we have selected patients older than years old. all of them had symptoms compatible with dermographism at the moment of medical evaluation. at least one third of patients additionally showed rhinitis and/or asthma symptoms. we performed:: -skin prick tests with our basic neumoalergens (mites d. pteronyssinus y lepidoglyphus destructor, pollen, molds, dog, cat and horse dander, latex and anisakis simplex). -determination of specific ige levels for dermatophagoides pteronyssinus, lepidoglyphus destructor, and anisakis were measured in serum by using the immunocap (thermo fisher scientific). -blood count, serum immunoglobulins, antithyroid antibodies, serine tryptase and proteinogram. results: blood count, serum immunoglobulins, antithyroid antibodies, serine tryptase and proteinogram were normal. we divided patient in different groups. background: urticaria results from the appearance of pruritic papules and/or erythematous plaques caused by substances from mastocytes present in the skin, notably histamine. chronic urticaria is defined as flare-ups that occur at least two or three days per week over a six-week period. in addition, affected subjects are often prone to an atopic or auto-immune profile that promotes urticaria [ ] . the association of polyphenols (ambora, green tea) and the soothing active ingredients slow down the itching biological process from the outset by reducing the release of pruritic mediators (e.g. histamine, cytokines, etc.) of immune cells such as mastocytes and lymphocytes, involved in urticaria. in this context, the purpose of the study was to evaluate the efficacy and the tolerance of an anti-pruritic spray containing the polyphenols and the soothing ingredient. the tested product aims to quickly calm the itching in subjects with chronic urticaria. results: on average, the product was applied . times per day with a significant decrease of d-pruritus scale (- %) and sensations of itching (- %) between d and d . in terms of quality of life, a significant decrease of the skindex score was observed (- %). the product soothed the pruritus within seconds for all subjects and the anti-pruritic effect lasts at least hours for % of subjects. the product also showed very good cosmetic properties and was well tolerated; no intolerance case was reported. showed near complete remission. in the week before omalizumab and for a few days after, her urticaria flared but on of weeks she was largely asymptomatic (uas - ). after years of successful treatment she reported an increase in csu activity. no trigger factors could be identified. add-on treatment with cyclosporine was refused, montelukast showed no, and prednisolone only transient benefit. over a period of months wheals occurred almost daily and a maximal score of was achieved on uas . we replaced omalizumab with cyclosporine but this was subsequently discontinued due to side effects. months later the patients' csu remained poorly controlled with up to wheals occurring almost daily despite rupatadine mg/d. due to the good initial response to omalizumab and lack of good treatment alternatives, a trial of re-treatment was considered. results: within week of re-commencing omalizumab she once again achieved near complete remission of csu with uas ≤ on of weeks. the mechanism of action of omalizumab and the development of resistance to it in csu, are incompletely understood. our case shows that some csu patients developing resistance to omalizumab may benefit from a subsequent trial of re-treatment, particularly if treatment alternatives are poorly tolerated. manipulate and store data by electronic means. this includes e-mail, sms text messaging, video chat and online social media as well as all the different computing devices that perform a wide range of communication and information functions. a rapid increase in the use icts in recent decades is an enormous contributing factor in the development of a number of novel clinical and public health intervention strategies. the aim of the present study is to assess the level of ict use and to examine patterns of preferences among patients with chronic urticaria (cu). method: we will conduct an anonymous multicentre cross-sectional study, starting from january , to investigate the use of icts in patients with cu, using a questionnaire as a survey method. this questionnaire will assess the frequency of use of social media and icts in patients, and their preferences for receiving and asking disease-related information. the survey will consist of items, evaluating demographical information, time with disease, medication currently used, and additional aspects of social network use. results: we will use a chi-squared test to assess the association between internet access or owning a cell or smartphone, and age, gender, type of urticaria, educational level and number of years since diagnosis. we will employ the same test to assess the association between the independent variables previously introduced and the frequency of use of each ict type (short messaging service [sms], facebook, twitter, youtube, email, internet, linkedin and skype) as well as agreement in receiving and seeking information (i.e. asking questions to the practitioner) through such icts. we will perform adjusted regression analyses between categories of age, gender, educational level, type of urticaria, years since diagnosis and the use and level of interest shown in communicating through icts. our aim is to report on remarkable findings from a registry of a large sample of patients, potentially providing clues for its approach and results: patients with a median length of months suffering from urticaria were registered, being % women; mean age . years. in % of patients no causal agent was identified. parasites were found in . % and thyroid peroxidase antibodies in . %, while autologous serum skin test was positive in % and igg to mycoplasma in % of evaluations. two thirds of patients reported wheals on uas , with just / having concomitant angioedema. almost / reported significant affection on quality of life because of itch by cu-q ol. just % of patients achieved total control on first anti-histamines provided, and less than half had good control of urticaria. cetirizine was the first choice in %, followed by fexofenadine ( %) and first generation anti-histamines ( %). method: cases at - years of age which were being followedup in our clinic with diagnosis of chronic urticaria and were not receiving any antihistaminic medication for last one month were included in the study. cu-q ol, uas- , psqi and psg results of the patients were evaluated. correlation of data with each other in regard to sleep disturbances was evaluated. results: patients were included in the study. patients' mean total score in cu-q ol was . ± . . patients' mean uas- value was . ± . . mean total psqi was . ± . , the ratio of total scores ≥ and those with poor quality of sleep was . %. mean epworth sleepiness scale (ess) score was . ± . , with total score ≥ in . %. in psg, mean apnea-hypopnea index (ahi) was . ± . , with . % of the patients having ahi ≥ . when patients having ahi< were compared with patients having ahi ≥ , no significant difference was determined in regard to total cu-q ol score, mean score for questions concerning status of sleep, uas- and psqi. when correlation analysis was performed between cu-q ol and total score for questions concerning status of sleep, a positive correlation was determined with psqi (p = . ). conclusion: it was demonstrated in our study that patients with chronic urticaria had poor quality of sleep and this disturbance was independent from ahi. omalizumab was discontinued due to absence of improvement in csu symptoms after three consecutive doses. the plasmapheresis without intravenous immunoglobulin replacement was initiated. results: the symptoms were relieved during the first procedure and the disease improved shortly thereafter. the following weeks the symptoms still occurred but with lower intensity and severity. (angioedema was gone). the second attempt with omalizumab was successful after this course ( procedures of plasmapheresis). case report: rosacea is a chronic skin disorder associated with flushing, erythema, dryness, burning and stinging, and inflammatory papules and pustules. new treatments available or in development target the inflammatory and erythematous components of the disease. these agents include the selective alpha- receptor agonist brimonidine. allergic contact dermatitis to brimonidine is an unusual condition. in addition to this, urticarias due to brimonidine are rarely reported. we report on a -year-old woman who, immediately after apply a thin layer of brimonidine gel as preparation for a rosacea treatment on her face developed facial urticaria, which reverted in approximately four hours with systemic steroids. she had previously tolerated this product without any problems, but has not use it again ever since. skin prick-tests with brimonidine ( . mg/ml) and latex were realized in the patient. skin prick-tests with brimonidine were realized in eleven healthy control subjects. results: skin prick-tests with latex was negative in the patient. skin prick-tests with brimonidine were positive in the patient ( x mm). the prick-test with brimonidine was negative in teen healthy control subjects. we report on a case of immediate urticaria due to brimonidine and triggered by an immediate, probably ige-mediated, hypersensitivity mechanism. we highlight this case because it is the only case described in the literature with a positive prick-test. method: the study was in accordance with the helsinki declaration and was previously approved by the national comity of ethics. this was a one dose study conducted on fasting young healthy volunteers, of which were females and five males. the mean age was ± years old and the body weight . + . background: cetirizine is a potent h -receptor antagonist indicated in the treatment of allergic rhinitis and urticaria. cetirizine is widely used due to its potent antihistaminic effects in yielding strong and fast relief of itchy sensation, sneezy and rhinorrhea and its unlikely probability to manifest anticholinergic side effects in therapeutic doses. histamine flare and wheal inhibition by anti-h are widely used as a standard to test and compare the effect intensity and duration. our study aimed to test these effects of cetirizine in young healthy adults. method: this was a double-blind, single dose study in healthy young adults, previously approved by the national comity of ethics. eleven females and five males with a mean age ± years participated in this study. histamine skin pricks were tested before and after they received a tablet of mg cetirizine as previously scheduled. twenty minutes after each test flare and wheal were drawn in a transparent paper which was then scanned and measured with a software. wilcoxon signed ranks test two-sided with significance at % level was used to analyze the differences. claims that the preparation relieves itch within seconds of its application. we performed a simple study to verify this claim. we used irp in consecutive subjects, males, median age , range - years, whose workup implied ast. their preliminary diagnoses were "asthma" ( subjects), "allergic rhinitis" ( subjects), "atopic dermatitis" ( subjects) and "food allergy" ( subjects). all of them had refrained from systemic antihistamines for at least one week. standard skin prick tests (spt) were applied as appropriate, including histamine controls to assess the level of their skin sensitivity. subjects were asked to mark their sense of itch in the area of the skin to be tested on mm visual-analogue scales (vas) starting from " "-"no itch" to " "-"unbearable itch". vas assessments were repeated minutes after ast was done; then irp was applied according to the manufacturer's instructions, and the vas assessments were repeated after seconds and minutes. results: there vas assessments are shown in table format: table irp did not affect the wheal and flare of the histamine control, nor did it abolish positive spt. no differences were outlined between subjects with different diagnoses. the commercially available itch relieving preparation not containing defined pharmacological antihistamine is effecting in relieving itch associated with allergen skin testing. before ast ( ) . ± . vs ( ) p < . represent the first-line treatment for osteoporosis-related mastocytosis. we report a case of sm with bone pain and with an area of osteolysis in the femur as first sign and symptom. we had to consider the risk of adverse reaction when we decided to treat the patient with bp, but the patient was under antihistaminic treatment and also we made a premedication to reduce the risk. the pk/pd model available was informed by data from clinical trials. the pd endpoint data was available from two studies and used to characterize the effect of bilastine on wheal and flare. moreover, food effect had been characterized in pk studies and the data was used to model the effect of food in the pk of bilastine. the pk parameters relative to the fed state were then used to simulate the temporal evolution of the wheal effect using the pk/ pd model. all analyses were conducted by nonlinear mixed effect modeling (nonmem v . ). using the pk model developed (food effect model) and the pk/pd model already available, monte-carlo simulations for plasma concentrations and pd over time were performed for both the fed and the fasting states. results: . a reduced bioavailability (f) and a slow absorption constant characterized the pk of bilastine when administered concomitantly with food (f = % relative to the fasting state and ka = . hour − , a -fold reduction compared to fasting conditions). the rest of the pk parameters remained unchanged. onset of action was hour for bilastine both in fed and fasted conditions. maximum wheal inhibition occurred at . hours (fasted % and fed %). from to hours, the percentage reduction with bilastine for both fasted and fed was between % and % after the third day of treatment. a % inhibition in wheal effect was maintained during hours for both conditions after the third day. the results of the simulations show that even if the pk is altered with food, the pd is maintained unchanged. conclusion: even if a significant food effect was described for bilastine at a pk level, the difference is not translated directly into the pd. therefore, the antihistaminic effect of bilastine remains unaffected by the concomitant administration with food. the results of these simulations will be further confirmed in a dedicated clinical trial. results: we also found no correlation between the different tgt parameters and other clinical and analytical parameters associated with uc (table ) results: both cetirizine products have no differences in respect to the pharmacodynamic and pharmacokinetic parameters analyzed. the % confidence interval of the mean ratios of the auc - , auc -inf , cmax, auce - , and e max , between the test and the reference, were within the bioequivalence ranges ( %- %) in both cases. no statistical difference was revealed when comparing the respective t max and te max too. the two cetirizine products tested were bioequivalent. the bioequivalence was evident even when tested with the pharmacodynamic parameters. there is strong evidence that supports the use of histamine skin prick test for the bioequivalence evaluation of different cetirizine products. | bradykinin-mediated angioedema associated with combination of angiotensinconverting enzyme and dipeptidyl peptidase iv inhibitors: a disproportionality analysis from the who database method: we performed a disproportionality analysis using data from the who pharmacovigilance database by a case-noncase study, until the / / . we extracted all individual cases safety reports (icsrs) included in the high level term "angioedemas", according to the medical dictionary for regulatory activities classification. given the absence of term "bma", we selected only the icsrs of angioedema without associated symptoms evoking another underlying mechanism, such as histamine angioedema (e.g. pruritus, urticaria, rash, etc.). drug class exposure was "acei" and "dpp i", considered suspect or concomitant, using the atc classification. we results: there was no correlation between mother's disorders such as periodontitis, rhinitis, diabetes etc. and the onset of ar (p > . ). a multivariate analysis showed, neonatal jaundice (p < . ), respiratory system infection (p < . ), diarrhea (p < . ), eczema (p < . ) in the first months of life and home environmental factors (house decoration (p < . ), mold environment (p < . ), keeping flowers (p < . ), passive smoking (p < . )) increased the risk of ar. besides, there was no significant difference in current height and birth weight of the participants between ar and control group. however, ar group had significantly lower current weight (p = . ) and age (p < . ) compared with the control group. paternal age and maternal age in the ar group were significantly higher than the control group (p < . ). conclusion: diseases in the first months of life and home environmental factors increased the risk of sequential ar. the older parents increased the possibility of ar in the offspring. the data of general characteristics of participants were statistic analysis by z text analysis. *significance at p < . . results: anosmia was more frequent in crs than in rhinitis ( . % vs . %, p < . ) and in crswnp than in crssnp ( . % vs . %, p < . ). lms was higher in crs than in rhinitis ( [ - ] vs [ - ], p < . ) and in crswnp than in crssnp ( [ - ] vs [ ] [ ] [ ] [ ] [ ] [ ] [ ] , p < . ). in addition, lms was associated with loss of smell in patients with hyposmia (or = . [ . , . clinics. patients were submitted to confirmatory exams including oral provocation test with aspirin. nasal polyps were removed by functional endoscopic sinus surgery and eosinophils in this tissue were quantitated. eosinophil counts in peripheral blood was obtained. serum periostin was measured by elisa and total ige was determined using immunocap. as control groups, ( f/ m) patients with par and healthy subjects ( f/ m) were selected. samples of nasal tissue and blood were collected from these subjects during elective surgery for correction of anatomical variations, and compared with the patients with aerd. results: ar symptoms were significantly improved in the treatment group compared with the control group ( . % ( / ) vs . % ( / ); p < . ). furthermore, the mean total vas score for patients in the treatment group was reduced from . ± . before treatment to . ± . after treatment (p < . ). moreover, the reduction in free ige levels was greater in the treatment group than in the control group. the results of this study suggest that the chinese herbal medicine ber may be effective for improving the symptoms of ar. a multicenter clinical trial is needed to confirm this finding. results: in patients with "eosinophilic" polypoid rhinosinusitis, mucociliary transport was . ± . minutes, ph . ± . , suction- . ± . minutes, excretory- . ± . mlg and in patients with "neutrophilic" polypous rhinosinusitis, mucociliary transport was . ± . minutes, ph . ± . , suction- . ± . minutes, excretory- . ± . ml. the study showed that disruption of the transport function, changing the concentration of hydrogen ions method: this prospective controlled study was carried out on crs patients underwent ess. patients participating in the study were divided into two groups-group : partial middle turbinectomy (n = ) and group : partial middle turbinectomy and middle turbinate fenestration (n = ). objective assessment of olfactory function using the university of pennsylvania smell identification test (upsit) and subjective assessment of symptom using visual analogue score (vas) were performed before and months after surgery. results: there were significant improvement comparing postoperative and preoperative upsit in both group ( . ± . vs . ± . , p = . ) and group ( . ± . vs . ± . , p = . ). the vas were also significantly improved postoperatively compared to preoperatively in both group ( . ± . vs . ± . , p = . ) and group ( . ± . vs . ± . , p = . ). patients undergoing partial middle turbinectomy and middle turbinate fenestration were more likely to show improvements in upsit ( . ± . vs . ± . , p = . ) and vas ( . ± . vs . ± . , p = . ) compared to those with only partial middle turbinectomy. conclusion: partial middle turbinectomy and middle turbinate fenestration during ess is an effective method for improving postoperative olfactory function. | nasal irrigation for the alleviation of nasal symptoms in pregnant women with allergic rhinitis we sought to determine specific ige responses to bacterial pathogens in sera from cystic fibrosis patients and analyze their kinetic during disease course. genes, respectively. in contrast, most of healthy donors had normal homozygous genotype with tt- . ± . %(n = ) and cc- . ± . %(n = ) with low frequency of mutations; gg- . ± . %(n = ) and tt- . ± . %(n = ) and heterozygous genotype tg- . ± . %(n = ) and ct- . ± . %(n = ) for il- and il- genes, respectively. following a month treatment, there was a significant reduction of cytokine levels in the il - . ± . and increased in the il - . ± . , when compared to the beginning of therapy and after months (p < . ) results: at baseline the st group had serum levels of il- ( . ± . ) pg/l; il- ( . ± . ) pg/l and ifn-γ ( . ± . ) pg/ l; nd group had il- ( . ± . ) pg/l; il- ( . ± . ) pg/l and ifn-γ ( . ± . ) pg/l vs il- ( . ± . ) pg/l; il- ( . ± . ) pg/l; ifn-γ ( . ± . ) pg/l in the control group. after months, there was a significant decrease in pro-inflammatory cytokine levels in the st (il- : ± . ; ifn-γ: . ± . ) pg/l and nd group (il- : . ± . ; ifn-γ: . ± ) pg/l, respectively. conversely, il- increased in st and nd groups to . ± . pg/l and . ± . pg/l (p < . ). conclusion: prior to the study initiation patients with tuberculosis had higher il- , ifn-γ and lower il- content than healthy controls. two-month chemotherapy produced significant reduction in proinflammatory cytokines and increase in anti-inflammatory il- , with levels approaching those of healthy controls. thus, tuberculosis drugs appear to have the anti-inflammatory effect in tuberculosis patients, which was predictive of positive clinical outcome. | antibiotic resistance: ligands of innate immunity take the challenge in this work, we aimed to perform an ex vivo hrsv infection in precision-cut lung slices (pcls) from human, rhesus, and cynomolgus macaques, comparing whenever possible the response with the viral surrogate poly i:c. method: pcls containing airways were prepared from lung sections of human, rhesus, and cynomolgus macaques. the slices were inoculated with hrsv-a iu/ml, uv-inactivated hrsv, or vehicle control for hours. macaque slices were also incubated with poly i:c μg/ml with and without the immunosuppressive dexamethasone μg/ml. viral replication, tissue viability, and immune response assays were assessed in supernatants, lysates, or slices. the inoculum infectivity of iu/ml as well the uv-inactivation were confirmed by plaque-assay on hep- cells. immunofluorescence staining using a fitc-labeled anti-rsv showed the presence of infected macrophages in pcls, but not in mock infected samples. hrsv stimulation slightly decreased tissue viability, as seen by live/dead staining and ldh assay. the viral infection increased ip- production in pcls of human, rhesus, and cynomolgus macaques, reaching respectively . , . , and . fold-increase in comparison to the vehicle controls. poly i:c stimulation caused ip- response comparable to hrsv in rhesus and cynomolgus pcls. the ip- production ratio comparing hrsv/poly i:c was . in rhesus and . in cynomolgus pcls. conclusion: hrsv infects ex vivo pcls of human and non-human primates, inducing the release of the pro-inflammatory chemokine ip- . this response is comparable to the viral surrogate poly i:c. in the future, these systems can be used to further investigate host response to hrsv, especially in the context of asthma development. however, a relatively small number of reports are related to the association of ebv with allergic diseases, in particular atopic ones. we found that among patients with activated ebv infection, polysensitization was found to be . times more frequent, chest syndrome was . times more common and hyper-ige syndrome occurred . times more frequently. in most of these patients, atopy was not detected in medical history. method: we evaluated the laboratory test results of five boys ( . %) and six girls ( . %), children ( with hbov and with cov). their average age at the study time was . ± months. nasal swab specimens were taken from these patients who admitted to our hospital with respiratory symptoms between - . patients are recalled after an average of ± . months. isaac questionnaire and skin prick test to common inhalated allergens were performed. results: only one patient had family history of atopy. forty percent of the patients with cov and % of the patients with hbov developed rhinitis. one patient with cov and one patient with hbov developed recurrent wheezing. one patient with cov developed atopic dermatitis. all skin prick tests were negative. it was noteworthy that . % of the patients were passive smokers. conclusion: hbov and cov may be associated with rhinitis but there is a need for more patient groups for a clear result. rna_lig (ccg-agg-aug-cga-ggc-uug-uu) . to study chemotaxis in vitro, a boyden chamber was used -wellfiltrationplatemultiscreentm -mic with a pore size of μm (millipore, usa). chemotaxis was studied in dynamics after , minutes and a day using the above ligands. as control, rpmi- medium without glutamine was used (paneco, russia). the statistical analysis was carried out using the computer statistical program biostat conclusion: with all the data provided, a drug induced hypersensitivity was diagnosed. we present a case of immediate allergic reaction with eosinophilia due to carbapenems, with tolerance to other beta-lactams antibiotics. written informed consent of patient has been obtained in the two cases. discussion: the first case shows cutaneous immediate hypersensitivity response to infbeta a. literature reports a few cases of urticaria and anaphylaxis but this is the first for the pegylated formulation. polyethylene glycol (peg) confers to a drug modified pharmacokinetics, solubility and immunogenicity. immediate reaction to peg (macrogol) have been described when combinated in vaccines or drug pils. dmf is a known cause of contact dermatitis related to footwear, wallets and furniture. flushig is a reported side effect of dms in ms managed with dose reduction. this case shows the possibility to immediate sensitization to dmf. as the armamentarium to treat ms now combines immunomodulatory and biologic drugs, the avaliability of diagnostic and desensitization protocols for hypersensitivity reactions must be keeped in mind. case report: drug rash with eosinophilia and systemic symptoms (dress) syndrome is an uncommon but serious hypersensitivity drug reaction, manifested with rash, fever, lymphadenopathy and visceral organ involvement. table) . drug withdrawal and prednisolone treatment leaded to attenuating of mentioned skin symptoms within days, associated by occurrence of a exfoliative dermatitis. one week after admission, the patient developed fever that lasted for days with enlarged lymph nodes on submandibular, paracervical, axillar and inguinal regions. a preventive antibiotic therapy is started and weeks later, the lymph nodes were not palpable and the skin got the normal appearance. corticoid therapy is reduced gradually according to symptoms resolvement. case : a -year old woman presented to our department with a -day history of pruritic, macular rash, periorbital swelling, cheilitis and fever. she had started some weeks ago the allopurinol for asymptomatic hyperuricemia, had longer history for treatment of arterial hypertension and type- diabetes mellitus (olmersartan, nitrendipine, methyldopa, furosemide, regular and glargine insulin), and experienced nephrectomy and cholecystectomy. the patient was febrile, while blood tests revealed eosinophilia, increased seric creatinine/urea levels (due to nefrectomy), and severely-altered liver parameters (see table) . the allopurinol withdrawal, topical and systemic corticoid therapy, and the liver protectors attenuated serologic transaminases levels and patient's skin lesions within few days, followed by substantial improvement of laboratory findings one week after therapy start. the treatment dosage was gradually tapered and finally stopped within a period of months in accordance with attenuating and complete resolvement of the clinical and laboratory abnormalities. our case demonstrated that dress syndrome is a severe drug reaction, but the immediate introduction of treatment and supportive measures can improve disease's outcome even after a temporary exacerbation or severe affection of internal organs. case report: a -years-old woman, diagnosed of ischemic cardiopathy, developed an anaphylactic shock minutes after the administration of ml sulphur hexafluoride intravenous during an echocardiogram. she was treated in emergency room with a total recovery. months earlier, she had developed an extensive erythematous-maculopapular rash converging in plaques in relation with adhesive dressings which had been placed during a hospitalization due to thoracic pain. an allergic contact dermatitis was suspected and recommendations thereon were given. interestingly, an arteriogram with iodixanol (icm) was carried out one week before skin reaction with good immediate tolerance. methods: blood test: blood count and serum chemistry were done during both reactions to contrast media. serum tryptase level was not measured during the anaphylaxis, but its baseline level was quantified later. conclusions: we present a patient with a double sensitization to parenteral contrast media: an anaphylactic shock due to sulphur hexafluoride and an atypical delayed exanthema related to iodixanol, and diagnose was obtained with st in both cases. this is the first documented case with a positive immediate st to sulphur hexafluoride. with the culprit drugs mixed with % and % petrolatum resulted negative. patient was suspected to have behcet's disease, and consulted to rheumatology department. oral colchicum dispert twice a day was prescribed. afterwards, patient achieved to take oral amoxicillin-clavulanate for a week without any hypersensitivity; and has been following by oral colchicum dispert maintenance therapy since then. the reported patient had one anaphylactic perioperative reaction to morphine and another anaphylactic reaction to tramadol during her diagnostic investigation. remain the question if this patient had two allergic anaphylactic reactions with cross-reaction between morphine and tramadol, or two non-allergic anaphylaxis due to "hypersensitive" mast cells. case presentation: a -year-old female was diagnosed with rectal adenocarcinoma. one year after radical surgery, progression with pulmonary metastasis was shown. in first line of systemic therapy she received premedication with pantoprazole, metoclopramide, clemastine and dexamethasone, followed by cetuximab infusion. during first minutes of infusion, grade anaphylactic reaction occurred. a reaction started with generalized pruritus, urticaria, rhinitis, followed by hypotension, bradycardia and loss of consciousness. she was treated with fluids, clemastine and methylprednisolone. next day she received same premedication followed by panitumumab. during first minutes she had grade reaction with generalized urticaria. the third day she had generalized urticaria minutes after metoclopramide application. skin prick tests with cetuximab ( mg/ml) were negative, but intradermal test were posi- bat response was highly positive for both cetuximab and alpha-gal, with comparable values and dose response curves. thus, we showed %, %, %, % and % of cd positive basophils for stimulation with cetuximab ( - . μg/ml), and %, %, %, and % for stimulation with alpha-gal ( . - . ng/ml). bat response to panitumumab was negative (< %; - . μg/ml). drug provocation with panitumumab was negative and patient received treatment with panitumumab. in the operating theatre, the skin is disinfected using povidoneiodine and pupil dilation is carried out with tropicamide (showing no immediate reaction in the surgery). method: as we are dealing with a late cutaneous reaction, the study of the medicine involved is carried out by means of epicutaneous medicine testing. in order to do the study of aflibercept, we wore gowns, two sets of gloves, a mask, eye protection and in a containment hood in the outpatients hospital. the patient diagnosed himself with dermatitis when in contact with povidone-iodine and despite the fact that the cutaneous provocation was negative, it is known that when there is surgery involved, there needs to be moistness and occlusion for it to show up clinically. the application of this antiseptic seems to lose its irritation and allergic properties when it dries on the skin and therefore tends to give a negative result in these patients, but this does not mean that they are not allergic to this antiseptic. we report the case of a year old man who experienced erythema and pruritus immediately after an intravenous injection of ranitidine and hyoscine butylbromide given for gastric pain treatment. results: spt and idt were performed for ranitidine ( mg/ml and . mg/ml respectively) and hyoscine butylbromide ( . mg/ml and . mg/ml respectively) being exclusively positive for ranitidine at idt dose with a × mm papule (histamine control × mm). oral provocation test for hyoscine butylbromide was negative. bat for ranitidine and famotidine were carried out, being negative for both drugs. conclusion: skin tests for h ra are the best option when studying a suspected reaction to h ra and are also useful for assessing cross-reactivity between other h ra. the sensitivity for bat in diagnosis of drug allergy is about %, and the specificity up to %, although these percentages make reference to the common drugs studied (beta-lactams, quinolones, pyrazolones, etc). specific studies for h ra are still to be done. in our case we had a negative result for the bat test, although we proved ranitidine was responsible for the reaction. conclusion: gentamicin is an aminoglycoside antibiotic used systemically for septicemia and as prophylaxis during surgery. immediate type allergy (type i) to gentamicin is rarely reported. since , approximately only five cases have been reported in literature. in our case, initial theories were pointed towards cefazolin as beta-lactams report a higher rate of allergic reactions. after an exhaustive allergological study, results disproved our initial theory indicating gentamicin as the responsible drug. giangrande n ; bobadilla-gonzález p ; garcía-menaya jm ; cámara-hijón c allergy department, infanta cristina university hospital, badajoz, spain; clinical immunology department, san pedro de alcántara hospital, cáceres, spain background: polyethylene glycol (otherwise known as macrogol or peg) is a polymer with a wide application as an excipient, solvent and dispersing agent in food, cosmetic and pharmaceutical industry. it presents distinct length polymer chains with a molecular weight from to g/mol conferring them specific properties. macroglol with a molecular mass between and g/mol is commonly used as osmotic laxative previously to colon endoscopy and radiologic examinations. after the introduction, anaphylactic reactions to macroglol are rarely reported, considering it safe and well tolerated. we report on a -year-old man who, immediately after of the topical application of benzindamine in left inferior limb developed acute urticaria in this limb, which reverted in approximately hours with systemic steroids. she had previously tolerated this product without any problems. skin prick-tests with benzindamine ( . mg/ml) and latex were realized in the patient. skin prick-tests with benzindamine were realized in eleven healthy control subjects. results: skin prick-test with latex was negative in the patient. skin prick-test with benzindamine was positive in the patient ( × mm). the prick-tests with benzindamine were negative in eleven healthy control subjects. we report on a case of contact urticaria due to benzindamine and triggered by an immediate, probably ige-mediated, hypersensitivity mechanism. some of the drug used in daily clinical practice can cause allergic contact urticaria and should therefore be borne in mind. background: the use of new oral anticoagulants which act as direct inhibitors of activated factor x is constantly increasing, due to lower rates of serious and fatal bleeding events than warfarin/acenocoumarol. rivaroxaban, the first commercialized drug in this group, is the most used for prevention of thromboembolic events. however, < cases of hypersensitivity reactions have been described so far, most of them delayed and severe. to present a case of delayed hypersensitivity to rivaroxaban, diagnosed by a positive ltt (lymphoblastic transformation test). a year old woman with hypertension and chronic atrial fibrillation (af) was referred to our clinic for suspected drug allergy. she reported that months before, for af she was started on oral amiodarone and rivaroxaban, presenting on the seventh day with both of them generalized erythema, pruritus, micropapular rash and facial angioedema. no oral or other mucosal were observed, neither pustules, vesicles or blisters. blood eosinophilia, enlarged lymph nodes, renal and hepatic injury were discarded in emergency, where the new drugs were discontinued and replaced by acenocoumarol. the rash subsided one week later, with oral antihistamines. before and after the episode the patient also has been taking losartan and hydrosalurethyl, with good tolerance. she denied other adverse reactions. in allergy department we performed skin prick tests and intradermal tests with amiodarone ( . mg/ml and . mg/ml) and rivaroxaban ( . mg/ml and mg/ml), and a ltt with both drugs, months after the reaction. background: patients with history of beta lactam allergy, often self-reported, are commonly encountered in the hospital setting. this frequently leads to increase use of broad spectrum and more expensive antibiotics that may be unnecessary or even less efficacious at times due to fear and concerns about potential disastrous outcomes. nonetheless, with increasing awareness, many patients are now being referred to allergy service for formal evaluation. we aim to look at patients who underwent evaluation for beta-lactam hypersensitivity and determine the number of patients that were successfully de-labelled. method: a retrospective analysis was conducted with patients referred for evaluation of questionable beta-lactam allergy to the allergy service in our institution from the years - . initial evaluation process included a thorough history to determine the type of hypersensitivity reaction and suitability for further testing. patients underwent skin prick test (spt) and intradermal (idt) with either (a) both major and minor determinants of penicillin, benzyl penicillin, amoxicillin and ampicillin, and/or (b) the culprit drug itself. if skin testing was negative, oral or intravenous (iv) drug challenge was then performed after informed consent. clinical details and reactions were documented. patients were also contacted post challenge to ensure no delayed reaction had occurred. results: a total of patients were evaluated for beta-lactam allergy in the year period, of these were females and were males. of the referred patients had presumed penicillin group allergy and had cephalosporin group allergy ( patients had both penicillin group+cephalosporin allergy). cases ( %) were successfully de-labelled. beta-lactam allergy was confirmed in patients ( %); identified by positive spt in two patients, positive idt in six patients and positive drug challenge in patients ( patients developed rash/urticaria, had respiratory symptom and patients developed anaphylaxis). patients were referred before any drug allergy labelling was done, out of which were confirmed not to have beta-lactam allergy. conclusion: in our study, % of patients were confirmed not to have true beta-lactam allergy. we were able to successfully remove beta-lactam allergy label from the electronic record for % of the patients. results: a total of % referred amoxicillin-clavulanic acid (ax-clv) as trigger for the hypersensitivity reactions (hrs), followed by ax ( %), penicillin ( %) and cephalosporins ( %). almost % of hrs were immediate (< minutes). positivity of skin tests was observed in % subjects, of bat in % and of rast in %. in conclusion: the label of penicillin allergy is quite often erroneous. this involves using of more expensive and less effective therapeutic alternatives, which also facilitate the emergence of multi-resistant micro-organisms. hence the importance of confirming the diagnosis of allergy. finally, we did not find differences in the study of penicillin allergy in patients older than years compared with the general population. background: severe cutaneous delayed drug reactions (toxic epidermal necrolysis -ten-, stevens-johnson syndrome -sjs-, acute generalized exanthematous pustulosis -agepand drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome -dress/dihs-) among others, are a rare but potentially fatal complications of drug treatment. although its epidemiology has been described in different latitudes, it is unknown in latin america. our aim was to describe the epidemiological characteristics of severe cutaneous reactions to drugs in countries of latin america. method: an online questionnaire was designed to report new and old cases (since ). it was a modified and adapted version of enda questionnaire for drug allergy interesting group. sociodemographic data, type of reaction (ten, sjs, dress-dihs, agep), culprit drug (s), treatment, complications, mortality and sequelae, were described. three centers from colombia, one from argentina, one from brazil and one from paraguay were included. an excel database was created, in which cases were recorded and analyzed. results: thirty seven cases were reported. ( %) were women. the median age was years. ( %) had dress/dihs, ( %) ten, ( %) sjs, ( %) agep, ( %) other not classified scars, and ( . %) overlapping ten/sjs. the main culprit drugs were aromatic anticonvulsants in cases ( %), beta lactam antibiotics in ( %), non-beta lactam antibiotics in ( %) and allopurinol in ( . %). in % of the patients the suspect drug was withdrawn. thirty one patients ( . %) received systemic corticosteroids. complications occurred in cases ( %) and death in one patient ( . %). seven patients ( %) had some type of sequelae. countries, dress/dihs was the most frequently reported clinical entity, and the anticonvulsants were the main triggers. complications were frequent, but mortality was low. | drug-induced cough: analysis of nationwide spontaneous reports in korea over ten years using who-adverse reaction terminology (who-art) indicative of cough. results: from cases of spontaneously reported adverse drug event cases, a total of cases ( . %) were identified as drug-induced cough. most cases occurred in adults ( . % of the subjects) and females were more common than males ( . % vs . %). regarding severity, only cases ( . %) were classified as serious based on who criteria. the most common causative drug category was antineoplastic and immunomodulating agents ( . %), followed by cardiovascular drugs ( . %). the most common causative drugs were ace inhibitors including perindopril and ramipril. conclusion: in the nationwide spontaneous reports of adverse drug events, many cases of drug-induced cough have been reported so far. much attention is needed to find new causative drugs of cough in the future. background: allergological assessment to determine the mechanism of the perioperative reaction and to identify the agent responsible and recommendation of a range of drugs or agents likely for future surgery is essential, but it often poses a significant challenge. in this study, we analyze our experience in the investigation of adverse reactions during anesthesia in the last years. method: a total of patients who attended our allergy unit with suspected perioperative reactions between january and december were reviewed retrospectively. the severity of the perioperative allergic reactions was graded according to ring and messmer system. results: grade iii, ii and i reactions were observed in , and patients, respectively. in patient we didn't know the reaction suffered. tryptase measurements were available for patients. of those, and patients had elevated and normal levels respectively and suffered grade reaction. ige mediated reactions was diagnosed in patients ( %): for ßlactam antibiotics ( . %), for patent blue ( . %), for neuromuscular blocking agents-nmbas ( . %), for latex ( . %), for colloids ( . %) and for ranitidine ( . %) . cefazolin was the ß-lactam antibiotics causing the largest number of reactions. non-ige-mediated reactions was diagnosed in patients ( %). the allergy tests were negative and tryptase levels were normal. conclusion: in our series, among the patients who suffered allergy reactions during anaesthesia and the cause was subsequently identified, ß-lactam antibiotics were the most common causative agent ( . %), followed by patent blue ( . %), nmbas, latex, colloids and ranitidine ( . % each agent). in contrast, data from other authors indicated that nmbas were the most common cause of anaphylaxis, followed by latex, hypnotics, antibiotics, plasma substitutes and opioids. these differences might be due to the small size of our study, which was limited to our centre over the last years and thus may not be representative. diagnostic evaluation. all patients signed an informed consent. we made a retrospective analysis of their clinical records and excluded patients whose records were missing or incomplete. it was analyzed each patient's medical history (focusing allergic disease) and clinical reaction to the suspect drugs. signals/symptoms at pcc were characterized. we also studied the variation of the dpt's results when it was performed after a pcc. aim: to define and quantify the ongoing pharmacy needs in sustaining a large drug allergy assessment program. method: a retrospective review of pharmacy files was used to identify and quantify the drugs and dosages most frequently used and to determine prescription trends within the allergy testing program over the last years. results: initially, this reaction was thought to be a result of a drug allergy, but upon further review and the onset of fever, we determined that it met the diagnostic criteria of jhr. his twin brother was diagnosed with penicillin and betalactamic allergy. neutrophilia % was to be underlined in the blood test. after this, drug oral challenge with penicillin was performed, ruling out penicillin allergy. conclusion: it is not uncommon to confuse drug allergy with jhr. jhr should be an anticipated reaction to early doses of antibiotic treatment for treponemal diseases, such as syphilis. antibiotic treatment should be continued; it is not a warrant to stop treatment. clinicians should be aware and anticipate jhr as a potential complication to early doses of antibiotic for spirochetal diseases such as syphilis or lyme, leptospirosis. the patient was unresponsive in oral drug provocation tests with amoxicillin-clavulanic acid, clarithromycin and trimethoprim sulfamethoxazole for months. the patient could use these drugs. results: chronic abacterial inflammation of the prostate gland was accompanied by a significant increase in concentration of slpi, il- , tnf-α, il- in the seminal plasma and serum concentration, and a decrease in the concentration of il- and tgf-β compared to healthy men (p < . ). there was no statistically significant difference between slpi, il- , tnf-α, il- , il- , and tgf-β in the ejaculate of patients with inflammatory and non-inflammatory forms of cap (p < . ). the concentration of il- in ejaculate of patients with inflammatory forms of cap was significantly greater than in patients with non-inflammatory form of cap (p = . ). the inflammatory and non-inflammatory forms of cap are pathologically similar with changes in the concentration of the studied cytokines except for il- in both forms with signs of inflammation. the terms "leukocytic" vs "non-leukocytic" chronic abacterial prostatitis are more correct than "inflammatory" and "non-inflammatory" when describing chronic abacterial prostatitis. results: the status of all patients after dc immunotherapy was evaluated as satisfactory. heart rate, blood pressure in patients remained within the age norm. skin had normal color without rash or peripheral edema. there were no local or systemic allergic reactions. the body temperature after the injection did not exceed °c. conclusion: these results show, for the first time, that among mastocytosis patients, besides the already known periodontal disease risk factors that include diabetes, age, osteoporosis and alcohol consumption, the bone marrow mast cell burden is also associated with increased periodontal disease severity. results: metformin at relatively low doses ( - μm) was shown to mildly suppress ige-mediated responses, including degranulation ( % reduction, p = . ), tnf-α ( % reduction, p = . ) and il- ( % reduction, p = . ) secretions in bmmcs. importantly, metformin at the same doses potently inhibited mast cell responses in all parameters ( % reduction, p < . for degranulation; % reduction, p < . for tnf-α; % reduction, p < . for il- ) in mast cells treated with an ahr ligand, , -dihydroindolo[ , -b]carbazole- -carbaldehyde (ficz). mechanistically, its inhibitory effect was mediated through the suppression of ficz-induced mapk activation, intracellular calcium release and ros generation. metformin also blocked ahr-mediated pca in vivo ( % reduction, p < . ). conclusion: metformin, a common anti-diabetic agent, was shown to exert inhibitory effect on ahr-mediated mast cell activation in vitro and in vivo, suggesting its potential utility as a newer form of therapy for asthma and allergic diseases; this is particularly relevant when considering the adverse effect of the exposure to environmental polycyclic aromatic hydrocarbons. gasser p ; brigger d ; zbären n ; jardetzky t ; pennington l ; eggel a results: in one of affected family members, we were able to identify the c. a>g mutation in the plasminogen (plg) gene that was recently described to be associated with hereditary angioedema. this mutation leads to a missense mutation with an amino acid exchange p.lys glu in the rd kringle domain of plasminogen. there is no direct relationship between the earlier described cases with this mutation and the family we report here. in all affected members of the family, the symptoms manifested in early adulthood, with swelling of the face, the tongue and the larynx. the frequency of attacks was variable, between once in a year to once in a month. in one of the three family members, we found a slightly decreased level of coagulation factor xii and of plasminogen. icatibant proved to be very effective for the treatment of acute attacks in the affected family. the occurrence of the same c. a>g (p.lys glu) mutation in the plg gene in many families with no or only unknown distant relationship suggests that the disease might have been inherited through the generations without being purged from the population. the mutated amino acid exchange appears to be significant for the function of plasmin or plasminogen. we found a decrease in plasma levels of coagulation factor xii and plasminogen, which may be beneficial markers for diagnosis and monitoring of this disease. several biomarkers are useful in the diagnosis (fibrin degradation products (fdps), d dimer (dd), and fragments of prothrombin + ). also, a correlation between the levels of biomarkers and activity phases of the disease has been detected. alterations in coagulation parameters have an etiopathogenic role in the ae attack, but have not been considered as biomarkers of activity phases. tgt is a global coagulation test which quantifies in vitro the ability of plasma to generate thrombin and estimates alterations in coagulation parameters. the objective is to assess the usefulness of the thrombin generation test (tgt) to characterize patients with hereditary angioedema (hae). method: seventeen hae patients from hospital la fe were recruited to obtain blood samples in remission and during ae attacks. none of them experienced thromboembolic events. plasma was collected in citrate tubes to obtain platelet rich plasma. hemostatic parameters were analyzed:. tgt was conducted using a calibrated automated thrombogram (cat) method and a fluoroskan ascent as a reader. results were analyzed via thrombinoscope v . citrated plasma was incubated with calcium, tissue factor, phospholipids and a fluorogenic substrate. a thrombin generation curve is generated, obtaining parameters: latency time (lagtime), thrombin generation maximum speed (vo), maximum peak of thrombin generated (peak), time to generate the maximum peak of thrombin (ttpeak), total quantity of generated thrombin (etp), and the end time of thrombin generation (starttail). tgt parameters from healthy donors were used as controls. results: thirty-eight samples were collected from seventeen hae patients ( . % female). fifteen ( . %) samples were collected during ae attacks. tgt parameters and fdps were significantly higher in hae patients compared with controls (p < . ), although no significant differences were found in tgt between acute attacks and remission. a decrease trend in tgt is observed in ae attacks. fdps were increased during ae attacks, but normalized at remission periods. these results support the involvement of coagulation in the pathophysiology of hae, although no increase in prevalence of thrombosis is observed during acute attacks. method: the repeated measures design study included patients in two groups: the slit group, patients- follow-ups per allergen (p), and the vit group, patients- p. the slit group had patients treated for hdm ( p), and patients on pre-coseasonal pollen ait (grass p, ragweed p, birch p). the vit group had patients on rush protocol ( for bee and for wasp) and patients on conventional protocol ( bee, wasp, and for both). the ige and igg levels were measured by the immunocap method. the friedman test was used to compare data. results: when compared to placebo group, slit+vitamin d group therapy was more effective in the reduction of nasal symptoms (p = . ), asthma symptoms (p = . ) and combined symptommedication score (p = . ); there was no significant difference between groups in medication and ocular scores. we observed a significant improvement of fev (vitamin d group p = . , placebo group p = . ) and fev %vc levels (vitamin d group p = . , placebo group p < . ), within both groups, between visits. feno results did not differentiate statistically significantly the study participants in terms of receiving slit along with vitamin d or placebo. significant increase in the percentage of cd + cd + foxp + and in tlr positive cells in children receiving slit+ vitamin d was observed compared to placebo group. increase in cd + cd + fox-p + induction, and in tlr positive cells recruitment were independently associated with better clinical effect of slit in children. conclusion: overall, ait with a high-polymerized ash pollen extract was well tolerated. as ash pollen are supposed to be an important allergen during spring time, it is recommended to include spt and npt with ash pollen in the test panel for allergological diagnostic. additionally, determination of ash pollen specific ige could be applied. furthermore, appropriate ait should be considered for ash pollen allergic patients. a | impact of sublingual immunotherapy with a five-grass pollen tablet on grass pollen allergic rhinitis and asthma: a real-life, long-term analysis in france background: data on the fulfilment of prescriptions of symptomatic medications in patients with grass pollen allergy were analysed to evaluate the long-term effectiveness of sublingual immunotherapy (slit) on allergic rhinitis (ar) and asthma. method: by using data in the lifelink ™ treatment dynamics database (iqvia, paris, france), we compared two cohorts of patients with ar: a group treated with oralair® (stallergenes greer, antony, france) slit tablets (n = ), and a matched control group having received symptomatic medications only (n = ). oralair®'s effectiveness was assessed as the change in symptomatic medication fulfilments between the pre-index period (before the initiation of slit) and the follow-up period (after slit), and as the onset of asthma or the progression of pre-existing asthma (based on fulfilments of prescriptions for asthma medication). the number of fulfilments per year was calculated for each patient and each period. results: in line with prescribing guidelines, the mean duration of treatment with oralair® was . months per season for either seasons or seasons. the mean number of symptomatic medications for ar fulfilled per patient and per year in the pre-index period was . ± . in the slit tablet group and . ± . in the control group. in the follow-up period, this value fell for the slit tablet group (to . ± . ) but did not change significantly in the control group ( . ± . ). when considering individuals not taking any asthma medications in the pre-index period, asthma onset during the treatment period was observed in . % of those in the slit tablet group and in . % of those in the control group. the corresponding values for the follow-up period were . % in the slit tablet group and . % in the control group. when considering individuals already taking asthma medications in the pre-index period, the mean ± sd number of asthma medication fulfilments in the pre-index period was lower in the slit tablet group ( . ± . ) than in the control group ( . ± . ). the corresponding values for the treatment period were . ± . and . ± . , respectively. in the follow-up period, the number of asthma medication fulfilments fell more in the slit tablet group (to . ± . ) than in the control group ( . ± . ). oralair® tablets have long-term effectiveness by relieving allergic rhinitis and slowing a progression to asthma. b | a real-life, retrospective analysis evidencing slower long-term progression of asthma in grass pollen allergy patients treated with sublingual immunotherapy tablets | an examination of the reasons for treatment discontinuation and non-compliance to allergen immunotherapy background: allergic rhinitis (ar) patients treated with subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit) may be non-compliant or discontinue treatment too early, which can negatively impact efficacy. therefore, understanding the reasons for non-compliance and treatment discontinuation is vital to help improve compliance, persistence and thus outcomes. this study reported reasons for treatment discontinuation to scit and slit and non-compliance to slit in patients with ar in published real-world studies. method: a literature review was conducted in embase, medline, ebm reviews, psycinfo and econlit ( - ) using key search terms for allergic rhinitis, scit, slit, non-compliance and non-persistence. across all studies,~ % of patients were non-compliant, and -year drop-out rates ranged from % to %. reasons for noncompliance and treatment discontinuation in this subset of patients were stratified according to the who dimensions for adherence (patient-related, treatment-related, or socio-economic). results: from the publications identified, six studies reported reasons for non-compliance to slit (n = ) or treatment discontinuation (n = ) to scit or slit, and the results were grouped for analysis. the majority of patients cited treatment-related factors as the primary reason for discontinuation ( % for slit, % for scit). common reasons were a length of treatment for slit and frequency of injections for scit. % of patients discontinued scit due to patient-related factors such as travel to doctors and waiting time for administration. only % of slit patients discontinued due to patient-related factors. socio-economic reasons for discontinuation were low for both therapies ( % slit and % scit). conversely, for non-compliance to slit, socio-economic factors were the most frequently cited reasons ( %), and included taking time off work and financial concerns. conclusion: of patients who discontinued therapy, treatmentrelated factors were the most cited reasons for scit and slit, reflecting concerns with administration and treatment length. noncompliant slit patients cited socio-economic factors as common reasons for non-compliance, suggesting financial concerns over a long treatment course. differences in reasons for non-compliance and treatment discontinuation may be due to patients assigning differing importance for compliance (a day-to-day decision) compared to the long-term decision to discontinue treatment. results: % of patients had monosensitization to rbet v component. the rest % had combinations ige to rbet v and ige to one, two or even three minor allergens ( %, %, % accordingly). after courses of slit by standardized pollen extracts symptoms of arc and pfas decreased in % and % patients accordingly. in group patients with monosensitization to rbet v : patients had a reduction of arc ( % had - degree by ado); patients had reduction of pfas. patients hadn't finished treatment due to allergic reactions. among patients with sensitization to rbet v /v : patients had a reduction of arc ( % - to degree by ado); had reduction of pfas. patient hadn't finished treatment due to allergic reactions. in patients with sensitization to rbet v /v : patients had a reduction of arc ( % - to degree by ado); had reduction of pfas. patients with sensitization to rbet v /v /v showed the similar results: patients had a reduction of arc ( % - to degree by ado); had reduction of pfas. patients had sensitization to all cra, and only patient who also received slit with grass allergens had reduction of arc only ( degree by ado). as the result of the study it was identified that beneficial effect of slit is highest in patients with monosensitization to rbet v . the increase of sige sensitization profiles to minor birch allergens caused less efficacy of slit treatment. dermatophagoides pteronyssinus immunotherapy is independent of sensitization to blomia tropicalis among children with allergic rhinitis and asthma method: children ( - years old) with allergic rhinitis and asthma sensitised to both dp and bt received years dp-scit. clinical symptom and medication scores, serum specific ige and specific igg were evaluated during dp-scit. in order to investigate whether the treatment outcome was dependent on the sensitisation pattern between dp and bt, patients were further grouped into dp and bt co-sensitisation and cross-reaction, according to positive or negative ige against bt major allergen (btma) blo t and blo t . btma+ group, with specific ige to either blo t or blo t , was defined as the co-sensitized group; btma-group, with no detectable ige to both blo t and blo t , was defined as the cross-reactive group in this study. results: all the recruited patients completed year of dp-scit, ( %) patients completed years of treatment. after years of dp-scit, compared to baseline, all patients had significant reduction in symptom and medication scores. lung function (fev ) was significantly improved as well. % of the patients were free of medication use and asthma symptoms, % of them were free of rhinitis symptom, and the fev % in all patients were higher than % of predicted. dp-scit induced significant increases in dp and bt specific igg . in % of patients, dp specific igg increased more than fold and bt specific igg increased more than . fold. further investigation in btma groups showed moderate correlation (spearman r = . , p = . ) between specific ige against dp and bt in the btma-group (n = ), indicating specific ige cross-reactivity. no specific ige correlation (spearman r = . , p = . ) was found in the btma+ group (n = ) indicating co-sensitisation to both dp and bt. the two groups showed almost identical change in clinical responses. dp and bt specific igg significantly increased during dp-scit, no difference was found between the two btma groups. conclusion: dp-scit can induce specific igg cross-reacting with bt allergens. patients with specific ige sensitisations to both dp and bt may have clinical benefit from dp-scit treatment. moreover, the clinical benefit of scit was independent of ige cross-reactivity or co-sensitisation to dp and bt. method: we investigated allergic rhinitis children who were basically sensitized to house dust mite and received house dust mite slit for year and months. among patients, patients were mono-sensitized to house dust mite (group ) and patients were poly-sensitized aside from house dust mite (group ). we also assigned another allergic rhinitis children who were only treated by medication as control group. nasal symptoms (rhinorrhea, sneezing, nasal obstruction, nasal itching, sleep disturbance) and anti-allergic medications use were assessed at every -month visit. results: the symptoms of allergic rhinitis started to improve after months of slit and significantly improved after a year and a half in group and group compared with control group. there was no significant difference between group and group . anti-allergic medication use in group and group significantly decreased after a year and a half compared with control group and there was no significant difference between group and group . conclusion: house dust mite slit was more effective than treatment only by medication. the effect of house dust mite slit was similar between mono-sensitized and poly-sensitized allergic rhinitis children. house dust mite slit could also be recommended to polysensitized allergic rhinitis children. method: a prospective, randomized, double-blind, controlled, multicenter phase ii study was conducted with four different concentrations of cluster allergoid clustoid wiesenlieschgras (group : tu/ml; group : tu/ml; group : tu/ml; group : tu/ml). out of patients screened, grass pollen allergic patients ( - years) were randomized. the cluster build-up phase was followed by four monthly maintenance injections of . ml. the efficacy was evaluated by the change of the threshold concentration step needed to induce a positive reaction in a titrated nasal provocation test (tnpt) before start and after end of the study (pre-post analysis). the safety profile was assessed for each treatment group by analyzing treatment-related adverse events. background: allergen immunotherapy relies on the consistent administration of allergen extract, therefore compliance to these treatments (subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit) tablets and drops) is vital for efficacy. as scit is administered as an injection by a healthcare professional, and slit is self-administered, compliance to scit may be perceived as superior. therefore, a review of real-world studies investigating compliance to scit, slit-tablets or slit-drops was conducted. real-world studies, instead of clinical trials, were included in this review as they are more likely to reflect actual clinical practice and patient compliance. method: a literature review was conducted in embase, medline, ebm reviews, psycinfo and econlit ( - ) using key search terms for ar, scit, slit-tablets and slit-drops, and real-world compliance. compliance was reported according to ispor medication compliance and persistence work group definitions. results: from the publications identified, eight studies (seven slit [one slit-tablets, two slit-drops, four unspecified], one both scit+slit-tablets) reported compliance rates and were included in the analysis. real-world compliance rates ranged from % to % for slit administration and % for scit administration. only one study compared compliance of slit to scit, with similar rates reported over three years ( % and % respectively). three studies reported "good" compliance (physician-reported or patients consuming > % of allergen extract) to slit-drops or slit-tablets. the good compliance rates were higher for slit-drops ( %- %) compared to slit-tablets ( %- %). observed compliance to slit-drops or slittablets did not vary by country or geographical region. the percentage of patients defined as having "good" compliance to slit-tablets or slit-drops did not vary by study length or patient population. conclusion: whilst compliance to scit may be perceived as superior to slit-tablets and slit-drops, comparable compliance rates between scit, slit-tables and slit-drops were identified across real-world studies. differences between perception and real-world results may be explained by a lack of direct comparisons between scit and slit administration. limitations included discrepancies in definitions of compliance, as well as methodology between studies. however, these are common to reviews analysing compliance, regardless of therapy area. conclusion: in the allergic rhinitis patients, successful compliance for -year slit compared with control was approximately %. method: igg inhibition elisa: rabbit igg antibodies specific for grass allergen allergoids are pre-incubated with different concentrations of alum-adsorbed grass pollen allergoid. the mix is added to an allergoid coated microtiter plate. unbound igg will bind to the allergoid coat and is subsequently incubated with anti-igg hrp labeled conjugate and stained with tmb. results are expressed as percentage inhibition relative to the uninhibited value. the concentration of alum-adsorbed allergoid that is required to inhibit % igg is used as read-out. circular dichroism: far-uv cd spectra ( - nm) were recorded on a j- spectropolarimeter. a cuvette with a stirring compartment was used to keep the suspension homogeneous during measurement. results: the igg inhibition elisa assay is specific for grass pollen allergoids (not for other allergen allergoids), has a good inter-and intra-assay precision and is robust for assay variation. thermally stressed alum-adsorbed grass pollen allergoids were used to show that the igg inhibition assay can be used as a stability indicating method. severe thermal stressing resulted in a higher % inhibition value, indicating a loss of igg epitopes. furthermore, far-uv cd analyses showed that there is a close relation between the decreasing igg binding capacity ( % inhibition values) and the loss secondary protein structures by unfolding (cd-ratio / nm values). the igg inhibition assay was demonstrated to be a valuable method to determine the stability of alum-adsorbed grass pollen allergoid preparations. in addition, a relation was shown between the igg binding capacity and the change in secondary protein structures. | design of a pivotal phase iii trial of allergen specific immunotherapy (ait) using a high-dose house dust mite (hdm) allergoid in patients with allergic bronchial asthma method: male and female outpatients (age - years) asthmatics allergic to hdm are enrolled. during the baseline phase, the patient's minimal dose of ics required to achieve asthma control will be assessed. after the baseline period, approx. patients will receive double-blind placebo-controlled treatment for approx. months, followed by a nd period of weeks to assess the minimal ics dose and further months of observation for the assessment of asthma exacerbation. based on the results of the dose finding study regarding the efficacy endpoints and the safety profile, the optimal allergoid dose is considered to be pnu. results: competent authorities and ethic committees in all participating eu countries, serbia, russia and ukraine approved the study design. the primary endpoint of the trial is the change in predefined dose steps of the minimal daily ics dose required to achieve asthma control after approximately months of subcutaneous ait. all efficacy data will be determined using daily questionnaires and the acq by e-diary for months from october to january. the aim of this clinical trial is to demonstrate efficacy and to evaluate safety of ait with an allergoid preparation of major allergens of dermatophagoides pteronyssinus in patients suffering from allergic bronchial asthma caused by house dust mites. asthma increases the burden of allergic disease and health care costs, especially when uncontrolled. with the development of a high-dose preparation we intend to treat asthmatic patients highly efficiently. romantowski j; jassem e; lata j; wasilewska e; chelminska m; specjalski k; niedoszytko m background: specific immunotherapy (sit) is the only causal treatment in patients allergic to airborne allergens. it has been proven to be widely effective in allergic populations, but individual patients vary in terms of response to the therapy. the aim of the study was to assess the factors that might affect the efficacy of sit. method: patients treated with sit for grass pollen or house dust mites were included. the efficacy of sit was assessed with the use of allergy control score (acs), performed before and at least after one year of sit. the following variables were assessed as potential risk factors for a poorer response to sit: age, gender, type of allergy, type of allergen, type of vaccine, type of sit and smoking history. background: specific immunotherapy (sit) is a suitable treatment option for asthma and allergic rhinitis (ar), but it is not commonly used in korea. in the achievement of the treatment, it is important that immunotherapy is applied with ideal dose and regular intervals and it is essential for the patient compliance. the aim of this study is to investigate evaluate compliance with immunotherapy protocols of patients who were treated with sit in clinic and their satisfaction of the treatment. we performed a multicenter, cross-sectional survey using a specially designed questionnaire that was given to allergy specialists and patient in korea. a member of the trained research group conducted face-to-face questionnaire interviews with each respondent. conclusion: this study shows that most patients are ar with asthma. in our study sit compliance and satisfy are found to be high in both groups. aim: to identify the frequency of regress claims in a dermatological setting and to assess its impact on general prescription behaviour and immunotherapy. method: all physicians of the psoriasis-praxisnetz süd-west e.v. (n = ) were invited to participate in a web based questionnaire study on the topics of dermatology and medical law. the survey was separated into two sub-polls which were carried out after a first poll deciding whether the topic of medical law is of any interest for the dermatological practice. the topic of interest was located in the second poll. results: overall, dermatologists participated in this study. most participants were form bavaria, baden-wuerttemberg or rhineland-palatinate and had more than years of experience as a dermatologist. out of the participating physicians . % (n = ) already experienced a previous regress claim. of these, . % (n = ) stated, that the experienced regress claim changed their prescription behaviour. half of these participants (n = ) further stated, that the fear of a possible recourse affects their prescription behaviour, whereas only out of the other participants declared a possible influence. missing values excluded, this leads to a substantial hesitation in physicians who experienced a prior recourse ( . % vs . %). nevertheless, this seems not to affect the usage of allergen immunotherapy, as all physicians who already experienced a regress claim, stated to use allergen immunotherapy. the fear of a possible regress can change physicians' prescription behaviour but does not seem to have an effect on the prescription of allergen immunotherapy. therefore, the topic should be addressed from another perspective such as providing trainings on relevant regulations for physicians who experienced a prior recourse claim. this approach could also improve patient centred care related to modern treatments. results: the sds were significantly reduced in patients subjected to slit (p < . ) year after the onset it. vas also was significantly reduced (p < . ) with satisfied control of sar and the same time with translation from moderate-severe to mild-moderate sar, after slit. nbh was also significantly reduced (p < . ) year after the onset slit. in patients receiving pht only, sds, vas and severity of sar did not change and significantly higher (p < . ) from the value obtained in the experimental group. nbh also remained unchanged and significantly higher (p < . ) then in experimental group. precoseasonal slit added to pht shows short-term beneficial clinical effects in polysensitized patients with sar and scuad phenotype. results: ten studies ( children, adults; median sample size, ) met the inclusion criteria. the risk of bias was moderate to high in all but one studies. low strength evidence supports the assumption that ait is effective in reducing symptoms and medication use, with only out of studies reporting higher benefit in the ait group vs comparator group. subgroup analyses of studies sharing similar characteristics did not explain inconsistency. safety does not appear was not major concern for alternaria ait. conclusion: this is not enough strength of evidence to suggest that mold ait is efficacious for the treatment of respiratory allergies. high-quality studies with an adequate sample size are needed. abstracts | | tolerability of a two week rush updosing with modified trees, modified grasses or modified grasses/trees mixture in pollen allergic subjects in the day-to-day practice table) severe systemic reactions (grade iii and iv) did not occur. conclusion: rush immunotherapy is an effective therapeutic method for patients with allergic rhinitis. it seems that in cases requiring faster response to treatment, this immunotherapy can be considered as a substitute for conventional immunotherapy. | design of a pivotal phase iii allergen immunotherapy study to assess the efficacy and safety of subcutaneously administered tyrosine adsorbed modified birch allergen+mpl results: the design of this study, including sample size and primary and secondary endpoints, will be discussed based on prior experience gained in two dose finding studies. in addition, the number of patients screened and randomized will be presented by country, gender and/or age and screen failures will be categorized. results: the primary analysis showed an absolute difference in tcs between placebo and du of . ( %, p < . ). the odds of experiencing a severe day during the bps were approximately doubled in the placebo group compared to the du group (or = . , p < . ) and the odds of experiencing a mild day were halved (or = . , p < . ). similar results were seen for the tree pollen season (tps), covering both alder, hazel and birch pollen seasons. the total rqlq score was improved for du compared to placebo during the bps and tps (p < . , except for the last week of the tps), with the most pronounced effects during week - of the bps (absolute difference: . - . , p < . ). treatment was well tolerated. the most frequent adverse reactions were mild or moderate local reactions related to the sublingual administration. no deaths were reported and no serious adverse events were assessed as related to the sq tree slit-tablet. conclusion: treatment with the sq tree slit-tablet improved arc symptoms and need for symptomatic treatment. the du group had less "severe days" and more "mild days" during the pollen seasons. the quality of life was similarly improved. these findings substantiate the clinical relevance of the sq tree slit-tablet for patients with arc induced by pollen from the birch homologous group. nagaraju k ; nagaraju k ; katare s ; kapatkar v ; shah a ; rathod r results: total n = subjects (mean age . ± . years, . % males) completed entire study. the mean incidence of artis reduced from . ± . episodes at baseline to . ± . (p < . ), with . % subjects not suffering from any episode. the mean duration of episodes reduced from . ± . to . ± . days (p < . ). % of episodes (vs % at baseline, p < . ) required antibiotics for mean duration of . ± . days (vs. . ± . days, p < . ). none of arti episodes in follow-up period required hospitalization as against . % episodes, (mean duration ± . days; p < . ) before pidotimod therapy. the number of school days lost & work days lost showed reduction of . ± . days(p < . ) & . ± . days(p = . ) respectively. the average expenses incurred in treatment of artis shows significant reduction of rs. ± (p < . ). adverse events were reported in ( %) subjects, which were mild in nature. a statistically significant increase in absolute counts of t-& nk cells was seen in explorative assessment of immune markers. the study shows pidotimod to be well-tolerated effective therapy in reducing the incidence and severity of recurrent artis, thereby providing additional benefit of reduction in discomfort & healthcare cost due to recurrent artis. thus, pidotimod can be considered as potential therapeutic option for treatment of recurrent artis in children. martignago i ; ridolo e ; incorvaia c department of medicine and surgery, university of parma, parma, italy; cardiac/pulmonary rehabilitation, asst pini/cto, milan, italy background: two registered sublingual immunotherapy (slit) products are available to treat grass-pollen induced rhinoconjunctivitis, consisting of the -grass (phleum pratense) and the -grass pollen tablets. no study of direct comparison of the efficacy of the two products was performed. we report the case of a patient who was treated in different years with the -grass or the -grass tablets with contrasting efficacy. the patient was a -year old woman suffering from years of grass pollen induced rhinoconjunctivitis. in slit was started with the -grass pollen tablets, but in , due to unavailability of the product, slit was performed by the -grass pollen tablets. in the third year of treatment slit with the -grass pollen tablets was resumed. for the -grass tablets slit was initiated before the pollen season and stopped after months of treatment, while for the -grass tablets the treated was prescribed to be continuous. the efficacy of slit was evaluated by symptom-medication scores as reported in diary cards by the patient during the month of may, when the grass pollen usually reach the higher concentration in the atmosphere in lombardy, where the patient lives. results: the mean symptom-medication score in the first year of treatment ( -grass tablets) was . , compared with a mean score of . in the second year ( -grass tablets). the patient was unsatisfied of the symptoms control and asked to resume for the last year of slit the -grass tablets. the mean symptom-medication score in such year was . . no clinically relevant adverse event was reported with any slit product. conclusion: based on the momentary unavailability of the -grass pollen tablet, it was possible to assess in a same patient the clinical outcome associated to either of the two registered slit products. a significantly different efficacy of slit with the -grass tablets compared with the -grass tablets was observed. | fusion proteins consisting of bet v and phl p form ige-reactive aggregates with reduced allergenic activity najafi n ; hofer g ; gattinger p ; smiljkovic d ; blatt k ; selb r ; stoecklinger a ; keller w ; valent p ; niederberger v ; thalhamer j ; valenta r ; flicker s background: bet v and phl p representing major allergens in birch and grass pollen, occur as monomeric proteins with high allergenic activity as assessed by clinical provocation testing in patients. she did not suffer more hymenoptera stings after the last reaction. one bee sting several years before bst resulted in no reaction. she has no symptoms with honey, vegetable or other food ingestion. methods: total ige and specific ige were determined using inmu-nocap system (thermofisher, scientific inc). apis, vespula and polistes (hørsholm, denmark) were also performed. prick tests showed negative results for all extracts tested. intradermal skin tests were positive for apis at μg/ml, but negative for vespula and polistes. our patient was diagnosed of anaphylaxis due to apis venom, thus bst was contraindicated and an epinephrine autoinjector was prescribed. she rejected hymenoptera venom immunotherapy. conclusion: to our knowledge, this is the first case of anaphylactic reaction after bee sting therapy. bee sting therapy should be considered a risk factor for anaphylaxis. patient reported good control of their disease, improved their quality of life, tolerating contact and exposure to numerous horses as well as contact with clothes of people who had been exposed. although ita is absolute contraindication on uncontrolled asthma with a degree of evidence ia, our case had only "transitory" con- and immunocap among wasp allergen components-i , i , i were %; . , %; . and %; . respectively and honey bee allergen components-i , i , i were %; . , %; . and %; . respectively. agreement between polycheck and immuno-cap i and i allergen components were %; . and %; . respectively. agreement between polycheck and euroline i and i allergen component were %; . and %; . respectively. based on wasp and bee components in all three systems, sensitization pattern was analyzed. similar test results were found between euroline and immunocap systems. the comparative studies carried out showed a markedly higher compliance of results with the euroline tests compared to polycheck with the immunocap system. percent agreement was extremely high and kappa value was substantial or almost perfect in the case of bee venom allergy between euroline results: a total of patients were included; ( %) males, with a mean age of (± ) years; ( %) beekeepers, ( %) were atopic, ( %) had asthma, ( %) rhinitis and ( %) cardiovascular disease, and of these patients were on ace/beta blockers. vit with honeybee was proposed in ( %), wasp ( %) and polistes ( %). the mean duration of vit was (± ) months. however, completed less than months. of the total, patients ( %) were not treated with vit. eighty-eight patients ( %) participated in the telephone interview: completed vit ( %), were still on vit ( %) and did not undergo vit ( %). of those who completed vit, ( %) were restung and went to the emergency department (er). twenty-four patients ( %) were stung while still on vit. of those never on vit, ( %) were re-stung and went to er. the severity of the reactions according to mueller of the patients who completed vit (mean follow-up time was months ( - months)) and were stung again was: local reaction in ( %), grade i in ( %); grade iii in ( %). one had a toxic reaction after multiple stings. in those who were stung during vit, ( %) had local reactions, ( %) grade i and ( %) grade iii. of those who were not treated and were re-stung: ( %) had grade i, ( %) grade iii and ( %) grade iv. in this series, the patients who did not undergo vit presented a greater number of systemic reactions when re-stung as well as more severe reactions (p < . ). conclusion: in this group with indication for vit, the reactions of the re-stings were less severe in the patients who had completed or who were on venom immunotherapy, as expected. three quarters of those who did not undergo treatment had severe anaphylactic reactions when they were stung again. this study reinforces the importance and the efficacy of immunotherapy in the treatment of hymenoptera venom allergy. method: this is a retrospective, descriptive study of cases diag- method: data were issued from the reference centre in mastocytosis of toulouse university hospital. ms diagnosis was determined using world health organization diagnostic criteria. hymenoptera venom immunotherapy was performed with an ultra-rush protocol (table) . results: seven patients were included ( women, men), median age years old. during the anaphylactic reaction, cutaneous signs missed in all cases. the reaction was most often severe: grade (n = ), grade (n = ), grade (n = ). three patients suffered from digestive symptoms and one from respiratory manifestations. basal tryptase in serum reached . - . μg/l. hymenoptera venom specific ige were low ( . - . kui/l) except for one patient ( . kui/l). ait was initiated with vespula venom in patients, polistis in patient, apis mellifera and vespula in patient, vespula and polistis in patients. no reaction was observed during ait. four restringing accidents led to increase the cumulative dose to μg and μg in patients. in these patients, the diagnosis of mastocytosis was made due to the resting. conclusion: hymenoptera venom ait using ultra-rush protocol seems well tolerated in patients with systemic mastocytosis. specific studies are necessary to determine the real tolerance profile of this protocol. collaboration with reference centres for mastocytosis should be considered for all patients with mastocytosis associated to hymenoptera venom allergy. dose (μg/ml) results: see table. conclusion: there is a shift or immunomodulation in terms of sige to vespids. even in patients double sensitised who were receiving venom of only one of the vespids. albanesi m background: slit has been suggested as an alternative route for allergen-specific immunotherapy. aim of this study was to investigate allergen-specific antibody responses in birch pollen allergic children who had received slit for two years using recombinant allergens. method: children (n = ; - y o) with respiratory symptoms of birch pollen and oral allergic syndromes (oas) were studied. ten children received slit with staloral, were treated by slit with microgen, and children received only symptomatic therapy (control group). sige and sigg levels to rbet v , rbet v , rbet v were measured twice (before therapy started and after two years) using quantitative immunocap and a panel of more than microarrayed allergens using immunocap isac technology. clinical efficacy of slit was evaluated by recording symptoms upon allergen contact and need of rescue medication. results: all children were sensitized to the major birch pollen allergen, bet v and one patient from each of the groups showed to bet v , no patient had sige to bet v . after two years of slit clinical improvement was observed in the slit patients. in the staloral group there were no respiratory symptoms in patients and a decrease of symptom severity in the other cases as well as a partial or complete tolerance to pr allergen-containing food in the patients. microgen treatment had no influence on oas symptoms but decreased of pollinosis severity in children. however, there were no statistically significant differences of bet v -specific levels measured before and after treatment in the slit and control groups (mann-whitney, p > . ). in this real-life study we found that birch allergic children who had been treated with slit showed a reduction of clinical symptoms but we did not find a significant induction of allergen-specific igg levels in the slit-treated group when compared with children who had only symptomatic treatment. conclusion: our study confirms the scarcity of food additives allergy. it also suggests that even when the diagnostic of allergy was excluded with a negative oral food challenge, families remain suspicious about industrials feeding products containing food additives. these results should reassure health professionals and parents who incriminate too frequently food dyes and conservators when a manifestation which mimics allergic reactions occurs. background: autumn/winter birth has been reported to be a risk factor of food allergy (fa) development. a putative mechanism is that dry/cold weather causes and exacerbates infant atopic dermatitis (ad), which is a major risk factor for food sensitization through inflamed/damaged skin. we investigated prevalence of fa among infants under well skin care in relation with seasons of birth (sob). we recruited full-term newborn infants without perinatal diseases at an obstetric/pediatric clinic. participants were followed up for skin status and food allergy symptoms until months of age. sob were defined as spring (march-may), summer (june-august), autumn (september-november) and winter (december-february). ad was diagnosed based on the united kingdom working party's criteria. use of moisturizer (mo) and topical corticosteroids (tcs) was recorded. primary outcome was fa based on apparent immediate allergic reaction after ingestion of causative food. we classified infants who avoided any food because of sensitization or mother's anxiety as suspected fa. results: six hundred and thirty-one infants were screened for month-period and infants were enrolled in this study. of them, infants were born in spring-summer (s-born) and infants were born in autumn-winter (w-born). fa developed in ( . %) infants and ( . %) infants had suspected fa. there was no difference (p = . ) in prevalence of fa and suspected fa between s-born and w-born. multivariate analysis revealed ad at and months of age was a significant risk factor for fa with or= . ( %: . - . ) and or= . ( % ci: . - . ), respectively. prevalence of ad at months of age was higher in w-born than s-born but prevalence promptly decreased thereafter and stayed low with early use of mo and tcs. prevalence of ad was rather higher at months in s-born than w-born. results: cases and controls were included. the median age was years, (q -q - ). men and women were almost equally represented ( . % males). alcohol consumption associated with the intake of mammalian meat or innards as the trigger factor. the overall prevalence of a positive result of sige to α-gal was abstracts | . % ic % ( . , . ); cases _ . % ic % ( . , . ) controls _ . % ic % ( . , . )_. among cases sige anti α-gal positivity rate ranged from . % (rural), to . % (half-urban) and . % (urban). the rates of positivity were . %, (northern) . % (center) and % (mediterranean). a positive result of sige to α-gal was more frequently observed among men ( . %) than women ( . %) and associated with history of tick bites, practice of outdoor activities, pet's ownership and the antecedent of having eaten mammalian meats or innards previously to the development of symptoms background: a special challenge in the st century for allergists is allergy to food, which is considered "the second wave" of epidemics of allergic diseases. panallergens occur in unrelated organisms and perform a similar function in them. in their structure, they have highly conserved amino acid sequence regions and a similar three-dimensional structure, and thus meet the requirements for cross-recognition by ige. results: in patients ( %) isac test has been shown to have specific ige for panallergen components. mostly, the presence of ige for pr- proteins has been shown in patients. in patients ige to ltp; patients ige to ccd; patients to profilin; patients to tropomyosin; patients to serum albumin, person to tlp. an important aspect is undoubtedly the occurrence of simultaneous sensitization to several panallergens. analysis of data from the study group showed that isolated sensitization to one panallergen concerned only pr proteins ( patients), tropomyosin ( patients) and profilin ( patient). in the remaining patients, the analysis of the isac test results showed that two or more panallergens were allergic. in the study group, asige for the component responsible for the occurrence of real food allergy was detected in ( %) patients. mostly, the presence of ige for jug r has been shown in patients. in the study group, panallergens were more likely to be responsible for food intolerance than specific food allergens. results: of children, children had peanut allergy only, children had tree nut allergy only, and children had both. the mean age was . ± . years in peanut allergy, . ± . years in tree nut allergy, and . ± . years in both. male to female ratio was significantly higher in tree nut allergy ( . %) than peanut allergy ( . %). among tree nut allergens identified, walnut ( . %) was most frequent, followed by almond ( . %), hazelnut ( . %), pine nut ( . %), chestnut ( . %), cashew ( . %), pistachio ( . %), and macadamia ( . %). mean serum total ige level was kua/l in tree nut allergy and kua/l in peanut allergy. mean serum specific ige level to peanut, walnut, almond, hazelnut, and pine nut was . , . , . , . , . , and . kua/l, respectively. children with peanut allergy had higher rate of co-sensitization with soybean and higher soybean-specific ige levels than children with tree nut allergy. however, there was no difference in co-sensitization rate with tree pollen between peanut and tree nut allergy. children with peanut allergy showed significantly increased co-sensitization rate with egg white and wheat compared to children with tree nut allergy. a . % of the children with peanut allergy and . % of tree nut allergy showed co-sensitization with aeroallergens. a total of % of the children with peanut allergy showed decreased specific ige levels within - years. conclusion: prevalence of peanut and tree nut allergy is similar. tree nut allergy develops later than peanut allergy and more common in male. children with peanut allergy showed higher co-sensitization rate with soybean, egg white and wheat compared to children with tree nut allergy. | natural history of egg allergy in a large cohort of infants with food allergy shows its high prevalence but also its transient nature in a months of follow-up background: the cohort of infants ( boys, girls, - months) with the food allergy has been followed for months. as more than % of infants manifested atopic dermatitis (ad), a condition closely linked to egg sensitisation, we focused our attention on egg allergy, following its natural history as well as a development of atopic march. method: the diagnosis of food allergy was based on a personal history, clinical examination, skin prick tests and/or atopy patch tests with native foods. laboratory tests were performed within year of age the latest. the specific ige levels against food allergens were measured using immunocap or immulite. patients with ad were scored according to scorad system. the oral food challenges (ofcs) with cooked/baked egg were done in children at the age of months except for children at risk of anaphylaxis. results: within the whole cohort the allergy to cow milk proteins was confirmed in pts ( . %), to egg in pts ( . %), to wheat in pts( %), to lentil in pts ( . %) to banana in pts ( . %), to soya in pts ( . %) and to potatoes in pt ( . %). in a cohort of egg allergy patients we found out that: % of pts presented the early onset of allergy-up to months of age, % of pts presented severe ad (scorad > ), % of pts showed cosensitisation to peanuts, % of pts had early sensitisation to inhaled allergens, and majority % of pts presented with early onset allergic rhinitis and/or asthma. we proved that egg allergy is closely linked with the early onset of allergy symptoms, with severe forms of ad, co-sensitization to peanuts, early sensitisation to inhaled allergens and an early onset of allergic rhinitis and/or asthma. we also proved that the egg allergy in infancy is transient. the tolerance to baked/cooked egg was achieved in about % of pts at the age of years, unlike previously published results claiming the reach of tolerance in % of pts at the age of years. in these patients we studied: sex, personal history, type of reaction they presented, time of onset of symptoms and food involved. results: out of a total of patients over years of age (from to years old) who have been attended the consultation for the first time during these period, patients ( . %) ask about possible allergic food allergies. of these patients who came for possible allergic pathology, patients ( %) presented positive results. these are the other item we have studied: . sex of patients: % of the patients are women. these patients because of that, it is important to remember that food allergy can also appear in old people. the food that is mainly involved in our population is fish and seafood. a much higher percentage than in other populations, probably due to the mediterranean diet of spain. the symptoms mainly involved are itching and skin lesion, which is the characteristic symptom of a mild allergic reaction. in our population, there was patients with a anaphylactic shock, a much higher percentage than in other studies. the experience with this group of patients is still limited. more studies are needed to know better this patient profile. background: cow's milk allergy (cma) is the first atopic disease in children. diagnosis suspicion in the emergency room (er) is increasingly frequent, however, further assessment by an allergist is often difficult to schedule. therefore, screening for cma through a blood test (specific ige) while the infant is still in the er has gained momentum in recent years. we set out to analyse (a) symptoms which had led the emergency physician to prescribe specific ige, (b) the prevalence of confirmed cma among infants screened in the er, and (c) the long-term outcome of the screened infants. method: a retrospective study of medical records and laboratory results was performed. patients were infants under months, without a previous diagnosis of cma, attending one of the two pediatric er of the university hospitals of marseille, france. allergy blood tests were specific ige to cow's milk extract (immuno-cap, thermofisher, sweden). in infants with specific ige to cow's milk extract of . kua/l or higher, ige directed to the main three individual proteins (casein, alpha lactalbumin et beta lactoglobulin) were also measured. results: infants were included from december to june . the sex ratio was . . % of infants were atopic et % were currently or had been breastfed. the most prevalent symptoms were vomiting and reflux. one third of infants were hospitalized after the er visit. following the er visit, % of infants attended a specialized consultation with an allergist. % of infants with a follow-up visit were diagnosed with an ige mediated cma. infants with cma developed further food allergies (egg, nuts, cashew…). it is difficult to diagnose it. the emergency pediatrician are increasingly confronted to infant with symptoms evoking cma. thus they prescribe sige and extensively hydrolysed proteins because they know that ige-positive infants can be ige-negative during the interval between the er visit and the follow-up one. after bad results interpretation of blood assay after er visit, cma was probably over diagnosed without prick test for ige positive allergy and no eviction/ reintroduction test for non ige. the lack of allergist is probably leading to over prescription of blood assay in er to diagnose cma and prolonged eviction of milk. results: a total of patients were included ( % female) aged from to yo with an average . ± years. % of patients had history of atopic disease: % rhinitis, % asthma, % prior food allergy, % eczema, % drug allergy, % eosinophilic esophagitis (ee) and % chronic urticaria. mean serum total ige was . ui/ml. sensitization to aeroallergens was present in % of patients, the most common were dust mites ( . %), pollen ( . %) or both ( %). in ( %) patients, first symptoms of fa appeared ≥ yo, with an average age of ± . yo. in this group, were diagnosed with ee, with eosinophilic colitis and with eosinophilic gastritis. from the remaining patients, had history of reaction with more than food group (fg). cutaneous reactions were referred in % of patients followed by anaphylaxis ( %) and gastrointestinal symptoms ( %). the fg most commonly implied were: fresh fruits (n = ), seafood (n = ) and tree nuts (n = ). fa diagnosis was confirmed in % of patients, the remaining had negative ofc. in ( %) patients, their symptoms started under yo, with an average age of . ± yo. from this group, ( %) were diagnosed ee. from the remaining patients, cutaneous complaints were the most frequent ( %) followed by gastrointestinal ( %) and respiratory symptoms ( %). the most common fg implied were: fresh fruits(n = ), seafood(n = ) and tree nuts(n = ). only one anaphylaxis was referred. fa was confirmed in %, the remaining had negative ofc. in patients with history of anaphylaxis of had positive st and/ or sige; one had negative sp and sige, with ofc positive. the blood donors were classified based on their clinical symptoms related to possible as contact via fish intake: allergic to as ( %), chronic urticaria ( . %), unspecific dyspepsia ( . %) and asymptomatic ( . %). the prevalence of sensitization (anti-as ige > . kua/l) were . % (ic: . - . %; mean . kua/l; median . kua/l) with a maximum value of . kua/l. raw fish consumption was the only variable associated with statistical significance (p < . ) to as sensitization ( . % vs . %, respectively). albacore and codfish were the most consumed species associated to seropositive results ( %), followed by hake ( %). coastal population ( . % vs . %), non-previously frozen fish consumption ( . % vs . %) and > times per week fish consumption ( . %) were other seropositive associated factors. background: oral allergy syndrome (oas) is an ige-mediated allergy caused by raw fruits and vegetables in patients with pollen allergy, which is known as the most common food allergy in adults. however, there has been no nation-wide study on oral allergy syndrome in korea. the aim of this study is to investigate the prevalence and clinical manifestations of oas in korea. method: twenty two investigators from hospitals and private clinics participated in this study. the patients with allergic rhinoconjunctivitis and/or bronchial asthma with pollen allergy were enrolled to the survey. the questionnaires include demographics, a list of fruits and vegetables, and clinical manifestations of food allergy. pollen allergies were diagnosed by positive results of one or more pollen allergens including birch, alder, hazel, beech, oak, willow, poplar, bermuda, meadow, orchard, rye, timothy, mugwort, ragweed, hop japanese on allergy skin prick tests (allergen/histamine ratio ≥ +) and/or serum specific ige levels using multiple allergen simultaneous tests (mast ≥ +) or immunocap (≥ . ku/l). conclusion: this is the first nation-wide study for oas in korea. the prevalence of oas in korea was . %, in which substantial proportion had anaphylaxis. these results will provide useful information for clinicians to apply in clinical practice. we conducted a self-administered, questionnaire-based survey in - during the -month checkup. children were considered to have food allergies if they were diagnosed by a physician or if they had been instructed to avoid a causative food after medical examination by interview. we divided the year into three periods. the months of march-june were considered spring, july-october as summer/fall, and november-february as winter. while the season of onset for the boys occurred in . %, . %, and . % in spring, summer/fall, and winter, respectively, it was . %, . %, and . %, respectively, for the girls. thus, the onset rate was the highest in winter for both genders. in boys whose mothers did not consume folic acid (fol − ), the food allergy onset rate was significantly higher for boys whose mothers ate no eggs and for boys whose mothers ate - eggs per week than for those whose mothers ate eggs daily according to the dunnet multiple comparison test. however, no relationship was observed with egg intake if the mother had consumed folic acid (fol + ). on the basis of seasons, fol − and egg intake by mothers affected only children born in winter, with a significant difference in the dunnet multiple comparison. among mothers who did not eat eggs, fol + was . % and fol − was . %; for mothers who ate - eggs per week, fol + was . % and fol − was . %; and for mothers who ate eggs every day, fol + was . % and folwas . %. thus, consumption of folic acid seemingly annulated the effects of eating eggs. however, for girls, neither folic acid nor eating eggs had any effect on the onset rate. conclusion: since this effect varied according to the birth season, consumption of folic acid, a methyl group donor, appeared to affect the allergy onset in children. results: skin prick test with commercial extracts of tuna ( mm), cod ( mm), rooster ( mm), hake ( mm), salmon ( mm), trout ( mm) and anisakis ( mm ige to shrimp, lobster, crab and mixed seafood were all undetectable. dermatophagoides pteronyssinus , to assess for tropomyosins was negative. outcome: the patient continues to react to both hdm and shrimp, despite undetectable ige levels to tropomyosin associated components. this is the only testing available in south africa currently and hence we are unable to look at other proteins. the relationship between tropomyosins in shellfish allergy and mite allergy has been well documented and investigated, but other allergens are now also being implicated in cross-reactions. we also established the level of ige specific to allergen components using the immunocap isac method. allergen-specific ige was not elevated to any shrimp allergens available in immunocap isac: n pen m (tropomyosin), n pen m (arginine kinase) and n pen m (calcium binding sarcoplasmic protein). the patient was diagnosed with a shrimp allergy. the molecular diagnostics used did not explain which allergen component is the patient allergic to. it is possible that the patient is allergic to hemocyanin, which can also cross-react with house dust mite allergens, but confirmation of this diagnosis requires further investi- results: the groups were identical in terms of the age and sex (table ) . ara h ige correlated (spearman test) with the cumulative protein dose (threshold dose) r = − . (p = . ) but not with reaction severity r = . (p = . ), or the use of adrenaline r = . (p = . ). patients with ara h ige < ku/l had higher threshold doses ( vs mg) than children whose ara h ige was ≥ ku/l (p = . ). there were no significant differences in severity of the reaction or in use of adrenaline (table ) . the level of ara h ige is relevant in predicting the threshold dose at peanut exposure. a low reaction threshold dose increases the risk of reaction at an accidental exposure leading potentially to a severe reaction. flaxseed allergy is uncommon and most of the cases reported involved anaphylaxis. cross reactivity has been described with other seeds. case report: a -year-old atopic girl diagnosed with egg allergy and rhinoconjunctivitis and asthma due to pollens. when she was eight, she presented two reactions consisting of conjunctival, periorbicular, malar erythema and abdominal pain after eating egg free french toasts cooked with flaxseed. she was treated with oral antihistamines. the allergic workup included prick-by-prick test with flaxseed which was positive and skin prick tests with mites, molds, cockroach, cat, dog, profilin, ltp and pollens with positive results for olive and grass pollens. the serum total immunoglobulin (ig) e was u/l, and specific ige to flaxseed was . kua/l. the flaxseed extract was resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and an ige immunoblotting was performed under nonreducing conditions. the patient's serum showed specific recognition of a -kda band in the immunoblot. proteins were identified using mass spectrometry (maldi-tof) that showed results highly consistent with conlinin, a s storage protein of flaxseed. we described for the first time a patient with allergy to flaxseed due to conlinin, a s storage protein of flaxseed. background: cyperus esculentus is an herbaceous plant that has edible tubers called tiger nuts. in spain, they are used mainly in the elaboration of the well-known "horchata" or tiger nut milk, which is obtained by macerating tiger nuts with water and sugar. tiger nut allergy has rarely been reported, despite of its widespread consumption. case report: we present the case of a -year-old male with a history of oral syndrome allergy with several fruits (peach, melon, banana, kiwi, apple, pear and plum) and seasonal allergic rhinoconjunctivitis due to grass pollen. he reported oral pruritus, vomiting and cutaneous itching in both arms and hands immediately after drinking tiger nut milk. he became asymptomatic without treatment after - hours. the allergic workup included skin prick tests with profilin, ltp, latex and fruits that were positive to melon and watermelon and negative to profilin, ltp, latex and the rest of the fruits. prick-by-prick tests with melon, banana, kiwi, apple, pear, tiger nut and tiger nut milk were positive. the serum total immunoglobulin (ig) e was . μg/l, and specific ige was negative to profilin, ltp, bet v and all the tested fruits. background: specific blood ige tests for food allergens are mainly used to confirm a suspect food allergy than to diagnose such an allergy. this is due to the low positive predictive value and the high negative predictive value they have. nevertheless, they are very helpful when they are interpreted in the context of medical history by an experienced allergist. we analyzed the prevalence of sige in children with suspected food allergies. we retrospectively analyzed the all the consecutive laboratory tests of sige for food allergens during the two-year period ( ) ( ) ( ) . tests were of children diagnosed or suspected to have food allergies. a quantitative immunoblot assay was used to measure the circulated different sige. t-test, wilcoxon signed rank test, and chi-square were used to make comparisons. results: fifty-six ( . %) men and ( . %) females, . + . years old with a maximum of years and a minimum of months old were part of children whose tests were analyzed. one-third of the tests ( . %) reveals more than one sige present and . % of the tests resulted negative for the sige for the allergens tested (table) . only ( . %) children have very high concentrations (> iu/ml) of egg whites sige, and ( . %) have egg yolk sige. concentrations of . % of sige positive cases were . - . iu/ml. in our study more than half of the children suspected of food allergies resulted negative for sige for most common food allergens. seventy-nine percent of the positive cases had relatively low sige. only a few positive cases have higher sige than iu/ml. our data support the recommendation that sige couldn't be decisive in food allergic diagnosis, but they may help if they are interpreted cautiously. method: seven patients with a mean age of . years and female to male ratio of: : , with a background history of hypertension treated with an acei presented with oro-pharyngeal irritation (itch, tingling), face and tongue angioedema and laryngeal constriction, on ingesting fresh fruits (cherries, apples, plums, peaches, apricots, strawberries, grapes), vegetables (parsnips) and/or nuts (peanuts, hazelnuts). two patients required admission to emergency department and three received adrenaline auto-injector. six out of seven patients underwent skin prick testing to common aeroallergens and the index foods. in five cases, immunocap/isac testing was undertaken. in one case the diagnosis was based on the history and in one other case it was based on history and skin prick tests. results: a diagnosis of pfas was confirmed in all patients through the clinical history, spt and/or specific ige serology to the offending food confirming predominant sensitisation pr- allergens. primary food allergy and spontaneous angioedema was excluded in all patients. in the cohort studied, the pfas symptoms were unusually severe. we therefore postulate that this was secondary to concurrent use of acei. the management of these patients to sensitization to a β-casein with high homology between only the first milks. more precisely, the allergen candidate could be γcasein, which is derived from β-casein by proteolysis, whose abundance increases during cheese production from fresh milk, and which is absent in cow's milk. a lactoglobulin specific to buffalo's milk may also be responsible. in case of ewe's and goat's milk allergy without cow's milk allergy, sensitization to buffalo's milk should systemically be seeked out. we recommend inclusion of all mammalian milks in the list of the mandatory allergens for declaration on food products. method: prick tests with fruit battery, ltp and profilin was made. analytical with blood count, immunoglobulins, triptasa, total ige and specific ige to banana, apple and orange. finally, open oral provocation with fruits was performed. the diagnostic key was given by the mother of the patient who attended consultations because the infant had erythema in the temporary zone after drinking sea water on the beach. associated with salivation, the patient presented erythema in the malar area lasting a few seconds many times. the prick tests with fruits, ltp and profilin were negative. hemogram: eosinophils/μl, total ig e: . iu/ml, specific ige to fruits were negative. triptase: . μg/l. method: here we describe a variety of cases of nsltp allergy presenting to a tertiary allergy centre in the north west of england. results: nsltps have been found to be major allergens in various foods and they are likely to produce severe and systemic allergic reactions. this is reflected in the cases we present here. these proteins are highly cross-reactive due to extensive sequence homology and are panallergens. nsltps are remarkably heat stable and retains its allergenicity in processed foods. it is assumed that nsltps may sensitise both by inhalation and ingestion. an intriguing aspect in nsltp hypersensitivity is the extreme variability of its clinical expression. co-factors are often needed for the clinical expression of nsltp hypersensitivity. conclusion: patients regardless of where they are from, presenting with multiple severe/systemic food allergies need to be investigated for nsltp allergy. these patients require specific dietary advice on foods to avoid and a tailored management plan on how to deal with their allergic reactions. cow's milk as well as cow's milk products are tolerated. material and methods: skin prick tests with different sorts of milk, cheese and milk proteins were performed, specific ige antibodies were measured, a basophil activation test with cow's and goat's milk was performed and an oral provocation test (opt) with cow's milk, cow's milk cheese, raw milk and raw milk cheese was conducted. results: skin prick tests were positive for sheep's milk, goat's milk, goat's milk casein, feta, pecorino and parmesan cheese. elevated specific ige against goat's milk ( . ku/l) and sheep's milk ( . ku/ l) were detected. activation of basophil granulocytes after incubation of the patient's blood with cow's milk and goat's milk was measurable but also in the non-incubated control blood. all cow's milk products were tolerated in the opt. conclusion: despite consistent homologies between whey and casein proteins of mammals and high cross-reactivity between cow's, goat's and sheep's milk an isolated goat's and sheep's milk allergy with tolerance of cow's milk is possible. skin testing and specific ige help to distinguish from allergy against cow's milk proteins. diet counseling is possible after opt. introduction: salmon roe's allergy, without concomitant fish allergy, is rarely described in western countries. there are few studies on its allergenicity. objective: to report of a case of salmon roe allergy without concomitant fish allergy in a western country. case report: a -year-old male with house dust mite allergic rhinitis and asthma, describes for the st time, in , an acute episode of dyspnoea, rhinorrhoea, ocular pruritus, epigastric pain and nausea, a few minutes after the ingestion of a sushi meal with rice, salmon, salmon roe, wasabi, soy and ginger. these complaints motivated observation in the emergency room, where it was still documented uvular oedema. he was prescribed intramuscular adrenaline, intravenous steroids and anti-histamines with complete symptoms resolution. the patient declares not had eaten other foods, taken any drugs including nsaid, been infected or practised exercise. skin prick tests with food extracts (salmon and other fish, shellfish, soy, rice, egg total, egg white, egg yolk, ovalbumin and ovomucoid) were negative. skin prick-prick tests were positive for salmon roe ( × mm) and negative for egg (white and yolk), ginger, salmon, flying fish roe (tobiko), sturgeon roe (caviar) and black scabbard fish roe. specific-ige (sige) to salmon roe extract was . kua/l (immu-nocap-phadia) and negative against extracts from salmon fish and other fish (< . kua/l). sds-page immunoblotting with salmon roes extract showed a kda-ige binding band, that may correspond to a lipovitelin. after the allergic reaction the patient have tolerated abstracts | salmon fish and other fish roes (tobiko, caviar and black scabbard fish). no oral provocation test with salmon roes was performed given the severity of the reaction. conclusion: this report is an example of a severe allergic reaction to salmon roe without concomitant fish allergy, where the clinical history and the in vivo and in vitro tests were important to an accurate diagnosis. the authors believe this is the first report of a salmon roe anaphylaxis in our country and highlight the importance of this allergen in the western countries, given the increase of sushi consumption in these countries. case report: the prevalence of food allergy is increasing worldwide and consumer habits are changing. pomegranate (punica granatum) was commonly consumed in the mediterranean area but in the last few years became also popular in the different parts of europe. a -year-old boy was admitted to our emergency department suffering from allergic reaction within minutes after consumption of pomegranate. he presented with skin pruritus, generalized urticaria and eyelid edema. symptoms resolved within an hour after oral intake of cetirizine. prick-to-prick test with pomegranate was positive (wheal diameter × mm). he had a history of anaphylaxis with egg (urticaria and wheezing) at the age of month, meanwhile consumption of egg is well tolerated. an episode of anaphylaxis with unknown origin appeared at the age of years (urticaria, wheezing and abdominal pain). skin prick tests with aeroallergens revealed birch pollen allergy and he was diagnosed with allergic rhinoconjunctivitis. dietary elimination of pomegranate was suggested and adrenaline auto-injector was provided. we have analyzed omalizumab effectiveness and safety in patients with csu from our database. clinical response was categorized as: no response, partial or complete response by using the urticaria activity score (uas ). furthermore, the dosage, administration frequency and any side effects were recorded. results: effectiveness: out of patients ( %) achieved complete response. ( . %) after a single dose of mg ( patients) or mg ( ); patients ( . %) after two doses of mg ( patients) or mg ( ) results: multi-ethnic adolescents accounted for approximately . % of the total sample of adolescents. prevalence of asthma was significantly higher in multi-ethnic group than non multi-ethnic group. we examined if maternal or paternal foreign born status had a differential effect: in multi-ethnic family with foreign-born father, prevalence of asthma was significantly higher. parental region of country at birth had a significant influence on the prevalence of asthma. adjusted logistic regression analysis was used to determine risk factors for occurrence of allergic disease. residential area, perceived household economic status, parental region of country at birth, and body mass index (bmi) had a significant effect on prevalence of asthma. conclusion: population admixing appears to have significant effect on the prevalence of asthma. further study will be needed to clarify the effect of population admixing on prevalence of allergic disease. several studies have aimed to explore the possibility of mirs as biomarkers for various diseases. in our study we examined six different mirs, previously shown to be involved in eosinophil development and other immune responses, in serum from non-allergic and allergic asthmatics and healthy control subjects in order to determine their potential ability to be used as biomarkers for varying forms of asthma. method: serum from healthy individuals as well as age matched non-allergic asthmatics (naa) and allergic asthmatics (aa) were utilized. additionally, the naas and aas subjects had high eosinophila (≥ . × cells/l) compared to healthy controls (≤ . × cells/l) and eosinophil cationic protein (ecp) in serum was measured. asthmatic subjects were included irrespective of inhaled corticosteroid usage. rna was extracted from serum, reverse transcribed and subjected to qpcr analysis. expression changes in six candidate mirs, mir- , - , - a, - , - , and - , were investigated. results: two mirnas, mir- and mir- a, were significantly upregulated in aas as compared to naa or healthy subjects. additionally, mir- was upregulated in naa, but not aa or healthy subjects. furthermore, the expression change observed in the aa mirs appeared to correlate with the use of inhaled corticosteroids, but not in the naa mirs. finally, mir- and mir- expression levels were altered based on the number of eosinophils, which correlated to ecp levels, in naa subjects. conclusion: using six mirs found in the literature to be involved in eosinophila or immune responses, we were able to detect expression changes in the serum of healthy and asthmatic individuals. moreover, were able to distinguish between healthy individuals, aas, and naas on inhaled corticosteroids or with differing eosinophil levels, leading to the possibility that these mirs may be valuable future biomarkers for asthma. background: some studies report that certain sensitization profiles may increase the risk of a more serious allergic respiratory disease. the aim of this study was to describe the sensitization patterns to major allergens of dust mites in our area and investigate the association of these patterns with a specific clinical picture. method: multicenter study performed in hospitals for months. we recruited patients older than years with rhinitis and/or bronchial asthma, with a history of allergy to dust mites and both skin test and specific ige to d. pteronyssinus, d. farinae or l. destructor positive. der p and der p were determined to all of them. we analyzed patients with an average age of . years, . % women, . % smokers, . % rhinitis and asthma, . % only rhinitis and . % only asthma. % of patients presented sensitization to d. pteronyssinus, . % to d. farinae and . % to l. destructor. the detected sensitization patterns were: both der p /der p positive . %; der p positive . %, der p positive . % and both der p /der p negative . %. it was observed that patients with higher specific ige levels had more severe forms of respiratory disease, with isolated asthma or associated with rhinitis. for d. pteronyssinus, d. farinae, der p and der p > ku/l there is a greater number of cases of asthma associated with rhinitis, while for l. destructor > . ku/l greater number of cases of asthma. no relationship was observed between a specific sensitization pattern and an increased risk of asthma. conclusion: . specific ige values greater than ku/l for d. pteronyssinus, d. farinae, der p and der p , were significantly associated with a higher probability of asthma and this association was significant for l. destructor> . ku/l (class ). . there are four well-defined sensitization patterns in our population that are influenced by geographic location, being the double sensitization to der p and der p the most prevalent and allowing the correct characterization of % of the cases. none of them increased the risk of asthma. kobori t ; nagao m ; ekenkrantz t ; borres m ; sjölander a ; fujisawa t national mie hospital, tsu-city, japan; thermo fisher scientific, uppsala, sweden background: reliable biomarkers for diagnosis and management of asthma in young children are needed since pulmonary function test and exhaled no measurement, good biomarkers for asthma in older children and adults, are difficult to perform in young age. we have developed a sensitive and stable assay system to measure eosinophil-derived neurotoxin (edn), an eosinophil granule protein, that is released upon activation, and that may serve as a marker for eosinophilic inflammation also in young children. method: volunteer children from - years old were recruited and an isaac-based questionnaire was filled out by their caregivers. venous blood was obtained to measure serum and plasma edn and eosinophil count. edn was measured with a research assay developed on the immunocap ® platform. conclusion: blood edn may be a reliable biomarker for diagnosis of asthma in preschool children. conclusion: feno measurement as an add-on option in asthma management to identify asthma patients with th driven airway inflammation is less costly than the use of standard diagnostic methods. new biologics may have an additional impact on overall asthma treatment costs. our model demonstrates that incorporating feno measurement may help to optimize asthma medication and reduction in physician visits as well hospitalizations due to severe exacerbations. | peripheral airway inflammation assessed by fractional measurement of the exhaled breath temperature is a leading feature of asthma background: airway inflammation is considered to be a hallmark of asthma. the potential clinical benefits of assessing it non-invasively has led us to develop a method and device for measuring the temperature of the exhaled breath (ebt=exhaled breath temperature) reflecting the thermal state of the airway mucosa. studies have demonstrated that ebt is increased in asthma, proportionately to the level of control of the disease. in an attempt to further increase the usefulness of this approach, we have further developed a device to allow the assessment of the relative contribution of the central and peripheral airways (caw and paw). now we present the frebt data gathered from patients with suboptimal control of their asthma and from healthy subjects. method: in this cross-sectional study we included volunteers: patients with suboptimally controlled asthma of mild to moderate severity (median age , range - years, men) and nonsmoking subjects without respiratory disease (median age , range - years, men). we measured the fractions corresponding to caw and paw sampled with a fast reacting inflatable balloon valve system operated by a computer during a single breathing cycle. it allows steering of the expired airflow through channels with sensitive temperature sensors. during an initial deep inhalation, the inspired volume is measured and the sequence of valve openings is adjusted so as to yield volumes of air characteristic of caw or paw during expiration. the ratios between [pawebt-cawebt] over the total ebt [%] measured during the same manoeuvre (fractional ebt, frebt) were calculated and compared between asthmatics and controls. results: there was high statistically significant difference between the frebt ratios of asthmatics and controls: . ± . (mean ± sem) vs . ± . , p < . . as the magnitude of the ratio depends on the difference between pawebt and cawebt, higher values of the frebt ratio point to bigger contribution of the peripheral lung tissues, presumably indicative of peripheral inflammation. multiple regression analysis with frebt ratio as dependent variable identified only asthma diagnosis as significant predictor (p < . ) and excluded all other anthropometric indices. conclusion: peripheral airway inflammation assessed by frebt measurement appears to be a leading characteristic in asthmatics compared to healthy subjects. method: we examined outpatients ( % male, aged - year, mean age . years) with severe asthma according to ers/ ats ( ) definition treated with high dose of ics/laba± tiotropium, antileukotrienes and omalizumab. some patients (n = ) had orally steroid-dependent asthma. they referred to our secondary care center by gps. pulmonary function tests were measured by dry spirometer ( , vitalograph ltd., uk). skin prick tests or serum specific ige to common inhalant allergens (house dust mite, animal dander, pollen) were used to assess atopic status. results: seventy five percent (n = ) of patients with severe asthma had fao in % of those was diagnosed concomitant copd. duration of asthma was . years in patient with reversible airway obstruction (rao) and . years in those with iao (p > . ). early (before age years) onset of asthma was established in % of patients with rao and in % of patients with fao (p > . ). prevalence of atopy did not differ between both groups ( % vs %, p > . ) but total ige level in serum was higher in severe asthmatics with rao than fao ( me/ml vs me/ml respectively, p < . ). most of atopic patients with severe asthma both with fao ( %) and roa ( %, p > . ) were sensitized to house dust mites (d. pteronyssinus and d. farinae). hypersensitivity to pollen was diagnosed in % patients with foa and in % with rao (p > . ), to cat and dog dander in % and % respectively (p < . ). the majority ( %) of patients with severe asthma had fao. hypersensitivity to house dust mites was most common in severe atopic asthmatics with foa and roa where as sensitization to animal danger was associated with presence of foa. | loss of smell as a clinical marker of severe asthma and its association with upper airway inflammatory diseases background: asthma is frequently associated with rhinitis and chronic rhinosinusitis (crs) while severe asthma is more associated with crs with (crswnp) than without (crssnp) nasal polyps. loss of smell (los) is associated with crs, mainly with crswnp. we aimed to assess loss of smell as a clinical marker to discriminate crs from rhinitis and severe from non-severe asthma. method: in a cross-sectional multicentric study, asthmatic patients (n = ) were evaluated by pulmonologists and ent specialists using gina, aria, and epos definitions. los was evaluated by severity [vas scale, - mm, median (iqr,inter-quartil range)] and by prevalence of anosmia (hyposmia vas > - mm, anosmia vas> mm). results: los was present in . % of asthmatics (hyposmia . %, anosmia . %). los was more severe [ mm ( - ), p < . ] and anosmia more frequent ( . %, p < . ) in severe persistent asthma than in moderate [ mm ( - ); . %] mild [ mm ( - ); . %], or intermittent [ mm ( - ); . %] asthma. in addition, los was more severe [ mm ( - ) vs mm ( - ), p < . ] and anosmia more frequent [ . % vs . %, p < . ] in crs than in rhinitis patients. in those asthmatic patients with crs, los was even more severe [ mm vs mm ( - ) p < . ] and anosmia more frequent ( . % vs . %, p < . ) in crswnp than in crssnp. conclusion: loss of smell and specially anosmia may clearly discriminate severe from non-severe asthma and crs (specially with np) from rhinitis alone in asthma patients. thus, los may be considered a significant clinical marker of severe asthma and its association with upper airway inflammatory diseases. | last station in the eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis march: eosinophilic asthma with radiological findings associated with blood eosinophilia yilmaz i ; nazik bahçecioglu s ; türk m ; tutar n ; oymak fs ; gülmez i erciyes university school of medicine, kayseri, turkey; department of chest diseases, kayseri, turkey; division of immunology and allergy, kayseri, turkey background: eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis (eacrs/np) is a subphenotype of adult-onset eosinophilic asthma. blood eosinophil levels are shown to be highly elevated in patients with ea-crs/np and have potential for tissue infiltration. we aimed to demonstrate the clinical features of the patients who have a blood eosinophil level above % and have thorax computed tomography findings due to blood eosinophilia. results: we identified patients who met the above criteria. we defined this group as "eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis with radiological findings related to blood eosinophilia" (earr). the mean age was . ± years and % was female. nasal polyps, aspirin exacerbated respiratory disease and atopy was present in %, % and % of the patients, abstracts | respectively. the mean blood eosinophil count was . cells/mm ( %). the majority of earr patients had upper lobe dominant ground-glass opacities. the mean follow-up period was . ± . years. earr patients did not evolve into eosinophilic granulomatous polyangiitis in the follow-up. method: we aimed to identify features more probably associated with asthma in a unselected group of patients with diagnostic criteria for aco. we consecutively selected the first consecutive patients with diagnostic criteria for aco. all patients were evaluated by accurate clinical history interview, assessment of asthma control test (act) and copd activity test (cat), lung function and exhaled nitric oxide (fe no ) measurements, sputum cytology, blood eosinophil count, serum total ige and periostin levels, methacholine and adenosine-mono-phosphate (amp) bronchial challenges. all these measures were repeated after an oral corticosteroid (ocs) trial of methylprednisolone mg/day for days. we defined parameters that we expected improved after the ocs trial, and therefore considerable as markers of asthma: fev , fef - , fev /fvc, fe no , act, sputum and blood eosinophilia, methacholine and amp challenges. patients with improvement of at least of these parameters after ocs trials were defined as "responders" to the treatment, and therefore more likely to be asthmatic than copd or aco. results: five ( %) patients were classified as responders and they were characterized by having basal higher fe no values ( . ± . vs . ± . ppb, p = . ), greater bronchial reversibility basal values of serum periostin and total ige, and blood eosinophils were higher in responders but without reaching the statistical significance. conclusion: fe no and the degree of bronchial reversibility (and possibly also the degree of response to an amp challenge) are reliable biomarkers to distinguish asthmatics among those with suspect aco. method: the preliminary case-control study included obese persons with asthma who were matched for age and sex and nonobese asthma subjects. non fasting serum levels of adiponectin, and leptin were measured by commercially available immune assay kits, and routine biochemical parameters were analyzed in both the study groups. the results show statistically significant lower levels of serum adiponectin and higher serum leptin levels in obese asthma subjects with respect to non-obese asthma patients (p < . ). moreover, an inverse correlation was also observed between serum adiponectin and serum leptin in obese asthma subjects (p < . ). our results indicate the association of these hormones might act as a significant predictor in the progression of asthma. moreover, the role of serum adipokines is promising and might potentially act as a meaningful drug target in the pathogenesis of asthma. background: overweight/obesity is known to be a possible factor for poor asthma control. the aim of the study is to determine the serum concentrations of leptin in atopic asthmatic patients and its relationship with body mass index (bmi), asthma severity defined by medical treatment and asthma control defined by the asthma control test (act). method: we randomly selected adult patients previously diagnosed with allergic asthma based on gina (global initiative for asthma) guidelines, returning for follow-up to an outpatient allergy/ immunology clinic during november . following an informed consent, the patients were asked to fill act, their bmi was recorded, and elisa blood assays for leptin were drawn. exploratory data analysis, spearman's correlation ( % ci by bootstrapping), and partial correlations were performed. results: female/male ratio was / , mean bmi was . ± . conclusion: leptin was significantly associated with overweight/ obesity in asthmatic subjects, and showed higher values for women. leptin inverse correlation with act did not reach statistical significance, likely owing to underpowered estimates, in a small sample characterized by an elevated mean bmi and severe allergic asthma. background: immunoglobulin lowering may be associated with recurrent wheezing symptoms and clinic by increasing the tendency to viral respiratory tract infections. in this study, it was aimed to investigate the frequency of immunoglobulinemia in preschoolers with wheezing. the study was conducted between . . and . . between. university of health sciences, ankara child hospital, the children allergy and immunology clinic included patients who had been followed up and treated for at least one year with recurrent wheezing attacks within younger than months. the immunoglobulin (g, a, m) values of the patients were retrospectively analyzed. immunoglobulin levels were determined to be normal and low according to age limits. the study included patients ( . % male, . % female) under the age of years with a mean age of . months. the mean follow-up period of the patients is . years. in . % of these patients, at least one immunoglobulin was found to be low. none of these patients had any signs or symptoms of immunodeficiency. immunoglobulin a was low in % of the patients, immunoglobulin g in %, and immunoglobulin m in . % of all patients. conclusion: immunoglobulin was found to be low in these patients when there was no immunodeficiency and preschool wheeze was diagnosed. this should be etiologically investigated as to whether if this is a special group in preschoolers with recurrent wheezing and hypogammaglobulinemia combination. method: asthma severe unit is formed by allergists, pneumologists, pediatricians and otorhinolaryngologists. hematologists and immunologists make specific collaborations. we present the partial results of our data collection, which include patients with severe asthma according to ers/ats task force, selected by peripheral eosinophils > according to wagener et al. we followed them up to assess control for year. we obtained cellularity in sputum using induced sputum technique. values of il of th , th , th pathway, periostin and ilc were not yet available. results: median age was ± , feno ± , exacerbations previous year . ± . , act . ± , fev % ± and dose of inhaled corticoids (budesonide equivalent) ug ± . most of the patients were sensitized ( %) and . % were polysensitized. the most frequent sensitization was dust mites ( %). % had received immunotherapy of whom . % with lack of response. not sensitized patients were older. sputum cell analysis of patients was performed, % had sputum eosinophils> %, mean sputum eosinophil value was . ± . and peripherally ± . correlation among sputum and peripheral eosinophilia was . (p = . ). the peripherally eosinophil value > had a sensitivity of % and a specificity of % for the detection of sputum eosinophils > %. no differences were observed in sputum cell count depending on allergic sensitization. % had an uncontrolled asthma. presence of polysensitization, rhinitis or polyposis were not statistically related with the control. different patterns were observed in function of cause of poor control: patients with obstructive pattern (fev < %) were older and received more inhaled treatment. patients with high rate of exacerbations had more sputum eosinophilia and neutrophilia. both groups had worse act and received more oral steroids. patients who received oral steroids were more often sensitized to fungi in some follow-up visits. not significant differences were observed in control according to the act. asthma had more sputum neutrophilia, were older, received higher inhaled steroids dose and had adult onset asthma. the only control variable related with sputum eosinophilia was exacerbation. fungi sensitization was more frequent among patients with oral steroids. method: a randomized, double blind, placebo controlled study with patients from the de la salle university medical center with a mean age of with partly or uncontrolled asthma. they were assigned to either cm glucan or placebo group for two months. act score and %fev postbronchodilator were assessed at the st visit, th week follow up, and th week follow up visits. an independent and paired t-test were used to determine mean changes in act scores and %fev between the groups. results: in the two treatment groups, those in the cm glucan group had a greater % fev mean change of − . compared to placebo which had only − . , a mean difference of − . , and a trend toward significance with a t-test p value of . . in terms of changes in act score, those in the cm glucan group had a mean change of . and . for placebo, a mean difference of . and was not significant at t test p value of . . the result of the emanuel trial showed a trend of improvement among patients on both groups in terms of act score and %fev postbronchodilator. however, it was not statistically significant. | lung function improvements with tiotropium in patients across all ages: impact of episodes of asthma worsening during phase trials vogelberg c ; casale tb ; bleecker er ; goldstein s ; szefler s ; engel m ; el azzi g ; dewberry h ; hamelmann e method: post hoc analyses involved phase iii, randomized, double-blind, placebo-controlled trials: in patients aged - years (rubatina-/canotina-/vivatina-/pensietina-asthma) who received tiotropium ( or . μg) or placebo, as two puffs once-daily via the respimat, as add-on to ics ± other controllers; and in adults (pri-motina-asthma replicate trials) who received once-daily tiotropium μg or placebo, as add-on to ics/laba ± other controllers. we analyzed change from baseline for peak fev ( - h) and trough fev at week in vivatina-and pensietina-asthma, and week in pri-motina-/rubatina-/canotina-asthma, comparing patients with and without episodes of asthma worsening during the trials. asthma worsening was defined as an episode of progressive increase in dayto-day asthma symptoms (recorded by patients and confirmed by the investigator) or a decrease of patient's best morning pef ≥ % from mean for ≥ consecutive days. as a post hoc analysis, p values are nominal. results: there were no differences in baseline disease characteristics between those who experienced episodes of asthma worsening and those who did not, within specified age and asthma severity groups. placebo-adjusted lung function improvements were observed with tiotropium μg in patients who experienced episodes of asthma worsening and those who did not during the trials (table ) . there was some variability in subgroups with low numbers of patients. conclusion: once-daily tiotropium add-on had a similar efficacy in adult and pediatric patients with symptomatic asthma, irrespective of whether they experienced episodes of asthma worsening or not during the trials. these data support the broad efficacy of tiotropium and show largely consistent improvements in lung function even in patients who experience episodes of disease worsening. | cochrane review of the use of antibiotics for acute exacerbations of asthma method: we searched the cochrane airways trials register, trial registries and reference lists of primary studies. we extracted outcome data and assessed risk of bias in duplicate and used current cochrane methodology throughout. our primary outcomes were intensive care unit (itu) admission, duration of symptoms/exacerbation and adverse events. we included six studies, including a total of adults and children. trials were of varied methodological quality and we were able to perform only limited meta-analysis. one study reported a single itu admission but no other studies reported admissions to itu. two studies investigating macrolides reported diary card symptom score and showed antibiotics improved symptoms (md − . , % ci − . to − . ). one study including participants reported more symptom-free days in the macrolide group than usual care. one study of a penicillin including participants reported asthma symptoms at hospital discharge; the between group difference was reported as non-significant. serious adverse events were rare; events were reported across the three trials (n = ). the pooled effect estimate for all adverse events from three studies was imprecise (or . , % ci . - . ). no deaths were reported. conclusion: our results confirmed that omalizumab significantly improves disease control and is a safe add-on therapy. also in appropriate patients with controlled disease over time, efforts to stepdown other asthma medications will be appropriate. ( ) aerd: aspirin exacerbated respiratory disease; sd: standard deviation. data are n (%), mean ± sd or n/n (%). c-act: asthma control test for children, fev : forced expiratory volume in one second; fev /fvc: the ratio of forced expiratory volume in one second to forced vital capacity, pef: peak expiratory flow; feno: fractional exhaled nitric oxide, vas: visual analogue scale *these included allergic rhinitis, asthma, eczema, atopic dermatitis, food allergy, etc. **there were , and missing data in treatment a, b, and c, respectively. this study examines the potential treatment effects of sq ® hdm slit-tablet on qol measured by sf- v in people with aa and ar. the analyses are based on data from the mt- trial (eudract no. - - ) and utilize data from the sq-hdm treatment group ( subjects) and the placebo group ( subjects). throughout the trial, qol was measured at each of visit - via sf- v . this yielded psychometrically-based physical and mental health summary measures, as well as a sf- v total score. according to trial design, the use of inhaled corticosteroid (ics) was reduced by % for a three months period (visit and ) and completely withdrawn for the last three months of the trial (visit and ). results: by estimating a simple regression on differences in sf- v total score from baseline measurements (visit ), a positive and statistically significant treatment effect on the overall qol of the sq-hdm treatment compared to the placebo group in visit and was found. further analyses show that the qol improvements are mainly driven by increases in the general mental health score, which are carried through to visit . in particular, the mental health and role emotional domains show statistically significant improvements. the results show that the sq ® hdm slit-tablet improves qol measured by sf- v in patients with hdm induced aa and that this effect is driven by improvements in the mental health domains. | impact of treatment prescription, adherence to treatment and use of inhalers in asthma control-results of the efimera study method: cross-sectional multicenter observational study conducted with patients who use any type of medication with inhaler devices. patients referred from primary care and seen by a pneumologist or allergist for the first time were evaluated. the following data was collected in a single visit: adequate prescription according to gina guidelines (gina); specific and general treatment adherence using morisky-green questionnaire (mg) and inhaler adherence test (tai); disease control with asthma control test (act) and assessment of inhaler use technique were measured with the extended tai. results: patients included in this study (n = ) had a mean age of ± years, an average disease evolution of . ± . years, % of which were women. according to gina recommendations, . % of patients have insufficient or inadequate prescription. when measured by the mg test the . % of patients showed bad adherence, meanwhile measured by the tai test adherence was . % measurements of inhaler use technique resulted in % of patients having one or more mistakes regardless of whether the device was a mdi or dpi. several factors showed to be related with bad asthma control: inadequate prescription (or: . [ . - . background: it is well known that the constant and prolonged tobacco smoking affects the natural history of asthma. vaping is the act of inhaling and exhaling the vapor produced by an electronic device called e-cigarette (e-cig), whose basic structure includes a power source and an atomizer. two types of vaping are the most popular ("mtl" and "cloud chasing"). we have created a web-survey with questions concerning epidemiological data, quality of life and symptoms worsening in asthmatic vapers. the survey has been advertised through various social networks and local press. people responded, including asthmatics ( %). the asthmatics were: males %, under %, - years %, - years %, - years % and over %. % used ecig-only, % smoked and vaped together, %. those who preferred mtl-type of vape were % and "cloud chasing" were %. results: to the question: "has vaping ever worsened asthma symptoms?" % answered no, % yes. to the question: "as asthmatic, would you suggest to an asthmatic smoker to start vaping instead of smoking?" . % answered no, . % yes. to the question: "how much nicotine do your vaping liquids have?" % answered mg/ml, % . mg/ml, % mg/ml, % mg/ ml, % mg/ml, % mg/ml and % mg/ml. to the question: "do you take medications for your asthma?" % declared to use a drug as needed, % used a single drug daily, % used more than one drug daily and % declared "i don't take any asthma medication". we related (χ test) the worsening of asthma symptoms with the nicotine content (p = . ), the type of vaping (p = . ), the current therapy (p = . ) and we did not find a statistically significant correlation. vaping has undoubtedly shown an advantage in terms of improvement of symptoms compared to cigarette smoking (p = . ), in particular . % subjects who smoke and vape did not have a worsening of symptoms, while . % of them had a worsening. the vaper-only users who never worsened were ( . %) and ( . %) had a worsening. conclusion: despite the limits related to the online survey as a data source, e-cigs seem to be a useful tool in the pathway to quit smoking. in fact, % of the asthmatics who smoked traditional cigarettes would recommend switching to e-cig and % did not worse their asthma symptoms. background: despite the success of pharmacotherapy, more than half of patients with persistent bronchial asthma (ba) do not achieve disease control. in recent years, the issue of approaches to treatment based on the identification of phenotypes of the disease has been increasingly discussed. this approach becomes the key to optimizing therapy for asthma, allowing the personification of treatment. anti-ige-therapy using omalizumab is one of the most researched variants of phenotype-specific treatment. method: aim of our study was to investigate of the causes of uncontrolled predominantly atopic asthma, the frequency and effectiveness of the personalized therapy in real clinical practice. patients with uncontrolled severe atopic asthma were examined in outpatient department of the city hospital during . all patients underwent physical examination, pulmonary function testing, and total serum ige evaluation. results: % of patients had uncontrolled asthma due to inadequate basic therapy of the disease. the change in therapy allowed them to achieve control of the disease. obstructive sleep apnea syndrome (osas) was revealed in . % of patients. these patients underwent cpap (continuous positive airway pressure) therapy. % of patients had gastroesophageal reflux disease (gerd). % of patients had an elevated level of serum ige level and needed anti-ige therapy. in . % of cases, the initial serum ige level was more than iu/ml which was a contraindication to therapy of omalizumab. patients received omalizumab therapy. this therapy led to relief of symptoms and decreased frequency of asthma exacerbations. results: it was found that the prevalence of obesity among the patients with asthma and being treated in inpatient conditions in - was . % of patients, which is comparable to the prevalence of obesity among the population in general. the data of the patients suffering from asthma and obesity treated both in inpatient and outpatient conditions, was analyzed and it is set that obesity does not affect the severity of the clinical course of asthma. it is shown that obesity does not affect the control of symptoms of asthma. thus, the control of asthma symptoms depends on timeliness of diagnosis, the adequacy and terms of appointment of basic asthma therapy, the presence, severity and adequate treatment of concomitant diseases, psychoemotional background of patients, their compliance and adherence to therapy. results: the causes of smoking in asthmatics were not significantly different from the control (p > . ). patients most often used smoking as "support for emotional stability". the motivation to smoking cessation was higher in the asthmatics group ( %) than in the control group. the main reason for smoking cessation was a deterioration in health - %. the majority of smokers - %, performed attempts for smoking cessation. low level of br was revealed in % asthmatics ( % non-smoking asthma patients and . % of cases in the control group, p < . ), cf had low values and was lower in asthmatics group in compare to the control group (p < . ). the pac values correlated with the level of br: a low level was determined in % in smoking asthmatics, in % in nonsmokers with asthma and . % in smokers of the control group obstructive sleep apnea (osa). all of the patients were on regular treatment with low dose inhaled corticosteroids for at months and start treatment with continuous positive airway pressure (cpap) .to assess quality of life, we used asthma symptom control tools (asthma control test) .patients performed daily peak flow meter and spirometry (once a week) during period of weeks after start using cpap. results: during the study, of the followed patients had no exacerbation of asthma. four of patients during this period had exacerbation, due to upper airway infection so they were excluded from study. results of following showed that there was improvement in quality of life in all patients included in study but there no statistically significant improvement in pulmonary function tests fpt. huang y ; yao t ; huang y ; chiu c ; tsai z ; kao p ; lu k ; fang h ; lin c ; gau c ; lee w ; tsai h results: the rate of preterm birth among the study subjects was . %. the prevalence of physician-diagnosed rhinitis was . %. there was no significant association between preterm birth and physician-diagnosed rhinitis (p = . ). when stratifying by atopy status, we found that preterm birth was associated with physiciandiagnosed rhinitis among children without atopy (adjusted or [aor] = . , % ci = . - . , p = . ), but not among children with atopy (p = . ). when further classifying by gender, greater protective effect of preterm birth on rhinitis was only found in boys without atopy (aor = . , % ci = . - . , p = . ). the results suggest that preterm birth may have a protective effect against the development of childhood rhinitis in our study population. the protective effect is only observed in boys without atopy. further investigations will be merited to confirm these findings and to investigate underlying mechanisms. background: folic acid supplementation (fas) during pregnancy has been suggested due to its protective effect against neural tube defects. at present the effect of fas during pregnancy on childhood rhinitis has remained unclear. we aimed to investigate the relationship between fas during pregnancy and childhood rhinitis. logistic regression analysis with covariate adjustment was performed. adjusted covariates included sex, age, number of older siblings, breast feeding duration, maternal smoking during pregnancy, maternal allergy, maternal education level, maternal age and socioeconomic status results: the prevalence of physician-diagnosed rhinitis was . %. there is a significant association between fas and physician-diagnosed rhinitis (adjusted odds ratio [aor] = . ; % confidence interval [ci] = . - . for fas ≥ months) compared to the group of never use. in the stratified analysis by atopy status, maternal fas during pregnancy was significantly associated with physician-diagnosed rhinitis in the atopic group (aor = . , % ci = . - . for fas < months; and aor = . , % ci = . - . for fas ≥ months), but not in the non-atopic group. when further stratified by gender, significant association between maternal fas during pregnancy and physician-diagnosed rhinitis was only found in boys with atopy (aor = . , % ci = . - . for fas < months; and aor = . , % ci = . - . for fas ≥ months). the results demonstrate that maternal folic acid supplementation during pregnancy might increase the risk of childhood rhinitis, especially among boys with atopy. further investigation will be needed to validate our findings and to understand potential underlying mechanisms. according to sequence data from detected adv (in all groups of patients) belongs to species f type and samples to species c type (rei group). bv type was identified in strongly positive (ct ≤ ) swab samples in ari group. conclusion: simultaneous testing of respiratory and stool samples together shown that at least . %/ . % of study subjects had dual/mixed infections, respectively, including %/ . % of respiratory disease patients, . %/ % of gastroenteritis patients and . %/ . % of patients with combined respiratory/enteric infections. we found no virus combination specific for different groups of patients. | neonatal respiratory supports and future asthma-like presentation in prematurity with bronchopulmonary dysplasia results: of all the tests analyzed . % were males and . % females with a mean age . ± . years old. half of the tests ( . %) reveals positive specific-ige to more than one allergen and . % ( ) have no serum specific-ige for the tested allergens (table ) . sixteen patients ( . %) have very high concentrations (> iu/ml) of derm. pteronyssinus specific ige, ( . %) of derm. farina, ( . %) of rey pollen and ( . %) of oak and timothy grass pollen. further studies are needed in order to elucidate the effect of these cytokines on allergy development and protection. shinohara m ; matsumoto k department of pediatrics, ehime university hospital, toon, japan; department of allergy and clinical immunology, national research institute for child health and development, tokyo, japan background: probiotics consumption during perinatal and postnatal periods reportedly reduces the risk of atopic dermatitis in the offspring, whereas such probiotics consumption did not affect ige levels or the risks of other allergic diseases; the precise mechanism how probiotics consumption reduces the risk of atopic dermatitis remains unknown. we hypothesized that probiotics consumption may reduce skin hypersensitivity to histamine. to test this hypothesis, we investigated whether perinatal/postnatal consumption of yogurt associates with skin hypersensitivity to histamine or not. method: this was a cross-sectional study enrolled motherinfant (≥ -months-old) pairs. physician-diagnosed allergic diseases and food consumption, such as milk, fermented drinks, and yogurt, by mothers during the third trimester of pregnancy and by infants during the first months of life were assessed using self-questionnaires. skin prick tests (spts) to saline and mg/ml histamine were performed using bifurcated needles, and wheal sizes were measured minutes after the puncture. the spt wheal sizes in infants with eczema/atopic dermatitis (n = ) were significantly larger than those in infants without eczema/atopic dermatitis (n = ; . ± . mm vs . ± . mm, respectively, p = . ), and thus these infants were excluded from the further analyses. the spt wheal sizes to histamine in infants with daily yogurt consumption during the first the aim of this study was to evaluate the prevalence and clinical relevance of sensitization to profilins in atopic patients with food allergy. the study was performed on a group of children age - years with sensitization to at least one plant-derived food allergen (ige > . ku/l). the included patients had never been treated with allergen immunotherapy before the study. the presence of ige to recombinant (r) rbet v , rart v and ramb a in serum was evaluated using elisa method as previously described (jbc ; : ) . in addition serum level of igg to rbet v , rart v and ramb a was also evaluated. results: sensitization to profilins was found in out of ( . %) patients (p+). sensitization to all studied profilins was demonstrated in each p+ patient. the remaining children, with pollenfood sensitization, were not sensitized to any of the studied profilins and they served as a comparator group (p−). analysis of the clinical status revealed that asymptomatic patients in regard to plant-derived food hypersensitivity were found more frequently among p+ ( %) than p− ( . ; p < . ) patients. sensitization to profilin was associated with positive ige to the same food allergens as in the control group. clinical manifestation of pollen-food sensitization expressed as allergic rhinitis, bronchial asthma and atopic dermatitis was comparable between groups, except of oral allergy syndrome, which was not seen among p+ children. similarly, history of anaphylaxis to plant-derived foods was registered only among p− ( . %) patients. interestingly, all patients with sensitization to profilins had also elevated level of serum igg against rbet v , rart v and ramb a . results: no significant difference of physician-diagnosed eczema (p = . ) or current eczema (p = . ) was observed between children born full-term and preterm. after stratifying by atopy status, we found that children born preterm had a more than three-fold higher risk of having physician-diagnosed eczema (adjusted or (aor) = . ; % ci = . - . ; p = . ) and current eczema (aor = . ; % ci = . - . , p = . ) than their counterpart in the non-atopic group. no statistical significance was observed for the association between preterm birth and eczema in the atopic group. no association between preterm birth and eczema was found when stratifying by gender. our results reveal that non-atopic children born preterm have a higher risk of developing eczema. the results suggest potential modifiable effect of atopy on the association between preterm birth and eczema. further studies with a larger sample size are needed to validate the findings in this study. background: there is a need for more knowledge about factors of importance for a successful transition from childhood to adulthood among adolescents with allergic disease and especially those with severe allergy. therefore the aim of this study was to describe experiences of living with severe allergy from the adolescents and their parent's perspective and thereby identify factors of importance for transition from pediatric to adult care. method: a qualitative study was performed based on six focus groups interviews, two with adolescents and four with their parents. in total adolescents (age - years old) and parents participated. the interview guide contained questions about experiences of living with severe allergy. the transcribed data was analysed using systematic text condensation. results: in total four themes were presented, two themes occurred in both the adolescent and the parent's focus groups, to be special and to be prepared. for two themes there was a difference between the adolescents and their parents. the theme, the importance of the parents, only occurred in data from the adolescents and the theme the meetings with health care only occurred in the parent's data. the adolescents felt that they had low priority in the class and several stated they were teased at school and their parents felt that focus on their child often was in a negative way. the adolescents described that they took responsibility for their diseases while their parents expressed a need to protect. the adolescents stated that one of the parents were always present or had been during the years, the reason being safety and security. only the parents mentioned experiences from healthcare. parents who described that they had continuity in healthcare meetings and where met by high competence and with a professional approach were more satisfied with the support from the health care. one factor that was felt to be important was whether the doctor involved the youth in the conversation or not. the teenagers in this study relied on their parents while also taking responsibility for their illness at the same time. parents, on the other hand, showed a tendency to overprotect their adolescents. for healthcare professionals it is important to involve the adolescents in the care to facilitate the transition. results: . % of the children used antibiotics currently and . % out of them used antibiotics ≥ times yearly. current wheeze (w) was established in . %, sleep-disturbing w in . %, exerciseinduced w in . %, dry night cough apart from a cold in . %, and asthma in . %. current antibiotics use ≥ times yearly was positively associated with current w (aor: . ; . - . ; p < . ), sleep-disturbing w (aor: . ; . - . ; p < . ), exercise-induced w (aor: . ; . - . ; p = . ), dry night cough (aor: . ; . - . ; p < . ), and diagnosed asthma (aor: . ; . - . ; p = . ) while antibiotics use < times yearly was positively associated only with current w (p = . ) and dry night cough (p = . ). the results suggest an aggravating role of antibiotics use on asthma in school age thus further supporting the recommended restriction of antibiotics exposure. results: after questioning . % % ci, . - . were suffering from respiratory diseases, having symptoms of chronic disease: cough- . %, wheezing- . %, tightness in the chest- . %. the risk factors (passive smoking, open fire house warming and no air conditioning) were commonly met in major cases at ill children rather than healthy ones ( . % % ci, . - . ). as a result of studies made of the equal to . ± . , comparative the end of lessons equal . ± . (p ≤ . ); air relative humidity varies during lessons equal with . ± . (norma toilet %- %); co concentration exceeds allowable limits − . ± . (mac − . %). conclusion: respiratory morbidity in high school examined has a tendency to increase. we noticed deviations from the hygienic norms: the indoor temperature and relative humidity was lower and the co level was twice higher than the normal one. the "asthma ever" outcome was reported in cohorts. cohorts defined this as parental reported asthma (with or without specifying that it was doctor-diagnosed), cohorts used gp records as the only source of diagnosis, and used parental report or gp records. the "current asthma" outcome was reported in cohorts. there was little consistency with how current asthma was defined or worded, with different definitions used. the most common definition of current asthma, reported times, was "asthma ever and either asthma symptoms in the last months or asthma medication in the last months". other criteria included in asthma definitions were bronchial hyper-responsiveness, reversible airway obstruction, positive exercise test, and asthma symptoms reported at a previous questionnaire. only one "current asthma" definition was based exclusively on prescription data: "dispensed two asthma medication during the past year". nine cohorts reported asthma outcomes without specifying how it was defined, and were categorized as "asthma unspecified". conclusion: "asthma ever" and "current asthma" are two main asthma outcomes used to define asthma in child cohort studies. definitions of asthma vary substantially across cohorts. case report: thereby we present two case reports of two children with impairment verbal communication as part of asd and allergic diseases. the first patient was a year old boy with sneezing, rhinorrhea night cough and eye redness. he had been suffering for almost years from the above mentioned symptoms. he had family history for atopic diseases and was for month breastfed. specific ige revealed sensitization to birch, alder, hazel, oak, mugwort pollen and dog epithelia and dermatophagoides farinae. specific ige resulted positive for nuts and rye flour. the second patient was almost year of age in the tame that he presented in our hospital. he cried and screamed all the time because of severe atopic dermatitis and typical symptoms such as itching all over the body and his impairment of verbal communication. specific sensitization showed sensibilization to egg white and egg yolk, to nuts, rye and wheat flour. the food specific ige leaded to positive results to alder, birch, hazel and oak pollen, but also to grasses, ragweed and mugwort. prick by prick test showed positivity to egg white and egg yolk. atopy patch test to pollens resulted negative. results: the first patient symptoms were well controlled after treatment with antileukotrienes. his verbal communication was also improved after a year or more. the second three year old patient after required a combination of specific treatment with antihistamines, corticosteroids, immunosuppressive drugs and diet recommendation. afterwards he had a reduced level of itching and anxiety but compared to other children he had a severe eczema. erythema multiforme (em) is an acute, immune-mediated, mucocutaneous condition that is most commonly caused by infection and drugs. it is characterized by targetoid lesions, sometimes accompanied by oral, genital or ocular mucosal erosions. there was no pediatric patient that had previously been reported in the literature with development of type reaction after omalizumab treatment. we presented a case who developed em to omalizumab therapy. an year-old female patient was admitted to pediatric allergy clinic with complaints of fever and rash. she had been diagnosed with chronic spontaneous urticaria (csu) years ago and she was planned to treat with omalizumab ( mg, subcutaneously every week) because of the inadequate response of antihistamines at a medical center. her complete blood counts, liver, renal, thyroid function tests and serum c ,c ,c esterase inhibitor protein levels introduction: celiac disease is an autoimmune disease triggered by exposure to gluten in genetically predisposed individuals and characterized by chronic inflammation of the small intestine. chronic urticaria is a skin disease, characterized by the appearance of pruritic wheals with or without angioedema, whose underlying mechanism cannot be identified. objective: to report a sporadic case of an -year-old boy with chronic urticaria associated with celiac disease. methods: an -year-old boy(weight kg, rd- th percentiles) was admitted to our clinic with a -year history of chronic urticaria. during the first three years, he was under antihistamine treatment(of incremental doses)and occasionally received preparations of cortisone according to the eaaci guidelines. he was asymptomatic for years until treatment was discontinued. eight months earlier, after a viral infection, a recurrence of urticaria, involving the trunk and extremities without angioedema was noted. subsequently, he was under antihistamine treatment with cetirizine but had an uas- score of . total laboratory investigations were performed. results: laboratory control was negative except for positive antibodies to celiac disease(anti-transglutaminase > u/ml, anti-endomysial, gliadin antibodies).further control with colonoscopy and biopsies (from duodenum and stomach) were obtained. the histopathological findings along with the clinical findings indicate celiac disease, type b marsch-oberhuber and grade b corazza-villanacci. in the past, similar cases have been reported. efforts have been made to associate chronic urticaria with celiac disease, although the mechanism remains unclarified. evidence suggests that the duration of gluten exposure, among otherwise asymptomatic patients with celiac disease, is related to the development of other autoimmune mechanisms. this can be explained by resolution of urticaria manifestations after the onset of gluten-free diet. in our case, three months after gluten-free diet, an improvement of urticaria with decreased uas- score of was observed. conclusion: he specific case of subclinical diagnosis of celiac disease in a child with chronic resistant urticaria further reinforces the suggestion that screening for celiac disease should be included in the diagnostic approach of chronic urticaria. | allergy to gingival balm in an infant with cow's milk protein allergy we report a case of an infant with a diagnosis of cmpa with an allergic reaction to a gingival balm caused by the presence of cmp in its constitution. furthermore, it is important to reinforce that milk proteins were labeled in an unusual form which might increase the risk of misunderstanding. these findings illustrate the difficulty in implementing total avoidance of common food allergens as well as the need to improve their labeling, particularly in non-food products. bakiri ah results: after specific treatment with corticosteroids, antihistamines, emollient creams, disinfectants and antileukotriens he was feeling better, he was smiling again and wished to have the chance to play with his classmates again. conclusion: this case report shows an association between level of stress and risk for atopic dermatitis. as previously showed children with low educational level parents and boys with higher stress have increased risk of having severe atopic dermatitis as compared to "no stress" boys. so early treatment and diagnoses are key important factors improving the children`s social life. results: the data cover immigrants (mean age . , range - ) and locals (mean age . , range - ). a slight difference in male prevalence ( . % vs %, p = . ), and pet possession ( . % vs . %, p = . ) were found between immigrants and locals, respectively. no differences were find in term of age and symptoms at presentation. the pattern of sensitization to the different allergens showed no statistically significant differences between migrants and controls. the rate of monosensitization resulted slightly higher in migrants ( . %) than controls ( . %). pollen-only sensitization was statistically higher among migrants than control ( . % vs . %, p < . ). monosensitization was more frequent among patients who have been living in italy for less than years ( . % vs %, p = . ). the opposite phenomena can be seen among polysensitized patients. conclusion: migrants are more frequently monosensitized than locals and tends to cluster towards either a pollen or dust mite sensitization. sensitization to house dust mite tends to appear early (< years of stay). pollen or mixed sensitization is more frequent the longer the residence time. | allergenonline.org: update of comprehensive allergen and celiac protein searchable databases for risk assessment of novel food proteins goodman re ; baumert jl ; taylor sl ; ebisawa m ; ferreira f ; bohle b ; van ree r ; kleine-tebbe j ; abdelmoteleb m ; koning f ; amnuaycheewa p conclusion: allergen and cd databases have been updated following a described review process. they can be used to identify proteins that might represent risks of food allergy or cd for affected consumers. han dh ; lee jw ; yim hj ; ko yk ; kim d ; rhee c seoul national university hospital, seoul, south korea; seoul national university bundang hospital, seoul, south korea background: stress can change the immune response and aggravate various allergic diseases. we already demonstrated in previous allergic rhinitis cohort (arco) kids study that stress might be a risk factor for pediatric allergic rhinitis (ar). the aim of this arco study is to investigate relationship between stress intensity, symptoms severity and quality of life as well as allergic markers in adult ar patients. results: as stress intensity increased, the proportion of moderatesevere ar patients was significantly increased. ar patients in high stress group was likely to belong to moderate-severe group (or, . ; % ci, . - . ). global vas of ar symptom was . ± . in high stress group and . ± . in low stress group, respectively. the each rqlq domain score was significantly higher in high stress group than in low stress group. total rqlq scores were . ± . in high stress group and . ± . in low stress group, respectively. however, as the level of stress increased, there were no significant changes in serum levels of allergic markers. our results suggest that stress may affect ar symptom severity and quality of life in ar patients. | skincare and synbiotics for the prevention of atopic dermatitis or food allergy in newborn infants: a × factorial randomized non-treatment controlled trial dissanayake e ; tani y ; sahara m ; mitsuishi c ; nagai k ; sato y ; suzuki y ; nakano t ; yamaide f ; shimojo n (n = ). the skin care group was advised to apply an emollient - times/day especially on cheeks and peri-oral area. the synbiotics group consumed a mixture of fos ( g) and bifidobacterium bifidu-mol ( × )/day. the last group received both. emollient application was not prohibited in the no-intervention group. interventions were carried out from birth to months of age. the development of ad was assessed at month, months and months by a pediatrician and at year by a questionnaire. ad was diagnosed using guidelines of the japanese society of dermatology. sensitization to food allergens was assessed by allergen-specific ige levels at months of age. results: skin care and synbiotics, alone or in combination, did not prevent the development of ad at year of age or the sensitization to food allergens at months of age. conclusion: our data suggest that skin barrier protection using emollients may be insufficient to prevent the development of ad as other factors affecting skin barrier integrity and trans-epidermal water loss such as the method of skin washing may have an additional effect. the probiotic bacterial species used may also affect the outcome as lactobacilli have been shown to be more beneficial. more studies are required to confirm the effects of skin care and synbiotics on ad. results: in the population number of girls exceeded the one of boys (p < . ), especially within the age group from to years. questioning, for months, symptoms of allergic rhinitis (rhinorrhea, sneezing, nose itch, nasal obstruction and eyes' itch) were identified in . (p < . ); symptoms of bronchial asthma (wheezing ( %), episodes of cough at night ( . %), intolerance to physical load ( . %), indoor and outdoor ( . %), coughing and rales in response to stimulus ( . %)) in . % of the population; atopic dermatitis (dermatitis, itch, revelation in early age, involvement of large areas in early age, damage of extremities bending and stretching surfaces in adults)- . % (p < . ); food allergy- . % (p < . ) etc. at the second stage of clinical studies, on the basis of prick-testing, average ige, in our case, was - times greater than normal level. results of study of allergens showed sensibilization to domestic dust (d.f. and d.p.) ( , %) (p < . ). in . % of cases there was stated sensibilization conditioned by cat and dog epidermal allergens results: among the women with available serum, . % were sensitized of whom . % were monosensitised, and . % polysensitised (to two or more allergens). sensitisation to inhalant allergens dominated ( . %), with grass being most common ( . %). only . % were sensitized to food allergens, most often to peanuts ( . %), while among the . % who reported ddfa, ige reactivity to foods were identified in . %. compared to women with no asthma, women with dda ( . %) were in a significantly higher background: regular exercise has been known as beneficial that it reduces the risk of chronic diseases including allergic diseases. however, little has known regarding the relationship between exercise and allergic diseases in korean adolescents. we analyzed the national data whether exercise is related to the prevalence of allergic diseases in the population of korean adolescents method: data from sixth korean national health and nutrition examination survey ( - ) that included adolescents from to years old was analyzed. we defined regular exercise according to physical activity guidelines for americans. multivariate regression analysis was performed to find whether lack of exercise could be a risk factor for allergic diseases. results: the prevalence of asthma, allergic rhinitis (ar) and atopic dermatitis (ad) were . %, . % and . % in korean adolescents, respectively. after adjusting for factors, lack of exercise was not associated with asthma and ar, but was significantly related to ad in korean adolescents (adjusted odd ratio . , . - . , results: it was found that over % of ch up to y.o. having the ad within allergic disease (ads). the most significant symptom was a long-lasting itchy rash lasting for month in . ± . % of g and . ± . % of g. the first morbidity of ad was noticed at the age of up to y.o. among . ± . %. at the age of ch - y.o. and older than y.o. the skin ads onset was noticed for . ± . % and . ± . % accordingly. the ad sl was determined as follows: %moderate (mo), %severity (s), % were ± kua/l, ± iu/l respectively. skin prick tests were positive in . % of the patients ( . % multiple allergens). grass pollens ( %) and dermatophagoides ( . %) were the most common allergens. average vitamin a and d levels were . ± μg/l ( - ), . ± . ( - ) respectively. thirty percent of the patients vitamin d levels were mildly low, . percent was low. in control group % was mildly low, vitamin a levels was low in . % of the patients. none of the children in control group had low vitamin a levels. we didn't find any statistical significant difference for both vitamin levels between patient and control groups. vitamin a deficiency was mostly found in asthma patients whereas vitamin d deficiency was mostly in allergic rhinitis and asthma groups. passive smoking and vitamin d deficiency was significantly related (p = . ). there wasn't any relation between asthma attacks and vitamin levels. conclusion: in conclusion vitamin a and d levels weren't found significantly related with allergic diseases but was found lower than control group. patients having chronic diseases are one of the population groups that are chronically exposed to drugs. this study aims at evaluate the impact of this factors in developing drug allergies in the medical staff. method: this was a cross-sectional study that included nurses from the uhc "mother theresa" of tirana. they were asked to fill up a questionnaire where questions about chronic diseases and drug allergies were included. . % were females and the mean age was . (+ . ) years old. relative risks with % ci were calculated for different groups. results: . % ( ) nurses reported to have at least a chronic disease. the most common non-atopic disease was hta followed by the groups of autoimmune and thyroid diseases. nurses who had one chronic disease have a rr of . ( % ci = . - . , p < . ) to develop a drug disease higher than those who didn't had any chronic disease, and those who have more than one chronic disease have a rr of , p < . ) to develop a drug disease. the presence of chronic diseases can be a risk factor to develop a drug allergy probably through the increased risk to drug exposure. these patients may be exposed to drugs not only through therapy but also through hospitalizations and other forms of health care. lapeere h ; oosterlinck p ; vermeir p ; vermeire i ; coppens m ; gevaert p ghent university hospital/ghent university, ghent, belgium; ghent university hospital, ghent, belgium; ghent university hospital/ghent university, ghent, belgium background: the key to managing latex allergies in healthcare professionals and patients lies in correct recognition and appropriate action. . million people are employed in the health care sector. while there are no overall statistics on the prevalence of latex allergy in that work force, studies do indicate that %- % of health care workers regularly exposed are sensitized, compared with %- % of the general population. latex allergy is defined as an immune mediated reaction to latex products (e.g. balloons, contact dermatitis for gloves, condoms, surgical catheters); these encompass immediate and delayed hypersensitivity reactions. method: based on the experience of the belgian dutch pathway network, a -phase method to develop, implement, evaluate and continuously follow up a care pathway for latex allergy was designed and implemented. the purpose of the study was to develop and implementation of latex allergy clinical care pathways to provide all staff at ghent university hospital with appropriate knowledge and skills to identify and manage patients who have a known latex allergy or those at risk of developing latex allergy. results: care pathways, also known as clinical pathways, are used all over the world to implement and monitor patient-centered care processes in a transparent way. care pathways are defined as a complex intervention. -phase method consists of: ) screening phase; ) project management phase; ) diagnostic-and objectification phase; ) development phase; ) implementation phase; ) evaluation phase and ) continuous follow-up phase. this phased approach is based on the deming cycle, better known as the "plando-study-act" (pdsa)-cycle. conclusion: this method can offer support to multidisciplinary teams (re)designing and implementing safe, efficient, effective, person-centered, timely, equitable, continuous and integrated care processes. however, the method is no guarantee to success. the key to success is the collaboration and critical attitude of the entire multidisciplinary team when implementing pathways. background: cord blood ige (cb-ige) were considered to be a useful predictive tool for allergic symptoms especially in early childhood. there is only sparse knowledge about their importance for health in later life. the aim of our work was to determine the importance of cb-ige for allergic symptoms in young adults. we also studied the possible modifying factors for cb-ige concentration. results: resutls shown as daily mean, pollen grains/m³: table . the daily means of pollen concentrations of cupressus arizonica, platanus acerifolia and plantago lanceolata in our area differs from other sites in madrid city. although cupressus arizonica and platanus acerifolia counting were lower, plantago lanceolata counts were higher, representing a relevant pollen in our area. the clinical relevance of these findings is under evaluation by our group. method: grass pollen counts were performed since - using a burkard days spore trap located in our allergy center in madrid. the beginning of the algid period of pollination was considered the first of three consecutive days with more than grains/m and the end, the last day of three consecutive days with more than grains/m . madrid, barajas meteorological station data, was used. skin prick tests (pt) to grass pollen was also studied in comparison conclusion: total grass pollen concentration did not suffer any increase or decrease in its counts despite the dramatic increase of the temperature. an advance at the beginning and the end of the season was seen. these changes significantly correlate with the temperature increase during may and july. discrete decrease in the sensitization prevalence. since several years, the reference method to monitor the biological particles concentrations has been the hirst method: a volumetric pollen trap, located on the roof of building for background measurements, sucks continuously l of air per minute, particles depositing by impaction on a coated tape. the tape is then analyzed by optical microscopy. the hirst method produces accurate but past data. nowadays, many researches are focused on the development on new devices to get real time information. method: rapid-e from plair sa is a device using red laser beam to determine the size and the shape of sucked particles and an ultraviolet ray to measure the fluorescence of these particles. the results: the correlation coefficients got between rapid-e and hirst trap are higher than % for most of calibrated pollens, this correlation reaching % for all pollen taxa: • plane % • pine % • birch % • oak % • plantain % • dactylus % • urticaceae % conclusion: new calibrations are planned for and a real time information will be set up. results: the quinquennial media concentrations since - were . ; . ; . ; . ; . ; . ; . and . grains/m . the quinquennial media temperatures were . ; . ; . ; . ; . ; . ; . and . °c. increase of . °c (r s = . p < . ). the beginning and the end of the actual season advanced days respectively in regard to the period from to . the annual prevalence of positive pt to platanus in was % an % in . the quinquennial media from to was , , and %. conclusion: platanus pollen counts had a dramatic increase that meaningfully correlates with the dramatic increase of the temperature. a discreet advance at the beginning and the end of the season was seen. these changes did not influence in a longer duration of the season. we observed a significant increase in platanus pollen sensitization prevalence whiting madrid pollinosis patients. results: the quinquennial media concentrations since - were , , , , , , and grains/m . the quinquennial media temperatures were . ; . ; . ; . ; . ; . ; . and . °c. increase of . °c (r s = . p < . ). the actual season beginning advanced in days and the end has results: over % of house dust samples collected between april and may from central european countries were found to contain bet v allergen at levels well above the limit of detection of . μg/g for elisa . ep kit and . μg/g on maria. samples were found to have much higher levels of bet v allergen from midto-late april, particularly those that were collected in germany, belgium and hungary. samples taken from outside of the pollination season were tested and found to be negative for bet v . in conclusion, we found that bet v allergen can be detected and quantified in house dust samples. these data suggest that household dust is a source of pollen allergen and could therefore be contributing to asthma and allergic rhinitis symptoms in individuals affected by pollen allergy. household dust may also be considered as a source of bet v allergen which could contribute to allergic sensitization. | cupressaceae pollen in the atmosphere of alentejo: disruption of pollen grain during air transport spring, depending on the temperature. despite being considered moderately allergenic, it might be responsible for winter allergic outbreaks. as ornamental trees, they are found scattered throughout the territory but are more abundant in pockets of wild forest, outside alentejo. despite being more common in mountain, this pollen type is captured in considerable amounts in alentejo, portugal, where its aerobiological features and allergenic impacts are poorly characterized. the aim of this work is to characterize the aerobiology of cupressaceae pollen, to evaluate the effect the meteorological conditions and the source of this allergenic pollen type in the atmosphere of evora, alentejo. method: pollen were collected using a hirst type -day pollen trap and pollen was identified following standard methodology. background: allergic rhinitis caused by pollen is one of the most common allergic diseases. the presence of pollen in the air is currently centrally monitored at roof top levels, and not in the direct living environment of sensitized subjects. in the current project we aimed to develop a handheld pollen sampler, called pollensniffer, that can collect pollen in the living environment of the allergic subjects. as a first step this device was validated against the standard burkard pollen sampler and used to monitor local pollen concentrations at street level in the city of leiden. method: rooftop level pollen were monitored routinely by a hirst type pollen sampler (burkard, uk). the pollensniffer ( | does the allergy risk due to pollen exposure information is useful for the allergy sufferers? sindt c; oliver g; thibaudon m background: in france the information for the allergy sufferers is not made with pollen counts, which have not a real signification, but with the allergy risk due to pollen exposure. method: since more than years, rnsa (réseau national de surveillance aérobiologique), the french aerobiology network, has measured the pollen exposure in the main cities of france, using background: the effect of environmental factors on allergic sensitizations is still unclear. rural areas vs cities have different exposure levels to pollutants and aeroallergens. these differences could give clues on the causes of higher allergic sensitization rates in children exposed to city air. method: two studies with children aged years old were initialized to analyse the airborne drives of allergic sensitization: seal (günzburg, children) and ae r kids (munich, children). capillary blood was collected and the parents filled in a questionary. sensitization rates were quantified using the immunocap ® isac sige array. pollen data were measured at both locations. results: in günzburg more children were sensitized to aeroallergens, however munich children showed significant higher sensitization to phl p (p < . ), despite the lower concentration of pollen. in günzburg % children had no sensitization at all compared to % in munich. % of the children in munich spend at least hour per day outside and % of the total have no animals at home. % felt symptoms of hay fever in the last months, the majority between march and june, which correlated with the pollen flight. results: the total rate of atopy in crd patients was . %, and asthma patients was the highest ( . %). the positive rate of phadiatop in urban asthma patients ( . %) was significantly higher than that in rural areas ( . %, p < . ) and the phadiatop positive rate of office staff ( . %) was significantly higher than that of outdoor workers ( . %, p < . ). the total rate of atopy in copd patients was . %, and in patients with acute exacerbation was . %. beside, atopy is a risk factor for dyspnea (or = . , p < . ), and the fvc levels in copd patients with atopy were significantly lower than those without ( . l vs . l, p < . ). optimal scaling analysis show that, there were a correlation between the tige and smoking coefficient (cronbach's alpha = . %). in addition, the correlation between the level of tige and phadiatop sige was so strong in the patients with mild to moderate asthma (r s = . , p < . ), but it was weak in severe asthma patients (r s = . , p < . ), and up to . % of the gold iii iv patients with low phadiatop level (≤ ku/l) had a high level of tige (≥ ku/l) compared gold i ii ( . %). conclusion: the rate of atopy in patients with crd is high, and atopy is an important factor affecting the process of crd. the patients with severe copd or asthma is likely to has high serum tige level but the level of common allergen sige is low, so the allergy screening strategy should be adjusted and we should pay attention to those patients, therefore, it is necessary to screen the sensitization situation of crd patients at first, and the results can guide the treatment, management and prevention of crd. background: due to a limited amount of epidemiological data [ ] it has been thought that many severe allergic asthmatics in germany remain unidentified and are therefore not adequately treated. a pilot project demonstrated that more than % of patients, having been mean total ige (sd) was . ( . ) ku/l. . % of the patients had no sensitization towards any of the specific iges tested, whereas % were positively tested on - allergens and further . % showed sensitizations towards > allergens. conclusion: approximately % of online recruited (severe) asthmatics had a total ige level of > ku/l and ≥ sensitization (allergen-specific ige) towards atopic allergens. this further supports the high prevalence of atopy in asthma. results: patients (mean age: ± . years, range - years, m/f ratio: . ) who suffered from allergic rhinitis or allergic rhinoconjunctivitis enrolled in this study. highest rate of skin sensitivity was for weeds/grasses pollen including salsola kali, amaranthus retroflexus, chenopodium album and compositae family ( . %, . %, . % and . % respectively). among tree's pollen; ash ( %), walnut ( . %) and mesquite ( . %) were the most common. less than % of patients showed skin reactivity to indoor allergens and storage mites, mix of cockroaches and house dust were the most common ( . %, . % and . % respectively). the results of current study confirmed the importance of weed/grass and trees pollen as the major source of allergic sensitization in our area. interestingly the rate of sensitization to indoor allergens was low which can be explained by geo-climatic situation. background: there are few studies of cutaneous sensitivity to gramineae in our region. mostly of them use allergens of foreign species. the study aims to estimate the prevalence of skin sensitivity to widespread grasses in our region. method: this is a retrospective observational study of patients with seasonal allergic rhinitis. patients were studied using skin tests with pollens extracts from pooideae, chloridoideae and panicoideae grass species. results: the prevalence of positive reaction to pollen from pooideae subfamily was . % (ic: . %- . %). in turn, prevalence of allergy to panicoideae subfamily pollens was . % (ic: . %- . %) and positive reaction to chloridoideae subfamily reach . % (ic: . %- . %). cochran test suggests that prevalence in those three groups is different (χ = . , p < . ). when comparing just the groups of allergens from pooideae and panicoideae differences are also significant (χ = . , p < . ). in particular, . % (ic: . %- . %) of patients were allergic to paspalum notatum. regarding cross-reactivity between subfamilies, we find a no crosscorrelation between pooideae and panicoideae (χ = . , p = . ). conclusion: in bahia blanca, patients with seasonal rhinitis are sensitive to pooideae, chloridoideae and panicoideae. paspalum notatum, belonging to panicoideae, has a significant prevalence, high reactivity and low cross-reactivity within the group of species studied. this last species is relevant because it is a native grass from the northwest region of our country, paraguay and the south of brazil. prevalence of grass positive skin tests in patients with seasonal rhinitis by species. allergen frequency percentage % ci results: correlation analyzes were performed between sige and spt and area results. the concentration with the highest correlation by diameter and area for blo t was μg/ml and for der f of μg/ml. in the case of der p the concentration with the highest correlation for the diameter was μg/ml and for the area of μg/ml. when evaluating the reproducibility of the results according to the area and the greater diameter of the spt, a strong agreement was observed for blo t in the concentrations of μg/ml and . μg/ml. results: among the patients, the majority of allergens-positive was t , accounting for . %, followed by f ( . %), f ( . %), ds ( . %) and ccd ( . %). the prevalence of plant-related allergens (t , w , f , f , w and u ) in ccd-positive patients were significantly higher than those in ccd-negative patients (all results: with our new point-of-care methods using a selected recombinant protein e other markers, we were able to detect the disease early as days post-infection and more than % of positive cases from chronic and low endemicity areas (which are characterized by hard to detect patients with extremely low parasite load, < eggs per gram of feces) were obtained. plus, chromatography poc-cca ® test was improved by our group with a urine concentration step that turned its sensibility from % to %. conclusion: monoclonal antibody and recombinant protein technologies allowed superior detection methods when comparing it to the conventional ones. in conclusion, data showed % of sensitivity of chronic patients and % of acute patients. marton c county hospital, oradea, romania background: allergic rhinitis is a disease that affects about a quarter of the population, a disease with an important negative impact on daily activities, both on learning and working ability, as well as spending leisure time or sleeping. in the western part of romania, the most popular and blamed allergen is ambrosia, in the late summer months. it is a plant of the compositae/asteraceae family, along with goldenrod, sunflower, dandelion, cocklebur, chamomile, wormwood, daisy, etc. allergen identification is important for applying prophylactic measures, but especially for determining the allergen to be desensitized. considering the possible cross-reactivity within the compositae plant family, as well as the possibility of co-sensitization, as well as the number of patients sensitized to these pollens, which is steadily increasing, i considered is necessary a broad screening for a more precise identification of the allergen and increase chances for a successful desensitization. method: the observational study includes patients who presented on october for testing with standardized allergen extracts, as recommended. criteria for inclusion: patients with specific symptoms of rhinoconjunctivitis in august and september, with or without asthma symptoms, who returned for allergic prick test after the end of treatment. criteria for exclusion: patients who disagreed with cutaneous testing, who did not discontinue antihistamine treatment or who had been treated for other diseases with drugs that influence skin testing. background: in recent years, cationic liposomes are thought to be the most effective and non-toxical nucleic acids`transport system, so most of gene therapy drugs are developed on their base. however, lipoplexes are quickly captured by reticuloendothelial system cells after the injection and taken out of a blood stream. there are many modification methods of liposomal surface for liposomes with prolonged pharmacokinetic properties production. addition of hydrophilic polymers (peg) is seemed to be the most promising approach, that is able not only to create steric barrier on the particle`s surface and prevent the interaction with blood plasma lipoproteins, but also inhibits the protein adsorption, opsonisation and subsequent degradation in human body. the aim of this study is the evaluation of liposomal surface modification by hydrophilic polymers influence on nucleic acids`lipoplexes conjugation and on their physico-chemical and biological properties. method: liposomes preparation (including peg-modified liposomes), size determination by photon-correlation spectroscopy, examination of transfection efficacy by luciferase assay. (c h ) ) were obtained. also the modified liposomes were produced by addition of % of peg (by mass) during thin lipid layer preparation step. the size distribution was analysed by photon-correlation spectroscopy. it was shown that peg addition does not increase the par- conclusion: it can be noted that addition of peg can change the lipoplex formation but the cationic liposomes still remain an effective rna delivery system. and peg modification will be able to impart prolonged properties for the vehicle in bloodstream. foundation (grant № - - ). ory c may cause a cross reaction with fel d (cat), can f (dog), equ c (horse), mus m (mouse) and rat n (rat). february eight patients that were treated at our institution were diagnosed with rabbit allergy. results: all eight patients with the diagnosis of rabbit allergy presented with signs of upper respiratory involvement. two patients had itching teary eyes, watery nasal discharge and sneezing while feeding farm rabbits. one of those also presented with dyspnea. four patients developed problems whenever in contact with domestic rabbits. one patient developed allergic rhinoconjunctivitis whenever she was home-her parents own a rabbit, but while away in her college room she had no problems. another patient had dyspnea whenever visiting his girlfriend's house. she owned a rabbit. two patients developed asthma-like symptoms, one also presented with angioedema. the other two had developed allergic rhinoconjunctivitis. two patients have problems in contact with cats, one of them also with cows, however skin prick tests were also positive to rabbit. three out of eight patients developed allergic asthma with a positive methacholine test. six patients had a positive house dust mite prick test. all patients were diagnosed with a positive prick tests to rabbit allergens. all were treated with a nasal steroid and antihistaminesic. they were also advised to avoid contact with the animal. conclusion: domestic rabbit-induced asthma and/or allergic rhinoconjunctivitis is possible, however it is still rare in our environment. it is very important to always ask the patient about their pets in general, not just focusing on cats or dogs. only with a thorough examination and history we can find the true cause of the patient's allergy where pets play an important role. korea. changes of protein and major allergen concentration were measured over one year by bradford assay, two-site elsia, and sds-page after reconstitution of the lyophilized allergen extracts in various buffer (normal saline, . % phenol saline, and or % glycerol with saline) and stored at room temperature (rt, ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) or refrigerated ( °c). results: more than % of the initial protein concentration in all four extracts examined was detected over one year when % glycerol was added and refrigerated, whereas . %- . % remained in the extracts at rt. the addition of % glycerol to the storage buffer was found to prevent protein degradation at rt. all four extracts were found to be stable when reconstituted in % glycerol. amb a , a major allergen of ragweed, was almost completely degraded in weeks at rt when reconstituted in a buffer without % glycerol. however, . %- . % of amb a content was detected after one year of incubation at °c in all buffer conditions except . % phenol. conclusion: addition of % glycerol as well as refrigeration was found to be the important to increase the shelf-life of allergen extracts from pollens of allergenic importance. results: this is the first genetic study of the bulgarian hae patients. genetic defects were identified in hae families are: nonsense, splice-site defects, frameshift mutations, indel non frameshift, missense, and large deletion of exon . novel mutations, not previously reported in human gene mutation databases were discovered, and were predicted to be deleterious due to the expected effect on dna transcript and protein. descriptive statistics were used to summarize the eq- d-y descriptive system responses and vas scores by treatment and visit. results: twelve patients with hae type i and a median (range) age of . ( ) ( ) ( ) ( ) ( ) years were enrolled, ( . %) of whom were female. during bop, treatment with u c inh, and u c -inh, ≤ . %, ≤ . %, and none of the patients, respectively, reported having problems with mobility, self-care, doing usual activities, pain or discomfort, and feeling worried, sad or unhappy. the mean [sd] eq- d vas scores increased from . ( . results: overall, the model-derived median exposure and peak concentration across all weight ranges in paediatric patients is predicted to be higher with wb vs weight-based dosing (table) . the effect was most pronounced in patients aged - years, where the wb dosing achieved approximately % higher values than weight-based dosing for median auc - ( ng hour/ml vs ng hour/ml, respectively) and c max values ( ng/ml vs ng/ml, respectively). the wb levels are closer to those in adults receiving mg icatibant (median auc - ng hour/ml; median c max ng/ ml) but never exceed them. results: samples from patients were analysed. lanadelumab concentrations in plasma increased with higher doses and dosing frequencies. steady state was reached around week (range week to week as evaluated by predose concentrations). at baseline, mean (sd) chmwk levels were . % ( . ), . % ( . ), . % ( . ) and . % ( . ) for patients in the placebo and lanadelumab mg q wks, mg q wks and mg q wks treatment arms, respectively. by week , mean (sd) chmwk levels decreased to . % ( . ), . % ( . ), and . % ( . ) following treatment with lanadelumab mg q wks, mg q wks, and mg q wks, respectively, and remained reduced throughout the treatment period. conversely, chmwk levels remained elevated at . % ( . ) at week in patients who received placebo. patients in the placebo group had the highest attack rates over the -week treatment period (mean . attacks/month), whereas the rates were markedly lower in patients treated with lanadelumab mg q wks ( . attacks/month), mg q wks ( . attacks/month) and mg q wks ( . attacks/month). dose-and frequency-dependent manner. exposure to lanadelumab was associated with decreased chmwk levels (indicating inhibition of plasma kallikrein activity) and lower hae attack rates, corroborating the efficacy findings and utility of chmwk as a bioactivity marker in the help study. with cyklokapron (tranexamic acid) since she was years old and took the medication very irregularly due to lack of efficacy. one year before presentation at our clinic, she married and moved to vienna. we started a treatment with the bradykinin- receptorantagonist icatibant sc at the beginning of the menses and if needed a second time at the time of ovulation. she responded well until she got pregnant. during pregnancy, she developed weekly attacks with increasing severity. therefore, a weekly treatment with humanplasma-derived, pasteurized, nanofiltered c -inhibitor (inh)-concentrate, units iv once a week was started and had to be increased to twice per week after one month of therapy due to increasing number and severity of attacks. results: with this treatment attack frequency and severity attenuated. in january she had a normal delivery at term and gave birth to an otherwise healthy son. treatment had to be continued during year of lactation period and also thereafter due to persistent attack severity. conclusion: there are only limited data for the use of humanplasma-derived, pasteurized, nanofiltered c -inh concentrate during pregnancy and lactation period. this case confirms the safety and efficacy of the named drug during these periods. wheals are yet classified. the best characterized stem from hereditary or acquired c inhibitor deficiency (c -inh-hae and c -inh-aae) . last year, the french angioedema network (creak) joined the registry of angioedema without wheals (cloud-r hae). here we present the contribution of the grenoble alpes university hospital (chuga) to this disease registry. the study population is composed of patients with a proved diagnosis of c -inh-hae/aae. the following items are collected: patients' personal-demographic data, clinical/laboratory/genetic characteristics, major comorbidities, treatments (prophylaxis/acute attacks). data from existing registries at chuga are merged into cloud-r hae and missing data obtained at follow-up visits. as from cloud-r hae structure, patients can directly provide information on angioedema attacks and their treatment through a dedicated electronic app, web connection or paper support, which is then transferred into the registry at chuga. method: in a retrospective study, we included a total number of patients, suffering from a chronic skin disease, whose lesions did not improve or even worsened under immunosuppressive treatment ( chronic ulcers/pyoderma gangrenosum, bullous autoimmune diseases, skin lymphomas). ffpe tissue was examined for the presence of cmv dna by pcr. next, within the framework of a small prospective study (n = ) we analyzed the seroprevalence of cmv as well as the presence of cmv dna in lesional skin in patients that had been diagnosed with a chronic skin disease and in whom longterm immunosuppressive therapy had been initiated. results: in the retrospective study cmv dna could only be detected in / chronic ulcers/pyoderma gangrenosum ( . %), but not in bullous autoimmune diseases and skin lymphomas. / patients ( . %) of the prospective study group were seropositive for anti-cmv-igg, as compared to / patients ( . %) in an ageand sex-matched control group. anti-cmv-igm could be detected in method: in this study, we aimed at testing the diagnostic potential of skin function measurements in ss. sixteen patients with conformed diagnosis ss were enrolled in the study. skin fibrosis was assessed by conventional rss and involvement of inner organs and serum inflammation parameters were determined. four objective criteria, namely transepidermal water loss (tewl), corneometry, ph and elasticity, were assessed at nine predefined sites of the body. results were compared to patients with atopic dermatitis (n = ) and acne vulgaris (n = ). method: a multicenter prospective observational study was conducted to investigate the clinical significance of serum scca in children as a biomarker for ad. patients with ad younger than years old and age-matched healthy children without any allergic disease were enrolled in this study. the severity of ad was evaluated using the objective scorad (o-scorad). the serum levels of scca , tarc and total ige were also measured. results: in total, patients with ad and non-allergic healthy children were recruited. the serum levels of scca had the strongest significant correlation with o-scorad, compared with tarc and ige (r = . , and . , respectively). after standard treatment with topical steroids and emollients resulting in an improvement of symptoms, the serum levels of scca and tarc decreased significantly. the area under the curve (auc) for the roc curve was higher for scca ( . ) than for tarc ( . ) or ige ( . ). the difference in aucs between a single cut-off value and age-dependent cut-off values was not significant for scca , compared with that for tarc ( . and . , respectively). conclusion: scca is a more reliable biomarker than tarc for the diagnosis of ad and for determining the clinical severity of ad in children. challenges in predicting severity of atopic eczema patients results: before modeling, we checked for significant differences between patients and controls. these were detected for the levels of ccl , ccl , cxcl , ige and ldh. next, we assessed whether single serum proteins already explain disease severity by calculating correlations. twelve of the proteins, namely gcsf, il- , il- , il- , ccl , il- ra, cxcl , ifng, ccl , il- ß, ccl , and il- , significantly correlate with severity (r range: . - . ). finally, we built a model for the severity of ae based on all measured serum proteins. ten of the proteins are included in the best-fit model (adjusted r = . ). the overall correlation between original and predicted severity scores is high (r = . ) nevertheless the cross validation prediction error is substantial with %. conclusion: applied in daily practice, a prediction error of % translates to a possible miscalculation of scorad points in both directions and therefore the model is of no practical use. aside from using model-based quality measures like cross validation prediction errors to infer the usefulness of predictive models, testing them in independent cohorts could validate these models. collaborations among scientists working on similar approaches would lead to an increase in statistical power and ideally to more robust models. only robust and validated models are going to have the chance to take the step forward from being a result of computational modeling to being applied in the clinical practice of assessing disease severity in patients. jargosch m ; lauffer f ; pätzold k ; krause l ; garzorz-stark n ; schmidt-weber c ; eyerich s ; eyerich k preclinical studies in cell cultures, mice, guinea pigs and rabbits, comprising sterility, cytotoxicity, systemic toxicity, skin irritation, delay contact sensitization and phototoxicity tests, demonstrated safety of this therapeutic agent. we next conducted a single-blinded, intra-individually controlled, phased clinical trial on patients with keloids. the aim was to determine the effects of -month therapy on keloid volume and symptoms of pain and itch. two similar keloids on each subject were selectedone was treated with once-daily, self-administered application of triamcinolone-loaded ( . mg/patch) microneedles for weeks, while the other served as control with no intervention. outcome measures were (a) keloid volume using a -dimensional high-resolution ( . mm) scanner and (b) pain and itch scores on - numerical rating scales. evaluations were performed at baseline, and weeks. in phase of the trial, the whole process was repeated using microneedles loaded with a higher dose of triamcinolone ( . mg/patch). case report: a -year-old girl was admitted to our department with a single round-shaped lesion in the popliteal fossa which spread to extremities, trunk and face and persisted for several weeks and then faded slowly to residual hyperpigmented patches. courses of antihistamines, antibiotics, cyclosporine a, fluconazole, hydroxychloroquine, prednisolone and topical steroids were ineffective. also patient has had history of itchy urticarial rash and angioedema since -year-old, suffered from the flares triggered by physical exertion, stress, cold air and water, spicy food, which resolved within - hours. the patient's father and -year-old brother also had chronic urticaria induced by the same stimuli. the physical examination revealed multiple pink-to-red non-scaly, non-pruritic papules coalescing into annular, arcuate, polycyclic plaques ( - cm) with central clearing, centrifugal spread, indu- | the role of humoral immunity in the pathogenesis of psoriasis results: we found significantly increased levels of iga in the serum of treatment-naïve psoriasis patients correlating with disease score. however, iga was only observed in dermal vessels of skin sections. we next performed in-depth analysis of peripheral b cell subsets using flow cytometry. among all investigated subsets, we only found a moderate positive correlation of cd + plasma cells with iga levels and disease score in untreated psoriasis patients. however, in the group of treated psoriasis patients, neither did iga levels drop nor did plasma cells correlate with iga levels and disease score, rather hinting at an epiphenomenal finding. confirming our hypothesis that psoriasis can develop in the absence of proper humoral immunity, we present a patient who suffered concomitantly from both psoriasis and a hereditary common variable immune defect (cvid). conclusion: here, we provide new insights in the immunology of psoriasis, demonstrating the clear dominance of t cells over shifts in b cell subsets. conclusion: allergic diseases show an increasing incidence in geriatric age. this is partly due to the growing emphasis on a more accurate and careful diagnosis of the aging population. we must also take into consideration the influence of other factors, besides comorbidities and therapeutic regimens in elderly that might affects the immune response, such as environmental pollution as well as food contamination and changing dietary habits of elderly such as easy access to exotic food. one of the challenges in the decades to come is recognizing and fulfilling the need for accurate and timely diagnostics of allergic manifestations in elderly patients, as important part of achieving the best possible quality of life for this growing age group. method: sixty-eight patients with various forms of psoriasis and healthy subjects (healthy control group) were assessed after informed consent was obtained. all subjects were asked to complete a questionnaire including age, gender, duration of psoriasis, concomitant diseases and medications. in the group of patients psoriasis was with only skin involvement with skin plus joints involvement ranging from moderate to severe. psoriaticplaques were evaluated by a specialized medical team using the psoriasis area and severity index (pasi). all patients were seen by a dermatologist and clinical immunologist, who collected data considering the demographic, health status and any other relevant details. blood samples included serum levels of -hydroxycholecalciferol and tnf-α using an elisa kit (germany). method: % ethanolic extract of sp (sp etoh ) and its five major chemical constituents are prepared. to elucidate whether human orai modulated by sp etoh and its chemical constituents, conventional whole-cell patch clamp performed in horai -overexpressing hek t cell. we also assessed whether sp etoh and its constituents could inhibit mast cell degranulation and t cell activation. results: in jurkat t lymphocytes, we found that mg/ml sp etoh inhibited orai current (i orai ) by . ± . %, while one of its constituents (compound v (com v ); μm) inhibited i oria by . ± . %. investigation of human primary t cell proliferation induced by co-stimulation with antibodies to cluster of differentiation and , and of rbl- h mast cell degranulation following ige-antigen complex stimulation, revealed that μm com v inhibited both t cell proliferation (by . ± . %) and mast cell degranulation (by . ± . %); these effects were concentrationdependent, and no cytotoxicity was observed. conclusion: considering that most regional plants have not been investigated chemically or pharmaceutically, they remain as untapped potential sources of topical agents for drugs and other application. our findings suggest that com v , which derived from sp etoh , represents a promising candidate compound for the development of therapeutic agents for the prevention and treatment of allergic diseases. results: the cohort consists of caucasian patients. eight of them ( %) are women. the mean age (and range) at the clinical presentation of disease was years ( - years). the mean age at diagnosis for men was years and years for women. all patients have a positive history of recurrent and/or persistent lip swelling, of them ( %) report oral ulceration, cases ( %) have history of previous or current facial palsy and patients ( %) present tongue fissuring. concurrent cd has been diagnosed in one patient. biopsy reports were available for patients ( %); in cases ( %) non-caseating granulomas were seen. various therapeutic approaches have been described: intralesional corticosteroids had a good response in patients, infliximab was partially effective in cases; oral corticosteroids and/or methotrexate seem to cause a partial symptoms improvement. conclusion: this is the first attempt, in our knowledge, to (a) centralize all data of patients with ofg in a national registry with the aim of carrying out epidemiological data and (b) develop italian guidelines including a diagnostic-therapeutic flow chart, shared by the participating centers. the registry will guide the clinicians in the identification and management of the ofg patients, reducing the diagnostic delay and hopefully improving quality of life. case report: a -year-old girl without personal history of atopy, got a temporary tattoo with henna. after three days, she developed a local exudative, erythematous eruption with painful blisters lesions that followed the contours of the tattoo. she had neither fever nor other lesions. she was treated with topic methylprednisolone-gentamicin showing an important improvement days after. as a liquenoid scar remained in tattoo area, trofolastin ® (centella asiatica, αtocopherol, hydrolysed collagen, elastin) patch was prescribed to be placed on the scar. forty-eight hours later, the patch was removed and was newly observed an exudative, erythematous and painful wound that required oral treatment with amoxicillin-clavulanic. after three days, the girl developed on a maculopapular, generalized and itching rash. she was treated with dexchlorpheniramine and methylprednisolone with a complete resolution in days and she was referred to our allergy unit to be studied because of a suspicion of drug allergy to amoxicillin-clavulanic acid. an allergy workup was performed after obtaining an informed consent. case report: it may be sometimes difficult to find the causing allergen in allergic contact dermatitis. face is a region on which various materials contact. in this manuscript a woman case is presented who shows patch test positivity to her husband's shaving product. a years old woman applied because of allergic contact dermatitis on her face. it is learnt that lesions have been continuing for a long time, occasionally getting well with corticosteroid creams; but continuing again. patch test was performed with european standard series and cosmetic products she was using. negative result was observed. following, patch test was performed for the products her husband was using. positive results were obtained for the shaving cream of her husband was using. in detailed anamnesis, it is learnt that the lesions developed approximately month after her husband started to use this cream. it is advised not to use this product to her husband. the disease did not repeat again. it should not be forgotten that cases with allergic contact dermatitis could get in touch with allergenic materials via individuals in close contact. gül Ü akdeniz university faculty of medicine, department of dermatology, antalya, turkey case report: tnf-alpha plays role in etiopathogenesis of allergic contact dermatitis (acd). in mice which lack tnf-alpha, the response of late type hypersensitivity is spoiled. in addition, tnfalpha blocker are also used in some cases with acd. in this poster the results of european standard patch test is given in which acd is observed and tnf-alpha blocker are used without dermatological indication. cases who use tnf-alpha blocker applied because of acd: there was lesion in one case on face, in other case on face and hand, and in the last case only on hand. european standard patch test was performed to patients who were continuing to use tnf-alpha blocker. in one case no positive response was observed, while in two cases positive response to more than one allergen were obtained. in conclusion, tnf-alpha blockages cannot suppress the response of delayed type hypersensitivity. | case of allergy to nickel on the background of its intake in food peredelskaya m case report: nickel is one of the most commonly used metals; it is used for the manufacture of jewelry, plates and dishes, and medical products. a patient n, years old, female, complains of pruritic rash on the body skin with the itch intensity up to - points and the number of lesions more than . allergic background: for quite some time now the patient noted occasional eruptions on her skin after a contact with jewelry made of non-precious metals. previously patch skin tests with nickel showed a positive reaction. the patient sought emergency medical care with complaints of a number of itchy lesions erupted on her whole body during the last hours. on admittance: state of moderate severity, the patient was emotionally labile, focused on her body sensations, tearful. on the skin of face, upper and lower extremities and torso a punctuate purpura with lesions up to . cm diameter, prone to confluent. a physical status was within normal limits. in order to control the itching, as well as to sedate the patient, antihistamines of the first generation were administrated parenterally; but the eruptions kept to progress and to intensify; lesions were spread throughout the whole body, merged in gigantic areas. system glucocorticosteroids therapy was administrated, with mg of prednisolone, but then new lesions kept appearing in a large number, including after-meal rash. water, tea, bakery products, thin yoghurts did not impact the skin condition, whereas the intake of pasta, cereals, and similar products provoked intensifying of eruptions. the patient observation revealed a sharp increase in the rash after such manipulations as intravenous injections or blood sampling from the vein, the process spreading from the injection site to the entire arm. a detailed anamnesis of the disease: on the eve of the start of hives, the patient purchased a coffee machine (with metal nickel-plated parts) and started to use it. diagnosis: a systemic contact dermatitis. an allergy to nickel. the injection treatment was discontinued and a therapy with per oral gcs and antihistamines of the second generation was administrated. a recommendation was given to cook and to eat food using ceramic or wooden utensils. three days later marked positive dynamics of the skin process has been noted. the episode of systemic contact dermatitis has developed due to exposure to nickel from ingestion in food, as well as during the parenteral treatment. background: anaphylaxis reactions during anesthesia can have a mortality of %- %. / of the anaphylaxis in the operating room are due to the use of neuromuscular blockers. rocuronium is frequently involved because is oftenly used. we present a case of a years old man with an anaphylaxis shock due to the administration of rocuronium. method: years old man with no personal history of interest that is going to undergo vertebral surgery. minutes after anesthetic induction with fentanyl, propofol and rocuronium he started with lowering of oxygen saturation. orotracheal intubation is performed and, with the suspicion of anaphylaxis shock, adrenaline, antihistamines and corticoids were administered. after minutes without improvement, mg of sugammadex was administered, given the possibility that the condition was secondary to the use of rocuronium. tryptasa level was . results: skin test to fentanyl, propofol, látex and rocuronium were done weeks after and only rocuronium test was positive. conclusion: in summary, the occurrence of anaphylactic shock after neuromuscular blockers is widely described in medical literature. there are conflicting data about the use of sugammadex as coadjutant treatment in case of anaphylaxis due to the use of rocuronium. we believe is a good option when conventional treatment is not useful. case report: a -year-old woman with past history of allergic rhinitis and hypertension was admitted to the obstetrics service in labor of first child in april . epidural anesthesia with ropivacain and sufentanil was administered. as there was no labor progression, eighteen hours later she was admitted to undergo cesarean section and epidural anesthesia was re-administered. metoclopramide, ampicilin and ranitidine were given intravenously. during ranitidine perfusion, the patient presented general cutaneous erythema and pruritus, tongue, lips and eyelids angioedema and dyspnea. perfusion was suspended and hydrocortisone and supplementary oxygen administered. she denied any type of previous adverse reaction to drugs and any symptoms with use of latex-containing material. allergic evaluation revealed negative latex skin prick test (spt) and negative penicillin, amoxicillin and ampicillin specific ige assay. skin prick and intradermal tests with sufentanil, ppl, mdm, amoxicillin and ampicilin were negative. oral amoxicilin and metoclopramide provocation challenge were negative. spt and subcutaneous provocation challenge with ropivacain were negative. spt with ranitidine was negative but skin intradermal test proved to be positive. the patient was taught to avoid histamine h receptor antagonists and use as a safe alternative proton pump inhibitors. conclusion: anaphylaxis during anesthesia is an unpredictable, severe, and rare reaction. the identification of responsible drugs is a complex task. we report a case in which a commonly used and generally safe drug caused a severe reaction, which demonstrated that even the least obvious culprit should not be disregarded. epidemiologic data suggest that the number of cases of chx allergy appears to be increasing. background: chlorhexidine is a synthetic chemical with excellent antiseptic and disinfectant quality frequently used in everyday products and medical devices. the prevalence of allergic reactions towards chlorhexidine is rare, though there is increasing evidence for its allergenic potential. in this case we report about a patient with serious perioperative anaphylaxis. next to multiple potential allergens that he was exposed to, a chlorhexidine containing lubrication gel has been used for urinary catheterisation. within minutes post-exposure, the patient developed generalized urticaria, bronchospasm, tachycardia and hypotension. material and methods: we performed skin prick tests and intradermal tests with all substances documented in the anaesthesia chart, further we analysed specific immunoglobulin e (sige) antibodies and performed oral provocation challenges for exclusion. results: in the skin tests all substances except for chlorhexidine (spt: mm wheal diameter/ mm erythema) were negative. a sensitization for chlorhexidine was further corroborated by chlorhexidine-specific ige antibody ( . ku/l) in the patient's serum. in addition, the challenges for the drugs without sensitization (cefuroxime, lidocaine) were tolerated. considering all potentially relevant allergens that the patient was exposed to and the proof of specific sensitization, we diagnosed an immediate-type allergy towards chlorhexidine. conclusion: with the ubiquitous use of chlorhexidine an increase in hypersensitivity reactions including immediate-type allergic reactions is observed. anaphylactic reactions are rare, but potentially life-threatening, the diagnosis is crucial. as a warning declaration in medical devices is missing, the diagnosis of chlorhexidine allergy might be easily under-recognized or misdiagnosed. unfortunately, until now validated provocation tests are not existent, but the evaluation of combined skin tests and sige is sensitive and specific. | an approach to incidence of death due to anaphylaxis in spain ( spain ( - background: reports about death due to anaphylaxis are still scarce because of its rarity and limited information to few countries. also, data source analysis is usually not included. we report incidence of death due to anaphylaxis in spain using two databases. method: we used a hospital series of anaphylaxis deaths from the spanish hospital system and a series from the national institute of toxicology and forensic sciences (intcf) predominantly formed by extra-hospital deaths. deaths from the spanish hospital system were extracted using codes from icd- -cm, related to anaphylaxis among all deaths occured in the - period. for extracting deaths due to anaphylaxis at the intcf in the same period, two allergist researchers identified these deaths among cases with suspicion of anaphylaxis cause. a regression logistic was run to discriminate the probability of anaphylaxis death belonging to each database. incidence rates were calculated for the different groups (age, sex) using the spanish population as the denominator. temporal trends were calculated from the hospital database using poisson regression models with the number of cases of anaphylaxis detected each year as the dependent variable, and age and sex as covariates. results: there were four positive predictors of fatal anaphylaxis after the logistic model (usual allergen, positive specific ige, suggestive symptoms and previous reaction to the same allergen case report: we were informed that a girl was admitted to the pediatric endocrinology department due to early breast development. she had been diagnosed as central precocious puberty (pp). later, triptorelin acetate (ta) therapy had been started monthly. within minutes after first sc injection of ta at home, she had developed shortness of breath, decreased air entry, and coughing for ten minutes and lastly she had developed vomiting for minutes. her symptoms were accompanied by a pruritic blanchable maculopapular rash on her ears, cheeks, lips, and eyelids approximately for two hours. although they had applied emergency department of the local hospital. based on the diagnosis of anaphylaxis she was immediately treated with adrenalin. she was subsequently hospitalized for possible recurrence and discharged next day without any further events. treatment with another preparation, leuprolide aseptate(la-lucrin), as an alternative treatment was started with premedication against anaphylaxis risk only at first time and the patient did not develop any reactions. the patient is still on this treatment with no complications. anaphylaxis is diagnosed in the presence of a detectable allergen accompanied by symptoms of two systems. our patient had symptoms of the three systems as described above, that is, dyspnea with coughing, hives, nausea, and vomiting. main treatment of anaphylaxis is the epinephrine use. early usage maximizes the likelihood of survival. diagnostic tests with culprit drug were not performed in our hospital if the patient had the anaphylactic drug reaction and grouped as "physician diagnosed anaphylaxis". there has been only one report regarding anaphylaxis to ta treatment in cpp in turkey. in the literature, anaphylactic reactions against ta have been reported only in few pediatric cases. gnrh analogues are important to ensure the physiological growth in precocious puberty. because anaphylaxis can be lethal, and gnrh analogues are similar structure; the present case suggests that one should bear in mind the possibility of anaphylaxis in all patients who receive gonadotropin-releasing hormone and anologs and monitor such patients carefully as needed. furthermore, we must provide sufficient information of adverse reactions, including anaphylaxis, to patients. hence, managements against anaphylactic shocks should be recognized and treatment should be given immediately. | an anaphylactic shock induced by the rocuronium anesthesia: a case report cabrera v; barrios j; callero a; gonzález ce; pérez e; martínez ja hospital universitario nuestra sra de la candelaria, santa cruz, spain background: the anesthetic act is a unique pharmacological situation, where the patient is exposed to a multitude of substances.among them, neuromuscular blocking agents are the leading cause of preanesthetic anaphylaxis, with a frequency of between %- %.followed by latex in second place and antibiotics in third place. among the neuromuscular relaxants, most reactions are due to suxamethonium or succinyl-choline in . %, followed by atracurium, rocuronium and verocuronium.the one that produces the least reactions is cisatracurium. method: a -year-old woman presented a type iii anaphylaxis of the brown classification during the anesthetic induction in a surgery scheduled for laparoscopic cholecystectomy. for which adrenaline, dexchlorpheniramine, hydrocortisone, ranitidine and sugammadex was administered and was transferred with orotracheal intubation to the anesthetic resuscitation room. due to good evolution of the patient, she was extubated within three hours. the drugs involved in the reaction were: rocuronium, amoxicillin-clavulanic, fentanyl, propofol, midazolam, lidocaine and atropine. there was a high suspicion by the anesthesiology and resuscitation service that the abstracts reaction could have been due to the neuromuscular relaxant used, in this case rocuronium, since the reaction was reversed with sugammadex. the patient had undergone surgeries under general anesthesia previously without incidents. a specific allergy study was performed with laboratory tests with tryptase, skin tests with drugs and basophil activation test for rocuronium, sugammadex-rocuronium mixture and cisatracurium. • serial measurement of serum tryptase: . u/l, . u/l y . u/l there is no activation of basophils for sugammadex-rocuronium mixture and cisatracurium. the patient is diagnosed with rocuronium allergy. sugammadex not only acts as an antidote to reverse the neuromuscular block against rocuronium, but also has antiallergic properties by inhibiting mast cells. as an alternative for future interventions, the patient can use cisatracurium, as the skin tests and the basophil activation test are negative. unal d yedikule chest disease, istanbul, turkey case report: tetracycline hydrochloride may rarely cause hypersensitivity reactions. (hrs). immediate type reactions are at the level of case presentations and anaphylaxis is reported. we report a patient with late onset anaphylaxis caused by tetracycline. a -year-old woman referred to our allergy outpatient clinic because of urticaria due to an antibiotic that she does not remember the name of. the patient reported that many years before she had presented urticaria on her arms and legs one hour after taking the drug. to confirm drug allergy invivo and invitro testing have to performed. for many drugs there was no validated skin test. for all that invitro tests are often less sensitive and more expensive. therefore single blind placebo controlled drug provocation tests (sbpcdpt) is the gold standard in the diagnosis of drug hypersensitivity reactions. we did not know which group of antibiotics were allergy to the patient. because the patient had history of asthma and atypical pneumonia we were performed the allergy tests with clarithromycin and she had tolerated. it was necessary to use tetracycline because of patient had vaginal infection. skin tests have not yet been validated for tetracyclines. for skin prick tests of tetracycline that is only available as tablet, not in a soluble form. therefore, the tablet was smashed and diluted with . % nacl. it was also tested. healthy controls to exclude irritation. because of skin prick tests with tetracycline negative. sbpcdpt was planned. sbpcdpt was performed by progressively increasing four divided doses at minute intervals. two hours after last dose the patient experienced dyspnea, palpitations, and hypotension. as the reaction was considered to be anaphylaxis, she was given . mg of intramuscular epinephrine, intravenous mg of pheniramine, and mg of methylprednisolone. the reaction resolved within hours. blood tryptase level was . ug/l taken at the nd hour of the reaction approximately months after the anaphylaxis, serum tryptase level was . ug/l the serum tryptase level and the patient's clinic confirmed anaphylaxis due to tetracycline. we had proved late onset anaphylaxis due to tetracycline with the patient's clinic and serum tryptase level. anaphylaxis due to tetracycline is limited to case reports and small series but to our knowledge, there is no previous report of late onset tetracycline anaphylaxis. | case series of ige mediated anaphylactic shock due to polysorbate case : an -year-old male patient with hypertension, hypothyroidism and episodes of sustained monomorphic ventricular tachycardia (smvt), developed an anaphylactic shock after the administration of injectable amiodarone due to smvt. serum tryptase levels reached . μg/l during the reaction (baseline . μg/ l). skin tests were positive to injectable amiodarone (prick mg/ ml, intradermal . mg/ml) and polysorbate and (prick-prick). skin prick-prick to amiodarone and dronedarone tablets were negative. the patient tolerated oral amiodarone. we report an anaphylactic reaction during the first intravenous administration of amiodarone in a female patient being treated for supraventricular tachycardia. bat was positive, suggesting a direct effect on basophil activation, as the patient was not previously exposed to the drug. | anaphylaxis during labor: don't forget to think of an amniotic fluid embolism case report: a -year old primigravida ( weeks of gestational age) was admitted with signs of pre-eclampsia and labor was induced. benzylpenicillin and ropivacaine (epidural anesthesia) was administered > hours before the event. eighteen minutes after starting an infusion with oxytocin ( ml/h) and a vaginal toucher, the patient developed a decreased level of consciousness, generalized edema/erythema and thoracic pain, followed within minutes by fetal bradycardia and maternal collapse. after resuscitation, an urgent sectio was performed, and a baby girl was born. patient was extubated the same day. serum tryptase, hours after the event, was . μg/l (basal tryptase level . μg/l). allergy workup demonstrated negative specific ige and skin tests for latex and chlorhexidine, negative skin and provocation testing for ropivacain. however, skin testing was hampered by dermographism: intradermal (idr) testing of benzylpenicillin ( iu/ml, / - / ) and oxytocin ( ie/ml, / - / ) showed extensive erythema. idr testing of oxytocin in healthy volunteers showed pallor around the injection site (n = ). intravenous provocation with benzylpenicillin was uneventful. a basophil activation test with oxytocin (patient and control) was negative. an additional bone marrow evaluation showed no evidence for mastocytosis. although clinical criteria for anaphylaxis were fulfilled, a diagnosis of an amniotic fluid embolism (afe) was concluded. no drugs were prohibited. patient gave consent for publication. conclusions: afe is one of the most devastating conditions in obstetrics, occurring typically during labor and delivery or immediately postpartum. the pathogenesis remains incompletely understood, however, it has been suggested that afe involves an anaphylactic reaction to fetal tissue exposure associated with breaches of the maternal-fetal physiological barrier, supported by transiently increased serum tryptase levels. the diagnosis is primarily clinical, and generally one of exclusion. no specific antemortem diagnostic tests are available to confirm afe. postmortem identification of fetal squames in the maternal pulmonary circulation gives final diagnosis. differential diagnosis includes drug-induced anaphylaxis or mastocytosis, which were ruled out in our case. method: the patient presented after hymenoptera stings dyspnoea, generalized erythema with pruritus, edema of the face that required emergency therapy in episodes. results: an angio-ct was performed at the inferior limbs with optiray and minutes after the end of the investigation, the patient presented an anaphylactic shock requiring admission to the icu for days. conclusion: the patient's progression was slowly favorable. results: thirty seven cases were reported. ( %) were women. the median age was years. ( %) had dress/dihs, ( %) ten, ( %) sjs, ( %) agep, ( %) other not classified scars, and ( . %) overlapping ten/sjs. in % of the patients the suspect drug was withdrawn. thirty one patients ( %) received systemic anti-inflammatory treatment. twenty six patients ( %) received intravenous (iv) corticosteroids alone, ( %) iv corticosteroids plus ivig, ( . %) iv corticosteroids plus ivig, infliximab and colchicine, and ( . %) iv corticosteroids plus infliximab and cyclosporin. there were complications in cases ( %), and death occurred in the patient with overlapping ten/sjs who had received corticosteroids plus immunoglobulin. in this study, our aim was to evaluate severe ihr to icm. method: we retrospectively analysed patient who consulted to our allergy unit between july and july reporting symptoms within hour after icm administration. from a total of patients, we selected eight that had suffered an anaphylactic reaction. a written informed consent had been obtained for diagnostic procedures. introduction: immediate type hypersensitivity reactions to pemetrexed have been reported as very rare case reports. as limited availability of alternative therapies in chemotherapeutic allergy, desensitization plays an important role in ensuring reuse of the culprit drug. we report a case of pemetrexed anaphylaxis and successful desensitization. case: years old female patient with lung adenocarcinoma had been treated with cisplatin-pemetrexed as second-line therapy. during the th cycle within minutes after the end of pemetrexed infusion she had chest pain, shortness of breath, cough, swallowing difficulty, erythema on face and body, nausea and vomiting. she was diagnosed as anaphylaxis and adrenaline was administered besides antihistamine and methylprednisolone. symptoms and findings of the patient were improved within minutes. oncologists decelerated no suitable alternative therapy for the patient. although skin tests (prick test with / concentration, intradermal test with / - / concentration) were negative with pemetrexed, taking into account the severity of the reaction, pemetrexed desensitization was applied with the consent of the patient. no reaction was observed during the procedure result: desensitization is a successful and safe method of reusing the culprit drug. successful desensitization of pemetrexed with immediate type hypersensitivity reaction is described. the years old man was admitted emergency department with fever, rash (maculo-papular) and pain in joints. it was the th day of taking of amoxicillin. the hematological abnormalities were revealed -eosinophilia, increased erythrocytes sedimentation rate. the level of serum ecp was μg/l. the liver functional tests were increased too. hepatomegaly and cervical lymphadenopathy were observed. the patient was treated as a dress syndrome (infusion therapy, systemic steroids) and discharged after weeks with improvement. all hematologic parameters were in normal limits. lymphadenopathies were resolved. the level of ecp was retaken - μg/l. patient was prescribed oral steroids till normalization of limits of ecp. it lasted weeks after discharging. the serum level of ecp can play key role in the management of dress syndrome and in the making of diagnostic processes. until now, allergic or anaphylactic reactions to peg have been rarely reported. although patient with hypersensitivity to peg should avoid peg-containing drugs or products, patient who needs colonoscopy has few alternative bowel cleansing methods. no successful desensitization to peg has been reported to date. we report a case of successful desensitization and subsequent safe colonoscopic examination in patient with allergic reaction to peg. method: a -year-old woman developed generalized urticaria, pruritus, throat swelling, and shortness of breath immediately after taking a bowel preparation solution for colonoscopy. she had the first symptoms years ago, and has had more experiences so far. the symptoms appeared within - minutes of taking cleansing solution, and the endoscopy was no longer possible. seven years ago, she underwent endoscopy with no specific symptom. when the last symptom occurred a year ago, she was treated at emergency room because of severe dyspnea and dizziness. the patient came to our clinic for the proper diagnosis of allergy reaction and possible colonoscopic evaluation. objectives: to describe a successful desensitization to vedolizumab in one patient diagnosed with ulcerative colitis, refractory to infliximab and intolerant to azathioprine and sulfasalazine. methods: our patient was a year old woman receiving treatment with intravenous vedolizumab ( mg/cycle). cycles and were well tolerated, but in cycles , and she experienced hypotension and dyspnea, in spite of premedication with oral dexamethasone and metoclopramide. during cycle , she also showed facial angioedema, systemic urticarial reaction and oropharyngeal pruritus treated with methylprednisolone and ebastine. the results of prick (vedolizumab concentration mg/ml) and intradermal skin tests ( : and : ) with vedolizumab were negative in our patient and in ten healthy controls. total ige level was . ui/ml and specific ige against dermatophagoides were positive, being negative for hamster epithelium and latex. since vedolizumab was the only therapeutic alternative, the patient was planned to undergo vedolizumab desensitization according to an -step protocol. patient informed consent was obtained previously. premedication consisting of ebastine, acetylsalicylic acid, montelukast and methylprednisolone one hour before desensitization was administered. desensitization protocol was performed with a total duration of hours and minutes and a total dose of mg. dose steps were . , . , . , . , . , , and . mg. conclusions: our -step protocol desensitization to vedolizumab resulted safe and effective in our patient and it has allowed the continuation of treatment with vedolizumab for her ulcerative colitis. montelukast, anti h and h blockers were used for the pretreatment of desensitization. all procedures (skin and blood tests, desensitization) were carried out with the informed consent of the patient. we present an exceptional, non-immediate case of fever after cisplatin and etoposide infusion with positive skin test. case report: a -year-old man, recently diagnostic of lung cancer stadium iv, in first line of treatment with cisplatin and etoposide, started hours after finishing the nd infusion: facial erythema that becomes generalized after - hours from infusion. twelve hours later, developed warmth sensation, shivering and fever ( °c) that persisted despite the use of several oral antipyretics treatment. infectious disease was discarded, so he was referred to our department in order to assess further administration of cisplatin and etoposide. methodology: skin testing was performed days after the last reaction to minimize false-negative results, as follows (a) cisplatin prick test ( mg/ml) and intradermal tests ( . mg/ml); (b) etoposide prick test ( mg/ml) and intradermal tests ( mg/ml); with histamine as the positive control and nacl-diluent as the negative control. the results of skin test were negative for immediate reading. but two hours later, intradermal test for cisplatin turned into red and itchy and hours later, still associated a wheal. the patient was classified as high-risk (lung diseases, forced expiratory volume in second < l) and underwent programmed inpatient desensitization according to the standardized birmingham women's hospital protocol. desensitization was performed in the medical intensive care unit. the patient received only standard oncology premedication. he tolerated the final dose of cisplatin with no breakthrough reactions followed by etoposide standard infusion. two additional desensitization procedures were performed, with no breakthrough reactions. therapy ended when the disease worsened. the importance of this case, lies in the fact that fever has not been described as a clinical hypersensitivity reaction for cisplatin but for oxaliplatin. although a non-immediate reaction at the nd infusion of cisplatin could scarcely suggest a hypersensitivity reaction, the positive skin test and successful desensitization with this drug, could suggest it. introduction: propylthiouracil is commonly used as the first treatment option in patients with hyperthyroidism. although it is generally a well-tolerated drug, it may lead to some side effects including liver damage, leucopenia and skin rash. among skin rash findings, urticaria is considerably common. nevertheless, in cases that developed urticaria, a rapid desensitization protocol specific to propylthiouracil has not been encountered. we represented a case in which we applied successful oral desensitization via a scheme in accordance with general desensitization principles in a case that developed propylthiouracil-induced urticaria. case report: propylthiouracil at a dose of mg/day was initiated for a year-old female patient with diagnosis of hyperthyroidism in internal diseases clinic. the patient developed widespread itching and swelling in the body - hours after she took the first dose of the drug. she had experienced a similar reaction with use of propylthiouracil in . the patient who was breastfeeding a baby and did not have any treatment option other than propylthiouracil was referred to us with pre-diagnosis of drug allergy. the patient was thought to have propylthiouracil-induced hypersensitivity reaction and desensitization was planned. we prepared a desensitization scheme in accordance with general desensitization principles (table ). in accordance with this prepared scheme, we successfully applied the desensitization protocol with propylthiouracil for the patient. the patient gave informed consent before testing and desensitization. results: spt was negative, but idt reaction was positive at : method: we present a desensitization protocol to intravenous etoposide used in a -year-old male for non-hodgkin's lymphoma who was referred to the department of allergy at sotiria general hospital of athens. within minutes after receiving the first dose of the drug, the patient complained for flushing, retrosternal pain, difficulty in breathing and weakness. the infusion was ceased immediately and the patient received proper treatment with gradual recovery of the symptoms. the next day, skin prick test (spt) and intradermal test (id) were performed with etoposide at dilution : ( mg/ml). both of the tests, spt and id, were negative. histamine and nacl . % were also used as positive and negative controls, respectively. a desensitization protocol of three-day cycle with intravenous etoposide was conducted. premedication for days was administered including methylprednisolone, cetirizine, ranitidine, paracetamol and montelukast. results: the desensitization protocol of the first day consisted of steps of rapid pulses administered at increasing infusion rates every minutes, and step of drip infusion at a final rate of ml/hour ( mg/ . ml) until completion of the infusion. the following days, the patient received a modified rapid protocol consisting of the administration of the calculated dose of mg in only one step of infusion rate of ml/hour completing in only hours and minutes. the same protocol was applied in another three-day cycle with no adverse reactions. conclusions: hsrs to etoposide are rarely described in the literature. we propose a three-day modified rapid desensitization protocol to intravenous etoposide that could be particularly useful compared to other time-consuming desensitization protocols. case report: imatinib, a tyrosine kinase inhibitor, sometimes causes cutaneous reactions that can be of various severity. we present a case of a patient who was started on imatinib mg daily and after months developed diffuse mildly pruritic rash with some desquamation of palms of the hands. the dose of imatinib was reduced to mg daily and therapy with prednisone mg was started. after resolution of rash, the dose of prednisone was tapered to mg daily, but the rash reappeared, although milder in intensity. the dose of prednisone was increased and levocetirizine added and rash resolved. prednisone was slowly discontinued and rash did not appear. in the case of reactions to imatinib the dose of drug can be reduced and short course of oral corticosteroid given. milder reactions can be treated with antihistamine or topical corticosteroid. therefore, when adverse skin reaction to imatinib occurs, induction of tolerance to this important drug should be attempted. method: the exosomes were collected from in vitro primary human sinonasal epithelia cell, which derived from three different groups (normal control, chronic rhinosinusitis and chronic rhinosinusitis with asthma). generation of exosomes in epithelia was confirmed by nanosight, tem and western blot. the proteins of exosomes were identified by proteomics analysis. the cellular proliferation and ciliogenesis were analyzed by cck and qpcr.the ciliary beat frequency was detected by sava system. we found that epithelial cellular exosomes from chronic rhinosinusitis and chronic rhinosinusitis with asthma could reduce the multiplication rate of normal epithelial cell at a certain concentration (≥ μg/ml).we found that exosomes from chronic rhinosinusitis with or without asthma could interrupt the cellular ciliogenesis and ciliary beat frequency. using mass spectrometric analysis we demonstrated that the epithelial exosomes contained different proteins in different disease states. conclusion: our findings first identified that exosomes could be secreted by nasal epithelial cells. we also demonstrated exosomes from chronic rhinosinusitis with or without asthma could be a pathogenic factor in the remodeling of sinonasal mucosa. it could be considered as a significant biomarker for detecting the progress of chronic rhinosinusitis and a alternative therapy target. background: mucociliary transport (mct) is a major respiratory tract host defense mechanism and chronic exposure to allergen can deteriorate the these defense mechanism. the aim of this study was to investigate the effects common allergen (dp/df) on human nasal mucociliary transport in allergic rhinitis patients, and to determine the pathophysiology of ciliary beat frequency (cbf) during allergeninduced change method: allergic nasal mucosa cells of allergic rhinitis patients were exposed to common allergen (dp/df), and cbf was analyzed using an optical flow technique with the peak detection method results: the allergen(dp/df) exposed group showed a decreased cbf when compared to the control group. in the cytotoxicity assay, difference in survival rates was not found between the two groups. in the allergen(df/df)-exposed group, protein kinase c (pkc) activity was increased during a pkc activity assay. the broad pkc inhibitor, calphostin c abolished the allergen(dp/df)-induced decrease of cbf. the allergen-induced decrease of cbf was abolished by gf x, a novel pkc (npkc) isoform inhibitor, whereas the decrease was not attenuated by g€o- , a specific inhibitor of conventional pkc (cpkc) isoform. conclusion: allergen may inhibit cbf via an npkc-dependent mechanism. therefore, we have confirmed that chronic exposure to allergen could decrease cbf by increasing pkc activity. method: ova-alum allergic rhinitis mouse model (ar model) and poly(i:c) induced il- dominant mouse model (neutrophil dominant model) were used. both mouse models were exposed to tio particles for hours twice daily for days, while the controls (n = ) were not. sirius red staining for eosinophil infiltration, immunohistochemistry for neutrophil and il- a, serum immunoglobulin (ig) g and e were assayed by using enzyme-linked immunosorbent assay. in addition, the expression of interleukin (il)- , il- , and interferon (ifn)-γ in the nasal mucosa and cervical lymph nodes was measured by immunohistochemistry, and real-time reverse transcription-polymerase chain reaction (rt-pcr), il- monoclonal antibody (secukinumab) was administered in vivo to evaluate il- a dependency. results: tio exposure did not influence eosinophil infiltration in both ar and neutrophil dominant model. however, tio exposure increased neutrophil infiltration in both models and neutrophil infiltration was correlated with il- expression in the nasal mucosa. serum igg and ige levels were changed significantly in the tio exposed group. th cytokines (il- , il- ) and th cytokine, ifn-γ were not changed significantly in both models after tio exposure, however, il- were increased in tio exposure group. and these increased type pathway and neutrophil infiltration were reversed after il- monoclonal antibody administration. conclusion: exposure to airborne tio induced neutrophil infiltration in the nasal mucosa. the type response seems to play a dominant role in the nasal immune response following airborne tio exposure. | toll-like receptor ligands increase type i interferon induced b-cell activating factor expression in chronic rhinosinusitis with nasal polyposis results: first: paf-r mrna expression was very low in fibroblasts from nm and np (data not shown). paf-r mrna expression was detected in whole sinonasal tissue, submerged and ali epithelial cell cultures from both controls nm and np. paf-r mrna was also detected in peripheral blood eosinophils. although no differences were found between nm and np tissues and cultures, paf-r mrna expression was significantly higher (p < . ) in eosinophils than in upper airway tissues and cells. second: protein paf-r was found expressed in whole tissue (predominantly in the epithelium and submucosal glands), submerged and ali epithelial cell cultures from both nm and np. peripheral blood eosinophils also showed paf-r protein expression. conclusion: both paf-r mrna and protein expression was found in sinonasal nm and np tissues (epithelium and submucosal glands) and in peripheral blood eosinophils. these findings suggest the paf/ paf-r system could play a pathophysiological role in crswnp through the modulation of structural and inflammatory cell functions. "this study was funded with a research grant from uriach group". background: allergic rhinitis (ar) is an increasingly more common nasal inflammatory disease in which an antigen such as pollen or dust mites triggers symptoms such as itching, sneezing, and rhinorrhea, which can lead to nasal obstruction. ar is mediated by thelper type cells together with mast cells, eosinophils, and several inflammatory cytokines and chemokines. for example, recent abstracts | research indicates that hypoxia-inducible factor α (hif- α) is involved in the mechanism of ar development. the anti-heart failure drug digoxin has a specific inhibitory effect on hif- α, and thus, the aim of the present research was to explore the anti-hypertensive effect and mechanism of digoxin in ar. method: an animal model of ovalbumin-induced ar was established in guinea pigs. the experimental group was treated with digoxin through the tail vein. for the comparison of symptoms between the experimental and control groups, the incidence of sneezing was recorded, and the eosinophilic interleukin il- and il- levels in nasal secretions were measured by enzyme-linked immunosorbent assays. western blotting and reverse transcription polymerase chain reaction analyses were conducted to evaluated hif- α expression in guinea pig nasal mucosa. results: the ar symptoms of guinea pigs in the experimental group were significantly improved after administration of digoxin. specifically, the experimental group exhibited a significantly lower numbers of sneezing times(average . ± . vs . ± . , p < . ) and lower il- and il- secretion levels (p < . ) compared with the control group. moreover, guinea pigs of the experimental group showed less severe nasal mucosa edema, lower hif- α production, and reduced eosinophil infiltration in nasal mucosa compared with the control group. conclusion: the anti-heart failure drug digoxin may ameliorate the symptoms of ar by inhibiting hif- α production. campo p ; eguiluz i ; bogas g ; gomez f ; ariza a ; espino t ; torres mj ; rondon c allergy unit-regional hospital of malaga-ibima, malaga, spain; allergy laboratory_regional hospital of malaga-ibima, malaga, spain background: similarly to what has been described in allergic rhinitis, there is an important association of local allergic rhinitis (lar) with lower airway symptoms suggestive of asthma, being selfreported in . % of lar patients after five years of follow-up, and increasing to . % after years. however, clinical suspicion alone it is not enough for asthma diagnosis and could overstate its real prevalence. the aim was to evaluate the real prevalence of asthma in lar patients based on validated objective methods. method: seventy-five patients ( with lar, with non-allergic rhinitis (nar), with allergic rhinitis (ar)), and healthy controls (hc) were included. all patients had perennial history of rhinitis and bronchial symptoms suggestive of mild-moderate asthma for at least two years. non-specific airways hyperresponsiveness (methacholine challenge test, using tidal breath method following ats guidelines) was performed in all subjects. results: subjects were mostly young females, non-smokers. median μg/day of inhaled corticosteroids (budesonide/equivalent dose) was similar in all groups. median fev % in ar group ( . %) was significantly lower compared to lar ( %, p = . ), nar ( %, p = . ) and hc ( %, p = . ). in the lar group, / ( . %) had a positive methacholine, / ( %) in the nar, / ( . %) in ar group and / ( %) in hc. patients with lar had a significant lower percentage of confirmed asthma than ar (p = . ) and similar to nar (p = . ). no differences were detected between ar vs nar (p = . ). conclusion: presence of objectively demonstrated asthma was lower in lar compared to ar, and with better lung function. conclusion: ambient air pollution influenced the hospital visit of patients with rhinitis, even, the level of pollutants, below the national standard. so , o , no , and pm could increase an incidence of rhinitis and/or induce an aggravation of rhinitis symptoms. health care provider might expect upraising patients with rhinitis in the clinic with increase of air pollutants, even under the standard levels. results: during relapse of erosive oral lichen planus mononuclear cells obtained from peripheral blood of patients showed increased number of nk-cells cd + cd + in acute ( . ± . %) and chronic ( . ± . %) disease periods, and cd + grb cells in acute ( . ± . %) and chronic ( . ± . %) disease periods, p < . . in patients with the non-erosive forms of olp there were cd + cd + and cd + grb cells in acute ( . ± . %) and ( . ± . %) and chronic disease ( . ± . %) and ( . ± . %), p < . . the number of cd + cd + and cd + grb cells in the controls were ( . ± . %) and ( . ± . %), p < . . conclusion: acute relapse of erosive oral lichen planus, unlike nonerosive forms, is characterized by increases in the number of cd + cd + and cd + grb cells. chronic disease in patients with erosive oral lichen planus showed a steady increase in the number of cd + cd + killer cells and cd grb lymphocytes. bite", were observed in patients ( %). typical hyper-and hypopigmentation were observed in six patients mainly on the fingers and the cheekbones. fibrosis of the skin of the fingers often leads to flexion contractions, which we observed in patients. we were watching two-sided swelling of the fingers, but it was very pronounced in patients ( %). % of our patients have impaired motility of the esophagus. accelerated esr and c-reactive protein were found in patients as follows- intensively accelerated and moderate. in our patients with positive ana, we observed patients -at low titer : at and titration : in patients. the spectrum of ana found by us in raynaud's syndrome patients is closer to scleroderma than to lupus. we underline the importance of ana ( %) and anti-cc antibodies ( %) for the early diagnosis of raynaud's syndrome and scleroderma, which is also seen in our patients. anti-scl- antibodies were observed in patients coinciding with other publications describing about % of the patients. low levels of complement were observed in patients. low hemoglobin levels were observed in patient, with no iron deficiency. conclusion: . we observed a typical fibrinoid necrosis and polymorphonuclear infiltration, and collagen accumulation in the walls of small and medium-sized blood vessels. results: statistically significant increase of il level ( . pg/ml [ . ; . ]; . pg/ml [ . ; . ] respectively) was determined in patients with uc both in acute stage and remission compared to controls ( . pg/ml [ . ; . ] , (p = . ; . respectively). statistically significant increase of il a level ( pg/ml [ . ; . ] ); . pg/ml [ . ; . ] respectively) was also observed in patients both in acute stage and remission compared to controls ( . pg/ml [ . ; . ], p = . , p = . respectively). besides statistically significant increase of ifnγ both in acute stage ( . pg/ml [ . ; . ] ) and remission ( . pg/ml [ . ; . ]) compared to controls ( . pg/ml [ . ; . ], p = . ; . respectively) was revealed. background: the presence of antinuclear antibodies (ana) is commonly associated with a broad spectrum of connective tissue diseases. low titres might be detected rarely also in healthy individuals, especially in higher age. an indirect immunofluorescence (iif) detection of ana antibodies on hep- cells is the most frequently used laboratory method in this respect. the method is quite reliable regarding sensitivity, however the specificity of this test is lower. we would appreciate a biomarker for clinical discrimination of ana- other autoantibodies were tested in relation to basic diagnosis. results: a cohort of patients was divided into groups according to main diagnosis: immunodeficiency, connective tissue diseases, bronchial asthma and allergic rhinitis, recurrent infectious diseases, gastrointestinal diseases, endocrinopathy and others and the last group was generated from healthy subjects. the presence of anti dfs antibodies was highest in the group of recurrent infections, mostly in females. in these subjects homogenous pattern of ana antibodies by iif was also detected quite often, probably induced by non-specific activation of immune system. on the other hand, in a group of connective tissue diseases, we have not found any anti dfs positive patient. the clinical impact of anti-dfs antibodies is not yet finally confirmed, but their low frequency in connective tissue diseases and presence in %- % of healthy subject suggests their potential role as a new biomarker to be used as a negative predictive factor in non aard. confirmation of presence or absence of anti-dfs antibodies seems to be helpful to exclude potential diagnostic errors in iif ana positive patients. background: multiple sclerosis is a debilitating autoimmune and degenerative condition of the central nervous system, that predominantly affects young adults. both genetic and environmental factors are associated with increased risk for this disease. we propose that the effect of environmental factors, particularly latitude of childhood, is mediated through epigenetic mechanisms. specifically, we propose that unfavourable gene methylation predisposes individuals to multiple sclerosis, that this is set in childhood and adolescence, and transmitted from haematopoietic stem cells to progeny. method: cd + , cd + and cd + cell subsets were isolated from peripheral blood of healthy controls. libraries enriched for cpg islands and promoter regions were generated using modified reduced representation bisulfite sequencing and subjected to next generation sequencing. site specific methylation profiling of genome wide cpg islands, including ms susceptibility genes was conducted using methpipe software. results: genomic coverage was consistent with other published methylomes using modified reduced representation bisulfite sequencing. the methylation signature of peripheral blood derived subsets showed greater differences in methylation compared to buccal cells than with each other. individuals displayed differences in cd + methylomes, and these were recapitulated in the progeny cd + and cd + cells for those individuals. methylation of specific genes regions (e.g. prf ), were consistent with the known biological function of these genes and their potential contribution to ms risk. the vast majority of cpg islands interrogated show recapitulation of their methylation signature from cd + to progeny. however, individual differences and cell subset differences identified, likely reflect the known biological function of these genes in progeny cells. our preliminary results are consistent with the hypothesis that the epigenetic signature (that predisposes to ms risk) is set in childhood and adolescence. the physiological basis underlying the setting of this epigenetic signature is still to be elucidated, but may involve uv light and/or vitamin d, and may provide novel therapeutic targets, especially at a personalised level, for treatment of ms. background: auto-inflammatory diseases are rare disorders characterized by recurrent episodes of fever/inflammation affecting serosal surfaces, joints, eyes and skin without autoantibody production or an underlying infection. innate immunity is implicated in their pathogenesis and the underlying genetic defect has been identified in a fraction of the syndromes. during last years, the increased knowledge about auto-inflammatory diseases and the difficulty in their characterization aroused great interest to better understand these pathologies. the acidic soluble fraction of salivary proteome of patients and controls (hc) were analyzed by rp-hplc-esi-ms. known salivary proteins (salivary acidic proline-rich phosphoproteins (aprps), histatins (hst), salivary cystatins s, sn and sa, statherin, p-b peptide, α-defensins - , cystatins b, c, thymosin β- , s a , s a , s a , and s a proteins) and several derivatives (acetylated, glutathionylated, phosphorylated, and oxidized forms) were searched in the chromatographic profiles by xic (extracted ion current) procedure. adult patients (mean age ± sd: . ± . ; f, m) were enrolled and compared with sex/age matched healthy controls (mean age ± sd: . ± . ; f, m). patients are classified on the base of clinical manifestations as follows: patients with fmf (mean age ± sd: ± . ; f, m), and with unclassified fever syndrome (uc) (mean age ± sd: . ± . ; f, m). results: fmf patients showed low levels of α-defensins , and , this last was absent, with respect hc, and high levels of the glutathionylated proteoforms of cystatin b, and s a , and of antileukoproteinase (slpi). similar results were obtained on saliva of unclassified patients, which showed also levels of cystatin c higher than controls. interestingly, proteins and peptides typically secreted by salivary glands (cystatin c, histatins, statherin, aprps) were found more abundant in uc patients than in controls, and in some cases also than fmf patients (see table) . an evaluation of relative abundance of phosphorylation of phosphorylated proteins/peptides highlighted a significant hypophosphorylation of hst- , prp- and prp- in uc patients with respect to controls, probably due to a less active fam c kinase responsible for their phosphorylation conclusion: we show by a top-down proteomics approach a wide salivary modification, highlighting dysregulation in neutrophil-derived proteins and significant differences between fmm and uc patients. the control group consisted of healthy donors aged - years. immunological methods of investigation included determination of membrane antigens b cells: cd − cd + cd + , cd + cd + cd + , cd + cd + cd , cd + cd + cd + , cd + cd + cd + , cd + cd + cd + , cd + cd + cd l + cd + , cd + cd ra + cd + cd + , by flow cytometry. results: in the study subpopulation composition of lymphocytes in seropositive variant form of ra visceral a statistically significant increase in relative amount as b -cells with immunophenotype cd + cd + cd + ( . ± . % . ± . %) and b lymphocytes with the phenotype cd + cd − cd − cd + ( . ± . % and . ± . %). in the analysis of the processes of maturation and differentiation of b cells detected statistically reliable increase of the relative number of mature cd + cd − cd + naïve b cells cd + cd ra + cd number of mature cd + cd − cd + naïve b cells cd + cd ra + cd − cd + ( . ± . % and . ± . %) compared to the control group. in the study of surface markers b lymphocytes revealed an increase of expression of costimulatory cd + cd + ( ± . % and . ± . %) molecules and increasing the relative amount of cd l . ± . % ( . ± . %) ligand on cd + cd + cd + subpopulation of t-lymphocytes. analysis of surface antigenic receptor b cells in the visceral form of ra showed an increased expression of early markers of cd + cd + cd + ( . ± . % and . ± . %), cd + cd + cd + ( . ± . % . ± . %) activation in comparison with the control group. case: a year-old male patient who works as a dental technician with a history of lung silicosis and recurrent sinusitis applied to an orthopedics clinic for left hip pain and difficulty in walking. he has a history of keeping a dog during childhood. hip mri revealed a × cm sized mass on left iliac wing extended to gluteus muscle and subcutaneous tissue. incisional biopsy was reported as chronic granulomatous osteomyelitis. the lesion was considered as tuberculous abscess. despite anti-tuberculous (fourdrug regimen) treatment for one year, the lesion showed no regression. excisional biopsy was carried out by the same orthopedics clinic. chronic inflammatory reaction and fibrosis was considered to be due to cyst hydatid in the detailed evaluation. antiechinococcus igg and igm was performed with elisa and found positive. no other lesion was detected in lungs and liver. albendazole mg twice a day was initiated and substantial regression observed after three months. atypical and sustained infections made us think of primary immunodeficiency disorders. immunoglobulin subgroups were as follows: iga: < mg/dl ( - case description: year-old male was firstly admitted to gastroenterologist due to intermittent diarrhea, abdominal pain and reactive lymphadenopathy. celiac disease was suspected as genetic test showed hla dq (hla-dqa * and hla-dqb * ), histological evaluation of duodenum biopsy provided picture of lymphoid hyperplasia and marsh iiia variant. however, laboratory testing for celiac disease showed very low amount of antibodies against transglutaminase. gluten free diet for almost one year was ineffective as patient had a continuous problem of gaining weight due to chronic diarrhea. additional questioning revealed recurrent respiratory tract infections with a need of antibiotics more than two times/year during last decade. lymphocyte phenotyping by flow cytometry showed that cd , cd , cd , cd are in normal ranges, but amounts of all immunoglobulins are low: igm < . g/l, igg . g/l and iga . g/l. based on clinical symptoms and immunological evaluation diagnosis of cvid was confirmed, and replacement therapy with subcutaneous immunoglobulin ( mg/kg/month) was initiated. after six months of treatment patient affirmed reduction of gastrointestinal symptoms; he gained kg of weight, has no more infections and stable sufficient level of igg ( . g/l). conclusions: this clinical case shows the importance of immune testing for primary immunodeficiency in all subjects (despite age) with unusual symptoms of autoimmune and/or infectious disorders. cvid may have manifestation of various symptoms, which can lead to misdiagnosis, as well as inadequate treatment. results: in our sample, all patients who progressed to hypogammaglobulinemia were receiving lymphomas. there is no immunoglobulin dosage record prior to treatment. of the cases, mean age was years ( men and women), lost follow-up, and of them also presented neutropenia. seventeen patients who continued in followup required ivig replacement, due to infectious exacerbations, mainly pneumonia and sinusitis. the mean serum igg dosage at the time of onset of ivig replacement was g/dl. the mean time between the first dose of rtm and the need for ivig replacement ranged from to years, with an average of years. the iga dosage was used as a parameter for the recovery of hypogammaglobulinemia, and it was observed that only of the patients presented recovery of the condition up to the moment. conclusion: given the data, we considered the immunoglobulin dosage to be important before initiating rtm treatment and periodically, in order to indicate the replacement of ivig or igsc in a timely manner avoiding complications such as potentially serious infections. background: steinert's disease, also known as type myotonic dystrophy (md ), is the most common dystrophy of the adult. it is inherited with an autosomal dominant mechanism. it causes myotonia, progressive muscles atrophy, muscular weakness, and problems at the heart's conduction tissue and at the respiratory muscles. in patients with myotonic dystrophy, hypogammaglobulinemia is frequently described. the associations and the pathogenesis between those affections are not totally clear, but it is recognized an increased catabolism of the immunoglobulin in these patients. in most of the cases, hypogammaglobulinemia affects only the igg class and does not become clinically manifest. however, replacement treatment is not always successful in these patients. we report the case of a patient with myotonic dystrophy and hypogammaglobulinemia. case report: a -years-old man with md came to our attention for a history of recurrent infections of the upper respiratory tract and persistent infection by helicobacter pylori. at the laboratory tests, we documented low serum igg levels ( mg/dl), normal igm and iga levels and protective antibodies against tetanus consisting with the diagnosis of hypogammaglobulinemia. due to the recurrent infections, he started replacement therapy with ivig ( . g/kg/ months), switched one year ago to facilitated subcutaneous ig (fscig) with achievement of protective serum igg levels (> mg/dl) and significantly reduction of infectious episodes. conclusion: hypogammaglobulinemia is frequently reported in patients with md . in literature most of the cases described does not become clinically manifest, but in our case, the patient was symptomatic with recurrent infections. the replacement therapy with fscig showed both clinical effectiveness and safety. | real-world experience of a novel, highly purified % liquid iv human immunoglobulin for the treatment of antibody deficiencies guidelines for immunoglobulin use (july ). a highly purified % liquid iv human immunoglobulin (ig), with low levels of iga, anti-a and anti-b haemagglutinins, factors xia, xiia, kallikrein and aggregates (i e) was recently approved for use in the uk. here i report our centre's experience in using this novel % i e in three patients with antibody deficiencies. case presentations: a patient who presented in clinic with a first diagnosis of pid, was initiated on % i e at g infused every four weeks. after starting i e, they experienced a decrease in the rate and frequency of infections, in line with expectations for igrt. a young patient on home therapy with a % subcutaneous ig for pid presented in clinic with low trough igg levels. non-compliance was identified as the cause of these low trough levels and therapy was switched to % i e at g infused every four weeks in a clinical setting. both the rate and severity of infections reduced and trough igg levels normalised. an older patient on igrt for sad was reviewed in clinic due to discontinuation of their current igrt product. they were switched to % i e at g infused every four weeks. the efficacy and tolerability of i e was comparable to their previous therapy. a detailed analysis of patient, clinical and safety parameters associated with the initiation of % i e will be presented, including infection rates, white cell counts, c-reactive protein levels, tolerability and infusion-related adverse events. conclusion: these cases highlight the real-world use of % i e in two patients with pid and one patient with sad. they show that i e was well-tolerated and efficacious in one treatment-naïve, and two previously-treated patients. method: prospective study of families with one or more members with c -inh-hae followed in hospitals in the northern area of spain. a cohort of patients from families with c -inh-hae was evaluated for familiar diagnosis of c -inh-hae one or several patients from the same family were chosen and were given a questionnaire to identify the total family members from the family branch affected by c -inh-hae that had been already studied (members with diagnosis of c -inh-hae and healthy members) and those that had not been previously studied. we also register the difficulties for obtaining these data. family members not previously studied and that consent to be contacted were asked for study of c -inh-hae. for c -inh-hae screening we use c blood levels results: we have studied families with c -inh-hae, ( %) type and ( %) type ii; families had all their known members already studied for c -inh-hae ( %): families have all their members studied ( . %), families have % or their known members studied ( . %) and families had less than % of their total known members studied. we have identified members from unrelated families that had not been previously studied for hae, healthy, had low c levels and had presented symptoms of hae; had not presented symptoms of angioedema, had normal c levels, and low antigenic and functional c -inh levels. difficulties for a complete family testing study have been: family dispersion, scarce or no family relationship, do not wish to know their possible pathology conclusion: it is crucial to insist on the study of the relatives of patients with hae. we propose to include a questionnaire to identify all patient's relatives at medical reviews of hae patients. case: a- year old boy was admitted to our clinic with the history of recurrent respiratory tract infections. his all immunoglobulins were low (igg < mg/dl, iga < . mg/dl, igm < . mg/dl) associated with the absence of b cells. his aunt cousin also had xlaa missense point mutation, c c>t in exon of the btk gene was identified in both affected cousins. the patient was commenced on regular ivig treatment every weeks. at the age of , he suffered from intermittent fever attacks, abdominal pain and weight loss. tests for giardia lamblia, clostridium difficile and cryptosporidium, noro virus or parasites were negative. mr-enterography revealed intra-abdominal fluid and thickened walls of his jejunum and cecum. histopathological examination of the biopsy material obtained from terminal ileum, colon and cecum showed crohn disease. initially, he was treated with prednisolone and infliximab. because of the lack of response, infliximab treatment was switched to adalimumab. terminal ileum was resected to relieve obstruction complication. although he had been treated with adalimumab for year, a significant improvement was not observed. vedolizumab (entyvio ™ ), is a humanized monoclonal antibody α β integrin-receptor antagonist, was commenced. induction dosing was mg infusions at , , and weeks followed by a maintenance phase at week intervals. at the month of the treatment, fever and abdominal pain attacks reduced, while his weight and oral intake increased. no side effects were observed. discussion: vedolizumab is effective for inducing and maintaining remission in adults with inflammatory bowel disease (ibd); however, there is limited pediatric data. this is the first immunocompromised child treated with vedolizumab. the symptoms of the patient receded and no side effect observed during months of the treatment. results: louis-bar syndrome is a multisystem progressive disease with polymorphic manifestations which varies by age. the locomotor disability of these children is determined by neurological disorders, the exitus being caused by respiratory infectious and malignancies. the children involved in the study, had frequent episodes of respiratory infectious (bronchitis, pneumonia, atelectasis, empyema, lung abscess), ent infections (otitis, mastoiditis, sinusitis), chronic pulmonary disease (pulmonary fibrosis, bronchiectasis). index of death in this group is high ( . %). in one of the boy, the pulmonary ct showed lymphadenopathy, later was confirmed non-hodgkin lymphoma, with subsequent death. another child died from pulmonary and systemic infectious complications. results: in this paper we present the clinical and morphological analysis of children with nezelof syndrome diagnosed post-mortem. clinically were predominantly the generalized intrauterine infections or their development in the postnatal period. at macroscopic examination all patients had thymic hypoplasia. later on the microscopic study of the thymus specimens determined dysplastic changes, defined by the presence of concentrically arranged epithelial cells. in all patients, was determined the total lack of hassall corpuscles and its predecessors. besides the above-mentioned modifications in all specimens, there was no cortico-medullary segregation. thymic parenchyma outside pseudorrhagia was made up of a reticular stroma with total lymphocyte depletion. conclusion: nezelof syndrome is a severe primary immunodeficiency associated with thymic dysplasia and alymphocytosis, which is manifested early with generalized infections and major risk of death in neonatal and infant. background: the study was aimed to evaluate the cytokine profile in nasal secretion and blood serum in patients with seasonal (sar) and perennial allergic rhinitis (par) with a potential for additional sensitization with microbial allergens. method: the inclusion criteria for ar were as follows: a diagnosis of ar for more than years, the absence of nonallergic disorders of the nasopharynx, age of patients from years to years.control group: healthy volunteers at the age of - years without any allergic disorders at examination.in order to evaluate the innate and adaptive immunity, the cytokine profile of blood serum (il- , il- , and tgf-β) and nasal secretion (tslp, il- β, tnf-α, and gm-csf) was determined. to determine tslp, tgf-β, il- , and gm-csf concentrations, enzyme-linked immunosorbent assay kits were used (ebioscience, bender medsystems, r&d systems, mn, usa). we have noticed a significant correlation (r = , p = ) between the tslp concentration in nasal secretion and as-ige level to staphilococcus aureus enterotoxin (allergen component m ) in patients with par. there was a significant correlation conclusion: staphylococcal superantigens might be one of the stimuli of local tslp hyperproduction by the epithelium. there was a significant correlation between gm-csf concentrations in nasal secretion and the intensity of sensitization to a staphylococcal enterotoxin (seb) in the patients with ar. seb is one of the polyclonal t cells activators, which may account for increased concentrations of cytokines such as gm-csf locally within the system of mucosal immunity. the patients with ar and additional high sensitization to ses demonstrated a higher tnf-α production profile due to macrophage and tcell activation by these toxins. | evaluation of circulating osteopontin level as potential biomarker of allergic asthma in patients with caucasian and south-east asian ethnicity background: osteopontin (opn) is a pleomorphic cytokine known to influence a wide range of immune cells; allergic asthma was previously associated with high circulating opn levels. in the present study, we aimed to verify if opn may qualify as biomarker of activated immune response in allergic patients belonging to two different ethnic groups: caucasians and south-east asians. method: serum opn levels were measured by elisa test (human osteopontin duoset, r&d systems) in a series of italian adult patients affected by extrinsic asthma, allergic rhinitis, hymenoptera venom allergy, food allergy, allergic contact dermatitis and ige mediated hypersensitivity to beta lactams. healthy subjects served as controls. ethnic chinese subjects were recruited at the national university of singapore (nus) as cross-sectional cohort of an ongoing epidemiological study on the national prevalence of allergic diseases, and opn levels were detected by luminex (milliplex map, merck) and elisa assays (r&d systems). results: in the italian cohort, opn levels were significantly higher in cases compared to controls (p = . by the mann-whitney test). statistically higher opn levels were found in asthma (p = . ) and food allergy (p = . ) groups in comparison to controls. no significant differences were found (p = . ) between singaporeans with lifetime asthma and healthy controls, only the highest opn levels were heterogeneously found to correlate with asthma. however, a strong gender effect was shown, in both cases (p < . ) and controls (p < . ), with males presenting higher opn levels in comparison to females. consequently, we checked the mrna expression levels of opn gene (spp ) with illumina chips in whole blood of males and females, and no difference was found (p < . ). several experiments with western blots and different gel types were performed to verify if possible post-transcriptional/posttranslational modifications of opn could explain these findings. conclusion: opn seems to be a promising biomarker for current, active allergic asthma in caucasians even though technical difficulties, due to opn intrinsically disordered structure, the complex enzymatic metabolism, and the low circulating levels, significantly affect the experiments. further studies are needed to confirm these data. | mortality, intubation, and healthcare cost in patients with allergic bronchopulmonary aspergillosis in a hospital setting: a nationwide study fan x; luo y; yue b background: abpa is a complex hypersensitivity reaction to aspergillus fumigatus that colonize in airways, it is almost exclusively seen in patients with asthma or cystic fibrosis(cf). this study is to estimate hospitalization outcomes and healthcare cost of hospitalized patients with abpa. method: we conducted the study using data from national inpatient sample(nis) from to . diagnosis were identified using icd- -cm codes. hospitalization with a primary diagnosis of abpa and hospitalization with a primary diagnosis of acute respiratory failure/acute and chronic respiratory failure/respiratory distress/ asthma/cf and a secondary diagnosis of abpa were included. the study population was divided into groups including abpa with asthma, and abpa with cf. mortality and intubation rate were the primary outcomes; length of stay and total hospitalization cost(adjusted to cost in based on medical care cpi) were secondary outcomes. student t-test and chi-square were used for univariable analysis, linear and logistic regression were used for multivariable analysis. results: a total of hospitalizations with abpa were included, with hospitalizations with abpa and asthma, and hospitalizations with abpa and cf. the overall mortality rate was . % ( % ci: . %- . %), the mortality for abpa with asthma was . % ( % ci: . %- . %) and for abpa with cf was . % ( % ci: . %- . %). the overall intubation rate was . % ( % ci: . %- . %); the intubation rate for abpa with asthma was . % ( % ci: . %- . %) and . % were early intubation (< days); the intubation rate for abpa with cf was . % ( % ci: . %- . %) and . % were early intubation. the overall mean length of stay(los) was . ( % ci: . - . ) days, while the los for abpa with asthma was . ( % ci: . - . ) days and the los for abpa with cf was . ( % ci: . - . ) days. the overall total cost was million usd, the total cost for abpa with asthma was . million usd with a mean of , while the total cost for abpa with cf was million usd with a mean of . conclusion: mortality among hospitalized patients with abpa is low< %. intubation rate is relatively low, intubation, especially early intubation (< days), is more common in patients with asthma. although abpa is not a common disease in inpatient population, it does have a high health care cost and despite lower intubation rate, patients with abpa and cf generally have a longer hospital stay with a higher hospitalization cost. | frequent exacerbations of bronchitis with wheezing in adults: is it possible to predict and prevent asthma? case report: frequent episodes of bronchitis, accompanied by wheezing and dry with a prolonged duration in adults, the clinical course may be similar to bronchial asthma. the aim is to assess the risk of asthma in adult patients with or more episodes of acute bronchitis per year, had a prolonged duration and accompanied by a dry wheezing. for years in two regional clinical pulmonology centers were observed in patients ( men and women) with average age ± . years. each had at least episodes of acute bronchitis per year, which was accompanied by prolonged cough and presence of wheezes. average number of acute episodes per year was . ± . . in the course of the observation the patients were divided into equal groups. the first group consisted of persons treated in acute episodes of the disease symptomatic therapy, including inhaled β -agonists short-acting short course. in the second group to the corresponding treatment added montelukast mg per day lasting for month. in all cases of exacerbation had a viral nature. held in the period of remission of allergic sensitization, the survey revealed. starting from the first year of follow-up all patients were vaccinated against influenza annually. by the end of the fifth year of observation in the first group in cases was diagnosed of bronchial asthma- cases easy persistent asthma and case moderate. the diagnosis was exhibited in accordance with the gina criteria. in the second group, the diagnosis of bronchial asthma were exposed to patient (hazard ratio of . ). prospective observation suggests that the use of anti-inflammatory potential antileukotriene medicines in complex therapy of recurrent acute episodes of bronchitis accompanied by a dry wheezing in adults may be a factor preventing the development of asthma. for more conclusive results require more extensive research. background: chemokine receptors play an important role in regulating the migration of t lymphocytes, monocytes and neutrophils from the peripheral blood into inflamed tissue, such as lung. however, little is known about their expression on natural killer (nk) and natural killer t (nkt) cells in patients with chronic obstructive pulmonary disease (copd). therefore the aim of the study was to determine the chemokine receptor profile of peripheral blood nk and nkt cells of copd patients. method: for analysis of lymphocytes subtypes the flow cytometry method was used. the study population consisted of smokers with copd, healthy smokers and healthy non-smokers. results: we observed an increase in blood nk cells expressing cxcr receptors in smokers with copd compared to healthy smokers (p = . ) and healthy non-smokers (p < . ). the percentage of nkt cells containing cxcr receptors was also significantly higher in blood of smokers with copd compared to healthy smokers (p = . ) and healthy non-smokers (p < . ). copd smokers had significantly higher proportion of ccr + nk cells than smokers without copd (p = . ) and healthy non-smokers (p < . ). increased proportion of blood nkt cells expressing ccr on their surface was observed in smoking copd patients compared to healthy smokers (p = . ) and healthy non-smokers (p < . ). there were no significant changes in the percentage of cxcr + and ccr + nk and nkt cells between healthy smokers and non-smokers. in addition, no differences were seen in the proportion of nk and nkt cells expressing cxcr , cxcr , ccr and ccr among all studied groups. method: patients with copd in stable condition (gold stage ) aged - years old, smoking history of ≥ pack-years, were studied. bmi of patients were divided into groups: obese (n = ) (bmi- . - . kg/m ) and non-obese (n = ) (bmi- . - . kg/ m ). ten subjects with normal lung function and bmi were the control group. the level of il- assessed in induced sputum (pg/ml) was measured using an elisa (raybiotech ® ). serum levels of crp were measured using the "vector-best" (russia federation). spirometry was performed according to american thoracic society and the european respiratory society (ats/ers) guidelines. results: obese copd patients had significantly increased concentrations of il- compared with healthy subjects and non-obese copd patients by . fold ( . ± . pg/ml vs . ± . pg/ ml) (p < . ) and . fold, ( . ± . pg/ml vs . ± . pg/ml)(p < . ), respectively. non-obese copd patients had higher levels of il- by . fold compared with healthy subjects ( . ± . pg/ml vs . ± . pg/ml) (p < . ). results: among these three groups, the level of cer in lung cancer patients ( . ± . g/l) was significantly higher than that in ild patients ( . ± . g/l) and healthy individuals ( . ± . g/l) (p < . ). meanwhile, the levels of c and c in healthy individuals, which are . ± . g/l and . ± . g/l respectively, were both significantly higher than that in lung cancer patients (c : . ± . g/l, c : . ± . g/l) and ild patients (c : . ± . g/l, c : . ± . g/l), (c : p < . , c : p < . ). results from optimal scaling demonstrated that lung cancer was closely associated with immune factors including crp, cer, c and c (cronbach's alpha = . %). conclusion: for ild patients, when the level of crp and cer is increased and the level of c and c is decreased simultaneously, the risk of the development of lung cancer should be considered for these patients. results: among pbmc subpopulations, endurance exercises impacted the number of nkt and activated t cells with nkt cell numbers greater in male bobsledders vs bullet shooting and biathlon ( . % and . %, respectively). the number of activated t cells (cd + ) was greater in bullet shooting and bobsleigh athletes vs the biathlon group ( . % and . %, respectively). in female athletes the number of cd + cells in the shooting and bobsled groups was greater by . % and . %, respectively vs the biathlon group (p < . ). increased il- occurred in bobsleds in comparison to bullet shooting and biathlon: % and % in male and . % and % in female, respectively (p < . ). the concentration of il- in male bobsledders and biathlon was % and % greater, respectively, compared with bullet shooting (p < . ). serum concentrations of ifnγ in male as well as female athletes showed an increase of . % and . %, respectively vs biathletes (p < . ). increased il- occurred in the male biathlon group by . % and %, respectively, vs the bullet shooting and bobsleigh athletes (p < . ). il- was increased in the male biathlon group, compared to bullet shooting and bobsledders by . % and . %, respectively (p < . ). conclusion: prolonged endurance exercises impacts secretion of pro-and anti-inflammatory cytokines in athletes of different sport specializations. concentrations of studied cytokines did not exceed reference values perhaps due to specialized sport nutrition, which may restore immune function during endurance exercises. background: oral immunotherapy (oit) is a promising therapeutic approach to treat food allergic patients. recently, we have shown that the use of a mixture of short-chain-and long-chain fructo-oligosaccharides (scfos/lcfos) improves the efficacy of oit in cow's milk and peanut allergic mice. however, concerns with regard to safety and long-term efficacy of oit remain and there is a need to identify novel biomarkers (panels) that predict, monitor and/or evaluate the effects of oit. here we present a method for the selection of candidate biomarkers by using the computational approaches bayesian networks (bn) and topological data analysis (tda). method: data were used from scfos/lcfos diet-supported oit studies performed in independent cow's milk allergy (cma) and independent peanut allergy (pna) experiments in mice. first, a subset of the data was used for learning the data structure and their interactions in terms of a bn. this bn was used to compare the key parameters in both experimental food allergy models. finally, the relations within the dataset in combination with the bn were explored to identify and rank candidate biomarkers for the effect of oit by applying tda. the bn was able to predict the efficacy of oit in the cma and in the pna model with % and % accuracy respectively, thereby identifying a set of parameters (allergen-specific ige and igg , body temperature, mmcp- , earswelling) being key in the mechanisms involved in both scfos/lcfos-aided oit food allergy models. the tda zoomed in on the full set of previously analyzed parameters and identified clusters of biomarkers closely linked to biologically relevant clinical symptoms but also unrelated and redundant parameters within the network. taken together, this enables the prioritization of candidate biomarkers. moreover, the tda indicated differences between pna and cma models in how the data are related to each other. here we provide promising bioinformatics methods to compare mechanistic features between two different food allergies and to determine the biological relevance of biomarker (panels) of oit for food allergy. we have shown that the key drivers that influence pna and cma are similar, but that these phenotypically similar diseases show mechanistic differences in their subnetworks. these new insights provide excellent starting points to generate new hypotheses to explain why cma has a different disease pattern than pna and to select biomarkers that are useful in future clinical studies. | functional and immunoreactive levels of igg correlate with clinical responses during the maintenance phase of house dust mite immunotherapy basophils were identified as ssc low cd high , and cd was used as an activation marker. reactivity was confirmed by anti-ige as a positive control. results: in patients, basophil reactivity and sensitivity was comparable for grass pollen extract and recombinant phl p , while phl p only caused a lower basophil activation. in one patient, recombinant phl p did not cause any basophil activation, while phl p elicited an even higher sensitivity and reactivity than grass pollen extract. conclusion: in patients, basophil reactivity was comparable for grass pollen extract and recombinant phl p , while phl p only caused a minor basophil activation. in one patient, recombinant phl p did not cause any basophil activation, while phl p elicited an even higher sensitivity and reactivity than grass pollen extract. reactivity of extract and the main sensitizing components correlated, while sensitivity did not. | in vitro assessment of hypersensitivity to allergen before and after allergen immunotherapy with whole blood basophil histamine release assay wbbhr assay in these patients was performed - days before and - days after ait. heparinized whole blood samples ( ml) of each patient after substitution of plasma with pipes buffer were incubated one hour at °c with different concentrations of birch pollen extract (t ) in u-shape -well micro-titer plates. after incubation plates were centrifuged and supernatants from each well of the plate were directly analyzed for histamine content by reversedphase high performance liquid chromatography with electro-spray ionization mass-spectrometry (rp-hplc-esi-ms). results were expressed as ng/ml released histamine. sensitivity (limit of quantification) was - ng/ml. to compare results of histamine release in patients before and after ait data were calculated as area under the curve (auc) values. results: in contrast to pre-immunotherapy activity of blood basophils there were significant decreases in hr induced by t extract after ait. according to auc values all patients demonstrated decrease in hr after ait in compare to hr before ait in a range of %- % demonstrating a decrease of hypersensitivity to birch allergens. analysis of basophil hr in patients received s.c. or s.l. the asthma and rhinoconjunctivitis symptom scores during and after pollination season decreased significantly and showed correlation with histamine release by t . | immunotherapy with the recombinant b cell epitope-based grass pollen allergy vaccine bm induces a biphasic allergen-specific igg and igg response background: immunotherapy with the recombinant b cell epitopebased grass pollen allergy vaccine has been shown to reduce symptoms of grass pollen allergy in a multicenter, double-blind, placebocontrolled study. aim of this study was to investigate the levels and kinetics allergen-specific igg responses in a double-blind, placebocontrolled phase iib combined field and exposure chamber trial studying the effects of three, four and five pre-seasonal injections of bm as compared to placebo. method: a quantitative elisa assay based on purified human monoclonal allergen-specific igg as well as igg antibodies as standards was developed to measure allergen-specific igg and igg concentrations induced by ait with bm . results: we found rises in levels of both tested allergen-specific igg subclasses in the actively but not placebo-treated patients. phl p -and phl p -specific igg levels up to μg/ml and μg/ml, respectively and phl p -and phl p -specific igg levels of up to μg/ml and μg/ml, respectively were measured in bm treated patients. five pre-seasonal injections induced the highest allergen-specific igg levels. interestingly, allergen-specific igg and igg antibodies showed a biphasic response with early rises of allergen-specific igg which declined quickly after the pollen season and a delayed but very sustained allergen-specific igg response. conclusion: treatment with bm induces a biphasic allergen-specific igg response consisting of an early igg and a sustained allergen-specific igg response which may be responsible for early and sustained protection against allergic symptoms. | t reg cd + cd high in peripheral blood in patient with grass pollen allergy during sublingual specific immunotherapy slit the aim of this study was to evaluate t reg cd + cd high in peripheral blood in patients with grass pollen allergy during sublingual immunotherapy slit. method: we examined adult patients, aged - , female and male. patients were qualified to slit after confirmation of allergy-by skin prick tests, specific ige and nasal provocation tests. we determined t reg cd + cd high from blood sampling of those patients (by flow cytometry method), before the slit, after reaching the maintenance dose, before the grass pollen season, during the grass pollen season, and after one year of slit. results: during slit the percentage of t reg cd + cd high increase after reaching the maintenance dose, then it decreased before and during the grass pollen season, and again increase after one year os slit. we observed, that in the group with significant improvement of symptoms, t regcd + cd high decreased during grass pollen season, comparing to group without clinical improvement. conclusion: slit as a method of immunotherapy influence on levels of t reg cd + cd high cells. the observed decreased levels of these cells during the grass pollen season might be consider as a prognosing marker of clinical improvement. | t reg cd + cd high from peripheral blood during subcutaneous specific immunotherapy (scit) for grass pollen hofman a; hofman j; hofman t centrum alergologii, poznan, poland background: specific immunotherapy is the only causal method for grass pollen allergic rhinitis. however, we don't observe in every patients satisfying clinical effects. because the treatment of allergic rhinitis takes - years, and is quite expensive, everyone is constantly looking for a perfect parameter, which may prognose the effectiveness of specific immunotherapy (sit). the aim of this study was to evaluate t reg cd + cd high lymphocytes from peripheral blood in patients during subcutaneous specific immunotherapy (scit) for grass pollen . method: we examined adult patients ( female, male), age - , who undergo scit for grass pollen allergy. we have done skin prick tests and specific ige in those patients. additionally, we confirmed the allergy by nasal provocation tests. we have determined lymphocytes t reg cd + cd high from blood sampling from those patients, with flow cytometry method: before immunotherapy, after reaching the maintenance dose of scit, before and during the grass pollen season, and after one year of scit. our control group was represented by adult healthy volunteers. after one year of scit we divided patients into two groups-with and without clinical improvement. results: in allergic patients we have observed decreased levels of t reg cd + cd high compared to control group before the start for scit. during immunotherapy, the percentage of t reg cd + cd high increased after reaching the maintenance dose, however it did not reached the level of healthy volunteers. again, levels of t reg cd + cd high decreased before and during the grass pollen season, and increased after one year of scit. we compared also patient with significant improvement of clinical symptoms, and without. and we observed that the level of t regs cd + cd high decreased during pollen season in improved group, and increased in the group without clinical improvement. conclusion: in patients with grass pollen allergy, during the grass pollen season, decrease of t reg cd + cd high cells might be con- results: in the group of patients treated with sit gene expression analysis revealed significant change in ifng expression (p = . ) (comparison between sample a and b). comparison between samples a and c showed significantly different expression in genes: afap l (p = . ), commd (p = . ), pik cd (p = . ), and twist (p = . ). duncan's multiple range test confirmed difference between sample a and c for commd (p = . ) and also revealed new significant difference in tbx in samples a and b (p = . ; in wilcoxon's test p = . ). k nearest neighbors algorithm was built based on ifng, pik cd, commd expression. the results of the study indicate, that there is a significant change in the expression of a few genes during the build-up phase of sit. it may be suspected, that this change contribute to the mechanisms involved in the building tolerance to allergen. k nearest neighbors algorithm may be useful for sit efficacy prediction. | regulation of cytokine thymic stromal lymphopoietin (tslp) in modulating tgf-ß induced interstitial inflammation and cellular fibrosis background: thymic stromal lymphopoietin (tslp) has previously been linked to allergic inflammatory diseases, tissue fibrosis and organ dysfunction. it remains unclear, however, whether tslp plays any role in the occurrence of renal fibrosis, so this study investigated that underlying mechanism. method: an in vitro fibrosis model was established by treating normal rat kidney fibroblast (nrk- f) cells with transforming growth factor-β (tgf-β ), after which the levels of various fibrogenic markers (e.g., fibronectin) and downstream fibrogenic signal proteins (e.g., smad ) were investigated. also, tslp shrna was used to silence the effects of tslp, while an elisa was conducted to evaluate the fibronectin secretions. results: the level of fibronectin in the nrk- f cells was doseand time-dependently increased by the administration of exogenous tslp (p < . ). tslp also significantly increased the level of fibrosis signaling, in addition to inducing a marked decrease in the down-regulation of smad . interestingly, the application of tslp shrna caused a dramatic reversal of the tgf-β -induced cellular fibrosis while simultaneously leading to the suppression of fibronectin and fibrogenic signal proteins. conclusion: taken together, these observations provide insights into how extracellular matrices develop and could lead to potential therapeutic interventions for the suppression of renal inflammation and fibrosis. abstracts | | effects of two years treatment with the recombinant b cell epitope-based grass pollen allergy vaccine bm on allergen-specific b and t cell responses background: bm contains recombinant fusion proteins of nonallergenic peptides from ige-binding sites of the four major timothy grass pollen allergens phl p , , and and pres protein from the hepatitis b virus as a carrier. in a multicentre, double-blind, placebocontrolled trial, grass pollen allergic subjects were treated for two years either with bm or placebo. here we investigated in detail the effect of immunization with bm on allergen-specific t and b cell responses. during the study from subjects treated in the vienna centre (bm : n = , placebo: n = ) were investigated regarding proliferation using h thymidine incorporation and cytokine production in response to various recombinant allergens at different time points. grass pollen allergen-specific ige, igg and igg levels were determined by immunocap and elisa. results: a significant increase of allergen-specific igg and igg levels was found in the bm -but not in the placebo group in both years (year > year ) after treatment. there was no difference regarding t cell proliferation in response to phl p and phl p after first grass pollen season between actively and placebo-treated patients whereas proliferation in particular of phl p -specific responses seemed to be blunted in the active group in the second year. no significant differences regarding allergen-specific th , th and tolerogenic (i.e., il- ) cytokines were observed between bm and placebo-treated patients. the findings indicate that the bm induces high levels of allergen-specific blocking antibodies which may reduce allergen-specific t cell proliferation but does not induce significant increases of regulatory cytokines in t cells. this study was supported by grants f , f and dk -b of the austrian science fund (fwf). hospital universitario ramón y cajal, madrid, spain; department of immunology iis-fundación jiménez díaz, uam, madrid, spain background: shiitake mushroom (sm) (lentula edodes) is an edible fungi native to east asia. it is traditionally cultivated and used in many asian countries and its consumption is increasing worldwide. direct skin exposure to sm can cause cutaneous reactions, including allergic contact dermatitis and urticaria, while its oral intake may prompt "shiitake flagellate dermatitis" (sfd), which is a distinctive itching linear erythematous eruption. sfd is usually considered a toxic reaction to lentinan, a thermolabile polysaccharide that increases interleukin- . we report cases (p , p , p ) of shiitake flagellate dermatitis studied in our centre. method: skin prick tests (spt) to environmental allergens-including moulds -, prick-by-prick and patch test with raw and cooked sm were carried out. total ige and specific ige to mushroom, white mushroom and environmental moulds were also determined. a raw and cooked shittake mushroom extracts were prepared. both extracts were analyzed in all the patients by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). results: skin prick tests, prick-by-prick, patch tests and specific ige were all negative except for p , who had positive prick-by-prick to raw shiitake mushroom. sds-page ige immunoblotting assays with the patient's sera revealed ige-reactivity with proteins ranging from kda to kda for p , p and p . we report cases of shiitake flagellate dermatitis with demonstrated ige-sensitization. physicians should take into account that some cutaneous reactions considered as toxic might be allergic reactions. vorozhko i ; sokolnikov a ; sentsova t ; donnikov a ; ilyenko l ; denisova s background: allergic diseases such as asthma, rhinitis and food allergy have increased in recent decades in tropical countries. the tropics has climatic, environmental and ecological peculiarities that allow us to emit several hypotheses that could explain this phenomenon. in one of them, it is postulated that the increase of the sensitization to food, is due to the presence of lower serum levels of vitamin d, product of the adoption of a western lifestyle, with lower sun exposure, which in its turn diminishes the immunomodulatory action of this vitamin at intestinal level, favoring the sensitization against food antigens. we evaluate differences between the titers of serum antibodies against food antigens between two populations with african ancestry but different environment (rural vs urban) and investigate the influence of vitamin d levels. method: an observational, cross-sectional and descriptive study was carried out on afro-descendant children living in san basilio de palenque (rural) or in the city of cartagena, bolivar (urban). the sensitization was determined by a positive skin prick test to allergen extracts, including foods, and, specific ige, iga and igg to egg, milk and peanut extract, as well vitamin d, were measured by elisa. antibody and vitamin d titers were correlated by spearman's test and comparisons between groups were done using the wilcoxon rank sum test. a p < . was considered significant. results: atopy was more prevalent in the urban population ( % vs %, p < . ). however, none participant tested was positive for food allergens. regarding vitamin d levels, these were found to be higher in the rural population compared to the urban group (p < . ). among the antibodies analyzed, only ige against peanut showed differences, which were higher in rural population (p < . ) as well as those of iga to peanut, which were higher in the urban population (p < . ). we observed only a significant correlation between peanut specific ige (rho . , p < . ) and iga (rho - . , p < . ) response and vitamin d. conclusion: in our study, we found differences between the peanut specific ige and iga response in urban and rural populations. the correlation between the levels of specific ige and iga to peanut and vitamin d, suggest that this vitamin may influence peanut sensitization in this population, besides other components like diet and genetic and environmental factors. | evaluation of inhaled allergen sensitivity in patients with food allergies younger than two years of age kulhas celik i ; aldemir es ; buyuktiryaki b ; ginis t ; toyran m ; dibek misirlioglu e ; kocabas cn ; civelek e carbohydrates and pyruvate was observed in the severe group compared to the rest of allergic groups. in addition, an increment in lactate was noticed. these metabolites were closely associated with the energy metabolism. other metabolic changes included increased levels of fatty acids such as myristate, palmitate and laureate. these fatty acids might be precursors of arachidonic acid, a key molecule in inflammation. finally, alterations in some amino acids and adenosine were found method: to evaluate these critical processes, five-week old germ-free c h/hen mice were split into two groups; were intraperitoneally sensitized to the peanut allergen ara h and remained ns. upon reaching weeks of age, mice were intragastrically challenged with purified ara h . mice were harvested in two groups: -minutes and -minutes post-gavage. upon harvest, the left lobe of the liver was collected and sera were removed. sera and livers were evaluated for drp-ara h using an in-house quantitative sandwich enzyme-linked immunosorbent assay (elisa). a sample of the proximal small intestine was monitored for drp-ara h using immunohistochemistry (ihc) and for mast cell degranulation using toluidine blue stain. results: sensitization does not have a large effect on the concentration of allergen present in the sera or liver. however, s mice allowed to digest ara h for -minutes were more likely to display tissues positive for detection of drp-ara h than ns mice at the same time point. conclusion: there is drastic biological variation among mice in their capacity to absorb and transport allergens. the elisa used in these analyses proved effective in the quantitative detection of drp-ara h in both liver and sera samples, while ihc provided inconsistent results for the detection of drp-ara h in tissues. however, in positive ihc samples, staining was indicative of paracellular transport across the epithelial barrier. | eczema induces a high ovalbuminspecific ige/igg ratio and affinity maturation during the lactation period irahara m; kido h; shinahara w inst. for enz. res., tokushima university, tokyo, japan background: recent articles have revealed that ingestion of foods induces oral tolerance and cutaneous sensitization induces food allergy. relationships with levels of immunoglobulin subclasses, affinity of allergen-specific ige, and development of food allergy have also been indicated. however, relationships with levels and affinity of specific immunoglobulins and eczema during early infancy remain poorly understood. therefore, the present study aimed to elucidate these relationships. method: this study enrolled women who visited naruto hospital (tokushima prefecture, japan) in late pregnancy and their children. blood samples and information on skin condition were taken every months from neonate to months old. egg white and milk allergen-specific immunoglobulin subclasses and affinity of ovalbumin (ova)-specific ige levels were measured using the densely carboxylated protein (dcp) microarray with μl of serum. results: this study included infants whose parents agreed to join this study. of these, infants ( %) were diagnosed with eczema by months old. egg white (ew) and milk-specific igg were detected in a few subjects at months old. however, these specific ige and igg were detected in some subjects at that time ew-and ova-specific ige levels and ige/igg ratios were significantly higher in participants with eczema than in those without eczema at months old. moreover, subjects with high ova-specific ige/igg ratios showed higher affinity ova-specific ige antibodies than subjects with low ova-specific ige/igg ratios. these results were not reflected in milk-specific ige levels. the milk-specific ige level differed between breast feeding and formula-fed infants, with no difference in the ige/igg ratio. conclusion: eczema contributed to high ew-and ova-specific ige levels and ige/igg ratios. high ova-specific ige/igg ratios involved high affinity ova-specific ige antibodies. however, the milk source during early infancy had no effect on the specific ige/igg ratio with eczema. these results suggest different sensitization routes provoke different results in levels and affinity of immunoglobulins. | purification and characterization of naturally occurring post-translationally cleaved ara h , an allergen that contributes substantially to the peanut allergome background: the s albumin ara h is one of the most important peanut allergens. a post-translationally cleaved ara h isoform has been described in the past but had not been characterized in detail, nor had its relevance for peanut allergy been investigated. method: post-translationally cleaved ara h (para h ) and intact ara h (intact ara h ) were purified from virginia type peanuts and the cleavage site was mapped using high-resolution mass spectrometry. biochemical characteristics were determined by sds-page, uv absorbance spectroscopy, far uv cd spectroscopy, and immunochemical reactivity of both forms of ara h was compared by igg immunoblotting and ige-elisa using sera from individuals sensitized to peanut. reversed-phase liquid chromatography was applied to study the occurrence and abundance of para h in various peanut types. results: compared to intact ara h , para h lacks a -amino acid stretch, resembling amino acids - (uniprot accession number q g ) in the non-structured loop. consequently, para h consists of chains; a n-terminal chain of approximately kda, and a c-terminal chain of approximately kda, held together by disulfide bonds. intermediate post-translationally cleaved products, in which this stretch is cleaved but not removed, are also present. the secondary structure and ige-binding of para h resembles that of intact ara h , indicating that the loss of the non-structured loop is not critical for maintaining conformational ige-epitopes. both forms of ara h were reactive with several commercially available igg antibodies. the peanut cultivars runner, virginia, valencia, and spanish contained para h at equivalent levels, suggesting para h is a consistent and important constituent of the peanut proteome. conclusion: a post-translationally cleaved form of ara h is abundant in the main peanut market types, and has ige-binding comparable to intact ara h . this should be taken into account when ara h is investigated in peanut-containing products. | release of major peanut allergens from their matrix at various ph and at saliva conditions; ara h and ara h are quickly bioaccessible background: the oral mucosa is the first immune organ that encounters allergens upon ingestion of food. peanut is often consumed in solid form, and it is not known if peanut allergens are released from the food already in the mouth. we set out to investigate the solubility of individual peanut allergens at conditions that mimic the first exposure site, i.e. the mouth. method: light roast peanut flour was suspended in buffers of various ph mimicking saliva. protein concentration was measured in supernatant, and release of major allergens ara h , ara h , ara h , and ara h was assessed by sds-page. also, the allergen profile of un-dissolved material was assessed. results: peanut protein solubility is poor in the ph range - , while at low ph ( . ) and at moderately high ph (> ), the solubility is higher. at all conditions tested, there was a substantial amount of un-dissolved protein. this indicates that the ph range of saliva, between . and . in healthy individuals, may be critical for the release of peanut protein from its matrix. in this ph range from . to . , ara h and ara h are readily released, while ara h and ara h are poorly released. increasing the ph from . to . slightly increased the release of ara h and ara h , but still the recovery was low (approximately % for both ara h and ara h ) compared to that of ara h and ara h (approximately % and %, respectively). this remarkable difference in extraction kinetics suggests that ara h and ara h are the first allergens an individual is exposed to upon ingestion of peanut-containing food. conclusion: based on our observations, we conclude that the peanut allergens ara h and ara h are quickly bio-accessible in the mouth upon ingestion of peanut. this new insight may contribute to the understanding of the extraordinary allergenicity of ara h and ara h compared to other peanut allergens. background: in proven cases of non-ige mediated cow's milk allergy clinical response can be partial even when treated with amino acid formulae e.g pain in infants. residual intestinal symptoms can be related to ongoing nerve hypersensitivity, changes in microbiome or motility disturbance. mast cells are thought to play crucial role in non-ige mediated food allergy. ketotifen is a first generation h antihistamine which has mast cell stabilising properties with pain blocking and anti tnf-a effect. hence ketotifen could have important role in symptom resolution where diet elimination has not been successful. we aim to find out effectiveness of ketotifen to unresponsive/partially responsive symptoms such as pain in non-ige mediated cow's milk allergy infants. method: children who presented to single specialist centre over years had their case notes reviewed retrospectively. inclusion criteria were those children with confirmed non-ige mediated cow's milk allergy by elimination of cow's milk with improvement of symptoms and worsening of symptoms on reintroduction of dairy. where symptoms partially responded e.g pain, ketotifen was used at . - mg once at night for weeks and symptoms were reassessed. statistical analysis was performed using r v . . with significance was set at p = . . results: patients were identified with patients excluded due to unconfirmed non-ige mediated allergy. of the case ( males, age - months), atopic co-morbidities were found in % children. common symptoms were abdominal pain ( %), vomiting ( %), back arching ( %), constipation ( %), bloating ( %), food aversion ( %) and diarrhoea ( %). we compared the children who had symptom improvement on ketotifen and cow's milk elimination against children who improved on cow's milk elimination alone. significant difference of symptom improvement was found with abdominal pain; % using ketotifen compared to % who did not use results: a total of analysis for ttgiga have been performed for a total of patients during the study period ( , , , ) . patients showed at least once a positive ttgiga. among these, had a negative result at first testing, and were positive at the second (n = ) or third testing (n = ). despite an increasing number of ttgiga requests, the number of positive results decreased. wige were rarely requested but were positive in about % of tested sera (table a and b). the amount of laboratory requests for ttgiga has increased, while those for wige remains stable and is rare. wheat allergy seems to be rarely investigated in our center and may deserve more attention. case report: eosinophil-associated gastrointestinal disorders (egids), including eosinophilic gastroenteritis (eog), are a inflammatory diseases, characterized by gastrointestinal symptoms and eosinophilic infiltration. patients with eoe have an increased incidence of allergy, with increased ige mediated food and inhalant sensitivities. use of either a targeted food allergen avoidance approach (based on allergy testing) or untargeted approach (based on food allergen avoidance) results in the resolution of eosinophilia in the gastrointestinal tract of %- % of adult. we describe a case of a -year-old patient diagnosed with eosinophilic enteritis, associated to protein-losing enteropathy. the patient experienced severe diarrhea, nausea, vomiting and weight loss, that caused a severe dysproteinaemia and electrolytes abnormalities. an upper and lower endoscopy was performed, showing an ulcerative ileitis. the histological pattern was characterized by eosinophilic infiltration of ileum and duodenum> hpf. she presented also high levels of total ige ( k/ui), high serum tryptase ( μg/l, n.v. ≤ . ) and sensitization to the lipid transfer protein (ltp) of peach. the patient was prescribed to a six-food elimination diet (sfed) and underwent high doses of oral and intravenously corticosteroids, but a satisfactory therapeutic response was not achieved. we hypothesized that ige has a role in the mechanism of aeg and that blocking ige would have improved disease symptoms and reduced allergic inflammation, as measured by a decrease in intestinal tissue eosinophilia. we started off-label administration of omalizumab mg/month subcutaneously, the same dosage schedule used in allergic asthma and, by other authors, in eosinophilic gastrointestinal disease after achieving informed consent by patient. except for an exacerbation of symptoms occurred months after starting the therapy, when a further endoscopy, showing a gastrointestinal eosinophilic infiltration> hpf, was performed, a significant improvement of both gastrointestinal and cutaneous symptoms was observed during therapy, together with a normalization of laboratory parameters. after months a clinical remission of disease was obtained and administration was stopped. although a histological remission during the first few months of treatment was not obtained, in a subset of aeg patients, ige plays a role in the pathophysiology of the disease and that anti-ige therapy with omalizumab may result in disease remission. | fullerene c reduces the allergic inflammation in food allergy mouse model background: a food allergy (fa) is an abnormal immune response to food. the signs and symptoms may range from mild to severe. they may include itchiness, swelling of the tongue, vomiting, diarrhea, hives, trouble breathing, or low blood pressure. food allergy is becoming increasingly common. fullerene c has the unique electronic properties making it an attractive candidate for allergic diseases therapy. the main purpose of our research was to assess therapeutic effect of fullerene c in a mouse model of fa. method: new efficient method for producing a water-soluble fullerene c has been developed. fa experimental model was induced in balb/c mice by the intragastrical (ig) ova administration after subcutaneous (sc) sensitization. fullerene c was administrated ig once a week, or twice a week, or daily. ova-specific antibodies were assessed by elisa. splenocytes cytokine production upon ova in vitro stimulation was detected by elisa. samples of jejunum of the small intestine were removed for histological examination immediately after the last ig allergen administration. results: it was shown that ova-specific ige and il- level were significant decreased in groups treated with water-soluble fullerene c . the greatest effect was observed in mice receiving fullerene c daily. the ifn-gamma level was significantly higher in ig c treated groups. the histologic analysis of jejunum of the small intestine samples showed that c -therapy improved the histologic picture. the greatest effect was observed in mice receiving fullerene c daily too. conclusion: taken together, these results demonstrate that the water-soluble fullerene c exhibits a significant anti-inflammatory effect in a mouse model of fa, and possesses a high therapeutic potential. background: a -year-old male reported eight episodes of anaphylaxis after exercise. all the ingested food eight hours before each episode was analyzed. before each episode, he had always eaten chicken or turkey meat, and he tolerated these foods without exercising. in one of the episodes the patient had taken a tablet of dexketoprofen a few hours before. since several years, he referred chest tightness after eating some fish (emperor, salmon and whiff), however he tolerated others. the patient denied having eaten fish before any of the episodes of anaphylaxis. method: commercial skin prick tests (spts) and prick by prick tests (pp) with all food ingested and fishes were performed. tryptase, total serum ige (ige) and specific ige (sige) (immunocap. thermo-fisher scientific, uppsala, sweden) to the foods involved were determined. a controlled oral provocation test (opt) with dexketoprofen was performed. results: spt was positive to tuna extract ( mm) and negative ( mm) for the rest of fish extracts. it was also negative for chicken meat extract and other foods tested. pps were positive for raw and cooked turkey meat ( mm), raw and cooked tuna ( and mm), raw emperor ( mm), raw and cooked whiff ( and mm) and raw hake ( mm). pps were negative to raw and cooked chicken meat. ige was ui/ml and tryptase . ng/l. sige was slightly positive to hake, cod and chicken meat. dexketoprofen opt was negative. at that moment, we recommended the patient to avoid chicken and turkey, as well as the fishes which he had symptoms with. since then, he has not suffered any new episode of anaphylaxis despite exercising daily. protein extracts from turkey meat, tuna, emperor, salmon, hake and whiff were prepared and analyzed by sds-page. conclusion: recently, triosephosphate-isomerase ( kda) has been identified as a new chicken meat allergen. this allergen could be responsible for the cross-reactivity between bird and fish meat and the episodes of anaphylaxis after exercise in our patient. the triosephosphate-isomerase has not been implicated previously as a cross-reactive allergen involved in the fish-chicken syndrome. results: twenty-three patients were included, % male, median age years (iqr . ), % atopic, % asthmatic. non-specific lipid transfer proteins (nsltp) were implicated in % (n = ) and ω- -gliadin in % (n = ). eighteen ( %) patients referred anaphylaxis in the reaction with co-factor, ( %) urticaria/angioedema, had both depending on the co-factor. all patients in which ω- -gliadin was the allergen involved had anaphylaxis in the presence of co-factor, with tolerance to wheat without it. in patients in which nsltp was the allergen involved, ( %) had anaphylaxis in the presence of co-factor. reaction with co-factor was more severe than without in ( %) patients; patients had no previous history of reaction and subsequently tolerated the culprit food. only patient had anaphylaxis in the absence of co-factor; the remaining presented oral allergy syndrome and/or urticaria. exercise was the main co-factor, present in patients. nonsteroidal anti-inflammatory drugs (nsaids) were the only co-factor in patients; all of them had anaphylaxis, with allergy to ω- -gliadin, to nsltps. all subsequently tolerated the nsaids involved. conclusion: nsltps and ω- -gliadin were the most frequently involved allergens in cefa, with exercise being the most frequent co-factor. nsaids were relevant co-factors, even when ω- -gliadin was the allergen involved. several patients subsequently tolerated culprit foods and nsaids, difficulting the diagnosis and further emphasizing the importance of a correct cofactor evaluation. molecular allergens had an important role in the diagnosis, avoiding unnecessary ofc. information about co-factors must be included in all patients with allergy to nsltps and ω- -gliadin. | allergy to wheat-dependent exerciseinduced anaphylaxis (wdeia) proteins, without? -gliadins as responsible ferreira a ; castillo m ; martins s ; pineda f unidade de imunoalergologia hospital das forças armadas., lisbon, portugal; departamento de aplicaciones. diater laboratorios, madrid, spain background: the second well-characterized form of allergy to wheat proteins is wheat-dependent exercise-induced anaphylaxis (wdeia), with the ω -gliadins (part of the gluten protein fraction) being the major group of proteins which are responsible, but other forms of food allergy have also been reported, with the proteins responsible including gluten proteins, cm proteins and non-specific lipid transfer proteins. the patient was a -year-old man who visited the hospital with acute urticaria just eat bread before run ( minutes). according the components of bread. it was formed by a mixture of wheat, rye and barley. with a history (for several years) of episodes of severe urticaria after intake a mixture of cereals and/or different kinds of beer. results: prick test and specific ige with wheat, rye and barley were negative and the proteins from allergenic extract from these cereals, and also the gliadins and glutenins fractions were transferred onto a pvdf membrane to carried out a western blot technique with the patient's serum. the patient's serum recognized several proteins from wheat and millet gliadins not compatible in molecular mass with a ω -gliadins. the association of w gliadin as responsible for the symptoms produced after the intake of products containing wheat and exercise is well referenced but in the case of this patient could have other proteins involved as triggers of their symptoms. suksawat y phramongkutklao hospital, bangkok, thailand background: food-dependent, exercise induced anaphylaxis (fdeia) is an anaphylactic condition that develops in patients who ingest specific food followed by exercise. a variety of foods have been described to be the cause including shellfish, wheat and vegetables. the mechanisms of fdeia is believed that exercise increases allergen absorption or decreases threshold of mast cell. the investigations such as skin prick test or specific ige for food are useful because food sensitization is demonstrated. however, a challenge test including ingestion of suspected food followed by exercise is the only method to diagnose this disease. we report a case of fdeia in a -year-old adolescent male. result: he presented with generalized urticaria and hypotension after eating a barbecue buffet which was one hour followed by playing taekwondo. after treatment with intramuscular adrenaline, antihistamine and systemic steroid, his condition was improved. the barbecue buffet consists of many kinds of food including shrimp, squid, salmon and pork meat which were previously tolerated. he had no past history of anaphylaxis or drug allergy. he was referred to our allergy unit for investigation. we performed skin prick test with food allergens and many kinds of fresh foods that he ate on that day and the result was positive to shrimp ( mm. in diameter). three-day challenge protocol was set up a month after recovery and we used aspirin as a cofactor. on the first day, open challenge for gram of shrimp was administered and the result was negative. on the second day, exercise challenge test based on the american thoracic society guideline was also negative. however, on the last day, he developed generalized urticaria five minutes after the same exercise challenge test which was hour preceded by aspirin intake and gram of shrimp ingestion. but his vital signs appeared to be stable. the patient was administered intramuscular adrenaline and antihistamine with full recovery. he was strongly advised to avoid shrimp for - hours before exercise and carry an adrenaline autoinjector. the-three day challenge protocol is a definite tool to confirm the diagnosis of fdeia. a correct diagnosis is important to avoid unnecessary restricted diet. | food dependent exercise induced anaphylaxis in peach allergic patient-case report consumption of different types of food (pancakes with cream cheese and fruit, peach, chinese dish, sandwiches-all eaten on other occasions without symptoms) and co-occurring physical exercise (dancing, shopping, walking). during diagnosis we performed spt with inhaled and food allergens (allergopharma), prick by prick tests with peach, banana, apple, pear and bread. we established the concentration of allergen specific ige (peach, wheat flour, peanuts, hazelnuts) and the level of ige specific to allergen components (immunocap isac). we performed exercise provocation test and open food challenge with peach. results: spt were negative with all tested food and inhaled allergens (inc.egg; milk; cocoa; tomato; carp; apple; banana; strawberry; rye flour; wheat flour; peanuts; hazelnut; citrus, d. farinae; d. pteronyssinus; grass; weeds; clad. herbarium; alt. tenuis; dog; cat; poplar; hazel; alder; birch; mugwort). prick by prick tests were positive with fresh peach. concentration of peach specific ige was . ku/l. in immunocap isac we found elevated levels of ige specific to ltps from different allergen sources (jug r - . ; pru p - . ; pla a - . ; tri a - . [isu-e]). open food challenge with a medium size peach was negative. exercise provocation test without allergen exposition was negative. exercise provocation test after eating a medium size peach concluded with severe lip and eyelids edema, followed by whizzing, dyspnea and urticarial. patient received adrenaline . mg im, steroids and antihistamines with good clinical effect. conclusion: patient was diagnosed with food dependent exercise induced anaphylaxis (fdeia) due to ltp allergy. she was advised to eat peeled fruit and vegetables, avoid cofactors of allergic diseases and carry rescue set (adrenaline, steroids and antihistamines). cases reports: we report case of fdeia and cases of wdeia. case : -year-old woman with intermittent severe allergic rhinitis and food allergies since childhood. in the last years she registered weekly episodes of fdeia (urticaria, angioedema, wheeze, drop of bp) especially when during effort and after alcohol intake. prick tests were positive for grass pollen, mugworth, ragweed, dust mites, celery, soy, sesame, pistachio, mango, honey and shellfish. she followed years of subcutaneous immunotherapy for grasses pollen. the fdeia episodes frequency and severity diminished during it. provocation test for celery and mango were positive, for alcohol, soy, sesame, pistachio, honey and shellfish were negative. case : -year old man with a -year history of acute gluten induced urticaria, recently developed episodes of wdeia (flushing, severe urticaria and angioedema, wheeze) when he went for gym. skin test was highly positive for wheat flour and dust mites, in vitro tests for omega gliadin and wheat-specific ige were positive. food challenge for wheat was positive. he followed oral immunotherapy for wheat. the wdeia episodes were rare and mild. case : -years old female with mild allergic rhinitis and controlled asthma, with wdeia (urticaria, angioedema, wheeze, drop of bp) in the last years. skin tests showed positive results for wheat flour, dust mites and dog hair. omega- gliadin and wheat specific ige were high, but the provocation test for wheat was negative meanwhile combined wheat and effort provocation test was positive. -year-old female athlete, otherwise healthy, experienced three episodes of fdeia following running sessions. two reactions were preceded by intake of salad containing lettuce, tomato and sunflower seeds and the third one occurred after eating celery salad. the patient denied occurrence of any symptoms with physical exertion or food ingestion alone. physical examination and blood testing did not reveal any abnormalities. the differential diagnosis was performed. in skin prick test and specific serum ige antibodies sensitization to house dust mite, grass and mugwort were found. the spt and specific ige assay to culprit and most common food allergens were negative. the molecular diagnostic has been applied (faber, caam, rome, italy). the test scored positive for art v, blo t, der f, der p, eur m, lol p, phl p. no positive results for available food molecules including celery, tomato, sunflower seeds and lettuce were found. the detection of culprit food in fdeia is of crucial meaning as the syndrome can be life-threatening. the molecular diagnostic has been applied already in diagnosis of wheat-dependent exercise-induced anaphylaxis (wdeia) proving ω- -gliadin sensitization in the majority of the cases. as the presented case did not reveal sensitization to culprit food in traditional allergy tests the molecular diagnostic was performed. this test did not show sensitization to culprit food either. however, not all of the molecules are available in molecular assays yet. cross-reactivity reaction to mugwort and grass has to be considered. the pathophysiological components of physical exertion has to be taken into consideration as well. this could contribute to the assessment of reasonability of molecular approach in diagnostic work-up for fdeia and to the establishment of standardized protocols for diagnosis and management of that syndrome. case report: food allergy to wheat is rare in adults, often reported in exercise-induced anaphylaxis. food-dependent exercise-induced anaphylaxis (fdeia) is a form of food allergy induced by exercise. fdeia symptoms can include urticaria/angioedema, respiratory and gastrointestinal manifestations and hypotension/shock. a -year-old male patient presented to the emergency department, was admitted after an episode of hives, hypotension and loss of consciousness. his consciousness was restored after treatment with epinephrine, glucocorticoids as well as fluids, and thereafter, the patient reported that the anaphylactic episode occurred when he started rapidly walking hour after eating a slice of pizza. he mentioned that the offended food was tolerated always when it was not followed by a physical exercise. review of his past medical history and family one were non-contributory with respect to this episode. the allergy skin prick testing for common foods revealed a positive response only to wheat, while and other laboratory test values were within normal ranges. the patient is discharged after instructions on the use of epinephrine auto-injector. he was also advised to avoid wheat containing products up to hours prior to physical exercise. our case demonstrated that fdeia can be characterized by the onset of anaphylaxis soon after physical exercise, when preceded by the ingestion of the responsible food. avoidance of the combination of the exposure to respective allergen and exercise is the most efficient precautive measure toward subsequent fdeia episodes. | residual exercise-induced allergic reactions after successful rush oral immunotherapies for milk and wheat method: we conducted roit for children (median . years old) with milk allergy and children (median . years old) with wheat allergy during - . after - days of the rush phase in the hospital and a slow-increasing phase at home, patients consumed the maintenance dose ( . g milk protein or g wheat protein). after at least three months of the maintenance phase without allergic symptoms, we conducted an exercise provocation test (ept) after eating the target food. if the ept was positive, we repeated it after a couple of years to check for remission. the presence or absence of eiars was based primarily on the results of epts but also on the clinical history in some cases. results: as of december , milk-and wheat-allergic patients were able to continue ingesting the maintenance dose (desensitization). in these patients, milk-and wheat-allergic patients underwent the first ept at a median of ( - ) days after roit, and the result was positive in ( . %) and ( . %) patients, respectively. among these ept-positive patients, milk-and wheat-allergic patients conducted a second ept at a median of ( - ) days after the first ept. the result of the second ept was positive in milk-and wheat-allergic patients. in addition, clinical histories of eiars were subsequently observed in milk-and wheat-allergic patients after negative results on an ept. altogether, ( . %) milk-and ( . %) wheat-allergic patients still had eiars even after getting desensitization as of december conclusion: patients with persistent milk and wheat allergy often have residual eairs even after three to five years of desensitization due to the administration of successful roit. abstracts | | anaphylaxis caused by omega- -gliadin initially diagnosed as idiopathic anaphylaxis: a case report tziotou m ; syrigos k ; syrigou e ; sinaniotis a department of allergy,, athens, greece; gpp,, athens, greece case report: we report the case of a -year-old man who experienced two episodes of wheat dependent exercise induced anaphylaxis, initially diagnosed as idiopathic anaphylaxis. first episode: the patient woke up in the morning and drove to his resort. while driving, he ate a piece of cheese and ham pie. when he arrived, he walked some meters to the garden and started feeling pruritus and dizziness. he lost consciousness and recovered by himself. he was carried to hospital where his vital signs were normal. the patient has a history of atrial fibrillation and has been on flecainide bid and aspirin at noon for the last four years. a month after the first episode he visited an allergist. skin prick tests to aeroallergens and prick to prick tests to the ingredients of the pie were negative. tryptase levels were within normal limits and skin biopsy was negative for mastocytosis. an endocrinology workup was also negative. the patient was prescribed an epinephrine autoinjector and was asymptomatic for eight months. second episode: that morning he had a cup of milk and two slices of toast for breakfast and started working in the garden. two hours later he experienced pruritus and urticaria and fell unconscious. his wife had to administer two epinephrine autoinjectors before he regained consciousness. after the second episode it was decided to start treatment with omalizumab. two months later he experienced an episode of urticaria while working in the garden. he could not recall what he had eaten before. based on history, we thought that a cofactor might contribute to the occurrence of anaphylaxis. we performed skin prick tests with peach (ltp) and gliadin, allergens associated with food dependent exercise induced anaphylaxis in our region. the test to gliadin was positive. specific ige in serum to omega- -gliadin was also positive, while specific iges to all ltps tested were negative. the patient was advised to avoid wheat and has been asymptomatic ever since. cases diagnosed with idiopathic anaphylaxis may actually be cases in which the culprit allergen has not been identified. detailed history and extensive workup may contribute to the successful management of these patients. written informed consent has been obtained from the patient. | wheat-dependent exercise-induced anaphylaxis (wdeia) and nsaids: clinical history is crucial conclusions :we present a case clinically compatible with wdeia with nsaids intake as augmenting factor. this case emphasizes that a carefully and thoroughly taken medical history is of crucial importance, otherwise wdeia can easily be unrecognized. as a result, non-allergic hyperreactivity to nsaids could be excluded and the diagnose of selective allergy to arylpropionic acids was made. exercise challenge test could not be performed in our case. case report: kounis syndrome (ks) has been defined as an acute coronary syndrome that manifests as unstable vasospastic or non-vasospastic angina, and even as acute myocardial infarction. it is triggered by the release of inflammatory mediators following an allergic insult. a -year-old woman with type ii diabetes and hypertension, and unstable angina pectoris as cardiovascular risk factors, has consumed chamomile tea. thirty minutes later, she developed generalized itching, skin rash, swelling of the face and the throat, chest tightness, dyspnea and syncope. the patient was transferred immediately to the emergency department, and sodium chloride, mg prednisolone, mg dexamethasone, mg methylprednisolone, ui heparin, mg voltaren were intravenously administered along the subsequent minutes under simultaneous treatment with oxygen therapy. an ekg examination is performed based on the patient disease's history, showing a . - mm st-depression on d -d leads, mm on v , and . mm on the v -v ones. in addition, . mm st-elevation on the avr and mm on the v derivation was observed, associated by a negative t-wave on the v , v , and avr leads. blood tests revealed a normal troponin i level (of . ng/ml). five hours later in the ekg was noticed: isolined st-segments, and negative t waves on the d , avr, and v leads. ultrasound examination revealed normal heart kinetics and function. following the heart changes, the patient was administered sol. heparin ui twice i.v., nebivolol mg, plavix mg, atorvastatin mg, abstracts | monocinque mg, ordinary insulin ui s.c., glargine insulin ui s.c., and sol. furosemide mg i.v. the patient progressed favorably, and four days after the anaphylactic episode the ekg revealed a . mm st-depression on leads v , and v , negative t-wave on avl lead, and normalized one on the v one. this case emphasizes the role of serious allergic reactions as cause of acute coronary syndrome in patients with altered coronary arteries and food intake as cause of kounis syndrome. | recall urticaria in two young patients with alpha-gal-syndrome after tick bites case report: patient a (m, age ) had suffered from - anaphylactic reactions (hives, nausea, dyspnea and dizziness) within the past months. all episodes occurred - hours after ingestion of red meat, once with alcohol as a co-factor. all episodes started with a wheal measuring about . cm in exact the same spot where he had been bitten by a tick one year before. specific ige to galactose-alpha- , -galactose (alpha-gal) was positive ( . ku/l). skin prick testing using raw pork kidney suspension and intradermal testing with gelafundin ® % diluted : also showed positive reactions. we performed an oral challenge with cooked pork kidney under careful monitoring being able to reproduce the recall urticaria as described above with a cumulative dose of g pork kidney. we stopped the challenge and treated the patient with antihistamines and corticosteroids. patient b (m, age ) reported on several anaphylactic reactions within the past years with symptoms including abdominal pain, diarrhea, as well as dyspnea and loss of consciousness in one of the episodes. all episodes occurred several hours after ingesting food. furthermore the patient remembered a tick bite about years before the first anaphylactic reaction, which repeatedly became inflamed and only healed completely over months. every episode started with pruritus and a wheal in the area of the former tick bite (in loco). specific ige to galactose-alpha- , -galactose was positive ( . ku/l) as well as the skin prick testing with cooked pork kidney and intradermal testing with gelafundin ® % diluted : . this patient refused performance of oral challenge tests. an elimination diet of red meat for months resulted in the absence of the symptoms as described. the diagnosis of alpha-gal-syndrome with recall urticaria in loco was made in both cases. this symptom may also be useful in evaluating results of oral challenge tests as well as an important clinical sign in medical history. pali-schöll i ; meinlschmidt p ; purschke b ; hofstetter g ; einhorn l ; mothes-luksch n ; jensen-jarolim e ; jäger h background: insects have gained interest as alternative nutrient source for humans and animals. however, being a "novel food" in the industrialized part of the world, several safety aspects, like allergenicity, need to be thoroughly addressed. in the present work we evaluated the cross-recognition of ige from patients allergic to crustaceans, house dust mite or stable flies, using house cricket acheta domesticus (ad), desert locust schistocerca gregaria (sg) and mealworm tenebrio molitor (tm). we further investigated changes of immune-recognition in terms of ige-binding in differently processed insect extracts. method: migratory locust locusta migratoria (lm) was subjected to different extraction methods, enzymatic hydrolysis or thermal processing, whereas tm larvae (tml) were evaluated after different centrifugation modes and ph levels. results: we revealed that ige from patients with crustacean allergy shows cross-recognition of acheta domesticus, schistocerca gregaria and stable flies. ige from house dust mite allergic individuals binds to acheta domesticus and schistocerca gregaria. importantly, the cross-reactivity to lm can be deleted by enzymatic hydrolysis with different enzymes or heat treatment (cooking, autoclaving), but not by different extraction methods. changes of ph and varying centrifugation steps are not sufficient to reduce ige-binding to tml. our results show that patients allergic to crustaceans, house dust mite or stable flies-allergic patients cross-recognize desert locust and house cricket proteins, and crustacean-allergic patients also flies proteins. furthermore, we confirm that the appropriate food processing method of insect proteins can reduce the risk of cross-reactivity for crustaceans-and house dust mite-allergic patients. the study was supported by the austrian science fund fwf (grant sfb f -b to ejj). results: the mean age at diagnosis was years in children and years in adults, more frequent in males ( : ). in the pediatric group, three had first-degree relatives with eoe and three had celiac disease. two children had performed milk oral immunotherapy and five adults aeroallergens subcutaneous immunotherapy. most of them were atopics with sensitization to aeroallergens ( . % of children and . % of adults) and food allergens ( % of children and . % of adults), without statistically significant differences. the most frequent foods were fruits and nuts in both groups. we found significant statistical differences in fruits ( % of children abstracts | and . % of adults; p = . ) and cereals sensitization ( % of children and . % of adults; p < . ). in the clinical presentation we observed significant statistical differences in impaction ( . % of children and . % of adults; p < . ), dysphagia ( . % of children and . % of adults; p < . ) and abdominal pain ( % of children and . % of adults; p = . ). in the endoscopic findings children had more frequently exudates ( . %; p < . ) and adults had esophageal trachealization ( %; p < . ). significant statistical differences were found in the treatment with topical corticosteroids ( % of children and . % of adults; p < . ) obtaining a variable positive response. . % of patients in both groups received food elimination diet, % with four or more foods. conclusion: eoe presents differences in the sensitization profile, clinical manifestations and endoscopic findings according to the age of presentation. the response to pharmacological treatment is variable and a high percentage of patients receive food elimination diets. it is a pathology difficult to control, therefore new non-invasive techniques would be useful in order to facilitate its management. | modulation of gut microbiota in patients with nickel allergy and ibs after diet and probiotics supplementation mb ; garcía-figueroa be department of allergy department of allergy fang l united states | bcx improves health-related quality of life in hereditary angioedema with c -inhibitor deficiency bygum a fang l former yugoslav republic of; division of clinical immunology huissoon a bygum a ; panovska vg united states callejas fdb alobid i ; muñoz-cano r izquierdo i icahn school of medicine at mount sinai method: the case report form was developed by experienced allergists, and the web-based registry was established in cooperation with a professional medical software team. twenty-two departments from hospitals took part during the first year %), whereas in adults, drugs ( . %) were more common than foods ( . %). the most common food triggers were eggs ( . %), milk ( . %), and walnut ( . %) in children, and shrimps ( . %), wheat ( . %), and crab ( . %) in adults. among drug triggers in adults, antibiotics ( . %) were the most common cause followed by nsaids ( . %), and h -blockers ( . %). the onset time was≤ minutes in . %. in children, home was the place of occurrence in more than half of the cases, whereas adults experienced anaphylaxis in out-of-home settings more often than children. cofactors were present in %. among the cases registered via the emergency department of participating hospitals, epinephrine was administered in . % ( . % in adults, . % in children) and the route of administration was im in . %, iv in . %, both im and iv in . %, and subcutaneous in . %. the number of epinephrine administration was single in % conclusion: this multicenter prospective registry would provide a better understanding of anaphylaxis, and provide visionary modalities to improve the management and prevention of anaphylaxis in future a case of kounis syndrome after chamomile tea consumption characterized by symptoms related to esophageal dysfunction and esophageal mucosal infiltration by eosinophils objective: characterize patients (pts) with eoe diagnosis and analyze the differences between pts with diagnosis at pediatric (ch, < years old) and adult age epicutaneous tests(epict)], serum total ige and eos, findings in upper digestive endoscopy (ude) and biopsies. the correlation between food sensitization, clinical severity (visits to er services or hospitalization due to complications of eoe, sclin) or severe histology results: pts ( % male, average age ± years) ad and , respectively. % ch and % ad were atopics. the most frequent symptoms of eoe were dysphagia ( %) and gastroesophageal reflux( %) in ch; impaction( %) and dysphagia( %) in ad. % ch and % ad had aeroallergens sensitization. % ch and % ad had food sensitization. the most frequent positive tests were for ch: spt to milk( %) and shellfish( %), epict to shellfish( %) and meat( %); for ad: spt to milk( %), fresh fruits and nuts both %), epict to shellfish( %) and meat( %). ude showed: % striation and white plaques in % ch shist ( %) was associated with sclin ( %), p = . in ch; but this was not observed in ad group there was no correlation between food sensitization and sclin or shist in both groups(p > . ). the average values of serum total ige (kua/l) were in ch and in ad; eos were and , respectively in ch and ad allergy unit-fondazione policlinico universitario a. gemelli, università cattolica del sacro cuore conclusion: ltp with a fixed dose ( iu in ml) of ready-touse shp led to fewer severe attacks, a higher proportion of attack-free patients, and a clinically meaningful and statistically significant reduction in cumulative attack severity and daily severity in hae patients relative to placebo. background: c -inh-hae is a rare, potentially life-threatening disease characterized by episodes of subcutaneous and/or submucosal swelling. apex- was a phase , double-blind, placebo-controlled study to evaluate the prevention of attacks with bcx , a once daily oral kallikrein inhibitor, in patients with c -inh-hae.method: patients with c -inh-hae with a history of at least hae attacks per month were randomized to receive four different doses of bcx ( mg, mg, mg, . mg) or placebo for days. blood samples for bcx concentrations and kallikrein inhibition were obtained from patients before dosing and for hours post-dose on day . pk analyses and pk-pd modeling were done in phoenix winnonlin v . and sas v . . the pk population included , , , and subjects in the mg, mg, mg, and . mg groups respectively.after daily dosing achieved steady state, c max was reached at a median of - hours after dosing. there was a greater than dose proportional increase in exposure (auc tau and c max ) over the . -mg to -mg dose range, with an approximate -fold increase in exposure with a . -fold increase in dose. at doses ≥ mg, which showed statistically significant and clinically meaningful reductions in hae attack rates, geometric mean plasma trough concentrations (c tau ) were maintained at or above the minimum target concentration ( -fold ec ) estimated to be required for adequate plasma kallikrein inhibition. percentages of study subjects at steady-state with bcx plasma concentrations> -fold ec were %, %, % and % in the . , , , and mg dose groups, respectively. a -mg dose provided a mean c tau of slightly above . fold ec , with a corresponding reduction in hae attack rate of % (p < . ) compared with placebo. consistent with the exposure data, a dose dependent inhibition of kallikrein was observed with bcx treatment over the dose range. the drug effect on kallikrein inhibition was highly correlated with exposure (r = . ). in patients with c -inh-hae, bcx treatment at doses ≥ mg resulted in clinically meaningful reductions in the mean weekly hae attack rate. concentrations of bcx at doses ≥ mg were maintained at or above a c tau of -fold the kallikrein inhibition ec in most patients, and kallikrein inhibition was highly correlated with bcx plasma concentrations.background: c -inh-hae is a rare, life-threatening disease characterized by recurrent episodes of subcutaneous and/or submucosal swelling that lead to considerable morbidity and a poor quality of life (qol). apex- was a phase , double-blind, placebo-controlled study to evaluate the prevention of attacks with bcx , a once daily oral kallikrein inhibitor, in patients with c -inh-hae.method: patients with c -inh-hae with at least hae attacks per month were randomized to four different bcx doses ( mg, mg, mg, . mg) or placebo. subject-reported qol assessments were conducted at the start and end of treatment using the disease specific angioedema quality of life (ae-qol) questionnaire that measures domains (function, fatigue, nutrition, fear/ shame) and has minimal clinically important difference (mcid) of points. the changes from baseline in total and domain scores was compared between the treatment and placebo groups. modified angioedema activity score (aas) values across domains (daily activities, appearance, physical discomfort, overall severity) were calculated for each attack and a total score was derived for each subject by summing scores from each attack. total scores were compared to placebo using an ancova model with adjustment for qualifying attack rate. reduction of attacks was statistically significant for all top doses and there was a dose related increase in adverse events.results: in the mg dose group, qol assessed by ae-qol was significantly improved after weeks of treatment compared to placebo for ae-qol total score (- . , p < . ) as well as across all domains (function: - . , p = . ; fatigue: - . , p = . ; fears/shame: - . , p < . ; nutrition: - . , p = . ). all other treatment groups showed a trend towards improvement. qol improved the most in the mg group, and % of subjects in the mg group showed ae-qol reduction of more than points. disease activity as assessed by the aas was significantly reduced in the mg, mg and mg dose groups as compared to placebo, whereas there was no significant reduction in the . mg dose group. results: there were no marked differences in the age, sex, total ige titer, comorbidity of bronchial asthma and atopic dermatitis or the starting dose of rush oit among the groups. all patients in the low-bmfi group achieved ≥ % of the target dose, whereas . % of the middle-bmfi group and % of the high-bmfi group failed to achieve the target dose (p < . ). results: a total of infants were diagnosed with food allergy (ige-mediated and mixed type) at our center during the study period, of these patients underwent prick test for inhalant sensitivity, and ( . % male) of these were younger than years of age. conclusion: sensitivity to inhaled allergen were found in of ( . %) patients with food allergy. therefore, we believe that inhalant sensitivity should be evaluated in these patients. there is also a requirement for further studies to identify the influence of inhaled antigens on the disease activity of patients with allergic conditions. method: in this study, we aimed to perform the metabolic profiling of severe profilin mediated food allergic patients looking for biomarkers that might both, predict the prognosis of the disease and understand the molecular mechanisms of inflammation underneath. other allergic patients (mild and moderate) and non-allergic were recruited in the study as comparative groups. the allergic patients class was predicted using a mathematical algorithm from non-allergic vs severe model results: plasma samples from non-allergic subjects, mild, moderate and severe allergic patients were measured using gas chromatography coupled to mass spectrometry (gc-ms). the samples were from different hospitals in spain covering the areas with the highest pollen exposure. the metabolic profile was composed of metabolites for each sample. results after the statistical analysis showed differences between the groups. firstly, a clear reduction of several abstracts conclusion: we found, as expected, a predominance of males with eoe diagnosis. ch were more frequently atopic and had aeroallergen and food sensitization. impaction and esophageal stenosis were more frequent in ad than ch. shist was associated with sclin only in ch. method: three children with ee, boys aged . - years, were assessed over months to years. results: cases developed in infancy. one had dyspepsia and low weight gain in infancy soon after feeding began. another had symptoms in infancy but not diagnosed until age with dysphagia and esophageal stricture.. the third case was diagnosed at years old after episodes of food impaction in the esophagus beginning at age . all cases had allergic comorbid diseases including atopic dermatitis in all and allergic rhinitis in . skin prick tests were positive to several food allergens (cow's milk, egg protein) in , to dust mite in and to pollens in . serum total ige levels ranged from to iu/ml. eosinophils in peripheral blood were elevated in all , reaching %- %. treatment included restricted diet and topical budesonide - mg daily depending with periodic endoscopic biopsy.in all cases clinical improvement occurred by one month of treatment, with endoscopic confirmation. morphological improvement fol- the study aimed to evaluate the effects of probiotic supplementation, in addition to diet, in ibs and snas patients, in terms of modulation of faecal microbiota population, reduction of gi and cutaneous symptoms, increase of patient's quality of life and modification of gut dysbiosis.method: forty patients aged between and years, affected by ibs, ni sensitization and ltp sensitization were enrolled to evaluate gut dysbiosis. dna extraction method (next generation sequencing) with commercial kit (microbiopassport ® ) was performed on stool samples. ibs patients were divided in two groups, according a gluten free diet prescription or a low fodmaps diet prescription for three months. similarly, (suspected) snas patients (confirmed by % ni sulfate in petrolatum patch test) were prescribed a low ni diet ( μg/kg nickel content during the first four weeks and then up to μg/kg up to three months). two ltp (lipid transfer protein) sensitized patients underwent a ltp free diet.gut dysbiosis was re-assessed after a fixed probiotic supplementation. a sex-age matched group of individuals without history of ibs, snas or any gastrointestinal disease was considered as control.gastrointestinal symptoms were evaluated using the visual analogue scale before and after treatment. conclusion: our preliminary findings suggest that probiotic implementation could be useful in patients with snas on a low-ni diet to increase population diversity, which could contribute to restore the intestinal homoeostatic conditions. key: cord- - sk wc authors: wu, jianping; mok, chee-keng; chow, vincent tak kwong; yuan, y. adam; tan, yee-joo title: biochemical and structural characterization of the interface mediating interaction between the influenza a virus non-structural protein- and a monoclonal antibody date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: sk wc we have previously shown that a non-structural protein (ns )-binding monoclonal antibody, termed as h , can significantly reduce influenza a virus (iav) replication when expressed intracellularly. in this study, we further showed that h binds stronger to the ns of h n than a/puerto rico/ / (h n ) because of an amino acid difference at residue . a crystal structure of h fragment antigen-binding (fab) has also been solved and docked onto the ns structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. in one of the models, the predicted molecular contacts between residues in ns and h -fab correlate well with biochemical results. taken together, residues n and t in h n ns act cooperatively to maintain a strong interaction with mab h by forming hydrogen bonds with residues found in the heavy chain of the antibody. interestingly, the pandemic h n - and the majority of seasonal h n circulating in humans since has n in ns , suggesting that mab h could bind to most of the currently circulating seasonal influenza a virus strains. consistent with the involvement of residue t , which is well-conserved, in rna binding, mab h was also found to inhibit the interaction between ns and double-stranded rna. residues - in h n -ns are sufficient for its interaction with mab h . as described previously , the deletion of residues - of ns abolished its interaction with mab h . these residues lie in the helix α (residues - ) of h n -ns (rbd) and are well conserved between h n , h n and h n viruses (fig. a) . this is consistent with the ability of mab h to bind to both avian h n and seasonal iavs . in order to determine if the helix α of h n -ns (rbd) is sufficient for the interaction with mab h , enzyme-linked immunosorbent assay (elisa) was performed by using a synthetic peptide h n -ns - mer corresponding to helix α . as shown in fig. b , mab h bound to h n -ns - mer in a dose dependent manner, indicating that the helix α of ns (rbd) is sufficient for its interaction with mab h . in contrast, there was no binding between mab h and an irrelevant control peptide of similar molecular weight. within helix α , there is only one amino acid difference between h n and h n -pr , namely n in h n -ns and s in h n -pr -ns (fig. a) . to determine if residue in ns is involved in its interaction with mab h , recombinant h n -ns (rbd) and h n -pr -ns (rbd) proteins were bacterially expressed and purified for elisa. the elisa readings were similar for h n -ns (rbd) and h n -pr -ns (rbd) when high concentrations of proteins were coated on the plate (fig. c) . however, the readings were significantly higher for h n -ns (rbd) at lower protein concentrations, indicating that that the single amino acid difference between helix α of h n -ns (rbd) and h n -pr -ns (rbd) affects their interactions with mab h (fig. c ). residue in ns is critical for its interaction with mab h . to further define the contribution of residue in h n -ns to the interaction with mab h , two mutant proteins in which n was mutated to a and s respectively, were generated. comparative elisa showed that mab h bound to ns (rbd)-wild-type (wt) stronger than ns (rbd)-n s when different amounts of protein were coated on the plate and analyzed with μ g/ml of mab h ( fig. a) . similarly, when a fixed amount of protein ( μ g/ml) was coated on the plate and analyzed with different concentrations of mab h , the binding to ns (rbd)-n s was significantly lower than ns (rbd)-wt (fig. b ). furthermore, when n was substituted with a at residue , the interaction between mab h and ns (rbd) was totally abolished in all the conditions tested ( fig. a,b) . this result suggests that the difference at residue is the main reason for the stronger binding of mab h to h n -ns (rbd) when compared to h n -pr -ns (rbd) (fig. ). since both n and s are polar amino acids while a has no side chain, it is probable that the formation of hydrogen bonds is important for the interaction between mab h and ns . mab h binds differently to h n -pr virus when compared with mutant viruses carrying substitution at residue in ns . since mab h binds to bacterially-expressed ns protein of h n and h n -pr with different affinities, it is important to investigate whether this holds true for ns expressed in infected cells. thus, recombinant pr (rgpr ) viruses expressing the ns protein containing a single amino acid substitution at residue (rgpr -ns -s a and rgpr -ns -s n) were generated using a reverse genetics system. subsequently, a cells were infected with each virus at a low multiplicity of infection (moi) of . respectively and plaque assay was used to determine the amount of virus secreted at different time points post-infection. as shown in fig. a , although the viral titer was slightly lower in the case of rgpr -ns -s n infection, the overall growth rates of wt and mutant viruses were similar from to hours post-infection (h.p.i.). this is consistent with a previous report showing that substitution of residue in ns does not affect viral replication in vitro . next, t cells were infected with moi of viruses and cell lysates were collected at and h.p.i. to determine the rate of viral protein synthesis. consistent with the virus growth kinetics, the expressions of structural proteins np and m were similar for all viruses at both time-points (fig. b ). the level of ns , as determined by using a rabbit anti-ns polyclonal antibody, was also comparable for all viruses. however, mab h bound to rgpr -ns -s n virus significantly stronger than rgpr -ns -wt virus containing s residue in mismatches are shown in white letters. ns (rbd) is composed of α -helices as shown above the sequence. the region corresponding to helix α (residues - ) is boxed. (b) peptide elisa was performed to determine the region of ns sufficient for binding to mab h . wells were coated with serially diluted h n -ns - mer or a negative control peptide and probed with μ g/ml mab h . * indicates statistically significant difference of p < . when compared with control peptide. (c) comparative elisa was performed to determine the ability of ns (rbd) of h n -pr and h n to bind to mab h . wells were coated with serially diluted proteins and probed with μ g/ml mab h . data shown represents result from three independent experiments and error bars represent standard deviation (sd) of the experiment carried out in duplicates. *indicates statistically significant difference of p < . when compared with h n -pr -ns (rbd). scientific reports | : | doi: . /srep ns , supporting the above results that n in ns is preferred over s for the interaction with mab h . as expected, mab h did not bind to rgpr -ns -s a virus. to further define the interaction interface between mab h and ns , attempts were made to co-crystallize the complex but failed. however, the h -fab alone was found to crystallize in space group p ( ) and the structure was solved by molecular replacement using the structure of bl - (pdbid: q q) as the starting model and refined to the crystallographic r-factor of . % (deposited in the pdb under accession code b m). the refinement statistics are shown in table . previously, we demonstrated that the intracellular expression of h -scfv in mammalian cells reduced the replication of pr virus. since the three dimensional structure of h -fab has been solved, a commercially available lipodin-ab reagent (abbiotec), which is a protein transfection reagent dedicated to the transport of antibodies into living cells, was used to deliver h -fab into a cells. the cells were then subjected to infection so as to determine if h -fab has an impact on viral replication. when either h -fab or a -fab (which is derived from a negative-control antibody binding to the spike glycoprotein of sars coronavirus) was transfected into a cells using lipodin-ab reagent, intracellular accumulation of fab was observed even up to h post transfection (fig. a) . the intracellular accumulation of fab was also observed at as early as h post transfection (data not shown). the transfection efficiency was about % and most of the fab molecules were evenly distributed in the cytoplasm but there were some punctate staining which could be due to aggregation of fab inside a cells. in contrast, no intracellular accumulation of fab was observed when a -fab or h -fab was added to the cells without lipodin-ab reagent. as shown in fig. b , the delivery of fab did not have any significant effect on cell viability at either or h post transfection. upon successful delivery of fab, its biological function was assessed in influenza a virus infected cells. as mab h binds strongly to rgpr -ns -s n virus (fig. ) , a cells were infected with rgpr -ns -s n virus after transfection of h -fab. cell lysates were collected at , , h.p.i. to determine the rate of viral protein synthesis. as shown in fig. c ,d, the level of viral m protein at h.p.i. was significantly reduced in h -fab transfected cells when compared to a -fab transfected cells. at h.p.i., the average reduction in normalized m expression in h -fab transfected cells was % when compared to a -fab transfected cells. at h.p.i., a slight reduction in m expression (~ %) was also observed in h -fab transfected cells but it is not statistically significant. this result suggests that the successful delivery of h -fab into living cells could reduce viral replication by affecting certain function(s) of ns in the infected cells. next, computational modelling was used to study the complex between this high resolution h -fab structure and the published structure of h n -ns (rbd) of h n /a/crow/kyoto/t / strain (pdb id: z a). this was conducted by using haddock on water-refined models, including an analysis of energy contributions from van der waals interaction, electrostatic interaction, restraints violation and buried surface area . as comparative elisa in this and previous studies showed that residues n and t in ns (rbd) are important for the interaction with mab h , they were defined as active residues involved in the binding interaction to generate a series of models of the ns (rbd) and h -fab complex. all the models of ns (rbd) and h -fab complex were found to cluster into groups, in which there were at least two conformations of the ensemble showing backbone root-mean-square deviations at the interface of less than . Å. as additional elisa results showed that residues, namely s , r , and g , in ns are not involved in interaction with mab h ( figure s ), this information was used to distinguish between the models in these clusters. of all energetically best models generated, predicted models were grouped into cluster . the average buried surface area was . ± . Å , and rmsd from the overall lowest-energy structure was . ± . Å. among them, the best predicted model from this cluster showed good agreement with our comparative elisa data, since only residues n and t were predicted to be involved in the interaction, while the side chains of s , r and g were either distal from the interface (s and r ) or inaccessible (g ) to the binding partner of h -fab (fig. ) . by analyzing the polarity of the amino acids and distance between them in this model, it is predicted that residues n and n in the variable domain of heavy chain (vh)-complementarity determining region (cdr ) of h -fab could form hydrogen bonds with the side chain of n in ns (rbd). in addition, the side chain of t in ns (rbd) could form hydrogen bonds with residue r in vh-cdr . in contrast, vh-cdr , vh-cdr and all the cdrs in the variable domain of light chain (vl) are unlikely to be involved in the interaction as they are distal to helix α of ns (rbd). on the other hand, predicted models were grouped into cluster . for these refined structures analyzed, the average buried surface area was . ± . Å , and rmsd from the overall lowest-energy structure was . ± . Å. based on the best predicted model from this cluster ( figure s ), it was predicted that t of ns (rbd) could form the hydrogen bond with n of vh-cdr while n of ns (rbd) could form the hydrogen bond with n of vl-cdr . these predictions are in agreement with the results shown in fig. and in our previous publication . however, this model also predicted that r of ns (rbd) could be involved in the interaction with h -fab because it was in close proximity to two residues in vl-cdr . in this model, the distance between r of ns (rbd) and y of vl-cdr was . Å while the distance between r of ns (rbd) and s of vl-cdr was . Å. thus, this model does not agree with the results from comparative elisa which showed that substitution of r of ns (rbd) with k did not affect its interaction with mab h ( figure s ). lastly, another predicted models were grouped into cluster . the average buried surface area was . ± . Å , and rmsd from the overall lowest-energy structure was . ± . Å. based on the best predicted model from this cluster ( figure s ), the contacts between n and t with residues in h -fab were the same as described above for the model from cluster . however, this model also predicted that r of ns (rbd) could be involved in the interaction with h -fab because it was in close proximity to three residues in vl-cdr . in this model, y , s and y of vl-cdr were found to be at . Å, . Å and . Å from r of ns (rbd) respectively. thus, this model does not agree with the results from comparative elisa which showed that substitution of r of ns (rbd) with k did not affect its interaction with mab h ( figure s ). overall, the predicted model from cluster is consistent with our comparative elisa data and suggests that residues n and t are important for the binding between ns (rbd) and h -fab because their side-chains could make hydrogen bonds with residues in the vh-cdr of the fab. in addition, r of ns (rbd) was distal from the antibody-antigen interface, which is consistent with the results from comparative elisa ( figure s ) showing that substitution of r of ns (rbd) with k did not affect its interaction with mab h . in contrast, the predicted models from cluster and cluster do not agree with the results from comparative elisa. mab h disrupts ns and dsrna interaction. ns (rbd) forms a symmetric six-helical homodimer, which binds to dsrna. the key residues in ns involved in the interaction with dsrna are t , d , d , r , r , r , l , s and t , most of which are positively charged residues and mainly clustered in the middle of helices α /α ′ of the rbd , . to investigate whether mab h hampers dsrna-ns interaction in vitro, an alphascreen assay was carried out. in this experiment, glutathione s-transferease (gst)-tagged h n -ns (rbd) protein, which was produced in e. coli, was incubated with synthetic -nucleotide sirna ( nt-sirna) followed by addition of streptavidin coated donor beads and anti-gst-conjugated acceptor beads which recognize biotinylated rna and gst-tagged protein respectively. if the interaction between ns and nt-sirna brings both beads to close proximity, transfer of excitation energy from donor beads into acceptor beads will yield a luminescent signal (fig. a ) . when ns (rbd) was pre-incubated with different concentrations of mab h followed by addition of nt-sirna, acceptor and donor beads, the luminescent signal decreased at high concentration of mab h (fig. b ). the luminescent signal was reduced by ~ % and ~ % at mab h concentrations of and μ m respectively (fig. b) , suggesting that the binding of mab h to ns (rbd) can block the interaction of ns with dsrna. on the other hand, the negative control mab a did not reduce the luminescent signal at all the concentrations tested. furthermore, when mab h , ns and nt-sirna were mixed simultaneously, μ m of mab h could reduce the signal by about % (fig. c) , which suggest that mab h also directly competes with dsrna to bind to ns . the ns protein of influenza virus is a multi-functional protein that is involved in key aspects of the virus replication cycle . the ns (rbd) consists of the first amino acids and contains residues critical for dsrna binding . the dimerization of the rbd is a prerequisite for its rna binding activity . since the ns protein is an intracellularly expressed viral protein, which is subjected to less host selective immune response and thus has lower mutation rate, antibodies targeting this protein could be helpful for the development of therapeutic treatment of influenza a infection. indeed, our previous study showed that mab h binds to the highly conserved t residue in ns and reduces viral replication of h n -pr in mammalian cell lines . in this study, comparative elisa showed that helix α of ns (rbd) is sufficient for its interaction with mab h (fig. ) . while mab h binds to ns (rbd) of both h n and h n -pr , the binding affinity to the homologous h n viral protein is higher. interestingly, a single amino acid difference at residue in helix α of ns (rbd) of h n and h n -pr is found to be critical for the interaction with mab h (figs and ). by using either purified ns protein or ns expressed in infected cells, our results showed that the interaction of mab h with ns is stronger when residue is n than when it is s and is abolished when the residue is an a. to understand the pattern of polymorphism at residue in ns , sequences were retrieved from niaid influenza research database (http://www.fludb.org) and analyzed ( table ). sequence analysis of avian h n isolates revealed that % of them have n and % have s suggesting that mab h has the ability to bind to the majority of avian h n isolates. interestingly, a recent computational study compared the viral proteins of highly and lowly pathogenic h viruses and identified s to n substitution as a potential marker of pathogenicity of avian influenza virus subtype h . as for human isolates, the majority ( %) of seasonal h n isolated before have s in ns like h n -pr . however, almost all the pandemic h n (pdmh n ) isolates have n in ns . as for seasonal h n , % of viruses isolated before have n and this percentage increased to % for those isolated from to . thus, mab h is expected to bind to the majority of circulating seasonal influenza viruses. by solving a crystal structure of h -fab and docking it onto the ns (rbd) of h n with the haddock program, molecular contacts made by residues t and n at the interface of antibody-antigen complex were predicted (fig. ) . based on one of the energetically best models generated, the side chain of residue n in ns (rbd) could form hydrogen bonds with the side chains of residues n and n of vh-cdr of h -fab. meanwhile, residue t in ns (rbd) seems to interact with residue r in vh-cdr . these predictions are consistent with the results of binding assays performed using substitution mutants of ns as described above and in our previous study . while the predicted d model of the antibody-antigen complex gives us some clues on how residues and in ns (rbd) interact with h -fab, it may be able to accurately reveal the contributions of all molecular contacts between ns (rbd) and h -fab. hence, further studies could focus on the use of other epitope mapping methodologies , besides crystallography, to obtain a more precise map of the molecular contacts at the antigen and antibody interface. furthermore, an alphascreen assay showed that mab h could inhibit the interaction between ns and dsrna (fig. ) . while both residues and are located in helix α of the ns (rbd), it has been shown that t , but not s , is one of the key residues involved in dsrna binding . thus, the interaction between t and h -fab predicted in the docked model is consistent with the ability of mab h to inhibit the interaction between ns and dsrna. although a previous study has demonstrated that mutation at residue did not affect the affinity of ns (rbd) for dsrna , our study suggests that the side chain of n makes crucial contacts with mab h to stabilize the antibody-antigen complex. as such, when the side chain is not present in the case of gst-tagged ns protein was incubated with biotinylated dsrna and the interaction between ns and dsrna could bring streptavidin-coated donor bead and anti-gst-conjugated acceptor bead close to each other. when excited at nm, singlet oxygen molecules ( o ) are produced from donor beads, which react with acceptor beads to produce light emission measured at - nm. (b) inhibition activity of mab h was determined by pre-incubating nm gst-tagged ns protein with serially diluted mab h or a negative control mab a . following that, luminescent signal was measured after the addition of nm biotinylated dsrna, acceptor and donor beads. (c) inhibition activity of mab h was determined by incubating nm gst-tagged ns protein, nm biotinylated-dsrna and serially diluted mab h or a simultaneously. following that, luminescent signal was measured after the addition of acceptor and donor beads. all readings obtained were normalized against that of samples in the absence of antibody. error bars represent sd of the experiment carried out in triplicates. *indicates statistically significant difference of p < . when compared to mab a . the n a substitution mutant, interaction with mab h is completely abolished (figs and ). this indicates that the interaction between the side chains of residue t in ns and residue r in h -fab is not sufficient to maintain the antibody-antigen interface. on the other hand, the t v and t a substitution mutants still retained some binding to mab h ( figure s ), presumably because of the hydrogen bonds between residue n and the heavy chain of the antibody. based the alphascreen assay, a high concentration of mab h (~ μ m) was required to reduce the interaction between ns (rbd) and dsrna by > % (fig. ) . on the other hand, the concentration of mab h required for binding ns (rbd) in elisa was in the nanomolar range (fig. ) . this discrepancy could be due to the differences between the two assays but it also suggests that the binding of mab h to ns could have other effects on ns besides disrupting its interaction with dsrna. indeed, our previous gel filtration and dynamic light scattering results suggest that the complex between h -fab and ns (rbd) is multimeric in nature and each oligomer could consist of molecules of ns (rbd) and molecules of h -fab . in contrast, ns (rbd) eluted out the gel filtration column as a dimer, as would be expected, in the absence of h -fab . in addition, the delivery of h -fab into a cells also caused a reduction in the replication of rgpr -ns -s n recombinant virus (fig. ) . while it is difficult to precisely define the biologically relevant quaternary structures of ns , several studies have shown that the ns has conformational plasticity and dynamic changes in the quaternary structure of ns are likely to be important for the different functions of ns in infected cells . hence, it is possible that the binding of h -fab to the ns expressed in infected a cells could affect the conformational plasticity of ns and/or its ability to interact with certain cellular factor, thus resulting in a reduction in viral replication. in future studies, advanced fluorescence microscopic techniques could be used to determine the effect of h -fab on the dynamic of ns inside infected cells. in summary, we have used biochemical and structural methods to characterize the interaction between ns and mab h . our results showed that helix α in ns (rbd) is sufficient for interacting with mab h and residues n and t in this helix are likely to make hydrogen bonds with the cdr of the antibody heavy chain. helix α is highly conserved and this is consistent with the ability of mab h to bind to different subtypes of iav. after solving a high resolution crystal structure of h -fab, a haddock-derived model of the antibody-antigen complex has been obtained and the molecular contacts predicted from this model are in agreement with results obtained from comparative elisa performed using ns mutants. this model may be used in antibody engineering experiments to increase the affinity of interaction between ns and mab h so as to increase mab h 's potency in viral inhibition. in addition, it may be useful in structure-based rational drug design to identify small molecule inhibitors of ns . cells. a , t and mdck cells were purchased from american type culture collection (manassas, va, usa). a cells were cultured in minimum essential medium (mem) (gibco). t and mdck cells were cultured in dulbecco's modified eagle's medium (invitrogen). both media were supplemented with % fetal bovine serum (hyclone), penicillin ( , units/ml)-streptomycin ( mg/ml) solution (sigma aldrich). all cell lines were maintained at °c with % co . ascites and rabbit polyclonal antibodies production. ascites were produced by injecting hybridoma cells into peritoneal cavities of pristine-primed balb/c mice. the protocol was approved by institutional animal care and use committee (iacuc) of the biological resource center, a*star, singapore (protocol number: ). in order to generate rabbit anti-h n -ns polyclonal antibody, gst-fusion ns protein was purified using the method as previously described . new zealand white rabbits were then immunized with this protein and bled as previously described . the protocol was approved by iacuc of the biological resource center, a*star, singapore (protocol number: ). all the animal procedures were performed in strict compliance with the recommendations of the naclar guideline in singapore. all efforts were made to minimize the suffering and euthanasia was performed using carbon dioxide. protein expression and purification. ns (rbd) (residues - ) of a/chicken/hatay/ (h n ) was pcr amplified from a full-length ns gene (accession no.: caj . ) and the ns (rbd) (residues - ) of a/puerto rico/ / (h n ) was pcr amplified from a full-length ns gene (accession no.: abd . ). the expression constructs were generated by inserting pcr product into pet sumo expression vector (invitrogen). ns mutants expression construct was generated by overlap pcr as described previously . all proteins were expressed and purified as previously described . the purified proteins were dialyzed against dialysis buffer ( mm tris-hcl, ph . , mm nacl) and concentrated to mg/ml in a centrprep- (amicon) for subsequent assays. crystallization of h -fab. mab h was purified from the ascites by using affinity chromatography and h -fab was obtained by papain cleavage as described previously . the purified h -fab was dialyzed against dialysis buffer and concentrated to mg/ml for subsequent crystallization. crystallizations were performed with the hanging drop vapor diffusion method at °c by mixing μ l of h -fab with μ l of reservoir solution and the mixture was equilibrated against μ l of reservoir solution. crystals of h -fab were grown against crystallization buffer containing % peg , . m nacl and . m bis-tris, ph . . these crystals grew to a maximum size of . mm × . mm × . mm over the course of days. single crystals were obtained by dissecting from multiple crystals. crystals were flash frozen ( k) in the above reservoir solution supplemented with % glycerol. a total of frames of a native data set with oscillation at . Å wavelength were collected for h -fab. all data sets were processed by hkl . most of the crystals diffracted rather weak and the scaled data sets were anisotropic with strong ice rings and high mosaicities. nevertheless, one of the data sets displaying scientific reports | : | doi: . /srep weak ice ring was able to scale to . Å at the mosaicity of . °. this data was used for structure determination and refinement with the statistic table listed as table . of h -fab were predicted using the online igblast tool (http://www.ncbi.nlm.nih.gov/igblast/). as the three-dimensional structure of ns (rbd) of a/chicken/hatay/ (h n ) has not been solved, the structure of ns (rbd) of a/crow/kyoto/t / (h n ) (pdb id: z a) was used instead. as shown in fig. a , there are amino acid differences between the ns (rbd) of these two strains but the sequence of helix α in the ns (rbd) is % identical. mutagenesis data from comparative elisa was used to generate a series of models for the complex between h -fab and ns (rbd) by using version . of haddock webserver , . along with the available individual structure, haddock utilizes the experimentally derived data to predict the complex structure. to achieve this, ns (rbd) was docked to h -fab using the easy interface of haddock webserver, where both residues n and t of ns (rbd) and amino acids in the cdrs of h -fab were defined as active residues involved in the interaction. in this docking experiment, only the variable domains of h -fab were used. enzyme-linked immunosorbent assay (elisa). elisa was performed as described previously . briefly, a synthetic peptide corresponding to residues - in h n -ns (apfldrlrrdqkslrgrgntlgld, chemically synthesized by gl biochem) or purified wt and mutant ns (rbd) proteins were serially diluted into . m carbonate-bicarbonate buffer, ph . . a -mer peptide corresponding to a fragment of the hepatitis c virus core protein (rpswgpidprrrsknlgkvidtltcgfap, chemically synthesized by genway biotech) was used as a negative control. proteins or peptides ( μ l) were then coated onto -well elisa plates (nunc) overnight at °c. the wells were blocked in % milk in pbs with . % tween (pbst) for h at °c followed by addition of μ l of mab h as primary antibody to each well and incubated at °c for - h. the wells were then washed in pbst followed by the addition of goat anti-mouse horse-radish peroxidase (hrp)-conjugated antibody (pierce) as secondary antibody and incubated at °c for h. tetramethylbenzidine substrate (pierce) was added and reaction was stopped using . m sulfuric acid. absorbance at nm was recorded using an absorbance reader (tecan infinite m ). recombinant viruses were generated with phw reverse genetic system as described previously . residue in ns was changed from s to a or n by pcr mutagenesis and the resulting dna were inserted into phw vector. this plasmid was co-transfected with another seven phw plasmids containing the other pr genomic dnas into t cells. days post transfection, culture supernatant was collected and used to infect mdck cells. when cytopathic effect (cpe) was visibly detected, culture supernatant was collected and used to infect naïve mdck cells. individual plaque was then amplified and viral titer was determined by plaque assay. virus infection and western blot analysis. % confluent t cells were infected with moi of wt or mutant viruses at °c for h. the medium was discarded and cells were rinsed with pbs. cell lysates were harvested at and h.p.i. in ripa buffer ( mm tris-hcl ph . , mm nacl, . % np , . % sodium deoxycholate, . % sds). then, μ g of total lysate were resolved using electrophoresis on an sds-polyacrylamide gel and transferred to a nitrocellulose membrane (bio-rad). antibodies against gapdh (santa cruz), np (millipore), and m (as described previously) were used. mab h and rabbit anti-h n -ns polyclonal antibody (as described above) were also used. after washing, the membrane was incubated with a hrp-conjugated secondary antibody (pierce) at room temperature for h. the membranes were then washed and detected with enhanced chemiluminescence substrate (pierce) using chemidoc ™ mp imaging system (bio-rad). multiple-cycle growth kinetics of recombinant virus. plaque assay was applied to determine the growth kinetics of rgpr -ns -wt, rgpr -ns -s a and rgpr -ns -s n recombinant viruses. % confluent monolayers of a cells were rinsed with pbs and subsequently adsorbed with . moi of wt and mutant viruses respectively for h at °c. the medium was discarded and the cells were rinsed using pbs and cultured in mem without serum at °c. supernatant containing virus was collected at , , , and h.p.i. respectively and subjected to plaque assay to determine viral titer. plaque assay in mdck cells. % confluent mdck cells were adsorbed with serially diluted supernatants containing viruses for h at °c. the medium was discarded and the cells were rinsed using pbs. the cells were overlaid with ml of dmem supplemented by . % agar and μ g/ml tpck-trypsin (thermo scientific). after incubation at °c for days, the cells were fixed using % formalin for h and stained using . % crystal violet solution. delivery of a -fab and h -fab into a cells. a cells were cultured in -well plate. the a -fab and h -fab were then transfected into % confluent cells by using lipodin-ab reagent (abbiotec) according to manufacturer's protocol with slight modification. briefly, μ l of fab solution ( μ g) was mixed thoroughly with μ l lipodin-ab solution and incubated for min at room temperature. μ l of serum-free mem medium was added to fab/lipodin-ab solution and then immediately added to the cells. the cells were washed with pbs once before the fab/lipodin-ab solution was added. the cells were incubated at °c in % co for h, washed once with pbs and subjected to viral infection and western blot analysis as described above. cell viability assay. cell viability was determined using wst- reagent (roche) according to manufacturer's protocol. briefly, μ l of wst- reagent was diluted in the μ l of culture media and added to a cells cultured in a transparent -well microplate and incubated for h, followed by measuring absorbance at nm. immunofluorescence assay. a cells grown on coverslip were transfected with fab as described above. approximately h after transfection, the medium was aspirated, washed with pbs once and replaced with serum-free mem medium and incubated at °c in % co for another h. the cells were then washed with pbs once and fixed with % paraformaldehyde for min and permeabilized with . % tritonx- for min, followed by blocking with % bsa in pbs for min. the cells were incubated with alexa fluor -conjugated goat anti-mouse igg antibody (invitrogen) for h. after washing, cells were stained with dapi before mounting. images were captured using an olympus fluoview fv laser-scanning confocal microscope. alphascreen-based dsrna binding inhibition assay. a nt-sirna previously reported to form complex with ns (rbd) was purchased from thermo scientific dharmacon (dharmacon, lafayette, co). the sirna was biotinylated at the ′ end of the sense strand and the sirna sequences were as follows: ′ -biotin-agacaccauuaugcugucuuu- ′ (sense) and ′ -agacagcauaauggugucuuu- ′ (antisense). the lyophilized sirnas were reconstituted in rnase-free water to a final concentration of nm. to express gst-tagged ns (rbd) of a/chicken/hatay/ (h n ) was pcr amplified from a full-length ns gene (accession no.: caj . ) and cloned into pgex- p- vector (ge healthcare). the expression and purification were performed as described previously . the purified gst-fusion protein was dialysed against pbs and the final concentration was determined using the coomassie plus protein assay reagent (thermo scientific). in vitro rna binding inhibition assay was carried out in -well proxiplate by using the alphascreen anti-gst kit (perkinelmer). in the first experiment, μ l of nm gst-tagged proteins were mixed with same volume of serially diluted mab h and incubated at room temperature for h. then, μ l of nm biotinylated nt-sirna was added into the binding mixture and incubated at room temperature for h before the addition of μ l of detection mixture containing . μ l of anti-gst (glutathione s-transferase) acceptor beads and . μ l of streptavidin-coated donor beads (perkinelmer). after another incubation at room temperature for h, luminescent signal was measured using an enspire multimode plate reader (perkinelmer) . in the second experiment, μ l serially diluted mab h was mixed with μ l of nm gst-tagged proteins and μ l of nm biotinylated nt-sirna simultaneously. after incubation at room temperature for h, detection mixture was added and measurement was made similarly. statistical analysis. two-tailed student's t test was applied to evaluate the statistical significance of differences measured from the data sets obtained in independent experiments. p < . was considered statistically significant. influenza-who cares orthomyxoviridae: the viruses and their replication new world bats harbor diverse influenza a viruses mixed infections of pandemic h n and seasonal h n viruses in outbreak advances in the development of influenza virus vaccines toward a universal influenza virus vaccine: prospects and challenges evasion of influenza a viruses from innate and adaptive immune responses viral m ion channel protein: a promising target for anti-influenza drug discovery influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance avian flu: isolation of drug-resistant h n virus influenza a virus strains that circulate in humans differ in the ability of their ns proteins to block the activation of irf and interferon-beta transcription virulence of h n avian influenza virus enhanced by a -nucleotide deletion in the viral nonstructural gene rna binding by the novel helical domain of the influenza virus ns protein requires its dimer structure and a small number of specific basic amino acids binding of influenza a virus ns protein to dsrna in vitro the influenza virus ns protein is a poly(a)-binding protein that inhibits nuclear export of mrnas containing poly(a) the influenza virus ns protein binds to a specific region in human u snrna and inhibits u -u and u -u snrna interactions during splicing the primary function of rna binding by the influenza a virus ns protein in infected cells: inhibiting the ′ - ′ oligo (a) synthetase/rnase l pathway influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i structural basis for a novel interaction between the ns protein derived from the influenza virus and rig-i binding of the influenza a virus ns protein to pkr mediates the inhibition of its activation by either pact or doublestranded rna loss of function of the influenza a virus ns protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol -kinase (pi k) influenza a virus ns protein activates the pi k/akt pathway to mediate antiapoptotic signaling responses identification of influenza virus inhibitors targeting ns a utilizing fluorescence polarization-based high-throughput assay synthesis and evaluation of quinoxaline derivatives as potential influenza ns a protein inhibitors novel influenza virus ns antagonists block replication and restore innate immune function antiviral activity of baicalin against influenza virus h n -pdm is due to modulation of ns -mediated cellular innate immune responses small interfering rna targeting the nonstructural gene transcript inhibits influenza a virus replication in experimental mice a new panel of ns antibodies for easy detection and titration of influenza a virus a monoclonal antibody binds to threonine in the non-structural protein of influenza a virus and interferes with its ability to modulate viral replication roles of the phosphorylation of specific serines and threonines in the ns protein of human influenza a viruses monoclonal antibodies targeting the hr domain and the region immediately upstream of the hr of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus haddock: a protein-protein docking approach based on biochemical or biophysical information conserved surface features form the double-stranded rna binding site of non-structural protein (ns ) from influenza a and b viruses structural basis for dsrna recognition by ns protein of influenza a virus assay optimization and screening of rna-protein interactions by alphascreen the multifunctional ns protein of influenza a viruses a complete map of potential pathogenicity markers of avian influenza virus subtype h predicted from expressed proteins current approaches to fine mapping of antigen-antibody interactions conformational plasticity of the influenza a virus ns protein comparing the antibody responses against recombinant hemagglutinin proteins of avian influenza a (h n ) virus expressed in insect cells and bacteria simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies haddock versus haddock: new features and performance of haddock . on the capri targets a dna transfection system for generation of influenza a virus from eight plasmids the pymol molecular graphics system, version we are grateful to r.g. webster for the pr based reverse genetics system. we thank members of the monoclonal antibody unit at institute of molecular and cell biology for technical assistance. this work was supported by the singapore ministry of health's national medical research council under its nmrc-cbrg scheme [grant no. nmrc/cbrg/ / ]. key: cord- - c wttxg authors: lin, huixing; zhou, hong; gao, lu; li, bin; he, kongwang; fan, hongjie title: development and application of an indirect elisa for the detection of antibodies to porcine epidemic diarrhea virus based on a recombinant spike protein date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: c wttxg background: as the major causative agent of swine viral diarrhea, porcine epidemic diarrhea virus (pedv) has caused massive losses to the economies of swine raising countries. accordingly, the serological detection of corresponding antibodies would be beneficial to diagnose pedv indirectly to control the disease. in this study, an indirect enzyme-linked immunosorbent assay (elisa) based on the recombinant truncated spike (s) protein of pedv was developed and validated. results: the reaction conditions of the developed indirect elisa were optimized. this indirect elisa was compared to indirect immunoinfluscent assay (ifa), and the overall coincidence rate was . % based on testing clinical serum samples with different pedv antibody levels. no cross-reactivity with other common swine pathogens was detected for the developed s indirect elisa. finally, the s indirect elisa was applied to detect serum antibodies of field samples collected from different pig farms in eastern china, and it presented an overall substantial agreement on the pedv infection status. conclusions: this established s indirect elisa is capable of detecting serum antibodies against pedv, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of pedv infection. porcine epidemic diarrhea (ped) is a highly contagious swine enteritis caused by porcine epidemic diarrhea virus (pedv), which belongs to the order nidovirales and family coronaviridae. the typical symptoms of ped are diarrhea, vomiting, and dehydration, which can be especially dangerous to suckling piglets [ , ] . the mortality of neonatal piglets younger than days old can approach % [ ] [ ] [ ] . ped first appeared in britain in , followed by an outbreak of diarrhea in several pig farms in belgium in [ ] . these outbreaks led to identification of a coronavirus-like particle named cv , which is now recognized as the classic pedv strain. in recent years, ped epidemics have become prevalent in swine-raising countries in asia, including south korea, china, japan, and vietnam, and can cause enormous economic loss [ , ] . pedv is a single-stranded rna virus composed primarily of four structural proteins: the spike protein (s, - kda), membrane protein (m, - kda), envelope protein (e, kda) and nucleocapsid protein (n, - kda). s protein is located on the surface of the virus particle. it is categorized as a type i membrane fusion protein and has the significant biological effect of binding to target cell receptors and entering the cell through plasma membrane fusion [ , ] . the s protein has higher antigenicity than any of the other pedv proteins, and anti-s antibodies detected in pedv-infected pigs persist longer than anti-n antibodies [ ] . the s protein can be separated into the s ( - aa) subunit and the s ( - aa) subunit [ ] . the s subunit is the extracellular domain and can recognize and bind to target cell receptors [ ] , and it is closely linked to the formation of neutralizing antibodies. therefore, this study selected a gene fragment within the s subunit as a coating antigen to develop an indirect enzyme-linked immunosorbent assay (elisa) method for the detection of pedv antibodies. the pedv yc strain was isolated on a breeding farm in yancheng city in (genbank: ku . ). the prokaryotic expression vector pet- a(+) was purchased from biovector ntcc inc. (beijing, china). the hrp-goat anti-pig iga, hrp-goat anti-pig igg, and fitc-goat anti-pig iga was purchased from abcam plc. (shanghai, china). the standard pedv negative serum were collected from specific pathogen free (spf) pigs. the standard pedv positive serum were collected from experimentally pedv immunized spf pigs, at , , , , , and day post-inoculation (dpi). these standard serum were identified of pedv-specific antibodies positive by both indirect immunoinfluscent assay (ifa) and seroneutralization assay (sn) as previously described [ , ] . the swine porv antibody elisa kit and the swine tgev elisa kit were obtained from ingenasa (madrid, spain). the gene sequence of truncated spike protein (named s ) was amplified from the genomic rna of pedv yc strain (genbank: ku . ) by reverse-transcriptase (rt)-pcr. the forward primer was ' cgcggat ccgtcactaggtgccagtccactattaa- 'and the reverse primer was '-cccaagctttcaattgtaaa tatccactttaagaaaacaataa- ′. underlined portions represent bamh i and hind iii restriction sites, respectively. the target gene was bp in length and subcloned into the prokaryotic expression vector pet- a(+), then transformed into a strain of competent e. coli cells, dh α. transformed colonies were selected from luria-bertani (lb) agar plates containing kanamycin ( μg/ml) and were identified by pcr. the resulting recombinant expression plasmid was named a-s and was identified by double enzyme digestion and dna sequence analysis. subsequently, the recombinant expression plasmid a-s was transformed into e. coli bl (de ). the positive transformants were cultured in lb medium containing μg/ml kanamycin with vigorous shaking at °c until the nm optical density (od ) of bacteria cultures reached approximately . . then, by adding isopropyl-β-d-thiogalactopyranoside (iptg), the recombinant protein s was induced for - h at °c. the expression of recombinant proteins was analyzed by % (v/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and the gels were stained with coomassie brilliant blue. induced cells were pelleted and washed in phosphate-buffered saline (pbs) three times, then lysed by sonication in an ice-water bath. the lysed cells were centrifuged at , g for min, then the precipitate (inclusion body) was dissolved with binding buffer containing m urea. the supernatant and the precipitate were then subjected to sds-page analysis. the recombinant protein was purified through affinity chromatography using a ni-nta spin column following the manufacturer's recommendations. purified recombinant protein s was subjected to % (v/v) sds-page, and the gel was prepared for western blotting as follows. recombinant proteins separated in the gel were electrically transferred to a nitrocellulose membrane and the membrane was blocked overnight at °c with tris-buffered saline containing tween- (tbst) which contained % (w/v) skimmed milk powder. the composition of tbst is as follows: mm tris-hcl, mm nacl, and . % tween- . the membrane was then incubated with pig anti-pedv polyclonal antibody ( : dilution in blocking buffer) for h at °c on a plate shaker. following this incubation, the membrane was washed three times with tbst buffer and reacted with hrp-goat anti-pig igg ( : dilution in tbst) at °c for min. after three washes, the final color reaction was developed with a solution of , ′-diaminobenzidine (dab). conventional indirect elisa was performed with the following steps. elisa plates with wells (costar, usa) were coated with μl purified recombinant s protein in bicarbonate buffer (ph = . ) for h at °c. then, plates were washed three times with pbst (pbs containing . % tween- ) and blocked with % skimmed milk in pbs for h at °c. after plates were washed, μl porcine serum samples diluted in pbs containing % (w/v) skimmed milk was added and incubated for min at °c. plates were washed four times and reacted with μl diluted secondary antibody (hrp-goat anti-pig iga or hrp-goat anti-pig igg) for min at °c for the purpose of detecting iga or igg against pedv in serum samples. plates were then washed four times and μl tetramethylbenzidine (tmb) substrate solution was added to each well for a chromogenic reaction at room temperature for min in complete darkness. the color reaction was stopped by the addition μl of m h so to each well. finally, the od was measured and recorded immediately using an infinite pro microplatereader (tecan, männedorf, switzerland). the optimal dilution of recombinant protein s and standard serum was determined by a checkerboard titration based on the method mentioned above. briefly, the concentration of s protein was gradually reduced in the following series: , . , , . , . , and . μg/ml. the standard pedv positive and negative serum were serially diluted in a -fold series from : to : . when the od ratio of positive serum to negative serum was highest, and the od of positive serum was closest to . , the corresponding dilutions of coated antigen and serum sample were considered optimal. in addition to optimal protein dilution, the coating conditions, blocking solution, and reaction time of various materials was explored. furthermore, the optimal concentration of hrp-goat anti-pig iga was tested using the following dilutions: : , : , : , : , and : . two hundred and seventy serum samples were collected for the purpose of determining the positive-negative cut-off value, of which serum samples were collected from spf pigs, serum samples were collected from grow-finish pigs from five farms between and . these five farms were located in areas with no previous history of enteric signs compatible with viral diarrhea, and were pedv rna negative by real-time rt-pcr based on a single collection. these serum samples were confirmed pedv-negative by both ifa and sn assays as previously described [ , ] , and then were used to define the cut-off value in the s indirect elisa. the od value of these pedv-negative serum samples obtained in this s indirect elisa were recorded to calculate the cut-off value. the mean od value of these negative samples (n) + × standard deviations (sd) was defined as the cut-off value. serum samples showing od value greater than or equal to this cut-off were considered pedv-seropositive. to assess the accuracy of this developed s indirect elisa, serum samples from different pig farms were tested using this elisa method. as a comparison, ifa was applied to test these samples and act as a reference method to distinguish positive or negative samples. briefly, vero cells grown on -well plates were infected with the pedv yc strain at multiplicity of infection (m. o. i) of . at h post-infection, cells were washed three times with pbst and fixed with cold methanol for min at − °c. cells were then washed three times with pbst and blocked with % bovine serum albumin (bsa) at °c for h. after been double diluted for six consecutive dilutions in dilution buffer ( % bsa in pbst), the serum samples with varied pedv antibody status were added in the wells of -well plate, and were incubated for h at °c. after three washes with pbst, cells were treated with a fitc-conjugated goat anti-pig iga (thermo scientific) at a : dilution with pbs for min at °c. after a final four washes with pbst, all wells were examined using fluorescence microscopy (axio observer z , zeiss, germany). the pedv antibody titers of the serum samples were expressed as the highest dilution of serum samples producing green fluorescent in the wells of -well plates. the results of this two methods were compared, and the sensitivity and specificity of detection were calculated to evaluate the accuracy of the s indirect elisa. sensitivity was defined as the ratio of positive tests from the developed s indirect elisa to the positive tests from the ifa. specificity was defined as the ratio of negative tests from the developed elisa to the negative tests from the reference ifa. serum cross-reactivity of s indirect elisa to other pathogens to validate the cross-reactivity, this s indirect elisa was utilized to test porcine serum positive for other swine pathogens, namely, porcine transmissible gastroenteritis virus (tgev), swine rotavirus (porv), porcine kobuvirus (pkv), porcine bocavirus (pbov), porcine norovirus (pnov), porcine circovirus type (pcv ), porcine reproductive and respiratory syndrome virus (prrsv), and enterotoxigenic e. coli (etec), jerson prand of the small intestine, clostridium welchii type c. the positive sera were prepared by our lab, by immunizing the specific pathogen free (spf) piglets with purified virus or bacteria. thirty positive serum samples for each virus were tested, and each sample was repeated in triplicate. to test the repeatability of this elisa, serum samples with different pedv antibody levels were chosen. for inter-assay variability, each sample was tested in replicates on plates of different occasions. for intra-assay variability, each sample was tested in replicates on plates within the same occasion. the results were presented as the coefficient of variation (cv), which is the ratio of the standard deviation (sd) to the mean od value of each group of samples (s). a cv value criterion of % was used to meet the repeatability requirement of the test. this s indirect elisa was applied to seroepidemiological analysis of a total of clinical swine serum samples collected from thirty seven farms in eastern china. the pedv infection status of a given farm was determined based on demonstration of pedv rna in fecal samples by real-time rt-pcr and presence of enteric signs [ ] . one thousand one hundred and twenty five serum samples were collected from nursery and grow-finish pigs from ten farms between and at - weeks after the start of pedv outbreaks in these farms. four hundred and eighty two serum samples were collected from nursery and grow-finish pigs from five farms between and . these five farms were located in areas with no previous history of enteric signs compatible with viral diarrhea, were pedv rna negative by real-time rt-pcr based on a single collection, and were considered non-exposed to pedv. three hundred and forty four serum samples from nursery pigs without pedv exposure were collected from six farms between and . these samples were confirmed to be positive for anti-porv antibodies ( farms, n = ) or anti-tgev antibodies ( farms, n = ) by both ifa and commercial elisa kits (obtained from ingenasa). one thousand three hundred and fifty three porcine serum samples with unknown pedv exposure status were randomly selected from sixteen farms from nursery and grow-finish pigs between and . the gene of pedv truncated s fragment ( - nt) was amplified by rt-pcr (fig. a) . a bp pcr product was obtained and subcloned to prokaryotic fig. amplification, sds-page and western blotting analysis of the recombinant protein s . a rt-pcr amplification of the truncated s gene fragment. lane m, dl dna marker. lane and , the truncated s gene fragment. b identifiction of the recombinant expression plasmid a-s by double enzyme digestion. lane m, dl dna marker. lane , the recombinant expression plasmid a-s digested by bamh i/sal i. c sds-page analysis of s protein. lane m, prestained protein molecular weight standard. lane , transformed cells of bl /pet- a(+) after iptg induction for h. lane , transformed cells of bl / a-s after iptg induction for h. lane , purified recombinant protein s by affinity chromatography of ni-nta spin column. d western blotting analysis of s protein. lane m, prestained protein molecular weight standard. lane , e. coli bl with empty vector pet- a(+) reacted with polyclonal mouse anti-pedv antibody. lane , purified s protein reacted with polyclonal mouse anti-pedv antibody. a prominent band with the expected size kda appeared after incubation expression vector pet- a(+), and the inserted gene was sequenced to ensure the correctness of the reading frame. as shown by sds-page (fig. c) , the recombinant protein s was expressed in the form of inclusion body, resulting in a × his-tag fusion protein whose molecular mass was approximately kda. sonicated lysates from recombinant e. coli were harvested, and the precipitate was dissolved in m urea and purified by affinity chromatography of ni + -nta agarose. the immunoreactivity of s protein was examined by western blotting. an obvious band revealed that s protein was specifically bound by pig anti-pedv polyclonal antibody (fig. d) . as expected with checkerboard titration, with concentrations of antigen and serum regularly decreasing, absorbance values of corresponding samples declined. the optimal dilution of coated antigen s protein was measured at . μg/well ( . μg/ml), and optimal serum sample dilution was : (table ) . furthermore, other reaction conditions of the developed elisa were optimized. in brief, the optimum coating condition was h at °c. the best blocking solution was selected as % skimmed milk in pbs. the optimal reaction times for serum, secondary antibodies, and tmb solution were min, min and min, respectively. finally, the best working dilution of the hrp-goat anti-pig iga was : , . to determine the cut-off value of the s indirect elisa, pedv-seronegative samples, verified by both ifa and sn assays, were tested by this elisa method. the average optical density of these negative serum samples (n) was calculated as . , and the standard deviation (sd) of these samples was . . consequently, the cut-off threshold value of s indirect elisa was calculated to be . , indicating that the sample od ≥ . was identified as pedv-seropositive and vice versa. this developed s indirect elisa was applied to serum samples with varied pedv antibody status ( table ). in these samples, the s indirect elisa detected pedv-positive samples, of which tested pedv-positive by ifa. on the other hand, of the remaining samples that tested pedv-seronegative by this s indirect elisa, of them were tested pedv-negative by ifa. hence, the sensitivity of s indirect elisa was . % among pedv-seropositive individuals, and the specificity was . % among pedv-seronegative individuals using ifa as standard evaluation method. in summary, the overall coincidence rate of the s indirect elisa to ifa was . %. to test the cross-reactivity of this s indirect elisa, other viruses known to cause swine diarrhea were examined. the average od of positive serum samples for tgev, porv, pkv, pbov, pnov, pcv , prrsv, etec, jerson prand of the small intestine, and clostridium welchii type c were . , . , . , . , . , . , . , . , . and . , respectively. the results showed that these serum samples were pedv-seronegative and non-cross-reactive with this s indirect elisa, indicating that the established elisa was an effective method for detecting pedv antibodies. the repeatability of s indirect elisa intra-assay variability of the s indirect elisa was assessed by testing swine serum samples, each with replicates. this analysis produced cvs ranging from . - . %, with an average value of . %. inter-assay variability of four batches using identical samples produced cvs ranging from . - . %, with an average value of . %. the results demonstrated that this elisa method yielded low levels of variation, and its repeatability was in the credible range. this s indirect elisa method was used on swine serum samples of different pedv exposure status collected from farms (table this s indirect elisa was applied to test the sera of pedv immunized pigs at , , , , , and day post-inoculation (dpi). the results (fig. ) showed that, both the iga and the igg were positive at dpi. at early infection stage ( dpi), the iga titer was significantly higher than the igg titer; at dpi the iga titer was equivalent to the igg titer; and after dpi, the iga titer was significantly lower than the igg titer. the igg could exist in the sera of infection recovered stage for more time than iga. since december , a large-scale outbreak of diarrhea has been observed in swine farms in china. accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent pedv variants [ , ] . serological assays can quickly detect large numbers of samples with both high sensitivity and specificity. several indirect elisa have been developed based on either whole pedv preparations or recombinant viral proteins [ ] [ ] [ ] . the s protein of pedv has numerous epitopes and highly antigenic index regions that induce the production of neutralizing antibodies [ , , ] , and anti-s antibodies detected in pedv-infected pigs persist longer than anti-n antibodies [ ] , thus, we choose s protein as the diagnostic antigen. the recombinant s protein was applied to establish an indirect elisa, and its reaction conditions were optimized. as pedv is an enteric virus, it directly infects and damages enterocytes. mucosal iga, but not systemic igg, plays a crucial role in protection [ , ] . in this research, the titers of iga in the serum were tested for serological evaluation and indirect diagnosis of pedv infection. of the serum samples which were pedv exposed, the overall positive rate of the antibody is . %, which were varied from to % between farms. of the serum samples which were pedv non-exposed, the overall positive rate of the antibody is . %, which were varied from . to . % between farms. the evaluated elisa presented an overall substantial agreement on the pedv infection status of the field swine serum samples. the intra-and inter-assay variability tests proved that this elisa method had good repeatability. when testing other positive serum related to swine viral pathogens, this established elisa demonstrated no cross-reactivity to them. further, the overall rate of coincidence of this elisa was calculated at . % compared with ifa, proving that the diagnostic sensitivity and specificity of this elisa method were favorable. of the numerous pathogens which can cause swine viral diarrhea, pedv, tgev, and porv account for the largest proportion [ ] . in the present study, / anti-porv antibody positive sample and / anti-tgev antibody positive samples were tested pedv antibody positive, which indicated there may be co-infection of pedv and other virus. in conclusion, this established s indirect elisa is capable of detecting serum antibodies against pedv, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of pedv infection. fig. determination of iga and igg in the sera of pedv immunized pigs. the sera of pedv immunized pigs were tested by this s indirect elisa at , , , , , and day post-inoculation (dpi). both the iga and the igg were positive at dpi. the igg could exist in the sera for longer time than iga. different letters (a, and b) indicate significant difference between the groups porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology nursery pig growth performance and tissue accretion modulation due to porcine epidemic diarrhea virus or porcine deltacoronavirus challenge in situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets poly (d,l-lactide-coglycolide) nanoparticle-entrapped vaccine induces a protective immune response against porcine epidemic diarrhea virus infection in piglets cross protective immune responses in nursing piglets infected with a us spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original us pedv strain an apparently new syndrome of porcine epidemic diarrhoea isolation of porcine epidemic diarrhea virus during outbreaks in south korea heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in china structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s fusion protein identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus an elisa for detection of antibodies against porcine epidemic diarrhoea virus (pedv) based on the specific solubility of the viral surface glycoprotein spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs. the veterinary quarterly epidemic strain yc of porcine epidemic diarrhea virus could provide piglets against homologous challenge multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus new variants of porcine epidemic diarrhea virus, china sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (pedv) neutralizing epitopes identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains the data analyzed during the current study are available from the corresponding author on reasonable request. authors' contributions hl, kh and hf designed the study. hl, hz, lg and bl performed and collected data from experiment and analyzed data. hl, hz wrote the manuscript. all authors read and approved the final manuscript. this study was performed in accordance with the recommendations in the guide for the care and use of laboratory animals of the ministry of health, china. all experimental protocols were approved by the institutional animal care and use committee of nanjing agricultural university (no. syxk - ) and performed accordingly. samples were collected only from animals for laboratory analyses, avoiding unnecessary pain and suffering of the animals. the owners gave their written consent for sample collection, and the locations where we sampled are not privately owned or protected in any way. the studies did not involve endangered or protected species. not applicable. the authors declare that they have no competing interests. key: cord- -qnbgywgy authors: yilmaz, huseyin; faburay, bonto; turan, nuri; cotton-caballero, maira; cetinkaya, burhan; gurel, aydin; yilmaz, aysun; cizmecigil, utku y.; aydin, ozge; tarakci, eda altan; bayraktar, erhan; richt, juergen a. title: production of recombinant n protein of infectious bronchitis virus using the baculovirus expression system and its assessment as a diagnostic antigen date: - - journal: appl biochem biotechnol doi: . /s - - - sha: doc_id: cord_uid: qnbgywgy the avian coronavirus-infectious bronchitis virus (avcov-ibv) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. sensitive diagnostics and efficacious vaccines are necessary to combat ibv infections in chickens. the aim of this study was to produce recombinant n protein of ibv in the baculovirus system to use in elisa diagnostic tests in order to enable the assessment of the sero-prevalence and risk of ibv infections in chickens in turkey. for this, the gene encoding the n protein of the beaudette strain of ibv was expressed using a recombinant baculovirus expression system. the recombinant n protein was purified using ni-nta affinity chromatography. an estimated -kda recombinant protein corresponding to the expected molecular weight of ibv n including the xhis tag was detected using an anti-his monoclonal antibody. specific immunoreactivity of the recombinant protein was confirmed by western blot using antiserum obtained from vaccinated and naturally infected chicken from turkey as well as using a monoclonal antibody raised against the n protein of the ibv massachusetts strain. the results obtained with the in-house elisa had high agreement with a commercial elisa. immunoreactivity analysis using antisera in western blotting and the in-house elisa suggests that the recombinant ibv n protein could be broadly cross-reactive with antisera produced against different ibv strains. we conclude that the recombinant baculovirus expressed ibv n protein could serve as a useful diagnostic antigen for detection of ibv infections in chickens by elisa. infectious bronchitis virus (ibv) is the causal agent of infectious bronchitis (ib), an acute and highly contagious disease, threatening the poultry industry worldwide [ , , , , ] . avian coronavirus-infectious bronchitis virus (avcov-ibv) is an enveloped, positive single-stranded rna virus, and belongs to the genus gamma-coronavirus within the family of coronaviridae in the order nidovirales [ ] . the virus is spread mainly by aerosol, consumption of contaminated feed and water, and contact with infected feces or equipment. ib is characterized by various clinical signs in broilers and layer hens: coughing, sneezing, and decreased weight gain [ , ] . specifically, in layers, egg production can drop up to % with eggs that have shells that are wrinkled, thin, and soft. in young chicks, ibv infection can lead to oviduct cysts and reduced laying potential [ , , ] . the to kb genome of ibv encodes nine genes, which includes spike (s), membrane (m), envelope (e), and nucleocapsid (n) [ ] . the spike protein, a viral surface glycoprotein, was shown to induce neutralizing antibody response, and the nucleocpasid protein was shown to elicit strong antibody responses [ , ] . despite the presence and application of ibv vaccines in poultry, there is a high rate of emergence of antigenic variants and recombinant strains, and the lack of cross-protection between different viral genotypes, making disease control difficult and vaccine development rather challenging [ , ] . therefore, genetic characterization of circulating strains of ibv, appropriate vaccination programs, and application of sensitive diagnostic tests to detect and assess disease risk are important regional, national, and international strategies to control ibv infections [ , , , , ] . several elisas have been developed for detection of antibodies to ibv in chickens. recombinant antigens used in these elisas were either based on the s protein [ ] , which is highly variable, or the n protein and often produced in an e. coli expression system [ ] . e. coli is a common flora or pathogen in chickens, thus creating the possibility of serological cross-reactivity and detection of false positives in these diagnostic assays. thus, it is necessary to assess the suitability of other expression platforms for production of diagnostic antigens for use in ibv serology. the objectives of this study were to clone and express ibv n protein using the recombinant baculovirus expression system and assess its use as diagnostic antigen for serological diagnosis of ibv infection in chickens in turkey. the complete coding sequence ( bp) of the ibv n gene of beaudette strain (genbank accession no. m . ) was initially amplified by pcr using primers, jar f: ′-cac cat ggc ttc cgg taa ggc tg- ′ and jar r: ′-cag ctc gtt ctc acc cag agc agc- ′. the pcr product was cloned into pfastbac vector (life technologies), to create a recombinant donor plasmid, pfastbac-n, which was transformed into one shot mach t chemically competent e. coli (life technologies). the donor plasmid was double digested with restriction enzymes bamhi and psti to determine the presence of the correct insert in the right orientation. the accuracy of the sequences was confirmed by dna sequencing. the donor plasmid was transformed into max efficiency dh bac competent e. coli to construct a recombinant bacmid via site-specific transpositioning. to rescue recombinant baculoviruses encoding the ibv n gene, recombinant bacmids were purified using highpure miniprep kit (life technologies) and used to transfect spodoptera frugiperda (sf ) cells grown in sf- ii serum-free medium (sfm) supplemented with % fetal bovine serum and % penicillin-streptomycin as manufacturer's instruction (life technologies). transfection was carried out using cellfectin ii reagent as previously described [ ] and according to the manufacturer's instructions (invitrogen-life technologies). recombinant ibv n protein was expressed using passage or higher passage recombinant baculovirus stocks (> pfu/ml). the protein was expressed with a carboxy-terminal xhis tag, and purification using ni-nta superflow resin (qiagen inc., valencia, ca) was performed as described previously [ ] . concentration of the purified protein was measured by the method of bicinchoninic acid (bca) assay (thermo scientific, rockford, il) at an absorbance of nm, using bovine serum albumin (sigma-aldrich, st. louis, mo) as the protein standard. aliquots of the protein were stored at − c until used. western blot analysis was performed to verify specific protein expression. for this, approximately μg of purified recombinant ibv n protein was resolved in % bis-tris polyacrylamide gel (life technologies) as described previously [ ] . following electroblotting of the proteins onto pvdf membrane, the blots were probed with mouse anti-his (c-terminal)-hrp monoclonal antibodies ( . mg/ml) (life technologies) diluted : to determine expression of the correct molecular size protein. further confirmation of expression was performed using a mouse anti-ibv n (massachusetts strain) monoclonal antibody ( mg/ml) diluted : (cat no. mbs ; mybiosource, san diego, ca) and antiserum (at : dilution) collected from chickens naturally infected with local turkish wild-type ibv strains. the membrane was then incubated with anti-species igg-hrp secondary antibody conjugate (santa cruz biotechnology, dallas, tx) at : dilution, and specific signals were detected using enhanced chemiluminescent (ecl) detection system. to assess the induction of specific ibv n-specific antibodies in the immunized chickens (described below), the antisera were tested in immunoblot assays. approximately, μl of sample containing approximately μg of recombinant ibv n protein was diluted in -μl laemmli sample dilution buffer (santa cruz biotechnology, dallas, tx) and resolved in % bis-tris polyacrylamide gel as described above. following transfer, the blot was probed with ibv n antisera obtained from the chicks immunized with the recombinant ibv n protein at a dilution of : and with antisera from wild-type ibv-infected chicken at : dilution [ ] . ibv negative chicken sera, at a dilution of : , and mouse anti-ibv n monoclonal antibody ( mg/ml) (thermo fisher, cat. no. ) at a dilution of : were used as negative and positive controls, respectively. thereafter, the blots were probed using goat anti-chicken hrp-conjugated secondary antibody ( μg/ . ml) (cat. no. sc ; santa cruz biotechnology, dallas, tx) at a dilution : , and goat anti-mouse hrp conjugated secondary antibody ( μg/ . ml) diluted : (cat. no. sc ; santa cruz biotechnology, dallas, tx). reactivity was detected using ′- ′-diamino benzidine peroxidase substrate system (sigma cat no: d - ) as recommended by the manufacturer. to assess immunogenicity of recombinant baculovirus-expressed ibv n protein, embryonated spf chicken eggs were obtained from the bornova veterinary control institute (izmir, turkey). the eggs were incubated in an incubator for hatching. after hatching, -day-old ibv antibody negative chicks (determined by commercial elisa-biochek, ibv elisa, cat no: ck ibv) were immunized with the recombinant ibv-n protein produced in this study. four chicks were immunized intramuscularly with μg each of the recombinant ibv-n protein mixed with isa vg adjuvant (seppic, france) at a ratio of : (adjuvant/ibv-n solution), and four chicks were given placebo as mock-immunized controls. on day , immunization was repeated using the same amount of adjuvant and antigen. chicks were bled on day (pre-immunization) and then on days , , and to test for presence of antibodies to recombinant ibv-n protein. optimization of elisa an indirect elisa protocol was first optimized by checkerboard titration following the methods as described previously [ , , ] . for this, two-fold dilutions of ibv-n protein were made starting from to ng in a carbonate bi-carbonate coating buffer (sigma, c- ). the negative control serum was from the spf chicks tested antibody negative by a commercial elisa (biochek). positive control was from chickens either vaccinated or had field infection tested ibv-positive by pcr. the ibv positive and negative sera were also diluted two-fold (from : to : ), and the conjugate (hrp-conjugated goat anti-chicken antibody, santa cruz, sc ) was diluted : , : , , : , , and : , , in blocking solution. per the results of the optimization, the optimal amount of the recombinant ibv protein for use in elisa ranged from to ng, whereas the optimal dilution of ibv positive chicken serum ranged from : to : , that of the conjugate at : dilution. following optimization of the elisa protocol, test sera were obtained from broiler chickens exposed to natural wild-type ibv infection, test sera from broiler chickens vaccinated with a live-attenuated commercial ibv vaccine, and sera obtained at different time-points from chicks immunized with recombinant ibv n protein (described above) were analyzed to detect ibv n-specific antibodies. for this, a -well plate (nunc maxisorb, thermo fisher scientific) was coated overnight at °c with ng ibv-n protein/per well in -μl carbonate-bicarbonate coating buffer (sigma, cat. no. c- ). the plate was then blocked for h at room temperature with a blocking buffer (pbs containing % skimmed milk and . % tween ) . a volume of μl of test sera, diluted : in blocking solution, was added and incubated for h at °c. each serum sample was tested in duplicate, and each test plate included duplicate positive and negative control sera. the negative control serum was from the spf chicks tested antibody negative by a commercial elisa (bochek). positive control serum was from chickens either vaccinated or had field infection and tested ibv positive by pcr. goat anti-chicken hrp-conjugated antibody (santa cruz biotechnology, cat. no. sc ), diluted : , was added to the wells and incubated at °c for h. between each step, the plate was washed four times by using the wash buffer (pbs containing . % tween ) . hundred microliters of tmb substrate solution was added to each well and incubated at room temperature for min. the reaction was stopped by adding μl of m h so and absorbance (optical density) was measured at nm using a microplate reader . (slt-spectra, slt lab instruments, germany). the elisa cut-off value was determined by addition of standard deviations to measurements of the mean od value of the negative control sera in each plate. od values above cut-off value were taken as positive. thus, a cut-off od value of . was determined for the in-house indirect elisa. to assess the reliability of the performance of our in-house indirect ibv n elisa, a panel of sera was obtained from chickens naturally infected with local wild-type ibv strains, chickens vaccinated with live-attenuated commercial ibv vaccine, and chickens immunized with recombinant ibv n protein (expressed in a baculovirus expression system). the serum samples were tested simultaneously in the in-house indirect elisa (using the procedure described above) and a commercial elisa (biocheck, san francisco, ca) per manufacturer's instruction. the cut-off value for the commercial elisa was defined according to the manufacturer and determined as samples with a sample to positive ratio (s/p ratio) of . or greater. a bp fragment representing the expected molecular size of the ibv n gene was successfully amplified by high fidelity pcr (fig. ) . the pcr amplicon was successfully cloned into pfastbac/ct plasmid resulting in the creation of a donor plasmid pfastbac-n ( bp), restriction enzyme analysis of the recombinant donor plasmid confirmed the presence of the correct insert in the correct orientation by release of two restriction fragments of the expected size ( and bp) (data not shown). the donor plasmid with the correct sequences was used to create recombinant bacmid for subsequent expression of the target protein. in order to express the recombinant n protein of ibv, sf cells were infected with a recombinant baculovirus encoding the n gene of ibv. at h post-infection, cells were harvested and the recombinant protein was purified via affinity chromatography. an estimated kda recombinant protein was overexpressed, which corresponded to the expected molecular size of ibv n protein as determined by coomassie staining (fig. a) and western blot analysis using anti-his (c-terminal)-hrp monoclonal antibody (fig. b) . to confirm specificity of the target protein, an immunoblot analysis was performed using monoclonal antibodies raised against the n protein of the massachusetts ibv strain (fig. a) and as well as antiserum obtained from local chickens naturally infected with turkish wild-type ibv strain (fig. b) . in both cases, specific reactivity was confirmed by detection of a single band ( kda) of the expected molecular size. to assess the ability of the recombinant ibv n protein to induce host specific antibody responses, four chicks ( -day old) were immunized with the recombinant protein and the sera examined for specific reactivity via immunoblot analysis. specific reactivity was detected with serum samples obtained from of the ibv n-immunized chickens at day post-immunization (example see fig. seen with the other two chicken sera. this result was supported by detection of specific ibv n-reactivity with the ibv-specific monoclonal antibody (fig. , lane ) and with antisera from a chicken exposed to a natural field infection (fig. , lane ) . no specific bands were detected in sera from mock-immunized chickens or sera taken from chicks before immunization (fig. , lanes , , , , , , ) . overall, sera from vaccinated and naturally infected (field-exposed) chickens that tested positive in the immunoblot assay were found to be correspondingly positive in the in-house indirect ibv n elisa (data not shown). to further assess the immunogenicity of the recombinant n protein and the reliability of our results, a panel of antisera from naturally infected ( samples plus negative and positive controls), from chickens vaccinated with live-attenuated ibv ( samples plus negative and positive controls) and from chickens immunized with recombinant ibv n protein ( samples), were tested using both the in-house indirect elisa and a commercial elisa. with the in-house elisa, cut-off value was set at od of . . there was relatively good agreement in performance between the two elisas in detecting ibv n-specific antibodies in the sera from naturally infected chickens (fig. a) and live-attenuated vaccinated chickens (fig. b) . of the serum samples obtained from the naturally infected chickens, % ( / ) tested positive in the in-house indirect elisa, whereas % ( / ) tested positive in the commercial elisa detected (fig. a) . all samples collected from vaccinated chickens tested positive in both the in-house and commercial indirect elisas (fig. b) . of the commercial elisa-positive field serum samples, tested positive in the in-house indirect elisa, indicating a sensitivity of % (using the commercial elisa as reference test). none of the sera obtained from preimmunized or naïve chicks tested positive in either elisas, indicating % specificity (n = ). of the four chicks immunized with the recombinant ibv n protein, two chicks seroconverted at day after immunization. all four chicks seroconverted at days post-immunization in both elisa tests, indicating a % agreement between the two assays. the od values of chicks immunized with recombinant ibv n protein were . , . , . , and . . production of immunogenic recombinant proteins derived from pathogenic organisms represents a good strategy for identification of antigenic proteins that may serve as targets for sero-diagnostic and vaccine development [ , ] . this represents the first study in turkey that expressed recombinant ibv n protein in baculovirus and examined its reactivity against antisera obtained from turkish chickens for potential use as antigen fig. detection of ibv n specific antibodies in sera obtained from naturally infected chicken (a) and vaccinated chickens (b) using an in-house ibv-n elisa and a commercial elisa. the cut-off value for inhouse indirect elisa is set at od . , and for the commercial elisa at s/p (sample to positive) ratio of . in serological investigation of ibv infection in domestic poultry. we have demonstrated that ibv n produced in a recombinant baculovirus expression system is immunogenic in local chickens and could detect ibv n-specific antibodies in sera obtained from chickens exposed to natural infection. the recombinant n protein was reactive in both immunoblot and indirect elisa assays. the performance of the in-house indirect elisa was comparable to the commercial elisa tested in this study, indicating the potential suitability of recombinant baculovirus ibv n as a diagnostic antigen that could be used for serological risk assessment of ibv in domestic chickens in turkey. indeed, the n protein of ibv, in contrast to the s protein, has been associated with high stability and immunogenicity and has been used by others as a diagnostic antigen [ , ] . the recombinant n protein used in the current study was based on nucleotide sequences of the beaudette ibv strain. the reactivity of the recombinant ibv n protein with a monoclonal antibody (b m) produced against the heterologous ibv massachusetts strain, and with sera from local turkish chicken indicate the conserved, cross-reacting nature of the n protein sequence and suggest potential broad cross-reactivity of the target protein among different ibv isolates and its suitability as a diagnostic antigen to detect ibv infection in local turkish poultry. other researchers have utilized recombinant ibv n expressed in various host systems in serological assays. for example, in a study performed by pradhan and others [ ] , ibv-n protein was produced using a prokaryotic expression system. the protein was used for diagnostic purposes and similar to this study, compared the performance with a commercial elisa kit (idexx). the authors reported a . % sensitivity and . % specificity for the in-house elisa based on the recombinant ibv n antigen and they concluded that an indirect elisa based on the recombinant antigen is suitable for use in development of serodiagnostic tools [ ] . in another study, the ibv-n protein was produced in both e. coli and baculovirus expression systems [ ] , and the recombinant antigens were used in elisas to analyze chicken sera collected from farms [ ] . their data from screening of sera for the presence of ibv antibodies indicated that using the recombinant n protein as coating antigen could achieve equivalent performance to an elisa kit based on extracts from infected material as coating antigen [ ] . in a study performed by ding and others [ ] utilizing a multi-fragment antigen composed of s (spike), m (matrix), and n proteins produced in e. coli [ ] , a good reactivity with an anti-ibv chicken serum was demonstrated. an in-house elisa using the multi-fragment protein exhibited a . % concordance with a commercial elisa [ ] . in the present study, recombinant ibv-n protein was produced using a baculovirus expression system and utilized as a diagnostic antigen in an indirect elisa. the chicken sera from farms and the sera from immunized chickens were reactive with the recombinant antigen with comparable performance to a commercial elisa test kit. this study describes the successful cloning and recombinant baculovirus expression of ibv nucleoprotein and its evaluation as a potential serodiagnostic antigen. the recombinant antigen was immunoreactive with hyperimmune sera from local turkish chickens and the performance of an in-house indirect elisa based on the antigen was comparable to a commercial elisa, suggesting the potential utility for serological detection of ibv infections in chickens in turkey. further studies to validate the assay using a larger sample size including comprehensive assessment of assay specificity will be performed. decreased neutralizing antigenicity in ibv s protein expressed from mammalian cells coronavirus avian infectious bronchitis virus evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus the long view: years of infectious bronchitis research infectious bronchitis virus variants: a review of the history, current situation and control measures development of an elisa based on a multi-fragment antigen of infectious bronchitis virus for antibodies detection rift valley fever virus structural and nonstructural proteins: recombinant protein expression and immunoreactivity against antisera from sheep. vector borne and zoonotic diseases a glycoprotein subunit vaccine elicits a strong rift valley fever virus neutralizing antibody response in sheep s gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel avian infectious bronchitis virus review of infectious bronchitis virus around the world expression and purification of the kda periplasmic protein of brucella abortus: a reagent for the diagnosis of bovine brucellosis cleavage of structural proteins during the assembly of the head of bacteriophage t expression, purification, and improved antigenic specificity of a truncated recombinant bp protein of brucella melitensis m - : a potential antigen for differential serodiagnosis of brucellosis in sheep and goats detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay immune responses to mucosal vaccination by the recombinant a and n proteins of infectious bronchitis virus molecular epidemiology and evolution of avian infectious bronchitis virus recombinant nucleocapsid protein based single serum dilution elisa for the detection of antibodies to infectious bronchitis virus in poultry s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification an elisa for antibodies against infectious bronchitis virus using an s spike polypeptide a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from detection of antibodies to equine arteritis virus in horse sera using recombinant chimaeric n/g(l) protein. the veterinary record phylogeny and s gene variation of infectious bronchitis virus detected in broilers and layers in turkey compliance with ethical standards national and international ethical rules were followed during this study. the authors declare that they have no conflicts of interest. key: cord- - cg yj authors: lassaunière, ria; frische, anders; harboe, zitta b; nielsen, alex cy; fomsgaard, anders; krogfelt, karen a; jørgensen, charlotte s title: evaluation of nine commercial sars-cov- immunoassays date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: cg yj due to urgency and demand, numerous severe acute respiratory syndrome coronavirus (sars-cov- ) immunoassays are rapidly being developed and placed on the market with limited validation on clinical samples. thorough validation of serological tests are required to facilitate their use in the accurate diagnosis of sars-cov- infection, confirmation of molecular results, contact tracing, and epidemiological studies. this study evaluated the sensitivity and specificity of nine commercially available serological tests. these included three enzyme-linked immunosorbent assays (elisas) and six point-of-care (poc) lateral flow tests. the assays were validated using serum samples from: i) sars-cov- pcr-positive patients with a documented first day of disease; ii) archived sera obtained from healthy individuals before the emergence of sars-cov- in china; iii) sera from patients with acute viral respiratory tract infections caused by other coronaviruses or non-coronaviruses; and iv) sera from patients positive for dengue virus, cytomegalovirus and epstein barr virus. the results showed % specificity for the wantai sars-cov- total antibody elisa, % for the euroimmun iga elisa, and % for the euroimmun igg elisa with sensitivities of %, %, and %, respectively. the overall performance of the poc tests according to manufacturer were in the rank order of autobio diagnostics > dynamiker biotechnology = ctk biotech > artron laboratories > acro biotech ≥ hangzhou alltest biotech. overall, these findings will facilitate selection of serological assays for the detection sars-cov- -specific antibodies towards diagnosis as well as sero-epidemiological and vaccine development studies. in december , a novel coronavirus causing severe acute respiratory symptoms emerged in wuhan, china [ ] . the world health organization (who) termed the disease, coronavirus disease , and the causative virus severe acute respiratory syndrome coronavirus (sars-cov- ). as of april, the virus has spread to countries and territories with confirmed cases and deaths worldwide [ ] . at present, the epidemic within the majority of countries have not yet reached its peak with the number of cases and deaths predicted to rise in the coming weeks and months. accurate diagnosis of covid- is essential, not only to ensure appropriate patient care but also to facilitate identification of sars-cov- infected people, including asymptomatic carriers, who need to be isolated to limit virus spread. the who recommends nucleic acid detection of sars-cov- in respiratory samples for the diagnosis of covid- . unfortunately, in the face of the rapidly growing epidemics worldwide, an increased demand for diagnostic tests has led to a critical shortage in operational material for respiratory sample collection and within the molecular diagnostic workflow [ , ] . this impedes rapid large scale testing, a necessity for controlling the epidemic. moreover, the heterogeneity of respiratory sample material and anatomical location of sample collection, for example throat swab, saliva or endotracheal aspirate, affect the sensitivity of sars-cov- viral nucleic acid testing [ , ] . overall, there is an urgent need to identify alternative diagnostic means. antibody testing, either using enzyme-linked immunosorbent assay (elisa) or point-of-care (poc) lateral flow immunoassays, may overcome some of these challenges. sars-cov- -specific antibodies can be detected in in serum of approximately % of covid- patients as early as seven days after the onset of symptoms, with seroconversion rates rapidly increasing to > % by day [ ] . in recent studies, antibody testing has been shown to be more sensitive than viral nucleic acid detection after approximately eight days of covid-protein subunit (s ) are detected in human serum or plasma. briefly, : diluted serum samples were added to wells coated with recombinant sars-cov- antigen and incubated for minutes at °c. wells were washed three times followed by the addition of hrp-conjugated anti-human iga or igg and subsequent incubation for minutes at °c. wells were washed three times and a chromogen solution was added. following minutes of incubation at room temperature, the reaction was stopped and the resultant absorbance was read on a microplate reader at nm with reference at nm. a ratio between the extinction of the sample and calibrator on each plate were calculated. according to the manufacturer's recommendations, a ratio < . is considered negative, ≥ . and < . borderline, and ≥ . positive. however, for sensitivity and specificity, . was used as a more stringent cut-off value for positive results and all values < . were considered negative. six poc tests for rapid detection of antibodies in blood, serum or plasma were evaluated: -ncov igg/igm for all tests, the recommended sample volume of μl serum was added to the specimen well on the individual test cassettes followed by the addition of the supplied buffer. the buffer volume differed by manufacturer and was added accordingly ( μl, three drops, two drops, two drops, two drops and μl, respectively). the result was read visually after minutes. weak signals for igm and igg, together or separate, was considered positive. sensitivity was defined as the proportion of patients correctly identified as having sars-cov- infections, as initially diagnosed using nucleic acid detection of sars-cov- in respiratory samples. specificity was defined as the proportion of sars-cov- immune naïve study participants accurately identified as negative for covid- . the clinical accuracies of the elisa assays were examined by using receiver operator characteristic (roc) plots with graphpad prism version . . (graphpad software, san diego, ca, usa). roc area under the curve (auc) were calculated as the fraction "correctly identified to be positive" and the fraction "falsely identified to be positive" determined according to manufacturer cut-off values for positive results. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint three commercial ce-marked elisa assays for detecting sars-cov- antibodies were evaluated using serum samples from pcr-positive cases with sars-cov- and control serum samples. twenty nine of the cases ( %) were positive for sars-cov- -specific antibodies by at least one of the three elisa assays. in one case, only a positive iga result was detected, while in another case antibody responses were negative by all tests. the distance of data points from the manufacturer recommended cut-off values and confidence in assigning a positive or negative status differed between the three assays ( figure a ). the distribution of positive and negative data points were distinct for the wantai total ab assay, with a cut-off value above all the control sera samples, which allowed for unequivocal interpretation. conversely, the euroimmun iga and igg assays data had a less distinct separation, resulting in a 'grey zone' of borderline data points to which a positive or negative status could not be assigned. both case and control sera had borderline or inconclusive data points. the sensitivities and specificities are shown in table . the sensitivity of the wantai total ab elisa was equivalent to that of euroimmun's iga elisa at % and was greater than that observed for euroimmun's igg elisa at %. the specificity of the wantai total ab elisa was % compared to % and % for the euroimmun iga and igg elisas, respectively. the positive predictive value and negative predictive value was ). the positive predictive value of these tests were %, while the negative predictive values were %, %, %, and %, respectively. the acro biotech was evaluated on five case serum samples and had a specificity of %. one case serum sample was tested with the hangzhou alltest biotech test and was positive for both igm and igg. the specificity of the six poc tests were evaluated primarily on control samples that showed some crossreactivity in the sars-cov- elisa assays; the number of control sera tested varied between the different poc tests ( table ). the poc tests manufactured by dynamiker biotechnology, ctk biotech, autobio diagnostics and artron laboratories had a % specificity, whereas the test from acro biotech and hangzhou alltest biotech had a specificity of % and %, respectively. for the latter two tests, crossreactivity was only observed for igm. the acro biotech test cross-reacted with a control serum sample from a human coronavirus hku patient. to evaluate the sensitivities of the assays at different stages of covid- disease, case sera were grouped according the duration of disease: early phase, to days after the onset of disease symptoms; middle phase, to days after the onset of disease symptoms; and late phase, ≥ days after the onset of disease symptoms. the sensitivities of the assays ranged from to % for the early phase samples, to % for the middle phase samples, and to % for the late phase ( figure ). in the early phase, the wantai total ab elisa had a sensitivity of % that plateaued at % after days of illness duration. the igg elisa had the lowest sensitivity at all three phases that showed a distinct increase with each consecutive phase i.e. % in the early phase, % in the middle phase, and % in the late phase. while the four poc tests evaluated according to illness duration were often weakly positive or detected only igg or igm during the early phase (data not shown), their sensitivities were comparable to the wantai total ab elisa and euroimmun iga elisa in all three phases. in the early phase, a case sample that was negative by both total ab and igg was positive in the iga elisa. to determine the agreement between the different elisas and poc tests evaluate, the proportion of case sera that shared the same result between two assays were calculated. despite comparable sensitivities of certain assays, the tests did not necessarily give the same result in all instances ( figure a ). the only tests that were % concordant were the dynamiker biotechnology and ctk biotech poc tests ( figure b) . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint in the present study, three sars-cov- -specific commercial elisa assays and six poc rapid tests were evaluated using sera from hospitalized adult patients with pcr-confirmed diagnoses for sars-cov- and a collection of control serum samples taken before the emergence of the virus in china in december . overall, the wantai total ab elisa had superior sensitivity and specificity compared to both euroimmun iga and igg elisas. the poc tests varied notably, with the best performance observed for the test produced by autobio diagnostics, followed by the tests produced by dynamiker biotechnology and ctk biotech. the differences observed for the sensitivity and specificity of the sars-cov- -specific total antibody testing and antibody type elisas correspond to previous reports. . this notably lower sensitivity for sars-cov- -specific igg detection is in agreement to that observed here for the euroimmun igg elisa ( %). in the present study the wantai igg and euroimmun igg elisas could not be compared due to unavailability of the former. the possibility that overall lower sensitivity of sars-cov- igg elisas may be a more universal occurrence rather than manufacturer dependent warrants further investigation. in addition to lower sensitivities, the euroimmun iga and igg elisas are also more prone to cross-react with negative sera as described in the present study and in a separate analysis of the beta-versions of these assays [ ]. the differences between the assays may, in part, be explained by the sars-cov- antigen targeted and the elisa format used. both kits detect antibodies to the s subunit of the sars-cov- spike protein; however, the wantai total ab elisa only targets the rbd within the s . the rbd represents approximately % of the s subunit, thus epitopes that may be recognized by cross-reacting epitopes outside of this domain are absent. furthermore, the rbd is highly diverse between sars-cov- and other beta-coronaviruses (hcov-oc and hcov-hku ), which may further reduce the likelihood of cross-reaction with these circulating coronaviruses. moreover, the wantai total ab elisa uses an antigen-antibody-antigen(peroxidase) format whereas the euroimmun elisas employ an antigen-antibody-antibody(peroxidase) format. the specificity of the former is determined by a single antibody, whereas the latter has a second antibody that may introduce additional specificities. the antigen-antibody-antibody format is required to distinguish between specific antibody types, but may not necessarily have lead to decreased specificity as shown for in-house elisas [ ]. the clinical sensitivity of igm for early diagnosis of covid- is currently unclear. sars-cov- -specific igm does not consistently appear before its igg counterpart, with some studies reporting detection of sars-cov-. cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. in conclusion, our findings show that in an elisa format the sensitivity of detecting total sars-cov- rbdspecific antibodies is higher than that of assays detecting spike-specific iga or igg only. it is important to note that the presence of sars-cov- -specific antibodies does not necessarily correspond to protection against sars-cov- infection and disease. in order to define antibody-mediated protection, further investigation of virus-specific antibody functions that include neutralization and fc-mediated effector functionality are needed. sero-epidemiological investigations together with longitudinal studies on sequential samples taken from sars-cov- patients are necessary to characterize the spread of the virus and the long term protection of the antibodies measured. due to comparatively poorer assay performance in an initial round of testing, further testing were suspended. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster european centre for disease prevention and control. novel coronavirus disease (covid- ) pandemic: increased transmission in the eu/eea and the uk -sixth update the standard coronavirus test, if available, works well-but can new diagnostics help in this pandemic? sci mag the research has been conducted using the danish national biobank resource, supported by the novo nordisk foundation, grant number - - and - - . exemption for review by the ethical committee system and informed consent was given by the committee on biomedical research ethics -capital region in accordance with danish law on assay development projects. the authors declare no competing interests. key: cord- -h ikjgic authors: ong, david s.y.; de man, stijn j.; lindeboom, fokke a.; koeleman, johannes g.m. title: comparison of diagnostic accuracies of rapid serological tests and elisa to molecular diagnostics in patients with suspected covid- presenting to the hospital date: - - journal: clin microbiol infect doi: . /j.cmi. . . sha: doc_id: cord_uid: h ikjgic objectives: to assess the diagnostic performance of rapid lateral flow immunochromatographic assays (lfas) compared to an enzyme-linked immunosorbent assay (elisa) and nucleic acid amplification tests (nats) in suspected coronavirus disease (covid- ) patients. methods: patients presenting to a dutch teaching hospital were eligible between march and april , , when they had respiratory symptoms that were suspected for covid- . the performances of six different lfas were evaluated in plasma samples obtained on corresponding respiratory sample dates of nats testing. subsequently, the best performing lfa was evaluated in patients and in sera of a historical patient control group. results: in the pilot analysis sensitivity characteristics of lfa were heterogenous ranging from / ( %; % confidence interval (ci) - ) to / ( %; % ci - ). in the total cohort, orient gene biotech covid- igg/igm rapid test lfa had a sensitivity of / ( %; % ci - ) and specificity of / ( %; % ci - ). sensitivity increased to / ( %; % ci - ) in patients with at least seven days of symptoms, and to / ( %; % ci - ) in patients with c-reactive protein (crp) > mg/l. sensitivity and specificity of wantai sars-cov- ab elisa was / ( %; % ci - ) and / ( %; % ci - ) in all patients, respectively, but sensitivity increased to / ( %; % ci - ) in patients with at least seven days of symptoms. conclusions: there is large variability in diagnostic test performance between rapid lfas, but overall limited sensitivity and high specificity in acutely admitted patients. sensitivity improved in patients with longer existing symptoms or high crp. lfas should only be considered as additional triage tools when these may lead to the improvement of hospital logistics. in december the outbreak of the severe acute respiratory syndrome coronavirus (sars-cov- ) started in wuhan in china [ ] , but the coronavirus disease rapidly spread to other countries as well [ ] . the first infected patient in the netherlands was detected on the th of february [ ] . accurate diagnostics are fundamental in the fight against this increasing pandemic. moreover, hospitals would benefit from rapid detection of this virus infection in patients who acutely present to hospitals with respiratory symptoms suspected for covid- . time delay in the establishment of diagnosis increases logistic challenges and causes stagnation of patient flow in emergency departments as these patients are unable to be transferred to appropriate hospital wards or intensive care units (icus) when results of the diagnostic tests are still pending [ ] . nucleic acid amplification tests (nats) are the gold standard because of the high specificity, although sensitivity may depend on the timing of disease presentation, sampling location and severity of illness [ ] . nevertheless, it usually takes about to hours before laboratory-based results become available depending on specific nat platforms and laboratory organisation. therefore, numerous lateral flow immunochromatographic assays (lfas) have been introduced into the market, and some countries have stocked up on such rapid tests. these lfas detect the presence of igm and igg against sars-cov- . this study aimed to assess the diagnostic performance of lfas, and compare these to an enzyme-linked immunosorbent assay (elisa) and nats in suspected covid- patients. patients presenting to a teaching hospital in the netherlands were eligible between march , and april , when they had respiratory symptoms that were suspected for respiratory tract infection. patients were sampled from the oral cavity and subsequently from the nasal cavity using the same nasopharyngeal swab, which was tested by nats. in some cases, sputum samples were tested, because of persisting clinical suspicion on covid- despite a negative nat on nasopharyngeal swabs. nats were performed according to the national reference method that was established after international collaboration [ ] , or by the ce-ivd kit genefindertm covid- plus realamp kit using the sample to result platform elite ingenius®. the institutional review board waived the need for informed consent as tests were performed on samples which had been acquired for routine clinical care (irb protocol number - ), and according to hospital procedure all patients were informed about the possibility of an opt-out if they had objections against the use of left-over material for research to improve or validate diagnostic testing procedures. the study was conducted in accordance with helsinki declaration as revised in . first, in a pilot phase nat-positive and nat-negative patients were retrospectively selected for which six lfas were performed on heparin plasma samples obtained upon hospital presentation ( figure s ), which corresponded to the dates of molecular testing. lfas were included from boson biotech, cellex, dynamiker biotechnology, orient gene biotech, prometheus bio, and wantai rapid test. any visible band for either igg, igm or unspecified ig was indicative for a positive result. second, based on the sensitivity and specificity results in the pilot study, the best performing lfa was further evaluated in an extended cohort of randomly selected patients. third, this lfa was prospectively tested in consecutive patients between april and april . fourth, specificity was additionally tested in a historical control group of randomly selected sera of adult patients in september as sars-cov- was not circulating at that time. finally, samples were also analysed by the wantai sars-cov- ab elisa kit, which detects total antibodies, and interpreted according to manufacturer's instructions. both clinical information and reference standard results were unavailable to the performers of lfas and the elisa. all analyses were performed using sas . (cary, north carolina). we compared groups using non-parametric tests for continuous variables and chi-square test or fisher's exact test for categorical variables as appropriate. p-values < . were considered to be statistically significant. in the pilot study sensitivity characteristics of lfa were very heterogenous ranging from / ( %; % confidence interval (ci) - %) to / ( %; %ci - %)) ( table ) . we decided to continue with the orient gene biotech covid- igg/igm rapid test (ogbrt) as it had the highest sensitivity. a total of patients (including the from the pilot study) were retrospectively selected between march and march . subsequently, consecutive patients were prospectively included between april and april . in total, patients were included with a median age of years (interquartile range (iqr) - ), ( %) were male, ( %) were admitted to the icu within hours and median c-reactive protein (crp) upon hospital presentation was (iqr - ) mg/l (table s ). median time from symptom onset to sample collection was (iqr - ) days. ogbrt had an overall sensitivity of / ( %; %ci - %) and specificity of / ( %; %ci - %) ( table ) . sensitivity increased to / ( %; %ci - ) in patients with at least seven days of symptoms, and to / ( %; %ci - ) in patients with crp > mg/l upon presentation. however, there was no significant difference between patients requiring icu figure s ). in the randomly selected historical control sera, the lfa and the elisa specificity was / ( %; %ci - ) and / ( %; %ci - ), respectively; lfa showed a very weak igg line in one sample. this study shows that the sensitivity of lfa was low in patients suspected for covid- presenting to the hospital, but it improved in patients with at least seven days of symptoms and in those with crp levels > mg/l upon presentation. specificity of lfas and the elisa was very high, and fulfilling a frequently used criterium of at least %. the elisa had a higher sensitivity compared to lfas. several countries, including spain and the united kingdom, have purchased one or more of these lfas. however, our study findings underline that cautiousness is required when considering implementation of such tests. interestingly, cellex rapid test, which is currently the only rapid diagnostic test that is fda approved, performed less than ogbrt in our pilot study. another rapid test was reported to have a sensitivity below % in acute patients referred to emergency department [ ] . other studies showed higher sensitivities of lfas up to % in unspecified patient groups with more time between disease onset and testing or missing information regarding timing of sampling [ , ] . test performance characteristics as provided by manufacturers were higher than those observed in our study, which is related to different selection of positive and negative controls. in our study we primarily included consecutive patients presenting to the hospital, which represents clinical practice and clinical sensitivity (i.e. diagnosing covid- upon hospital presentation) rather than analytical sensitivity (i.e. detecting the presence of antibodies at that moment). the observed higher sensitivity in patients with at least seven days of symptoms is in line with findings from other studies [ , ] . there are some study limitations to consider. this study included a wide comparison of six different lfas, an elisa and nats, but more tests are available on the market. nevertheless, both lfas and the elisa were limited in sensitivity, suggesting that antibody production is not always detectable or at least not yet detectable during the early phase of infection. second, nat as reference standard remains suboptimal, and it remains possible that in some cases actual infections were missed. in some patients nats were only positive in sputum and negative in nasopharynx, whereas the majority of patients were only tested by nasopharyngeal swabs. third, the subgroup of patients admitted to the icu was limited, precluding definite conclusions in this group. in conclusion, the high specificity of lfas may contribute to rapidly confirm covid- , accelerate decision-making in emergency rooms and routing to appropriate hospital wards. yet, negative lfa results are unreliable to exclude covid- due to the limited sensitivity of these tests. therefore, these lfa tests cannot replace molecular diagnostics in acute care settings, but should only be used as an additional triage tool when improvement of hospital logistics is expected and their limitations are carefully considered. the authors declare no conflicts of interest. clinical characteristics of coronavirus disease in china rapidly increasing cumulative incidence of coronavirus disease (covid- ) in the european union/european economic area and the united kingdom current information about the novel coronavirus (covid- ) bilthoven: rivm better tests, better care: improved diagnostics for infectious diseases sars-cov- viral load in upper respiratory specimens of infected patients detection of novel coronavirus ( -ncov) by real-time rt-pcr performance of vivadiag covid- igm/igg rapid test is inadequate for diagnosis of covid- in acute patients referring to emergency room department evaluation of a covid- igm and igg rapid test; an efficient tool for assessment of past exposure to sars-cov- development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis antibody responses to sars-cov- in patients of novel coronavirus disease antibody detection and dynamic characteristics in patients with covid- boson biotech rapid -ncov igg/igm combo test card / ( % cellex qsars-cov- igg/igm cassette dynamiker biotechnology -ncov igg/igm orient gene biotech covid- igg/igm rapid test cassette / ( % prometheus bio -ncov igg/igm in the subgroup of patients with time from symptom onset to sample collection >= days, sensitivity and specificity of the lfa were / ( %; %ci - ) and / ( %; %ci - ), respectively, whereas sensitivity and specificity of the elisa of note, sensitivity was / ( %) in patients with >= days from symptom onset to sample collection and crp >= mg/l we would like to thank our laboratory technicians and team managers for their assistance in performing the serological tests.contribution: dsyo and jghk contributed to the conception and design of the study. dsyo, sjm and fal acquired the data. dsyo and sjm analysed the data. all authors contributed to the interpretation of the data. dsyo drafted the first manuscript and all other authors revised it critically for important intellectual content. all authors approved this manuscript version to be submitted. key: cord- -abanr authors: brigger, d.; horn, m.p.; pennington, l.f.; powell, a.e.; siegrist, d.; weber, b.; engler, o.; piezzi, v.; damonti, l.; iseli, p.; hauser, c.; froehlich, t.k.; villiger, p.m.; bachmann, m.f.; leib, s.l.; bittel, p.; fiedler, m.; largiadèr, c.; marschall, j.; stalder, h.; kim, p.s.; jardetzky, t.s.; eggel, a.; nagler, m. title: accuracy of serological testing for sars‐cov‐ antibodies: first results of a large mixed‐method evaluation study date: - - journal: allergy doi: . /all. sha: doc_id: cord_uid: abanr background: serological immunoassays that can identify protective immunity against sars‐cov‐ are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. to date, however, the utility of such immunoassays remains unclear. in a mixed‐design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various sars‐cov‐ proteins and assessed the neutralizing activity of antibodies in patient sera. methods: consecutive patients admitted with confirmed sars‐cov‐ infection were prospectively followed alongside medical staff and biobank samples from winter / . an in‐house enzyme‐linked immunosorbent assay utilizing recombinant receptor‐binding domain (rbd) of the sars‐cov‐ spike protein was developed and compared to three commercially available enzyme‐linked immunosorbent assays (elisas) targeting the nucleoprotein (n), the s domain of the spike protein (s ) and a lateral flow immunoassay (lfi) based on full‐length spike protein. neutralization assays with live sars‐cov‐ were performed. results: one‐thousand four‐hundred and seventy‐seven individuals were included comprising sars‐cov‐ positives (defined as a positive real‐time pcr result; prevalence . %). igg seroconversion occurred between day and day . while the elisas showed sensitivities of . % for rbd, . % for s , and . % for n protein, the specificity was above % for all tests. out of sars‐cov‐ positive individuals, . % showed full neutralization of live sars‐cov‐ at serum dilutions ≥ : , while none of the sars‐cov‐ negative sera revealed neutralizing activity. conclusions: elisas targeting rbd and s protein of sars‐cov‐ are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against sars‐cov‐ . governments worldwide are facing a unique challenge: to save thousands of lives threatened by coronavirus disease (covid- ) , while minimising economic and social damage caused by lockdown and other strict measures. serological immunoassays will play a central role in addressing these challenges for the following reasons . first, serological tests might improve the rate of diagnosis as real-time rt-pcr is associated with a high number of false-negative results due to pre-analytical and other issues . second, antibody assays may support intensive surveillance measures such as universal testing, active case-finding, contact tracing, and linking clusters and thereby may facilitate an exit strategy from lockdown [ ] [ ] [ ] [ ] , . in patients with severe disease extensive activation of cytokine-secreting cells from the innate and adaptive immune system has been reported to result in a cytokine storm contributing to acute respiratory distress syndrome and multiorgan failure [ ] [ ] [ ] [ ] [ ] . antibody responses against different sars-cov- antigens have been described in serological samples of infected patients. few patients with anti-viral antibodies have been identified in the first days following symptom onset but the positive rate rapidly increases thereafter , . to date, antibody testing has focused primarily on two highly abundant structural antigens of sars-cov- , specifically the nucleoprotein (n) protein this article is protected by copyright. all rights reserved and the spike (s) protein . while the n phosphoprotein ensures the linkage of the viral rna to the membrane , the s glycoprotein binds to ace and thereby initiates viral entry into the host cell , [ ] [ ] [ ] . neutralizing antibodies (nab) are typically generated against the s protein and often target the receptor binding domain (rbd) , . as demonstrated in a vaccination approach using inactivated virus, the rbd represents an immunodominant viral antigen since at least half of the detectable anti-s igg antibodies were directed against the rbd . in contrast, the amount of anti-n antibodies was -fold lower. lateral flow immunoassays (lfi) , as well as enzyme linked immunosorbent assays (elisa) , have been developed but not yet adequately evaluated. while lfis are remarkably fast and only require minutes to perform, significant concern regarding their sensitivity and specificity has been raised . elisas are considered more robust but require highly specialized laboratories with the capacity to run automated high-throughput measurements. at the time of compiling this paper, the diagnostic performance of different immunoassays as well as their predictive value for protective immunity remains unclear. before a broad implementation of immunoassays can be justified, the following points need to be carefully assessed in adequately powered and designed diagnostic studies: ( ) diagnostic accuracy (or sensitivity/ specificity respectively) in the acute and subacute phase of the disease, ( ) antibody kinetics over time in patients with confirmed covid- , ( ) extent of cross-reactivity with other pathogens and patients with autoimmune disorders, ( ) reliability between different assay settings and material characteristics, as well as ( ) correlate of protective immunity . with the present study, we aimed to comprehensively establish the utility and diagnostic accuracy of serological immunoassays for sars-cov- infection and to explore protective immunity as predicted by such immunoassays in a mixed-method observational study of hospital inpatients as well as medical personnel. this article is protected by copyright. all rights reserved international guidelines on study design were strictly followed and cross-sectional, prospective observational, as well as case-control designs were used. participants were recruited via three different routes: (i) inpatients with a sars-cov- test result (real-time pcr; rt-pcr), (ii) medical personnel of the inselspital, and (iii) residual material from patients stored at the liquid biobank bern (www.biobankbern.ch). inclusion criteria of inpatients are (i) hospitalisation in inselspital, (ii) tested positive for sars-cov- using rt-pcr (nasopharyngeal swab), (iii) aged or older and (iv) signed general consent (exemption was granted for a few patients). for this manuscript, only inpatients who had tested positive for sars-cov- with more than days of residual material available were considered. the temporal pattern of antibody response and seroconversion rate was assessed in a subgroup of inpatients; the first consecutive patients were selected. inclusion criteria of medical personnel were (i) medical staff at inselspital since february , (ii) aged or older, and (iii) signed informed consent. the personnel were recruited via mailing lists. a limited number of fully anonymized, residual biobank samples were also used for the purpose of this study with the inclusion criterion of having been collected from inpatients between december and february . a total of randomly selected sera from individuals who were tested positive in either of the three elisa immunoassays as well as negative controls were assessed in a live sars-cov- neutralization assay (all collected in april ). the university hospital bern (inselspital) is one of the largest tertiary hospitals in switzerland covering a catchment area of more than million inhabitants. with several associated smaller hospitals, it provides the full spectrum of general as well as highly specialised medical services. more than , employees work at the insel gruppe ag. the study was supported by the local covid- task force. the study protocol was approved by the appropriate ethics committee and the authorities of the university hospital and conducted in accordance with the declaration of helsinki. the manuscript was prepared according to the standards for reporting diagnostic accuracy studies (stard) guideline . this article is protected by copyright. all rights reserved blood was taken following an established in-house protocol to ensure adequate preanalytical conditions and samples were collected using plastic syringes (serum or lithium heparin respectively, s-monovette®, sarstedt, nümbrecht, germany). only residual material was used in the case of inpatients. two tubes (serum and lithium heparin respectively) were drawn in the case of medical personnel. samples were immediately transported to the central laboratory, processed using a glp laboratory track and centrifuged within minutes with an established protocol . with regard to inpatients, pseudonymized demographical, clinical as well as laboratory data were extracted and transferred by the insel data science center (idsc) from electronic patient documentation. limited data were collected for the purpose of this substudy: age, gender, time interval since rt-pcr (nasopharyngeal swab). a positive sars-cov- rt-pcr result was used as additional inclusion criterion. with regard to medical personnel, a redcap database survey was constructed collecting demographical data, covid- symptoms (presence, extent and date), comorbidities and risk factors, professional exposure, and date of rt-pcr. the s protein and rbd are regarded as ideal candidates for the development of diagnostic tests and vaccines targeting sars-cov- . the pcaggs plasmid containing the human codon-optimized sequence of the sars cov- s protein receptor binding domain (rbd, amino acids r -f ) with native s signal sequence (amino acids m -s ) and a c-terminal hexahistidine tag was kindly provided by prof. florian krammer. plasmid dna was prepared using the gene elute hp plasmid maxiprep kit (sigma-aldrich). prior to transfection expi f cells (thermo-fisher) were grown to a density of . x cells/ml in culture medium (a mixture of % expi and % freestyle- media from thermo-fisher). for each liter of transfection, . mg of plasmid dna was diluted in ml of culture medium, mixed with . ml fectopro transfection reagent (polyplus), and incubated for minutes at room temperature prior to addition to cells. immediately following transfection cells were supplemented with x d-glucose ( g/l) this article is protected by copyright. all rights reserved and x valproic acid ( mm) boost solutions. three days post transfection the cell culture supernatants were harvested by centrifugation at , x g for min. supernatants were passed through a . µm filter and : diluted with pbs containing mm imidazole. for purification of his-tagged rbd protein ml ni-nta resin (hispur ninta thermofisher) was washed three times with washing buffer (pbs with mm imidazole) and incubated on a stir plate at °c for hour. subsequently, the mixture was poured into a glass column with a frit and washed times with column volumes of washing buffer. the protein was then eluted three times with ml pbs containing mm imidazole. elutions were pooled and dialyzed overnight against pbs using . kda cutoff snakeskin dialysis tubing. the final protein concentration was determined by nanodrop measurement at a . the quality of recombinant rbd protein was analyzed by sds-page and analytical size-exclusion chromatography. all elisa assays were performed on a dsx automated elisa system device (dynex technologies). the in-house assay was prepared as follows: -well plates were coated overnight at °c with µl of µg/ml rbd protein in pbs. the following day, each well was blocked with µl of pbs/ . % casein at °c until use and at least overnight. subsequently plates were washed twice with pbs and µl sera were added at a : dilution in pbs/ . % casein for hour at rt. after five washes with µl pbs/ . % tween µl of hrp-labeled secondary polyclonal anti-human igm (sigma, a ) and anti-human igg (sigma, a ) antibodies were added in a : ' dilution for minutes at rt. again, the plates were washed times with pbs/ . % tween and µl of tmb substrate solution (sigma, t ) was added for minutes at rt. the development was stopped by adding µl of . m h so and results were measured at od - nm. all samples with an od > . were assigned as positive. several commercial tests were conducted according to the manufacturers' instructions. an elisa produced by euroimmun ag, lübeck, germany targeting the s protein as the this article is protected by copyright. all rights reserved immobilized antigen for the detection of igg antibodies was employed. briefly, samples were diluted : in sample buffer and l of diluted samples, pre-diluted positive and negative controls, as well as pre-diluted calibrator were added for hour at °c. after three wash steps with µl wash buffer, µl of hrp-labeled secondary anti-human igg antibodies were added for minutes at °c. the plates were washed again three times with wash buffer and µl of tmb solution was added for minutes at rt. the development was stopped by adding µl of . m h so and results were measured at od - nm. antibody values were expressed as a ratio (od sample /od calibrator ). all samples with a ratio > . were assigned as positive. comorbidities and risk factors, which will be used as covariables in subsequent phases of this study, will be extracted from electronic patient records and asked in the redcap this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved at university hospital bern we first established a carefully designed mixed-method diagnostic accuracy study (fig. ) . this article is protected by copyright. all rights reserved breathlessness, coughing, or loss of smell recombinantly expressed rbd has been used to establish an in-house elisa for the detection of igm and igg anti-sars-cov- antibodies in human serum samples (supplementary fig. a,b) . optimal serum dilutions were determined by titration of sera derived from six sars-cov- + and six sars-cov- -individuals. the serum dilution of : allowed efficient discrimination between positive and negative outcome (supplementary fig. ) . after automatization on a dynex dsx device, the intra-assay (within-run) and inter-assay (day-to-day) precisions of the in-house rbd elisa was assessed (supplementary fig. a- e,f) . overall, the in-house rbd elisa assay showed high intra-and inter-assay reproducibility and demonstrated a high degree of agreement between plasma and serum samples. among a subgroup of sars-cov- + inpatients, seroconversion for igm and igg antibodies was observed between day and day after the rt-pcr result and between day and day after the start of symptoms (fig. ) . interestingly, igm and igg antibody responses against rbd and s were substantially more pronounced as compared to n. assessment of the longitudinal dynamics of patient sera revealed a marked and consistent increase of igg antibodies for rbd and s (fig. a) . igm antibodies were measured in the rbd and n elisa and detectable at least for two weeks after seroconversion (fig. b) . interestingly, the individual temporal igg and igm patterns showed a high degree of inter-individual variability with one group of patients this article is protected by copyright. all rights reserved of these samples, all were negative for anti-rbd igm and igg, as well as anti-s igg. however, two biobank samples tested positive for anti-n igg (elisa; . %), and one tested positive for anti-n igm (elisa; %). all samples were negative for anti-s igg and igm ( %) as tested by lfi. the pooled study population consisted of individuals, of whom tested as rt-pcr positive (prevalence . %). sera from all individuals were tested in the three different elisa setups for igg and igm anti-sars-cov- antibodies (fig. a) . a subgroup of samples (n= ) was additionally assessed on lfi (fig. b) . both assay formats showed high specificity above % for igg and igm measurements (supplementary table ). however, the sensitivity between assays and formats varied considerably. the highest sensitivities were reached for igg measurements with the s ( . %) and rbd ( . %) elisa, followed by igg measurements on n ( . %) elisa. sensitivities for igm measurements were all considerably lower for both elisa and lfi formats, which could be due to the more transient detectability of igm upon infection. to detect potential sources of variability, we additionally studied the antibody response in salient subgroups of rt-pcr positive individuals (fig. c) this article is protected by copyright. all rights reserved in the tested inpatient population, we observed three "false-negative" (negative in s elisa despite positive rt-pcr) outcomes. among three false-negative inpatients (p , p , and p ), two were measured at an early time-point (patient and ), and one patient (p ) might have experienced seroconversion at a very late time-point because of a significant increase of antibody titers at day (supplementary fig. and supplementary fig. ) . in the assessed hospital staff, seven were classified as "falsenegative". all of these reported mild diseases and had symptoms clearly associated with covid- (fever, breathlessness, cough, and loss of taste or smell). twenty-two individuals in the hospital staff group tested "false-positive" (positive s elisa results despite negative rt-pcr). fourteen of them experienced one or more symptoms clearly associated with covid- . the remaining eight individuals were clearly positive in at least three assays. all other individuals were either classified as "true-positive" (positive in s elisa, and positive in rt-pcr), or as "true-negative" (negative in s elisa, and negative in rt-pcr). in terms of performance, the calculated area under the receiver operating characteristic (fig. b) . a total of randomly selected sera from individuals who were tested positive in either of the three elisa immunoassays as well as negative controls were assessed in a live sars-cov- neutralization assay using ace -expressing vero-e cells ( inpatient samples, and samples of medical personnel). full neutralization of viral infection has been determined based on % inhibition of the cytopathic effect in a serial dilution of the sera (supplementary fig. ) . the means of highest serum dilutions at which full neutralization was observed correlated remarkably well with the measured antibody responses in the elisa immunoassays (fig. a-c) . importantly, . % of the sera from elisa positive individuals showed full inhibition at serum dilutions ≥ : . the two sera that did not show neutralization (p and p ) were drawn at an early time point this article is protected by copyright. all rights reserved where the patients did not yet show antiviral antibodies. both patients, however, fully neutralized the virus after seroconversion at a later time point (fig. d) . further, all sera from elisa negative individuals showed no neutralizing activity. of note, one or two elisa assays were negative in samples with full neutralization. we report first results of a large, mixed-design evaluation study which was implemented to compare the diagnostic accuracy of serological immunoassays for sars-cov- antibodies. while the time to seroconversion varied substantially between infected individuals, the mounted igg responses were robust and stable over time in all assays relying on rbd, s as well as n. with regards to the elisa assays, the overall diagnostic accuracy was adequate with a high specificity. some "false-positive" results are likely due to a rather narrow diagnostic window and limited sensitivity of the rt-pcr as well as asymptomatic disease course . "false-negative" results may be caused by a long seroconversion period observed in some patients and mild disease course in other individuals. the accuracy measures of lfi and n were inferior compared to elisa targeting s and rbd. strikingly, there is a high degree of correlation between antibody responses to these viral surface proteins and the neutralizing activity against live sars- a few other studies have previously assessed the diagnostic accuracy of serological immunoassays. recently, long and colleagues studied the antibody response in patients with covid- using a magnetic chemiluminescent immunoassay. in accordance with their results, we observed high inter-individual variation in the time to seroconversion. in contrast to their study, we confirmed these findings with an appropriate diagnostic accuracy protocol using different serological immunoassays. in another case-control study, infantino et al. analyzed covid- inpatient samples and selected patients collected before using a magnetic chemiluminescent immunoassay . in agreement with their results, we found limited sensitivity but high specificity of the serological sars-cov- immunoassays. in further study conducted at the geneva university hospital, samples of covid- patients were included as well as controls collected before , and analyzed with the same s elisa that we this article is protected by copyright. all rights reserved used in our study. similar to our results they report a high specificity for igg, particularly with an adjusted cut-off value . in line with other studies the accuracy and performance lfis was rather weak , . the study presented here adds important value to previous reports as it (i) was designed as a comprehensive diagnostic accuracy study combining different research methods, (ii) directly compares major assay approaches, (iii) was fully approved by all appropriate authorities, (iv) was independently conducted at a university hospital, (v and indicate that such serological tests might even be used to predict protective immunity in near future , . to draw further conclusions, however, sars-cov- positive patients have to be followed over an extended time period in future studies. this article is protected by copyright. all rights reserved in line with previous studies , we observed that the antibody response is more pronounced in patients with severe disease than patients without (figure , panel c; inpatients, hospitalized patients, older patients). however, the response was similar in patients with mechanical ventilation and hospitalized patients. this is most likely due to limitations in sensitivity, which does not contradict our general observations. in summary, we report the first results of a large, mixed-design evaluation study that has been conducted in an independent academic setting at the university hospital bern to assess the diagnostic accuracy of various immunoassays to determine antibody responses against sars-cov- . while antibody responses of individual covid- patients against rbd and s protein were similar, a weaker reactivity against n protein became apparent. the time to seroconversion varied substantially between covid- patients but the igg response was robust and stable in all three elisa setups. their overall diagnostic accuracy was adequate with a high specificity but limited sensitivity. the antibody responses measured in these elisas correlated remarkably well with sars-cov- neutralizing activity of the sera. on the other hand, accuracy measures of s protein based lfis were poor. together, our results emphasize that appropriate serological immunoassays represent a valuable tool to identify a good portion of patients with previous sars-cov- infection, will help to facilitate exit strategies from lockdown and might even be used to predict immunity to sars-cov- in near future. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved accepted article serology assays to manage covid- the laboratory diagnosis of covid- infection: current issues and challenges the important role of serology for covid- control connecting clusters of covid- : an epidemiological and serological investigation investigation of three clusters of covid- in singapore: implications for surveillance and response measures universal weekly testing as the uk covid- lockdown exit strategy immunology of covid- : mechanisms, clinical outcome, diagnostics and perspectives -a report of the european academy of allergy and clinical immunology (eaaci) distribution of ace , cd , cd , and other sars-cov- associated molecules in tissues and immune cells in health and in asthma, copd, obesity, hypertension, and covid- risk factors sars-cov- , covid- , skin and immunology -what do we know so far? allergy clinical characteristics of patients infected with sars-cov- in wuhan is global bcg vaccination-induced trained immunity relevant to the progression of sars-cov- pandemic? immune response to sars-cov- and mechanisms of immunopathological changes in covid- a compendium answering questions on covid- and sars-cov- accepted article this article is protected by copyright. all rights reserved clinical characteristics of pediatric covid- patients with different severities and allergic status blood myeloperoxidase-dna, a biomarker of early response to sars-cov- infection? allergy a preliminary study on serological assay for severe acute respiratory syndrome coronavirus (sars-cov- ) in admitted hospital patients. medrxiv antibody responses to sars-cov- in covid- patients: the perspective application of serological tests in clinical practice evaluation of nucleocapsid and spike protein-based elisas for detecting antibodies against sars-cov- the coronavirus nucleocapsid is a multifunctional protein the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor sars-cov- receptor ace protein expression in serum is significantly associated with age neutralizing antibodies against sars-cov- and other human coronaviruses characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine. cellular & molecular immunology rapid development of an inactivated vaccine for sars-cov- designs, formats and applications of lateral flow assay: a literature review accepted article this article is protected by copyright. all rights reserved cross-sectional pilot study exploring the feasibility of a rapid sars-cov- immunization test in health and nonhealthcare workers a serological assay to detect sars-cov- seroconversion in humans. medrxiv sars-cov- seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup evaluation of antibody testing for sars-cov- using elisa and lateral flow immunoassays studies for evaluating diagnostic and prognostic accuracy stard : updated reporting guidelines for all diagnostic accuracy studies rapid centrifugation in the routine hemostasis laboratory sars-cov- immunogenicity at the crossroads detection of novel coronavirus ( -ncov) by real-time rt-pcr distinct characteristics of covid- patients with initial rrt-pcr-positive and rrt-pcr-negative results for sars-cov- antibody responses to sars-cov- in patients with covid- diagnostic accuracy of an automated chemiluminescent immunoassay for anti-sars-cov- igm and igg antibodies: an italian experience validation of a commercially available sars-cov- serological immunoassay accepted article this article is protected by copyright. all rights reserved rapid point-of-care testing for sars-cov- in a community screening setting shows low sensitivity evaluation of sars-cov- serology assays reveals a range of test performance a comparison of four serological assays for detecting anti-sars-cov- antibodies in human serum samples from different populations sars-cov- seroconversion in health care workers lack of reinfection in rhesus macaques infected with sars-cov- . biorxiv serological and molecular findings during sars-cov- infection: the first case study in finland severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease patients antibody responses to sars-cov- at weeks postinfection in asymptomatic patients the dna plasmid encoding the sars-cov- receptor binding domain of the spike all authors declare that there is no conflict of interests. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord- -ysgz adr authors: arabi, yaseen m.; hajeer, ali h.; luke, thomas; raviprakash, kanakatte; balkhy, hanan; johani, sameera; al-dawood, abdulaziz; al-qahtani, saad; al-omari, awad; al-hameed, fahad; hayden, frederick g.; fowler, robert; bouchama, abderrezak; shindo, nahoko; al-khairy, khalid; carson, gail; taha, yusri; sadat, musharaf; alahmadi, mashail title: feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: ysgz adr we explored the feasibility of collecting convalescent plasma for passive immunotherapy of middle east respiratory syndrome coronavirus (mers-cov) infection by using elisa to screen serum samples from potential plasma donors: patients with suspected or laboratory-confirmed mers-cov infection, healthcare workers, and household contacts exposed to mers-cov. elisa-reactive samples were further tested by indirect fluorescent antibody and microneutralization assays. of the tested samples, ( . %) had a reactive elisa result, and of the had reactive indirect fluorescent antibody and microneutralization assay titers. undertaking clinical trials of convalescent plasma for passive immunotherapy of mers-cov infection may be feasible, but such trials would be challenging because of the small pool of potential donors with sufficiently high antibody titers. alternative strategies to identify convalescent plasma donors with adequate antibody titers should be explored, including the sampling of serum from patients with more severe disease and sampling at earlier points during illness. we explored the feasibility of collecting convalescent plasma for passive immunotherapy of middle east respiratory syndrome coronavirus (mers-cov) infection by using eli-sa to screen serum samples from potential plasma donors: patients with suspected or laboratory-confirmed mers-cov infection, healthcare workers, and household contacts exposed to mers-cov. elisa-reactive samples were further tested by indirect fluorescent antibody and microneutralization assays. of the tested samples, ( . %) had a reactive elisa result, and of the had reactive indirect fluorescent antibody and microneutralization assay titers. undertaking clinical trials of convalescent plasma for passive immunotherapy of mers-cov infection may be feasible, but such trials would be challenging because of the small pool of potential donors with sufficiently high antibody titers. alternative strategies to identify convalescent plasma donors with adequate antibody titers should be explored, including the sampling of serum from patients with more severe disease and sampling at earlier points during illness. m iddle east respiratory syndrome coronavirus (mers-cov) was initially identified in september when a patient in saudi arabia with a severe, acute respiratory infection and acute renal failure died ( ) . as of june , , more than , mers-cov cases and at least associated deaths had been identified; > % of the cases occurred in saudi arabia ( ) . more than countries outside of the arabian peninsula have reported mers-cov cases, and the outbreak in south korea with attendant mortality has reinforced concerns about international outbreaks ( ) . no specific treatment has been proven effective for mers-cov infection. convalescent plasma containing mers-cov-specific antibodies from recovered patients has been suggested as a potential therapy for infected persons ( ) . convalescent plasma has been used to treat several other viral infections, including those caused by the severe acute respiratory syndrome coronavirus (sars-cov), avian influenza a(h n ) virus, and influenza a(h n )pdm virus ( ) ( ) ( ) ( ) ( ) ( ) . a recent metaanalysis of studies using passive immunotherapy for treatment of severe acute respiratory infections of viral etiology suggests that the timely use of convalescent blood products, particularly those with neutralizing antibodies, results in a reduced death rate ( ) . public health england and isaric (the international severe acute respiratory and emerging infection consortium) published a decision-making support tool on potential therapies for mers-cov that highlights convalescent plasma and other neutralizing antibody-containing immunotherapeutics (e.g., hyperimmune immunoglobulins and monoclonal antibodies) as the most promising potential treatments for serious mers-cov illness and deserving of evaluation in human clinical trial(s) ( ) . however, no data support the feasibility of obtaining convalescent plasma from patients who have been exposed to mers-cov or recovered from infection with the virus. camels are the likely source for most animalto-human transmission and appear to have long-lasting antibody responses; in preclinical models, such antibodies appear effective in reducing the severity of pathologic plasma immunotherapy for mers-cov infection, saudi arabia changes in infected lungs ( ) . however, the antibody response to mers-cov infection in humans is poorly defined. thus, we planned a -phase study to ) determine the feasibility of collecting high-titer convalescent plasma from mers-cov patients and contacts and, if successful, to ) conduct a pilot therapeutic study using convalescent plasma in symptomatic mers-cov patients with moderate to severe illness. herein, we report on the feasibility study. in collaboration with the king abdullah international medical research center, the gulf cooperation council infection control center, and the world health organization (who)-international severe acute respiratory and emerging infection consortium mers-cov working group, we developed a study protocol to screen potential donors, collect high-titer convalescent plasma, and administer the plasma in a clinical trial ( ) . the study was approved by the ministry of the national guard health affairs institutional review board (approval no. irbc/ / , june , ) and registered in clinicaltrials.gov (nct ). we conducted the study at king abdulaziz medical city, a , -bed tertiary care center in riyadh, saudi arabia. the hospital is accredited by the joint commission international, and the hospital's department of pathology and laboratory medicine is accredited by the college of american pathologists and the american association of blood banks. we screened potential convalescent plasma donors from cohorts: ) patients with acute respiratory illness who were suspected of having mers-cov or who were confirmed mers-cov-positive by real-time reverse transcription pcr (rrt-pcr) of upper or lower respiratory secretions; ) healthcare workers exposed to a laboratoryconfirmed mers-cov patient, as identified by ongoing active surveillance of the hospital infection prevention and control department; and ) household contacts of patients with laboratory-confirmed mers-cov infection. we obtained written informed consent for mers-cov serologic testing from all healthcare workers and household contacts. medical teams ordered serologic testing as part of the clinical care for patients with suspected or confirmed mers-cov infection; no additional informed consent was required. healthcare workers completed a self-administered survey that asked questions about the nature, duration, and degree of exposure to patients with laboratory-confirmed mers-cov infection. for all study participants, we documented the time that had elapsed from symptom onset or exposure to the collection of samples for testing. during july-october , we screened serum samples from study participants by using a spike protein subunit (s )-based elisa. to confirm results of elisa-reactive samples, we used indirect immunofluorescent antibody (ifa) and microneutralization (mn) assays ( ) ( ) ( ) . for mers-cov patients with a nonreactive elisa result, we collected a follow-up sample - days later for repeat elisa. study participants were considered candidates for plasma donation if they ) had a reactive elisa result; ) had a mn assay titer of > ; ) had no clinical or laboratory evidence of ongoing mers-cov infection; and ) met the eligibility for plasma donation according to the institutional criteria, which were in accordance with who guidelines ( ) . persons who met all criteria were eligible for plasma donation according to the position paper of the who blood regulators network ( ) . the primary outcome of this first phase of the study was the feasibility of conducting the second phase. feasibility was measured by our ability to screen and identify a sufficient number of potential plasma donors to provide enough high-titer, fresh-frozen plasma to enroll and provide transfusions to patients over months. each phase -enrolled patient would require fresh-frozen plasma units ( - ml/unit). we first conducted testing for mers-cov by rrt-pcr. we extracted rna from respiratory specimens (nasopharyngeal swab, tracheal aspirate, bronchoalveolar lavage) using the magna pure viral na kit (roche applied science, indianapolis, in, usa). we tested the extracted nucleic acids by rrt-pcr targeting the upstream envelope protein gene (upe) and open-reading frame a (orf a) regions of the mers-cov genome on a lightcycler system (roche diagnostics, mannheim, germany) realtime pcr ( ) . a positive control for orf a and upe rrt-pcr was performed according to the manufacturer's instructions. to be consistent with the cutoff used by the saudi arabia ministry of health reference laboratory, we considered a cycle threshold (c t ) of < the cutoff for upe and orf a. for c t s > , we repeated the testing using different samples, preferably from the lower respiratory tract, to avoid false-positive results. we detected mers-cov antibodies by elisa (euroimmun ag, lubeck, germany), using wells coated with mers-cov s antigen ( , ) . serum samples were diluted ( : ) and incubated with antigens according to the elisa manufacturer's instructions. positive and negative control serum and calibration samples were included. antibodies were detected by adding peroxidase-labeled rabbit anti-human igg (euroimmun ag, lubeck). results were reported as the optical density (od) ratio, which was calculated as the od value of the patient's sample divided by the calibrator od value. we used cutoff values recommended by the elisa kit manufacturer: a ratio of < . was considered negative, > . to < . was considered borderline, and > . was considered positive. we used an ifa (euroimmun ag) according to the manufacturer's instructions to detect mers-cov antibodies. serum samples were diluted in doubling dilutions, starting with : and ending with : , , in sample buffer and then incubated with vero b cells infected with hcov-emc (euroimmun ag). mers-cov igg was detected by adding fitc-labeled goat anti-human igg (euroimmun ag); positive and negative controls were included. samples with an ifa titer of > : were considered reactive according to the ifa manufacturer's instructions. our original protocol used an ifa cutoff of > to define suitable donors for plasma ( ) . in the course of the study, mn became available, and we revised the criteria for plasma donation to be based on mn assay results. the presence of neutralizing mers-cov antibodies was also assessed using a mn assay ( ) . in brief, × vero cells/well were plated onto a -well microtiter plate. after h, -fold serial dilutions of serum samples (heat-inactivated at °c for min) were incubated with an equal volume of the mers-cov strain jordan-n / ( tcid [ % tissue culture infectious doses]) for h at °c ( ) . medium was aspirated from the microtiter plate, and ml of the serum-virus mixture was added to the wells in triplicate. the plate was incubated for h at °c in a humidified chamber with % carbon dioxide, after which the serum-virus mixture was aspirated and the cells were fixed by adding ml of a : mixture of cold ethanol and methanol. the plate was then incubated at - °c for min, washed times with pbs, and processed as described above for elisa, using rabbit anti-coronavirus spike protein antibody and horseradish peroxidase-conjugated goat anti-rabbit igg secondary antibody. plates were developed using abts substrate (kpl inc., gaithersburg, md, usa); od was measured at nm. controls consisted of uninfected cells and cells infected with tcid of mers-cov. the highest dilution of serum sample that resulted in a > % reduction in od, compared with the control containing no antibody, was reported as the % virus neutralization titer. rrt-pcr, elisa, and ifa testing for mers-cov were performed at the king abdulaziz medical city laboratory. mn was performed at the naval medical research center (silver spring, md, usa). we used descriptive statistics (i.e., numbers and proportions, means ± sd, and medians with quartile [q ] and q values) for measurements for eligible donors and participants with seroreactive test results. we used the pearson correlation to test for correlations between elisa od and ifa and mn titers. the identity of study participants with mers-cov was known only to investigators listed on the approved king abdullah international medical research center protocol. all samples were delinked from any identifiable personal information when provided to nonlisted investigators. we contacted healthcare workers who had a history of exposure to or a diagnosis of mers-cov infection. of those healthcare workers, ( %) consented to serum sampling and were tested (table ) ; had a history of laboratory-confirmed mers-cov infection, and had a history of exposure but were mers-cov rrt-pcr negative during their asymptomatic or potential immediate postincubation period. only ( . %) of healthcare workers who had a history of laboratory-confirmed mers-cov infection had elisa-reactive serum samples after a median of days (q days, q days) after infection ( figure ). the confirmatory ifa was reactive for all of those healthcare workers, and mn was reactive for (table ) . however, only healthcare worker (participant no. ) had a high mn titer ( ) ( table ), but she was not considered a candidate for plasma donation because of a previous pregnancy. exposed healthcare workers who had negative mers-cov rrt-pcr results also had nonreactive elisa results. a total of patients with suspected or laboratoryconfirmed mers-cov infection were tested; ( . %) were hospitalized in the emergency department, ( . %) in the intensive care unit, and ( . %) in the medical wards (table ) . two ( %) of patients with laboratoryconfirmed mers-cov and ( %) of who were mers-cov rrt-pcr negative had elisa-reactive serum samples. ifa and mn assay results were positive for ( %) of patients who had elisa-reactive serum samples; the patients who had nonreactive ifa results also had nonreactive mn results. one of the patients (no. ) had high ifa ( : , ) and mn ( ) titers (table ) . this patient, a -year-old man, was admitted to the intensive care unit with mers-cov infection resulting in acute respiratory distress syndrome, acute kidney injury, and shock. he required mechanical ventilation, renal replacement therapy, and vasopressors ( figure ). the high titer occurred while he was in intensive care, days after symptom onset. his serologic titers by elisa, ifa, and mn declined progressively as he recovered clinically; elisa and ifa were nonreactive by months after hospital admission ( figure ). of the patients, (nos. - ) had mn titers > (table ), but these patients did not meet clinical criteria for plasma donation because of age, concurrent conditions, or previous pregnancy. of note, patients with laboratory-confirmed mers-cov infection had a nonreactive elisa; these samples were collected , , and days after symptom onset. two of the patients died before the test was repeated. for the third patient, repeat elisas at and weeks after the first nonreactive elisa were negative. a median of days (q days, q days) after patients received a laboratory diagnosis of mers-cov infection, we tested household contacts for of the patients and for the other (table ) . serum samples for all contacts were nonreactive by elisa (figure ; table ). elisa and mn results were highly correlated (pearson correlation coefficient . , p = . ) (figure ) . however, elisa and ifa results showed only a modest correlation (pearson correlation coefficient . , p = . ), and ifa and mn results were not statistically table . characteristics of participants in a study for the feasibility of collecting convalescent plasma from persons who had been infected with or exposed to mers-cov, saudi arabia, july-october * characteristic value healthcare workers exposed to laboratory-confirmed mers-cov patients, n significantly correlated (pearson correlation coefficient . , p = . ). our results indicate that it would be possible to obtain quantities of convalescent plasma large enough to use in therapeutic studies or in a large number of mers-cov patients; however, large-scale screening would be required because of the limited availability of eligible potential donors with sufficient levels of antibody. our findings suggest that recently recovered mers patients may be suitable potential donors, provided they meet other plasma donation criteria. of note, none of the seropositive persons in our study met our clinical and laboratory criteria for plasma donation. our findings show that serum antibody to mers-cov was infrequently reactive by elisa; however, reactivity may have been affected by the timing of sample collection or severity of the illness. most of the small subset of participants with elisa-reactive serum samples had mers-cov antibodies as assessed by ifa and mn. elisa results and mn titers were highly correlated; ifa and mn were not. one healthcare worker had high mn titers, but she did not meet the clinical criteria for plasma donation. another critically ill patient had high antibody titers by the assays, but antibody titers declined quickly as the patient recovered clinically, and he was not eligible to donate plasma. in accordance with who and us centers for diseases control and prevention guidelines, we used elisa (figure ). values shown are the highest values for the patient. to screen for mers-cov igg and ifa and mn assays to confirm positive results ( , ) . the elisa is based on the virus s protein as antigen, and the ifa is based on detection of virus-specific antibodies, using cell cultures infected with the virus. the mers-cov spike protein is a glycoprotein that forms the spikes of the virus, and the n terminal component (s ) is believed responsible for the first step of virus entry into the host cell ( ) . our original protocol used ifa as a confirmatory test; however, we switched to mn when that assay became available. our findings showed a high correlation between elisa and mn results but not between ifa and mn results, which may indicate that mn is a better confirmatory test. however, for patient in our study, the tests showed similar results (figure ), and other studies have shown good correlation between the tests ( ) , which may indicate that the lack of correlation shown between ifa and mn in our study was associated with sample size. our findings suggest that the low prevalence of seroreactivity for mers-cov, even among persons with confirmed or suspected infection, may be a reflection of a short-lasting antibody response. it is possible that some of the study participants were seronegative at the time of testing because the window for positive serologic results had passed. a short-lasting immune response may also partly explain why negligible or low levels of mers-cov seroreactivity have been detected in persons at risk for the disease (i.e., camel and abattoir workers) in saudi arabia and elsewhere ( ) ( ) ( ) ( ) . a seroprevalence study conducted during december -december showed mers-cov antibodies in only . % of the general population (n = , ) in all provinces in saudi arabia ( ) . seroprevalence was also low among camel shepherds ( . %, n = ) and slaughterhouse workers ( . %, n = ), albeit higher than in the general population ( ) . the clinical relevance of antibody titers in protecting against subsequent mers-cov infection is uncertain. similar findings have been described with other coronaviruses. cao et al. ( ) studied specific and neutralizing antibody titers in patients who recovered from sars-cov infection. their findings showed that sars-cov igg and neutralizing antibodies peaked at months and then began diminishing, reaching undetectable levels in . % (igg) and . % (neutralizing antibodies) of patients at months. xie et al. ( ) showed that sars-cov igg decreased over year in recovering sars patients. in an experiment of intranasal inoculation of cov e in human volunteers, callow et al. ( ) studied the time course of specific antibody response and found that those antibodies peaked week after the inoculation and then began declining. furthermore, it appears that the antibody immune response to mers-cov in humans differs from that in camels. alagaili et al. ( ) showed that % of camels from different parts of saudi arabia have antibodies to mers-cov by elisa, and the prevalence of antibodies is higher in older camels ( %). two patients in the mers-cov outbreak in south korea were reported to have received convalescent plasma collected from recovered patients ( ) . it is unclear whether the plasma was tested for the presence of mers-cov antibodies. our study demonstrates that such testing should be mandatory for donated convalescent plasma because of the low prevalence of mers-cov antibodies, even in patients with past laboratory-confirmed mers-cov infection. without such testing, the presence of antibodies to mers-cov cannot be confirmed, and the convalescent plasma may not be associated with a protective effect. our study also highlights the need for prospective serology studies to better understand the humoral response to mers-cov infection. the strengths of our study are that we screened a large number of persons, including patients with laboratoryconfirmed mers-cov infection, and used screening and confirmatory antibody assays. a study limitation was the small number of household contacts who were screened, although, based on our findings and those of others ( ) , a small proportion of household contacts are likely to show seroreactivity. only one third of invited healthcare workers participated in the study. we used an s -based elisa for screening, and our study did not address the magnitude and duration of other antibody isotypes in the immune response. the interval between illness and recovery was prolonged in most of the exposed healthcare workers (median interval > year). it is possible that earlier sampling would have resulted in the detection of more reactivity and higher antibody titers. our study, which was designed to screen for antibodies in convalescent plasma, was not designed to characterize the immune response to mers-cov infection or to identify clinical correlates of the presence or absence of mers-cov antibodies. further testing is needed to determine whether the antibodies in convalescent plasma are clinically effective against mers-cov infection. our findings suggest the need to explore other passive immunotherapeutic approaches, such as monoclonal or polyclonal human antibodies from transchromosomic bovines ( , ) and, possibly, polyclonal antibodies from camels ( ) . our findings also raise questions about whether naturally occurring infections and potential mers-cov vaccines will offer longlasting immunity. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus outbreak in the republic of korea treatment of mers-cov: information for clinicians. clinical decision-making support for treatment of mers-cov patients hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe influenza a(h n ) infection sars: systematic review of treatment effects convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h n ) virus infection meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h n treatment? use of convalescent plasma therapy in sars patients in hong kong successful treatment of avian influenza with convalescent plasma the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol centers for disease control and prevention. cdc laboratory testing for middle east respiratory syndrome coronavirus world health organization. laboratory testing for middle east respiratory syndrome coronavirus: interim recommendations (revised) human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo world health organization. guidelines on assessing donor suitability for blood donation position paper on collection and use of convalescent plasma or serum as an element in middle east respiratory syndrome coronavirus response detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction transmission of mers-coronavirus in household contacts presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a -residue region in the spike protein that efficiently elicits neutralizing antibodies mers coronaviruses in dromedary camels lack of mers coronavirus neutralizing antibodies in humans investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during . influenza other respir viruses disappearance of antibodies to sars-associated coronavirus after recovery dynamic changes of serum sars-coronavirus igg, pulmonary function and radiography in patients recovering from sars after hospital discharge the time course of the immune response to experimental coronavirus infection of man middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia south korea tries old weapon to fight mers pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection key: cord- -bmq zcb authors: martínez-sernández, victoria; perteguer, maría j.; hernández-gonzález, ana; mezo, mercedes; gonzález-warleta, marta; orbegozo-medina, ricardo a.; romarís, fernanda; paniagua, esperanza; gárate, teresa; ubeira, florencio m. title: comparison of recombinant cathepsins l , l , and l as elisa targets for serodiagnosis of bovine and ovine fascioliasis date: - - journal: parasitol res doi: . /s - - - sha: doc_id: cord_uid: bmq zcb infections caused by fasciola hepatica are of great importance in the veterinary field, as they cause important economic losses to livestock producers. serodiagnostic methods, typically elisa (with either native or recombinant antigens), are often used for early diagnosis. the use of native antigens, as in the mm -sero elisa (commercialized as bio k , bio-x diagnostics), continues to be beneficial in terms of sensitivity and specificity; however, there is interest in developing elisa tests based on recombinant antigens to avoid the need to culture parasites. of the antigens secreted by adult flukes, recombinant procathepsin l (rfhpcl ) is the most commonly tested in elisa to date. however, although adult flukes produce three different clades of cls (fhcl , fhcl , and fhcl ), to our knowledge, the diagnostic value of recombinant fhcl and fhcl has not yet been investigated. in the present study, we developed and tested three indirect elisas using rfhpcl , rfhpcl , and rfhpcl and evaluated their recognition by sera from sheep and cattle naturally infected with f. hepatica. although the overall antibody response to these three rfhpcls was similar, some animals displayed preferential recognition for particular rfhpcls. moreover, for cattle sera, the highest sensitivity was obtained using rfhpcl ( %), being equal for both rfhpcl and rfhpcl ( . %), after adjusting cut-offs for maximum specificity. by contrast, for sheep sera, the sensitivity was % for the three rfhpcls. finally, the presence of truncated and/or partially unfolded molecules in antigen preparations is postulated as a possible source of cross-reactivity. fascioliasis (or fasciolosis) is recognized as one of the most important food-borne trematodiasis in the veterinary field because of the economic impact it has on livestock (mainly sheep and cattle) producers (schweizer et al. ; charlier et al. ; charlier et al. ; mezo et al. ). the genus fasciola includes two species, f. hepatica and f. gigantica, the former of which is distributed worldwide and the second of which is restricted to several regions of africa and asia ). the broad distribution of f. hepatica seems to be associated with its ability to adapt to new definitive hosts and with its capacity to infect different snail species (intermediate hosts) living in diverse habitats and under different environmental conditions (correa et al. ) . when humans or animals are infected by ingestion of the metacercariae (present in vegetables or contaminated water), the parasites excyst in the intestine, transverse the intestinal wall and the peritoneal cavity, and then migrate to the liver where they feed and grow for - weeks (andrews ) . finally, the parasites move into biliary ducts where they mature and begin producing eggs. nevertheless, sometimes a small proportion of parasites reaches ectopic locations (mas-section editor: xing-quan zhu * florencio m. ubeira fm.ubeira@usc.es coma et al. coma et al. , , thus preventing elimination of the eggs through the biliary ducts. considering the characteristics of the biological cycle of fasciola, diagnosis of infections by either f. hepatica or the more pathogenic f. gigantica (valero et al. ) has traditionally been carried out by microscopic observation of parasite eggs in fecal samples from infected hosts (mas-coma et al. ). however, these methods are tedious, have poor sensitivity, and depend to a greater or lesser degree on the experience of the examiner. moreover, fecal examination cannot detect acute infections when the parasites feed on liver parenchyma, before egg production has started (charlier et al. ; mas-coma et al. ). in the past two decades, considerable effort has been made to develop rational elisa methods for the diagnosis of human and animal infections caused by f. hepatica and f. gigantica in order to prevent the limitations inherent in microscopic examination of fecal samples (espino and dumenigo ) . such methods include the use of whole or purified natural antigens from fasciola as well as recombinant antigens, to detect anti-fasciola circulating antibodies (cornelissen et al. ; carnevale et al. b; espinoza et al. ; mezo et al. ; gonzales santana et al. ; gottstein et al. ) . they also include elisa methods capable of detecting secreted antigens present in serum or fecal samples from infected humans and animals (espino and finlay ; abdel-rahman et al. ; mezo et al. ; ubeira et al., ; george et al., ) . because antibody titers may remain elevated for long periods of time after treatment of the infection, elisa methods for the detection of specific antibodies cannot distinguish between current or past infections. nevertheless, these elisas offer the advantage of being able to diagnose early prepatent (acute) infections (salimi-bejestani et al. ; mezo et al. a,b; mas-coma et al. ) , and, for this reason, they are frequently used to screen herds of domestic animals in serum and mostly in milk samples (mezo et al. b; duscher et al. ; mezo et al. ). most of these methods are indirect or capture elisas that include fasciola cathepsins l (cls) as antibody targets, as these antigens probably induce the highest proportion of anti-fasciola antibodies during natural infections. however, except for elisas based on the use of single recombinant cls (rcls) or recombinant procathepsins l (rpcls), typically l clade members, the suitability of each of the several cathepsins secreted by fasciola for detecting anti-fasciola antibodies by elisa has not previously been investigated. this aspect is relevant, as it has been reported that the pattern of cathepsins that are secreted by f. hepatica varies during its biological cycle, so that immature parasites begin producing fhcl , fhcl , and cathepsins b, but that expression is gradually replaced by fhcl , fhcl , and fhcl in adults (robinson et al. ; smooker et al. ; cwiklinski et al. ) . moreover, production of these fhcls by adult flukes has been reported to be imbalanced, with proportions of , and % for fhcl , fhcl , and fhcl , respectively ). it is therefore important to determine which of these molecules are most relevant from the point of view of their antigenicity and suitability as target antigens for serodiagnosis of human and animal infections by fasciola. in the present study, we developed and tested indirect elisas based on recombinant procathepsin l (rfhpcl ), rfhpcl , or rfhpcl , in order to investigate which of these targets are best recognized by sera from sheep and cattle naturally infected with f. hepatica. we also evaluated if any of these rfhpcls is advantageous over the use of native cls using as reference method of this study the mm -sero elisa, in which the monoclonal antibody (mab) mm captures native fasciola cls (mezo et al. ). cattle a slaughterhouse that processes cattle from the whole region was visited fortnightly during a year. at each visit, adult cows (over years old; friesian breed, females) were selected at random, and their livers as well as stool and blood samples were collected and transported to the laboratory promptly. in total, samples from naturally f. hepatica-infected and f. hepatica-free adult cattle, as determined, respectively, by the presence or the absence of flukes in livers (gold standard), were collected. for recovering and counting all flukes, each liver was thoroughly examined. in the first step, we proceeded to the opening of the bile ducts and the gallbladder to obtain the most of intact adult flukes and then the livers were cut into slices (approximately cm thick) which were manually squeezed to obtain the immature flukes inhabiting the parenchyma. this procedure prevented any possible problems of low sensitivity when liver inspection is not performed thoroughly, as has been described in previous studies (rapsch et al. ; charlier et al. ) . in addition to fluke counts, samples of feces from all animals were subjected to sedimentation (anderson et al. ) and flotation (maff ) procedures to concentrate eggs from fasciola or other parasites, and then they were microscopically examined. the remaining fecal material was frozen at − °c for subsequent further quantitation of fasciola antigens by the mm -copro elisa (mezo et al. ; martínez-sernández et al. ) . as we prioritized testing simultaneously the sera with the four f. hepatica antigens in a single run, the sample size was limited to f. hepatica-infected and f. hepatica-free cows, which was optimal for the conditions of our laboratory. for the first group, the infected animals (n = ) were stratified in three categories according to their parasite burden (low: - flukes, middle: - flukes, and high: ≥ flukes) and animals from each group were selected by random sampling using epidat . software (consellería de sanidade, xunta de galicia, spain). to assure a better comparison of the performance of the different elisas evaluated in the present study, animals with low parasite burdens represented the % (n = ) of the sample, while animals from the remaining categories constituted both the ≈ % (n = and ) of the sample. of the f. hepatica-free cattle, were selected by simple random sampling. serum and fecal samples used in the present study were obtained from the herd of a commercial farm suffering from fascioliasis (infected sheep) and from the fluke-free herd maintained at ingacal (non-infected sheep). the sheep in both herds were an autochthonous galician breed (braza gallega^). the sample size was limited by the availability of animals to conduct the study. in the first herd, the sheep suffering chronic fascioliasis (using both data from coprology and mm -copro elisa as gold standard) were sampled. in the second herd, samples were taken from another sheep chosen completely at random. as for cattle samples, the feces from all sheep were examined for the presence of eggs from fasciola and other parasites. fecal aliquots were also frozen at − °c for further quantitation of fasciola antigen by the mm -copro elisa (mezo et al. ; martínez-sernández et al. ). the f. hepatica excretory-secretory antigens (esas) used in the mm -sero elisa (see below) were obtained as previously reported (mezo et al. ) . briefly, live adult flukes were collected from the bile ducts of naturally infected cows and washed, first in sterile saline solution containing antibiotics (penicillin/streptomycin) and glucose ( g/l), at °c, and then in rpmi cell culture medium supplemented with mm hepes, . g/l l-glutamine, g/l sodium bicarbonate and antibiotics, at °c under % co in air. the flukes were then transferred to -cm tissue culture flasks and maintained in culture medium ( ml/fluke) at °c under % co in air. after h incubation, the medium containing the secreted antigens was removed and centrifuged at , g for min at °c in the presence of protease inhibitors (sigmafast protease inhibitor tablets, sigma-aldrich, madrid, spain). the supernatant was then passed through a . -μm pore filter disk, concentrated in an amicon ultrafiltration cell (amicon, inc., beverly, ma) equipped with a ym membrane ( kda molecular weight cut-off), dialyzed against pbs, sterilized by filtration and, finally, stored at − °c until required. the protein concentration was measured using the micro bca protein assay kit (pierce; thermo fisher scientific, barcelona, spain). the recombinant ani s allergen (rani s ) (anadón et al. ; cuéllar et al. ) included in the trisakis kit (lin et al. ) , was produced in e. coli and purified and refolded as previously described (anadón et al. ). hybridoma cells secreting mab mm were obtained as previously described (mezo et al. ; martínez-sernández et al. ) . the secreting hybridoma cells were grown intraperitoneally in pristan-primed balb/c mice, and the anti-f. hepatica igg /ĸ antibodies were isolated from the ascitic fluid by affinity chromatography on a protein g column (hitrap protein g, ge healthcare, madrid, spain) according to the manufacturer's protocol. cloning of the genes encoding fhpcl , fhpcl , and fhpcl and expression of recombinant proteins messenger rna (mrna) was obtained from adult specimens of f. hepatica as previously reported . briefly, whole f. hepatica mrnas were obtained using the fast track mrna isolation kit (invitrogen, san diego, ca), according to the manufacturer's instructions, and the mrna concentrations were determined by spectrophotometry (nanodrop; thermo fisher scientific). a collection of cdna was prepared with one microgram of mrna using the marathon cdna amplification kit (clontech, palo alto, ca), according to the manufacturers' instructions. the double-strand cdnas were subsequently ligated to the marathon adaptors (ap ). the procedures used to clone and subclone fhpcl are reported elsewhere . the deduced amino acid sequence was annotated under genbank accession number cca . . for expression of the corresponding protein, the fhpcl gene was directionally cloned into the pqe- expression vector (qiagen; qiagen iberia sl, madrid, spain) and further transformed into the e. coli strain m [prep ]. primers corresponding to sequences described by kofta et al. ( ) were synthesized to amplify the full cathepsin molecules: f-kofta ( ′ atgtggttcttcgtattagc ′) and rkofta ( ′ ccaagtatttttaacaatccaata ′). pcr reactions were carried out under conditions of low stringency, in order to amplify genes corresponding to different types of cathepsins. the cdna described above was used as template. the pcr products were cloned into the pgem-t vector (promega biotech ibérica sl, madrid, spain), and dna from recombinant plasmids was automatically sequenced using fluorescence-base labeling with the abi prism system (perkin elmer, langen, germany) and the universal primers d and sp . a clone similar to the fhcl (gb|cca . ) was obtained. the fhpcl without the signal peptide was directionally cloned (sac i -sma i) into the pqe- expression vector (qiagen) using primers f-pcl ( ′ tcgaatga cgatttgtg ′) and r-pcl ( ′ ttcacggaaatc ′), and was then further transformed into the e. coli m [prep ] cells. the selected fhpcl sequence was annotated in genbank under accession number ky . generic primers, f-pcl ( ′ tcaaatgacgattt g t g g c at c a at g g a a g ′ ) and r-pcl ( ′ tcacggaaattgtgccaccatcgggac ′), were designed to directly clone the fhpcl gene using the cdna collection as template. considering that the fhpcl , fhpcl , and fhpcl sequences were almost identical at the amino terminal region and quite similar at the carboxy terminal region (both used to design the different sets of primers), and as fhpcl is the least abundant member of the fhcls expressed by adult worms ), numerous clones had to be sequenced to obtain a sequence compatible with the characteristics previously reported by norbury et al. ( ) for the cl clade. the selected fhpcl sequence was annotated in genbank under accession number ky . for protein expression, competent e. coli m [prep ] cells were transformed with the fhpcl gene directionally cloned (bam h i -sac i) into the pqe- expression vector (qiagen) with the primers f -pcl ( ′ tcaaatgacgatttgtgg ′) and r -pcl ( ′ tcacggaaattgtgc ′). expression, purification, and refolding of rfhpcl , rfhpcl , and rfhpcl recombinant fhpcls expression was induced by adding mm iptg to the transformed e. coli cultures . once the expression was induced, cultures were maintained h at °c. then, e. coli cells were harvested by centrifugation, and the insoluble recombinant proteins were purified with b-per reagent (thermo fisher scientific), solubilized and purified by affinity chromatography with his-select nickel affinity gel (sigma-aldrich) under denaturing conditions ( m urea), as indicated by the supplier. the different rfhpcls were refolded as previously described ) with a few modifications. briefly, each elute from the affinity column was pretreated with mm dtt for h at room temperature (rt), and subsequently diluted at a ratio of / in tbs ( mm tris, mm nacl, ph ) plus mm cysteine, mm cystine, and mm edta. once dialyzed against tbs (ph ), each rfhpcl was dialyzed against pbs and concentrated by membrane-filtration in an amicon-stirred ultrafiltration cell equipped with a filtron omega series membrane ( kda nominal molecular weight limit; pall corporation, port washington, ny). finally, the protein concentration was determined with the micro bca protein assay kit, and the refolded recombinant proteins were stored at − °c until use. to investigate the optimal concentration of each rfhpcl for use as target in indirect elisa, we tested several concentrations of each antigen in the range of - μg/ml in pbs. polystyrene microtiter plates (greiner bio-one; soria-melguizo, madrid, spain) were coated with μl/well of each rfhpcl dilution and incubated for h at °c. the plates were then washed three times with pbs and blocked with μl/well of . % sodium caseinate in pbs for h at rt. aliquots of μl of an appropriate dilution of mab mm ( / ) in pbs containing . % tween and % skimmed dry milk (pbs-t-sm) were then added to each well, and the plates were incubated for min at rt with shaking at rpm. the plates were then washed five times with pbs-t, and bound mm antibodies were detected after incubation with hrp-labeled goat anti-mouse igg secondary antibodies (bio-rad, madrid, spain) diluted / in pbs-t-sm for min at rt with orbital shaking at rpm. the plates were then washed, as above, and incubated for min at rt with μl/well of the enzyme substrate opd (sigmafast opd, sigma-aldrich). finally, the optical density (od) was measured at nm. recombinant procathepsin dilutions containing the same equivalent concentration (i.e., yielding the same od signal with mab mm ) were selected for use in an indirect elisa, as indicated below. indirect elisas with rfhpcl , rfhpcl , rfhpcl , and rani s the wells of elisa plates were coated with μl of each rfhpcl (rfhpcl , rfhpcl , or rfhpcl ) or with the anisakis simplex allergen rani s , all at a concentration of . μg/ml in pbs, and incubated for h at °c. the plates were then washed three times with pbs and blocked with μl/well of . % sodium caseinate in pbs for h at rt. aliquots of μl of sheep or cattle sera (from f. hepaticainfected and f. hepatica-free animals) diluted / in pbs-t-sm were added to each well of the plates in duplicate, and incubated for min at rt with orbital shaking at rpm. the plates were then washed five times with pbs-t, and bound igg antibodies were detected with either hrpconjugated mouse anti-sheep/goat igg monoclonal antibodies ( / , in pbs-t-sm; sigma-aldrich), or hrp-conjugated sheep anti-bovine igg polyclonal antibodies ( / in pbs-t-sm; bio-rad), and opd, as indicated above. the mm -sero elisa was performed as previously described (mezo et al. ) but with some modifications. briefly, elisa plates were coated with purified mab mm ( μl/well at μg/ml), incubated on at °c, washed three times with pbs, and blocked with μl/well of . % sodium caseinate in pbs for h at rt. aliquots of μl of f. hepatica esas at μg/ml in pbs or pbs only were added to each well in odd (ag+) and even (ag−) plate rows, respectively. the plates were incubated for h at rt and then washed three times with pbs, before μl of each serum sample (from sheep or cattle) diluted / in pbs-t-sm was added to each ag+ and ag− well in duplicate. the plates were then incubated for min at rt with shaking at rpm, washed five times with pbs-t, and specific sheep or bovine igg was detected as described above. the od value for each sample was calculated as od -od , where od is the value for the ag+ well, and od is the value for the ag− well. the significance of the differences in coproantigen values for fecal samples obtained from parasitized cattle classified according to their parasite burden (low, middle, high; see above) was determined by the kruskal-wallis test (nonparametric anova) and dunn's multiple comparison test. the analysis was conducted using the graphpad instat statistical package (graphpad software inc., san diego, ca). differences were considered significant at p < . . pearson's correlation coefficients (r) were calculated to compare the data obtained analyzing samples from infected animals with the different indirect and capture elisas using originpro . (originlab corporation, northamptom, ca). the cut-off values for each elisa and species tested (sheep or cattle) were calculated from the od values obtained testing the sera from f. hepatica-free animals. two methods were used to calculate the cut-off values for the different indirect elisas. in method a, designed to guarantee maximum specificity (i.e., %; see martínez-sernández et al. ) , the cut-off values were calculated as the sum of the highest od value obtained on testing the negative sera plus sd. these cut-offs were used to obtain the sensitivities of each test, which were calculated using epidat . software as the number of true positives (correctly identified by each test), divided by the total number of infected animals. in method b, the cut-off values were obtained by receiver operating characteristic (roc) analysis. the roc curves were generated using the medcalc software (medcalc software, ostend, belgium) following the methodology proposed by delong et al. ( ) . the cut-off values defined by the youden index (i.e., that maximize the sum of sensitivity and specificity) were used to estimate the sensitivity and specificity of each test. finally, the cut-offs and the corresponding sensitivities of the mm -sero elisa were determined considering method a only. comparison of the fhpcl , fhpcl , and fhpcl amino acid sequences the sequence alignments of the three fhpcls used in this study are shown in fig. . the displayed sequences corresponded to the full-length propeptide (residues s -r/ l ) lacking the deduced signal peptide (residues - ). the alignments revealed a high percentage of sequence identity among the three fhpcls, although the percentage of identity was higher between fhpcl and fhpcl ( %), in comparison with the % calculated for fhpcl and fhpcl , and the % for fhpcl and fhpcl . assignation of the sequence gb|cca . to fhcl was previously reported . regarding the sequence gb|ky , we observed that it differs by three nucleotides, but only by one residue (l s), from sequence gb|abq . (baspinar et al. unpublished results) and by seven nucleotides, which translate into five residue changes (l s, f y, n d, p t, l f), from the sequence gb|aac . (dowd et al. ) , both of which were classified as fhcl . interestingly, these three sequences totally coincide with the key residues y , m , a , l , t , a , and l (mature protein numbering) reported by norbury et al. ( ) as being typical of the fhcl clade. the sequence gb|ky was classified as fhcl because of its similarity to the sequence gb|aaf reported by smooker et al. ( ) and classified as fhcl by norbury et al. ( ) . our sequence has four nucleotide changes, two of which translate into two amino acid substitutions (i t and m t), relative to gb|aaf . however, both sequences share the characteristic residues l , m , a , l , n , g , and l reported by norbury et al. ( ) in the mature fhcl . as indicated in the previous section, fasciolosis in cattle was determined by liver necropsy and further confirmation by fecal egg counting and coproantigen detection (mm -copro elisa). the non-infected group tested negative in fluke, egg, and coproantigen determinations. coprology also revealed that most cows in both groups had intestinal nematodes from one or more genera of trichostrongylidae, trichuridae (trichuris spp.), and strongylidae. additionally, the farm records for individual cows revealed that most were routinely treated with albendazole during the dry period and vaccinated against infectious bovine rhinotracheitis and bovine respiratory disease (bovine respiratory syncytial virus, parainfluenza virus type , and mannheimia haemolytica). some were also vaccinated at the end of the gestation period against coronavirus, rotavirus, and e. coli. regarding sheep, f. hepatica eggs (ranging between . and eggs per gram of feces) and coproantigens (fecal antigen concentration ranging from . to . ng/ml) were detected in all sheep from the infected herd but were absent in the fluke-free herd. as for cattle, infections by gastrointestinal nematodes were detected in most animals from both infected and non-infected herds. specifically, n e m a t o d es i n t h e f a m i l i e s tri c h o s t r on g y l i d a e , molineidae (nematodirus spp.), ancylostomatidae, strongylidae, and trichuridae (trichuris spp.) were identified. the fecal antigen concentrations, parasite load and other characteristics from the infected animals included in the study are shown in table . recognition of rfhpcl , rfhpcl , and rfhpcl by sera from infected and non-infected cattle and sheep the antigens rfhpcl , rfhpcl , and rfhpcl were evaluated in indirect elisa testing sera from cows and sheep naturally infected with f. hepatica and from non-infected animals. as indicated in the previous section, the optimal concentration of the target antigens was determined in elisa plates by their recognition by mab mm , which binds to an epitope that is present in the three fhcl clades. moreover, as the mm epitope is conformational in nature , their recognition by this mab in elisa strongly suggests correct folding of these recombinant molecules. the results presented in fig. a (fasciola-infected) and fig. b (fasciola-free) show the individual igg antibody response of the and cows, respectively, to the three recombinant cathepsins in indirect elisa, and to native cathepsins in capture elisa (mm -sero). most of the sera from fasciolainfected cows produced od signals higher than . to the three rfhpcls although, considering the samples individually, the antibodies in some sera displayed a preference for certain rfhpcls. for example, this was the case with serum # , which reacted preferentially with rfhpcl and rfhpcl , and serum # which produced the highest od value with rfhpcl . finally, only two serum samples (# and # ) produced low responses with ≤ . od (fig. a) . with respect to sera from fasciola-free cattle (fig. b) , unlike for the elisa mm -sero, several serum samples produced high backgrounds in indirect elisa, with od values ≥ . . the highest background values were obtained with rfhpcl (sera # , # , and # ) and with rfhpcl (sera # , # , and # ), although one sample (serum # ) also produced a considerable background (od > . ) with rfhpcl . the antibody responses of sera from fasciola-infected and fasciola-free sheep are shown in fig. a , b. regarding infected sheep, the response to the three recombinant antigens follows the same pattern as indicated above for cattle, with good elisa signals and only one sample yielding an od signal diagnostic value of the indirect elisa with rfhpcl , rfhpcl , or rfhpcl versus the mm -sero capture elisa for a better comparison of the signals obtained in indirect elisa with rfhpcl , rfhpcl , and rfhpcl with those of the classical mm -sero elisa, we elaborated a graph grouping the response given by each individual serum in each of the four elisas tested (fig. a, b) . in these figures, the corresponding cut-offs, calculated as the maximum od value from negative sera plus one sd (method a) or obtained by roc analysis (method b) are represented, respectively, by horizontal dashed red and blue lines. the sera from infected cattle produced similar mean od signals (p > . for all comparisons), ranging from od = . to od = . for rfhpcl -elisa, from od = . to od = . for rfhpcl -elisa, from od = . to od = . for rfhpcl -elisa, and from od = . to od = . for mm -sero elisa (fig. a ). however, due to the big differences observed between the cut-off of the reference test mm -sero elisa (od = . ) and the calculated cut-offs for the indirect elisas (od = . , . , and . , respectively, with method a, and . , . , and . , respectively, with method b) several positive sera were incorrectly classified by these latter. specifically, n = , n = , and n = sera from infected cattle were misclassified by rfhpcl , rfhpcl , and rfhpcl elisas, respectively, considering the cut-off obtained by method a, while n = , n = , and n = of these sera were incorrectly classified using the cut-off obtained by method b (roc analysis). these results yielded sensitivity values of . , . , and . %, respectively, considering the former cut-offs (method a), and sensitivities of . , . , and . %, respectively, considering the latter cut-offs (method b). although the sensitivity values of the rfhpcl and rfhpcl elisas obtained using cut-off values calculated with roc curves (method b) are apparently better than those obtained with method a, it is relevant to consider that this increase in sensitivity was done at expenses of lowering the specificity of the assays by about % (see table ). with respect to infected sheep, the distribution of od signals obtained in indirect elisa with the three rfhpcls was similar to that obtained for infected cattle (the lowest od signal obtained with rfhpcl (od = . ) and the highest with rfhpcl (od = . )). interestingly, as the cut-off values calculated with method a (rfhpcl , od = . ; rfhpcl , od = . ; rfhpcl , od = . ; mm -sero, age (range) . ( - ) . ( - ) . ( - ) . ( - ) a statistic differences (p < . ) with group harboring - flukes b statistic differences with group harboring - flukes (p < . ) and with group harboring - flukes (p < . ) c animals in production, not sacrified at the moment of sample collection od = . ) or the method b (rfhpcl , od = . ; rfhpcl , od = . ; rfhpcl , od = . ) were much lower than for cattle, the three elisa variants and the mm -sero elisa were capable of correctly classifying all sera from infected and non-infected sheep, i.e., with a sensitivity and specificity of % (table ) . nevertheless, the comparison of the od signals obtained in any of the indirect elisas with those yielded by the mm -sero capture elisa (either for cattle or sheep) revealed that the signal-tonoise ratio was much more favorable in the latter. finally, the r values obtained on comparing the four elisa methods for fasciola-infected cattle and sheep sera are shown in table . the correlation was strongest between the data obtained with rfhpcl and rfhpcl elisas for both cow and sheep sera, while the lowest r value corresponded to the comparison between the od values of the tested cow sera in the mm -sero and rfhpcl elisas. the relatively large proportion of sera from fasciola-free cattle with cross-reactive antibodies to one or more of the recombinant rfhpcls used in this study (see fig. b ) led us to investigate whether these cows also had cross-reactive antibodies to other recombinant or non-recombinant antigens. for this purpose, we evaluated the presence of igg antibodies reactive with a recombinant a. simplex allergen (rani s ) expressed in e. coli and purified using the same procedure as for rfhpcls, or reactive with mouse igg (purified mab mm without f. hepatica antigens, which is used as control for each individual serum in the mm -sero elisa). a relevant proportion of sera from cattle infected with f. hepatica had igg antibodies reactive with rani s , three of which produced od signals above . (see fig. a ). although the reactivity of sera from the fasciola-free population of cattle was much lower (fig. b) , these samples still showed a notable reactivity to rani s (fig. b) . moreover, anti-mouse igg crossreactivity was also observed for a significant number of sera within the samples from fasciola-infected and fasciola-free cows (fig. e, f) . on the other hand, regarding serum samples from sheep, the population infected with f. hepatica did not recognize the rani s (fig. c ) allergen, and only two serum samples from non-infected sheep produced od values > . (fig. d) . also, no reaction to mouse igg antibodies was observed when testing sheep sera from both populations (fig. g, h) . this is the first study that comparatively evaluates the performance of three rfhpcls (rfhpcl , rfhpcl , and rfhpcl ) as targets antigens in elisa for the serodiagnosis of fasciolosis in sheep and cattle. these molecules were selected since (i) they are produced by adult flukes and thus continue stimulating the immune system during the chronic phase of the illness, and (ii) fasciola cathepsins l , l , and l contain a common epitope recognized by mab mm , which is the capture antibody in the mm -sero elisa (mezo et al. ) . although rfhpcls from the l clade have already been successfully used to develop a very sensitive and specific lateral flow test for immunodiagnosis of human fascioliasis , as well as to design elisa tests for human (o'neill et al. ; carnevale et al. a, b; gonzales santana et al. ; gottstein et al. ) and animal use (cornelissen et al. ; kuerpick et al. ; selemetas et al. ; bloemhoff et al. ) , the performance of rfhpcls from l and l clades as elisa targets has not previously been investigated. from a functional point of view, a single amino acid substitution may be sufficient to affect substrate specificity in cysteine proteases from f. hepatica (smooker et al. ) . the comparison of the new fhpcl (gb|ky ) and fhpcl (gb|ky ) sequences described in the present study with other reported cathepsins l (gb|abq . ; gb|aac . ) and l (gb|aaf ) revealed some nucleotide and amino acid changes. however, as none of the amino acid changes were in the positions referred by norbury et al. ( ) as being typical of the fasciola l and l clades, the assignation of our recombinant molecules as fhpcl and fhpcl was probably correct. the alignment of the sequences of the three fhpcls used in this study showed a mean of amino acid changes of %, which, besides affecting substrate specificity, it may be enough to induce variations in the level of circulating antibodies produced in infected animals. the results showing preferences in procathepsin recognition in the population of sera from fasciola-infected animals, mainly cattle (see fig. a ), are consistent with this hypothesis. more importantly, our results indicated that indirect elisas based on rfhpcl , rfhpcl , and rfhpcl show differences in sensitivity, despite the fact that the target antigens were expressed and purified in the same way. specifically, considering the od values obtained with the different immunoassays, the cut-off values calculated to maximize specificity (method a), and the number of false negatives obtained for each rfhpcl, the rfhpcl might be a better target antigen than the other rfhpcls. however, the mm -sero elisa had a better performance, as this method classified correctly all samples from sheep and cattle. there are at least two explanations for the superiority of the mm -sero elisa over the elisas based on the use of individual rfhpcls: (i) mab mm is able to virtually capture all native cathepsin isoforms of the cl , cl , and cl clades from fasciola ; and this study), including those from the more pathogenic f. gigantica species valero et al. ) . this may explain the fact that od values obtained in mm -sero elisa tended to be higher than in indirect elisas among infected animals with low anti-fasciola antibody levels (see fig. a ), and (ii) the mm -sero elisa includes an individual control for each serum, which ensures that particular animals with anti-species cross-reactive antibodies or antibodies against other irrelevant proteins present in the elisa plate (see fig. e , f) can be easily detected, thus allowing this signal to be subtracted from that of the corresponding positive well. consequently, good od signals and low backgrounds (which translates into lower cut-offs) are normally achieved, yielding an excellent signalto-noise ratio, for both cattle and sheep sera. these results suggest that the use of a single recombinant cathepsin/ procathepsin as target antigen in elisas for serodiagnosis of fascioliasis may limit the sensitivity of the assay when testing sera from some species, particularly cattle. considering the above characteristics of the mm -sero elisa, its highest sensitivity (see table ) over the indirect elisas based on the use of a single f. hepatica cathepsin/ procathepsin l , l , or l is not surprising. however, considering that representatives of all mature fasciola cls are captured in mm -sero, the high backgrounds obtained with the rfhpcl , rfhpcl , and rfhpcl antigens for fasciola-free cattle (see fig. b ) may appear unexpected at first sight. moreover, this phenomenon was not limited to the samples investigated in this study, nor to the expression of rpfhcls in e. coli. in agreement with this, a similar finding was recently reported in a study where the performance of an indirect elisa based on a rfhpcl expressed in the yeast pichia pastoris was compared with that of a commercial indirect elisa test containing a purified fraction from fasciola esas (f antigen; institute pourquier, montpellier, france) (kuerpick et al. ). cross-reactivity was also observed by cornelissen et al. ( ) who tested sera from sheep and cattle harboring other parasites, mainly nematodes, suggesting that the cross-reactivity may be due to common epitopes between recombinant fasciola cathepsins and antigens present in other parasites. however, as such cross-reactivity did not occur with the three tested rfhpcls (see fig. b ), this explanation seems unlikely. another possible source of cross-reactivity is that rfhpcls produced in e. coli were contaminated by bacterial proteins retained in the imac column during the affinity purification process. coelution of native e. coli proteins (e.g., proteins containing clusters of histidine residues) with recombinant proteins is a recognized feature, mainly when recombinant proteins are expressed at low level (robichon et al. ). however, this seems also unlikely for several reasons: (i) rfhpcls are produced as inclusion bodies, and contaminating proteins are removed during the washing procedures before denaturation and subsequent imac purification of rfhpcls; (ii) as indicated above, the reactivity of a particular serum does not occur with the three rfhpcls, despite the fact that all of them were produced and purified following the same procedure; and (iii) preincubation of the cross-reactive sera with a bacterial extract prior to the incubation in elisa plates, or the use of a rfhpcl expressed with a second tag in order to eliminate possible remaining contaminants in a capture elisa did not eliminate the observed backgrounds (data not shown). conversely, we hypothesize that the phenomenon may be related to a greater opportunity of cross-reactive antibodies to bind non-specific linear epitopes present in rpfhcl preparations than in native cls. while in native antigens, the availability of non-specific linear epitopes may be low due to the correct folding of the protein, such epitopes may be exposed for antibody-binding when truncated, partially folded, or unfolded molecules are present, as occurs frequently when recombinant proteins are overexpressed in bacterial or yeast cell systems (lilie et al. ; cornelissen et al. ). in addition, an unexpectedly high number of samples from fasciola-infected and non-infected cattle reacted with a recombinant form of the a. simplex ani s allergen (fig. a, b) , an antigen that is present in a fish parasite never naturally in contact with cattle, or with other herbivorous animals (anadón et al. ). such allergen is structurally related with kunitz-type serine protease inhibitors (audicana and kennedy ) . thus, with respect to fasciola-infected cows, it could be hypothesized that cross-reactivity with rani s is due to the presence of common epitopes between this allergen and the kunitz-type molecules secreted by fasciola (bozas et al. ; di maggio et al. ; smith et al. ) . nevertheless, the fact that the response was irregular among these cows, and that it also occurred, to a lesser extent, among fasciola-free cows, suggests again that cross-reactive antibodies may be induced against antigens not related with fasciola but which share some linear epitopes. considering the above argumentations and the sensitivity obtained in the indirect elisas tested, the usual purification conditions of recombinant proteins expressed in bacterial or yeast systems may render these antigens not suitable for use as target in elisas for serodiagnosis of fascioliasis in certain animal species. besides, there is also experimental data suggesting that cross-reactivity may not occur in the same way in different host species. in this sense, we observed that the od responses to rfhpcls were more homogeneous in infected sheep than in cattle (see figs. a and a, respectively), which was also reflected by the higher r values obtained for sheep sera relative to cattle sera, when comparing the od values obtained on testing the different rfhpcls (see table ). moreover, interspecific differences in the generation of cross-reactive antibodies to fasciola procathepsins can also be deduced from a previous study in which no cross-reactivity was observed for human sera in a lateral flow test based on the same rfhpcl ) used in the present study. this reflects that some species may be more prone to induce cross-reactive antibodies than others or that the antigens to which humans are exposed have fewer common epitopes with fasciola cls than those to which ruminants are exposed. the limited number of positive and negative sera tested in this study may not be sufficient to obtain precise values of sensitivity and specificity for the three indirect elisas based on recombinant rfhpcls and to obtain cut-off values valid for use under field conditions. however, this did not prevent drawing conclusions about their performance with respect to the mm -sero elisa, as they were tested simultaneously with the same sera. moreover, it did not condition the fact that recombinant cls purified by imac may be more prone to be recognized by cross-reactive antibodies than the corresponding natural antigens. finally, although liver necropsy and coproantigen detection may not be perfect gold standards, since some animals may only have migrating or ectopic flukes, considering the age of cattle in our study ( - years old), the probability of having a single contact within the previous weeks before slaughtering is minimal. in summary, our results show that rfhpcl may be more suitable than rfhpcl and rfhpcl as target antigen in indirect elisa for the serodiagnosis of fascioliasis in ruminants, particularly in cattle, as these molecules, unless purified to homogeneity and correctly folded, may be more frequently recognized by cross-reactive antibodies than the corresponding natural antigens. these results reflect that further research into methods of producing and purifying recombinant fasciola cathepsins is required to overcome the need to collect live adult parasites in abattoirs, or from experimental infections, to obtain natural antigens. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. blood and fecal samples were collected from naturally infected sheep and cattle by veterinarians from the bcentro de investigaciones agrarias de mabegondo^(ingacal, a coruña, spain). the samples were collected either during routine control and treatment of herds (sheep) or in the slaughterhouse (cattle). all procedures were carried out in strict accordance with spanish and eu legislation (law / , r.d. / , and council directive / /eu). evaluation of a diagnostic monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of a -to -kd fasciola hepatica coproantigen in cattle diagnosing human anisakiasis: recombinant ani s and ani s allergens versus the unicap fluorescence enzyme immunoassay the sensitivity and specificity of two methods for detecting fasciola infections in cattle the life cycle of fasciola hepatica anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity determining the prevalence and seasonality of fasciola hepatica in pasture-based dairy herds in ireland using a bulk tank milk elisa characterisation of a novel kunitztype molecule from the trematode fasciola hepatica immunodiagnosis of human fascioliasis by an enzyme-linked immunosorbent assay (elisa) and a micro-elisa immunodiagnosis of fasciolosis using recombinant procathepsin l cystein proteinase associations between anti-fasciola hepatica antibody levels in bulk-tank milk samples and production parameters in dairy herds qualitative and quantitative evaluation of coprological and serological techniques for the diagnosis of fasciolosis in cattle measurement of antibodies to gastrointestinal nematodes and liver fluke in meat juice of beef cattle and associations with carcass parameters recent advances in the diagnosis, impact on production and prediction of fasciola hepatica in cattle use of a pre-selected epitope of cathepsin-l in a highly specific peptide-based immunoassay for the diagnosis of fasciola hepatica infections in cattle early immunodiagnosis of fasciolosis in ruminants using recombinant fasciola hepatica cathepsin l-like protease bridging gaps in the molecular phylogeny of the lymnaeidae (gastropoda: pulmonata), vectors of fascioliasis ani s and ani s recombinant allergens are able to differentiate distinct anisakis simplex-associated allergic clinical disorders the fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach across intramammalian stages of the liver fluke fasciola hepatica: a proteomic study isolation of a cdna encoding fasciola hepatica cathepsin l and functional expression in saccharomyces cerevisiae fasciola hepatica -monitoring the milky way? the use of tank milk for liver fluke monitoring in dairy herds as base for treatment strategies international handbook of foodborne pathogens sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis evaluation of fas -elisa for the serological detection of fasciola hepatica infection in humans application of a coproantigen elisa as an indicator of efficacy against multiple life stages fasciola hepatica infections in sheep the diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (elisa) using recombinant cathepsin l protease comparative assessment of elisas using recombinant saposin-like protein and recombinant cathepsin l- from fasciola hepatica for the serodiagnosis of human fasciolosis successful dna immunisation of rats against fasciolosis evaluation of a recombinant cathepsin l elisa and comparison with the pourquier and es elisa for the detection of antibodies against fasciola hepatica advances in refolding of proteins produced in e. coli an extended study of seroprevalence of anti-anisakis simplex ige antibodies in norwegian blood donors development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fasciolosis mf p/fhhdm- major antigen secreted by the trematode parasite fasciola hepatica is a heme-binding protein rapid enhanced mm -copro elisa for detection of fasciola coproantigens fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control direct and indirect affection of the central nervous system by fasciola infection diagnosis of human fascioliasis by stool and blood techniques: update for the present global scenario optimized serodiagnosis of sheep fascioliasis by fast-d protein liquid chromatography fractionation of fasciola hepatica excretory-secretory antigens an ultrasensitive capture elisa for detection of fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (mm ) the use of mm monoclonal antibodies for the early immunodiagnosis of ovine fascioliasis kinetics of anti-fasciola igg antibodies in serum and milk from dairy cows during lactation, and in serum from calves after feeding colostrum from infected dams field evaluation of the mm -sero elisa for detection of anti-fasciola igg antibodies in milk samples from individual cows and bulk milk tanks association between anti-f. hepatica antibody levels in milk and production losses in dairy cows molecular and immunological characterization of fasciola antigens recognized by the mm monoclonal antibody analysis of fasciola cathepsin l by s subsite substitutions and determination of the p -p specificity reveals an unusual preference short report: immunodiagnosis of human fascioliasis using recombinant fasciola hepatica cathepsin l cysteine proteinase estimating the true prevalence of fasciola hepatica in cattle slaughtered in switzerland in the absence of an absolute diagnostic test engineering escherichia coli bl (de ) derivative strains to minimize e. coli protein contamination after purification by immobilized metal affinity chromatography zoonotic helminth infections with particular emphasis on fasciolosis and other trematodiases proteomics and phylogenetic analysis of the cathepsin l protease family of the helminth pathogen fasciola hepatica: expansion of a repertoire of virulenceassociated factors an integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen fasciola hepatica: proteins associated with invasion and infection of the mammalian host evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to fasciola hepatica in milk estimating the financial losses due to bovine fasciolosis in switzerland weather and soil type affect incidence of fasciolosis in dairy cow herds unexpected activity of a novel kunitz-type inhibitor: inhibition of cysteine proteases but not serine proteases a single amino acid substitution affects substrate specificity in cysteine proteinases from fasciola hepatica cathepsin b proteases of flukes: the key to facilitating parasite control? mm -elisa detection of fasciola hepatica coproantigens in preserved human stool samples mm -elisa evaluation of coproantigen release and serum antibody production in sheep experimentally infected with fasciola hepatica and f. gigantica higher physiopathogenicity by fasciola gigantica than by the genetically close f. hepatica: experimental long-term follow-up of biochemical markers the authors declare that they have no competing interests. key: cord- - c xw authors: petrosova, a.; konry, t.; cosnier, s.; trakht, i.; lutwama, j.; rwaguma, e.; chepurnov, a.; mühlberger, e.; lobel, l.; marks, r.s. title: development of a highly sensitive, field operable biosensor for serological studies of ebola virus in central africa date: - - journal: sens actuators b chem doi: . /j.snb. . . sha: doc_id: cord_uid: c xw we describe herein a newly developed optical immunosensor for detection of antibodies directed against antigens of the ebola virus strains zaire and sudan. we employed a photo immobilization methodology based on a photoactivatable electrogenerated poly(pyrrole-benzophenone) film deposited upon an indium tin oxide (ito) modified conductive surface fiber-optic. it was then linked to a biological receptor, ebola virus antigen in this case, on the fiber tip through a light driven reaction. the photochemically modified optical fibers were tested as an immunosensor for detection of antibodies against ebola virus, in animal and human sera, by use of a coupled chemiluminescent reaction. the immunosensor was tested for sensitivity, specificity, and compared to standard chemiluminescent elisa under the same conditions. the analyte, anti-ebola igg, was detected at a low titer of : , and : , , for subtypes zaire and sudan, respectively. while the same serum tested by elisa was one order ( times) less sensitive. infection with ebola virus causes an acute hemorrhagic fever in human and some non-human primates resulting in high mortality rates. there are four species of ebola virus that have been identified: zaire, sudan, ivory coast, and reston. all four species differ in their virulence; thus acute mortality in human outbreaks is typically greater than % for ebola virus zaire strain and greater than % for sudan. ebola zaire and sudan viruses are the most widespread and have been responsible for the majority of the recent hemorrhagic fever outbreaks in humans [ ] [ ] [ ] . the last large outbreak occurred in [ ] [ ] in the gulu district of uganda. in total, there were cases with deaths. this case fatality rate of % was similar to that observed for two previous outbreaks of ebola virus sudan in and in which % and % of cases died, respectively [ ] [ ] [ ] . ebola virus has affected populations primarily throughout central africa. although it has only affected a limited geographic region its effects have reverberated world-wide due to its extremely high mortality rate. in addition, since the reservoir for this virus has not been identified it has evoked much fear in populations living outside central africa and has become a biodefense concern [ , , ] . the elisa was proved as a more sensitive and specific test for ebola virus than the indirect fluorescent antibody test (ifat) [ ] . therefore the elisa has been the most commonly used serologic technique for ebola virus serology studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] . recently other assays for ebola virus have been developed and described. they include reverse transcriptase polymerase chain reaction (rt-pcr) [ , ] , antibody phage indicator assay [ ] , and immunohistochemical assay [ ] . in this study we present a newly developed optical immunosensor for detection of antibodies to ebola virus strains zaire and sudan, by using a photoimmobilization methodology based on a photoactivable electrogenerated polymer film. this newly developed immunosensor relies on luminescencebased technology that couples antigen targets to a fiber-optic and employs chemiluminescence for detection of minute quantities of specific antibodies. in the field of optical fiber immunosensors, we have previously demonstrated that optical fibers, which are made of doped silica and hence are electrically inert, can be modified by an electrically conductive layer [ ] [ ] [ ] [ ] . this fiber-optic electroconductive surface modification was achieved by deposition of a thin layer of indium tin oxide (ito). ito is an indium oxide (in o )-based material that has been doped with tin oxide (sno ) to improve its electrical quality. tin acts as a cationic dopant in the in o lattice and substitutes indium sites to bind with interstitial oxygen. the presence of sno results in n doping of the lattice because the dopant adds electrons to the conduction band [ ] . we must take care to produce thinner films of indium tin oxide even though there is a correlation between thicker films and lower resistivity, as lower light transmission may occur due to absorption by the film [ ] . since sputtering is one of the most extensively used techniques for deposition of transparent conducting oxide films, it was selected as the deposition technique. this method renders far better control over the stoichiometry, obviating, in most cases, the heat treatment step, which is usually performed after deposition of the film. highly transparent and conductive films of ito have been previously deposited by this technique [ , ] . after deposition of the ito film, monomers of pyrrolebenzophenone were electropolymerized onto the conductive metal oxide surface. the advantage of the electrochemical polymerization method is that the film can be prepared easily in a rapid, reproducible, and well controlled one-step procedure, that enables production of a thin homogeneous layer with well defined thickness of the desired polymer [ , ] . modification of an optical fiber with pyrrole-benzophenone film allows the photoactivable linking of a biological receptor, inactivated ebola virus, to the tip of the fiber-optic surface (fig. ). this innovative photoelectrochemical method for immobilization of biological macromolecules combines the advantages of photolithography with those of the electrochemical deposition of polymer films. the immunosensor we constructed was tested for sensitivity, specificity, and compared to standard luminescence elisa under the same conditions. our results suggest that the detection of antibodies to the ebola virus using our newly developed immunosensor will contribute significantly to serological and epidemiological studies in central africa by increasing the sensitivity of the tests dramatically. when modified into an "easyto-use" procedure, this technology might be used in the future in a field operable clinical tool for ebola virus antibody screening. in addition, our newly developed fiber-optic immunosensor can be reversed and transformed into an antigen detection biosensor for viral agents. the production of an ultrasensitive immuno-biosensor for the ebola virus is indeed extremely important since: ( ) the reservoir of this virus is unknown and it can therefore suddenly reemerge from the environment; ( ) the virus has an extremely high mortality rate and it is therefore important to diagnose potential victims as soon as possible; ( ) the development of an environmental sensor for ebola is important as an early warning of a potential outbreak, especially since ebola is a biodefense concern [ ] . we present herein a study that employs an immunosensor for evaluation of sera samples obtained in the field from survivors of ebola and close contacts. this work will serve as a blueprint for future biosensors, especially viral biosensors, and is important for biodefense and control of this and other viruses. the pyrrole monomer with a benzophenone functional group was prepared as previously described [ ] . acetonitrile, % (cp, biolab ltd.), and lithium perchlorate ( . %, aldrich) were used as received for electropolymerization. bovine serum albumin (bsa, a , fraction v) and polyoxyethylene-sorbitan monolaurate (tween ® , p ) were purchased from sigma. luminescence measurements were carried out using the western blot chemiluminescence reagent plus kit from nentm life science products (nel , containing enhanced luminol reagent and oxidizing reagent). all work with infectious ebola viruses was performed in the bsl- facility of the institute of virology, philipps university of marburg, germany, by elke mühlberger and the bsl- laboratory at vector laboratories, koltsvo, novosibirsk, by alex chepurnov. for preparation of viral stocks, vero cells were infected with ebola virus sudan, strain maridi, or ebola virus zaire, strain mayinga, respectively, at an m.o.i. of . pfu per cell. following an adsorption period of h, cells were incubated in dulbecco's modified eagle's medium (dmem) supplemented with % (v/v) fetal calf serum for days at • c, % co . subsequently, culture fluid was clarified by centrifugation in a minifuge at rpm for min at • c and used either as virus inoculum or further purified for isolation of antigen. clarified culture supernatant was centrifuged through a % (w/v) sucrose cushion in tne ( . % (v/v) of . m tris-hcl (ph . ) and . % (v/v) of . m edta in m nacl) buffer at , rpm for h at • c in a sw rotor (beckmann). pelleted virus was resuspended in tne and further purified by gradient centrifugation through - % (w/v) potassium tartrate, - % (w/v) glycerol in tne at , rpm for h at • c. the virus band is isolated, diluted in tne, and pelleted by centrifugation at , rpm for min at • c in a sw rotor (beckmann). the virus pellet is resuspended in phosphate buffered saline (pbs). for the immunosensor assay and elisa tests the virus was inactivated with % (w/v) sds and boiled according to standard techniques [ ] . this inactivated viral preparation was the ebola antigen used for all our assays. the sera of ebola immunized animals, provided by dr. alex chepurnov (vector laboratory, koltsvo, novosibirsk), was used for igg tests on the optical fiber biosensor and comparative chemiluminescence elisa. for ebola virus zaire and sudan subtypes, goat and rabbit anti-ebola antisera were used, respectively. the human sera samples were taken from survivors (in ) of the ebola sudan outbreak in gulu, uganda - and from healthy volunteers that live in this district but were not clinically ill with ebola. all patients were healthy at the time of phlebotomy. all animal sera tested negative for ebola virus and human survivors were healthy at the time of phlebotomy and tested negative for the ebola virus by pcr. horseradish peroxidase (hrp)-labeled polyclonal secondary antibodies were used for sera antibody detection. goat antirabbit igg conjugated hrp (a- , sigma) and donkey antigoat igg conjugated hrp (jackson laboratories) were used for anti-ebola sudan and zaire calibration curve construction, respectively. for human samples, testing was performed with goat anti-human igg conjugated hrp secondary antibody (jackson laboratories). elisa was performed according to standard procedures. a volume of l of inactivated viral preparation diluted : (this material was used for all our assays), was added to each well of a -well microtiter plate (maxisorp, nunc). the plate was sealed and allowed to incubate overnight at • c. after incubation, the coating solution was removed and the plate was washed in pbs (ph . ). a blocking step involved adding l per well of a blocking solution (bsa % (w/v) in pbs (ph . )), which reduced overall background and increased the sensitivity of the assay. the plate was then incubated for h at • c and the wells washed thrice with pbs tween ® , . % (v/v) (pbs-t), ph . . then l per well of diluted serum solutions (at dilutions ranging from : to : , for ebola zaire strain and from : to : , , for ebola virus sudan) were added, along with control serum samples, in triplicate and the plate incubated h at • c. additional empty wells and non-coated wells were used to check the level of the background signal. the wells were then washed thrice with pbs-t and l per well of a secondary antibody solution was added and the plate incubated h at • c. donkey anti-goat igg and goat antirabbit igg antibodies labeled hrp were used for the ebola virus zaire and sudan elisa, respectively. after incubation, the plate was washed three times with pbs-t and then read in a luminometer. the microtiter plate was read with a standard luminometer (thermolabsystems-luminoskan ascent . ) where, before each well measurement, oxidizing reagent and enhanced luminol reagent solutions were injected into the well with an automatic dispenser in a : ratio. a total of measurements were performed in a kinetic measurement type with a gap of s between each reading. the data were collected with the luminoskan ascent software (version . ); the mean and the standard deviation of the triplicates were calculated for each point. all fibers used were sfs / b silica fibers (fiberguide industries, nj, usa) with an original numerical aperture (na) of . . their core was m in diameter (refractive index of . at nm) and was surrounded by a m silica cladding (refractive index of . at nm), followed by a m thick silicon buffer and finally a m thick black tefzel jacket. the detailed protocol of the optical fiber tip preparation is described by konry et al. [ ] . the depositions were carried out in a radio frequency (r.f.)-sputtering varian system. the sputtering target was a in. diameter circular disk of hot pressed powder . % purity ito ( % wt. in o + % wt. sno ), made by testbourne ltd. (hampshire, uk). the sputtering chamber was evacuated to a pressure lower than − torr. argon and oxygen gases were introduced into the sputtering chamber with partial pressures of × − and . × − torr, respectively. the r.f. supply was then switched on and stabilized to a power of w. the fiber tips were exposed to the sputter beam for - . h. as a result, the fiber core end-face (the important area where electro-polymerization must occur) and at least one side of the cylindrical surface (for contacting and voltage supply) were covered with the ito coating. all electrochemical experiments were performed with an electrochemical instruments autolab with pgstat (eco chemie bv) operated by general purpose electrochemical system (gpes) for windows (version . ) software. the electropolymerization of pyrrole monomers and characterization of the resulting polymers on the modified electrode surfaces was carried out in a conventional three-electrode cell at room temperature. the poly-pyrrole films were prepared by controlled potential oxidation of monomers ( mm) in . m liclo in ch cn. the working electrode was the ito coated optical fiber; an electrical contact was established with a steel wire above the acetonitrile electrolyte solution. a platinum wire ( m diameter) was used as a counter electrode and ag/ag + of mm agno in electrolyte solution was used as a reference electrode. electropolymerization of pyrrole-benzophenone was accomplished by controlled potential at . v until the appearance of the characteristic apex in an amperometric plot. scanning electron microscopy (sem) micrographs were taken by a jeok jsm f system with ev resolution of detector and exposure time of s. the specimens were analyzed using energy dispersive x-ray spectrometry microanalysis analytical equipment installed in the scanning electron microscopy. the optical fibers coated with poly(pyrrole-benzophenone) were soaked in diluted solution containing inactivated ebola virus antigen (approximately . g/ml, the concentration was determined by micro bca protein assay kit, pierce) and irradiated with uv light. benzophenone and most of its derivatives absorb a photon at around nm resulting in the promotion of one electron from a nonbonding sp -like n-orbital on oxygen to an antibonding * -orbital of carbonyl group. the actual electron-deficient oxygen n-orbital becomes electrophilic and therefore interacts with weak c-h -bonds, resulting in a hydrogen abstraction to complete the half-filled n-orbital. when amines or similar heteroatoms are in the vicinity of the excited carbonyl, an electron-transfer step may occur, followed by proton abstraction from an adjacent group. in biological systems, the most effective h-donors include backbone c-h bonds in amino acids; thus, methylene groups of amino acid side chains are good candidates providing abstractable hydrogens through the general mechanism shown in fig. [ ] . the steps carried out for the construction of the immunosensor are visualized in fig. . a poly-pyrrole with a benzophenone functional group was applied for photo immobilization of viral antigens on the optical fiber tip surface. in order to produce the desired activation radiation we used a w xe lamp (oriel ) mount connected to a light condenser (oriel ). the light was reflected through a dichroic mirror (oriel ). the spectrum radiation was then condensed into the monochromator (oriel ) using the appropriate lenses (oriel, plano-convex lenses). after this, a nm wavelength light output, with a light intensity of mw cm − (as measured by an ophir optronics power-meter nova reader, pd -uv) was projected for min into the far end of the optical fiber that was coated at its nearend with poly(pyrrole-benzophenone) and soaked in l of an inactivated ebola virus solution during irradiation. the excited benzophenone radicals could then bind to neighboring proteins in the solution, in our case ebola virus antigens (approximately . g/ml). later, the optical fibers were rinsed profusely and washed with pbs-t, ph . and transferred to blocking solution ( % (w/v) bsa in pbs-t) for min to prevent the nonspecific binding of anti-ebola igg. the fibers were then rinsed and washed with pbs-t for min, before being incubated in l of antibody solution for min. next the antigen conjugated fibers were placed in diluted serum solutions, ranging from : , for ebola virus zaire and from : , , for sudan up to : . sets of three replicas were prepared for each concentration measured and a series of fibers were set aside for use as controls. thereafter, all fibers were rinsed and washed once with pbs-t for min. subsequently, the optical fiber tips were dipped into l of a solution containing the hrp labeled secondary antibody for min, and finally, rinsed and washed once with pbs-t for min. all materials were disposed according to standard biosafety regulations. the instrument set-up for the chemiluminescence measurements has been previously described [ ] . a hamamatsu hc - photo multiplier tube (pmt) sensor module was used for chemiluminescence measurements, combining the sensitivity of a photomultiplier tube with the intelligence of a microcontroller. the detector was optimized to the blue light region and included a mm diameter active area convenient for gathering light radiation without any optical focusing elements for luminescence measurements. the instrument set-up and the chemiluminescence's procedure were fully described by konry et al. [ ] . the detection of anti-ebola virus antibodies was achieved using a sandwich fiber-optic immunoassay (fig. ) . for this purpose the prepared tip of an optical fiber was coated with an ito transparent semiconductor film that afterwards enables electropolymerization of the photoactive polymer-poly(pyrrolebenzophenone) and photoimmobilization of viral antigens. fig. shows sem micrographs of ito coated optic fiber tip conjugated with poly(pyrrole-benzophenone) film. animal anti-ebola virus antisera were serially diluted down to − and a fiber-optic luminescence assay was performed along with a standard chemiluminescence elisa for comparison. the subsequent binding of peroxidase-labeled secondary antibodies enabled detection via a chemiluminescence reaction by adding an enhanced luminol solution. the light, emitted as a side reaction, is transduced by the optical fiber to the measuring instrument. the standard curve for the goat anti-ebola subtype zaire sandwich immunoassays was constructed by collecting data in triplicate at sera titers ranging from : to : , . fig. shows the typical sigmoidal behavior for the standard calibration curve. for the characterization of the immunosensor calibration curve, the linear range curve fit was carried out using the equation of the form y = a + bx, where x is the dilution of serum sample and y is the corresponding response signal obtained. the dynamic linear range of the curve most useful in the quantization of titers was found to be from : to : , , exhibiting in this range an acceptable square correlation coefficient, r = . and a satisfactory sensitivity of . photocounts/s fig. . the calibration curve obtained from the optical fiber immunosensor and elisa for the detection of anti-ebola subtype zaire antibodies. (determined within the linear range of the biosensor as the slope b, of the calibration curve). at higher concentrations, the curve levels off with a response saturation observed from titers : and up. as small inaccuracies in such measurements occur at such low dilutions, these measurements can lead to large errors in prediction, so such samples should be diluted for accurate quantization. the lower detection limit of the immunosensor, defined as the amount (or concentration) of the analyte that gives a response (ydl) that is significantly different, is three standard deviations (sdbr) from the background of the analysis that is itself obtained from negative sera (ybr). therefore, the lower detection limit (yldl) was calculated using eq. ( ): the background signal recorded for the blank (in the absence of the analyte) and its calculated standard deviation enable quantization with dilutions as high as : , for the immunosensor and : , for the elisa. tests of samples were performed in triplicate and under identical conditions, with the same materials, in both immunosensor and elisa assays. therefore, this enables a direct comparison of results, and reveals that the immunosensor is much more sensitive than chemiluminescence elisa and is likely capable of recognizing virus specific antibody presence in the sera of patients in earlier stages of disease. finally, the optical fiber immunoassay showed a greater lower detection limit than elisa. since elisa uses absorption as the immobilization methodology for the antigen, a change in protein conformation may occur. therefore the loss of putative epitopic binding sites would reduce their potential for binding to antibodies, while the covalent immobilization would offer a reduction in such a loss. the same experiment was repeated with the sudan subtype of ebola virus (fig. ) . in this case diluted sera of rabbits immunized with ebola virus sudan was used as the analyte for antibody detection. as expected, the behavior of the assay was similar to the first one. the linear range fit for the immunosensor curve was also found to be in the titer range between : and : , with an acceptable square correlation coefficient r = . and a satisfactory sensitivity of . photocounts/s (determined within the linear range of the biosensor as the slope b, of the calibration curve). the detection limit of the immunosensor for the sudan strain of the virus was calculated using the same statistics methods as in the previous case. the very low background signals allow measurements of very low concentrations of ebola specific antibodies in the analyte solution. this is significant as following the emergence of ebola hemorrhagic fever there is an undetectable or very negligible elevation in the concentration of the igg antibodies in the sera using conventional techniques. as a result, the immunosensor has great potential for clinical and serological studies of ebola hemorrhagic fever. in addition, the fiberoptic immunosensor assay is less time consuming than standard elisa (approximately h versus h, respectively, after antigen binding, thus constituting a real gain in time for high-throughput diagnosis). therefore the immunosensor will enable an earlier detection of the production of antibodies, whereas the standard elisa will only detect them at a more advanced stage, when the level of the antibodies reaches a high concentration [ , , [ ] [ ] [ ] . to assess the precision of our technique, the coefficient of variance (cv) was calculated for the replicates measured in triplicates. the fiber-optic biosensor shows a lower cv percentage and better precision between replicates, than does the elisa assay for nearly all the presented ranges of concentrations used. in addition, the natural host for ebola virus, presumed to be an animal, has not been identified [ , , ] . a straightforward approach that might be used to determine animal contact with the ebola virus, or identification of an animal reservoir, is to assess the presence of specific antibodies in the serum. finally, many studies have demonstrated the long-term persistence of igg antibodies against filoviruses in sera, which suggests that this new serologic tool will be useful for field studies of the ebola virus [ , ] . there is some evidence that some cross-reactivity between ebola subtypes is observed in elisa assay tests [ , , ] . fig. demonstrates the serological cross-reactivity between two different ebola strains, zaire and sudan, using the optical fiber immunosensor. in this experiment immobilized antigen of sudan strain was placed in a serum sample of goat immunized against the zaire strain of ebola virus and vice versa. the signals received under such condition were very low and close to the background of the assay, however they indicate a true cross-reactivity. this data demonstrates the high sensitivity of the biosensor and its ability to identify very low levels of crossreactivity between the two strains of the ebola virus, zaire and sudan. to demonstrate further the practical application of the immunosensor, we tested the human sera samples from the survivors of the outbreak in the gulu district of uganda, - . all sera samples, from ebola survivors and from healthy volunteers serums from the same geographical region, were initially checked by pcr and found to be currently negative for the virus. as a valid negative control for this test we used the serum of a healthy individual from israel, and in addition, the sera samples of healthy volunteers from the gulu district of uganda. although these data are limited by the number of sera samples available for verification, one of the healthy volunteers from the same geographical region showed a positive reaction to inactivated ebola virus when tested with the immunosensor. an elisa test for the same person was negative due to relatively low sensitivity of the method as was reviled in standard calibration curves for elisa and immunosensor (figs. and ) . also due to limited number of sera samples, the one negative serum (in elisa) that was converted to positive by our method could be a false positive result. this data may indicate the presence of igg antibodies in a healthy individual due to exposure to antigen in an endemic area without any clinical evidence of infection (or perhaps a subclinical case). in addition, the prevalence of this seropositivity within the population, in areas of prior epidemics, could be found among the latest surveys performed using elisa assays [ , , ] . using a more sensitive detection method, such as the immunosensor presented herein, will enable serological studies among the population of endemic areas that may strengthen the hypothesis that one can develop some immunity to the ebola virus without developing clinical illness. consequently the ebola virus might leave its footprint in some individuals without causing an active infection. finally, our sensitive immunosensor can be used effectively for tracking the reservoir of ebola. we have demonstrated herein that one can electropolymerize the conductive surface of a fiber-optic tip with a photosensitive layer, which will enable a one-step immobilization procedure of proteinaceous biospecific entities such as inactivated ebola virus. the model analyte, anti-ebola igg, was detected at a low titer of : , and : , , for subtypes zaire and sudan, respectively. even though our ability to evaluate the sera of known infections with this group of viruses was limited by the availability of appropriate sera, we believe that the results are encouraging and strongly suggest that this immunosensor will prove a preferable alternative to elisa for measurement of antibodies to ebola viruses. dr. alex chepurnov, phd, heads the biosafety level virus laboratory at russia's state research center of virology and biotechnology in novosibirsk, commonly referred to world-wide as "vector". he is one of the leading russian experts on the ebola virus and has studied the host response to this virus as well as methods for treatment. most of his own research focuses on ebola zaire, the deadliest strain of ebola. science with qualification for tenure professorship in virology from the university of marburg, germany. currently she works as assistant professor and group leader of the department of virology in university of marburg, germany. her current research is concentrated on replication and transcription of filoviruses, host-virus interactions of filoviruses and interaction of sars-coronavirus with the type i interferon system. dr. leslie lobel, md phd, a graduate of columbia college of columbia university where he received ba degree summa cum laude. his graduate education was in the medical scientist training program at the college of physicians and surgeons of columbia university. he graduated with the md, phd degrees in . dr. lobel is now in the department of virology in the faculty of health sciences of ben gurion university of the negev in israel. his current work focuses on immunovirology and he studies the human humoral immune response to a variety of infectious diseases that include smallpox, hepatitis c, ebola and west nile virus. he has isolated a library of fully human monoclonal antibodies from patients immunized against smallpox and from patients that were infected with hepatitis c. currently dr. lobel focuses on more deadly diseases, such as ebola, for development of immunotherapeutic tools and synthetic vaccines. sciences and his msc in cell biology and physiology from the university of california, santa barbara, usa. he then completed his phd in chemical immunology at the weizmann institute of science, rehovot, israel, in on an enhanced mucosal vaccination method using silica microparticles coated with synthetic peptides. from to , he was a research associate at the university of cambridge, uk designing a fluorescent adiabatically tapered optical fiber immunosensor. in , he moved to the the ben gurion university of the negev, where he is now a tenured senior lecturer and heads a biosensor group with projects on bioluminescent whole-cell bioreporter biosensors, chemiluminescent fiber-optic immunosensors to viral antigens, phagocyte fiber-optic biosensor, amperometric biosensors, biomaterials, enzyme nanolithography and other projects. filoviridae: marburg and ebola viruses an introduction to ebola: the virus and the disease clinical virology of ebola hemorrhagic fever (ehf): virus, virus antigen, and igg and igm antibody findings among ehf patients in kikwit, democratic republic of the congo outbreak of ebola haemorrhagic fever outbreak of ebola hemorrhagic fever uganda containing a haemorrhagic fever epidemic: the ebola experience in uganda anthrax, tularemia, plague, ebola or smallpox as agents of bioterrorism: recognition in the emergency room elisa for the detection of antibodies to ebola viruses prevalence of igg antibodies to ebola virus in individuals during an ebola outbreak, democratic republic of the congo serological reactivity of baculovirus-expressed ebola virus vp and nucleoproteins development, characterization and use of monoclonal vp -antibodies for the detection of ebola virus diagnosis of ebola haemorrhagic fever by rt-pcr in an epidemic setting rapid detection protocol for filoviruses detection of antibodies against the four subtypes of ebola virus in sera from any species using a novel antibody-phage indicator assay a novel immunohistochemical assay for the detection of ebola virus in skin: implications for diagnosis, spread, and surveillance of ebola hemorrhagic fever, commission de lutte contre les epidemies a kikwit indium tin oxide-coated optical fiber tips for affinity electropolymerization physico-chemical studies of indium tin oxidecoated fiber optic biosensors optical fiber immunosensor based on a poly(pyrrolebenzophenone) film for the detection of antibodies to viral antigen development of an "electroptode" immunosensor: indium tin oxide-coated optical fiber tips conjugated with an electropolymerized thin film with conjugated cholera toxin b subunit properties of rf magnetron sputtered ito films without in-situ substrate heating and post-deposition annealing calculation of the figure of merit for indium tin oxide films based on basic theory transparent conductors-a status review an electrogenerated poly(pyrrole-benzophenone) film for the photografting of proteins benzophenone photoprobes for phosphoinositides, peptides and drugs optical fiber bioluminescent whole-cell microbial biosensors to genotoxicants human asymptomatic ebola infection and strong inflammatory response unexpected ebola virus in a tertiary setting: clinical and epidemiologic aspects early immune responses accompanying human asymptomatic ebola infections ecology of marburg and ebola viruses: speculations and directions for future research search for the ebola virus reservoir in kikwit, democratic republic of the congo: reflections on a vertebrate collection ebola virus can be effectively neutralized by antibody produced in natural human infection recombinant human monoclonal antibodies to ebola virus haemorrhagic fever virus activity in equatorial africa: distribution and prevalence of filovirus reactive antibody in the central african republic the authors thank the north atlantic treaty organization (nato), yakov yuzhelevski from the department of physics at bgu for his technical assistance in running the ito r.f. sputtering instrumentation, and luba burlaka from the center for meso, nanoscale science and technology, department of chemical engineering (bgu) that carried out the sem measurement. key: cord- -z fpy authors: kusano, nario; hirashima, keita; kuwahara, minoru; narahara, kenji; imamura, tadashi; mimori, tomohiro; nakahira, ken; torii, kuniaki title: immunochromatographic assay for simple and rapid detection of satsuma dwarf virus and related viruses using monoclonal antibodies date: - - journal: j doi: . /s - - - sha: doc_id: cord_uid: z fpy a simple and rapid immunochromatographic assay (ica) to detect satsuma dwarf virus (sdv) was developed using colloidal gold conjugates of anti-sdv monoclonal antibodies. of six homogenization buffers tested, . m citrate buffer (ph . ) gave the best results for the ica. in the ica, addition of . % thioglycolic acid in the homogenization buffers that have been widely used in enzyme-linked immunosorbent assays (elisa) was deleterious to the reaction because of undesirable coagulation of the colloidal gold. ica using the anti-sdv monoclonal antibodies was times and times more sensitive than double antibody sandwich-elisa and ica using the anti-sdv polyclonal antibody, respectively. the analysis is complete in only min. furthermore, ica using the anti-sdv monoclonal antibodies could also detect sdv-related viruses. satsuma dwarf virus (sdv), a graft-transmissible pathogen of citrus trees, causes satsuma dwarf disease, which reduces tree vigor and fruit yield in japan (yamada and sawamura ) . this disease is characterized by a dwarfed tree, boatshaped or downward-cupped leaves, and fruit with lower sugar content and higher juice acidity than uninfected fruit ). based on fi eld observations, sdv is transmitted through a soil source, but no vector has been identifi ed (koizumi et al. ; katagi and ushiyama ; iwanami and koizumi ) . during the s, a group of sdv-like viruses, citrus mosaic virus (cimv), natsudaidai dwarf virus (ndv), and navel orange infectious mottling virus (nimv) were also reported in japan. based on a comparison of symptoms on various citrus plants and herbaceous indexing plants, these viruses seemed to be related to sdv and were thus classifi ed as sdv-related viruses (sdv-rvs). recently, novel strains of sdv-rvs (e.g., az- , lb- ) were reported in japan. iwanami et al. ( iwanami et al. ( , suggested an alternative classifi cation for sdv-rvs based on serology, nucleic acid sequence, and the symptomatology of citrus and herbaceous plants. iwanami suggested that sdv-rvs could be classifi ed into three groups, sdv (s- , mie- ), cimv (ci- , lb- , az- , nd- ), and nimv (ni- ). based on serological relationships, viruses of the cimv group are relatively close to the sdv group, while nimv is a distinct virus. satsuma dwarf disease has usually been diagnosed using an enzyme-linked immunosorbent assay (elisa), although elisa needs expensive equipment such as a microplate reader, uses many reagents, and is time consuming. therefore, we focused our attention on developing rapid, simple detection methods for sdv. a rapid immunofi lter paper assay (ripa) has been developed and is often used to detect plant viruses (tsuda et al. ; ohki and kameya-iwaki ) . although we tried this method for sdv, we could barely detect the virus in : diluted extracts of sdvinfected plants. an immunochromatographic assay (ica) was also developed to detect pathogens of humans such as hepatitis b virus, infl uenza virus, and others (sato et al. ; vaughn et al. ; guan et al. ; weinberg and walker ) . recently, ica has been used in plant detection devices, the pocket diagnostic (central science laboratory, york, uk) and immunostrip (agdia, elkhart, in, usa), for viruses of vegetable and ornamental plants. ica is a very simple and rapid test with a one-step sample application, as opposed to elisa with multiple steps such as washing, incubation, and secondary color development. therefore, we developed a new system and a versatile plastic assay device for ica for plant viruses, and we report here on the fi rst application of the ica system in detecting sdv in the fi eld. virus isolates and antibodies sdv and sdv-rv isolates were collected in japan and maintained by grafting on trifoliate orange or rough lemon rootstocks under greenhouse conditions. three isolates (mh, yo, and hv) were supplied by dr. t. ito (national institute of fruit tree science), two isolates (hs- and az- ) were supplied by dr. t. iwanami (national agricultural research center for kyushu and okinawa regions), one isolate (sw- ) was supplied by k. shimazu (wakayama research center of agriculture, forestry, and fisheries), and four isolates (cs- , ku- , sa- , and cik- ) were collected in fukuoka prefecture. three clones of monoclonal antibodies (mabs) that recognize a different epitope on sdv (hirashima et al. ) and a polyclonal antibody (pab) (elisa kit; japan plant protection association; jppa) against sdv were used. the pab against sdv (s- ) was supplied by dr. iwanami (national agricultural research center for kyushu and okinawa regions). double antibody sandwich enzyme-linked immunosorbent assay (das-elisa) using an elisa kit for sdv (jppa) was performed according to the manufacturer's instructions with some modifi cations. the test samples were prepared from young shoots of infected citrus in mid-april and at the end of july. for the mab, the biotinylated conjugate was modifi ed and das-elisa was carried out as in a previous report (kusano and shimomura ) . immunochromatographic assay (ica) and homogenization buffers the underlying principle and the test device for our ica system are illustrated in figs. a,b and respectively. the immunochromatographic devices were prepared as follows ( fig. ) : mg/ml of anti-sdv monoclonal antibody (i.e., µl/cm) was deposited onto a nitrocellulose membrane (millipore, billerica, ma, usa) to make a test line. similarly, . mg/ml of casein fl uorescein isothiocyanate (fitc c , sigma, st. louis, mo, usa) was deposited to make a reference line. the membrane was then soaked in . % casein (kishida, osaka, japan). finally, the membranes were dried at °c to fi nish the antibody-conjugated membrane. the colloidal gold solution was prepared by reduction of tetrachloroauric acid with trisodium citrate (frens ) . the ph of the colloidal gold solution was adjusted to . with . m sodium carbonate buffer. anti-sdv monoclonal antibody was added to the colloidal gold solution for a fi nal concentration of . µg/ml. the mixture was incubated at room temperature with gentle stirring, and % bovine serum albumin (bsa) was added. the preparation was centrifuged at rpm for min, and fi nally the supernatant was removed to make anti-sdv monoclonal antibody-colloidal gold conjugate for the test line. anti-fitc monoclonal antibody was conjugated as mentioned to make the anti-fitc monoclonal antibody-colloidal gold conjugate for the reference line. the anti-sdv monoclonal antibody-colloidal gold conjugate and anti-fitc monoclonal antibody-colloidal gold conjugate were mixed at a concentration of optical density at nm of . and . , respectively. the preparation was deposited at µl/mm onto glass fi ber (whatman, middlesex, uk). then the prepared glass fi ber was dried in a vacuumed desiccator to make a conjugate pad. filter paper (the adsorption pad), the membrane, the conjugate pad, and glass fi ber (the sample application pad) were assembled on an adhesive plastic sheet. the sheet was cut into -mm wide strips with a hand cutter, and every strip was inserted into a plastic device (fig. ) . finally, the assembled devices were sealed with a desiccant in moistureresistant protective foil. six homogenization buffers for ica were prepared: . m citrate buffer ph . ( . m cb), . m cb, . m tris buffer ph . ( . m te), . m te, . m phosphatebuffered saline ph . (pbs), and . m pbs. effects of thioglycolic acid, which is often added to the homogenization buffer in elisa, on ica were tested. ica was simply performed by the addition of µl of supernatant of plant extract directly onto the sample application pad. after the sample application, the result was read by visual inspection for staining of the test and reference lines. a positive result was indicated by a typical purple line, which is produced when virus particles are trapped on the test line. the reference line ensures that the test result is valid. the intensity of any line was scored as negative (fig. a) , weak positive (fig. b) , moderate positive (fig. c) , or strong positive (fig. d ). comparison of the sensitivities of ica and das-elisa out of the three combinations of using three mabs for ica, clone g and clone e , which had a different paratope group (pg), were more sensitive and suitable to the colloidal gold conjugate and coating antibody on the nitrocellulose strip for detection of sdv, respectively (data not shown). to determine which of the six homogenization buffers was the most suitable for ica, the ica results using four different virus isolates and the buffers were compared. of the six buffers, . m cb gave the strongest, most stable staining (table ) . when using . % thioglycolic acid in the homogenization buffers, none of the virus isolates produced any positive test lines and the intensities of all reference lines were very weak. sensitivities of ica and das-elisa were compared using young shoots from an sdv-infected tree at the end of july (table ) . ica using mabs remained sensitive up to a : dilution, while das-elisa remained sensitive to a dilution of : , indicating that ica using mabs is eight times more sensitive than das-elisa. the amount of colloidal gold conjugate in ica using pab was optimized for the highest sensitivity (table , cases - , - , - ). suspensions of colloidal gold conjugate at various concentrations were applied to the reagent pad, and ica the immunochromatographic devices were prepared under the following conditions: case - , mg/ml of anti-sdv polyclonal antibody was deposited onto a nitrocellulose membrane to make a test line. the anti-sdv polyclonal antibody-colloidal gold conjugate suspension was prepared at a concentration of optical density at nm (od) of . , and was deposited onto glass fi ber; case - , the immunochromatographic device conditions were as described in case - , except that the concentration of optical density at nm (od) was . ; case - , the immunochromatographic device conditions were as described in case - , except that the concentration of optical density at nm (od) was . ; case , the immunochromatographic device conditions were as described in case - , except that all the antibodies were monoclonal and the concentration of optical density at nm (od) was . a s- polyclonal antibody b test line intensities for ica: + +, strong (positive); +, moderate (positive); ±, weak (positive); −, negative was performed. the concentrations of the conjugate in cases - , - , and - were adjusted to give absorbances of . , . , and . at nm, respectively. in case - , the ica detected sdv up to a dilution of : of the infected young shoot and was negative for the uninfected shoot, while ica in cases - and - produced falsepositive lines for the uninfected shoot. on the other hand, ica using the mabs detected sdv up to a dilution of : and produced no false-positive lines for the uninfected shoot. this indicated that the sensitivity of ica using mabs was times higher than ica with the pab. the effect of the concentration of the anti-sdv monoclonal antibody on the nitrocellulose membrane was also evaluated. from . to . mg/ml, the sensitivity reached a plateau at mg/ml. therefore, we selected mg/ml as the optimum concentration (data not shown). the reactivity of three isolates of sdv and seven isolates of sdv-rv in ica with mabs and in das-elisa using the jppa kit and mabs was studied. the results showed that ica is superior to the commercial jppa kit and elisa with mabs (table ). ica and das-elisa gave strong positive reactions and high elisa values, respectively, for three sdv isolates, cs- , ku- , and sw- . ica produced higher positive reactions for sdv-rv isolates compared with the jppa sdv elisa kit and elisa with mabs. ica detected hs- and az- , while the jppa kit did not. furthermore, ica was completed within min, while das-elisa required about days. the addition of . % thioglycolic acid into the homogenization buffer is known to stabilize the reaction in elisa. however, the reagent should not be used in the homogenization buffer for ica because it caused an undesirable coagulation of the colloidal gold, leading to inaccurate results. in our ica system, sdv(-rv) is also stable in the plant homogenate without thioglycolic acid for at least several weeks at °c or several years at − °c (data not shown). homogenization buffers without thioglycolic acid have been reported by salomone and roggero ( ) and salomone et al. ( ) , who tested homogenization buffers (pbs ph . containing . % tween- , % polyvinylpyrrolidone, . % triton-x , and mm sodium n,ndiethyldithiocarbamate trihydrate for pepino mosaic virus; pbs ph . containing . % tween- , % polyvinylpyrrolidone, and . % triton-x for citrus tristeza virus) in ica. of the six homogenization buffers tested, . m citrate buffer (ph . ) gave the best results. assembled on an adhesive plastic sheet cut into -mm wide strips inserted in a plastic device citrus tristeza virus in ica using polyclonal antibody returned % false positives in the total test results when compared with elisa; thus, this level was acceptable only as a screening method (salomone et al. ). porter et al. ( ) reported that ica and elisa were similar for sensitivity and specifi city in the detection of dengue virus. guan et al. ( ) developed ica and elisa to detect immunoglobulin g antibodies to severe acute respiratory syndrome (sars) coronavirus in sars patients with excellent correlation between ica and elisa. we evaluated the sensitivity and specifi city of ica using ten virus isolates. cs- , ku- , and sw- were shown serologically to belong to sdv by das-elisa using pab (table ) . iwanami et al. ( ) reported that az- belongs to the cimv group. furthermore, ito et al. ( ) suggested that mh is a strain of nimv, based on a sequence analysis of part of the coat protein region. iwanami et al. ( ) indicated that sdv-related viruses (sdv-rv) could be classifi ed as sdv, cimv, or nimv by sequence analysis. ito et al. ( ) showed hyuganatsu virus (hv) is a new sdv-rv by sequence analyses of the partial rna-dependent rna polymerase region in rna and the coat protein region in rna of the virus. thus, the ten virus isolates used in this experiment are probably various types of sdv-rvs. the higher sensitivity of ica with mabs over that with pab or the elisa kit (tables , ) for sdv is supported by previous results with different pg mabs in elisa, which was also more sensitive than with pab to detect sdv (hirashima et al. ) . as for specifi city, the reactivity of ica was clearer than the elisa kit for sdv-rv (seven virus isolates) detection and elisa with the same combination of mabs (table ) . we think the % specifi city (seven of seven samples) was the reason that ica requires no washing process and that the complex of the colloidal gold conjugates and virus moves on the nitrocellulose strip by capillary action. ica detecting sdv and sdv-rv offers advantages, such as the sensitivity and specifi city, over elisa. in conclusion, ica using mabs is a simple, reliable, and rapid detection method for sdv and sdv-rv in citrus fi elds; results can be obtained within min after dropping µl of the sample homogenate on the nitrocellulose membrane. controlled nucleation for the regulation of the particle size in monodisperse gold suspensions recombinant-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin g antibodies of severe acute respiratory syndrome (sars) coronavirus in sars patients detection of satsuma dwarf virus by using monoclonal antibodies partial nucleotide sequences of satsuma dwarf related viruses detected from mihocall and hyuganatsu a new virus related to satsuma dwarf virus: the nucleotide sequence of the ′-terminal regions of hyuganatsu virus rnas and satsuma dwarf virus group diversity of properties among satsuma dwarf virus and related viruses sequence diversity and interrelationships among isolates of satsuma dwarf-related viruses studies on satsuma dwarf virus iii. survey on the extension of satsuma dwarf virus disease and effect of root isolation or soil fumigation china laurestine: a symptomless carrier of satsuma dwarf virus which accelerates natural transmission in the fi elds improvement of enzyme-linked immunosorbent assay (elisa) for detection of citrus tatter leaf virus simplifying of rapid immunofi lter paper assay for faster detection of plant viruses: simplifi ed ripa evaluation of commercially available immunogloblin m capture enzyme-linked immunosorbent assay kit for diagnosing acute dengue infection host range, seed transmission and detection by elisa and lateral fl ow of an italian isolate of pepino mosaic virus reliability of detection of citrus tristeza virus an immunochromatographic fl ow assay in comparison with elisa evaluation of immunochromatographic assay systems for rapid detection of hepatitis b surface antigen and antibody, dainascreen hbsag and dainascreen ausab changes in fruit character and quality in satsuma mandarin infected with satsuma dwarf virus a novel detection and identifi cation technique for plant viruses: rapid immunofi lter paper assay evaluation for a rapid immunochromatographic test for diagnosis of dengue virus infection evaluation of three immunoassay kits for rapid detection of infl uenza virus a and b studies on the dwarf disease of satsuma orange acknowledgments the authors thank dr. t. iwanami, national agricultural research center for kyushu and okinawa regions, for providing the sera and dr. y. takanami, kyushu university, for critical reading of the manuscript. key: cord- - qcoxl authors: nicklas, werner; bleich, andré; mähler, michael title: viral infections of laboratory mice date: - - journal: the laboratory mouse doi: . /b - - - - . - sha: doc_id: cord_uid: qcoxl viral infections of laboratory mice have considerable impact on research results, and prevention of such infections is therefore of crucial importance. this chapter covers infections of mice with the following viruses: herpesviruses, mousepox virus, murine adenoviruses, polyomaviruses, parvoviruses, lactate dehydrogenase-elevating virus, lymphocytic choriomeningitis virus, mammalian orthoreovirus serotype , murine hepatitis virus, murine norovirus, murine pneumonia virus, murine rotavirus, sendai virus, and theiler’s murine encephalomyelitis virus. for each virus, there is a description of the agent, epizootiology, clinical symptoms, pathology, methods of diagnosis and control, and its impact on research. in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but clinically inapparent infections may also have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system, or both at the same time but on different parts of the the laboratory mouse Ó elsevier ltd. all rights reserved. isbn - - - - doi: . /b - - - - . - immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase interindividual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells or tumours [ , ] . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals [ ] . mouse antibody production (map) testing or polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to find. to address this problem, several meetings have been held on viral complications on research. the knowledge available is summarized in conference proceedings [ , ] and has later repeatedly been reviewed [ ] [ ] [ ] . viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. the nomenclature and taxonomy of viruses is described based on recent nomenclature rules by the international union of microbiological societies [ ] and the universal virus database of the international committee on the taxonomy of viruses (http://www. ictvdb.org). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with the methods presently available. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via the germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with a virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of the virus. an animal may have been infected, mounted an effective antibody response and cleared the virus, but remains seropositive for weeks or months or for ever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct detection methods such as virus isolation, electron microscopy or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and, depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary, including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpesviruses and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices [ ] [ ] [ ] [ ] [ ] [ ] . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, these animals are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can cause natural infections in mice (mus musculus). mouse cytomegalovirus (mcmv- ) or murid herpesvirus (muhv- ) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus (muhv- ) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both are enveloped, double-stranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar [ ] . mcmv- is very uncommon in european and american colonies of laboratory mice and is found at a very low rate [ ] or reported as not found [ , ] . seropositivity has, however, been reported from asian countries [ , ] . testing for mtv is not frequently reported, and no sample tested positive in recent studies [ ] . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv- and mtv are frequently found in wild mice, which may be coinfected with both viruses [ , [ ] [ ] [ ] . mouse cytomegalovirus (mcmv- ) or murid herpesvirus (muhv- ) natural infection with mcmv- causes subclinical salivary gland infection in mice. like other cytomegaloviruses, mcmv- is strictly hostspecific. it persists in the salivary glands (particularly in the submaxillary glands) and also in other organs [ ] [ ] [ ] . the virus can be cultured in mouse fibroblast lines like t cells, but primary mouse embryo fibroblasts are more sensitive to infection and produce higher virus titres. however, passage in cell culture results in its attenuation. to maintain virulence, the virus is best propagated by salivary gland passages of sublethal virus doses in weanling mice of a susceptible strain (e.g. balb/c) [ ] . most information concerning the pathogenesis of mcmv- infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history [ ] , virus dose and route of inoculation [ ] . in general, newborn mice are most susceptible to clinical disease and to lethal infection and develop higher levels of resistance with increasing age. infection of neonates leads to abnormal brain development [ , ] . virus replication is observed in newborn mice in many tissues and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days and of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains [ ] . mice of the h- b (e.g. c bl/ ) and h- d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h- k haplotype (e.g. c h), which are approximately -fold more resistant to mortality than those of the b or d haplotype [ ] . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone) or critical illness such as sepsis [ ] . reactivation of mcmv- also occurs after implantation of latently infected salivary glands into prkdc scid mice [ ] . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn nu and lyst bg mice are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver and other organs, and multinucleate syncythia with inclusion bodies in the liver [ ] . similarly to aids patients infected with human cytomegalovirus, athymic foxn nu mice experimentally infected with mcmv- also develop adrenal necrosis [ ] . the virus also replicates in the lungs, leading to pneumonitis, whereas replication and disease are not seen in heterozygous (foxn nu/þ ) littermates [ ] . the pathogenesis of mcmv- infection in immunocompetent and in immunocompromised mice, as well as the role of the immune system, have been reviewed by krmpotic et al. [ ] . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and are found also in other organs such as liver, spleen, ovary and pancreas [ ] . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis or splenic necrosis in susceptible strains [ , , ] . virus is transmitted by the oronasal route, by direct contact and is excreted in saliva, tears and urine for several months. the virus is ubiquitous in wild house mice worldwide. they serve as a natural reservoir for infection and can even be infected with different virus strains [ ] . the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. sexual transmission and transmission with tissues or organs is also possible. the virus does not cross the placenta in immunocompetent mice, although infection of pregnant females results in fetal death or resorption and wasting of borne pups. however, fetal infection is possible by direct injection of mcmv- into the placenta [ ] and also occurs by transplacental transmission in mice with severe immunodeficiency [ ] . vertical transmission is also possible by milk during lactation [ ] . it is generally assumed that mcmv- has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, different serological tests, including multiplex fluorescent immunoassay (mfia) [ ] , are used for health surveillance of rodent colonies. as the virus persists, direct demonstration of mcmv- in infected mice is possible by pcr [ ] [ ] [ ] or by virus isolation using mouse embryo fibroblasts ( t cells). although mcmv- does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection [ ] . these aspects have been discussed in detail by shellam et al. [ ] . mcmv- has also been used as a vaccine vector aiming at a disseminating mouse control agent by inducing immunocontraception in mice [ ] . the virus is known to influence immune reactions in infected mice [ , ] and may therefore have an impact on immunological research [ , ] . mtv was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, mtv is difficult to culture in vitro and is usually propagated by intraperitoneal infection of newborn mice. the thymus is removed - days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason, and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice [ , ] . further, mtv obviously represents a significant source of contamination of mcmv- (and vice versa) if virus is prepared from salivary glands, since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about days. infection results in nuclear inclusions in thymocytes and their almost complete destruction within weeks. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. the virus persists here and is shed via saliva for months. mtv also establishes a persistent infection in athymic foxn nu mice, but virus shedding is reduced compared to euthymic mice, with virus recovery possible only in a lower percentage of mice [ ] . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion [ ] . lymphoid necrosis may also occur in lymph nodes and spleen [ ] , with necrosis and recovery similar to that in the thymus. in mice infected during the first days after birth, necrosis of thymus becomes evident within - days, and the animals' size and weight are markedly reduced at day - . intranuclear inclusions may be present in thymocytes between days - after infection. the thymus and the affected peripheral tissues regenerate within weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t-cell mitogens) returns to normal as the histological repair progresses [ ] . selective depletion of cd þ t cells by mtv results in autoimmune disease [ , ] . information about additional influences on the immune system is given in textbooks [ , ] . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion and virus shedding, suggesting that the oral-nasal route is likely to be involved in natural transmission [ ] . the virus spreads to cagemates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mtv is not transmitted to fetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion [ ] . mtv and mcmv- do not cross-react serologically [ ] . serological monitoring of mouse populations for antibodies to mtv is possible by indirect immunofluorescent assay (ifa) testing, which is commercially available; enzyme-linked immunosorbent assays (elisa) tests have also been established [ , ] . elisa and complement fixation yield similar results [ ] . serological tests based on recombinant proteins and direct detection of virus by pcr are currently not possible because the genome of the virus has not yet been sequenced. it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns, whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes and spleens for lymphoid necrosis [ ] . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa [ ] and map testing, although this is slightly less sensitive than infectivity assays [ ] . there is very little experience of eradication methods for mtv because of its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses will likely eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction of the virus into laboratory animal facilities. murid herpesvirus (muhv- ) or rat cytomegalovirus infects rats and is also a member of the genus muromegalovirus. murid herpesvirus (muhv- ) is a member of the genus rhadinovirus in the subfamily gammaherpesvirinae and is also known as mouse herpesvirus strain (mhv- ). other murid herpesviruses are not yet assigned to a subfamily within the family herpesviridae. among these is murid herpesvirus (mouse thymic herpesvirus), but also murid herpesvirus (field mouse herpesvirus) which infects voles (microtus pennsylvanicus), murid herpesvirus (sand rat nuclear inclusion agent), and murid herpesvirus [ ] . furthermore, a gammaherpesvirus of house mice (mus musculus) has been described recently which is clearly distinct from mhv- [ ] . experimental infection of laboratory mice with mhv- is a frequently used model system for the study of human gammaherpesvirus pathogenesis, e.g. of kaposi's sarcoma-associated herpesvirus or epstein-barr virus (ebv) [ , ] which are members of the same subfamily. they are also important models to study viral latency and immune mechanisms controlling latency [ ] [ ] [ ] . mus musculus is not the natural host for this virus; it was first isolated in slovakia from bank voles (myodes glareolus). additional closely related strains (mhv- , mhv- ) exist from the same host species, and similar strains (mhv- , mhv- ) were isolated from wood mice (apodemus flavicollis and apodemus sylvaticus). apodemus sp. seem to be the major hosts for mhv- in great britain [ ] . different virus strains exhibit different genetic and biological properties and also differ in their pathogenicity, e.g. for prkdc scid mice [ ] . infections in laboratory mice take the same course as in their natural hosts [ ] . there are, however, some differences as, e.g. higher virus levels are reached in the lungs of balb/c mice, and wood mice develop higher titres of neutralizing antibodies [ ] . house mice develop an acute infection in the lungs after intranasal infection. a latent infection develops within weeks and the virus persists lifelong in epithelial cells in the lungs and also spreads to the spleen and other organs (e.g. bone marrow, peritoneal cells) where it persists in different cells of the immune system. it behaves like a natural pathogen in inbred strains of mice and persists without causing disease. the mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses, with a diameter of nm and a length of - nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of nearly nucleotides [ ] . it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to days of age) is possible [ ] . unlike various other orthopoxviruses, ectromelia virus does not infect humans [ ] . the virus is resistant to desiccation, dry heat and many disinfectants. it is not consistently inactivated in serum heated for min at c [ , ] and remains active for months when maintained at c in fetal bovine serum [ ] . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite and iodophores [ , ] . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between and almost individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in - and has been described in great detail [ ] . severe outbreaks were also described in various european countries [ ] [ ] [ ] . a more recent outbreak in the usa, which resulted in the eradication of almost mice in one institution, was described by dick et al. [ ] . another recent and well-documented case of mousepox was published by lipman et al. [ ] . few additional but unpublished cases of ectromelia have been observed since then; the latest report of an outbreak was published in [ ] . in general, positive serological reactions are occasionally reported from routine health surveillance studies [ ] but the virus is extremely rare in european and american colonies of laboratory mice [ ] [ ] [ ] . natural infections manifest differently, depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs and may therefore be very difficult to diagnose [ ] . the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form) or subacute to chronic infection (cutaneous form) [ , [ ] [ ] [ ] . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent [ ] [ ] [ ] . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/ , dba/ , balb/c, a and c h/hej. immunodeficient mice may also be very susceptible [ ] . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (> %). clinical disease may not be evident in resistant strains such as c bl/ and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice [ , ] . killer cells are necessary to control mousepox infections [ ] . mice that are resistant to mousepox may lose their resistance with increasing age, most likely due to the decreased number and activity of nk cells [ ] . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h d b (termed rmp , resistance to mousepox), on chromosome [ ] ; the c genes (rmp , on chromosome ); rmp , localized to a region on chromosome encoding the nk cell receptor nkr-p alloantigens [ ] ; the nitric oxide synthase locus on chromosome [ ] ; and the signal transducer and activator of transcription locus on chromosome [ ] . mousepox infections are controlled for several days during the initial course of infection by the complement system until the adaptive immune system can react. loss of the complement system results in lethal infection [ ] . clearance of the virus by the immune system is dependent upon the effector functions of cd þ t cells while nk cells, cd þ t cells and macrophages are necessary for the generation of an optimal response [ , ] . t-and b-cell interactions and antibodies play a central role during recovery from a secondary infection [ ] . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes [ , , ] . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages, resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis and skin lesions (papules, erosions or encrustations mainly on ears, feet and tail; figure . . ). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen [ , , , ] . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure . . ), lymph nodes, peyer's patches, thymus and liver [ , , , , ] . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viraemia ( natural transmission of ectv mainly occurs by direct contact and fomites [ , , , ] . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved [ ] . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection [ ] . intrauterine transmission is possible at least under experimental conditions [ ] . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about - weeks, even though the virus can persist for months in the spleen of an occasional mouse [ , ] . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding [ ] . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays [ ] . in the s, diagnosis relied on clinical signs, histopathology and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also used. serology is currently the primary means of routine health surveillance for testing mouse colonies for exposure to ectv. the methods of choice are mfia, elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay [ , , ] . serological tests based on virus particles detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus or other orthopoxviruses, respectively. vaccinia virus is commonly used as an antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding [ ] . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. [ ] depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical signs suggestive of mousepox are observed. pcr assays to detect different genes of poxviruses in infected tissues have been used [ , , ] . other pcr tests which were developed to detect smallpox virus have also been shown to detect ectromelia virus and can be used as well [ , ] . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines [ ] , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last three published outbreaks of ectromelia were introduced into the facilities by mouse serum [ , , ] . lipman et al. [ ] found that the contaminated serum originated from a pooled lot of l that had been imported from china, but in both other cases, serum was obtained from animals in the usa. because mouse serum is commonly sold to the end user in small aliquots (a few millilitres), it has to be expected that aliquots of the contaminated lot may still be stored in freezers. these published cases of ectromelia outbreaks provide excellent examples of why testing should be performed on all biological materials to be inoculated into mice. in the case of ectromelia virus it was shown that pcr is more sensitive, and map testing failed to detect contamination [ ] . eradication of mousepox has usually been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method because of the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious [ , , ] and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring, with removal of clinically sick and seropositive animals, are a potential alternative. the period from the last births until the first matings after cessation of breeding should be at least weeks [ ] . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population [ , ] . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs and thymus [ ] . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals and financial resources can be substantial. experimental mousepox infections are frequently used as a model to study various aspects of smallpox infections of humans [ ] [ ] [ ] . mousepox shares many aspects of virus biology and pathology, and models the course of human smallpox. experimental mousepox infections are used to study vaccination procedures [ , ] or anti-poxvirus therapies [ ] . murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae. two distinct strains have been isolated from mice. the fl strain (madv- ) was first isolated in the usa as a contaminant of a friend leukaemia [ ] and has been classified as a member of the genus mastadenovirus. the k strain (madv- ) was isolated in japan from the faeces of a healthy mouse [ ] and has not yet been assigned to a genus. both strains are considered to represent different species [ ] [ ] [ ] . they are host species specific and are not infectious for infant rats [ ] . madv- can be cultured in vitro in mouse fibroblasts (e.g. t or l cells), madv- is usually cultured in vitro in a mouse rectum carcinoma cell line (cmt- ). in laboratory mice, seropositivity to adenoviruses was reported to be very low [ , , , , , ] or negative [ , ] . antibodies to madv are also found in wild mice [ , ] and in rats [ , ] . neither virus is known to cause clinical disease in naturally infected, immunocompetent mice. however, madv- can cause a fatal systemic disease in suckling mice after experimental inoculation [ , , ] . disease is characterized by scruffiness, lethargy, stunted growth and often death within days. experimental infection of adult mice with madv- is most often subclinical and persistent but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia and paralysis in susceptible c bl/ and dba/ j mice [ ] . balb/c mice are relatively resistant to this condition. athymic foxn nu mice experimentally infected with madv- develop a lethal wasting disease [ ] . similarly, prkdc scid mice succumb to experimental infection with madv- [ ] . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv- include small surface haemorrhages in the brain and spinal cord of c bl/ and dba/ j mice [ ] , duodenal haemorrhage in foxn nu mice [ ] and pale yellow livers in prkdc scid mice [ ] . histologically, experimental madv- infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas and brown fat [ , , , ] . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns) [ ] . histopathological manifestations in madv- -infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes [ ] . in contrast to madv- , the tissue tropism of madv- is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum [ , , ] . transmission of madv primarily occurs by ingestion. madv- is excreted in the urine and may be shed for up to years [ ] . madv- infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice [ ] ; immunodeficient mice may shed the virus for longer periods [ ] . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross-reactivity of madv- with madv- [ ] . serum from mice experimentally infected with madv- yielded positive reactions in serological tests with both viruses, while serum from mice infected with madv- reacted only with the homologous antigen [ ] . smith et al. [ ] reported that sera might react with madv- or madv- or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv- ) indicate that the infection may not be detected if only one virus is used as an antigen [ ] . it is therefore usual to test sera for antibodies to both madv- and madv- . the commonly used methods are ifa, elisa and mfia. the low prevalence in colonies of laboratory mice indicates that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance, which is also indicated by the fact that recent publications about murine adenoviruses are very rare. however, the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only a few influences on research attributable to madv have been published. for example, it has been shown that madv- significantly aggravates the clinical course of scrapie disease in mice [ ] . natural infections with madv could also interfere with studies using adenovirus as a gene vector. a novel murine adenovirus classified as a mastadenovirus has recently been isolated from a striped field mouse (apodemus agrarius) [ ] . it was cultured in vero e cells and named madv type (madv- ). it revealed the highest similarity to madv- but it represents a separate serotype. however, there is some cross-reactivity between madv- and both other mouse viruses [ ] . in addition to serological and antigenic differences it also shows a unique organotropism and infects predominantly the heart tissue of c bl/ n mice after experimental infection. experimentally infected mice show no clinical signs. the virus is not easily transmitted from experimentally infected mice to contact sentinels [ ] . polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) was formerly known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related, but antigenically distinct, from each other [ ] , and also viruslike particles from the major capsid protein (vp ) do not cross-react [ ] . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible [ ] . seropositivity to these viruses was not reported in a recent survey conducted in the usa [ ] , and other publications also indicate that these viruses do not presently play a significant role in laboratory mice [ , , ] . because of their low prevalence, neither virus is included in the list of agents for which testing is recommended on a regular basis by felasa [ ] . although polyomavirus genes, especially those of sv , are widely used in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last - years. the reader is therefore referred to previous review articles for details [ , ] . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence [ , ] . clinical signs are observed only after infection of infant mice less than - days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium (figure . . ). endothelial cells in other organs are also involved in virus replication [ , ] (figure . . ). in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn nu mice, experimental infection of adults is clinically asymptomatic, although virus is detectable for a period of several months [ ] . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays [ ] . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available [ ] . furthermore, a pcr test for demonstration of mptv in biological samples has also been published [ ] . mpyv was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours [ ] . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs when mice are experimentally infected at a young age or when inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage [ ] . parotid, salivary gland and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels and thyroid also occur. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c bl and c br/cd lineage are considered to be the most resistant strains; athymic foxn nu mice are considered to be most susceptible; c h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn nu ) also supports the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses [ ] . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney and colon become infected [ ] . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection [ ] . mpyv persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about weeks [ ] . however, virus may persist and can be reactivated by prolonged immunosuppression [ ] or during pregnancy, at least in young mice [ ] . it has been shown that interferon-gamma is an important factor of the host defence against tumour formation and mpyv infection [ ] . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice, emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa, mfia and ifa; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing [ ] . while mpyv infections are of low importance for laboratory animal medicine, the virus is used in models of persistent virus infection [ , ] . virus-like particles from both murine polyomaviruses have been used as a vector for gene therapy or vaccines [ , ] . parvoviruses are non-enveloped small viruses (approximately nm in diameter) with a singlestranded dna genome of approximately nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. up to date, two distinct species that infect laboratory mice are officially listed: the minute virus of mice (mvm), previously named mice minute virus (mmv), and the mouse parvovirus (mpv). non-structural proteins (ns- and ns- ) are highly conserved among both viruses whereas the capsid proteins (vp- , vp- , vp- ) are more divergent and determine the serogroup [ ] . both viruses require mitotically active cells for replication. severe clinical signs are therefore not found in mature animals because of the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed [ , , [ ] [ ] [ ] [ ] [ ] . already in the mid- s mouse colonies were identified that gave positive reactions for mvm by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses [ ] and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and subsequently characterized on a molecular basis [ , ] . on the basis of epidemiological analyses, further parvoviruses were recently identified in mice, sequenced, and tentatively named serially mpv- and mpv- [ ] , mpv- (genbank fj ) and mpv- (genbank fj ). in addition, several variants are published for mpv- [ , , ] . at present, mpv is among the most common viruses found in colonies of laboratory mice. the prevalence of sera positive for parvoviruses ranged from % to nearly % in western europe and north america, with the majority of sera being positive for mpv in studies differentiating between the two parvovirus species [ , , , ] . these prevalence data are based on testing at commercial laboratories and do not reflect that, despite highly specific and sensitive test methods, enzootic parvovirus infections are difficult to detect due to virus-associated characteristics [ , ] . a recent survey conducted in the usa showed that during a - month period mouse parvoviruses were detected at almost all facilities that responded to a questionnaire, with mpv being more often diagnosed than mvm [ ] . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals [ , ] . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice [ , ] . humoral immunity to mpv does not protect against mvm infections, and vice versa [ ] . serological surveys have indicated that mpv naturally infects only mice, with the exception that mpv- shows genetic similarity to hamster parvovirus, suggesting that a cross-species transmission has occurred, where the mouse probably served as the natural host [ , ] . differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection with mpv- b, seroconversion occurred in all c h/hen mice, fewer balb/c, dba/ and icr mice, and seroconversion could not be detected in c bl/ mice [ ] . upon mpv- f inoculation, antibody response was absent in balb/carc mice [ ] . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/ even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than months [ ] , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for - weeks [ ] , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice might not transmit the virus under similar experimental conditions [ ] . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi is a practical screening test for mpv because they require large quantities of purified mpv, which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mvm hi-negative result coupled with an mvm ifa-positive result. ifa provides the opportunity to detect both serogroup-specific vp proteins as well as ns proteins that are conserved among mouse parvoviruses. a generic rodent parvovirus elisa using a recombinant ns- protein as antigen has been developed [ ] , but mpv ifa and mpv hi assays are more sensitive techniques than the ns- elisa and the mvm ifa [ ] . in contrast, elisa tests that use recombinant vp- provide sensitive and serogroup-specific assays for the diagnosis of mpv infections in mice [ , ] , although considerable cross-reactivity with heterologous capsid antigens exists [ ] . nevertheless, when using the elisa technique, one needs to consider that mpv- may not consistently be detected by mpv- vp- elisa [ , ] , especially when antibody titres are low (own observations). therefore, elisas using mpv- vp- and mpv- vp- antigens are also used for diagnostics. as parvovirus diagnostics using recombinant assays should be based on a combination of antigens, bead-based mulitplex assays are a convenient extension of traditional elisa, allowing the use of multiple antigens simultaneously. in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv [ , ] and other parvoviruses. mpv has been shown to persist for at least weeks in the mesenteric lymph nodes [ ] . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success [ ] . for antemortem detection, shedding of parvoviruses can also be detected by pcr of faecal samples [ ] . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigen-based serological assays. the pcr is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours [ ] . in addition, the map test has been reported as a sensitive tool to detect mpv [ ] . given the high environmental stability of the virus and the potential fomite transmission, together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations by embryo transfer or by hysterectomy. it should be noted that recent studies suggest a risk of virus transmission by embryo transfer, though successful sanitation of immunodeficient mice was achieved despite antibody response in recipients and progeny after embryo transfer [ , ] . although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts [ ] . mvm is the type species of the genus parvovirus. the virus was intermediately named mice minute virus (mmv). it was originally isolated by crawford [ ] from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma [ ] and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. mvmp infects fibroblast cell lines and does not cause clinical disease [ , ] . both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mvmp. a third strain, the cutter strain mvmc, was isolated from bhk- cells [ ] . in contrast to these three strains detected as cell culture contaminants, an isolate was obtained from naturally infected mice with a b-cell maturational defect maintained at the university of missouri and therefore denominated mvmm [ ] . serological surveys show that the mouse is the primary natural host [ , , ] , but the virus is also infective for rats, hamsters [ , ] , and mastomys [ ] during fetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis [ ] observed mild growth retardation a few days after experimental infection of neonatal mice with mvmp. studies of transplacental infection yielded no pathological findings in mice [ ] . the immunosuppressive variant, but not the prototype strain, is able to produce a runting syndrome after experimental infection of newborn mice [ ] . depending on the host genotype, experimental infections of fetal and neonatal mice with mvmi produce various clinical presentations and lesions. infection in c bl/ mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/ mice. infection of strains such as balb/c, cba, c h/he and sjl is also lethal and mice have renal papillary haemorrhage [ ] . the mvmi also infects haematopoietic stem cells and mediates an acute myelosuppression [ , ] . because of its dependence on mitotically active tissues, the fetus is at particular risk for damage by parvoviruses. mvm and other parvoviruses may have severe teratogenic effects and cause fetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in fetal death. adult prkdc scid mice develop an acute leukopenia month after experimental infection with mvmi and die within months. the virus persists lifelong in the bone marrow of these mice [ ] . during a natural concurrent outbreak of mvmm and mpv, a runting syndrome with lymphohistiocytic renal inflammation and inclusion bodies in cells resembling splenic haematopoietic progenitor cells was reported in b-cell (ighm)deficient mice [ ] . mmv is shed in faeces and urine. in faecal samples, mvm was detected for up to - weeks by pcr [ , ] , although shorter periods ( - days) have been observed [ ] . notably, shedding re-occurred after immunosuppression by irradiation [ ] . contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mvm can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. hi is a highly specific diagnostic test whereas ifa always exhibits some degree of cross-reactivity with mpv and other closely related parvoviruses. elisa is probably the most frequently used test, but depending on the purity of the antigen preparation, cross-reactions with mpv may occur due to contamination with non-structural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp- antigen [ ] . by using serological methods, one needs to consider that the mouse strain has a considerable effect on seroconversion so that an antibody response might not be detectable despite infection; while c bl/ j mice showed good antibody response, seroconversion was observed only in some balb/c, akr/n, dba/ j, fvb/n and c h/hen, but not in nmri and icr mice upon contact exposure to mvmi-inoculated mice [ ] . viral detection is also possible by pcr in biological materials, organs (intestine, mesenteric lymph node, kidney, spleen) and faeces from infected animals [ , , , , ] . although mvm was not thought to cause persistent infection in immunocompetent mice, recent data show that it can be detected in spleens for up to weeks after exposure in some mouse strains [ ] . therefore, pcr may be considered as a confirmatory method for serology. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. however, certain precautions such as careful washing and accompanying testing need to be minded, as mvm has been detected in reproductive organs and gametes and this virus firmly attaches to the zona pellucida or might even cross it [ , ] . in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found to be free from viral contamination. both allotropic variants of mvm have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published [ , , , , ] . because of their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect [ ] . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mvm is a common contaminant of transplantable tumours, murine leukaemias and other cell lines [ , , ] . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. the genome organization and replication of ldv and other arteriviruses, their cell biology and other molecular aspects have been reviewed by snijder and meulenberg [ ] . ldv has repeatedly been detected in wild mice (mus musculus), which are considered to be a virus reservoir [ , ] . after infection of mice, virus titres of - particles per ml serum are found within - h after infection. the virus titre drops to particles per ml within - weeks and remains constant at this level for life. it persists in infected mice for the whole lifetime although it stimulates various immune mechanisms [ ] [ ] [ ] [ ] . the virus can be stored in undiluted mouse plasma at À c without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. only mice and primary mouse cells are susceptible to infection with ldv. it replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues [ ] . as suitable cell systems have not been available for virus production, routine serology has not been easily possible so that testing for ldv was not included in serological health monitoring programs. the prevalence of ldv in contemporary colonies of laboratory rodents is likely to be very low but detailed information about its prevalence comparable to most other agents is not available. ldv was first detected during a study of methods that could be used in the early diagnosis of tumours [ ] . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host [ ]. ldv has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours or matrigel prepared from such materials [ , , , ] , monoclonal antibodies or ascitic fluids [ ] , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after serial passage in an infected and viraemic mouse. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials [ , ] . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is poliomyelitis with flaccid paralysis of hindlimbs developing in c and akr mice when they are immunosuppressed either naturally with ageing or experimentally. it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis [ ] . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes [ , ] , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only h after infection and peaks - days after infection at - -fold normal levels, or can even be up to -fold in sjl/j mice. the enzyme activity declines during the next weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli [ ] . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brainstem. ldv is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cagemates, most likely via blood and saliva. infected females transmit the virus to their fetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection [ , ] . maternal immunity protects fetuses from intrauterine infection. immunodeficient prkdc scid mice also transmit virus to their offspring during chronic infection [ ] . an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principle, serological methods such as ifa may be used for detecting ldv infection [ ] but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests, and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle [ ] . however, it is meanwhile possible to use recombinant viral proteins of ldv as antigens [ ] in elisa and mfia tests so that routine testing by serology is possible. in the past, diagnosis of ldv infection has primarily been based on increased ldh activity in serum or plasma of mice. ldv activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within - days is measured. an - -fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear non-haemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. this assay was usually included in a 'map test', but antibody detection similar to other viruses was not involved for reasons mentioned earlier. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates [ , ] . however, reports exist that pcr may produce false-negative results and should be used cautiously [ ] . just as important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogenfree mice [ , ] or by pcr [ , , [ ] [ ] [ ] . ldv spreads slowly in a population because direct contact is necessary. therefore, ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations may be indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages [ ] . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats [ ] or other animal species. another method to remove ldv from contaminated cells, which is based on cell sorting, has recently been described [ ] . ldv is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. ldv may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice); [ , , , [ ] [ ] [ ] [ ] [ ] and enhance or suppress tumour growth [ , , ] . interaction with other viruses has also been described [ ] . lymphocytic choriomeningitis virus (lcmv) is an enveloped, segmented single-stranded rna virus of the genus arenavirus family, arenaviridae. it can easily be propagated in several commonly used cell lines like bhk- cells. however, cells are not lysed and a cytopathic effect (cpe) is not visible. the virus name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir [ ] , but syrian hamsters are also important hosts [ ] . additional species such as rabbits, guinea-pigs, squirrels, monkeys and humans are susceptible to natural or experimental infection [ ] . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' [ , ] . antibodies to lcmv have been found in wild mice in europe [ , ] , africa [ ] , asia [ ] , australia [ ] and america [ ] . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice [ ] . seropositivity to lcmv in laboratory mice was reported to be low during the last decade [ , , , ] or negative [ ] [ ] [ ] . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters [ , ] . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lcmv is frequently transmitted to humans from wild mice and is also endemic to a varying degree in the human population [ ] [ ] [ ] [ ] [ ] due to contact with wild mice. it has also been transmitted to humans by infected laboratory mice [ ] and by pet and laboratory syrian hamsters [ ] [ ] [ ] [ ] . in addition, contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours [ , ] . transmission of lcmv to humans also occurred repeatedly by organ transplantation and was most likely transmitted to organ donors by close contact with infected pets [ , ] . lcmv can cause mild-to-serious or fatal disease in humans [ , , ] . congenital infection in humans may result in hydrocephalus, or fetal or neonatal death [ ] . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation [ , , ] . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) non-tolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and viral shedding. mice may show growth retardation, especially during the first - weeks, but they appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at - months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites and occasional deaths. this immunopathologic phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs [ ] . the non-tolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung and kidney) of mice for at least weeks after infection as determined by sensitive assays such as nested reverse transcriptase (rt)-pcr or immunohistochemistry [ ] . such non-lethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg a antibodies [ ] . in experimentally infected mice, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease [ ] . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day after inoculation, and animals die within - days after the onset of symptoms, or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmvinduced disease by immunosuppression [ ] . lcmv disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis, clinical and pathological features of lcmv infection, the reader is referred to review articles [ , , ] . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection [ , ] . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv, indicating that oral infection is possible, e.g. by ingestion of contaminated food or by cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). the virus is not easily transmitted to dirty-bedding sentinels, and it is important that colony animals or animals having had direct contact with a population are tested to exclude lcmv infection [ ] . lcmv is most commonly diagnosed by serological methods such as mfia, ifa and elisa [ ] . all strains show a broad cross-reactivity and are serologically uniform. however, subclinical persistent infections may be difficult to detect because they may be associated with minimal or undetectable levels of circulating antibody. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals [ ] [ ] [ ] . both map test and pcr can also be used to detect contamination of biological materials [ , ] . specifically for exclusion of contamination by lcmv, it was requested by different authorities that virus is inoculated intracerebrally at a lethal dose - weeks after administration of the material to be tested. in case of contamination by lcmv and subsequent seroconversion, animals survive the challenge infection. vertical transmission of lcmv by transuterine infection is efficient so that this virus cannot reliably be eliminated by caesarean rederivation [ ] . caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (non-tolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting non-viraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals, or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn nu and prkdc scid mice may pose a special risk because infections are silent and chronic [ ] . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, hydrogen peroxide or other effective disinfectants. prevention of introduction into an animal facility requires that wild mice cannot get access to the facility. similarly important is screening of biological materials originating from mice and hamsters because these can be contaminated by lcmv. finally, it has been shown that the virus can also be introduced into a population by mice with an undetected infection [ ] . appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) will be considered to be sufficient in most cases. bsl practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching or aerosol formation from the bedding. animal biosafety level (absl) practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or absls by cdc [ ] . lcmv is frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro [ ] [ ] [ ] . accidental transmission may have a severe impact on various kinds of experiments [ , , , ] and also affect infection with other agents [ ] . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes, numbered - . mammalian orthoreovirus- (synonyms: hepatoencephalomyelitis virus; echo virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice [ , , ] . a study in france reported antibodies to mrv- in % of mouse colonies examined [ ] . in more recent studies in north america and western europe, such antibodies were detected in . - . % of mice monitored [ , , ] . schoondermark-van de ven et al. [ ] found antibodies to mrv- in . % of mouse samplings from western european institutions; and in a survey conducted by carty [ ] , about % of responding institutions in the usa reported mrv- infection in their mouse colonies. in addition, contamination of mouse origin tumours and cell lines by mrv- has been reported many times [ , , ] . experimentally, mrv- infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus and lymphoma [ , ] . the literature on mrv- infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice [ ] [ ] [ ] . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented [ , ] . the mechanisms of viral pathogenesis and their interactions with the host cell as well as the host's immune response are reviewed in detail by tyler et al. [ ] , schiff et al. [ ] and ward et al. [ ] . natural infection by mrv- in a mouse colony is usually subclinical, although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted [ , , [ ] [ ] [ ] . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia) and neurological signs such as incoordination, tremors or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv (murine hepatitis virus) or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns and intestine. the outcome of mrv- infection depends on age and immunological status of mouse, dose of virus and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease [ , ] . experimental infection of adult prkdc scid mice is lethal [ ] . depending on the route of inoculation, experimental infection of adult foxn nu mice is subclinical or results in liver disease [ , ] . histological findings reported to occur after experimental mrv- infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia [ , [ ] [ ] [ ] ] . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route [ , ] . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with mfia, elisa or ifa is in widespread use for detection of antibodies to mrv- in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice [ ] , although a recent report indicates that some ifa-positive mrv infections in mice may not be detected by commonly used elisas [ ] . the hi test does not detect such cross-reacting antibodies but is prone to give false-positive results due to nonspecific inhibitors of haemagglutination [ , ] . rt-pcr methods for the detection of mrv- rna [ , ] or mrv rna [ , ] are also available. reports on contamination of mouse origin tumours and cell lines by mrv- and its interference with transplantable tumour studies [ , ] emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv- without clinical disease is also observed in laboratory rats, hamsters and guinea-pigs [ , ] . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv- infection [ , ] . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, single-stranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). mhv is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens, by use of monoclonal antibodies, or by sequencing [ ] . the beststudied strains are the prototype strains mhv- , mhv- , mhv- , jhm (mhv- ), a , and s, of which mhv- is regarded as the most virulent. like other coronaviruses mhv mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism and cell tropism [ ] . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) also exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating ( c for min) and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity [ ] . given the environmental conditions present in mouse rooms, mhv might remain infective for several days, at low humidity ( % relative humidity) or low temperatures ( c) even for weeks on surfaces [ ] . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. monitoring results for research institutions across north america and europe indicate that the prevalence of mhv has decreased in the past, though it seems to have remained quite stable since the s [ , ] . recently . % of north american laboratory mouse serum samples tested positive [ ] . in europe, prevalence rates ranged from . % to % [ , , ] . a retrospective study in france covering the period from to reported antibodies to mhv in % of mouse colonies examined [ ] , and a survey performed in revealed that almost half of north american research institutions detected mhv in their mouse populations [ ] . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease [ ] . similarly, deer mice seroconverted but showed no clinical disease after experimental infection [ ] . mhv is also a common contaminant of transplantable tumours [ , ] and cell lines [ , ] . the pathogenesis and outcome of mhv infections depend on interactions between numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status) [ , , , , , ] . mhv strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv- , mhv- , mhv- , a , s and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than weeks of age, infection of genetically susceptible strains of mice or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of the brain, liver, lymphoid organs, bone marrow and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant [ ] . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells [ ] , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages [ ] . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain [ ] . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are self-limiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within - weeks [ , , ] . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement [ ] [ ] [ ] [ ] . therefore, immunodeficient mice such as foxn nu and prkdc scid mice cannot clear the virus [ , ] . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection [ ] . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv [ ] [ ] [ ] . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains [ , ] . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine [ ] . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice [ , ] . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach % in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hindlimbs, convulsions and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn nu and prkdc scid ) mice, infection with virulent polytropic mhv strains is often rapidly fatal while less virulent strains cause chronic wasting disease [ ] . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease [ , ] . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by coinfections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed in [ , ] ). in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach [ , , ] . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver [ , , ] and thymus involution [ , ] may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis [ ] . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow [ , , , ] (figure . . ) . recently, pulmonary inflammation has been observed in susceptible mouse strains (c h/hej and a/j) after intranasal inoculation with polytropic mhv- [ , ] . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brainstem and periependymal areas. lesions in peritoneum, bone marrow, thymus and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t-cell mediated [ ] . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytium arising from endothelium, parenchyma or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferongamma-deficient mice infected with mhv [ ] . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues [ , , , ] . the most common sites are terminal ileum, caecum and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells) and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within - h, but are usually more severe at - days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity [ ] , but other agents such as helicobacter spp. may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. in t-cell deficient mice, multisystemic lethal infection was observed after experimental infection with the enterotropic strain mhv-y [ ] . mhv is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol and fomites [ , ] . vertical (in utero) transmission has been demonstrated in experimental infections [ ] but does not seem to be of practical importance under natural conditions. mhv was transmitted by ovarian transplantation after reproductive organs became infected [ ] . however, risk of mhv transmission by sperm or oocytes (ivf) or by embryo transfer seems to be low, though thorough washing of gametes and embryos is required [ , [ ] [ ] [ ] . diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry [ ] or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc or dbt [ ] . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn nu mouse inoculation [ , ] . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay [ ] and a number of rt-pcr assays [ ] [ ] [ ] [ ] . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. multiplex fluorescent immunoassay, elisa and ifa are well established and sensitive, and all known mhv strains cross-react in these tests [ , , ] . the magnitude of antibody response depends on mhv strain and mouse genotype [ , ] . dba/ mice are poor antibody responders whereas c bl/ mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least months [ , ] . infected mice may not develop detectable antibodies for up to days after initial exposure [ ] . in such cases, a direct diagnostic method, as discussed above, may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are weeks of age [ ] . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas [ ] and embryonic stem cells [ , ] these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test and rt-pcr can be used for this purpose. therefore, surveillance programmes should combine careful evaluation of clinically ill animals, testing of biological materials and routine health monitoring. soiled-bedding sentinel mice, which are frequently used for routine monitoring, are likely well suited for detecting enterotropic strains of mhv, but might not indicate the presence of less contagious respiratory strains of mhv [ ] equally well. the mouse strain used as sentinel should be considered as a critical factor. furthermore, duration of mhv shedding and stability of the virus, which seems to be lower in static microisolator cages than in ivc cages, might interfere with detection. the amount of bedding transferred seems not to be as critical as for, e.g. parvoviruses, at least for enterotropic strains [ ] . use of contact and exhaust air sentinels and testing of exhaust filters by pcr was also shown to be effective at detecting mhv [ ] . the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchasing mice from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing has also been reported to be effective [ ] . quarantine of an affected colony with no breeding and no introduction of new animals for approximately months has been effective in immunocompetent mice [ ] . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains [ ] . it may be practical to select a few future breeders from the infected population and quarantine them for approximately weeks [ ] . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the -week interval should permit recovery from active infection, and the additional -week gestation period effectively extends the total quarantine to weeks. it is advisable to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion [ ] . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation is likely to be the most cost-effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters, as well as stringent personal sanitation, are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic foxn rnu rats [ ] . mhv is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed in [ - , , , ] ). noroviruses are non-enveloped, single-stranded rna viruses with high environmental resistance and belong to the family caliciviridae, genus norovirus. they were first identified after an outbreak of acute gastroenteritis at a school in norwalk (ohio, usa) in and cause about % of non-bacterial epidemic gastroenteritis in humans. noroviruses found in animals include bovine, porcine and murine noroviruses. noroviruses are not known to cross species. murine norovirus (mnv) is endemic in many research mouse colonies and currently the most commonly detected viral agent in laboratory mice [ , , ] . in the hitherto largest survey [ ] , about % of mouse serum samples examined had antibodies against mnv. the first norovirus to infect mice was described in [ ] . experimental inoculation studies with this murine norovirus (mnv- ) show that duration of infection and disease manifestation vary depending on the mouse strain [ ] [ ] [ ] . in immunocompetent strains, mnv infection is variable in length (e.g. ! - days in s mice, ! weeks in hsd:icr mice) and does not induce clinical signs. infection is associated with mild histopathological alterations in the small intestine (increase in inflammatory cells) and spleen (red pulp hypertrophy and white pulp activation) of s mice. in certain immunodeficient strains, however, infection can cause lethal systemic disease (encephalitis, vasculitis, meningitis, hepatitis and pneumonia in interferon-alpha-beta-gamma-receptor-deficient and stat tm mice) or persist without symptoms (! days in rag À/À and rag À/À mice). these findings indicate that components of the innate immune system are critical for resistance to mnv- induced disease. consistent with this hypothesis, it was demonstrated that mnv- replicates in macrophages and dendritic cells [ ] . meanwhile, many additional strains of mnv with diverse biological properties were isolated [ , ] . an analysis of mnv isolates revealed distinct mnv strains that comprise a single genogroup and serotype [ ] . experimental inoculation studies show that several mnv strains are able to persist in various tissues (small intestine, caecum, mesenteric lymph node, spleen) of immunocompetent (c bl/ j, hsd:icr, jcl:icr) and immunodeficient (cb -prkdc scid ) mice with viral shedding in faeces for the duration of at least - days [ ] [ ] [ ] . murine norovirus is transmitted via the faecaloral route and is efficiently transferred to sentinel mice by soiled bedding [ , ] . mnv infection can be detected directly by rt-pcr on faecal pellets or tissue specimens (see above) and indirectly by serology (mfia, elisa, ifa) [ , , ] . detection is facilitated by high stability of mnv rna in faeces (at least weeks at room temperature) [ ] and by broad serological cross-reactivity among different strains of mnv [ , ] . embryo transfer [ ] and hysterectomy [ ] are most likely effective means of eliminating mnv from mouse colonies. since -to -day-old pups are resistant to infection, elimination of mnv may also be achieved by transferring neonates from infected dams to uninfected foster dams ('cross-fostering') [ ] . this transfer should ideally be performed within h after birth. mnv is used as a surrogate to evaluate resistance of human noroviruses to disinfectants. the impact of mnv on animal experiments remains to be evaluated. recent studies show that mnv is immunmodulatory and may alter disease phenotypes in mouse models of inflammatory bowel disease [ ] [ ] [ ] and other experimental mouse models [ , ] . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies [ ] ; however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus (mpv). pvm infection remains prevalent in contemporary colonies of mice and rats throughout the world. a serological survey in france demonstrated antibodies to pvm in % of mouse colonies examined [ ] . in more recent studies in north america and western europe, the prevalence of pvm-specific antibodies in mice ranged between % and . % [ , , ] . schoondermark-van de ven et al. [ ] found antibodies to pvm in . % of mouse samplings from western european institutions. antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea-pigs and rabbits [ , , ] . experimentally, pvm infection of mice is used as a model for hrsv infection and has therefore been extensively studied (reviewed by rosenberg and domachowske [ ] ). in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings [ , , ] . however, natural disease and persistent infection may occur in immunodeficient mice [ ] [ ] [ ] . in particular, athymic foxn nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation and death due to pneumonia [ , ] . similar clinical signs have been reported for experimentally infected immunocompetent mice [ ] . necropsy findings in naturally infected foxn nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation [ ] . pulmonary consolidation (dark red or grey in color) has also been found after experimental infection of immunocompetent mice [ ] . histologically, natural infection of foxn nu mice with pvm presents as interstitial pneumonia [ , ] . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia [ , ] . hydrocephalus may result from intracerebral inoculation of neonatal mice [ ] . susceptibility to infection is influenced by age and strain of mouse, dose of virus, and a variety of local and systemic stressors [ , , ] . in terms of the extent of the alveolar inflammatory response, /sv and dba/ mice are susceptible to pvm infection, while balb/c and c bl/ mice are relatively resistant [ ] . in terms of the control of viral replication, mice of strains /sv, dba/ , balb/c and c bl/ are susceptible to pvm infection, while sjl mice are relatively resistant. pvm is labile in the environment and rapidly inactivated at room temperature [ , ] . the virus is tropic for the respiratory epithelium [ , ] , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol [ , ] . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (mfia, elisa, ifa or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections [ , ] ; however, proper sampling (see chapter . , 'health management and monitoring') is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported [ ] . however, the use of direct methods requires good timing because the virus is present for only up to about days in immunocompetent mice [ ] . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus [ , ] . pvm could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease [ , ] or become more susceptible to the deleterious effects of other agents such as p. murina [ ] . murv-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a non-enveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis [ ] . murv-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. large commercial laboratories found . % to % of mouse sera from north american and european facilities to be positive for antibodies against murv-a/edim [ , , , ] , and up to % of mouse colonies in the usa were identified as affected in a survey performed in [ ] . experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies [ ] . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first weeks of life [ , , , , ] . diarrhoea usually begins around h after infection and persists for about week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region (figure . . ) . in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrhoeic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea (figure . . ). they are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells; the large intestinal surface mucosa may also be affected. though inflammation is minimal, the lamina propria may be oedematous, lymphatics may be dilated and mild leukocytic infiltration in the large intestinal mucosa and submucosa has been observed in a recent outbreak of disease [ , ] . hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system [ ] and through the effects of a viral enterotoxin called nsp (for non-structural protein ) [ ] . it is hypothesized that nsp is released from virus-infected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between and days of age [ , , , , ] . mice older than about weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus and diarrhoea does not occur. the exact reason for this agerelated resistance to disease is unknown. pups suckling from immune dams are protected against edim during their period of disease susceptibility [ ] . in general, the infection is self-limiting and resolves within days. successful viral control and clearance is promoted by an intact immune response [ ] [ ] [ ] [ ] , and some immunodeficient mice (e.g. prkdc scid and rag tm fwa mice) may shed virus for extended periods or become persistently infected [ , ] . protection against murv-a/edim reinfection is primarily mediated by antibodies [ , ] . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route [ , , ] . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols and is facilitated by the general property of rotaviruses that they remain infectious outside the body, show resistance to inactivation (e.g. low ph, non-ionic detergents, hydrophobic organic liquids, proteolytic enzymes), and are shed in high quantities (> particles/g faeces) [ ] . murv-a/edim is stable at À c but otherwise tends to be susceptible to extreme environmental conditions, detergents and disinfectants containing phenols, chlorine or ethanol [ ] . mfia, elisa and ifa are in widespread use for detection of serum antibodies to murv-a/ edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also used [ ] . as murv-a/edim shares the vp protein determined group a antigen, for example, with human, simian or bovine rotavirus strains, commercially available elisa assays utilizing polyclonal or monoclonal antibodies have been used to detect rotavirus antigen in mice; however, great care must be taken in interpreting the results because some feeds have been reported to cause false-positive reactions with certain elisa kits [ ] . electron microscopy of faeces of diarrhoeic pups should reveal typical wheel-shaped rotavirus particles, - nm in diameter. rt-pcr also can be used to detect rotavirus rna in faecal samples [ ] . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks [ ] . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy for - weeks combined with excellent sanitation, filter tops and conscientious serological testing of offspring and sentinel mice has also been reported to be effective, and prolongation of breeding cessation up to weeks resolved infection even in immunocompromised mice [ ] . murv-a/edim has the potential to interfere with any research using suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice [ ] . a disease-induced stress-related thymic necrosis may occur and alter immunology experiments [ ] . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus . the virus was named after sendai, japan, where it was first isolated from mice. historically, infections were relatively common in mouse and rat colonies worldwide. in addition, there is evidence that hamsters, guinea-pigs and rabbits are susceptible to infection with sev [ , , , ] ; however, some apparently seropositive guinea-pigs may in fact be seropositive to other parainfluenza viruses instead of sev. a study in france reported antibodies to sev in % of mouse colonies examined [ ] . a low rate of seropositive mice ( . %) was found in a survey in north america [ ] . schoondermark-van de ven et al. [ ] also found antibodies to sev in . % of mouse samplings from western european institutions. in more recent surveys in north america and western europe, sev infection was not detected [ ] [ ] [ ] , indicating that sev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. sev can contaminate biological materials [ ] . sev is pneumotropic and can cause significant respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein [ ] . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type [ , , ] . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after - months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high, resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings and runting in young mice. breeding colonies may return to normal productivity within months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport and copathogens are important in precipitating overt disease. dba and strains of mice are very susceptible to sev pneumonia, whereas sjl/j and c bl/ /j and several outbred stocks are relatively resistant. resistance to sev infection is under multigenic control with epistatic involvement [ ] . there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia [ , ] . clearance of a primary sev infection is mediated by cd þ and cd þ t-cell mechanisms [ , ] . heavier than normal, consolidated, plumcolored or grey lungs are a characteristic gross finding in severe sev pneumonia [ , , , ] . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative and resolution phases [ , ] . lesions of the acute phase, which lasts - days, are primarily attributed to the cellmediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils and mononuclear cells. atelectasis, bronchiectasis and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week after infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial, rather than alveolar, as they are in the acute phase. the resolution phase may be complete by the fourth week after infection and lesions may be difficult to subsequently identify. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present [ ] . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens [ ] . sev has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice [ ] . sev is extremely contagious. infectious virus is shed during the first weeks of infection and appears to be transmitted by direct contact, contaminated fomites and respiratory aerosol [ , ] . serology (mfia, elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only - weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry [ ] and rt-pcr [ , ] . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently [ , ] . map testing and rt-pcr can be used to detect sev in contaminated biological materials. sev infection in mouse colonies has proved to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies is probably the most appropriate means of eliminating the virus in most situations. embryo transfer, or caesarean derivation, followed by barrier maintenance, can also be used to eliminate the virus [ , ] . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding and prevent addition of other susceptible animals for approximately months until the infection is extinguished, and then breeding and other normal activities are resumed. vaccines against the virus have been developed [ , , ] , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sev has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and fetal growth; alterations of macrophage, nk-cell, and t-and b-cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed in [ ] [ ] [ ] ). pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev), or murine poliovirus, is a member of the genus cardiovirus in the family picornaviridae. members of this genus are non-enveloped viruses with single-stranded rna. the virus is rapidly destroyed at temperatures above c. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev [ ] . the best-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates, this virus is not pathogenic for rats and mice after intracerebral inoculation [ ] . recently, another rat isolate has been characterized and shown to be most closely related to, but quite distinct from, other tmev viruses [ ] . antibodies to tmev (strain gdvii) have been detected in guinea-pigs and are considered to indicate infection with another closely related cardiovirus [ ] . seropositivity to tmev was reported in approximately % of french mouse colonies in a retrospective study [ ] . in more recent studies, the prevalence of tmev infections was found to be lower. schoondermark-van de ven et al. [ ] detected antibodies to tmev in . % of mouse samplings from western european institutions. in a survey conducted by carty [ ] , about % of responding institutions in the usa reported tmev infection in their mouse colonies. further surveys in north america and western europe revealed antibodies in . - . % of mice monitored [ , , ] . tmev is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to weeks, sometimes intermittently [ ] , and transmission under natural conditions is via the faecal-oral route, by direct contact between mice, as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only in - infected immunocompetent animals [ ] . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher [ ] . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hindlimbs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virusinduced demyelinating disease including multiple sclerosis [ ] . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at - months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains, while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age [ , , ] . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is also effective. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice - days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling, rolling and weakness. animals may develop typical flaccid paralysis of hindlimbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralyzed. experimental infections with low-virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr and dba/ strains are most susceptible to this chronic demyelinating disease. cba and c h/he are less susceptible strains, and strains a, c bl/ , c bl/ and dba/ are relatively resistant [ ] . differences in humoral immune responses play a role in resistance to tmev infection [ ] , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h- d region of the major histocompatibility complex [ ] . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low-virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of - weeks while adult mice often show no clinical signs of infection. the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralyzed limbs, or wasting or atrophy of the hindlimbs in long-term survivors. tmev infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis and poliomyelitis with neuronolysis, neuronophagia and microgliosis in the brainstem and ventral horns of the spinal cord [ ] . demyelination in immunocompetent mice is considered to be immunemediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by t cells that release cytokines leading to recruitment of monocytes and macrophages as a consequence of infection of macrophages and other cns-resident cells [ ] [ ] [ ] . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation [ , ] . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes [ ] . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. [ ] , kim et al. [ ] and lipton et al. [ ] . experimental infection of foxn nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs, including the typical hindlimb paresis, develop weeks after inoculation, and most animals die within weeks. in foxn nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes [ ] . demyelination and lethality are reduced after administration of neutralizing antibodies [ ] . histopathological changes in prkdc scid mice are very similar to those in foxn nu mice [ ] . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by days of age. intrauterine transmission to fetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection [ ] . all tmev isolates are closely related antigenically and form a single serogroup, as determined by complement fixation and hi [ ] . hemelt et al. [ ] demonstrated cross-reactions among four strains used in experimental infections, but differences were evident in homologous and heterologous titres. the viral strain most commonly used as antigen for serological testing is gdvii. this strain agglutinates human type o erythrocytes at c, and hi has been the standard test for routine screening of mouse populations. meanwhile, hi has been replaced by mfia, elisa or ifa, all of which are more sensitive and specific. virus isolation is possible from brains or spinal cords of mice with clinical disease or from the intestinal contents of asymptomatic mice. pcr techniques are also available to test for virus-specific nucleotide sequences in biological samples [ ] . mice that have been shown to be free from tmev by serological testing can be selected for breeding populations. if the virus is introduced into a mouse population, depopulation of infected colonies may be the most appropriate means to eliminate tmev. embryo transfer or caesarean derivation is the method of choice for eliminating virus from valuable breeding populations. foster-nursing has been reported to be effective in generating virus-free offspring [ ] , although transplacental transmission has been demonstrated with experimental infection early in gestation. lesions of demyelination in cns of mice with clinically inapparent chronic infection may interfere with investigations that require evaluation of the cns [ ] . conceivably, such lesions could also affect neuromuscular responses or coordination, and affect neurological and behavioural evaluations. murine virus contaminants of leukemia viruses and transplantable tumors contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses mousepox resulting from use of ectromelia virus-contaminated, imported mouse serum viral and mycoplasmal infections of laboratory rodents: effect on biomedical research complications of viral and mycoplasmal infections in rodents to toxicology research and testing natural pathogens of laboratory animals: their effects on research implications of infectious agents on results of animal experiments committee on infectious diseases of mice and rats. infectious diseases of mice and rats virus taxonomy: eighth report of the international committee on taxonomy of viruses ten-year long monitoring of laboratory mouse and rat colonies in french facilities: a retrospective study diagnostic testing of mouse and rat colonies for infectious agents prevalence of naturally occurring viral infections, mycoplasma pulmonis and clostridium piliforme in laboratory rodents in western europe screened from opportunistic infections of mice and rats: jacoby and lindsey revisited a serological survey to evaluate contemporary prevalence of viral agents and mycoplasma pulmonis in laboratory mice and rats in western europe contemporary prevalence of infectious agents in laboratory mice and rats biology of mouse thymic virus, a herpesvirus of mice, and the antigenic relationship to mouse cytomegalovirus microbial contaminations of laboratory mice and rats in taiwan from microbiological quality assessment of laboratory mice in korea and recommendations for quality improvement the prevalence of viral antibodies during a large population fluctuation of house mice in australia serological survey of virus infection among wild house mice (mus domesticus) in the uk infectious diseases in wild mice (mus musculus) collected on and around the university of pennsylvania (philadelphia) campus persisting murine cytomegalovirus can reactivate and has unique transcriptional activity in ocular tissue james (lawson) cm. characterisation of murine cytomegalovirus myocarditis: cellular infiltration of the heart and virus persistence murine cytomegalovirus and other herpesviruses a mouse model for cytomegalovirus infection laboratory strains of murine cytomegalovirus are genetically similar to but phenotypically distinct from wild strains of virus murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain effects of cytomegalovirus infection on embryogenesis and brain development acute murine cytomegalovirus infection induces lethal hepatitis antiviral prevention of sepsis induced cytomegalovirus reactivation in immunocompetent mice mouse cytomegalovirus reactivation in severe combined immune deficient mice after implantation of latently infected salivary gland experimental murine cytomegalovirus infection in severe combined immunodeficient mice murine cytomegalovirus adrenalitis in athymic nude mice murine cytomegalovirus replication in the lungs of athymic balb/c nude mice pathogenesis of murine cytomegalovirus infection pathology of laboratory rodents & rabbits mixed infection with multiple strains of murine cytomegalovirus occurs following simultaneous or sequential infection of immunocompetent mice long-term impact of intrauterine mcmv infection on development of offspring nervous system transplacental murine cytomegalovirus infection in the brain of scid mice transmission of murine cytomegalovirus in breast milk: a model of natural infection in neonates simultaneous serodetection of highly prevalent mouse infectious pathogens in a single reaction by multiplex analysis development of a highly sensitive quantitative competitive pcr assay for the detection of murine cytomegalovirus dna quantitative measurement of infectious murine cytomegalovirus genomes in real-time pcr improved detection and quantification of mouse cytomegalovirus by real-time pcr acute murine cytomegalovirus infection: a model for determining antiviral activity against cmv induced hepatitis viral vectored immunocontraception: screening of multiple fertility antigens using murine cytomegalovirus as a vaccine vector latent cytomegalovirus infection exacerbates experimental colitis occult cytomegalovirus in vivariumhoused mice may influence transplant allograft acceptance mouse thymic necrosis virus: a novel murine lymphotropic agent mouse thymic virus (mtlv; murid herpesvirus ) infection in athymic nude mice: evidence for a t lymphocyte requirement neonatal infection with mouse thymic virus: spleen and lymph node necrosis a mammalian herpesvirus cytolytic for cd þ (l t þ) t lymphocytes virus and autoimmunity: induction of autoimmune disease in mice by mouse t lymphotropic virus (mtlv) destroying cd þ t cells thymic necrosis following oral inoculation of mouse thymic virus transmission of mouse thymic virus critical factors in an enzyme immunoassay (elisa) for antibodies to mouse thymic virus (mtlv) evaluation of mouse thymic virus antibody detection techniques detection of mouse thymic virus (mtlv) antigens in infected thymus by competition immunoassay comparative sensitivity of infectivity assay and mouse antibody production (map) test for detection of mouse thymic virus (mtlv) the order herpesvirales identification of novel rodent herpesviruses, including the first gammaherpesvirus of mus musculus a mouse model for infectious mononucleosis murine gammaherpesvirus : a model for the study of gammaherpesvirus pathogenesis viral latency and its regulation: lessons from the gammaherpesviruses immune mechanisms in murine gammaherpesvirus- infection immune control of mammalian gammaherpesviruses: lessons from murid herpesvirus- the wood mouse is a natural host for murid herpesvirus analysis of genomic homology of murine gammaherpesvirus (mhv)- to mhv- and impact of mhv- on the survival and tumorigenesis in the mhv- -infected cb scid/scid and cb þ/þ mice comparative study of murid gammaherpesvirus infection in mice and in a natural host, bank voles pathogenesis of a model gammaherpesvirus in a natural host the genomic sequence of ectromelia virus, the causative agent of mousepox variable resistance to ectromelia (mousepox) virus among genera of mus mousepox detected in a research facility: case report and failure of mouse antibody production testing to identify ectromelia virus in contaminated mouse serum mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. i. clinical responses prevention and control of mousepox observations of an outbreak of mousepox in laboratory mice in at the university of utah medical center demonstration of an ectromelia enzootic in hairless mice reaction of mouse strains to skin test for ectromelia using an allied virus as inoculum mousepox in the netherlands mousepox outbreak in a laboratory mouse colony mousepox (infectious ectromelia): past, present, and future clinical, pathologic, and serologic features of an epizootic of mousepox in minnesota kinetics of ectromelia virus (mousepox) transmission and clinical response in c bl/ j, balb/cbyj and akr/j inbred mice mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. v. genetics of resistance to the moscow strain the mouse in biomedical research. diseases pathology and diagnosis of mousepox induction of natural killer cell responses by ectromelia virus controls infection agedependent susceptibility to a viral disease due to decreased natural killer cell numbers and trafficking h- -linked control of resistance to ectromelia virus infection in b congenic mice differential pathogenesis of lethal mousepox in congenic dba/ mice implicates natural killer cell receptor nkr-p in necrotizing hepatitis and the fifth component of complement in recruitment of circulating leukocytes to spleen identification of nitric oxide synthase as an innate resistance locus against ectromelia virus infection enhanced resistance in stat -deficient mice to infection with ectromelia virus surviving mousepox infection requires the complement system innate resistance to lethal mousepox is genetically linked to the nk gene complex on chromosome and correlates with early restriction of virus replication by cells with an nk phenotype different roles for cd þ and cd þ t lymphocytes and macrophage subsets in the control of a generalized virus infection correlates of protective immunity in poxvirus infection: where does antibody stand? transmission of mouse-pox in colonies of mice mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. iii. experimental transmission of infection and derivation of virus-free progeny from previously infected dams intrauterine infection of mice with ectromelia virus mouse pox threat serological detection of ectromelia virus antibody evaluation of an enzyme-linked immunosorbent assay for the detection of ectromelia (mousepox) antibody administration of vaccinia virus to mice may cause contact or bedding sentinel mice to test positive for orthopoxvirus antibodies: case report and follow-up investigation specific detection of mousepox virus by polymerase chain reaction real-time pcr system for detection of orthopoxviruses and simultaneous identification of smallpox virus detection of human orthopoxvirus infections and differentiation of smallpox virus with real-time pcr observations on the replication of ectromelia virus in mouse-derived cell lines: implications for epidemiology of mousepox reexamination of the efficacy of vaccination against mousepox effect of vaccination on the clinical response, pathogenesis and transmission of mousepox pathogenesis of vaccinia (ihd-t) virus infection in balb/cann mice mouse models for studying orthopoxvirus respiratory infections animal models of orthopoxvirus infection ectromelia virus: the causative agent of mousepox a protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost postexposure immunization with modified vaccinia virus ankara or conventional lister vaccine provides solid protection in a murine model of human smallpox mousepox in the c bl/ strain provides an improved model for evaluating antipoxvirus therapies a new mouse virus apparently related to the adenovirus group an adenovirus isolated from the feces of mice i. isolation and identification genotypic differences between the mouse adenovirus strains fl and k molecular cloning, physical mapping and cross-hybridization of the murine adenovirus type and type genomes genetic relationship between mouse adenovirus- (strain k ) and human adenovirus- mouse adenoviruses microbiological contamination of laboratory mice and rats in korea from to a serologic survey for viruses and mycoplasma pulmonis among wild house mice (mus domesticus) in southeastern australia comparative biological characterization of mouse adenovirus strains fl and k and seroprevalence in laboratory rodents pathogenesis of experimentally produced mouse adenovirus infection in mice age and susceptibility of swiss mice for mouse adenovirus, strain fl mouse adenovirus type causes a fatal hemorrhagic encephalomyelitis in adult c bl/ but not balb/c mice duodenal lesions associated with adenovirus infection in athymic 'nude' mice murine adenovirus infection of scid mice induces hepatic lesions that resemble human reye syndrome experimental adenovirus infection of the mouse adrenal gland. i. light microscopic observations electron microscope study of experimental enteric adenovirus infection in mice experimental infection with mouse adenovirus in adult mice intestinal resistance in the experimental enteric infection of mice with a mouse adenovirus. i. growth of the virus and appearance of a neutralizing substance in the intestinal tract fluctuation of antiviral resistance in the intestinal tracts of nude mice infected with a mouse adenovirus biological and biophysical characteristics of mouse adenovirus, strain fl serological relationship between mouse adenovirus strains fl and k a naturally occurring intestinal mouse adenovirus infection associated with negative serologic findings acceleration of scrapie disease in mice by an adenovirus a novel cardiotropic murine adenovirus representing a distinct species of mastadenoviruses serological diagnosis of murine adenovirus characterization of k virus and its comparison with polyoma virus murine pneumotropic virus vp virus-like particles (vlps) bind to several cell types independent of sialic acid residues and do not serologically cross react with murine polyomavirus vp vlps recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units polyoma viruses the major site of murine k papovavirus persistence and reactivation is the renal tubular epithelium distribution of k-papovavirus in infected newborn mice morphological and immunohistochemical studies of the central nervous system involvement in papovavirus k infection in mice chronic infection of nude mice by murine k papovavirus serial passage of murine k-papovavirus in primary cultures of mouse embryo cells comparison of an enzymelinked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to k-papovavirus in mice diagnostic polymerase chain reaction assays for identification of murine polyomaviruses in biological samples a model for mixed virus disease: co-infection with moloney murine leukemia virus potentiates runting induced by polyomavirus detection of dna and rna virus genomes in organ systems of whole mice: patterns of mouse organ infection by polyomavirus transplacental transmission of polyoma virus in mice persistence of polyomavirus in adult scid c.b- mice immunosuppression and murine polyomavirus infection reactivation of polyoma virus in kidneys of persistently infected mice during pregnancy ifn-g controls mouse polyomavirus infection in vivo heterogeneity among viral antigen-specific cd þ t cells and their de novo recruitment during persistent polyomavirus infection long-term infection of adult mice with murine polyomavirus following stereotaxic inoculation into the brain murine polyomavirus virus-like particles (vlps) as vectors for gene and immune therapy and vaccines against viral infections and cancer cd þ, and cd þ t cells can act separately in tumour rejection after immunization with murine pneumotropic virus chimeric her /neu virus-like particles molecular characterization of a newly recognized mouse parvovirus the rodent parvoviruses viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research. diseases lurking in the shadows: emerging rodent infectious diseases coping with parvovirus infections in mice: health surveillance and control identification and propagation of a putative immunosuppressive orphan parvovirus in cloned t cells molecular characterization of newly recognized rodent parvoviruses identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice temporal transmission studies of mouse parvovirus in balb/c and c.b- / icr-prkdc scid mice serologic prevalence of mpv in mouse strains in a commercial laboratory mouse colony determined by using vp antigen serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant vp proteins in vivo studies with an 'orphan' parvovirus of mice characterization of mouse parvovirus infection by in situ hybridization humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus experimental infection of mice with hamster parvovirus: evidence for interspecies transmission of mouse parvovirus effect of mouse strain and age on detection of mouse parvovirus by use of serologic testing and polymerase chain reaction analysis strainand age-associated variation in viral persistence and antibody response to mouse parvovirus in experimentally infected mice characterization of mouse parvovirus infection among balb/c mice from an enzootically infected colony expression of recombinant parvovirus ns protein by a baculovirus and application to serologic testing of rodents validation of an enzyme-linked immunosorbent assay for detection of mouse parvovirus infection in laboratory mice detection of newly recognized rodent parvoviruses by pcr detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays antemortem detection of mouse parvovirus and mice minute virus by polymerase chain reaction (pcr) of faecal samples polymerase chain reaction for detection of rodent parvoviral contamination in cell lines and transplantable tumors embryo transfer rederivation of c.b- /icr-prkdc scid mice experimentally infected with mouse parvovirus detection of mouse parvovirus in mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft rejection a minute virus of mice immunosuppressive activity of a subline of the mouse el- lymphoma. evidence for minute virus of mice causing the inhibition pathogenicity of fibroblastand lymphocyte-specific variants of minute virus of mice pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype minute virus of mice. ii. prevalence, epidemiology, and occurrence as a contaminant of transplanted tumors electron microscopic localization of virions in developing teeth of young hamsters infected with minute virus of mice experimentally induced infection with autonomous parvoviruses, minute virus of mice and h- , in the african multimammate mouse (mastomys coucha) pathogenicity of minute virus of mice (mvm) for rats, mice, and hamsters fetal infections of hamsters, rats, and mice induced with the minute virus of mice (mvm) in vitro myelosuppressive effects of the parvovirus minute virus of mice (mvmi) on hematopoietic stem and committed progenitor cells myeloid depression follows infection of susceptible newborn mice with the parvovirus minute virus of mice (strain i) severe leukopenia and dysregulated erythropoiesis in scid mice persistently infected with the parvovirus minute virus of mice reduced fecundity and death associated with parvovirus infection in b-lymphocyte deficient mice minute virus of mice: antibody response, viral shedding, and persistence of viral dna in multiple strains of mice presence of minute virus of mice in immunocompetent mice despite the onset of host immunity gender influences infectivity in c bl/ mice exposed to mouse minute virus a rapid and simple procedure to detect the presence of mvm in conditioned cell fluids or culture media risk assessment of minute virus of mice transmission during rederivation: detection in reproductive organs, gametes, and embryos of mice after in vivo infection transmission of mouse minute virus (mmv) but not mouse hepatitis virus (mhv) following embryo transfer with experimentally exposed in vivo-derived embryos antineoplastic activity of parvoviruses experience with viral contamination in cell culture the molecular biology of arteriviruses lactic dehydrogenase virus isolation of lactate dehydrogenase-elevating viruses from wild house mice and their biological and molecular characterization lactate dehydrogenaseelevating virus induces systemic lymphocyte activation via tlr -dependent ifnalpha responses by plasmacytoid dendritic cells distinct gamma interferon-production pathways in mice infected with lactate dehydrogenase-elevating virus lactate dehydrogenase-elevating virus replication persists in liver, spleen, lymph node, and testis tissues and results in accumulation of viral rna in germinal centers, concomitant with polyclonal activation of b cells transmissible agent associated with types of experimental mouse neoplasms from bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics lactic dehydrogenase virus (ldhv) contamination in human tumor xenografts and its elimination contamination of a monoclonal antibody with ldh-virus causes interferon induction infection of central nervous system cells by ecotropic murine leukemia virus in c and akr mice and in in utero-infected ce/j mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus lactate dehydrogenase-elevating virus regulation of transplacental virus infection by developmental and immunological factors: studies with lactate dehydrogenaseelevating virus transplacental lactate dehydrogenase-elevating virus (ldv) transmission: immune inhibition of umbilical cord infection, and correlation of fetal virus susceptibility with development of f / antigen expression regulation of maternal-fetal virus transmission in immunologically reconstituted scid mice infected with lactate dehydrogenase-elevating virus immunofluorescent antibody response to lactic dehydrogenase virus in different strains of mice characterization of lactate dehydrogenase-elevating virus orf protein expressed by recombinant baculoviruses enzymatic amplification of lactate dehydrogenase-elevating virus detection of lactate dehydrogenase-elevating virus in transplantable mouse tumors by biological assay and rt-pcr assays and its removal from the tumor cell false negative results using rt-pcr for detection of lactate dehydrogenase-elevating virus in a tumor cell line comparison of the sensitivity of in vivo antibody production tests with in vitro pcr-based methods to detect infectious contamination of biological materials detection and typing of lactate dehydrogenase-elevating virus rna from transplantable tumors, mouse liver tissues, and cell lines, using polymerase chain reaction comparison of the mouse antibody production (map) assay and polymerase chain reaction (pcr) assays for the detection of viral contaminants relationship between the lactic dehydrogenase-elevating virus and transplantable murine tumors removal of lactate dehydrogenase-elevating virus from human-in-mouse breast tumor xenografts by cell-sorting infection of mice with lactate dehydrogenase-elevating virus leads to stimulation of autoantibodies suppression of development of diabetes in nod mice by lactate dehydrogenase virus infection natural killer cell activation after infection with lactate dehydrogenase-elevating virus effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen lactate dehydrogenase-elevating virus infection at the sensitization and challenge phases reduces the development of delayed eosinophilic allergic rhinitis in balb/c mice suppression of acute anti-friend virus cd þ t-cell responses by coinfection with lactate dehydrogenase-elevating virus mammalian reservoirs of arenaviruses risk to humans through contact with golden hamsters carrying lymphocytic choriomeningitis virus (author's transl) lymphocytic choriomeningitis virus first outbreak of callitrichid hepatitis in germany: genetic characterization of the causative lymphocytic choriomeningitis virus strains arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin- expression and hepatocyte proliferation lymphocytic choriomeningitis virus spatial and temporal dynamics of lymphocytic choriomeningitis virus in wild rodents, northern italy antibodies to lymphocytic choriomeningitis virus in wild rodent sera in egypt seroepidemiological survey of lymphocytic choriomeningitis virus in wild house mice in china with particular reference to their subspecies lymphocytic choriomeningitis virus infection and house mouse (mus musculus) distribution in urban baltimore contamination of transplantable murine tumors with lymphocytic choriomeningitis virus human-rodent contact and infection with lymphocytic choriomeningitis and seoul viruses in an inner-city population seroprevalence of lymphocytic choriomeningitis virus in nova scotia lymphocytic choriomeningitis virus infection in a province of spain: analysis of sera from the general population and wild rodents mouse-tohuman transmission of variant lymphocytic choriomeningitis virus exposure to lymphocytic choriomeningitis virus lymphocytic choriomeningitis outbreak associated with nude mice in a research institute laboratory studies of a lymphocytic choriomeningitis virus outbreak in man and laboratory animals lymphocytic choriomeningitis virus in southern france: four case reports and a review of the literature lymphocytic choriomeningitis in laboratory personnel exposed to hamsters inadvertently infected with lcm virus pet rodents and fatal lymphocytic choriomeningitis in transplant patients outbreak of lymphocytic choriomeningitis virus infections in medical center personnel virus zoonoses and their potential for contamination of cell cultures transmission of lymphocytic choriomeningitis virus by organ transplantation lymphocytic choriomeningitis virus: an unrecognized teratogenic pathogen lymphocytic choriomeningitis virus: reemerging central nervous system pathogen lymphocytic choriomeningitis virus: emerging fetal teratogen persistence of lymphocytic choriomeningitis virus at very low levels in immune mice mechanisms of antibody-mediated protection against lymphocytic choriomeningitis virus infection: mother-to-baby transfer of humoral protection murine hepatitis caused by lymphocytic choriomeningitis virus. ii. cells involved in pathogenesis biology and pathogenesis of lymphocytic choriomeningitis virus infection lymphocytic choriomeningitis infection of the central nervous system murine infection with lymphocytic choriomeningitis virus following gastric inoculation timed appearance of lymphocytic choriomeningitis virus after gastric inoculation of mice lymphocytic choriomeningitis infection undetected by dirty-bedding sentinel monitoring and revealed after embryo transfer of an inbred strain derived from wild mice detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by elisa using purified recombinant nucleoprotein development of a reverse transcription-polymerase chain reaction assay for diagnosis of lymphocytic choriomeningitis virus infection and its use in a prospective surveillance study detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain reaction analysis quantitative pcr technique for detecting lymphocytic choriomeningitis virus in vivo centers for disease control and prevention and national institutes of health mechanisms of humoral immunity explored through studies of lcmv infection lymphocytic choriomeningitis virus and immunology viral persistence: parameters, mechanisms and future predictions stage of primary infection with lymphocytic choriomeningitis virus determines predisposition or resistance of mice to secondary bacterial infections reovirus type infection, liver, mouse the mouse in biomedical research. diseases reovirus infection in laboratory rodents direct spread of reovirus from the intestinal lumen to the central nervous system through vagal autonomic nerve fibers type reovirus neuroinvasion after intramuscular inoculation: direct invasion of nerve terminals and age-dependent pathogenesis reoviruses and the host cell orthoreoviruses and their replication passive immunity to fatal reovirus serotype -induced meningoencephalitis mediated by both secretory and transplacental factors in neonatal mice infectivity, disease patterns, and serologic profiles of reovirus serotypes , , and in infant and weanling mice reovirus-induced liver disease in severe combined immunodeficient (scid) mice. a model for the study of viral infection, pathogenesis, and clearance histopathological characterization of the naturally occurring hepatotropic virus infections of nude mice detection methods for the identification of rodent viral and mycoplasmal infections reverse transcriptionpolymerase chain reaction detection and nucleic acid sequence confirmation of reovirus infection in laboratory mice with discordant serologic indirect immunofluorescence assay and enzyme-linked immunosorbent assay results diagnosis of murine infections in relation to test methods employed reovirus not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease detection of reovirus type by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral l genome segment isolation of a non-pathogenic tumour-destroying virus from mouse ascites an oncolytic virus recovered from swiss mice during passage of an ascites tumour mouse hepatitis virus enterotropic mouse hepatitis virus effects of air temperature and relative humidity on coronavirus survival on surfaces asymptomatic infection of mouse hepatitis virus in the rat effects of experimental infection of the deer mouse (peromyscus maniculatus) with mouse hepatitis virus isolation of a latent murine hepatitis virus from cultured mouse liver cells induction of lytic plaques by murine leukemia virus in murine sarcoma virus-transformed nonproducer mouse cells persistently infected with mouse hepatitis virus mhv-s mouse hepatitis virus biology and epizootiology the cellular and molecular pathogenesis of coronaviruses enterotropic coronavirus (mouse hepatitis virus) in mice: influence of host age and strain on infection and disease response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice effective clearance of mouse hepatitis virus from the central nervous system requires both cd þ and cd þ t cells role of cd þ and cd þ t cells in mouse hepatitis virus infection in mice antibody prevents virus reactivation within the central nervous system mouse hepatitis virus enterotropic mouse hepatitis virus infection in nude mice persistent transmission of mouse hepatitis virus by transgenic mice duration of challenge immunity to coronavirus jhm in mice virus strain specificity of challenge immunity to coronavirus duration and strain-specificity of immunity to enterotropic mouse hepatitis virus passively acquired challenge immunity to enterotropic coronavirus in mice epizootic coronaviral typhlocolitis in suckling mice isolation of mouse hepatitis virus from infant mice with fatal diarrhea thymus involution induced by mouse hepatitis virus a in balb/c mice adverse effects of mouse hepatitis virus on ascites myeloma passage in the balb/ej mouse murine hepatitis virus strain produces a clinically relevant model of severe acute respiratory syndrome in a/j mice tolllike receptor deficiency increases disease and mortality after mouse hepatitis virus type infection of susceptible c h mice granulomatous peritonitis and pleuritis in interferon-gamma gene knockout mice naturally infected with mouse hepatitis virus pathogenesis of enterotropic mouse hepatitis virus in immunocompetent and immunodeficient mice vertical transmission of mouse hepatitis virus infection in mice tissue distribution and duration of mouse hepatitis virus in naturally infected immunocompetent icr (cd- ) and immunodeficient athymic nudenu mouse strains used for ovarian transplantation and in vitro fertilization rederivation of inbred strains of mice by means of embryo transfer risk assessment of mouse hepatitis virus infection via in vitro fertilization and embryo transfer by the use of zona-intact and laser-microdissected oocytes mouse hepatitis virus immunofluorescence in formalin-or bouin's-fixed tissues using trypsin digestion comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses monoclonal antibody solution hybridization assay for detection of mouse hepatitis virus infection detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection detection of rodent coronaviruses by use of fluorogenic reverse transcriptase-polymerase chain reaction analysis an immunofluorescence test for detection of serum antibody to rodent coronaviruses simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay differences in antibody production against mouse hepatitis virus (mhv) among mouse strains maternally-derived passive immunity to enterotropic mouse hepatitis virus mouse hepatitis virus: molecular biology and implications for pathogenesis maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus replication of murine coronaviruses in mouse embryonic stem cell lines. in vitro reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus efficacy of three microbiological monitoring methods in a ventilated cage rack rederivation of mhv and mev antibody positive mice by cross-fostering and use of the microisolator caging system elimination of mouse hepatitis virus from a breeding colony by temporary cessation of breeding evolution of mouse hepatitis virus (mhv) during chronic infection: quasispecies nature of the persisting mhv rna the elimination of mouse hepatitis virus by temporary transplantation of human tumors from infected athymic nude mice into athymic nude rats (rnun/rnun) development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase pcr assay to detect murine norovirus infection in mice stat -dependent innate immunity to a norwalk-like virus murine norovirus infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat -dependent interferon responses replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages persistent infection with and serologic cross-reactivity of three novel murine noroviruses murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence molecular detection of murine norovirus from experimentally and spontaneously infected mice naturally occurring murine norovirus infection in a large research institution soiled-bedding sentinel detection of murine norovirus the use of cross-foster rederivation to eliminate murine norovirus, helicobacter spp., and murine hepatitis virus from a mouse colony impact of murine norovirus on a mouse model of ibd virus-plus-susceptibility gene interaction determines crohn's disease gene atg l phenotypes in intestine murine norovirus: an intercurrent variable in a mouse model of bacteria-induced inflammatory bowel disease investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection effects of murine norovirus infection on a mouse model of diet-induced obesity and insulin resistance mouse adenovirus, k virus, and pneumonia virus of mice sendai virus and pneumonia virus of mice (pvm) pneumonia virus of mice: severe respiratory infection in a natural host pneumonia virus of mice infection, lung, mouse, and rat persistence of pneumonia virus of mice and sendai virus in germ-free (nu/nu) mice fatal pneumonia with terminal emaciation in nude mice caused by pneumonia virus of mice respiratory disease and wasting in athymic mice infected with pneumonia virus of mice pathogenesis of pneumovirus infections in mice: detection of pneumonia virus of mice and human respiratory syncytial virus mrna in lungs of infected mice by in situ hybridization differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains experimental pneumovirus infections: . hydrocephalus of mice due to infection with pneumonia virus of mice (pvm) detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis lethal exacerbation of pneumocystis murina. pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice handbook of animal models of infection mouse rotavirus murine rotavirus infection, mouse successful sanitation of an edim-infected mouse colony by breeding cessation role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhoea age-dependent diarrhoea induced by a rotaviral nonstructural glycoprotein the immunology of rotavirus infection in the mouse murine model of rotavirus infection evidence that resolution of rotavirus infection in mice is due to both cd and cd celldependent activities ifn-l determines the intestinal epithelial antiviral host defense persistent rotavirus infection in mice with severe combined immunodeficiency role of b cells and cytotoxic t lymphocytes in clearance of and immunity to rotavirus infection in mice comparison of methods for detection of serum antibody to murine rotavirus identification of nonspecific reactions in laboratory rodent specimens tested by rotazyme rotavirus elisa removal of inhibitory substances from human fecal specimens for detection of group a rotaviruses by reverse transcriptase and polymerase chain reactions synergistic rotavirus and escherichia coli diarrhoeal infection of mice infection of rabbits with sendai virus pathogenesis of sendai virus infection in the syrian hamster determinants of organ tropism of sendai virus sendai virus infection, lung, mouse, and rat multigenic control of resistance to sendai virus infection in mice naturally-occurring sendai virus infection of athymic nude mice signs and lesions of experimental sendai virus infection in two genetically distinct strains of scid/beige mice cooperation between cytotoxic and helper t lymphocytes in protection against lethal sendai virus infection. protection by t cells is mhc-restricted and mhc-regulated; a model for mhc-disease associations delayed clearance of sendai virus in mice lacking class i mhc-restricted cd þ t cells naturally occurring sendai virus disease of mice affinity of sendai virus for the inner ear of mice detection of nucleoprotein gene of sendai virus in the lungs of rats by touchdown nested reverse transcription polymerase chain reaction the effectiveness of a microisolator cage system and sentinel mice for controlling and detecting mhv and sendai virus infections the efficacy of a dirty bedding sentinel system for detecting sendai virus infection in mice: a comparison of clinical signs and seroconversion serological evidence that mus musculus is the natural host of theiler's murine encephalomyelitis virus comparison of mhg virus with mouse encephalomyelitis viruses genetic analysis of a theiler-like virus isolated from rats a serological indication of the existence of a guineapig poliovirus duration and patterns of transmission of theiler's mouse encephalomyelitis virus infection a spontaneous outbreak of theiler's encephalomyelitis in a colony of severe combined immunodeficient mice in the uk axonal loss results in spinal cord atrophy, electrophysiological abnormalities and neurological deficits following demyelination in a chronic inflammatory model of multiple sclerosis the theiler's murine encephalomyelitis viruses susceptibility of inbred mice to chronic central nervous system infection by theiler's murine encephalomyelitis virus role of the humoral immune response in resistance to theiler's virus infection the genetics of the persistent infection and demyelinating disease caused by theiler's virus infection with theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via nf-kappab activation: potential role for the initiation of demyelinating disease innate immune response induced by theiler's murine encephalomyelitis virus infection cardioviruses: encephalomyocarditis virus and theiler's murine encephalomyelitis virus effect of immunization with theiler's virus on the course of demyelinating disease protection of sjl/j mice from demyelinating disease mediated by theiler's murine encephalomyelitis virus immunosuppression promotes cns remyelination in chronic virus-induced demyelinating disease pathogenesis of theiler's murine encephalomyelitis virus mechanism of theiler's virus-induced demyelination in nude mice survival of athymic (nu/nu) mice after theiler's murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody vacuolar neuronal degeneration in the ventral horns of scid mice in naturally occurring theiler's encephalomyelitis evolution of the placental barrier to fetal infection by murine enteroviruses enhanced detection of theiler's virus rna copy equivalents in the mouse central nervous system by real-time rt-pcr spontaneous demyelinating myelopathy in aging laboratory mice key: cord- -tj ye mx authors: nan title: abstract book date: - - journal: ann allergy asthma immunol doi: . /s - ( ) -x sha: doc_id: cord_uid: tj ye mx nan introduction: the effect of anti ige has been described as a function of complexing free ige to reduce mast cell implantation and consequent reduction of mast cell degranulation upon exposure to antigen. theoretically, as total free ige drops below ng/ml, improvement occurs as free and cell bound ige equilibrate and allergic reactions subside. initial observations ( togias a et al. j allergy clin immunol. ; :s )suggested that prick skin tests consistently reached their low point at days with significant reduction in size. laynadier (leynadier f, doudou o, gaouar h, le gros v, bourdeix i, guyomarch-cocco l, trunet p : effect of omalizumab in health care workers with occupational latex allergy.j allergy clin immunol ; : - ) noted some reduction in skin testing in sixty percent of patients treated over a six month period with monoclonal antiige. but clinicians since approval of the drug in have noted that even in the presence of good clinical response to asthma, skin test reactivity is still obvious, and in many cases unchanged. methods: following the suggestions for standard clinical follow up by lanier and marshall ( annals may ) , routine skin prick skin testing was completed after informed consent on patients at onset of anti-ige therapy, and repeated at - months. in all instances were skin tests done and photographed in a standard manner. in a few cases, skin tests were repeated on patients receiving anti ige for as long as five years. results: photographic evidence will be presented. in of patients on current therapy, prick skin testing remained at an almost identical photographic level to the baseline analysis, but there was a correlation between the patients with the lowest serum ige and the likelihood of apparent reduction. there was no correlation to reduction of skin testing and clinical response to anti ige since all patients had experience significant quality of life improvements. conclusion: anti ige has variable effects on reducing prick skin testing, with a greater likely hood associated with low or extremely low initial total ige levels. skin testing is more sensitive for detection of allergen-specific ige than is immunoassay for allergen-specific ige. for some assay methods, the qualitative cutoff of . ku/l is inappropriately high. this study was conducted in order to determine whether a particular assay system can be used to measure lower levels of allergen-specific ige. the pharmacia unicap system was studied. all reagents were purchased from the manufacturer, and the study was approved by a local human assurance committee. to determine background of the calibration curve, the fluorescent unit (fu) response of replicates of assay diluent measured with an anti-ige solid phase was determined. the background responses of arbitrarily selected allergen solid phases (oak, timothy and short ragweed pollen; cat; peanut; yellow jacket venom; penicillin g) were also measured. lower detection limits (lld) were calculated using the mean background measurement + standard deviations (sd). to examine linearity of a low-range calibration curve, : dilutions to extinction were made, starting with the . ku/l calibrator. the mean background of the anti-ige solid phase was . fu, sd . and coefficient of variation (cv) of %; the lld was . this lld corresponded to a level of approximately . ku/l of ige. the lld of the allergen solid phases ranged from . fu (yellow jacket venom) to . fu (short ragweed); this corresponded to about . ku/l ige. the low level dilution curve exhibited parallelism with a curve constructed using a zero (diluent) calibrator as a th point in the assay's reference curve. a low level method is capable of measuring specific ige levels lower than the manufacturer's . ku/l cutoff. this would seem to be particularly important in research applications, and in the analysis of certain highrisk allergens. the clinical significance of very low levels of specific ige in the serum warrants study. allergists/immunologists are often consulted on patients with rashes or recurrent infections. the differential diagnosis can comprise a variety of disorders, some of which are rare syndromes that require early diagnosis and specific management. a mo old wm developed persistent rash and worsening eczema. later, he had recurrent severe skin infections, skin abscesses, recurrent sinusitis, and chronic otitis media that required multiple placement of tympanostomy tubes, yet had a persistent tympanic membrane perforation. recurrent wheezing was noted from - yr of age. he had several episodes of pneumonia since yr of age affecting different lobes. he failed to shed the primary teeth and had history of periodontitis and oral thrush. he developed scoliosis and multiple fractures of the upper extremities and ribs. at yr he had staphylococcal bacteremia and osteomyelitis of the acetabulum. in spite of antibiotics prophylaxis, he continued to have recurrent skin infections and lymphadenitis. laboratory evaluation revealed eosinophilia (up to /mm ), normal levels of igg, iga, igm & igg subclasses, except for decreased igg ( mg/dl). he had protective levels of anti s. pneumoniae and h. influenzae, but low anti-tetanus and anti-diphtheria titers. at yr, total ige level was , iu/ml which supported the diagnosis of hyper ige (hie) syndrome; it decreased to iu/ml by yr. rast was positive to hd mite, cat, dog, egg, milk, and pollens. flow cytomerty, nbt and phagocytic index were normal; ch was low ( mg/dl). dexa scan showed osteopenia. chest x-ray showed bilateral infiltrate, atelectasis and scoliosis of the thoracic spine. during his hospitalization at yr, his skin was fair, lichenified with widespread maculopapular erythematous rash. the palms and fingers showed open cracks and pustules, but no weeping lesions. in addition to coarse facial features, his face was eczematous. conclusion: hie syndrome is an autosomal dominant disorder that mimics atopic dermatitis but has severe course, multiple infections, and several complications. although a markedly elevated total ige level introduction: mannose-binding lectin (mbl) is a serum protein in the lectin complement pathway. it is important in innate immunity, and is thought to be particularly relevant in young children during the development of adaptive immunity. deficiency in mbl has been reported in population-based studies as a risk factor in children for infection and hospitalization. additionally, age-dependent variability of mbl has been previously noted. this study evaluates the prevalence of mbl deficiency in children with established recurrent infection who were referred for evaluation of immunodeficiency. method: we prospectively evaluated mbl status of children referred for recurrent infection. serum was collected from october to september . children with known primary or secondary immunodeficiencies were excluded. mbl analysis was performed by standardized elisa using mbl oligomer assays. we performed chart and laboratory review for comorbid diagnoses, quantitative immunoglobulin levels and subclasses, and complement function with ch and ah . results: two-hundred thirty five children were evaluated. mean age was . years (range . - years). mean mbl was ng/ml (range - ng/ml). thirty-one of children ( . %) were mbl deficient, levels < , and among this group the mean mbl level was . ng/ml (range - ng/ml). mean age in this group was . years (range . - years). of the children with mbl deficiency, ( . %) children had levels < . the m:f ratio of children with abnormal mbl levels was . . linear regression analysis showed no correlation of mbl level with age. comorbid diagnoses were variable, including asthma, allergy, and atopic dermatitis. none of the children had symptoms compatible with connective tissue disease. no child with an abnormal mbl level was found to have significant deficiency of igg, iga, or igm. several children had low igg , which were within normal physiologic range. no other concomitant complement pathway disorders were detected. conclusions: our study did not reveal any correlation between mbl level and age. in children with recurrent infections, mbl deficiency was approximately twice the estimated rate of the general population. contrary to previous studies, no other significant immunologic disorders were identified. therefore, mbl deficiency alone is a risk factor for infection in children. t.b. fausnight * , hershey, pa. introduction: the incidence of perioperative anaphylaxis is estimated to be between in , and in , procedures. approximately % of cases of perioperative anaphylaxis are attributed to neuromuscular agents. very little information is reported in the literature regarding pediatric perioperative anaphylaxis. i describe a pediatric patient with suspected perioperative anaphylaxis to rocuronium. methods: a year-old girl with a history of sacral agenesis and neurogenic bladder was scheduled to have bladder augmentation surgery. the patient was taken to a latex-free operating room. during induction of general anesthesia, she was found to be difficult to ventilate. she also became hypotensive. examination of the patient revealed urticaria on her right arm. she was given epinephrine, diphenhydramine, and dexamethasone. the procedure was aborted. the reaction was believed to be from either rocuronium or propofol. results: because of the high incidence of anaphylaxis to neuromuscular agents, allergy skin testing was performed for rocuronium, vecuronium, and succinylcholine. the patient had negative percutaneous skin tests ( : ) for rocuronium, vecuronium, and succinylcholine. she had a negative intradermal skin test to rocuronium at the : dilution. she had a positive intradermal skin test to rocuronium at the : dilution. she had negative intradermal skin tests to both vecuronium and succinylcholine at the : , : , and : dilutions. she underwent surgery weeks after skin testing. she received a test dose of vecuronium and had no reaction. she received doses of vecuronium and multiple doses of morphine without any adverse reactions. propofol was avoided. the surgery was completed without difficulty conclusions: this case illustrates the usefulness of skin testing for neuromuscular agents in a pediatric patient. introduction: extrapulmonary pneumocystis jiroveci infection is rare in non-hiv infected individuals and, to our knowledge, it has not been reported before in a patient with good's syndrome. we report a case of extrapulmonary pneumocystis in a patient with good's syndrome. case report: a -year-old african american male patient with a history of good's syndrome presented with left flank pain of months duration. the pain was constant and associated with night sweats, fever, and chills. furthermore, the patient also reported a -pound weight loss. on exam, patient was found to have multiple hypopigmented areas over his abdomen and left lower extremity, bitemporal wasting, sunken eyeballs, and oral thrush. the patient was also found to have left costovertebral angle tenderness with a palpable solid fixed mass along the left mid axillary line overlying the splenic area. computedtomography (ct) scan of the chest and abdomen showed a . x . cm soft tissue mass at the left lateral aspect of the t vertebral body and . x . cm soft tissue parasplenic mass. the parasplenic mass involved the inferior anterior aspect of the left th and th ribs (see figure) . the giemsastained biopsy of the parasplenic mass revealed pneumocystis jiroveci and was confirmed using immunohistochemical stain with monoclonal antipnumocystis antibodies. the patient's symptoms of night sweats and loss of appetite improved within hours following initiation of trimethoprim-sulfamethoxazole therapy. a ct of the chest and abdomen, repeated six months post treatment, confirmed the resolution of both the paravertebral and the parasplenic masses. conclusion: to our knowledge, this is the first case of extrapulmonary pneumocystis presenting in a patient with good's syndrome. although it is rare, extrapulmonary pneumocystis should be considered in the differential diagnosis of patients with good's syndrome and chest or abdominal mass. introduction: t cells are regulated by cellular interactions with the environment during development. they play a critical role in the regulation and development of autoimmune diseases. we report inflammatory myositis in a -year-old caucasian female with primary t cell-deficiency since infancy. methods: at nine months of age, our patient developed lymphoid interstitial pneumonitis. immunologic evaluation revealed isolated t-cell deficiency. during the first years of life, she had failure to thrive, recurrent sinusitis, oral candidiasis, fungal skin infections, and pseudomonas pneumonia. at years, autoimmune conditions, characterized by a purpuric rash over the nose; face and exposure area of elbows; knees and fingers; raynaud's phenomenon; and nasal septum perforation, developed. inflammatory markers were persistently elevated and autoantibodies detected. by age , she developed proximal muscle weakness with distal joint contractures. dry gangrene of the fingers from a thrombotic incident occurred. results: serial immunologic evaluation revealed low cd - %, # - (normal - %, # - cell/mm ), low cd - %, # - (normal - %, # - cell/mm ) , decreased lymphocyte transformation to antigens but appropriate to mitogens. igg - (normal - mg/dl); iga - (normal - mg/dl), and igm - , (normal - mg/dl) with appropriate antibody responses to vaccine antigens. bone marrow and thymus biopsies were normal. further evaluation was not consistent with known primary or secondary immunodeficiencies. several skin biopsies revealed nonspecific dermatitis without vasculitis. mri of lower extremities showed abnormal signals involving bilateral muscles of thighs and calves. muscle biopsy revealed inflammatory myositis with plasma cell predominance, inconsistent with dermatomyositis or polymyositis. aldolase - iu/l (< ), crp - mg/dl (< . ), esr - (< ), rf : (< : ) and type ii collagen ab - ei/ml (< ) . ana, anca and ace were within normal range. antibodies for myositis and myositis related-antibodies were negative. diagnosis of nonspecific plasma-cell inflammatory myositis was made. a trial of monthly ivig gm/kg resulted in clinical improvement. conclusion: this novel plasma cell myositis occurring in a child with primary t cell deficiency underscores the role t cells play in the development of autoimmune diseases. introduction the incidence of hypersensitivity reactions (hr) is increased in patients treated with multiple courses of carboplatin. the purposes of this investigation were to evaluate the effectiveness of a -hour, -step desensitization protocol and to characterize the immune mechanism of carboplatin hr. methods we analyzed ten consecutive patients over a two year period with documented hr to carboplatin who required continued treatment with a platinum agent. the patients were treated with carboplatin using a -hour, -step desensitization protocol. skin tests were performed on five patients. results ten patients successfully completed planned courses of desensitizations to carboplatin, of which were without reactions. four patients had symptoms during their first (n= ) and third (n= ) desensitizations but tolerated the re-administration of infusions without further reactions. for subsequent courses, the protocol was modified for two patients who had extracutaneous symptoms during desensitization and was unchanged for the patient who had mild urticaria. these three patients tolerated subsequent courses of desensitizations without reactions. the fourth patient with symptoms during desensitization no longer required carboplatin. of the five patients who were skin tested to carboplatin, four had positive wheal and flare reactions. in one patient, the skin test response to carboplatin became negative after desensitization. conclusions the -hour, -step desensitization protocol is safe and effective for treating patients with carboplatin hr. positive skin tests to carboplatin suggest a mast cell/ige-mediated mechanism. conversion of the positive skin test to a negative response after desensitization supports antigen-specific mast cell desensitization. hypothesis: there is an association between food allergies and acid reflux in atopic adults study design: a retrospective chart review of patients was conducted. people who tested positive and people who tested negative for food allergy were included. the prevalence of gastroesophageal reflux disease (gerd) in the total group and each of the study arms was compared to population prevalence. methods: results of allergen specific ige tests (pharmacia immunocap, nj) run for food allergies were reviewed to help locate charts of atopic subjects, with positive, and with negative food allergy results. we reviewed charts for history of heartburn and acid regurgitation or the diagnosis of gerd, laryngopharyngeal reflux (lpr) or peptic ulcer disease (pud). population prevalence ( . %; ci . - . ) of acid reflux was estimated from a historical comparison that studied adults in a similar geographical location. results: in the food allergy positive group, . % of the subjects ( % ci . - . ) had either a history of heartburn and/or a diagnosis of gerd, lpr or pud. in the food allergy negative group, % ( % ci . - ) had acid related disorders. in the total study group of atopic subjects, the prevalence of heartburn, gerd, lpr or pud was . % ). on chi square analysis, the prevalence of acid reflux disorders was significantly higher in the total study group when compared with population prevalence (p= . ). the prevalence of acid reflux disorders between the food allergy study group and in the population shows no significant difference (p= . ). the prevalence of acid reflux disorders was significantly higher in the food allergy negative group when compared with the prevalence in the population (p= . ). there was no significant difference in the prevalence of acid reflux disorders between the two study groups with and without food allergy (p = . ). conclusions: the prevalence of acid reflux disorders was higher in people with food allergy when compared to the population, but missed statistical significance. patients without food allergy had a significantly higher prevalence of acid reflux disorders compared to the population. atopic individuals have a statistically significant higher prevalence of acid reflux disorders, but there is no statistically significant difference between atopics with and without food allergy. abstract background: analysis of dispensing patterns of injectable epinephrine offers a method to study the characteristics of school children with allergic or anaphylactic reactions. objective: to analyze the demographics of children prescribed injectable epinephrine in massachusetts school districts with diverse racial and ethnic enrollment patterns. methods: school nurses in schools (grades pk- ) enrolling , students recorded the characteristics of students prescribed injectable epinephrine including the number and racial mix of students in each school, student age, grade level, race, sex, and allergic disorder requiring epinephrine. surveyed school districts were two predominately white ( %) suburban districts enrolling students and one urban area with a minority population of % enrolling , students. the use of epinephrine for peanut allergy was analyzed in detail. results: a total of of , ( . %) students in three school systems were dispensed injectable epinephrine. males outnumbered females ( to ). whites outnumbered non-whites to . two thirds (n= ) of children dispensed epinephrine had peanut allergy. the second most common allergy was stinging insect allergy (n= ). the rate of dispensed epinephrine for peanut allergy was lowest in the urban school district ( . %) versus the two suburban districts, . % and . % respectively. whites with peanut allergy outnumbered nonwhites to . males outnumbered females to . the lowest rate of prescribed injectable epinephrine for peanut allergy in all three school districts was found in non-white school children- . %. eighty-nine of ( %) school children with peanut allergy were enrolled in grades pk through . there were twice as many white versus non-white ( to ) school children in the urban system with prescribed injectable epinephrine for peanut allergy. only eight (. %) of hispanic and asian students in the were dispensed injectable epinephrine for peanut allergy. conclusions: this is the first study to suggest that there may be a racial disparity in the prevalence of childhood peanut allergy. one possible explanation for this disparity is that varied feeding practices in minority infants and children may induce a state of tolerance and lead to a lower incidence of peanut allergy. f.i. hsu * , h.j. burstein, m.c. castells, boston, ma. introduction: trastuzumab is a humanized igg kappa monoclonal antibody specific for human epidermal growth factor receptor protein, her , used in the treatment of her /neu positive breast cancer. we present the first report of desensitization to trastuzumab in a patient with an ige-mediated reaction to trastuzumab and documented igg-anti-igg human antibodies. meth-ods: a year old woman with metastatic invasive ductal breast cancer (er/pr positive, her- strongly positive), unresponsive to conventional therapy, presented with an anaphylactic reaction to trastuzumab and was evaluated for rapid desensitization by skin testing and serum specific igg antibodies. the patient had previously received trastuzumab. results: the anaphylactic reaction to trastuzumab consisted of diffuse erythema, urticaria, respiratory distress and laryngeal edema. premedication with steroids, h and h blockade, and slow infusion did not prevent a subsequent reaction. skin prick testing with trastuzumab ( mg/ml) was positive with negative controls, confirming an ige-mediated mechanism. igg anti-human igg antibodies (haha) to trastuzumab were confirmed by elisa. the patient was desensitized to trastuzumab mg/kg with an intravenous protocol that started at . mcg/h, and increased in rate every minutes until a final rate of mg/h, for a total infusion time of to hours. premedication included diphenhydramine, prednisone, famotidine, and montelukast. desensitization was confirmed by negative skin prick and intradermal tests ( mg/ml and mg/ml), with positive histamine control. the patient tolerated a total of weekly doses of trastuzumab mg/kg. intradermal skin testing prior to her th and th courses showed reactivity, indicating resensitization. serum levels of tratuzumab were undetectable at week after desensitization. conclusion: allergic reactions to trastuzumab are rare but can include anaphylaxis. in patients with documented haha and/or ige-mediated reactions to trastuzumab and clinical symptoms of type i/mast cell mediator-related symptoms, desensitization can allow continued administration of this treatment by providing tolerance. the clinical effectiveness of desensitizations in patients with ige and haha antibodies remains to be defined. in contrast to reports in the medical literature, details of the medical aspects were limited. there were attacks on infants bringing the total to reported infant attacks to date. two of the infants suffered long term morbidity and died. like those reported in the medical literature, the majority of adults were in long term care facilities, although were in hospitals. overall, of the individuals stung died within one week of stings. no significant medical consequences of stings were reported in of of the newspaper reports as opposed to of reports in the medical literature. conclusion: our data suggest that increasing numbers of fire ant attacks are occurring in medical facilities, where chronically ill, frequently immobile patients come in contact with foraging ants. unattended infants in private homes in fire ant endemic areas also appear at risk. the factors that determine why individuals are stung and the severity of injury after attacks remain uncertain. the presence of fire ants inside health care facilities and homes is a harbinger for fire ant attacks of disabled or infant occupants. we hypothesize that morbidity is determined by the number of stings, the condition of the patient and the type of treatment administered. purpose: to determine if gender confers a risk for positive penicillin (pcn) skin test. method: rates of positive pcn skin tests, according to gender, were determined in patients with a history of pcn allergy undergoing an allergy pre-operative evaluation from june to june . during this period, , patients were seen in the pre-operative evaluation clinic in which had a history of pcn allergy and comprise our study population. a univariate logistic regression analysis was employed to calculate the odds ratio (or) and the % confidence interval (ci) for gender differences in the rates of positive pcn skin test and a multivariate logistic regression analysis was used to adjust for age and history of multiple drug allergies. p-value of . or less was considered statistically significant. results: of the patients, underwent skin testing for pcn, patients did not, and charts were not available for review. the mean age of the study group was years. sixtyfour ( . %) patients had a positive skin test to pcn. of these, ( %) were females and ( %) were males (or . , % ci . - . , p = . ). of those with a negative/equivocal pcn skin test, ( %) were females versus ( %) males. in a multivariate logistic regression analysis adjusted for age and history of multiple drug allergies, female gender again was more likely to have a positive pcn skin test (or . , % ci . - . p < . ). patients did not undergo pcn skin testing, ( %) were male and ( %) were female. conclusion: this is the first report showing that a greater risk for a positive skin test to pcn exists in association with female gender. age and a history of multiple drug allergies are unlikely to be confounding factors to the observed gender risk. further studies are needed to identify other possible confounding factors and/or mechanisms that can explain this risk. a. fiocchi * , p.a. restani , s. cucchiara , g. lombardi , g. magazzu' , g.l. marseglia , k. pittschieler , s. tripodi , r. troncone , a. vierucci , . milan, italy; . roma, italy; . pescara, italy; . messina, italy; . pavia, italy; . bolzano, italy; . napoli, italy; . firenze, italy. background: cow milk substitutes for children allergic to cow milk proteins (cmp) include soy-based formula and cow s milk hydrolysates. neither can rule out a sensitisation risk. objective: prospective assessment of clinical tolerance to a rice-based hydrolysate formula by children allergic to cow milk proteins (cmp) who consume rice openly. patients and methods: ninety-seven children aged to months with immediate reactions to cow milk confirmed during dbpcfc were assessed for clinical tolerance to cow milk by spt with whole milk, -lactalbumin (ala), -lactoglobulin (blg), -and -casein ( -cas, -cas) (sigma chemical, st. louis, mo). whole milk, ala, blg and cas specific ige determinations were performed using cap test (pharmacia, uppsala, sweden) . similarly, sensitisation to rice and hrf were investigated by spt and cap test. patients sera were investigated by immunoblotting for cmp, rice and hrf (heinz-plada, milan, italy). dbpcfc was carried our with g rhf powder masked in neocate tm (equivalent to ml reconstituted formula). results: spt: all patients were positive to cow s milk and/or cmp fractions (> mm wheal diameter). ige determinations gave positive results with cow milk and/or cmp fractions ( / patients tested), rice ( / ) and with hrf ( / ). immunoblots (n= ) were positive for -cas (n= ), -cas (n= ), ala (n= ), blg (n= ) and bovine serum albumin (n= ). similarly, although patients sera largely recognized rice ( / ), only one weakly reacted with rhf. challenge with rhf was negative in all cases. conclusions: we conclude that, despite their frequent sensitisation to rice, children with cma tolerate both rice and rhf clinically. hydrolyzed rice formulas may thus represent an alternative protein source for children with cma. r.c. cartwright * , w.k. dolen, augusta, ga. introduction: conventional treatment of human seminal fluid allergy includes abstinence, barrier protection, or subcutaneous immunotherapy with fractionated seminal fluid. treatment using local intravaginal desensitization with unfractionated seminal fluid has recently been described, but experience with this desensitization method is still limited and little has been reported as to its long-term results. methods: a -year-old woman with a history of allergic rhinitis and asthma experienced anaphylaxis following her first unprotected intercourse since the birth of her first child. ige-mediated sensitization was demonstrated through the use of specific ige testing (pharmacia cap system) and skin prick testing using undiluted seminal fluid obtained from the patient's husband. after approval from the human assurance committee, the patient underwent rush intravaginal desensitization with steadily increasing concentrations of seminal fluid, beginning with a : , v/v concen-tration. results: her specific ige level to human seminal fluid was . ku/l. skin prick testing was positive with a wheal of mm diameter with pseudopods and a flare of mm diameter. desensitization was successful and the patient tolerated local application of whole semen without significant reaction. following desensitization, the patient reported that she and her husband engaged in unprotected intercourse without local or systemic symptoms. over the last year since the desensitization, she has not experienced further anaphylaxis, but she has developed local symptoms if her exposure to seminal fluid was delayed past days. the longest time period between exposures has been weeks. she became pregnant months ago and has not had any pregnancy complications. conclusions: local intravaginal desensitization was a safe and effective treatment for human seminal fluid allergy in this patient. a delay in seminal fluid exposure greater than days was associated with the return of symptoms emphasizing the need for frequent seminal fluid exposure to maintain desensitization. l. terracciano, t. sarratud, a. fiocchi * , p. restani, s. guerci, milan, italy. background kiwifruit allergy in children has been seldom reported and the reactions observed are usually mild. anaphylaxis has not been described. we document the case of an infant who developed anaphylacic symptoms within minutes of his mother eating two kiwifruits and initiating breastfeeding. case history in his fourth month, the boy was admitted into an emergency department with dysphonia, breathing difficulties, generalised urticaria and angioedema of the lips and face. this episode was treated as an anaphylactic reaction with epinephrine, chlorpheniramine and hydrocortisone sodium succinate administered via a percutaneous catheter and symptomatic control was achieved within minutes. after days a second anaphylactic episode of similar severity occurred under the same circumstances. neither episode required critical care. the boy presented two months later for clinical evaluation in our paediatric allergy unit. skin prick tests (spt) were positive only with egg white and yolk while negative to cow s milk (and protein fractions), beef, chicken, pork, codfish, rice, wheat, soybean, maize, potato, carrot, tomato, bean, pea, celery, peanut, dermatophagoides pteronyssinus and d. farinae, grass and banana. spt with kiwifruit was specifically ordered because exquisite contact was suspected, and induced a positive wheal (> mm diameter). positive specific ige determinations (cap-feia from pharmacia, sweden) with kiwifruit ( . ku/l), cat dander ( . ku/l) and egg white ( . ku/l) were returned but determinations with other inhalant and food allergens were all below the cut-off point of . ku/l and total ige levels were ku/l. strict avoidance of kiwifruit by the breastfeeding mother proved effective and nothing untoward happened in the intervening year. currently aged . years, the boy is free from food-related symptoms. prick-byprick tests with native allergens and spt carried out with commercial extracts of allergens associated in the literature with kiwifruit allergy were all negative. comment there are no reports of immediate reactions to kiwifruit under two years. in this case, severe reactions via breastmilk in an infant monosensitised to kiwifruit indicate that nursing may represent a hidden source of exposure. in older children monosensitisation without prior sensitisation to pollen has been described as the major risk associated with severity of symptoms. m. sikora * , j.w. sleasman, n. tangsinmankong, st. petersburg, fl. introduction: treacher-collins syndrome (tcs) is an autosomal dominant disorder with an abnormality of craniofacial development during early embryogenesis. there is a known relationship between new bone formation and development of lymphocytes and cytokines. the association of tcs and humoral immunodeficiency has not yet been reported. we present a novel case of a year-old caucasian female patient with tcs and common variable immunodeficiency. methods: our patient presented with midface hypoplasia, micrognathia, microtia, conductive hearing loss and cleft palate with a history of recurrent upper and lower respiratory infections. patient had a -year history of chronic sinusitis, recurrent otitis media and pneumoniae which resulted in bronchiectasis. haemophilus influenza and staphylococcus aureus were persistently isolated from bronchial fluids. laboratory work-up for immunodeficiency was initiated. results: immunoglobulin analysis revealed low igg ( - mg/dl); low iga ( - mg/dl); igm ( - mg/dl); igg ( - mg/dl); low igg ( - mg/dl); igg ( - mg/dl); and low igg < . ( - mg/dl). patient had no detectable response to protein-derived vaccines (diphtheria and tetanus) and to polysaccharide-derived vaccines (pneumococcal) at baseline and at - weeks after immunization. t and b lymphocyte enumeration, ch , ah , and mannose-binding lectin were all within normal limits. laboratory findings were consistent with the diagnosis of common variable immunodeficiency. patient began receiving monthly doses of mg/kg/dose of intravenous immunoglobulin resulting in clinical improvement. our patient no longer requires antibiotic therapy for her respiratory infections; pulmonary function has improved and bronchiectasis resolved months after initiation of ivig. conclusion: our finding shows that tcs can be associated with common variable immunodeficiency which suggests a link between skeletal dysplasia and immunodeficiency. a.s. hartel * , j.w. sleasman, n. tangsinmankong, st. petersburg, fl. introduction: streptococcus pneumoniae is the most common cause of invasive bacterial infection in humans. however, incidence of s. pneumoniae infections in healthy adolescents is low ( / , per year). mannose-binding lectin (mbl) is a c-type lectin which plays a central role in the innate immune response by activating the classical complement pathway and acting as an opsonin by binding c q receptors. several studies have demonstrated an association between invasive bacterial infections, including s. pneumoniae and a homozygous mutation of the mbl gene. however, this association has not previously been described in patients with the heterozygous mutation. methods: a -year-old caucasian female presented with a second episode of meningitis over a -year span. streptococcus pneumoniae was isolated from peripheral blood cultures on both occasions. lumbar puncture revealed wbc , ; % bands; % pmns; glucose mg/dl, protein mg/dl; these results consistent with bacterial meningitis. extensive evaluation for predisposing causes revealed benign arnold-chiari type malformation. further immunological evaluation was initiated. results: evaluation revealed igg mg/dl (normal - ); iga ( - ); igm ( - ), and normal igg subclasses. antibody responses to diphtheria and tetanus vaccines were adequate. antibodies response to pneumococcal vaccine given years prior were protective to all common serotype tests (> . mcg/ml). lymphocyte subset analysis was within normal range. howell-jolly bodies were not detected from peripheral blood smear. evaluation for secondary immunodeficiency was negative, including hiv antibody by elisa. complement analysis showed ch u/ml ( - ); ah % ( - %), and markedly low mbl of ng/ml (> ). genetic analysis of her mbl revealed heterozygous mutation in codon and mutations in two promoter regions (homozygous mutation on h/l variants and heterozygous mutation on p/q variants). conclusion: heterozygous mutation of mbl codon when associated with mutation of promoter regions can result in severe impairment of mbl protein production, and may lead to increased susceptibility to invasive bacterial infections and meningitis. rationale: patients with similar symptoms may have allergic, non-allergic, or mixed rhinitis triggers and can differentially respond to distinct medications. we assessed st in our clinic patient population to characterize factors that influence appropriate diagnosis and treatment of rhinitis type. methods: we used a validated commercial questionnaire and chart review of patients seen in an academic allergy clinic, to assess the number of allergic vs. irritant st and demographic and historical factors (including pharmacotherapy) associated with differences in symptoms. results: the population (n= : female, male) consisted of individuals with a mixed rhinitis history. women had higher st scores than men, including: total scores [ . +/- . vs. . +/- . , p= . (unpaired t-test)]: allergen scores ( . +/- . vs. . +/- . , p= . ); and irritant scores ( . +/- . vs. . +/- . , p= . ). further, patients treated with both azelastine (az) and intranasal steroids (ins) had lower st scores than patients treated with either alone (total st: az . +/- . , ins . +/- . , both . +/- . , p= . ; allergen st: az . +/- . , ins . +/- . , both . +/- . , p= . ; irritant st: no significant difference). conclusions: female patients have significantly higher symptom scores; total, allergic and irritant. rhinitis monotherapy (ins or az) is minimally effective in reducing st but is more effective when used in combination. these data demonstrate rhinitis population heterogeneity and address differential responsiveness to similar medications. increased response to combined drug therapy support the heterogeneous pathophysiology of mixed rhinitis features and may relate to a combination of multiple aeroallergen and air pollution exposure in the houston area. introduction: interaction between eye & nose warrants attention with regard to the propagation and treatment of allergic reactions. the role of topical therapy for rhinitis and conjunctivitis is becoming more widely considered and questions of therapeutic route have been raised. purpose: to elucidate the anatomic and pharmacokinetic interactions of conjunctival & nasal mucosa leading to more rational selection of routes of medication administration. methods:our studies have investigated the effect of ocularly instilled allergen inducing signs and symptoms of rhinoconjunctivitis. study compared effects of allergen administered via conjunctival allergen challenge (cac) or nasal allergen challenge (nac) and analyzed tear & nasal secretions for ecp & tryptase. studies have also used the ability of the cac model to induce rhinitis signs & symptoms to evaluate the relative effects of medication routes: ) ocular v. nasal spray v. systemic; ) ocular + nasal spray v. systemic + nasal spray; ) ocular v. placebo. results: the nac/cac study revealed that, following cac (n= ), significant ocular and nasal signs & symptoms were noted; following nac (n= ), only nasal symptoms were clinically significant. nasal symptom scores between cac & nac differed significantly at timepoint, representing lag in allergen & mediator movement from eye to nose. measurable levels of ecp & tryptase were found in tears and nasal secretions for cac, but only in nasal secretions (with exception of subject) for nac. in cac studies: ) ocular therapy exhibited greater efficacy in ocular itching relief (n= ;p< . ) and was not significantly different from nasal or systemic therapy in nasal symptom relief. ) eyedrop+nasal spray combination exhibited significantly greater prevention of overall rhinoconjunctivitis signs and symptoms than nasal+systemic therapy (n= ). ) ocular therapy offered greater protection from nasal signs & symptoms compared to placebo (n= ;p< . ). conclusion: nac and cac results support the unidirectional flow from eye to nose, the ability of cac to yield nasal symptoms, and the efficacy of topical therapy. tearing, due to inflammation of the inferior turbinate would be the only ocular symptom resulting from nasal challenge. these results offer insight to the nature of the connection between ocular and nasal mucosa and the efficacy of varied medication administration routes for management of rhinoconjunctivitis symptoms. introduction: asthma disease management programs frequently target high utilizers because they are responsible for a large amount of asthma costs. once identified, such "frequent fliers" usually are offered interventions including case management. programs using this approach usually demonstrate reductions in utilization. though the interventions may cause this decline, it could also be due to regression to the mean (rtm) which is a tendency for outliers to become more like the mean over time. in this study we measured rtm in a medicaid hmo asthma population to determine whether targeted case management is effective independent of this phenomenon. we hypothesized that directed case management provides additional utilization reduction beyond rtm. methods: a weighted asthma utilization score was determined quarterly for members of an hmo with asthma from to . rtm was measured by determining how many patients were persistent high utilizers quarterly for year after baseline. to determine the effect of utilization-directed case management, the number of frequent fliers at baseline for each quarter also was determined. results: a total of asthma frequent fliers were identified on january , . by september , only of these individuals continued to be frequent fliers. similar decreases in utilization were seen for frequent fliers identified at the beginning of each quarter of . the mean decrease in the number of frequent fliers is shown in the table. this represents a substantial regression to the mean. the total number of high utilizers at baseline decreased by % after implementation of utilization-directed case management in independent of regression to the mean. this also represented a decrease from . % of health plan members with asthma to . % by the start of suggesting that the decline is not a result of diminishing health plan membership. the benefit appears to be the result of early intervention with members before they become frequent fliers. conclusions: utilization-directed case management can reduce overall asthma utilization by preventing members from becoming high utilizers at an early stage. programs that claim to intervene with plan members who already are high utilizers are likely to be taking advantage of regression to the mean. studies indicate a high incidence of asthma(as) among school-aged children. with the prevalence increasing, significant numbers remain unidentified. they experience morbidity, including school absenteeism, which may be preventable, in part, by adherence to national asthma guidelines. we implemented a pilot study to detect as, as control and initiation of appropriate health care among th grade students in a suburban population. the study was coordinated with the middle school staff and the local county health department. the screen parameters included: student questionnaires, peak flows, exercise with peak flow assessment (frast) , and spirometry as indicated. environmental tobacco smoke (ets) was recorded. results: students screened. no as history and negative screen (d). as history and negative screen (e). as history and positive screen (a). no as history with suggestive written screen and negative screen on exercise (b). no as history and positive screen (c). ets in groups a, b, c: %, %, %. ets in groups d, e: %, %. individual student results were mailed home with medical follow-up recommended for a positive screen. parents of the children who failed the questionnaire or exercise screen (groups a, b, c) were phoned at a - wk interval. no student in-group a or b had medical follow-up. ( %) students in group c had medical follow-up. conclusion: this as screen of th grade students identified ( %) as having significant, undetected as. ( %) with known as had uncontrolled as at the time of screen. ( . %) students with strong suspicion of as based on questionnaire had an acceptable exercise screen. only of the children with a screen suggestive of as or uncontrolled as had medical follow-up. ets was increased in the homes of students with a positive screen and remains a serious health issue. school screening for as has merit, but mail and phone follow-up was insufficient to intervene or initiate acceptable as treatment. references . redline s. et. al. development and validation of school-based asthma and allergy screening instruments for parents and students. ann allergy asthma immunol. ; : - . tsanakas.j.n., et al. free running asthma screening test. arch diseases in childhood. ; : - . american college of allergy, asthma, and clinical immunology screening test ages ( - ) acaai.org. introduction. some laboratory evidence suggests that desloratadine is effective in inhibiting of inflammatory mediators, which play an important role not only in allergic but also in virally induced inflammation. the purpose of this study was to assess its efficacy in acute bronchiolitis developed in young children suffering from concomitant atopic dermatitis (ad). meth-ods. participants were young boys and gilrs aged - who suffered from acute bronchiolitis as established by wheezing, rhinitis and fever. all patients also had concomitant ad as established by hanifin and rafka criteria. patients were allocated to receive syrup formulation of desloratadine (erius, schering plough) , mg/day for days (active group n= ) or no desloratadine treatment (control group n= ) using quota allocation system. we calculated physical global symptom score (combination of cough, wheezing, chest retractions, nasal flaring, blocked nose, runny nose, sore throat - - scale) and respiratory distress assessment instrument (rdai) to examine the patient at baseline and on day th after therapy was initiated, day of normalization of body temperature, respiratory rate, heart beat rate, and use of albuterol. results. both group of patients were comparable on age, gender, family history of asthma, time of onset of the acute respiratory illness, extent of medication taken prior to entering the study, global symptom score and rdai index. all children were evenly treated with oxygen, oral theophylline ( mg/kg/day) and adequately rehydrated. on day th global symptom score of patients receiving desloratadine was , ± , vs , ± , in control group (p= . ). on day th it was also significant difference between active and control groups on rdai index ( , ± , vs , ± , ; p= . ) especially for significant drop in wheezing score in active group. day when respiratory/heart rate normalized, temperature returned to normal, use of albuterol did not differ significantly in these groups. conclusions. our preliminary study is the first to show that desloratadine is an effective agent in viral lower respiratory tract infections of young children suffering from ad. it encourages further gcp trials to explore action of desloratadine in infantile bronchiolitis and concomitant ad. h.j. su * , w.t. lin, p.j. tsai, c.y. huang, p.c. wu, tainan, taiwan. increasing prevalence of childhood asthma has been observed across the world, and found to be associated with, partly, indoor pollution, including bioaerosol exposure. meanwhile, adequate ventilation is shown to be effective in diluting most indoor air pollutants, while only limited data are available addressing directly how the ventilation rate is implicated with childhood respiratory symptoms. this study aimed to examine the concentration distribution of selected indoor air pollutants, including bioaerosols, in domestic environment, and further to assess the effects of ventilation rate, characterized by a co trace gas concentration decay method, on concentration variations of the above-mentioned air pollutants. study subjects were chosen from a prior city-wide questionnaire survey based on positive response to inquiry for physician-diagnosed asthmatic status and wheezing symptoms in the past months. environmental assessments, including ventilation and air quality measurements, were conducted twice, in early winter and the other on early summer. respiratory health diary and pefr (peak expiratory flow rate) were recorded for week concurrent with the sampling activity. increasing ventilation rate is statistically associated with decreasing indoor concentrations of co and tvocs. after adjustment for sex, age, and selected housing characteristics, the or between tvocs concentrations and reporting cough of study children is . , and . between co and nasal congestion. the or between indoor bacterial concentration and the morning pefr less % is . in the similar multivariate logistic regression for data collected from winter study. in summer, the only significant relationship is between ventilation rate and the morning pefr less % of study children, or= . , in a multivariate logistic regression. this study has identified less reporting of childhood respiratory symptoms, especially for coughing and the morning pefr less % are associated with increasing ventilation rate, and higher levels of indoor air pollution appear to be present with greater frequency of the above symptoms. this study suggest quantitatively that proper management of ventilation efficiency may be beneficial for the control of childhood respiratory illnesses. *adjusted for: sex, age, environmental tobacco exposure, use of incense and air conditioner **:p< . ns:no statistical significance with whole model test background: moderate-to-severe allergic asthma can have a substantial impact on a patient's asthma-related quality of life (arql). in addition to asthma symptoms, allergic rhinitis, rhinosinusitis and other related comorbidities are often apparent in patients with more severe disease making it difficult to treat. despite guideline-consistent care, many patients still experience variability in asthma control signaling an unmet need within this population. omalizumab (xolair®) has recently demonstrated clinical efficacy and safety in treating asthma. objective: the aim of this paper is to summarize the arql outcomes associated with omalizumab therapy in moderate-to-severe allergic asthma. methods: we performed a systematic review of arql data from the clinical study reports and published clinical trials on omalizumab. arql was measured by the juniper-asthma quality of life questionnaire (aqlq). results: statistically significant results for arql endpoints consistently favored omalizumab over placebo. the magnitude of the changes in arql were consistently aligned with clinical endpoints. moderate to large effect sizes in the omalizumab groups were maintained throughout the -week clinical trial program and during the -week double-blind extension phase. however, the placebo groups also experienced within-group improvements and moderate effect sizes. a meta-analysis indicated a . to . fold increase in large (> . point) improvements in overall aqlq scores in the omalizumab-treated group compared with placebo during the stabilization and steroid-reduction phases of the clinical trials. conclusions: the consistently positive impact of omalizumab on arql outcomes during the clinical trial program provides evidence of its value as an adjunct therapy in patients with moderate-to-severe allergic asthma. significant differences and large effect sizes were observed despite the fact that the control group received active, guideline-consistent treatment producing a substantial placebo effect. improvements were observed in overall, symptom, activity, emotional and environmental dimensions of the aqlq indicating that omalizumab produced benefits in arql in patients with moderate-to-severe allergic asthma. background: omalizumab, a monoclonal anti-ige antibody, significantly improves asthma-related quality of life (arql) for patients with moderatesevere allergic asthma who express symptoms despite moderate-high inhaled corticosteroids (ics) doses. mean scores can mask underlying variability in arql outcomes. this investigation examined variability in outcomes to elucidate the specific impact of omalizumab treatment. methods: aqlq data (n= ) from two randomized, double-blind, placebo-controlled clinical trials were pooled to assess underlying variability in the mean scores and to identify key drivers of arql treatment-effect differences (juniper-asthma quality of life questionnaire (aqlq)) between omalizumab and placebo (active control) patients. results: correlations between aqlq and other clinical outcomes were low to moderate at best (r= . to r= . ). aqlq assessment captures patient benefit that supplements clinical outcome measures. across all component items of the aqlq patients receiving omalizumab improved more than patients receiving placebo (active control) (p< . ). omalizumab patients reported the greatest improvement for reducing waking with symptoms in the morning (symptoms domain: . vs. . ; p< . ), limitations in all activities done (activities domain: . vs. . ; p< . ), the fear of not having medication available (emotions domain: . vs. . ; p< . ), and symptoms from being exposed to dust (environment domain: . vs. . ; p< . ), compared to placebo (active control) patients. conclusion: arql assessment provides complementary and non-overlapping information on clinical benefit that is distinct from other clinical outcome measures. examination of underlying variability in aqlq mean scores and item-level response extend previously published results on omalizumab treatment effect by showing that aggregate arql improvements for omalizumab patients are strongly influenced by symptom and activity improvement. a. brimer , k. malhi, c. adams, c. dinakar, kansas city, mo. introduction: the desire to belong to a group is a very powerful motivator and is exceptionally strong in the adolescent population. asthma is a disease that makes people feel different. this perception may impinge on patient adherence with medication regimens. we hypothesize that belonging to a club where every member has asthma may encourage identification of the adolescent asthmatic with their peers, and mitigate the perception of being different. objectives: ( ) to investigate the factors that make the asthmatic adolescent feel different ( ) to explore the hypothesis that group activities, such as an asthma club, would help adolescent asthmatics feel less different. methods: as part of an ongoing survey, children with asthma between the ages of - years were offered an anonymous questionnaire in the primary care and adolescent clinics at our hospital. the questionnaire included both multiple-choice and open-ended questions designed to explore the feelings of the respondents. the responses were numerically tallied and reported as percentages. results: at the present time, surveys out of a proposed have been completed. one third of the youth with asthma answering the survey had negative feelings regarding their asthma. nearly forty percent of the respondents reported that their diagnosis made them feel different from their healthy peers. forty-five percent responded that they have felt restricted or excluded from school activities, athletics, and clubs due to their asthma. over one-third of them feel uncomfortable taking their inhaler in front of their friends. most respondents ( . %) indicated that they enjoy group activities. the majority of respondents ( . %) rated playing sports as their favorite group activity. conclusion: almost forty percent of asthmatic youth surveyed indicated that having asthma made them feel different and resulted in restriction/exclusion from school activities, athletics, and clubs. an overwhelming majority expressed a preference for participation in group activities, particularly recreational sports. this social preference towards group activities may be incorporated into an intervention, such as an asthma club, to help asthmatic youth adjust to the disease and its treatment regimen. introduction: airway hyperresponsiveness (ahr) is an exaggerated narrowing of the airways in response to stimuli, such as allergens, histamine, and cold air. ahr is a characteristic feature of asthma linked to chronic airway inflammation. phosphodiesterase (pde ) is an enzyme found in key inflammatory cells involved in the pathophysiology of asthma. inhibitors of pde prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde inhibitor, which has shown anti-inflammatory activity in vitro and in vivo. this study examined the effect of a single dose of roflumilast on changes in ahr following allergen-induced asthmatic reactions in patients with mild asthma. methods: this double-blind, randomized, crossover study consisted of treatment periods separated by a -to -week washout period. a total of patients (forced expiratory volume in one second [fev ] % predicted) who were hyperresponsive to histamine (provocative concentration causing a % drop in fev [pc fev ] mg/ml) were randomized to receive a single dose of oral roflumilast μg or placebo on day of each treatment period followed by an allergen challenge min after medication. histamine provocation was performed before and h after intake of study drug. results: there was no change in fev min after roflumilast administration, suggesting the absence of direct or acute bronchodilation. allergen challenge elicited early and late asthmatic reactions that were attenuated by a single dose of roflumilast μg. the histamine pc fev in patients treated with roflumilast decreased to a lesser extent than in those patients treated with placebo. the change in pc fev from baseline after challenge was . ± . mg/ml (mean ± sd) with placebo and . ± . mg/ml with roflumilast. the magnitude of the change in pc fev was statistically significantly different between the placebo and roflumilast treatment groups (p= . ). thus, roflumilast decreased the development of ahr by approximately . doubling-dilutions. conclusions: a single dose of oral roflumilast μg attenuated allergen-induced ahr. these data provide further evidence that roflumilast may provide anti-inflammatory activity in vivo and may be an effective treatment for patients with asthma. introduction for the past years we have been tracking pediatric asthma admissions and evaluations at the huntington memorial hospital. during the fall and winter months (oct-march) from to , we noted a . x increase in the number of asthma admissions and evaluations compared with the spring and summer (april -september; , patient encoun-annals of allergy, asthma & immunology ters in years). we have analyzed potential causes for this increase. diesel particulate was estimated in - , and a . -fold increase in the fall versus spring was found in burbank, a city adjacent to pasadena. pollutant particles from the combustion of fossil fuels may act as immuno-adjuvants to allergen exposure as has been demonstrated experimentally in mice and in human nasal exposure studies. methods computerized analysis of asthma admissions and evaluations were calculated according to established codes for acute and chronic reactive airways disease. infectious diseases in patients were analyzed by standard asthma questionnaire. also, pollen and mold counts, as well as diesel particulates and meteorological conditions were studied. results similar numbers of viral infections occurred in the fall of and spring of . among the most prevalent pollens and molds are chinese elm pollen (sept -oct) weed pollens (july -dec), and a newly recognized aureobasidium mold (nov -march). pollen grains can fragment and release respirablesized debris that are loaded with allergens (taylor et al (taylor et al , . the conidia of aureobasidium are also of respirable size and were identified by sequence analysis. fine particulate air pollution (pm . ), containing diesel particles, is increased in the fall and winter in southern california. con-clusions the incidence of asthma outbreaks may vary with air pollution levels in the immediate environment. pollen fragments and particles from fossil fuel combustion can deposit in similar regions of the lower airways. rainfall increases in the fall and winter and this coincides with increased molds and aureobasidium. aureobasidium was recently discovered in high concentrations in pasadena, is known to be allergenic and could be a factor in increased asthma. a mixture of pollens and molds and fine combustion particles may be relevant to this increased incidence of asthma in the fall and winter months in pasadena. background we have previously noted that tree and ragweed pollen grains can be airborne for most if not all daily periods during their respective seasons (aaaai, ) , (acaai, ) . because grass levels are lower than those of trees & weeds (aaaai / nab), we chose a longer daily interval to examine (six hours) that we had for trees (one hour) and ragweed (three hours). methods twenty-four hour microscope slide samples were collected using a burkard day air sampler from / / to / / . the slides were examined @ x along a single longitudinal traverse. the presence or absence of grass pollen was noted per six-hour segment (six successive fields). results the presence of airborne grass pollen during the daily segments was sparse the first two weeks of may. it gradually increased during the next days, and then was detected during most of the daily segments through to the end of june. # ammophila arenaria (aa) is a highly resilient grass with habitat in europe, north america, australia, new zealand, south africa and the mediterranean, used predominantly for dune stabilization. patients with atopic diseases including allergic reactions to pollen are transferred to coastal regions in order to minimize pollen exposure. in spite of the presence of aa pollens, patients experience allergy relief at the coast. our study examines the contradiction between the presence of strong grass allergens and the absence of allergic symptoms in atopic patients, and whether allergic cross-reactions among different types of grass pollen include aa pollen. adult patients presenting symptoms of grass pollen allergy underwent prick testing and rast testing to grass pollen mix and extracted aa pollen. subjects showed positive results in prick and rast testing to aa extracts and were subsequently tested via rhinomanometry for reactions to aa using prick test solutions. subjects showed positive reactions to prick tests for aa extract and grass pollen mix, but did not show specific antibodies. all other sera showed elevated specific ige levels, with specific reactions on protein bands of aa. in sera incubated with grass pollen extract, almost all specific bands previously detected with aa were now inhibited. patients with high levels of total and specific ige to aa showed weak bands after inhibition, demonstrating the allergenicity of aa. comparing data from coastal and mainland weather stations in june and july from to , coastal wind speeds average . to . m/s while inland wind speeds average only . to . m/s. high wind speeds likely dilute pollen concentrations. based on findings of an inverse relationship between wind speed and pollen concentration in the dispersion of ragweed pollen, we hypothesize that concentrations of aa pollens may be similarly influenced. no sensitized patient reported allergic symptoms in coastal areas inhabited by aa plants, indicating that under natural conditions, either the plant modifies its pollen allergenicity or that environmental conditions including salt aerosols, wind patterns, topography or light exposure cause the modification. as such patterns are likely to become more dramatic as the effects of global climate change transform the natural environment, these findings may also become relevant for other grass allergy types. asthma is the most common chronic inflammatory disorders of the airways with increased prevalence in the past decade. it is characterized by airway obstruction, airway inflammation and airway hyperresponsiveness (ahr) to non-specific stimuli. in this study, we examined the effect of a novel class of molecule, ocid , in inhibiting antigen-induced early and late allergic response (ear and lar), ahr, and airway eosinophilia in mouse and guinea pig models of asthma. pulmonary functions were measured in conscious unrestrained animals by whole-body plethysmography. animals were sensitized with ovalbumin (ova; mg, intraperitoneally) with mg alum followed by treatment with ocid ( mg/kg, i.p. twice daily for days). after the last dose, animals were challenged with ovalbumin. pulmonary functions were recorded for ear and lar and hrs later for ahr. this was followed by bronchoalveolar lavage, blood and lung tissue collection. treatment with ocid ( mg/kg) significantly attenuated lar in ova-sensitized and challenged mice or guinea pigs with no effect on ear. ahr to methacholine in mice and to histamine in guinea pigs were prevented by treatment with ocid . ocid attenuated the rise in total number of inflammatory cells in the lung and almost ablated the rise in bal eosinophilia in the lung due to antigen sensitization and challenge. effect of ocid on pulmonary functions and airway inflammation in ova-sensitized and challenged mice were comparable to that of dexamethasone in both models of asthma. these data suggest that ocid prevents the underlying pathophysiological changes in allergic airway inflammation and therefore could prove beneficial in the treatment of bronchial asthma. ma; . madison, wi; . portland, or; . milwaukee, wi; . normal, il; . denver, co; . los angeles, ca; . fremont, ca. introduction. daclizumab (zenapax®), a humanized monoclonal antibody directed against the il- receptor chain (cd ), is approved for prevention of renal allograft rejection and is under evaluation for treatment of asthma. daclizumab inhibits activation of human t lymphocytes by blocking il induced proliferation, and by reducing production of th -and th -associated cytokines. we recently reported that daclizumab improved pulmonary function in a phase ii trial of patients with moderate to severe chronic persistent asthma (jaci, , ( ):s ). we now report additional data from this trial on the possible role of daclizumab as an anti-inflammatory agent that affects asthma outcomes. methods. non-smoking asthmatics age - with baseline fev - % predicted despite use of mcg daily inhaled triamcinolone (taa) or equivalent were enrolled in a randomized, multi-center, double-blind, placebo-controlled trial. patients were randomized ( : ) to i.v. daclizumab ( mg/kg followed by mg/kg every weeks) or placebo added to stable dose taa. starting at week , patients underwent % reduction of taa every weeks while continuing study drug to week . results. patients on daclizumab demonstrated a prolonged time to exacerbation requiring systemic steroid rescue compared to placebo patients (p= . ). exacerbation rates were reduced in the daclizumab group compared to the placebo group for the -week steroid-stable and steroid-taper phases ( . % vs. . %, p= . ). patients on daclizumab demonstrated decreased peripheral eosinophil counts from baseline to week (- ± /mm vs. placebo + ± /mm , p= . ). daclizumab-treated patients with elevated baseline serum eosinophil cationic protein (secp) had a significant reduction in secp from baseline to day compared to placebo patients (p< . ). peripheral eosinophils decreased significantly in daclizumab patients who had no asthma exacerbations compared to an increase in daclizumab-treated patients with at least one exacerbation (p= . ). conclusions. daclizumab reduces time to and frequency of exacerbation in treated asthmatics. the mechanisms of this effect remain to be fully elucidated, but initial findings suggest that associated reductions in circulating eosinophil and eosinophil products may play a role in these therapeutic effects. hae is a genetic deficiency causing a decrease in c esterase inhibitor levels. it is a rare condition (approximate prevalence : to : individuals). it is manifest by acute attacks of swelling which can involve the larynx (a potentially life threatening condition), the gi tract (causing a syndrome similar to an acute abdominal catastrophe) or the skin and soft tissue of the patient including the limbs, face and external genitalia. there is no approved therapy for acute attacks in the usa. pathogenesis of this potentially fatal condition is thought due to excessive activity of plasma kallikrein. dx- is a specific and highly active (ki pm) recombinant inhibitor of human plasma kallikrein undergoing evaluation in hae and cardiopulmonary bypass. it is an animal free product. a double blind, randomised ( : drug to placebo) placebo controlled dose ascending study of dx- in acute attacks of hae was run in the usa and the european union. four dose groups of patients were to be included in the study. the doses were , , and mg/m . the primary outcome variables were proportion of patients achieving a significant clinical response within hours of administration of therapy, and safety. secondary endpoints included site response, dose response, and median time to significant response by site and dose group. dx- met its primary endpoint; % of dx- patients had a significant improvement within hours, (placebo response rate %, difference % p= . ). patients receiving dx- responded in a median time of minutes. there was no difference in proportions of cases that responded within hours between the three anatomical sites. time to significant response did not significantly differ between dose groups and anatomical sites. the safety profile was comparable for patients treated with dx- and placebo, in terms of saes and aes. in conclusion, dx- in this double blind study was shown to be statistically superior by % to placebo and to be at least as safe as placebo. the drug is effective at treating any hae site, including the larynx. dx- represents an important advance in the management of hae. ar is among the most common chronic childhood disorders, and is most effectively treated with intranasally inhaled glucocorticosteroids (ics). recent evidence suggests that intranasal ics therapy may suppress hpa axis function and decrease growth velocity. this study reports -year follow-up data on children ( female, african american), aged to years (mean . years) at entry, enrolled in a long-term growth trial of intranasal taa for treatment of ar. primary measures included height velocity, bone mineral density (bmd), serum osteocalcin and salivary cortisol levels. all subjects followed their expected age-appropriate growth velocities. average growth velocities were . and . cm/year for girls and boys aged < years, respectively, and . and . cm/year for girls and boys aged > years, respectively. mean (+ std) bmd was . + . and . + . gm/cm , mean serum osteocalcin levels were . + . and . + . ng/ml, and mean salivary cortisol levels were . + . and . + . nm/l at entry and -year follow-up, respectively. these results demonstrate no significant effect of one year of intranasal treatment with taa on growth velocity or hpa function in children with ar. patients males, females ranging in age from years to years, received (o) in doses ranging from to injections over to weeks. patients were evaluated with symptoms scores, pulmonary function (pft), blood eosinophil count (bec), medication use, and allergy skin test. st were initially performed by prick and if negative, by intradermal (id) ( / w/v) and if further negative by id ( / w/v). skin tests were performed immediately before and minutes after the administration of o. there was no change in pft or bec. however there were significant reductions in symptom scores, including nocturnal asthma (<. ), exercise tolerance (<. ), sense of well being(<. ). in addition, parenthetically there was a reduction in nasal symptoms (<. ). there were also significant declines in medication use especially for albuterol (<. ). st declined in all patients evaluated and dramatically in many. for several individual allergens st went from prick positive to id ( / w/v) negative. moreover st declined in all instances further, minutes after the administration of o except for patients. in addition to asthma one of the patients had allergic bronchopulmonary aspergillosis and another had allergic fungal sinusitis. three of the patients tested positive on prick test to mouse antigen and in each instance this became negative after administration of o with all patients tolerating o without problem. we conclude: ( .) administration of o is associated with an improvement in symptoms and decreased use of medications ( .) in addition there is a prominent decline in st which is more marked minutes after administration of o compared to immediately prior to administration the explanation of this finding is not apparent. ( .) o appears to be safe to administer to patients even if they demonstrate specific ige to mouse antigen by st a.p. baptist * , t.s. tang, j.l. baldwin, ann arbor, mi. introduction: academic medical institutions are under pressure to shorten or eliminate resident elective rotations due to federal work-hour restrictions. it is unknown if this will affect knowledge and referral patterns for resident and faculty physicians. the objective of this study was to examine the factors associated with resident and faculty perceived knowledge and referral patterns towards allergy/immunology (a/i). methods: a questionnaire was sent to primary care physicians at one academic center. it addressed past history of a/i referrals, referral intentions for conditions that may be seen by allergists, and perceived a/i knowledge. independent variables included history of an a/i rotation, gender, department, years in training/practice, and history of referral to an allergist. results: ( %) completed surveys were returned. using logistic regression with forward selection, we found in the resident physician cohort those with advanced years in training or history of an a/i rotation were more likely to have increased past history of referral to an allergist (or = . , or = . ), increased referral intention for chronic sinusitis to an allergist (or = . , or = . ), and increased perceived a/i knowledge (or = . . for the faculty physician cohort, those with a history of an a/i rotation were more likely to have an increased perceived knowledge about a/i (or = . - . ). in addition, pediatric faculty physicians were more likely to refer asthma (or = . ) and chronic eczema (or = . ) to an allergist than faculty from other departments. finally, faculty physicians with a past history of referral to an allergist were more likely to have refer asthma (or = . ) and allergic rhinitis (or = . ) to an allergist. conclusion: the factors associated with resident and faculty physician perceived knowledge, referral history, and referral intentions towards a/i differ. for residents, senior training level and a history of an a/i rotation appear most important. for faculty, history of an a/i rotation, pediatric department, and past history of referral to an allergist appear most important. given that a history of an a/i rotation may increase perceived knowledge and referral patterns towards a/i, eliminating a/i rotations in medical institutions may have important consequences for patients and the field of a/i. because recent anecdotal reports suggest that hbv may be an effective treatment for patients with multiple sclerosis (ms), there has been a movement of patients to zealous lay practitioners to receive multiple and repeated bee stings. since this practice has real risk of possible fatal allergic reactions as well as emotional and economic costs, properly conducted studies of safety and efficacy are needed. the purpose of the present phase i pilot study was to evaluate the safety of hbv extract in patients with pfms. a total of evaluable bee venom non-allergic patients with pfms ( - years) were enrolled and were randomly divided into groups, each receiving an increasing allergen dose immunization schedule for one year. hyperreactivity to hbv was evaluated by questionnaire, px and hematologic, metabolic and immunologic tests including skin tests. any possible beneficial responses to therapy were evaluated by questionnaire, functional neurologic tests (fnt) and changes in measurement of somatosensory-evoked potentials (seps) prior to and at the completion of the study. no serious adverse allergic reactions were observed in any of the subjects even at the highest doses of bee venom therapy. although of subjects had worsening of neurologic symptoms during the course of bee venom therapy, requiring termination, this response could not be ascribed to side effects of the hbv or to a spontaneous worsening of the neurologic disease independent of treatment since there was no correlation of these events with the bee venom injections. of the remaining , felt that the therapy was beneficial.there were no changes either in fnt or seps measured during the study. the present report represents the first controlled study evaluating the safety of hbv as a possible adjunctive treatment for multiple sclerosis. while this preliminary safety study suggests that administration of repeated injections of hbv in a step-up dosage regimen in appropriately selected non-hbv allergic patients with ms had no serious adverse allergic reactions, because of the small number of subjects studied, it does not permit conclusions regarding efficacy and therefore provides little evidence to support the use of hbv in the treatment of ms. much larger carefully conducted multi-center studies would be required to establish efficacy. i. finegold * , new york, ny. rush immunotherapy (rit) has benefits for inducing immunologic desensitization in shorter periods of time than conventional immunotherapy. however, there is an increased risk of systemic reactions utilizing accelerated schedules. in the past radiocontrast media reactions have been significantly reduced using a standardized protocol. a modification of this protocol was used prior to rit. the oral premedication consisted of mgm prednisone hours, hours and hour prior to rit, fexofenadine mgm at hours and again hour prior to the procedure, and ranitidine mgm, hours prior to rit. patients tolerated the premedications well. a signed consent was obtained prior to rit. patients were desensitized in an office setting with appropriate therapy for anaphylaxis available without an indwelling intravenous line. injections generally began with / dilution of the anticipated maintenance solu-tions and were doubled every - minutes for about - hours and then the patients were observed for hour or more prior to discharge. in this manner patients were desensitized in - half day sessions. the patient was injected again in week, weeks and then weeks. doses were increased if they were not at a full maintenance dose. if reactions occurred this process was appropriately decreased. utilizing this technique patients were treated. the average age was . years old ( - years of age) % were males. % of patients had allergic rhinitis, % asthma, % insect allergy. patients with complicated medical illnesses were excluded. nine patients had systemic reactions requiring an injection of epinephrine. among the symptoms experienced were wheezing, nasal congestion and flushing. no patient required a second injection of epinephrine, intravenous fluids, or hospitalization. the patient reaction rate was % of patients treated or in injections or % of all injections given during the rush protocol. these results are typical of the increased rate of reactions to rit. however, these systemics were mostly very mild and responded to therapy and in only one case was there a delayed hour reaction. thus premedication while not preventing systemic reactions seemed to modify them to make rit in an office setting a practical and effective therapy. we have previously shown that oligonucleotides consisting a novel '- 'linked structure and synthetic immunostimulatory motif cpr (r is a synthetic purine moiety), referred to as second-generation immunomodulatory oligonucleotides (imos), induce potent th immune responses and prevent ovainduced allergic asthma in mouse models. in the present study, we examined local and systemic immune responses of imos following intranasal (i.n.) administration to naive mice. these studies showed that imos produce higher levels of serum and local il- compared with il- . we next examined the ability of s.c. and i.n. administrated imos to reverse ova-induced th immune responses in a murine model of asthma. treatment of ova-sensitized and challenged mice with imos by either route of administration suppressed il- and il- levels with an increase in ifn-gamma secretion in spleen cell cultures and lung homogenates. imos decreased levels of serum ige and igg and induced higher levels of total and ova-specific igg a titers in serum and balf. while both routes of imo delivery showed similar efficacy on serum and balf immunoglobulin levels, s.c. delivery resulted in stronger systemic effects on the spleen cell cytokine production and the i.n. route of administration produced stronger local effects on the lung cytokines. imos also decreased eosinophils in balf and suppressed inflammatory cell infiltration and goblet cell hyperplasia in lungs. these effects in lung were superior with i.n. administration of imo than did with s.c. administration. further studies to understand the effect of low multiple doses against a single higher dose of i.n. administered imos in naive mice suggest that low multiple doses induce strong th responses locally while the single higher dose produces higher systemic responses. these findings suggest that second-generation imos containing cpr dinucleotides potently reverse ova-induced th immune responses with strong th type cytokine induction in ova-sensitized and challenged mice and i.n. delivery is superior to s.c. delivery in reversing ovainduced airway inflammation and mucosal secretion. showed ait was medically inappropriate, lacked documentation of necessity for initiation or continuation and/or had strong contraindications in / ( %) of the reviewed records. method: we audited a convenience sample of our own records for documentation of necessity for (continuing) ait, using a checklist based on the oig report which included the following criteria: an appropriate diagnosis; specific justification; exclusion of strong contraindications; signed, informed consent; a written physician order, and at least annual re-evaluation for continuing ait. results: the first charts in our alphabetized file of total ait charts were reviewed. the patients ( m, f) ranged in age from - yr (mean, yr). the mean duration of ait was . yr (range, . - yr). indications for ait included allergic rhinitis alone ( %) or with asthma ( %) or chronic sinusitis ( %), asthma alone ( %) and venom anaphylaxis ( %). all had initial documentation of necessity. seven charts ( %) had no written order to initiate ait. nine ( %) lacked a signed consent form. no strong contraindication was found. (peak flow documentation showed the asthma cases were well-controlled. three episodes of ait-induced anaphylaxis requiring epinephrine had occurred, but ait was subsequently resumed and well-tolerated.) seventeen ( %) charts had no documentation of re-evaluation during the preceding - months, including with no documented re-evaluation during the previous months or more, of ait. discontinuation of ait was "considered" for patients after . , , and yr of ait, respectively, but all were still receiving maintenance doses. the estimated time for completing this review was - min per chart. inter-pretation: an internal chart review for adherence to ait practice parameters using a checklist such as ours can quickly and easily assess documentation for medicare reimbursement and patient safety. objective(s): immunotherapy is an accepted mode of treatment for children suffering from allergic rhinitis and allergic asthma. this study demonstrates that rapid allergen vaccination (rav) can be as safe as conventional allergen vaccination (cav) in children. rav may also improve patient adherence. study design: pediatric patients, - years of age, diagnosed with allergic rhinitis and/or mild to severe asthma underwent rav over a / hour period in an office-based setting. all patients were premedicated with prednisone and h antagonists for days prior to the procedure. patients were monitored for reactions during the procedure and continued on a cav schedule. adherence was reviewed subsequently. results: a total of pediatric patients underwent rav utilizing a / hour protocol. of the patients, of them were male ( . %) and of them were female ( . %). ( . %) of the patients had allergic rhinitis, ( . %) had asthma, and ( . %) had chronic sinusitis. during the procedure, patients ( . %) experienced systemic reaction, and none experienced true anaphylaxis. seven of the eight patients who suffered a systemic reaction were patients with asthma on inhaled corticosteroids, three of the eight patients were male ( . %) and five of the eight were female ( . %). every systemic reaction occurred within minutes of the injection. treatment usually included one or a combination of the following: nebulized breathing treatment with albuterol, two sprays in each nostril of azelastine, and diphenydramine taken orally or intramuscularly. every patient that was treated was discharged within two hours, and no one required treatment with subcutaneous epinephrine, treatment for recurrent symptoms or hospitalization. all of the patients continued with a conventional allergen immunotherapy regimen following the rapid protocol to reach their maintenance dose. typically, months of build-up period was saved. patients reached efficacious dosages almost immediately. adherence rates were . %, . %, and . % at , , and months respectively. conclusions: effective doses of allergen vaccine can be safely reached using a / hour protocol annals of allergy, asthma & immunology for children. advantages of rav over cav include improved adherence with almost immediate clinical efficacy, and decreased costs. introduction: studies involving allergen immunotherapy (ait) have furthered our understanding of cat allergen, proteases, and dust mite allergen. the impact of these data has not been assessed. objective: to evaluate ait prescribing trends over years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) focusing on new prescriptions before and after published data regarding specific prescribing recommendations. methods: a retrospective review of , ait prescriptions from a centralized allergy and extract laboratory database was performed with respect to published literature on findings and recommendations regarding the ubiquitous nature of cat allergen, effective dosing of cat antigen, combining protease containing extracts with those susceptible to degradation, dust mite antigen dosing, and the use of house dust versus dust mite antigen. results: ) in , cat allergen was included in % of new allergy immunotherapy prescriptions reviewed; in , it was included in % of new prescriptions, a statistically significant increase (p< . ). ) over the past years, the mean quantity of cat antigen included in new prescriptions has been unchanged at ml/prescription. only % of new prescriptions were written at the manufacturer recommended maintenance dose of ml, significantly less than those written below this level (p < . ). ) there was no significant change in the percentage of extracts combining antigens with proteases with antigens susceptible to degradation. in , % of prescriptions containing alternaria and/or cockroach antigen were mixed with one or more antigens susceptible to degradation; in , % of these extracts were still being combined. ) the mean volume of dust mite mix contained in these prescriptions was between - . ml/ ml. ) prescriptions containing dust mite rose from % in to % in . the use of house dust was unchanged over the same period at . % of prescriptions. conclusions: although the percentage of cat containing extracts has increased significantly, dosing of cat allergen has remained unchanged despite published literature recommending higher volumes. protease-containing allergen prescribing patterns have not significantly changed from [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] young stage of life is a crucial period for aeroallergen sensitization, considering the immaturity of the neonatal immune system which may lead to t-cell anergy or a deviation towards th -type response in mice. the aim of this work was to evaluate the influence of oligodeoxynucleotides containing cpg motif (cpg-odn) on ovalbumin (ova) and blomia tropicalis (bt) immunization in early life compared to adult stage. three days old a/sn mice were immunized with ova in al(oh) and boosted on th and th day after immunization (dai) and bled on th dai. groups of mice received mg of cpg-odn or control-odn associated with ova on immunization and boost. some animals from the three groups (ova, ova+cpg, ova+co) were killed at days old and the spleen was collected and prepared to culture. eight to weeks old female a/sn mice were immunized with ova in al(oh) , boosted on th dai and bled on th dai. groups of mice received mg of cpg-odn or control-odn associated with ova on immunization. animals from the three groups were challenged months after immunization and bled days later. similar protocols were performed using blomia tropicalis (bt) extract in the place of ova. neonate and adult co-administration of cpg-odn with ova or bt were able to significantly decrease specific ige antibody levels and increase igg a production. moreover, cpg-odn decreased specific igg levels in the bt immunization. the co-administration of control-odn in neonates decreased anti-ova igg production. after ova-challenge months later of adult immunization, a similar response was detected in the group that received cpg-odn associated with ova, which showed a decreased anti-ova ige and igg and enhanced igg a levels. analysis of cell division of neonate lymphocytes by flow cytometry showed a decreased proliferation in ova+cpg mice group under ova stimulation. this effect was seen either in b cells and t cells. the results showed that immunization with both allergens, ova and bt, associated with cpg-odn decreased the type i hypersensitivity response. the pattern of antibody production suggests th -type response induced by cpg-odn, and the establishment of memory th response. this findings may imply that the use of cpg-odn in early life seems to be beneficial as an strategy to modulate or to prevent the development of allergic diseases. n. horne * , a. capetandes, m. frieri, east meadow, ny. introduction: it has been shown that dust mite allergens can affect the functioning of airway epithelial cells (winton et. al. br j pharm : , . previous experiments showed ca aggregate in the presence of dermatophagoides pteronyssinus (dp) (capetandes et. al. am j clin path, : , ) . it is hypothesized that epithelial damage is associated with airway remodeling characterized by fibroblast dysregulation. to evaluate the response of fibroblasts to damaged airway epithelial cells, the bioactivity of serum-free conditioned media from dp-treated ca cells (dpcm) was assayed with nhlf. methods: all experiments used insulin-transferrin-selenium supplemented dmem (its). dpcm (generated with ca treated with au/ml dp; alk-abello) was added to % confluent nhlf for hours at oc and % co . its from cultured ca without dp (its cm) was added to % confluent nhlf (control). cell morphology and density were evaluated by microscopy and mtt assay, respectively. data were analyzed by anova followed by student-neuman-keuls (snk) at p< . with power analysis. results: % confluent nhlf treated with dpcm showed decreased cell density (figure ) , and increased aggregation relative to its cm or its alone. nhlf in its alone or with direct addition of au/ml dp to its (dp its) showed no or weak aggregation. aggregated nhlf showed % viability and grew to confluence when subcultured to serum-supplemented media. conclusion: nhlf aggregation was greater, and cell density was lower with dpcm than with its, its cm, and dp its. this suggests that dp-treated ca cells release an unidentified factor or factors that contribute to nhlf aggregation and possible apoptosis. identification of these factors would increase the understanding of the fibroblast response to epithelial cell exposure to dp which may be involved in airway remodeling, a feature of chronic severe asthma. j.t. zimmermann * , y.c. huang, m. frieri, east meadow, ny. introduction: budesonide has been demonstrated to inhibit il- , tgf and gm-csf in ragweed and dust-mite (dm) stimulated human alveolar epithelial cells (j allergy clin immunol : a, ; ann allergy asthma immunology : p , ) . rantes production and expression in dm and il- -stimulated a cells has recently been shown to be inhibited by budesonide (allergy asthma proc, in press ). histamine is known to influence immune response by regulating cytokine synthesis (allergy : - ) . in this study we examined the effect of budesonide in the presence of histamine on il- production and expression by a cells. methods: a pulmonary epithelial cells were cultured in dmem for hours in % co in the presence of : of , au/ml dm, - m histamine and - m budesonide. il- production was measured by a sensitive elisa and il- mrna was measured by qualitative rt-pcr using standardized primers for il- with a gapdh control. results: a cells were stimulated by dm ( - pg/ml) (p< . ). budesonide alone decreased il- levels to pg/ml (p< . ), and in combination with histamine further decreased il- levels to pg/ml (p< . ). relative intensity of il- mrna expression was reduced -fold in dm-stimulated cells and -fold in control cells by budesonide. conclusion: budesonide in combination with histamine showed greater inhibition of il- production by a cells than budesonide alone possibly by increasing cell membrane permeability. c.r. oliveira * , a.e. fusaro, j.r. victor, e.a. futata, c.a. brito, a.j. duarte, m.n. sato, são paulo, brazil. antigen-driven bystander suppression induced by oral tolerance could be an interesting approach in the allergy field, considering the high incidence of new allergens sensitization in atopic individuals. to address the influence of non-related allergen exposure on the type i hypersensitivity response to the mite blomia tropicalis (bt) or ovalbumin (ova) in mice and to verify oral tolerance effect in the bt/ova co-immunization model. groups of mice were immunized with bt extract and two weeks later submitted to ova-immunization, or first immunized with ova or co-injected with bt and ova. ova feeding was performed five days prior co-immunization. ige abs were estimated by means of passive cutaneous anaphylaxis reaction and specific ab and cytokines secretion by elisa. mice sensitized with bt and then exposed to ova developed an enhanced ige response to itself and to ova, but such response has not been observed when ova-immunization was prior to btimmunization. co-injection of bt and ova led to a dominant ige response toward ova over bt and vice-versa for the igg response. ova feeding prior co-immunization decreased ige, igg and igg a ab levels against ova in parallel to a bystander suppression, which avoided the outcome of bt-sensitization. these mice showed increased ifn-?secretion levels induced by antigen-specific stimulus. furthermore, effectiveness of oral tolerance was also related with ova amounts employed in the co-immunization since ova feeding prior co-immunization with low amounts of ova led to inhibition of ige ab response to both allergens, whereas inhibition of specific and non-related antigen igg ab response was broken. the results evidenced that, depending on allergenic potential, new allergen exposure may exert an adjuvant effect on the primary sensitized allergen. the bystander effect to non-related allergen by oral tolerance should be an interesting mechanism to control new aller- rationale. cd + cd + regulatory t cells from patients with atopic asthma undergo apoptosis when stimulated by specific allergen. antihistamines are used to control allergic inflammatory diseases, but their influence on treg cells is currently unknown. the present study investigates the influence of desloratadine (d) on apoptosis of cd + cd + regulatory t cells in atopic patients having dust mite induced allergic disease, including allergic asthma. methods. patients (n= ) with positive skin prick tests to house dust mite allergens and a clinical history of allergic upper and/or lower respiratory symptoms were treated with d mg (schering-plough, usa) once daily for days. no patients used systemic corticosteroids, while theophylline and other medications were stopped at least hours before blood collection. blood was sampled before and after treatment with d. the control group was comprised of individuals without allergic respiratory symptoms and with negative skin prick-tests. peripheral blood mononuclear cells (pbmc) were isolated over a ficoll density gradient and stimulated by specific allergen (dermatophagoides farinae) and in a control series by phorbol myristate acetate (pma). pbmc were labeled with anti-cd , cd , cd , and bcl- monoclonal antibodies. the annex-inv-propidium iodide (anv-pi) test was done. lymphocyte subpopulations and apoptosis were analyzed by flow cytometry. results. after treatment with d, atopic patients had no significant differences in the number of cd + cd + cells. during co-culturing of pbmc from patients treated with d with specific allergen a significant increase in cd + cd + cells was observed. simultaneously, treatment with d led to a significant increase in expression of the antiapoptotic protein bcl- in cd + cells. this data paralleled the decrease of cd expression on cd + cells. there was also a decrease in treg cells in the late stages of apoptosis (anv + pi + cells) in d treated patients. conclusions. the antihistamine preparation desloratadine prevented allergen-specific apoptosis of cd + cd + t regulatory cells in patients with atopic asthma. the type histamine receptor may be involved in the regulation of apoptosis and/or survival of cd + cd + t cells. a.g. palma-carlos * , m.l. palma-carlos, lisboa, portugal. introduction: solar urticaria porphyrin corresponds to sun sensitivity to a a wavelengths and is due to disturbances of posphorin metabolism. photosensitivity can cause urticaria, erythema, polymorphic solar eruption and in more severe cases vesicles and bullae. photosensitivity was confirmed by light test. methods: protoporhyrin in rbc, uro and coproporphyrins in urines, copro and protoporhyrins in faeces and porphyrin precursors have been studied un all the cases of solar urticaria seen in the last few years. results: in patients, female, males, a diagnosis of porphyria has been confirmed by laboratory methods. clinically patients presented solar urticaria and solar erythema or polymorphic solar eruption. patients, female, males ( - ) presented also neuro visceral symptoms: abdominal pains ( ) vomiting ( ) asthenia ( ) constipation ( ) muscle pains and paresia ( ) depression ( ) anesthesic reactions ( ). in cases the symptoms were associated with anticonceptional drugs. the conjunction of clinical history with laboratory data has allowed to confirm the diagnosis of cases of protoporphyria erytheropoietica, one triggered by anticonceptionals, cases of coporporhyria hereditaria and cases of porphyria variegata. this group comprises one case of erythropoietic protoporphyria induced by estrogens not previously reported. conclusions: research of porphyrins must be done in all the patients with solar urticaria and erythema. the incidence of sun sensitivity in mixed hepatic porphyriaalso presenting neuro-visceral symptoms which can be drug dependent suggests that porphyria study must be mandatory in suspected cases. a.r. narayan * , j. kaplan , v. sirvan , a. chandrasekaran , c. goodwin , l. goodwin , l. guida , m. frieri , . east meadow, ny; . manhasset, ny; . bayshore, ny. rationale: our division has reported that nitric oxide (no) from tracheal epithelium can mediate induction of il- (american journal of respiratory critical care medicine : a, ) . increased levels of il- , ltb- , and no in mononuclear cells (mnc) from cystic fibrosis (cf) patients were noted.(pediatric asthma allergy immunology : - ) . no in mnc from cf was increased by stimulation with aspergillus fumigatus (asp) and rhdnase (j. clin allergy immunol : p ). tnf-is produced from bronchial epithelial cells (bec) in the presence of mnc of normal controls (nc). rantes, a chemokine that facilitates leukocyte migration, is associated with airway inflammation in cf, and asp sensitization in cf patients could amplify pro-inflammatory cytokines such as il- , tnf-, and rantes. methods: we studied the mrna and protein expression of tnf-, il- and rantes in a year-old cf patient and a nc. x mncs from the patient and nc were stimulated with μg/ml of asp with bec at x and μg/ml rhdnase. mrna expression was studied by real time pcr (taqman chemistry abi prism ) and protein levels by elisa. results: tnf-production in supernatants of mnc with bec from nc increased from pg/ml to pg/ml with rhdnase. whereas in cf patients the tnf-mrna expression was greatly enhanced over nc but declined in the presence of rhdnase (fold difference: . - . ). there was no difference in expression levels of nc (fold difference: . - . ) with rhdnase treatment. il- expression in mnc with bec of patients increased . fold compared to nc ( . fold). rhd-nase decreased asp stimulated levels to . fold in patients compared to nc ( . fold). mnc and bec rantes production in patients increased from to pg/ml and decreased to pg/ml with rhdnase, but increased from pg/ml to pg/ml in nc. in contrast, rantes mrna expression in all groups was higher in nc but decreased with rhdnase in both cf and nc ( - fold vs. - fold). conclusion: rhdnase can increase no and decrease pro-inflammatory tnf-and rantes in cf patients stimulated with asp. rhdnase could lead to augmented bactericidal activity and epithelial defense by enhancing no production and decreasing the expression and production of tnf-, il- and rantes. this is a case report of two steroid dependent atopic dermatitis patients who both responded to treatment with omalizumab. patient a is a year-old white male who presented with a history of severe atopic dermatitis for years along with concomitant mild persistent asthma and allergic rhinitis. he had previously received monthly triamcinolone injections, methotrexate and doxycycline with limited response. on presentation he had mild improvement on a regimen of alternate day prednisone mg, fexofenadine mg bid, and zafirlukast mg bid. however, attempts at weaning prednisone were unsuccessful. omalizumab was initiated at a dosage of mg every four weeks. the patient remains prednisone dependent yet free from atopic dermatitis. patient a's diagnostic work up determined a total ige of . ku/l. the patient's specific ige tests were positive for grass mix, weed mix, maple, white pine, peanut, strawberry, gluten, soybean, wheat, oat, and dog dander. specific ige tests were negative for cat dander, egg white, milk, goose feathers, chicken feathers, mold mix, dust mix, and fish mix. patient b is a year-old white male who presented with a history of severe full body atopic dermatitis along with mild persistent asthma and allergic rhinitis. he also was receiving triamcinolone injections with limited success. he responded to alternate day prednisone mg, desloratidine mg bid, and zileuton mg bid. attempts at weaning prednisone failed until initiation of omalizumab mg every two weeks. his atopic dermatitis is now under control with once daily desloratidine and omalizumab every two weeks. he was successfully weaned off of corticosteroid medication and has begun an immunotherapy regimen. patient b's diagnostic work up determined a total ige level of ku/l. patient b's specific ige tests were positive for weed mix, tree mix, maple, peanut, strawberry, dust mix, cat dander, dog dander, egg white, milk, oat, wheat, goose feathers, and chicken feathers. specific ige tests were negative for grass mix, white pine, mold mix, gluten, soybean, and fish mix. these two case reports reveal significant response to omalizumab in severe steroid dependent atopic dermatitis. further research is strongly indicated considering the quality of life issues with severe atopic dermatitis and potential side effects to long-term corticosteroid treatment. eosinophilic esophagitis (ee) has been described in children and is characterized by high levels of eosinophils (> - eosinophils/high powered field [hpf]) in the esophageal mucosa. a recent report indicates that the combined prevalence of the eosinophilic gastrointestinal disorders may be higher than that of inflammatory bowel diseases (ibd). the presenting symptoms of ee mimick those of gastroesophageal reflux disease (gerd), and patients of all ages may experience a delay in time from onset of symptoms to diagnosis of ee. there is a paucity of data in the literature regarding the delay in time from symptom onset to diagnosis of ee. we report the data on four children three years of age and under with ee who experienced a significant diagnostic delay. four young children were referred to the pediatric allergy clinic with a diagnosis of ee (table) . three of the patients were male and one was female. the patients were - months of age at the time of diagnosis, and the diagnostic delay was . - months (average delay . months). the most common presenting symptom was vomiting, and three of the four patients had a history of respiratory obstructive symptoms. all of the patients had received conventional treatment for reflux disease, and one had undergone a nissen fundoplication prior to diagnosis. in all four patients the esophageal biopsy revealed > eosinophils/hpf. three out of four patients had a family history of atopy, and two patients had a known history of a food allergy. skin tests to foods were positive in two patients and rast (radioallergoabsorbent) testing to foods was positive in one patient. recent data suggests that the diagnostic delay in ibd is decreasing as much as %. it is hypothesized that this may be due in part to an increased index of suspicion by health care providers. there is little data available regarding the diagnostic lag in children with ee. it is currently believed that chronic ee can lead to progressive esophageal scarring and dysfunction. stricture formation has also been described in children less than two years of age. given the reported increased incidence of this allergic gastrointestinal disease over the past decade, more data regarding the diagnostic delay of these conditions may be instrumental improving health care providers' awareness of this disorder. a. kohli-pamnani * , e. cooney, p. huynh, f. lobo, new haven, ct. introduction: cutaneous hypersensitivity reactions to amprenavir, an hiv- protease inhibitor, are reported in up to % of treated patients, of whom % have severe or life-threatening rashes, with treatment discontinuation required in % of cases. we report a case of successful desensitization to amprenavir, for recurrent maculopapular exanthem, in an hiv-infected patient with late stage disease and limited antiretroviral (arv) agent options. methods: the patient is a year-old caucasian female with late stage hiv disease (absolute cd count cells/mm , hiv rna , copies/ml) and multiple arv intolerances, who developed a severe generalized maculopapular eruption sparing mucous membranes six days following initiation of a regimen comprised of amprenavir, lopinavir/ritonavir, zidovudine, and lamivudine. a similar reaction occurred following re-challenge with amprenavir alone ( mg oral formulation bid via percutaneous endoscopic gastrostomy (peg) tube), despite concurrent administration of oral prednisone mg/day and loratidine mg/day. results: skin prick testing with amprenavir . mcg/ml was negative, whereas intradermal testing with . mcg/ml was positive with a mm wheal and mm flare. percutaneous and intradermal testing with normal saline was nonreactive. the positive histamine control (skin prick only) yielded a mm wheal and mm flare. subsequently, incremental doses of . mg, . mg, . mg, mg, . mg, . mg, mg, mg, mg, mg, mg, and mg of amprenavir oral solution were administered via peg tube at to minute intervals (table) . the patient successfully tol-erated amprenavir desensitization and has remained on therapy without recurrence of rash out to months of follow-up. conclusions: desensitization may permit continued use of amprenavir in patients with a history of amprenavirinduced maculopapular eruptions who have limited alternate treatment options. efforts aimed at characterizing the mechanism of amprenavir cutaneous hypersensitivity reactions seem warranted, given the frequency of reactions and the limited number of arv agents available for patients with late stage hiv disease. doses of oral solution were administered by peg tube at twenty to thirty minute intervals. introduction: beta-lactam (bl) allergy is the most common drug allergy. in most cases, ige antibodies are specific to the bl nucleus. however, sidechain-specific ige (scsige) to bl has been described. despite this, skin testing (st) to bl other than penicillin (pcn) is not commonly performed. we report a case of a year-old female with a selective allergy to pip, and describe the utility of st to this agent to confirm scsige. methods: st to prepen (pp) and pcn g was carried out with percutaneous (pc) testing followed by intradermal (id) testing. st was also carried out with ampicillin (amp; mg/ml), pip ( mg/ml at pc level; mg/ml at id level-a non-irritating concentration), and pip/tazobactam (tbm; mg/ml at pc level; mg/ml at id level) to exclude the possibility of selective allergy to tbm. case report: a year-old white female with crohn's disease was hospitalized for treatment of intra-abdominal abscesses. she had immediate flushing and urticaria during an infusion of pip/tbm approximately years prior. the allergy/immunology service was consulted for pcn st because her primary service preferred to empirically treat with pip/tbm. she had received imipenem approximately months prior without untoward reaction. st to pp and pcn g was performed with negative responses and adequate controls. she subsequently received pip/tbm, but developed flushing and urticaria during her initial infusion, consistent with an ige-mediated reaction. two months later, st to pp and pcn g was repeated. in addition, st to amp, pip, and pip/tbm was performed. she had positive responses to pip and pip/tbm with negative responses to pp, pcn g, and amp, implying selective ige-mediated potential to pip. she tolerated an oral challenge with pcn vk mg immediately following st without untoward reaction. conclusion: we have described a case in which st to pip was useful for diagnosing scsige in our patient, who had prior reactions consistent with ige-mediated pathogenesis. this information will be helpful in identifying antibiotics she can safely receive in the future. this case supports the utility of st to bl in addition to pcn, in evaluation and management of patients with a history of adverse reaction to bl that may reflect presence of scsige. a.r. vaishnav * , b.s. bochner, baltimore, md. we report the case of a -year-old caucasian male with a -year history of asthma, two episodes of eosinophilic pneumonia and chronic peripheral eosinophilia with baseline eosinophil counts around /μl who developed amnesia and elevated cardiac enzymes in february . another physician had added montelukast in january and because of a good response advair was decreased from / to / bid. in february his eosinophil count increased to /μl and soon he developed left arm numbness, diplopia as well as left arm and leg weakness. at his local hospital, he was found to have elevated troponins and cpks. cardiac catheterization showed normal coronary arteries and good left ventricular function. soon after discharge, he developed confusion and global amnesia. brain mri showed diffuse uptake consistent with global inflammation and/or vasculitis. upon hearing this story, we told him to take mg of prednisone and urgently come to our hospital for admission. within hours, his mental status and visual symptoms improved. physical examination was unremarkable except for subungual splinter hemorrhages. montelukast was stopped and gm/day solu-medrol iv was started. endomyocardial biopsy days later showed mild hypertrophy and fibrosis without eosinophils. repeat brain mri and mra were normal. chest ct showed multiple patchy infiltrates with a new central cavitary lesion in the right lobe. other labs included a negative anca, ana of : , normal complements and csf. our differential diagnosis included churg-strauss syndrome (css) versus hypereosinophilic syndrome (ihes) with myocardial, neurological and pulmonary involvement. serum tryptase was normal as was fluorescent in situ hybridization for the fip l -pdgfr fusion gene. based on this, along with the new pulmonary cavitary lesion on chest ct, the diagnosis of css was made. after days of iv steroids, he was switched to mg/day of prednisone. upon discharge his eosinophil count was /μl on mg of prednisone. after all procedures were completed, he was also started on aspirin mg daily to prevent thrombotic and thromboembolic complications. he was seen in follow-up in clinic and cytoxan mg daily was started. his prednisone is being tapered. he is tolerating the treatment well and so far has had an excellent clinical and laboratory response. introduction-c -esterase inhibitor (c -inh) deficiency is a rare disorder classified into acquired and hereditary forms. both entities are distinguished by recurrent angioedema without pruritus or urticaria. the upper airway, head, neck, extremities and gi tract are typically involved. the inherited form usually presents in the first or second decade accompanied by a family history. the acquired form more commonly presents after the fifth decade. in acquired c -inh deficiency, serological evaluation reveals low levels of c , c -inh, and c q and a normal c level. levels of c q are normal in the hereditary form. the acquired form may be associated with lymphoproliferative disorders and autoimmune disease. acquired c -inh deficiency is typically recognized before the underlying malignant condition is diagnosed. clinical regression has been reported in patients whose underlying disorder responds to treatment. methods-a case report of a -year old female who developed repeated episodes of facial angioedema requiring hospitalization preceded by one year of vague abdominal pain and cramping. data-laboratory investigation of this patient revealed c < mg/dl ( - mg/dl), c q< . mg/dl( . - . mg/dl), ch < u/ml( - u/ml), and c -inh at mg/dl( - mg/dl) with a % activity (> % normal). c was mg/dl( - mg/dl). other laboratory evaluation was notable for a normal cbc with diff, comprehensive metabolic panel, amylase, lipase and spep pattern. ige was elevated at ku/l. hematology was consulted. ct of chest, abdomen, and pelvis were nondiagnostic. peripheral blood flow cytometry revealed a small distinct pop-ulation consistent with a clonal b-cell lymphoproliferative disorder. a repeat test showed skewing towards lamba light chain expression. bone marrow biopsy and aspirate were essentially normal except for erythroid hyperplasia on danazol treatment. the physical examination was without any masses or lymphadenopathy. the patient remained asymptomatic after beginning danazol although elevations in rbc, hgb, hct and absolute lymphocyte count have developed. conclusion-acquired c -inh deficiency is rare cause of recurrent angioedema that has been reported in small cohorts and case presentations. we report another case report that reinforces the need for physicians to evaluate for concomitant lymphoproliferative disorders. chronic urticaria is a distressing condition usually associated with poor quality of life and poor response to symptomatic therapy. a wide variety of causes and mechanisms have been described and in some patients the cause remains unknown. rare cases have been associated with thyroid antibodies and some responded to thyroxin, even in the absence of overt thyroid disease. case report: a -yr-old obese white female presented with a history of persistent urticaria/angioedema for mo. urticaria involved various parts of the body and individual lesions usually lasted < hr. it was often accompanied by facial edema. oral diphenhydramine mg qid caused only little improvement. the patient could not suspect any offending factors. review of systems and past medical history were unremarkable, except for hyperthyroidism at age yr that was treated with "medication" for yr. on physical examination there were several urticarial lesions though she has been taking diphenhydramine. the thyroid appeared normal regarding size, shape and texture. various second generation antihistamines and doxepin were of little help. laboratory evaluation revealed total serum ige of iu/ml (normal < iu/ml) and ch of u/ml (normal - u/ml), but high tsh of . iu/ml (normal . - . iu/ml) and low free t of . ng/dl (normal . - . ng/dl). her antithyroglobulin titer was iu/ml (normal < iu/ml) but the antimicrosomal titer was highly elevated at iu/ml(normal < iu/ml) consistent with hashimoto's thyroiditis. thyroxin therapy was initiated at a dose of mcg/d which resulted in marked improvement within one week and the patient was able to discontinue doxepin and other antihistamines. because of marked drop in tsh to . iu/ml, the thyroxin dose was reduced to mcg/d. her thyroid function tests became normal within two months. she did not experience any recurrence of urticaria or angioedema during over mo of follow-up so far. conclusion: patients with chronic urticaria/angioedema, especially women, should be screened for thyroid autoantibodies and if positive, thyroxin therapy might bring impressive remission in urticaria. hypersensitivity pneumonitis (hp) results from an abnormal immunologically mediated response to an environmental antigenic trigger. typically patients with hp experience transient fever, hypoxemia, muscle and joint pain, difficulty breathing, fatigue, weight loss, and cough. there are two clinical presentations of hp differentiated by onset and resolution of symptoms. patients experiencing acute hp experience the onset of symptoms to hours following exposure to a particular antigen and resolve in to days without specific treatment. however, patients with chronic hp experience symptoms that persist for months to years when exposed to a recognized cause of hp. a year-old caucasian male was referred to our practice with acute bronchitis. his referral was secondary to a history of allergic rhinitis, chronic asthma, gastro-esophageal reflux disorder, and sinus surgery. this non-smoking male was a machine operator at a local factory. he was doing well until he began working in the presence of a heat induction machine and metal lubricating spray called multan ea (hinkle surface technologies), i.d. no.: . multan ea is a product containing naphthenic petroleum distillates, amine salts, amine soap, triethanolamine hexyl, hexylene glycol, ethanol, sodium petroleum sulfonate, and triazine. this lubricant is known to irritate the eyes, skin, and respiratory tract. recent studies indicate that thermal decomposition of triazine, which readily occurs during metalworking, results in the production of formaldehyde. formaldehyde is a known carcinogen, as well as respiratory, skin, eye and digestive tract irritant. the patient developed serious respiratory health problems resulting in work absence and eventually hospitalization. while hospitalized he received a high resolution ct scan which showed diffuse ground glass appearance, and multiple attenuation areas of the lung which are consistent with hypersensitivity pneumonitis. the patient was treated with corticosteroids, and antibiotics with eventual normalization of lung parenchyma by chest ct. this represents the first reported case of hypersensitivity pneumonitis resulting from exposure to the metalworking fluid and respiratory irritant multan ea . a.j. ham pong * , f. chan , s.l. bahna , . ottawa, canada; . shreveport, la. sexual intercourse has been known as the route for semen hypersensitivity reactions to the seminal fluid protein or to a contaminating drug or food. we report a case of anaphylaxis to cephalexin-containing semen by ingestion. history of present illness: a -year-old woman developed a systemic reaction after swallowing semen. since the couple frequently practiced fellatio without any reactions, her husband suspected cephalexin that he was taking over days ( mg qid). her reaction began in less than min and peaked over min; first as itchy oral mucosa followed by wheezing, flushing of the face and upper chest, and nausea. diphenhydramine mg was administered po at min and the reaction subsided over hr. past medical history: she had generalized urticaria to oral penicillin more than yr earlier and had positive penicillin skin test. she also had allergic rhinoconjunctivitis, mild intermittent asthma, and positive skin test to house dust mite, cat and dog epidermals, and pollens of grasses, trees, and ragweed. she also had oral allergy syndrome to certain fresh fruits and tree nuts; hazelnuts caused also diarrhea and colic. neither the patient nor her husband ate any nut-containing food on the days preceding the reaction. family his-tory: allergic rhinoconjunctivitis in the mother, sister and brother. eval-uation: the patient sought allergy evaluation about yr after the reaction. she and her husband gave a consent. her serum ige was iu/ml and skin test positive to penicilloyl polylysine x - mol id, but negative to cefazoline mg/ml for prick and mg/ml for id. she had negative prick test to her husband's seminal plasma both before and after his intake of cephalexin mg qid for days. using a cephalexin bioassay sensitive down to . mcg/ml, the medication level in the husband's urine at min post last dose was mcg/ml and in his serum at min was mcg/ml, but was undetectable in the semen collected at min. conclusion: this is probably the first case report of systemic reaction to ingested semen. the reaction occurred in an atopic, penicillin-sensitive woman and seems to be caused by cephalexin excreted in the semen or through urine contamination ( drop = mcg). in addition to her avoiding penicillin and cephalosporins, she was advised to avoid her husband's semen while him taking such drugs and for at least days afterwards. background:asthma causes serious morbidity & mortality all over yet most asthma educational evaluations and programs have an urban rather than rural focus.there is lack of data on rural north dakota(nd). this nd survey aimed to study knowledge & awareness of management strategies for asthma like use of metered dose inhalers, spacer devices and peak flow meters. methods:the qualitative survey addressed attitudes and beliefs on asthma & its triggers;knowledge of medication delivery routes & use of hospitals and provider visits.a simple questionnaire was self-administered at rural clinics in small towns in southeastern north dakota. questions explored asthma knowledge, extent of family history of asthma & factors aggravating asthma.it was voluntary & the population surveyed included patients, family, teachers, employees at nursing homes, the local hospital & clinics. results:of surveys completed % had no asthma in the family.of those with a family history( %), % had person with asthma, . % had & . % had or more.respondents were aged - years & . % were female.see table. discussion:our survey was rural & comparison with urban areas may help in planning for asthma education & resource allocation. % denied asthma in their family. similarly, nonurban alaskan natives have a lower incidence of asthma compared to nonnatives.our results show a lack of awareness in our area.grain dust exposure was perceived as an asthma trigger but without evidence, local research is needed.in our study smoking was considered a trigger by . % & this may be real.there was lack of awareness of peak flow meters & their role in asthma care.a study showed that compared to other areas even rural nurses used peak flow meters less often to assess and monitor asthma.this suggests a need for comprehensive asthma educational programs in rural areas that are based on national guidelines.we had only a moderate followup rate at %.in comparison, one study had % missed scheduled follow-ups. exercise induced asthma was known to only % of our group. conclusion:the need for better patient-provider communication has been highlighted by the lack of awareness of proven strategies to combat asthma.the use of a simple survey has revealed many unknown facts about asthma awareness in rural nd.further study to determine cost effective solutions to achieve national targets for asthma management are essential. aim: in most studies of asthma, the emphasis was on changes in large and middle airways. the aims of this study:( )to observe the morphologic changes in small airways and lung tissue(salt) in guinea pig asthma models(gpam); ( )investigate the role of vcam- , eotaxin, nf-b and ap- in the inflammation of asthma; ( )explore the functional variation of alveolar type cells in asthma; ( )evaluate the effects of inhaled glucocorticoids on the above annals of allergy, asthma & immunology parameters in salt of asthma models. methods: ( )the gpam were established by ovalbumin challenge. five groups were divided: control, asthma day and day , intraperitoneal dexamethasone, and budesonide inhalation group. ( )the expression of vcam- , eotaxin, nf-b and ap- was determined by immunohistochemical technology and rt-pcr; the dna binding activity of nf-b and ap- by electropharetic mobility shift assay. ( )the balf phospholipide concentration was measured by phosphorus detection. results: ( )significant inflammatiom with infiltration of eosinophils and lymphocytes in salt was observed in asthma groups. ( )the protein levels of vcam- , eotaxin, nf-b and ap- expressed in the salt were significantly elevated in asthma groups than those in control. ( )the mrna expression of vcam- and eotaxin of lung tissue homogenate was significantly increased in asthma group. ( )the dna binding activity of nf-b and ap- of lung homogenate was significantly increased in asthma group. ( )the balf surfactant represented by phospholipides was significantly decreased in asthma groups. ( )glucocorticoids in different ways of intake provided significant effects on the inflammatiom of salt. conclusion: ( )widespread and significant inflammation with eosinophilic infiltration existed in the salt in gpam, indicating asthma is a disease involving the whole airway and lung system. not only there were structural changes, but also impaired function of alveolar cells. the role of small airway inflammatiom may be of great importance. ( )eotaxin and vcam- actively mediated the process of inflammatiom, nf-b and ap- played important roles in the regulation of vcam- and eotaxin. the up-regulation of the above mediators in gpam was expressed in salt similar to that in central airways. we have recently reported that immunomodulatory oligonucleotides (imos) consisting of a novel structure and synthetic cpr or r'pg (r and r' are synthetic purine moieties) stimulatory motif effectively prevent ovainduced asthma in mouse models. in the present study we examined whether these novel imos (hyb and hyb ) can reverse established allergic airway inflammation in mice. balb/c mice sensitized and challenged with ovalbumin (ova) were evaluated for airway hyperresponsiveness (ahr) to methacholine. following ova-sensitization, mice were randomized and treated with placebo or mg or mg/dose of hyb or s.c. during the following days. two days after the final treatment, mice were rechallenged with ova and pulmonary functions were recorded. mice treated with either imo were significantly protected from both early (ear) and late (lar) allergic response, airway hypersensitivity and hyperreactivity to methacholine, bal and peribronchial eosinophilia, bal and serum il- , and total serum ige as compared to vehicle-treated ova-sensitized and challenged animals. there was a significant increase in immature lung dendritic cells (cd c+cd r+) together with increase in serum il- levels and significant decrease in lung dc type cells (cd c+ cd b+cd a-) as compared to vehicle-treated ovalbumin-sensitized animals in the lungs following treatment with either imo. these data suggest that both imos are effective and potent in attenuating key features of established allergic airway inflammation in bronchial asthma, and this effect could be mediated via decreasing lung dc cells and increasing immature dendritic cells. additionally, imos contain a novel '- '-attached structure that provides higher metabolic stability and may permit lower and/or less frequent dosing. hyb was evaluated for its safety and immunopharmacology in a phase clinical trial in healthy human volunteers. introduction -hemoglobinopathies are common in southern europe and mediterranean area the more frequent being thalassemia and sickle cell disease. aside of the major hematological diseases of homozygotic patients minor forms are frequent, thalassemia minor and sickle cell trait. in these cases mycrocytosis with decrease of red cell volume and mean corpuscular volume or abnormal erythrocytes are found and can lead to hemorheologic disturbances in bronchial circulation and bronchial hypereactivity. the rationale of this study is to evaluate the incidence of asthma in hemoglobinopatic patients allergic to house dust mites.methods-from patients seen in the last years in an out-patient allergy clinic cases of hemoglobinopathies have been confirmed by red cell count, hemoglobin electrophoresis, assays of hemoglobin a , f, and s, and sickle cell test.all these patients had allergic disease characterized by clinical history, skin prick test to aeroallergens total and specific ige (rast-cap-feia) and respiratory function evaluation. results -hemoglobinopathies: betathalassemia cases, betadelta thalassemia , sickle cell trait , hemoglobin c. . aside of cases of urticaria, all the patients presented respiratory allergy, rhinitis in cases of thalassemia and hemoglobin c, asthma with or without rhinitis in cases of thalassemia and cases of sickle cell trait. therefore asthma was present in , %. in a group of respiratory allergic patients without hemoglobinopathies, % had asthma and % only rhinitis. conclusions -the prevalence of asthma is higher in hemoglobinopathies.(p< square chi test). hemorheological changes probably greater rigidity of red blood cells and capillary bed can contribute to bronchial hypereactivity. detection of hemoglobinopathies must be done in asthmatic patients with slight anemia or mycrocytosis. we investigated the role of aspirin-exacerbated respiratory disease (aerd) as a risk factor for the development of airway remodeling. patients with aspirin intolerance develop hyperplastic sinusitis with fibrosis and nasal polyposis. we speculated that similar mechanisms could be acting in the lower airway and that these individuals would demonstrate more severe asthma and evidence for airway remodeling. the epidemiology and natural history of asthma: outcomes and treatment regimens (tenor) study is a multicenter observational study of subjects with severe or difficult-to-treat asthma. baseline data were compared between subjects years who reported asthma exacerbation following aspirin ingestion and those who did not. the primary measure of asthma remodeling was the maximally achieved post-bronchodilator spirometry. adult subjects with aerd (n= ) were compared with aspirin tolerant subjects (n= ). subjects with aspirin intolerance demonstrated evidence for airway remodeling as shown by lower post-bronchodilator predicted fev . in addition, they were more likely to have physician-assessed severe asthma, to have been intubated, and to have required high-dose inhaled corticosteroids or oral corticosteroids in the previous months. we conclude that aspirin intolerance is associated with remodeling of both the upper and lower airways. asthma is a complex and variable disease with two main components, airway inflammation and smooth muscle dysfunction. according to national and international asthma guidelines, subjects with persistent asthma can be classif ied into one of three categories (mild, moderate, or severe) based upon lung function, symptoms, nighttime awakenings, medications and exacerbations. though it is widely believed that there is a high degree of variability in both pediatric and adult subjects with asthma, few studies have compared variability between them. therefore, an analysis of previously conducted asthma studies was undertaken to evaluate pediatric subjects aged - years (n= ) and adult subjects (n= ) previously receiving short-acting beta -agoinsts alone in seven double-blind, randomized, -week trials. the analysis is limited to subjects who were randomized to placebo in these trials. during the study, subjects exhibited marked fluctuations in asthma severity. despite the fact that all subjects met criteria for moderate or severe asthma at baseline, %, %, % and % of weeks for pediatric subjects and %, %, %, and % of weeks for adults were spent in the intermittent, mild, moderate and severe categories, respectively. a summary of severity classification based upon symptoms and albuterol use is presented below. in addition, based upon pef % predicted, % and % of weeks were spent in the intermittent/mild category for pediatric and adult subjects, respectively. however, fluctuations in pef occurred frequently, with % of pediatric subjects and % of adult subjects experiencing changes in severity based on pef over weeks, indicating more variability in pediatric subjects. this analysis clearly demonstrates that asthma is a variable condition and that both pediatric and adult subjects frequently move between severity categories. furthermore, there are marked differences in severity classifications between pediatric and adult subjects. asthma severity, and consequent optimal therapy, cannot adequately be assessed by discrete, point-intime assessments of lung function, frequency of albuterol use, or asthma symptoms, especially in the pediatric age group. studies suggest that there may be an association between single nucleotide polymorphisms (snps) in the beta -adrenergic receptor gene (adrb ) and the response to beta -adrenergic bronchodilators. polymorphisms at codon have been at the center of this debate. therefore, a retrospective analysis of six large, randomized trials was conducted to evaluate clinical responses to salmeterol administered with fluticasone propionate (fsc) / mcg bid for weeks in patients ( yrs) with moderate or severe asthma and differing adrb polymorphisms at codon . baseline demographics were similar for all genotype subgroups. all measures of asthma improved over baseline and were similar across arg /gly subgroups at weeks. pairwise comparisons were conducted between genotypes and there were no differences other than fev as noted in the table. findings from this retrospective analysis show that regardless of arg /gly genotype, clinical response to salmeterol with an ics was similar during chronic dosing. although prospective studies are needed to fully understand the effect of adrb polymorphisms on response to therapy with a long-acting beta -agonist, this analysis suggests that therapy with salmeterol and an ics together is appropriate for caucasian patients with differing arg /gly genotypes. coronary artery disease(cad) and asthma commonly coexist in adults. iv dipyridamole thallium scintigraphy is considered a safe non-invasive technique in the evaluation of cad when patients can't exercise. dipyrdamole is a purine that blocks reuptake of extracellular adenosine increasing serum adenosine levels after iv administration.this results in a transient coronary vasodilation increasing the sensitivity of the thallium study. methylxanthines reverse the effects of endogenous adenosine by competitively antagonizing adenosine at local purinoreceptors. we report a case of a yr.old female with a year history of stable mild persistant asthma treated with daily salmeterol and low-dose fluticasone. she was referred to cardiology for atypical chest pain. within minutes of a standard iv dose of dipyridamole the patient reported chest tightness, cough, and wheezing. all these symptoms, typical of her past asthma flares, were abolished within minutes of an aminophylline infusion and before albuterol was administered.the stress test was completed and was normal. inhaled adenosine induces bronchconstriction and is used as a probe for bronchial hyper-responsiveness. commercial iv adenosine preparations used in treating supraventricular tachycardia are reported to induce bronchospasm in known asthmatics. since serum levels of adenosine are transiently increased following iv dipyridamole, bronchospastic symptoms during dipyridamole stress tests should not be unexpected. an earlier study found an increased incidence of wheezing in % of patients with known copd/asthma undergoing dipyridamole stress tests despite pretreatment with beta agonists. the marked decline of theophylline use for chronic asthma in recent years may actually be increasing the incidence of this risk. cardiologists performing thallium stress tests are familiar with the risk of asthma flares after dipyridamole. they use iv aminophylline frequently to reverse several types of clinical dipyridamole reactions, including bronchospasm. on the other hand, we are impressed that allergists are relatively unfamiliar with this association. allergists and asthma specialists as well as asthmatic patients should be aware of risk of acute dipyridamole induced bronchospasm during elective cardiac stress testing. it has been shown that adhesive molecules are involved in inflammatory diseases of the lungs such as bronchial asthma. the purpose of the study was to measure and establish possible difference in serum levels of soluble icam- in atopic patients (patients with allergic rhinitis and patients with bronchial asthma) in comparison with patients without atopy (patients with asthma without rhinitis); whether there is a difference in sicam- levels between groups of patients with allergic rhinitis and asthma in comparison with group of patients with allergic rhinitis only and also in comparison with healthy controls. results of the study have substantiated statistically significant difference in sicam- levels between all groups of patients in comparison to healthy control, but no statistically significant difference in sicam- levels between patients with and without atopy (z=- . ) or between patients with allergic rhinitis and bronchial asthma in comparison with group of patients with allergic rhinitis only (z= . ). conclusion: icam- is an important marker of inflammation in patients with allergic rhinitis as well as in those with bronchial asthma. atopic status does not influence differences in sicam- levels. although mean sicam- levels were higher in patients with allergic rhinitis and bronchial asthma ( . ng/ml) in comparison with mean sicam- levels in patients with allergic rhinitis only ( . ng/ml), no statistically significant difference was noted in sicam- levels between these groups of subjects, i.e. asthma itself did not contribute to statistically significant increase of sicam- levels. k. nadarajah * , g.r. green, m. naglak, abington, pa. objective: to study the various clinical outcomes of penicillin skin testing (pst) in a community-based hospital and to determine the percentage of patients who have an antibiotic modification and the choice of antibiotic used following the result of pst. method: this study is a retrospective chart review of all in-patients who were penicillin skin tested during a period of . years(jan to july ). information was collected on patients using a detailed data collection form. data was summarized using descriptive statistics including frequencies and percentages. results: of the patients penicillin skin tested, had a negative test, five had a positive test and in four patients the test was indeterminate(histamine controls were negative). eighty six percent of the patients had a history of penicillin allergy, . % had a history of cephalosporin allergy and . % had a history of both penicillin and cephalosporin allergies. the duration of antibiotic prior to pst ranged from zero to days. there was a . % ( / ) reduction in the use of vancomycin, a . % ( / ) reduction in the use of floroquinolones and a . % ( / ) reduction in the use of aminoglycosides following pst. aztreonam was used in . % ( / ) of patients before pst and in zero patients after pst. use of penicillin-based drugs was % ( / ) after pst and cephalosporin use increased from . % ( / (all third generation cephalosporins)) to . % ( / ( second and third generation cephalosporins)). i.e., % of patients with a negative pst received a penicillin or a cephalosporin. vancomycin usage was higher among pst positive patients. endocarditis was the diagnosis in % of all patients skin tested and staphylococcus aureus ( . %) and enterococcus ( %) were the most common organisms on culture. there were no serious adverse reactions to the use of penicillins or cephalosporins when used following a negative pst, and there were no adverse reactions to penicillin skin testing. conclusion: in the population studied, pst lowers the usage of vancomycin, floroquinolones and aminoglycosides and increases the use of penicillins. third generation cephalosporin usage was increased by pst in our study. overall, pst results in antibiotic modification that could lower the emergence of multi-drug resistant organisms and vancomycin resistant enterococcus. the aim of the study was to find a new marker which could be easily done to predict about possible allergy development in infants. preventive procedure is always economically better than treatment itself.food allergens are able to stimulate lymphocytes even in prenatal period. immunological maturity of newborns is still in the center of our interest because its dependence on many different factors. the question was if cd activity in cord blood and in -year-old children correlates with development or not of allergy? the examined group consists of newborns( , % of boys). breastfed were only children until -months-old and until they were -monthsold. total ige from cord blood and mothers' blood taken at delivery were measured using unicap. immunological detection was done using fluorocytometry(becton nad dako). additionally parents were questionaired( family atopy). our reaults show that positive family atopy history(group a) or elevated level of ige( group b) or symptoms of allergy in first months of life(group c) does not correlate with cd antigenicity. in all subgroups cd frequency was %. our findings show unfortunately that cd cannot be a predictive marker for allergy development. rationale: the etiology of eosinophilic esophagitis (ee) is unknown and the relationship to a type i allergic response is unclear. comparison of patients with positive and negative type i allergy testing may further clarify ee and determine what are the most common food allergens. methods: this is a retrospective chart review of medical records from january to january of children year of age with biopsy confirmed ee (n = ). ee was defined as having eosinophils per high power field on esophageal mucosal biopsy. patients were grouped according to positive (n = ) and negative (n = ) allergic responses on skin or radioallergosorbent testing (cap rast, pharmacia). skin testing with commercial extracts (hollister-stier laboratories and greer laboratories) was performed in an allergist's office. a wheal of mm greater than the negative control on skin testing or ige > . ku/l on rast was considered positive. we performed fisher exact analysis to determine if associations existed between a type i allergic response and factors such as age, symptoms, peripheral eosinophilia, and personal and family history of atopy (asthma, allergic rhinits, and atopic dermatitis). results: ee patients with a positive type i allergic response were significantly younger than those with a negative response (mean . yo, median . yo, range to . yo versus mean . yo, median . yo, range to yo; p = . ). vomiting as a presenting symptom was significantly increased in the allergic population ( %, %; p = . ) and abdominal pain as a chief complaint was significantly increased in the non-allergic population ( %, %; p = . ). personal and family history of atopy and peripheral eosinophilia were similar between groups. the most common allergens were cow s milk ( %), peanut ( %), egg ( %), soybean ( %), and wheat ( %). conclusion: patients diagnosed with ee who present at a young age or who present with symptoms of vomiting may have a type i allergic response contributing to the esophageal eosinophilic inflammation. consistent with food allergies in children, milk, peanut, egg, soybean, and wheat were the most common foods found with allergy testing. because the positive predictive accuracy of food annals of allergy, asthma & immunology testing is only about %, the challenge remains to develop a specific plan to confirm causative foods such as through oral challenges or elimination trials. rationale: the relationship between specific ige and igg levels in atopic individuals is unknown. increasing antigen exposure is expected to increase igg production, however, the influence on ige levels is unclear. to further clarify this relationship the following studies were conducted. methods: three hundred and sixty tandem determinations of specific ige and igg levels in individuals were collected using the immunocap instrument (pharmacia diagnostics) and commercially available reagents. only subjects who had ige levels > . ku/l and igg determinations > mg/l to the same specific allergenic species were included in the study. specific ige and igg determinations were to alternaria alternata, aspergillus fumigatus, penicillium notatum, cladosporium herbarium, felis domesticus, canis familaris, dermatophagoides farina, and periplaneta americana. correlation coefficients were calculated using excel (microsoft). results: the review of this data set yielded tandem determinations of specific ige and igg levels in atopic individuals. the most common ige sensitization was to alternaria. sixtyfour percent of individuals had specific ige directed against alternaria. the lowest ige response rate was for cat and roach. only % of individuals had an ige response to those specific species. correlation coefficients (cc) were calculated between specific ige and igg levels and were positive for all eight species tested. the highest cc was for dog (n = , cc = . ) followed by aspergillus (n = , cc = . ), penicillium (n = , cc = . ), alternaria (n = , cc = . ), cladosporium (n = , cc = . ), cat (n = , cc = . ), and roach (n = , cc = . ). the lowest correlation was for dermatophagoides farina (n = , cc = . ). conclusion: when individuals with measurable specific ige and igg levels are considered, there appears to be a positive correlation between specific ige and igg levels for many allergenic species. factors that influence the correlation are likely to be both genetic and environmental. identifying this link and a clinical application are our next challenge. recently, protein microarray tests are competing with traditional allergenspecific in vitro assays. while the advantages of microarrays in allergy testing are attractive, microarray analysis alone has not been very effective in reducing analysis time and automated microarray equipment typically is much larger than its benchtop counterparts. we have incorporated microarray technology into an automated microfluidic cartridge that provides rapid allergen testing using a compact, low-cost, desktop instrument. the injection molded microfluidic cartridge (fig. a) contains arrays of miniature pumps and valves that direct reagents independently to a solid phase reaction area. standard ige (nibsc) as well as allergens were immobilized within the cartridge to form a protein microarray. a small desktop analyzer actuated the pumps in the cartridge to automatically carry out a chemiluminescence-based elisa reaction. total ige quantitation was performed in a minute reaction. fig.b shows the resultant image of a dilution series of immobilized ige. concentrations ranged from . to iu/ml corresponding to . fg to ng ige per spot. the average cv value for ige quantitation was % showing good linearity (r = . ) and fg sensitivity. this quantitative ige curve is used to eliminate the effects of cartridge variability. specific ige detection was demonstrated using extracts, d. pteronyssinu(dp), a.fumigatus and b. verrucosa(bv). a -step, minute reaction elisa technique was employed. fig. c and d show the resultant images for bv and dp positive serum, respectively. the sensitivity of the dp specific ige test was further investigated using a positive class serum sample confirmed by mast. dilution of the class serum with negative control serum could then be detected on the cartridge down to an fold dilution, resulting in a limit of detection in the range of . - . iu/ml for dp specific ige. we have demonstrated total ige and allergen-specific ige detection with total analysis times less than minutes in a convenient, low cost system using a microfluidic-based microarray cartridge. such rapid diagnosis allows physicians to provide treatment while the patient is still in the office. background: accurate allergen skin tests (ast) are the basis of optimal care of the allergic patient. completing these tests on the first visit can expedite the diagnosis and treatment and offer patients(pts.) efficient use of their time and increase satisfaction. appropriate pre-visit preparation is necessary to reduce the variables that affect ast outcomes. both positive and negative skin test controls are necessary to assure the ast are reliable. methods: we conducted a retrospective chart review of , sequential pts. skin tested at an allergy practice, in order to examine the success of our pre-visit instructions and to identify other medications that may affect ast. pts. were given verbal and written instructions to discontinue antihistamines and other histamine- receptor antagonist medications (h- a) days before their visit. they then underwent ast only if adequate positive (histamine) and negative (saline/vehicle) controls were obtained. results: only ( . %) of the , pts. had inadequate histamine response (ihr). of these pts., ( . %) had used h- a within the prior days. ( . %) pts. had taken h- a in the recent past, but stopped them at least days prior to testing. ( . %) pts. had no exposure to h- a. the use of psychiatric medications in the pts. with an ihr was also examined. of the pts. who discontinued h- a for more than days, ( %) were also taking various medications drugs used in the treatment of psychiatric disorders (ssris, benzodiazepines, atypical antidepressants & antipsychotics) which are not usually associated with h- a. the group of of the pts. with no prior use of h- a included ( %) who were also on various psychiatric medications. the high prevalence of psychiatric medication use in these two groups is in contrast to the to pts. with recent use of h- a in whom % were on psychiatric medications. conclusion: pre-visit patient education about discontinuation of h- a can lead to successful first visit ast in the overwhelming majority of patients ( . %).failure to stop h- a at the appropriate time occurred in only . % of the pts. . % of pts. discontinued h- a for the recommended day interval, but still had ihr. another % of pts. had an ihr yet no obvious use of h- a. a few pts. taking psychiatric medications not normally associated with h- a had ihr. further investigation into this issue is warranted. a. blaziene * , a. chomiciene, l. jurgauskiene, n. ciaponiene, vilnius, lithuania. background: sublingual specific immunotherapy (sit) is accepted as an alternative treatment to subcutaneous sit of seasonal allergic rhinitis. the aim of this study was to evaluate changes in allergen-specific ige and basophil degranulation test (bdt) before and after year of sit with a grass pollen mix. methods: patients ( male and female, aged - years) having sensitization to grass pollen with seasonal allergic rhinitis were treated with standardized grass pollen extracts. the blood samples were collected before and after year of sit assessing allergen-specific ige using an elisa method and bdt using a direct immunofluorescent method employing monoclonal antibodies. results: the sit was well tolerated by all patients. an increase in total ige after sit was observed in all patients. specific ige concentration was increased in ( . %) patients, while the bdt result was greater in ( %) patients after year of sit. conclusions: allergen-specific ige and bdt may serve as markers to indicate the clinical efficacy of sit for seasonal allergic rhinitis patients. a.g. palma-carlos * , s.l. silva, m.l. palma-carlos, lisboa, portugal. introduction -the incidence of primary immunodeficiencies in patients attending an out-patient center for clinical allergy and immunology depends on the recruitment and on patients refered. methods -in the last years patients have been observed in lisbon clinical allergy center reporting allergic diseases or repeated infections or refered by specialists (ent, gynaecology, internal medicine, pediatrics, or gp.). screening for primary immunodeficiencies has been done by electrophoresis, assay of immunoglobulins and complement, igg subclasses and flux-cytometry for t, b and nk cells. results - cases ( , %) of primary immunodeficiencies have been diagnosed. humoral: deficits of iga ( , % of immunodeficiencies), of igg ( , %) of igg subclasses ( , %) of common variable immunodeficiency cvi-( , %), of igm ( , %). complement: cases of c esterase inhibitor deficiency ( , %) cellular: cases of mucocutaneous candidiasis ( , %) in fertile females, cases of cd deficiency ( , %). combined: cases of deficiency of the couple cd /c ligand with igg deficiency ( , %).conclusions -in the present series chronic mucocutaneous candidiasis is the prevalent primary immunodeficiency, due to the number of patients refered by gynaecologists. aside of this group where a decrease of nk cells was the more common immunologic pattern, humoral immunodeficiencies are frequent, mainly cvi, iga, igg and igg subclasses deficiencies. the search for immunodeficiencies must be done in all patients with repeated infections attending immuno-allergology departments. a case of atypical c complement deficiency. c.c. randolph * , waterbury, ct. introduction:c complemennt deficiency occurs in in , individuals .it results in decreased opsonization and chemotaxis with increased prevalence ofsle and other rheumatic disordersas well as enhanced vulnerability to pyogenic infection.there are two types of c complement deficiency :type ( %associated with sle)with no protein translation and type ii with absence of protein secretion.we present a year old white female otherwise healthy with recurrent urticaria and angioedema with c complement deficiency. method:case report:a y/o w/f athlete presented with a two month history of recurrent hives and angioedema which she associated with ingestion of halloween candy .one week before evaluation she had hives with coconut as well.her history was othewise unremarkable except for recurrent uti's, annual sinusitis, pneumonia in as well as migraines.she denied sexual activity.her physical exam was normal.results:an evaluation for autoimmune disease revealed normal esr, ana, dsdna, mono and hepatitis serology as well as lyme titers however her ch was low u/ml(normal - u/ml)and evaluation of complement revealed c mg/dl(normal - mg//dl)and c < . mg/dl(normal . - . mg/dl)with normal c , c -c .her father had nor-malc but c was . mg/dl (normal . - . mg/dl)her sister had c of . mg/dl and normal c and her mother had normal c and c .her workup included positive prick skin test to ragweed, ash and grass and she was started on rhinocort and clarinex seasonally.she has been followed for one year with resolution of hives and is asymptomatic.her diagnosis had been confirmed by a pediatric rheumatologist.conclusion;we present an atypical case of c complement deficiency in an currently asymptomatic individual. background: we diagnosed a month old male with cgd and aspergillus brain abscesses and pulmonary infiltrates. review of the literature suggests he is one of the youngest cases of cgd complicated by cerebral aspergillosis. case report: a ten month old male previously seen for unexplained persistent pulmonary infiltrates since month of age presented to our hospital with lethargy, fevers, and increased irritability. ct scan of the head revealed severe hydrocephalus. multiple brain abscesses were found on surgical exam and cultured for aspergillus fumigatus. he had a history of failure to thrive and hypotonia since birth. family history was significant for parents that were second cousins. there was no family history of recurrent infections or childhood deaths. physical examination was significant for a child with weight and height in the % and %, respectively with persistent fevers > . c, hypotonia, hepatosplenomegaly, and left lower lung field rales. initial immune workup was normal except for elevated total ige of ku/l. cd + ( /m ) and cd + ( /m ) t cells were initially low, but later increased to normal levels without intervention. neutrophil oxidase activity assessed by dihydrorotamine (dhr) oxidation was % of control. superoxide production assessed by cytochrome c reduction was zero. the patient was treated with interferon ? for presumed cgd. his aspergillus brain abscesses improved with intravenous voriconazole and caspofungun. the responsible organism for his recurrent pulmonary infections was not identified, but improved with antifungal therapy and broad spectrum antibiotics. the dhr test of both parents and siblings were normal. antibody testing showed a complete deficiency of the p protein with normal levels of gp , p , and p proteins. evaluation of the exons of the ncf- gene that encodes the p protein were normal. conclusion: mutations due to p protein deficiency are responsible for % of cases of cgd. there has been only one previously reported case of p deficiency without ncf- exon mutations in which a single point mutation in an intron of the ncf-ii gene was identified. fungal infections may complicate cgd, however cerebral aspergillosis is seldom encountered. this case annals of allergy, asthma & immunology reports a month old with cgd due to a p deficiency without exon mutations complicated by cerebral aspergillosis. introduction: lymphoid interstitial pneumonia is an uncommon condition which is considered both as a disease per se or as an inflammatory pulmonary reaction to various external stimuli or systemic diseases; however, at the present time most of the cases remain idiopathic. we present a case of an infant who developed a lymphoid interstitial pneumonia associated to a deficit of interferon-production. methods: we describe a clinical case and review the medical literature. results: we present the case of a five months old male with a history of cough, respiratory distress and four previous hospital admissions regarded as a 'pneumonic' events from two months of age and four siblings dead (two of them with 'abdominal disease' and one of them with candidiasis) during their first year of life. the parents referred no consanguinity. the physical examination showed a malnourished child with signs of bilateral lung consolidation. oxygen supplementation and intravenous antibiotics were started. our patient didn't have neonatal history related to the present compliant. the chest film revealed a diffuse bilateral interstitial pattern which was confirmed by pulmonary ct scan. sweat chloride test, hiv and epstein barr virus serology test were all negative. an initial immunologic evaluation reported slightly increased leukocytic count, hypergammaglobulinemia, lymphocityc flow citometry and nitro blue tetrazolium reduction test in normal parameters. the open lung biopsy showed a thicked pulmonar interstitium with moderate number of lymphocytes with not atypical pattern. our patient was discharged with clinical improvement, ambulatory oxygen supplementation and inhaled fluticasone-salmeterol. in an ambulatory follow-up we evaluated lymphocytic production of cytokines and we found no levels of interferon-. we started weekly administration of oral transfer factor. the patient showed an excellent clinical improvement evidenced by no more oxygen dependence, weight gaining and absence of further hospital admissions. con-clusions: our patient represent a new linkage between lymphoid interstitial pneumonia and primary immunodeficiency characterized by interferonproduction deficit, maybe with a non previously described molecular defect and a probably autosomic recessive inheritance pattern; also we confirmed the transfer factor usefulness to induce gamma interferon endogeous production. objective: marijuana is a schedule class psychoactive control substance more frequently used by the oriental societies( in the event of jubilation) causing altered state of conscience, euphoria, its prolonged use is followed by addiction and dependence.it contains more than chemical entities with over are of the cannabinoid i.e, cannabidiol (cbd), cannabinol (cbn), and delta- , tetrahydrocannabinol (thc) the later on use has been associated with craving for further use of narcotics just to enhance the euphorient effects .the active ingredients i.e, delta- -tetrahydrocannabinolthc) and other cannabinoids(thc) are liquid soluble at high concentrations which alters membrane function, resulting in alterations in immune cell response. cannabinoid has immunosuppressant properties causing impaired cell-mediated and humoral immune system activities, cytokine production, leukocyte migration and natural killer-cell(nk) activity resulting in reduction in the host resistance to bacterial and viral infection, (pms)with hiv infection are at higher risk of developing aids, infection by opportunistic bacteria, fungi, or viruses (pms), when compared to non marijuana smokers, have more respiratory ill-ness.cannabinoids also characteristically as being immunomodulators i.e, generally suppressing but occasionally enhance some immunological responses, some have suggested that the immunosuppressive effects of cannabinoids might be useful clinically; for example, in treating multiple sclerosis.as per clinical response cannabinoids had been found exacerbating existing allergies from antigenic complex (eliciting formation of specific antibodies/metabolites as a hapten, combining with a body proteins). methods .in the follow up studies including individuals(all males age - years) with(pms).of more than months having serum level of tch > um, there have been times reduction in the proliferation of t lymphocytes with a proportionate increase in the proliferation of b lymphocytes, reduction in the cytotoxic activity of t lymphocytes, reduction in macrophage activities i.e, phygocytosis rationale: patients with xla are subject to arthritis and cellulitis. these three relatives have had similar courses, suggesting infectious etiology. methods: two brothers and a cousin, from an african-american family known to carry xla, have been diagnosed with xla and chronically treated with ivig. all three have developed arthritis and cellulitis of a lower extremity. the eldest, now years old, had a -month waxing and waning arthritis and cellulitis that was refractory to the oral antibiotics attempted. his younger brother had not yet accomplished pubertal changes at the age of , and was below rd percentiles for weight and height. he also had several months waxing and waning arthritis and cellulitis refractory to oral antibiotics. he and his cousin have had esophogastroduodenoscopies that show gastritis and duodenitis with heavy growth of an organism visualized by specific staining for h. pylori. the cousin has also experienced more recent weight loss and arthritis/cellulitis. results: the younger brother had a blood culture that grew a curved gram-negative rod. a subsequent culture at the state lab grew a similar organism that was urease-negative. the two brothers are at or near completion of a six-month course of ertapenem and gentamicin; they have had resolution of their arthritis and cellulitis. the younger has gained . kg and experienced his pubertal changes at the age of years. con-clusions: there are prior reports of several different related organisms that can cause arthritis and cellulitis in patients with xla. these include helicobacter, campylobacter, and flexispira species. the urease-negative organism would not be flexispira, but may be a campylobactor species or helicobacter canis. the results from this family suggest that long-term combination iv antibiotics may be indicated for patients with this syndrome. slow virus infections have been reported to affect the central nervous system. parvovirus b , herpes and cmv are usually not included in the diagnosis of cns diseases nor has been associated with cns manifestation in an immuno-compromised patient. in this abstract, we report on a neurological manifestation that can be linked to ebstein-barr/herpes virus and parvovirus-b in an immune deficient patient. a year old female presented to our clinic with a history of chronic fatigue symptoms, daily arthralgias, frequent sinus infections and idiopathic tremors for the past years. the patients neurological evaluation, including mris was found to be within normal limit, despite worsening tremors of her head and arms. a full clinical and labora-tory evaluation was performed which revealed igg subclass, low t-cell numbers and functioning, low response to specific antibodies and positive igm for ebv, parvo and cmv. the patients common immune deficiency coupled with chronic viral infection and worsening tremors led us to try high dose ivig ( g/kg divided over days on cycles of every weeks). within three months of starting ivig, the patient had a large reduction in her tremors and negative igms for parvovirus, herpes and cmv. given our findings, we suggest that ivig may play a role as a neuro-immune modulator, as has been shown in other neuro-immune diseases. we further suggest that the interaction between parvovirus b- , herpes and cmv as part of the slow viruses, combined with underlying immune disorder may lead to neurological presentation and high dose of ivig can reverse the syndrome. purpose: to date, only two patients older than fifty years of age have been diagnosed with velocardiofacial syndrome (vcfs); this case represents the third such patient. methods: physical and laboratory findings of the patient are presented as a case report. results: we describe a sixty-six-year-old male, recently diagnosed with vcfs, confirmed by fluorescent in-situ hybridization (fish), was referred by his psychiatrist for comprehensive care. he had a history of cleft palate and learning disabilities as a child, with the onset of psychiatric illness in his late teens. although there was no history of cardiac disease, the patient had agenesis of the left renal artery as well as other vascular anomalies, including hypoplasia of the left a segement of the anterior cerebral artery. other findings included hypothyroidism, hypoparathyroidism, sensorineural hearing loss, and typical facies. he also suffered from chronic candidiasis. immunologic laboratory studies revealed over a : ratio of cd :cd t cells with a decreased cd percentage, markedly diminished b cell percentage, decreased t cell mitogen responses, and markedly decreased b cell mitogen responses. nevertheless, total serum igg and specific antibody responses remained normal, with a low serum igm. conclusions: this patient meets the criteria for the diagnosis of vcfs; only the third reported case diagnosed over the age of fifty. though his immunologic findings may be consistent with his diagnosis, they may be due in some part to immunologic senescence given his age. background: the classic presentation for patients with xla and mutations in the btk gene includes marked hypogammaglobulinemia, absent b cells, and significant sinopulmonary infections beginning at - months of age. typically, mds presents with anemia, leukopenia, monocytosis, and thrombocytopenia with recurrent infections, skin rash, and hepatosplenomegaly. many of these subjects demonstrate chromosomal abnormalities including monosomy . we describe a case of mds presenting with some features of both conditions. clinical history: we present a year old male with a suspected diagnosis of xla, chronic sinopulmonary disease beginning at mo, diffuse bronchiectasis, and agammaglobulinemia with absent b cells. he had wbc = cells/ul, with % monocytes and platelet count of , /ul. he had no anemia, hepatosplenomegaly, or skin rash. bone marrow biopsy showed mds with absent plasma cells and monosomy in % of his cells. btk expression and sequence analysis were normal. conclusion: while xla is the most likely primary immune deficiency that would present in a male with the clinical picture described above, this case illustrates the importance of maintaining an expanded differential diagnosis in patients with presumed primary immunodeficiency. j. wang * , l. mayer, c. cunningham-rundles, new york, ny. background: chronic granulomatous disease (cgd) usually results in acute or chronic infections with a defined spectrum of bacteria or fungi. other characteristics include inflammatory disorders, including genitourinary or mucosal inflammation resembling inflammatory bowel disease. gm-csf has been used in the treatment of mucosal inflammation in glycogen storage disease ib and crohn's disease with some success. objective: to explore the use of a novel therapy in the treatment of mucosal inflammation associated with cgd. methods: the patient was treated with daily gm-csf ( . mcg/kg/dose subcutaneous injection) along with parenteral antibiotics and total parenteral nutrition. after days on gm-csf, hydrocortisone enemas were added due to continued pain and swelling. results: rectal and abdominal pain significantly improved, blood streaked stools decreased; he tolerated a regular diet and was discharged home days after starting gm-csf. he now has no rectal or abdominal pain and no blood in the stool. conclusions: gm-csf may be a useful therapy for the management of mucosal inflammation in cgd patients. it appears safe and well tolerated and permits avoidance of immune suppressive alternatives. introduction: management of autoimmune cytopenias in patients with cellular immunodeficiency may be very challenging. treatment of these cytopenias with corticosteroids may potentiate the immune dysfunction leading to further infections. methods: we describe a month-old female with a cellular immunodeficiency and autoimmune thrombocytopenia / hemolytic anemia being treated with steroids and ivig without improvement. rituximab, a monoclonal antibody that binds to b-lymphocyte cd surface antigens, was initiated. results: this patient presented at months of age with failure to thrive, eczema and recurrent bacterial pneumonias. she was diagnosed with a t lymphocyte immunodeficiency with decreased absolute numbers of cd ( ), cd ( ), and cd ( ) lymphocytes. t cell function was markedly decreased as measured by pha, cona, and pwm. she developed chronic thrombocytopenia with platelets of less than , and coombs positive anemia with hemoglobin of g/dl. she was placed on mg/kg/day of prednisone with minimal improvement and continued to develop severe pneumonias. rituximab mg/m /week was initiated for doses. her cytopenias improved with an increase in both platelet count ( , ) and hemoglobin ( . g/dl). her absolute cd count fell from to . she was weaned off steroids permanently. she has also continued on ivig, prophylactic antibiotics, and as needed rituximab pending bone marrow transplant. conclusions: rituximab, a monoclonal anti-cd antibody, may be helpful in treating autoimmune cytopenias in patients with cellular immunodeficiency in which steroids need to be avoided. ivig antibody replacement substitutes for the subsequent humoral antibody depletion due to induced secondary b cell lymphopenia. introduction: the patient is a month old male born at full term. during his prenatal course he was noted to have a pulmonary abnormality in his left lower lobe at approximately weeks of age. serial ultrasounds and fetal mri were consistent with the diagnosis of congenital cystic adenomatoid malformation (ccam). histology from elective resection of the lesion performed at months of age revealed no evidence of ccam. silver stain revealed florid pneumocystis jiroveci (carinii) infection. the patient was completely asymptomatic. the differential diagnosis for this infection in the newborn period includes hiv infection, severe combined immunodeficiency, and x-linked hyper-igm syndrome. the infection has also occurred in newborns with apparently normal immune systems. methods: immunology evaluation was performed to evaluate the patient for the listed diagnoses. a complete blood count was performed. lymphocyte subsets were performed by flow cytometry. quantitative immunoglobulins g,a, m and e were measured by nephelometry. specific antibodies to tetanus and diptheria were determined by elisa. hiv status was ascertained by western blot and dna pcr. dichlorofluorescein assay was performed. fluorescenceactivated cell sorter technique was used to evaluate cd ligand. lymphocyte stimulation to mitogens and tetanus were performed. results: the patient had negative hiv / antibodies and a negative hiv dna pcr. the result of the complete blood count was normal with an absolute lymphocyte count of , . the patient had normal percentages and numbers of cd , cd , cd , cd , and natural killer cells. lymphocyte stimulation to mitogens and tetanus was normal. dcf assay was normal. facs revealed normal levels of cd ligand. at months of age he had protective titers to diptheria and tetanus titers of . iu/ml (protective > . iu/ml). at months igg, a, m and e were (normal - ), < , , and < respectively. conclusion: immunologic testing revealed no evidence of severe combined immunodeficiency, hiv infection or x-linked hyper-igm syndrome. the patient remains well clinically with normal growth and development with no subsequent infections. pneumocystis jiroveci pneumonia can be seen in infants with apparently normal immune systems. commercial preparations of igg produced for iv use (ivig), must fulfill regulatory requirements. the method of preparation, however, may produce alterations in the content or function of igg subclasses such as disturbed composition which can be reflected as a lowered clinical effectiveness of the product. we report patients with specific igg immunodeficiency, that, when changed to a new ivig preparation, had lessening of clinical effectiveness of the new product but who also experienced recovery after they were changed to a different ivig product. case . a y/o male with hx of cervical lymphadenopathy associated with recurrent uri, fatigue, cognitive alterations and hypercholesterolemia. he was dx in with a specific igg immunodeficiency for which he was started on ivig infusions mgxkg q/ w which brought his igg levels to normal values mg/dl, ( - mg/dl) with good clinical improvement. when he was changed to a different ivig product with lower iga content his initial symptoms returned with decrease of his igg to mg/dl ( - mg/dl) he was changed to a different ivig product with higher iga content with disappearance of all his previous symptoms. case . a y/o female with history of chronic yeast infection, fatigue, joint pain, asthma, ge reflux, depression, hypothyroidism and shunt for hydrocephaly. she was diagnosed with igg and igg immunodeficiency and started on ivig mg x kg x q/ w. when changed to another ivig with lower iga content she experienced worsening of her symptoms during the months that she was receiving it. she was changed to a different ivig product with a higher iga content with complete improvement of her symptoms. although the content of iga in product ( - ug/ml) was significantly lower than in prod-uct ( ug/ml), the total igg and igg subclass distribution (including igg ) were comparable therefore, the lack of clinical effectiveness of product would appear to be related to qualitative alterations in the product related to methodological preparation possibly in removal of the iga or other physicochemical alterations affecting biologic activity which contributed to a lessened clinical efficacy of the product. we present these cases to alert the allergist-immunologist to these observations when treating patients with igg or igg subclass deficiencies. background: development of atopy has been known to occur in nonatopic recipients of bone marrow transplants ( bmt) from atopic donors. the reverse condition, with atopic recipients losing evidence of specific ige after bmt from non-atopic donors is quite unique. case report: ph is a year old male referred for recurrent nasal congestion. as a teenager, the patient was treated for chronic allergic rhinitis and asthma, atopic dermatitis diagnosed as severe, and multiple food allergy. skin testing done as a teenager showed + reactions to multiple pollen, dog, cat, most seafood, cashew, walnut and pecan. at years of age, he underwent an allogeneic bmt from his non-atopic brother for acute myelogenous leukemia. asthma and atopic dermatitis were observed to clear almost completely and allergic rhinitis improve after the bmt. his blood type changed from + to -. skin testing done on his current visit showed unremarkable responses to all pollen, cat, dog, dust mites and foods with good histamine control. discussion: the negative skin test results suggests that his current nasal symptoms are vasomotor in nature. considering the past severity of his symptoms, it appears unlikely that he lost his specific-ige sensitivity spontaneously. transfer of atopy is known to occur in non-atopic recipients of bmt from atopic donors. in a study of patients undergoing allogeneic bmt for hematologic malignancies, nonatopic recipients developed positive skin tests with profiles similar to atopic donors. a possible mechanism is passive transfer of memory b-cells which initiate specific-ige production with a donor pattern. a reverse of this mechanism could occur with the recipient gaining lymphoid precursors with no atopic tendency leading to clearing of atopy. the literature yielded one report of asthma resolving after high-dose chemotherapy and autologous stem cell transplantation the authors suggested that chemotherapy could have resulted in immune system reconstitution, normalization of the t-cell repertoire and resolution of asthma. introduction: primary central nervous system (cns) lymphomas constitute only a small percentage of central nervous system tumors in adults and are seen more frequently in aids patients and immunodeficiency states. they are extremely rare in children, even those with primary immunodeficiency. we present a -year-old female with combined immunodeficiency who developed an eber+ (ebv-associated) large b-cell lymphoma that was confined to the cns. methods: the patient is a -year-old female that was being evaluated for combined immunodeficiency who presented to our institution with acute fever and seizure activity. results: the patient had a history of recurrent pneumonia, otitis media, sinusitis, and upper respiratory infections. she also exhibited failure to thrive, chronic diarrhea, and sen-sorineural deafness. she was found to have a combined immunodeficiency with severe neutropenia and lymphopenia, low iga, low igm, and poor specific antibody response to protein and polysaccharide antigens. she had no response to a candida delayed hypersensitivity skin test. hiv tests were negative. she was thought to have acute meningoencephalitis causing the fever and seizure activity, however bacterial, viral, and fungal studies were negative. she received multiple anitbacterial, antifungal, and antiviral agents. she died secondary to brainstem compression due to severe cerebral edema. autopsy revealed an eber+ large b-cell lymphoma that involved about % of her brain and spinal cord. no tumor cells were found elsewhere. con-clusions: this case represents a rare manifestation of combined immunodeficiency: the development of a primary cns lymphoma. the tumor cells were found to be eber+ (ebv-associated). ebv is known to be associated with lymphoma, especially in aids and immunocompromised patients. only . % of primary tumor sites in severe combined immune deficiency patients involved the cns in the immunodeficiency cancer registry that was instituted in the s. v. litvinova * , g. muzlaev, krasnodar, russian federation. introduction: glyoma is one of the most prevalent diseases among all brain tumors and more than % of them are malignant. in the previous investigations it has been shown the immune disorders in patients with glyoma tumor. at the same time it was suggested the possible anti-tumor activity of some proinflammatory cytokines (tnf, il , il ), which can penetrate via haematoencephalic barrier and induce the lysis of tumor cells. the aim of this investigation was to study the serum and spinal fluid concentrations of one of the key immunoregulatory cytokines -gamma ifn and il in patients with glyoma tumor. methods: the concentrations of the gamma ifn and il have been studied in serum and spinal fluid of patients with glyoma by eliza method. in control, the same parameters have been studied in patients with brain trauma. results: it has been shown that concentrations of gamma ifn and il in serum and spinal fluid was . - times higher than in trauma patients (p< . ). the serum levels of gamma ifn and il in glyoma patients were . and . pg/ml, accordingly. in trauma patients- . and . pg/ml. the concentration in spinal fluid of the studied cytokines was . time higher than in control patients. conclusion: proinflammatory cytokines gamma ifn and il may be involved in pathogenesis of glyoma and can participate as possible anti-tumor factors of immunity in glyoma patients. a. arrey-mensah * , r.u. sorensen, new orleans, la. rationale: immunodeficiencies need to be ruled out in infants that present with failure to thrive. patients with t-cell lymphopenia, hypogammaglobulinemia and pancytopenia may have a primary immunodeficiency such as scid, or a secondary immunodeficiency that may need to be managed differently. methods: evaluation of cellular and antibody-mediated immunity of a month old aaf admitted with failure to thrive and chronic diarrhea. ohistory of duodenal atresia corrected by creating a blind duodenal loop and anastomosis of the stomach to the jejunum at birth. o mass in hypogastrium. results: immunologic evaluation revealed: " lymphopenia ranging to cells/ul " anemia: hemoglobin . g/dl, retic . " ancs ranging to " thrombocytopenia l to l " cd +: " cd +: " cd /cd r: . " cd : " cd : " mitogen responses were normal: " pha , cpm " con-a, , cpm " pwm, , cpm immunoglobulins: igg mg/dl iga mg/dl igm mg/dl ige iu/ml total protein was . g/dl, albumin was . g/dl the immunoglobulin-g half-life study was days in our patient: metabolic imbalance: hypokalemia ( . mmol/l) hyponatremia ( mmol/l) hypocalcemia ( . mg/dl) blood, stool, urine cultures normal. stool ova, parasite, virus were negative. hiv-pcr (-) radiological evaluation: superior mesenteric vein thrombosis, chronic malrotation and volvulus, dilated duodenal blind loop with possible lymphatic obstruction. the patient improved clinically and all immunological markers normalized after surgical removal of adhesions and obstruction. conclusion: the patient had a secondary immunodeficiency due to lymphangiectasia. lymphangiectasia should be considered in the presence of a lymphopenia with normal lymphocyte function and hypogammaglobulinemia accompanied by hypoproteinemia and hypoalbuminemia. hypogammaglobulinemia and hypoproteinemia were secondary to gastrointestinal losses. the immunological component of q . deletion syndrome (also known as digeorge syndrome;dgs), hypoplasia of the thymus, is quite variable among patients and provides the opportunity to determine the relationship between thymic function and t cell receptor (tcr) repertoire diversity. using a novel measure of repertoire diversity based on cdr length polymorphism called hamming distance, tcrbv repertoire diversity in dgs was found to differ from control subjects by having an overall skewing of receptor expression. as expected from clinical outcomes, there was also great intra-individual variability in dgs tcr repertoires. the degree of repertoire diversity was directly correlated with thymic output as measured by t cell receptor excision circles (trecs), i.e. greater thymic output resulted in more diverse repertoires (see figure below). this result demonstrates a quantitative relationship between thymic function and repertoire diversity in humans and may reflect the balance between thymic output and peripheral expansion to maintain an adequate tcr repertoire. in addition, it may suggest a basis wherein limited repertoire diversity mediates both immune deficiency and autoimmunity. tcr repertoire diversity in dgs, as measured by hamming distance, correlates with thymic output, as measured by trecs (plotted on a logarithmic scale) a. sabra * , s. sabra , h.j. castro , j. malka-rais , j.i. mendez-inocencio , g. santos , j.a. bellanti , . rio de janeiro, brazil; . washington, dc. a major investigative effort of our laboratory has been directed to the study of non-ige mechanisms and their role in the immunopathogenesis of food allergy (fa). we have previously reported th and th cytokine alterations associated with several clinical entities that had overlapping disease manifestations affecting the mucosal-associated lymphoid tissues (malt), of the gi tract (galt), skin (salt), nasal (nalt) and bronchial tissues (balt). the objective of the present study was to evaluate specific immunologic alterations in a group of patients with non-ige fa. peripheral blood cd and cd lymphocyte subset analyses were performed in patients with fa documented by double-blind placebo-control food-challenge (dbpcfc). all patients were treated with an amino-acid based formula (aabf) and then received an open challenge using a panel of commonly offending allergenic food allergens in a normal diet.the clinical picture of all patients with fa were linked to malt-related manifestations; with galt symptoms of diarrhea, vomiting, abdominal pain and ftt, with balt symptoms of asthma, with salt symptoms of eczema, with nalt symptoms of rhinitis and with edema, ascites and anaphylaxis. all subjects had normal serum ige and eosinophil levels, negative ige food rast tests, normal cd (mean + ) and very low cd ( + ) levels in peripheral blood. abnormal cd /cd ratios > . were observed in all patients. age-and gender-matched controls revealed cd /cd ratios ranging from . to . . both patients with anaphylaxis had cd /cd ratios > . all patients responded well to aabf. open challenge to common offending foods from a regular diet (milk, soy, wheat, egg, nuts, beef and chicken), revealed multiple food allergies. after two years of follow-up on an aabf regimen, the patients remain well but allergic to multiple foods. very low cd levels remain as the unique immunological alteration in all patients.these studies suggest that abnormalities of very low cd distribution may play a pathogenetic role in non-ige mediated fa. since cd lymphocytes play a role in immunologic tolerance (it), it is tempting to speculate that the very low cd levels may signal the failure of development of it in our patients predisposing them to the development of allergies to multiple food components. background: mixed connective tissue disease (mctd) is a disease taht have caused controversy, while some authorities consider it a distinct rheumatic disease, others believe that it's an early stage of a fully defined autoimmune disease. the distinctive feature is the presence of the u small nuclear rnp autoantibodies. clinical features involve raynaud's is phenomenon, swollen hands, sclerodactyly, esophageal hypomotility, polyarthritis, and myositis, allof them can be present in others well deined rheumatic diseases. only aew cases have been described in the pediatric population. objetive: to determine the frecuency of mixed connective tissue disease in mexican.children in a tertiary level institution according to kasakawa's criteria, alarcon-segovia's criteria and sharp's criteria and establish if a diagnosis of a well defined autoimmune entity was made during the follow up. methods:medical charts were assessed with the diagnosis of mctd between and in our hospital. results: between and , , cases of autoinmune diseases were treated; with systemic lupus erythematosus(sle), with rheumatoid arthritis(ra), , with scleroderma and , with dermatomyositis. we found only four cases, of them did not complete the kasakawa's criteria but the diagnosis was probable according to alarcon-segovia and sharp's criteria. one of them developed sle after one year of follow up. the case that fulfilled the diagnosis criteria of mctd; raynaud's phenomenon, rnp antibody positive, arthritis, lymphadenopathy, restrictive pulmonary disease, sclerodactily and muscle weakness developed a full blown sle at years follow up. discussion: the disease is extremely rare in children. we conclude that at least in pediatric age the disease may not exist by it's own, but it's an early stage of a defined autoimmune disease, and the terminology "un differentiated connective tissue disease" is more appropriate to define this cases. a. baysan * , h.y. song, s. gupta, l. yel, irvine, ca. hereditary angioedema (hae) is an autosomal dominant disease characterized by episodic angioedema of the skin or mucosa of the respiratory and the gastrointestinal systems. hae is caused by a quantitative (type i) and/or functional (type ii) deficiency of plasma protein c inhibitor, an early component of the classical complement pathway. attacks of hae may be lifethreatening and even cause death when the airway is involved. here, we report a year-old female patient with hae type ii, who has seven affected family members, two of whom died of laryngeal angioedema. the patient had a history of recurrent swelling of the eyelids, lips, tongue and extremities, and respiratory distress since five years of age. she was hospitalized on several occasions one of which required intubation and assisted ventilation. laboratory studies, between the attacks, revealed normal serum ch and complement c levels. complement c level was decreased to mg/dl (n: - mg/dl). c esterase function was impaired, % (n: greater than %) in contrast to normal quantitative c esterase. the patient was on long-term danazol treatment for ten years and experienced side effects, most notably hirsutism. currently, there are limited efficacious and safe treatment options for hae. the treatment of choice for acute attacks and prophylaxis appears to be the c inhibitor concentrate, which is not yet licensed in the us. the present patient emphasizes the need to establish the correct diagnosis and an appropriate management plan in recurrent angioedema. j. hajsam * , l. ponomarjev, krasnodar, russian federation. background: the previous investigations indicate on the positive effects of ncir in different chronic inflammatory and infectious diseases. nevertheless, the mechanisms of the clinical efficacy of ncir remain still unknown. the aim of this investigation is the study of the immune disorders in patients with chronic tonsillitis and the immunomodulatory effects of the complex treatment in combination with ncir. methods: children with chronic tonsillitis in the age from to years old were under the observation. the t cell receptors (cd +, cd +, cd +, cd +, cd +, hla-dr+) have been studied using flow cytometry method. the cytokines (il , gamma ifn and il ) have been investigated by eliza method. the treatment include traditional methods in combination with ncir. control group (without tonsillitis) consists of children of the same age. results: it has been shown that in chronic tonsillitis was dcrease of the number of cd +, cd + and hla-dr+ cells. the decrease of cd + cells was mainly due to decrease of cd + cells. at the same time it has been shown the increase in number of cd + cells. in chronic tonsillitis have been determined the increase in serum proinflammatory cytokine il level in . times (p< . ). at the same time the levels of gamma ifn and il were lower than in control up to - times. the studied treatment method including ncir resulted in increase in the number of cd + and cd + cells. the level of il decreased from pg/ml to . pg/ml. at the same time the serum concentration of gamma ifn and il increased up to . and . times, accordingly. conclusion: in chronic tonsillitis it has been shown the immune disorders in t cell's subpopulations and cytokine production. the efficacy of ncir mainly depends on the normalisation in t cellular subpopulations and increase in the concentration of gamma ifn and il . introduction: viral hepatitis is characterized by suppression of cd +th cell function related to disease severity. the aim of our research was the determination of total ige concentration and anti-c q-autoantibodies levels in patients with viral hepatitis c (vhc) and mixed viral hepatitis (vhb+c) and the investigation of their correlation with t cell parameters. methods: patients with vhc and patients with vhb+c confirmed by pcr were examined. evaluation of serum total ige level was performed by elisa kits produced by "vector-best", russia. immunophenotyping of peripheral blood mononuclear cells (pbmc) was done by cytometry with monoclonal antibodies to cd , cd , cd , cd b, cd , cd , cd , cd and hla-dr-antigens. results: the level of serum total ige in patients with vhc and vhb+c was significantly increased in all groups studied, especially in acute hepatitis c ( . ± . mu/ml, p< . ) compared with the control group. analysis of pbmc subpopulations in patients with vhc and vhb+c was done related to the total ige level: group i - - mu/ml; and group ii -> mu/ml. in patients with viral hepatitis and high total ige level, the content of cd +t-lymphocytes, cd +t-cells and cd /cd ratio were significantly decreased compared with the control group (p< . ). more notable changes were found in patients with acute vhc and vhb+c. the increase in cd +-cell content was seen in groups with both low and high levels of total ige. the content of cd +-cells in patients with high levels of total ige was also increased (p< . ). conclusion: patients with vhc and vhb+c had significantly greater levels of total ige compared with the control group (p< . ). the increase in total ige levels in patients with viral hepatitis is associated with imbalances of t-lymphocyte subpopulations, including a decrease in cd +t-cells, and an increase in b-and nk-cells. title: occupational airway allergy among health care workers (hcw) with latex allergy. introduction: for the last years the frequency of latex allergy has increased. health care workers (hcw) is the risk group of this diseases. latex allergy symptoms occure in different organs -including skin, conjunctive, nose and bronchi. study aim:the aim of this study was to estimate the incidence of airway allergy in group of hcw with latex allergy. materials and methods: the investigations were carried out in a group of hcw, aged - (average age , ). there were two hundred and eight women and forty four men in the group. investigation consisted of questionaire examinations, skin prick tests (latex), patch tests (rubber additives), sige (latex) and spirometry. results: seventy eight ( %) hcw reported undesirable effects after the contact with latex products. fourty ( , %) hcw reported symptoms of airway (nose and bronchi). latex allergy (spt and/or sige and anamnesis positive) was diagnosed in ( , %) cases. the symptoms concerned with: skin, nose and conjunctive ( cases, %); skin and conjunctive ( cases, %); nose and conjunctive ( cases, %); nose and skin ( cases, %); bronchi, skin, nose, and conjunctive ( cases, %) and only skin in cases ( %). conclusions: the incidence of occupational airway allergy in group of health care workers with latex allergy is high ( % of all cases). latex allergy symptoms usually affect skin, but sometimes it includes also other organs -conjunctive, nose and seldom with bronchi. the incidence of anaphylaxis is increasing. the food allergy and anaphylaxis network wishes to expand the use of injected epinephrine by first responders to allergic emergencies. in indiana, there are , first responders, , basic emts, , advanced emts, and , paramedics. the use of injected epinephrine was only permitted by paramedics and advanced emts ( % of all responders). in a bill was introduced to expand the use of injected epinephrine by ems personnel in the event of an allergic emergency. it was sponsored by the indiana allergy asthma and immunology society and the emergency medical services commission of indiana. on july st senate bill no. took effect permitting all first responders to administer, without restrictions, injected epinephrine. once the legislation passed it became the responsibility of the medical director (md) of each ambulance service in the state to implement the change. in an attempt to assess the impact of this legislation we surveyed each of the mds in the state - months after the passage of the bill. mds were notified of the legislation by memo and public meetings. in addition each md received a copy of the legislation. of the mds, ( %) responded to the survey. results: . unaware of the legislation: ( %) . aware of legislation but no implementation: ( %) . aware of legislation with implementation restricted to basic emts: ( %) . aware of legislation and implementation at all levels: ( %) conclusions: twenty-five percent of mds were unaware of legislation. among those that implemented changes the majority ( %) permitted use by basic emts only. allergists, lay organizations, and ems commissioners need to assure implementation of legislation with all mds within the state. a. khadavi * , b. silverman , a. schneider , . great neck, ny; . brooklyn, ny. rabbit anaphylaxis is extremely rare, with one documented case upon inhalation only. we describe a patient with severe anaphylaxis upon consumption of a rabbit. a -year-old female with a -year history of asthma and allergic rhinitis was presented for evaluation. the patient complained of worsening asthma and rhinitis symptoms in the home and upon exposure to outdoor pollen. further history revealed the family had a rabbit as a pet. laboratory testing showed her total ige was ku/l, rast results were normal for tree, ragweed and molds (< . kiu/l). positive results were found for ragweed, dust, cockroach, cat, dog, mouse, peanut and rabbit epithelium ( . kiu/l, class iv). among other environmental control measures, the family was advised to eliminate the rabbit from the home environment, as it could be a potential trigger for their daughters allergy symptoms. two days later, the patient presented to the emergency room with wheezing, coughing and angioedema of the hands and face. she was treated with albuterol, diphenhydramine and oral steroids. the parents said they followed all of our instructions and did not know why anaphylaxis occurred. further questioning revealed that the family consumed the pet rabbit on the same night preceding the anaphylactic reaction. this was the first time the daughter ate rabbit. the parents assumed that only exposure to the live rabbit would worsen her allergy and asthma symptoms, never expecting an allergic reaction via ingestion. they were advised not to feed their daughter rabbit again and were given a prescription for self-injectable epinephrine. this demonstrates either complete identity, partial similarity or cross reactivity between inhaled and food allergens, as has been noted previously with certain other foods such as garlic and crustacean proteins. physicians need to advise food allergic patients that an allergic reaction can develop upon inhalation of the food, as seen with peanuts. but also respiratory sensitization to an allergen can lead to allergic symptoms upon its ingestion. t. nsouli * , j. scheiner , j. malka-rais , j.a. bellanti , . burke, va; . washington, dc. the safety of selective cyclooxygenase- (cox-ii) inhibitors in patients with known nsaid-induced allergic reactions has not been definitely established and is still open to debate. in the present report, we describe two patients with hypersensitivity reactions to naproxen, the first characterized by a systemic anaphylactic reaction and the second by localized urticaria following ingestion of the drug. the first case was a y/o wf with a history of osteoarthritis who, minutes after the ingestion of mg of naproxen, developed generalized pruritus, urticaria, laryngeal edema and hypotension. she was treated in the er with epinephrine, diphenhydramine and iv corticosteroids. epicutaneous and intradermal skin testing to celecoxib revealed negative results similar to a control. an oral challenge with celecoxib was well tolerated by the patient without any adverse responses. the second case was a y/o wf with a known history of chronic urticaria and osteoarthritis requiring regular use of naproxen. a complete allergic and immunologic workup was negative. upon discontinuation of the drug there was resolution of the urticaria. an oral challenge with celecoxib was well tolerated by the patient without adverse sequelae. these two case reports exemplify two extremes in the spectrum of adverse allergic reactions to naproxen, a non-selective cox-i and cox-ii inhibitor, one systemic, the second localized and suggest that the pathogenesis of these symptoms was most likely pseudo-allergic in nature and not immunologically-mediated. the dissimilar chemical structures of naproxen and celecoxib together with their differing mechanisms of action suggest that the absence of allergic reactions to challenge with celecoxib, a cox-ii inhibitor, may be related to a lack of cross-reactivity between the two drugs. although these findings suggest that oral celecoxib could be a possible safe alternative in patients with naproxen-induced drug reactions, it would be prudent to first conduct careful challenges with the drug in a well-equipped medical setting where clinical acceptability and tolerability could be safely assessed. introduction: most fixed and removable dentures are made from casting alloys. many orthodontic appliances are also fabricated from metallic biomaterials. it has been documented in vitro and in vivo, that metallic restorations release metal ions mainly due to corrosion. those metallic ions may be distributed systemically and locally and could pay a significant role in the induction of oral or/and systemic immunoinflammatory conditions. this study determines the frequency of sensitization to metal salts and clinical characteristics in the group of patients with complaints related to adverse effects of dental alloys. methods: patients ( women and men) aged - years with symptoms assumed to adverse effects of dental alloys were studied. all patients were studied with base metal salts, including cu + , co + , cr + , mg + , mn + , ni + , ti + , zn + using patch tests. all patch tests substances were % salts in petrolatum. the patch tests were conducted in accordance with recommendations of the icdrg. occlusion time was days, and patch tests were read when removing the patches (finn chambers on scanpor, epitest ltd oy, tulusa, finland) and to days later. the total number, percentage of irritant and allergic patch test reactions were calculated. results: in of patients ( %) sensitivity to base metals used in dental restorations was noted. symptoms included burning mouth ( %), metal taste ( %), electrical sensations ( %), dry mouth ( %), and taste irritation ( %), but / patients ( %) had no symptoms. local gingivitis ( . %), anomalies of the tongue ( %), stomatitis ( %) and lichenoid reactions ( %) were often seen. the most frequent patch test reactions were caused by ni ( %), cr ( %) and co ( %) . conclusion: this study demonstrated higher frequency of hypersensitivity reactions to ni, cr and co in this group of patients often having symptoms such as burning mouth, metallic taste, electrical sensation, local gingivitis, and anomalies of the tongue. w.y. mak * , s. kearney, b. silverman, a. schneider, brooklyn, ny. the process of intravenous drug desensitization often involves simple but tedious calculations. miscalculations may arise due to human error. for patients who are truly allergic to the drug in question, such errors can be life threatening, if not fatal. we propose the use of a spreadsheet to minimize such calculation errors. spreadsheets, such as microsoft excel, ibm lotus - - , and corel quattro pro, are used extensively in finance for tedious and repetitive calculations. this property of a spreadsheet makes it an ideal tool to assist with any drug desensitization protocol. we chose microsoft excel for its availability at our institution. a general template was initially created with equations in specific cells which were based on the protocols listed in patterson's allergic diseases, th edition and middleton's allergy: principles and practice, th edition. by inputting specific numbers, such as the amount of drug and volume of diluent into key cells of the spreadsheet, the program will automatically calculate the protocol for the given intravenous drug. an example of our pipericillin protocol is listed below. we find that the use of a spreadsheet not only minimizes calculation errors and hence reduces the likelihood of avoidable reactions; but also decreases our preparation time for the desen-sitization protocol. we recommend its use for all allergists performing drug desensitizations. states years ago, the infestation of homes across the northeast, southeast and midwestern states with these insects during the fall and winter months has become increasingly common. in the last years, several investigators have published case reports of patients with allergic rhinitis, conjunctivitis and asthma symptoms associated with suspected inhalational exposure to high levels of proteins from these beetles. we report a series of patients who presented with a spectrum of various allergy complaints ranging from symptoms of allergic rhinitis, asthma, urticaria, angioedema and acute anaphylaxis (with documented elevated serum tryptase) on exposure to high numbers of multi-colored asian ladybeetles(malb.) methods: we performed western blotting with the patients' serum and a whole-body ladybeetle extract made from confirmed h. axyridis obtained from one of the infested homes in that region. results: blots with the patients' serum revealed ige binding to different proteins with molecular weights approximately , and kd. the serum from two patients bound a kd protein possibly similar to the kd protein previously identified by yarbrough et al. jaci ; ; - . ige from four patients bound to an kd protein not previously reported. lastly, serum from two patients revealed ige binding to a kd protein possibly similar to the heavier proteins mentioned in an abstract by magnan et al. jaci ; ; . conclusion: we present five patients with specific ige to malb and allergic symptoms on exposure to high levels of malb. we conclude that malb are likely a significant source of several different allergenic proteins and that malb are increasingly becoming a significant cause of a wide variety of hypersensitivity reactions across the united states. as multiple exposed subjects and several entomologists report that these beetles bite humans, we speculate that a wide range of ige-mediated symptoms including urticaria, angioedema and anaphylaxis can occur by exposure to proteins by inhalation, direct contact and possibly by inoculation of these proteins into the skin by the bite or scratch of this beetle. a case report and literature review. c.s. taylor * , s. ramesh , . getzville, ny; . buffalo, ny. introduction: lentils belong to the legume family and are the staple ingredient of the ethnic indian diet. lentils have been shown to cause allergic reactions and even anaphylaxis. however, little research has been done to distinguish the types of lentils used commonly in the indian diet and their various hypersensitivities. some of these lentils include the black gram (phaseolus mungo), mung bean and split chick peas (bengal gram). case report: we report a -month-old indian boy who presented with severe atopic dermatitis since the age of four months. his dermatitis was exacerbated by the ingestion of peas and beans and resolved with the avoidance of such. at nine months of age, the child was introduced to boiled mung beans and subsequently developed lip swelling within a few minutes. a similar reaction occured with split chick peas. physical exam at consultation revealed severe eczema. skin tests using commercial extract revealed + response to peanut (he had never eaten peanuts), soybean, pea and egg. subsequent skin tests at the follow up visit to prepared reagents revealed much greater than + response to mung bean, chick pea, split chick peas, and black gram. the patient developed a systemic response during testing which required epinephrine. discussion: legume allergy (other than peanut, soy and peas) is rare in the unites states but has been reported in asia and the mediterranean area. it has been reported in one case series that patients with lentil allergy react to more than one lentil. there is a five percent cross reactivity between peanut and other legumes such as soy and peas. however, the extent of cross reactivity between peanut and the other legume sub-groups (ie. lentils) is not well documented. there also appears to confusion in the labeling of the various lentils used in ethnic diets. conclusion: this case has been presented to make allergists aware that there are many differnt types of lentils that can cause hypersenstivity reactions and the importance of considering ethnicity and dietary habits in evaulating for food allergy. background: epinephrine is the drug of choice for the treatment of anaphylaxis however it is frequently underutilized. one possible reason is the fear of adverse cardiac effects. the most common side effects of epinephrine are palpitations, tachycardia, sweating, nausea, vomiting, respiratory difficulty, pallor, dizziness, weakness, tremor, headache, nervousness, anxiety and arrhythmias. a previous study showed that administration of epinephrine, in a small number of healthy adults did not cause any significant cardiac adverse events and vitals sign changes were not clinically significant. the purpose of this retrospective analysis is to determine the safety profile of epinephrine in a large number of patients that were administered epinephrine for treatment of anaphylaxis that occurred in a physician's office. methods: all patients in an allergy office, that were administered epinephrine for acute anaphylaxis between - were selected for this retrospective analysis. a chart review for adverse events and vital was conducted. vital signs and side effects had been monitored every to minutes for minimum of to minutes after the administration of epinephrine. results: patients between the ages of yrs to yrs received injections of epinephrine. after an injections of epinephrine % of the patients had a pulse between - , % between - , and % between - . systolic blood pressure determined % of patients to be between - , with %, between - , and % between - , and % between - . concurrently, diastolic blood pressure was found to have % between - , % between - and % betweem - . the most common reported side effects were tremor, headache and pallor. any rise in blood pressure and heart rate after epinephrine was transient and returned to normal within minutes of observation. there were no serious side effects. all patients responded to the epinephrine and were able to go home. conclusions: this retrospective analysis revealed that the administra-tion of epinephrine is safe and effective for the treatment of acute anaphylaxis in an outpatient setting. the use of epinephrine for treatment of anaphylaxis is life-saving and its use to treat non-cardiac or elderly patients who have anaphylactic reactions should be encouraged. purpose: a clinical pathway was established to decrease the use of vancomycin in surgical patients with self-reported penicillin (pcn) allergy. methods: in , our institution developed a preoperative evaluation (poe) clinic for elective surgical patients. in june , on-site, same-day allergy consultation and penicillin skin testing was made available for preoperative patients with self-reported pcn allergy. we reviewed the antibiotic recommendations, the actual administration of vancomycin, and compliance by the surgeons with our recommendations from july , -september , . results: during this time, , patients were seen for their preoperative medical exam at the poe clinic, and were evaluated for pcn allergy. of these, patients met irb standards to participate in the study. the mean age of the patients was years. the top three surgical specialties represented in the poe clinic were orthopedic ( . %), urology ( %), and neurosurgery ( %). of the patients, underwent skin testing for pcn allergy. eighty-five percent ( %) or of these patients with a history of pcn allergy were recommended to use -lactams such as cefazolin, and ( %) were recommended to avoid -lactams. forty-three ( ) or % had one or more positive skin tests to pcn. of the patients, patients actually received pre-operative antibiotics. of these patients, ( %) of those received cefazolin, ( %) received clindamycin, ( %) received ciprofloxacin, ( %) received levofloxacin. some patients received more than one antibiotic for prophylaxis. only ( %) patients received vancomycin. conclusions: establishment of a clinical pathway in a preoperative clinic that includes allergy testing and consultation reduced vancomycin use to only % in surgical patients with a history of penicillin allergy. h. yarmohammadi * , a. nowak-wegrzyn, new york, ny. introduction: anticonvulsant hypersensitivity syndrome is a potentially fatal drug reaction with cutaneous and systemic reactions to the arene oxideproducing anticonvulsants. in most cases, the hallmark features of fever, rash, and lymphadenopathy are accompanied by multi organ-system abnormalities. fatal outcomes are most often associated with liver failure. recognition of the syndrome, which may have variable presentations, is the key to prompt discontinuation of the drug, close monitoring, and management. case : a y/o male received dilantin for seizure prophylaxis following craniopharyngioma resection. fourteen days later he was readmitted for fever and csf leak from surgical site. he was started on ceftriaxone and vancomycin empirically; an lp and pan culture was done. he continued to have low grade fever and, on the th day of admission, developed a maculopapular rash on trunk. on day th lfts increased and cbc showed eosinophilia. antibiotics were stopped but lfts continued to rise. on day , dilantin was stopped and lfts normalized in days, fever stopped and rash disappeared. case : a y/o girl was admitted to the hospital with generalized rash, facial edema and fever ( degrees c). she developed a pruritic maculopapular erythematous rash over her trunk and face weeks prior to admission. she then developed high fever. three months prior to this visit she had been started on phenytoin ( mg/kg) for control of grand mal seizure. physical exam revealed cervical and axillary lymphadenopathy. she had elevated wbc ( , ), eosinophilia ( %), and elevated lfts. phenytoin was stopped upon admission but hours later she continued to have fever, elevated lfts and devel-oped erythema in her mouth. treatment with ivig and prednisone was initiated and resolution of symptoms was seen within - days after therapy. conclusions: the timely recognition of anticonvulsant hypersensitivity syndrome is important, because accurate diagnosis prevents potentially fatal re-exposure and influences subsequent anticonvulsant treatment options. ivig and prednisone should be considered in situations where prompt improvement of the lfts or clinical symptoms is not observed. the recently published results of a telephone survey about the prevalence of seafood allergy in the us indicate that seafood allergy is a significant health concern. aim of the present study was to retrospectively review our data on the topic. we have interviewed subiects attending our allergy outpatients about any suspected food allergy. over % were years or older. any reaction to food was reported by % of the patients, more frequently by women. of these patients, reported reactions to seafood ( %) with an overall prevalence of seafood reactions of . %: to fish ( . %), to shellfish (including mollusks) ( . %) and to both ( . %). in the case of the reactions to fish it is quite possible that shellfish is also included, as the patients were often unable to tell the role of finfish from shellfish apart. this is most probably due to the fact that seafood dishes or a complete seafood meal in italy generally include both shell and finfish. in only less than half cases there was a convincing relationship between the ingestion of seafood and the reaction. the reactions reported were: gastrointestinal ( ), non life-threatening angioedema ( ), mild oral allergy syndrome ( ), acute urticaria ( ), asthma ( ), rhinoconjunctivitis ( ). the gastrointestinal reactions might not all be true (ige-mediated) allergic reactions, but also toxic. in conclusion, even in a selected population like that attending an allergy clinic and using a broad definition of adverse reaction, seafood allergy appears to be a rare phenomenon and of limited clinical impact, the prevalence of moderate to severe reactions (angioedema and asthma) in the whole selected population being . %. clopidogrel (plavix) is an antithrombotic agent currently used to prevent thrombotic cardiovascular events by inhibiting adenosine diphosphate (adp)dependent platelet activation. clinically, it has been used in patients with aspirin intolerance or who developed neutropenia from ticlopidine. there have been reported cases of clopidogrel causing rash and urticaria. often these patients are told they are allergic to clopidogrel and should not take it again, despite the fact that no workup was ever performed to elucidate whether the reaction was indeed immunologic in nature. a review of the literature revealed no reported attempts at desensitizing a patient to clopidogrel after a presumed immunologic drug reaction. we present the case of a year-old female who initially took clopidogrel ( mg. p.o. daily) for transient ischemic attacks. after tolerating the medicine for five days, it was replaced with an aspirin/warfarin combination, which she took for the next five days. reintroduction of clopidogrel a week later was uneventful, until she developed a pruritic, maculopapular rash on the th, through th days of restarting therapy. at that time, she was not taking any other medications, and had no cardiopulmonary or gastrointestinal complaints. she had discontinued the clopidogrel and was asymptomatic during our consultation. skin testing was done using clopidogrel prepared at concentrations of mg/ml and / mg/ml. at the same time, control skin testing on three non-atopic subjects was performed and produced no reaction to either epicutaneous or intradermal testing. by contrast, the patient had a positive skin reaction at both concentrations on intradermal testing. the patient was hospitalized for an oral desensitization where clopidogrel dilutions were prepared by the hospital pharmacy. the patient tolerated an initial dose of . mg. serial three-fold increases in concentration were given every minutes, until a total cumulative dose of mg. was reached. the patient tolerated the entire procedure and suffered no adverse reactions upon continuation of the treatment. this demonstrates the utility of oral desensitization to clopidogrel in patients with a positive skin test who require this medication. lamotrigine is a non-aromatic anticonvulsant with desirable pharmacological profile. the most common idiosyncratic reaction in children is rash ( - %). severe cutaneous adverse reactions and systemic hypersensitivity reactions are uncommonly reported. method: case presentation an -yearold caucasian female was prescribed lamotrigine in escalating dose for her first generalized seizure. a week later she developed a pink rash on her cheeks gradually progressing to her trunk, hands and feet. three days later she was seen in the er and treated for possible streptococcal-pharyngitis with fever and rash. oral penicillin-v treatment was initiated. the rash continued to spread all over her body with mild pruritis and sores on lips and mouth. few blisters were noted in left ear, hands and feet. later lamotrigine was discontinued. oral steroids were prescribed. she continued to develope new blisters, rash and hemorrhagic plaques and dysuria. she was later transferred to our institute. the physical examination was significant for afebrile girl without pallor and icterus. bilateral conjunctivitis without keratitis evident. tender cervical lymphadenopathy was palpable. multiple ulcers on lips and gingiva were seen. several targetoid lesions on trunk and extremity were noted along with few hemorrhagic plaques on extremities. superficial sloughing of distal fingers and toes was noted. nikolskys sign was not demonstrable. the systemic examination was unremarkable. laboratory tests: hemoglobin . g/dl, wbc-normal range without eosinophilia. normal pt/aptt and inr. liver function tests showed elevated sgpt- iu/ml and sgot- iu/ml with normal bilirubin. urine analysis was normal. serology for cmv, ebv, hsv and hepatitis a and b was negative. skin biopsy showed full thickness epidermal necrosis and sub epidermal clefts. aggressive supportive therapy was initiated. corticosteroids were continued for five more days. prior to being discharged from hospital the rash had dried and most lesions faded. the blisters and oral ulcers had healed. the liver enzymes had decreased. conclusions: we report an occurrence of stevens-johnson syndrome with lamotrigine in a young child. in the literature severe reactions are associated with higher doses or rapid escalation of the dose, and concomitant use of valproate. early recognition and withdrawal of medication is necessary to improve the outcome. introduction: in patients with a history of penicillin (pcn) allergy and negative pcn skin tests to major and minor determinants, - % of patients will tolerate pcn administration without risk of an immediate reaction. in fact, middletons allergy principle & practice reported that no life-threatening false-negative reactions have been reported when pcn was administered after a negative pcn skin test. we describe a case in which a patient with a history of pcn allergy and negative pcn skin tests to the major and minor determinants experienced life-threatening anaphylactic shock when administered piperacillin/tazobactam. case history: a -year-old woman with crohns disease was admitted for treatment of an enterocutaneous fistula. three months before admission, the patient reported a severe reaction to either piperacillin/tazobactam or intravenous (iv) lorazepam needing respiratory support in the intensive care unit. in order to delineate this problem, an allergy consultation was obtained. serum ige antibodies to pcn, measured by a commercial cap system radioallergosorbent test (rast) fluoroenzymeimmunoassay (feia; pharmacia and upjohn, uppsala, sweden), and pcn skin tests to major and minor determinants were negative. a skin test (prick and intradermal) to piperacillin/tazobactam at . mg/ml was also negative. five minutes into receiving . grams of piperacillin/tazobactam iv, the patient reported feeling lightheaded, flushed, nauseated, and diaphoretic. the blood pressure decreased to / from a baseline of / , heart rate /minute and appeared toxic. no wheezing or rash was noted on physical examination. patient was treated with iv epinephrine, diphenhydramine, and dexamethasone and recovered without sequela. serum tryptase, drawn hour after the beginning of the reaction, was elevated at . ng/ml but decreased to . ng/ml hours later. complement levels were normal. conclusion: despite a very high negative predictive value of a negative pcn skin test to the major and minor determinants and reports that no life-threatening false-negative reactions have been reported when pcn is administered after a negative pcn skin test, physicians need to be very cautious in administering piperacillin/tazobactam and other b-lactams in patients with a history of severe reactions such as anaphylaxis to past pcn administrations. a. majmundar * , d.a. khan, dallas, tx. introduction: a variety of adverse drug reactions have been reported after therapy with allopurinol. successful protocols for desensitization to allopurinol have been developed. we report a case of a patient who was successfully desensitized to allopurinol only to develop dress after four months of allopurinol therapy. methods: a year old man was referred to our department with a remote history of rash and fever resulting in hospitalization after initiation of allopurinol. due to severe gouty arthritis unresponsive to colchicine and requirement of chronic steroids, it was recommended to attempt allopurinol desensitization. based on the history of his prior reaction, a slow desensitization protocol was performed beginning with allopurinol μg and increasing the dose weekly to μg, μg, μg, μg, μg, mg, mg, mg, mg, mg, and mg. the patient tolerated the entire desensitization protocol without adverse reaction and was initiated on daily allopurinol at mg per day. results: four months after desensitization and daily allopurinol use, the patient developed fevers and an acute maculopapular eruption on his back and abdomen without mucosal involvement. laboratories demonstrated eosinophilia of cells/mm , elevated ast of , alt of , and ggt of u/l. he was treated with prednisone mg a day which was tapered weekly over weeks with successful resolution of his symptoms, eosinophilia and transaminitis. conclusion: while desensitization to allopurinol can be safely accomplished, allergists need to be cognizant of the potential for delayed serious drug reactions such as dress. wiskott aldrich syndrome is an immunodeficiency characterized by eczema, pyogen infections, and mixed immunodeficiency.we describe a male seven-month old, perinatals antecedents without importance who had an ulcer in site of application of bcg. non-blood relatives, healthy parents. at twomonths-old, he initiated with continuous fever, not quantified, treated with multiples antibiotics without resolution, he was hospitalized, plaquetopenia and anemia were diagnosed, he received a red globules and plateles transfusion, one week after he presented a disseminated dermatosis characterized by erythema and desquamation, at four-month-age presented right axillary adenomegaly, hepatoesplenomegaly and recurrent bilateral media otitis for what was hospitalized in our institute. he was malnutrition, bad general condition, febrile, with disseminated dermatosis affecting the head, trunk and extremities characterized by erythema and desquamation, in addition he had left ankle cellulitis, right axillary adenomegaly, hepatoespenomegaly. during his hospitalization he presented left hand cellulitis, hairy skin abscess, oral candidiasis, required surgical treatment by econdary compartimental syndrome because cellulitis of left ankle. persistent hemogram reported thrombocytopenia with normal platelet volume, blood cultives were positive for grampositive bacterias, isoaglutinines, were normal. immunoglobulines were elevated for the age range, he received antimicrobial and antifungic treatment. but he died. in the autopsy timic alymphoplasia was reported, cortical lymphoid depopulation in lymphatic ganglia and spleen, disease by disseminated cytomegalic inclusion, multifocal pulmonary pneumocystosis, bcgitis, disease graft versus guest. . with the previous features we concluded in a compatible mixed immunodeficiency with wiskott-aldrich syndrome with particular characteristics that make this case interesting. the patient course with cellular immune deficiencie with thrombocytopenia and eczema, even when he didn't have platelet sizing diminished, we consider that the patient had a severe of wiskott s aldrich syndrome and at the moment we are awaiting result of genetic study uasp gene. background : hypersensitivity to mosquito bites (hmb) is a disorder characterized by necrotic skin reaction and systemic generalized symptoms subsequent to mosquito bites. it has been suggested that hmb is associated with chronic epstein-barr virus (ebv) infection and natural killer cell leukemia/lymphoma. we describe here a korean child who had hmb associated with chronic ebv infection and natural killer cell lymphocytosis. case : a -year-old male was admitted with well-demarcated necrotic skin lesions and severe swelling on right ear lobe developed after mosquito bites. he had suffered several similar symptoms since last summer, which complicated as deep scars on skin. hepatosplenomegaly or peripheral lymphadenopathy was not detected. laboratory tests showed wbc , /mm (neutrophil %, lymphocyte %), total eosinophil count /mm , ige by prist above , iu/m. immunoglobulin levels were normal. specific ige for aedes communis by cap was negative. lymphocyte subset analysis demonstrated increased nk cells (cd +cd , %) and decreased cd and cd cells. igm for anti-nuclear antigen (ebna), igm for viral capsid antigen (vca) and igm for anti-early antigen (ea) dr to ebv were negative. but the levels of anti-vca igg (> u/ml), anti-ea dr igg (> u/ml) and anti-ebna igg ( u/ml) were increased. type a eb virus was demonstrated in blood mononuclear cells by dna pcr method, and eber in situ hybridization was negative in necrotic tissues. immunostaining with nk-cell marker (cd ) revealed many immunoreactive cells with the perivascular inflammatory infiltrates in tissue. skin patch tests for mosquito allergen (aedes togoi and culex pipiens) showed positive response to c. pipiens. introduction: scimitar syndrome is a congenital anomaly resulting in anomalous pulmonary venous return from the right lung to the inferior vena cava. recurrent respiratory infections have been associated with scimitar syndrome. methods: we report on a year-old adolescent female who presented to immunology clinic with recurrent pneumonias. results: the patient presented with numerous recurrent pneumonias, multiple er visits, and hospitalizations. she complained of intermittent chest pain, cough, fatigue and exercise intolerance. the patient had a large secundum atrial septal defect surgically corrected at years of age. a cardiac echo obtained at years of age revealed rvh, but was normal at years of age. she was a known atopic asthmatic. previous immunologic workup was normal with iga ( - ), igg ( - ) and igm . at presentation to our clinic pulmonary functions were fvc of % and fev of %. review of previous chest radiographs showed chronic changes with an opacity partially obscuring the right hemidiaphragm. a high resolution chest ct-scan showed a large irregular venous structure extending through the right chest joining the inferior vena cava above the liver consistent with scimitar syndrome. the patient was referred to pediatric cardiology who recommended surgical correction. conclusion: scimitar syndrome may present as recurrent pneumonias and chronic lung disease. a high resolution chest ct scan may be useful in delineating this disorder. background: laryngomalacia is the most common cause of stridor in infants but only rare reports exist of clinically relevant laryngomalacia in adults. objective: to present a case of laryngomalacia in an adult with significant respiratory symptoms initially attributed to asthma. methods: an year-old female with a history of allergic rhinitis and gastroesophageal reflux disease presented to the allergy clinic for further recommendations regarding a prior diagnosis of asthma poorly controlled on inhaled fluticasone, montelukast and albuterol. the patient was clinically diagnosed with asthma at age due exercise related symptoms. the symptoms progressed and intermittent trials of various inhaled steroids provided minimal relief. on evaluation, the patient described constant wheezing which occurred only on inhalation and originated from the throat. symptoms did not respond to albuterol use three to four times a day, rhinitis control and long-term, high-dose, antireflux therapy. baseline spirometry was normal. histamine bronchoprovacation and fiberoptic laryngoscopy were performed for further evaluation. results: histamine challenge was positive with a % decrease in fev with mg/ml histamine. however, laryngoscopy revealed redundant airway tissue most notable over the right arytenoid cartilage, consistent with laryngomalacia, which prolapsed into the laryngeal vestibule significantly obstructing the airway on inspiration only. the patient was referred to otolaryngology and surgical excision using a carbon dioxide laser was performed with subsequent improvement in symptoms and decreased asthma medication use. conclusions: we report an unusual case of laryngomalacia in an adult presenting as asthma, which was successfully treated with laser surgical excision. laryngoscopy of the patient revealing redundant airway tissue most notable over the right arytenoid. c. so * , s. kuhl , . davis, ca; . mather, ca. c. so, s. kuhl u.c. davis medical center, sacramento, ca & sacramento va hospital, mather, ca background: wheezing is an uncommon manifestation of phrenic nerve dysfunction. objective: we offer a description of wheezing attributable to phrenic nerve dysfunction to remind clinicians that dyspnea and wheezing can be caused by cor pulmonale which can be caused by phrenic nerve dysfunction. methods: a -year old male non-smoker presented with chronic dyspnea and occasional wheezing for the last decade. the patient had a remote history of pericardial stripping for presumed tuberculous pericarditis. he also had a non-productive cough and orthopnea. dyspnea was exacerbated by putting his hands over his head and bending over. he had been treated with inhaled corticosteroids and bronchodilators without relief. results: repeated chest x-rays showed a chronically elevated right hemidiaphragm. pulmonary function tests showed a restrictive pattern with: fev . ( %), fvc . ( %), tlc . ( %), dlco/va . ( %) and no bronchodilator response. left heart catheterization was normal while right heart catheterization showed elevated pulmonary artery pressures ( / ) and he was subsequently diagnosed with cor pulmonale. cardiopulmonary exercise testing showed an increase in minute ventilation which was achieved predominantly by an increase in respiratory rate rather than to an increase in tidal volume suggesting restrictive or interstitial lung disease. chest ct showed left ventricular enlargement and no evidence of interstitial lung disease. a fluoroscopic sniff test was performed and showed paradoxical movement of both diaphragms. he was diagnosed as having diaphragmatic dysfunction as a result of phrenic nerve injury from prior pericardial stripping. conclusions: wheezing and chronic dyspnea can be related to phrenic nerve dysfunction. a review and discussion of various causes of phrenic nerve dysfunction, including autoimmune causes, is offered. patients with a history of prior cardiothoracic surgery who present with recalcitrant dyspnea and wheezing may benefit from evaluation for phrenic nerve dysfunction. background: sudden sensorineural hearing loss (ssnhl) is defined by a loss of at least db in contiguous frequencies over a time course of hours or fewer. etiologies of ssnhl include viral infections, ototoxic drugs, autoimmune diseases, trauma, neoplasms, and vascular occlusion, but viral labyrinthitis is the most common cause. in cases of sudden hearing loss, herpes infections can be reported in approximately % of cases caused by viral infections. typically, sensorineural hearing loss is not recurrent. we report a case of recurrent ssnhl is which oral herpes lesion preceded the onset of symptoms on three consecutive episodes. case report: a -year-old male with a history of hypertension, hypothyroidism and chronic tinnitus of the left ear (since a gunshot wound years prior) presented to clinic with a complaint of recurrent episodes of sensorineural hearing loss. the first episode of bilateral hearing loss occurred months prior. an otolarnolgologist treated with a steroid taper and valacyclovir and the hearing loss resolved after one day of therapy. since the initial presentation, the patient reports three subsequent episodes of ssnhl, with two of the episodes responding to prednisone and valacyclovir and one episode responding to steroids alone. oral herpetic lesions preceded at least three of the episodes one day prior to the hearing loss. laboratory data was significant for an elevation of varicella-zoster igg, and of hsv igg. hsv igm was not performed. rpr was non-reactive and ana was negative. a mri of the head was normal. audiogram demonstrated db increase and speech discrimination improved from % to %. the rest of the laboratory data was unremarkable. the patient has been maintained on daily valacyclovir therapy and has had no further episodes of hearing loss. conclusion: our patient experienced hearing loss with concomitant evidence of hsv- stomatitis. this suggests a cause and effect relationship. we found that the antiviral therapy was effective for treatment of the ssnhl in this patient as demonstrated by the absence of further symptoms while on antiviral prophylaxis and conclude the most likely etiology of the recurrent hearing loss was secondary to the recurrent herpes simplex infections. abstract the illness was described for the first time in in the chinese literature by kimm, and szeto, the definitive histological description was published by kimura in , this illness is endemic in asia, but rare, about cases had been reported. kimura disease is extremely sporadic in the rest of the world. the etiology of this disease is ignored but it is believed that there is an aberrant immune reaction to an unknown antigenic stimulus, however epstein barr's virus, human herpes virus and candida albicans had been involved in certain cases. the mast cells had been implicated in its pathogenesis and a th cytokine pattern with the production of interleukin , and rantes which regulate the synthesis of ige and orchestrate the eosinophilic infiltration. on the other hand it is suggested that the eosinophils had undergone an accelerated apoptosis in this illness. case report. it is a year-old boy with a months evolution with the presence of bilateral subcutaneous nodules of x cm in parotid and submaxillary glands, presenting hypereosinophilia ( total eosinophils ) and high ige ( total ige), with normal renal function and negative mycotic and parasitic tests.the histopathologic findings revealed the presence of eosinophilic infiltrates with capillary proliferation and fibrosis. discussion. for the clinical characteristics of the nodules together with the hypereosinophilia, extremely high ige and the characteristic histological lesions the diagnosis is kimura disease. the usual clinical presentation consists on several indolent subcutaneous nodules that grow very slowly in volume, located in the neck and head, accompanied by satel-annals of allergy, asthma & immunology lites adenophaties, and with increment in the salivary glands, there is renal affectation in half of the patients, and the laboratory detects hypereosinophilia and elevation of the total ige. the histological lesions, shows hyperplastic lymphoid tissue with proliferative germinal centers, with infiltration of eosinophils in their interfollicular and perivascular zones sometimes forming an eosinophil abscess and proliferation of poscapillary venules. at the moment he have been treated with prednisone ( mgkdia), with great improvement in the clinical evolution. this is the first case reported in the literature in mexico. introduction: the incidence of cow's milk protein allergy (cmpa) is approximately - % and presents primarily during the first year of life. manifestations of cmpa in the neonatal period include gastroenteritis, colic, lethargy, metabolic acidosis, and hematochezia or melena and appear to be non-ige mediated. ige mediated reactions in the neonatal period, such as urticaria or angioedema, are unusual. case: a -day-old african-american male presented with a day history of rash, swelling, and erythema overlying multiple joints. there was no history of fever, diarrhea, or eczema. he had been on no medications prior to admission. besides mother with a history of childhood asthma, there was no family history of atopy or food allergy. he had been fed cow's milk-base formula (similac ® ) since birth exclusively. physical examination revealed a diffuse erythematous, raised, macular-papular rash, with areas of duskiness and exfoliation. there was angioedema overlying the joints and periorbital areas (figure ). laboratory evaluation included cbc with diff, hgb electrophoresis, lumbar puncture, urinalysis, c q (qualitative and quantitative), c , c , c , and cultures of the blood, csf, and urine which were within normal. percutaneous allergy skin testing for cow's milk allergy was performed revealing a + reaction to cow's milk extract (greer, lenoir, nc) with positive histamine and negative saline controls. rast testing showed . ku/l for -lactoglobulin and . ku/l for cow's milk. he was placed on an elemental formula. skin lesions and swelling resolved completely within hours. at week follow-up the patient was thriving without complaint. conclusion: early sensitization to cow's milk protein in the neonatal period may occur, resulting in ige mediated urticaria and angioedema. the rashes may be misdiagnosed as erythema multiforme or anaphylactoid purpura, since hemorrhagic lesions and cockade pattern are common. physicians should be aware that these reactions may occur so that early recognition and management may be initiated. neonate with periorbital angioedema and exfoliating, urticarial rash. rationale: two patients, presenting with invasive fungal cns infections, were found to have nk cell dysfunction and hypogammaglobulinemia. methods: case reports results: patient # : a year old caucasian male presented with headache, double vision, periorbital swelling, and bilateral sinus disease on ct scan. biopsies showed invasive rhinocerebral mucormycosis. he continued to deteriorate in spite of iv and intrathecal amphotericin b, hyperbaric oxygen, many debridement procedures, and a left orbital exenteration. immune evaluation revealed low igg, low igm and a barely detectable nk cell killing activity. neutrophil respiratory burst was normal. he continued to worsen despite ivig replacement. he had intolerable side effects to ifn-a. gm-csf ( mcg qod) was initiated, in order to boost nk cell activity. this resulted in stabilization of his infection. he was discharged on oral anti-fungal therapy (posaconazole), ivig, and gm-csf. sixteen months later, he continues to remain stable clinically and radiographically on ivig and gm-csf. patient # : a -year-old caucasian male presented with headache, mental status changes, and ataxia. a head ct scan showed a left mass effect and edema. he underwent surgical debridement for ventriculitis and zygomycetes (the same family as mucormycosis) was found. he was started on iv amphotericin b and oral posaconazole. his immune evaluation revealed low igg and igm and low nk cell activity. neutrophil respiratory burst was normal. he was started on ivig and gm-csf after which his nk cell function improved significantly. he remains clinically stable on ivig and gm-csf with persistent radiographic evidence of enlarged ventricles. conclusion: hypogammaglobulinemia is usually not associated with invasive cns fungal infections, suggesting that nk cell dysfunction was likely responsible for the clinical courses of these patients. nk cell deficiency has been reported to be associated with recurrent mucosal candidiasis, suggesting an important role for these cells in the control of some fungi. the spectrum of the clinical presentations of nk cell deficiency is not known since it is not normally included in immune system evaluations. these patients illustrate the importance of including functional nk cell assessment in any evaluation of immune function. introduction: to report a case of successful systemic hydrocortisone desensitization, since allergic reactions and systemic desensitization to corticosteroids have rarely been documented. method: we present a patient with multiple medical problems who has a history of both radiocontrast induced anaphylactoid reaction and corticosteroid allergy. this patient had to undergo cardiac catheterization and corticosteroid desensitization was performed prior to the procedure. results: skin testing to radiocontrast is not considered helpful, therefore patients with suspected reactions to radiocontrast are generally premedicated with corticosteroids and antihistamines to decrease the risk and severity of a reaction. , since this patient experienced an allergic reaction to a corticosteroid previously, she was skin tested to two different corticosteroids. the least reactive skin test revealed a + positive immediate reaction to hydrocortisone. cardiac catheterization with contrast was considered absolutely necessary in this case and corticosteroid desensitization was performed. the half-life of hydrocortisone is the shortest among the tested corticosteroids ( - minutes), so a protocol was developed with short intervals of escalating doses. each dose was diluted yielding a total volume of ml to avoid fluid overload because patient has renal failure. during desensitization, patient developed pruritus and erythema between the rd and th dose that resolved immediately with mg diphenhydramine. after desensitization, the patient continued to be on hydrocortisone mg intravenously every four hours. one hour prior to the procedure, the patient was premedicated with hydrocortisone and diphenhydramine and was administered radiocontrast without any adverse reactions. conclusion: we successfully desensitized our patient to a corticosteroid and premedicated her with hydrocortisone and diphenhydramine before administering radiocontrast. this case illustrates that intravenous desensitization may be a suitable approach to therapy in corticosteroid allergic patients who require systemic corticosteroids administration. autoimmune neutropenia is defined as a decrease in the absolute number of peripheral neutrophils caused by an immunologically-mediated mechanism. autoantibodies directed to neutrophils can lead to the peripheral destruction of neutrophils and/or inhibit myelopoesis in the bone marrow. although recurrent aphthous stomatitis is often the heralding sign of neutropenia, the diagnosis of ain requires the demonstration of specific antineutrophil antibody which acts by promoting the immune destruction and clearance of neutrophils by mononuclear phagocytes. the present case report describes the rare clinical association of ras in an adult. a yr-old black male presented with a year history of recurrent and repeated painful multiple oral ulcers. initially the oral ulcers occurred on a monthly basis then over time the ulcerations on the oral mucosa and tongue began to appear weekly and later continuously. past medical history revealed no drug allergies but a positive history of hypertension. physical exam revealed an otherwise healthy male with no lymphoadenopathy or splenomegaly. laboratory workup revealed : hiv negative, viral culture for h. simplex negative, mild increase in cd , cyroglobulins negative, anti dsdna negative anti-smith and rnp negative, wbc count /ml, neutrophils % (anc ), lymphocytes %, monocytes %, and platelets: , . bone marrow biopsy revealed a normal production of neutrophils. a direct neutrophil antibody assay: revealed an elevated value of , (n= < , ). although following initiation of prednisone ( mg) an immediate increase in anc was seen, the levels fell as the dosage was tapered. the figure below shows the time course and dose-response relationship of anc and prednisone dosage. this case report illustrates the importance of recognition of the relationship of ras and neutropenia, an association that can masquerade as other clinical entities. background: dyspnea, wheezing, and decreased fev are suggestive of asthma. it is essential for the clinician to consider a broad differential diag-nosis as the outcome could be catastrophic if the correct diagnosis is missed. case presentation: we present a case of a y.o. filipino female who was referred to our clinic for the evaluation of cough, shortness of breath, and wheezy respiration associated with changes in voice quality, nasal and palatal pruritus, and postnasal drainage. her initial evaluation revealed mold spore hypersensitivity by prick puncture testing and spirometry with an obstructive pattern with fvc- . l ( %) and fev - . l ( %) predicted and a % reversibility post nebulized albuterol. an initial diagnosis of allergic rhinitis with adult onset asthma was made and she was started on salmeterol, budesonide, montelukast, and pirbuterol. her symptoms persisted and rabeprazole was added to treat possible laryngopharyngeal reflux. repeat spirometry revealed fev - . l prompting treatment with systemic corticosteroids again with no improvement. fiberoptic laryngoscopy was within normal limits. a high resolution computed tomography was obtained and showed a mass in the left side of the trachea which was obstructing % of the airway. bronchoscopy revealed a tumor - cm below the vocal cords with the appearance of adenoid cystic carcinoma which was confirmed by pathology. the tumor was resected by removal of cm trachea with re-anastomosis, followed by a week course of radiation therapy. all medications were discontinued. her symptoms of wheezing, dyspnea, and cough completely resolved. repeat spirometry was within normal limits and she remained asymptomatic. surveillance bronchoscopies have been negative for any recurrence. discussion: adenoid cystic carcinoma (acc) is an uncommon form of malignant neoplasm that occurs within the salivary glands. tracheobronchial acc typically presents with symptoms of cough, dyspnea, and hoarseness. due to its slow growth, acc has a relatively indolent course. in a recent study of a cohort of acc patients, survival was % at years but only % at years. standard therapy is surgical resection often followed by radiotherapy. conclusion: in patients who fail conventional therapies for asthma it is important to entertain other diagnosis and have a systematic approach to establish the correct diagnosis. ipex is an extremely rare, hereditary condition characterized by immune dysfunction, polyendocrinopathy, enteropathy and x-linked recessive inheritance that leads to death without prompt diagnosis. patients usually present by months with severe diarrhea, failure to thrive, and early onset iddm. most children die by one year without a bone marrow transplant. immunologic evaluation is typically normal except for elevated ige, eosinophilia, and autoantibodies. we report a case of ipex in an infant who presented at birth and died at days of multi-organ system failure. this male infant was born at weeks due to chorioamnionitis and prom. at birth, copious green fluid appeared from his rectum and ng tube which evolved into a secretory diarrhea. workup for fistula was negative. at weeks, the patient developed ascites and explorative laparotomy revealed an inflamed appendix. after the laparotomy, the patient had no further stool output and never tolerated enteric feeds. he remained intubated and had problems with apnea and coagulapathy. pathology of the appendix showed an excess of lymphocytes. immune system investigation showed elevated ige ( ) and igg ( ). serum anti-enterocyte igg antibody was positive in the patient and negative in his mother. based on this data, ipex was suspected which autopsy seemed to confirm. autopsies are scarce in patients with ipex. the findings revealed many organs affected by fibrosis but lymphocyte infiltration of only the pancreas and gi tract. the pancreas revealed almost complete loss of the exocrine structure, with invasion of fibrosis and chronic inflammatory cells. the mucosa of the gi tract from the stomach to rectum showed columnar epithelium taken over by fibrosis, capillaries and chronic inflammation. these inflammatory cells were cd + lymphocytes and plasma cells on immunochemistry. the lymphoreticular system was consistent with lymphoid paucity in the thymus, lymph nodes and spleen. preliminary data suggests this patient has a splice mutation of the foxp gene. ipex is an extremely rare disease, often difficult to diagnose while a patient is alive. infants will present in early infancy with diarrhea and endocrinopathies. without prompt diagnosis, death is inevitable. as a result, one must be aware of atypical presentations and considered in patients with total villous atrophy and one other clinical feature such as iddm or autoimmunity. introduction: digeorge syndrome (dgs) is characterized by thymic hypoplasia, parathyroid hypoplasia, and conotruncal cardiac defects, but has a wide variety of clinical manifestations. there is also an increased risk for autoimmune phenomena in later life due to thymic hypoplasia. case report: a year-old aa girl with tetralogy of fallot (repair at age ), developmental delay, asthma, recurrent sinopulmonary infections/skin abscesses, gerd, arthralgias and seizure disorder (due to hypoxic encephalopathy during cardiac surgery) presented to allergy/immunology clinic for immune workup. there was no history of documented hypocalcemia. at age , she developed persistent annular patches on her left leg. a skin biopsy appeared consistent with sarcoid dermatitis. there was no other evidence of sarcoidosis except for slightly elevated ace level. given lack of systemic involvement, the skin lesions were not treated, but resolved spontaneously. at age , she developed swelling of the right mandible, and a bone biopsy revealed garre's osteomyelitis (sterile hyperproliferative osteomyelitis), likely triggered by dental caries, with elevated esr ( ) and polyclonal hypergammaglobulinemia (igg ), but normal crp. immune workup revealed a decrease in cd and cd cells (cd /cd ratio . ), slightly elevated b cells, high-normal range nk cells, normal range t cell cytokine production in response to mitogens and il- , but excessive production of proinflammatory cytokines in responses to lps. in conjunction with facial dysmorphism, dgs was suspected, and fish analysis revealed q . microdeletion. a repeat workup did not reveal evidence of systemic sarcoidosis, and autoantibody screening was negative including lupus anticoagulant. she was treated with a cox- inhibitor, secondary to gerd and mild thrombocytopenia, and her joint symptoms resolved with a concurrent decline in esr (to ) and igg level (to ) after months. conclusion: recurrent infections with fragmented care and resultant chronic inflammation (hence overstimulation of the immune system) may have led this dgs patient to develop atypical autoimmune phenomena and other unusual clinical manifestations. this case illustrates the importance of recognizing the phenotypic features of dgs early in life, and of providing close monitoring and coordinated care. rationale: we report a case of a patient with celiac disease who continues to have symptoms of fevers, nausea, vomiting, night sweats and fatigue despite being on a gluten free diet whose symptoms have been responsive to antihistamines. methods: case report. results: in , this year-old white male suffered from mononucleosis and episodes of prostatitis. he began suffering from fatigue and fevers and was followed at a chronic fatigue syndrome center in . in november , patient was placed on famvir alleviating his sore throat. he was later placed on interferon gamma which was discontinued due to increased fevers, nausea and vomiting. after stopping medication, symptoms abated for sometime. in , patient's father was diagnosed with celiac sprue. in april of , the patient developed a pruritic rash on his right arm. biopsy revealed subepidermal vesicles with pmn's at the tips of dermal papillae. in may of , he developed oral ulcers as well as joint pains. patient underwent endoscopy with biopsies revealing intraepithelial lymphocytosis in the duodenum. patient has been on a strict gluten free diet since october . he reports that some symptoms have improved: skin lesions, itchy eyes, and oral ulcers. however, he has begun to suffer from constipation and continues to intermittently have night sweats, fevers, nausea and vomiting. in february of , he had pruritus that was not alleviated by hydroxyzine. a regimen of benadryl and pepcid was started to aid with the pruritus. the patient reported in the improvement of his symptoms of pruritis, sweats, and emesis. conclusions: diagnosis: celiac disease(cd) patient with persistent nausea, vomiting, sporadic fevers, and fatigue despite maintaining a strict gluten free diet has shown improvement of symptoms with antihistamines. patient has history of chronic infections: prostatitis and chronic ebv. patient also reported alleviation of symptoms after interferon therapy suggesting some autoimmune component to his current illness. cd prevalence in the united states is much higher than once thought . - % of the u.s. celiac disease has been associated with increased risk of lymphoma and malabsorption leading to neurological diseases. this is a rare case of cd associated pruritus responding to antihistamines. rationale: eosinophilic cystitis is a relatively rare condition in children and adults with a varied course. it usually responds to short-term nsaid and steroid therapy. we report a man with a severe case who responded to prolonged oral steroids. case report: a year old caucasian man with a prior esophageal cancer s/p esophagectomy, diabetes, hypertension, allergic rhinitis and chronic obstructive pulmonary disease presented with suprapubic pain, urinary frequency, dysuria, and hematuria. urinalysis showed numerous red blood cells and bacteria but no malignant cells or eosinophils. he was treated with antibiotics with resolution of symptoms. several weeks later he experienced severe suprapubic pain and hematuria resulting in a symptomatic drop in his hemoglobin. he underwent a cystoscopy and biopsy that revealed a chronic cystitis with an inflammatory infiltrate containing numerous eosinophils. he was unresponsive to treatment with nsaid and intravesicular dmso and was then referred to our service. we started prednisone mg/day but the dysuria and hematuria persisted. prednisone was doubled plus a third generation quinolone was added. three weeks later the hematuria and dysuria resolved and he was asymptomatic. the antibiotic was stopped and prednisone was gradually tapered over several weeks. whenever his steroid dose dropped below mg/day he experienced an exacerbation of his symptoms. over the last three years this dose of prednisone has kept his symptoms abated and his renal function stable. conclusion: eosinophilic cystitis is an uncommon diagnosis of unknown etiology. we describe a patient with debilitating suprapubic pain and symptomatic anemia from the hematuria associated with eosinophilic cystitis. for over three years since we first saw him, continued therapy with mg of prednisone/day has prevented exacerbations. this case is unique in the severity of the hematuria experienced and the prolonged relatively low steroid dose needed to suppress exacerbations. we propose that in eosinophilic cystitis patients with severe hematuria who may otherwise be candidates for a cystectomy, a trial of prolonged oral prednisone may be beneficial. rationale: allergic reactions to insulin occurred more frequently in the past, with porcine and bovine preparations. in contrast, allergic reactions to human insulin preparations are now reported in < % of patients treated with insulin. insulin allergy may be manifested as an immediate-type ige-mediated reaction, delayed type hypersensitivity or as serum sickness, varying in severity from mild discomfort to life-threatening events. we present a case to illustrate that an insulin allergy may complicate hospitalization and often be misdiagnosed. methods: a y.o. diabetic female with a history of "insulin allergy" was evaluated. the patient is a long standing diabetic controlled on oral hypoglycemics but required insulin during acute illnesses and hospitalizations. the patient was unable to recall previous types of insulin she had received. during previous hospitalizations, the patient complained of vague symptoms of fatigue, parasthesia, sweating, anxiety, and palpitations. on one occasion the patient had a syncopal episode with hypotension and on another occasion the patient developed a rash and dyspnea after receiving sq insulin. the physcian intrepreted the findings may be due to hypoglycemic. an allergic reaction was not suspected and the patient never underwent any testing. during a recent hospitalization, the patient's history was reviewed and a formal allergy consult was obtained. she underwent epicutaneous and intradermal testing to human insulin preparations. insulin antibody levels were also obtained. results: the patient had negative epicutaneous testing to all human insulin preparations. however, on intradermal testing, the patient had positive reactions to lispro, nph, and lente and negative intradermal tests to insulin glargine and regular insulin. igg and ige insulin antibody test results were < . conclusion: hospitalized patients receiving multiple medications commonly experience pharmacologic, adverse and/or allergic reactions. it is necessary to obtain a thorough history and document all reactions that patients experience to determine what type of event occurred. with a suspicion of drug allergy, skin testing and/or rast assay may provide insight into possible ige mediated reactions. in this case we advised the patient that she may use regular insulin during hospitalizations and that insulin glargine can be used to help achieve optimal glycemic long term control. background: hereditary angioedema type iii (hae iii) is a recently described form of angioedema occurring exclusively in females and characterized by normal c , c inhibitor (c inh) protein and function. hae iii is thought to have an x-linked or autosomal dominant mode of inheritance. we evaluated a year old female with recurrent facial swelling, abdominal pain and laryngeal edema with a family history of similar symptoms in several female relatives. case report: a year old african american female presented with a two year history of recurrent lip, tongue and facial swelling. she also had abdominal pain, diarrhea and shortness of breath. episodes were not associated with hives. her symptoms predated menarche and did not correlate with her menstrual cycle. at the time of presentation she described an increase in frequency of her symptoms that did not respond to antihistamines (diphenhydramine, cetirizine) or prednisone. the patient had no other medical problems. family history was significant for recurrent episodes of facial, lip and tongue swelling in a maternal aunt, grandmother and great grandmother. the patient's great grandmother required tracheal intubation with ventilator support for upper airway compromise. the patient's physical exam was unremarkable. laboratory values drawn during an acute episode of swelling revealed: c inh function = > % (normal > %), c inh protein = mg/ml (normal - mg/ml), c = . mg/dl (normal - mg/dl). dna sequencing at exon to investigate the possibility of an unusual c inh mutation with normal c s binding but abnormal kallikrein inhibition was negative. other lab tests included a normal mast cell tryptase of . mcg/l, a negative rf and ana, a normal angiotensin converting enzyme of u/l (normal - u/l) and positive skin prick tests to a variety of foods which the patient tolerates. based on two case reports of treatment of hae iii with androgens, the patient was started on danazol mg daily with symptomatic improvement. conclusion: hae iii is a rare disease that affects females exclusively. clinically it is indistinguishable from c inh deficiency. the mechanism of inheritance remains to be elucidated. it is unclear why danazol appears to ameliorate symptoms even though there is no evidence for c annals of allergy, asthma & immunology inhibitor dysfunction in this disorder. we believe this to be the first kindred of hae iii reported in the united states. r. dworski * , m. peters, nashville, tn. a four-month-old female identical twin was evaluated for noisy breathing and recurrent cyanosis. she was delivered at weeks of an estimated gestational age after an uncomplicated pregnancy. at birth she was intubated for hours for respiratory distress but the remainder of her neonatal period was uneventful. at age weeks she developed a noisy breathing often associated with cyanosis, particularly in a supine position or while crying. treatments with inhaled albuterol and prednisolone were ineffective. she had no respiratory infections or symptoms of gastroesophageal reflux disease. her growth was normal. her twin sibling was well. the family history was negative for allergies or respiratory conditions. initially she was diagnosed with tracheomalacia. however, the history of cyanotic episodes prompted a search for a definitive diagnosis. she underwent bronchoscopy which showed tracheomalacia when breathing spontaneously and circumferential narrowing of lower trachea likely due to compression. echocardiogram revealed a double aortic arch. the finding was confirmed by computed tomography angiography which demonstrated the presence of a vascular ring composed of double patent aortic arches, each giving rise to the ipsilateral carotid and subclavian arteries. the airway was normal at the aortic inlet, but narrowed markedly at the level of the two arches. a surgical division of the ductus ligamentous and distal anterior vascular arch was performed. aortic arch abnormalities should be suspected in all infants with hoarse coughing or noisy breathing, especially during inspiration but sometimes also during expiration. the diagnosis should also be considered in older children with recurrent bronchitis or pneumonia. respiratory symptoms are more frequent than gastrointestinal manifestations. diagnosis usually occurs in the first year of life. a surgery is often the treatment of choice. postoperative complications are relatively rare. outcome of surgery should be judged after months, including at least one winter season. malacia can delay extubation and recovery following surgery. surgical cure occurs in approximately % of patients. surgery is probably less successful in children with double arches and malacia. a. thatayatikom * , a.j. white, st. louis, mo. background: common variable immunodeficiency (cvid) is the commonest symptomatic primary antibody deficiency syndrome in which b lymphocytes produce low levels of immunoglobulin, leading to recurrent bacterial infection. although hypogammaglobulinemia and susceptibility to the recurrent infection are seen in all patients, other associated conditions such as a non-infectious granulomatous disease have been well described in cvid. corticosteroid therapy has been used with improvement in a subset of cvid with granulomatous disease; however, its treatment remains problematic and a new therapeutic agent is needed. tumor necrosis factor (tnf ) has been demonstrated as a primary mediator in granuloma formation and maintenance. therefore, anti-tnf medications are potentially therapeutic agents of the granulomatous disease. case report: a -year-old caucasian male with cvid and severe granulomatous disease was treated successfully with infliximab, a chimeric anti-tnf monoclonal antibody. the patient initially presented with high fever, chills and abdominal pain; subsequently, he developed acute respiratory failure and adult respiratory distress syndrome. the patient was hospitalized and he required intensive care with ventilatory support. his diagnostic tests revealed elevated sedimentation rate and positive epstein-barr virus (ebv) capsid igm antibody. imaging studies demonstrated bilateral diffuse pulmonary infiltrates and hepatosplenomegaly. open lung and liver biop-sies revealed non-caseating granulomatous lesions without evidence of ebv or other infections. high dose corticosteroid therapy was given with partial improvement, then high dose infliximab ( mg/kg) was given weekly with remarkable improvement. the patient was able to wean off ventilator successfully within weeks and his prednisone dose was dramatically decreased. infliximab infusion ( mg/kg) every weeks and low dose prednisone were continued. a follow-up liver biopsy after months of the infliximab showed no granulomatous lesions. infliximab was discontinued after months of the treatment. there was no serious infection or complication during the period of treatment. conclusion: anti-tnf therapy may be a safe and effective treatment and may allow corticosteroid dose reduction. future clinical studies of anti-tnf therapy in cvid with granulomatous disease are warranted. background: chrug-strauss syndrome is a disorder characterized by hypereosinophilia and systemic vasculitis occurring in individuals with asthma. objetive: to present a pediatric case suffering from a systemic vasculitis. this case fulfilled the churg-strauss syndrome clinical criteria and the histophatology findings were compatible with the diagnosis. case: a year female came to our institution with the diagnosis of severe asthma, chronic sinusisits and polyps requiring high doses of steroids. there was no history of administration of antileukotriene receptor antagonists. months before her admission she presented weight loss, fatigue, cephalea and cough. on physical examination, pallor, respiratory difficulty and signs of bronchospasm were evident. tachycardia and hepatomegaly were also documented. the laboratory test showed anemia, eosinophilia /dl, anca+, sgot , stgop , ige elevated ui/ml. the chest-x ray showed patchy opacities in both lungs and cardiomegaly. pulmonary scintigraphy reported low perfusion in both lungs, predominantly in the left lung.echocardiography demonstrated signs of myocarditis and eyection fraction of %. an open lung biopsy was executed and vasculitis with fibrinoid changes affecting small and medium vessels was reported. treatment was started with oral prednisone mg/kg/d and cyclophosphamide pulses with a satisfactory evolution. discussion: to our knowledge this is the first pediatric case of css reported in mexico. she fulilled the following criteria. asthma, eosinophilia and systemic vasculitis involving the heart, liver and lungs. the disease is extremely rare, specially in mexico. aggressive treatment is necessary as in this case, with a favorable outcome. introduction "all that wheezes is not asthma" is a well-known aphorism among physicians. this same principle exists for patients that present with lip swelling in the allergist's office. we describe a patient who presented with fluctuating lip swelling who was ultimately found to have cheilitis granulomatosa. case history a -year-old male with a history of allergic rhinitis and asthma presented to the allergy clinic with lower lip swelling and occasional upper lip swelling. he was receiving allergen immunotherapy for dust mites and trees. the swelling had been waxing and waning for years, but became more persistent for the previous six months. he denied tongue/throat swelling, dysphagia, respiratory distress, or any triggers for the swelling. chapstick® and vaseline® were used topically on his lips. failed treatments included loratadine, ranitidine, cetirizine, fexofenadine, and montelukast. he was placed on a one-week course of prednisone, which decreased his lip swelling, but it recurred after completion of the treatment. physical exam was significant for diffuse, firm lower lip edema to - times the normal size. there were no oral lesions or tongue swelling. patch testing to a standard panel, preservatives, oral flavors, and dental acrylate was negative. punch biopsy revealed a noncaseating epithelioid granulomatous inflammation consistent with granulomatous cheilitis. he was placed on minocycline mg by mouth twice daily with little benefit. an -week course of oral prednisone resulted in improvement of the lip swelling. conclusion melkersson-rosenthal syndrome (mrs) is a rare syndrome that is characterized by a triad of recurrent facial paralysis, chronic edema of the face and lips, and hypertrophy and fissuring of the tongue. cheilitis granulomatosa is considered a monosymptomatic form of mrs and manifests as a chronic swelling of the lips caused by granulomatous inflammation. the swelling is typically not tender and may be either soft or firm. allergists are often consulted for lip swelling thought due to angioedema. however, as this case illustrates, not all lip swelling is angioedema and one must consider other diagnoses such as melkersson-rosenthal syndrome and cheilitis granulomatosa. progressive multifocal leukoencephalopathy (pml) is a disorder of the nervous system that affects individuals with immune suppression. it has been associated with hiv infection and is present in nearly % of patients with acquired immune deficiency syndrome. the jc virus, a common human polyomavirus, causes this demyelinating disorder. progressive symptoms reflect the multifocal distribution of brain lesions, and include mental deterioration, vision loss, speech disturbances, ataxia, paralysis, and, ultimately, coma. in rare cases, seizures may occur. there is no known treatment for pml. we report a year-old (y/o) male with months weight loss and a sudden onset of confusion, lethargy, and progressive loss of cognition requiring hospitalization. upon questioning he was found to have had recurrent upper respiratory tract infections since infancy successfully treated with antibiotics. as a child he had atopic dermatitis, exercise induced asthma, and myringotomy tubes placed twice. at y/o, he underwent a nasal polypectomy. in , at y/o, a squamous cell carcinoma was removed, and he developed benign cervical lymphadenopathy and common warts. a year later he developed hsv esophagitis. the remainder of his history was unremarkable with normal development and growth and no history of drug abuse, multiple sexual partners, or homosexual contacts. on physical examination, he was thin, ill appearing, with oral ulcers, generalized scanty lymphoadenopathy, multiple common warts on both feet, and occasional ronchi. a brain mri showed a demyelinating process consistent with pml. pcr for jc virus was positive, while hiv pcr was negative; total immunoglobulins and cd counts were low ( we are reporting a year old female with a year history of moderate persistent asthma, who was started on xolair mg sq q wks., and who then presented with a presumed allergic reaction. three hours after her second xolair injection, patient reported developing dizziness, shortness of breath, and felt like she was having an allergic reaction. she was evaluated and observed for two hours in an emergency department. the treating physician reported no wheezing and felt there was no need for treatment. to rule out psychogenic factors, she was given a placebo injection at the next scheduled visit. ten minutes later she reported developing throat tightness and shortness of breath. she had no changes in her peak flows and her lungs were clear. her "symptoms" resolved completely within minutes of receiving placebo epinephrine and nebulized normal saline. patient was informed she had reacted to a placebo injection, as well as placebo epinephrine and albuterol, and counseled. the patient returned to the office every week for the next three weeks to receive blinded injections. she subsequently did not react to either doses of placebo or xolair. she has since tolerated her monthly xolair injections without incident. this case illustrates the importance of ruling out psychologic causes of presumed allergic reactions. introduction :latex allergy, type i ige mediated hipersensitivity, occurs specially in high risk populations, like in patients that have undergone various surgeries. case: a year old boy with asthma and allergic rhinitis since , penicillin allergy and retrospectively, his mother refers lip edema with balloon inflating.at years of age ( ): left orchiorrhaphy because of cryptorchis. between - : surgeries because of sacral giant melanocitic naevus.during the fourth surgical intervention ( ct - ) to collocate a tissue expander, the patient presents perioral and fingertip cyanosis, generalized cutaneous rash and severe bronchospasm.ap: / mmhg, hr x/min. he was treated with iv fluids, steroids, antihistamines and inhaled racemic epinephrine.at the icu his final outcome is satisfactory. laboratory: total ige : . iu/l, skin prick test with glove extract, raw and natural latex extract and purified latex proteins(pseudoeveine, molecular hevein, hev b . and modified hevein)all positive +++. western blot with protein extract of latex positive and elisa with purified latex proteins ( same as above) positive. the last surgery to withdraw the tissue expander was performed with latex free surgical equipment without any problems. discussion: the patient's risk factors for latex allergy are atopy and repetitive exposure to latex articles because of surgery. he presents mild manifestations of latex allergy, till he finally develops full-blown anaphylaxis. diagnosis was made based on clinical history, skin prick test, western blot with protein extract and elisa with purified latex protein. he had a favorable outcome withdrawing latex during the last surgery. introduction: churg-strauss syndrome (css) is a form of primary vasculitis that is a rare diagnosis in an elderly patient. case report: a year old woman presented with a week history of fatigue, vomiting, diarrhea, abdom-inal pain, and right lower leg paresthesia. prior to admission she was being treated for left neck erythema and adenopathy presumed to be cellulitis. she was in good health with no history of atopy until the age of , when she developed both chronic sinusitis, requiring bilateral sinus surgery and polypectomy, * and new onset asthma, * that required systemic steroid control. prednisone was tapered month prior to admission. objective findings included coalescent non-blanching petechiae on her abdomen, peripheral eosinophilia of ( %), * normochromic normocytic anemia, rf= , esr= , ige= , igg= . ana, p and c-anca were negative. ct showed ascites and pleural effusions. egd revealed duodenitis with ulceration and eosinophilic infiltration on biopsy. echo showed pericardial effusion and septal motion abnormality. troponin of without cad was consistent with subepicardial myocarditis. emg confirmed right peroneal neuropathy.* skin biopsy revealed a dense superficial and mid-dermal perivascular and interstitial eosinophilic infiltrate. additional evaluation excluded malignancy, infection, and abpa. treatment with prednisone, mg/kg/day was initiated. a rapid clinical improvement ensued and has persisted. {*acr criteria for css}. discussion: churg-strauss syndrome is a rare form of vasculitis with mean age of onset within the third and fifth decades. it is an uncommon cause (< %) of systemic vasculitis in patients older than . the formes frustes of css is a variant in which early manifestations of the syndrome are hidden by oral and systemic steroids employed in the treatment of worsening asthma often associated with css. this variant makes expedient identification of css more challenging. delayed recognition contributes to a relentless progression of this entity resulting in a systemic vasculitis with multi-organ involvement. early diagnosis, especially in the prodromal and eosinophilic phases, is essential. untreated, css has a high rate of morbidity and mortality. therefore, css and the formes frustes variant must be an integral component in the differential diagnosis of patients presenting with adult onset asthma and/or recurrent sinusitis. common variable immunodeficiency (cvid) is the most prevalent of the primary immunodeficiency diseases. cvid is a heterogeneous group of immunologic disorders of unknown etiology, characterized by impaired antibody responses, hypogammaglobulinemia with normal b cells. the common immunologic defect in patients with cvid is defective antibody formation, and many different immune system defects have been reported in this group of patients. most patients, really, have no identified molecular diagnosis. cvid consists of several different genetics defect. the immunologic defect in cvid is a failure of b-lymphocyte differentiation into plasmacells. b lymphocytes from these patients failed to differentiate into ig-producing cells when stimulated with pokeweed mitogen in vitro, even when cocultured with normal t cells. an overwhelming body of literature suggests that most patients with cvid have intact b lymphocytes of immature phenotype. however the functional classification of cvid patients on the basis of in vitro ig production is time consuming. recently has been proposed a new classification based on the quantitative repartition of memory b cell according to the dual expression of igd and cd . we present a case of a yr old boy. he presented soon in his life frequent infections, of particular severity: pneumonia, meningoencefalitis, sepsis, bronchitis, otitis, linfoadenitis. he also had a -thalassemia intermedia. this clinical manifestations suggested an immunodeficiency. for this reason at years of age serum immunoglobulin and antibody detection showed a reduction in igg subclass and in cd + cells, with normal total igg, iga, igm, normal cd /cd ratio, normal cd , isohemagglutinins and in vitro t cell function. there was also a defective antibody production after tetanus, diphtheria, pertussis immunization. these laboratory findings did not allow, however, a sure diagnosis for cvid. a new immunological evaluation at the age of years old, after the onset of splenomegaly, and enlarged lymph nodes, demonstrated a b memory defect, with a severe deficit of t lymphocytes function in vitro. it was also possible to find a severe deficiency of cd + cells, meaning a defect in memory b cells: we can therfore label this condition as a cvid. in the past two decades there have been conflicting views regarding the clinical importance of igg subclass deficiencies in children. igg subclass plays a vital role in the immune response to polysaccharide antigen. isolated igg subclass deficiency may be widespread and often asymptomatic in children. however, in association with other subclasses and/or other immunoglobulin classes, there may be a significant, symptomatic outcome. the reported patient was diagnosed with familial dysautonomia (fd) at the age of five weeks, presenting with severe hypotonia and tachypnea. hindered by poor pulmonary function, he was hospitalized over fifteen times for recurrent pneumonias, including four lengthy intensive care admissions. daily inhaled-corticosteroids and brochodilator therapies were initiated, along with chest therapy via a high frequency chest wall oscillator. immunoglobulin levels were measured recently and point to low levels of igg , igg and total iga antibodies: igg mg/dl (n - ), igg mg/dl (n - ), igg . mg/dl (n . - . ), igg < . mg/dl (n . - . ), and total iga mg/dl (n - ). since receiving monthly ivig therapy he had no further recurrence of pulmonary infections and was slowly weaned off daily brochodilator therapy. the currently accepted theory is that low igg subclass levels may be associated with increased risk of bacterial infections only in selective groups. fd patients may be in a distinctively susceptible population in which igg levels are critical. the older brother, who was also diagnosed with fd, demonstrated igg , igg and iga levels that were slightly higher but nevertheless on the lower end of the normal range. he suffers from less invasive and less frequent bacterial infections. this may support a genetic association between the fd and hypogammaglobulinemia. alternatively, it may signal that fd patients may have a prolonged variant of transient hypogammaglobulinemia of infancy. follow-up immune profile studies, post-ivig trough levels and broader investigations of the fd population are necessary to determine the severity and prevalence of these findings. pulmonary failure is the dominant cause of death in patients with fd. prompt diagnosis and effective treatment of the associated immune deficiency may be proven essential in the effort to enhance and prolong their lives. s. hassan * , j.a. grant, galveston, tx. rationale: common variable immunodeficiency (cvid), a rare primary immunodeficiency presenting in young adults with repeated sinopulmonary infections as a result of profound hypogammaglobulinemia, was first described by suri et al. (ann acad. med. singapore, ) . we describe a young man with diagnosed but untreated cvid and its eventual course. case description: a -yr-old caucasian male hospitalized secondary to chronic pneumonia and respiratory failure was noted to have non-existent levels of immunoglobulins. history revealed ivig treatment at age , stopped after a year due to non-compliance. although untreated, he denied recurrent sinusitis, otitis media, or bronchitis for almost years but notes a recent inability in keeping up with baseball practice. he is the last of ten healthy siblings. patient started monthly ivig infusions but was noted to have hypertension ( / ), tachycardia ( - ), tachypnea ( - ) on the th month with o saturation of - % and po of % on room air. with a -day history of acute dyspnea, calf muscle and right abdominal pain, he was admitted to the hospital and pulmonary thromboembolism was ruled out. laboratory data: humoral functions (pneumococcal, tetanus toxoid, and hepvac) -undetectable t cell function (mumps, candida, ppd) -normal immunoglobulin (igg, iga, igm) -undetectable flow cytometry -b and nk cell markers normal, mild decrease in the cd /cd ratio high resolution ct thorax -bronchiectasis, bronchial wall thickening, and obstructive changes with airtrapping. minute walk -desaturation to % on l o by nc bnp - echocardiogramestimated ef - %; severe pulmonary hypertension. cardiac catheterization -normal coronary arteries, severe pulmonary hypertension (pa pressure / ). conclusion: cvid patients have a reasonably good prognosis on treatment. untreated cvid is associated with chronic lung infections, bronchiectasis, pulmonary hypertension and right heart failure. although, lung transplant became available during the early eighties (nejm ), this extremely invasive but life saving procedure was undertaken in a patient with cvid and end-stage pulmonary hypertension in (thorax ). lung transplant may be the only way of ensuring survival for this patient. introduction chronic eosinophilic pneumonia (cep) is a rare disorder of unknown etiology characterized as a chronic and relapsing interstitial lung disease with blood or tissue eosinophilia. cep occurs more often in women with preexisting atopic disease. patients with cep respond rapidly to systemic corticosteroids, but often relapse with short, low-dose courses of therapy. while uncommon, extrapulmonary involvement, such as arthralgia, cutaneous purpura, pericarditis, and hepatitis, have been reported. we present a case of a patient who has cep with pericardial effusion. case report the patient is a non-smoking -year-old woman with a past medical history significant for allergic rhinitis and asthma who initially presented with a four-month history of worsening shortness of breath, dyspnea on exertion, dry, non-productive cough, and weight loss (approximately five pounds). her symptoms were refractory to increased dosages of inhaled fluticasone. she then developed intermittent fever up to °c. chest x-ray revealed bilateral apical infiltrates. treatment with levofloxacin for seven days resulted in no improvement of symptoms or roentographic findings. thereafter, she presented with chest and abdominal pain, hypotension, and hypoxia. blood work revealed a white cell count of c/mm with a differential significant for % eosinophilia (absolute eosinophil count of c/mm ). chest ct showed dense consolidation predominantly along the peripheral aspect of the upper and superior segment of the lower lobes, and pericardial effusion. moderate pericardial effusion without evidence of tamponade was confirmed on echocardiogram. left upper lobe wedge biopsy findings included significant tissue eosinophilia, scattered foci of active organizing exudates, and no evidence of granulomatous or necrotizing vasculitis. the patient was treated for cep with a six-month course of prednisone, starting at mg daily, resulting in rapid improvement of her pulmonary and systemic symptoms. background: immunologists are consulted for evaluation of immunodeficiency in patients with recurrent skin infections. disorders of the phagocyte system may be associated with cutaneous infections. methods: case report case: a -month-old african american girl (twin a) presents with recurrent skin abscesses. at months of age, she had her first buttocks abscess, which required incision and drainage with oral antibiotics. a month later, she developed another abscess and was found to be neutropenic, with an absolute neutrophil count (anc) of /μl. cyclic neutropenia was considered and her pediatrician monitored cbcs, which all showed persistent neutropenia (ancs between and ). at months of age, she was hospitalized for fever with neutropenia. immunology was consulted for evaluation of neutropenia. the rest of the past medical history was unremarkable. she was healthy appearing and growth parameters were appropriate for age. her physical examination was unremarkable. immunoglobulins, b-and t-cell markers, nitroblue tetrazolium, complement assay, hemoglobin, platelets and the peripheral smear were normal. antibodies for hiv, cmv, ebv and parvovirus were undetectable. anti-neutrophil antibodies were positive, establishing the diagnosis of primary autoimmune neutropenia (ain). the abscess healed with oral antibiotics. severe neutropenia (anc - ) persisted for three subsequent months without further infections. twin (b) was also found to have persistent severe neutropenia without morbidities, suggestive of primary ain. primary ain is less known among physicians and is typically diagnosed after extensive investigations that exclude other causes of neutropenia. the exact incidence of ain is unknown. it is usually seen in children between and months of age, often with severe neutropenia and self-limiting bacterial infections. the clinical course and presence of antibodies to neutrophil antigens (na , na or cd b/ ) is diagnostic. familial occurrence of primary ain is not reported in the literature. conclusion: primary ain may remain under diagnosed due to lack of characteristic clinical features. although a benign clinical course is likely, severe infections, including pneumonia, sepsis and meningitis, have been reported. genetics may play a role in this disease, as we present primary ain in twins. background: atopic dermatitis (ad) is a chronic inflamatory disease of skin that affects % of the wordl population.the natural history of ad in some patients, evolve to the coexistence with other allergic diseases: allergic rhinitis, allergic conjunctivitis and asthma. the sublingual immunotherapy has demonstrated utility in some patients; however, semi-rush immunotherapy to pollens has only showed utility in one animal case published few years ago. case report: a -year-old infant was referred to us. he began one year before with skin lesions compatible with ad, six months later began perennial rinhorrea and nasal itching. multiple treatments with topic corticosteroids, moisturizing, antihistamines and topic/systemic antibiotics did not demonstrate utility. our evaluation revealed ad lessions that affected % of the total body surface and clinical features of allergic rhinitis (ar).the lab tests revealed eosinophylia ( cell/mm ) in blood cell count, normal levels of total ige but specific ige to dermatophagoides pteronissinus (dpt) was high. the skin prick test reveales the same results. we added environmental control, oral costicosteroids and transfer factor. despite our treatment no improvement was observed. we considered dpt as the principal factor in the maintenance of ad lessions and ar episodes. in the absence of sublingual immunotherapy in our hospital, we decided for semi-rush immunotherapy schedule. we began from . ml of : , w/v concentrations of dpt until , ml of : w/v concentrations in two months receiving three doses per week. no local or systemic adverse events was reported and the ad lesions and ar symptomatology disappeared in the first month of treatment. at this time, no exacerbations have been documented. conclusion: the semi-rush immunotherapy can be useful and safe for treatmente of ad in some patients in whom specific ige to aeroallergens has been demonstrated. introduction: eosinophilic gastrointestinal disorders are a rare group of disorders that can involve the entire gastrointestinal tract. presentations are varied but may include vomiting, dysphagia, abdominal pain, diarrhea and failure to thrive. the diagnosis is made by endoscopic biopsies which reveals eosinophil rich inflammation in the absence of known causes for eosinophilia. peripheral eosinophils and ige may be elevated but can be normal. we report a patient with eosinophilic gastroenteritis associated with an ampullary tubulovillous adnenoma. methods:a -year-old white male with allergic rhinitis and a family history of atopy was admitted for profuse watery diarrhea. he denied any new medications, eating raw foods or recent travels. eosinophils were elevated to . ( % of wbc) and ige was elevated to . multiple stool specimens were negative for ova & parasites and enteric pathogens. serology was negative for strongyloides, trichinella, e. histolytica and toxocara. ast, alt & bilirubin were elevated and a ct scan showed dilated billiary ducts with a possible ampullary mass. biopsy of the ampullary mass revealed a tubulovillous adenoma. biopsies of the duodenum, terminal ileum, colon and rectum were remarkable for focal eosinophilic cryptitis & chronic inflammation in the lamina propria consisting of eosinophils, scattered lymphocytes and histiocytes. all specimens were negative for parasitic infections including duodenal aspirates. he was empirically started on metronidazole and singulair with gradual improvement of symptoms, eosinophils and liver tests. two months after discharge, the patient remained diarrhea free with a normal eosinophil count. conclusion: we report a patient with eosinophilic gastroenteritis and an ampullary tubulovillous adenoma with obstruction of the biliary system. this unique presentation illustrates the diverse nature of gastrointestinal manifestations seen in eosinophilic gastroenteritis. background: zonisamide is an anti-seizure medication chemically classified as a sulfonamide and unrelated to other ant seizure agents. we report a case of hypersensitivity to this agent in a child. method: case report results: this patient is a -month-old girl who developed "peeling of her lips" three weeks after starting zonisamide. one week later she developed a rash that began on her face and generalized over several days to her neck, trunk and extremities. there was no respiratory distress, joint complaints and no angioedema associated with the episode, but fever to prompted referral to our hospital on day of the rash. the rash was macular-papular without discrete urticarial lesions. the rash coalesced with underlying erythema on face, chest and neck. the patient has a known history of seizure disorder, asthma, mild eczema, gastro esophageal reflux, development delay, lactose intolerance and failure to thrive. her other medications were, lansoprazole, topiramate, and albuterol. labs revealed a normal white cell count, an elevated sed rate ( ) and elevated lft's, i.e. sgot and sgpt . the only new medication was zonisamide that was discontinued. she was treated with iv steroids and hydroxyzine. the rash started fading by day and the patient's fever resolved by the third hospital day. liver enzymes returned to normal by day . conclusion: this relatively new anti-seizure agent can be associated with hypersensitivity reactions in children. introduction: kawasaki disease (kd) is an acute chilhood vasculitis. in addition to the diagnostic criteria a broad range of nonspecific clinical features may be observed including aseptic meningitis, vomiting, diarrhea, abdominal pain, sterile pyuria, arthralgia and arthrtis, pulmonary infiltration, pleural effusion and nonspecific paralytic ileum as manifestation of gastrointestinal vasculitis. we describe a child who developed all features of kawasaki disease included the most rarely reported. patient report: a year-old female presented days of high fever, nonsuppurative cervical lymphadenopathy, petequial rash in legs, swelling of feet, distended abdomen, vomiting, obnubilated and hypoactive, incongruent speech, fisured lips and distended abdomen. lab tests showed: anemia ( . g/dl), high wbc count ( , cells/mm ), thrombocytopenia ( , /mm ), hypoalbuminemia ( . g/dl), lactate dehydrogenase (ldh) mcg/l, glutamin transferase (ggt) . csf total proteins , glucose , cells , pm %, mn %, seric complement , cultures were negatives. she developed myocarditis, aneurysms in the right and left coronary arteries. on the th day presented cardiac failure, pleural effusion, paralytic ileum, hydrops vesicular, mechanic ventilatory assistance was required. the first dose of intravenous immunoglobulin (ivig) ( g/k) was infused, heparin and hydrocortisone. on day a second doses of ivig was infused, because of fever, and abdominal vasculitis. steroids ware discontinued, heparin was suspended and aspirin was added as antiaggregant. discussion: our patient presented an unusual and severe presentation of kd with pleural effusion, nonspecific ileum, cardiac failure secondary to myocarditis, aseptic meningitis and thrombocytopenia; all those manifestations had rarely been reported at the same time. she presented with a devastating evolution. complications were resolved. the lastest studies have shown that treatment with ivig plus corticosteroids significantly reduce serum concentrations of proinflamatory cytokines. in this case antiinflamatory doses of aspirin were contraindicated and steroids were used with satisfactory outcome. limited data is available for nonresponding patients to guide therapy. although multiple doses of ivig are sufficient in some patients, some of them remain refractory to therapy and they need corticosteroids to control the vasculitis process eosinophilia is defined by an absolute eosinophil count above . x and can be seen in association with a broad spectrum of disorders ranging from allergic to malignant. idiopathic hypereosinophilic syndrome (hes) should be considered in any patient with an eosinophil count above . x for more than months without commonly recognized causes of eosinophilia and with evidence for organ damage not otherwise explained in the clinical setting. it is potentially an aggressive disease with mean survival of less than a year without treatment. we present a year old male horse breeder, with eosinophilia lasting more than years. prior to coming to our institution, he underwent extensive medical evaluation including bone marrow and gi biopsies, multiple imaging studies with mri and ct scans of the chest and the head as well as detailed evaluation of his cardiac status. all these tests were normal and the patient was empirically started on treatment for possible hes including trials of prednisone, hydroxyurea, interferon alfa, and imatinib, all without lasting resolution of his eosinophilia and causing significant side effects with profound depression of his immune status. finally, in light of the patient's profession, a strongyloides enzyme immunoassay was done and found to be remarkably positive. duodenal drainage confirmed the infestation with this nematode. within a week of starting treatment with ivermectin, his eosinophil count came down by % and eventually normalized. we learn that one should be persistent in excluding all common causes of eosinophilia before considering hes. in case of parasite infestation, premature treatment with immunosuppressors can result in worsening of the infection with potential for poor and even fatal outcome. stool evaluation might not be sensitive enough for detection of parasites and it is therefore necessary to complete a diagnostic work up with appropriate serology. doxil is the pegylated liposomal form of doxorubicin and has been used successfully as a cancer chemotherapy agent in many types of tumors. a hypersensitivity reaction can occur, usually during the first infusion, and appears to be rate related. the symptoms include dyspnea, tachypnea, facial swelling, chills, hives, chest pain, and back pain. we report a case of a hypersensitivity reaction to doxil in a year-old woman with hodgkin's lymphoma. during her first outpatient doxil infusion at mg/min, she developed urticaria, chest tightness, dyspnea, and back pain. these reactions persisted despite being medicated with antihistamines and steroids and immediately resolved during pauses in the infusion. after mg of doxil, the infusion was discontinued. six days later, she was admitted to complete the other half of the dose. she had a negative intradermal skin test to a : dilution of doxil. she was then premedicated with diphenydramine, dexamethasone, acetaminophen, and famotidine. the doxil infusion was started at . mg/min and was soon stopped due to flushing of the hands and face. the infusion was decreased to . mg/min. she was able to tolerate this slow infusion with only mild and tolerable symptoms. when her symptoms worsened, the infusion was stopped for to minutes. she completed the mg infusion of doxil after hours. pre-infusion and post-infusion complement levels were drawn during this second administration of doxil. her pre-infusion c , c , c a, c , c , c a, and bb levels were all normal. her pre-infusion c a and sc b- levels were high, indicating that she might have had some residual or persistent complement activation caused by her first doxil infusion. her post-infusion c , c , c a, c , c , c a, and bb levels were all normal. however, her post-infusion sc b- levels significantly increased, suggesting complement was activated during the second doxil infusion. given her reaction during her first doxil infusion and a negative skin test to doxil, it is highly unlikely that her doxil hypersensitivity was an ige-mediated process. therefore, in patients with a similar doxil hypersensitivity, we suggest a slow rate of infusion of . - . mg/min, toleration of mild symptoms, to minute pauses during more severe symptoms, and continuation of premedication during the entire lengthy infusion. introduction eosiniphilic esophagitis (ee) is an isolated, severe esophageal eosinophilia. patients are usually young males presenting with vomiting, epigastric or chest pain, dysphagia and obstructive respiratory problems. they are often initially misdiagnosed with and treated for gastroesophageal reflux disease (gerd). distinction between the two diseases can be made with biopsies of the esophageal mucosa. while any eosinophils in the esophageal mucosa indicate pathology, gerd typically presents with up to eosinophils per high powered field (hpf, x) while ee most often presents with greater than - eosinophils per hpf. case report the patient is a and one half year old boy who has experienced severe symptoms of gerd from the age of months. he vomits after meals at least - times per day. he coughs when he lies down at night and regurgitates phlegm in the early morning. he has complained of discomfort in his lower chest after eating. his weight has remained at pounds for the last six months. the patient was diagnosed with asthma at age and a half; however, there is no family history of asthma or allergies. the patient initially experienced improvement on lansoprazole, but his symptoms recurred when the medication was discontinued and subsequent courses were ineffective. his physical exam was normal barring slightly edematous nasal turbinates. an endoscopic biopsy showed "numerous eosinphils" (later clarified to eosinophils per hpf) in his distal esophagus. the patient showed allergy to milk and wheat on radioallergosorbent (rast) testing. the patient was started on swallowed fluticasone puffs twice daily and advised to see a nutritionist regarding a wheat and milk elimination diet. conclusion ee is an important differential of gerd-like symptoms in childhood. to avoid misdiagnosis it is critical to evaluate the number of eosinophils present in a biopsy specimen to help differentiate between gerd and ee. children with ee are at increased risk of developing esophageal dysmotility and esophageal strictures. patients often have positive skin prick or rast tests to foods and aeroallergens. alternative treatments such as food elimination diets and glucocorticoids (systemic or topical) are effective in treating symptoms which may not respond to reflux medications. introduction in asherton introduced the term catastrophic antiphospholipid syndrome (caps) to describe patients sharing clinical evidence of multiple (three or more) organ involvement and/or histopathological evidence of multiple vessel occlusions. case report we present a year old female, with lumbar pain, arthralgias, weight loss, fever, malaise, raynaud phenomenom, alopecia, oral ulcers and hepatomegaly. on arrival, she was polypneic with tachycardia, basal hypoventilation, and hepatomegaly was evident. hb: . , leucocytes: . , total lymphocyctes: , , total neutrophyles: , , platelets , . coombs positive. urin exam: proteinuria, leucocyturia and erythrocyturia. creatinine . . diagnosed as sle with pericarditis, cardiac failure and acute pulmonary edema, urinary tract infection and pneumonia, requiring mechanical ventilation and inotropic support, intravenous gammaglobulin and hydrocortisone. acute renal failure and hemodialysis was begun with improvement. suddenly she presented seizures crisis, stuporous, livido reticularis skin and acrocyanosis, external opthamalplexia, bilateral facial diplexia, ocular fundus with arteriolar vasospasm. right facio-corporal hemiparesia, cortical and progressive medular annals of allergy, asthma & immunology segment changes. ct showed multiple left fronto-occipital parietal hypodensities suggestive of lacunar infarcts. diagnosed as neurolupus and caps. initially anti b glp and anti clp were negative with later positivization. urinary tract infection contraindicated high doses of steroids and, intravenous gamaglobulin and anticoagulation were started. with significant improvement. currently the patient is evolving in a satisfactory way. discussion the literature establishes that catastrophic aps is characterized by elevated mortality. in this patient damage was evident to the cns, pns, kidneys and the skin. with the presence of positive antiphospholipid antibodies with an energic antiinflamatory, immunoregulatory and immunosupressive therapy, the function of each affected organ completely recovered. this case exemplifies that the therapy of caps should be undertaken in a sufficient and early manner given the elevated mortality of this syndrome. patient, a -year-old white, male non-smoker physician on high-dose regimen of advair and singulair presents with an -day history of progressive chest tightness. pulmonary function tests showed normal (fev of %) lung function. in the past, whenever inhaled steroid dosages were lowered, patient experienced a reoccurrence of asthma symptoms, despite consistently normal lung function results. to rule out the possibility of a psychologically induced asthma exacerbation, the patient's fractional concentration of exhaled nitric oxide (feno) levels were measured. the patient's feno level was elevated at . ppb. values greater than ppb have been described as consistent with airway inflammation. with an increase in the patient's inhaled steroids, the patient had a remission of symptoms within a week. this case is illustrative of an increasingly common clinical picture where a symptomatic asthmatic may have normal spirometry but elevated feno. as devices for measuring feno become more available in the outpatient clinic setting, elevated feno may be an excellent diagnostic marker in assessing whether airway inflammation is being adequately treated in situations where spirometry values are within normal ranges. introduction the autoimmune thrombocytopenic purpura (atp) is characterized by thrombocytopenia and megakaryocytic hyperplasia. the first choice of treatment consists of intravenous gamma globulin (ivig), corticosteroids and anti-d antibodies and the second line measures are immunosuppressant drugs, splenectomy and danazol. case report a years old male presented in the first year of life ecchymosis in several parts of the body intermittently. in april he presented ephistaxis and lower gastrointestinal bleeding. complete blood count showed platelet count of , /ul. prednisone was started ( mg/kg) with no improvement. at that time, danazol, anti-d antibodies, and ivig ( g/kg) were added. subsequently, the platelet average count diminished to , . the patient was transferred to our hospital. at his admission he presented cushingoid habitus (figure ) and acanthosis nigricans. the immunological tests (aan, ch , c , c , immunoglobulins, anticardiolipins and b glycoprotein) showed no alterations. bone marrow aspirate demonstrated megakaryocytic hypercellurarity. prednisone dose was tampered when the patient presented secondary glaucoma. patient continued his treatment with danazol and ivig ( dosages of g/kg each), hydroxychloroquine and cyclophosphamide with no improvement. thus, months after the immunosuppressant treatment, splenectomy was performed, obtaining partial improvement. at that time ranitidine and hydroxychloroquine were suspended and cyclophosphamide was changed to azathioprine. last platelet count was , /ul. conclusion chronic atp in childhood as the present case account for approximately - % of the total atp patients. chronic atp that does not respond to conventional treatment is a therapeutic challenge. in this patient we used first choice and second choice drugs with no improvement, consequently a splenectomy was indicated, not obtaining the desired response at first. the use of immunosuppressant drugs is not common for this disease, but it could be a good alternative for atp resistant to conventional treatment. this are the most important laboratory test of our patient. background: budesonide is the only corticosteroid available for inhalation by jet nebulizer and is indicated for the treatment of asthma in children. objective: to evaluate the distribution and clinical efficacy of inhaled budesonide administered by nebulization with a modified commercial device. methods: a year old male with severe persistent asthma underwent a lefort procedure for multiple craniofacial abnormalities. the external device maintained his mouth open impeding the proper use of controller medications. as a result he developed an increase in his asthma symptoms. he was subsequently treated with nebulized budesonide delivered with a modified jet nebulizer through the end of a plastic tube in a flow-by manner. we then performed dynamic ventilation imaging after administering . mci of nebulized technetium m-dtpa diluted in two ml of normal saline. images were obtained for five minutes at three seconds per frame. spirometry monitoring was not possible given the obstructive nature of the facial device. results: the patient demonstrated improvement of his asthma symptoms. the ventilation scan showed that the patient breathed the technetium dtpa through the specialized device with delivery to his full lung volumes within seconds. conclusions: we report the successful treatment of a patient unable to use the commercially available methods for administration of inhaled steroids. rationale: hp is a non-ige mediated inflammatory response in the lungs due to a variety of organic antigens, including metal working fluids. the wideranging clinical features include acute, subacute, and chronic forms. elevated antineutrophilic cytoplasmic antibodies and platypnea, defined as dypsnea induced by the upright position and relieved with recumbency, have not been previously reported in patients with hp. case: -year-old white male tool and dye machinist presented with progressive cough, weakness, dyspnea on exertion, and platypnea. symptoms began weeks earlier with coryza and diffuse myalgias. he had lost % of his body weight since symptom onset. examination revealed a pulse of , respiratory rate , and right basilar crackles. resting pulse ox on room air was %, but dropped to % upon ambulation. pft's revealed severe obstruction (fev % predicted) with significant reversibility (fev + %), and a dlco of % (adjusted for va and hemoglobin). hemoglobin was . with a normal leukocyte count and differential. c-anca (anti-pr ) was . (< ), and p-anca (anti-mpo) was . (< ), both performed by elisa. echocardiogram showed an ef of - % with mild pulmonary hypertension; no shunt was present. chest roentogram was normal, but a chest ct revealed a diffuse ground-glass pattern with scattered centrilobular opacities. biopsy was consistent with hp. he was removed from his work exposure to metal working fluids with full recovery of lung function and resoulution of symptoms. conclusions: hp presents as a constellation of symptoms without a single, unique identifying pattern. platypnea and elevated anca's have been observed in a wide range of disorders, all of which were excluded in this patient. platypnea has been associated with hereditary hemorrhagic telangiectasia, pulmonary avm, hepatopulmonary syndrome, recurrent pulmonary emboli, and patent foramen ovale. elevated anca's has been associated with wegener's granulomatosis, goodpasture's syndrome, churg-strauss vasculitis, drug-induced vasculitis, inflammatory bowel disease, and others. elisa is a more specific modality for anca's than indirect immunofluorescence, but it is less sensitive. the possibility of hp should be considered in patients with either of these two abnormalities. rationale: heart disease is the leading cause of death in america and % of persons between and years of age have coronary athersclerosis at autopsy. the presentation in the elderly is commonly atypical. % of myocardial infarctions were silent or unrecognized in the framingham cohort. in patients and older, it is estimated that - % will present with dyspnea without any associated chest pain. we present a case of unstable angina presenting as exercise-induced asthma. case: an year-old white male with moderate persistent asthma, hypertension, dyslipidemia, and a long history of allergic rhinitis presented having had an abrupt increase in his usual exercise-induced asthma symptoms four weeks prior. asthma had been diagnosed at age with spiromety showing moderate obstruction and significant but incomplete reversibility. he had a remote history of pipe and cigar smoking, but had quit at age . he had been well-controlled since that time with his most recent regimen consisting of fluticasone mcg/salmeterol mcg diskus, one inhalation twice daily. his typical exercise-induced asthma symptoms included dyspnea on exertion and chest tightness without radiation, and were relieved with rest and albuterol. the amount of exertion needed to trigger his symptoms, however, was much less than he had previously experienced, and this remained constant during the four weeks prior to his presentation. one week prior he had a normal ecg evaluation by his primary care provider. six months prior he had a normal nuclear cardiac stress test. physical examination was unremarkale except for moderately decreased aeration and + pitting edema of his lower extremities. cardiology performed a nuclear stress test the next day which was abnormal. catheterization revealed % steonsis of his proximal lad. he succefully underwent cabg and is doing well on follow-up. conclusion: the elderly present many challenges to the diagnostician; these include multiple co-morbidities as well as atypical and often late disease presentations. the key to raising suspicion for cardiac involvement in this case was the recognition of the patient's cardiac risk factors in the setting of an abrupt onset of exercise symptoms while lacking other asthma symptoms such as nocturnal cough. cold urticaria is an uncommon form of physical urticaria. this case of a -year-old with cold urticaria and angioedema provides additional information regarding an unusual disorder in the pediatric population. an otherwise healthy -year-old female presented with a complaint of urticaria precipitated by cold exposure over the preceding weeks. she had no recent illnesses and a past medical history significant only for cat allergy. on multiple occasions the patient noted erythema and pruritus of her arms and face after walking through the freezer aisle of a grocery store. urticaria would then develop on regions where she scratched, spontaneously resolving in - hours. on one occasion, urticaria appeared diffusely while taking a shower after the patient had been swimming. the urticaria resolved within a few hours after the patient was given diphenhydramine by her mother. three days prior to presentation the patient experienced upper lip angioedema with erythema, globus sensation and difficulty swallowing after drinking a strawberry slushy. the patient denied any respiratory complaints at that time and her symptoms again annals of allergy, asthma & immunology resolved spontaneously. family history was significant for a maternal history of seasonal allergies. upon physical exam the patient was well appearing. she had - discrete urticaria on each calf. the patient's mother noted that recently these would appear on "cold and rainy" days, attributing them to the fact that the patient's pants left her lower legs exposed. the remainder of her exam was normal and dermatographism was absent. laboratory evaluation consisted of cryoglobulins and strawberry rast, both of which were negative. application of an ice cube to the patient's forearm for minutes resulted in a x centimeter wheal noted minutes after ice removal. a diagnosis of cold urticaria with associated angioedema was made. the patient's mother opted to use diphenhydramine as needed and an epinephrine autoinjector was dispensed. by months after symptom onset, the patient's only complaint was pruritus of her hands if they became too cold. no urticaria were noted. at month follow-up the patient denied any symptoms for the preceding months and had a negative ice cube test. cold urticaria in the pediatric population is a rare entity and not well understood. this case of a -year-old with cold urticaria and angioedema offers additional insight into this unusual disorder. a. khuntia * , m. mcmorris, ann arbor, mi. introduction: chronic granulomatous disease(cgd) is a heterogeneous group of disorders characterized by genetic defects in the ability of phagocytes to generate microbicidal reactive superoxide anions and its metabolites. it manifests early in life, primarily as recurrent infections, caused by catalase-producing bacteria such as staphylococcus aureus, burkholderia cepacia, and serratia marcescens and fungus such as aspergillus fumigatus. the disease may be inherited in an x-linked or autosomal recessive manner, with x-linked disease accounting for - % of cases. the us incidence of cgd is / , live births with an average age at presentation of years for xlinked and . years for autosomal recessive disease. case report: year old male with history of three separate episodes of pneumonia beginning at age . each episode resulted in a hospital admission and intravenous antibiotic therapy after failed oral antibiotic therapy. an extensive pulmonary evaluation was initiated after the third episode of pneumonia, including a chest ct, viral, bacterial and immunodeficiency studies. cbc, complement, immunoglobulins, flow cytometry, viral and bacterial studies were all normal. the ct scan revealed dense nodular opacities in the right upper lobe with surrounding ground-glass opacification and mild bronchiectasis. a subsequent bronchoscopy demonstrated necrotizing granulomatous inflammation. open lung biopsy grew burkholderia cepacia on tissue culture. the clinical history, tissue histopathology and atypical organism found on culture were all suggestive of an underlying immunodeficiency. a neutrophil oxidative burst assay was performed which demonstrated minimal neutrophil activity upon stimulation, suggestive of the diagnosis of cgd. a chemilluscence assay verified the diagnosis of cgd, with minimal fluorescence noted on flow cytometry after neutrophil stimulation. dna analysis demonstrated a p -phox deficiency, resulting in one of the autosomal recessive and less clinically severe forms of the disease. conclusions: this case of cgd is unusual because of the delayed presentation. it demonstrates the importance of a complete immunological evaluation including an evaluation for neutrophil disorders such as cgd in patients of all ages who present with recurrent and recalcitrant episodes of pneumonia, especially when atypical organisms such as burkholderia cepacia are found on culture. autoimmunity may play a role in the development of premature ovarian failure (pof), but the exact mechanism is not well understood. pof is mainly diagnosed after years of age and most women complain of secondary amenorrhea. pof has been reported in combination with presence of ana and autoimmune diseases, but rarely with jra. here we report two cases of pof and positive ana in pediatric patients, one with clinical features of jra. the first patient, an african american -year-old girl, height in th percentile, weight in th percentile, with tenosynovitis of wrists and arthritis of knees and elbows for the past two years, was referred for further evaluation and management. she did not have her menarche yet and laboratory investigation revealed positive ana. the second patient, an african american -year-old girl, th percentile for height and weight, was referred to the immunology clinic with chief complaint of primary amenorrhea and delayed development of secondary sexual characteristics and was found to have positive ana without clinical findings of jra. both patients were tanner stage for breasts and tanner stage - for pubic and axillary hair. both had elevated fsh and lh, karyotype xx and small uterus and small ovaries on pelvic ultrasound. antiovarian antibody was not detected in any of the two patients. the bone age was significantly delayed. pof in karyotypically normal women is frequently seen in combination with elevated serum ana. the clinical spectrum of rheumatoid disease associated with pof ranges from asymptomatic ana positivity to typical presentation of jra. women with pof should be monitored for the emergence of autoimmune disorders including jra, and women with jra should be followed for menstrual irregularities and signs of pof. introduction: many u. s. military personnel deployed to the middle east continue to develop infection with leishmaniasis, a parasite transmitted by the sand fly. pentavalent antimonials have been used as an effective treatment for leishmaniasis for many years. in the united states, sodium stibogluconate (pentostam) is the pentavalent antimonial of choice, and is currently being administered under an ind protocol. side effects of therapy include myalgias, arthralgias, rash, malaise, abdominal pain, pancreatitis, and hypersensitivity reactions. the true incidence of hypersensitivity reactions is not currently known. case reports: two u. s. soldiers receiving pentostam for the treatment of cutaneous leishmaniasis were evaluated in our clinic at walter reed army medical center after experiencing urticaria, angioedema, wheezing and dyspnea after medication infusion during the -day course of daily therapy. due to the concern of a potential ige-mediated reaction, skin testing was performed. after informed consent, skin testing included a prick test at full strength, followed by intradermal (id)testing. soldier # was a -year old male reporting lip swelling, throat tingling, dyspnea and chest pain - hours after treatment # / . skin testing to both lots used during the treatment course showed positive values of x mm and x mm respectively at id : . soldier # was a -year old male reporting diffuse pruritus, hives, dyspnea, and chest pain minutes after infusion # / . skin testing showed positive values of x mm and x mm respectively at id : . due to clinical symptoms and positive skin testing, therapy was discontinued in each case. one control individual showed negative testing to prick at full strength, id : , and id : . conclusion: skin testing with pentostam may provide an objective tool for accurate classification of adverse reactions as igemediated. reliance on skin prick testing alone may not be sufficient to detect pentostam skin test reactivity, as both of these patients reacted to id testing only. a prospective study including pre-and post-treatment skin testing should provide more information on the value of skin testing in providing objective evidence for an ige-mediated process and determining the incidence of hypersensitivity to pentostam. introduction: a -year-old girl with a history of multiple infections was hospitalized with respiratory distress and hypoxia. her past medical history was significant for hypothyroidism, psoriasis, asthma and multiple pneumonias. a cbc revealed an absence of lymphocytes. laboratory: t and b cell subsets showed no b-cells and very low t cells (< cells). inadequate lymphocytes were present for mitogen and antigen studies. serum immunoglobulins were normal and she had antibodies to streptococcus pneumoniae and tetanus. antibodies to rsv, influenza and mycoplasma were absent. chest x-ray revealed interstitial infiltrates bilaterally. a high resolution ct scan revealed septal thickening and confirmed interstitial infiltration. open lung biopsy was consistent with non-specific inflammation with extensive lymphocytic infiltration. bal fluids and biopsy were negative for bacteria, fungi and opportunistic pathogens. clinical course: the patient did not improve with antibiotics and steroids were started. the patient improved rapidly and was discharged to home. biochemical analysis demonstrated a deficiency of adenosine deaminase (ada), a form of severe combined immune deficiency (scid). the patient was started on replacement ada, adagen. a repeat high resolution ct scan showed some improvement. however, the patient continues to be steroid dependant to control her pulmonary symptoms. lymphocyte numbers remain low despite effective ada replacement and the absence of serum datp. she remains clinically stable and receives adagen injection twice weekly. discussion: ada deficiency is a condition, which leads to accumulation of the metabolite datp, which is toxic to lymphocytes. this disease most commonly presents in the first year of life as scid and is fatal unless treated. our case is unusual due to the late onset of severe disease, normal serum immunoglobulin and the presence of some protective antibodies. despite adequate replacement of ada the patient continues to be profoundly lymphopenic, most likely due to steroids. although we do not yet completely understand the underlying lung disease we suspect that the patient has an autoimmune process causing interstitial inflammation. background: atopic dermatitis has a broad range of differential diagnoses. human sarcoptic infestation is characterized by severely pruritic lesions of variable appearance and distribution and may masquerade as eczema. infestation may be difficult to confirm and eradicate. animal transmission has been reported as a source of human infection. objective: to report a case of persistent sarcoptic infestation masquerading as eczema and associated with delusional parasitosis. methods: a -year-old female was referred to allergy clinic for evaluation with a two year history of recurrent, pruritic rash thought to be refractory atopic dermatitis. previous ineffective treatments included topical steroid creams, lindane, topical anti-fungals and multiple otc antiitch preparations. at initial evaluation, she had widespread excoriated papules in various stages of healing over % of her body. she reputed her dog was diagnosed with recalcitrant mange, which necessitated giving him medicated baths twice daily. results: a clinical diagnosis of subacute sarcoptic infection was made and the patient was prescribed two courses of elimite followed by oral ivermectin. her rash quickly resolved except for post-inflammatory hyperpigmentation. three weeks after resolution of primary lesions, she again complained of pruritic eruptions occurring on easily accessible areas and began bringing in medicine bottles of skin debris and scabs for examination. scrapings, koh preparation and skin biopsy examined microscopically showed no evidence of sarcoptic re-infestation. a diagnosis of delusional parasitosis was made. conclusions: the animal to human transmission of sarcoptic infection seen in this patient is rare and responded quickly to appropriate treatment. despite eradication of the infection, she developed delusional parasitosis, a rare psychiatric disorder characterized by fixed, false belief of an infestation by insects or other creatures. she displayed the classic matchbox sign in which samples of skin, scabs and other detritus are brought in for examination. she is currently receiving psychiatric care. background: thimerosal is a mercury derivative that has been used since the s. it is a commonly used preservative in ophthalmic solutions, otic drops, and vaccines due to its bactericidal property. objective: to report the first case of a generalized reaction to thimerosal found in an influenza vaccine. methods: we present a patient who developed a generalized maculopapular eruption after receiving a thimerosal containing influenza vaccine. patch testing was performed to determine if there was an allergy to thimerosal. results: patch testing confirmed a type iv (t cell mediated) sensitivity to thimerosal, further supported by the prior history of a reaction to a thimerosal containing contact lens solution. the patient was asked to avoid thimerosalcontaining products, including vaccinations, unless the benefit clearly outweighed the potential risk of a reaction. conclusion: physicians need to be aware that thimerosal is found in many products including vaccinations. clinicians should also be aware that allergic reactions do occur with exposure to thimerosal even in vaccines. this is the first case report in the literature of a generalized reactions to thimerosal from an influenza vaccine angioedema is a rare condition that has been described in the literature and exists in both inherited and acquired forms. a defect of the innate immune system, more particularly the complement system, is the inciting culprit. the acquired form of c esterase inhibitor deficiency has been divided into two classes and is generally not commonly seen until after the forth decade of life. type acquired angioedema has been described in association with lymphoproliferative disorders, while type is related to excessive complement activation and consumption due to autoantibodies. our case is a -year old man referred from an outside physician for further management of idiopathic edema. he first experienced facial edema in april attributed to sweet myrrh root ingestion. subsequently, in november he developed diffuse swelling of his upper extremities and tongue without airway compromise. a minimal work-up at that time was inconclusive. he fortunately remained asymptomatic until january at which time he experienced two episodes of tongue swelling without etiology. he was seen in his local emergency department and treated with diphenhydramine, corticosteroids, and epinephrine on both occasions with gradual improvement of his symptoms. the patient was not on medications commonly associated with angioedema and did not report insect envenomation. subsequently, he was seen by his primary care physician who ordered a number of laboratory tests including a ch , which was significantly suppressed. in light of this finding, he was referred to our clinic for further evaluation. at the time of presentation to our office, the patient was symptom free. his physical exam was within normal limits. further laboratory evaluation was essentially unremarkable with the exception of comple-ment studies, which are listed in the table. our case demonstrates an elderly patient with evidence of idiopathic swelling. we arrived at our diagnosis of acquired angioedema based on clinical presentation and confirmatory serum complement levels. a low ch at time of presentation allowed us to further delineate the etiology of complement deficiency. the fact that our patient did not present with swelling until after age and the paucity of a family history of idiopathic angioedema makes the diagnosis of acquired angioedema probable. measure of serum c q level confirmed the diagnosis. introduction: certain diseases, widely believed to be of allergic etiology, including atopic dermatitis and episodes of wheezing might be the result of interplay of genetic and environmental factors, at least partially. we describe a child with a rare chromosomal disorder presenting with typical features of atopic dermatitis and recurrent mild wheeze. materials and methods: a new born african-american male was noted to have unusual facial features immediately after birth. the baby was born naturally, without antenatal and neonatal problems. on physical examination, the infant had unusual facial characteristics with tight and taut facial skin, relatively diminished facial pad of fat, pointed chin, and markedly hypertonic extremities. additionally, cardiac exam revealed mumur consistent with uncomplicated asd. frequent reassessments of the infant in the outpatient clinic were done. the child had repeated bouts of wheezing attacks, and facial rashes compatible with the diagnosis of atopic dermatitis. the wheezes were treated in the clinic with nebulaized bronchodilators, and the atopic dermatitis responded reasonably to topical steroid applications and moisturizers as needed. chromosomal analysis of the peripheral blood of the chid confirmed the diagnosis of deletion of long arm (q) of chromosome [ , xy, del ( )(q q )]. karyotypic analyses of the mother and father were normal. the child exhibited features of developmental delay, and seizure activities. anti-epileptic drugs (aed) were instituted. initial eeg was normal and the subsequent eeg showed findings of static encephalopathy. ct scan of the brain revealed absent corpus callosum. neurologic evaluations and physiotherapies were requested for improvement of fine motor skills. he did not seem to suffer from any serious infectious or immunodeficient illnesses. his cbc was normal. he continued to have fair gains in his weight, height, and head circumference. his atopic dermatitis and wheezing episodes remained under control. there were a few hospitalizations for break-through seizures and dehydration from gastroenteritis. a subsequent echocardiography confirmed closure of asd. con-clusion: a child with chromosomal anamoly, atopic dermatitis, and mild intermittent wheeze is reported. despite multiple congenital problems, the child continued to have a stable clinical course. etoposide is a chemotherapeutic agent used to treat many solid tumor malignancies. hypersensitivity reactions have been well described and there are a few reported deaths from anaphylaxis. some suggest that the hypersensitivity is an anaphylactoid type reaction as it may occur during the first dose. case reports of cutaneous complications include stevens-johnson syndrome, radiation recall and diffuse erythema. there are four cases in which diffuse erythematous papules developed after etoposide therapy. all rashes spontaneously resolved within three weeks and biopsies demonstrated keratinocytes in a starburst pattern. we report the first case of an immediate etoposide induced skin reaction that evolved into long lasting hyperpigmented plaques. pretreatement was able to prevent this immediate and late reaction on subsequent exposure to etoposide. a year old female with ovarian cancer was treated with bleomycin, etoposide and vinblastine. three hours after initiation of the third dose of etoposide, patient developed pruritic, erythematous macules on her chest, abdomen, face and extremities. the infusion was stopped and decadron administered. over forty-eight hours, the pruritic macules became hyperpigmented plaques on her chest, abdomen, extremities and face. pruritus resolved after a slow taper of prednisone. the darken plaques were treated with multiple topical preparations but persisted for about three months. biopsies demonstrated superficial perivascular infiltration of lymphocytes and a few eosinophils suggestive of a drug eruption. etoposide was considered essential for this patient so allergy was consulted. the literature supports cautious readministration of etoposide with pretreatment and slower infusion rate to prevent immediate hypersensitivity. we could not guarantee prevention of the late hyperpigmented reaction. the patient was pretreated with prednisone and cetirizine. etoposide was administered in an icu with a slower rate of infusion. etoposide was tolerated without immediate pruritus or erythematous reaction. the patient did not develop the delayed darkened plaques. cautious administration of etoposide after premedication and a slow rate of infusion prevented both the immediate and late reaction previously experienced by this patient. eosinophils normally comprise - % of peripheral white blood lymphocytes. peripheral blood eosinophilia is defined as an absolute eosinophil count > cells/mm and is most often caused by atopy, helminth infections, or collagen vascular diseases. less common causes include adrenal insufficiency and neoplastic processes. eosinophilia can be characterized as mild (< cells/mm ), moderate ( - cells/mm ) or severe (> cells/mm ). although severe eosinophilia has been reported in association with adult hiv infection, studies of hiv-infected children have not shown peripheral blood eosinophilia to be a feature of pediatric hiv infection. we report an unusual case of severe peripheral blood eosinophilia in an adolescent male who was subsequently found to be hiv-infected. a previously healthy -year-old male presented with week history of fever, diarrhea, cough, vomiting, abdominal pain, anorexia and a lb weight loss over the previous months. the patient had recently emigrated from guyana and denied sexual activity, intravenous drug abuse, or other hiv risk factors. there was no known maternal hiv infection. repeated stool specimens were negative for ova and parasites. serologies for e. histolytica, t. canis, and s. stercoralis were negative. an abdominal ultrasound, chest x-ray, serum electrolytes, and liver function tests were all within normal limits. total white blood cell was , with % eosinophils (absolute eosinophil count of , cells/mm ). elisa and western blot were positive for human immunodeficiency virus (hiv- ). cd + tlymphocyte count was cells/mm with an hiv- rna level of , copies/ml. the patients symptoms resolved over the next days without treatment. he was started on combination antiretroviral therapy (zidovudine, lamivudine, abacavir, and efavirenz). absolute eosinophil count continues to slowly decrease with the last count of , cells/mm three months following the introduction of antiretroviral therapy. severe peripheral blood eosinophilia may be a presenting feature of hiv infection in adolescents. hiv testing should be considered in cases where more common causes of eosinophilia such as atopy and parasitic infections are excluded. triad asthma is well described in adults but not in the pediatric literature. this case highlights the successful treatment of a severe pediatric asthmatic with nasal polyps and aspirin sensitivity. a year-old female presented with severe persistent asthma, eib, allergic rhinitis, chronic sinusitis, nasal polyps, and a history of pneumonia. her asthma symptoms were minimal from age until age . she then started flovent and required increasing amounts of inhaled and oral steroids. she required courses of oral steroids per year by age . in addition, she had snoring, fatigue, chronic nasal congestion, rhinorhea, and sneezing despite treatment withallegra and rhinocort. she had received immunotherapy from age to with no clinical improvement. since age she had recurrent sinusitis and had required polypectomies. she noted aspirin caused nasal stuffiness and mild wheezing and therefore avoided it. on exam she had allergic shiners, dennie lines, boggy turbinates, nasal polyps, and diffuse wheezing. on evaluation she had severe airway obstruction. her fev increased from % to % with albuterol. her chest ct had a mosaic pattern due to severe air trapping, and her no level was . a sleep study showed severe hypopneas, and she had pan-sinusitis on ct. she had multiple positive spts, an eosinophil count of and an ige of . she had a normal sweat chloride and ph probe.the differential diagnosis included churg-strauss, abpa, cystic fibrosis, bronchiolitis obliterans, and triad asthma. after a thorough evaluation, she was diagnosed with triad asthma. with weeks of treatment with oral prednisone, her fev improved from % to %. her inhaled controller therapy was increased and she was placed on xolair. she had a polypectomy and then underwent aspirin desensitization. one year after starting treatment her fev remains %. she requires albuterol x/month and has not required prednisone. her eib, allergic rhinitis and congestion are markedly improved. she has had no further episodes of sinusitis, and a repeat sleep study was normal. in conclusion, we treated a severe pediatric asthmatic with nasal polyps and aspirin sensitivity. this is not frequently reported in the pediatric population, and raises questions about the incidence, optimal long term treatment of triad asthma, and the differences from the adult onset of this disease. a.a. white * , r.a. simon, la jolla, ca. background: human disease from mold has traditionally been isolated to infection, allergic disease, or hypersensitivity pneumonitis. specific diseases or pathologic findings other than those listed above have not been well described. we report a case of acute eosinophilic pneumonia related to mold exposure. case presentation: a year old woman developed dry cough and shortness of breath after stachybotrys mold was discovered in her home. a chest radiograph showed bilateral upper lobe infiltrates which worsened two weeks later. treatment with macrolide and flouroquinolone antibiotics was ineffective. a white blood cell count was , /cumm with % eosinophils. an erythrocyte sedimentation rate was mm/hr. aspergillus ige was negative. treatment with prednisone led to complete resolution of the chest radiographic abnormalities and improvement in pulmonary function testing. this patient was given a diagnosis of acute eosinophilic pneumonia with mold as a likely causal factor. discussion: there is dispute amongst health care professionals regarding the significance of environmental mold contamination in the etiology of human disease. this case represents well characterized disease occurring in the setting of mold exposure. while a causal relationship cannot be established, the temporal relationship of mold contamination, symptom onset, and disease progression is compelling. interestingly, a recent report of mold contamination leading to nonspecific interstitial pneumonia/fibrosis has been described. we have observed a patient in our clinic with similar pathology on biopsy and temporal relationship to mold exposure. to our knowledge, acute eosinophilic pneumonia has not been reported in conjunction with mold exposure. conclusion: this case highlights a new condition in which mold may have a causal role. the mechanism is unknown, but likely is through a non-ige mediated immunologic pathway. public awareness of mold as a health concern is increasing. perhaps similar cases will emerge as a history of mold exposure is offered by patients at the time they are evaluated for lung disease. a stronger correlation may then emerge. scuba diving is a commonly practiced activity which normally carries only minimal risks. any severe allergic reaction such as anaphylaxis, however, occurring during this activity could be a particularly dangerous not only because the swimmer is submerged but also because of the lack of proximity to medical care. we report a case of anaphylaxis occurring during scuba diving resulting from an unsuspected hypersensitivity to a latex component of the scuba diving suit. a yr-old white male developed a severe generalized urticarial rash with angioedema of his lips and eyelids, and difficulty breathing within minutes of his applying the suit and entering the water. after being rescued, he was transported to a nearby emergency department where he received epinephrine, antihistamines, corticosteroids and iv fluids with gradual improvement over a hour period. the patient denied being stung by a marine aquatic organism and there was no prior history of allergy or medications usage prior to his reaction. since subsequent investigation revealed that the scuba diving suit contained latex (brazilian rubber), a specific ige rast was found to be strongly positive (class v) to latex. therefore, the patient was advised to use a suit made of neoprene synthetic rubber (polychloroprene) which is a nonlatex containing product. this case report illustrates the importance of a diligent search for hidden sources of latex products which can produce life-threatening allergic reactions in sensitized patients. there has been considerable debate concerning the safety of immunizing egg-allergic children with the combined mmr vaccine. this concern derives from the possibility of an anaphylactic reaction since the mmr vaccine is prepared from attenuated viruses grown on chick embryo fibroblast cell cultures. we have previously reported the safe administration of the mmr vaccine to severe egg-allergic children without development of an adverse reaction (nsouli, tm, et al. ann allergy asthma immunol. ; : ) , a finding consistent with the current report of the committee on infectious diseases, american academy of pediatrics, . the present case report describes an anaphylactic reaction in an egg allergic yr-old-white male consisting of generalized urticaria, angioedema, wheezing immediately following the administration of a second mmr vaccine. the history of hives following ingestion of eggs was confirmed by positive specific ige rast testing. the patient's anaphylactic reaction necessitated emergency treatment including epinephrine, diphenhydramine, and corticosteroids in addition to iv fluids. although the administration of mmr vaccine in egg-allergic children is not considered as an absolute contraindication, the present case report suggests that caution should be observed when administering the mmr vaccine in such patients and that careful medical observation be included together with the ready availability of emergency medical equipment. p. buddiga * , r. turbin, a. baisre, l. bielory, newark, nj. introduction: sarcoidosis is a chronic granulomatous disease of unknown etiology that may have a multi-organ system manifestation and is characterized by the histopathological evidence of nonnecrotizing granulomas. case report: a year old african american woman with a year history of type ii diabetes mellitus, hypertension and sinusitis recalcitrant to multiple courses of antibiotics over months was admitted to the hospital with complaints of right eye proptosis, diplopia, headache and right facial numbness.her exam was consistent with an ipsilateral mild optic neuropathy, complete sixth (vi)nerve palsy, trigeminal, ophthalmic and maxillary division numbness.ct and mri of the face, orbit and brain revealed an extensive process infiltrating ethmoid, maxillary and frontal sinuses;orbits and deep facial structures.chest ct revealed bilateral interstitial nodules and symmetric hilar adenopathy. differential diagnoses included sarcoidosis, lymphoma/tumor, wegener's granulomatosis or fungal infection.labs-purified protein derivative test=negative(neg), antineutrophil cytoplasmic autoantibodies=neg. angiotensin converting enzyme= [ - u/l], fungal & anaerobic and acidfast bacilli culture of sinus secretions=neg.ethmoid, adenoid and maxillary sinus biopsies=multiple nonnecrotizing granulomas. on the basis of compatible clinical and radiographic findings, histopathological evidence and exclusion of other diseases with similar findings, the diagnosis of sarcoidosis was established. she was started on parenteral methylprednisolone and subsequently tapered to oral prednisone after days.concomitantly she was started on methotrexate as a steroid sparing agent.most recent chest x-ray after months of treatment reveals near complete resolution of the hilar lymphadenopathy.at months her optic neuropathy and facial dysesthesia had resolved, and she was left with mild persistent vi nerve dysfunction. conclusion:sarcoidosis that manifests itself as sinusitis is an uncommon presentation and the mechanism involved is thought to be a consequence of the destruction of cilia and mucus producing glands by the granulomatous process.review of the literature indicates that this is the eighth case reported and illustrates that a high index of suspicion of other etiologies must be maintained when there is a refractory response to multiple courses of antibiotics in the treatment of sinusitis. j.b. hein * , p. patel, l. bielory, newark, nj. introduction: cid may result in opportunistic infections such as cryptococcal meningitis. we present an unusual case of cryptococcal meningitis in an hiv-negative patient with severe cd lymphocytopenia. case: a yearold male with a three year history of sarcoidosis presented with acute onset of right-sided body numbness. the patient had been on prednisone mg/day for months prior to presentation. ct and mri scanning showed no vascular defects, meningeal enhancement, hydrocephalus or mass lesions. gallium scanning revealed normal uptake in the liver and spleen, mild uptake in the lungs and nasopharyngeal region, and asymmetric uptake in bilateral kidneys. the patient's initial wbc was , cells/mm , composed of neutrophils/mm , eosinophils/mm , basophils/mm , lymphocytes/mm , and basophils/mm . repeat studies revealed the total lymphocyte count decreased at cells/mm with cd (pan b) cells decreased at cells/mm and cd (pan t) cells decreased at cells/mm . cd count was cells/mm and cd count cells/mm with a cd /cd ratio of . . igg levels were normal at mg/dl. elisa was negative for hiv, and hiv- rna by pcr was not detected. serum ace level was u/l and csf ace level was u/l. csf obtained by lumbar puncture stained positive with india ink and culture revealed cryptococcus neoformans. the patient responded to amphotericin b lipid complex mg/kg/day, and his neurological status eventually returned to baseline. conclusions: the severe lymphopenia in this patient caused predisposition to infection with cryptococcus neoformans. the etiology of the profound lymphopenia likely was multifactorial, including mild sarcoidosis activity (lung uptake on gallium scan) and chronic corticosteroid therapy. however, once cryptococcus became entrenched in the patient's csf, the infection itself likely depressed peripheral lymphocyte numbers even further. this case demonstrates the importance of exploring a broad differential diagnosis when faced with lymphocytopenia. background: isosulfan blue % is a common dye used in sentinel lymph node dissection. the usage of the procedure and dye has increased in numbers, and although rare, several cases of anaphylactic reaction have been reported. objective: we are reporting a patient who had an anaphylactic reaction to isosulfan blue while undergoing breast cancer excision with sentinel lymph node biopsy. methods: the patient is a year-old woman with breast cancer. she underwent breast mass excision with sentinel lymph node biopsy using the lymphazurin % blue dye (isosulfan blue). she has a history of penicillin induced hives but has no other drug allergy. as soon as the dye was injected, she became flushed, hypotensive, and tachycardic. hypotension was refractive to fluid challenge. she was then treated with epinephrine as well as intravenous steroids and cimetidine with relief of symptoms. subsequently, she was evaluated in allergy and skin tests with isosulfan blue % at : , : dilution, and undiluted were performed using histamine as positive control and saline as negative control. this procedure was also performed on two control subjects. results: the patient had a positive skin test (wheal and flare) to isosulfan blue % (undiluted) with the control being appropriately positive for histamine and negative for saline. control subjects had negative response to dye and saline and positive response to histamine. conclusions: isosulfan blue -% dye may cause anaphylactic reactions in patients undergoing sentinel lymph node dissection. . the positive skin test result to the dye plus the negative skin test responses in the controls suggests that the reaction may be immunoglobulin e (ig e) mediated. eosinophilic duodenitis (ed) and gluten-sensitive enteropathy (gse) or celiac disease (cd) are distinct clinical entities with well-defined clinical and laboratory parameters. ed is a rare condition of unknown etiology, which is manifest by eosinophilic infiltration of the gastrointestinal tract and peripheral eosinophilia. gse or cd is a form of non-ige food allergy caused by immune hypersensitivity to ingested gluten. the simultaneous occurrence of the two entities, however, is a rare event. the following presentation is a case report in which both entities were found in the same patient. an y/o white hispanic male presented with severe, chronic, colicky abdominal pain and headache of months duration. hematologic and immunologic studies were within normal limits. serum ige levels were iu/ml (n= < iu/ml), anti-endosomial ab (+), igg antigliadin: u/ml (n=: - u/ml), skin tests for food and inhalant allergens were weakly positive ( +). biopsy of the inferior third of esophagus revealed chronic moderate esophagitis; biopsy of gastric antrum revealed lymphatic hyperplasia; duodenal biopsy showed shortening of the villi with the presence of eosinophils. following treatment with esomeprazole mg bid, famotidine mg qd, montelukast md qd, lactobacillus, a diet free of gluten, rofecoxib mg qd and betamethasone for days, betamethasone the patient improved with partial resolution of symptoms. the presence of duodenal eosinophils persisted despite a gluten free diet, and continued to require repeated bursts of prednisone. since to our knowledge this is rare finding in which ed occurred in association with gse, a high index of suspicion for simultaneous occurrence of ed should be raised in any case of gse that fails to respond to a conventional gluten free regimen. we report the evaluation of a month old male presenting with a pustular rash and choking episodes from birth. by age weeks, he had experienced two pneumonias, one with fleeting infiltrates requiring intubation. persistent eosinophilia ( - /ml) and eosinophils on pustule biopsy were noted. persistence of these and subsequent rsv pneumonia and recurrent draining otitis media led to referral. physical examination showed a thriving male infant with multiple erythematous papules and pustules along the scalp, face, axilla, and trunk. eosinophilia was confirmed. quantitative immunoglobulins were abnormal for igg and ige iu/ml. peanut cap rast was kua/l. hib post-vaccination titer, t cell enumeration/stimulation, and nbt were normal. hiv testing, stool eosinophils, o&p, and hemoccult were negative. cxr, hrct, ekg, and echocardiogram were normal. pustule cultures for bacteria, virus, and fungus were negative. skin biopsy revealed numerous eosinophils in the pustule, dermis, epidermis, and perifollicular region. cd a+ and s- staining were negative for histiocytic infiltration. nemo defect/incontinentia pigmenti were considered but testing was negative and karyotype , xy. bone marrow biopsy revealed numerous eosinophils at different stages of maturation, and no myeloproliferative or neoplastic changes. bronchoscopy was remarkable for % eosinophils on balf. -hour ph probe and impedence evaluation was negative. egd with biopsy was normal except for mild eosinophilic infiltration of the proximal and distal esophagus. with the clinical picture of recurrent pruritic crops of sterile pustules and characteristic skin biopsy demonstrating eosinophil infiltration, the diagnosis of eosinophilic pustulosis (ep) of childhood was made. despite no longterm sequelae or other end-organ involvement in ep, the degree and duration of eosinophilia, and presence of eosinophils in the airways and esophagus, raises the concern for other eosinophilic syndromes. following cardiac, cns, pulmonary, and gi systems is warranted. the infants pneumonias were felt to be due to aspiration, and ear infections the result of humoral immunity nadir or draining pustules in the external auditory canals. immunoglobulin levels will be monitored. this case illustrates the heterogeneity of eosinophilic diseases and raises the question of what drives the mechanisms behind malignant and benign disease. introduction: behcet's disease is a chronic, relapsing vasculitic disease characterized by recurrent oral, genital, and gastrointestinal ulcerations, a wide variety of skin lesions, uveitis, and arthritis. pediatric cases of behcet's disease are uncommon with an estimated prevalence of in , children under the age of . although the disease in children shows similar characteristics as adults, the diagnosis of behcet's disease in the pediatric population remains a challenge. this report describes an year old girl referred to our pediatric immunology clinic for evaluation of recurrent painful oral ulcerations since the age of with no genital ulcerations. the oral ulcers would last for two weeks and heal spontaneously but would reappear to weeks later. the patient had a history of raynaud's phenomenon for the last to years and the recent onset of joint pain. physical examination revealed - white elliptoid lesions - mm in diameter on an erythematous base on the tongue and soft palate. skin examination was significant for hyperpigmented patches over the neck, abdomen, and back. there were erythematous, serpigenous lesions on the palms and punctuated necrotic lesions on the finger-tips. pathergy test was positive. laboratory investigation was unremarkable and cultures from the oral ulcers remained negative. the patient was diagnosed with juvenile behcet's disease. she was started on immunosuppressive therapy with low dose prednisone and responded. conclusion: behcet's disease is a difficult diagnosis to make in the pediatric population. this case demonstrates that in children, recurrent oral ulcerations may be the only initial manifestation of behcet, and an important clinical marker for the disease. rationale: relapsing polychondritis is an uncommon severe inflammatory condition with unknown etiology that can present in a variety of manners. we report a year old patient who initially presented with signs and symptoms of anaphylaxis to shellfish and was later diagnosed with relapsing polychondritis. case report: a year old african-american boy with a year history of asthma and fish and shrimp allergy presented to the pediatric intensive care unit on / / after an episode of severe shortness of breath. his symptoms started shortly after accidental exposure to shrimp. in the intensive care unit, he was noted to be wheezing and stridorous. an initial chest xray as well as subsequent fiber optic laryngoscopy showed sub-glottic stenosis. he was intubated and received mechanical ventilation for greater than a week. after extubation, he stated that he felt fine, but continued to demonstrate audible stridor. pulmonary function tests demonstrated extra-thoracic obstruction, laryngoscopy continued to show sub-glottic stenosis, and esophagogastroduodenoscopy showed erosive esophagitis consistent with reflux. initial lab tests demonstrated a normal eosinophil count, elevated ige ( iu/ml), and positive immunocaps testing to multiple foods. the patient was discharged home with minimal stridor and no wheezing. over the next nine months, the severity of his stridor waxed and waned, but in general it worsened in spite of a strict elimination diet and anti-reflux therapy. on / / he received an emergency tracheostomy. on / / , the patient was again admitted to the hospital. at this time he complained of bilateral knee pain as well as significant weight loss. physical exam demonstrated arthritis with effusions of both knees as well as unilateral auricular chondritis and bilateral episcleritis. antibodies against type ii collagen were positive ( . eu/ml). the diagnosis of relapsing polychondritis was suggested. conclusion: relapsing polychondritis is a rare inflammatory disorder of the cartilage and connective tissue with an unknown etiology. the wide array of presenting complaints as well as the relapsing nature of this disorder often causes significant delay in diagnosis and treatment. in our case there was a period of nine months between the initial presentation and the time when the diagnosis of polychondritis could be made. d.k. geller * , m. ballow, buffalo, ny. introduction: an yo male previously diagnosed with pandas presented to our immunology clinic for ivig. the patient was healthy until age when he developed facial motor tics associated with group a beta hemolytic strep pharyngitis. he was treated with antibiotics and the tics resolved completely. he has had multiple recurrences associated with strep and other viral infections. the tics improve or resolve completely when he is infection free. he has no history of vocal tics, attention deficit/hyperactivity disorder, or obsessional thinking or compulsive behaviors. laboratory: multiple throat cultures positive for group a beta hemolytic strep, positive antistreptolysin and antideoxyribonuclease b titers. discussion: pandas identifies a subgroup of children with an obsessive compulsive disorder and/or tic disorder whose symptoms seem to be triggered by streptococcal infections. the proposed pathophysiology is an immune-mediated mechanism in which antistreptococcal antibodies, antistreptolysin and antideoxyribonuclease b, cross react with the basal ganglia of genetically susceptible hosts. a few studies have looked at immunomodulatory therapies including ivig for neuropsychiatric symptoms including tic disorders secondary to post-streptococcal autoimmunity. we plan a trial of ivig for this patient given the recurrent nature of his tics and the relation to streptococcal and other viral infections. introduction: the chronic granulomatous disease (cgd) is an inheritable disorder of phagocyte cell respiratory burst that result in life-threatening infections. the pulmonary infection is the primary cause of death in greater that % of the cases and the role of surgery in management of this entity remains undefined. case report: a lobectomy was performed in a year-old male who presented a persistent opacity of the medial right lobe with fever, cough, malaise an rapid onset of respiratory failure; skin abcesses were concomitant. his familiar history was unremarkable. a chest tube was placed in a first instance in order to drain empyema, but no progress was observed. a ct scan showed necrotizing pneumonia of the right lung, mediastinal and retroperitoneal limphadenopathy and bronchopleural fistula. a second surgical intervention was undertaken to correct the fistula and pleural debridement was done. at this time, serratia marcenses was isolated from blood culture and lung; a primary immunodefiency was suspected. our evaluation showed nbt reduction on % and dyhidrorhodamine assay (dhra) didn't demonstrate and effective oxidative burst. the subsecuent management was based on specific antibiotic to serratia and prophylaxis with tmp-smz and itraconazole. transfer factor to improve the ifn levels was initiated. the patient's mother showed on dhra two granulocyte populations, with and without oxidative burst. a x linked cgd was consistent. medical progress was observed in the next days. conclusion: despite the poor utility showed by the surgical procedures in the complicated pneumonia in cgd patients in many series, attending to the unusual nature of the pulmonary infections and the high mortality and morbidity associated with thoracic surgery; the management of this case showed that an aggressive approach in the diagnosis combined with some procedures used in immunocompetent patients pneumonia may improve the clinical condition in cgd patients. background: takayasu arteritis is a large vessel vasculitis primarily affecting the aorta and its branches. it is most prevalent in women in the second and third decades of life. initial symptoms are systemic and often self-limited, but the disease may progress undetected over years until signs of vascular insufficiency develop. arteritis is commonly seen in the aortic arch and its branches, although the disease is a panarteritis and vascular compromise can occur in many organs. pathologically, involved vessels show intimal proliferation and fibrosis, and scarring of the elastic lamina. case report: we report the case of a year old southeast asian woman who presented with diplopia, ptosis, dizziness, and progressive dyspnea. past medical history was significant for rheumatic fever complicated by aortic valve insufficiency and hypertension. physical findings on admission included hypertension, asymmetric upper extremity blood pressure (left arm / mm hg, right arm / mm annals of allergy, asthma & immunology hg), wide pulse pressure and a harsh diastolic murmur. admission labs showed anemia (hemoglobin: g/dl, hematocrit %) and an elevated erythrocyte sedimentation rate of mm/hr. transesophageal echocardiography revealed severely dilated aortic root aneurysm with secondary severe chronic aortic valve insufficiency and calcified aortic leaflets not typical of rheumatic heart disease. resection of the aortic valve and aneurysm was performed. pathologic examination of the aortic aneurysm revealed intimal and adventitial fibrosis with focal chronic inflammation and partial loss and fibrosis of media, findings compatible with healed takayasu arteritis. the patient was started on prednisone mg by mouth daily for takayasu arteritis and discharged in stable condition. discussion: this is a case of takayasu arteritis masquerading as rheumatic heart disease. the episode of rheumatic fever was likely the initial presentation of takayasu arteritis, as the systemic symptoms of these diseases can overlap. takayasu arteritis is an insidious disease that often leads to a delayed diagnosis with considerable morbidity for the patient. since takayasu arteritis may go undiagnosed until ischemic symptoms develop, physicians should be alert to the possibility of this disease in young women to avert end organ damage and achieve early remission with drug therapy. we present a fifteen year old boy with disseminated papillomatosis and idiopathic cd + lymphocytopenia (icl). the warts have been present since he was one year old and have progressively worsened. there are more than fifty warts on each of his hands. his legs have multiple warts and about ten warts recently appeared on his lips and around his mouth. his past medical history is significant for no hospitalizations and no blood transfusions. he has never had problems with other infections, and he had a benign course of chicken pox that spontaneously resolved. he has been on cimetidine for six months without improvement. several topical therapies, including imiquimod, have failed. he has a total lymphocyte count of /ul (normal range, - /ul) and a cd + count of /ul (normal range, - /ul). the cd +, natural killer cells, and cd + cells were normal. his hiv test is negative. the serum immunoglobulins were normal as well. lymphoctye proliferation studies reveal an absent response to antigens (candida and tetanus) with a normal response to mitogens. further studies showed no other cause of cd + lymphocytopenia. this case shows that the diagnosis of idiopathic cd + lymphocytopenia should be considered in any patient with widespread viral infection whose hiv test is negative. appropriate evaluation of the cd + count should be pursued. j. ko * , a. nowak-wegrzyn, new york, ny. introduction: approximately % of children with moderate to severe atopic dermatitis (ad) have food allergies. complementary and alternative medicine (cam) is utilized by an estimated - % of patients with allergies. we present a case of a year-old boy with ad referred after receiving a diagnosis of food allergy by a cam practitioner. methods/case: a year-old boy was referred for evaluation of mild ad and possible food hypersensitivity. his ad started at year of age and was limited to the face. he was initially breast-fed and was switched to milk-based formula at months of age. solids were introduced at months of age without difficulty. he had no immediate reactions or noted worsening of ad due to foods. the patient presented to a naturopath for allergy evaluation. based on electrodermal skin testing results, he was reported to be allergic to milk, egg, peanut, rice, and beef (all of which he had tolerated previously). he was restricted from milk and egg for weeks without noticeable change in his ad, which was limited to the cheeks and controlled on topical pimecrolimus. results: his weight and height were both th percentile. physical exam was normal except for - cm dry, erythematous patches on both cheeks and mildly dry skin on flexor/extensor surfaces of both arms. skin prick testing revealed negative tests to milk, egg, peanut, and dust mite. serum ige testing was also negative. since he had no evidence of ige sensitization to commonly allergenic foods, he was recommended to continue an unrestricted diet and discontinue use of a "sippy" cup. his ad subsequently improved on a skin care regimen of postbathing moisturization and topical pimecrolimus prn. asthma and allergies are the # reason for cam use in the us. electrodermal skin testing is a method utilized by naturopathic doctors to detect allergen "sensitivity". however, in two double-blinded trials examining atopic and non-atopic patients, electrodermal testing did not correlate with skin prick testing results, regardless of inter-operator variability. conclusions: allergy patients are seeking cam treatment, and it is important for physicians to be aware of the safety and efficacy of alternative medical practices. the use of electrodermal skin testing has not been proven to detect allergen sensitivity and use of this modality should be discouraged. e.e. mcgintee * , k.e. sullivan, philadelphia, pa. introduction: hematopoietic stem-cell transplantation causes profound tcell immunodeficiency due to the rigorous conditioning involved. following transplant, the t-cell compartment is initially reconstituted through expansion of mature donor-derived t-cells. however, thymopoiesis is necessary to generate naive t-cells with a diverse repertoire. factors affecting thymic function impact the ability of the body to reconstitute the immune system. we report a case of severe t-cell immunodeficiency after two stem-cell transplants for neuroblastoma in a patient with a history of thymic hemorrhage secondary to mediastinal surgery. case: a -year-old female with a history of high-risk neuroblastoma was referred for immunologic evaluation due to a significant infectious history since completing her neuroblastoma therapy. she initially presented at months of age with a right atrial mass that was presumed to be an atrial myxoma. she underwent mediastinal surgery to resect the tumor, which was found to be neuroblastoma extending from her right adrenal gland along the vena cava. a ct scan following her surgery revealed an area of hemorrhage in her right thymus. she subsequently received high-dose chemotherapy and two autologous stem-cell transplants. her infection history was significant for multiple episodes of upper respiratory illnesses, sinusitis, and otitis media requiring myringotomy tube placement on two occasions. immunologic evaluation revealed a dramatically low absolute lymphocyte count with deficits primarily in the t-cell compartment, and reduced proliferation in response to mitogens. immunoglobulin levels were normal; she formed protective titers to diphtheria and tetanus but not to any of pneumococcal serotypes. conclusion: stem-cell transplantation often results in severe t-cell immunodeficiency. the thymus plays an important role in reconstituting the t-cell compartment with naive t-cells generated from hematopoietic stem-cells, which restores tcr diversity. as this case illustrates, damage to the thymus can significantly impair the ability to reconstitute the t-cell compartment. a.k. knight * , c. cunningham-rundles, new york, ny. rationale: this is the first case report of oxcarbazepine induced immunoglobulin deficiency. methods: a y/o white female was referred for further investigation of low serum immunoglobulin found as part of an evaluation for chronic bacterial vaginitis. she was taking oxcarbazepine for chronic pain. this was discontinued and immunoglobulin levels and b cell numbers were tested at intervals. specific antibody responses to pneumococcal vaccine were tested. results: at presentation, on oxcarbazepine, immunoglobulin levels were low [igg , iga < , and igm < mg/dl] and she had a b cell deficiency [ %, b cells (normal - %, - cells/cu mm)]. antibody response one month after pneumococcal vaccine was poor (protective antibody to / serotypes). ( %)] with protective antibody responses to / pneumococcal serotypes. she continued to have iga deficiency. conclusions: the patient's initial evaluation suggested the diagnosis of common variable immune deficiency with hypogammaglobulinemia and specific antibody deficiency. however, these defects reversed when oxcarbazepine was discontinued. it is unclear if persistent iga deficiency was a pre-existing condition, possibly predisposing her to this adverse reaction to oxacarbazepine, or induced by the oxacarbazepine. immunoglobulin deficiency is a known, though rare, reaction to carbazepine, the parent drug of oxacarbazepine; this adverse reaction can occur with its derivative oxcarbazepine as well. secondary hypogammaglobulinemia should be considered before diagnosing primary immunodefiencies such as cvid and committing the patients to lifelong immunoglobulin therapy. introduction: interferon and glatiramer acetate (copaxone) are indicated for the treatment of relapsing-remitting multiple sclerosis (ms). anaphylactic reaction has been reported as a rare complication of interferon and copaxone use. we report a case of interferon - a (rebif) and copaxone hypersensitivity associated with positive skin tests and desensitization to interferon - b (betaseron). case report: the patient is a year-old woman with asthma and allergic rhinitis who was diagnosed with ms in oct ' after an uncomplicated pregnancy and was subsequently placed on rebif. one month after starting therapy, patient developed wheezing and generalized urticaria after receiving a dose of rebif. the symptoms recurred the next day. her neurologist stopped the rebif and started her on copaxone. two months later she developed episodes of generalized urticaria. the patient was then evaluated in our center and underwent skin testing with interferon - a intramuscular (avonex), interferon - a subcutaneous (rebif), betaseron, and copaxone. she had a positive skin prick reaction to copaxone and positive intradermal skin tests to avonex ( : strength), rebif ( : ), and betaseron ( : ) after hrs. the intradermal reactions to all interferon formulations continued to progress upto hrs. her neurologist felt that she would benefit from betaseron therapy. the positive intradermal skin test to betaseron was sufficient to warrant desensitization to prevent immediate hypersensitivity reactions. she underwent subcutaneous desensitization as shown in the table. the desensitization was halted at minutes after the patient developed itching of the hands.* she returned in hrs and we restarted by administering . ml of : strength betaseron. increasing doses were given until the patient received a full therapeutic dose ( . mg in ml). she has since done well with daily doses of betaseron ( . mil units). conclusion: we report a ms patient who developed urticaria and asthma exacerbation in response to rebif and urticaria to copaxone. skin tests confirmed an ige-mediated allergic reaction. she underwent successful desensitization to betaseron. to our knowledge, this is the st report of desensitization to betaseron as well as extension to previous reported cases of systemic reaction to interferon and copaxone. pentoxifylline (ptx) is a phosphodiesterase inhibitor that has been used for ischemic vascular disease because of its effects on red blood cells and platelets. it has been used for systemic inflammatory diseases such as sarcoidosis because of its ability to inhibit production of cytokines such as tumor necrosis factor (tnf-a). we decided to offer a trial to a patient with chronic urticaria since agents that increase intracellular camp have been shown to inhibit mast cell degranulation. we report a case of a year-old female with a history of allergic rhinitis, hypothyroidism, and diabetes mellitus with recurrent episodes of chronic urticaria since adolescence. she had known allergies to various antibiotics but otherwise had an unremarkable history, family history and social history. she was being treated with fexofenadine mg qd, azelastine nasal spray, zafirlukast mg bid, hydroxychloroquine mg bid, levothyroxine mcg qd, doxepin mg qhs, metformin mg bid, and spironolactone mg qd at the time of her initial evaluation. her physical exam was notable only for scattered - cm urticarial skin lesions as well as areas of hypo and hyperpigmentation from previous excoriations. her nasal turbinates were mildly congested with dull membranes. a previous skin biopsy showed only urticaria with increased numbers of mast cells. her workup included normal cbc, urinalysis, spep and complete chemistry profile, as well as negative ana and anti-thyroid antibodies. pentoxifylline mg tid was added to the patient`s regimen. her urticaria resolved within - weeks and was no longer visible at months follow-up. pentoxifylline is a safe medication with few side effects that may benefit patients with chronic urticaria via several mechanisms, including the inhibition of mast cell degranulation and secretion of various cytokines and chemokines. the beneficial effects of pentoxifylline in the treatment of this patient`s urticaria have been seen in other patients. further study is indicated to determine which patients with urticaria are most likely to benefit from pentoxifylline, as well as elucidate the mechanisms of action by which pentoxifylline ameliorates symptoms of chronic urticaria. c.m. mjaanes * , m. boguniewicz, denver, co. we report the case of an -year-old male who since the age of years has suffered from recurrent episodes of left tongue swelling. onset of the swelling is usually abrupt, and generally occurs during the night, awakening him from sleep. he describes a sensation of pain in his tongue but denies any pruritus, throat, lip or eyelid swelling. he has no cough, dyspnea, wheezing, rash, or itchy/watery eyes. the episodes occur infrequently, approximately once every spring . they tend to resolve spontaneously within to hours, however, on occasions, the swelling has lasted for - days. the patient's current episode has been present for weeks and is progressing. today he reports more pain and increased swelling. the child reports no benefit from oral antihistamines. he was treated with dexamethasone during his initial episode and experienced gradual resolution of the swelling. he has never been treated with topical or systemic epinephrine, or additional courses of systemic steroids. there is no family history of angioedema. the child was referred for evaluation of an immunologic/allergic etiology of his unilateral tongue swelling. laboratory studies obtained early in the course of his current episode revealed the following: c level mg/dl; c level . mg/dl; c level . mug/ml; c d level . mug/ml; c esterase inhibitor function % of normal. c esterase inhibitor level . mg/dl; ch units/ml; ana : , negative; beta-tryptase < . ng/ml; total tryptase . ng/ml (all normal). initial evaluation revealed marked swelling along with erythematous and violaceous discoloration of the left lateral and anterior portions of the child's tongue. the remainder of his examination was normal. the patient was referred to the pediatric otolaryngology clinic with a presumptive diagnosis of a glossal hemangioma versus lymphangioma. he underwent a magnetic resonance imaging study which revealed a poorly defined, mass-like enlargement of the anterior and left aspect of the tongue. he was treated with a five day course of prednisolone. after initial regression of the lesion a planned surgical excision was carried out without any complications. in conclusion, this rare case illustrates the unique presentation of glossal hemangioma presenting as recurrent angioedema. highlighted are the differential diagnoses, laboratory studies and clinical features of oropharyngeal swelling. severe tongue swelling. rare skin conditions such as leprosy need to be considered in the differential diagnosis of apparent urticaria and angioedema that is atypical in appearance or response to therapy. a year old male originally from laos presented with chronic recurrent nonpruritic indurated plaques on his face and trunk, and was referred to allergy by his primary physician with a diagnosis of urticaria and angioedema to rule out an allergic reaction to foods. he was otherwise healthy and on no medication, and had a negative personal and family history of atopy. he had moved to the united states from southeast asia in , and visited for several weeks again in . he was treated with prednisone and hydroxyzine without benefit; skin testing to foods was negative, and cbc, differential, sedimentation rate, antinuclear antibody, and liver function testing was normal or negative. a skin punch biopsy from an indurated area on the back showed granulomas with clusters of acid-fast organisms on afb stain consistent with tuberculoid leprosy. as the patient was g pd deficient, he could not be treated with dapsone and was treated alternatively with minocycline mg, rifampin mg, and clofazimine mg daily. he was subsequently started on prednisone as he was determined to be having a reversal reaction; his lesions continue to improve. this patient had a rare condition, leprosy, which was confused with urticaria and angioedema. in patients presenting with atypical apparent urticaria or angioedema, especially if response to standard pharmacologic treatment is poor, a skin biopsy is indicated, and may be diagnostic as in this case. introduction: cutaneous flushing about the face of a toddler within minutes of eating specific foods prompts parents and clinicians alike to pursue food allergy testing. auriculotemporal syndrome, also known as frey syndrome, is an isolated and transient facial erythema about the distribution of the auriculotemporal nerve following mastication. we report a child who was referred for food allergy testing and was diagnosed with auriculotemporal syndrome. case history: a yo wf, prior full-term forceps-assisted delivery, has a history of facial flushing within minutes following ingestion of specific foods. the flushing can be induced by a variety of foods, specifically spaghetti, crackers, potato chips and hard candies, and typically resolves within minutes after eating. the child has no respiratory or gastrointestinal distress during the flushing episode. there is no angioedema, urticaria, or other rash elsewhere on her body. within ten minutes of eating a lollipop in clinic, an erythematous, warm macular eruption appeared in a band-like region anterior to her ear extending from her temporal bone to her mandible. this response occurred bilaterally, with a mildly greater intensity on the right side of her face. no sweating or other symptoms developed, and the flushing began to diminish after thirty minutes of observation. discussion: auriculotemporal syndrome is commonly seen in patients who have undergone facial surgery, specifically about the parotid gland. it is believed to be secondary to a disruption in the fibers of the auriculotemporal nerve. it is theorized that the nerve regeneration following an injury may join the parasympathetic fibers of the salivary gland with sympathetic fibers of eccrine sweat glands. mastication then could result in flushing and sweating over the cutaneous distribution of the auriculotemporal nerve that extends from the temporal region to the mandible, anterior to the ear. although rare in children, auriculotemporal syndrome has been associated with forceps delivery. therapy is seldom of benefit or needed, and the syndrome often resolves spontaneously over many years. conclusion: auriculotemporal syndrome leads to a cutaneous eruption temporally related to the ingestion of foods. allergists should maintain an awareness of this rare syndrome to minimize food allergy testing or eliminate unnecessary food provocation challenges or restrictive diets. m. al-ahmad * , s.s. mace, toronto, canada. introduction: ranitidine is a well-known h -receptor antagonist. anaphylactic reactions are seldom reported despite widespread use of the drug. we report a patient with anaphylactic reaction to ranitidine methods: we report a case of anaphylactic reaction with ranitidine. results: a -year-old female with a history of urticaria over one year period, presented with episodes of anaphylactic reactions of increasing severity over year, after ranitidine ingestion. the first two episodes, occurred one hour after ranitidine ingestion, began with a sensation of burning, and itching in the head followed by dizziness, hypotension and near loss of consciousness. the patient had no significant pulmonary or laryngeal symptoms. the fourth episode, occurred minutes after taking ranitidine tablet, was characterized by severe hypotension. two episodes were managed at the emergency department. a skin prick test with a crushed ranitidine tablet demonstrated a positive mm wheal response, which was negative in a control. investigations for carcinoid syndrome and systemic mastocytosis were negative. conclusion: an ige mediated allergy to ranitidine is possible. anaphylactoid reaction to ranitidine is a well recognized entity. however, to our knowledge, few cases of anaphylactic reaction to ranitidine have been described. hypersensitivity reaction to ranitidine should be considered in patients suspected of having drug-related allergy. introduction: severe combined immune deficiency(scid) and cystic fibrosis(cf) may both present in infancy with a history of failure to thrive, diarrhea, and recurrent respiratory infections. although cf is the most common genetic disease occurring in caucasians with an incidence of in births, scid is a rare condition estimated to occur between in , to in , births. we report a case of x-linked scid where the diagnosis of cf was originally entertained due to similar presentation and misleading laboratory results. case: -month-old caucasian male with month history of recurrent respiratory illnesses, fever, loose stools, thick nasal discharge and failure to thrive. this was his second admission for a respiratory infection. relevant lab results consisted of cultures which grew parainfluenza a virus on np wash and pseudomonas aeruginosa on deep throat culture, chest xray showed patchy infiltrates and a borderline sweat test of mmol/l. despite being treated with broad spectrum antibiotic coverage, albuterol and pulmozyme, his respiratory symptoms were not improving. physical exam revealed a thin infant with palpable though small lymph nodes cervical and groin, diffuse scattered rhonchi and liver edge palpable cm below the costal margin. quantative immunoglobulins were obtained and resulted as an igg of , iga of < , and igm of . review of his chest x-ray was significant for absent thymic shadow. lymphocyte count since admission ranged from - . flow cytometry revealed a cd /cd count of %, cd /cd count of %, cd of % and cd of % of all lymphocytes. t-cell stimulation tests were markedly diminished to both antigen and mitogen. a presumed diagnosis of x-linked scid was made and patient undrewent haploidentical bone marrow transplantation. subsequently, cf gene analysis resulted as negative. discussion: as outlined in the case it is essential to recognize the similarities between the presenting symptoms of cf, a relatively common genetic disease and scid, an uncommon one. although pseudomonas is seen in increased frequency in patients with cf, it is essential to recognize that it is also a leading pathogen affecting patients with antibody deficiency. review of the literature did not reveal any previous cases of individuals with scid reported as having an increased sweat chloride, nor any explanations of why this would occur. while immunodeficiency has previously been associated with some forms of dwarfism, we report the first described case of common variable immune deficiency in a patient with seckel syndrome (bird headed dwarfism). the diagnosis of seckel dwarfism, an autosomal recessive disorder, was made at months of age in this boy based on intrauterine growth retardation, proportional dwarfism, microcephaly, micrognathia, and narrow face with "bird-like" large eyes and beaking of the nose. hematologic abnormalities are reported in an estimated % of such patients, and this child developed thrombocytopenia at the age of years. by age , he had pancytopenia with hypocellular bone marrow. a low cd count ( /ul) was noted and both bactrim and neupogen were begun. his only significant infections, rotavirus and salmonella gastroenteritis, occurred at that time along with frequent otitis leading to pe tube placement at ages and but which subsequently resolved. he has never had opportunistic infections or thrush. by the age of years, he required monthly red cell transfusions for severe anemia. after several months of transfusions, the blood bank reported that he no longer had detectable blood group antibodies during crossmatch; this led to measurement of immunoglobulins. igm was undetectable with low iga ( mg/dl) and low igg ( mg/dl). he demonstrated no protective titers to any pneumococcal serotypes after either conjugated or unconjugated pneumococcal vaccines. response to diphtheria, tetanus, rubeola, and influenza vaccines was adequate. total hemolytic complement was normal. he remains lymphocytopenic (alc = /ul) with low cd ( /ul). after five doses of ivig at mg/kg monthly, platelets have remained between - k/ul and there has not been any improvement in the anemia. although the mechanism for the bone marrow failure in these patients is presumed to be chromosomal breakage, this has not been demonstrated in this child. his karyotype is xy with no chromosomal fragility or deletions detected to date. hypogammaglobulinemia should be investigated in any patient undergoing frequent transfusions who loses blood group antibodies, and suspicion of immune deficiency should remain particularly high in patients with severe growth retardation or dwarfism. n.l. rider * , t. craig, hershey, pa. the objective of this study was to assess the effectiveness of physicians at a university primary care clinic in diagnosing and managing adult patients with asthma. a retrospective chart review of adult patients from this clinic was performed. each chart was evaluated using a survey instrument developed from the national asthma education and prevention program (naepp), which consisted of queries. of the charts evaluated met our entry criteria. overall compliance with the naepp recommendations was %. adherences to the asthma guidelines in the following areas were: diagnosis ( %), treatment/disease control ( %), monitoring ( %), and education ( %). these data suggest that there is an opportunity to improve the care provided to patients with asthma, especially in the areas of asthma education and monitoring, even in a university outpatient facility. these data also suggest that naepp guidelines have not been effectively incorporated into primary care practices. b.m. wahlers * , l. sherwood, t. craig, hershey, pa. objective: our aim was to evaluate adherence to the national heart lung and blood institute (nhlbi), national asthma education and prevention program (naepp) diagnosis and management guidelines, regarding asthma admissions at a teaching hospital. methods: a retrospective chart review of patients over the age of years admitted to the hershey medical center between and with a primary diagnosis of asthma or asthma exacerbation was conducted using a -item questionnaire focused on key aspects of asthma care as outlined in the naepp guidelines. results: overall compliance to the guidelines was . %. maximal areas of compliance were noted in regards to pharmacologic treatments utilized ( %, %). much lower levels of compliance were noted in areas of patient education ( %), instruction in written action plan prior to discharge ( %), provision of peak flow meter for home use ( %), assessment of asthma symptoms and severity ( %), and inhaled steroid prescription on discharge ( %). conclusion: adequate care in terms of pharmacologic treatments is being given but sub-optimal cares in other areas of the guidelines exist. there continues to be room for improvement in regards to asthma management, most notably in the area of education, documentation of triggers, and preparation for discharge with adequate tools to monitor and control symptoms. background: asthma is one of the most common problems of childhood, responsible for a significant proportion of absence from school because of chronic illness. objective: this study was carried out among the school-aged children ( - years) in tehran schools during - , in order to determine the frequency of asthma. methods: according to the recommendation of who, a questionnaire was designed, containing standard questions, and the students were given necessary information to complete the questionnaires. the guidance and high school students completed the questionnaires but the parents of primary school students completed them by themselves. results: seven hundred and eight children out of children had asthma ( . %); this prevalence was higher in the boys ( . %), when compared to the girls ( . %). the prevalence of this disease has been estimated about . % in guidance schools, . % in high schools and . % in primary students. based on this survey, the most common clinical manifestations in asthma were included: prolonged cough lasting more than days ( . %), and exerciseinduced wheezing or dyspnea ( . %), followed by repeated dyspnea or wheezing ( . %). based on the drug responses after receiving solbutamol, the prevalence of asthma was evaluated in the range of . % in primary schools, . % in guidance schools and . % in high schools. conclusions: the prevalence of asthma is high among the students of tehran schools and it needs more careful screening programmes and either more information given to the patients and parents about the disease. in the us, asthma is the most common chronic disease of childhood and affects approximately million children. reasons for inadequate asthma control include: inappropriate therapy, incorrect inhaler technique, poor compliance with treatment, and exposure to environmental triggers. rates for asthma prevalence, hospitalization, and death are highest among children residing in inner cities, and important risk factors for asthma-related mortality include being poor and black. the communities of braddock, north braddock and rankin are among the poorest in allegheny county and are designated areas of greatest health risk. recent statistics have demonstrated an increase in the number of asthma hospitalizations in these communities: . children per , which is double the average for allegheny county and greater than seven-times the overall us rate of asthma hospitalizations. our goals were to assess asthmatic severity and appropriateness of treatment, provide asthma education, and to ensure access to healthcare for asthmatic children living in these medically underserved and predominantly african-american communities. twenty-seven asthmatic children were identified. of the children assessed % were under the age of years, % were aged - years, and % were - years of age. based on nhlbi guidelines the severity of each child's asthma was determined: % had mild-intermittent asthma, % had mildpersistent asthma, % had moderate-persistent asthma, and % had severepersistent asthma. the appropriateness of treatment based on severity was also determined. we found that % of the children with mild-intermittent asthma, % of children with mild-persistent asthma, % of children with moderate-persistent asthma and % of children with severe-persistent asthma were receiving inappropriate treatment. furthermore, of the inappropriately treated children % were undertreated. following the assessment, patients and parents were educated with regard to asthma triggers, pathophysiology, use of peak flow meters, inhaler technique and pharmacologic treatment. patients were also scheduled for outpatient follow-up care. these results demonstrate that inappropriate treatment, especially undertreatment, may be a significant cause of the increased asthma morbidity in this population and underscore the need for aggressive education and innovative methods of ensuring health care. w. li-ling * , t. keng-leong, f.c. stephanie, e. philip, singapore. background: asthma admission is an important indicator of asthma morbidity. little is known about the risk factors associated with asthma admission among high-risk asthmatics. purpose: ) to characterise the profile of high-risk asthmatics who had previous asthma hospitalization. ) to identify predictors and risk factors that are associated with asthma hospitalization. methods: data for consecutive high-risk asthmatics who were enrolled into our asthma program from october to may were retrieved from our prospective database. high-risk asthmatics were defined as patients who had a clinical diagnosis of asthma and who had at least one hospital admission for exacerbation of asthma in the preceding months or at least one severe exacerbation of asthma in the preceding months requiring unscheduled visit for beta -agonist nebulisation. a comparison was made among high-risk asthmatics who had at least one or more hospitalization for asthma in the past months prior to the asthma program enrolment with those who were not hospitalized. statistical analyses were performed using spss version . for windows. results: among high-risk asthmatics, ( . %) had at least one or more asthma hospitalization in the past months prior to the enrolment into the asthma program. hospitalization was significantly associated with female gender ( . % with asthma admission v . % without asthma admnission). a significantly higher proportion of asthmatics with no formal education ( . % v . %), primary education ( . % v . %) and secondary education ( . % v . %) were hospitalized for asthma as compared to the those who had tertiary education ( . % v . %). those admitted were predominantly in lower income group, had poor inhaler technique and have no ownership of an asthma action plan ( . % v . %). those hospitalized were also younger, median age years ( , ) v years ( , ) and had a lower first peak flow reading, l/min ( , ) v l/min ( , ) . conclusion: among high-risk asthmatics, certain population subgroups are at greater risk for hospitalization for asthma. intervention aimed to reduce asthma morbidity should take the above findings into consideration. introduction an association between asthma symptoms and air quality is well established, but controversy exists concerning the role of various air quality indicators. the association of san antonio air quality with emergency department (ed) visits for asthma at our military medical center has not been defined. the objective of this study is to determine the correlation of selected air quality indicators in san antonio with ed visits for asthma for both the general population and for the pediatric population. methods selected air quality indicators were the -hr air quality index (aqi) for ozone, -hr aqi for particulate matter < . μm, pollen counts, mold spore counts, and daily high temperature. the number of daily ed visits coded for asthma was obtained retrospectively. pediatric visits (patients < years-old) were evaluated as a subgroup. pearson correlation coefficients (r) were calculated for all data pairs. results during the days of the study period, there were visits for asthma. of these visits, were pediatric. the number of daily visits ranged from to for all visits and from to for the pediatric visits. r values for all visits were - . , - . , . , - . , and - . ; for pediatric visits, - . , - . , . , - . , and - . for the -hr aqi for ozone, -hr aqi for particulate matter < . μm, total pollen grains/m , mold spores/m , and daily high temperature, respectively. in addition to calculating r values for same-day air quality indicators and ed visits, r values were also calculated for a -day average of air quality indicators with the ed visits. for the -day averages, r values for all visits were - . , - . , . , - . , and - . ; for pediatric visits, - . , - . , . , . , and - . for the -day averages of the -hr aqi for ozone, -hr aqi for particulate matter < . μm, total pollen grains/m , mold spores/m , and daily high temperature, respectively. conclusion there was no significant correlation between the selected air quality indicators and ed visits for asthma, either for the general population or for pediatric visits. this study provides no evidence that air quality is linked to ed visits for asthma in san antonio but the degree to which air quality in san antonio affects asthma symptoms remains unclear. introduction: the aim of this study was to evaluate the level of no in the exhaled air of patients suffering from chronic cough. methods: the study was performed on young (mean age . +/- . years), nonsmoking patients with chronic cough referred to the allergy clinic for evaluation. bronchial provocation challenge with histamine was performed according to the method of ryan. exhaled no was measured on-line using a chemiluminescence analyzer (sievers, usa). all patients had resting spirometry within normal values. results: significant bronchoconstrictive response to histamine at a concentration equal to or lower than mg/ml (pc mg/ml) was found in patients ( . %). these patients had a significantly greater mean concentration of no in the exhaled air ( . +/- . ppb) than those with a pc > mg/ml ( . +/- . ppb; p< . ). receiver operating characteristic (roc) curve analysis revealed that it is possible to identify from among patients suffering chronic cough those who have significant bronchial hyperreactivity (pc mg/ml). using ppb as a cut-off value for the exhaled no concentration, the specificity for bronchial hyperreactivity was . % and sensitivity . %. conclusion: assessment of no concentration is helpful in the assessment of bronchial hyperreactivity in patients having chronic cough. l. abetz * , j. bousquet , e. juniper , e. bateman , h. boushey , w. busse , t. clark , r. pauwels , s. pedersen , . manchester, united kingdom; . montpelier, france; . ancaster, canada; . cape town, south africa; . san francisco, ca; . madison, wi; . london, united kingdom; . ghent, belgium; . kolding, denmark. introduction: the acq has been developed as a measure of asthma control for use in clinical practice or in clinical trials. the reliability, validity and responsiveness have previously been demonstrated for the original item acq, the item (minus fev question) and item (minus fev and short acting bronchodilator use questions) versions. data from the -year gaining optimal asthma control (goal) study was used to assess the predictive value of the acq s , , and item versions in determining asthma control status. method: receiver operating characteristic curves (roc curves), plotting true positive rates versus false positive rates for different acq cut-points, were used to determine optimum cut points for totally controlled and well-controlled asthma. scores for the , , and item versions of the acq were examined at . intervals. results: when predicting totally controlled asthma, the areas under the roc curves (arocc) were . / . / . for the , and item versions, respectively; and when predicting well-controlled asthma the arocc were . / . / . for the , and item versions. in both predictive models, the and item versions provided the best arocc. for the and item versions, the best cut-off point to predict totally controlled asthma was <= . , with arocc of . / . , and corresponding sensitivity of . / . and specificity of . / . , respectively. for the item version the best cut-off point was <= . , with arocc of . , sensitivity of . and specificity of . . the best cut-off points for predicting well-controlled asthma were <= . for the item version (arocc= . ; sensitivity= . ; specificity= . ), and <= point for the and item versions (arocc= . / . ; sensitivity= . / . ; specificity= . / . , respectively). conclusion: results indicate the sensitivity and specificity of the acq in predicting asthma control status. a yo black male with asthma and allergic rhinitis developed gradually worsening cough productive of clear to yellow sputum despite treatment with high dose fluticasone, salmeterol, monteleukast, and prn albuterol. asthma history was significant for an intubation at age and pneumonia at age . physical exam revealed pale turbinates and expiratory wheezes. pulmonary function test showed a severe obstructive lung defect with fev . ( %), improving by % after bronchodilator and fev /fvc ratio %. a chest ct revealed significant bronchiectasis in the right middle lobe, lingula and both lower lobes. evaluation for bronchiectasis showed normal quantitative immunoglobulins, subclasses, and humoral responses, rendering innate immunodeficiency less likely. the diagnosis of allergic bronchopulmonary aspergillosis was entertained, but ige was ku/l and aspergillus titers were negative. a sinus ct failed to show active disease, yet the patient reported chronic yellow nasal discharge despite courses of antibiotics. although he was sexu-ally active with several male partners, hiv testing was negative times, months apart. evaluation for cystic fibrosis (cf) showed no symptoms of malabsorption and a negative sweat test. other tests revealed normal - antitrypsin level, p-anca, esr, ace level, and negative sputum for afb and mycobacteria, helping to exclude other conditions such as -antitrypsin deficiency, vasculitis, sarcoidosis, and chronic infection. with no definitive answer, the diagnosis of cf was pursued, but genetic screening as well as mapping were negative. the diagnosis of primary ciliary dyskinesia was then entertained; however, a mucosal biopsy of the nose did not reveal any structural abnormalities. finally, a sperm analysis revealing azoospermia confirmed the diagnosis of young's syndrome, a rare disease with features similar to cf including bronchiectasis, chronic sinusitis and azoospermia. in young's syndrome however, there is an obstructive process rather than a lack of vas deferens resulting in azoospermia and normal sweat chloride, genetic, and pancreatic testing. this case demonstrates the importance of investigating a persistently productive cough in an asthmatic patient and the differential diagnosis as well as the appropriate work up for bronchiectasis. a final diagnosis is important since appropriate management in each situation may affect long term outcome. a.g. palma-carlos * , m.l. palma-carlos, lisboa, portugal. introduction: bronchial challenges are currently done in allergic asthma with allergen, histamine or metacholine.bronchial reactivity to allergens and histamine can probably be variable. purpose to evaluate if the dose of allergen and histamine triggering a significant bronchial obstruction are always correlated in asthmatic patients. methods: a vitalograph model compact has been used throughout the study. allergen challenge has been done with an acqueous solution of house dust mite. bronchial challenges with placebo (sodium chloride) histamine or allergens have been done in all patients. for the provocations with histamine or allergens the threshold dose, inducing a decrease in fev greater than % has been determined in all the cases, as well for histamine as for allergens. a second challenge has been done in all patients with a supra-threshold dose of histamine or allergen corresponding to the double of the threshold. results: provocations have been done in patients either with histamine or allergen. were allergic and non allergic. the threshold dose for histamine were between and ug. in allergic patients and and ug. in non allergic patients.in allergic patients after histamine threshold challenge mean decrease was for fev , % and for fev /vc , %. for supra-threshold dose fev decreases , % and fev /vc , %. after allergen challenge fev decreases , % and fev /vc , %. with supra threshold doses fev decreases , % and fev /vc , %. in non allergic patients there was no changes after allergen challenge. a good correlation in constriction induced by histamine or allergen either for threshold or supra threshold doses (p< . ) was observed but not between the threshold doses of histamine and allergen. conclusions: these results confirm that bronchial obstruction can be triggered by allergens and mediators that the sensitivity of fev and fev /vc for evaluation are roughly comparable after challenge and points to a better sensitivity of a supra threshold dose of allergen or mediators. allergen and histamine challenges don't give the same information. introduction: peak expiratory flow rate (pfr) and forced expiratory volume in the first second (fev ) are currently used in functional evaluation of asthmatic patients but his degree of correlation in obstructive, restrictive and mixed spirometric ventilatory patterns are not well known. the purpose of this study was to evaluate if the correlation between pfr and fev markers of global airways obstruction is comparable in asthmatic patients with obstruction restriction or both. methods: asthmatic patients, males, females have been studied by spirometry with a vitalograph compact in absence of bronchodilatory or anti-inflammatory therapy. patients have been classified in functional patterns according to vc, and fev /cv: normal : vc greater than % of expected value and fev /vc greater than %, obstructive:vc greater than % and fev /vc greater than % restrictive vc, less than % and fev /vc greater than % and mixed vc less than % and fev /vc less than % presenting a so called functional emphysema. results: according to these criteria patients were normal, restrictive, obstructive and mixed. in all the group the correlation between pfr and fev was statiscally significant (p< ) but the correlation coefficient variable. in functionally normal patients r =+ , in restrictive patients + , in obstructive + , in mixed + , and in the sub-group with functional emphysema + , . for this sub-group the correlation coefficient r allows to define fev = + , pfr. conclusions: the report between the two most usual assays of global bronchial obstruction depends on the balance between restriction, (either by interstial fibrosis or hyperinflation) and obstruction and is more close when a more marked obstruction coexist with a restrictive pattern (functional emphysema). in practice pfr has the same value that fev only in more severe cases of ventilatory failure. the dispersion of values and the standard error or regression do not allow to correlate both data in most patients. introduction: the tenor study longitudinally observes the natural history of subjects with severe or difficult-to-treat asthma. this analysis assessed the association between previous and future asthma exacerbations in tenor. methods: subjects eligible for this analysis were years of age at baseline and had at least one follow-up assessment in year and year . recent asthma exacerbations in the past months were defined as: asthma-related emergency room (er) visits, nights of hospitalization, unscheduled physician office contacts; or as a composite measure of at least one of the previous exacerbation events. relative risks (rrs) and % confidence limits ( %ci) were generated comparing exacerbations in year relative to year . exacerbation rates were defined as the number of events divided by total patient follow-up time. no adjustment was performed for baseline characteristics. results: subjects were eligible for this analysis. rrs in year vs. year were: . ( %ci: . - . ) for unscheduled office contacts, . ( %ci: . - . ) for the composite exacerbation measure, . ( %ci: . - . ) for er visits and . ( %ci: . - . ) for nights of hospitalization. over % of subjects with or more exacerbations (composite measure) in year had a similarly high rate in year compared to % of patients without an event in year who went on to have or more exacerbations in year (see table) . conclusion: tenor subjects who experienced an asthma exacerbation in year were at statistically significantly increased risk of subsequent exacerbations the following year compared to subjects not experiencing an exacerbation in year . this increased risk was consistent for all asthma exacerbation outcomes and most elevated for hospitalizations due to asthma. assessment of recent asthmarelated healthcare use is a critical component of the clinical evaluation of subjects with severe or difficult-to-treat asthma. funded by genentech inc. and novartis pharmaceuticals corp. background: preliminary studies investigating yoga and breathwork for asthma have been promising. several randomized controlled trials have shown a benefit from yoga postures and/or breathing versus control, but the control in these cases involved no intervention other than usual care. this study advances the field by providing an active control. methods: a randomized, controlled, double-blind clinical trial was conducted from october to march to determine the effectiveness and feasibility of a yoga and breathwork intervention for improving clinical indices and quality of life for adult patients with mild to moderate asthma. random assignment was made to either a -week yoga intervention that included both postures and breathwork, or a stretching control condition. outcome measures were assessed at , , and -weeks and included the mini asthma quality of life questionnaire (mini-aqlq), rescue inhaler use, spirometry, symptom diaries, and health care utilization. results: sixty-two participants were randomized into the intervention and control groups, and completed the final follow-up measures. intention-to-treat analysis was performed. significant within-group differences in post-bronchodilator fev and morning symptom score were apparent in both intervention and control groups at and weeks; however, no significant differences between groups were observed on any outcome measures.conclusions: iyengar yoga conferred no appreciable benefit in mild to moderate asthma. circumstances under which yoga is of benefit in asthma management, if any, remain to be determined. in order to investigate asthma mortality trends for the u. s. hispanic population, i have collected and graphed data from the national center for health statistics. mortality data for the hispanic population have been available for the entire united states only since and readily available for asthma only since . these data indicate decreases in deaths from asthma (j -j ) from in to in with decreases in the non-hispanic population from in to in . rates of death from asthma decreased for the hispanic population from . per , in to . in , while that for the non-hispanic population decreased from . to . . rates for non-hispanic whites decreased from . to . in . rates of death from asthma have been twice as high among hispanic women as men. rates of death from asthma for non-hispanic blacks have been more than twice as high as those for non-hispanic whites. (national center for health statistics data dictate racial terminology). data for people of hispanic origin include people of any race. these data are affected by both underreporting of hispanic origin on death certificates and undercoverage in the census and resultant population estimates for a net correction of . %. thus, rates of death from asthma have been lower and have been decreasing faster among hispanics than the rest of the population in the united states. allergic rhinitis and asthma are both highly prevalent diseases and often coexist in patients. rhinitis symptoms have been shown to impact the lower airway. as such, the impact of rhinitis on asthma control may be the greatest in patients with the most severe rhinitis symptoms. therefore, this analysis was performed to determine the impact of baseline rhinitis severity on asthma control in patients with both asthma and allergic rhinitis who received either fluticasone propionate aqueous nasal spray (fpans) or montelukast (mon) added to fluticasone propionate/salmeterol / mcg (fsc). a total of patients (> years) with persistent asthma and seasonal allergic rhinitis received fpans mcg qd, mon mg qd or placebo (pbo) in addition to fsc / mcg bid for weeks. at baseline, total nasal symptom scores (tnss) ranged from to . the analysis was restricted to patients whose pre-enrollment asthma therapy did not include fsc. regardless of baseline rhinitis severity, in patients with both asthma and allergic rhinitis, mon added to fsc resulted in no additional improvements in overall asthma control compared with fsc alone. these data suggest that optimal treatment of the individual conditions should be the goal of treatment for patients with coexistent allergic rhinitis and asthma. (sam ) introduction recent studies have demonstrated that parainfluenza and coronaviruses are as important triggers of asthma. parainfluenza viruses are one of the main triggers of virus-induced asthma at summer. parainfluenza viruses often cause severe low respiratory tract infections in immuncompromised patients and in patients with bone marrow transplantation. a retrospective study found that % of pediatric bmt patients with hpiv infection ( % of viral respiratory infections) developed pneumonia and % died. both viruses contributed to fatal asthma. materials and methods for amplification of these viruses, primers which anneal to specific regions of viral genomes: hn-gene for piv , piv and piv and spike glycoprotein gene for coronavirus oc and nucleocapsid glycoprotein gene for coronavirus e were used. pcrdiagnostics was performed on nasal and throat swabs taken from children with atopic asthma exacerbation. results from / to / a total of samples (including negative controls) were tested by pcr. positive for respiratory viruses samples noted in children having asthma exacerbations including . % of all tested samples. piv was detected in . % of all positive samples, piv in . %, and coronavirus oc in . %. piv and coronavirus e were not detected during this study. for negative controls samples were taken from children with atopic asthma in remission. all controls were found to be negative by viral pcr. conclusions respiratory viruses are an important factor in asthma exacerbations in children. piv was the most frequently detected among the positive samples. although hpiv- was less often positive than hpiv- and hpiv- infections in this study, the frequency of the detected coronaviruses may have been influenced by the seasonal activity of these viruses. introduction. in budapest all children come under the regular supervision of a specially trained paediatrician. each paediatrician is responsible for an average of children, often seen up to the age of years. methods. a questionnaire was sent to the district paediatricians of budapest in budapest in , budapest in and . the total number of children in their practice and the number of the asthmatics was assessed. the diagnosis of asthma in every case was established in a paediatric hospital, or a special allergy or pulmonology outpatient clinic. results. in , replies were received from paeditricians, who were responsible for the supervision of , children, of which . ± . % had been diagnosed as having asthma. in replies were sent by physicians, who had a total of , children under their care, including , asthmatics, a prevalence of . ± . %. at the end of , paediatricians having , children answered, noting , asthmatics, a . ± . % prevalence of asthma. conclusion. the current survey of prevalence of asthma in childhood is by far the largest that has been made. an increasing prevalence of the diagnosis of asthma based on clinical investigations was noted, the differences between the investigated years being highly significant. panel report guidelines for the diagnosis and management of asthma have been available since , to standardize and improve the quality of asthma care. however, asthma remains the leading cause of hospitalization for new york city children. implementation of the guidelines has not been fully embraced in the primary care setting. we attempted to increase primary care practitioners' compliance with the guidelines through extensive training. a hospital based pediatric clinic and school based health clinics in the brooklyn inner city, participated from april to march in the program as part of new york city's education and quality improvement project (equip). initially an asthma physician specialist and asthma educator trained the primary care physicians and nurse practitioners in the guidelines. the training was reinforced during monthly luncheon sessions. clinical exam rooms were also supplied with asthma education materials, peak flow meters, and asthma action plans. goals were to identify asthmatics, document asthma severity, give persistent asthmatics written management plans, and utilize appropriate controller medications. compliance was measured by tracking asthma visits with an asthma intake form. prior to this project no consistent attempt had been made in the clinics to regularly classify asthmatic severity, provide written asthma action plans or to place patients on controller medications. after onset of the project a total of asthma visits were evaluated; were from school based health centers and from hospital based pediatric clinic. severity was classified in %( / ) of asthma visits. patients classified as persistent received updated written management plan during %( / ) of asthma visits and %( / ) were placed on controller medications. this asthma management program demonstrated success in improving primary care practitioners' compliance with the naepp asthma guidelines. further studies are needed to determine if compliance with the guidelines persisted after cessation of the extensive training and if such compliance improves asthma outcomes. numerous studies have associated asthma and obesity. although obesity has been identified as a risk factor for asthma, there is limited information on the impact of obesity on childhood asthma. this study examines the effect of body composition on asthma severity and pulmonary function in children. we retrospectively reviewed charts from asthmatic children ages to years, referred to a community-based pediatric pulmonology practice between and . data were included if asthma was the the primary diagnosis and a baseline spirometry was available. asthma severity was classified and body mass index(bmi) was classified as normal, overweight and obese. spirometry results were reviewed. eighty-five patients were reviewed. thirtyone( . %) patients were normal weight; ( . %) were overweight and ( . %) were obese. baseline characteristics such as age, gender and race/ethnicity were similar. asthma severity classification did not differ between normal, overweight and obese children. overall, % were classified as intermittent, % as persistent mild asthma and % as persistent moderate-severe asthma. % of children with intermittent asthma were receiving controller therapy compared to . % with persistent mild and . % with moderate-severe asthma(p= . ). although healthcare utilization for asthma(emergency room visits/admissions) were significantly different between children with intermittent, persistent mild and moderate-severe asthma(p= . ), there was no difference when normal, overweight and obese children were compared. spirometry results showed comparable peak expiratory flow rates(pfr) in all groups; % of predicted in normal weight and % of predicted in both overweight and obese. mean forced vital capacity(fvc) was . %, . % and % and the mean forced expiratory volume in second(fev ) was . %, . % and . % respectively. there was no difference in fev /fvc; . %, . % and . % and forced expiratory flow in mid-lung volumes (fef - ); . %, % and . % of predicted. in this study we retrospectively reviewed asthmatic children and compared bmi, asthma severity and spirometry. we found no difference in asthma severity classification between normal and overweight/obese patients. regardless of the bmi, all indices of spirometry were similar. further studies are needed to examine the impact of obesity on different asthma related disease outcomes. our objective is to further investigate the effect of budesonide, formoterol, and levalbuterol, alone, and in combination, on tgf- production and expression in ragweed (ra) stimulated a cells. meth-ods: a cells were cultured in duplicate for hours at degrees celcius with % co in serum free dmem with or without the following: μg/ml of ra, . x - m of budesonide, . x - m formoterol, and . x - m levalbuterol. tgf- production and expression was measured by a sensitive elisa and mrna assay in cell supernatants and pellets. results: tgf- production and expression from a cells rose markedly in the presence of ra ( . - pg/ml) ( - amol/ml) with significant inhibition following the addition of budesonide, and the combinations of budesonide and formoterol, budesonide and levalbuterol, and budesonide, formoterol, and levalbuterol (p< . ). combined treatment with budesonide and levalbuterol versus budesonide alone reached significance for production and expression (p< . ). budesonide and formoterol compared with budesonide alone reached significance for tgf- production (p< . ). conclusion: combination therapy with budesonide and a long or short acting beta-agonist showed greater effect of tgf- inhibition compared with budesonide alone. this synergistic effect on the distal airways may prevent a subset of asthmatics from developing airway remodeling. g. tamura * , y. sano , k. hirata , s. ishioka , m. nakashima , t. miyamoto , . sendai, japan; . tokyo, japan; . osaka, japan; . hiroshima, japan; . hamamatsu, japan. tulobuterol tape is the world's first long-acting transdermal preparation of a beta -agonist designed to release tulobuterol in an optimal fashion. when it is applied once daily at bedtime, the blood concentration of tulobuterol peaks early in the morning and is kept at effective levels for hours. tulobuterol tape has milder and less frequent adverse events than conventional oral tulobuterol preparations, and when used at bedtime can prevent marked drop in peak expiratory flow (pef) early in the morning. consequently, tulobuterol tape has been commonly used in japan as a long-acting beta -agonist. in the present study, to evaluate its efficacy as a long-acting beta -agonist and to compare its effects at two different doses, we administered tulobuterol tape at doses of or mg/day to patients with persistent asthma already using inhaled corticosteroids. this study was a randomized, double-blind, double-dummy, parallel-group, multicenter trial of -week duration. mean pef in the and mg/day groups were significantly increased from the baseline value by . and . at week , . and . at week , . and . at week and . and . l/min at week , respectively. the increase in mean morning pef in the mg/day group was significantly higher than that in the mg/day group at every point of determination. the mean evening pef was significantly increased in both treatments groups compared with baseline values at every point of determination. although the increase in the mg/day group was greater than that in the mg/day group at each point of determination, the difference between groups was statistically significant only at week . the safety profiles of the two treatments were similar. in patients with persistent asthma who require inhaled short-acting beta -agonists while receiving inhaled corticosteroids, tulobuterol tape mg/day significantly improved pef compared with tulobuterol tape mg/day. introduction: on an average, % of the children admitted annually for asthma to children s hospital require admission to the pediatric intensive care unit (picu). these admissions are highest in the months of august, september and october. aeroallergens such as alternaria and ragweed predominate during this season, and allergies to these allergens may account for the increased number of picu admissions for asthma. objective: we hypothesized that the seasonal increase in the admission rate could be related to allergen sensitivity, particularly to alternaria and/or to ragweed that predominate during late summer and fall. method: data was collected from a retrospective chart review of all patients admitted to the picu for asthma exacerbation between and . skin prick testing (spt) within one year of picu admission was the criteria for inclusion in this study. results: there was a higher prevalence of admissions to the picu for asthma exacerbation during august, september and october ( out of admissions = % of all admissions, p< . ). sixty-one subjects met the one-year spt criteria for analysis. subjects admitted during august, september and october were categorized as group a, and all others were categorized as group b. sensitivity to a total of aeroallergens (tress, grasses, weeds, ragweed, outdoor molds, indoor molds, alternaria, cat, dog, roach and dust mites) was compared between the two groups. there was no statistical significance in aeroallergen sensitivity to alternaria or ragweed between subjects admitted to the picu in august, september and october (group a) and subjects admitted during the other months of the year (group b). conclusion: asthma exacerbation requiring picu admissions is more prevalent during the months of august, september and october. the increased rated of picu admissions did not correlate with the aeroallergen sensitivity to the prevalent allergens during august, september and october. therefore we suspect other factors such as, viral upper respiratory infections, in addition to aeroallergen sensitivity, which may contribute to severe asthma exacerbation during the late summer and fall months. k. kuzume * , shigenobu, ehime, japan. infantile wheezing may be the result of different phenotypes. the aim of this study was to evaluate the risk factors for allergies and the results of allergyrelated blood tests among infants with different types of allergy symptoms. infants were examined physically at birth and at , , , , and months of age, and questionnaires about number of siblings, the family history of allergy, and feeding methods were given. allergic diseases were diagnosed at months of age, and the subjects were divided into groups: infants with atopic dermatitis (ad) only (a group, n= ), infants with both ad and wheezing (a/w group, n= ), infants with wheezing only (w group, n= ), and infants without allergic symptoms (c group, n= ). risk factors were then evaluated. patients ( from the a group, from the a/w group, and from the w group) were given allergy-related blood tests, total serum ige levels, and rast with egg white, milk, and house dust mites at and months of age. the results of the blood tests in control subjects at months of age were compared to the other groups. as shown in table , there were more male infants in the a/w and a groups than in the c group (p< . , a/w vs. c; p< . , a vs. c). numbers of siblings were greater in the w and a/w group than in the a or c group (p< . , w vs. a and w vs. c; p< . , a/w vs. a; p< . , a/w vs. c). the rate of positive family history of allergic diseases was high in the a and a/w groups, compared to the w or c group (p< . , a vs. c and a/w vs. c; p< . , a/w vs. w). the rate of breastfeeding at months of age was high in the a group compared to the others (p< . , a vs. c). patients in the w group had lower levels of total serum ige at months of age, compared to patients in a or a/w (p< . , w vs. a/w and w vs. a). also patients in the w group had lower levels of rast with egg white at months of age, compared to patients a or a/w (p< . , w vs. a/w and w vs. a). the median of total serum ige levels at months of age was higher in a and a/w but not in w, compared to c (p< . , a/w vs. c and a vs. c). in conclusion, there were two different phenotypes of wheezing in infants before months of age. wheezing with ad might be "allergy-related", while wheezing without ad may be related to other factors. as compared to c group: a), p< . ; b), p< . ; c), p< . ; d), p< . as compared to a group: ), p< . ; ), p< . ; ), p< . ; ), p< . as compared to a/w group: f), p< . ; g), p< . ; h), p< . a group, infants with atopic dermatitis only; a/w group, infants with atopic dermatitis and wheezing; w group, infants with wheezing only; c group, infants without allergic symptoms. rast, radioallergosorbent test, n/a, data not available r. amelio * , c. capristo , a. capasso , m. miraglia del giudice , n. maiello , f. decimo , . naples, italy; . napoli, italy. forced oscillation technique (fot) is considered a sensible and specific method to estimate breathing functionality in asthmatic children aged to years, because it doesn't need special collaboration. the aim of our study was to value baseline respiratory resistances with the fot in preschool-aged children with asthma under high dose of budesonide and flunisolide treatment. at this purpose, asthmatic children aged to years were selected: at a first examination (t ) all the patients showed basal value fot at hz frequency (rr ) changes major or equal than % under mcg of salbutamol treatment. days before the study, children selected didn't take systemic and inhaled steroids, cromons, leukotrienes antagonists, teophylline or anti-histamines and they didn't present upper and lower airways infections; then, two weeks before run-in, all selected patients took beta- -agonists at least times per week. all the patients were divided in two groups: group i ( children) under inhalation of flunisolide mcg/kg/dose + salbutamol mcg b.i.d. for days and, for the following two weeks, under inhalation of flunisolide mcg/kg/dose + salbutamol mcg by pmdi+spacer prn.; group ii ( children) under inhalation of budesonide , mg b.i.d. + salbutamol mcg b.i.d. for days, and for the following two weeks, under inhalation of budesonide , mg b.i.d. + salbutamol mcg by pmdi+spacer prn. moreover, we gave diary-card with a simple symptoms score (at t and t ), to get the real perception of the symptoms in the course of study. however, during the treatment, children have ritired from the study. the results obtained were statistically analysed (t student's test). significant rr pre beta- -agonists and ? value reduction was obtained at t and t vs. t . group i had (at t ) such a quick action than group ii reducing airways resistances (p< , ). children treated with flunisolide had lower frequency of breathless and nocturnal cough at t and lower salbutamol use from t to t than budesonide group (p=n.s.). in conclusion, fot is actually a safety method to value efficacy of inhaled steroids in preschool-aged children with asthma. introduction: approximately . % of u.s. adults and . % of louisiana (la) adults have current asthma, and over % currently smoke cigarettes, according to the behavioral risk factor surveillance system (brfss). the purpose of this study is to compare asthmatic smokers vs. nonsmokers in la and the u.s. here we address whether asthmatic smokers utilize more medical care services in the er and outpatient clinics, and whether they were able to fully participate in activities of daily living compared to nonsmoker asthmatics. methods: brfss is a state-based, telephone survey of u.s. adults. the survey collects self-reported information about modifiable risk factors for chronic diseases. this study analyzed data from the brfss survey using sas software. data: out of the current asthmatics, % are cigarette smokers in the u.s. in louisiana, % of asthmatics are currently smoking, which is significantly higher than the national average (p< . ). within the past year, the average number of emergency room visits due to asthma was similar for both locations (la . , us . , p= . ) . nationally, smokers visited the er significantly more frequently than nonsmokers (s . , ns . , p< . ). in la, smokers visited the er twice as often as nonsmokers (s . , ns . , p< . ) the number of annual routine outpatient checkups for asthma was similar based on location (la . , us . , p= . ). smokers and nonsmokers had a similar number of checkups nationally (s . , ns . , p= . ) and in louisiana (s . , ns . , p= . ). the number of days where asthmatics were unable to perform their usual activities was significantly fewer in la than nationally (la . , us . , p< . ). smokers had a significantly higher number of missed days nationally (s . , ns . , p< . ). in la, there was not a significant difference in the number of missed days based on smoking status (s . , ns . , p= . ). conclusion: even though louisiana has a lower prevalence of asthmatics compared to the national average, significantly more of our asthmatics are smokers. asthmatic smokers in la and the u.s. utilized the er more than their nonsmoking counterparts. smokers and nonsmokers had similar numbers of routine outpatient visits. asthmatic smokers missed more days, but this was only significantly different at the national level. other research has shown that young age is highly associated with improper use in children using a mdi and holding chamber (arch pediatr adolesc med ; : ) . the objective of this study was to compare health outcomes achieved by children with asthma using inhaled corticosteroids (icss) delivered by a nebulizer compared with outcomes of children using icss delivered by non-nebulized device. methods: using a managed care organization database (pharmetrics integrated outcomes database), we identified children aged years with an asthma diagnosis and asthmarelated hospitalization/emergency department (ed) visit (july -june and with a prescription claim for an ics within days of discharge. we compared relative risk of hospitalization/ed recurrence from day - (cox proportional hazards regression, covariates=sex, age, current and prior asthma medications, prior oral corticosteroid and short-acting -adrenergic agonist use, initial type of index event) for patients receiving nebulized ics vs other ics by age groups ( - years and - years). results: of patients with claims for ics, received nebulized ics, of which were aged years; received non-nebulized ics, of which were aged years. postindex hospitalization/ed rates were . %. refill rate was higher for patients using nebulized ics. after model risk adjustment, patients using nebulized ics had a % risk reduction for hospitalization/ed recurrence vs those not using nebulized ics (hr: . , % ci: . , . ). in the -to -year age group, patients on nebulized ics had a % risk reduction compared with patients not using nebulized ics (hr: . , % ci: . , . ). in the -to -year age group, patients on nebulized ics had a % risk reduction (hr: . , % ci: . , . ). conclusion: in actual practice, treatment with nebulized ics after an asthma exacerbation is associated with a significant reduction of recurrent hospitalization/ed visits in young children, possibly due to improved technique and compliance. introduction: tenor is a -year, multi-center, cohort study of patients with severe or difficult-to-treat asthma. the objective of the tenor study is to better understand the natural history of patients with severe or difficult-totreat asthma. this analysis assessed the frequency of skin testing in this population and characterized the differences between the positive (st+) and negative (st ) subjects. methods: subjects were asked whether they had ever been skin tested and, if so, the test results. those who were st+ were compared to both st and those never tested (stnd) using clinical and other asthma-related characteristics. subjects years of age were included in this analysis. results: subjects were eligible for this analysis. . % were skin tested in the past, with stnd frequency of . % from allergist sites and % from pulmonologist/other sites. of those tested, . % were positive (allergist . %, pulmonologist/other . %). baseline ige for st+ subjects was . iu/ml vs. . iu/ml for st ; p< . (table) . as shown, the age at asthma onset, duration of asthma, rate of atopic disorders, and the rate at which asthma was triggered by aeroallergens differentiated the st+ from the st group. disease severity as evaluated by fev , healthcare utilization, and medication use, however, was similar between the two groups. in general, stnd were more likely to have values closer to the st+ group, suggesting that the majority of those not tested would have been st+, if administered a test. conclusions: the prevalence of st+ subjects from both allergy and pulmonary practices was high, demonstrating that the majority of these severe or difficult-to-treat patients have allergic asthma. st+ patients showed differences in clinical characteristics compared to st , including a greater likelihood of their asthma symptoms being triggered by aeroallergens. these data also show that the clinical profile of stnd patients may be similar to that of st+ patients, suggesting the utility of a more universal allergic evaluation in severe asthmatics. funded by genentech and novartis pharmaceuticals corp. background current national guidelines for the diagnosis and management of asthma such as the naepp epr , classify asthma severity based on frequency of asthma symptoms, medication use, and measurements of lung function by spirometry or peak expiratory flow variability. recently, the utility of spirometry measurements for assigning asthma severity has come into question. here we evaluate the correlation of spirometry measurements with asthma severity in school-aged children who are mostly naive to anti-inflammatory therapy. methods children were participants in a school-based lowincome asthma mobile van program, the breathmobile. recruitment was through referrals by school nurses and community public health clinics, parental response to flyers, and asthma screening questionnaires. spirometry was performed on all children greater than years of age as part of a comprehensive asthma evaluation. asthma severity was assigned based on symptoms only, according to the naepp epr . results from april to april of the children evaluated on the breathmobile, patients were diagnosed with asthma. classification of severity by symptom frequency according to naepp epr guidelines resulted in % as mild intermittent (mi), % as mild persistent (mip), % as moderate persistent (mop), and % as severe persistent (sp). the percentage of patients and their mean lung functions at baseline evaluation without bronchodilator challenge are shown in the table. there were statistically significant differences (p<. ) between severity groups which was most pronounced for the fef - % when comparing the severe persistent group and the intermittent group. however, mean values appear to be "normal" for all degrees of asthma given current accepted cut-off values, fvc %, fev %, fev /fvc %, and fef - %. conclusion in our study, airflow impairment did correlate with asthma severity, although it was "normal" (fev %) even in children with severe persistent asthma. therefore, a "normal" lung function measurement may be misleading as sole criteria for asthma severity classification. perhaps a higher cutoff point (fev < %) might be a better indicator of asthma severity in children. asthma is the most frequently chronic disease in childhood.our main was to know the prevalence of bronchial asthma in children ( - and - years) in the north zone of mexico city and to compare the prevalence obtained by the written questionnaire versus the video questionnaire in the group of to years, according to methodology proposed by isaac. material and methods: by means of a validated and standardized questionnaire (isaac) that was applied to children from to years and from to . the questionnaires of and years were filled out by their parents. the group from to years answered a questionnaire and a video questionnaire. according to isaac specification and based on a studied population, it was calculated by statcal program the size of the sample. it was choose a haz-ardous sample of children between - years and of the - group years, both sexes in elementary and high school. measures of central tendency and chi were used. results: the final sample size for both groups was children ( of - and adolescents), . % men and . % women. the prevalence of the asthma diagnosis for teenagers was of % (p< . ic . - . ) and it was . % in children (p< . ic . - . ), wheezing in the last months was . % (p< . ic . - . ) in children and of . % (p< . ic - . ) for - years. wheezing induced exercise appeared in . % (p< . ic - . ) in teenager and . % (p< . ic . - ) in the other group. the severity of asthma showed by nocturnal awakness was . % and . % (p< . ), number of crisis in the last year . % and . % for children and teenagers respectively (p< . ). in the video questionnaire of teenagers . % (ic . - . ) answered affirmative on have displayed a shaken breathing on the last year resting and only . % (ic - . ) in the last month. about exercise question . % confirmed have displayed symptoms, . %(ic - . ) in the last year and . % (ic . - . ) in the last month. wide-awake at night . % (ic . - . ) in the last year and . % ) in the last month. conclusions: the prevalence of asthma by diagnosis was higher in teenagers group than in children group. the prevalence of asthma was higher also in this group. rationale: we investigated the relationship of pet exposure and healthcare utilization (hcu) in a cohort of pediatric patients with severe or difficult-totreat asthma in the epidemiology and natural history of asthma: outcomes and treatment regimens (tenor) observational study. we hypothesized that pet exposure would increase hcu due to increased airway inflammation. methods: tenor is a -year multicenter cohort study of patients with severe or difficult-to-treat asthma. children ages - were interviewed at baseline regarding asthma-related hcu in the previous months. patients with pet exposure (ppe, n= ) were compared with patients with no pet exposure (pnpe, n= ). responses were analyzed using fisher's exact test. results: ppe were less likely to have severe asthma by physician assessment ( % vs. %; p= . ). in addition, ppe were less likely to have an emergency room (er) visit ( % vs. %; p= . ) or to have been hospitalized ( % vs. %; p= . ) in the previous months. more pnpe had an ige level > iu/ml ( % vs. %; p= . ). ppe and pnpe were similar with regard to presence of allergic rhinitis and skin test positivity. compared with pnpe, fewer ppe with two or more pets had er visits and hospitalizations (table) . among ppe, there were no differences in ige levels or in asthma severity related to the number of pets. conclusions: pet exposure was associated with reduced hcu in the tenor pediatric cohort, with the most pronounced effect seen in reduced er visits and hospitalizations needed by those with multiple pets. ppe had significantly lower ige levels compared to pnpe, regardless of the number of pets. these data support the hypothesis that exposure to pets may paradoxically protect individuals from the development of severe asthma. alternatively, these data may reflect a self-selection in patients with severe or allergic asthma who are likely to avoid having a pet. however, there may be other confounding factors in families with pets that confer a protective effect on asthma severity. introduction: in asthma and other chronic obstructive airway diseases, phosphodiesterase (pde ) is involved in the pathophysiology of the disease and is expressed abundantly in key inflammatory cells. inhibitors of pde are investigational, anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde inhibitor with demonstrated in vitro and in vivo anti-inflammatory activity, which may translate into clinical efficacy in asthma therapy. this study examined the ability of roflumilast to exert direct or acute bronchodilatory activity in patients with mild to moderate asthma. methods: this was a double-blind, randomized, placebo-controlled, crossover study consisting of three treatment periods of one day each, separated by a -to -day washout period. during each treatment period, patients with a forced expiratory volume in one second (fev ) of % to % of predicted received either oral roflumilast μg, roflumilast μg, or placebo. the fev was measured twice prior to administration and periodically up to h following treatment. after h, patients inhaled μg salbutamol from a metered-dose inhaler; fev was measured min later. adverse events, vital signs, and electrocardiogram results were monitored throughout the study. results: median baseline fev was similar for the three treatment periods. both doses of roflumilast did not lead to statistically significant differences versus placebo in the time-averaged fev over the first or up to h after drug intake (p > . ). thus, there was no evidence of a direct or acute bronchodilatory effect with either single doses of roflumilast μg or μg. in contrast, inhalation of the short-acting bronchodilator salbutamol led to a distinct improvement in fev versus baseline in all treatments groups with median fev increases of %, %, and % in the roflumilast μg, roflumilast μg, and placebo groups, respectively. roflumilast was well tolerated. conclusions: oral, once-daily roflumilast exhibits no direct or acute bronchodilatory activity in patients with mild to moderate asthma as could be achieved with short-acting -agonists. these data support the proposed mechanism of action of roflumilast as an antiinflammatory agent. south africa; . guadalajara, spain; . munich, germany; . weinheim, germany; . budapest, hungary; . konstanz, germany. introduction: phosphodiesterase (pde ) is found in key inflammatory cells involved in the pathophysiology of chronic obstructive pulmonary disease and asthma. inhibitors of the pde enzyme are anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, oncedaily pde inhibitor with demonstrated in vitro and in vivo anti-inflammatory activity. this study examined the dose-related efficacy of roflumilast in patients with asthma. methods: patients with stable asthma (forced expiratory volume in second [fev ] % to % of predicted) were enrolled in this randomized, double-blind, dose-range finding study. after a single-blind placebo run-in period of up to three weeks, patients received oral roflumilast μg, μg, or μg once daily (n = , , and , respectively) for weeks. efficacy was assessed by change from baseline in spirometric lung function fev and forced vital capacity (fvc), as well as morning and evening peak expiratory flow (pef) recorded in patient diaries. safety and tolerability parameters were monitored throughout the study. results: treatment with roflumilast led to dose-dependent and statistically significant increases in fev , fvc (both p < . ), and in morning pef (p < . ). at last visit, fev improved from baseline by ml ( %), ml ( %), and ml ( %) in patients treated with roflumilast μg, μg, and μg once daily, respectively. similarly, dose-dependent improvements of l/min, l/min, and l/min in morning pef from baseline were reached. roflumilast was well tolerated at all dose levels tested. the most frequent drug-related adverse events were headache followed by gastrointestinal disorders such as diarrhea and nausea. there were no clinically relevant changes in vital signs, electrocardiogram, or laboratory parameters. conclusions: oral, once-daily roflumilast was associated with dose-dependent, clinically relevant improvements of lung function in patients with stable asthma. roflumilast was well tolerated. j.l. izquierdo * , e.d. bateman , p. magyar , u. harnest , p. hofbauer , a. varga , c. schmid-wirlitsch , d. bredenbroeker , t.d. bethke , . guadalajara, spain; . cape town, south africa; . budapest, hungary; . munich, germany; . weinheim, germany; . tatabanya, hungary; . konstanz, germany. introduction: phosphodiesterase (pde ) inhibitors are a new class of anti-inflammatory agents for therapeutic use in inflammatory airway diseases. in several studies, the investigational, oral, once-daily pde inhibitor roflumilast has provided clinically meaningful improvements in patients with chronic obstructive pulmonary disease and asthma. this study examined the long-term safety and tolerability of roflumilast over weeks in patients with asthma. methods: patients with persistent, stable asthma (fev % to % of predicted at randomization) participated in a double-blind, randomized dose-range finding study and were treated with oral roflumilast μg, μg, or μg once daily for weeks. patients completing the -week study per-protocol, then continued treatment in this open-label -week extension study. all patients received oral roflumilast μg once daily during the extension period. adverse events (aes), clinical laboratory parameters, vital annals of allergy, asthma & immunology signs, and ecg were assessed throughout the extension period. results: a total of patients were enrolled in the -week extension study. overall, the most common aes were related to the respiratory system. only a small number of patients ( %) experienced aes that were assessed as at least likely related to study medication. the most frequent drug-related adverse events were headache, diarrhea, and nausea as reported by %, %, and % of patients, respectively. most aes were mild to moderate in intensity and transient in duration; % of patients discontinued the study due to aes. most ( %) of these aes leading to discontinuation were assessed as not related or unlikely related to study drug. out of patients, reported serious aes, which were all assessed as not related or unlikely related to roflumilast. no clinically relevant changes in clinical laboratory parameters, vital signs, ecg, or physical examination occurred. conclusions: oral, once-daily roflumilast μg administered for weeks was well tolerated in patients with persistent, stable asthma. this study provides evidence that roflumilast is associated with a low incidence of aes, which are mostly mild to moderate in intensity and transient in nature, thus, supporting the potential therapeutic use of roflumilast in asthma. paris, france; . madrid, spain; . guadalajara, spain; . harrow, united kingdom; . weinheim, germany; . augsburg, germany; . munich, germany; . kaufbeuren, germany; . konstanz, germany. introduction: in asthma, inhaled corticosteroids (ics) have been the mainstay of maintenance treatment. new therapeutic agents are needed that target key inflammatory processes in asthma as effectively as ics but overcome known limitations of ics. phosphodiesterase (pde ) inhibitors are anti-inflammatory agents that prevent the breakdown of cyclic adenosine monophosphate, a natural modulator of inflammation. roflumilast is an investigational, oral, once-daily pde inhibitor for potential use in anti-inflammatory asthma therapy. this study compared the standard corticosteroid treatment of twice-daily, inhaled beclomethasone dipropionate (bdp) with oral, once-daily roflumilast in patients with asthma. methods: in a randomized, double-blind, double-dummy study, patients (forced expiratory volume in one second [fev ], % to % of predicted) received either oral roflumilast μg once daily (n= ) or inhaled bdp μg twice daily ( μg/day; n= ) for weeks. mean change (lsmean and sem) in lung function parameters fev , forced vital capacity (fvc), and morning and evening peak expiratory flow (pef) from baseline was determined after weeks of treatment. further, symptom score and rescue medication use were assessed. results: both roflumilast μg and bdp μg improved fev from baseline to clinically meaningful levels ( ± ml and ± ml, respectively; both p< . ). similarly, roflumilast and bdp improved fvc ( ± ml and ± ml, respectively; both p< . ) as well as morning and evening pef (p . ). roflumilast μg was statistically non-inferior to bdp μg. roflumilast and bdp led to statistically significant and comparable improvements in asthma symptoms and use of rescue medication. adverse events were generally mild to moderate; the most common drugrelated adverse events reported in the roflumilast group were nausea, headache, and diarrhea. there were no clinically relevant changes in vital signs, ecg, or laboratory parameters. conclusions: roflumilast μg provided clinically meaningful improvement of lung function parameters, reduced asthma symptoms, and decreased the need for rescue medication. oral, once-daily roflumilast was as effective as inhaled, twice-daily bdp in the treatment of asthma. oral roflumilast μg was well tolerated. rationale: evaluation of medical claims from commercial health plans permits assessment of therapeutic interventions on resource utilization. this study investigated the impact of controller therapy in children starting asthma maintenance medications. the risk of obtaining prescriptions for oral corticosteroids (ocs), short-acting beta-agonists (saba) or adding another asthma therapy was evaluated. methods: this was a retrospective observational study utilizing medical and pharmacy claims from a large representative managed care plan from / / - / / . children aged - years old with an asthma diagnosis (icd- , .xx) and an initial pharmacy claim for one of the following regimens: fluticasone/salmeterol in a single inhaler, n= , (fsc), fluticasone propionate alone, n= , (fp), montelukast, n= , (mon), any inhaled corticosteroid plus montelukast, n= , (ics+mon), and an ics plus salmeterol from separate inhalers, n= (ics+sal) were included in the analysis. subjects were excluded if they had received any asthma controller medication in the months prior to the initiation of therapy. regression was used to estimate the incidence rate ratio (irr) for ocs and saba use (negative binomial) and the odds ratio (or) of adding a controller (add) and an ed or hospitalization (ip) event (logistic). all models controlled for demographics, pre-controller asthma-related medications and events, and baseline total health care costs. results: the ratios in the table with a confidence interval excluding unity indicate significantly increased use or likelihood of an event compared to the fsc cohort. controller naïve children started on fsc were less likely to add another controller than mon, fp or ics+sal, and had less use of ocs or saba than the cohorts when studied for months after the initiation of controller therapy. in addition, children treated with ics + mon had a significantly greater or of an ed/ip visit compared to fsc. conclusion: use of fp + sal (fsc) in a single inhaler assures that children are getting more optimal inhaled corticosteroid therapy as well as the benefits from the long acting bronchodilator while avoiding the potential for selective discontinuation of ics in the multiple controller cohorts. a.s. nayak * , r. nathan , j. williams , s. kundu , m. lloyd , d. banerji , . normal, il; . colorado springs, co; . bridgewater, nj. introduction: inhaled corticosteroids (ics) are recommended firstline therapy for severe, persistent asthma. systemic exposure to ics may suppress hypothalamic-pituitary-adrenal (hpa)-axis function, particularly at doses required to control severe asthma. ciclesonide (cic) hydrofluoroalkane (hfa) metered dose inhaler (mdi) is a novel and effective ics that is converted in the lungs to its active metabolite, desisobutyryl ciclesonide (des-cic). previously, in short-term studies, cic has been shown to have no effect on serum or urinary cortisol levels, which may be attributed to the low oral bioavailability, high serum protein binding and high clearance rate of cic and its active metabolite. this hpa axis analysis was part of a long-term study evaluating the safety of cic and beclomethasone dipropionate (bdp) hfa-mdi in patients with severe persistent asthma. methods: this was a multicenter, double-blind, parallel-group, -month extension of a -week double-blind trial of patients years with severe persistent asthma. patients were randomized in a : ratio (cic:bdp) to receive cic ( g bid) exactuator or bdp ( g bid) ex-actuator. after weeks, the doses of both medications could be titrated to g bid as needed for asthma control. hpa-axis function was assessed at selected centers in a subset of patients at baseline and end of study (at month or early termination) by measuring both peak serum cortisol induced by low dose ( -μg) cosyntropin and -hr urinary free cortisol corrected for creatinine. results: data were available for a small number of patients at centers evaluating hpa-axis (table) . mean baseline levels and change from baseline to end of study values in low-dose cosyntropin peak serum cortisol levels for cic and bdp were comparable. likewise, baseline levels and mean change from baseline to end of study values in -hr urinary free cortisol corrected for creatinine were comparable among the two treatment groups. conclusion: these findings demonstrate that cic to μg daily for months is safe and has no significant effect on hpa-axis function. a. long * , a. rahman , . boston, ma; . wilmington, de. introduction: little information is available regarding the relationships between asthma severity, disease control, and compliance with medications. methods: physicians' perceptions regarding these variables were determined by internet-based general asthma medication usage survey and patient-specific asthma medication usage surveys between december and , , for physicians ( pcps, pediatricians, allergists and immunologists, and pulmonologists). the patient-specific surveys were based on chart information of randomly chosen patients per physician on controller medication. results: of patients, %, %, %, and % were categorized by their physicians as having mild intermittent or mild, moderate, or severe persistent asthma, respectively. overall, % of patients were categorized as having "very controlled" asthma and % as being "very compliant" with their current regimen. worse asthma control, decreased compliance, and increased physician visits were all associated with increased asthma severity classification. physicians reported that only % of patients with severe persistent disease (n= ) had "very controlled" asthma in contrast to % of patients with mild intermittent asthma (n= ). patients with mild intermittent asthma, and mild, moderate, and severe persistent asthma, had a mean of . , . , . , and . physician visits for asthma, respectively, within the past year. physician perception of patient compliance varied less across the groups, with % of patients with severe persistent asthma and % of patients with mild intermittent asthma being rated as "very compliant." the incidence of allergic rhinitis was similar among all severity levels (range: %- %), but patients with severe persistent asthma were more likely to have comorbid conditions, including hypertension, chronic obstructive pulmonary disorder, chronic bronchitis, and osteoporosis, compared with patients in other asthma severity levels. conclusions: decreased asthma control and rates of com-pliance, as well as increased physician visits and comorbid medical conditions, may be associated with increased asthma severity classification. rationale: children with asthma frequently have co-morbid allergic conditions requiring medications. the purpose of this study was to assess whether the controller asthma medication selected would reduce the utilization of intranasal steroids (ins) and prescription nonsedating antihistamines (nsa) for children treated for co-morbid allergy. methods: this was an observational retrospective study that utilized the pharmetrics integrated outcomes database that contains administrative medical and pharmacy claims data from over managed care plans across the united states. children age - years old with a new diagnosis of asthma (icd- , .xx) were identified and observed for months after their initial diagnosis and treatment of asthma (post-index). four cohorts were established based on their controller medication dispensed: montelukast, n= (mon), fluticasone propionate, n= (fp), fp+mon n= , fp/salmeterol in a single inhaler, n= (fsc). results: baseline comorbid diagnosis of allergic rhinitis was observed in - % of the children. the patterns of use of ins and nsa were similar in the months before and the months after the initial asthma diagnosis and treatment. children with a diagnosis of allergic rhinitis were nearly twice as likely to receive a nsa or ins. all treatment cohorts used a similar amount of each allergy medication. the presence or absence of a diagnosis of allergic rhinitis did not alter the findings. the adjusted odds ratio ( %ci) of using a nsa or ins relative to the use of fsc in the post-index period was mon: . ( . , . ); fp: . ( . , . ); fp+mon: . ( . , , ). the table below shows the percent of children dispensed a nsa and/or an ins. the number of units of each allergy medication was also similar across all cohorts. conclusion: the use of mon, fp, fp+mon or fsc for asthma did not alter the rate or quantity of either intranasal corticosteroid or nonsedating antihistamines dispensed to children over the -month period. although allergic rhinitis is a common comorbidity of pediatric asthma, the selection of a regimen containing montelukast did not reduce the use of either nsa or ins compared to asthma treatments with fp or fsc. rationale: medication compliance is recognized as a significant challenge in pediatrics.the purpose of this study was to compare inhaled corticosteroid (ics) persistence in pediatric patients using a single inhaler, containing both an inhaled corticosteroid (fluticasone propionate, fp) and an inhaled long-acting beta-agonist (salmeterol, sal) (fsc), to patients receiving fp alone, fp + sal from separate inhalers, or ics + montelukast (mon). methods: retrospective -month pre-post database study utilizing medical and pharmacy claims. a total of subjects - years of age with a diagnosis of asthma (icd ) were grouped into cohorts: fsc [n= ]; fp only [n= ] ics+sal [n= ]; and ics+mon [n= ]. patients were controller naïve for months prior to the index event (the first prescription of the medication of interest). subjects were required to have continuous enrollment of months pre-and months postindex. subjects with cystic fibrosis, copd, bronchopulmonary dysplasia or respiratory distress syndrome were excluded. ics refill rates, as a measure of persistence, were compared between fsc and the ics components of the other cohorts over a -month follow-up period. results: mean refill rates over the -month follow-up period was significantly greater (p=< . ) for fsc ( . ) compared with the mean ics refill rate in the fp alone ( . ) cohort, the ics + sal ( . ) and the ics + mon ( . ) cohort. patients on fsc filled % more ics prescriptions than patients on fp alone, % more than ics+sal and % more than ics + mon. mean refills were generally higher than the median fills as a considerable number of children had only one fill: fsc ( . %), fp ( . %), ics ( . %) + sal ( . %), ics ( . %) + mon ( . %). conclusion: patients using fsc, are likely to fill their inhaled corticosteroids more often over -months compared with patients using ics + sal in separate inhalers, ics+mon, and fp as monotherapy. improved persistence with ics has been shown in other studies to decrease morbidity and mortality of asthma. use of fsc, a single inhaler, assures that children are getting more optimal inhaled corticosteroid therapy as well as the benefits from the long acting bronchodilator while avoiding selective discontinuation of ics that may occur when two medications are dispensed. the national health interview survey (nhis) estimates . million united states residents with asthma. noncompliance with treatment has been estimated as between and %. the consequences of noncompliance include absences from work or school, increased emergency room visits, more severe attacks, increased drug side effects, greater cost of care, and death. we questioned compliance in a yo man with severe, prednisone dependent eosinophilic asthma because he had persistent symptoms despite treatment with fluticasone propionate/salmeterol xinafoate / bid, prednisone mg to mg qd, and budesonide ( mcg/puff) puffs bid. after discontinuation of budesonide and prednisone and initiation of methylprednisolone mg bid for hours and then mg qd and beclomethasone dipropionate puffs ( mcg) hfa bid with a spacer, clinical improvement was unexpectedly abrupt. fev increased from % to % of predicted. sputum eosinophil counts decreased from % to %. because of the sudden improvement with the change to methylprednisolone, compliance to prednisone and fluticasone was questioned. to evaluate compliance, the patient's blood, urine, and sputum were tested for synthetic corticosteroids, without his knowledge, using mass spectrometry. the blood level of methylprednisolone was . mcg/dl and prednisolone was . mcg/dl, documenting use of methylprednisolone and the recently discontinued prednisone. the urine levels were mcg/dl of methylprednisolone, . mcg/dl of prednisolone and . mcg/dl of prednisone that further confirmed recent use of prednisone. the sputum testing revealed . mcg/dl beclomethasone, mcg/dl fluticasone and . mcg/dl methylprednisolone confirming compliance to inhaled fluticasone and beclomethasone at the time of the test. thus, contrary to our clinical suspicion, the patient was indeed compliant with his inhaled glucocorticoids and prednisone and subsequently with the methylprednisolone. to our knowledge, this is the first case report to document compliance to inhaled and oral corticosteroids by analysis of synthetic corticosteroid concentrations in blood, urine, as well as sputum. if compliance could be documented with certainty, it could potentially minimize the adverse effects of needless escalating steroid doses, reduce the cost of treatment, and minimize morbidity and mortality as well. rationale:the burden of pediatric asthma extends beyond those children with an asthma diagnosis. children with wheezing frequently have a delay in the diagnosis of asthma that may impede instituting appropriate therapy and reducing disease morbidity. this observational study was designed to assess the treatment patterns of children - years old receiving asthma medication(s) without a diagnosis of asthma compared with children diagnosed with asthma and children without claims for asthma. methods:this was a retrospective cross-sectional study that utilized the pharmetrics integrated outcomes database containing administrative medical and pharmacy claims data from over managed care plans across the united states. three cohorts were selected: children with an asthma diagnosis (icd- , .xx) (dx cohort), children with prescription claims for asthma controllers or rescue medications without an asthma diagnosis (rx cohort) and children with neither an asthma diagnosis nor prescription claims for asthma medications (control cohort). utilization of asthma medications, asthma specific costs and total costs of care were assessed. results: a total of , children were identified: . % in the dx cohort, . % in the rx cohort and . % in the control cohort. the potential asthma cohort was . %. total annual non-asthma related costs for the dx cohort was $ , for the rx cohort was $ , and for the control was $ . only % and % of the total healthcare charges in the dx and rx cohorts retrospectively were asthma related. non-asthma charges were significantly higher in both the dx and rx cohorts compared with the control cohort. in the rx cohort, the ratio of saba prescription claims to asthma controller claims were . -fold higher than in the dx cohort. conclusion: children treated with asthma medications without an asthma diagnosis consume greater health care resources resembling the pattern of health care utilization of children with an asthma diagnosis more closely than controls. further, children with treatment in the absence of an asthma diagnosis appear to be under-treated with controller therapy as recommended by national and international guidelines. background: a recent retrospective study assessed asthma variability in inner-city patients months before and months after enrollment into an naepp guidelines-directed clinical management and educational program. while guidelines-directed care improved asthma symptoms and outcomes, significant variability in disease indices were observed over the -month period. the present analysis evaluates resource utilization associated with this variability. method: economic end points, including hospital/emergency department (ed) visits, total unscheduled office visits, sick visits, and days lost from work or school, were collected from inner-city patients aged years ( % female, % minority, % treated by primary care physicians) enrolled in a guidelines-directed asthma clinical management and education program aimed at minimizing barriers to adherence. patients were stratified into groups: those with high variability in asthma (n= ) and those with low variability in asthma (n= ), with variability defined as number of fluctuations in naepp symptom class in the -month postintervention period (high variability = patients with fluctuations higher than the mean; low variability = patients with fluctuations lower than the mean). results: guidelines-directed therapy was associated with improvements in asthma during the -month treatment period for both groups. patients in the high-variability group had more ed visits, sick visits, total unscheduled visits, and office visits compared with patients in the low-variability group (see table) . conclusions: despite guidelines-directed therapy and overall improvement in asthma control, patients experienced variability in asthma, indicating that even when their disease is stable, their symptoms continue to fluctuate. as a result, asthma variability may contribute to increased resource utilization. introduction: inhaled corticosteroids (ics) are considered first-line therapy for patients with persistent asthma. ics can lead to oropharyngeal adverse events, which may affect treatment outcomes and adherence. the occurrence of these local adverse events is dependent upon several factors, including dosage and duration of ics treatment. ciclesonide (cic) hydrofluoroalkane (hfa)-metered dose inhaler (mdi) is a novel and effective ics, with relatively low oropharyngeal deposition. cic is converted in the lungs to its active metabolite, desisobutyryl-ciclesonide (des-cic). furthermore, cic undergoes limited conversion in the oropharynx, which may account for its improved safety profile. this oropharyngeal safety profile analysis was part of a long-term study evaluating the safety of cic hfa-mdi vs beclomethasone dipropionate (bdp) hfa-mdi in patients with severe persistent asthma. methods: this was a multicenter, double-blind, parallelgroup, -month extension of a -week double-blind study of patients years with severe persistent asthma. patients were randomized in a : ratio (cic:bdp) to receive cic ( g bid) or bdp ( g bid) (both ex-actuator). after weeks, the doses of both medications could be titrated to g bid as needed for asthma control.) oropharyngeal adverse events were monitored, and suspected oral fungal infections were verified by positive culture. results: the incidence of oral candidiasis was lower in subjects receiving cic ( . %), compared with those receiving bdp ( . %) ( table) . the incidence of pharyngitis and hoarseness was . % and . %, respectively, for the cic group, and . % and . %, respectively, for the bdp group. all local adverse events resolved without sequelae, and there were no withdrawals due to oropharyngeal treatment-emergent adverse events. conclusion: after -months of treatment with cic μg or μg twice-daily, the incidence of oropharyngeal adverse events was low. cic treatment resulted in a much lower incidence of oral candidiasis, compared with similar doses of bdp, reflecting a better local tolerance. objective: to determine the prevalence of asthma and asthma-related morbidity, treatment and asthma-risk factors in a selected population. method: a routine health screening, including tests for asthma, was conducted at the new orleans center for creative arts high school. participants ( ) identified their medications and asthma tools in addition to completing the isaac questionnaire. prevalence data included lifetime wheezing, month wheezing, and previous asthma diagnosis. asthma-related morbidity included sleep disturbance, wheezing with exercise, asthma attack rate and night awakening. results: " participants were aged - years ( . % african american, . % white) with . % male, . % female. " cumulative asthma prevalence was . %, lifetime wheezing ( %), -month wheezing . % with girls reporting . times more often ( % ci: . , . ), and wheezing more than times in months . %. " there was a significant association between race and wheezing in the last months (p < . ) with whites reporting . times more often ( % ci: . , . ). asthma morbidity was reported as follows: a. night cough - . % b. wheezing with exercise - . % " parents with a less education (high school or less) were . times more likely ( % ci: . , . ) to have children who developed wheezing in the past months. " higher body mass index (bmi = ) was associated with wheezing after exercise (p < . ) with a . -fold increase ( % ci: . , . ). " among those with current asthma ( / ), were not on any medication, were using bronchodilators, and were on anti-inflammatory medications. three reported using a spacer and reported using a peak flow meter. conclusion: asthma prevalence is higher in this school population than the national average. less parental education, female gender, and bmi = were associated with greater asthma morbidity. the majority of the participants with current asthma reported receiving episodic and inadequate treatment with inhaled corticosteroids. objectives: to determine the prevalence of asthma and asthma related symptoms; to assess its severity among new orleans school children. methods: seven elementary, middle, and high schools in new orleans participated in the screening of students, % female, % male, ages - in the fall, . the -item questionnaire was designed by the international study of asthma and allergies in childhood (isaac) to determine asthma prevalence and severity in children. the isaac questionnaire is a validated protocol used on over , children in countries. asthma is defined as cur-rent wheezing for the purposes of this study. data was entered using spss and sas software for analysis. results: table: presence of asthma symptoms by ethnicity conclusion: asthma prevalence is significantly higher in inner-city school children in new orleans when compared to the national prevalence ( . % to . % vs. . %). asthma prevalence and severity do not differ significantly between african-american and caucasian children in new orleans even after removing the effect of gender and age. video is an effective method of teaching pollen identification. projected digital images offer three-dimensional views of unique pollen detail similar to focusing a microscope. the national allergy bureau™(nab) currently provides pollen counts to the media. these reported levels are obtained by standardized counting methods rather than forecasts based on historical pollination predictions and weather patterns. the american college of allergy, asthma and immunology and the american academy of allergy, asthma and immunology have an interest in ensuring consistent counting and accurate reporting practices. both organizations offer pollen identification training in conjunction with their annual meetings and have used the video for initial training sessions and for experienced counters in advanced courses. there are certified pollen counting stations in the united states and canada with approximately currently active locations. the nab requires the demonstration of ability to count and accurately identify pollen on test slides. an updated recertification process using an interactive web site is currently being developed. ongoing pollen identification training is essential for the continued success of this aeroallergen network. most observers found that the video provided a quality emulation of microscopic viewing and enhanced the depth and detail of individual pollen characteristics. the video received the highest possible ratings by course attendees. in conclusion the video plays a positive role in pollen identification training and the continuing education of experienced counters. the diagnosis and treatment of mold allergies are complicated by the genetic diversity of individual fungal species and the influence of endogenous fungal proteases on extract compositions and potencies. studies examining the biochemical comparability of mold extracts from different sources and their compatibility with other allergens in immunotherapy mixtures are essential to the clinical effectiveness of these products. compositional comparisons of extracts prepared from alternaria, aspergillus and penicillium source materials by u.s. allergen manufacturers revealed variable sds-page protein banding patterns and noticeable differences in total protein and carbohydrate concentrations. alt a levels in alternaria extracts did not correlate closely with total protein levels or with ige-binding potencies measured by elisa inhibition. immunoblot profiles of fungal extracts were more closely related to one another compared to sds-page patterns, suggesting that similar allergenic or antigenic epitopes may be retained in molecules of varying size. parallel elisa inhibition dose-response curves provided statistical evidence that a repertoire of ige epitopes is conserved in many of these products. variations in the potencies of fungal extracts from different manufacturers may result from differences in source materials and/or conditions of extraction and storage. the stability and compatibility of allergen mixtures containing mold extracts were assessed after storage for up to months at - °c using specific immunoblot and elisa procedures. grass and mite allergens compromised by mixing with mold extracts were stabilized by glycerin. alternaria extracts from different sources produced similar effects on grass allergens when added at comparable strengths. degradative effects caused by aspergillus or penicillium products were more closely related to total fungal protein concentrations than to extraction ratios. structural epitopes on cat, ragweed and fungal allergens were stable after mixing with protease-containing mold extracts, indicating the presence of natural protease inhibitors or allergenic protein sequences distinct from those recognized by these enzymes. allergenic extracts labeled as weight/volume or by pnu have no regulatory requirement for determination of activity. alum-adsorbed allergenic extracts are labeled in pnu and are felt to be depot formulations. the purpose of this study was to develop methods to measure important allergen proteins and specific ige binding capability of these alum-adsorbed allergenic extracts and to begin to assess the potency of these non-standardized extracts. allergen proteins are adsorbed to alum particles making their measurement difficult. in this study allergens were released from center-al ® alum precipitated allergenic extracts using . m citrate buffer, ph . the major allergen contents of several lots of each product were measured using monoclonal antibody elisas for group grasses, cyn d bermuda, bet v birch, ole e olive, and pla l english plantain. these assays were developed and validated by alk-abello and optimized for the citrate releasing buffer. direct binding ige was performed by immobilizing serial dilutions of extract to microtiter plates and then adding specific atopic sera and enzyme labeled anti-ige. the methods used were able to measure in vitro allergen activity in the alum-adsorbed extracts. major allergen content was successfully measured in the extracts and the amount was related to the pnu content. as expected, the variability of the major allergens within a particular extract species was higher than in standardized extracts that are adjusted for potency. the extracts demonstrated specific ige binding ability in the binding assay. the ige binding capability at various dilutions was related to the major allergen content. in conclusion, methods were developed to release adsorbed allergen proteins from alum extracts. the extracts were then analyzed for major allergen pro-teins and the ability to bind ige. major allergen content is currently used to monitor the activity of aqueous and glycerinated extracts and this study demonstrates that implementing these testing procedures will improve the consistency and quality of alum-adsorbed extracts. introduction there are over species of smut and rust fungi. ustilago maydis, or corn smut, is a basidiomycete fungus that infects ears of corn. it is responsible for a percentage of grain loss in the united states but eaten as a delicacy in mexico. it grows into large tumor-like structures, called galls, dispersing soot-like spores responsible for the common name, "smut." it is readily airborne and believed to be a causal agent for allergic rhinoconjunctivitis. "corn smuts" was added to the standard screening panel for patients presenting for evaluation of rhinitis in the region of middle tennessee. methods using greer laboratories™ corn smut extract : w/v, patients were tested by prick/puncture method with the hollister-stier quintip™ device. if negative, intradermal testing was performed. positive and negative controls were assessed. based on current national recommendations, wheals mm greater than negative control were considered positive for prick/puncture testing and wheals mm greater than negative control were considered positive for intradermal testing. results patients were tested between november, and march, . / ( . %) met criteria for prick/puncture positivity. / ( . %) met criteria for intradermal positivity. patients prick positive to corn smut had the following prick positive tests: dust mites / ( . %); cat / ( . %); local grass mix / ( . %); local tree mix / ( . %); local weed mix / ( . %), mold mix / ( . %); cockroach / ( %); and dog / ( . %). discussion allergy testing for aeroallergens is available to a limited number of antigens, of which, only a few are standardized. as time passes, relevant antigens are discovered and their importance further elucidated. this assessment reveals a significant presence of corn smut ige largely accounted for by intradermal positivity which may or may not reflect clinical sensitivity. these numbers are provided so that other clinicians may compare to prevalence in their geographical area. further studies are needed to determine the association of corn smut ige with clinical symptoms. h antihistamines are the mainstay of therapy for allergic disorders, including skin diseases. the most common side-effect of marketed antihistamines is sedation, especially when the clinical symptoms require a higher dose than recommended, a condition commonly encountered in dermatological disorders. the present study describes the pharmacology of a new non-sedating h antihistamine, r (hivenyl™). r binds to the cloned human h receptor with a similar affinity (ki: nm) as the reference antihistamines cetirizine (ki: nm) and loratadine (ki: nm). in vivo, r protects rats and guinea pigs from lethal shock, induced by compound / and histamine, respectively (ed : . and . mg/kg respectively). the compound is at least as effective as cetirizine and loratadine. in rats, r inhibits the histamine-and allergen-induced cutaneous reactions (ed : . and . mg/kg) with a similar potency as cetirizine (ed : . and . mg/kg) and loratadine (ed : . and . mg/kg). even so, in guinea pigs the compound inhibits the histamine-and allergen-induced skin reactions (ed : . and . mg/kg) to a similar extent as loratadine and cetirizine. however, in dogs r is more potent in inhibiting the ascaris allergen-induced wheal reaction (ed : . mg/kg) than cetirizine (ed : . mg/kg) or loratadine (ed : . mg/kg). r fails to occupy central h receptors in the guinea pig cerebellum up to mg/kg, in contrast to loratadine (ed : . mg/kg). in vitro and in vivo cardiovascular safety experiments indicate that r lacks the intrinsic capacity to prolong the qt-interval, even at high doses. as such, r (hivenyl™) is characterized as a potent, non-sedating and cardiosafe h antihistamine. it has been selected for further clinical development, mainly in the field of dermatology. the compound will be a suitable tool to explore the activity of a selective h antihistamine in various indications, without the contamination of the sedative activity often observed with other marketed antihistamines when increasing the dose. ciu represents one of the most clinically perplexing disorders which the allergist-immunologist is faced with. we have previously reported the clinical efficacy of fxt (prozac), an ssri widely used for the treatment of depression, in the management of ciu (nsouli tm, et al. ann allergy asthma immunol ; : ) . in this presentation we report additional cases of ciu which responded dramatically following the use of fxt. a yr-old female and a yr-old male presented with ciu of and months duration, respectively, requiring oral corticosteroid (cs) therapy for control of their ciu after failure of high dose anti-h (hydroxyzine) and anti-h (ranitidine) agents (table) . testing for food and latex allergy, viral hepatitis, autoimmune thyroid disease and parasitic infection were all negative. a skin biopsy performed in subject # was consistent with an urticarial vasculitis. following one week of oral fxt ( mg qd [subject # ] mg qd [subject # ]) a striking and complete resolution of urticaria in each patient was observed within to days during which time it was possible to successfully taper the cs. both patients have been maintained on fxt therapy with complete control of their ciu. the very favorable response to fxt therapy in these patients in addition to our first case report suggests that this drug may have a new therapeutic application in the management of recalcitrant ciu. background: ipratropium bromide nasal spray (ib) is indicated for treatment of rhinorrhea caused by common cold (cc), seasonal and perennial allergic rhinitis (ar) in adults and children age and up. symptoms of rhinorrhea from cc or ar in children are similar to those in adults, yet there is little data on the use of ib in children under years of age. objective: evaluate the safety and efficacy of ib nasal spray . % in - year-old children with symptoms of rhinorrhea from cc or ar. methods: a total of children ( cc and ar) were treated in an open-label, multi-center study. the cc patients received ib nasal spray ( mcg per nostril) tid for days, the ar patients received ib nasal spray ( mcg per nostril) tid for days. effectiveness was measured using a global assessment questionnaire and daily symptom scores reported by the parent. results: from the global assessment questionnaire, % and % of the parents found ib either "very useful" or "somewhat useful" in the cc and ar groups respectively. regarding effectiveness, % (cc) and % (ar) of the parents reported that ib had either a "good effect" or "excellent effect" in treating rhinorrhea. moreover, % (cc) and % (ar) of parents found administration of a nasal spray either "extremely easy" or "very easy." eighty-one percent (cc) and % (ar) of the parents reported they would use ib again for their child's allergy symptoms. the daily symptom score ( = none to = unbearable) for rhinorrhea decreased from . to . (- %, p< . ) for cc and from . to . (- %, p< . ) from baseline compared to the average on-treatment score, with decreases also seen for stuffy nose and sneezing. the nasal spray was well tolerated with adverse events (ae) reported in % of cc and % of ar patients. the aes were mostly mild to moderate and no potentially systemic anticholinergic or serious aes were reported. conclusions: ib nasal spray . % administered at a dose of either or mcg per nostril tid is an easy to use, safe, and effective therapy for control of rhinorrhea in children to years of age with common cold or allergic rhinitis. chronic idiopathic urticaria (ciu) represents one of the most clinically perplexing disorders which the allergist-immunologist is faced with. the pathogenesis of the condition derives from the release of potent vasoactive substances including histamine, and products of arachidonic acid metabolism, e.g., prostaglandins and thromboxanes formed by the action of the enzymes cyclooxygenase (cox) of which cox- is responsible for pseudoallergic and other inflammatory responses. although a number of therapeutic options exist owing to the plethora of mediators produced, treatment has focused largely on the use of h antihistamines and, unsurprisingly, at times without complete resolution of symptoms. in this presentation we report the successful use of a cox- inhibitor for the treatment of chronic urticaria. a y/o white hispanic female presented with a month history of chronic urticaria predominantly on the soles of the feet, with unknown precipitating factors, and fail-ure to respond to prior treatment with: cetirizine, steroids, benadryl, ebastine and epinastine. the patient received a blood transfusion years ago and there was no prior history of allergies. skin testing revealed + reactions to dust mite, pollen, cockroach, shellfish, + reactions to corn, milk and ige = iu (nv < iu), aso = ui (nv < ui). after weeks of treatment with rofecoxib (vioxx) mg, and benadryl, the rash resolved. vioxx was continued for more weeks until complete resolution of symptomatology. this case illustrates that the addition of a selective cox- inhibitor (i.e., rofecoxib) with an h antihistamine may be a more effective regimen for patients with ciu who fail to respond adequately to conventional therapy. introduction: elderly asthmatic patients whose symptoms are controlled by inhaled corticosteroid (ics) therapy may still have breathlessness on exertion. we randomized elderly asthmatic patients stabilized by medium-dose ics therapy into two groups, treated one group with medium-dose ics therapy plus montelukast, a leukotriene receptor antagonist, and the other with increased-dose ics therapy, and compared the effects of the treatment regimens on exercise tolerance. methods:the subjects were patients with bronchial asthma ( males and females, . ± . years) stabilized by ics therapy (with fluticasone proprionate, fp; mg/day) for three months or more with low peak expiratory flow (pef) rates (< %predicted, variability< %). they were randomly divided into two groups ( patients each) to be treated with ics therapy plus montelukast ( mg/day fp + mg/day montelukast; m group) or with increased-dose ics therapy ( mg/day fp; f group). pulmonary function tests, a six-minute walking test, respiratory gas analysis during incremental ( w/min) cycle ergometer exercise were conducted, and exhaled nitric oxide (no) levels were measured before and after two weeks of the study treatment. results: pulmonary function tests showed significant increases in maximal mid-expiratory flow (mmf) and forced expiratory flow at % vital capacity (v ) in the m group (p< . ) but no significant changes in the f group. exhaled no levels decreased significantly in both groups ( . ± . to . ± . ppb in the m group and . ± . to . ± . ppb in the f group; p< . ). the six-minute walking distance extended from ± to ± m in the m group and from ± to ± m in the f group.the peak oxygen uptake (peakvo ) increased significantly in the m group (%peakvo /w from . ± . to . ± . %; p< . ) but not in the f group (%peak vo /w from . ± . to . ± . %). the peak exercise load also increased significantly in the m group ( . ± . to . ± . w; p< . ) but not in the f group ( . ± . to . ± . w). conclusions: the results indicate that concomitant administration of montelukast is more effective than dose escalation of ics on exercise tolerance in elderly asthmatic patients under medium-dose ics therapy. e. meltzer * , y. luo , l. shen , z. guo , c. schemm , y. huang , k. chen , p. king , r. nave , d. banerji , s. rohatagi , . san diego, ca; . bridgewater, nj; . konstanz, germany. introduction: inhaled corticosteroids (ics) are first-line treatment for persistent asthma. ciclesonide (cic) is a novel and effective ics under development. freely circulating, unbound ics is available to cause systemic adverse effects, such as hypothalamic-pituitary-adrenal (hpa) axis suppression. hence, it is important to determine the free fraction of ics in plasma. in separate studies, the protein binding of the active metabolite of cic, desisobutyryl-ciclesonide (des-cic), was evaluated, and the effects of inhaled cic on hpa axis function were determined. methods: human plasma protein bind-ing of des-cic ( . - ng/ml) was determined using equilibrium dialysis. dialyzed samples were analyzed by liquid chromatography with tandem mass spectroscopy to determine free and bound des-cic. in separate clinical studies, the effects of cic (hfa; - μg daily) and placebo (pbo), via metered-dose inhaler (ex-actuator doses), over days, or weeks, on basal hpa axis function ( -hour area-under-the-curve [auc - h] serum or urinary cortisol corrected for creatinine) or stimulated (low-dose [ μg] cosyntropin) serum cortisol were investigated in patients ( years) with persistent asthma. results: the mean % of human plasma protein binding for des-cic was %. in studies measuring serum cortisol auc - h, there was no difference between pbo and cic ( - μg). similar results were observed for -hr urinary cortisol corrected for creatinine. in the rd study measuring low-dose ( μg) cosyntropin-stimulated peak serum cortisol, after weeks of treatment, there was no significant difference in the mean change from baseline versus placebo for either cic (p= . ) or cic (p= . ). conclusions: the favorable pharmacokinetic profile of cic, in particular the high protein binding of des-cic, may explain the lack of hpa-axis suppression. this appears to result in greater systemic safety. purpose: unscheduled office and ed visits for urgent asthma care are an ongoing point of concern. determining preventive variables regarding these unscheduled visits could have a significant impact on asthma costs and quality of life. objective(s): this research addresses the following question: when stratifying by race, gender, age, metropolitan/rural place of residence and comorbidity, do adults with asthma have fewer ed or unscheduled office visits for urgent asthma care if they: a) have an identified primary care provider, or b) have health insurance? method(s): univariate and stratified bivariate contingency table analyses were performed on weighted behavioral risk factor surveillance survey (brfss) data. result(s): adults with asthma who had an identified primary care provider were more likely to have no unscheduled office visits (or= . ) or ed visits (or= . ) for urgent asthma care. this was also true for adults with asthma who had health insurance (or= . for no unscheduled office visits and or= . for no ed visits). these relationships held when stratifying by race, gender and age. the relationships also held for metropolitan residents. the analysis was inconclusive for rural residency and the existence of co-morbidities. conclusion(s): despite race, gender, age and metropolitan residency, having a primary care provider or having health insurance impact whether or not adults with asthma are more likely to have unscheduled office or ed visits for urgent asthma care. further investigations are needed to examine how these factors impact adults with asthma who are rural residents or who have co-morbidities. d. bukstein * , g.a. cherayil , . madison, wi; . brookfield, wi. introduction: costs for plans to process prior authorizations for non-formulary medications, has been estimated to be $ to $ per request. costs for physicians to process these requests has not been extensively studied. methods: dr.bukstein, board certified allergist, developed a data collection tool. the form was utilized by physicians and nurses to document time spent on processing prior authorizations. data collected included, class of medicines requiring the pa, nursing time spent on calling the patient, pharmacist, health plan, nursing time spent completing forms, nursing time clarifying the information for the pa, as well as physician time spent completing pa activities. results: data was collected over weeks in and requests were processed.the class of medicines most often processed was antihistamines, comprising % of requests. nursing calls were tracked and calls to and from patients were the most common call documented. they averaged . +/- . calls per day per nurse. the nurses spent an average of minutes per patient call. calls to health plans averaged . +/- . calls per nurse per day and time spent on these call was . +/- . minutes per call. physician calls documented included calls averaging . +/- . calls per physician per day. these calls averaged . +/- . minutes per call.often the results of the prior authorizations were not known on the day of the request. originally . % of requests were granted the same day. retrospective review revealed . % were approved the first time they were processed. salary and benefits were calculated for nurses and physicians. the hourly rate was defined as $ . per hour for nurses and $ per hour for physicians. the costs for the time spent on the prior authorizations were calculated. during the week study period, over hours was spent by nurses on calls and over hours was spent by physicians on calls during the same time period. the total nursing and physician cost in this specialty practice was $ . per prior authorization. conclusion: there are substantial costs with processing of prior authorization requests for non-formulary drugs on the physician office side of managed care as well as on the insurance side of the process. specialty physicians should have a different process for obtaining non-formulary medications since almost % of their requests are granted. introduction: diabetes mellitus can adversely impact the course and outcome of myocardial infarction (mi). one of the mechanisms underlying this phenomenon is alteration of the course of inflammation and the reparative process following myocardial necrosis. abnormal wound healing, tissue reparation and immune responses in diabetic patients have been intensively studied, but the cellular and the molecular mechanisms are still unclear. transforming growth factor-beta (tgf-beta) is a multifunctional cytokine which plays a critical role in coordination of the course of inflammation and reparation, acting as a potent depressor of inflammation and a stimulator of regeneration. this study investigates the dynamics of serum concentrations of the active form of tgf-beta during the period up to the th day after the onset of a mi in diabetic and non-diabetic patients. results: in non-diabetics a significant increase was observed in tgf-beta on day ( -fold greater than in controls; . ± . and . ± . pg/ml respectively) with further increases reaching a peak on day ( . ± . pg/ml). on day tgf-beta decreased to levels less than on day , but was still greater than in healthy controls ( . ± . pg/ml). in diabetics, concentrations of tgf-beta on days ( . ± . pg/ml) and ( . ± . pg/ml) after mi were similar to diabetics without mi ( . ± . pg/ml). only on day was tgf-beta increased to levels ( . ± . pg/ml) which were fold greater than in diabetics without an mi. thus, in diabetic patients serum concentrations of the active form of tgf-beta are much greater than in non-diabetics. mi in patients with diabetes mellitus is associated with a reduced and significantly delayed increase in tgf-beta . conclusion: tgf-beta deficiency may be a factor associated with low activity of tissue reparation after mi in diabetic patients. introduction helicobacter pylori (hp) is the most common gastrointestinal infection worldwide, but only - % of those infected develop chronic gastritis or peptic ulcer disease. the pathogenesis of ulceration, mechanisms of hp lifelong persistence in gastric mucosa and local immune disturbances induced by this infection are well known, but the mechanisms of resistance and elimination of this infection have not been extensively studied. most study is based only on phenomenological findings, such as absence or low hp colonization in subjects spontaneously producing high levels of il- . the aim of this investigation was the analysis of the efficacy of the recombinant interleukin (ril- ) roncoleukin (biotech, russia) in treatment of hp-associated gastric ulcer disease. methods patients were randomly divided into two groups. the first group of patients was treated with standard therapy including of two antibiotics (claritromycin and amoxicillin), proton pomp inhibitors and h receptor antagonists. patients of the second group were treated with the same therapy, but instead of antibiotics they received . mg roncoleukin into four to six areas submucously using a gastroscopic method and . mg roncoleukin dissolved in ml of . % nacl with ml % human albumin infused intravenously. this procedure was performed three times at an interval of hours. results immunological findings demonstrated that roncoleukin results in an increase of cd +, hla-dr+ and cd +cd + cell levels. there was an increase in the serum concentrations of il ( fold), il ( fold) and ifn (more than fold) while the level of tnf and il profoundly decreased. one month after the end of treatment, the group treated with ril- had hp eradication achieved in . % in comparison to . % of the control patients. in the ril- treated group, the ulcer epithelization period was . ± . days while in the normal treatment control group it was . ± . days (p< . ). conclusion immunotherapy with ril- is a more effective method of treatment of helicobacter pylori-associated gastric ulcer disease when compared with traditional methods of treatment employing only antibiotics. introduction the role of different cytokines and the growth factors is now appreciated in progression of essential hypertension. the participation of et- and tgf in pathogenesis of essential hypertension especially in the process of fibrosis, hypertrophia of vascular smooth muscular fibers, and cardiomyocytes, and the activation of renin-angiotensin system is now recognized, in addition to effects on myocyte cultures. the aim of the investigation was the study of serum et- and tgf levels in patients with essential hypertension. materials and methods patients with essential hypertension ( males and females) with average age . ± . years were studied. all patients suffered from left ventricle hypertrophy documented by echocardiography. the control group consisted of healthy volunteers similar to the investigated patients in sex and age. in all patients the serum concentrations of et- and tgf were determined using elisa (biomedica, biosource). results an increase in the serum concentrations both growth factors were noted in the studied group when compared with the control group. the average concentration of et- in patients with essential hypertension was . ( . - . ) pmol/l. the level of et- in the control group was . ( . - . ) pmol/l (p= . ). the concentration tgf in essential hypertension patients was . ( . - . ) pg/ml and in control group was . ( . - . ) pg/ml (p= . ). there was a positive correlation between the concentrations (spearmen's rank coefficient of correlation was . ; p= . ). con-clusion the increase of the serum concentration of growth factors et- and tgf and their co-influence in patients with essential hypertension, suggests a role of these growth factors in the pathogenesis of arterial hypertension. u. kaza * , c. lauter , . bloomfield hills, mi; . royal oak, mi. introduction: few studies have examined the referral patterns for allergy and immunology inpatient consultations in a community hospital. consequently, an invaluable part of physician and housestaff education is missing. our objective was to examine the number of inpatient allergy and immunology consultations, the reasons for consultations and the outcome of the patients in order to improve physician education. methods:we performed a retrospective chart review of all inpatient allergy and immunology consultations in the years - and in - to determine the reasons for consultation, the recommendations made and if they were followed and the outcomes of the patient. results:we reviewed a total of inpatient allergy and immunology consultations. in the - time period % of inpatient consults were for asthma, % for drug allergy, % for rash. in the - time period % of inpatient consults were for rash, % for drug allergy, % for asthma. the top three reasons for consultations remained the same although the order changed. consultations for immune deficiency, angioedema and rashes increased, whereas consultations for asthma and allergies decreased. there were a total of consultations in - and in - . the number of consultations remained the same despite an increase in overall number of hospital admissions from , in to , in . in greater than % of consultations, allergists' recommendations were followed. in both of the time periods studied, greater than % of patients improved, with less than % having no improvement, in the remainder of cases improvement was not applicable. conclusion:in conclusion, we believe that identifying the reasons for inpatient allergy and immunology consultations and examining the most common recommendations, as well as the outcomes of patients will be a valuable guide in the education of our physicians. by incorporating this information into grand rounds and resident conferences, physicians will benefit from learning about when an allergist can be helpful and how to manage some of the more common allergic and immunologic problems in patients that are hospitalized. the high percentage of providers who follow the advice of allergists indicate that allergists have a great deal of educational value to offer other physicians. while complementary and alternative medicine (cam) has generally experienced increased popularity, its utilization by allergy/asthma patients remains uncertain. our private allergy practice surveyed the use of cam in allergy/asthma patients in ( ). using a similar questionnaire, we assessed the current interest in cam with our allergy/asthma patients and compared the data to our survey. we analyzed completed questionnaires from sequential surveys administered. the results were compared to questionnaires reported in . they indicated that in both study periods ( & ) , the majority of patients wanted to discuss cam ( and % respectively). an equal number ( %) in each study period discussed cam with their primary care provider. sixteen percent ( %) of our respondents sought a cam practitioner for general medical needs in vs. % in . however, there was an increase from % to % of our patients seeing a cam practitioner for their allergies and/or asthma from to . sixty-two percent ( %) would like to consider pursuing cam through our allergy spe-cialty practice or other provider. when asked regarding preferred treatment, % stated combination traditional with cam, % preferred traditional only, % cam only, and % did not know/doctor s choice. more patients are now seeking chiropractic care ( % to %) compared to our results. acupuncture was the first choice cam modality at % in surpassing vitamin/mineral therapy in . currently, the second and third choices were vitamin/mineral therapy and deep tissue massage. while these numbers were small, we were impressed that currently % (two and a half times more since ) of patients in our practice were seeking cam allergy/asthma care from outside of our practice. these results demonstrated that more than half of our patients were interested in pursuing traditional with cam options within our office. given this trend, we have begun discussing the concept of integrative allergy, which to us means integrating evidenced-based cam modalities within our traditional allergy/asthma practice. ( ) introduction: clinical immunization knowledge is complex and demands ongoing training. nationally, basic immunization and vaccine safety education is limited within traditional medical, nursing, and provider education. project immune readiness (pir), a peer-reviewed, web-based, interactive course, was developed in response to this deficit and the need for standardized resources to provide initial and sustainment training for safe and effective immunization services. it is designed for medical personnel with diverse educational preparation. currently, pir offers course modules addressing hours of instruction on specific vaccines, their respective diseases, and general information on immunization healthcare and vaccination procedures. methods: users completed a pre-test (establishes baseline knowledge), an interactive module, followed by a post-test (to observe change from baseline) for each course in sequence. anonymous user and score data were collected as part of a quality assurance and course validation process. learning gain indices (lgi) were calculated based on average mean pre-test and post-test scores for each module lgi of all modules demonstrated substantial increases in user vaccination knowledge. comparing pre and post-testing is an effective method to assess learning gains. the findings support pir as a successful and valid distance-learning tool that establishes and documents core knowledge of medical personnel administering vaccinations. further research is needed to assess the effectiveness of knowledge acquisition and retention, in addition to variance in vaccine delivery after training. this approach to learning may have value as a resource that supports smallpox and influenza pandemic emergency preparedness plans for just in time training. introduction: variances from practice guidelines for the prescription of auto-injectable epinephrine are well documented among practicing physicians, families, and patients. effective patient education requires provider competency. the current study was designed to survey resident physician perceptions regarding auto-injectable epinephrine education, use, and patient education requirements. methods: residents from primary care disciplines at a tertiary care medical center were invited to complete a voluntary, anonymous questionnaire. a total of surveys were distributed and returned: emergency medicine, family practice, internal medicine, and pediatric residents completed the questionnaire. due to the small sample size, responses from physicians in various disciplines were reviewed as a whole. results: respondents ( %) reported that they had previously prescribed auto-injectable epinephrine for allergic emergencies. the majority ( %) of these prescribers reported that their training was inadequate. respondents ( %) reported no training, while respondents ( %) reported that their training was less than that needed to ensure proficiency. respondents ( %) reported that their training was adequate to ensure proficiency. none reported expertise. only of prescribers ( . %) reported that either they or their staff always demonstrated proper medication use to the patient. interestingly, of these providers reported they had not been trained on proper use. the table below summarizes resident training and patient education practices. additional physician knowledge deficits included the proper site of medication administration, the proper interval for replacing medication, and the proper place for medication storage. conclusions: of the residents surveyed, who have previously prescribed auto-injectable epinephrine, % identified training deficiencies. only . % of prescribers reported that the standard of care requirement to demonstrate proper medication use was always met. there is a clear need to improve auto-injectable epinephrine education in all residency training programs. r. bloebaum * , r.k. calabrese , m. . houston, tx; . new york, ny. introduction: pneumocystis carinii pneumonia (pcp) is a major cause of morbidity and mortality in patients with aids. adverse reactions occur frequently to the most effective medication for both the prevention and treatment of pcp, trimethoprim-sulfamethoxazole (tmp-smx). we looked at the immediate safety and efficacy of three protocols for desensitization in aids patients. methods: by retrospective chart review, we identified patients with aids who had experienced previous mild to moderate hypersensitivity reactions to tmp-smx and required desensitization. patients received one of three desensitization protocols based on illness severity or ward attending preference: a -hour intravenous (iv) desensitization, an -day oral desensitization, or a -day oral desensitization. results: of the patients that received desensitization, ( . %) completed successfully. of these, subjects had no reaction during the desensitization process; however, seven of these subjects were on steroids for treatment of other diseases. the remaining five successful patients had mild reactions which were treated symptomatically with acetaminophen, antihistamines or both. eleven patients failed to complete the desensitization: six stopped by the attending physician and four dropped out voluntarily. one patient expired during desensitization from extensive disease complications related to the admitting diagnosis. all protocols were equally successful when comparing the immediate success rates, / ( %) of the hour protocol, / ( %) of the -day protocol and / ( . %) of the day protocol. the -hour iv desensitization protocol was most frequently used in the intensive care unit on critically ill patients. two of these patients, counted as successfully desensitized, died and days post protocol completion secondary to extensive comorbid conditions unrelated to the desensitization. conclusion: given the insignificant differences between the success of the hour iv desensitization and the oral desensitization protocols, we believe that either may be used effectively. further, in patients with mild to moderate hypersensitivity reactions to tmp-smx, an oral desensitization protocol may be used safely in the outpatient setting if given appropriate lab follow up. a. morales * , e. gonzalez, a. contreras, d. lopez, g. lopez, mexico city, mexico. introduction in davis describes job syndrome in two women, in dr buckley reports two children, being known as hyper-ige syndrome (job's syndrome or buckley's syndrome). defined as a primary immunodeficiency, dominant autosomic, characterized by multi-systematic alterations (immunological, skeletal, dermal and dental). it's diagnostic criteria are levels of ige ui/ml, chronic dermatitis, recurring respiratory infections, cold abscesses, pneumatoceles, infections caused by candida, and finally craniofacial alterations. on another front, extraordinary high levels of ige have been reported in patients with allergic illnesses that increase the risk of anaphylaxis, but do not have the job's syndrome criteria. objetive to determine if allergic patients with ige levels higher than ui/ml have diagnostic of job's syndrome. material and methods a retrospective revision was realized from may to april in files of patients treated in allergy services at the instituto nacional de pediatria with a total ige greater than ui/ml, by means of a page with recollected dates. results nine women and men were included with an age range of to years, and an average age to years; ( . %) were diagnosed with allergies; of which . % rhinitis allergy, . % asthma, and . % topical dermatitis of which co-existed in patients. . % presented hereditary antecedents of atopic. the cutaneous tests were positive in ( . %) with a greater reactivity of dermatophagoids. one syndrome of hyper ige was detected. others diagnosed were found as not allergic were hunter's syndrome and toxocariasis. conclusion there are patients with allergies that have total levels of ige as elevated as , ui/ml without correspond to job's syndrome. by which a multi-allergic clinical entity is proposed with levels greater than ui/ml. introduction home monitoring of lung function in asthmatic patients is used extensively in both clinical and research settings, however, little attention is given to device quality control. objective the purpose of this study was to determine the accuracy, precision and usefulness of the airwatch system (enact health management systems, palo alto, ca, usa). methods the subjects included in this study were submitted to spirometry following american thoracic society guidelines (ats ), using collins gs g pft system. afterwards, peak expiratory flow rate (pef) and forced expiratory volume in one second (fev ) were determined using airwatch. fev and pef measures from both devices were compared, by using two sample t-test and pearson correlation coefficient. results a total of patients ( females) were enrolled, and their mean age was . years ( to years). fev measures ranged from . to . l (mean . l), and from . to . l (mean . l), in collins and airwatch, respectively. pef ranged from to l/min (mean l/min) in collins and from to l/min in airwatch (mean l/min). significant difference was noted for pef measures (p< . and pearson correlation r= . ) between the two devices; however, we did not observe this difference when fev measures were concerned. regarless the degree of obstruction (high or low flow rates), these results did not change. conclusion airwatch showed great utility for fev measures when compared to collins spirometer. although airwatch is able to assess lung function at home, on a daily basis, it is not reliable for pef measures. introduction: epidemiologic data show that poorly controlled asthma is a serious public health problem. the degree of implementation of the naepp guidelines in primary care practice remains to be defined. the objective of this survey was to determine if introduction of an assessment tool into primary care practices along with a specially designed program to implement the guidelines would improve diagnosis and therapy. methods: the asthma care network (acn), a program designed to assist healthcare providers in the assessment and management of their patients with asthma, employs a team of specially trained respiratory care associates (rcas), rns and rts, who visit primary care offices to inform staff about various components of the naepp guidelines and assist in their implementation. .a total of primary care providers in sites were recruited as part of the acn program. data from more than , patient visits were collected and analyzed between march and january . the program assessment tool surveyed asthma control and medication prescribing patterns. outcome measures included degree of symptom control, limitation of activity, sleep disruption, use of rescue medication and utilization of urgent care services. these data were collected on an office visit assessment form (ova) completed by both patient and physician. the rcas provided information, education, device training in the use of inhalers and spacers, and a ce course for the staff discussing pathophysiology, assessment and management of asthma. results: a total of , ova forms were completed. among all patient including adults (older than years of age) and children (< - years of age), % (range % to %) reported symptoms consistent with lack of asthma control. approximately % of the survey group had more than markers of uncontrolled asthma. as a result of this assessment, controller medication use increased by over %, % of which was an ics-containing medication. conclusion: the information provided to the primary care health care providers resulted in a considerable increase in prescription of controller therapy, and in particular, increased use of ics controller medication consistent with naepp guidelines. background: patients with allergic rhinitis (ar) demonstrate symptoms of allergy to fruits, vegetables and nuts in - % of cases. oral allergy syn-drome (oas), typical for hypersensitivity to plant food, is based on cross-reaction of pollen-allergen specific immunoglobulin e (ige) antibodies with homologous food proteins. the production of th and th cytokines in allergic rhinitis patients with or without sensitization to food (fruits and vegetables) allergens was assessed. methods: fifty five patients aged - years with allergic rhinitis were observed. group i - patients with ar; group ii - patients with ar and oas. sensitivity to pollen allergens was tested by skin prick tests. the allergic reaction to food in patients with oas was proved by a positive history of oral symptoms caused by eating fruits and vegetables and a positive skin prick test with respective food allergens. blood eosinophil counts and total ige levels were determined during the peak of allergic rhinitis symptoms. il- , il- and -interferon levels were measured by elisa. results: . % of patients were sensitized only to grass pollen, . % only to tree pollen and . % reacted to pollens of grasses, trees and weeds. in patients with oas, skin tests were more often positive to birch ( . %), alder ( . %), and mugwort ( . %). the most common food products implicated in oas were hazelnut ( . %), apple ( . %), carrot ( . %), and peanut ( . %). the allergy to fruits and vegetables was confirmed by positive prick test in . %. during the season blood eosinophil count and total ige levels were elevated in all patients. there was an increase in the production of il- to . ± . pcg/ml in group i and to . ± . pcg/ml in group ii (nor-mal= . ± . pcg/ml). the levels of il- increased to ± pcg/ml in group i and to ± pcg/ml in group ii (normal= . ± . pcg/ml); the level of -ifn decreased to . ± . pcg/ml in group i and to . ± . in group ii (normal= ± pcg/ml). after sit with pollen allergens the clinical manifestations of allergic rhinitis and oas decreased in . % of patients. conclusion: allergic rhinitis patients' sensitivity to food allergens may cause oas in these patients associated with increased functional activity of th responses. patient knowledge and improvement with aller-gen immunotherapy. c.c. randolph * , waterbury, ct. introduction: there is no consensus on objective parameters for improvement in immunotherapy. similarly little is known regarding patient knowledge of immunotherapy. utilizing two published questionnaires ( - ), we assessed clinical improvement based on symptom and medication scoring and knowledge of immunotherapy. methods: the charts of patients of an estimated ( %), ( %) with allergic rhinitis only and ( %) with concomitant asthma, were retrospectively or prospectively reviewed who had been on immunotherapy to inhalants for months to years, mean . years, age range y- y, mean y, male( %), female( %) with caucasian ( %), oriental ( %) and ( %) afroamericans .they completed symptom and medication survey ( ) every months with range of improvement using a decline in likert scale (+) to (-) , mean . ( %) indicated improvement in their symptoms and/or medication . ( %) completed ( ) a question survey of knowledge of immunotherapy. there were questions regarding the outcome of immunotherapy, the years to onset of immunity, the duration until onset of immunity, the danger of immunotherapy and the extract in the vial. the correct responses were recorded to / ( / = . %), ( . %) / , ( %) / , ( %) / , ( %) / . ( %) had a perfect response to all questions. ( %) had no correct responses. conclusion: the majority of immunotherapy patients improved ( %) by symptom and medication scoring over the mean of . years but only % had complete knowledge of the rationale for immunotherapy. further education and repeated surveying of patients on immunotherapy to assure comprehensive knowledge of immunotherapy and achieve better outcomes is recommended by this investigation. as is unique in this study symptom and medication scoring using the rhinitis /sinusitis questionnaire approved by the acaai and a comprehensive questionnaire assessing knowledge should be conducted periodically ie every months to provide objective parameters for improvement. references: .santilli j, nathan r, glassheim j .patient receiving immunotherapy report it is effective as assesses by rhinitis outcomes questionnaire(raq) in private ( )) is a protein involved in the parasite invasion of host erythrocytes and is a leading vaccine candidate for the erythrocyte stage of malaria infection. an increasing number of vaccine clinical trials are being undertaken using various formulations of msp- ( ). comparison of humoral responses among these trials has been limited by the lack of a universal reference standard for specific antibody. the purpose of this study was to develop a human reference standard for msp- ( ) antibody measured in absolute quantity units that could facilitate comparison of interstudy vaccine response. method: we formulated the reference standard by pooling human plasma samples known to contain high titers of msp- ( ) antibody. the specific antibody within the pooled plasma was captured by msp- ( ) adsorbed to nickel resin in a process of immobilized metal affinity chromatography (imac). the intact msp- ( ) antibody-antigen complexes were separated from the nickel resin and total igg in the complexes measured by enzyme-linked immunosorbent assay (elisa). results: our antibody quantitation method yielded a concentration of . mcg/ml of msp- ( ) antibody in the reference standard. conclusion: the reference standard characterized in this study may be useful as a quantitative working standard for msp- ( ) antibody response in future vaccine clinical trials involving msp- ( ). this standardization may facilitate the clinical development of msp- ( ) as a candidate vaccine for malaria infection. background. specific immunotherapy (sit) is currently one of the most effective and widely used treatment methods of allergic diseases including asthma. efficacy of sit depends on the correct choice of patients, severity of asthma and patient's condition when the sit is begun. according to who recommendations, sit is approved for use in patients with mild to moderate asthma. methods. sit with a saline extract of house dust allergen was administered to patients with mild persistent allergic asthma. injections were performed subcutaneously - per day. the course consisted of pnu of allergen and lasted - days. patients were repeatedly surveyed at , and months after sit was completed. dyspnea attacks occurring during daytime and at night were assessed, as was the influence of physical exertion on dyspnea and lung function, and also the number of utilizations of short-acting beta-agonists per day. pulmonary function measurements were performed as was assessment of concomitant allergic rhinitis. % of patients received treatment including cromones, and % utilized inhaled corticosteroids among whom . % had a dose of mcg per day and . % mcg per day. sit efficacy was assessed by clinical symptoms, pulmonary function measurements and amount of concomitant therapy received. results. the number of daytime dyspnea attacks reduced from . ± . per month to . ± . during the months following sit, and . % of patients reduced concomitant asthma therapy. at months, dyspnea attacks increased to . ± . per month, still significantly less then prior to sit. nocturnal symptoms followed the same pattern, occurring . ± . per month prior to sit, . ± . per month months after sit and . ± . per month at the end of the sixth month after sit. . % of patients had no symptoms of bronchial obstruction during the months after sit and their pulmonary function approached normal values, although . % of patients returned to asthma therapy. nasal congestion decreased from . ± . points to . ± . (p< . ), clinical improvement still present for months after sit, but recurring at months after sit. conclusion. sit may be an effective treatment method that improves both asthma and allergic rhinitis for more than months following a brief course, allowing a decrease in the amount of symptomatic treatment. a safe therapeutic vaccine that can alter the allergic response to peanuts would serve a serious unmet medical need. peanut allergy responses are largely associated with downstream events related to antigen specific ige crosslinking of ige receptors and subsequent degranulation of mast cells and basophils. our clinical studies with ragweed have shown that linking immunostimulatory dna (iss) to allergens can decrease ige recognition of the allergen and generate an immunogen that generates protective th responses and reduces harmful th responses. to test this approach to peanut immunotherapy, we focused on the clinically relevant allergen, ara h , as a proof of concept. iss oligonucleotides were linked to ara h at two different ratios: pic ( iss per protein) and hpic ( iss per protein). immunogenicity of pic and hpic was evaluated in c h/hej female mice immunized twice with ug of pic, hpic, or ara h . sera were analyzed for anti-ara h igg and igg a responses. spleens were harvested and ara h -specific ifng and il- responses were measured in vitro. mice were immunized with ara h elicited predominantly igg and il- responses, indicative of a th -type response. animals immunized with pic showed significantly enhanced igg a responses and strong ifng responses, indicative of th -type responses. hpic immunized mice elicited little antibody response, presumably due to iss blocking b cell epitopes, but did induce ifng responses. to assess allergenicity of pic and hpic, histamine release was measured in blood from peanut allergic donors treated in vitro with pic, hpic, and ara h . histamine release was detected at very low concentrations of ara h ( . ng/ml), -fold higher concentrations for pic ( . ng/ml) and was undetectable at concentrations up to fold higher for hpic ( ng/ml). an ige binding competition assay also confirmed reduction in allergenicity following the same trend for the iss-linked allergens. linking iss to ara h increases th responses to the allergen, blocking ige recognition of epitopes on ara h . thus, iss-linked ara h appears to be a promising candidate for a safe immunotherapy product for treating peanut allergic subjects. introduction: immunotherapy has been studied for its adverse effects in some sensitive patients as with worsening of therapeutic response in some amounting to withdrawal of the later. methods: in the allergy center therapeutic trials of immunotherapy(house dust, mixmite) had been undertaken since , in therapeutic dose of . pnu/ml, . pnu/ml, pnu/ml, pnu/ml, pnu/ml pnu/ml, pnu/ml(housedust), . au/ml, . au/ml, au/ml au/ml au/ml au/ml au/ml(mixmite).patients age - years, both sex after diagnostic scratch/prick tests with appropriate antigenic preparations reported for local/systemic adverse effects the details of which were as under, results: please see the attached table for details, in some cases worsening of the existing allergic status was noted, which on analysis revealed initially a slow rise in the allergen -specific ige to be later on fol-lowed by rise of igg & igg & gradual fall of ige.only / patient could not complete immunotherapy for fear of adverse effects. conclusions: with utmost care/follow-up, no incidence of mortality outcome was reported from to late the incidence of adverse effects were significantly lowered on pre-medication with anti-histamines significantly more with elderly than younger age. even with all these documented hazards the beneficial effects far exceeded the harmful effects. background: the prevalence of allergic disease in most human population is steadily increasing. seafood allergy is a serious food allergy, although hypersensitive reactions caused by seafood has long been know, biochemical and immunological studies on seafood allergies had only begun lately. shrimp and abalone are the most frequently reported causes early asthmatic response. objective: to investigate cross-reactivity of shrimp, abalone and derp . methods: shrimp and abalone extracts were prepare from raw seafood. sera from patients from hongkong were studied who had asthma after consumption of seafood. ige elisa analyses comfirmed the combined sensitization to shrimp, abalone and derp . specific-ige elisa assays were accomplished for shrimp and abalone extracts inhibited by derp and derp elisa and immunoblot assays inhibited by shrimp and abalone extracts. results: elisa inhibition showed that most ige antibodies against shrimp and abalone were cross-reactive with derp and the same time, derp elisa was inhibition by shrimp and abalone extract. the elisa inhibition percent (%)of shrimp extract (gm: . %) and abalone extract(gm: . %) by derp were significantly higher than derp by shrimp extract(gm: . %) and abalone extract (gm: . %). (p< . ). furthermore, and there was a significant correlation of elisa inhibition percent between shrimp extract, abalone extract and derp inhibited by each other; sds-page and immunoblot of shrimp and abalone is the and kd allergen respectively. conclusion: this indicates that derp was the sensitive agent. shrimp, abalone and derp demonstrate significant cross-reactivity. these findings confirm that the primary crossreactive allergen of shrimp and abalone is the and kd allergen respectively. b. sun * , a. wu , n. zhong , . guangzhou, china; . hong kong. background: the house dust mites (dermatophagoides farinae (derf) are a major source of aeroallergens implicated in the expression of atopic disorders, including asthma, allergic rhinitis, and atopic dermatitis . in particular, strong circumstantial evidence suggests that house dust mites antigens are important precipitating factors of asthma. many house duse mite allergens are proteases that can elicit airway inflammation by stimulating the release of cytokines in bronchial epithelial cells. objectives: we have investigated whether der f allergen proteases induced cytokine production from the epithelial cell line beas- b. methods: cells were exposed to four different concentrations with serial additions of der f ( . , . , , ug/ml) were incubated for h to h. and compare with those without incubation of allergen. cytokine in the supernatants were assayed by elisa, reverse transcription?pcr was also performed. results: cells treated with der f allergen showed serial changes in the cohesiveness of the monolayer. there was a significant increase in the level of cytokine production compared with the untreaed sample. statistically significantly increased with addition of der f caused the release of il- and il- in time and concentration-dependent manner (p< . , respectively). levels of il- and il- were elevated h and h after allergen exposure, increasing with time, continued increased levels to be present of il- and il- in the supernatants at h and h. at the same time show the concentration dependence of induction of il- and il- expression as well as an increase in the expression of il- and il- mrna. conclusion: hdm-induced airway inflammation may include der f-mediated release of inflammatory mediators, and the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium. suggesting that il- and il- production by bronchial epithelial cells may play a role in the pathogenesis of allergic asthma. the purpose of this study is to delineate the immune injury mechanisms that involved in the autoimmune inner ear disease by introducing plasmid dna encoding of th cytokines (inf-g) into the inner ear. b-tubulin is a microtubular protein which we found as an important autoantigenic in meniere's disease as well as other autoimmune hearing loss. hearing loss was induced in mice and guinea pigs when they are immunized with the tubulin molecules. autoimmune hearing loss could be the results of th cytokine responses from autoimmune injury. to test the hypothesis, guinea pigs were immunized with mg of tubulin in cfa and boosted once more. two weeks later, we introduced ug ( ul) of naked dna encoding inf-g was injected into the left side inner ear through round window. same volume of . m pbs was injected into right side as control. abr was recorded before and after the injection. weeks after the injection, the animals were sacrificed and temporal bones were examined with h-e and inf-g immunocytochemical staining. the ears injected with the plasmid dna-inf-g had an enhanced hearing loss ( db), and degeneration of the spiral ganglion was found in these ears. however, the injection of the naked dna encoding inf-g did not change the expression of the inf-g in the inner ears. these results suggest that autoimmune hearing loss could be the result of th responses to inner ear autoantigens. -tubulin is an important molecule in the hair cells supporting cells within the sensory epithelium of organ of corti and found to be an auto autoantigen in autoimmune hearing loss including mèniére's disease. the object of the study is to induce hearing loss in mice with varying doses of antigen and evaluate the pathogenesis of autoimmune hearing loss induced by -tubulin in mice. mice were immunized with , or μg of -tubulin and hearing was evaluated by auditory brainstem responses (abr) and distortion product of otoaccoustic emission (dpoae). all mice had hearing loss by abr and dpoae tests and morphological study of temporal bone showed spiral ganglion degeneration and tunel staining positive cells were noted in these immunized mice. thus this study showed that -tubulin is an autoantigen for hearing loss in animal model as in human autoimmune hearing loss patients including mèniére's disease. supported by nih r dc - introduction: oral polio vaccination (opv) in the united states is currently being replaced with inactivated polio vaccination (ipv) given parentrally. while past studies have looked into the relationship of vaccination and asthma prevalence, none have investigated this relationship with regards to vaccination route namely orally versus parentrally. this study investigates the relationship between vaccination rates for the live attenuated orally administered polio vaccine and parentrally administered vaccines(dtp, mmr)and two potentially dependent factors; asthma prevalence rate and asthma-caused death rate. methods: we looked at data from the national center for health statistics yearly publications of health, united states ( - and the morbidity and mortality weekly report surveillance summaries . two databases were compiled to cover the - age population in the united states since this is the primary period of childhood vaccination. one database contained information for asthma related deaths and vaccination rate (dtp, mmr, opv) covering the years - and the other database compiled information for self reported asthma prevalence and vaccination rates(dtp, measles, rubella, opv) covering the period - . a t-test was used for statistical analysis. results: statistically significant correlation was found between vaccination rates and asthma prevalence rates. data for dtp (p = . ), measles (p = . ), and rubella (p = . ) indicated a statistically significant positive correlation with asthma prevalence. the oral polio vaccine was the only one of the vaccines that failed to display a significant relationship with asthma prevalence rates (p = . ). statistical analysis proved that a correlation between vaccination rates of united states children ages - and asthma-caused deaths was insignificant (p > . ). conclusions: the opv, which is administered orally rather than parentrally, displayed no relationship with asthma prevalence. this could be due to the fact that live attenuated orally administrated polio vaccine may induce mucosal immunity, simulating a normal pathogen route of entry into the body. childhood vaccination had no relationship with asthma-caused death rates. a.s. alfrayh * , riyadh, saudi arabia. introduction: heredity plays a major role in asthma and other allergic diseases, mechanisms underlying the inheritance of these disorders are poorly understood. this study therefore analyzed the risk conferred by family history of asthma and atopy for having childhood asthma. methods : a total of children between - yrs selected randomly in three cities in saudi arabia ( hail, taif and gizan )in - . the questionnaire which is similar to the one used in the international study of allergy and asthma in childhood isaac. were self administerd under medical supervision.apart from the demogrphic details, the questionnaire included questions on symptoms and physician diagnosis of asthma, rhinitis, eczema and family history of these conditions. the family members were grouped as immediate family and relatives. asthma and atopy were defined as ever having had physician diagnosis of such conditions information was also available about exposure to cigarette somke at least one member was a smoker in the household and having pets. relative risk for developing asthma was estimated in terms of odds ratio by bivariate analyses using chi square test and p value was considered significant when less than . . results : history of asthma in the immediate family and relatives conferred a fold and fold risk for development of childhood asthma odd ratio ( or )= . , % confidence interval(ci)= . to . , p= . and or= . , %ci= . to . , p= . respectively. rhinitis in immediate family and the relatives was associated with folds increased risk for childhood asthma or = . , % ci = . to . , p= . respectively whereas history of eczema conferred over folds risk or= . , % ci = . to . , p= . for childhood asthma when present only in the immediate family. history of eczema in relatives was not associated with any risk. of the environmental factors, exposure to cigarette smoke conferred folds risk of developing childhood asthma or = . , % ci = . to . , p= . , whereas exposure to pets was not a significant risk foctor. conclusion : presence of asthma and atopy either in the biological parents or relatives constitute a significant risk for childhood asthma.paricularly in the presence of evironmental risk factors. the murine local lymph node assay (llna) has been developed as an alternative to guinea pig models for the assessment of the contact sensitization potential. however, there is a need to develop a non-radioisotopic endpoint for the llna, because of the radioisotopic method's requiring the use of special facilities. in this study, we investigated to evaluate the lymphocyte subpopulations in the lymph node cells following allergen and irritant treatment. female balb/c mice were treated by the topical application on the dorsum of both ears with sensitizers, , -dinitrochlorobenzene (dncb), toluene diisocyanate (tdi), and a-hexylcinnamaldehyde (hca), and an irritant, sodium lauryl sulfate (sls), once daily for three consecutive days. the lymph node (ln) cells were harvested h after the final treatment. phenotypic analysis of lymphocytes subsets was performed with a flow cytometry. the allergens dncb, tdi, and hca and an irritant, sls increased cell number compared to the vehicle. there was an increase in the percentage of b + cells in mice treated with dncb and tdi compared to the vehicle control, but not in those treated with sls. mice were treated with dncb, hca and tdi showed a preferential increase in the percentage of b +cd + cells compared with vehicle and irritant-treated mice. there was an increase in b +cd + cells of mice treated with dncb, tdi and hca, but no significant increases were observed in mice treated with sls. mice were treated with dncb, and tdi showed an increase in the percentage of b +cd + cells compared with vehicle and irritant-treated mice. these results suggest that analysis of b cell activation marker, cd on b cells may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice. m. frieri * , y.c. huang, east meadow, ny. introduction: nitric oxide (no) is an important biomarker for inflammation in airway epithelial cells and in exhaled breath of asthmatic subjects. we have previously demonstrated no production in antigen-stimulated human bronchial epithelial cells and an effect of omalizumab (monoclonal anti-ige antibody) on no production in those cells [leyko bt et al. j allergy clin immunol ; (suppl ) :a ]. in this study, we investigated the potential role of ige and its receptors on a cells by characterizing the effect of omalizumab on antigen, egf, and il- stimulation of a cells in a medium containing atopic serum. methods: a human alveolar epithelial cells were stimulated with iu/ml of il- , μg/ml of ragweed (ra), au of dust mite (dm), and ng/ml of egf, and exposed to either - m budesonide or . μg/ml omalizumab for or hours. no production was measured in duplicate by a highly sensitive elisa. results: omalizumab but not budesonide inhibited no production at hours in a cells stimulated with il- ( - μm, p<. )and il- +dm ( - μm, p=. ), but not with ra alone. however, at hours omalizumab and budesonide each significantly inhibited no production stimulated by il- ( - μm; - μm), il- +dm ( - μm; - μm), and egf ( - μm; - μm) (p<. ). conclusion: no production is a marker for inflammation. omalizumab demonstrated a significant anti-inflammatory effect in distal alveolar cells by inhibiting no production stimulated by antigen, il- , and egf in a medium containing atopic serum. as persistent inflammation in asthma may play a role in airway remodeling, treatment with omalizumab may have a beneficial effect on chronic airway inflammation in patients with asthma. oral tolerance trigger regulatory mechanisms able to down-modulate antigen-specific t and b cell response. to address the lasting effect of several regimens of oral tolerance to ovalbumin, in naive or antigen primed mice, b-cell function has been focused. furthermore, we analyze specific antibody response up to eight months of post-immunization, proliferative response, b (cd /cd ) expression on b cells and t cell ctla- involvement in oral tolerance. a/sn mice were immunized by intraperitoneal route with μg of ova/ , mg-alum and boost days after priming (dap). ova feeding was done with mg at different days before or after antigen priming. in others protocols, mice were fed twice, before and after priming with a total of mg of ova. all groups were boosted at or or or dap. the results showed that only mice fed at naive status and those fed twice before and after immunization demonstrated a long lasting of ige ab response inhibition up to months of immunization. these mice showed a marked inhibition of antigen-specific proliferative response that was restored with anti-cd mab in vitro stimulation. evaluation by flow cytometry of spleen cells cultured for hours upon ova stimulation, showed an important decrease of b . expres-sion on b cells of naive fed mice, which remained inhibited until months of immunization. after addition of anti-ctla- mab an enhancement of b . expression was detected on b cells of naive fed mice. ctla- molecules expression on cd + t cells of naive fed mice remained unchanged following ova stimulation while a peak of expression was detected at h of ova stimulation in control group. this finding reinforce the t cell anergic status of naive fed mice, due to the less cell division and consequently a low rate of cd +/cd high activated cells. the results showed that antigen feeding before immunization induce a long lasting anergy mediated by an impaired t-b cell cooperation and ab production due to the decrease of costimulatory molecules expression on b cells and negative signaling effects by ctla- expression. introduction: vascular endothelial growth factor (vegf), basic fibroblast growth factor (bfgf) and fibronectin (fn) can promote angiogenesis, a putative component of airway remodeling. (s)-albuterol can exacerbate airway hyperresponsiveness, bronchospasm and release pro-inflammatory cytokines from small airway and smooth muscle cells. our study evaluated the effects of (s)-albuterol ((s))-and (r)-albuterol ((r)) on secretion of these factors on normal human lung fibroblasts (nhlf) and myofibroblasts (myonhlf) in the presence or absence of tgf , il- , or allergens. methods: nhlf were stimulated to differentiate to myonhlf with pg/ml tgf . dose-dependent effects of (s) and (r) [ - to - m] were evaluated for secretion of vegf, bfgf and fn by nhlf and myonhlf with and without au/ml d. pteronyssinus (dp), pnu/ml ragweed (ra), u/ml il- , or pg/ml tgf into serum-free media (its) at o c with % co collected at hr and assayed by elisa. results: in nhlf the following was observed: vegf secretion was -fold higher with - m (r) relative to (s), p< . ; bfgf secretion was increased %- % by - m (s) relative to (r), p< . . a lower concentration of (s) ( - m) in the presence of either dp or il- caused a -fold increase in secretion of bfgf relative to (r), p= . . in the presence of myonhlf the following was observed: - and - m (s) caused a %- % increase in secretion of fn relative to (r). at - m (s), this effect was further increased with the addition of il- , p= . ). conclusion: in a dose-dependent manner, (s)-albuterol stimulated the release of bfgf and fn by nhlf and myonhlf, respectively. this was enhanced by dust mite and/or il- , potentially contributing to the matrix remodeling observed in chronic asthma. vegf over-expression can have a protective effect against chronic hypoxia and can recruit immune cells to the alveoli. increased vegf by (r)-albuterol could contribute to such an effect in vivo in asthmatics. bfgf, in bal and sputum of asthmatics, and fn which contributes to subepithelial fibrosis, can promote angiogenesis. increased bfgf and fn by (s)-albuterol could be detrimental over time by enhancing matrix deposition and remodeling in a subset of asthmatics. o. ozdemir * , c. moore , y. ravindranath , s. savasan , . detroit, mi; . new orleans, la. background: mast cells (mc) have been shown to demonstrate natural cytotoxicity against mouse fibrosarcoma cell line in culture when incubated for - h. this effect has been postulated to be mediated through soluble and/or membranous tnf-. more recently; fasl, mc chymase and serine protease granzyme h with its chymase activity were proposed as mediators of mast cell-mediated cytotoxicity. thus, both 'granule-exocytosis' through chymase, granzyme h and soluble tnf-and 'death receptors' through membrane-bound tnf-and fasl pathways appear to be operative in this process. aims: following our earlier observations on long-term liquid culture-grown human bone marrow mast cell cytotoxicity against human leukemia cells in - h co-incubation experiments, we investigated mast cell-mediated cytotoxicity against natural killer/lymphokine-activated killer cell-sensitive cells in short term ( h) cultures without any stimulation for the first time. methods: human bone marrow mononuclear cells were cultured in methyl cellulose supported with il- , il- and scf. mast cell colonies that developed in six weeks were transferred to liquid medium and maintained for weeks before experiments. cytotoxicity was investigated against k , raji and daudi cells at , and hours of co-incubation by our established flow cytometric cell-mediated cytotoxicity assay. results: after h co-incubation, % ( % early apoptotic and % late apoptotic or necrotic death) and % ( % early apoptotic and % late apoptotic or necrotic death) target cell kill was demonstrated in daudi and raji cells, respectively. daudi cell killing has stayed stable at h ( %; % early apoptotic and % late apoptotic). despite a small numbers of experiments, daudi cell kill was statistically significant at h (p: . ) and h (p: . ) compared to control. however, k cell elimination ( %) has not occurred until h. mast cell-daudi cell conjugates were seen on the wright/giemsa slides (figure) . conclusion: our results demonstrate that human mc can cause cell-mediated cytotoxicity against certain cells in relatively short-term. this further suggests possible contribution of 'granule-exocytosis' pathway to mc natural cytotoxicity, indicating a faster mc response in immune surveillance. o. ozdemir * , m. buyukavci , y. ravindranath , s. savasan , . detroit, mi; . erzurum, turkey. background: the effect of melatonin (mlt) on cellular immunity has been controversial. recently, mlt has been demonstrated to activate t and nk cells through its membrane or nuclear high affinity receptors. it was also shown that pharmacological concentrations of mlt (>nm) could be cytotoxic against different human cancers. aims: our aim was to investigate the effect of mlt alone or in combination with il- on peripheral blood lymphocytes (pbl), lymphokine activated killer (lak) cell generation and its cytotoxicity. methods: pbl were cultured for days in the presence of mlt at different concentrations ( - , - , - m) with or without il- ( u/ml). cell viability was determined by trypan blue exclusion test. cell-mediated cytotoxicity of lymphocytes/lak cells against k- and daudi cells was studied using our established flow cytometric cell-mediated cytotoxicity assay. results: although - m concentration of mlt did not affect cell proliferation much on day , it significantly inhibited proliferation by day (p< . ) consistent with known anti-proliferative effect of mlt. - and - m concentration of mlt also mildly inhibited proliferation on day ; however there was a minimal rebound with - m concentration by day . consistent with the reported mlt-treated pbl's reduced response to mitogens, il- and mlt ( - m) combination suppressed proliferation on days and ; however, with - m mlt concentration pbl counts increased gradually from day to . although mlt treatment alone did not enhance cell-mediated cytotoxicity, il- and mlt combinational treatment at both concentrations ( - and - m) increased it significantly compared to baseline activity (figure) . conclusion: il- and mlt combination at - m concentration resulted in superior lymphocyte proliferation and lak cell-mediated leukemia cell kill. mlt-induced increase in il- r-expression of pbl shown earlier might be the mechanism for our observations. mlt can be considered in immunotherapy as an adjunct to il- treatment. a.e. fusaro * , j.r. victor, c.r. oliveira, c.a. brito, e.a. futata, m. maciel, a.j. duarte, m.n. sato, são paulo, brazil. the maternal exposure to allergens during pregnancy or even in postnatal period may influence the allergy onset to newborns, through antigen or antibody transmission. we sought to verify the effect of maternal antigen exposure before conception, during gestation or in the breastfeeding period on the offspring type i hypersensitivity response. female balb/c mice were immunized or not with ova extract/alum, boosted twice at th and th and mated with normal balb/c male on the th day after sensitization (das). others groups of immunized mothers also received oral administration with ova along pregnancy, or non-immunized mothers received ova only during breastfeeding. offspring from immunized or normal mice were immunized intraperitoneally with ova at do and boosted on the th das. the results showed a important decreased of tgf-levels in the amniotic fluid and milk from immunized mothers before mating in comparison to obtained from normal mothers. ova exposure during pregnancy of immunized mothers decrease significantly the transference of tgf-by breastfeeding, while both tgf-isoforms were founded at high levels in the amniotic fluid. similar levels of tgf-transference by placenta to the newborn was detected in both immunized mother groups. pups from mothers exposed with ag during pregnancy showed an increased spleen cell number, whereas did not produced il- , il- , il- e ifn-secretion induced by antigen neither altered responsiveness to anti-cd or mitogen. maternal ova-immunization induced a marked inhibition of spe-cific ige antibody response in the immunized pups, contrasting to the enhancement of ige responsiveness detected in the immunized pups from mothers which were exposed to ova only at postnatal period. these results showed that preconceptional immunization exert a protective effect on the offspring ige development and an exacerbation of ige responsiveness due to mother antigen exposure during breastfeeding. the findings suggest that rather than in utero antigen priming occurrence, postnatal period may contribute to offspring early life sensitization. financial support: fapesp and lim -fmusp introduction: the reasons for the increased incidence of allergic diseases in westernized countries are still unknown. mercury is an important pollution factor to which humans are increasingly exposed. prior studies on the effect of mercury on mitogen stimulated human lymphocytes indicated a th weighted immune response, but the results were inconsistent and difficult to reproduce. phorbol myristate acetate (pma) is a direct activator of protein kinase c, which has been shown to play a role in mercury induced il- production in animals. therefore we investigated the effect of mercury on pmaactivated human peripheral blood mononuclear cells (pbmc). methods: pbmc from individuals were cultured for days in culture medium containing pma and ionomycin in the presence or absence of mercuric chloride (hgcl ). il- and gamma-ifn concentrations were measured by elisa of culture supernatants. cell death and apoptosis were determined by -aad and annexin staining and fluorescence activated cell sorting (facs). cell-proliferation was assessed by h-thymidin-incorporation. results: after days of culture, pma/ionomycin-stimulated a small amount of il- compared to untreated pbmc ( . +/- . pg/ml versus . +/- . pg/ml). however, mercury induced a more than fold increase in il- production when added to pma-activated cells ( . +/- . pg/ml, p< . ). gamma ifn production was strongly increased in pbmc that were treated with pma/ionomycin (> pg/ml versus +/- pg/ml in unstimulated cells) but dropped markedly in cells treated with mercury plus pma/ionomycin. in addition, mercury induced increased cell death, apoptosis and reduced cell proliferation. conclusions: hgcl strongly stimulates il- production in pma/ionomycin treated pbmc while cell viability and gamma-ifn production drop significantly. these preliminary results suggest that human exposure to mercury may be playing a role in the observed increased incidence of allergic disease in the industrialized world background: mast cells (mc) have been shown to induce natural cellmediated cytotoxicity in long-term ( - hrs.) in vitro assay systems. the cytotoxicity is mediated by at least two pathways: secretory via exocytosis of mc granules containing serine proteases such as granzymes, chymase and soluble tnf-and nonsecretory (cell-to-cell contact) via membranous tnfand fasl. chymase induces apoptosis in neonatal rat cardiomyocytes and human vascular smooth muscle cells. the objective of this study was to investigate mc mediated cytotoxicity against nk/lak-sensitive cells in short term ( hrs.) unstimulated cultures. methods: human bone marrow mononuclear cells were cultured in methylcellulose supported with il- , il- , and stem cell factor. mast cell colonies developed at weeks and were transferred to liquid imdm and maintained for weeks. a flow cytometric cytotoxicity assay was used to determine cytotoxicity against daudi, raji, and k cell lines at hrs., hrs., and hours. the controls consisted of cell lines without mast cells at hrs., hrs., and hours. results: rationale: to establish the antibody response rate in children with recurrent infections and fully immunized with the pneumococcal -valent conjugate vaccine. methods: we have analyzed patients referred to our clinic with recurrent infections despite complete immunization with the pneumococcal -valent conjugate vaccine for age, according to acip guidelines. we assessed the patients by checking their immunization status and the antibody titers to all streptococcus pneumoniae serotypes included in the vaccine ( , b, v, , c, f, f) assessed by standardized elisa. the patients were assembled into groups, a non-immunized group with laboratory data prior to the vaccine, and an immunized group consisting of responders and nonresponders according to their antibody titer (< . or > . iu/ml respectively). the data were analyzed using epi info and spss. results: the mean age was . years for non-responders and . years for responders. there was no significant statistical difference between the groups regarding age, race and sex. ten patients were identified who failed to respond to all serotypes included in the pneumococcal -valent conjugate vaccine. there was no significant statistical difference between the non-immunized and the immunized nonresponders to all serotypes. conclusions: we have identified a special immunological phenotype of specific antibody deficiency (sad) patients with normal total immunoglobulins and normal responses to protein antigens, but who failed to respond to conjugate pneumococcal polysaccharides. a. yates * , r. deshazo , j. butler , g. howell , j. farley , h. liu , n. nanayakkura , g.b.yi , r. rockhold , . jackson, ms; . oxford, ms. introduction: venom from s. invicta consists of % disubstituted piperidine alkaloids and is toxic to insects, birds and farm animals. recent reports of morbidity and mortality in elderly patients after massive fire ant stings suggest the potential for systemic mammalian toxicity. we evaluated the toxic responses to systemic administration of two structurally verified, synthetic s. invicta venom alkaloids, solenopsin a (trans- -methyl- -n-undecylpiperidine) and its cis-isomer, isosolenopsin a, in rats. methods: sprague dawley rats were anesthetized with isoflurane, paralyzed with gallamine, artificially ventilated and instrumented to record arterial blood pressure (bp; mm hg), heart rate (hr; bpm) and % change in left ventricular contractility (lvc; p/ t). in addition, a group of rats was chronically instrumented to record bp and hr while the animals were conscious and freely-moving. results: solenopsin a at to mg/kg iv dose-dependently lowered bp, hr and lvc. at mg/kg iv, hypotension (- ± mmhg), bradycardia (- ± bpm) and decreased lvc (- ± p/ t) were marked. isosolenopsin a, mg/kg iv, produced responses similar to solenopsin a mg/kg iv. solenopsin a mg/kg iv elicited tonic-clonic convulsions and respiratory arrest in conscious, freely-moving rats. hematuria was seen with solenopsin a, but not isosolenopsin a. superfusion of a working, isolated, perfused rat heart with um of solenopsin a elicited a marked, reversible decrease in lvc, and cardiac arrest occurred with um of solenopsin a. conclusion: the results demonstrate that these alkaloids possess significant depressant activity on the cardiac and respiratory systems of rats. the neurologic and cardiorespiratory effects can account for lethality to small mammals in the wild, and may contribute to adverse cardiovascular effects noted in humans after massive fire ant stings. introduction: during asthma attacks, the ph of exhaled breath condensate (ebc) decreases two log orders (ph= . ), returning to normal levels after corticosteroid therapy (ph= . ). ion channels, once thought to participate only in the transport of ions, are now suspected of mediating airway inflammation in asthma as well. to determine whether allergen directly alters airway mucosal ph and ion function, we measured nasal ph and nasal potential difference (pd) before and after nasal allergen challenge (nac). methods: ten allergic rhinitic subjects (mean±sem age . years± . , females) underwent a crossover, single-blinded, placebo-controlled study, where they were challenged with allergen (dust mite, grass, cat) and control diluent in two different occasions in random order via nasal spray. nasal ph was measured on the surface of nasal mucosa with a ph probe. nasal mucosa pd was measured between nasal mucosa and forearm skin. subjects also filled out a nasal symptom scale. measurements were taken at baseline, hour, hours, and hours after nac. results: the nasal symptom score increased significantly immediately after allergen compared to control challenge ( . ± . vs. . ± . respectively, p= . ). there were no statistically significant differences in the nasal ph at h and h, but a significant decrease at h compared with baseline (- . ± . vs. + . ± . , p= . ). the change in ph from h to h correlated significantly and inversely with change in symptoms (r=- . , p= . ) and number of sneezes (r=- . , p= . ) after challenges. nasal pd did not change significantly after challenges. conclusion: allergen decreases nasal mucosal ph in the very late phase after challenge in allergic rhinitic subjects. decrease in airway mucosal ph during asthma exacerbations may be caused by aggravation of allergic inflammation. nasal potential difference does not change after allergen challenge. funding: niaid, acaai foundation introduction: uv induces differentiation of t-and b-lymphocytes, suppresses natural killer cells, renders a tolerogenic effect and induces apoptosis resulting in local and system immunosuppression. methods: lymphocytes were studied using indirect immunofluorescence methods employing monoclonal antibodies to cd-markers cd , cd , cd , cd , cd , hla-i, and hla-ii, from the blood of volunteers (aged - years) before uv exposure (control), after an exposure of blood in vitro to a dose . kj, and after hours of exposure of the medial surface of the elbow joint in vivo (s= cm ), to a dose of . kj. results: the direct exposure of blood results in a significant reduction in the number of lymphocytes, probably due to direct phototoxic effects. significant modifications both of the aggregate number of lymphocytes and amount of t-helpers after uv exposure of the skin were not seen. after uv the number of cells which express the marker cd is sig-nificantly increased. exposure to uv in vitro induces reduced number of cells bearing cd . however on in vivo exposure, a significant increase in number of cells which express cd was observed. uv exposure of blood reduced significantly the hla-i ( . ± . reduced to . ± . ) and hla-ii (dr) ( . ± . reduced to . ± . ) expression. uv exposure of skin increased significantly hla-i to . ± . , while hla-ii (dr) was insignificantly decreased to . ± . . cd expression increased from . ± . to . ± . due to blood uv exposure, while there were insignificant reductions in cd ( . ± . reduced to . ± . ) and cd ( . ± . reduced to . ± . ) expression. conclusion: uv starts a cascade-like response, including apoptosis, leading to changes in nk cell, helper and suppression lymphocyte numbers consistent with an immunomodulating effect of uv radiation. background: previous investigations have shown the involvement of histamine and histamine receptors (h , h , h , h ) in ige synthesis in atopic and lymphoproliferative diseases. ige responses may depend on the concentrations of histamine and histamine receptor antagonists and agonists. the goal of this investigation was to evaluate the role of the concentration of histamine and h /h antagonists along with the importance of pre-existing levels of ige in determining ige responses. methods: ige synthesis was studied in mnc cultures of patients ( - years old) having mild atopic asthma and rhinitis. all patients had high levels of total ige and specific ige to ragweed pollen, house dust mite, epidermal or mold allergens. patients were divided into two groups according to the serum ige and levels of spontaneous ige synthesis: group a -with low levels ( . ± . iu/ml) and group b -with high levels ( . ± . iu/ml). the serum level of total ige in group a was . ± . iu/ml and in group b was . ± . iu/ml. fub hydrogenmaleate was used as an h /h specific antagonist. results: histamine in high concentrations ( - m) suppressed and in low concentrations ( - m) stimulated spontaneous ige synthesis. the h /h antagonist fub activity depends on the pre-existing levels of ige. in high concentrations ( - m), the antagonist increased ige synthesis only in group a, but not in group b having high spontaneous levels of ige synthesis. the synthesis in group a increased . fold (p< . ), but h /h blockade cancelled the ige suppressive effect of high concentrations of histamine ( - m). the addition of histamine into the mnc culture stimulated ige synthesis . fold. fub had no effect on ige-stimulatory effects of low concentrations of histamine ( - m). in allergen (ragweed)-stimulated mnc culture, fub had a co-stimulatory effect on ige synthesis induced by histamine. conclusions: the ige stimulating response depends on the concentrations of histamine and the h /h antagonist as well as pre-existing levels of serum ige and ige spontaneous synthesis. introduction: preliminary data has shown that oral contraceptives can precipitate or worsen attacks of hereditary angioedema (hae). it is thought that estrogens affect the synthesis and degradation of bradykinin with resulting edema. objective: our objective is to determine if there is a difference among the various oral hormonal preparations, namely, combined monophasic, combined multiphasic, progesterone only, and hormone replacement therapy, in regards to their effect on frequency of hae exacerbations. methods: patients in this study consisted of women over the age of diagnosed with hae who receive their care at our institution and women meeting the same criteria who are active in the hae foundation and have access to this association's web page. all patients answered a sixteen item questionnaire about past or present birth control pill or hormone replacement therapy usage and its impact on the frequency of exacerbations. results: of the patients who completed the questionnaire, % had taken or were currently taking oral contraceptive pills or hormone replacement therapy. of the women whom had taken monophasic birth control pills, % worsened with increased number and severity of exacerbations. however, only % of the women that had used combined multiphasic birth control pills worsened. all of the women who had tried progesterone only birth control pills worsened. of those on isolated estrogen for hormone replacement, % had an increased number of exacerbations. when androgens were used concurrently with oral contraceptive pills or hormone replacement therapy, exacerbations decreased to %. conclusion: we are not able to demonstrate a statistical benefit of one oral contraceptive pill preparation over another because of our sample size. however, our preliminary data supports that the oral contraceptive pill which produces less exacerbations and less symptoms is a combined multiphasic pill with concurrent androgen treatment. introduction atopic dermatitis (ad) is associated with multiple immunological abnormalities including imbalances in the subsets of blood circulating lymphocytes forming cellular infiltrates in inflamed skin. many studies of the functional and phenotypic properties of lymphocytes in ad have been limited to either peripheral blood or skin-infiltrating lymphocytes. the purpose of our study was to evaluate and compare the content and phenotypic properties of cd + lymphocytes from blood and inflamed skin of ad patients. materials and methods adult (age - years) patients with chronic ad were selected by the criteria of hanifin. all patients gave written informed consent. the cd + lymphocytes of peripheral blood were phenotyped by flow cytometry. skin biopsies were obtained from eczematous areas, then cryosected and double immuno-histochemistry was performed. for phenotyping of lymphocytes in blood and skin a panel of monoclonal antibodies was used including cd , cd , cd , cd , hla-dr and cla. results immunophenotype analyses of the peripheral blood lymphocytes showed a predominance of cd +cd + cells ( %± . ). most cells were cd +hla-dr+ ( %± . ) and cd +cd + phenotype ( %± . ). double immunohistochemistry of the skin biopsies revealed in the epidermis rare but constant presence of cd +cd +cells ( . ± . cells/mm ) and cd +hla-dr+ cells ( . ± . cells/mm ). in the dermal inflammatory infiltrates the predominant cells were cd +cd + ( . ± . cells/mm ) and cd +hla-dr+ ( . ± . cells/mm ). the dominant inflammatory infiltrate consisted of cd +cla+ phenotype ( . ± . cells/mm ) cells. conclusion in ad skin inflammation is associated with the appearance in the circulation of cd + lymphocytes in activated form (cd +hla-dr+) and lymphocytes with regulatory properties (cd +cd +). these lymphocytes are recruited from the circulation, having the skin homing properties (cd +cla+), and form the main constituent of the dermal inflammatory infiltrate. contact dermatitis (cd) comprises a spectrum of inflammatory skin reactions usually caused by exposure to non-immunogenic low molecular weight substances (haptens). failure to diagnose the condition may result in a chronic and disabling condition with impaired quality of life. a yr-old white male presented with a severe, symmetrically distributed facial maculopapular rash with secondary excoriated lesions. prior to the appearance of the rash, the patient had been applying lubriderm as a skin moisturizer for dry skin. a standardized thin layer rapid use epicutaneous (true) test containing chemical agents suspended in a vehicle and attached to an adhesive backing was applied to the patient's back. at hrs, a positive reaction was observed at the skin site of the paraben mix application. the lubriderm preparation used by the patient contained paraben, in contrast to a newer preparation, advanced therapy lubriderm, which is paraben-free. complete resolution of the facial lesions occurred following discontinuation of the lubriderm and the use of oral and topical corticosteroid therapy. this case report illustrates how a commonly used moisturizer can contain a sensitizing agent that could be detected by standardized patch testing which should be a part of the diagnostic armamentarium of every allergist-immunologist. we report how a commonly used and effective moisturizer (lubriderm) can cause severe allergic cd and that the use of a standardized thin layer rapid use epicutaneous (true) patch test panel can be an effective diagnostic tool for the detection of the offending agent. rationale: this study aimed to establish the possible differences of cutaneous sensitization to common aeroallergens in children under years old. methods:the study was conducted between - and included infants from silesia/southern part of poland/.these infants were refered to our allergy unit due to respiratory symptoms like rhinitis, otitis, pharyngitis, cough, bronchitis recurring;eye's symptoms/ conjunctivitis/ and oral symptoms. the skin prick test/ spt/ to major aerollergens in our environment(hdii, birch, alternaria, cladosporium and trees)were done to -months-old and -yearold children. a spt of mm or larger was considered as positive. patients were classified into three groups: i:positive family atopy ii:smoking ciggarettemother/ or father iii:cat or dog at home iv: coexisting food allergy results: , % of examined children were boys. the prevalence of sensitisation found were as follows:latex , %, birch , %, dpii- , %, grass pollen , %, alternaria- , %. positive spts to latex were first seen in the -months-old group and this prevelance doubled until they finished year. conclusion: in our study, cutaneuos sensitisation start to appear in -months-old chlidren. background: propylene glycol (pg) may induce allergic contact dermatitis (acd) and skin irritant reactions. topical formulations frequently contain pg. it is present in low concentration ( %) in pimecrolimus cream %, a non-steroid inflammatory cytokine inhibitor. objectives: this study was designed to assess the incidence of cutaneous responses to pimecrolimus cream in patients allergic to pg and to determine their nature (acd or irritation) and severity. methods: in this double-blind, randomized, vehicle-controlled, withinpatient study, subjects allergic to pg underwent -hour patch-testing (pg and %, pimecrolimus cream and vehicle) followed by a -day repeated open application test (roat). application sites were assessed by the investigator using a scale ranging from (no reaction) to (spreading bullous reaction) at , , and hours after patch removal and at the completion of the roat. results: pg allergy was confirmed by patch-testing in patients. two patients showed a positive patch-test reaction with pimecrolimus cream and vehicle, indicating an acd. in contrast, no patient demonstrated signs of acd when pimecrolimus cream was applied under normal conditions, i.e. without occlusion (roat). the statistical analysis showed that there were significantly less reactions at pimecrolimus patch-test sites than at the pg patch-test sites (p< . for both pg concentrations, one-sided exact binomial test) and that the median severity was lower with pimecrolimus cream vs. pg (p= . and p< . for pg % and % respectively, wilcoxon signed-rank test). conclusions: this pilot study suggests that pimecrolimus cream %, when applied under normal conditions, can be used safely in patients with pg allergy. introduction: chronic idiopathic urticaria is not always responsive to antihistamine therapy. multiple alternative agents have been tried. we report two cases where tacrolimus has been successful in treating this condition. methods: this is a case report of two patients who have chronic idiopathic urticaria and have been treated with tacrolimus. patient # is a y/o wm who pre-sented with urticaria that had been going on several months. the duration of his urticarial lesions was < hours his urticaria was treated with several medications including: hydroxyzine, cyproheptadine, ceterizine, montelukast, colchicine, and dapsone. he was intolerant to hydroxychloroquine. montelukast seemed to provide some modest benefit but he continued to have daily urticaria with > lesions/day after two months of therapy. tacrolimus was added to montelukast at mg bid. patient # is a y/o wf with a history of biopsyproven urticaria. she had suffered from episodic urticaria since the age of six that was responsive to systemic steroids. after thirty years of having no urticaria, she developed recurrent urticaria at age . at this time, she was treated with hydroxyzine, doxepin, montelukast, loratadine, ceterizine, fexofenadine, colchicine, dapsone, and even prednisone with very little improvement in symptoms. she was then started on tacrolimus mg bid as monotherapy while she was having daily urticaria. results: after one month of treatment with tacrolimus mg bid, patient # had a decrease in the number of urticaria. his dose of tacrolimus was increased to mg/day, and after four weeks, his urticaria resolved. he remained on tacrolimus for a total of six months which was tapered and discontinued. he continues to be in remission from urticaria. patient # had complete resolution of her hives after just two doses of tacrolimus. after two months, she remains free of urticaria on tacrolimus mg bid. conclusion: for patients with severe chronic urticaria unresponsive to multiple therapies, tacrolimus, like cyclosporine, may be efficacious. tacrolimus may also be truly immunomodulatory and capable of inducing remission of urticaria. s.v. gerasimov * , lviv, ukraine. introduction. recent studies suggest that probiotics can be useful in prevention and treatment of atopic dermatitis (ad) in children. as clinical effect of probiotics varies greatly depending on the specific strain, we are currently conduct a search for the most promising probiotic to be used as complementary therapy in infants and young children with ad. methods. we studied infants aged - weeks ( ± ) with ad established using hanifin and rafka criteria. patients were evenly allocated among probiotic (n= ) and nonprobiotic (n= ) treatment groups using quota allocation system. severity of ad was defined using scorad index and infant`s quality of life was assessed using idqol index. patients were examined before and weeks after the treatment with/without probiotic powder formulation (b. infantis, l. acidophilus dds- , billion cfu/day, dds-junior, uas laboratories). we compared pre/posttreatment values of indices above and amount of mometasone furoate ( , %) used employing a paired or unpaired t-test. results. at baseline, infants in either groups were comparable on age, gender, duration of the disease, scorad and idqol indices, and medication taken for the previous weeks. before entering the study all patients were maintained on allergen elimination diet due to milk and egg white allergy. after the treatment with/without probiotics the mean scorad index decreased from , to , (p= , ) and from , to , (p= , ), respectively. however, the mean amount of mometasone furoate used in non-probiotic treatment group was significantly greater ( , g vs , g, p= , ), despite the same recommendation on the use of the drug was given. additionally, parents of children taking probiotics reported an improvement in infant`s quality of life as seen on the decrease of idqol index from , to , (p= , ). in non-probiotic group decrease in idqol was not significant ( , to , ; p= , ) . there were no adverse events in any of the treatment groups. conclusions. our preliminary results suggest that some probiotics may have a corticosteroid-sparing effect and improve quality of life of infants with ad. there is a clear trend towards reduction of scorad index, which did not change significantly, probably due to a limited number of patients involved. introduction : the aim of this study was to analyze the risk factors of severe atopic dermatitis(ad) in the first months of life. methods : the children aged less than months with ad were divided into two groups according to six area six sign in atopic dermatitis(sassad) score. children with score less than was classified as mild ad(n= ) and those with score above as severe ad(n= ). these patients were fed with breast milk or cow's milk formula, and no allergenic food was given except rice and some vegetables. we analyzed the gender, feeding patterns, family history of allergy, number of siblings, total ige and specific ige to common food allergens (egg white, cow's milk, wheat, soy) by cap-feia assay. total eosinophil count was ± /ul in severe ad and ± /ul in mild ad(p< . ). results : ) total ige was . ± . u/ml in severe ad and . ± . u/ml in mild ad(p= . ). specific ige to egg white, wheat and soy ( . ± . u/ml, . ± . u/ml, . ± . u/ml in severe ad; . ± . u/ml, . ± . u/ml, . ± . u/ml in mild ad; p< . ) were associated with severe ad, but cow milk( . ± . u/ml in severe ad, . ± . u/ml in mild ad) showed no difference. ) gender, feeding patterns, family history of allergy and the number of siblings were not significantly associated with severe ad. conclusion : severe ad is associated with sensitization to food allergens in the first months of life, although they are not fed with those foods. background: atopic dermatitis (ad), an inflammatory skin disease diagnosed primarily in children, has been shown to have a negative impact on quality of life (qol). pimecrolimus cream % is a non-steroid, topical calcineurin inhibitor with demonstrated efficacy in the acute treatment and longterm management of pediatric and adult ad. in a -week, double-blind, vehicle-controlled study, a secondary aim was to evaluate the impact on parent's quality of life of a pimecrolimus-based or corticosteroid (cs)-based treatment regimen of children with ad. methods: children aged months to years (mean years) with mild to severe ad were randomized : to receive treatment with pimecrolimus or vehicle cream. emollients for dry skin and pimecrolimus or vehicle bid were applied at the first signs of ad. for severe flares, a mid-potency topical cs (fluticasone or mometasone) indicated for once-daily use in ad replaced the evening study drug application for a maximum of weeks or until all ad resolved. parents completed the parent's index of quality of life-atopic dermatitis (piqol-ad), a -item validated questionnaire, which measures parent's needs-based qol, at baseline, week , and study completion. change in piqol-ad scores at baseline and week were compared between treatments. a negative change indicated improvement. results: parents of patients in both groups reported improvement in piqol-ad scores at week , with greater improvement for the pimecrolimus group. the mean change in piqol-ad score from baseline to week was - . in the pimecrolimus group vs. - . in the cs-based treatment group ( . % vs. . % improvement, respectively). ancova analysis, using treatment and center as main effects and baseline score as a covariate, compared the change in piqol-ad score in two treatment groups. pimecrolimus treatment demonstrated a more favorable change (- . ) which approached statistical significance [p= . ; %ci= (- . , . )]. conclusion: a treatment regimen utilizing pimecrolimus cream % had a beneficial effect on parent's quality of life compared to a corticosteroid-based regimen. this benefit was consistent with other measures of efficacy studied. introduction: atopic dermatitis is a chronic, inflammatory and recurrent skin disorder . its prevalence has increased in recent decades but little is known about it in mexico city. the aim of this study was to evaluate the prevalence and severity of atopic eczema in a pediatric population. methods: a cross-sectional questionnaire survey (isaac phase i questionnaire) was conducted on random samples of schoolchildren aged to years and to years from educational centers of four northern counties from mexico city. those children with a positive response to being questioned about the presence of an itchy relapsing skin rash in the last months were considered to have atopic dermatitis. children whose symptoms resulted in sleep disturbance for or more nights per week were considered to have severe atopic eczema. statistical analyses were done with spss . for windows, chi square . the size of the sample was determined folowing the isaac specifications and calculated with the statcal program. results: complete data was available for children aged to years in schools and children aged to years in high schools. . % males and . % females. response rates were high ( . % for those aged to years and % for those aged to years). the prevalence of symptoms of atopic dermatitis in the last months was . % at age to and % at age to years. medical diagnose of atopic eczema was % and . % for children aged to years and to years, respectively. reported eczema accounted for . % and . % at age to and to years, respectively. children with symptoms of severe atopic dermatitis accounted for % of all those with symptoms of atopic dermatitis. we also found that the majority of the children begun with skin manifestations of atopic dermatitis before the age of years old. conclusions: the prevalence of atopic eczema was very similar for both groups of ages. our results agree with those found in other two studies acomplished in other mexican cities (cuernavaca and chihuahua) and with those from other countries of latin america like brazil, chile and costa rica. although, we found lowest values than those from sweden, japan and australia. studies that include objective skin examination are required to confirm these findings. a. kaplan * , s. meeves , y. liao , s.t. varghese , g. georges , . charleston, sc; . bridgewater, nj. introduction: the symptoms of chronic idiopathic urticaria (ciu) can have a profound impact on patient health and quality of life. the safety and efficacy of fexofenadine (fex) bid for the treatment of ciu has been previously established in two multicenter, double-blind, randomized, placebo-controlled trials. this study evaluated the efficacy and safety of a qd dose of fex hcl mg, as this dosing schedule could offer advantages in terms of patient compliance and convenience. methods: this multicenter, randomized, double-blind, parallel-group, placebo-controlled study consisted of a single-blind placebo run-in period of - days, followed by a (± )-day treatment period. males and females aged years with a diagnosis of ciu and with active disease were enrolled. patients were randomized : to receive either fex hcl mg qd or placebo qd. the primary endpoints were change from baseline in mean daily number of wheals (mnw score) and mean daily severity of pruritus (measured on a -point scale) over the -day treatment period, as assessed reflectively by the patient. secondary efficacy measures included a modified total symptom score (motss), comprising sum of number, frequency, size and duration of lesions, and severity of pruritus. mnw and pruritus severity were also assessed instantaneously at trough drug levels (immediately prior to dosing). results: over the -day treatment period, patients treated with fex (n= ) experienced significantly greater improvements in mnw and pruritus scores compared with the placebo group (n= ) (p< . for both). similarly, over the treatment period, and at individual weekly timepoints, the mean reductions in am reflective, pm reflective and mean daily motss were significantly greater for patients in the fex group compared with those in the placebo group (p . for all comparisons). the mean reductions in instantaneous mnw and pruritus scores were greater with those who received fex than those who received placebo (mnw: p= . ; pruritus score: p= . ). there were no significant differences in the frequency of treatment emergent adverse events between the two treatment groups, and no clinically relevant changes were observed with respect to clinical laboratory data, vital signs or ecgs. conclusion: this study demonstrated that a qd dose of fex hcl mg offers effective and well-tolerated relief from the symptoms of ciu. introduction: the wheals and pruritus associated with chronic idiopathic urticaria (ciu) are often so debilitating that they have a profound impact on patient quality of life. specifically, ciu has been shown to negatively affect patient mobility, sleep, energy levels, social interaction and emotional wellbeing. the purpose of this study was to examine the impact of treatment with fexofenadine hcl (fex) mg on health-related quality of life (hrql) among patients with ciu. methods: as part of a multicenter, randomized, double-blind, parallel-group, placebo-controlled study, designed to evaluate the efficacy and safety of a once-daily dose of fex mg in ciu, the impact of treatment on hrql was also examined. patients were asked to complete the dermatology life quality index (dlqi) and the work productivity and activity impairment questionnaire (wpai) at baseline and at weeks and (final visit or early termination). the primary endpoint was mean change from baseline in dlqi total score, using the mean data of evaluations performed at weeks and as the post-baseline measure. secondary endpoints included change from baseline in individual dlqi domains and wpai scores. additional analyses were conducted to examine the reliability, validity and responsiveness of the hrql measures. results: a total of patients were included in the hrql population: n= fex, n= placebo. patients in the fex group experienced significantly greater improvements in the mean dlqi total score than those in the placebo group (p= . ). this pattern was repeated with respect to the individual domains of symptoms and feelings (p= . ), daily activities (p= . ), leisure (p= . ) and personal relationships (p= . ). both treatment groups reported improvements in productivity, as measured by change from baseline using the wpai. patients randomized to fex experienced significantly less impairment while working (p= . ) and performing activities (p= . ) than those who received placebo. the dlqi was demonstrated to be reliable (cronbach's alpha= . ); both the dlqi and wpai were found to be valid and responsive instruments in this ciu population. conclusion: this study demonstrated that a once-daily dose of fex mg improves the hrql of patients with ciu, as assessed by change in dlqi total score. t. algozzine * , a. lee , s. wong , l. anzisi , . manchester, nh; . centerport, ny; . syosset, ny; . mamaroneck, ny. symptoms of allergic and non-allergic rhinitis may significantly impact a patient's quality of life, by causing fatigue, headache, cognitive impairment and other systemic symptoms. appropriate management of allergic rhinitis is important in the effective management of coexisting or complicating respira-tory conditions (e.g. asthma, sinusitis, or otitis media). in addition, many commonly used over-the-counter (otc) antihistamines can cause performance impairment that may impact daily activities. we designed a brief ten-question allergy survey in an attempt to determine how patients were being treated for their allergies, evaluate a patient's self-assessed impact of their allergies on their daily activities, and identify any difference in treatment outcomes between primary care and allergist practices. patients were invited to complete the surveys anonymously. completed surveys were collected and entered into a microsoft access database for evaluation. minitab was utilized for statistical calculations. four hundred and thirty seven patients completed the survey. fifty-eight percent of survey respondents were women and % were adults (age > ). of those patients surveyed, % (n= ) were under the care of an allergist. compared to primary care, patients treated by allergists reported more allergies ( . vs. . , p < . ) and received more medications on average ( . vs. . , p< . ). pollen was the most common allergy type reported among all patients. while the majority of all patients ( %) received prescription medications, more primary care patients received no therapy or only otc treatment when compared to allergist patients ( % vs. %, p< . ). seventy-four percent of primary care patients reported their allergy symptoms were controlled compared to % of allergist patients (p< . ). patients receiving treatment from an allergist reported less impact of their allergies on daily activities (a lower score signified less impact) compared to patients seen in primary care ( . vs. . , p< . ). the results of our survey suggest that allergists treat more complex patients. these patients had multiple allergies, more uncontrolled symptoms, and utilized more prescription medications. despite these findings, patients treated by allergists reported less impact of allergies on their daily activities. introduction the patogenic mechanism of nasal polyps are unknow.they frecuently are associated with aspirin intolerance, intrinsic asthma, chronic sinusitis, young sindrome, cystic fibrosis, kartagener syndrome and churg-strauss syndrome.chemical mediators found in nasal polyps are as follows: histamine, serotonin, leukotrienes, norepinefrine and possibly pgd .recently, leukotrienes have been implicated in mediation of bronchoconstriction and inflammatory leukotriene levels have also been shown to be elevated in some patients with sinonasal polyposis (figure ) hypothesis antileukotrienes might play a significant role in controlling polyposis and symptoms secundary to sinonasal disease, and they might viable alternative to longterm, oral steroid therapy and repeat surgical debridement purpose this study was undertaken to evaluate the potential role of leukotriene receptor antagonist on recurrent polyposis associated with asthma and improvement of some factor implicated with them design of study:clinical assay, prospective, triple blind. materialsand methods:the study involved patients ( %) males and ( %) women, mean age . years, with a range - years selection criteria:inclusion criteria: nasal polyps associated with astha, allergic or non allergic.exclusion criteria: patients with any concurrent illness, use of sistemic or local corticosteroidsor any kind of antileukotriene within month prior to the beggining of the study.we made an alleatory selection patients recived montelukast ( mgs/day), and nasal steroids, beclometasone ( mgs/day) for months. patients recived loratadine plus pseu-doefedrin mgs/ mgs/day and nasal steroids, beclometasone, patints were operated of fees and transnasal endoscipic polypectomy plus montelukast and nasal steroids, beclometasone ige serum levels .skin prick test .sensitivity in vitro .ct scan . celullarity on nasal lavage (eosinophils and neutrophils) .proinflamatory cytokines (il -il -il ) on nasal lavege by elisa test. results ther was a tendency for more improvement of the group ( surgery-montelukast-local steroid) of symptoms and objective measurments, in second place of improvement the group montelukast-local steroid) and the worse on improvement subjective and objetive was group local steroid and loratadine-pseudoefedrine use as placebo introduction: sensory perceptions of intranasal corticosteroids (ins) vary among products and can be unpleasant and affect adherence to therapy. methods: we conducted a cross-sectional study of patients across allergy and immunology clinics in the united states. respondents were asked to choose between pairs of hypothetical ins that differed by sensory attribute composition. based on prior research, we measured salient sensory attributes: smell, taste, aftertaste, throat rundown, nose runout, and feel of spray in nose/throat. each attribute was described in intensity levels, such as "no taste" (low), "weak taste" (moderate), and "strong taste" (high) ( table) . other outcomes included an importance score for each sensory attribute and patients' willingness to adhere to an ins having the lowest levels of each sensory attribute compared to one with moderate levels. results: preferences decreased with increasing intensity level of each sensory attribute. the most important attribute was aftertaste in % of patients, taste in %, throat rundown in %, nose runout in %, smell in %, and feel of spray in %. only % had more than one attribute tied for most important. if instructed to take ins daily for months, % of patients stated that they would definitely be able to follow their doctor's advice (willing to adhere) if given an ins containing the lowest level of each sensory attribute compared with % for one having moderate levels (p< . ). conclusions: patients' preferences decrease with increasing intensity levels of each sensory attribute and affect patients' willingness to adhere. tailoring ins to patient preferences may lead to improved treatment satisfaction and adherence. introduction:a high prevalence of rhinitis and asthma comorbidity has been persistently noticed in epidemiological studies in last years. our objective was to evaluate the prevalence of rhinitis and asthma comorbidity in a portuguese population including both atopic and non-atopic patients. methods: a retrospective study was performed. clinical records from patients attending an immunoallergology outpatients'clinic during months (from january until june ) were reviewed. data were collected concerning clinical history of rhinitis and/or asthma, aeroallergens'skin prick tests and respiratory function evaluation. results: among the patients attending an appointment during the study period, ( . %) had rhinitis and/or asthma ( . % f; . % m;mean age +/- years). patients with respiratory disease included atopic ( . %) and non-atopic %). diagnosis of asthma, rhinitis or of both diseases was made in ( . %), ( . %) and ( . %), respectively, in atopic patients and in ( . %, ( . %) and ( . %), respectively, in non-atopic patients. rhinitis was diagnosed in . % of atopic asthmatic and in - % of non-atopic patients with asthma. in patients with rhinitis, asthma was also diagnosed in . % of atopic patients and . % on non-atopic patients. conclusions: in this study population rhinitis was frequently diagnosed in patients with asthma and vice-versa, thereby leading to a high prevalence of this comorbodity. this association was more frequent in atopic patients. our data are comparable to those we found in recent literature. these results suggest that clinical investigation of asthma should be mandatory in management of patients with rhinitis as well as asthmatic patients should be routinely submitted to clinical evaluation of rhinitis. introduction: cetirizine hcl (c) has been shown to be more effective than fexofenadine hcl (f) in controlling seasonal allergic rhinitis (sar) symptoms at - hr post-dose, however, the efficacy of c and f was comparable between - hr post-dose. response to treatment in the middle of the dosing interval needed to be addressed. methods: this randomized, double blind, placebo (p)-controlled study was designed to compare the efficacy and tolerability of a single dose of c mg, f mg, and p between - hr postdose in ragweed-sensitive sar subjects. subjects meeting entry criteria were exposed to pre-determined controlled levels of ragweed pollen in the eeu during priming and double blind treatment. subjects assessed rhinitis symptoms at half hr intervals during pollen exposure; at priming, at the qualifying period, at baseline, and from - hr post-dose. the primary efficacy endpoint was the change from baseline in total symptom severity complex (tssc) score at hr post-dose. tssc score was the sum of severity ratings ( =absent to =severe) for symptoms: runny nose, sneezing, itchy nose/palate/throat and itchy/watery eyes. results: a total of subjects (mean age . y; . % females) were randomized: c, ; f, ; p, . baseline characteristics were comparable among groups; tssc: c= . , f= . , p= . . c produced a % greater reduction in tssc score at hr (- . , p= . ) and a % greater reduction overall (i.e., average over the - hr post-dose period) (- . , p= . ) compared with f (- . , - . , respectively). both c and f reduced tssc score more than p ( hr, - . ; overall, - . , p< . ) including all individual symptoms (p< . ). c however, was more effective than f for runny nose and sneezing at hr and overall, itchy/watery eyes at hr, and itchy nose/throat/palate overall (p< . ). rates of discontinuation due to adverse events (aes) were low: c, . %; f, %; p, . %. the incidence of treatmentemergent aes was similar: c, . %; f, . %; p, . %. somnolence occurred in . % of subjects on c, and % on f or p. conclusion: c produced a greater improvement in rhinitis symptoms compared with f and p, at hr post dose and over the - hr post-dose period. all treatments were safe and well tolerated. background: medication utilization patterns of patients suffering from seasonal allergic rhinitis (sar) are not well documented, and although many anti-allergic medications are prescribed for daily use, actual usage is unknown, but recognized to be quite variable. methods: subjects with positive ragweed skin tests were mailed a survey during the third week of ragweed season, soliciting the nature and severity of sar symptoms, usage patterns and reasons for choice of anti-allergic medication. results: subjects completed the survey ( . %). the prevalence of symptoms were, in decreasing order: sneezing ( . %), runny nose ( . %), itchy/gritty eyes ( . %), stuffiness ( . %), itchy nose ( . %), watery eyes ( . %), itchy palate/throat ( . %), post-nasal drip ( . %), red/burning eyes ( . %), headache ( . %), itchy ears ( . %), cough ( . %), shortness of breath ( . %), and wheeze ( . %). sar patients used antihistamines most frequently ( . %), followed by decongestants ( . %), combination (antihistamine/decongestant) products ( . %), and intranasal corticosteroids ( . %). medications were mostly taken intermittently rather than daily (antihistamines . %; nasal corticosteroids . %), conclusion: a constellation of nasal symptoms were the most common seasonal allergic manifestations, followed by ocular, palatal and ear irritation. antihistamines were the most frequently used medication to treat symptoms, succeeded by decongestants (alone or in combination). a significant proportion of subjects took their allergy medication, including nasal corticosteroids, intermittently rather than regularly, underscoring the relevance of single-dose evaluations of drug efficacy. a.k. ellis * , e. rafeiro, j.d. ratz, j.h. day, kingston, canada. background: traditional assessment of seasonal allergic rhinitis (sar) medication efficacy utilizes randomized controlled trials over - weeks in season. an additional study method employs single-dose responses using controlled allergen challenge such as the environmental exposure unit (eeu). a comparison of allergic symptoms generated by controlled allergen challenge to those occurring in ragweed season symptoms has not been done. methods: subjects with known sar to ragweed were mailed a survey during the third week of ragweed season, soliciting the nature and severity of sar symptoms. subjects participating in a subsequent controlled allergen challenge study using the eeu, were again asked to complete a similar survey that documented symptoms generated in this model. those who completed both surveys comprised the primary analysis group. results: subjects completed the ragweed season survey, subjects completed the eeu survey, and completed both. symptoms generated by eeu exposure were similar to those elicited during ragweed season, with the exception of cough ( % vs. %, respectively, p < . ). subjects reported that symptoms were more severe in the eeu than those experienced on a typical ragweed season day, but less severe than those during peak ragweed season days. conclusion: allergic upper respiratory tract symptoms produced during controlled ragweed pollen exposure in the eeu were similar in nature and degree to those expe-rienced during ragweed season, supporting evidence that the eeu is a valid model for studying sar. r. nave , m.a. wingertzahn * , s. brookman , s. kaida , t. shah , . konstanz, germany; . florham park, nj; . princeton, nj; . tokyo, japan. rationale: ciclesonide (cic) is a new corticosteroid under development for treatment of allergic rhinitis (ar). cic is a pro-drug that is hydrolyzed to the active metabolite desisobutyryl-ciclesonide (des-cic) in the target tissue. cic has low oral bioavailability and is highly bound to plasma proteins; therefore cic administered as a nasal spray is expected to have minimal local and systemic effects. objective: the primary objective was to evaluate the safety and tolerability of repeated escalating doses of cic ( - mcg/day) given as a nasal spray for days to healthy and asymptomatic subjects with sar. secondary objectives were to determine pk of cic and des-cic and to evaluate the effect of cic on endogenous cortisol. methods: this was a single-center, randomized, placebo-controlled, double blind, modified sequential dose study. six cohorts were randomized. cohorts i-v consisted of healthy subjects given doses of cic up to mcg bid. cohort vi (asymptomatic sar subjects) received mcg bid. each cohort was comprised of subjects who received cic and subjects who received placebo. safety assessments were conducted by recording adverse events (aes), clinical laboratory, eye, and nasal examination findings. serum and urine samples were taken for evaluation of cortisol levels. cic and des-cic serum concentrations were determined by lc-ms/ms. results: no trend was observed for aes when comparing cic and placebo treatment. additionally, no subject experienced a serious ae or withdrew from the study due to an ae during the trial. cortisol levels showed no differences between the dose groups or between activetreated subjects and placebo-treated subjects. additionally, serum concentrations of cic and des-cic in the majority of serum samples were shown to be below the lower limit of quantification (lloq; pg/ml for cic and pg/ml for des-cic), therefore no descriptive statistics could be calculated. con-clusions: ciclesonide nasal spray, at the doses evaluated, was safe and well tolerated in healthy and asymptomatic sar subjects with no detectable effects of cic on serum or urinary free cortisol concentrations. additionally, no pk parameters could be calculated for cic nasal spray, as it was virtually undetectable in serum despite the use of a sensitive assay. the preferred treatment for allergies is avoidance. air filtration is logical but room air purifiers have been of limited efficacy. zephyr™ is a new device which creates an envelope of air % free of allergenic particles around the head of the sleeping person. allergic rhinitis frequently causes daytime somnolence. this is a pilot study of the effectiveness of zephyr on symptoms of seasonal allergic rhinitis (ragweed hay fever) and on daytime sleepiness. methods: subjects age to with ragweed hay fever were studied in the ragweed season using each subject as his/her own control. usual allergy medicines were not allowed, except loratadine for rescue. outcome measures were a symptom score, juniper rhinitis quality of life questionnaire, a tolerability rating, and epworth sleepiness scale. during the first week subjects qualified by symptom scores, then entered the one-week treatment period with zephyr. the third week was a post-treatment observation period. results: of participants, ( %) showed symptom improvement. the whole group averaged % reduction in morning symptoms and % reduction in evening symptoms. sleepiness scores improved %. rhinitis qol improved %. the zephyr system was well tolerated as evidenced by the response to several statements regarding zephyr use and tolerability: (scoring: = strongly disagree, = strongly agree) "the system did not bother me while i was sleeping." (mean score . ) "the noise level did not affect my ability to sleep." (mean score . ) "the temperature was just fine for me." (mean score . ) "the system did not get in my way during sleep." (mean score . ) conclusions: zephyr significantly reduced seasonal hay fever symptoms and daytime sleepiness, improved quality of life, and was well-tolerated by subjects. zephyr may provide maximal environmental control of bedroom allergen exposure irrespective of ambient airborne allergen levels in the room. nasal congestion is an important symptom that is associated with significant morbidity in the rhinitis sufferer. disrupted sleep leading to daytime fatigue, loss of concentration, and decreased productivity are potential results of this symptom. a study employing online interviews with rhinitis sufferers from harris interactive's online database was conducted in order to understand the symptoms they identify with most and to determine the impact nasal congestion had on their daily activities. respondents were at least years of age and experienced nasal congestion from seasonal allergic, perennial allergic, and/or perennial non-allergic rhinitis. the results showed that % of the respondents agreed nasal congestion is the most bothersome symptom of rhinitis; % experienced nasal congestion daily, while % experienced it several times per week. not surprisingly, sleep was the most important factor affected by nasal congestion. two-thirds ( %) of the respondents felt their sleep had been moderately or significantly impacted by nasal congestion, and more than half ( %) felt it was difficult to get a good night's rest because of congestion. sleep was interrupted or disturbed by nasal congestion approximately nights per week, on average. furthermore, sleep disruption from nasal congestion contributed to daytime fatigue; % of the respondents indicated that they were typically tired or fatigued during the day when they experience nasal congestion. in addition, % felt that activities requiring concentration, such as reading, were moderately or significantly impacted by nasal congestion. this study confirms that nasal congestion causes the rhinitis sufferer significant problems beyond that of just a stuffy nose. consequently, healthcare providers need to ensure that their rhinitis patients who have nasal congestion as their primary complaint are managed effectively. desloratadine (dl, clarinex ® ) is a non-sedating oral antihistamine that is metabolized to -oh dl. however, a phenotypic polymorphism has been observed in some patients that results in reduced formation of -oh dl. the prevalence and safety profiles of such poor metabolizers of dl were examined in pharmacokinetic and clinical trials. a poor metabolizer was defined as a subject having a -oh dl to dl auc ratio of < . , or a dl half-life of hours. in pediatric studies, where a sparse sampling approach was uti-lized to screen for poor metabolizers, a plasma concentration ratio of -oh dl to dl of < . at hours classified a subject as a poor metabolizer. a total of , adult and pediatric subjects ( - years old) were phenotyped with a single dose of dl or loratadine. the overall prevalence of the poor metabolizer phenotype was % ( / ). this prevalence was comparable for adult ( / , %) and pediatric subjects ( / , %), and greater in both populations among blacks ( % pediatric, % adult) than caucasians ( % pediatric, % adult). pharmacokinetic analysis found that exposure to dl was approximately -times higher in poor metabolizers than in normal metabolizers. there was no apparent difference in dl exposure among poor metabolizers in different age groups ( -months to < -years, -to < -years, -to < -years, and -years) when treated with age-appropriate doses. the multiple-dose ( - days) safety profile of dl was examined in pediatric poor metabolizers ( - years) in placebo-controlled trials, and in adult poor metabolizers ( - years) in pharmacokinetic trials. pooled ae rates were low and comparable between the poor metabolizer and placebo subjects, as shown in the table below with all aes that appeared in > % of subjects in any treatment group (or > % in adult poor metabolizers). there was no difference in cardiovascular safety profile or ecg results (including qtc interval) among these groups. in conclusion, ( ) expression of the dl poor metabolizer phenotype is independent of age, but higher in blacks than in caucasians, ( ) exposure to dl in poor metabolizers is independent of age when administered at age-appropriate doses, ( ) the safety profile of dl poor metabolizers is not different from that of placebo at all ages down to at least -years old. these results are consistent with the high therapeutic index of dl. desloratadine (dl, clarinex®) is extensively metabolized to -oh dl and subsequently glucuronidated. the enzyme responsible for the formation of this active metabolite is unknown. poor metabolizers of dl represent a subset of the population that has a reduced ability to form -oh dl. a poor metabolizer was defined as a subject having a -oh dl to dl exposure ratio of < %, or dl half-life of hr. pk parameters from adult and pediatric poor metabolizers following repetitive administration of dl were characterized in clinical pharmacology trials and are summarized in the table below. exposure to dl [auc( - hr)] in poor metabolizers was approximately -fold greater than the corresponding values in normal metabolizers; cmax was to -fold greater. the magnitude of the reduction in the formation of -oh dl and the concurrent increase in exposure to dl associated with the poor metabolizer phenotype was similar in pediatric and adult subjects at age-appropriate doses. despite the increased exposure to dl in poor metabolizers, there was no increase in adverse event frequency or changes in electrocardiographic parameters. a: dose normalized to mg. b: least squares mean ratio: anova of log-transformed data extracting sources of variation due to age group and metabolizer status. the ratio is a contrast of metabolizer status. c: lower and upper % confidence interval based on log-transformed data. introduction: many patients with seasonal allergic conjunctivitis (sac) complain of symptoms of dry eyes. we have previously reported that patients with sac experience discomfort from dry eyes anywhere between to days per week and that the overall severity of the dry eye symptoms tend to range from mild to severe. this preliminary cross over study evaluated the benefits of nedocromil sodium % ophthalmic solution used twice daily, compared with nedocromil sodium % ophthalmic solution used with refresh (ocular lubricant), for the treatment of dry eye symptoms in association with sac during weeks. methods: patients who had a minimum of two-year history of sac and dry eyes, a positive skin prick test towards grass pollen and between the ages of to were enrolled. patients were evaluated on four visits and completed a daily diary, which included scales for grading allergic conjunctivitis and dry eye symptoms. at each clinic visit they completed the osdi and the rqlq (rhinitis quality of life questionnaire). the osdi (ocular surface disease index) was developed to assess dry eye symptoms and the impact on vision related functioning. the rqlq evaluates quality of life in patients with allergic conjunctivitis. at the last visit, both the patient and the physician assessed the treatment and the effectiveness, if any, of the addition of refresh to nedocromil sodium % ophthalmic solution. daily grass pollen counts were preformed using a burkhard sampler. results: patients ( male and female) were enrolled and dropped out. patients experienced minimal symptoms at the start of the season due to low concentration of grass pollen. preliminary analysis carried out using a paired t-test was preformed on the ocular average scores for the two treatment periods. there was no significant difference between the treatment groups for redness, light sensitivity and tearing of the eyes. a reduction in dry eye symptoms was reported in all patients using refresh eye drops and the number of drops required varied between patients. during the treatment periods there was improvement in patients rqlq. conclusion: patients preferred the addition of refresh eyedrops in alleviating sac and dry eye symptoms. further studies need to be carried using refresh eye drops in larger number of patients with sac and dry eye symptoms. c. laforce * , raleigh, nc. introduction: the objective of this study was to determine the ability of azelastine nasal spray to improve rhinitis symptoms and quality of life parameters in seasonal allergic rhinitis patients remaining symptomatic after treatment with fexofenadine. methods: this placebo-controlled, double-blind study began with a -week, open-label lead-in period, during which patients received fexofenadine mg bid. after days, patients who improved less than % to % on fexofenadine were randomized to treatment for weeks with: ( ) azelastine nasal spray, ( ) azelastine nasal spray plus fexofenadine, or ( ) placebo. the primary efficacy variable was the change from baseline to day in thetotal nasal symptom score (tnss), which consisted of runny nose, sneezing, itchy nose, and nasal congestion scores recorded twice daily in patient diary cards. in addition, quality of life was assessed using the rhinitis quality of life questionnaire (rqlq). results: after weeks of treatment, azelastine nasal spray (p<. ) and azelastine nasal spray plus fexofenadine (p<. ) significantly improved thetnss compared to placebo. based on patients with complete tnss and rqlq data, the overall rqlq score also was significantly (p<. ) improved compared to placebo. conclusions: azelastine nasal spray was an effective treatment for patients with seasonal allergic rhinitis who did not respond well to fexofenadine and significantly improved quality of life parameters compared to placebo. the results of this study indicate that azelastine nasal spray is an important alternative to oral antihistamines and should be considered in the initial management of seasonal allergic rhinitis. w. berger * , mission viejo, ca. objective: to evaluate improvement over time with azelastine nasal spray in the treatment of patients with moderate-to-severe seasonal allergic rhinitis (sar) who remained symptomatic after treatment with loratadine or fexofenadine. methods: the studies were -week, multicenter, double-blind, placebo-controlled trials that began with a -week, open-label lead-in period in which patients received either loratadine mg qd (study no. ) or fexofenadine mg bid (study no. ). patients who improved < %- % with loratadine were randomized to treatment with: ( ) azelastine nasal spray sprays/nostril bid, ( ) azelastine nasal spray sprays/nostril bid plus loratadine mg qd, ( ) desloratadine mg qd, or ( ) placebo. patients who improved < %- % with fexofenadine were randomized to treatment with: ( ) azelastine nasal spray sprays/nostril bid, ( ) azelastine nasal spray sprays/nostril bid plus fexofenadine mg bid, or ( ) placebo. the primary efficacy variable was the change from baseline to day in the total nasal symptom score (tnss), consisting of runny nose, sneezing, itchy nose, and nasal congestion. symptom severity was recordedam and pm in diary cards on a -point scale ( =none; =mild; =moderate; =severe). results: in both studies, patients treated with azelastine nasal spray experienced increasing improvement intnss over days of treatment.the improvements were approximately -fold greater than placebo at each day of the study, and the differences from placebo were statistically significant (p<. ) at days , , , and overall. in study no. , % of patients treated with azelastine had > % improvement in tnss compared to % in the placebo group. in study no. , % of patients treated with azelastine had > % improvement in tnss compared to % in the placebo group. conclusions: azelastine nasal spray was effective in treating patients with moderate-to-severe sar who remained symptomatic after treatment with either loratadine or fexofenadine. azelastine demonstrated first-day effectiveness, and patients treated with azelastine experienced increasing improvements in rhinitis symptoms over the -day study periods.azelastine nasal spray is an effective treatment alternative to oral loratadine or fexofenadine and an appropriate first-line therapy in the management of sar. introduction: a large, open-label, azelastine (astelin) nasal spray patient experience trial was conducted in patients with sar, vmr, or mixed rhinitis (allergic rhinitis with nonallergic triggers). this analysis evaluated the effect of azelastine nasal spray in treating rhinitis symptoms in a subset of patients with a history of asthma or sinusitis. methods: patients were entered into an open-label protocol and treated for weeks with azelastine nasal spray at a dosage of sprays per nostril bid. after weeks, the patients completed a questionnaire that assessed onset of action, symptom improvement, satisfaction with therapy, and quality of life. results: from a total of rhinitis patients who received azelastine monotherapy during the -week study period, data were analyzed for patients with asthma (n= ) or sinusitis (n= ). a greater percentage of these patients had severe rhinitis compared to the overall population. nasal congestion and postnasal drip were reported as the most bothersome rhinitis symptoms. after weeks of treatment with azelastine nasal spray, > % of patients with asthma and > % of patients with sinusitis reported some or complete control of congestion and postnasal drip. in addition, > % of patients with asthma reported chest tightness, shortness of breath, and wheezing were somewhat or completely controlled, and > % of patients with sinusitis reported that headache and facial pain were somewhat or completely controlled during treatment with azelastine nasal spray. conclusion: azelastine nasal spray provided effective control of rhinitis symptoms, including nasal congestion and postnasal drip, in patients with a history of asthma or sinusitis. introduction: epinastine is an antihistamine with mast cell stabilization and anti-inflammatory properties. epinastine . % ophthalmic solution was evaluated for treatment of allergic signs and symptoms elicited by feline dander in a cat exposure room. methods: participants (n= ) were aged years, with a history of ocular allergy to cats, a positive skin prick reaction to cat dander, and an ocular itching score of (on a - scale) within minutes of entering the cat room. subjects wore a tb mask while in the cat room to reduce the effects of inhaled allergen. after minutes of exposure, drop of epinastine hcl . % was instilled in one eye, and olopatadine . % was instilled in the fellow eye. environmental exposure to cat dander continued for another minutes. prior to instillation, and at , , , and minutes after instillation, conjunctival hyperemia and chemosis (scales of - ), and ocular itching, tearing, ocular burning, nasal itching and rhinorrhea (scales - ) were assessed. results: instillation of a single drop of ophthalmic epinastine significantly reduced ocular itching from a mean pre-instillation score of . to a mean score of . at minutes after instillation (p<. ), despite continuous exposure to cat dander during the entire period. similarly, ocular burning, tearing, and hyperemia were significantly reduced by epinastine treatment (p<. ). chemosis was only weakly induced by cat room exposure, with a mean pre-instillation score of . ; however, epinastine treat-ment decreased that to (p=. ). instillation of epinastine also significantly reduced nasal itching and rhinorrhea scores (p<. ). results for olopatadine were not statistically different; however, the change from baseline in itching scores in the epinastine-treated eyes was greater than that seen in the olopatadine-treated eyes at the majority of timepoints. conclusion: ophthalmic epinastine is indicated for the prevention of itch associated with allergic conjunctivitis.this study shows that treatment of cat-sensitive subjects with ophthalmic epinastine following environmental exposure to cat dander significantly reduced ocular itching and other signs and symptoms of allergic conjunctivitis. objective: the objective of this study was to evaluate azelastine (astelin®) nasal spray, cetirizine (zyrtec®), fluticasone (flonase®), and placebo in the treatment of patients with symptomatic seasonal allergic rhinitis. methods: this was a double-blind placebo-controlled pilot trial in patients with seasonal allergic rhinitis. the study began with a -week, placebo lead-in period, followed by a -week blinded treatment period (day to day ). efficacy variables were: ( ) change from baseline to day in the total nasal symptom score (tnss; consisting of rhinorrhea, sneezing, itchy nose, and nasal congestion); ( ) onset of action based on tnss over the hours following initial administration of study drugs; and ( ) change from baseline to day ( -hour change) in tnss. tnss was scored twice daily (am and pm) on a -point rating scale ( =none, =mild, =moderate, =severe). patients recorded a minimum -hour tnss of on at least days during the lead-in period, and a congestion score of on at least days to qualify for entry. qualified patients were randomized to treatment with: ( ) azelastine nasal spray sprays per nostril bid plus placebo capsules qd; ( ) cetirizine -mg tablets qd plus placebo nasal spray; ( ) fluticasone sprays per nostril qd plus placebo capsules qd; or ( ) placebo nasal spray plus placebo capsules. results: azelastine significantly (p<. ) improved the tnss compared to cetirizine, fluticasone, and placebo beginning minutes after initial administration; cetirizine significantly improved tnss versus placebo at minutes; fluticasone showed no significant differences from placebo over the -hour evaluation period. azelastine significantly (p<. ) improved the tnss at day ( -hour change from baseline) compared to cetirizine, fluticasone, and placebo. conclusions: azelastine nasal spray improved the tnss in patients with moderate-to-severe seasonal allergic rhinitis. in addition, azelastine nasal spray demonstrated a -minute onset of action and significantly improved tnss versus cetirizine, fluticasone, and placebo hours after initial administration. introduction: epinastine, an antihistamine with mast cell stabilization and anti-inflammatory properties, has been developed for the treatment of allergic conjunctivitis. the efficacy and tolerability of epinastine was assessed and compared with levocabastine. methods: eligible patients for this randomized, double-masked, parallel-group, active-controlled environmental clinical trial were - years old with a recent diagnosis of seasonal allergic conjunctivitis. patients instilled drop epinastine hcl . % or levocabastine . % ophthalmic solution in each eye bid for weeks. patients and investigators assessed efficacy and tolerability at study visits on days (baseline), , , , and , and assessed overall efficacy and tolerability at study exit. adverse events were monitored. results: epinastine provided superior itch relief compared with levocabastine (p=. ; see figure) ; mean ocular itch scores over treatment visits were . for epinastine and . for levocabastine ( - scale; =worst; baseline scores were . for epinastine and . for levocabastine). the mean summed score (ocular itching, tearing, and foreign body sensation) over treatment visits was significantly better for epinastine than levocabastine (p=. ).at study exit, % of epinastine-treated patients rated overall efficacy "very good" (the highest possible rating), versus % of levocabastine-treated patients. at minutes postinstillation, % of epinastine-treated and % of levocabastine-treated patients rated tolerability "very good". at study exit, % of epinastine-treated and % of levocabastine-treated patients rated overall tolerability "very good", with investigator ratings being similar. treatment-related adverse events occurred in . % of epinastine-treated patients (most frequent: eye pain and skin itching, . % each) and . % of levocabastine-treated patients (most frequent: double vision, . %; influenza-like symptoms, . %); most aes were mild or moderate. conclusion: our results confirm the therapeutic potential for epinastine as an efficacious treatment suitable for long-term use throughout the allergy season. ophthalmic epinastine was superior to levocabastine for relief of ocular symptoms in patients with allergic conjunctivitis and was well-tolerated. since chronic inflammation is the histopathologic landmark of otitis media with effusion, clinical observations have led us to believe that the combination of a cysteinyl leukotriene receptor antagonist montelukast with an oral antibiotic may be more efficacious than monotherapy with an oral antibiotic in the treatment of serous otitis media. we studied twenty pediatric patients (age years to years) in a randomized open labeled -week trial to compare the efficacy of the combination montelukast ( mg or mg chewables tablets qd dosed according to patient's age) with an oral antibiotic amoxicillin/clavulanate potassium ( mg/kg/day in divided doses every hours) to a monotherapy with an oral antibiotic amoxicillin/clavulanate potassium for the treatment of otitis media with effusion. the efficacy of treatment options was assessed using pneumatic otoscopy, impedance tympanometry, and audiometry to monitor the clinical course of the middle ear effusion in both treatment groups. in the combination group montelukast and antibiotic a resolution of otitis media with effusion occured at the th day. in contrast in the group treated with monotherapy with the oral antibiotic the resolution of otitis media with effusion occured on the th day. in conclusion, the combination of montelukast plus an oral antibiotic is more effective than monotherapy with an oral antibiotic.the combination of montelukast plus an antibiotic may be a safer and shorter therapy given the safety issues with long term use of systemic antibiotics. introduction: a randomized, double-blind, placebo-controlled study was conducted in an environmental exposure chamber (eec) to compare the efficacy of three doses of olopatadine nasal spray, a topical anti-allergy treatment for seasonal allergic rhinitis (sar), versus placebo spray. methods: patients aged - years old with a history of sar were screened, consented, evaluated for a positive skin test to ragweed allergen, and enrolled in this irbapproved study. a total of "primed" patients were exposed to ragweed allergen in the eec and randomized to olopatadine . % (n= ), olopatadine . % (n= ), olopatadine . % (n= ), or placebo (vehicle) (n= ) spray, sprays/nostril once in the morning. symptoms were self-assessed using a point scale (total nasal symptom score, tnss, comprised of sneezing, runny, itchy and stuffy nose) via diaries at periodic intervals during the -hour study period. safety was also assessed. results: obvious trends indicated a dosedependent response to olopatadine . %, . % and . %, though concentrations were not statistically different from each other. all three concentrations of olopatadine were clearly more efficacious than placebo spray at the first time point, minutes, continuing to the end of the -hour session. olopatadine exhibited a safety profile comparable to placebo. conclusions: olopatadine nasal spray . %, . % and . % exhibited dose-dependent responses. onset of action for all three concentrations of olopatadine was apparent at the first post-dose timepoint, minutes, and efficacy was maintained throughout the next hours. olopatadine nasal spray was safe, well-tolerated, and effective for the treatment of sar in the eec chamber. c. slonim * , tampa, fl. objective: to assess patient subjective responses to the treatment of allergic conjunctivitis using azelastine hydrochloride ophthalmic solution. methods: participating physicians selected patients from their practice to receive azelastine hydrochloride . % ophthalmic solution drop per affected eye twice daily for days. patients on prior ocular allergy medications were allowed to participate. after days of treatment with azelastine hydrochloride, patients (n= , ) rated their experiences with azelastine hydrochloride via a self-administered survey. not all questions were answered by each patient. results: at baseline, % of patients reported that their ocular itching affected their work, school, and/or leisure activities at least "somewhat". after using azelastine hydrochloride, % of patients achieved either "moderate" or "complete" relief from ocular itching, including % who reported "complete" relief from itching. seventy-one percent ( %) of patients had been treated previously with a topical prescription anti-allergy medication for their ocular itching. seventy percent ( %) of patients (n= ) who had not been previously treated with a topical prescription anti-allergy medication treatment and % of previous medication users (n= , ) experienced at least "moderate" relief of itching when treated with azelastine hydrochloride. sixty-five percent ( %) of the previously-treated patients rated azelastine hydrochloride as "somewhat better" or "much better" than their previous medication. conclusions: results suggest that azelastine hydrochloride ophthalmic solution is an effective treatment for the ocular itching associated with allergic conjunctivitis as rated by the patient, regardless of whether they had been treated previously with a topical prescription anti-allergy medication. objective: to determine an association between food allergy and acid reflux in adults. method: we conducted a retrospective chart review of atopic adult patients in an academic otolaryngic allergy practice. adults who tested positive and adults who tested negative for food allergy were included in the study. charts were reviewed for a diagnosis of gastroesophageal reflux disorders (gerd). this included a history of laryngopharyngeal reflux (lpr) and peptic ulcer disease (pud). population prevalence ( . %; ci . - . ) of acid reflux was estimated from a historical study (locke gr et al. prevalence and clinical spectrum of gastroesophageal reflux: a population-based study in olmstead county, minnesota. gastroenterology ; : - ) that had a similar subject population. the prevalence of gerd in each study arm, as well as in the total study group of atopic adults, was compared to the historical control. results: subjects testing positive for food allergy had a diagnosis of gerd in . % ( % ci . - . ) of cases. those subjects who did not demonstrate a food allergy were positive for gerd in % ( % ci . - ) of cases. in the total study group, the prevalence of gerd was . % ( % ci . - . ). on chi square analysis, the prevalence of gerd was significantly higher in the total study group when compared with the historical control (p= . ). those subjects who tested negative for food allergy had a statistically significant higher prevalence of gerd than the control population (p= . ). the prevalence of gerd between the group testing positive and the historical control did not show a significant difference (p= . ). when comparing the study arms to each other, there was no significant difference in the prevalence of gerd (p= . ). conclusions: the prevalence of acid reflux disorders was not higher in subjects with food allergy when compared to a historical control. although a statistical significance was noted between adults who tested negative for food allergy and the control population with regards to the prevalence of gerd, this may reflect the fact that individuals examined in an otolaryngologist's office are more likely to have acid reflux disorders than the general population. overall, the atopic subjects did have a higher incidence of acid reflux disorders, but there was no statistical significance between subjects with and without food allergy. a. suryadevara * , d.l. hamilos, boston, ma. introduction: recent studies suggest that crs without nasal polyposis (crssnp) and crs with nasal polyposis (crscnp) represent distinct pathologic entities. we wished to determine whether these conditions differed in their clinical presentation. methods: over a two-year period, new patients coming to a university based specialty clinic meeting criteria for crs were enrolled in an outcomes study. patients indicated which of four major (facial pain/pressure/headache, nasal obstruction, nasal purulence/discharge, and hyposmia/anosmia) and four minor (fever, halitosis, dental pain, cough) criteria for crs they were experiencing. rhinoscopy was performed to look for nasal polyps or polypoid tissue in any sinus area. the prevalence of each symptom was compared in the groups by chi square analysis. results: the population (n= ) had a mean age of +/- . and was % female, % caucasian, . % african-american, . % asian and . % hispanic. most patients ( %) were non-smokers. all had at least major criteria or major + minor criteria for crs at enrollment. forty-one patients ( . %) had crscnp. the mean number of major criteria was greater in the crscnp than crssnp ( . vs . , p= . ). nasal obstruction and hyposmia/anosmia were more prevalent in crscnp (p= . , . respectively). facial pain/pressure/headache was more prevalent in crssnp (p=). ). the most prevalent symptoms in crscnp were: nasal purulence/discharge ( . %)>hyposmia/anosmia ( . %)>facial pain/pressure/headache ( . %)>nasal obstruction ( %). in contrast, the most prevalent symptoms in crssnp were: facial pain/pressure/headache ( . %)>nasal purulence/discharge ( . %)>hyposmia/anosmia ( . %)>nasal obstruction ( . %). none of the symptoms were absolutely distinguishing of these conditions. no differences were found between the two groups for fever, halitosis, dental pain or cough. conclusion: we conclude that patients with crscnp have a greater burden of symptoms of crs and a much higher prevalence of hyposmia/anomsia. these findings are consistent with other studies showing that, in comparison to crssnp, crscnp tends to be more difficult to treat and have a higher rate of relapse after intensive medical therapy (subramanian et al, am j rhinology ; : ). l.e. mansfield * , e.e. philpot , c. posey , . el paso, tx; . research triangle park, nc. daytime sleepiness is a common complaint in sar. patients often complain of mental slowness, difficulty in concentrating, and thinking. the present study evaluated whether effective therapy of sar would decrease dss and improve an objective measure of cp. dss was measured using the epworth sleep scale (ess). objective cp was measured using the test of variables of attention (tova), a validated test. thirty two adults ( males, females with a year history of sar, a compatible physical exam, and corresponding positive allergy testing) volunteered for this week randomized double blind placebo controlled study. after a one week inf placebo (pl) baseline, the subjects received either active inf or continued pl. they maintained daily nasal symptom dairies and ess. the subjects took the tova test at the end of week and week . weekly nasal symptom scores significantly improved with the inf, but not the pl. w vs. w nasal congestion inf , p=. , pl , p=ns; runny nose inf . , . p=ns; pl . , . p=ns; sneezing inf . , p=. ; pl . , . p=ns. total weekly ess was abnormal and decreased significantly in the inf group, but not in pl group. w vs. w inf . , . p=. , pl . , . p=ns. response time of the tova testing, initially somewhat slow, significantly decreased in inf but not pl treatment. w vs. w inf msec, msec p=. ; pl msec, msec p=ns. these results demonstrate that sar is associated with dss and cp problems. the mechanism is likely to be sleeping disordered breathing associated with nasal congestion and obstruction. effective treatment of nasal congestion with inf led to decreased dss and improved cognitive performance. l.e. mansfield * , c. graham , . el paso, tx; . new york, ny. there is increasing recognition that sleep disturbance and daytime tiredness occur during active ar. the mechanism appears to be nasal congestion and obstruction leading to sleep disordered breathing and resultant poor quality of sleep. in our practice, as part of the initial history, questions regarding fatigue, snoring, sleep problems, and tiredness are addressed. sleep problems and tiredness are graded according to the following scale: effect on daily activity ; =not troubled; = a little trouble; =somewhat troubled ; = trou-bled a lot ; = total disruption. we reviewed consecutive charts of patients with allergic rhinitis documented by history, physical examination and allergy testing. there were females and males; age range y to y. ( %) of patients or parents recognized they commonly snored. ( %) stated they were chronically fatigued. the graded answers concerning sleep problems and tiredness were even more revealing of ar patient's perception of their problems see table in general, the higher sleep problem responses were associated with higher tiredness scores. national surveys of unselected populations suggest that about percent of adults consider themselves to have sleep problems. the high frequency of sleep related problems and tiredness in our sample has prompted us to add more detailed questions regarding sleep related events to our intake history. it is our opinion that questions specifically related to sleep and daytime tiredness should be included in all evaluations for allergic rhinitis. we conclude that sleep related problems and tiredness may be more common in patients with allergic rhinitis than previous reported. r.w. weber * , j. garcia , r. faruqi , d. banerji , g. georges , the study investigator group , . denver, co; . bridgewater, nj; nj. introduction: the intranasal glucocorticosteroid triamcinolone acetonide (taa) is a safe and effective treatment for persistent allergic rhinitis (par). a new hydrofluroalkane- a (hfa) propellant delivery system (taa-hfa) has recently been developed. this study primarily assessed the long-term safety of taa delivered via this new device, as well as its long-term efficacy. methods: patients aged - yrs (mean= ) with par enrolled in this -yr, open-label study at centers in the us. patients received taa-hfa μg once daily for a -week run-in period before adjusting the dose to μg or μg once daily based on symptom severity. doses were standardized to μg once daily across all patients at ~ months to ensure sufficient long-term safety data at the maximum dose. physical exams, including measurement of vital signs and laboratory measurements were taken at baseline, months and study end. independent patient and physician global symptom evaluations were performed at baseline, week and months - thereafter. patients recorded any adverse events (aes) on daily diary cards. results: of the patients included in the study, ( . %) reported aes. the incidence of aes was similar to that of other comparable allergic rhinitis long-term studies. the most frequently reported aes were pharyngitis, rhinitis, local reactions, headache, epistaxis and sinusitis (table ) . most aes were mild-to-moderate in intensity; patients withdrew from the study due to aes. there were no clinically relevant changes in physical exams, vital signs or laboratory measurements. a total of serious aes (saes) were reported; breast carcinoma (n= ), depression (n= ), post-operative spinal fluid leak (n= ) and staphylococcal infection (n= ). saes were thought to be not related to the study drug. at final visit, % of patients had either moderate or marked/complete relief of symptoms using global symptom scores. similarly, % of physicians rated their patients as having either moderate or marked/complete relief. conclusions: long-term administration of taa-hfa μg exhibited a good safety and tolerability profile, while providing moderate-to-complete symptom relief in more than % of patients treated for par. introduction: second-generation, 'non-sedating' antihistamines (ahs) have lower tendency to cross the blood-brain barrier and cause central nervous system side effects than first-generation agents. however, studies have suggested differences between second-generation ahs regarding cognitive function impairment. methods: a medline literature search was performed using the search terms 'antihistamine and impairment', 'antihistamine and psychomotor' and 'antihistamine and central effects', as well as with individual ah names (acrivastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, mequitazine and mizolastine). the findings for second-generation ahs were reviewed and well-designed, placebo-and positive-controlled studies in humans using objective measures of cognitive impairment were included. results: publications were identified as the inclusion criteria. all included objective assessments such as driving performance, critical flicker fusion and divided attention tasks. there was a large variation in the numbers of available well-designed studies; the most rigorously assessed agents were fexofenadine and cetirizine. a number of the ahs (ebastine, acrivastine, loratadine, mequitazine and mizolastine) were not impairing at recommended doses, but were at higher doses. in a small number of available studies, the newer ahs desloratadine and levocetirizine produced no impairment at recommended doses ( mg); however, higher doses were not investigated. cetirizine studies were variable; impairment at the recommended mg dose or higher was seen in a number of studies. in contrast, fexofenadine hcl, up to a dose of mg, was non-impairing in a large number of studies. only one study indicated impairment in one task (critical tracking) and this was only observed with the first doses of fexofenadine hcl ( and mg) and not subsequent doses; however, the authors concluded that doses up to mg/day should be safe for patients who drive. conclusion: while second-generation ahs are less impairing than the first-generation, some newer ahs produce impairment at or above the recommended dose. these differences become important to the patient when agents cause sedation or if patients over use beyond the recommended dose. in conclusion, fexofenadine was the only ah found to be non-impairing in all studies, even at double the recommended us dose. s. shaver , r.b. berkowitz * , c. lutz , p. jones , c. qiu , s. meeves , s.t. varghese , g. georges , . woodstock, ga; . bridgewater, nj. introduction: fexofenadine (fex) is a h -receptor antagonist, with proven efficacy in the treatment of allergic rhinitis (ar). to date, no live cat-room challenge studies have assessed the efficacy of fex in cat-allergen induced ar. this study assessed the efficacy of a single dose of fex hci mg in preventing and controlling cat-allergen induced ar using the cat-room challenge model. methods: this single-center, prospective, randomized, doubleblind, placebo-controlled, two-way crossover study consisted of a screening visit, one or two priming visits and two treatment periods, separated by a (± )-day wash-out. qualifying subjects were randomized to treatment sequence (placebo followed by fex) or (fex followed by placebo). baseline endpoints were obtained prior to study drug administration, and minutes before entering the cat challenge room for allergen challenge. allergen challenges were initiated . hours post-dose, for both treatment periods. the primary endpoint was the change from baseline in total symptom score (tss; sum of rhinorrhea, itchy nose/palate/throat, sneezing and itchy/watery/red eyes) after minutes of allergen exposure at hours post-dose, compared with placebo. other endpoints included changes in individual symptom scores, including nasal congestion. levels of airborne felis domesticus allergen (fel d ) were determined. results: of subjects screened, were randomized and completed the study; and in sequence and , respectively. mean change in tss from baseline was significantly less with fex compared with placebo at minutes after initiation of the cat allergen challenge (p= . ). significantly greater percentage reductions in the individual symptom scores for sneezing (p= . ) and nasal congestion (p= . ) were observed with fex compared with placebo, minutes after challenge. although levels of fel d varied widely, they were balanced between treatment groups. the overall incidence of treatment-emergent adverse events (aes) was low and comparable between groups; no serious aes occurred. conclusion: this study demonstrated that a single dose of fex hci mg is effective and well tolerated as a prophylactic agent for alleviating the ar symptoms induced by exposure to cat allergen. further large-scale clinical trials are warranted to confirm these findings. rationale: allergic rhinitis in children is thought to be associated with several co-morbid disorders. we conducted the following study to assess whether children with diagnosed hypertrophy of tonsils and/or adenoids evaluated by an allergist were more likely to demonstrate objective evidence of allergen sensitization than children similarly evaluated without evidence of these upper airway obstructions. methods: records from the past ten years were identified in the hospital database by presence of an allergy clinic visit and an icd- code for hypertrophy of adenoids and/or tonsils. we reviewed these records to confirm that the diagnosis was accurate. we included subjects if reliable skin prick or in vitro testing for specific ige to aeroallergens was performed. for comparison, we randomly obtained an age and testing-type similar sample. first group subjects were excluded from the second group. skin testing was performed with commercial allergen extracts applied with a dermapik©. in vitro testing was by unicap© feia. positive skin testing had at least a wheal mm or flare mm greater than the negative control. positive in vitro values were . ku/l or greater or a positive pollen mix. additionally, we performed a search in an insurance database to determine how many local children had a code for allergic rhinitis. results: of pediatric allergy patients with adenoid/tonsillar hypertrophy tested, . % ( % ci +/- . %) had at least one positive test. in without adenoid/tonsillar hypertrophy, . % ( % ci +/- . %) had at least one positive test. the observed difference was . % ( % ci +/- . %). the standard error of the difference in percentage was . ; the z score was . . the corresponding p value is . . the percentage of , children with an allergic rhinitis icd- was . % ( % ci +/- . %). conclusion: the number of allergist-referred children with adenoid and/or tonsillar hypertrophy testing positive for an aeroallergen is slightly less than the number of similar children without these diagnoses testing positive. it is not clear that adenoid/tonsillar hypertrophy is associated with a higher risk of allergic rhinitis. a prospective study with allergy testing of all children with adenoid and/or tonsillar hypertrophy might provide information that could alter referral patterns. w.e. berger * , w. storms , s. kimura , m. beck , s. galant , t. westbrook , . mission viejo, ca; . colorado springs, co; . pensacola, fl; . miami, fl; . orange, ca. background: patients having allergic rhinoconjunctivitis are often treated with nasal spray or systemic allergy therapy, forgoing therapy specifically targeting ocular symptoms. the rhinitis quality of life questionnaire (rqlq) and allergic conjunctivitis quality of life questionnaire (acqlq) instruments can be used to quantify the relative benefit of varying medication regimens. objective: to determine the extent of benefit gained in quality of life when an eye drop treatment for ocular allergy (olopatadine) was added to rhinitis patients' preexisting regimen of nasal or systemic allergic treatment. methods: this was a four week prospective, multi-center, open-label crossover, quality of life study occurring during allergy season. at visit , patients completed the rqlq and acqlq questionnaires to assess baseline quality of life. patients were randomized to receive ocular allergy therapy (olopatadine, patanol bid) concomitant with their systemic or nasal rhinitis treatment(s) for weeks between either visit and visit (group a) or between visit and visit (group b). at visit and visit , patients completed the rqlq and acqlq. results: a total of patients completed this study: in group a, in group b. of these, ( . %) experienced eye allergy symptoms at least day during the previous week as reported in the baseline acqlq. baseline scores of the rqlq and acqlq for both groups were comparable. clinically significant improvement from baseline in global rqlq and acqlq was seen following addition of ocular therapy for both groups (rqlq: - . , - . ; acqlq: - . , - . ). similar improvement was seen across all domains of both questionnaires. the acqlq correlated with the rqlq in the applicable domains. conclusion: many allergic rhinitis patients using nasal or systemic medication also suffer from ocular allergic symptoms. for these patients, quality of life improvement is not maximized; the addition of a topical antiallergy eye drop can result in beneficial effects on quality of life. in this study, the addition of olopatadine eye drops to these patients' medication regimens resulted in significant improvement in not only eye symptom related quality of life domains but in overall quality of life. h. milgrom * , r. lanier , f.c. hampel , b. kittner , . denver, co; . fort worth, tx; . new braunfels, tx; . bridgewater, nj. introduction: fexofenadine, a non-sedating, selective h -receptor antagonist, is currently indicated for use in children aged - years with seasonal allergic rhinitis in a number of countries, including the us, and has an excellent safety profile in this age group. this study was designed to assess the safety and tolerability of fexofenadine in children aged - years with allergic rhinitis (ar). methods: the study had a multicenter, double-blind, randomized, placebo-controlled, parallel-group design. children aged - years (n= ) with ar were randomized : to either placebo twice daily (bid; n= ) or fexofenadine hcl mg bid (n= ), for weeks. both treatments were given orally as granulated powders (capsule content) mixed with two teaspoons of apple sauce. treatment-emergent adverse events (teaes) were recorded for all subjects by parents/caregivers and assessed by investigators. clinical laboratory variables, physical examinations, vital signs and ecg evaluations were also assessed in a subgroup of children (placebo, n= ; fexofenadine, n= ). results: baseline demographics were similar in both treatment groups. while approximately % of children in both groups experienced at least one teae, no unusual or unexpected teaes were observed in the fexofenadine group. when the total group was analyzed for teaes by body system, or assessed separately by age groups of -, -, -and -year-olds, no clinically meaningful differences were observed between the two treatment groups. in both treatment groups, the majority of subjects overall and in each age group experienced teaes rated as mild or moderate in intensity. few subjects experienced teaes considered possibly related to study medication (placebo: . % [ / ]; fexofenadine: . % [ / ]). the percentage of discontinuations due to teaes was also comparable between treatment groups (placebo: . % [ / ]; fexofenadine: . % [ / ] ). in the subgroup assessment, no clinically relevant changes were seen from baseline for laboratory variables, vital signs, ecgs or physical examinations in either treatment group. introduction: the efficacy and safety of fexofenadine hcl mg bid has been proven in two large phase iii studies in pediatric subjects aged to years with seasonal allergic rhinitis. in addition, the safety of fexofenadine has been demonstrated in pediatric subjects to years of age with allergic rhinitis (ar). subsequently, two studies (t/ ; t/ ) have assessed the pharmacokinetics, safety and tolerability of fexofenadine and mg bid in pediatric subjects months to years of age. methods: both studies were of multicenter, randomized, double-blind, placebo-controlled, parallelgroup design and enrolled pediatric subjects aged months to < year weighing . kg and aged ± year to < years weighing > . kg. all subjects had a diagnosis of ar as assessed by previous medical history, pattern, or suggestive physical findings. subjects were randomized to receive fexofenadine hcl mg (t/ ), or mg (t/ ) granulation powder twice-daily, or placebo for a minimum of days. safety was evaluated based on adverse events (aes), vital signs, -lead electrocardiograms (ecgs), and physical examinations. results: a total of and subjects were randomized in studies t/ (fexofenadine hcl mg bid: n= , placebo: n= ) and t/ (fexofenadine hcl mg bid: n= , placebo: n= ), respectively. in t/ , . % ( / ) of children receiving fexofenadine and . % ( / ) receiving placebo experienced at least one treatment-emergent ae (teae). in t/ , the incidences of teaes were . % ( / ) and . % ( / ), respectively. in both studies, most of the teaes experienced were mild or moderate in intensity. the incidence of possibly-related teaes was also similar for both treatments in each study. no clinically relevant changes from baseline to study end were observed for vital signs, ecgs and physical examinations. conclusions: the findings of this study show that fexofenadine mg and mg bid are well tolerated and have a safety profile comparable to placebo in pediatric subjects aged months to years. for seasonal allergic rhinitis (sar) patients that remain symptomatic on an h -receptor antagonist, cetirizine, and a nasal glucocorticosteroid, mometasone furoate, the addition of omalizumab, a recombinant, humanized, chimeric, anti-ige monoclonal antibody, may provide additional efficacy in sub-optimally controlled seasonal allergic rhinitis patients. in this open labeled week trial, patients with symptomatic sar currently using cetirizine, mg qd, + mometasone furoate, mcg/nostril qd, were randomized to continue the existing therapy cetirizine + mometasone or to add-on omalizumab subcutaneously (every weeks to weeks) dosed according to patient's weight, and baseline ige levels to the existing therapy cetirizine + mometasone furoate. the endpoints of the trial include: rhinomanometry, nasal symptom score (composite score of nasal congestion, rhinorrhea, sneezing, post nasal drip and itching) and flexible rhinopharyngolaryngoscopy examination. mean efficacy measurements at the end of the -week trial revealed a significant improvements in all parameters examined in the treatment group receiving omalizumab (as add-on to the existing therapy), compared to the other group. in conclusion, the addition of omalizumab to the combination of cetirizine plus mometasone furoate, is more effective than the combination of cetirizine plus mometasone furoate, for the treament of seasonal allergic rhinitis patients. it appears that when omalizumab is added to the combination h receptor antagonist, cetirizine, and nasal corticosteroid, mometasone furoate, the primary end points (rhinomanometry and symptom scores) are significantly improved. background: based on the official statistic data, the prevalence of allergic diseases in russia has increased more than times during the past - years, but still the lowest of all european countries. in some studies done in some regions of russia based on isaac program, it has been shown that the prevalence allergies is significantly higher than indicated in official statistics of health departments. the goal of this investigation was to study the prevalence of allergic diseases, in children of south russian region. meth-ods: this study was done in two steps according to isaac program guidelines. in the first step a questionnaire was given to a total of school children, ages to (group a) and to years old (group b), from the two cities of krasnodar and novorossiysk in southern russia. in the second step a physical exam, skin prick tests with different allergens (pollens, molds, cat, dust mites, foods etc.), and pulmonary function tests were performed. results: wheezing was reported in . % of group a and . % of group b children within the past months. the severity of asthma reported was mild in % (group a)- % (group b); moderate in . % (group a)- . % (group b) and severe in . % (group a)- . % (group b) in studied children). majority of the cases of asthma was noted to be allergic asthma. in both groups of children there was a high incidence of allergies to perennial allergens such as dust mites, cats, molds and other indoor allergens, . % (group a) and . % (group b). the percentage of allergies to pollens was . % and . %, accordingly. allergic rhinitis symptoms were noted in . % (group a) and in , % (group b). atopic dermatitis symptoms with skin itching observed in . % ( - years old)and . % ( - years old). family history of atopy was noticed in . % of group a and . % of group b children. conclusions: the prevalence of allergic diseases in south russia is similar to the ones in most of the european countries. the prevalence of asthma has been associated with increased incidence of allergies, due to indoor and pollen allergens. the prevalence in rhinitis and atopic dermatitis in south russia is parallel to asthma. acknowledgement: we would like to thank hollister-stier lab., greer and antigen lab. for allergen samples. objective: to examine the cost and effectiveness of telithromcyin vs. azithromycin for treatment of mild acute sinusitis under current levels of antimicrobial resistance. methods: we considered an adult with mild acute sinusitis, and symptom duration of days. a decision analytic model was created to compare strategies of st-, nd-and rd-line therapies: azithromycin/amoxicillin-clavulanate/levofloxacin (azi/amc/lev), and telithromycin/amoxicillin-clavulanate/levofloxacin (tel/amc/lev). we considered outcomes: response to initial therapy, cost, and time to completion of all antibiotic therapy. clinical resolution was due to response to antibiotic, or to spontaneous resolution. we assumed bacteria that were resistant in vitro would only be resistant in vivo % of the time. resistance levels were based on the - us protekt surveillance study. model parameters, such as prevalence of bacterial infection, frequency of causative pathogens, and rates of spontaneous resolution, were based on the published literature. those failing to improve after days were switched to next line therapy. we analyzed claims data from managed care organizations to estimate non-drug charges for initial and follow-up care, and applied a % cost-to-charge ratio. drug costs were estimated using the wholesale acquisition cost plus $ for overhead and dispensing. all costs were adjusted to year $us. results: the model predicted the tel/amc/lev strategy would result in . % improving by days compared to . % for azi/amc/lev. the telithromycin strategy had lower mean cost, $ vs. $ . although telithromycin cost more than azithromycin, the tel/amc/lev strategy had slightly lower total drug costs ($ vs $ ) due to less need for additional antibiotic courses. mean time to completion of therapy was . days for tel/amc/lev and . days for azi/amc/lev. sensitivity analyses showed that tel/amc/lev had superior health outcomes even when only % of in vitro resistant organisms were also resistant in vivo. tel/amc/lev also had lower cost if at least % of cases were bacterial, or if in vitro resistant organisms were at least % resistant in vivo. conclusion: based on results obtained using this decision analytic model, initial treatment of mild acute rhinosinusitis with telithromycin may result in improved outcomes and lower cost than initial treatment with azithromycin. rationale: "delta crud" is the term used to describe rhinosinusitis syndromes in the mississippi delta region. local perception is that it is associated with chemical crop dusting, regional produce, especially cotton, or high rates of mold allergy. the climate is extreme with mild winters, hot humid summers, and large volume rain. thirty-five percent of inhabitants live below the poverty level, % are african american. patients from the agriculturally intensive delta suffer from asthma at almost twice the rate of those in the neighboring hills. rates of pesticide use are among the highest in the nation. there are no prevalence estimates for rhinosinusitis in this region. "delta crud" is chronic in many patients with exacerbations occurring in the summer and fall, especially during chemical spraying and defoliation. we conducted this cross-sectional questionnaire based pilot study to begin characterization of rhinosinusitis in this population. method: the sinusitis treatment outcome questionnaire, with two modifications, was given to consecutive patients presenting to the north sunflower regional hospital er and the sunflower outpatient clinic one month prior to the beginning of summer crop dusting. the question, "do you have delta crud?" was added. the survey was anonymous, allowing irb exemption. results: consecutive patients returned completed questionnaires. ( %) admitted to having "delta crud". patients who claimed to have delta crud had significantly more sinus headache, nasal pruritus, conjunctivitis, and chest symptoms (see table) . there was no significant difference in antibiotic prescriptions, er visits, and missed work. six patients with delta crud had been hospitalized for "allergy reasons" compared with three patients without delta crud. despite severe symptoms just patients received sinus ct scans. only / patients were prescribed intranasal corticosteroids. conclusion: in our pilot study, the prevalence of chronic rhinosinusitis symptoms (delta crud) is %, with severe sinus, pulmonary and ocular symptoms. evaluation and treatment may be suboptimal in this economically disadvantaged rural region. further characterization of "delta crud", pollen counts, ige mediated disease, environmental contributions and comparative studies of related regions are needed. introduction: patients with rhinitis commonly experience sinus pain and pressure (sp+p). many patients with recurrent acute bacterial sinusitis (rabs) base the presence of a recurrence on the severity of sp+p and other nasal symptoms. the ability of patients to differentiate between rhinitis and rabs was evaluated by reviewing the subject screening logs of previously reported clinical trials of flonase (fluticasone propionate) nasal spray, mcg (fp). methods: the efficacy of fp was studied in two trials in subjects with sp+p due to allergic rhinitis and in two trials of subjects receiving cefuroxime axetil to treat an acute episode of rabs. sp+p screening logs were examined to determine how many subjects who thought they had sp+p were excluded from the study for an upper respiratory or sinus infection. rabs screening logs were examined to determine how many subjects with symptoms of an acute episode of rabs were excluded from the study for a negative sinus x-ray or ct scan. rabs symptoms experienced at the screening visit by subjects who qualified for the study were also evaluated. results: of subjects screened in the sp+p studies, only ( . %) were excluded for evidence of sinus or upper respiratory tract infections, as clinically diagnosed by the investigator. out of subjects with screening log data in the rabs studies, ( . %) were excluded for a negative sinus x-ray or ct scan. of the subjects who qualified for the rabs study, the most prevalent symptoms included nasal congestion ( %), mucopurulent discharge ( %), facial pain or tenderness in the sinus area ( %), sinus headache ( %) and malaise ( %). only % of subjects had a fever. only one symptom, nasal congestion, was rated by clinicians as severe for greater than % of subjects in either study ( % and %). conclusions: while most subjects can determine when sp+p is due to allergic rhinitis, many cannot determine when symptoms progress to a sinus infection. symptoms experienced by subjects with rabs were consistent with inflammation, but did not include fever as may be expected with an untreated bacterial sinus infection. under appreciation by patients for the role of inflammation in sinus disease may result in over self-diagnosis of sinus infection and the demand for antibiotics. liposomes are small particles consisting of lipid bilayer membranes, which are used to deliver drugs including amphotericin b, more efficiently and with less toxicity. very few cases of allergy to liposomal amphotericin b (lab) have been reported to date. although there is a report on conventional amphotericin b (cab) desensitization, to our knowledge no case of lab desensitization has been described yet. a y/o patient with lymphoma was treated with lab for pulmonary aspergillosis. within minutes of infusion he developed urticaria, hypotension and tachycardia for which he was treated with the appropriate drugs for anaphylaxis. a second attempt to re administer lab resulted in a similar anaphylactic reaction. desensitization to lab was then successfully completed using a modified protocol based on desensitization to cab (jaci ; : - ) : . mg infusion over minutes . mg infusion over minutes . mg infusion over minutes . mg infusion over minutes mg infusion over minutes mg infusion over minutes mg infusion over minutes mg infusion over minutes conclusion: desensitization to lab, a lifesaving drug for invasive fungal diseases, can be performed successfully. adverse events (aes) were recorded throughout the study. therapeutic comparability was defined a priori as a clinical response within % for hfa vs cfc for the primary efficacy measures. results: baseline demographics were similar between treatments for the intent-to-treat population. mean -h baseline symptom scores for nasal stuffiness, nasal discharge and sneezing were . , . and . , respectively, out of a possible score of . all taa-cfc and taa-hfa doses significantly (p< . ) reduced -h, and am and pm -h reflective scores for nasal stuffiness, nasal discharge, sneezing and ni vs placebo (except cfc μg for sneezing). taa-hfa and taa-cfc were statistically comparable (within . and . for the one-sided % confidence interval) at doses of μg and μg for the primary variables of nasal discharge, sneezing and ni for the -h, as well as the am and pm -h reflective assessment, over the -wk period * , * agrawal p , p , , p , p , p , p , p * , p , p , p * p , p , p , p p * finegold, i. * fink p * * sienra monge * , , * so, c. p * soane * , p * staveren, a.m rates of cardiovascular mortality are higher during peak pollen times. (brunkreef, lancet ) . for this reason, mast cell stabilizing agents and leukotriene antagonists are under development for the treatment of myocardial infarction. conversely, ige mediated disease may be protective against sudden cardiac death.(szczeklik, coron art dis ) ar has not been studied in this context. we conducted a retrospective case control pilot study of va patients who were diagnosed with allergic rhinitis and had an acute myocardial infarction. methods:the electronic medical record of a large va hospital was queried for patients diagnosed with an acute myocardial infarction (ami) in the past six years. patients were divided into groups with and without ar. patients with chronic urticaria, asthma, and eczema were excluded from this study. primary outcome was all-cause mortality. secondary outcomes included lv systolic function and peak troponin levels. results: patients were diagnosed with ami, ( %) of which were also diagnosed with ar. patients with and without ar did not differ in terms of age, ethnicity, tobacco status, hypertension, diabetes, or lipid levels. / ( %) patients with ar died, compared to / ( %) control patients, (p-value: . ). ar patients had lower mean peak troponin values ( . vs. . ), but these were not significant. both groups had similar lv function. conclusion: this pilot study suggests an association between allergic rhinitis and a decrease in ami allcause mortality independent of other factors. atopic patients have been shown to have prolonged bleeding times, reduced platelet aggregation, and delayed thrombin generation which can result in delayed clot production. (szczeklik, thromb haem ) possibly, patients in our study diagnosed with allergic rhinitis were more likely to monitor their health symptoms or had physicians who carefully addressed multiple issues. rigorous prospective studies are needed to determine the role of ar in myocardial infarction. purpose and methods. a formulation of olopatadine hydrochloride ophthalmic solution (olopatadine . %) was evaluated in a randomized, placebocontrolled, double-masked, hybrid environmental study intended to determine efficacy and safety in subjects with histories of seasonal allergic conjunctivitis or rhinoconjunctivitis. in this -week trial, subjects regularly assessed their ocular signs and symptoms. additionally, subjects evaluated both the frequency and severity of their nasal symptoms. daily throughout the study, ragweed pollen counts were obtained from each investigative center. repeated measures analysis of variance was used to compare treatment differences in the slopes for nasal symptoms as a function of pollen counts. results. the nasal results are presented herein. specifically, relative to placebo, olopatadine . % significantly reduced the frequency of pollen effects on sneezing (p= . ) and itchy nose (p= . ), and reduced the severity of pollen effects on sneezing (p= . ), itchy nose (p= . ), and runny nose (p= . ). in this study, the most frequent ocular adverse event that was related to therapy with olopatadine . % was ocular dryness, occurring at an incidence of . %. no treatment-related, clinically relevant changes were observed for visual acuity, intraocular pressure, ocular signs, or fundus parameters. conclusion. olopatadine ophthalmic solution, . % is safe, well-tolerated, and effective in reducing some effects of pollen on the nasal symptoms associated with allergic rhinoconjunctivitis. author index key: cord- - wnmdvg authors: nan title: p – p date: - - journal: clin microbiol infect doi: . /j. - . . _ _ .x sha: doc_id: cord_uid: wnmdvg nan resistance rates (rr) over a period of years were generally better than those reported for a total of icus. the mean mrsa rr was . %, for all the sari icus, whereas it was only . % in the study icu. by the end of duration of treatment for pneumonia had been reduced to - days and written guidelines on empiric antibiotic treatment and prophylaxis were revised with respect to the resistance situation of the study icu. the significant decrease between and in total antimicrobial ad from , to in the study icu resulted mainly from the reduced consumption of nd generation cephalosporins, carbapenems and imidazoles. ni did not change significantly over time. compared to the year , the costs for antibiotics were halved from € , to , , which corresponds to € . /pd and € . /pd, respectively. the percentage of antibiotics in the total icu budget for pharmaceuticals decreased from . % to . %. conclusion: surveillance and feedback of antibiotic use and resistance can serve as a valuable quality control instrument and can have an impact on antibiotic treatment. from to , antibiotic use was reduced by % and costs for antibiotics/pd were cut by two third in the icu study without any increase in device associated nosocomial infection rates. the resistance situation was generally better than in all sari icus, but showed heavy fluctuations. similar illness burden but different antibiotic prescription to children: a population-based study k. hedin, m. andre, a. håkansson, n. rodhe, s. mö lstad, c. petersson (växjö, falun, malmö, linköping, se) objectives: respiratory tract infections are the most common reason for antibiotic prescription in sweden as in other countries. the prescription rates vary markedly in different countries, counties and municipalities. the reasons for these variations in prescription rates are not obvious. the aim of the study was to find possible explanations for different antibiotic prescription rates in children. therefore a prospective population based log book study was conducted in four municipalities which, according to official statistics, had high and three municipalities which had low antibiotic prescription rates. methods: during one month, parents recorded all infectious symptoms, physician consultations and antibiotic treatments, from -month-old children in a log book. the children's parents also answered a questionnaire about socioeconomic factors and concern about infectious illness. results: antibiotics were prescribed to . % of the children in the high prescription area and . % in the low prescription area (crude or . ( % ). after multiple logistic regression analyses taking account of socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations, differences in antibiotic prescription rates remained (adjusted or . ( %ci . - . )). the variable that impacted most on antibiotic prescription rates although it was not relevant to the geographical differences was a high level of concern about infectious illness in the family. conclusion: the differences in antibiotic prescription rates could not be explained by socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations. the differences may be attributable to different prescription customs, in which case physicianś prescription patterns are not always rational. decreasing outpatient antibiotic prescribing in germany, germany, - , does not include newer macrolides, fluoroquinolones and extendedspectrum beta-lactams w.v. kern, k. de with, k. nink, h. schrö der (freiburg, bonn, de) objective: the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project has shown that outpatient antibiotic prescribing in germany has been comparatively low among european countries. we assessed trends over time and regional variation of outpatient antibiotic use in germany, and wondered if the observable decreasing trend included all drug classes to a similar extent. methods: prescription data (compulsory health insurance covering > % of the population, sample of . % until the year , all prescriptions thereafter) were analysed using the atc/who methodology and current ddd definitions. we specifically defined the following drug groups: ''basic'' penicillins (bpens, oral penicillin or aminopenicillins), extended-spectrum betalactams (esbls, oral cephalosporins, staphylococcal penicillins, aminopenicillin/betalactamse inhibitor combinations, parenteral cephalosporins and broadspectrum betalactams), newer macrolides (nmls, roxithromycin, clarithromycin, azithromycin) versus older macrolides (omls). quinolones (fqs), folate synthesis inhibitors (t/ss) and tetracyclines (tets) were also assessed. data were expressed in yearly ddd/ persons covered by the insurance (ddd/ ). findings: outpatient prescribing in was ddd/ (corresponding to . did = ddd/ and day) and decreased to ddd/ in the year and to ddd/ in . the decreasing trend over the last years was observed in all regions. the decrease was most significant for omls () %), t/ss () %), tets () %), and bpens () %) while there was no decreasing use of esbls (± %) and increases in the rate of prescribing nmls (+ %) and fqs (+ %). tets and bpens, however remained the most prescribed antibiotics in . regional variations in remained large for bpens (> -fold) with very low prescribing rates in the eastern region, but were small for t/ss, nmls and fqs (< -fold). conclusions: over a decade we observed a % decreasing outpatient antibiotic prescribing that included relevant antibiotic drug classes except esbls, nmls and fqs. the relative increase was most significant for fqs. severe community-acquired pneumonia admitted to the intensive care unit: impact of antibiotic therapy delay on hospital mortality antibiotic therapy were enrolled in the study. pts were divided in groups according to time to treatment (< h gi, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . baseline severity scores (apache ii, sofa, psi, curb- ), microbiological documentation, and hospital outcome were compared for all groups. results: pts were included in the study. microbiological documentation was achieved in % of all pts, positive blood cultures in / ( . %), s. pneumoniae in / ( %). mean age was ± , apache ii . ± . , sofa . ± . , psi ± , curb- . ± , mechanical ventilation in . % and vasopressors use in . %. overall icu and hospital all-cause mortality were . % and . %, respectively. baseline severity scores were comparable in all groups and their respective hospital mortality is provided in table . conclusions: in severe cap, treated with a combination therapy, time to treatment seems to have an impact on hospital all-cause mortality. based on our results, antibiotic treatment should be initiated within the first hours after hospital admission. (period ii) -cefoperazone/ sulbactam ( g a day) as monotherapy were used as the empirical antibacterial therapy of vap. the rotation from cefepime to cefoperazone/sulbactam was performed due to our previous study demonstrated high frequency of esbl producers among enterobacteriaceae. the samples from the lower respiratory tract were obtained by mini-bal. the sensitivity of microorganisms to the antibiotics studied (ceftazidime, cefepime, cefoperazone/sulbactam and carbapenems) was determined by the disk diffusion method. results: the main pathogens of vap were s. aureus ( %), p. aeruginosa ( %), enterobacteriaceae ( %) and this structure did not changed during both periods. the antibiotic sensitivity of p. aeruginosa and enterobacteriaceae (k. pneumoniae and e. coli), was studied separately. a high level of resistance of enterobacteriaceae to cefepime can be explained by the strains prevailing in the given icu, which produced extended spectrum beta-lactamases (ctx-m). the resistance of enterobacteriaceae to cefepime was . % in i period and . % in ii period, to ceftazidime - . and %, meropenem - and %, imipenem - . and . %, cefoperazone/sulbactam - . and . %, respectively. a change of cefepime for cefoperazone/sulbactam was not followed by any decrease of enterobacteriaceae resistance level to cefepime during ii period. the resistance level of p. aeruginosa to cefepime was . % in i period and . % in ii period, to ceftazidime - . and . %, meropenem - . and . %, imipenem - . and . %, cefoperazone/ sulbactam - . and . %, respectively. conclusion: the exclusion of cefepime for months didn't improved the sensitivity of enterobacteriaceae to this medication. the level of resistance of p. aeruginosa and enterobacteriaceae to cefoperazone/sulbactam did not increased despite a wide use of this antibiotic during months. antibiotic consumption in german acute care hospitals m. steib-bauert, k. de with, e. meyer, p. straach, w.v. kern (freiburg, frankfurt, de) objective: outpatient antibiotic use in germany differs substantially between eastern and southern parts of the country (relatively low use) and western part (relatively high use). there is no nationwide estimate of hospital antibiotic use and its geographic variation if any. the aim of the present study was to provide an estimate of recent hospital antibiotic use density in germany and to identify basic unit/hospital characteristics associated with excess use. methods: data on hospital consumption of systemic antibiotics in anatomical therapeutic chemical (atc) class j were obtained from a convenience sample of acute care hospitals in germany that participated in an ims survey in the year and had complete data (dispensed drugs and patient-days per year) for at least one non-paediatric, non-psychiatric department or ward. a total of non-icu surgical departments/wards, non-icu non-surgical (general medicine, oncologyhaematology, neurology/stroke) departments/wards, and icus covering > million patient-days were analysed. data were expressed in ddd (who/atc definition version ) or ''prescribed/recommended daily doses'' (pdd, better reflecting [high] dosages given to hospitalized patients) per patient days (ddd/ and pdd/ ). findings: the weighted mean over all departments/wards incl. icus was . ddd/ ( . pdd/ ). as expected, icu antibiotic use density was much higher than use in non-icu areas, and use in haematology-oncology was higher than in other non-surgical departments/wards. in univariate analyses, bed-size category and university affiliation (icus, surgical wards), region (icu, surgical and non-surgical wards) and haematology-oncology as specialty (non-surgical wards) were associated with use density, but these associations were only partly confirmed in multivariate logistic regression analyses of factors associated with excess ( ‡ %) use density which showed university affiliation and haematology-oncology to be independently associated with high use. conclusions: based on this hospital sample, antibiotic use in german hospitals shows little, non-significant regional variation and appears to be similar to what has been described from other european countries. adjustment of the data at least for university affiliation and haematology-oncology is important in comparative analyses of hospital antibiotic consumption. impact of formulary change in medical intensive care unit on outcome of infection and antimicrobial resistance sought to evaluate a formulary change and impact it has on infection and resistant. methods: prospectively, all patients in a -bed icu were followed for a period of months in phase i ( patients per patient days) and to collect baseline data after a decrease in the use of piperacillin-tazobactam (pt) when substituted by cefepime for a period of months in phase ii ( patients per patient days). results: total infections in phase i vs. phase ii were lower respiratory tract (lrti) patients ( %) vs. patients ( %); urinary tract infection (uti) patients ( %) vs. patients ( %); and sepsis of undetermined aetiology patients ( %) vs. patients ( %), respectively. there were no significant differences in death ( % vs. %) , cure or improvement of infection ( % vs. %), readmission to the unit ( . % vs. . %), hospital risk of death ( . % vs. . %), mean length of icu stay ( . days vs. . days), or rates of nosocomial infection ( . % vs. . % for lrti; . % vs. . % for uti; . % vs. . % for soft tissue infection; . % vs. . % for bacteremia; . vs. . per patient days for intravenous catheter infection) in phase i and ii respectively. the cost of antimicrobial acquisition in phase i and ii were $ and $ per patient respectively (p < . ). the mean antimicrobial treatment costs per patient for pt were $ vs. $ and cefepime were $ vs. $ in phase i and ii respectively (p < . ). the in vitro susceptibility and rate of infection and colonization with escherichia coli were unchanged in both study periods. there were vs. staphylococcus aureus (p < . ); of these percnt vs. percnt were methicillinresistant s. aureus and vs. enterococcus faecium ( % vs. % vancomycin-resistant enterococci) in phase i and ii respectively. there were % vs. % pseudomonas aeruginosa and % vs. % klebsiella pneumoniae extended spectrum beta lactamases in phase i and ii respectively. conclusion: the implementation of formulary substitution of pt to cefepime in the medical icu had resulted in a decrease in the use of pt. in addition, there were decreased costs and less s. aureus infections without adversely affecting the outcome of infection or antimicrobial resistance. intravenous antibiotic use in scottish hospitals; evaluation of the glasgow antimicrobial audit tool r.a. seaton, d. nathwani, p. burton, e. douglas (glasgow, dundee, uk) introduction: there are few data on antibiotic prescribing within scottish hospitals and a coordinated multisite point prevalence survey had not been performed before. there is concern that antimicrobials are overused in hospitals. methods: antibiotic use in acute medical and surgical units in scottish hospitals across trusts, was investigated using a point prevalence survey. data were collected by pharmacists. appropriateness of the iv route of administration was determined by review of data by an infectious diseases physician (idp) and compared with a specifically designed computerised algorithm. the idp also judged the appropriateness of the chosen iv agent against local guidelines. patients from hospitals in regions were surveyed on a single day. ( . %) were receiving an antibiotic, ( . %) intravenously. receiving oral antibiotics had received an iv previously. median duration of iv therapy was days (iqr - days) and time from iv to oral switch was . ( ) ( ) ( ) ( ) ( ) . the idp judged appropriate iv route in % patients compared with . % by the algorithm. the sensitivity of the algorithm was . % and specificity . %. the positive predictive value was . % and the negative predictive value was . %. the idp judged iv agents to have been chosen and administered appropriately in %. most frequently prescribed iv agents were rd generation cephalosporins ( gc) ( . %), co-amoxiclav ( . %), metronidazole ( . %), glycopeptides ( . %). significant regional differences were seen for most antibiotic groups including gcs ( . % (site ) vs . % (sites , , , ) , p < . ) and glycopeptides [ . % (site ) vs . % (site , , , ) , p < . ]. it is possible to coordinate, collect and compare data from scottish hospitals. the gaat gives a good estimate of the appropriateness of iv therapy. significant differences in prescribing patterns between similar patient groups across different hospital sites were demonstrated. such data may usefully inform local and national audit and support prescribing initiatives. associations between continuous variables were tested in univariate analysis with the spearman correlation test (r). multiple linear regression analysis was performed in a backward stepwise approach. results: the median rate of total hospital glycopeptides use was . (range . to . ) ddds per , pd with higher consumption in large public hospitals. consumption was higher in intensive care areas (median . ; range . to ) than in surgery areas (median . ; range . to . ) and in medicine (median . ; range to ). glycopeptides use correlated with number of central line per , pd (r: . ; p: . ) and with size of the various areas in the hospital (for intensive care, r: . ; for medicine areas, r: . and for surgery areas, r: . ; p < . ). median incidence of mrsa was . per , pd. incidence of mrsa explained a small proportion of the variation in hospital glycopeptides consumption (r : . ). in a multivariate linear regression model, incidence of mrsa and number of beds in surgery areas were independent predictors of total glycopeptides use in the hospital (r adjusted: . ). after controlling for these factors, number of central-line per , pd was no more associated with glycopeptides use. conclusion: in our hospitals, total glycopeptides use was not heavily determined by incidence of mrsa. although glycopeptides use in surgery areas was not the highest, the total number of surgery beds in the hospital explained a large variation of the total hospital glycopeptides use. therefore we had to take it into account to interpret these consumption and to decide further evaluation. antibiotic management of acute lower respiratory tract infections among dutch elderly patients in primary care j. bont, c. birkhoff, t. verheij, e. hak on behalf of esprit objectives: acute lower respiratory tract infection (lrti) can cause various complications leading to morbidity as well as mortality notably among elderly patients. antibiotic treatment of lrti is common, despite dutch clinical guidelines recommending antibiotics only in case of pneumonia or high risk of serious complications. we assessed the course of illness and outcome of pneumonia, acute bronchitis and exacerbations of copd or asthma among dutch elderly patients in primary care and assessed whether gps were inclined to prescribe antibiotics more readily to patients with potential risk factors for complications in acute bronchitis or exacerbations of copd/ asthma. methods: we retrospectively analysed medical data from , episodes of lrti among patients ‡ years of age presenting in primary care to describe the course of illness and outcome. the relation between prescriptions of antibiotics and patients with risk factors for a complicated course was assessed by means of multivariate logistic regression. risk factors for a complicated course included heart failure, history of myocardial infarction, angina pectoris, diabetes, history of stroke, dementia, malignancy, and history of pneumonia or hospitalisation in preceding year. results: one or more complications arose in % of episodes of lrti. among these, % suffered from pulmonary complications, % had cardiovascular complications (heart failure, myocardial infarction etc.), % had a protracted course and . % had a diabetes event. in . % of the patients complications led to hospital admission and in . % lrti were fatal. antibiotics were more readily prescribed to patients aged ‡ years, when heart failure was present and in patients with diabetes. no significant association was observed in patients with other co-morbid conditions. patients diagnosed with an exacerbation of copd or acute bronchitis with a history of pneumonia or hospitalisation in the preceding year were not more likely to receive antibiotics. conclusions: a considerable part of elderly patients with a lrti suffers from a severely complicated course in primary care. although gps are inclined to prescribe more readily antibiotics in the very old and those with heart failure or diabetes, other potential risk factors are not taken into account. objectives: in this study it was aimed to analyse the infectious diseases (id) trainees' night/weekend shift consultation process in terms of patient and consultant characteristics, types of recommendations, and compliance with recommendations. methods: all consultations performed by id trainees in night shift and at the weekends between june th-august th were analysed in terms of consultation type [treatment continuation (tc), consultation for surgical antibiotic prophylaxis (pa), and consultation with or without a request of a specific antibiotic (others)]. appropriateness of recommendations was assessed the day after the consultation by infectious diseases specialists (ids). adherence to recommendations was assessed days after the consultation by idss. recommendations including antibiotics were considered appropriate, if they were appropriate according to national and international guidelines. recommendations were considered complied, if they were done in up to hours after the consultation (except the consultations in the emergency medicine and the consultations in which antibiotics were started by the counselling idss). results: of consultations was for tc, was for pa and was for others. the clinic where id consultations were requested mostly was general surgery clinic ( / , . %) . in % of all consultations trainees consulted the specialists. overall consultations ( for sp, for a clinical infectious disease diagnosed clinically, for an infectious disease diagnosed microbiologically) were for requesting spesific antibiotic(s). pa were approved in of consultations. antibiotic was not recommended in of other consultations. in six of consultations for pa antibiotic was changed for a clinically diagnosed infectious disease. in one of consultations for tc antibiotic was changed due to lack of response to the given antibiotic, in others tc was approved. inappropriate antibiotic recommendation rate was . % ( / , inappropriate choice, inappropriate dosage, one antibiotic unnecessary). overall compliance to id recommendations was . % ( / ). rate of compliance to antibiotic recommendations was evaluated in consultations and was found . % ( / ) and was higher than compliance to other (microbiology etc.) recommendations ( . %, / , chi square p < . ). conclusion: methodologies to improve the compliance to nontreatment based recommendations and optimizing antibiotic selection is necessary. study of the influence of online practice guidelines on the appropriateness of antibiotic prescribing in a university-affiliated psychiatric hospital j.f. westphal, c. nonnenmacher, d. gregoire, m. hittinger, c. oulerich, f. jehl (brumath, strasbourg, fr) background: problems with the dissemination of guidelines are frequently cited as a major reason for failure to impact practice. reviews of the effectiveness of various methods of guideline dissemination show that the most predictable impact is achieved when the guideline is made accessible through computer-based reminders that are integrated into the clinician's workflow. we report a time-series prospective investigation aimed at comparing the appropriateness of antibiotic (ab) orders for pneumonia at the treatment initiation level after vs. before having embedded our current ab guidelines for pneumonia in the computerized physician drug-order entry system of our teaching psychiatric hospital comprising adult beds. methods: in total, consecutive ab orders for pneumonia were evaluated by the pharmacy department, including orders just before and orders just after implementation of online ab guidelines. appropriateness of ab orders relative to the guidelines was assessed according to criteria: ( ) the choice of ab with respect to the mode of acquisition (community-or hospital-acquired) of pneumonia or the presence of clinical risk factors for involvement of gramnegative bacilli, ( ) the daily dosage, ( ) the planned duration of treatment. data were extracted from the computerized infection declaration system that recorded all ab-requiring infections in our hospital. results: the number of ab orders with at least criterion of inappropriateness tended to decrease, yet not significantly (p = . ), after vs. before implementation of online guidelines: / ( . %) and / ( . %), respectively. the number of criteria of inappropriateness relative to all ab orders for pneumonia was significantly lower in the post-implementation period: . % vs. . % before implementation (difference . %, % ci . - . , p < . ) , with a trend to a decreased number of orders containing more than criterion of inappropriateness. analyzed separately, the numbers of inappropriate orders for the choice of the ab, or the daily dosage, or the planned duration of treatment decreased, yet not significantly (p > . for each criterion), in the post-vs. preimplementation period : vs. , vs. , vs. , respectively. conclusion: in this study, the moderate impact on ab prescribing practices of online guidelines available at the time of drug order shows that additional types of intervention are needed to improve further the quality of ab prescribing. material: the pilot hospitals had a median capacity of (range, to ) beds; their regional distribution was representative of population size; were general hospitals, teaching hospitals and general hospitals with teaching beds. results: ams were internists ( ), microbiologists ( ) and pharmacists ( ). amts included a mean of members who met every weeks on average. all hospitals irrespective of size or affiliation had undertaken a wide range of antibiotic management interventions in , which increased in ; these included (in and , respectively) : major review of formulary (in and hospitals), development of clinical guidelines ( and topics), restricted access to selected antibiotics (carbapenems, glycopeptides, quinolones, new drugs; in and hospitals). in , antibiotic consumption databases were established in hospitals and antibacterial susceptibility databases in hospitals. in , cross-analysis of these databases was performed in hospitals. in , prescribing assistance, antibiotic stop orders, treatment streamlining and iv/po therapy switch were implemented in , , and hospitals, respectively. in , hospitals reported a better use of target antibiotics, hospitals a decrease in consumption of restricted antibiotics, hospitals a decrease of total antibiotic consumption, hospitals a decrease in high consumer departments. conclusion: all hospitals participating in the amt pilot scheme have developed multiple antibiotic policy interventions and established monitoring and guidance of antibiotic prescription. preliminary data from some hospitals indicated success in meeting self-defined targets of appropriate use and reducing the consumption of selected antimicrobial agents. more systematic evaluation using standard quantitative and qualitative indicators is planned. antibiotic prescribing practices at two linked london teaching hospitals p comparison of different antibiotic consumption measurement methods in large multidisciplinary hospital e. pujate, i. apine, u. dumpis (riga, lv) objectives: antibiotic selection pressure is determined by the total amount of antibiotics, number and density of patients treated with antibiotics in the particular geographical area. several antibiotic consumption detection methods should be combined in the hospital setting. our objective was to evaluate efficacy of different approaches in large multidisciplinary hospital. methods: point prevalence studies were repeated annually at [ ] [ ] [ ] in stradins university hospital ( beds) in latvia. all patients receiving antibiotics on the day of the survey were identified and their medical records were reviewed. data on antibiotics, dose and route of administration were collected. in addition, annual data on antibiotics dispensed to the departments were collected from pharmacy. total used grams for each antibiotic were expressed into defined daily doses (ddd-who). bed days (bd) and admission days (ad) were used as denominators. results: table total use of antibiotics in stradins university hospital hospital - the most commonly used antibiotic groups in the pharmacy study were st generation cephalosporins ( . ddd/ bd in , . in , . in ) and penicillin's with extended spectrum ( . , . , . ) followed by fluoroquinolones ( . , . , . ) and metronidazole ( . , . , . ). there was no significant difference between distribution of different antibiotics from prevalence and pharmacy studies if calculated in ddds. in contrast, distribution of antibiotics calculated per patient in the prevalence study was quite different; st generation cephalosporins ( . %, . %, . % in , , respectively) and fluoroquinolones ( . %, . %, . %) with smaller proportion of extended spectrum penicillins ( . %, . %, . %) and metronidazole ( . %, . %, . %). conclusions: there were no differences in the distribution of antibiotics calculated in ddds per bed days and admissions. distribution of antibiotics in annual pharmacy studies and point prevalence studies if calculated in ddds were also similar. in contrast, the prevalence data expressed as a proportion of patients with selected antibiotics showed quite different distribution. studies using only ddds may overestimate use of certain antibiotic groups in our setting where who ddds are significantly different from actual pdds used. a study of prescribing patterns and errors of antibiotics in a saudi hospital m. al-jamal, m. al-barrak (riyadh, sa) background: the term ''prescribing patterns'' has been used extensively in studies to describe different aspects of the prescribing process. antibiotics as well as other drugs are prescribed for the purpose of achieving definite therapeutic outcomes that improve a patient's quality of life while minimizing risk. in the clinical literature, the incidence of antibiotics prescribing errors ranges between . % and . %. objective: in this study we will address antibiotics prescribing patterns and the incidence of prescribing errors in a tertiary hospital and the potential relationship between them. methods: a prospective study of all prescriptions in a -month period (june to august ) in a tertiary hospital has been analysed. the hospital provides both primary and secondary levels of care. criteria used include frequency of selected prescribed drugs, average number of items per prescription, compliance to the hospital formulary, frequency of prescriptions for antibiotics, generic prescribing and diagnosis. the prescribing patterns and the incidence of prescribing errors were performed. results: total number of prescriptions for the -month study was , . emergency room (er) and primary care have the highest number of prescriptions ( . %). the average number of items per prescription is . . the most prescribed drugs by primary care ( . % errors), emergency are antibiotics ( . %), medicine ( . ), ophthalmology ( . ), gynaecology ( . ), and paediatrics ( . ). the prescription errors were . % in primary care and . % in emergency department. discussions and conclusions: over prescriptions were included in this study. the incidence of prescribing errors was . % the average number of items per prescription was . . total prescription errors are also related to frequency of prescribing antibiotics. there was a relation between prescribing of antibiotics and prescribing of trade names (p < . ), and compliance to the hospital formulary (p < . ). several factors influence prescribing patterns and variations in prescribing rates has been identified. these include general physician behavior, differences in morbidity and mortality patterns, social perception toward illness, and physician clinical skills, experience and qualification, as well as physician continuing education and training. special antibiotic prescribing guidelines and restrictions should target primary care and emergency department physicians. effect of a policy for restriction of selected classes of antibiotics on antimicrobial drug cost and resistance of the non-restricted antibiotics. the logistic regression model we performed showed that the new policy had an independent positive effect on the in vitro antimicrobial susceptibility of pseudomonas aeruginosa (p = . ) but not of acinetobacter baumannii and escherichia coli isolates. conclusion: our data suggest that there are considerable limitations of the programs aiming to reduce the consumption of restricted antibiotics through the approval of their use by specialists, at least in a proportion of settings. education programs that aim to involve the medical staff directly responsible for the care of patients in voluntary decisions regarding the appropriate use of antimicrobial agents may have more profound and sustainable success, and thus, deserve to be studied. estimating hospital versus ambulatory care consumption of antibiotics in southwestern germany k. de with, m. steib-bauert, h. schrö der, k. nink, w.v. kern (freiburg, bonn, de) objective: preliminary data from the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project indicated that the proportion of hospital care (hc) antibiotic use on total antibiotic use in several european countries ranges between and %. only few countries, however, have so far been able to report representative countrywide information on both hc and ambulatory care (hc) antibiotic consumption. we estimated ac versus hc consumption of antibiotics for one of the german federal states located in the southwestern part of the country with a . million population. methods: data on hc consumption (atc class j ) were obtained from a convenience sample of acute care general hospitals (n = ), extrapolated to state-wide consumption (using official statistics for the total state-wide general plus special non-psychiatric/non-paediatric/non-radiotherapy hospitals), expressed in defined daily doses per inhabitants and day (did), and finally compared to ambulatory care antibiotic use density in the same region and period of time (years and ) . findings: the estimated state-wide hc consumption of antibiotics was . did ( % confidence interval, . to . did) in both years. state-wide antibiotic consumption in the ac setting during the same time was did (~ % of total consumption). ac consumption of fluoroquinolones ( . - . did, %) and macrolides/clindamycin ( . did, %) made up a major proportion of total use of that drug classes. conclusions: hospital antibiotic use in the southwestern part of germany can be estimated to contribute~ % to overall antibiotic consumption in the general population. antibiotic use profile and temporal trends during a -year period at a greek university hospital: implications for antibiotic policy changes e.i. kritsotakis, p. assithianakis, p. kanellos, n. tzagarakis, m.c. ioannides, a. gikas (heraklion, gr) objectives: to investigate the profile and temporal trends of inpatient antimicrobial use over a -year period at the university hospital of heraklion crete, greece. further, to examine the way in which frequency of data collection and stratification by different patient-care areas provides guidance to antibiotic policy changes. methods: retrospective monitoring of antimicrobial consumption was carried out according to the who anatomic therapeutic chemical classification (atc) and defined daily dose (ddd) measurement methodology. pharmacy records were used to obtain aggregate data of drug deliveries to individual wards. results were expressed as usage density rates in ddds per bed-days (ddd/ bd). linear regression was used in order to assess the statistical significance of a temporal trend in usage densities. results: during - , hospital-wide antimicrobial use (atc group j ) significantly increased by %, from . to . ddd/ bd. the annual average increase rate was . ddd/ bd. stratification by clinical service demonstrated differences in the intensity and profile of class use, as well as varying temporal trends (figures , ) . pooled usage rates in ddd/ bd, overall percentage increases and annual average increase rates were respectively . , . %, . for medical wards; . , . %, . for icu's; and . , . %, . for haemato-oncology wards. surgical wards had a fairly constant usage rate ( . ). a shift towards the newer broad-spectrum antibiotics to the detriment of the older penicillins and cephalosporins was noted in all hospital areas. conclusion: surveillance of aggregate data on the consumption of antimicrobials using the atc/ddd system provided a clear picture of the profile of hospital usage. monthly data over a sufficient surveillance period allowed the assessment of temporal trends. stratification of usage rates by clinical service allowed areas of concern to be specified. thus, surveillance of monthly antimicrobial consumption rates stratified by patientcare area can provide a simple, rapid and efficient tool for triggering antibiotic policy changes in the hospital and targeting more detailed quality-of-use audits. appropriate use of aminoglycosides: the impact of an antibiotic control team c. rioux, p. lesprit, j.r. zahar, a. hulin, a. bernier-combes, c. brun-buisson, e. girou (créteil, paris, fr) objectives: many factors are involved in the appropriate use of aminoglycosides (ag), such as modalities of administration, serum monitoring and duration of treatment. we assessed prospectively the risk factors and the impact of an antibiotic control team on the appropriateness of ag prescriptions. methods: in a setting of a restricted delivery system of ag in our hospital, we first performed an observational audit (oa) to assess the appropriateness of prescriptions including justification of prescribing, adequacy of drug choice, adequacy of administration modalities, modalities of serum monitoring and duration of treatment. after implementation of specific guidelines hospital wide, we then performed an interventional audit (ia) where an antibiotic control team could interfere when ag prescriptions were considered inappropriate. appropriateness of ag prescriptions between the audits was then compared. results: prescriptions were analysed. during the ia, % of prescriptions were modified by the control team. as compared to the oa, prescriptions in the ia were significantly more appropriate with regard to treatment duration ( vs %, p = . ) and serum monitoring ( vs %, p = . ). median treatment duration was shorter in the ia ( d) than in the oa ( d) (p < . ). a logistic regression model showed that risk factors for appropriate treatment duration were (adjusted or, % ci, p value): hospitalization in intensive care unit ( . , . - . , . ) , polymicrobial infection ( . , . - . , . ) and antibiotic control team intervention ( . , . - . , . ) . table: conclusions: despite a restricted delivery system, ag use was frequently associated with excessive treatment duration and errors in monitoring modalities. reinforcing practice guidelines through direct counselling improved appropriateness of prescriptions. hospital antibiotic consumption in southern and eastern mediterranean countries: preliminary results from the armed project p. zarb, m.a. borg, h. goossens, m. ferech for the armed participants introduction: armed is an international research project investigating antimicrobial resistance and consumption in southern and eastern mediterranean countries through the collection of comparable and validated antimicrobial resistance data as well as information about antibiotic consumption patterns and infection control initiatives. objectives: the consumption part of the study aims to collect data on antimicrobial use within participating hospitals in the region, which information is currently unavailable. methods: data collection is planned over a -month period using anatomical therapeutic chemical (atc) classification, a validated methodology adopted by the european surveillance of antimicrobial consumption (esac -www.ua.ac.be/esac). hospitals are participating: cyprus ( hospitals); egypt ( ); jordan ( ); malta ( ); tunisia ( ); turkey ( ). results are expressed in ddd/ bed-days. results: data from , the first year of data collection, indicates that turkish hospitals seem to show the lowest overall consumption [ - ddd/ bed days], whilst the cypriot hospitals show highest values [ - ddd/ bed days]. the most common antibiotics used are the beta-lactams, especially the penicillins although in jordan and turkey cephalosporin consumption is very close to the penicillins. broad-spectrum penicillins [j ca] are the mostly utilised penicillins in cyprus, jordan and tunisia whereas in malta and turkey the combination penicillins [j cr] are the most widely used. there is more variability where cephalosporin consumption is concerned. cyprus utilises mainly first generation, jordan and malta the second generation. in egypt, tunisia and turkey there is significant variability between hospitals; nevertheless use of third generation cephalosporins appears to be significant. conclusion: a significant variability was evident between countries. this is likely to be multifactorial depending on the antibiotics licensed in a country, the national and/or hospital formulary, the type of hospital as well as any antibiotic donations that are relevant in some of the study hospitals. nevertheless, the preliminary results suggest that trends within hospitals of the same country tend to be similar. furthermore, the region as a whole seems to utilise a considerable quantity of broad-spectrum antimicrobials. this can be a factor in the high prevalence of resistance already documented in the study. russian pharmacoepidemiology study of the antibiotic prescription during pregnancy results: mean age of the patients was . ± . (min - , max - ) years, mean gestational ages at admission to hospital was . ± . ( to ) weeks. most often ( . %) infection was community acquired and . % -nosocomial, in % patients there was not to estimate origin of the infection. the most prevalent infections during pregnancy in russia was urinary tract infections - . %, std - . %, candidiasis - . %, rti - . % therefore the most interest was analysing the antibiotic prescription for uti in pregnancy (table) . in % cases were used topical (intravaginal) antimicrobial administration. most often of topically administrated antimicrobials ( . % of all prescriptions) were prescribed combined drugs included antibacterials and amtimycotics. in . % cases antimicrobials were prescribed systemically. mostly prescribed antimicrobials were beta-lactams ( . % for outpatients and . % for inpatients), ampicillin was prescribed more often ( . % for outpatients and . % for inpatients). amoxicillin + clavulanic acid was prescribed in . % of outpatients and . % inpatients pregnant women with uti. cephalosporins were prescribed in . % and . % for outpatient and inpatient uti (mainly iii-and ist generations). mitroimidazoles - . - . % (in general metronidasole), nitro-furanes - . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . %, aminolglycosides - . - . % were prescribed quite often but unjustified. other antimicrobials (fluoroquinolones, doxicicline, antiviral drugs, antifungals) were prescribed relatively rarely. despite the fact that most prescribed drugs were class b by fda, . % all antimicrobials prescribed to pregnancy were class c, . % class d and . % were unclassified. conclusions: most often prescribed antimicrobials for uti (the most prevalent infections during pregnancy in russia) are betalactams and combined topical antibacterials. in . % cases were prescribed antimicrobials of class c, d or unclassified by fda. in % outpatient and . % inpatient were used antibiotics with low in vitro activity for uropathogens. objectives: to study the dynamics of the antibiotic usage in children from orphanages located in different russian cities as the result of interventions with the increased use of the most active antimicrobials and restrictions on use of the least active. methods: the study was performed in orphanages ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) from cities of european russia (moscow, saint-petersburg, smolensk, karachev, bryansk) . use of antimicrobials during the previous months was analysed upon reviews of medical records of children < years in . appropriate recommendations on predominant use of selected beta-lactams (e.g. amoxicillin/clavulanate -amc) with restriction of antimicrobials of other classes (e.g. co-trimoxazole -sxt) were made where applicable on the basis of the expert analysis of antibiotic usage and pneumococcal nasopharyngeal resistance rates. repeated antibiotic usage analysis was performed months later in upon reviews of medical records of children < years. results: total usage of antimicrobials increased . increase of resistance to pen and aminopenicillins. enhanced use of cephalosporins led to increase of resistance to these drugs. in spite of recommendations to restrict usage of am/ox, aminoglycosides and sxt, the analysis showed that these antimicrobials still accounted for . %, . % and . % of all prescriptions, respectively, thus dictating the need for further enforcement measures. antibiotic consumption in ambulatory care in latvia, s. berzina, m. ferech, g. ozolins, h. goosens (riga, lv; antwerp, be) objectives: to collect data on antibiotic consumption in ambulatory care (ac) in latvia according to the esac data collection protocol. esac (european surveillance of antimicrobial consumption, granted by dg sanco of the ec) is an international network of national surveillance systems, aiming to collect reliable and comparable data on antibiotic consumption in europe. methods: the data on ac antibiotic consumption for have been collected using atc/ddd classification (who, version ) and expressed in defined daily doses per inhabitants per day (did). data were obtained from the state medicinal agencybased on the reports of the wholesalers for ac. results: the overall use of antibiotics in ac was . did in , which positions latvia to countries with comparatively low antibiotic consumption in europe. the mostly used class of antibiotics in ac were penicillins with extended spectrum (mainly amoxicillin) - . did ( . %). other frequently used antibiotics were tetracyclines (mainly doxycycline), representing . did ( . %), combinations of penicillins/with betalactamase inhibitors (essentially co-amoxiclav) - . did ( . %), macrolides (mainly clarithromycin) . did ( . %), fluoroquinolones (essentially ciprofloxacin) - . did ( . %) and combinations of sulphonamides and trimethroprim, incl.derivatives - . did ( . %). the most frequently used antibiotics in ac in latvia, in , were amoxicillin ( . did) , doxycycline ( . did) , and co-amoxiclav ( . did) . conclusions: valid data on outpatient antibiotic use in latvia has been for the first time collected and delivered to european surveillance of antimicrobial consumption. this allows international comparison of the pattern of antibiotic consumption in latvia with other european countries. trends in glycopeptide antibiotics consumption over a -year period in a general hospital, athens, greece introduction: glycopeptide use is under restriction in hellenic hospitals since late ' s. the aim of our study was to record trends in their consumption over the last years in our hospital (''a. fleming'' general hospital - beds) and to correlate these data with the numbers of important gram (+) strains isolated in our hospital during the same time period. methods: we measured glycopeptide use for the period - by using data from the pharmacy computer. consumption was expressed as ddds/ patient days (abc calc . ). furthermore we correlated these data with data from the microbiology department concerning numbers of mrsa, mrse and enterococci isolated during the same period. results: glycopeptide consumption was . , . , . , . , . and ddds/ patient days for the years , , , , , ( % increase) . at the same time the cumulative number of mrsa, mrse and enterococci isolated were , , , , , respectively ( % increase) . when both types of data were put on the same graph, glycopeptide consumption correlated well with the number of important gram(+) strains isolated (figure). furthermore vre percentage among enterococci was , , . , . , . , for the study years respectively. it is worth noting that % of our mrsa strains were sensitive to rifampin, % to clindamycin, % to cotrimoxazole, % to clindamycin + rifampin and % to cotrimoxazole + rifampin. linezolid has not been introduced in our hospital yet. conclusions: (a) despite the restriction policy, a tremendous increase in glycopeptide use was recorded in our hospital during the study period and this correlated to the number of the important gram(+) strains isolated; (b) nevertheless, vre is not a significant problem for our hospital yet; and c. the huge increase in glycopeptide use could be avoided at least in part, since other, older and simpler antibiotics could substitute for glycopeptides in many cases. an audit of linezolid use in a university teaching hospital, galway, ireland objective: to audit linezolid use over a six-month period among the in-patient population of a -bed teaching hospital that includes most medical and surgical specialties with the exception of nephrology, rheumatology and orthopaedics. methods: a prospective audit was carried out of the prescribing of linezolid to in-patients from october to april . the ward pharmacist recorded the details of all patients who were prescribed linezolid. a chart review was performed to assess the profile of patients prescribed linezolid, clinical and microbiological indications for treatment, adherence to treatment guidelines and documented adverse events. results: over the -month period courses of linezolid were prescribed. fifty two percent of the patients for whom linezolid was prescribed were from surgical specialties; half of these patients were under the care of one surgeon. pneumonia was the clinical indication for use in % of cases and soft tissue infection in % of cases. the microbiological indication was clear in % of cases where mrsa or vre had been isolated. in % of cases therapy was either ( ) empiric with no significant organisms isolated prior to prescription of linezolid or ( ) therapy was directed against an organism that could have been treated with an alternative agent. duration of treatment exceeded to days in % of courses. an adverse event was recorded in the case of only one course of linezolid. conclusion: in more than a third of cases linezolid use was prescribed without clear justification. avoidable use of linezolid is associated with increased costs and risks of acquired resistance. participants prior to on an oral interview during which the interviewer filled in the answers. results: a total of cm specialists completed the inquiry. this represents approximately % of the national quorum. mean age was years ( - ). of the interviewed cm specialists worked full-time with more full-time employment in hospital labs (hl). next to routine microbiology, other activities performed by cm specialists are mainly the other domains of clinical biology, hospital hygiene and to a lesser extent quality control and lab management. almost two thirds of the interviewed cm specialists believes that their training hasn't prepared them properly for the tasks they are performing now. most desired changes include more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. cm does not exist as a separate speciality in belgium but is included in the 'clinical biology' speciality training. the majority of the respondents thinks that cm should become a sub-speciality (still part of clinical biology) but with a specific minimal training that needs to be defined. the majority of the cm specialists also believes that cm can share lab infrastructure with other disciplines and that the essential aspect of cm lies predominantly in the medical expertise. conclusion: cm training should put more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. the majority of the respondents feels that cm should become a sub-specialty (still part of clinical biology), with a well defined training curriculum. objectives: since more than years, all infectious disease consultations have been recorded in a computerized database (epi info . d, cdc). here we report on consultations of a fellow, conducted during year, compared with consultations conducted by two veteran board-certified infectious disease consultants during the same period. methods: we analysed computerized consultation records, including demographic details of patients; referring department; initiative for, route and purpose of the consultation; and recommendations; and compared between the different consultants. results: a larger percentage of veterans' compared to the fellow's consultations, were requested by attending physicians ( % vs. %, p < . ), while follow-up ( % vs. %, p < . ), laboratory results ( % vs. %, p < . ) or prescription for a restricted antimicrobial agent ( . % vs. . %, p < . ) were more prevalent in fellow consultations. the fellow had a higher rate of additional consultations (in which the patient was seen more than once) ( % vs. %, p < . ), and performed more bedside consultations ( % vs. %, p < . ) or consultation by curb side discussion ( % vs. . %, p < . ), and less consultations by telephone ( . % vs. . %, p < . ). diagnosis and prophylaxis were more often the purposes of the veterans' consultations ( . % vs. . %, p < . , . % vs. . %, p < . , respectively), and they also offered new diagnoses more frequently (p < . ). the veteran consultants more often conducted consultation for communityacquired infections ( % vs. %, p < . ), and more often started antibiotic treatment ( % vs. . %, p < . ). conclusions: significant differences were detected between consultations conducted during the first year of a fellow compared to those of veteran infectious disease consultants. these differences reflect the changing demands and activities in the consultant's work as experience and knowledge accumulate. periodic analysis of computerized data of consultations facilitates supervision as well as direction of consultants' work, addressing issues such as antibiotic use and patterns of microbial resistance. bridging the gap between health care and public health; capacity building in infectious disease control objectives: in recent years, the european union (eu) has developed and supported many activities in the field of communicable diseases. these activities not only concern surveillance networks of specific infectious diseases (e.g. enter-net for salmonella and escherichia coli infections, ewgli for legionella infections, eiss for influenza infections), but also eu training programmes like epiet (european programme for intervention epidemiology training) and a eu communicable disease bulletin. even more recent are the eu's initiative bichat to improve preparedness and response to bioterrorism, and the development of a eu cdc. moreover, a major part of the new programme of community action in the field of public health (ph) ( ) ( ) ( ) ( ) ( ) ( ) concerns id, with not only a commitment to improve information and information exchange, but particularly to strengthen the international rapid response capacity.all this, to illustrate the importance of idc on the eu agenda. methods: the european public health association (eupha) is an umbrella organisation for ph associations in eu. in eupha has created an eupha section idc bringing together eupha members with expertise in this field and representatives of the various above mentioned eu initiatives in order to: promote and strengthen research in the field of idc; provide a platform for the exchange of information, experience and research in idc; bring together researchers, ph practitioners and policymakers active in idc; encourage joint activities in idc; and improve idc training. results: by now the section has members from different countries. as of the section is represented in the ecdc advisory forum. different section activities: organising workshops, a breakfast meeting and a pre-conference meeting on timely idc topics during eupha conference. objectives: to establish a cross-border dutch-german network (www.mrsa-net.org) providing a user-friendly knowledge centre for hospitals, public health authorities, gps, nursing homes and laboratories. primary purpose is to aid in the reduction of mrsa-rates and limit the cross-border transmission of mrsa. guidelines and their implementation play a significant role in reaching these aims. cross-border (ca-) mrsa guidelines will be redesigned according to international standards and socio-cultural differences between the nations. methods: based on quality standards for safety and healthcare documentation used in high risk chemical organizations, a framework for a systematic content analysis of national mrsaguidelines was developed. national guidelines were analysed on the basis of this framework. results: a content analysis of the current national mrsaguidelines showed five dominating mrsa-perspectives: rule-, expert-, risk-, demand-and community-driven. german guidelines are mainly dominated by the rule-and expertdriven perspectives (guidelines are literally derived from law and follow the infection transmission route), in contrast to the dutch which focus on the demand of the user and the community (addressed to public health and acceptability of guidelines by users). conclusion: the analysis showed that the fact that there are different guideline-perspectives results in an enormous, confusing set of guidelines. the management and use of guidelines becomes uncontrollable and leads to an illusory organisation where healthcare workers don't act in accordance with the guidelines and start applying their own insights. this might lead to cost-increasing and contrasting situations. to implement guidelines successfully in a cross-border situation, a cultural and technical synchronisation alongside an integrated approach of the different perspectives of guidelines is necessary, inline with the current disease management models. further research about the redesign and the evaluation of those guidelines in practice will help achieving this. r. tsiklauri (tbilisi, ge) background: animal bites are a common but under recognized public health problem. it has been estimated that there are - bites each year in georgia, and based on an average visit and post-exposure treatments cost at list $ per year. despite the frequency and expense of these injuries, there is little information about the incidence of animal bites because of a lack systematic reporting and a lack of measurement of the quality and completeness of reported data. objectives: to investigate animal bites and rabies reported cases, revealed unreported cases, analyse and based on study results find more effective epidemiological measures of animal bites and deaths (due to rabies) prevention in georgia. methods: the capture-recapture method was used, along with log-linear modelling. for sources were used to identify victims: policlinic/ambulatory reports, hospital reports, animal control reports and victim reports. results: in - years dog and other animal bites were reported. the capture-recapture method estimated that there were unreported bites. during these period deaths due to rabies was registered in georgia and ( %) cases among them have been registered during the last years. the reasons of fatal cases were untreated ( %), uncompleted treated ( %) and late began post-exposure treated ( %) cases of bites (mostly dog bites). about % of bitted persons did not know about rabies and it's prevention measures. about % had incorrect information about prevention and only % of them knew epidemiological and clinical aspects of disease. about % of physicians who were responsible on quality post-exposure treatment had not an adequate knowledge. conclusion: dog and other animal bites are common but preventable injuries. to improve surveillance and prevention of rabies in georgia, the focus should be on educating the general public about the serious consequences of animal bite injuries and developing the animal's vaccination strategy. pharmacoeconomics and electronic resources p the expected economic burden of methicillinresistant staphylococcus aureus in complicated skin and skin structure infections: a modelling approach a. kuznik, r. mallick, d. weber (collegeville, chapel hill, us) objective: to model the expected rate of clinical failure of initial empiric therapy and economic burden likely to be associated with the increasing prevalence of methicillinresistant staphylococcus aureus (mrsa) in patients hospitalised with complicated skin and skin structure infections (csssi) in the united states. methods: using published data on ( ) the prevalence of mrsa and other bacterial pathogens causing csssi in the us, ( ) the in-vitro susceptibility rates of commonly used regimens in csssi in the us in relation to the most pervasive pathogens identified above, and ( ) estimated costs of failure of initial, empiric treatment from a recent study of a large us multi-hospital database, we developed a model to predict the expected clinical and economic impact of increasing prevalence of mrsa. specifically, clinical failure of of the more commonly used initial regimens in csssi was modeled in terms of their in-vitro susceptibility rates with respect to mrsa, weighted by mrsa prevalence. varying the rate of mrsa further yielded projected clinical failure rates and costs attributable to increasing levels of methicillin resistance over time. results: given current % prevalence of s. aureus pathogens in csssi, half of them methicillin-resistant (base case mrsa = %), the model projected an overall clinical failure rate of . % for of the more commonly used initial regimens, with an expected overall treatment cost (in us dollars) of $ , per patient (range, $ , - , ) . if none of the s. aureus pathogens were resistant (mrsa = %), clinical failure rate was projected to be . % and treatment cost to be $ , per patient. the differences in the two scenarios translated to an expected clinical failure rate of . %, an incremental cost of $ per patient, and for the , patients hospitalised for csssi annually in the us, an expected health care system burden of $ million attributable to mrsa. under a "worst-case" scenario in which mrsa was the only causative pathogen (mrsa = %) in csssi, clinical failure rate was projected to be . %, and treatment cost per patient was expected to be $ , . conclusions: going beyond existing estimates, our model generated a substantial expected clinical failure rate and economic impact attributable to current mrsa levels, as well as simulations of the expected impact of increasing mrsa prevalence over time, varying levels of mrsa across regions and choice of initial empiric regimens. treatment of complicated skin and skin structure infections in the us: expected cost differences between tigecycline and vancomycin/aztreonam r. mallick, a. kuznik, d. weber (collegeville, chapel hill, us) objective: to compare tigecycline and vancomycin/aztreonam in terms of treatment-related costs for patients hospitalised in the united states with complicated skin and skin structure infections (csssi). methods: we conducted a retrospective analysis of pooled data from us centres in two randomized, double-blind clinical studies comparing tigecycline and vancomycin/aztreonam in the treatment of csssi. using regression analysis, we estimated the effect of tigecycline treatment on hospital length of stay (los), controlling for other significant predictors. using published estimates of daily hospitalisation cost of csssi in the us from a multi-hospital audit, we then translated the estimated impact on los into economic terms. this analysis was repeated for the subgroup of patients in which the primary pathogen was methicillin-resistant staphylococcus aureus (mrsa). clinical efficacy (tigecycline %, vancomycin/aztreonam . %; p = . ) was similar across treatments and was not included as a model parameter. results: our retrospective analysis of the pooled clinical data from us centres found that tigecycline was associated with a shorter los [) . days (p = . )] compared with the combination of vancomycin/aztreonam in the treatment of patients with csssi. at a mean daily hospitalisation cost (in us $) of $ , excluding antibiotic costs, this translated into expected medical cost savings of $ , per patient for tigecycline compared with vancomycin/aztreonam. in the mrsa subgroup, comprising % of the clinical study sample, tigecycline was associated with a greater reduction in los [) . days (p = . )] compared with vancomycin/ aztreonam, translating to expected medical cost savings of $ , per patient treated with tigecycline. these expected medical cost savings more than offset the higher average daily drug acquisition costs of tigecycline ($ /day) relative to the vancomycin/aztreonam combination ($ /day). conclusion: in a retrospective analysis of pooled clinical data of patients with csssi treated at us centres, tigecycline was associated with a significantly reduced length of hospital stay relative to vancomycin/aztreonam; this translated into substantial cost savings, especially in the subset of csssi patients with mrsa. the economic impact of linezolid in the treatment of skin and soft tissue mrsa infections in italy m. eandi, p. dale, s. sorensen, t. baker, m. procaccini, s. duttagupta (turin, it; london, uk; bethesda, us; rome, it; new york, us) objective: linezolid has been shown to be highly effective against infections caused by methicillin-resistant staphylococcus aureus (mrsa) in patients with complicated skin and soft tissue infections (cssti). the objective of this study was to evaluate the clinical and economic consequences of using linezolid for the empiric treatment of cssti from the italian hospital perspective. methods: a decision-analytic model was developed to calculate the clinical and cost outcomes of empiric treatment of hospitalized patients with cssti in italy prescribed linezolid, vancomycin or teicoplanin. efficacy data were derived from clinical trials. costs from published sources were applied to tests, adverse events, and days of intravenous and oral (linezolid only) treatment and hospitalization by ward type (general, intensive-care). resource use and utilization patterns were obtained from a combination of clinical trial data and expert opinion. outcomes included total costs per patient, cost per cure and cost per death avoided. uncertainty surrounding the ce ratio was tested using one-way sensitivity analysis. results: starting empiric treatment with linezolid resulted in . % of patients cured from mrsa compared to . % with vancomycin. the average cost per patient treated with linezolid was € , versus € , for patients treated with vancomycin. this resulted in a cost per cure of € , . in a separate analysis more patients were cured using linezolid ( . %) compared to teicoplanin ( . %). the average total cost per episode was € , for linezolid treated patients versus € , for teicoplanin treated patients, resulting in a cost per cure of € , . the most sensitive parameters included hospital los and mrsa resistance rate. conclusions: in the treatment of cssti due to suspected mrsa in italy, the empiric use of linezolid is cost-effective when compared to vancomycin and teicoplanin p outpatient and home parenteral antimicrobial therapy for the treatment of cellulitis: evaluation of efficacy and cost h. ziglam, r. tilley, c. wootton, j. morrison, d. nathwani (dundee, uk) objective: outpatient and home parenteral antibiotic therapy (ohpat) programmes are effective, well tolerated and economically advantageous in carefully selected patient populations. skin and soft tissue infections represent a high burden disease which in amenable to treatment by ophat programmes. we retrospectively analysed our outcomes registry to evaluate the clinical and health economic impact of treating cellulitis in this setting. methods: we have reviewed patients with cellulitis and erysipelas who were treated with ohpat. each patient treatment has a full integrated care pathway (icp). the icp documents the microbiological outcome, drug and vascular access complication rates, impact on drug costs and in-patient bed days on the number of patients treated from april to march are presented here. we also reviewed using the smr o inpatient discharge diagnosis data from the information statistics division scotland (isd) and the dundee infectious diseases units (didu) outcomes registry database. the key diagnosis (icd codes) groups considered were cellulitis (lo , , , , , ) and erysipelas (a x) over eight consecutive years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: the patients received intravenous antibiotic therapy for a mean duration of . days. the two primary agents administered were once-daily ceftriaxone in %of patients and teicoplanin in . % of patients. of the patients, ( . %) were cured or improved; worsened and required surgery. tinea pedis was found in % of patients treated for cellulitis. economic benefits were realized despite use of more expensive agents. data from the dundee outcomes registry revealed a mean reduction in length of hospitalization from . days ( / ) to . in - -a reduction of % compared to scottish data from isd which did not show any changes in length of hospitalization between year / ( . days) and year - ( . days) . conclusions: we have found that ohpat is clinically effective and can be administered safely and successfully in an outpatient setting. the majority of complications were minor, and % of patients were cured. tinea pedis and were found to be significant risk factors for acute cellulitis and indicate that improved awareness and management of toe web intertrigo might reduce the incidence of cellulitis. this analysis also supports the premise that an adult ohpat programme can substantially reduce healthcare resource use in the european healthcare setting. cost-effectiveness analysis of intravenous moxifloxacin compared to levofloxacin in hospitalised elderly patients with communityacquired pneumonia objective: to evaluate the cost-effectiveness of moxifloxacin compared to levofloxacin in hospitalised patients aged ‡ with community acquired pneumonia (cap). methods: a randomised double-blind parallel group study was conducted in us hospitals. patients had radiological evidence of bacterial pneumonia confirmed by at least other signs, were aged ‡ years and were managed as inpatients on initiation of treatment. patients initially received moxifloxacin mg iv o.d. or levofloxacin mg iv o.d., and once stabilised were switched to oral therapy with the same agent. the effectiveness endpoint for the economic analysis was the percentage of patients successfully treated, defined as patients with marked improvement, resolution or clinical cure at test of cure visit after - days of therapy who did not experience a serious cardiac adverse event. total costs were estimated from the perspective of the treating hospital and included antibiotic drugs, hospital stay, hospital re-admission within days and cost of managing treatment failures. results: patients were included in this analysis, randomised to moxifloxacin and to levofloxacin. % ( % ci: - %) of moxifloxacin and % ( - %) of levofloxacin treated patients were successfully treated (resolution, clinical cure at toc and no serious drug related aes). in the moxifloxacin group patients reported a mean of . days of iv antibiotic treatment and . days inpatient stay with . days iv antibiotic treatment and . days inpatient stay in the levofloxacin group. mean per patient drug cost was $ in the moxifloxacin group, $ in the levofloxacin group. mean total cost was $ , in the moxifloxacin group and $ , in the levofloxacin group. findings were consistent across a range of patient subgroups. costs were sensitive to length of hospital stay. conclusions: patients in the moxifloxacin group had higher rates of successful treatment at slightly lower average costs than the levofloxacin group. this confirms the results of the target study where moxifloxacin showed superior clinical efficacy in comparison to co-amoxiclav with or without clarithromycin in hospitalised cap patients. antibiotic costs were slightly higher in the moxifloxacin group than the levofloxacin group but total costs were slightly lower, due to reduced hospital stay. economic impact of invasive fungal infections in icu patients in a tertiary care hospital in switzerland a. imhof, w. zingg, r. laffer, c. ruef (zurich, ch) objectives: invasive fungal infections (ifi) cause significant morbidity and mortality. the management of invasive fungal infections is currently undergoing important changes due to the availability of new therapeutic agents with improved safety profiles but the acquisition costs of these new agents are high. we evaluated the average overall cost of management (microbiological diagnosis and treatment) of invasive fungal infection in critically ill patients at a large university hospital. methods: a retrospective ( ) ( ) , pairwise-matched cohort study was performed on surgical icus and one medical icu at our university hospital. icu patients with documented ifi (n = ) were matched with control subjects (n = ) on the basis of disease severity, sex and age (± years). clinical outcome was principally evaluated by in-hospital mortality. the economic impact of microbiological studies and antibiotic treatment was assessed. calculations were based on the period between admission and diagnosis of ifi in cases and the duration of hospital stay in controls, respectively. results: the median length of hospital and icu stay differed significantly between cases and controls ( vs days, vs days, p < . , respectively). ifi occurred after a median hospital stay of (range - ) days. the mortality rate for patients with ifi and matched control subjects were . % and . %, respectively (p = . ). there was no significant difference between cases and controls for charlson index, mccabe and saps ii score. median number of antibiotic treatment courses was for cases and for controls (p = . ), with a median duration of therapy of days vs day (p < . ), respectively. microbiological studies (mis) were conducted times/ patient-days (pd) in cases and times/ pd in the control group (p = . ). the most frequent samples were bloodcultures in both groups. swabs were ordered significantly more frequently in cases (median (range: - ) vs. ( - ); p = . ). the cost for mis was ' euro/ pd in cases vs. euro/ pd in controls, and the costs for antifungal therapy ' euro/ pd vs. euro/ pd, respectively. conclusions: ifi is associated with excess length of icu and hospital stay, increased use of antibiotics and microbiological diagnostics. the microbiological studies have a significant economic impact on the treatment of ifi. cost-effectiveness of voriconazole to amphotericin b deoxycholate in early and late treatment of invasive aspergillosis r. greene, j. mauskopf, c. roberts, t. zyczynski, h. schlamm (boston, research triangle park, new york, us) objective: we estimate the cost-effectiveness of alternative initial drug treatments of invasive pulmonary aspergillosis (ipa) in suspected earlier and later lung involvement, based on the presence or absence of the halo sign on thoracic computed tomography (ct). methods: we constructed a decision analysis model comparing -week treatment outcomes for a subset of patients enrolled in a clinical trial of initial treatment of ipa with amphotericin b dexoycholate (ambd) vs voriconazole (vor). patients included those with suspected lung involvement who underwent a baseline thoracic ct. the subset was subdivided into two groups based on the presence or absence of a characteristic ct halo sign, a perimeter of ground glass ct opacity surrounding a solid lung nodule ‡ cm diameter, known as an early indicator of ipa. healthcare resource use and survival data were obtained directly from the clinical trial. us unit costs for drugs and health care services were applied from standard data sources. cost and survival at -weeks were estimated for those with and without a halo sign at baseline. incremental cost-effectiveness ratios comparing vor to ambd were calculated for both patient subgroups. sensitivity of results to uncertainty in health care use and cost estimates was tested. results: patients in the halo subgroup had better survival than those in the no-halo subgroup ( . % vs . %), with lower total treatment cost ($ , vs $ , ) . survival was higher for vor than for ambd in both patient subgroups (halo: . % vs . %; no-halo: . % vs . %). in the halo subgroup, total costs were lower for those treated with vor than for those treated with ambd ($ , vs $ , ) . in the no-halo subgroup, total cost per patient was slightly higher for those treated with vor ($ , vs $ , ) . accounting for the difference in survival, the incremental cost-effectiveness ratio for vor compared to ambd was $ , per additional -week survivor in this subgroup. conclusions: earlier identification and treatment of ipa appears to result in better survival and potentially lower costs than later treatment. initial treatment of ipa with vor improves survival in patients with early or late disease compared with ambd, is cost saving in the halo sub-group, and is cost-effective in the no-halo subgroup, within the constraints of our analysis. objective: to describe and compare the nursing labor time required for preparation and administration of liposomal amphotericin b (l-amb), amphotericin b deoxycholate (ambd), and voriconazole (vor). methods: activities associated with nurse preparation and administration of the three study drugs were timed by trained observers at five hospitals (one in italy, three in france, and one in the united kingdom). target tasks were classified as those likely to be affected by the difference between the drugs and excluded those tasks likely to differ because of site-specific factors (e.g., travel time to a patient room in different hospitals). target tasks included: obtain supplies and medications; prepare medications; educate patient; administer medications; monitor for adverse events; and prepare follow-up medications. the mean times for administration of a single day of study drug were summarised and compared, accounting for a single daily dose of l-amb and ambd and daily doses of vor iv or oral. results: sixty-nine patients were observed receiving doses of study medications at the five hospitals. time of administration in minutes per day was , , , and for l-amb, ambd, vor iv, and vor oral, respectively. administration time was significantly lower for vor iv compared with l-amb (p < . ) and for vor oral compared to all iv regimens (p < . ). the task of preparation of medications required the most time for iv formulations, and was longer in the l-amb group than the others (l-amb: mins vs ambd: mins; vor iv: mins). ambd required more time for patient monitoring and administration of followup drugs than other formulations (ambd: mins vs l-amb: mins; vor iv: mins). conclusion: vor iv required significantly less time to prepare and administer on a daily basis compared to l-amb. measurements of iv antifungal versus oral vor administration suggest the opportunity to save - minutes per day by switching to oral therapy when possible. need of cost-effectiveness investigation focused on diagnosis, management and prevention of osteopenia and osteoporosis in the setting of hiv disease treated with haart: when to act, how to act, which patients are the first target of intervention r. manfredi, l. calza, f. chiodo (bologna, it) background: osteopenia/osteoporosis are emerging untoward effects of hiv infection/haart. the pathogenesis is multifactorial, involving all classes of anti-hiv drugs, although protease inhibitor use, overall haart duration, and the male sex, seem related to a greater risk.epidemiologicalclinical data. in an ongoing study at our centre where > hiv-infected patients (p) are followed, bone mineral density was assessed in lumbar spine/femural head by a dual energy xray absorptiometry (dexa) exam to estimate the prevalence of osteopenia/osteoporosis. in a screening of~ p, the frequency of osteopenia and osteoporosis (based on lumbar t-score) was % and~ %, respectively. an increased risk was found in p treated with protease inhibitors versus those receiving nonnucleoside reverse transcriptase inhibitors or triple nucleoside/ nucleotide combinations. discussion and future insights: prospective studies of extensive p samples are needed, to elucidate the epidemiology, pathogenesis, clinical issues and evolution of hiv-associated bone metabolism anomalies. when planning strategies for their early diagnosis, prevention and management also cost-effectiveness issues should be considered, since no pharmacoeconomic data still exist in this setting. although severe consequences (e.g. pathological fractures, prosthetic implants) are expected to be infrequent their consequences in terms of length and intensity of hospitalization, related costs, and especially severe consequences on the p's quality of life, play a notable role. anyway, the most reliable diagnostic procedure (dexa) has affordable costs (around eur . for a total-body scan which also offers a body composition assessment), as well as the first-line drugs for osteopenia, e.g. supplementation with calcium (eur - . /month), and vitamin d (eur /month). these costs cannot be compared with the costs of a standard care of an asymptomatic haart-treated p (eur to /month) and the immunological, virologic, laboratory and clinical controls made at least quarterly. like postmenopausal osteopenia/osteoporosis (burdened by a greater risk of bone mass anomalies) also hiv disease should be investigated from multiple cost-effectiveness points of view to establish which p are the preferred candidates for a dexa screening when this examination is more useful during hiv disease course and therapy, when the exam should be repeated and when and how to intervene pharmacologically to prevent serious and potentially invalidating complications. a comparative study on the cost of new drugs in different therapeutic categories m. falagas, k. fragoulis, g. zouglakis, i. karydis (athens, gr) objectives: drug treatment is becoming more expensive due to the increased cost for the introduction of new drugs and there seems to be an uneven distribution of medication cost for different therapeutic categories. we hypothesized that the cost of new antimicrobial agents may differ from that of other therapeutic categories and this may play a role in the stagnation of development of new antibiotics. methods: we performed a pharmaco-economical comparative analysis of the drug cost of treatment for new agents introduced in the united states drug market in various therapeutic categories. we calculated the drug cost [in us dollars (usd)] of a -day treatment of all new drugs approved by the fda during the period between january and july , according to the red book pharmacy's fundamental reference. results: new anti-neoplastic agents were found to be the most expensive drugs in comparison to all other therapeutic categories with a median -day drug-treatment cost of usd compared to the median -day drug-treatment costs of all other categories ranging from to usd (table) . on the other hand, new antimicrobial drugs were found to be much less expensive with a median -day drug-treatment cost of and usd for all anti-microbial agents and for anti-microbial agents excluding anti-hiv medications, respectively. conclusion: the drug-treatment cost of new medications varies considerably by different therapeutic categories. this fact may influence industry decisions regarding the development of new drugs and may play a role in the shortage of new anti-microbial agents in the fight against the serious problem of anti-microbial resistance. usage and expenditure of f-quinolones in a tertiary hospital in t.a. peppas, k. malengou, n. zachos, d. voutsinas, o. kosmopoulou, n. galanakis (piraeus, athens, gr) objective: our aim was to assess -f-quinolone ( fq) usage, distribution and expenditure over years. objective: to analyse antiobiotic utilization in croatia using anatomical-therapeutic-chemical (atc) drug classification system and number of defined daily doses (ddd). methods: data on the number of packages and purchase price were collected for each individual drug. these data were used to calculate the number of defined daily doses (ddd) and ddd per inhabitants per day (ddd/tid). data obtained from % of pharmacies and % of hospitals were extrapolated to the total number of pharmacies and hospitals in croatia. drug utilization % (du %) segment was used as a prescribing quality indicator. results: in , the overall utilization of antibiotics in croatia amounted to . ddd/tid. according to drug groups, penicillins (j c) showed highest utilization ( . ddd/tid), predominated by the subgroup of penicillin combinations (including beta-lactamase inhibitors, j cr) with . ddd/ tid, within which the combination of amoxicillin + clavulanic acid accounted for . % with . ddd/tid. broad-spectrum penicillins (j ca) accounted for . %( . ddd/tid) of total penicillin utilization, with a . % predominance of amoxicillin ( . ddd/tid). cephalosporins (j d) ranked second with . ddd/tid, followed by macrolides and lincosamides (j f) with . ddd/tid, with an . %predominance of macrolides (j fa) with . ddd/tid. among the latter, azithromycin showed highest utilization with . ddd/tid, accounting for . % of total macrolide utilization. tetracyclines (j a) ranked fourth with . ddd/tid, accounting for . % of overall antibiotic utilization, followed by quinolones with . ddd/tid, other antimicrobials with . ddd/tid, and aminoglycosides with . ddd/tid. sulfonamides (j e) accounted for a negligible proportion of overall utilization. du % segment included of antibiotics registered in croatia, with amoxicillin + clavulanic acid as the leading one, followed by cephalexin with . , cefuroxime with . , azithromycin and norfloxacin with . each, nitrofurantoin with . and clarithromycin with . ddd/tid. hospital utilization accounted for . % of overall antibiotic utilization expressed in ddd/tid and . % of the respective financial cost, predominated by aminoglycosides (j g) with % and . %, and lowest proportion of tetracyclines (j a) with . %, and . %, respectively. conclusion: the utilization of antibiotics in croatia is among the highest in europe, mostly due to overuse of amoxicillin + clavulanic acid, which has no rational ground in professional guidelines. objective: the objective of the research was to analyse antibiotics prescripsion behavior by family doctors and specialists treatments prior to and after the introduction of the health care reform in poland. materials and methods: prescriptions from the first six months of and were compared. the data was collected from two randomly chosen pharmacies in the city of zabrze that supply the citizens of the silesian agglomeration from various social backgrounds. taking into account the value of a single antibiotics package and the price a patient has to pay for it an average price of medications prescribed by family and specialist doctors was calculated. results: a total of prescriptions were analysed out of which dated from and from .in the first half-year of the percentage of prescriptions for antibiotics reached . % on average, and in the year -the average was . %. in the first half-year of family doctors mostly prescribed: penicyllins ( %), makrolids ( %), cephalosporins ( %), tetracyclins ( %), chinolons ( %). in the same period spcialist doctors prescribed: penicyllins ( %), cephalosporins ( %), makrolids ( %), tetracyclins ( %), lincozamids ( %), chinolons ( %). in the first half-year of family doctors most often prescribed penicyllins -( . %), makrolids -( . %), cephalosporins -( . %), tetracyclins -( . %) and lincozamidsbased ( . %) treatments specialist doctors, on the other hand, prescribed penicllins ( . %), makrolids ( . %), cephalosporins ( . %), tetracyclins ( . %), lincozamids ( . %) and chinolons ( %). the average prices of the prescribed medications in the years and were, respectively: for family doctors-eu . and . , for specialist doctors eu . and . . conclusions: there has been a considerable increase in the percentage of prescriptions for antibiotics from . % (in ) to . % (in ). the tendency towards prescribing antibiotics in the specific groups of doctors has not changed significantly. in both years prescriptions for antibiotics were in line with the recommendations. also, prices of medications prescribed by family doctors have risen. internet guide on antimicrobial resistance a. sosa, f. traub, s. valovic, p. chea (boston, us) objectives: ( ) to organize the plethora of information available, providing clinicians the tools to easily access available online resources that include academic institutions, professional societies as well as sites maintained by private individuals; ( ) to inform clinicians of new advances in the epidemiology, diagnosis, treatment, and prevention of most common infections; ( ) to inform on subjects such as clinical trials in antimicrobial resistance, information about specific pathogens and their infections, genomic resources, culture collections, electronic images of pathogens and antimicrobial agents, antimicrobial resistance lecture and teaching materials, environmental health and safety information, and a listing of websites of infectious disease and clinical microbiology professional societies. methods: we defined four inclusion criteria after extensive consulting with apua staff and scientific advisory members: ( ) recognized/reputable source; ( ) high quality of information presented; ( ) potential usefulness to medical professionals and the general public; and ( ) ease of navigation. ideal parameters were determined for the guide's scope and the appropriate sources identified online were subsequently reviewed. a set of broad categories was established to organize the topics and the online resources. sites reviewed included those maintained by the federal government, academic institutions, nonprofit organizations, and commercial entities. some personal websites were included because of their quality and their association with academic institutions. this review is intended as an introduction to amr websites. results: with use of popular search engines, such as google, yahoo!, and altavista, we initially identified a great number of websites. using broad search terms, such as "antibiotic resistance," we identified , , web addresses. the term "antimicrobial resistance" generated , hits, and the term "drug resistance" generated , , hits. conclusions: websites found were classified following a systematic topic structure. each website listed describes: ( ) full citation of the resource: author/editor and title of website; ( ) date of publication or last revision; ( ) methods and results: the portal is free to use but requires registration and has more than registered users as of november . of these, % are in-training positions, % hold a faculty position in infectious diseases (id), microbiology or hemato-oncology, % are specialists in a non-university, but a teaching setting. the remaining are a mixed group of primary physicians, other speciality doctors and pharmaceutical company workers. running costs of the portal are partially covered by educational grants from pharmaceutical sponsors who have no role in organization of the site, but their names are acknowledged. articles chosen by the two faculty members, one id physician and one haematologist, are sent to registered users by daily e-mail postings. these are selected from toc alerts of various core clinical journals and well known educational web sites (e.g. cdc, who, medscape). a short turkish summary is provided and the reader is referred to the abstract and if available, to the free full text of original article with a link. other materials included guidelines, free slide sets, study protocols and updates from the group, cme activities and meeting announcements. registrants may also use the site for expert opinion. during the trial period, the site has been visited times with hits. approximately . gb material was downloaded. the frequency of readings are related with the time (highest between . - . am during weekdays and lowest during weekends), the type of documents (i.e. educational materials and guidelines), popularity of the news (e.g. peaked during an epidemic of avian influenza when related news and articles announced). the pages are most frequently visited by id specialists followed by clinical microbiology, haematology and pharmaceutical company workers ( %, %, % and % respectively). conclusion: timely published medical data have high attraction rates among physicians. our results also indicated that, a web page gets ''old'' after about a month of publishing, emphasizing the importance of well-timed announcements of the portal material. neli and nric survey: information needs of infectious disease professionals p. kostkova, s. d'souza, g. madle, j. mani-saada, s. wiseman, a. roy, j. weinberg (london, uk) healthcare professionals are increasingly facing the problem of information overflow. it is getting impossible to keep up-to-date with the latest research findings, care guidelines and pathways, government strategies and national and local policies. internet enables an instant information dissemination enabling access to the latest results at any time as well as informal knowledge exchange by using chat rooms and discussion forums. however, it is getting increasingly difficult for busy professionals to find reliable quality-assured information on the internet when they need it. national internet libraries in the uk are addressing this problem: the umbrella portal national electronic library of infection neli (http://www.neli.org.uk) providing a single access portal to quality-assured information on treatment, diagnosis, prevention and management of infection diseases, and the national resource for infection control nric (http:// www.nric.org.uk) -a single-stop shop for policies, guidelines and research around infection control, hosted by neli. to better meet the information needs of these internet portals, accessed by unique users per month, we are conducting an information needs study to explore clinical questions, user needs and disease priorities of users seeking answers on neli and nric. a pilot qualitative online questionnaire-based study revealed that our users come from the variety of professionals: clinical scientist, consultant, registrar, psychotherapist, lecturer, gp, medical librarian, information scientist, health protection. these have questions mainly around hiv, tinea, molluscum contagio, meningitis, cold, mrsa, lyme, toxoplasma, chicken pox, influenza, diarrhoea and vomiting, rash, staph. aureus, traveller infections antibiotics resistance, malaria, mmr, meningitis, viral myocarditis, anthrax, smallpox, and tb. this is in line with our quantitative weblog-based evaluation of the most commonly access topics on neli by nhs-based users: antimicrobial resistance and hae ( . %), tb ( . %), meningitis ( . %), hiv ( . %), chlamydia ( . %), e. coli ( . %), staph. aureus ( . %), adenovirus ( . %), blood borne infections ( . %). the results of the ongoing analysis of google search keywords that brought users to neli and nric will be discussed. further results identifying the needs specific to the infection disease professions will be discussed in relation to differences in the national variations in information needs and priorities. training in infection s. d'souza, p. kostkova, f. cooke, a. holmes (london, uk) specialists today require prompt access to quality information in order to work effectively. the diversity of specialist interests in the field of infection has led to the formation of a large number of professional and scientific societies. these play an increasingly important role in ensuring that the trainee is effectively supported, not only during the period of training, but also in longer-term personal development. details of relevant societies, conferences, grants etc are, on most society websites, confined to those for either that society or others in that specialty only, and knowledge of the numerous places in which to look for this information is necessary to find out the latest information. training in infection (tii -www.trainingininfection.org.uk) is an online resource, primarily aimed at infection specialist trainees but useful throughout the career path, which brings together this information into one central access point, so that users from all infection specialties can find the appropriate information for their specialty quickly and easily. it identifies and links to the key relevant resources covering a broad range of infection related disciplines in a dynamic database structure. information on societies, conferences, grants, journals, textbooks and more are available on the site, and have been put together to create a one-stop infection training portal. online discussion forums to be implemented will allow trainees' to share ideas and make the most of their combined expertise, and users will be able to receive alerts on new information in their specialty as well as be reminded of conference deadlines, journal submission deadlines etc. the ability to discuss regional issues online within specialties also aims to promote greater local and international collaboration. training in infection is endorsed by the national electronic library of infection (neli -www.neli.org.uk), an established digital library bringing together the best available online evidence-based resources on the investigation, treatment, prevention and control of infectious diseases. research designs and statistical methods in medical abstracts m. kompoti, m. matsagoura, a. koutsovasilis, a. koutsovasili, s. drimis (athens, gr) statistical methods used in biomedical research articles are being increasingly scrutinized in medical journals. however, no such strict policy is generally applied in abstracts presented in medical congresses. objective: this study aimed at assessing the frequency of research designs and statistical methods reported in abstracts presented in two successive years of the european congress of clinical microbiology and infectious diseases (eccmid). material and methods: we reviewed all abstracts included in the abstract book of the th eccmid (prague ) (pg) and the th eccmid (copenhagen ) (cp). all abstracts of original research studies but no abstracts of lectures were included in our study. two independent investigators read all abstracts and extracted information concerning origin, type (clinical, laboratory, animal model), research design, sample size and statistical methods used in the study. data analysis was performed with logistic regression and pearson's chi-square test for categorical variables and student's t-test for continuous variables. statistical significance level was set at p < . . results: a total of abstracts were included in the analysis according to eligibility criteria ( from pg and from cp). laboratory studies prevailed ( %) followed by clinical studies ( %) and experimental studies with animal models ( %). the majority ( . %) of the studies were observational (retrospective, prospective, cross-sectional) of which . % concerned diagnostic accuracy testing of laboratory methods and . % were pharmacological studies, . % were randomized controlled trials. statistical evaluation was clearly described in . % of abstracts ( . % in pg and . % in cp, p < . ), while the rest of abstracts included only descriptive statistics or no statistics at all. the proportion of statistical methods reporting varied according to the type of the study (animal model studies . %, clinical studies . % and laboratory studies . %, p < . ). multicentre research studies reported statistics more frequently than single-center studies ( . % vs. . %, respectively, p = . ). conclusions: statistical analysis is an inseparable part of original research. research design as well as the implemented statistical methods should always be reported in an adequate manner, thus improving the scientific quality of abstracts. antimicrobial pk/pd p nxl -oral streptogramin: a phase i, doubleblind, single escalating oral dose study to evaluate safety, tolerability and pharmacokinetics in healthy adult male volunteers m. rangaraju, j. rey, j. hodgson (romainville, vitry sur seine, fr) background: nxl (formerly xrp ) is a novel semisynthetic oral streptogramin that consists of a / (w/w ratio) association of a pristinamycin ia (pi) derivative and a pristinamycin iib (pii) derivative. nxl is being developed for the treatment of respiratory tract and skin and skin structure infections. methods: healthy male subjects were enrolled in this study. subjects in each of cohorts ( mg, mg, mg, mg, mg and mg) received either nxl ( ) or placebo ( ) . an additional cohort of subjects received a single dose of mg nxl in fasting and fed conditions. blood and urine samples for pk analysis were collected at multiple time points. safety was assessed via adverse events, physical examination, clinical laboratory data, ecg and cardiac monitoring. results: nxl administered as mg capsules at single doses from mg to mg was well tolerated and safe. there was no serious or severe adverse event, no dosedependency in the number of aes or their severity, no significant variation in blood pressure or heart rate, no abnormality on ecg recording, and no clinically significant changes compared to baseline for laboratory parameters. both components were rapidly absorbed; pi being slightly more rapidly absorbed than pii. the cmax and auc ( -t) increased approximately in proportion with dose. the proportion of pi and pii components estimated on mean exposure values was approximately comparable to that administered ( / ), indicating that the relative bioavailabilities of pi and pii are simila. elimination half-life ranged from between to hours for pi to to hours for pii. food increased the bioavailability of pi and pii by approximately %. conclusions: nxl is safe, well tolerated and exhibits predictable pk properties in healthy volunteers in doses up to mg administered as a single dose. correlation of vancomycin and daptomycin susceptibility in staphylococcus aureus in reference to accessory gene regulator polymorphism and function w. rose, m. rybak, b. tsuji, g. kaatz, g. sakoulas (detroit, new york, us) objective: polymorphism at the accessory gene regulator (agr) locus in s. aureus (sa) defines groups (i-iv). agr group ii sa have been associated with glycopeptide treatment failure in patients. sa with loss of agr function appear to have a higher tendency to become vancomycin (v) resistant. it is unknown whether this association only pertains to glycopeptides. we examined the effect of varying v and daptomycin (d) against agr+ and agr null pairs in an in vitro pharmacodynamic model (ivpm). methods: agr group i and ii wild-type prototype and knockout (tetm::agr) pairs were evaluated. mic values were determined according to clinical laboratory standards institute. ivpm glass and hollow fibre models were used to simulate dosages and auc/mic exposures for v ranging from . mg- g q h fauc/mic range - mg/l*h, and d . mg/kg- mg/ kg/day fauc/mic range . - . the dosage regimen and auc/mic breakpoints that produced resistance was then evaluated in the hollow fibre ivpm. all ivpm simulations were performed in duplicate over h. resistance was evaluated using and x mic screening plates at , , , , and h. results: pre-exposure mic values for agr i± and agr ii± were . / and for v and . lg/ml for d respectively. vintermediate resistance (mic = mg/l) was detected in both agr i and ii null strains at a simulated v dosage of . mg q h (auc/mic ), representing an mic increase of - fold. this breakpoint for resistance was verified in the hollow fibre model. although significant regrowth was noted with suboptimal dosing of d, no resistance was detected on d screening plates for any daptomycin regimen evaluated. conclusions: exposure of sa to v approximating / of optimal serum concentrations resulted in the development of heteroresistance in the agr null group i and ii. loss of agr function did not correlate with the development of d resistance despite suboptimal simulations of d exposures. these results implicate loss of agr function important to the development of glycopeptide resistance but not to loss of susceptibility to d. teicoplanin efficiently penetrates into the rabbit infected vitreous but may enhance expression of virulence factors at sub-inhibitory concentrations e. forestier, f. jehl, c. gallion, r. andres, s. bronner, l. leininger, g. prévost (strasbourg, fr) objectives: fluoroquinolones are the antibiotics that most efficiently penetrate inside vitreous. however, alternative treatments for endophtalmitis may be required in some cases for example resistant bacteria. we used a rabbit experimental model of endophtalmitis to evaluate the penetration of teicoplanin in different conditions. the influence of subinhibitory concentrations of teicoplanin was also evaluated on the expression of s. aureus virulence factors. methods: new zealand rabbits (> kgs) received one or repeated doses of intra-venous (iv) teicoplanin ( mg/kg) every hours for days plus one dose a day for more days. another group of rabbits was infected by cfu of a methicillin resistant s. aureus ibs (cmi = . mg/l) producing enterotoxin a, panton-valentine leucocidin and luke-lukd. they were administrated - hours later with mg/kg teicoplanin, as a single dose or as to reach the steady state. vitreous ( ll) was sampled before new injections of teicoplanin or at indicated time as well as blood before or min after teicoplanin injection. teicoplanin concentrations were measured by hplc. bacterial counts were recorded and expression of virulence factors was semi-quantified by dedicated competitive rt-pcr tests. results: in safe eyes, teicoplanin penetration remains moderate reaching about mg/l within about - h after one iv injection. the half-life of teicoplanin in the rabbit vitreous is about hours. after days of repeated injections, intra-ocular concentration stabilises around mg/l while residual blood concentrations were comprised between - mg/l. in infected eyes, teicoplanin, when repeatedly administrated after the beginning of clinical signs, i.e. h postinfection = cfu/ml, reaches intra-vitreal concentrations of . ± . mg/l h post-infection, and increases to . ± . mg/l h post-infection and h after a fourth injection. however, at sub-inhibitory concentrations (~ mg/l), it may be responsible for a significant increase of agr, gammahemolysin hlga, luked and panton-valentine leucocidin luk-pv expressions with ratio ranging from to folds. conclusion: these preliminary results strongly suggest that teicoplanin iv administration constitutes an interesting alternative therapy for endophtalmitis provided high intraocular concentrations are rapidly obtained. investigations now concern optimisation of teicoplanin dosage regimen. pharmacokinetics of temocillin in intensive care patients and monte carlo simulations to evaluate susceptible breakpoints j.w. mouton, r. dejongh, v. basma, p. tulkens, s. carryn (nijmegen, nl; genk, brussels, be) background: temocillin (tmo) is a narrow spectrum penicillin with good activity against gram negative micro-organisms including esbl and ampc producers. little pharmacokinetic data are available however. we performed a pharmacokinetic study in icu patients receiving tmo g q h. parameter estimates were used to predict concentrations during continuous infusion (coinf) and compared with data obtained from other icu patients receiving coinf to validate the model. the model was then used to perform monte carlo simulations (mcs) to determine probabilities of target attainment (ptas) for pharmacodynamic indices (pdi) in order to evaluate and suggest clinical breakpoints. methods: blood samples were taken from icu patients prior to (t = ) and after (t = , , , , h) a m infusion of g tmo (n = ) or after h during coinf with g/ h (n = ), and then cooled, centrifuged and stored at ) °c until analysis by hplc. protein binding was determined using an ultrafiltration method. results were used to estimate population pharmacokinetic parameters by winnonmix including the covariance matrix. miclab was used to perform simulations for coinf as well as to perform mcs ( cycles) and obtain ptas for the unbound fraction including % confidence intervals (ci) for the target concentrations. ft>mic was chosen as the pdi because of the pharmacodynamic properties of tmo. results: protein binding was %. a one-compartment model best fitted to the data, with estimates (se) of vc = . ( . ) l and k = . ( . ) /h corresponding to a mean half-life of . h. using these estimates, the predicted unbound concentration during coinf was . mg/l, while the mean concentration in the other patients was . mg/l, a bias of less then %. the breakpoint mic for a mean ft>mic of % was mg/l. however, mcs -taking the variation in the population into account -indicated that % ptas of a g q h dose were obtained at , , and mg/l for , and % ft>mic, respectively. the % ci at % ft>mic indicated a clinical breakpoint of mg/l. the % ci was relatively large, as expected from data obtained in patients rather than volunteers. conclusion: the population pharmacokinetic estimates from icu patients were very well in agreement with the validation study, with a bias of < %. the mcs indicate a susceptible breakpoint for temocillin of £ mg/l provided an administration of g q h is used. tissue penetration and pharmacokinetics of moxifloxacin in diabetic foot infections:an interim analysis j. majcher-peszynska, k. karrasch, m. saß, r. mundkowski, a. gussmann, p. kujath, b. ruf, w. schareck, h. koch, b. drewelow (rostock, bad saarow, lubeck, leipzig, beeskow, de) objectives: with its broad spectrum of activity against grampositive, gramnegative and anaerobic organisms moxifloxacin covers the pathogens of the mainly polymicrobial infections associated with the diabetic foot. inflammatory and fibrotic processes in diabetic foot infections (dfi) contribute to impaired tissue penetration of antibiotics. in addition, diabetic patients represent a pharmacological risk population, physiological changes in diabetic patients may alter the pharmacokinetics of antibiotics. the study was designed to investigate the penetration of moxifloxacin into perinecrotic tissue in patients with dfi and the pharmacokinetic properties of moxifloxacin in diabetic patients. methods: the interim analysis of this open, multicentre study included adult, hospitalized male and female patients (mean age: . years) with type diabetes mellitus and dfi. the pharmacokinetic parameters of moxifloxacin and penetration into dfi tissue at steady state (day to ) following once daily administration of mg iv or po were evaluated. correlations between penetration of moxifloxacin and clinical and laboratory parameters were examinated. results: in all patients the moxifloxacin concentrations measured in infected diabetic foot wounds hours after administration exceeded the in vitro mic values of susceptible staphylococci ( . mg/l). the moxifloxacin concentrations achieved in dfi tissue correlated more strongly with the auc - (r = . ; p < . ) than with the corresponding plasma concentrations (r = . , p < . ), but not with the extent of the systemic inflammation and the blood glucose level. taking into account the predictive pk/pd parameters for moxifloxacin (based on an in vitro mic value of . mg/l for staphylococcus aureus) a therapeutic success can be expected (auc /mic: . ; cmax/mic: . ). significant differences between the routes of administration (iv vs po) were only observed for tmax (p < . ) and t / (p < . ), but not for other clinically relevant parameters (auc - , cmax, moxifloxacin tissue concentration). this allows sequential therapy i.v./p.o. in this indication. conclusion: based on adequate plasma concentrations in diabetic patients, the sufficient penetration into dfi tissue and the possibility of a sequential therapy, moxifloxacin representsfrom a pharmacological point of view -a valuable therapeutic option in the treatment of diabetic foot infections caused by susceptible organisms. fluoquinolones effects on patient lymphocytes during prolonged treatments e. bertazzoni minelli, a. benini, d. doria, p. franceschetti, m.e. fracass (verona, it) fluoroquinolones (fq), widely used in clinical practice, are well tolerated. the most common adverse reactions are those affecting gastrointestinal tract, phototoxicity and allergy. the aim of the study is to evaluate the possible cellular damage in lymphocytes of patients treated with different fqs according to pharmacokinetic data. blood samples obtained from thirty-six patients treated with ciprofloxacin (cpx, pts), levofloxacin (lvx, pts.) and moxifloxacin (mfx, pts) at different doses were analysed. patients treated with cpx and lvx were in therapy with other drugs (diuretics, cardiovascular drugs, omeprazole, antiinflammatory drugs, etc.). mxf treated patients were not in therapy with other drugs. samples were collected at time (before fqs administration) and after and days of treatment. serum levels of fqs were determined with microbiological method and hplc. comet test was performed on lymphocytes, to evaluate dna damage. gsh levels were determined as efficiency marker in metabolic process of detoxification. cpx showed good serum concentrations; its levels increases proportionally with administered doses (from to mg). lvx concentrations resulted in good inhibitory levels after treatments ( mg) both per os and i.v. patients orally treated with mg showed similar serum levels (from . to . mg/l). mfx levels were between . and . mg/l after and days. repeated cpx administration induced a dose-dependent increase in all dna damage parameters, with statistical differences after treatments. mfx ( mg) and lvx administration didn't induce dna damage after and days. intracellular levels of gsh were similar in all treated groups, even if cpx treated patients showed the lowest concentrations. no statistical correlations were found between all parameters studied. these data indicate that cpx induce dna damage in lymphocytes in combination with a reduced efficiency in detoxification system. this effect does not seems to depend on high intersubject variability for fqs administered doses, co-administration of other drugs, different ages of patients and low samples numbers. effects of single fqs molecules seems to be structurespecific and selective. objectives: ertapenem is a carbapenem commonly used to treat intra-abdominal infections. the antibacterial spectrum includes the major causative pathogens. clinical trials proved excellent clinical and microbiological efficacy in peritonitis. on the other hand in inflammatory pancreatic diseases sufficient antibiotic concentration in the inflammatory tissue is vital for the outcome of the disease. we therefore investigated ertapenem concentrations in pancreatic tissue and juice in comparison to the plasma levels measured at the same time. methods: in a prospective clinical trial ertapenem was given in a dosage of g i.v. minutes prior to operation in patients ( - years) suffering from chronic pancreatitis or pancreas carcinoma undergoing pancreas resection. blood samples were collected every minutes during the operation. moreover we collected pancreatic tissue and pancreatic juice shortly before resection and shortly before finalisation of the anastomosis. the samples of ertapenem (blood, juice, tissue) were determined by hplc. results: in patients ( female, male, mean age . ± . years) ertapenem blood concentrations were determined and demonstrated intraoperatively high concentration ( ± mg/l) above mic values for major expected pathogens. concomitantly in of these patients ertapenem concentration was determined also in pancreas tissue and pancreas secretion (in further patients in pancreas secretion only). in / patients sufficiently high ertapenem levels were detected in pancreatic tissue. in patients with chronic pancreatitis no accumulation was seen. mean pancreas tissue concentration was . ± . lg/g tissue. of patients with pancreas carcinoma had increased ertapenem levels in pancreas secretion but only of patients with chronic pancreatitis. conclusion: in patients with pancreas carcinoma, ertapenem levels were measured in pancreatic tissue as well as in pancreatic secretion and penetration seems to be similar to imipenem. due to chronic inflammation and possibly altered microcirculation only in one half to one third of chronic pancreatitis patients ertapenem levels were detected. bacterial strain-independent pharmacodynamics of linezolid/doxycycline combinations with staphylococcus aureus: -day simulations using an in vitro dynamic model m. smirnova, i. alferova, i. lubenko, y. portnoy, s. zinner, a. firsov (moscow, ru; cambridge, us) objective: to delineate the possible advantages of linezolid (l)/doxycycline (d) combinations over monotherapy, the pharmacodynamics of l, d and l+d were studied with s. aureus. methods: s. aureus atcc and a clinical isolate s. aureus were exposed to twice-daily l (half-life h) and once-daily d (half-life h), alone and in combination ( : ratio based on -h auc/mics), for five consecutive days. to provide simultaneous mono-exponential elimination of l and d with different half-lives, a previously described dynamic model was modified according to blaser and zinner. the mics of l were . and . mg/l and mics of d were . and . mg/l for s. aureus atcc and s. aureus , respectively. nine dosing regimens were simulated with each organism exposed to different auc/mics (in hours): l , l and l ; d , d and d ; l + d , l + d and l + d . the cumulative antimicrobial effect was expressed by its intensity (ie) measured from the start of treatment to the time after the last antibiotic dose when numbers of antibiotic-exposed bacteria reached at least cfu/ml. emergence of resistance was monitored daily by quantitating surviving organisms on agar plates containing x and xmic of l or d. results: with both s. aureus atcc and s. aureus exposed to l or d, ie increased with increasing simulated auc/ mic ratios, although significantly higher ies were produced with l , l and l treatments relative to d , d and d treatments. each of the combined treatments, i.e., l + d , l + d and l + d , produced much greater ies than the sum of l and d ies observed in the respective mono-treatments with both s. aureus strains. based on population data, a pronounced selection of s. aureus resistant to d occurred in all mono-treatments with d. it was also observed with l + d and, to a lesser extent, with l + d but not with l + d . no resistance to l was observed with l mono-or combination treatments. conclusions: these data predict a synergistic interaction of l with d against s. aureus. anti-staphylococcal effects of telavancin in an in vitro dynamic model: impact of different half-lives and initial concentrations that simulates normal (nek) and impaired elimination kinetics (iek). materials and methods: a glycopeptide intermediately susceptible strain of s. aureus (gisa) mu- with a telavancin mic of . mg/l was selected for the study. with both nek and iek simulations at a starting inoculum of log cfu/ml, gisa mu- was exposed to different ratios of the peak concentration (cmax) to the mic of telavancin (as a single dose), i.e., . , . and . based on time-kill data, the intensity of the antimicrobial effect (ie -the area between control growth and time-kill curves) was determined from time zero to the time when the effect no longer could be detected, i.e. the time after the last dosing at which the number of antibiotic-exposed bacteria reached log cfu/ml. results: in each treatment, bacterial regrowth followed gradual reduction in the starting inoculum during the first h (similar in nek and iek simulations) that led to significantly lower minimal numbers of surviving organisms in iek simulations compared to nek simulations. despite similar rates of initial killing, times to regrowth were much longer in iek than nek simulations. at a given cmax/mic ratio, the ies observed in iek were greater than in nek simulations (figure). conclusions: these findings demonstrate pharmacokineticdependent pharmacodynamics of telavancin with staphylococci. pharmacokinetics of amoxicillin in pregnant women with pre-term premature rupture of the membranes objectives: amoxicillin is widely used during pregnancy, in particular to treat group b streptococcus. insufficient knowledge on the pharmacokinetics just before and during delivery, could pose patients with preterm premature rupture of the membranes (pprom) at serious risk for under dosing. we investigated the pharmacokinetics in patients with pprom in this critical situation. methods: seven healthy women at - weeks of gestation were included. they received g (first dose g) amoxicillin for pprom according to local guidelines. from each patient - blood samples were taken. antibiotic serum concentrations were determined by a validated hplc method. pharmacokinetic parameters were estimated by population pk modeling using nonmem. to discriminate between various models the minimum value of the objective function (mvof) was used. a reduction of > in mvof was considered significant. results: a three-compartment pharmacokinetic model best described the time course of amoxicillin. the clearance and volume of distribution of the central compartment (vc) were estimated at respectively ± . l/h and ± . l (mean ± se). estimates of the parameters and model discrimination improved when we assumed the size of the third compartment to be equal to the first compartment. the residual error was found to be proportional to the serum concentrations. most of the inter-individual variability could be explained by variation of clearance. the mean volume of distribution at steady state (vss) and terminal half-life were . l and . h respectively. estimated values of elimination and distribution rate constants were: k = . h- , k = . h- , k = . h- , k = . h- and k = . h- . as was to be expected due to the small population size, no significant relationship was observed between the individual posthoc estimates for clearance and patient characteristics. conclusion: here we describe the pharmacokinetics of amoxicillin in pregnant women with pprom. it was found that the pharmacokinetics clearly differs from that in nonpregnant individuals. clearance and vss were significantly higher and the terminal half-life was shorter. furthermore, a -compartment model was found to describe the data better than a -compartment model. it is an intriguing question whether this rd compartment is a unique feature associated with pregnancy. these data offer a theoretical basis to make proper dose-adjustments in a particular patient group in a critical condition. penetration of piperacillin and tazobactam in severe acute pancreatitis objectives: acute necrotizing pancreatitis is still related to an extremely high mortality rate, based on local infectious complications, particularly in necrotizing areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotizing pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of betalactamases. on that score, the penetration of co-administrated pip and bli into inflamed or necrotic pancreatic tissue has not been investigated yet. methods: adressing the penetration capability of bsp and bli a clinical trial was designed to investigate the penetration of piperacillin (pip) and tazobactam (taz) in patients with severe necrotizing pancreatitis undergoing pancreas surgery. samples (n = ) were taken from plasma (pl), necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of . g pip and . g taz. concentrations of pip/ taz were determined by hplc/ uv. results: mean plasma concentrations at . h after application were . ± . mg/l (pip) and . ± . mg/l (taz). exceeded in pl and bs, nearly reached in pn but not in pft. the concentration of pip in combination with taz exceeded or reached the mic in pl, pn and bs against e. coli, klebs. spp., enterobacter, proteus spp. and clostr. spp., in pl and bs even against pseudomonas and bacteroides. conclusion: given in combination both -pip and taz -have been demonstrated to reach rapidly effective inhibitory concentrations in inflamed and necrotic compartments of pancreatic and peripancreatic tissue. co-administration of piperacillin and tazobactam may have a potential clinical benefit in prevention and treatment of local infectious complications of severe necrotizing pancreatitis. pk/pd challenges of in vitro time-kill curves -a new modelling approach s. schmidt, o. burkhardt, w. treyaprasert, h. derendorf (gainesville, us; bangkok, th) objective: in vitro pk/pd models, based on time-kill curve data, have become a powerful tool to predict the in vivo situation. up to date, several modelling approaches have been undertaken to develop suitable pk/pd models that fit in vitro data sufficiently well. widely used simple sigmoid emax models meet these criteria only partly. a further approach was undertaken to address the weak points of currently used models and applied to model the effects of ceftriaxone against escherichia coli. methods: constant concentration time-kill curves were performed in mueller-hinton broth (mhb, difco) with and without bovine serum albumin (bsa) g/l. using concentrations of ceftriaxone, ranging from . mic to mic, the change in number of bacteria (cfu/ml) versus time was linked to its effect. escherichia coli atcc was employed as the test organism. samples were taken at , . , , . , , , , and hours. the data were modelled simultaneously, using a modified sigmoid emax model and the software scientist Ò for windows tm . results: a differential equation, characterized by growth rate constant (k ) times the starting number (n) of bacteria represent the simplest case. barging from log-growth phase to stationary phase can be described by an additional nmax term. however, bacteria do not necessarily start growing in the loggrowth phase. this delay in onset of growth can be modeled by an exponential term, characterized by a factor beta (b) and time (t). to describe the overall change in number of bacteria not only growth but also concentration (c) dependent kill has to be taken into account. from certain drug specific concentrations on, a maximum effect is reached, described by the maximum kill rate constant kmax. however, it may be necessary to model a delay in the onset of kill with an additional exponential term, characterized by a factor alpha (a) and time (t). finally, a hill factor/shape factor (h) is necessary to smooth the predicting curves out. as shown in figure this new model meets the in vitro time-kill curve data sufficiently well. the final equation including all parameters described above is: conclusion: the proposed model was able to describe the observed data much better than a simple emax-model. incorporating two additional terms into the model, the in vitro situation could be described much better, taking the delay in onset of growth and kill into account. objectives of this study were ( ) to describe the pharmacodynamic (pd) profiles of bpr mg iv q h as a hr infusion & mg iv q h as a -hr infusion; ( ) to determine the overall probability of target attainment (pta) by weighting for the expected distributions (dis) of renal function (rfx) in the populations (pop) of interests; ( ) to determine the organism-specific pta against the pathogens encountered in phase ii trials. methods: subjects (total samples) were studied (phase i/ii subjects). samples were analysed using bignpod. to assess the impact of differing degrees of rfx impairment on pta, crcl (crcl-cockcroft & gault method) was employed as a covariate in the pop pk analysis. monte carlo simulation (mcs) ( subjects) was performed with adapt ii. overall pta was calculated for - % ft>mic. weighting for the expected dis of rfx in the pop of interests was accomplished by using the dis of crcl observed in previous registration studies of the same indications (cssti and np). dis of mics for pathogens was supplied by sponsor. results: in the pop pk analysis, the pop mean (sd) values for volume, clslope, clintercept, kcp and kpc were: . ( . ) l, . ( . ) l/hr, . ( . ), . ( . ) hr- and . ( . ) hr- , respectively. the obs-pred plot was obs = . x pred + . ; r = . after the bayesian step. in the mcs analysis of bpr mg iv q h, the pta of achieving % ft>mic & % ft>mic exceeded % for mics values £ mg/l & £ . mg/l, respectively. for bpr mg iv q h, the pta of achieving % ft>mic exceeded % for mics values £ mg/l. in the organism-specific analysis, the pta of a static effect ( % ft>mic) exceeded % for both mssa & mrsa for bpr mg iv q h. bpr mg iv q h provided a > % pta of a cidal effect ( % ft>mic) for both mssa & mrsa. for gnb, the pta of bpr mg iv q h in achieving a cidal effect ( % ft>mic) exceeded % for non-ampc-bearing gnb. for ampc-bearing gnb, the pta of achieving a cidal effect was . %. conclusions: an extensive evaluation of the pd of bpr was performed to estimate the overall activity of bpr against target pathogens. these findings need to be validated in the clinical trial arena. investigation of different levofloxacin regimens in patients with acute complicated urinary tract infections p. tenke, r. benko (budapest, szeged, hu) objective: in the present study we aimed to find out if a continuous or an intermittent levofloxacin ( · mg) treatment is more advantageous for patients with acute complicated urinary tract infection (uti) caused by urinary obstruction. we investigated if levofloxacin adsorbs to the surface of the foreign body, which was inserted with the aim of temporary resolution of ureteral obstruction. preventive effect of levofloxacin on bacterial biofilm formation and incrustation was also evaluated. methods: we enrolled and randomised patients who had acute uti caused by urinary obstruction. obstruction was resolved with double j stent (djs) insertion or percutaneous nephrostomy (pcn) and meanwhile, antibiotic treatment was started in all patients. patients (group ) were on antibiotics till the day of definitive curative operation when all foreign bodies were removed. in the other % of the patients (group ) the antibiotic therapy was stopped days after the djs or pcn insertion. short term antibiotic course -which is advisable for prevention of uti before invasive endoscopic treatmentwas administered in both groups from the day of the operation (after djs or pcn removal) and it was continued until the removal of all possible urinary foreign bodies used during the operation. in both groups of patients we recorded and evaluated early and late clinical and microbiological recovery. retrieved stents were sectioned for further laboratory examinations. adsorbed levofloxacin in the conditioning film layer and on the stent surface was detected by hplc. rasterelectron microscopy (rem) was used to examine biofilm formation and encrustation. results: we did not find any significant differences between the two groups of patients, neither in clinical (presence of fever, back pain, flank pain, leukocyte count) nor in microbiological recovery. statistical analysis showed that significantly greater amount of levofloxacin adsorbed to the conditioning film than to the stent surface in both groups of patients ( . ± . vs. . ± . in group and . ± . vs. . ± . in group ). no viable, adherent bacteria were recovered by sonication and culture in any of the patients, and no biofilms or encrustation were seen under rem either. conclusion: our data prove the hypotheses that continuous antibiotic treatment does not have any clinical or microbiological advantages in patients with indwelling ureteral stents compared to intermittent therapy. objective: the prophylaxis of bacterial infections during cardiac surgery is widely used in clinical practice. staphylococcus aureus, staphylococcus epidermidis and enterococcus spp are the pathogens most frequently involved in infective complications of cardio-pulmonary bypass (cpb) surgery. it is generally agreed that the success of prophylaxis is dependent on the ability to reach and maintain free antibiotic concentration in tissues higher than the mics for the most common pathogens. so we estimated the tissue concentrations of linezolid into sternal bone of patients undergoing cpb surgery. methods: six patients undergoing routine cpb surgery were given mg linezolid as a min iv infusion along with conventional prophylaxis of . g of cefuroxime immediately before surgery. two hours after the end of infusion blood samples and sternal bone tissues were collected. the local medical research ethics committee approved this study and all patients gave written informed consent. samples were assayed for the presence of linezolid by a high-performance liquid chromatography (hplc) method. results: following a mg infusion of linezolid, mean serum concentration for the six patients were . mg/l (range . - . mg/l) hours after the end of infusion. the concentration of linezolid into sternal bone was . mg/l (range . - . mg/ l) hours after the end of infusion. the penetration of linezolid into sternal bone was . %. conclusion: the penetration of linezolid into bone was . % of the simultaneous blood levels. in all bone samples the concentration of linezolid exceeded the mic for susceptible pathogens (< mg/l). although these data have been obtained from healthy, well-perfused bone the values suggest that linezolid may be a useful agent in the management of multidrug-resistant gram-positive bone infections. the antibacterial effect of daptomycin, teicoplanin and vancomycin against s. aureus studied in an in vitro pharmacokinetic model of infection a. noel, k. bowker, a. macgowan (bristol, uk) objectives: daptomycin (dap) is the first cyclic lipopeptide antibiotic approved for parenteral use in gram-positive infection. as yet, no comparative pharmacodynamic studies have been performed using dap and the two most common iv therapies teicoplanin (tei) and vancomycin (van). we used a pharmacokinetic (pk) model to study the antibacterial effect (abe) of these agents against two typical mrsa strains (ukemrsa & ) and a hetero vancomycin intermediate mrsa (hvisa). methods: an in vitro dilutional model was used to simulate the free drug concentration over h associated with doses of -dap mg/kg hrly (cmax . mg/l, t / h); tei mg hrly (cmax . mg/l, t / h); van g hrly (cmax . mg/l, t / h). an inoculum of cfu/ml was used and the experiments performed in triplicate. abe was assessed by area-under-the-bacterial-kill-curve - h (aubkc ) and logcfu/ml.h objectives: linezolid, the first oxazolidinone, is active against methicillin resistant staphylococcus aureus and has been effective in a variety of acute infections. however long-term administration, although desirable in bone infections caused by resistant gram-positive organisms, is hampered by the occurrence of anaemia and thrombocytopenia. administration of vitamin b has been reported to prevent myelosuppression. methods: patients attending the infectious disease clinic with bone infections caused by resistant gram-positive bacteria and treated with linezolid ( mg b.d. orally), received vitamin b ( mg o.d. orally) for the period of administration of linezolid. full blood counts were followed-up weekly. linezolid treatment was discontinued if haemoglobin declined below mg/dl or platelets below /ll. data from sixteen patients with osteomyelitis and with prosthetic joint infections were evaluated. comparisons were performed with matched historical controls receiving linezolid without b by kaplan-meyer curves with the log-rank test. results: the median follow-up of patients receiving b was . weeks and of controls weeks. in the b group % of the patients discontinued while in the control group % of the patients discontinued treatment because of side effects (p ns). % of patients receiving b discontinued due to thrombocytopenia and % due to anaemia. respective percentages in the control group were % and % (p ns for all comparisons). mean time to the occurrence of thrombocytopenia was weeks in the patients who received b and . weeks in the control patients. respective times to occurrence of anaemia were . and . weeks. all cases of myelosuppression were reversible. conclusions: administration of b failed to prevent or delay both thrombocytopenia and anaemia in patients receiving linezolid. other methods should be investigated to facilitate longer administration of linezolid in this group of patients. therapeutic drug monitoring of colistin -a -year review from a uk clinical antibiotic assay service k. bowker, a. noel, s. tomaselli, a. macgowan (bristol, uk) objectives: over the last years there has been increased use of colistin (col). however, little clinical data is available on the therapeutic levels of colistin. monitoring of col is useful in terms of therapeutic levels and avoidance of toxicity for patients with cystic fibrosis and complicated gram-negative infections. previous in vitro data has shown that col is bactericidal at col concentration is ‡ mg/l. here we assessed col data collected from our antibiotic assay service from the last seven years in order to establish if such levels were obtained. methods: col levels were determined by bioassay. data was retrospectively collected from the hospital information management system. data was assessed collectively and stratified by known cystic fibrosis patients, sex and age. objectives: ßlactams like ceftriaxone (cfx) and quinolones such as moxifloxacin (mox) are widely used to treat pneumococcal infection. we studied the antibacterial effect of (abe) after the first dose exposure to free drug serum concentrations of iv cfx g and po mox mg against s. pneumoniae strains with typical mics at low and high inocula. methods: a hollow fibre in vitro model was used to simulate free drug concentrations over h of cfx ( g h iv, cmax mg/l, auc mg/laeh, t / h) and mox ( mg, po, cmax . mg/l, auc mg/laeh, t / . h). the cfx mic was . mg/l (t>mic cfx %) and mox mic . mg/l (auc/ mic ). initial abe was measured by the slope of the log viable count - h and total abe over the dose interval ( h) by log reduction in viable count at h (d ) and the area-underthe-bacterial-kill-curve (aubkc ). inocula of cfu/ml and cfu/ml were used. results: the initial and total abe at low and high inocula were: given the pk/pd indices modelled both drugs showed a maximal effect. clearance from the model occurred at h ( inoculum) and h ( inoculum). there were no significant differences in speed or extent of abes comparing cfx and mox. conclusion: the abes of iv cfx and po mox against s. pneumoniae is similar in the first hrs of drug exposure. emergence of resistance in e. coli and ent. cloacae after exposure to ceftriaxone or ertapenem in an in vitro model of infection k. bowker, a. noel, a. macgowan (bristol, uk) objectives: emergence of resistance (eor) is an emergent factor in therapeutic choice. we studied eor to ceftriaxone (cfx) and ertapenem (ert) in e. coli (ec) and ent. cloacae (entclo), a more challenging species within inducible blactamases. methods: an in vitro dilutional model was used to simulate free drug concentrations associated with g hrly cfx (cmax mg/l, t½ h) and ert (cmax mg/l, t½ h) over h. two inocula and cfu/ml were used and eor assessed by population-analysis-profiles (pap). the area under the pap (auc-pap) was used to measure eor. ert mics were . mg/l ec and . mg/l entclo, cfx mic . mg/l ec and . mg/l entclo. experiments were performed in triplicate and mean values presented. results: observations at h were similar to those at h, hence data to h is given. at and cfu/ml, ec viable counts were reduced by fi logs, there was no eor. against entclo inoculum and , cfx resulted in an initial - log drop, then regrowth, ert produced a > log reduction. eor as measured by the mean auc-pap (n = ) with entclo is shown below: dosing with cfx resulted in eor to cfx and ert at high and low inoculum. dosing with ert resulted in no eor at inoculum, at resistance emerged to both cfx and ert. conclusions: eor depends on species (entclo > ec); duration of exposure (long > short) and agent (cfx > ert). ert appears to induce less eor both to itself and cfx than cfx does to itself and ert. however, initial use of cfx may reduce the effectiveness of ert. comparative serum activity of telithromycin, azithromycin, and amoxicillin/clavulanate against aerobic and anaerobic respiratory pathogens objectives: the purpose of this investigation was to study the clinical potential of telithromycin, a new ketolide antibiotic, for the treatment of mixed aerobic/anaerobic respiratory infections. in this study, we compared the pharmacodynamics (duration of inhibition/killing) of telithromycin (tel) to azithromycin (azi) and amoxicillin/clavulanate (a/c) against aerobic and anaerobic pathogens associated with mixed respiratory infections. methods: following written informed consent, ten healthy adult subjects (ages, - yrs) received single doses of tel ( mg), azi ( mg), and a/c ( mg) one week apart following a -h fast. venous blood samples were obtained at , , , and h after the dose and stored at ) °c. inhibitory and bactericidal titre s were determined by microdilution (s. pneumoniae & h. influenzae) and agar dilution (peptostrep. magnus, peptostrep. micros, prev. bivia, & prev. melaninogenica) procedures following clinical & laboratory standards institute methodology. bactericidal titres in serum endpoint was determined as the highest dilution of serum yielding . % killing. the median titres at each time point were calculated and the duration of activity was used for comparison of these agents. conclusions: in this ex-vivo study, we found that tel can provide prolonged ( % of the dosing interval) inhibitory activity in serum against macrolide-resistant strains of s. pneumoniae, bl pos. and neg. strains of h. influenzae, and common respiratory anaerobic pathogens. these findings suggest that tel could have clinical utility in the treatment of community-acquired mixed aerobic-anaerobic respiratory tract infections, including sinusitis, bronchitis, and pneumonia. objectives: increasing resistance in isolates of e. coli ( %) and p. aeruginosa ( %) to fluoroquinolones (fq) is a concern since these antibiotics are commonly used in the treatment of complicated urinary tract infections (utis). currently, no interpretive standards exist for ''susceptible'' isolates in urine for the newer fq. the purpose of this investigation was to evaluate the activity of high-dose levofloxacin against fqresistant urinary pathogens. methods: in this study, we determined the serum and urine levels of high dose ( mg) levofloxacin (l) as well as its bactericidal activity in urine (uba) against l-resistant isolates of e. coli (mics = to lg/ml) and p. aeruginosa (mics = to lg/ml). following written informed consent, blood and urine samples were collected from healthy adult (ages, - y/o) fasting subjects ( m and f) prior to and at . , , , , and hours after a single mg dose of l. serum and urine concentrations were measured by a validated hplc assay ( . - . % cv). the testing methodology for uba was similar to the microdilution assay used for serum bactericidal testing (clsi) with the exception that antibiotic-free urine was used to dilute these samples. the median titre ( : - : ) at each time point for the subjects was used to determine uba. results: the mean serum pharmacokinetic parameters were similar to previously published values: cmax = . lg/ml, auc = mgaeh/l, and t / = . h. mean urine concentrations ranged from lg/ml ( h) to lg/ml ( h). uba (titres > : ) was maintained for at least hours in all subjects for e. coli isolates with mics = , , and lg/ ml. for the e. coli strain with a mic = lg/ml, subjects exhibited uba at h but only subjects exhibited uba at h. similar results were observed against the p. aeruginosa isolates. conclusions: the results from this ex-vivo pharmacodynamic study in healthy volunteers found that mg of l provides prolonged (at least half the dosing interval) uba against l-resistant strains of e. coli and p. aeruginosa up to lg/ml. this suggests that a separate urinary susceptibility breakpoint is indicated for urine isolates treated with mg doses of l. objectives: exposure of methicillin-resistant s. aureus (mrsa) to acid ph restores its susceptibility to beta-lactams (sabath and al., aac, ) . in macrophages, s. aureus is mainly confined within phagolysosomes where the ph is acidic. we showed that meropenem (mem) displays similar intracellular activity against mrsa atcc and mssa atcc in macrophages. in the present study, we have investigated the intraphagocytic activity of mem and cloxacillin (clx) against mrsa clinical isolates (including one visa strain), in comparison with the reference mrsa atcc and mssa atcc strains. methods: mic's were determined in mhb (plus nacl %) by micro-dilution method. meca expression was examined at neutral and acidic ph by a semi-quantitative rt-pcr ( s rrna as housekeeping gene). intracellular activity was assessed in human thp- macrophages exposed to extracellular concentrations equivalent to human cmax (total drug; mem: mg/l; clx: mg/l) by examining the decrease in cellassociated cfu after h from the original, post-phagocytosis inoculum (controls [no antibiotic]; approx. log cfu increase). results: the table shows the mics (in broth) at neutral and acid ph and the intracellular activity for the strains studied. in atcc , meca expression was similar for bacteria maintained in broth at ph . conclusions: the intracellular environment markedly enhances the activity of beta-lactams against mrsa, probably through exposure to acid ph, although the latter does not affect meca expression. comparative activity of dalbavancin against european gram-positive isolates i. morrissey, c. dencer, j.w.t. dallow, j. childers, a. brook, j. cowan (london, uk) objectives: dalbavancin (dal) is a new semisynthetic lipoglycopeptide with a half-life of . days, enabling onceweekly dosing. this study compared the activity of dal with other agents against gram-positive isolates from europe. methods: isolates from belgium, the czech republic, denmark, finland, france, germany, hungary, italy, the netherlands, poland, spain, sweden and the uk were included. the clsi broth microdilution method was used to determine mic using dried microtitre plates. the following antimicrobial agents were evaluated: dal, vancomycin (van), teicoplanin (tei), daptomycin (dap), linezolid (lzd), dalfopristin/quinupristin (syn), erythromycin (ery), levofloxacin (lev) and tetracycline (tet). results: selected data are shown in the objectives: dalbavancin (dal) is a next generation lipoglycopeptide antibiotic in development for the treatment of complicated skin and skin structure infections (csssi). a population pharmacokinetic (pk) analysis was performed to estimate patient parameters and to determine significant covariates. incorporating the pk model, pharmacodynamic (pd) parameters were simulated to support the effectiveness of a weekly dosage. methods: the pk analysis included dal concentrations from patients across clinical trials. most patients received mg on day and mg on day . possible covariates examined included demography and concomitant medications, including medications that are considered inhibitors, inducers, and substrates of cytochrome p enzymes. the pk-pd analysis employed monte carlo simulations of time-dependent and concentration-dependent parameters. distributions of mics were obtained directly from clinical studies, and were also simulated to explore the effect of higher mics. results: dal pk fit a -compartment model with interpatient variability (ipv) on all parameters. the typical value and ipv (cv%) of clearance (cl) was . l/h ( . %), influenced by body surface area (bsa) and creatinine clearance (clcr). volume of distribution (v ) was . l ( . %) and influenced by bsa. the inter-compartmental clearance and peripheral volume were . l/h and . l, respectively. free drug concentrations were simulated using a dal protein binding of %. for a weekly -dose regimen, free dal remained above mg/l for the majority (> %) of patients for more than days. using previously described area under the curve (auc)/mic targets for staphylococcus aureus, a proposed mic of at least . mg/l was associated with a greater than % probability of target attainment. conclusions: dal pk were predictable, demonstrating low ipv. bsa and clcr were the only sources of variability, but described less than % of the ipv. pd simulations support the use of dalbavancin in a weekly regimen. objectives: methicillin-resistant staphylococcus joint infection due to peri-operative contamination is a complication after arthroplasty. the objective of this study was to assess the distribution of radioactivity in bone and related structures using quantitative autoradiography after administration of [ c]dalbavancin in rabbits. methods: new zealand white male rabbits were given a single intravenous (iv) bolus dose of mg/kg [ c]-dalbavancin (n = ) or control vehicle (n = ). plasma, cerebrospinal fluid (csf), bone, bone marrow, and nucleus pulposus were collected at , , , , and h post-dose by necropsy, homogenized, combusted, and analysed for total drug-derived radioactivity using liquid scintillation counting (lsc). in addition, the left hindlimb from rabbit/time point was flash-frozen and cryosectioned for quantitative autoradioluminography. results: [ c]-dalbavancin-derived radioactivity was rapidly and widely distributed into bone, bone marrow and, to a lesser extent, in csf and nucleus pulposus. autoradioluminography data indicated that concentration of radioactivity was highest in bone marrow, whole blood, articulate cartilage, ligament, epiphyseal plate, periostium, and meniscus. at h postdose, [ c]-dalbavancin-derived radioactivity was measurable in all tissues, and remained at relatively high concentrations in bone marrow ( . lg equiv/g), epiphyseal plate ( . lg equiv/g), periostium ( . lg equiv/g), and articular cartilage ( . lg equiv/g). in homogenized bone using lsc, mean concentration after hours was . lg equiv/g. conclusion: [ c]-dalbavancin-derived radioactivity rapidly penetrated knee joint tissues and persisted at relatively high concentrations for at least h after a single iv dose in rabbits. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound ( %) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution ( . l/kg) means that it may be removed by cvvh. cvvh is widely used in the management of critically ill patients. the objective of this study was to determine cvvh telavancin transmembrane clearance (cl) with commonly used hemofilters (an , polysulfone) at conventional ultrafiltrate flow rates. methods: tlv cl was assessed in our in vitro cvvh model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run using an (m , gambro) and polysulfone (f nr, fresenius) hemofilters. ultrafiltrate (uf) flows were , , and l/hr with sufficient blood flows [(qb) - ml/min] to maintain uf rates. blood samples were collected from the pre-filter line and uf samples from the post-filter uf port. concentrations of tlv in plasma and uf samples were assayed using validated lc-ms/ms methods. tlv cl was determined using the following formula:cl = (uf flow rate) [tlv]uf/[tlv]arterial. cl differences between the filter types were compared using a two-tailed, unpaired t-test. conclusion: tlv is substantially cleared by cvvh and cl increases significantly with increasing uf rate. cl did not differ by hemofilter type. cvvh cl at higher uf flows exceeds the total cl reported in patients with normal renal function. tlv likely will require dose adjustments in patients receiving cvvh. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound ( %) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution ( . l/kg) means that it may be removed by cvvhd. cvvhd is used in the management of critically ill patients. the objective of this study was to determine cvvhd tlv transmembrane clearance (cl) with commonly used hemodialyzers at conventional cvvhd dialysate flow rates. methods: tlv cl was assessed in our in vitro cvvhd model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run times using an (m , gambro) and polysulfone (f nr, fresenius) hemodialyzers. dialysate flows were , , and l/hr with sufficient blood flows [(qb) - ml/min] to maintain appropriate transmembrane pressures. blood samples were collected from the pre-filter port (a) and post-filter port (v), and spent dialysate samples (d) from the post-filter d port. plasma tlv concentrations (arterial and venous) and dialysate samples were assayed using validated lc-ms/ms methods and tlv cl was determined using the following formula: cl = (d flow rate) [tlv]d/(([tlv]arterial+[tlv]venous)/ ). dialytic cl between filter types was compared using a two-tailed, unpaired t-test. conclusion: tlv is effectively cleared by cvvhd. the higher permeability polysulfone dialyzer was associated with significantly increased cl vs. the an dialyzer as dialysate flow increased. the degree of tlv cl seen with cvvhd suggests that dose adjustments will be necessary in patients receiving cvvhd. objective: it is still a subject of controversy that only free, unbound drug is responsible for antibacterial activity of antibiotics. to provide further proof, that only free drug contributes to antimicrobial efficacy a comparative, doseranging time-kill curve study was performed. to exclude influence factors resulting from different mechanisms of action this was done within the antibiotic class of carbapenems, using compounds with different serum protein binding. methods: constant concentration time-kill curves were performed in % serum for the slightly serum protein bound mer-openem (~ %) and imipenem ( %) as well as for the highly serum protein bound ertapenem ( %) and faro-penem ( - %), ranging from · mic to · mic. the change in number of bacteria (cfu/ml) versus time was linked to their effect. escherichia coli atcc , klebsiella pneumoniae bay , staphylococcus aureus bay and streptococcus pneumoniae atcc were used as the test organisms. samples were taken at , . , , , and hours. the data were modelled simultaneously using the software scientist Ò for windows tm and a modified sigmoid emax model characterized by growth rate constant (k ), maximum kill rate (kmax), and concentration at half maximum effect (ec ). results: for all four bacterial strains investigated, there were dramatic increases ( - %) in ec for the highly se-rum protein bound carbapenems (ertapenem, faropenem) in the presence of serum proteins (fig. ) . for both substances no significant differences in k and kmax were determined. in contrast, imipenem and meropenem showed only minor differences in ec in the presence and the absence of % serum. conclusion: only free, unbound drug is responsible for the antimicrobial activity. analysis of these time-kill curves clearly showed that the antibacterial efficacy was significantly decreased in the presence of % serum for the highly bound ertapenem and faropenem while being unaltered for the slightly bound meropenem and imipenem. objective: numerous in vitro experiments have shown that protein binding (pb) is an important factor for antimicrobial activity, especially for highly bound antibiotics. however, the experimental conditions that simulate the in vivo situation best are still subject of controversy. therefore, an in vitro microdialysis experiment was performed that evaluates various influence factors on the pb of the highly bound betalactams ceftriaxone (pb - %). methods: a comparative, dose-ranging in vitro microdialysis study was conducted to determine free, unbound ceftriaxone concentrations in lactated ringer's solution and todd hewitt broth (thb) both with and without bovine serum albumin (bsa; sigma, st. louis) g/l and human plasma at °c. furthermore, in vitro constant concentration time-kill curves were performed, using escherichia coli atcc , streptococcus pneumoniae atcc and streptococcus pneumoniae atcc as the test organisms. the data was analysed using an appropriate pk/pd model, characterized by growth rate constant (k ), maximum kill rate (kmax), and concentration at half maximum effect (ec ) and correlated to free ceftriaxone thb concentrations determined by hplc-uv. results: there were only minor differences in both unbound drug concentrations and anti-infective activity when bsa g/ l was added to either lactated ringer's (pb % and . ± . %, with and without bsa respectively) or thb (pb % and . ± . %, with and without bsa respectively). no significant changes in k , kmax and ec were observed. however, using human plasma, unbound concentrations (pb %) %) were altered dramati-cally. conclusion: only free, unbound drug is responsible for the antimicrobial activity. however, one cannot rely on that binding to commercially purchased bsa is consistent with reported protein binding values. unbound concentrations should be measured under the respective experimental conditions to be able to correctly interpret the experimental results. in vitro postantibiotic effect of faropenem on penicillin-resistant streptococcus pneumoniae and beta-lactamase-producing haemophilus influenzae c.l. young, i.a. critchley, u.a. ochsner, n. janjic (louisville, us) objectives: faropenem (far) is an oral penem with potent activity against respiratory pathogens such as penicillin (pn)resistant streptococcus pneumoniae (sp) and beta-lactamase (bla)-producing haemophilus infleunzae (hi). the postantibiotic effect (pae) is a pharmacodynamic (pd) parameter that monitors suppression of bacterial growth following short exposure and removal of the drug. paes are clinically important for agents such as far with short half-lives ( h) . the aim of the study was to determine the pae of far on resistant phenotypes of sp and hi. methods: nine clinical isolates of sp, pn-s, pn-i, and pn-r and six clinical isolates of hi, bla-negative and blapositive were tested in pae studies. paes were determined in cation-adjusted mueller-hinton broth with - % lysed horse blood for sp and haemophilus test medium for hi. exponential cultures ( cfu/ml) were exposed to far at , and x mic. far was removed by serial washing ( , -fold dilution) prior to transfer to fresh media. control cultures were treated in the same way. bacteria were incubated with shaking and viable cfus determined at , , , , and h. counts of log cfu were plotted against time and pae defined as the difference is the time required for count in test culture and control (untreated culture) to increase log above the count observed immediately after removal. results: significant paes of > . h were observed for all strains of sp at and x mic. however, the pae was more prolonged on the pn-r strains with mean paes of . h at and x mic. among hi, little or no pae was observed on bla-negative strains but a significant pae was observed on the bla-positive isolates (mean paes of . h and . h at and x mic respectively). conclusions: far demonstrates a prolonged pae on key resistant phenotypes of sp (pn-r) and hi (bla-positive) compared with susceptible strains. the observation of pae in bla-positive hi is unique in the class of beta-lactams. far exhibits in vitro pd properties that may contribute to its clinical efficacy against pn-r sp and bla-positive hi. telavancin is more efficacious than vancomycin in a murine model of bacteraemic peritonitis induced by methicillin-resistant staphylococcus aureus s. hegde, n. reyes, b. benton, r. skinner (south san francisco, us) objective: telavancin (tlv) is a novel lipoglycopeptide that operates through multiple mechanisms to produce potent and rapid bactericidal activity against clinically relevant grampositive bacteria including methicillin-resistant staphylococcus aureus (mrsa). the present studies evaluated the in vivo efficacy of tlv vs vancomycin (van) in a model of mrsa induced peritonitis in neutropenic mice. methods: female nsa immunocompromised mice were inoculated intraperitoneally with atcc mrsa and treated, beginning at h post-infection, with subcutaneous doses (q h) of vehicle (veh) or test compound. mouse pharmacokinetic data were generated and used to choose doses of tlv ( mg/kg) and van ( mg/kg) in order to equate clinical exposures (auc's (free drug) of and lg.hr/ml, respectively). in survival studies, deaths were recorded for days post-infection and survival curves were compared using log-rank test. in bacterial titer determination studies, designated groups of control and drug-treated surviving animals were humanely euthanized at various times post-treatment and their blood and spleen were harvested to determine bacterial titers. results: mics of tlv and van were . and . lg/ml, respectively. mortality was % in animals treated with veh or van. mortality was % in tlv-treated animals (p < . vs veh and van). the pre-treatment bacterial titres were . log cfu/ml and . log cfu/g in the blood and spleen, respectively. analysis of the time kill curves for both blood and spleen revealed that tlv exhibited significantly greater killing activity than van (p < . , two-way anova). at hrs after the first dose, the titers in the blood were reduced to a greater extent by tlv () . log cfu/ml) compared to van () . log cfu/ml). at hrs after the second dose, the splenic titers were reduced to a greater extent by tlv () . log cfu/g) when compared to van () . log cfu/g). conclusions: the data described here demonstrate that tlv's in vivo bactericidal activity is superior to that of van against mrsa and results in successful infection resolution and, consequently, improved survival in the murine peritonitis model. proper use of carbapenems for blood-derived clinical isolates of pseudomonas aeruginosa y. kobayashi (tokyo, jp) methods: regimens of carbapenems were given to healthy adult subjects. changes in their blood concentrations of carbapenems were compared by using pharmacokinetic parameters (two-compartment model analysis) of meropenem (mepm), imipenem (ipm), and panipenem (papm) and by applying the lognormal distribution to the probability distribution of distribution volume and plasma half-life with monte carlo simulation (mcs). based on the data on distributions of the minimal inhibitory concentrations (mic) of various carbapenems for blood-derived clinical isolates of pseudomonas aeruginosa isolated/identified at keio university hospital between october and october (mic : mepm mcg/ml, ipm mcg/ml, papm mcg/ml), the mics in the subjects were obtained with mcs. from the changes in blood concentrations and mics in the subjects, the probability of achieving t>mic was calculated for each carbapenem regimen, using the formula reported by kuti et al.: %t>mic = ln (dose/vd*mic)*(t / / . )*( /di) <<ln: natural logarithm, dose: dose (mg), vd: distribution volume (l), t / : plasma half-life (h), di: dosing interval (h)>>. based on craig's data, the maximum bactericidal effect on gram negative bacilli is attained when %t>mic is approximately %. we focused on this information and analysed our data. results: the probability of achieving t>mic % was . % for mepm mg bid, followed by . % for ipm mg bid and . % for papm mg bid. when the dose was increased from mg to mg, it was . % for mepm mg bid, followed by . % for ipm mg bid and . % for papm mg bid. when the dose remained at mg and the dosing frequency was increased to three times daily, it was . % for mepm mg tid, followed by . % for ipm mg tid and . % for papm mg tid. regarding mepm, it was . % for mg gid? and . % for mg tid showing higher probabilities. discussion: in severe sepsis caused by pseudomonas aeruginosa, remarkably higher t>mic % was achieved with carbapenems at mg tid, although the daily dose ( mg) was lower, compared to mg bid. carbapenems with a low mic distribution, i.e. a superior antibacterial activity, showed higher probability of achieving t>mic. therefore, the optimal treatment for such sepsis is mepm mg tid. mepm mg qid appeared to provide comparable therapeutic effects with those at mg tid, the usual dose in foreign countries. penetration of moxifloxacin into normal and infected subcutaneous tissue in patients with spinal cord injury measured by microdialysis background: skin breakdowns, also termed decubitus ulcers or pressure sores, are a major complication associated with spinal cord injury, resulting in infection and tissue death. moxifloxacin (mfx) is approved for the treatment of sssi. our objective was to construct a population pk model for mfx disposition in plasma, normal and infected subcutaneous tissue in spinal cord injured patients with infected decubitus ulcer. methods: patients receiving mg mfx orally daily were enrolled in this study. blood, saliva and interstitial tissue fluid samples (microdialysis in normal and infected tissue) were collected over a time period of hrs. mfx concentrations were measured by a validated hplc. concentration-time data obtained in the present study were pooled with previously published mfx data (n = ). population pk modelling was performed with nonmem. results: the concentrations of mfx achieved in plasma, saliva, normal subcutaneous tissues and infected decubitus ulcers showed parallel profiles versus time. the pk was best described by a -compartment model with a link to interstitial tissue fluid. the population pk parameters were as follows (given as estimate with percent interindividual variability in parentheses): cl . l/h ( %); central vd . l ( %); intercompartmental cl . l/h ( %); peripheral vd . l ( %); and elimination rate constant for interstitial tissue fluid . h) ( %). with a conservative mic of . mg/l, the peak/mic ratios were higher than and the auc /mic ratios were higher than for plasma, saliva and interstitial tissue fluids. conclusions: this study showed the good diffusion of mfx into subcutaneous tissue in spinal cord injured patients with decubitus ulcers. the interstitial tissue fluids reached bactericidal levels for common bacteria found in infected skin lesions. objective: investigations of pharmacodynamic parameters such as postantibiotic effect and postantibiotic subminimum inhibitory concentration effect have been employed for design of dosing schedules of antimicrobial agents. in this study we compared postantibiotic effect and postantibiotic subminimum inhibitory concentration effect of ciprofloxacin, levofloxacin, and moxifloxacin for clinical isolates of methicillin susceptible staphylococcus aureus, methicillin resistant staphylococcus aureus and pseudomonas aureginosa. methods: the following strains were tested in this study: methicilline-susceptible staphylococcus aureus (n: ), methicilline resistant -staphylococcus aureus (n: ) and pseudomonas aeruginosa (n: ). the pae was determined by viable plate count method using mueller hinton broth. tubes containing ml of broth and the antibiotic to be tested at , , and x the mic were inoculated with approximately · cfu/ml. growth controls with an inoculum but not antibiotic were included with each experiment. result: postantibiotic effects of ciprofloxacin, levofloxacin and moxifloxacin increased with increasing concentration of the drug. the longest postantibiotic effect was observed for moxifloxacin. moxifloxacin showed no postantibiotic effect one p. aureginosa at all concentration and had no post antibiotic effect to another p. aureginosa at x mic and mic. in our study the longest postantibiotic subminimum inhibitory concentration effect against mssa was determined with moxifloxacin. similarly the moxifloxacin induced the longest effect against mrsa. however, this time frame was shorter than that of mssa. conclusions: all three antibiotics, showed for longer postantibiotic subminimum inhibitory concentration effect in all submic concentrations, immeasurable within the study period i.e. hours. lack of horizontal transmission of fluoroquinolone resistance between s. mitis and s. pneumoniae objectives: fluoroquinolone (fq) resistance can arise in s. pneumoniae through acquisition of dna from s. mitis and subsequent homologous recombination. the frequency at which this occurs is unknown, and while likely a rare event, increases in fq resistance among s. mitis may increase the rate at which horizontal transmission occurs. we sought to determine the frequency at which fq resistance could be transferred from s. mitis to s. pneumoniae or from s. pneumoniae to s. mitis. methods: s. mitis (either fq^r,tetracycline[tet^s], fq^r,penicillin[pen^s], or fq^s,pen^r) and s. pneumoniae (either fq^s,tet^r, fq^s,pen^r or fq^r,pen^s) were grown in co-culture using a pharmacodynamic model in the presence of either moxifloxacin (mxf) or levofloxacin (lfx) at salivary drug concentrations. after incubation, aliquots were plated onto either tet or pen containing sba plates to select for the recipient strains. fq susceptibility was performed using microbroth dilution. the entire parc and gyra genes were amplified and sequenced to determine if horizontal transmission occurred. results: in initial experiments tet was used as the selective agent. however tet resistance was transferred and therefore pen^r was used as a selective marker. an increase in the lfx mic in was observed in s. pneumoniae and s. mitis strain. sequencing of the parc gene revealed the selection of ser phe and ser tyr mutations in s. pneumoniae and ser phe in s. mitis consistent with fq resistance. sequencing of the entire gene failed to uncover evidence of horizontal transmission. no mutations were detected in gyra. selection of st step parc clinical microbiology and infection, volume , supplement , mutations occurred only after exposure to lfx. mxf eradicated both s. mitis and s. pneumoniae and failed to either select for resistance or support horizontal transmission. conclusions: although st step parc mutations were selected in strains ( s. pneumoniae, s. mitis), we failed to find evidence of horizontal transmission between s. pneumoniae and s. mitis under our laboratory conditions. the phenomenon of horizontal transfer resulting in fq resistance has been described, however, based on our results, we must speculate that it is an extremely rare event and not likely to be a major driver of fq resistance. of interest, the parc mutations were selected only under the selective pressure of lfx. mxf completely eradicated both s. pneumoniae and s. mitis and did not select for the development of fq^r mutations. objectives: the aim of the present study was to assess the killing activity of ertapenem (ert) and metronidazole (mtr) against four selected bacteroides fragilis strains with different mic values in an in vitro pharmacokinetic/pharmacodynamic (pk/pd) model. since anaerobes are often present in mixed infections, kill kinetics were also established for mixed inocula employing the b. fragilis strains together with four selected escherichia coli strains. the killing activity was analysed for kinetic concentrations of the antimicrobial agents simulating human serum kinetics. methods: a pk/pd in vitro model was established by adding appropriate amounts of broth every half hour. at the same time intervals samples were obtained and plated. after incubation colony forming units were counted. human serum concentrations were simulated with cmax = mg/l and t / of hours for ert and cmax = . mg/l and t / of hours for mtr. mann trend test was used for statistical analysis. results: as to be expected the e. coli strains were not killed by mtr both in pure as well as in mixed cultures whereas the susceptible e. coli strains were effectively killed by ert. in pure cultures the b. fragilis strains were effectively killed by mtr and the growth of the susceptible b. fragilis strains was reduced by ert by about two to four logs. however, in some mixed cultures the killing activity of mtr against the b. fragilis strains was significantly reduced. conclusion: the in part moderate in vitro activity of ert against the b. fragilis strains and the reduced activity of metronidazole in mixed cultures against the b. fragilis strains may explain some of the difficulties in treating mixed aerobic/ anaerobic infections. penetration of ciprofloxacin into human cerebrospinal fluid and brain tissue a. tsona, s. metallidis, e. koumentaki, j. nikolaidis, p. kollaras, g. lazaraki, p. nikolaidis (thessaloniki, gr) objectives: the aim of the present study was to determine the penetration of ciprofloxacin into cerebrospinal fluid (csf) and brain tissue of humans. methods: a total of patients undergoing brain tumor excision were evaluated. the patients received a single intravenous dose of mg ciprofloxacin. samples of blood, cerebrospinal fluid and brain (brain-adjacent tumour tissue) were collected during surgery h after drug administration. ciprofloxacin concentrations in serum, cerebrospinal fluid and brain homogenate were analysed by means of a validated hplc method. results: ciprofloxacin concentrations in plasma (mcg/ml), cerebrospinal fluid (mcg/ml) and tissue homogenate (mcg/g), respectively, after h ranged . - . mcg/ml, . - . mcg/ ml and . - . mcg/g. csf-to-serum ratio ranged between . and . . tissue-to-serum ratio ranged between . and . . mean (±s.d.) csf/serum concentration ratios and brain tissue/serum concentration ratios were respectively . ± . and . ± . . conclusion: these findings suggest that valuable informations on brain tissue penetration can be obtained only from brain material. data from csf penetration cannot be extrapolated to the brain since the blood: sf barrier differs from the blood:brain barrier. concentrations of ciprofloxacin in cerebrospinal fluid were lower than those in serum, in contrast to the brain tissue concentrations that exceeded serum concentrations. the achieved concentrations in brain tissue were generally above the mic of common pathogens in central nervous system infections (h. influenze, n. meningitidis, s. pneumoniae, l. monocytogenes, escherichia coli, aerobic gram-negative bacilli, group b streptococci, mssa). cerebrospinal fluid concentrations exceed the mics of neisseria meningitidis and most gram-negative aerobic bacilli. our findings suggests that ciprofloxacin may be an acceptable alternative for the treatment of meningitis due to susceptible gram-negative aerobic organisms and for the treatment of brain abscesses. objective: to model the performance of imipenem (imi), meropenem (mem), and ertapenem (etm) against esbl producing e. coli and klebsiella spp in order to identify possible pd differences among compounds. methods: minimal inhibitory concentrations (mics) were generated for randomly selected esbl producing isolates of ec (n = ) and kl (n = ) collected during from brazilian hospitals as part of the mystic program. mic testing for imi, mem, etm, ceftazidime (ctz), and cefotaxime (ctx) were done by e-test methodology. esbls were confirmed via ctz/clavulanate and ctx/clavulanate e-test. pd exposure, measured as percent time above the mic for free drug (ft>mic), was modelled via a subject monte carlo simulation for the following -minute infusions:imi gram every hours, mem gram every hours, and etm gram every hours, using pharmacokinetics from healthy volunteers. the bactericidal cumulative fraction of response (cfr) was calculated for each regimen against the populations of ec, kl, and against all esbl isolates together. bactericidal cfr was defined as % ft>mic for all agents. results are reported as cfr ( % confidence interval). results: isolates were % susceptible (s) to imi and mem (mic range . - . and . - mg/l, respectively), and % s to etm (mic range . - mg/l conclusions: these findings support other data that although etm is likely to be an effective empiric agent against most esbl producing ec and kl, its ability to achieve high bactericidal pd exposure will be dependent on the presence of less susceptible organisms in the population. imi and mem should remain first line for esbl infections. objectives: this study analyses eradication and resistance selection in streptococcus pneumoniae with moxifloxacin, levofloxacin and azithromycin, using a parental serotype infecting strain (a) and subsequent resistant step-mutants (isolates b, c and d) selected in vivo in a patient with pneumonia. methods: moxifloxacin, levofloxacin and azithromycin mics were , and . lg/ml for the parental strain, , , and lg/ ml for isolate b, and , and > lg/ml for isolates c and d, respectively. a pharmacokinetic computerized device was used to simulate serum and epithelial lining fluid (elf) concentrations. initial inocula was approx. cfu/ml. population analysis profiles were performed using plates with increasing antimicrobial concentrations on a mic basis. results: in serum, moxifloxacin eradicated the parental isolate (isolate a), with an auc - h/mic value of . . serum auc - h/mic values of . and . for levofloxacin and azithromycin, respectively, were not able to eradicate isolate a. in elf, moxifloxacin showed a bactericidal pattern against all isolates with a minority (approx. cfu/ml) of the survival population (isolates b, c and d) growing in plates with moxifloxacin concentrations higher than those obtained in elf. levofloxacin and azithromycin showed a bactericidal pattern only against isolate a, with the whole population of isolates b, c and d growing in plates with levofloxacin concentrations higher ( - lg/ml) than those obtained in elf, and in plates with azithromycin concentrations as high as lg/ml (for isolates c and d). in elf, moxifloxacin auc - h/mic values were . for isolate a, and . for isolates b, c and d. levofloxacin auc - h/mic values were . for isolate a, and . for isolates b, c and d. azithromycin auc - h/mic values were . for isolate a; . for isolate b; and for isolates c and d. conclusion: if prevention of resistance depends more on the eradication of possible emerging mutants in pulmonary tissues than of the parental susceptible strain, moxifloxacin concentrations in elf may provide advantages over previous quinolones and macrolides in preventing clinical failures. objectives: to explore how antimicrobial pressure influences the evolution of streptococcus pneumoniae populations sharing the same ecological niche. methods: an in vitro computerized pharmacodynamic model simulating physiological concentrations obtained over h after mg o.d levofloxacin, mg b.i.d ciprofloxacin, and mg o.d azithromycin was used to investigate its effect on a mixed culture of five s. pneumoniae serotypes (s) as an approach to ecology of population dynamics. resistance patterns were: s was susceptible to study drugs, s was low-level macrolideresistant (efflux phenotype), s was high-level macrolideresistant (erm genotype), s v was low-level quinoloneresistant, and s was high-level quinolone-resistant. initial mixed inocula (time ) included similar percentages of each serotype. results: mean colony counts in antibiotic-free plates (whole pneumococcal population) increased (from to h) from log . to . in drug-free simulations (control), from log . to . in levofloxacin simulations, from log . to . in ciprofloxacin simulations, and from log . to . in azithromycin simulations. at h of control drug-free experiments, dominant strains were s v ( . %) and s ( . %) with marginal populations of s , s and s . azithromycin selected in a much higher extent the strain with low-level resistance to macrolides (s ) than the strain with high-level resistance (s ) (accounting for . % vs. . % of total population at h). ciprofloxacin selected in a higher extent low-level (s v) than high-level (s ) quinolone resistance ( . % vs. . %). levofloxacin decreased the proportion of the predominant s v in controls to . % (an intermediateresistant strain with mic = lg/ml), and unmasked the highlevel resistant strain (mic = lg/ml) up to . %. conclusion: strain distribution in antibiotic-free environment depends on bacterial fitness in mono-and multi-strain niches. the selective pressure of antimicrobial regimens eradicate some populations and unmask minor populations, thus redistributing the whole population. selective potential only for resistance phenotypes with very low prevalence (as high-level quinolone resistance) in the community should be preferred to that selecting more prevalent resistance phenotypes. re-evaluation of the role of broad-spectrum cephalosporins against staphylococci applying contemporary in vitro results and pharmacokinetic-pharmacodynamic principals h. sader, s.m. bhavnani, p.g. ambrose, r. jones (north liberty, us) objectives: to re-evaluate the current in vitro activity and to assess the pk-pd target attainment of cefepime (cpm), ceftriaxone (cro) and ceftazidime (caz) against staphylococcus spp. methods: the potency of cpm, cro and caz against staphylococci was accessed through the sentry antimicrobial surveillance program database, worldwide. during the - period , s. aureus (sa; % oxacillin [oxa]-susceptible [s]) and , coagulase-negative staphylococci (cons; % oxa-s) were s tested against cpm, cro, caz and numerous comparators by clsi broth microdilution methods. using volunteer pk data and a linear intermittent intravenous infusion model, and an animal-derived pk-pd target of % time above mic, expected probabilities of target attainment (pta) for cephems were evaluated using monte carlo simulation. pta were determined for the following dosing regimens: cpm gm q and q hours, caz gm q hours and cro gm q hours, each representing the most common dosing patterns applied clinically. cephem susceptibility (%s) was calculated based on the current clsi ( ) breakpoints (bkps) and also on bkps derived from a pta > %. results: against oxa-s sa, mic / values were (in mg/l): / for cpm, / for cro and / for caz, respectively; and against oxa-s cons mic / values were (in mg/l) . / for cpm, / for cro, and / for caz, respectively. the calculated %s of these cephems are summarized in the table: twenty year-old clsi bkps would rank the tested agents cpm ‡ cro > caz and by pk-pd pta cpm ‡ caz > cro. cpm has a potency advantage over caz ( -to -fold) and superiority at the usual dosing over cro ( . - . %) for oxa-s staphylococci. caz pk overcomes by-weight activity disadvantages, while a low proportion (< %) of active freedrug penalizes cro in the pta calculations. pta remained at > % to a bkp of mg/l for cpm ( gm q ) and caz and to a bkp of mg/l for cro. conclusions: regardless of applied bkp (clsi or pk-pd), cpm has the widest and more potent anti-staphylococcal activity among commonly used ''third-or fourth-generation'' cephems. when used at doses ‡ gm/day, cpm assures maximal coverage of oxa-s staphylococci whether using existing (clsi) or modified (pk/pd) bkps. cro should be used with caution. methods: the mic for all strains were determined by serial two-fold macrodilutions. an in vitro kinetic model was used to investigate the antibacterial efficacy of constant drug concentrations during hours. the selection of the doses of azithromycin tested in each bacterial strains was based on their mic values. bacterial counts were determined on appropriate agar plates using an adapted drop-plate method. twelve different pk/pd models were fitted and compared to the time-kill data by using non-linear regression. results: a simple pk-pd model was not sufficient to describe the pharmacodynamic effects for the four bacterial strains. appropriate models that gave good curve fits included a saturation term for the number of bacteria (nmax), delay terms ( -e-zt) for the initial bacterial growth phase and/or the onset of anti-infective activity as well as a hill factor (h) to capture the steepness of the concentration-response relationship. azithromycin had high potency against s. pneumoniae strains and m. catarrhalis while the potency of azithromycin against h. influenzae was poor. conclusions: the developed pk/pd models are suitable for describing the pharmacodynamics of azithromycin. applications of these pk-pd models will eventually provide a tool for rational antibiotic dosing decisions. objectives: optimal antimicrobial dosage regimens aim to achieve successful clinical outcomes without drug toxicity or emergence of bacterial resistance. for concentration dependent antibiotics, such as the fluoroquinolones, in humans a cmax:mic ratio of > is considered more important for efficacy and reduced selection of resistance than prolonged antibiotic concentrations just above the mic. fluoroquinolone resistance in zoonotic bacteria is a matter of public health concern, and fluoroquinolone treatment of poultry can rapidly select for bacteria with reduced fluoroquinolone susceptibility. in this study we compared basic pharmacokinetic parameters for the recommended dose of baytril (enrofloxacin) % oral solution in poultry to . x this dose for birds dosed by continuous water (standard) compared to pulsed water treatments and dosing by gavage.methods. for the pulsed versus continuous water treatments, groups of chickens received baytril % oral solution at (recommended) or ppm continuously in the water or at (recommended) or mg/kg pulsed in the water. for each group, three birds were killed at , , , , , and hours after start of antibiotic treatment and caecal contents, liver, lung and sera were taken and the concentration of fluoroquinolone determined by fluorescence hplc. for gavage treatment, dosing was at and mg/kg by crop intubation and four birds were killed in each group at , and hours after gavage; caecal contents, liver and sera were taken and analysed as above. basic pharmacokinetic parameters were determined using pk solutions software. results: the mean fluoroquinolone cmax in caecal contents (and sera) for gavage, pulsed water and continuous water treatments respectively was . ( . ), . ( . ) and . ( . ) mg/ml after the recommended dose and . ( . ), . ( . ) and . ( . ) mg/ml after . x the recommended dose. cmax of antibiotic in liver and lung was increased by the modified regimens in similar proportions to above. both pulsed water and gavage treatment not only resulted in higher cmax values, but also a faster rate of fluoroquinolone clearance than continuous water treatment ( figure ). dosing by gavage is not practical for thousands of chickens. however, pulsed dosing at . x the recommended dose can increase cmax values about fourfold and so could improve efficacy and reduce selection of resistance, compared to the current recommended treatment regime. objectives: nephrotoxicity is the major concern arising with the use of intravenous colistimethate sodium. methods: a prospective cohort study was performed at ''henry dunant'' hospital, a -bed tertiary care center in athens, greece. patients who received intravenous colistin for at least days for the treatment of multidrug resistant gram-negative bacterial infections were included in the study. the development of nephrotoxicity through evaluations of serum creatinine, blood urea, serum electrolytes, urinalysis, and creatinine and sodium in -hour urine collection during intravenous colistin therapy was the primary end point of the study. results: twenty-six patients were included in the study, of whom received colistimethate sodium (cms) for at least days and were evaluated further. the mean (± sd)/median daily dose, cumulative dose, and duration of treatment of intravenous cms was . (± . )/ million iu, . (± . )/ million iu, and . (± . )/ days (range - days), respectively. three of the evaluable patients ( . %) developed nephrotoxicity during the intravenous treatment with cms. the cumulative dose of the administered cms was statistically correlated with the difference between the end and start of cms treatment values of serum creatinine (r = . , p = . by spearman's test). a statistically but not clinically significant decrease of the mean baseline serum sodium concentration was observed between start and end of treatment [mean . (± . ) to . (± . ) mmol/l, p = . ]. no other toxic events were noted during the intravenous administration of colistimethate sodium. conclusion: although this is an evaluation of a small number of patients, our prospective study shows that nephrotoxicity was not commonly observed in this group of patients who received intravenous colistimethate sodium. however, caution should be taken to avoid the prolonged administration of the antibiotic. objectives: the objective of the present work is quantitative structure-activity relationship (qsar) analysis of antimicrobial activity of the -thiazolidone derivatives and consequent computational design of new antimicrobials. methods: for the achievement of the formulated objectives the qsar investigation has been carried out using computational chemistry approach based on simplex representation of molecular structure (sirms). on the framework of sirms it is possible to develop the molecular design of the new effective antimicrobials. results: systematic researches of relationship between antimicrobial activity (staphylococcus aureus -methicillin-sensitive (mssa) strain, pseudomonas aeruginosa -r and s strain, klebsiella pneumoniae, candida albicans s and Ñ itrobacter freundii) and a structure of about one hundred fifty compounds ( -thiazolidone derivatives and analogs). the elucidation of structure-activity relations allows predicting biological properties of such compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of their biological effect. completely adequate statistical partial least squares models (r = . - . , q = . - . ) have been obtained for all of the studied cultures. on the base of the first ones the molecular fragments both promoting and interfering the given antimicrobial activity have been determined. they give a possibility to realize the computer high throughput screening and molecular design of active compounds. the results of prognosis are verifying by the experimental investigations. also the influence of heterocycle system evolution on antimicrobial activity has been revealed. conclusion: qsar analysis of antimicrobial activity of -thiazolidone derivatives allows us to discover that the presence of naphthalene-substituted fragment (independently on its location in molecule) has distinctly negative influence on antimicrobial action. the requirements to molecular design have been formulated. for example, high active compounds must include -indolyl fragment. computational design of the new antimicrobials based on the substituted crown ethers activity of the row of substituted crown ethers and consequent molecular design of new antimicrobials. methods: the well-known hierarchic system of qsar models based on simplex representation of molecular structure has been used for the solution of the formulated problem, within the framework of which one it is possible to develop the molecular design of the new effective antimicrobial agents. results: we tried to conduct systematic researches of relationship between antimicrobial activity (planococcus citreus, streptococcus lactis, micrococcus lysodeiktious, staphylococcus aureus, streptococcus faecalis, bacillus subtillum) about two hundred fifty crowns ethers including aromatic, cyclic and heterocyclic etc. fragments and a structure of these molecules, in particular -macro cycle size, it dentacy, lipophily, nature of the substituents, and other factors. the elucidation of similar relations allows predicting biological properties of crown compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of biological effect of such kind of compounds. completely adequate qsar models (r = . - . , q = . - . ) have been obtained using partial least squares method for all of the studied cultures. on the base of the first ones the molecular fragments with positive or negative influence on the explored properties have been determined. they give a possibility to realize the virtual screening and molecular design of compounds with the high level of target activity. the results of prognosis are verifying by the experimental investigations. conclusion: qsar analysis of antimicrobial activity of crown ethers allows us to suppose the presence of two different mechanisms of their antimicrobial action. it is discovered that the presence of diphenyloxide and tert-butyl fragments promotes; diphenyl-sulphide and diamino-biphenyl -prevents the antimicrobial action. it is shown that the hexadenthal crown ethers containing aromatic fragments with a tert-butyl group are the most perspective antimicrobials. objectives: methionyl trna synthetase (mrs) catalyses the covalent attachment of methionine to its cognate trna. rep is a synthetic inhibitor of mrs with potent antibacterial activity against staphylococcus aureus including clinically-relevant resistant strains (mic equals . to . lg/ml). we determined the biochemical potency and mechanism of action of rep and related compounds with respect to s. aureus mrs enzymatic activity. we also evaluated the enzyme kinetic properties of mutated forms of s. aureus mrs. methods: the mets gene from s. aureus was expressed in e. coli and mrs was purified to near homogeneity by ammonium sulfate fractionation and anion exchange chromatography. aminoacylation of trnamet was measured using scintillation proximity assays (spa). the kinetics of the atp:ppi exchange were determined using thin layer chromatography (tlc). mutants of s. aureus mrs were selected by serial passage and spontaneous resistance in the presence of rep . results: rep exhibited strong inhibition of s. aureus mrs in the aminoacylation reaction, having an ic limited by the enzyme concentration. in order to estimate the true inhibition constant (ki), we utilized an atp:ppi exchange assay. rep showed potent inhibition of s. aureus mrs, with a ki of pm. related inhibitors were analysed, and a correlation was observed between the ki for mrs and the mic for s. aureus. rep was found to be competitive with methionine binding, but uncompetitive with atp binding (i.e., increasing the atp concentration resulted in tighter binding of rep ). the majority of analogs exhibited comparable mechanism of action; altered mechanism of action was observed with a subset of analogs. mutated s. aureus mrs variants (derived from strains with elevated mics) showed substantially weaker binding by rep . all of the mutated enzymes exhibited impaired trna aminoacylation activity, with defects ranging from reduced turnover rates to weaker affinities for one or more substrates. conclusions: rep is a potent inhibitor of s. aureus mrs. enzymatic potency of this class of inhibitors correlates with microbiological potency. mutations that confer resistance to rep result in functionally impaired mrs, encompassing a wide variety of enzymatic phenotypes. we report here the antibacterial and antifungal activity of newly synthesized and physico-chemically characterised thioureides of -( -chlorophenoxy)-benzoic acid. the new compounds were prepared in three stage. firstly, the -( -chlorophenoxymethyl)-benzoic acid was prepared by treating the phtalide with p-chlorophenol potassium salt in xylene. the second stage was the synthesis of -( -chlorophenoxymethyl)benzoyl chloride by treating the corresponding acid with thionyl chloride using anhydrous , -dichloroethane as solvent, followed in the third stage, by the treatment of the above-mentioned chloride with ammonium thiocyanate. the -( -chlorophenoxymethyl)-benzoyl isothiocyanate resulted after refluxing the reaction mixture in dry acetone. the new compounds were prepared by refluxing the isothiocyanate with primary aromatic amines in dry acetone. the obtained compounds have been characterized by their physical properties and their chemical structures were confirmed using the spectral analysis. the aim of this study was also to evaluate the in vitro antimicrobial activity of the new compounds. the in vitro antimicrobial testing was performed by binary microdilution method, in multi-well plates, in order to establish the minimal inhibitory concentration (mic), against gram-positive (listeria (l.) monocytogenes, staphylococcus (s.) aureus, bacillus (b.) subtilis), gram-negative (psedomonas (p.) aeruginosa, escherichia (e.) coli, salmonella (s.) enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, depending on the nature of the substituents and their position on the benzene ring, both concerning the microbial spectrum and the mic value. the mics values widely ranged between mcg/ml and mcg/ml. the most active proved to be n-[ -( -chlorophenoxymethyl)-benzoyl]-n'-( , -dichloro-phenyl)-thiourea and n-[ -( -chlorophenoxymethyl)-benzoyl]-n'-( -bromo-phenyl)-thiourea, showing a large spectrum of antimicrobial activity against enterobacterial strains (e. coli and s. enteritidis), l. monocytogenes, s. aureus and candida sp. all the tested compounds were highly active against s. aureus (mic = mcg/ml). four of the tested compounds exhibited antifungal activity (mic = - mcg/ml), and p. aeruginosa as well as b. subtilis were resistant to all tested compounds. in vitro antimicrobial activities of novel dianthraquinones produced by a marine streptomyces sp. against clinical staphylococcus aureus and enterococcus faecium isolates k.l. laplante, k. lor, a. socha, d.c. rowley (providence, north kingston, us) objectives: the escalation of antibiotic resistance among grampositive pathogens presents increasing treatment challenges and requires the development of new therapeutic agents. recently we discovered a new class of dianthraquinone antibiotics produced by a marine streptomycete. the inhibitory and bactericidal activity of four dianthraquinone secondary metabolites and four semi-synthetic derivatives were measured against clinical strains of vancomycin resistant e. faecium (vre), methicillin susceptible and methicillin resistant s. aureus (mssa and mrsa, respectively). two compounds, daq a and daq , were tested against an expanded panel of pathogens. methods: thirty-two clinical strains of vre (n = ), mssa (n = ) and mrsa (n = ) were obtained from patients at the veterans affairs medical center in providence, ri. mic's were performed using methodologies described by clsi. control isolates were atcc and atcc . the bactericidal activity of each antimicrobial agent was evaluated with time-kill experiments using randomly selected mssa (n = ), mrsa (n = ), and vre (n = ) isolates tested at times the respective mic. conclusions: the potent activities and unusual structures of the dianthraquinones tested here suggest that these may provide a new molecular scaffold for the development of novel antimicrobial agents. more biological testing is warranted to more fully explore the clinical potential of these antibiotics. efficacy of the novel antimicrobial peptide plectasin to staphylococci objective: the purpose of the investigation was to investigate the in vitro efficacy and kill kinetics of plectasin against staphylococcus aureus. plectasin is a newly discovered defensintype antimicrobial peptide found in the fungus pseudoplectania nigrella which showed activity against several gram-positive bacteria including drug resistant strains (mygind ph. et al. plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. nature ; : - ). methods: all experiments were determined according to clsi/ nccls guidelines. bactericidal activity was characterized by time kill experiments at and times the mic. staphylococcus aureus (s. aureus) atcc were used as the test organism and vancomycin was used for comparison. the kill kinetics and post antibiotic effect (pae) were evaluated by cfu determination. inoculum sizes ranging from e to e cells were used to test the inoculum effect. e cells were employed for determination of mutant prevention concentration (mpc) and the frequency of spontaneous resistance. results: plectasin is bactericidal as evidenced by kill kinetics showing a . log reduction in cfu/ml after hour of incubation and a reduction of . log cfu/ml after hours. this is superior compared to the activity of vancomycin. no inoculum effect was observed in the employed range of cells. the observed pae had a duration of hours and minutes. no spontaneously resistance mutation was observed among e cells of staphylococci and the mpc were determined to be times mic. conclusions: plectasin is a novel antimicrobial peptide that shows potent antimicrobial activity against gram-positive bacteria including drug-resistant organisms. the potent, excellent bactericidal activity in vitro, lack of cross-resistance to clinical used antibiotics, low spontaneously resistance mutation frequency and good pae properties, suggest that plectasin may have potential as a therapeutic agent against staphylococci. in vitro antimicrobial activity of the novel polymeric guanidine akacid plus Ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: cationic antimicrobials are widely used for disinfection within clinical settings. in the present study the bactericidal and fungicidal activity of akacid plus Ò , a novel polymeric compound of the cationic family of disinfectants, was evaluated against quality control strains of staphylococcus aureus, enterococcus hirae, escherichia coli, pseudomonas aeruginosa, candida albicans and aspergillus niger in comparison to chlorhexidine digluconate. methods: the in vitro activity of akacid plus Ò and chlorhexidine was determined by quantitative suspensions tests according to the european committee for standardization at concentrations of . - . % against bacterial strains and c. albicans and at concentrations of . - % against a. niger after exposure for , and min in the presence and absence of . % bovine albumin and dilution in distilled and hard water. results: in the basic quantitative suspension test akacid plus Ò destroyed all bacterial pathogens at a concentration of ‡ . % in £ min contact time. chlorhexidine was also highly active against s. aureus, e. coli and p. aeruginosa, but failed to eliminate e. hirae within min. under high organic burden, the bactericidal activity of both disinfectants was slightly reduced. akacid plus Ò showed fungicidal activity against c. albicans within - min and eliminated a. niger at a concentration of ‡ % in min contact time. chlorhexidine was fungicidal against c. albicans, but did not achieve biocidal activity against a. niger. conclusion: the novel polymeric guanidine akacid plus Ò when compared to chlorhexidine digluconate showed similar bactericidal activity against s. aureus, e. coli and p. aeruginosa and superior biocidal activity against e. hirae and a. niger. investigation of emergence of bacterial resistance to the novel antibacterial photodynamic agent xf- are novel, light activated antibacterial agents ( ) active against gram-positive bacteria, which have greater potency than antibiotics. the emergence of resistance to xf- has been investigated. methods: . mg/l of xf- was added to cells/ml of mrsa. after minutes incubation in the dark the unbound xf- was removed and the culture illuminated with . j/cm of light at nm and cfu analysis undertaken to determine the number of viable cells remaining. surviving clones of the treatment were cultured and subjected to further treatment. cycles were undertaken to determine whether the number of surviving cells increased, suggesting resistance build up to xf- . results: the survival of methicillin-resistant staphylococcus aureus (mrsa) (atcc baa- ) is expressed as log n /n, where n and n are the cfu of untreated and treated suspensions, respectively. the results demonstrate that no detectable resistance build up to the activity of xf- was seen after successive treatments. a low propensity for emergence of resistance is a valuable attribute for new anti-bacterial agents. xf- might be effectively employed in the clinical setting for prophylactic use to decolonise skin and nares and therapeutic use to treat infected wounds/ulcers. objectives: the xf drugs are novel, light activated antibacterial agents ( ) active against gram-positive bacteria which have superior potency to antibiotics but possess a low propensity to induce resistant bacterial strain emergence. a novel ex-vivo porcine skin model has been developed to test the antibacterial activity of xf- on the surface of skin. methods: x cells of methicillin-resistant staphylococcus aureus (mrsa) were inoculated onto a . cm area of ex-vivo porcine skin samples, immobilised in agar. after drying, solutions of xf- were applied and after minutes, the samples were illuminated for minutes with blue light ( nm) with various total light doses using a lumacare tm lc- m lamp. cfu analysis were undertaken to determine the number of viable cells remaining after treatment. controls of drug alone and light alone were included. results: using . mg/l of xf- , cfu analysis demonstrated that at a total light dose of j/cm , there was~ % kill of bacteria. at j/cm , there was . % kill of bacteria, and . % at j/cm and j/cm . at a total light dose of j/ cm , it was found that there was a < % kill by xf- at concentrations of . , . and . mg/l. at a concentration of . mg/l, there was a > . % kill. this kill did not significantly increase at . and . mg/l. conclusions: the results demonstrate that xf- has exceptional activity at low concentrations against mrsa on the surface of porcine skin. xf- and light are non-toxic to skin at therapeutic concentrations. work is in progress to clinically evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objectives: the rise of epidemic methicillin-resistant staphylococcus aureus (emrsa) and the emergence of mupirocin resistance means that it is essential to develop new therapies that cannot be readily overcome by microorganisms. the xf series of novel light activated antibacterial agents ( ) active against gram-positive bacteria addresses this issue and have superior levels of activity to antibiotics but with less likelihood of resistance emergence. the antibacterial activity of the xf drugs against emrsa has been investigated. methods: mic and mbc assays were used to investigate the antibacterial activity of xf- , a novel antimicrobial photodynamic agent against a range of staphylococcus aureus strains. a concentration range of - . mg/l was investigated. minutes of nm light activation ( j/cm ) was applied. light alone had no effect. results: conclusions: the results demonstrate that xf- has exceptionally low mic and mbc values against all of the s. aureus strains tested. the results also demonstrate that xf- is equally effective against mrsa and methicillin-sensitive staphylococcus aureus (mssa) indicating its mode of action is independent of antibiotic resistance. xf- may therefore be useful in prevention and treatment of emrsa. xf- is non-toxic to skin at prophylactic/therapeutic concentrations and has potential for the treatment of skin sepsis and the eradication of nasal and skin mrsa carriage. work is in progress to evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objective: nxl is a novel antibacterial currently in preclinical development. the mechanism of action is directed against topoisomerase, and the spectrum of activity is exclusively against gram positive organisms. the goal of the study was to characterise the activity and time/kill kinetics against common aerobic cocci in comparison to currently marketed molecules: linezolid (lin), vancomycin (van), quinupristin/dalfopristin (q/d) and moxifloxacin (mox). methods: (i) in vitro susceptibility tests: the strains used were from the culture collection of novexel and were of clinical origin. mics were determined by an agar dilution technique. mueller hinton agar medium was used, supplemented with % horse blood for group a streptococci (gas), group b streptococci and s. pneumoniae. overnight cultures were diluted to obtain the final inoculum of cfu/spot. the mic was the lowest concentration which inhibited all visual growth ( or less colonies were ignored). (ii) time/kill kinetics: experiments were performed against strains of s. aureus (n = ) and s. pneumoniae (n = ) in ml volumes of appropriate growth medium with initial inoculum of around cfu/ml of logarithmically growing culture. timed samples over a hour period were enumerated using a spiral plating method. nxl was compared to linezolid and vancomycin and the concentrations tested were , and -fold the mic for both species. results: (i) the mic s of nxl versus comparators are shown in the table. (ii) time kill experiments showed that nxl was bactericidal against s. aureus, including methicillin resistant strains (> log reduction within - hours) compared to a slowly bactericidal effect for vancomycin ( hours). nxl and vancomycin were both bactericidal against s. pneumoniae within - hours. linezolid was bacteriostatic against all strains tested. conclusion: nxl exhibits bactericidal activity against common gram positive cocci, including strains which exhibit resistance to methicillin, vancomycin and fluoroquinolones. nxl warrants further investigation. objectives: the aim of this study was to identify bacterial proteins as targets of the endogenous antiseptic n-chlorotaurine (nct), which is a promising microbicidal agent for topical treatment of infections. in addition, a combination of nct with ammonium chloride which enhances the microbicidal activity significantly was investigated. methods: escherichia coli and staphylococcus aureus were treated with nct and nct plus ammonium chloride for different incubation times between and min -a period where killing takes place. to find out protein changes, d-page of bacterial proteins followed by mass spectrometry was performed. results: incubation in % nct revealed a change of the charge and a separation of numerous proteins into a series of spots with a different isoelectric point. moreover, in e. coli heat shock protein appeared, while ribosome releasing factor, d-ribose periplasmic binding protein, and malonyl-coa transacylase spots decreased. in s. aureus, enolase and a translation elongation factor decreased. these changes appeared more rapidly in the presence of ammonium chloride, which can be explained by formation of the more lipophilic and microbicidal monochloramine. molecular mechanisms of attack comprised mainly oxidation of thio and amino groups as confirmed with model peptides. conclusion: these results fit very well to previous preclinical and clinical findings. they indicate both surface attack and penetration of oxidation capacity into the bacteria and destruction of essential proteins by nct and nct plus ammonium chloride, respectively. objectives: ceftobiprole is a new extended-spectrum cephalosporin with activity against methicillin-susceptible and methicillin-resistant staphylococci, as well as against most enterobacteriaceae. in this study the anti-staphylococcal activity of ceftobiprole is reported from a set of isolates from a recent clinical trial. methods: consecutive clinical isolates of staphylococci from patients enrolled in a multicentre clinical trial involving complicated skin infections were examined for their susceptibility to ceftobiprole and selected anti-gram-positive agents. mics were determined using clsi methodology. results: among these isolates, staphylococcus aureus and coagulase-negative staphylococci (cons) were identified. the percentages of methicillin-resistant strains were % for s. aureus and % for cons. all strains (except one cons with a linezolid mic of mg/l) were susceptible to vancomycin and linezolid, with mics < mg/l.against methicillin-susceptible s. aureus, ceftobiprole mic and mic values were . and . mg/l, respectively, and against methicillin-resistant s. aureus, ceftobiprole mic and mic values were . and mg/l, respectively. ceftobiprole mics ranged from £ . to mg/l against methicillin-susceptible-cons (ms-cons) and methods: consecutive, non-duplicate bacterial isolates ( , strains) acquired from patients with bloodstream, respiratory, and skin and skin structure infections both nosocomial and community acquired were submitted from > medical centres in europe, the americas and the asia-pacific region. all isolates were tested using clsi/nccls broth microdilution methods against grn, the currently marketed fluoroquinolones (fq) including cipro, levofloxacin (levo), gatifloxacin (gati) and representative comparator agents. oxa-and cipro-s and -r subsets were included. a grn-s breakpoint of £ . mg/l was applied for comparative purposes only and was based upon the mic population distributions of strains that included quinoloneresistance determining region (qrdr) mutations. results: potency for grn and comparator fqs tested against sa: (see table) . key resistance patterns (%) among this sa collection included oxa ( . ), cipro ( . ), erythromycin ( . ), clindamycin ( . ), tetracycline ( . ), and trimethoprim/ sulfamethoxazole ( . %); gram-positive-targeted comparator including vancomycin, linezolid, daptomycin and quinupristin/dalfopristin all remained > % s. compared with currently marketed fqs when tested against all sa, grn was -to -fold more active (mic , £ . vs. . or . mg/ l). against both oxa-s and -r sa, grn displayed markedly enhanced potency compared with cipro and levo ( ‡ -fold), and gati ( -to -fold). among cipro-r isolates, grn also maintained ‡ -fold greater potency (mic , vs. ‡ mg/l) although overall s for all fqs was - %. compared to the fq agents tested against sa, grn was the most potent agent and maintained the broadest coverage against oxa-and cipro-r strains even when applying a very conservative epidemiologic breakpoint. when a fq is indicated for staphylococcal coverage, this des-f( ) quinolone may represent a superior alternative among fq class agents, while minimizing selection of resistance. objective: to assess the garenoxacin (grn) potency against a vast number of international respiratory tract infection (rti) pathogens, especially versus phenotypic (high mic) or genotypic (sequence change) qrdr mutants. a total of , isolates from continents were analysed ( ) ( ) ( ) ( ) ( ) ( ) ( ) table) conclusions: grn maintains clinically usable activity (mic, £ mg/l) against important community-acquired rti pathogens having r to presently marketed fluoroquinolones and against those isolates with documented qrdr mutations. continued development of this novel des-f( ) quinolone agent appears desirable. in vitro activity of garenoxacin tested against ciprofloxacin-susceptible and -resistant enterobacteriaceae and acinetobacter spp. strains collected worldwide by the sentry antimicrobial surveillance program ( ) ( ) h. sader, t. fritsche, p. strabala, r. jones (north liberty, us) objective: to evaluate the contemporary activity of garenoxacin (grn) against ciprofloxacin (cipro)-susceptible (s) and cipro-resistant (r) enterobacteriaceae (ent) and acinetobacter spp. (asp). unlike recently marketed fluoroquinolones (fq), grn, a des-f( ) quinolone lacks the c- fluorine. methods: a total of , isolates ( , ent and asp) were consecutively collected from > medical centres from bloodstream, respiratory, urinary and skin and soft tissue infections and tested by reference broth microdilution methods according to clsi/nccls methods and interpretative criteria. a grn s breakpoint of £ mg/l was applied for comparison purposes only. results: the results of the major organism groups tested: (see table) . grn showed excellent activity against this large collection of ent (mic , . mg/l) and . % of isolates were inhibited at £ mg/l. objectives: garenoxacin (grn) is a novel, broad-spectrum des-f( )-quinolone with activity against gram-negative and grampositive aerobes and anaerobes including quinolone-resistant staphylococcus aureus. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in complicated skin and skin structure infections (csssi). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn ( mg iv to po qd) or piperacillin/tazobactam ( . g iv q h) with transition to po amoxicillin/clavulanate ( mg po q h). in the second study, subjects received grn ( mg po qd) or ciprofloxacin/metronidazole ( mg q h/ mg q h). all antimicrobials were administered for to days. subjects were adults ( ‡ y) newly hospitalized or ambulatory outpatients with evidence of csssi who did not have underlying osteomyelitis. microbiologic efficacy was determined to days post-therapy. results: a total of subjects were microbiologically evaluable (grn, n = ; comparators, n = ). the disease diagnosis was similar between grn and comparators and included infected pressure sore ( % vs %), infected diabetic foot ulcer ( % vs %), major abscess ( % vs %), or postsurgical wound infection ( % vs %). the majority of common skin pathogens were eradicated by grn background: acute bacterial sinusitis (abs) is a common infection world-wide, with many patients having an associated an allergic component/history. however the role of antibacterials in these patients (pts) has not been examined. as some fluoroquinolones (fq) have an in-vitro immunomodulatory effect (ie) the clinical efficacy of gem was compared other agents in abs pts with or without allergic rhinitis (ar). methods: phase clinical trials were pooled and pts where allergic rhinitis was identified ( pts) were compared with pts not reporting ar ( pts). clinical response (success or failure) at end of therapy (eot) & at follow-up (fu, approx. - weeks after treatment) was studied. comparators (cmp) were cefuroxime (cef) and trovafloxacin (tro). results: % success based on clinical outcome at eot and fu for ar and non ar pts are shown in the table. for all treatments eot success was high for the non ar pts, but at fu this was reduced, especially with both fqs. in contrast, gem retained a high clinical success rate in pts with ar unlike cef or tro. conclusion: gem has been shown to be very efficacious in a sub group of problematic abs pts. this advantage may be due to the high antibacterial activity of gem vs key abs pathogens and/or a stimulatory ie. both being important with pts having decreased local immune defences. these data also show that not all fluoroquinolones have immuno-stimulatory properties. garenoxacin efficacy against multidrug-resistant streptococcus pneumoniae: retrospective analysis of community-acquired pneumoniae isolates obtained from nine phase ii and iii clinical studies ( ) ( ) ( ) ( ) ( ) t. black, h. waskin, r. hare (kenilworth, us) objective: garenoxacin (grn) is a novel, des-f( )-quinolone with excellent activity against s. pneumoniae, one of the most common pathogens causing community-acquired pneumoniae (cap). the incidence of infections caused by antibiotic-resistant isolates of streptococcus pneumoniae is on the increase, therefore information regarding the activity of new anti-infective drugs against populations of s. pneumoniae that are multi-drug resistant (mdr) is critical. mdr s. pneumoniae (mdrsp) includes isolates previously known as prsp (penicillinresistant s. pneumoniae), as well as strains resistant to two or more of the following antibiotics: second-generation cephalosporins, macrolides, tetracyclines, and trimethoprim/ sulfamethoxazole. methods: pretreatment sputum and blood isolates collected worldwide during grn phase / clinical cap trials ( ) ( ) ( ) ( ) ( ) were retrospectively analysed for the mdrsp phenotype. of the s. pneumoniae isolates originally identified, from subjects were subjected to secondary mdr susceptibility testing by central laboratories. confirmed mdrsp isolates were matched to individual subjects to assess clinical and microbiological outcomes for mdrsp-infections treated with grn. results: expanded susceptibility testing identified / mdrsp isolates from unique subjects. the lowest mic and mic values for mdrsp isolates tested against a panel of representative drugs were observed for grn (table ; . lg/ ml and . lg/ml, respectively). the incidence of resistance to the five classes of drugs was %, %, %, % and % for penicillin, nd generation cep., macrolides, tetracycline and tri/sulf, respectively. no isolates were resistant to grn using a proposed susceptibility breakpoint value of £ lg/ml. thirtyfive percent, %, %, % and % of isolates were resistant to , , , and drug classes, respectively. the worldwide incidence of mdrsp was % with an equivalent geographic distribution of %, % and % among north america, europe and the rest of world. overall, grn provided clinical and bacteriological success for / ( %) cap evaluable subjects with mdr infection, which was similar to clinical success for evaluable subjects with non-mdrsp cap infections / ( %). conclusions: these data demonstrate the ability of grn to successfully eradicate mdrsp associated with cap. . per cent success is shown in the table (ab, antibiotics, copd, chronic bronchitis and obstructive lung disease, hd, heart disease). results: although gemifloxacin showed lower % success than comparator against cap patients with no defined risk factor, gemifloxacin was considerably more successful than comparator against patients associated with risk factors, especially diabetic patients where comparator success was low. this advantage was often more prominent at fu than at eot. patients with other comorbidities such as renal failure or malignancy were not recruited in sufficient number for analysis. conclusions: these data support the use of gemifloxacin in the treatment of cap, especially where the patient has recognised idsa risk factors. microbiologic efficacy of garenoxacin vs. comparators against common pathogens associated with community-acquired pneumonia objectives: garenoxacin (grn) a novel, broad-spectrum des-f( )-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. grn has dual sites of inhibition (dna gyrase and topoisomerase iv) and may be less likely to promote resistance. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in community-acquired pneumonia (cap). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn ( mg po qd for d) or amoxicillin/clavulanate (a/c; mg po q h for - d). in the second study, subjects received grn ( mg po qd for - d) or levofloxacin (lev; mg po qd for - d). adults ( years of age or older) were enrolled with clinical and radiologic evidence of cap [new infiltrate(s) on chest radiograph and fever, leukocytosis, cough, chest pain, auscultatory findings, or sputum production]. the majority of subjects were fine class i/ii in both studies. bacteriologic eradication was assessed to days post therapy. results: a total of treated subjects had pretreatment pathogens (grn, n = ; comparators, n = ) . the overall eradication rate in all treated subjects was % ( / ) for grn and % ( / ) for the comparators. eradication rates for s pneumoniae were % ( / ) for garenoxacin and % ( / ) for the comparators. eradication of s pneumoniae was % and % for a/c and lev, respectively. in strains with reduced susceptibility to penicillin eradication rates were % ( / ) vs % ( / ) in favour of grn. eradication rates for h. influenzae were % ( / ) and % ( / ) for grn and comparators, respectively. lev eradicated % of h. influenzae isolates and a/c eradicated % of the strains isolated. there were very few isolates ( ) of moraxella catarrhalis in the studies. in study grn was % effective against strains of m. catarrhalis and in the other a/c was % effective against the strain isolated. grn eradicated % ( / ) of the staphylococcus aureus isolates vs % ( / ) for the comparators. conclusions: grn was highly active against pathogens commonly associated with cap including drug-resistant strains of s pneumoniae and represents an effective therapeutic option for this patient population. objectives: garenoxacin (grn) a novel, broad-spectrum des-f( )-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. there is a growing problem of resistance in strains of s pneumoniae, with multi-drug-resistant s pneumoniae (mdrsp) becoming increasingly more common. the objective of this study was to evaluate the clinical and microbiologic efficacy of grn in the treatment of communityacquired pneumonia (cap) caused by mdrsp. methods: this was a multinational, open-label, noncomparative study. subjects were adults ( ‡ and < y) with clinical (clinical signs, sputum production), radiologic (new infiltrates on chest radiograph), or microbiologic (predominance of gram-positive cocci in pairs on sputum gram-stain or a positive blood culture for s. pneumoniae) evidence of cap caused by s. pneumoniae. subjects received grn mg po qd or grn mg iv with transition to mg po qd for to days. clinical and microbiologic responses were determined at a test-of-cure visit to days posttherapy. results: a total of subjects were enrolled. of these, ( po only, iv to po) were clinically and microbiologically evaluable. clinical and microbiologic success rates were % ( / ) and % ( / ), respectively. clinical success rates were % ( / ) and % ( / ) for po and iv to po, respectively. documented s. pneumoniae bacteremia was present in % (n = ) of subjects with a clinical success rate of %. among evaluable subjects, resistance rates for s. pneumoniae were penicillin %, second-generation cephalosporin %, macrolides %, tetracyclines %, and trimethoprim/ sulfamethoxazole %. twelve evaluable subjects had pneumonia caused by mdrsp. clinical success rate was % ( / ) in subjects with mdrsp and % ( / ) in non-mdrsp subjects. clinical success of grn for strains resistant to , , , or antimicrobial drug classes, were % ( / ), % ( / ), % ( / ), and % ( / ), respectively. microbiologic success was % ( / ) and % ( / ) for mdrsp and non-mdrsp (susceptible or resistant to class) strains, respectively. grn was generally well tolerated with drug-related adverse events (ae) reported in % ( / ; po) and % ( / ; iv to po) of subjects. conclusions: grn (po or iv to po) is an effective treatment for cap caused by mdrsp and non-mdrsp. grn is well tolerated. in vitro bactericidal activity of daptomycin against staphylococcus aureus and enterococcus spp.: comparison with vancomycin, teicoplanin and linezolid h. drugeon, m. juvin (nantes, fr) objectives: the aim of this study was to evaluate the bactericidal activity (by killing kinetics) of daptomycin (dap) against staphylococcus aureus (sa) clinical isolates with different teicoplanin mics and against enterococcus faecalis (efl) and e. faecium (efm) with different mechanisms of glycopeptide resistance. dap has been compared with teicoplanin (tei), vancomycin (van) and linezolid (lin). methods: sa strains ( mssa and mrsa) with tei mic distributed from . to mg/l, enterococcus ( efl and efm) with glycopeptide phenotypes [s, r-vana, r-vanb] were studied using a killing curve method. antibiotic concentrations were used from mg/l to mg/l in two fold dilutions. surviving bacteria were counted at t , t ', t , t , t , t and t hours using agar plates with inhibitors to prevent antibiotic carry-over. antibiotics tested were daptomycin (dap), teicoplanin (tei), vancomycin (van) and linezolid (lin). results: all the sa isolates were susceptible to dap (mic = . - mg/l), to lin (mic = - mg/l), to van (mic = - mg/l) regardless of susceptibility to methicillin. dap showed the same strong concentration dependent bactericidal activity with mssa and mrsa: at t ' bactericidal activity (ba) (decrease of log cfu/ml) was observed with - mg/l of dap; at t hours, - mg/l of dap was sufficient and at t hours, ba was obtained with mg/l of dap. the other antibiotics showed a time dependent bactericidal activity but ba was observed only with long exposure ( ‡ hours) and with high concentrations. all the enterococcus isolates were susceptible to dap (mic = - mg/ l) and to lin (mic = - mg/l) regardless of the resistance to glycopeptides. ba of dap was also concentration dependent. ba was obtained with - mg/l after hours of contact and with mg/l after hours of contact for efl. ba was observed with - mg/l after hours of contact and with - mg/l after hours of contact for efm. the other antibiotics had a time dependant activity but didn't show bactericidal activity with concentrations mg/l. conclusion: the bactericidal activity of daptomycin was very strong, concentration dependent, and not influenced by the level or mechanism of glycopeptide resistance the bactericidal activity of linezolid was time dependent and observed only with the highest concentration and the bactericidal activity of vancomycin and teicoplanin was time dependent but was influenced by the mechanism of glycopeptide resistance. objectives: telavancin (tlv) is a bactericidal lipoglycopeptide with multiple mechanisms of action that is in phase clinical trials for the treatment of complicated skin and skin structure infections and hospital-acquired pneumonia with a focus on infections due to methicillin-resistant staphylococcus aureus (mrsa). this study evaluated and compared the antibacterial activity of tlv with that of other antibacterial agents against recent gram-positive clinical isolates from germany. methods: a total of aerobic gram-positive bacterial strains recently collected were included. antibiotics tested were tlv, vancomycin (van), teicoplanin, penicillin, oxacillin, ampicillin, cefuroxime, ceftriaxone, daptomycin (dap), linezolid (lzd), quinupristin-dalfopristin, clindamycin, ciprofloxacin, levofloxacin, gentamicin, streptomycin, erythromycin, telithromycin, co-trimoxazole and tetracycline. mics were determined by the broth microdilution procedure according to the guidelines of the clsi. results: tlv exhibited potent activity against all grampositive bacteria including resistant isolates such as mrsa, van-resistant enterococci, pneumococci (including multiple resistant strains with various antibiotic resistance phenotypes) and other streptococcal species. tlv showed excellent in vitro activity against the species irrespective of the antibiotic phenotype tested. for methicillin-susceptible s. aureus (mssa, n = ) and mrsa (n = ) mic of tlv for both phenotypes were . mg/l. for coagulase-negative staphylococci (n = , incl. msse, mrse, mssh, mrsh and others) mic s were . or mg/l. mic s of tlv for enterococcus faecalis (n = ) and e. faecium (n = ) were and mg/l, respectively. for van-resistant strains of e. faecalis (n = ) or e. faecium (n = ) mics for tlv ranged from . to mg/l. against streptococcus pneumoniae (n = ) tlv mics ranged from £ . to . mg/l. all streptococcus pyogenes, streptococcus agalactiae and all viridans group streptococci (n = ) had mics of £ . mg/l. conclusion: based on mic , tlv was more potent than van, dap or lzd against staphylococci, streptococci and e. faecalis. it was superior to dap and lzd against e. faecium and at least as active as dap or lzd against most van-resistant enterococci. tlv appears to be a promising new antimicrobial agent for the treatment of infections caused by gram-positive organisms including multiply resistant isolates. the extent of protein binding (pb) of dap is still under investigation and data available so far indicate pb of either % or %. therefore we tested two fscs: . (corresponding to % pb) and . (corresponding to % pb). the activity of dap was determined in mueller-hinton broth supplemented with mg/l calcium. viability counts were performed at . , . , , , , and h. one methicillin-susceptible staphylococcus aureus (mssa), two methicillin-resistant s. aureus (mrsa), one vancomycin-susceptible (van-s) and one van-resistant (van-r) enterococcus faecalis, one van-s and one van-r enterococcus faecium were tested. bactericidal activity was defined as > . % killing during incubation. results: dap was bactericidal at concentrations of . mg/l and . mg/l in all seven strains. the concentration of . mg/l was bactericidal against the two mrsa and against the van-s e. faecium. in the other four strains the maximum reduction of initial inoculum ranged from . to . log cfu/ml. in six strains a bactericidal effect at . mg/l and . mg/l of dap, respectively, occurred between minutes and h and after h in the van-s e. faecalis. van at . mg/l or . mg/l was bactericidal in only two strains after h ( mssa, mrsa). against the other five strains, van was bacteriostatic with maximum reduction of initial inoculum between . and . log cfu/ml at mg/l after h, respectively. both tpl and lzd were consistently bacteriostatic against the test strains. conclusion: dap at psc of . mg/l as well as at fsc of . mg/l showed a pronounced bactericidal effect within h in / strains. van was bactericidal in only / strains after h. compared to van bacterial killing by dap was very rapid. tpl and lzd were bacteriostatic only. the effect of human serum on the bactericidal activity of daptomycin and comparators against staphylococcus aureus and enterococcus spp. background: daptomycin is a new cyclic lipopeptide antibiotic that shows rapid bactericidal activity and has high protein binding when assessed by standard methodology. this study investigated the bactericidal activity of daptomycin and the effect of protein binding by the addition of % human serum (hs). methods: exponentially-growing methicillin-susceptible andresistant s. aureus (mssa, mrsa) and vancomycin-susceptible enterococcus faecium (vse) and -resistant enterococcus faecium (vre) (ca. cfu/ml) were exposed to daptomycin (dap), vancomycin (van), teicoplanin (tei), piperacillin-tazobactam (ptz) or linezolid (lzd) at peak (p) and trough (t) serum concentrations in mueller hinton broth supplemented with ca + to mg/l with or without hs. viable count was determined at . , . , , & h. plots were made of log reduction in viable count over time and the area-under-thecurve measured to calculate bactericidal indices (bis) from these plots (j antimicrob chemother , : - ). results: daptomycin reduced viable count of mssa & mrsa by approx. logs or more within . h and vse or vre within h at p. other agents either did not achieve this or required h to do so (not shown). bi data are shown below (>represents kill beyond the limit of detection). hs had little effect on dap kill, except against the vre at t. nevertheless, dap at t against vre was more bactericidal than any other antibacterial except dap at p. conclusions: dap was the most bactericidal agent tested as measured either by bi or rate of kill. dap at p reduced mssa and mrsa to below detection within min. the effect of hs was minimal which suggests that protein binding is either weak or highly reversible. these data support the use of dap in the treatment of infections caused by these organisms. daptomycin activity against multi-resistant staphylococcus haemolyticus bloodstream isolates from severe infections objectives: daptomycin, a new cyclic lipopeptide with activity against multidrug-resistant gram-positive pathogens including mrsa, is approved for use in cssst infections (us-fda) and is being reviewed by emea for approval in eu member countries. the rapid bactericidal activity of daptomycin, due to its unique mechanism of action, makes it an attractive antibiotic for serious gram-positive infections. the study was performed: (i) to evaluate the activity of daptomycin and other drugs against multi-resistant clinically relevant staphylococcus haemolyticus (mrsh), isolated from bloodstream infections in various hospitals in italy (ii) to determine epidemiologic and genetic correlation among strains, and (iii) to characterize the sccmec dna of these strains. methods: the mrsh strains were tested against a panel of antimicrobial agents, by broth microdilution method performed according to clsi (clinical laboratory standards institute) guidelines, including supplementation of mg/l calcium for daptomycin. moreover, phenotypic tests and antibiotic susceptibility profiling were carried out and the results compared with molecular typing analysis by using smai-pfge fingerprints and pcr to characterize the mec-complex. results: all isolates were resistant to erythromycin, gentamicin, ciprofloxacin, strains showed reduced susceptibility to vancomycin (mics mg/l), strains were resistant to cotrimoxazole, strains to clindamycin, strains to chloramphenicol and strains to tetracycline. almost all isolates were inhibited by £ mg/l of daptomycin, and only four strains exhibited a mic value of mg/l. pfge analyses showed the existence of at least two multi-resistant s. haemolyticus clones widespread in different hospitals. methicillin-resistance was correlated to the presence of the meca and preliminary results regarding the genetic element carrying the gene, showed an organization of the mec-complex of class a and class c. conclusions: our results suggest that daptomycin has excellent activity against multiresistant mr s. haemolyticus isolates, which represent a serious threat in catheter-related bloodstream infections. furthermore, the emergence of s. haemolyticus exhibiting reduced susceptibility to vancomycin is of particular concern, probably due to the common use of vancomycin as initial therapy for such infections. moreover, the use of additional molecular techniques to fingerprint isolates makes this study of clinically important cons more accurate. objectives: ceftobiprole is a new cephalosporin with a broad spectrum of action including methicillin-resistant staphylococci (mrs) as well as many other gram-positive and gram-negative pathogenic bacteria. this study investigates the structural basis for the good activity against mrs. methods: the primary beta-lactam resistance determinant of mrs, penicillin-binding protein pbp ' (or a) has been cloned and expressed as a soluble form in which the amino-terminal residues forming a membrane-anchor have been deleted. the soluble form has been crystallized and the structure of the complex formed after soaking crystals in a solution containing ceftobiprole has been determined at . angstrom resolution. additional data on the structure of the ceftobiprole-pbp ' complex formed in solution has been obtained using spectroscopic methods such as uv-circular dichroism. results: ceftobiprole reacts rapidly with pbp ' to form a stable acyl-enzyme complex. the ceftobiprole moiety is positioned deep within the active site of the acyl-enzyme complex formed with pbp ', where it forms several hydrogen bonds and hydrophobic interactions. in particular, the -aminothiadiazolylhyroxyiminoacetyl side chain of ceftobiprole sits more deeply within the side-chain binding pocket of pbp ' than does the -acylamino side chain of nitrocefin in the previously determined complex structure. the additional interactions probably add to the enhanced stability of the acyl-enzyme complex formed with ceftobiprole, compared to complexes formed with other betalactams that are inactive against mrs. significant structural rearrangements between apo-enzyme and acyl-enzyme are evident in the crystal structure and in solution. conclusion: ceftobiprole readily forms a stable inhibitory acylenzyme complex with the pbp ', the beta-lactam resistance determinant of mrs. this, together with potent inhibition of the normal complement of beta-lactam sensitive penicillin-binding proteins, accounts for its excellent activity against staphylococci and probably accounts for the low rates of resistance development observed in experimental conditions. incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in a french hospital (november -april c. morate, a. charron, c. bebear, j. maugein (bordeaux, fr) staphylococcus aureus are a major cause of nosocomial infections around the world. glycopeptides remain the drug of choice for severe infections caused by mrsa. however, after the emergence of vancomycin resistance in enterococcus and in the coagulase negative staphylococcus, strains of staphylococcus aureus with reduced susceptibility to glycopeptides (gisa) have been reported in different countries like japan, france, spain, the uk and the united states. the aim of our study was to determine the proportion of vancomycin resistance in clinical s. aureus isolates in a french university hospital, between november and april , then we wanted to define if there was an epidemic clone and study the clinical impact of these gisa strains. the protocol of detection was, first, a screening test on bhi agar containing mg/l of teicoplanin, then, the vancomycin and teicoplanin mics were determined by the method of etest with an inoculum of . mcf on the selected strains. finally, the isolates with mic of the teicoplanin ‡ mg/ l and mic of the vancomycin ‡ mg/l or mic of the teicoplanin ‡ mg/l and mic of the vancomycin £ mg/l were studied on population analysis. after that, pulsed-field gel electrophoresis (pfge) was performed on the different isolates and the pulsotypes were compared. from november to april , s. aureus isolates were collected from patients and screened for glycopeptide resistance on an initial agar screening test containing mg/l of teicoplanin. the teicoplanin mic was > mg/l for isolates ( . %) from patients and these strains were selected for the determination of the mics by ''macromethod'' etest. by this technique, strains were selected and studied by population analysis. all the profiles were compared to the reference strain mu profile. this procedure detected isolates (from patients) with heterogeneous reduced susceptibility to glycopeptides (hgisa). so the incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in our hospital was found to be . %. four strains were resistant to methicillin and were also resistant to gentamicin. the diversity of the strains was confirmed by pfge: there was not an epidemic clone in the hospital. the clinical history showed that patients had received a prior treatment with vancomycin, and that patients had a failure in treatment: of them had cystic fibrosis. objectives: enterococcus faecalis was the most prevalent organism ( . %) involved in enterococcal infections at tehran hospitals followed by e. faecium ( . %). due to widespread expansion of aminoglycoside modifiying enzymes (agmes) genes, the rate of resistance to high level concentration of aminoglycosides has increased in these years. the rate of high level gentamicin resistant isolates of enterococci (hlgr) is high in iran ( %). the aim of this study was to determine the genes encoding resistance to aminoglycosides among enterococci in iran. methods: disks containing lg gentamicin were used to detect hlgr isolates. primers specific for aac ( ') aph ( ") and aph ( ') iiia genes were used in pcr to possibly detect acetyltransferases and phosphotransferas, the common agmes among isolates of enetrococci. theses isolates were resistance to different concentration of gentamicin. results: a bp region of the aac ( ')-aph ( ") gene was amplified by pcr in % hlgr isolates as well as in % of low level getamicin resistant isolates (llgr). moreover the gene aph ( ') iiia was detected in . % and % of isolates of hlgr and llgr respectively. differences between isolates of e. faecalis and e. faecium were found in term of prevalence of aph ( ') iiia gene. conclusion: the bifunctional enzyme aac ( ')-aph ( ") is the main cause of resistance to high concentration of aminoglycosides in our collection of enterococci. this enzyme confers resistance to all clinically useful aminoglycosides with the exception of streptomycin. in the absence of aac ( ')-aph ( "), gentamicin could be used in combination therapy. prevalence and genetic analysis of methicillinresistant staphylococcus aureus expressing highlevel and low-level mupirocin resistance m. kural, t. us, y. akgun (eskisehir, tr) objectives: to investigate the genetic location of mupa gene which encoded mupirocin resistance and characterize mupirocin-resistant methicillin resistant staphylococcus aureus (mrsa) isolated from patients in a turkish university hospital by polymerase chain reaction (pcr) and plasmid analysis. methods: methicillin and mupirocin resistance were detected by disk diffusion (oxoid, uk). the etest (ab biodisk, sweden) was performed to determine mupirocin minimum inhibitory concentrations (mics). the presence of mupa and meca were detected by pcr using specific primers. plasmid analysis were used to study the genetic location of mupa gene. results: a total of ( . %) mrsa strains were identified by disk diffusion in s. aureus. of the clinical isolates ( . %) were from wound, ( . %) from blood, ( . %) from catheter, ( . %) from lower respirator tract (bronchoalveolar lavage, pleural fluid and transtracheal aspirates), ( . %) from sputum, ( . %) from urine and ( . %) from other (serebrospinal fluid, parasynthesis fluid, peritoneal fluid, and bone marrow) clinical samples. among the mrsa isolates, mupirocin resistance was detected in ( . %) strains with disk diffusion and etest. of the mupirosin-resistant isolates ( . %) expressed high-level (muh) and ( %) expressed lowlevel (mul) mupirocin resistance. all isolates were vancomycin, teicoplannin susceptible and chloramphenicol resistant with disk diffusion. isolates with high-level and low level mupirocin resistance due to the mupa gene were also detected with pcr. plasmids were detected in all of the isolates. however only the muh isolates contained a kb plasmid that encoded highlevel resistance. all of the isolates contained a . kb plasmid and resistant to chloramphenicol. conclusion: our results indicated that the mrsa clones detected in the hospital had acquired a high-level mupirocin resistant plasmid. the past observations and recent studies suggested that the numbers of such strians have increased following extensive topical use of mupirocin. the usage of mupirocin in our hospital has not yet been systematically implemented. it is frequently prescribed for the treatment of staphylococcal skin infections and less to eliminate nasal carriage of mrsa. in our hospital we should be aware of the possible emergence and increase of mupirocin highly resistant mrsa strains in the future so that we should be considered when using mupirocin to control the spread of mrsa in hospital. emergence and spread of acquired fusidic acid resistance in staphylococcus aureus objectives: a major route to fusidic acid resistance (fusr) in s. aureus involves acquisition of fusb, a resistance determinant first clinical microbiology and infection, volume , supplement , identified on plasmid pub . here we show that (i) the two currently-circulating major clones of fusr s. aureus identified to date have acquired fusb from pub (or from the same ancestral source as pub ), and (ii) that the pub -encoded fusb is only one of at least three lineages of this protein that appear to have evolved since recruitment of the original, ancestral fusb to the staphylococci. methods: plasmid purification, dna sequencing, pcr amplification, and cloning in s. aureus rn using shuttlevector pcu , were all performed using established methods. antibiotic susceptibility testing was performed by agar dilution. results: the epidemic european fusidic acid-resistant impetigo clone (eefic) and community-acquired mrsa strain st have been shown to carry chromosomal and plasmid-encoded fusb, respectively. dna sequencing of fusb and its surrounding regions in these backgrounds revealed that they are identical to sequences on pub . however, acquired fusr does not always result from acquisition of the prototypical fusb gene. a gene encoding a fusb homologue was recently identified during sequencing of s. aureus strain mssa , and we identified an additional homologue encoded in the genome of s. saprophyticus strain atcc . the products of these genes exhibit~ % homology to fusb and to each other. cloning of pcr amplicons corresponding to these genes and their upstream expression signals into s. aureus established that they both confer resistance to fus. since these functional homologues are more closely related to each other than to those from other gram-positive organisms, it is highly likely that they evolved from an ancestral fusb after its recruitment to the staphylococci. conclusions: the three members of the staphylococcal family of fusb proteins appear to have evolved from the same ancestral protein, which, based on the low level of sequence homology between fusb genes at the nucleotide level, clearly occurred well before the introduction of fus into the clinic. of the three, the fusb protein encoded by pub is by far the most successful, and this gene/plasmid represents the source of (or shares a source with) the major fusr strain lineages. telithromycin activity is reduced by efflux in streptococcus pneumoniae c. benvenuti, r. koncan, g. bahar, a. mazzariol, g. cornaglia (verona, it; ankara, tr) objectives: telithromycin shows an excellent activity against m-type erythromycin-resistant streptococcus pneumoniae, thus is commonly regarded as being capable of overcoming the efflux resistance mechanism. nevertheless, telithromycin mic values in those strains appear to be distinctly higher than in the erythromycin-susceptible ones. the possibility of telithromycin acting as an actual efflux substrate, as it was already demonstrated in streptococcus pyogenes, seemed worth investigating. methods: telithromycin mic distribution was analysed in a collection of italian s. pneumoniae strains originating from multi-centre studies ( ) ( ) ( ) ( ) . the effect of an efflux mechanism was investigated using [ h]-telithromycin. results: telithromycin mic ranges were £ . - . mg/l (mic . mg/l and mic . mg/l) in erythromycinsusceptible strains (lacking both mef and erm genes) and . - mg/l (mic . mg/l and mic . mg/l) in strains endowed with the m phenotype. a distinct telithromycin efflux was detected in the strains expressing the mef gene, but not in those expressing the erm(b) gene, nor in the susceptible strains lacking mef or erm genes. efflux reversibility by addition of an inhibiting compound (sodium arsenate) was demonstrated. an msr-like sequence was also found in all strains effluxing telithromycin, but not in the others. conclusions: this is the first time that telithromycin has been shown to be effluxed by s. pyogenes isolates. that the efflux is related to the presence of both the mef and the msr-like genes is clearly demonstrated, but -owing to the increasingly evident complexity of s. pneumoniae efflux systems -other genes might also contribute to the efflux. an unusual phenotype of enterococcus faecalis in greece expressing low-level resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin m. maniati, f. kontos, p. liakos, e. petinaki, i. spiliopoulou, a. maniatis (larissa, patras, gr) objectives: to investigate the resistance mechanism of a new described phenotype among enterococcus faecalis expressing lowlevel resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin (q-d). methods: in greece, during , three enterococcus faecalis isolates, expressing this unusual phenotype, were recovered from urine samples. the isolates were studied by pcr for the lsa-gene and by pfge. nucleotide sequencing analysis of lsa and bp of the upstream region was performed. the isolates were also tested by rt-pcr for the expression of the lsa-gene. results: the isolates belonged to three distinct clones and carried the lsa-gene. no stop codons were found in any strain, while some point mutations in the lsa-gene were detected. comparing the lsa mrna production of these unusual strains with that obtained from fully q-d resistant ones no quantitative differences were found. conclusions: the findings of the present study clearly show that the resistance mechanism of quinupristin-dalfopristin is not only correlated with the presence and the expression of the lsagene. some mutations detected in the lsa gene probably are responsible for the production of an lsa protein with decreased activity, resulting to the q-d susceptibility. the presence of erm tr gene is responsible for the macrolide-resistance of streptococcus agalactiae objectives: to investigate the mechanism of resistance to macrolides in strains of streptococcus agalactiae in the area of thessalia, greece during the period - . methods: the subject of this study were strains of s. agalactiae which were collected from clinical specimens ( % vaginal swabs) from pregnant and non pregnant women. the strains were identified by gram stain, the lancefield b antigen, and by api strep system (biomerieux, france). susceptibility to macrolides, lincosamides and streptogrammines b was studied by the disk diffusion method. the mics were also measured by the use of e-test. the differentiation between m and mlsb inducible type was tested by the double disk synergy test (ddst). the detection of the genes mef a, erm tr, and erm b was performed by polymerase chain reaction (pcr). the clonality of the resistant strains was studied by pulse-field gel electrophoresis. results: of the strains, were resistant to erythromycin, lincosamid and streptogrammines b. none was found to be resistant to erythromycin only (m-phenopype). % of the strains were mlsb constitutive phenotype, while % were mlsb inducible. all strains were found to carry the erm tr gene. only one strain was found to carry both erm tr and erm b genes. pfge analysis revealed the emergence of multiple resistant clones. conclusions: the resistance of s. agalactiae to mlsb antibiotics is related with the presence of erm tr gene in central greece. emergence of novel clindamycin resistance phenotype among invasive streptococcus pyogenes isolates in sweden a. jasir, b. luca, c. schalen (lund, se) objectives: in some recent throat group a streptococci (gas) isolates from our diagnostic laboratory total resistance to clindamycin but susceptibility to erythromycin and other -as well as -membered macrolides was found. the isolates were susceptible to -membered macrolides and streptogramin b. these atypical strains thus did not agree with previously known mls resistance phenotypes. the main objective was to characterize theses resistance phenotype and genotypes. method and results: the isolates were examined for resistance genes by pcr. out of strains one harboured an erma gene. the gene was sequenced and showed a mutation in regulatory part and was localized on a transposons. all other strains were negative for any erm genes and were also tested for s rrna mutations with negative outcome. strains were t-and emm typed and showed to belong to different types. conclusions: gas account for common human infections such as acute pharyngotonsillitis and impetigo, which untreated may be followed by the nonsuppurative complications rheumatic fever and acute poststreptococcal glomerulonephritis gas may also give rise to invasive, often life-threatening acute disease, such as scarlatina, erysipelas, endometritis, necrotising fasciitis and sepsis, often accompanied by toxic shock. without known exceptions, gas are fully susceptible to betalactams, which are first-choice drugs for treatment. in cases of allergy or intolerance to penicillins, macrolides are most used, and possibly as a consequence, a significant resistance development to these agents has evolved in many parts of the world. though the role of clindamycin for treatment of streptococcal disease is more limited this drug was shown to be particularly effective in eradicating streptococci after penicillin treatment failure of pharyngotonsillitis. clindamycin, often as a supplement to betalactams, also may have a life-saving effect in the treatment of fulminant streptococcal infections. due to its important role in the treatment of invasive streptococcal disease, resistance development to clindamycin in gas is considered highly undesirable. the alarming finding of a possibly new phenotype of selective clindamycin resistance in gas will motivate a thorough analysis of the phenotype as well as identification of its resistance determinants. a. al-lahham, m. van der linden, r.r. reinert (aachen, de) objectives: telithromycin is a novel ketolide antibiotic with significant in-vitro activity against streptococcus pneumoniae. the aim of this study is to characterize the resistance mechanisms of clinical isolates of s. pneumoniae with reduced susceptibility to telithromycin (> mg/l) and to perform the time-kill kinetics with telithromycin. methods: determination of mics was performed by the microbroth dilution method according to the clsi and the serotyping by the neufeld quellung reaction. multilocus sequence typing, sequencing of the s rrna, sequencing of genes encoding ribosomal proteins (l and l ), and ermb were performed according to standard methods. four isolates were selected for time-kill, two of which with a telithromycin mic mg/l and two strains with a telithromycin mic of mg/l. results: in two nation-wide studies and one european surveillance study (n = ) performed at the national reference center for streptococci (nrcs) in germany, reduced susceptibility to telithromycin (> mg/l) was detected in isolates ( . %). mic /mic (mg/l) of the strains to other antibiotics were as follows: telithromycin / , penicillin g / , cefuroxime / , erythromycin a > /> , clindamycin > /> , tetracycline / , and gatifloxacin . / . . two major serotypes were observed, serotype ( . %) and serotype a ( . %). all isolates possess the cmlsb phenotype (ermb positive). the isolates showed a wide range of combinations of resistance determinants including multiple alterations in the s rrna (a g, c t, a g, a t, and c t), a s n alteration in the ribosomal protein l (n = ), and a n s alteration in the erm(b) gene (n = ). the predominant clone was serotype sequence type ( of isolates), which was seen in france (n = ) and germany (n = ). telithromycin-resistance has also spread to the spain f- clone (st ; n = ) and its serotype a variant. in vitro time-kill showed a minimal kill from - hours and then regrowth. bactericidal activity was achieved only with times the mic in all strains. conclusions: although the incidence of telithromycin resistance remains rare world-wide, the spread of telithromycin resistance to multi-drug resistance clones with world-wide distribution is worrisome. gbs obtained from non-pregnant women. the erythromycin resistant-gbs were identified, phenotypically analysed, screened by pcr for mre(a) gene and for erythromycin resistance genes: erm(b), erm(tr), mef(a) and mef(e), and serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of gbs, ( . %) were erythromycin-resistant: ( . %) erythromycin-resistant gbs were isolated from vaginal swabs of pregnant women and ( . %) from non-pregnant women. the frequency of serotypes in erythromycin-resistant gbs tested, the distribu-tion of their resistance genes and the distribution of serotypes among the different genotypes are illustrated in the table. nt, nontypeable, the mre(a) gene was found in all the gbs strains tested. mics of erythromycin in erythromycin-resistant gbs were: mic and mic , > mg/l; range, to > mg/l for gbs harbouring erm(b) and erm(b)+erm(tr) and mic and mic , mg/l and > mg/l, respectively; range, . to > mg/l for gbs harbouring erm(tr). conclusion: erm(b) was the erythromycin-resistant gene most prevalent among the gbs isolates and these isolates showed the highest mics of erythromycin. the commonest serotypes among erythromycin-resistant gbs isolated were iii, ii and i, and showed genotypic variability harbouring either of the two most prevalent genes, erm(b) or/and erm(tr). methods: we studied the rates of resistance to tetracycline and minocycline among erythromycin-resistant gbs strains isolated at the university hospital lozano blesa of zaragoza, spain. isolates were subsequently phenotypically analysed by means of the disk diffusion method and screened by pcr for erythromycin and tetracycline resistance genes [erm(b), erm(tr), mef(a/e), tet(m) and tet(o)]. the susceptibility to erythromycin, josamycin, tetracycline and minocycline was tested by the agar dilution method according to the nccls. the strains were serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of isolates of macrolide-resistant gbs collected from may to april in our hospital ( . % of the total sgb isolated), ( . %) were tetracyclineresistant. the distribution of tet(m) and tet(o) among the erythromycin-resistant gbs harbouring erm(b) was ( . % and . %, repectively) and harbouring erm(tr) was ( . % and . %). the distribution of tetracycline resistance genes and serotypes among the different genotypes in gbs are illustrated in the table.*nt: non typable isolates carrying tet(m) or tet(m)+tet(o) presented the following mics: tetracycline (mic , mic , range - mg/l); minocycline (mic , mic , range - mg/l). isolates carrying tet(o) presented the following mics: tetracycline (mic , mic , range - mg/l); minocycline (mic , mic , range - mg/l). conclusion: the majority ( . %) of tetracycline-resistant gbs harboured tet(m) alone or in combination with tet(o). the most prevalent serotypes among the total of tetracycline-resistant gbs was the serotype iii ( %) and the serotype ii ( %). serotype iii was more prevalent among the gbs harbouring the tet(m) gene and serotype ii was more prevalent among the gbs harbouring the tet(o) gene. objectives: to know the prevalence of resistance to macrolides in viridans streptococci, its mechanism and the genetic elements which are involved. methods: we studied viridans streptococcus pharyngeal isolates from different patients. mics for macrolides were determined by the agar dilution method. the presence of mef and erma, ermb and ermtr genes, and the presence of mega (macrolide efflux genetic assembly) or tn in resistant, mef + isolates was determined by pcr with specific primers. the similarity to mef genes first described in pneumococci (mefe) and streptococcus pyogenes (mefa) was determined by sequencing. results: viridans streptococci isolates were resistant to macrolides ( . %). out of the resistant isolates harboured mef genes ( . %), one harboured ermb ( . %), and isolates harboured both mef and ermb genes ( . %). no isolates harboured erma or ermtr genes. we studied genetics elements which harbour mef genes in other streptococci, in mef (+) isolates. we found mega insertion element in of isolates ( . %), all of them harbouring mefe. the only isolate in which we found mefa, did not harbour mega, but tn . conclusions: m phenotype is frequent in viridans streptococci, and all of them harbour mef genes. most mlsb phenotype viridans streptococci do not harbour erm genes alone; most of them combine erm and mef genes. most isolates contained the mef sequence corresponding to mefe, and the genetic element (mega) usually described in pneumococci as harbouring this gene. one isolate contained the sequence corresponding to mefa, and the genetic element usually described in s. pyogenes (tn ). the increasing presence of macrolide-resistant pneumococci harbouring mega element might be related with its wide presence in viridans streptococci. the acquisition of mega by pneumococci from viridans streptococci through transformation is being studied. objectives: the principal mechanism of macrolide resistance in streptococcus pneumoniae in italy is target site modification mediated by erm(b). the erm(a) gene is common in streptococcus pyogenes but rare in s. pneumoniae, even if recent studies have demonstrated an increased detection of this resistance determinant. recently, a clinical s. pneumoniae isolate carrying erm(a) has been obtained from a patient with meningitis in italy. the aim of this study is the molecular characterization of this isolate. methods: antimicrobial susceptibility tests were determined by etest. the presence of erythromycin resistance determinants was detected by pcr assays. genotyping was performed by pfge and mlst. the flanking regions of erm(a) were analysed by sequencing a fragment amplified by inverse pcr. transformation and conjugation experiments were carried out. transformants were analysed by pfge and hybridization with an erm(a) probe. results: the isolate belonging to serotype (st) a, was resistant to erythromycin, inducibly resistant to clindamycin and susceptible to penicillin and tetracycline. by pcr, the only macrolide resistance determinant detected was erm(a). pfge analysis revealed the genetic correlation of this strain with other st a s. pneumoniae italian isolates. mlst confirmed this data since the isolate belonged to st , which is a single-allele variant of st , the most common st among italian st a isolates. a bp dna fragment, obtained by inverse pcr and containing the erm(a) gene, was sequenced. this fragment contains an orf upstream erm(a), the gene erm(a) identical to that described in s. pyogenes and orfs, downstream erm(a), one homologous to a hypothetical kinase and the other two to transposases of other gram-positive species. in transformation experiments the gene erm(a) was transferred to an erythromycin susceptible recipient. hybridization analysis of one transformant revealed that the size of the transferred dna fragment was approximately kb. no transconjugant was obtained in mating experiments. conclusions: this is the first italian report of an s. pneumoniae isolate carrying erm(a). erm(a) appears to be contained in a genetic element that includes two transposases, although the gene is not transferable by conjugation. objective: treatment with the first oxazolidinone antibiotic, linezolid, of infections caused by staphylococci has proved effective in most cases. in the present study we present the first three cases of linezolid resistant staphylococci in our hospital. methods: we examined three coagulase negative staphylococcus strains isolated from blood cultures (bactec, becton dickinson). identification and susceptibility testing were performed by the vitek ii automated system (biomerieux) and the results were confirmed by the api system (biomerieux) and e-test (ab biodisk, solna, sweden), according to nccls guidelines. results: three linezolid resistant staphylococci were isolated from blood cultures. identification showed that all three isolates were staphylococcus cohnii subsp. urealitycum and the mic values were lg/ml (n = ) and lg/ml (n = ) which are much higher than the value of lg/ml that characterizes sensitive strains. the isolates derived from three patients in different wards of our hospital. the first two isolates were recovered from two icu patients in april and august and the last staphylococcus cohnii was isolated from a patient in the neurosurgery ward, who is still hospitalized. all patients received prolonged treatment with linezolid. conclusions: although six linezolid resistant clinical isolates of s. aureus were previously reported in the literature, these three isolates are the first coagulase negative staphylococcus isolates resistant to linezolid. it is imperative to screen for resistance to linezolid all staphylococci and take the necessary precausions in order to prevent the spread of a linezolid resistant strain in other wards of our hospital. correlation between mic and number of mutated s rrna genes in oxazolidinone-resistant staphylococcus aureus objectives: to determine the number of mutated s rdna alleles present in clinical and laboratory-generated linezolidresistant staphylococcus aureus isolates. methods: linezolid-resistant isolates were tested, of them clinical isolates (mics - mg/l) and mutants selected in-vitro (mics - mg/l). the mutants were raised by repeated passage on increasing linezolid concentrations and their parentage was verified by pfge. mics were determined by agar dilution. genomic dna was digested with ecori and hybridized with a bp probe corresponding to domain v of the genes encoding s rrna, to determine gene copy number. pyrosequencing was used to quantify the proportions of wildtype and mutated alleles present; assays were designed to detect the presence of mutations conferring oxazolidinone resistance. pyrosequencing and hybridization data were combined to determine the number of mutated alleles present. results: resistance selected in-vitro proved less stable than that in the clinical isolates. pyrosequencing showed that all clinical isolates had the g t mutation, of the in-vitro selected mutants had g t, had t c, had t a, had a g and had g a. the s rdna copy number in the oxazolidinone-resistant clinical isolates varied from - , and from - in the laboratory-generated mutants; / laboratoryselected mutants had changes in copy number, compared with their parent strains, and had changes in fragment size, but not number. the number of mutated copies in lin-resistant isolates ranged from - in laboratory-selected mutants and from - in clinical isolates. an increasing number of mutated genes correlated with increasing linezolid mic. conclusions: in combination, pyrosequencing and hybridization successfully determined the number of mutated s rdna alleles. exposure to linezolid selected changes in s rrna gene copy number as well as sequence in % of in-vitro selected mutants. there was a positive correlation between both the number and proportion of mutated s rdna copies and mic, previously unproven for staphylococci. objectives: linezolid (lzd) is an important antibiotic for the treatment of enterococcal infections, especially when the corresponding strain possesses multiresistance including resistance to vancomycin (van). we report the emergence lzd resistance in clonally related van-susceptible and vanresistant enterococcus faecium isolates originated from an icu patient only days after initiation of linezolid therapy. patient and methods: van-resistant e. faecium was repeatedly isolated from intraabdominal cultures of a -year-old female icu-patient with infected necrotizing pancreatitis after pancreaticoduodenectomy (whipplés procedure). antibiotic susceptibility testing of the bacteria was performed by e-tests; vana gene were detected by pcr. the possible lzd resistance mechanism (mutation in the s rdna of one or more of the six s rrna alleles of e. faecium) was examined by a pcr-based method. molecular typing of the strains was performed by smai macrorestiction analysis. results: van-resistant but lzd-sensitive e. faecium (vrlse) were initially detected in intraabdominal cultures, however, already twelve days after initation of lzd therapy, van-and lzd-resistant e. faecium (vrlre) strains were detected. resistance to lzd was confirmed: mics ranged from to mg/l. all e. faecium isolates showed identical or closely related pfge patterns. throughout the icu period, van-and lzd-susceptible e. faecium (vslse) strains were repeatedly detected in the same specimens from which the vrlse and vrlre were isolated. additionally, van-susceptible e. faecium isolates with resistance to lzd (vslre) were detected. mutations in the s rdna of three out of six alleles led to lzd resistance in the e. faecium isolates examined. two weeks after termination of the lzd therapy, no lzd-resistant strain could be detected in follow-up swabs. conclusions: resistance to lzd in e. faecium can occur already shortly after the initiation of lzd therapy. assessment of antibiotic susceptibilities of all isolates at the start of therapy and regularly during the therapy is advisable, especially during therapy of severe infections. the epidemiological and clinical repercussions of resistance to lzd among enterococci cannot be predicted at this time. attention to proper dosing and prompt removal of infected devices, when feasible, could limit occurrence and spread of lzd-resistant e. faecium. objectives: to investigate the mechanisms of resistance to tetracycline in shigella spp. methods: one hundred and eleven tetracycline-resistant shigella spp strains ( s. sonnei, s. flexneri), were isolated as a cause of enteritis in our geographical area and the remaining recovered from patients with traveller's diarrhoea. antimicrobial susceptibility to tetracycline was determined by the kirby-bauer method. presence of teta, tetb and tetg genes was established by pcr. sequencing of amplified products were used to corroborate the reliability of the pcr results. maentel haenszel test was used to establish the statistical significance. results: the statistical analysis showed that the teta gene was more frequent in s. sonnei (p < . ), while tetb was more usual in s. flexneri (p < . ). although without statistical significance (p: . ), presence of non-determined mechanisms of tetracycline-resistance seems to be more frequent among s. sonnei. conclusions: species-specific differences in the distribuition of the teta and tetb genes has been shown. moreover . % of the analysed strains did not show any of the analysed determinants of tetracycline-resistance. the concomitant presence of more than one of the analysed genes is a rare event. distribution and genetic determinants of tetracycline resistance in laribacter hongkongensis isolates from humans and fish objectives: to study the distribution of tetracycline resistance and to clone and characterize a tetracycline resistance determinant in laribacter hongkongensis, a recently discovered bacterial genus and species associated with communityacquired gastroenteritis. methods: twenty-four l. hongkongensis strains isolated from patients with community-acquired gastroenteritis and l. hongkongensis strains isolated from freshwater fish in hong kong were used in this study. genetic determinants for tetracycline resistance were looked for by screening a genomic dna library of l. hongkongensis. the prevalence of teta gene in other strains of l. hongkongensis was studied by pcr using laboratory-designed primers. the presence of the tetracycline resistance determinants in plasmid was examined by southern blot analysis. results: among human and fish isolates tested, human and fish isolates were tetracycline-resistant. a -bp gene cluster, which consists of putative transposases, a tetr and a teta gene, was cloned by inserting restriction fragments of genomic dna from a resistant strain, hlhk , into pbk-cmv. the -bp teta and -bp tetr genes shared significant nucleotide sequence homology with known teta and tetr genes. while the flanking regions and ' end of the teta were identical to the corresponding regions of a tetc island in chlamydia suis, the teta was almost identical to that of transposon tn and plasmids found in many gram-negative bacteria, suggesting that illegitimate recombination may have occurred to produce the present tetracycline resistant determinant. southern hybridization suggested that the teta gene of hlhk was plasmid-encoded. the tetracycline resistance in l. hongkongensis was associated with teta. pcr amplification of the teta gene in the isolates of l. hongkongensis, including hlhk , showed the presence of teta in all the four tetracycline resistant isolates but none of the tetracycline susceptible ones. in contrast to strain hlhk , the teta of two strains were identical to that of tn , while that of the other strain was more closely related to other gram-negative bacteria plasmids. conclusion: our results indicate that horizontal transfer of genes, especially through tn and related plasmids, between l. hongkongensis and other gram-negative bacteria is probably a frequent event and is an important mechanism for acquisition and dissemination of tetracycline resistance in l. hongkongensis. succesful treatment of infective endocarditis with linezolid t. hryniewiecki, u. lopaciuk, j. stepinska (warsaw, pl) objectives: there is an increasing proportion of resistant strains causing infective endocarditis in recent years. it has changed the approach to choice of antibiotic therapy. linezolid (zyvoxid Ò ) is a new bacteriostatic antibiotic with a wide spectrum of activity against gram-positive organisms and with good efficacy in experimental animal models of endocarditis. unfortunately clinical experience with linezolid in the treatment of endocarditis is limited. the aim of the study was to observe efficacy of linezolid in the treatment of infective endocarditis. methods: the study group consisted of patients hospitalised in institute of cardiology in warsaw ( warsaw ( - due to clinically resistant infective endocarditis. the diagnosis of endocarditis was established according to the duke criteria by clinical examination, echocardiography, laboratory investigations and positive blood cultures with vancomycin mic estimation (in pts). all patients were treated surgically (valve replacement, artificial material removal) in conjunction with different conventional antibiotics and afterwards with mg of linezolid every hours intravenously. results: infective endocarditis was diagnosed as caused by mrcns in pts, mssa in pt, enterococcus faecalis in pt and staphylococcus epidermidis mr in pt. vancomycin mic vary from to mg/l. in pts culture-negative endocarditis was diagnosed. all patients were treated with linezolid intravenously to weeks (average , ). clinical response and eradication of bacteremia were achieved in all patients. leukopenia nad thrombocytopenia as an adverse reaction occurred in patient. conclusions . linezolid is effective in patients with grampositive endocarditis. . linezolid could be also effective in some patients with culture-negative endocarditis. . linezolid may provide an alternative in the treatment of infective endocarditis due to multi-resistant bacteria, in patients with resistant course or with adverse reaction to conventional antibiotics. objectives: to evaluate the safety and efficacy of lzd in a chinese population. methods: this randomized, double-blind, multi-centre study was conducted in china. after obtaining written informed consent, patients from to years of age with pneumonia (pneu) or skin and soft tissue infection (ssti) known or suspected to be caused by a gram-positive pathogen were randomized : to receive either lzd, mg, or vancomycin (van), g, each given iv q h. patients were to be treated for to days, and outcomes were assessed at end-of-treatment (eot) and follow-up (f-u) visits. results: one hundred forty-two patients were enrolled and received study medication, with pneu and with ssti. clinical assessments (effective = ''cured'' plus ''marked improvement'') for patients in the fully evaluable population are summarized in the table. the most frequently isolated pathogen was staphylococcus aureus: all isolates were susceptible to both study drugs. the eradication rates for all pathogens in evaluable patients at the f-u evaluation were / ( . %) in lzd-treated patients and / ( . %) in van-treated patients (p = . ). all patients receiving study drug were evaluated for safety. drug-related adverse events (aes) were reported in ( . %) lzd-treated and ( . %) van-treated patients. the most commonly reported drug-related aes in lzd-treated patients were mild abnormalities in liver function tests and leucopenia ( . % each); rash ( . %) was the most commonly reported ae in van-treated patients. seven ( . %) lzd-treated and ( . %) van-treated patients discontinued study drug because of an ae. conclusions: linezolid is an effective drug for the treatment of infections caused by gram-positive pathogens and is welltolerated. eradication in one patient, by rifamycinlinezolid, of a methicillin-resistant staphylococcus aureus producing panton-valentine leukocidin, responsible for relapses over months, and decolonisation of her family by mupirocin objective: we report the case of the mother who experienced relapses over the period. methods: pvl-mrsa were isolated on routine and mrsa agars (biorad). antibiotypes were studied by disk diffusion method. genetics and pulsotypes were studied by the french centre national de référence des staphylocoques in lyon (cnr). results: mrs kym had her th child on october , in the hospital of orléans. she was healthy and presented no risk factor for delivery. three weeks later she was addressed for surgical treatment of an abscess on buttocks. cultures yielded the special antibiotype: methicillin-r, kanamycin-r and tobramycin-s, of the pvl-mrsa currently spreading across europe and maghreb. the cnr found the luks-pv and lukf-pv-genes.through march , she relapsed times and was treated by pyostacin for a total of weeks. two of her children were addressed for abscesses ( buttocks, thumb) yielding the same bacteria. in march , mrs kym was addressed to the infectious diseases ward because of nasal furonculosis. samples yielded a pvl-mrsa with mls-b phenotype. treatment by rifamycin-linezolid d was initiated. the whole family was screened. the father and the boy, , who had the infected thumb months earlier, were carriers. the girl, , who had an abscess on buttocks months earlier was not. in april the whole family accepted an attempt for decolonisation by % nasal mupirocin or /d for d. cure and decolonisation were confirmed by nares and cutaneous folds samples in may and june. they missed an additional appointment in the beginning of term, but a phone call to the social worker confirmed none of them relapsed. the cnr studied strains ( from mrs kym , , from children abscesses in and , from boy's and father's nares ), and confirmed that they were all identical along the period and across the family. conclusion: short treatment with linezolid-rifamycin in the relapsing case associated with familial decolonisation by nasal mupirocin was an effective strategy to stop a time-prolonged familial outbreak of pvl-mrsa infection. multiple brain abscesses and purulent meningitis by listeria monocytogenes in an otherwise healthy man. favourable linezolid response, hampered by a suspected early drug myelotoxicity introduction: l. monocytogenes cns infection in immunocompetent adults remains rare. meningitis is the most common cns manifestation, with brain abscesses being < % of overall episodes. anecdotal episodes of cns l. monocytogenes infection were reported from immunocompetent patients where both diagnosis and treatment may be hampered by low clinical suspicion and a frequent non-specific presentation. in a -yearlong survey conducted in dallas (us), only cases of nonneonatal l. monocytogenes meningitis were found (estimated incidence rate: . %). case report: a -year-old male with a negligible history and no obvious exposure to l. monocytogenes was hospitalized owing to dizziness. a brain ct scan showed a small, late ischemic lesion. a few days later hyperpyrexia, headache, vomiting and altered mentation occurred. the csf study detected an elevated albumin content ( mg/dl), low glucose ( mg/dl) and a wbc count of cells/ll ( % neutrophils) so that ceftriaxone-chloramphenicol were immediately started. clinical-neurological conditions deteriorated while l. monocytogenes was cultured from the csf so that treatment was changed towards high-dose ampicillin-gentamicin. the persistence of severe clinical-neurological conditions and altered csf assay prompted the introduction of rifampicin-cotrimoxazole after days, but days later other focal neurological deficits appeared and a mri showed small, hyperintense focal lesions involving the medulla oblongata, interpreted as multiple abscesses. the introduction of linezolidmeropenem, despite anemia (requiring rbc transfusions after - days) led to a progressive clinical-csf improvement. our patient recovered completely and a control mri carried out month after discharge confimed the complete disappearance of the multiple brain listeria abscesses. discussion: the l. monocytogenes meningitis and multiple subtentorial abscesses (including rare localizations at cerebellum, bulb, and pons varolii), had an evolving cumbersome presentation. despite the in vitro activity of a broad spectrum of agents, multiple therapeutic changes became necessary, until the last linezolid-meropenem combination, which was proved very effective, although it was affected by relapsing anemia probably attributable to linezolid. linezolid, due to its elevated csf-brain penetration, and its activity against a broad spectrum of cns pathogens (including the intracellular l. monocytogenes), is expected to become a key antimicrobial compound, waiting for rct. discrepancy between favourable in vitro microbiological data and a severe clinical course of a staphylococcal knee and soft tissue infection responsive to oxazolidinone linezolid only after failure of all other therapeutic attempts introduction: to offer therapeutic alternatives for the emerging, multiresistant, serious gram-positive infections, novel molecules (quinupristin/dalfopristin, linezolid, daptomycin) were introduced and are made available when multiresistant gram-positive cocci are documented as no more susceptible to all available drugs including glycopeptides. however, inezolid encompasses unique tissue penetration and diffusion features (regarding soft tissues, lungs, joints and central nervous system) which make this last drug extremely promising in all circumstances where the penetration rate into infectious foci becomes critical. clinical experience: a very intriguing case report of a severe, staphylococcal knee arthtiris associated to an extensive local cellulitis/fasciitis and haematogenous dissemination occurring after a surgical curettage was characterized by a complete lack of response to a prolonged vancomycin/teicoplanin plus rifampicin therapy based on the apparently favourable in vitro sensitivity assays of methicllin-resistant staphylococci, but rapidly responded to i.v. (followed by oral) linezolid administration. the complete lack of clinical activity of a -week glycopeptiderifampicin administration cannot be explained by the in vitro measured mic values of isolated pathogens which showed complete sensitivity of staphylococcus aureus against vancomycina/teicoplanin and rifampicin and susceptibility of a concurrent hematogenous s. epidermidis strain to glycopeptidesrifampicin. since an abscess formation and an underlying osteomyelitis were carefully excluded by adequate instrumental examinations, from a theoretical point of view the active glycopeptide-rifampicin molecules should have been provided appropriate cure. on the other hand, from a strictly clinical issue, only a -week administration of i.v. linezolid followed by one more week of oral linezolid allowed to obtain a complete clinical-bacteriological cure and a complete function recovery without any sequelae after a . -year follow-up. conclusions: when the management of severe, multiresistant gram-positive infections is of concern, the in vitro activity of single drugs and therapeutic classes should be carefully evaluated in relation with the expected penetration and diffusion rates of these drugs into the relevant organs and tissues involved by the ongoing infectious localizations. otherwise, apparently unexplained failures may occur also when in vitro studies point out a complete activity of the tested compounds. epidemiology of resistance to antibiotics -ii p contemporary prevalence of bro betalactamases in m. catarrhalis: report from the sentry antimicrobial surveillance program (usa; l. deshpande, h. sader, r. jones (north liberty, us) objectives: to evaluate the prevalence of bro- and bro- among b-lactamase (bl)-producing m. catarrhalis (mcat) in the usa. although the bl-mediated penicillin (pen) resistance (r) in mcat has been stable at %, the bro- and - occurrence has not been determined in usa isolates since . bro- rates have been reported at < ( s), . ( - ), . ( - ) and . % ( - ) . methods: community-acquired mcat isolates (sentry program were tested by clsi broth microdilution methods including: , worldwide and , in north america (na). bro- and - was detected by pcr methods (levy and walker; ), compared to epidemiologic tests, and mic values. b-lactamase-positive (bl+) mcat samples per year from usa ( sites) and canada (ca; sites) were tested for the odd-numbered years. results: the bro- rate was , , , and % for , , and , respectively; rates in ca ( isolates) > usa ( ). several agents remained active: amoxicillin/clavulanate (mic , £ . mg/l), ceftriaxone (ctri; . ), cefuroxime ( ), erythromycin (£ . - . ), levofloxacin (£ . - . ), tetracyclines ( ) and trimethoprim/sulfamethoxazole (tmp/ smx; £ . / . ). pen mic distribution was tri-modal (£ . , - , > mg/l) and ctri bi-modal ( . , . ), yet bro- and - mic/zone distributions overlap (best discriminated by methicillin (mean zone, . vs. . mm) and pen ( . vs. . ) disks). possible bro- epidemic clusters could not be excluded due to a very common ribotype in centres (ca, sites; usa, ). conclusions: this bro- and - enzymes na prevalence update in mcat isolates ( ) ( ) ( ) ( ) ( ) ( ) ( ) shows stability at - % and - %, respectively. phenotypic tests (zones or mics) cannot easily distinguish between these b-lactamase types, necessitating the use of molecular applications. objective: although resistance to penicillin in beta haemolytic streptococci has not been reported yet, increasing resistance rates for alternative drugs, such as erythromycin, clindamycin or tetracycline is an emerging concern which brings the necessity to carefully monitor penicillin susceptibility. materials and methods: in order to detect any changes in penicillin mics, we performed antimicrobial susceptibility testing for all isolated beta haemolytic streptococci in our hospital between january and november . identification to serogroup level was done using a commercial latex agglutination kit (avipath strep, omega diagnostics ltd., scotland, united kingdom). results: a total of isolates were identified, distribution of groups for serogroup a, b, c and g were . %, . %, . % and . %, correspondingly. penicillin susceptibility was determined using etest (ab biodisk solna, sweden) strips according to manufacturers' instructions. when results are evaluated in year periods, mic increased from . to . mg/ml for group a, from . to . mg/ml for group b, from . to . mg/ml for group c and from . to . mg/ml for group g (table) . conclusions: even though highest mic values were to be found in group b ( . mg/ml), our results indicate the steady increase in penicillin mic for all serogroups. three group a and six group b isolates with penicillin mic of . mg/ml, reaching susceptibility breakpoint concentration according to clsi, and also highly elevated mic concentrations for group b streptococci may be messengers of possible forthcoming resistant strains. objective: to study trends in macrolide resistance rates among s. pneumoniae isolated from children aged to months attending day-care centres in france following implementation of prudent antibiotic use campaigns (alpes maritimes , france and pneumococcal conjugate vaccine (pcv) ( ) . method: nasopharyngeal aspirates were obtained from a random -stage cluster sample of children attending day-care centres in the nord (n) and alpes maritimes (am) areas during consecutive surveys between january and march march , march and . susceptibility to erythromycin and clindamycin and resistance phenotype were analysed by disk diffusion method. serotypes were determined using the quelling reaction. pneumococcal immunization status and antibiotic prescriptions over the previous months were recorded. results: sp was isolated from / , / and / children in , and , respectively (p < ) ). resistance to macrolides declined overall from . % to . % of strains between and (p < ) ). among erythromycin-resistant (e-r) isolates, percentage of erm-b phenotype increased from . % to . % (p = . ). while the proportion of penicillin non-susceptible strains declined from . % to . % of sp isolates (p < ) ), erythromycin resistance remained stable among these strains at . %. overall proportion of treated children fell from . % to . % (p < ) ) between and ; in am this reduction was observed in ( . %; p < ) ), while in n it occurred in ( %; p < ) ) and the percentage of macrolides among prescriptions fell from . % to . % (v for trend: p = . ). serotype distribution showed most e-r isolates were b, , f and f. a % reduction in serotype f was observed in am in and in n in . immunisation with pcv concerned at least . % of children in . conclusion: macrolide resistance has followed a parallel decline with penicillin resistance as a result of antibioticprescription reducing campaigns and pneumococcal immunization against the most prevalent macrolide-resistant serotypes. objective: to evaluate the prevalence of resistance of invasive strains of s. pneumoniae to erythromycin after decline in macrolide consumption. methods: the number of packages of antibiotics was obtained from the institute of public health of slovenia. for the period - the data on outpatient antibiotic consumption were collected using the atc/ddd classification (who version ) and the results were expressed in ddd/ inhabitants per day (did). all invasive strains of s. pneumoniae isolated from sterile body fluids in all slovenian hospitals were included in the study. susceptibility testing was performed using nccls approved disk diffusion test. results: during - the total use of antibacterials in slovenia decreased for . % from . did to . did. the consumption of macrolides which constituted . - . % of total use of antibacterials decreased for . % ( . to . did). short-acting (erythromycin, miocamycin), intermediate-acting (midecamycin, roxithromycin, clarithromycin), and long-acting (azithromycin) decreased for . %, . % and % respectively. in all years the use of intermediate-acting macrolides was the most prescribed subclass of macrolides corresponding for . - . did, followed by long-acting ( . - . did) and short-acting ( . - . did). the resistance of s. pneumoniae strains to erythromycin increased from . % ( / ) to . % ( / ); in children from . % ( / ) to . % ( / ) and in adults from . % ( / ) to . % ( / ) respectively. rates of the isolates resistant to erythromycin and at least of the following agents: penicillin, tetracycline, tmp/smx, chloramphenicol increased from . % ( / ) to . % ( / ); in children from % ( / ) to . % ( / ) and in adults from . % ( / ) to . % ( / ) respectively. conclusion: despite a reduction of macrolide consumption in outpatients the resistance of invasive strains of s. pneumoniae was increasing during the observation period especially in children. multiple drug resistance explains best the changes in s. pneumoniae resistance in a ten-year surveillance study in belgium objective: belgium is located between countries with very high and very low antibiotic resistance rates. modeling how resistance changes over time and place in belgium provides insights into correlates of s. pneumoniae resistance at the population level. methods: surveillance data consists of , s. pneumoniae invasive isolates from - , identified by postal code as well as clinical and demographic information. antimicrobial consumption (ims health services) is expressed in defined daily doses (ddd) per inhabitants per day. changes in resistance by month and postal code were evaluated using mixed effects models for repeated measures, using mathematical models of transmission for the curve shape, and taking into account seasonality. resistance to penicillin, erythromycin, tetracycline, and ofloxacin was considered in the analysis. results: resistance to penicillins, macrolides and tetracyclines peaked in the year , and their levels in were . %, . % and . % respectively. the shape of the curves is similar for most of the antibiotics studied, with a steep rise from to and a plateau thereafter. resistance to two or more antibiotic classes corresponded to % of all resistant isolates and in a multivariate model explains most of the variability through time and place of the antibiotics studied. resistance to only one antibiotic (any) decreased from . % in to . % in , while resistance to two or more increased . times ( % ci . - . , p < . ) from . % in to . % of all isolates years later. more than nine out of ten isolates that were macrolide or tetracycline resistant were also multiply resistant (mr). mr increases . % for each ddd of overall cumulative antimicrobial consumption, and out of all antibiotic classes, macrolides and broad-spectrum penicillins are most associated with resistance. conclusion: resistance to two or more antibiotics is the most important factor in understanding the changes over time for all studied antibiotic classes in belgium. the cumulative impact of antimicrobial exposure of separate antibiotic classes at the population level facilitates the survival and transmission of any isolate that is resistant to two or more antibiotic classes. methods: the isolates were identified by biochemical tests and specific serotyping. antimicrobial susceptibility to ampicilllin (amp), amoxicillin plus clavulanic acid (auc), cloramphenicol (cm), gentamicin (gm), cotrimoxazole (sxt), nalidíxic acid (nal) and tetracycline (tc) were established by the method of kirby bauer. the presence of beta-lactamases encoding genes (tem, carb, oxa -like) as well as the teta, tetb, tetc, tetg, cmla and flor genes, and integrons type was established by pcr, while the presence of plasmid-mediated dhfr was determined by pcr-rflp and the cat activity by a colorimetric assay. results: seven different resistance patterns were identified: i. susceptible ( strains); ii. amp, sxt, gm, a/c ( ); iii. amp, tc, cm ( ); iv. amp, tc, sxt, cm ( ); v. amp, a/c ( ); vi. amp, sxt, a/c ( ); vii ( ). -sxt. no isolate resistant to nalidixic acid was detected. resistance to beta-lactam agents was due to the presence of beta-lactamases type tem-like (pattern v), carb- (iii) and tem-like plus oxa- (ii, v, vi). meanwhile resistance to cloranphenicol and tetracycline was associated to cat activity (iii, iv) and flor (iii), and tetb (iv) and tetg (iii) respectively. no mechanism of cotrimoxazole resistance was detected in the isolates of the patterns ii, vi and vii, while dfra was detected in the isolates of the group iv. resistance to gm was associated to the presence of the gene aadb, detected in the analysis of integrons type . type integrons were detected in isolates belonging toi the pattern ii ( bp -aadb; bp -oxa , aada ), iii ( bp -carb , -aada ), iv ( bp -dfra , aada ), v and vi ( bp -oxa , aada ). conclusions: a great diversity of resistance mechanisms has been detected. those mechanisms might spread among microorganisms resulting in a serious health problem due to the limited number of antibiotic treatments available in the area. small outbreaks of veb- esbl producing acinetobacter baumannii in belgian nursing homes and hospitals through cross-border transfer of patients from northern france methods: from / to / , all belgian acute hospitals were invited to report cases of nosocomial infections/colonisations due to mdr ab isolates presenting a resistance profile similar to the french epidemic strain (resistance to all agents except carbapenems and colistin) and to send such isolates to the reference laboratory for phenotypic confirmation and for genotypic characterization (pcr of veb- and class integron, pfge typing). guidelines for detection of the epidemic strain, screening for carriage in patients transferred from hospitals or nursing homes (nh) close to the french border as well as infection control measures were sent to all hospitals. results: overall ab strains from hospitals were sent to the reference laboratory. only, of these fulfilled the phenotypic resistance patterns and were definitely confirmed as veb- ab and had a pfge pattern identical to the french epidemic clone. two mini-outbreak clusters (each involving cases) were documented in hospitals from two cities (tournai and chimay) closed to the french border. two patients died from their infection. in the first outbreak, all patients were residents who lived in the same nh. two of them were french citizens who had been hospitalised in different acute care hospitals in the north of france within the last year. in the second outbreak, the index case had also been previously hospitalised in a french hospital. secondary transmission to two other hospitalised patients occurred in this outbreak. conclusion: despite the large extension of the veb- ab outbreak in france no similar problem occurred in belgium. however, this national alert allowed to detect two small outbreaks in belgian institutions located close to the french border. in both outbreaks the epidemic strain was imported from france through patient circuits. this study illustrates that transfers between acute care hospitals and nh may explain cross-border spread of multi-resistant epidemic strains. types of extended-spectrum beta-lactamases in salmonella spp. and decreased susceptibility to fluoroquinolones objectives: the aim of this study was to determine the rate of esbl production in clinical isolates of salmonella spp. and to detect decreased susceptibility to fluoroquinolones in esbl positive isolates in turkey. methods: a total of salmonella spp. isolated from clinical samples from thirteen centres between and were included in the study. in vitro susceptibility to ampicillin, amoxicillin/clavulanic acid, cefotaxime, gentamicin, chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and ciprofloxacin were determined using the agar dilution method on mueller-hinton agar following the clinical and laboratory standards institute (clsi) guidelines. decreased susceptibility to ciprofloxacin was defined as an mic of . - mg/l. salmonella isolates were screened for esbl production by double disk synergy method using amoxicillin/clavulanic acid, cefotaxime and ceftazidime disks. types of esbl enzymes were analysed by pcr for tem, ctx-m, shv and per- genes. results: in salmonella spp. the highest level of resistance was observed against ampicillin ( . %) followed by chloramphenicol ( . %), tetracycline ( . %) amoxicillin/ clavulanic acid ( . %), trimethoprim/sulfamethoxazole ( . %), gentamicin ( . %), and cefotaxime ( . %). ciprofloxacin resistance was observed in one isolate ( . %). among salmonella isolates, ( . %) were shown to produce esbl by double disk synergy testing. these isolates were salmonella typhimurium (n = ), serogroup c (n = ) and salmonella enteritidis (n = ). three isolates were from fecal samples two were from urine and one was from blood. one of the esbl producing isolates were susceptible to cefotaxime in vitro. two isolates showed decreased susceptibility to ciprofloxacin. all the esbl producers were resistant to ampicillin, amoxicillin/ clavulanic acid, chloramphenicol and harbored ctx-m type enzymes. in three isolates a tem-type enzyme was also present. conclusion: albeit being rare, esbl production is an important resistance factor among salmonella spp. in order to prevent treatment failures, decreased susceptibility to fluoroquinolones should be investigated routinely in invasive isolates as well as esbl production. incidence of faecal carriage of esbl-producing enterobacteriaceae in hospital and community patients during two non-outbreak periods of time the identities of the esbl-producing isolates recovered during were: e. coli (n = ), k. pneumoniae (n = ), p. vulgaris (n = ) and e. cloacae (n = ), and isolates recovered during were: e. coli (n = ), k. pneumoniae (n = ), k. oxytoca (n = ), p. vulgaris (n = ), p. mirabilis (n = ), e. cloacae (n = ) and e. aerogenes (n = ). conclusions: a dramatic, significant increase in the frequency of faecal carriage of esbl-producing isolates was demonstrated in among hospitalized ( . %) and ambulatory patients ( . %).the results revealed that the prevalence of faecal carriage among ambulatory patients and hospitalized patients was not significantly different in both periods of time. outpatients came from the community carrying enterobacteria harbouring esbl in the intestinal tract, suggesting that the community could be a reservoir for these microorganisms and enzymes. methods: a total of k. pneumoniae, k. oxytoca, e. coli, c. freundii, s. marcescens, and e. cloacae from university hospitals, isolated from blood, wound, urine, sputum and other clinically significant specimens were proven to produce esbls. antimicrobial susceptibility was determined according to clsi, ; conjugation on a solid medium was performed; isoelectric focusing was followed by bioassay; pcr with beta-lactamase group-specific oligonucleotides was applied, followed by nucleotide sequencing; rapd with eric- a and eric primers was performed. results: mic of ceftazidime varied from to > mg/l, mic of cefotaxime - - mg/l; the addition of sulbactam : reduced mic > -fold. transconjugants exhibited resistance both to extended-spectrum cephalosporins and aminoglycosides in of strains. according to their pi, two clusters of betalactamase producers could be described: first one -esbls focussed at pi . , and the second -pi at . . results from pcr confirmed the presence of two groups esbls: tem and shv. sequencing of representative strains showed the presence of shv- in two participating hospitals and of shv- in only one strain e. cloacae, while tem- like enzyme was found in centres and had a clonal dissemination. objectives: during treatment with selective decontamination of the digestive tract (sdd), four strains of multidrug-resistant (mdr) gram-negative bacteria (three escherichia coli strains and one klebsiella pneumoniae) were isolated at the intensive care unit (icu) in the academic medical center (amc) in amsterdam. these isolates were extended spectrum betalactamase (esbl) positive. we investigated whether this was due to interspecies transfer of resistance genes. methods: the strains were typed by amplified fragment length polymorphism analysis. the plasmids from these strains were characterized by restriction fragment length polymorphism. resistance genes of the mdr-strains were characterized by pcr and sequence analysis. results: aflp analysis confirmed that the three mdr e. coli isolates represented three different strains. the mdr-strains were shown to harbour the same plasmid with identical extended-spectrum â-lactamase (esbl) genes; ctx-m- and shv- . conclusions: identification of the emergence of such mdr gram-negative bacteria and recognition of resistance plasmid transfer during sdd treatment is crucial for optimal application of this regimen in icus. the use of the third generation cephalosporins in sdd may associate with emergence and increase in the prevalence of esbls. therefore, for optimal screening of resistance to cephalosporins in icus, the screening for esbls should be included. objective: carbapenems are the drugs of choice for the treatment of serious infections caused by esbl-producing enterobacteriaceae and the emergence of carbapenem resistance is rarely documented. we investigated pairs of carbapenem-susceptible and resistant k. pneumoniae isolates from three patients, collected before and after therapy with carbapenems. methods: pre-and post-therapy pairs of esbl-producing k. pneumoniae isolates were from three patients with urinary catheter-associated infections who were treated with ertapenem (erp, cases) or meropenem (mem, one case) in a district general hospital with a low incidence of esbl-producing organisms ( . / bed days), and meropenem use of ddd/year. isolates were compared by pfge of xbai-digested genomic dna. mics were determined and interpreted by british society for antimicrobial chemotherapy methodology. blactx-m alleles were sought by multiplex pcr. outer membrane proteins (omps) were extracted, and analysed by sds-page. results: the three patients relapsed following erp or mem therapy, and the post-therapy isolates from repeat urine samples were resistant (table), with mics erp>mem>ipm. all six isolates from the three patients belonged to the same pfge strain, but transmission of the resistant variants is unlikely as the patients were geographically and temporally unrelated and separate selection of resistance in individual patients seems more likely. all isolates had a group ctx-m esbl; the resistant isolate in each pair had lost a major omp, consistent with a porin, compared with its susceptible 'parent'. all three patients were successfully treated with amikacin. the emergence of carbapenem resistance in ctx-m-producing k. pneumoniae following therapy severely limits treatment options. whilst unusual in general, such selection has occurred repeatedly with this strain. wide geographic spread of diverse acquired ampc beta-lactamases in escherichia coli and klebsiella spp. in the uk and ireland objective: to determine the distribution of genes encoding acquired ampc beta-lactamases in cephalosporin-resistant isolates of e. coli and klebsiella spp. submitted to the uk national reference laboratory. methods: mics were determined by agar dilution and interpreted according to breakpoints of the british society for antimicrobial chemotherapy. isolates of e. coli or klebsiella spp. resistant to cefotaxime and ceftazidime, irrespective of addition of clavulanic acid, were inferred to have possible ampcmediated resistance. genes encoding six phylogenetic groups of acquired ampc enzymes were sought with a multiplex pcr assay (perez-perez & hanson. j clin microbiol ; : - ) . selected isolates were compared by pfge, and selected blaampc amplicons were sequenced. results: e. coli isolates and klebsiella spp. from separate patients yielded pcr amplicons indicating the presence of genes encoding acquired ampc enzymes. forty of these e. coli isolates (from hospitals) produced cit-type enzymes, (from irish hospitals) produced acc types, and a dha type. the klebsiella spp. produced acc ( isolates from irish hospitals), fox ( isolates from welsh hospitals) or dha ( irish isolate) enzymes. genes encoding ebc-/ent-and moxtype enzymes were not detected. twelve e. coli isolates from one hospital all produced a cit-type enzyme; these isolates belonged to an epidemic uk strain, designated strain a; isolates also contained blactx-m- linked to an upstream copy of is , as is characteristic of strain a; isolates lacked blactx-m- . sequencing of a representative blaampc amplicon indicated production of a novel cmy- variant in these isolates. conclusions: diverse acquired ampc enzymes are present in e. coli and klebsiella spp. in the uk and ireland, with cit-types the most common, and acc types linked to ireland. the broad resistance profiles of ampc enzymes compromises patient management. hence, the acquisition of a cmy- -like enzyme by epidemic e. coli strain a suggests that acquired ampc enzymes are poised to become an important public health issue in the uk. objective: to characterize the spectrum of activity and potency of dor (formerly s- ) and comparator agents against contemporary wild-type bacterial isolates from medical centres in europe and the middle east in . dor is a novel parenteral -b-methyl carbapenem in late stage clinical development whose molecular structure confers stability to b-lactamases and resistance (r) to renal dehydropeptidases. methods: the collection included non-duplicate, consecutive clinical isolates from patients in medical centres in europe ( ), turkey ( ) and israel ( ) that were submitted to the dor surveillance program ( ) for identification confirmation and susceptibility (s) testing. mic values for > antimicrobials were determined using nccls broth microdilution methods ( ) . a tentative dor susceptible (s) breakpoint of £ mg/l (£ . mg/l for s. pneumoniae) was used for comparative purposes; clsi ( ) criteria were used for other tested agents. results: antimicrobial activities of dor and other carbapenems vs. selected isolates. dor consistently displayed activity against staphylococci and streptococci (mic , . and . mg/l) most similar to that of imipenem, and against e. coli and klebsiella spp. (mic , . and . mg/l, respectively, including . and . % of strains that met esbl screening criteria), most similar to that of meropenem. enterobacter spp. isolates, including . % that were ceftazidime-r (indicative of ampc production), were also highly s to dor and other carbapenems ( . to . % r). dor also provided slightly enhanced coverage against p. aeruginosa ( . % s) and acinetobacter spp. ( . % s) compared to other carbapenems. carbapenem r among these latter strains is, however, a particularly worrisome development. conclusions: dor is a new carbapenem with a competitive profile that incorporates both potent gram-negative and grampositive activity, with enhanced activity against the commonly occurring non-fermentative gram-negative bacilli. carbapenems are assuming a greater therapeutic role in many nations as multi-drug resistance (including emergence of ambler class a, c and d b-lactamases) spreads, necessitating their accelerated development. phenotypic and genetic characterisations of enterococcal isolates in tehran sewage, with emphasis on detection of vana and vanb genes objectives: enterococci are members of the normal gut flora of animals and humans and are thus released into the environment directly or via sewage outlets, where they can survive for long time periods. during the last decade the concern has been focused on enterococci that are resistant to the glycopeptide antibiotic vancomycin [vancomycin-resistant enterococci (vre)]. the aim of the study was to detect and to analyse the biochemical diversity of the entrococci strains in tehran sewage and to determine the genetic characterization of vre. methods: a total of isolates of enterococci were selected on me agar medium. all of the isolates were identified at the species level by the common biochemical tests. drug susceptibility test of isolates was done by disk diffusion method with antibiotics vancomycin, erythromycin, gentamicin, tetracycline, chloramphenicol and ciprofloxacin. the mic was also done by macrobroth dilution assay. analysis of the plasmid profiles and the pcr tests for vana and vanb genes were done. methods: we studied vre isolates collected in the north and center of portugal ( portugal ( - from: (i) clinical isolates from hospitals in different cities, (ii) faecal samples from healthy volunteers, (iii) river water samples, (iv) samples collected downstream of hospital sewage water, (v) samples from urban sewage water, (vi) swine faeces (vii) poultry food samples for human consumption. identification and characterization of vancomycin resistant genes vana, vanb, vanc and vanc were determined by a multiplex pcr. the backbone structure of tn was characterized by a pcr overlapping assay ( overlapping fragments), and further sequencing. conclusion: beta-lactamase production among hi strains has declined significantly since among children attending daycare centres as antibiotic prescriptions fell among this population. results: the mic distribution of am showed % of strains (n = ) with a mic > mg/l and % with a mic of > mg/l, indicating that resistance to am is still relatively rare and does increase as compared to nethmap . the lognormal distribution of both am and amc ( strain r) extended to mg/l but showed tailing to mg/l. this may indicate hidden less susceptible strains but could equally well be explained by testing circumstances, since the left part of the mic distribution showed comparable tailing. all strains were susceptible to moxifloxacin, levofloxacin and cefotaxim. the lognormal distribution of sxt extended to . mg/l with % of strains showing higher values. doxycyclin resistance was less than %. most of the strains were resistant to clarithromycin and azithromycin with a mic > . mg/l for both. conclusions: resistance of hi to common antimicrobials in the netherlands is still low and does not increase. objectives: s. pneumoniae (sp) and h. influenzae (hi) are the two most common pathogens associated with community-acquired pneumonia. changes in the prevalence of resistance or multidrug resistance (mdr) among these pathogens have important therapeutic ramifications. the global surveillance initiative is a longitudinal study that benchmarks antibacterial resistance among respiratory pathogens. methods: during , sp and hi were isolated from patient specimens collected at hospital laboratories in france (fr), germany (ger), italy (it), spain (spa), and the united kingdom (uk). isolates were centrally tested by broth microdilution against lev, penicillin (pen; sp only), azithromycin (azi), erythromycin (ery), clindamycin (cli), ceftriaxone (ctx), cefuroxime (cfx), and trimethoprimsulfamethoxazole (tmp-smx) (nccls, ) . data were analysed according to pen resistance, mdr, and b-lactamase status. mdr was defined concurrent resistance to ‡ of the following agents: ctx, cfx, ery, lev, pen, and tmp-smx. results: for sp, pen r was . % in ger, . % in the uk, . % in it, . % in spa, and . % in fr. azi r was . % in the uk, . % in ger, . % in spa, . % in it, and . % in fr. overall, lev r was rare (£ %) and mic s = mg/l in all countries. . % of isolates were susceptible to all of the drugs tested, the most common phenotype encountered. the prevalence (%) of mdr among sp ranged from . in uk to . in fr. resistance to pen, ery, cfx, and tmp-smx was the most prevalent mdr phenotype found in europe. overall . % of mdr sp were susceptible to lev. for hi, b-lactamase rates varied by country from . % in it to . % in fr. based on mic lev and ctx were the most active agents tested against hi, regardless of b-lactamase status. conclusions: lev showed potent activity against sp and hi. for sp, lev activity was independent of resistance to pen or mdr phenotype. lev maintained consistent activity against sp based on mic , regardless of country studied. antimicrobial surveillance data from studies such as the global offer guidance to physicians for empiric prescribing. sxt was obtained ( sxt was obtained ( - . conclusions: our results suggest that beta-lactamase production does not constitute a threat in hi therapy since values were almost constant. although with an unregulated fluctuation on arnblp percentages, it seems that this mechanism is gaining importance in relation to beta-lactamase production. thus, we conclude the need to be aware of arnblp, as these strains are difficult to detect using the nccls ( ) breakpoints. further molecular studies of the resistance genes responsible of this resistance mechanism are needed. resistance of beta-lactamase producer strains, to other antibiotics decreased during the period of study, due to the diminished use of these antibiotics. this study shows the importance of monitoring antibiotic resistance in hi in order to detect emerging mechanisms. antimicrobial susceptibility of respiratory haemophilus influenzae strains in northern greece k. koraki, p. karapavlidou, d. sofianou (thessaloniki, gr) objectives: to investigate the antimicrobial susceptibility of haemophilus influenzae, one of the most frequent bacterial pathogens of respiratory tract infections. treatment of these infections is most often empirical and considerable geographical resistance variation has been reported. methods: eighty h. influenzae strains were collected from respiratory tract specimens (sputum, bronchoalveolar lavages, endotracheal secretions) in a -year period ( ) ( ) ( ) ( ) ( ) . identification was made by colonial morphology, gram staining characteristics, x-and v-factor requirements and api nh (biomerieux, france). antibiotics were selected to reflect representative current treatment options and susceptibility was determined by kirby-bauer disc diffusion method on haemophilus test medium according to nccls guidelines. results: out of the h. influenzae strains were isolated from children and from adults. % of isolates came from children admitted to the intensive care units and . % from cystic fibrosis patients. a seasonal trend was reported for infections since . % of isolates were collected during springtime and % during autumn months. overall ampicillin resistance was . % and resistant strains were isolated exclusively from children. ampicillin resistance was doubled among cystic fibrosis patients ( . %). all isolates were susceptible to amoxicillin/clavulanate, chloramphenicol, ciprofloxacin and imipenem. the rank order of cephalosporin activity was cefotaxime and ceftriaxone ( %) followed by cefuroxime and cefaclor ( . % and . % respectively). trimethoprim/ sulfomethoxazole was active against . % of isolates while erythromycin was the least potent antimicrobial agent with % of isolates being susceptible to it. no multiresistant phenotypes were detected. conclusion: our results demonstrated that ampicillin resistance among h. influenzae in our area is still relatively low and overall antibacterial susceptibility rates are high. knowledge of antimicrobial resistance among these pathogens is imperative for physicians to choose the most appropriate therapeutic agent. results: nosocomial gram-negative uropathogens were studied. most common uropathogens were p. aeruginosa ( . %), e. coli ( . %), k. pneumoniae ( . %), followed by a. baumannii ( . %), enterobacter spp. ( . %), s. marcescens ( . %), proteus spp. ( . %) and other gram-negative rods ( . %). resistance rates (i+r, %) among p. aeruginosa were: gentamicin - %, levofloxacin - %, ciprofloxacin - %, cefoperazone - %, cefoperazone/sulbactam - %, cefepime - %, piperacillin - %, amikacin - %, ceftazidime - %, imipenem - %, meropenem - %, piperacillin/tazobactam - %, polymyxin b - %. resistance rates (i+r, %) among e. coli were: piperacillin - %, ticarcillin/clavulanic acid - %, amoxicillin/clavulanic acid - %, ciprofloxacin - %, gentamicin - %, moxifloxacin - %, levofloxacin - %, cefoperazone - %, ceftriaxone - %, cefepime - %, ceftazidime - %, cefoperazone/sulbactam - %, piperacillin/tazobactam - %, amikacin - %, all strains were susceptible to ertapenem, imipenem, meropenem. resistance rates (i+r, %) among k. pneumoniae were following: piperacillin - %, cefoperazone - %, ceftriaxone - %, gentamicin - %, amoxicillin/clavulanic acid - %, cefepime - %, ceftazidime - %, ciprofloxacin - %, piperacillin/ tazobactam - %, moxifloxacin - %, cefoperazone/sulbactam - %, levofloxacin - %, amikacin - %, ertapenem - %, imipenem and meropenem were active against all isolates. conclusion: p. aeruginosa, e. coli and k. pneumoniae are the main gram-negative uropathogens in russian icus patients. imipenem, meropenem, ertapenem showed prominent activity against e. coli and k. pneumoniae. cefoperazone/sulbactam, piperacillin/tazobactam, amikacin exhibited considerable activity versus e. coli, while k. pneumoniae were more resistant to them. p. aeruginosa were highly resistant to all tested antimicrobials except polymyxin b, thus leaving virtually no choices for therapy in terms of acceptable patient safety. results: overall gram-negative anaerobic bacteria from patients were studied. isolation sites were represented by intraabdominal - ( . %), soft tissue - ( . %), prostate fluid - ( . %), bone - ( . %), and dental - ( . %) infections. susceptibility of ( . %) prevotella spp., ( . %) bacteroides spp. (predominantly b. fragilis group - strains), ( . %) fusobacterium spp., ( . %) porphyromonas spp., and ( . %) veillonella spp. to ampicillin, clindamycin, metronidazole, imipenem, ertapenem, amoxicillin/clavulanic acid and cefoperazone/sulbactam was determined. all species were susceptible to carbapenems. in prevotella spp. there were % and % strains resistant to ampicillin and clindamycin and % of strains with intermediate resistance to metronidazole. among bacteroides spp. % of strains were resistant to ampicillin and % to clindamycin. no resistance to metronidazole was detected in bacteroides spp. objectives: the objectives of this study were to: analyse our current blood culture practice; describe the frequency of occurrence and antimicrobial susceptibility of bloodstream infections (bsi) isolates; determine the contamination rate. methods: we performed a prospective survey of all positive blood cultures received in the department of microbiology of tartu university hospital ( beds) in . blood culture system used was bactec . duplicates within one week were excluded. isolates were identified using conventional microbiology methods and susceptibility tests were those recommended by nccls. to determine extended spectrum beta-lactamase (esbl) producers an e-test with cefepime and cefepime combined with clavulanic acid was used. nosocomial infections were defined according to cdc criteria. results: during study period blood culture bottles were received, comprising blood culture sets ( . sets per patient-days). these resulted in ( . %) positive blood cultures, ( . %) were considered contaminants and contamination rate was . %. a total of bsi episodes involving patients were identified and ( %) of these were nosocomial. the incidence of nosocomial bsi (n-bsi) and community-acquired bsi (ca-bsi) was . and . per patient-days, respectively. polymicrobial bsi was detected in patients. among n-bsi dominated coagulase-negative staphylococci ( / . %), staphylococcus aureus ( / . %), klebsiella spp. ( / %), and escherichia coli ( / . %). the most frequent pathogens of ca-bsi were e. coli ( / . %), s. aureus ( / . %), haemophilus influenzae ( / . %), and streptococcus pneumoniae ( / . %). susceptibility to oxacillin of s. aureus and cons was % and . %, respectively. all s. pneumoniae isolates were susceptible to penicillin. . % of e. coli strains were susceptible to ciprofloxacin, . % to ampicillin, and % to gentamicin. susceptibility of klebsiella spp. to both ciprofloxacin and gentamicin was . %, and to ampicillin . %. . % of klebsiella spp. and none of e. coli isolates were esbl-producers. the susceptibility patterns of n-bsi and ca-bsi pathogens were similar to each other. conclusion: compared to west and north european countries our number of blood culture sets per patient-days is low. this may explain the relatively low incidence of bsi. the interventions to reduce contamination rate need to be implemented. the susceptibility among bsi isolates was high. recent outbreaks of c. difficile associated diarrhoea (cdad) reported in north america, united kingdom and the netherlands have emphasized the importance for an ongoing surveillance of cdad. the aims of the present study was to determine the epidemiology of cdad over the past years and the rate of nosocomial transmission in our acute care hospital ( -beds). materials and methods: all the cases of cdad diagnosed between january st and december st were retrospectively reviewed. a cdad case was defined as diarrhoea in hospitalised patients with a positive result for c. difficile cytotoxin or with a positive toxigenic culture. cdad was considered as severe if patient fulfilled at least of the following criteria: fever > . c abdominal pain or leukocyte count > , /mm or if the patient had an endoscopically proven pseudomembranous colitis or complications (toxic megacolon, perforation…). cdad was considered as community acquired if the diarrhoea occurred in patients within h after admission and if the patient had no history of hospitalisation in the previous months, otherwise cdad was considered as nosocomial. all the strains were serogrouped and characterized by toxinotyping and pcr-ribotyping. detection of toxin a, toxin b and binary toxin was performed by pcr. results: cases of cdad were diagnosed: clinical charts could be reviewed and strains were studied. global incidence of cdad was . per thousand discharges with higher rates in and . diarrhoea was community acquired in % of patients. for patients with nosocomial cdad, transmission of the strain from patient to patient (i.e. strain with the same serogroup and pcr-ribotype than the strain from another patient hospitalised in the same ward in the previous months) was demonstrated in . % of cases. binary toxin was positive in % of strains. binary toxin was associated to a more severe diarrhoea (p < . ) and to a higher case fatality (p < . ). a specific clone accounted for % of all the strains (serogroup h, pcr-ribotype '' '') but this clone was found both in nosocomial or community cases. three strains belonged to toxinotype iii but further investigations are needed to know whether these strains correspond to the hypervirulent strains involved in recent outbreaks. conclusion: incidence of cdad is low in our hospital and cross infection is limited. these results also suggest that strains with binary toxin might be more virulent. the development and application of a new exact typing method for clostridium difficile: multilocus variable number of tandem repeat analysis objectives: to study the epidemiology of clostridium difficile, a typing method with a higher discriminatory power, typeability and reproducibility than currently available methods is required. multi-locus variable number of tandem repeat analysis (mlva) is a new candidate technique, that has already been tested successfully on a number of bacterial and fungal species. using the whole genomic sequence, we developed mlva for c. difficile and compared the method to standardized pcr-ribotyping. additionally, mlva was tested on a collection of the new emerging hypervirulent pcr-ribotype strains. methods: short tandem repeat loci ( to bp) were identified using tandem repeat finder v . on the genome of c. difficile strain . amplification of the repeats was performed using a single pcr-protocol. pcr-fragments were analysed using multicoloured capillary electrophoresis on an abi , with a rox -marker as internal marker for each sample. the number of repeats per fragment was subsequently determined.the discriminatory power of the mlva was tested on reference strains representing serogroups and toxinotypes. the ability to subtype specific pcr-ribotypes was investigated with subtypes of pcr-ribotype (rep-pcr types - ), tcda-/tcdb+ strains of pcr-ribotype , and strains belonging to pcr-ribotype . of these type strains, were isolated from outbreaks and from endemic cases. results: a total of regions with short tandem repeats were identified. mlva discriminated all reference strains and the known reference strains of pcr-ribotype (rep-pcr - ). two mlva-types were recognized among tcda-/tcdb+ strains; the differences were present in only one of the repeat-regions. of pcr-ribotype strains, outbreak-related strains were identical to each other. interestingly, two endemic type strains differed from the other strains in of the regions. conclusion: mlva is a highly discriminatory genotyping method for c. difficile and is capable to subtype various crribotypes. mlva is also an important new tool to study the epidemiology of the emerging pcr-ribotype strains. comparative study of clostridium difficile diarrhoea in elderly patients treated with moxifloxacin versus amoxycillin for lower respiratory tract infections l. mooney, m. wilcox (leeds, uk) fourth generation fluoroquinolones such as moxifloxacin have improved anti-anaerobic activity. consequently, these new agents could induce c. difficile infection (cdi) by inhibition of 'protective' anaerobic flora. recent reports have suggested such an association. however, further studies are warranted to determine the risk of cdi in elderly in-patients treated with these agents, and notably where exposure to cd is measured/ controlled. methods: we prospectively investigated the propensity of moxifloxacin (mox) or amoxycillin/macrolide (aml/mac) to induce cdi when used to treat lower respiratory tract infections (lrtis) in elderly in-patients, using a -ward, crossover design ( months total). patients prescribed mox or aml/mac were monitored for gastrointestinal symptoms. diarrhoea was assessed as due to cd, viral or other cause. relevant clinical data were collected. concurrent epidemiological surveillance was also performed to determine environmental exposure to cd. results: patients were studied, receiving mox and had aml/mac. univariate analysis indicated that there was no significant difference between mox and aml/mac patients in gender, age ( . vs . mean years, respectively), or duration of hospitalisation (total, prior to and post diarrhoea). duration of antibiotic therapy did not differ significantly between mox and comparator patients (either total days or days before diarrhoea onset). there was a significant association between mox and overall risk of diarrhoea. however, there was no significance between mox treatment and cd, viral or other cause of diarrhoea. risk factor analysis to inform on possible confounders was performed. initial epidemiological survey results indicate that there was no change in environmental exposure levels to cd on each hospital ward. molecular typing of all clinical and environmental isolates of cd is ongoing. conclusions: although recent reports have highlighted a risk of cdi associated with fluoroquinolones (and increased age), none have specifically studied hospitalised elderly populations prospectively and controlled for exposure to cd. diarrhoea occurs relatively frequently after antibiotic therapy in the elderly. mox was associated with an increased rate of diarrhoeal symptoms, but causes other than cdi explained this association. mox treatment was not significantly associated with cdi when compared with amox/mac treatment for lrti in elderly in-patients. prevalence and association of macrolidelincosamide-streptogramin b resistance with resistance to moxifloxacin in clostridium difficile strains isolated from symptomatic adults and children hospitalised in two university hospitals in warsaw h. pituch, d. wultanska, g. nurzynska, p. obuch-woszczatynski, f. meisel-mikolajczyk, m. luczak (warsaw, pl) objectives: clostridium difficile is the main aetiological agent of nosocomial diarhoea. clindamycin, penicillins, and cephalosporins have been associated with cdad. however, several case reports of fluoroquinolone-associated c. difficile diarrhea have been published. c. difficile strains usually exhibits susceptibility to metronidazole, and vancomycin. we describe prevalence and association of macrolide-lincosamide-streptogramin b (mlsb) type resistance with resistance to moxifloxacin of c. difficile strains isolated from adults and children. methods: eighty-three c. difficile strains recovered from adults and children hospitalised in two university hospitals were investigated (hospital : adults n = , and children n = ; hospital : adults n = ). toxin types were determined by commercial test for toxin a and cytotoxicity test for toxin b. tcda, tcdb were detected by pcr. mics of erythromycin, clindamycin, moxifloxacin, vancomycin and metronidazole were determined by e-test (ab biodisk, sweden). the ermb gene was detected by pcr. results: sixty-seven ( %) c. difficile strains were toxigenic. among these, were a+b+, and were a-b+. all strains were susceptible to vancomycin and metronidazole. high level resistance to erythromycin, clindamycin and moxifloxacin was found in %, %, % of the tested strains, respectively. twenty-one c. difficile strains harboured high level resistance to erythromycin, clindamycin and moxifloxacin, simultaneously. among these, all were a-b+ and were isolated from adults, only. twenty-one of the macrolide-lincosamide-streptogramin b (mlsb)-resistant a-b+ strains carried the erythromycin resistance methylase gene (ermb). conclusion: resistance against clindamycin, erythromycin and moxifloxacin among polish a-b+ c. difficile strains was very frequent. fluoroquinolone resistance is associated with resistance to mlsb antimicrobials. we suggest that increasing use of fluoroquinolones is selective pressure for clonal dissemination of a-b+ c. difficile strains. fluoroquinolones use is a strong risk factor for cdad in our hospitals. acknowledgement: this work was supported by the ministry of scientific research and information technology, grant no. p d . national surveillance to the incidence of clostridium difficile-associated diarrhoea in the netherlands s. paltansing, r. guseinova, r. van den berg, c. visser, e. van der vorm, e.j. kuijper (leiden, amsterdam, nl) objectives: the recent outbreaks of clostridium difficileassociated diarrhoea (cdad) due to the new emerging pcrribotype , toxinotype iii strains has renewed the interest of cdad as an important nosocomial infection. to determine the incidence of cdad in the netherlands, we conducted a prospective surveillance study in hospitals in the netherlands. clinical microbiology and infection, volume , supplement , methods: from may st to july st of , participating hospitals registered all patients diagnosed with cdad. a standardized questionnaire was devised to obtain patient information. faeces samples or isolated strains were sent to the reference laboratory at the lumc for culture and the presence of genes for toxins a and b (tcda and tcdb). pcrribotyping was performed according to the method of bidet and toxinotyping as described by rupnik et al. results: routine methods to diagnose cdad in laboratories included combinations of cytotoxicity tests ( %), enzymeimmunoassays ( %) and culture of toxinogenic strains ( %). in total, patients with cdad were reported. the overall incidence (median) of cdad was for , patient admissions and varied from to . of patients with cdad, % was community acquired. the median age of patients with nosocomial acquired cdad was years. of patients with cdad, ( . %) died during the study period. at least different pcr-ribotypes could be recognized among strains. type was identified in patients from hospital. toxinotyping revealed the presence of at least different types. of strains, % were tcda+/tcdb+, % tcda-/tcdb-and % tcda-/tcdb+. conclusions: the incidence of cdad in the netherlands is lower than reported in usa and canada, but varied considerably per hospital. the new emerging type was found in patients from hospital with a high incidence of cdad ( per , admissions). outbreak of clostridium difficile pcr-ribotype toxinotype iii in harderwijk, the netherlands objectives: since , several epidemics of clostridium difficileassociated diarrhoea (cdad) caused by c. difficile pcr-ribotype toxinotype iii have occurred in usa, canada, and the uk. in april , the first outbreak encompassing patients was observed in a medium large hospital of beds in the netherlands. the isolated strain was completely resistant to erythromycin and ciprofloxacin. the patient characteristics, predisposing factors and outcome of cdad were studied. methods: a case-control study was performed in patients and at random selected controls without diarrhoea who stayed at the same department as the patients when the diagnosis of cdad was made. standardized questionnaires were designed to collect data from the patient records and all surviving patients were interviewed months after the diagnosis. faeces samples were cultured for the presence of c. difficile and isolates were typed. results: the incidence of cdad increased from per , patient admissions in to . per , admissions in . between april and september , patients with cdad due to type were identified. of patients, ( %) died of which ( %) as a direct result of cdad. eleven ( %) patients experienced one or more relapses. the average age of the cases was yrs, . % of the patients was male. in a multivariate analysis, antibiotic use (or . , p < . ), duration of hospital stay (cases days, controls days; p < . ) and tube feeding (or . , p = . ) were found to be significantly associated with cdad. in particular, the use of ciprofloxacin (or . , p < . ) and cephalosporins (or . , p < . ) were associated. no association was found between the use of protonpump inhibitors and the risk of cdad. the use of erytromycin was significantly higher in cases ( . %) than in controls ( . %) in a univariate analysis (p < . ), but this relation was not significant in a multivariate analysis. conclusion: antibiotic use (especially ciprofloxacin and cephalosporins), duration of hospital stay and tube feeding were significantly associated with cdad caused by c. difficile type , toxinotype iii in the netherlands. we could not confirm the previously described relation between use of protonpump inhibitors and risk of cdad. clostridium difficile pcr ribotype , toxinotype iii in the netherlands objectives & methods: shortly after the reports in june of clostridium difficile pcr ribotype , toxinotype iii in england, this more virulent type was also detected in the netherlands. in response, the dutch centre for infectious disease control has undertaken measures to monitor and control the outbreak. c. difficile guidelines for infection control and treatment were formulated, separately for hospitals and nursing homes. the leiden university medical centre serves as a reference centre for diagnostics and typing of c. difficile. laboratories are encouraged to send in samples for typing in case of a clear rise in the incidence in c. difficile, rapid spread, or several clinically suspect cases.organisation-based surveillance was set up: questionnaires are sent monthly to institutions with c. difficile associated diarrhoea (cdad) outbreaks to obtain information on incidence, c. difficile testing strategies, antibiotics use and control measures taken.measures taken in hospitals dealing with an outbreak of type include: treatment of cdad with vancomycin in stead of metronidazole, emphasis on frequent and thorough cleaning and disinfection, isolation of all patients with diarrhoea until tested negative for c. difficile toxin, cohort isolation of cdadcases if individual isolation capacity is exceeded and strong restriction of certain antibiotics, including fluorochinolones. results: until november st, , samples from institutions have been sent in for typing, resulting in type positives. epidemic spread of type has been detected in hospitals and one nursing home. furthermore, in retrospective studies in four hospitals isolated cases of type were detected. it became clear that in one region with three hospitals, the cdad incidence had already risen in , and , respectively . unfortunately, no samples from that period were available for typing. in the hospitals with epidemic spread of type , a wide range in the monthly incidence of cdad was observed, from to per , admissions during the outbreaks. the incidence in the pre-epidemic period varied from to (see figure) . conclusions: the outbreaks in hospitals are difficult to control: most hospitals continue to have new cases for a long period, although the incidence is decreasing in several hospitals. fortunately, once a c. difficile outbreak in a hospital is recognised, spread to other hospitals has not been observed. objectives: c. difficile is a major cause of antibiotic associated diarrhoea (aad) and colitis (c). the aim of this study was to determine the incidence of these infections in our hospital ( beds), during a period of months (march-october ) . methods: a number of liquid stools from equal adult patients (mean age y, m: , f: ) receiving broad spectrum antibiotics (especially cephalosporins) were plated in ccfa (oxoid) and anaerobic brucella agar (ba), after alcohol shock procedure. if the culture was positive, an immunochromatographic test was performed for toxin a (colorpactm toxin a, bd, usa). if the last test was negative, a rapid enzyme immunoassay was performed for toxins a+b (immunocard, meridian bioscience inc. cincinatti, ohio). results: c. difficile was isolated in / ( . %) samples. seventeen men (pathological -p, pneumological -pn, surgical -s, urologic -u, outpatients -o, , , , , respectivly) and women (p: , pn: , o: ) harbored c. difficile in their intestin. twelve out of strains ( %) produced toxin a, while the remaining ( %) produced toxin b. eleven patients had severe diarrhoea ( - days). one patient got endoscopic examination, which confirmed colitis findings. the two outpatients received oral cefuroxime in the preceding week of the positive culture. conlusions: ( ) the incidence of c. difficile infections in this study is among these reported in international bibliography ( . %). ( ) since toxigenic b c. difficile strains were demonstrated in half cases, the use of the tests detecting both toxins a and b by clinical laboratories is recommended.( ) molecular technics application (e.g. pfge and ribotyping) will offer a better knowledge of c. difficile spread in our hospital. assay of the cytotoxicity of stool samples to cells in tissue culture is commonly considered the 'gold standard' for detection of c. difficile toxin. however the method is slow and therefore its use can result in delayed patient treatment and implementation of infection control measures. we undertook a comparison of two microtitre plate-based elisa kits (techlab c. difficile tox a/b ii and meridian premier toxins a & b) and three rapid immunoassay card kits (remel xpect clostridium difficile toxin a/b, meridian immunocard toxins a & b and techlab tox a/b quik chek) with an in-house cytotoxin assay. all samples tested had been referred for routine microbiological examination. toxin tests were done on unformed samples from adult hospital in-patients and bone marrow transplant recipients and on samples where c. difficile toxin testing was requested by the referring clinician. all kits were used according to manufacturers' instructions. three hundred and thirty three specimens were tested using all five kits and cytotoxin assay. sensitivities and specificities were calculated both (a) assuming the cytotoxin assay to be the 'gold standard' (universally correct) test and (b) taking a concensus view that any sample with at least two tests positive is truly positive. data are shown in the table below. overall, the microtitre plate-based elisa kits were more sensitive than the rapid immunoassay card kits. the cytotoxin assay was negative for seven samples that were positive by at least two other tests. thus the plate-based elisa kits were also more sensitive (but less specific) than the cytotoxin assay if consensus data was used to judge true positivity. we conclude that some immunoassay kits offer an acceptable alternative to cytotoxin assays for the detection of c. difficile toxin, allowing more rapid diagnosis. location of the enterotoxin gene in strains of clostridium perfringens associated with gastroenteritis objectives: clostridium perfringens type a is a common cause of food poisoning and is also associated with non-food borne gastroenteritis including antibiotic associated, infectious and sporadic diarrhoea. the disease symptoms are due to an enterotoxin produced when the organism sporulates in the human small intestine. the c. perfringens entertoxin gene (cpe) has been shown to be located either on the chromosome or on one of two large plasmids and it is generally accepted that c. perfringens strains associated with food poisoning have a chromosomal cpe gene whilst strains isolated from non-food borne diarrhoea have a plasmid encoded cpe gene. spores from strains possessing a chromosomal cpe gene have been found to be far more heat resistant than spores from strains with a plasmid encoded cpe gene. heat resistant spores are more able to survive the cooking process and go on to cause food poisoning, thus explaining why most food poisoning strains have been found to have chromosomally located cpe genes. the purpose of this study was to determine the location of the cpe gene in a range of c. perfringens strains from the uk, including those from both food borne and non-food borne illness. method: a multiplex pcr assay described by miyamoto et al., ( ) was used to determine the location of the cpe gene in strains of c. perfringens isolates associated with food borne illness and strains associated with non-food borne illness. results: by multiplex pcr assay % of c. perfringens strains associated with food borne outbreaks in the uk were found to have a plasmid encoded cpe gene. these findings have not been described before. all strains associated with non-food borne illness had the cpe gene located on one of two plasmids, as anticipated. conclusions: a significant number of food borne outbreaks of c. perfringens food poisoning were found to be caused by strains of c. perfringens carrying a plasmid encoded cpe gene. since strains of c. perfringens with a chromosomal cpe and plasmid cpe genes have different physiological characteristics this may have a profound impact on their mode of transmission. references miyamoto, k., wen, q. and mcclane, b. a. ( ) multiplex pcr genotyping assay that distinguishes between isolates of clostridium perfringens type a carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an is -like sequence, or a plasmid cpe locus with an is sequence. journal of clinical microbiology , - . novel multiplex-pcr method for simultaneous detection of clostridium difficile toxin a and toxin b and the binary toxin (cdta/cdtb) genes applied on a danish cohort k.e.p. olsen, s. persson (copenhagen, dk) objectives: a new multiplex pcr method was developed for the detection of the clostridium difficile toxin genes: tcda, tcdb, cdta and cdtb. this method was applied on clostridium difficile strains isolated from danish hospitalised patients with diarrhoea in the period from april to october , in order to investigate the present toxin profiles and their correlation to sex and age. method: a -gene multiplex pcr method was developed for the simultaneous amplification of the four clostridium difficile toxin genes tcda, tcdb, cdta, cdtb and s rdna as an internal positive control. template dna was prepared from plate grown bacterial colonies by a simple boiling procedure, and amplicons were visualized by standard gel electrophoresis. results: three different toxin profiles were detected in the danish cohort: tcda+, tcdb+, cdta+/cdtb+; tcda+, tcdb+, cdta-/cdtb-and non-toxigenic tcda-, tcdb-, cdta-/ cdtb-. the prevalence of the binary toxin genes in this study was % of the clinical isolates.more than half of the strains ( %) were isolated from the elderly part of the population (> years), and % of these strains displayed the tcda+, tcdb+, cdta+/cdtb+ profile. of the non-toxigenic strains, % of the patients were females. one fourth of the strains isolated from children under years of age were non-toxigenic. in four patients, two different toxin profiles were obtained from independent faecal samples. conclusion: this method offers a one-step, rapid and specific identification of clostridium difficile toxin genes. this specific toxin profiling allows an evaluation of the pathogenic potential of the isolated clostridium difficile and surveillance of emerging toxin profiles. further studies of the isolated toxigenic clostridium difficile strains will include gene deletion analyses of the tcda and the tcdc (toxin regulating gene) which independently have been observed to cause enhanced pathogenicity. prevalence of clostridium difficile-associated diarrhoea in hospitalised patients with nosocomial diarrhoea in university of medical sciences hospitals, tehran, iran objectives: this study was aimed at determining the prevalence of clostridium difficile associated diarrhoea in hospitalized patients with nosocomial diarrhoea at three university hospitals in tehran from december to august . methods: during the study period, the stool samples of hospitalized patients with nosocomial diarrhoea were cultured and tested by stool cytotoxin assay, toxigenic culture and also of the samples were examined by enzyme immunoassay. results: in ( . %) of samples c. difficile grew and stool samples (prevalence: . %) were toxin positive by stool cytotoxin assay, enzyme immunoassay or toxigenic culture. there were no significant relationships between c. difficileassociated diarrhoea and sex and age of patients. the results of the present study showed that among requested samples the highest percentage of c. difficileassociated diarrhoea was observed from the transplantation department ( . %), followed by icu and paediatric section. objectives: the prevalence of toxigenic clostridium difficile (c. difficile) has been reported about - % in korea. toxin a(-)/ toxin b(+) variant c. difficile strain is also important in nosocomial c. difficile infection. however, characterization of clostridial toxin (toxin a, toxin b) had not been studied. methods: we used pcr for toxin a and toxin b genes in c. difficile isolates from patients admitted in three tertiary hospitals during january to december, . primers for toxin a genes were nk -nk , nk -nk and nk -nk and toxin b gene was nk -nk . results: toxin a and toxin b positive rates using nk -nk , nk -nk and nk -nk were concordant and ranged from . % to . % in hospitals. the proportions of non-toxigenic strains were - %. however, we could differentiate toxin a(-)/toxin b(+) variants using nk -nk primers. the proportion of toxin a(-)/toxin b(+) c. difficile variants were . %, . % and . % in hospitals respectively. objective: administration of antibiotic drugs has long been known to cause alterations in the gut ecosystem. in some patients, these alterations may create a niche that allows the overgrowth of some pathogens such as clostridium difficile, the main causative agent in nosocomial infectious diarrhoea. a predictive tool to assess the risk of development of clostridium difficile, would be of utmost clinical relevance. it remains to be determined whether specific patterns in pre-existing gut microbiota can predict the risk of onset of clostridium difficile, upon initiation of antibiotic treatment. using samples from subjects enrolled in a previously published clinical study on antibiotic-associated diarrhoea (aad), we investigated the potential relationship between their dominant faecal microbiota and the subsequent development of clostridium difficile when subjects received antibiotics. methods: temporal temperature gradient gel electrophoresis (ttge) was used to assess dominant species distribution in gut microbiota. each electrophoregram was digitised from the migration distances and a regression model [partial least square-discriminant analysis (pls)] was built to investigate the correlation between pre-treatment dominant faecal microbiota and the acquisition of clostridium difficile during antimicrobial chemotherapy. results: this pls model could explain % of the subsequent onset of clostridium difficile. this result supports the concept of ''permissive'' flora with preliminary data focusing on clostridium coccoides-phylogenetic group. conclusion: to our knowledge it is the first time that dominant faecal microbiota is found to heighten susceptibility to the subsequent onset of clostridium difficile upon initiation of antibiotic treatment. these findings insinuate that strategies reinforcing the control of dominant faecal microbiota at homeostasis would be of clinical relevance. this study has been partially financed by biocodex laboratories. objectives: selective therapy of c. difficile diarrhea (cdd) requires the reduction of pathogen counts in the colon, but spare the normal flora. to determine if par is selective for cdd, serial stool samples were collected at study entry, at day , and weekly x during the conduct of a phase a study of cdd treatment. methods: patients (n = ) were randomized to receive , or mg twice daily of par for days. no prior therapy was given to patients; receive or doses of standard therapy. as treatment controls, additional patients were treated with vancomycin mg qid for days. five well persons donated stools as normal flora controls. fresh stool samples were cultured - , , , for c. difficile vegetative and spore forms; faecal filtrates were tested for cytotoxin b by cell assay. strains were characterized by tcda/b, ermb, cdta/b pcr and by ribotyping. at study entry and day , aerobic and anaerobic faecal flora cultures, diluted - , , , , were examined for major floral shifts. since bacteroides group organisms are ubiquitously present and cultivable, this genera was selected as a indicator of the integrity of the microbial flora. results: at study entry, mean log cfu + sd vegetative counts of c. difficile (all par patients) were . + . , range - . ; at day , with the exception of one patient receiving mg, all other patients had c. difficile quantitative counts reduced < log /gm faeces. vancomycin was similarly effective. at study entry, bacteroides group counts were < , - , & . - log cfu/gm in~ / each of patients. all normal stools showed complex, multi-genera in high counts, with - bacteroides group species > log cfu/g. mean + sd of log cfu of bacteroides group counts/g feces wet weight at study entry and day for mg/day (n = ) were . + . / . + . (p = . , wilcoxon matched pairs signed-ranks test, tailed); for mg/day (n = ) were . + . / . + . (p = . ); for mg/day (n = ) were . + . / . + . (p = . ); and for vancomycin (n = ) . + . / . + . (p = . ). conclusion: patients with cdd have variably impaired normal flora. par was effective in all dosages in eradicating c. difficile. a dose-dependent reduction in bacteroides counts was not observed. vancomycin significantly reduces bacteroides counts during cdd treatment. par is effective against c. difficile in-vivo, and is relatively sparing of the normal flora. results: the three rt pcr assays were able to detect all enterovirus strains in cell culture supernatants. however the detection limit of the mgb rt pcr was to log more sensitive in out of dilutions assays of vc supernatants compared to the rab and ver rt pcr. all ver and mgb negative csf were vc negative. thirty-two csf specimens from patients suspected of viral meningitis were positive by all rt pcr ( . %), whereas only were found positive by vc ( . %). the rab rt pcr failed to detect csf confirmed positive by vc ( echo and non typable ev). among samples positive by rt pcr, sensitivity of ver, mgb and rab was respectively %, % and . %. conclusion: in our laboratory, mgb rt pcr has a good correlation with ver rt pcr whereas rab rt pcr is less sensitive especially for the detection of echovirus . the mgb rt pcr seems to be the most sensitive of the rt pcr. further studies, including more ev strains should help to precise the sensitivity of this assay. a. dalwai, s. ahmad, e. hussein, a. pacsa, w. al-nakib (kuwait, kw) objectives: enteroviruses generally share tissue tropism and present with overlapping disease spectrum, however certain enteroviruses may be over represented in certain diseases than others. coxsackievirus a though has been reported to cause several diseases such as febrile illness, herpangina, aseptic meningitis and acute flaccid paralysis, the frequency was very low. the study aimed to determine the prevalent enteroviruses causing non-specific febrile illness, aseptic meningitis, encephalitis, neonatal disease and myositis, in kuwait. it also aimed to study the association between a certain enterovirus and a particular disease and its severity. methods: diagnosis of enteroviral infection was based on detection of enteroviral rna by semi-nested rt-pcr of a portion of the 'utr of the enteroviral genome followed by southern hybridization with an enterovirus specific probe to confirm the results. the enterovirus was genotyped by sequencing of the 'utr, the vp and a portion of the vp encoding regions, and the sequence was analysed by blast analysis, clustalw alignment and phylip phylogenetic analysis package. results: enteroviruses were the only etiological agents detected in % ( ) of disease cases investigated. coxsackievirus a was identified to be the second most predominant enterovirus ( %; of cases genotyped) associated with disease, after only echovirus ( %; / ). although identified in all the diseases investigated, coxsackievirus a occurred less frequently in cns disease cases ( %; / ) than in febrile illness cases ( %; / ). in a preliminary study, it was also predominantly detected in % ( / ) of myositis cases. the 'utr of this virus showed % homology with that of coxsackievirus a prototype strain (parker strain) whereas the vp and the adjoining region showed greater homology to human enterovirus b genotype sequence. conclusions: coxsackievirus a was determined to be an emerging enterovirus associated with different diseases in kuwait. it was frequently represented in mild febrile illness and myositis cases than in cns disease suggesting that the isolate might be less neurovirulent. molecular analysis suggests that the isolate might have emerged due to recombination between coding and non-coding segments of coxsackievirus a and human enterovirus b group genomes. acknowledgement: supported by research administration project grants mi / , ym / and college of graduate studies, kuwait university. the new proposed enterovirus type is causing meningitis in spain introduction: several new proposed enteroviruses (ev) have been recently described, including the named ev [ ] . a total of isolates of this serotype were identified from to in america, africa or asia associated mainly with acute flaccid paralysis or unspecified disease. objective: to determine if this new serotype circulates in spain and what type of disease produces. methods: a total of ev isolates coming in to the spanish enterovirus reference laboratory were studied both by micro neutralization assays and by typing pcr [ ] . in the isolates in which ev was suspected by the mentioned methods complete vp gene was amplified and sequenced with specific designed primers. results: four isolates from two different regions of spain were identified as ev (more than % of homology with the published sequences). three of them corresponded to aseptic meningitis in children and were isolated from csf. discussion: the present work demonstrates that this new proposed virus circulates also by europe and is associated to aseptic meningitis. till the moment it seems that is represented in a minor proportion ( / studied), however the possibility of spreading of this viral infection should be considered, as evs may behave in that way, as previously have been demonstrated [ ] . objectives: rotavirus is the most important cause of severe gastroenteritis in infants and young children through the world and is responsible of , deaths annually, mostly in developing countries. therefore, development of rotavirus vaccine is a high priority. rotavirus strains with g types account for the majority of the diarrhoea episodes. recently, a monovalent g attenuated rotavirus vaccine was licensed in mexico. in view of a hypothetical introduction of such vaccine in europe, we investigated the variability over time of vp antigenic genes of g rotavirus strains in our area. methods: fifty strains were selected from a total of g strains obtained from children of less than years of age hospitalised with acute gastroenteritis at the ''g. di cristina'' children's hospital of palermo in the period - . the selected strains were genotyped by rt-pcr and of them were submitted to vp gene sequence analysis. results: all but one of the strains were genotyped as g p( ). the vp sequences of of them were distributed into lineages i and ii. lineage i included strains from different years in the range - . lineage ii included strains from different years in the range - . the degree of similarity among the nucleotide sequences of italian strains in each lineage were comprised between % and %. an alignment of the deduced amino acid sequences showed major lineage specific amino acid changes in the variable antigenic regions with respect to the reference wa strain. conclusions: sequence analysis indicated that in palermo there was co-circulation of g strains belonging to two different lineages. overall, the g strains showed a high degree of similarity inside each lineage and shared specific amino acid modifications. the antigenic differences between circulating strains might permit them to escape neutralization and persist in the infantile population. our results suggest that rotavirus strains belonging to the two g lineages should be both included in a rotavirus vaccine preparation. epidemic spread of recombinant noroviruses with four capsid types in hungary objectives: noroviruses (''winter vomiting diseases'') are the predominant etiological agent in hungary and common pathogen worldwide in outbreaks of gastro-enteritis in humans. noroviruses are genetically diverse group of viruses with multiple genogroups (gg) and genotypes. more recently, naturally occurring recombinant noroviruses were identified. these viruses had a distinct polymerase gene sequence (orf , designated ggiib/hilversum) and were disseminated through waterborne and food-borne transmission in europe. our aim was to characterize these emerging recombinant noroviruses causing outbreaks of gastro-enteritis in hungary. methods: stool and rna samples -from norovirus outbreaks between january and may -containing ''ggiib/ hilversum polymerase'' (ggiib-pol) were selected for analysis of the viral capsid region (orf ) by reverse transcriptionpolymerase chain reaction (rt-pcr) followed by sequence-and phylogenetic analysis. results: forty ( . %) of confirmed norovirus outbreaks were caused by the new-variant lineage with the ggiib-pol. viral capsid region was successfully characterized in ggiibpol outbreaks. four different recombinants were detected with capsids of hu/nlv/ggii- /mexico/ (n = , . %), hu/ nlv/ggii- /snow mountain/ (n = , . %), hu/nlv- /ggii/hawaii/ (n = , . %) and hu/nlv/ggii- / lordsdale/ (n = , . %). interestingly, outbreaks caused by recombinant ggiib-pol strains mostly associated with outbreaks among children ( . %) and had non-winter seasonality. conclusions: epidemic spread of emerging multiple recombinant norovirus strain ggiib-pol were detected in hungary that became the second most common norovirus variants -next to the endemic ggii- /lordsdale virus -causing epidemics of gastroenteritis in the last . years. the respiratory infections are the most common diseases in the world being the origin of a great morbidity and mortality especially in infants and elderly. ( ) human metapneumovirus (hmpv) was first described in dutch children with acute respiratory tract infections (artis) in june . ( ) very limited studies data are available from tropical and developing countries. we sought to determine the role of hmpv in upper and lower respiratory tract infections in cuban patients and correlated the presence of virus with clinical characteristics of the disease. between october to september clinical samples received from the national surveillance program of artis at the national reference laboratory of respiratory viruses, for virological study, were used to detect hmpv by rt-nested pcr, amplifying a conserved fragment of nucleotides in the polymerase gene. we found rna hmpv in . % of samples from the patients with artis. . % of individuals who tested positive for hmpv were under months of age. patients with evidence of hmpv had symptoms consistent with either upper or lower respiratory tract disease or both. . % of hmpv positive individuals were detected during august-october (table ). the results of this preliminary study shows that hmpv is present among cuban patients with arti. constitute the first report of the frequency of hmpv infection in a non-preselected group of cuban patients with ages ranged from months to years old. it should be noted that this is the first report of hmpv infection in central america and in the caribbean region, further confirming the worldwide distribution of the virus ( ) ( ) ( ) . detection of human metapneumovirus in paediatric nasopharyngeal aspirates by a taqman minor groove binder probe assay: a one-year prospective study in belgium w. verstrepen, p. bruynseels, a. de smet, a. mertens (antwerp, be) objective: human metapneumovirus (hmpv) has a relative high incidence in acute respiratory infections in children but is difficult to isolate in culture. the aim of the study was to decrease the number of undiagnosed viral respiratory infections in our hospital by means of a taqman minor groove binder (mgb) probe assay. methods: from october to september a total of nasopharyngeal aspirates from children presenting at our paediatric facility were analysed. rna extracts from specimens negative for rsv, parainfluenzavirus and influenzavirus with an (in) direct immunofluorescence assay (ifa) were subjected to a taqman mgb probe assay in parallel with a previously published taqman assay. results: of the specimens, ( %) were positive by ifa for either rsv ( ), parainfluenzavirus ( ), influenzavirus a ( ) or influenzavirus b ( ). hmpv was detected in ( . %) of the remaining specimens subjected to the newly developed pcr. of the patients with a positive hmpv assay, / ( . %) presented with respiratory symptoms. % of the positive specimens were from children less than year as compared to only % from children older than years. viral load was highest in children less than year. a prominent seasonal variation was noted since more than half of the positive specimens occurred during the months march and april. there was no significant difference in the proportion nor viral load of positive specimens from ambulatory patients, patients admitted to a general ward or patients requiring intensive care. as compared to the published taqman assay, diagnostic sensitivity and specificity were . % and . % respectively, whereas ppv and npv were . % and . %. method comparison (nccls guideline ep- a) failed to demonstrate a significant difference between both assays when the threshold cycle (ct) was between and . strongly positive specimens (ct < ) were associated with a lower ct using the published taqman assay. however, the new taqman mgb probe assay appeared to be more sensitive for weakly positive specimens (ct > ). conclusion: the number of viral respiratory infections confirmed in our hospital was substantially increased by means of the hmpv taqman mgb probe assay. the new assay is a reliable alternative to the previously published taqman assay for detection of hmpv in nasopharyngeal aspirates. nucleic acid sequence based amplification and molecular beacon detection for the real-time identification of respiratory syncytial virus in paediatric respiratory specimens r. manji, f. zhang, c. ginocchio (lake success, us) background: respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract infection in infants and young children, with bronchiolitis and pneumonia being the major clinical manifestations. the rapid diagnosis of rsv infections is of central importance for individual patient management (rational use of antibiotics and antiviral agents), hospital infection control and monitoring epidemiological disease patterns. this study included a technical validation and a retrospective clinical evaluation of a real time nasba assay for the detection of rsv a and rsv b in paediatric respiratory samples. methods: samples tested included: dilution panels of in vitro transcribed rna, local rsv isolates, isolates of common respiratory pathogens, and frozen respiratory specimens (nasopharyngeal aspirates, washes or swabs) from children (age range: d to yr) who were evaluated in the paediatric emergency department for respiratory disease. nucleic acid (na) isolation, amplification and detection were performed using the nuclisens easyq basic kit and nuclisens easyq rsv a+b reagents (biomérieux). specimen nas and a rsv specific internal rna control (ic) were co-extracted using nuclisens magnetic extraction reagents and the nuclisens minimag instrument (biomérieux) and co-amplified using a single rsv specific primer pair. included in the reaction were a rsv specific molecular beacon ( '-fam) and an ic specific molecular beacon ( '-rox). target amplification and continuous monitoring of emitted fluorescence were performed using a nuclisens easyq analyzer (biomérieux). results were compared to direct immunofluorescence (dfa) and/or viral culture using r-mix cells (diagnostic hybrids, oh). results: the limit of detection for rsv was rna copies/rxn and the % detection rate was copies/rxn. the assay was % specific for rsv with no cross reactivity to other respiratory pathogens. the nasba assay detected % more positive specimens than dfa and % more positive samples than vc. the npvs of the assays were: nasba . %, dfa . % and vc . %. the nuclisens easyq rsv assay demonstrated superior sensitivity to both dfa and viral culture for the detection of rsv a and b from respiratory specimens. the assay was easy to use, required minimal hands on time ( hr) and a faster time to results as compared to rapid culture ( hr vs. - hr). respiratory syncytial virus (rsv) is a major cause of acute lower respiratory tract infection in infants and young children. it has previously been shown that hrsv isolates can be divided into two antigens groups a and b. the g protein is the most divergent both between and within the two subgroups and appears to accumulate amino acid changes with time, suggesting evolution under selective pressure. our knowledge of the molecular epidemiology of rsv has so far been based mainly on studies done in the developed world with e temperate climate. very limited epidemiological data are available from tropical and developing countries, where rsv infections may follow a different pattern. in this report we examine the molecular epidemiology and evolutionary pattern of the g protein of both subgroups a and b rsv through consecutive epidemics in cuba. sixthly four nasopharyngeal swabs were collected from children under years of age with respiratory disease to different hospitals in cuba between and , to examine the molecular epidemiology and evolutionary pattern of the g protein of rsv. all samples collected from to were rsv subgroup a; however both subgroups co-circulate during . the cuban isolated from to showed a great homogeneity between them and were resemble to an ancient strain (long) with only five nucleotide differences, this also occur in and with two strain. furthermore was detected different size of g protein ( or for rsv a and for rsv b) due to change in stop codon used he genetic homogeneity of the cuban isolates ( ) ( ) ( ) and their resemble to an ancient strain such as long was an unusual finding in our country. in both subgroups was observed the predominance of strains with almost similar sequences. phylogenetic analysis for subgroup a strains showed that strains were cluster in different genotypes with virus isolated in different geographic regions. both subgroups co-circulated during and clustered whit south african strains that circulate during at the same time. point mutations in respiratory syncytial virus detected by lightcycler pcr and melting curve analysis u. germer, l. nielsen, k. boye, h. westh (copenhagen, dk) objective: the objective was to analyse rsv real-time pcrpositive isolates from clinical samples, which appeared to belong to three different groups according to melting temperature (tm) of the amplicons. the analysis was done according to genotypic and phenotypic difference and related to geographical distribution. materials and methods: nasopharyngeal aspirates were collected from children with respiratory distress in the city of copenhagen. viral rna extracted using the magnapure lc automated extraction system was amplified in a real-time rt-pcr previously described ( ) . five samples from each of the three groups with different tm's were selected for bidirectional dna sequencing using the rsv primers. sequences were analysed using chromas lite version . . results: a total of clinical samples were analysed. ( %) of the samples were positive and ( %) were negative for rsv. three distinctive groups with different tm's could be identified from the melting curve analysis. group (n = ) had a tm with a median of . °c, group (n = ) and (n = ) had lower tm's with a median tm of . °c and . °c respectively. sequence analysis of amplicons showed that the difference in tm was due to differences in genotype between the three groups. genotype and were closely related, differing only in two nucleic acids in position (c to t) and (a to t). both were silent mutations. only position is targeted by the probe. genotype and were both blasted to a complete genome sequence of respiratory syncytial virus subgroup a (genbank rsu ) with the highest identity score for genotype . genotype sequences were blasted to human respiratory syncytial virus mutant cp subgroup b (genbank af ). geographical analysis showed a higher prevalence of the mutant strain (genotype ) in the northern areas of the greater copenhagen area compared to central, southern and western areas (p = . ). conclusion: we found three genotypes of rsv according to the tm of the pcr product. two of the genotypes were closely related with only two point mutations and the same phenotype. genotype was mainly found in clinical isolates from the northern part of copenhagen, suggesting a local epidemic spread. objectives: biomerieux is developing a real-time nasba assay to detect influenza a and b rna in different kind of respiratory clinical samples, by using the nuclisens Ò easyq basic kit in combination with specific primers and molecular beacons. methods: nasal/throat swabs in transport medium from hospitalised children ( - yrs from edouard herriot hospital, lyon, france) were used for this evaluation. influenza rna is isolated using the nuclisens Ò minimag extraction. an internal control is added to the sample prior to nucleic acid extraction. the assay is designed to detect in a single tube, using a three-label approach, the internal control and both influenza a and influenza b rna. amplification reactions were performed in a nuclisens Ò easyq analyser allowing real-time detection. the results of the clinical samples were compared to cell culture results. results: among swabs tested, real-time nasba detected ( . %) samples for influenza a and ( . %) samples for influenza b. comparatively, by cell culture only ( . %) samples were identified as influenza a and non as influenza b. interestingly, influenza a positive sample identified by cell culture was found negative in real-time nasba. conclusions: the data showed that nuclisens Ò easyq influenza a/b assay detected % more influenza a virus than cell culture method. moreover, real-time nasba detected influenza positive samples, which were not detected by cell culture. with this assay a qualitative detection of influenza a and influenza b viruses in a single reaction can be done within hours. it provides a valuable alternative to cell culture method for the clinical management of patients with influenza infections. results: patients have developed mumps meningitis and patients were diagnosed with mumps meningoencephalitis. age limits were from to years and sex ratio m/f was / .clinical manifestations involved fever ( %), stiff neck ( %), nausea and vomiting ( %), headaches ( %), photophobia ( %) and neurological manifestations such as: equilibrium disorders and drowsiness ( %), convulsions ( . %), cerebellum syndrome ( . %). meningeal symptoms have occurred shortly after parotiditis in % of cases and before parotiditis in % of cases; the other cases have evolved without parotid swelling. other localizations of the mumps infection were: parotiditis ( %), pancreatitis ( %), submaxillitis ( %) and orchitis ( %). lumbar puncture yields csf containing between and wbc/mm . the predominating cells were usually lymphocytes, but % of the patients have polymorphonuclear leukocyte predominance at the first puncture. protein levels are normal to mildly elevated in all cases and hypoglycorrachia was founded in % of the patients. therapy for mumps meningitis was symptom-based (analgesics and antipyretics) in % of cases and glucocorticoid therapy in % of cases. conclusions: ( ) neurological involvement in mumps occurred in . % of cases; ( ) men are afflicted two times more often as women, but the age distribution is the same as for uncomplicated mumps; ( ) mumps meningitis was the only localization of the mumps infection in % of cases. mumps is acute generalized infection occurs primarily in schoolaged children and adolescents. most prominent manifestation of mumps is swelling and tenderness of salivary glands especially parotid gland. uveitis is a rare manifestation of mumps. here we present a mumps case complicating with uveitis. years old paediatric nurse was admitted to our emergency department because of headache and malaise. on physical examination bilateral parotid enlargement was noticed. opthalmology consulatation revealed anterior uveitis. local prednisolon and cyclopentholate treatment were prescribed. lumbar puncture revealed lymphocytic pleocytosis without hypoglychorachea and elevated protein levels. mumps igm was found positive. differential diagnosis made with other viral infections and sarcoidosis. her headache diminished day after the hospitalisation. uveitis responded very well to local therapy and patient got well in weeks. clinical and epidemiological aspects of a measles epidemic, bucharest, romania results: there were cases; sex ratio m/f: / . the mainly affected age group is under months ( . %) followed by months- years ( . %), - years ( . %), > years ( . %) and - years ( . %). . % cases were hospitalacquired (mostly in paediatric clinics), . % were communityacquired; in . % cases the source was unknown. the most common clinical features were fever ( %), rash ( %), conjunctival hiperemia ( . %), cough ( . %), micropoliadenopatia ( . %), diarrhoea ( . %). pulmonary complications were described in . % of cases; . % of them were bacterial pneumonia, . % were viral pneumonia. in . % of cases we diagnosed acute stomatitis, in . % bacterial conjunctivitis; in . % of cases -otitis; in . % of cases -pharingitis, and in one case ( . %) -urinary tract infection. . % of the patients were previously diagnosed and treated for pulmonary tb. all cases were confirmed serologically through detection of specific igm antibodies. patients ( . %) had severe clinical forms of measels. the evolution was good in all cases. conclusion: . this year in the south-east part of the country, evolves a measels epidemic with different features comparing to the previous one ( ) ( ) we investigated the recombinant proteins np and hn to develop new antigen with useful properties for applied in elisa test systems. methods: significant antigenic epitopes of nucleoprotein (np) and haemagglutinin (hn) measles virus strain edmonston were generated by computer analysis. using standard geneengineering techniques was evaluated two fusion peptides np and hn consist from only linear t-cell antigenic determinants. the virus-neutralization activity of hyperimmune serum on recombinant proteins was determined by plaque reduction neutralization test (prn). the level of specific igg in serum to genotypes a, d , and d of measles virus was determined by enzyme-linked immunosorbent assay (elisa). we used recombinant proteins np and hn as antigens for elisa. results: hyperimmune serum was collected from mice after immunization by np and hn recombinant proteins. the level of neutralize activity was measured in the prn assay with strain edmonston. the titre reached up to : . and : . for np and hn recombinant proteins, respectively. interestingly that, hyperimmune serum on recombinant protein np in elisa reacted both with np (titre : ) and with hn (titre : ), and in turn serum on recombinant protein hn reacted only with hn (titre : ). the estimation immunological properties of proteins with use of the panel of serum ( samples) collected from patients. the diagnosis of measles infection was confirmed in laboratory (by rt-pcr). the nucleotide sequences of rt-pcr products used for genotyping of mv. selective interaction of antibodies in elisa with recombinant proteins in relation to various genotypes is revealed. the interaction with genotypes a and d was expressed with high level of correlation whereas with genotype d any serum did not react authentically (as the control was used recombinant protein n of sars virus). conclusion: we have shown that neutralize antibodies formed hot only on superficial proteins such as hn, f and sh but also on core proteins such as np. our data demonstrate that the recombinant proteins np and hn could be a cost-effective alternative to current whole virus based elisas for surveillance for immunity to measles and could more efficient in detecting susceptibility to measles in relation to genotypes a and d . episode : a pregnant woman with thirty-eight week of gestation was hospitalized in obstetrics clinic with the complaints of fever, malaise, and severe vaginal bleeding. on admission, white blood cell count was /mm , haemoglobin was . g/l, platelet count was /mm . the level of ast was iu, alt iu, lactate dehydrogenase iu, and creatinin phosphokinase iu. the baby was delivered by cesarean section. in serum cchf igm was positive by elisa, and per oral ribavirin was administered after delivery. at the first day of delivery, the clinical and laboratory of findings of the baby were found to be normal. however, on his th day, he died because of massive bleeding. his cchf igm was found to be negative. episode : a pregnant woman with week of gestation was admitted to the hospital. her complaints were fever, malaise, headache, myalgia, nausea, vomiting, diarrhoea, and subconjuntival bleeding. in her laboratory investigation, white blood cell count was /mm , haemoglobin level was . g/l and platelet count was /mm . the level of ast was iu, alt iu, and ldh iu. in her serological analysis cchf igm and cchf virus -pcr was found to be positive. at the twenty six week of gestation in obstetric ultrasound, fetal intraabdominal fluid was visualized and amniocentesis was performed. in serological analysis of amniotic fluid cchfv-pcr was found to be negative. intraabdominal fluid had increased and scrotal edema was visualized at thirty eighth weeks of the gestation. after her vaginal delivery, baby was severely ill and was operated with the diagnosis of necrotizing enterocolitis. his laboratory findings were normal except high white blood cell count. on his fifth day, thrombocytopenia occurred and he died because of massive bleeding. his cchf igm and pcr were negative. conclusion: to our knowledge, these are the first episodes of intrauterine cchf infection. these episodes show that cchfv can transmit through placenta. obstetricians in endemic countries should consider cchf infection among the patients with massive bleeding and thrombocytopenia. objective: to detect the asymptomatic crimean congo hemorrhagic fever virus (cchfv) infections in an endemic area, and calculate the attack and the infection rate. methods: the study was performed in a cchf endemic region. the household members of the index cases were screened for cchfv igg and igm by elisa. the data related to risk exposure were obtained by a structured form. results: eleven index cases were admitted to the clinic, household members of these cases were screened. all the index patients had positive igm or pcr for cchfv. among the household members, three individuals had igg positivity (%), and only one patient had igm positivity. none of the screened individuals had symptoms. the mean age was (sd ), and % of the subjects were female. tick bite was detected a risk factor (p = . ) for cchv infection, whereas patient care and contact with body fluids of the patients were not (p > . ). eighteen patients had the history of tick bite, and became infected ( %), and five ( %) became ill. among the infected eight individuals, five became ill ( %). conclusion: although we consider that some of the patients do not notice tick bite, we can still suggest that the infection rate of the virus is rather high compared to similar diseases. tick bite is the major risk factor, in comparison to exposure to blood and body fluids of the infected cases. results: children were included in our study. distribution according sex was: . % female and . % male. median of age was years (iqr = ). during the follow-up study we recorded years when the number of cases increased. the distribution of cases among the study was: . % in , . % in , . % in , . % in , . % in and . % in . the proportion of paediatric patients also varied from; . % in , . % in , . % in , . % in , . % in and . % in . in panama city we recorded . % of the infants. we detected an increase in the number of patients in the rain season, from may till november. the mean of days between the onset of symptoms and the first blood sample was . days (ds: . ) a second sample was obtained in . % of our infants with an average time of . days (ds: . ). the frequency of classical symptoms related to dengue virus infection was: fever ( . %), severe headache ( . %), chill ( . %) rash ( . %), myalgia ( . %), retro-orbital pain ( . %), arthralgia ( . %), gastrointestinal symptoms ( . %), inflamed pharynx ( . %), cough ( . %), mild respiratory symptoms ( . %) and diarrhoea ( . %). in our infants the symptoms which were detected first were; fever, severe headache, chill, myalgia, retroorbital pain, arthralgia, mild respiratory symptoms, cough and inflamed pharynx. we did not observed differences on clinical features between girls and boys. however, we detected detected significative differences among symptoms when we compared infants who were £ years old with those who were older (p < . ). four of our patients died because of dengue hemorrhagic fever. conclusion: dengue is endemic in panama as in most tropical countries and is one of the world s major emerging infectious disease. more data about this illness are needed to elaborate sanitary programmes which contribute to control this infection. diagnosis of dengue infection by enzyme-linked immunosorbent assay and reverse transcriptionpolymerase chain reaction from oral specimens dengue. salivary elisa has been shown by various investigators to be useful for dengue diagnosis. we sought to perform a pilot evaluation of diagnostic value of elisa and pcr of oral brushes and saliva for dengue diagnosis in adults. methods: adults with acute fever and suspected of dengue infection admitted to our university hospital were enrolled. dengue diagnosis was made by standard elisa using serum or plasma. patients with negative elisa served as controls. buccal mucosal cells were collected for rt-pcr and saliva for both rt-pcr and elisa at least twice, - days apart. our elisa criteria for saliva were single igm > units or single igg > units or -fold increase in igg titre with the second titre > units for secondary dengue infection. criteria for primary dengue infection were the same as secondary infection plus igm:igg ratio of over . . results: cases and controls were enrolled. our country is endemic for dengue and thus there was no primary dengue adult case in this study. as the study was performed in hospitalized patients, most of the first samples were collected one day before or on the day of defervescence. the specificities of either methods and the sensitivity of elisa method for saliva were %. sensitivities were approximately - % for rt-pcr using buccal cells or saliva specimens. however, a combination of rt-pcr results for both types of oral specimens gave a sensitivity of %. the results are summarised in the table. conclusions: collection of oral specimens is less invasive and may be more acceptable in certain situations. a single, acute specimen is adequate for diagnosis by rt-pcr. our specimens, however, were collected late in the course of illness which affected the sensitivity of rt-pcr's. earlier specimens may give a better yield. a study in paediatric patients is needed to assess the value of these methods for primary dengue infection. objective: the aim of this study was to assess the proportion of seropositives against hantaviruses among healthy blood donors. methods: volunteer donors were recruited by the institute of transfusion medicine, representing the demographic situation in the tyrol regarding gender and residence. sera were tested for igg with a commercially available elisa. positive samples were confirmed by a commercially available dot blot which was also used for identification of the serovar. setting: the study area comprises north tyrol (austria, north of the main ridge of the alps), south tyrol (italy) and east tyrol (austria, both south of the main ridge of the alps). south tyrol belongs to the catchment area of the etsch river, which drains into the adriatic, while north-and east tyrol are part of the catchment area of the danube, which drains to the black sea. results: none of samples from the italian part of the study area yielded a positive result, wherein of donors of the austrian part turned out to be seropositive. two patients were positive for hantaan, patients were positive for puumala, one patient was positive for dobrava and one patient had antibodies against hantaan and dobrava. only one of those patients reported extensive travelling abroad. conclusions: evidence was found for the occurrence of hantaviruses in the austrian part of the region covering the catchment area of the danube, but not in the italian part of the study area covering the catchment area of the etsch river. seropositivity to hantaviruses differs by hydrogeographic areas. objectives: canine coronavirus (ccov) is an enveloped, singlestranded rna virus, belonging to group i coronaviruses within the family coronaviridae. two different ccov genotypes have been recognised, that are designated ccovs type i and type ii on the basis of their genetic relatedness to feline coronaviruses (fcovs) type i and type ii, respectively. ccov is usually responsible for mild, self-limiting infections restricted to the enteric tract. we report the molecular characterisation of a pantropic variant of ccov that caused fatal disease in pups. methods: ccov type ii strain cb/ was isolated from an outbreak of fatal disease affecting seven dogs housed in a pet shop in apulia region, italy and characterised by fever, lethargy, inappetance, vomiting, haemorrhagic diarrhoea, neurological signs, and severe lesions in the parenchymatous organs. in all tissues, ccov antigen was detected by immunoistochemistry and ccov type ii rna was identified by genotype-specific realtime rt-pcr. the ' end of the genome of strain cb/ was determined by amplification of seven partially overlapping fragments. the pcr-amplified products were subjected to direct sequencing and the obtained nucleotide (nt) sequences were assembled and analysed using the bioedit software package and the ncbi's and embl's analysis tools. genbank accession number dq was assigned to the sequenced . -kb fragment. the inferred amino acid sequences (aa) were compared to the analogous proteins available in the online databases. results: the structural proteins s, e, m, n of strain cb/ displayed a high degree of aa identity to the cognate orfs of ccov type ii, although the s protein showed the highest identity to type ii fcovs. while the nonstructural protein (nsp) a had the same length of known ccovs, the nsp b was -aa shorter than expected due to the presence of a -nt deletion at position and to a frame shift in the sequence downstream the deletion that introduced an early stop codon. conclusions: association of strain cb/ to a severe, fatal disease of dogs, together with virus isolation from organs with remarkable lesions, strongly suggests that this virus has changed the tropism, acquiring the ability to spread from the enteric tract to the internal organs. by sequence analysis of the viral genome, the only striking change was the truncated form of nsp b, but the role of the deletion in the orf b in determining the patho-biological change deserves more in-depth investigation. objectives: to perform a surveillance study for sars coronavirus (sars-cov)-like virus in non-caged wild animals from the wild of hong kong special administrative region (hksar). methods: from summer to spring , bats, rodents and monkeys from locations in hksar were captured. nasopharyngeal and anal swabs and blood samples were collected and tested for sars-cov-like virus rna by rt-pcr using conserved primers targeted to a -bp fragment of the rna-dependent rna polymerase (pol) gene. the complete genome of the sars-cov-like virus from bats (bat-sars-cov) was sequenced using rna extracted from three anal swabs of three bats as template. phylogenetic tree construction was performed using neighbor-joining method with growtree using jukes-cantor correction. prediction of signal peptides and cleavage sites was performed using signalp, transmembrane domains using tmpred and tmhmm, potential n-glycosylation sites using scanprosite and protein family analysis using pfam and interproscan. antibodies were detected using a recombinant bat-sars-cov nucleocapsid protein enzyme immunoassay and neutralization assay for human sars-cov. results: we identified a coronavirus closely related to sars-cov (bat-sars-cov) from ( %) of anal swabs of wild chinese horseshoe bats by rt-pcr. sequencing and analysis of three bat-sars-cov genomes from samples collected at different dates showed that bat-sars-cov is closely related to sars-cov from humans and civets. phylogenetic analysis showed that bat-sars-cov formed a distinct cluster with sars-cov as group b coronaviruses, distantly related to known group coronaviruses. most differences between the bat-sars-cov and sars-cov genomes were observed in the spike gene, orf and orf , which are the regions where most variations were also observed between human and civet sars-cov genomes. in addition, the presence of a -bp insertion in orf of bat-sars-cov genome, not in most human sars-cov genomes, suggests that it has a common ancestor with civet sars-cov. antibody against recombinant bat-sars-cov nucleocapsid protein was detected in % of chinese horseshoe bats using an enzyme immunoassay. neutralizing antibody to human sars-cov was also detected in those with lower viral loads. conclusion: our data support the existence of sars-cov-like virus in chinese horseshoe bats in hksar. noroviruses are genetically heterogeneous and form at least genotypes within genogroups, gi, gii, giii, giv, and gv, based on the capsid genes. human novs cause an estimated million cases of illness annually in the united states alone and > % of nonbacterial epidemic gastroenteritis worldwide. porcine calicivirus have been found to be genetically similar to human gii novs or to sapoviruses but calicivirus rna has been detected at low frequency by rt-pcr in adults or fattening pigs. the close genetic relationships between human and porcine novs raise public health concerns regarding their potential for zoonotic transmission and as a potential source of new epidemic human strains. methods: a total of faecal samples of nursing and weaning piglets with enteritis were collected during - in porcine herds in italy. an additional samples were include in the analysis, that had been collected during a rotavirus (rv) surveillance study in - , all which tested positive to rv by electron microscopy and by rt-pcr. viral rna was extracted by the guanidine thiocyanate/glass milk method to eliminate enzyme inhibitors. primer pair con -con , targeted to the vp outer capsid protein, was used for rv detection. a degenerated version of primer pair / was used for nv detection, that targets a conserved region in the rnapolymerase. results: nov rna was detected in / of the screened samples, while rvs were detected in / samples. mixed infections nov+rv were found in samples. screening of the rv positive samples allowed detection of mixed infections with novs. conclusions: in previous investigations novs were detected in of , normal slaughtered pigs in japan, in of pooled pig faecal samples of -to -month-old fattening pigs in the netherlands and in out of healthy adult and finisher pigs in the united states. interestingly, in this study a high rate of positivity to novs ( / ) was found in nursing and weaning piglets with diarrhoea, a finding that may suggest a higher frequency of infection by nov in young pigs or an association between nov infection and occurrence of enteric disease. altogether, these findings demonstrate that novs are common in porcine herds in italy and provide new insights into the ecology of novs. detection of calicivirus genome in calves using ni/e primers m. mahzounieh, t. zahraeisalehi, e. moghtadaei khorasgani (shahrekord, tehran, ir) caliciviruses may cause a wide spectrum of disease in animals and are important etiological agent of viral gastroenteritis in humans. members of the family caliciviridae are small nonenveloped viruses to nm in diameter. they possess a single stranded poly adenylated rna genome. caliciviruses have been isolated from mink, dog, cattle and non-human primates. "norwalk-like viruses" (nlvs) are the most common cause of acute non-bacterial gastroenteritis in humans. cattle may be a reservoir of nlvs although never bovine nlvs have been found in humans. in this study, we try to detect enteric caliciviruses genome from faecal samples of dairy cattle herds in shahrekord area using reverse transcriptase polymerase chain reaction (rt-pcr) assays specific for nlvs found in humans. the primers used for pcr amplification were ni and e , which amplify a -bp product for the detection of both genogroups i and ii srsv rna in fecal material. our results showed that nine specimens ( %) were positive. these findings suggest that calicivirus infection is endemic in dairy herds in shahrekord, iran and may be have an important role in calf diarrhea. objectives: reoviruses are non-enveloped, -segmented dsrna viruses. in humans and mammalians three distinct serotypes exist, whose prototypes are strains lang (t ), jones (t ) and dearing (t ). although reoviruses have been isolated both from the enteric and respiratory tract, no diseases has been clearly associated to reovirus infection in humans. the potential association with extra-hepatic biliary atresia, myocarditis, and, above all, neurological and cutaneous diseases require further investigations. reoviruses are ubiquitous and scarcely speciesspecific. reovirus identification is usually based on electronic microscopy or gel electrophoresis and reovirus incidence seems to be very low in humans and most mammalians. in this study, we investigated the presence of reoviruses in dogs by means of molecular methods. methods: one hundred ninety-two rectal samples from dogs with diarrhoea, ocular swabs, nasal swabs and oropharyngeal swabs from dogs with ocular/nasal discharge were subjected to an rt-pcr assay targeting a conserved region of viral genome segment l (primers l -rv /l -rv ). positive samples were characterised by polyacrylamide gel electrophoresis (page), serotype-specific rt-pcr assays targeting segment s and sequence analysis. to increase the sensitivity, a nested pcr using primers l -rv /l -rv was performed on samples tested rt-pcr negative. results: only faecal swabs ( . %) were found positive (rt-pcr product of bp). by using a serotype-specific rt-pcr assay and/or sequence analysis, two strains was characterised as type and the other ones as type . page of viral dsrna confirmed the genetic characterisation. unexpectedly, in secondround pcr faecal samples ( %), ocular swabs and nasal swabs yielded a bp product, while no oropharyngeal swab was positive. conclusions: these data suggest a wider distribution of reoviruses in dogs than previously thought, even if most reovirus infections were detected only by nested pcr. the ability of reoviruses to induce disease in dogs, alone or in synergism with other pathogens, is still unclear, since attempts to reproduce a specific disease in germ-free dogs have given contradictory results. due to their poor species-specificity, reoviruses may be easily transmitted from animals to humans (and vice-versa). further studies are required to understand reovirus ecology and their potential zoonotic impact. objectives: parvovirus b is a member of a family parvoviridae. on the basis of genetic distances and evolutionary relationships, human parvoviruses are divided into three genotypes: genotype i corresponding to b -related isolates, genotype ii to lali-related isolates, and genotype iii to v -related isolates. parvovirus b causes a common exanthematous disease in childhood or adult age, arthropathy, hydrops fetalis, various haematological disorders and myocarditis. up to now, we have had no data of the prevalence of b virus in slovenian population. consequently, we also lack information on the genotypes of parvovirus b that are involved in the patients who suffer from the infection. methods: to gather information of the genetic variants of b virus present in slovenia, we extracted dna from serum samples that were sent for serologic diagnostic of parvovirus b infection and were positive for specific igm in the period from january to june . nearly half of all patients were children and young adults up to years. the ns region of parvovirus b was amplified by the nested pcr (primers pb f , r and pb f , r ). all pcr products were directly sequenced. the results of our study show that dna of parvovirus b was present in all samples that tested positive for specific igm antibodies. after the first round of pcr reaction, samples were positive, and after the second reaction, all samples were positive. altogether unique genotype variants of parvovirus b were identified and all were clustered in the genotype i group of b -related isolates. most of the distinct genetic variants differed in % to % from the sequences deposited in gene bank. the majority of sequences obtained from the b virus epidemic in represents a single variant of genotype i with the gene bank acc. no. aj . we also found that different genetic variants of parvovirus b were circulating in and were % or % identical to the genotype i variant with the gene bank acc. no. z . in our study, we were not able to identify any variants of other rare genotypes (lali or v ). conclusion: parvovirus b dna was successfully amplified from all igm positive serum samples of the patients. the genotype i of parvovirus b is dominating in infections with parvovirus b in slovenia. objectives: great britain has been free of animal brucellosis since (european commission decision / ). the main source of infection for uk residents is through contact with infected material in foreign countries. the objective of this study is to type human samples received in the uk since using variable number tandem repeats (vntr) molecular typing to confirm results obtained by classical typing and relate these results to the suspected source of infection. methods: classical typing is traditionally used and is based on the phenotypic attributes of each strain and biovar. vntr typing is a recently developed molecular method, which is based on short repeats contained in the dna that can be amplified to give a banding pattern specific to each strain. results: results found using both methods are consistent. the results show geographical differences, consistent with observations of strain genotype distribution found in animal brucellosis. conclusion: patient history has been gathered where possible giving information on recent travels. along with results found by classical typing and confirmed by vntr typing we can draw a picture of the sources of infection. these results illustrate the potential of vntr typing as a tool to aid conventional approaches to epidemiological traceback that, in the presence of a suitably comprehensive database of strain genotypes, could help identify the source of an infection. is -fingerprinting of brucella isolates from humans e.stubberfield (surrey, uk) objective: brucellosis is a zoonotic disease usually associated with cattle, sheep, goats and pigs. human infection has been attributed to b. melitensis, b. abortus, b. suis, b. canis and b. maris. although the uk is officially bucellosis free there are a number of human cases due to travel and occupation that are submitted to our laboratory for diagnosis. definitive diagnosis of brucella is by bacteriological culture and microbial tests (classical typing), however these require skilled personnel and the results can be subjective. there are a number of molecular tests that have been developed to assist with diagnosis more rapidly and in some cases to strain level less subjectively. is -fingerpinting is a molecular technique that has proved useful for the identification of brucella isolates to species and in some cases stain level. is -fingerprinting relies on the variable number and location of the is mobile genetic element found in all brucella isolates. method: brucella isolates from humans have been tested. genomic dna was extracted, digested using restriction endonuclease ecor , and electrophoresed. southern blotting was performed, hybridising with a dig-labelled is probe. results: the number of brucella is copies range from to more than . brucella melitensis remains the most commonly acquired brucella species of travellers, while occupational infections have included b. abortus isolated from cattle farmers and b. suis associated with pig butchers. two marine brucella strains have been isolated originating from an occupational perspective (a laboratory worker) and a natural setting from an unknown source. unusual patterns have been observed, of which are unique. one of the new patterns has been observed only in isolates originating in east african countries. conclusion: although the diagnosis of brucella to species and strain level is not essential for the treatment of human brucellosis, it is useful for epidemiological studies. is fingerprinting is able to identify the three biovars of b. melitensis, many other techniques do not offer this capability, because of this it may be a useful test in epidemiological studies. this method remains an important diagnostic tool for brucella identification. rapid diagnosis of brucellar epididymo-orchitis by real-time pcr assay in urine samples objectives: to study the diagnostic yield of a real-time pcr assay in urine samples for the rapid diagnosis of brucellar epididymo-orchitis, in comparison with conventional microbiological techniques. methods: ten consecutive patients with brucellar epididymoorchitis were included in the study. the diagnosis of brucellosis was established according to one of the following criteria: first, isolation of brucella spp in blood or any other body fluid or tissue sample or, second, the presence of a compatible clinical picture together with the demonstration of specific antibodies at significant titers or seroconversion. epididymo-orchitis was diagnosed in patients with scrotal enlargement, swelling and pain not due to other causes.for dna amplification we used a sybr green i lightcycler-based real-time pcr assay. the assay amplifies a bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein (bcsp ). the pair of nucleotide primers b ( ' tgg ctc ggt tgc caa tat caa ') and b ( ' cgc gct tgc ctt tca ggt ctg ) were used in the amplification process. after dna amplication, we performed melting curve analysis to verify the specificity of the pcr products. in order to study the specificity of the technique, all the samples from the patients with brucellosis were paired with an equal number of samples from controls with urinary tract infection. (e. coli four cases, k. pneumoniae two cases, p mirabilis two cases, and c. freundii and p. aeruginosa one of each). results: the mean age was . years (range - ). the duration of the symptoms prior to diagnosis was . ± . days (range: - ). b. melitensis was isolated from blood cultures in nine cases ( %). wright's seroagglutination was negative or inconclusive in % of cases. brucella was isolated from urine in only one case whereas real-time pcr assay in urine was positive in nine ( %) cases and the results were available in four hours, whereas the mean time to availability of the final blood culture results was . days (range . - days). real-time pcr was negative for all the control samples from patients with urinary tract infections. conclusion: sybr green i lightcycler-based real-time pcr assay in urine samples is highly sensitive and specific, easy to perform and could provide the clinician with the results in under five hours. the technique could be a practical and useful tool for the rapid diagnosis of genitourinary complications of human brucellosis. objectives: although the united kingdom remains brucellosisfree, there are more than , new cases of human brucellosis reported each year according to the world health organisation. uk residents returning from worldwide travel may have encountered exposure through contact with infected animals and animal products such as dairy produce and meat. phenotypic characterisation or classical methods remain the definitive diagnosis though require skilled personnel and have their limitations. the increasing range of molecular techniques can aid the rapid detection and characterisation of brucella species and their biovars and may have significance in epidemiological studies. methods: a study of brucella reference and field strains of mainly human isolates from different geographic locations were analysed for diversity of their genes encoding the outer membrane proteins (omps) , a and b. pcr products of the three genes digested with seven restriction enzymes were analysed for polymorphisms. results: a re-occurring unique pattern profile seen only in human isolates was observed originating in some european countries and beyond. a growing database of strain types giving a recent overview of brucella infection of humans of many countries. conclusions: molecular typing methods may have an advantage over classical typing concerning brucella melitensis, the most common brucella infection of humans. the characterisation of human brucella isolates may be useful in epidemiological studies for a variety of purposes. objectives: a study to demonstrate the rapid detection and speciation of campylobacter jejuni and of campylobacter coli isolates directly from enrichment broth using a taqman Ò assay. single nucleotide polymorphism analysis of mapa positive strains was used for rapid identification of c. jejuni clonal complexes. methods: thermotolerant campylobacter species were initially confirmed by culture according to the modified draft iso method, where water samples were filtered through . mm pore size nylon membrane. the filters were transferred to selective enrichment in preston broth to improve their recovery and therefore detection of any campylobacter cells present. dna was extracted directly from the enrichment broth culture for real-time detection of c. jejuni and c. coli using the taqman Ò . samples, which were map a positive were, further characterise by single nucleotide polymorphism profiling for rapid recognition of c. jejuni clonal complexes. results: environmental samples, which were confirmed by culture were also map a positive by taqman Ò . snp profiling of mapa positive isolates identified clonal complexes, which are predominantly contained in isolates of human disease and chicken. conclusions: this study has demonstrated the feasibility of rapid detection and identification of c. jejuni and c. coli following short enrichment incubation using a taqman Ò assay. a rapid turnaround time of between - h per batch of samples was achieved. snp profiling offers important epidemiological grouping at strain level, enabling accurate and phylogenetically valid strain identification for c. jejuni, which may have important host associations for tracing sources of infection and consequently improve public health responses. objectives: campylobacter jejuni and c. coli are recognized as the most common causes of acute bacterial gastroenteritis in humans, c. jejuni being the predominant species in most developed countries. the hippurate hydrolysis test is widely used to differentiate c. jejuni from other campylobacter species. about % of c. jejuni isolates fail to hydrolyze hippurate under laboratory conditions. molecular methods represent an alternative to the phenotype-based methods. we tested two multiplex pcr assays for species identification of human campylobacter strains and compared the results with the hippurate hydrolysis test. methods: campylobacter strains isolated from patients were tested for hippurate hydrolysis with rosco diagnostic tablets. hippurate-negative and hippurate-positive strains were selected for two multiplex pcr assays. one pcr-method was based on distinctive ceue-genes of c. jejuni and c. coli, the other pcr-method detected genes from five major clinically relevant campylobacter species: hipo from c. jejuni, glya from c. coli, c. lari and c. upsaliensis, sapb from c. fetus subsp. fetus, and s rrna gene from campylobacter spp. as an internal validation control. results: the c. jejuni hipo gene was detected in all of the hippurate-positive strains and of the hippurate-negative strains. the c. coli glya was detected in of the hippuratenegative strains. in one hippurate-negative strain, sapb from c. fetus subsp. fetus was detected. species-specific genes were detected in of the strains with the ceue-based pcr assay. c. jejuni ceue was detected in hippurate-positive and hippurate-negative strains. c. coli ceue was detected in hippurate-negative strains. conclusion: all hippurate-positive strains were identified as c. jejuni. of the hippurate-negative strains, % were identified as c. coli, whereas % were identified as c. jejuni and one strain as c. fetus subsp. fetus. the results of the two pcr assays were concordant, although some strains could not be identified with the ceue-based pcr assay. the results suggest that molecular species identification should be performed on hippuratenegative strains after the hippurate hydrolysis test for accurate species identification. multiplex-pcr is quick and easy to perform. using the pcr assay that simultaneously detects five campylobacter species also diminishes the need for further phenotypic testing. phenotypic typing of cryptosporidium species isolated from children in kuwait: a role in unique transmission j. iqbal, p. hira (safat, kw) background: cryptosporidiosis is recognized worldwide as a significant cause of diarrhoeal diseases in both adults and children especially in children less than years of age. objective: cryptosporidium spp. isolated from young children in kuwait were characterized at the molecular level to understand the transmission of infection. the study was approved by the ethical committee, faculty of medicine, kuwait. methodology: over a period of years, faecal specimens from kuwaiti children with persistent diarrhoea found to be positive for cryptosporidium spp. by microscopy were genotyped and sub-typed with a small subunit rrna-based pcr-restriction fragment length polymorphism analysis. informed consent was taken from all individuals included in the study. results: the median age of infected children was . years, and the majority of the infections (> %) occurred during the cooler months january-april, indicating a marked seasonal variation. more than % of the children with cryptosporidiosis had only cryptosporidium infection. socio-demographic information did not reveal any particular mode of transmission of infection. genotyping of the organisms isolated showed that ninety-two ( %) of the children had c. parvum, ( %) had c. hominis, and ( %) had both c. parvum and c. hominis. altogether, subtypes of c. parvum and c. hominis were observed. objectives: the intracellular respiratory pathogen chlamydophila pneumoniae (cp) might be involved in the pathogenesis of atherosclerosis. several studies have demonstrated a serological association between cp and cardiovascular disease and dna from the bacteria has been found in various atheromatous vessels. after infection in the respiratory tract, cp is believed to be disseminated systemically within alveolar macrophages. the prevalence of cp within peripheral blood mononuclear cells (pbmc) has in some studies been shown to be higher in patients suffering from cardiovascular disease than in control patients. we investigated the presence of cp dna in aortic heart valves and pmbc in patients ( men; women; mean age years) undergoing aortic valve replacement because of aortic stenosis. also, the presence of cp mrna was investigated in the sclerotic aortic heart valves as a marker of viable bacteria. methods: dna was extracted from aortic valve biopsies and pbmc using the qiaamp dna mini kit (qiagen). mrna and dna were extracted from another piece of the same biopsy using trizol (invitrogen). real-time pcr directed against the chlamydia momp gene was used to detect cp-specific dna and mrna. patient sera were tested for cp-specific igm, igg and iga antibodies by the microimmunofluorescence technique. results: cp dna was found in aortic heart valves from % ( / ) of the patients and in pbmc from % ( / ) of the patients. in one patient cp dna was found in both pbmc and heart valve. no patient had cp-specific igm antibodies. in patients that were pcr-positive for cp dna in the aortic heart valves, % had igg ‡ : and % had iga ‡ : . in patients that were pcr-negative in the aortic heart valves, % had igg ‡ : and % had iga ‡ : . cp-specific mrna in aortic heart valves will be presented on the poster. conclusion: cp-specific dna was found in sclerotic aortic heart valves from % of patients undergoing aortic valve replacement. this confirms previous investigations supporting a role for cp in the pathogenesis of aortic valve sclerosis. the prevalence of cp in pbmc was % which is comparable to that reported in healthy blood donors and lower than that recorded in patients suffering from other cardiovascular diseases. if the bacteria are involved in the pathogenesis of aortic sclerosis they have likely been spread to the aortic valve long before the patient is in need of surgery because of the stenotic valve. introduction: diarrhoea is one of the most common causes of morbidity and mortality among young children in developing countries. diarrheagenic e. coli strains include several emerging pathogens of worldwide public health. six important categories are entero-aggrigative e. coli (eaec), entropathogenic e. coli (epec), enterotoxigenic e. coli (etec), enterohemorrahgic e. coli (ehec), entroinvasive (eiec) and shigatoxin-producing e. coli (stec). this study investigated the role of different diarrheagenic e. coli in iranian children with acute diarrhoea by molecular methods and the antibiotic susceptibility of isolated strains. methods: from april to january , one thousand eighty five children with acute diarrhoea in tehran hospitals in were enrolled in the study. the fecal samples were cultured on macconkey for conventional bacterial pathogen and sorbitol macconkey agar for non sorbitol fermenting phenotype, than they were incubated in ordm;c. the primary stool cultures were subjected to six different pcr reactions targeting stx and stx gene, heat-labile enterotoxin (lt) producing gene, heatstable enterotoxin (st) producing gene, eae gene and pcvd plasmid. the kirby -bauer disc diffusion method was used for antibiogram of isolated strains from different diarrheagenic e. coli by different antibiotics. results: two hundred seventy one diarrheagenic e. coli strains were detected. stec was the most prevalence with ( . %). the frequency of other strains was . %, . %and . % for etec, eaec and epec, respectively. out of stec isolated strains (% . ) had stx or stx gene, and strains had stx and stx gene. the eae gene was found in ( . ) stec strain. out of tested strains, ( . %) were resistance to ampicllin and cefalotin, and ( %) to streptomycin. conclusion: in this study stec was the most frequent associated with diarrhoea. the strong association between use of antibiotics and colonization with antibiotic resistant e. coli, suggest a major role for selection of resistant strains while using antibiotics. the existence of other unknown intestinal adherence factors has been suggested by the isolation of stec strains that lack the eae gene but are still associated with bloody diarrhoea or hemolytic ureamic syndrome (hus). since there is no specific treatment, there is an urgent need for effective preventive measures based on detailed understanding of the epidemiology of stec infections. identification of shiga toxin-producing escherichia coli in raw beef using dna hybridization with digoxigenin-labelled probes and multiplex pcrs m. weiner, j. osek (pulawy, pl) shiga toxin-producing escherichia coli (stec) is an important cause of bloody diarrhoea, haemorrhagic colitis, haemolytic uremic syndrome and thrombotic thrombocytopenic purpura. transmission of stec occurs through consumption of contaminated food, especially meat, dairy products and water. objectives: to develop a three-steps procedure based on two multiplex pcrs and dna hybridization with digoxigeninlabelled probes for identification of stec in raw beef. methods: beef samples inoculated with different number of e. coli o :h cells were incubated in tsb medium at °c for h. the cultures were then transferred to tsb with mitomycin c and incubated for another h. the resulted cultures were used as a source of dna template. the mpcr- was established to identify shiga toxins genes (conserved sequence). the positive culture samples were subjected to dna hybridization with dig-labelled probes as follows: the culture was diluted and inoculated onto agar plates supplemented with tergitol Ò and incubated at °c for h. then, the nylon membranes were put on agar plates, carefully removed and incubated in denaturation, neutralisation and equilibration solutions following incubation with the stx-specific dig-labelled probes, anti-dig conjugates and finally developed with enzyme substrates (bcip and nbt). dark spots visible on the membranes were compared with the respective bacterial colonies on the original agar plates. the corresponding bacterial colonies were isolated and characterized using the mpcr- test which allows amplification of stx (shiga toxin type ), stx (shiga toxin type ), rfbo (e. coli o ) and flich gene (h antigen). an internal control of amplification (e. coli s rrna gene) was also included in both mpcr tests. results: the first mpcr resulted in two amplification products: bp for stx and bp for s rrna genes. the positive meat samples were further tested with dna probes and positive colonies were then characterized with the second test (mpcr- ), generating the amplicons either of bp (stx ), bp (stx ), bp (rfbo ), bp (flich ) or bp ( s rrna). the specificity of this procedure was confirmed by testing e. coli o :h , o :h-and non-stec bacteria. the sensitivity of the method was estimated as cfu/g of meat. conclusion: the obtained results demonstrated the high specificity of the procedure developed and the possibility of using it for identification of shiga toxin-producing e. coli in raw beef. correlation between virulence pattern, phylogenetic group and extended spectrum betalactamases genes in escherichia coli strains isolated from blood cultures m. damian, c. usein, d. tatu-chitoiu, s. ciontea, d. jardan, a. palade (bucharest, ro) e. coli, heterogeneous species consisting of commensal and pathogenic strains, is causing a broad spectrum of human diseases, including extra intestinal and enteric infections.the strains isolated from invasive infections were documented to be carriers of a large number of genetic structures coding for virulence, as well as for resistance to antimicrobial agents. the aim of this study was to evaluate the virulence of strains in comparison with the presence of esbl genes and their distribution among the different phylogenetic groups. a total of e. coli strains, isolated from blood cultures, in hospitalised patients, adults and children, were screened for virulence factors-encoding genes (pap, sfa/foc, afa, hly, cnf, aer and fimh), for genes encoding resistance to extended spectrum betalactam antibiotics (bla shv and bla tem genes) and the appurtenance to one of the main four phylogenetic group based on presence or absence of markers chua, yjaa and tspe .c three strains, negative for all virulence genes, were included in the phylogenetic group a. ten strains, which were positive for five or six virulence genes, were identified as b group. no matter the phylogenetic grouping, the remaining strains possessed at least one virulence gene. no strain was pcr positive for all seven virulence genes targeted. among the strains which were positive in the double disk test, strains exhibited both bla shv and bla tem genes and strains only bla tem gene. restriction with pst i and dde i and sequencing of the amplicons were performed in order to identify the type of esbl gene expression product. taking into account the link between phylogenetic group and virulence, we obtained a good correlation for the bacteremic e. coli strains analysed, but there was no relationship with the production of esbls. isolation of shiga-toxin producing escherichia coli from meat samples, phenotypic and genotypic characterisation of isolated strains f. baghbani-arani, f. jafari, m.r. zali, s. salmanzadeh-ahrabi (tehran, ir) objective: shiga-toxin producing escherichia coli (stec) is an emerging foodborne pathogen of worldwide public health importance. this bacterium has been reported as an etiological agent of many outbreaks and sporadic cases. definition of the diversity and antimicrobial susceptibility of (stec) may be helpful in the management of sporadic cases and outbreaks. studies in different countries show that food items maybe contaminated by this pathogen. the present study was carried out to determine the frequency of contamination of meat samples by stec collected in tehran as well as defining genotypes, serotypes, antibiogram susceptibility patterns and molecular diversity of isolated bacteria. methods: from july to june , beef samples were collected from different part of tehran. a grams of each samples was enriched in ec broth and subculture on mac-conkey agar. dna was extracted from a loop full of bacteria taken from primary first streaking area of mac-conkey agar and was subjected to three different pcr reactions targeting stx , stx and eae genes. as much as colonies required were tested for finding the colony responsible for positive results in the first pcr. antibiogram susceptibility patterns of isolated strains were determined by standard disk diffusing method. the antimicrobial agents were used at this study. all isolates were serotyped by slide agglutination test using standard antisera (mast groups) subtyping of strains was done with rapd-pcr by primer. results: among samples, ( %) samples were positive and their genotypes were as follow: ( . %) stx +, stx -, eae-, ( %) stx -, stx +, eae-, ( . %) stx -, stx + and eae+. ( . %) stx +, stx +, eae-, ( . %) stx +, stx +, eae+. among these positive samples strains were isolated. according to the antibiotic susceptibility tests, all isolates were resistance to erythromycin (e) and oleandomycin (ol), and were sensitive to imipenem (i); gentamicin (g) norofloxazin (nx) enterofloxazin (ex) ciprofloxazin (cf) and ceftazidim (ca). in otyping and htyping the most frequency were o ac and h serotypes. analysis of isolates by rapd-pcr yielded different patterns. conclusion: our results show that contamination of meat samples by stec is a life-threatening health problem. combinational analysis of antibiogram susceptibility patterns and serotypes with rapd-pcr patterns can aid to survey the characteristics of stec strains. factors affecting the conjugative transfer of plasmid pip in enterococcus faecalis a.m. al-qurashi (dammam, sa) objectives: factors which are known to influence plasmid transfer were studies using the conjugative plasmid pi , which encodes erythromycin resistance, in enterococcus faecalis. methods: the donors strains streptococcus a agalactiae v (group b) is resistant to in rifampicin and fusidic acid, non hemolytic and b-lactamase-negative. it contains the broad host range plasmid pi , which confers resistance to erythromycin and chloramphenicol. the recipient is enterococcus faecalis strain jh - group d. results: transfer of pip occured on a agar, on filters and in broth cultures at relatively high densities ( - bacteria/ml). transfer frequency was largely unaffected over a wide range of temperatures ( - °c). the ph of the medium, in the range ph - had little effect on the transfer frequency. log phase cultures and donor: recipient ratios of : - : were required for optimal for plasmid transfer. conclusion: factors which modified the transfer efficiency of the conjugative plasmid pip were mating media, solid or liquid environment, mating time, mating temperature, selection temperature, growth temperature of donor and recipient of prior to mating, ph culture age, and donor/recipient cells ratio, to obtain a better understanding of this plasmid and its transfer process will help understand what role they may have in the dissemination resistance among streptococcal and enterococcal populations. enterococcus faecium blood-culture isolates collected during a five-year period h. billströ m, Å . sullivan, b. lund (stockholm, se) background: enterococcus faecalis and enterococcus faecium have during the last years become a significant nosocomial problem. this could be due to the enterococcus hardy nature combined with intrinsic and acquired antibiotic resistance. since most individuals harbour enterococci in their normal intestinal microflora there has been a discussion regarding the origin of these isolates. during the last ten years the isolation ratio between e. faecalis and e. faecium have shifted from : to : . this could be because of increasing antibiotic resistance among infectious e. faecium isolates compared to infectious e. faecalis ones. it is possible that this increase also depends upon different virulence genes such as enterococcal surface protein (esp), hyaluronidase variant gene (hylefm) and e. faecalis antigen a variant (efafm). objectives: the objectives in this study were to determine the presence and frequencies of seven different enterococcal virulence genes in infectious isolates. further objectives were to see if the number of virulence genes in these isolates vary or increase over time. methods: a total of strains isolated from bacteraemia patients during year - at the karolinska university hospital, huddinge were used. all isolates were screened for seven different virulence genes using a multiplex pcr. these seven virulence genes were aggregation substance (asa), cytolysin (cyt), collagen binding protein (ace), e. faecalis antigen a variant (efafm), enterococcal surface protein (esp), gelatinase (gel) and hyaluronidase (hyl). results: according to the results about half of all isolates were esp-positive. the prevalence of the other virulence genes asa, efafm, gel and hyl were detected, but in low frequencies (< %). conclusion: it seems like the esp gene is the most dominant virulence gene in e. faecium isolates. the occurrence of virulence traits in these isolates further indicates that the potential to cause infection is potentiated among this enterococcal population the data from this investigation supports the hypotheses that enterococci causing infection in hospitalized patients are probably of nosocomial origin rather than endogenous. objectives: the ability of l. monocytogenes to tolerate alkaline stress is of particular importance, as this pathogen is often exposed to such stress in food processing environments cleaned with alkaline detergents or in the mildly alkaline ph values which prevail within engulfing phagolysosomes. this study aims to investigate the alkaline tolerance response (altr) in listeria monocytogenes s using dna microarray technology. knowledge of the alkaline-induced stress response will be useful in understanding how this pathogen tolerates alkaline stress. methods: transcription profiling of l. monocytogenes s was carried out at , and min at high ph in order to capture an early, an intermediate and a prolonged expression response to alkaline stress using oligo arrays from the pathogen functional genomic resource centre. to verify the microarray results the regulation of some ph stress response genes were confirmed by real time quantitative polymerase chain reaction (rt-pcr). results: about genes were upregulated and genes (of open reading frames represented on the arrays) were down regulated at least . fold upon alkaline shock. many of the repressed genes encode enzymes that are involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating a metabolic adjustment of the cells to the high ph. notably, the strongest alkaline-inducible genes were involved in the membrane transport systems. conclusion: the analysis of the data revealed that cells sense and respond to alkaline stress with an extensive program of changes in gene expression. interestingly, there is a strong correlation between the altr and virulence gene expression. comparison to various microarray data already in the literature revealed similarity between the response to alkaline stress and the transcriptional response to stresses such as osmotic shock. engineering improved listerial stress tolerance "with a twist"! r. sleator, c. hill (cork, ie) objectives: to engineer listeria monocytogenes strains with a significantly improved ability to tolerate stresses encountered in the external environment and during gastrointestinal transit, thus, improving listeria's efficacy as a potential vaccine and drug delivery platform. methods and results: using a directed evolution approach, based on a random mutagenesis strategy involving the e. coli xl -red mutator strain, we generated a mutant variant of the listerial betl gene (designated betl*), encoding a secondary betaine uptake system. the mutant betl* promotes a dramatic increase in resistance to a number of biologically relevant stresses when expressed in a variety of different surrogate hosts. using a luciferase (lux) reporter system in combination with the ivis imager system (xenogen corporation, alameda, ca), we tracked betl* expression, in real time, both in vitro under various environmental stresses and in vivo in animal models of infection. in each case strains expressing betl* demonstrated a marked improvement over those expressing wild type betl, both in terms of gene expression and bacterial growth. sequence analysis of the mutated gene revealed a single nucleotide deletion in the spacer region between the - and ) promoter elements upstream of the betl coding region. this deletion presumably introduces a conformational 'twist' in the putative promoter, thereby increasing its transcriptional output. furthermore, the betl* mutation appears to counter the heretofore unreported 'twisted' cell morphology observed using scanning electron microscopy of l. monocytogenes grown at elevated osmolarities. conclusions: it is possible to selectively improve genes required for bacterial stress survival both inside and outside the host. such mutated genes systems may ultimately be used for the construction of more physiologically robust bacterial based vaccine and drug delivery platforms. a.r. samarbaf-zadeh, s. tajbakhsh, s.m. moosavian (ahwaz, ir) introduction: peptic ulceration following infection of stomach with h. pylori is a common disease. accurate and rapid detection of the bacteria can lead to implementation of appropriate treatment and recovery. this research was undertaken to evaluate the sensivity and specificity of fluorescent in-situ hybridization (fish) in the detection of h. pylori in patients who were suffering from dyspepsia. methods: for this purpose, one hundred gastric biopsy samples taken from antrum and corpus of stomach by endoscopy were tested by fish and compared with conventional culture method complemented with biochemical tests. results: fish detected h. pylori in clinical samples while conventional method detected samples. the sensivity and specificity of fish for detection of h. pylori were calculated as % and % respectively. conclusion: the findings of this study suggest that fish is a highly suitable and rapid method for diagnosis of h. pylori, especially when the samples are taken from the antrum and the corpus of the stomach this technique potentially can be applied routinely for detection of this bacterium in clinical samples. objective: numerous studies have demonstrated that h. pylori is ubiquitous; approximately % of the world's population is infected with the organism. gastroduodenal diseases associated with h. pylori infection are manifested principally in adults. however, it's usually during chilhood that the infection is acquired, and it is possibile that mucosal and humoral responses at this time may determine, at least in part, the course of the natural infection. our study will describe the prevalence of the h. pylori oral carriage in children resident in bari, south of italy, using the pcr method. methods: the evaluation was performed in children, with ages ranging from to years, from primary school district of local health unit of bari, italy (ausl ba/ ). the school and the class have been selected using the cluster sampling method. a standardized questionnaire was used to verify socio-economic standard, hygiene and history of previous gastrointestinal disorder. a standard full-mouth examination was made to detect periodontal diseases, then dental plaque and saliva collected from children were placed in pbs and transported in laboratory. h. pylori infection status was checked by pcr method. dna was extracted from oral samples by the boiling method and evaluated for the presence of h. pylori caga and urea genes using commercial kit (ab analitica, padova). results: a total of children ( females and males) partecipated to the study. the presence of gene coding for caga was found in children ( %), but gene urea was detected only in ( %). the bacteria was detected in saliva, supragingival and subgingival plaque, suggested that these sites may be considered reservoirs for h. pylori in ureasi-positive patients. there was statistically significant relationship between who didn't wash their hands frequently and the presence of urea gene (o.r. . ). conclusions: current knowledge implies that acquisition of h. pylori seems to occur predominantly in childhood and that once acquired the infection persists life-long in most infected subjects. it has been reported at a worldwide level that h. pylori infection prevalence in children varies between % and % and increases with low socio-economic and educational levels and age. the results of this study suggest that oral carriage of h. pylori may play a role in the transmission of infection and that the hand may be instrumental in transmission. the role of helicobacter pylori in otitis media with effusion t. yilmaz, m. ceylan, y. akyon, o. ozcakir, b. gursel (ankara, tr) objectives: otitis media with effusion (ome) is such a common disease of childhood and its pathogenesis still remains unsettled. pepsinogen and pepsin has been shown in the middle ear fluid of patients with ome, indicating that gastric juice could reach as far as middle ear. if gastric juice could enter the middle ear, helicobacter pylori, a common inhabitant of gastric juice and mucosa, would also be expected to be found in the middle ear of patients with ome. the objective of this study was to evaluate possible role of helicobacter pylori in pathogenesis of otitis media with effusion. methods: the study group consisted of children who are to undergo bilateral ventilation tube insertion, adenoidectomy, tonsillectomy with a diagnosis of ome, adenoid hypertrophy and chronic tonsillitis. the control group consisted of children who are to undergo adenoidectomy, tonsillectomy with a diagnosis of adenoid hypertrophy and chronic tonsillitis. for the study group, middle ear fluid was aspirated and a small biopsy was taken from the promontorium mucosa. for the control group, myringotomy was done and a small biopsy was taken from the promontorium mucosa. for both groups, mm deep tissue specimens were obtained from tonsil and adenoid. for all the specimens taken from the patients, culture and a nested-pcr were performed to show helicobacter pylori. results: middle ear fluid culture was positive for h. pylori in patients and mucosa culture was positive in patient only. in the control group middle ear mucosa cultures were always negative. when culture and pcr results were combined together; the middle ear was positive for h. pylori in patients in the study group and in patients in the control group. this difference was statistically significant. h. pylori presence in the tonsillar and adenoid tissues by culture and pcr was also significantly more frequent in the study group compared to the control group. conclusion: this study is the first to grow h. pylori in the middle ear in ome. significantly increased colonization by h. pylori of the middle ear, tonsillar and adenoid tissue in patients with ome indicates that the bacteria reaching the middle ear through gastroesophageal reflux might be involved in the pathogenesis of ome. for ome cases resistant to medical treatment it may meaningful to evaluate the patient for gastroesophageal reflux and h. pylori. distribution of the serine-aspartate repeat protein-encoding sdr genes among nasal carriage and invasive staphylococcus aureus strains objectives: this study was designed to examine the distribution of the sdr genes among nasal carriage and invasive staphylococcus aureus strains as well as methicillinsensitive s. aureus (mssa) and methicillin-resistant s aureus (mrsa). methods: the presence or absence of the sdr genes using dna from s. aureus strains was determined by a novel triplex pcr procedure. the two-tailed fisher's exact test was used to analyse the distribution of the sdr genes among s. aureus strains originating from different hosts. p values less than . were considered a statistically significant difference. results: the sdr locus was found in all investigated s. aureus strains although in strains it contained only the sdrc gene (sdrd -sdre-). the sdrc + sdrd -sdre-gene profile was exclusive to mssa strains (fisher's exact test; p = . ) and was not found in the strains collected from bone infections (p = . ). we also found a strong association between the presence of the sdrd gene and mrsa strains (p < . ). conclusion: our findings suggest that mssa strains with the newly uncovered sdrc + sdrd -sdre-gene profile have a substantially decreased potential to establish bone infection. sequencing of luks-pv and lukf-pv in methicillin-sensitive and methicillin-resistant staphylococcus aureus of diverse genetic backgrounds in a swedish county c. berglund, b. sö derquist (Ö rebro, se) objectives: community-aquired methicillin-resistant staphylococcus aureus (ca-mrsa) have been reported to carry the loci for panton-valentine leukocidin (pvl) in high frequency. the aim of this study was to describe variations within the pvl genes (luks-pv and lukf-pv) in methicillinsensitive and methicillin-resistant s. aureus of diverse genetic backgrounds. methods: twelve pvl-positive s. aureus were characterised by multilocus sequence typing (mlst) and mrsa also by staphylococcal cassette chromosome mec (sccmec) typing. ten of these were isolated between - in Ö rebro county, sweden. oligonucleotide primers were designed to yield a product size of~ bp including luks-pv and lukf-pv and flanking regions by pcr amplification. cyclic sequencing was performed with several sets of primers to overlap the sequences on both strands and was separated on abi prism Ò genetic analyzer (applied biosystems). the nucleotide sequences were analysed using abi prism Ò autoassembler tm dna sequence assembly . . software and compared using bioedit . . . results: analysis with mlst differentiated the pvl-positive ca-mrsa into six different sequence types (st , , , , and ) with either sccmec type iv, iv c, v or unknown types. six additional sts (st , , , , and new) were detected among the pvl-positive methicillin-sensitive s. aureus. sequencing luks-pv and lukf-pv revealed eight point mutations among these isolates with twelve different origins. five substitutions had occurred in luks-pv and three in lukf-pv. only one substitution was nonsynonymous (histidine fi arginine). conclusion: the pvl-genes were well conserved despite the different genetic origins of the isolates analysed. the pvl is an extracellular product and the genes are not subject to any selective forces and thereby diversify very slowly. additional nonsynonymous mutations might result in a non-functional toxin. the first case of staphylococcus pseudintermedius in humans isolated from an icd lead l. van hoovels, a. vankeerberghen, k. van vaerenbergh, a. boel, h. de beenhouwer (aalst, be) introduction: staphylococcus pseudintermedius is recently described as a new coagulase-positive species from animals (devriese et al., ) . the pathogenic significance of this novel species remains unclear and to our knowledge no human infection due to s. pseudintermedius has been reported to date. here, we present the first isolation of s. pseudintermedius in humans with important clinical significance. patient and methods: a -year old male patient was referred to our centre for an ischemic cardiomyopathy and ventricle tachycardia for which he recieved an implantable cardioverterdefibrillator (icd) in january . in august he presented with complaints of migration of the icd device. clinical examination revealed perforation of the icd pocket. infection was suspected and confirmed by the presence of pus in the pocket. the infected icd was completely removed and several samples (ventricular lead, pus and a tissue sample from the pocket) were sent for culture.bacteria obtained by routine culture were further characterised by phenotypical identification, pastorex Ò staph-plus (biorad), api staph Ò (biomérieux) and phoenix Ò (bd). for molecular analysis, pcrs were performed targeting the nuclease (nuc) and coagulase (coag) genes of s. aureus. additionally, sequencing of the s rrna gene was performed and further analysed using blast. results: staphylococci with identical phenotypical appearance were isolated from of the icd samples (lead and pus). colonies were beta-hemolytic on sheep blood agar, dnase and coagulase positive but clumping factor, mannitol and pastorex Ò negative. biochemical identification by api staph Ò and phoenix Ò gave a presumptive identification of s. aureus with a confidence value of respectively , % and %.the pcrs for the nuc and coag genes were both negative. s rrna gene sequencing resulted in the identification of s. pseudintermedius based on a % sequence similarity with a previous reported sequence by devriese et al. conclusion: this case report describes the first identification of s. pseudintermedius as a significant pathogen in human. growth characteristics and commercial identification systems misidentify the organism as s. aureus. when confronted with an inconsistent phenotypical identification pattern, clinical labs should consider the use of s rrna gene sequencing for final confirmation. characterisation of staphylococcus aureus isolates recovered from dairy sheep farms (agr group, adherence, slime, resistance to antibiotics) e. vautor, m. sabah, g. mancini, m. pepin, h. carsenti-dellamonica (sophia-antipolis, nice, fr) objectives: the purpose of this study was to investigate staphylococcus aureus natural isolates associated with dairy sheep mastitis for epidemiological key features (agr group, adherence, slime production and antibiotics resistance). methods: the s. aureus isolates (n = ) were recovered from a field study in the southeast of france in - ( from subclinical mastitis, from clinical mastitis, from the environment of the dairy sheep farm). a total of thirteen dairy sheep farms, producing cheeses manufactured with raw ewe's milk, were involved. the agr group were determined by multiplex and real-time pcr. the evaluation of adherence and slime production were assessed with methods previously described by christensen et al. ( ) . the susceptibility patterns to antibiotics were determined using the discdiffusion method on mueller-hinton agar plates. oxacillin susceptibility testing was performed on all the isolates. the others antibiotics susceptibility was only studied on the isolates recovered from subclinical mastitis as they represent the major source of cheese contamination. results: % ( / ) of the isolates belonged to agr group , regardless of clinical findings. % ( / ) were adherent, strongly adherent or with maximal adherence (biofilm producers). % ( / ) were slime producers (moderate or strong producers). all the isolates (n = ), but seven, were susceptible to all the antibiotics tested. two isolates recovered from subclinical mastitis were resistant to oxacillin and partly resistant to ampicillin and penicillin-g. the five other isolates were found: partly resistant to erythromycin (n = ), cefoperazone and penicillin-g (n = ), erythromycin (n = ), neomycin (n = ) or resistant to enrofloxaxin and partly resistant to ampicillin and penicillin (n = ). conclusions: s. aureus isolates recovered from sheep mastitis in the southeast of france are mainly related to agr group suggesting a role for agr-regulated proteins in the persistence of this bacteria in the sheep udders. biofilm and slime production may also be an important aspect for intracellular survival of s. aureus which could promote the development of persistent intramammary infections. finally, ewe's milk does not appear to represent a source of resistant s. aureus and specially methicillin (oxacillin)-resistant s. aureus (mrsa) for human health. detection of virulence genes in staphylococus aureus isolates from dairy sheep, goats and cows mastitis, using single-dye dna microarray e. vautor, v. magnone, g. rios, m. pepin, p. barbry (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy farms animals. although many putative virulence factors have been identified in s. aureus genomes (kuroda et al., ) , the differences in pathogenic potential between naturally occurring isolates remain largely unaddressed. the relative importance of host (tissue) factors versus bacterial virulence determinants in disease pathogenesis is not well known, but it is widely accepted that bacterial factors including toxins, cell wall-associated adhesions, and secreted exoproteins are involved in the process. in this study, we use a single-dye dna microarray assay to investigate the presence or absence of putatives virulence genes in s. aureus isolates recovered from cases of ovine, caprine and bovine mastitis. methods: mastitis s. aureus isolates: sheep (n = ), goats (n= ), cows (n = ).dna microarray: the arrays were spotted with long oligonucleotides ( -mer) representing known virulence genes and new candidates identified in mu genome (a human strain) and other s. aureus genomes. each gene were spotted four time. dna extracted from the strains were labelled with fluorescent cy using the bioprime Ò array cgh (invitrogen). control strains with known genetic and phenotypic characteristics were used to normalize the data. results: (i) the majority of the virulence gene was detected in all the isolates (e.g. coa, ica adbc operon, htra, hysa, nuc, sbi, sdre, ssp, feob, fnb, sib, spa). (ii) genes were not detected in the majority of the isolates (e.g. cna, edin, lukf-pv, sav ,…). (iii) genes were not found in isolates, depending on the herd (e.g. aur or sav absent in isolates from some dairy sheep farm), on the isolates whatever the species (i.g. bsap, caph, entk, eta, fnbb, hsds, lpl , lukd, …) . but we found gene mainly related to species (e.g. agriii, sav ,…) comprehensive results will be given in the poster. conclusions: the present study indicated that the prevalence of virulence genes among s. aureus isolates recovered from dairy farm species depends on the gene. these observations suggest a common occurrence of host-adapted (or tissueadapted) s. aureus strains in which particular virulence genes may play a significant role. when taken with complementary methods such as pcr or/and southern hybridisation, singledye dna microarrays may provide a powerful tool to type s. aureus strains for epidemiological and possibly pathogenesis studies. detection of dna sequences distinguishing two closely related genomes of staphylococcus aureus from subclinical versus gangrenous ewe mastitis strains n. chevalier, c. huard, r. thiery, e. vautor (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy sheep. the severity of mastitis ranges from subclinical to gangrenous forms. subclinical mastitis is an inflammation that is not readily detected clinically whereas gangrenous form is an acute necrotizing mastitis. with the ain to find genetic markers or virulence factors that are only present in gangrenous strains a suppression substractive hybridisation (ssh) method was used in the present study to compare two strains of s. aureus respectively recovered from subclinical or gangrenous mastitis in the same dairy sheep herd. methods: ewes were held in the investigated farm. the subclinical strain was recovered in january from the milk of ewes. the gangrenous strain was recovered in december from an primipare dairy sheep that subsequently died from this acute mastitis. dna extracted from the strains were first compared by pulsed field gel electrophoresis (pfge). then, ssh was performed by using dna from the subclinical strain (driver), as described in a commercial kit (clontech pcr-select bacterial genome substraction kit). results: using pfge, four band differences were found between the two strains. two dna fragments, presumably specific from the gangrenous strain were detected by ssh and sequenced: (i) a bp ( % of homology with the sulfide quinone reductase contained in orf pathogenicity island of the mrsa strain) (ii) bp ( % homology with a gene coding a bacteriophage holine contained in the s. aureus n genome). control pcr tests using primers designed from these specific gene candidates confirmed that they were only present in the s. aureus gangrenous strain. conclusions: according to tenover et al. ( ) , a band difference using pfge indicates that the strains may possibly be related genetically. although genes classically involved in the virulence of s. aureus were not detected in the present study, two putative virulence factors were detected. the sulfide quinone reductase allows s. aureus to growth on sulfide (found in animal manure). the holine protein breaks the internal membrane of s. aureus to release daughter phages suggesting that a mechanism of horizontal gene transfer could have been mediated by bacteriophages and could explain the acquisition of virulence factors. antimicrobial clinical trials p outpatient treatment of acute pyelonephritis in pregnancy after weeks. a randomised controlled trial z. ahmadinejad, s. hantooshzadeh (tehran, ir) objectives: the purpose of this study was to compare the safety and efficacy of outpatient and inpatient treatment of acute pyelonephritis in pregnancy. methods: this was a randomized controlled, clinical trial. one hundred twenty eight gravidas past weeks' gestation admitted in imam khomeini hospital, tehran & sahid dr bahonar hospital, kerman, divided by random blocks to outpatient or inpatient therapy, received two -g doses of intramuscular ceftriaxone at -hour intervals while hospitalized, then were discharged and reevaluated within - hours or remained hospitalized until afebrile for hours. all patients completed a -day course of oral cephalexin. we performed urine cultures on admission and - days after therapy. results: the two groups were similar with respect to age, parity, temperature, estimated gestational age, initial white blood cell count, and incidence of bacteremia. there were not any significant differences between two groups about the clinical improvement after - hours, bacteriuria - days after treatment, relapse of pyelonephritis, requirement to change in antibiotic, date of pregnancy at delivery and preterm labor. the relapse of bacteriuria and preterm labor in inpatients were significantly more than outpatients (pv = . and . respectively). the birth weight of neonate in outpatients were significantly more than inpatients (pv = . ). conclusion: outpatient antibiotic therapy is effective and safe in selected pregnant women with pyelonephritis. however in this study, the neonatal outcomes were better in outpatients and the maternal outcomes in inpatients. experience with daptomycin in patients with renal insufficiency investigators collected demographic, disease state, clinical and microbiological data; outcomes were defined using standard definitions. patients nonevaluable for outcome were excluded. core data were divided and data on cohorts of pts with a creatinine clearance (crcl) ‡ or < ml/min were examined. results: of the pts enrolled, ( %) had evaluable pt outcomes and either crcl ‡ ml/min (nml, n = ) or crcl < ml/min not yet requiring renal replacement therapy (ri, n = ). the distribution of males and females was equal in both groups. ri pts were older ( % ‡ yrs vs %, p < . ). the groups did not differ in the percent coming from the community setting prior to starting dap (nml %, ri %). nml had more frequent history of fractures/orthopaedic procedures ( vs %, p < . ) and haematological cancers ( vs %, p < . ) while ri had higher rates of any renal disease ( vs %, p < . ), chf ( vs %, p < . ) and other immunologic/ inflammatory disease ( vs %, p < . ). ri had higher rates of skin infections ( vs %, p < . ) and endocarditis ( vs %, p < . ). infections that were frequently reported for nml and ri were bacteremia, non-catheter-related ( vs %), bacteremia, catheter-related ( vs %), osteomyelitis ( vs %), and foreign body-orthopaedic ( vs %), all p > . . methicillin-resistant staphylococcus aureus was the most common pathogen; nml %, ri %. ri had higher rates of coagulase-negative staphylococci ( vs %, p < . ) and viridans streptococci ( . vs . %, p < . ). there was no difference in the percentage receiving antibiotics prior to dap; nml %, ri %. the mean dap dose and duration were similar; nml . mg/kg for d, ri . mg/kg for d. the most frequent dose was mg/kg; nml %, ri %. ri initial dap dosing was more frequent than recommended (q h) in %. the mean time to clinical response was similar; nml . d, ri . d. more pts in nml received concomitant antibiotics with dap; vs %, p < . ). the clinical success (cure and improved) rates were; nml %, ri %. conclusion: dap shows favourable clinical success rates in pts regardless of the presence of renal insufficiency. in vitro activity of second line antibiotics against helicobacter pylori infection objective: the aim of our study was to determine the in vitro activity of levofloxacin, ciprofloxacin and rifampicin in clinical strains of h. pylori. material and methods: isolates of h. pylori from biopsies of dyspeptic patients were obtained following standard methodology. in vitro activity of metronidazole, clarithromycin, levofloxacin, ciprofloxacin and rifampicin was determined by e-test using % sheep blood agar and incubated at ordm;c during - days in a co atmosphere. mic was determined as the point of complete inhibition of growth. breakpoint of the nccls for other microorganisms were considered for fluorquinolones: resistant if mic > mg/ l. for rifampicin we considered the strain susceptible if mic < mg/ l, as same studies reported. results: . % of the strains were resistant to metronidazole and % to clarithromycin. mic , mic and range (mg/l) was: . , . and . fi for levofloxacin, . , . and . fi for ciprofloxacin and . , . , and < . - for rifampicin. all the strains were susceptible to rifampicin and only % of them were resistant to fluorquinolones. conclusions: the fluorquinolones tested and rifampicin showed an excellent in vitro activity against h. pylori, despite the high resistance rate to metronidazole and clarithromycin. however, in vitro susceptibility test should be done before the use in clinical practice. vibrio antibodies in serum and breast milk samples of parturient women in calabar, nigeria objectives: serum and breast milk samples from parturient women and serum from non-parturient controls were analysed for prevalence and titres of vibrio antibodies. methods: v. cholerae agglutinins and vibriocidal antibodies in serum samples were analysed by direct agglutination and immune bacteriolysis techniques respectively, using well microtitre plates. the protective value of breast milk was evaluated by haemagglutination inhibition and rabbit intestinal mucosal attachment of v. cholerae cells. results: vibrio agglutinins were detected in serum samples of ( . %) parturient and ( . %) non-parturient subjects (p < . ). high prevalence rates of . % and . % occurred among parturient and control subjects of - years of age respectively. at : cut off titre to evaluate vibrio cholerae specific bacteriocidal antibodies, activity was detected in samples of ( . %) and ( . %) parturients and controls respectively aged - years. breast milk from ( . %) parturients contained vibrio agglutinins with titres ranging between : and : , while milk samples from subjects showed haemagglutination inhibition (hi) activity titres of p : . of the hi positive milk samples ( . %) showed inhibition of v. cholerae adherence to rabbit intestinal mucosa at titres p : , and - % reductions in cell attachment. conclusion: our study confirms that parturient women in calabar may benefit from significant serum titres of v. cholerae antibodies and provide immune protection for their babies through breast milk secretions. moxifloxacin vs clarithromycin for treatment of community-acquired pneumonia associated with common respiratory pathogens: a pooled analysis objectives: streptococcus pneumoniae and haemophilus influenzae are pathogens commonly associated with community-acquired pneumonia (cap). this study compared the clinical and bacteriologic efficacy of moxifloxacin (mxf) to clarithromycin (clar) in cap patients with these pathogens. patients and methods: data were pooled from three doubleblind, multicenter, phase iii trials comparing oral mxf mg qd to clar mg bid for days. all patients included had mild-to-moderate cap. clinical and bacteriologic success rates were identified for s. pneumoniae and h. influenzae isolated from these studies. data for the efficacy-valid population was recorded at the test-of-cure (toc) visit ( - days post-therapy). results: patients were entered, of which were microbiologically evaluable. infection with s. pneumoniae and/or h. influenzae was documented in ( %) of patients ( mxf and clar patients had mixed infection). within this cohort, the two treatment groups were well balanced based on demographic/baseline medical characteristics ( % male, mean age yrs, % smokers, % recent antimicrobial therapy). clinical success and bacteriologic eradication rates (one response per patient) at toc are presented in the table. conclusions: in cap associated with s. pneumoniae and h. influenzae there was a trend towards greater bacterial eradication for mxf vs clar. clinical success rates were significantly higher for mxf monotherapy vs clar. variability of creatinine clearance measurements in inpatients with community-acquired pneumonia r. grossman, s. choudhri, d. haverstock (mississauga, ca; west haven, us) objectives: moxifloxacin, levofloxacin and gatifloxacin have been recommended as empiric therapies for patients with community-acquired pneumonia (cap). levofloxacin and gatifloxacin require dose-adjustment for renal insufficiency while no dose adjustment is required for moxifloxacin. this study was designed to determine the frequency and underlying variability of renal insufficiency in patients with cap. methods: a pooled analysis of data from patients with mild to moderate or severe cap entered into one of six randomized, controlled clinical trials was undertaken. renal function (calculated creatinine clearance; crcl) was assessed in each patient prior to treatment with mxf and then again during and post-treatment. results: baseline crcl levels in this pooled population of patients with cap were: < ml/min in ( . %) of patients, - . ml/min in ( . %) and ‡ ml/min in ( . %) patients. after the pre-treatment crcl measurement patients ( %) were lost to follow-up, so there was no during or post treatment value. in patients with cap the crcl improved from baseline in many patients during or post-treatment, while some patients experienced a worsening of renal function (see table) . conclusions: renal function (crcl) is highly variable in cap patients with baseline evidence of renal insufficiency. renal function should be monitored closely to permit appropriate dose adjustments if levofloxacin or gatifloxacin is used in this patient population. moxifloxacin may be a better empiric choice in this setting as it does not require dose adjustment in patients with renal insufficiency or renal failure. a prospective, controlled, randomised, nonblind, comparative study of the efficacy and safety of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice-daily ceftazidime plus amikacin in the empirical therapy of febrile neutropenic patients objective: empirical antibiotic treatment for febrile neutropenia is well established. the best regimen is still controversial. the purpose of this study was to evaluate the efficacy, safety and cost of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice daily ceftazidime plus amikacin in neutropenic febrile patients. patients and methods: ninety-five patients with febrile neutropenia were included in a prospective, controlled, randomized, non-blind, comparative study. patients were randomly assigned to either treatment group ( in the ceftriaxone/ciprofloxacin group and in the ceftazidime/ amikacin group) and evaluated as successes or failures according to defined criteria. daily assessments were made on all patients all adverse events were record. results: the overall incidence of documented infections was . %: / ( . %) in the ceftriaxone/ciprofloxacin group and / ( %) in the ceftazidime/amikacin group. there was significant difference in clinical efficacy between groups (p = . ) at the end of therapy. ceftriaxone/ciprofloxacin group had an overall incidence of resolution and improvement of , % in comparison to the % of the ceftazidime/amikacin group. thirty-nine organisms were isolated, ( . %) gramnegative and ( , %) gram-positive. there was low incidence of adverse events in both groups. conclusion: the combination of high dose single daily ceftriaxone plus ciprofloxacin was more effective than the standard combination of thrice daily ceftazidime plus amikacn with no significant adverse events in either group. objective: in past studies of azithromycin in children, a posttreatment (pt) benefit was observed at day . in recent phase trials in adults, single-dose zmax was at least as effective as standard comparators for treatment of respiratory tract infections (rtis), including cap. our objective is to demonstrate a pt benefit in this adult population. methods: post-hoc analyses, including respiratory adverse event burden (raeb), were conducted on the all treated population (n = ; az-m, comparators) in the phase studies. the raeb is the sum of duration, in days, of all respiratory adverse events, divided by total number of observation days of all patients, normalized to year. the overall and per study day raeb were calculated for zmax and the pooled comparators for the studies combined. results: raeb, in days/patient year, was . for az-m patients vs . for comparator patients (p = . ). the difference in raeb consistently and progressively favoured zmax, beginning at day and achieving statistical significance between days and , when the upper limits of the % cis around the differences were below zero (figure). faropenem medoxomil (fm) is an oral penem with potent activity against streptococcus pneumoniae and haemophilus influenzae. this integrated analysis was conducted to summarize the efficacy of days of mg bid of fm compared with other beta lactams in the management of community acquired pneumonia (cap). methods: efficacy was determined in three multicenter randomized double-blind controlled trials (rct) and a single uncontrolled study of faropenem medoxomil. comparators were days of cefpodoxime (c), days of amoxicillinclavulanate (ac), or days of amoxicillin (a). the analysis allowed examination of treatment effects by age, race, gender and study site subgroups. results: a total of subjects were studied. studies and were conducted in n. america, studies and in europe, latin america, israel, and s. africa. n. american vs. other studies included subjects at least (vs. at least ) years of age and only out patient (vs. outpatient and hospitalized) subjects. the clinical responses for fm in both per protocol and intention-to-treat populations were non-inferior to comparator for each study and for the three trials combined. no differences were found in treatment effect by age, race, gender, or country. recovery of an etiologic agent from initial respiratory or blood culture varied between . and . % of cases in the studies for a total of microbiologically evaluable subjects. s. pneumoniae was eradicated or presumed eradicated in / ( . %) and / ( . %), h. influenzae in / ( . %) and / ( . %), s. aureus in / ( %) and / ( %), h. parainfluenzae in / ( %) and / ( %), and m. catarrhalis in / ( . %) and / ( %) fm and comparator recipients, respectively. clinical response for s. pneumoniae bacteremic patients was / ( . %) for fm. conclusions: fm efficacy was consistent across studies, within subgroups, and non-inferior to comparators. it is efficacious against the most common bacterial pathogens and in the most severe form (bacteremic) disease. fm is a good option for the treatment of cap. propionibacterium acnes strains isolated from acne vulgaris and severe infections c. oprica, c.e. nord (stockholm, se) propionibacterium acnes is a member of the resident flora of the skin and is an important factor involved in inflammatory reactions in acne patients. during the last years the prevalence of different severe infections due to p. acnes has increased. objectives: ) to detect the prevalence of resistant p. acnes strains isolated from acne patients in stockholm and different severe infections in europe; ) to identify the mechanisms of resistance and the genetic diversity among resistant strains. methods: p. acnes strains isolated from acne vulgaris and severe infections were tested against clindamycin, erythromycin, linezolid and tetracycline and pulsed-field gel electrophoresis was used for further characterization. pcr and sequencing of the genes encoding domain v of s rrna for clindamycin and erythromycin resistant strains and s rrna for tetracycline resistant strains were performed. results: i) antibiotic-resistant strains were more often isolated from antibiotic treated patients with moderate to severe acne area than from non-antibiotic treated acne patients. an individual might harbor different pulsotypes of p. acnes with various degrees of resistance. ii) among the clinical isolates from european countries were found resistant strains to tetracycline, clindamycin, and erythromycin. overall, in the southern europe a higher prevalence of erythromycin-resistant strains was noticed and in southern and eastern europe a higher prevalence of resistance to clindamycin. it was noticed a high genomic diversity and the geographical spread of some clones in related areas but also in geographically distant countries. most clindamycin or erythromycin resistant p. acnes isolates, were found to be members of a single clone that has spread in different geographically countries. iii) p. acnes clindamycin and erythromycin resistant strains carrying one of the described mutations within the s rrna were predominantly isolated from swedish acne patients compared to strains from other infections. forty-four per cent of tetracycline resistant strains were found to carry a mutation in the s rrna. these strains were isolated from swedish acne patients, were highly resistant and were clustered in one pulsotype. conclusion: surveillance of both the prevalence of resistant p. acnes strains and associated resistance mechanisms is important due to the rapid variation in resistance patterns, both in acne patients and other severe infections. antimicrobial activity of unisepta quick and deconex solarsept on the surface contamination and dental instrument in dental clinics in iran f. shahcheraghi (tehran, ir) objectives: quaternary ammonium compounds (qacs) are amphoteric surfactants that are widely used for the control of bacterial growth in clinical and industrial invironment.unisepta quick and deconex solarsept are new generation of qacs is widely used as adjuncts in iran to hygine in dental clinics.the aim of present study was to investigate clinical efficiency of these substances on the surface and instruments in dental clinics. material and methods: the following bacteria and fungi on the base of aoac standard were used.pseudomonas aeruginosa (atcc ), staphylococcus aureus (atcc ) bacillus. subtilis atcc ( ), mycobacterium bovis atcc ( ) and wild types of trichophyton mentagraphit, p. aeruginosa and salmonella typhimorium (a common fungi in iran). a stock solution of deconex solarsept (borer chemie) and unisepta quick (micro unident) was prepared as recommended by the manufacturer. the concentration of bacterial suspention was . macfarland and the results were reported on the base of decreasing in (cfu) colony forming unit from to . results: the results shows that both of these disinfectants have bactericidal and fungicidal activity on the standard p. aeruginosa, s. aureus, s. typhimurium and trichophyton mentagraphit, the number of bacteria decreased significantly (p < . ), but no significant difference was seen with b. subtilis, wild type of p. aeruginosa and m. bovis. conclusion: the results confirm that these qacs are not able to sterilize or disinfect medical and dental instruments, and they can not be used lonely, and it must be used with the other methods for sterilization of surface and dental instruments. macrolide as long-term treatment in patients with bronchiectasis colonised by p. aeruginosa background: a certain efficacy of macrolide against p. aeruginosa has been described in vitro, mainly through mechanisms such disruption of quorum sensing and suppression of inflammation. aim: to evaluate the efficacy of macrolide in patients with bronchiectasis colonised by p. aeruginosa. methods: the study prospectively included patients with bronchiectasis and p. aeruginosa isolated in sputum in stable state. all subjects received either azithromycin mg · days/week or clarithromycin mg daily on long term and completed daily diary cards for symptoms and pef values until the end of therapy. follow-up period was year. results: patients with bronchiectasis and p. aeruginosa evidence in sputum were included ( men, mean age . ± . yrs.). patients received azithromycin and patients clarithromycin, with a mean duration of . ± . months. five ( . %) patients discontinued treatment after less than weeks because of adverse events. at the end of therapy, ( . %) patients showed no evidence of p. aeruginosa in sputum while ( . %) patients still had ps. aeruginosa in sputum. an improvement in the following parameters could be observed in all patients: sputum volume ( . ml/day before therapy versus . ml/day after therapy, p = . ); pef ( . ± . l/min before therapy versus . ± . l/min after therapy, p = . ); number of exacerbations/year ( . in the previous year versus . in the follow-up year, p = . ). conclusion: the study shows that macrolide may be an effective therapy in patients with bronchiectasis colonised by p. aeruginosa. independently of the microbial eradication, an improvement of the clinical symptoms and a reduction of exacerbations were observed in all patients. fungal pathogens from haematoncology patients and their susceptibility to new and old antifungal drugs the expanding population of immunocompromised hosts has been infected with many established and emerging opportunistic fungi. most pathogens can be treated empirically whereas for an increasing number of species proper treatment starts once the mic becomes available. though invasive aspergillosis remains the principle life threatening complication in the haematoncology patients (hop) other pathogens cannot be ignored as selection and resistance during prophylaxis increases the risk of treatment failure.in order to understand the frequency of rare fungal pathogens, selection and emergence of resistance in our trust all fungi from hop were identified using standard mycological techniques and the mics to amphotericin b (amb), flucytosine ( fc), fluconazole (fcz), itraconazole (itz), voriconazole (vcz) and caspofungin (cfg) were determined using the nccls method. specimens were processed, % respiratory, . % blood, . % oral, . % other sterile (bile, csf, drains, lines and tissue biopsies) and . % nonsterile sites. yeasts accounted for % and filamentous fungi (ff) for %, representing candida sp, other types of yeast, aspergillus sp and other types of ff. c. albicans represented . %, c. glabrata . %, c. krusei . %, a. fumigatus % and other aspergillus sp % of all isolates. the mic s for all isolates were amb . , fc , fcz > , itz , vcz and cfg . mg/ l. with the exception of acremonium sp, a. versicolor, a. terreus and scedosporium apiospermum all isolates including the isolates of c. lusitaniae were sensitive to amb. most but not all ff and only one isolate of c. albicans from the yeasts were resistant to fc. all ff, rhodotorula sp, c. albicans %, c. glabrata % and c. krusei % were resistant to fcz. only absidia corymbifera, acremonium sp %, c. albicans %, c glabrata % and saccharomyces cerevisiae % were resistant to itz. for vcz a. corymbifera, acremonium sp %, c. albicans %, c. glabrata %, c. krusei %, c. tropicalis %, rhodotorula sp . % and p. aecilomyces variotii % had an mic ‡ mg/l. with cfg the effective concentration was ‡ . mg/l for a. corymbifera, fusarium solani, geotrichum capitatum, sporobolomyces salmonicolor, acremonium sp % and c. parapsilosis %.the data show that hop are exposed to many different fungal pathogens some of which are resistant to the old and the new antifungals and that amb is still the drug with the broader spectrum and less developed resistance for both yeasts and ff. faropenem medoxomil in the treatment of acute bacterial sinusitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial agent with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute bacterial sinusitis (abs) was evaluated in phase iii trials; , , and . study was conducted in n. america, study was conducted in europe and israel and study was conducted in the us and argentina. and were prospective, randomized, double-blind, active comparator trials and was an open-label ''sinus tap'' trial. the dose of fm was mg bid in all studies. the comparator in and was cefuroxime axetil (cfx) mg bid. the duration of fm treatment in was days and days vs cfx for days. in , fm or cfx were given for days. in , fm was administered for days. the primary efficacy variable in all studies was clinical response at the test-of-cure (toc). microbiologic response at the toc was a secondary efficacy variable in (sinus puncture and endoscopic collection) and (sinus puncture and aspiration). non-inferiority was defined as the difference in cure rates (fm minus comparator) where the lower boundary of the % ci was greater than - %. results: the cure rates at the toc are shown in the table for the valid per protocol (vpp) and the intent-to-treat (itt) populations. the frequency of isolation of key pathogens and the rate of eradication in samples obtained by endoscopicallyguided swab and in samples obtained by tap were consistent across studies. the eradication rates for s. pneumoniae, h. influenzae, and m. catarrhalis were . % vs. . % (fm / d vs. cfx / d), . % vs. . % (fm vs. cfx) and . % vs. . % (fm vs. cfx), respectively. conclusions: fm mg bid x days was shown to be noninferior to cfx in clinical efficacy in two prospective, doubleblind, comparative trials. a third, open-label trial, demonstrated similar efficacy in microbiologically documented abs caused by key pathogens. longer ( d treatment) with fm provided no additional efficacy. faropenem medoxomil in the treatment of acute exacerbation of chronic bronchitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute exacerbation of chronic bronchitis (aecb) was evaluated in phase iii trials. study was conducted in europe, israel, mexico, and south africa. study was conducted in the us and argentina. both were prospective, randomized, double-blind, active comparator trials. the dose of fm was mg bid for days in both studies. the comparators were clarithromycin (clr) mg bid for days and azithromycin (azi) qd for days ( mg on day and mg on . the primary efficacy variable was clinical response at the test-of-cure (toc). microbiologic response at the toc, in subjects with a baseline pathogen was a secondary variable. non-inferiority was defined as the difference in cure rate (fm minus comparator) where the lower boundary of the % ci was greater than ) %. results: the cure rates are shown below for the valid per protocol (vpp), intent-to-treat (itt) and modified itt populations (all itt subjects who met inclusion/exclusion criteria). in both the individual studies and the pooled analyses, for all populations, treatment with fm was not less effective than either comparator. % of treated subjects in and % of subjects in were evaluable for microbiological response. in , the eradication rates for the microbiologically evaluable population was higher in the clr group ( . %) compared with the fm group ( . %) ( % ci ) . , . ) . in contrast, the eradication rate in was similar in the fm ( . %) and azi ( . %) groups ( % ci - . , . ). when the data were pooled across studies, the response rates were similar with fm ( . %) and combined comparator ( . %) groups ( % ci - . , . ). the combined eradication/presumed eradication rates in the pooled fm and comparator groups were . % vs. . %, respectively for s. pneumoniae and . % vs. . %, respectively, for h. influenzae. conclusions: fm was shown to be non-inferior to either azi or clr in clinical efficacy in two adequate and well-controlled trials. pooled analysis further strengthened the clinical noninferiority conclusion. the difference in eradication rates observed in study (clr) was not supported by study (azi). an integrated safety analysis of faropenem medoxomil: results of , subjects from phase ii/iii clinical trials r. echols, r. tosiello (milford, us) objective: to evaluate the safety profile of faropenem medoxomil (fm), a novel oral penem antibiotic. methods: , subjects from phase ii and phase iii clinical trials received fm, mg bid for - days for treatment of acute bacterial infections. randomized controlled trials (rcts) included , fm and comparator treated subjects. analyses were conducted to identify possible disparate adverse event (ae) reporting based on type of infection, subject age ( - , - , , - , > ) and gender, duration of treatment ( / d v. / d), geography (na, eu, row), study design (open label v rct), relationship to treatment. comparisons were made to control treatment based on antibiotic class (b-lactam v. other), and individual antibiotic treatments. results: fm compared favourably to penicillins, cephalosporins and macrolides. fm was better tolerated than tmp/smx and co-amoxiclav. open labeled trials had higher aes reported v. rcts. aes reporting na = row > eu except serious aes and deaths where row = eu>na. aes for fm / d = fm / d. underlying infection did influence ae reporting. female gender had higher ae reporting than male gender. fm was tolerated equally well across age ranges, although deaths and saes were more common in > age group. common aes (> %/related from rcts) were diarrhoea, nausea, fungal vaginosis and headache and were generally less frequent with fm than control rx. no evidence of neuro or cardio toxicity was identified. laboratory tests identified no hepatic, renal or hematopoietic signals. conclusion: faropenem medoxomil, a novel oral penem antibiotic, has the safety profile expected of a b-lactam but is better tolerated than co-amoxiclav with approximately one-third the gi side effects. the efficacy of non-surgical and systemic antibiotic treatment regimens in smoking and non-smoking patients e. pähkla, k. lõ ivukene, p. naaber, m. saag (tartu, ee) periodontitis is a chronic infectious disease, which leads to the destruction of periodontal ligament fibres and alveolar bone until tooth loss. the objective of this study was to compare the longitudinal effect of combination of non-surgical periodontal therapy with systemic antibiotics in smoking (s) and nonsmoking (ns) patients. methods: there were total of patients with severe generalized chronic periodontitis involved in this study ( s, ns), who did not respond well to previous mechanical periodontal treatment. the clinical examination included recordings of visible plaque index (vpi), modified gingival index (mgi), bleeding on probing (bop) and suppuration after probing (sup), probing pocket depths (pd) and clinical attachment levels (cal). the non-surgical periodontal therapy was performed within weeks. clinical parameters were recorded at baseline, - weeks after the first mechanical treatment and months after combined treatment, during a regular check-up visit. as the patients did not respond to the conventional periodontal therapy, the microbiological analyses were taken and a combination of systemic amoxicillin mg · and metronidazole mg · for days, was prescribed. results: the results suggested that the combined systemic antibiotic therapy is effective in case of severe generalized chronic periodontitis, as vpi, bop, sup, cal, and mgi improved significantly after the treatment. in the ns group all parameters, except cal, improved significantly after the treatment. the s showed markedly smaller reduction in sup, mgi, and cal.after instrumentation, no periodontal pathogens were isolated in ( %) patients, while patients ( %) were infected with one to three different pathogens. among the pathogens, prevotella intermedia/nigrescens ( patients) and actinobacillus actinomycetemcomitans ( patients) were dominating. the total level of microbial load (log cfu/ml) as well as the spectrum of pathogens in s and ns patients remained similar. conclusions: despite of positive treatment effect in general, there were insignificant improvements in any clinical parameters in the smoking group. smoking has adverse effect on periodontal therapy; therefore the dentist should cooperate with patients in counselling of smoking cessation to achieve better results in the treatment of periodontitis. objectives: laminin (ln), which is a large multidomain glycoprotein of the extra cellular matrix, has attracted much attention because of its importance in many cellular functions, including induction of cell adhesion, growth promotion and mediation of cell communication. the target of this study was to find out whether there is any relation between the levels of serum ln and the inflammatory activity of a microbial infection. patients/methods: from june to october , immunocompetent adults, with confirmed bacterial infection were admitted to our hospital ( with pneumonia, with pyelonephritis and with cholecystitis) (group ). at the same time hospitalised patients for non-infectious causes (stroke, gastrointestinal bleeding, anaemia) were also studied (group ). the levels of serum ln and crp were measured on the day of admission in both groups. the levels of ln were measured using an enzyme immunoassay kit (takara laminin eia kit) and healthy volunteers were used to determine its normal limits ( - ng/ml). plasma crp concentration was assessed by immunoturbidometric method (using randox, uk kits). normal values were considered those below . results: the mean serum ln levels of patients of group were . ± . (much higher that the normal limits), while the mean crp value was . ± . . the mean corresponding values in group were . ± . for ln (within normal limits) and . ± . for crp. there is a statistically significant difference between the mean ln levels of the two groups (p < . ). additionally, there is a statistically significant correlation between the levels of ln and crp (a well studied serum inflammatory marker) in patients with bacterial infection (group ) (pearson correlation coefficient r = . , p = . ). conclusions: the definition of the ln levels could constitute a new reliable, simple, direct serum marker for the confirmation of an active bacterial infection. additionally, as the crp levels are above normal in group too (patients without infection) while ln lies within normal limits, maybe ln is even more specific than crp. more studies are required in the future, with more patients included, in order to confirm the outcome of this study. performance and clinical significance of a direct tube coagulase test using serum separator tubes for rapid identification of staphylococcus aureus from blood culture broth d. kwa, t. schü lin-casonato, p. sturm (nijmegen, nl) objective: blood cultures are important in the diagnosis of serious infections. early administration of effective antibiotics is associated with improved patient outcome. the performance of the direct tube coagulase (dtc) using serum separator tubes (ssts) for rapid identification of s. aureus from blood culture broth (bcb) was investigated. the clinical significance of rapid identification was assessed. methods: consecutive blood cultures with gram-positive cocci in clusters were tested. bcb was collected in ssts using a subculture-venting unit. after centrifugation, the supernatant was discarded and ml rabbit plasma was added to the remaining pellet of bacteria. coagulation was evaluated after and hours incubation at °c, and after overnight incubation at room temperature. in parallel, a direct tube coagulase test was performed using a : saline dilution of bcb as described previously. isolates were identified by standard microbiology procedures. clinical significance was measured by comparison of antimicrobial prescription based on gram stain results, direct coagulase results, and culture results. results: over a -week period, bcbs from patients were tested. s. aureus was present in bcbs. using the serum separator tube method and the saline dilution method, the sensitivity of the dtc after hours incubation was % and %, and after hours % and %, respectively. the specificity of both methods was %. rapid identification of s. aureus resulted in initiation (n = ) or streamlining (n = ) of antimicrobial therapy in of patients with s. aureus bacteremia. rapid identification of coagulase-negative staphylococci resulted in changes in antimicrobial therapy in of patients. conclusion: the dtc using ssts for bacterial enrichment is a very reliable, rapid, cheap and easy to perform method for identification of s. aureus from bcb. implementation of this test can improve antimicrobial therapy. evaluation of the results of the spanish seimc external quality control program for the diagnosis of enterococcus faecalis and klebsiella pneumoniae infections r. guna, j.l. pérez, n. orta, c. gimeno on behalf of seimc objectives: to evaluate the results obtained from four shipments of two different strains by the participants in the seimc external quality control program (eqcp). these controls were intended to analyse the percentages of correct species identification and the ability of the participants in detecting some special features of the control strains: vanb phenotype in the case of e. faecalis, and the production of extended spectrum betalactamase (esbl) in k. pneumoniae. methods: the same strain of each microorganism was sent in two different shipments. the vanb e. faecalis strain was sent both in a control of year as well as in other of , while the esbl-producing k. pneumoniae was sent in and in to an average of laboratories. the results obtained were compared with those of a reference laboratory that certified both the species identification and the resistance features. results: in the control, . % of participants identified correctly e. faecalis, while . % did it in . as for the glycopeptide resistance pattern of the enterococcal strain, . % and . % of participants detected the vanb phenotype in and , respectively. overall, the k. pneumoniae strain was correctly identified in both separate controls by most of the participants ( . % and . %, respectively). interestingly, the percentage of laboratories that detected the presence of the esbl in the k. pneumoniae strain sharply increased from . % in to . % in . the overall percentages of correct species identification were high for the two microorganisms and for both control points. most important, the ability of the spanish clinical laboratories in detecting the special resistance features of these strains clearly improved along the study period. these data confirm the importance of implement a continuous surveillance of the diagnostic training in the clinical laboratory, as well as the possible positive intervention of the seimc external quality control program in such improvement, since the analysis of results is accompanied of updated reviews on the subject of each control. a. bonnet-pierroz, a. resenterra, o. péter (sion, ch) objective: to evaluate new elisa ridascreen Ò borrelia igg and igm for antibody response in patients with confirmed lyme borreliosis and to compare to the results of vidas lyme (igg-igm) and in-house immunoblots (b. garinii igg and igm for early cases or b. burgdorferi sensu stricto, b. afzelii, b. garinii, b. valaisiana igg for late cases). methods: elisa ridascreen Ò borrelia igg and igm was used to screen sera from patients with clinically confirmed erythema migrans em (n = ). patients with confirmed neuroborreliosis by intrathecal antibody synthesis (n = ) were evaluated for igg antibodies to borrelia. sera from patients with acrodermatitis chronica atrophicans aca (n = ) and sera and synovial fluids from patients with lyme arthritis (n = ) were also evaluated for igg antibodies. patients with syphilis (n = ) and infectious mononucleosis (n = ) were screened for igg and igm antibodies to borrelia in order to estimate the specificity. conclusion: the elisa ridascreen Ò borrelia igg and igm have shown a good sensitivity for the serological diagnosis of lyme borreliosis. the short evaluation for the specificity of the igg test revealed a good assay with few false positive reactions, whereas the igm assay was, as expected more prompt to give false positive results with sera from patients with infectious mononucleosis. so far any equivocal or positive tests should be confirmed by immunoblots. is it necessary to incubate the bact/alert blood culture bottles more than days? objective: to assess the incubation time reduction of the aerobic and anaerobic bact/alert system bottles from to days. methods: from to we processed . blood culture sets and detected . ( %) positive blood cultures with clinical significance. we retrospectively examined the detection time of positive bottles and assessed the clinical significance of the bottles that were positive between the fourth and fifth day. results: out of positive blood cultures with clinical significance, ( . %) were detected within the first days of incubation. out of the positive blood cultures detected between the fourth and fifth incubation days, were recovered in concurrent cultures within the first days. chart reviews were conducted from patients with the remaining isolates. only in patients ( . % positive blood cultures) changes in antimicrobial therapy based upon the positive blood culture results on day to were made, in the other patients the empirical treatment was adequate. the isolated microorganisms in those patients were: gram-positive cocci ( staphylococcus spp. not s. aureus, staphylococcus aureus, streptococcus viridans and streptococcus pyogenes), anaerobes, enterobacteriaceae, pseudomonas aeruginosa, campylobacter spp., candida spp. cryptococcus neoformans, brucella spp. and haemophilus influenzae. conclusions: incubation of bact/alert blood cultures bottles only for days would have represented a detection loss of . % of the clinically significant isolates, which led to antimicrobial therapy changes. although we keep employing a -day incubation for routine blood cultures, we could reduce the incubation time to days depending on current instrument capacity. an enzyme immunoassay for anti-diphtheria antibodies: a practical alternative to the vero cell assay r. budd, e. harley, r. george, a. efstratiou, k. broughton, a. bradwell (birmingham, london, uk) introduction: in this extended study, results from an anti-diphtheria toxoid enzyme immunoassay (eia), specifically designed to detect higher affinity antibodies, were compared with those from a vero cell assay (vca). methods and results: serum samples with antibody concentrations ranging from . - . iu/ml on the vca from the respiratory and systemic infectious laboratory (rsil) were assayed by eia (the binding site ltd, uk). a further samples from rsil selected on the basis of being close to the protective level, were assayed to confirm the performance of the eia. the eia was calibrated, against the nibsc reference material / and the assay measuring range was . - . iu/ml results were compared using the who guidelines of . - . iu/ml as minimum protective level, and > . iu/ml as protective. relative agreement, sensitivity and specificity for the first samples were: . %, . % and . % respectively, for the second set of samples performance was: . %, . % and . %, and for the combined samples results were: . %, . % and . % respectively. roc analysis of the total samples confirmed the highest sensitivity . % and specificity . % occurred at a cut-off of precisely . iu/ml for the elisa assay. conclusion: of the total discrepant samples, had vca and eia values < . iu/ml, therefore we suggest the possibility of establishing an equivocal zone for the interpretation of the eia results. if the test is part of a general immune status assessment a grey zone is not required. if undertaken to determine the requirement for immunization, the use of the equivocal zone is recommended. by applying these criteria in the eia, only one sample would have suggested inappropriate immunization, as indicated by a vca result > . iu/ml. because of the > % agreement between the two assays, significant advantages of cost and speed, ease of use and the potential for automation, the eia could therefore be considered as an alternative to the vca. evaluation of accuracy limits of countable colony-forming units on agar plates j. arbique, a. rendell, k. forward (halifax, ca) objectives: accurate colony counts are an essential component of many microbiology research projects and clinical laboratory processes. the suggested range of accuracy of colony-forming units (cfu) extends from to (standard methods for the examination of water and wastewater). this recommendation dates to , and fails to adequately address the numerous sources of inter-and intra-variability. without more detailed analysis it is difficult to estimate the sample size and number of replicates necessary to ensure accurate results. the purpose of this study was to determine the validity of accuracy limits for quantifying cfus on agar plates. methods: escherichia coli (atcc ) and staphylococcus epidermidis (atcc ) were used to prepare series of four organism densities ranging from approximately - cfu, on three different days. on each day, each of the densities for both organisms was plated on sba and viable organisms were counted following incubation. an average of the margins of error obtained over the days of testing was used to determine the reproducibility of agar plate counts, and to estimate the optimum number of replicate plates (sample size) required for each organism at each concentration. results: margins of error for both organisms were greatest with suspensions yielding approximately cfu, and lowest for suspensions yielding and cfu. nine replicate plates were required for a suspension of s. epidermidis yielding cfu to achieve the same margin of error as obtained with replicate plates at concentrations yielding - cfu. seven replicates plates were required for a suspension of e. coli yielding and cfu to achieve similar margins of error to those obtained with replicate plates at concentrations yielding cfu, and replicate plates at concentrations yielding cfu. conclusion: we found that the greater the concentration ( and cfu), the fewer replicate plates necessary to reliably estimate organism concentrations. the lower the organism density ( cfu), the more plates necessary to reliably estimate cfus. contrary to the recommendations described in standard methods for the examination of water and wastewater, cfu of were reliably reproducible. for greatest accuracy, experiments should be conducted so as to assure that colony counts are in the range of - . direct microscopy: a valuable instrument for diagnosis and prognosis of periodontal disease objective: to appreciate the composition of micro flora from periodontal pockets, using light microscopy and to compare it with clinical status. introduction: it is generally accepted that periodontal disease occurs when anaerobic gram-negative flora increase in number with the subsequent decrease of facultative anaerobe grampositive bacteria. in other words, the switch from gram-positive to gram-negative of sub-gingival flora has a pathologic significance and could be observed using direct microscopy. materials and methods: specimens sampled with sterile paper points from periodontal pockets and samples from clinical healthy persons were included in this study. each sample was diluted in . ml saline solution and, with a calibrated loop, was taken ll aliquots in order to prepare a smear for microscopic examination and for inoculation on solid media (columbia with % sheep blood). the smears were gram stained and the culture plates were incubated in anaerobic conditions ( h, °c) and in air ( h, °c). results: in % of samples from patients with periodontal disease, easily notable, high number of gram-negative bacteria at direct microscopy, associated with abundant growth in anaerobic condition and poor growth in air. in from healthy patients, the gram-negative flora was almost absent and gram-positive bacteria were in high number, correlated with the absence of bacterial growth in anaerobiosis and some growth in air.the presence of treponema spp. at direct microscopy was associated with deep and bleeding periodontal pockets. after few days of proper therapy, the good clinical status was well correlated with an increasing number of gram-positive bacteria. conclusions: ) using a diluted sample for microscopic examination, the value of the method increase, offering important information about the composition of sub-gingival flora. ) the good correlation between the clinical status and microscopic finding recommend it as an easy to use diagnostic method in dentistry. identification of species and glycopeptide resistance among enterococcal isolates by bd phoenix objectives: vancomycin resistant enterococci are emerging in europe necessitating their fast and accurate identification by the laboratory. there was an attempt to evaluate the performance of the bd phoenix automated microbiology system (bd diagnostic systems, sparks, md.) for the correct identification of species and glycopeptide resistance in comparison to the gold standard of diagnosis, pcr, using a large collection of clinical strains. methods: a total of enterococcal isolates were tested by the bd phoenix sytem. these strains were isolated from faecal, urine, pus, blood and samples from other body sites cultures. a multiplex pcr was applied using different pairs of primers, specific for the identification of e. faecium, e. faecalis and the vana, vanb, vanc, vand, vang, vane glycopeptide resistance genotypes. susceptibility to the glycopeptides was also confirmed by the etest (ab biodisk, solna, sweden). results: according to the pcr, there were e. faecium (including vana-positive strains), e. faecalis (including vana-positive and vanb-positive strains) and e. cass/gall isolates. two strains were not identified and were excluded from the analysis. discrepant results between the multiplex pcr and the phoenix system were obtained for / isolates ( %) with similar rates amongst faecal ( / , . %) and the rest of the isolates ( / , . %). the most common discrepancies were the misidentification of e. faecium vana strains and e. faecalis strains as e. cass/gall by phoenix. two e. faecalis strains were incorrectly characterized as vancomycin resistant, two e. faecium strains were misidentified as e. hirae and e.cass/gall, respectively, and one e.cass/gall strain was reported as e. faecium resistant to both glycopeptides. thus, the sensitivity and specificity for the identification of e.cass/gall by phoenix were . % ( / strains) and . % ( / strains), respectively, while . % of vana strains ( / strains) were not recognized by this system. conclusion: this study demonstrates that the new identification system, phoenix, similarly to other automated or manual systems, presents with problems regarding correct identifica-tion of enterococcal species and glycopeptide resistance. specifically, laboratories should be aware that clinically significant isolates identified as e.cass/gall should be confirmed by another method. an audit of sputum requisition practices p. lal, i. balakrishnan (london, uk) objectives: to analyse the indications and rationale for the processing of sputum specimens in a london teaching hospital. methods: sputum samples received from / / - / / were included in this study. data were obtained from the patient requisition forms and the winpath systems and were analysed further as per the objectives. results: a total of specimens were received during this period. ( %) from hospital in-patients and ( %) from general practitioners. out of the total of samples received from hospital-in patients ( . %) had > epithelial cells/ lpf. no clinical details were mentioned in ( . %) and ( . %) were from patients already on antibiotics. repeat specimens within one week were sent in ( . %) cases and ( %) had atypical serology also sent. out of the hospital-in patient samples ( . %) had a significant isolate and ( . %) had normal respiratory tract flora isolated. others were reported as: ''gross oral contamination'' [ ( . %)], ''no growth'' [ ( . %)] and ''no significant growth'' [ ( . %)]. there were a few specimens reported as ''inappropriate specimen -two days old'' [ ( . %)] and ''leaking'' or ''saliva only' ' [ ( . %) ]. out of a total of samples received from gp patients ( . %) of samples had > epithelial cells/lpf, ( . %) had no clinical details provided, ( . %) samples were sent while patients were on antibiotics and ( . %) samples were repeated within one week. only ( . %) had atypical serology also sent. conclusions: -less than one-third of specimens yielded a significant pathogen.-adequate clinical details were lacking in about one-fifth of specimens.-nearly one-third of specimens were repeated within one week, without a clear indication.-about % of specimens were of poor quality.-atypical serology was only performed in . % of outpatients, as compared with % of in-patients.this audit brings forth the fact that the clinical indications for which sputa are being sent for culture need to be clearly defined and an educational campaign instituted amongst relevant healthcare professionals. sputum collection techniques need to be rigorously applied if good-quality specimens are to be obtained. indications for performing atypical serology need to be defined and reinforced, particularly in primary care. a new approach to laboratory diagnostic of infectious gastroenteritis -a follow-up objectives: in order to optimize use of laboratory facilities and ensure flexibility in relation to current epidemiology, a new approach to laboratory diagnosis of infectious gastroenteritis was applied: from an algorithm the decision of which organisms to test for was defined by the demographic, clinical and epidemiological information submitted to the laboratory on paper/electronic request forms. methods: from april , -june , , hospitals and general practitioners submitted a request form with the following information together with the stool sample (s): ( ) acute or persistent diarrhoea (duration > weeks); ( ) bloody stools; ( ) recent history of foreign travel; ( ) > patients within same epidemiological setting; and ( ) nosocomial infection. provision of data is mandatory when submitting electronically. based on these data, analyses were performed according to an algorithm. examination for salmonella, shigella, yersinia, campylobacter, and clostridium difficile was done by culturing. verotoxin producing e. coli (vtec), enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec) and enteroinvasive e. coli (eiec) were identified by pcr for virulence genes and serotyping. rota and adenovirus were detected by antigen tests and parasites by microscopy. results: in total we examined , samples from , patients. a pathogen was isolated in % of patients. in cases ( %) clinical/epidemiological data were missing. • , patients had diarrhoea < weeks: % with campylobacter, % with salmonella, % with etec, % with giardia, and % each with eiec and vtec • , patients had diarrhoea > weeks: % with campylobacter, % with giardia, % each with epec and vtec • patients had a history of foreign travel: % with campylobacter, % with etec, % with salmonella, and % with giardia • patients had bloody stools: % with campylobacter, % with salmonella, and % with vtec • patients were < years: % with campylobacter, % with epec, % each with giardia, vtec and salmonella, and % with etec conclusions: campylobacter was the most common bacterial pathogens in all groups and rotavirus was the most common pathogen in children < years. the new approach had a number of advantages: more relevant microbiological analysis, collection of data on defined patient groups, and flexibility regarding adaptation to current epidemiological knowledge. increasing use of electronic submission of request forms will optimize the approach used. objectives: small colony variants (scvs) are an emerging infectious disease problem, presenting as a naturally occurring, slow-growing subpopulation of staphylococcus aureus that are characterized by tiny colonies on solid media. studies on scvs recovered from patients with persistent infections are hampered due to their frequent unstable phenotype. in particular, scvs are not easily distinguishable from the normal phenotype in broth media and a reversion of scvs into the normal phenotype is not traceable. methods: a set of isogenic s. aureus isolates comprising the (i) normal and the (ii) scv phenotype (isogenic to the isolate with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding mutants mimicking the scv phenotype (knock-out of hemb), and (iv) their complemented mutants were used to investigate the feasibility of fourier-transform infrared (ftir) spectroscopy to trace the expressed phenotype in broth media. the respective isolates cultured on solid media served as controls. in addition, all isolates were genotyped by pulsed-field gel electrophoresis and spa typing. results: using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information in three different spectral ranges resulted in a dendrogram that showed a clear discrimination between both staphylococcal phenotypes. distinct clusters comprising the clinical and mutant scv phenotype on one hand and the normal phenotype (isolate with normal phenotype and complemented mutant) on the other hand were found. thus, scvs from different clonal lineages gave spectra that were more similar to one another than to their normal growth parent. ftir was also shown to be able to trace the switch of the phenotypes in broth when the medium was supplemented. conclusion: ftir spectroscopy allows a rapid, reproducible and clear discrimination of different phenotypes of s. aureus in fluid media for diagnostic and research purposes. in contrast to genotyping approaches, ftir staphylococcal fingerprinting is only reliable for typing purposes if the isolates exhibit the same phenotype. in future studies, this technique may also provide an approach for tracing the scv phenotype in infected tissues. objectives: triggering receptor expressed on myeloid cells- (trem- ) is a recently discovered cell surface molecule whose expression on phagocytes is up regulated by exposure to bacteria or fungi. a soluble form of trem- (strem- ) can be measured in various body fluids. we studied whether strem- in cerebrospinal fluid (csf) could serve as a biomarker for the presence and outcome in patients with bacterial meningitis. methods: in this retrospective study on diagnostic accuracy we used an elisa to determine levels of strem- in csf from adults with bacterial meningitis, confirmed by csf culture, who participated in the prospective dutch meningitis cohort study; patients with viral meningitis, confirmed by polymerase chain reaction of csf; and healthy control subjects, who underwent lumbar puncture to exclude the diagnosis of subarachnoid haemorrhage. the mann-whitney u test and the chi-square test were used to identify differences between groups. a receiveroperating-characteristic curve (roc) was constructed to illustrate various cut-off csf levels of strem- in differentiating between the presence and absence of bacterial meningitis and diagnostic accuracy was quantified by % confidence intervals ( % ci). results: levels of strem- in csf were higher in patients with bacterial meningitis as compared to those with viral meningitis [median, pg/ml (range, to pg/ml) versus . pg/ml (range, to pg/ml); p = . ] and controls [ pg/ml (range, to pg/ml); p < . ; fig]. patients with viral meningitis and controls had similar csf strem- levels. the area under the roc curve for discriminating between patients with and without bacterial meningitis was . ( % ci, . to . ; p < . ). at a cut-off level of pg/ml, strem- yielded a sensitivity of . ( % ci, . to . ) and a specificity of . ( % ci, . to . ). in patients with bacterial meningitis, csf strem- levels were associated with mortality [survivors versus nonsurvivors: median pg/ml (range, to pg/ml) versus pg/ml (range, to pg/ml); p = . ]. conclusions: measuring strem- in csf may be a valuable new approach to accurately diagnose bacterial meningitis and identify patients at high risk for adverse outcome. therefore, a prospective study on strem- as biomarker in bacterial meningitis is needed. systematic review of rapid diagnostic tests for enterohaemorrhagic e. coli i. abubakar, l. irvine, l. shepstone, s. schelenz, c. aldus, p. hunter (norwich, uk) objective: a variety of rapid tests for the detection of enterohaemorrhagic escherichia coli (ehec) have recently emerged. culture on sorbitol macconkey (smac) agar and biochemical identification, while easy to use and inexpensive, is slow and lacks sensitivity in the detection of non o :h serotypes. this study sought to determine the accuracy of rapid serological or polymerase chain reaction (pcr) assays which have been evaluated for the detection of all ehec serotypes compared to culture. methods: a systematic review and meta-analysis of articles, identified via searches of electronic databases, hand searching of selected journals, and through contact with experts and commercial test manufacturers. the majority of these needed to be excluded due to low quality or lack of accuracy data. sensitivity and specificity of each method was calculated using full biochemical identification as the reference standard. twenty-one studies met the inclusion criteria, of which used pcr methods and used serological assays and were based on culture. a summary receiver operator curve (sroc) was constructed from these data and the area under the curve (auc) calculated (using the trapezium rule). results: serological tests had individual sensitivities ranging from . to . and specificities ranging from . to . . pcr tests had individual sensitivities ranging from . to . and specificities ranging from . to . . additional analysis comparing smac agar culture with toxin detection methods showed poor sensitivity compared to pcr and serological tests (ranging from . to . ) yet the specificity was very good ( . for all studies considered). our results suggest that both molecular and serological tests may have a potential role in detecting ehec infection. whilst there is very little difference in the effectiveness of these techniques, both are faster and have improved sensitivity when compared to traditional culture methods. fast, reliable diagnosis could lead to more informed treatment choices and improved outbreak control measures. however, given the substantial extra cost of these assays, an assessment of economic feasibility is necessary prior to use in everyday practice. antibodies against bordetella pertussis detected by slow agglutination test and elisa in two agerelated groups of vaccinated people suspected of acute pertussis: a comparative study objective: the aim of presented study was to describe differences between results of two tests used for detection of antibodies against bordetella pertussis (slow agglutination and elisa) in two age-related groups of patients suffering from respiratory infection. each of the people has undergone vaccination against b. pertussis. methods: paired sera obtained from two age-related groups of patients [( ). age - years, n = ; ( ). age above years, n = ] suffering from acute respiratory infection were tested. the first group comprised the children who were vaccinated earlier than one year before testing; the second group was determined by longer interval between the vaccination and the testing. the criterion of positivity of the slow agglutination was based on quadruple increase/decrease of the titer of specific antibodies; the criterion of serodiagnosis of the illness was the same. each of the patients was tested by elisa iga,igg,igm (virotech) during the same period, positive results of each of class of immunoglobulins were evaluated as positive elisa. the differences of results obtained by the two tests were assessed inside and between the groups. results: there were found . % (respective . %) concordant positive and . % (respective . %) concordant negative results between the tests in the first (respective second) group. there were found the following discrepancies in the frame of non equal results: agglutination positive/elisa negative sera were present in . % (respective . %) persons and agglutination negative/elisa positive samples were present in . % (respective . %) persons. conclusion: ( ) . the frequency of serologically confirmed infection based on results of slow agglutination is higher in the group of people older six; the interpretation of the results in the younger group is limited by the influence of actual vaccination. ( ) . the elisa evaluated as described above shows extremely high frequency of positivity in both groups, thus, the usefulness for diagnostics of acute infection seems to be low. ( ) . the study will be continued to asses relationships between the positive results detected by slow agglutination and the positive ones detected by elisa in separate classes of specific immunglobulins. accuracy of the microscan walkaway system to identify coagulase-negative staphylococci a. sáez, b. ruiz, l. martínez-martínez (santander, es) objective: to determine the reliability of the identification of coagulase-negative staphylococci (cons) with the microscan walkaway (wa, dade behring) system at species level when a > = % probability is obtained, considering as a reference the results of molecular identification. methods: one hundred and sixty-eight isolates of cons from clinical samples (october -may for which the identification with the wa system was ‡ %, and atcc type strains were evaluated. bacteria were identified with the wa system using pos combo s panels. absence of coagulase was determined with a latex assay (pastorex Ò staph-plus, bio-rad). reference identification was established by sequencing of the s rrna; when identification with wa and s rrna disagree, definitive identification was defined after sequencing of the soda and tuf genes, as previously described (drancourt et al. jcm ; : - and heikens el al. jcm ; : - ) . for identification, the sequences of s rrna, soda and tuf were compared with those in genebank. homologies values above % were considered reliable. results: all type strains were correctly identified by s rrna sequencing as named by the atcc. among the clinical isolates, the molecular method identified the following species (number): s. hominis ( ), s. haemolyticus ( ), s. saprophyticus ( ), s. epidermidis ( ), s. lugdunensis ( ), s. schleiferi ( ), s. capitis ( ), s. simulans ( ), s. pasteuri ( ), s. warneri ( ), s. intermedius ( ) and s. equorum ( ). the wa system correctly identified out of the atcc strains. s. pasteuri is not included in the wa database, and the corresponding atcc strain was misidentified as s. warneri. one hundred and fiftyseven out of the ( . %) clinical isolates were correctly identified by the wa. five s. haemolyticus were identified by wa as s. auricularis ( ), s. simulans ( ) and s. warneri ( ) . other errors corresponded to: two s. pasteuri misidentified as s. warneri, one s. epidermidis as s. hominis, one s. lugdunensis as s. schleiferi, one s. hominis as s. haemolyticus and one s. equorum as s. cohnii. all isolates of s. saprophyticus, s. schleiferi, s. capitis, s. simulans, s. warneri and s. intermedius were correctly identified by the wa system. conclusions: the microscan walkaway is reliable to identify cons at species level when a probability of > = % is obtained. s. pasteuri should be incorporated to the wa database in order to improve its performance. objectives: the aim of this study was to analyse the results of proficiency testing obtained by polish microbiology laboratories participating in polmicro. haemophilus influenzae is an important pathogen causing a variety of community-acquired respiratory tract infections, acute otitis media and purulent meningitis. two mechanisms of ampicillin (amp) resistance in this organism are described. one is mediated by the production of beta-lactamases tem- and rob- ; these amp-resistant strains are termed beta-lactamase-producing, amp-resistant (blpar). the second mechanism involves development of altered penicillin-binding proteins (pbp) with decreased affinity to amp and other beta-lectam agents. strains with resistance mechanisms mediated by pbp alterations are termed beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae. methods: four hundred seventy eight laboratories participated in this part of the scheme. each participating laboratory received haemophilus influenzae (pm- )-beta-lactamase negative, ampicillin-resistant strain (blnar). the laboratories were asked to provide identification to the species level and of the susceptibility results and interpretation. results: correct identification to the species level of this strain was reported by laboratories ( . %) of the labs involved. thirteen laboratories reported the analysed strain as haemophilus parainfluenzae. three hundred ninety eight laboratories ( . %) of correctly detected the mechanism of resistance to beta-lactams. only three laboratories incorrectly reported the organism as beta-lactamase producer. the greatest dispersion of inhibition zone was observed in the susceptibility of h. influenzae to ampicillin, amoxicillin-clavulanic acid and clarithromycin. conclusions: over % of the laboratories correctly identified and interpreted beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae strain. purpose and methods.the architect syphilis tp assay is a chemiluminescent eia that employs three recombinant antigens of treponema pallidum on the solid phase and an anti-human igm and igg conjugate. we evaluated this assay in comparison with a conventional eia (diesse enzywell syphilis screen recombinant) on unselected routine serum samples and on repository specimens for whom the results for specific igg and igm and of the rapid plasma reagin (rpr) assay were already known. in both instances an immunoblot (ib: inno-liatm syphilis score, innogenetics) has been employed on discordant specimens as a confirmatory assay. the precision and robustness of the architect assay were also evaluated.results.on . routine samples ( from volunteer blood donors and from in and outpatients) the concordance between architect and eia was high ( . samples, or . %; positives, , negatives). one of the discordant, positive by architect and negative by eia, was confirmed by ib. the specificity of the architect assay was . % ( % confidence limits: . - . ). the repository samples assayed belonged to three groups: ) biological false positives from subjects: all negative by architect; ) true positives, all positive by architect, with a significantly stronger signal (average s/co: . vs. . ) on the igm positive samples, all of whom were also positive by rpr; ) samples positive by rpr and negative by eia igg: of them were negative by architect as well and for igm, while two specimens were strongly positive by architect and positive also for specific igm and with three specific bands by inno-lia, suggesting a pattern of recent infection. the reproducibility of the architect assay was good, with cvs of . %, . % and . % on replicates over weeks of the assay's negative and positive control and of an internal control; finally, the s/co distribution of negative specimens confirmed the robustness of the assay, with a mean of . , a median of . , standard deviations between the mean and the cut-off value and the th percentile at a s/co value of . .conclusion.the automated assay for anti-treponema pallidum antibodies on the architect system has an excellent sensitivity and a good specificity. the analytical performances, coupled with the elevated throughput and minimal samples handling, make this method a first-choice option for syphilis screening and diagnosis in medium and large volume laboratories. objective: quantitative urine culture is the gold standard for defining the diagnosis of urinary tract infection (uti), because it allows identification of the uropathogenic species. however, this method is time consuming and expensive. approximately, up to % of urine cultures are negative with high cost for unnecessary testing. thus, we have evaluated the usefulness of two automated analysers for uti screening to quickly identify the negative samples that can be prompt reported to the clinicians, improving in the quality of patient care and allowing the laboratory to direct more effort into positive samples. methods: . of midstream urine samples submitted for microbiological examination were analysed by conventional urine culture plates (mcconkey agar + trypticase soy agar + bile esculine azide), sysmex uf- (sysmex, japan) and coral uti screen (coral biotechnology, ca, usa) automated analysers. uti was defined positive as follows: one or two strains of bacteria with at least ufc/ml for the culture plates, more than . bacteria/ll and more than wbc/ll for the uf- and/or an rlu value grater than % of the calibrator value for the coral. when more than two strains of bacteria were found, the culture was classified as contamined. results: the diagnostic performance of sysmex uf- and coral uti screen are shown in table . the sensitivity ( . %) and negative predictive value ( . %) confirm that sysmex uf- and coral uti screen are an excellent screening for uti. after this evaluation, we decide the use of the sysmex uf- and coral uti screen on our routine workflow for uti screening. the results of both the analysers are sent to a software system (labfinity dasit, italy) connected to the lis. if the results are lower than the cut-off values, uti can be excluded and directly reported to the physician. positive results are submitted to microbiological culture and reported within or hours depending on negative or positive bacterial growth. in our experience, evaluated on further . samples, this means that % of samples are immediately reported within very few hours. of the % of positive samples, ( %) were confirmed by culture and reported within hours, ( %) were not confirmed and reported within hours. comparison of the blood and bone marrow culture positivity rates for the diagnosis of brucellosis objectives: brucellosis is a common disease, seen worldwide as well as in our country. the diagnosis of brucellosis is made with certainly when brucellae are recovered from blood, bone marrow. in our study, we aimed to compare the blood and bone marrow culture positivity rates in patient with brucellosis. methods: this study was performed in the infectious diseases and clinical microbiology department of ankara research and training hospital between and . the diagnosis of brucellosis was made on the history, physical findings, serologic findings and the isolation of the organism. the number of patients with brucellosis included to the study was . blood and bone marrow samples were taken from all of the patients on admission and cultured by using the bactec system. results: blood culture positivity for brucellosis was % ( / ), while bone marrow culture positivity was % ( / ). the difference between those positivity rates was found to be statistically significant (p < . ). the isolation ratio from blood cultures among acute cases was % ( / ) while it was % ( / ) among subacute cases. brucella isolation from blood was not detected in chronic cases. the isolation rates of the microorganism from bone marrow of acute, subacute and chronic cases were . %, . %, . % respectively. among our patients, had history of medical therapy for brucellosis before admission and of them was treated inadequately. of those cases, the organism was isolated in ( %) from blood and in ( %) from bone marrow.in the cases with high standard tube agglutination titers, the rate of positivity was also high both in blood and bone marrow cultures. however when compared with low standard tube agglutination titers, that difference was not statistically significant.the mean growing time for the positivity of cultures was . days for bone marrow and was . days for blood cultures. the difference between the mean growing times of two culture types was found statistically significant (t-test. p < . ). conclusion: premedication, subacute and especially chronic phases decrease the possibility of isolation of the microorganism from blood culture. therefore we suggest taking bone marrow culture only for these kinds of patients as it. is a traumatic process. serological findings in blood sera of patients with yersinia-triggered arthritis e. golkocheva, r. stoilov, h. najdenski (sofia, bg) objectives: immunoblot analysis of iga and igg antibody response of blood sera from patient with yersinia triggered reactive arthritis and with undifferentiated arthritis were made. patients and methods: serum samples were obtained from patients admitted to clinic of rheumatology at medical university, sofia, bulgaria with suspicion of yersinia triggered reactive arthritis, based on diagnostic criteria. a total of blood serum samples were analysed by immunoblot analysis with specific antigens-yops (yersinia outermembrane proteins). when y. enterocolitica is cultivated at o c under calcium restriction ( . mm ca + ), large amounts of yops are secreted into medium. these proteins were separated by d-sdselectrophoresis. results: immunoblot analysis of iga and igg antibody response against yops in blood sera from patients with arthralgias and polyarthropathies was carried out. yersinia enterocolitica, serotype o: , was used as source for yop. seven strong bands of the molecular weights kda-yope, kda-yopn, kda-yopd, kda -v-ag, kda-yopb, kda-yopm and kda-yoph were visualized. for immunoblot assay the optimal concentration of antigen was established by analytical electrophoresis. of the blood sera from the patients with yersinia triggered reactive arthritis igg antibodies were detected against yoph, yopm, yopb, yopd, yopn and yope. iga antibodies were established against yopm, yopb, yopd, yopn and yope. all sera from the patients with other rheumatic diseases were negative for the presence of anti-yersinia iga antibodies and two of them were positive for igg against yopd. antibodies from two classes were not detected in sera samples from healthy people. conclusions: yops are borne by the virulence plasmid, which mean that they are clearly associated with virulence properties of pathogenic strains. moreover, yops is not restricted to single serotype and this made them a specific antigen in diagnosis of different yersinia infections. conventional techniques such as culture and demonstration of serum agglutinins prove to be insufficient to demonstrate invasive or chronic yersiniosis in contrast with the determination of specific serum iga and igg antibodies by immunoblot analysis and antigen detection. the detection of anti-yops igg and iga antibodies by immunoblot can be used for diagnosis of yersinia triggered arthritis. acknowledgements: this work was sponsored by natoreintegration grant . objectives: to evaluate the identification and susceptibility results by using suspensions obtained directly from positive blood cultures. methods: during the period between st august and st october we selected all positive cultures grown in bact/ alert Ò sa and sn bottles (biomérieux) from gram-negative bacilli. only the first culture positive from each patient was included. we inoculated ml fluid from a positive bottle into a serum separator tube (bd vacutainer systems, plymouth, united kingdom) and centrifuged at x g for minutes and the supernatant was carefully aspirated. using a cotton swab the bacteria were removed from the top of the separator layer to be suspended in . % saline solution to get . mcfarland. the suspension was processed according to standard inoculation procedure for gn and ast-n vitek Ò cards. positive bact/alert d bottles were also sub cultured and after an overnight incubation several colonies were used to make a . mcfarland suspension in . % saline. the suspension was processed according to standard vitek Ò inoculation procedure for gn and ast-n cards. results: identification: a total gram-negative bacillus from positive blood cultures were investigated. fifty ( . %) strains were correctly identified to the species level, four ( . %) strains were not identified and two ( . %) strains were misidentified. antimicrobial susceptibility testing: in all, mics were determined for isolated by both methods. the unidentified strains ( ) were excluded. the overall mic agreement between direct and standard inoculation was . %. all individual antimicrobial agents scored > %. the overall minor error rate was . % ( of ). the overall major error rate was . % ( of ). the overall very major error rate was . % ( of ). the highest rate of mic agreement was for amikacin, norfloxacin ( %), meropenem ( %), gentamicin and ofloxacin ( . %). conclusion: the direct method from positive bact/ alert&# ; cultures cannot totally replace the approved methods of identification and susceptibility but in some cases provides earlier information which allows a better patient management and also reduce cost in patient care. investigation of listeria monocytogenes "o" antibodies in maternal and cord sera with the agglutination test e. us, a.t. cengiz, o. gelisen (ankara, tr) objectives: listeria monocytogenes is a gram-positive food borne pathogen that is responsible for listeriosis, a human infection with a mortality rate of %, which could cause severe motherto-child infections. this serious pathogen in pregnancy could be treated if diagnosed, but there is no routine screening test for susceptibility to listeriosis during pregnancy. therefore, we investigate different l monocytogenes serotype o antibodies for diagnosis of listeriosis in maternal sera with agglutination test. of them had spontaneous abortion, premature labour or stillbirth (group i), while had no obstetric patology (group ii) in their previous pregnancies. cord bloods were also obtained at the delivery and tested. methods: all sera were being tested against antigens with the o formulation of serotype / c, b, ab, c and d. the antigens were prepared by the method of osebold, and larsen et all. the bacterial suspensions were trypsinized for min at °c to prevent cross-reactions and contaminations. sera were diluted by doubling serially in saline followed by addition of an equal volume of antigen. a positive titre of greater than or equal to : was chosen as positive test result to maximize the sensitivity and specificity. results: . % of cases have ingested raw milk and diary products, . % ready-to-eat foods, and . % developed nonspecific febrile illness (nfi) during their pregnancies. % of group i were found positive ( . % developed nfi) while at group ii % had positive ( . % developed nfi) agglutination titres to one ore more serotypes. all the cord blood sera of group i were found negative, whereas two in group ii (all ab) were positive, with the positive maternal sera of the same serotype. it's evaluated as transmission of the antibody from mother to foetus. at group i the frequent serotypes were / c = ab, at group ii ab, / c, respectively. the newborns showed no symptoms or signs of listerial foeto-maternal infection. conclusion: the women encountered the antigens of l monocytogenes in any period of their life time (most - years of age) and produce antibodies against this pathogen. there is a relationship between nfi and positive titres. if the disease is recognized, it is possible to treat the mother and allow the birth of a healthy infant. we propose the less time consuming and easy to perform agglutination test as a routine screening test for susceptibility to listeriosis during pregnancy to prevent bad pregnancy outcomes. objectives: to evaluate the performance of a real time pcr assay (with a fluorogenic target-specific probe), mrsa-idi (geneohm sciences) for mrsa detection directly from mucocutaneous swabs in hospitalized patients. methods: clinical swabs ( to samples with a median of . samples per patient) from nares (n = ) and skin (n = ) were prospectively collected for mrsa screening from patients admitted to a -bed teaching hospital. swabs were inoculated onto selective mrsa agar (mrsa-id, biomérieux), into the buffer extraction solution for idi-mrsa pcr assay and into enrichment broth (bhi with . % nacl). after h, bhi broths were subcultured onto mrsa-id agar. selective agars were incubated for h at ordm;c and examinated daily. suspected colonies were identified by coagulase testing; oxacillin resistance was tested by cefoxitin disk diffusion according to clsi recommendations. the pcr assay was performed according to the manufacturer's instructions. pcr results were compared with phenotypic identification test results. in case of discordant results, the assay was repeated, but only results from first testing were considered for calculating test performance. results: mrsa was detected by culture in specimen ( . %) from patients. the sensitivity and specificity of the pcr compared with culture was . % and . %, respectively. positive predictive value and negative predictive value were . % and . %, respectively. the sensitivity of pcr ( %) was higher on nasal swabs than on swabs from other sites ( . %, p < . ). the pcr assay detected mrsa in patients ( . %). the pcr assay provided results in to versus to hours for conventional method. conclusion: in our hospital, the id-mrsa pcr assay detected . % mrsa carriers in less than hours when performed on multiple specimen. the assay appeared more sensitive in testing nasal swabs than other clinical specimens. prospective studies are needed to evaluate the impact of this assay for rapid implementation of infection control procedures and its global costs and benefits. the purpose of this study was to establish a rapid and sensitive real-time polymerase chain reaction (pcr) method for detection of methicillin-resistant staphylococcus aureus (mrsa) from blood culture bottle. as a result of over use of broad-spectrum antibiotics after the s in whole the world, an outbreak of mrsa infection has been seen. severe nosocomial infections with mrsa such as bacteraemia and sepsis may lead to multiple organ failure and high mortality in the hospital. although standard method took at least hours to identify mrsa by the blood culture method, the presence of meca and nuc genes which is specific for methicillin resistance and s. aureus was determined by real-time pcr method within only hours after blood culture signal positivity. nineteen s. aureus and coagulase negative staphylococci positive blood culture bottles were studied retrospectively for detection of s. aureus and methicillin resistance. staphylococci were identified with classical methods and mics of oxacillin were determined by etest (ab biodisk) on mueller-hinton agar supplemented with % nacl. real-time pcr was performed to all positive blood culture samples for s. aureus and methicillin resistance determination. nineteen ( %) s. aureus were determined correctly by real-time pcr method. forty-four methicillin resistant and methicillin sensitive staphylococci were detected by etest. using the real-time pcr method, the meca gene was detected in staphylococci except . when compared with etest and realtime pcr method gave sensitivity, specificity, and positive and negative predictive values of %, %, %, % for both positive and negative tests, respectively. agreements between two methods were high ( %); there were discrepant results among the strains were tested. detection of mrsa bacteraemia and methicillin resistance with real-time pcr definitely is useful for reducing mortality and morbidity of this type infection. in conclusion, this method, as many as sensitive and specific for detection of mrsa bacteraemia and clinically should be beneficial for prevention of unnecessary antibiotic use and determination of appropriate antibiotic treatments of mrsa infection. pcr detection of class b, c and d betalactamases in environmental and clinical aeromonas strains t. fosse, c. giraud-morin, f. la louze (nice, fr) objectives: aeromonas spp. strains are waterborne opportunistic pathogens. they are able to produce different types of beta-lactamases (class b, c and d). the determination of beta-lactamase content is not easy by phenotypic methods. we have developed a pcr tool to study diversity and distribution of class b, c and d beta-lactamases in a set of representative clinical and environmental aeromonas species. method: a total of references, environmental and clinical strains were tested. identification was realized by conventional tests and gyrb sequence analysis. beta-lactam antibiotic susceptibility was determined by diffusion agar and micro broth dilution methods. three sets of specific primers were defined for the pcr amplification of the internal region of class b beta-lactamase (mei and mei , bp size), class c beta-lactamase (aercp and aercp , bp) and class d betalactamase (aerd and aerd , bp). all pcr products were sequenced. results: class d pcr was positive with most strains except a. trota, a ticarcillin susceptible species ( strains) . class c pcr was positive with most cephalothin resistant strains (mic > mg/l; / strains, %) including a. hydrophila and a. caviae phenospecies. class b pcr was positive with most strains of a. hydrophila and a. veronii phenospecies ( / ; %) including three imipenem susceptible strains (mic < mg/l). beta-lactamase type distribution was species related and was particularly useful to better characterize environmental species such as a. bestiarum, a. popoffii and a. allosaccharophila. partial beta-lactamase gene sequence analysis allowed phylogenic studies. some cephalosporinase gene from environmental species was probable progenitor of ampc plasmidic beta-lactamase. conclusion: pcr with specific primers was a good method to detect class b, c and d beta-lactamase in aeromonas species. beta-lactamase type distribution and sequence analysis phylogeny were largely species related and could be helpful for molecular diagnostic and taxonomic purpose. objectives: the aim of this study was to develop a convenient dna extraction method and to optimise a pcr reaction in order to detect enterotoxin b producing s. aureus strains directly from milk. methods: we applied a chemical extraction method of bacterial dna from milk samples artificially inoculated with s. aureus. a pcr based method was used for the detection of seb gene (coding for enterotoxin b) and nuc gene (coding for termonuclease). a protocol for the multiplex pcr was developed and optimized. the sensitivity of the reaction was checked by determining the minimum number of organismsaeml - , which can be detected in the multiplex pcr and in each single pcr reaction. amplification specificity of the seb gene was verified by amplicon digestion with restriction endonucleases. results: the specific bands for both genes in the multiplex pcr were detected in samples containing a dna quantity corresponding to organismsaeml ) . in the same reaction, the amplicon for nuc gene was visible for as little as the dna concentration corresponding to organismsaeml ) . the sensitivity of each single pcr reaction was similar with those of multiplex pcr reaction. conclusion: the applied dna extraction method allowed us to obtain a good quality dna and can be used for a direct milk extraction. multiplex pcr reaction is a simple, rapid and reliable method for detecting enterotoxin b producing s. aureus strains from milk. objective: to detect the resistance to fluoroquinolones in acinetobacter baumannii strains by a pcr-rflp assay. methods: thirty a. baumannii clinical isolates were obtained from different specimens (bronchial aspirates, blood-cultures, catheters, etc.) . the mics (minimal inhibitory concentrations) for ofloxacin were determined by agar dilution following standard methodology.a pcr-rflp method using one primer pair for amplification of a bp fragment related to gyra gene (which codifies subunity a of dna-gyrase) and using one restriction enzyme hinf i was developed to study the resistance to ofloxacin in the different a. baumannii strains. when an a. baumannii strain is resistant to fluoroquinolones, a mutation in the position ser of the dna-gyrase has been detected, decreasing the affinity for the antimicrobial. agarosa gel was used to determine the dna pattern: fragments of bp and bp when there is not mutation and fragment of bp when the ser to leu mutation is present. results: the relationship between the pcr-rflp pattern and the mic to ofloxacin is shown in the table . the results of pcr-rflp analysis of most strains were in agreement with the results of mic. one isolate was susceptible to ofloxacin by agar dilution (mic = . mg/l) whereas by pcr-rflp this isolate seems to be resistant because it presents the mutation in gyra gene. two isolates with intermediate mic ( mg/l) showed mutation in gyra. the genotypic study by pcr-rflp proved that ofloxacin resistant a. baumannii strains showed a punctual mutation in gyra gene, in the same position inside the sequence of gene. evaluation of a rapid amplification-detection assay for the identification of vancomycinresistant enterococci j. fuller, l. turnbull, s. shokoples, b. lui, l. rosmus, r. rennie (edmonton, ca) objective: the routine identification of vancomycin-resistant enterococci (vre) in clinical laboratories often yields a lengthy turn-around-time that may impede infection control efforts, particularly in an outbreak situation. in search of an improved vre test, we evaluated the genotype Ò enterococcus assay (hain lifescience, germany), which provides both species and van gene identification for vre, and compared the results to conventional methods. methods: forty clinical enterococcal strains isolated on vrescreen agar media were selected for study. lactococcus and pediococcus were used as negative controls. conventional testing involved basic culture and identification tests, e-test susceptibility testing for vancomycin and teichoplanin, and pcr for vana, b, and c genes. the genotype Ò enterococcus assay involved multiplex dna amplification and reverse hybridization of amplified product on an immobilized dna strip-blot containing probes for e. faecium, e. faecalis, e. casseliflavus, e. gallinarum, vana, vanb, vanc , and vanc / . the genotype Ò enterococcus assay produced correct species and van gene identification for all ( %) vre isolates, including e. faecalis vanb, e. faecium vana, e. faecium vanb, e. gallinarum vanc , e. gallinarum vana-vanc , and e. casseliflavus vanc / . the only minor discrepancy was an e. casseliflavus that hybridized very weakly with the vanc probe in addition to the expected vanc /c probe. the costs per specimen were comparable for each test method. however, the genotype Ò enterococcus assay could be completed within a normal working day in contrast to conventional testing, which required a minimum of two days from the point of isolation on the vancomycin-screen media. conclusion: from this preliminary evaluation, the genotype Ò enterococcus amplification-detection assay provides vre species and van genotype identification in a rapid and costeffective manner, superior to conventional culture methods. although further study is required, this kit may have clinical utility during a vre outbreak. application of minimal sequence quality values prevents misidentification of blashv type in single bacterial isolates carrying different shv extended-spectrum beta-lactamase genes background: detection of extended spectrum beta-lactamase (esbl) genes by pcr and sequence analysis is the gold standard for detection of shv-type beta lactamases. usually, quality values of sequence analyses are not reported. during a study on esbl epidemiology, three strains for which the default sequence assembly showed an shv) or shv- gene, showed low quality values at certain positions in individual sequence traces. we investigated the reason for these lower values. methods: shv genes were amplified by pcr from three isolates (escherichia coli, enterobacter cloacae and pseudomonas aeruginosa). individual sequence traces were analysed with the computer programs phred and codon code. pcr products were ligated in vector pcr . and transformed to e. coli. sequence analysis was performed on eight individual clones from each transformation. results: visual inspection of the low quality positions in the sequence traces showed signals for two different nucleotides at three positions in the shv sequence: a or t at position , a or g at position and a or g at position . the polymorphisms at positions and lead to aminoacid substitutions, the four different combinations would give shv types , a, or . the double signals suggested that two or more blashv alleles were amplified. pcr amplicons were cloned in e. coli, in the sequences of individual clones only two combinations of the three polymorphisms were present: a g a and t a g . these two combinations correspond to shv- and shv- , respectively. conclusions: (i) in isolates of three different species, two different shv genes were present: shv- and shv- . (ii) genotypic detection with default sequence assembly parameters may lead to misidentification of the number and type of shv genes carried by a single strain. (iii) careful interpretation of sequence data of shv genes, including analysis of low quality positions, may further improve our understanding of the epidemiology and evolution of these esbl genes. antimicrobial susceptibilities and epidemiological analysis of salmonella typhimurium human isolates in slovakia by phage typing and pulsed-field gel electrophoresis v. majtán, l. majtánova, m. szabó ová (bratislava, sk) objectives: salmonella typhimurium is a common cause of salmonellosis among humans and animals in many countries. in the last few decades the incidence of multidrug-resistant s. typhimurium infections appears to pose a particular health risk. the objectives of this study were analysis by antibiotic susceptibility, phage typing and pulsed-field gel electrophoresis (pfge) of s. typhimurium human isolates. methods: a total of strains isolated during -september were analysed. the susceptibility of isolates to ten antibiotics was evaluated by a disk diffusion method. the phage types were identified according to anderson et al. ( ) in the national reference center for phage typing of salmonellae. pfge was used to resolve xbai macro restriction fragments from all strains. results: of human isolates ( . %) were resistant to more than two antibiotics. sixty-three of isolates ( . %) showed a classic dt resistance profile to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline (acssut). among this resistance type . % were dt , . % were dt and one strain was dt a. isolates encompassed phage types. the majority of isolates was found to be definitive phage type dt , representing . % of all isolates. other phage types were mainly dt , dt and dt a. nine pulsotypes and subpulsotypes were obtained using xbai restriction enzyme, but pattern x with its subtypes predominated ( . %). a major pulsotype x was represented by . % of dt isolates and was also found among dt isolates. conclusion: results indicated the spread of different clones of the multidrug-resistant s. typhimurium in the slovakia, but with predominance of one clone represented mainly by dt isolates. the phage typing as well as pfge may offer an improved level of discrimination for the epidemiological investigation of s. typhimurium human strains. novel reverse hybridisation assay to identify ctx-m genotype in cephalosporin-resistant isolates from uk and india to validate the assay results by dna sequencing. methods: isolate collection : enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in london and south-east england. these isolates were known to carry phylogenetic group blactx-m, but precise genotypes had not been determined. isolate collection : enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in aligarh, north india. resistance determinants had not been investigated previously. a novel multiplex pcr was used to amplify blactx-m. reverse hybridisation was carried out using biotinylated pcr amplicon and sequence-specific oligonucleotides designed to identify members of ctx-m phylogenetic group . hybridisation results were validated by dna sequencing for representative isolates from each collection. results: / london and se england isolates known to carry group blactx-m gave a consistent profile, corresponding to that for ctx-m- and ctx-m- ; / gave a profile corresponding to ctx-m- and ctx-m- . / indian isolates had blactx-m genes, all of which belonged to group , and all these gave a hybridisation profile corresponding to ctx-m- or ctx-m- . ctx-m- and ctx-m- are rare variants, suggesting that the enzymes present were more likely to be ctx-m- and ctx-m- , and this was confirmed by dna sequencing. conclusions: this is the first reported application of this novel reverse hybridisation assay to the analysis of large numbers of cephalosporin-resistant enterobacteriaceae. results were validated by dna sequencing. the assay is cheap and convenient, enables reasonable throughput, provides results within one day and can be used in place of dna sequencing. we believe it will be valuable for monitoring the prevalence and genotypes of blactx-m genes in enterobacteriaceae. detection of mexa and mexx efflux genes in p. aeruginosa: correlation between qc-rt-pcr and real-time pcr objectives: efflux systems are rarely identified as such in clinical microbiology laboratories. yet, over expression of transporters such as mexab-oprm and mexxy-oprm are likely to cause antibiotic multi-and cross-resistance in pseudomonas aeruginosa, leading to potential clinical treatment failures because of their inducible character. we have previously developed and validated with reference strains a qc-rt-pcr method to quantify mexa and mexx expression levels (eccmid . in the present study, we have developed a real-time-pcr assay and present here the correlation between both methods using control strains and clinical isolates. methods: expression levels of mexa and mexx were measured by both techniques in (i) reference strains expressing only one of these efflux mechanisms [mexa ( ) or mexx ( ) ]; and (ii) clinical isolates, in comparison with the wild-type strain pao (basal mexa and mexx expression levels). results: real-time pcr showed an inter-day reproducibility of ± . % (triplicates of strains). among the clinical strains, over expressed mexa and mexx. the table shows (i) the mean level of overexpression of mexa and mexx in comparison with the wild type strain pao (set at ), as detected by real-time pcr for all strains; (ii) the ratio of these values to those observed by qc-rt-pcr for the corresponding transporters. conclusions: both qc-rt-pcr and real-time-pcr are potentially useful in clinical laboratories as sensitive and rapid diagnostic tools to quantify the expression level of mexa and mexx in p. aeruginosa. combined with phenotypic characterization, this approach may help in a better understanding of the resistance mechanisms and epidemiology of resistance in this difficult-to-treat nosocomial pathogen. molecular detection of penicillin resistance in streptococcus pneumomiae n.g. rizk, n.a. abo khadr, s.m. abdel salam, n.m. gamil, m. hassan (alexandria, eg) objectives: the aim of the study was to detect penicillin resistant streptococcus pneumoniae by using seminested polymerase chain reaction (pcr) and to compare it with minimum inhibitory concentration (mic) of penicillin g. methods: fifty clinical isolates of streptococcus pneumoniae where isolated from patients admitted to alexandria main university hospital in egypt and were recovered from sputum ( strains), throat swabs ( strains), and pleural effusion ( strains) . two species-specific primers a- and a- , which amplified bp region of the pbp a penicillin-binding gene, were used for pneumococcal detection. two resistance primers, a-r and a-r , were used to bind to altered areas of pbp a gene which, together with the down stream primer a- , amplify dna sequences of bp and bp from isolates with penicillin mic > . objective: lipopolysaccharide-binding protein (lbp) is an acute phase protein produced in the liver. the objective of our study was to evaluate lbp as a marker of severity and prognosis in patients with bacteraemia. methods: adult patients with community-acquired bacteraemia were included in a prospective manner. daily blood sampling for lbp and interleukin- (il- ) was performed. the patients were classified according to the systemic inflammatory response syndrome (sirs) criteria. demographic data, co-morbidity, microbiological aaetiology, routine biochemical parameters, focus of infection, severity score and mortality on day were recorded. lbp and il- levels were analysed on plasma samples with a chemiluminescent immunometric assay (immulite- Ò ). results: the median age was yrs. the mortality rate on day was . %. patients had bacteraemia without sirs, patients had sepsis and patients had severe sepsis. lbp concentrations are presented as medians and range: . lg/ml ( . - . ) in patients without sirs, ) in patients with sepsis and . lg/ml ( . - . ) in patients with severe sepsis (p < . ). lbp levels correlated to levels of il- (rs . ), c-reactive protein (rs . ), leukocytes (rs . ) and neutrophils (rs . ) (p < . ). lbp did not predict the outcome of the patients with bacteraemia. conclusion: lbp levels increased with the severity of sepsis in patients with bacteraemia. lbp correlated to il- , c-reactive protein, leukocytes and neutrophils. lbp did not predict the outcome of the patients in this small cohort. pyrosequencing of the gra gene to discriminate type i, ii and iii toxoplasma gondii in clinical samples b. edvinsson, b. evengård on behalf of the esgt objectives: infection with toxoplasma gondii in immunocompromised transplant recipients is rare but often fatal. to increase our knowledge about the significance of the genotype of the parasite during infection, methods suitable for routine use need to be developed. pyrosequencing is a rapid sequencing-bysynthesis method performed in real-time. it is developed for detection of short nucleotide polymorphisms (snps), and is suitable for molecular genotyping of microorganisms. we here present a pyrosequencing assay for rapid and reliable discrimination of toxoplasma gondii type i, ii and iii in clinical samples. methods: twenty-two isolates of t. gondii were used for pyrosequencing analysis of the gra gene. real-time pcr was performed using a lightcycler . instrument to amplify a bp fragment of the gra gene. pyrosequencing analysis of two different snps contained within a bp fragment of the amplified product was preformed to identify t. gondii type i, by detection of nucleotides g and a at these respective positions. type ii was g and g, and type iii was a and a. to test the assay in a clinical context, blood samples and lung tissue from an immunocompromised patient was analysed. results: the detection limit of the assay is parasitic genomes in a sample. reproducibility (r) was calculated as r = nr/n (nr = the number of isolates assigned the same type on repeat testing and n = the number of isolates tested). r was determined using three independent runs, and was , suggesting clearly interpretable results with little variation. typeablility (t) of the assay was calculated as t = nt/n (nt = the number of typeable strains and n = the number of isolates tested). t was determined using three independent runs, each including four atypical isolates. t was . , suggesting that the assay discriminates correctly between the three main genotypes of t. gondii, but does not detect atypical strains. analysis of the clinical samples revealed type ii t. gondii in blood samples and lung tissue. conclusion: when preceded by real-time pcr, pyrosequencing is a rapid process with a high reproducibility and throughput. this makes it a good candidate for routine use. the method does, however, not detect atypical or recombinant strains. more than one gene may have to be analysed for that purpose. acknowledgement: in particular, we want to thank marie-laure dardé and hervé pelloux for provision of the t. gondii isolates. virulence genes in escherichia coli isolates from calves in shahrekord area, iran shiga toxin-producing escherichia coli (stec) strains, also called verotoxin-producing e. coli (vtec) strains, represent the most important recently emerged group of food-borne pathogens around the world. members of this group are a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (hc) or the hemolytic uremic syndrome (hus), which is the main cause of acute renal failure in children. domestic ruminants, mainly cattle, sheep, and goats, have been implicated as the principal reservoir. transmission occurs through consumption of undercooked meat, unpasteurized dairy products and vegetables, or water contaminated by feces of carriers because stec strains are found as part of the normal intestinal floras of the animals.we studied the prevalence of shiga toxinproducing escherichia coli (stec) in stool specimens of calves with diarrhoea or other gastrointestinal alterations from dairy cattle farms of shahrekord city (central of iran). the virulence genes, stx , stx , eae, intimin hly, enterohemolysin, st, lt, were detected by multiplex pcr method. stec strains were detected in ( . %) of e. coli from cases investigated. stec o was isolated in cases ( . %), whereas non-o stec strains were isolated from animals ( %). stec strains were the most frequently recovered enteropathogenic bacteria. pcr showed that ( . %) isolates carried st gene. none of isolates carried an ehxa, eae, and lt (labile toxin) genes. our results suggest that stec strains are a significant cause of calf infections in this area and confirm that, infections caused by stec non-o strains are more common than those caused by o :h isolates. the high prevalence of stec strains (both o and non-o strains) also found in human patients by other investigators, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of stec in spanish clinical microbiology laboratories. objectives: shiga toxins are a-b holotoxin including one enzymatically active a subunit associated non-covalently to five identical receptor binding b subunits. each subunit can cause different signalling pathways in different cells. to assess the effect of each single subunit the specific clones for expressing the single subunit was designed. periplasmic expression yielded native ab holotoxin or b pentamer. methods: o was used as bacterial strain for pcr amplification of shiga toxin gene. each subunit was amplified by specific primers and the amplified genes were cloned in pbad expression vector. the expression of the cloned genes was induced and optimized by different concentration of arabinose. the expressed proteins was assessed on sds-page and detected by elisa and western blotting. the expressed recombinant ab holotoxin and b subunit were purified and assessed for its biological activity on cells. cell cytotoxicity was shown by the expressed (ab ) holotoxin. moreover inhibition was observed by b subunit and antibody against it. results: e. coli clones expressing recombinant shiga toxin a and recombinant shiga toxin b subunits were established to release the toxin to periplasmic space. expressed toxin was examined by sds-page to visualize two subunits. the whole structure of these expressed subunits was checked in native gel. active ab structure expressed in periplasmic space was extracted by polymyxine b. the biological activity of the constructed recombinant shiga toxin showed both vero cell cytotoxicity and inhibition of in vitro protein synthesis. conclusion: in this study it was shown that for b subunit assembly and secretion to periplasmic space as b pentamer homologous leader sequence is not needed. although for biological active holotoxin (ab ) secretion to periplasmic space the presence of homologous leader sequence of gene is essential. these subunits can be used for studying on cell cytotoxicity and also as a vector for antigen presentation in immunotherapeutic approaches. characterisation of gram-positive anaerobic cocci by biochemical tests and partial s rrna sequencing a. bryk, a. kanervo-nordstrom, m. hyvonen, e. kononen (helsinki, fi) objective: gram-positive anaerobic cocci, which are common findings in various infections, are difficult to identify in clinical microbiology laboratories, where identification is based only on few phenotypic tests. in recent years, this group of organisms (traditionally known as peptostreptococci) has encountered several taxonomic changes. the aim of the present study was to compare the characterization made by a selection of key phenotypic tests to that by partial sequencing of the s rrna gene. methods: fifty-nine clinical isolates sent to our laboratory as gram-positive anaerobic cocci were examined for their colony and cell morphologies and biochemically characterized using spot catalase and indole reaction, enzyme reactions by individual diagnostic tablets (rosco), sodium polyanethol sulphate susceptibility, glucose fermentation, and determination of metabolic end products. in addition, commercial identification test kit (rapid id a) patterns were performed. the sequencing of the s rrna gene of the clinical isolates and reference strains comprised of about bp, and the sequences obtained were compared to those in genbank database by using the multisequence advanced blast comparison software from the national center of biotechnology information. results: the biochemical characteristics of the isolates were consistent with those of peptostreptococcus anaerobius (n = ), peptostreptococcus (micromonas) micros (n = ), finegoldia magna (n = ), peptoniphilus asaccharolyticus (n = ), peptoniphilus sp. (n = ) and anaerococcus sp. (n = ), whereas isolates remained as unidentified gram-positive anaerobic cocci. biochemical identification correlated with that obtained by partial s rrna sequencing in / ( %) isolates at genus level and in / ( %) isolates at species level. the agreement of the biochemical and sequence-based identification was % for p. micros and f. magna. of isolates biochemically identified as p. asaccharolyticus, isolates were identified as peptoniphilus harei and remained as peptoniphilus sp. by sequencing. according to the sequence data, the unidentified isolates were peptoniphilus ivorii. conclusion: most isolates from human infections proved to be f. magna. a relatively good agreement of identification was obtained using biochemical testing and partial s rrna sequencing. objectives: molecular methods for identification of infectious agents in patients with clinical infectious disease are increasingly being used. especially in cases where antibiotics have been given prior to sampling or when fastidious bacteria difficult to grow are the aaetiology of the infection. infectious arthritis is a serious disease where identification of the etiological agent is mandatory for optimal antibiotic treatment as well as indication of the primary focus if not the joint it self. methods: in the present prospective study, synovial fluids taken from patients in elucidation of affected joints and sent to a clinical microbiological laboratory in the copenhagen area, denmark, were examined by conventional (culture, phenotypic tests) and molecular methods (pcr/sequencing of s ribosomal genes). conventional methods included gramstaining and microscopy, aerobic and anaerobic culture and identification. pcr/sequencing included dna extraction, pcr assay which produced a bp fragment of s rdna, and sequencing of both dna strands of the amplicons. sequencing data were edited and a blast search in the ncbi database was done. results: overall a microorganism was identified in of the synovial fluids ( . %). in synovial fluids from nine patients bacteria were identified by either methods [staphylococcus aureus (n = ), streptococcus pneumoniae (n = ), streptococcus dysgalactiae (n = ), citrobacter freundii (n = )]. six synovial fluids were only culture positive; in four of those six specimens coagulase negative staphylococci were isolated. in three of the synovial fluids a microorganism was identified by s pcr only. in two synovial fluids s pcr identified only one microorganism, whereas culturing resulted in two isolates. conclusion: the present study indicates a significant contribution by molecular methods (pcr/sequencing of s ribosomal genes) in recognizing and identification of microorganisms from foci normally considered sterile like synovial fluids. continued suspicion of infected arthritis despite of negative cultures should result in use of molecular diagnostics. direct detection of cardiobacterium hominis by broad-range s rrna pcr and sequencing in the serum of a patient with infective endocarditis e. malli, d. klapsa, a. vasdeki, m. morava, m. pitsitaki, e. petinaki, a. maniatis (larissa, gr) objectives: to describe the detection of cardiobacterium hominis directly in the serum of a patient with infective endocarditis, by employment of broad-range s rrna pcr followed by sequencing. methods: a series of blood cultures were taken from the patient before starting empirical treatment. in addition, ml whole blood was collected in rubber sealed pyrogen-free tubes for direct detection of bacterial dna. bacterial dna was detected by a broad range pcr reaction and sequencing process allowed identification of bacteria species. results: cardiobacterium hominis was identified as the causative agent of infective endocarditis, on two days after the serum collection. blood cultures, simultaneously obtained with the serum sample, remained negative after days of routine incubation; however, after a prolonged incubation of twelve days a gram negative bacterium was isolated from the aerobic bottles, that was identified as c. hominis species, by the usual phenotypic studies (catalase, oxidase reaction, indole, nitrate, etc) which are time-consuming. conclusions: to our knowledge this is the first report of direct detection of c. hominis in the serum using molecular methods, emphasizing the need for the establishment of such methods especially for infections caused by fastidious organisms. identification of dangerous bacterial pathogens by s ribosomal rna gene sequence analysis w. ruppitsch, a. stoeger, a. indra, d. schmid, k. grif, c. schabereiter-gurtner, a. hirschl, f. allerberger (vienna, at) to assess the usefulness of partial s rrna sequence analysis for identification of dangerous bacterial pathogens, a total of isolates comprising bacillus anthracis, brucella melitensis, biovars melitensis, suis, abortus and bovis, burkholderia mallei, burkholderia pseudomallei, francisella tularensis, yersinia pestis, and genus-related and unrelated control strains were sequenced and analysed using the genbank database (blast . . , national institute of health, u.s.a), the microseq database (version . . and v . , applied biosystems, foster city, u.s.a.) , the ribosomal database project-ii database (rdp-ii, release , update , michigan state university, u.s.a), and the ribosomal differentiation of medical microorganisms database (ridom, university of wuerzburg, germany). on genus level all isolates were identified using genbank, rdp-ii, and microseq v . . the older microseq . . database identified % of the tested samples correctly on genus level. the ridom database did not include sequence data of the tested species even on genus level, the ridom database none (''there seems to be, at least currently, no close relative available''). genbank and rdp-ii identified all dangerous pathogens correctly. the microseq v . database identified four of the six species of dangerous pathogens. on species level none of the dangerous pathogens was correctly identified using microseq . . or ridom. as previously noted by various other authors, the most important reason for failure of databases in identifying a bacterium is a lack of the s rrna gene sequence of the particular bacterium in the database rather than misidentification because of poor sequence quality. one must also be aware that the following bacterial species or subspecies have the same s rrna gene sequence, which makes differentiation by sequence analysis impossible: b. anthracis and b. cereus, y. pestis and y. pseudotuberculosis, all brucella subspecies, and francisella tularensis ssp. holarctica and mediasiatica. in addition to s rrna gene analysis complementary methods are essential to discriminate between these bacteria on species or subspecies level. identification of nontuberculous mycobacteria by sequence analysis of the s ribosomal rna, the heat-shock protein and the rna polymerase beta-subunit genes s. shin, j.h. yoon, e.c. kim (seoul, kr) objectives: the diagnosis of diseases caused by nontuberculous mycobacteria (ntm) is difficult because ntm are prevalent in the environment such as soil and water and because they have fastidious properties. in this study, we investigated the distribution pattern of ntm clinical isolates and the identification to the species level. methods: among the presumptive ntm clinical isolates, cultured in a third referral hospital from -jan- to -jan- in seoul, south korea, which were negative by probe hybridization method for mycobacterium tuberculosis complex, we selected those of more than colonies or those cultured more than twice in a same patient. a total of isolates were studied for the distribution of ntm including isolates recruited for species identification by direct sequencing of s rrna, hsp and rpob gene segments. ( . %) were also identified in the presumptive ntm isolates. the identification rate by sequencing of s rrna, rpob, and hsp were %, % and %, respectively. hsp or rpob gene was more efficient than s rrna in identification of ntm by sequencing. conclusions: some ntm are considered to be the causative organisms of clinical diseases even in the countries with intermediate burden of tuberculosis, so accurate identification method by direct sequencing can be adapted to clinical laboratories. evaluation of the genotype mtbdr assay for the simultaneous detection of resistance to rifampicin and isoniazid of mycobacterium tuberculosis clinical strains f. brossier, c. truffot-pernot, n. veziris, v. jarlier, w. sougakoff (paris, fr) objectives: the rapid determination of drug resistance in mycobacterium tuberculosis is an important challenge to ensure a rapid effective chemotherapy. the genotype mtbdr test is a commercially available dna strip assay enabling the molecular genetic identification of the m. tuberculosis complex and its resistance to rifampicin (rif-r) and isoniazid (inh-r) by detecting the most commonly found mutations in the genes rpob (asp val, his tyr, his asp, ser leu) and katg (ser thr). here, we report the evaluation of the genotype mtbdr assay from a set of clinical isolates of m. tuberculosis. methods: clinical isolates were collected in france over a years period ( ) ( ) and were included in the study: were rif-r, were inh-r (of which were also rif-r) and were susceptible to both drugs. the susceptibility tests were carried out by the standard proportion method. the mutations involved in rif-r and inh-r in rpob, katg, inha and his promoter region, were characterized by dna sequencing. results: the genotype mtbdr assay identified % of the rif-r strains harbouring mutations in the rpob gene, of which ( %) showed a ser leu mutation and ( %) a his asp or tyr mutation. of the inh-r strains ( %) harboured a ser thr mutation in katg, all identified by the genotype mtbdr assay. of this strains displayed a high level of inh-r. among the other inh-r strains, showed a katg mutation at the level of the regions, which was different from ser thr ( of which showing a low level of inh-r), and one harboured a deletion in katg (with a high level of inh-r). these mutations were also detected by the strip. finally, among the remaining inh-r strains not detected by the mtbdr assay, were characterized by a mutation in position - of the promoter region for the maba-inha regulon ( with a low level of inh-r), by a ser ala mutation in inha (all with a low level of resistance) and by other mutations. conclusions: the mtbdr assay, which can readily be included in a routine laboratory workflow, identified % and % of the strains resistant to rif and inh, respectively. interestingly, of the inh-r strains showing a high level of resistance ( %), but only of the inh-r strains with a low level of resistance ( %), were detected by the mtbdr assay, indicating that complementary tests are necessary for detection of the m. tuberculosis strains having a low level of resistance to inh. variation in the streptococcal s rdna detected by pyrosequencing m. haanperä, p. huovinen, j. jalava (turku, fi) originally the aim of this study was to identify alpha-haemolytic streptococcal isolates to the species level by pyrosequencing the v and v regions of the s rdna and comparing the results to the sequences of type strains that have been determined earlier. however, the isolates could not be unambiguously identified due to sequence variations detected in the alpha-haemolytic isolates. materials and methods: invasive s. pneumoniae isolates (n = ), alpha-haemolytic streptococcal blood culture isolates (n = ) and alpha-haemolytic streptococcal isolates from the normal pharyngeal microbiota (n = ) of six elderly persons were analysed by pyrosequencing the v and v regions. results: varying degree of genetic variation was found in different types of streptococcal isolates. in the pneumococcal isolates, no sequence variation was detected as all the isolates contained the sequence specific for s. pneumoniae in both regions. also the sequences of the alpha-haemolytic blood culture isolates were well in agreement with the sequences of the streptococcal type strains. however, most of these isolates could not be unambiguously identified, as they contained sequences belonging to different species in the v and v region. consequently, only five of the isolates could be unequivocally identified as s. gallolyticus (n = ), s. anginosus (n = ), s. mitis (n = ) and s. sanguinis (n = ). the commensal streptococci contained numerous sequences to which an identical type sequence could not be found. also sequences identical to type strains were found; but similarly to the blood culture isolates, the results enabled the identification of only four isolates: s. mitis (n = ), s. parasanguinis (n = ), and s. salivarius or s. vestibularis (n = ). moreover, the pyrograms of three blood culture isolates and ten pharyngeal isolates indicated heterogeneous s rdna alleles. one such pyrogram of the v region is presented in the figure. interestingly, four of the eight different nonheterogeneous v and v sequence combinations of the blood culture isolates were also present among the pharyngeal isolates. the results of this study indicate that the variation in commensal streptococci is greater than that of the streptococcal type strains and pathogenic isolates. the presence of identical sequence combinations among the blood culture and pharyngeal isolates supports the assumption that potentially pathogenic isolates are present in the normal microbiota. evaluation of partial s rrna gene sequencing for identification of clinical isolates of nocardia species m. marín, m. sánchez, m. del rosal, e. cercenado, p. martín-rabadán, e. bouza (madrid, es) new species of nocardia are being described. conventional identification based on biochemical characteristics and pcrrestriction enzyme analysis is frequently unable to distinguish them. partial sequencing of s rrna gene has proven useful in the identification of bacteria. objective: to evaluate the utility of 'end s rrna gene pcr and sequencing in the identification of clinical isolates of nocardia sp. compared with conventional methods and pcr-rflp of hsp . methods: clinical isolates of nocardia sp. were characterized by biochemical reactions and disk diffusion susceptibility testing. molecular identification was performed by hsp pcr-rflp and pcr of 'end of s rrna gene followed by sequencing. the sequences obtained were compared with those included in genebank. only alignments with similarities higher than % were considered. a comparison of sequences of our nocardia isolates with those deposited in genebank and well characterized phenotypically was performed using clustal x . software. results: distribution of species after pcr-rflp of hsp was n. asteroides vi ( ), n. farcinica ( ), n. nova ( ), n. asteroides i ( ), n. otitidiscaviarum ( ) and n. asteroides iv ( ) . partial sequence analysis of s rrna revealed a great heterogeneity between the isolates of n. asteroides vi, as follows: n. cyriacigeorgica ( isolates), n. abscessus ( isolates) and n. carnea ( isolate). for isolates, no genebank sequence was found with more than % similarity. all n. farcinica isolates had the same sequence and showed % similarity with those deposited in genebank. n. nova, n. asteroides i and n. otitidiscaviarum also showed sequence heterogeneity. three n. nova isolates matched with the recently described n. veterana and with n. nova. n. asteroides i isolates were identified as n. abscessus ( ) and n. beijingensis ( ) . all n. otitidiscaviarum were identified properly. the isolate of n. asteroides iv was identified as n. transvalensis. conclusions: sequencing of 'end s rrna gene is a useful and rapid molecular tool for the identification of nocardia clinical isolates. this method could provide more accurate results than the conventional ones used routinely in our laboratory. sequence analysis of the 'end s rrna has enabled us to recognize great diversity and new species among our nocardia isolates. several species would have gone unnoticed using non-sequencing-based methods. antibacterial susceptibility studies-iii p anaerobic bacteraemia due to fusobacterium necrophorum and clostiridium cadaveris: a case report m. panopoulou, e. alepopoulou, e. chrisafidou, a. tsaroucha, c. simopoulos, s. kartali (alexandroupolis, gr) introduction: anaerobic bacteremia is uncommon accounting . - % of bacteremias and it is associated with a high mortality rate, which is strongly and independently associated with underlying liver disease. case report: a year-old man presented to our hospital with a -day fever and rigor. he had a history of cancer of the extrahepatic biliary tree, which was found incidentally during an operation for the treatment of echinococcal cyst of the liver. physical examination reveals high fever ( c) and tachycardia. blood tests showed the following results: hb: . gr/dl, wbc: . /ul, plt: . /ul, tprot: . each colony type subcultured to blood agar plates and incubated aerobically and anaerobically (aerotolerance test). after hours of incubation the two organisms grew only in anaerobic conditions. they identified by the api a system (bio-merieux-france) as fusobacterium necrophorum and clostiridium cadaveris. the patient's treatment started with metronidazole, amikacin and ceftriaxone and followed by metronidazole and imipenem. he was discharged after weeks in a good condition. conclusions: although anaerobic bacteremia is rare, there is value in performing separate anaerobic blood cultures. the early recognition of anaerobic bacteremia and administration of the appropriate antimicrobial therapy play a major role in preventing mortality especially in patients with underlying disease. fluoroquinolone resistance among enterobacteriaceae strains isolated from urinary tract infections v. skandami-epitropaki, p. fostira, a. tsiringa, a. xanthaki, k. zampitha, m. toutouza (athens, gr) objectives: to study the frequency and antibiotic susceptibility of quinolone resistant bacterial stains isolated from patients with community-aquired bacteriuria and compare it with urinary pathogens from hospitalized patients. methods: during a -month period (october -october a total of bacterial strains were isolated out of urine samples submitted for culture in our hospital laboratory from the community and from hospitalized patients with urinary tract infection symptoms. cultures and bacterial identification were obtained by conventional methods. antibiotic susceptibility testing was done by kirby-bauer disk diffusion method according nccls criteria. results: of the bactrial strains studied (escherichia coli , klebsiella pneumoniae , proteus mirabilis ), . % of them were found to be quinolone resistant. the percentage of quinolone resistance was . % for hospitalized patients (hp) and . % for community patients (cp). the quinolone resistance for e. coli was . % ( . % for hp and . % for cp), for k. pneumoniae . % ( . % for hp and . % for cp) and for p. mirabilis . % ( . % for hp, . % for cp). susceptibility pattern of the quinolone resistant isolates to other antimicrobial agents was for hospitalized patients and community patients respectively as following: for e. coli ampicillin (am) %- . %, amoxicillinclavulanate (amc) . %- . %, piperacillin-tazobactam (tzp) . %- . %, cefuroxime (cxm) . %- . %, trimethoprimsulfamethoxazole (sxt) . %- . %, ceftazidime (caz) . %- . %, cefepime (fep) . %- . %, gentamicin (gm) . %- . %. for k. pneumoniae am %- %, amc %- %, tzp . %- %, cxm . %- %, sxt . %- %, caz . %- %, fep . %- %, gm . %- %. for p. mirabilis am . %- %, amc . %- %, tzp . %- %, cxm %- %, sxt . %- %, caz . %- %, fep %- %, gm . %- %. seven strains of k. pneumoniae ( . %) were carbapenem resistant and metallo-beta lactamase producing. conclusions: high resistance rates to fluoroquinolones were observed in uropathogen bacteria isolated not only from hospitalized patients but also from patients with communityacquired urinary tract infections in greece. increasing resistance rates to the rest antibiotic agents make the treatment of urinary tract infections a very difficult problem. susceptibility of pseudomonas aeruginosa isolated from the mystic programme to the carbapenems: meropenem and imipenem p.j. turner (macclesfield, uk) objectives: the meropenem yearly susceptibility test information collection programme (mystic) was initiated in in order to track the susceptibility of organisms in centres that were prescribing meropenem. this poster seeks to examine the susceptibility of pseudomonas aeruginosa isolates over this period to the carbapenems; meropenem and imipenem and, in particular, records the susceptibility of imipenem-resistant isolates to meropenem and vice versa. methods: pseudomonas aeruginosa isolates were speciated by the methods in current use at the participating centres. minimum inhibitory concentrations of meropenem and imipenem were determined using reference methods described by clsi. results: a total of isolates of pseudomonas aeruginosa have been tested globally, of these . % were susceptible to meropenem at the breakpoint of < mg/l and . % to imipenem. globally, susceptibility to the two carbapenems has remained stable over the period - , however when imipenem-resistant isolates were examined (n = ) . % proved to be susceptible to meropenem, conversely of the meropenem-resistant isolates only . % proved to be susceptible to imipenem. a similar pattern was seen when isolates were separated into global regions:usa imipenem-resistant isolates, . % susceptible to meropenemusa meropenem- results: bacteroides fragilis group (bafg) accounted for % of the isolates, fusobacterium spp. for %, other gram negative bacilli (ognb) for %, clostridia (clos) for %, nonsporeforming gram-positive bacilli (nsfgpb) for % and cocci for %. beta-lactamases (bl) were detected in % of isolates. most bl + strains belonged to bafg ( %) and ognb ( %). at nccls-recommended breakpoints, more than % of isolates were susceptible to tzp, mtz, chl and mem, % to amc but only %, %, % and % to fox, ctt, cli and pen respectively. no nccls-breakpoints for anaerobes are available for mxf, lzd and tig. mic and mic for mxf were and mg/l, for lzd and mg/l and for tig . - mg/l. in comparison with similar surveys conducted in and - susceptibility of bafg to clindamycin decreased from % in , to % in - and % in in bafg % of b. fragilis and % of non-b. fragilis were susceptible to amc in this study; in - susceptibility in these groups was % and % and in - % and % respectively. all isolates, except bafg and clos, were susceptible to mem. % of the isolates were susceptible to chl. susceptibility to mtz remains stable and is high in all groups except nsfgpb where mtz is active on merely % of the isolates. conclusions: tzp, mem and mtz remain very potent antimicrobial agents in the treatment of anaerobic infections. although still rare, resistant organisms were detected to each of them. therefore susceptibility testing of anaerobic isolates is indicated in severe infections to confirm appropriateness of antimicrobial therapy. further monitoring of background susceptibility is necessary to guide empiric treatment. comparative in vitro activity of levofloxacin against escherichia coli isolated from acute pyelonephritis in france in c.j. soussy, c. lascols, c. dib-smahi and the multicenter group study. objectives: the objective of this study was to evaluate the in vitro activity of levofloxacin (lvx) comparatively to other antibiotics against escherichia coli strains isolated from acute pyelonephritis in women consulting emerging rooms by french hospitals in . methods: mics of lvx, ofloxacin (ofx), ciprofloxacin (cip), nalidixic acid (nal), amoxicillin-clavulanic acid (amc), ceftriaxone (cro), cefixime (cfm), amikacin (an), gentamicin (gm) and cotrimoxazole (sxt) were determined by agar dilution according to the eucast breakpoints approved by recommendations of the comité de l'antibiogramme de la société française de microbiologie. quality control was performed with e. coli strain atcc . results: a total of strains were collected. . % of strains were isolated from urinary samples, . % from blood culture and . % from the two specimens. mics / (mg/l), the range of mics and the percentage of susceptibility (%) are presented in the following table: concerning the fluoroquinolones, mics / of lvx were one/two dilution lower than those of ofx and two/one dilution higher than those of cip. for the other antibiotics, a higher percentage of susceptibility was observed with cro and an, when a lower percentage of susceptibility was observed with amc and sxt. conclusions: levofloxacin exhibited good in vitro activity against e. coli strains isolated from acute pyelonephritis with . % of susceptible strains. in vitro activity of double and triple combinations of colistin, imipenem, rifampicin and linezolid against epidemic strains of multidrug-resistant acinetobacter baumannii producing oxa carbapenamases d.w. wareham, d.c. bean (london, uk) objectives: a. baumannii has emerged as an important cause of nosocomial infection in critically ill patients worldwide. in the uk three strains in particular exhibiting multi-drug resistance and producing oxa carbapenamases have been responsible for ongoing outbreaks. treatment options for infection with these organisms are limited as only colistin and tigecycline retaining significant activity in vitro. animal models and in vitro studies using other multi-resistant strains suggest that drugs in combination with colistin may be effective. we assessed the activity of colistin in combinations including imipenem, rifampicin and linezolid against epidemic strains from a recent uk outbreak. methods: isolates of a. baumannii exhibiting resistance to carbapenems were recovered from patients at barts and the london nhs. isolates were referred to the health protection agency and confirmed as belonging to clones producing oxa carbapenemases. activities of polymyxin, imipenem, rifampicin and linezolid alone and in double and triple combinations were determined using standard chequerboard assays with increasing concentrations of drug on the x axis, drug on the y axis and drug three in multiple replicate plates. after incubation at hours wells were examined for growth and mic's determined for each combination. synergy between agents was defined as a fixed inhibitory concentration index (fici) of < . . results: the isolates tested belonged to the oxa- clone , oxa- clone and the south east clone, as confired by the hpa. colistin was the most active agent alone with mics from - mg/l. imipenem mic's varied from - mg/l. the most active combinations were colistin plus rifampicin (fici = . ) and colistin, rifampicin and imipenem (fici = . ). synergy was not seen with colistin in combination with imipenem alone. linezolid in combination with colistin (fici = . ), or imipenem (fici = . ) was synergistic but at therapeutically unobtainable linezolid concentrations ( mg/l). conclusion: multidrug resistant strains of a. baumannii from the uk producing oxa carbapenemases remain susceptible to polymyxin in vitro. polymyxin exerts its effect on the bacterial cell wall; theoretically assisting other antibiotics to reach their respective targets, and seems a logical choice for inclusion in combination therapy. we have shown that rifampicin is synergistic with polymyxin against these isolates in vitro and may be effective in treating severe a. baumannii infections in man. a comparative in vitro evaluation of resistance development after exposure to teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin in staphylococcus spp. and enterococcus spp. mssa, mrse, msse, e. faecium and e. faecalis strains was determined on agar plates containing each antibiotic at clsi resistance breakpoints and at peak blood concentrations. after incubation at °c for h colonies were counted and compared to the inoculum to calculate frequency of mutation. colonies grown in plates containing antibiotics were sampled for determination of mic values. results: frequency of mutation was less than - for all the tested antibiotics at peak blood concentrations. same results were obtained when breakpoint concentrations for each drug were used. conclusion: this one-step in vitro study demonstrated the ability of teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin to prevent growth of resistant mutants of staphylococci and enterococci, thus suggesting no occurrence of mutational events leading to resistance when bacteria are exposed to blood concentrations of these drugs. in order to establish the development of resistance after in vitro serial exposure to the same antibiotics simulating different in vivo concentrations, further studies are needed and are now in progress (multi step induction of resistance). in vitro activity of antimicrobial agents against legionella obtained from hotel water systems in turkey objectives: the aim of this study was to evaluate the in vitro activity of colistin against endemic pan-resistant acinetobacter baumannii (including resistance to imipenem) isolated during a year period in a university hospital. methods: imipenem-resistant acinetobacter spp. isolates were collected between january and october , from a variety of clinical specimens of different patients attending distinct wards in a university teaching hospital. isolates were identified by api gn and by sequencing the s rrna gene. mics of colistin were determined by agar dilution method, according to nccls susceptible breakpoint (£ mg/l). pfge (apai restriction enzyme) was performed. results: of a. baumannii isolates ( %) were susceptible to colistin. colistin resistance (mic ‡ mg/l) was observed in isolates ( isolates with a mic of ‡ mg/l and isolates with a mic of mg/l) recovered from different patients in distinct wards. among these imipenem-and colistin-resistant isolates, distinct pfge patterns were identified (clones a, b, and c). resistance to almost all beta-lactams (including carbapenems) and variable susceptibility to aztreonam, amikacin and tobramycin was a common feature of clone a. isolates belonging to clone b showed resistance to imipenem, amoxicillin and its association with clavulanic acid (amc), ureidopenicillins and their associations; susceptibility to ceftazidime; and variable behaviour to meropenem, cefepime, cefpirome and aztreonam. the susceptibility profile to aminoglycosides was variable, differing from clone a in its susceptibility to netilmicin and minocycline. clone c was resistant to imipenem, amoxicillin, amc, piperacillin, piperacillin + tazobactam, ticarcillin and ticarcillin + clavulanic acid, but remained susceptible to meropenem, aztreonam, cefpirome, ceftazidime and cefepime. conclusion: only colistin, one of the few effective drugs available against multi-drug-resistant acinetobacter infections, showed in vitro activity against the majority of acinetobacter spp. strains isolated within the sampled hospital. the observed % a. baumannii resistance to the recently re-introduced colistin seems like the first chapter of a novel repeatedly told for several antibiotics. emergence of high-level gentamicin resistance in clinical enterococcal isolates of companion animals in portugal objectives: to characterize in vitro gentamicin susceptibility among enterococci causing infections in cats and dogs, in order to evaluate the impact of high-level gentamicin resistance in small animal therapeutics. methods: the samples were collected at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. from january until november , a total of enterococci were isolated from dogs and cats with urinary tract infection (uti), otitis externa (oe) and pioderma. bbl crystal gram positive id system was used for identification at the species level. minimal inhibitory concentrations (mic) were determined by the microdilution method according to nccls ( ) . the bifuntional enzyme gene that confers high-level gentamicin resistance (hlgr) was detected using pcr ( ) . results: enterococcus faecalis was the predominant isolate (n = ), followed in frequency by enterococcus faecium (n = ). mic cumulative data analysis showed that mic values were lg/ ml and mic lg/ml. six ( %) hlgr clinical enterococcal isolates were detected, with mic ranges between - lg/ml. four of these enterococci were isolated from uti and from oe. four of the phenotypically high-level gentamicin resistant isolates carried the aac( ')-ie-aph( '')-ia gene. conclusions: the importance of enterococcal infection in small animal clinical samples has increased over the last years. mic cumulative data points out low-level gentamicin resistance among clinical enterococci isolates of veterinary origin and the emergence of high-level isolates, as previously detected ( ). this fact compromises cell-wall active agents (such as ampicillin or vancomicin) and aminoglicoside in vivo synergy. the aac ( ')-ieaph( '')-ia gene carriage is of concern because its expression confers resistance also to tobramicin, netilmicin, amikacin and kanamicin. our findings are of critical importance, as they may have a direct impact in therapeutic decision in the management of companion animal's infections by enterococci. furthermore, transfer of resistance genes and resistance strains between animals and owners/caretakers by direct contact is a concerning probability. references: ( ) results: interpretative criteria were used according to nccls .during the study period penicillin resistant strains of s. pneumoniae was noted as follows: % in sputum or ta, % in blood, %in csf,and % in others,against cefuroxim resistant strains: % in sputum or ta, % in blood, % in csf.regarding the susceptibility to ofx,penicillin resistant s. pneumoniae strains from sputum or ta revealed . %.the penicillin resistant strains coming from sputum or ta showed resistance as follows; % to em and % to sxt,against strains isolated from others: % to em and % to sxt.no resistant strain to va was found. conclusion: the percentage of the penicillin resistant s. pneumoniae isolates from the lower respiratory tract, middle ear fluid, eye fluid and sinus was markedly higher than that of the isolates from blood and csf. the most efficient drugs against penicillin resistant pneumococci were cefuroxim and ofloxacin. these results from romania also underline the previous observations regarding the higher emerging rates of resistance in s. pneumoniae worldwide. penicillin resistance in streptococcus agalactiae objectives: streptococcus agalactiae has become recognized as a cause of serious illness in newborns, pregnant women, and adults with chronic medical conditions. heavy colonization of the genital tract with streptococcus agalactiae also increases the risk that a woman will deliver a preterm low-birthweight infant. early-onset infections (occurring at < days of age) are associated with much lower fatality than when they were first described, and their incidence is finally decreasing as the use of preventive antibiotics during childbirth increases among women at risk. penicillin or ampicillin remains the drug of choice for intrapartum antibiotic prophylaxis for streptococcus agalactiae colonization in pregnant women. erythromycin and clindamycin are the drugs of choice for women with serious penicillin allergy who are colonized with streptococcus agalactiae. the objective of this study is to estimate the insorgence of penicillin resistance among streptococcus agalactiae. methods: all streptococcus agalactiae were tested against penicillin by agar dilution method according to clinical and laboratory standards institute (clsi); breakpoints for resistance were those recommended by the clsi. antimicrobial agents were obtained from their manufacture as laboratory grade powder. results and discussion: four hundred seven ( ) clinical isolated were analysed during . streptococcus agalactiae resulted resistant to penicillin in case; and about % resulted borderlines.the present findings indicate a probable evolution in s. agalactiae toward penicillin resistance this finding suggest the need a continuous national and international surveillance programs to provide timely data on the evolution of incidence of penicillin resistance in this pathogen. ciprofloxacin susceptibility of the most common isolates at bacterial conuctivitis conclusion: according to the average numerals we concluded that all the isolated strains are highly susceptible at ciprofloxacin. its application in the conuctivial saccus is especially important in curing the conuctivial infections with resistent strains like pseudomonas aeruginosa. we successfully cure the bacteria chronic conuctivitis with the adequately used therapy according to antibiogram. antimicrobial resistance patterns of acinetobacter baumannii in clinical isolates g.t. tsilika, v.p. pliatsika, m.t. tsivitanidou, d.s. sofianou (thessaloniki, gr) objectives: a. baumannii is a nosocomial pathogen, commonly isolated from critically ill and immunocompromised patients. the aim of the present study was to evaluate the antimicrobial resistance of a. baumannii strains isolated in a tertiary care hospital througout a three-year period. methods: a total of a. baumannii strains were selected from january to december .the specimens were obtained from inpatients hospitalized in intensive care unit (icu) and pediatric intensive care unit (picu) and other departments of our hospital.the identification and the antimicrobial susceptibility testing were performed using the vitek automated system(biomerieux,france conclusions: the emergance and rapid spread of multidrug resistant a. baumannii isolates are of a great concern worldwide.imipenem was one of the most potent agents for treatment of those infections caused by multiresistant strains.the increasing prevalence of imipenem resistance limits therapeutic options and leads to outbreaks of carbapenems resistant strains. tigecycline in vivo studies objectives: antibacterial agents disrupt the ecological balance of the normal human microflora. disturbances may lead to the emergence of antibiotic resistance and/or to infections by potentially pathogenic bacteria. tigecycline, a member of a new class of antibiotics (glycylcyclines), has been shown to have a potent expanded broad-spectrum activity against most grampositive and gram-negative aerobic and anaerobic bacteria. the aim of the study was to investigate the ecological effects of tigecycline on the normal oropharyngeal and intestinal microflora in healthy subjects. methods: thirteen ( ) white subjects ( women, men) aged to years, received mg of tigecycline in the morning on day as a -minute intravenous (iv) infusion, followed by mg doses of tigecycline given every hours as a -minute infusion for days. one ( ) subject was withdrawn on day because of an adverse event. serum, saliva, and faecal samples were collected before, during, and after administration for microbiologic cultivation and for assays of tigecycline. all new colonizing bacteria were tested for susceptibility (resistance > mg/l) during the investigation period. results: the serum concentrations on day , hours after dosing, were . to . mg/l (mean value . mg/l, median value . mg/l, and sd . mg/l). the faecal concentrations on day were . to . mg/kg (mean value . mg/kg, median value . mg/kg, and sd . mg/l). saliva concentrations were generally low, with highest mean value . mg/l, median value . mg/l, on day , hours after dosing. a minor effect on the oropharyngeal microflora was observed. the numbers of enterococci and escherichia coli in the intestinal microflora were reduced at day , while other enterobacteria and yeasts increased. there was a marked reduction of lactobacilli and bifidobacteria but no impact on bacteroides. no clostridium difficile strains were isolated. two ( ) klebsiella strains and enterobacter strains resistant to tigecycline were found. conclusion: tigecycline had a minor effect on the oropharyngeal microflora. tigecycline's effect on the intestinal microflora was due to its spectrum of antibacterial activity and intestinal concentrations. objectives: to examine and report the use of tigecycline (wyeth) in the treatment of multidrug resistant acinetobacter (mdra) culture positive sepsis in patients requiring mutiorgan support. methods: all patients were managed within the liver intensive care unit. physiological data was collected prospectively and entered onto a specialist database. patients received standard intensive care management; antibiotic and antifungal therapy administered as indicated by microbiological cultures. systemic inflammatory response (sirs) features initiated blood cultures (vascular lines and peripheral), drain fluid culture and broncoalveolar lavage (bal). screening swabs were undertaken weekly and samples sent for culture at laparotomy. mdra positive cultures from blood, bal, drain fluid or samples taken at laparotomy in the context of sirs resulted in the initiation of tigecycline treatment. results: patients received tigecycline treatment for mdra infections. the underlying disease states were necrotizing pancreatitis ( ), post hepatectomy ( ), polytrauma ( ), all with postive intra-abdominal cultures. acute and acute on chronic liver failure ( ), mdra +ive broncho-alveolar lavage ± blood cultures and post liver transplant patients (necrotising pancreatitis in one, with recurrent small bowel perforation and with retroperitoneal haemorrhage) all with positive blood cultures and in positive intra-abdominal tissue/clot. mean time from admission to treatment for mdra was days. mean duration of treatment was days (range - ). mean apache ii score at initiation of therapy was (range - ); / patients survived to intensive care discharge and / to hospital discharge. microbiological clearance of mdra was observed in / cases. in those who did not achieve microbiological clearance cause of death was intra-abdominal haemorrhage, recalcitrant organ failure with recurrent small bowel perforation and vasopressor resistant shock. in these patients one remained culture positive for intraabdominal sepsis despite full treatment (small bowel perforation x ). the drug was well tolerated with the only side effect being that of hypercalcaemia observed in / patients, mean corrected calcium . mmol/l, range . - . . in all cases this resolved on drug discontinuation. conclusion: tigecycline appears to be an efficacious agent in the treatment of deep seated mdra infections. objectives: nausea (n) and vomiting (v) have been reported with tigecycline, a new glycylcycline with expanded broad spectrum activity. exposure-response relationships and patient covariates predictive of the first n and v occurrence were evaluated in patients with complicated intra-abdominal infections (ciai). methods: data from patients from ciai trials (one phase and two phase ), receiving mg loading dose and mg every hours, were pooled for analysis. n and v (definitely, possibly, or probably related to tigecycline) reported from the start of infusion until hours after the last dose were included. individual exposure measures [auc - and cmax] were calculated using a previously developed population pk model. logistic regression was used to evaluate predictors of first n and v occurrence. covariates included age, weight, sex, region of treatment, and baseline n and v. results: the dataset included patients ( with pk). mean (sd) age and weight were ( ) years and ( ) kg. % of patients were men and %, %, and % were enrolled in north america, europe, and latin america, respectively. baseline nausea or vomiting was reported in % and %. overall, n and v occurred in % and % of patients receiving tigecycline, however most ( %; %) of first n and v events were mild in nature. women had more n and v ( %; %) than men ( %; %). n and v were lower in europe ( %; %) than in other regions. auc - and cmax were not predictive. the final nausea model included weight, sex, region, baseline nausea, and the interaction of weight/region as predictors of the first nausea occurrence (p = . , . , . , . , & . , respectively objective: because hospitalisation for community-acquired pneumonia (cap) is associated with substantial morbidity and health resource utilisation, we evaluated the predictors of prolonged hospital length of stay (los) and treatment duration. methods: we conducted a retrospective analysis of data from a double-blind, randomised, multicentre clinical study that compared the efficacy and safety of tigecycline with that of levofloxacin in the treatment of patients with cap requiring hospitalisation. patients were stratified by the fine pneumonia severity index and randomly assigned to receive tigecycline or levofloxacin via iv administration for at least days. treatment duration and hospital discharge were based on physician assessment of signs and symptoms of infection and patient condition. we used cox proportional hazards modelling with stepwise selection to identify statistically significant predictors (p < . ) of treatment duration and hospital los. results: among patients with cap in the clinical intent-totreat population with complete hospitalisation data, mean age was . years (range - ) and . % of patients were aged ‡ years. diabetes ( . %), chronic obstructive pulmonary disease ( . %), and congestive heart failure ( . %) were leading co-morbidities. about . % of patients were smokers and . % were characterised by alcohol abuse. median fine pneumonia severity index score was ; . % of patients had a score > . . there were no significant differences between the groups in treatment duration or los. conclusions: tigecycline, a first-in-class glycylcycline, was associated with treatment duration and los similar to that of levofloxacin, adjusting for several identified risk factors. tigecycline effective in treating patients with intra-abdominal or skin/skin structure infections who have bacteraemia e.j. ellis-grosse, r. maroko (collegeville, us) objectives: the treatment of bacteraemia, which is a potentially fatal complication of infections originating at other body sites, is complicated by increasing resistance. tigecycline, a first-in-class glycylcycline, has an expanded spectrum of activity against gram-positive, gram-negative, anaerobic, and atypical bacteria including resistant strains. tigecycline is safe and effective in treating complicated skin and skin structure (csssi) and intraabdominal infections (ciai). this analysis examines tigecycline clinical trial experience in patients with ciai or csssi who had bacteraemia (presence of bacteria in blood) at baseline. objectives: treatment of complicated intra-abdominal infections (ciai) is challenging due to diverse bacteriology and bacterial resistance. the efficacy and safety of tigecycline (tgc), a first-in-class glycylcycline approved in mexico, brazil, peru, colombia and usa for treating ciai and complicated skin and skin structure infections, was compared with imipenem/cilastatin (imi/cis) in adult hospitalised patients with ciai in two double-blind, phase multinational trials. this analysis evaluated tgc efficacy and safety in the european region of the integrated results of these two trials. methods: one study was conducted in centres ( countries) and the other study was conducted in centres ( countries). patients were stratified by disease severity (apache ii score £ vs > but £ ), and randomly assigned to iv tgc ( mg loading, then mg q h) or iv imi/cis ( / mg q h) for - days. clinical response at test-of-cure (toc, - days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) were co-primary efficacy endpoints where cure/failure responses were determined. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, patients were mitt (received ‡ dose), m-mitt ( tgc, imi/cis) and me ( tgc, imi/cis). treatment groups were balanced with respect to demographics. patients were mostly white ( . %) men ( %) with a mean age of years. for me, clinical cure rates at toc were . % ( / ) for tgc vs . % ( / ) for imi/cis ( % ci = ) . , . ; test for non-inferiority p < . ). clinical cure rates for m-mitt were . % ( / ) for tgc vs . % ( / ) for imi/cis ( % ci = ) . , . ; test for non-inferiority p < . ). most commonly reported treatment emergent aes (teaes, mitt) for tgc and imi/cis were nausea ( . % and . %, p = . ) and vomiting ( . % and . %, p = . ). the imi/cis group had significantly higher teaes of fever ( . % imi/cis vs . % tgc, p = . ), hyperglycaemia ( . % imi/cis vs tgc, p = . ) and dyspnoea ( . % imi/cis vs . % tgc, p = . ) where tgc had significantly higher amylase increase ( . % tgc vs . % imi/cis, p = . ) and bun increase ( . % tgc vs imi/cis, p = . ). conclusions: similar to the overall integrated analysis of the two phase trails, in the european analysis, tgc was safe and effective in the treatment of hospitalised patients with ciai in comparison with imi/cis. tigecycline is safe and effective in the treatment of complicated skin and skin structure infections: european experience of two double-blind phase comparison studies with vancomycin/aztreonam r. maroko, n. dartois, d. sarkozy, j. goodrich, e.j. ellis-grosse on behalf of the tigecycline and study groups objectives: tigecycline (tgc) a first-in-class expanded spectrum glycylcycline, has been approved in mexico, brazil, peru, colombia and usa for treating complicated skin and skin structure infections (csssi) and complicated intraabdominal infections. two phase , randomised, double-blind studies were conducted in hospitalised men and women with csssi to determine tgc safety and efficacy compared with vancomycin/aztreonam (v/a). the objective of this analysis was to evaluate the efficacy and safety seen in the european population of the integrated analysis of these phase trials. methods: one study was conducted in centres in countries while the other study was conducted in centres in countries. patients were randomly assigned ( : ) to receive either tgc ( mg, followed by mg iv twice daily) or vancomycin ( g iv twice daily) plus aztreonam ( g iv twice daily) for up to days. clinical response at test-of-cure (toc, - days after therapy) for clinically evaluable (ce) and clinical modified intent-to-treat (c-mitt) populations were coprimary efficacy endpoints in which cure/failure responses were determined. secondary objectives included determination of in vitro susceptibility to tgc of a range of bacteria that cause csssi and microbiological efficacy. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, patients comprise mitt (received ‡ dose of study drug), comprised ce ( tgc, v/a/cis) and comprised c-mitt ( tgc, v/a/ cis). treatment groups were balanced with respect to demographics. patients were mostly white ( . %) men ( . %) with a mean age of years. in the european region, clinical responses to tgc and v/a at test-of-cure were similar: c-mitt, . % ( / ) versus . % ( / ), difference tgc-v/a was - . % ( % ci - . , . ). similar results were noted in the ce population with tgc curing . % ( / ) and v/a curing . % ( / ), difference tgc-v/a was - . % ( % ci - . , . ).most commonly reported treatment emergent aes (teaes, mitt) for tgc and v/a were nausea ( . % and . %, p < . ) and vomiting ( . % and . %, p = . ). the v/a group had significantly higher teaes of sgpt increase ( . % v/a vs . % tgc, p = . ) and rash ( . % v/a vs tgc, p = . ). conclusion: in the european analysis of the integrated phase worldwide clinical studies, tgc monotherapy is as safe and efficacious as the combination of v/a in the treatment of patients with csssi. safety and tolerability of tigecycline r. maroko, n. dartois, g. rose, e.j. ellis-grosse (collegeville, us; paris, fr) objectives: tigecycline (tgc), a glycylcycline, is a first-in-class, extended, broad-spectrum iv antibiotic that has demonstrated clinical activity in patients with complicated intra-abdominal infections (ciai) and complicated skin and skin-structure infections (csssi). the safety of tigecycline was evaluated in four phase iii trials. methods: a total of hospitalized patients from these trials were pooled and evaluable for safety analysis. in the ciai trial, patients received tgc mg q hrs (following a -mg loading dose) or imipenem mg and cilastin mg q hrs. those in the csssi study were treated with either tgc (same dose/schedule) or vancomycin gm with or without aztreonam gm q hrs. results: the most frequently reported adverse events (aes) in both tgc-treated groups were nausea (n) and vomiting (v). the incidence of n was . % while v was approximately . %; these were generally mild to moderate in severity. infection-related serious aes were slightly more frequent with tgc versus comparators ( . % vs . %). discontinuations due to treatment-emergent aes (including n/v) occurred at similar rates with tgc and comparators ( . % vs . %). six patients ( . %) treated with tgc presented with intestinal perforations and developed sepsis/septic shock compared with ( . %) for imipenem/cilastatin, with higher baseline apache ii scores in the tgc group; the relationship to treatment could not be determined. in the overall efficacy analysis, subjects with ''perforation of the intestines'' were balanced between the two groups, and overall efficacy was not statistically different. no clinically significant renal, hepatic, cardiac (qtc), bone marrow, or cns toxicities were noted with tgc. conclusion: tgc appears to be safe and tolerable for patients with ciai and csssi. n/v were generally mild to moderate in severity, self-limiting, and did not result in increased overall drug discontinuation. there did not appear to be clinically significant renal, hepatic, cardiac, bone marrow, or neurological toxicities related to tgc treatment. all-cause mortality rates did not statistically differ between those treated with tgc and the comparators. its demonstrated efficacy and favourable toxicity profile make tgc a good monotherapy option for selected serious infections. tigecycline as effective as imipenem/cilastatin in the treatment of complicated intra-abdominal infections: experience in india objective: due to diverse bacteriology and bacterial resistance, treatment of complicated intra-abdominal infections (ciai) is a challenge. in a double-blind, phase , multinational trial, the efficacy of tigecycline, a first-in-class glycylcycline, was compared with imipenem/cilastatin (imi/cis) in hospitalised patients with ciai. this subanalysis evaluated tigecycline safety and efficacy from investigational sites in india. methods: patients were stratified by disease severity (apache ii score £ vs > but < ), and randomly assigned to iv tigecycline ( mg loading, mg q h) or iv imi/cis adjusted for body weight ( / mg q h for ‡ kg) for - days. clinical response at test-of-cure (toc, - days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) populations were co-primary efficacy endpoints where cure/failure responses were determined. safety evaluations included vital signs, laboratory tests and record of adverse events (aes). results: in india, patients received at least dose (mitt, tigecycline, imi/cis), patients were clinically evaluable (ce), were me, were m-mitt. treatment groups were balanced with respect to demographic/baseline medical characteristics. primary diagnoses (mitt) were complicated appendicitis ( %), gastric/duodenal perforation ( %), perforation of intestine ( %), cholecystitis ( %), peritonitis ( %), and intraabdominal abscess ( %). cure rates at toc in me in india were / ( . %) tigecycline and / ( . %) imi/ cis, which are consistent with overall me results [ . % ( / ) tigecycline vs . % ( / ) imi/cis ( % ci = ) . , . ; non-inferiority p < . )]. in india m-mitt, cure rates at toc were / ( . %) tigecycline and / ( . %) imi/cis, similar to the overall m-mitt results [ . % ( / ) tigecycline vs . % ( / ) imi/cis ( % ci = ) . , . ; non-inferiority p < . )]. noninferiority of tigecycline among india patients could not be statistically demonstrated because of insufficient sample sizes, however, magnitude of response to study drugs in patients treated in india was comparable to that in overall patients. in india, treatment aes were similar with significantly higher incidence of dyspnoea in tigecycline ( . %) vs imi/cis ( . %), p = . . conclusions: efficacy results in india are consistent with findings from the overall study and results at other centres, suggesting tigecycline is noninferior to comparator in treating ciai. nosocomial infection: control of environment, viral infections p bacterial flora contamination of blood pressure cuffs in use on hospital wards n. walker, r. gupta, j. cheesbrough (preston, uk) blood pressure cuffs are a plausbile vehicle for the transmission of nosocomial infection between patients. despite this, few studies have examined the level of bacterial contamination and tested for the presence of common nosocomial pathogens on their surface. we swabbed cuffs currently in use on hospital wards. using sterile gloves, a disposable template measuring · cms was placed onto the cuff and a moistened sterile swab was rubbed onto the defined area for minute and then transported in mls of buffer medium. from each sample, . mls of the buffer was plated onto different media which included a non-selective agar medium for total viable count (tvc) and selective media for s. aureus, mrsa, c. difficile, coliforms and vancomycin resistant enterococci (vre.) bacterial growth was recovered from all cuffs. pathogenic organisms were isolated from cuffs ( %). mssa from , mrsa from and c. difficile from . the remaining three cuffs grew more than one pathogenic organism; mssa + mrsa + c. difficile from one and mssa + c. difficile from cuffs. colifroms and vre were not isolated from any of the cuffs. the range of total viable counts recovered per cm area of the cuff varied from > cfu and the cuffs with the highest counts tended to have more pathogens present. mssa and c. difficile were isolated from % of the cuffs sampled and mrsa from %. while the actual importance of this potential route of transmission for nosocomial pathogens remains unclear, it can not be dismissed. the impracticality of decontaminating blood pressure cuffs between patients suggests that single patient use cuffs or a barrier between cuff and skin would be a more viable option on a busy general ward. needlestick and sharp injuries of health care personnel in a newly founded tertiary hospital: a prospective study m. falagas, i. karydis, g. georgoulias, p. hatzopoulou, d. nikita, i. kostogiannou (athens, gr) objectives: needlestick and sharp injuries of health care workers are a major cause of anxiety and may expose susceptible employees to the risk of infectious diseases. however, the incidence of such injuries has not been examined in a newly founded hospital while preventive programmes are taking place. methods: we prospectively studied the needlestick and sharp injuries of employees in a newly founded tertiary hospital in athens, greece while a vaccination program against hepatitis b virus as well as educational activities for avoidance of injuries were taking place. serologic studies for hepatitis b and c virus as well as human immunodeficiency virus (hiv) were performed in all injured employees and the source patients (when known). results: sixty-eight needlestick, sharp injuries, and splashes were reported during the study period ( / / to / / ) in nurses, housekeepers, technicians, and ambulance workers. the overall incidence (percutaneous injuries and splashes) per full-time employment-years ( fteys) was . % whereas the incidence of percutaneous injuries alone per fteys was . %. a higher incidence of injuries was noted during the first than the second half of the study period ( . % versus . %, p = . ). no source patient was found positive for hepatitis c or hiv. the use of high-titre immunoglobulin after adjustment for the incidence of injuries was higher in the first than the second half of the study period ( . % vs . %, p = . ). conclusion: although we did not adjust for possible confounders, our data show that educational and vaccination preventive programs for needlestick and sharp injuries led to a statistically significant decrease in the incidence of such injuries and use of high-titre immunoglobulin. epidemiology of occupational needlestick and sharps injury among healthcare-workers in turkey s. hosoglu on behalf of the occupational infections study group, turkey background: health care workers (hcws) are frequently exposed to the danger of infectious agents through needle stick and sharps injury (nssi) in their occupational efforts. in turkey, the hepatitis b and c viruses cause an essential threat to the hcws because of their prevalence rate ( %- % and . %- %, respectively). a cross-sectional countrywide survey study was performed on the epidemiology of nssi among hcws at hospitals in cities throughout the country. data relating to the epidemiology of nssis were collected using a standard questionnaire in . results: totally hcws completed the questionnaire forms. nurses are the leading group ( persons) that joined into the study were followed by doctors ( persons) andlaboratory technicians ( ). totally of them ( . %) declared an occupational exposure or nssi in the last months related their job. needle stick injury was reported in of them ( . %), splash into the eye in ( . %), sharp injury in ( . %), and the other injuries in ( . %). the hepatitis positivity was reported in cases ( . %) objectives: to assess the microbiological status of reprocessed single-use devices for interventional cardiology by testing bioburden, sterility and pyrogenic load. methods: a total amount of electrophysiology non-lumen catheters (ep) were collected after the first clinical use on patient. devices were contaminated with bacteria spiked human blood and underwent four different pre-sterilization protocols including chlorine, polyphenol, and enzymatic agents. treated samples were assayed by cultural quantitative methods (cqm) for bactericidal properties and electron microscopy (em) for biologic residuals. ep were tested for sterility. by the repetition of simulated-use (bacteria spiked blood) and regeneration (enzymatic and chlorine treatment, gas plasma sterilization) we obtained , , , , , samples respectively reprocessed , , , , , times. devices were cultured for days in trypticase soy broth. the pyrogenic status of ep was monitored after clinical use, after decontamination-cleaning treatments and after complete reprocessing by lal test. results: high-resolution em and cqm confirmed the superior properties of chlorine releasing agent added to enzymatic detergent for devices treatment before sterilization. hypochlorous acid based protocols were more biocide (> . log cfu reduction) than polyphenolic ( . - . log cfu reduction). sterility tests showed no positive sample to inoculated strain until the fourth cycle of reprocessing. catheters showed the growth of the inoculated strain, bacillus subtilis in / and / samples after five cycles and six cycles respectively. every reprocessed device was non-pyrogenic (< eu/catheter). in addition, tests conducted on in-vitro spiked catheters showed that pyrogenic loads of eu/device were reduced to less than eu/device. conclusions: reprocessing procedures following the adopted regeneration protocol were able to satisfy the fundamental microbiological requirements until five in-vitro reuses. sterility tests showed that devices' sterility was not guaranteed after five reuses. pre-sterilization treatments including enzymatic solutions and chlorine revealed high cleaning properties with effective bioburden reduction. storage intervals among reprocessing steps longer than hours should be avoided in order to limit contamination and pyrogenic load. technical considerations suggest to consider the introduction of reprocessing procedure only in hospitals with a considerable workload. room disinfection in the hospital setting using akacid plus Ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: akacid plus Ò , a novel polymeric guanidine with broad antimicrobial activity also against multi-resistant bacterial strains, was used in the present study as room disinfectant. methods: disinfection of closed rooms experimentally contaminated with antibiotic-susceptible and multi-resistant staphylococcus aureus (mrsa), pseudomonas aeruginosa and escherichia coli was performed using akacid plus Ò at concentrations of . %, . % and . % for minutes. bacterial suspensions were distributed on stainless steel plates and placed in a test and control room. recovery of the test bacteria was determinedbefore nebulizing, and minutes after the beginning and hours after the end of room disinfection by a modified simple swab-rinse technique. for the detection of mrsa in isolation units, surface samples were collected by direct swab and enrichment culture. results: the swab-rinse method demonstrated a dose-and time-dependent effectiveness of akacid plus Ò in eradicating s. aureus, e. coli and p. aeruginosa on stainless steel plates. nebulizing of . % akacid plus was successful in eliminating all hospital pathogens in min contact time, while mrsa was still detectable after use of . % akacid plus Ò . . % akacid plus Ò achieved a reduction > cfu of s. aureus and p. aeruginosa, but was only able to eradicate e. coli during the observation time. the results suggest that nebulized akacid plus Ò at a concentration of . % is a potent substance for eradication of pathogenic organisms in the hospital setting. study on the antiviral efficacy of citrofresh Ò , a flavonoid based organic acid complex sanitizer z. nack (north-geelong, au) objective: determine the antiviral efficacy of this organic sanitizer against enveloped and non-enveloped viruses using a carrier based method. seeking registration for citrofresh Ò in australia and in the eu as a hospital grade antiviral sanitizer. methods: the study was performed according to the american society of testing and materials (astm) designation (e - ) recommended by the australian therapeutic goods administration (tga) to determine the efficacy of a disinfectant intended to use on inanimate, environmental surfaces. we tested citrofresh Ò (diluted in standard hard water) in three different concentrations: %, % and % on adherent cell lines (pk- , mrc- , mdck, a , l ) in four replicates against five different viruses including: porcine parvovirus (non-enveloped, high resistant against sanitizer); human rhinovirus- (non-enveloped, high resistant against sanitizer); human adenovirus- (non-enveloped, moderate resistant against sanitizer); human influenza type a (h n ) virus (enveloped, moderate resistant against sanitizer); human herpes simplex virus type (enveloped, low resistant against sanitizers). prior to the viral testings, acute toxicity assay was carried out to determine the adherent cells viability against citrofresh Ò . results: cell lines exhibited > % viability after exposure to all three concentration. herpes simplex type , human influenza type a and human adenovirus- exhibited the most significant viral log reduction of log to at % concentration of citrofresh Ò followed by the human rhinovirus- and porcine parvovirus log reduction at % concentration. the reduction of viable virus load was exhibited after minute exposure time to citrofresh Ò , which means no time-dependant activity. citrofresh Ò clearly exhibited concentration and ph dependent viral load reduction activity against influenza type a and the human adenovirus - and human herpes simplex type virus. the reduction in viral titre for porcine parvovirus and human rhinovirus- is probably ph dependent (the ph of % citrofresh Ò is . , % is . and % is . ). conclusion: our investigation shows that citrofresh Ò is an effective disinfectant on environmental surfaces, eliminating enveloped and non-enveloped viruses and sufficient to achieve the minimum -log reduction with complete viral inactivation which is prerequisite for registration. rapid environmental recontamination of an intensive care unit after decontamination with hydrogen peroxide vapour objectives: to evaluate the effectiveness of hydrogen peroxide vapour (hpv) to reduce the levels of total bacterial and methicillin resistant staphylococcus aureus (mrsa) environmental contamination on an intensive care unit (icu), and to establish the rate of environmental recontamination. methods: the study took place on a bed open plan icu. on each environmental screen sites in each bed space (under the bed, the workstation and the monitor) were examined using broth enrichment for the detection of mrsa. in addition total bacterial counts were determined for under the bed and workstation using rodac plates. environmental screening was carried out monthly for the months preceding the usage of hpv, increasing to weekly for the weeks prior to usage. additional sampling was carried out immediately before patients were discharged from icu, following the subsequent terminal clean and then immediately after hpv use. after readmission of patients sampling was carried out at h, h and then weekly for a period of weeks. patients were screened for mrsa on admission and then weekly. results: sampling of the environment prior to the usage of hpv revealed contamination of the environment with mrsa on / occasions, with mrsa colonised patients being present on only / occasions. after discharge of the patients and terminal cleaning of the environment, mrsa was isolated from ( %) environmental sites. after the use of hpv, mrsa was not isolated from any environmental sites upon immediate sampling, but h after patients were readmitted, including patients known to be colonised with mrsa, mrsa was isolated from sites. these sites were not clustered around the colonised patients but were widespread across the icu. in the weeks post hpv usage mrsa has been isolated every week. the mean total bacterial counts prior to the use of hpv were . / cm underneath the beds and . / cm on the workstations, this was reduced after hpv to . / cm and . / cm respectively. after patients readmission the counts were . / cm underneath the beds and . / cm on the workstations after h and returned to pre-hpv levels of . / cm and . / cm at each site respectively after week. conclusion: hydrogen peroxide vapour is effective in eliminating bacteria from the environment. the rapid rate of recontamination of the environment suggests that the use of hpv is not an effective means of maintaining low levels of environmental contamination on an open plan icu. objectives: the nosocomial infections are more serious and dangerous than community acquired infections since they have high rate of morbidity and mortality as well as they increase the cost of therapy. recently many precautions have been taken to prevent these infections. one of these applications is that covering of the floor of the wards, clinics, intensive care units and operating rooms of the hospitals with vinyl flooring material, which is believed to be cleaned easily and effectively. in this study it was aimed to determine the duration of survive of the staphylococcus aureus, enterococcus feacalis, escherichia coli and pseudomonas aeruginosa, which were most common encountered as nosocomial infection agents, on the surface of flooring materials such as vinyl flooring, ceramic laminated wood and galvanized sheet at room temperature. methods: four kinds of flooring materials were prepared approximately in - cm coupons and sterilized. separate bacterial suspensions equal to mc farland turbidity were swapped to the surface of each flooring materials by sterile cotton swabs. all contaminated test materials were put in sterile petri dishes with cover and kept at room temperature without subjecting to the direct sunlight. on the third day, culture samples were taken from the surface of each material by sterile cotton swaps soaked with sterile saline and streaked on the blood agar surface. culturing procedure was repeated every other day until no growth detected. in case of three consequently, negative culture results obtained culturing was ended. results: overall results of the study were presented on table . conclusions: among the four flooring materials, galvanised sheet seemed to be the most unsuitable one for the bacteria to survive long period. in other words this material should be preferred as to laminated wood for covering benches and laboratory tables. as for the flooring of the floors the vinyl flooring material is better than ceramic. covering the complete cmv ie- and pp proteins.results: cmv seropositive transplant recipients had significantly hightened ie- and pp specific t cell frequencies compared to seronegative individuals. patients withevidence of cmv antigenemia or dnaemia could not be discriminated based on cmv-and donor-reactive t cells or serum creatinine. however, recipients of seropositive grafts with low ie response showed a tendency towards more frequent cmv infection. cmv disease was observed in only / individuals. had no detectable ie or pp -t cell response, the third presented with a dominant pp response. interestingly, ie -specific t cells correlated inversely with early post-tx donor-reactive t cell frequencies during weeks - post-tx. most importantly, ie -specific t cell frequencies correlated inversely with serum creatinine at and months at several times post-tx. in patients without acute rejection, even pre-transplant ie- specific t cells correlated inversely with and months creatinine. conclusion: these data suggest subclinical control of cmv infection by ie- specific t cells and subsequently less graft injury by (cmv-induced) alloimmunity. universal precautions: knowledge, attitude and practice of healthcare workers regarding hiv, hepatitis b and c v. gupta, s. bhoi, a. goel, p. aggarwal (new delhi, in) objectives: increasing incidence of hiv, hepatitis b (hbv) and hepatitis c (hcv) in the patients expose the healthcare professionals of acquiring these infections during occupational exposure. we studied the knowledge, attitudes and practices of healthcare workers regarding hiv, hbv, hcv and the risk of occupational transmission of these diseases. methods: an interview survey was conducted among all the health care workers (hcw) using a standardised questionnaire comprising of items in english and local language, as suitable, by an expert in the emergency ward of a tertiary care teaching hospital of a developing nation. data analysis (bivariate and multivariate analysis) was done using spss version . results: (response rate: %) hcw participated in the study. the mean age was ± years, were females. the study population comprised of % doctors, % nurses, % lab technicians and % support staff. respondents had adequate knowledge about causative ( %) usual transmission ( %), symptoms ( %) of aids but poor knowledge about hbv and hcv ( %, % and % respectively). inadequate knowledge was also revealed about the infectious bodyfluids ( %), disinfection of equipments ( %), pregnancy in hcw as a susceptibility factor ( %), post exposure prophylaxis ( %) and comparative infectivity of hiv and hepatitis ( %). % of hcw became anxious while treating these patients. poor compliance with universal precautions was noticed. high compliance was reported for wearing masks ( %) and wearing gloves ( %). doctors were more likely to suffer needlestick injury (p = . ) occupational exposures was found to be high ( %) with poor declaration rate ( %). guidelines adherence was influenced by profession (p < . ), availability or adequacy of protective equipments but not by work experience as hcw (p = . ). all of the respondents urged for an interactive information session. conclusions: results from this study reveal that there is a fair level of knowledge about hiv/aids but hepatitis b and c have not generated adequate concern among the hcw. incongruity between perceived knowledge and reported practice suggests that there is a need for an interactive awareness course about the universal precautions. the educational programmes need to consider attitudes in conjunction with empirical knowledge. objectives: the sero-prevalence of hepatitis a (hav) antibodies are known to be low in young adults in korea. recently, seventeen cases of hepatitis a have been reported in health-care workers (hcw) of icu in a university hospital from may to july . we performed surveillance, and determined molecular identification of outbreaks. methods: . we checked the hav igm from all the patients of sicu with elevated ast/alt retrospectively and screened ast/alt level from all the nurses and the doctors in contact with suspicious index case. . when we determined the existence of outbreak, the molecular subtypes of hav from a blood of hcw were determined to provide the data for epidemiologic study. we determined the index case, a transmission route and the intervention for control an outbreak were planned. results: . seventeen hcw including nurses and doctors who are to years old, suffered from acute hav over weeks period. . the possible transmission of hav was fecaloral route from the bed-ridden patients with diarrhea to the exposed hcw. . seventeen hcw were identified with a positive anti-hav igm. the eight hcw had a positive hav rna. analysis of the vp -p a region of each isolate showed genotype a in five strains and co-circulation of a and b in others. conclusions: the occurrence of hav outbreak highlights the importance of standard precaution in a hospital. the hav vaccination is considered in young aged-hcw. the genotype identification of blood would be useful for the epidemiologic study of suspicious hav outbreak in a hospital. management of a norovirus-associated gastroenteritis outbreak on two psychiatric wards a. buehling, u. arnold (magdeburg, de) objectives: we report a norovirus-associated outbreak of gastroenteritis on a closed psychiatric and a gerontopsychiatric wards from december to february . during this time patients and healthcare workers (hcws) were affected. introduction and results of hygiene measures based on published guidelines on psychiatric wards are described. methods: effective and adapted measures had to be implemented to stop the outbreak and to prevent the spread of disease to other areas of our hospital. isolation or cohorting of the psychiatric patients was excluded for therapeutic reasons. regular hand disinfection in patient rooms was impossible because of the high risk of abuse. the following measures have been introduced: use of gowns, masks and gloves by hcws during care of infected patients-frequent hand disinfections with alcohol-based disinfectants by hcws using ''pocket bottles''; recommendation for all persons entering the station to use gowns, gloves and masks and to disinfect their hands frequently, distribution of handouts describing the measures; hand disinfection by all patients after using toilet, before and after taking meals (distribution of disinfectants by hcw); increased frequency of routine surface disinfection ( times daily) instead of routine cleaning once daily; routine disinfection of door handles, handrails, wash-basins and -fittings and light switches - times a shift; avoidance of patient transfer via hospital; visitor restriction during outbreak time; daily evaluation of recommended measures and adaptation to the current situation; exclusion of affected staff from the ward until h symptom free. results: the hygienic measures have been explained to the local hcws in daily meetings. they have been fully accepted only after a severe staff shortage in the fifth week of outbreak because of new cases of gastroenteritis during hcws and newly infected patients. because of the restrictive application of the adapted guidelines for these special wards the outbreak has been stopped within further weeks. conclusion: in case of norovirus-based gastroenteritis outbreaks on closed psychiatric wards hygienic measures which are adapted to the concrete situation are necessary. especially in these cases the compliance with guidelines can be increased by daily meetings and daily evaluation of recommendations. staff shortage during the outbreak forced the strict compliance with the recommended measures. regional spread of antibiotic resistance methods: we performed surveillance of patients, healthcare stuff and icu environment and we registered the infections of ab during periods of days each one. the interval between st- nd period was months and nd- rd period was year. rectal, oropharyngeal swabs tracheal aspirates from patients, handswabs from stuff and samples from environment were taken weekly. the identification of ab was performed using vitek ii system the susceptibility was tested by kirby-bauer and mic methods and the <<dna fingerprints>>obtained by pulsed field gel electrophoresis (pfge). results: during the st nd and rd period, patients ( men, women), patients ( men, women) and patients ( men, women) were hospitalized in icu respectively. ab was isolated in from samples ( . %) at the st period, from ( . %) at the nd and from ( %) at the rd period. totally ab was isolated in from specimens ( %) at the st nd and rd period among the patients carrying ab, / ( %), / ( %) and / ( %) were infected respectively. the infections observed during the study period were: sepsis ( ), urinary tract infection ( ), pneumonia ( ), meningitis ( ), thrombophlebitis ( ) . all the isolated ab strains were multiresistant to antimicrobial agents. molecular analysis of isolated strains by pfge distinguished the following types: a ( , subtypes a -a ), b ( ) at the st period a( ), c( ), d( ), e( ), f( ), g( ), h( ), i( ). j( ) at the nd period a( ), b ( ), d( ), h( ), k( ). l( ) at the rd period. infections were caused mainly by a and d types while the same types were isolated from the environment and the hands of the icu stuff. conclusion: there was a high rate of colonization and infection of icu patients by multiresistant clones of ab. the persistence of clone a of a. baumannii and the appearance of b type at the rd period after its disappearance at the nd period despite the application hygiene measures, indicates the need for more strict reinforced infection control in icu. the transmission via the hands of stuff to patients has become the most important contributor factor in patient colonization and/or infection. objectives: the antibiotic resistance and its mechanism of group a streptococci (gas) varies according to nations or study period. we have investigated antibiotic resistance and mechanism of macrolide resistance for the strains isolated from korean children and compared to the previous ( ) results. methods: throat cultures were taken from elementary school children in jinju, korea from october to december, to isolate gas. antibiotic susceptibility test to erythromycin (em), clindamycin (cc), and tetracycline (tc) was performed by disk diffusion method. macrolide resistance phenotype and genotype as well as emm genotype were studied. results: isolation rate of gas was . % ( / ). resistance rates of em, cc, and tc were . %, . %, and . % respectively, which were dramatically decreased from %, %, and % in at the same area. emm / was prevalent ( %), while emm was the most common type ( %) in . cmlsb, m, and imlsb were observed in . %, . %, and . % respectively, compared to %, %, and % in . the strains with cmlsb and imlsb had ermb gene and the ones with m phenotype were positive with mefa gene. conclusion: the resistance rates to em and cc were dramatically decreased compared to the past ( ). education to the public and physicians, decreased consumption of antibiotics, acquisition of immunity to the resistant strains, or change of prevalent emm types could be considered to explain the reason of decrease of antibiotic resistance. although antibiotic resistance rate was decreased, cmlsb type which has high mic was prevalent suggesting treatment failure for those children carrying these resistant strains in jinju, korea. analysis of skin and soft tissue infections in european medical centres: report from the sentry antimicrobial surveillance program ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) g. moet, p. strabala, h. sader, t. fritsche, r. jones (north liberty, us) objective: to analyse the skin and soft tissue infections (ssti) or wound infections in hospitalized patients in the sentry program for pathogen prevalence and resistance (r) variations in european (eu) medical centres for the years to ( years). this program also included north america (na) and latin america (la) for the same years, except . methods: consecutively isolated pathogens/site were collected from each centre per year and varied in number of sites each year in eu: eu: ( ), eu: ( ), eu: ( ), eu: ( ), eu: ( ), eu: ( ), and . susceptibility testing was determined by clsi (formerly the nccls) broth microdilution methods and interpreted by current ( ) breakpoints. results: table of all years total of ssti pathogens. (see table) sa was the predominant pathogen in eu ranging from . % of ssti isolates in to . % in . the top most prevalent organisms accounted for . % of isolates in all years with psa and ec ranking second and third, respectively with . % combined, and enc and ent ranked fourth and fifth with . % of the total isolates. compared to the americas, mrsa and vre isolation was at a lower occurrence rate in the eu; but between the rates of other monitored continents for ctz-r psa, cipro-r ec and ctz-r ent (ampc). vre increased in eu over the year. conclusions: pathogen prevalence in ssti for eu has been consistent over the monitored years although sa (with mrsa) appears to be increasing. eu is not a world leader in any key r marker compared to the americas. however, the r rates are evolving which suggests continued need for surveillance programs at regular intervals to detect mobile genetic r elements. objectives: carbapenems play an important role in the therapy of pseudomonas aeruginosa infections. the aim of our study was to characterize the molecular alteration responsible for changed susceptibility towards carbapenems in multiresistant p. aeruginosa strains from germany. methods: multiresistant p. aeruginosa strains from cystic fibrosis and non cystic fibrosis patients were collected in german hospitals in . the strains showed reduced susceptibility (intermediate or resistant; din guidelines) to imipenem, piperacillin, ciprofloxacin and gentamicin. clonality was tested using pfge. a pcr screening for vim and imp was carried out. effluxpump overexpression was detected using an effluxpump inhibitor (epi) test. oprd and for strains with positive results in the epi test the effluxpump repressorgenes mexr and nfxb were sequenced. results: pfge patterns revealed no clonal relationship among the multiresistant strains. neither vim nor imp was detected. the geno-and phenotypes found are depicted in table . defective oprd genes caused by premature stopcodons or frameshifts were found in strains. among those had no mutations in mexr or nfxb and showed the highest mics found ranging from to > and to mg/l of imipenem and meropenem, respectively. additionally had defective mexr genes, but intact nfxb genes, also had modifications in mexr and nfxb, and showed only in nfxb additional alterations. for strains no alterations in oprd but in mexr were proven. conclusions: the predominating mechanism of carbapenem resistance in multiresistant p. aeruginosa strains from germany was the loss of oprd. accessory overexpression of mexaboprm due to modifications in mexr did not result in significantly elevated mics of meropenem. moreover, the additional overexpression of mexcdoprj did not lower the mic of imipenem. in strains with modifications only in mexr only elevated mics of imipenem indicate a reduced expression of oprd accompanied by overexpression of mexefoprn as conferred by nfxc-type mutants. objective: mart (study for monitoring antimicrobial resistance trends) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intraabdominal infections (iai). the aim of this sub-analysis was to assess antimicrobial susceptibility patterns among gramnegative bacilli from different regions of the world during . methods: pa total of major medical centres in north america, latin america, europe, middle east/africa, & asia/ pacific tested the in vitro activity of antimicrobial agents commonly used to treat iai against consecutive unique aerobic and facultative gram-negative bacilli from iai using microdilution techniques according to clsi guidelines & breakpoints. results: enterobacteriaceae were recovered from ( %) & non-enterobacteriaceae were recovered from ( %) of the patients in the study worldwide, constituting ( %) & ( %) of the total isolates, respectively. e. coli (n = ; %) and klebsiella spp. (n = ; %) were the most commonly isolated enterobacteriaceae. pseudomonas spp. (n = ; %) and acinetobacter spp. (n = ; %) were the most commonly isolated non-enterobacteriaceae. isolates from asia/pacific and latin america were generally more resistant. ( %) of the enterobacteriaceae & ( %) of the non-enterobacteriaceae were recovered < hours after hospitalization. the % susceptible isolates are reported below: conclusion: in this study, enterobacteriaceae were the predominant intraabdominal isolates recovered both < h and > h after hospitalization. carbapenems were overall the most active agents against enterobacteriaceae worldwide. resistance rates varied among geographic regions, with the asia/pacific and latin america regions generally having the most resistance. characterisation of streptogramin resistance genes among enterococcus faecium isolates from austrian animal husbandry a. eisner, g. gorkiewicz, g. feierl, f. dieber, e. marth, j. kö fer (graz, at) objectives: the streptogramin virginiamycin has been widely used as a growth promoter in animal husbandry in the european union but was banned in because of concerns about evolving cross-resistance to the streptogramin quinupristin-dalfopristin used in human medicine. the aim of the present study was to investigate the prevalence of streptogramin resistance genes of enterococcus faecium recovered from animal faecal specimens collected in southeast austria. methods: we analysed e. faecium isolates of cattle (n = ), pig (n = ), and poultry (n = ) for the presence of streptogramin resistance genes. we used selective enterococcal broth for isolation. species identification was done on basis of gram stain, catalase and pyrrolidonyl arylamidase activity, motility, lancefield group d antigen typing, and by the strep apitest (biomérieux). detection of the resistance genes vat(e), vat(d), and erm(b) was done by pcr. results: the erm(b) gene encoding macrolide, lincosamine, and streptogramin b (mlsb) resistance was found in each e. faecium isolates recovered from pig and cattle, none of the isolates from these animals carried genes coding for streptogramin a resistance. on the contrary, e. faecium isolates from broiler specimens contained the vat(e) gene and one isolate contained the vat(d) gene. all of these isolates also contained the erm(b) gene. conclusion: our data indicate that the use of the meanwhile banned antimicrobial feed additive virginiamycin has created a reservoir of streptogramin-resistant e. faecium in southeast austrian poultry. characterisation of macrolide-resistant streptococcus pneumoniae isolates from russia objectives: streptococcus pneumoniae (spn) resistance to macrolide antibiotics continues to be of major concern. the aim of present study was to analyse phenotypic and genotypic characteristics of macrolide resistant spn isolates. methods: eighty one macrolide resistant spn isolates were collected in moscow, moscow, - . the susceptibility testing was performed according to the clsi guidelines. macrolide resistance phenotypes were characterized by triple-disk diffusion test, using erythromycin, clindamycin and rokitamycin disks. detection of genes, coding resistance to macrolides, was done by rt-pcr. sequencing for qrdr mutations was performed on levofloxacin resistant spn isolates. selected isolates were analysed by mlst. results: by the triple-disk test, isolates were assigned to the m phenotype, of them were carrying mefa gene, and one was negative. twenty eight isolates were cmlsb-phenotype, of them were carrying both ermb and mefa genes, in two isolates only ermb was detected, and one isolate was negative for both genes. imclsb phenotype was demonstrated by isolates, both ermb and mefa genes were detected in of them, and only ermb in . three isolates didn't demonstrated blunting of zone of inhibition around rokitamycin disk. associated resistance to penicillin g, tetracycline, chloramphenicol and co-trimoxazole was observed in . %, . %, . % and . % of spn isolates respectively. nine multidrug resistant isolates, harbouring both mefa and ermb genes, were subjected to mlst. among them one isolate was found to share the allelic profile st (spain f- clone), and four isolates were single-allele variants of st . in four isolates new allelic profiles were detected. three isolates were resistant to levofloxacin (mic ‡ mg/l), in two of them with levofloxacin mic > mg/l (st single-allele variants) e k, s f and i v substitutions were detected in gyra, parc and pare, respectively. d n and i v substitutions were detected in parc and pare of one isolate with new allelic profile. conclusion: high prevalence of macrolide resistant spn, harboring both ermb and mefa genes is observed in moscow, macrolide resistance is associated with resistance to other groups of antibacterials. some multidrug resistant isolates are highly related to internationally disseminated multiresistant clone spain f- . strains with fluoroquinolone resistance in moscow were all single locus variants of the spain f- clone. occurrence of tet(w) gene in a clostridium difficile clinical isolate p. mastrantonio, f. barbanti, p. spigaglia (rome, it) objectives: to investigate the presence of tet(w), a tetracycline resistance gene recently identified in anaerobic commensal bacteria from animals and humans, in c. difficile clinical isolates. methods: several c. difficile clinical isolates from different italian hospitals were analysed for the presence of a tet(w) gene by pcr assays. the primers used were designed on the tet(w) sequences available in genbank. pcr fragments obtained by these amplifications were sequenced. tet(w) dna flanking regions were also examined with a set of pcrs constructed on the sequence of the conjugative transposon tnb of butyrivibrio fibrisolvens . , that is the only element carrying a tet(w) partially characterized so far. tet(w) positive isolates were also examined for the tet(m) gene and for the presence of int and tndx, markers for the tn and the tn like elements, respectively. the tn -like elements were further characterized by pcrs designed on enteroccus faecalis tn sequence. tetracycline mic values were determined by the e-test method. tetracycline resistance gene transfer was evaluated by filter mating experiments, using c. difficile p r strain as recipient. results: a tet(w) gene was found in only one isolate, c. difficile cd , also positive for the tet(m) gene. this isolate was resistant to tetracycline with a mic of mg/l. sequence analysis of the tet(w) pcr fragment (about bp) showed that this gene had an identity of % with the genes found in clostridium spp strain k , mitsuokella multacida and butyrivibrio fibrisolvens. no amplifications were obtained with the primers designed on tnb , indicating the presence of a different genetic support for tet(w) in c. difficile. tet(m) gene of c. difficile cd was carried by a tn -like element that showed nucleotide sequence mutations in the region containing orf - compared to the element of e. faecalis. conjugative transfer of tet(w) was not observed, whereas the tet(m) gene was transferred to the recipient strain. c. difficile transconjugants were resistant to tetracycline with a mic of mg/l. conclusion: the results obtained in this study demonstrate for the first time the presence of a tet(w) gene in a clinical isolate of c. difficile, providing further evidence of the spread of this resistance determinant among gastrointestinal bacteria. macrolide resistance determinants are prevalent and readily selected for in viridans group streptococci among healthy norwegian adults background: norway has a low prevalence of antimicrobialresistant bacteria including macrolide resistant (mr) respiratory tract pathogens. we have observed an increase in macrolide consumption in norway and there is a lack of knowledge on the reservoir of macrolide resistance determinants among viridans group of streptococci (vgs) in the pharyngeal flora. objectives: examine the occurrence, selection and persistence of macrolide resistance determinants in vgs pharyngeal flora in healthy norwegian adults before and after treatment with azithromycin. methods: throat samples were collected before (day ), after treatment (day ) and after months (day ) from healthy volunteers. the samples were plated directly as a lawn on pdmii agar plates with % defibrinated blood with an erythromycin etest strip. photos were used as quantitative comparisons. up to morphological different colonies with erythromycin etest mic ‡ lg/ml from each specimen were collected; day (n = ), day (n = ) and day (n = ). in total representatives mr, vgs-isolates were selected for further studies: (i) mics of erythromycin, tetracycline and penicillin were determined by etest. (ii) pcr's for erm(b), erm(tr), and mef(a/e), and subsequent sequence-typing of mef. species identification was performed by soda sequencing. results: a total of / persons carried a low number (< ) of mr vgs in day specimens, while / had a significant higher number (> ) of mr strains in day specimens. in day specimens, / carried a low number of mr, resembling day . reduced susceptibility to penicillin was observed in / ( %) isolates. tetracycline resistance was found in / ( %), and mainly in erm(b)-positive strains. mef(a/e)-positive dominated day ( %) and erm(b) day specimens %. sequence typing revealed mef(e) (n = ) and mef(a) (n = ). soda sequence; s. mitis (n = ), s. oralis (n = ), s. parasanguinis (n = ), s. salivarius (n = ), and s. sanguinis (n = ). conclusion: there is a pool of vgs carrying macrolide resistance determinants in the normal pharyngeal flora of healthy adults that are readily selected for during azithromycin exposure. the mef(e) and erm(b) were the most prevalent resistance genes and co-resistance to tetracycline was frequently observed, resembling the findings in norwegian clinical isolates of s. pneumoniae. these vgs may provide a pool of resistant bacteria that may transfer resistance determinants to more pathogenic organisms. relationships in genotype, phenotype, t type and pfge type among macrolide-resistant streptococcus pyogenes strains isolated in the czech republic v. jakubù , p. urbášková, l. straková (prague, cz) objectives: to determine relationships between phenotypic and genotypic methods among erythromycin-resistant s. pyogenes strains. methods: a total of clinical isolates of s. pyogenes resistant to erythromycin were collected in microbiology laboratories during - . erythromycin susceptibility was tested by the disk diffusion method. strains with an inhibition zone < mm around the erythromycin disk ( lg) were sent to the national reference laboratory for antibiotics (nrl). presences of mlsb resistance genes (ermtr, ermb and mefa) were tested by pcr. t serotypes were determined in randomly selected representatives of each phenotype (n = ). pfge type were determined in strains from year only (n = ). results: the rate of the most prevalent phenotype (constitutive mlsb resistance) was %, % in the year and , respectively. the major prevalent t types among the analysed strains were serotype t ( %), t ( %), t ( %) and t b ( %). gene ermb was the most frequent ( %). the results of pcr method was highly congruent with observed phenotype of resistance. pfge patterns of strains with constitutive mlsb resistance were highly identical. conclusion: m phenotypes, constitutive and inducible resistance to mlsb antibiotics were found and ermtr, ermb and mefa genes were detected among the analysed strains. the t serotype was identified the mainly prevalent in our collection. the majority of strains harbouring t serotype were constitutively resistant to macrolides. the study showed close relationships among genotypes, t types, specific resistotypes (phenotype) and pfge types. objectives: since recognition of transferable clindamycin and tetracycline resistance in bacteroides, we have undertaken a us national survey on the susceptibility of b. fragilis group to analyse emergence of resistance and trends, since these species are not routinely tested for susceptibility in hospital clinical laboratories. methods: agar dilution mics were determined for isolates from - for b. fragilis and related species from geographically diverse centers in the us. antibiotics included carbapenems, b-lactam/b-lactamase inhibitors, quinolones, a tetracycline, clindamycin, metronidazole, chloramphenicol, a glycylcycline and linezolid. isolate identity was confirmed by api a. results: analysis of resistance trends from - showed a decrease in geometric mean mic's (geomic) for imipenem ( . mcg/ml to . mcg/ml, p < . ) and meropenem ( . mcg/ml- . mcg/ml, p = . ) for the bacteroides species. ertapenem geomic remained unchanged ( . mcg/ ml). for the b-lactamase inhibitors, piperacillin-tazobactam geomic declined from . mcg/ml to . mcg/ml (p < . ). ampicillin-sulbactam geomic did not change. few isolates were resistant to any carbapenem or b-lactamase inhibitor combination. clindamycin resistance increased, especially for b. fragilis, b. ovatus and b. thetaiotaomicron (all p < . ). among quinolones, resistance of bacteroides to moxifloxacin increased (geomic went from mcg/ml to . mcg/ml, p < . ). b. fragilis remains the most sensitive bacteroides species to moxifloxacin, although approximately % of stains have mic's ‡to mcg/ml in . tigecycline susceptibility, tested over years, did not change. the first confirmed metronidazole-resistant isolate (mic = mcg/ml) obtained in the us was noted in but none were noted in or . conclusion: improved susceptibility of bacteroides species to some carbapenems and the b-lactamase inhibitor combinations is unexplained but significant. clindamycin resistance continues to increase, especially for b. fragilis. moxifloxacin susceptibility for the non fragilis species shows that the majority of strains are resistant. the first metronidazole resistant isolate has been reported from the us. since resistance trends are associated with species, the differentiation within the species is of extreme importance, since it may impact the choice of antimicrobial agent for the treatment of infections caused by this group of anaerobes. observed duration of nasopharyngeal carriage of penicillin-resistant pneumococci: relations to age and serogroup p. geli, l. hö gberg, h. ringberg, e. melander, m. lipsitch, k. ekdahl (solna, malmö, lund, stockholm, se; boston, us) background and objectives: knowledge of how the duration of pneumococcal carriage varies with age and serogroup is essential to understanding how immunity to carriage arises throughout the course of life, and designing appropriate models for the effects of vaccination or other public health initiatives aiming to reduce the pneumococcal transmission in the community. using data from an ongoing swedish intervention project, the duration of nasopharyngeal carriage of penicillinresistant pneumococci (mic pcg > . mg/l) stratified by both serogroup and age of the carrier were estimated. methods: the mean duration and corresponding % confidence interval was estimated by fitting a gamma distribution to the observed duration of carriage for each serogroup and age stratum. results: the mean duration of carriage for all cases was days ( % ci - ). children below the age of years carried prp for significantly longer periods ( days, % ci - ) compared with older individuals ( days, % ci - ). there were also differences within the group of cases below the age of years, as the duration of carriage became significantly shorter for each year older the cases were. serogroup and were carried for significantly shorter periods compared with serogroup . serogroup also had significantly shorter carriage duration compared with serogroups and for cases - years. for cases years or older, no significant difference in carriage duration for different ages or serogroups could be noted. conclusions: even though the estimate does not cover any correction for the censored carriage duration and therefore not yield an estimate of the total length of carriage, the results highlight the importance to take both serogroup and age of the p exploring the molecular basis for differences in phenotype of salmonella enteritidis typing phage n. delappe, d. morris, m. cormican (galway, ie) objectives: the salmonella enteritidis phage tying scheme of the laboratory of enteric pathogens, health protection agency, uk, is a widely used method for subtyping this important pathogen. the method is rapid and highly discriminatory. interpretation of results can be subjective and the typing phage which are central to the method have not been well characterised. complete sequence data is available for the salmonella typhimurium podovirus phage p . methods: the typing phage were propagated on s. enteritidis pt b (pb ). phage were visualised by electron microscopy. phage dna was extracted and digested with hindiii. consensus pcr primers were designed based on sequences of p and other s. typhimurium phage. additional primers were designed based on the sequence of the s. enterititidis typing phage (a siphovirus). amplification, sequencing and dna probe hybridisation of various phage genes were performed using standard techniques. results: on em the typing phage comprise podoviridae (phage , , , , and ) , siphoviridae (phage , , , , and ) and myoviridae (phage , , and ). digestion with hindiii subdivided each morphotype into groups. the podoviridae contained genes homologous to p while the siphoviridae contained genes homologous to the sequenced s. enteritidis typing phage . some sequence variation was detected in podovirus and siphovirus genes however in some cases phage, which differ in their phenotype had no difference detected in hindiii digestion pattern or partial sequence. conclusions: the s. enteritidis typing phage set comprise distinct phage morphotypes. in some instances distinct phage that contribute to differentiation between s. enteritidis phage types had no dna sequence variation detected. variations in phage typing reactions may in part be due to epigenetic difference in typing phage, e.g. due to methylation of phage dna. salmonella enteritidis typing phage biology could provide a model for developing approaches to phage therapy. tularemia is a zoonotic bacterial disease. the causative agent, francisella tularensis, is spread to humans by direct contact with infected rodents, inhalation, ingestion of contaminated water or by arthropod bites. in some endemic regions, outbreaks occur frequently, whereas nearby rural parts may be completely free. we presented two cases of tularemia in non endemic region of the turkey. case : a year old female patient referred to tertiary hospital due to swollen on the neck for months. before admission beta lactam antibiotics had been prescribed to her for tonsilopharyngitidis. but her complaints had been continued. so she admitted to our hospital. she had been suffered fever sore throat and neck pain. she had a palpable and painfull cervical lymphadenopathy which was not suppurated. leukocytosis and elevated c reactive protein were predominant. at screening there were not any lymphadenopathy detected elsewhere. she had been examined about cytomegalovirus epstein barr virus and brucellosis. they were negative. fine needle aspiration from neck was negative considered as malignancy. cultures were negative for routine bacteriologic examination. microagglutination test for tularemia was / positive. then we decided to treat her with gentamycin for days. after treatment cervical lymphadenopathy became small. leukocyte count and c reactive protein levels were reach normal range. case : a year old female patient referred to university hospital due to cervical lymphadenopathy and fever and sore throat. before admission beta lactam antibiotics were prescribed to her for weeks. but no apparent benefits had been detected. there was a palpable and fistulated cervical lymphdenopathy. drainage was examined microscopically and cultured for bacteria, mycobacteria and fungi. on routine cultures no microorganisms were grown. fine needle aspiration was done. it was reported that suppurative granulamatous lympadenitis. so we were examined for tularemia, cat scratch disease. microaggltunation test for tularemia was / positive. then streptomycin had been given for days and excision of lymphadenopathy had been done. no complications or recurrence occur. results: both patients were applied to us from non endemic and different regions of the turkey. they had no known insect bite history. both of them were diagnosed by serological tests. conclusions: in the differential diagnosis of tonsillopharyngitidis, tularemia also must be considered in the non endemic regions. tularaemia presenting with tonsillopharyngitis and cervical lymphadenitis: two case reports b. kandemir, i. erayman, m. bitirgen, e. turk aribas, a.c. inkaya, s. guler (konya, tr) tularemia is a zoonotic disease caused by francisella tularensis. francisella tularensis is transmitted to humans by direct contact or ingestion of infected animal tissues, through the bite of infected arthropods, by consumption of contaminated food or water, or from inhalation of aerosolized bacteria. in this report we describe two cases of oropharyngeal tularemia who presented with tonsillopharyngitis and cervical lymphadenitis. case i: a years old woman with multiple cervical lymphadenitis has been admitted to our clinic. her complaints started months ago with signs and symptoms of tonsillopharyngitis. she had received non specific treatment (ampicillin+sulbactam) and ten days later cervical lymph nodes appeared. the diagnosis was made serologically. the antimicrobial therapy (streptomycin · g im) was given for fourteen days. the patient recovered completely. case ii: a years old girl with multiple cervical lymphadenitis was admitted to hospital. her complaints started months ago with throat ache after which multiple cervical lymphadenitis appeared. she was admitted to our out patient clinics and diagnosed to have tularemia. anti-microbial therapy (streptomycin · g im+doxycyciline · mg) was given for four weeks but no clinical response was achieved. patient was admitted to the hospital and surgical drainage was performed. treatment against tularemia was prolonged. patient was finally recovered at the end of nine weeks of therapy. it can be concluded that early diagnosis and treatment of tularemia are important. some patients may benefit from surgical drainage and prolonged therapy. a case of nonclostridial crepitant cellulitis which is due to escherichia coli c. ayaz, m. ulug, m.k. celen, m.f. geyik, s. hosoglu (diyarbakir, tr) objectives: this condition is caused by gas forming bacteria that involve the skin, either or as an extension from deeper structures. the origin of infection is an abdominal wound, perianal disease, or operative incisions that have become secondarily infected. tracking of gas-forming organisms from deeper sites of infection may also present as crepitant cellulitis without a break in the skin. diabetics are more likely to acquire such infections, especially in the lower extremites. among the bacteria isolated are anaerobic organisms such as bacteriodes or anaerobic streptococci, or coliform bacteria, especially escherichia coli and klebsiella. because of this reason we reported a case of a nonclostridial crepitant cellulitis which is due to escherichia coli. case: a year old man who was previously healthy, has come with fever, pain, oedema, erythema, crepitant and limitation of movement at the right lower extremity. in his history he had no complaint until weeks ago. perianal abscess has developed at this time and it has drainged spontaneously days later. than his complaints has comprised day duration. on physical examination, the temperature was . °c, pulse rate / minute, respiratory rate /minute and blood pressure was / mmhg. laboratory evaluation showed a haemoglobin . g/dl, leucocyte count of /mm (neutrophils %). serum electrolytes, renal and liver function tests were within normal limits. c reactive protein was elevated up to mg/dl, esr was mm/h. escherichia coli was isolated from wound and blood cultures. he was treated initially with ampicilinsulbactam ( g/day) and required attempt. even with optimal surgical and medical therapy, he dies at the third day of the treatment from septic shock. conclusion: the onset is generally gradual, and there is usually mild local pain and systemic toxicity, allowing clinical differentiation from the more fulminant clostridial myonecrosis. the surgical approach should be aggressive, but tailored specifically to the underlying cause of infection. antibiotic therapy is directed at a mixed aerobic-anaerobic flora, until culture reports are available. a case of iliopsoas abscess which is due to pseudomonas aeruginosa objectives: pyogenic psoas abscess, a rare but life-threatening infection, results from primary suppuration or is secondary to the spread of infection from an adjacent structure. primary iliopsoas abscess occurs probably as a result of hematogenous spread of an infectious process from an occult source in the body. primary iliopsoas abscess can occur in diabetus mellitus, intravenous drug abuse, aids, renal failure and immunosupression. ultrasound is diagnostic in only % of the cases. computed tomography should be done for definitive diagnosis and is considered the gold standard. stapylococcus aureus is the causative organism in patients with primary iliopsoas abscess, but pyogenic psoas abscess caused by pseudomonas aeruginosa is uncommon. because of this reason we reported this case. case: a previously well year old woman presented with a month history of right loin to groin pain, limping or limitation of hip movement, fever and nausea. she was a diabetus mellitus patient for years. on her physical examination, the temperature was . °c, pulse rate /minute, respiratory rate /minute and blood pressure was / mmhg. examination of the respiratory system, cardiovascular system and abdomen were found to be normal. laboratory investigations revealed total leucocyte count of /mm (polymorphs %), c reactive protein was elevated up to mg/dl, esr was mm/h. serum electrolytes, renal and liver function tests were within normal limits, but serum glucose level was elevated to mg/dl. her blood cultures were sterile, but abscess culture yielded pseudomonas aeruginosa which was taken during the surgery. she was treated imipenem ( g/ day) + amicasin ( . g/day) and required surgical drainage. she was treated and followed up days, and discharged at the end of the treatment. conclusion: in these patients treatment involves the use of appropriate antibiotics along with drainage of the abscess. an adequate knowledge of the causative organisms should guide the initial choice of antibiotics. depending on the results of the abscess fluid culture and sensitivity, adjustments should be made. percutaneous drainage or surgical drainage may be done in them. in conclusion early recognition, empiric antimicrobial coverage and aggressive drainage or debriment are indicated in these patients. cervical lymphadenitis in a diabetic woman f. Ç okça, a. azap, s. gö çmen, h. erdi sanli, s. gü l (ankara, kirikkale, tr) objective: rhodococcus equi infections are commonly seen in immunocompromised patients. exposure to domestic animals, such as horses and pigs may play a role in some cases. two thirds of the r. equi infections in immunocompromised were reported in hiv infected patients, and the rest divided between transplant recipients, immunosupressive medications and other kinds of immunosupression. the clinical picture presents with pulmonary infection in % of patients. here, we report a rare case of cervical lymphadenitis in a diabetic women due to r. equi. case: a sixty-year-old diabetic woman was admitted with the complaints of fever, right cervical erythematous swelling with tenderness and warmth. on physical examination; inflammation beginning from the right submandibular region and descending to the upper chest was detected. a tender mass of · · cm. was palpated on the right cervical region. ampicillin/sulbactam g/day was given emprically for a week with no improvement. the ct scan of the neck showed conglomerated lymphadenopathy extending from the submandibular area to the supraclaviculary region with . · cm in size. the mass began to fluctuate and cc abscess material was drained surgically. gram's stain of the purulent material showed polymorphonuclear leukocytes with pleomorphic gram positive coccobacilli. the cultures of the material grew r. equi. therapy was changed to teicoplanin and ciprofloxacin combination and surgical care of the wound with antiseptics was performed. after a month, intrevenous medical therapy was changed to oral route with roxythromycin and ciprofloxacin and was continued to months with complete resolution. conclusion: increased awareness and improved laboratory techniques help for the early diagnosis of rhodococcal infections. timely diagnosis is important because the microorganism is usually resistant to penicillin g, oxacillin, ampicillin, carbenicillin and cefazolin. the use of at least one antibiotic with intracellular activity is necessary in the treatment of r. equi infections. empirical two drug regimens with erythromycin, rifampin and/or ciprofloxacin are recommended. objectives: to analysed the features of spondylodiscitis (sd), their clinical presentation, the commonest diagnostic methods and the kind of treatment applied according to the different groups of the study. methods: a retrospective and descriptive study taking place amongst the patients diagnosed as having sd from till . in each case we studied the presence of underlying disease, primary infectious sources in the prior months, the way symptoms started, location, diagnostic methods, treatment and evolution, comparing between different aetiologies. results: patients with sd were studied. of them had pyogenic sd,( had spontaneous sd and had an sd after spinal surgery) and patients had tuberculous sd. were men ( to years; mean . ). patients with postoperative sd were the youngest (mean . y, p = . ). underlying diseases were found in % of patients, mainly in postoperative sd ( % of cases) (p = . ). an episode of previous bacteremia or infectious source was found in % and % respectively of patients with spontaneous pyogenic sd, significantly higher than in surgical sd ( % had bacteremia and % other infectious source, p < . ). the most common presenting symptoms were back pain ( . %) and neurological deficits ( %). frank fever occurred in % of cases, being more frequent in spontaneous sd ( %) than in postoperative sd ( %) or tuberculous sd ( %), p £ . . leukocytosis was found only in % of patients. postoperative sd presented the lowest levels of esr (p = . ). s. aureus was the most frequent bacteria isolated ( %) in pyogenic spontaneous sd, as coagulase negative staphylococci was in surgical sd. lumbosacral localization was detected in % of spontaneous pyogenic sd and in % of postoperative sd. tuberculous sd predominate in dorsolumbar region. paravertebral abscess formation was observed in % of pyogenic sd and in % of tuberculous sd (p = . ). surgical treatment was required in . % of tuberculous sd and in % of pyogenic sd (p = . ). outcome of patients with spontaneous sd was worse (sequelae in %), than in patients with surgical sd ( . %) or tuberculous sd ( %) (p = . ). conclusions: ) spontaneous sd was the most frequent and it occurred mainly in patients suffering from underlying diseases; ) nearly all patients had pain but only in / of them was accompanied by fever; ) the lumbar zone was the most frequent location; ) the majority of patients had a complete resolution of their symptoms only with medical treatment. background: the ethiopathogenesis of cns abscess includes a broad spectrum of pathogens and predisposing conditions, so that a polymicrobial flora is a quite frequent event. capnocytophaga spp. includes fastidious gram-negative organisms, usually underestimated in the common clinical practice, and poorly tested in vitro for antimicrobial susceptibility. surprisingly, also agents usually active on gram-positive pathogens demonstrated some efficacy against capnocytophaga spp. (i.e. erythromycin, rifampin, tetracyclines, cotrimoxazole, chloramphenicol, and glycopeptides), which is usually responsible of anecdotal episodes of cns infection (meningitis, brain abscess, and subdural empyema). methods and results: the fourth case report of capnocytophaga spp. brain abscess is herewith reported. a probable origin from a recent cat bite and a mandibular granuloma is suspected. due to the lack of clinical and neuroradiological response to neurosurgical debridement and an association therapy including imipenem, amikacin, clindamycin and fluconazole, empiric administration of linezolid ( mg/day) was attempted, and a rapidly favorable clinical, microbiological, and neuroradiological response was achieved. notwithstanding the identification of capnocytophaga spp. as the sole microorganism yielded by purulent drainage of a cns abscess, patients with multiple risk factors and recent surgery are expected to suffer from a polymicrobial cns infection. due to its favourable cns penetration and its dual mode of administration (both i.v. and oral), linezolid may represent an alternative option in the event of cns diseases borne by numerous risk factors and a suspected polymicrobial origin, especially when a lack of response to first therapeutic attempts is of concern. in the management of a cns abscess where the role of microorganisms with an unpredictable sensitivity profile remains of concern, chemotherapy should be directed also against potentially multiresistant organisms. considering also the relevant limitations given by the often poor cns penetration, the activity of glycopeptide agents is limited, compared with that of linezolid. aetiologies and antimicrobial resistance profiles of purulent meningitis study carried out in a hospital of infectious diseases, algiers objectives: bacterial meningitis is a serious clinical and medicolegal consequences if management is incorrect. meningitis protocols have recently been published by the british infection society/meningitis research foundation and are widely disseminated in our institution. local guidelines are also available on the hospital intranet and in the emergency department and acute medical admissions wards. this study investigated the level of understanding about meningitis and knowledge of the guidelines in medical staff of different grades working in the emergency department and the acute medical admissions unit in a large teaching and emergency hospital. methods: medical staff were interviewed faced to face and asked a series of questions on the management of meningitis. results were stored on a database and responses were analysed. results: general knowledge about meningitis was variable. although % knew that bacterial meningitis was a notifiable disease only % knew the procedure for informing the health protection agency and only % would notify viral meningitis. only % of responders were aware that guidelines could be viewed on the hospital intranet. only % correctly identified the indications and cautions for lumbar puncture. although the majority recognised the need for urgent administration of antibiotics % would omit antibiotics until further assessment and lumbar puncture results. only % were aware of the need to consider adding ampicillin to cover listeria in patients over years of age and there was uncertainty about the management of patients with penicillin resistance. conclusions: although protocols and guidelines for meningitis have been produced and are easily accessible the majority of medical staff were uncertain how to access and utilise this information. the level of knowledge and expertise in managing meningitis amongst medical staff working in a and e and the acute medical unit was poor and there is a need for further education to improve patient management. guidelines are of no value if they are not disseminated to front-line medical staff. objectives: the aim of this study was to evaluate the prevalence of penicillin resistant and multi-drug resistant pneumococci isolates in streptococcus pneumoniae meningitis. methods: a retrospective study was carried out on clinical records between january and october . among the csf samples the pneumococcal ethiology was confirmed by % positive cultures and % latex agglutination. antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. isolates of pneumococci with oxacillin zone sides of > mm are susceptible (mic < . microg/ml) to penicillin, while at those of < mm the mic has to be determined (by e -test). results: isolates from patients ( %) were found with penicillin-resistance (prp) -of which % were multi-drug resistant-and ( %) with penicillin susceptibility -of which % were resistant to other drugs. an abrupt onset of disease was found in % prp patients and % from non-prp ones. chest x ray pulmonary determinations were found in % prp patients and % non-prp ones. sixty-six per cent of prp patients and % of non-prp ones had a prior hospitalization. only % of non prp patients had a positive blood culture. antibiotic switch was made in % cases with prp isolates and % cases with non prp ones. the overall rate of mortality was %, with % for prp patients and % for non-prp ones. conclusions: non-prp isolates were the prevalent ethiology of s. pneumoniae meningitis. % of non -prp strains developed other drug resistance, and % prp strains were multi -drug resistant. prp meningites evolved more as a hospital-related pathology, with an abrupt onset, frequently associated with pulmonary determinations and higher mortality rate. background: although vaccination strategies have shifted the age distribution of meningitis to older age groups, few studies have specifically examined bacterial meningitis in the older adult. methods: from october to april , we prospectively included episodes of community-acquired bacterial meningitis, confirmed by culture of cerebrospinal fluid, which occurred in patients aged > years. we dichotomized the cohort with respect to age: patients aged ‡ years were defined as older adults and patients aged - years as younger adults. predictors for an unfavourable outcome (defined as score - on the glasgow outcome scale) were determined by logistic regression. we tested for statistical interaction between age group and potential prognostic factors by adding multiplicative interaction terms to the model. the mann-whitney u test and the chi-square test were used to identify differences between groups. results: of episodes ( %) occurred in older adults and episodes in younger adults ( %). streptococcus pneumoniae was the most common pathogen in older adults ( %). meningitis in younger adults was caused by neisseria meningitidis and s. pneumoniae in % and % of the episodes, respectively. older adults were more likely to present with the classic triad of bacterial meningitis (fever, neck stiffness and altered mental status) than younger adults ( % versus %; p < . ). the prognostic value of independent risk factors for unfavourable outcome was similar in both age groups. older adults had more complications during clinical course, resulting in a higher mortality rate than in younger adults ( % versus %; p < . ). sepsis was the most common cause of death in both age groups ( % in older adults versus % in younger adults; fig) . whereas older adults tended to die more often due to cardiorespiratory failure ( % versus %; p = . ), younger adults more often died due to brain herniation ( % versus %; p = . ). conclusions: bacterial meningitis in older adults is associated with high morbidity and mortality rates. elderly patients often present with classic symptoms and s. pneumoniae is the most common pathogen within this age group. whereas older adults often die due to cardiorespiratory failure, younger adults more often die due to brain herniation. incidence of serogroups and penicillin susceptibility in neisseria meningitidis isolates ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) objective: the aim of this study was to analyse the serogroup incidence and penicillin susceptibility in n. meningitidis before and after spanish epidemic outbreak in . in this year the public health service decided a massive vaccination in our sanitary area (galicia, north-west of spain, . inhabitants) and in autum of the inclusion of vaccine against n. meningitidis serogroup c in vaccination programme. methods: retrospective study of all cases of meningococcal disease confirmed by culture and/or pcr in the health care area of santiago de compostela (galicia) from to . results: in the period - we identified meningococcal disease episodes by microbiologic diagnosis ( . %, . % and . % to b, c and w respectively). in , serogroup c were the % of the isolates. in and the serogroup incidence was almost the same (b = . %, c = . %). from an increase in b serogroup cases were detected, in ( . %), in ( . %) and in ( . %). the most frecuent phenotype has been b p : . in c serogroup. during this period an increase in penicillin susceptibility was observed (in b serogroup % in and % in of the isolates were susceptible and in c serogroup % in and % in ). conclusions: the b serogroup is the most frecuent isolate during this period except in the years and . the strain that cause the epidemic outbreak in (c: b p : . ) was not isolated since . in our health care area, c: a serotype, was isolated for first time in , and since then, is the unique serotype isolated in c serogroup. incidence rate in c serogroup has changed from . / . in to . / . in . this decrease was caused by the drop of incidence rate on the youngest groups (< years and - years). the incidence rate in b serogroup during these years was modified from . / . in to . / . in . a four-year retrospective analysis of infective endocarditis in a belgian university hospital n. de visscher, b. delaere, b. krug, y. glupczynski (yvoir, be) objectives: to establish the epidemiology of infective endocarditis (ie) and determine the prognostic factors for adverse outcome in patients admitted to a university hospital with a cardiovascular surgery department. methods: between / and / , the clinical and laboratory features of all consecutive adult patients with a definite diagnosis of ie (duke criteria) were evaluated retrospectively by two infectious diseases physicians on the basis of clinical data charts and microbiological laboratory. results: patients ( men, women) presented with a definite diagnosis of ie. mean age was , yrs; cases ( %) were native valve endocarditis (nve) and ( %) were prosthetic valve endocarditis (pve); % of patients with nve had underlying valvular abnormalities ( bicuspidies, mitral prolapsus or regurgitation, others). ten out of cases of pve were late-onset episodes (> year after surgery). global mortality was % ( / pts), including patients ( %) still under antibiotic therapy. a higher mortality rate was observed in pve [ / ; ( %)] than in nve [ / ; ( %)]. overall, pts ( %) underwent surgery (mean: days following admission). valvular replacement was contra-indicated in pts because of critical status and/or major co-morbidities. the distribution of isolated pathogens was: streptococci: cases ( %) including cases of s. bovis, s. aureus: cases ( %, including mrsa), enterococci: cases ( %), miscellaneous: cases. the affected valves were: only aortic: ( %), only mitral: ( %), only tricuspidal: , aortic and mitral: , mitral and tricuspidal: , aortic, mitral and tricuspidal: . a high mortality rate was observed in s. aureus ie ( / [ %]), especially in the subgroup of patients with a pve ( / pts [ %] ). the mortality rate in patients with ie episodes caused by streptococci amounted % ( / pts). clinical microbiology and infection, volume , supplement , related, % (n = ) had previous surgery and % (n = ) were related to urinary or digestive tract procedures. only patients had illegal substance abuse. the most frequent predisposing acquired cardiac condition for native valve endocarditis was degenerative valvular disease in % ( / ). twelve percentage (n = ) had prior ie. the most frequent predisposing congenital cardiac condition was a bicuspid aortic valve in % (n = ). in % (n = ), no predisposing heart disease was discernible. causative microorganisms included: staphylococci in % (n = ) with s. aureus in % (n = ), cons in % (n = ), streptococci in % (n = ) with s. viridans in % (n = ), s. bovis in % (n = ), enterococci in % (n = ) and other pathogens in % (n = ). culture negative ie was reported in % (n = ). both in community-acquired and nosocomial ie, s. aureus was the most frequent causative agent. twenty-three percentage ( / ) were methicillin-resistant s. aureus. s. viridans ie was mainly community-acquired while enterococcal ie was nearly equally distributed between community and nosocomial origin. conclusion: compared to older series, we observed a high proportion of nosocomial ie and of prosthetic valve ie. s. aureus and e. faecalis were the most prevalent causative microorganisms. enterococci were nearly equally distributed between community and nosocomial origin, suggesting that nosocomial enterococcemia should be added as a major criterion, as proposed before for s. aureus. the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis: a meta-analysis of comparative trials m. falagas, d. matthaiou, p. papastamataki, i. bliziotis (athens, gr) objectives: the addition of an aminoglycoside to a beta-lactam for the treatment of patients with infective endocarditis has been supported mainly from data from laboratory and animal studies. we sought to review the evidence from the available comparative clinical trials regarding the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis due to gram-positive cocci. methods: the studies for our meta-analysis were retrieved from searches of the pubmed database and from references of relevant articles. included studies were trials that provided comparative data regarding the effectiveness of the treatment and/or mortality in patients receiving monotherapy with a betalactam or beta-lactam/aminoglycoside combination therapy. two independent reviewers performed the literature search, study selection, and extraction of data from relevant studies published in english during the period / - / . results: no clinical trial comparing beta-lactam monotherapy to beta-lactam/aminoglycoside combination therapy for the treatment of enterococcal endocarditis was found. we performed a meta-analysis of available comparative trials ( randomized controlled trials and comparative prospective trial) that included patients with bacterial endocarditis in native valves due to staphylococcus. aureus ( studies) or streptococcus viridans ( study). there was no statistically significant difference between the compared arms regarding mortality (or . , ci % . - . ), treatment success (or = . , ci % . - . ), treatment success without surgery (or = . , , and relapse of endocarditis (or = . , . nephrotoxicity was less common in the beta-lactam monotherapy arm compared to the beta-lactam/aminoglycoside combination therapy (or = . , ci % . - . , p = . ). conclusion: the limited evidence from the available prospective comparative studies does not offer support for the addition of an aminoglycoside to beta-lactam treatment of patients with endocarditis due to gram-positive cocci. a large multicenter randomized controlled trial may be necessary to reach a definitive conclusion on this issue. outpatient antimicrobial therapy for infective endocarditis. single-centre experience objectives: to evaluate the characteristics and outcome of infective endocarditis (ie) patients included in a outpatient antimicrobial therapy (opat) program. methods: from january to may all patients who received opat therapy for an ie were prospectively evaluated. inclusion in opat program require clinical stability and agreement of patients. active drug addiction was contraindicated for inclusion. antibiotic treatment was administered in bolus for once-daily antibiotics regimens. we used cadd-legacy tm plus (deltec, inc. st paul. usa) portable infusion system for either continuous or intermittentprogrammed bolus infusion. results: we included patients, male ( %), mean age years old (sd: . years). the diagnostic of ie was definite in cases ( with pathologic diagnosis), probable and possible. mostly of the cases were community-acquired ie ( %). mitral valve ie was the most frequent anatomical site involved ( %), followed by aortic ( %). native-valve ie represent the majority of cases ( %), but % were prostheticvalve and % were pacemaker lead ie. viridans group streptococci was the most frequent isolate ( patients, %) with cases of s. bovis ie. eleven patients had s. aureus ie ( %). at the time of the diagnosis, patients had valve rupture and patients had periannular abscess. a total of patients required some surgical intervention for the ie [ valvular replacement ( of them associated with aortic graft), pacemaker extraction and aortic graft]. the majority of the patients received outpatient monotherapy ( %). the most frequent antibiotic used was ceftriaxone ( % of the cases), followed by cloxacillin %, gentamycin %, vancomycin %, teicoplanin %, ampicillin % and other antibiotics in %. in % of the patients the vascular access was a perifericallyinserted venous central catheter and in % we used a portable infusion system. twelve patients ( %) had some complication during opat that require hospital readmission, of which could return to opat program. three patients had a fatal outcome (deaths) during admission, not related to ie complications. the mean duration of opat was . days per patient, and globally supposed . days of hospital admission savings. conclusion: opat for ie can be a good therapeutic option for ie stable patients. this procedure can represent a considerable amount of hospital admissions savings, improving also patients' well-being, and must be take into account for the treatment of this disease. objectives: botulism, a neuroparalytic illness, is caused by toxin produced by clostridium botulinum. food born botulism, a potentially lethal neuroparalytic disease, is caused by ingestion of preformed toxin. clinical illness is characterised by cranial nerve paralysis, followed by descending flaccid muscle paralysis. in this article we report a case series including a family group of type e botulism after ingestion of an iranian traditional soup. methods: in january , patients of a family group developed clinical manifestations of botulism - hours following ingestion of a traditional soup. their main clinical presentations were severe weakness ( . %- case) and lethargy ( . %- case). other signs and symptoms were blurred vision, fixed and dilated pupils, diplopia, dry mouth and decreased gag reflex. based on clinical finding, all patients received monovalent antitoxins (a, b, c). stool, gastric fluid and serum samples were sent for toxicological evaluation using the standard mouse bioassay. results: type e toxin was detected in the stool and serum sample of only one patient. all patients recovered and discharged one week after admission. conclusion: this study confirmed that prompt administration of antitoxin can prevent progression of disease based on clinical judgment and also may be life saving. in this case series study, we observed a short incubation period of - hours only in type e botulism. an outbreak of group g streptococcal pharyngitis among hospital personnel considered to be foodborne n. karabiber, a. gurbuz ertas, m. karahan, e. aykut arca, z.c. karahan, a. tekeli (ankara, tr) introduction: food-born outbreaks of streptococcal pharyngitis are relatively rarely reported,and while group a streptococci are the main causative agents, only a few epidemics caused by group g streptococci have been published. we describe here an outbreak of group g streptococcal pharyngitis occurred among the staff of a teaching hospital in ankara.the outbreak: an explosive outbreak of pharyngitis occured mainly among the staff working in certain departments (i.e. intensive care units, operation rooms) of tü rkiye yü ksek ihtisas teaching hospital, in january . methods: a total of ( and ; and from catering firm personel) throat cultures were evaluated in days,and bhs strains were isolated, and on the first and the second days, respectively. presumptive identification by nbacitracin and trimethoprim/sulfametoxazole disk diffusion test showed that strains were non-group a, strains were group a streptococci. in definite grouping by streptococcus grouping kit (avipath-strep,omega), strains were found to be lancefield group g, strains were found to be group a streptococci (gas). one of the gas strains was isolated from a catering staff on the first day, the other two were isolated from two health care personnels on the second day. during the outbreak, of catering firm personel ( %) were found to positive for group g streptococci. all the bhs tested were found sensitive to penicillin g and erythromycin by agar disc diffusion method. conclusions: the configuration of the epidemic curve suggested a common source of exposure. since respiratory spread of streptococci in such a rapid fashion would be highly unlikely and that of positive throat culture were from the staff of the catering firm that provide all the food services for the hospital, and that most of them were working at the departments in which the outbreak occurred, we considered that the outbreak might be food-borne. prompt treatment with penicillin all the ill personnel and -day holiday coming consequently january, terminated the outbreak. all the strains were cryopreserved for further typing studies.we are now typing these strains by pulsed field gel electophoresis (pfge) after digestion with smai restriction endonuclease. our initial results show that these strains are of the same origin. outbreak of acute gastroenteritis in an air force base in western greece e. jelastopulu, t. constantinidis, t. kolokotronis, d. venieri, g. komninou, c. bantias (patras, andravida, gr) objectives: on september , an operative training day at the air force base in western greece, soldiers and staff experienced an outbreak of acute gastroenteritis. the purpose of this study was to determine the causes of the outbreak and develop control measures. methods: following the assessment of descriptive epidemiology, a case-control analytic approach was utilized with randomly selected cases and controls. patients completed a questionnaire pertaining to the presence and severity of gastrointestinal disturbances, date and time of symptoms onset and consumption of food items served in the base on the implied training day. adequate questionnaire was administered to the controls. odds ratios were calculated and statistical significance was determined using x test. samples of food items were collected for bacteriological examination. results: the overall attack rate was at least % among the approximately attendees. the outbreak started abruptly in the late afternoon on september, peaked at midnight and ended about hours later. from the interviews and the analysis it was established that the lunch (beef, macaroni, tomato sauce and grated cheese) consumed several hours prior to onset of symptoms by affected military personnel was the likely source of the outbreak with a strong statistical association. there was only one subject who did not eat lunch. among the symptoms the most prominent were watery diarrhoea ( %) and abdominal pain ( %). relatively few indicated vomiting ( %) and nausea ( %). the mean incubation period was h. in the bacteriological examination, staphylococcus aureus was detected in a sample of raw beef and in two samples of grated cheese (rest-cheese from lunch and an unopened package). conclusion: the short incubation period with abrupt onset, the symptomatology and the short, self-limiting nature of the illness, are suggestive of gastroenteritis caused by an enterotoxin-producing bacterium. s. aureus is considered to be the most likely cause. although mortality and longer-term morbidity are uncommon with food poisoning caused by enterotoxin-producing bacteria, this outbreak highlights its capacity to cause short term, moderately-severe illness in a young and healthy population. it underscores the need for proper food handling practices and reinforces the importance of appropriate microbiological specimen collection from cases, as well as the public health importance of timely notification of such outbreaks. occurrence, characterisation and antimicrobial resistance pattern of staphylococcus aureus strains isolated from dairy products in southern italy g. la salandra, e. goffredo, c. pedarra, m.c. nardella, a. parisi, a. dambrosio, n.c. quaglia, g.v. celano, g. normanno (foggia, valenzano, it) objectives: the ingestion of food contaminated by enterotoxins (ses) synthesized by staphylococcus aureus is responsible of one of the most common foodborne diseases (staphylococcal food poisoning-sfp). since s. aureus is often involved in cases of subclinical mastitis of ruminants, milk may results contaminated. infact, the dairy products are frequently related to cases of sfp, expecially in areas characterized by a high level of consumption of these products. consequently an active microbiological surveillance is needed in order to control the risk of sfp and to allow the improvement of the public health standards. s. aureus also show a large antimicrobial resistance pattern. in this work are reported the results of a survey conducted on the occurrence of s. aureus in dairy products from apulia region (southern italy). furthermore, the isolated strains were characterized in order to determine their ability in synthesizing ses and to evaluate their antimicrobial resistance pattern. methods: samples of dairy products (milk, cheese, mozzarella cheese, ricotta cheeses) were analysed for the detection of s. aureus. the isolated strains were tested for the detection of ses, using the reverse passive latex agglutination test (sea to sed) and submitted to pcr to detect enta, entb, entc, entd and ente genes. furthermore, the strains were tested for susceptibility to ampicillin, tetracycline, gentamicin, eritromycin, enrofloxacin, co-trimoxazole, teicoplanin and vancomycin, by the agar diffusion method. results: out of samples analysed, ( . %) resulted contaminated with s. aureus and, among these, ( . %) have been recognized as enterotoxigenic strains ( samples of milk, samples of mozzarella cheese, samples of cheese from ovine milk and sample of cheese). all the strains tested (one per each positive sample) showed antimicrobial resistance properties but none of these was resistant to teicoplanin and vancomicin. conclusions: the results obtained from this survey show that milk and dairy products from southern italy are frequently contaminated by enterotoxigenic strains of s. aureus and highlighted the need to implement strict hygienic control measures along the food chain in order to decrease the risk of spf. furthermore, the presence of antimicrobial-resistant strains of s. aureus in food may be considered a source of communityacquired infections, with the direct risk of transfer of the antimicrobial-resistance to intestinal human microflora. objectives: infection accounts for about one-third of cases of fever of unknown origin (fuo), which remains a major diagnostic challenge. recently, f- -fluorodeoxyglucose (fdg) positron emission tomography (pet) has entered the field of clinical infectious diseases. fdg accumulates in tissues with a high rate of glycolysis, which is present in malignant cells and in all activated leukocytes. the aim of this prospective multi-centre study was to validate the use of fdg-pet as part of a structured diagnostic protocol in the general patient population with fuo. methods: from december to july , patients with fuo, defined according to the revised petersdorf criteria, were recruited from one university hospital and five community hospitals. a structured diagnostic protocol was used. fdg-pet was performed after certain obligatory laboratory tests, chest xray and abdominal ultrasound. the final clinical diagnosis was used for comparison with the fdg-pet results. results: a final diagnosis was established in patients ( %): infections, malignancies, non-infectious inflammatory disorders and miscellaneous causes. of the total number of fdg-pet-scans, % were helpful. positive predictive value of fdg-pet was % and negative predictive value was %. fdg-pet was helpful in all patients diagnosed with an infection except for one case of pyelonephritis. contribution of fdg-pet to the final diagnosis did not differ significantly between the university hospital and the community hospitals. fdg-pet was not helpful in any of the patients with normal erythrocyte sedimentation rate (esr) and c-reactive protein (crp). conclusion: in addition to the apparent value of fdg-pet in diagnosing different infectious diseases as described in several case series, fdg-pet is a valuable imaging technique as part of a diagnostic protocol in the general patient population with fuo and a raised esr or crp. based on previous studies comparing gallium- -citrate or labelled leukocyte scintigraphy and fdg-pet in patients with fuo and resulting from favourable characteristics of fdg-pet, conventional scintigraphic techniques may be replaced by fdg-pet in institutions where pet is available. emergence of clindamycin-resistant streptococcus pyogenes causing cellulitis epidemiology of viral respiratory infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus as a cause of community-acquired respiratory illness seroprevalence of human metapneumovirus in japan - . carriers into account when studying the dynamics of pneumococcal transmission and modelling the effect of pneumococcal vaccination in young children erythromycin-resistant streptococcus pneumoniae isolated in spain: serotypes, clones and mechanisms of resistance ( %) and f ( %) that accounted for % of eryr strains. among eryr strains ( %) had mlsb phenotype ( % constitutive and % inducible) and ( %) had m phenotype. the genes detected in mlsb isolates were: ermb in isolates, and ermb and mefe genes in isolates. all ( %) mlsb isolates with resistance to tet had tetm gene and of them had int gene (related to tn -like). seven positive ermb strains susceptible to tet had int gene spain f- , spain b- , sweden a- and st - f) accounted for % of these strains. capsular switching was observed in two clones, spain f- (serotypes f and a) and sweden a- (serotypes a, f suggesting the spread of tn -like elements. the ermb positive strains, related to spain f- , spain b- , sweden a- and st - f clones, were more frequently isolated in adults us) objective: to characterize changes in the frequency of occurrence of bacterial pathogens responsible for pneumonia in hospitalized patients in europe for the years - and examine select antimicrobial susceptibilities (s) for predominant pathogens. the emergence of resistance (r) among pathogens responsible for pneumonia has resulted in changes to empiric therapy, with increasing reliance upon third-and fourthgeneration cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, carbapenems and fluoroquinolones. methods: participating european medical centres ( - /year) referred consecutive, non-duplicate pathogens ( isolates) from lower respiratory tract sites determined to be significant by local criteria as the probable cause of pneumonia. all identified isolates were tested for s by the broth microdilution method and . %, respectively), increased significantly in ( . % s), and returned to near- levels in . esblphenotypes (cro or caz or aztreonam mic ‡ mg/l) remained essentially unchanged among ec between and ( . % and . %, respectively), whereas among ksp increases were more substantial ( . % and . %). metallobeta-lactamase-producing pa were identified during the study from italy vim- ) methods: four-hundred consecutive mrsa isolates were collected at centres (max. isolates per centre) as part of a multicentre study conducted throughout germany in . isolates were collected from various sources, including colonization sites as well as infectious foci. only one isolate per patient was included and all isolates were spa-genotyped. cps were determined by slide agglutination with cp-specific antibodies (anti-t -dt, anti-t -conjugate, anti- -repa). the serotypes were confirmed by immunodiffusion using lysostaphin-digested cell lysates. results: in the present study, we serotyped mrsa isolates collected most recently in a german multicentre study. all mrsa isolates evaluated were one of the serotypes tested invasive pneumococcal disease in adults in north-rhine westphalia methods: surveillance for our current study focused on north-rhine westphalia, the largest federal state in germany ( million inhabitants). ( . %) acute care hospitals microbiological laboratories serving these hospitals agreed to participate. we studied hospitalized patients older than years of age. a case of ipd was identified by the isolation of s. pneumoniae from an otherwise normally sterile site. isolates were verified for species diagnosis by optochin testing and bile solubility, and for serotyping by the neufeld quellung reaction. mics of penicillin g, amoxicillin, cefotaxime, cefpodoxime, cefuroxime, clarithromycin us) objectives: carbapenems are the most reliably active betalactam antibiotics against enterobacteriaceae and are often the treatment of choice for infections caused by multi-drug resistant isolates. while carbapenem resistance has occasionally been reported in enterobacteriaceae, there are limited data on its frequency and distribution. methods: two large ongoing surveillance databases were searched for imipenem (imp) and ertapenem (etp) resistance in enterobacteriaceae: smart (study for monitoring antimicrobial resistance trends), a worldwide program focusing on community-and hospital-acquired intra-abdominal pathogens, and iss (icu surveillance survey), a us program focusing on icu isolates from any sterile body site. results: the overall frequencies of carbapenem-resistant enterobacteriaceae remained < % in smart and < % in iss throughout the periods of observation (see table). for % of esbl producing k. pneumoniae and e. coli were resistant to etp or imp and rates varied by geographic region. all isolates studied to date have exhibited multiple resistance mechanisms. conclusion: carbapenem resistance was uncommon among clinical isolates of enterobacteriaceae in these surveillance studies. its observed frequency varied by species and geographic region no significant seasonal variability in the prevalence of emergence of streptococcus b-haemolyticus strains in swabs was observed. conclusions: . seasonal fluctuations of pharyngeal discussion: with regard to high prevalences of giardiasis and enterobiasis it increase the prevalence that intestinal parasitic infections. it is suggested to decrease the rate of these parasitic infections in the region by strict programes that help to increase the knowledge of students, their parents and teachers about hygen. results: the study included patients, with a mean age of . ± . . ( . %) were women. the predisposing factors were: renal lithiasis patients ( . %), prostatic adenoma ( . %), vesical structure disease ( . %), vesical functional disorder ( . %), chronic kidney failure ( . %). the underlying diseases included: diabetes mellitus ( . %), immunosuppression ( . %), previous urinary tract instrumentation ( . %), permanent catheter ( . %). the mean hospital stay was . ± . days. the mean duration of symptoms was . ± . days the absence of leukocyturia or mictional syndrome does not exclude the presence of complicated upper uti. ) the high percentage of bacteriemia necessitates blood cultures, with e. coli being the most common pathogen. ) the associated morbidity and mortality are important in association with sepsis or septic shock gr) objectives: to estimate the incidence of streptococci in community acquired urinary tract infections (uti) and also to carry out the in vitro antibiotic resistance of streptococci in urinary tract infections %) were enterococcus avium, ( . %) were enterococcus gallinarum and ( . %) were streptococci group b. the in vitro antibiotic resistance of enterococcus faecalis was: penicillin g . %, ampicillin . %, gentamicin %, streptomycin . %, nitrofurantoin %, ciprofloxacin . %, tetracyclines . %, vancomycin . %, linezolid %. the in vitro antibiotic resistance of enterococcus faecium was: penicillin g %, ampicillin . % tremolieres for the french aup study group background: short-course therapy for acute uncomplicated pyelonephritis (aup) is the newly suggested standard. talan et al. have demonstrated that oral ciprofloxacin (cip) for days, was associated with greater cure rates than a -day trimethoprim-sulfamethxazole regimen. we assessed efficacy and tolerance of a -d. regimen of cip (study i), then of levofloxacin (lvx) in study ii results: of (i) and (ii) enrolled pts. aged . ± . y., and , aged . ± . y. were retained for itt analysis; . % and . % had positive blood cultures. escherichia coli was the uropathogen in . % and % of cases. finally (i) and (ii) were retained for per protocol (pp) analysis. at v bacterial eradication rates were . % and . %. global cure rates were . and . % at v and . % and . % at v with only less than % of lost to follow-up between v and v in both cases. side effects were observed in . % and . % of pts. who received or more fq doses. conclusions: aup treatment with lvx mg hiv-infected patients and drug addicts were excluded. antimicrobial susceptibilities of all s. pyogenes isolates were studied by microdilution method, and macrolide resistance phenotype by double disc test. macrolide resistance genes were detected by pcr. results: over the -year study period, there were episodes of cellulitis. the infection was microbiologically ). of note, all cases of cellulitis due to clindamycin-resistant strains occurred during the last years of the study. five ( %) patients presented with stss and died ( due to an erythromycin-resistant strain). overall mortality (< days) was % this resistance might become a problem when treating s. pyogenes infections, especially stss cases. p risk factors for community-acquired bacteraemic gram-negative cellulitis an administrative database was used. then we selected the patients with blood cultures obtained at the time of the cellulitis episode using the microbiology laboratory database. nosocomial cellulitis were excluded. a standardized data collection form was used to review the hospital records. in statistical analyses, student's t test was used for the comparison of mean values and chi square test and fisher's exact test for the comparison of categorical data (two tailed). results: of the patients with limb cellulitis identified in the study period, patients had blood cultures and were selected for the analysis. bacteremia was detected in of the patients ( . %), of them due to gram-negative microorganisms hemorrhagic rash was present in . % cases. koplick spot was found in . % cases. measles was associated with streptococcal tonsillitis in . % cases, with oral candidiasis in . % cases and with pulmonary tuberculosis in . % cases. severe forms of evolution were observed in complicated cases with: encephalitis ( . %) or bronchopneumonia ( . %), which required intensive care unit survey. we registered only one deceased, in a case of measles encephalitis in gipsy collectivities even it's very difficult, it's necessary to was performed. respiratory samples were tested routinely for twelve common respiratory pathogens. results: over the study period, of samples processed, -six cases were community-acquired and ( %) patients had significant co-morbidities. cough was the predominant reported symptom. chest x-ray was performed in cases, of which showed abnormalities. bronchiolitis ( / ) was the commonest initial clinical diagnosis. the majority ( %) of patients received antibiotic therapy, but a convincing bacterial pathogen was isolated in less than half of these cases. thirty patients were admitted for management. more than one virus was identified from patients, with rhinovirus being the predominant co-infection. overall, the average length of stay was . days. however, where hmpv was the sole pathogen identified, average length of stay was . days. conclusion: our data suggests that hmpv infections are more common in children with underlying co-morbidities. the rate of radiological imaging was higher than expected and perhaps is a reflection of the patient population or the degree of severity of illness. nosocomial acquisition occurred in cases, which has implications for patient cohorting acknowledgements: the financial support for this study was provided by kuwait university research grant / . evaluation of infection control practices in haematopoietic stem-cell transplant facilities in german-speaking countries: variation of measures reflects lacking evidence s. wenzler-rö ttele, a. conrad, w.v. kern, h. bertz, f.d. daschner, m. dettenkofer (freiburg, de) objective: haematopoietic stem cell transplant (hsct) recipients are highly immunocompromised during pre-and postengraftment. thus, they are cared for in specialised facilities and versatile precautions are practised in order to prevent nosocomial infections. however, there is a lack of evidence whether these interventions are effective. furthermore, most of the measures are cost-intensive and restrict the patients' comfort. for evaluation of precautions, a survey was performed to assess the spectrum of measures commonly practised. methods: a questionnaire was compiled asking in detail for infection control measures differing according to allogeneic and autologous hsct recipients. the questionnaire was sent to hsct facilities in germany, austria and switzerland. results: questionnaires ( %) were filled in and sent back. among the centres, were university hospitals, and teaching hospitals. the overall number of transplantations that were performed by the facilities varied considerably and ranged from to /y for auto hscts and from to /y for allo hscts. % of the institutions performing allo and auto hsct have implemented different precaution standards for each group. some measures regarding allo hsct were routinely adhered to in practically all institutions: accommodation in single rooms ( %), interdiction of plants and opening of windows ( % each) and protection from waterborne bacteria by use of terminal tap water filters ( %). % of hsct facilities perform their allo transplantations in hepa-filtered rooms and % are providing laminar air-flow for this population. there was a broad spectrum of different measures regarding barrier precautions: gowns when entering the room (required in % of centres for allo and % for auto hsct) and face masks ( % allo and % auto hsct). precautions to be followed by the patient varied among centres, e.g. specification of the face mask/respirator to be worn outside the isolation room (for allo hsct: % surgical mask, % ffp , % ffp and % ffp ). conclusion: the broad variety of different preventive measures performed by the different facilities reflects lacking evidence for many infection control precautions that are commonly practised in the care of hsct recipients. this survey provides the basis for further studies within the onko-kiss project (hospital infection surveillance system for patients with haematologic/ oncologic malignancies). objectives: in this study it was our aim to evaluate the microbiological contamination of physiological serum flasks in use in medical day center for wound cleaning and to identify the isolated microorganisms. methods: we have collected saline solutions from health care centres localized in the health sub-region of coimbra. from each centre we have recovered aleatory flasks in current use.the samples were transported at ordm;c and maintained at this temperature until its processing. saline solutions were seeded by the pour-plate technique in plate count agar and plates incubated at and ordm;c for h. the saline solutions were evenly spread over the surface of blood agar and sabouraud chloramphenicol agar (sab chl-d). the transfers of saline solutions flasks were also tested for microbiological contamination with a sterile cotton swab that was rubbed vigorously, over the transfer surface and directly applied on blood agar media. blood agar plates were incubated at °c for h and sab chl-d plates were incubated at °c and °c and examined daily for a period of days before declared as culture negative. microbial identification was firstly accomplished by employing conventional morphological and biochemical tests. when identification was not possible by these methods, s rrna gene sequence determination and phylogenetic analysis were used for bacterial strains and in the case of moulds we performed the amplification and sequencing of internal transcriber spacers region of . s gene. results: from the saline solutions analysed, . % were contaminated. a total of strains were isolated, % could be identified to species level using morphological and biochemical tests, the remaining % were identified by gene amplification and sequencing. about . % of the identified strains were gram-positive cocci, the second dominant type of strain were gram-positive bacilli ( %), and the third dominant type of strains were gram-negative bacilli and moulds, both with . %. the most frequent contaminants belong to human normal flora ( %), supporting the idea that the source of contamination of saline solutions analysed was human, in contrast with % of contamination due to the environment. conclusions: the contamination of the saline solutions is due to inadequate clinical practices. these results claim for more strict hygienic measures and for the replacement of big flasks by single use flasks with an incorporated overture used for wound irrigation. frequencies of cmv-ie specific memory t cells are inversely correlated with alloimmune memory and serum creatinine in kidney transplant patients p. nickel, g. bold, f. presber, c. schö nemann, j. pratschke, d. bitti, f. kern, h.-d. volk, p. reinke (berlin, de) background/aims: cytomegalovirus infection is a significant cause of morbidity in transplant patients and has been associated with allograft rejection. in this study frequencies of ifng-producing t cells following ex-vivo stimulation with protein-spanning peptide pools for cmv proteins pp and ie as well as donor-reactive t cell frequencies were serially determined during the first months after renal transplantation (tx) to analyse the relation of cmv specific t cells, virus control and alloimmunity. patients: kidney transplant recipients were included. immunosuppression generally consisted of anti-il- r mab, calcineurin inhibitor, mmf and steroids. presensitized patients received an induction by x low dose okt- , anti-tnf mab, anti-cd mab and x plasmapheresis. patients received fty- , cyclosporine and steroids. methods: pbmcs from renal transplant recipients were analysed in a computer-assisted elispot assay before and at multiple times (mean ) post-transplantationfor ifn-yproducing t cells following in-vitro stimulation for hrs by irradiated donor cells and pools of overlapping peptides conclusions: although temporary r declines were seen among some european pneumonia pathogens, all showed increasing r to most class agents during the study period. the increase in esbl among enterobacteriaceae, and r among pa to most agents except polymyxin b, are especially worrisome. continued longitudinal comparisons of emerging pathogens and changing susceptibility profiles are critical elements in guiding empiric therapies and epidemiologic interventions. week, all participating hospital inpatients were swabbed on three anatomical sites: throat, nose and groin. we investigated the molecular epidemiology of the mrsa isolates collected from patients in hospitals using the pfge method with the smai restriction enzyme. cluster analysis was carried out using bionumerics software. band-based similarity dice coefficients were used for dendrogram construction, which provides a quantitative assessment of strain similarity. samples were defined to belong to a cluster using a similarity coefficient of % or higher. pfge profiles were compared with the most similar strains from the harmony iums global mrsa database.results: different restriction profiles were observed among the mrsa isolates and patients. isolates from the same patient but from different anatomical sites had similar pfge profiles. clusters of mrsa strains could be identified with the two largest clusters containing ( %) and ( %) patients, respectively. strains from these major clonal clusters occurred in and out of the hospitals, respectively. isolates from the cluster with patients were most similar to the well-known iberian clone: france a ( strains), belgium e ( strains), france b, france c and northern germany i ( strain each). isolates from the next largest cluster of patients correlated with a group of strains previously found in finland and belgium: belgium e ( strains), finland e ( strains) and finland e ( strain). the remaining strains were most closely related to belgium ec ( strains), berlin iv ( strain), southern germany ii ( strains) and uk e ( strains). conclusion: two major clonal clusters of mrsa strains were found to be dominant among hospitals inpatients in luxembourg. the molecular diversity of circulating strains was fairly diverse and profiles were very similar to previously described patterns in neighbouring countries and europe. further sequence-based genotyping is warranted to gain a better understanding of the clonal structure and elucidate transmission patterns. enterococci were identified by basic tests and by pcr amplification of ddl genes. susceptibility testing was performed using the icls broth microdilution method. resistance genes were detected by pcr, selected vana, vanb and vanc amplicons were sequenced. macrorestriction analysis (smai) resolved by pulsed-field gel electrophoresis (pfge) was performed. results: during the study vre isolates with different phenotypes of resistance to glycopeptides were obtained from specimens. the prevalence of vre in the gastrointestinal tract was . %. one e. faecalis (isolated from patient arrived from us) and e. faecium isolates, harbouring vana genes, demonstrated mic's of vancomycin (van) and teicoplanin (tec) - and - mg/l respectively. three e. faecium and four e. gallinarum isolates were vanb-positive, with van and tec mic's > and . - mg/l respectively. all stains were susceptible to linezolide. among e. faecium isolates with vana genes one predominant pegf type was observed, differentiated in nine pegf sub-types. each of three other pegf types detected seemed to be unique. among six vana genes sequenced, four demonstrated similarity to vana gene from e. faecium (genebank af ) and two to -vana gene from e. faecalis (genebank ay ). in two sequenced vanb genes from in e. gallinarum nucleotide substitutions, resulting in seven new amino acid substitutions, were detected. conclusions: heterogeneity of glycopeptide-resistance genes, circulating in haematological centre, leads to the conclusion that their spread is not a local phenomenon. spread of vre is an emerging and, possibly, underestimated problem for russia. study of resistance and clonal relatedness of clinical isolates of stenotrophomonas maltophilia from a hospital in northern spain c. valderrey, e. sevillano, f. calvo, l. gallego (bilbao, es) objectives: the aim of this work was to study the antibiotic resistance and genetic relatedness among clinical isolates of s. maltophilia isolated from patients with tract respiratory infections. methods: the study included s. maltophilia isolates obtained in a hospital from bilbao (northern spain) during (from january to october). susceptibility to antimicrobial agents was determined by the disk diffusion method following the nccls recommendations. the antibiotics tested were imipenem, meropenem, cefotaxime, ceftazidime, cefepime, aztreonam, amikacin, tobramicin, ciprofloxacin, ofloxacin and trimethroprim/ sulfamethoxazole. total dna was used as target for pcrfingerprinting experiments with primers rd , eric , ap , m and rnar and . to detect class integrons, primers cs and cs were used in amplification experiments.results: resistance to antibiotics tested was the following: imipenem ( %), meropenem ( %), cefotaxime ( %), ceftazidime ( %), cefepime ( %), aztreonam ( %), amikacin ( %), tobramicin ( %), ciprofloxacin ( %), ofloxacin ( %) and trimethroprim/sulfamethoxazole ( %). pcr-fingerprinting technique was only useful when eric primer was used identifying distinct genotypes. the other primers were not able to produce reliable band patterns. patients with several isolates maintained the same clone along time, although there are two patients from which two different genotypes have been isolated, and two clones that have been isolated from more than one patient. class integrons were detected in % of isolates ranging in size from of to bp ( isolates bored combinations of two structures). conclusions: trimethroprim/sulfamethoxazole and amikacin showed the best activity against the isolates tested. for pcrfingerprinting experiments the best primer was eric which produced reliable and reproducible band patterns. there was a high clonal diversity since different genotypes were identified among the patients included in the study. many isolates bored class integrons with sizes similar to those detected in other nonfermenters bacilli from the same environment.methods: -identification: the strains were identified by colonial morphology, haemolysis on blood agar plates, biochemistral and antigenic identification; antibiotic susceptibility testing: all the strains were tested by disk diffusion according to the national committee for clinical laboratory standard methods. mics were determinated by screening test or mic evaluation in solid media. results: our study concerns bacterial strains isolated from january to june . among the strains isolated, neisseria meningitidis represented the most number of cases with . %.these were distributed among all different age groups. serogroup a was the most predominant and represented . % of total strains while groups b and c represented . % and . % respectively. streptococcus. pneumoniae represents the second causes of purulent meningitis with . % while haemophilus. influenzae b is the third causative bacterial agent with . %.this last agent is most predominant among infants less than years of age in % of cases. neisseria. meningitis is susceptible to all types of antibiotics tested. however, haemophilus. influenzae b produced an inactivating enzyme (penicillinase) in . % of cases. the resistance was associated to cotrimoxazole in . % of cases. the results of mic done on streptococcus. pneumoniae show that . % of strains has an intermediate resistance to penicillin and high level of resistance in . %. the amoxycillin is active in . % of the strains,in the opposite cefotaxim has an intermediate resistance in . % and a high level of resistance in . % of the strains. the resistance to penicillin was associated with resistance to erythromycin, cotrimoxazole or to both in some cases. conclusion: streptococcus. pneumoniae represents the second causative bacterial agent responsible of purulent meningitis and showed an increasing prevalence of resistance profiles to penicillin and cefotaxim in our hospital. this implicates an effective microbiological and epidemiological control.conclusion: as expected in a referral hospital with a cardiac surgery department, the prevalence of s. aureus ie was elevated as well as the attributable mortality rate. the high global mortality rate may be explained by the high frequency of severe co-morbidities and by the late referral of patients to hospital. our data suggest that there is room for improvement in the diagnosis and management of ie in a multidisciplinary collaborative approach. objective: to determine the clinical, epidemiological, diagnostic, and therapeutic characteristics of a series of cases of prosthetic valve endocarditis. methods: we undertook a retrospective, descriptive study of cases of prosthetic valve endocarditis obtained from a series of definite or probable left sided infectious endocarditis from six second-or third-level andalusian hospitals from to . results: of the cases of prosthetic valve endocarditis, ( . %) were definite and ( . %) possible. the mean age was ± years, and they were more common in men ( %). late infection was more common than early involvement ( vs. cases). the aortic valve was involved in cases ( %) and the mitral valve in cases ( %. most ( %) of the valves were made of metal and prior handling had taken place in cases ( %). clinical characteristics were fever %, constitutional syndrome %, murmur %, vascular events %, and immune phenomena %. complications included left ventricular failure %, kidney failure %, peripheral embolism %, cns embolisms % and heart block %. the etiology was as follows: in early prosthetic valve endocarditis the three most common pathogens were s. coagulase-negative ( %), s. aureus ( %) and enterococcus ( %). late prosthetic valve endocarditis involved s. viridans ( %), s. coagulase-negative ( %) and s. aureus ( %). transesophageal echocardiography alone in cases ( %), and transthoracic followed by transesophageal echocardiography in cases ( %). medical therapy was applied in cases ( . %) and surgery in ( . %). a cure was achieved in cases ( %), the other ( %) dying. of those who underwent surgery, . % died and . % of those who were treated medically died. the death rate from early prosthetic valve endocarditis was greater than that for late prosthetic valve endocarditis ( % vs. %). conclusions: ) prosthetic valve endocarditis is a very serious infection which is still associated with an excessively high mortality, despite advances in diagnosis and treatment. ) early prosthetic valve endocarditis has a worse prognosis than late prosthetic valve endocarditis, due to its distinguishing pathophysiological features. ) the greater mortality seen in patients who underwent surgery is probably associated with the fact that they had more complications, such as perivalvular abscesses or persistent infection. outcome of infective endocarditis e.e. hill, s. vanderschueren, p. herijgers, m-c. herregods, p. claus, w.e. peetermans (leuven, be)objectives: despite progress in diagnosis and therapy, almost half of patients with infective endocarditis (ie) has at least one complication and overall mortality remains high. the aim of the present -year prospective observational study was to define predictors of outcome in patients with ie. methods: from june through december , all first episodes of definite ie by the modified duke criteria, encountered in a single tertiary-care medical center, were registered and followed-up for months. results: overall, patients suffered ie episodes. sixtyone percentage were males. the median age was years (range - ). fifty-five percentage of episodes were referred from another hospital. at least one complication occurred in %. surgical intervention was performed in % and was mainly indicated because of congestive heart failure. the median time from diagnosis to surgery was days (range - ). six-months mortality was % (n = ). in bivariable analyses, factors associated with -months mortality were: age, female gender, causative microorganism, nidus of infection and therapeutic policy. six-months mortality was % for native valve ie and % for prosthetic valve ie; twenty-five% for nosocomial ie and % for community-acquired ie. six-months mortality rates for microorganisms were: staphylococci % (n = ) [s. aureus % (n = ) and cons % (n = )], enterococci % (n = ), streptococci % (n = ) and other microorganisms % (n = ). the -months mortality for patients with a contraindication to surgery was % (n = ), for patients conservatively treated without a contraindication % (n = ) and for combined surgical-medical treatment % (n = ). in multivariable logistic regression predictors of -months mortality were age (or, . ; % ci, . - . ; p = . ), causative microorganism (or, . ; % ci, . - ; p = . ) and a contraindication to surgery (or, . ; % ci, . - ; p < . ). conclusion: in the present prospective single centre study of patients with definite ie, -months mortality rate was . , and was especially high in patients with preestablished contraindications to surgery, in the elderly and in patients with staphylococcal ie. six-months mortality in patients with combined surgical-medical treatment versus exclusively medical therapy in patients without a contraindication to surgery was not statistically significant. staphylococcal and enterococcal ie had a worse prognosis compared to streptococcal ie. epidemiology and aetiology of infective endocarditis e.e. hill, p. claus, m-c. herregods, p. herijgers, s. vanderschueren, w.e. peetermans (leuven, be)objectives: the epidemiological features of infective endocarditis (ie) have changed. we report the results of a -year prospective observational study investigating trends in the epidemiology and etiology of ie. methods: from june through december , we registered definite ie episodes according to the modified duke criteria in patients older than years, hospitalized in a single tertiary-care center. results: sixty-one% of episodes involved males. the median age was years (range - ).fifty-five percentage (n = ) were referred from another hospital. forty-four percentage (n = ) were nosocomial. thirty-four percentage (n = ) involved prosthetic valves and % (n = ) thereof were of early postoperative onset. the mitral valve was most frequently involved. exposure to ie risk factors during the previous months was recorded in % (n = ) of the episodes. twenty-four percentage (n = ) were intravascular catheter- objective: to determine the eco-epidemiology of cryptosporidiosis in the health services executive -western area (formerly the western health board).concerns about the incidence of cryptosporidiosis in the western area prompted the department of public health to undertake further investigation of potential links between cryptosporidiosis and environment by focusing on farming activity and water supplies in the first instance. background: cryptosporidiosis was not notifiable in the republic of ireland prior to , unless cited as a cause of gastroenteritis in a child less than two years old. as a result the incidence of cryptosporidiosis in the republic of ireland at the time was unknown. nationally it was estimated that up to % of cases of gastroenteritis in children less than two years old could be attributed to cryptosporidium. in the western area from to the proportion of cases of gastroenteritis in children less than two years old attributable to cryptosporidium ranged from . % to . %. this was cause for concern.many rural locations in the western area are served by voluntarily-operated water schemes. water quality from these schemes is often microbiologically unsatisfactory. the department of public health methods: initial research involved analysis of notification records for cases of cryptosporidiosis received from to inclusive. crude incidence rates for cryptosporidiosis in the western area were compared with crude incidence rates in england & wales, northern ireland, and scotland for the same time period. cases of cryptosporidiosis from the western area were geo-coded and mapped to visualize the geographic spread of cases, and are being contrasted with geographic data for farming activity, and also with available data on water supplies. the results of the initial phase of this research indicated the incidence of cryptosporidiosis in the western area may be cause for concern. the geographic spread of cases and potential links to farming practices and water supplies will be presented. objective: the evaluation of epidemiology and seasonal fluctactions of bacterial flora in pharyngeal swabs taken from family doctors' patients. material and methods: a total of of positive pharyngeal swabs ordered by primary care physicians from silesia were examined during the - period. the microbiological analysis was performed in silesian analytic laboratories. the intake of material, its transport and final identification complied with laboratory standards. results: the most common pathogens were, in order of prevalence: streptococcus viridans ( . %), moraxella catarrhalis ( . %), staphylococcus aureus along with mrsa ( . %) and mrsa alone ( . %), e. coli ( . %), klebsiella pneumoniae ( . %), streptococcus b. haemoliticus ( . %). candida albicans was identified in . % of positive specimens. considering seasonal fluctuation, the number of positive swabs in each month tended to gradually increase in spring with its culmination in may ( . %). as for the most common pathogens streptococcus viridans and moraxella catarrhalis mirrored the general tendency and dominating in spring season (up to . and . %, respectively) and having less stronger impact in automn (up to . and . %). the frequency of isolation of the other pathogens revealed seasonal fluctuations confined to either spring, as in the case of klebsiella pneumoniae, escherichia coli and staphylococcus aureus strains (up to . , . clinical microbiology and infection, volume , supplement , aim: the aim of this study was to identify the microorganisms isolated from corneal and conjuntival samples, isolated from patients attending the ophtalmology department of a spanish hospital. material and methods: a total of corneal scrapes and conjunctival swabs were obtained since october of to october of in an university hospital of madrid. samples were cultured into blood and chocolate agar plates and incubated at ordm;c in o and co atmospheres, respectively, for two days (conjunctival swabs) and fifteen days (corneal scrapes). identification and susceptibility tests were performed following standard methodology. results: thirty four ( . %) out of corneal samples and ( . %) out of conjunctival swabs yielded positive cultures, respectively. results are summarized in the following table:conclusions : corneal scrapes yielded a higher number of positive cultures than conjunctival swabs. gram-positive microorganisms were more prevalent both from corneal scrapes and conjuntival swabs although the difference was more evident in corneal scrapes. s. aureus was the specie most prevalent in conjunctival samples meanwhile cns were the most prevalent in corneal scrapes. methods: vitreous fluid samples (n = ) were obtained from patients ( male, female) undergoing vitrectomy for endophthalmitis between january and october . specimens of undiluted aqueous and vitreous fluid were cultured for aerobic, anaerobic bacteria and fungi by conventional methods. identification and antibiotic susceptibility were performed by the api system, vitek ii system (biomerieux) and the agar disk diffusion methods according to clsi recommendations. results: ninety one isolates were recovered from the samples. gram stain was positive in / ( . %), while cultures were positive in / ( . %) samples. gram-positive bacteria were the most common isolates ( / , %), followed by gramnegative bacteria ( / , %) and fungi ( / , %). staphylococci coagulase-negative were isolated in / ( %). the next most common species isolated among gram-positive bacteria were s. aureus ( . %), streptococcus spp ( . %), propionibacterium acnes ( . %), bacillus spp ( . %), streptococcus. pneumoniae ( %) and enterococcus faecalis ( %). among gramnegative bacteria eight isolates were enterobacteriaceae, two were non fermenters and one was haemophilus inlfuenzae. two of the fungal isolates were candida albicans, one acremonium spp and six aspergillus fumigatus. polymicrobial growth was observed in six patients with two at least isolates. of staphylococci coagulase-negative / ( %) were resistant to methicillin. only one strain of staphylococcus aureus was methicillin resistant. all gram positive isolates were susceptible to vancomycin. all isolates were sensitive to amikacine and ceftazidime while resistance was observed in / ( %) isolates to fluoroquinolones. conclusion: a variety of microorganisms was isolated from the vitreous fluid of patients. the predominant isolates were grampositive bacteria, especially staphylococci coagulase-negative with low resistance rate to methicillin. so, therapy should be based on the isolation and identification of the infecting agent and the in vitro antibiotic susceptibility to the appropriate antibiotics. the prevalence of intestinal parasitic infection in the students of primary schools in nazloo region in urmia during [ ] [ ] k. hazrati tappeh (urmia, ir)background: intestinal parasitic infections are of the most important hygienic and economical problems of millions of people in all over the world, mostly from developing countries. understanding their epidemiological situation and relation to environmental and social factors is necessary for struggling with them in every society. this investigation was designed to study the prevalence of parasitic intestinal infections among primary school attending students in nazloo region of urmia district in . materials and methods: students were chosen randomly from schools upon their population. having their questionnaires filled, two faecal samples were taken from each student and examined with direct wet mount and formalinether sedimentation technique. scotch tape was also applied in order to detect the enterobiasis and taeniasis. students completed the test. all infected persons by e. vemicularis, h. nana were treated by mebandazole and giardia lamblia were treated by metronidazole. results: overall prevalence of parasitic protozoan infections was . %. giardia lamblia was found in cases ( . %), entamoeba coli in cases ( %) and blastocystis hominis in cases legionella pneumophila as an occupational risk factor for inter-city bus drivers y. polat, Ç . ergin, i. kaleli, a. pinar (denizli, ankara, tr)objectives: legionellaceae are ubiquitous aquatic microorganisms that usually isolated from evaporative condensers. various man-made sources such as cooling towers, whirlpools and spas are sources for legionella pneumophila. in hot climate, bus air-conditioning and aircirculating systems are possible sources for the organism. in this study, serologic status of bus drivers and their assistants for legionella infections as well as bus air-conditoner moisture exit samples for legionella species were investigated.methods: serum samples were collected from bus drivers (n = ) and their assistants (n = ). samples were tested for anti-legionella antibodies by indirect immunofluorescence technique. / dilution was accepted as a positive result for anti-legionella pneumophila antibodies. results were analysed according to risk factors based on hot/cold climate route (aegean and mediterranean parts of the turkey were accepted as hot climate region), immundeficiency, chronic diseases and work hours. according to serologic test results, air-conditioners of buses which has been driven by / dilution seropositive persons, were investigated. air-conditioner moisture exit samples were cultured on bcye-alpha agar supplied with bmpa. same samples were tested by pcr targeting a -bp fragment of the s rrna gene of legionella. results: anti-legionella pneumophila antibodies were positive in ( . %) bus-persons. bus drivers' seropositivity was higher than assistants (p < . ). in hot climate route, seropositivity was higher than cold climate route (p < . ). no positive pcr result was detected. coclusion: in conclusion, higher seropositivity rates in bus drivers were pointed out a newer occupational risk factor for legionellosis. although pcr positivity was not detected for bus air-conditioners, high seropositivity rates show that bus drivers have been somehow exposed to legionella. further legionellosis surveillance studies for bus drivers may help to understand legionella exposure during travel. objective: asymptomatic bacteriuria is an important risk factor contributing to pyelonephritis and renal disfunction in diabetic patients. in this study, the relationship between microalbuminuria and age, body mass index, duration of the disease, the level of glycohemoglobin, glycosuria and glomerular filtration rate is studied prospectively in diabetic patients who have asymptomatic bacteriuria. methods: a hundred and twenty-three type diabetic outpatients who were admitted to baskent university konya medical and research center between january-october were included in the study. ages of the patients were within the range of - years. the diagnosis of asymptomatic bacteriuria was established according to the cdc criteria. concurrent samples for urinary culture, glomerular filtration rate, microalbuminuria and glycohemoglobin were obtained. results: twenty-two of ( . %) patients had significant bacteriuria. of these patients % were female. although age, body mass index, creatinine clearence and presence of microalbuminuria were similar, there was a significant difference in glycohemoglobin levels, duration of diabetes and glycosuria between the two groups (p < . ). e. coli was the most common microorganism obtained from urinary samples. risk factors for asymptomatic bacteriuria were shown in the table. conclusion: the frequency of asymtomatic bacteriuria was found to be similar with the previous studies. high glycohemoglobin levels and long duration of diabetes were found to be the risk factors contributing to asymptomatic bacteriuria in type diabetic patients. descriptive study of complicated pyelonephritis objective: the evaluation of prevalence and contributory factors associated with the development of urinary tract diseases among women with urinary incontinence. material and methods: women aged from to years had their urine culture examination performed. the material was taken from the central stream of first catch urine and transported on uromedium. antibiogram was carried out with the use of becton-dicinson's discs. results: in cases the urine culture tested positively which accounted for . % of subject women. the most common pathogens of urinary tract were, in order of prevalence:e. coli- . %, staphylococcus aureus - . %,citrobacter diversus- . % and klebsiella pneumoniae- . %. candida albicans strains were isolated in one patient. e. coli had the highest sensitivity to norfloxacin - % and cefuroxim - %, amoxicillin with clavulonian acid - . %, ampicillin nitrofurantoin and trimethiprim -sulfamethoxazole - . % in each case, cefalothin - . %, tetracycline - . %, and amikacin - . % but only in . % to amoxacillin. staphylococcus aureus proved sensitivity only to gentamicin ( %) and nitrofurantoin ( %). in the case of citrobacter diversus % sensivity to norfloxacin, nitrofurantoin, tetracycline, trimethoprim / sulfamethoxazole, ceftazidim and cefotaksym was confirmed.klebsiella pneumoniae also proved sensitivity to amoxicillin with clavulonian acid, cefuroksime, nitrofurantoin, norfloxacine, tetracyclin and trimethoprim / sulfamethoxazole. when considering the sensitivity of pathogens to antibiotics in the family practise setting of higher reliabilty are nitrofurantoina, norfloksacyna.after the administration of guided therapy complete release from symptoms was observed in women ( %).conclusions: women with urinary incontinence relatively seldom suffer from urinary tract infections. the most common pathogen among women with urinary incontinence was e. coli sensitive to floxacins and cephalosporins but with impaired reaction to amoxycillin. incidence and in vitro antibiotic resistance of streptococci in community-acquired urinary tract infections uncomplicated community-acquired urinary tract infections (ca-utis) and non-pregnant women in london hospital in kuwait over a period of two years. methods: eighty-six pregnant and non-pregnant women with signs of ca-utis were enrolled in the study. the strains isolated from the patients who had significant bacteriuria were included in the microbiological analyses. the identification of the strains was performed using the api e system (biomerieux), while their susceptibility was determined by disk diffusion method. the interpretation of the results was realized according to nccls guidelines. quality control was performed using reference strain e. coli atcc . oserotyping was carried out with polyvalent and monovalent antisera. hemolysin production was tested on human blood agar plates. possession of k antigen by e. coli was tested with agglutination by murine monoclonal antibodies to the group b meningococcal capsule. results: we found o serogroups o , o , o , o , o , o and o among strains isolated from pregnant and non-pregnant women. hemolysin was presented in % and % respective. k antigen was presented in % of strains in studied groups.there are some statistically significant differences in antimicrobial resistance between both groups. amoxicillinclavulanate (amx-clv) resistance was higher among uti haemolytic isolates of e. coli in pregnant women ( %) then in non-pregnant women ( %). similar distinction in cefuroxime resistance was found - % and . amikacin resistance was higher among uti isolates of e. coli in non-pregnant women ( %) then in pregnant women ( %).conclusions: there are no significant differences in expression of virulence factors of e. coli from pregnant and non-pregnant women with ca-utis in london hospital, kuwait. the resistance rates of e. coli from pregnant women to amx-clv and cefuroxime are significantly higher than in non-pregnant women. the penetration of telithromycin in gynaecological tissues and activity in cervicitis patients h. mikamo (gifu, jp)objectives: chlamydia trachomatis and neisseria gonorrhoeae are major causative organisms for sexual transmitted infections in japan. although several oral antimicrobial agents are active against c. trachomatis, few effective oral antimicrobial agents against n. gonorrhoeae exist in japan. two studies were conducted: a clinical pharmacology study examining penetration of telithromycin (tel), an oral ketolide antibiotic, in female genital organ tissues and a clinical study examining tel mg once daily (qd) in cervicitis patients (pts chronic prostatitis (cp) is believed to be an infectious disease in most cases. both aerobic and anaerobic bacteria are involved in the polymicrobial microbiocenosis found in prostate specific specimens. coryneform bacteria form a remarkable part of this community, yet scarce knowledge exists about their clinical significance, species composition and antibiotic susceptibility.our aim was to compare the corynebacteria of the seminal fluid of cp patients and controls and to evaluate their antibiotic susceptibility.material and methods: semen samples from controls and cp patients (nih iiia or iv category) were analysed. corynebacterium seminale was identified by beta-glucuronidase activity, the rest of coryneforms using api coryne (biomerieux). e-test method was used for susceptibility testing.results: coryneforms were found from % cp patients and % controls (p > . ). twelve species and genera were found among strains identified, the most frequent being c. seminale (in % cp patients and % controls). cp patients harboured significantly more arthrobacter sp. ( % vs %, p = . ) andcorynebacterium group g ( % vs %, p = . ), the latter association was especially eminent in case of patients with serious inflammation (> wbc/ml): % vs %, p = . . all tested strains were susceptible to ampicillin-sulbactam, single strains were resistant to doxycycline ( %) and tmp/smx ( %), however, moderate resistance was common to doxycycline ( %). resistance to clindamycin ( %), benzylpenicillin ( %), nitrofurantoin ( %), erythromycin ( %) and norfloxacin ( %) was observed as well. half of cp-related corynebacterium group g strains showed resistance to nitrofurantoin and benzylpenicillin. in addition, they were often moderately resistant to clindamycin, erythromycin and, finally, norfloxacin frequently used to treat cp. conclusions: most of men have coryneforms in their semen, more than half harbour c. seminale. corynebacterium group g and arthrobacter sp are more frequently found in cp patients than the controls. in the treatment of cp of unknown etiology it is useful to take into consideration the susceptibility profile of corynebacterium group g. objective: to evaluate the role of cmv and listeria monocytogenes in abortion.methods: this descriptive prospective study was done on women, women with spontaneous abortion before th weeks of pregnancy as a case group and healthy woman with full term delivery as a control group. serum samples were taken from all patients. elisa test was done for evaluation of cmv (igg and igm) and listeria antibodies in both groups. prevalence of seropositivity was determined. data were analysed by x and chi-square test.results: seologic tests were done on samples. average age in case group was . ± . and in control group was . ± . years. in cases with abortion ( . %) and in control group ( . %) were seropositive for listeria monocytogenes. difference in seropositivity between groups is statistically significant (p = . ). cmv igg antibodies were positive in ( %) of case group and in ( %) of control group; the difference is significant statistically (p = . ). cmv igm antibody was positive in ( . %) of case group and none in control group. difference is significant (p < . ) there was no correlation number of previous abortion and seropositivity for listeria and cmv. conclusion: the present study showed an important role of listeria monocytogenes and cmv infection in abortion. serum and prostatic tissue concentrations of moxifloxacin ( mg) after a single intravenous infusion in patients with benign prostatic hyperplasia undergoing transurethral resection of the prostate background: the spectrum of bacterial prostatitis comprises gram-negative, gram-positive and atypical pathogens. because of its broad spectrum of activity, moxifloxacin might be a suitable antibiotic for the treatment of bacterial prostatitis. aim: in this study the penetration of moxifloxacin into prostatic tissue after intravenous application of mg as single dose was investigated.methods: in a prospective, multicentric study patients with benign prostatic hyperplasia received a single dose of moxifloxacin mg in an hour lasting infusion ( ml) for perioperative prophylaxis before undergoing transurethral resection of the prostate (tur-p). serum concentrations were determined in all patients before infusion, at the end of infusion (time point ), . , and h after the end of infusion. patients were randomized for tissue sampling either , . , or h after the end of infusion. at the beginning of tur-p approximately g of tissue was sampled for analysis. concentrations of moxifloxacin in serum and tissue were determined by hplc. results: patients were evaluated in the study. the concentrations (mean, sd, median, / % quantile) are shown in the table. the prostatic tissue concentrations of moxifloxacin were approximately twice as high as in serum. at the end of infusion the tissue and serum concentrations were already equilibrated, because the tissue-serum ratios did not differ significantly from the end of infusion until h after the end of infusion. after an intravenous infusion of mg the serum and prostatic tissue concentrations of moxifloxacin were well above the mic values of the most important prostatic pathogens until h after the end of infusion. therefore, moxifloxacin might be a good alternative for the treatment of bacterial prostatitis and/ or perioperative prophylaxis for tur-p. statistical significant differences were detected between patients with and without bgnc in the proportion of patients older than years ( . % vs . %), the antecedent of recent animal bite ( . % vs . %), the presence of immunosuppression ( . % vs . %), the presence of haematological illness ( . % vs . %), and the degree of leukocytosis at admission ( ± vs ± cel/ll). conclusions: bgnc is frequently detected in our patients. age older than years, the existence of immunosuppression, the existence of haematological illness, and the antecedent of animal bite are more frequent among patients with bgnc. patients with bgnc had a lower degree of leukocytosis at admission. these factors should be borne in mind to select empiric therapy for patients with cellulitis. is erysipelas-associated tinea pedis a site of streptococcal colonisation? objectives: tinea pedis is considered the most frequent portal of entry of erysipelas of legs (sel) but whether it is the site of streptococcal colonisation is unknown. methods: from june to october we prospectively searched for clinical tinea pedis in patients hospitalised in our infectious diseases ward for sel (acute and unilateral feature with fever were only retained). all patients had bacteriological samples on inter-digital spaces of both feet (sel side and contra lateral side).results: fifteen patients were included. all but one were treated by intra-venous penicillin-g followed by oral amoxicillin. on sel side: tinea pedis was found in / ( %) and, when present, streptococcal colonisation (c or g streptococcal groups) was found in / ( %), although streptococcal colonisation was never found ( / ) in its absence. on contra lateral sides : no streptococcal colonisation was found without tinea pedis, which was observed in / , with streptococcal colonisation in / . then there is a strength statistical association between streptococcal colonisation and tinea pedis, on sel side (p = . ) as well as on contra lateral side (p = . ). in one patient blood-cultures yielded with the same streptococcus than found in foot samples. discussion: streptococcal colonisation of tinea pedis is a common finding on both feet of patients hospitalised for sel. whether inter-digital colonisation is a primary stage of invasive disease remains unproved. in our experience, a strain of streptococcus that colonised inter-digital space was isolated in patient's blood, suggesting this hypothesis may be true in some cases. if confirmed, this concept could lead to a new strategy for secondary prophylaxis of recurrent sel by decontaminating streptococcal colonisation of tinea pedis. among ggs, different emm types were found; stg , stg and stg predominated. among gas, types were found, emm predominated. one patient had the same ggs isolate in throat and skin. six patients had recurrent infections during the study; two of them with disease episodes. of the culture positive skin samples, were taken from the erysipelas infection focus ( % positive for ggs) and from another site ( % positive for ggs), e.g. wound, intertrigo, between toes or an unknown site.conclusion: a predominance of ggs was seen in the throat of erysipelas patients and their families whereas ggs was not present in control subjects. ggs, instead of gas, also seems to predominate in erysipelas skin lesions. several emm types were present in both groups and there was no clear predominance of a distinct type. the recurrent nature of erysipelas became evident also during this study. the evaluation of fournier's gangrene severity index score in patients m. ulug, m.k. celen, m.f. geyik, c. ayaz, s. girgin (diyarbakir, tr)objectives: fournier's gangrene is synergistic necrotizing fasciitis of the perineum and abdominal wall along with the scrotum and penis in men and the vulva in women. it is rare but life-threatening process. in this study we identify effective factors in the survival of patients with fournier's gangrene and to determine the accuracy of the fournier's gangrene severity index score (fgsis). methods: we evaluate patients with fournier's gangrene who were threated and follewed up from us between january and september in the department of general surgery prospectively.results: the results were evaluated in two groups: those who died (n: ) and those who survived (n: ). no statiscally significant difference was found between the age of the survivors and those who died. the admission and final laboratory parameters that correlated statiscally signinificant with outcome includes leucocyte count, hematocrit, urea, creatinine, lactate dehydrogenase, bicarbonate and albumin. sites of culture were skin/soft tissue ( , and %), respiratory tract ( , , and %) , blood ( , and %), urine ( , and %) , and other ( , and %) . -day mortality was % in this population. % of patients received antibiotic therapy alone, % surgery alone, % antibiotics + surgery, % other therapy, and % no treatment. the most common antimicrobial classes received were vancomycin ( %), beta-lactams ( ), fluoroquinolones ( ), and cotrimoxazole ( ) with % of patients receiving multiple agents. median duration of antibiotic therapy was , , and days, in the ca-mrsa, ha-mrsa and ca-mssa groups respectively. , , and % received adequate antimicrobial therapy (p < . ). hospital admission was required in , , and % of patients (p < . ). clinical success rates of initial therapy were , , and % (p < ), and recurrences were more common in the ca-mrsa group, ( , , and %, p < ). characteristics associated with outcome are listed in table . in multivariate analysis, presence of mrsa and diabetes were predictive of clinical failure.conclusion: in the community setting, mrsa infections are associated with an adverse impact on outcome compared to mssa infections and patients with ca-mrsa are significantly less likely to receive adequate antibiotic therapy. microbiological analysis of root canals associated with periapical abscesses and the antimicrobial susceptibility of isolated bacteria s. ozbek, a. ozbek, m. koseoglu, s. evcil, a. erdogan, a. ayyildiz (erzurum, tr)objective: the periapical abscess is a collection of pus in the pulp or around the root of teeth. many odontogenic infections can be managed without antimicrobial therapy or bacteriologic investigation. however, when an acute bacterial infection has progressed or antimicrobial therapy might be of benefit to patients, antibiotics are prescribed. we aimed to identify microorganisms in root canals with periapical abscess and the antimicrobial susceptibility profile of them and to revise antimicrobial treatment protocols when antimicrobials is used empirically. methods: patients with odontogenic infections included in this study. the microbiologic investigation was performed under strict aseptic conditions. a standardize routine of root canal therapy was instituted, and in each case a single root canal was sampled. in multirooted teeth only the largest canal was sampled to preserve the identity of a single endodontic/ microbiologic ecosystem. for microbial sampling, two sequential paper points were introduced into the full length of the canal, and kept in place for min. one of the paper points was used for aerobic culture and the other one for anaerobic culture. to identify isolated bacteria, whole bacterial fatty acid profiles were evaluated by using microbial identification system. antimicrobial susceptibility results were obtained by disc diffusion test for aerobics, and e-test for anaerobics. results: totally bacterial strains were isolated. of them were aerobic and of them were anaerobic. or % of cultured specimens yielded mixed (aerobic and anaerobic) species. the most prevalent bacteria were staphylococcus spp. as aerobic, peptostreptococcus prevotii and streptococcus morbillorum as anaerobic. conclusion: beta-lactam antibiotics combined with beta-lactam inhibitor (amoxicillin-clavulanic acid) had a quite effect on gram (+) and (-) aerobics. when we take into consideration that beta-lactam antibiotics stimulate production of beta-lactamase, amoxicillin-clavulanic acid combination appears a good first step antimicrobial. clindamycin may be second alternative for that purpose. for anaerobics, cefoxitin and metronidazol had well effect. although imipenem and piperasilin-tazobactam are perfect, they should not be first step of therapy. due to the frequency of mixed infections, a combination of amoxicillinclavulanic acid and metronidazol or a combination of clindamycin and metronidazol considered to have well effect for mixed infections. clinical microbiology and infection, volume , supplement , study is to review the spectrum of p. multocida infections in our centre. methods: we studied the medical records of all patients who had positive cultures for p. multocida between and . demographic, epidemiological, clinical and microbiological data including age, sex, animal exposure, site of infection, underlying diseases, type of therapy and outcome were evaluated. all isolates were identified by standard conventional microbiological methods. antibiotic susceptibility testing was performed by the disk diffusion method onto muller-hinton agar supplemented with % sheep blood and the mics of the antibiotics tested were determined by the e-test method. results: thirteen cases of p. multocida infections were diagnosed during this period. the male to female ratio was : and most patients ( %) were > years of age. respiratory tract infections were most commonly encountered ( . %), followed by soft-tissue infections ( . %) and septicemia ( . %). underlying disease was present in ( . %) patients. among them, presented a kind of malignancy. bullous pemphigoid, mitral valve stenosis, coronary disease, chronic obstructive pulmonary disease, and intracranial haemorrhage served also as predisponding factors. a traumatic animal exposure was reported in only patients and non-traumatic in cases. all isolates were susceptible to beta-lactams (penicillin, amoxicillin, amoxicillin/clavulanic acid, cefepime, cefuroxime, ceftriaxone, imipenem, and meropenem), quinolones (ciprofloxacin, norfloxacin, levofloxacin, and sparfloxacin), chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and % were intermediately resistant to aminoglycosides (gentamicin). appropriate antibiotic therapy was administered to all patients and a clinical response was observed in ( %) of them. mortality rate was %. conclusions: pasteurella multocida must be considered as a possible etiology for a variety of infections, even without an obvious animal exposure. although this organism is susceptible to a large spectrum of antibiotics, a failure to treatment may be recorded especially in severe infections and in compromised patients. infections caused by nocardia cyriacigeorgici in zaragoza, spain: identification and antibiotic susceptibility c. villuendas, b. moles, v. rodriguez-nava, a. couble, f. laurent, m. revillo, p. boiron (zaragoza, es; lyon, fr)objectives: nocardia species known to date differ in their clinical presentation, antibiotic resistance patterns and geographic distribution. nocardia cyriacigeorgici is a recently described species.the aim of this study is to analyse the identification results, antimicrobial susceptibility together with the clinical data, of n. cyriacigeorgici clinical isolates, recovered from to in our laboratory. methods: identification of nocardia spp. isolates was achieved in our laboratory on the basis of the following: visualization of the colony, gram stain and parcial acid-fast positivity by modified acid-fast staining, casein, xanthine and tyrosine hydrolysis, opacification of middlebrook h agar, production of arylsulphatase after days incubation and antimicrobial susceptibility pattern.identification at species level was achieved by s rdna gene sequencing (laboratoire de mycologie. faculté de pharmacie. lyon. france)antimicrobial susceptibility tests included commercial broth microdilution (emiza ef sensititre Ò ) and gradient strip agar dilution (e-test ab biodisk Ò ). interpretation of results was done according to nccls standard guidelines. in the six years of study, isolates of nocardia spp. were recovered, of them belonging to n. cyriacigeorgici species ( %). n. cyriacigeorgici represents the third species in frecuency in our serie, after n. abscessus and n. farcinica. the strains were recovered from patients, from respiratory specimens and one from blood-culture.pneumonia was the most frequent clinical manifestation, being copd and previous corticosteroid therapy the most common predisposing conditions. all n. cyriacigeorgici isolates showed susceptibility to: amikacin, tobramycin, cefotaxime, imipenem, trimethoprimsulfamethoxazole and linezolid, and resistance to: amoxicillinclavulanic acid and ciprofloxacine. conclusion: n. cyriacigeorgici is not an infrequent cause of nocardiosis in our geographical area. the uniformity showed in the antimicrobial profile can be useful for its identification. in our hospital, patients with copd and receiving corticoid therapy is the most important group of risk for adquiring n. cyriacigeorgici infection. whit the technics available in our laboratory the isolates were identified as nocardia spp. and identification at species level was only possible by phylogenetic analysis using rdna sequencing. high frequency of single-step resistance mutations in nocardia farcinica exposed to quinolones u.s. jensen, j.d. knudsen, k. schønning (hvidovre, dk)objectives: nocardia farcinica infections often require prolonged antibiotic therapy and perorally administered agents are desirable. isolates commonly display in vitro susceptibility to quinolones when tested by disc diffusion methodology. in the present study, we investigated the activity of three different quinolones (ciprofloxacin, levofloxacin and moxifloxacin) against n. farcinica and assessed the robustness of their activity by determining the frequency of single step resistant mutants when exposed to inhibitory concentrations of quinolones. methods: isolates of n. farcinica were used in the study; correct identification to the species level was verified by s rdna sequencing. mics of ciprofloxacin, levofloxacin and moxifloxacin against n. farcinica as well as s. aureus atcc and e. coli ccug were determined by the agar dilution method using inocula of approximately . cfu and h of incubation. single step mutation frequencies were determined by heavily inoculating selective agar plates containing quinolone at a concentration of x mic and counting resistant colonies after days incubation. inoculum was quantified by seeding a dilution series of the inoculum employed on unselective plates and counting colonies after h of incubation and frequencies were calculated by dividing the number of resistant colonies by the number of cfu present in inoculum. results: when mics were determined by agar dilution method all quinolones displayed roughly the same potency against n. farcinica isolates (mics between . and ). as expected moxifloxacin were the most potent quinolone against s. aureus. however, all three quinolones selected for single step resistant mutants, the frequency of which was higher for ciprofloxacin (~ ) ) than for levofloxacin ( ) - ) ), which again was higher than for moxifloxacin ( ) - ) ). however, even for moxifloxacin the frequency against n. farcinica was comparable to the single step mutation frequency of ciprofloxacin against s. aureus ( - ).conclusions: although quinolones may exhibit activity against n. farcinica, n. farcinica is capable of rapid development of resistance. therefore, quinolones should probably be avoided, at least as single agents, in the treatment of nocardia infections. correlation between clinico-laboratory findings and a positive igm elisa test for leptospira: a retrospective study e. mendrinou, p. goudas, a. regli (patra, gr) objective: to correlate a positive elisa test for igm antibodies against leptospira with the clinical and laboratory findings in patients with suspected leptospirosis. method: we retrospectively analysed the history, clinical course and laboratory findings in a total of patients, with suspected leptospirosis. all patients fulfilled the criteria for clinical diagnosis of leptospirosis. from the patients, had to be transferred to the dialysis unit for haemodialysis and patients had to be admitted to the intensive care unit (icu) due to severe pulmonary haemorrhage. serum samples from all patients were tested for igm antibodies against leptospira. results: from the total of patients death occurred to only four, due to respiratory failure from severe pulmonary haemorrhage. the rest of the patients recovered completely. from the total of patients had a positive elisa igm test for leptospirosis ( . %). however, from the patients that were transferred to the dialysis unit, had a positive leptospirosis test ( %) and from the six patients admitted to the icu, three had a positive test ( %). among other laboratory findings there was a stronger correlation between very low platelet levels (< . mm ) and very high blood bilirubin levels (> mg/dl) with a positive test for leptospirosis. all patients with a positive test had less than . platelets per mm and had blood bilirubin over mg/dl. the differential diagnosis of icterohemorrhagic fevers includes a vast number of pathogens, some of which are untraceable with the common laboratory methods. in our study, from the total of patients, only . % had a positive test for leptospirosis. in of the rest patients, many different pathogens were traced, most of them being several kinds of viruses (cmv, ebv), brucella and coxiella. in of the patients no pathogen was traced. conclusions: taking into consideration the high sensitivity of the elisa test we conclude that: . icterohemorrhagic leptospirosis comprises only a small subtotal of icterohemorrhagic fevers; . there is a correlation between higher levels of bilirubin and/or very low platelet levels with leptospira infection; . there seems to be a correlation between leptospira infection and severity of icterohemorrhagic fevers. evaluation of continuous ambulatory peritoneal dialysis-related peritonitis attacks in ankara s. tekin koruk, m.a. yetkin, i. koruk, f.s. erdinc, s. sahan, n. tulek, m. duranay, a.p. demirö z (ankara, konya, samsun, tr) objectives: peritonitis is a common clinical problem that occurs in patients with end stage renal disease treated by peritoneal dialysis. the aims of this study were to assess demographic aspects, rates of peritonitis, causative organisms, clinical outcomes and treatment approach for continuous ambulatory peritoneal dialysis (capd) -related peritonitis cases. methods: seventy cases of peritonitis occurred in patients treated in infectious diseases and clinical microbiology department between may and april were enrolled into this study. the mean age of the patients was . years (range - years). cloudiness of the peritoneal dialysis fluid and/or abdominal pain were considered suggestive of peritonitis and were confirmed by cell count and culture. baseline cell count, gram stain, and cultures were obtained, and repeated with periodic follow-up. results: the overall incidence of peritonitis was . ± . episodes/patient-year. in . % of patients there were only one peritonitis attack, where as in . % of them had two or more attacks. age, gender, education and profession of the patients have not been found as a risk factor in peritonitis attacks.the most common presenting symptoms of the patients were abdominal pain, cloudiness of the peritoneal dialysis fluid, nausea and vomiting. peritoneal dialysate fluid white blood cell count was ± /mm in episodes. cultures were positive in ( . %) peritonitis episodes; coagulase-negative staphylococci was the most common organism (% . ), followed by staphylococcus aureus (% . ), episodes (% . ) had negative culture results. there was a statistically significant decrease in serum crp and esr levels and at the end of the treatment when compared with the levels on admission.at the end of the study, episodes of peritonitis cases were treated with ip cefazolin and gentamicin protocol. seven of the patients did not respond initiate therapy and the therapy was converted to iv protocol. seven episodes were treated with iv antibiotics on admission for medical reasons (systemic infection and/or concurrent exit-side or tunnel infection). there were two deaths. two catheters were removed and the patients were transferred to haemodialysis programme. conclusion: despite all technical improvements during recent decades peritonitis is still the major complication of capd. for the accurate treatment of complications, causative organisms and their antimicrobial susceptibilities must be known. objective: viruses are a frequent cause of upper respiratory tract infections in children. among the respiratory viruses, influenza viruses are known to cause outbreaks globally. the present study was carried out to identify the influenza virus serotypes causing acute respiratory infection in children attending univesity hospital in konya in turkey. methods: thorat swabs were collected from acute viral upper respiratory infection suspected children attending the out patient clinic of meram medical faculty hospital. two swabs were taken fron each chidren and one of the swabs was used for bacteriological cultures and if these were negative the other one was used for viral diagnosis. totally bacteriological cultures negative swabs were investigated by real-time pcr for the presence of parainfluenza , and , influenza a and b. results: one or more viral pathogens were detected in children, with parainfluenza % being the most commonly identified virus. parainfluenza in % and parainfluenza in %, influenza a were identified in % and influenza b in %. from the specimens of children more than one virus detected. conclusion: the influenza viruses cause morbidity and mortality among children and elderly. this study analysed the occurrence of influenza and paranfluenza respiratory ifections due to influenza and paranfluenza viruses. molecular methods used directly on clinical material have an important role in the rapid diagnosis and surveillance of influenza viruses and can be applied in clinical practice for correct diagnosis and administration of effective treatment. , , , , , and . the demographics, clinical presentations and laboratory findings of the patients with serotype were presented. results: the mean age was y m, ranging from months to y m. seventy percents of children with serotype infection clustered between october and january . the mean duration of a positive culture result was . days. the mean duration of fever was . days, with days before admission. forty ( %) children were treated as outpatients. the mean length of hospital stay was . days. the most common diagnoses were exudative tonsillitis ( %), pneumonia or bronchopneumonia ( %) and pharyngoconjunctival fever ( %). the most common symptoms and signs were fever ( %), cough ( %) and coryza ( %). neurologic complications were noted in children. eighteen children had documented coinfection (including virus, bacteria and mycoplasma pneumoniae). leukopenia (wbc < /microliter) was noted in two of cases while leukocytosis (wbc > /microliter) in ( %). six ( . %) of cases had a normal serum c-reactive protein (crp) level (< mg/l), while % of children had a serum crp greater than mg/l. seventy ( . %) of children ever received antibiotics therapy. the outcomes were excellent in these cases. conclusion: recognizing that children with adenoviral serotype infection may present with prolonged high fever, leukocytosis and elevated crp, which mimics bacterial infection, the clinician may not prescribe unnecessary antibiotics for these children. the infectious mononucleosis like syndrome (im) is an acute febrile disease of older children and young adults, and is characterized by lymphadenopathy, tonsillitis, splenomegaly, liver dysfunction and by the presence of peripheral lymphocytosis with > % atypical lymphocytes. epstein -barr virus (ebv) is responsible for over % of the cases, cytomegalovirus (cmv) for %- % and toxoplasma gondii < %.herpes simplex, rubella and adenovirus are rare. the infection is usually characterized by mild symptoms. however in some cases the clinical manifestations may be rather atypical and severe. objective: to determine the prevalence of im like syndrome among patients in a children's hospital and its possible association with etiologic factors, age, major symptoms and atypical manifestations. material and methods: during a one-year period (january to december ) a total of samples were examined in our laboratory. the study population was children between - years old, which either examined in the outpatient's clinic or hospitalized. all serum specimens were examined by . indirect immunofluorescence for the presence of igg and igm antibodies against the viral capsid antigen (vca) ebv, .immuno chemistry luminescence for the detection of igg and igm antibodies to cmv and .eia for the detection of igg,igm abs of herpes simplex i and ii and toxoplasma gondii. results: of the children examined ( . %) were found positive for igg and igm vca antibodies and ( %) showed positive specific igg and igm antibodies for cmv. these patients had one or more of the primary following symptoms: fever ( %), lymphadenopathy ( %), pharyngalgia ( %), cough ( %), skin eruption ( %). atypical manifestations as meningoencephalitis were found in two children one aged months (caused by ebv) and the other of years old (caused by hsv i) confirmed by pcr. the laboratory data showed positive serology for ebv and cmv infection, the existence of atypical lymphocyte ( %), ldh, asat and alat were moderately elevated ( %) and crp increased ( %). conclusion: the frequency of im like syndrome in greece, though it's relatively low, it's not rare. the above results suggested that ebv, cmv, hsv should be considered in any young patient with im and acute neurological illness of uncertain etiology. objectives: enterovirus, parvovirus b and human herpes virus type (hhv- ) are a common cause of infection in young infants. the objective of this study was to determine what portion of the infants who received a clinical diagnosis of febrile syndrome have a viral etiology by these three genera of viruses. methods: ninety-six patients were included in the study, all of them were admitted to the pediatric casualty of a tertiary care hospital, and all of them presented a febrile syndrome without a clear focus of infection (urinary tract, lung and meningeal infections were discarded). the assay was carried out in blood samples by real-time pcr. dna was isolated from ll of blood by semi-automated system magna pure lc total nucleic acid isolation kit (roche diagnostics, nederland bv). pcr was performed in a lightcycler instrument (roche molecular biochemicals) by a uniform cycling parameters: min at °c for polymerase activation, and cycles of s at °c and s at °c for amplification of the specific target sequence ( utr gene for enterovirus, vp gene for parvovirus b and dna polymerase gene for hhv- ). pcr product formation was detected continuously by the use of taqman probes. results: a viral amplification was detected in ( %) of the patients included in this study. enterovirus was detected in ( . %) of the patients, parvovirus b in ( . %) and hhv- in ( . %). in five cases two viral amplifications were detected at the same time: parvovirus b /hhv- and enterovirus/hhv- . the mean age of the patients was years old (range from days to years). in group of infants < months old (n = ) there were enterovirus and hhv- . in the infants from months to years old (n = ) there were enterovirus, parvovirus and hhv- . in the last group of infants > years old (n = ) there were enterovirus and parvovirus b . conclusions: viral infections are an important cause of sepsis in infants admitted to hospital. enterovirus was the most frequent virus detected in infants < months, parvovirus b the most frequent in children > years old, and the hhv- was detected in all age groups. qualitative real-time pcr in blood is a rapid and sensitive method for diagnosis of enterovirus and parvovirus. however, is not the better method for diagnosis of hhv- , a latent virus, in which this technique is not capable of distinguish between recent and acute infection. objectives: group a rotaviruses are a major cause of acute gastroenteritis in infant and young children worldwide. in this study, the molecular epidemiology and clinical features of rotavirus infection in iranian children was investigated. methods: between february to january , thirty hundred and seventy two diarrhoea stools from children under -years-old with acute diarrhoea that attended the biggest paediatric hospital in tehran (iran), were analysed using elisa, electropherotyping and reverse transcriptionpolymerase chain reaction (rt-pcr). results: ninety-four samples ( . %) were positive for the presence of rotavirus either by page, elisa, or both. according to page, the predominant electrophoretic pattern detected was the long profile of ( . %) followed by the short electropherotype five of ( . %). out of the positive samples, were further characterized by rt-pcr typing assay for identification of g types, resulting in strains of g genotype while samples could not be assigned a g type. all of g genotypes had a long rna electropherotype. among the patients with rotavirus infection, ( . %) required hospitalization. watery diarrhoea ( . %), vomiting ( . %) and fever ( . %) were significantly more frequent in children suffering from rotavirus gastroenteritis. seven out of rotavirus-positive patients had severe dehydration (p < . ). rotavirus infection mostly affected children under years of age with a peak incidence of % in children - years of age and it occurs year round with a seasonal pattern: more frequently during winter ( . %). conclusion: this study revealed that rotavirus is an important etiological agent of acute gastroenteritis in tehran. we found that a major proportion of the specimens were untypeable. improved detection and characterization of incompletely typed strains will help to develop comprehensive strain information that may be required for tailoring effective rotavirus vaccines. serological study of prevalent rotaviruses in tehran e. habibi, s. ghorbani, a. jarollahei, z. habibi (tehran, zanjan, ir)objectives: rotaviruses are icosohedral and non-enveloped viruses that belong to reoviridae family which consist of three layers of protein surrounding segments of dsrna. rotavirus is one of the most important agents of acute gastroenteritis in children. in this survey, the most prevalent serotypes in tehran and seasonal distribution in a year were detected. methods: in this study, a total number of specimens of faecal samples of children and infants with acute gastroenteritis were collected from two children hospitals in tehran. the samples were tested by elisa procedure. serotyping investigation of iranian rotavirus isolates, using serotypes monoclonal antibodies (g -g -g -g -g -g -g ) in elisa tests and immunosorbent electron microscopical studies using trapping and decoration techniques were performed. results: rotavirus type a infection was identified in samples ( %). serotyping investigation in elisa tests proved that serotypes g and g were the most common serotypes circulating among infected children and infants in tehran. by electron microscopic studies the characteristic of rotavirus particles were observed in the faecal samples of infected children. the maximum incidence of infection was determined to occur among the cold months of the year. conclusion: it was approved that g and g serotypes are the main rotavirus serotypes present among children in tehran. it was detected that rotavirus diarrhoea was most prevalent among children of under years of age. results: from patients with varicella presented neurological manifestations (sex ratio m/f: / ). had acut cerebellar ataxia and one had encephalitis. we estabilished the diagnosis on the basis of clinical aspects (including neurological examination), cerebrospinal fluid examination and electroencephalogram. the age interval was between months and years. most cases were diagnosed in children and teenager ( ); one case toddlers, and cases in adults. neurological manifestations appeared in most cases among and days after the onset of rash ( cases). in the order of frequency: gait disorders ( ), cerebellar ataxia ( ), fever ( ), vomiting ( ), nistagmus ( ), seizures and coma ( ) . csf showed limphocytic pleiocytosis and elevated levels of protein ( cases); in cases csf had normal aspects. electroencephalogram had dominant theta wave with totally or partially suppression of alpha activity in all patients. all cases showed clinical and eeg improvement at the end of the treatment. conclusions: the most frequent neurological manifestation was cerebellar. the evolution was good under treatment, with no sequelae at month of follow up. key: cord- -dnsdg n authors: nan title: poster sessions date: - - journal: eur j immunol doi: . /eji. sha: doc_id: cord_uid: dnsdg n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx , the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th -oriented adaptive response. actually, ptx -deficient mice are highly susceptible to aspergillosis and develop th skewed responses; moreover, ptx -deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx , which does not show direct activity on fungal cells. finally, ptx alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx -mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx -opsonised conidia, cd b activation, internalization, recruitment to the phagocytic cup and cd b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx , fcgriia/cd mediates inside-out activation of cd b and consequently phagocytosis of c b-opsonised conidia. in vivo phagocytosis experiments performed with c q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx ) and the cellular arm (fcgrs, cr ). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx -/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd , immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx . we found high concentration of ptx in human colostrum ( . ± . ng/ml at day post-delivery) compare to the one found in human serum ( x ng/ml). the presence of ptx in human colostrum seems to be due to the secretion of ptx by human mammary gland since we report the production of ptx by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx supplying by breast feeding: (i) soluble ptx in colostrum (ii) hmc that can secrete ptx upon stimulation in the specific tissue, (iii) an increase of ptx production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx may participate to the beneficial role of breast feeding on the newborn health. a. m. baru , j. stephani , h. wagner , t. sparwasser twincore, institute for infection immunology, hannover, germany, technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of receptors in humans (tlr - ) and in mice (tlr - , - ). as transmembrane receptors, tlrs are expressed on the cell surface (tlr , , , , , ( ) ( ) ( ) ( ) and at endosomal membranes (tlr , , and ) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about years later to this, toll-like receptor (tlr ) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr (hutlr ) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr (mutlr ) is also expressed on conventional dendritic cells (cdcs). consequently, tlr ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr (henceforth called as hut mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr knock-out background. we expect that hut -mutlr -/mice mimic the human specific expression pattern of tlr , i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr . by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin- is a heterodimeric cytokine consisting of the two subunits p and p . the main inducers of il- p are microbial components activating toll-like receptors with the magnitude of il- p induction depending on the specific tlr engaged. differential induction of il- p upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il- p due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il- p promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il- p promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone and acetylation in specific regions of the proximal promoter region. acetylation of h showed a specific distribution pattern and occured mainly in regulatory elements within the il- p promoter, whereas acetylation of h took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue on h turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue , w. ellmeier medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi , m. buxadé , j. minguillón , r. berga , j. aramburu , c. lópez-rodríguez universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat , the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat . our work indicates that nfat is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr , tlr , tlr and tlr and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p and p nfxb subunits. interestingly, we observed that tlr stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr , tlr and tlr stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl- expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr , tlr , tlr and tlr . finally, in vivo tlr stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr , tlr or tlr triggering can directly favor tumor development whereas tlr signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms- ) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd + and cd + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd + and cd + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd + and cd + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: . innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . . adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni , r. oatuni , m. collini , m. caccia , p. castagnoli , g. chirico , f. granucci university of milano-bicocca, biotechnology and bioscience, milan, italy, university of milano-bicocca, physics, milan, italy, singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin- (il- ) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il- production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca +/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca + influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr engagement, depending instead exclusively on cd . we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase- (cox- ) that, by generating prostaglandins (pgs), such as pge , regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd . given the essential involvement of cd in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd represents a major step towards the development of potential treatments with modes of action involving interference with cd functions. we have examined the interaction of cd , a -kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd . human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd antibody coupled to biotin visualised by qd- -streptavidin and anti-cd antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd and cd , suggesting a new functional role of cd as a member of a multimeric lps receptor complex. l. lundvall , r.r. schumann charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase- induction leads to an increased release of mature il- b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d ) was established for assessing il- b induction during this disease. the murine raw . cell line, the human astroglial u- mg and the murine microglial cell-line bv- were stimulated with the tlr ligand lps, the tlr ligand pam cys, the nod ligand mdp, or the nod ligand c -ie-dap, and, as control, atp alone or in combination. we assessed il- b by elisa and caspase- and pro-il- b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il- b induction pathway. s. pneumoniae (d ) infected mice showed a significant increase in il- b release after hours. in vitro, an increase in il- b levels after costimulation with lps or pam cys, and mdp or c -ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il- b by tlr-and nlr-ligands was observed in raw cells, bv- cells, u- cells and primary astrocytes. active caspase- (p ) was induced by mdp or c -ie-dap, corresponding with high il- b responses when lps or pam cys was added. sirna experiments show that a knock-down of nod leads to a diminished il- b release after lps-and mdp-stimulation. the precursor forms of il- b and caspase- seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il- b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il- b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il- b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd and cd activation model system -tlr presence on cd + cells is found in mouse t cells, human t cells and jurkat cell lines. following cd /cd activation for hours we have identified a small but distinct populationof tlr + cells. further characterization indicates these cells to be cd +cd + cells. further characterization of the expression and functional acitvity of the tlr + t cells indicates co-expression of tlr with md- indicating a functional tlr receptor. in addition lps activiation did not lead to upregulation of tlr expression in t-cells. the data indicate that tcr activation leads to tlr expression. the expression appears to be associated with cd +cd + cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann , d. kaul , c. krüger , f. zipp , r. nitsch , s. lehnardt charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna ) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr (and human tlr ) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr is expressed in these cells. incubation of microglia with all three of the above mentioned tlr ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il -b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr knock out (ko) microglia by real- because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin- was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin- is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin- -transfected plb cells, coronin- overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd b labeling. concerning apoptosis, increased coronin- expression in dmf-differentiated plb significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin- significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase- and caspase- activities, but not caspase- or bid truncation suggesting that coronin- interfered with mitochondria-related events. to validate the prosurvival role of coronin- in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin- expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin- immunolabeling. we concluded that coronin- could constitute a potential target in resolving inflammation. p.-n. hsu national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw . macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf- sirna and traf- decoy peptide, indicating this pathway is traf- dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b -h and b -dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il- was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il- and ifn-g which could not be overcome by restimulation with acd . this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd t cells into t helper (th ) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il- receptor-related protein st could be implicated in lps tolerance. the natural ligand of st was recently identified as interleukin- (il- ), a new member of the il- family. in this study, we investigated whether il- triggering of st was able to induce lps desensitization of mouse macrophages. we found that il- actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il- enhancing effect of lps response is mediated by the st receptor since it is not found in st ko mice. the biochemical consequences of il- pretreatment of mouse macrophages were investigated. our results show that il- increases the expression of the lps receptor components myeloid differentiation protein- (md ), cd and tlr- and the myeloid differentiation factor (myd ) adaptor molecule. in addition, il- pretreatment of macrophages enhances the cytokine response to tlr- but not to tlr- ligands. thus, il- treatment preferentially affects the myd -dependent pathway activated by the tlr. c. klotz , b. lenz , r. lucius , s. hartmann humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., ) . the cystatin effect was blocked by the application of anti-il- receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin- (il- ). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il- in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il- production after avcystatin stimulation. application of specific inhibitors revealed that the il- induction was p and erk dependent, and inhibitor titration indicated a higher sensitivity towards p . western blotting experiments confirmed the phosphorylation of p and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il- is also regulated by the phosphatidylinositol- -kinase (pi k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il- and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose- -phosphate (s- p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose- p and xylulose- p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p /jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s- p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts , y. morias , p. de baetselier , a. beschin vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m ) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd , ifng, il- , ccr and nf-kb to the development and/or recruitment of pathogenic m in the liver was investigated using knock-out mice or by delivering il- in infected mice. results: we established that cd b+ly c+cd c+ tnf and inos producing dcs (tip-dcs) represent the major m liver subpopulation. tip-dcs differentiated in an ifng/myd -dependent manner from cd b+ly c+ inflammatory monocytes in the liver of infected mice. ccr promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il- enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il- in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p and il- play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m , in particular tip-dcs. the inflammatory activity of liver m is controlled by il- and/or p nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov , j. driesen , z. abdullah , a. niño castro , t. chakraborty , m. krönke , o. utermöhlen , c. wickenhauser , j.l. schultze limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, institute of molecular medicine and experimental immunology, bonn, germany, institute of medical microbiology, university of giessen, giessen, germany, institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine , -dioxygenase, cyclooxygenase- and cd and production of prostaglandin e (pge ) and interleukin . all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd , expressed by regulatory myeloid cells, acts as an il- scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd + ido + cox- + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin (il- ) and il- in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il- and il- or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il- and il- . in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il- and il- . therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel , m. rodriguez gomez , m. niedermeier , y. talke , n. göbel , k. schmidbauer , m. mack unversity hospital regensburg, internal medicine ii, regensburg, germany, university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il- and il- . activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp- cells infected with m. bovis bcg, the avirulent m. tuberculosis h ra strain and the virulent m. tuberculosis h rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva , l.d. miteva , s.a. stanilova trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il- is a heterodimeric cytokine composed of a p subunit associated with the il- / p subunit. like il- , il- is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il- p -related cytokines controls the appearance of normal th and pathological th mediated immune responses. in this study, we examined the dynamics of inducible il- p and il- p mrna expression and protein production in purified human monocytes and how jnk and p mapks inhibitors influenced il- p and il- production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il- p gene expression was higher than those observed for il- p gene at all time-points. the level of il- p mrna increased after th h and reaching a maximum level at th h ( . fold for c bgp and . fold for lps). c bgp and lps triggered il- p gene transcription were almost equal at the rd h ( . and . fold) and at th h ( . and . fold, respectively) after stimulation. the higher level of il- p gene expression was detected at th h in lps compared to c bgp stimulated monocytes ( . vs. . fold). however, il- p and il- protein production was increased in the highest level after c bgp stimulation. the inhibition of p led to the statistical significant augmentation of c bgp stimulated il- p production. the inhibition of the same map kinase enhanced lps stimulated il- p production without significant difference. the inhibition of jnk and p mapks significantly decreased c bgp stimulated il- production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c bgp and lps to induce the expression of il- p and il- p mrnas in purified human monocytes. we showed that inhibition of p mapk down regulated il- and upregulated il- p production in stimulated monocytes. we concluded that in human monocytes p map kinase activation has an opposite effect on the il- p and il- p expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd , cd and cd , and secrete il- , thus acquiring the ability to induce proliferation and th polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd c, bdca- , and cd . by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd + subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd + dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd + dc, including their survival and their ability to produce il- p . besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il- , il- and mcp- involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg , s. wolke , a. brakhage , m. gunzer otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and -photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor (irf ), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr . in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens , s. vander beken , p. bogaert , j. grooten ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th -driven immunological counterpart of the th -driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th -and th -driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd . + ram remained constant in cell number for the first days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker , a. stojanovic , a. cerwenka german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr- + cd b + f / + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas , f. marchesi , m. fabbri , s. schiarea , c. chiabrando , a. mantovani , , p. allavena istituto clinico humanitas, rozzano, italy, istituto mario negri, milano, italy, università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m and m . m macrophages act as a first line of defence against pathogens whereas m cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about % of the monocytes differentiated after days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd , cd and low levels of mhc class ii. tc-macro produced il- , il- , tnf but not il- , even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl , cxcl , ccl and cxcl . the transcriptional profile of tc-macro revealed that several genes in line with an m polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g . kda). il- and il- were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl- h rat mast cell line. we demonstrate that gr nuclear translocation begins within minutes and completes after minutes in dx treated rbl- h cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within minutes as non-genomic. studying gc-caused apoptosis, rbl- h cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl- h cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. minutes of dx treatment inhibited ca + -signaling in antigen stimulated rbl- h cells in the concentration range of nm - mm. moreover, minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl- h cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box (hmgb ) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin ( ) . extracellular hmgb can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb exerts antibacterial functions in human adenoid and testis ( ) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity ( ). we asked whether hmgb from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for or minutes with nm phorbol ester (pma), ng/ml interleukin (il- ), or ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h a-histone h b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb localizes on nets. we hypothesize that net-bound hmgb might exert a direct antimicrobial function, or that nets might concentrate hmgb locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation ( °c, h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor- induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr- and anti-vegfr- antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il- . altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd a and cholesterol-dependent lipid rafts impact on cd a surface expression and cd a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd -restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd molecules in multiple sclerosis. we first analyzed cd expression on monocytes from ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd a was not expressed on ms patient monocytes, while the other members of the cd family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd molecules in this context. c. ohnmacht , d. voehringer ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il- reporter ( get) mice as well as in vivo depletion of basophils are used to uncover their role during type immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th cells. these results demonstrate that basophils play a crucial role as effector cells in type immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl- in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il- beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl- -transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water ( mg and mg daily) for days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il- and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il- and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant ). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type- macrophages, mph ), and during resolution of inflammation (type- macrophages, mph ). methods: mph / were generated by culturing cd + human monocytes for days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph / of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph / were stimulated with lps and secreted il- , il- , il- , il- p and tnf were quantified by elisa. results: mph are relatively efficient phagocytes for apoptotic neutrophils whereas mph are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph and mph which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il- , il- and tnf production is suppressed while il- secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of years old and older (long-livers). intracellular ros and hsp levels were registered by flow cytofluorimetry upon labeling with ', '-dichlorofluorescin diacetate (invitrogen) and anti-hsp antibody (brm- , sigma), respectively. intracellular level of hsp was also estimated in neutrophils after heat shock (hs) performed at °c for min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at - min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for min at °c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp (hsp basal ) and ros level, both intracellular and extracellular. at the same time increased hsp level immediately after hs (hsp ( min)) correlated negatively with intracellular ros (initial and after hs). the hsp increase value (hsp ( min) -hsp basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp increase was registered in min after hs (hsp ( min) -hsp basal ). thus it was found that within this age group the alteration in hsp induced by hs in neutrophils but not basal hsp itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant # . d. goyeneche-patiño , z. orinska , f. mirghomizadeh , s. bulfone-paus forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type ifn. two novel genes, receptor transporter protein (rtp ) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during and h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine . stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr and , as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd and trif and the pathways that they represent are not relevant in the expression of rtp and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano , e. erba , r. frapolli , m. d'incalci , a. anselmo , s. pesce , p. allavena , a. mantovani , humanitas clinical institute, rozzano, italy, mario negri institute, milan, italy, institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et- ) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl , cxcl and the inflammatory protein pentraxin (ptx ) either at transcriptional and protein level, especially after tnfa/il b stimulation. down-regulation of ccl , cxcl , ptx and also of il and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd + vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin- (il- ) is a key cytokine of the t helper cell response. il- has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il- production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il- producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day - after birth, a small population of il- producing cells was observed in the absence of any stimuli. the il- + population was not detectable at or weeks of age. other cytokine producing cells (ifn-g, il- ) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il- + cell population. phenotyping of the neonatal il- + cells showed that they were ige + /mhcii -/cd cells. the occurrence of cd + il- producing cells after pma stimulation increased slowly with age and did not reach adult levels by weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il- at day after birth. neonatal pbmc collected before colostrum uptake did not produce il- in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il- responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il- response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel , d. voehringer ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat -dependent manner when stimulated with the th cytokines il- or il- . alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat -dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat -deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat -deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il- exposed macrophages from wild-type and stat -deficient mice. candidate genes will be expressed using retroviral transfections of stat -deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of pleural effusions, including malignant and nonmalignant tumors. the following human malignant cell lines were tested: a , ht , hct , sw , mcf , mda-mb , jurkat and hl . results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m , and the tams isolated from the malignant effusions similar to m . among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms a a and ms a a: highest trascriptional level after h of stimulation with - m dexamethasone. ms a murine genes are differently expressed respect to the human counterpart and only the homologs of ms a a (ms a b, c and d) have a similar regulation. finally, egfp-tagged ms a a, ms a a, and ms a expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein- (mcp- )/ccl and the gpcr ligand fmlp or the tlr agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il- b or ifn-g, significantly and dose-dependently synergized with ccl in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin- /cxcl , but not of the ccl receptor ccr in monocytic cells. in turn, the induced cxcl synergized with ccl for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr /cxcr , because cxcl receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, ) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for min at °c with gentle shaking, followed by spinning down of pmns for min, at °c and g. the supernatant was sedimented at g for min, °c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd low cd + blood monocytes (mo) in healthy individuals and the increase in cd +cd + blood mo in patients with inflammation has been reported. the functional activity of human cd + blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd and activation markers such as hla-dr and trem- . percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a . fold increase (p x . ) in the total number of circulating cd low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd + blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd + blood mo expressed increased levels of cd on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem- +) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd on cd high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m -and m -polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m and m macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m type and were cd negative but cd and mhcii positive and produced high levels of il- , tnfalpha and il- following lps stimulation. culture in the presence of mcsf resulted in induction of the m phenotype. these cells were cd positive with intermediate expression of cd and mhcii expression and produced high levels of il- , il- and il- following lps stimulation. interestingly, already baseline il- production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m -and m -polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma ( ng/ml) treatment for h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl cells expressed antigen receptors cd , tlr , tlr and cd , and displayed enhanced phagocytic activity and production of ros. expression of cd , cd and cd was also maintained however the cells were hla-dr and cd a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl cells to differentiate into macrophages in response to pma. hl may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta , n. ferrer , a. gómez , j. gonzalo , a. arbués , a. anel , c. martín , j. pardo , apoptosis, immunity and cancer university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so , a m. tuberculosis phop mutant strain that was shown (perez et al ) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al ) . in the present study, we compare the time course and phenotype of cell death induced by so , bcg and wild type m. tuberculosis on the murine macrophage cell line j and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase- activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl x deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type receptor for vip (vipr ) gene is highly conserved through species and, in humans, is highly polymorphic. vipr has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr after , , and h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from donors that had been typed for the relevant vipr snps. the down-regulation of vipr correlates with the presence of a t at rs mapping in the '-end of the gene (p= . ). the vipr protein level was decreased about % in monocytes of subjects typed as t/t at rs whereas subjects typed as c/c at rs maintained a high level of expression after h of lps treatment. the data show that different haplotypes of the vipr gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska , t. vavrochova , d. filipp , immunobiology institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e . - . ) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd in the early mouse embryo (me). facs analysis of cell suspension prepared from . day me showed that about . - % of cells were positive for cd b. these cells exclusively were also positive for cd , tlr , and cd antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days , - , . reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e , ), embryo-derived mhcii + macrophages start to appear in the embryo around day . multicolor facs analysis of cd b, cd , cd , f / , tlr , tlr , c-kit and mhcii surface markers revealed differential expression of tlr and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd b + tlr + cells isolated from the e , embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il /il -dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in out of these patients mutations in several ifng pathway genes, other candidate genes are being investigated for the other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp . we did focus on the study of genes which are considered as important pathogen recognition receptors of the innate immune system: tlr is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il- cytokine. using a case/household-contact cohort we did investigate polymorphisms of these genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to - % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, . hsp are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp were used. exogenous hsp was used in concentration - ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio : and : directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp on their surface. results demonstrating effect of exogenous hsp on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp on amplitude of respiratory burst. the cells expressing surface hsp impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd /cd and cd /cd double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd / subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd / subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević , i. pilipović , s. stanojević , k. mitić , k. radojević , v. pešić , g. leposavić , institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h o ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to -day-long propranolol treatment and measured both no and h o production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b -and a -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h o production. an increase in h o production in the presence of the a -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h o production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b -and a -adrenoceptors on peritoneal macrophages (a stimulatory effect on b -adrenoceptors and a suppressive effect on a -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd ++ cd -('classical') and cd + cd + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd + cd + monocytes display higher tlr and - expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd ++ cd monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr stimulation. methods: cord blood (n= ) and peripheral-blood (n= ) mononuclear cells were stimulated in vitro for hrs with peptidoglycan and subsequently analysed for cd and cd and intracellular il- p and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il- p , both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il- p was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il- p (% positive cells and geomfi) was significantly higher for cd + cd + cells than for cd ++ cd cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd ++ cd cells were positive for tnf to a significantly higher extent than the cd + cd + cells. in particular the tnf response to tlr stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd ++ cd monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a -year-old boy who admitted with complaints of seizures during the previous months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g ( mgr/m /day, subcutaneously every other day) was started. after months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf gene concerns the well-known gt deletion in the second exon of ncf gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = ) subjected to moderate brain injury were randomized, and received either . mg/kg ubiquitin or vehicle (placebo) intravenously within min after cci. levels of tnf-a, il- b, il- , il- and il- receptor antagonist were analyzed in brain tissue using real time rt-pcr at and hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at h and days. data were analyzed with the mann-whitney u test and a two-tailed p x . was considered significant. all cytokines were highly up-regulated hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il- were significantly lower in the ubiquitin treated animals, whereas the levels of il- and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day . the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight ( ) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p= . ), tpp (p= . ), osi (p = . ), mda (p = . ) were significantly raised but taa (p = . ) was significantly reduced in ptb patients compared with controls. the levels of mda (p = . ), neopterin (p= . ) and tpp (p= . ) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p= . ), mda (p= . ), neopterin (p= . ), osi (p= . ) were significantly reduced while taa (p= . ) was significantly raised in c+m after weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas , e.m. cunha , m.j. oliveira icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles ( nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage ( %) of macrophages contained the metal particles than neutrophils ( %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap- is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap- regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., ; sivalenka and jessberger, ) . swap- -/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., ; sivalenka and jessberger, ; sivalenka et al., ) . crucial regulators of these processes are members of the rho family of small gtpases such as rac and rhoa. swap- interacts with rac in vitro and preferentially binds the active gtp-bound rac . swap- supports the increase of active rac in vitro by a yet to be defined mechanism (shinohara et al., ) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap- and rac . it was found that fulllength swap- preferentially binds to constitutively active rac (rac q l) but not to its dominant negative form (rac t n). binding assays with swap- truncated mutants showed interaction of swap- 's n-terminus with gtpgs rac or rac depleted of guanine nucleotide, whereas swap- central or c-terminal regions do not bind to any form of rac . preliminary competitive-binding assays with overlapping mer peptides, spanning the entire swap- sequence, mapped the rac binding site near the n-terminus of swap- . full-length swap- site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap- with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap- . v. c. barbosa , c. d. polli , m.c. roque-barreira , m.c. jamur , c. oliver , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl- h cells were sensitized with ige anti-tnp and stimulated with dnp -hsa or artinm. artinm binding to rbl- h cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp- and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp- release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand (psgl- ), b and b -integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il- b, il- and tnf-a. herpes simplex virus (hsv- ) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il- and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv . methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) ( ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv- for h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot . software. results: in the first itraq experiment over human proteins were identified in the hsv infected cell supernatants. from these proteins had at least fold increase after poly(i:c) + hsv infection compared to the uninfected cells. hsv infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of . fold increase in the protein amount in the poly(i:c) + hsv infected cell supernatant and a . fold increase in the hsv infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand , il- , tnf-a induced protein , complement factor b, galectin- and mxa. at present, further experiments are on-going for more detailed analysis of the hsv infected macrophage secretome. h. p. prakash , german cancer research centre, translational immunology, heidelberg, germany, max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein- (ciap- ) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap- and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal- ( mg/ml) binding to monocytes, in the presence or absence of mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal , laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal or rpmi and monocytes ( x ) were added into each insert. when necessary, hrgal was pre-incubated with mm lactose or sacarose. mcp- ( ng/ml) was used as positive control. we observed that hrgal- binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal- is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal- , we observed that the association between these glycoproteins and hrgal- resulted in a % increase in the number of migrating cells. both n-and c-terminal domains of hrgal- are involved in the association between laminin or fibronectin and hrgal- , since the presence of lactose resulted in % and % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal- induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for weeks in the presence of bmp ,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd +cd + cells. the sorted cd +cd + cells were further cultured for - days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd , m-csf receptor cd and the scavenger-receptor cd , we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd and cd (fcgammariii) : the cd lowcd -and cd + cd +. trancscriptional, phenotypic and functional assays suggest the alternative (m ) polarization of cd +cd + embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi , p. kropf imperial college london, immunology department, london, united kingdom the balance between t helper (th) and th cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th or th responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg vd t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f -atpase) is expressed on many cell types and is a possible specific ligand for the vg vd tcr. the present study aims at understanding the role of f -atpase in antigen regognition. using video microscopy calcium imaging in single vg vd t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f -atpase. purified f -atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg vd cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f -atpase. thus the f -atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg vd t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f -atpase. by using purified f -atpase and peptides derived from vg vd tcr sequences, interaction sites between f -atpase and tcr were identified on both ligands. based on these findings a generalized model for vg vd t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg d activation in vivo. by transiently overexpressing the nkg d ligand rae- -beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma vdelta gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae induction. the primary systemic th response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg d receptor suggest that different ones may play unique roles. a novel nkg d-ligand, h c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae- induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th (ifn-g, tnf) and th (il- , il- ) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd -nk . -inkt cells producing high levels of the pro-inflammatory cytokine il- has been identified (inkt cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt cells in type diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il- as compared to c bl/ mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt cells present in nod mice were mainly cd -nk . -, express the ror-g transcription factor and il- receptor, both molecules being usually associated with th commitment. we are currently analyzing, using co-transfer experiments, whether these inkt cells play a beneficial, a deleterious, or any role in the development of type diabetes in nod mice. j. s. dodd , r. muir , s.s. affendi , p.j. openshaw imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd d-deficient mice with poor nkt cell responses have inefficient induction of cd t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th immunity (increasing il- and il- ), promoting pulmonary eosinophilia and ablating cd t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd t cell activity (as measured by cd expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d ) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d ) weight loss by a cd t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd + ab t cells, are regarded as immature or t h biased. vg + vd + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)- -hydroxy- -methyl-but- -enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg + vd + t cells towards these activators. because il- is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg + vd + t cells. herein, we observed that zoledronate induced neonatal vg + vd + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h -like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h -like cytokines such as il- and il- were not induced. addition of il- to zoledronate selectively costimulated ifn-g production from neonatal vg + vd + t cells. furthermore, zoledronate/il- treatment resulted in neonatal vg + vd + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il- (il- r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il- resulted in a further increase of t-bet expression in neonatal vg + vd + t cells. these changes in the expression of il- r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il- treatment. of note, in contrast to adult peripheral blood vg + vd + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg + vd + t cells. altogether, these observations show that neonatal vg + vd + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il- is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e ligases such as hhv encoded k , k or mhv encoded mk . these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e ligases manipulate them. we discovered that both the cellular march and viral e ligases ubiquitinate cd molecules. however, whereas viral molecules inhibit cd -antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd molecules. in contrast mhc class ii was only targeted by cellular and not by viral e ligases. furthermore cd molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd neg clones in vivo, we generated hypodermal ht tumors in immunodeficient mice. concomitant injections of vd neg clones, in contrast to vd + cells, prevented the development of ht tumors. vd neg clones expressed chemokine c-c motif receptor (ccr ) and migrated in vitro in response to chemokines secreted by ht cells, among which were the ccr ligands macrophage inflammatory protein (mip)- d and monocyte chemoattractant protein (mcp)- . more importantly, a systemic intraperitoneal (i. p.) treatment with vd neg clones delayed the growth of ht subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd neg clones were not able to inhibit the growth of a hypodermal tumors. our findings suggest that cmv-specific vd neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht cells expressing the luciferase and realized orthotopic injection of ht -luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint , currently the only known determinant of a canonical iel compartment, that is selectively required for vg vd + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint function; for example, we demonstrate that the constitutive expression of wild-type skint fully restores detc development in a skint mutant mouse, but does not rescue normal detc function. thus, skint provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd + cd hi cd lo cd c -) and splenic conventional dendritic cells (cdcs) (cd c hi cd a +/-cd b +/-b -) from wt and cd d -/mice as apcs for nkt cells from va -ja transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il- by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il- producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h response, whereas cdc-primed nkt cells rather favor a t h response. objectives: il- is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il- give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd d -/-) mice. we set out to investigate the activated b cells in il- injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il- ( mg) for days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day was evaluated by flow cytometry and immunohistology. results: mice injected with il- developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il- was increased in inkt cell deficient (cd d -/-) mice, in contrast to published data. an increased response to il- was also observed in ja -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il- induced antibody responses. further characterization of the recruitment of b cells in il- injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il- induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd d, are prone to autoantibody production and often involved in early immune responses. the il- induced antibody response in mzb deficient (cd -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il- induced antibody responses. we conclude that the role for inkt cells in il- induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c bl/ mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha -/-and cd d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il- . moreover, ifn-g production required the presentation of ehlppg by cd d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il- . similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd in cmv-infected individuals. cd is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that . ± . % of gamma-delta t cells from cmv-infected ktr expressed cd , when compared with only . ± . % in non cmv-infected ktr (p x . ). similarly, . ± . % of gamma-delta t cells from cmv-seropositive blood donors expressed cd compared to . ± . % in cmv-seronegative donors (p x . ). cd + gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd -dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il- and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd a on cd + gamma-delta t cell lines. cd is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd + gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a skin carcinoma cell line pre-incubated either with rituximab (anti-cd ) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd -dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il- , ifn-g, tnf-a, osm, ccl , cxcl , cxcl , and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd , cd , hla-dr, and dc-sign, and lost cd , ccr , ccr , and cxcr . addition of further microbial stimuli (lps, peptidoglycan) induced ccr and enabled these inflammatory dcs to trigger antigenspecific cd + effector ab t cells expressing ifn-g and/or il- . importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg /vd t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg /vd t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg /vd t cells stimulated with hmb-pp in the presence of il- express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il- regulate expression of the b cell attracting chemokine cxcl /bca- , its receptor cxcr , and co-stimulatory molecules involved in b cell help. purified peripheral vg /vd t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to days with and without hmb-pp, in the absence or presence of il- or il- , or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl protein were detected in co-culture supernatants only when both il- and hmb-pp were provided, implying an il- -dependent and tcr-dependent expression. vg /vd t cells were confirmed as producers of cxcl by flow cytometry and immunofluorescence. under the same conditions, activated vg /vd t cells expressed cd , cd , cd l, cd , icos and ox . in contrast, neither cxcr nor ccr changed markedly by il- stimulation of peripheral vg /vd t cells. conclusion: our findings confirm on the protein level that stimulation of vg /vd t cells with hmb-pp and il- induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto , m. emoto gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)- . although downmodulation of surface t cell receptor (tcr)/nkr-p c (nk . ) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il- stimulation. to determine whether failure to detect inkt cells after il- stimulation is caused by dissociation/internalization of tcr and/or nkr-p c or by block of de-novo synthesis of these molecules, and to examine the role of il- in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p c by inkt cells after stimulation with a-galcer or il- , and influence of il- neutralization on down-modulation of stcr/snkr-p c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il- neutralization. whereas s/cnkr-p c + inkt cells became undetectable after in-vivo administration of il- , s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p c remained unaffected by a-galcer or il- treatment, despite the down-modulation of ctcr and/or cnkr-p c protein expression. in contrast, ctcr + cnkr-p c + stcr -snkr-p c -inkt cells and cnkr-p c + snkr-p c -inkt cells were detectable after in-vitro stimulation with a-galcer and il- , respectively. our results indicate that tcr and nkr-p c expression by inkt cells is differentially regulated by signaling through tcr and il- r. they also suggest that il- participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg -gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region d (cdr d ) and cdr d repertoire, with a striking enrichment in a specific germline-encoded cdr d sequence. differentiated gd t cells and the enriched cdr d sequence were detected as early as after weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd -jd and vgi-jg . / . rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd -jd and vg -jg . selection was seen in pb tcrgd cells. detailed analysis of the cdr motifs revealed selection determinants in both vg -jg . (canonical length and cdr motif) and vd -jd (minimal cdr length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚ ) and tcrgd cb cells ( - ) to tcrgd pb cells (˚ or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type diabetes (t d). we have previously shown that transgenic expression of a cd d-restricted, va . -vb tcr in nod mice lead to an increase in cd d-restricted type ii nkt cells ( abnkt cells), and prevention of the development of t d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc . cd + t cells, in the presence or absence of selected cells from abnkt cell transgenic mice. results: in ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, abnkt cells expressed a high level of cxcr and a low level of ccr and cd l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd + bdc . t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from ab transgenic mice with bdc . cd + cells resulted in the prevention of diabetes development. the protection from disease required a minor cd + subset of ab+ nkt cells, but was independent of cd + t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs , induces in vitro inkt cell expansion and ifng and il- secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il- with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th -type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th immune response is characterized by the secretion of il- a and il- f. the il- locus encodes the highly conserved il- a and il- f cytokines that are syntenic in kb distance to each other. besides cd + th and nkt cells, approximately % of the il a producers are gd t-cells. like cd + th cells, il- producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il- production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il- or ifn-g. combined with the well known classification of il- producing gd t-cells along the markers cd and cd , our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th -type immune responses in vivo. in this context, the potential redundancy of il- a and il- f may complicate the analysis. so far, most studies were carried out with il- a single-deficient or il- f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il- a and il- f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg vgd t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b and il- differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp ) and, similarly to ab tregs, suppress the proliferation of anti-cd /anti-cd stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il- was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd b in the liver following a-galcer treatment, numbers of cells lacking cd b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma /vdelta t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma /vdelta t cells expansion upon stimulation with zoledronic acid (za) and interleukin- (il- ). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by ) the bioinformatic analysis of gene expression profiling data ) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in patients ( %) (responders, r), whereas patients ( %) were non-responders (nr). vgamma /vdelta t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt , whereas temra of r patients had an higher expression of the costimulatory molecule nkg d. the proliferative response of vgamma /vdelta t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, % of r patients were m, whereas % of um patients were nr (p x . ). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c bl/ mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il- p (clone r - f ; atcc) or anti-cd- d (clone b ) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il- p , a component of both il- and il- ; pretreatment with anti-cd d mab had no effect. il- rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il- was not a critical factor. this suggestion is supported by the increased expression of il- p and il- p (but not il- p ) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il- production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb ) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd expression. gd + cells secrete interferon-g, while gd cells are capable of producing il- . this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd -defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine . cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚ % of gd + cells but not at all in gd cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, - % of gd + cells were positive for pgp activity, although their gd counterparts remained largely negative (p x . ). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd . as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd + and gd cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma vdelta t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma vdelta t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma vdelta t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma vdelta t cells from healthy donors were stimulated with different compounds (zol, ipp) for hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma vdelta t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma vdelta t cells. moreover, mcp- depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma vdelta -mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma vdelta tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma vdelta t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg vd t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg vd t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg vd t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg vd t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg vd t cells and their effects on the viability of glioma cells, we expanded in vitro vg vd t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg vd t cell lines to kill three different glioma cell lines (t , u , u ) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg vd t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg vd t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg vd t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg vd t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e -induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e -expressing transplanted skin is dependent on interactions between populations of cd d-expressing cd c+/f + myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il- and a-galcer. expression of cd a, cd d and the costimulatory molecules cd and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il- ) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd d expression on the cell surface in comparison with control cells. in contrast cd a expression was decreased. cd and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd a, cd b and cd c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il- producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, . - % of circulating lymphocytes express a vg vd t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg -enriched genes compared to conventional mhc-restricted cd + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf & crtam in gd and ab t cells respectively. because igsf binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd + ab t cells express crtam, and that engagement of igsf on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf /crtam. we therefore sought to answer: . what is the function of igsf /crtam on gd t cells? . how is the igsf -crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf enrichment on resting gd t cells, with expression also detected on˚ % of ab t cells. the properties of those cells are being examined. however, igsf generally correlates with markers of activation/antigen experience such as cd ro. thus, igsf cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf and crtam within hours. however, engagement of igsf by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf interactions are sufficient for cd t cells to kill gd t cells and/or vice versa. instead, crtam-igsf interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd z is a kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately - % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd tcr variable segment associated with the vg segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg vd t cells without antigen presentation. in vitro stimulated vg vd t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg vd t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg vd t cell anergy observed in hiv+ patients. to this aim, cd z expression and ifn-g production by vg vd t cells from hiv+ and hiv-subjects were analyzed. we show that vg vd t cells from hiv-infected patients expressed lower level of cd z compared with healthy donors. a direct correlation between cd z expression and ifn-g production capability by vg vd t cell was found. however, pkc activation by pma is able to restore cd z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg vd t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner , m. swamy , s.l. clarke , a. hayday king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd aa or cd -cd cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to days. the cells are initially activated by plate-coated acd antibody and a cytokine cocktail and maintained further in medium containing low levels of il- . after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il- ) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg molecules in heterodimer with cd , we screened the presence of these receptors on t cell subsets in bd. the expression of nkg a/c/d molecules on gd and cd + t cells were analyzed in active and inactive patients with bd and healthy controls. expression of nkg molecules was evaluated on cd +, gd t and cd + nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls ( . vs. . %, p= . ). in addition to the increase of gd t cells, increased expression of activating nkg c molecules was also observed on gd t cells ( % vs. %, p= . ). nkg a expression on gd t cells was found to be higher than nkg c expression in patients and controls; but nkg a expression on the t cells was not statistically different in both groups ( . vs. %). nkg d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd + cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th and th cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg /vd + lymphocytes. we have screened a panel of lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg /vd + cells, and selected two susceptible ("target") cell lines (over % death in the assay) and two vg /vd + resistant ("non-target") cell lines (under % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs (non-target), and validated the results by rt-qpcr quantification. results: we identified commonly up-regulated and commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp , ifitm and prame, for example, are up-regulated, whereas cd and clec d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg /vd + target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd d, in the rat. mice and rats have very similar cd d and inkt tcr genes, with the exception of the va gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd d revealed a very similar pattern of cd d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il- production was - fold lower in the best responder rat strain (f ) compared to mouse (c /bl ). since nkrp a (rat homologue of mouse nk . ) and tcr are not appropriate markers for rat inkt, cd d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g d t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g d t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g d t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g d t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp- . a significant (p x . ) reduction in intracellular bacterial numbers was observed (n= ) in the presence of pbmcs cultured with picostim+il- in comparison with pbmcs cultured with il- or media alone. picostim+il- caused significant expansion and activation of gd t cells following culture of pbmcs for - days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers -fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il- , reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma /vdelta (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: ) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; ) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); ) to establish whether the same issues could be solved using a simplified protocol of dc generation. ) dc were generated from cd + cells of healthy donors/mm patients; immaturedc on day were induced to fully mature by incubation for hours with tnfa + il- b + pge in the presence or absence of mm za. after days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; ) idc generated from cd + cells of hla-a* + healthy donors/patients were pulsed with sv-peptide and stimulated for hours with tnfa + il- b + pge in the presence or absence of mm za; after rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd + t cells was determined by svpentamers staining; ) the same experiments were performed both with dc generated following a standard protocol and a h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [ - h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd + cd + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin- on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd + have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n= ) and inactive (bdna) (n= ), versus healthy controls (hc) (n= ) and patients with recurrent oral ulcerations (ru) (n= ). to determine gd cytokine profile and surface markers treg-related in bd (n= ) and hc (n= ). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta , vdelta , cd alpha, cd beta, nkg d, nkg a and cd . -intracellular expression of ctla- , and foxp . -intracellular expression of il- , il- , ifngamma, il- and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta + cells were significantly increased in ru. vdelta + and gdcd + lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg d was slightly increased in gd from bda. -nkg a expression by gdcd + was not different in bd versus hc. -most of gdcd + presented cd alpha-alpha homodimers in bd and hc and were negative for cd , foxp and ctla- . gdcd + and gdcd -subsets were (in bd and hc): -high ifngamma-producers without differences. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd + than in gdcd --very low producers of il in most of cases. the hallmark in bd was the increase of gdcd /vdelta +. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd +, except a lower percentage of il- + cells than in the gdcd -subset. gdcd + from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd in humans. a subgroup, the invariant nkt cells (inkt), expresses the va vb tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd + cd + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from healthy umbilical cord blood samples and stimulated with ifn-g ( ng/ml), anti-cd ( ng/ml) and il- ( ui/ml). these cells were cultured for days and the expanded cd + cd + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd + c + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x , was considered significant. results: we could significantly expand cord blood cd + cd + nkt cells from , ± , % to achieve an enrichment of , ± , % (p= , ). table shows the percentage (mean±sd,n= ) of phenotypic markers in cd + cd + cells at baseline (day ) and after days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb and vb families (p x , ). conclusion: our results show that cord blood-derived nkt cells are mainly cd + and cd + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb and vb families in these cells. l. marischen , d. wesch , p. rosenstiel , a. till , d. kabelitz institute of immunology, kiel, germany, institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod . peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp and pvama proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd + and cd + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd a + (lysosomal associated membrane proteins: lamp- ) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th and th cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th -like and th -like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n= ) and healthy controls (n= ) were determined by flow cytometry using anti-cd and monoclonal antibody specific for the cdr loop of the invariant tcr a chain of inkt cells (clone: b ). furthermore, after pma/ionomycin stimulation for hours, intracellular ifng and il- cytokines were detected in cd +cd -, cd -cd -(dn), cd -cd + and cd +cd + subsets of inkt cells by five colour flow cytometry in patients with ad (n= ) and healthy controls (n= ). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x . ) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x . ). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r= . and p x . ) and healthy controls (r= . and p x . ). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il- level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x . ). the frequency, the number of inkt cells and the cytokine producing capacity of the cd /cd inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il- that promotes the th differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting . %, . %, . %, . %, . % and . % of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells ( . %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd + nkt and cd -cd -nkt cells. nk . + nkt cells may release large amounts of il- , il- , ifn-g and il- after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il- had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il- . there was a significantly increase in the percent of cd + nk . + and tcrvb + nk . + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd molecule existed in . % of the cells from large-scale selection. the percent of cd + nk . + and tcrvb + nk . + nkt cell subsets in the giant lymphocytes were enhanced to . and . folds, respectively. under a light microscope at x magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than . and they are non-adherent cells. the differentiation pathway of the seb-activating cd + and tcrvb + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd + nkt cell and tcrvb + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo- am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h begfp reporter mice. stimulation with anti-gd clone gl or anti-cd clone c elicited activation of gd t cells suggesting that tcr gd and cd molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl in vitro produced ccl , ifng and tnfa. therefore, we were interested whether the ccl production of gd iiel influenced the homing of ccr cells such as lamina propria (lp) cd + foxp + cells (tregs). to test this, wt mice were i. p. treated with gl mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il- production in g reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il- gene. in the absence of any manipulation gfp was expressed from the il- locus in populations of immature thymic nkt cells (predominantly cd +cd lotcrhi cells on a balb/c background, and cd +cd lonk . -on a c bl/ background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il- production was induced predominantly in cd + nkt cell subsets of the liver and spleen, and after i. n. administration, in cd + nkt cells of the airways. spontaneous and a-galcer-induced expression from the il- locus occurred in the absence of stat signalling, and did not require initial exposure to il- protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin- (il- ) plays an important role in neutrophil recruitment. herein, we investigated the role of il- receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c bl/ (wt) and il- receptor gene-deficient (il- r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated hours after surgery. the ability of il- mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il- r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il- induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il- r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il- receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il- showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il- also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il- receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin- receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il- r associated kinases to activate nfxb and p mitogen-activated protein kinase. the il- -induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il- in different mast cell subsets. methods: different mast cells subsets (hmc- , human cbmcs and murine bmmcs) were stimulated with il- . the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk / , pakt, pnfxb, p and pjnk). additionally, we studied the signal transduction of il- in il- r transfected hek t cells. results: we found, that a tir family member, il- r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il- -induced cytokine production depends on c-kit transactivation. il- r and il- r accessory protein (il- racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il- r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il- -induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il- in mast cells in absence of iger activation. ( ) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase- activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease- (ho- ) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho- expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin and . recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr shows a bimodal kinetic, with the first peak at ''/ ' and the second at ' after stimulation. rna interference-mediated depletion of beta-arrestin specifically inhibits the occurence of the second wave of rap activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin is involved in rap activation and that the oscillations in the formation of rap -gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c g (rap gef) and spa (rap gap): preliminary results suggest that spa- has probably a role in the early activation peak. since this oscillatory chemokine-induced rap activation is present on other myeloid cell lines (hl , d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin , localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of different sh domains that revealed over putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)- and il- stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il- and il- receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il- , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: ( ) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna- expression is induced by activation of the toll-like/interleukin- receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna- a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna- a transfected cells showed significantly reduced levels of the active proapoptotic caspases and (casp / ). in line with this, mirna- a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna- a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna- a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna- a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il- , il- r a-chain and il- r a -chain genes in hiv disease progression. methods: we studied antiretroviral treated patients (progressors) and long term non progressors (ltnp). we analyzed snps in the il- gene, snps in the il- r gene and snps in the il- r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il- r aa and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il- r and il- r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd and cd t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art , and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art transcripts by rt-pcr analysis in human blood leukocytes. soluble art , released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek cells with art and tnf resulted in modification of tnf at at least distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r , a site that has previously been implicated in binding to tnfr . binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik , t.v.poroshina institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and control men were assessed for slpi, tnf-alpha, il- and free tgf-b in ejaculate by elisas using stat-fax plus. a to day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at - degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients ( . ± . pg/ml, p x . ) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid ( . ± . pg/ml; p x . ). the il- concentration was elevated in all patients ( . ± . pg/ml; p x . ) in seminal fluid. free tgf-b was present in normal seminal plasma in high concentrations ( . ± . pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b concentrations were . ± . pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il- and free tgf-b are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi , e. a. elgaaeid faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th or th cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod /card protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod /card gene in cd. we performed a cases /controls study upon cd patients and healthy controls. this study suggests that in northen tunisian population, insc mutation in nod /card gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il- polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il- cytokine genetic profile in patients with ibd. we examined the contribution of il- gene promoter polymorphisms (- and - ) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card /nod gene mutations in cd presentation and location. in tunisian population, the insc insertion in nod /card gene is a marker of susceptibility to cd, while the a allele at position - in the il- promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il- gene polymorphisms and card /nod gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il- -dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il- -driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il- molecule by ganglioside. but gangliosides apparently can also form complexes with il- r; such complexes influence on the signal transduction through il- r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il- r subunits. in our work we use il- -dependent cytotoxic t-cell murine line ctll- . two different approaches for study of possible interaction types between exogenous gangliosides and il- r subunits were applied: antibody staining of il- r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with i-dcp-gm followed by immunoprecipitation of il- r subunits. the fluorescence intensity of the antibody-labeled il- r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by . % after cells incubation with ganglioside gm , and by . % after incubation with gm . labeling of the cells with antibodies to the il- r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm ( . %) or with gm ( . %). to determine the mode of this impressive masking influence of ganglioside gm , photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm with subunits of il- r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il- r, but not for il- r a-subunit. these results demonstrate that exogenous ganglioside gm can interact with a-and bsubunits of il- r in different modes. interaction of il- r b-subunit with ganglioside gm requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa , j. bukur , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak . results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak expression due to a gene deletion on chromosome , whereas the expression and functionality of other ifn-g signal transduction components like stat and jak were not affected. jak blockade by two jak -specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak leading to a lack of jak expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of controls of the spanish population. methods: two il polymorphisms located on the il- promoter region, snps - a/c (rs ) and - g/c (rs ) were genotyped using taqman snp genotyping assays. the functional il- gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il- - p= , . il - p= , ) but may contribute to disease onset and aggressiveness: the genotype il- - cc genotype, which is associated with higher il- production, was significantly associated with higher tumor size (p= , ), grade (p= , ), t (p= , ), m (p= , ) and stage (p= , ). the influence of the il- - gg genotype was less relevant, however was correlated with higher tumor size (p= , ), grade (p= , ), t (p= , ) and stage (p= , ). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il- polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il- gene (il- - and - ) may be associated with an worse prognosis of rcc. high levels of il- production can play an important role in grow, invasion and metastasis of renal cancer. interleukin- (il- ) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il- is its ability to induce the production of ifn-gamma in presence of il- . moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l . then it seems that il- has a crucial role in immunity against brucella infection. since the expression of il- can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il- gene polymorph isms and brucellosis. methods: a total of patients with brucellosis and healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il- polymorphisms at positions - , - , , + , + and codon / using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il- polymorphisms at position - g/+ t/+ c (correlated with high production of il- ), codon / c and - g/- c (correlated with higher production of il- ) were significantly higher in the healthy controls than the patients (p= . , p= . and p= . , respectively). discussion: as data revealed genotypes that have correlation with higher production of il- are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il- at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor (ifngr ) mouse mutant. we have generated a mouse line carrying a conditional ifngr allele using the loxp -flp recognition target (frt) approach. a targeting vector with loxp sites flanking exons - and frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez , r. carretero , p. saenz-lopez , j. cantón , j. carretero , f. ruiz-cabello , l.m. torres , cts- hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca) allele of ifgn is significant more frequent in healthy control than in cin patient (p= , ) in contrast homozygosis for (ca) allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p= , and p= , respectively). conclusions: our study suggest that ifng (ca) allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin- (il- ), a th -related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il- single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: patients with brucellosis and healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il- (c t, g a, c a and a t) polymorph isms using pcr-rflp. results: at position , the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p= . and p= . , respectively). at position , the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p= . , p= . ). discussion: as shown the frequency of cc genotype and c allele at position were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position is lower in patients than the healthy controls and the frequency of c allele at position is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf- and skbr- , as well as their ability to respond to several cytokines and to the g- gpr agonist. the mcf- and skbr- human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il- ra, il- rg, il- /il- /gm-csfr, il- /il- r, il- ra, il- ra, il- r (gp ), il- ra, il- r, tnfr i, tnfr ii, ifngra, cxcr , cxcr , cxcr , cxcr , cxcr . cells were incubated with il- , ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr agonist. cytosolic ca + concentrations [ca + ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr- cells were found positive for a larger number of receptors than the mcf- cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf- cells with il- reduced the calcium response to g- , while pretreatment with ifn-g and tnf-a potentiated the calcium response to g- . tnf-b had no effect on mcf- g- stimulation. no direct effect on basal [ca + ] i stimulation could be noticed when administering the cytokines alone. in skbr- cells, pretreatment with il- or tnf-b had no effect on basal [ca + ] i and did not significantly alter the calcium response to g- , while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g- . pretreatment with tnf-a produced calcium oscillations and reduced the response to g- . conclusions: mcf- and skbr- cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd a -pdcs and cdcs were infected with mcmv, whereas after h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag -deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg , respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt -l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f / + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd t cells in response to an adova infection due to an impaired cd t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il- . to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp and vacv-gp expressing the gp epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p t cell receptor recognizing the gp epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p ifnar -/mice. upon adoptive co-transfer of p ifnar -/and p ifnar wt/wt t cells and subsequent challenge with mva-gp , a massive expansion of p ifnar wt/wt t cells was observed, whereas p ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp induced a rather similar expansion of ifnar competent and ifnar deficient p t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h n -specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h n induced an exceptionally nf-kb dependent antiviral response. irf is essential part of this interferon-response of human endothelia. furthermore, we identified hmga as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h n . finally, nfatc was found to be a transcriptional regulator for specifically h n -induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h n which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns , delta-ns influenza virus (a recombinant influenza virus lacking the ns gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting days post-infection, reached its maximum at day , and triggered t cell priming in vivo. a direct comparison of delta-ns virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns protein. thus, we propose that the virally encoded ns protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that - - proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of - - target proteins. thus, these results suggest that - - proteins have a regulatory role in host defence against viral infections. i. wessels , d. fleischer , l. rink , p. uciechowski institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)- b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il- b expression is not elucidated, yet. it is known that the il- b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il- b when stimulated. b-cells which are il- b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il- b promoter and the impact of methylation on il- b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl- cells were differentiated into monocytic cells after dihydroxyvitamine d treatment. the monocytic phenotype was confirmed by flow cytometry. the il- b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with -aza- -deoxycytodine (aza) and changes in il- b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl- cells, differentiated cells displayed upregulation of cd antigen and acquired the ability to express il- b. by comparing the accessibilities of il- b promoter we detected that the il- b promoter was not accessible in undifferentiated hl- cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl- cells, demonstrating that the chromatin remodeling of the il- b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il- b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il- b expression were found. our data indicate that the il- b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il- b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg , g. wetzel , h. arnold , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il- b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp , , , and mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. ( )). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. aug ; ( )). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. dec; ( ) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il- b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory t breast cancer cells over expressing il- b ( t /il- b) tumor cells, but rarely in untransfected t cells. secretion of il- b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il- ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array . having p x - in two independent cohorts each consisting of blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for h and we measured the levels of il- , il- , il- , tnf-alfa and il -ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed snps with p x , * - . to identify/replicate the association of cytokine production for these we reanalysed these on a cohort. a combined analysis revealed snps with p x , * - .these results are not genome wide significant. the snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp on adipocytes. for the first time we could show a hsp -stimulated release of the proinflammatory cytokines il- , cxcl and mcp- in a time-and concentration-dependent manner from murine t -l adipocytes. analyses of hsp -signalling in these adipocytes revealed that members of the mapk-family (erk / , p ) and the transcription factor nfkb are involved in hsp -mediated induction of the mediators il- , cxcl and mcp- . binding-studies with fluorescence-labelled hsp demonstrated that the interaction of hsp with adipocytes exhibits basic features of a receptor-mediated binding. hsp -binding to adipocytes was saturable and reached its maximum at . mm. binding was inhibitable only by the unlabelled ligand ( %), but not by unrelated proteins, thereby proving the specificity of hsp -binding. further analyses to characterize hsp -receptor structures on adipocytes revealed the presence of toll-like receptor (tlr) on adipocytes. tlr has been found to be expressed on macrophages and to interact with hsp , therefore suggesting tlr as a potential receptor candidate for hsp on adipocytes. in order to identify the responsible binding-epitope of hsp we investigated the effect of specific antibodies directed against different epitopes of the hsp -molecule. incubation with antibodies directed against the n-terminus of hsp (aa - ; - mg/ml) were capable of inhibiting the hsp -binding to adipocytes ( - %) indicating that the n-terminal region of hsp is involved in receptor binding. our experiments demonstrate that hsp stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp -mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf . we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with % of fetal calf serum at °c and % co . bone marrow nonadherent cells were removed after h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd , cd , cd , cd and stro- and were negative for cd . intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost % of msc. interleukin- , ifn-gamma and tnf (th cytokines) increased msc contractility, whereas il- (a th cytokine) decreased msc contractility. by immunofluorescence, we observed that il- , ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il- decreased that incorporation. our results suggest that th and th cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca + -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of × /ml in complete rpmi- medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as nm and nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents ( mm) at the -h culture interval reached the values of approximately ng/ml and ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of - h in rat pecs. the -h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p and erk / . it was not suppressed by the calcium chelating agents bapta-am and tmb- . the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr / / . pin is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin substrates and targeting sites. recent data show that pin interacts with apo-bec g (a g). the pin /a g interaction results in a reduced a g expression and a diminished a g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin gene (- g/c and - t/c) modulate pin expression; in particular, the - gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, ; - , ) . the - c/g and - t/c polymorphisms in the promoter of pin gene as well as pin protein levels were analyzed in exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; hiv-infected patients (hiv) and healthy controls (hc). the genotype and allele distributions of the - snp was skewed in esn (genotype: p= . ; allele: p= . ). in particular esn showed a significantly lower frequency of the - gg genotype compared to hiv and hc (p= . and p= . , respectively) and consequently a lower g allele frequency (p= . and p= . , respectively). no significant differences were found for the - snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, -(r)- [ -(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, -[ -(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of × /ml in complete rpmi- medium. secretion of cytokines was determined after the -h culture by elisa. production of no was assayed at the interval of h using griess reagent. approximately compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. -[ -(phosphonomethoxy)ethlyl]- , -diaminopurine derivatives, c) -[ -hydroxy- -(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) -heteroalkyl substituted -amino- -guanidinopurines, and f) -amino- -(purin- -yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip- a and cytokines tnf-a and il- . although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as to mm. the remarkably enhanced secretion of chemokines was reached within - h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant m . host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and million units of ifn-a treatment three times a week for months was initiated. before and after treatment: percentages of the il- and ifn-g in cd + t cells were assessed to determine intracellular t helper cell (th ) type cytokine expression. similarly, percentages of intracellular il- and ifn-g were detected to verify cytotoxic t cell (tc ) type cytokine expression in cd + t cells. percentage of th and tc type cytokine expression, (il- and il- ) were determined in cd + and cd + t cells, respectively. six ( %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th cells with respect to healthy controls before treatment. tc percentages, both tc and tc , were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il- expression was higher and the percentages of th type cells were significantly low. il- expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency ( , %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects ( , % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt , f. juengerkes , b. schumak , g. gielen , j. kalff , p. knolle , b. holzmann , a. limmer university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, university hospital bonn, department of surgery, bonn, germany, department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il- , the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il- and il- in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, h. pylori-infected du patients ( patients were positive for anti-caga antibody and patients were negative for anti-caga antibody), h. pylori-infected as carriers ( subjects were positive for anti-caga antibody and subjects were negative for anti-caga antibody) and healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il- and il- was measured by elisa method. the mean serum levels of il- in total du patients ( . pg/ml ± . ) was significantly higher than those observed in total as subjects ( . pg/ml ± . , p x . ) and healthy control group ( . pg/ml ± . , p x . ). in du group, it was found that the mean serum levels of il- in subjects with positive test for anti-caga ( . pg/ml ± . ) was significantly higher than those observed in subjects with negative test for anti-caga ( . pg/ml ± . ; p x . ). the mean serum levels of il- in du ( . pg/ml ± . ) and as groups ( . pg/ml ± . ) was significantly higher than those found in uninfected control group ( . pg/ml ± . , p x . and p x . , respectively). however, no significant difference was observed for mean serum levels of il- between du and as groups. moreover, in both du and as groups the mean serum levels of il- was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il- and il- in h. pylori-infected subjects as compared with control group. in du group the expression of il- influenced by the bacterial caga factor. a. aral , , a. atak gazi university faculty of medicine, department of immunology, ankara, turkey, gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il- levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il- elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il- levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the th hour and reached to maximum levels at the st week and decreased again at the rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the st week and started to decrease at the rd week. il- reached to its peak at the rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic , m. jurisic university of kragujevac, school of medicine, kragujevac, serbia, university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in radicular inflamed cysts and odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd , cd and cd . tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x . ) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. ) drawings blood from patients and healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. ) detection of the gene expression of prolactin and tlr- in cd + peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p . ) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr- and peripheral prolactin expression in cd + monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table- ). when tb household contacts and healthy controls were compared, cfp and esat seemed to be more useful than tst in tb contacts for displaying ltb (table- ) . although cfp spot numbers were much more than esat spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: , )( table- ). both esat and cfp spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g analytes immediately prior to liver transplantation and at sequential time points up to days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip- , il- and il- . notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/ / (h n ) (hk) differs from its putative avian precursor by amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk- aa-i r, n d, k n, s n, g a, human ( - ); rhk-r -l q, s g and rhk- aa-i r, n d, k n, s n, g a, l q, s g, avian ( - )). among these variants, the double mutant rhk-r and the seven mutant (rhk- aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l q and s g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip- and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r and rhk- aa as compared to rhk and rhk- aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il- , shedded adhesion molecules (cd , vcam- , icam- ), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk- aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il- , and neutrophilic cd levels, procalcitonin and il- were measured by elisa technique while, neutrophilic cd by single colour flowcytometric technique. of these "infected" infants, had positive blood culture (subgroup ia: culture-positive sepsis), and infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il- , cd , and crp. il- had the highest sensitivity and specificity, % and %, respectively, using cutoff n . pg/ml. for pct, the highest sensitivity and specificity, % and %, respectively, were at a cutoff value of n . pg/ml. neutrophilic cd had maximal sensitivity and specificity of % and %, respectively, at cutoff value of . %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il- and neutrophilic cd , which together provided sensitivity and specificity of % and %, respectively, and npv %. the combination of il- and crp had high sensitivity and moderate specificity, % and %, respectively. conclusions: il- and neutrophilic cd levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp , naip or ipaf and the adaptor asc are involved in caspase- activation in response to bacterial infection, triggering the processing and secretion of il- b and il- . recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi k, vps and can be inhibited with the pi k inhibitors wortmannin and methyladenine ( ma). in contrast, activation of akt, via class i pi k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il- b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with ma, wortmannin or an akt inhibitor. supernatants were analysed for il- b by elisa. results: ma enhanced il- b secretion by bmdc treated with the tlr and tlr ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il- b secretion was greatly reduced in bmdc from nalp -/mice compared to wild type c /bl controls. treatment with the akt inhibitor had no effect on lps-induced il- b secretion by bmdc. tlr-dependent secretion of il- a was also enhanced by treatment with ma. conclusions: these data demonstrate that il- b secretion by bmdc in response to treatment with pi k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp inflammasome. this response is limited to tlr and tlr agonists. inhibition of akt had no effect on lps-induced il- b production, suggesting that the effect of wortmannin and ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr signaling by the inflammatory lipid mediator sphingosine -phosphate (s p) through receptors and in human monocytes-macrophages, which could explain some of the s p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s p, and later analyzed by flow cytometry. a pharmacological analysis of the s p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam- expression was increased following lps and s p concomitant stimulation in both venous and arterial cells, suggesting that tlr and s p receptors cooperate in the expression of icam- . conversely, no cooperation was observed when tlr ligands were used. in order to elucidate which s p receptor subtype was involved in the increase of icam- expression, we used a pharmacological approach with s p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam- after lps and s p challenge was significantly reduced by blocking s p receptor and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s p receptors and , which suggest that s p receptor might be involved in the effect. conclusions: altogether these data demonstrate that tlr and s p receptors can interact to increase adhesion molecules such as icam- in human endothelial cells, and the s p receptor subtype involved in the effect differs between arterial and venous cells. with ssc without pah) and a pool of sera of healthy controls (hc) were tested. results: in dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with - , - and - protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized ± , ± , ± and protein spots respectively. twenty one protein spots were recognized by more than % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein and peroxyredoxin and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: patients suffering from different diseases were enrolled in our study. patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam- and vcam- were assesed by an enzyme immunoassay (elisa). results: out of the patients ( , %) had elevated levels of the intercellular adhesion molecule. out of the patients suffering from bone diseases ( , %) had raised values (mean value ng/ml) whereas patients out of the suffering from soft tissue diseases and diabetes ( , %) had raised values (mean value , ng/ml). reference value for icam- was - ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients ( %) having increasd levels of icam- . high icam- levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes ( , %) than in patients with bone diseases ( . %). mean values were found , ng/ml and ng/ml accordingly. those findings verify the positive correlation between icam- and inflammatory diseases and tissue damage but not for vcam- . colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: cases of ulcerative colitis (urc), adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, tubular or tubulo-villous adenomas with low grade dysplasia, and infiltrating adenocarcinomas. immunohisto- objectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). mg of pravastatina oral hours they were administered before the procedure (group study, n= ) or placebo (group placebo, n= ), and control (group control, n= ). samples of outlying veined blood were extracted to the hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in degrees: degree . without expression. degree . weak; degree . moderate; degree . intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree - . the placebo group: mixed pattern, degree - . group study ( mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree - . the percentage of cells that expressed cd was greater in the group study ( mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam and adam . we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam . these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam activity in the tissue. in the presence of the adam inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam -mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf- / vegf+ is associated with rcc risk ( p= , ), metastases ( p= , ), nuclear grade ( p= , ), tumor stage ( p= , ), and tumor size (p= , ). on the other hand, the polymorphism vegf - a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol , , -trisphosphate. therefore, pten is one of the main antagonists of the pi -kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il- as well as il- levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il- as well as il- and il- mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th- t cells, we measured th- cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il- and a strong reduction of il- mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il- exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il- ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il- . based on our previous observation that support a direct role of il- -activated stat in the enhancement of il- ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il- ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il- . quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il- ra promoter. crosslinked nuclear lysates were immunoprecipitated and min after il- addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il- ra promoter. chip assays showed that the pol ii recruitment to the il- ra promoter induced by lps is significantly increased by il- , further strengthening the concept that the rapid enhancement of lpsinduced il- ra gene expression by il- initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat immunoprecipitated dna showed statistically significant levels of stat binding to the il- ra promoter only in cells stimulated with lps in the presence of il- . surprisingly, anti-p and anti-p chip assays revealed enrichment of both p and p recruitment to the il- ra promoter when il- was added to lps-stimulated cells, suggesting that il- enhances the recruitment of nf-kb to the il- ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il- -treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il- ra promoter site is dependent on il- -activated stat , since it is greatly reduced when stat activation by il- is impaired. the molecular mechanism through which il- -activated stat promotes the recruitment of nf-kb to the il- ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il- acts as a specific negative transcriptional regulator of mouse mast cell protease- (mmcp- ). we examined the mechanisms underlying the repression of mmcp- gene expression. our data show that the "repressor" effects of il- on mmcp- promoter activity are still operating on the mmcp- bp long minimal promoter. moreover, il- deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy . furthermore, chromatin immunoprecipitation revealed that il- promoted specific reciprocal recruitment of c/ebpb but not yy to the mmcp- promoter. finally, il- deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp- . thus, we proposed that the expression of mmcp- and possibly other immunoregulatory genes may be regulated by il- through epigenetic modification and by balancing the content and binding of c/ ebpb and yy in mast cells. i. nagy , k. filkor , a. vörös , l. kemény , a. szász bzaka, baygen, szeged, hungary, university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir- , mir- a and mir- , which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir- expression; in contrast, pgn re-stimulation had no further effect on mir- a and mir- expression. next, we investigated the correlation between the expression of mir- and its two known direct targets: regulatory protein p and suppressor of cytokine signalling- (socs- ). although the gene-expression profile of neither p nor socs- changed, we found that the expression of mir- reversibly correlates with both p and socs- protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir- prior to pgn-treatment completely abolished both p and socs- down-regulation, revealing the involvement of mir- in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir- expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb +/-had a lower incidence and burden of benign papillomas when compared to tgfb +/+ animals. however, more scc developed in the tgfb +/-mice. after acute and chronic promotion, tgfb +/-skin showed a reduced proliferative response with no increase in epidermal tgfb or nuclear p-smad compared to tgfb +/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb gene dosage. further, pharmacological inhibition of alk suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb +/-skin, tgfb +/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb +/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb +/+ but not tgfb +/-keratinocytes, indicating that tgfb switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp- ) and fibroblast (mrc- ) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of ra patients and controls the levels of survivin correlated to urokinase (upa) (r= . ), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that / ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc- and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol , , -trisphosphate -kinase type b (itpkb) phosphorylates inositol ( , , ) trisphosphate (ins( , , )p ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca + responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h and h receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il- b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il- b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase- to generate the mature secreted active form. caspase- is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of genes involved in splicing with an average -fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified genes that significantly affect the production of il- b by thp- cells after a h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il- b secretion. tissue transglutaminase (tg ) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg inhibitor, in a transgenic mouse model cf and in the taz transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg in generating inflammation in two very different pathologies. this work underlines the critical role of tg in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge stimulated a-dc. preliminary results indicate no accumulation of hif- alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif- alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p but not erk / or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il- and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt a -but not wnt a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp in te. after infection with t. gondii, mice lacking neuronal gp (synapsin-cre gp fl/fl ) died significantly earlier in the chronic phase of infection than control gp fl/fl mice. death of synapsin-cre cre gp fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp -deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il- b, il- and il- have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il- , il- and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb further may generate more effective therapies. as tnfa mrna ' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb suppresses tnfa protein production by upregulating the rnabinding protein fxr , which can bind to tnfa mrna and inhibit translation. methods: using raw . cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb , we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb and luciferase expression was quantified. cells treated with lps and tgfb were also examined for fxr expression using pcr and western blot. following fxr knockdown using sirna, the influence of tgfb and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb . using the luciferase-tnf- 'utr vector we show that tgfb targets the 'utr of tnfa. furthermore, tgfb and il- both upregulate fxr mrna and protein; and treatment with tgfb and lps can synergistically upregulate mrna expression, more than tgfb alone. following sirna inhibition of fxr , tgfb can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp- and mmp- secretion from saecs, nhbes and fibroblasts to a peak of . +/- . ng/ml, at hours. interleukin- augmented comtb-stimulated up-regulation of mmp- and mmp- secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin- down-regulated mmp- secretion from saecs by %. interleukin- up-regulated mmp- and mmp- secretion from fibroblasts but not from saecs. timp secretion from saecs was enhanced by interleukin- but there was no effect of interleukin- . mmp up-regulation by interleukin- and comtb was inhibited by the pi kinase inhibitor ly and on western analysis akt (protein kinase b) was phosphorylated at minutes. chemical inhibition of the p d isoform of pi kinase with ic abrogated the il- and comtb driven secretion of mmp- from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome ) accentuated mmp- secretion. these inhibitory effects were confirmed with sirna. mmp- up-regulation was secondary to increased gene expression with promoter activity peaking h after stimulation. in summary, interleukin- and interleukin- drive transcription dependent mmp- and mmp- secretion from airway epithelial cells and fibroblasts. interleukin- also increases timp but down-regulates mmp- gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi kinase pathway is central in interleukin- driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto , l.s. moreira , e. gonzalez-rey , m. delgado institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper response. cholesterol metabolism is regulated by factors such as pparg (proliferator activated receptor g), srb (a class b scavenger receptor), cd or abca , that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg , srb , cd , and abca . we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il- in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il- and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il- , tnf-a, il- b, il- and il- , and the anti-inflammatory cytokine il- , in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il- was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il- and il- , and in some cases, of il- . the maximal plasma levels of il- and il- were found at hours and of il- between - hours. modest plasma levels of il- were also observed, with maximal production at various time points ( - hours). by contrast, production of tnf-a, il- b and il- did not occur to a significant extent, while production of il- occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il- , il- , il- and il- also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il- , il- , il- and il- , i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il- , activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il- is a novel il- cytokine family member that is expressed as an intracellular precursor (pro-il- ) and is thought to be cleaved by caspase- to yield a mature bioactive form of the molecule (mat-il- ). to date however, evidence of cell-associated proteolytic processing and caspase- dependent secretion of mat-il- has not been reported. here we show that pro-il- but not mat-il- is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il- to the il- r and also il- r-dependent bioactivity of pro-il- on mast cells. we propose a previously unrecognized role for pro-il- as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il- p mrna expression in myeloid cells, and markedly increase secretion of il- , but not il- , by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il- promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop . chromatin immunoprecipitation (chip) assays using anti-chop and isotype control mab were performed using nuclear lysates from u cells and il- promoter dna measured by qpcr. chop binding on the il- promoter was detected following stimulation of u cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il- promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop in il- gene transcription, u cells expressing shrna's specific for chop or non-specific gene target were tested for their ability to express il- following tlr and er stress stimulation. u expressing three independent shrna targets for chop exhibited significant reductions in il- p mrna (up to % reduction of the response to lps+tp) compared to u expressing a control shrna. chop shrna expression did not affect the expression of other lpsresponsive genes, including il- , il- , ccl and sod . to identify if er stress induction of il- mediated by chop expression plays a role in a more physiological setting, we examined the role of chop in the induction of il- p gene expression following chlamydia trachomatis (ct) infection. infection of u cells with live but not g-irradiated ct induced expression of er stress response genes, including chop . u infected with live ct exhibited increased il- p mrna expression compared to u infected with nonviable bacteria. chop silencing significantly reduced the ability of live ct to induce il- p mrna, confirming the important role of chop in this response. these data suggest that er stress induction of chop could contribute significantly to the pathogenesis of diseases in which il- plays an major role, through induction of il- and il- producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd b maintained a sustained activation of p kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd b-deficient mice showed only transient p activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd and cd were elevated on gadd b-deficient thymocytes. thus, we provide evidence that gadd b and a resulting persistent activation of p constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo institut pasteur, paris, france, monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il- is recognized as an essential factor for thymopoiesis, the nature of the thymic il- niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il- promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il transcripts (il- hi cells). il- hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl , ccl , cxcl ) and cytokines (il ) that are critical for normal thymopoiesis. in the adult thymus, il- hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il- hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il- levels. conversely, the frequency of il- hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il- -expression by tecs. > together, our temporal-spatial analysis of il- -expressing cells in the thymus suggests that thymic il- levels are dynamically regulated under distinct physiological conditions. this novel il- reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il- expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl -tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl -tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl -tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg plasma cells and serum igg levels were˚ - fold increased. importantly, serum from young slc-tg;em-bcl -tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in / and / cases, respectively. these values were increased when compared with control groups: / in fcgriib -/and / in bcl -tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd + cd + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd hi cells, representing multipotent progenitors, or cd + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb and ephb expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b and/or ephrin b , the ligands of ephb and ephb receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb or ephrinb genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb /b double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd + cells in the cortex, increased proportion of k +k + cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb and ephrinb in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink , d. vanhecke university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op -dl cell culture system ( ) . using this in vitro assay we obtain large numbers of human cycd + and cd + cd + double positive thymocytes starting from umbilical cord blood (ucb) derived cd + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il- and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd + cells upon notch signaling is cd followed by cd . t cell specification is accompanied by the induction of cd a and loss of cd on cd + cd + cells. these cd -cd a + cd + cells become dependent on continuous il and notch signaling for sustained survival and further differentiation into cd + cd ab + dp thymocytes. we found that flt l is not essential for the differentiation of cd + cd + human thymocytes but that addition of exogenous flt l in the co-cultures increases the number of cd + precursors and consequently result in higher yields of developing cd + cd ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd + cd + cd ab + dp subset in this in vitro assay suggesting that op stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn to dn stage, where bdnf and its receptor p are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga , c. lópez-rodríguez pompeu fabra university, barcelona, spain nfat is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat -null mice are unable to mount cd +-and cd + -immune responses. data from our laboratory indicate that nfat -null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat during the development of t lymphocytes, we developed mouse models that delete nfat at early (lck-cre + /nfat flox/flox ) or late (cd -cre + /nfat flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p , is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues - of the proline-rich domain of p . methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p null (p -/-) and wild type (p +/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p -/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p is not important for preventing thymic t-cell tumors. s. myrczek , r. pardi , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, vita-salute san raffaele university school of medicine, milano, italy jab is the catalytic subunit of the highly conserved cop signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab regulates the activity of ap transciption factors. to date jab is thought to be essential for every cell type as jab knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab . to investigate the function of jab in b cells we established a mouse strain deficient for jab selectively in b cells. mice with floxed alleles of jab kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb -locus (m. reth, freiburg). ablation of jab expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b and b cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab -deficient, bcl -transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab when apoptosis is prevented. t. nitta , s. murata , k. tanaka , y. takahama university of tokushima, tokushima, japan, university of tokyo, tokyo, japan, rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th and th antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd + t cells or functional deficits that selectively interfere with th or germinal centre responses. in this talk i will present data from some of the first strains that have been identified including the first strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn c-kit high (etp), dn and dn thymocyte populations was hybridised to affymetrix mouse a- . genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors ( out of genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included signal transducers (out of genes) such as acvr , bmpr , fzd , chemokine receptors cx cr , cxcr and integrins a , a , a , ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata- , tcf- , notch- , rag- , rag- and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks out of of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by - days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd + cd ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged month to years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd + cd precursors with recombinant wnt a ( ng/ml) or with licl ( mm) for hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab ( e ) under conditions of phosphatase activity inhibition. wnt a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il- and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st cells suplemented with il- and flt l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il- and flt l and modify the transcription factor profiles of cd + cd thymocytes mainly increasing hes- and id expression levels. human th clones and circulating th cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th and th clones or circulating th oriented t cells, respectively. accordingly, human th cells exhibited lower expression of clusterin, and higher bcl- expression and reduced apoptosis in the presence of tgf-beta, in comparison with th cells. umbilical cord blood naï ve cd (+)cd (+) t cells, which contain the precursors of human th cells, differentiated into il- a-producing cells only in response to il- beta plus il- , even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd (+) t cells but it increased the relative proportions of cd (+) t cells differentiating into th cells in response to il- beta plus il- , whereas under the same conditions it inhibited both t-bet expression and th development. these data suggest that tgf-beta is not critical for the differentiation of human th cells, but indirectly favors their expansion because th cells are poorly susceptible to its suppressive effects. m. irla , w. reith university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd (+) or cd (+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd (+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd on mtecs by cd l induced on the positively selected cd (+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd (+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu , i. ravens , g. bernhardt hannover medical school, institute of immunology, hannover, germany cd is originally identified as human poliovirus receptor (pvr) and as rodent tage , which is overexpressed in rodent colon carcinoma. cd is also known as necl- , a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd is overexpressed in transformed cells and promotes the cell cycle. thus, cd seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd and cd ; the dn subset is further subdivided into four stages (dn - ) by differential expression of cd and cd . thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd + sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd lineage. here we show that the frequency of terminally matured cd + sp cells but not that of cd + sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd , a selective deficiency of mature cd + sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd + sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd + t cells. in cd deficient animals, a shift in the tcr repertoire displayed by the pool of cd + sp cells was found demonstrating that cd is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. , , . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht , propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd + ter + erythroid and cd + cd b + myeloid cells are simultaneously cycling (s/g /m) in the post-gastrulation mouse embryo (e - ). the peak of lymphohematopoietic cell proliferation occurs at e in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence - hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e - that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd -cd interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd , we hypothesised a role for cd -cd interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd -/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd on reconstitution of b cell numbers following depletion. we show that cd is expressed by pro-b cells, and these cells proliferate in response to cd signalling in vitro. pcr identified a source of cd , negative for cd eta, in the bm of wt mice showing this cd is not provided by activated re-circulating t cells. we have shown that when cd -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn (cd -/cd -/cd + /cd -) to the dp (cd + /cd + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn stage on. in the bone marrow we found yfp + /b + and yfp + /b populations. thus these pta expression analyses show closely similar pattern to those observed with hucd preta-reporter transgenic mice (gounari f. et al. , martin et al. . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. , - ( ) martin c. h. et al., nat. immunol. , - ( . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz , g. klein zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm- which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x kda.mmp- , a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm- . in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp- . by western blotting the zymogen and the activated form of mmp- can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm- and mmp- can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp- which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp- , timp- and timp- , are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp- plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of - % compared to only % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd /cd expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd +cd + double positive (dp) tcrab low cells entering selection, and their cd +cd + dp tcrab-immediate precedents followed by underrepresentation of the selected cd +cd + dp tcrab high and the most mature cd -cd + and, particularly, cd +cd -single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd -cd -double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd +cd -/cd -cd + sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd / cd /tcrab), in adult wistar rats subjected to -day-long treatment with a -ar blocker urapidil ( . mg/kg body weight/day s. c.). the a -ar immunoreactivity was found in both thymocytes (mainly less mature cd and cd low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed -postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd / cd /tcrab expression were observed. these changes comprised of an increase in the percentage of cd + + tcrabthymocytes, which was accompanied by the reduction in that of cd + + tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd + -tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd - + tcrab high was reduced. in addition, the percentage of cd + t regulatory and cd +tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c bl/ mice infected with m. avium ( cfus, iv) were sacrificed at different time points after infection ( , and weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il- ) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il- in this organ. in the spleen, ifn-g reaches a peak of expression earlier ( weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm and ai from nih and grant i- from the robert a. welch foundation to wtg, and by grants hl and hl from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. ) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c bl/ x balb/c)f mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski , s. lang , m. stein , t. winkler friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h ac) or methylation of h on lysine (h k me / ). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h ac and h k me / ) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar # ) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 'rlm race at the ivar # element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo locus, is thought to be accessible only during the double-negative (dn) and thymocyte stages based on mrna expression, implying that translocations between lmo and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx (hox ), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed ) to evaluate lmo and tlx breakpoint-site accessibility during thymocyte development; ) to determine in which stage of development there is an increased chance for lmo or tlx translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo and tlx loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn and dn development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn , dn , dn and immature single positive (isp) stages for both lmo and tlx . conclusion: our findings show that both the lmo and tlx loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn and dn stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that to successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e - ), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd cell depletion. however, cd depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd t cells has important implications for vaccine development against neonatal infections. ( ) showed that irf knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs . conclusions: although recent findings indicated that irf function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez , t. boehm max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd and cd t cells from tuberculosis patients highly express pd- when compared to healthy uninfected individuals. in addition, analysis of pd- expression in lung biopsies from tuberculosis patients revealed that pd- is expressed on cd and cd t cells confined to lung granulomatous lesions. finally, blocking of the pd- /pd-l axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd- /pd-l pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p to p . among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th and th cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd + t cells, nfatc and c are predominantly expressed. nfatc is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc /a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc /c. as demonstrated by y h screen and co-ips, nfatc /c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc /c -but not the unsumoylated nfatc /a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc target gene interleukin- . other lymphokines like ifng and il are reversely regulated. interestingly, ntreg cells which do not express il exerted only nfatc /c, but no nfatc /a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc function. therefore, especially ntreg cells and anergized cd + t cells might be regulated by the long sumoylatable isoform nfatc /c. lnk/sh b and aps/sh b , two members of the lnk/sh b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b- cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il- signalling in pre-b cells overexpression of lnk dramatically inhibits il- -dependent growth demostrating that lnk negatively regulates il- pathways. furthermore, we showed that il- stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav . to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh or sh domains known to mediate protein-protein interactions are key players in these processes. sly (sh domain protein expressed in lymphocytes ) was identified as a putative adaptor protein containing a sh and a sam domain as well as a bipartite nls. sly belongs to a family of three molecules: sly , sly and sash .in humans, the sly gene is located on chromosome , in mice on chromosome . sly is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin -associated polypeptide p (sap ) as a putative interaction partner of sly . sap is a conserved member of the sin a-hdac corepressor complex that contains histone deacetylase (hdac ) and histone deacetylase (hdac ) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected t cells. in addition, we could show a direct interaction between sly and hdac . to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly increases the activity of hdac in whole cell lysates and, more precisely, in nuclear extracts of t cells. the interaction of sly with sap and hdac indicates a transcriptional function of this protein. within the sin a-hdac corepressor complex sly might act as a switch for the activity of hdac . cd -cyt and cyt are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd (b cell marker) fused to the transmembrane and intracellular domain of cd -cyt or cyt in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt coengagement controlled il- secretion, while cyt coligation inhibited ifng production. moreover, our preliminary data suggest that cd -cyt inhibits the phosphorylation of several molecules known to be activated by cd stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh domain and three sh domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut cells with gst fusion proteins containing full length nck, the three sh domains or the individual sh and sh domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp and cd epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam ) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g , results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g ) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g ) degranulation as measured by cd a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose- -phosphate receptor which exhibits structural and functional similarity to the vps p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose- -phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla- (sctla- ) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla- in immune responses. using a specific anti-human soluble ctla- monoclonal antibody, jmw- b that selectively binds the soluble isoform but not membrane bound ctla- , or cleaved fragments of it, we demonstrate that sctla- plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla- , secreting increased amounts of cytokines including interferon-g, il- and tnf-a, but lower amounts of il- . soluble ctla- was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla- induced secretion of the immunoregulatory cytokine il- by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine , dioxygenase enzyme cascade was also initiated by sctla- . it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla- it is crucial to t cell inhibition. membrane-bound ctla- exists as a homo-dimer on t cells but sctla- is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b ligands on antigen presenting cells. a third important observation from this study is that sctla- exists both in serum and culture supernatants as a natural kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla- , concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il- dependent regulation is most critical, boosting sctla- secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin- /efhd- , in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin- /efhd is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin- /efhd- in the immature murine b cell line wehi enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin- /efhd- impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g cell cycle arrest. to understand how swiprosin- /efhd enhances pro-apoptotic bcr signals, we analyzed whether swiprosin- /efhd is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin- /efhd enhanced bcr-induced calcium flux in wehi cells, whereas shrna-mediated down-regulation of swiprosin- / efhd impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin- /efhd interacts with phospholipase cg (plcg ) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg and swiprosin- /efhd was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin- /efhd silenced wehi cells with swiprosin- /efhd- was inhibited by the syk inhibitor bay - . in analogy, swiprosin- /efhd regulated syk activity positively. moreover, swiprosin- /efhd re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg and of syk tyrosine residue , which is involved in syk activation. finally, reconstitution of swiprosin- /efhd knock-down cells with swiprosin- /efhd mutants revealed that the n-terminal putative sh -binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi cells. interestingly, swiprosin- /efhd re-expression in swiprosin- /efhd -silenced cells induced already in unstimulated cells raft partitioning of syk, plcg and the bcr, which was reversed after min of bcr stimulation. in summary, swiprosin- /efhd is an accelerator of proximal bcr signalling and acts through syk and plcg by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p mice die within hours after a second challenge with p peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e ligase dead mice can spontaneously reject tc tumors. conclusion: cbl-b e ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd + cd ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal ) and co-stimulatory signals (signal ), provided by beads coated with anti-cd /cd antibodies. gene expression patterns were compared for cells stimulated with anti-cd /cd beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip , itac). the enhanced expression of granzyme-b, ifn-g, trail and ip were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd -coated p cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal- to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd + foxp + and cd + cd high t cells. moreover, t cell receptor activation with combined anti-cd and anti-cd stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk- , but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv- infection leads to immune dysfunction owing to a successive loss of the cd + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv- virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk / . this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk / directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap- , nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk / is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal , t. brdicka , v. horejsi institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd are key regulators of src-family kinases in leukocytes. while cd is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh domain of csk binds phosphotyrosine of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd -csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie- kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa- integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi , s. parusso , b. frossi , g. gri , c. pucillo university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il- -/-mcs and anti-il- receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il- derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd on naï ve b cells and the interaction of cd on b cell surface and cd l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd l e cd on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves , n. bercovici , a. caignard inserm u , paris, france inhibitory killer ig-like receptors (kir dl - / ) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd + t cell subsets. these receptors suppress cd + t cell activation through recruitment of the src homology domain-containing protein tyrosine phosphatase (shp- ). to further analyse the yet largely unclear role of inhibitory kir receptors on cd +t cells, kir dl transfectants were obtained from a cd + t cell line and primary cells. the transfection of cd + t cells with kir dl dramatically increased the t cell receptor (tcr)-induced production of il- independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir dl -itim phosphorylation, shp- recruitment, zap- and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp- and p-pkc-v but not of shp- . in contrast, the kir dl /hla-cw interaction led to a strong synaptic accumulation of kir dl and the recruitment of shp- / , inhibiting tcr-induced il- production. kir dl may induce two opposite signaling outputs in cd + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir dl receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg antibody during these conditions. the observed igg switching behaviour mimics that of b cells responding to lps and il- , but is mediated by a different, stat -independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana , c. schwindling , m. pasche , c. junker , c. kummerow , u. becherer , e.c. schwarz , j. rettig , m. hoth saarland university, biophysics, homburg, germany, saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of days. under tolerogenic conditions (ova alone), cd + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, - bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd + t cell expansion and contraction kinetics in the early phase of the t cell response (days - ). in the late phase of the primary response (days - ), under immunizing conditions, the large majority of transgenic cd + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il- , ifn-g, and il- a, and only few ova-specific foxp + regulatory t cells ( x %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells ( %) was substantially increased. on day , both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells ( %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il- a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin- , interferon-g and macrophage inflammatory protein- by cd tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog - protein. our results provide direct evidence that mc contribute to cd -specific priming in eae and show that the tc proliferation failure is specific for cd tc from mog - -immunized w/w sh mice. the role of mc-cd tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla- , but not via any conventional motifs in this region. overexpression of trim augments ctla- surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla- localisation, mainly restricted to the tgn. ctla- vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla- trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla- expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla- transport to the cell surface. it is imperative to reveal the mechanisms by which ctla- is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd molecules, nkt cells are activated and release cytokines, including ifn-g, il- and il- . nkt cells are efficiently recruited to the liver via cxcr -dependent chemotaxis toward cxcl and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd t cell tolerisation via interaction with lsec. to this end we analysed cd d expression on lsec and their ability to activate nkt cells by presentation of the cd d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd d, as agalcerpresenting-lsec were capable to induce tnf-a, il- , il- and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd and b -h on lsec. as naï ve cd t cell tolerisation by lsec critically depends on b -h , we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg , jg . , and vd gene products. they recognize nonpeptide antigens like (e)- hydroxy- -methylbut- -enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd- . one of the inhibitory receptors, pd- , is a member of cd /ctla- family and contains a single ig v-like domain in its extracellular region. pd- can bind to two b homologue molecules, pd-l and pd-l . it has been reported that interaction of pd- with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd- is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with -methyl- -butenyl- -pyrophosphate plus il- to obtain gd t cells. pd-l + and pd-l -human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l mabs, the pd-l extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd- in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd- /pd-l interaction. results: gd t cells expressed pd- upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l . we first examined whether or not the engagement of pd- receptor could modulate the cytotoxic activity of gd t cells. pd-l -expressing tumor cells tempered cytotoxic activity of pd- + gd t cells, and cytokine production such as tnf-a was down-regulated by pd- engagement. in addition, inclusion of anti-pd-l mab reversed cytotoxic activity and cytokine production when pd-l -expressing tumor cells were challenged by pd- -expressing gd t cells. conclusion: pd- delivers inhibitory signals in gd t cells upon engagement with pd-l . peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il- production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova - peptide)-specific t cells from do . tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler , p. aichele immh, university freiburg, immunology, freiburg, germany interleukin (il- ) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il- has a direct influence on cd + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal ) and costimulation (signal ). we analysed direct il- signaling to cd t - signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd t cells lacking il- signaling failed to up-regulate klrg and to down-regulate cd in the context of listeria but not viral infections. thus direct il- signaling to cd t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd + t cells are tightly controlled in their effector functions by cd (ctla- ). we demonstrate that signals induced by cd reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd + t cells. for this novel function cd specifically represses the transcription factor eomes, but not t-bet. a cd mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd -mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd + t cells differentiated in the absence of cd signaling could be demonstrated in vivo. the novel insights that cd -mediated signal transduction in vivo indeed alters cd + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p receptors. p x - receptors open to non-selective ion channels, whereas p y , , , , - are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p x antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca( +)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek , a. brouckova , d. filipp institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih t cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y flck. comparative -d gel analyses followed by ms/maldi identified rack as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih t cells of ha-tagged rack with either a wild type lck or constitutively active y flck revealed a significantly enhanced complex formation between y flck and rack compared to that of wtlck. ectopic expression of y flck with its domain-inactivating mutations showed that lck-rack interaction depends on functional sh , sh and the c-terminal tail sequence of lck. lck-rack interaction is readily detectable also in primary cd + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack in the context of lck translocation to lr is further strengthen by the observation that rack is associated with elements of cytoskeleton. these results are the first to characterize rack as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin (il- ) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd +cd +cd -cd -igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th response to alumova inducing large early germinal centres and massive plasma cell formation with more than % of these switching to igg . the plasma cells up-regulate cxcr , but not cxcr , a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th -associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚ %), igg a (˚ %), igg b (˚ %) or igg (˚ %). in addition to cxcr , some % of these plasma cells strongly express cxcr . the induction of cxcr in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg a switching during th responses. this is functionally significant for oti-dependent cxcr expression, as well as induction of switching to igg a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg b. t-bet is known to be induced in b cells exposed to ifng or tlr stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd . objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd ) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd + t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd + t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla- pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa- and cd in the psmac of untransformed human t-cells. in marked contrast, tcr/cd accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl- regulates interclonal t cell competition during acute and chronic immune activation. we found p -independent noxa gene induction and mcl- downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl- , which was delayed in noxa -/cells. using ot- cells and altered peptide ligands we observed that the level of mcl- downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl- -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np ) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl- axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen (ebag ) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag , which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g -adaptin in t cells. both interactions suggested an involvement of ebag in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag -/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag -deficient ctls. these data imply a role for ebag in regulating the formation of mature ctl granules and identify ebag as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag -related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd / and il- . in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd /acd or pma/ionomycin could not significantly activate cd and cd t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca + influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena , s. carrasco , i. merida centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp -ras-erk signal intensity is critical to determine the final cell outcome. rasgrp is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. . analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. . develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd cells expressing gfp-c domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h , cd ), a cd -like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th biased responses and high production of the anti-inflammatory cytokine il- , and was essential to the development of germinal centres. however, icos can also help in the il- -dependent differentiation of inflammatory th cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p subunit of class ia pi- kinases. these can complex with one of the three kda catalytic subunits (p a, p b, and p d) expressed by leukocytes that generate pip affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi -k regulatory (p a, p b, p a) and catalytic (p a, p b, and p d) pi -kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p a g p a g g p b and p a g p d g g p b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi -kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd -induced secretion of il- and il- . during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap , a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap construct that acts as a dominant negative, or sirna knockdown of endogenous akap expression in t cells prevents the correct organization of cd z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa- localization was also analyzed to assess p-smac architecture and, interestingly, confocal d reconstruction revealed that lfa- ring was not clear in the akap -disrupted cells. moreover, akap was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap decrease the phosphorylation of molecules such as lat, plcg and pkcv. these defective activation events as reflected in a reduction of il- production. together, our results underscore a key role for akap in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin /cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca + mobilization and cell activation involving the pi k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc d c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr ) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as ) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. ) we then studied the phenotype of carabin kd (shrna expressing) a b cells after bcr stimulation. ) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t tot b cells and to follicular mature b cells. ) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. ) bcr simulation, but not lps stimulation, of carabin kd a b cells shows an acceleration of ras target erk / phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk / pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor (tlr ) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd + t cells express tlr and respond to the well characterized synthetic tlr ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr . tlr stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor (irf ) as revealed by realtime-rcr analysis of ifn b and irf , whose transcription depends on the activity of irf . combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd + t cells. this study was supported by dfg spp "innate immunity" (ka / - ). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd e cytoplasmic domain (cd e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd e cd and a fluorescent membrane dye (r ). with this assay, we show that the cd e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd e cd to lipid bicells. membrane binding by the cd e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia , y. ge , u. quitsch , p. beckhove german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd + t cell help is believed to contribute to optimal cd + memory expansion via cd l on cd + t cells binding cd on dendritic cells. however, a few reports suggest that cd l-cd engagement may mediate direct cell-cell contacts between cd + and cd + t cells. in this study, we investigated the importance of cd -cd co-operation and cd l-cd interactions for t em proliferation. methods: we isolated human cd + and cd + t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd + and cd + populations were activated in vitro using anti-cd /cd beads. proliferation was measured by [ h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd or cd l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd + or cd + t em cells, demonstrating that optimal t em expansion requires direct cd -cd interactions. surprisingly, not only cd + but also cd + t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd + t em proliferation depends on signals from cd + t em cells. activation induced the expression of cd on both populations and cd l on subsets of cd + and cd + t cells. blocking of cd l on cd + t em cells impaired significantly cd + t em proliferation, which confirms that the improved expansive potential of cd + t em cells in mixed populations depends on cd l co-stimulation by the cd t em subset. conclusions: our data demonstrate for the first time that activated cd + t em cells deliver help to the cd + t em subset via cd l-cd signalling and may play an important role for cd + t em expansion upon stimulation. the t cell surface glycoprotein cd , a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd localization upon t cell:apc conjugation. we have questioned which domains of cd mediate the localization within the is, and for this we have expressed cd mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd depends on sequences within the cytoplasmic domain, as a cd deletion mutant lacking most of the cytoplasmic tail, cd .k stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd translocation was mapped within amino acids glu and his since the cd .h stop mutant, just short of aa is still able to translocate to the is, whereas cd .e stop , that lacks important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu -his ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna- is missing, but lmp- is still expressed. using a hl derived cell line, we have shown that the cytokine il- can induce lmp- expression in vitro and can replace ebna- . we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is , or . a high affinity stat binding site is spaced by nucleotides. we found three potential stat binding sites in the lmp- promoter, which we named lrs, tr and edl . they were spaced by , and nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il- -treated or non-treated kmh -ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat binding site, or lrs-stat . a stat complex binding to the gl-epsilon promoter and lrs-stat was induced by il- . the specificity of the stat complex was shown by supershift experiments with anti-stat , but not anti-stat antibodies. when gl-epsilon or lrs-stat was used as cold competitors in a -fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat contains a functional stat binding site. oligonucleotides, corresponding to lrs in which the stat site had been mutated, could not compete for stat binding. interestingly, the unlabeled lrs-tr with nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by nucleotides (lrs-edl ), it could not compete. thus, expression the transforming protein lmp- can be induced directly by the t cell derived cytokine il- in a stat dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp- and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji (ji . : . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd + t cells primarily affect the proportion of cd + t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd , cd and cd on parameters of cd + t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd , a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd leads to an enhancement of ig and cytokine production. the current dogma postulates that these signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd l (rmcd l) can increase proliferation induced by tlr (poly ic) and tlr (lps) agonists. by contrast, we never observed any synergy between rmcd l and tlr / (pam csk ) or tlr / (pam csk ) agonists. to go further in the study of cd l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd l, named mini-cd ls, based on a c -symmetry core holding cd -binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd ls bind to immobilized human cd and compete with the binding of cd l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd l, mini-cd ls synergize tlr (lps), tlr (poly ic) and tlr / (r ) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd ls and tlr / (pam csk ), tlr / (pam csk ) and tlr (odn ) agonists. synergy between cd l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd + t cell tolerance. we have defined a phenotypic profile for cd + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd and cd , and high levels of expression of cd l and ly c. whereas, cd + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd and cd , and loss of cd l and ly c expression. ly c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly c, expressed on naïve cd + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to days after infection. results: daily injection of paraoxon induced g % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining weeks of treatment. mice exposed to paraoxon exhibited g % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with % of mice surviving the infection compared to % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th and th cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide -kinases (pi k) constitute a family of enzymes that generate -phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi k are divided into two types: class ia p /p heterodimers, which are activated by tyr kinases, and the class ib p g (p gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p g deletion affects tcr-induced t cell stimulation. mice lacking p g show a partial defect in t cell differentiation, activation and survival. p g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd +/cd + t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi kg-specific inhibitor causes a reduction in the number of cd + memory t cells that mediate renal injury. similarly, pi kg deletion in p pi k transgenic mice also reduces the numbers of cd + memory t cells. there is therefore evidence that pi kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi kg regulates this process is not well understood. we studied the specific role of p g in t cell activation. methods: we studied whether the tcr activates p g and the consequences of interfering with p g expression or function on t cell activation. results: we found that after tcr engagement, p g interacts with and forms a complex with ga q/ , lck and zap . tcr stimulation activates p g, which affects -phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p g controls rac activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p g-/-t cells. our observations clarify the activation mechanism and mode of action of p g in the control of t cell activation, confirming a crucial role for p g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a b and a , expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c bl/ j mice. they were stained with fluorescently labeled igm-, cd -or cd specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by h-thymidine incorporation upon stimulation with anti-cd and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a or b nachr subunits inhibited binding of igm-and cd -specific antibodies but facilitated that of cd -specific antibody. in contrast, antibody against a subunit prevented binding of anti-cd but not of anti-igm or anti-cd suggesting that a nachrs are located close to cd , while a b ones are close to bcr/cd . consequently, anti-cd -induced b lymphocyte proliferation was increased by mla much stronger than by a b -specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine- . in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd -mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd + t h -lymphocytes to antigen presenting cells or of cd + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim and trpc ), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about %. down-regulation of stim was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, ) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be - pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein (hsp ) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd + cytotoxic t-lymphocyte (ctl) and cd + t-helper cell (th ) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k cells as well as targetindependent cytotoxicity. cd + cells exhibited a strong increase in proliferation after stimulation with hsp , with rates of up to %. in the presence of target cells, a -fold up-regulation of granzyme b mrna was observed after stimulation of cd + t-helper cells with hsp in combination with il- , - and - . the target cell-independent secretion of granzyme b by cd + cells was greatly augmented after stimulation with hsp plus il- or il- , - and - . in this study, we have shown that hsp is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd + and cd + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd , which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk overexpression in a model system was achieved by transfection. pjnk / expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk , but not jnk activation. cd ligation on primary tonsillar b cells also resulted in jnk activation. however, bcr crosslinking did not affect the level of jnk / phosphorylation. cd -mediated jnk activation was independent from sh d a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase (hpk ) was associated with cd in primary b cells as well as in b cell lines. cd -hpk association was independent from cd tyrosine phosphorylation and sh d a expression. overexpression of hpk in a model system significantly enhanced cd mediated jnk phosphorylation. it is known that tnf family receptors such as cd , cd , rank trigger survival signals in hrs cells. we observed the expression of pjnk / in hrs cells of primary classical hl. cd could be involved in sustained jnk activation in primary hrs cells, and this may reflect the role of cd receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk is activated via cd in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk is involved in cd -mediated jnk activation. objectives: cd has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd are involved in c-cbl phosphorylation and association. methods: el thymoma cell line was stably transfected with wild-type human cd or hcd cytoplasmic tail mutants: cd .k stop (maintaining only a pseudo itim); cd .h stop (lacking the distal s and y in the carboxy-terminal region); cd . ¿ e -l stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd in combination or not with anti-human cd biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py ) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x . ). in murine thymocytes, co-crosslinking of cd with cd induces an increase in c-cbl phosphorylation compared to cd alone. analysis of the el- transfectants showed that mutants cd .k stop and cd .h stop lost the ability to costimulate cd -mediated phosphorylation of c-cbl. in contrast, cd . ¿ e -l stop mutant, was able to efficiently costimulate cd -mediated c-cbl phosphorylation, similarly to the hcd wt. our results indicate that the absence of the pseudo itam in cd does not interfere with c-cbl phosphorylation in response to cd plus cd crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd cytosplasmic tail, but rather, may indirectly associate with cd through the interaction with other sh -sh domain-containing molecules, that may be recruited to cd through its carboxy-terminal region. l. kolly , s. narayan , j. tschopp , a. so , n. busso chuv, rheumatology, lausanne, switzerland, unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase- into inflammasomes and thus plays a key role in regulating capase- -dependent il- b and il- production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd + and cd + t cells were activated in vitro through anti-cd stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd activated asc -/-t cells predominantly displayed a more th phenotype, producing more il- ( vs. pg/ml; asc +/+ vs. asc -/-t cells respectively; p= . ) and less ifn-g ( , vs. , pg/ml; asc +/+ vs. asc -/-t cells respectively; p = . ). when asc +/+ and asc -/-t cells were purified into cd + and cd + t cell fractions and activated individually using anti-cd , no inhibition in proliferation was observed amongst activated asc -/-cd + and cd + t cells. interestingly, the activated asc -/-cd + t cell fraction produced significantly more il- when compared to activated asc -/-cd + t cells and asc +/+ cd + and cd + t cells (asc -/-cd + t cells = pg/ml il- ; asc -/-cd + t cells = undetectable il- ; asc +/+ cd + t cells = pg/ml il- ; asc +/+ cd + t cells = undetectable il- ). cd + and cd + t cell mixing experiments revealed that asc -/-cd + t cells are able to inhibit the proliferative ability of asc -/-cd + t cells, asc +/+ cd + and cd + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd + t cells. collectively, these results demonstrate that the absence of asc drives cd + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge and pge suppress some t-cell functions including proliferation, activation and cytokine production. pge signals through four types of gpcrs called the ep receptors. at low concentrations, pge is believed to be necessary for t cell function, whereas at higher concentrations, pge inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd + t cells with ep receptor antagonists was found to impair cell surface expression of cd , cd , cd and ox but not cd . suppression of t cell proliferation by pge has already been widely studied. however, blocking ep receptors in cd + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd + t cells to the chemokine sdf- b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd + cd + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd + t cells. our results also suggest that considering pge -mediated camp signaling in cd + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd + human t-cells was examined. oxidation affects several ca + signalling pathways by altering the activity of ip receptors, trp channels and store operated ca + channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca + signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd knockout mouse possess enhanced mixed lymphocyte reactions and cd monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd in jurkat t cells augments the secretion of the t cell growth-factor interleukin- (il- ) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il- promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd , we identified the immunomodulatory sub-domain of cd . supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd and il (simulates t cell-dependent activation). microarray assays identified about mirnas in unstimulated b cells. of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor- (irf- ). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf- transcripts differs from that observed for irf- protein abundance after b cell stimulation. further analysis identified the irf- transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk and the dfg forschergruppe for . objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey , f. hauck , i. berberich , f. berberich-siebelt , gk -immunomodulation institute for virology and immunobiology, university of wuerzburg, würzburg, germany, university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd + t lymphocytes, the transcription factor is predominantly expressed in t helper (th ) compared to t helper (th ) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp- encoded by prdm is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp- is expressed in differentiated effector t cells where it is higher in th than th cells. the regulation of the blimp expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm promoter and activates blimp- expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp- in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp- in b cells using cre recombinase. moreover we found a new putative blimp- isoform lacking exon . currently, we analyze the expression of c/ebpb and blimp- in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd + t cells but not transitional b cells or cd + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein- a (mip- a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy revealed constitutively high background levels in cd low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas , v. courtois , k. de luca , r. sodoyer sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il- , il- or il- could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il- , il- and il- . furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a- - - ). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen- (ctla- ) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla- during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il ) in the tissue and the released eggs and first stage larvae (l ) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla- during the infection, a neutralysing antibody (a-ctla- ; f ) was administered intraperitoneally ( mg) two hours before subcutaneous infection with s. ratti il . the in vivo neutralisation of ctla- -signalling by applying a-ctla- during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th cytokines, such as il- and il- and a reduction of the proinflammatory cytokines ifn-g and il- . the investigation of the humoral response showed a remarked increase of the igg -titer in the serum during secondary infection in mice that had been treated with a-ctla- during primary infection. furthermore, the blockade of ctla- resulted in a diminished worm burden as indicated by reduced release of l in the faeces. these results suggest that the blockade of ctla- during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg during secondary infection also reflects the induction of a potent th response. objectives: cd is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd expression. cd knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd ko compared to heterozygous mice. since reduced levels of cd are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd ko mice. after one injection of apoptotic cells, cd ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd on b-cells are involved in setting this threshold. conclusion: our data suggest that cd is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd acts as a major check-point of immune responses, but the mechanism by which cd controls peripheral t cell responses is unknown. the consequences of cd signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd in th cells reduced migration towards ccl , cxcl and ccl . crosslinking of cd together with cd and cd stimulation on activated th cells increased expression of the chemokine receptors ccr and ccr , which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd expression and cd -mediated migration-enhancing signals. importantly, migration of cd positive th lymphocytes in in vivo experiments increased, as compared to cd negative counterparts, showing that indeed cd orchestrates specific migration of selected th cells to sites of inflammation and antigenic challenge in vivo. these data show that cd signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd adds to the already significant role of cd in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd signaling on the longevity of cd null t cells. using a sensitive staining method for cd , we show that human cd + cd null and cd + cd null t cells rapidly express surface cd . serological inactivation of cd using specific fab fragments or blockade of cd ligands using ctla ig in cd + cd null and cd + cd null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd crosslinking on activated cd null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd is mediated by pi 'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd act directly on activated cd null t lymphocytes and, due to its exclusive expression as a receptor for cd /cd on cd null t cells, prevention of cd mediated signaling is likely a major target mechanism taking place during therapy with ctla ig. objectives: cd is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd (ptprj, dep- ) which acts as a positive regulator of sfk in cd -/-b cells and macrophages and can compensate for cd deficiency in these cells. indeed, combined deficiency of cd and cd in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd -/mice were reported so far. however, in human t cells the role of cd may be different since naive human t cells express cd at a level comparable to b cells. using cd -/-/cd -/human t cell line (jurkat-derived js- cells) we tested the ability of cd to complement cd deficiency in t cells. we used retroviral transduction to express human cd or cd in js- cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js- cells. expression of wild type cd or cd in js- cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js- cells expressing cd when compared to control cells. finally, cd substrate trapping mutant expressed in js- cells interacted with lck in vivo suggesting that lck is a direct substrate of cd in js- cells. the results suggest a level of redundancy between cd and cd in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam . ) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr and tyr we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca + release-activated ca +) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa- (leukocyte function-associated antigen ) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa- prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al. ) we describe a non-viral vector based approach (vockerodt et al. ) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd signaling cascade was conducted. after activating this particular signaling cascade (cd ) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. ). a rat thymic epithelial cell (tec) line (r-tnc. ) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc. cell line in vitro. it was found that a number of adhesion molecules, such as cd , cd , cd , cd a, cd , cd , cd was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc. line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il- activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc. cells. we found that both the adhesion ( min and h) of activated thymoytes were partially blocked by mab to cd and cd molecules (ifn-g unstimulated and ifn-g stimulated r-tnc. cells). early adhesion was inhibited by mab to cd , abtcr, mhc class i molecule (ifn-g stimulated r-tnc. cells) and cd molecule (ifn-g unstimulated r-tnc. cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc. cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay ( h), namely mab to cd , cd , cd , cd molecule (ifn-g unstimulated and ifn-g stimulated r-tnc. cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc. cells). our results also suggest the involvement of cd a/cd dependent -cd independent pathway in adhesion and cd a/cd dependent -cd dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc. cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin- (il- ), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il- favours naï ve cd t cell differentiation into th cells to the detriment of th or th differentiation. the il- receptor (il- r) is a heterodimer composed of tccr, which confers ligand specificity, and gp , a signal transducing chain that is utilized by several other cytokines. il- has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd t cells, but the potential impact of il- on human cd t cells has not been elucidated. our goal is to investigate the impact of il- on human cd t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il- r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd than cd t cells expressing the complete surface il- r (gp +tccr). however, we detected high amounts of intracellular tccr in both, cd and cd t cells, but only polyclonal activation (anti-cd ) of cd t cells led to an actual increase of il- r surface expression. purified cd t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il- activated stat and stat signalling with rapid kinetics in both cd and cd t cells, indicating the capacity of il- to signal through these molecules. addition of il- to anti-cd activated cd t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il- on human cd t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd- /pd-l pathway is associated with production of the immunoregulatory cytokine il- , the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l and pd , as well as myelin basic protein (mbp)-stimulated il- production, pakt inhibition, and apoptosis (annexin v), in ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); had a diagnosis of stable disease (sms). results showed that: ) pd-l -expressing cd + and cd + cells are reduced in ams compared to sms individuals (p= . ); and ) pd expression is increased in cd + t cells of sms individuals and is comparable on cd + t cells of ams and sms patients. this is associated with a significant decrease in il- production by mbp-stimulated cd + and cd + cells of ams patients (p= . ). additionally, cd + anexin v+ (av+) cells were diminished and cd + pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd +av+ and cd + pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l /pd- pathway seen in ams patients result in a reduced mbp-specific il- production by cd + and cd + cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd + t. the pd /pdl pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti- - bb in cd cells. this difference could be due to down regulation of cd by activated lymphocytes and possible preferential response of cd cells to anti- - bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin (stx ) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx . rna interference technique was also used to down regulate stx expression in primary human ctls. results: we identified stx in ctls by pcr and immunocytochemistry. stx accumulates at the is after ctl/target cell contact. when stx function was blocked by overexpression of a dominant negative stx mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the years history of mouse t h and t h subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h , t h or t h phenotypes, based on their cytokine production (il- , ifng or il- ). a comparative analysis of t-bet, ifng and il- mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca +response, membrane raft expression/organization, k + -and ca + -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h g t h g t h , although the membrane cholesterol level (detected with anti-cholesterol ab, ac ) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h cells upon activation than in t h cells. t h cells produced a more sustained calcium response with higher amplitude than t h cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav . and kv . ion channels, major functional determinants of the sustained calcium influx. t h cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd -dynabeads. cd + isolated cells were facs sorted into cd + cd naïve b or cd + cd + memory b cells. dna synthesis was measured by h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd + cd naï ve b cells, of which bmp- and - were most efficient ( % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd + cd + memory b cells by - %. to induce differentiation, both naï ve and memory b cells were stimulated with cd l and il- . this increased the production of igm, iga and igg - -fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad / / in cd + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd l/il- -induced production of igs in mature human b cells. s. gutenberger , k. warnatz university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases and (erk / ). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of hd and cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk / phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk / . to increase the signal intracellular phosphatases were inhibited by h o . as markers of activation and initiation of proliferation, cd and ki expression were measured after days of in vitro stimulation. k. theil , p. aichele immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p cd t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b -recipient mice and compared their expansion with wild-type (wt) p t cells after viral infection. we could demonstrate a severe impairment in the capacity of p t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p t cells into h mice. h mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp - . therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h mice are infected with lcmv . . this is a lcmv variant that has got a point mutation in gp - and consequently cannot be recognized by the p t cells. s. frischbutter , r. baumgrass deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap which are important for expression of cytokines such as il- , ifng and il . it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl- was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl- phosphorylation but rather an inhibition of bcl- dephosphorylation. furthermore, calcineurin and bcl- co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl- /malt signaling complex and dephosphorylates bcl- and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop , j. lamoureux , c. fortin université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla- has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla- expression was higher after stimulation in t cells of elderly. there was differences between cd and cd t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh and ww domain proteins. since fasl surface expression is regulated by adam -mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh domain proteins that potentially interact with the riped fasl prd, we used a sh domain phage display library containing all sh domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conse- optimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b family members on antigen-presenting cells with cd on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound , with classical gcs regarding their suppressive effect on cd -costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd and anti-cd , and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd -costimulated human memory/effector cd + t cells by compound than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound than prednisolone. when evaluating possible mechanism for the increased activity of compound in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound which was not prevented by the partial gc receptor antagonist, ru- , in vitro. moreover, in vivo we observed less induction of il- beta and tnf-alpha by pre-treatment with compound than with prednisolone. our data indicate that the non-steroidal segra, compound , may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b mice elicits robust cd + t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il- + cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa -specific cells also express tnfa, but only about half of the ifng+ np -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd + t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd + t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova - peptide shows a close correlation with division in vivo. early after antigen encounter ( - divisions) the vast majority of cells express only tnfa. after - divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions ( - divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd + t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd + t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd + t cells, expansion plays a very important role in the regulation of cd + t cell effector function. in addition to its chemo-attractant function, sdf- a (stromal-cell derived factor- a, cxcl ) has been described to costimulate cd + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd + t cells was increased by immobilized sdf- a to a level similar to that obtained with the costimulatory molecule cd . as visualized by real time confocal microscopy, t cells entering in contact with sdf- a formed a tether and displayed an active scanning activity. since sdf- a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd mabs, it is conceivable that the sdf- a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf- a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf- a in the context of cd + t activation by antigen-presenting cells secreting sdf- a. this study should help us to better define how sdf- a modulates cd + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd and anti-cd antibodies and cape for days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il- production and lymphoproliferation in cd + t cells stimulated by anti-cd /cd . cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd t cells express a variety of molecules on their surface, such as mhc-class ii, cd , cd , cd , whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd t cells might induce t cell proliferation and differentiation from cd resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd , cd , and cd . analysis of the cytokine profile of these cells revealed the differentiation of il- -and ifn-g-double-producing cells in response to contact with th effector cells, and il- -producing cells in response to contact with th effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd /cd stimulation. whereas neutralization of ifn-g or il- during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd , cd , and cd could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to - %. conclusion: interaction of cd memory t cells with activated t cells resulted in the production of the cytokines il- , il- , and ifn-g. given the immunomodulatory capacity of il- and il- , these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir , s. thompson , j.j. murphy king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il- and il- and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp l by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp l has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl differentiation was observed to be associated with downregulation of zfp l protein. in an attempt to determine whether zfp l downregulation was directly linked to bcl differentiation, a zfp l shrna expressing lentivirus (psicor) was employed to knockdown zfp l expression. this reagent downregulated zfp l expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp l shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp l downregulation in promoting bcl plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd (fas, apo- ) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd /cd -triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il- and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk / phosphorylation, the expression of various activation markers, the il- production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb ) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb , galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd + and cd + t cells. in addition, mitogenic stimulus increase -fold the all binding to cd + t cells. previous studies in human pbmc showed that all binds to human cd + t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd + t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd -dependent activation of purified cd + t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd expression, but enhanced the anti-cd -dependent proliferation and cd expression of purified cd + t cells. analisis of calcium influx showed that all enhanced anti-cd dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd + t cells by up-regulating t cell activation mediated by anti-cd stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in ) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd receptor. it has been previously demonstrated that cd ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp- , slap- and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il- r, il- r, il- r, il- r, il- r, il- r, cd , cd , cdw ( - bb), cd (fas) and cd (fasl) on hpbls in different cultured time, i. e. d, d, d, d, d, d, d and d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. . the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. ) the expressions of membrane immune molecules before cultured. ) expressions of the membrane molecules on hpbls during culturing. % fbs rpmi group ( group), il- group, pha group... ( ) mtt assay. ( ) proliferative times and growth curves of hpbls... . during cultured in vitro, there are expression changes of the il- rs (il- ra, il- ra, il- rg, il- r, il- r, il- r, il- r), co-stimulatory molecules (cd , cd , - bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in group, il- group and pha group, but the rests are different. . our data also suggest that the hpbls cultured in cd mcab+cd mcab+il +il a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. . celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun , r. tauber zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p lck and the ras/rac signalling pathway ( ) followed by mitogen-activated protein kinases ( ) and c-jun n-terminal kinase ( ), which leads to an enhanced binding of l-selectin to soluble ligands ( ). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues ( ). here we show that the protein phosphatase a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer , e. glasmacher helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago - genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm and rck or expression of dominant-negative gw . interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd + t-cell activation and differentiation. naïve cd l hi cd lo cd + t cells were sorted and stimulated by anti-cd and anti-cd antibodies. results: firstly, we show that the activation and differentiation of cd + t cells require il- provided by activated cd + t cells at the initial priming stage after stimulation. secondly, this critical il- signal is delivered through il rbg of cd + cells and is independent of il- ra. besides promoting cell proliferation, il- stimulation increases the amount of ifng and granzyme b produced by cd + t cells. conclusion: therefore, our studies demonstrate that a full cd + t-cell response is elicited by a critical temporal function of il- released from cd + t cells, providing mechanistic insights into the regulation of cd + t cell activation and differentiation. most antigenic peptides recognized by cd t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a - is a tumor antigenic peptide presented by hla-a and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a - we setup an in vitro digestion assay using a -mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a - by tumor cells. by electroporating hla-a cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart- /melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart- /melan-a - specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart- /melan-a - ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart- expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart- /melan-a - epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart- /melan-a - ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev : - , ) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h -m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity : - , ) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide - , whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from - peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the - epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, ), we found, after an initial induction at hrs p. i., a strong inhibition of tapasin transcription at hrs p. i. furthermore, also reduction of tap and tap transcription was observed contrasting to the elevated levels of erp and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß , ß and ß , which are replaced by their ifng-inducible counterparts ß i, ß i and ß i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß i is replaced by a thymus-specific subunit ß t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß i, ß i, ß i and ß in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß ,ß and ß i (single intermediate proteasome), and the other comprises ß i, ß and ß i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent - % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about % of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a* ) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a* binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a* from tapasin, but only ttp-ha dissociated hla-a* from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl- . -a showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a* from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a* . m. basler , , c. lauer , m. groettrup , biotechnology institute thurgau, kreuzlingen, switzerland, university of constance, division of immunology, department of biology, konstanz, germany two lmp -dependent antigens have been described that relied on the 'structural presence' of lmp in the proteasome but not on the activity of lmp . here we have investigated processing of the h- d b -restricted uty - epitope of the male minor antigen uty reported to be lmp -dependent. using splenocytes from lmp -/-, lmp -/and mecl- -/mice we found that the uty - epitope requires lmp and lmp but not mecl- . curiously, a selective lmp inhibitor did not interfere with uty - presentation. objective: we investigated why the deletion but not the inhibition of lmp interferes with uty - presentation. we hypothesized that the 'structural' requirement for lmp is based on replacement of the caspase-like activity of b in the proteasome. methods: it was determined if t a mutants of lmp and/or b can rescue the uty - epitope. we used a b -selective inhibitor to determine if the inhibition of the caspase-like activity of b preserves the epitope. finally we determined by mass spectrometry if the uty - epitope embedded within a mer precursor peptide is differentially cleaved by lmp -deficient and proficient immunoproteasomes in vitro. results: we found that t a mutants of lmp and b rescue presentation of uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . also inhibition of cells with a b -selective inhibitor preserves uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] presentation. an aspartate in position of the uty - sequence wmhhnmdli is preferentially used as a cleavage site by lmp -deficient but not half as frequently by lmp -proficient immunoproteasomes. the generation of the uty - epitope relies on the replacement of the caspase-like activity of b by lmp because the b activity destroys the uty - epitope. this is the first example for the 'structural' requirement of lmp for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp and perhaps also lmp and mecl- exert their function in antigen processing. a. linnemann , a. musiol , r. lindner hannover medical school, cell biology, hannover, germany, hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf -regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf ) were overexpressed in t fibroblasts. we show that antibody-mediated oligomerization of mhc i in t fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf : endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf -regulated to a novel, arf -independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv virus and since sv binding triggers mhc i oligomerization, this novel pathway may be involved in sv uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab b, c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta -microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab b and rab c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab b or c positive vesicles. furthermore, the rab b, c positive compartment were colocalizd with a fraction of rab a at a juxtaposition of phagosomes. together these data demonstrate that rab b and rab c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd + t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf a and bglf have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf , has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens ). this represents a novel function for a virally-encoded gpcr. bilf is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf can hinder the recognition of virally-infected cells by cytotoxic cd + t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe natural ligands, including two peptides derived from semenogelin- , a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg is transcribed in thymus from both male and female individuals. finally, we detected the semg mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta , beta , beta ) and immuno-subunits (beta i, beta i, beta i). deficiency in beta i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta i-deficient cells could be restored by treatment with d t, which increases the amount of proteasomes independent of beta i via induction of mixed proteasomes containing beta i, beta i and beta . consequently, not the lack of the specific proteolytic activity of beta i or immunoproteasomes, but the reduced proteasome quantity in beta i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr , and , which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd + t cells. the presence of exogenous tlr and tlr ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james , i. bailey , t. elliott university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct colon carcinoma. this response is long lasting and mediated by both cd and cd t cells. importantly, the treg depleted ct specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a , c , bcl , renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw , which is h -d d restricted. analysis of the generation of gsw in ct revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct cells substantially increased the amount of gsw present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct and immunised balb/c mice. greater than % of mice injected with eraap kd ct were found to reject the tumour. analysis of t cell responses revealed the presence of gsw -specific t cells, however, these responses were small ( . - %). this compared to a much larger response to ct (˚ %). preliminary results indicate that the majority of the t cell responses (non-gsw -specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [ ] . the di-allelic hla-a restricted minor histocompatibility antigen ha- locus codes for the highly immunogenic ha- his and the non-immunogenic ha- arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha- arg immunogenicity was the estimated numbers of cell surface presented copies i. e. /cell for ha- his and less than / cell for ha- arg [ ] . as ha- his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha- his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha- mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha- allelic peptides, we investigated the impact of the ha- his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha- his and ha- arg peptides were performed. moreover, the crystal structures of hla-a in complex with either ha- his , ha- arg or a ha- variant with a citrulline residue at position were determined in order to obtain atomic level insights into the conformation of the hla-a /ha- peptide complexes. our results exclude a role for antigen processing in preventing ha- arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha- arg allele in the hla-a peptide binding groove [ ] . they provide a rationale for the lack of ha- arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd t cell response. on the other hand, immunosubunit expression is not essential for development of cd t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp (ib ) and mecl- (ib ) develop a variety of autoimmune responses, including a latent form of t d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd but not cd t cells. a cd t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose- -phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr and dr . analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type (hsv- ) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd + and cd + t cells in hsv- immunity have yet to be clearly elucidated. to better understand the role of hsv- -specific cd + t cells in immune control we have identified a amino acid epitope derived from glycoprotein d of hsv- . following flank infection, gd-specific cd + t cells were first detected in the draining brachial and axillary lymph nodes (ln) -days post-infection (pi), peaking at day and declining thereafter. gd-specific cd + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day pi and peaked at day . while hsv-specific t cells were first observed in the draining ln at day pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as days pi, with peak activity days pi before declining to background by day . however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd + t cells was observed up to days post-infection in the brachial ln. ex vivo analyses suggest that only cd c + cells were involved in gd antigen presentation at days , and post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd a + dcs can present the gd antigen to cd + t cells at day pi, whereas by day pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv- infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap and erap unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap /erap in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp . upon assembly of the heavy chain with b m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp . both crt and erp are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp point mutant that fails to bind to cnx or crt was just as effective as wild type erp in normalizing rates of disulfide formation. we conclude that erp does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp point mutant, class i heavy chains, crt and the tapasin-erp disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp have no effect on plc stability or class i surface expression, suggesting that erp plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies - % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb * /dqb * haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr* transgenic ab nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd + and cd + t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr* transgenic mice (cd + t cell competent) develop aip even unprovoked, similar to ab nod mice (cd + t cell deficient), while hla-dr* , hla-dq or hla-dr* /dq (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr* fails to protect from aip, likely due to defects in the induction of cd + regulatory t cells. our results identify hla-dr* as a prominent risk factor for aip on the hla-drb * /dqb * haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan , c. britten , h. overkleeft , g. van der marel , k. melief , d. filippov , f. ossendorp leiden university medical center, section tumorimmunology, leiden, netherlands, leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd and cd t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi , , x. hu , , a. kawana- tachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping -mer and -mer epitopes (rypltfgwcf (nef - ) and rypltfgw (nef - )) were found to be presented by hla-a* and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y f, y w, t c, f l, w r, f r, f y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef - (wild type) and its four mostly common immune escape mutants (y f, t c, y f&t c, f l), and also nef - (wild type). we found that there was little difference between the nef - (wild type) and nef - (y f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y or f . interestingly the central bulge region of the peptide was becoming very flexible for the nef - (t c) and nef - (y f&t c), which may affect the binding of peptide and the recognition of t cell receptor. for nef - (f l), the side chain of l was more flexible compared to the nef - (wild type). alignment of the nef - and nef - showed that the nef - became flat and the side chain of f was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef - was featured, while the peptide nef - was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef - was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd + and cd + t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a restricted cmvpp derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a - log reduction of activation efficiency. this pattern was seen for / epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to - log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b restricted cmvpp rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv -mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd + t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp , and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor- (fgf- ) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf- peptide was produced in vitro after incubation of proteasomes with a -amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf- spliced peptide by cells transfected with mutant constructs encoding fgf- proteins where the intervening segment was shortened from amino acids to , or residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b* , are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b* , appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b* and b* are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us protein. to address this hypothesis, us was stably coexpressed in b lymphoblastoid cell lines expressing hla-b* or hla-b* . in the presence of us , the surface expression of hla-b* was substantially reduced whereas hla-b* expression was relatively unaffected. although us was able to form complexes with both hla class i allotypes, only hla-b* was retained intracellularly in an immature form whereas hla-b* was transported to the cell surface. accordingly, in the presence of us , hla-b* , but not hla-b* , constitutively presented a hla-b restricted alloantigen to reporter t cells, suggesting that us binds hla-b* without interfering with peptide loading. us has been reported by others to bind the plc but surprisingly we have not detected such us -plc complexes in our system. rather, in the presence of us we identified a pool of class i molecules distinct from the plc and only present in us expressing cells, implying that us may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa , b. seliger martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il p . since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for h with the gold standard of maturation (tnfa, il b, il and pge ) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase- (lap ) had a more than -fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap and erap and of the immunoproteasome subunits lmp and lmp was enhanced between and -fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr ligand mpla in comparison to the tlr- and / ligand polyi:c and r . these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il- induced surface expression of mature mhc class ii molecules and cd . when ifn-g/il- primed pcmc were used as antigen presenting cells for cd + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type -diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b - to specific cd + t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd + t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd + tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd + t cell clones, faure, , eur j immunol ( ): - ). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd + t cells after their in vitro differentiation into dc, days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv- genome contains arfs within gag, pol and env genes encoding for a panel of hla-b* restricted epitopes. qprsdthvf (q vf/ d) is one such epitope but its parental epitope qprsnthvf (q vf/ n) has a significant higher frequency among hiv- isolates. strikingly, q vf/ d-or q vf/ n-specific ctls recognize apcs infected with hiv strains encoding for q vf/ d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad ) encoding for q vf/ n do not activate ctl responses raising the possibility that q vf/ n epitope is not presented by infected cells. we asked whether introducing mutations within q vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q vf/ n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q vf mhc-i presentation. we performed in vitro proteasomal digestions of mer peptides encompassing q vf/ d or q vf/ n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q vf/ n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q vf/ n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q vf/ d or q vf/ n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q vf/ d peptide. we cloned and sequenced hiv- genomes from the three donors. surprisingly, out of hiv proviral genomes isolated from pbmcs of q vf/ d reactive donors, we could not find any virus bearing the q vf/ d sequence. the isolated hiv sequences either encoded for q vf/ n or had a stop codon within the epitope. in contrast, viruses encoding for q vf/ d were isolated from pbmcs of the q vf/ d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv- seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch , y. wang , d.c. tscharke the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h- bxd f mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f progeny, with some peptides being almost equally immunogenic in f and inbred mice, while others elicit responses that are reduced by more than % in f mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd + t cells with a restricted vb usage in f mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr agonist, s-[ , -bispalmitoyiloxy-( r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp- ), as an adjuvant for cross-priming against cellular and soluble antigens. malp- has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd + and cd -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr / results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap ) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, and % as compared to control rma cells, were injected s. c. in the flank of c bl/ syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to days after injection. all mice injected with rma clone with a % level of eraap expression developed a tumor and died within days after injection. surprisingly, any animal injected with rma clone with a % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd + t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor ( bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor ( . bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb * -restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with -mer synthetic fviii peptides to stain epitope-specific cd + cells.cd + t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a domain: fviii - , fviii - and fviii - , as well as the c domain peptide fviii - , but not any c domain peptides. the c domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost. : - , ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni , s. albini , m.z. limongi , l. cifaldi , d. fruci ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap and erap , we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap , erap and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap and erap . this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p nf-kb subunit, we demonstrated that nf-kb is involved in erap and erap expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap and erap and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap , erap and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch , s. tenzer , h. schild , e. von stebut-borschitz uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c bl/ mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th immunity, whereas ifng secretion of both cd + th and cd + tc cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il- -dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm (lmp -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c bl/ mice. in addition, ex vivo co-cultures with infected dc from either lmp -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc cell ifng secretion and the dc restimulatory capacity of cd + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd + t cells, isolated from low dose infected c bl/ wildtype or lmp -/mice, were not detected. in summary, our data indicate that despite the fact that cd responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd + t cells against l. major is independent of the lmp subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a , generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd + t cell epitope ( - ) from the g domain of aggrecan , times more efficiently than non-specific b cells and over times more efficiently than the macrophage line j . however, despite this highly efficient aggrecan capture, processing and presentation of the - epitope took at least hours, comparable to the time required for presentation of aggrecan by j . treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the - epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions , and at the peptide binding groove. position also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b* , peptide loading is relatively independent of this chaperone. we analyzed the effect of b subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b subtypes. the association of b heavy chain with tapasin was analyzed in c r cells transfected with hla-b subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta- , which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me monoclonal antibody that recognizes b properly folded b /peptide complexes. the formation of fully assembled b molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc , which recognizes mhc class i free heavy chains (hc), or with me . maturation was analyzed by pulse-chase analysis, immunoprecipitation with me and treatment with endoglycosidase h (endo h). hla-b polymorphic positions other than , both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c bl/ mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately % of vacv global presentation in the context of h- b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd + and cd -dc, which differ in their mhci antigen presentation capacities. cd + dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd -dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd + and cd -dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd + and cd -dc was determined. surprisingly, cd + dc exhibit rapid surface mhci turnover compared to cd -dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd + and cd -dc were stabilized and no longer underwent rapid turnover. this suggests that cd + and cd -dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd + and cd -dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h- kb loaded with the ova-derived peptide, siinfekl. cd + and cd -dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd + dc, but not the cd -dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd +, and not cd -dc. this data further validates the role for cd + dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp- cytosol using gelatinase b/mmp- as a model enzyme. in the first dimension, ion exchange chromatography separated the thp- proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp- . in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp- on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp- cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp- -cleaved or intact thp- cytosol. proliferation was assessed by measuring incorporated hthymidine. results: - mmp- candidate substrates were isolated, of which were identified, revealing many novel mmp- (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about / of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp- decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e - k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue in hla-a and residue in e - k are highly critical for association of both proteins. this defines a putative interaction surface between e - k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e - k and the class i antigen presentation pathway. moreover, because soluble e - k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap and tap . tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap and tap were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap on tap was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap mutants suggested that the lack of tap protein expression is associated with a strong reduction of tap protein levels, which could be restored by tap gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap on tap different shrna plasmids specifically targeting tap or tap , respectively, were stably transfected into constitutively tap and tap expressing hacat keratinocytes and colo melanoma cells. in both cell types the shrna-mediated tap and tap inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap caused not only an almost complete loss of tap , but also a strong decrease of tap protein expression. in contrast, the tap knock down exhibited no influence on the tap expression. specific inhibition of the proteasome prevented the degradation of tap in the tap knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap on tap protein expression is not restricted to rare tap mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of healthy volunteers was exposed twice to . minimal erythema dose of uv. blister roofs were then collected before and , and days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd + cd + and of a macrophagic cd -cd + cell subsets that emerge and days post exposition, respectively. more importantly whereas classical cd a hi cd + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd a low cd and cd a low cd + that emerged and days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd and hladr. finally, days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey , e. reeves , t. elliott , e. james university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd + cd + regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct , stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct -derived cross-protective antigen, gsw , which was found to be encoded within the ectotropic murine leukaemia virus (emv- ) envelope protein, gp . this protein has previously been shown to encode ct -specific cd and cd antigens, implicating it as a 'hot-spot' for ct tumour antigens. interestingly, we have identified a truncated version of gp which may be responsible for generation of gsw . expression studies have revealed increased gp expression in ct compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw ) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p , p and p tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp - , bound to hla-dp with high affinity ( - nm). binding studies of mbp - derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f a, f a and k a) were affected, and only by a - fold reduction in affinity for hla-dp . the observation that none of the substitutions resulted in a complete loss of binding to hla-dp indicates that ) hla-dp binding to peptides does not depend on a large hydrophobic residue accomodated in p , or ) mbp - can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp molecule binds the immunodominant epitope in ms, mbp - , possibly in more than one register. additional studies are required to resolve the hla-dp peptide binding properties, and to determine whether expression of hla-dp affects the disease course in ms patients. results: depletion of mncs for cd + cells abrogated the tg-induced cytokine production and proliferation of cd + t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd + t cell proliferation was significantly reduced in cd /cd -depleted mnc cultures, as compared to cultures depleted for cd + monocytes alone. the same applied to the production of il- , il- and tnf-a. production of ifn-g and il- was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa , , p. pala , g. miiro , j. todd , p. kaleebu , , n. imami , f. gotch , mrc uganda, basic science, entebbe, uganda, imperial college, london, united kingdom, mrc uganda, entebbe, uganda background: hiv- -specific t-cell responses are preserved in hiv- infected individuals with non-progressing hiv- disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv- for g years, maintaining cd + t-cell counts g , and with minimal cd + decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: art naï ve hiv- infected patients from the entebbe cohort in uganda were recruited and stratified by cd + t-cell count, cd + decline slopes, and time of enrolment, into groups - ltnp and rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd t-cell count, and ifn-g, il- and il- elispot responses to pools of to peptides ( -mers overlapping by aa). peptides were based on consensus sequences of gag and nef from hiv- clades a , a and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il- responses were significantly higher in the ltnp than in rp (p= . , ifn-g responses to gaga pool ; p= . , il- responses to gaga pool ). il- responses were low and not significantly different between ltnp and rp. there was a positive correlation between il- responses to gaga pool and cd t-cell counts (r = . , p= . ), but no correlation between either il- or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv- specific responses were higher in ltnps than in rps confirming previous results. non-specific il- responses were high possibly reflecting baseline th responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her /neuand her /neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using -aad for dna analysis and specific antibodies directed against the proliferation marker ki- , the m-phase specific phistone h as well as for the h- l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her /neuversus her /neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g /g -, s-and g /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g /g -phase, which continuously raised and peaked within the s-phase. however, h- l d surface antigen expression was not altered in her /neucells during cell cycle progression. in contrast to her /neufibroblasts the her /neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h- l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd ) and ccr were cultured from cd + stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd + derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il- -dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mØ). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mØ than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mØ have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, ) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, , ) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x , ), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd +, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for of them. for of these patients hla-drb data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p= . ). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb- a preparations, whereas no differences were shown for ifnb- b. an association between hla-drb * and nab-negativity was detected (p= . ). the known associations between hla-drb * and csf-positive ms and hla-drb * and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb- a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb- b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart- and gp more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni , s. lorenzi , f. mattei , f. spadaro , l. gabriele istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg thymoma line, we show that type i ifn promote the ability of cd a + dc to capture apoptotic eg cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd a + dc and the persistence of apoptotic eg -cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg -derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd a + dc. as a result, eg -loaded dc become competent at inducing ot-i cd t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. ( ). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd + dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam csk , and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd + dc but not cd -dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm , which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a human lung cancer cell line. methods: dm induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a cells. we examined the morphological change using optical microscope. and gfp-lc punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm was showed high cytotoxicity on various cancer cell line, especially a cells. dm treated a cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc and beclin- protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc was increased concentration and time-dependently. beclin- also was increased. and then, we examined gfp-lc punctation on a / gfp-lc stable cells using confocal. a cells were formed gfp punctuation after dm treatment. to confirm these data, we measured cell death ratio using -methyladenine ( -ma), an autophagocytosis inhibitor. after -ma treatment, dm induced cell death was decreased comparing with dm treatment. conclusion: dm did not induce apoptotic cell death. but dm showed several characteristics of autophagic cell death. taken together, dm induced autophagocytosis on a cells. these finding indicated that therapeutic potential of dm by triggering autophagic cell death. s. b. rasmussen , s. r. paludan university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr) , which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv- infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv- , we found that tlr dependent ifn regulatory factor activation was abrogated. in addition, inhibition by -methyladenine of phosphoinositol -kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr dependent recognition. in the ongoing project we are examining how hsv- triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko , i. berberich , gk -immunomodulation universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl- family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl- family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a and its human homologue bfl- are anti-apoptotic members of the bcl- family. a is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a . unless the c-terminal ends of other bcl- proteins the tail of a does not function as a strong membrane anchor. nevertheless, the last amino acids of a are important for the protein. in fact, the c-terminus of a serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl- factor bimel results in increased stability of a . this is due to reduced ubiquitination of a after binding of bimel. we conclude that the cterminus of a /bfl- serves as a docking site for e ubiquitin ligase(s) that control the stability of a by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e ubiquitin ligase that interacted with a in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs- in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs- is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs- significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs- was manifest not only by the suppression of jaks acting upstream of p mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak / activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs- overexpressing cells. the results strongly suggest that socs- acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl /sdf- a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u- , and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p and fas-l expression was carried out by western-blot. results: cxcl induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p -ser levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after h of tnf-a treatment. conclusions: cxcl induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p function/levels. s. y. demiroglu , r. dressel universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp does not protect against cell death mediated by ctl. acute overexpression of hsp can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res : ) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp on granzyme b-induced apoptosis. methods: hsp was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type . results: hsp did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase- were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g peak measurements (p= . ). in contrast, a partial protection of ge-tet cells was observed after acute hsp overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood : ) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk and dr / - . the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr and tnfr , both belonging to the tnf receptor superfamily. tnfr -mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr -associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr , resulting in depletion of traf from the cytoplasm. here we confirm that tnfr induced depletion of cytosolic traf by the use of tnfr -and tnfr -specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf in the alternative nfkb pathway, we show that tnfr induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr and membrane tnf. j. c. morales , d. ruiz-magaña , d. carranza , c. ruiz-ruiz universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase- . in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp l is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp l may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl- protein is an important cell survival protein at different stages of b cell development. bcl- mrna also contains are regions that could possibly be targeted by zfp l protein. in the present study, we have tested the ability of zfp l protein to bind to a bcl- mrna are probe and to degrade bcl- mrna. recombinant bacterially expressed zfp l protein was shown to bind specifically to a bcl- are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp l also provided evidence that endogenous zfp l in b cells could bind to the bcl- are. in order to examine whether zfp l binding to bcl- are resulted in bcl- mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp l knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl- mrna was expressed in both wild-type and knockout mefs. the half-life of bcl- mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp l does play a role in degradation of bcl- mrna. overall, our data are consistent with a role for zfp l in degradation of bcl- mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp . since lmp is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi -kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te ) and lymphocytic (nc ) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes or tes (transforming effectors site) derived from the c-terminal intracellular part of lmp , in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp 's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase- . using this nc tumorigenic model in scid mice, we showed that induction of the dn lmp -tes prevent development of tumours and mouse death. these dominant negative derived from lmp could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs and socs differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p mapk activities, while socs promoted apoptosis with an increase in p activities. in contrast, both socs and socs display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs or socs induced a slight decrease in g or s phase cells and a prominent increase in g /m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g /s transition and g /m arrest induced by socs or socs . socs promoted dna damage-induced p induction and g /m cyclin b expression, while socs induced decrease in g cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein (hsp ) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp is released during necrotic cell death and proinflammatory effects of extracellular hsp have been observed. thus, we asked whether apoptozing cells cleave hsp during apoptosis or how hsp is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp protein content. we observed that hsp is degraded during apoptosis resulting in the formation of a fragment of about kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp (hsp alpha and hsp beta) we could show that the kda fragment is formed after degradation of the alpha isoform of hsp . further, we were able to show, that hsp cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp was inhibited or if exogenous hsp was added. these results demonstrate that cleavage of hsp represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc -i is processed to lc -ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf- gfp-lc cells were therefore incubated in starvation medium for hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl- /beclin- control of autophagy. recently, jnk was shown to be necessary for beclin- upregulation, and jnk-mediated phosphorylation of bcl- is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf- cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol -angelate (pep ), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb turned their response to pep from an increased to decreased rate of apoptosis. furthermore, our data show that pep inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl- and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p , trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch and analysed by flow cytometry. expression of the caspase inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases and . diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase- abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor (tnf-r ), and tumor necrosis factor (tnf)-a receptor (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid -o-octadecyl- -o-methyl-rac-glycero- -phosphocholine (et- -och ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd independent of its ligand fasl/cd l. fas/cd is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin- , an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl -family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment ( -fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo , sw , lovo, caco- , ht- ) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki- , pcna, p , bcl- ) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl- , bax, caspase , , , nfkb, parp / kda, tnfr /cd a and cd /fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl- , bax, nfkb, parp was significantly lower and expression of caspases, tnfr as well as percentage of cd cells significantly higher as compared with control group. caspase expression was significantly higher as compared with nfkb, parp and tnfr . in comparison to the standard nutrition preoperative immunonutrition increased bcl- and nfkb expressions and decreased caspases and parp expressions. in addition, we found a significant down-regulation of bcl- expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega- fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x . ). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x . ). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with healthy volunteers receiving either la ( cfu/day) or placebo, during days prior to uv ( × . med). blister roofs, liquid and skin biopsies were collected , and days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p ). while a similar decrease of lc for both groups was observed on day after uv exposure compared to placebo, la- group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd a low cd -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il- and a tendency to inhibit il- was observed in la- group compared to placebo. on day , la- group presented a greater recruitment of early lc precursors and a trend to increase cd a low cd + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il- , tnfa, il- , and il- ) was observed in la group compared to placebo. we show that la limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la supplementation for photoprotection at a lower dose ( log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn appears to be expressed in all epithelial cells of the early thymic rudiment starting around e . . previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn in a nude (foxn -deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn . if true, it should be possible to target this cell type by use of foxn -promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either or glutamine residues under the control of foxn promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei , , y. toriumi , k. kimura european university viadrina, frankfurt (oder), germany, shimane institute of health science, izumo, japan, shimane university faculty of medicine, department of pediatrics, izumo, japan, japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a -minute-rest period, followed by a -minute yoga exercise called asana, a -minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a -minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp ). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r= . , p x . ), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r= . , p x . ) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd (r= . , p= . ). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd , within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd + cd + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox l expressed on mcs and the constitutive expression of ox (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca + responses and degranulation. the cross-talk between t reg cells and mcs through ox -ox l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th or th effector cells and the induction of antigen specific foxp + regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th and treg subset, gata and foxp respectively, counter regulate each other (mantel y et al., ) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp + tregs and high gata + th cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost million allergic patients are sensitized to the major birch pollen allergen bet v , which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g %) of patients' ige binding to bet v was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa - (p ) and aa - (p ) of bet v . cross-reactive ige epitopes between bet v and related pollen allergens and plant food allergens involved primarily p . p and p are not adjacent peptides in the bet v sequence but define a surface-exposed patch on the three-dimensional structure of bet v . as determined by surface plasmon resonance, monoclonal antibody mab specific for p and mab specific for p showed high affinity, i. e., dissociation constants, k(d) = . e- m and k(d) = . e- m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p and anti-p antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th cells as well as tc cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th -mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th /tc cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd + and cd + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd (gk . ) or anti-cd ( . . ) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr- monoclonal antibody rb - c or monoclonal antibody nimp-r . one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd + t cells, cd + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd + or cd + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th and tc cells operative in the elicitation of airway inflammation. conclusions: robust type immune responses, although highly effective in the counter-regulation of local th -mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h . the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p , we were able to show that the major allergen of phleum pratense, phl p , although sharing molecular similarities with der p , does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p . chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il- and il- from nci-h cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p . in contrast to hdm extract grass pollen allergens induce the release of mcp- from respiratory epithelial cells, as well as moderate levels of il- . detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr ) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): . ± . ) and of il- (mean±sd: ± pg/ml), and reduced il- (mean±sd of il- -spot forming units/ x cells (sfu): ± ), compared to healthy subjects ( . ± . si, p x . ; ± pg/ml, p x . ; ± sfu, for proliferation, il- and il- , respectively); children with non-ige mediated cma had a significant reduction of il- ( ± pg/ml), compared to ige patients (p x . ) and an increased, although not statistically significant, production of ifn-g ( . ± . sfu) compared to control ( . ± . sfu) and to ige-allergic patients ( . ± . sfu). finally, tolerant patients showed reduced il- ( ± pg/ ml, p x . ) and proliferation ( . ± . si), compared to acute ige-cma children. interestingly, the high level of il- observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il- was undetectable in all patients even blocking the il -receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th -like response with il- and proliferation is dominant in ige-mediated cma patients; by contrast a th -skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang , b. chua , f.c. lew , a. ho , k. wong , k.l. wong , d. m. kemeny national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd th t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd t cells inhibits the induction of the th response. here we have investigated the effect of cd t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd + t cells ( . %± . % to . %± . %). when ifn-gamma -/-ot-i cd t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced ( . %± . %), suggesting an important role for cd t cell ifn-gamma. cd c + cd + cd blung dcs from cd transferred mice secreted higher levels ( pg/ml) of il- p following ex-vivo stimulation as compared with animals that were not given cd t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd t cells can subsequently divert the local lung environment to one that favors th immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in % of patients with hiv infection. this reaction is strongly associated with hla-b* and appears to be mediated by cd + t cells producing inflammatory cytokines. we show that cd +t cell responses can be primed in vitro, in normal blood donors who are hla-b* +, but not in non-b* + donors. cd t cells, but not cd t cells, are expanded by abacavir pulsed autologous apc over a -day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b* . similar responses were detected in abacavirhypersensitive hlab* + patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b* since they were undetectable using apc expressing closely related hla-b or b allotypes. responses to apc expressing mutants of hla-b* demonstrated a crucial role for residue . isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b* triggering cd t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b* may be relevant to the role of other disease-associated class i allotypes such as hla-b and b . a. jenckel , s. bulfone-paus , n. föger research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin a (coro a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro a during mast cell activation. cell fractionation experiments demonstrated that the association of coro a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro a phosphorylation. a functional correlation between coro a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro a. thus, coro a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin a (coro a) deficient mice have demonstrated that coro a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin b (coro b) is a closely related homolog of coro a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro b deficient mice and crossed them with coro a deficient mice to obtain coro a/coro b double deficient mice. analysis of t lymphocytes from coro a/coro b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro a/coro b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro a/ b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro a/ b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn (also known as hacs or sly ) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology (sh ) domain and a predicted bipartite nuclear localization signal. the samsn gene is located on mouse chromosome and encodes a well conserved protein with amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn in all tested hematopoietic cell types. the highest expression level of samsn mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd + and cd + t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly (hacs ) and sly (sash ) -were expressed only at a very low level in mast cells. the high level of samsn mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn deficient mast cells. in additional studies we are now investigating the effects of samsn deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., ). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st siai-v; st galnac i-iv, st gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg abs. results: we observed that the expression of st gal i, iii and v coding genes was similar in both hybridomas, while the st gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg . in addiction, the expression levels of st sia and st galnac genes in the hybridoma producer of anaphylactic igg were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg . conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il- and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il- and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf mediates the differentiation into th cells by activating multiple genes which are independently crucial for the development of naive t cells into th cells. because irf is expressed in many different tissues, it can be considered as a master switch factor for th cell differentiation. methods: here, we tested mice deficient in irf in the murine acute asthma model to evaluate its importance in this th cell-mediated disease. the protocol setup was the following: sensitizations s. c. with ova, followed by challenges via ova inhalation and adoptively transferred wildtype cd + t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf deficient mice with the help of adoptively transferred cd + t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by . fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. ) were also significantly higher in irf deficient mice. conclusion: interferon regulatory factor plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd + t cells. mechanistically, a potential counterregulation of asthma by th cells is not available in irf knockout mice. together with our previous report that irf represents a susceptibility gene for allergy in the human, our data highlight irf as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c bl/ mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk -receptor and ppt mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd c expressing apc substets characterized by cd and cd expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th- cytokine profile and increased levels of il- mrna. further the number of cd + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: ) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase , ) asthma and ee are prototypic th diseases in which the cytokines il- and il- play a principal role through the activation of the transcription factor stat , and ) we have recently demonstrated that some chloromethyl ketones can downregulate stat by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat , we analyzed its effect in the activation of stat by il- and il- . we found that treatment of cells with ppis inhibited the ability of il- and il- to signal stat activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd and cd high (treg)), that might inhibit the development of a th response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd or cd at hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h r. the level of intracellular ccl production in human lc was reduced after stimulation with h r agonists and basal production could be restored when h r was blocked with the specific antagonist jnj . moreover histamine and a h r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h r in allergic skin diseases and encourage further exploration of the h r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g p , an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g p was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g p which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th to chronic th -predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received weekly intravenous infusions of rituximab at a dose mg/m body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g , ; g , ; g , mg/dl, respectively). all patients underwent prior treatment with omalizumab for months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the nd week after initiation of rituximab therapy up to year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up year), ige levels decreased from , to , mg/dl. the other two patients are in the and -months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) and th cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either %dncb, %tma or to vehicle alone for . - h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il- (mean= pg/ml, n= , p x . ), il- (mean= pg/ml, n= , p x . ) and il- a ( pg/ml, n= , p x . ) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of ng/ear of murine recombinant il- or of the known regulator of lc migration; il- b. interleukin- b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after ( %) and h ( %) (n= , p x . ). in contrast, il- or control injections were without effect. however, il- administration caused an increase in cutaneous il- production ( pg/ml, n= , p x . ) compared with control injection and naï ve tissue ( and pg/ml, n= , ns). in addition, systemic treatment with anti-il- antibody failed to impact on lc migration provoked by dncb ( % reduction; n= , p x . ). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il- a ( pg/ml, n= , p x . ) and il- ( pg/ml, n= , p x . ) expression was reduced to baseline levels by anti-il- treatment. these data demonstrate that il- is not involved in the regulation of lc migration, unlike il- b and tnf-a. however, il- is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il- may influence lc and dermal dc maturation, via the expression of il- a and il- . allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey ), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p -mapk-pathway related protein (p prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct- - , www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il- receptor antagonist (il- ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il- b and of il- -like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il- -related genes by real-time pcr on mrna from blood cd + monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il- , il- bp, and caspase- , both before and after therapy with kineret. il- b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il- b, tnfa, il- ) nor anti-inflammatory cytokines (il- , tgfb) were detected. as expected, il- ra was only detectable after therapy. il- was detectable in schnitzler's sera at higher levels than in controls ( . vs. . pm) and decreased after therapy ( . pm). the circulating il- inhibitor il- bp was lower than in controls and not affected by therapy. thus, free il- levels were increased in schnitzler's patients as compared to controls ( . pm vs. . pm in controls) and decreased after therapy ( . pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il- -related cytokines (il- b, il- ), nor of the il- /il- -converting enzyme caspase- in blood monocytes. however, the high circulating levels of il- suggest an increased activity of caspase- , as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr signal via myd to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c bl/ myd -deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd -/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg -titers in ova-treated myd -/-mice compared to wildtype mice, although the nacl-treated myd -/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over , species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies ( mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = ) or from non-infested contra-lateral sites. expression of mip- a, igf- , mcp- and ip- genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd , cd , cd , cd , mhc class ii, and p than that of holsteins. lymphocytes expressing wc and cd antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x . ). infested skin of nelores contained significantly more message for mip- a, igf- , mcp- and ip- than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd + t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il- plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa- in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam- /lfa- binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th than a th local response pattern by virtue of il- secretion and icam- /lfa- interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th like micromilieu in acute towards a th dominated milieu in chronic lesions. the genotyping of ccl l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi / ). psoriasis is an inflammatory dermatosis with % prevalence among caucasians. hla-cw allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in - and - positions. strong association was found between polymorphisms in the - region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b , and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to years of age) were selected. hla-a -b -c -dr -dq alleles and tnf- and - snp were differentiated by pcr/ssp. analyzing the total group, patients ( . %) were male, ( . %) were female. in the male group, the mean age at disease onset was , years. severe forms were seen in this group (psoriatic arthritis in cases and erythroderma in ). seven patients ( , %) had a favorable evolution of the disease, but ( , %) developed extensive psoriasis, covering over % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw* allele was positive in cases ( , %), tnf g/a genotype was found in ( , %) and tnf g/a in ( , %). in the female group, the mean age at disease onset was , years, one case of psoriatic arthritis and one of erythroderma. twenty-nine ( , %) had a favorable evolution of the disease and ( , %) an unfavorable evolution. cw* allele was positive in cases ( , %), tnf g/a genotype was presented in ( , %) and tnf g/a in ( , %). severe disease was seen in male patients. there was no difference in frequency of cw* allele between male and female groups, but there was a tendency of significant difference in tnf g/a genotype. we found that c bl/ mice were more susceptible than balb/c and dba/ mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il- and mrp / , among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: cases, which were studied retrospectively, included children ( boys and girls), aged - years, who had an anaphylactic reaction, out of the that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food ( %-particularly sea food and dried fruit), drugs ( %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites ( %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema ( %), and gastrointestinal symptoms ( %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g was found in out of the severest cases ( , %), in which the adequate control was held, while in their vast majority ( out of ) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in , % ( cases) there was a hereditary family history of atopy, while in children ( %) there was also a personal history of asthma. finally, at a great percentage ( %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that )the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. ) in particular, in many cases it is proved that there is a personal but also a family history of atopy. ) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd we could observe dose dependent upregulation of programmed death ligand (pdl- ) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since years. in march a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters ( mg) every second day. in april the patient presented himself in the consult with multiple livid papules with a diameter of mm in the area of the auricle. the histological examination showed an hhv- positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs , socs and foxp in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n: ), the patients who showed recurrence or progression after treatment (non-responders, n: ) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n: ) were evaluated for socs , socs ve foxp mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd , cd , cd , foxp , cd + cd high , cd + foxp + . expression of socs and foxp- mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd , foxp , cd +cd high , cd + foxp + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs , socs and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs and socs molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of silicosis patients, with mild ( ), moderate ( ) and severe ( ) silicosis, coal workers, exposed to inorganic dust containing fcs (exposed), and healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than . was considered significant.neopterin level was significantly elevated in exposed ( , ± , ng/ml) compared to controls ( , ± , ng/ml; p= , ). moreover, the neopterin level in exposed was similar to silicosis patients ( , ± , ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls ( , ± , au vs , ± , au p= , ) and to silicosis patients ( , ± , au p= , ). in contrast, igmcic was significantly elevated in silicosis than in exposed ( , ± , au vs , ± , au; p= , ).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p= , and , respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a* , b* , drb * (these of good prognosis) * /* /* /* ) or tbc (drb * /* /* /* and associations with dqa * /* /* ) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a* , b* and drb * (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a* , drb * . as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd + , cd ++ , foxp + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein (hhsp ). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il- , il- , il- , tgf-b and ifn-g production prevailed, pointing to a th /th weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp . importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th /th cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices ( ) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c -complement, myeloid related protein and two new proteins, integrin-ß and fibrinogen. data from more than patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than % and %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least mice per group with distinct cytokine pattern: th (c bl/ , c bl/ ) and th (balb/c). muscular injury was performed by injection of bupivacaine. both th -dominant strains presented more areas with regenerating myofibers and macrophages at dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at dpi. balb/c mice showed increased collagen expression and decrease of mmp- activity associated with more mrna for tgf-b . this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th cytokines) or myonecrosis and collagen deposition (th cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß -adrenergic receptor (anti-ß ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß ec ii -specific rat monoclonal antibody (clone f ), and showed by elisa that it binds to the linearized ß ec ii peptide. additionally, we confirmed with flow cytometry that f also binds the ß ec ii in its native conformation, i. e. directly labeled circular ß ec ii (dyl -ß ec ii ) peptide. moreover, we demonstrated activation of the ß -adrenoreceptor by f using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß ec ii -specific peptide-based therapy, by intravenously applying a circular ß ec ii peptide in the dcm lewis rat model to neutralize the anti-ß ec ii antibodies. while the peptide therapy strongly reduced the anti-ß ec ii titers in the serum by up to % and consecutively lead to clinical remission, elispot assays for the detection of ß ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca - epitope elicits autoreactive cd + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca - peptide. in a first step, hybridoma cells were generated by fusing bw tcra -cd lymphoma cells with myhca - -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca - -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle , a. schwarting , p.r. galle university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase (pr ), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr -positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il- . pr -mrna from the murine cancer cell line wehi- was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr -promoter in the second intron of the mouse pr gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr transcript and its promoter indicates that the murine pr may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n= ) or pbs (n= ) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr activation, in the presence/ absence of och. subsequently we transferred . x mdcs (n= ) or och-primed mdcs (n= ) ( times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a . % reduction in plaque size compared to mice treated with mdcs (p x . ). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant . % (p x . ) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a to -fold increase in these cells was detected days after the last treatment with och-primed dcs (p x . ). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il- . discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c bl/ background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c bl/ and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg c were isolated only from fcgriib deficient mice, but not from c bl/ control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box protein (hmgb ), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb . we investigated if hmgb -containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb remains associated with ncs released from late apoptotic cells in vitro. hmgb -ncs complexes were detected also in the blood of patients with sle. hmgb containing ncs from apoptotic cells induced secretion of il-b, il- , il- , and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd and toll-like receptor . neither hmgb -free ncs from living cells nor from apoptotic hmgb -or hmgb / -deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il- release by macrophages. immunizations with hmgb -containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr -dependent manner. in conclusion, hmgb in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher , k.p. hoefig , e. kremmer , v. heissmeyer stretches and helps in the selection of the correct splicing borders. a allele of (r h) creates a strong binding site for a splicing enhancer protein srp according to bioinformatics. our findings indicate that, the putative branch point, r h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank . finally, we believe that bank delta protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta protein may contribute to the observed reduction in sle susceptibility. s. beermann , r. seifert , d. neumann hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h receptor (h r), h r, h r, and h r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or -methylhistamine, a h r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and -methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c bl/ mice induced by either a-cd antibodies or cpg-odn. this histamine effect was completely inhibited by the h r-specific antagonist famotidine, while h r-, h r-, and h /h r-selective antagonists had no or only moderate effects. interestingly, the h r-selective reagent jnj , which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h r, jnj may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h r and, to a much lesser extend, the h r. using this assay system, cells obtained from control c bl/ mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega- fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of ng/ml resolvin e (rve ) on the release of tgfb, il- and il- in the culture of peripheral mononuclear cells ( x /ml) stimulated by phorbol ester (pma) ( nm), and the combination of pma and ionomycine ( mg/ml) for hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from healthy subjects and from patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = . ) and sjögren's (p= . ) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p= . ) or pma+ionomycin (p= . ), however rve was ineffective at reducing tgfb release in the sle and ss patients. rve caused a non-significant decrease in il- release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il- was not significantly modified by rve in any of the groups tested. the release of tgfb by ng/ml of rve can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve tested, il- and il- release were not significantly affected in healthy or autoimmune patients. omega- fatty acid derived rve may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis , a.k. tsirogianni , m. herrmann , h. m. moutsopoulos , m.n. manoussakis university of athens, dpt pathophysiology, athens, greece, university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included with primary ss (american-european criteria ) and with sle (acr criteria ). age-and sex-matched healthy blood donors to the ss and sle groups ( donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, ) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma ( patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd -staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p= . ). in ss patients, defective mb-phagocytosis involved only monocytes (p x . ) and significantly correlated with the presence of extraglandular manifestations (p= . ). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x . ) and of ss (p= . ). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: healthy persons have been included in our study and sle patients ( women and men) with various clinical signs ( persons had st degree of disease activity, persons - nd degree of pathological process activity). women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in , %, aphl of igg class -in , % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x = , ; p x , ). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x = , ; p x , ). the increased levels of anti-ada were revealed in of women with hnp, and the combination of anti-ada and aphl ( / ) was found out more often than isolated anti-ada ( / , x = , ; p x , ) or isolated aphl ( / , x = , ; p x , ). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc and mdc , and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc , mdc and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from pss patients fulfilling the american european consensus group criteria (aecc) and gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p= , ) and mdc (p x , ) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd + b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin (il- ) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd + b cells and increased il- levels. we therefore hypothesized that il- may have similar biological effects on cd + b cells in autoimmune diseases. here we demonstrate that the amount of il- in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to % of cd + b cells in sle individuals expressed grb. in-vitro experiments revealed that il- was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor . importantly, il- significantly decreased the cd +/cd -b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd + b cell death. these results suggest that il- -induced grb may play a regulatory role for cd + b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il- and grb levels in sle and showing that il- reduces the cd +/ cd -b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il- may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il- in sle and other autoimmune diseases. r. de palma , e. d'aiuto , s. vettori , g. abbate , g. valentini second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd + t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il- beta, il- and il- , cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed igg antibodies from single facs purified cd +cd +cd +cd + bone marrow plasma cells of patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran , e. moazemi godarzi , e. kamali sarvestani , e. aflaki shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin (il- ) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il- gene at positions - g/c and - g/c have been described that are key regulators of il- gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il- gene polymorphism at - position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the - position significantly differed in sle patients and controls. at this position gg genotype was observed in . % of patients compared to . % in the control group (p x . ). the frequency of - g allele in patients ( . %) was also higher than in controls ( . %) (p= . ). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. - polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x . ). at - polymorphism, a significant difference with regard to photosensitivity in male patients (p= . ) was found. in conclusion, results of this study showed that - polymorphism plays an important role in susceptibility to sle and that - polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained patients with ibd ( with cd, with uc, with gluten sensitive enteropathy, gse) and healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd -ve cd b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd (+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling- (rgs- ) protein, we have now found substantial over-expression ( - fold) of rgs- in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs- decreases primary t cell responses to cxcl and ccl , strongly implying that it may regulate t cell localisation. thus, rgs- may be a novel target for modulating t cells in ibd, consistent with which snps in rgs- have been associated with both coeliac disease and type diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c bl/ j mice were divided in control (c) and experimental (e) groups. c -c received peanut protein immunization, animals of the control groups c were sham immunized, and control group c received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a day peanut containing cd, the experimental group was divided into groups (e -e ). ova feeding began days prior cd (e ) on day (e ), (e ), (e ) and (e ) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e ) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase and secretion of bioactive il -b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il -b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying % destrane sulphate sodium (dss) in the drinking water for days to wildtype mice. when ultrafine nanoparticles were administered on day and by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl , n. schneiderhan-marra , m. blum , g. stein , m. schmolz , t. joos nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [ ] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco- cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of -well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h -associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h -like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il- production and t h polarization and decreases recruitment of cd + cd + foxp + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen , , g. van assche , p. rutgeerts , a. liston , j. l. ceuppens k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, k. u. leuven, experimental immunology lab, leuven, belgium, k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp- allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced types of colitis dss (th /th ), tnbs (th ) and oxazolone (th ) in hp ko mice. neutralizing anti-il- mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th /th cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. ) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; ) in dss but not in oxa colitis mice, il- , ifn-g, tgf-b and il- were significantly increased (p x . , dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x . , ko vs wt). in tnbs colitis, we found elevated il- and ifn-g (p x . , tnbs vs control). although not significant, il- was also somewhat upregulated; ) in dss colitis we observed that il- enhanced differentiated th cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il- and il- , more mln-t cells from hpko mice differentiated into th cells; ) anti-il- mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il- showed reduced il- , il- and ifn-g in both mln and lp (p x . , anti-il- vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il- , supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann , g. talabér , g. süt" o , p. németh , t. berki university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that . million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd +cd hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th and th cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd + and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd + inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells ( . ± . %, mean ± sem), while cd + inkt cells ratio was moderate ( . ± . %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: . ± . %; cd: . ± . %, mean ± sem), while the percentage of cd + inkt cells was elevated (uc: . ± . %; cd: . ± . %, mean ± sem) in both disease groups. proportions of foxp + treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b adenovirus (tgf-b -exo). the t cell inhibitory function of tgf-b -exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b -exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b -exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd + foxp + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b -exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b -exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd + foxp + regulatory t cells (treg) revealed that the relative numbers of cd + foxp + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd + foxp + treg. tgf-b -exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd + t-cells was similar in the four vaccinees as observed by ifng, il- production-profiles. however, the killing capacity of expanded cd + t-cells was distinct as observed by the competence to kill ns -peptide presenting transfectants in vitro. as depicted in figure , cd + t-cells cells from both vac (cleared ) and vac (chronic) produced il- and ifng after stimulation with ns -peptide . however, specific killing of the peptide loaded transfectants was only observed in vac , who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure ] killing of ns peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd + t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr -expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns or eda-ns under the control of cmv promoter were prepared. recombinant ns and the fusion protein eda-ns were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns . results: immunization of mice with the plasmids expressing eda-ns , but not ns alone, induced strong t cell responses against the main hla-a restricted cytotoxic t cell determinants from ns . the recombinant eda-ns fusion protein, but not ns , was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp- monocyte cell line. immunization of hhd mice with eda-ns fusion protein induced both cd + and cd + t cell responses against ns and, when immunized with poly(i:c) and anti-cd antibodies, was able to down-regulate the intrahepatic expression of hcv-ns rna. the recombinant eda-ns fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd +cd a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd + responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd + t cells by mature dcs transduced with melan-a-recombinant human coronavirus e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin (il ) and interleukin (il ) will be involved, and fms-like tyrosin kinase ligand (flt l) will be expressed to modulate dendritic cells. il is known to enhance early t cell expansion and limits t cell overshoot, whereaes il guarantees survival of high affinity t cells during memory phase. on the other hand flt l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik , ag waisman uniklinik mainz, . med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il- production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il- produced by b cells by conditional deletion of the il- gene in these cells. we found that b cell secreted il- has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for days in the presence of il- and il- with exposure to overlapping peptide pools of latent ebna- and lytic bzlf- antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd , cd , cd , ccr , cd ra, il- , tnfa, ifng and cd a. the majority of ex vivo ebv-reactive cd + t cells as well as ebna- -reactive cd + t cells were il- and tnfa-producing memory cells, the later being more frequent in bone marrow (cd +, median, ebna- : bm . %;pb . %; bzlf- : bm . %;pb . %, p= . ). after in vitro expansion a major subset of ebv-specific cd + and cd +t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd + and cd + t cells retained, however, a tnfa single, tnfa/il- or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd + and cd + t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr /cd ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il- delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd + and cd + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd + and cd + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd + and cd + t cells in peripheral blood with a hexon protein-spanning pool of synthetic -mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to - % in the t cell lines and the absolute numbers of both hexon-specific cd + and cd + t cells were to log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd + and cd + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype (ad ) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad vaccine vectors expressing whv core protein was first examinated in c bl mice. groups of mice were immunized with a dna prime-ad boost regimen or with dna and ad alone. ad was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd + t cell responses against peptide pools and in spleens of dna and dna-ad immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd + t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad very weak cd + t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad level of antibodies did not change. those antibodies were only igg a subclass what indicates th t helper type of response. ad -immunized mice had over -fold lower level of anti-whc: both igg a and igg subtypes were detected. the weak response induced by ad may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal ) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m e serum ab titers against peptide and native m e. this correlated with a large number of m -specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h n ), which contains a variant m e-sequence different in amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x (h n ) and the highly pathogenic pr/ (h n ) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m e as a potential "universal" influenza vaccine. this research is supported by a nih t fellowship ca - , the nih grant ai and a grant from the commonwealth of pa. l. yu zhejiang university, zhangzhou, china interleukin- (il- ) is a cytokine produced by stimulated mononuclear macrophage system. in this report, -day-old chicken embryos were vaccined with the plasmid dna (pci-chil- ) encoding chicken interleukin- and the copy numbers of chil- in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil- in -day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil- could accelerate high concentrations of chil- in nonage peripheral blood, accelerate high expression of chil- in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil- could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil- (p x . ). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on volunteers and a phase i study on volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv- neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) and the transmembrane protein gp . two sites on gp and gp are attractive targets for vaccine design: the epitope in the third hypervariable region (v ) is recognized by the human monoclonal antibody - d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies e and f . in order to elicit anti-hiv- neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp -v region or the gp -mper. the vlps are based on the acyltransferase component (e chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e chain self-assembles into a nm protein scaffold resembling a vlp and that contains copies of e . efficient display and refolding of the v and mper regions in e vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the ' of the gene coding for e . the priming and boosting with a combination of vlps and specific hiv- envelope dna were used to immunize mice and rabbits. results: the v -e and mper-e vlps were purified as stable mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of - d, e and f antibodies to hiv-e monomers was confirmed by western blot. we obtained high titers of hiv- gp -specific antibodies in mice immunized with a combination of vlps plus dna (hiv- sf gp ). these antibodies generated a low ( %- %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e vlps plus dna elicited a low titer of hiv- gp specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e vlps are able to display antigenic determinants of hiv and to induce high titers of hiv- -specific antibodies. the e vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h n whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h n influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [ ] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h n whole virus wild-type vaccine, could induce an immune response and protect mice against h n influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h n antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h n virus. the protective efficacy of the trivalent vaccine derived mainly from the h n component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h n virus, suggesting that antibodies are the main contributor to protection. h n specific serum did not inhibit neuraminidase activity of h n virus suggesting that protection was not mediated by neuraminidase n -specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h n whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h n vaccine enhanced anti-h n antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h n vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h n candidate vaccine. [ ] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of children in the first grade of primary school in the area of central and west macedonia. there were greek and foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following ) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to %. ) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). ) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. ) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om- , a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om- . cytokine secretion, activation and proliferation of b cells and foxp + tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr , tlr or the myd adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om- . two synthetic tlr agonists used as adjuvant in human (om- -dp and om- -mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om- -induced protection of diabetes required natural tregs, as nod-cd -/mice were not protected. remarkably, om- activated b cells and not t cells, promoting their proliferation and il- secretion, two phenomena that were tlr -and myd -dependent. om- -dp and om- -mp-ac two synthetic murine tlr agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd + cd + foxp + t cells and the proliferation of il- secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr signaling in the protective effect of om- on development of diabetes and show that two other tlr agonists induce proliferation of b cells and their secretion of il- as well as stimulation of regulatory cd + cd + foxp + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit , a. legat , s. thomas , m. van mechelen , p. hermand , m. goldman institute for medical immunology/université libre de bruxelles, gosselies, belgium, glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide -phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor (tlr ). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd (scd ) for its tlr ligand activity, we investigated the involvement of both forms of cd in the responses elicited by crx- , a prototypical agp. first, we found that crx- efficiently induces nf-kb and irf activation in hek cells transfected with tlr and md- genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd . likewise, crx- efficiently induces the synthesis of nf-kb and irf- dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd respond to crx- but not to lps in serum-free medium. the addition of the soluble form of cd did not modify the levels of il and tnf produced by crx- stimulated dc but increased the levels of interferon-b (ifn-b). when scd was added to hek cells expressing tlr /md- , nf-kb activity was not modified but irf activity was increased in a dose-dependent manner in response to crx- . we will further compare the responses induced by crx- in wild-type and cd deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h- d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled and days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin- gene polymorph isms (c t, g a, c a and a t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [ ] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n= ) have higher amount of both nonactivated (subtype infg+/cd -, p= . ) and activated (subtype infg+/cd +, p= . ) cbmc, producing ifn-g, as compared with newborns from urban mothers (n= ) exposure to pets and the risk of allergic symptoms during the first years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc / to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity ( - %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: months - years (n= ), - years (n= ) and - (n= ) tb has the highest diagnostic value in children n years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp- antigen is more sensitive for detection of tb-specific t cells compared to esat- antigen. . in children with tst - mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr and tlr in the development of spontaneous lupus disease by creating tlr or tlr deficient c bl/ lpr/lpr mice. methods: tlr and tlr deficient lupus prone mice have been generated by crossing c /bl -tlr -/-or c /bl -tlr -/-mice with c /bl lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr or tlr deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr or tlr in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr deficient mice suggesting an important role of tlr in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr pc / expression of full length mcl- and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl- , an anti-apoptotic protein. a splice variant of mcl- arises by removal of exon and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl- (mcl- l) and its splice variant (mcl- s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl- . method: neutrophils were isolated from children (diagnosed x years) with jsle (n= ) and non-inflammatory conditions (control, n= ) and incubated with control serum, jsle serum alone or with jsle serum plus pg/ml gm-csf. quantitative real time pcr was used to assess mcl- l and mcl- s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl- s to mcl- l was also higher in jsle patients compared to controls (p x . ). the addition of gm-csf to jsle serum was associated with an increase in mcl- l ( . ± . ) and a decrease in mcl- s ( . ± . ) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl- compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl- s, and less anti-apoptotic full length mcl- cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex / mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd + cyld ex / t cells were transferred into rag -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex / cd + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex / mice are capable to control inflammatory responses. for this purpose cd + cd + cells were co-transferred with naïve wt cd + t cells into rag -/-recipients. interestingly, rag -/-recipients of cyld ex / t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc / the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a* in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb * , * and * alleles, and a decreased allele frequency of drb * in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb * is associated with the development of severe disease and positive association of cd with drb * and drb * . indeed, drb * was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb * frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb * in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb * were strongly increased with respect to cd and controls. however, the frequency of drb * was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb * is associated with uc in european, north american, japanese and korean populations methods: a total of children were studied, ( boys and girls), up to years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, positive cases of children were found ( %) with active infection : boys ( x years of age, - years of age and g years of age) and girls ( x years of age, - years of age and g years of age). pharyngitis was present in children ( , %), had fever ( , %) and had lymphadenitis ( %). the lab tests revealed leukocytosis up to . leukocytes in cases ( , %) and leukocytosis g . in cases ( , %). the most frequent complication documented was streptococcal superinfection in children ( , %) and thrombocytopenia in children ( , %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and ) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il- ra , previously believed to be a decoy for il- only, is able to transmit a signal via il- . our results support this and may suggest that il- / il- ra signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il- ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il- or il- ra and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd + cd -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il- and il- b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd + cd -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi- , l. acidophilus ncfm tm and b. bifidum bi- ) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi- strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il- or il- b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed pediatric cd patients ( active, remission), pediatric uc patients ( active, remission), and age-matched non-ibd controls. nkg d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x . was considered significant. results: nkg d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd +cd + nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg d ligands on circulating monocytes is also observed. the dramatic increase of nkg d+ lymphocytes, and the strong upregulation of nkg d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc / peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp + t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd + t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec- antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa - ) or hcv-ns (aa - ) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns and core we successfully optimized culture and protein purification conditions. briefly, ns was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec- to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec- / hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd / mva-nef vaccination induces polyfunctional cd t-cells and increases the proliferative capacity of cd t-cells in hiv- infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv- protein nef (mva-nef) was safe in hiv- infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il- and mip- b, the expression of the activation marker cd and the differentiation marker cd ra in nef-specific cd and cd t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il- and mip- b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd t-cell response and a significant increase of polyfunctional nef-specific cd t-cells, simultaneously expressing ifn-g, il- and cd . using the standard ics no increase of nef-specific cd t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il- and cd expressing cd t-cells and the increase of proliferating cd t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd t-cells in hiv- infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv- mediated membrane fusion and p -detected replication. db was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd + and cd + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns ( - aa) and rns a ( - aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/ j mice were immunized intraperitoneally times at a month interval with different doses ( . - mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns protein. immunization with rns a-immunomax conjugate and rns in cfa ( . mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd / degree of cross-genotype reactivity of hcv-specific cd t cells directed against ns the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd t cell response targeting hcv genotype (gt ) and genotype (gt ) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: subjects ( with gt , with gt and anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns from gt or gt . individual reactive peptides and the degree of cross-reactivity between the gt and gt variants were determined by ics. complete ns is sequenced from all viremic patients pd / anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes , , or suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes , or , but not led to a significant reduction in viral loads. decreased splenic viral load after ifna treatment correlated with an expansion of activated fv-specific cd + t cells and nk cells in the spleen, whereas in ifna -and ifna -treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna , and are under investigation pd / elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a* -or k b -restricted cd t cell responses by these dna vaccines differed. k b /ova - -and k b /s - -specific cd t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd + and cd + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least healthy, randomly chosen blood donors were cultured for days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. out of tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il- -exposed cd + t cells showed at least five-fold increase of survival rate in vivo than il- -exposed cd + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il- -exposed cd + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il- and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x . ) . there was a significant elevation of t ada and ada activities in iga deficient patients as compared to healthy individuals (p x . ) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd / hiv- sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after - weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in out of patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, -day-old ross broiler chickens divided randomly into groups, experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/ strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/ and h strains, via the drinking water at days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, & days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd t cells from patients with active tb and patients with successfully treated tb were analysed for simultaneous expression of ifng and il at the single cell level using multi-colour flow-cytometry after hours stimulation with ppd. moreover, cells were stimulated with esat- and cfp- receiver operator characteristics analysis revealed that a percentage of ifng /il dual positive cells x % served as an accurate marker for active tb patients (specificity %, sensitivity %), while frequencies g % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd / necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately inhabitants) is . to . among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, out of children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently . to inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of to led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd / novel analogues of thalidomide inhibit cd expression and production of tnf-a, il- , ifn-g, cxcl- this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta -dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta r k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd and cd . these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il- a and il- b, while il- levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il- b production. using bmdc from nlrp -/-mice we examined il- b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il- b production is dependent on nlrp . collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho- expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from % in wild type (hmox +/+ ) mice to % in ho deficient (hmox -/-) mice. hmox -/-but not hmox +/+ mice developed end-stage multiorgan failure. mortality of hmox -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h o or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb . similarly, circulating and cytoplasmic hmgb was increased in hmox -/-relative to hmox +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b- , -linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at - years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and - weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were . and . iu/ml and those of dtwp-pasteur were . and . iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were . eu/ml for dtwp-local and . eu/ml for dtwp-pasteur vaccines, respectively (p x . ). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd / united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b: , a gram-negative coccobacillus. jrmt , an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of cfu of jrmt . a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b: on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for h before adding concanavalin-a (cona) pd -vaccination and immunotherapy against parasitic diseases pd / evaluation of simian adenoviral vector adch expressing msp- as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype (adhu ) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp- and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho- expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho- is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp expression was confirmed by sds-page and elisa using monoclonal antibody against gp . discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a - recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a - recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at - °c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r= . , p= . ) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il- also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il- on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il- to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il- and il- -related inhibition of cfu-e growth were dependent on p mapk activity, since the p mapk inhibitor, sb , markedly downregulated il- -induced activation of nos and reversed the growth inhibitory effects of il- on cfu-e. the in vivo stimulating effect of il- on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il- effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il- acts on bone marrow cells and also revealed a link between the il- , no and hematopoiesis. further studies on il- -mediated induction of both inos and enos methods: a total of blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of samples ( , %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of iu/l anti-hbs in all the blood units, which also goes for our study pd / neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen , national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa- a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa- a iggs we monitored children ( boys and girls) in ages from . to . years with average age of . years. in of them all was diagnosed for the first time. subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest ( subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc h gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. , nature , - ). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 'utr of icos, in which a bp sequence, containing a possible mir- binding site, was sufficient (yu et al. , nature, , - ). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. , j biol chem , - ). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia- -and ago -deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r ai . we have recently reported that -hydroxyl- -methylindole- -acetonitrile ( -hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw . macrophages. in this report, we investigated the effect of -hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin- (il- ), monocyte chemotactic protein- (mcp- ) in ht- human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed -hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated -hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. -hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p . in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of -hma significantly reduced the mrna levels of il- and mcp- stimulated by tumor necrosis factor-a (tnf-a) in the ht- cells. pretreatment of -hma also significantly blocked the ixba degradation and nf-xb p nuclear translocation stimulated by tnf-a in the ht- cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of -hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, -hma could be new potential therapeutic agent for inflammatory bowel disease.cd serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd igg immune response in hiv- -exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp -binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp , reacted with the external domains of soluble human cd , in the absence of the target cells expressing the co-receptor ccr . the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd -gp complex and trigger the membrane fusion between effector (gp expressing) cells and target (ccr expressing) cells. thus, in the absence of ccr we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv- infections. a conventional protocol for the generation of monoclonal antibodies was used. db- (igg , x), one of the anti-cd antibodies obtained, recognized preferentially cd complexed to gp , as compared to cd alone, not competed for the gp binding site on cd and was specific for the second extracellular domain of cd . g. röder , l. geironson , a. darabi , m. harndahl , c. schafer-nielsen , k. skjödt , s. buus , k. paulsson copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, lund university, immunology bmc d , lund, sweden, lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, schafer-n, copenhagen, denmark, department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a* . to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn - / , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn / was demonstrated to be located on tapasin [ ] [ ] [ ] [ ] [ ] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd + t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region - of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b* -restricted np-flu epitope (aa - ) was replaced by hla-b or hla-a -restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard cr release assay and through the ifn-g production. results: using hla-b or hla-a restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b as a remarkable hla-class i molecule that, differently from hla-a , can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv- )-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv . consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p -gag and p region was determined in vitro. mer peptides were digested with i s and c proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv- p -and p -gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers , g. koster , o. landsverk , f. skjeldal , o. bakke university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi kinases and thus ptdins( )p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier , l. visvabharathy , j. fu university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e - k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e - k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type e - k and class i molecules.results: we showed that e - k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a molecules, with the mature form being more tightly associated. we also provided evidence that e - k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e - k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e - k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il- , il- , tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands and , but also with the t-celldependent stimulus cd l (cd ) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd , cd , and cd was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p and erk- / . strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd + t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th (il- +) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th / th balance and to identify potential t cell target antigens (ags), we analyzed circulating cd + t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il- expression in peripheral blood cd +cd + lymphocytes were measured in ccs patients and healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il- /ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il- and lower ifn-g intracellular expression were detected in cd + t cells from css patients, as compared to hs (il- : . ± . % vs. . ± . %, p x . ; ifn-g: . ± . % vs. . ± . %, p x . ). similar results were obtained by elisa (il- : . ± . vs. . ± . pg/ml, p x . ; ifn-g: . ± . vs. . ± . pg/ml, p x . ). elispot counts of hexavalent vaccine-stimulated cd + cells were positive for il- in / ( %) css patients and in / ( %) hs, and for ifn-g in / ( %) css patients and / ( %) hs. mpo stimulation determined significant ifn-g release in / ( %) css patients, but not in hs ( / ) no il- response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd + cells from css patients show a th -polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd + t cells, and responses towards it are instead th -polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd + t cells in css. a. voigt , e. opitz , k. savvatis , k. klingel , k. stangl , u. kuckelkorn , p.-m. kloetzel charité -universitätsmedizin berlin campus mitte, berlin, germany, charite -campus benjamin franklin, berlin, germany, universität tübingen, tübingen, germanymurine models of coxsackievirus b (cvb )-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp of the stress-induced ip, were infected with x e pfu cvb nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp as early as day p. i. in lmp -deficient mice. however, lmp -deficiency was linked to less severe myocarditis at day p. i. (he stain of cardiac tissue sections: wt . ± . vs. lmp -deficiency . ± . (grade of myocarditis; scale - ; p x . ). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x . ), there was no difference in lmp -deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb infection in lmp -deficient mice. likewise, diastolic function was preserved in lmp -deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp -deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp -immunosubunit function in regulatory processes of viral replication. absence of lmp confers host protection in enterovirus myocarditis. h. w. liao , j. xu , j.q. huang sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype (den- ) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr and tnfr - were constantly very low whereas trailr - decreased after den- infection. fasl was expressed at similar levels on huvecs throughout den- infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase , caspase were also observed by western blot after by den- infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for days into wis rat paw web tissue in saline containing . x cells. group ii. s.epidermis was injected as in group after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day .they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors , and , respectively (p x . ). immunohistochemical pictures showed increase in percentage of ox (migrating dendritic cell), mhc ii, his (granulocytes), ox (stem cells) and cd (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed (macrophages) and ox cells. popliteal lymph nodes contained evidently less of ox , his and mhcii cells than in group i (p x . ). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega- fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega- fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega- fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega - fatty acids consumption. the results indicate that omega- fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega- fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega- fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density . and , g, top % layer contained lipoproteins only and - % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin- . objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e f mutation, and other affecting b cell apoptosis control, such as bcl- over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c bl/ (b ) genetic background. e f -/-bcl- tg were obtained backcrossing e f -/-and e f +/+hbcl- tg mice. e f -/-bcl- tg, e f -/-, e f +/+hbcl- tg and control mice (e f +/+) were followed up to mo-old. serologic studies: serum samples obtained at , and month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of , and mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h ]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl- tg in b lymphocytes of e f -/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e f -/-bcl- tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e f -/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e f or the overexpression of a bcl- tg in the b genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor (tlr ) abolishes the generation of antinucleosome igg a and igg b autoantibodies but exacerbates lupus disease. however, the tlr -dependent tolerance mechanism is unknown. here we show that loss of tlr in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th and th t cells. transfer of a synthesized monoclonal polyreactive igm to tlr deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr -dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n= ) (vitali et al. ) . ninety-seven of ( %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g (fs+), while biopsies from / ( %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in / ( %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x . ). interleukin (il) , il- ra, il- beta, il- p , il- , macrophage inflammatory protein (mip) alpha, mip- beta, eotaxin, interferon (ifn) alpha, and il- levels were significantly increased in the gc+ patients (n= ) compared with the gc-patients (n= ). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il- found in these patients (gao et al. ) . increased titers of th -associated cytokines, il- , il- beta and the il- subunit il- p , may indicate a higher activity of these cells in gc+ patients (nguyen et al. ) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il- . the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg . production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd -dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd -dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/- baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il- . objectives: increased levels of il- , an innate and inflammatory cytokine of the il- family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il- and other genes of the il- /il- r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from patients and healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il- and il- bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il- are higher than in controls. serum il- correlates with disease activity and decreases upon remission. monocyte expression of the receptor il- rb is increased and correlates with disease severity, while expression of tir /sigirr (a down-regulatory receptor of the il- r/il- r family) is reduced. in mrl lpr/lpr mice, expression of il- , caspase- and il- rb genes precedes disease onset in lymph-nodes. in other organs, changes in il- -related genes (il- and tigirr- up-regulation, tir /sigirr down-regulation) occur after disease onset. free il- levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il- levels correlate with autoimmune lupus both in mice and humans. free il- may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il- rb is a marker of pathology, suggesting increased il- -dependent activation. both in mouse organs and human monocytes, tir /sigirr expression decreases with disease, suggesting impaired control of il- r activation. thus, il- may be involved in autoimmune lupus pathology, and il- -related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f (amino acid (aa) - ) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f was recognized by all patients. fragment f (aa - ) was recognized by of dcssc patients. fragment f (aa - ) was recognized by of sle patients. analysis of clinical data revealed a significant difference between the f negative and f positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f and f could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril- . by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd + t cells enriched in tregs were co-cultured with activated cd + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd + t cells anergy was also evaluated based on cbl-b, grail and foxp mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b- cells through targeting p d by shrnas strategy. methods: we used the drugs, ly and wortmannin, pan-specific inhibitors against pi ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p d. we then introduced either pan-specific inhibitors against all pi ks or p d-targeting shrnas into an sle-prone animal model, nzb/w f mice, for therapeutic purposes. the results suggested that pi ks are not only important for the development of b- cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p d. either pan-specific inhibitors against pi ks or p d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f mice. one inhibitor, ly , and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi ks are critical for the maintenance of b- cell populations might shed light on future treating other diseases associated with b- cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool , m. alarcón-riquelme , s. kozyrev institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs , r h). we identified that bank gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon (delta ). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs ), which is in complete ld with r h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia , which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a -year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti- gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow cnrs , strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed patients, with minor clinical and/or biological manifestations of their disease, for at least monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below . b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd surface protein for all patients. this cd lower expression is associated with cd lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf lacking an exon in the c -domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf in t-cells. smurf expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf expression was upregulated by tgf-beta stimulation in t-cells and smurf was markedly upregulated in tumor infiltrating cd + lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day , smurf transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf showed an upregulation of the tgfbrii and an activation of smad and as compared to wild-type t-lymphocytes, which were previously described as typical smurf targets for degradation. in addition the transfection of smurf and a caga-luc plasmid into cos-cells for smad -promotor studies yielded the same effect as shown by upregulation of the smad activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf has beneficial effects on mucosal inflammation and tumor development. smurf thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat has important functions in cytokine signalling in a variety of tissues. however, the role of stat in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat activation in dss colitis is dependent on il- rather than il- . il- was secreted by colonic cd c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat activity were highly susceptible to experimental colitis, indicating that epithelial stat regulates gut homeostasis. stat iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il- and epithelial stat was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat activation regulates immune homeostasis in the gut by promoting il- -dependent mucosal wound healing. stat seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd and tlr expression at day and day after infection. mycobacterial infection did not result in differential tlr expression as compared to uninfected cells. cd is involved in stimulating th pro-inflammatory responses, although map may interfere with cd signalling ( ) . tlr signalling elicits anti-inflammatory responses, which can contribute to bacterial replication ( ) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd and tlr receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd t cells by exogenous antigen leads to colitis. methods: eight million naïve cd + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h -kb, were transferred i. v. into b mice. at day and , mice were treated intra-rectally (i. r.) with % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day after the injection of ot-i cells. the phenotype of effector cells was evaluated at day by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd , the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd + t cells. our study demonstrates that the local activation of antigen-specific cd t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of patients, ( male and female), with age average , years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x and t-test programmes. results: in patients ( , %,with age average , years of age) no antibodies were detected. on the contrary, in the remaining , male and female, ( , %, with age average , years of age), antibodies were detected. out of them, in cases the results were strong positive ( male and female) and weak positive in cases ( male and female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: ) helicobacter pylori infection is relatively common in the general population ( , %). ) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women). ) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova , t. ulmannova , k. stechova , k. tesarova-flajsmanova , v. stavikova , j. nevoral charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of mothers whose infants were diagnosed with allergic colitis and mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis ( - weeks, average . weeks of infant's age) or at the age of weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il- , il- , il- , il- , il- , ifn-gamma, tgf-beta , egf and eotaxin. man-whitney u test was used for statistical analysis, p x . was considered statistically significant.results: il- as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x . ) in breast milk of mothers whose infants were suffering from allergic colitis (range - . pg/ml, mean . pg/ml) than in control group (range - . pg/ml, mean . pg/ml). higher concentrations of il- and lower concentration of tgf-beta were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. - . background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il- (murine homologs kc and mip- ) and its receptor cxcr are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at , , and h post-transfer. anti-kc antibody or its isotype control was administered at mg/mouse one hour before transfer followed by whole body and organ imaging hours post-transfer. results: facs analysis revealed % neutrophil purity, % of which were cxcr + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model ( . % dss in drinking water for week, followed by pure drinking water for week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell ), treatment of commensal-depleted mice with the tlr ligand lps in drinking water protected against the lethality of % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of . % they may become pathogenic and drive an intestinal inflammation. at % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by . % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il- production which is an important pathological factor. neutralizing il- or il- prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th cytokines in colitis remain undefined, we used mice deficient in il- /il- or the key receptor through which they signal, il- ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il- ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il- /il- double deficient mice which were protected from colitis. removing il- production from il- ra -/mice, by using il- ra/il- double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il- mediates susceptibility in an il- ra independent manor. recent evidence pc / introduction: the activation of cd + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th -like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th cytokines, such as il- ,- and il- . however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc was found in uc and cd colonic tissue compared to control specimen. transmitted to the th -mediated oxazolone-induced colitis model, nfatc -production is significantly increased in both diseases, too. nfatc deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc deficient mice compared to control mice, which can be observed by tunel assays, caspase and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl- and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il- , ifn-gamma, il- and il- by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc regulates il- /il- in an indirect way. last, administration of il- blocked the protective effects of the nfatc deficiency in experimental colitis, suggesting that nfatc through il- signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc in colitis by controlling mucosal t cell activation in an il- dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd cd rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd cd rb high t cells also regulatory cd cd rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at different time points during colitis progression. after weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at weeks. microrna was isolated from colons of mice in different stages of colitis progression ( , and weeks) and control mice that do not induce colitis (n= for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from micrornas that demonstrated an induction during the development of disease we selected micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd cd rb high transfer model. objective: the purpose of this clinical trial (id: nct of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in groups: group a (n= ) receiving haart+ gh (for months) + tt+hva/b vaccines (at month post gh adminsitration); group b (n= ) receiving haart+gh but not vaccines; and group c (hiv control group, n= ) with haart+vaccines (at month ) but without gh. all patients are followed up months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after weeks of administaring hormone the absolute numbers of cd incresase from ± to ± cells per mm (mean and sem; p x . ). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd absolute numbers from ± to ± cells per mm (p x . ). viral load remained undetectable in all patients. despite the increase in cd counts the percentage of recent thymic emigrants (as assessed by the expression of cd ) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv ( , - , ), hepatitis c virus (hcv; . - . million) and hepatitis b virus (hbv; - million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv- infected patients with g cd + t-cell counts and plasma hiv-rna levels of x . log copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g and when the responses were g times the standard deviation above the mean of replicate negative controls (mock electroporated dc). / patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in / patients before and / patients after vaccination. for rev, / patients showed a pre-existing rev specific response and / patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already / patients responding before and / patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in out of patients before vaccination and / patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus (ev ) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev inactivated virus. methods: mice were immunized intraperitoneally with mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after , , and weeks. blood samples were collected at , , , and days. the total serum anti-ev igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il- from splenocytes was also measured. results: immunization with ev /ps-g showed that the anti-ev igg levels were significantly increased compared with ev alone or ev /cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev /ps-g group compared to ev alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev /ps-g-immunized mice compared to those of ev or ev /cfa/ifa-immunized mice, indicating a th cells response elicited by heat-inactivated ev vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd + t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd + t-cell responses against hcv-ns , studying induction of il- , ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il- and ifng production by cd + t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv- -recognizing cd + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv- replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd + t cells by electroporation, and chose tcrs which were able to recognize the hla-a restricted hivpol-peptide iv , or the hivgag-peptide sl . results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il- , tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd , after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt l pretreated balb/c mice were incubated for hrs with hcv ns -coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g % dcs and was used for immunization. dc expression of the maturation markers cd , cd and cd was determined before and after ingestion of ns -coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns expressing sp / cells). results: in immunized animals, the ctl activity was increased -fold compared to mock immunized mice. accordingly, tumor challenge with ns expressing tumor cells showed a significant reduction in tumor growth. the number of cd + ifn-g + cells was increased g -fold and the number of cd + il- + increased g fold in the dc-ns -bead immunization group. these results paralleled the proliferative response of splenocytes to ns protein obtained from immunized animals with the most significant response in the cd + population of dc-ns -bead immunized animals. the use of ns coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec- antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec- induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec- promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec- antibody can be developed. we screened the anti-dec- sequence computationally for putative hla dr -restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec- antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec- as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih r ai a live oral vaccine based on human adenovirus (ad) has proved safe and effective in us military recruits for nearly years. in these experiments, we have investigated whether replication-deficient ad can be an efficient potential vaccine carrier for oral vaccination. ad vectors were used throughout to provide a benchmark for efficacy. we generated novel ad and ad vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad than ad . ad routinely infected and provided transgene expression in˚ % of human cd + and cd + t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to - % of cd + t cells present, showing that ad infected a surprisingly large proportion of t cells. in comparison, ad provided egfp expression in x % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad and ad vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad and ad induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day after dosing, and transgene expression being reduced below detectable levels by day . interestingly, when delivered together ad and ad vectors targeted the same subset of cells. together, these data show that ad is a viable alternative to ad -based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies ( recently commercialized; g in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that -treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, ; gros et al, ; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp ) of rhdv at two different locations: ) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp- ), and ) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp- ). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp- ) was more immunogenic than insertion at position for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp- at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr- ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd + t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd + t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np - specific cd + t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr- targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd +, cd +, cd +, cd + t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by , - -fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns and ns a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h n influenza virus inhibits the ifn-g synthesis (mibayashi et al., ) and causes a decrease in cd + and cd + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., ) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions - (tat'kov c. i. et al., ) . deltaferon was i. p. administered to male non-inbred mice in doses of - * iu once or twice at an interval of hours, alone or in combination with double-stranded yeast rna preparation ( mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., ) . results: when deltaferon was administered in doses of - * iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of * iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon ( * iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd responses (tetramer+ hiv- gag p cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd responses after pla-p immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas , c. prego , s. vicente , m.j. alonso , a. gonzález-fernández university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer , with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with or immunizations to balb/c female mice ( weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day to post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease ( miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigen- background: infection with human immunodeficiency virus type (hiv- ) is characterized by dysfunction of hiv- -specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p antigen from hiv- and the tlr ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p microcapsules containing p and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p peptides. intracellular staining of interferon-gamma (ifn-g), interleukin- (il- ) and cd a after p stimulation was also performed. results: mddc from hiv(-) subjects, incubated for hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il- , upon p peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd b between our model cytokine il- and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il- dependent proliferation assays of il- variants. results: murine il- attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il- dependent cell line ht- . we found that the membrane anchors comprising one and four ig-like domains (il- :: iggpi and il- :: iggpi) resulted in severely reduced vlp production by producer cells and despite of an increased targeting of il- :: iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il- fused to the minimal gpi-anchor acceptor sequence of hcd b (il- ::gpi). il- :: iggpi, however, showed comparable particle production and biological activity in vitro when compared to il- ::gpi. furthermore, il- fused to ig::gpi showed an increased capacity to co-stimulate primary p tcr transgenic t-cells specific for lcmv-gp - in the context of h -d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il- ::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f -b of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild , interdisciplinary transplant laboratory charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects - % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of - months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from , month to days. t cell lines are composed of cd and cd cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker , m. assenmacher , a. richter miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp -specific cd + and cd + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd ro -cd -) cd + and cd + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp peptide pool and cd -depleted autologous pbmc as feeder cells in the presence of il- , il- , and il- . already - days after primary activation pp - /a -tetramer + cd + t cells were detectable for hla-a + blood donors. to analyze cd + t cells having other specificities than for the peptide pp - as well as probably primed cd + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to . % of the cd + t cells and up to . % of the cd + t cells after restimulation with pp peptide pool, but not with either irrelevant ie- peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for days led to a - fold expansion of pp -specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd and cd only if restimulated with the pp peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il- , indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd + and cd + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf , k. mozdzanowska , l. otvos , j. erikson the wistar institute, philadelphia, united states, temple university, philadelphia, united statesthe influenza virus a matrix protein ectodomain (m e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine ; virology journal ), we generated a novel peptide and investigated its efficacy in inducing an anti-m e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd + t cells, have been considered. clinical data confirm a crucial role for antiviral cd + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a* and hla-b* previously. considering other frequent hla alleles cmv specific cd + t cells were monitored longitudinally in hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a* /pp ( - ) ( . x cells/l) and hla-b* /pp ( - ) ( . x cells/l) specific cd + t cells are significantly lower than those for hla-a* /pp ( - ) ( . x cells/l) and hla-a* /pp ( - ) ( . x cells/l) specific cd + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. ( , - mcg) . no adverse effects were indicated during trials (up to month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after th immunization with mcg of vichrepol.no differences were detected in cd + and cd + t cell counts and cd +/cd + ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp or gp alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb f hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb f mice with adenoviral nanoparticles expressing fusion proteins containing gp resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted - % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf did not show any increase in their levels of ifn-gamma. conclusion: caf enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from children aged - years old ( boys and girls) -group , and children ( boys and girls) - years old -group were isolated on a gradient of density before vaccination ( or revaccination) with priorix, week, and months after and incubated with cfse. then million/ ml pbl were incubated in rpmi- supplemented with % fcs (the negative control), at presence of mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing % cÎ at °c within day. intensity of a fluorescence estimated on fl by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control % pbl in both groups did not enter mitosis. in the positive control % of cells have passed one and more mitoses. in group measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in months - % of lymphocytes demonstrated antigen-specific proliferation. in group , on the contrary, before the vaccination the most part of cells ( - %) has not entered division, but - % of cells have passed and more mitoses. in a week specific lymphocyte proliferation decreased and in months it was increased up to - %. production of the interleukin (il) , ifn-g, tnf-a, il- , il- was more informative than il- , il- , il- , il- . measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp cd -binding site (cd bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv- neutralizing polyclonal iggs directed against the cd bs. the mabs were shown to react with an anti-cd bs human neutralizing mab (b ), to elicit antibodies that recognize the gp molecule and an anti-hiv- neutralizing response in rabbits, confirming them as cd bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p and p ) reacted in elisa only with the cd bs-directed igg fraction. the clones were also recognized in elisa by b . p and p -immunized rabbit sera showed a strong anti-gp titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb strain glycoproteins. in particular, / rabbits in the p group and / in the p group showed an % hiv neutralization at dilutions ranging from : to : . the immunoenzymatic assay used, allowed to detect a p and p reactivity in hiv-positive sera and was able to detect a b concentration equal to ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines ( d and dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a -year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following d- vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd , cd and cd ) along with increased levels of nkt-cells (cd + cd +/-cd +/-/cd + ratio) and activated t-cells (cd + and cd + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il- , il- , il- , il- and il- ) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il- + ), cd + t-cells (il- + and il- + ) and b-lymphocytes (tnf-a + , il- + and il- + ). the analysis of cd + t-cells revealed a complex profile with increased frequency of il- + and ifn-g + and decreased percentage of tnf-a + , il- + and il- + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester , h.-g. rammensee , s. stevanović eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd t cell epitopes for the frequent mhc class i alleles a* , a* , and a* from the proteins pii (hexon), pviii, and e a of adv strains ad and ad by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a -day recall stimulation of pbmcs from at least healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd t cells. we could identify new peptides eliciting ifn-g responses, several of which were confirmed as novel cd t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle ( ara criteria) was diagnosed in a -year-old african female patient with hiv- (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd t-cell count x cells/mm . initiation of mg mmf bid was associated with biological and clinical remission of sle and cd t-cell increase. no opportunistic infections or cancers were noted during a -year follow-up and the patient remained always naive to art. hiv- -specific cd and cd t-cell responses were analyzed after months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il- production following stimulation with a panel of hiv- -derived optimal epitopes ( / -mers) covering various hiv regions and a pool of hiv- -derived peptides ( -mers overlapping by aminoacids) encompassing the entire gag protein. all peptides are derived from hiv- consensus strain iiib. results: highly polyfunctional hiv- specific cd and cd t-cell responses against gag were detected. epitope-specific cd t-cell responses were identified: except for one response restricted by hla a* and another one by hla cw* , all the others were restricted by hla-b alleles and mostly by b* (n = ). seven out of responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il- +) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv- specific cd and cd t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv- t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of dl /ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf- of - g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt- , cofaa and cgpi- . . state of vaccine-induced measles immunity was determined by means of elisa in - , - and - years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in - years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p . ) measles immunity and antibody level much lower (p p . ) than among children with mt conversion. in - years the comparison group kept decreased (p p . ) measles immunity, the majority ( ± . %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p . ) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p . ), the majority ( . ± . %) of persons lost protected antibody level; among children with mt conversion in - years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p . ) to low values; among children with hyperergic mt igg level decreased (p p . ) and reached low (in - years), minimal protected (in - years) and lower than protected (in - years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il- producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein (dnahsp ). methods: balb/c female mice were infected by intra-tracheal route with h rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp genetic vaccine was done at days , , and post-infection. each dose consisted of micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd +, cd + and gamma-delta t cells from lungs were determined and days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x . were considered significant (t test). results: at day after the end of the immunotherapy, dnahsp treated mice exhibit increased numbers of absolute cd + and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il- producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp treated mice showed more ifn-gamma producing cells in both cd + and cd + cell populations. at day after the end of the therapy, the main observation in mice which received dnahsp treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd + and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd + cells, together with a more frequency of gamma-delta t cells producing il- . finally, the immunotherapeutic effects of dnahsp vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd + cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il- , are the main effects of dnahsp immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il- and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of - log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput ( -well format), requires only ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to % in pb, % in rx and % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd l, a co-stimulatory molecule preferentially expressed on activated cd + t cells, is the ligand of cd . cd -cd l interaction induces the production of il- and the initiation of a th -type immune response. several studies show that cd l is required for the activation of macrophages and the maturation of dcs. moreover, cd l enhances the capacity of cd + t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd l. we have constructed the recombinant bcg strain expressing cd l (rbcg ) by electroporation of bcg with pgfm /signalsequenceag b-cd lec and an another recombinant strain with empty vector pgfm (rbcg ) as a control. the expression of cd l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd l weeks after vaccination but not at and weeks. rbcg seems to be more protective against paratuberculosis than rbcg , but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il- a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x . ) of tnfa, ifng and il- a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x . ) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd mab and ag a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd binding on cd transfected l fibroblasts. the conjugates were tested in vivo in wild type and cd + cell-depleted mice for the induction of specific anti-ag a serum antibodies. splenocytes were challenged ex vivo with ag a and were examined for their ability to produce th -related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il- . we developed a method to successfully crosslink anti-cd mab to ag a. serum antibodies against ag a were detected after immunisation with this conjugate vaccine in both wild type and cd + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag a alone. production of two other th -related cytokines, tnfa and il , was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer , r. burger robert koch-institute, cellular immunology, berlin, germany, robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd -positive and cd -positive t-lymphocytes. cd -positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd + t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd + t-lymphocytes in vivo. similarly, the cd -positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd + and the cd + subpopulation depended on the presence of apc. stimulation oft cd + cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd -positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target (esat- ) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, iranian and afghan adults ( patients with sputum smear and culture positive tuberculosis, recovered patients during months after full course of chemical treatment and healthy individuals) were recruited to quantify the frequency of esat- and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of spot forming unit ( g spots per million), we found detectable response to esat- in almost % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and % of these individuals had detectable esat- specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in %, % and % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th immune response, against esat- , in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal , p.k. upadhyay national institute of immunology, pdc- , delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c bl/ mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: . % alginate, . % pva concentration gives particles with size of - micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with % trehelose and . % pvp (poly vinyl pyrollidone). there was fold increase in proliferation index of spleenocytes and releases pico-gram of interferon gamma after week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by % increase in cd and . % in ccr expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b s. by cell cycle analysis we determined that b s is able to induce apoptosis in up to % of jurkat and molt- t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase is the first to be cleaved, followed by caspases and , thus suggesting that b s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd , cd and cd , and of the inflammatory cytokines il- , il- and mcp- . using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th and th /th skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd t cell responses. microcapsule based vaccination resulted in a marked induction of il- secreting th cells, without inducing strong th (cfa) or th (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg , igg c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd , cd and cd and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il- and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr -dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr -defective c h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il- b secretion by dc, through its effects on caspase- processing, which is also tlr -independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc (st ) or lactobacillus rhamnosus ncc (lpr), each at x cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week to week of life. fresh feces were collected at , and weeks of life for determination of iga levels (assessed by elisa). pups were bled and weeks after immunization for determination of measles-specific igg and igg a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st -fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, ( - )- -amino- -deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at : ration and washed times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw . by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of extracts from euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc /propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that up to euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd + cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il- and ige serum levels were increased on animals from group i when compared to control.the enhancement on th immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd + ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd +t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg a and igg b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren , , n. almqvist , a. lönnqvist , s. Östman , c. rask , e. telemo , a.e. wold university of göteborg, department of clinical bacteriology, göteborg, sweden, university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; . mg/ml) in the drinking water for days and, days later, mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin- and interleukin- . examination of gut sections from sea treated donor mice revealed increased density of cd a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp , proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna , a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp , or groel together with cpg-dna reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp together with cpg-dna . the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp microspheres induced protective ifng-secreting cd + t-cells and raised pulmonary pmp -specific iga levels in vivo. also, pmp microspheres caused lower il- serum levels upon administration than the injection of pmp together with cpg-dna , indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in cases of s. aureus bacteraemia in iv drug users and cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen ).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day ) and four weeks thereafter (day ). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter ) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st (spa-type t , agr , and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p= . ).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at and years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in and ds children, respectively. samples were taken before and - weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg , igg and igg ). at years, ds children had lower postvaccination geometric mean igg anti-tt-titers only. post-vaccination igg -anti-tt avidity levels were decreased in / and / ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. kda; , kda; , kda; , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda and , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an -month period. sera were obtained from healthy subjects from the same community ( months to years of age). the highest incidence of salmonella-associated diarrhea, / , occurred in children under years of age. the lowest incidence, / , was observed in the population aged to . whereas serum from individuals ranging - years of age showed maximum igg , igg and iga anti-s. typhimurium titres, children less than years-old did not show detectable igg and igg titres and had weak igg , iga and igm antibody levels; only their igg levels were comparable to those detected in adults. moreover, the levels of igg and igg antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs - , a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses ( %), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a- , linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after -fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková , s. bystrický institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd , expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age - years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg , igg and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after to days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers : ). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones ( : vs. : ). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was . fold of normal control). subcutaneous immunization gave a weaker stimulation: . fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv - and vega / / grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a- , -linked d-mannopyranose units and many branches composed of a- , a- , and/or b- , -linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant ( mg oligosaccharide per one conjugate dose) two times in days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs ( g- g) were treated orally with mg respivax five consecutive days. after the last application, on days , , , , and six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on mm thick serial sections, stained with hemalauneosin. the populations of cd , cd and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd positive cells, which number reached maximum at the end of the second week. on day b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd and cd positive cells. on days and the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta , s. majumdar bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein- (ip- ) and interleukin- (il- ) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip- mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase (inos ) expression and was linked to the mapk signaling pathway via antagonistic regulation of p mapk and erk / . further, ip- was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th cytokines and no. thus this study strongly demonstrates that ip- , like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th /th cytokines mediated by cd + t cells and activating cd + t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x . ) following immunization and after challenge with leishmania major. il- values were increased in all groups, but there was no statistical difference between the groups(p g . ) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x . ) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x . ) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x . ). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at week intervals. three weeks after booster injection, × stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg a/igg was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin (il- ), and cellular expression of cd and cd . results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd + lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with , infective n. brasiliensis larvae on day post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg and igg a). this was independent of level of me supply. feeding regime did not affect levels of the type- cytokines il- and il- . conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp of l.major strain mrho/ir/ /er. methods: l.major promastigotes were grown in rpmi supplemented with % fcs. l.major rna extraction and cdna synthesis were carried out. gp gene segment was amplified by specific primers and cloned into ptz r to construct ptz r/gp . the presence of gp into ptz r was confirmed by pcr. then, ptz r/gp was sent to determine the sequence of its nucleotides. after that the gp gene segment was sub-cloned into pet a (+) expression vector and transformed into e.coli bl (de ) plyss and gp protein was expressed in presence of mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, ) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including volunteers. male volunteers ( and years) for the adult group, and children of both sexes ( - years) were enrolled. subjects received virosomal formulations containing mg of ama -c (pev t), an apical membrane antigen- derived synthetic phospatidylethanolamine (pe)-peptide conjugate and ug of uk (pev t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days , and . results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling % of the treated mice to survive till days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only . % of the amount fed and it remained in circulation in the blood only for minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to . % ( of the amount fed but it's circulation was sustained till hrs post feeding. under such conditions when mgm of curcumin bound to mgm of chitosan nano particles were fed one time daily for days post infection to plasmodium yoelii infected mice % of mice were cured and survived atleast for days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma vdelta t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il- responses, to epitopes on the dominant rbc autoantigen, anion exchanger- (ae- , or band ) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla- + regulatory t cells or soluble forms of ctla- , may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =- . ) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b (r medium =- . ) and il- (r medium =- . ) in isolated splenic hscs ex vivo. analysis of effector cd + t cells in spleen showed decrease of t h cells quantity and simultaneous t h cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd + cd + ctla- + -and cd + cd -il- + il- regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h , t h and regulatory t cells were correlated (r th =- . , r th = . and r th = . and r tr = . ) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th = . and r tr = . ) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b -and il- -produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p , jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin- , t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il- , as compared to non-infected controls. the infection also altered the effect of il- on mapk activation by preventing its stimulating effect on p mapk. moreover, in s.obvelata-infected animals il- markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il- induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. .-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from ⁄ to / with a normal serum pool. .-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r g . . the analytical range was from g/l to g/l. the coefficient of variation (cv) was x % for [mc] n g/dl, and x % for [mc] x g/dl. this procedure was successfully implemented to quantify the mc in serum samples between march and february, . among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: . we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. . the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from day and reached plateau level after day of teratocarcinoma insertion. moreover, cd + cd cells showed in spleen and main lymph nodes from day and achieved plateau level after day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at day and had a maximum pick at day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than , times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b , il- , pge and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number = . and r activity = . ) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd -ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd l is cleaved to soluble (s)cd l. we sought to examine the levels of scd l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd l -along with other products -were assayed by quantitative elisa. levels of il , cd p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l . to examine if scd l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il- secretion. the il- concentration was consistently below - pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il- . the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il- production over control (p x . ) at d after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l (p x . ). pre-incubation of b cells with cd -blocking antibodies substantially abrogated il- secretion, unlike isotype-matched control. the partial blocking of cd binding on cd + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd l (under investigation) and indicates a sustained role for pc-derived scd l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule- (dnam- ) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from aml patients before specific anti-leukemia therapy and healthy donors. all results were analyzed statistically using spss version . results: aml patients under years showed a significant reduction in the expression of dnam- , nkp and nkp compared with age-matched controls. both healthy individuals and aml patients older than years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam- on nk cells and its ligand cd on aml blasts has been found in aml patients under years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam- expression on aml patients and confirm previous reports showing a significant decrease of nkp and nkp on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel , e. gorska , u. demkow , m. wasik medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for or hours with or without dexamethasone in concentration - m. analysis of: p-gp surface expression, p-gp function (rhodamine test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was , %± , . after hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine efflux, which characterized p-gp activity, was , %± , . after hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine ( , %± , , p= , ). average phi in lymphoblasts was , ± , . acidification of cells incubated hours with dexamethasone was seen in - % percentage of cells ) . rest of lymphoblasts showed alkalization (phi - , ).the percentage of lymphoblasts in early stage of apoptosis after hours incubation with dexamethasone (annexin-v test) was higher than in control cells ( , % vs , %; p= , ). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd d/cd integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd and anti-cd (clon huts ) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures ( hours). results: cd active form was expressed in the majority of normal and tumoral bm pc from healthy subjects ( . ± . , n= ) and mm patients in the early stages of the disease ( . ± . , n= ). in these cells, huts epitope was clearly upregulated by mn + . in contrast, circulating pc were almost all huts negative, and levels did not significantly augment when these cells were exposed to mn + . moreover, not only pb but also bm malignant cells from pcl patients were also huts negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index ( . ± . brdu+ cells, n= ) in comparison to pc from mm patients in the first stages of disease ( . ± . brdu+ cells, n= ). these results suggest that the active form of cd must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either % fetal bovine serum (fbs) with and without fgf ,or % human platelet lysate (hpl), %hpl, ( % fbs + % hpl), ( % fbs + % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day ). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: ) ten cases with fa diagnosed on the basis of dna breakage analysis, ) ten cases with aaa, and ) ten normal control cases. the presence of p dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p dna was demonstrated in bone marrow of % of children with fa, compared to % in children with aaa (p x . ), while, no p dna was seen in normal control. a positive correlation between dna breakage and presence of p dna was seen in bone marrow from fa (p x . , r . ). the presence of p tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord- - dannjjm authors: nan title: research abstract program of the acvim forum denver, colorado, june – , date: - - journal: j vet intern med doi: . /j. - . . .x sha: doc_id: cord_uid: dannjjm nan clinics Ãà also see infectious disease abstracts id- -id- (thursday, june , : pm - : pm) Ãà also see pharmacology abstracts p- -p- (thursday, june , : pm - : pm) Ãà also see gasteroenterology abstracts gi- - june , hypertrophic cardiomyopathy (hcm) is the most commonly observed myocardial disease in cats. beta-blockers and calcium channel inhibitors are frequently administered drugs to cats with preclinical hcm despite the fact that neither drug category has been proven to slow disease progression or improve survival. ivabradine (procorolan s , servier, france) is a novel negative chronotropic agent used in the treatment of ischemic heart disease in people. little is known about its efficacy and safety in cats. the purpose of this study was to determine the short-term effects of ivabradine on heart rate (hr), blood pressure, left ventricular (lv) systolic and diastolic function, left atrial (la) performance, and clinical tolerance in healthy cats after repeated oral doses. ten healthy laboratory cats were involved in the present study. physical examination, systolic blood pressure measurement, and transthoracic echocardiography were performed in all cats at baseline and after oral administration ( weeks each) of ivabradine ( . mg/kg, q h) and atenolol ( . mg/cat, q h; . - . mg/kg) in a prospective, double-blind, randomized, active-control, fully crossed study. a-priori non-inferiority margins for the effects of ivabradine compared to atenolol were set at % (f . ) based on predicted clinical relevance, observer measurement variability, and in agreement with fda guidelines. variables were compared by use of -way repeated measures anova. ivabradine was clinically well tolerated with no adverse events observed. hr (ivabradine, po . ; atenolol, po . ; ivabradine vs. atenolol, p . ) and rate-pressure product (ivabradine, p o . ; atenolol, p . ; ivabradine vs. atenolol, p . ) were not different between treatments. at the dosages used, ivabradine demonstrated more favorable effects than atenolol on echocardiographic indices of left ventricular (lv) systolic and diastolic function and left atrial performance. ivabradine is non-inferior to atenolol with regard to effects on hr, rate-pressure product, lv function, la performance, and clinical tolerance. clinical studies in cats with hcm are needed to validate these findings and further assess safety. the aim of this study was to compare outcome from cpa in dogs following initial administration of either epinephrine or vasopressin during cardiopulmonary resuscitation (cpr). dogs having cpa in the er or icu of a university hospital were randomized to receive either iv epinephrine ( . - . mg/kg) or vasopressin ( . - . u/kg) in a blinded fashion immediately following establishment of iv access and again three minutes later. a standardized cpr protocol was followed. other vasopressors were not permitted during the six minute study period, at the end of the study period additional cpr interventions were at the discretion of the managing clinician. the primary end point was return of spontaneous circulation (rosc) within the study period; secondary end points included rosc at any point, survival to minutes, and survival to one hour. sixty dogs completed the study, received epinephrine and received vasopressin. rosc within six minutes was % ( vasopressin, epinephrine p . ), rosc at any time was % ( vasopressin, epinephrine p . ). survival to minutes was % ( vasopressin, epinephrine p . ), survival to one hour was % ( vasopressin, epinephrine p . ). five dogs survived to hours, one survived to hospital discharge. of animals dying after rosc, / were euthanized and / rearrested. no advantage of routine substitution of vasopressin for epinephrine was seen for rosc, a small survival advantage at one hour was seen in the group receiving epinephrine. the study also demon-strated that prospective clinical cpr research in animals is both possible and practical. three dogs were evaluated in phases. phase- : single-dose diltxr at approximately mg/kg po. phase- : same dose q h for . days. phase- : after a day wash-out the single-dose protocol was repeated using cut tablets to assess affect on extended release properties. blood pressure (bp), -lead ecg, echocardiogram, and h-ambulatory ecg were performed at baseline, and conclusion of phase- . blood samples and bp was obtained , , , , , , and h after final dosing. peak median plasma diltiazem concentrations (mcg/ml) measured by hplc for each phase were . , . , and . respectively. diltiazem concentrations were below the limit of detection in the majority of samples in phase- . median diltiazem concentration reached purported therapeutic concentrations ( . - . mcg/ml) by h post-pill in phase- and h post-pill in phase- . therapeutic concentrations were maintained for h in phase- , but only h in phase- . median bp (mmhg) was . at baseline and . at peak concentration in phase- . median heart rate (bpm) was . at baseline. h-ambulatory ecg analysis revealed the median hourly heart rate was . at baseline and during phase- . median heart rate at peak concentration in phase- was . . lack of detectable plasma diltiazem during phase- may be due to up-regulation of drug metabolism via p-glycoprotein (abcb - ) mutations. ongoing data collection and analysis will include mutation testing. adiponectin (adpn) is a cytokine produced by fat cells which has been shown to be correlated with adverse cardiac conditions in humans. in the heart, adiponectin activates several pro-survival reactions, including the ampk pathway and cox receptors, which protect the heart following ischemic injury. recent studies have shown that higher levels of adpn influence cardiac remodeling signaling, inhibiting protein synthesis and suppressing pathological cardiac growth. in humans, adpn plasma levels rise with decreased activity of the sympathetic nervous system and b-adrenergic agonists inhibit adpn at the level of gene expression. in contrast c-reactive protein (crp), a marker of systemic inflammation is elevated in humans with congestive heart failure (chf) and correlates to the severity of disease. first, we hypothesized that dogs with chf would have reduced adpn and elevated crp compared to normal dogs and that cytokine concentrations would predict severity of chf. second, we hypothesized that adpn receptor- (r ) and adpn protein would be elevated in the myocardium of chf dogs reflecting a compensatory process. we collected serum from dogs ( healthy and chf). circulating adiponectin and crp levels were quantified using a mouse/rat adiponectin elisa and a canine crp elisa. we found lower mean crp concentrations in normal dogs ( . ae . mg/ ml) than dogs with chf ( . ae . mg/ml), however, the results were not statistically significant due to the large variability seen among the chf dogs (p . ). we found greater mean adpn concentrations in normal dogs ( . ae . mg/ml) than chf dogs ( . ae . mg/ml) (p . ). in general, the greater the severity of the heart failure, the lower the level of serum adpn. when the tests the purpose of this study was to determine if there are any clinically important differences between the approaches (including devices) used in non invasive transvascular (interventional) closure of patent ductus arteriosus (pda) in dogs in our institution. initial and follow up records from all dogs (n ) that underwent attempted transvascular pda occlusion from january -december were examined. dogs were placed into groups depending on the device and route of vascular access (transvenous or transarterial). group : amplatz s canine ductal occluder (acdo) (transarterial) - dogs; group : gianturco s or mreye flipper s detachable embolization (flipper) coil (transarterial) - dogs; group : amplatzer s vascular plug (avp) (transarterial) - dogs; group : flipper coil (transvenous) - dogs. statistical comparisons were made using the kruskal-wallis test with mann-whitney tests to compare pairs of groups when significance was detected. p o . was considered significant. there was no significant difference in ages between the groups. there was a significant difference in body weight between groups with dogs receiving a coil either transarterially or transvenously (groups and ) being significantly smaller than dogs receiving an acdo or avp. this was by design since the acdo and avp cannot be used in small dogs. overall, the success rate of the total procedure (including vascular access and satisfactory pda occlusion) was high ( %) with success rates being comparable between groups ( - %). there was a significant difference in complication rate between groups (p o . ) with the acdo group having a markedly lower complication rate than the remaining groups ( % for acdo versus - % for the other groups). total fluoroscopy time ranged from - minutes (median minutes). fluoroscopy time for the transvenous method was significantly longer (median minutes; range - minutes) than in the remaining groups (median minutes; range - minutes) (p o . ). number of dogs with residual flow immediately following the procedure and hrs later was significantly less in the acdo group than in the remaining groups ( dogs from group and from group had moderate persistent flow while dog from group and from group had severe persistent flow hours after the procedure). the acdo appears superior in ease of use, complication rate, completeness of occlusion and fluoroscopy time than other devices. the remaining limiting factor with this device is patient size. until a smaller acdo device is marketed, coils remain the only choice for interventional closure in very small dogs ( o . kg). previously presented at the university of california davis, house officers seminar day. subvalvular aortic stenosis (sas) is one of the most commonly reported canine congenital heart defects and is inherited in newfoundland dogs and human beings. the golden retriever and rottweiler are breeds over-represented in dogs with subvalvular aortic stenosis; however, a genetic cause of this disease in these breeds has not been described. we performed genome wide association analysis in both normal and sas affected rottweilers and golden retrievers to identify chromosomal regions of interest that could implicate a causative mutation by high density single nucleotide polymorphism (snp) array. ( unaffected/ affected) adult golden retrievers and ( unaffected/ affected) adult rottweilers were included in this study. criteria for affected included a subcostal continuous-wave doppler aortic velocity ! . m/s and presence of a left basilar systolic ejection murmur; criteria for unaffected included a doppler aortic velocity . m/s. dna samples were obtained from anticoagulated blood. genotypes were obtained using high density ( , ) snp arrays, and genome wide association with sas was evaluated for each breed. significance cut-off was set at p  À , and all snps meeting this criterion were plotted within each breed and compared across breeds using plink. affected golden retriever data implicate the most significant region of genetic variation on chromosome at location (p .  À ; odds ratio . ) with other significant surrounding snps . affected rottweiler data also implicate the most significant region of genetic variation on chromosome at location (p .  À ; odds ratio . ) with other significant surrounding snps . other regions of statistical significance were on chromosomes and in the golden retriever and and in the rottweiler. genome wide association with subvalvular aortic stenosis in the golden retriever and rottweiler implicate overlapping chromosomal regions of interest for causative mutations on chromosome . the different secondary chromosomal regions of interest (chr , in golden retrievers and , in rottweilers) supports the known familial nature of this disease within different breeds and may suggest the presence of multiple mutations or breed specific disease modifiers. these data highlight the need for candidate gene evaluation on chromosome in golden retrievers and rottweilers with sas. heart valves share developmental signaling pathways with bone and cartilage. degenerative aortic valve disease in humans is characterized by valve stenosis and calcification. recent evidence suggests that degenerative aortic valves are undergoing pathologic processes that mimic osteogenesis. degenerative mitral valves in dogs and humans are characterized by valve regurgitation, and rarely undergo calcification. we tested the hypothesis that canine and human degenerative mitral valves might be undergoing pathologic processes that mimic chondrogenesis. to test this hypothesis, expression of bone morphogenic protein (bmp ), a chondrogenic growth factor; sox , a chondrogenic transcription factor; aggrecan, a proteoglycan abundant in cartilage; and type ii collagen were evaluated utilizing immunohistochemistry. normal canine mitral valves, different stages of canine degenerative mitral valves (early, intermediate, and late), and late-stage human degenerative mitral valves were studied. canine and human degenerative mitral valves showed focal areas that co-expressed all four markers of chondrogenic signaling and phenotype. valve interstitial cells and surrounding extracellular matrix in these focal areas adopted a morphologic appearance reminiscent of cartilage. focal chondrogenesis was present in all stages of canine degenerative mitral valves, but not normal canine mitral valves. focal areas of chondrogenesis did not coincide with nodular areas of glycosaminoglycan accumulation on the leaflet edge, but rather seemed to occur at points of chordae attachment to leaflets. in conclusion, canine and human degenerative mitral valves undergo pathologic processes that mimic chondrogenesis. this finding suggests that mitral valve degeneration may be recapitulating developmental signaling pathways shared by heart valves and cartilage. the triggering events for chondrogenesis in mitral valves remain unknown; as does the reason why aortic and mitral valves appear to be undergoing different pathologic processes. the fact that humans exhibit degeneration of both the aortic and mitral valve, and that dogs commonly exhibit only the latter could eventually provide insight into both processes. arrhythmogenic right ventricular cardiomyopathy (arvc) is a familial cardiomyopathy characterized by right ventricular fibrofatty infiltration and ventricular ectopy of left bundle branch block morphology (vpc) . a deletion in the striatin gene has been associated with arvc in at least some boxer families. syncope and sudden death (sd) occur in some affected dogs, although many affected dogs survive for years. the objective of this study was to define clinical characteristics of arvc in boxers that experienced sd, and compare them to those of a contemporaneous group of arvc boxers that had not died suddenly (nsd). data for both groups were collected from adult boxers enrolled in a long term prospective study of arvc in which echocardiograms and hour ambulatory ecg (aecg) are evaluated annually. aecg are quantitated for vpc numbers and arrhythmia grade ( - ). arvc diagnosis requires at least vpcs/ hours in the absence of other disease. forty three adult boxers that entered the study had died suddenly at the time of analysis (sd defined as the absence of observed clinical signs within hours prior to an unexpected and unexplained death). striatin genotype was available for of the sd dogs ( heterozygotes, homozygotes); were female ( intact) and were male ( intact). sd occurred at a mean age of years (range, - ); sd dogs ( %) had no prior history of syncope. twelve sd dogs ( %) were on antiarrhythmics at the time of death (metop-p oooooooooooprolol ( ), sotalol ( ), amiodarone ( ), procainamide ( ), mexiletine & atenolol ( ), atenolol ( )). eleven sd dogs ( %) had decreased myocardial systolic function defined by a shortening fraction (%fs) o % (range - , mean ) on the most recent echocardiogram prior to sd. median vpcs/ hours on annual aecg was , (range - , ) with a median arrhythmia grade of (range - ). twenty one contemporaneously entered arvc boxers that had survived to at least the median age of the sd group with nsd were available for comparison; / were genotyped ( heterozygous, homozygous, negative), were female ( intact) and male ( intact). twelve nsd dogs ( %) had no prior history of syncope. median nsd group age was years (range, - ); / ( %) were on an antiarrhythmics (sotalol ( ), mexiletine & sotalol ( ), mexiletine & atenolol ( )). one nsd dog had decreased %fs (nsd group %fs range - , mean ). the nsd median number of vpcs was , (range - , ), median arrhythmia grade was (range - ). striatin genotype was significantly associated with sd. no significant differences were found between groups with respect to vpc numbers or arrhythmia grade. shortening fraction was significantly lower in the sd group (p o . ). sd in arvc appears to be associated with the presence of the striatin mutation and reduced % fs, it does not appear to be associated with number of vpcs or arrhythmia grade. coughing in the small breed dog may be related to cardiac causes associated with myxomatous mitral valve degeneration (mmvd) including pulmonary edema and compression of the mainstem bronchus by a severely enlarged left atrium, or due to respiratory causes such as tracheal and/or bronchial collapse or chronic bronchitis. the purpose of this study was to evaluate the association between left atrial enlargement and large airway collapse in dogs with mmvd and chronic cough. we hypothesized that airway collapse was independent of degree of left atrial enlargement. twelve dogs with mmvd and a chronic cough in the absence of congestive heart failure were prospectively evaluated with thoracic and cervical radiography, echocardiography, fluoroscopy, bronchoscopy and bronchoalveolar lavage (bal). group dogs (n ) had moderate to severe left atrial enlargement based on an echocardiographically calculated left atrial:aortic surface area [la:ao(a)] . group dogs (n ) had no to mild left atrial enlargement [la:ao(a) ]. the site and severity of airway collapse was graded on bronchoscopy and bal cytology was assessed for evidence of inflammation or infection. the occurrence of bronchoscopic abnormalities was compared between groups using fisher's exact test. p o . was considered significant. age and body weight did not differ between groups. left atrial size was interpreted radiographically as moderately to severely enlarged in of dogs in group and as moderately enlarged in of dogs in group . fluoroscopy revealed variable degrees of airway collapse during normal respiration and induced cough in both groups. radiography and fluoroscopy were not accurate in identifying site and degree of collapse in either group when compared to bronchoscopy. cervical tracheal collapse was identified during bronchoscopy in both group ( of ) and group ( of ) dogs but was subjectively less severe in group dogs. bronchial collapse % was evident at multiple sites in both groups of dogs with no difference between groups. all dogs had suppurative and/or lymphocytic inflammation on airway cytology. infection was not present in either group of dogs, although non-specific light bacterial growth was detected in of group dogs and of group dogs (p . ). preliminary results failed to identify an association between left atrial enlargement and airway collapse in dogs with mmvd but did suggest that airway inflammation is common in affected dogs. further studies are needed to identify factors contributing to airway collapse in dogs with and without mmvd. atenolol is often used empirically in cats with asymptomatic hcm, even though clinical and experimental evidence of efficacy is lacking. cardiac biomarkers play a critical role in the early detection of subclinical cardiac disease, in the prediction of long-term prognosis, and in monitoring the response to therapy in humans. we hypothesized that serum concentrations of the biomarkers, nterminal pro-brain natriuretic peptide (nt-probnp) and cardiac troponin i (ctni), would improve following the chronic administration of atenolol po to asymptomatic cats with hcm. six maine coon or maine coon cross cats with severe hcm from the research colony at ucdavis were administered atenolol ( . mg po twice a day) for days. no cat had severe left ventricular dynamic outflow tract obstruction due to systolic anterior motion of the mitral valve. the concentrations of nt-probnp and ctni were assayed prior to drug administration and on the last day of drug administration. there was no statistically significant difference identified in nt-probnp [median before: pmol/l (range: - pmol/l), median after: pmol/l (range: - pmol/l); p . ] or ctni [median before: . ng/ml (range: . - . ng/ml), median after: . ng/ml (range: . - . ng/ml); p . ] concentrations before and after drug administration using the wilcoxon matched pairs test. the ctni finding suggests that atenolol does not reduce chronic myocyte death in cats with hcm. the lack of improvement in nt-probnp suggests that atenolol does not improve myocardial wall stress in cats with hcm. a clinical trial is warranted to confirm or refute the findings from this study. therefore, leptin-gene expression was investigated in blood samples of dogs with congestive heart failure (chf; n ) in comparison to dogs presented for cardiac screening (n ) without abnormalities. additionally myocardial samples (interventricular septum, right and left atrium and ventricle) of dogs with no cardiac abnormalities (controls), seven dogs with acquired and three with congenital cardiac diseases were investigated using quantitative rt-pcr. leptin blood levels were significantly higher in dogs with chf in comparison to dogs without diseases (p . ). there was an association with gender with higher myocardial leptin levels in female dogs with cardiac diseases (p . ). differences between cardiac regions were present (p o . ) and cardiac diseases resulted in an increase in atrial leptin levels in both sexes (p . ). interestingly, a significant reduction of myocardial leptin was present in dogs with congenital cardiac diseases (p . ), whereas acquired cardiac diseases resulted in an increase in leptin (p . ) in comparison to controls. these results suggest that the heart might be a target of leptin action in the dog and myocardial leptin production might play a role in regulating cardiac function in an auto-and paracrine manner. predicting risk of chf in asymptomatic dogs with mitral valve disease (mvd) is challenging. we examined ability of nt-probnp to identify asymptomatic dogs at high risk for chf. dogs with isachc- b (la:ao . ) were prospectively recruited; dogs with current or previous chf or diuretic therapy were excluded. physical examination, radiography, echocardiography, and nt-probnp were performed at - mo intervals for dogs (median follow-up d, range, - d). thirty-one patients reached a study endpoint of radiographic pulmonary edema; remained asymptomatic. parameters from the visit immediately previous to onset of chf (future-chf) or prior to the most recent visit without chf (remain-asympt) were analyzed. median nt-probnp of future-chf ( pmol/lpmol/l, iqr - ) was significantly different from remain-asympt ( pmol/l, - ; p . ). median time to chf of future-chf was d (iqr, . groups also differed in median la:ao (future-chf . [ . - . ]; remain-asympt . [ . - . ], p . ); vhs (future- ]; remain-asympt . [ . - . ], p . ); and lvidd:ao ]; remain-asympt . [ . - . ], p . ). roc analysis to predict if chf would occur prior to the next visit yielded auc . ( %ci, . - . ). sensitivity was . % or . % and specificity . % or . % for nt-probnp pmol/l or pmol/l, respectively. mean increase in nt-probnp between penultimate visit and two visits prior to endpoint: future-chf . pmol/l vs. remain-asympt . pmol/l. within mo, . %, . %, . %, and . % of dogs with nt-probnp o , , and pmol/l developed chf. nt-probnp and heart size helped assess risk of chf in asymptomatic mvd. increasing the assay's upper limit of detection would likely improve utility of nt-probnp. piiinp is a serum biomarker of collagen biosynthesis and is described as a marker of myocardial fibrosis in human patients. we hypothesised that piiinp concentrations would vary according to the degree of remodelling demonstrable in dogs with naturallyoccurring myxomatous mitral valve disease (mmvd). serum piiinp concentrations (mg/ml) were measured in dogs with mmvd and healthy controls using a validated commerciallyavailable radioimmunoassay. results are reported as (mean ae sd). non-normally distributed variables were logarithmically transformed. comparisons of continuous variables were made between groups using t-tests and one-way anovas with tukey's post-hoc comparisons. univariable analyses were used to evaluate associations between piiinp and clinical characteristics (age, breed [cavalier king charles spaniel (ckcs) yes/ no], sex, weight, heart rate [measured from ecg], treatment with acei [yes/ no], treatment with diuretics [yes/ no] and echocardiographic measurements [la/ao, lvedd/ lvfwd, lveddn, lvesdn]). multivariable analysis was initially performed with all dogs included and then repeated excluding all dogs receiving therapy. dogs with mmvd were divided into those with no cardiomegaly (nc) (la/ao o . and lveddn o . ), those with cardiomegaly (la/ao ! . and/ or lveddn ! . ) but no clinical signs (c) and those dogs with cardiomegaly requiring treatment for congestive heart failure (chf). one hundred and fifty-four dogs with mmvd and control dogs were studied. there was no difference in age (p . ) or weight (p . ) between the mmvd and control groups. there was a significant difference in serum piiinp (p . ) between normal ( . ae . ), nc ( . ae . ), c ( . ae . ) and chf ( . ae . ) groups. post-hoc comparisons demonstrated a difference between nc and chf groups (p . ). there was no difference in serum piiinp between genders (p . ). in the univariable analysis ckcs (yes/ no) (p . ) was positively associated with serum piiinp. age (p o . ), log (la/ao) (p . ) and lveddn (p . ) were negatively associated with serum piiinp. in the multivariable model including all dogs, lveddn (p o . , b À . ( %ci À . to À . )), age (p . , b À . ( %ci À . to À . )) and ckcs (yes/ no) (p . , b . ( %ci . to . )) were independently associated with serum piiinp. in the multivariable model including only dogs not receiving therapy (n ), lveddn (p . , b À . ( %ci À . to À . )), age (p . , b À . ( %ci À . to À . )) and ckcs (yes/ no) (p . , b . ( %ci . to . )) were independently associated with serum piiinp. in conclusion, serum piiinp decreases with age and with increasing lveddn. ckcs have higher serum piiinp measurements independent of age and lveddn, which may reflect a difference in collagen turnover in this breed. left atrial (la) chamber dilation and congestive heart failure (chf) are common consequences of cardiac conditions in cats. in some cases chf is manifest as right-sided chf (r-chf) or pleural effusion, in other cases chf manifests as left-sided chf (l-chf) or pulmonary edema. it is not always readily apparent as to which cats will develop what form of chf. a general impression has been that la enlargement is associated with the average burden of elevated filling pressures, but little attention has been paid to the function of the la chamber itself. since chf is classically preceded by abnormal atrial chamber dilation and alterations in atrial chamber function, we want to understand how these changes may help us manage or predict chf in the cat. we hypothesized that cats manifesting r-chf have la failure with the la acting primarily as a conduit, resulting in greater pulmonary hypertension, whereas l-chf cats maintain some booster pump and reservoir function. we measured la maximum and minimum areas from right parasternal long axis four-chamber views on d echo, and la m-mode dimensions at maximum, minimum, and beginning of atrial contraction. la area change, fractional shortening, active emptying fraction, and expansion index were calculated from these measurements. right ventricular internal diastolic diameter was also measured on m-mode views. preliminary data revealed that maximum left atrial size is not significantly different between r-chf and l-chf cats on d or m-mode measurements due to high variability. however, total left atrial fractional shortening is significantly reduced in r-chf cats ( . % ae . ) compared to l-chf cats ( . % ae . )(p . ), and r-chf cats have reduced left atrial active emptying fraction ( . % ae . ) as compared to l-chf cats ( . % ae )(p o . ). left atrial expansion ability is poorer in r-chf cats ( . % ae . ) than in l-chf cats ( . % ae )(p . ). these findings may suggest that atrial stiffness and poorer atrial function is associated with a greater degree of pulmonary venous and thus secondary pulmonary arterial hypertension resulting in pleural effusion (r-chf). right ventricular diameter on m-mode was increased in r-chf cats ( . mm ae . ) when compared to l-chf cats ( . mm ae . )(p . ) and normal cats ( . mm ae . )(p . ), which may also be evidence for a greater degree of pulmonary arterial hypertension in these cats. episodic weakness and syncope are common in boxer dogs. reported causes include rapid ventricular tachycardia (vt) and exertion-excitement triggered neurally-mediated bradycardia (nmb) .the purpose of this retrospective study is to describe the features of presumed nmb in boxers. to be included in the study, each dog must have been overtly healthy with a history of exertion-excitement triggered syncope or presyncope; had a normal echocardiogram (ec); had absence of vt and fewer than ventricular premature complexes (vpc) on an initial and subsequent hour holter recordings; and been alive and overtly healthy for at least six months following the initial evaluation. a total of boxers were identified. sixteen were male and were female. most ( %) dogs were either less than or more than years of age. most dogs had multiple, but infrequent, episodes and heart rhythm was documented at the time of an episode in only ( %) and only once (bradycardia) on the first holter recording. owners were instructed to attempt to precipitate episodes. bradycardia related episodes were subsequently recorded in : during the nd ( ), rd ( ) or th ( ) day of hour holter recordings and during the th day of an event recording ( ). collapse and bradycardia were documented during auscultation in additional dogs. the heart rate during syncope was never documented in ( %) dogs. a presumptive diagnosis of nmb was based on the absence of initial and follow-up of ec abnormalities and the presence of no or few vpc during extended ecg monitoring. multiple holter recordings ( - hours) were performed in of ( %) dogs and event monitoring of days ( ) and days ( ) was performed in additional dogs. in conclusion, documentation of the heart rhythm during episodes of collapse was difficult, accomplished in only % and was unlikely during the first holter recording. in boxers with suspected nmb, extended ecg monitoring and implantable loop recorders may be best for hr documentation. arrhythmogenic right ventricular cardiomyopathy (arvc) is an inherited myocardial disease with high prevalence in the boxer dog population, and is associated with sustained monomorphic ventricular tachycardia, sudden cardiac death, and replacement of myocardium with fatty or fibro-fatty tissue. though several genes have been linked to the disease both in humans and in boxers, the etiology of arvc is still unclear. several mechanisms for the development of arvc have been suggested, including dysfunction of the canonical wnt pathway, which results in an arvc phenotype in the mouse. the canonical wnt pathway has been linked to many cellular functions, including growth and differentiation of adipocytes. with the recent discovery that the gene encoding striatin, a protein involved in wnt signaling, may be involved in the development of boxer arvc, we hypothesized that changes in the wnt pathway may also play a role in the etiology. here, we show changes in the localization and decreased amount of proteins affiliated with the canonical wnt pathway. afflicted boxers were identified by -hour holter monitoring and histopathological examination of the heart. samples from the right ventricle rv) of arvc afflicted boxers, and unafflicted dogs ( beagle, mongrels, and german shepherds) were collected, fixed in % formalin, processed, treated with antibodies recognizingcatenin, striatin, and calnexin, and examined using confocal microscopy. western blots were performed on unafflicted rv samples, and arvc afflicted rv samples. frozen tissue samples were homogenized in laemmli buffer, and mg of protein was loaded into each well of a - % gradient gel. -catenin, an integral modulator of the wnt pathway, and striatin were colocalized with the endoplasmic reticulum (er) marker, calnexin. in the unafflicted animals, -catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern, with no detectable localization in the er. in contrast, in the arvc boxers, bothcatenin and striatin were colocalized with calnexin in an er pattern. in the afflicted samples, -catenin and striatin were not visualized to the intercalated disc and intracellular space, respectively. western blots of striatin and -catenin revealed no changes in the amount of protein. interestingly, a western blot for the wnt protein revealed a decrease in the amount of protein in arvc samples, compared to unafflicted samples. our preliminary data suggest that disturbances of the canonical wnt pathway may play an etiological role in the development of arvc in the boxer dog. there are numerous benefits of omega- fatty acid supplementation in human heart disease, including reduction in arrhythmias, decreased incidence of sudden death, and improved survival in heart failure. antithrombotic effects of omega- fatty acids have been demonstrated in people, which may have particular benefit in cats given their risk of thromboembolic complications with cardiac disease. benefits also have been found in canine heart disease, and reduced serum concentrations of the omega- fatty acids, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) have been found in dogs with congestive heart failure (chf) secondary to dcm. to the authors' knowledge, no studies to date have investigated fatty acid concentrations in cats with cardiomyopathy. the purpose of this study was to measure serum fatty acid concentrations in normal cats and cats with cardiomyopathy. serum fatty acid concentrations were measured in normal cats and cats with cardiomyopathy using gas chromatography. cats with cardiomyopathy and at least mild left atrial (la) enlargement (la to aortic ratio . on two-dimensional echocardiography from a right parasternal short axis view) were candidates for study. normal cats had a normal history, physical examination, echocardiogram, packed cell volume, total solids and platelet count. cats with evidence of other systemic disease or those receiving anticoagulants were excluded from the study. normally distributed and skewed data were compared between the cardiomyopathy and control groups with independent t tests or mann whitney u tests, respectively. statistical significance was set at p o . . thirty cats with cardiomyopathy ( neutered males and neutered females) and healthy controls ( neutered males and neutered females) were enrolled. median age was yr (range, - yr) in the cardiomyopathy group and yr (range, - yr) in the control group (p . ). cats in the cardiomyopathy group were classified in the international small animal cardiac health council stage b (n ), (n ), a (n ) and b (n ). compared to control cats, cardiomyopathic cats had higher concentrations of palmitic acid (p . ) and dha (p o . ), and lower concentrations of linoleic acid (p . ). among cats with cardiomyopathy, there was no significant correlation between any serum fatty acid concentration and left atrial size or age. these findings warrant further investigation into the role of fatty acids in cats with cardiac disease. platelet mapping is an application of thromboelastography that relies on the generation of at least three tracings: ma thrombin (maximum platelet activity),ma fibrin (fibrin activity only), an-dma aa or adp (platelet activity not inhibited by arachidonic acid or adp receptor antagonists, respectively). using these three tracings, the % inhibition of platelets can be calculated. the purpose of this study was to evaluate the platelet mapping assay in normal cats and assess platelet inhibition in cats receiving clopidogrel. employee-owned cats with normal history, physical exam, echocardiogram, thromboelastography, packed cell volume, total solids and platelet count were eligible. clopidogrel ( . mg po q h) was administered for days with platelet mapping performed on days and . platelet mapping values were compared using a paired t test, with significance set at p o . . seven cats ( fs, cm, aged - years) were enrolled. compared to day , ma adp (p o . ) and ma fibrin (p o . ) were lower on day . the latter unexpected result prompted measurement of fibrinogen concentrations at day and in the last of these cats. fibrinogen was not different from day to in these cats. these results suggest that platelet mapping may be a simple, outpatient clinical tool to measure antiplatelet activity in cats receiving clopidogrel. this clopidogrel dose resulted in significant platelet inhibition as measured by ma adp in all cats. further studies correlating antiplatelet effects measured by platelet mapping with clinical outcomes in cats with cardiomyopathy are warranted. this study investigated the hemodynamic effects of application of an itd in a canine model of cardiopulmonary arrest. laboratory beagles which were part of a separate terminal study were anesthetized and instrumented for continuous measurement and recording of right atrial pressure, arterial pressure and carotid blood flow. following euthanasia, cpr was performed for one minute, then a pause of one minute followed by a second one minute period at a compression rate of - /minute, ventilation with % oxygen was delivered at eight breaths/min and ml/kg tidal volume. cpr was performed with the itd in place (itd-cpr) and without the itd (s-cpr) for one period each in a randomized fashion with the rescuer blinded to its application. baseline, s-cpr and itd-cpr data were assessed for normality, a kruskal-wallis one-way anova on ranks was used (baseline v cpr). when appropriate a pairwise multiple comparison procedure (dunn's method) was used. percentage of baseline s-cpr v itd-cpr was assessed using the student t-test. the right atrial diastolic pressure was significantly more negative with the itd attached than without (p . ), the coronary perfusion pressure and carotid blood flow were significantly higher during cpr with the itd than standard cpr (p . , p . ). no significant differences in diastolic, mean or systolic arterial pressure or end tidal co were seen. application of the itd resulted in significantly improved hemodynamics during cpr in dogs. clinical evaluation of the device is warranted to examine whether this translates into improved success in resuscitation and survival. left ventricular (lv) systolic dysfunction is a common problem in dogs and can be due to a variety of etiologies. one potential etiology for systolic dysfunction is persistent or paroxysmal tachyarrhythmias, leading to tachycardia-induced cardiomyopathy (ticm). in humans, ticm carries a relatively good prognosis in that remodeling may be reversible with normalization of heart rate. differentiating between primary and secondary tachyarrhythmias in dogs with systolic dysfunction is critical for prognostic purposes as primary tachyarrhythmias may be associated with a better outcome. the goal of our study was to describe a population of dogs with ticm and to determine if treatment of arrhythmias was associated with reversible cardiac remodeling as indicated by standard echocardiographic parameters. medical records of dogs referred to the cardiology service of ksu vmth from to were reviewed. ticm was defined as the presence of severe tachyarrhythmias that were reversible with treatment, systolic dysfunction or ventricular enlargement that improved with treatment of the arrhythmia, or dogs with severe tachyarrhythmias and systolic dysfunction of breeds with that are atypical for idiopathic dilated cardiomyopathy. exclusion criteria were dogs with congenital heart disease, severe mitral regurgitation, and endocarditis. transthoracic echocardiography, thoracic radiographs and electrocardiography (ecg) were performed in all dogs. ventricular enlargement and systolic dysfunction were defined according to standard echocardiographic parameters. arrhythmias were confirmed with a holter monitor in dogs. a total of dogs were included in the study. mean age was years (range - years) with males ( intact, castrated) and females ( spayed, intact). four dogs had pulmonary venous congestion or pulmonary edema and two dogs had ascites. at initial presentation, the meanaesd values were as follows: heart rate ae bpm, m-mode fractional shortening (fs) . ae . %, ejection fraction (ef) using the area-length method . ae . %, and left atrial to aortic root ratio (la/ao) . ae . . initial meanaesd m-mode derived lv internal dimensions corrected for body weight were as follows: diastolic . ae . and systolic . ae . . at least one of the following tachyarrhythmias were identified in each dog: atrial fibrillation ( ), supraventricular tachycardia ( ), junctional tachycardia ( ), and ventricular arrhythmias ( ). ten dogs were available for follow up. seven dogs improved in at least one of the following parameters: resolution of tachyarrhythmia ( ), improved systolic function ( ) antidiuretic hormone (adh) has been shown to be elevated in humans with congestive heart failure (chf). recently, antidiuretic hormone antagonists were successful during investigational treatment of refractory congestive heart failure in humans. adh levels have been only modestly investigated in dogs with cardiac disease, primarily due to the technical difficulty in measuring adh levels via radioimmunoassay. based on the homologous structure of canine and human adh, we aimed first to determine the feasibility of measuring adh in dog plasma using a human elisa kit, and secondly to investigate the level of adh in dogs with congestive heart failure due to acquired cardiac disease. elisa assay kit validation was performed using six healthy dogs with normal clinical and echocardiographic examinations. pooled canine plasma was spiked with synthetic adh and intra-assay precision, dilutional parallelism, and linearity were assessed. to address the second aim of the study, samples were collected from normal dogs and dogs with heart failure due to one of two types of acquired cardiac disease: chronic degenerative valve disease (cdvd) or dilated cardiomyopathy (dcm). patients underwent clinical, radiographic, and echocardiographic examination to confirm diagnosis, assess severity of disease, and determine presence of pulmonary edema. whole blood was collected into edta tubes containing protease inhibitors, cold centrifuged, and plasma was stored at À until analysis. following ether extraction, plasma adh in each sample was measured in duplicate using a human elisa kit. statistical analysis included a d-agostino and pearson test for normality; group results were compared using a nonparametric mann-whitney test. adh was measured in canine plasma using the human elisa kit with acceptable intra-assay precision, linearity, and dilutional parallelism. intra-assay coefficient of variation was %. twenty-four dogs were recruited for the second phase of the study. six normal dogs and twelve dogs with radiographic evidence of pulmonary edema due to either cdvd or dcm were selected for participation. the remaining six dogs were excluded due to lack of definitive radiographic evidence of congestive heart failure. median adh values were . ae . pg/ml for the normal group (n ) and . ae . pg/ml for the heart failure group (n ). median adh values for the two groups were statistically different (p . ). our preliminary results indicate that measuring canine adh using a human elisa kit is feasible and provides results with an acceptable coefficient of variation. we also showed that dogs with congestive heart failure due to cdvd and dcm have elevated adh levels in comparison to normal dogs. our findings motivate further investigation to assess the degree of plasma adh level elevation and the possible use of adh antagonists as an adjunct treatment for refractory congestive heart failure in dogs. aortic thromboembolism (ate) occurs in both cats and dogs. whereas ate in cats is strongly associated with structural heart disease and typically has an acute catastrophic presentation; the pathogenesis and presentation of ate in dogs is less well known or understood. further, an effective antithrombotic strategy for ate in dogs has not been reported. medical records of dogs diagnosed with ate between and were examined retrospectively. diagnosis of ate was based on ultrasonography, doppler flow studies, and diminished or absent femoral pulses. dogs were treated with various acute and chronic antithrombotic therapies. the severity of ambulatory dysfunction was graded as none, mild, moderate, severe, or non-ambulatory at presentation and after therapy. a cohort of dogs in this study received a standardized protocol of chronic warfarin therapy with or without antiplatelet drugs. target international normalized ratio for warfarin therapy was to . twenty-six dogs were diagnosed with ate. all had an apparent mural aortic thrombus caudal to the renal arteries with most having evidence of embolization to the iliac and femoral arteries. none had structural heart disease at diagnosis. twenty dogs ( %) were still ambulatory at diagnosis. the median duration of ambulatory dysfunction prior to presentation was . weeks (range day - weeks). a majority of dogs ( %) had no concurrent conditions at diagnosis. nine dogs ( %) had protein-losing nephropathy. four dogs ( %) were hypothyroid. fourteen dogs were treated with a standard warfarin protocol for a median period of . months (range . - months). eight dogs were treated concurrently with aspirin, dogs were treated concurrently with clopidogrel, and dogs were treated with warfarin only. ambulatory function improved between and grades in dogs treated with chronic warfarin. the median period until clinical improvement was . days (range - days). two dogs treated with chronic warfarin therapy had documented resolution of ate, and dogs had complete resolution of ambulatory dysfunction. none of the dogs treated with chronic warfarin became nonambulatory, died, or underwent euthanasia because of ate. the median period of freedom from an adverse event was . months. no serious hemorrhagic events were reported. four dogs were treated with tpa. three of these had an unfavorable outcome. two dogs were ambulatory before tpa and become non-ambulatory after treatment. two dogs underwent surgical thrombectomy. one had a favorable outcome until ate recurred months after surgery. in conclusion, the pathogenesis of ate in dogs is not associated with structural heart disease or left atrial thrombus formation. the presentation tends to be for chronic ambulatory dysfunction. most dogs are still ambulatory at presentation. warfarin, with or without concurrent antiplatelet therapy, is an effective antithrombotic treatment strategy for dogs with ate. information is known. through previous work, investigators have encountered norfolk terriers (nt) with echocardiographically apparent dmvd in the absence of a heart murmur. in order to more fully understand dmvd in this breed of dog, we sought to characterize findings from the physical and echocardiographic exam, biochemical, biomarker, and nutritional profile, and select environmental variables from a cohort of apparently healthy nt. overtly healthy nt ! yrs old were recruited by different veterinary hospitals and underwent historical, physical, ecg, and d/color-flow doppler echocardiographic exam. anterior mitral valve length, maximal thickness, area, and prolapse were measured from d images. presence of dmvd was defined as thickened, prolapsing, or flail mitral valve leaflets in the presence of color flow doppler evidence of mitral regurgitation. blood samples were obtained for serum biochemistry and serotonin, plasma nt-probnp, amino acid profile, c-reactive protein, and cardiac troponin-i. forty-eight dogs were entered into the study (median age, yrs iqr [ - ]; gender, f, m; median bcs, ). of the dogs, ( %) had murmurs, ( %) had mid-systolic clicks, ( %) had ecg p-pulmonale, and ( %) were deemed to have echocardiographic evidence of dmvd, including nt without a murmur. seven ( %), ( %), and ( %) dogs were classified as isachc , a, and b, respectively. mean indexed echocardiographic mitral leaflet length (p o . ), thickness (p . ), prolapse (p . ), and la:aod (p . ) were significantly different between isachc a/b vs . between isachc a/ b and , there were no differences in serum amino acids, c-reactive protein, troponin, diet, or environmental factors; however different amino acids (ala, gly, phe, pro, trp, tyr) were significantly higher in isachc b vs. a. median serum serotonin was increased in dogs with a/b vs. (p . ). dogs whose diets contained some canned food (p . ) and dogs residing in suburban environments (p . ) had higher serotonin concentrations. nt-probnp tended (p . ) to be higher in isachc a/ b vs. . dmvd appears to be relatively common in nt and echocardiographic changes consistent with mild dmvd can be seen in dogs without a heart murmur. the results of this study establish a foundation of useful information upon which additional prospective studies can be developed. left ventricle (lv) evaluation is one of the most important contributions of echocardiography in the assessment of cardiac function. however, lv analysis can be made from images obtained by different modes and views of the heart. the aim of this study was to compare lv measurements, shortening fraction (sf) and ejection fraction (ef) obtained from four methods: m mode in short-axis and in long-axis, bidimensional mode in short-axis and in long-axis views of the heart. forty normal adult german shepherds were selected. echocardiographic study of lv of each animal was performed by the four methods described above. ancova test was used to examine the effects of axis, mode, weight and gender over lv measurements. isolated effect of the axis was observed for lv end-diastolic diameter (lvedd), with greater values obtained from short-axis views. there was isolated effect of mode over ef and sf, with greater measurements derived from bidimensional mode methods. weight correlated with all linear lv measures at least in one method, but not with ef and sf. weight had positive effect over lv endsystolic diameter and lv end-diastole posterior wall thickness in all methods, except from m mode in short axis in the last one. gender had isolated effect over lvedd, males showing greater values than females in bidimensional mode in short and long axis. the combined effect of axis, gender and weight was identified in interventricular septal end-diastolic thickness. we concluded that normal reference values obtained by different echocardiographic modes and planes should not be used interchangeably. abstract c- assessment of left ventricular diastolic func-tion by color tissue doppler imaging echo-cardiography in maine coon cats tested for mypbc-ap mutation hypertrophic cardiomyopathy (hcm), characterized by increased cardiac mass and diastolic dysfunction, is the most common feline heart disease. myocardial analysis by color tissue doppler imaging (tdi) is more sensitive than conventional echocardiography. this study evaluated diastolic dysfunction in various stages of feline hcm. maine coon cats (n ) were screened for the mybpc-a p mutation and examined with both echocardiography and tdi. then, were phenotypically classified in: normal (n ), suspects for hcm (n ) and hcm group (n ); and genotypically classified in: negative (n ), heterozygous (n ) and homozygous group (n ). myocardial velocities, measured in the basal and mild ventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw) and in radial segment of left ventricular wall, was compared among different groups. a significant decreased (p , ) longitudinal em/am at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. a significant increased (p , ) longitudinal e/em at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. and a significant decreased (p , ) longitudinal sm at the basal segment of the lvw was observed in heterozygous cats compared with negative cats, both without hypertrophy. there was a significant positive correlation between summated early and late diastolic velocities (emam) and heart rate (p o , ); and a positive correlation between sm and em velocities and heart rate (p o , ). the mybpc-a p mutation is not consistently associated with ventricular hypertrophy and negatives cats can also develop hcm. the tdi alone is not able to identify cats with mutation before myocardial hypertrophy. diastolic dysfunction occurs in many cats with hypertrophic cardiomyopathy (hcm) but less is known about systolic function in various stages of hcm. myocardial strain analysis by tissue doppler imaging is a noninvasive echocardiographic method to assess systolic function. this study evaluated systolic function in various stages of feline hcm. maine coon cats (n ) were screened for the mybpc-a p mutation an examined with both echocardiography and strain. then, were phenotypically classified in: normal (n ), suspects for hcm (n ) and hcm group (n ); and genotypically classified in: negative (n ), heterozygous (n ) and homozygous group (n ). peak myocardial strain, measured in the basal and mildventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw), left ventricular anterior wall (lvaw), left ventricular posterior wall (lvpw) and radial segment of left ventricular wall, was compared among different groups. whereas conventional echocardiography demonstrated an apparently normal contractile state based on fractional shortening, myocardial strain (at mildventricular segment of ivs) in hcm cats was significantly decreased compared with normal group (p , ). myocardial strain (at basal segment of lvaw) also was significantly decreased in heterozygous cats compared with negative group (p , ); and was significantly decreased in heterozygous cats compared with negative group, both without ventricular hypertrophy (p , ). there was a significant negative correlation between strain values and wall thickness (p o , ). this method allows detection of abnormal systolic deformation in maine coons cats with hcm mutation despite apparently normal systolic function. the abnormal systolic deformation already can be present in heterozygous cats without hypertrophy and increased with progressive ventricular hypertrophy. recently, multiple advanced resting electrocardiographic (ecg) techniques have been applied in humans for detection of cardiac autonomic and repolarisation function. this has improved the diagnostic and/or prognostic value of short-time ecg in detection of common human cardiac diseases even before onset of symptoms or changes in the standard ecg. therefore, this study investigates, if advanced ecg can predict the severity of mitral regurgitation (mr) in dogs with myxomatous mitral valve disease (mmvd) and thereby improve the diagnostic value of ecg. the study included privately owned cavalier king charles spaniels (ckcss) (age . ae . years; males and females). all dogs were examined by echocardiography and a short-time ( - min) high-fidelity -lead ecg, with the dog in a resting position and in sinus rhythm. dogs were divided into groups according to the degree of mr estimated as the percentage of the left atrium area using color doppler mapping ( %; % o jet %; % o jet %; jet %; jet % and with clinical signs of congestive heart failure). ecg recordings were evaluated via custom software programs to calculate different parameters, including heart rate variability (hrv), qt variability (qtv), t-wave complexity, wave morphology and -d ecg. one-way anova determined ecg parameters, which were significantly different (p o . ) between the dog groups. principal component factor analysis identified a factor model with . % explained variability. qrs dipolar voltage and two repolarization indices of qtv increased significantly with mr severity, whereas total power of the frequency spectrum of rr interval and the standard deviation of qtv decreased significantly with mr severity. for the selected parameters the prediction of mr jet value was tested by multiple linear regression. a correlation coefficient (r) of . indicated that the prediction value was significant (p o . ). if age was included in the multiple linear regression the prediction value was further increased (r . ). our results indicate that for a cut-off criteria of mr ! % jet the five selected ecg parameters could predict the severity of mr caused by mmvd in ckcss with sinus rhythm with sensitivity % ( % with age inclusion) and specificity % ( % with age inclusion) (p o . ). nt-probnp concentration is increased in canine patients with heart disease. relatively little is known about the stimuli for release of nt-probnp in dogs. physical activity independent of cardiac disease and the stress of being in the hospital could influence nt-probnp release and affect diagnosis and management of patients. we hypothesized that nt-probnp concentration in healthy dogs would not exceed the normal reference value ( pmol/l) following a period of exercise. the goal of this study was to examine whether physical activity could elevate plasma nt-probnp and cause false positive results in healthy dogs. the study population included healthy dogs yr of age without heart murmur or known systemic disease, and normal d/color flow echocardiographic exam. plasma samples for nt-probnp were obtained before, immediately after, and hour after a standardized -minute submaximal exercise regimen. the study included dogs with a median age of . yrs and included females and males. there was no statistical difference in median plasma nt-probnp concentration across the three time points (baseline median, [iqr, immediately post, ; p . ). the average coefficient of variation of nt-probnp concentration across the exercise regimen was . ae . %. in of dogs ( . %), nt-probnp increased from to pmol/l immediately after exercise. the results of this study demonstrate that submaximal exercise does not significantly change median nt-probnp concentration and the incidence of false positive results is low. further studies should investigate effects of exercise on nt-probnp concentrations in dogs with heart disease. obesity is an increasing problem in veterinary medicine. obese human patients are shown to present lower levels of natriuretic peptides, regardless of an increased volume and pressure load, what raises the possibility that the natriuretic response is impaired in these individuals. considering the controversial findings in obese humans, and the lack of studies reported in dogs, this study proposed the evaluation of nt-proanp concentration in obese dogs. nt-proanp concentration was determined prospectively in obese dogs ( females; males; - months) and in non-obese dogs (controls; females; males; - months) from a veterinary hospital population. obesity was determined by body condition score [ ( / ); ( / ); ( / ); ( / ); ( / ); ( / )]. dogs were excluded if they had any primary cardiac disease, renal insufficiency, endocrine disease, or if they were receiving diuretics, vasodilators, antiepileptic drugs or corticosteroids. commercial kits were used (vetsign s canine cardio screen nt-pro-anp vc -guildhay/biomedica). mann whitney test was used for group comparison. results are presented as median; interval; p and p ). nt-proanp was significantly lower in obese dogs [ . fmol/ml ( . - . ); p . ; p . ] than in controls [ . fmol/ml ( . - . ); p . ; p . ]; (p . ). results were similar to what has been found in obese humans. lower levels of natriuretic peptides are also seen in obese heart failure patients. this study provides important information regarding nt-proanp concentration in obese dogs, which should be better explored characterizing the behavior of natriuretic peptides after weight loss, and also in obese dogs with primary heart disease. left-to-right shunting patent ductus arteriosus (pda) is one of the most common canine congenital cardiovascular defects. human studies have shown that bnp and nt-probnp concentrations are elevated in patients with pda, and can be used to detect hemodynamically significant pda. the purpose of this study was to measure nt-probnp concentrations in dogs with pda, and to assess whether additional indicators of hemodynamics correlate with ntprobnp. we hypothesized that nt-probnp will serve as a simple non-invasive marker of hemodynamic significance in dogs with pda prior to and following transcatheter ductal occlusion. nt-probnp was measured in client-owned dogs with echocardiographically normal hearts. ten dogs with pda were initially evaluated with thoracic radiographs, transthoracic and transesophageal echocardiography, pulmonary capillary wedge pressure (pcwp) and nt-probnp. nt-probnp and echocardiography were repeated at day and months following ductal occlusion. pcwp was repeated at months. baseline nt-probnp was significantly higher in pda dogs compared to control ( ae pmol/l (mean ae sd), ae ; p o . ). at day and months following ductal occlusion, nt-probnp was ae and ae , respectively. the following decreased significantly from baseline: pcwp ( . ae . to . ae . mmhg; p . ), and indexed left ventricular internal dimensions in diastole ( . ae . to . ae . ; p . ) but not significantly in systole ( . ae . to . ae . ; p . ). nt-probnp is elevated in dogs with pda and transductal closure is associated with a reduction in nt-probnp, pcwp and left ventricular size. cardiac biomarkers, particularly nt-probnp, are becoming more commonly used in dogs and cats as part of a diagnostic work up. multiple studies already have documented the correlation of this peptide with cardiac disease status and potential clinical implications. in a portion of these reports the manner in which samples were handled was placement of whole blood into an edta tube, followed by centrifugation and decanting of the supernatant that was ultimately stored at À c or À c prior to shipment, either with or without protease inhibition. our objective was to compare the nt-probnp concentrations in feline plasma collected using the previously reported methods to the california animal hospital (cah) collection method using tubes containing a protease inhibitor. this study compared nt-probnp concentrations using the protease inhibitor tubes vs. edta tubes from privately owned feline patients, with confirmed cardiac disease, and control feline patients. for all study participants, we performed a full history and physical examination, a hematology and chemistry panel, thoracic radiographs, ecg, and echocardiogram. in each study participant, at least ml's of whole blood was drawn from a peripheral vein, and transferred to a plastic edta tube. the sample was centrifuged within hour after collection. ml of plasma was then transferred to a tube containing a protease inhibitor, which was stored at c until being shipped within hours of collection. the remaining plasma was placed into separate microtubes, which did not contain a protease inhibitor. one microtube was then stored and shipped as previous studies have reported (À c, styrofoam container, shipment within hours), and the second microtube was frozen at À c. all samples were shipped, received and analyzed within hours of collection. results of this study showed that no difference was found between the frozen sample methods ( pmol/l and pmol/l p . ). it was determined that both frozen methods had lower nt-probnp levels ( and pmol/l) when compared to plasma samples shipped in protease inhibitor tubes ( and pmol/l). the findings of this trial demonstrate that the nt-probnp levels are significantly different between samples placed in edta tubes vs. contain protease inhibitor (p . and p . ). utilizing protease inhibitor tubes allows more accurate measurement of plasma nt-probnp. as for its relevance for future research and publications, authors should take care to investigate the manner in which blood samples were handled and the conclusion/results of these studies should be taken in light of the methodologies used in collecting, storing, shipping and analyzing the samples. degenerative mitral valve disease (dmvd) is one of the most common heart disease and is present approximately % of the canine heart disease. although the high prevalence exists in small dogs, the underlying molecular mechanism of its pathophysiology is rarely known. dmvd is functionally and pathologically similar in humans and dogs, thus, there will be a common pathogenesis in human and dogs with naturally occurring dmvd. serotonin and serotonin-related mechanisms have been implicated as a cause of valvular disease in human and animals, including spontaneous dmvd in dogs. increased circulating ht concentration as a potential source of heightened ht signaling is demonstrated in small dogs with dmvd. the aim of this study was to investigate whether serum ht concentrations were associated with severity of naturally occurring dmvd in small dogs and to investigate potential associations of dog characteristics on serum ht concentrations in our study population. forty-eight dogs were included in this study and were classified into control and dmvd groups according to the results of physical and echocardiographic examinations. based on the la:ao ratio, dogs with dmvd were classified as follow: control (la:ao ratio . and no mr), mild (la:ao ratio . and mr), moderate ( . o la:ao ratio . and mr), severe (la:ao ratio . and mr). serum serotonin concentrations were measured by elisa. an overall significant difference (p o . ) was found among groups and ht concentrations (control, . ng/ml [ . - . dmvd, ). significantly higher ht concentrations were observed in dogs with moderate (p o . ) and severe (p o . ) dmvd, compared with concentration in control group. additionally, ht concentration in dogs with moderate dmvd were significantly higher (p o . ) than concentration in dogs with mild dmvd. also, dogs with severe dmvd had significantly higher ht concentration than dogs with mild (p o . ) and moderate (p o . ) dmvd. there was no significant association of age, platelet, and lvidd, on serum ht concentration, however, weak correlation between serum ht increased significantly and la:ao ratio (r . , p o . ) was observed. the results of this study indicate that serum ht concentrations were higher with increasing severity of spontaneous dmvd, which may be the potential cause to advance the progression of dmvd. further studies should be performed to reveal the role of ht in inducing and accelerating spontaneous dmvd and to investigate if lowering serum ht concentration could alter the progression of dmvd. the objective of this prospective study was to evaluate the utility of cardiac troponin i (ctni) in differentiating between underlying etiologies of pericardial effusion in the canine patient. patients were prospectively recruited at time of diagnosis of novel pericardial effusion. serum samples were collected prior to pericardiocentesis. patients were evaluated by echocardiography and classified with the diagnosis of hemangiosarcoma (hsa), heart base tumor (hbt), or unknown etiology at the initial evaluation based on established characteristic echocardiographic findings. idiopathic pericardial effusion (ipe) was defined by histopathology, echonegative for a mass lesion with no recurrence of pericardial effusion months, or symptom free months from time of enrollment. patients were excluded from analysis if a diagnosis could not be established based on above criteria or concurrent moderate azotemia (creatinine . mg/dl) was present at time of sample collection. serum samples were frozen and analyzed in batches within days of collection by a ctni assay with a . ng/ml lower limit sensitivity. sixty-three patients were recruited over a one year period with patients excluded due to lack of diagnosis ( ) or azotemia ( ). median ctni levels of dogs with hsa (n ), hbt (n ), and ipe (n ) were . ng/dl (interquartile range (iqr) o . - . ), . ng/dl (iqr o . - . ), and o . ng/dl (iqr o . -o . ) respectively. concentrations of ctni differed significantly between dogs with hsa and hbt (p . ) and ipe (p . ). there was no difference between ctni concentrations between hbt and ipe dogs (p . ). receiver operating curve analysis to determine the optimal cutoff for differentiation of dogs affected with hsa and both hbt and ipe revealed a significant (p o . ) area under the curve ( . ). a cut-off point of ctni of . yielded a sensitivity of % ( % ci, - %) and specificity of % ( % ci, - %). utilizing a higher cut-off point of . yielded a lower sensitivity of % ( % ci, - %), but a higher specificity of % ( % ci, - %) which may have more clinical utility given the disparity in prognoses of the etiologies compared. in conclusion, this study supports the diagnostic utility of ctni concentrations to delineate between patients with hsa and other etiologies of pericardial effusion, but does not reliably differentiate between dogs with ipe and other neoplastic etiologies. the pathogenesis of degenerative mitral valve disease (dmvd) in dogs remains to be fully elucidated. the high sheer stress caused by mitral regurgitation damages the endothelial surface of the valve, and a previous study demonstrated increased transcription of intercellular adhesion molecule- (icam- ) and e-selectin in affected mitral leaflet tissue. we hypothesized that this may be responsible for platelet recruitment and adhesion, and initiation of a proliferative cascade, resulting in further myxomatous changes. the goal of this study was to compare plasma levels of icam- and e-selectin in healthy dogs and those with dmvd. the study population included dogs with echocardiographic evidence of dmvd and healthy control dogs year old with no heart murmur or known systemic diseases. dmvd dogs underwent d/color-flow doppler echocardiographic exam. blood samples were obtained for plasma icam- and e-selectin analysis using commercially available elisa kits. the study included dogs, of which had dmvd and were normal. the dmvd group had a median age of . yrs ) and included females and males. two ( %), ( %), ( %) and ( %) dogs were classified as isachc a, b, and a, respectively. of the control dogs, median age was . yrs [ - . ], with females and males. there was no statistical difference in plasma e-selectin between control dogs (median . [ . - . ]) and those with dmvd ( . [ . - . ]); p . . plasma icam- concentrations were higher in dmvd dogs ( . [ . - . ]) than controls (median . [ . - . ], but this difference did not reach statistical significance (p . ). linear regression analysis showed no significant correlation between icam- or e-selectin and serum serotonin level, nt-probnp or echocardiographic measures of dmvd severity (la:ao, lvidd:ao, lvids:ao). the results of this study demonstrate no significant difference in circulating adhesion molecules icam- and e-selectin in dogs with dmvd as compared with healthy controls. further studies investigating adhesion molecules within the mitral valve tissue itself are likely needed if icam- and e-selectin play a role in the pathophysiology of dmvd. the rate of glucose utilization in the heart is greater than in other tissues, and impaired glucose uptake may play a major role in the pathogenesis of heart failure (hf). glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (gluts), which includes glut- , the major cardiac isoform, and glut- , a recently discovered isoform, the role of which is unknown in the heart. in addition, despite the wellknown regional differences in myocardial structure and function, potential regional patterns in glucose transport have not been investigated. thus, we hypothesized that glut- and - protein and gene expression would be chamber specific in healthy dogs and during chronic hf. using a canine model of tachypacing induced chronic hf, glut protein and messenger rna in both ventricles and atria were investigated by immunoblotting and real time pcr. in control dogs, glut- , but not glut- , protein expression were significantly higher in the atria compared to the ventricles, with the highest content in the right atrium (ra, p o . ). glut- and - mrna were homogeneously expressed in all the cardiac chambers. during chronic hf, glut - and - expression was highest in the left ventricle (lv, by . and . fold, respectively, p o . ), with a concomitant increase in glut- and - mrna (p o . ). glut - , but not glut- , was decreased in ra during chronic hf (p . ). our data suggest that glut- protein was differentially expressed across the cardiac chambers in the healthy heart. during chronic hf, lv was the primary site dependent on both glut and glut -mediated glucose transport, which was transcriptionally regulated. in addition, the paradoxical decrease in glut content in ra may induce perturbations in atrial energy production during chronic hf. some obese dogs are suspected to have cardiac disease because they have enlargement of the heart on thoracic radiograph. it has been reported in cats that the fat increases the cardiac silhouette, while echocardiograms revealed normal cardiac dimensions. the purpose of this study was to determine whether obesity overestimates cardiac dimension in radiographs compared to echocardiographic findings in dogs. twenty three obese dogs and controls were included based on a - body condition scoring (bcs). computerized radiography was obtained and vhs measurement was performed as previously described. echocardiographic measurements were interpreted based on reference values according to lean body weight regression equations. results for echo and vhs measurements were classified in scores as normal, mild, moderate or severe increase. student's t test was used for comparison of vhs between groups. mann-whitney rank sum test was used to assess echocardiographic scores between groups. spearman rank order correlation was used to assess relationships between any pairs of variables between echo and vhs scores, echo vs bcs and vhs vs bcs. groups were similar regarding age [obese ( ae ); control ( ae ); p . ], breeds and gender distribution. obese dogs had significantly higher vhs and echo scores compared with controls [vhs: ( . ae . ) vs ( . ae . ); p o . ; echo score: range ( - ) vs ( - ); p . ]. there were no relationships between any pair of variables analyzed. these results show that there are changes both in echo and radiographic appearance of the heart in obese dogs, but vhs overestimates cardiac silhouette compared to echo, probably related to pericardial fat accumulation. heart rate variability (hrv) is an indirect measurement of the autonomic modulation of heart rate (hr). reduced hrv measured from short-time electrocardiography is seen in dogs with heart failure (hf) secondary to myxomatous mitral valve disease (mmvd). however, hrv is suggested to increase with disease severity at early stages of mmvd. the aims of this study were ) to associate hr and hrv with severity of mmvd in cavalier king charles spaniels (ckcs) and ) to compare hr and hrv between ckcs and other dog breeds in a group of dogs in hf secondary to mmvd. one-hundred dogs were examined by echocardiography and hour electrocardiography. the dogs were divided into five groups: ) ckcs with no/minimal mitral regurgitation (mr) (mr jet % of the left atrial area using color doppler mapping) and no murmur, ) ckcs with mild mr ( % o jet %), ) ckcs with moderate/ severe mr (jet %) and no clinical signs of hf, ) ckcs in hf (hf defined as left atrium to aortic root ratio (la/ao) . , clinical signs of hf and furosemide responsiveness) and ) non-ckcs in hf. dogs in hf were allowed hf therapy. both hr and hrv were analyzed over a -hour period, while hrv were also analysed over a -hour nightly period. analyses of variance were performed with hr or hrv as response variables and the explanatory variables dog group and echocardiographic indices of mmvd were included separately. all p-values were bonferroni corrected. minimum-and mean hr were significantly higher in ckcs with moderate/severe mr and in hf compared to ckcs with no/ minimal and mild mr (all p o . ). seven out of hrv variables were significantly decreased in ckcs with moderate/ severe mr and in hf compared to ckcs with no/minimal and mild mr (all p o . ). another hrv variables showed the same groupwise differences (all p o . ), except that the difference between ckcs with mild mr and ckcs with moderate/severe mr did not reach statistical significance. mminimum hr, mean hr and the hrv variables ( and ) differing between dog groups, also consistently decreased with increasing mr, la/ao and the proximal isovelocity surface area in ckcs. non-ckcs in hf had a lower minimum hr compared to ckcs in hf (p . ) and a higher triangular index measured in both periods (all p o . ). in conclusion, hr increased and most hrv variables decreased with increasing severity of mmvd in ckcs, even prior to the development of hf. other breeds in hf secondary to mmvd had lower minimum hr, but higher triangular index compared to ckcs in hf. although the cells in the specialized conduction system in the heart are capable of initiating their own impulse, the rate in which those impulses are generated can be influenced by autonomic nervous system. different types of respiratory patterns can stimulate autonomic nervous system in different manners. thus, non-sedated rabbits were studied during forced respiration aiming to evaluate the influence of this breathing pattern on heart rate. twenty male, one-year-old healthy new zealand rabbits were enrolled in the study. animals were set in right lateral recumbency and maintained that way by physical contention. chemical sedation was not used. partial nasal obstruction by digital compression was applied to those rabbits for five seconds, eliciting a forced inhaling and exhaling against semi closed nostrils. heart rate was obtained by measurement of two consecutives rr intervals in the computerized electrocardiography, recorded continuously prior and during the maneuver. heart rate before the intervention was ae bpm (mean ae standard deviation). all rabbits submitted to the maneuver showed a dramatic reduction in this parameter. after nasal partial obstruction, heart rate was ae bpm. data was submitted to statistical analysis by paired student's t test and a significant difference between the heart rate before and after the maneuver was observed (p o . ). although the exactly mechanism involved in this response was not elucidated, the presented data support the applicability of this maneuver as an efficient method for non-pharmacological heart rate reduction in rabbits. obesity can affect cardiac function due to effects on cardiac rhythm, ventricular volume and blood pressure. the purpose of this study was to determine the effects of obesity and overweight on noninvasive systemic blood pressure and doppler echocardiographic parameters in cats without others causes of cardiac hypertrophy. the study groups comprised fifteen obese cats with mean body score index (bsi) of , , seven overweight cats (bsi , ) and seven cats with ideal bsi ( , ). the blood pressure was measured by doppler method and the doppler echocardiography was performed in conscious animals. the statistical analysis was performed by analysis of variance followed by tukey's test and pearson's correlation. the blood pressure values of the obese cats were superior ( , ae , mmhg, p o , ) than in overweight ( , ae , mmhg) and normal cats ( , ae , mmhg) and % of the obese cats had blood pressure higher than mmhg. there were observed differences on the ratio of early (e) and late (a) left ventricular filling velocity (p , ) of obese animals (e/a , ae , ) compared to overweight ( , ae , ) and normal cats ( , ae , ). seven obese cats ( %) had inversion of e/a compatible with diastolic dysfunction and there were negative correlation (r À , , p , ) between the e/a ratio and blood pressure values. other differences observed were increases in left ventricular septum in diastole (p , ) and in free wall in diastole (p , ) and systole (p , ) of the obese animals compared to overweight and normal cats. these results demonstrate the possibility of cardiovascular effects related to obesity in cats, such as systemic arterial hypertension and secondary diastolic dysfunction. diuretic therapy reduces preload, and relieves congestion secondary to cardiac dysfunction. torsemide (torasemide) is a loop diuretic with longer duration of action, less diuretic resistance, and adjunctive aldosterone antagonism as compared to furosemide. we hypothesized that torsemide was no less effective than furosemide at diuresis, control of clinical signs, and maintenance of quality of life in dogs with congestive heart failure. a double-blinded, randomized, crossover clinical trial was performed in dogs with stable heart failure receiving bid furosemide and adjunctive medications. dogs were randomized to their current furosemide dose or torsemide (calculated as / of the daily furosemide dose divided into bid dosing). crossover occurred at day and the study ended on day . clinical, laboratory, radiographic, and owner-perceived quality of life variables were evaluated on days , and . no dog developed recurrent heart failure during the study. average furosemide dose on day was . mg/kg/day (range, . - . ). following torsemide treatment, blood urea nitrogen (p . ), albumin (p . ), and albumin:globulin ratio (p . ) were significantly increased, and urine specific gravity (p . ) and chloride (p . ) were significantly decreased as compared to baseline and/or furosemide dosing (one-way anova with bonferroni correction). no differences in qol were found. results indicate that torsemide is equivalent to furosemide at controlling clinical heart failure in dogs, and might in fact, achieve greater diuresis vs. furosemide. larger clinical trials evaluating furosemide resistance and/or torsemide as a first-line loop diuretic for congestive heart failure in dogs with heart failure are warranted. the purpose of this study was to investigate the feasibility of speckle tracking echocardiography (ste) in healthy cats and to determine whether or not it can detect myocardial dysfunction in cats with diseased heart. radial and circumferential strain and strain rate were measured by ste using left ventricle short-axis view in clinically healthy cats. eighteen cats with hcm whose lv thickness at end-diastole with mm or more were evaluated with ste analysis, and compared with healthy cats. index of left ventricular synchrony (trs-sd) was also assessed in cats with hcm, and compared to healthy subjects. ste resulted in technically adequate images in % of the cats. fusions of early and late diastolic (e and a) wave in strain rate were seen in of cats. percent errors in analysis with or without simultaneous ecg monitoring were . - . % in all parameters. inter-and intraobserver variability of ste parameters in healthy cats was minimal ( . - . %) except for the systolic circumferential strain rate. sedation using buprenorphine and acepromazine did not affect any ste parameter. e wave in radial and circumferential strain rate of hcm cats was significantly decreased compared with healthy cats. no significant difference was seen in trs-sd. ste analysis was considered clinically feasible to assess cardiac function in cats, and could detect myocardial dysfunction in cats with hcm. further study is warranted to investigate to assess whether or not ste can differentiate the etiology of left ventricular concentric hypertrophy since it is clinically important. carvedilol, a rd generation non-selective beta-blocker with ancillary alpha -blocking and antioxidant properties may have therapeutic implications for multiple diseases in cats; however, pharmacokinetics and bioavailability of commercially prepared oral carvedilol has not been determined. hplc for carvedilol measurement in feline plasma was validated and standardization curves created. the pharmacokinetics (pk) of carvedilol was evaluated in apparently healthy male neutered adult cats (average weight of kg) following single dose intravenous (iv) of . mg/kg and single dose oral administration of . to . mg/kg. concentrations of the active parent compound, carvedilol, were detected in plasma using hplc analysis. lower limit of quantification was ng/ml. the mean peak concentration after iv administration of carvedilol was ng/ml (range, to ), elimination half-life was . hours (range, . to . ), and clearance was . l/hr/kg. the volume of distribution was . l/hr. after a single oral administration of carvedilol, the time to peak plasma concentration was minutes (range, to minutes) and the mean residual time was . hours. the half life was . hours. maximal concentration ng/ ml and the mean bioavailability was . % with a median of . % (range, . % to %). these data demonstrate a low bioavailability of oral carvedilol and a wide variation in cats. all cats tolerated the oral dose of carvedilol with no major adverse effects. also, a mean residual time of . hours would suggest that a more frequent dosing schedule may be required to maintain therapeutic plasma levels. pharmacodynamic studies investigating beta-adrenergic blockade duration may provide a more accurate dosing interval of carvedilol. abstract c- effects of sildenafil citrate on dogs with ei-senmenger's syndrome. k nakamura, m yamasaki, h ohta, m takiguchi. department of veterinary clinical sciences, graduate school of veterinary medicine, hokkaido university, sapporo, hokkaido, japan. sildenafil has shown to be effective for dogs with pulmonary hypertension; however, its efficacy for dogs with eisenmenger's syndrome (es) and secondary erythrocytosis has not yet been determined. the objective of this study is to determine the effect of sildenafil for dogs with eisenmeger's syndrome and secondary erythrocytosis. this was a prospective, single arm, open-label study. five clinical dogs with es and secondary erythrocytosis were included. new york heart association (nyha) functional class, pcv, and pulmonary artery acceleration time to ejection time ratio (pa at : et) were evaluated before and after sildenafil therapy ( . mg/kg, bid). nyha functional class was significantly improved after (median ; range - , p . ) and months (median ; range - , p . ) of sildenafil therapy, compared with the baseline (median , range - ). pcv was significantly decreased after month ( . ae . %, p . ) and months ( . ae . %, p . ) of therapy, compared with the baseline ( . ae . %). at : et was significantly increased after month of therapy ( . ae . , p . ) from the baseline ( . ae . ). sildenafil resolved the clinical signs and secondary erythrocytosis in dogs with es. sildenafil therapy could be the treatment of choice for dogs with es. sepsis is the number one cause of mortality in neonatal foals. the role of the raas and hpaa in systemic inflammation and response to stress is well documented in critically ill human neonates, but limited information exists in foals. we hypothesized that in septic foals the raas and hpaa will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality. blood samples were collected on admission from septic (sepsis score ), sick non-septic (sns), and healthy foals of o days of age. blood concentrations of corticotropin-releasing hormone (crh), adrenocorticotropin (acth), cortisol, aldosterone, angiotensin-ii (ang-ii), arginine vasopressin (avp) and plasma renin activity were determined by radioimmunoassays. acth, cortisol, aldosterone, ang-ii and avp concentrations were higher while crh was lower in septic and sns foals compared to healthy foals (p o . ). septic non-survivor foals had higher concentrations of aldosterone, cortisol, acth and avp and lower concentrations of ang-ii and crh than survivors. avp was associated with ang-ii in septic, and with acth in septic and sns foals (p o . ). there was no difference in renin activity and ang-ii concentrations among foal groups. septic foals had a higher acth:aldosterone ratio than healthy foals (p o . ). this study shows that in response to sepsis there is raas and hpaa activation in critically ill foals. we propose that in sick foals avp is more important than crh in regulating acth secretion. the increased acth:aldosterone ratio further supports relative adrenal insufficiency in septic foals. this prospective, cohort study aimed to characterize alterations in coagulation and blood-derived inflammatory biomarkers in adult horses that developed diarrhea during hospitalization. physical and hematological parameters were evaluated at times (onset of diarrhea), , , and h, then every h until resolution of diarrhea or death. each hematological analysis included a complete blood count (cbc), thromboelastography (teg), partial-thromboplastin-time (ptt), prothrombin-time (pt), plasma concentrations of lactate, tumor necrosis factor alpha (tnf-a), interleukin (il)- , il- , il- and nt-proc-type-natriuretic peptide (pcnp). horses were categorized into three groups based on the duration of diarrhea and evidence of systemic inflammation. group : diarrhea o h without systemic inflammation (si); group -diarrhea ! h without si; group -diarrhea with si. assessment of vital parameters and cbc established a diagnosis of si as previously described (levy, ) . descriptive and univariate outcome analyses were based on data normality. horses were enrolled, of which ( . %) survived to discharge. the mean age was . /- . years. eight horses ( . %) were categorized as group- , ( . %) as group- and ( . %) as group- . two horses developed thrombophlebitis. based on the results of teg, / ( . %) were normocoagulable, / ( . %) were hypocoagulable and / ( . %) were hypercoagulable, at one or more time points. of these, / ( . %) group- horses were coagulopathic. additionally, group- horses had a significantly lower ma than group- horses at baseline ( . ae . vs. . ae . ) and h ( . ae . vs. . ae . ). biomarker analyses are pending. in conclusion, si was associated with coagulation disorders in horses with hospital acquired diarrhea. clostridium difficile and clostridium perfringens are commonly associated with colitis and diarrhea in equines but asymptomatic carriers exist. reported carrier rates of toxigenic c. difficile and c. perfringens strains in feces range between - % and - % respectively. toxigenic c. difficile has also been isolated from the small intestine of diseased foals and is implicated as etiologic agent of duodenitis/proximal jejunitis in adult horses however scarce information is available on prevalence in gastrointestinal compartments other than feces in healthy horses, and it is unclear whether fecal samples are good predictors of the status of proximal intestinal sites. the objectives of this study were to investigate the presence of c. difficile and c. perfringens in various intestinal compartments of healthy adult horses and to molecularly characterize isolates. intestinal contents were collected from the stomach, duodenum jejunum, ileum, cecum, right dorsal and left ventral colon, small colon and rectum of euthanized horses free of apparent gastrointestinal disease. enrichment culture was performed for c. difficile and c. perfringens and c. difficile isolates were further characterized via toxin gene detection and ribotyping. c. difficile was isolated from / ( %) samples from / ( %) horses. between zero and three sites were positive per horse, and multiple sites were positive in three horses. isolates were recovered from duodenum (n ), right dorsal colon (n ), small colon (n ) and rectum (n ). in one horse, the rectal sample was negative but c. difficile was isolated from a proximal site, all other horses were positive on the rectal sample if a more proximal compartment was positive. in three horses multiple compartments were positive however different strains were always present within the same horse (n ). all isolates possessed genes encoding toxins a and b. five isolates were ribotype and also possessed genes encoding the binary toxin. the other isolates were ribotype and were negative for the genes encoding the binary toxin. despite using a method with a detection level as low as cfu/g of feces, no c. perfringens was recovered. rectal samples were a good predictor of overall c. difficile carrier status ( / horses), however rectal samples were not always representative for the ribotype carried in more proximal compartments. the presence of variable strains within the same horse suggests transient passage of the bacterium through the gastrointestinal system rather than actual colonization although further study testing multiple colonies per site is needed. the predominance of ribotype is consistent with recent emergence of this strain in this region, as earlier studies found other strains ( , ) to be more prevalent and a variety of ribotypes were typically recovered from horses. interestingly ribotype has recently emerged as a hypervirulent strain in humans in our area. clostridium difficile, clostridium perfringens and salmonella are important enteric pathogens in horses, however some healthy animals also harbour these pathogens. point prevalence studies have reported these carriage rates, but there are no data regarding longitudinal prevalence of these enteric bacteria, information that would be useful to better understand the epidemiology of these pathogens. additionally, antimicrobial resistance is a pressing concern. commensal e.coli is often used as an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data from horses on farms. the objectives of this study were to longitudinally investigate the above enteric pathogens over the course of one year, molecularly characterize obtained isolates and determine the antibiotic susceptibility profile for e. coli. fecal samples were collected from adult horses from five farms on a monthly basis over the course of one year. selective cultures were performed for c. difficile, c. perfringens, salmonella and e. coli. c. difficile isolates were characterized via toxin gene pcr and ribotyping. broth microdilution was performed to assess antimicrobial susceptibility profiles of e. coli. clostridium difficile was isolated from / ( . %) samples from / ( %) horses. four horses were positive on more than one occasion, three were positive in two consecutive months. different ribotypes were found in two of the latter horses. most isolates were ribotype (n ) with ribotype (n ) and ribotype c (n ) also identified. ribotypes and c possessed genes encoding toxins a, b and binary toxin, while ribotype only possessed toxin a and b genes. despite a detection threshold of cfu/g feces, c. perfringens was not detected in any samples, nor was salmonella. e coli was isolated from / ( %) samples. resistance to ! antimicrobial was present in only / ( . %) isolates. multidrug resistance (! antibiotics) was present in / ( %). most commonly, isolates were resistant to sulfisoxazole ( / ) and trimethoprim sulfamethoxazole ( / ). the overall detection rate for toxigenic c. difficile in fecal samples of healthy horses was . % which is consistent with previous studies. the cumulative prevalence of % was striking but only one horse shed the same strain for more than one month, indicating c. difficile shedding is a transient and dynamic state. the predominant isolation of ribotype is consistent with the suspicion that this strain has emerged and become widely disseminated in this region in recent years. the low prevalence of c. perfringens and salmonella is in agreement with some other studies. the low prevalence of antibiotic resistance in commensal e. coli was encouraging and suggests that healthy horses on pleasure horse farms are not likely a major reservoir of resistance in enteric bacteria. type polysaccharide storage myopathy (pssm) in horses is associated with a dominant missense mutation (r h) in the skeletal muscle glycogen synthase gene (gys ). since disease severity varies between affected horses, we hypothesised that some clinical variability could be accounted for by the underlying genotype. belgian / percheron horses were genotyped using a validated restriction fragment length polymorphism assay enabling grouping of horses as homozygotes (hh), heterozygotes(hr) or normal (rr). subsequently, semimembranosis muscle samples were biopsied from each of six matched sedentary horses from each group; one sample was formalin-fixed and one fresh frozen. sections were stained using haematoxylin and eosin, periodic acid schiff /diastase. anti-dystrophin, nnos and myosin heavy chain immunohistochemistry was performed to examine sarcolemmal intregrity, there were significant differences in resting ck activity (p . ) (median hh u/l interquartile range(ir) - ; hr u/l ir - ; rr u/l ir - ) and ast activity (p o . ) (ast mean hh u/l sd ; hr u/l sd ; rr u/l sd ) and muscle pathology between the groups, with severity increasing rr o hr o hh. there were significantly more type a (p . ) and fewer type x fibres (p . ) in homozygotes ( a % sd . ; x % sd ) compared with the other groups (hr a % sd . , x % sd . ; rr: a . % sd . x % sd . ). more type a fibres contained polysaccharide inclusions in homozygotes ( % sd . ) than in heterozygotes ( . % sd . ) (p o . ). both dystrophin and nnos expression was normally localised to the sarcolemma in pathologically normal and vacuolated fibres from mutant horses. in conclusion, sedentary homozygotes have more severe skeletal muscle pathology and higher resting plasma ck and ast activities than heterozygotes, and pssm is associated with a fibre type shift towards type a. although subsarcolemmal vacuolation likely disrupts the contractile apparatus's attachment to the sarcolemma, the latter's integrity appeared intact. the recumbent horse presents a logistic, diagnostic, and therapeutic challenge to the equine practitioner. there is currently very little data available on the prognosis and outcome of horses that are recumbent. therefore, the purpose of this study was to investigate the outcome of hospitalized horses that had been recumbent in the field or in the hospital and the factors affecting their survival. records of horses admitted to the school of veterinary medicine, university of california davis from january of to december of with a history of recumbency or horses that became recumbent while hospitalized were evaluated. a horse was defined as recumbent if it was unable to stand on its own. the medical record was examined for the following criteria: history pertaining to the current illness including treatment by the rdvm, breed, age, weight, date of presentation, physical and neurological examination findings, cbc and biochemical profile results, initial drugs administered on arrival, time spent recumbent, time spent in a sling, diagnosis, and hospitalization costs. statistical analysis correlating factors associated with survival was performed using logistic regression. overall there were non survivors and survivors. factors that favored survival included early initiation of treatment in the field by the rdvm, horses that tolerated a sling and spent more time in a sling, increased duration and costs of hospitalization, horses that were recumbent post anesthesia, and those recumbent due to disease of the musculoskeletal system. factors that increased likelihood of non survival included horses that were ataxic on presentation, horses with increased bun, horses that spent more time recumbent, those that did not tolerate a sling, and horses diagnosed with botulism and spinal cord disease. in conclusion, this retrospective study demonstrated that both the cause of recumbency and the ability of horses to tolerate a sling had a direct effect on survival. abstract e- plasma peak and trough gentamicin concentra-tions in hospitalized horses receiving once daily gentamicin. jr read , pa wilkins , rd nolen-walston . university of pennsylvania, new bolton center, kennett square, pa. university of illinois, champaign-urbana, il. gentamicin is often used to provide gram negative antimicrobial coverage in horses at . mg/kg iv every hours. therapeutic drug monitoring in our hospital suggests larger doses are required in many clinical cases to achieve the desired concentration ( -  minimum inhibitory concentration) for common bacterial isolates (peak target range - mg/ml). the aim of this study was to determine the correlation between gentamicin dose and plasma concentration in hospitalized horses receiving gentamicin treatment in order to identify an optimum dose range for this population. review of records ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) identified horses ! months old receiving once-daily gentamicin with peak and trough assays performed (n sets). spearman rank correlation coefficient analysis revealed a weak (r . ) but statistically significant correlation (p . ) between gentamicin dose and peak plasma concentration. horses receiving . - . and . mg/kg gentamicin (groups and ) had higher median peaks ( mg/ml) than horses receiving . - . mg/kg (group ; . mg/ml). higher doses were more likely to result in peaks mg/ml ( and %, groups and respectively) than horses receiving . - . mg/kg (group ; %). all hour post-gentamicin administration trough values were o mg/ml. no correlation was found between dose and change in plasma creatinine during treatment, nor dose and trough level. these data suggest that gentamicin dosage in horses should be individually determined by therapeutic monitoring. additionally, these data support an initial dose of . - . mg/kg iv every hours in order to achieve desired peak concentration and an appropriately low trough concentration. heaves is a common respiratory inflammatory disease, characterized by a pulmonary neutrophilia. this disease is also characterized by an activation of circulating neutrophils after antigen challenge but their specific role in heaves is not well understood. also, there are anecdotal studies concerning heaves-affected horse to be more susceptible to infection. however, to our knowledge, the antibacterial host defense role mediated by circulating neutrophils was not investigated in heaves-affected horses. the objective of this study was to compare phagocytosis activity and bacterial killing by circulating blood neutrophils of heaves-affected and control horses. peripheral neutrophils were isolated from heaves-affected (n ) and control (n ) horses using a density gradient technique. the killing capacity was assessed by incubating neutrophils with streptococcus equi spp equi and spp zooepidemicus. after h of bacterianeutrophil coculture, total viable bacterial cells were measured by quantitative plating. the phagocytosis was evaluated by flow cytometry using fluorescent beads and gfp-transformed streptococcus suis suilysin-negative mutant strain. circulating neutrophils from heaves-affected horses showed a significant decrease in their killing capacity toward s. zooepidemicus (p . ). a reduced, although not significant (p . ), killing capacity of s. equi by these neutrophils was also observed. the phagocytosis activity was not different between groups. this impairment of blood neutrophil bactericidal activity in heaves-affected horses could contribute to an increase susceptibility to infection. obesity is a common disorder of the horse, with current prevalence estimated at %. in people, obesity is associated with dyslipidemia, insulin resistance, mitochondrial dysfunction and downregulation of lipid and glucose metabolic pathways. in the horse, obesity is similarly associated with insulin resistance and alterations in lipid profiles; however, metabolic regulatory gene expression profiles have not been fully characterized. we hypothesized that obese horses have decreased expression of metabolic regulatory genes and decreased mitochondrial content in skeletal muscle compared with non-obese horses. sixteen light breed horses, - years of age were included. body condition score (n ) and neck circumference (n ) were recorded. post-mortem biopsy samples of the semi-membranosus muscle were obtained. dna and rna were isolated. relative expression of the metabolic genes, peroxisome proliferator activated receptor g (pparg), pparg coactivator- a (pgc- a), fatty acid translocase (fat) and estrogen related receptor a (erra) was determined by quantitative polymerase chain reaction (qpcr). mitochondrial content was assessed by determining mitochondrial dna/nuclear dna ratio by qpcr, using nadh-dehydrogenase subunit and cytochrome oxidase subunit as mitochondrial genes and beta actin as the nuclear reference gene. non-normal data was log transformed for analysis and a pearson coefficient of correlation was calculated for relative gene expression, body condition score and neck circumference. a value of p o . was considered significant. body condition score was strongly correlated with neck circumference (n , r . , p . ). relative expression of erra and glut- increased with body condition score (erra: n , r . , p . ; glut- : n , r . , p . ). copy number of the mitochondrial genes (nadh-dh and cox- ) was not related to body condition score or metabolic gene expression. expression of glut- , erra, pparg, and fat were strongly correlated to each other, but not pgc- a. there was a strong trend towards correlation between pparg and pgc- a in horses with body condition score (n , r . , p . ). in this study, there was no change in mitochondrial content in obese horses. assessment of mitochondrial function in obese horses and horses with ems is under way. the strong correlation between pparg and pgc- a observed only in horses with high body condition scores suggests this pathway is activated with obesity. the role of pparg and pgc- a in equine obesity should be further investigated to determine their potential as therapeutic targets. upregulation of erra and glut- in horses with increasing body condition score is unexpected, and may indicate a compensatory response to dysfunction of a downstream pathway. further studies to better define the role of metabolic regulatory gene expression in obese horses and those with ems are ongoing. previously presented at the th annual harold hamm diabetes center research retreat, oklahoma city, ok. inflammatory bowel disease is a cause of weight loss, decreased performance, and colic in horses. this condition is difficult to diagnose and clinicians rely upon absorption tests to document malabsorption. the purpose of this study was to compare glucose and xylose absorption tests in normal horses and determine the repeatability of these procedures. eight horses received mg/kg dextrose or d-xylose powder mixed as a % solution in water or water alone via nasogastric intubation on three different occasions within the same week for three consecutive weeks ( tests/horse). a crossover design was employed and the order of treatments was randomized. blood samples were collected at time , , , , , , and min. data were analyzed by repeated measures anova and t-tests. results showed that the xylose response over time differed significantly from the glucose response over time (test  time; p o . ). mean time to maximum concentration differed (p o . ) between tests (glucose min; xylose min). within-horse area under the curve, maximum concentration, and time to maximum concentration values for dextrose and xylose did not differ significantly when tests were repeated. results indicate that glucose and xylose absorption tests are repeatable within the same horse, but plotted curves differ between tests, with peak concentrations occurring at a later time point for the glucose absorption test. we conclude that both tests provide repeatable measures of intestinal absorption, but glucose and xylose appear to differ in their rates of absorption and clearance. the purpose of this study was to examine the records of a population of thoroughbreds with cervical vertebral malformation (cvm) and to determine which factors have an effect on these horses achieving athletic function. this was a retrospective case study of thoroughbreds with cvm treated medically from to . forty-one were euthanized after diagnosis, while the remaining were discharged for treatment. racing records were reviewed to determine which horses raced after treatment. horses were separated into groups based on whether or not they raced. medical records were reviewed, and results of neurologic examination, radiographic and laboratory findings, treatments, and outcome were assessed and compared between groups. twenty-one of horses treated medically ( %) improved enough to race. median neurologic grade between groups was significantly different (p o . ), with a hind limb grade of . (range - ) for the raced group and . (range . - ) for the unraced. intravertebral sagittal ratios measured from standing lateral cervical radiographs were equivocal between groups. radiographs of all horses were examined for kyphosis, dorsal over-riding arch, caudal epiphysitis, degenerative joint disease, cystic bone lesions, and cranial stenosis of the vertebral canal. horses with kyphosis (p . ), degenerative joint disease (p . ), or cranial stenosis (p . ) at any site were less likely to return to racing. racing prognosis for horses with cvm treated conservatively is equivalent to that of those treated surgically as reported by rush moore et al (javma, ) . radiographic changes and neurologic grade may help serve as indicators for whether a horse will respond to conservative therapy. since pain assessment is vital for management of colic, a valid, reliable and feasible tool for assessing the severity of acute abdominal pain in horses is urgently needed. our aim was to construct and validate a behavior-based pain scale by methodology utilized in construction of pain scales in non-verbal humans. the project consisted of four stages. firstly, behaviors to include in a scale were empirically identified. thirty equine clinicians noted behaviors in each of random film clips of horses with colic using a checklist. nine behaviors (e.g. rolling, pawing, and flank watching) demonstrated good inter-observer agreement without bias (multi-rater kappas: . - . ). secondly, the clinical judgment of experts was utilized to identify and to weight behaviors. six expert clinicians independently expressed opinions as to which of behaviors to include and the severity of pain they indicate. two contending scales (equine acute abdominal pain scales (eaaps) & ) were constructed based on both the empirical and the judgmental approaches. each included identical behaviors with a - point score range; eaaps- with gradations to some of the behaviors and eaaps- without. in the third stage, blood cortisol and lactate levels and heart rate were shown to only approximate pain since they correlate poorly with degree of pain as assessed by visual analog scale (vas) in horses with colic and controls (spearman rho; lactate . ; cortisol . ; heart rate . ). finally, reliability and validity of the pain scales were evaluated including constructs of pain; face validity, convergent and discriminate validity and extreme groups. thirty of films of horses with colic were randomly presented to expert equine clinicians internationally who were randomly allocated into three groups to score pain; one group by both vas and numerical rating scale (nrs)), and two groups, each by one of the two eaaps scales. inter-observer reliability of both eaaps scales was excellent (intraclass correlation . ). intra-observer reliability based on scores given for identical films demonstrated; % and % agreement, kappa . and . , and spearman's rho . and . for eaaps- and , respectively. both scales varied by score between observations. face validity; each group reported their scale to be valid ( % & %). convergent validity; the scales compared favorably with vas/nrs scores (spearman's rho: . - . ). discriminate validity; correlation to heart rate, lactate and cortisol levels was predictably low (rho . - . ). extreme group validity; colic horses scored significantly higher than control horses; scores of . - . in controls versus . - . in cases. in conclusion, methodology established in human medicine but novel in veterinary medicine was used to construct and validate two clinically feasible equine abdominal pain severity scales that showed excellent reliability and validity. further refinement of the eaaps scale is advised prior to introduction into clinical practice. aortic valve prolapse (avp) is a common echocardiographic finding in horses, but when compared with mitral valve prolapse in dogs, little is known about the natural progression of this condition. previously published data has shown that echocardiographic identification of avp in horses is reliable, diagnostic criteria have been established and that development occurs with training. the aims of this study were to evaluate the different rna and protein expressions of smooth muscle actin (sma), transforming growth factor-b (tgf), nitric oxide synthase (nos) and the concentrations of elastin and collagen in normal, prolapsing and diseased cusps to evaluate what structural changes may predispose them to prolapse. valve cusps were harvested and processed from a group of horses at a commercial abattoir following disease classification using echocardiography. horses were aged . ae . years, weighing ae kg and with a median body condition score of / . cusps were collected in rnalater s and stored at À c prior to processing. cdna was produced from half a valve using a standard protocoland qrt-pcr performed to assess relative rna expression of sma, tgfb , endothelial (enos) and inducible nos (inos) and compared with the housekeeping gene s. a quarter of cusp was processed using an adapted commercial protocol to evaluate protein expression of sma, tgf b , enos and compared to vimentin. specific antibody binding was assessed with western blotting and protein expression evaluated using dot blots. the remaining quarter cusp was used to measure soluble collagen and elastin concentrations using commercial assays . statistical analyses included one way anova with post-hoc bonferoni, paired student's t-test, linear and logistic regression. there was no effect of gender or age on any of the measurements. valves from animals with avp had lower expression of sma and elastin compared to normal and diseased valves, increased expression of tgfb and enos, whereas inos expression was greater than normal valves (table ) . collagen content of valves from horses with avp was increased compared to normal but lower than horses with valve disease. prolapsing cusps appear to be a different phenotype from diseased cusps. further studies will help to elucidate the significance of these findings in vivo. a clear association between heart rate (hr) and body mass has been observed across a wide range of mammalian species. furthermore, it is well known that electrocardiographic (ecg) time intervals vary with heart rate and body mass. within the equine species, small breeds are generally thought to have higher heart rates than large breeds. however, despite the large differences in size among different equine breeds, there is little information about normal heart rates and normal ecg time intervals in horses and ponies of different body size. similarly, the relationship between hr and body mass in dogs of various breeds and sizes is still under debate. the goal of this study was to investigate the relationship between heart rate and ecg time intervals to body mass in apparently healthy horses and ponies and to calculate normal ranges for different weight groups. adult horses and ponies at an age of . ( - ) y [median (range)] and a body weight of ( - ) kg were included in the study. all animals were considered clinically healthy based on history and physical examination. a standard base-apex ecg was recorded at a speed of (n ) or mm/s (n ) using a multiparameter monitor (datascope passport). during the procedure, the horses were unsedated, standing quiet in a box stall. mean hr over sec was determined for each recording. the following ecg time intervals were measured in triplicate and averaged for further analyses: pq interval, qrs duration, qt interval, and difference between qt and qrs (qt-qrs duration). the relationship between hr, ecg time intervals, and body mass was assessed using linear regression analyses. normal ranges ( . % to . % percentile) were calculated for different weight groups. the level of significance was p . . heart rate was inversely related to body mass (p o . , r . ). the pq interval (p o . , r . ), qrs duration (p o . , r . ), qt interval (p o . , r . ), and qt-qrs duration (p o . , r . ) were directly related to body mass. normal ranges for hr, pq, qrs, and qt within the different weight groups were - bpm, - ms, - ms, - ms (o kg); - bpm, - ms, - ms, - ms ( - kg); - bpm, - ms, - ms, - ms ( - kg); - bpm, - ms, - ms, - ms ( - kg); and - bpm, - ms, - ms, - ms ( kg). we conclude that in healthy horses there is a significant but weak relationship between body mass and hr and between body mass and ecg time intervals, respectively. this study therefore supports the hypothesis that within the equine species, small breeds have faster heart rates and shorter ecg time intervals than large breeds. therefore, body mass has to be considered when comparing hr and ecg time intervals to normal ranges in horses. horses with pituitary pars intermedia dysfunction (ppid) often have elevated plasma acth concentrations. however, ppidaffected horses rarely have resting serum cortisol levels above the reference range or adrenal hyperplasia. we hypothesized that this apparent dissociation between plasma acth levels and adrenal response in horses with ppid is due to the secretion of acth that is less biologically active than that from normal horses. to test our hypothesis, a bioassay to evaluate acth activity was developed. adrenocortical explants were harvested aseptically from normal horses at euthanasia and stimulated with plasma from healthy (n ) and ppid-affected horses (n ). the assay was performed three times with explants obtained from different horses. cortisol secreted by the explants and plasma acth levels were measured with commercially available radioimmunoassays. cortisol secretion stimulated by each sample was standardized to the respective explant protein concentration. cortisol data was normalized for acth concentration in each plasma sample and expressed as a cortisol:protein:acth ratio. ratios from horses with ppid and normal horses were compared by unpaired t-test. horses with ppid had significantly lower cortisol:protein:acth ratios compared to normal horses. (assay : . ae . vs. . ae . , p o . ; assay : . ae . vs. . ae . , p o . ; assay : . ae . vs. . ae . , p o . ). these results suggest that plasma acth from ppid horses is less biologically active than plasma acth from normal horses. our findings give further insight into the pathophysiology of ppid and may aid in the development of novel diagnostic testing protocols. an online survey was conducted to determine perceived needs of potential employers of new acvim-laim diplomates. the survey was designed as the first step in determining what is needed for success in the various sectors of practice employing acvim-laim diplomates. demographic and background data were collected using questions and drop-down menus on the first page. the survey evaluated skills or concepts in areas of veterinary practice. participants answered questions about each skill or concept using drop-down ranked lists. those participants that had completed an acvim-laim training program were asked additional questions about whether they were taught the skill or concept during their own residency. data were collated and descriptive statistics calculated. the mean scores or frequencies of use for each skills or concepts were ranked to determine which of the skills or concepts were most important for an entry-level diplomate. eighty-eight individuals participated in the survey with respondents being acvim diplomates, respondent was not board-certified and respondent was an act diplomate. nineteen respondents were diplomates of acvim and an additional specialty. eighty-three respondents had completed an acvim residency. the majority of respondents were in academia ( %) with % being in private practice. equine specialists prevailed ( %) followed by mixed large animal ( %) and then food animal only specialists ( %). the distribution of years post-residency was slightly skewed toward younger diplomates, but overall there was a good distribution of diplomates across years of experience. most respondents stated that they did not make hiring decisions in their practice. competency in disciplines other than internal medicine was expected with ultrasonography and radiology being the most desirable followed by theriogenology and lameness. surgical skills, both equine abdominal ( %) and food animal general surgery ( %) were considered important by some respondents. thirty-six per cent of respondents thought that a new diplomate should expect to make o $ , per annum, while only % of respondents thought that a new diplomate should expect to make ! $ , per annum. not all respondents answered questions on all skills or concepts. the mean number of skills or concepts evaluated was (sd ) with only respondents answering all . all skills or concepts evaluated were found to be at least somewhat important, were estimated to be used at least occasionally, were recommended for inclusion in training programs as core or elective, and some level of knowledge was expected. at least some of the respondents were taught each of the skills or concepts during their residency, practiced the skill or concept at least occasionally during their residency, and some degree of competency was expected at the time of completion of their residency. these data will provide a framework for designing future laim residency programs. abstract this study evaluated pharmacokinetics and clinical safety of an oral paste formulation of a commercially available cox -sparing nsaid in clinically healthy pony foals in a randomized controlled clinical trial. values for complete blood count, serum chemistry profile, urinalysis, pharmacokinetic assay, and gastric endoscopy were evaluated in eighteen shetland pony foals treated with firocoxib ( . mg/kg, po, q h) or placebo for days. foals were divided into treatment groups. group and foals received firocoxib while a rd group was administered an oral placebo. gastric endoscopy was performed on group and foals prior to treatment and on days and to monitor for the presence of gastric ulcers. group and foals had blood and urine samples taken sequentially for pharmacokinetic analysis, cbc, serum chemistry evaluation, and urinalysis. physical examinations were performed prior to treatment and daily for days. data were analyzed using anova and paired t-tests (p o . ). none of the foals presented adverse clinical effects. there were no significant changes in cbc, biochemical profiles within groups, or differences between groups. pretreatment gastric endoscopy scores were not significantly different from evaluations at and days. firocoxib was quickly absorbed with an observed maximum concentration at hr, the first sampling interval, for the majority of animals. firocoxib plasma concentrations decreased in a log-linear manner after reaching the maximum concentration and steady state concentrations were achieved by the th dose. based on the sampling times after the final and th dose, an average half life of . days was estimated. administration of firocoxib did not cause any adverse effects on gastrointestinal, or hematological or serum biochemical variables, appears to have been well tolerated, and follows a predictable pharmacokinetic pattern in - week old foals. equine herpesvirus (ehv- ) is highly prevalent in most horse populations. horses are routinely vaccinated against ehv- , and neutralizing antibodies have helped to prevent disease. however, the usda has recently classified ehv- myeloencephalopathy (ehm) as an emerging disease, in response to the apparent increase in incidence, morbidity, and mortality of ehm that suggests a change in virulence of the virus. it has been reported that cellular immune mechanisms, in particular cytotoxic t-cells (ctls), are important in controlling ehv- viremia. interferon-alpha (ifn-a) has a key function in innate immune regulation by inducing the differentiation and maturation of ctls. here, we investigated the influence of abortogenic (racl , ny ) and neuropathogenic (ab ) ehv- virus strains on ifn-a, il- and il- secretion in equine pbmc. equine pbmc were infected with racl , ny or ab ehv- strains or kept in medium for hours. ifn-a, il- and il- secretion was detected in the supernatants by a fluorescent bead-based cytokine assay. the production of ifn-a increased with increasing viral doses and similarly for all three ehv- strains. the production of the antiinflammatory cytokine il- was significantly decreased after ab infection compared to racl and ny strains at viral infection doses of moi . - . at high doses (moi ), il- production was suppressed by all three ehv- strains. the results suggested that abortogenic and neuropathogenic ehv- strains equally induce antiviral ifn-a production in equine pbmc. they also illustrated the differences in the ability of ehv- strains to modulate anti-inflammatory il- . neuropathogenic ab strain had an increased potential to down-regulate il- production suggesting specific viral mechanisms that interfere with the control of inflammation in the host. the variations in innate il- secretion might influence the development of protective immunity and might offer an explanation why neuropathogenic ab induces more severe disease, including myeloencephalopathy, than abortogenic ehv- strains. previously presented at a conference of research workers in animal disease. rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. treatment protocols are long, expensive and do not always produce good results. rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. recent studies are based on the use of the virulence associated protein a (vapa) which has been described as an important inducer of immunity against r. equi. the present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of s. enterica typhimurium expressing vapa antigen (test group) or s. enterica typhimurium without the vapa gene (control group), previous to and following experimental challenge. two experimental phases were established according to the immunization route: intranasal or oral vaccination up to hrs following birth and at days of age. the experimental and control groups were challenged on day with a virulent stain of r. equi. clinical examination, cbc and image complementary exams were used to evaluate the development of clinical signs. immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation assays, isolation of r. equi and cytological profiles of tbw. clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group ( / ) and was due to r. equi pneumonia. the test group produced a more intense iggb response when compared to controls however no statistical difference was observed. lymphoproliferation and th cytokine expression were higher in the test group. in contrast, controls produced an il- response. local iga was significantly higher in animals immunized with salmonella carrying vapa. immunization protocols produced no severe toxic effects. the vaccination of neonatal foals with s. enterica typhimurium expressing vapa was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental r.equi infection. this study was conducted to test the hypothesis that the kd protein, myristolated alanine-rich c-kinase substrate (marcks), is involved in equine neutrophil migration and adhesion. in other species, marcks phosphorylation and dephosphorylation causes the protein to cycle between the cell membrane and cytosol, respectively. to investigate marcks phosphorylation in horses, neutrophils were isolated from whole blood and stimulated with platelet activating factor (paf), leukotriene b (ltb ) or phorbol myristate acetate (pma). western blot was performed using specific phospho-marcks and total marcks primary antibodies. these results determined marcks phosphorylation is maximal seconds following stimulation and that dephosphorylation occurs within minutes. to investigate the requirement for marcks in equine neutrophil chemotaxis, isolated neutrophils were pre-treated with mans (a cell permeant peptide identical to the n-terminal amino acids of marcks), rns (a control peptide) or vehicle control (vc) prior to a migration assay toward known neutrophil chemoattractants (ltb or paf). pre-treatment of equine neutrophils with mans significantly inhibited migration while rns pre-treatment had no effect. to investigate marcks requirement in equine neutrophil adhesion, mans, rns or vc treated cells were stimulated to adhere to immulon plates coated with % fbs. pre-treatment of equine neutrophils with mans significantly inhibited adhesion while rns pre-treatment had no effect. inhibition of marcks using a cell permeant peptide identical to the protein's n-terminus significantly inhibited equine neutrophil adhesion and migration. these results indicate that marcks is an important regulator of equine neutrophil chemotaxis and represents a potential target for anti-inflammatory therapy. amongst other tests, a thorough neurologic examination of horses may include walking with the head elevated and during blindfolding, in order to help differentiate normal from abnormal and to help with neuroanatomically localising any lesion(s) i.e. in the ataxic horse. consensus amongst equine neurologists suggests that gait abnormalities associated with these specific tests are often exacerbated in horses with underlying proprioceptive deficits however the effect of these tests on temporal gait characteristics in normal horses has not previously been assessed quantitatively. we hypothesized that head elevation or blindfolding, in comparison with walking in a straight line would result in a compensatory decrease in lateral (left front-on to left hind-on and right front-on to right hind-on) and diagonal coupling intervals (left front-on to right hind-on and right front-on to left hind-on) in normal horses. four thoroughbreds without any history or clinical signs suggestive of neurological disease (age range to years) were included in the study. retroreflective markers were applied to the withers, to the sacrum and to left and right tuber coxae; for each limb, lateral fetlock markers and dorsal and lateral hoof wall markers were used. a minimum of trials each with - walk strides for each task were analysed as horses walked across an -force-plate runway i surrounded by a -camera kinematic system. ii force-plate data were processed with semi-automated custom written matlab iii scripts. data were analysed with a mixed model using the statistical software r. there was a significant fixed effect of normal walk on a straight line and head elevation on left and right lateral coupling intervals (p o . ) and of the left and right diagonal coupling intervals (p o . ). there was no significant effect of blindfolding on neither lateral nor diagonal coupling intervals. the random effect of horse had no influence on the coupling intervals. the decrease of the lateral coupling intervals indicates a tendency towards a pacing gait during head elevation. we conclude that there is a significant change in temporal gait characteristics of non-neurologic horses when the head is elevated but not during blindfolding compared to normal walking. current results suggest that pacing and increased variation in foot-placement during head elevation should be interpreted with caution however further work is required to determine whether the change differs between horses with neurological disease and non-neurologic disease. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder associated with a mutation in cyclophillin b that leads to impaired collagen folding, aberrant wound repair, and corneal abnormalities. it affects young quarter horses, appaloosa, and paints. herda shows similarities to the human hereditary connective tissue syndrome ehlers danlos (eds). many eds patients suffer from joint pain and osteoarthritis (oa) as adults. the similarity between eds and herda raises the question whether horses suffering from herda develop oa. in oa, excess production of inflammatory mediators such as prostaglandin e (pge ) activate enzymes that degrade cartilage as well as impede wound healing. the present study examined articular cartilage from yearling horses afflicted with herda. we hypothesized that chondrocytes from these horses are continually activated to produce inflammatory mediators. to test this hypothesis, articular cartilage from carpal and tarsal joints of herda horses were evaluated using histology. pge production by chondrocyte cultures was measured by elisa and analyzed by one-way anova, tukey post-hoc test, p o . significance. we also determined the antiinflammatory effects of an avocado/soybean unsaponifiables (asu), glucosamine (glu), and chondroitin sulfate (cs) mixture (ingredients found in cosequin s asu) and phenylbutazone (pbz) on chondrocytes. cosequin s asu and pbz are used alone or in combination for the management of oa. chondrocyte cultures were incubated for hrs with control media alone, a clinically relevant concentration of pbz ( mg/ml), or the combination of asu (nmx s , . mg/ml) glu (fchg s , mg/ml) cs (trh s , mg/ml). articular cartilage from joints of five herda-afflicted horses showed gross and histologic evidence of osteoarthritic lesions. chondrocyte cultures from cartilage of horses suffering from herda spontaneously produced greater pge than chondrocytes from normal horses ( -fold). pbz significantly decreased pge production by $ % (p o . ). the combination of asu -glu cs also significantly reduced pge production by $ % (p o . ). the present study supports anecdotal findings that horses suffering from herda are likely to develop oa. the inhibition of pge synthesis by asu glu cs suggests that this combination may be beneficial for the management of oa in herda. research supported by nutramax laboratories, inc. equine inflammatory airway disease (iad) is a common condition often treated empirically with corticosteroids. gene expression analysis in the bronchoalveolar lavage fluid (balf) may help understand the effects of corticosteroids in iad. the first part of the study aimed at identifying reference genes in the balf of iad horses treated with corticosteroids. the second part of the study investigated the effects of dexamethasone and fluticasone propionate treatments on the mrna expression of il- b, il- , il- and il- . the expression stability of seven candidate reference genes was determined in balf taken pre-and post-treatment with dexamethasone and fluticasone propionate in horses with iad. primers' efficiencies were calculated using linregpcr. normfinder, genorm and qbaseplus softwares were used to rank the genes according to their stability. gapdh was the most stably expressed gene whereas b m was the least stable gene. in addition, genorm analysis revealed that the number of genes required for optimal normalization was four (gapdh, sdha, hprt, rpl ). in the second part of the study the mrna expression of il- b, il- , il- and il- was measured in balf samples from seven iad horses treated in a randomized cross-over design with dexamethasone ( . mg/kg sid, days) or inhaled fluticasone propionate ( mcg bid with aerohippus, days). the balf samples were taken at baseline and after each treatment period. there was no significant effect of the corticosteroids treatment on the mrna expression of il- b, il- and il- in the balf. the mrna expression of il- was suppressed by dexamethasone and fluticasone propionate treatments. pneumonia is observed in horses after long distance transportation in association with confinement of horses' head position leading to a reduction in tracheal mucociliary clearance time (tmct). we hypothesize that clenbuterol, a beta- agonist shown to increase tmct in the horse, will ameliorate the affect of a fixed head position on large airway contamination and inflammation in a long-distance shipping model. six adult horses were enrolled in a cross-over design prospective study. horses were housed with their heads in a fixed position for hours to simulate long distance transport, and treated with clenbuterol ( . ug/kg po q h) or a placebo starting hours before simulated shipping. tmct was measured using a charcoal clearance technique. data were collected at baseline and hours, and included tmct, tracheal wash cytology and quantitative culture, rectal temperature, cbc, fibrinogen, and serum tnfa, il- and il- levels. there was a -week washout between study arms, and each horse served as its own control. the data was analyzed using regression analysis and wilcoxon rank-sum tests. no statistically significant difference was seen between treatment and placebo groups for any of the variables investigated. tmct did not differ after treatment ( . ae . cm/min) versus placebo ( . ae . cm/ min; p . ), and intratracheal bacterial counts were similar for treatment (  ae  cfu; p . ) and placebo (  ae  cfu) groups. a reduction of tracheal b hemolytic streptococcus. spp. after clenbuterol versus placebo was also nonsignificant ( % versus %; p . ). in conclusion, treatment with clenbuterol does not appear to combat the deleterious effects of this long-term shipping model. breathing cold air during strenuous exercise is associated with airway inflammation. under these conditions, warming and humidification of inspired air occurs in the lower respiratory tract resulting in mucosal cooling, desiccation, and hyperosmolarity. the purpose of this research was to test the hypothesis that airway hypertonicity causes inflammatory cell migration and alterations in cytokine expression associated with exercise induced airway inflammation. horses (n ) were examined in a randomized crossover design after exposure to hypertonic aerosols ( minute nebulization with solutions of either isotonic or hypertonic mannitol). airway leukocytes were harvested and hours post aerosol challenge via bronchoaveolar lavage, and were used to determine total and differential nucleated cell count and expression of cytokinespecific mrna. hypertonic aerosol challenge resulted in an increase in total number of cells hr after challenge, characterized by increased macrophage (p . ) and neutrophil (p . ) concentrations, but there was no effect on airway leukocyte concentrations hours after nebulization. no significant changes in the relative quantity of mrna for airway cytokines were noted at either time point. these data demonstrate that transient airway hypertonicity can cause airway leukocyte influx and may be responsible for the airway inflammation commonly found in athletes that exercise in cold conditions. however, our data do not support the hypothesis that hypertonicity is the sole initiating cause of changes in cytokine expression secondary to cold weather exercise. it is likely that factors such as airway temperature, shear stress or epithelial damage also play a role in this phenomenon. we studied the importance of abdominal sonograms in neonatal foals suffering from gastrointestinal conditions. we hypothesized that there would be a subgroup of neonates with sonographically detectable pneumatosis intestinalis (pi) as a reflection of a necrotizing component of the disease. records of foals days of age hospitalized between and with signs of gastrointestinal disease were evaluated (n ). the association of sonographic, clinical, pathological and clinicopathological signs with outcome and severity of disease was determined. pneumatosis intestinalis was imaged in foals. twenty-seven foals were classified as having necrotizing gastrointestinal disease based on the presence of gastrointestinal signs (colic, diarrhea, gastric reflux or abdominal distension) and pi detected sonographically ( ), surgical ( ) or pathological ( ) evidence of gastrointestinal necrosis. there was a difference between overall survival rate ( %) and survival rate in foals with necrotizing disease ( %, p . ) or foals with pi detected sonographically ( %, p . ). pneumatosis intestinalis was the only sonographic finding associated with outcome. sonographic abnormalities in peritoneal fluid, stomach, duodenum, jejunum, cecum, umbilicus or the presence of meconium were associated (p o . ) with surrogates of severity of disease (hospitalization cost or days of hospitalization). hypoproteinemia was associated with pi (p . ). the presence of blood in the feces, reflux and abdominal distension was associated with necrotizing gastrointestinal disease (p o . ). abdominal sonograms have prognostic value in neonatal gastrointestinal disease. pneumatosis intestinalis was a common sonographic sign that worsened the prognosis. the therapeutic implications of detecting a necrotizing component of the gastrointestinal disease deserve further study. the interaction of insulin and the microvascular endothelial insulin receptor (irc) plays an important role in the normal and insulin resistant (ir) individual. while endothelial irc signaling is normally vasodilatory, this effect is well-documented to reverse in the ir individual, resulting in vasoconstriction. although vascular dysfunction has been reported in sepsis-associated equine laminitis, the role of the laminar microvasculature in endocrinopathic laminitis remains poorly characterized. the purpose of this study was to characterize the pattern of irc expression in digital laminae in ponies subjected to a dietary carbohydrate challenge that mimicked abrupt exposure to pasture rich in nonstructural carbohydrates (nsc). mixed-breed ponies (body weight . /- . kg) received a diet of hay chop (nsc $ % on a dm basis) for weeks prior to initiation of the experimental feeding protocol. following conditioning, ponies either remained on the control diet (n ) or received the same diet supplemented with sweet feed and oligofructose (total diet $ % nsc; n ) for a period of days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including dorsal digital laminae, were collected immediately following euthanasia. the samples were formalin-fixed for hours, transferred to % ethanol, and paraffin-embedded. laminar sections were stained immunohistochemically for irc using a commercially-available antibody (abcam); the number of irc ( ) cells was quantified in x light microscopy fields (n ) for each section. the total number of irc ( ) cells was greater in the laminae of challenged ponies than control ponies (p . ), and there was a significant correlation between the change in serum basal insulin concentration and number of laminar irc ( ) endothelial cells (r . ; p o . ). while the number of irc ( ) endothelial cells was significantly greater in the dermal laminae of challenged ponies (p . ), there was no difference in the number of interstitial irc ( ) cells (p . ). no epithelial irc ( ) cells were observed in any laminar section, and irc ( ) cells were conspicuously absent from the deep dermal tissue (including vessels). up-regulation of irc expression in the laminar vasculature occurs acutely in response to dietary carbohydrate challenge and accompanies hyperinsulinemia in ponies. the dramatic increase in endothelial irc expression in the laminar microvasculature in nutritionally challenged ponies, with no apparent epithelial irc present, suggests that hyperinsulinemia associated with exposure to increased dietary nsc may induce laminar injury by causing a similar vasoconstriction in ir equids as described in the microvasculature of ir humans. glucose transport from the blood stream into cells, the limiting step in whole-body glucose utilization, is regulated by a family of glucose transporter (glut) proteins in insulin-sensitive (i.e., muscle and adipose) tissues. we previously demonstrated that glut , the major isoform, is a key factor in the pathogenesis of equine insulin resistance (ir). while it has been recently demonstrated that glut (a newly discovered isoform) increases insulin-stimulated glucose transport in human muscle, its role in other tissues, particularly in the setting of ir, is not well characterized in any species. in addition, as has recently emerged as a key downstream signaling molecule regulating translocation of glut to the cell surface, the rate-limiting step in glucose uptake. we hypothesized that glut content would be differentially expressed across tissues and that ir would induce alteration in glucose transport by affecting active cell surface glut . biopsies of skeletal muscle, and subcutaneous and visceral adipose tissue were collected from light-breed horses, characterized as either insulin sensitive or compensated ir, based on the results of an insulin-modified frequently-sampled intravenous glucose tolerance test (n /group). we specifically quantified active cell-surface glut in these biopsies, using an innovative exofacial bismannose photolabeled assay, which has not been previously applied to glut . total glut protein expression was measured by western blots, as well as total and phosphorylated (indicating activation of) as . glut was expressed in all the depots with a significant regional effect. total glut protein content was increased (p o . ) in visceral (omental and mesenteric) compared to subcutaneous (nuchal ligament and tailhead) adipose tissue and skeletal muscle of healthy horses. ir did not induce alterations in active cell-surface and total glut content nor in total and phosphorylated as in any of the tissues evaluated. our data suggests that glut is abundant in visceral adipose tissue and is therefore likely to play a substantial role in the regulation of glucose transport. however, neither glut translocation nor as activation are impaired in insulin-sensitive tissues of ir horses. it is concluded that, in contrast with glut , glut does not appear to contribute to glucose transport alterations during naturally-occurring equine ir. insulin resistance (ir), characterized by exaggerated glycemic or insulinemic responses to glucose challenge, is a key metabolic disturbance in horses that develop obesity-associated laminitis. in addition to obesity, diet and age have been demonstrated to affect tissue sensitivity to insulin in other species but these factors have received limited investigation in horses. we hypothesized that there would be greater glycemic and insulinemic responses to a sweet feed meal in aged horses, as compared to adult horses, as well as in horses adapted to a forage-only diet. three diets, grass hay only, grass hay plus sweet feed (starch and sugar-rich, ss), and grass hay plus a fat and fiber (ff) feed, were fed to mares, adult ( - yr) and aged ( yr), for a -week adaptation period in a randomized design. glycemic and insulinemic responses to a standardized meal of sweet feed ( g/kg bw offered for hour) were determined for hours from the onset of feeding. peak glucose and insulin concentrations and areas under the glucose or insulin vs. time curves (auc-g, mg/ dl/ min, and auc-i, mu/ml/ min) were determined and data were analyzed by one-and two-factor repeated measures anova. there were no differences between age groups in glycemic responses to any of the diets. however, in aged horses peak glucose concentration (p o . ) and auc-g (p o . ) were greater after adaptation to the forage-only diet, as compared to the other two diets. in contrast, aged horses had a greater peak insulin concentration (p o . ) and auc-i (p o . ) than adult horses on all diets but no differences in peak insulin concentration or auc-i was found between diets within age groups. as hypothesized, the insulin response, but not the glycemic response, to a sweet feed meal was greater in aged horses, regardless of background diet. further, the glycemic response was greatest after adaptation to a forage-only diet, but this finding was only significant in aged horses. morbidity, mortality, and economic loss to the equine industry. in obese humans and rodent models of nutritional obesity, systemic insulin resistance and hyperinsulinemia are followed temporally in a majority of individuals by decreased glucose tolerance, pancreatic bcell failure, and type ii diabetes mellitus. in stark contrast to humans, obese horses and ponies chronically remain in what is termed a ''prediabetic'' state in human ir, characterized by hyperinsulinemic euglycemia. few data exist describing the biology of the equine endocrine pancreas in the chronically ir animal that may both: ) explain this unique equine endocrine physiology and ) characterize the animal at-risk for hyperinsulinemia-associated laminitis. the purpose of the study reported here was to characterize the morphology and physiology of the equine endocrine pancreas in response to a dietary carbohydrate challenge. twenty-two mixedbreed ponies (body weight . ae . kg) were conditioned to a diet of chopped hay (nsc $ % on dm basis) for weeks; following conditioning, ponies either remained on the control diet (n ), or received the same hay supplemented with sweet feed and oligofructose (total diet $ % nsc; n ) for days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including pancreas, were collected immediately following euthanasia. the samples were formalin-fixed for hours, transferred to % ethanol, and paraffin-embedded. immunohistochemistry was performed on pancreas sections using a commerciallyavailable anti-insulin antibody (abcam), and measurements of islet surface area and b-cell surface area were performed (n islets per tissue section) using a commercially-available computer software program (image j). there was a trend for greater total islet surface area in pancreatic tissue from ponies fed the high nsc diet when compared to the ponies on the hay diet (p . ); however, no difference was noted in b-cell surface area between diet treatments (p . ). the change in serum insulin concentration was significantly greater in the high nsc-fed ponies than in controls ( . /- . miu/l vs. . /À . miu/l; p . ); however, this variable was not correlated with total islet surface area (r . ; p . ) or b-cell surface area (r . ; p . ). due to the relatively modest changes in pancreatic islet surface area that accompany marked increases in serum insulin concentrations in ponies fed a high nsc diet, it is important to assess both b-cell function and insulin clearance mechanisms in future studies to delineate the mechanism(s) of hyperinsulinemia in this model. humans that suffer from obesity show exaggerated inflammatory responses and this may be relevant to the association between increased adiposity and laminitis in horses with equine metabolic syndrome (ems). this study was performed to test the hypothesis that inflammatory responses to endotoxemia differ between healthy horses and those affected by ems. six healthy adult mares and horses with ems received an intravenous infusion of lipopolysaccharide (lps; ng/kg in ml sterile saline) or saline alone. a crossover design was employed with a -day washout period. physical examinations were performed hourly for h and whole blood was collected at , , , , , and min for assessment of inflammatory cytokine gene expression. a liver biopsy was performed between and min postinfusion. data were analyzed using mixed model anova. mean rectal temperature, heart rate, and respiratory rate increased following lps infusion (treatment  time; p o . ), with higher heart (group  treatment; p . ) and respiratory rates (group; p . ) detected in ems horses. lipopolysaccharide infusion significantly increased whole blood gene expression of tumor necrosis factor a (tnfa), interleukin (il)- b (p o . ), il- (p o . ), il- (p o . ), and il- (p . ), and hepatic gene expression of il- (p o . ), il- (p o . ), and il- (p . ). inflammatory gene expression did not differ significantly between groups, so our hypothesis was not supported. heart rates tended to be higher when lps was administered to horses with ems. elevated serum concentration of cardiac troponin i (ctni) is a biomarker for myocardial damage in horses. preferred times to test blood for ctni levels following athletic performance or other events that may cause myocardial injury are not yet established and would be affected by time of release from the myocytes, location of release within the myocytes, duration of release and half-life of ctni in the horse. this information would be necessary to more accurately and reliably test horses for myocardial injury. the aim of this study was to determine the elimination half-life (t / ) of equine ctni. to establish the t / of equine ctni in horses, ctni was recombinantly expressed in e.coli. two healthy ponies received intravenous injections of recombinant equine ctni and plasma ctni concentrations were measured with a point-of-care ctni analyzer at multiple time points after injection. standard pharmacokinetic analysis was performed to establish the elimination half-life of ctni. for comparative purposes, data were subjected to pharmacokinetic models describing a single versus biphasic elimination profile. elimination of recombinant equine ctni following intravenous administration exhibits a short half-life. establishing the t / of troponin provides critical information in understanding the clinical application of this cardiac biomarker in clinical practice. this study describes a true biological ctni t / , which has not been documented in any species thus far. stall-side assessment of this cardiac biomarker in horses should enhance the ability of clinicians to detect myocardial damage and aid in the management and treatment of horses with cardiac disease. the objective of the study was to evaluate the between-pony, within-pony, between-analyser and within-analyser variation of flow-mediated vasodilation (fmd) measurement in healthy ponies, to investigate the hypothesis that fmd occurs in healthy ponies. six healthy, native breed, unrelated pony mares of varying weight ( - kg), body condition score ( / - / ) and age ( - years) were used. the median artery was occluded for minutes. twodimensional ( d) ultrasonographic images of the artery were recorded for seconds prior to and for minutes after occlusion. the peak luminal diameter was compared to baseline diameter to calculate the relative percentage increase in luminal size (fmd). images were obtained from six ponies on one occasion and from one pony on six occasions. analysis of images was performed by two independent analysers and by one analyser twice. the mean (sd) fmd in ponies was . % ( . %) and in pony ( occasions) was . % ( . %). coefficients of variation were . % and . % respectively. agreement between analysers was fair (icc . ) and within analyser was poor (icc . ). fmd is used to assess endothelial function in humans and has recently been assessed for its use in canine subjects. fmd occurs and measurement is feasible in ponies. fmd could be used to assess endothelial function, in the context of laminitis or other cardiovascular diseases. current state-of-the-art technique for measuring blood pressure (bp) in the horse is invasive and involves cannulation of the facial artery. indirect techniques, such as oscillometry, have proven useful in the anaesthetised horse, but have not become routine in the standing horse. monitoring bp can be indicated for the diagnosis and treatment of the hypotensive patient (ie. caused by endotoxemia, hypovolemia, systemic inflammatory response syndrome and cardiac failure) or the hypertensive patient (ie. due to equine metabolic syndrome or pain). the objective of this study was therefore to a) describe the methodology for application of oscillometric bp using a cuff applied to the tail in the standing horse and b) and to determine accuracy and precision of this method applied to the normotensive standing horse. the oscillometric method is simple to apply in a clinical setting. a pneumatic cuff is snugly applied to the unclipped tail-base with the cuff bladder centered over the middle coccygeal artery. the tail circumference must match the manufacturers description of the cuffs diameter range. the oscillometric apparatus inflates the cuff and obtain systolic, diastolic and mean arterial bp (sap, dap and map). at least consecutive measurements must be obtained. a correction of . mmhg/cm vertical distance between cuff and heart level is added to the measurement to correct for hydrostatic pressure difference. for determination of accuracy and precision of indirect sap, dap and map, eight healthy horses (age to years), was equipped with an intra-arterial catheter ii in the facial artery and a commercial tail-cuff oscillometric apparatus. i measurements were recorded every minutes for minutes. the data were analysed with the statistical software r using a mixed model with repeated measurements and a bland-altman analysis corrected for repeated measurements. oscillometric bp was accurate and precise for map (mean bias, lower confidence level, upper confidence level, variation in difference, all mmhg) (À . , À . , . , . , respectively) in the conscious horse but not for sap (À . , À . , . , . , respectively) and dap ( . , . , . , , respectively) . there was no significant contribution to the statistical model of either horse or measurement number. all horses tolerated the tail-cuff well and the method was simple to apply. only map could be measured with acceptable accuracy and precision in the normotensive standing horse using the described oscillometric method. reference intervals for thyroid hormone (th) concentrations have not been established for donkeys. therefore, clinicians must use reference ranges from horses, potentially leading to misdiagnosis of thyroid diseases. we hypothesized that th concentrations are different between donkeys and horses. the purposes of this study were: a) to compare th concentrations between donkeys and horses and, b) to determine whether the age may influence th concentrations. thirty-eight healthy donkeys ( . ae . years), mixed breeds, and healthy andalusian horses ( . ae . years) were used. donkeys were divided into three groups: o years (n ), - years (n ), and years (n ). serum concentrations of total triiodothyronine (tt ), free triiodothyronine (ft ), total thyroxine (tt ), free thyroxine (ft ), reverse triiodothyronine (rt ) and thyroid-stimulating hormone (tsh) were quantified by radioimmunoassay. all blood samples were collected the same day. neither horses nor donkeys had received any treatment for days before sampling and both farms had similar production conditions. total t , ft , ft and tt concentrations were higher (p o . ) in donkeys than horses. in contrast, no statistical differences were found for rt and tsh concentrations. young donkeys ( o years) had higher ft , tt and rt concentrations compared to other donkey groups (p o . ). old donkeys ( years) had lower tt and ft concentrations than both younger donkeys groups (p o . ). this study shows that there are differences in th concentrations between donkeys and horses, raising awareness on the possibility of misdiagnosis of thyroid gland dysfunction when using values from horses, being necessary to determine exclusive reference intervals for donkeys. ovariectomy is associated with alterations of responses to many hormones, not just those associated with reproductive function. in humans and rats, ovariectomy leads to insulin resistance, increased adiposity and altered fat mobilization. the effects of ovariectomy on energy metabolism have not been reported in horses. ovariectomized mares have been shown to respond normally to an acth stimulation test, but the response to suppression of the hypothalamo-pituitary-adrenal axis has not been previously described. the aim of this study was to evaluate the effect of ovariectomy on insulin response in mares and to determine if mares exhibit alterations in response to dexamethasone administration after ovariectomy. six healthy mares underwent an intravenous glucose tolerance test (ivgtt), an insulin sensitivity test (ist) and a dexamethasone suppression test (dst) before and weeks after bilateral ovariectomy. body weight, cortisol values at baseline, and hours after dexamethasone injection and acth values at baseline, and hours after dexamethasone injection, basal insulin/glucose ratio, time to reach a % decrease in blood glucose in the ist, time to reach baseline glucose concentration in the ivgtt and area under the curves plotting blood glucose and time to injection of glucose or insulin were compared before and after ovariectomy using a paired t-test or an anova for repeated measures. significance level was p o . . average body weight was decreased after surgery ( kg ). the injection of dexamethasone resulted in a serum cortisol concentration of less than mg/dl in all mares before ovariectomy, whereas after ovariectomy, dexamethasone injection resulted in a serum cortisol concentration of less than mg/dl in out of mares. in all cases, acth concentration was within the reference range ( - pg/ml) before and after ovariectomy. however, acth concentrations at t and at t were significantly higher after ovariectomy. each mare had a normal ivgtt, both before and after ovariectomy. additionally, no significant differences were observed in basal blood glucose ( ae mg/dl before and ae mg/dl after) or in the time to reach glucose baseline ( ae min before and ae min after). serum basal insulin concentration and insulin/glucose ratio was not significantly different before or after ovariectomy ( . ae . miu/ml and . ae . miu/ml and . ae . and . ae . , respectively), nor was the average time to reach a % decrease in blood glucose after insulin injection ( ae min and ae min, respectively). these findings suggest that, as reported in other species, the shortterm effect of ovariectomy may modify dexamethasone response in mares and that, contrary to other species, it may not modify insulin response. equine gastric ulcer syndrome (egus) is a common medical problem in horses. the high prevalence of gastric ulcers, vague clinical signs and negative effect on performance make it a significant clinical and economic problem within the horse industry. current pharmaceutical treatments are expensive and alter the acidic environment of the stomach. berries and pulp from the seabuckthorn plant (hippophae rhamnoides) are a rich source of vitamins, trace minerals, amino acids, antioxidants, and other bioactive substances and have been used successfully to treat stomach ulcers in man and rats. the purpose of this study was to evaluate the efficacy of a commercially sold, liquid extract of seabuckthorn berries (seabuck tm sbt gastro-plus) for treatment and prevention of gastric ulcers in horses. eight thoroughbred and thoroughbred-cross horses ( - years of age, geldings & mares, - kg) were used in a blinded two-period cross-over study. treatments consisted of control (untreated) and treatment (seabuck tm sbt gastro-plus) twice daily mixed with the grain meal. horses were treated for weeks followed by a week alternating feed-deprivation period to induce or worsen existing ulcers. gastroscopies were performed on all horses on day , week , and week (at the end of the alternating feed-deprivation period). gastric juice was aspirated and ph was measured. during gastroscopy, gastric ulcer scores were assigned to each stomach based on lesion number and severity. horses acted as their own controls, and between each treatment period the horses had a -week washout period. data was analyzed by anova for repeated measures via the glm procedure (sas inst. inc., cary, nc). when significant differences (p o . ) were observed, a post-hoc tukey's test was used to determine differences. non-glandular gastric ulcer scores significantly increased in all control and sbt-treated horses from week to week , after the feed-deprivation phase of the study. there was no significant difference in the non-glandular gastric number (p . ) and nonglandular gastric severity (p . ) scores in sbt-treated horses compared to non-treated controls. glandular ulcer number (p . ) and glandular ulcer severity (p . ) was significantly lower in the sbt-treated horses compared to the control horses. there was no significant difference in the ph (p . ) in sbt-treated horses compared to non-treated controls. thus, seabuck tm sbt gastro-plus, mixed in the feed twice daily, may be efficacious in controlling the severity of glandular ulcers in horses during stress, without increasing stomach ph. the availability of rapid and accurate quantitative fibrinogen measurements may be useful for evaluation of hospitalized equine patients. the abaxis vspro analyzer was evaluated for precision using two levels of human fibrinogen controls ( mg/dl and mg/dl), four different vspro machines, and two different lots of cartridges, assessed over subsequent days. the coefficients of variation of the assay ranged from % ( mg/dl) to % ( mg/ dl). we subsequently evaluated the abaxis vspro fibrinogen assay compared to fibrinogen concentration measured using the beckman coulter acl- in equine samples of varying fibrinogen concentrations obtained from horses with gastrointestinal disease. all samples were measured in citrated plasma. fibrinogen samples measured on the acl- ranged from to mg/dl (median mg/dl). vspro samples were run in duplicate, and the mean compared to the acl values. pearson correlation coefficient analysis generated an r value of . (p o . ). duplicate measurements on the vspro were strongly correlated to each other with an r value of . (p o . ). bland-altman analysis of these samples for the vspro compared to the acl- noted a bias of À ae mg/dl the results of this study indicate that the vspro benchtop fibrinogen analyzer provides accurate and precise fibrinogen data compared to the acl- reference analyzer. the immune response of foals to r. equi is incompletely understood and believed to be responsible for clinical disease caused by this pulmonary pathogen. in a recent study foals receiving a large inoculum exhibited th skewing with pneumonia and a small inoculum exhibited th skewing without clinical disease. we hypothesized that cytokine/chemokine production by pulmonary alveolar macrophages, in vitro, would increase with the infective dose and that the magnitude of the response would differ between foals and adults. alveolar macrophages were obtained by bronchoalevolar lavage from healthy mares and their -week-old foals. macrophage cultures were infected with r. equi ( or -) at a multiplicity of infection (moi) of or . total rna was harvested and hours post-infection, reverse transcribed and used as template for quantita-tive pcr. the ddct method was used to calculate relative gene transcripts for il- , il- p , tnfa and cxcl . cellular infections at moi resulted in significantly higher expression of il- , il- p and tnfa mrna transcripts compared to moi . however, the dose-effect was reversed for cxcl with significantly lower expression at the higher moi. there was no difference in magnitude of cytokine/chemokine responses by the alveolar macrophages between adults and foals. dose-dependent responses of alveolar macrophages may represent a novel mechanism by which r. equi could modulate immune responses and therefore disease. significant down-regulation of cxcl mrna transcripts associated with a higher dose is of particular interest as this chemokine plays a role in development of protective th responses. the intent of this study was to develop likelihood ratios (lrs) for infection attributable to corynebacterium pseudotuberculosis in horses based on synergistic hemolysis inhibition (shi) test titers. medical records for horses presented to the uc davis veterinary teaching hospital with serum submitted for shi titer determination were evaluated and cases met study inclusion criteria. these cases were grouped based on evidence of internal and/or external infection attributable to c. pseudotuberculosis and likelihood ratios with % confidence intervals determined. results showed increasing lrs indicating increasing odds for any form of active disease as titer increased with all cases considered. lrs for internal infection were for titers ! overall and for titers with external abscess cases excluded. no difference from (and therefore no significant change in pre-test to post-test odds) was seen in any lrs for internal disease when only cases with external disease were examined (external and internal disease vs. external only). overall, the shi test results showed usefulness in determining internal c. pseudotuberculosis infection in horses with no evidence of external abscessation. overall, however, higher titers were more indicative of active external or internal disease than internal disease specifically in contrast to previous reports. the shi test was unable to distinguish internal infection when external abscesses were present. salmonella enterica is a zoonotic pathogen that has tremendous impact on many different animal production and management systems. rapid detection of s. enterica in fecal samples may facilitate effective infection control practices. current detection methods require - hours (polymerase chain reaction or pcr) or - hours (enriched aerobic culture) to obtain results. alternatives have been developed, lateral flow antigen detection systems (lfads), which are currently marketed for salmonella detection related to food safety microbiology. the objective of this study was to evaluate two commercially available rapid salmonella detection systems in equine feces. fecal samples collected from repeatedly culture-negative horses were inoculated with known concentrations of salmonella enterica serotype typhimurium (five uninoculated control samples, and samples of each -fold dilution [ .  - .  cfu/gram of feces]). all samples were aerobically cultured using a standard enrichment technique. in a blinded fashion, samples were tested using two different lfads as well as plated on agar media for confirmatory testing. at hours of incubation, using bacterial culture as the reference method, test was correctly identify % of samples ( bacterial contamination of stalls with salmonella sp. is a serious problem in equine hospitals. salmonella sp. exposure to horses in the facility can result in nosocomial infections which results in temporary facility closure, until the organism is eradicated. hospital closure can result in loss of revenue, damage to reputation and interference with patient care. the purpose of this study was to evaluate three stall cleaning methods on eradication of salmonella sp. at an equine veterinary teaching hospital (vth). horses admitted to the vth were assigned to salmonella sp.negative stalls within areas of the vth during the study period (september -january . when the horses were discharged stalls were randomly assigned to one of three cleaning methods (pressure-washing only [pw] , pressure washing and hand scrubbing [pws] , or hand scrubbing only [s]) in a single period, non cross-over design. all stalls were stripped of bedding and surfaces sprayed with tap water and cleaned with a disinfectant quaternary-ammonia solution (super hdq neutral, spartan chemical co., inc, maumee, oh). the pressure-washing system (psc cleaning systems, inc., toronto, canada) used, provided a pressure of psi and a temperature range of - f. following cleaning, each stall was allowed to air dry and within hours, stall surfaces were sampled using three  sponges moistened with sterile saline. the person collecting the samples was masked to the method of cleaning. sponges were submitted to the louisiana animal disease diagnostic laboratory (laddl) for culture of salmonella sp. a chi-squared analysis was used to determine significant differences (limit p o . ) between cleaning methods and salmonella sp. isolation. during the study period, stalls (pw [n ]; pws [n ]; s [n ] were included. all stalls had negative environmental salmonella sp. cultures prior to beginning the study. for pw cleaned stalls, / ( . %) were salmonella sp.-positive, for pws cleaned stalls, / ( %) were salmonella sp.-positive, and for s cleaned stalls, / ( . %) were salmonella sp.-positive. although, there were fewer salmonella sp.-positive stalls ( . %) in the handscrubbed stalls, cleaning method did not significantly (p . ) affect the isolation of salmonella sp. from the stall environment. in conclusion, power washing alone, power washing and hand scrubbing, and hand scrubbing alone, using a quaternary-ammonia solution did not significantly affect environmental isolation of salmonella sp. from stalls surfaces in the vth during this study. the objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against streptococcus equi subsp. zooepidemicus (s. zooepidemicus) and rhodococcus equi isolates. a single dose of gamithromycin ( mg/kg of body weight) was administered intramuscularly. concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (pelf), bronchoalveolar lavage (bal) cells, and blood neutrophils were determined using hplc with tandem mass spectrometry detection. the minimum inhibitory concentration of gamithromycin required to inhibit growth of % of r. equi and s. zooepidemicus isolates (mic ) was determined. additionally, the activity of gamithromycin against intracellular r. equi was measured. mean peak gamithromycin concentrations were significantly higher in blood neutrophils ( . ae . g/ml) and bal cells ( . ae . g/ml) compared to pelf ( . ae . g/ml) and plasma ( . ae . g/ml). mean terminal half-lives in neutrophils ( . h), bal cells ( . h), and pelf ( . h) were significantly longer than that of plasma ( . h). the mic of s. zooepidemicus isolates was . g/ml. the mic of gamithromycin for macrolide-resistant r. equi isolates ( g/ml) was significantly higher than that of macrolide-susceptible isolates ( . g/ ml). the activity of gamithromycin against intracellular r. equi was similar to that of azithromycin and erythromycin. intramuscular administration of gamithromycin at a dosage of mg/kg would maintain pelf concentrations above the mic for s. zooepidemicus and phagocytic cell concentrations above the mic for r. equi for approximately days. eight western stock yearling horses were infected with ehv- (ab ) by nasopharyngeal instillation. venous blood samples for collection of plasma were collected in na-citrate tubes on the day prior to infection (d - ) and on d through d . in addition, clinical data, nasal swabs and peripheral blood mononuclear cells (pbmc) for detection of viremia were collected on the day before infection (d - ) and on d through d post-infection. d-dimer concentrations were determined in citrated plasma samples using a latex agglutination test (minutex d-dimer, biopool, ireland). viral load in pbmc was determined using quantitative pcr. all horses showed bi-phasic fevers typical for ehv- infections. one horse developed acute ehm on d and was euthanized after samples were collected. in all horses d-dimers were undetectable on d - and on d , and . in contrast, all horses had increased ddimer concentrations for to consecutive days starting on day post-infection. d-dimer concentrations in horses increased to ug/ml and one of these horses was the horse with acute ehm. interestingly, mean increased d-dimer concentrations showed timely overlap with the mean fever curve and, delayed by day, with the mean viremia curve. because plasma samples for d-dimer measurements were not collected during the first days post-infection, which are typically associated with a primary fever, conclusion on the association of d-dimers with fever of viremia await analysis of a second study currently conducted in our laboratory. in conclusion our data indicates that during ehv- infection with neuropathogenic strains activation of the coagulation cascade and production of cross-linked fibrin is wide-spread; not limited to horses with clinical signs of ehm, and can be expected between days and post-infection. lawsonia intracellularis is an emerging pathogen in horses and the causative agent in equine proliferative enteropathy (epe). the goal of this study was to evaluate the exposure of pre-weanling foals and broodmares to lawsonia intracelluaris on several farms in louisiana with a history of epe and compare the results to several farms with no known clinical cases of epe in foals. an additional goal of the study was to identify whether a relationship exists between lawsonia intracelluaris and other gastrointestinal pathogens in foals. whole blood and fecal samples were collected from mares and foals from four breeding farms in louisiana. farms a and b had no known clinical cases of epe, while farms c and d had previous know cases of epe in . serum samples were examined for the presence of antibodies against lawsonia intracellularis using an immunoperoxidase monolayer assay (ipma). dna was extracted from fecal samples using a commercial dna kit and molecular detection of lawsonia intracelluaris was assayed using real-time pcr. fecal ova were determined using quantitative sucrose floatation. the presence of fecal clostridium difficile toxin was measured using a commercial enzyme linked immunosorbent assay (elisa). three of the farms examined had foals and mares with exposure to l. intracellularis as evidenced by serum antibodies against the organism. of the total population sampled, foals ( . %) and mares ( . %) had evidence of antibodies to l. intracellularis based on serology. three foals ( . %) tested positive for l. intracellularis organism by fecal pcr, and all of these foals were located on farm c. of these, one of the foals was seronegative, while the other two were seropositive. farm c also had the highest percentage of mares ( . %) serologically positive for l. intracellularis, while farm a had the highest percentage of foals ( . %) with antibody titers against l intracellaris. farm c also had the only pairs (n ) of serologically positive mares with seropositive foals. while farm a and b had seropositive mares and/or foals, none of the foals were positive for l. intracellularis fecal shedding by pcr. all serum and fecal samples were negative for evidence of l. intracellaris on farm d. ten foals ( %) had fecal egg counts greater than egg per gram and foals ( %) were positive for c. difficile toxin. this study demonstrated evidence of natural exposure to l intracellularis on farms both with and without a history of epe in louisiana. further, this study failed to establish a relationship between l intracellularis and other gastrointestinal pathogens. the objective of this study was to examine the clinical, hematological, biochemical, and outcome data from equids infected with anaplasma phagocytophilum presented to a primary care field setting in southeastern pennsylvania. computerized medical records from febrile equids with confirmed anaplasma phagocytophilum infection were reviewed. confirmation of anaplasma phagocytophilum was defined by the presence of granular inclusion bodies seen within leukocytes or eosinophils on microscopic blood smear evaluation and/or a positive polymerase chain reaction (pcr) for anaplasma phagocytophilum. horses and donkey presented with a mean fever of . f and mean fever duration of hours. the mean age at presentation was . years and the mean pack cell volume was . %. / cases were diagnosed in the months of may to december. equids ages to years had significantly lower platelet counts. / cases were positive on blood smear for inclusion bodies and / cases were positive for anaplasma phagocytophilum on pcr. treatments included intravenous oxytetracycline, oral doxycycline, or both. mean treatment duration was . days and mean treatment cost was $ . / cases were normothermic within hours. the treatment used in the two remaining cases was changed from oral doxycycline to intravenous oxtetracycline and was successful. this is the first case series of equine granulocytic anaplasmosis in the mid-atlantic states. all cases were examined and treated in the field. in order to make a definitive diagnosis, some cases required pcr. treatment failures were documented with the use of oral doxycycline alone. % of the cases survived. a high incidence of clinical and possibly genetic abnormalities has been reported amongst friesian horses including dwarfism, hydrocephalus, dissecting aortic aneurism and esophageal dysfunction. the purpose of the current study was to develop a new electromyography (emg) method to assess neurophysiological function of the esophagus especially for friesian horses. five friesian horses with esophageal dysfunction were included (ranging in age from . - years and comprising mares and a stallion) and two friesian control horses (a -and -year-old gelding). all five horses with esophageal dysfunction had a history of recurrent esophageal obstruction and were examined histopathologically post-mortem. barium contrast radiography was used as the gold standard to distinguish the diseased from the control horses. an endoscopically-guided percutaneous needle emg procedure (viking quest r ; software version . ) was performed just caudal to the larynx and just cranial to the thoracic inlet (to monitor striated and smooth muscle, respectively) to visualize esophageal motility. esophageal contractility in both control horses was predominantly reflected by interference patterns associated with longer duration and lower amplitude in smooth muscle compared to striated muscle. mean (ae sd) values were . ae . ms and . ae . mv (n readings) and . ae . ms and . ae . mv (n readings), respectively. in diseased horses, aperistalsis in smooth muscle was the most remarkable finding suggesting a loss of inhibitory neurogenic input resulting in aperistalsis and thus esophageal dysfunction. preliminary findings suggest that endoscopically-guided percutaneous needle emg might become a valuable method in elucidating the pathophysiology of dysfunction of esophageal motility especially in friesian horses. lymphoma affects horses of all ages. unlike in humans, no etiologic agent has been discovered. a year old thoroughbred/warmblood cross mare presented with signs of upper and lower respiratory disease and was subsequently diagnosed with lymphoma and equine multinodular pulmonary fibrosis (empf) and was positive for equine herpes virus (ehv- ) in both the pulmonary tissue and the lymph nodes. retrospective polymerase chain reaction (pcr) testing of six lymphoma cases found that of of the cases were positive on pcr for ehv- ( . %, p . , rr . ). electron microscopy was performed on one sample and herpes virus particles were identified. of the samples in which immunohistochemistry was performed ( of ), only t-cell rich b-cell lymphoma was identified. samples of mesenteric or submandibular lymph nodes from clinically healthy horses were submitted for ehv- pcr analysis; % were positive. gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as kaposi's sarcoma and burkitt's lymphoma. equine herpesvirus , also a gamma herpesvirus, is found in association with equine lymphoma; although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia remains unknown. pathologic events reported to occur in the digital laminae in early stages of sepsis-related equine laminitis include leukocyte extravasa-tion into the laminar interstitium, pro-inflammatory cytokine expression, and epithelial stress. while these events have been documented early in the disease process at both a developmental stage and at the onset of obel grade (og ) lameness in the carbohydrate overload (cho) model of laminitis, the later events occurring at the onset of obel grade lameness(og , time point at which structural failure of the laminae usually occurs) have not been determined. we hypothesized that the inflammatory events described above are sustained through og lameness, likely playing an injurious role culminating in laminar failure. our objectives were to determine pro-inflammatory gene expression, leukocyte extravasation, and epithelial stress at og induced using the cho model. archived laminar tissue samples (snap frozen and paraffin embedded sections) were used from a previous cho study at louisiana state university (control group [n , water], cho group [n , corn starch]. calprotectin (cp) immunohistochemistry (ihc) was used to assess both laminar myeloid leukocyte numbers and epithelial stress; rt-qpcr was used to assess inflammatory gene expression. minimal inflammatory changes were present at og compared to published values at og stage in the cho lameness model including decreased mrna concentrations of cytokines (i.e. -fold increase in il- at og vs. -fold increase at og , no increase in il- b at og vs. -fold increase at og ), chemokines (no change in mcp- at og vs. fold increase at og , -fold increase in il- at og vs. fold increase at og ) and adhesion molecules (no change in e-selectin at og vs. -fold increase at og ). laminar leukocyte emigration was also decreased at the onset of og lameness compared to previously reported leukocyte infiltration at og . interestingly cox- , underwent a greater increase at og (approx. -fold) compared to that reported at og lameness ( -fold). finally, epithelial stress at og evidenced by cp ihc did not follow the uniform widespread distribution reported at og lameness, but instead was present in focal areas in which secondary epidermal laminae on either side of a common primary dermal vascular supply demonstrated increased cp signal. overall, laminar inflammation appears to be subsiding at og lameness, with epithelial stress possibly more dependent on vascular dysregulation instead of inflammatory events. the sustained increase in cox- , central to the induced production of vasoactive prostanoids in disease processes, may play a role in vascular dysregulation. this study was conducted to characterize clinical, laboratory and postmortem findings associated with oleander toxicosis in equids and to determine factors predictive of survival in these cases. retrospective analysis of medical records from our veterinary medical teaching hospital from january , to july , was completed. records of equids demonstrating detectable oleandrin in serum, plasma, urine or gastrointestinal fluid samples or detectable serum digoxin in the absence of pharmaceutical cardiac glycoside administration were included. descriptive statistics were used to evaluate the history, physical examination, and laboratory and postmortem data of affected individuals. logistic regression analysis was used to detect physical examination and laboratory factors significantly associated with survival. thirty equids met inclusion criteria of the study. three of subjects were dead on arrival or died immediately upon arrival ( %). of the remaining equids, % presented with gastrointestinal abnormalities, % were azotemic and % had cardiac arrhythmias. mortality was % for all subjects and % for those treated. the predominant cause for non-survival was cardiac dysfunction. factors significantly associated with survival included relatively decreased hematocrit and serum glucose, relatively increased serum chloride, absence of cardiac arrhythmias, and increased duration of hospitalization. equids with oleander toxicosis frequently present with gastrointestinal upset and may develop cardiac and renal disturbances. patients with cardiac arrhythmias and relatively increased hematocrit and serum glucose and decreased serum chloride are significantly less likely to survive. oleander intoxication is a differential diagnosis for colic in endemic areas, particularly with concurrent azotemia or cardiac dysrhythmia. the quantitative physicochemical approach emphasizes the importance of strong ions (na, k, cl, lactate), pco , and the plasma protein concentrations in determining plasma ph. serum concentrations of strong ions, proteins, and total co are reported on modern biochemical profiles. we hypothesized that the results of serum biochemical analysis can be used for acid-base interpretation in horses. the objective was to determine whether blood ph, anion gap, and strong ion gap could be quantitatively estimated and clinically used based on the results of serum or plasma biochemical analysis. horses ( adults and foals) presented to the isolation unit of our veterinary teaching hospital for suspected infectious diseases were prospectively enrolled. a venous serum sample was analyzed using a hitachi or copas c automated machine. measured parameters included strong ion difference (sid {na k}-{cl lac-tate}), total protein concentration (tp), and total co (tco ), with lactate being measured by blood gas analyzer. a second venous blood sample was collected into a na-heparin blood gas syringe and analyzed for ph (ph m ), pco and concentrations of na, k, cl, and lactate using a radiometer flex blood gas analyzer; sid was calculated from the measured values, and total solids (ts) were estimated using refractometry. serum/ plasma ph (ph calc ) was calculated using stewart's factor equation from the results of serum or plasma biochemical analysis, assuming pco mmhg for serum and pco accurate for plasma. anion gap (ag) was calculated as: ag (na k)-(cl tco ). strong ion gap (sig) was calculated as: sig . x[total protein, g/l]/ ( {pka-ph} )-ag. linear regression analysis was used to compare ph calc to ph m, as well as ag and sig to blood lactate concentrations. measured ph ranged from . to . ( . ae . ). measured sid from serum biochemistry (sid sb ) ranged from . to . meq/l ( . ae . meq/l) and sid from blood gas analyzer (sid bg ) from . to . meq/l ( . ae . meq/l; r . ; sid bg .  sid sb ). sid sb and sid bg showed small variability in measurements. tp ranged from to g/l ( . ae . g/l) and ts from - ( . ae . g/l; r . ; ts .  tp). using sid sb and tco values with constant pco , ph calc was poorly associated with ph m (r . ; ph calc . . ). in contrast, using sid bg with accurate pco , ph calc was closely associated with ph m (r . ; phcalc . . ) and the equation was not different from the line of identity. anion gap and sig (meq/l) calculated were significantly linearly correlated with lactate concentrations (mmol/l); ag .  [lactate] . (r . ), and sig À .  [lactate] . (r . ). we conclude that ph calc using sid sb , tco and constant pco values is not accurate. however, variability of measured biochemical parameters between machines was small, permitting use of serum biochemistry for clinical metabolic acid-base abnormalities interpretations of patients. these results reemphasize the importance of strong electrolytes and proteins in acid-base balance. metalloproteinases (mmps) are critically important in remodeling processes and in wound healing. however, excessive activation of mmps by pro-inflammatory mediators including cytokines, prostaglandin e , and nitric oxide lead to tissue breakdown. this is observed in osteoarthritis (oa) which is characterized by erosive lesions in articular cartilage. in hereditary equine regional dermal asthenia (herda), afflicted horses exhibit collagen abnormalities and can have associated chronic inflammation and aberrant wound repair. herda affects horses with quarter horse bloodlines and is similar to the human hereditary connective tissue syndrome ehlers danlos (eds). many adult eds patients suffer from joint pain and oa. we hypothesized that chondrocytes from articular cartilage of herda horses have increased activity of mmps. to test this hypothesis, chondrocytes were retrieved from articular cartilage of homozygous herda carpal and hock joints. chondrocytes from normal horses were also obtained for comparison. chondrocytes were seeded at x /ml into -well plates and incubated at c, % co for up to seven days. activity of secreted mmps was determined by zymography using equal amounts of proteins for loading. secreted mmps were analyzed by western blot. zymography showed that normal chondrocytes secreted two major bands with gelatinolytic activity observed at and kda suggestive of the latent form of mmp- and mmp- , respectively. less intense bands of gelatinolytic activity were observed at about and kda suggestive of the active form of mmp- and mmp- . another band of activity was also seen at kda which is suggestive of a dimer of mmp- that has been reported when mmps are in excess of tissue inhibitors of metalloproteinases (timps). chondrocyte cultures from homozygous herda cartilage showed a similar profile but with decreased activity by % at kda and - % increased activity at kda compared to normal chondrocytes. western blot analysis detected mmp- and mmp- immunoreactivity in chondrocyte culture media of herda-afflicted and normal horses. the present study demonstrates for the first time that horses suffering from herda have increased mmp activity which may predispose them to the development of lesions in articular cartilage. research supported by nutramax laboratories, inc. equine polysaccharide storage myopathy (pssm) type is a dominantly inherited glycogenosis caused by a mutation in the gene coding for skeletal muscle glycogen synthase type (gys- ). the disease has been reported to affect the haflinger breed but so far its prevalence is unknown. aim of this preliminary study was to estimate the occurrence of the gys- mutation in austrian haflingers and establish which of the seven haflinger sire lines appear mostly affected. gys- genotyping of randomly chosen haflingers was performed with a validated restriction fragment length polymorphism assay. resting and post-exercise muscle enzyme activities (creatine kinase (ck), aspartate aminotransferase (ast), lacate dehydrogenase (ldh)) and blood lactate concentrations were compared between horses with and without the mutation. among the horses were heterozygous (hr) carrier of the mutation. no homozygotes (hh) were identified. all horses with the gys- mutation were descendents of the a-or w-sire lines. the estimated hr prevalence was % ( % ci: . - . %). ck activity after exercise (p . ) was significantly higher in hr horses compared with horses not carrying the mutation (rr). ast activity was significantly higher in the hr group at rest and after exercise (p o . ). there was no statistically significant difference in resting ck, resting and post exercise ldh activity or blood lactate between hr and rr. results suggest that the prevalence of hr in the austrian haflinger population is higher than in the overall quarter horse population and might be as high as %, similar to some draft horse breeds. further research is needed to establish the prevalence within the different breeding lines. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder affecting quarter horse lineages. although a mutation in the gene encoding cyclophilin b has been genetically linked to herda, its causal association with the disease is not yet documented. previously, we demonstrated reductions in ultimate tensile strength (uts), modulus of elasticity, and energy to failure (toughness) of skin from many corporal regions of herda animals. given the presumed relationship between her-da and abnormal collagen structure, and the predominance of type i collagen in skin, we hypothesized that altered biomechanical properties would be detected in tendons which are rich in type i collagen. to evaluate this hypothesis we compared the uts, modulus of elasticity, and energy to failure of forelimb deep digital flexor tendons (dft) from six herda horses to six age-matched controls. isolated dft was secured and pulled to failure on an instron s universal testing instrument using purpose-built cryogenic clamps. analysis of variance was executed using sas . proc glimmix program (sas institute, ). p-values . were identified as significant. uts and modulus of elasticity were significantly lower in herda dft when compared with controls (p o . ); energy to failure did not differ between groups. these findings document abnormal biomechanics in herda tendon, leading us to postulate that lower uts and modulus of elasticity associated with the herda defect could convey a competitive advantage in the athletic disciplines in which this defect has segregated. (references on request). a proprietary herbal biocontamination product (bios) approved for cosmetic use in france, inhibits proliferation of medically relevant bacteria, mold, and viruses. these properties make bios potentially useful as a topical wound medication, prompting us to compare bios to silver sulfadiazine (ssd) in a distal extremity wound healing model in horses. using general anesthesia, two . cm wounds were aseptically created on the dorsomedial aspect of all limbs. for the duration of the study, two contralateral limbs were randomly chosen to be bandaged; the other two limbs were un-bandaged -with one limb of each group being treated with % bios and the other with ssd. for each limb the most proximal wound served as an untreated control. every hours wounds were evaluated, digitally photographed, and perimeter and area determined using morphometric software (imagej, nih). analysis of variance did not identify significant differences between ssd or bios treatment for wound perimeter (p . ) or area (p . ). at individual time points the effect of bandaging was significant when area was evaluated (p . ) and trended toward significance for perimeter (p . ) comparisons, substantiating published reports that bandaging modifies wound healing. difference in perimeter and area between control and treatment were highly significant (p o . ), substantiating the importance of topical treatment. over the study duration, effects of bandaging (p o . ) and topical treatment (perimeter p o . ; area p . ) continued to be highly significant. bios performance in the equine distal extremity wound model was equivalent to ssd. both bandaging and topical treatment significantly impacted wound healing. this effect was compounded when both variables were evaluated over time. radiolabeled leukocytes are the only scintigraphic method currently available for identifying sites of infection and/or inflammation in horses; however the clinical applicability of this technique is limited by expense and poor efficacy. this pilot study compares the accumulation of m tc-labeled igg, peg-liposomes and leukocytes in an equine muscle abscess model. three mixed breed adult horses had  cfu s. equi zooepidemicus inoculated into the right semitendonosis to create an abscess. peg-liposomes were prepared via the film hydration method and labeled using mci m tc-hexamethyl-propylene-amine-oxime ( m tc-hmpao). autologous leukocytes were obtained from ml whole blood and labelled using mci m tc-hmpao. commercial equine polyclonal igg was conjugated with the chelator hydrazinonicotinamide (hynic) and labelled with mci m tc. radiopharmaceutical administration was initiated hours after inoculation. horses and received mg m tc-igg, . mmol/kg m tc-liposomes and m tc-leukocytes, with a hour interval between each radiopharmaceutical. horse received only m tc-leukocytes. scintigraphic examinations were performed at and hours post injection (p.i.) with each radiopharmaceutical. after the final study, horses were euthanized and tissue samples collected. the percentage of injected dose per kilogram of tissue (%id/kg) was calculated for the region of the abscess, normal muscle and multiple organs. scintigraphic examinations demonstrated increased radiopharmaceutical in the region of the abscess with all three techniques at both time-points. at hours p.i. abscess-to-background ratio was highest using m tc-igg ( . ae . ). at hours p.i. abscess to background ratio was highest using m tc-liposomes ( . ae ). tissue biodistribution data revealed abscess to muscle ratios of ( m tc-igg), ( m tc-liposomes), and . ( m tc-leukocytes). this preliminary data demonstrates that m tc-liposomes, m tc-igg and m tc-leukocytes exhibit long circulating characteristics and accumulate at inflammatory/infectious foci after intravenous injection in horses. m tc-igg and m tc-liposomes appear to be superior to m tc-labelled leukocytes in this model. due to its low cost and ease of preparation, m tc-igg has great potential for clinical use where identification of infectious or inflammatory foci is necessary. digital hypothermia is used clinically to decrease the incidence of sepsis-related equine laminitis, a disease causing structural failure of digital laminae resulting in crippling lameness. due to the fact that hypothermia was recently reported to effectively decrease laminar expression of inflammatory molecules including pro-inflammatory cytokines, chemokines and cox- in equine laminitis, our laboratory is investigating the effect of hypothermia on central upstream signaling cascades which may induce expression of these diverse inflammatory molecules. the p mapk pathway has recently been reported to be a central component of inflammatory signaling in multiple diseases including human sepsis, and is currently being assessed as a therapeutic target. we thus hypothesized that ) p mapk is upregulated and activated in affected laminae in equine laminitis and ) digital hypothermia inhibits inflammatory mediator expression by blocking p mapk phosphorylation (indicator of p mapk activation). western hybridizations using both a total p mapk and a phospho-p mapk antibody were performed on archived pooled laminar samples from black walnut extract (bwe) model ( control, developmental (dev) groups [ . h & h post bwe administration] and the onset of obel grade lameness (og ) [n each]) and carbohydrate overload (cho) models (con [n ], dev [n ], og [n ]) of laminitis, and individual laminar samples from two groups of horses from a digital hypothermia (dh) study. in the dh study, one forelimb of each horse was kept at approximately c in ice water and the other at ambient temperature following administration of g/kg oligofructose (of). dorsal laminae were harvested for snap freezing at either hours after of administration (dev, n ) or at the onset of lameness (og , n ) using protein extracted from treated and untreated digital laminae of each horse. increased laminar concentrations of phospho-p mapk were present in the developmental periods ( . h and h) in the bwe model, and in both the dev and og periods in the cho laminitis models. however, digital hypothermia had no effect on laminar phospho-p mapk concentrations. thus, p mapk is activated in affected laminae in multiple models of laminitis, but does not appear to be the central signaling cascade through which hypothermia works to block the expression of inflammatory molecules. therefore, p mapk is not likely to be a viable therapeutic target as a sole source for blocking the multiple inflammatory signaling mechanisms inhibited by local hypothermia. abstract e- does cefquinome penetrate the blood brain barrier in the normal horse? hollis ar duggan ve and corley ktt . scott dunn's equine clinic, berkshire, uk; university college dublin, dublin, ireland; anglesey lodge equine hospital, the curragh, ireland. meningitis is a rare but serious condition that occurs in both foals and adult horses. there is currently a restricted choice of antimicrobials that are both safe to use in horses and penetrate the blood brain barrier. cefquinome is a fourth generation cephalosporin that has activity against streptococcus, the most commonly reported causative organism in adult horse meningitis. therefore, if cefquinome were to achieve therapeutic concentrations in cerebrospinal fluid following routine administration, this would be an exciting advance for the treatment of meningitis in the horse. mature, healthy horses were used on separate occasions, seven days apart, in a crossover design. each horse was administered either cefquinome ( mg/kg) or saline (equivalent volume). cerebrospinal fluid was collected via atlanto-occipital puncture under general anaesthesia and hours after administration of cefquinome or saline placebo. blood samples were collected prior to, and and hours after administration of cefquinome or placebo. all samples were analysed for the presence of cefquinome by a laboratory masked to treatments administered. cefquinome was detectable in the cerebrospinal fluid in all horses hours after intravenous administration, and in horses hour after administration. cefquinome penetrates the blood-brain barrier and it is therefore a potential treatment for equine meningitis. further investigation of the pharmacokinetics and pharmacodynamics of cefquinome in the cerebrospinal fluid is warranted to establish the optimum intravenous dose. the purpose of this study was to determine if enrofloxacin alters the pharmacokinetics of firocoxib in the horse. firocoxib is a coxibclass nonsteroidal anti-inflammatory drug (nsaid) approved for use in horses to control pain and inflammation associated with osteoarthritis. dosages of firocoxib are species dependent, with the recommended dose for horses being . mg/kg as an oral paste every h. the main elimination pathway of firocoxib is hepatic; however the effects of concurrent administration of drugs that may inhibit its metabolism have not been evaluated. enrofloxacin is a synthetic antibacterial agent from the flouroquinolone group developed for veterinary use. it is primarily used for gastrointestinal, urogenital, skin and respiratory tract infections in various animals. a well acknowledged problem associated with flouroquinolone usage is their effect on the metabolism of other drugs. co-administration of multiple drugs can result in unpredictable therapeutic outcomes. often it is either diminished therapeutic efficacy or increased toxicity of one or more of the administered drugs. various pharmacokinetic interactions between antimicrobials and nsaids have been described. six healthy, adult mares were administered . mg/kg of firocoxib orally. samples were collected by direct venipuncture of the jugular vein at (control), , , and min, , , , , , , , , and h after administration. after a day washout period the six horses were pretreated days with enrofloxacin mg/kg intravenously every h then on the fourth day given . mg/kg of firocoxib orally. samples were collected at (control), , , and min, , , , , , , , , and h after administration. all samples were stored at À c until analysis using a validated hplc method. the t / , c max , t max , auc - and auc -f after firocoxib administration were . angiotensin converting enzyme (ace) inhibitors improve survival and quality of life in humans and small animals with cardiovascular and renal disease. there is limited information regarding their effects in horses. the purpose of this study was to determine the pharmacokinetics of quinapril and its effects on ace inhibition in horses. six healthy horses were administered quinapril at mg iv, mg po or mg po in a -way crossover design. blood was collected at predetermined times for measurement of quinapril and quinaprilat concentrations using high pressure liquid chromatography, as well as ace concentrations using a radioenzymatic assay. normally distributed data were analyzed with one way repeated measures analysis of variance (rm-anova) and non-normally distributed data were analyzed using friedman rm_anova on ranks. significance was set at p o . . no adverse effects were observed during the study period. plasma quinapril concentrations were low and rapidly declined after iv administration. quinaprilat concentrations were below the limit of quantification ( . mg/ml). ace activity was significantly decreased from baseline at . and hour after iv dosing and at all timepoints after oral dosing. maximum % ace inhibition was , and % with the iv, high and low oral doses, respectively. these results suggest that, despite low plasma concentrations, quinapril has sufficient oral absorption and results in inhibition of ace in healthy horses. controlled studies in clinically affected horses are indicated. this study determined the pharmacokinetic profile of firocoxib in healthy neonatal foals. foals are more sensitive to the side effects of nsaid, primarily due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. firocoxib, a novel, second generation nsaid, is reported to have reduced side effects due to cox- selectivity. the pharmacokinetic profile of firocoxib in neonates has not been established. we hypothesized that firocoxib given po to neonatal foals would achieve therapeutic concentrations in plasma. seven healthy foals of mixed gender were administered . mg/kg firocoxib po q h for nine consecutive days, commencing at h old. blood was collected for firocoxib analysis at (dose # only), . , . , , , , , and h after doses # , and . for all other doses ( , , , , and ) blood was collected immediately prior to the next dose ( h trough). elimination samples were collected after dose # . plasma was stored at À c until analysis. physical examinations were performed on foals daily and body weight obtained every two days during the sampling period. analysis of plasma samples by liquid chromatography-mass spectrometry revealed firocoxib was rapidly absorbed. after the initial dose, a maximum plasma concentration was reached in min, minimal accumulation after repeat dosing occurred and steady state was obtained after approximately four doses. after the final dose, plasma drug concentration decreased in a linear manner with an estimated terminal t / of h. seventy-two hours after the final dose, firocoxib was not detectable (o ng/ml). erythrocytosis is reportedly a rare finding associated with hepatocellular carcinoma in horses. the purpose of this study was to determine the relative frequency of erythrocytosis and the clinicopathologic abnormalities and hepatic histopathology associated with erythrocytosis in horses with liver disease. ninety-seven horses aged ! year with clinicopathologic or clinical signs of liver disease, a complete blood count (cbc), and hepatic histopathology were included. information on cbc, biochemical variables, and hepatic histopathology was collected from records. data from horses with erythrocytosis (packed cell volume %) were compared to those without using the mann-whitney rank sum test with significance set at p o . . there were no differences between groups in white blood cell count, gamma-glutamyl transferase, sorbitol dehydrogenase, aspartate aminotransferase, and alkaline phosphatase activities, total protein, albumin, globulin, blood urea nitrogen, or glucose concentrations. fibrosis ( %), biliary hyperplasia ( %), inflammatory infiltrate ( %), megalocytosis ( %), degeneration ( %), necrosis ( %), cholestasis ( %), anisocytosis and anisokaryosis ( %), and lipidosis ( %) were observed in livers of horses with erythrocytosis. neoplasia ( %) was observed rarely. this study reports a high frequency of erythrocytosis in horses with liver disease. erythrocytosis is associated with higher total bilirubin and serum bile acids concentrations. common histopathologic changes include fibrosis, biliary hyperplasia, and inflammatory infiltrate. hepatic neoplasia was rare. this study was performed to determine if horses diagnosed with equine proliferative enteropathy (epe) from lawsonia intracellularis (li) infection had long term effects from disease based on their sale price as yearlings and race earnings. a retrospective review of medical records of thoroughbred horses that were treated for lawsonia intracellularis infection between january , and january , at hagyard equine medical institute in lexington, kentucky was performed. three criteria were used for inclusion in this study. first, each horse had presumptively been diagnosed with li based on physical examination findings such as ventral edema, diarrhea, lethargy, or poor body condition. second, horses had hypoalbuminemia of less than . mg/dl (normal reference range: . - . mg/dl). third, each horse had a positive fecal polymerase chain reaction (pcr) for li, a positive serum immunoperoxidase monolayer assay (ipma), or both. an ipma titer greater than or equal to was considered positive for disease. horses met the initial criteria. of the horses sold at public auction as yearlings. the sale price of these horses was compared to the average sale price of all yearlings by the same sire as the affected horse (control group). of the horses raced in the united states. their monetary earnings from racing were compared to the average monetary earnings of all progeny by the same sire as the affected horse (control group). earnings of horses that were between and years of age ( / horses) at the conclusion of the study were compared to the lifetime average earnings of the stallion's progeny. earnings from horses that were two years of age ( / ) at the end of the study were compared to the two year old average earnings of the stallion's progeny. monetary earnings from all races prior to december , were included in the study. horses both sold at public auction and raced. as well as being included in the total number of horses that sold and raced, their sale records and monetary earnings were compared to the averages from their respective sire as a separate group. this retrospective study indicated that yearling horses previously infected with li do not sell for as much at public auction as their herdmates, but their monetary earnings from racing are not significantly different from other horses. these results should assist practitioners in guiding owners in determing if treatment of horses with epe is appropriate and it may aid in reassuring owners that despite the poor condition of the horse during and shortly after the course of disease, horse may still have future athletic potential. this abstract was presented at the aaep in december . bronchopneumonia caused by streptococccus equi subsp. zooepidemicus (s. zooepidemicus) is one of the most important causes of morbidity in weanling foals. ceftiofur crystalline free acid (ccfa) is a long acting third-generation cephalosporin antimicrobial recently approved for the treatment of bronchopneumonia associated with s. zooepidemicus in adult horses. the objective of the present study was to determine the disposition of ccfa in plasma and pulmonary epithelial lining fluid (pelf) of weanling foals. six healthy -to month-old weanling foals were administered a single intramuscular injection of ccfa at a dose of . mg/kg of body weight. concentrations of desfuroylceftiofur acetamide (dca) and related metabolites were measured by use of ultra-high performance liquid chromatography and tandem mass spectrometry. following im administration, median time to maximum plasma and pelf concentrations was h ( - h) . mean (ae sd) peak dca concentration in plasma ( . ae . mg/ml) was significantly higher than that in pelf ( . ae . mg/ml). terminal half-life of dca in plasma ( . ae . h) was not significantly different from that of pelf ( . ae . h). time above the therapeutic target of . mg/ml was significantly longer in plasma ( ae h) than in pelf ( ae h). based on the results of the present study, intramuscular administration of ccfa at a dose of . mg/kg would be appropriate for the treatment of bronchopneumonia caused by s. zooepidemicus and other susceptible pathogens in weanling foals. fgf- is secreted by osteocytes and osteoblasts in response to hyperphosphatemia. fgf- enhances phosphaturia and is postulated to have a central role in the development of secondary renal hyperparathyroidism. hyperthyroid cats have elevated plasma phosphate and parathyroid hormone concentrations, which may in part be associated with underlying chronic kidney disease (ckd). the aim of this study was to determine if plasma fgf- concentrations were associated with the presence of underlying ckd in hyperthyroid cats, and to investigate the changes in plasma fgf- concentrations that occur following treatment of hth. hyperthyroid cats were recruited from two london-based first opinion practices between and . cats that were azotemic at diagnosis were excluded. hth was treated with anti-thyroid medication alone or in combination with thyroidectomy. cats were included in the study if they had a plasma total thyroxine concentration o nmol/l documented for a six month period following commencement of treatment. cats were classified as having azotemic ckd if they developed renal azotemia within six months of establishment of euthyroidism. otherwise cats were deemed to have normal renal function. stored edta plasma samples were assayed for fgf- using a recently validated elisa. the mann-whitney u test and the wilcoxon signed rank test were used to compare between the groups and assess the response to treatment respectively. results are reported as median [ th , th percentiles]. correlations were made using spearman's correlation coefficient. thirty one cats with hth ( azotemic and non-azotemic) were included in the study. plasma phosphate concentrations decreased following treatment in cats that did not develop azotemia ( . [ . , . ] mg/dl vs. . [ . , . ] mg/dl; n , p . ) whereas plasma phosphate concentrations did not change significantly following treatment in cats that did develop azotemia ( . [ . , . ] mg/ dl vs. . [ . , . ] mg/dl; n , p . ). plasma fgf- concentrations were significantly higher in cats that developed azotemia than cats that did not at both pre treatment ( . [ . , . ] pg/ml vs. . [ . , . ] pg/ml; p . ) and post treatment ( . [ . , . ] pg/ml vs. . [ . , . ] pg/ml; p . ) timepoints. plasma fgf- concentrations increased following treatment in both azotemic (p . ) and non-azotemic groups (p . ). plasma fgf- concentrations and plasma phosphate concentrations were not correlated at baseline (r s . , p . ) or following treatment (r s . , p . ). plasma fgf- concentrations were higher in pre-azotemic cats than non-azotemic cats and increased following treatment of hth. the reason that fgf- concentrations increased following treatment, particularly in the face of decreasing plasma phosphate concentrations in cats that remain non-azotemic, is unclear but may be related to the decline in glomerular filtration rate. hyperthyroidism is a disorder resulting from the excessive production and secretion of t and t by the thyroid gland. although the disorder and its pathological lesions have been well studied and described the cause remains illusive. whole blood and solid tissue samples from non-diseased, severe disease and mild disease cats based on t levels and thyroid histology were used in this study. whole blood samples from non-disease cats, severe disease cats and mild disease cats as well as solid thyroid tissue samples from non-disease cats, severe disease cats and mild disease cats were collected and processed. the resulting total rna samples were used for genechip analysis using our custom feline gene chip designed by affymetrix. data analysis was performed using the partek s gs software for gene expression data. the robust multichip average algorithm was used for background adjustment, normalization, and probe-level summarization of the raw data. anova analysis was performed to find significant differentially expressed genes with a minimal false discovery rate control of . and a fold change of . in each direction. during the mild disease state, pathways associated with dna damage and apoptosis are most prominent. at later stages when the histopathological disease is more severe in addition to the aforementioned pathways others associated with tgf-beta signaling, cell adhesion and extracellular matrix remodeling take more prominence. the analysis of this unique data set generated from the use of our proprietary genechip revealed molecular mechanisms that are associated with the transition from non-disease, to mild disease to severe disease, in the thyroid tissue as well as the blood. these mechanisms could provide insights into the causes of the disease and identify potential new therapeutic and diagnostic targets. although it is well established that concurrent chronic kidney disease (ckd) develops in about % of hyperthyroid cats, no one has reported the use of the iris staging system for ckd before and after treatment of these hyperthyroid cats. the purpose of this study was to compare the effects of treatment in hyperthyroid cats with known stage and ckd in order to determine the effects of restoring euthyroidism or inducing hypothyroidism has on the iris stage in these cats. we evaluated hyperthyroid cats (median age, years) in this study. one day prior to treatment, serum t concentration, serum chemistry analysis, complete urinalysis, and urine protein-to-creatinine ratio (upc) were measured. all cats were again evaluated with the same parameters again months after treatment with i. prior to treatment, ( %) of the cats had no evidence of azotemia (serum creatinine o . mg/dl), whereas cats ( %) had stage ckd (serum creatinine, . - . mg/dl). in the cats, iris staging revealed proteinuria in cats ( %), with borderline proteinuria (upc, . - . ) and with overt proteinuria (upc . ). hyperthyroidism was cured in all cats (median post-t , . mg/dl). all cats had a good response to treatment; there were no signs of ckd except for polyuria and polydipsia in some cats. a significant (p o . ) increase in median values for both serum urea nitrogen ( mg/dl to mg/dl)) and creatinine ( . to . mg/dl) occurred after treatment. nine of the cats ( . %) classified as nonazotemic or iris stage prior to i progressed to stage ckd after i. all cats with stage ckd before treatment remained azotemic after i, with cats remaining in stage ckd, and cats progressing to stage ckd (serum creatinine, . - . mg/dl). there was a significant inverse relationship (p . ) between pretreatment urine specific gravity (usg) and post-treatment serum creatinine in the cats. of the cats with post-treatment serum creatinine values . mg/dl (stage to ckd), ( %) had pretreatment usg of o . . in contrast, in the cats with post-treatment serum creatinine values o . mg/dl, only ( %) had pretreatment usg of o . . a significant (p o . ) decrease in median upc from . to . occurred after treatment, but there was no relationship between degree of proteinuria and iris stage in these cats. two cats developed iatrogenic hypothyroidism after i, diagnosed by finding low serum t and high ctsh concentrations. both hypothyroid cats had progressed from stage before treatment to stage and ckd, respectively, after i; after thyroxine replacement, serum creatinine decreased to near pretreatment concentrations in both cats. conclusions: ) iris stage ckd is common in untreated hyperthyroid cats. ) progression to next higher iris stage is common after treatment, but most cats with remain relatively asymptomatic for ckd. ) usg may be helpful in predicting which cat's iris stage will progress after i. ) iatrogenic hypothyroidism worsens azotemia, an effect that appears reversible with replacement therapy. home blood glucose monitoring (hbgm) of diabetic pets is likely to result in superior glycaemic control, minimizing episodes and impact of dangerous hypoglycaemia and reducing costs. nevertheless, it has proven difficult to objectively establish a clear benefit of hbgm using biological parameters (clinical signs, blood glucose, fructosamine). the current study aimed to assess the impact of hbgm on owner perceived quality of life (qol) aspects of diabetes mellitus (dm) treatment, using the recently validated psychometric tool diaqol-pet. owners of insulin treated diabetic cats were recruited to complete the -item tool, evaluating areas affecting the cat's and owner's qol, including: worry about pet's dm, hypoglycaemia, costs, owner's desire for autonomous control over the pet's dm, etc. item-weighted-impact-scores (iwis), reflecting frequency and importance ratings of each item, were calculated, as well as averageweighted-impact-scores (awis; average iwis of all items), as an overall measure of diabetes dependent qol. frequencies, iwis and awis were compared between owners practising hbgm and those who did not using mann whitney u test (significance p o . ). two hundred and eleven owners of insulin treated diabetic cats completed the diaqol-pet; owners practised hbgm, whereas the remaining did not practise any form of home monitoring (including urine glucose). iwis for 'excessive drinking' and 'owner wanting more control' were significantly different between the hbgm-group (mean /-standard deviation: À . /À . and À . /À . ) and the non-hbgm-group (À . /À . and À . /À . ). there was no significant difference between the groups with regards to the iwis for other items, including 'worry about hypoglycaemia' or 'worry about pet's dm'. polydipsia was reported significantly more frequently in the non-hbgm-group and this was the reason for the difference between groups in this item's iwis as it was considered of equal importance. frequency and iwis of reported occurrence of hypoglycaemia signs were not significantly different. awis for both groups was not significantly different (hbgm: À . /À . ; non-hbgm: À . /À . ). the current study suggests that hbgm is predominantly practised by owners who desire more autonomous control over their cat's dm. the frequency of polydipsia was lower in the hbgm-group perhaps suggesting superior control. however, hbgm did not detectably affect the impact of the majority of qol-items, nor the frequency of hypoglycaemic episodes. overall diabetes dependent qol of diabetic cat and owner, as measured per diaqol-pet, was unaffected by hbgm. these data argue for the use of hbgm in selected pet-owner combinations rather than as part of a practice's standard dm management protocol, although further studies are indicated. insulin resistance is associated with impaired activation of the insulin signaling pathway in peripheral tissues such as skeletal muscle, visceral and subcutaneous (sc) adipose tissue. high plasma glucose, fatty acid and endotoxin levels are three major causes of insulin resistance in feline and human obesity and in type diabetes mellitus. however, the mechanisms by which these factors influence insulin action are still unclear. therefore, our aim was to investigate the tissue-specific expression of crucial mediators of insulin action such as the insulin-receptor substrate (irs ), the serine/threonine protein kinase b (pkb/akt) and of the principal insulin-dependent glucose transporter protein (glut ) in feline models of hyperglycemia, hyperlipidemia and subacute endotoxemia. healthy cats were infused through the jugular vein with glucose (n ), lipids (n ) or lipopolysaccharide (lps; n ) for days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: - mmol/l; triglycerides: - mmol/l) or to induce a systemic low-grade inflammation (lps; rectal temperature: . - . c), respectively. healthy control cats were infused with saline (n ). on day , specimens were collected from skeletal muscles, visceral and sc fat and processed for irs mrna expression, total and phosphorylated pkb/akt and glut protein expression. gene transcripts of irs were not different between the groups. compared to controls, skeletal muscle pkb/akt phosphorylation was % lower in cats infused with glucose (p o . ); lipid-infused cats showed a trend for a decrease in pkb/akt phosphorylation ( % lower than saline) and had decreased glut expression (p o . ) in muscle. total (p o . ) and phosphorylated (p o . ) pkb/akt protein expression were decreased in the sc adipose tissue of lps-infused cats compared to controls. in these cats, phosphorylation of pkb/akt protein was also decreased in visceral fat (p o . ). sustained hyperglycemia and, to a lesser extent, hyperlipidemia impaired insulin signaling and glucose transport pathways primarily in skeletal muscle; endotoxemia reduced insulin sensitivity mainly in adipose tissues. thus, the development of insulin resistance in response to hyperglycemia, hyperlipidemia or endotoxemia might be affected by tissue-specific mechanisms in cats. separately used, single photon emission computed tomography (spect) and computed tomography (ct) both lack sensitivity and are additionally hampered by a poor anatomical location capacity and a lack of specificity, respectively. these drawbacks suggest an interest in the fusion of images obtained by the techniques. the aim of this study is to test spect/ct fusion performance in dogs with insulinoma. inclusion criteria were: / a biological diagnosis of insulinoma; / an examination by high resolution ct scan and in-pentetreotide spect followed by spect/ct fusion; / a surgical or post mortem examination completed by histopathological analysis. spect examination showing abnormal foci and ct scan showing pancreas, lymph nodes (ln) or liver abnormalities were considered positive. in case of double positivity, presence (imp ) or absence (imp-) of superimposition of abnormal images was noted. ten dogs were included. in / dogs, superimposition of abnormalities couldn't be tested. ct scan detected abnormal images [ pancreatic nodules (pn), enlarged ln (eln)] while spect failed to show any abnormal uptake. both dogs became euglycemic after removal of pn and ln designed by ct scan. in / , all abnormal images were classified as imp [ pn, eln and diffuse hepatic infiltration (dhi)]. surgery performed on / resulted in euglycemia in ; dog remained hypoglycemic after partial removal of pn. pn localization and dhi were confirmed after necropsy in the th dog. in / dogs imp and imp-images were both recorded. in dog, a dhi was classified as imp but pn localization was imp-: localized in the left lobe by ct scan and in the corpus by spect, the latest localization being confirmed after necropsy. in the other dog pn localization was imp but a diffuse spect signal superimposing to the liver considered as normal on ct scan was noted. hepatic biopsy confirmed spect results. this study confirms an imperfect sensitivity of both ct scan and spect. it confirms that ct scan can be associated with unspecific abnormal images. subject to a confirmation on a larger cohort of dogs, it indicates that imp images provide specific detection and accurate localization of canine insulinomas' primary lesions and metastasis. the majority of dogs with primary hypoadrenocorticism (ph) reveal clinical and laboratory abnormalities of gluco-and mineralocorticoid deficiency. in some of them sodium and potassium levels are normal, a phenomenon currently called atypical addison's. it has been postulated that in those cases adrenal destruction is confined to the zona fasciculata/reticularis, resulting in isolated glucocorticoid deficiency. however, there are no histological studies confirming a normal zona glomerulosa and in most reported cases diagnosis was based solely on low post-acth cortisol levels. the aim of the study was to evaluate aldosterone (aldo) levels in dogs with ph with and without electrolyte abnormalities. seventy dogs with newly diagnosed ph were included. aldo concentrations (ria, coat-a-count s , siemens) were measured before and min after administration of mg synthetic acth (synacthen s , novartis) iv. results were compared to those of healthy dogs and dogs with diseases mimicking ph. to confirm that peak concentrations were not missed aldo was additionally measured , and min after acth in dogs ( with ph, with ph mimicking diseases). results were analysed by means of non-parametric statistical methods (p o . ). post-acth aldo was significantly lower in dogs with ph ( - pg/ml, median pg/ml) than in healthy dogs ( - pg/ml, median pg/ml) and in dogs with ph mimicking diseases ( - pg/ml, median pg/ml). low post-acth aldo was found in / dogs with ph, in / of them levels were below the detection limit of the assay. normal sodium and potassium levels were found in / dogs ( %), / dogs ( %) had hyponatremia and normal potassium, / dogs ( %) had hyponatremia and hyperkalemia. electrolyte abnormalities ranged from mild to severe. there was no correlation between post-acth aldo and sodium and a weak correlation between post-acth aldo and potassium (r À . ). aldo concentrations were not different , and min after acth. the results demonstrate that aldo levels are low in most dogs with ph independent of the degree of electrolyte abnormalities. this implicates that all three zones of the adrenal cortex are compromised and that there are mechanisms which allow maintenance of a normal electrolyte balance without aldo. definitive diagnosis of canine hypoadrenocorticism (ha) is based on inadequate cortisol secretion following adrenocorticotropic hormone (acth) administration. an abnormal serum sodium to potassium (na:k) ratio can be used to determine whether an acth stimulation test is warranted. the aim of this study was to examine the utility of combining the na:k ratio with white blood cell counts to determine whether an acth stimulation test is warranted. a retrospective review of medical records of dogs examined between and was performed. dogs diagnosed with ha and control dogs, in which a diagnosis of ha was excluded during the study period, were included. inclusion criteria for all dogs were hospitalization with intravenous fluid therapy, a complete blood count, and serum na and k measurements at the time of initial examination. dogs were included in the ha group if they also had pre and post acth stimulation serum cortisol concentrations . mg/dl. dogs were included in the control group if they had resting or post acth stimulation serum cortisol concentration . mg/dl. exclusion criteria were recent administration of glucocorticoids, prior treatment of hyperadrenocorticism, or serum cortisol concentration . mg/dl but . mg/dl. continuous variables were compared between groups using the mann-whitney u test. receiver operating characteristic (roc) curves were produced to assess the sensitivity and specificity of detecting ha with various cutoffs for each variable. data is presented with % confidence intervals (ci) and statistical significance was defined as p o . . the na:k ratio, neutrophil count and neutrophil:lymphocyte ratio were significantly lower in dogs with ha than in dogs without ha (p o . for each). lymphocyte and eosinophil counts were significantly higher in dogs with ha compared to dogs without ha (p o . for each). the areas under the curve by roc analysis were largest for na:k ratio ( . , ci: . - . ) and lymphocyte count ( . , ci: . - . ). a na:k ratio . was % sensitive (ci: - %) but only % specific (ci: - %) for detecting ha. a lymphocyte count ! . x cells/ml was % sensitive (ci: - %) and % specific (ci: - %). conversely a na:k ratio . was % sensitive (ci: - %) but % specific (ci: - %) and a lymphocyte count ! .  cells/ml was % sensitive (ci: - %) but % specific (ci: - %). a na:k ratio . was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha and a lymphocyte count ! .  cells/ml was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha. a combination of this na:k ratio ( . ) and lymphocyte count (! .  cells/ml) was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha. these results indicate that the combination of lymphocyte count and na:k ratio results in a better screening test for ha than the use of the na:k ratio alone. pheochromocytoma is a malignant, catecholamine-producing, adrenomedullary tumor. clinical signs resulting from excessive catecholamine secretion are typically non-specific, making differentiation from other adrenal tumors a challenge. elevated plasma concentrations of the catecholamine breakdown products metanephrine (mn) and normetanephrine (nmn) are used to identify pheochromocytoma in humans. this study tested the hypothesis that plasma metanephrine concentrations are greater in dogs with pheochromocytoma than in dogs with other adrenal neoplasms, healthy dogs and dogs with non-adrenal illness. edta plasma was collected from healthy dogs and unwell, hospitalized dogs with non-adrenal illness, pheochromocytoma and cortical tumors between april and october . samples were stored at À c before measurement of free mn and nmn concentrations using high pressure liquid chromatography at the central laboratory for clinical chemistry at the university of groningen ( samples) or the mayo clinic, rochester, minnesota ( samples). kruskal-wallis tests followed by dunn's multiple comparison analysis were used to compare results between groups. significance was set at p o . . results are reported as median [range] . eight dogs with pheochromocytoma, healthy dogs, dogs with non-adrenal illness and dogs with cortical tumors were sampled. pheochromocytoma was diagnosed histologically ( dogs) or cytologically ( dog). cortical tumors were diagnosed histologically ( dogs) or by response to trilostane treatment after obtaining consistent endocrine test results ( dogs occult hyperadrenocorticism (hac) has been theorized to exist in which excess adrenal sex hormone secretion induces the clinical signs and laboratory changes associated with classic hac. however, the ability of sex hormones to cause such alterations has never been closely evaluated. if sex hormones can cause a syndrome similar to classic hac, they should be able to induce expression of classic glucocorticoid-induced genes. the purpose of the study was to determine if in vitro expression of the gene for corticosteroid-induced alp (cialp) could be induced by clinically relevant concentrations of cortisol and sex hormones believed to cause occult hac. canine hepatocytes were purchased from a commercial source (cellzdirect or invitro) in -well plates. upon arrival ( - plates per shipment), the cells were allowed to recover in general media per supplier recommendations. after hrs, media was changed to william's e media (-l-glutamine) containing concentrations of cortisol or sex hormones that have been documented in the literature in dogs with hac or with purported occult hac. each plate was treated with a different hormone (cortisol, -hydroxyprogesterone [ ohp], progesterone, estradiol or androstenedione), and each well contained a different concentration (starting with no hormone added as a negative control) to evaluate a dose response. media was changed daily. after days of hormone exposure, rna was extracted. reverse transcription was performed and the product used for quantitative pcr for cialp and beta-actin (roche lightcycler) using a gene-specific fluorescent probe for detection. standard curves were created for each gene. all samples and standards were run in duplicate. using the lightcycler software (vers . ), cialp expression was normalized to that of beta-actin. fold change in expression was determined relative to the negative control. each sex hormone was used to treat plates; one plate in each shipment was treated with cortisol as a positive control. for cortisol, a dose response was seen in expression of the cialp gene. compared to no cortisol, , , , , and nmol cortisol increased expression . , . , . . . , . and . fold, respectively. a -fold increase is considered significant (j.vandesompele et al genome biol ). expression of cialp was not significantly induced in response to any concentration of ohp ( nm maximum), progesterone ( nm maximum), estradiol (max pm maximum) or androstenedione ( nm maximum). we conclude that in vitro these sex hormones do not induce expression of the cialp gene which is classically induced by cortisol in vivo; indeed, elevated serum cialp activity is a hallmark of classic hac. thus, the ability of the sex hormones to induce the gene in vivo must be questioned and evaluated. measurement of sex hormones has been advocated as an adjunct means for diagnosing typical hyperadrenocorticism (hac), i.e. disease due to excess cortisol secretion, as well as for diagnosis of atypical hac, i.e. disease due to excess adrenal sex hormone secretion. however, measurements in either setting have not been widely studied. therefore, our objectives were: . to determine the sensitivity of -hydroxy-progesterone ( ohp) and estradiol concentrations pre-and post-acth for diagnosis of typical hac. . to determine the specificity of ohp and estradiol concentrations preand post-acth for diagnosis of occult hac. dogs that had pdh (n ), dogs that were suspected to have hac but proven not to (had non-adrenal illness [nai, n ]) or dogs that were healthy (n , used to establish reference ranges [rr]) were enrolled. acth stimulation tests were performed ( mcg/kg cosyntropin iv); blood samples were drawn pre and min post; ohp and estradiol were measured by previously validated radioimmunoassays. a kruskal-wallis rank sum test was used to compare values between the groups. significance was set at p o . . for basal and acth-stimulated ohp concentrations, the rr were determined to be . - . ng/ml (mean ae s.d.; range . - . ) and . - . ng/ml (range . - . ), respectively. in pdh dogs, and had basal and post-acth ohp concentrations above the rr, respectively; in the nai group, and dogs had concentrations above the rr, respectively. thus, the sensitivity of basal and post-acth ohp measurement for diagnosis of hac is % and %, respectively. specificity of diagnosis is % and %, respectively. post-acth ohp concentration was significantly different between groups. for basal and stimulated estradiol concentrations, the rr were determined to be - pg/ml (range - ) and - pg/ml (range - ), respectively. for both basal and stimulated estradiol, pdh dogs (n ) had concentrations above the rr; for those with nai (n ), and had concentrations above the rr, respectively. thus, the sensitivity of estradiol measurement for diagnosis of hac is % for both pre-and post-acth. specificity of estradiol for diagnosis for hac is % and % for pre-and post-acth, respectively. overall, dogs with nai had at least one elevated estradiol concentration (total specificity %). post-acth estradiol concentration was not significantly different between groups. we conclude that use of ohp and estradiol concentrations for diagnosis of hac can be problematic. sensitivity and specificity are relatively low, potentially leading to misdiagnoses. diabetes mellitus is one of the most common feline endocrinopathies and is considered to have a similar pathophysiological basis to human type diabetes. several studies have identified risk factors for development of diabetes mellitus in cats, which include age, obesity, inappropriate diet and physical inactivity. however, to date, no specific genetic risk factors have been identified. genome-wide association studies in humans have identified several genes that predispose to obesity and/or diabetes mellitus, one of which is the melanocortin receptor (mc r) gene. the aim of the current study was to identify polymorphisms (snps) in the feline mc r gene and to use these to perform a case:control study to determine whether these candidate gene snps were associated with diabetes mellitus in cats. genomic dna from cats ( domestic short hair [dsh], burmese) was initially analysed by pcr and direct sequencing using felmc r-specific primers, which identified a missense mutation (mc r:c. c t) in the region encoding the extracellular domain of the receptor protein in dsh cats only. one hundred and nineteen dsh cats were subsequently recruited into the case:control study. fifty nine cats were obese diabetic ( male, female), mean age . years (range - y); mean weight . kg (range . - kg). sixty lean cats were used as controls ( male, female), mean age . years (range - y), mean weight . kg (range . - . kg). the t to c base change alters a restriction site in the sequence recognized by the enzyme bstoi, such that dna from cats with the mutant (c) allele can be cut, whereas that from the wild-type (t) allele cannot. primers were designed that flanked the mutation to allow pcr amplification of this region of mc r from genomic dna obtained from edta blood. the pcr products were purified and subject to restriction fragment length polymorphism (rflp) analysis. bstoi digestion products were then analysed by agarose gel electrophoresis. of the diabetic cats, ( %) were homozygous for the mutation (cc), compared to ( %) of control cats. statistical analysis (two tailed fisher's square test) revealed that this difference between groups was statistically significant (p . ). in conclusion, this pilot study has identified a missense mutation in the coding sequence of mc r. this could be an important predisposing factor for development of diabetes and/or obesity in dsh cats. polymorphisms in a similar region of human mc r predispose to obesity, which in turn is a major risk factor for type diabetes. hyperadrenocorticism (hac) is one of the most common endocrine disorders of dogs. the two most effective medical treatments are trilostane (vetoryl s ) and mitotane (lysodren s ). previous studies evaluating the effect of treatment on aldosterone secretion measured the hormone at min post-acth administration. however, the optimal sampling time would be at the time of maximal secretion, which occurs minutes after the mg/kg dose commonly used for the test (carlson et al, jvim, ). thus, the true effect of either medication on aldosterone secretory capacity is unknown. our objectives were: ) to assess and compare the effect of treatment with trilostane and mitotane in dogs with pituitarydependent hac (pdh) on aldosterone secretory reserve at min post-acth stimulation and ) to determine if changes in aldosterone concentration at that time correlate with changes in serum sodium and potassium concentrations. forty-six dogs being treated for pdh with either mitotane (n ) or trilostane (n ) have been enrolled. the dogs could be treated for any length of time. all had acth stimulation tests performed ( mcg/kg cosyntropin iv); blood samples were drawn before and at and min post-acth for monitoring of cortisol and aldosterone concentration using previously validated radioimmunoassays. ten historical normal controls were also included. serum sodium and potassium concentrations were measured in the basal samples. a kruskal-wallis rank sum test was used to compare values between normal dogs and those treated with mitotane or trilostane. linear regression analysis was used to determine if a correlation existed between electrolyte and aldosterone concentrations or between cortisol and aldosterone concentrations. significance was set at the p o . level. acth-stimulated aldosterone concentrations in mitotane-treated but not trilostane-treated dogs were significantly lower than that in normal dogs at both the and min time points. no difference was detected between aldosterone concentrations at and min after acth injection in either treatment group. a positive correlation existed between the -min cortisol and -min aldosterone concentrations in the trilostane-treated group (r . ), i.e. the peak post-acth concentration for each hormone, but not in dogs treated with mitotane. basal serum sodium and potassium concentrations were not correlated with the basal aldosterone concentration in either treatment group. in conclusion, treatment with mitotane resulted in decreased aldosterone secretory reserve, but this did not correlate with hyperkalemia or hyponatremia. measurement of aldosterone concentrations is not predictive of electrolyte concentrations. previously presented at the auburn university phi zeta research emphasis day, november , . antioxidant depletion is documented in humans with hyperthyroidism, and is reversible with treatment. in addition, antioxidant depletion has been shown to increase the risk of methimazole toxicity in rats. the primary aim of this study was to determine whether deficiencies in glutathione (gsh), ascorbate (aa), or vitamin e, along with increases in urinary -isoprostanes, were present in hyperthyroid cats, and were reversible after radioiodine treatment. a secondary aim was to determine whether antioxidant abnormalities were associated with a prior history of methimazole toxicity. ongoing prospective, controlled, observational study. otherwise healthy client-owned hyperthyroid cats presenting for radioiodine therapy (n to date) and healthy age-matched controls (n to date) were recruited. all cats were screened with cbc, biochemical panel, urinalysis, and t , as well as red blood cell (rbc) gsh, plasma aa, plasma vitamin e, and urinary -isoprostanes. hyperthyroid cats were re-evaluated months after radioiodine treatment. unlike in humans, median blood antioxidants were not significantly different in hyperthyroid cats (gsh . mm; aa . mm, and vitamin e, g/ml) compared to controls (gsh . mm; aa . mm, and vitamin e, g/ml). results for urinary isoprostanes are pending, and associations with methimazole toxicity will be investigated after full recruitment. rbc gsh concentrations did increase significantly (to . mm; p . ) after radioiodine treatment. however, this modest change is unlikely to be clinically significant. preliminary data do not indicate clinically significant blood gsh, ascorbate, or vitamin e deficiencies in hyperthyroid cats. with appropriate insulin therapy and a low carbohydrate diet, up to % of newly diagnosed diabetic cats are eventually able to maintain euglycemia without insulin administration, and these cats are considered to have achieved remission. there are currently no published data reporting the glucose tolerance status of cats classified as being in remission, and it is unknown whether these cats are truly in diabetic remission, or should be classified as non-insulin dependent diabetics, or having impaired glucose tolerance, and/or impaired fasting blood glucose. the aim of this study was to determine fasting blood glucose concentrations and glucose tolerance status of cats in remission. the study was a prospective study in a feline-only clinic. for inclusion, diabetic cats had to have achieved remission through insulin therapy, and insulin withheld for a minimum of two weeks. five diabetic cats in remission and five matched non-diabetic cats were enrolled in the study. blood samples were obtained via the ear vein but where the cat's temperament precluded this, from the jugular.glucose concentration was measured using a meter calibrated for feline blood (abbott alphatrak). a simplified glucose tolerance test was performed after food was withheld for hours. a g catheter was placed in a cephalic vein three hours before the gtt was commenced, to minimize the effects of stress on blood glucose concentration. blood glucose concentration was measured at time and then a g/kg dose of glucose was administered slowly via the intravenous catheter. further blood glucose measurements were made at hours and then hourly until glucose had returned to o mg/dl (o . mmol/l). in the control group, all cats had a fasting blood glucose below mg/dl, and following glucose administration, glucose had returned to o mg/dl by hours. fasting blood glucose in the remission group was o mg/dl ( mmol/l) in all cats except one, which had fasting blood glucose of mg/dl ( . mmol/l). following glucose administration, all five cats in remission had blood glucose above mg/dl ( . mmol/l) at three hours, four were o mg/dl at four hours, and one returned to o mg/dl at five hours. the cat with impaired fasting glucose subsequently became diabetic after steroid administration. the results of this study show that these cats, while no longer diabetic, have mildly impaired glucose tolerance compared to nondiabetic cats, and a minority have impaired fasting glucose. the objective of this study was to determine the role of iodine restriction in the nutritional management of cats with naturally occurring hyperthyroidism. five domestic shorthair cats ranging in age from - years were confirmed to have hyperthyroidism based on persistently increased serum total thyroxine concentrations (tt ), palpable thyroid nodule and weight loss. serum tt concentrations ranged from - nmol/l (reference range - nmol/l). the cats were then fed a low iodine containing food ( . ppm iodine dmb, as measured by epiboron neutron atomic activation). serum tt concentrations were measured every weeks. biochemistry parameters were also evaluated at weeks , and . at weeks, serum tt concentrations had decreased in all cats with of cats ( %) being euthyroid (mean nmol/l; range - nmol/l). the remaining hyperthyroid cat had an initial serum tt of nmol/l, which decreased to nmol/l after being fed the iodine-restricted food. mean decrease in tt for all cats was nmol/l (range - nmol/l). renal parameters remained stable in all cats. these cats along with additional newly diagnosed hyperthyroid cats were transitioned to a similar food that contained less iodine ( . ppm dmb). baseline serum tt concentrations in the new cats ranged from - nmol/l. serum tt and other biochemical parameters were monitored every weeks for weeks, and then every weeks for an additional weeks. with the . ppm iodine food the four new cats became euthyroid with a mean tt concentration of nmol/ (range - nmol/l). the euthyroid cats from the earlier feeding study had a further decrease in tt concentration (mean tt nmol/l, range - nmol/l). the single non-euthyroid cat from the first study had a serum tt concentration of nmol/l, a decrease from the baseline concentration of nmol/l. the average decrease in serum tt for all cats was nmol/l (range - nmol/l). finally, of the cats were fed a third iodine-restricted food ( . ppm dmb) along with one other newly diagnosed hyperthyroid cat ( nmol/l serum tt ) and evaluated every weeks. all cats in this evaluation were euthyroid (mean tt nmol/l; range - nmol/ l). this result included the cat whose serum tt remained in the hyperthyroid range in the first two evaluations. the average decrease in tt was nmol/l (range - nmol/l). biochemical features of renal function remained stable and no other biochemical abnormalities were observed. in summary, the results of these three feeding studies demonstrate that feline hyperthyroidism can be managed effectively with dietary iodine restriction. we have shown previously that restriction of dietary iodine (i) is a safe and effective method for decreasing serum thyroxine concentrations (tt ) in cats with hyperthyroidism. the objective of this study was to determine the maximum level of iodine in a nutritionally balanced feline mature adult food required to maintain normal serum tt concentrations in hyperthyroid cats currently being controlled on a food containing . ppm i (dmb) as measured by epiboron neutron atomic activation. all cats were previously diagnosed at least months prior to the start of the study and their tt concentrations were maintained in the normal range by dietary iodine restriction for a minimum of months (range months- years). serum tt concentrations ranged from - nmol/l (reference range - nmol/l) at the beginning of the study. the cats were divided into two groups each containing cats. groups were similar in age and gender distribution (mean age . years, range - years). one group (group a) was placed on a food that was formulated for mature adult cats containing . ppm i (dmb). the other group (group b) was placed on a similar food that differed only in that it contained . ppm i (dmb). blood was collected from all cats every three weeks and analyzed for serum tt concentration. biochemistry parameters were also evaluated at weeks , and . all group a cats exhibited increases in serum tt concentration (mean increase of nmol/l above baseline, range - nmol/l). seven of the cats remained in the euthyroid range (mean serum tt nmol/l, range- - nmol/l). two cats exceeded the upper limit of the reference range ( and nmol/l respectively). the cats in group b also exhibited increases in serum tt concentration but to a greater degree than the cats in group a (mean increase nmol/l, range - nmol/l). four cats remained in the euthyroid range (mean serum tt , range - nmol/l). the five remaining cats all exceeded the upper limit of the reference range (mean serum tt nmol/l, range- - nmol/l). all cats returned to a euthyroid state within month of being returned to a diet containing . ppm i (dmb). it was determined that serum tt concentrations are not ideally controlled in the normal range in hyperthyroid cats fed a food containing ! . ppm i (dmb). hyperthyroidism is a common disease in old cats. excessive production of thyroid hormones is the hallmark of the disease. three main treatments for feline hyperthyroidism include radioactive iodine, thyroidectomy, and antithyroid drugs such as methimazole. previously we have shown that limiting dietary iodine to or below . ppm induces euthyroidism in cats with hyperthyroidism compared with a similar diet containing . ppm iodine. the objective of this study was to test whether dietary iodine at . ppm would induce euthyroidism in cats with naturally occurring hyperthyroidism. fourteen cats with hyperthyroidism confirmed by serum tt and ft measurements were stratified into two groups based on gender and age. one group (control: males and females, age ranged from to years) was given a positive control dry cat food ( . ppm iodine) while the other group (test: males and females, age ranged from to years) was fed a commercial dry cat food ( . ppm iodine) for at least weeks before the study. afterwards (week ), the control cats continued to receive the same food while cats in the test group were given a test food ( . ppm iodine) for additional weeks. all cats had free access to their food and deionized water during the study. blood samples were collected during weeks , , , and of the study. the control cats maintained euthyroidism during the study. the test food significantly reduced serum tt ( ae , ae à , ae à , ae à nmol/l in weeks , , and , respectively; à : p o . compared with week , dunnett's t test). it also significantly reduced ft at the end of the study ( ae vs. ae pmol, week vs. week ; dunnett's t test, p o . ). serum ft was within the reference range ( - pmol/l) in cats in both groups. serum tt , ft , and tsh were not affected by the test food and were within the reference ranges (tt : . - . nmol/l, ft : . - pmol/l, and tsh: - mu/l) in cats of both groups during the study. this study demonstrates that dietary iodine at or below . ppm provides an effective and inexpensive therapy for cats with naturally occurring hyperthyroidism. radioactive iodine ( i) is a widely used treatment for feline hyperthyroidism. prior to i administration, many cats receive methimazole therapy. it has been suggested that recent withdrawal of methimazole prior to i may increase the risk of hypothyroidism, inhibit the response to therapy, or have no effect. to further address this question, a retrospective medical records search was performed to identify hyperthyroid cats that received i therapy after methimazole treatment. inclusion criteria included documentation of the time interval between discontinuation of methimazole and i administration, and measurement of thyroxine (t ) at - days after i. cats were divided into groups: those receiving i within day of stopping methimazole, and those receiving i treatment or more days after stopping methimazole. sixty cats met the inclusion criteria. forty received i within day of stopping methimazole. of those, ( %) had a low t (o . mcg/dl), ( . %) had a normal t ( . - . mcg/dl), and ( . %) had an elevated t ( . mcg/dl) at - days after i therapy. fourteen cats received i or more days after stopping methimazole: ( %) had a low t , ( %) had a normal t , and ( %) had an elevated t at - days after i therapy. the results were compared with a fisher's exact test and there was no difference between the groups (p . ). these findings indicate that stopping methimazole therapy within day of i therapy does not inhibit the response to therapy. pharmacokinetic studies evaluating synthetic insulin analogs such as glargine necessitate the ability to measure the blood concentrations of glargine without cross-reactivity to endogenous insulin. although the cross-reactivity between endogenous human insulin assays and synthetic analogs is often known for commerciallyavailable assays, the degree of cross-reactivity of human insulin assays with feline insulin is not. the purpose of this study was to evaluate the cross-reactivity of feline insulin with a commerciallyavailable human insulin elisa with known cross reactivity to several synthetic analogs. pre-and post-prandial blood samples were collected from four healthy cats immediately prior to and approximately minutes following a meal, for a total of samples. dextrose was added to the meals given to two of the cats. blood samples were immediately centrifuged and the serum was collected, aliquoted, and stored at À c until analysis. serum insulin levels were determined in parallel with commercially-available feline insulin and human insulin elisas. the elisas were run in duplicate and according to the manufacturer's instructions. concentrations of serum insulin measured by the feline insulin elisa ranged from . ng/l to ng/l. despite the wide range of concentrations of feline insulin, all samples evaluated with the human insulin elisa yielded absorbance readings equal to or lower than the absorbance of the negative control, indicating no crossreactivity between the evaluated human insulin assay and feline insulin. since this assay is reported to cross-react significantly with glargine, it is a great candidate for determination of serum glargine concentrations in cats. the aim of this prospective, controlled study was to compare the efficacy of two trilostane protocols for treatment of canine pituitary-dependent hyperadrenocorticism (pdh). among the client-owned dogs diagnosed with pdh, only the dogs weighing o kg were selected (n ). group a (n ; low-dose treatment group) and group b (n ; high-dose treatment group) received . ae . mg of trilostane/kg orally every hours and mg of trilostane/ body orally every hours, respectively. all of the dogs were reassessed at , , , and weeks after the initiation of treatment. the improvement in post-acth stimulation serum cortisol concentration, as well as clinical signs in group a, required more time than group b; however, of dogs in group b had clinical signs and abnormal laboratory findings consistent with hypoadrenocorticism after treatment for weeks. twenty-four weeks later, all of the dogs of both groups improved the abnormal clinical findings. the present study suggests that twice daily, low-dose administration of trilostane is effective in the management of canine pdh and may be safe without the potential adverse effects of once daily, high-dose treatment. however, because this study involved only a small number of dogs, a population-based control study will be needed to clarify the efficacy of low-compared to high-dose trilostane treatment. cobalamin is essential for a variety of metabolic processes in many tissues and organs, and has effects on cell growth and peripheral and central nervous system function. chronic distal small intestinal disease in humans, cats, and dogs has been shown to cause cobalamin deficiency. an immunoassay for the measurement of serum cobalamin concentration in these species is being used in routine practice for the diagnosis of cobalamin deficiency. in pigs, the role of cobalamin has not yet been extensively investigated. thus, the aim of this study was to analytically validate an immunoassay, labeled for use in humans, for the measurement of cobalamin in porcine serum samples and secondly to determine serum cobalamin concentrations in weaned pigs. for the analytical validation of the assay, serum cobalamin concentrations were measured using the commercially available immulite s cobalamin immunoassay (siemens healthcare diagnostics ltd., deerfield, il, usa) in surplus porcine serum samples from a variety of studies. validation of the assay consisted of determination of dilutional parallelism, spiking recovery, and intra-and inter-assay variability. additional surplus serum samples from piglets from four litters at a texas a&m university farm were obtained. each piglet had been bled twice, the first at weaning ( days of age) and the second one days later. to investigate results in comparison between age groups, serum cobalamin concentrations were compared using a wilcoxon matched pairs test. significance was set at p o . . observed to expected ratios (o/e) for serial dilutions ranged from . to . % (mean ae sd: . ae . %) for four different serum samples at dilutions of : , : , and : , and from . to . % (mean ae sd: . ae . %) for one serum sample at dilutions of : , : , and : . o/e for spiking recovery ranged from . to . % (mean ae sd: . ae . %) for five different porcine serum samples that had been spiked with each other in a : dilution. intraassay coefficients of variation (%cv) for five different serum samples were . , . , . , . , and . %. inter-assay %cvs for five different serum samples were . , . , . , . , and . %. serum cobalamin concentration was significantly lower in piglets post weaning (median: ng/l) compared to those at the time of weaning (median: ng/l; p . ). the immulite s cobalamin immunoassay labeled for use in humans is linear, accurate, precise, and reproducible for measurement of serum cobalamin concentrations in pigs. this study also showed that piglets that differ in age by only days have significantly different serum cobalamin concentrations. further investigations of cobalamin concentrations in both sows and piglets at different stages of weaning are warranted. primigravid dairy heifers can be infected with mastitis pathogens during the periparturient period. the prevalence of intramammary infection (imi) ranges from - % of quarters pre-partum and - % at parturition. some pre-partum infections self-cure before parturition, however a number of these imis persist into early lactation. these imis may impact milk production and quality and may serve as a reservoir for contagious pathogens. no study has specifically investigated the risk of an imi persisting from the prepartum period into early lactation. the objectives of this study were to describe the prevalence of mastitis pathogens in heifers on a grazing dairy before and after parturition and calculate the relative risk (rr) and attributable fraction of population (afp) for the association between a post-partum and pre-partum imi. two-hundred-ninety-four heifers were systematically assigned to of groups: g ) pre-partum secretions from all mammary quarters (n ), g ) no pre-partum secretions collected (n ) and g ) pre-partum secretions from two diagonal quarters (n ). group assignments were designed to assess whether pre-partum sampling increased the likelihood of imi at calving. mammary quarter secretions were collected for bacterial culture approximately weeks prior to expected calving date. quarter milk samples were collected for bacterial culture once weekly during the st -weeks of lactation. bacterial isolates were classified as staphylococci, non-agalactiae streptococci and gram-negatives. mammary quarter samples yielding different bacteria were classified as mixed infections and those yielding ! bacterial types were classified as contaminated. bacterial isolates were speciated using gene sequencing methods and strain-typed using pulse-field-gel-electrophorysis to evaluate the relatedness of bacteria isolated from pre-and post-partum samples from the same mammary quarter. relative risk and afp were calculated using  tables. forty-five percent of mammary quarters had a pre-partum imi. during the st weeks of lactation the mean prevalence of imi was . % of quarters. staphylococci were most frequently isolated bacteria from pre-partum secretions and milk with s. chromogenes and s. aureus being the most common species. using data from mammary quarters, the rr and afp for the association between a post-partum and pre-partum imi were and %, and %, and and % for all staphylococci, s. aureus only and cns only imis, respectively. mammary quarters sampled pre-partum were no more likely to have a post-partum imi than those not sampled (chisquare, p ! . ). these data demonstrate that pre-partum imis persist into early lactation and that pre-partum secretion cultures may be a useful, not only in predicting imi at calving, but also in assessing risk of introducing new contagious mastitis pathogens, e.g., s. aureus, into the lactating herd. despite concerns about antimicrobial resistance and clostridium difficile in food animals, there has been little study of the prevalence or mechanisms of resistance. this study evaluated the impact of tetracycline treatment on c. difficile shedding in veal calves and the impact on resistance. calves arriving on veal farm received oral oxytetracycline for days as per farm protocols. calves were sampled at arrival and days later. selective culture for c. difficile was performed. isolates were ribotyped, and tested for tetracycline susceptibility and the presence of tetracycline resistance genes. multivariable logistic regression models were used to determine the relationship between tetracycline resistance and the presence of tetracycline resistance genes. clostridium difficile was isolated from % ( / ) and % ( / ) calves, at the first and second samples, respectively. the percentage of tetracycline resistant isolates increased from % to %. isolates from the second sample were times more likely to be tetracycline resistant (p . ) and times more likely to possess tet(m) (p . ). tet(m) was detected in % ( / ) and % ( / ), tet(o) in % ( / ) and % ( / ) and tet(w) in % ( / ) and % ( / ) of isolates from first and second samples, respectively. tet(l), tet(k) and tet(s) were not detected. resistant isolates were not carrying any of the genes investigated. routine tetracycline use may have had an impact on both the prevalence of c. difficile, as well as the strain distribution and resistance patterns. this is the first report of presence of tet ( the objectives of this study were to ) estimate the prevalence of antimicrobial resistance in the study population and ) to investigate the associations between exposures to antimicrobial drugs and antimicrobial resistance in fecal non-type specific e. coli (ntsec) recovered from individual feedlot cattle. two-stage random sampling was used to identify cattle for enrollment at western canadian feedlots. a fecal sample was collected per rectum from each individual at arrival and in the middle of the feeding period when cattle were rehandled as part of standard feedlot protocol. from samples collected at this second time point, a total of , ntsec isolates were tested for susceptibility to antimicrobial drugs by disk diffusion. parenteral and in-feed exposures to antimicrobial drugs were recorded for each individual enrolled in the study. the least square means estimates and % confidence intervals for the prevalence of resistance at each time point were modeled using poisson regression. multivariable logistic regression was used to investigate associations between antimicrobial resistance and exposure to antimicrobial drugs. regression models were adjusted for clustering of observations among individuals and pens. the most common resistances identified in arrival samples were sulfisoxazole ( . %; %ci: . - . ), streptomycin ( . %; %ci: . - . ) and tetracycline ( . %; %ci: . - . ). at the second sampling point, resistance prevalence was . % ( %ci: . - . ) for sulfisoxazole, . % ( %ci: . - . ) for streptomycin, and . % ( %ci: . - . ) for tetracycline. logistic regression modeling identified weak associations of exposures to tetracycline and macrolide classes of drugs with antimicrobial resistance at the second time point. abstract fa- premature/dysmature syndrome in cria: a ret-rospective study of cases ( ) ( ) ( ) ( ) ( ) ( ) ( ) . c. gerspach, d. anderson. the ohio state university, columbus oh. prematurity is widely acknowledged as risk factor for subsequent morbidity and mortality in llama and alpaca cria. a review of medical records for premature cria alive at the time of admission to the veterinary teaching hospital between and was performed to determine risk factors of prematurity and to report the outcome and related conditions or diseases in affected cria. medical records for premature or dysmature cria were included in this study. of these cria, were alpaca and llama, were female and were male. reasons for referral were prematurity, failure of passive immunity, dyspnoea, weakness and failure to gain weight. cria were presented at a mean age of . days and were premature by a mean estimated time of . days. overall survival rate was . %, with all llama cria surviving. a multivariate logistic regression model was used to identify risk factors associated with not surviving. cria receiving camelid colostrum had a significant better outcome than cria receiving no colostrum or colostrum from different species. dyspnea and tachypnea was associated with a poor outcome. all cria that were able to nurse, without assistance prior to referral, survived. clinical pathology parameters most commonly associated with death were hyperphosphatemia and acidosis. enrofloxacin is approved for the treatment of swine respiratory disease, however there are no published studies describing the pharmacokinetics of enrofloxacin at the approved dose and route in pigs ( . mg/kg subcutaneously). furthermore no studies have assessed the unbound concentrations of enrofloxacin at its site of action, the extracellular tissue fluid. therefore the objective of this study was to use an in-vivo ultrafiltration method to measure the active fraction of enrofloxacin, and the metabolite ciprofloxacin, at tissue sites relevant to pigs, and to compare these concentrations with plasma concentrations collected at similar time points. six healthy pigs were used in this study. pigs were recently weaned and weighed an average . kg. on the day before the experiment, pigs were anesthetized for the placement of jugular vein sampling catheters and interstitial fluid collection probes. three ultrafiltration probes were placed in each pig in a subcutaneous site near the right shoulder, an intramuscular site along the epaxial muscles, and in the pleural space of the chest cavity. each pig received an injection of enrofloxacin (baytril , bayer animal health) at a dose of . mg/ kg subcutaneously behind the left ear. plasma and interstitial fluid samples were collected at pre-determined time points, and enrofloxacin and ciprofloxacin concentrations were measured using hplc with fluorescence detection. protein binding was determined with a microcentrifugation system. pharmacokinetic data was analyzed using a one compartment model. the analysis of plasma and isf showed that only a small fraction of ciprofloxacin was produced in these pigs, therefore ciprofloxacin concentrations were not used in pharmacokinetic measurements. the plasma half-life (t / ), volume of distribution, clearance, and peak concentration (c max ) for enrofloxacin was . hr (ae . ), . l/kg (ae . ), . l/kg/hr (ae . ), and . mg/ml (ae . ), respectively. the concentrations from each of three tissues were not different in each pig. when pharmacokinetic values from all tissues were combined for the isf, the t / was . hr (ae . ) and the c max was . mg/ml (ae . ). the enrofloxacin plasma protein binding was . % (ae . ) and . % (ae . ) at a high and low concentration, respectively. this study has demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. the half-life of enrofloxacin is longer in tissues and plasma than has been reported in previous studies. the high tissue concentrations and long half-life produce an auc/mic ratio sufficient for the pathogens that cause respiratory infections in pigs. ceftiofur crystalline free acid (ccfa), a long-acting ceftiofur formulation labeled for use in cattle, pigs, and horses for treatment of respiratory disease has been used for treatment of ovine respiratory infections in clinical practice. pharmacokinetic data, however, do not exist for ccfa administered subcutaneously in sheep. the present pharmacokinetic study evaluated the single dose subcutaneous administration of ccfa in sheep (n ) at . mg/kg body weight. concentrations of ceftiofur free acid equivalents (cfae) in plasma were measured by high performance liquid chromatography for days following drug administration. pharmacokinetics of subcutaneous ccfa in sheep were best described using a single compartment model with the following average (ae sd) parameters: area under the concentration time curve ! ( . hÃug/ml ae . ), observed maximum plasma concentration ( . ug/ ml ae . ), and observed time of maximum plasma concentration ( . h ae . ). no significant adverse drug reactions were observed. adequate cfae plasma concentrations were attained to effectively treat respiratory tract pathogens associated with pneumonia in sheep. the purpose of this study was to assess, using thoracic ultrasonography, the prevalence of lung lesions in pre-weaned dairy calves. subsequent aims were to describe ultrasonographic changes within the lung, clinical respiratory score, and treatment of respiratory disease. a longitudinal study was performed using female dairy calves from commercial dairy farms in new york state. calves were enrolled based on age. thoracic ultrasound and clinical respiratory scoring were performed on each calf at time points. a standard mhz linear ultrasound probe was utilized to evaluate intercostal spaces through of each hemi-thorax with the calf in lateral recumbency (us ) or standing (us ). lesion appearance, size, and location were recorded. respiratory score (rs) was assigned based on a previously published protocol incorporating fever, nasal discharge, cough, ocular discharge and ear droop, with a higher numerical score corresponding to more severe disease. abnormal lung on ultrasound was defined as one or more areas of ! cm width or depth of non-aerated lung. farm records were evaluated to identify treated calves. calves were treated for respiratory disease at the farm manager's discretion, not based upon ultrasound findings or rs. non-parametric methods were used to evaluate the data. ninety-one calves were enrolled into the study, with lost to follow-up. an average of minutes was spent performing the rs and ultrasound on each calf. the median ages at first (us ) and second (us ) examination were (interquartile range - ) and (interquartile range - ) days, respectively. the majority of calves had a low rs (o ) and only . % of calves had a rs high enough to warrant treatment based on previous recommendations (rs! ). the prevalence of calves that had abnormal lungs on ultrasound but a low rs (o ) was . % (us ) and . % (us ). the prevalence of calves that had abnormal lungs on ultrasound and a high rs (! ) was % (us ) and . % (us ). of the calves that had abnormal lungs on ultrasound but a low rs, % were treated with antimicrobials within days of examination. none of the calves with high rs and abnormal lungs on ultrasound were treated with antibiotics within days of examination. this study demonstrates a high prevalence of abnormal lungs, as detected by thoracic ultrasonography, without significant clinical signs in pre-weaned dairy calves. the relatively low treatment rate in these calves may suggest an area of opportunity for improvement in calf health, welfare, and herd longevity. further studies and follow up are needed to elucidate the significance of these findings and whether or not treatment is indicated. literature regarding diseases causing lameness in beef cattle is limited. this retrospective study was undertaken to examine beef cattle presented for lameness. medical records of beef cattle having a lameness examination done during the period to were reviewed and descriptive statistics generated. lameness was classified based on clinical diagnosis. the medical records of beef cattle were reviewed of which . % were male and . % were female. beef cattle presented for lameness most often during the summer months ( %) and least during autumn ( %). causes of lameness were categorized as infectious ( . %) or non-infectious ( . %) and infectious lameness subcategorized as either a primary disorder or a secondary infection. all cases of a primary infectious disorder were interdigital phlegmon. secondary infections diseases included sole abscess ( . %), septic arthritis ( . %), tenosynovitis ( . %), and pedal osteitis ( . %). non-infectious lameness included proximal limb lameness ( . %), foot trauma ( . %), hoof horn cracks ( . %), hoof defects ( . %), interdigital fibromas ( . %), overgrown hooves ( . %), sole bruise ( . %), subclinical laminitis ( . %), white line disease ( . %), osteoarthritis ( . %), heel erosion ( . %), sole ulcers ( . %), and sole hemorrhage ( . %). the most frequently affected claw was the lateral digit of the hind limb ( . %), followed by the medial digit of the front limb ( . %), lateral digit of the front limb ( . %), and the medial digit of the hind limb ( . %). the findings of this study suggest significant differences in the frequency of disease causing lameness in beef cattle compared to published reports for dairy cattle. in people, endoscopic ultrasound (eus) has become the technique of choice for assessing pancreatic disease and eus-guided fineneedle aspiration (eus fna) has proven a useful and safe modality for characterizing pancreatic lesions. reported complications include infections, bleeding and acute pancreatitis. in dogs, laparoscopic-assisted pancreatic biopsy has been suggested to be a safe procedure, however eus and eus fna have not been evaluated in dogs so far. thus the aim of the present study was to assess the practicability and safety of eus examination of the abdominal cavity as well as pancreatic eus fna in healthy dogs. this study was approved by the cantonal committee for the authorization of animal experimentation, zurich, switzerland. the study population consisted of healthy beagle dogs with a median bodyweight of . kg ( . - . ). eus was performed with an olympus gf-uc p-echoendoscope and fna were performed using g needles (cook echotipultra). after completion of the eus-examination of the abdominal cavity from the stomach (liver, gallbladder, bile ducts, kidneys, adrenals, pancreas), the scope was advanced into the duodenum and eus fna of the pancreas was performed. fna tissue acquisition was made applying negative pressure and to needle passes were made. all dogs received mg/kg metimazole im after eus fna and were re-checked ultrasonographically minutes post eus fna. postoperative activity was assessed using a standardized scoring system. a cbc, serum biochemistry, urinalysis and spec cpl s were measured before, as well as and h after eus fna. the eus examination was complete in / dogs, the pancreas could not be visualized in dog. the pancreas was hypo-( / ) to isoechoic ( / ) to the surrounding mesenterium in all cases. in / dogs parts of the pancreas presented hyperechoic. the mean measured thickness was . cm. the pancreas was aspirated in dogs using a transgastric approach ( ) or transduodenal approach ( ). duodenal transmural puncture was not accomplished in dog where a re-sterilized needle was used. a minimal amount of peripancreatic fluid was observed in / dogs after eus fna. all dogs recovered uneventfully and required no further analgesia. all laboratory results including the spec cpl s measurements were within reference ranges on all three time points. cytologically, conglomerates of exocrine pancreatic cells were seen in / cases, duodenal villous epithelial cells were seen in / cases. in dog the aspirated pancreatic material was sufficient for a histological assessment. the aspirates with exocrine pancreatic cells on cytology were obtained by transgastric ( ) and transduodenal ( ) aspirations. in conclusion, ( ) eus examination of the abdomen is feasible in medium-sized dogs, ( ) the healthy canine pancreas can be difficult to visualize completely, and ( ) eus-guided pancreatic fna using a g needle is a safe procedure in healthy dogs. studies evaluating its use in dogs with pancreatic disease are warranted to assess its clinical utility. miniature schnauzers have a high prevalence of idiopathic hyperlipidemia, which is characterized by an increased serum triglyceride (tg) concentration, with or without an increased serum cholesterol (chol) concentration. a common initial therapeutic approach for the management of hyperlipidemia is the use of a low-fat diet. also, it is believed that low-fat diets may be beneficial in the treatment of pancreatitis in dogs. however, the efficacy of this approach has not been evaluated for either condition. the aim of the present study was to evaluate the effect of a commercially available low-fat diet on serum concentrations of tg, chol, and canine pancreatic lipase immunoreactivity (cpli; measured as spec cpl s ) in apparently healthy miniature schnauzers with hypertriglyceridemia. blood samples were collected from apparently healthy miniature schnauzers with hypertriglyceridemia (serum triglyceride concentrations mg/dl). common causes of secondary hyperlipidemia were excluded based on historical information, physical examination findings, and the measurement of serum glucose, total t , and free t (by ed) concentrations. the owners of the dogs were asked to switch their dog to the study diet (royal canin gastrointestinal low fat s ; fat content: . g/ , kcal) and have a second blood sample collected weeks after their dog had been on the new diet. all blood samples were collected after food had been withheld for hours. serum tg, chol, and spec cpl concentrations were measured both before and after the diet change. results were compared between the two time-points using the wilcoxon signed rank and fisher's exact tests. serum tg concentrations were significantly higher before (median: mg/dl) than after the diet change (median: mg/dl; p . ). the proportion of dogs with hypertriglyceridemia was significantly higher before ( / ) than after the diet change ( / ; p . ). also, the proportion of dogs with serum tg mg/dl was significantly higher before ( / ) than after the diet change ( / ; p . ). serum chol concentrations were significantly higher before (median: mg/dl) than after the diet change (median: mg/dl; p . ). the proportion of dogs with hypercholesterolemia was significantly higher before ( / ) than after the diet change ( / ; p . ). finally, the difference in serum spec cpl concentrations before (median: mg/l) and after the diet change (median: mg/l) approached but did not reach significance (p . ). also, the proportion of dogs with high serum spec cpl concentrations before ( / ) and after the diet change ( / ) was different, but this difference was not significant (p . ). in summary, a commercially available low-fat diet was effective in reducing serum tg and chol concentrations in miniature schnauzers with hypertriglyceridemia. toll-like receptor (tlr ) is an extracellular pattern recognition receptor which recognizes flagellin present in motile bacteria. we have previously demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the tlr gene (g a, c t and t c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). recently, we have confirmed that two of these tlr snps (c t and t c) are significantly associated with ibd in other canine breeds. to further substantiate the role of tlr in canine ibd functional analysis of these polymorphisms would be needed. therefore the aim of this study was to determine the functional significance of the tlr snps by transfecting wild-type and mutant receptors in to human embryonic kidney cells (hek) and carrying out nuclear factorkappa b (nf-kb) luciferase assay and il- elisa. the tlr gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) as determined by the case-control analysis in gsds with ibd were cloned into plasmids expressing yellow-fluorescent protein (yfp). these were then stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were then stimulated with various ligands ( . mg/ml flagellin, . mg/ml flagellin, mg/ml lps, mg/ml pam csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk) according to the manufacturer's recommendations. the supernatants were harvested and used in an il- elisa (r&d systems). human tlr transfected hek cells (invivogen) served as positive controls in all experiments. independent t-test was used to determine the significance of relative luciferase activity and il- concentration between wild-type and mutated tlr cells. although there was no significant difference between the wild-type and mutated receptor when they were stimulated with . mg/ml of flagellin (p . ), there was a significant increase when the cells with mutated tlr were stimulated with . mg/ml of flagellin compared to the cells expressing wild-type tlr (p . ). similarly, there was a significant increase in il- concentration in the supernatants in the cells with the mutated tlr receptor when stimulated with . mg/ml flagellin compared to the wild-type (p . -one-tailed, . -two-tailed) but not with . mg/ml flagellin (p . ). we show for the first time that polymorphisms associated with ibd are functionally hyper-responsive to flagellin compared to the wild-type receptor. this suggests that tlr may play a role in canine ibd and that blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. however, further in-vivo functional analysis of tlr , especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions. tlr has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (ibd). similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the canine tlr gene (g a, c t and t c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). therefore the aim of this study was to determine the functional significance of the tlr snps in the breed of gsds. the tlr gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) were stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were stimulated with various tlr ligands ( . mg/ ml flagellin, . mg/ml flagellin, mg/ml lps, mg/ml pam csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk). the supernatants were harvested and used in an il- elisa (r&d systems). peripheral whole blood from dogs carrying the wild type and mutant tlr genes was cultured and stimulated with tlr ligands as above. canine tnf-alpha was measured in the supernatant by commercially available elisa (r&d systems). t-test was used to determine differences of relative luciferase activity, il- concentration and tnf-alpha concentration between wild-type and mutated tlr cells. there was a significant increase in nf-kb activity when the cells with mutated tlr were stimulated with . mg/ml of flagellin compared to the cells expressing wild-type tlr (p . ), which correlated with il- expression in the supernatant (p . ). similarly, in the whole blood assay the tlr risk haplotype for ibd in gsds (acc) was significantly hyperresponsive to flagellin at a concentration of . mg/ml compared to the tlr wild-type haplotype (gtt) (p . ). we show for the first time that polymorphisms associated with canine ibd in gsds are functionally hyper-responsive to flagellin compared to the wild-type receptor. blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. proton pump inhibitors (ppi) are widely used in human and also veterinary medicine. side-effects of ppi treatment reported in people are atrophic gastritis, gastric and esophageal cancer, and rebound hyperacidity following cessation of treatment, which has been speculated to be due to a sustained increased in circulating gastrin concentration. moreover, long-term ppi treatment has been associated with an increased risk for osteoporosis in people. little is known about the effect of ppi treatment on serum gastrin concentration or calcium metabolism in dogs. eight healthy adult research dogs ( males and females) were enrolled into the study. the dogs received an average dose of . mg/ kg of omeprazole orally twice daily for days. blood samples were collected prior to initiating the treatment and every days during the days of treatment and during the days after discontinuation of treatment for determination of serum gastrin, ionized calcium, pth, and oh vitamin d . gastric fluid was collected via gastroscopy after an overnight fast for measurement of gastric ph prior to, during, and after the omeprazole treatment period. normally distributed data were compared with a repeated measures anova and post hoc dunnett's test. data that were not normally distributed were compared with a friedman's test and a post-hoc dunn's test. gastric fluid ph was significantly higher (p o . ) at the end of the treatment period (median: . ; range: . - . ) when compared to pretreatment values (median: . ; range: . - . ). serum gastrin concentrations increased significantly from a median baseline of . ng/l (range: . - . ) to a maximum median of . ng/l (range: . - . ) at day of treatment (p o . ). serum gastrin remained significantly increased above baseline values from day to day of the treatment, but was not different from pre-treatment values days after the end of the treatment. omeprazole treatment had no effect on ionized calcium or pth for the duration of the study. marginal, but significant changes of oh vitamin d were observed at day (end of the treatment period -increased by . %) and day ( days after the end of the treatment -decreased by . %). this study shows that treatment with omeprazole for weeks results in a profound and sustained increase in serum gastrin concentration in dogs. this effect is rapidly reversible after cessation of the treatment. no effect on calcium metabolism was observed. however, this study documents only the effect of short-term treatment and it is possible that the effects of long-term administration are different. omeprazole treatment has been associated with small intestinal bacterial overgrowth and a higher risk for infectious enteropathies in humans. using a semi-quantitative sequencing approach, we have previously shown that omeprazole treatment may lead to alterations in both duodenal and gastric bacterial populations in healthy dogs (acvim ). however, a sequencing approach can only estimate relative proportions of genomic bacterial targets. therefore, significant changes in the total number of bacteria could not be evaluated. the aim of this study was to quantify gastric and duodenal bacterial populations in dogs undergoing omeprazole treatment. eight month-old healthy research dogs ( males and females) were enrolled. the dogs received an average dose of . mg/kg of omeprazole orally twice a day for days. endoscopic gastric and duodenal biopsies were harvested and days before starting omeprazole treatment, on the last day of treatment (day ), and days after the end of treatment (day ). all biopsies were fixed in % formalin for hours, processed, and embedded in paraffin blocks. fluorescent in situ hybridization was used to quantify mucosa-associated bacteria using fluorescently-labeled probes targeting the s ribosomal rna. statistical analysis aimed to compare changes in helicobacter spp. in gastric biopsies and total bacteria in both gastric and duodenal biopsies using the glimmix and npar way procedures in sas s . . bacteria were counted in , and microscopic fields ( Â) obtained from and gastric and duodenal biopsies, respectively. in the stomach, omeprazole treatment led to a decrease in helicobacter spp. (log of average counts ae standard error: . ae . at day ) when compared to the counts ( . ae . , p . ) and ( . ae . , p . ) days before treatment. after completion of omeprazole treatment, helicobacter spp. increased and returned to baseline counts ( . ae . at day , p . vs day ). also, in the stomach, non-helicobacter spp. bacteria were observed more often during omeprazole treatment (median: , range: - ) than on days (median: , range: - ) and (median: , range: - ) before and days after (median: , range: - ) omeprazole treatment; however, statistical comparison across time points did not reach significance. in the duodenum, while the median number of bacteria for all time points was zero, non-parametric comparison of median scores (number of points above median) revealed significantly higher numbers of bacteria during omeprazole treatment (p . ). our results suggest that omeprazole treatment for weeks leads to a lower abundance of helicobacter spp. organisms in the stomach of healthy dogs. also, this transient decrease in helicobacter spp. was accompanied by a higher abundance of other bacteria in both the stomach and the proximal duodenum. the smartpill ph.p s capsule (the smartpill corporation) is a wireless motility capsule that measures ph, pressure, and temperature as it passes through the gastrointestinal (gi) tract. analysis of this data allows the calculation of gastric emptying time (get), small and large bowel transit time (slbtt), and total gi transit time (tgtt). this study evaluated the variability associated with repeated measurement of gi transit times and the effect of oral administration of ranitidine (zantac s ) on gi transit times in dogs using this system. it was hypothesized that ranitidine would reduce gi transit times. six privately owned healthy adult dogs weighing between . kg and . kg were used. on occasions each dog was fed a standard meal followed by oral administration of a capsule. data were recorded until the capsule had passed in the dog's feces. on a th occasion each dog was given mg of ranitidine po q hrs starting hrs prior to testing. the dogs were then fed the test meal and the capsule was administered as above. ranitidine was given until the capsule had passed in the dog's feces. proprietary smartpill software was used to calculate get, slbtt, tgtt, and the median gastric ph (mgph). mean intra-individual and inter-individual coefficients of variation (cv%) were calculated for get, slbtt, and ttt for the first time points. transit times and gastric ph recorded at all time points were compared using a repeated measures anova. where significant differences were identified, post-hoc testing was performed using a bonferroni's multiple comparisons test. significance was set at p o . . a sharp rise in ph indicating exit of the capsule from the stomach was identified in each experiment. mean (ae sd) get, slbtt, and tgtt without ranitidine were ae , ae , and ae min, respectively. mean get, slbtt, and tgtt during treatment with ranitidine were ae , ae , and ae min, respectively. mean intra-individual cv% before ranitidine for get, slbtt, and tgtt were . , . , and . %, respectively. mean inter-individual cv% before treatment with ranitidine for get, slbtt, and ttt were . , . , and . %, respectively. no significant differences in get, slbtt, or tgtt were found at any of the time points. the mean mgph during treatment with ranitidine (ph . ) was significantly higher than at all other time points (overall mean ph for the time points: . ; p o . ). the smartpill system is an easy to use, ambulatory, non-invasive, non-radioactive method for assessing gi transit times in medium to large breed dogs. measurements of gi transit times, especially slbtt, were subject to considerable intra-individual and interindividual variation. no significant effect of oral ranitidine on gi motility was identified in this group of dogs. however, as expected, oral ranitidine caused a significant increase in gastric ph. the intestinal microbiota has been implicated in the pathogenesis of various gastrointestinal disorders in both humans and dogs. recent metagenomic data suggest that specific bacterial groups, including bacteria within the clostridium clusters iv and xiva (i.e., faecalibacterium spp., ruminococcaceae, and lachnospiraceae) and bifidobacterium spp. are decreased, while proteobacteria are increased in dogs with clinical signs of gastrointestinal disease. the objective of this study was to establish quantitative polymerase chain reaction (qpcr) assays for these specific bacterial groups and evaluate their abundance in healthy dogs and dogs with clinical signs of gastrointestinal disease. fecal samples were collected from healthy dogs ( females and males) and dogs with clinical signs of gastrointestinal disease ( females and males). novel quantitative pcr assays were established for faecalibacterium spp., ruminococcaceae, and lachnospiraceae by aligning respective group specific sequences against canine specific sequences obtained from s rrna gene clone libraries and sequences available from the ribosomal database project. primers for bifidobacterium spp. and proteobacteria were selected from previously published studies. the specificity of the qpcr assays was confirmed by sequencing of obtained qpcr amplicons. the bacterial dna abundance in fecal samples was compared between healthy dogs and dogs with clinical signs of gastrointestinal disease using a mann-whitney u test. significance was set at p o . . a significantly lower abundance of faecalibacterium spp. (p o . ) and ruminococcaceae (p . ) was observed in dogs with clinical signs of gastrointestinal disease when compared to healthy dogs. proteobacteria were more abundant in dogs with clinical signs of gastrointestinal disease, but this difference did not reach statistical significance (p . ). there was no significant difference in the abundance of lachnospiraceae (p . ) and bifidobacterium spp. (p . ) between both groups. in conclusion, we established novel qpcr assays for faecalibacterium spp., ruminococcaceae, and lachnospiraceae. we observed significant decreases in the abundance of faecalibacterium spp. and ruminococcaceae in dogs with clinical signs of gastrointestinal disease. these bacterial groups are considered major short-chain fatty acid producers and studies are warranted to determine if a decrease in these bacterial groups is associated with decreases in short chain fatty acid production. further studies are also needed to determine if these bacterial shifts are associated with specific gastrointestinal disorders. the pathogenesis of chronic enteropathies (ce) in dogs likely involves complex interaction between the mucosal immune system and the intestinal microbiota. while the application of bacterial s rdna sequence-based analysis has shown an association between altered microbial composition and duodenal inflammation in dogs, relatively little is known about alterations in non-invasive mucosal and luminal bacteria seen with diseases involving the ileum and colon. the present study sought to evaluate the relationship of enteric bacteria to type and severity of mucosal inflammation affecting the ileum and colon of dogs with ce. eleven client-owned dogs with ce involving both the small and large intestines were prospectively enrolled. ce was diagnosed on the basis of a history of chronic gastrointestinal signs, exclusion of identifiable underlying disorders, and histopathologic evidence of intestinal inflammation. mucosal bacteria were detected in formalinfixed ileal and colonic tissue sections with fluorescence in situ hybridization (fish) using s rdna-targeted probes directed against all bacteria, enterobacteriaceae, e. coli, eubacterium rectale-clostridium coccoides group, bacteroides/prevotella, and helicobacter spp. sections were examined by epifluorescence microscopy and the number of bacteria and their spatial distribution (luminal, superficial mucus, epithelial adherent, within mucosa) was determined in ten x fields of each section. microbial composition in ce dogs was compared to the ileal/colonic microbiota of healthy control (hc) dogs using a mixed effect anova model. p values o . were considered significant. the final diagnoses for dogs with ce included ibd (n ) and lymphosarcoma (n ). when compared to hc dogs, dogs with ce showed regional (ileum versus colon) imbalances in microbiota composition characterized by selective enrichment of mucosa-associated populations. evaluation of colonic biopsies in dogs with ce showed that the total number of bacteria (p o . ), clostridium (p o . ), enterobacteriaceae (p o . ) and e. coli (p o . ) were increased in the adherent mucus regions of dogs with ibd as compared to hc dogs. total bacteria (p o . ) and e. coli (p o . ) were also more numerous in dogs with lsa versus hc and ibd dogs (p o . for e. coli). ileal biopsies from ce dogs similarly showed variable dysbiosis with increased total bacteria (p o . ) but decreased helicobacter spp (p o . ) and bacteroides (p o . ) observed within inflamed intestines as compared to hc tissues. the spatial distribution of these bacteria was also appreciably different from hc dogs, with higher numbers of bacteria generally found within the adherent mucus compartment as compared to other ileal regions. our data demonstrate that dogs with ce affecting the ileum and colon have altered microbiota composition that may be a cause or consequence of mucosal inflammation. recognition of these microbiota imbalances may provide new opportunities for therapeutic intervention. trichomonads have been rarely reported in the feces of dogs and their pathogenicity remains uncertain. although pentatrichomonas hominis (ph) is considered to be a commensal that may overgrow in dogs with other causes of diarrhea, little is known regarding the history, clinical presentation or prevalence of concurrent gi infections in dogs with trichomonosis. the aim of this study was to determine whether dogs with diarrhea and trichomonosis could be distinguished from dogs having diarrhea without trichomonosis on the basis of clinical signs or presence of concurrent enteric infections. fecal samples from dogs were submitted to ncsu from - for trichomonas spp. pcr testing. dna was extracted using a zr fecal dna mini-prep kit and absence of pcr inhibitors verified by amplification of bacterial s rdna. pcr for ph and tritrichomonas foetus (tf) was performed as well as real-time pcr assays for possible concurrent enteric infectious agents. obtainable medical records were reviewed. all submitted fecal samples were submitted from dogs with diarrhea that was variably described as soft, mucoid, hemorrhagic, or watery. mean age of the dogs was . years (median . ; range: . - months) and represented a total of breeds. ph, tf, or concurrent ph and tf were diagnosed in , , and dogs respectively (group a). the remaining dogs were negative for ph and tf by pcr no dogs were identified as infected with canine distemper virus or parvovirus. five samples from each group had insufficient quantity or quality of dna for concurrent infectious disease testing. in this large study of canine trichomonosis, no differences in age, clinical signs, or prevalence and identity of concurrent enteric infection between diarrheic dogs with or without ph were identified. thus, these findings do not appear to support a primary pathogenic role for ph as a causative agent of diarrhea in dogs. gastrointestinal motility disorders are a common clinical problem in domestic animals. many of the g.i. motility disorders have been treated previously with -ht agonists although limited availability of drugs in this classification have stimulated interest in the use of new (and old) drug therapies. the dopaminergic antagonists are a group of drugs with well-known anti-emetic effects at central dopamine d receptors, and putative gastrointestinal prokinetic effects at peripheral d receptors. domperidone has been shown, for example, to reverse gastric relaxation induced by dopamine infusion in the dog. similar studies have not been reported in the cat or rabbit, two species at risk for distal gastrointestinal motility disorders. our aim was to study the effects, mechanisms, and sites of action of domperidone in feline colonic and rabbit gastrointestinal smooth muscle contraction. portions of stomach (fundus and antrum), intestine (duodenum and ileum), cecum (rabbits only), and colon (ascending and descending) were obtained from healthy cats and rabbits from - months of age. longitudinal and circular smooth muscle strips from each site were suspended in physiologic (hepes) buffer solution, attached to isometric force transducers, and set to optimal muscle length (l o ) using acetylcholine (ach; À m). muscle strips were treated with domperidone (d; À to À m) in the presence or absence of ach ( À to À m), and maximal force output (p max ) was normalized for cross-sectional area (n  newtons/m ). domperidone (d) had a minor direct effect of inducing feline and rabbit gastric, cecal, and colonic smooth muscle contraction. direct effects were similar whether in the longitudinal or circular muscle orientation. the direct effect of domperidone was dose-dependent and maximal (feline colon p max . - . n; rabbit colon p max . - . n) at a dose of À m. domperidone had a much greater indirect effect in augmenting cholinergic (ach; À m) contractions in feline and rabbit gastric, cecal, and colonic smooth muscle. domperidone-augmented cholinergic contractions were - % (feline colon p max . ae . n ach only; feline colon p max . ae . n ach d) of baseline cholinergic contractions. domperidone contractions were of a similar magnitude to those induced by cisapride. domperidone effects were similar in mucosaintact and mucosa-dissected preparations. domperidone contractions were unaffected by prazosin (a receptor antagonist), yohimbine (a receptor antagonist), or terbutaline (b receptor agonist), but were somewhat attenuated by dopamine (d receptor agonist) and a non-specific cholinergic antagonist (atropine). in vitro studies show for the first time that domperidone has minor direct and major indirect effects in augmenting cholinergic contractions of feline and rabbit gastrointestinal (stomach, cecum and colon) smooth muscle. as recognition of acute and chronic pain in dogs has increased, so too has the use of non-steroidal anti-inflammatory drugs (nsaids) often in conjunction with tramadol. in people and rats, co-administration increases the risk of perforation and gastric injury over nsaids alone. using an ex vivo model of acid injury in canine gastric mucosa, we examined the effects of indomethacin and tramadol on gastric permeability and concentrations of gastroprotective prostaglandin e (pge ). mucosa from the gastric antrum was harvested from shelter dogs immediately after euthanasia, and mounted on ussing chambers. the tissues were equilibrated for -minutes prior to addition of acidic ringer's solution (ph, . ). after -minutes of injury, the acid was replaced with neutral ringer's and the tissues were treated with indomethacin, tramadol or both. tissues were maintained for minutes total, during which time permeability was assessed electrically. prostanoid concentrations were quantified using a commercially available elisa. western blots were performed for cox- and À . recovery of gastric barrier function after acid injury was inhibited by co-administration of tramadol and indomethacin ( figure ) but not by tramadol or indomethacin alone (data not shown). prostaglandin e increased with acid injury. the increase in pge was inhibited by co-administration of indomethacin and tramadol (in pg/ml: acid injury . ae . , indo tramadol . ae . ). there was no significant effect of treatment on cox- or À expression. co-administration of tramadol with a non-selective nsaid inhibits the return of gastric mucosal barrier function after acid injury in canine tissue, suggesting that caution is required in prescribing concurrent use of these drugs in dogs at risk for gastric ulcers. these drugs may exert this effect by decreasing levels of gastroprotective prostanoids. further study is needed to understand the mechanism of this drug interaction. an increased intestinal permeability (ip) has been suggested to be both cause and consequence of gastrointestinal (gi) disease, such as inflammatory bowel and celiac disease, in people. a novel tight junction regulator, larazotide acetate (alba therapeutics, baltimore, md) has been shown to significantly decrease ip in rats and in humans with celiac disease. the purpose of this study was to determine if larazotide acetate reduces ip in soft coated wheaten terriers (scwt) and norwegian lundehunds (nl) with chronic gi disease and ameliorates clinical signs. four nl ( females, males; median age: . yrs, range: . - . yrs) and scwt ( females, males; median age: . yrs, range: . - . yrs) were enrolled based on presence of clinical signs of gi disease and hypoalbuminemia, increased fecal alpha proteinase inhibitor (a -pi) concentrations, and/or hypocobalaminemia. scwt with protein-losing nephropathy were excluded. dogs were fed q hrs and received . mg ( nl and scwt) or . mg ( scwt) of larazotide acetate po before each meal for days. prior to start of treatment (day ) and at the end (day ), ip was evaluated by calculating the lactulose/rhamnose (l/r)-ratio in serum samples obtained at , , , and min after oral dosing. also, consecutive fecal samples each were collected prior to day and day for n-methylhistamine (nmh) measurement. pre-and post-treatment data were compared using a wilcoxon signed rank test. the . mg vs. . mg dose groups were compared using a mann-whitney u test. statistical significance was set at p o . . l/r-ratios (medians) for the min sampling time point were significantly lower on day ( . ) than on day ( . ; p . ). dogs treated with . mg q hrs had significantly lower min l/r ratios on day than dogs treated with . mg ( . vs. . ; p . ). no difference was found between breeds. fecal nmh concentrations were not different between time points, treatment groups, or breeds. fecal a -pi concentrations were available for of the dogs and were significantly higher on day compared to day (p . ). no differences were found between pre-and post-treatment serum albumin or cobalamin concentrations. weight gain was seen in all nl. resolution of diarrhea, vomiting, hyporexia, as well as an increased activity was seen in scwt. another scwt had resolution of diarrhea and a decrease in pruritus. no changes in clinical signs were reported in the remaining scwt. this study indicates that larazotide acetate might be able to reduce ip in dogs. this effect may be dose-dependent. however, not all dogs showed an improvement in clinical signs, suggesting that factors other than increased ip might have been responsible for the clinical signs in these dogs. breed-related effects cannot be ruled out, and further studies are warranted to determine the efficacy of larazotide acetate in dogs of other breeds with gi disease. to analyze different biochemical markers, calculate clinical activity scores, and assess survival in dogs with ple and compare them with those in dogs with food-responsive diarrhea (frd) without protein loss. dogs with ple and dogs with frd, referred to the university of bern, ch, were enrolled. selection criteria included a history of chronic diarrhea ( weeks), exclusion of identifiable underlying causes, and histopathologic evidence of intestinal inflammation, but not neoplasia. underlying disorders were excluded based on cbc, chemistry profile, urinalysis, fecal analysis, trypsinlike immunoreactivity, cobalamin, folate, and transabdominal ultrasound. also, canine pancreatic lipase immunoreactivity (spec cpl s ), c-reactive protein (crp), calprotectin and alpha -proteinase inhibitor (a -pi) were measured in serum from dogs and compared with dogs with frd without ple. all dogs were scored using the canine ibd (cibdai) and the canine chronic enteropathy (cce) clinical activity index (ccecai). total protein, albumin ( - . g/l), and total calcium ( . - . mmol/l) were decreased in all dogs. cobalamin was decreased in all but dogs ( o - ng/l). spec cpl was mildly increased in / dogs with ple and normal in / ple and all frd dogs. crp was normal in / dogs with ple ( / frd), mildly increased in / ( / frd), and moderately increased in / ple dogs ( / frd). calprotectin was slightly higher in dogs with ple, but all ple and frd dogs yielded values in the normal range. serum a -pi was significantly lower in dogs with ple than in those with frd (p o . ), with / ple dogs below the reference range ( / frd). cibdai ranged from to and ccecai from to . at the end of the study, / dogs were still alive with survival times between and days. / dogs died with a median survival of days (range - days). dogs with mildly increased crp died earlier than dogs with a normal or moderately increased crp (p . ), whereas albumin, calcium, spec cpl, calprotectin, cibdai, and ccecai had no significant impact on outcome and survival. in conclusion, dogs with ple have a significantly lower a -pi in the serum than dogs with frd. furthermore, most dogs with ple have an increased crp and a decreased cobalamin. a mild increase in crp appears to be a poor prognostic factor. while hypoalbuminemia is a common finding associated with chronic enteropathies, its impact on survival in this population is poorly defined. the aim of this study was to compare dogs with chronic enteropathies on the basis of their serum albumin concentration at the time of presentation. we hypothesized that dogs with a protein losing enteropathy (ple) have a significantly shorter survival time compared to dogs with chronic enteropathies which are not hypoalbuminemic (controls). information obtained from the medical records included signalment, duration and characteristics of clinical signs, physical examination findings, clinicopathologic data and survival time. one hundred seventeen cases fit the inclusion criteria; in the ple group and controls. there was no statistical significance between groups for age (p . ), weight (p . ), weight loss (p . ) and body condition score (p . ). compared to control dogs, ple dogs had decreased serum concentrations of cobalamin (p . ), total calcium (p o . ), globulin (p o . ), cholesterol (p o . ) and ionized calcium (p o . ). survival analysis revealed a significantly decreased survival time for ple dogs (p . ); median survival was days for ple dogs and , days for controls. while the ple group did not survive as long, survival was not directly associated with severity of hypoalbuminemia; patients with albumin concentration o . g/dl survived longer than those with mild hypoalbuminemia ( . - . g/dl). this study supports the observation that chronic enteropathy patients have decreased survival time when presented with hypoalbuminemia; however this study suggests the severity of hypoalbuminemia is not a reliable indicator of survival. cobalamin (vitamin b ) deficiency in the chinese shar pei (shar pei) is suspected to be hereditary. inherited causes of cobalamin deficiency have been reported in humans and may affect absorption, transport, or cellular processing of cobalamin. based on human and veterinary studies, an increased serum methylmalonic acid (mma) concentration has been suggested to reflect cobalamin deficiency at the cellular level. in this context, it has been shown in humans that mma concentrations are higher in patients with genetic disorders affecting intracellular processing than in patients with genetic defects affecting gastrointestinal processing and extracellular transport of cobalamin. therefore, the aim of this study was to evaluate serum mma concentrations in shar peis and dogs of six other breeds with cobalamin deficiency. from in conclusion, serum cobalamin deficient shar peis had a times higher median serum mma concentration compared to cobalamin deficient dogs of six other dog breeds. further studies are needed to investigate the intracellular processing of cobalamin in shar peis with cobalamin deficiency. chinese shar peis (shar peis) have a high prevalence of cobalamin deficiency. two other conditions reported frequently in this breed are shar pei fever and cutaneous mucinosis. shar pei fever is an autoimmune disorder causing periodic flare-ups and is associated with increased serum concentrations of c-reactive protein (crp), a nonspecific marker of inflammation. cutaneous mucinosis is characterized by excessive deposition of mucin in the dermis. also, hyaluronic acid (ha), the main component of mucin, was shown to be significantly higher in serum from shar peis with cutaneous mucinosis than in healthy controls. to date, a possible association between shar pei fever and/or cutaneous mucinosis on one side and cobalamin deficiency on the other has not been investigated in shar peis. thus, the aim of this study was to compare serum concentrations of ha (an indicator of cutaneous mucinosis) and inflammatory markers (crp, calprotectin, and s a ), assumed to be increased in episodes of shar pei fever, in shar peis with and without cobalamin deficiency. serum samples from shar peis, collected from to , were analyzed. serum ha and crp (reference interval (ri): . - . mg/l) were quantified by using commercial elisa kits (echelon biosciences, salt lake city, ut, usa and tridelta, maynooth, ireland; respectively). serum calgranulin concentrations were measured using an in-house elisa (calprotectin; ri: . - . mg/l) and ria (s a ; ri: . - . mg/l), respectively. mann-whitney u tests were used to compare serum ha, crp, calprotectin, and s a concentrations between shar peis with and without cobalamin deficiency. significance was set at p o . . fourteen shar peis were severely cobalamin deficient, defined by an undetectable serum cobalamin concentration ( o ng/l). in the remaining dogs, serum cobalamin concentrations were within the reference interval ( - ng/l). serum concentrations of ha, crp, calprotectin, and s a were not significantly different between cobalamin deficient shar peis (medians: . ng/ml, . . fifty percent of cobalamin deficient shar peis had serum calprotectin concentrations above the upper limit of the reference interval, and % had serum s a concentrations above the suggested upper reference limit. in this study, serum concentrations of ha, crp, and the calgranulins did not differ between cobalamin deficient shar peis and shar peis with a normal serum cobalamin concentration. this finding leads us to speculate that increased ha and/or inflammatory markers are not associated with cobalamin deficiency in shar peis. further studies are needed to investigate serum cobalamin concentrations in patients with shar pei fever or cutaneous mucinosis. cobalamin deficiency (cd) has been associated with gastrointestinal and pancreatic disease in dogs. hereditary cd has been demonstrated in giant schnauzers and single case reports have suggested congenital cd in the border collie (bc) breed. clinicopathologic findings of cd vary and can be unspecific as cobalamin acts as a co-factor for a multitude of enzymatic reactions. the two most important reactions concern the conversion of methylmalonyl-coa to succinyl-coa and the re-methylation of homocysteine (hcy). these two metabolites increase when cobalamin is lacking and act as markers for cobalamin availability on a cellular level. preliminary data from dogs suggested that measurement of methylmalonic acid (mma) may be a better diagnostic test for cd than serum cobalamin concentration. therefore the goals of the study were ( ) to establish reference values for serum cobalamin, urine mma and plasma hcy in healthy pet dogs, ( ) to screen a larger bc population from switzerland for cd, and ( ) to perform genomic analyses on bc with cd. for determination of reference values healthy pet dogs were used. serum cobalamin was measured using an automated chemiluminescence assay (immulite ), urine mma was determined using gas chromatography and expressed as a ratio to urine creatinine and plasma hcy was measured using high pressure liquid chromatography and fluorimetric detection. to calculate reference ranges the th and th percentile were used. data were analyzed using non-parametric tests. reference ranges for cobalamin, hcy, and mma were: cobalamin . - . ng/l; urine mma - . mmol/mol creatinine; and plasma hcy . - . mmol/l. the screened bc population comprised purebred dogs and bc (median . months; range - ) suffering from congenital cd could be identified. clinical signs differed and consisted of tiredness ( ), stunted growth ( ), anemia ( ), dysphagia ( ) and persistent fever ( ). median (ranges) results for healthy bc and bc with cd were: for cobalamin ( - ) and . ( - ) ng/l; for urine mma ( - ) and ( - ) mmol/mol creatinine; for hcy . ( . - . ) and . ( - . ) mmol/l. strikingly, healthy bc with cobalamin concentrations well within the reference range had significantly higher urine mma concentrations compared to control dogs. under the assumption that the four affected bc are inbred to a single founder animal, first results of genotyping on the k illumina canine_hd snp chip suggest that mutations in the cubn and amn gene can be excluded to cause the observed cd in these dogs. we conclude that cd is a rare familial disease in bc with variable clinical signs. to define the genomic region responsible for cd further genetic analysis is in progress. it remains to be determined why some bc have high urine mma concentrations despite a serum cobalamin concentration within the reference range. calprotectin is a protein complex that plays an important role in the innate immune response. preliminary data suggest that canine calprotectin (ccp) is a useful marker for the detection of inflammation in dogs. recently, a radioimmunoassay for the measurement of ccp has been developed and analytically validated, but this test requires the use of a radioactive tracer. therefore, the aim of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (elisa) for the quantification of ccp in serum and fecal specimens from dogs. canine calprotectin (ccp) was purified, antiserum against purified ccp was raised in rabbits, monospecific antibodies were purified by affinity chromatography, and a sandwich-elisa was developed. purified antibodies were used for capturing and, after coupling with horseradish peroxidase (hrp), for reporting. a hrp substrate was used for color development. the assay was analytically validated by determination of analytical sensitivity and specificity, dilutional parallelism, spiking recovery, and intra-and inter-assay variability. control intervals for serum and fecal ccp were established from and healthy pet dogs, respectively, using the central th percentile. sensitivity of the assay for serum samples assayed in a : dilution and for fecal extracts assayed in a : , dilution was . mg/l and . mg/g, respectively. over a wide range of the assay, there was no cross-reactivity with cs a , the closest structural analogue of ccp available. observed to expected ratios (o/e) for serial dilutions ranged from . - . % (mean ae standard deviation [sd]: . ae . %) for four different serum samples, and from . - . % (mean ae sd: . ae . %) for five different fecal extracts. o/e for spiking recovery ranged from . - . % (mean ae sd: . ae . %) for four different serum samples and different spiking concentrations, and from . - . % (mean ae sd: . ae . %) for different fecal extracts and different spiking concentrations. intra-assay coefficients of variation (cv) for different serum samples were . , . , . , and . %, and . , . , . , and . % for different fecal extracts. inter-assay cv for different serum samples were . , . , . , and . %, and . , . , . , and . % for different fecal extracts. the control intervals for serum and fecal ccp were established as . - . mg/l and . - . mg/g, respectively. we conclude that this new elisa for the measurement of ccp is analytically sensitive, linear, accurate, precise, and reproducible, and does not cross-react with canine s a . further studies evaluating the clinical usefulness of measuring serum and/or fecal ccp are currently under way. the syndrome of hemorrhagic gastroenteritis (hge) is characterized by a peracute onset of hemorrhagic diarrhea, vomiting, depression, and anorexia, and can be associated with a high mortality if untreated. the etiology of hge is unknown, but it is speculated that an abnormal response to bacterial endotoxins, bacteria, or dietary components may play a role. hge is characterized by an increased vascular/mucosal permeability, thought to represent a type i-hypersensitivity reaction, whereas inflammation and necrosis appear to be rare. however, markers of gastrointestinal (gi) inflammation and changes in the intestinal microbiota have not been studied extensively in dogs with hge. therefore, the aim of this study was to evaluate fecal canine calprotectin (cp) and s a (a ), a -proteinase inhibitor (a -pi, a marker of gi protein loss), and bacterial groups that have previously been shown to be decreased (i.e., faecalibacterium spp., ruminococcaceae, bifidobacterium spp.) or increased (i.e., proteobacteria) in fecal samples from dogs with hge. fecal samples from consecutive days were collected from dogs with hge. fecal cp, a , and a -pi concentrations were measured by in-house immunoassays. bacterial dna was extracted from each fecal sample and was analyzed for faecalibacterium spp., proteobacteria, rumino-coccaceae, and bifidobacterium spp. using quantitative pcr assays. concentrations of fecal cp, a , and a -pi, and the abundance of bacterial dna were compared using a friedman test with dunn's post-hoc tests. significance was set at p o . . at the time of diagnosis (day ), fecal cp, a , and a -pi were above the suggested reference intervals in , , and of the dogs, respectively. until day , this number decreased to , , and , respectively. decreases in concentrations were significant between days and for a (p . ), and between days and for a -pi (p . ), but not for cp despite a trend (p . ). no differences in the abundance of faecalibacterium spp. (p . ), bifidobacterium spp. (p . ), or proteobacteria (p . ) were observed. however, the abundance of rumino-coccaceae was significantly lower on day when compared to day (p . ). in this study, fecal markers of inflammation and gi protein loss were increased in dogs with hge. although the number of patients was small, following initiation of treatment, two of the markers decreased significantly. these results suggest a loss of protein into the gi tract at the onset of hge. the lack of significant increases of faecalibacterium spp., bifidobacterium spp., and ruminococcaceae, and decreases in proteobacteria may suggest gi dysbiosis. further longitudinal studies are needed and are currently under way to evaluate gi dysbiosis in canine hge patients. the most recent antiemetic approved for use in dogs is maropitant citrate (cerenia s , pfizer animal health). maropitant is a selective nk receptor antagonist that acts by blocking the binding of substance-p within the emetic center and chemoreceptor trigger zone. label dosage recommendations for maropitant citrate are mg/ kg sc or mg/kg orally once daily for up to consecutive days (acute emesis) and mg/kg orally once daily for up to consecutive days (motion sickness). the study objective was to determine when steady-state is reached and the pharmacokinetics of maropitant administered at label oral dosages once daily for consecutive days. two groups of eight healthy beagles were administered maropitant citrate at or mg/kg orally once daily for days. concentrations of maropitant and its metabolite were measured in plasma using a lc-ms/ms assay. pharmacokinetic parameters were estimated using non-compartmental pharmacokinetic techniques and a modeling approach was used to estimate steady-state. the accumulation ratio for maropitant was . (auc - ) and . (cmax) for the mg/kg dose; and . (auc - ) and . (cmax) for the mg/kg dose after days. the model estimate for the number of doses required to reach % of steady-state was . for mg/kg and . for mg/kg. three dogs experienced a single episode of vomiting. dosing maropitant citrate beyond the label duration was well tolerated by healthy dogs. steady-state was reached after approximately doses for daily mg/kg and doses for daily mg/kg oral dosing. previously presented at the veterinary cancer society, november . cobalamin (vitamin b ) is involved in a variety of metabolic processes. altered serum cobalamin concentrations have been observed in dogs with gastrointestinal disorders, such as exocrine pancreatic insufficiency (epi) or severe and longstanding ileal disease. this study was conducted to identify breeds with a higher proportion of a decreased serum cobalamin concentration that were submitted to the gastrointestinal laboratory. the study was also aimed at investigating serum trypsin-like immunoreactivity (tli) concentrations that were diagnostic for epi in the dogs with a decreased serum cobalamin concentration. except for csp, breeds identified here, have not previously been identified to have a higher rate of a decreased serum cobalamin concentration. also, a possible association between an undetectable serum cobalamin and a decreased serum tli in ai needs to be further investigated. calprotectin (cp) is a widely used marker for the diagnosis and monitoring of gastrointestinal (gi) inflammation in humans. studies in humans usually report fecal cp concentrations based on a single stool sample although considerable day-to-day variability of fecal cp was found in patients with gi disease and in healthy controls. intra-individual variation of canine cp (ccp) was also substantial in a small number of healthy dogs but has not been determined in dogs with chronic gi disease. thus, the aim of this study was to compare the day-to-day variation of fecal ccp in dogs with chronic gi disease before and during treatment to that in healthy dogs. we hypothesized that fecal ccp would be less variable in patients with chronic gi disease than in healthy controls, and thus collection of a single fecal sample would be sufficient. fecal samples from consecutive days were prospectively collected from dogs (group a; median age: . years) referred for diagnostic work-up of chronic signs of gi disease, from dogs (group b; median age: . years) with stable gi disease while being treated, and from healthy adult dogs (group c; mean age: . years). fecal samples were extracted and ccp was measured by an in-house immunoassay. mean ccp, standard deviation, coefficient of variation (cv), and difference between maximum and minimum ccp for the -day sample collection period were calculated for each dog and were compared among groups using a kruskal-wallis test. fecal ccp ranged from . - . mg/g (median: . mg/g) in dogs with gi disease (group a), from . - . mg/g (median: . mg/g) in dogs of group b, and from . - . mg/g (median: . mg/g) in healthy controls (group c). cvs were - . % in group a (median: . %), . - . % in group b (median: . %), and - . % in group c (median: . %), respectively. patients in group a appeared to have less variable fecal ccp than dogs in group b and c, but this difference was not significant (p . ). the difference between maximum and minimum ccp for the -day sample collection ranged from - . mg/g in group a (median: . mg/g), from . - . mg/g in group b (median: . mg/g), and from - . mg/g in group c (median: . mg/g), and were not significantly different between any of the groups (p . ). in this study, considerable day-to-day variation of fecal ccp was found in dogs with chronic gi disease (regardless of treatment) and was comparable to that in healthy dogs. results of this study suggest that for evaluating fecal ccp in dogs with clinical signs of gi disease, three consecutive fecal samples rather than a single fecal sample should be analyzed. because we did not intend to evaluate the clinical usefulness of fecal ccp as a marker of gi disease in dogs, disease severity, quality, and location differed among dogs in groups a and b. the diagnostic utility of fecal ccp in dogs with gi disease is currently being investigated. it has been suggested that diagnosis of clostridium perfringens related enteropathy should be based on the detection of the c. perfringens enterotoxin gene (cpe-gene) by pcr and/or c. perfringens enterotoxin (cpe) by elisa in feces. however, the prevalence of the cpe-gene and cpe in dogs and especially cats with gastrointestinal disease has not yet been reported. also, there is limited information about the stability of cpe in fecal samples at various storage conditions. the aim of this study was to evaluate the prevalence of the cpe-gene and cpe and the stability of cpe in fecal samples from dogs and cats. to evaluate the prevalence of the cpe-gene, a total of fecal samples from dogs and cats with clinical signs of gastrointestinal disease ( dogs and cats) and fecal samples from those without such signs ( dogs and cats) were examined using pcr. to evaluate the prevalence of cpe, a total of fecal samples from dogs and cats with clinical signs of gastrointestinal disease ( dogs and cats) and dogs without such signs were evaluated using a commercially available elisa kit (techlab, blacksburg, va). the results were analyzed using a fisher's exact test. significance was set at p o . . to evaluate the stability of cpe, fecal samples from dogs and from cats with clinical signs of gastrointestinal disease that were positive for cpe were examined. also, cpe negative samples from dogs were evaluated as negative controls. each sample was subdivided into aliquots and evaluated on day ; on days , , and after being stored at room temperature (rt) or c; and on day after being stored at À c. the prevalence of the cpe-gene was not significantly different between dogs with signs of gastrointestinal disease ( / ; . %) and dogs without ( / ; . %; p . ). also, the prevalence of the cpe-gene in cats with signs of gastrointestinal disease ( / ; . %) was not significantly different compared to cats without ( / ; . %; p . ). pcr and elisa results were available for samples. of the pcr positive samples, only ( . %) were elisa positive. of the pcr negative samples, only ( . %) was elisa positive. the prevalence of cpe was not significantly different between dogs with clinical signs of gastrointestinal disease ( / ; . %) and those without ( / ; . %; p . ). the prevalence of cpe in cats with signs of gastrointestinal disease was / ( . %), but no samples from cats without such signs were available. when evaluating the stability of cpe, results for all aliquots were consistent with the initial result, except for one sample (on day , stored at rt, which was initially cpe positive). these results indicate that only a small proportion of samples that are pcr positive for the cpe-gene are also positive for cpe. studies are warranted to further compare the prevalence of cpe among animals with gastrointestinal disease and those without. furthermore, the results indicate that cpe is relatively stable in fecal samples at various storage temperatures. clostridium perfringens has been implicated as a cause of diarrhea in dogs. the main study objective was to compare two culture methods for the identification of c. perfringens. a secondary objective was to evaluate c. perfringens toxin genes a, b, b , e, ı and cpe from canine isolates using a multiplex pcr and determine their prevalence in a group of normal and diarrheic dogs. fecal samples were collected from clinically normal (nd, n ) and diarrheic dogs (dd, n ) at a primary care veterinary facility. isolation of c. perfringens was performed using direct inoculation of feces onto % sheep blood agar (sba) as well as enrichment of stool in bhi broth followed by inoculation onto sba. isolates were tested by multiplex pcr for the presence of a, b, b , e, ı and cpe genes. c. perfringens was isolated from % ( / ) of nd fecal samples using direct culture and . % ( / ) with bhi enrichment (p . ). in the dd, corresponding isolation rates were . % and . % (p . ). all isolates possessed a toxin gene. b, b , e, ı and cpe toxin genes were identified in . %, . %, . %, . % and . % of nd isolates, respectively. in the dd group, b and b were identified in %, e and ı were not identified and the cpe gene in . % of isolates. bhi enrichment did not significantly increase the yield of c. perfringens compared to sba but increased time and cost involved. c. perfringens (p . ) and c. perfringens toxin genes were present in equal proportions in nd and dd groups (p ! . ). culture of c. perfringens and pcr for toxin genes are of limited diagnostic utility due to the high prevalence of c.perfringens in normal dogs and the lack of apparent difference in toxin gene distribution between normal and diarrheic dogs. endoscopic biopsies are a relatively convenient, non-invasive test for feline infiltrative intestinal disorders. commonly, only the duodenum is examined due to cost, risks and time required to prepare the colon using lavage solutions, cathartics and/or enemas. the purpose of this study was to evaluate the consistency between endoscopic biopsies of the duodenum and ileum in cats. endoscopic biopsies from cats which had duodenal and ileal tissue specimens were evaluated retrospectively. all slides were randomized and reviewed by a single pathologist (jm) for quality, number of biopsies, and diagnosis according to wsava standards. no information regarding history, clinical signs, endoscopic findings, or previous histological diagnosis was made available to the pathologist. statistical comparison of the diagnosis of sc-lsa and ibd by intestinal location was conducted using fisher's exact test (p o . significant). of cats ( . %) were diagnosed with sc-lsa in the duodenum and/or ileum. of these cats, ( . %) were diagnosed with only duodenal sc-lsa, ( . %) were diagnosed with only ileal sc-lsa, and ( . %) had sc-lsa in both duodenum and ileum. in cats with only ileal sc-lsa, had severe ibd in duodenal biopsies, possibly consistent with early sc-lsa. of these had duodenal biopsies without evidence of sc-lsa. our results suggest there is a population of cats in which diagnosis of sc-lsa may only be found by evaluating ileal biopsies. clinicians should consider performing both upper and lower gi endoscopic biopsies in cats with suspected infiltrative small bowel disease. periodontitis is one of the most common diseases in cats and is mainly due to the presence of plaque and calculus. in this study, we investigated putative correlations between dental tartar and gingivitis and also between gingivitis and subgingival bacteria in cats. twelve cats (median age: years; range: - years; dsh and persians; females and males) were enrolled. dental tartar was obtained during scaling for a dental prophylactic procedure. all cats were negative for felv and fiv infection as assessed by a commercial elisa test (snap s fiv/felv combo test). severity of gingivitis (scores: - ; normal, mild, moderate, and severe) and dental tartar (scores: - ) were scored in each cat. endodontic paper points were applied for collecting a bacterial sample from the subgingival area and transferred to thioglycollate transporting media for bacterial culture. the relationship between gingivitis and tartar thickness scores was analyzed by spearman correlation. a student's t-test was used to compare the mean differences (gingivitis and tartar thickness scores) between upper and lower teeth. the association between severity of gingivitis and bacterial type was tested by chi square test. the spearman correlation coefficient for the average gingivitis score and the average tartar thickness score was . (p o . ). interestingly, the average tartar thickness scores from the upper jaw were significantly higher than those from the lower jaw (p o . ). the highest scores were found for the molar teeth in all cats. bacterial culture revealed . % anaerobic bacteria species (i.e., bacteroides spp., peptostreptococcus anaerobius, and eubacterium aerofaciens) and . % aerobic bacteria species (i.e., pasteurella multocida, streptococcus spp., enterococcus spp., staphylococcus spp., bacillus cereus, escherichia coli, and pseudomonas aeruginosa). anaerobic bacteria were found mostly in cats with higher gingivitis scores ( - ; chi square: p o . ), while pasteurella multocida was found mostly in cats with lower gingivitis scores ( - ; chi square: p o . ). antimicrobial sensitivity testing indicated that all of the anaerobic bacteria were sensitive to clindamycin, chloramphenicol, metronidazole, cefoxitin, or tetracycline, % were sensitive to erythromycin, and % were sensitive to penicillin. the most abundant aerobic bacterial species, pasteurella multocida, was sensitive to cefoxitin in all cases in which it had been cultured. these results suggest that anaerobic bacteria may be associated in the pathogenesis of severe gingivitis. these data warrant further studies of the prophylactic use of antibiotics in cats undergoing dental prophylactic procedures. inflammatory bowel disease is the most common cause of vomiting and diarrhea in dogs. although it can occur in any canine breed, certain breeds are more susceptible. we have previously shown that polymorphisms in the tlr and tlr gene are significantly associated with inflammatory bowel disease (ibd) in the german shepherd dog (gsd), a breed at risk of developing this disease. it would be useful to determine if these polymorphisms are significant in other canine breeds as this may allow the development of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. therefore the aim of this study was to investigate whether polymorphisms in canine tlr and tlr genes are associated with ibd in other non-gsd canine breeds. four non-synonymous snps in the tlr gene; t c, g a, a t and g a and three non-synonymous snps in the tlr gene; g a, c t and t c previously identified in a mutational analysis in gsds with ibd were evaluated in a case-control study using a snapshot multiplex reaction. sequencing information from unrelated dogs with ibd consisting of different non-gsd breeds from the uk were compared to a breed-matched control group consisting of unrelated dogs from patients treated for noninflammatory disease at the royal veterinary college, london, uk. as in the gsd ibd population the two tlr snps; c t and t c were found to be significantly protective for ibd in other breeds included in this study (p . and p . respectively). this study confirms the protective effects of the two tlr snps (c t and t c) in other canine breeds with ibd. this highlights the importance of tlr in the pathogenesis of canine ibd and may represent common pathological pathways of ibd in different canine breeds due to the high degree of haplotype sharing seen among breeds. this may allow for the future expansion of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. further functional studies looking at the role of tlr in the pathogenesis of canine ibd are needed to confirm these findings. toll-like receptor (tlr ) is an extracellular pattern recognition receptor belonging to the innate immune system. we have recently shown that three non-synonymous single nucleotide polymorphisms (snps) in the tlr gene (g a, c t and t c) are significantly associated with inflammatory bowel disease (ibd) in german shepherd dogs. in addition, we confirmed that two of these tlr snps (c t and t c) were significantly associated with ibd in a population consisting of different dog breeds. in order to determine if other novel snps exist in the tlr gene in addition to the ones identified in the gsd population, mutational analysis was carried out in seven boxer dogs with ibd. polymerase chain reaction was carried out to amplify the tlr coding region in the seven dogs with ibd. sequencing was carried out using sequence based typing with the abi prism sequencing kit (applied biosystems, uk) and analyzed using an abi automated sequencer (pe applied biosystems). sequencing information from seven boxer dogs with ibd from the uk were compared to the reference sequence published on the ensemble webserver (www. ensembl.org/canis_familiaris). in addition to the three snps identified previously in the tlr gene, a novel non-synonymous snp; t c was identified in the boxer dog population with ibd. this snp has never been reported before and was present as the homozygote genotype in three dogs with ibd and in one dog as the heterozygote genotype. using the simple modular architecture research tool (smart) web server (http:// smart.embl.de/) we were able to map the t c snp to the leucine rich repeat domain of the tlr protein. the leucine rich repeat domain is involved with ligand binding and therefore a change in the amino acid in this region may affect function, especially as the t c snp results in a change in the amino acid from non-polar to polar. our study further confirms the role of tlr in the pathogenesis of canine ibd. our results suggest that in addition to shared risk polymorphisms amongst breeds, individual breeds may harbor unique snps arising after breed formation which may further affect their susceptibility to this disease. however, a case-control study would be needed in the boxer dog to confirm the significance of the tlr t c snp and further functional data would be needed to elucidate the exact role of this polymorphism in canine ibd. an automated power driver device (oncontrol, vidacare) has recently become available for bone marrow aspiration (bma) and bone marrow biopsy (bmb) in humans. the purpose of our study was to compare this automated technique to the traditional manual technique for bone marrow sampling in cats. twelve healthy research cats were anesthetized using a standardized protocol on different occasions, days apart, to have bmas and bmbs performed by the same operator. on day , half of the cats were randomized to have a bma performed at both the proximal humerus and the iliac crest, and a bmb performed at the iliac wing, using the oncontrol device ( -gauge needle for bma; -gauge needle for bmb). the other half of the cats had the same procedures performed using a manual technique ( -gauge illinois needle for bma; -gauge jamshidi needle for bmb). on day , each cat had bma performed at the opposite humerus and iliac crest, and a bmb performed at the proximal humerus using the opposite technique from day . for each procedure, the operator was given a maximum of attempts to successfully collect a sample. the rate of success, as well as the number of attempts were recorded. the ''ease of use'' of the device was rated by the operator on a -point scale after each procedure. using previously determined criteria, the macroscopic and microscopic qualities of the bma and bmb samples were assessed by a board-certified pathologist, blinded to the technique used. the level of pain experienced by each cat was evaluated , , , , and hours following each set of procedures, using a previously validated pain scoring system. two sample t-tests were used to compare the automated technique to the manual technique and to compare the humerus to the iliac crest site for bmas and the humerus to the iliac wing site for bmbs. for all procedures, at all sites, the ''ease of use'' was better for the automated technique than for the manual technique (p o . ). the duration of the procedure and the number of attempts to collect a sample were significantly lower with the automated technique for bma at the proximal humerus (p o . ). there was no significant difference in the level of pain at any time point following each set of procedures with either technique. performing bma at the proximal humerus was associated with a higher rate of success (p o . ), a lower number of attempts (p o . ), a shorter duration of the procedure (p o . ), a higher-rated ''ease of use'' of the technique (p o . ), and a better quality sample (p o . ) when compared to sampling from the iliac crest, in conclusion, we found the automated bone marrow sampling technique suitable for use in adult cats. this technique was easier to use than the manual technique for both bma and bmb, and reduced the duration of the procedure and the number of attempts for successful bma at the proximal humerus. performing bma at the proximal humerus was faster, easier and allowed collection of better quality samples than at the iliac crest, independently of the technique used. the fractious nature of some feline patients sometimes makes sedation or general anesthesia necessary for routine procedures such as blood collection for hematologic analyses. it has been anecdotally reported that sedation or general anesthesia could induce variations in hematologic parameters in cats, making it important for the clinician to be able to anticipate potential changes on hematologic parameters that could result from chemical restraint. this study evaluated the effects of a standardizecd anesthetic protocol using ketamine ( mg/kg, iv), midazolam ( . mg/kg, iv) and buprenorphine ( mg/kg, im) on the hematologic parameters of healthy adult research cats. each cat had blood samples collected before and after induction of anesthesia on different occasions, days apart. in total, pairs of complete blood counts were obtained. analyses were performed at a certified veterinary laboratory. paired sample t-tests were used to determine whether there were any statistical differences between hematologic parameters before and after induction of general anesthesia, for each cat, on different occasions. compared to preanesthetic values there was a significant decrease in red blood cell count, hemoglobin concentration, hematocrit, lymphocyte count and plasma total protein concentration after induction of anesthesia. there was no significant difference in the segmented or band neutrophil, eosinophil, basophil, monocyte and platelet counts between the samples taken before and after induction of anesthesia. on average, there was a . % decrease in the red blood cell count ( .  /l to .  /l) (p o . ), a % decrease in hemoglobin concentration ( . g/l to . g/ l) (p o . ), a . % decrease in the hematocrit ( . l/l to . l/l) (p o . ), a . % decrease in the lymphocyte count ( .  /l to .  /l) (p . ), and a . % decrease in the plasma total protein concentration ( . g/l to . g/l) (p o . ) when samples taken before and after induction of anesthesia were compared. if only the hematocrit was considered as a marker of anemia, % of the samples from these healthy cats, taken while they were under general anesthesia, would have been misinterpreted as belonging to anemic patients (hematocrit o . l/l), using the reference interval established in our laboratory. none of the cats would have been considered anemic before induction of general anesthesia. in practice, the decrease in lymphocyte count following anesthesia is unlikely to be of clinical relevance, as all the samples except had a lymphocyte count that was within the reference interval for cats established by our laboratory. this study suggests that complete blood counts performed on blood taken under general anesthesia with this combination of anesthetic drugs in cats should be interpreted cautiously in order not to make a false diagnosis of anemia. the mechanism responsible for the decrease in circulating red blood cell mass following anesthesia induction in cats is unknown and requires further investigation. rivaroxaban is an oral inhibitor of activated coagulation factor x (xa). it is expected to have similar coagulation effects as low molecular weight heparin, without the need for injection, making it an attractive alternative for long-term anticoagulant therapy in cats. citrated blood obtained from five healthy adult cats was exposed in vitro to varying concentrations of rivaroxaban, followed by coagulation testing. the rivaroxaban was extracted from commercially available tablets (xarelto s ) and dissolved in dmso prior to addition to the blood. tests performed included kaolin-activated thrombelastography (teg), prothrombin time (pt), dilute pt (dpt), activated partial thromboplastin time (aptt), and anti-factor xa (axa) activity. dose-dependent prolongations were seen in all coagulation parameters. similar to human data, therapeutic axa levels (between . - . axa units) were achieved at in vitro concentrations between and mg/l. at mg/l, dpt measurements were clinically prolonged in all cats ( . ae sec vs. . ae . sec, p . ), while aptt values were only mildly prolonged from baseline ( . ae sec vs. . ae sec, p . ). significant prolongations were seen in dpt at ( . ae ec, p . ). teg r time did not prolong from baseline values until concentrations of mg/l were reached ( . ae min compared to . ae . min, p . ). rivaroxaban has similar coagulation effects in the cat as in other species and may play a role in feline thromboprophylaxis. kaolinactivated teg does not appear to be sensitive to low concentrations of rivaroxaban in the cat. anticoagulated blood is required for platelet function studies. sodium citrate, a calcium chelater, is the most commonly used anticoagulant to measure coagulation parameters including platelet aggregation but it may have a negative effect on platelet responsiveness. dogs are generally considered moderate responders to collagen on platelet aggregation and are notorious for being poor or inconsistent responders to adp-induced platelet aggregation using citrated whole blood. hirudin, a selective thrombin inhibitor, can also be used as an anticoagulant for coagulation assays and is the anticoagulant of choice for certain assays including the multiplate s platelet function analyzer. ten adult healthy dogs were used to compare whole blood platelet aggregation between citrated and hirudinated blood samples. venous blood was collected atraumatically from the external jugular vein directly into tubes containing . % trisodium citrate or hirudin. whole blood platelet aggregation was performed (whole-blood lumi-aggregometer, chrono-log corporation, havertown, pa, usa) with collagen ( mg/ml) and adp ( mm) as agonists. maximal platelet aggregation (ohms) was recorded. there was a significant increase in collagen-induced platelet aggregation from the hirudinated samples compared to the citrated samples ( . ae . vs. . ae . o, p o . ). there was also a significant increase in adp-induced platelet aggregation from the hirudinated samples compared to the citrated samples ( . ae . vs. . ae . o, p . ). the results of this study show a significant difference in platelet responsiveness between citrated and hirudinated whole blood using the chrono-log impedance aggregometer. while both collagen and adp-induced platelet aggregation was attenuated from citrated blood samples, this was most notable for adpinduced aggregation where almost all samples had no objective measurable platelet aggregation. it is suggested from this data that future whole blood platelet aggregation studies performed on the chrono-log impedance aggregometer should use hirudinated blood samples although new reference limits would need to be established. low-molecular-weight heparin (lmwh) is now used to prevent thrombotic complications in dogs. a functional assay such as the calibrated automated thrombogram (cat) may provide a new approach for monitoring lmwh therapy. we hypothesized that cat would detect decreased endogenous thrombin potential (etp) in healthy dogs receiving lmwh (fragmin s ). twenty-four healthy adult beagles were included in this study and divided equally in four groups. one dose of u/kg, u/kg or u/kg of lmwh were given subcutaneously to healthy dogs and compared to a control group. platelet poor plasma (ppp) was collected over a hour period. using a repeated-measure linear model, effect of lmwh on etp was time and dose dependent with a significant interaction (p o . ). compared to control dogs, significant differences were obtained for group u/kg at t (p . ), for group u/kg at t (p . ) and between t -t minutes (p o . ) respectively, and for group u/ at t (p . ), between t -t minutes (p o . ) respectively and at t (p . the cat assay can be employed to measure the effects of lmwh at different doses in healthy dogs, resulting in significant time and dose-dependent decreases in etp and warrants further investigation as a tool for monitoring lmwh therapy in dogs. the purpose of this study was to determine the effects of prednisone and prednisone plus ultralow-dose aspirin on coagulation parameters in healthy dogs, with an emphasis on thromboelastography (teg). this was a prospective, randomized, blinded study utilizing fourteen dogs determined to be healthy based on normal physical examination, complete blood count, biochemistry, urinalysis, and fecal floatation. dogs were evenly divided into either prednisone plus aspirin (pa) or prednisone plus placebo (pp) groups. baseline values for teg parameters (r, k, angle, ma, ly , ly , g, ci) were measured twice two days apart, and thrombin-antithrombin complexes (tat), and traditional coagulation parameters (prothrombin time, activated partial thromboplastin time, d-dimer, antithrombin (at), fibrinogen) were measured once. each dog received mg/kg/ day of prednisone, and either . mg/kg/day of aspirin (pa group) or placebo (pp group) for days. a complete blood count, biochemistry profile, teg, tat, and traditional coagulation parameters were then repeated on each dog. day to day variation was calculated for the teg parameters using the two baseline measurements. the change from baseline between and within each group were compared using t-tests, or wilcoxon sample test where appropriate, for teg, tat, traditional coagulation parameters, and hematocrit. day to day variation in teg was acceptable ( %) for ma, g, and angle, unacceptable ( %) for r, k, ly and ly , and not meaningful for ci. within both groups, ma, g, ci and fibrinogen significantly increased from baseline (p o . ). within both groups, ly and at significantly decreased from baseline (p o . ). for the pp group, ly significantly decreased from baseline (p . ), and approached significance for the pa group (p . ). all other within group changes from baseline were not statistically significant (p-values . ). for all parameters, there was no difference between groups for change from baseline (p values . ). day to day variation in some teg parameters is high and may preclude their clinical utility. prednisone causes hypercoagulability in healthy dogs based on increased g, ma, and ci. the addition of ultra-low dose aspirin to prednisone has no effect on the parameters measured in this study. 'aspirin resistance' has been identified in people and dogs that develop thrombi despite low dose aspirin therapy. variability in platelet cyclooxygenase (cox) isoform expression is one proposed mechanism for aspirin resistance in people. two isoforms (cox- and cox- ) have been identified in canine platelets. high (antiinflammatory) dose aspirin inhibits platelet function and alters expression of both cox isoforms in most dogs. this study evaluated the effects of low dose aspirin on platelet function and cox expression in normal dogs. twenty-five healthy client-owned dogs were evaluated before and at two time points (days and ) during aspirin therapy ( mg/kg po sid). platelet response to aspirin (siemens pfa- s ; collagen/ epinephrine cartridges), was stratified into one of three groups [aspirin responders ( dogs), non-responders ( dogs), or inconsistent responders ( dogs)]. flow cytometry identified platelet cox- and cox- expression. an elisa was used to measure urine -dehydro-thromboxane b ( -dtxb ). there were no significant differences between groups for cox- , cox- or -dtxb at any time point. when all dogs were considered as a single group, there was a significant increase (p o . Ã) in cox- and cox- mean fluorescent intensity (mfi) from baseline to day , . % ae . (mean ae sd) and . % ae . , respectively. there was a significant decrease in mean urine -dtxb :creatinine on day and by . % (p . à ) and % (p o . à ). as with our previous high dose studies, cox- expression was increased with aspirin exposure. however, there was a significant increase in cox- expression with low dose aspirin in contrast to the decrease seen at higher doses. our study suggests that levels of platelet cox- and cox- expression do not influence aspirin response in dogs. although thromboxane levels decreased in most ( of ) dogs on low dose aspirin, platelet function was consistently affected in only % of dogs, suggesting that differences in response to thromboxane may play a role in the variable affects of low dose aspirin on canine platelet function. delayed postoperative bleeding is common in retired racing greyhounds (rrgs), despite normal results of routine hemostasis assays. the excessive postoperative bleeding in the rrgs is not due to primary or secondary hemostatic defects, and may be due to enhanced fibrinolysis or to a clot maintenance dysfunction. providing a method to prevent or minimize the severity of postoperative bleeding in rrgs will not only have major economic impact for owners, but also will markedly decrease the associated complications of minor or major surgeries in the breed. epsilon aminocaproic acid (eaca) is a potent inhibitor of fibrinolysis that also supports clot maintenance due to unknown mechanisms. the objective of this double-blinded, prospective, randomized study was to evaluate the effects of eaca versus placebo on the prevalence of bleeding in rrgs, and to investigate its mechanism of action by using thromboelastography (teg). we compared the effects of eaca and placebo in rrgs that underwent elective ovariohysterectomy or orchiectomy at the veterinary medical center, the ohio state university during years. the main endpoint was bleeding (prevalence and severity); minor endpoints included most teg parameters. thirty percent ( / ) of the rrgs in the placebo group had delayed postoperative bleeding starting to hours after surgery, compared to % ( / ) in the eaca group (p . ). on the teg parameters, the r time (clot formation time) was significantly different between treatment groups (p . ). the postoperative administration of eaca significantly decreased the prevalence of postoperative bleeding in rrgs. thromboembolism associated with protein losing nephropathy (pln) has been long recognized as a serious and unpredictable complication in dogs, however its prevalence remains unknown. in humans, surrogate indicators are frequently used to assess thromboembolic risk. this study aimed to investigate the prevalence of hypercoagulability in pln dogs based on thromboelastography (teg), and to determine whether hypercoagulability in these patients could be predicted by clinical assessments that identify systemic hypertension (systolic blood pressure mmhg), hypoalbuminemia (serum albumin o . mg/dl), antithrombin activity (o %), and degree of proteinuria (urine protein:creatinine ratio [upc] ! ). between march -september , twenty-seven dogs were identified with pln at the animal medical center. the prevalence of hypercoagulability based on a teg g-value . was . %. there was no statistically significant relationship, either categorically or continuously, in univariate as well as multivariate analyses of all variables. univariate logistic regression (odds ratio; lower and upper confidence limit; p value) for hypertension was À . ; . , . ; . ; for albumin - . ; . , . ; . ; and for antithrombin activity - . ; . , . ; . . thus, in this patient population, in the absence of teg, prediction of hypercoagulability using abnormalities in commonly measured clinicopathologic variables was not helpful. however, given the documented high prevalence of hypercoagulability in patients with pln, early institution of prophylactic anti-platelet or anticoagulant therapies should be considered. thromboelastography (teg) is a test of global hemostasis. due to the effects of extrinsic factors on whole blood coagulation, sample collection method (scm) may influence results. the purpose was to determine if scm influenced teg using kaolin-activated citrated whole blood (wb). healthy dogs with normal platelet counts were prospectively enrolled. three wb samples were obtained from each dog at least hours apart from alternating jugular veins in a randomized order of three methods: ) vacutainer s into citrated tube (vac), ) citrated syringe with transfer into plain tube (cit), or ) plain syringe with transfer into citrated tube (plain). draw time was recorded in seconds. kaolin-activated teg was performed, with measurement of reaction time (r), clot formation time (k), maximum amplitude (ma), and alpha angle. eleven dogs were enrolled. there were no significant differences in teg indices between vac samples and either cit or plain samples. cit samples had a significantly higher k value (p . ) and a lower alpha angle (p . ) compared to plain samples. draw times ranged from - seconds. a longer draw time was significantly correlated (p . ; r À . ) with a shorter r time. higher platelet count was significantly correlated (p . ; r . ) with a higher ma. scm did not have a significant effect on teg parameters when comparing vac samples to either cit or plain samples. minimizing sample collection time and trauma during venipuncture may be important in minimizing hypercoagulable changes in teg indices. liquid plasma (lp) is defined as either plasma collected and refrigerated immediately after collection or fresh frozen plasma (ffp) that is thawed and stored refrigerated until use. stability studies in people have shown that adequate clotting factor activity is preserved for at least days. lp is transfused in human level i trauma centers to critically ill people requiring rapid infusion of clotting factors as the time required to defrost ffp is considered prohibitive. the use of lp has not been described in veterinary critical care. the purposes of this study were to ) determine the length of time required in a water bath for a unit of canine ffp to thaw and ) describe the use of lp in a busy university emergency room (er). for part : six units ( ml) of canine ffp were individually thawed in a c water bath. the duration of time (in minutes) to thaw was recorded. for part : the transfusion log was reviewed for dogs receiving lp in the last months. the indications and outcome were recorded. the mean time ae sd thaw time was . ae . minutes. ten units of lp were transfused to critically ill or injured dogs during the study time. indications for lp transfusion included hypovolemic shock due to intra-abdominal hemorrhage in dogs ( traumatic, non-traumatic) and rapid correction of hemorrhage following parenteral tissue plasminogen activator administration in dog. lp volume transfused ranged from . to . ml/kg. no transfusion reactions were identified. effect on coagulation was not consistently evaluated. time required to thaw a unit of ffp is greater than minutes which could be detrimental in a bleeding, coagulopathic dog. lp was transfused without incident to critically ill and injured dogs and represents a potential new addition to the armamentarium of treatments in a veterinary er setting. further investigation of canine lp is warranted including evaluation of in vitro factor stability and in vivo efficacy in correcting coagulopathy. immune mediated thrombocytopenia (imt) is associated with increased morbidity and mortality. large prospective research studies in dog platelet antibodies and clinical utilization of platelet immunoglobulin assays are limited. potential explanations include limited availability and low specificity due to nonspecific binding. the focus of this study is to evaluate optimized direct and indirect platelet surface associated immunoglobulin (psaig) and staining with anti-cd antibodies (cd ab) for the utilization in classifying thrombocytopenic dogs. one hundred clinically ill and apparently healthy dogs were prospectively evaluated. data collected included a history of hemorrhage, physical examination evidence of bleeding, complete blood count, and measurement of psaig and cd ab. the psaig assay utilized polyvalent antibodies with correction for non-specific binding by subtraction of background fluorescence with control antiserum. thrombocytopenia was defined as o , /ml and all dogs were clinically classified into of groups (g): g imt, n , g thrombocytopenia from non-immune mediated diseases, n , g ill with normal platelet counts, n , g healthy dogs, n . median platelet counts, by groups, were g , , ; g , , ; g , , ; and g , , /ml. for the direct and indirect psaigs in dogs with itp (g ), more dogs (n and n ) with clinical evidence of bleeding had antibodies compared to those who were not bleeding (n and n ). considering only direct psaig the sensitivity and specificity was % and %, respectively for the diagnosis of imt. for indirect psaig the sensitivity and specificity was % and %, respectively, for the diagnosis of imt. when considering both direct and indirect psaig together, the sensitivity was % with a specificity of %. in g interference from high control antiserum background staining was noted in . % of dogs and resulted in a negative direct psaig classification. minimal background interference was noted in g , g , or g . the percentage of platelets stained with cd ab was significantly less in g (median , p . ) vs. g (median , p . ) vs. g (median . , p . ) and g (median . ). these findings indicate the optimized platelet surface associated immunoglobulin assay has a high specificity, however poor sensitivity, for the diagnosis of imt. the decreased cd staining in g (imt group) may reflect decreased surface gpiiia expression, blocking by anti-gpiiia antibodies or other proteins, clearance by macrophages, or increased non-platelet debris and has potential applications in the diagnosis and treatment of imt. greyhounds have lower serum concentrations of a-globulin than other breeds, explained by negligible levels of haptoglobin (hp) measured using different methods (colorimetric, immunoturbidimetric and protein electrophoresis). the purpose of the present study was to characterize the hp gene in greyhounds. we isolated dna and rna from blood samples of akc-registered and retired racing greyhounds (akcg, rrg), and a german shepherd dog (gsd). we sequenced the hp exons and splice sites, and conducted array comparative genomic hybridization to identify associated dna structural variation (custom m agilent oligonucleotide array). additionally, we tested for the presence of one or multiple haplotypes spanning hp in greyhounds using a high density snp array ( k illumina hd). sequencing results of hp in both dna and cdna revealed three synonymous snps in the racing greyhound. we did not identify structural variation overlapping or near the hp gene. notably, we detected that the rrg and akcg do not appear to share a specific haplotype spanning hp. despite having low or undetectable serum concentrations of hp, we did find that rrg hp mrna is expressed and lacks amino acid variation. this suggests that the clinical absence of the hp is attributable to post-transcriptional hp effects or to an unknown physiological interaction. finally, given the existence of distinct rrg and akcg haplotypes spanning hp, it is important to characterize serum levels of hp in akcg in follow on studies. we reported that hemoglobin in retired racing greyhounds (rrg) has higher oxygen carrying properties and affinity than other breeds. surprisingly, very little is known about canine hemoglobin genetics. the purpose of this study was to characterize genetics of canine beta globins. using computational blast analysis of the dog genome, we identified five beta globin genes in a single locus: two human hbelike followed by three hbb-like genes. we isolated dna and rna from blood of rrgs, akc registered greyhounds (akcg), and german shepherd dog (gsd). all beta globin exons and splice sites were sequenced, and the beta globin locus was examined by array comparative genomic hybridization (custom m agilent array). additionally, we determined the number of common haplotypes that span this locus in rrgs and akcgs using high density snp array ( k illumina hd). expression and sequence analysis of cdna showed all five beta globin genes are actively expressed in adults. canhbb and were created by relatively recent segmental duplication and have identical protein sequence. canhbb / are abundantly expressed in adults; canhbb is expressed at greatly reduced levels. sequencing results revealed one rare non-synonymous single nucleotide polymorphism (snp) in hbe of rrgs, but no variation that could explain their abnormal hemoglobin. we did not detect structural variation overlapping or near the beta globin locus. notably, rrg and akcg do not share haplotypes spanning the beta globin locus. this is the first characterization of canine hemoglobin genetics, and the first report of canine embryonic hemoglobins and their expression in adults. sampling of the bone marrow in the dog from the costochondral (cc) junction can be performed with minimal to no sedation and readily available equipment but is not in widespread use in the united states. the aim of this study was to compare the number of attempts needed to successfully obtain a sample, the time needed for the procedure, and the sample quality between aspirates obtained from the cc junction and more traditional sites (humerus or femur) in healthy dogs when performed by novice and seasoned practitioners. samples were obtained from healthy anesthetized laboratory reared adult dogs after undergoing terminal endoscopic surgery. paired samples from separate dogs were obtained by each practitioner using either a gauge needle and cc syringe at the cc junction or an gauge rosenthal needle and cc syringe from either the proximal humerus or femur (clinician preference). three small animal veterinary interns, one experienced technician and one boarded internist were monitored for number of attempts to success and length of time needed for success of each procedure. slides were prepared by a single investigator and read by a blinded clinical pathologist. data were compared using the paired t-test for normally distributed data and wilcoxen signed rank test for non-gaussian distributions. five pairs of samples from three dogs were evaluated. two dogs had two pairs drawn from opposite limbs and ribs. mean number of attempts to success for traditional sampling sites ( . /À . ) and time to success ( . minutes /À . ) did not differ significantly from attempts ( . /- . , p . ) or time ( . /- . , . ) needed when aspirating from the cc junction. subjectively, samples were of similar quality with regards to cellularity and number of particles present when compared within practitioners. myeloid: erythroid ratio and percentage of lymphocytes were also not significantly different between sites (m:e ratio p . , lymphocyte % p . ) and were within normal limits. while there were no significant differences between the two sites in terms of number of attempts or time to success, it should be noted that the ''seasoned'' practitioners had never performed an aspirate at the cc site and had an increased number of attempts compared to the traditional sites. if the number of attempts needed for success decreases with experience, it is likely the time required would decrease as well. both subjectively and objectively, there were no significant differences in quality or cell populations between the two sampling sites in healthy dogs. based on this data, bone marrow aspiration from the cc junction appears to be equivalent to more traditional sampling sites in healthy dogs. larger studies in clinically ill dogs should be performed before routinely using the site in the clinical setting. recent research on iron homeostasis has elucidated the tightly controlled intestinal uptake of iron. hepcidin, the major hormone limiting iron absorption and release from macrophages, is downregulated by matriptase- , a transmembrane serine protease (tmprss ) produced by the liver. while iron deficiency is commonly caused by chronic blood loss anemia and rarely dietary deficiency or intestinal disorders in dogs and other species, we report here the clinical to molecular investigations of a dog with iron-refractory iron deficiency anemia (irida) caused by a matriptase- deficiency homologous to a recently described autosomal recessive disorder in humans. the proband, a spayed female cocker spaniel without any clinical signs except for recent occasional idiopathic seizures, exhibited a lifelong history of microcytosis and hypochromasia but not anemia. there was no evidence of any blood loss and the dog was receiving an appropriate meat-based diet. mean values of complete blood cell counts, performed from . - years of age, were: hematocrit % (normal reference range - ); rbc count .  /ml ( . - . x ); mcv fl ( - ); mchc g/dl ( - ). serum iron parameters revealed severe iron deficiency with serum iron mg/dl ( - ); total iron binding capacity mg/dl ( - ); serum iron saturation o % ( - %), and ferritin ng/dl ( - ). prolonged courses of oral ferrous sulfate supplementation and several short courses of intramuscular (dextran) injections and intravenous iron infusions did not result in improvement of any red cell or serum iron parameters. however, this dog was never anemic and the partial seizures could not be directly related to the iron deficiency status. no family members were available for further studies. genomic dna was extracted from the proband's edta blood and the exons of the tmprss gene were amplified with flanking primers and then sequenced. in comparison to the normal canine tmprss sequence and that of a sequenced control dog we found a homozygous missense muation, r h, toward the c-terminal end of the protein in the proband's gene. in conclusion, the severe microcytosis and hypochromasia, low serum iron parameters and lack of a response to oral and parenteral iron therapy led to the diagnosis of irida. the missense mutation in the matriptase- at position from an arginine, which is conserved across all species currently deposited in the genbank, to a histidine is likely the disease-causing mutation. this is the first report of an irida in the dog with features very similar to those observed in humans. dogs with naturally-occurring irida may be helpful in developing and assessing novel therapies. accidental ingestion of copper-coated zinc pennies minted after is the most common causes of zinc toxicity anemia in the dog. zinc toxicity anemia may also be seen with ingestion of zinc from other sources as ingestion of metallic foreign material other than pennies, medicines containing zinc, and zinc supplements. the purpose of this study was to determine if there is a weight below which dogs are more susceptible to zinc toxicity anemia secondary to metallic foreign body ingestion. records of dogs presented to the internal medicine service at the veterinary medical center of long island for metallic foreign body ingestion were reviewed for signalment, weight, presenting pcv, and type of metallic foreign body ingested. eighteen dogs met the inclusion criteria and were compared. of the dogs, there were cases of coin ingestion ( %), with ( %) involving ingestion of or more pennies. the other cases involved ingestion of a metallic object ( ), decorative garland ( ), and bb pellets ( ).of the dogs exposed to zinc, ( %) were less than pounds ( . kgs). of those cases ( %) had ingested one or more pennies. eleven out of the ( %) zinc exposure dogs were anemic at presentation. the average weight of the dogs was . pounds ( kg). this study showed that dogs less than pounds appear to be more susceptible to developing anemia secondary to zinc toxicosis, with the majority of cases due to ingestion of pennies minted after . zinc toxicity anemia secondary to penny ingestion is more commonly seen in small dogs. we suspect larger dogs are able to pass pennies through the pyloric sphincter and thus not develop clinical signs. although thrombocytopenia is common in hospitalized dogs, canine cryopreserved platelet concentrate (pc) is used infrequently. data suggest in vitro efficacy of pc and when administered to research dogs, but efficacy is unknown in clinical patients. study objectives were to determine clinical characteristics of dogs receiving pc as well as safety and efficacy of pc in thrombocytopenic dogs. medical records were evaluated retrospectively to identify dogs that received pc. information evaluated included patient characteristics, platelet count, acute transfusion reactions, and survival. twenty six dogs met study criteria. dogs receiving pc ranged in age from - years (mean . years) and / ( . %) were spayed or intact females. hemorrhage was reported in / dogs ( . %) prior to pc transfusion. platelet counts prior to transfusion ranged from to  /ul (mean . /À .  /ul). change in platelet count was measured in dogs and the mean change was . /À .  /ul. dose of pc administered ranged from . to ml/kg with a mean of . /À . ml/kg. no acute adverse reactions were reported. there was no correlation between transfusion dosage and platelet count change post transfusion. survival to discharge occurred in / ( . %) of dogs. the only variable correlated with survival was age with survivors being younger than non-survivors ( . years-old ae . vs. . years-old ae . .; p . ). efficacy of cryopreserved pc transfusions for improving clinical outcome in dogs with thrombocytopenia is yet to be determined; however, pc is well tolerated in clinical patients. fresh frozen plasma (ffp) is used to treat coagulopathies in dogs. current transfusion guidelines recommend that ffp be administered within hours of thawing to avoid decreasing clotting factor function and bacterial contamination. the purpose of this study was to assess clotting factor activity and bacterial contamination of ffp that had been thawed and refrigerated for days. blood was collected from client-owned healthy dogs with no known history of coagulopathy or of administration of drugs affecting coagulation. plasma was separated from whole blood and frozen (À c) within minutes of collection. thawed plasma was maintained at c ( /À c). aerobic and anaerobic bacterial culture, prothrombin time (pt), activated partial thromboplastin time (ptt), and factor ii, vii, ix, and x analyses were tested at time of whole blood collection, ffp thaw, hours post-thaw, hours post-thaw, and hours post-thaw. there were no statistically significant differences in pt and ptt at any of the measured time points. statistically significant differences occurred between initial measurements of factors ii, vii, ix, and x and subsequent time points, but there was no difference in activity levels of the factors once ffp was thawed. one bacterial colony was grown from each of two samples from post-thaw plasma. thawed plasma protocols do not significantly decrease the function of factors ii, vii, ix, and x or prolong pt and ptt. bacterial contamination of the plasma supply seems unlikely, but strict aseptic technique should be used when obtaining plasma for patient use. erythrocyte pyruvate kinase (pk) deficiency is the first and most common erythroenzymopathy described in dogs, cats, and humans. the pk enzyme plays a crucial role in the erythrocyte energy metabolism and its absence causes severe hemolytic anemia, often misdiagnosed as autoimmune hemolytic anemia. the disease is inherited as an autosomal recessive trait and affected dogs also develop osteosclerosis. in dogs, the enzymatic diagnosis is complicated by the anomalous expression of malfunctioning m -pk expression, but breed-specific r-pk mutation tests have been reported for basenjis, west highland white terriers (whwt), and beagles. we report here on a survey of canine pk deficiency studied at the penngen laboratory. a biased group of samples were received for screening from dog breeds with known mutations as well as from dogs with chronic, prednisone-and antibiotic-resistant hemolytic anemia and their relatives. edta blood samples and/or cheek swabs as well as medical record information were received and genomic dna and/ or enzyme activity testing were performed. among the whwts % and % were found to be homozygous deficient dogs or carriers, respectively, with a mutant allele frequency of . . the average age at the time of diagnosis was . years ranging from months to years of age; some samples came from europe and south america. of the beagles studied, % were affected and % were carriers (mutant allele frequency . ). the average age at the time of diagnosis was years ranging from months to years. surprisingly, very few samples from basenjis were received for screening, and none showed the mutant allele. while pk-deficient basenjis lived o years, whwt and beagles often show milder signs and can reach years of age. several dogs from other breeds were also examined because of chronic regenerative anemia and none had any of the known mutations seen in the other breeds. however, based upon pk enzyme activity studies, chihuahua, dachshund, miniature poodle, spitz, eskimo toy, and labrador retriever dogs were found to be affected; they also had osteosclerosis and at least one labrador retriever developed severe hemochromatosis (hepatic iron , ppm; normal o , ppm, analyzed on a dry weight basis). moreover, sequencing of the r-pk cdna from a pk-deficient labrador retriever revealed a new nonsense mutation in exon . in conclusion, pk deficiency appears to be a common cause for hemolytic anemia in certain breeds, and mutation testing makes screening simple. pk deficiency should also be considered in dogs of other breeds which may require the more cumbersome enzyme testing. studies to identify new mutations will confirm and simplify the diagnosis. supported in part by nih grant rr . immune-mediated hemolytic anemia (imha) is a common hematological condition observed in dogs. the diagnosis is based on clinical history, presenting signs and hematological evidence of imha such as regenerative anemia, leucocytosis and presence of spherocytes. the definitive diagnostic procedure is the coomb's test (direct antiglobulin test, dat) which is known to be highly specific but lacks diagnostic sensitivity. direct flow cytometric assay (fca) for igg, igm or c coated red blood cells (rbcs) detection might be more sensitive and thus could be introduced as an alternative diagnostic tool. to investigate the usefulness of fca for imha diagnosis, evaluation of igg, igm or c coated rbcs was performed from dogs presented at the veterinary hospital at usp that fulfilled clinical and hematological criteria for imha. thirty eight healthy dogs were included as controls. dat was performed with polyvalent and monovalent anti-dog sera with twofold serial dilutions of each one, incubated with % rbcs suspension at c and c. for fca, % rbcs from anemic and healthy dogs were incubated with fitc anti-dog igg, anti-dog igm and anti-dog c and submitted to flow cytometry evaluation. specific software and mann whitney u test were used for data analysis. five dogs showed positive results for dat with polyvalent coombs reagent at c (titer to ) and c (titer to ) but only three of them had agglutination titer for anti-igg at c ( to ) and c ( to ). no positive results were observed for anti-igm and anti-c dat. by fc, percentage of igg, igm and c coated rbcs in normal and anemic dogs were, respectively, , % and , % (p o , ); , % and , % (p o , ); , % and , % (p o , ). igg coated rbcs percentage were higher in dogs showing dat positive results (min. , %; max. , %; median , %). direct flow cytometric erythrocyte immunofluorescence assay is more sensitive than dat for detection of antibodies coated rbc in anemic dogs and may provide quantitative data useful for laboratorial diagnosis of imzha. bone marrow aspirates from cats are typically obtained from the ilium, humerus or femur, but may be difficult to obtain and/or of poor quality. in this study the feasibility, safety, and nature of sternal aspiration in cats was investigated. under general anesthesia, bone marrow aspirates were obtained in a randomized order by a single investigator from the sternum and ilium of healthy cats weighing . - . kg, with body condition scores of - (on a scale of - ). for sternal aspirates, cats were positioned in sternal recumbency and a -inch, - ga hypodermic needle attached to a cc syringe was inserted into the cranial manubrium and directed caudally along the long axis of the sternum. aspirates were also obtained from the right iliac crest using an ga illinois needle attached to a cc syringe. difficulty of site localization, needle insertion and advancement, and specimen aspiration, were scored from (easiest) to (hardest). bone marrow smears were prepared by one investigator and reviewed by a pathologist blinded to aspiration site and cat. sample quality was scored from (no marrow particles) to (excellent) based on the number of wellsmeared marrow particles on the slide. particle cellularity was scored from ( % fat) to (o % fat). post-procedure, cats were treated with tramadol ( - mg/kg, po, q h) for days, and assessed for post-biopsy pain (colorado state university feline acute pain scale, range [no pain] - [maximum]) and site swelling (range [none] - [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. pneumothorax was not identified. it was significantly easier to perform sternal than iliac aspiration, but the quality of the sample was significantly better for iliac than for sternal aspirates. because of limitations due to sample quality, bone marrow morphology in sternal samples could not be compared to iliac samples in all cats. for samples that could be compared, cellularity was identical for sternal and iliac samples from cat but underestimated in the sternal sample from another cat. myeloid:erythroid ratios and lymphocyte numbers were the same for sternal and iliac samples in and cats, respectively. megakaryocyte numbers were the same in one sample, less in sternal samples compared to iliac samples from cats, and overestimated in the sternal sample from cat. bone marrow cell morphology was normal in all acceptable samples. it was concluded that sternal aspiration of bone marrow using a - ga hypodermic needle is ) easier to perform than iliac aspiration; ) safe; but ) provides samples of lower quality than iliac aspiration in cats. the diameter of - ga jamshidi-type needles makes bone marrow core biopsy difficult in cats. in this study, biopsies of the left humeral head were taken under anesthesia using a -inch, ga needle (ez-io s intraosseous infusion system, vidacare) from healthy cats weighing . - . kg with body condition scores of - (on a scale of - ). humeral biopsies were compared to biopsies taken from the left iliac crest using a -inch, ga jamshidi needle. biopsies were performed in randomized order by one investigator. biopsy was repeated to a maximum of attempts until a specimen ! mm long was obtained. difficulty of site localization, needle insertion and needle advancement were scored from (easiest) to (hardest). specimens were wrapped in tissue paper and placed in davidson's fixative for min and then transferred to formalin. biopsy sections were reviewed by a pathologist blinded to biopsy site and cat. biopsy length on the slide was measured, and biopsy quality was scored from (no hematopoietic tissue) to (! intertrabecular spaces free of artifact). post-procedure, cats were treated with tramadol ( - mg/kg, po, q h) for days, and assessed for postbiopsy pain (colorado state university feline acute pain scale, range [no pain] - [maximum]) and swelling of biopsy sites (range [none] - [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. there were no significant differences between ga and ga biopsies except for post-biopsy swelling, and there were no significant effects of body weight and body condition. six ( %) of ga and ( %) of ga biopsies were considered acceptable specimens for assessment of bone marrow architecture and morphology; all intact spaces in these biopsies had normal hematopoiesis and cell morphology. comparison of acceptable ga to ga biopsy specimens from cats showed no significant differences for cell density and lymphocytes/plasma cells, while cellularity, assessed as high in of the ga biopsies, was assessed as medium in corresponding ga biopsies; and megakaryocytes, assessed as - /low-power field in one ga biopsy, were assessed as /low-power field in the ga biopsy. myeloid:erythroid ratios were greater in ga biopsies compared to ga biopsies in cats, and less in the ga biopsy in one cat. discordant results between biopsies were not related to differences in quality. in conclusion, ga bone marrow biopsy of the humerus was as likely to yield a specimen of acceptable quality as was ga biopsy of the ilium, and resulted in less post-biopsy swelling. reports on canine acute liver failure (alf) include individual or small case series of animals with a specific diagnosis. the aim of this study was to describe the clinical course, outcome and etiology of alf in dogs presenting to a referral hospital. medical records ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) were reviewed for a diagnosis of alf (elevated serum bilirubin or icterus with concurrent coagulopathy or hepatic encephalopathy (he)). fifty cases were identified representing breeds: labradors retrievers, golden retrievers, german shepherds, and cocker spaniels. median age was years ( m to humerus, ga . ae . . ae . . ae . . ae . à ( . - . ) ( - ) ( - ) ( - ) ilium, ga . ae . . ae . . ae . . ae . . ae . . ae . à ( . - . ) ( - ) ( - ) ( - ) ( - ) ( - ) yrs). presenting signs included anorexia ( / ), vomiting ( / ), polydipsia ( / ) and neurologic signs ( / granulomatous hepatitis (gh) is a histopathological diagnosis characterized by focal aggregations of activated macrophages mixed with other inflammatory cells that is usually part of a systemic disease process (wsava). published case reports describe many potential infectious causes, but only one retrospective study involving nine dogs with gh has detailed clinically relevant findings. the aims of this study were to describe the clinical and clinicopathologic findings in dogs with a histopathological diagnosis of gh, and to identify infectious agents using differential staining techniques, pcr, and fluorescence in-situ hybridization (fish) in archival paraffin-embedded tissues from dogs with gh. medical records of dogs with a histopathological diagnosis of gh (n ) were reviewed and signalment, historical toxin exposure or evidence of other systemic diseases, clinical signs, physical exam findings, clinicopathologic test results, imaging findings, concurrent diagnoses, treatments administered, and case outcome, when available, were extracted and summarized. twelve archival formalin-fixed, paraffin-embedded hepatic tissue samples were available for special staining and molecular diagnostic testing. two of these samples had sufficient tissue for only pcr. the mean age of dogs with gh was years (median . years; range to years) and included males and females representing different breeds. common presenting complaints included inappetance or anorexia (n ), weight loss (n ), lethargy (n ), fever (n ), and vomiting (n ). high mixed liver enzyme activity ( / ) was the most common clinicopathologic abnormality. leukemia was diagnosed in one dog and copper-associated hepatopathy in dogs. no infectious agents were identified using differential staining techniques. bartonella species dna was not pcr amplified from the extracted archival tissue. furthermore, no bacteria were identified by means of fish using a universal eubacterial probe. these data suggest a possible role for copper accumulation in the genesis of gh in dogs and support further evaluation of dogs with gh for evidence of copper-associated hepatopathy. future studies should include detailed environmental histories, the collection of adequate sample volumes for quantification of hepatic copper content and the examination of frozen tissues using novel molecular diagnostic platforms. hepatocyte copper and iron accumulation contribute to cell loss, inflammation, and fibrosis. the purpose of this study was to compare copper and iron accumulation in feline liver samples with different disease processes. liver biopsies (n ) submitted between july , and june , were evaluated using wsava guidelines and categorized as non-hepatic/normal, congenital, inflammatory/infectious, neoplastic, and other. copper (by rubeanic acid) and iron (by prussian blue) accumulation were graded by increasing amounts ( - ) and location (centrilobular cl, midzonal mz, periportal pp, random r). associations between metal scores and diagnosis category were assessed using the kruskal-wallis test. histologic diagnoses were non-hepatic/normal (n ), congenital (n ), neoplastic (n ), infectious/inflammatory (n ), and other (n ). ninety-two samples were negative for copper; remaining samples were graded (n ), (n ), and (n ). histologic diagnoses (pattern) for positive samples were congenital ( cl), infectious/inflammatory ( : cl, mz, pp, r), neoplastic ( pp), and other ( cl). iron staining was negative in samples; remaining samples were graded (n ), (n ), and (n ). distribution was primarily cl (n ) or r (n ), though mz (n ) and pp (n ) distribution occurred. there were no significant differences by kruskal-wallis analysis for amount or location of hepatocellular iron or copper for the different disease categories. in this study, copper accumulation was rare, had variable distribution and occurred primarily in samples with inflammatory/ infectious disease. in contrast, iron accumulation was common and did not correlate with disease category. further prospective evaluation of copper and iron accumulation in feline liver disease and association with outcome may be warranted. chronic hepatitis (ch) in dogs is a progressive condition without clearly defined treatment. glucocorticoids are commonly used to stop progressive inflammation and fibrosis but are associated with significant side effects including a steroid hepatopathy that complicates enzyme monitoring. cyclosporine is proposed as an alternative therapy, but there are no published reports of its use for canine ch. patient records at the csu veterinary teaching hospital were searched for histologically confirmed cases of ch treated with cyclosporine. data were compiled on cyclosporine dosing, concurrent medications, clinical course and biochemical parameters. patients over a -month period were identified. serum alanine aminotransferase (alt) decreased by an average of % in dogs. the alt normalized completely in of dogs treated for days. in of dogs on mg/kg/day, the alt also normalized. five of the patients that exhibited clinical signs prior to treatment showed measurable improvement (weight gain, fewer gastrointestinal signs). eight patients had hyperbilirubinemia or ascites prior to treatment; these resolved in . post-treatment histopathology, available in one patient, revealed decreased severity of ch. five patients exhibited adverse effects including gastrointestinal signs ( ), gingival hyperplasia ( ), and papillomatosis ( ). cyclosporine was discontinued in dogs with gastrointestinal signs. cyclosporine was an effective therapy for many cases of ch and should be considered for patients who are refractory to or cannot tolerate glucocorticoids. prospective clinical trials with histological documentation are needed to better define cyclosporine's effectiveness in ch. insertion of the veress needle and establishment of pneumoperitoneum is associated with to % of all laparoscopic complications in humans. the purpose of this study was to determine the accuracy of interpretation of tissue impedance measurements for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in cadaverous dogs euthanized for reasons unrelated to the study. placement order was randomized. a third individual evaluated impedance measurements using a handheld device (sensormed, knoxville tn) to determine correct versus incorrect needle placement. veress needle locations were marked using contrasting colors of india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified / correct and / incorrect placements, respectively. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. agreement was moderate (kappa . , p . ) for placements by operator and very high (kappa . , p o . ) for placements by operator . results for tissue impedance measurement interpretation are superior to published data for currently available tests. impedance measurements accurately detected all incorrect needle placements. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether it increases operator detection of inappropriate veress needle placement and decreases installment phase complication rates. delayed detection of intestinal perforation during veress needle insertion is associated with high mortality. the purpose of this study was to evaluate the accuracy of tissue impedance measurement interpretation for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in cadaverous cats. placement order was randomized. a third individual evaluated impedance measurements (sensormed, knoxville, tn) to determine placement location. needle locations were marked using india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified / correct and / incorrect placements. all undetected incorrect placements were located within the retroperitoneal fat pad. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. correlation was absent (kappa À . , p . ) for placements by operator and substantial (kappa . , p o . ) for operator . there was no association between correct or incorrect placement and operator on chi-squared analysis. failure of impedance measurements to identify placement in the retroperitoneal fat pad resulted in poor accuracy and discordant kappa statistics. small cat size limited the number of appropriate placement sites, perhaps resulting in excessively dorsal placement. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether impedance measurements increase detection of inappropriate veress needle placements or decrease installment phase complication rates. best clinicopathologic tests detecting portosystemic shunting (pss) in dogs remains controversial. this retrospective study examined performance of single random "fasting" and paired serum bile acids (sba; pre-and -hr post-feeding) in a large population of non-icteric dogs with confirmed pss (abdominal ultrasound, colorectal scintigraphy, radiographic or spiral-ct portography, laparotomy, or necropsy). sba were measured by enzymatic colorimetric method with normal o mmol/l. dogs meeting inclusion criteria (n ) included portosystemic vascular anomalies (psva; extrahepatic [e-psva], intrahepatic [i-psva]), and acquired pss (apss). signalment and laboratory parameters were recorded. non-parametric statistical analyses were used, two-tailed p o . applied with bonferroni corrections. median age and weight of breeds were . ( . - ) yrs and . ( . - ) kg, with equal gender distribution. random "fasting" sba detected % psva and % apss, whereas post-feeding sba detected % psva and % apss. low protein-c (o % activity) occurred in % psva and % apss. low mcv and creatinine occurred in % and % of psva dogs, respectively; other tests were less helpful. in apss, post-feeding sba was superior. compared to apss, psva had significantly (p . ) lower mcv, cholesterol, bun, creatinine, glucose, and protein-c. compared to e-psva, i-psva had significantly (p . ) lower post-feeding sba, mcv, albumin, urine specific gravity, and protein-c but higher cholesterol and glucose. post-feeding sba reflect physiologically provoked bile acid challenge and should be the preferred sba test in non-icteric dogs for pss detection. protein-c assists in identifying psva but its utility in apss may be complicated by concurrent coagulopathies and inflammation. this study compared outcomes of treatment with adjunctive nonsteroidal anti-inflammatory drugs (nsaids) or anti-inflammatory glucocorticoids in dogs with severe pulmonary blastomycosis. medical records were reviewed for dogs diagnosed with blastomycosis at the university of illinois veterinary teaching hospital between and . dogs with a presenting pao of mmhg, and clinical or radiographic signs of respiratory blastomycosis were included. all dogs were treated with either itraconazole, fluconazole, amphotericin b, or a combination of these. group (g ) dogs were treated with nsaids and group (g ) dogs were treated with glucocorticoids as anti-inflammatory adjunctive therapy. the following comparisons were made: days of oxygen supplementation, days in hospital, survival to discharge, and long term patient survival. mann-whitney u tests and chi-squared tests were performed on continuous and categorical data, respectively. p o . was considered significant. sixty-eight dogs fit the inclusion criteria. g consisted of dogs and g consisted of dogs. the two groups were found to be similar in weight, age, and sex distribution. there was no significant difference between the two groups with regard to duration of oxygen supplementation, duration of hospitalization, survival to discharge, and patient survival. there does not appear to be a difference between the clinical course or patient outcomes between groups of dogs with severe pulmonary blastomycosis treated with nsaids or anti-inflammatory glucocorticoids. further studies need to be performed to fully evaluate the impact these adjunct treatments have on prevention of ards and additional respiratory complications. diagnosis of feline histoplasma capsulatum infection traditionally relies upon identification of organisms in circulating monocytes or affected organs. in recent years, an antigen assay (aa) was developed for the diagnosis of disseminated histoplasmosis in human patients, but there is little information describing this test in cats. the goal of this study was to determine the sensitivity and specificity of h. capsulatum aa in cats with clinical disorders suggestive of histoplasmosis. urine and serum h. capsulatum aa results for feline patients from veterinary hospitals were evaluated. medical records were reviewed for confirmatory evidence of histoplasmosis (based on cytological or histopathological findings) or an appropriately supported alternate diagnosis. aa results were available for cats; initial testing was performed on urine samples, serum samples, and unspecified sample. of these cats, / had a definitive diagnosis of histoplasmosis based on organism identification, and had a definitive alternate diagnosis (e.g., neoplasia, other infection) based on necropsy findings (n ) or other clinical data (n ). an additional cats had a clinical alternate diagnosis with no cytological or histopathological evidence of histoplasmosis in the affected body system(s). the remaining cats had unverified histoplasmosis (n ) or an open diagnosis (n ). of the cats with confirmed histoplasmosis, were positive on initial urine aa. one cat (with rectal involvement) was negative, indicating a test sensitivity of %. one cat was positive on urine aa but negative on serum aa. all of the cats with definitive or clinical alternate diagnoses had negative results on the aa, suggesting an excellent specificity ( %). however, this result should be interpreted with caution, as the possibility of primary or concurrent histoplasmosis was only definitively excluded in the patients who underwent necropsy examination. these findings suggest that the aa for h. capsulatum is a reliable diagnostic tool in this species. a positive result appears to reliably support the presence of infection, but a small percentage of infected cats may be negative on aa. in addition, tests performed on urine may be more sensitive that those performed on serum. the purpose of this study was to evaluate the sensitivity and specificity of an aspergillus galactomannan antigen enzyme immunoassay (ga-eia) for the diagnosis of canine systemic aspergillosis. serum and urine samples were collected from sick dogs at hospitals (ucd and tamu). group dogs were diagnosed with systemic aspergillosis using culture (sterile site) or microscopy and culture (non-sterile site). group dogs had clinical findings suggestive of aspergillosis but an alternate diagnosis was established. group dogs were not suspected to have aspergillosis. samples were tested using the ga-eia and results expressed as a galactomannan index (gmi). gmis . were considered positive. comparisons were performed using the mann-whitney test. there were dogs in group , in group , and in group . serum was collected from all dogs, and urine from , , and dogs, respectively. serum gmis did not differ from urine gmis across groups. serum gmis of group dogs were higher than those of group and group dogs (p o . ). results from dogs in group did not differ from those in group (p . ). two dogs in group tested negative, but had localized pulmonary infections. one dog in group , which had paecilomycosis, tested positive. two dogs in group tested positive. one was being treated with plasmalyte. the other had a cutaneous opportunistic mycosis. these data support the utility of this assay to aid in the diagnosis of systemic aspergillosis in dogs. anaplasma phagocytophilum, an ixodes tick transmitted rickettsial bacterium has a wide mammalian host range that is not commonly reported in cats. clinical signs in humans, dogs and cats are often vague and include lethargy, anorexia and malaise. the purpose of this retrospective study was to describe the clinical signs, laboratory data and response to treatment in cats that tested positive for a. phagocytophilum on a commercially available pcr of peripheral blood (fastpanel tm ). this study describes and reports the appearance of intracellular morulae in feline neutrophils contributing to the diagnosis of a. phagocytophilum. the a. phagocytophilum real-time pcr (rt pcr) assay consists of four multiplexed primer systems designed to detect a total of three distinct genes. amplicons were confirmed as a. phagocytophilum by dna sequencing. clinicopathologic data was obtained by review of medical records and interview of primary veterinarians. complete blood counts were available from / cats and / blood smears were reviewed. the cats included in this study were all positive for a. phagocytophilum by real-time pcr. the cats ranged from months to years of age with an average age of . years. fifteen of cats had a history of tick exposure and lived in the northeastern region of the us, an ixodes endemic area. all cats presented with lethargy, / were anorexic and / had a fever (temperature o f). other clinical findings included hepatomegaly, splenomegaly, ataxia and ocular changes of conjunctivitis and elevation of the nictitating membrane. hematologic findings included leukopenia ( / ), neutropenia ( / ) and lymphopenia ( / ). thrombocytopenia was not noted in any case. morulae were seen within neutrophils in / cases. all cases in this report responded to treatment with doxycycline. this is the first report of the identification of morulae within neutrophils via peripheral blood smear review in cats confirmed by rt pcr to be infected with anaplasma phagocytophilum in north america. infection with anaplasma phagocytophilum should be considered in a clinically ill cat with tick exposure, living in an ixodes endemic area that presents to a veterinarian for lethargy, anorexia and fever. the spectrum of disease manifestations and the accompanying clinicopathological abnormalities indicative of bartonellosis in dogs have not been thoroughly characterized. the objective of this unmatched case-control study was to compare signalment, clinical and pathologic findings in clinically-ill dogs suspected of a tick-borne disease that were negative for bartonella sp. dna (controls) as were the dogs diagnosed with bartonellosis by pcr amplification, dna sequencing and the bapgm (bartonella alpha proteobacteria growth medium) enrichment culture approach. both groups were tested under the same laboratory conditions and in the same time frame. medical records were reviewed for information regarding signalment, medical history, physical examination findings, clinicopathological abnormalities, microbiological data and treatment. the study population consisted of bartonella-infected dogs and non-infected dogs. healthy dogs with no historical illnesses, such as blood donors, were excluded. the following species were amplified: b. henselae (n , . %), b. vinsonii subsp. berkhoffii (n , . %), b. koehlerae (n , . %), b. volans-like (n , . %), b. bovis (n , . %). nineteen ( . %) bartonella-infected dogs were febrile and lethargic and ten ( . %) had neurological signs. laboratory abnormalities for both groups are summarized below (number of affected dogs provided in parenthesis): multivariate logistic regression using confounding factors was performed to establish potential associations between specific variables and bartonella sp. infection. there were no differences in signalment, age, sex, body weight and duration of clinical signs between the two groups. compared to the control population, infection with the genus bartonella was associated with a diagnosis of endocarditis (p . , or . , %ci . - . ) and hypoglobulinemia (p . , or . , % ci . - . ). controls were more likely to have joint effusion (p . , or . , % ci . - . ) and azotemia (p . , or . , %ci . - . ) than were the bartonella sp. infected dogs. bartonella was detected in dogs with signs such as fever, anemia, thrombocytopenia, hyperglobulinemia and proteinuria that are typically associated with tick-borne diseases. when endocarditis or hypoglobulinemia are detected, testing for bartonella should be prioritized. likewise, the detection of bartonella should prompt further testing for endocarditis, if not already investigated. surveillance studies in other species depend on detection of antibodies to the highly conserved influenza a nucleoprotein (np); however, no such antibody detection assay is approved for canine use in the u.s. the purpose of this study was to determine the diagnostic accuracy of a commercial blocking elisa used for avian species in detecting influenza a np antibody in dogs. since the blocking elisa is not a species-specific or viral subtype-specific format, we hypothesized that it would detect np antibodies in dogs infected by influenza a virus. serum samples from uninfected dogs (n ) and dogs naturally infected with canine influenza h n (n ) were tested using the idexx flockchek blocking elisa for influenza a np antibody according to manufacturer instructions. the sample/negative control (s/n) absorbance ratios for infected dogs ranged from . to . compared to . to . for uninfected dogs. a receiver operating characteristic (roc) curve analysis determined optimum diagnostic sensitivity ( . %) and specificity ( . %) at a s/n cutoff ratio of . . using this cutoff ratio, the overall diagnostic accuracy was . %. coefficients of variation for intra-assay ( . %) and inter-assay ( . %) testing demonstrated good repeatability with canine sera. the excellent diagnostic accuracy of the commercial blocking elisa makes it a suitable tool for large-scale surveillance of influenza a virus exposure in dogs. upper respiratory disease (urd) can affect a majority of cats in shelters and is one of the leading reasons for euthanasia of otherwise adoptable cats. the purpose of this study was to determine prevalence and risk factors for upper respiratory pathogens in four different models for managing unowned cats: short-term animal shelters (shel), long-term sanctuaries (sanc), home-based foster care (fost), and trap-neuter-return (tnr) programs. conjunctival and oropharyngeal swabs were collected from cats, half of which had clinical signs of urd, and tested for feline herpesvirus (fhv), feline calicivirus (fcv), chlamydiophila felis, bordetella bronchiseptica (bb) and mycoplasma felis by real-time pcr. management model, vaccination, sex, age, body condition, and clinical signs were evaluated as risk factors for infection. a majority of cats in all management models carried one or more organisms capable of causing urd. in many cases, prevalence was similar in cats with or without clinical signs. unlike diseases that can be controlled by segregation of symptomatic animals, the lack of strong correlation between the presence of pathogens with the presence of clinical signs suggests that feline urd control should be managed by vaccination before or at the time of intake ,biosecurity protocols that presume all cats may be shedding pathogens, and minimizing stressful conditions that contribute to disease susceptibility. depending on geographical location, sex, age and environment, - % of cats worldwide are infected with the feline immunodeficiency virus (fiv). knowledge of the fiv status of cats is important to limit the spread of disease and to institute appropriate health management. however, like all lentiviruses, fiv is highly variable in nucleotide sequence, and viral load in cats is variable during different disease stages. detection of antibodies is the most widely employed diagnostic approach, but does not distinguish fiv-infected from fiv-vaccinated cats. in this study, samples from fiv-seronegative cats, fivseropositive cats, and fiv-historically seronegative but vaccinated cats, were analyzed by a commercial quantitative pcr (qpcr) assay and virus isolation. replicate blood samples were coded, and then submitted for ) qpcr (idexx); and ) mononuclear cell isolation with -day culture and viral p antigen detection by elisa. for the p antigen elisa, cutoff absorbance values were established from analysis of fiv-negative samples. fiv infection status was pre-determined based on antibody-elisa results and vaccination history. results indicated that qpcr had a sensitivity of % for samples from fiv-seropositive cats, and a specificity of % and . % for samples from fiv-seronegative and fiv-vaccinated cats, respectively. at a cutoff value of standard deviations above the mean absorbance for p antigen elisa, results from fiv-negative samples yielded a sensitivity of . % for samples from fiv-seropositive cats, and a specificity of . % and . % for samples from fiv-seronegative and fiv-vaccinated cats, respectively. conclusions from this study are ) the commercial fiv qpcr assay has high specificity but limited sensitivity for diagnosis of fiv infection; ) -day virus culture has limited sensitivity and specificity. hence, detection of antibodies remains the most reliable test for diagnosis of fiv infection, but qpcr may be suitable to rule out infection. oral disease is an important clinical problem in feline medicine and includes common painful conditions such as oropharyngeal inflammation (formerly known as gingivostomatitis) and tooth resorptive lesions. a number of infectious agents have been associated with private veterinary clinics in the u.s. were recruited to test feline patients presenting with oral disease. presenting cases included cats with plaque, calculus, gingivitis, stomatitis, periodontal disease, tooth resorptive lesions and other oral diseases as defined by the practitioner. all cats were tested using a commercially available point-of-care elisa test (idexx snap combo). confirmatory tests were not performed as part of the study. seroprevalence was calculated as the percentage of positive tests in the study population for each virus. a total of , cats were tested. seroprevalence for felv was . % and for fiv was . %. of these, cats ( . %) were infected with both viruses. seroprevalence was higher in cats with inflammatory oral disease than in cats characterized with other types of oral disease. of , cats with gingivitis, seroprevalence for felv was . % and for fiv was . %, with . % of cats co-infected. of , cats with stomatitis, seroprevalence for felv was . % and for fiv was . %, with . % of cats co-infected. the seroprevalence for felv and fiv reported in this population of cats with oral disease was higher than in a recent large study where samples from u.s. cats not specifically selected for oral disease were tested (felv . %, fiv . %). results of this study indicate that further investigation of the role of retroviruses in cats with oropharyngeal inflammation is warranted. reliable tests and preventive vaccines and medications for feline retroviral and heartworm (hw) infections are available, but compliance with protocols to reduce transmission is unknown. no largescale longitudinal studies evaluating prevalence over time have been reported. the purpose of this study was to determine the prevalence and risk factors for infection compared with a similar study completed for the first time years previously. veterinary clinics and animal shelters in the us and canada submitted results of testing using a point-of-care elisa for felv antigen, fiv antibody, and hw antigen (idexx snap triple) and risk factor information for cats tested during march-september . bivariable and multivariable analyses were used to evaluate risk factors for infections. a total of , cats were tested. only % of owned cats were prescribed hw preventive. risk of retroviral infections was increased by outdoor access, adulthood, and male gender. the most important risk factor associated with all infections was clinical disease; in particular, respiratory and oral diseases and abscesses or bite wounds. multivariate analysis revealed differences among geodivisions and across infection types. feline retroviral and heartworm infections are easily prevented, but difficult to treat. despite availability of effective management protocols, compliance remains inadequate to reduce the prevalence of these infections. improved use of preventive care and testing to identify and segregate contagious cats, particularly those at high-risk, is required to reduce the morbidity of these preventable infections. infectious disease outbreaks are common in animal shelters and are frequently managed by depopulation when risk-assessment tools are not available. during a canine distemper virus (cdv) and parvovirus (cpv) outbreak in sheltered dogs, we used a cdv/cpv point-of-care antibody titer elisa, a cdv quantitative rt-pcr test, and a cpv fecal antigen test as risk assessment tools to guide release of exposed dogs from quarantine and euthanasia of diseased dogs. serum samples (for antibody titers) and swabs of the conjunctiva and upper respiratory tract (for cdv pcr) were collected from asymptomatic dogs starting on day of the outbreak. dogs with positive cdv pcr tests were retested every weeks until euthanized for progressive disease or released following recovery from infection. dogs with clinical signs of parvoviral infection were tested using a cpv fecal antigen test. for dogs ! months old, protective antibody titers correlated with resistance to clinical disease, but % of dogs shed cdv. lack of protective cdv antibody titers correlated with susceptibility to clinical infection, but most dogs recovered. risk assessment and outcome in dogs ! months of age feline herpesvirus (fhv- ) is a common ocular and respiratory pathogen of cats that can have clinical illness exacerbated by stress. cyclosporine (csa) is commonly used for the treatment of a number of inflammatory diseases in cats and can induce immune suppression. a small number of cats administered csa to block renal transplant rejection have developed clinical signs of upper respiratory tract disease that may have been from activated fhv- . in this study, young adult cats experimentally inoculated with fhv- several months previously were divided into three groups and administered methylprednisolone acetate ( cats, mg/kg, im, day and day ), csa ( cats, . mg/kg, po, daily for days), or a placebo ( cats, corn syrup; . ml/kg, po, daily for days). each cat was assigned a daily individual clinical score by a trained, masked observer using a standardized score sheet during the initial pre-treatment time period (day - to day ) and throughout the day treatment period. each individual clinical score (conjunctivitis, blepharospasm, ocular discharge, sneezing, nasal discharge, nasal congestion, and body temperature scores), the total clinical score (sum of all parameters), the total ocular score (sum of conjunctivitis, blepharospasm, ocular discharge), and total respiratory score (sum of ocular discharge, sneezing, nasal discharge, nasal congestion) were analyzed using sas proc glimmix with 'treatment', 'time', and the two-way interaction 'treatment by time' all as fixed effects. statistical significance was defined as p o . . on day of the study, all of the csa treated cats had detectable concentrations of csa in serum (mean . ng/ml; standard deviation . ng/ml; median . ng/ml). when group mean values for clinical signs were compared over time as described, significant differences in individual clinical score measurements, in total score, total ocular score, or total respiratory score were not detected over time among any of the treatment groups. while clinical signs of activated fhv- occurred in some cats administered methylprednisolone or csa, disease was mild and self-limited in most cats and there were no significant csa sideeffects. these results suggest that the csa protocol described here is unlikely to reactivate latent fhv- infection and cause significant clinical illness. the purpose of this study was to determine the prevalence and risk factors for enteropathogens in four different models for managing unowned cats: short-term shelter, long-term sanctuary, home-based foster care, and trap-neuter-return (tnr) programs. fecal samples were collected from cats, half with diarrhea (d) and half with normal feces (n), and tested for a panel of feline and zoonotic enteropathogens by polymerase chain reaction, antigen, and fecal flotation. risk factors for infection evaluated include management practices, fecal consistency, and signalment. a majority of cats had at least one enteropathogen of feline or zoonotic importance, regardless of management model or preventive healthcare protocol. for most enteropathogens, the presence or absence of diarrhea did not correlate with infection, the exceptions being t. foetus in sanctuary cats and fcov in foster cats. prevalence of specific enteropathogens varied between management models, reflecting differences in preventive healthcare and housing conditions. management protocols for unowned cats were inadequate for elimination of infections present at the time of intake and for prevention of transmission of enteropathogens among shelter cats. improved compliance with effective vaccination, deworming, sanitation, and housing protocols is needed to reduce zoonotic and feline health risks. several allergic diseases of cats, including atopy and gingivostomatitis, can be resistant to glucocorticoids but responsive to cyclosporine. toxoplasma gondii infection occurs in approximately % of cats and the effect cyclosporine therapy has on the t. gondii oocyst shedding period is unknown. the objective of this study was to determine whether administration of cyclosporine before or after t. gondii infection influences the oocyst shedding period. the young adult cats were t. gondii seronegative when administered , t. gondii tissue cysts orally on day . group cats (n ) were never administered cyclosporine; group cats (n ) were administered cyclosporine ( . mg/kg, po) daily on days - ; and group cats (n ) were administered cyclosporine ( . mg/kg, po) daily from days - . available feces from individual cages were collected daily and fecal flotation by sugar centrifugation was performed for days after t. gondii inoculation. group shed oocysts for a significantly shorter period than groups or and had a significantly lower oocyst shedding scores than groups and on days - after t. gondii inoculation. group cats had completed the oocyst shedding period prior to being administered cyclosporine and repeat oocyst shedding was not detected during administration of the drug. administration of cyclosporine prior to t. gondii infection lessened oocyst shedding which is likely from the anti-t. gondii effects of the drug. administration of cyclosporine using this protocol is unlikely to induce repeat t. gondii oocyst shedding in client-owned cats. à group with diarrhea significantly different than group with normal feces p o . known about its metabolic pathways or mechanism of pathogenicity and whole genome sequencing of feline hemoplasmas has not yet been reported. the aim of this study was to completely sequence the genome of m. haemofelis to further characterise this important pathogen. mycoplasma haemofelis genomic dna was purified and subjected to whole shotgun roche sequencing. gaps were closed using targeted pcr and amplicon sequencing. ribosomal genes and potential open reading frames (orfs) were predicted in silico. putative orfs were annotated and orthologous groups identified. analysis showed a circular genome of . mbp with a gc content of . %. thirty-one transfer rnas (trnas) were identified, accounting for all amino acids, including a tryptophan trna for the opal codon (uga). of the , putative proteins identified, ( . %) matched to proteins from other bacterial species. in common with the pneumoniae group of mycoplasmas, the closest phylogenetic relatives of the hemoplasmas, genes involved in carbohydrate metabolism were limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source for m. haemofelis. the majority of the pentose phosphate pathway genes present in other cultivatable mycoplasmas appear to be incomplete or absent in m. haemofelis, suggesting an alternative mechanism for sourcing purine and pyramidine bases such as scavenging from the host. a gene encoding a glyceraldehyde- -phosphate dehydrogenase homolog of the immunogenic msg protein of mycoplasma suis was present. of the uncharacterized hypothetical proteins, , were arranged in series of orthologous repeats, or comprised fragments there-of, encoding putative proteins of approximately amino acids. the predicted motifs of the majority of these putative proteins were consistent with these proteins being presented on the cell surface; an n' terminal signal peptide or transmembrane region followed by a non-cytoplasmic tail. these data have provided valuable information as to why this pathogen remains highly fastidious; it lacks some of the metabolic pathways found in cultivatable mycoplasmas. we have also identified a homolog of a known m. suis immunogenic protein, and identified a potential mechanism for host immune system evasion by way of highly repetitive, putatively surface-expressed hypothetical proteins with variable sequences. canine leptospirosis has been recognized as a re-emerging disease in the u.s. over the past years, and several serosurveys of the prevalence of leptospiral antibodies in dogs have been published during that time. the role of cats in the epidemiology of leptospirosis has received little attention. serosurveys of cats for exposure to or infection with leptospires have been published from other geographic areas, but none for cats in the u.s. in the past four decades. the new england states have been found to have a high incidence of canine leptospirosis. the purpose of this pilot study was to determine the prevalence of leptospiral antibodies in a population of feral cats in central massachusetts. blood was collected from sexually intact feral cats presented to a spay and neuter program. microagglutination titers to leptospira serovars autumnalis, hardjo, bratislava, icterohaemorrhagiae, canicola, pomona, and grippotyphosa were determined. three of cats ( . %) had a positive titer to one or more serovars, with autumnalis being the most common. these results are consistent with previously published prevalence rates in feral cats. further studies are required to determine the role of leptosporosis in clinical disease in the domestic cat. since years the rivalta's test is routinely used in several european countries as a tool to diagnose feline infectious peritonitis (fip) in cats with effusion. it is inexpensive and easy to perform in private practice. there is, however, only little information about mode of action or its diagnostic value. the objectives of this study were to evaluate sensitivity, specificity, positive (ppv) and negative predic-tive values of the rivalta's test to diagnosis of fip and to examine if there is a correlation with any effusion or blood parameters. medical records of cats with effusion in which the rivalta's test was performed between and were reviewed concerning diagnosis, blood and effusion parameters, and survival time. effusion and blood parameters were compared between rivalta-positive and -negative effusions using the mann whitney u test. prevalence of fip in cats with effusion was . %. the rivalta's test showed a sensitivity of . %, a specificity of . %, a ppv of . %, and a npv of . % for the diagnosis of fip. the ppv improved, when cats with lymphoma or bacterial infection were excluded (ppv . %) and also, when only cats younger than years (ppv . %) or year (ppv . %) of age were included. the most important significantly different parameters between rivalta-positive and -negative effusions were specific gravity as well as cholesterol, triglyceride, and glucose concentration in the effusion. the rivalta's test in general is a useful tool to diagnose fip, but its sensitivity and specificity are not as high as previously assumed. if the rivalta's test, however, is performed in young cats or if certain diseases have been ruled-out, its diagnostic value is high. effusion total protein is not highly correlated with test outcome. therefore, it is still unclear, which components in the effusion of cats with fip lead to a positive rivalta's test. canine parvovirus (cpv) and canine distemper virus (cdv) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. our study aimed to determine the antibody protection status of dogs at the time of admission into an animal shelter (pre-vaccination) and over the following weeks after vaccination. serum samples were obtained from incoming shelter dogs aged months and older with no known history of vaccination. immediately following serum collection, the dogs were vaccinated against cpv and cdv using a modified live vaccine (mlv). cpv and cdv antibody protection status was determined using synbiotics titerchek. dogs with unprotective serum antibody levels against cpv and/or cdv were retested at - days post-vaccination and again at - days post-vaccination, if antibody levels were still unprotective against cpv and /or cdv. at the conclusion of the study, stored duplicate sera were submitted for batch 'gold standard' testing to determine canine distemper virus serum neutralization and canine parvovirus hemagglutination inhibition antibody titers. based on the synbiotics titerchek results, / dogs ( . %) were protected against cpv and / ( . %) were protected against cdv at intake. older incoming dogs were more likely to be protected against cpv (p o . ) and cdv (p . ). dogs that were spayed/neutered were more likely to be protected against cpv on intake than intact animals, although this result was not statistically significant (p . ). the number of dogs with protective titers against cpv/cdv was increased at - days post-mlv (cpv - / , . %; cdv - / , . %) and further increased at - days post-mlv (cpv - / , . %; cdv - / , . %). we conclude that incoming shelter dogs often do not have protective antibody titers against cpv and cdv, but older shelter dogs are more likely to be protected against cpv. based on this population, we further conclude that a large percentage of dogs develop protective antibody titers to cpv and cdv within to weeks when vaccinated with a mlv. mycoplasma spp. are common inhabitants of the feline oral cavity and so likely contaminate many cat bite abscesses. mycoplasma spp. are cell-wall deficient and so do not respond to beta-lactam class antibiotics, the class most commonly use for the treatment of cat bite abscesses. the objectives of this study was to determine whether mycoplasma spp. are common contaminants of cat bite abscesses and are associated with beta-lactam resistant clinical disease. privately owned cats with clinical evidence of an acute abscess suspected to be from a cat bite were included in the study. participants were given a free aerobic and anaerobic culture as well as mycoplasma spp. culture and polymerase chain reaction using mycoplasma genus specific primers. mycoplasma spp. amplicons were sequenced to determine the species. all cats were initially treated with appropriate wound management, were administered an antibiotic in the beta lactam class (amoxicillin-clavulanate or cefovicin), and were rechecked in person or by phone days after beginning treatment. of the cats entered into the study to date, mycoplasma spp. were amplified from cats ( . %). of the positive samples with adequate dna for sequencing, one was consistent with m. felis and the other was consistent with m. equigenitalium. of the cats, responded by day to the initial treatment, including of the mycoplasma spp. positive cats. the cat that failed initial treatment was positive for m. equigenitalium on both day and day and ultimately responded to administration of a fluoroquinolone. the results suggest that while mycoplasma spp. commonly contaminate cat bite abscesses, routine wound management and antibiotic therapy is adequate for control. however, as mycoplasma spp. infections do not respond to beta lactam class antibiotic therapy, these organisms should be on the differential list for cats with abscesses that fail treatment with this antibiotic class. molecular diagnostic assays are frequently used in clinical practice to aid in the diagnosis of suspected infectious respiratory diseases in dogs. however, most currently available assays cannot distinguish strains of the organisms used in vaccines from naturally occurring strains. our prior studies demonstrated that previously immune adult dogs are unlikely to shed nucleic acids of vaccine strains of adenovirus , parainfluenza, or bordetella bronchiseptica. however, whether this is true for puppies is unknown. puppies (n ) at a breeding facility were moved into area without other dogs at weeks of age. swabs of the nasal and pharyngeal mucosa were collected prior to vaccination and on days , , , , , , , , , , , , , and after vaccination with an intranasal adenovirus , parainfluenza, and b. bronchiseptica vaccine (intratrac , schering plough). the swabs were shipped on cold packs by overnight express for dna/rna extraction and assay in the fastpanel tm pcr canine respiratory disease profile at antech diagnostics. all puppies were negative for the infectious agents prior to vaccination. after vaccination, positive assay results for parainfluenza and b. bronchiseptica were first detected on day and on day for adenovirus . by day , dna or rna of the agents were amplified from all puppies from both sample sites and most samples were positive for all agents through day . by day , only one dog was still positive for b. bronchiseptica. the results indicate that intranasal administration of adenovirus , parainfluenza, or bordetella bronchiseptica vaccines commonly leads to positive molecular diagnostic assay results for a short time period after primary vaccination. these findings should be considered when assessing the results of these assays in client-owned puppies with respiratory disease. antimicrobial resistance in escherichia coli is an increasing concern in both human and veterinary hospitals' patients. the choice drug for treatment in dogs is enrofloxacin, a second generation fluoroquinolone (fq) whose activity reflects, in part, ciprofloxacin. among the difficulties in effective e. coli treatment is rapid detection of fq resistance. the purpose of this study was to determine the specificity and sensitivity of a fret based assay for the rapid detection of urinary tract infections caused by fq associated multi-drug resistant e.coli. clinical e. coli isolated from canine urine and clinical veterinary urine samples being examined for e. coli were subjected to susceptibility testing for drugs representing drug classes. pure isolates were designated ndr (no drug resistance), sdr (single drug resistance) and mdr (multi-drug resistant) (n mdr, sdr and ndr). minimum inhibitory concentration (mic) for enrofloxacin ranged from . mg/ml to mg/ml, with high mic generally associated with mdr. extracted dna from culture and from urine were subjected to fret-pcr targeting single nucleotide polymorphisms in gyra. the resulting product was sequenced to detect other polymorphisms. further, to determine the level of detection, microbial free canine urine was inoculated with to cfu/ml of isolates characterized by variable susceptibility to enrofloxacin (mic enro . , . , . , , , , mg/ml). of pure isolates, were confirmed positive for enrofloxacin resistance (mic enro mg/ml), of which were positively identified by the fret-pcr assay giving a sensitivity of . %. only isolate that was resistant was not detected (specificity of . %). however, of the isolates expressing high level resistance (mic x breakpoint [ mcg/ml]), and mdr (n ), sensitivity . %. of the urine samples contained e. coli of which determined to be fq-resistant by the assay. colony dilutions of e. coli confirmed the assay able to detect enrofloxacin resistance at as low as cfu/ml. the relationship between cfus and the peak of the -(d/dt) fluorescence of the melting curve was r . . these results conclude that the assay is capable of detecting not only the presence of escherichia coli in clinical samples, but also detecting severity of fluroquinolone resistance and infection. the fluoroquinolones (fqs) are a key class of synthetic antimicrobial agents with an established history in both humans and companion animals of efficacy for treatment of urinary tract infections (utis) caused by e. coli, and fluoroquinolones are common therapy. among the commonly used fqs in dogs and cats are the nd generation drugs, enrofloxacin, marbofloxacin, orbifloxacin (all veterinary approved) and the human drug ciprofloxacin; no rd and th generation fq is routinely used. the purpose of this study was to assess the in vitro activity of different generation fqs toward e.coli uropathogens whose phenotype ranges from no resistance to multidrug resistance. a total number of canine uropathogenic canine or feline e.coli isolates had been subjected to susceptibility testing to drugs classes ( drugs) and phenotyped as to resistance: none (ndr, n ), single (sdr, n ), or multiple, mdr (resistance to - drug classes; n ). mdr included isolates susceptible (enr s -mdr, n ) or resistant (enr r -mdr) to enrofloxacin. the minimum inhibition concentrations (mics) for quinolones ( - st generation, - nd generation, - rd generation and - th generation) were determined for these isolates using broth microdilution methods according to clsi guidelines (e. coli atcc s served as a negative control). mic statistics were generated for each drug among phenotypes. the results showed that companion animal e. coli expressing ndr or sdr are largely susceptible to nd to th generation fqs. however, isolates expressing resistance to st or nd generation quinolone also express high level resistance based on the mic to rd and th generation fqs. the overall potency (mic) for the drugs for isolates not expressing enr resistance (that is, ndr, sdr and enr s -mdr) is gat canine leproid granuloma (clg) was first reported in brazil in . over the past years, cases of clg were diagnosed in sa˜o paulo, brazil, and clinical and epidemiological findings were similar to those reported in australia. all dogs presented with one or more, uni or bilateral, ulcerated or not, papular, nodular or tumoral lesions, mainly observed in the dorsal surface of the ear, site usually more affected. in general, the lesions are painless and confined to the subcutis and skin, and it does not involve regional lymph nodes, nerves or internal organs, and systemic clinical signs frequently are absent. short-coated breeds show a marked predisposition for this disease. the definitive diagnosis of clg was obtained by histological examination of skin biopsies that were stained with acid fast (ziehl-neelsen) and diffquik s . thirty one ( . %) of the dogs were purebred; in this study the breed pattern comprised ( . %) boxers, ( . %) german shepherd and labrador retriever, ( . %) dobermann, ( . %) brazilian terrier, ( . %) golden retriever, ( . %) bulldog, ( . %) american pitbull, ( . %) mastiff, ( . %) fila brasileiro and ( . %) cocker spaniel, ( . %) were of unknown breed. nineteen ( . %) of the thirty seven dogs were males. twenty ( . %) dogs were - years old. in most cases, dogs presented with unilateral or bilateral ear lesions, but rarely thoracic, foot and caudal lesions. the animals were successfully treated by use of rifampicin orally (''the brazilian protocol'') or enrofloxacin orally and topical rifamicin. anaplasma phagocytophilum is being recognized more frequently in dogs in endemic areas. currently, most suspected cases are evaluated for a. phagocytophilum antibodies by immunofluorescence assay (ifa) or elisa. since a. phagocytophilum is an acute disease, detection by antibody measurement may be negative on initial evaluation. it is possible that a. phagocytophilum dna can be amplified from blood or synovial fluid prior to seroconversion. wild caught ixodes scapularis adult ticks from rhode island were allowed to feed on young adult ( - years), mixed sex beagles for up to days. blood (weekly for weeks), serum (weekly for weeks), and synovial fluid (radiocarpal joint; alternating arthrocentesis weekly for weeks) were collected prior to tick attachment and then weekly after tick attachment. joint fluid cytology was performed and total dna was extracted from blood and synovial fluid and assayed in a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii. serum was assayed for a. phagocytophilum antibodies by ifa. time to first positive results for serology and pcr were compared by paired student's t test. none of the beagles developed clinical evidence of disease, and no major changes in synovial fluid cytology were detected over time. of the beagles, were positive for a. phagocytophilum dna in blood or synovial fluid or ifa antibodies in at least one sample after tick attachment. antibody titers appeared in of dogs from weeks to (median to st positive weeks ae ). titer magnitude ranged from : to : , . anaplasma phagocytophilum dna was amplified from the blood of of dogs with positive test results ranging from to weeks (median to st positive weeks ae . ). anaplasma phagocytophilum dna was amplified from synovial fluid from of dogs between weeks to (median to st positive weeks ae ). of the dogs, were pcr positive for only one week and dog was pcr positive for two consecutive weeks. of the dogs, were positive for a. phagocytophilum in both blood and joints by dna analysis. anaplasma phagocytophilum dna was amplified from blood more quickly than seroconversion was detected by ifa antibody titer (t À . , p o . ) or dna was amplified from synovial fluid (t . , p o . ). anaplasma phagocytophilum dna can be amplified from the blood prior to development of detectable antibody titers by ifa. amplification of a. phagocytophilum dna from synovial fluid does not occur in all dogs, appears to be transient in most dogs, and a negative test result does not preclude a diagnosis of a. phagocytophilum infection. canine granulocytic anaplasmosis and granulocytic ehrlichiosis are tick-transmitted infections caused by anaplasma phagocytophilum (aph) and ehrlichia ewingii (eew), respectively. both organisms induce an acute clinical disease, frequently accompanied by fever, polyarthropathy and thrombocytopenia. however, aph and eew have different tick vectors, i.e. ixodes scapularis and ambylomma americanum, respectively, with different, but overlapping geographic distributions. in addition, infection outcome may be affected by other regional ticktransmitted pathogens, such as borrelia burgdorferi (mn) or ehrlichia chaffeensis (ar). therefore, we compared serology and pcr results derived from dogs examined at two private practices located in highly endemic areas for either aph or eew. serum collected between april-december, from minnesota dogs (n ) was tested by snap s dx s and whole blood was tested by aph pcr. serum collected from arkansas dogs (n ) for year beginning in august was tested using microtiter plate elisas for antibodies to eew, e. canis, and e. chaffeensis (ech) while whole blood was tested by ehrlichia pcr. comparisons were evaluated using chi square (Ã) and binomial (w) tests with an alpha of %. the above results indicated that dogs are frequently exposed to both aph and bb in mn, whereas ar dogs are often exposed to eew, but less frequently to ech. antibodies to e. canis peptides were found infrequently in both mn and ar with only seroreactive dogs detected in both locations. active eew infection, as determined by pcr, was four times more frequent in ar pet dog seroreactors as compared to active aph infections among aph seroreactors. although both organisms induce acute disease, the number of aph and eew pcr positive dogs that were also seropositive was relatively high suggesting that both organisms induce persistent infections or that dogs are frequently re-infected, despite the presence of a measurable humoral immune response. additional studies are needed to determine regional infection profiles in other areas that are endemic for these pathogens. anaplasma phagocytophilum and ehrlichia canis are two of the most common vector borne disease agents that infect dogs and cats. while pcr assays that amplify the dna of these agents from blood are currently available, there is minimal information concerning the performance of these assays in different commercial laboratories that utilize different techniques. the purpose of this study was to compare the e. canis and a. phagocytophilum results of two different laboratories on the same samples collected from client-owned animals. veterinarians in states (az, md, ct) were recruited to participate in the study based on high prevalence rates for e. canis or a. phagocytophilum infection. blood in edta was collected from dogs or cats with fever, thrombocytopenia, or clinical evidence of polyarthritis and an equal volume of the same blood sample was simultaneously shipped on cold packs by overnight express to colorado state and to antech diagnostics. standard operating procedures at each laboratory were followed for total dna extraction and amplification of gapdh as the dna control. at colorado state university, a previously published pcr assay that amplifies the dna of ehrlichia spp., anaplasma spp., neorickettsia spp., and wolbachia was performed on each sample with positive amplicons sequenced to determine the species. at antech diagnostics, a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii was performed. in the study to date, samples from animals ( dogs and cats) have been assayed at both laboratories. dna of a. phagocytophilum ( cats and dogs) and e. canis ( dog) were amplified at both laboratories with a percentage agreement between laboratories of %. the results to date suggest that the assay results of the two laboratories for a. phagocytophilum and e. canis are comparable. ehrlichiosis and bartonellosis are zoonotic diseases caused by extremely small, obligate intracellular bacteria that require a mammalian reservoir and a blood sucking arthropod vector. human ehrlichiosis is present in peru, with a seroprevalence as high as % in the highlands. bartonella species in humans were also identified in peru since (b. bacilliformis). recently, a new species (b. rochalimae) was isolated from an american woman who became febrile after travelling to peru. dogs can become infected with the same ehrlichia species, and the majority of bartonella species that affect human beings. the role of dogs as reservoirs for human infections has not been clearly established, but exposure and/or infection in dogs has been used to monitor human exposure to tick-borne disease (tbd), since they share the same environment. the objective of this study was to determine the serological and molecular prevalence of anaplasmosis, ehrlichiosis and bartonellosis in rural dogs in the highlands of peru. a total of healthy adult dogs were enrolled in this study from four communities in the central highlands of peru: ondores, pachacayo, san juan de pachayo, and canchayllo. edta-blood samples were collected from dogs, whereas serum samples were available from dogs. serum samples were tested for ehrlichia canis, anaplasma, borrelia burgdorferi and dirofilaria immitis infections using a qualitative dot-elisa (snap s dx). the edta-blood samples were screened by conventional pcr for the groel gene of the genus anaplasma and ehrlichia, and for the intergenic transcribed spacer of the genus bartonella. speciation was conducted by nucleotide sequencing. bartonella genus dna was detected from seven of the dogs ( . %) and ehrlichia canis dna was detected and sequenced from one dog ( . %). four of the bartonella positive samples were identified by dna sequencing as b. rochalimae (genbank accession numbers hq and hq ). the other three bartonella positive samples were identified as b. vinsonii subspecies berkhoffii, the causative agent of endocarditis in dogs and humans. no dog was infected with anaplasma species by dna amplification, but one dog was seroreactive for this genus ( . %). no specific antibodies against ehrlichia canis and borrelia burgdorferi and no antigens of dirofilaria immitis were detected. this study expands the current knowledge about tbd in peru and describes for the first time the infection of b. rochalimae in dogs in peru. the results suggest that dogs may play an important role in the epidemiology of this infection in humans, since they can be asymptomatic but bacteremic. bartonella spp. dna is commonly amplified from the blood of cats exposed to ctenocephalides felis. in previous work, it was shown that cats administered imidacloprid and experimentally exposed to b. henselae infected cats and c. felis did not become pcr positive for b. henselae whereas untreated cats all developed infection. the purpose of this study was to determine if administration of imidacloprid to clientowned cats likely to be exposed to bartonella spp. and c. felis in the field lessens prevalence of bartonella spp. infection. veterinary students in tennessee and florida that owned cats that spent at least days per month outside and that were willing to apply imidacloprid to their cats monthly for six months were recruited for the study. blood for bartonella spp. pcr assay was collected from the cats seven months after starting imidacloprid administration and assayed at colorado state university. to serve as a control group that was unlikely to have been administered flea control products in the previous months, blood was collected from feral cats during tnr programs in each of the two cities and assayed for bartonella spp. dna. the bartonella spp. dna prevalence rates between the groups were compared by chi square analysis with significance defined as p o . . the overall prevalence rates for bartonella spp. dna in the blood of veterinary student cats ( . %) and the feral cats ( . %) were significantly different (p o . ). the distribution of results is shown in table . the results suggest that florida feral cats were more commonly exposed to c. felis than tennessee feral cats. while the cats in the groups were not exactly matched, the student cats were allowed outdoors for approximately days per month and lived in the same cities as the feral cats, so c. felis exposure rates were likely similar. as previously shown in experimentally-exposed cats, the use of imidacloprid monthly may influence transmission rates of bartonella spp. amongst naturally-exposed cats. in an endemic area for leishmaniosis and filariosis, coinfection can occur and immunomodulation produced by wolbachia might influence the clinical signs and progression of both diseases. the aims of the present study were ) to determine the prevalence of wolbachia in dogs infected with dirofilaria immitis (di) and other filarial nematodes, ) to evaluate the level of coinfection of leishmaniosis and filariosis by molecular assays and ) to evaluate any associations between leishmania infantum (li) infection, filariosis with or without wolbachia and clinical presentation and outcome. statistical differences between groups were tested for significance by the fisher exact test using spss v. . software (significance: p-value o . ). one-hundred and eighteen owned dogs from southeastern spain presenting for clinical evaluation were included in the study. criteria the results of this study highlight the increased sensitivity of pcr for diagnosis of filariosis, confirm the presence of wolbachia in dogs from the mediterranean basin, show the increased severity of hwd when li-filaria coinfection is present and suggest that wolbachia could play a protective role for leishmaniosis. wolbachia antigens can stimulate a th -type immune response, as has been previously described. however other factors (as treatment with doxycicline) might be responsible for the lower prevalence of wolbachia among filaremic dogs infected with li and further studies must be done to clarify this interaction. the purpose of the present study was investigate the occurrence of leishmaniasis in cats in the municipality of arac¸atuba, sa˜o paulo, brazil, an endemic area for canine visceral leishmaniasis. animals were evaluated by direct parasitological examination of lymphoid organs and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). thirteen ( . %) out of cats studied were diagnosed with visceral leishmaniasis; eight ( %) by parasitological diagnosis through cytological examination of lymphoid organs, six ( %) were considered positive by elisa and one ( . %) by ifat. only two ( . %) out of the thirteen infected cats had clinical signs, characterized by the presence of crusty lesions on the dorsal cervical region and hepatosplenomegaly. regarding age five cats ( . %) had between six months and two years, being the others older than years ( . %). only one cat ( . %) was positive for the three employed methods. pcr confirmed leishmania sp infection in nine ( . %) cats, of which six were diagnosed previously by cytological examination, two by elisa and one by the three techniques employed. since its first description in feline leishmaniosis has been reported in several countries. the purpose of this study was to assess the prevalence of leishmania chagasi infection in cats showing dermatologic lesions from an endemic area for visceral leishmaniasis in brazil. animals were evaluated by direct parasitological examination of lymphoid organs, immunohistochemical technique for detection of amastigotes in lesioned skin and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). twenty seven ( . %) out of the cats studied were diagnosed with visceral leishmaniosis. twelve ( . %) were positive by parasitological diagnosis; amastigote forms of leishmania sp were identified in lymphoid organs from / ( . %) infected cats, and immunohistochemical technique allowed the identification of nine ( . %) positive animals. the seroprevalence of leishmaniosis was . % ( / ) by elisa and . % ( / ) by ifat. fiv specific antibodies were found in / cats ( . %), of which / ( . %) had leishmaniosis. real time pcr confirmed leishmania chagasi infection in three cats. based on the evidence of the high occurrence of leishmaniosis in cats in this study, this disease should be included in the differential diagnosis of skin diseases of felines living in endemic areas. blastomyces dermatiditis is a dimorphic fungus that commonly affects large-breed hunting dogs. a recent advancement in diagnosis has come with the advent of a urine antigen screening test that has both high sensitivity and moderately high specificity. therapy for the disease involves use of antifungal agents, usually itraconazole, and length of treatment is based chiefly on resolution of clinical and radiologic signs. with the new urine antigen test, however, a noninvasive route of monitoring treatment progress is available and could be an adjunct device utilized to determine treatment efficacy and may even reveal a need for prolonged treatment. therefore, the purpose of this study was to determine if monitoring the blastomyces urine antigen test and comparing to pulmonary radiographic signs would elucidate the necessity for prolonged antifungal therapy, even after resolution of radiologic signs. to this end, a retrospective case review was performed that identified a series of client-owned animals with naturally occurring blastomycosis. the inclusion criteria were radiographic pulmonary parenchymal signs consistent with fungal disease and urine antigenconfirmed blastomycosis with repeated testing of both radiographs and urine antigen quantification as monitoring parameters until negative results achieved in each. ideally, intervals between testing dates would be between two and five months. radiographs were considered negative if all radiographic changes had resolved or if repeated radiographs separated by at least one month were considered static after documented improvement had occurred from original diagnostic radiographs (suspected scarring). urine antigen testing was considered negative if concentrations were less than . enzyme immunoassay units, a reference interval set by the testing laboratory. preliminary data analysis reveals resolution of radiographic signs of blastomycosis occurred earlier in many of the cases presented than did attaining a negative urine antigen concentration. ceasing treatment month after radiographic resolution of signs as has been recommended in the past might have resulted in premature discontinuation of therapy in many of the cases. monitoring of urine antigen concentrations may be of additional clinical use for determining when cessation of treatment should occur in cases of blastomycosis. persistent elevation of urine antigen concentrations after radiographic resolution of infection may account for apparent recrudescence of blastomycosis after suspected clinical resolution. giardia spp. and cryptosporidium spp. are both known to cause infections in dogs and humans in the united states. nevertheless, prevalence rates for dual infection in dogs had not been widely reported. in this study, fecal samples from dogs housed in a northern colorado animal shelter (n ), dogs owned by veterinary students in northern colorado (n ), and dogs from the pine ridge reservation in south dakota (n ) were collected. each sample was assayed with a commercially available fluorescent antibody assay that detects giardia spp. cysts and cryptosporidium spp. oocysts. those samples that were positive for giardia spp. or cryptosporidium spp. with adequate dna available for sequencing were genotyped by the glutamate dehydrogenase [gdh] and by the heat shock protein- [hsp- ] genes, respectively. overall, ( . %) of the dogs had current evidence of a protozoal infection ( table ). the dogs from pine ridge reservation had the highest prevalence rates for giardia infection and also for dual infections. from the student dogs, sequencing was successful for the three giardia isolates (assemblage d from dogs; assemblage c from one dog) and one cryptosporidium isolate (c. canis). from the reservation dogs, sequencing was successful for nine giardia isolates (assemblage d from dogs; assemblage c from dogs) and one cryptosporidium isolate (c. canis). cryptosporidium and giardia co-infections are commonly detected in dogs; in this study dual infections were more common than cryptosporidium infections alone. further studies will be required to determine the clinical importance of this finding. although the giardia and cryptosporidium isolates that were sequenced were the dog specific assemblages/genotypes, more samples should be analyzed in order to assess the potential for zoonotic transmission of either parasite. the current study was conducted to determine the prevalence of intestinal parasites in dogs visiting the veterinary teaching hospital, chiang mai university, northern thailand. fecal samples (n ) were collected and submitted by owners between august to february . demographic and geographic data were recorded. intestinal parasitic infection was diagnosed by both microscopic examination after zinc sulfate centrifugation flotation and commercially available ifa for giardia spp. and cryptosporidium spp. polymerase chain reaction and dna sequencing were performed on all giardia and cryptosporidium positive samples to provide genotyic information. overall prevalence of intestinal parasitic infection in dogs in chiang mai was . %. the most prevalent parasite was giardia spp. ( . %) followed by ancylostoma spp. ( . %), cryptosporidium spp. ( . %), cystoisospora spp. ( . %), toxocara canis ( . %), trichuris vulpis ( . %), coccidian-like ( . %), toxascaris leonina ( . %), and strongyloides spp. ( . %). the prevalence of having at least one parasite in dogs o year, - years, and years were . %, . %, and . %, respectively. of these infected dogs, . %, . %, . %, and . % were infected with one, two, three, and four organisms, respectively. available dna sequences from giardia spp. positive samples were shown to be dog specific. only one adequate dna sequence was available for cryptosporidium spp., which was shown to be c. canis. the findings suggested that intestinal parasitic infection was common in dogs in chiang mai, thailand. dogs could be potential source for zoonotic intestinal parasitic infection since dogs in this area are allowed for free roaming. regular deworming program is indicated to prevent not only transmission among dogs but also to human. a retrospective study was conducted on parasite positive fecal specimens consisting of canine, feline, equine and from other host species, comparing recovery of eggs, protozoan cysts and coccidian oocysts using standardized methods of parasite concentration: the formalin/ethyl acetate (f/ea) sedimentation concentration and the commercial fecalyzer (flotation) kit procedures. specimens were processed by each technique either according to manufacturer's instructions or according to standard laboratory procedures. formalin/ethyl acetate concentrations used at a ratio of ml normal saline to ml ethyl acetate for extraction of lipophilic material from pelleted stool samples, previously fixed in sodium acetate/acetic/acid/formalin (saf) solution. flotations with the fecalyzer kit were performed with concentrated zinc sulfate solution (s. g. . ) . the range of parasites recovered from these specimens included flagellate cysts ( total), coccidian oocysts ( total), ova and larvae of nematodes ( total), and ova of trematodes ( total) , and cestodes ( total). recovery rates by fecalyzer flotation were good for protozoan cysts, coccidian oocysts and nematode eggs and larvae, but very poor for cestode and trematode eggs. formalin/ethyl acetate concentration showed excellent recovery of all parasites and consistently outperformed fecalyzer in recovery rates. recoveries by f/ea concentrations were higher by . % for giardia, by . % for coccidia and by . % for nematode eggs and larvae. with the exception of coccidian oocysts, based on z-test analyses, recovery rates were significantly higher, at a confidence level of at least %, for all parasites, using formalin/ethyl acetate sedimentation concentration. although capc recommends the use of flotation with centrifugation methods for standard fecal ova and parasite examination for veterinary patients, sedimentation concentration methods are widely and effectively used in human diagnostic parasitology laboratories. these results provide good evidence for the use of f/ea concentration as a preferred method to flotation procedures for stool ova and parasite examinations in veterinary laboratories. cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases. cyclosporine inhibits calcineurin-dependent pathways of t-cell activation and the resultant cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. little work has been done comparing the effects of these agents on cytokine production in dogs. our study assessed these effects by measuring t-cell cytokine production using flow cytometry, and cytokine gene expression using quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) in activated canine t-cells treated with cyclosporine and dexamethasone. for flow cytometric assays, peripheral blood mononuclear cells were separated using density gradients and cultured for hours in the presence of cyclosporine ( , , or ng/ml), dexamethasone ( À , À , À m), or cyclosporine plus dexamethasone. for qrt-pcr, whole blood was cultured for hours with the same drugs at the same concentrations, and rna was then extracted from leukocytes. expression of cytokines il- and ifn-g was analyzed in pma/ionomycinactivated t-cells by flow cytometry, and gene expression for il- and ifn-g in activated t-cell populations was assessed via qrt-pcr. flow cytometry and qrt-pcr both demonstrated inhibition of il- and ifn-g that was generally dose-dependent in response to both cyclosporine and dexamethasone. flow cytometry results from the average of samples collected from different dogs are shown in figure a . similar results were achieved using qrt-pcr ( figure b ). suppression of il- and ifn-g in activated t-cells has potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine t-cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients. idiopathic eosinophilic diseases are described in several breeds, but are over represented in rottweilers. the immunopathogenesis of idiopathic eosinophilic disorders is poorly characterised. studies in people highlight the importance of cytokines, particularly interleukin- (il- ), in mediating eosinophil maturation, differentiation, egress from the bone marrow, migration and polyclonal expansion. eotaxin- and eotaxin- also appear important for induction of chemotaxis and release of reactive oxygen species from eosinophils. the aim of the current study was to establish whether definable differences in specific cytokines associated with mediation of eosinophil production and survival are present between healthy rottweilers, non-rottweilers and rottweilers with non-parasitic eosinophilia. secondly, by evaluating cytokine profiles the study aimed to improve understanding of the pathophysiology of eosinophilia therefore assisting development of potential molecular treatment options. quantitative real-time reverse transcriptase polymerase chain reaction (qrt-pcr) assays were used to quantify messenger rna (mrna) encoding cytokines il- , il- , il- , il- p , il- p , il- p , il- , interferon gamma (ifn-g) and chemokines eotaxin- and eotaxin- from peripheral blood mononuclear cell (pmbc) samples obtained from healthy non-rottweiler dogs with normal eosinophil counts (n ) and rottweilers with normal (n ), mildly increased (n ) and high (n ) eosinophil counts. quantification of serum ifn-g was also performed using a commercially available canine-specific elisa. all samples were positive for housekeeping genes and all cytokines could be quantified with the exception of eotaxin- and - . results were normalised using three stably expressed housekeeper genes (rpl a, sdha and ywaz) and a relative copy number was calculated for each sample with the sample with the fewest copies given a value of . no significant differences were found between groups but there was a tendency for ifn-g mrna expression to be lower in the rottweilers with moderate to severe eosinophilia versus control dogs (p . ). this trend was not seen in the concentration of serum ifn-g quantified by elisa as there were no significant differences between normal and diseased animals. in conclusion, there were no significant differences in cytokine mrna profiles between normal dogs and rottweilers with varying degrees of eosinophilia. additional studies including larger numbers of affected dogs are warranted before any accurate conclusions can be made. the presence of large amount of antibody on erythrocyte membrane can accelerate red blood cell (rbc) removal process by the mononuclear phagocyte system. an antigenic stimulus such as the one promoted by vaccines, for example, can induce hypersensitivity reactions and may accelerate rbc destruction. the study objective was to evaluate the erythrocytic membrane potential in inducing lymphocyte proliferative response of recently immunized dogs. healthy adult dogs (n ) were immunized with multiple antigens (commercial vaccine with eight antigens: distemper virus, parvovirus, coronavirus, parainfluenza virus, adenovirus, infectious hepatitis virus and leptospire; and anti-rabies). blood samples from each animal were collected into edta tubes in two moments: pre (immediately before vaccination) and pos ( to days after vaccination). mononuclear cells were separated by gradient, marked with cfse-fitc and cultured. the stimuli for lymphocyte proliferation used were autologous erythrocytic membrane (aem) and concanavalin a (cona). aem was obtained by hypotonic lysis and tested in two concentrations (m : . ug/ ul; m : . ug/ ul). the proliferation assay was evaluated by flow cytometry and analyzed with specific software. the proliferation index (pi) was calculated dividing the fluorescence intensity of the basal sample by the stimulated one. statistical analysis was performed using paired t-test for parametric samples and wilcoxon test for non-parametric samples (a . ). the for the tested concentrations, autologous erythrocytic membrane does not constitute a stimulus for lymphocyte proliferation in vitro, either before or after vaccination procedure. additionally, there was no evidence of self-reagent lymphocytes to erythrocyte membrane after vaccination. e. coli is a common cause of canine urinary tract infection. current treatment emphasizes eradication of established infection rather than infection prevention but increased antibiotic resistance necessitates strategies to prevent infection. proanthocyanidins found in cranberry juice inhibit e. coli attachment to human uroepithelial cells, impairing bacterial adherence and colonization. we hypothesized that purified cranberry extract (ce) inhibits bacterial adhesion to canine uroepithelial cells. five healthy female dogs received an oral ce supplement (vetoquinol; mg ce/tablet) according to body weight for days. voided urine collected from each dog before (pre) and after ( -day) completion of the protocol was membrane filtered ( mm) and stored frozen (- c). bacterial adhesion was determined using an in vitro assay. briefly, urine samples were incubated with an uropathogenic e. coli strain that had been subcultured to promote fimbriae expression. urine samples containing e. coli were next incubated in -well plates containing methanol-fixed madin-darby canine kidney (mdck) cells for -hr ( c) to permit bacterial attachment. after incubation, plates were washed to remove nonadherent bacteria and fresh media added. plates were incubated ( c) for -hr to grow attached bacteria to detection level. bacterial concentration in each well was determined using a spectrophotometer ( nm). results were analyzed using the chi-square test. ce significantly reduced bacterial adhesion by % (n ; p . ) in -day urine samples compared with pre samples. the results show that ce supplementation can reduce adhesion of uropathogenic e. coli to canine uroepithelium and suggests one mechanism by which ce might improve urinary tract health. the purpose of this study was to determine prevalence of urovirulence factors (uvfs) and antimicrobial resistance in canine uropathogenic e. coli (upec) and to evaluate associations between uvfs and antimicrobial resistance. two hundred and twenty-one upec isolates from samples collected from different canine patients submitted to the university of tennessee microbiology laboratory in were evaluated. a multiplex pcr assay was used to detect cnf, hlyd, sfa/foc, and papgiii in dna lysate. in vitro susceptibility was evaluated and if the isolate was resistant to any antimicrobial in a class, it was considered resistant to that class. of the samples, the number of uvf expressed per isolate was: / ( %), / ( %), / ( %), - / ( %), and / ( %). expression of uvf was sfa ( %), hly ( %), cnf ( %), and pap ( %). presence of uvfs was associated with less resistance (p o . ). the combination of hly, cnf, and sfa was associated with less resistance (p o . ). when sfa was present alone, resistance was less (p o . ). average resistance to antimicrobial class by number of uvfexpressed was: uvf . ae . classes, uvf . ae . classes, uvf . ae . classes, uvf . ae . classes, and uvf . ae . classes. urovirulence factors were present in a moderate number of upec and correlated negatively with resistance. neither individual nor combinations of uvfs were associated with increased resistance. obesity is associated with several comorbidities in dogs including pancreatitis, osteoarthritis, oral disease, neoplasia, and lower urinary tract disease. investigator observations led to the hypothesis that morbidly obese dogs are more likely to have asymptomatic bacterial urinary tract infections (abuti) than overweight and moderately obese dogs. therefore, a pilot study was conducted to screen for abuti in obese dogs. urinalysis with urine culture and dual energy x-ray absorptiometry (dxa) were performed on fortythree dogs with body fat (bf) percentages ranging from to %. following dxa, subjects were categorized as obese (o)(bf - %, n ) and morbidly obese (mo)(bf %, n ). no dogs had owner-reported symptoms indicative of uti. the prevalence of abuti in o dogs was % (n ) and % (n ) in mo dogs. the dog in the o group with abuti was close to being mo with a bf equaling . %. of the nine dogs with positive cultures, were neutered males and were spayed females. the prevalence ratio of abuti in mo dogs was . , indicating dogs with % or greater bf are . times more likely to have the condition then dogs o % bf. the results of this pilot study coincide with other surveillance data describing an increased prevalence of lower urinary tract disease in obese dogs. in conclusion, dogs with body fat percentages greater than % are at risk for abuti, and veterinarians should consider screening all morbidly obese patients for urinary tract infections. calcium carbonate (cac) is recommended to decrease phosphate intake in chronic kidney disease. however, its effect is poorly documented in dogs. our objectives were to assess within-day, postprandial and cac effects on phosphatemia variations in healthy dogs. phosphatemia was measured every hours for hours in eight adult healthy beagle dogs in i) fasted condition and ii) a  crossover design. one group received cac mixed with maintenance diet ( . % phosphorus), while the second group received the diet alone. after a -week wash-out period, groups were switched. a general linear model was used to test the period, sequence, treatment, dog and time effects on phosphatemia and the area under the phosphatemia versus time curve (auc - ). a significant (p o . ) circadian variation existed in fasted dogs. the maximum difference (mean: À . mg/dl; % c.i.: À . mg/dl; À . mg/dl) was observed between a.m. and midnight. the auc - with cac ( ae mg.min/dl) was mildly but significantly lower (p . ) than without cac ( ae mg.min/dl). however, it was similar to the auc - in fasted conditions. feeding, with and without cac, has minor effect on phosphatemia. however, circadian variation of fasted phosphatemia might affect its interpretation. gfr measurement permits diagnosis of kidney injury prior to development of azotemia, and is the gold standard for kidney function assessment. accurate and rapid (o min) gfr measurement has been performed in rats by simultaneous transcutaneous assay of two intravascular fluorescently-labeled markers. a recently developed analyzer assays fluorescence via a fiberoptic cable introduced through a peripheral catheter, and thus should also allow rapid gfr determination in larger species. the purpose of this study was to determine correlation and agreement between fluorescent ratiometry (fr) and iohexol plasma clearance (ipc) in dogs over a range of gfrs. acute kidney injury (aki) was induced in female hound-type dogs ( mg/kg gentamicin iv q h), and fr and ipc gfr were simulta-neously determined on days , , and . a -sample, -hr protocol was used for ipc; fr was determined following bolus injection of a dextran conjugate mixture ( -sulfohexamine rhodamine-carboxymethyl kd dextran, -aminofluorescein-carboxymethyl kd dextran) with fluorescence measured over min. gfr was calculated using -compartment model concentration-vs.-time curves for both techniques. correlation was determined via spearman's rho; agreement was analyzed via bland-altman plots. ipc gfr and serum creatinine confirmed progressive aki in all dogs. correlation between fr and ipc was . (p o . ). bland-altman plots confirmed good agreement between techniques with slight underestimation of gfr by fr across most observed values. these results suggest fr is suitable for gfr determination in dogs with aki. importantly, the portable analyzer allowed for point-of-care gfr determination in o min using a peripheral vein. previously presented at the american society of nephrology renal week (related but not identical abstract). dogs with protein-losing nephropathy (pln) are at risk of thromboembolic disease, but the mechanism of hypercoagulability and the population of dogs at risk are unknown. the purpose of this study was to characterize thromboelastography (teg) in dogs with pln. twenty-eight client-owned dogs with pln (urine protein:creatinine ratio (upc) . ) and control dogs were enrolled. teg parameters, antithrombin activity, serum biochemical profiles, and upc were measured. teg analyses were run in duplicate with kaolin activation; reaction time (r), clot formation time (k), maximal amplitude (ma), and g (global clot strength) were analyzed. a wilcoxon sum rank test was used to evaluate differences between groups. twelve pln dogs ( . %) were azotemic. nineteen pln dogs ( . %) were hypoalbuminemic [serum albumin (salb) o . g/dl]; had salb o . g/dl. dogs with pln had higher k (p o . ), ma (p o . ) and g (p o . ) than controls. r was similar between the two groups. pln dogs with salb o . g/dl had higher g (p o . ) values than dogs with salb . g/dl; however, even pln dogs with normal salb ( . g/dl) had significantly higher g values than controls (p o . ). no significant relationship between upc and g, salb and g, antithrombin and g, or salb and antithrombin was noted using linear regression analysis. these results indicate that antithrombin, salb, and upc cannot be used alone to predict hypercoagulability as assessed by teg in dogs with pln. a comprehensive evaluation of the coagulation system in individual patients may be necessary to predict the point at which to initiate anti-thrombotic therapy. cystinuria is a hereditary renal tubular reabsorption defect of cystine, ornithine, lysine and arginine (collectively, cola). the low solubility of cystine in acidic urine predisposes to the formation of uroliths. type i cystinuria in newfoundland and labrador retriever dogs is an autosomal recessive trait caused by mutations in the slc a gene, whereas in other breeds, the cause of cystinuria has not yet been determined. we report here on the clinical, biochemical and molecular features of cystinuria in irish terriers. urine and edta blood were collected from irish terriers from europe and australia. a nitroprusside screening test was used to identify increased cystine in urine. urinary amino acid concentrations were determined by high-pressure liquid chromatography. cystinuric dogs were defined as having cystine calculi, a positive nitroprusside result, urinary cystine ( mmol/g creatinine) and/ or a cola concentration of mmol/g creatinine. all females tested nitroprusside negative and had normal urinary cystine (o mmol/g creatinine) and cola (o mmol/g creatinine) concentrations. the intact males that formed calculi as adults exhibited cystine concentrations ranging from - and cola from - mmol/g creatinine. an additional males had similarly high cola values with cystine levels from - mmol/g creatinine. among the affecteds tested, % were nitroprusside positive. the negative nitroprusside results and/or low urinary cystine levels of affecteds may be due to precipitation of cystine in acidic urine. sequencing the coding regions of the slc a and slc a genes from edta blood identified no mutations. the mode of inheritance remains undetermined. however, castration appears to lower the urinary cystine and cola concentrations and to prevent cystine calculi formation, while diet changes have lesser effects. in conclusion, non-type i cystinuria in irish terriers (and several other breeds like mastiffs and scottish deerhounds) is a unique form characterized by increased aminoaciduria only in males, with lower cystine and cola excretion and fewer and later urolith formation compared to type i cystinuria. castrating cystinuric irish terriers lowers their cystine and cola excretion and thus their risk for calculi formation. cats and dogs that are diagnosed with acute kidney injury (aki) and resultant uremia that is not responsive to standard medical therapy are likely to benefit from renal replacement therapies, such as intermittent hemodialysis (ihd). the purpose of this study was to evaluate the long-term outcome of patients with aki treated with ihd, and to establish whether renal function, as determined by serum or plasma creatinine concentrations, is associated with longterm survival. medical records of cats and dogs that were diagnosed with aki, treated with ihd, and survived longer than days following the last ihd treatment were retrospectively analyzed. standard methods of survival analysis using kaplan-meier product limit curves and the log-rank test were performed. for all-cause mortality, the median survival time was days ( % confidence interval: , ) for cats and days ( % confidence interval: , ) for dogs. when only renal-related causes of death were taken into account, the median survival time was not reached for cats or dogs. survival time for all-cause mortality was inversely associated with the lowest creatinine concentration within the to day period following the last ihd treatment (p o . for cats, p o . for dogs). this study demonstrates that veterinary patients that are diagnosed with aki, treated with ihd, and survive greater than days after the last ihd treatment have a good longterm prognosis and frequently die from causes that are unrelated to renal impairment. renal fine-needle aspiration (r-fna) is oftentimes attempted during evaluation of dogs and cats with renomegaly, mass lesions, or suspected infiltrative processes. diagnostic utility of fna is dependent upon the organ being sampled; additionally, in some organs, certain diagnostic imaging findings are associated with improved concordance of fna with final diagnosis. objectives of this study were to evaluate the diagnostic utility of r-fna and determine whether concordance with final diagnosis is associated with specific clinicopathologic or diagnostic imaging findings. we hypothesized that r-fna is most useful in patients with diagnostic imaging results suggestive of renal neoplasia (i.e. masses or suspected infiltrative processes). dogs and cats that had undergone r-fna from jan , to dec , were identified by database search. patient signalment, serum creatinine and blood urea nitrogen concentration, urine specific gravity, dipstick protein, r-fna result, and final diagnosis were recorded. patients were excluded if abdominal radiographs or sonographic images were not available for review, or if diagnostic test results were insufficient for determination of final diagnosis. a single coauthor blinded to final diagnoses interpreted all abdominal images using a pre-set list of descriptors and grading criteria. radiographic kidney shape, margin distortion, and ventrodorsal kidney-to-l ratio were evaluated. sonographic kidney margin distortion, cortical echogenicity, and corticomedullary junction distinction were described, and presence of nodules or masses, peri-renal effusion, or a peripheral sonolucent rim was noted. concordance of r-fna and final diagnosis was determined, and the chi-squared or fisher's exact test were used to determine association of concordance with the above variables; p o . was considered significant. dogs and cats ( animals) met all inclusion criteria. r-fna results were concordant with the final diagnosis in ( . %) patients, discordant in ( . %) patients, and inadequate for cytologic interpretation in ( . %) patients. neoplasia or fip were the final diagnoses in of ( . %) and of ( . %) patients with concordant results, respectively. renal lymphoma (p . ), renal carcinoma (p . ), and renal neoplasia in general (p . ) were not associated with a higher likelihood of r-fna and final diagnosis concordance. there was no association noted between likelihood of r-fna and final diagnosis concordance when patients were stratified by species, serum creatinine or blood urea nitrogen concentration, urine specific gravity, dipstick proteinuria, or any diagnostic imaging variables. this study failed to identify concurrent clinicopathologic or diagnostic imaging findings that enhanced the diagnostic utility of r-fna. future studies should use standardized criteria to prospectively identify patients in which r-fna will be performed, evaluate additional variables that may be associated with increased r-fna diagnostic utility, and directly compare the utility of r-fna with that of other diagnostic techniques. feline lower urinary tract disease (flutd) is a disease with increasing prevalence in private practices and veterinary teaching hospitals. although several underlying causes can cause the obstructive form in male cats, the idiopathic form (feline interstitial cystitis) often is diagnosed as underlying reason in cats o years. the goal of this retrospective study was to identify possible predisposing factors in order to optimize the therapy of these patients. as a study group, cats hospitalized with obstructive flutd at the veterinary university of vienna were examined during a year period ( ) ( ) ( ) . as a control group cats presented for other reasons were randomly chosen during the same time period. the data were examined concerning the signalment and history. furthermore, the long-term outcome was evaluated with a questionnaire. based on assumptions a student's t-test or a chi-square test was used. there were no significant differences in age and breed. the body weight was significant higher in the flutd group than in the control group (p o . ). we could observe a significant risk for the disease of a weight of kg (p o . ). there were significant less cat toilets in the flutd group compared to the control group (p o . ). furthermore we could observe that in the households of flutd cats there was significant less than one toilet per cat (p o . ) and more cats diseased on flutd lived strictly indoor than outdoor (p . ).there were no significant differences at the time of hospitalization in age, breed, number of cats per household or season of the year between the two groups. in summary, we could observe that cats over kg body weight kept indoor with less than one toilet per cat have a significant higher possibility to be affected by obstructive flutd. further studies with an extensive history of animal husbandry are needed to identify risks predispoing cats to this frequent and cost-intensive disease. although purine uroliths (ammonium urate, sodium urate, xanthine, uric acid, etc.) represent the third most common stone type in cats, purine uroliths have the highest rate of recurrence ( % in months). in dogs, mutation of the urate transporter (slc a ) and portovascular anomalies are common risk factors. however the underlying cause(s) for purine urolith formation in cats is unknown. the purpose of this study was to test the hypothesis that hyperuricosuria without alterations in liver function is common in cats with urate uroliths. urine concentrations of purine metabolites were measured by high-performance liquid chromatography in cats with ammonium uroliths (cases), clinically healthy, breed and gender matched cats (negative controls), and cats with naturally occurring xanthine uroliths (positive controls). prior to urine collection, all cats were fed a standard maintenance food (protein g/ kcal) for weeks. urinary xanthine, uric acid, and allantoin concentrations and concentration to creatinine ratios were calculated and compared between groups. also, serum pre-and post-prandial bile acid concentrations were measured. when compared to control cats, urinary uric acid concentration was significantly higher in case cats (p . ). xanthine was not detected in the urine of cases or negative controls. a significant difference in fasted and post-prandial serum bile acid concentrations was not detected in cases or controls (p . , . ).hyperuricosuria without increased concentrations of urinary xanthine or allantoin appears to be a risk factor for ammonium urate urolith formation in cats. an association between portovascular shunts and purine urolithiasis was not observed in this population of cats. studies indicate that proteinuria is predictive, on a population basis, of those cats at risk of developing azotemia. seldi-tof-ms is a sensitive, high-throughput, proteomic technique utilising chromatographic surfaces to facilitate separation and detection of proteins and peptides within biological fluids such as urine. individual low molecular weight (lmw) urinary proteins have been considered as potential biomarkers for renal damage but provide only a limited representation of the urinary proteome; seldi-tof-ms may provide a more global assessment. normotensive, non-azotemic geriatric cats ( years) were recruited prospectively from two first-opinion clinics for routine health screening. at entry cats received a full physical examination, plasma biochemistry, evaluation of total t concentration and urinalysis including urine protein to creatinine ratio. re-examination was offered at and months. cats were divided into two groups based on clinical status at the month re-examination (azotemic; creatinine concentration ! . mg/dl and non-azotemic). optimisation studies were performed to facilitate the automated preparation (biomek ) of cm (weak cation exchange) arrays for seldi-tof-ms analysis (ciphergen enterprise ) of urine samples from cats at entry to the study. results are reported as median [ th , th percentile]. mann whitney u-test and wilcoxon signed rank test were used to compare variables between groups and between timepoints, respectively. ciphergen express ( . ) software was used to analyse spectral data and a mann whitney u-test was used to identify clusters which differed significantly between groups (p o . ) at entry to the study. twenty non-azotemic cats were recruited, of which cats developed azotemia by months. no significant differences in age, body weight, biochemical or urinalysis variables were identified between groups at entry to the study. as might be expected creatinine increased significantly ( . mg/dl [ . , . ], . [ . , . ], p . ) between study entry and months in the cats that developed azotaemia and there was a commensurate increase in phosphate concentration ( . mg/dl [ . , . ], . [ . , . ], p . ). creatinine and phosphorus did not change significantly over time in the cats that did not develop azotaemia. seven clusters with m/z values of , , , , , were found to differ significantly between groups at entry to the study. the low protein concentration of feline urine makes the use of proteomic techniques challenging. however, this pilot study indicates that seldi-tof-ms can be utilised to examine the feline urinary proteome and that differences in low molecular weight protein patterns may be useful to differentiate those cats which are at risk of the development of azotemia. further work is necessary to identify these proteins/peptides. fibroblastic growth factor (fgf- ) is a phosphotonin with an important physiological role in the regulation of phosphorous and vitamin d metabolism, and may therefore play a part in the development of renal secondary hyperparathyroidism. previous studies in cats have shown parathyroid hormone (pth) to be elevated prior to the development of azotemia. the study objectives were to explore the hypothesis that fgf- is a mediator of the development of renal secondary hyperparathyroidism in the nonazotemic stages of feline ckd. healthy, non-azotemic (plasma creatinine concentrations (cr) o . mg/dl) geriatric cats were recruited into the study prospectively and followed for months. at the study end point cats were categorised into the following groups: group (n )-cr . mg/dl, group (n )-cr ! . mg/dl but did not meet the criteria for group and group (n )-cr . mg/dl in association with reduced urine concentrating ability (usg o . ) or demonstration of persistent azotemia (cr . mg/dl). plasma samples were subjected to routine biochemical analysis, intact pth, calcitriol and intact fgf- assay. variables were compared between the groups at the baseline time point. gfr was measured in an additional group of cats ( non-azotemic, iris stage ii, iris stage iii) using a corrected slope-intercept iohexol clearance method. relationships were explored using linear regression analysis and determining the coefficient of determination (r ). results are presented as median [range] . at the baseline time point fgf- concentrations were significantly higher in group ( . [ . - . ], p . ) and group ( . [ . - . ], p . ) compared to group ( . [ . - . ] ). weak positive relationships were identified between fgf- and pth (r . , p . , n ) and fgf- and cr (r . , p . , n ). however, the positive relationships between fgf- and phosphate (r . , p . , n ) and fgf- and calcitriol (r . , p . , n ) were not significant. the additional group of cats in which gfr measurement was performed there was an inverse relationship between fgf- and gfr (r . , p . ). in conclusion, fgf- was elevated in cats prior to the development of azotemia. the role of fgf- in the development of feline renal secondary hyperparathyroidism remains to be determined and should be explored through interventional studies. however, considering the relationship between fgf- and gfr, it cannot be excluded that the phosphotonin is simply a marker of reduced filtration. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to the progression of this disease. the aim of this study was to evaluate urinary vascular endothelial growth factor (vegf) as a marker of renal hypoxia. cats were recruited through geriatric clinics held at two first opinion london practices. vegf was measured in stored samples using a canine elisa kit validated for use on feline urine and indexed to creatinine concentration to yield a vegf to creatinine ratio (vcr). two studies were undertaken -firstly a cross-sectional analysis of clinical variables associated with vcr in cats with ckd. diagnosis of ckd was based on concurrent findings of plasma creatinine ! mg/dl and usg . , with persistence of azotemia for ! weeks. only patients receiving no medical therapies were included. normotensive and (pre-treatment) hypertensive cats were included, but borderline cases (mean systolic blood pressure - mmhg on the date of sampling) were not. hyperthyroid cats were also excluded from this cross-sectional study. associations between vcr and clinical data were initially assessed using the spearman's coefficient and mann whitney test. linear regression was then used for multivariate analysis. the second study used samples from a trial in which hypertensive cats that had been treated with amlodipine for at least months were entered into a randomised cross-over study where they received placebo or benazepril ( . to mg/kg daily) for weeks in turn. vcr on placebo was compared with that on benazepril using the wilcoxon signed ranks test. cats with well controlled hyperthyroidism were included in this intervention study. results are reported as median [ th, th percentile]. vcr was higher ( . [ . , . ] vs. . [ . , . ] fg/g, p . ) in untreated hypertensives (n ) than normotensives (n ). vcr was correlated with pcv (r À . , p . , n ), upc (r . , p o . , n ), plasma phosphate (r . , p . , n ), and usg (r À . , p . , n ), but not plasma creatinine concentration. in the best multivariate model, pcv was associated with vcr independently of upc (r . , n ). vcr was significantly reduced by benazepril therapy ( . [ . , . ] fg/g) compared with placebo ( . [ . , . ] fg/g; p . , n ) with a reduction seen in % of cases. these results suggest urinary vegf excretion is associated with proteinuria in cats with ckd and might be a marker of renal hypoxia induced by low pcv. ace inhibitor therapy might reduce urinary vegf excretion because angiotensin ii causes constriction on efferent arterioles resulting in tubular hypoxia. fgf- is a phosphaturic hormone. fgf- concentrations increase with declining renal function in humans. the objectives of this study were to validate a method for fgf- quantification in feline plasma and to assess the association between fgf- concentration and plasma creatinine or phosphate concentration in cats with chronic kidney disease (ckd). non-azotemic and azotemic (plasma creatinine concentration (cr) . mg/dl) geriatric ( yrs) cats were recruited into the cross-sectional study from two london first opinion practices. cats were excluded from the study if they were fed a phosphate restricted diet, or had evidence of concurrent disease. the cats were categorized, using a modified iris staging system, into the following four groups: group (cr . mg/dl), group (cr . - . mg/dl), group (cr . - . mg/dl), group (cr . mg/dl). groups and were further subdivided based on the iris targets for plasma phosphate concentration (po ): group a (po . mg/dl), group b (po . mg/dl), group a (po mg/dl), group b (po mg/dl). fgf- concentrations were measured in feline edta plasma using a human intact fgf- elisa, validated by intraand inter-assay variability and assessment of dilutional parallelism. comparisons between groups were made using the kruskal-wallis test and mann-whitney u test, with statistical significance defined as p o . . bonferroni correction was applied where appropriate (statistical significance then determined as p o . ). results are reported as median [ th, th percentiles]. fgf- concentrations ! pg/ml (upper limit of quantification) were assigned the value of pg/ml. intra-and inter-assay variability of fgf- measurements were o . % and dilutional parallelism between feline samples and the calibration curve were demonstrated. plasma fgf- concentrations increased with increasing creatinine concentrations (group : [ , ] , n , group : [ , ] , n , group : [ , ], n , group : [ , ], n ). fgf- measurements were significantly different between all groups (p . to o . ) except between groups and (p . ). fgf- concentrations were significantly higher in cats with higher plasma phosphate concentrations (group a: [ , ] , n vs. group b: [ , ], n ; p . ) and (group a: [ , ] , n vs. group b: [ , ], n ; p . ). in conclusion, fgf- concentrations were higher in cats with more severe ckd or higher plasma phosphate concentrations as would be predicted from its known biological actions. further work is warranted to explore the role of fgf- in the development of renal secondary hyperparathyroidism by measuring parathyroid hormone (pth) and calcitriol in cats at different stages of ckd. progressive non-cardiogenic edema and lung dysfunction are common complications of acute kidney injury (aki) in people. pulmonary abnormalities have not been systematically reviewed in dogs with renal azotemia, but anecdotal reports of dogs with aki and concurrent non-cardiogenic pulmonary edema are suggestive of uremic pneumonopathy (up), a centrally-distributed pulmonary edema syndrome associated with kidney disease in people. we therefore hypothesized that pulmonary-associated clinical signs or thoracic radiograph abnormalities are more common in dogs with renal azotemia than in non-azotemic dogs, and that this association is more likely in dogs with aki than dogs with chronic renal failure (crf). our study objectives were ) to describe thoracic radiograph and lung histopathologic abnormalities in dogs with renal azotemia, ) to compare the occurrence of these findings in dogs with aki, crf, or non-systemic illness, and ) to determine if these abnormalities are associated with shorter survival times. records of dogs with renal azotemia evaluated from / / to / / were reviewed; dogs which could be classified as having aki or crf and which had complete thoracic radiograph studies available for review were included. dogs with primary intracranial disease and normal serum creatinine and a complete thoracic radiograph study were selected as controls. signalment, weight, presence of pulmonary-related clinical signs, azotemia duration and severity at time of radiography, and leptospirosis antibody titer were noted. alveolar, bronchial, interstitial, or nodular lesions were described using a -point scale, and lung tissue collected at time of necropsy was reviewed; both the radiologist and pathologist were blinded to final diagnoses. significance was p o . for all analyses. the final study population included aki, crf, and control dogs. crf dogs were older (p o . ) than aki and control dogs. pulmonary-related clinical signs were more commonly diagnosed at first evaluation in aki dogs ( / dogs, . %) than in crf ( / , . %; p . ) or control dogs ( / , . %; p o . ). presence of an alveolar pattern was the only radiographic finding which differed amongst groups (more common in aki [n , . %, p . ] and crf [n , %, p . ] dogs than in control dogs [n , . %]). there was no association between presence of an alveolar pattern and any other variable. alveolar mineralization was the most common lesion in aki dogs ( / dogs; . %), with concurrent alveolar space concretions or mineralization of vessels or bronchioles noted in dog each. necropsies had not been performed in any of the crf dogs, but mineralization was not seen in lung tissues from any control dogs (n ). neither pulmonary-associated clinical signs nor alveolar pattern were associated with median number of days from discharge until death in dogs with aki (p . and . , respectively) or crf (p . and . , respectively). in this group of dogs, presence and type of radiographic pulmonary abnormalities were associated with renal azotemia but not with median time until death. the association between and clinical relevance of alveolar mineralization in aki dogs were not determined, but both the radiographic and histopathologic abnormalities reported here differ from up in people. chronic kidney disease (ckd) is a common cause of morbidity and mortality in cats. the purpose of this study was to investigate the effects of chinese rhubarb (rheum officinale) supplementation on the progression of feline ckd. cats with stable iris stage ii or iii ckd and without comorbidity were included in the study. cats were divided into treatment groups and administered rhubarb extract (group , rubenal s , vetoquinol, mg tablet po q h), benazepril as a positive control (group , . mg/kg po q h), or both (group ). cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. body weight, laboratory data, and blood pressure were recorded every months for up to months. variables between groups at enrollment and within groups over visits were compared with anova and repeated measures ano-va, respectively. a treatment by visit interaction term was included in all repeated measures models. significance was set at p . . except for body weight there was no significant differences between treatment groups at enrollment. there was no significant change in body weight, hematocrit (hct), upc, or creatinine over time as compared to baseline within any group. there was no significant difference between groups over time in regards to change in weight, hct, upc, or creatinine. the treatment by time interaction was non-significant in all models. although there was no benefit associated with combination treatment, the results for rhubarb treatment alone were not different from benazepril treatment. azodyl, an encapsulated, enteric-coated probiotic/prebiotic nutraceutical, is marketed for reduction of azotemia (bun & creatinine) in dogs and cats. cat owners often sprinkle contents onto cat food to facilitate administration. however, exposure to air and stomach acid are thought to inactivate the lyophilized bacteria within the product. therefore, we examined the ability of foodsprinkled azodyl to reduce azotemia in cats with ckd. cats with ckd were enrolled in the study and randomized receive azodyl or placebo. owners were provided with - capsules of azodyl prior to enrollment to ensure compliance with administration. baseline blood samples were obtained month apart, and then & months after beginning therapy. clinicians and owners were masked as to medication assignment. we hypothesized that a % decrease in bun and/or creat in the azodyl group would be significant, and set a . . in order to maximize the probability of detecting a difference, we determined the % change as being the difference between the maximal baseline analyte concentration and minimal therapeutic concentration. we compared the % change between groups by mann-whitney u test. bun and creatinine did not differ between groups. based on these results, azodyl, applied by sprinkling onto food fails to reduce azotemia in cats with ckd. whether intact capsule administration reduces azotemia in cats with ckd remains unknown. lower urinary tract disease (lutd) occurs commonly in cats, and idiopathic cystitis (fic) and urolithiasis account for over % of cases in cats less than years of age. although several strategies have been recommended, a common recommendation is to induce dilute urine resulting in more frequent urination and to dilute calculogenic constituents. in addition to conventional therapy using modified diets, traditional chinese and western herbs have been recommended, although only one, chorieto, has published data. we evaluated commonly used herbal treatments recommended for use in cats with lutd including ( ) san ren tang, ( ) wei ling tang, and ( ) alisma. we hypothesized that these chinese herbal preparations would induce increased urine volume and decreased urine saturation for calcium oxalate and struvite. six healthy, spayed female, adult cats were evaluated in a placebocontrolled, randomized, cross-over design study. cats were randomized to of treatments including placebo (p), san ren tang (srt), wei ling tang (wlt), or alisma (a). treatment was for weeks each with a week washout period between treatments. at end of each treatment period, a -hour urine sample was collected using modified litter boxes. urine volume and biochemistries were measured, and urine saturation for struvite and calcium oxalate was estimated using equil . b. analysis of variance (anova) was used to analyze data statistically if distributed normally and kruskal-wallis was used to analyze data statistically if data were not distributed normally. a p o . was considered significant. body weights were not different between treatments. no differences were found in -hour urinary analyte excretions, -hour urine volume, urine ph, or -hour urinary saturation for calcium oxalate or struvite between treatments (table) . urolithiasis is a multifactorial disease, frequent and recurrent in dogs in the worldwide, in which breed, sex, age, diet, some anatomical abnormalities, urinary tract infection, urine ph and some geographical and hereditary features in the populations studied have been implicated as risk factors. the effective long-term management of urolithiasis depends on identification and control of the pathophysiological mechanisms involved, which, in turn, depend on accurate knowledge of the mineral composition of the uroliths. the aim of this study was to determine for first occasion the main epidemiological data of canine urolithiasis in mexico. this study was developed with dogs with urolithiasis from of the states of the country. chemical composition of the uroliths was determined by stereoscopic microscopy, infrared spectroscopy, scanning electron microscopy and x-ray microanalysis. urolithiasis affected nearly the same number of males and females; with ages ranging from two months to years with a median age of years. adult animals were the most affected. breeds more affected were schnauzer miniature, poodle, dalmatian, yorkshire terrier, scottish terrier, chihuahua and bichon frisee´. uroliths were found in the lower urinary tract in . % of the cases. mineral composition of the uroliths was: struvite . %, followed by calcium oxalate . %, purines . %, silicate . %, others . %, mixed . % and compound uroliths . %. struvite uroliths affected females in most cases, whereas calcium oxalate, purines and silicate uroliths, were mainly observed in males. our results are similar to studies developed in other countries and continents, though we found a higher frequency of uroliths containing silicate, either pure, mixed or compounds uroliths ( . %); in mexico city the frequency reached %. this high frequency may be due to high consumption of silicate in home-made food or in the groundwater derived from aquifers. acknowledgments: this work has been partially supported by a project of waltham foundation in mexico and the consejo nacional de ciencia y tecnologı´a (conacyt) of mexico. voiding urohydropropulsion is a non-invasive method for removing small urocystoliths from the dog, most commonly used in females due to the relatively wider and shorter urethra. this procedure is typically performed under general anesthesia to allow complete relaxation of the urethra, however, anesthesia results in longer procedure times and difficult endotracheal tube stabilization due to the vertical positioning of animals, especially in larger dogs. the aim of this study was to devise a novel injectable sedation protocol for urohydropropulsion when cystoscopy was not concurrently required. an intravenous catheter was placed, and a combination of medetomidine ( to mg/kg iv) and hydromorphone ( . to . mg/kg iv) was administered, with the addition of ketamine ( mg/ kg iv) in fractious animals; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this protocol was considered in cardiovascularly healthy, non-diabetic dogs without evidence of urinary obstruction. monitoring equipment included electrocardiography, blood pressure measurement, and pulse oximetry, and supplemental flowby oxygen was provided. two dogs received the proposed sedation protocol in order to perform urohydropropulsion. dog one was a year old female spayed shih tzu cross, and dog was a year old female spayed standard poodle. ultrasonography revealed a moderate number of urocystoliths present in both dogs, measuring up to mm in dog and . mm in dog . urohydropropulsion was performed and resulted in retrieval of urocystoliths in dog , and approximately urocystoliths in dog . repeat ultrasonography revealed no uroliths present after urohydropropulsion in both dogs. the time from administration of sedation to administration of reversal agent was minutes for dog , and . minutes for dog . records were obtained from dogs that had traditional general anesthetic protocols for urohydropropulsion with cystoscopy for confirmation of urocystolith removal, performed within the last years, and the average anesthetic time was minutes. subsequent to the use of medetomine-based sedation protocols for the above dogs, cystoscopy was performed in a year old neutered male golden retriever with prostatomegaly. medetomidine ( ug/kg iv) and butorphanol ( . mg/kg iv) were administered; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this sedation allowed adequate immobilization for cystoscopy of the urethra and urinary bladder, and endoscopic biopsying of the prostatic urethra and urinary bladder. the time from administration of sedation to administration of reversal agent was minutes for this dog. in conclusion, a novel sedative protocol for urohydropropulsion is proposed which allows for an appropriate level of sedation along with a short procedure time and rapid recovery. this sedation protocol may also be useful for certain cystoscopic procedures. analysis may be delayed for a variety of reasons, including the need for sample batching within the laboratory or shipping to an outsourced location. therefore, it is important to know how storage of the sample may affect enzyme activity. we hypothesized that urinary nag and ggt activity would be affected differently in samples stored by refrigeration vs. freezing. thirty-four canine urine samples submitted to the clinical pathology laboratory at kansas state university were included. samples were collected from clinical patients with a variety of medical/surgical disorders and were selected based on the day of the week and a minimum volume of ml. a complete urinalysis was performed on each sample; however there were no exclusion criteria based on urinalysis results. nag and ggt activity in the urine supernatant was assessed by colorimetric assay. aliquots of each supernatant were refrigerated for days and frozen at À c for and days at which time enzyme activity was re-assessed. compared to baseline values, enzyme activity for both nag and ggt were stable after days of refrigeration, however there were significant (p o . ) declines in ggt and nag activity when urine supernatants were frozen for and days. treatment for canine urinary tract infections (uti) typically consists of - days of antimicrobial drugs in primary care veterinary practice. compliance with this drug regimen can be difficult for some clients. enrofloxacin is a veterinary approved fluoroquinolone antimicrobial and is useful for treatment of canine uti. fluoroquinolones are often used in human medicine to treat uncomplicated utis in women and can be prescribed for as little as days. the primary objective of this study was to determine if dogs with naturally occurring uncomplicated uti have equivalent microbiologic cure with a high dose short duration protocol of enrofloxacin, compared to a standard antimicrobial protocol. client-owned adult dogs with naturally occurring, uncomplicated uti were prospectively enrolled in a multi-center clinical trial and assigned to of groups in a randomized blinded manner. group received treatment with - mg/kg oral enrofloxacin once daily for consecutive days. group dogs were treated with . - mg/kg oral amoxicillin-clavulante twice daily for days. both groups had urinalyses and urine cultures submitted on day , , and . at the time of this interim analysis, thirty-six dogs have completed the trial. bacteriological cure was achieved in dogs ( %) treated with enrofloxacin and dogs ( %) treated with amoxicillinclavulante, respectively. these data suggest that the high-dose, short-duration enrofloxacin protocol was equally effective to the standard protocol in treating uncomplicated canine uti in the sample patient population. and may represent a viable alternative therapeutic regimen for similar patients. azotemia is frequent in dogs with dmvd (nicolle et al; jvim ; : - ) and could result from renal hemodynamic alterations. renal resistive index (ri) allows assessment of renal vascular resistance. the aim of this prospective study was to assess ri in dogs with different dmvd stages. fifty-five dogs with dvmd were used (isachc class (n ), (n ), and (n )). physical examination, renal ultrasonography and echo-doppler examinations were performed in awake dogs by trained observers. plasma creatinine, urea and nt-probnp were measured. statistical analyses were performed using a general linear model. whereas ri of renal and arcuate arteries were unaffected by isachc class, left interlobar ri increased (p o . ) from . ae . (mean ae sd) in class to . ae . in class . left interlobar ri was also higher (p o . ) in azotemic ( . ae . ) than in non azotemic ( . ae . ) dogs. similar findings were observed for right interlobar ri. a positive effect of nt-probnp (p . ), urea (p o . ), creatinine (p . ), urea-to-creatinine ratio (p o . ), left atrium-to-aorta ratio (p o . ), regurgitation fraction (p . ), systolic pulmonary arterial pressure (p o . ) and shortening fraction (p . ) on ri was also observed. in conclusion, interlobar ri increases with the severity of dmvd and azotemia. a cause-effect relationship remains however to be established. antibodies against alpha-enolase are associated with immunemediated nephritis in people. it was previously shown that vaccinated cats commonly develop antibodies against alpha-enolase. the purpose of this study was to assess for associations between alphaenolase antibodies and azotemia in privately-owned cats. clinically stable privately owned cats ! years of age, with and without azotemia (creatinine mg/dl), and with an available vaccine history for ! years were recruited for the study. sera were assayed for creatinine concentrations and alpha-enolase antibodies by use of previously validated techniques. results from cats with and without azotemia were compared by student's -tailed t test or fisher's exact test with significance defined as p o . . median ages were years (range: - ) and years (range: - ) for cats with (n ) and without azotemia (n ), respectively. there was no significant difference in vaccine events (number, type, or route of administration) between groups. azotemic cats ( . %) were more likely than normal cats ( . %) to be positive for antibodies against alpha-enolase (p . ). in addition, alpha-enolase antibody concentrations were greater (p . ) in azotemic cats (mean % elisa . %) than cats with normal creatinine concentrations (mean %elisa . %). results of this study suggest that antibodies against alpha-enolase in cats may be associated with renal disease. additional prospective evaluation in a larger number of cats is indicated. aki is used in human medicine as a predictor of mortality based on the akin (acute kidney injury network) scoring system which utilizes relative increases in creatinine to determine stage. with this scheme, mortality has been shown to increase as the stage of kidney injury (indicated by akin score) increases. accordingly, we hypothesized that this system would improve predicting prognosis in dogs and cats. we retrospectively evaluated dogs and cats ( ) ( ) ) that had ! creatinine measurements within days, and whose first creatinine was o . mg/dl. patients were categorized as: level (no aki); level (second creatinine value o . mg/dl, but creatinine increased ! . mg/dl); or level (second creatinine . mg/dl with a creatinine increase ! . mg/dl). thirty and day survival for each level was compared to level . adjusted odds ratio (or) in dogs for day survival was . for level (ci %, . - . ) and . (ci %, . - . ) for level ; or for day survival was . for level (ci %, . - . ) and . (ci %, . - . ) for level . for cats, or at days was . (ci %, . - . ) for level and . (ci %, . - . ) for level ; or for day survival was . (ci %, . - . ) for level and . (ci %, . - . ) for level . thus, detecting increasing stage of aki helps predict mortality in dogs and cats. abstract n/u- feline urate urolithiasis: cases ( - . j dear , r shiraki , a ruby , j westropp . william r pritchard veterinary medical teaching hospital, university of california, davis, ca, gerald v. ling urinary stone analysis laboratory, university of california, davis, ca and the department of veterinary medicine and epidemiology, university of california, davis, ca. feline urate urolithiasis accounts for % of the feline stones our laboratory analyzes each year; little information is known about this disease, particularly the incidence of those cats with hepatopathies. the objective of the study was to characterize the signalment, clinicopathologic data, and diagnostic imaging of cats with this disease as well as the salts of uric acid present. a retrospective analysis of feline urate uroliths submitted to the stone lab between january -december were included. from these data, primary veterinarians were solicited to submit records. furthermore, all records from cats with urate uroliths from the vmth were analyzed separately. records were received from the primary care veterinarians. sixteen cases were identified from the vmth. median values for the cbc and chemistry panels available were within the reference ranges provided, with only a few outliers present. of the cats with radiographic reports, ( %) had visible evidence of uroliths. two external cases had confirmed pss; five cases from the vmth had a pss. cats with urate uroliths and pss were younger than cats without a documented hepatopathy ( years vs. years). the siamese breed was overrepresented. all stones were ammonium hydrogen urate. the pathogensis of urate uroliths in cats is poorly understood. most cats were not completely evaluated for pss, however, there were few clinicopathologic parameters which indicated hepatopathies were present. further studies are warranted to evaluate genetics and purine metabolism in cats with urate uroliths to help tailor proper management and breeding strategies. -indoxyl and p-cresyl sulfate (is, and cs, respectively), small protein-bound molecules derived from gastrointestinal protein metabolism, are among the most important uremic solutes affecting morbidity and mortality in human chronic kidney disease (ckd). in the blood stream, these compounds are predominantly bound to protein, but their debilitating effects on prognosis and quality of life in ckd appear to be driven by the free fraction. the objectives of the present study were to assess the normal, physiological levels of is and cs in healthy cats and to evaluate the correlation of the respective free and protein-bound levels. blood samples were taken from clinically healthy adult cats enrolled at five participating veterinary practices in germany. after centrifugation, the serum was deep frozen until transport on dry ice to the analytical laboratory. serum creatinine and urea levels were quantified by vettest s (idexx laboratories, inc.). total and free is and cs, respectively, were quantified by turbulent flow chromatography coupled with a tandem mass spectrometry detector. statistical analysis of the results comprised i) a descriptive report of the median with upper and lower bounds of the % confidence interval for reference values of is and cs, ii) a calculation of various pearson correlation coefficients r, also tested with reference to the null hypothesis of no relationship, and iii) wilcoxon-mann-whitney utest for an estimation of the effect of hemolysis on serum is or cs levels. six animals with serum creatinine or urea levels outside the reference range were excluded from the calculation of reference values. median levels of is in cat serum were . mg/l with upper and lower bound % confidence intervals at . and . mg/l, respectively. the corresponding median levels of cs were . mg/l (median) and . vs . mg/l (upper vs lower bound levels, respectively). these values showed a low, non-significant correlation with serum creatinine or urea levels. however, is and cs serum levels were moderately correlated (total levels r . , p o ). their respective free levels constituted about % of the total serum levels (r ! . , p o . ). non-hemolytic samples tended to yield lower values than hemolytic samples. due to the low number of hemolytic samples (n ) , the group difference could, however, not be statistically confirmed. the results indicate that it is sufficient to determine total levels of either is or cs in serum while studying the effects of therapeutic or dietetic interventions on the evolution of these parameters in feline ckd. reference values are provided for orientation towards clinically relevant changes. disrupted urothelial differentiation has been implicated in the pathogenesis of feline idiopathic cystitis (fic). studies of cultured human urothelium have shown that abnormalities in urothelial differentiation and repair may be mediated by persistent -hydroxy-prostaglandin dehydrogenase (pgdh) activity and subsequent metabolism of cytoprotective prostaglandins. the goal of this study was to confirm persistent pgdh expression in fic bladders compared to desmoplakin i ii expression, a marker of urothelial differentiation. urinary bladder biopsy specimens were obtained by cystotomy from symptomatic cats with chronic fic. cats with a history of another major disease, previous cystotomy, or recent treatment with corticosteroids, nsaids, antihistamines, antidepressants, or glycosaminoglycans were excluded. urinary bladder tissue specimens were also obtained from untreated clinically normal specific-pathogen-free cats. tissue specimens were fixed in buffered % formalin and embedded in paraffin. tissue sections were deparaffinized and subjected to citrate buffer microwave antigen retrieval. tissues were stained for pgdh using a rabbit anti-pgdh antibody, an isotype negative control or goat anti-desmoplakin i ii and developed using the avidin-biotin peroxidase complex method. all fic ( / ) and normal ( / ) cat bladder samples showed similar staining of urothelial cytoplasm for pgdh. however, desmoplakin i ii staining, found on the luminal cell surface in / normal tissues, was disrupted in / fic bladder samples. desmoplakin i ii staining confirmed altered urothelial differentiation in fic cats. however, pgdh expression remained intact in fic samples. we hypothesize that pgdh expression in fic may contribute to its pathophysiology due to breakdown of prostaglandins essential for urothelial healing. additional studies will explore this hypothesis. the university of tennessee college of veterinary medicine's picture archiving and communication system was searched over a month period for cats that had undergone both abdominal radiographs and ultrasound during the same visit. one hundred and three cats were identified (age range o to yrs; median yrs). kidney size was determined based on radiographic and ultrasound findings. of the included cats, . % had two normal sized kidneys, . % had one small and one normal, . % had one large and one normal, . % had two small, . % had two large, and . % had one small and one large kidney. the presence of mineralization, uroliths and hydronephrosis was also noted. medical records were reviewed for clinical chemistry data and historical information concerning previous urinary disease. no significant differences were found between kidney size and renal function, kidney size and the presence of uroliths, renal mineralization and function or the presence of uroliths and function. the presence of uroliths was significantly associated with hydronephrosis. of the cats with at least one large kidney, ( %) had hydronephrosis. of the cats with current or previously diagnosed uroliths, urinary tract infections or other uropathies, ( . %) had at least one small kidney. small kidneys were commonly found in older cats, however, this correlation was not statistically significant. based on these findings, small kidneys are more likely to be the result of urinary disease as opposed to being either congenital or due to aging. this study aimed to evaluate ife, which has been advocated for treatment of lipid-soluble drug intoxication, in the treatment of clinically-occurring canine ivermectin toxicosis. one australian shepherd and two miniature australian shepherds were included. all three dogs were homozygous for the mdr- gene mutation. two dogs roamed on horse ranches where ivermectin-based deworming products had recently been used. ivermectin was administered to the third dog ( mg/kg po). all three dogs exhibited tremors, ptyalism, and cns depression, which progressed over several hours to stupor in two dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. a % formulation of ife (liposyn ii, hospira) was administered as a bolus ( . ml/kg) followed by a slow iv infusion ( . - ml/kg over minutes). no change was observed in the neurologic status of any patient. lipemia visible upon blood sampling persisted for hours in one dog. no other adverse effects were noted. serum ivermectin levels confirmed ivermectin exposure in each case. in this study, ife administration did not result in clinical benefit in cases of ivermectin toxicosis. brain ivermectin concentrations in mdr mutant/mutant genotype dogs may be too high to be overcome by ife. additionally, these dogs may lack p-glycoprotein-mediated biliary clearance mechanisms needed for optimal ife function. further investigation is needed to determine the utility and optimal dosing of ife in canine toxicoses, to characterize its safety, and to determine how mdr- status may alter the efficacy of ife in treatment of canine ivermectin intoxication. rufinamide is a recently approved antiepileptic drug used for the treatment of seizure disorders in human patients. rufinamide is administered at a dose of mg/kg divided twice daily to achieve therapeutic concentrations of mg/ml. the objective of this study was to determine the pharmacokinetic properties and short-term adverse effects of single-dose oral rufinamide in healthy dogs in preparation for a possible clinical trial evaluating the efficacy of rufinamide in the treatment of canine epilepsy. six healthy adult dogs were included. the pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of . mg/kg (range . - . mg/kg), extrapolated from the dose used in human patients. dogs were monitored by repeat physical examinations, electrocardiograms and blood pressure assessments during the course of the study. plasma rufinamide concentrations were determined using high-performance liquid chromatography. pharmacokinetic data were analyzed using winnonlin version . . no adverse effects were observed. the mean terminal half-life was . /À . hours. the mean maximum plasma concentration was . /À . mg/ml and the mean time to maximum plasma concentration was . /À . hours. mean clearance was . /À . l/hr. auc inf was . /À . mgÃh/ml. results of this study suggest that rufinamide given orally at mg/ kg twice daily in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted. the aims of this study were to investigate the abg for (i) the prevalence of skull abnormalities; (ii) the prevalence of sm; (iii) an association between lateral ventricular size, cerebellar size and sm; and (iv) associations between sm, skull abnormalities, csf pleocytosis and clinical signs. seventy-six abgs, recruited as part of a larger epidemiological and genetic study, underwent brain and spinal mri evaluation ( . t general electric signa hdx, milwaukee, wi). all dogs were evaluated neurologically, recording deficits and the presence of spinal pain. sequences acquired included t w, t w pre-and postcontrast, and t w flair, sagittal and transverse. cervical spinal cord central canal (cc) and or syrinx size and its percent area of spinal cord was measured using osirix s . the presence of chari-like malformation (cm) was assessed by recording the presence of caudal cerebellar deviation and/or foramenal vermal herniation. lateral ventricle and cerebellar volume was expressed as a percent of the cerebrum and intracranial volume qa respectively. forty-five dogs underwent atlanto-occipital cerebrospinal fluid tap at the time of mri and the white blood cell (wbc) count was recorded. student's t-tests were used to compare the measured variables between groups with and without skull abnormalities, spinal pain and neurological signs. the mean age of the males ( intact) and the females ( intact) was . months (range - ; median months). neurological deficits and neck pain were noted in ( %) and ( . %) of dogs respectively; dogs ( . %) exhibited both. cerebellar deviation and vermal herniation were present in ( . %) and ( . %) dogs respectively; twenty-three dogs ( . %) had both. mean height of the cc was . mm ( - . mm). forty ( . %) ccs were greater than mm in height; the mean length of these lesions was . vertebrae ( . - ). mean csf wbc count was . /ml ( - ). syrinx height and extent were significantly higher in dogs with neurological signs (size p . ; extent p . ). there were no significant differences in syrinx sizes and extent in dogs with or without skull abnormalities or spinal pain. there were no associations of syrinx height or extent with csf wbc count or age of dog. intact females had a significantly lower syrinx extent than intact males (p . ). there were no significant differences in presence of spinal pain or neurological signs between dogs with or without skull abnormalities. there was a significant negative association of ventricular percentage and cerebellar percentage (p o . ). there was a significant association of ventricular percentage with syrinx percentage (p . ) and height (p . ). this study suggests that sm and cm are prevalent in abgs. syrinx size and extent are associated with neurological signs and ventriculomegaly is associated with both small cerebellar size and large syrinx size. however, sm may not be associated with cm as defined by cerebellar herniation and deviation and is not associated with csf inflammation. the power tissue resection device (ptrd) is a hand-piece comprised of an outer cannula with motor driven vacuum-assisted inner cutting blade. this device was designed and is marketed for human neurosurgical brain/spinal cord tumor resection. the purpose of this study is to describe the use of the ptrd for intervertebral disc fenestration and to compare the effectiveness of manual fenestration to that of the ptrd. fifteen cadaveric lumbar spines were randomly placed into three study groups: group was the control group on which no fenestrations were performed, group was the manual fenestration group and group was the ptrd fenestration group. the effectiveness of fenestration via both manual and ptrd was assessed by calculating the ratio of remaining nuclear weight post fenestration to total nuclear volume. discs with lower ratios were more effectively fenestrated. results showed a smaller ratio of post fenestration remaining nuclear weight to nuclear volume following fenestration with the ptrd ( . ae . ) as compared to manual fenestration ( . ae . ). these results did not show statistical significance. when fenestrated samples were compared to control samples ( . ae . ), there was a statistically significant reduction in ratios. in conclusion, the ptrd is easy to use and is as effective as the manual technique for canine intervertebral disc fenestration. according to the human who classification gliomatosis cerebri (gc) is a rare astrocytic tumor affecting at least three lobes of the brain with extensive infiltration, but relative preservation of brain architecture. gc has not been reported to occur as a hereditary disease, neither in man nor in animals. here, we report the temporally clustered occurrence of gc in a family of bearded collies. a years old female bearded collie with forebrain signs was presented. differentials included inflammatory/ infectious, metabolic/ toxic, and neoplastic diseases. within a time period of months, offspring of this bitch were presented with similar clinical signs. two dogs were full siblings ( males). the remaining female dog originated from a match with a different male dog. mri was performed in all dogs and revealed a diffuse and extensive intra-axial lesion with moderate mass effect and midline shift. the ill defined lesion showed mainly a white matter distribution with hyperintense signal in t -w and flair images and iso-to hypointense signal in t -w images without contrast enhancement. the lesion was bilateral in all cases, continued along the white matter extending partially into the gray matter with contact to the brain surface. neuropathology revealed a diffuse and extensive infiltration of the brain and spinal cord by a neoplastic glial cell population involving white and gray matter of both hemispheres, thalamus, brainstem and cerebellum in all dogs. based on the cell morphology and immunoexpression of glial fibrillary astrocytic protein by neoplastic cells diagnosis of gc was made. this is the first report of familial occurrence of gc, which is likely the result of a germ-line mutation. several human hereditary cancer syndromes are associated with cns tumors including amongst others the li-fraumeni cancer family syndrome (p mutation), neurofibromatosis (type and ) (neurofibromin, merlin mutation), and tuberous sclerosis (hamartin, tuberin mutation). furthermore, familial clustering of human gliomas unassociated to the known inherited cancer syndromes has been described. in the dog, hereditary cns tumors are not known. the exact mode of inheritance and putative gene mutations of gc in this bearded collie family are currently under investigation. preliminary results are consistent with a monogenic autosomal dominant mode of inheritance, although a recessive inheritance cannot be completely ruled out at this time. mutations in the tp gene were not found following amplification and sequencing of exons - in affected dogs. previously presented at the ecvn annual meeting in cambridge, uk. the gm gangliosidoses are characterized by a deficiency of bhexosaminidase. there are two isoforms: hex a composed of an a and b subunit encoded by hexa and hexb genes respectively and hex b with two b subunits. hex a requires an activator encoded by gm a. two japanese chin dogs with confirmed gm gangliosidosis showed elevated total hexosaminidase and normal hexosaminidase a activity, a pattern associated with the ab variant in humans and consistent with prior reports in the breed. this study was performed to identify the mutation responsible using resequencing with an applied biosystems xl dna analyzer as previously described (awano ). mutations in gm a cause the ab variant in humans, but resequencing gm a revealed no mutation that could account for the disease. resequencing hexa and hexb revealed a c. g a mutation in hexa which was homozygous in both affected dogs. sixty-five normal japanese chin dogs were screened for the mutant allele; were homozygous for the ancestral allele and heterozygous. this mutation predicts a p. e k substitution affecting one of two primary active-site amino acids that participate in the hydrolysis of gm ganglioside. substitution of a lysine residue at this site is likely to eliminate subunit a enzymatic activity. the apparently normal levels of hexosaminidase a activity in affected dog samples may be a result of b subunit overexpression. human hex b possesses low levels activity against the artificial substrate used to assess hex a activity, but specificity of activity of the canine enzyme is not known. previously presented at the american society for neurochemistry: additional data in this abstract. phenytoin (pht) is the intravenous drug of choice in humans for seizure emergencies following benzodiazapines. iv fosphenytoin (fos) is a pht pro-drug which causes less administration related adverse events. while the short half-life of pht is not suitable for chronic oral therapy in dogs, iv fos has not been studied. two dogs received mg/kg phenytoin equivalent (pe) and two dogs received mg/kg pe of fosphenytoin intravenously at a rate of mg pe/min. blood for plasma levels were drawn at time-points over hours; total and unbound drug levels were measured by hplc. vital signs including ekg, blood pressure, and neurological examination were monitored. the half-life of metabolism of fos to pht was $ min, with % of fos metabolized to pht by minutes. eighty to % of pht was protein-bound during the first minutes after dosing, compared to - % in humans. the elimination half-life for total pht ranged from . - . hours and for unbound pht ranged from . - . hours. dogs receiving mg/kg pe intravenously achieved unbound pht plasma maximum concentrations of . - . ug/ml at minutes, consistent with human loading dose levels. adverse events observed in some dogs included vomiting, mild ataxia, and short lived tremors, the severity of which appeared dose dependent. all dogs were clinically normal within minutes of all doses. a mg/kg pe dose of iv fos appears adequate for production of pht levels predicted to be effective for the treatment of canine seizure emergencies. further studies in clinical canine patients are warranted. acquired myasthenia gravis (mg) is caused by antibodymediated inactivation of the acetylcholine receptor on the neuromuscular endplate causing focal, regional or generalized muscle weakness. many medical treatments have been reported; however, responses to therapy and outcomes are unpredictable and death often results from aspiration pneumonia. therapeutic apheresis is an extracorporeal procedure that separates blood into its components for removal or specific alteration prior to return to the patient. therapeutic plasma exchange (tpe) is an apheresis treatment in which plasma (containing pathologic antibodies) is removed and exchanged with donor plasma. tpe is used routinely to treat mg in human patients with severe disease or disease unresponsive to conventional therapy. we report the successful use of tpe to treat large breed dogs with confirmed mg (aceytlcholine receptor antibody concentration: . and . nm/l, respectively; normal concentration: o . nm/ l) that was severe and not adequately responsive to traditional therapies. both dogs were non-ambulatory, recumbent, and demonstrated megaesophagus and aspiration pneumonia. three tpe treatments ( plasma exchange each) were performed over and days, respectively, in each dog without complication. both dogs became ambulatory within days of starting tpe treatment with subsequent resolution of regurgitation and megaesophagus. pyridostigmine was continued during tpe sessions and discontinued in both dogs within - months. both dogs remain asymptomatic and have had no recurrence of mg during and months of follow-up, respectively. tpe is a viable treatment option for dogs with mg that have severe disease, life-threatening complications or that remain unresponsive to traditional therapies. tpe may alleviate clinical signs more rapidly, and improve long-term outcomes when compared to historical experiences in patients with comparable disease. clinical findings, clinicopathologic data, imaging features, and treatment of canine spinal meningiomas have been described in the veterinary literature, but histological characteristics and tumor grading have less commonly been reported. the aims of this retrospective case series were to describe the clinical, imaging, and histologic features of seven canine spinal meningiomas including a cervical spinal cystic meningioma that had imaging and intraoperative features of a subarachnoid cyst. medical records from dogs with a histopathological diagnosis of spinal cord meningioma presented to the veterinary teaching hospital between and were reviewed. signalment, presenting clinical signs, physical and neurologic examination, clinicopathologic data, surgery reports and available images were reviewed. all meningiomas were histologically classified and graded following the international who human classification for cns tumors. seven dogs were included, males and females. median age at presentation was . years (range, . - . years), and median weight was kg (range, - kg) . median time between onset of clinical signs and diagnosis was days (range, days - year). cerebrospinal fluid (csf) analysis was performed in dogs, showing increased protein concentration in cases, and being normal in the other . spinal radiographs revealed vertebral canal widening in one case. myelography ( / ) showed intradural/extramedullary lesions in three cases, one of them consistent with a csf-filled subarachnoid cavity, and an extradural lesion in one case. magnetic resonance imaging (mri) was performed in all cases and revealed mild to marked hyperintensity on t w and precontrast t w images and homogeneous contrast enhancing (ce) intradural/extramedullary masses ( cervical and thoracic) in six cases, with one of these showing an additional intramedullary ce pattern. a dural tail was identified in two dogs. one dog had a fluid-filled subarachnoid enlargement located dorsally to the spinal cord. this lesion was hyperintense on t w, hypointense on t w and flair images, and did not enhance. it was diagnosed as a spinal subarachnoid cyst, but the histopathological study of the surgically resected mass revealed a grade i cystic meningioma. five other cases underwent cytoreductive surgery, two transitional meningiomas (grade i) that survived (alive at the time of writing) and months; and three anaplastic meningiomas (grade iii) that survived - . months before neurological deterioration and euthanasia. another anaplastic meningioma was euthanized right after diagnosis. there are few reports grading canine spinal meningiomas, with most being grade i or ii. of the few grade iii tumors reported, only one had been treated surgically and was euthanized days later because of neurological deterioration. we report four grade iii (anaplastic) meningiomas, three of which surgically treated and with longer survival times. finally, cystic meningioma should be considered in the differential diagnosis of cases with imaging features consistent with arachnoid cyst because of their similar appearance, making histopathological analysis essential for a definitive diagnosis. head trauma is a common veterinary emergency, but few prognostic indicators have been studied in dogs, making it challenging for clinicians to counsel clients about the odds of recovery. a recent meta-analysis showed that higher plasma glucose, lower plasma ph and lower hemoglobin at admission were associated with increased risk of death in human head trauma. the goal of this retrospective study was to investigate the association between admission point of care blood gas parameters and survival to discharge in dogs with head trauma. fifty one dogs presenting to the cornell university hospital for animals with head trauma from to that had a blood gas analysis done within hour of presentation were eligible for inclusion. parameters assessed included glucose, base excess (be), anion gap (ag), ph, hemoglobin, and sodium. biochemical data were found to be normally distributed using the kolmogorov-smirnov test. t-tests or welch tests were used to compare parameters between survivors (s,n ) and non-survivors (ns, n ). of glucose, be, ag, ph, hemoglobin, and sodium, only mean glucose (s mg/dl, ns . mg/dl, p . ) was significantly different between groups, although there was a trend for a difference in mean be (s À . , ns À , , p . ). logistic regression analysis showed that of the parameters, only be was independently associated with outcome (odds ratio . , % ci . - . , p . ). these results suggest that two easily measured biochemical parameters (glucose and be) may yield useful prognostic information in dogs with head trauma, but further studies are needed to further elucidate these findings. type i intervertebral disc disease (ivdd) commonly affects chondrodystrophic dogs. neurological recovery and outcome following surgical decompression may be unpredictable due to suspected ischemic neuronal injury. hyperlactatemia has been associated with spinal cord injury in humans and experimental animals. the purpose of the study was ) to determine the relationship between serum and csf lactate levels and ) to compare lactate levels with neurological outcome following decompressive surgery in dogs with ivdd. healthy, chondrodystrophic dogs diagnosed with ivdd localized to the t -l spinal cord were included. serum lactate levels were obtained at: anaesthetic induction, skin incision, muscle dissection, and extubation. in patients with hyperlacatemia at extubation, additional samples were obtained. csf was analyzed for lactate concentration. neurological status was recorded at presentation and multiple times during the recovery period. dogs were included in the study ( - years old). / dogs had normal lactate levels throughout the study. / dogs had serum hyperlactatemia prior to anaesthetic induction; / dogs returned to normal during anaesthesia and / dogs had continued hyperlactatemia until the end of the observation period. neurological status of the dogs varied similarly between all groups. in / dogs where csf lactate levels were measured, initial serum levels were lower than csf lactate levels; in / dogs where csf and serum were collected simultaneously, serum lactate concentration was consistently lower than csf lactate. no association between presenting neurological status or neurological outcome and serum or csf lactate concentration was made. neither serum nor csf lactate concentration is useful for predicting neurological outcome in dogs with ivdd. chiari-like malformation (cm) has been associated with syringomyelia (sm) in cavalier king charles spaniel (ckcs) and is postulated to result from a mismatch between the volume of the caudal cranial fossa and the brain parenchyma contained within. the objective of this study was to assess the role of cerebellar volume in caudal cranial fossa overcrowding and syringomyelia. three dimensional models were created using t -weighted transverse magnetic resonance images in the commercial software package mimics s . volumes of cerebellar parenchyma were analyzed as percentages of caudal cranial fossa volume (cerebellar caudal cranial fossa percentage) and total brain parenchyma volume (cerebellar brain percentage). data was assessed for normality and the appropriate statistical test was used to compare means/medians between groups. forty-five small breed dogs (sb), ckcs and labradors (ld) were compared. as sm is thought to be a late onset disease process, two subgroups were formed for comparison: ckcs younger than years with sm (group ) and ckcs older than years without sm (group ). ckcs had a larger cerebellar caudal cranial fossa percentage than the other groups . %] vs. sb . % [ . - . %] and ld . % [ . - . %]; p o . ). the cerebellar brain percentage was also larger in ckcs compared to the other groups (ckcs . % [ . - . %] vs. sb . % [ . - . %] and ld . % [ . - . %]; p o . ). group had a significantly larger cerebellar caudal cranial fossa percentage than group ( . % ae . vs. . % ae . , p . ) and a significantly larger cerebellar brain percentage ( . % ae . vs. . % ae . , p . ). our findings show that the ckcs has a relatively larger cerebellum than small breed dogs and labradors and there is an association between increased cerebellar volume and sm in ckcs. chiari-like malformation (cm) is nearly omnipresent in the cavalier king charles spaniel (ckcs) breed. the mis-match of the caudal cranial fossa and the parenchyma within is thought to lead to syringomyelia (sm). there is currently a lack of information if the morphological changes seen in ckcs with cm are progressive or non-progressive. in this retrospective study we used established measurements of cerebral volumes, foramen magnum height and cerebellar herniation length to assess if there is a significant difference between subsequent magnetic resonance (mr) imaging of the brain of the same dog. electronic patient records were reviewed for ckcs with cm which had two separate mri scans, which were a minimum of months apart. ckcs with diseases affecting measurements were excluded. for the volumetric measurements three-dimensional models were created using t -weighted transverse mr images in the medical imaging software (mimics v . , materialise n.v, ) . volumes of the caudal cranial fossa parenchyma were analyzed as percentages of caudal cranial fossa volume and caudal cranial fossa volume was analyzed as a percentage of total cranial cavity volume. the volume of the ventricular system was recorded as a percentage of total parenchymal volume. data was assessed for normality and the appropriate statistical test was used to compare means/medians. twelve ckcs were included with a median scan interval of . months ( - months). the size of the foramen magnum increased significantly between the first and second scan ( . ae . cm vs. . ae . cm; p . ), as did the length of cerebellar herniation ( . ae . cm vs. . ae . cm; p . ) and the caudal cranial fossa percentage ( . % [ . - . %] vs. . % [ . - . %]; p . ). there was no significant difference noted between the two time points in any of the other volumetric measurements ( this work could suggest that overcrowding of the caudal cranial fossa in conjunction with the movements of cerebrospinal fluid and cerebellar tissue secondary to pulse pressures created during the cardiac cycle causes pressures on the occipital bone. this leads to a resorption of the bone and therefore an increase in caudal cranial fossa and foramen magnum size allowing cerebellar herniation length to increase. the cord dorsum potential (cdp) is a stationary potential arising in dorsal horn interneurons after stimulation of sensory nerves. cdps have been recorded in normal anesthetized dogs previously, and normal latency values have been determined for tibial and radial nerves. this study was undertaken to determine whether cdps could be reliably recorded from the caudal nerves in normal dogs, thus allowing electrophysiological assessment of the cauda equina, and whether neuromuscular blockade improved recording quality. ten adult dogs weighing from . to . kg were anesthetized and cord dorsum recordings were compared before and after administration of atracurium. recording needles were placed onto the dorsal lamina at intervertebral sites from l /s to l / . stimulations were made on the lateral aspect of the caudal vertebrae approximately - cm from the tail base. recordings from stimulations were averaged. cdps were recorded successfully in all dogs. onset latency varied from . to . ms. the cdp was largest when recorded closest to the site of entry of the stimulated nerve into the cord, as determined by post-mortem examination immediately after testing in dogs. administration of atracurium did decrease muscle artifact, and in some cases helped isolate the origin of the cdp. these data show that cdps can be readily assessed from the caudal nerves of anesthetized dogs, with or without atracurium. cord dorsum potentials from caudal nerves may add important information about the integrity of the cauda equina in dogs with suspected degenerative lumbosacral stenosis. canine intracranial glial tumors and many human brain tumors express heat shock proteins (hsps) associated with their degree of malignancy. the up-regulation of hsps during tumor cell growth helps keep tumor proteins stable and therefore makes them a reasonable target for therapy. ki expression and ec have been strong indicators of cell proliferation and dedifferentiation, respectively.the aims of this study were to determine (i) if canine meningiomas express hsp and/or hsp ; (ii) whether the expression of the hsps was associated with ki and/or e-cadherin (ec) expression; and (iii) whether peritumoral edema was associated with hsp, ki and/or ec expression. forty-one formalin-fixed, paraffin-embedded canine intracranial meningiomas underwent immunohistochemical staining using anti-hsp , or antibodies. these tumor samples were also immunohistochemically stained for ki and ec expression. canine mammary carcinoma and squamous cell carcinoma tissues served as the control samples, as both have previously been shown to express hsps. skin was used as control for ki and ec. four non-overlapping high power fields of each stained sample were selected and cell staining was analyzed using a semi-quantitative method for hsps and ki ; a qualitative assessment was used for ec. all analyses were performed using sas v . (cary, nc). descriptive statistics of staining percentages were calculated for all tumors tested. simple pearson's correlation was used to test for correlations of ec area with hsp areas and ki- percent positive cells and of ec intensity with hsp intensities and ki- percent positive cells. all hypothesis tests were sided and the significance level was a . . thirteen meningiomas had mr images quantitatively evaluated for peritumoral edema using t flair sequences. the edema index (ei) was evaluated for an association with hsp , hsp , ec and ki expression. hsp was expressed in % (mean . % of cells; range - %), hsp in % (mean . % of cells; range - %) and ec in % of meningiomas. there was no association demonstrated between either hsp expression variable and ec or ki- expression. there was also no association between the ec expression variables and ki- . however, there was a significant negative association between hsp extent (p . ) and area (p . ) with ei. in conclusion, hsp and expression was demonstrated in canine intracranial meningiomas but was not associated with ki- or ec expression. this study suggests that hsps may not have a significant role in the maintenance of canine meningiomas and so do not represent a novel treatment target for this group of tumors unlike canine glial cell tumors. however, hsp may be involved in the pathogenesis of peritumoral edema in meningiomas and warrants further investigation. an extended release (xr) formulation of levetiracetam, a second generation antiepileptic drug, was recently approved for human use on a once daily basis. although levetiracetam is clinically effective for seizure control in dogs, it requires a three times daily administration. the potential benefits of the xr formulation include reduced daily dosing leading to improved compliance and relatively constant plasma concentrations. the aim of this study was to compare the pharmacokinetics of levetiracetam xr tablets with immediate release (ir) tablets following single dosing in dogs. five clinically and neurologically normal mixed breed dogs were used in a cross-over design. all dogs (mean body weight . kg; range . - . ) had normal hematology, serum chemistry and urinalyses. following a hour fast, each dog was administered oral ir levetiracetam ( mg; mean dose . mg/kg; range . - . ). heparinized blood for drug analysis was taken from each dog prior to administration and . , . , . , , , and hours after. blood was immediately centrifuged and supernatant plasma was stored at À c until analysis. after a day wash-out period, each dog was administered mg oral xr levetiracetam and blood samples were taken at identical timings. plasma samples were thawed at room temperature before preparation by solid phase extraction for hplc analysis. reverse phase chromatographic separation was performed. levetiracetam and an internal standard were detected using ultraviolet spectroscopy at nm. concentrations of levetiracetam were determined by peak area comparison to the internal standard. mean data were fit to a one compartment pharmacokinetic model with first order elimination and absorption and included a lag-phase for xr formulation. no adverse clinical effects were noted in any of the dogs. the auc associated with xr was hr ug/ml, a . fold increase over that with ir ( . hr ug/ml). the absorption half-life was . hr with xr and . hr with ir, a . fold difference. the elimination halflife was . hr with xr and . hr with ir, a . fold difference. the tmax associated with xr . hr and . hr with ir, a . fold difference. the cmax associated with xr was . mg/ml and . mg/ml with ir, a . fold difference. the plasma concentration of ir levetiracetam was not detectable at hr after administration whereas it was greater than mg/ml at hr after xr administration. based on the auc data, there is an approximately fold increase in bioavailability of the xr compared to the ir formulation. the cmax was approximately times greater following xr administration and a high plasma level in excess of the suggested canine therapeutic concentration ( mg/ml) for at least hours. although specific dosing recommendations cannot be made from this data, the favorable pharmacokinetics of xr over ir suggests that single, daily administration could be efficacious. thoracic and lumbar vertebrae are frequently affected by fractures and or luxations in dogs following trauma. surgical repair is part of the emergency treatment described for this disorder but does not guarantee improvement of the associated clinical signs. multiple surgical repair techniques have been described but have not been compared in terms of their success and the factors associated with a positive outcome. the aims of this study were to retrospectively evaluate the effect of different types of vertebral repair, injury type and injury location on outcome in dogs with thoracolumbar (tl) and lumbosacral (ls) spinal trauma. medical records were searched for dogs with radiographic evidence of a tl or ls vertebral fracture and or luxation ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; signalment, body weight and duration of disease were recorded. dogs were retrospectively scored neurologically ( - ; normal to plegic with absent pain perception) on admission and at re-evaluation following surgery. lesion location was classed as t -l and l -s ; dogs were evaluated as one group and as two separate groups with respect to outcome. a subset of lesions were classed as cord compression or not based on advanced imaging. three repair techniques were evaluated (i) pins and polymethylmethacrylate (pmma); (ii) screws and pmma; and (iii) spinal stapling. regression analysis was applied to test for an association between the type of surgery and a successful outcome (non-painful and ambulatory). simple bivariate analyses were performed to investigate for variables predictive of a successful outcome. fifty-nine dogs were included. twenty-eight dogs were classed as t -l and were l -s . there were dogs with fractures and with luxations; dogs had both. thirty-one of dogs evaluated had spinal cord compression. ten dogs were repaired with im pins and pmma, dogs with screws and pmma and dogs with spinal stapling. overall, there was a . % success rate; there was no significant difference in outcome between the anatomic sites (p . ). all dogs initially graded as - pre-operation were classed as a successful outcome after at least one week following surgery; % of dogs initially graded as (plegic with pain perception) were classed as successful recovery. one dog ( . %) initially as graded as (plegic with no pain perception) had a successful outcome. a low admission score was statistically predictive of a successful outcome (p o . ). surgery type was not associated with a successful recovery (p . ). signalment, body weight, location of injury, injury type (fracture, luxation or both), presence of compression, and duration of disease did not predict outcome. from this study, the successful recovery of dogs following surgical fixation is high and is only dependent on the neurological score at the time of admission. the choice of surgical technique does not seem to influence outcome although a prospective study comparing two surgery types is warranted to further investigate this issue the results of which can be confounded by surgeon experience and variable follow-up. cranial thoracic intervertebral disc disease (ivdd) is extremely rare due to the presence of the intercapital ligament, although anecdotic data suggest german shepherd dogs (gsd) can share some predisposition for this disorder. the objective of the study was to retrospectively evaluate through mri if cranial thoracic ivdd is significantly more common in gsd compare to other large breed dogs. a search was done through database of the ontario veterinary college. any gsd were a spinal mri including t -t spine was performed was recruited. a group of large-breed non-gsd was used as a control. in the midsaggital t wi plane, three variables were assessed and graded for each intervertebral disc space t -t : spinal cord compression (scc), disc degeneration (dd), and herniation. wilcoxon sign rank test was used to assess if scores were different between groups. exact conditional logistic regression was used to determine whether any intervertebral disc space was a risk factor. gsd and large breed non-gsds were recruited. the gsd group had significantly higher scores than the non-gsd for scc, and herniation. regarding the individual intervertebral discs, in the gsd group t - , t - , t - discs had significantly an increased risk for scc, and t - for herniation. the results of this study show that gsd have a higher risk of cranial thoracic disc ivdd than other large breed dogs. that risk was higher in discs t -t , t - , and t - , particularly in t - . genetic and/or conformational factors, such as weakness of the intercapital ligament, may predispose gsd to this lesion. diskospondylitis is a common disease of the canine spine; however, few reports of mr imaging findings in dogs are available. the purpose of this study was to describe the signalment, clinical and mr imaging features in affected dogs. twenty-three dogs with a diagnosis of diskospondylitis based on clinical signs, mr imaging, and urine, blood, csf and/or intervertebral disk cultures were included. large breed dogs ( kg) accounted for of the cases. the mean age was . years with males and females equally represented. most dogs ( / ) were ambulatory with varying degrees of pain and paresis. mr imaging characteristics of sites were reviewed. on t w images, vertebral endplates were of mixed signal intensity ( / ) while the vertebral body was hypointense ( / ). the intervertebral disk space was hyperintense on t w ( / ) and stir ( / ) images and mixed signal intensity ( / ) on t w images. paravertebral soft tissue hyperintensities were noted on / t w and / stir images. contrast enhancement occurred at / endplates and / intervertebral disk spaces. only / vertebral bodies and / parvertebral soft tissues contrast enhanced. intramedullary spinal cord t w hyperintensity was noted at / sites. spinal cord or cauda equina compression occurred at / sites. based on the spearman correlation coefficient, a significant direct correlation was found between the degree of spinal cord or cauda equina compression and the patient's neurologic status (p . ). the incidence and severity of spinal cord compression in canine diskospondylitis may have prognostic value and may have been previously underestimated using other imaging modalities. hemilaminectomy and pediculectomy are both well described and commonly utilized techniques to access the spinal canal. these procedures are most often performed to approach a compressive lesion, such as intervertebral disc disease and neoplasia, the goal being adequate visualization of the spinal canal and access to the offending lesion. a proposed benefit of pediculectomy is preservation of the articular facets and thus better maintaining stability of the vertebral column, but at the cost of reduced access to the spinal canal. the purpose of this study was to describe standardized anatomical limits of each technique and report any observed differences that could be considered during presurgical planning. ten canine cadavers had both procedures performed on opposite sides to access the t - , t -l , and l - spinal canal. measurements were obtained after performing a computed tomography study of the spine and recorded from the transverse slice most representative of the defect. the surgical technique, vertebral site, and side of vertebral column were compared with the mean spinal canal and defect height using a covariate model. dorsal and ventral remnant lamina heights were also compared. the height of the defect relative to the spinal canal was - % with hemilaminectomy and - % with pediculectomy. the observed difference in defect height of - % (p o . ) and varied with spinal canal height. dorsal remnant lamina height was . - . % of spinal canal height with hemilaminectomy and - % with pediculectomy. ventral remnant lamina height ranged from - % and . - . %, respectively, though the difference was not statistically significant. while a larger defect is expected with a hemilaminectomy procedure, our results demonstrate that this difference increases with increasing spinal canal height. interestingly, the proportion of exposed spinal canal decreases with increasing canal height for both procedures. the difference in defect height between techniques was due to greater removal of the dorsal spinal canal, possibly making the hemilaminectomy technique better suited for more dorsal lesions, while no statistically difference in access to the ventral canal is observed. no effect of vertebral site was detected. of note was the involvement of articular facets in half of the pediculectomy defects, involving an average of % of the articular facet height. this result questions the suggested benefit for the vertebral stability, but further biomechanical studies would be required. low level laser therapy (lllt) is a treatment used in human and veterinary medicine for a variety of clinical syndromes. some uses in human medicine include acute pain associated with osteoarthritis, rheumatoid arthritis, tendonitis, tmj disorders, chronic joint disorders, and wound healing. research is currently on-going to determine the adequate wavelengths to promote effective treatment results with lllt in these conditions. it is purported that lllt acts via the mitochondria to increase cellular metabolism promoting wound healing and a decrease in pain and inflammation. in this study, we hypothesized that dogs treated with lllt in conjunction with hemilaminectomy would display quicker recovery times regardless of the presence or absence of deep pain sensation. seventeen dogs ( dachshunds, chihuahuas, french bulldogs, lhasa ahpsos, and each of a pembroke welsch corgi, and a miniature poodle) were selected and divided into two groups. the dogs ranged in age from to years old, weighed between and pounds, and underwent hemilaminectamies after acute onset of paraplegia secondary to intervertebral disc disease (surgically confirmed). one group received laser treatments on days through of hospitalization. the second group did not receive lllt, but followed the same peri-operative medication protocol. the laser used in this study was an erchonia laser model pl ( nm). the hertz setting was similar for each patient using the previously established protocol for intervertebral disc disease (ivdd) with pulse rate ranging from hz to hz. all dogs received advanced imaging pre-operatively with myelogram or mri. results of the study revealed that treatment with lllt of nm wavelength did not shorten or improve recovery times for dogs with acute onset paraplegia secondary to ivdd after hemilaminectomy procedures. dogs that showed recovery to ambulation at the two week recheck were consistently dogs that were deep pain positive on presentation. a lengthened recovery time or no recovery was seen in the majority of those dogs with absent deep pain on presentation as has been revealed historically in past studies. lllt did not appear to have an effect on this result. however, there are few data describing normal glucose uptake of the canine brain for comparison with suspected or confirmed disease. thus the purpose of this study was to assess the normal distribution of fdg uptake of canine brain structures using a high-resolution research tomography-pet and t-magnetic resonance imaging (mri) fusion system. fdg-pet and t -weighted mr imaging of the brain were performed on healthy laboratory beagle dogs. acquired pet and mr images were automatically co-registered by the image analysis software. on mr images, regions of interest (roi) were manually drawn over intracranial structures, including gross structures (whole brain, telencephalon, diencephalon, mesencephalon, dorsal metencephalon, ventral metencephalon and myelencephalon). a standard uptake value (suv) and relative suv ratio (rsuv suv of roi/suv of whole brain) were calculated for each roi. t-mr images compensated the low anatomical resolution of pet qj;by proving good spatial and contrast resolution for the identification of the clinically relevant brain anatomy. among gross structures, mesencephalon and ventral metencephalon had the highest (suv: . ae . ; rsuv: . ae . ) and the lowest (suv: . ae . ; rsuv: . ae . ) fdg uptake respectively. when suvs were calculated on detailed regions, rostral colliculus and corpus callosum had the highest (suv: . ae . ; rsuv: . ae . ) and the lowest (suv: . ae . ; rsuv: . ae . ) value respectively. these data acquired from normal dog brain will be used in clinical neurology to investigate various intracranial diseases such as inflammation, neoplasm and behavioral disorders. degenerative lumbosacral stenosis (dlss) is a multifactorial condition affecting predominantly large breed dogs. the combination of stenosis and compressive neuropathy cause lumbar pain, lameness and neurologic dysfunction. previous reports describe urinary and fecal incontinence in severely affected dogs. the objectives of this retrospective case series were to describe the clinical signs associated with dysuria and eventual diagnosis of dlss in dogs, and to describe factors associated with regained micturition following prompt diagnosis and treatment. medical records from the university of georgia and the university of missouri between and of dogs were reviewed. inclusion required observation of dysuria, urine retention, absence of structural lower urinary tract disease and concurrent presumptive diagnosis of dlss. dysuria was defined as inability to initiate or sustain a urine stream. urine residual volume was evaluated postvoiding. dysuria was further evaluated using urethral contrast studies, urodynamic testing (urethral profilometry ( ) and cystometry ( )), ultrasonography ( ), and urine culture ( ). presumptive diagnosis of dlss was based on imaging using plain radiography and epidurography ( ), computed tomography ( ) or magnetic resonance imaging ( ). breeds represented included the german shepherd dog (n ), golden retriever (n ), burnese mountain dog (n ), and each labrador retriever, weimaraner, rottweiler and mixed-breed. all dogs were male. were intact at onset of clinical signs. median body weight was . kg (range . - ) and median age was years (range - ). median duration of clinical signs prior to admission was months (range . - ). other pertinent presenting clinical signs included dyschezia ( ), fecal incontinence ( ), general proprioceptive ataxia ( ), weakness ( ), and difficulty rising ( ). physical examination findings included pelvic limb muscle atrophy ( ) and prostatomegaly ( ). abnormal neurologic examination findings included postural reaction deficits ( ), hyporeflexia ( ), decreased tail tone ( ) and lumbosacral hyperesthesia ( ). neurologic examination was normal in dogs. dorsal laminectomy was performed and diagnosis confirmed in dogs; recovery was monitored for a median of . months (range . - ). three of the dogs ( %) regained normal micturition within . - . months of surgery. though not statistically significant, dogs that regained micturition tended to have a shorter duration of clinical signs (median . months, range . - ) versus dogs that remained dysuric (median months, range - ). two of the dogs that regained micturition were neutered at the onset of clinical signs, but only of dogs that remained dysuric was neutered. signs improved in all dogs with postural reaction deficits and decreased tail tone. hyperesthesia resolved in of dogs ( %) and fecal continence returned in of dogs ( %). these findings suggest that following prompt diagnosis and surgical decompression, normal micturition could be regained in dlss affected dogs presenting with signs of dysuria. glycogen storage disease type ia (gsdia; von gierke disease) is an inherited metabolic disorder resulting from a deficiency of glucose -phosphatase-a (g pase). previous reports indicate that clinical manifestations of gsdia occur only in individuals with homozygous expression of a p.i l mutation. heterozygote dogs (het) have been previously reported to exhibit an overall normal outward phenotype. the purpose of this report is to briefly describe some differences that have been observed between het and homozygous wild type (wt) dogs. a colony of dogs at the university of florida contains a mix of affected, wt, and het individuals. in the course of studies designed to determine the effectiveness of gene therapy for correction of gsdia in dogs, both wt and het dogs have been utilized as controls. available information about body weights, clinical pathology tests, fasting studies, and liver biopsies was retrieved from records for both wt and het dogs and compared. although birth weights are similar, het dogs have a slower average rate of weight gain than wt dogs and this difference is especially prominent during the first few months of life (figure ) . in contrast to affected dogs, both wt and het dogs are able to maintain normal blood glucose concentrations for up to - hours of fasting, however, after longer fasts of - hours, het dogs have lower glucose and higher lactate concentrations (table ). in addition, liver biopsy samples from het dogs had greater apparent levels of glycogen suggested by pas staining than did samples from wt dogs, and this correlated with the results of proton magnetic resonance spectroscopy which demonstrated . times greater glycogen content in a liver biopsy sample from a het dog compared to a sample from a wt dog. together, these findings suggest that the level of g pase activity in heterozygote dogs does not provide a completely normal physiological, biochemical, or histological phenotype as previously reported. the glucokinase gene (gck) encodes an enzyme involved in cellular glucose-sensing mechanisms in pancreatic beta cells and hepatocytes. gck mrna is present in feline pancreas but the gene is not expressed in feline liver. hepatic gck expression is abundant in omnivores so its absence may reflect an evolutionary adaptation of strict carnivores, like feline species. we hypothesized speciesspecific features in the gck hepatic promoter may underlie the gene expression pattern observed in cats. the putative feline gck (fgck) promoter region was located using bioinformatic software to identify homology with human gck (hgck). genomic dna from a dsh cat was subjected to direct sequencing using a series of pcr reactions with speciesspecific primers. dna clones thus obtained were aligned to generate the feline sequence. direct sequencing yielded . kb of genomic dna sequence with high homology with sequences (acbe , acbe ) archived in the feline genome project. the feline sequence had six regions homologous with non-coding regions of hgck; four of these conserved regions are upstream of the putative fgck start. a . kb segment immediately upstream of feline hepatic exon is not present in hgck. the . kb insert is the reverse complement of a conserved sequence located downstream of exon in feline and human sequences. in conclusion, the putative hepatic promoter of fgck shares extensive homology with the hgck promoter but contains a . kb insert not found in hgck. functional studies are needed to confirm the role of the unique insert in regulation of fgck gene expression. deuterium oxide (d o) dilution has been proposed for quantifying body water content, but remains difficult to perform routinely. the objective of this study was to assess if the volume of distribution (vd) of creatinine could be proposed as an alternative in dogs for such a measurement. creatinine and d o vd were measured before (c) and after induction (o) of obesity (by giving an hypercaloric diet ( kcal/ kg) for months) in six healthy adult beagle dogs. creatinine ( mg/kg) and d o ( mg/kg) were simultaneously injected by bolus iv. blood was collected before administration and then at , , , , , , , , and min post-injection (creatinine), and , , , , , , and min (d o) . plasma concentrations of both markers were determined. vd was calculated using pharmacokinetic equations. the body weight increased from . ae . (c) (mean ae sd) to . ae . kg (o). d o vd decreased from ae (c) to ae (o) ml/kg. similarly, creatinine vd decreased from ae (c) to ae (o) ml/kg. the individual difference between creatinine and d o vd (expressed in % of d o vd) ranged from À . to . % (c) and from À . to À . % (o). in conclusion, creatinine vd provides a good estimate of d o vd in both normal and obese conditions. a wk double blinded study was conducted comparing the affect of two foods on mobility in dogs. all work was approved by an iacuc. healthy beagle dogs ( - years old, mixed gender) were used. affected (a) and non-affected (na) dogs were identified based on orthopedic examination and radiography as having or not having evidence of naturally occurring joint pathology (presence of osteophytes, dysplasia, effusion, pain on manipulation etc) in one or more joints. a and na dogs were evenly distributed between two locations. foods had nutrient profiles adequate for maintenance according to the aafco official publication. the test food contained greater amounts of methionine, manganese, carnitine, vit. e,, vit. c, alpha linolenic acid (ala), and eicosapentaenoic (epa) acid: the food provided mg n fatty acids and mg n fatty acids per kcal. all dogs were fed the control food for wks followed by a wk feeding period where a and na dogs consumed the test food and a and na dogs the control. blood and urine were collected at weeks , , and and analyzed for serum fatty acids and urine thromboxane:creatinine ratios were determined. evaluators in this study were different than those making the original diagnosis and so were blinded as to treatment and diagnosis. orthopedic exams were performed by two veterinary surgeons at each site on weeks , , and . the same two evaluators examined the same dogs throughout. the data was evaluated for the difference between a and na dogs and between foods with age, gender and location as covariates. body weight, disease status, age and gender were blocked. analysis included anova repeated measures mixed procedure (sas version . ) to determine treatment effects over time.serum epa was greater and arachidonic acid lower at weeks , and in the test food fed dogs (p o . ). urine thromboxane:creatinine ratios were decreased in the a dogs fed the test food compared to the a dogs fed the control food at wks (p o . ). lameness score was significantly improved (p o . ) within and between groups of dogs fed the test food. a significantly greater proportion of a dogs fed the test food had improvement in total het (n ) blood glucose (mg/dl) (ae ) (ae ) blood lactate (mmol/l) . (ae . ) . (ae . ) joint score, lameness, functional disability and overall assessment score at wks compared to a dogs fed the control food. % of a dogs had an improved overall assessment score on the test food after wks and at wks compared to % at wks and % at wks of a dogs consuming the control food. this study shows that a food with moderate amounts of added linolenic acid and epa can have a positive impact on systemic inflammation and mobility in - weeks. a similar abstract will be presented at the orthopedic research society meeting in january to an audience largely of orthopedic researchers interested in human orthopedics. fat is an important dietary component, serving both as a source of energy and as a supplier of essential fatty acids (fa). medium-chain triglycerides (mct) contain intermediate length fa that do not rely on l-carnitine for transport across the inner mitochondrial membrane, bypassing this rate-limiting step in fa oxidation. longchain (n- ) polyunsaturated fatty acids (pufa) from fish oil (fo), and in particular eicosanoids derived from eicosapentaenoic acid (epa), may protect against excessive inflammatory reactions, which may be exacerbated by eicosanoids derived from (n- ) arachidonic acid (aa). this study investigated the effects of adding mct:fo and l-carnitine to a control diet (prescription diet s k/d s ) on lean body mass, and serum fa and metabolites. forty healthy beagles ( . to . y) were fed one of three foods (n to dogs each) for mo. the study protocol was reviewed and approved by iacuc, hill's pet nutrition, inc. all foods were complete, balanced, and sufficient for maintenance of adult dogs; and had similar concentrations of moisture, protein, and fat (approx. . %, . %, . %, respectively). composition of serum fa was determined by gas chromatography of fa methyl esters. metabolomic profiles of serum samples were determined from extracted supernatants that were split and run on gc/ms and lc/ ms/ms platforms, for identification and relative quantification of small metabolites. body composition was determined by dual energy x-ray absorptiometry. serum concentrations of lauric and myristic fa increased; epa and dha increased in a dose-dependent manner; and aa decreased in dogs fed treatment food (proc-mixed procedure in sas; all p . ) when compared to dogs fed treatment foods or . serum concentrations of acetylcarnitine and succinylcarnitine increased, indicating lcarnitine incorporation, in dogs fed treatment foods and . thus, a diet enriched with mct:fo significantly altered serum fa composition, enriching (n- ) pufa and lowering aa concentrations. there was no change in lean body mass for any of the diets compared to baseline values, and no difference between treatments, showing that all three treatment foods met protein requirements. ten owned dogs, obese for more than months (body condition score [bcs] of ; fat mass [fm] . ae . %) were studied. these dogs had their weight reduced by % (bcs ; fm . ae . %; p o . ) being designated weight reduced (wr) group and then were fed to maintain constant body weight during days (bcs . ; p . ), designated maintenance (main) group. a control (ct) group of beagles was also included (bcs . ; fm . ae . %; p o . ). in all groups the glucose postprandial response test was performed after hours of fasting. blood samples were taken prefeeding and after , , , , , , , , , and minutes of the consumption of cooked rice enough to the ingestion of g of starch/kg body weight. tnf-a and il- were dosed in milliplex tm map panel, insulin and leptin by radioimmunoassay. statistical analysis included paired or non-paired t-tests and wilcoxon (p o . ). the regimen normalized meal glucose response, the area under the curve (auc) of glucose for wr was lower than for obese (p o . ) and similar to main and ct (p . ). insulin secretion did not normalize immediately, as obese and wr exhibited similar auc of insulin and higher values than for ct (p o . ). main, however, presented similar auc of insulin than ct, with lower values than obese and wr (p o . ), suggesting that dogs require some time to adapt their metabolism. leptin, tnf-a, and il- presented significant reductions after weight loss (p o . ), without differences between wr, main and ct (p . ), suggesting an improvement of the pro-inflammatory state consequent to obesity. studying food base excess (be) modification, methionine intoxication was described. in a basal kibble dog diet (be meq/kg; . g/kg of s) two dosages of ammonium sulphate and methionine was added, resulting in diets with be of meq/kg ( . g/kg of s) and À meq/kg ( . g/kg of s), or be of meq/kg ( . g/kg of s) and meq/kg ( . g/kg of s), respectively. a  factorial plus a control diet design, resulting in five treatments, and adult health beagle dogs were used, in a completed randomized design with six dogs per diet. a -d adaptation phase followed -d of total urine collection (in bottles with mg of thymol). urine were pooled by dog and analyzed for density, volume and ph. food macroelements were determined by standards methods (aoac, ) and used for be calculation. dog's acid-basic status was studied by blood gas analysis of venous blood, at : h (pre feeding) and hours after meal. a dose-dependent reduction of urinary ph was verified for both compounds (p o . ). blood bicarbonate (r . ; p o . ), and blood base excess (r . ; p o . ) were highly correlated with food be. acidemia and reduced blood be were verified in diets with be close to zero (higher dose of both compounds, or . g/kg of s), resulting in daily or each other day vomiting episodes in the dogs. ataxia, seizures, and vomiting were previously describe in dogs fed g/kg of methionine, but our results suggest that a much lower value ( . g/kg) was toxic and that the safe upper limit should be between this value and . g/kg (the lower evaluated dose). in people with chronic kidney disease and heart failure, obesity is associated with longer survival times. this association, called the "obesity paradox," also has been recognized in dogs and cats with heart failure. excess weight appears to modulate the serious deleterious effects of muscle loss in these diseases. the purpose of this study was to determine the effects of body condition and body weight changes in dogs with naturally-occurring chronic kidney disease (ckd). dogs diagnosed with ! iris stage ii ckd between and at iowa state university and tufts cummings school of veterinary medicine were eligible for the study. dogs o year of age and those with acute renal failure or suspected congenital renal diseases were excluded. medical records were reviewed using a standardized data form, and data were collected for initial body weight and body condition score (bcs, - scale), clinicopathologic values, changes in body weight and bcs, comorbidities, and treatments. dogs were classified as underweight (bcs - ), moderate weight (bcs - ), or overweight (bcs - ). a change in body weight was defined as . kg. survival times were determined for all dogs that were discharged from the hospital and lived day. associations between survival and bcs or body weight changes were analyzed using cox proportional hazards models. one hundred two dogs were enrolled in the study. at the time of diagnosis, dogs were classified as iris stage ii, dogs were stage iii and dogs were stage iv. median body weight at baseline was . kg (range, . - . kg). for dogs with body condition scores recorded (n ), were underweight ( %), were moderate ( %), and were overweight ( %). for dogs that had at least two body weights recorded over the course of their disease, gained weight, lost weight, and had no change in weight. changes in body weight were not associated with survival; however, bcs at the time of diagnosis was significantly associated with survival. dogs classified as underweight had a significantly shorter survival time compared to both moderate (p o . ) and overweight dogs (p o . ). these results suggest that body condition is an important consideration in dogs with acquired chronic kidney disease. further studies are warranted to evaluate the relationship between obesity and longer survival in dogs with ckd. protein restriction is the cornerstone of dietary management of kidney disease. the national research council recommends % crude protein and the american association of feed control officials (aafco) recommends a minimum of % crude protein for maintenance for healthy adult cats. protein requirement is unknown for adult cats with kidney disease. most commercially produced cat foods for adult maintenance contains % or more crude protein on a dry matter basis. a typical therapeutic food for cats with kidney disease contains about % crude protein. the objective of the present study was to investigate whether dietary crude protein at . % would be adequate for the maintenance for adult cats with impaired kidney function. seven adult cats, female and male, with age ranging from to . years old (mean: . years) were used in the study. all cats had elevated serum creatinine concentration ( . mg/dl, range: . - . mg/dl) and reduced glomerula filtration rate (mean: % reduction; range: - % reduction) during the study. they did not have other systematic diseases, e.g., hyperthyroidism, at the beginning of the study. cats were fed an expanded dry food made with ingredients commonly used in commercial dry cat foods. the food contained . % crude protein (chemical analysis) and kcal/kg (calculated) on a dry matter basis, or . g protein/ kcal. each essential amino acid in the food was at least % of that recommended by aafco. other nutrients in the food also exceeded aafco's recommendations for maintenance for adult cats. cats were fed the food for weeks. lean body mass (dual x-ray absorptionmetry; hologic, hologic, inc, ma) and serum albumin concentration were measured periodically to monitor protein status of cats. the average lean body mass (mean ae sd) was . ae . kg, . ae . kg, . ae . kg, and . ae . kg in weeks , , , and of the study, respectively. paired t-test did not detect statistical difference (p . ) when comparing the lean body mass in weeks versus weeks , , and , respectively. serum albumin concentration were within the normal reference range during the study (mean ae sd: . ae . %, . ae . %, . ae . %, and . ae . % in weeks , , , and , respectively) . these data show that . % dietary crude protein in a dry food with kcal/kg on a dry matter basis, or . g protein/ kcal, is adequate for maintenance for cats with impaired kidney function. in humans, several disease conditions exist that involve abnormal patterns of polyunsaturated fatty acids and similar abnormalities may be present in companion animals. indeed there have been reports of decreased plasma arachidonic acid and reduced delta- desaturase activities in dogs with atopy and other skin disorders. the present study investigated serum fatty acid profiles in dogs and cats presented to the texas a&m university veterinary teaching hospital, clinical pathology laboratory over the past one year period. results were compared with normative data generated among dogs and cats from earlier feeding studies. sera used were residual samples submitted to the laboratory for other diagnostic procedures and stored frozen for no more than months after collection. the samples were grouped according to presenting disorders involving liver, kidney, digestive, and cardiac diseases. total lipids were extracted using chloroform:methanol ( : v/v) and fatty acid methyl esters were prepared for capillary gas chromatography. relative percentage distribution of individual serum fatty acids for each animal were then compared with average normative serum phospholipid fatty acid values (dogs, n ; cats, n ) by calculating the ratio of the value in the diseased individual to the normal mean value and used as an index of normalcy. normalcy ratios were then plotted on a logarithmic scale with normal at . . the ratio was then compared to changes greater than , and standard deviations of the normal mean values. in this way a graphical presentation of resultant values was obtained. although the animals had been fed various commercial diets and some home-prepared foods, a number of noteworthy patterns emerged from this analysis. dogs showed increased linoleic acid, decreased arachidonic acid, increased total monounsaturated and decreased saturated fatty acids at p o . ; oleic acid was increased at p o . . remarkably, these findings were similar for all canine disease categories evaluated (n , heart; n , kidney; n , liver, and n digestive disorders). in cats, a slight decrease in arachidonic acid and large decrease in : was observed but only in heart disorders. by contrast, modest elevations of arachidonate were observed in kidney, liver, and digestive disease groups but at p o . . sample sizes of the feline sera were considerably smaller (range of - per group). a limitation of this analysis is that variability of normal data may exist depending on diet fed making comparisons less reliable however, these preliminary data suggest that metabolic diseases of dogs may depress plasma arachidonic acid independent of diet fed suggesting either reduced conversion from linoleic acid or increased utilization of arachidonate for eicosanoid production during times of metabolic stress. conversely, in cats, increases in arachidonic acid may be associated with diet arachidonate or other mechanisms. additional studies to verify these findings are warranted. the objective of this study was to determine whether or not lalanyl-l-glutamine (ala-gln) supplementation in dogs with parvoviral enteritis improves the survival rate and ameliorates clinical signs without side effects. this randomized, double-blinded, placebo-controlled clinical trial included client-owned dogs. the dogs were randomly assigned into two groups and administered ala-gln solution (dipeptiven; . g/kg) or an equivalent volume of placebo orally twice a day. all of the dogs (ala-gln group [n ] and placebo group [n ]) received standard treatment while hospitalized and were monitored daily according to a clinical scoring system and diagnostic evaluation for days. among the dogs, (ala-gln-treated group [n ] and placebo group [n ]) were vaccinated and (ala-gln-treated group [n ] and placebo group [n ]) were not vaccinated. the population consisted of purebreds and mixed breed dogs, with a mean age of . ae . weeks. the survival data were compared statistically by means of a log-rank test for the kaplan-meier survival curves. the clinical scores of ala-gln-treated dogs improved significantly relative to the placebo group. there was a significant difference between the two groups in the survival distribution (p . ); specifically, of the ala-gln-treated dogs ( . %) died, whereas of the dogs in the placebo group ( . %) died. no side effects were associated with the administration of ala-gln. these results suggest that the oral administration of ala-gln is effective in improving clinical signs and survival rate in dogs with parvoviral enteritis. bleeding disorders, thrombocytopenia and alterations in platelet function have been documented in humans receiving lipid-containing parenteral nutrition formulations. despite a lack of evidence in the veterinary literature, it is believed that parenteral lipids are contraindicated in critical illness when the development of bleeding disorders is likely. the objective of this study was to determine if there is an in vitro effect on platelet function and thromboelastography (teg) in normal dogs with varying concentrations of a % soybean oil emulsion (intralipid s ). twelve clinically healthy dogs were used for this study. whole blood platelet aggregation, using adp and collagen agonists, was measured using multiple electrode aggregometry in hirudinated blood with final lipid concentrations of , , , and mg/ml. the teg parameters r, k, a-angle, and maximum amplitude (ma) were evaluated from citrated whole blood with equivalent final lipid concentrations as platelet aggregation. there was no significant difference between groups with collageninduced platelet aggregation. there was a significant increase in the area under the curve (auc) with adp-induced aggregation at a lipid concentration of mg/ml (p . ). the ma was significantly reduced at both the mg/ml (p o . ) and mg/ml (p o . ) lipid concentration. there was no statistical difference between groups evaluating the other teg parameters. while platelet aggregation appeared enhanced at the highest concentration evaluated, this concentration is not clinically relevant. the reduction in ma seems discordant but both fibrinogen and platelets contribute to the ma. therefore the higher lipid concentrations may be interfering with fibrinogen kinetics or fibrinogenplatelet interaction. in vivo studies are indicated to determine if any of these changes are clinically significant. rosiglitazone is a peroxisome proliferator-activated receptor gamma (pparg) agonist and an fda-approved anti-diabetic agent in humans that has been investigated for its ability to reduce tumor cell growth. specifically, the combination of rosiglitazone and carboplatin has demonstrated enhanced tumor control. the purpose of this study was to determine the peak plasma concentrations and side effect profile of rosiglitazone after oral administration in dogs with spontaneously occurring cancer. all dogs received carboplatin intravenously concurrently with oral rosiglitazone. ten cancer-bearing dogs with normal pre-treatment hepatic and renal function were enrolled. complete pre-treatment hematological and biochemical parameters were available in ten dogs and post-treatment parameters in nine dogs. peak plasma concentrations varied with dose and ranged from . - . ng/ml and occurred between minutes and hours post administration and rapidly declined after the peak. the dose limiting toxicity was hepatic at a dose of mg/m . there was one grade iii, two grade i alt, and one grade iii ast elevations noted. no changes in total bilirubin, alkaline phosphatase, or ggt values were noted. blood glucose values remained within normal limits. mild, self-limiting gastrointestinal and hematologic toxicities were observed when rosiglitazone was administered in combination with carboplatin. based on this study, the recommended dose of rosiglitazone in cancer-bearing dogs with normal hepatic function is mg/m orally once daily. side effects of the combination appear similar to side effects noted with carboplatin alone. further study is needed to determine efficacy of this combination and if more frequent dosing is required to maintain plasma concentrations. carboplatin has shown little activity as a single agent for the treatment of canine transitional cell carcinoma (tcc). however, gemcitabine has shown synergism with carboplatin in human cell lines. the purpose of this study was to evaluate the activity of gemcitabine against canine tcc cell lines alone or in combination with carboplatin. we hypothesized that gemcitabine in combination with carboplatin would have synergistic effects in vitro. the results of this study could provide a rationale for treatment of canine tcc with the combination of these drugs. tcc cell lines tcc-kiss, tcc-knapp-js, tcc-axa, tcc-hxc, and tcc-sh were treated with gemcitabine, carboplatin, or the combination. cell proliferation was assessed using cyquant assay, cell cycle was evaluated using propidium iodide staining, and apoptosis was assessed by measuring caspase- / activation. synergy was quantified by combination index analysis using compusyn software. treatment of canine tcc cell lines with carboplatin or gemcitabine decreased cell proliferation, induced cell cycle arrest, and apoptosis. when tcc cell lines were treated with gemcitabine and carboplatin in combination at a therapeutically relevant concentration (gemcitabine o um, carboplatin o um), a significant decrease in cell proliferation was observed compared to gemcitabine or carboplatin alone, and the drug combination was synergistic in of cell lines, and additive in the remaining lines. gemcitabine exhibits biologic activity against canine tcc cell lines and carboplatin combined with gemcitabine exhibits synergistic activity at biologically relevant concentrations. our results support further evaluation of these drugs in dogs with tcc to determine the clinical efficacy of this combination. metronomic chemotherapy has been shown in murine models and humans to improve tumor control by inhibiting tumor angiogenesis and suppressing regulatory t cells (treg). treg are a subset of t lymphocytes demonstrated to be increased in humans and dogs with cancer and are thought to suppress cellular immune responses against tumors. the purpose of this study was to determine whether metronomic cyclophosphamide therapy depletes treg and/or exhibits antiangiogenic activity in dogs with soft tissue sarcoma. client owned dogs with histologically confirmed grade i or ii soft tissue sarcoma were administered cyclophosphamide at . mg/m or mg/m orally once daily for days. whole blood and tumor biopsies were obtained on days , , and . flow cytometric analysis of blood was performed to assess changes in t lymphocyte subsets, including cd and cd cells as well as cd foxp treg. tumor microvessel density (mvd) was assessed by performing immunohistochemistry for cd . five dogs were enrolled in the . mg/m /day dose cohort and six dogs were enrolled in the . mg/m /day dose cohort. in patients that received cyclophosphamide at . mg/m /day, the mean number of treg decreased from day to but there was no change in the mean percentage of treg or mvd. for patients that received . mg/m /day, both the mean number and percent of treg as well as mvd decreased over the day time period. cyclophosphamide at . mg/m /day or greater selectively depletes treg and inhibits angiogenesis in dogs with soft tissue sarcoma. arsenic trioxide (ato) is used to treat leukemias, multiple myeloma, and relapsed lymphoid malignancies in humans; its use has not been explored in veterinary oncology. prior therapy with glucocorticoids decreases likelihood and duration of remission for dogs with lymphoma treated with chemotherapy. we hypothesized that ato will re-sensitize glucocorticoid-resistant canine lymphoma cells to glucocorticoid-induced apoptotic death. the osw canine lymphoma cell line was cultured with um dexamethasone. remaining viable dividing cells were considered resistant. resistant cells were exposed to . um and . um of ato without dexamethasone, after which cells were washed and re-exposed to um dexamethasone. after , and hours of dexamethasone exposure, cells were counted using trypan blue stain. apoptosis was assessed by tunel assays on cytospin preparations collected at , , and hours from ato-exposed and control groups. statistical analysis was performed using way anova and tukey's test. the proportion of dead cells increased over time in both . um and . um ato exposed groups. the proportion of dead cells was greater for . um ato (p o . ) and . um (p o . ) groups compared to control. apoptosis increased with increasing ato concentration and duration of dexamethasone exposure compared to control. these results support the effectiveness of ato at re-sensitizing glucocorticoid-resistant canine lymphoma cells to apoptotic death following re-exposure to glucocorticoids. ongoing gene expression studies aim to elucidate this mechanism. additional studies to determine if this effect is seen with other chemotherapeutic agents are warranted. lymphoma is the most common hematopoietic tumor of dogs. protein disturbances may be associated with this disease including monoclonal gammopathies in a low percentage of cases. serum protein electrophoresis (spe) is routinely used to aid diagnosis of various canine diseases including lymphoma when total protein concentration is elevated. the purpose of this study was to compare spe changes in lymphoma patients without elevated total proteins with a population of healthy dogs. agarose gel electrophoresis was performed on residual serum from healthy control dogs and untreated dogs with multicentric lymphoma (stage iii -v) after measuring total protein (tp) using the biuret method. densitometric traces of the protein bands were obtained using computer software (totallab ) and the albumin, alpha- , alpha- , beta- and gamma globulin subfractions were identified by visual inspection. the total protein concentration, the number of subfractions and the relative and absolute protein subfraction concentrations were then compared statistically between the two populations. in lymphoma dogs, tp, absolute albumin, beta- and gamma globulin concentrations and both relative and absolute concentrations of the alpha- globulins were significantly lower however relative and absolute alpha- globulin concentrations were significantly elevated. no monoclonal gammopathies were identified in any of the dogs and not every patient with lymphoma had the above changes in their electrophoretogram. this study has demonstrated that significant changes occur in the albumin and globulin fractions of canine lymphoma patients despite no obvious increase in tp. further investigation is required to identify the proteins responsible for these changes. it is well known that immunophenotype has a prognostic value for the outcome of canine lymphoma, with t-cell lymphomas having a worse prognosis than b-cell lymphomas. the recent advent of flowcytometric techniques allowed easy detection of many different markers on lymphoma cells and therefore, not only distinguish between t and b cells, but also estimate possible aberration on immunophenotype. in human oncology, although some controversy persists, it seems that non-hodgkins lymphoma and acute leukemia carrying aberrations have a worse prognosis. the aim of this study was to evaluate the role of immunophenotype aberration in canine high-grade lymphoma considering outcome and time span to achieve complete response under chemotherapy. samples of bone marrow, blood and lymph node suspensions from twentythree dogs were evaluated with flow-cytometry. eleven dogs had aberrant expression of neoplastic lymphocytes and twelve were non-aberrant. the most common aberrations found were: positivity to cd , biphenotypes, double expression of tantigens (cd , cd ), diminished expression of cd . all dogs were treated with a chop-based protocol. there was a significant difference for the time to achieve response to chemotherapy (partial or complete). / non aberrant lymphomas went into cr or pr after the first treatment (l-asparaginase), while aberrant lympho-mas needed more than treatment to reach cr or pr. there was a trend for a prolonged disease free interval with non-aberrant versus aberrant, although it was not statistically significant. aberration of immunophenotype may be a prognostic factor for canine lymphomas, but further studies with larger groups are needed. class ii major histocompatibility expression is a significant and independent predictor of prognosis in human b cell lymphoma. low class ii mhc is consistently associated with poorer outcome. the mechanism underlying this relationship is not clear, but one hypothesis is that high class ii mhc allows for better antigen presentation and tumor-specific immune responses. in the this study, we investigated whether that class ii mhc expression in canine b cell lymphoma was associated with remission and survival times. a total of patients were categorized by level of class ii mhc,expression of cd and cell size for on neoplastic b cells. multivariable cox-proportional hazard analysis was used investigate this research question using a randomly selected subset of the data, and the predictive ability of this model was validated on the remaining / of patient data. results suggested that low class ii mhc expression was associated with decreased times to relapse and death as is seen in human b cell lymphoma, and that large neoplastic cells were associated with decreased survival time. cd expression was not associated with patient outcomes. these findings have implications for the use of dogs to model human lymphomas, for the study of tumor vaccines, and for prediction of mortality in dogs with b cell lymphoma with a high level of specificity. one of the reasons for the failure of canine lymphoma treatment is related to the resistance of tumor cells against chemotherapy drugs. the major form of this resistance is provide by multidrug resistance abc transporters. abc transporters proteins comprise a large superfamily of transmembrane proteins, atp-dependent, that extrude a large variety of drugs from the cells. multidrug resistance phenotype in cancer cells is associated with overexpression of these transmembrane proteins. abcg , also known as bcrp, is a residue half-transporter protein that protect hematopoetic stem cells against toxic compounds. the aim of this study was to investigate the expression of bcrp (abcg ) in canine multicentric lymphoma. samples were collected by fine needle aspiration of an enlarged lymph nodes, from dogs with multicentric lymphoma (stage iii to v) at diagnosis, and normal lymph nodes (control). dogs that were previously treated with prednisone or chemotherapy were excluded from the study. quantitative rt-pcr was used to measure the mrna expression level of bcrp and flnb expression as a endogenous reference canine gene. a widely range expression value for abcg expression was found for canine multicentric lymphoma. high gene expression was observed in % ( / ) canine lymphoma, but % of dogs had a lower expression when compared with normal lymph node. gene expression was not associated with clinical staging, complete or partial remission, relapse and survival time. in conclusion, abcg was expressed in canine lymph node and canine multicentric lymphoma at the diagnosis, and it was not correlated with clinical response. osteosarcoma (osa), the most frequent primary malignant bone tumor of dogs, is both locally aggressive and highly metastatic. prognostic factors for canine osa include tumor location, distant metastatic disease, and serum alkaline phosphatase (alp) concentration. an increased serum alp concentration is associated with poor prognosis; however the mechanisms underlying this phenomenon are currently unclear. during normal bone development alp may be used as a marker for osteoblasts. additionally, alp is a downstream target of activated canonical wnt/b-catenin signaling. therefore, we hypothesized that increased serum alp would be associated with increased expression of b-catenin in canine osa. the goals of this study were: ( ) characterize and compare cellular alp expression in osa tissue from patients with normal and high serum alp; and ( ) assess b-catenin expression in those same patient populations. we used frozen osa samples collected from patients with either high alp (n ) or normal alp (n ). total rna was isolated from the frozen tissue, converted to cdna, and analyzed using quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) with either target gene alp (aim ), or target gene b-catenin (aim ). additionally, b-catenin expression was analyzed by western blot. qpcr data for bcatenin and alp expression were normalized to s, and relative expression was calculated by the ddct method. the relative expression of cellular alp was higher in high serum alp samples compared to normal serum alp samples: . ae . (mean relative expression ae standard deviation; p o . ). further, the relative expression of b-catenin was also increased; b-catenin expression of high serum alp samples relative to low serum alp samples was . ae . (p o . ), which is also seen by western blot. this study begins to clarify the mechanism behind high serum alp in canine osa, and suggests the wnt signaling pathway may be active in this population of patients. further work will focus on elucidating the role active wnt signaling plays in the biology of osa. in the future, serum alp status of osa patients may help identify patients that would benefit from therapies targeting this pathway. accurate assessment of abdominal lymph node status is of vital importance for appropriate treatment planning and determining prognosis in dogs with apocrine gland adenocarcinoma of the anal sac (agaas). pretreatment knowledge of lymph node status is helpful for determining prognosis and planning the optimal extent of lymphadenectomy. in addition, pretreatment knowledge of lymph node status may help in selecting patients who might benefit from adjuvant chemotherapy and radiation therapy. abdominal ultrasound is currently the most commonly employed test to screen for abdominal lymphadenopathy in dogs with agaas. imaging studies in people indicate that magnetic resonance imaging ( to determine and compare the plasma concentration of cyclophosphamide and its metabolite -ohcp, within the plasma of lymphoma bearing dogs being treated with either oral or intravenous cyclophosphamide. in this prospective study, patients were randomly assigned to either receive oral or intravenous cyclophosphamide, at a dose of mg/m . based on a priori power calculation eight patients per treatment group were enrolled. plasma was obtained at times , , , minutes, and then at , , , , hours post administration for evaluation of -ohcp concentrations by liquid chromatography-dual mass spectrometry (lc/ms/ms). average values were obtained for both cyclophosphamide and -ohcp concentrations within the plasma of both groups. the following values were obtained, half life (hl), time to maximum concentration (tmax), maximum concentration (cmax), and area under the curve (auc). the mann-whitney statistical test was used to compare the groups. the auc for cyclophosphamide was statistically significant (p o . ) when compared between the two groups. the auc for -ohcp was not statistically significant between the groups. the difference between cmax for cyclophosphamide and -ohcp was statistically significantly (p o . ) between the groups. although the auc for cyclophosphamide was statistically significant between the two groups, the auc for the active metabolite -ohcp was not different when administered intravenously or orally. thus drug exposure to the active metabolite of cyclophosphamide is the same when administered intravenously or orally. previously the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g power driver by a wide spectrum of novice participants in feline cadavers were evaluated. novice users' mean io catheter insertion time using the ez-io g driver was also compared to the mean iv catheter insertion time in normovolemic feline and canine patients presented to the western college of veterinary medicine (wcvm) small animal hospital. novice users included wcvm personnel ( technicians, veterinary students, interns, residents, clinicians). after watching a -minute ez-io g training video, each participant inserted io catheters using the ez-io g driver. site (proximal humerus or trochanteric fossa of the femur) and side of cat (right or left) were randomized for each attempt for each participant. a gauge x mm long needle and a gauge x mm needle were used for io catheter insertion in the humerus and femur, respectively. participants then graded the ''ease of use'' of the ez-io g device on a visual analog scale (vas) that was converted to a -point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by wcvm small animal hospital personnel ( technicians, veterinary students, intern, resident, clinicians) were then timed and compared to the mean io catheter insertion time in feline cadavers by study participants using the ez-io g device. the io catheter was inserted correctly on every attempt by % ( / ) of participants. no difference was found between participant groups for mean io catheter placement confirmation percentage (p . ). percentage of io catheter ''slippage off the bone'' at the time of placement did not vary across participant groups (p . ). mean io catheter insertion times were all less than seconds and did not differ significantly as a function of attempt number (p . ) or as a function of participant group (p . ). participants rated the ez-io g 's ''ease of use'' favorably and subjective scores did not differ across participant groups with varying levels of clinical experience (p . ). compared to the mean insertion time for iv catheterization ( sec), mean io catheter insertion by participants using the ez-io g ( sec) was significantly faster (p . ). regardless of their level of clinical experience, participants rated the ez-io g device favorably in terms of its ''ease of use'' and their willingness to use the device in the future. regardless of their level of clinical experience, study participants successfully placed io catheters using the ez-io g device and did so significantly faster than the reported iv catheter insertion time in normovolemic feline and canine patients in the wcvm small animal hospital. intraosseous catheterization using the ez-io g has the potential to provide very rapid vascular access and is a skill that can be easily learned. previously presented at the western college of veterinary medicine undergraduate poster competition. multicavitary effusion is a common cause of presentation for dogs to emergency medical centers. the goal of this study was to identify common underlying causes of multicavitary effusion as well as determine their relative importance. a retrospective analysis of cases of multicavitary effusion admitted to the icu of a tertiary referral center (ontario veterinary college) from to was performed. twenty-three different breeds, with golden and labrador retrievers ( . % and %, respectively) being most commonly seen, were included in the study. ages ranged from to years with a median age of years and a mean of . years. . % of cases were males ( / cases). most common presenting signs included lethargy ( . %), anorexia ( . %), vomiting ( %) and dyspnea ( %). cavitary effusion was detected by either ultrasonography (pericardial, pleural or abdominal) or radiographs (pleural). bicavitary effusion was present in cases ( . %) whereas cases ( . %) had tricavitary effusion. neoplasia was found to be the most common underlying cause overall ( . %), with hemangiosarcoma being the leading type ( . % of neoplasia cases), followed by congestive heart failure ( . %), gastrointestinal lymphangectasia ( . %), peritonitis/pancreatitis ( . %), cirrhotic liver disease ( . %) and acute renal failure ( . %). in cases ( . %), no underlying cause could be found. of these, ( . % of all cases) were diagnosed as having idiopathic pericardial effusion. taken together, these findings suggest a strong association between multicavitary effusion and diseases carrying a guarded prognosis in dogs. infection control practices in veterinary clinics and hospitals are becoming increasingly important, with rising client expectations, growing concern about the spread of antimicrobial-resistant pathogens, and the potential for zoonotic transmission of disease. surgical patients are at increased risk of developing infections, and can serve as sources of these pathogens for other animals and people with whom they have contact within and outside the clinic. taking all reasonable precautions to reduce the risk of surgical site infections, beginning with preoperative preparation of the surgeon and patient, is therefore an important part of any infection control program. while guidelines are available for preoperative preparation procedures, there has been no objective investigation of compliance with these guidelines in veterinary practices. the objectives of this pilot study were to describe a range of preoperative hand scrub and surgical site preparation practices in veterinary clinics, and to determine if there were any areas that consistently require improvement. observation of preparation practices was performed in each of ten clinics over - days using - small wireless surveillance cameras. data was coded for surgical patients, and surgeons performing a total of hand scrubs. patient hair removal was most commonly performed after induction of the animal ( / , %) and using clippers ( / , %) . steps in surgical site aseptic preparation ranged from - . contact time with soap ranged from - s (mean s, median s), and with alcohol from - s (mean s, median . s). application of alcohol or antiseptic using a ''cleanest to dirtiest'' pattern was infrequent ( / ( %) and / ( %), respectively). potential contamination of the surgical site occurred most frequently when the animal was moved to the surgery table after initial preparation ( / , %). preoperative alcohol hand rub was used in / facilities, but soap and water hand scrub was still more commonly used even at these clinics. proximal-to-distal scrubbing was noted in / ( %) of soap and water scrubs. contact time during surgeon hand preparation ranged from - s (mean s, median s) for soap and water and from - s (mean s, median s) for alcohol-based hand rub. approximately % of the variation in contact time was due to inter-surgeon variation. no significant changes in practices were identified over the course of the observation period. some preoperative preparation practices were fairly consistent between clinics in this study, while others varied considerably. contact times with preparatory solutions were often far shorter than recommended, and there was a high frequency of non-sterile contact with the surgical site during movement of patients to the surgical suite. the camera system used to perform this study did not have a significant time-dependent effect on the behavior of participants, and could be useful for performing similar field-based observational studies in the future. this prospective randomized study compared the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g power driver to manual io catheterization in feline cadavers. the io catheter insertion time in cadavers using the ez-io g device was also compared to iv catheter insertion time in normovolemic feline and canine patients. after a purposely limited training period, a preclinical veterinary student was timed and video-recorded as she performed io catheter placements in feline cadavers ( io insertions by placing an illinois needle manually and io insertions using the ez-io g ). order of technique (manual or ez-io g ), site of io placement (proximal humerus or trochanteric fossa of the femur), and side of cat (right or left) were randomized for each attempt. when using the ez-io g , a gauge x mm long needle and a gauge x mm needle were used for io catheter insertion in the humerus and femur, respectively. after each attempt, the student graded the ''ease of use'' of each technique on a visual analog scale (vas) that was converted to a -point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by western college of veterinary medicine (wcvm) small animal hospital personnel ( technicians, veterinary students, intern, resident, clinicians) were then timed and compared to the student's mean io catheter insertion time using the ez-io g .median io catheter insertion times for the techniques were significantly different (manual io technique sec; ez-io g sec) (p o . ); the manual method took seconds longer ( % confidence interval of to seconds) than the ez-io g method. insertion time was more variable for the manual technique than for the ez-io g . percentage of catheter ''slippage off the bone'' and extravasation around the inserted catheter were significantly higher for placement of the manual io catheter compared with placement of the ez-io g catheter (p o . ). student's subjective ratings were more favorable and more consistent for the ez-io g technique compared to the manual technique for io catheter insertion. compared to the mean insertion time for iv catheterization in the wcvm small animal hospital, io catheter insertion by the student using the ez-io g was significantly faster (iv catheter sec; ez-io g io catheter sec) (p o . ). intraosseous catheter insertion using the ez-io g can be said to be significantly faster, less traumatic, more user-friendly, and as effective as io catheter placement using the manual technique. vascular access via io catheter insertion using the ez-io g device may be suggested to be faster than iv catheter insertion. previously presented at the western college of veterinary medicine undergraduate poster competition. computed tomography (ct) has been widely investigated and applied as a means for non-invasive quantitative bone mineral determination in human medicine. the aim of this study was to assess age-related changes and anatomic variation in bone mineral density (bmd) using quantitative ct in normal cats. seventeen normal cats were included in this study and divided into the following age groups: o year (n ); - years (n ); and years (n ). a computed tomographic scan of each vertebra from the th thoracic to the th lumbar spine, and the pelvis, was performed with a bone-density phantom ( , , and mg/cm , calcium hydroxyapatite, cirs phantom s ). on the central transverse section, the elliptical region of interest (roi) was drawn to measure the mean hounsfield unit value. those values were converted to equivalent bmd by use of the bone-density phantom and linear regression analysis (r . ). the mean bmd value of the thoracic vertebrae ( . ae . mg/cm ) was significantly higher than of the lumbar vertebrae ( ae . mg/cm ). the maximum bmd occurred at the t , t , and l levels in all age groups. there was a statistically significant difference in the mean bmd value among the age groups at the t (p o . ), t (p o . ), and l levels (p . ), respectively. in addition, there was no significant difference between the mean bmd value of the left and right iliac bodies ( . ae . mg/cm and . ae . mg/cm , respectively). the present study suggests that age-related changes and anatomic variation in bmd values should be considered when assessing bmd using quantitative ct in cats with bone disorders. dynamic contrast-enhanced computed tomography (dce-ct) is a rapid and widely available method of cerebral perfusion imaging. however, there is no established reference value of cerebral blood flow (cbf) measured by dce-ct according to a dog's age. the purpose of this study was to identify the correlation between regional cbf and aging in clinically normal dogs using dce-ct. fourteen dogs with no evidence of hemodynamic disorders and central nervous system dysfunction were included in this study. dogs were assigned to the following age groups: o year (group ); - years (group ); and o years (group ). dce-ct scans were performed at the level of the third ventricle and mesencephalic aqueduct. cbf in the gray and white matter was calculated using stroketool-ct s software. the overall mean ae standard deviation quantitative estimate for regional cbf in clinically normal dogs was . ae . ml/min/ g, . ae . ml/min/ g, and . ae . ml/min/ g in groups , , and , respectively. there was no significant regional cbf difference between the right and left sides of the brain in each group. also, a statistically significant difference in the regional cbf was observed between groups and (p o . ). thus, aging affects the regional cbf in normal dogs and the values should be considered assessing the results of dce-ct. according to several clinical behavior guidelines, ''toileting'' type inappropriate urination (i.e. large amounts of urine deposited in horizontal surfaces) can arise in cats suffering from a medical problem (typically lower urinary tract disease). by contrast, ''spraying'' type behaviour (i.e. possibly smaller amounts of urine deposited on vertical areas) is more typically associated with anxiety brought about by a threat to local resources, arising from either a change in the physical environment or threat to these resources from another cat. however, there is some evidence that ''sprayers'' may also be presented with a medical problem, which might be linked to the disease (e.g. painful voiding associated with crystalluria may lead to a standing posture being adopted and small amounts eliminated at a given time). this might be associated with an apprehensive state or simply a co-morbid state. as part of a larger research project aimed at investigating behavioral and physical aspects of cats presented with inappropriate urination, owners of ''spraying'' and ''toileting'' cats with appropriate control subjects from the same households were recruited throughout local media coverage and the internet. the case-control dyads were brought by the owners to the veterinary hospital of the university of sa˜o paulo, at the same time, for a medical work-up (i.e. physical examination, complete blood count, biochemical profile, urinalysis, urine culture and abdominal ultrasound). no significant differences between the ''sprayers'' and ''toileters'' regarding the occurrence of medical problems were found. both groups had a similar proportion of cats affected by medical illnesses (sprayers: . %, toileters: . %; chi , p . ), directly or indirectly relating to the urinary system (e.g. diabetes, chronic kidney disease). in both groups, control cats also had a relatively high occurrence of medical concerns ( . % and . %, respectively for each control group). these results emphasize the importance of careful medical evaluation of cats presented for a urinary housesoiling problem. the relatively high prevalence of medical concerns among apparently healthy cats in multi-cat households may have arisen, at least in part, as a result of an inability/failure of owners to monitor individuals, thus allowing some early signs to pass unnoticed. the way in which medical and behavioral elements are linked (if at all) remain unknown but deserve further investigation. considered as a semi-social species, domestic cats appear to be highly sensitive to the effects of social stress, especially when living in high density populations. cats are capable of adapting to live ingroup; nonetheless, they do not appreciate living in close proximity with others as result of an environment lacking of great opportunities of escaping and hiding. this study aimed at testing the following hypotheses: (a) owners' perceived quality of life affects cats' global levels of stress; (b) cats' global levels of stress are influenced by cats' personality; (c) cats' living style (single housing versus large group housing) does affect stress levels in cats. to our knowledge, this is the first study investigating stress levels of domestic owned cats, under natural conditions, throughout measurement of faecal glucocorticoids metabolites concentration, and taking into consideration cat personality, cat living style and owner's subjective life quality. in this study, adrenocortical activity, as a valuable physiological indicator of emotional stress, was evaluated throughout the measurement of faecal glucocorticoids metabolites in fourteen single and sixteen in-group housed cats. cat personality as well as owners life quality was evaluated by self reported questionnaires given to the owners to answer. significant differences in mean glucocorticoids metabolites concentrations (mgcm) between the two populations (i.e. single versus in-group cats) were not detected (random effect model, p . ). however, when mgcm were taken as a function of cat personality, there were differences regarding single catstimid cats showed higher levels in comparison to easy-going (random effect model, p . ) and bossy (random effect model, p . ) cats. as to owner subjective life quality, a direct association between the scores given by the owners to the social dimension and mgcm was found for single cats only (i.e. the better the owner felt itself social wise the higher the mgcm of the cat; random effect model, p . ). social stratification may compensate the stress resulting from spatial restriction in large in-group living cats. other underexplored factors such as feline personality and owner life style seem to play an equally important role in domestic cats' day to day levels of stress, especially in the cats kept as single pets. in dogs, raas activation is a major feature of congestive heart failure (chf). benazepril (fortekor s ) is a potent ace inhibitor with well-documented effectiveness in canine chf. although ace activity (ace a ) has been used in preclinical studies as a surrogate marker of efficacy, some authors have reported a poor correlation between plasma ace a and changes in angiotensin ii (aii) or aldosterone (al). the purpose of this study was to investigate the effect of benazepril on canine plasma renin activity/concentration (pra/prc), angiotensin i (ai), aii, al, and fractional excretion of potassium (ufek), sodium (ufena) and aldosterone (ufeal). sixteen beagle dogs were fed a low-sodium diet and dosed with placebo or benazepril tablets ( mg po, q h) for days. blood and urine samples were collected on day (d ) and day (d ) over -hour periods. data were analyzed by repeated measures anova of baseline corrected values, and anova of auc hours . compared with placebo, benazepril induced a significant increase in pra and ai at d (p-value [pra] : . , p-value [ai] : . ) and d (p-value [pra] : . , p-value [ai] o . ). no differences in prc were noticed. based on auc hours, aii levels were % lower in the benazepril group at d (p-value [aii] : . ). ufeal and al decreased by up to % and % at d and d , respectively, though differences did not reach statistical significance. benazepril markedly influences raas dynamics in dogs. decreased exposure to aii and al are likely to be the key events required to counteract pathological remodeling of the heart in chf. this study compared two intravenous anesthetic agents, alfaxalone (alf) (alfaxan s , jurox pty. ltd.) and propofol (ppf) (rapinovet s , schering plough animal health) and their effects on spontaneous ventilation after induction of anesthesia in dogs at various doses. this randomized, crossover, dose-escalation study used six dogs in weight and gender-matched pairs ( m- f). for each drug, each dog was dosed incrementally at , , , and times the labeled anesthetic induction dose rate (alf mg/kg, ppf . mg/kg) or until a dose was reached that rendered the dog apneic. a minimum of three days was allowed between doses. for each dose administration, the entire calculated dose was delivered constantly over min. the primary variable was apnea, defined as an absence of spontaneous ventilation for minute. apneic dogs were manually ventilated with oxygen until they resumed adequate spontaneous ventilation. once the apneic dose was determined for an individual dog for one drug, the dog began incremental doses with the alternate drug. for each anesthetic episode times were recorded from completion of induction dose to; removal of endotracheal tube, dog lifting head, dog attaining sternal recumbency and dog standing. pulse rate, respiratory rate, spo and etco were each measured every min. within-dog comparisons were made using the paired student's t-test. for both alf and ppf all dogs respired voluntarily at the labeled ( x) dose. for ppf at and x doses, and dogs respired voluntarily respectively. for alf at , and x doses, all , and dog respired voluntarily respectively. for all six dogs to become apneic required x dose of ppf and x dose of alf. the mean no observable adverse effect dose (noael) expressed as a multiple of the labeled dose was higher for alf ( . x) than for ppf ( . x) (p . ). there were no significant differences between times to extubation, head lift or attaining sternal recumbency after alf and ppf at , and x doses. at the x dose, dogs took longer to stand after alf ( . ae . min) than ppf ( . ae . min). we concluded that based on anaesthetic duration, the manufacturer's labeled dose rates of mg/kg for alf and . mg/kg for ppf were equivalent. however, based on the dose escalation, the number of dogs becoming apneic at each dose-multiple is consistent with ppf having a narrower safety margin, i.e., ppf caused more respiratory depression than alf. parenteral levetiracetam (lev) has been shown to rapidly attain therapeutic levels in dogs when given iv or im, and has been used offlabel for the treatment of seizure emergencies. the purpose of this study was to determine the safety and pharmacokinetics of subcutaneously administered levetiracetam in healthy dogs. potential application of these results would be use of sq lev instead of or in addition to rectal diazepam for the treatment of cluster seizures at home. lev was administered sq between the shoulder blades to healthy, purpose-bred hound dogs, at a dose of mg/kg (undiluted). blood samples were collected at , and minutes after lev administration via jugular venipuncture. plasma lev concentrations were measured by high pressure liquid chromatography. none of the dogs became sedated, nor was there pain evident on palpation of the injection site. mean (standard deviation) lev concentration was . ( . ), . ( . ) and . ( . ) mg/ml at , and minutes, respectively. administration of sq lev was well tolerated, and exceeded the suggested therapeutic range ( - mg/ml) within minutes of administration, and remained above the range for at least hours. these data indicate that sq lev administration may be an alternative for the at-home treatment of cluster seizures in dogs, and prospective studies in epileptic dogs are warranted. the purpose of this study was to assess the effects of cyp inhibitors (ketoconazole, chloramphenicol, fluoxetine, trimethoprim, cimetidine, and medetomidine) in varying combinations on the bioavailability of oral methadone in healthy greyhound dogs. the iacuc approved this study. cyp inhibitors were administered po for hours prior to methadone administration. methadone hydrochloride was administered po at a targeted dose of mg/kg. blood was obtained for the determination of methadone plasma concentrations by mass spectrometry. the area under the curve (auc) of methadone for each treatment group was compared statistically to the auc of methadone administered without inhibitors using the mann-whitney rank sum test. significant increases (p o . ) in the methadone auc occurred in all treatment groups which included chloramphenicol, including chloramphenicol as the only inhibitor. the magnitude of increase was at least fold. mean concentrations of methadone exceeded ng/ml for at least hours in all groups administered concurrent chloramphenicol. no significant increases in the auc occurred in any of the groups which did not include chloramphenicol. in conclusion, chloramphenicol significantly inhibits the metabolism of methadone in greyhound dogs. as a result, the oral bioavailability of methadone is significantly increased and plasma concentrations are achieved that are reported to be effective in humans for - hours after a single oral administration. doxycycline hyclate is used frequently in small animals, horses and exotic animals for treatment of a wide variety of infections. because doxycycline hyclate tablets may not be suitable for oral administration in some animals, particularly horses and cats, it has been compounded into liquid suspensions. the commercially available doxycycline calcium mg/ml oral suspension, vibramycin s (pfizer) is not suitable for use in animals due to its low concentration and flavoring that animals find unpalatable. because of the known inherent instability of doxycline in aqueous vehicles under storage, this study was conducted to determine the potency of two formulations stored in dark and light conditions. a high pressure liquid chromatography (hplc) assay with uv absorption at nm was developed for analyzing doxycycline in formulations, in comparison to a reference standard from the united states pharmacopeia (usp). doxycycline hyclate mg tablets were first tested for potency. the tablets were then crushed and mixed with a pharmaceutical vehicle to make two concentrations: . mg/ml and . mg/ml. the vehicle used was a : mixture of a vehicle for oral solution (ora-sweet, usp-nf) and vehicle for oral suspension (ora-plus, usp-nf). the suspensions were prepared in replicates of . each replicate was divided, with one aliquot stored at room temperature in lighted conditions, and the other aliquot stored at room temperature in the dark. doxycycline was extracted from the formulations and measured by hplc at day , , , , , , and . each replicate was tested and the potency reported as the percent doxycycline relative to the usp reference standard. on day , , , and , the potency of each formulation was within - % of the reference standard (range . - %). this value is within the accepted range cited in usp o on pharmaceutical compounding-non-sterile preparations. however, starting at day , the potency declined dramatically and remained low for the tests performed on day and . the potency on day , , and was below % of the reference standard (range - %). there was also a noticeable change in the quality of the formulation starting on day , and a change in the color of the formulation to a dark brown. these results indicate that when doxycycline hyclate tablets are compounded as a suspension in an aqueous vehicle as described in this study, at . and . mg/ml under the storage conditions used in this study, potency of the formulation cannot be assured beyond days. we recommend a beyond-use-day (bud) of days for formulations prepared and stored at room temperature in light or dark conditions. therapeutic options for multidrug resistance (mdr) escherichia coli urinary tract infections (uti) are limited. fosfomycin (fos) tromethamine is an oral, broad-spectrum, cell-wall active, bactericidal drug approved for treatment of uncomplicated uti in humans. the purpose of this study was to determine time dependency of fos and the disposition of fos tromethamine in dogs. using a randomized, double crossover design, client-owned dogs received fos sodium iv ( mg/kg) and fos tromethamine (po, mg/kg) either with (n ) or without food (n ). serum and urine were collected for hr; fos was quantitated with a bioassay (atcc e. coli , serum or atcc proteus vulgaris , urine). in-vitro killing curves (cell counts through hours) were performed at (control), . , , , , and x mic for mdr e. coli canine fos susceptible (e-test s ) uropathogens. killing curves indicated fos to be time dependent. after iv administration, clearance (mlÃkg/hr), volume of distribution (l/kg), elimination half-life (hl; hr) and mean residence time (mrt, hr) were (mean ae sd): ae , . ae . , . ae . , . ae . and . ae . , respectively. for po, c max , hl and mrt were ae , . ae . and . ae . , respectively. serum fos exceeded the mic reported for multidrug resistant (mdr) e. coli ( . mg/ml) for hr (iv; . mg/ml) and hr (po, mg/ml diminazene is an aromatic diamidine, anti-protozoal drug that has shown promise in a small number of cases of cytauxzoonosis. in a noncontrolled case series, of cats with clinical cytauxzoonosis given mg/ kg of diminazene aceturate survived infection. dosage frequency was two intramuscular injections given one week apart. commercial formulations contain the diminazene diaceturate salt. the active base is diminazene with the salt consisting of two aceturate molecules. currently there is no data available on the pharmacokinetics of either diminazene compound in cats. the objective of this study was to determine the pharmacokinetics of diminazene diaceturate in healthy cats. four purpose bred cats with normal physical examination, cbc, chemistry and urinalysis were used. a powdered commercial drug formulation (veriben s , ceva sanet animale) was freshly reconstituted with sterile water to a concentration of mg/ml prior to administration and sterile filtered solution. heparinized blood samples were collected just before (hour ) or . , , , , , , , , , , , , and hours after intramuscular administration of mg/kg ( . mg/kg of diminazene base) diminazene diaceturate. the plasma was separated by centrifugation within minutes of collection and frozen (À c) until analysis. concentrations of diminazene were measured by hplc analysis using uv absorption and ion-pairing conditions. the pharmacokinetic profile was analyzed using a simple one-compartment model. in these cats, diminazene had a mean terminal half life (t / ) of . ( /- . ) hrs and mean peak plasma concentration (c max ) . ( /À . ) mg/ml. the mean residence time (mrt) of diminazene was . hrs ( /À . ). systemic clearance (cl/f) was . ( /À . ) l/kg/hr. the volume of distribution per fraction absorbed (vd/f) was . ( /À . ) l/kg. a single intramuscular dose of diminazene diaceturate was well tolerated by all cats. without knowing the concentration required to inhibit or kill cytauxzoon felis, it is not yet possible to make suggestions regarding optimum dosing schedules for this drug. additional toxicology data and studies to assess clinical efficacy for the treatment of cytauxzoonosis are indicated before routine clinical use can be considered. meloxicam has been shown to accumulate in areas of inflammation in both the rat and human. the objective of this study was to investigate the concentration of meloxicam in synovial fluid of inflamed joints versus that of non-inflamed joints in dogs. eight male dogs were treated with . mg/kg of meloxicam on day one and . mg/kg of meloxicam on day two. all treatments were administered orally. on day three reversible acute synovitis was induced in one stifle by aseptic, intra-articular administration of ml sodium urate crystal suspension ( mg/ml). in four dogs synovitis was induced in the l stifle and in four dogs the same procedure was used in the r stifle. in each dog the stifle without induction of synovitis served as the ''normal'' joint sample. a synovial fluid sample was collected from both the r and l stifle of each dog. sample collection occurred eight hours after administration of sodium urate and twenty four hours after the last administration of meloxicam. synovial meloxicam concentration was analysed using high performance liquid chromatography-mass spectrometry (hplc/ ms-ms). the concentration of meloxicam in the inflamed versus non-inflamed joint in each dog was compared using the paired t-test. the results indicate that meloxicam preferentially accumulates in inflamed joints in the dog as meloxicam concentrations are statistically significantly higher in inflamed joints than in non-inflamed joints. no national surveillance system exists for monitoring emergent resistance in companion animals. however, e. coli resistance is an increasing therapeutic and public health concern in these in dogs and cats. the purpose of this study was to describe current resistance patterns of canine and feline pathogenic e. coli throughout the united states and identify risk factors of antimicrobial resistance. isolates (n ) of clinical e. coli collected from dogs or cats from may through may located in different regions. susceptibility was determined to drugs ( drug classes) by broth microdilution methods. pharmacodyamaic statistics were described regionally. phenotypes were determined and type of resistance was based on the number of drug classes to which resistance was expressed: none (ndr), single (sdr) and multi (mdr). the majority of isolates were from urinary tract ( . %) and dogs ( . %). the proportion of resistance type for each drug was: ndr ( . %), sdr ( . %) and mdr ( . %). the proportion of mdr was greatest in the southwest ( . %) and least in the northwest ( . %) (p o . ). for all regions, the proportion of resistance was: cephalothin (cph, . %) amoxicillin-clavulanic acid (amx, . %), ampicillin (amp, . %), tricarcillin-clavulanic acid (tcx, . %), doxycyline (dxy, . %) cefoxitin (cfx, . %), cefpodoxime (cpx, . %), chloramphenicol (chp, . %), enrofloxacin (enr, . %) , ciprofloxacin (cif, . %), trimethoprim-sulfamethoxazole (tmx, . %), ceftazidime(cfz, . %), gentamicin (gtm, . %), cefotaxime (cft, . %) meropenem ( . %) (p o . ). the mic exceeded the resistant breakpoint for amp, amx, cpx, cph, cif, cfx, dxy and enr whereas mic did not surpass the susceptible breakpoint. beta-lactams ( . %) was the most and aminoglycosides the least ( . %) sdr. the drug class most frequently involved in mdr was beta-lactams ( . %) and least, gen ( . %). resistance differs regionally, being greatest in the southwest. cph is the most and meropenam is the drug least associated with resistance; these patterns are consistent with current drugs used by veterinarians. the fluoroquinolones (fqs) are common choices for treatment of e. coli urinary tract infections (utis) in animals and humans. nd generation drugs approved in animals include enrofloxacin (enr), marbofloxacin (mar), orbifloxacin (orb); human drugs include ciprofloxacin (cip). rd and th generation fq for humans include moxifloxacin (mox), gatifloxacin (gat) and ofloxacin, (ofl]), its lisoform levofloxacin (lev). for animals, pradofloxacin (pra) is approved for use in europe. the purpose of this study was to assess the in vitro activity of st (naladixic acid [nal] through th generation fqs (n ) toward dog or cat e.coli uropathogens (n ). isolates were subjected to susceptibility testing to drugs classes ( drugs). isolate phenotypes included no (ndr; n ), single (sdr; n ) or multidrug (to more than drug classes; mdr; n ) resistance (including enr resistant [enr r -mdr; n ] or enr susceptible (enr s -mdr, n ). the minimum inhibition concentrations (mics) were determined for each isolates using broth microdilution (e. coli atcc s served as a negative control). mic statistics were generated for each drug among phenotypes. the overall potency (mic ) for all enr susceptible isolates (ndr, sdr and enr s -mdr) was gat pra, mox, mar, lev, cip sar, orb, ofl enr nal. each e. coli isolate expressing ndr or sdr was susceptible to all fq. however, isolates expressing resistance to st or nd generation fq were also resistance to later generation drugs. glucocorticoids (gc) are standard therapy for allergic asthma but do not reverse the underlying type i hypersensitivity. allergenspecific immunotherapy (asit), a process of ''desensitization'', is potentially curative but requires identification of offending allergens. the purpose of this study was to determine if oral or inhaled gc administered at routinely used dosages would interfere with allergen identification. we hypothesized that oral but not inhaled gc would interfere with accurate identification of allergen-specific ige using skin and serum testing in experimentally asthmatic cats. asthma was induced in eighteen cats using bermuda grass allergen (bga). cats (n /group) were randomized to receive oral gc ( mg prednisolone q hr po), inhaled gc ( ug budesonide q hr) or placebo (gelatin capsule q hr po) for one month. intradermal skin testing (idst) and bga-specific ige amounts were measured prior to, during (weeks one and four) and every two weeks after treatment until both tests were positive. a paired t test was used to compare serum ige among groups pre-and post-treatment (p o . significant). idst reactivity was eliminated in / cats on oral gc, / on inhaled gc, and / placebo-treated cats. within two weeks after stopping treatment, idst was again positive in all cats. contrary to our hypothesis, serum ige reactivity to bga was not significantly diminished by any treatment. in conclusion, a two week withdrawal from gcs is adequate for idst identification of allergen but no withdrawal is required prior to serum ige testing to identify the sensitizing allergens. previously in people, increasing severity of asthma is associated with low serum concentrations of -hydroxyvitamin d ( -oh-d). -oh-d is thought to ameliorate lower airway inflammation primarily by decreasing the production of pro-inflammatory mediators, and by increasing the production of the anti-inflammatory cytokine il- . in people, serum -oh-d concentration is associated with sunlight exposure as well as dietary intake. cats do not rely on sunlight for vitamin d synthesis; all vitamin d comes from dietary intake. cats have a naturally occurring lower airway disease syndrome (lad) that shares many features with human asthma. the goal of this study was to evaluate serum -oh-d concentrations in cats with lad. cats with naturally developing lad were enrolled. criteria for a diagnosis of lad included a history of cough, wheeze or respiratory distress, radiographic evidence of a bronchial pattern and hyperinflation, negative heartworm antigen and antibody test, and a resolution of clinical signs in response to glucocorticoids. dietary history was obtained. -oh-d concentrations were determined on serum samples by a commercial laboratory. twelve cats with lad were enrolled. all cats ate commercial cat food. the median -oh-d concentration was nmol/l with a range of - nmol/l which is within the reported reference range of - nmol/l. in contrast to human asthma, lower airway disease in cats is not associated with low serum concentrations of -oh-d. interstitial lung diseases (ild) are uncommon in dogs, with the most commonly recognized ild idiopathic pulmonary fibrosis (''westie fibrosis''). in human medicine, ild represent a large umbrella of pulmonary diseases, with ipf only a subset. other, more treatable, ilds are also identified, and may respond to either the removal of a stimulus (hypersensitivity) or steroid therapy. the goal of this report is to describe the clinical course, including outcome, computed tomography and histopathology of dogs affected with an ild. the computed tomography (ct) log was reviewed for dogs that underwent thoracic ct scanning for evaluation of respiratory signs, and had changes consistent with ild as the primary abnormality, including the presence of diffuse disease in all lobes, and at least of the following: reticulation, ground glass opacity, consolidation, or traction bronchiectasis. survival time from ct date was calculated. the presence of moderate pulmonary hypertension [phtn] ( mmhg) as estimated by tricuspid regurgitant jet, was also reported and survival times were compared with a mann-whitney rank sum with p o . considered significant. thirteen dogs were identified. terriers and chihuahuas were the most commonly affected breeds. two dogs were adolescents, the remaining dogs ranged from - years, with a median of years. histopathology results (n ), including moderate to severe interstitial fibrosis ( ) alveolar proteinosis with fibrosis ( ), and interstitial eosinophilic pneumonia ( ). one had suspected cryptogenic organizing pneumonia and had a good response to glucocorticoids. eight dogs died of respiratory failure, with a median post ct survival time of days (range - ), two dogs died of non-pulmonary disease, dogs had severe lower respiratory infections as puppies with persistent respiratory signs, and both are still alive at years since diagnosis, terrier is alive at months and was lost to follow up. dogs had phtn, with a median survival of days ( - ), while the dogs without had a survival of days (range - ), [p . ]. interstitial lung disease in dogs is not just idiopathic pulmonary fibrosis. following respiratory infection, young dogs may develop an ild with a relatively indolent course and rare ild is steroid responsive. ct is useful to identify ild but further research correlated with echocardiography and histopathology is advised to use it to prognosticate. idiopathic pulmonary fibrosis (ipf) is an interstitial pulmonary disease, mainly described in west highland white terriers (whwt). identification of molecular pathways important in the pathogenesis of ipf would improve our understanding of this disease and may help identify therapeutic targets. the aim of the present study was to investigate gene expression in lungs of whwt with ipf using oligonucleotide microarray. total rna was extracted from post-mortem pulmonary samples from five whwt with ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. genes with a minimum of a two-fold difference in expression between the two groups were selected for further analysis. the most significant biological functions for these genes were identified using ingenuity pathways analysis. more than genes were identified as having greater than twofold difference in expression. the significant biological functions associated with these genes were related to cellular movement, cellular proliferation and apoptosis. most notable among these were genes encoding the leukocyte chemotactic proteins: ccl (fold change . ), ccl ( . ) and il ( . ); the proteins involved in fibroblast migration; and the matrix metalloproteinases (mmps) involved in matrix degradation: mmp (À . ), mmp (- . ), mmp (À . ). this study has identified genes which may be important in pathogenesis of ipf, e.g. proteins involved in leukocytes chemotaxis, fibroblast recruitment and activation, regulation of apoptosis, and extracellular-matrix turn-over. however, real-time quantitative rt-pcr studies are needed to confirm these results before any definitive conclusions can be drawn. idiopathic pulmonary fibrosis (ipf) is an interstitial disease, mainly described in west highland white terriers (whwt). defini-tive diagnosis ultimately relies on lung histopathology. identification of specific biomarkers would be very helpful. expression microarray is a powerful screening tool to study local gene expression in a disease state. the aim of the present study was to measure gene expression profiles in lungs of whwt with ipf to identify potential blood or bronchoalveolar lavage fluid (balf) biomarkers. total rna was extracted from post-mortem pulmonary samples from five whwt with histopathologically confirmed ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. ipa-biomarkers analysis (ingenuity system) was used to filter and prioritize biomarkers candidates using the three following criteria: a minimum of a two-fold difference in expression between ipf and ctrl; expression of the gene in lung tissue; possible detection of the protein in blood or in balf. fifty-four molecules met all the criteria. based on difference in expression, promising proteins included ccl (fold change . ), a -actinin ( . ), ccl ( . ), serum amyloid a ( . ), il ( . ), plunc (À . ), mmp (À . ). some are well-known biomarkers of ipf in humans either for diagnosis (mmp , il ) or prognosis (ccl ). these results provide novel potential biomarkers of canine ipf. measurement of these proteins in blood and balf of healthy dogs, dogs with ipf and with other respiratory diseases is needed to assess their use as biomarkers of canine ipf. heliox is a mixture of helium and oxygen that has been used therapeutically in human medicine for treatment of airway obstruction. helium's low density and other physical properties have been shown to reduce the work of breathing by limiting turbulence. the purpose of this study was, therefore, to evaluate respiratory parameters in response to inhaled heliox in dogs with meso-and brachycephalic conformation. eleven healthy dogs were recruited, five were mesocephalic and six were brachycephalic. flow-volume loops were collected using commercial software (buxcor) while breathing : helium: oxygen (heliox) and : nitrogen:oxygen (nitrox) in a randomized order via a low dead-space face mask. due to the intrinsic gas properties, gas flow rates and volumes were corrected in-vitro by a conversion factor for the effect of helium on the pneumotachograph. respiratory rate, tidal volume (ml), minute ventilation (l), inspiratory time (ti), expiratory time (te), peak inspiratory flow (pif) and peak expiratory flow (pef) were recorded while breathing heliox or nitrox. values were compared using a paired sample t-test, with p o . considered significant. all dogs cooperated with testing. there was no significant difference in respiratory rate, tidal volume, minute ventilation, inspiratory or expiratory times, or peak inspiratory flow. peak expiratory flow was significantly higher (p . ) while breathing heliox than when breathing nitrox in brachycephalics but not in mesocephalics (p . ). heliox is well-tolerated in healthy dogs and results in an increased expiratory flow rate in brachycephalic dogs. further investigation of heliox is warranted in dogs with airway obstruction. of this prospective multicentric study is to assess the effects that surgical correction has on the severity of clinical signs and levels of acute phase proteins (c-reactive protein [crp] , haptoglobin [hp]) and cardiac troponin i (ctni). thirty three brachycephalic dogs with boas were included and evaluated before and, approximately two months, after surgical correction. the most common components of boas found were elongated soft palate ( / ; %), stenotic nares ( / ; %) and everted laryngeal saccules ( / ; . %). staphylectomy was performed by means of two different surgical techniques: laser (n ) or electrical scalpel (n ). there were significant differences between dogs depending on the surgical technique used, with a higher reduction of respiratory signs (p o . ) and a better postsurgical improvement (p o . ) with the use of laser. the levels of crp, hp and ctni were categorized into normal or elevated. before surgical treatment three ( . %), six ( . %) and thirteen ( . %) dogs had elevated values of crp, hp and ctni, respectively. two months after surgical correction, five ( . %), eleven ( . %) and fourteen ( . %) dogs had elevated values of crp, hp and ctni, respectively. there were no statistical differences between values of crp and ctni before and after surgical correction but the levels of hp increased significantly after surgical treatment (p o . ), probably due to postsurgical treatment with corticosteroids. as previously suggested by others, there was a statistically significant reduction of respiratory and gastrointestinal signs in dogs with boas submitted to surgical correction (p o , ). according to the results obtained in the present study, the determination of crp, hp and ctni before and two months after surgical treatment do not have a prognostic value in dogs with boas. even though, near half of the dogs studied had elevated levels of ctni ( . %) that persisted after surgical treatment ( . %), suggesting some degree of myocardial damage is present. further studies are needed considering the influence of breed and age. to the authors' knowledge, this is the first description of crp, hp and ctni determination in dogs with boas. overweight and obesity are common conditions that lead to alterations in respiratory mechanics, airway resistance, pattern of breathing and gas exchange in humans. the objective of the present study was to investigate if there are significant differences on respiratory parameters and arterial gas analysis of obese and overweight cats, in conscious state and under general anesthesia. twenty nine adult cats were arranged in three groups: obese (n ), overweight (n ) and with ideal body score index (bsi) (n ). mean of bsi in the groups were: , (obese), , (overweight) and , (ideal bsi). cats did not had respiratory, cardiac or others systemic diseases. the respiratory parameters were evaluated with a ventilometer equipment coupled to facemasks in conscious cats and directly to the endotracheal tube in anesthetized cats under spontaneous respiration. the anesthesia was performed with propofol ( ae , ml/kg) and the cats were maintained in the same anesthetic plan. the three groups were compared by analysis of variance followed by tukey's test and conscious and anesthetized cats were compared by student's t test, with a % significance level. there were not observed differences on the respiratory parameters evaluated on ventilometry (tidal volume, expiratory and inspiratory times and peak pressures, respiratory rate and partial pressure of end tidal co (petco )) and on arterial gas parameters (pao e paco ) in the three groups. the pao of cats with ideal bsi was , ae , mmhg, although was not significantly different (p , ) from overweight ( , ae , mmhg) and obese cats ( , ae , mmhg). comparison of anesthetized to conscious cats, it was detected decreases in tidal volume, expiratory and inspiratory times and peak pressures and increase in petco in respiratory rate in the anesthetized cats. only petco , inspiratory time and respiratory rate in overweight cats did not differ in anesthetized cats. these results suggest that obesity and overweight did not result in impairment of respiratory function in cats and propofol induced respiratory depression. osteosarcoma (osa) is the most common bone tumor in dogs, however, little is known regarding the mechanisms underlying malignant transformation in these tumors. breeds such as rottweilers and greyhounds are at higher risk for developing osa, suggesting that heritable factors play a role in this disease. mirnas have tumor/tissue specific roles in regulating gene expression and dysregulated mirna expression is found frequently in cancer. we hypothesize that canine osa is characterized by a unique mirna expression profile(s) with dysregulation of some mirnas being associated with specific breeds. mirna expression profiling of primary osa tumors from greyhounds and rottweilers was performed using the nanostring technologies ncounter mirna expression assay kit, interrogating the mirna expression profile of human mirnas, of whose mature sequences are % conserved between human and dog. mirnas were differentially expressed in greyhound versus rottweiler tumors (p o . ), suggesting that breed-specific dysregulation of mirnas may contribute to the development and progression of spontaneous osa. hierarchical clustering revealed distinct mirna expression signatures in greyhound osa tumors as compared to rottweilers. based on these preliminary results, we are evaluating a larger cohort of osa tumor samples including greyhounds, rottweilers, golden retrievers, and a mixed population of other breeds. statistical analysis will be performed to determine the association of mirna transcript levels with specific breeds and overall outcome. characterization of mirna expression in canine osa will facilitate our understanding the biology of this disease and has the potential to identify targets for therapeutic intervention. originally combination therapies using drugs with documented single-agent activity and lack of overlapping toxicities could potentially improve outcome. the hypothesis intended to be tested is that palladia s can be safely administered concurrently with a standard weekly protocol of vinblastine (vbl), at dosages known to have activity against mast cell tumors. dogs with histologically confirmed measurable mast cell tumors were evaluated for eligibility to enter a standard phase i dose-finding trial ( cohort), at a starting dose of . mg/m iv vbl (weekly for a total of treatments) and . mg/kg po palladia s eod, concurrently. dose escalation of palladia s was scheduled in . mg/kg increments until mtd was established or fda label dose completed ( . mg/kg). safety evaluation was performed weekly throughout the week study period. dose-limiting toxicities were described following established vcog-ctcae(v . ) criteria. while antitumor response is not a primary endpoint of phase i trials, activity was documented prior to vbl treatments - , and monthly thereafter, based on recist criteria. nine dogs have been enrolled; cohort is filled and approaching completion of the evaluation period. hematologic dose limiting toxicity led to de-escalations of vbl. the current safe combination appears to include vbl at . mg/m every other week and palladia s at . mg/kg eod. response was seen in all but one dog. without head to head trials comparing efficacy of bi-weekly vbl combined with palladia s and vbl alone, choice of therapy should remain at the clinician's discretion. originally prostate specific membrane antigen (psma) is a transmembrane protein expressed by tumor-associated neovasculature, but not normal blood vessels. based upon its selective expression in endothelial cells associated with cancer, psma may serve as a conserved angiogenic target shared by macroscopic solid tumors of various histologies. to investigate the feasibility of targeting a homogenous population of psma-expressing endothelial cells as a novel anticancer strategy, we have investigated psma expression in several canine hemangiosarcoma (chsa) cell lines, and subsequently developed self-assembling nanoparticles containing diagnostic (near infrared dyes) and therapeutic (doxorubicin) cargo which selectively bind to psma by means of the a aptamer, a commercially-available oligonucleotide. the expression of psma by chsa cells was confirmed transcriptionally and translationally by real time pcr and immunohistochemistry, respectively. selective binding and endocytosis of a decorated nanoparticles was studied by fluorescent microscopy. the ability of a decorated nanoparticles encapsulating doxorubicin to exert in vitro cytotoxic effects in chsa cells was assessed by colony forming assays. using a chsa xenograft murine tumor model, clinically-relevant anticancer effects of a decorated nanoparticles encapsulating doxorubicin were tested. all chsa cell lines expressed psma mrna and protein. a decorated nanoparticles were selectively endocytosed by psma-expressing cells, and when these nanoparticles encapsulated doxorubicin, significant cytotoxic effects were exerted in vitro. finally, a decorated nanoparticles encapsulating doxorubicin significantly reduced the size of macroscopic chsa tumor burdens in transplanted mice. diagnostic and therapeutic nanoparticles can be targeted to psma-expressing endothelial cells, and chsa provides a comparative model for the future study of nanoparticle therapeutics. canine transitional cell carcinoma (tcc) is the most common tumor of the urinary tract, and is similar to human invasive tcc in histopathologic characteristics, molecular features, sites of metastasis, and response to medical therapy. prevalence is increasing, and novel therapies and strategies are needed to effectively treat this aggressive form of cancer in both species. personalized medicine techniques intend to improve treatment outcome by using patient tumor profiling to identify potential and individualized therapeutic targets. a genomic algorithm has been developed termed ''coexpression extrapolation'', or coxen, that aims to use expression microarray data to predict drug activity in patient tcc samples. the utility of this predictive methodology has been established in other types of cancer in vitro, however its clinical utility has not yet been determined. validation studies of coxen in canine tcc cell lines were conducted. the goal was to determine the value of coxen in predicting baseline sensitivity of canine tcc to chemotherapy agents (gemcitabine, mitoxantrone, carboplatin, vinblastine and cisplatin) that would then be used in a proposed clinical trial. additionally, expression data from canine treatment-naı¨ve primary tumor samples were generated on an affymetrix array platform (canine genome v . ). both the expression data and tcc cell line data (antiproliferative effects, % growth inhibition or gc ) were used to establish a canine specific predictive coxen algorithm. coxen scores for canine tcc cell-line drug activity were then analyzed. scores predicted the activity of cisplatin, gemcitibine, and mitoxantrone in all cell lines, and of carboplatin in cell lines. because all of the cell lines were sensitive to vinblastine (gi o . mm), the coxen score was not predictive of its potency. interestingly, coxen fails to predict vinblastine response in human tcc cell line data as well. in concurrent work, comparative genomic studies to define and compare the gene expression signatures of tcc in dogs and humans provides further evidence that canine tcc is a valuable genomic model of the human disease. current studies involve testing the chemo-predictivity of this derived canine coxen algorithm in additional canine tcc cell lines. canine tcc offers an excellent model for in vitro and in vivo studies of the coxen approach. this preclinical work will be used to guide the feasibility of future coxen clinical trials in dogs and humans with tcc. a small molecule complex (aminoact) isolated from bovine milk is a natural peptide mixture with multi-kinase inhibitory effects against epidermal growth factor receptor (egfr) and insulin-like growth factor receptor- (igfr- ). ingestion of aminoact in people with cancer results in lower serum tnf-alpha, an increase in antioxidant superoxide dismutase (sod) enzyme activity, and subjects' blood serum causes apopsotis in cancer cell lines. this study was designed to first assess safety and secondly the efficacy of three dosage levels of ax- in sustaining progression free survival (pfs) for dogs with refractory advanced and/or metastatic cancer. the prospective, open label study included dogs of different breeds with naturally occurring histologically confirmed malignancies. the first dogs received aminoact at g/m ; the second group of dogs subsequently received the same dosage mg of aminoact; and the third group of dogs subsequently received g/ m . each dog was treated orally daily for six weeks along with mg betaine hcl, that aids in peptide absorption. all patients were evaluated for toxicity using the vcog-ctcae and efficacy using the recist criteria via assessment of clinical parameters, blood work and client questionnaires. no toxicity other than mild, transient (grade i) nausea was noted, nor were there any changes in hemograms or biochemical profiles in any patient. dogs with tumors that were confirmed as responders ( % reduction in size) include pulmonary adenocarcinoma, mast cell tumor, trichoepithelioma and soft tissue sarcoma. it appears in limited studies that the response rate may be more durable at higher dosages. the response to aminoact is dose dependent and only transient mild toxicity was observed, which suggest the maximum effective dosage has not been reached. further clinical studies will be valuable in determining the effective dosage and response duration. treating cancer in dogs with aminoact offers a unique opportunity as a model for human cancer biology and translational cancer therapeutics. stereotactic radiation therapy (srt) combines patient immobilization, image guidance, and intensity modulated delivery to achieve ablative radiation doses within the tumor, while preferentially sparing surrounding normal tissues. the purpose of this study was to evaluate the efficacy of srt as a means of achieving local tumor control for canine nasal tumors. retrospective analysis was performed on dogs with a nasal tumor confirmed by histopathology and computed tomography, no previous surgical or radiation therapy, at least six months of follow-up, and completion of three fractions of srt at csu.srt was administered via the varian trilogy linear accelerator once daily for three consecutive days. the varian eclipse treatment planwas reviewed to determine the planned target volume (ptv) and dose to % of the ptv. kaplan-meir survival analysis was performed for disease free interval (dfi) and overall survival (os). sixteen patients with nasal tumors ( adenocarcinoma/carcinomas, squamous cell carcinomas, chondrosarcomas, osteosarcomas, and undifferentiated sarcoma) were treated with srt. a median dose of . gy was administered to % ptv with a median ptv of . cc. srt was well tolerated by the normal tissues with minimal, manageable side effects. to date, the median dfi is days, while the median os is days. based upon the initial clinical experience, stereotactic radiation therapy is an emerging modality in the management of canine nasal tumors. canine leptospirosis can vary from subclinical infection to illness that ranges from mild to severe, including death, depending on the susceptibility of the dog, virulence of the organism, and route and degree of infection. the objective of this study was to evaluate the ability of a canine leptospira bacterin to prevent infection and disease following challenge with virulent leptospira canicola, l. pomona, l. grippotyphosa, or l. icterohaemorrhagiae. groups of week-old beagles were vaccinated (day ) and boosted (day ) with placebo (n ) or the -way bacterin (n ! ) and subsequently challenged with each serovar. the results demonstrated that blood and various tissue samples from placebo-recipients became reliably infected, and the dogs developed typical clinical signs of leptospirosis including loss of appetite, ocular congestion, depression, dehydration, jaundice, hematuria, melena, vomiting, petechiae, and death. in addition, placebo-recipients developed kidney and liver dysfunction. in contrast, some vaccine-recipients became infected, but the organisms were cleared quickly from the blood. vaccinated dogs failed to develop severe clinical disease requiring medical intervention, and no animals died (p ! . ). a few of the vaccinated dogs developed clinical abnormalities, but the clinical signs remained mild and were self-limiting (p o . for each serovar). administration of the bacterin also prevented thrombocytopenia ( ciprofloxacin, a synthetic fluoroquinolone antimicrobic, is not fda-approved for veterinary use. however, due to recent availability of less expensive generic formulations, extra-label use of ciprofloxacin by veterinarians appears more common. although ciprofloxacin crystalluria and uroliths have been reported in humans, we are unaware of any published reports in dogs. this is surprising since mean urine ciprofloxacin concentration ( . mg/ml) in dogs following a modest iv dose ( mg/kg) was times higher than the solubility of ciprofloxacin in water ( . mg/ml). to identify the occurrence of ciprofloxacin uroliths in dogs, records from the minnesota urolith center were reviewed. between january and december , ciprofloxacin was identified in uroliths from dogs; uroliths were composed of % ciprofloxacin in , mixed uroliths containing ciprofloxacin were identified in , a shell of ciprofloxacin was observed in , and ciprofloxacin surface crystals were identified in . based on an experimental study in which % of human volunteers consuming mg of ciprofloxacin with nahco exhibited ciprofloxacin crystalluria (urine ph . ), while no volunteers consuming mg of ciprofloxacin and nh cl to acidify urine formed crystals; we postulated that ciprofloxacin uroliths could be dissolved in acidic urine. to test this hypothesis, canine uroliths composed of % ciprofloxacin from a single source ( -yr-old male, english bulldog receiving mg/kg of ciprofloxacin po, q hr to manage superficial pyoderma; turbulent flow chromatography/tandem mass spectrometry detected mg of ciprofloxacin/g of urolith) were incubated in urine at selected ph's and monitored for dissolution. urine obtained from multiple dogs not receiving fluoroquinolones, was pooled and divided into aliquots. aliquots were adjusted with hcl or naoh to a ph of , , , , or . aliquots were capped and preserved by refrigeration; ph was monitored and readjusted weekly. ten uroliths of approximately equal weight were randomly assigned to individual flasks containing mls of urine. flasks were constantly agitated and maintained at c. every hours, urine was discarded and replaced with mls of urine of identical ph until stone dissolution was complete. ciprofloxacin urolith dissolution times at each urine ph are reported below. ciprofloxacin uroliths are a newly recognized disease and a potential adverse effect of ciprofloxacin administration in dogs. in vitro dissolution of ciprofloxacin uroliths was achieved in canine urine, supporting the premise that in vivo dissolution is possible. urolith dissolution times were shortest at lower and higher ph's, which is consistent with the pka ( . and . ) of this amphiprotic antimicrobic (more soluble at ph below the acidic pka and above the alkaline pka). foods designed to promote struvite urolith dissolution may be designed for short term feeding facilitating rapid dissolution or may be formulated with a more moderate target urine ph to allow for dissolution and then life-long maintenance feeding minimizing recurrence. the purpose of this study was to compare the efficacy and rate of dissolution of a maintenance food with a struvite dissolution food. sixteen client-owned adult cats ( fs, mc) with naturally occurring struvite urocystoliths (mineral composition based on history, radiographs, urinalysis, urine culture and physical examination) were randomized to either a dry maintenance food (test) or a dry food known to dissolve struvite uroliths (control). the clinical care team and owner were blinded to treatment assignment. the test food was formulated to provide . % mg (dm), . % p, % protein, and a calculated target urine ph value (uph) of . - . . the control food was formulated to provide . % mg (dm), . % p, % protein, and a targeted urine ph of . - . . owners were advised to feed the assigned diet exclusively in an amount to maintain body condition. after diet assignment radiographs were performed at eight weekly intervals until there was no evidence of uroliths or until there was evidence that the uroliths were the same size or larger. a physical examination, complete blood count, serum chemistry profile, urinalysis and urine culture were repeated at the conclusion of the study. statistical analysis was by anova. all uroliths dissolved in all cats and both foods were palatable. radiographs of cats fed the control food indicated the uroliths dissolved in a significantly shorter time (mean ae std dev of . ae . weeks) compared to cats consuming the test food (mean . ae weeks; po . ).). cats in the control group finished the study at (n ), (n ) and weeks. cats in the test group finished the study , , (n ), , , , and weeks. all the minnesota urolith center occasionally receives uroliths for analysis that are immersed in formalin. results of quantitative analysis of these uroliths revealed that some submitted in formalin consisted of newberyite (magnesium hydrogen phosphate trihydrate). because newberyite is uncommonly found in uroliths formed by cats and dogs, we hypothesized that this mineral was an in vitro artifact caused by exposure of struvite (magnesium ammonium phosphate hexahydrate) to formalin. the purpose of this study was to determine if formalin alters the mineral composition of uroliths. urolith submissions containing stones of either % struvite (n dogs and cats), % calcium oxalate (n dogs and cats), % calcium phosphate apatite (n dogs and cats), % cystine (n dogs and cats), % ammonium urate (n dogs and cats), and % silica (n dogs) preserved by only air drying were tested. one urolith from each submission was quantitatively analyzed by polarized light microscopy or infrared spectroscopy. a subsequent urolith from the same submission was immersed in ml of % buffered formalin for hours at room temperature. uroliths were then air dried for minutes and the analysis was repeated. after exposure to formalin, portions of all struvite uroliths were transformed into newberyite. three ( dog and cats) of ammonium urate uroliths were completely dissolved. newberyite was not detected in any of the remaining uroliths. likewise quantitative mineral analysis of non-struvite uroliths remained unchanged. to avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis. we previously reported that transfusion to normal dogs of autologous erythrocyte concentrates (prbcs) that had been stored for days causes a profound inflammatory response ( x increase in leucocyte count and fibrinogen, x increase in c-reactive protein). we speculated that inflammation was due to cytokines produced during the storage period, and hypothesized that transfusion of fresh (f) prbcs would elicit less inflammation than would stored (s) prbcs. a whole blood unit was collected from healthy dogs (n ) for prbcs on day , then again on day . on day dogs received an autologous transfusion of prbcs stored for either days (s, n ) or days (f, n ). cbcs and in-tem thromboelastometry (ct:coagulation time, cft:clot formation time, a:alpha, mcf:maximum clot firmness) were evaluated on blood samples collected at (pre) and , , , , and hours after transfusion. fresh prbcs did not elicit any change in leucocytes, platelets, or thromboelastometry. stored prbcs elicited a degenerative left shift ( hr) followed by a regenerative left shift ( - hr), thrombocytopenia ( % decrease at hr), and marked hypocoagulability characterized by prolonged ct ( , , hr) and cft ( , hr), and decreased a ( , hr) and mcf ( , , hr). data are mean(sd). a: p o . between groups f and s by t test. b: p o . compared to '' '' by rm anova. transfusion of autologous stored prbcs elicits a greater inflammatory response than fresh prbcs, and results in hypocoagulability on thromboelastometry. clopidogrel is a potent antiplatelet drug that is gaining popularity in veterinary medicine for antithrombotic therapy. the parent molecule is an inactive prodrug that must be converted by hepatic isozymes to an active metabolite. the majority of the parent molecule is directed to the formation of inactive metabolites with only an extremely small proportion of parent molecule directed to the formation of the active metabolite. there are multiple hepatic isozymes responsible for the formation of the active metabolite. a non-specific hepatic isozyme inducer such as rifampin could increase the formation of the active metabolite of clopidogrel thereby increasing the pharmacodynamic response which may allow a reduced drug dose to achieve a clinical effect. we have previously presented data supporting the increased pharmacodynamic response of clopdiogrel after rifampin therapy. the goal of this study was to demonstrate an increased pharmacokinetic response of clopidogrel after rifampin induction of hepatic isozymes. six healthy, purpose-bred dogs were used for this study. the pharmacokinetics of clopidogrel were determined by measuring the parent molecule, primary inactive metabolite and active metabolite through lc/ms/ms. the pharmacodynamics of clopidogrel were determined by measuring collagen-induced whole blood aggregation. blood samples were collected prior to clopidogrel administration (baseline), after days of mg/kg clopidogrel po q hrs, and after days of mg/kg clopidogrel po q hrs mg/ kg po q hrs rifampin. given the absence of a known standard for the active metabolite, only a semi-quantitative assessment of active metabolite concentration can be made. there was no identifiable active metabolite peak noted at baseline or after clopidogrel treatment. however, with clopidogrel and rifampin combined administration there was an active metabolite peak identified in all dogs with a mean area of . ae . . the development of the active metabolite peak was associated with an increase in the pharmacodyamic response of clopidogrel in the dogs. this is the first study in any species to document the increased formation of the active metabolite of clopidogrel in response to a strong, non-specific hepatic isozyme inducer. this increased pharmacokinetic response was associated with an increased pharmacodynamic response of clopidogrel. this data provides supportive evidence to develop therapeutic protocols to improve the pharmacodynamic response to clopidogrel in dogs that may reduce dosing requirements or correct subtherapeutic pharmacodynamic response. critical illness-related corticosteroid insufficiency (circi) has been identified in humans, foals, dogs and cats with lower-thanexpected circulating cortisol concentrations, and/or by a blunted cortisol response to acth stimulation. our purpose was to determine if circi exists in critically ill horses. endogenous plasma acth and serum cortisol concentrations, and cortisol at t and t min after . mg/kg cosyntropin, were measured by radioimmunoassay from horses with colic or systemic illness on admission, and days , and of hospitilization. horses were divided into mild, moderate, or severe illness groups based on clinicopathologic data. inappropriately low cortisol was defined as endogenous cortisol o mean- sd achieved after administration of . mg/kg cosyntropin to normal horses ( o nmol/l). inadequate delta cortisol was defined as o mean delta cortisol in normal horses after . mg/kg cosyntropin ( o nmol/l). cortisol, acth and delta cortisol were compared using anova between groups, with p o . considered significant. fifty-eight horses classified as having mild ( ), moderate ( ) and severe ( ) disease at admission had survival rates of %, % and % respectively. admission acth and cortisol concentrations were highest in severely ill horses ( ae pg/ml, ae nmol/l) compared to moderate ( ae , ae ) and mildly ill horses ( . ae . , ae ). admission cortisol concentrations were higher overall in severely ill horses (p . ), but were low in % ( / ). admission delta cortisol was low in % ( / ) of severely ill horses, and was associated with marked adrenal hemorrhage in non-survivors. severely ill horses have high cortisol and acth, but low cortisol and delta cortisol may indicate circi secondary to adrenal hemorrhage. equine pituitary pars intermedia dysfunction (ppid) is a common endocrinopathy of aged horses that results from neurodegeneration of the dopaminergic periventricular neurons that innervate the intermediate lobe of the pituitary. factors that initiate spontaneous dopaminergic neurodegenerative disease remain elusive, however accumulation of misfolded a-synuclein protein and dysfunctional protein clearance have been implicated. misfolded protein accumulation occurs due to increased protein production or decreased clearance of damaged macromolecules through the process of autophagy. while have previously demonstrated that horses with ppid have increased asynuclein in the periventricular neurons compared to controls, it remains unknown whether the protein accumulates due to increased production or decreased clearance. we hypothesized that autophagy is decreased in the pituitary neurointermediate lobe from horses with ppid compared to controls. neurointermediate lobe pituitary tissue was from collected from horses with ppid (n ) and healthy horses (n , - years). realtime pcr was used to determine the relative expression of autophagy genes (mtor, beclin , atg , atg , atg , pink, lamp ) and a-synuclein relative gene expression from horses with ppid were compared to healthy horses by t-test following log transformation. a pearson coefficient of correlation was calculated comparing a-synuclein expression with autophagy gene expression. the expression of a-synuclein, autophagy-related genes (atg , beclin, lamp ), and mtor was greater in horses with ppid than in healthy horses. age was not correlated to a-synuclein or autophagy gene expression. there was a significant positive correlation between expression of a-synuclein and beclin , atg , atg , atg , and pink, but not mtor expression. accumulation of a-synuclein protein in horses with ppid may result from increased a-synuclein expression. autophagy genes are upregulated in horses with ppid, suggesting a compensatory response, although these findings need to be confirmed by demonstrating an increased functional response. asynuclein expression was positively correlated to expression of autophagy genes except mtor, suggesting a-synuclein may stimulate autophagy in an mtor independent manner. acvim forum session a efficacy of delayed antiviral therapy against ehv- challenge. lk maxwell , ll gilliam , n pusterla , r carmichael , rw eberle , jw ritchey , tc holbrook , t gull , gb rezabek , d mcfarlane , cg macallister . oklahoma state university, stillwater, ok. university of california, davis, ca. equine herpes virus type- (ehv- ) outbreaks are often not recognized until exposed horses are at immediate risk for developing equine herpes myeloencephalopathy (ehm). the objective of this study was to determine whether delayed therapy with the antiviral drugs valacyclovir or ganciclovir could protect those horses most at risk for ehm. eighteen aged ( years) mares were randomized to treatment: no therapy (control), oral valacyclovir therapy, or intravenous ganciclovir therapy. drug administration was initiated at the onset of the second febrile phase, between days - after ehv- inoculation (pi), and continued for one week. neurological examinations were performed prior to the study and for three weeks pi. one horse was excluded from the study for failure to become febrile. body temperature was significantly lower in the ganciclovirtherapy horses as compared to control horses on days - pi (p o . ), whereas valacyclovir-therapy horses did not differ from control horses. viremia in whole blood, as determined by pcr, was also lower in the ganciclovir-therapy horses on days - pi and on day pi in the valacyclovir-therapy horses (p o . ). although antiviral drug administration did not reduce the risk of ataxia (p . ) or nasal shedding, ganciclovir therapy did decrease the severity of ataxia (p o . ) as compared to valacyclovir-therapy and control horses, where / , / , and / horses, respectively, developed at least a two grade change in ataxia. in summary, ganciclovir administration provided better protection against ehm than did valacyclovir when therapy was initiated just prior to the onset of neurological disease. equine vaccination is amongst the most important method of prophylaxis against equine influenza virus (eiv), a pathogen in which continuous antigenic drift can lead to vaccine failure. a month duration of immunity (doi) challenge infection study was conducted using commercial inactivated vaccines containing different strains of a/equine/ /influenza virus's, including innovator tm , containing kentucky/ (pfizer animal health, new york, ny), and calvenza, containing a combination of ohio/ , kentucky/ , and newmarket (boehringer ingelheim vetmedica, st. joseph, ms) . the challenge virus strain was colorado/ , the most contemporary challenge strain currently in use. the study design was a blinded, randomized challenge trial. three groups of yearling ponies, with no history or serological evidence of eiv infection were established. each group received one of three treatments: vaccination with innovator tm ; vaccination with calvenza tm ; or injection with a saline placebo. each treatment was administered times, at intervals of month between the first two treatments, and months between the second and third treatments. all ponies were challenged by nasal nebulization of x eid influenza virus a/eq/ /colorado/ months after the third treatment. clinical signs of disease, including rectal temperature, nasal discharge, anorexia, coughing, and depression, were recorded daily for days prior to challenge infection, and days post-challenge. nasal shedding of eiv was measured on the same days, using a realtime pcr test procedure. eiv-specific antibody responses were measured by elisa. differences between groups were analyzed by non-parametric repeated measures anova, and differences were declared significant when p o . . all control group ponies demonstrated clinical signs of disease consistent with eiv infection post-challenge infection, including pyrexia, nasal discharge, inappetance and partial anorexia. these signs were significantly lower in both vaccine groups; mean body temperature was elevated ( . f) for days in controls, but only days in vaccine groups. nasal shedding of eiv was detected in all ponies in all groups: over the duration of the study the calvenza group shed significantly less virus than innovator and control. over time antibody titers were significantly higher in the calvenza than the innovator group, and both were significantly greater than controls. this study demonstrated that both current commercial inactivated eiv vaccines have a duration of clinical protection of at least months after a highly pathogenic challenge with a recent eiv isolate. both antibody responses and virological protection differed between the vaccines. formulation difference between the vaccines, including the eiv antigens employed, may have contributed to this performance difference. degenerative myelopathy (dm) may be homologous to a form of amyotrophic lateral sclerosis in humans which has excitotoxic and immunologic pathogeneses described. the aims of this study were to determine (i) presence or absence of abnormalities in concentrations of csf amino acid (aa) neurotransmitters (glutamate, glycine and gÀaminobutyric acid (gaba)) and cytokines in dogs with dm and if present (ii) investigate associations with disease severity. twenty-two dogs histopathologically confirmed for dm and dogs with suspected dm based on thorough diagnostic investigations and clinically normal age-matched control dogs were included in the study. the neurological severity of the dm dogs was graded ( - ) using an established scale. csf was evaluated for presence of glutamate, glycine and gaba by high performance liquid chromatography and for gm-csf, ifn-g, il- , il- , il- , il- , il- , il- , il- , il- , ip- , kc (keratinocyte chemoattractant), mcp- (monocyte chemotactic protein- ) and tnf-a using a commercially available, canine multiplex immunoassay (millipore, billerica, ma). all data analyses were performed using sas v . (cary, nc). analyte levels were compared between dm confirmed, dm suspected and control dogs by an analysis of variance (anova). spearman correlation was used to test for correlations of analyte levels and neurological grades. all hypothesis tests were -sided with a . . there were no significant differences between individual csf analytes in dm confirmed and dm suspected dogs. glutamate levels were not significantly different between dm affected (mean . mg/ ml; range . - . ; sd . ) and control dogs (mean . mg/ ml; range . - . ; sd . ). control dogs (mean . mg/ml; range . - . ; sd . ) had significantly higher levels of gaba (p o . ) than dm dogs (mean . mg/ml; range . - . ; sd . ). control dogs (mean . mg/ml; range . - . ; sd . ) also had significantly higher glycine concentrations (p o . ) than dm dogs (mean . mg/ml; range . - . ; sd . ). dm-affected dogs also had significantly higher levels of il- (p . ), kc (p o . ) and mcp- (p . ) than control dogs. neurotransmitter levels were not significantly associated with neurological grade. kc levels were significantly higher in the least affected dogs (p . ). there were no associations with disease severity and analyte concentrations. dm affected dogs have an imbalance of csf aa concentrations creating a relatively excitotoxic environment. reports in human als confirm an imbalance between csf excitatory and inhibitory aas suggesting a pathogenic role for excitotoxicity in als. it also appears that dm affected dogs have increases in csf cytokines and chemokines suggestive of an immunologic component to the pathogenesis as is similar to als. further prospective analysis of dm is warranted to evaluate the role of treatment on csf variables. the pathogenesis of neuropathic pain (np) and syringomyelia (sm) in association with chiari-like malformation (clm) in dogs has focused on the anatomical anomalies and secondary cerebrospinal fluid (csf) flow abnormalities. neuropathic pain in humans has been associated with abnormalities of neurotransmitters such as glutamate and serotonin as well as immunologic mechanisms. the aim of this study was to investigate the csf neurotransmitter and cytokine levels in brussels griffon dogs (bgs) with clm, sm and np. as part of an mri study investigating the prevalence of sm in bgs, atlanto-occipital csf was acquired from dogs and stored at - c until analysis. all dogs underwent a neurologic exam prior to mri; osirix s software was used to measure sm and the presence of cerebellar herniation and deviation were recorded. deproteinized csf samples were analysed for presence of serotonin (ng/ml), glutamate, glycine and gaba (mg/ml) by high performance liquid chromatography. all csf samples were evaluated simultaneously for gm-csf, ifn-g, il- , il- , il- , il- , il- , il- , il- , il- , ip- , kc, mcp- and tnf-a. a commercially available, canine multiplex immunoassay (millipore, billerica, ma) was used for the cytokine analysis (pg/ml). student's t-tests were used to compare the means of neurotransmitter and cytokine values between groups with and without skull abnormalities or spinal pain. simple pearson's correlation was used to test for correlations of neurotransmitter and cytokine values with syrinx dimensions and correlations of neurotransmitter with cytokine values. all hypothesis tests were -sided and the significance level was a . . np was detected in dogs ( %); sm was present dogs ( %); and cm was detected in dogs ( %). ifn-g levels were significantly lower in dogs with np than without (p . ). there were significant positive correlations between syrinx size and il- (p . ), kc (p . ) and mcp- (p . ). there were significant negative correlations between ifn-g and syrinx height (p . ) and extent (p . ). there was a significant negative correlation between il- and syrinx height (p . ). neurotransmitter levels were not associated with skull abnormalities or spinal pain, but there was a positive correlation of glycine with il- (p . ) and mcp- with glutamate (p . ) and serotonin (p . ). the size of the syrinx in bgs with sm is associated with several cytokine elevations but only a decrease of ifn-g was associated with np. based on this study it does not appear that excitotoxicity plays a role in either sm development or np. further work is justified on the role of the immune system in cm, sm and np. current knowledge about the conservative management of disk associated cervical spondylomyelopathy (da-csm) is rather limited and mainly based on retrospectively retrieved data. the goals of this study were to prospectively evaluate the evolution of clinical signs in dogs treated conservatively for da-csm. additionally, several potential prognostic parameters and the correlation of initial clinical signs with magnetic resonance imaging (mri) and transcranial magnetic stimulation (tms) were investigated. twenty-one dogs were included. after neurological evaluation, neurological status was graded from ( normal) to ( tetraplegia). all animals underwent low-field mri and tms with measurement of onset latencies and peak-to-peak amplitudes from the extensor carpi radialis and cranial tibial muscles. from the mr images, the following dimensions were calculated: remaining spinal cord area; compression ratio; vertebral occupying ratio of the spinal cord; canal height to body height ratio (cbr); canal height to body length ratio (cblr); and the canal compromise ratio. intraparenchymal intensity (isi) changes were graded from to . all dogs were reevaluated by the same person after , , , , and months. eight of dogs ( %) experienced a positive clinical evolution with improvement of clinical signs or stabilization of mild clinical signs. all dogs with a negative clinical evolution month after diagnosis experienced a further progression of clinical signs resulting in a poor outcome. the opposite was true for all dogs with a positive clinical evolution after month. outcome was further significantly associated by the remaining spinal cord area and the vertebral canal compromise ratio. prognosis was not significantly affected by clinical presentation or tms. progression of clinical signs, in unsuccessfully treated dogs, was generally characterized by a rapid and dramatic deterioration of neurological status. there were no significant correlations between clinical presentation, mri and tms. two dogs underwent necropsy and histopathological examination. this revealed in both cases chronic wallerian degeneration and segmental myelomalacia. the results of this study suggest that conservative treatment of da-csm is associated with a rather guarded prognosis. clinical evolution month after diagnosis and selected mri parameters can be considered as prognostic indicators. the lack of correlation between clinical presentation and outcome, medical imaging and electrophysiological evaluation is disturbing and warrants further investigation. a mri-guided stereotactic brain biopsy system has not been clinically evaluated in dogs. the purpose of this study was to determine the ability of the brainsight tm system to obtain histologically diagnostic samples and access the impact of this procedure on neurologic status for hours after the biopsy. five dogs with mri definable lesions in the brain have been enrolled. breeds included a pitbull mix, pembroke welsh corgi, french bulldog, border terrier and west highland white terrier. age ranged from - years. weight ranged from . - . kg.dogs presented with seizures (n ), ambulatory paresis(n ), unilateral blindness(n ) and head tilt(n ). one dog had a normal neurologic exam. lesions chosen for biopsy were in the olfactory and/or frontal lobes (n ), parietal lobe(n ), and pyriform lobe(n ). lesions were between - mm in diameter. all lesions were well-circumscribed and contrast enhancing except for one. histologic diagnosis of meningioma(n ) and granulomatous meningoencephalitis(n ) were made. the poorly-circumscribed, non-contrast enhancing frontal mass yielded non-specific necrosis. following biopsy, three dogs returned to pre-biopsy neurologic status within hours. the french bulldog took hours to return to previous neurologic status due to brachycephalic syndrome that required oxygen support. one dog had acute respiratory arrest hours post-biopsy. necropsy is pending. these results suggest that this mri-guided biopsy system can provide an accurate histologic diagnosis of brain lesions. biopsies of poorly-circumscribed and non-contrast enhancing brain lesions may be less diagnostic. further evaluation is on-going to determine the true diagnostic yield and complication rate of this procedure. concurrent malformations of the craniocervical junction are commonly identified in humans with chiari type i malformation. recent evidence suggests such craniocervical junction abnormalities (cjas) also occur in dogs suspected of having chiari-like malformation (clm). the purpose of this study was to objectively describe morphometric features of the craniocervical junction region of dogs with suspected clm and to investigate for associations between these features and the occurrence of other malformations in this region. magnetic resonance (mr) and computed tomographic (ct) images from dogs with clm were evaluated. three regions of neural tissue compression were assessed: cerebellar compression (cc); ventral compression at the c /c articulation, termed ''medullary kinking'' (mk); and dorsal compression (dc) at the c /c articulation. a compression index (ci) was calculated for all abnormal regions for each dog. multiple logistic regression analysis was performed (p o . ) to ascertain whether ci values for the different regions of compression were associated with the incidence of other craniocervical junction abnormalities. % of dogs had mk and % of dogs had dc. % of dogs also had evidence of atlanto-occipital overlapping (aoo medical infrared imaging (mii) is a non-invasive diagnostic imaging technique that measures skin surface temperature and generates thermal pattern maps based on predetermined color scales. because skin temperature, dependent on regional perfusion, is under direct control of the sympathetic nervous system, mii provides information about the function of the autonomic nervous system. because of recent advances in technology and lack of sedation needed to image patients, mii has potential use as a screening test for a variety of conditions that may result in autonomic dysregulation like chiari-like malformation in dogs (clm). the purposes of this study were to establish a mii protocol for dogs suspected of having clm, to identify thermal imaging patterns for various regions of interest (roi), to evaluate changes in thermal patterns and compare the results to those of mri findings, considered the standard for diagnosing clm in dogs. one hundred and five cavalier king charles spaniel dogs with clinical signs attributable to clm and confirmed clm with mri were evaluated with a complete blood count and chemistry profile, examination by a board certified surgeon/neurologist, multidetector ct scan of the craniocervical junction, whole body mri and mii. the protocol for thermal imaging included cranial and caudal views of the body, full lateral right and left body views, dorsal views of the head and body, and right and left lateral views of the head. thermal patterns were assessed with custom image recognition software. after each dog was imaged awake, general anesthesia was administered and the dogs re-imaged using the same protocol. mri findings in dogs with severe or moderate cerebellar compression and cerebellar herniation were compared with mii results. the top of head and front of head roi were . % and . % successful in identifying dogs with clm. based on these preliminary findings, mii may be a viable screening tool to detect clm in dogs. medical infrared imaging (mii) is an imaging technique that measures skin surface temperature derived from cutaneous perfusion and generates thermal pattern maps based on color scales. mii has been used as a test for a variety of conditions that cause autonomic dysregulation resulting in altered cutaneous perfusion. acute thoracolumbar intervertebral disk disease (tlivdd) is common in dogs. the purpose of this study was to: ) determine the success of mii in identifying dogs with tlivdd, ) compare the mii localization with mri results and surgical findings ) determine if the mii pattern returns to that of normal dogs following decompression surgery. small breed chondodystrophic dogs with tlivdd confirmed with mri and dogs with no tlivdd were evaluated with mri and mii. regions correlating with the intervertebral disk spaces were analyzed for average temperatures and thermographic patterns. thermal patterns were assessed with computer recognition pattern analysis (crpa) software. dogs were re-evaluated weeks after surgery using the same protocol. when analyzing temperature averages over a region, no significant difference was found between control and affected dogs. crpa was % successful in differentiating normal from affected dogs. crpa was % successful in identifying the intervertebral disk space when compared with mri and surgical findings. based on these findings, mii may be a viable screening tool to detect tlivdd in dogs. microglia physiologically shows regional topographical differences in immunophenotype and function within the central nervous system indicating the endowment for a prompt response to pathological stimuli such as trauma. spinal cord injuries (sci) consist of a primary injury encompassing the mechanical impact and the ''secondary wave'' of injury occurring minutes to weeks later and comprising various consecutive effects such as increased production of free radicals, excessive release of excitatory neurotransmitters and inflammatory reactions. activated microglia has the potential to perform some of these reactions, their contribution to the secondary wave is therefore controversially discussed. it has to be considered a double-edged sword as both, beneficial and deleterious effects have been attributed to these cells. the purpose of the presented study was to assess microglial involvement, particularly in the ''secondary wave'' following sci. microglia from dogs with sci was isolated and characterized ex vivo in terms of morphology, immunophenotype, and function by flow cytometry. the results were compared to region-specific findings obtained from healthy control dogs (n ). the histopathological exam confirmed the diagnosis of sci in the cervical (n ) and thoracolumbar (n ) spinal cord, and revealed a significant activation of microglia/ macrophages and upregulation of myelinophagia in dogs with sci days or longer prior to euthanasia. microglial ex vivo examination showed significantly increased expressions of b - , b - , mhc ii, cd c, icam- , cd , cd , and cd , and significantly enhanced phagocytosis and generation of reactive oxygen species (ros) in sci compared to healthy controls. microglial cells seem to be highly activated following sci with an immunophenotype indicating their active role in co-stimulation of t cells, in leukocyte adhesion and aggregation, and in lipid and glycolipid presentation. microglial phagocytosis might play a pivotal role in removal of injured or damaged cells and initialize subsequent healing processes. however, as ros can be directly neurotoxic an enhanced microglial generation might lead to bystander damage of the traumatized spinal cord and might therefore add to the deleterious effects of the secondary wave. modulating the microglial response in sci might be a valuable novel therapeutic strategy alleviating further damage to the spinal cord. thymidine kinase (tk) is a soluble biomarker present in s-phase of a salvage pathway for dna synthesis, and can be measured in serum. tk activity correlates with stage, prognosis, and relapse in canine and human lymphoma. we previously reported the results of a pilot study evaluating tk activity in archived canine osteosarcoma, transitional cell carcinoma, and hemangiosarcoma (hsa) sera, and found elevated tk activity in % of canine hsa sera evaluated. the purpose of this study was to prospectively evaluate serum tk activity in a large number of dogs presenting to emergency clinics with hemoabdomen and a splenic mass, to determine if tk activity could be used as a noninvasive means to distinguish hsa versus benign conditions in this population. dogs presenting with hemoabdomen and a splenic mass identified on ultrasound examination were studied. serum was collected prior to anesthesia, euthanasia or surgical intervention and frozen until batch analysis. tissue from all patients was evaluated histologically by a single pathologist. sera from age-matched normal dogs comprised a control population. an elisa using azidothymidine as a tk substrate was used. comparisons between groups were made using -tailed student t-tests, and receiver-operator characteristic (roc) curves were generated. sixty-two patients and normal controls were studied. there were dogs with hsa, dogs with other splenic neoplasia, and dogs with benign diseases. using a training set of normal dogs, a cutoff of . u/l was established from the roc curve. tk activity was significantly higher (p o . ) in dogs with hsa than in the validation set of normal dogs (mean /Àsd . /À . and . /À . respectively), but not between dogs with hsa and benign splenic disease (mean /Àsd . /À . , p . ). using a cutoff of . u/l, tk activity demonstrated a sensitivity of . , specificity of . , positive predictive value of . and negative predictive value of . for distinguishing hsa versus benign splenic disease. when interval thresholds of o . and . u/l were used together, diagnostic utility was markedly increased for distinguishing both hsa versus normal and hsa versus benign disease. in conclusion, serum tk evaluation may assist in detection of canine hsa, and may also discriminate between benign disease and hsa in dogs with hemoabdomen and a splenic mass. t cell chronic lymphocytic leukemia (cll) is a heterogeneous disease that affects a number of dog breeds. cll patients have variable disease outcomes. the objectives of this study were to use gene expression profiling of cd t cell leukemias with variable outcomes in order to identify markers that can be used in routine diagnostic tests to distinguish good from poor prognosis disease, and to identify potential targets for novel therapy. gene expression profiling of cd t cell leukemias ( good, poor prognosis) was conducted. samples from normal dogs were also profiled. several differentially expressed genes were found including cd , cd , and cd . these were selected for further study using flow cytometry to determine expression of protein on the cell surface. seventy nine cases of cd t cell leukemia were screened for cd expression. forty seven had associated outcome information. based on analysis to date, cd expression as assessed by flow cytometry does not appear to provide prognostic information. a monoclonal antibody to cd was recently made available. to date patients with cd t cell leukemia have been profiled. cd is variably expressed on t cell leukemias compared to normal cd t cells. cd is the receptor for interleukin . cyclosporin, a commonly used immunosuppressive drug, inhibits il- production, and has been used to treat a subset of t cell leukemias in people. thus, the finding that cd is up regulated on t cell leukemias compared with normal t cells suggests a possible new therapeutic avenue. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of dogs. there is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (ibd). similarly, compositional changes of the intestinal bacterial ecosystem have been associated with ibd in humans. the aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders using a next generation sequencing technique. fecal samples were obtained from healthy dogs (n ), dogs with acute uncomplicated diarrhea (n ), dogs with acute hemorrhagic diarrhea (ahd; n ), and dogs with active (n ) and therapeutically controlled ibd (n ). the bacterial composition was analyzed by massive parallel s rrna gene -pyrosequencing. differences between groups were analyzed using mann-whitney u tests and kruskal-wallis tests followed by dunn's multiple comparison tests. statistical significance was set at p o . . significant differences in the proportions of several bacterial groups were identified between healthy and diseased dogs. dogs with gastrointestinal disease had significantly higher proportions of proteobacteria (p o . ). proportions of firmicutes were lower in diseased dogs, but this difference did not reach significance (p . ). within the firmicutes the most notable findings were decreases in bacterial groups belonging to clostridium clusters iv and xiva (i.e., ruminococcus, dorea, and faecalibacterium spp.; p o . for all). dogs with ahd had the most profound changes of the microbiota, followed by dogs with acute uncomplicated diarrhea, and dogs with active ibd. faecalibacterium spp. was the bacterial group most prominently depleted in dogs with active ibd, but was not significantly different between healthy dogs and dogs with therapeutically controlled ibd (p . ). results of this study revealed bacterial dysbiosis in fecal samples of dogs with various gi disorders. bacterial changes were more profound in dogs with severe disease, but were not identified in dogs with therapeutically controlled ibd, suggesting that the microbiota is stable in non-active disease. the bacterial groups identified are considered to be important short chain fatty acid producers and may serve as candidates for the diagnosis or therapeutic monitoring of gi disease. future studies are necessary to determine if these microbial changes correlate with functional changes in the intestinal microbiota. ciprofloxacin oral tablets, available in a generic formulation for people, are widely used for treatment in dogs. oral absorption data for ciprofloxacin in dogs has been variable, and too limited to guide accurate dosing. subsequently, published doses for dogs in veterinary formularies have varied from to mg/kg. this study was undertaken to explore the factors that may affect oral absorption of generic ciprofloxacin in dogs, and to derive a pharmacokinetic-based dose for treating susceptible bacteria. six healthy adult beagle dogs were used for the study ( . kg mean weight). after placing jugular vein catheters for collecting blood samples, these dogs were administered either a single oral dose of ciprofloxacin ( mg tablet; mean dose mg/kg), or an intravenous (iv) dose ( mg/kg; mg/ml solution). a randomized crossover design was used with a washout time between treatments. blood was collected for plasma drug analysis for hours. ciprofloxacin concentration in plasma was analyzed using high pressure liquid chromatography (hplc) and pharmacokinetics analyzed using a computer program. oral absorption was also evaluated via deconvolution analysis. the oral dose was well-tolerated, but the iv dose produced transient vomiting and depression in some dogs. after the oral dose, the peak plasma concentration (c max ) was . mg/ ml (cv . %), terminal half-life (t / ) . hr (cv . %), auc . mg Á hr/ml (cv . %), and systemic absorption (f) . % (cv . %). after the iv dose, the t / was . hr (cv . %), systemic clearance . l/kg/hr (cv . %), and volume of distribution . l/kg (cv . %). after examining the pharmacokinetic results from the oral dose, it was apparent that oral ciprofloxacin was absorbed well in some dogs (approximately %), but poorly in others (approximately %). to explore the factors that may have affected oral absorption, two high absorbers and two low absorbers were administered an additional oral dose as a mg/ml solution ( mg total dose) via gastric tube. after administration of the oral solution, the plasma concentrations were more uniform and consistent among dogs. absorption of the oral solution of ciprofloxacin was . % (cv %) with a t / of . (cv . %) hr and c max of . mg/ml (cv . %). therefore, it appears that inconsistent oral absorption of ciprofloxacin in some dogs may be formulation-dependent, and affected by tablet dissolution in the canine small intestine. doses were calculated using the data for oral tablets in these dogs. the pharmacokinetic-pharmacodynamic (pk-pd) target was an auc/ mic ratio of . because of the wide range in oral absorption of tablets, a dose to reach the pk-pd target ranged from canine distemper (cd) is a highly contagious, acute or subacute systemic viral disease of dogs and other carnivores which can be controlled efficiently by the use of modified live-virus (mlv) vaccines. however, mlv strains do cross-react with molecular diagnostic tests and cause significant confusion for clinicians. the purpose of this study was to use quantitative real-time pcr viral load information to differentiate between vaccine virus used in mlv vaccines and wildtype infections in dogs. a real-time pcr test for cd virus (cdv) based on the p gene for phosphoprotein was used to determine viral loads in vaccinated and wildtype infected animals. a total of respiratory mucosal swab samples from mlv vaccinated and asymptomatic dogs were obtained within the first weeks after mlv vaccination. based on the viral load in vaccinated animals, a cutoff value was established for the differentiation of dogs with clinical signs of respiratory distress and presumably infected with a wildtype strain of cdv. two hundred clinical cases with known clinical and vaccination histories were analyzed to validate the cutoff value. the cdv real-time pcr proved to be of high analytical and diagnostic sensitivity: a standard curve was established using known numbers of cdv molecules to allow absolute quantitative cdv viral load data. the limit of detection was in the single molecule range while the limit of quantitation was established at around molecules per pcr reaction. a comparison to ifa showed real-time pcr to be % more sensitive. the cdv viral load in vaccinated animals averaged , viral particles per swab. a cutoff value of , viral particles was calculated by adding standard deviations to the average value. this cutoff value correctly detected . % of the vaccinated samples. acutely infected dogs with cdv compatible clinical signs have high viral loads normally several logs higher than the cutoff value. in dogs with clinical distress, recent cdv mlv vaccination but viral loads below the cutoff value, other infectious agents were detected by using a panel of real-time pcr tests. testing additional infectious agents in clinical settings is important in order to explain clinical signs when viral loads below cutoff values indicate that cdv is not the cause of clinical signs. in conclusion, quantitative real-time pcr is a sensitive, rapid and reliable test regardless of recent vaccination. the use of a cutoff value will be of significant help to discriminate between vaccine interference and wildtype infection in clinical settings. feline ureteral obstructions are a common urinary dilemma and traditional therapy is associated with substantial morbidity/mortality. feline nephrostomy tubes are reported as being effective when pelvic drainage is required. the biggest limitation is externalized drainage, requiring careful management to prevent infection/dislodgement. the development of an indwelling ureteral bypass using a combination locking-loop nephrostomy/cystostomy tube was modified from humans, resulting in permanent indwelling drainage, reduced complications, and improved quality of life. the objective is to describe the technical and clinical outcome using a novel device called a subcutaneous ureteral bypass (sub) in cats with ureteral obstructions. fifteen cats ( kidneys) had a sub placed for: ureterolithiasis ( ), ureteral stricture ( /À stones) ( ), and ureteral stent rejection ( ). the median pre-and post-procedure creatinine was mg/dl (range: . - ) and . mg/dl (range: . - ), respectively. the median pelvis diameter pre and post-procedure were (range: - ) and mm (range . - ), respectively. six french tubes were placed in , and fr. in . the bypass remained indwelling for a median of days (range - ). there were major complications resulting in nephrostomy tube dislodgement ( ) and port leakage ( ) days after surgery. one patient with severe coagulopathy developed a clot which resolved with tpa infusion through the port. no sub got occluded/obstructed long-term. overall, the use of a sub for cats with ureteral obstructions can be considered a functional option when other therapies have failed or are contraindicated, but shtime. oxidative stress is considered central to the pathogenesis of many systemic diseases. in humans, biomarkers of oxidative stress, antioxidant depletion and lipid peroxidation, have been correlated with disease severity and associated with poor clinical outcomes. therapeutic antioxidant supplementation with nac in glutathione (gsh)-deficient patients has shown clinical benefits, including repletion of intracellular gsh levels. we have shown that clinically ill dogs are gsh-deficient, and that gsh deficiency correlates with mortality, but it is not clear whether there are direct benefits of antioxidant intervention in these patients. the purpose of this randomized, investigator-blinded, placebo-controlled, prospective study was to evaluate the effect of nac to normalize blood antioxidants (rbc reduced gsh (rbc gsh), plasma cysteine (cys), serum vitamin e (vit e), and whole blood selenium (se)), reduce lipid peroxidation (urine isoprostane/creatinine ratio (u i/ c)), and improve illness scores (spi ) and outcome (survival to discharge) in clinically ill dogs. clinically ill client-owned dogs, admitted to the uw veterinary medical teaching hospital that did not receive blood transfusions, tpn, vitamins, or antioxidants were eligible for the study. dogs enrolled in the study were randomized to receive iv infusions q. h. of either nac (  mg/kg and  mg/kg) or equal volumes of % dextrose (placebo) over hours. at the time of enrollment, and hours following the final hour infusion, blood and urine were collected to quantify rbc gsh, cys, vit e, and se concentrations; u i/c ratios; and calculate spi scores. rbc gsh and cys concentrations were quantified by hplc. commercially available hplc, atomic absorption spectroscopy, and eia were used to quantify vit e, se, and u i/c ratios, respectively. nonparametric statistical analyses were used, with results reported as medians and p o . considered significant. sixty-one ill dogs were randomized to either nac (n ) or placebo (n ). overall this group of ill dogs had significantly decreased rbc gsh ( . vs. . mm; p . ), vit e ( vs. mg/ml; p . ), and se ( . vs. . mg/ml; p . ) levels and elevated u i/c ratios ( vs. pg/mg; p . ) in comparison to healthy control dogs. dogs in the placebo group showed a significant further decrease in rbc gsh over the next hours ( . to . ; p . ). nac supplementation significantly increased plasma cys levels ( . to . mm; p o . ), and prevented a further decline in rbc gsh ( . to . mm; p . ). however, serum vit e ( vs. mg/ml), se ( . vs. . mg/ ml), u i/c ratios ( vs. pg/mg), spi scores ( . vs. . ), and outcome ( % vs. %) were not significantly different between the nac and placebo groups after treatment. the results of this study further support that clinically ill dogs experience oxidative stress, and suggest that antioxidant supplementation with nac within the first hours of hospitalization prevents further rbc gsh depletion. further studies are necessary to investigate whether longer duration or combined antioxidant supplementation normalizes the redox state and impacts long-term outcome. diabetes mellitus in cats is very similar to type ii diabetes in humans, preceded by a period of insulin resistance. evaluating insulin resistance in a cat is a time consuming, expensive, and difficult procedure. there is a need for a simple biomarker based test predictive of insulin resistance. there is a biomarker based assay predicative of insulin resistance in humans. the purpose of this study was to evaluate the utility of this assay in overweight cats and show improvement in insulin sensitivity following weight loss and weight maintenance. the insulin resistance assay is based on the quantitative analysis of metabolites ( -hydroxybuterate, creatine, palmitate, decanoylcarnitine, and oleoyl-lpc). a proprietary algorithm (metabolon, inc, durham nc) was used to generate a predictive rd (rate of disposal) value (normal range in cats . - . ). individuals with an rd value less than will have a greater than % chance of being insulin resistant and an rd value less than will have a greater than % chance of being insulin resistant. initial studies demonstrated that the rd values indicating insulin resistance in cats correlated with age, obesity and severity of diabetes as determined by histopathology and blood glucose levels. in a feeding study of cats ( % vs. o % body fat) rd values improved from . ae . to . . (p . ). during weight maintenance, % body fat for months, further improvement was observed (rd, . . (p . e- )). these results demonstrate that long term weight maintenance following weight loss is critical for increasing insulin sensitivity in cats. the use of monoclonal antibodies and antibody fragments to directly target tumor antigens and neutralize their growth factors has shown promising results in human clinical trials. however, these targeted approaches have not been possible in dogs since specific tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses. to overcome these obstacles, we have generated canine antibody phage display libraries from canine splenocytes. these libraries consist of single chain variable fragments (scfv) comprised of canine variable heavy (vh) and variable light (vl) immunoglobulin chains displayed on the surface of bacteriophage (fig. ) . the antigen specificity within these libraries is diverse and recapitulates the antigen-experienced immunoglobulin repertoire of the dog. we can now use simple panning techniques to isolate scfv of canine origin that bind to either known targets or unknown targets which can then be identified using standard molecular techniques. canine hsa is a highly aggressive malignancy of vascular endothelial cells that affects large breed dogs. although there are no confirmed immunological targets for hsa, serum levels of vascular endothelial growth factor (vegf) are elevated in these patients and, as in many human cancers, vegf may represent an important therapeutic target for neutralization. we used simple panning techniques to screen canine scfv libraries generated from the spleens of dogs with hsa against canine vegf and successfully isolated scfv clones that bind and neutralize canine vegf in vitro. these scfvs are now being taken into a murine model of canine hsa to determine whether they can inhibit tumor growth and metastases. in addition, we have panned the same antigen-experienced scfv phage display libraries against allogeneic primary canine hsa cells of low passage number to isolate canine-derived antibody fragments that can target malignant endothelial cell surface molecules. early results demonstrate enrichment of scfv phage libraries for malignant endothelial cell binders. these scfv can be readily linked to chemotherapeutic agents or other toxins and used to deliver high doses directly to the malignant cell. this novel approach aims to reduce side effects of systemic chemotherapy and augment therapeutic response. calcitriol, (vitamin d ), has antineoplastic activity and acts synergistically to potentiate the antitumor activity of a diverse array of chemotherapeutics. ccnu, vinblastine, corticosteroids, and tyrosine kinase inhibitors, are used to treat canine mast cell tumors (mct). vitamin d receptor is expressed in the majority of canine mcts, suggesting a role for calcitriol in the management of dogs with these tumors. the purpose of our study was to examine the in vitro effects of calcitriol in combination with ccnu, vinblastine, imatinib, or toceranib on canine mastocytoma c cells. also, we evaluated the antitumor activity of dn , a highly concentrated oral formulation of calcitriol, as single-agent treatment in dogs with naturally occurring mcts. c cells were incubated with serial dilutions of calcitriol ( . - nm). twenty-four hours later, cells were then treated with vehicle control or serial dilutions of ccnu ( . - um), vinblastine ( . - nm), imatinib ( . - . nm), or toceranib ( . - nm). cell viability was assessed with an mtt assay after hours and data was used to derive a combination index (ci: values o , , indicate synergism, additivity, antagonism, respectively). in the phase ii clinical trial, dogs were eligible if they had at least measurable, histologically confirmed, mct. calcitriol was administered orally. recist criteria were used to assess tumor response. calcitriol, ccnu, vinblastine, imatinib, and toceranib each suppressed c cell viability in a dose-dependent manner. ci values o were obtained for calcitriol ( . - . nm) combined with ccnu ( and um), vinblastine ( . and nm), imatinib ( . - . nm) and toceranib ( . - . nm). due to the occurrence of toxicity (vomiting, anorexia, hypercalcemia), the phase ii trial was terminated early; only of planned patients were treated. one dog with a metastatic muzzle mct had a complete response that lasted days. three dogs achieved partial response lasting from - days. in summary, our in vitro data demonstrate that calcitriol combined with ccnu, vinblastine, imatinib or toceranib has synergistic effects on c mastocytoma cells. antitumor responses were observed in dogs with spontaneously occurring mcts treated orally with single-agent calcitriol, but the frequency of adverse effects was high. together these results suggest calcitriol combination therapies might have significant clinical utility in the treatment of canine mcts but refinement of the calcitriol-dosing regimen must be done. cyclosporine is a potent immunosuppressive agent used to treat many canine inflammatory and immune-mediated diseases. cyclosporine has gained popularity as an immunosuppressive agent because of a favorable toxicity profile compared to many other immunosuppressive agents. optimal dosing regimens for cyclosporine in the dog remain unclear, primarily because standard methods that monitor effectiveness of immunosuppression have not been established. pharmacokinetic testing is currently used during treatment with oral cyclosporine to adjust doses based on measurement of blood drug levels. individual patients, however, often demonstrate marked variations in blood drug levels while on similar oral doses of cyclosporine, and can also demonstrate different clinical responses even at comparable drug levels, making correlation of blood cyclosporine levels and degree of disease control extremely difficult. pharmacodynamic testing offers an alternative method for regulating cyclosporine dosing by objectively measuring the effects of cyclosporine on t-cells, the drug's main cellular target in the body. our acvim foundation-funded research has focused on developing and evaluating a comprehensive panel of biomarkers of immunosuppression that can be utilized for pharmacodynamic monitoring during treatment with cyclosporine and other immunosuppressive agents that affect t-cell function. we have completed several studies using flow cytometry to evaluate activated t-cell expression of surface molecules (cd & cd ) and cytokines (il- , ifn-g & il- ) as potential biomarkers. our first study was an in vitro study evaluating expression of surface molecules and cytokines in canine t-cells exposed to varying concentrations of cyclosporine. this study established consistent drug-associated suppression of the cytokines il- , ifn-g and il- . our second study was an in vivo study in normal dogs evaluating the effects of two doses of oral cyclosporine, a high dose considered to be reliably immunosuppressive (starting dose mg/kg bid, titrated upwards as needed to attain trough drug blood levels of at least ng/ml) and a lower dose used to treat atopy ( mg/kg sid), on t-cell expression of these three cytokines. significant suppression of il- and ifn-g expression was seen at the high cyclosporine dose, while at the lower dose only ifn-g expression was suppressed. because tcell expression of il- was not significantly suppressed at the high cyclosporine dose, il- was not evaluated at the lower drug dose. because of specialized sample handling requirements, flow cytometry is not as practitioner friendly as other assays (such as pcr) for routine use in pharmacodynamic testing. we have therefore conducted an in vitro study comparing the effects of cyclosporine on activated t-cell expression of il- and ifn-g using flow cytometry and qrt-pcr, and demonstrated dose dependent and comparable suppression of il- and ifn-g using either methodology. we are currently evaluating, using qrt-pcr, the effects of oral cyclosporine on t-cell expression of il- and ifn-g in normal dogs prior to moving on to pharmacodynamic trials in our clinic patients. effect of hypothyroidism on reproduction in bitches. dl panciera , bj purswell , ka kolster , sr werre . departments of small animal clinical sciences, large animal clinical sciences, and laboratory for study design and data analysis, virginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va. numerous reproductive abnormalities, including irregular interestrous period, anestrus, and infertility have been attributed to hypothyroidism. we previously documented reduced fertility and lower birth weight and increased periparturient mortality in pups born to bitches with experimentally-induced hypothyroidism for a median duration of weeks. the purpose of this study was to evaluate reproductive function in these same bitches after hypothyroidism was treated with a replacement dose of levothyroxine. twelve multiparous bitches were studied. hypothyroidism was induced in dogs by administration of mci/kg i. hypothyroidism was confirmed by finding serum t concentrations before and hours after iv administration of human recombinant tsh that were o nmol/l. levothyroxine ( . mg/kd q h) was administered to all hypothyroid bitches. six bitches served as euthyroid, untreated controls. dogs were evaluated daily for signs of estrus and were bred by of males when serum progesterone was ! ng/ml. interestrous interval, gestation length, strength and duration of contractions during whelping, time between pups, number of live pups and stillbirths, viability of pups at birth, weight of pups, and periparturient mortality were recorded. the student's t-test and anova were used to compare differences between control and hypothyroid bitches for continuous, normally distributed data. the wilcoxon rank sum test was used to analyze data between groups that was not normally distributed. the mean duration of hypothyroidism prior to levothyroxine administration was ae . weeks. breeding took place after levothyroxine treatment for ae . weeks in the hypothyroid group. all dogs in the hypothyroid group and / control dogs were pregnant, while / hypothyroid and all control bitches became pregnant prior to levothyroxine administration. no difference in interestrus interval or gestation length was noted between groups. during whelping, no difference in strength of contractions, contraction duration, interval between pups, or viability scores of pups was found between groups. litter size, birth weight and peirparturient mortality were similar between groups. levothyroxine administration reverses the detrimental effects of hypothyroidism on fertility and neonatal health. racing sled dogs have a high prevalence of exercise-induced gastric erosions/ulcers, with reports ranging from - % of dogs running at least miles in a day or less. omeprazole reduces the severity of, but does not completely prevent, gastritis under racing conditions, and can be difficult to administer under these conditions. famotidine can be administered in food, but has only demonstrated efficacy under less intense training conditions. the purpose of these studies was to evaluate different acid suppression strategies under racing conditions for the prevention of exercise-induced gastritis. experiment # was a randomized placebo-controlled study using sled dogs ( - years) competing in a mile race over - h. treatment groups were famotidine (approx mg/kg qd) or no treatment, beginning days prior to the start of the race and proceeding until gastroscopy was performed h after the race. experiment # was a randomized positive-control study using sled dogs ( - years) running a mock race of miles in h. dogs were divided into omeprazole (approx mg/kg qd, administered min prior to a meal) or famotidine (approx mg/kg bid) groups beginning days prior to the exercise challenge and continuing for h after completion. gastroscopy was performed immediately prior to the start of dosing and h after completion of the exercise. in all cases, mucosal appearance during gastroscopy was blindly scored using previously described scoring system. famotidine ( mg/kg qd) reduced the prevalence of clinicallyrelevant, exercise-induced gastric lesions compared to no treatment ( / vs / , p . ). compared to famotidine at mg/kg bid, omeprazole significantly decreased the severity ( . vs . , p . ) and prevalence ( / vs / , p . ) of gastric lesions. although famotidine provides some benefit in the prevention of exercise-induced gastric lesions, neither the recommended dose nor the higher dose were considered acceptable in the prevention of exerciseinduced gastritis as between - % of the dogs receiving famotidine had clinically significant lesions. a previous study examining omeprazole under racing conditions, but without careful administration on an empty stomach, resulted in a % prevalence of clinically significant gastric lesions. however, the bioavailability of omeprazole is reduced in the presence of food, and when the daily administration of the drug is carefully scheduled to coincide with an empty stomach, the resulting prevalence of clinically significant lesions induced by racing-intensity exercise is reduced to just over %. the conclusions of these studies are that omeprazole is superior to famotidine in preventing gastritis in racing sled dogs during competition. routine administration of omeprazole is recommended to prevent stress-associated gastric disease in exercising and racing alaskan sled dogs. mares may be an important source of environmental contamination with rhodococcus equi on breeding farms. attempts to reduce fecal shedding of r equi by the mare and the effects of the mare's fecal r equi concentration on airborne concentrations in the foaling stall have not been previously reported. twenty-one arabian mares were treated daily with either oral gallium nitrate or placebo in a randomized double-blind study. fecal samples were collected at day of gestation (time ), the week before foaling (time ), and the week after foaling (time ). airborne concentration of r equi were measured in the stall within hours post foaling using a microbial air sampling system into which standard ( -mm) culture plates with a media selective for r. equi have been loaded. concentration of total r equi were determined by morphological characteristics. the concentration of virulent r equi was determined using a modified colony immunoblot method. concentrations of total and virulent r equi were compared among mares to examine effects of treatment, time, and treatment by time interaction. there were significant (p o . ) effects of treatment that depended on time of sample collection. at sample times and there were no significant differences between groups in the fecal concentration of virulent r equi. at time concentrations of virulent r equi were significantly lower among mares in the treatment group (p o . ) compared to control. effects of time depended significantly on groups: for the control group, there were no significant effects of time. for the treatment group, concentrations tended to decrease over time, and concentrations at time were significantly (p o . ) lower than those at time . no other differences among times for concentrations in the treatment group were statistically significant. there were no significant effects of treatment, sample time, or their interaction on the concentration of total r equi between groups; however, the pattern for these data was similar to that observed for the virulent isolates. no significant differences were determined between treatment groups for airborne concentrations of virulent or total r equi. treatment of mares with oral gallium nitrate significantly reduced the fecal concentrations of virulent r equi over time, but had no impact on the airborne concentration of r equi shortly after foaling. the purpose of this study was to evaluate the protein profile of bronchoalveolar lavage fluid (balf) in horses affected with recurrent airway obstruction (rao) and in control horses using proteomics and western blot techniques. rao-affected (n ) and control horses (n ) were subjected to an experimental exposure trial; when the rao-affected horses showed clinical signs of disease, balf was collected from all horses. balf was also collected from client-owned rao-affected horses (n ) with naturally-occurring clinical signs of disease and client-owned control horses (n ) from the same environments. the balf from the experimental exposure trial horses was subjected to trypsin digestion and proteomics analysis with mass spectrometry (ms). peaks detected with ms were identified using tandem ms analysis and database searches. western blot was used to confirm the identity and expression levels of two proteins identified using proteomics techniques in the balf of all horses. data from ms experiments were analyzed with the student's t-test to compare peak intensity between rao-affected and control horses. western blot band density data was analyzed with the kruskal-wallis anova for comparison between groups of horses. significance level was set at p o . . with ms proteomic analysis of the balf from the experimental exposure trial horses, total peaks (peptides) were identified. of these peaks, were differentially expressed between the rao-affected ( over-expressed) and control horses ( over-expressed). identifications were made for balf proteins. transferrin and secretoglobin were chosen for validation with western blot. proteomics indicated that secretoglobin was not differentially expressed between the experimental exposure trial group; this was confirmed with western blot analysis. western blot also showed that clientowned rao-affected horses had lower secretoglobin expression than client-owned control horses and control horses before experimental exposure. according to the proteomics data, transferrin was over-expressed in control horses after experimental exposure compared to rao-affected horses. while the western blot analysis did not show a statistically significant difference in this comparison, transferrin was significantly over-expressed in control horses before experimental exposure compared to client-owned rao-affected horses. in addition, both secretoglobin and transferrin band densities on western blot were negatively correlated with airway obstruction and neutrophilic pulmonary inflammation. this study demonstrates that proteomics techniques can be used in the investigation of equine balf proteins. the proteins identified as differentially expressed between rao-affected and control horses in this study including, but not limited to, secretoglobin and transferrin should undergo further evaluation for their use as biomarkers of rao, and as potential targets of new therapeutic agents for rao. cardiotoxic effects of rattlesnake venom in the horse are not well defined. the first aim of this study was to document cardiac damage in naturally envenomated horses. twenty horses with clinical diagnosis of snake bite were included. a snake venom elisa was utilized to confirm envenomation when possible. serum and plasma were collected at selected intervals. plasma was assayed for cardiac troponin i (ctni) using a flurometric assay (stratus cs s , dade behring). holter monitors (zymed s , philips) were placed at admission, week and month post presentation. echocardiography was performed on available horses - months after envenomation. the second aim of this study was to investigate potential mechanisms of the cardiac damage. serum samples were assayed for tnfalpha using a commercial assay (endogen). antibody titers to crotalus atrox venom were measured at admission, week and month after natural envenomation and compared to titers in vaccinated horses (crotalus atrox toxoid, red rock biologics). a significant number of horses showed elevations in ctni (p o . ) at one or more time point indicating myocardial damage. holter readings revealed the presence of arrhythmias or persistent tachycardia in horses. five of twenty horses were available for echocardiography; no abnormalities were noted. horses with increased ctni tended to have greater tnfalpha concentrations compared with horses without increased ctni. peak venom titers in bitten horses were significantly higher than peak titers in vaccinated horses (p o . ). rattlesnake envenomation was associated with evidence of cardiac damage in a significant proportion of bitten horses. further studies are needed to determine the cause as well as mechanisms to treat and/or prevent its occurrence. little is known about the gastric mucosal flora in healthy horses and its role in gastric disease has not been critically examined. our laboratory previously reported that a diverse microbial flora with a predominance of streptococcus spp. and lactobacillus spp. exists in healthy horses using fluorescence in situ hybridization (fish). the present study sought to further characterize the gastric mucosal flora of healthy horses using massive parallel srrna bacterial tag encoded flx-titanium amplicon pyrosequencing (btefap). biopsies of the squamous, glandular, antral and any ulcerated mucosa were obtained from healthy horses via gastroscopy after a -hour fast and horses immediately post-mortem. dna was extracted from the mucosal biopsies and btefap and data processing was performed. hierarchical cluster analysis based on relative abundance data on the genus level were performed to look for trends in bacterial diversity among the individual horses. pyrosequencing yielded between , and , reads per horse with , , reads in the antrum, squamous and glandular regions, respectively. the microbiome segregated into two distinct clusters: cluster comprised of horses that were stabled, fed hay and sampled at post-mortem and cluster consisted of horses that were pastured on grass, fed hay and biopsied gastroscopically after a -hour fast. samples from different antomic regions clustered by horse rather than region. despite being very similar at the higher taxonomic level (phyla) differences in the distribution of bacteria were seen at the genus and species level. the dominant bacteria in cluster horses were firmicutes ( % reads/sample) consisting of mainly streptococcus spp., lactobacillus jensenii, l. fornicalis and sarcina maxima. cluster had more diversity with a predominance of proteobacteria, bacteroidetes and firmicutes and genera identified such as streptococcus spp., moraxella spp., actinobacillus spp., and others. though the relative abundance of the individual taxonomic groups was significantly different between individual horses, no significant differences in the overall diversity could be found (as assesed by shanon weaver, ace and choa i diversity indices). helicobacter spp. sequences were not identified in any sample (out of , reads). the ulcerated mucosa from horse (group ) had lower diversity and higher numbers of bacteria predominated by lactobacillus equigenerosi. this data shows that the equine gastric mucosa harbors an abundant and diverse microbiome which is unique to each individual and differs by sampling method, fasting prior to sampling and diet. seasonal pasture myopathy (spm; atypical myopathy [am] in europe), typified by nonexertional rhabdomyolysis, occurs in pastured horses during autumn or spring. clinical signs rapidly progress from muscular weakness to recumbency and frequently death. extensive myonecrosis and intramyofiber lipid storage occur in highly oxidative respiratory and postural muscles. recently, a defect of lipid metabolism called madd has been identified in european horses with am. this report documents the first cases of equine madd in the united states. six midwestern us horses suspected of having spm in the spring or fall of were evaluated for madd by urine organic acids, plasma acylcarnitines and/or muscle carnitine and histopathology. five horses had clinical signs and clinicopathologic data consistent with severe rhabdomyolysis. one horse was found dead on pasture after days of rear limb stiffness and inappetance. urinary organic acid profiles revealed markedly elevated ethylmalonic and methylsuccinic acids, butyrylglycine, isovalerylglycine, and hexanoylglycine, consistent with equine madd. plasma acylcarnitine profiles from horses had marked elevations of short chain acylcarnitines, while the third horse and only survivor had minor elevations of short chain acylcarnitines. affected muscle showed extensive degeneration with intramyofiber lipid accumulation, a marked decrease in free carnitine, and high levels of carnitine esters. spm appears to be a highly fatal emerging disease of pastured horses in the us characterized by weakness, colic-like signs and myoglobinuria. the disease is associated with a defect in muscular lipid metabolism that can be diagnosed by performing lipid staining of muscle samples and urine organic acid profiles. candidatus mycoplasma haemolamae (cmhl) is a common red blood cell parasite of new world camelids. the high degree of parasitemia that develops in an infected splenectomized animal allows for the efficient collection of parasitic dna. this dna can then be used in the development of genetically-derived tools such as pcr and in-situ hybridization. thus, one splenectomized animal can replace many immunologically intact animals within a research setting. the purpose of this study was to track the natural progression of cmhl parasitemia and associated clinical signs in a splenectomized alpaca. an intact, -month-old, . kg male alpaca was used in this study. he had tested positive via pcr for cmhl on three different occasions, although no organisms were seen on peripheral blood smears. the alpaca was placed under general anesthesia and a ventral midline incision was made. the spleen was located, the vessels ligated, and the organ removed. buprenorphine and flunixin meglumine were given for and days after surgery respectively. body weight, attitude, rectal temperature, blood glucose, and pcv were recorded daily. in addition, a peripheral blood smear was examined daily and the percent of red blood cells that were infected with mycoplasma organisms was determined. the alpaca was not parasitemic prior to surgery. one percent of the rbc's contained mycoplasma on days and after splenectomy. parasitic bloom developed on day with % of the red blood cells infected, and over % containing or more organisms. the alpaca was treated with mg/kg oxytetracycline i.v. on day . on postoperative day no parasites were seen in the peripheral blood. the peripheral blood remained free of parasites for days. on the morning of the th day, % of the peripheral red blood cells contained mycoplasma. by late that afternoon, % of the observed rbc's contained - organisms. the alpaca again received oxytetracycline. there were no more parasites observed from that time until the alpaca was euthanized days later. the alpaca lost . kg between days À and after surgery. his weight fluctuated between . and . kg for the remainder of the study period. blood glucose ranged between and mg/dl there was no major change in pcv (range - %), a finding that was expected as the spleen was not available to remove infected red blood cells. body temperature ranged between . and degrees celsius except for days and when more than % of red blood cells contained parasites. on those days rectal temperature reached . and . degrees respectively. this study confirmed that a non-parasitemic, yet pcr positive alpaca did indeed harbor cmhl. the time from splenectomy to parasitic bloom was shorter, and the length of oxytetracycline suppression longer than has been observed in other species. gastro-intestinal (gi) disease frequently results in increased wall thickness in many species. identification of changes in gi wall thickness using ultrasound has proved to be a useful diagnostic tool and is widely used in human patients, small animals and horses. although gi motility has been evaluated in cattle, normal reference ranges for wall thickness has not been reported in ruminants. the aims of this study were to report normal values for wall thickness of various gi structures and to assess the repeatability of this technique in adult dairy cows, sheep and goats. eight healthy adult holstein friesian (hf) cattle ( ae kg), eight jersey (j) cattle ( ae kg), thirteen adult sheep ( ae kg) and eleven adult goats ( . ae kg) were recruited and examined on three consecutive days. ultrasonographic images were optimised for the structure of interest. structures were identified based upon appearance and anatomical position. a minimum of three cineloops were obtained of the abdominal organs per intercostal space (ics) and three along the ventral midline in each ics; images were analysed offline. data were analysed using anova and post-hoc bonferoni, student's ttest and intra-class correlation coefficients. each structure was measured per ics per species; if no differences were noted for structures in different ics, then measurements were pooled. no differences were noted between hf and j cattle so data were pooled. data are displayed in table . good repeatability (icc . ) was obtained for all measurements and no differences were noted between animals of the same species or between days. these measurements for assessment of normal gi thickness are repeatable and may allow valuable additional information to be gained from ruminants with gi disease. ocular infections with the infectious bovine keratoconjunctivitis (ibk) agent moraxella bovis (m. bovis) are associated with significant economic loss in the cattle industry.although antibiotic therapy is the treatment of choice for ibk, treatment failures are common and current vaccines are not optimally effective mainly due to antigenic variation. as a result, our laboratory has been actively investigating the therapeutic potential of bdellovibrio bacteriovorus j (b. bacteriovorus); a predatory bacterium capable of attacking and inducing lysis of gram-negative bacteria, as a new treatment for ibk. we have previously shown that b. bacteriovorus can reduce the number of m. bovis attached to bovine epithelial cells in an in vitro model of ibk and that b. bacteriovorus can be trained to kill m. bovis as effectively as e. coli using serial passages. in this study, we hypothesized that b. bacteriovorus can remain viable in bovine tears without its prey for up to hours. this hypothesis was addressed by incubating inocula of active b. bacteriovorus in its preferred media peptone yeast extract (pye) and comparing b. bacteriovorus viability in bovine tears or phosphate buffered saline (pbs) at time , , , , and hours. using a plaque assay to quantify the mean amount of plaque forming units (pfus) of b. bacteriovorus exposed to each treatment, it was determined that viability of b. bacteriovorus over time was comparable between treatment groups. overall, the results supported that b. bacteriovorus can remain viable in tears for up to hours in the absence of prey bacteria. further studies are needed to determine the therapeutic potential of b. bacteriovorus in an in vivo model of ibk. correction of the measured ionized calcium concentration (cca ) to a ph . is routinely applied in experimental studies in order to assist in the interpretation of measured values relative to a reference range. the equation most commonly used for ph correction in bovine plasma is: cca ph . cca  (- . Â{ . -ph}) . the validity of this equation for bovine plasma is unknown. accordingly, our first objective was to characterize the in vitro relationship between cca and ph for bovine plasma. feeding rations with a low dietary cation-anion difference (dcad) during late gestation mitigates periparturient hypocalcemia in dairy cows, particularly when chloride containing acidodgenic salts are fed. the mechanism for this beneficial effect remains unclear. our second objective was to determine whether hyperchloremia displaces calcium from binding sites to albumin, thereby increasing cca . the in vitro relationship between plasma log(cca ) and ph in was investigated using lithium heparin anticoagulated blood from healthy holstein-friesian calves. plasma was harvested and tonometered with co at c over a ph range of . - . . plasma chloride concentration (ccl -) was altered by equivolume dilution of plasma with electrolyte solutions of varying ccl -( ae , ae , and ae meq/l; mean ae sd). the slope of the linear regression equation relating log(cca ) to ph for tonometered plasma samples from the calves was - . ae . at normal values for cca ( . ae . meq/l), albumin concentration ( . ae . g/l), and ccl -( . ae . meq/l). the experimentally-determined value for the slope for bovine plasma was identical to that determined previously for human plasma. the formula for correcting cca in bovine plasma for change in ph from . is therefore: cca ph . cca  (À .  { . -ph}) . this equation is only valid at normal concentrations of albumin and chloride in plasma. equivolume dilution of plasma by electrolyte solutions of varying cclindicated that cca ph . increased by . meq for every meq/l increase in ccl -. in other words, plasma cca at a given ph increases directly in response to an increase in plasma ccl -, presumably because the additional chloride displaces calcium that is electrostatically bound to albumin. furthermore, the increase in cca is independent to the change in plasma ph induced by an increase in ccland decrease in plasma strong ion difference. our finding that hyperchloremia directly increases plasma cca provides an additional mechanism by which ingestion of high chloride (acidogenic) rations prevents the clinical signs of periparturient paresis. our finding is consistent with the results of other studies that indicate acidogenic salts that contain chloride as the predominant anion (ie, nh cl, cacl ) are more effective in increasing cca than equimolar quantities of acidogenic salts such as mgso . coagulase negative staphylococci (cns) are among the most common bacteria isolated from the bovine mammary gland. historically, these bacteria were lumped together as minor mastitis pathogens. modern molecular techniques have allowed accurate speciation and fingerprinting of the cns species. these methodologies have recently been applied to the study of cns in bovine mastitis. the aim of the studies presented here was to evaluate the role of individual cns species on milk somatic cell count (scc) and duration of intramammary infection (imi). in the first study, mammary quarter foremilk samples were aseptically collected from all lactating cattle ($ head) at the university of missouri dairy research center once monthly for months for bacterial culture and milk scc. staphylococcal isolates were speciated by sequencing the rpob gene and strain-typed using pulsed-field gel electrophoresis (pfge). using species and fingerprint data along with published definitions for staphylococcal imi, cns imis were identified. overall, species of cns were identified with staphylococcus chromogenes, s. cohnii, s. epidermidis, and s. simulans being most prevalent. duration of imi and scc data were analyzed using regression models accounting for repeated measures. mean milk scc and duration of imi were found to differ between cns species (p o . ). although most imis were of short duration ( month), staphylococcus capitis and s. chromogenes imis had longer mean durations of infection than or more of the other species isolated. mean sccs were under , cells/ml in most cases. however, staphylococcus simulans and s. xylosus imis were more inflammatory (mean , cells/ml) and had a higher mean scc than s. cohnii, s. epidermidis, and s. haemolyticus. to examine the relationship between cns imi and milk scc in a larger population of cattle, cns isolates from the canadian bovine mastitis research network (cbmrn) culture collection were obtained for speciation. speciation and fingerprinting were performed as above. isolates were from subclinical imi from before and after the dry period and from subclinical imi during lactation. data associated with each isolate were obtained from the cbmrn database. nine-hundred-thirty-eight isolates from mammary quarters in herds were successfully speciated. twenty-two different species of cns were identified. staphylococcus chromogenes was the most frequent species identified accounting for % of the infections. three species, s. chromogenes, s. xylosus, and s. simulans accounted for % of all infections. data were analyzed using a linear hierarchical repeated measures mixed model. differences in mean scc were found between some cns species and culture negative control quarters and also between different species of cns (p o . ). overall, our data demonstrate potential differences in pathogenicity between strains of cns that cause bovine mastitis. passive transfer of maternally derived antibodies via ingestion of good-quality colostrum within the first hours of life is crucial for the health and future productivity of dairy calves. however, infectious diseases can be transmitted via colostrum feeding, which may require use of a colostrum replacement product or pasteurization to decrease disease transmission. while pasteurization of colostrum is effective for sterilization, heating during pasteurization can alter the viscosity of colostrum, destroys important nutritional biomolecules, and has been shown to decrease colostral igg concentrations. the purpose of this study was to investigate the effect of high pressure processing (hpp) on the viscosity, igg concentration, and bacterial contamination of bovine colostrum. first milking colostrum samples were collected from cows from different farms, and ml aliquots of each sample were pooled for analysis. pooled colostrum was processed in triplicate using an isostatic press at mpa ( , psig) for , , , , and minutes. samples were tested for the effects of hpp on the viscosity, bacterial load (cfu/ml), and igg concentration. there was a significant decrease (p o . ) in bacterial load at each time point when compared to time . no significant difference in igg concentration was found between any time points. subjectively, the colostrum viscosity appeared to increase with the processing time, though the rheologic assessment has not been completed at this time. hpp appears to be an effective method to decrease bacterial contamination of colostrum while maintaining appropriate igg concentrations. minimizing the processing time or pressure may be necessary to maintain an acceptable viscosity of the colostrum. based on these results, additional studies are justified in order to determine the optimum combination of processing time and pressure and the effect of hpp on specific bovine pathogens. the heme-associated iron-binding apoprotein lactoferrin (lf) is known for its, anti-inflammatory, anti-parasitic, antimicrobial and bactericidal effects. lactoferrin demonstrates ubiquity throughout mammalian host biological fluids: saliva; tears; mammary secretions, as well as at mucosal surfaces. it is also released from immune cells under pathogenic stimulation. the purpose of this study which has been approved by western university's institutional animal care and use committee is to further characterize the mechanisms through which lf modulates inflammation in the face of bacterial endotoxin. it was hypothesized that lf would inhibit p phosphorylation. numerous studies speak to the ability of lf to alter leukocyte function, inhibit cytokine production, and bind lipopolysaccharide (lps); mechanisms through which it is believed to achieve its anti-inflammatory effects. recently, investigators demonstrated its ability to interact with host dna while others describe regulation of granulocyte adhesion and motility; elucidating its roles in the apoptotic signaling. in earlier studies, dawes me, et al. demonstrated lactoferrin's ability to limit the expression of inducible cyclooxygenase- and the gelatinase, matrix metalloproteinase - by lps-induced macrophages. the generation of these inflammatory mediators is modulated by pro-inflammatory cytokines such as interleukin- b (il- b) and tumor necrosis factor-alpha (tnf-a), the production of both being dependent on signaling through the p mitogenactivated protein kinase (mapk) pathway. peripheral mononuclear cells (  )isolated from buffy coat cells of healthy neonatal to -month old holstein calves were cultured in the presence and absence of lf ( ng/ml), lps ( mg/ml), anisomycin ( mg/ml), a known p activator -the positive control, and mm of sb , a known p inhibitor -the negative control. sample lysates obtained post culture was subjected to immunoprecipitation and kinase reactions. reactions were terminated under reducing conditions and evaluated using western immunoblotting. phosphorylation of activated transcription factor- (atf- ) by phosphorylated p served as the marker of investigation. immunologically reactive atf- expression by lps and anisomycin-treated cells was compatible with a prominent band at kd. evidence of lf-induced inhibition of lps-induced p- activation was observed in lanes representative of co-cultures of lf lps; lf anisomycin; and anisomycin sb , which was demonstrated by decreased immunological reactivity at kd. the findings here, suggest that lf interferes with lps-induced p- activation of transcription factor atf- , in vitro. this serves as additional proof of its potential use in attenuating the systemic effects of lps. six ( ) clinically normal, purpose-bred cats of similar age and body condition were imaged with [ f] fluorodeoxyglucose ([ f]fdg) and [ f]ftha by using dynamic cardiac-gated fused pet/ct for kinetic assessment of myocardial glucose and fatty acid uptake and metabolism, respectively. kinetic tracer uptake within the myocardium was achieved by initiating image data acquisition simultaneously with tracer injection. pet data were acquired over a hour period with the heart in the center of the scanner field of view. regions of interest were drawn in the left ventricular wall and thoracic aorta for the purpose of measuring the kinetics of tracer redistribution. serial blood samples were also taken during pet imaging for comparison with image data. the equilibrium biodistribution of both tracers was documented hour post-injection in a whole body pet/ct image. standard echocardiographic examination of cardiac structures was also performed. both radiotracers remained in the plasma fraction; however, [ f]ftha was cleared from the more rapidly than [ f]fdg (t / $ and $ min, respectively). the tracers were readily visualized within the feline myocardium in dynamic pet images and analysis of the blood pool clearance from the kinetic image data agreed with blood sampling data. myocardial uptake of each tracer was best described by a double exponential analysis and was rapid but variable among animals (range - bq/cc/min), although blood glucose levels were similar in all cats during image acquisition. physiologic [ f]fdg was observed in the brain, salivary tissue, gastrointestinal tract, renal pelves and urinary bladder, with [ f]ftha seen in the myocardium, liver and renal cortex. all cats were normotensive with normal echocardiographic parameters. this study demonstrates the utility of kinetic imaging using the left ventricle (lv) shape has been suggested to change from elliptical to more globular in response to chronic volume overload. real-time three-dimensional echocardiography (rt de) offers new modalities for lv assessment. the aim of the study was to investigate left ventricular changes in shape and volume occurring in response to different severities of naturally acquired myxomatous mitral valve disease (mmvd) in dogs using rt de. privately owned dogs were classified by standard echocardiography into: healthy ( ), mild ( ), moderate ( ) and severe mmvd ( ). a lv cast was obtained using semi-automated endocardial border tracking from rt de dataset, from which global and regional (automatically acquired basal, mid, and apical segments based on lv long-axis dimension) end-diastolic (edv) and endsystolic volumes (esv), lv long-axis dimension and rt de sphericity index, were derived. global and regional edv and esv increased significantly with increasing mmvd severity, assessed by mmvd group-wise comparisons and linear regression analyses using left atrial to aortic root ratio, and lv end-diastolic and end-systolic dimensions. all three segments contributed to the overall increased global volumes, but the mid edv segment was strongest associated with increasing lv end diastolic dimension (p . ). furthermore, lv long axis distance and lv sphericity index increased with increasing mmvd severity. the basal and apical edv segments were strongest associated with sphericity index (p o . ). in conclusion, this rt de study showed that increased lv edv, primarily in the mid segment, leads to rounding of lv apical and basal segments in response to increasing mmvd severity in dogs. dogs from shelters in florida with naturally acquired di infection were euthanized and necropsied. all adult di in each dog were sexed using morphological features. total worm burdens and numbers of males and females were recorded. no other information was available for any dog. all data, raw and transformed, were examined visually and descriptively. raw numerical data were further examined by a paired t-test; log-odds transformed data were examined by logistic regression. we also conducted a binomial distribution goodness of fit analysis assuming a null hypothesis of a m:f . . worm intensities ranged from to di per dog. eight dogs had unisex infections: / had all-female infections. dogs with lowintensity dual-sex infections were more likely to have greater numbers of female di. overall, sex ratios were equal (paired t-test, p . ). however, logistic regression demonstrated that the probability of being female is strongly affected by the total worm intensity, with lower intensities increasing the probability of having a predominance of female worms. our data show that di sex ratios in naturally-infected dogs equal when examining the entire dog population, but deviate to favor female worms at low worm intensities. these data could impact adulticide treatment strategies. the reasons for sex ratio distortion in di are unknown. we evaluated cardiac reverse remodeling after mitral valve repair under cardiopulmonary bypass (cpb) for mitral regurgitation in small breed dogs. fifty dogs (body weight . - . kg, age - years) with mitral regurgitation were treated between august and november . the cardiac murmur was grade / - / . the preoperative chest x-rays showed cardiac enlargement (vertebral heart scale (vhs) . - . ). echocardiography showed severe mitral regurgitation and left atrium enlargement (la/ao . - . ). after inducing anesthesia, a thoracotomy was performed in the fifth intercostal space. cpb was started by using a cpb circuit connected to carotid artery and jugular vein catheters. after inducing cardiac arrest, the left atrium was sectioned and chordae tendineae rupture confirmed. the chordae tendineae were replaced with expanded polytetrafluoroethylene. a mitral annulus plasty was also done, and the left atrium was closed. after de-clamping for restarting the heart, the chest was closed. heart rate decreased from - bpm to - bpm. the grade of cardiac murmur was reduced to / - / three months postoperatively, and the heart shadow was reduced (vhs . - . ) in the chest x-rays. echocardiography confirmed the marked reduction in mitral regurgitation and the left atrial dimensions (la/ao . - . ). mitral valve repair reduced enlarged cardiac size by reduction of regurgitant rate. pulmonary arterial hypertension (pah) is a well recognized condition in dogs leading to considerable morbidity and mortality. the majority of therapeutics has focused on endothelial dysfunction causing reduced production of vasodilators, such as nitric oxide and prostacyclin, coupled with overproduction of vasoconstrictors, such as endothelin- . more recently, it has been shown that the mitochondria play an important role in the development of pah as oxygen sensors and regulators of cellular proliferation. in pah, pulmonary artery smooth muscle cells undergo a metabolic shift from oxidative phosphorylation in the mitochondria to glycolysis in the cytoplasm as the major energy source and this leads to suppression of apoptosis and increased proliferation. dichloroacetate (dca) inhibits pyruvate dehydrogenase kinase to activate pyruvate dehydrogenase which catalyzes the rate limiting step for entry of pyruvate into the krebs cycle, thus increasing mitochondrial respiration. in three different rat models of pah, dca has been shown prevent and reverse pah by normalizing molecular pathology, stimulating apoptosis of pulmonary artery smooth muscle cells, and reducing pulmonary artery hypertrophy. dca has known toxic effects, including reversible hepatotoxicity and peripheral neuropathy, and has not been studied in any species with naturally occurring pah. the objective of this open label pilot study is to evaluate the therapeutic and toxic effects of dca in naturally occurring canine pah. three dogs with pah diagnosed by doppler echocardiography and no correctable underlying cause are enrolled in the study. dogs are orally administered mg/kg of dca divided daily for weeks, and then . mg/kg of dca divided daily for the remainder of the study. at baseline, , , , and weeks, an echocardiogram, cbc, serum chemistry profile, urinalysis, nt-probnp, blood uric acid, blood lactate, noninvasive blood pressure, nerve conduction study, and trough dca level ( hr post-dose) are obtained. the measured echocardiographic parameters include peak and mean tricuspid regurgitant flow velocity and pressure gradient, peak and enddiastolic pulmonary regurgitant flow velocity and pressure gradient, pulmonary valve flow velocity acceleration time and ejection time, pulmonary valve flow velocity time integral, right ventricular myocardial performance index, tricuspid annular plane systolic excursion, and systolic tricuspid annular tissue velocity. variables are inspected for normalcy and equality of variances and a two-sided paired t-test is used to compare the variables before and after treatment at each evaluation time. the basis for the role of the mitochondria in pah and the results of this pilot study will be presented to determine if dca warrants further study as a therapy for dogs with pah. study produced the strongest associations between the ncl phenotype and cfa markers. all ncl-affected tibetan terriers were homozygous for the same haplotype which extended for consecutive snps spanning . mb. none of the annotated genes within this target region had previously been associated with human or rodent ncl. we used dna from ncl-affected tibetan terriers to resequence the coding regions and intron-exon borders of several genes harbored within the target region and found a single base pair deletion, c. delg, in exon of positional candidate atp a . this deletion produces a frame shift and a predicted premature termination codon. we genotyped all tibetan terrier dna samples in our collection and found all ncl-affected tibetan terriers to be homozygous for the c. delg allele. eleven additional c. delg homozygotes were either less than years old, or lost to follow up. there were no known cases of ncl in the remaining tibetan terriers which were either heterozygous (n ) or homozygous for the ancestral allele (n ). atp a is a member of group of ion transport genes and has been associated with lysosomes. mutations in human atp a cause kufor-rakeb syndrome (krs), a rare neurodegenerative disorder with clinical features that include parkinsonism plus spasticity, supranuclear upgaze paresis, and dementia. post-mortem findings in krs have not been reported. we conclude that ncl in tibetan terriers is caused by a mutation in atp a . our results suggest that krs may be a form of adult onset ncl in humans. niemann-pick type c (npc) disease is a progressive neurological disorder characterized by dementia and ataxia, hepatic and pulmonary disease, and death typically within the first or second decade. despite the identification of causative mutations, the pathogenesis is not clear and therapies to successfully treat npc disease have been ineffective to date. the recent use of intravenously administered -hydroxypropylbeta-cyclodextrin (hpbcd), an fda-designated orphan drug (may ), in a small number of children with npc disease is based on favorable treatment outcome data in subcutaneously treated mouse and cat models. to rigorously evaluate the mechanistic, pharmacologic, and toxicity issues associated with hpbcd therapy in npc disease, we have utilized the spontaneous feline npc model harboring a missense mutation in npc (pc s), orthologous to the most common mutation in juvenile-onset patients. the feline npc model has clinical, neuropathological and biochemical abnormalities similar to those present in juvenile-onset patients making this model homologous to the most common disease form seen in human patients. we identified that intrathecal administration of hpbcd ameliorated all clinical aspects of neurological disease at least up to weeks of age (an age when untreated cats die) but had no effect on hepatic disease. we identified that while subcutaneous therapy with hpbcd at all doses ameliorated liver disease, only mg/kg substantially affected neurological disease but also resulted in early death due to pulmonary toxicity. finally, we identified a dose-related toxic effect of hpbcd on hearing function that had not been described in any other species. leukodystrophies are disorders of myelin synthesis and maintenance that affect cns myelin. they are subdivided as leukodystrophies, hypomyelinating disorders and spongy degenerations. although infrequently seen, several forms have been described in various dog breeds. we present a novel form of complex leukodystrophy consisting of hypomyelination and spongy degeneration that presents primarily with hind end tremors in border terrier puppies. three border terriers from two different litters (and lineages) are described here that presented with a history of shaking movements. the youngest dog was a -week old male. it was the only dog affected in the litter. the other two dogs were -week old female littermates. there were two unaffected males in the same litter. physical examination revealed no abnormalities. on neurological examination, the affected dogs displayed severe hind end tremors, with a characteristic swinging side-to-side movement (best described as ''rumpshaker''). the tremors also involved the head and thoracic limbs but to a lesser degree, and disappeared when the dogs were asleep or at rest. severe cerebellar ataxia was observed when the dogs ambulated. proprioceptive positioning was delayed in the pelvic limbs of all dogs. spinal reflexes and nociception appeared normal. necropsy was performed in all puppies. no macroscopic changes were observed. histologic evaluation of the cns revealed spongy degeneration and hypomyelination in all funiculi of the cervical and thoracic spinal cord. white matter of the frontal, temporal and parietal cortices had mild multifocal spongy degeneration and hypomyelination, whereas white matter of the cerebellum, medulla and pons showed severe diffuse spongy degeneration and hypomyelination with gliosis. the combination of reduced myelin formation combined with spongiform white matter changes in the absence of microglial responses suggest a complex pathogenesis affecting both oligodendrocytes' capacity to synthesize myelin and the stability of the myelin that was formed. the number of oligodendrocytes and axons appeared subjectively normal indicating a primarily hypomyelinating process. the clinical and pathological features of this disease have not been described in any other canine leukodystrophy. the primary and most striking clinical feature is the presence of severe tremors in the hind end, causing the ''rumpshaker'' pheynotype. genetic studies are underway to determine if the disease is inherited and the inheritance mode. a syndrome of border collie collapse (bcc) appears to be common in dogs used for working stock. this syndrome has also been called malignant hyperthermia, heat intolerance, exerciseinduced collapse and ''wobbles''. a presumptive diagnosis of bcc can only be made by eliminating other causes of exercise intolerance and weakness. the purpose of this study was to describe the clinical features of collapse in affected dogs and determine if there were characteristic clinical or laboratory features at rest or after exercise that could aid in diagnosis. seven adult border collies with a history of collapse during sheep herding (affected) and adult border collies regularly used for sheep herding but showing no signs of exercise intolerance (normal) were evaluated before and after participating in a videotaped minute exercise protocol consisting ofa series of continuous short outruns and fetches of three sheep in an outdoor pen. exercise was halted at minutes or earlier if there were signs of gait or mentation abnormalities. pre-exercise evaluation included physical examination, orthopedic and neurological exam. pre and immediate post exercise rectal temperature, pulse and respiration, patellar reflexes, ecg, cbc, serum biochemistry profile, cortisol, arterial blood gas and plasma lactate and pyruvate concentrations were measured. clinical parameters (gait, temperature, reflexes) and lactate and pyruvate concentrations were evaluated at intervals up to minutes after exercise. additional testing in affected dogs included measurement of acetylcholine receptor antibodies (achrab) and dna testing for dynamin-associated exercise induced collapse (deic) and the ryanodine receptor mutation associated with canine malignant hyperthermia(mh). one week after exercise affected dogs had thoracic radiographs and echocardiography performed and were anesthetized for emg and muscle biopsies. there were no significant differences in temperature, pulse, respiration, or any laboratory parameter at any time point between normal and affected dogs. no arrhythmias were detected. affected dogs were negative for the dna mutations tested and for achr ab. thoracic radiographs, echocardiograms, emgs and muscle biopsies were normal. the normal dogs had no alterations in mentation or gait during or after exercise. three of the affected dogs had exercise halted early ( min- min) because of altered gait or mentation. all of the affected dogs were abnormal in the minutes following exercise. abnormalities seen in all dogs included disorientation, dull mentation, swaying, falling to the side, exaggerated lifting of limbs each step, choppy gait, delayed limb protraction, scuffing of rear and/or forelegs, and crossing legs when turning. all dogs returned to normal by minutes. bcc appears to be an episodic nervous system disorder that can be triggered by exercise. genetic testing excluded deic and the described canine mh mutation. common causes of exercise intolerance were eliminated, but the cause of collapse in bcc was not determined and no clinical or biochemical marker to aid diagnosis was established. equine cushing's disease (ecd) is common in older horses. the purpose of this study was to determine the frequency of diagnosis, identify prognostic factors and assess owner satisfaction with treatment. the study was a retrospective cohort design evaluating equine accessions reported to the veterinary medical data base (vmdb) and the ohio state university from - . proportional accessions, annual incidence and demographic characteristics of horses with ecd were compared with all accessions in the vmdb. medical records for a subset of horses were extracted and owners contacted to obtain long-term follow up information. two hundred seventeen new cases of ecd were reported to the vmdb. incidence increased from . / , in to . / , in . eighty-one percent of horses were ! years of age. average delay from onset of signs to diagnosis was days (range to , days). hirsutism ( %) and laminitis ( %) were the most common clinical signs. improvement in one or more signs months after diagnosis was reported by / ( %) of horse owners. none of the clinical or laboratory data were associated with survival and, % of horses were alive, . years after diagnosis. / ( %) of horses were euthanatized and / ( %) were euthanatized due to conditions associated with ecd. twenty-eight of ( %) of horse owners said they would treat a second horse for ecd. ecd is becoming a more frequent diagnosis. fifty percent of horses survived . years after diagnosis and owners were satisfied with the horse's quality of life. supported by centers of excellence in livestock diseases and human health, college of veterinary medicine, university of tennessee. the role of the hypothalamic-pituitary-adrenal (hpa) axis in sepsis has been a subject of a great deal of research. the role that the somatogenic axis plays in sepsis is less well understood and how these two axes interact during critical illness is not clear. the purpose of this study was to assess inter-relationships of adrenocorticotropin (acth), cortisol, and insulin-like growth factor-i (igf-i), in septic and non-septic term foals. blood samples were obtained from term septic foals less than days of age (n ) admitted to texas a&m university veterinary medical teaching hospital or mid-atlantic equine hospital. the foals were classified as septic by a sepsis score ! and/ or a positive blood culture. non-septic term foals less than days of age (n ) and having a sepsis score o and a negative blood culture, were obtained from texas a&m university veterinary medical teaching hospital and mid-atlantic equine hospital. plasma and serum were processed from whole blood collected by jugular venipuncture upon admission, at hours post admission and at days post admission or at the time of discharge. plasma concentrations of acth, and serum concentrations of cortisol and igf-i were determined by specific rias. data were analyzed using linear mixed-effects modeling with foal modeled as a random effect and day of admission modeled as an ordered categorical variable; post-hoc testing of pair-wise comparisons was made using the method of sidak. significance was set at p o . , and analyses were performed using s-plus software (tibco, inc., seattle, wa). plasma concentrations of acth were not significantly different between septic and non-septic foals whereas septic foals had greater serum cortisol ( ae ng/ml vs ae ng/ml) but lower serum igf-i ( ae ng/ml vs ae ng/ml) relative to non-septic foals pooled overall sampling times. the positive association of the peripheral blood concentrations of acth and cortisol depended on disease status of the foals. specifically, cortisol and acth were positively correlated for the septic foals (p . ) but not significantly correlated in the non-septic foals. peripheral concentrations of acth and igf-i were not significantly correlated whether data were pooled overall or stratified by sepsis status. however, peripheral concentrations of cortisol and igf-i were negatively associated (p . ); disease status did not influence this association, although it appeared to be a stronger association for the septic than the non-septic foals. the negative correlation between serum concentrations of the adrenal axis steroid cortisol and the somatogenic axis peptide igf-i may reflect interactions of these homeorhetic hormones. further studies of these and other metabolic hormones in a greater number of foals are warranted to better understand how these factors contribute to survival or non-survival of critically ill foals. botulism is a potentially fatal paralytic disorder which definitive diagnosis is difficult. the purpose of this study was to investigate if repetitive stimulation of the common peroneal nerve will aid in the diagnosis of suspected botulism in foals. four healthy foals were used for its comparison with foals with suspected botulism. controls were anesthetized and affected foals were sedated to avoid risks of anesthesia. the common peroneal nerve was chosen for its superficial location and easy access. stimulating electrodes were placed along the common peroneal nerve. for recording, the active and reference electrodes were positioned over the midpoint and distal end of the extensor digitorum longus muscle, respectively. repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies ( to hz). amplitude, area under the curve and percentages of decrement or increment for each m wave over subsequent potentials for each set of stimuli were analyzed. baseline m waves were decreased in affected foals compared to controls. a decremental response was seen at all frequencies in control foals. decremental responses were also observed in affected foals at low frequencies. however, an incremental response in amplitude and area under the curve was seen in all affected foals at hz. reduced baseline m waves with incremental responses at high rates are supportive of a presynaptic neuromuscular disorder which botulism was the most likely cause in these foals. repetitive nerve stimulation is a safe, simple, fast, and non-invasive technique that can aid in the diagnosis of suspected botulism in foals. this study examined the frequency with which dogs are exposed to e. chaffeensis and e. ewingii relative to e. canis, which is transmitted by the more ubiquitously distributed brown dog tick (rhipicephalus sanguineus). a total of , canine serum samples, ranging from to from each of the participating institutions, collected at random from clinical accessions, diagnostic laboratories and/or shelters were evaluated. all serum samples were tested by three microtiter plate elisas using species-specific peptides for antibodies to e. canis, e. chaffeensis and e. ewingii. zip code information for sample origin was provided by the collaborator and was used to assess seroprevalence by region. comparisons were evaluated using the chi-square test. seroreactivity for at least of ehrlichia spp was found in samples from every institution both mississippi and oklahoma had greater than a % samples from ohio had the lowest aggregate seroprevalence ( . %) with only dogs e. canis seropositive, one e. ewingii seropositive and no e. chaffeensis seroreactors. the geospatial pattern of e. chaffeensis and e. ewingii seropositive samples was similar to that previously reported based on modeling seroreactivity to e. chaffeensis in white-tailed deer as well as the distribution of human monocytic ehrlichiosis (hme) cases reported by the cdc. this study provides the first large scale regional documentation of canine exposure to these three ehrlichia spp., highlighting where infections most commonly occur and thus identifying areas where heightened awareness about these emerging vector urinary incontinence (ui) occurs in approximately % of spayed female dogs. the most common cause is urethral sphincter mechanism incompetency (usmi). pharmacological agents are effective, however, not all dogs respond, and dogs may become refractory to treatment over time. urethral bulking, where a compound is injected submucosally in the urethra, has been used in women and in female dogs with urinary incontinence. new synthetic compounds have been used in human medicine; the most promising is polydimethylsiloxane (pdms), which has been shown to be more effective than glutaraldehyde cross-linked collagen. the purpose of this descriptive clinical trial is to evaluate the safety and effectiveness of pdms urethral bulking agent (pdms uba) in client-owned, spayed female dogs with naturally-occurring ui due to usmi.twenty-two, spayed female dogs were included. dogs had a median age of years ( to years). eighteen dog breeds were represented, and dogs weighed a median of . kg ( . to . kg). average length of time of ui was . ae . years; / dogs had been treated medically, of which / were continent, / were improved, and / had no improvement. dogs were deemed healthy based on results of physical examination, complete blood cell counts, plasma biochemical analysis, and urinalysis; urine cultures were negative.dogs were anesthetized, positioned in dorsal recumbency, and cystoscopy performed using a . mm, -or -degree, cm rigid cystoscope. urethral bulking was performed with pdms uba. on average, . ae . ml were injected in to locations approximately to . cm distal to the trigone submucosally in the proximal urethra. good coaptation was achieved in all dogs. the procedure took on average . ae . minutes. one dog experienced urethral obstruction after the procedure; a foley catheter was inserted for approximately hours and removed at which time she urinated normally and was continent. three dogs experienced an acute allergic reaction characterized by blepharedema and urticaria treated successfully with diphenhydramine. dogs were discharged on day of procedure except for the one dog that experienced urethral obstruction. all dogs were treated with meloxicam ( . mg/kg po q h for days).owners were contacted on day after discharge and / dogs were continent; / dogs was improved. dogs were re-evaluated week after discharge and / dogs were continent and / dogs polyneuropathy in large breed dogs is a relatively common clinical problem for which the genetic basis is generally unknown. the first cases of polyneuropathy in the leonberger breed (leonberger polyneuropathy or lpn) were identified in by one of the authors (gds) and a report published in (musclenerve : - ) . in this report a spontaneous, distal and symmetrical polyneuropathy with onset between to years of age was described and characterized clinically, electrophysiologically, histologically and morphometrically. there were striking similarities between lpn and the charcot-marie-tooth group of human inherited sensory and motor polyneuropathies, which have many known genetic mutations.a genome-wide case-control association study for lpn was performed with cases and controls on high-density k canine snp arrays and revealed a significantly associated region on cfa (p raw .  À p genome .  À ). a clear association of an approximately mb cfa haplotype with cases (p .  À ) was observed, particularly with those cases that were affected more severely and at a younger age (p .  À ). a positional candidate gene, arhgef , which has previously been associated with peripheral nerve abnormalities in humans, was sequenced, revealing a deletion that results in a frame shift and premature stop codon. of all leonbergers with young onset lpn (before years), . % ( of ) have two copies of this deletion, and, of all young onset leonbergers that are nerve biopsy positive for lpn, . % ( of ) have two copies of this deletion. importantly, nearly all dogs carrying two copies of the deletion ( of or . %) are affected with lpn by the age of years.the leonberger breed was generated from crossing several breeds, including the st. bernard, and a polyneuropathy clinically and histologically similar to lpn occurs in this breed. to determine if the arhgef mutation was associated with polyneuropathy in the st. bernard, dna was extracted from archived frozen muscle biopsy specimens from clinical cases (n ). the identical arhgef startle disease or hyperekplexia is caused by defects in mammalian glycinergic neurotransmission resulting in an exaggerated startle reflex and extensor hypertonia triggered by noise or touch. in humans and animals, startle disease is typically caused by mutations in one of three genes (glra , glrb, and slc a ) encoding postsynaptic glycine receptor subunits (a and b) or a presynaptic glycine transporter (glyt ). a litter of seven irish wolfhounds was recently identified in which two puppies developed muscle stiffness and tremor beginning at - days of age post-partum. signs were dramatic when the puppies were handled and resolved when the puppies were relaxed or sleeping. both puppies were euthanized due to ongoing stiffness, tremor and breathing difficulties. necropsies were performed, but no microscopic pathological abnormalities were identified in the peripheral or central nervous system.based on the clinical signs, exons from the three candidate genes were amplified from genomic dna isolated using pcr and directly sequenced. no deleterious polymorphisms were identified in either glra or glrb. however, difficulties were experienced in amplifying slc a exons and from affected animals, although control samples were positive, suggesting that the pcr primer designs and conditions were not at fault. further pcrs revealed that the reason for this anomaly was the presence of a homozygous . kb deletion encompassing exons and of the glyt gene in both affected animals. this deletion is predicted to result in the loss of part of the large cytoplasmic n-terminus that is vital for trafficking of glyt to synaptic sites, and a loss of all subsequent transmembrane domains via a frameshift. this genetic lesion was confirmed by defining the deletion breakpoint, southern blotting and multiplex ligationdependent probe amplification (mlpa). this analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, suggesting recessive inheritance of this disorder. wider testing of related animals has identified a total of carriers of the slc a deletion and enabled the identification of non-carrier animals to guide future breeding strategies. insulin resistance (ir), obesity, and type diabetes affect glucagon-like peptide (glp- ) concentrations in humans and rodents, but this incretin hormone has not been examined in horses. we therefore hypothesized that glp- concentrations would change in horses as obesity and ir were induced or exacerbated by overfeeding. six horses previously diagnosed with equine metabolic syndrome were provided with twice the amount of digestible energy required for maintenance as sweet feed and hay for weeks. intravenous and oral glucose tolerance tests (ogtts) were performed at and weeks. effects of time and period ( and weeks) were assessed by repeated measures anova.mean body weight increased from ae kg (range, to kg) to ae kg (range, to kg) over weeks, with individual horse weight gain varying from to %. mean body condition score increased (p . ) from ae (range, to . ) to ae (range, to ). three horses developed mild laminitis. glucagon-like peptide concentrations increased over time during ogtts (p . ), but the period  time effect was not significant (p . ). area under the glp- curve remained unaffected by weight gain, whereas area under the insulin curve increased (p . ) over time, indicating a reduction in insulin sensitivity. obesity and ir were induced or exacerbated when horses previously diagnosed with ems were overfed, but glp- concentrations did not change as a result. hypertonic saline solution ( . %) (hss) is an intravenous fluid used for the emergency treatment of intravascular volume deficits. the use of this fluid in horses with severe dehydration is controversial. the purpose of this study was to compare the use of hss and isotonic saline solution ( . %) (iss) for the emergency treatment of endurance horses.endurance horses eliminated from competition and requiring intravenous fluid therapy were eligible for enrollment in the study. twenty-two horses were randomly assigned to receive ml/kg of either hss or iss along with l lactate ringer's solution (lrs). following this bolus, all horses were treated with an additional l of lrs. blood and urine samples were collected before, during and after treatment. data was compared using two-way anova with repeated measures.as compared to iss, hss horses showed a greater decrease in pcv (p . ), total protein (p . ), albumin (p . ), and globulin (p . ). hss horses showed a greater increase in sodium and chloride (p o . ) as compared to iss horses. horses receiving hss had a shorter time to urination (p . ) and lower specific gravity (p o . ) than those receiving iss.results of this study indicate that hss may provide faster restoration of intravascular volume deficits than iss in endurance horses receiving emergency medical treatment. more profound electrolyte changes should be expected with hss however. b -adrenergic receptor agonists have been shown to increase erythrocyte carbonic anhydrase activity, which may stimulate the jacobs-stewart cycle and increase pulmonary circulation transvascular fluid fluxes during exercise. increase in pulmonary transvascular fluid fluxes (j v-a ) and consequent increase in the pulmonary interstitial fluid would be detrimental for alveolar o exchange during the fast erythrocyte transition time across the pulmonary capillaries. therefore, we hypothesised that treatment with inhaled b -adrenergic receptor agonist will increase j v-a and the alveolar-arterial po difference (aado ) during exercise.six stb horses were exercised on a high-speed treadmill at % vo peak until fatigue. horses were randomly assigned to treatment with salbutamol (sal: mcg) or placebo (control: con) inhalation via aeromask à min prior to exercise, with cross over treatment used at the repeated exercise test ( days later). arterial and mixedvenous blood, as well as co elimination and o uptake, were sampled simultaneously at rest, during exercise at sec intervals until fatigue, and into recovery. blood gases were analyzed. aado was calculated using the inspired po ( mmhg), and blood partial pressure of o and co . blood volume (%) changes across the lung were calculated from changes in hemoglobin and hematocrit values in venous and arterial blood. cardiac output (q) was calculated using the fick equation. j v-a was calculated using q and blood volume changes across the lung. variables were analyzed using two-way repeated-measures anova (p o . ).the duration of exercise to fatigue was . ae . min and . ae . min in both con and sal, respectively. at rest sal had no effect on j v-a , oxygen consumption (vo ), blood oxygen saturation (so ) or aado (p . ). at the onset of exercise j v-a increased in con and sal (p o . ) and at fatigue reached . ae . l/min and . ae . l/min, respectively. treatment with sal had no effect on j v-a during exercise (p . ). at the onset of exercise so and vo increased in con and sal (p o . ). treatment with sal had no effect on so or vo during exercise (p . ). aado increased during exercise in con and sal (p o . ) and at fatigue reached . ae . mmhg and . ae . mmhg, respectively. treatment with sal had no effect on aado during exercise (p . ).inhaled b -adrenergic receptor agonist salbutamol at the dose of mcg given min before exercise did not affect the duration of exercise to fatigue, j v-a , vo , so or aado . therefore, it had no detrimental effect on alveolar-capillary diffusion distance and the ventilation/perfusion mismatch in exercising horses. inflammatory airway disease (iad) and recurrent airway obstruction (rao) represent two classes of equine lung inflammatory diseases that may share some similar immunologic mechanisms. there is evidence that th cytokines and il- play some role in rao. iad is a common condition in horses, but its pathophysiology is still not understood. the aim of the present study was therefore to determine the mrna expression of th , th and th inflammatory cytokines, to understand the immunological mechanisms of iad.the mrna expression of ten inflammatory cytokines and chemokines was measured in the bronchoalveolar fluid (balf) of seventeen horses with iad and compared with ten control horses. the horses were selected based on -their clinical signs, -the inflammatory cells count in the balf, -their physical examination and -their medical history. the mrna expression of il- , il- b, il- , il- and il- was significantly up-regulated in balf from horses with iad.furthermore, the balf samples were subdivided in two groups based on the differential cells count -balf with increased mast cells (iad-mast) and -balf with increased neutrophils (iad-neutro). il- was significantly down-regulated in the iad-neutro group compared to the iad-mast group. il- , il- and il- were significantly up-regulated in the iad-neutro group compared to the iad-mast group.the present study shows that iad in horses is characterized by a th and a th mrna inflammatory expression profile and that different immunological mechanisms are involved in mast cells or neutrophils accumulation in the balf of horses with iad. b -adrenoreceptor (b -ar) agonists are a class of medications that promote smooth muscle relaxation and bronchodilation in horses and humans with airway disease. activated human peripheral blood lymphocytes (pbls) also respond to b -ar agonist stimulation by attenuating the production of cytokines associated with the pathogenesis of asthma and recurrent airway obstruction (rao). the aim of this study was to develop an in vitro technique for measuring the response of equine pbls to stimulation with salbutamol, a b -ar agonist. this method was then used to compare the response of pbls from rao-affected and non-affected horses to b -ar agonist stimulation. pbls from rao and nonaffected horses were cultured ( x /ml) in rpmi complete media with concanavalin a (cona, ug/ cells) for , , or days then stimulated with salbutamol ( minutes). using flow cytometric techniques, response was measured by detecting protein kinase a phosphorylation of vasodilator stimulated phosphoprotein (vasp). results were verified by western blot analysis. activated pbls were then incubated with cona for one day were pre-incubated with b or b-adrenoreceptor antagonist (ici , , sigma s ; atenolol, sigma s ) for minutes, followed by minutes salbutamol ( nm) stimulation. results were analyzed by anova or ancova and differences were considered significant when p o . .response to b-antagonist was only observed in activated pbls (pre-cultured with con a) and was greater in cells from rao horses as compared to cells from non-affected horses. the addition of b-antagonist attenuated the response of pbls to salbutamol while the addition of a b -antagonist had no effect. these findings indicate that activated pbls from rao-affected horses have a greater response to salbutamol as compared to pbls from non-affected horses, and this response is mediated mainly through the b -ar.human b -ar are known be polymorphic and this polymorphism results in a variable response to b -agonist binding that affects long term outcome in human asthmatics. further studies are required to determine if the difference in response of pbls from rao affected as compared to non-affected horses is due to genetic polymorphism in the equine b -ar, and whether this difference is associated with a propensity for horses to develop equine rao. key: cord- -pol qm authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: pol qm nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on - march . when, in the mid- s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in provided stateof-the-art information and consensus on factors involved in injury and sepsis. in , the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until or . however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il- to il- , il- and il- and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in , to be dealt with at this symposium and congress? ( ) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. ( ) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. ( ) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. ( ) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. ( ) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. ( ) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~ ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha- macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro -iarg] ]aprotinin (bay ) or the b kinin antagonist, hoe on the hypotensive response to dxs. in the first study, anesthetized miniature pigs ( pigs/group, randomly assigned) were given one of the following treatment protocols: ) dxs ( mg/kg), - ) dxs plus bay ( , , , or rag), or ) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c adesarg and fibrin mom)mer were all increased. bay produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay . llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs ( /group) were given either dxs alone ( mg/kg) or dxs minutes after a bolus injection of hoe ( #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse , d- munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion ( , mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to u/ . in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to u/ . in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs ( ). when very small plasma kailikrein activities ( , u/kg bodyweight) were given intravenously a % decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c -inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin- ( x iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of , or u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion ( - u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c -inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; thrombosis research center, temple university, penn., usa; oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c b which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c b/c and c b/c, and a significant reduction was observed in animals that had received a lethal dose of e. coli together with mab c (treatment group), compared to animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-= antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c a, c , and c a were measured in patients from an internal intensive care unit. patients were clinically septic defined by the criteria of bone et al.(l) . the remaining patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l days period. during the first days blood samples were drawn every h, on day - every h and the last days once daily. mean plasma concentrations of c a within the first h after clinical onset of sepsis were + pg/ml, whereas non-septic-patients exhibited mean values of only +_ p_g/m/. c levels were lower for septic-patients ( + lag/ml) than for non-septic-patients ( _+ lag/ml). the most profound difference between both groups was found, when the c a/c ratio was compared ( . + . for septic-patients and . _+_ . for the control group). no significant differences between both patient groups were observed in c a plasma levels ( . + . ng/ml in septic-patients vs. . _+ . ng/ml in control patients). in of cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n= ) and non-survivors (n=l ) within the septic-group revealed, that the c a/c ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c a and c in only - minutes ( ) and the significant difference of the c ajc ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of amino acids with cysteine residues (mr daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw , behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of . p.g/kgxh thromboplastin for hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to . % and , % respectively, and by an increase of fibrin monomers from . to > . ~tg/ml. we administered rec. hirudin to rabbits in different concentrations ( . , . and . mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin ( . , . and . mg/kg i.v. bolus) as late as hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, marburg, germany $ novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r- ) and its effect on sepns-lnduced organ injury in rat atsuo murata , hitoshi toda , ken'ichi uda , hidewaki nakagawa , takesada mori , hideaki morishita , tom yamakawa , jiro hirese , atsushi ni~ , nariaki matsuura osaka university medical school, osaka, mochida pharmaceutical co. ltd. tokyo, wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je which lacks the membrane lipoprotein. the recombinant second domain (r- ) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r- was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r- significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai., ) . the cathepsin l-like and cathepsin b-like activity were measured in serum of patients with chronic bronchitis ( -with obstructive, -with nonobstructive bronchitis),acute bronchitis ( ) and healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai., ) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls ( - fold) .after treatment there was a tendency to normalization of indices,but the increase was about - % more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis - - -times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/ - diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il- / , ifn-a, il- , il- , il- , paf, pge, ltb and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur ( , ), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/ selectively t cells to produce lymphokines including i - and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/ + t cells accumulate within - min mrna for the ii- , i - , tnfai~ and ifn- ,. parallel in time high concentrations of bloodborne i - and tnf are noted. anti tnf t~// neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within - h distinct levels of tolerance to seb become discernable on ligand reactive v/ t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd , cd , cd accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c h/hej mice. the absence of sensitization to lps in lps d c bl/ sccr mice was identified as being due to their inability to produce interferon- (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c bl/ sccr mice concerns only the ifn response to bacteria, since the response to the t-cell mitogen con a and to cd monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c bl/ sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il- levels, whereas treatment with aztreonam resulted in progressively increasing levels of il- and a significant increase in mortality. the increased mortality in pbp- specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as -to lo -fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf- dgaln-treated mice with e. coli olll:b resulted in an lds of approximately . x cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection ( fold vs fold respectively, p< . ). parallel studies with endotoxin hyporesponsive c h/hej mice indicate significantly greater levels of protection with imipenem (> fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since , when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe ; pollack ) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: ) enhancing of opsonization and phagocytosis(antibactericidai activity) ) synergistic effects with [ - actam antibiotics ) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis ) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il- release in mice (baumgartner , corriveau and danner ) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger (berger , , in animal models (stephan , berger and also in prospective, randomized, controlled clinical trials (schedel , poynton , behre . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg ak) has been selected and chimerized into a human igglk (sdz - ). in elisa and in immunoblots on purified lps both sdz - and wni - show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz - and wni - have biological activity as they inhibit the lal assay and the secretion of monokines (il- and tnf) by mouse and human macrophages. moreover, sdz - and wni - inhibit the release of il- and tnf in vivo. in vivo sdz - as well as wni - neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni - as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch basel, $chweiz $ presentation of lps to cd by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd (mcd ). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd (scd ). these complexes of lps with scd are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd , either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd . these results show that the delivery of lps to scd is catalysed by lbp, i.e., lbp is not included with the lps-scd complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd . the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm , ai , ai , gm , and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm- , n. torrey pines rd. la jolla, ca usa . modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d- borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i _- , and il- , which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound ) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin- and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a % amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd receptors. a recombinant protein (rbpi ), corresponding to the amino terminal kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi will have to successfully compete with relatively high serum levels of lbp ( - ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of . nm for rbpi and nm for rlbp. in a competition assay format rbpi was approximately -fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi at . ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a -fold weight excess of rlbp over rbpi . and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions - , - and - . a single synthetic peptide ( - ) was bactericidal. these results suggest that rbpi contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p and p receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only % of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il- [ -inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered ( kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in % plasma +/-bpi ( ng/ml). pbmc were incubated for hours at °c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n= , mear~+sem, *p< . vs control). control . _+ + bpi . + % plas. . _+ + bpi . _+ pmp (ng/ml) lps (ng/ml) . _+ . _+ . _+ . _+. . +. . _+. . _+. " _+ " . _+ " . _+ " . _+. . + _+ _+. * _+ " . +. " . + -+ . -+ " . _+. " cba, c bl/ , balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection - % of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats ( - g) were studied [no operation (n= ) , sham operation (n= ), and bile duct ligation for days (bdl)(n= )]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n= ), sham op(n= ) or bdl(n=i ). blood was taken at this time (to) and days later(t at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp< . , mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t [p< , ,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp< . , paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than . pglml. polymyxin b was administered at a dose of , u every hours. plasma endotoxin rapidly decreased to the normal range in of the patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. - uchimaru, morioka , japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, n o r w e g i a n b r e d landrace pigs ( - kg) of either sex, pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a / air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e . m g / k g h a n d d i a z e p a m . m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n . x l cfu of e colt intraperitoneally as a bolus in ml saline, the a n t i b o d y g r o u p received in a d d i t i o n m g / k g e a n t i e n d o t o x i n i n t r a v e n o u s l y over h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for hours. results : a t a n d hours, the o x y g e n c o n s u m p t i o n increased by % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po . in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h- szeged~ hunbary involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit des toxines microbiennes and "unit d'immuno-allergie, institut pasteur, , rue du docteur roux - paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il- d and ~, tnf ~ and il- in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il- and il- . however, the most preponderant cytokine was tnf~, which peaked at i ng/ml after stimulation with i ~g eta. comparable amounts of tnfd (ca ng) were induced by .i ~g eta and .i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i, -glucan (ram ) was injected twice and h before the shock i.v. in a dose of ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli bs, mg/kg. aiiofcmg pretreated ruts survived during first haher ¢ndotoxine, while in controi shock group the lethality was %. the concentration of ~col)terin in serum was significantly elevated hafterthc second cmginjection (appare~tly % if compare with the control rats), but didu't chartged rain and s rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i and min after endotoxine onset ( i % trod ~, respectively of initial level) than in the control shock group ( % and % at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; . modify records; . erase records; . searches; . reports; . configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd ) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of organisms and the chance of identifyin organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group ( cases), the total effective rate was , %, in the contral group (combined administration of gentamycin and dexamethasone, cases) the total effective rate was %. however the obviously effective rate in sa group % was significantly higher than in contral group % (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o ). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and -glucuronidase ( -gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c h/hen mice at i and h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n= /grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at h the thymic cell yield from clp mice was significantly decreased (sham: . ! . x l vs clp: . i . x * cells; *,p< . ), the % of cells apoptotic by facs analysis increased from . i . % in sham to . ± . %* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from to patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). patients ( %) out of patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all patients with chronic septic knee joints showed poor results. additionally, in of these patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco- cell line) is grown on a micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco- monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco- system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco- monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. ) caco- cells are actively involved in the transcellular passage of bacteria across the caco- monolayer, since inhibition of caco- microtubule or microfilament function decreases the magnitude of st. ) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco- cell monolayer. ) caco- cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco- bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco- cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers ( . x colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about . % within the first minutes and are totaiy cleared within minutes. % offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about cfu of viable e.eoli per ml blood could still be counted hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with % respectively % of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at (n~ ), g (n ~ ), and (n~ ) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker - % at hour~ with return to % of gut leugth (similar to ~m) at hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at ,; hours with return to sham control at hours ( log cfu/g veraus log cfu/g ia ileum). translocation to mesenteric nodes - , , and percent at , , and fi hours compared to b in sham controls (n=l of ). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il- , soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs ( mmhg mean arterial pressure: map for - h terminated at an accumulated oxygen debt of - ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of - mmhg - rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at min and were still markedly increased at rain postshock. plasma tnf, il- , and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at min following resuscitation in rats subjected to hs for min, similarly at h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at min, while in baboons higher levels up to pg/ml were found at the end of shock and h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the -hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a (txa ) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky- , a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup , an a- receptor antagonist as a posthum treatment modality was evaluated. treatment with dup prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup from % to % (p< . , fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf -dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il- and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus , lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. male swiss (icr) mice were used weighing - g elimination of liver and spleen macrophages was accomplished by intravenous injection of / suspension of ci mdp-liposome suspension. control mice received an i.v. injection with pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either mg/g, . mg/g or . mg/g body weight (n= in each group), signs of st and bacterial translocation bt were measured hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n= in each group) were tested to verify that neither paraffin nor pbs or ci mdpqiposome suspension alone induced bt or st. furthermore survival over a -day period was assessed in a control and macrophage-depleted group with a dose of d mg/g z. results. neither the paraffin nor ci mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: / vs. / at . mg/g zymosan (p~o. ); vs. at mg/g zymosan (p~q ); / vs. / st . mg/g zymosan (p~o.o ).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of . mg/g zymosan ( . -+ . vs. . -+ , p~o. ) and . mg/g zymosan ( . -+ . vs. . -+ . , p<_.o. ) . the day mortality after . mg/g zymosan was % in the control group and % in the macrophage depleted group (p=o. ). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n= ) and two experimental (n= each). a portal vein catheter was placed and remained in situ hrs. a femoral artery and vein catheter were in place hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of . hrs., given more hours of anesthesia, and studied for further hrs. % of the estimated blood volume was withdrawn from each experimental animal over min. these animals were kept in shock for . hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for hrs. samples from portal blood for cultures and measurement of leukotriene b (ltb ), prostaglandin e (pge ), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to hrs. after hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb , pge and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha- a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c hb-iej mice). five days post-transfusion the animals were gavaged with lxl viable escherichia coil and randomized into groups (n= /group) to receive a sham burn (sb group) or a % thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies ( mg/kg) (ha- a group) or a % burn injury and administration of equal volume of sterile saline (c group). all groups were resuscitated by intraperitoneal injection of . ml of saline. the animal survival rate was observed for days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n= /group) were gavaged with viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline . and hr prior to sacrifice, group ih treated mice receiving ip of utinastatin . hr and endotoxin ( mg/kg) hr prior to sacrifice,group ilk sham reated mice receiving ip of saline . hr and endotoxin ( mg/kg) hr prior to sacrifice,group iv: saline control mice receiving ip twice during rain intervals,group v: mice received ip of utinasmtin, and min later they were received ip of endotoxin ( mg/kg), group vi: mice received ip of saline,and min later they received ip of endotoxin ( mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu - ,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (> points at days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range . - . mind) was studied after i.v. injection of mbq rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results . + + liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r= . ) and between l/m and ela (r= . ). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il- release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and , , , min, . the plasma concentrations (,pc) of -keto-pgft,, txb: and-ki- -pgf ~ (stable metabolites of pgi , txa and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il- levels by elisa. data are given as mean+sem (* p< . placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t . + . e[ . + . ] eu/ml). il- pc demonstrated an increase continuously from t to t (t + [ + ] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of -keto-pgf~,, pc up to h after mt in untreated patients with a peak of *[ ] ng/ at tl. also txb: and kh pge pc showed a considerabe increase up to h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il- release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into groups. group received standard laboratory chow feeding ad lib. in group a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group % of the calculated daily required calory intake (drci) ( /kcal/kg) was given by iv-tpn ( % glucose, , % amino acids) and % by limited chow administration. groups and received % and % of the drci by i.v. tpn and % and % respectively by chow feeding. group received iv-tpn only. after days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. , + , , - , , / + ~ " , -+ , , -+ ~ - , / +~ + _+ , + , , - , -+ + , ~ , , -+ ~ conclusions: the administration of % of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal ( %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and % in animals subjected to % bepatectomy and lreatment with fermented oatmeal, while - % and - %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < . vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < . ) in salinetreated animals following % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. _+ " +_ " . " data=mean_+sd, * stats anova p< . vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<. ) in lps gut translocation compared to control and l+co . this correlated with a significant increase in peritoneal inflammatory responses (o -,tnf) above that of the control and l+co groups, while mac- and cr opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin , ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), -keto-prostaglansin, leueotriene c , interleukin and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of patients showed a significant increase of endotoxin plasma levels during colonoscopy (p= . ), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after hours. ~hromboxane b o levels also increased after five min. from to pgyml peaking at min. with pg/ml. -keto-prostaglandin,leucotriene c , ii- and crp remained unchanged. a control group of i volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into groups were subjected to , and minutes of %, % and % of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p . ). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p . ). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: .long term( - days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. .in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. athol. . . .) . there was a increasing infiltration of the mucosa c m~nating at the d to th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. ~in isehemia was followed by a hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at m_in, i, ~ , and hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the th hour in the arterial blood .and at the d and th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for and minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion , and hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr ). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats hours after reperfusion in ischemia- rain group and and hours after reperfusion in the ischemia- rain group. delayed small intestinal transit time was seen from hours and on after intestinal ischemia for both and rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < . ). the distribution of radioactivity in segments and in the group with repeffusion hours after intestinal iscbemia for rain was significantly higher than that noted in the group with repeffusion hours after intestinal ischemia for min (p < . ). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy ( %) in the rat. water-soluble ehec was administered orally and hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a - % translocation rate to mlns or blood and hours after operation, while only - % of rats subjected to sham operation or animals with % hepatectomy and pre-treatment with ehec (p < . ). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal -hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s- lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca ÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca + concentration, [ca *]i, and ca + fluxes were measured by the use of fluorescent ca + chelating dyes (fura- or indo- ) and ca respectively. to assess sepsis-related changes in ca + dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca + i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response ( % ¢) in the liver, and a blunted insulin mediated sugar utilization ( % ) and increased proteolysis ( % ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca +]i by - % was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm ). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca + from intracellular stores. this protein kinase dependent process of [ca+ ] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca + signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn- (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute biochemistry, albert-ludwigs-university, hermann-herder-str. , d- freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a and kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: "-gggpunnf'ypycc- ") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il- b, tnf, [l- , il- , gm-csf etc), cell adhesion molecules (icam- , elam- , vcam- ), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p . upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a / kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g cells showed that lbp transcripts could be induced to ford within hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter . kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il- responsive elements hstf-hsp . and h-apf- rs as well as the transcription factor hnf- and the glucocrticoid-responsive elements oct- -d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g cells by il- , il- and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd .transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd , a kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd , which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd (cho/cd ). stimulation of cho/cdi with as little as ng of lps/ml resulted in the release of h-arachidonic acid ( h- : ) into the culture supernatant. in addition to h- : release, we examined lps-imiuced activation (transl cation) of the transcription factor nf-~b. similar to lps-induced h- : release, these responses were observed only in cho cells lransfected with cd closely resembling the same response in the marine macrophage cell line raw . . maximum nf-~cb expression occurred at minutes. in contrast to lps-induced h- : release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd , suggesting that nf-r,b activation begins early after lps binding to cd and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd , lps-induced tk activity in cho/cd was not observed. although the tk inhibitor herbimycin inhibited the release of h- : in cho/cd and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd -related lps signal transducer in cho/cd is not a tymsine ldnase. dimished cardiac inotropic function and response to -adrenergic receptor ( -ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin ( : gm/kg). to stimulate various components of the transmembrane signal cascade of -ar was isoproterenol (i -ar), acetylcholine (m ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia ( °c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c b and cdb was given to rats at a dose of mg/kg by intravenous bolus, min before reperfusion of the ischemic gut. a control group was given pbs buffer. at h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri ( . ± . to . _+ . ,p< . ) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced ( . ± . to . ± . u/g). c was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced ( . ± . x i to . ± . x , p<o. ). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia ( hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc , interleukin-i ( l-i) and il- plasma levels increased significantly, reaching maximum levels after hours ofreperfusion. polyclonal antibodies to tnfet and l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by % and % respectively and mpo was reduced by % and %. antml- yielded a protection in muscle and lung vascular permeability of . °, and . % respectively whereas muscle mpo was reduced by . % and lung mpo by . °, . these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using mabeled antmcam-i, constititive expression of icam- was evident only in lung but not in muscle. during reperfusion icam- expression increased by . %. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by . °, and . % respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following min of ischemia (mi) and . h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb . to p-selectin in a feline model of mi+r. pb . ( mg/kg) administered rain post-occlusion ( min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp . ) for p-selectin ( + vs + % of area-at-risk, p< . ). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb . compared to nbp . ( + vs + , p< . ). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb . treated cats ( + vs + pmns/mm z, p< . ). also, normal coronary endothelium activated with thrombin ( u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p< . ) blocked by pb . , but not by nbp . . furthermore, p-selectin is markedly upregulated - min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested , , , , , , , and h after transplantation (n= /time point) . snap frozen sections of organs were stained for cd- (w / ), cd- (ox ), t-lymphecytes (tl) (ox- ), macrophages (ed , ed ), neutrophils (pmn) (mom), icam-i (iap ), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w / , ox , ox , ed , ed and mom, staining for icam , lain and fib was assessed visually using a scale (ve= - ). as early as h after transplantation, fibrinogen and laminin expression increased in i, peaking h after transplantation (ve= . ; nc: ve=i. ) . at and h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i. ) but rose again at h, peaking at h (ve= ) and remaining elevated thereafter (ve= ). the high expression at h correlated well with the maximal pmn infiltration at that time (cf= ) in i returning to baseline at h (cf= . ) and thereafter. the second peak at h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il- , tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the minute interval (il-lb: , __+ , to , __+ , pg/ml; il- : . - , to , __+ , pg/ml; tnf-a: , __+ , to , + , pg/ml; p< . ). immediately after the beginning of reperfusion, soluble icam- and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam- , lfa- or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n= /group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within rain, ischemic hearts were stored in cold ringer's solution for hours before revascularization. representative grafts were removed after . , hrs, and days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts ( vs rain). after hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked ( vs c/f in non-ischemic grafts), whereas edl+macrophages ( vs c/f) and tnfe expression peaked by hrs. by hrs t-lymphocytes began to enter ischemic myocardium and icam- was moderately increased. after days cellular infiltration had returned to baseline, and no differences were seen among both groups after days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il- for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd treatment in the same model. in other species similar controversial results with anti-cd therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a -fold increase in neutrophil numbers associated with dispersed lullg tissues hours after lps treatment, while macrophage levels increased by -fold at the hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin- (il- ). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by hours, while mip-i levels were maximal at hours ( , ng/ml), with a second peak at hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a -fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip- . in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by -integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of -integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib and dreg , baseline values were determined immediatley after drawing blood. in addition cells were incubated min at °c to allow for spontaneous regulation of adhesion molecules. blood specimens from septic patients were obtained during the course of their illness. control values were determined in healthy volunteers. results: baseline expression of -integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g -integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to pg/ml tnfc~ or nm fmlp (but not at pg/ml tnfc~ or nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n = , bacteremia = ); group b : sepsis syndrome without septic shock (n = , bacteremia = ). serial plasma samples obtained on day , , , , and , were assayed using elisas method (british biotechnology), normal control levels of soluble icam- and e-selectin, obtained from healthy volunteers, were respectively ± . ng/ml and ± . ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis, ) . compared to control mean values, initial levels of groups a and b were significantly higher for icam- (p < - ) and e-selectin (p < - ). comparisons of group a and [] (* = p< . ; ** = p< . t) soluble icam- levels of group a enhanced significantly (p< . ) during the first hours, and a sustained high levels was of bad prognosis ( % of survivors at day ). the evolution of soluble icam- and e-selectin levels were significantly correlated with murray's score (spearman test : p < . ). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam- and e-selectin are correlated with endothelial lung damage, and loam- seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il- and il- , govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il- , have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam- . such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: ) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, ) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and ) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room ; barnhill drive; indianapolis, in usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd) chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd is expressed on all leukocytes, whereas cd b/cd and cd c/cd . are restricted to cells of myeloid origin. cd i / cd interacts with intracellular adhesion molecule- (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd , however, would affect all leukocytes, as would antibodies to cdlla/cdi . targeting cdllb/cd would affect cells of the myeloid lineage only, which could prove to be beneficial. cd b/cd is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd lb/cd may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt gs r.he, t focus on replacing slslic ~sd ~nd fuc s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c .an does sle:, s=h compounds are m ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the ~peutics for i~fi~anmat ry disease. lr:rng maimai models of acute lung lu ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi or its endothelial ligaud, intercellular adhesion molecule- (icam- , cd ). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two x -mm zones were produced by applying brass probes heated to °c to the animals' backs for sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd r . prior to burn injury (pre-r . ); animals given r . min after burn injury (post-r . ); animals given a monoclonal antibody to icam-i, r . prior to burn (pre-r . ); and animals given the r . min postburn injury (post-r . ). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies min after injury was as effective as preburn administration in preserving blood flow. at hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to . _+ % of baseline (p < . by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy- , a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but patients who died during the first hours were excluded, as this was part of the protocol. cy- was administered as a single intravenous bolus of . mg/kg (n= ), . mg/kg (n= ) or i mg/kg (n= ) mg/kg. the antibody was well tolerated. none of the patients died during the day follow-up period. organ failure was assessed for organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially . ± . , decreased to . ± . at the end of the study (p<o.o ). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin- (il- ) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh /sh domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb- and shc, via direct binding of the shc sh domain to the tyrosine-phosphorylated chain of the tcr/cd complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/ t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~// t cell population further segregates into cd t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a// t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of /$ t cells. moreover, these studies show that the relative contribution of cd or cd t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl- and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl- does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il- , il- and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format -d page, autoradiography of s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at and hrs. following clp. serum il- levels were determined at , , and hours by the tdi bioassay. by hrs. % of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant % elevation (p < . ) in serum il- levels at hrs. there was no difference in il- at and hrs. when compared to the control groups. d page studies examined splenic macrophage total protein distribution and biosynthetic activity at and hrs. post-clp. at hrs. macrophages from clp rats showed a substantial decrease in total protein pattern ( protein spots vs protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< kd) was observed. at hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early ( hrs.) staining with cd and cd mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd antigen (cd ++) and a minor population with weak expression of cd and expression of the cd antigen lcd +/ cd + cells). in healthy control donors(n= ) the latter cells account for + cells/mm and %+ % of the monocytes. in sepsis patients, the cd] +/cd + cells can become a major population with more than % of all monocytes in of patients and with more than cells/mm in of cases. there was no correlation of cd +/ cd + cells to any clinical parameter except for cd +/cd + percentage and body temperature (p= . ). the cd ++ monocytes showed a substanttial decrease in cd antigen density in of patients. three-color-if shows that the cd +/ cd + cells in sepsis patients when compared with the cd ++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd antigens, consistent with a more mature nature of the cd +/cd + cells. levels of il- were increased in sepsis patients: of patients with high numbers of cdi +/cd + cells ( /mm ) had high levels of il- ( u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il- . human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl ) and production of the cytokines il-lb, il- , tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il- production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il- and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il- -production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps ) on mo mediator release with a second endotoxin stimulus (lps ). methods: in vitro cultures of marine peritoneal exudate mo were exposed to hr. of hypoxic or normoxic conditions, then incubated hr under identical normoxic conditions _+ ng/ml of lps pretreatment. during the final hours all m~l were exposed to a range of lps concentrations. m~i supernatants were assayed for tnf, il- , il- , pge , nitric oxide (no), and superoxide release. results: lps markedly inhibited mo tnf release by lps but hypoxia had no effect on lps -triggered tnf release. hypoxia increased mo il- production in the absence of lps , but inhibited lps augmentation seen under normoxic conditions. pretreatment with lps increased no production from lps under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps signifcantly augmented mo release of il-i and pge , but hypoxia had no significant effect (data not shown) superoxide production was increased by lps under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. ( ) . h materials and methods. different groups of icu patients, postoperative (n= ), sepsis (n= ), septick shock (n= ), and healthy control (n= ), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf pl oflps ( ng/ml) to id of whole blood in presence of various concentrations of ptx ( , " , - , " , i " m). after hrs incubation at °c, the tubes were centrifuged and supematants frozen at - °c. tnfa and/l were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h . the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and , , corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being %, ) stimulation by lps. the control group is much more stimulated (> % for il , > % for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (> % for il , > % for tnfa ). the lowest stimulation was observed in patients with septic shock (median = %), some patients being not stimulated at all. )effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at - m (reduction to less than '¼ of the median values), and is almost complete at " m. the septic shock group has a decreased sensitivity to ptx. il production exhibits a lesser reduction at - m (~ 'a to ½ of the median values), further increased at - m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at - m and the inhibition is nearly complete at - m. surprisingly, there is a lesser, but significant suppressive action of ptx on il , not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. ( ) lymphokine research ( ) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp- was induced with phorbol ester ( h) and activated for h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il- and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received ml of either saline or blood from brown norway donors,iv. the animals were killed or days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day ; -+ vs + , p< . ). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure ( + , p< . vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from % in ileum on day to %, p< . ). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb i, hb nijmegen, the netherlands. leukemia cell line, teip- . robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. . adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa , an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa may alter monocyte adhesiveness. we selected the thp- cell line, a human monocytic leukemia cell line to further investigate the effect of txa on adhesion. we tested the hypothesis that txa alters lpsinduced adhesion of thp- cells and that txa exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa mimetic i-bop ( . .tm,) or txa receptor antagonists bms and l ( ~m). cells were allowed to adhere for hours and adherent protein/well was determined. lps-induced a significant (p< . ;n= ) increase in adherence of thp- cells (basal, . + . gg protein/well; lps, . +_ . p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between . - . mm, and is reduced in septic patients up to % ( . - . mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than %. subsequently cells were cultured in phenolred-free rpmi medium with various concentrations of glutamine ( . , . , . , . , . , , mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between and %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam- /cd on monocytes in a dose-dependent manner. the receptor for fc'/rucd as well as the receptors for complement cr /cdllb and cr /cdllc were down-regulated. cr /cd which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi , the receptor for transferrin cd and fc'friii/cd were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up - times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> days) of low hla-dr expression (< %) predicts fatal outcome (mortality > %). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [ of patients recovered from "immunoparalysis" following repeated plasmapheres; of them survived ( %). patients recovered temporarely and patients did not respond (all died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was of ( %; p< , ). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study : in vivo stimulation:cd- mice (n= ) were randomized to receive taurolidine ( mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after hours and were assessed for pm function [superoxide anion generation (o -), nitric oxide (no), tumor necrosis factor (tnf), fc/cr -mediated phagocytic function (phago) study : control pm were harvested and cultured in vitro with taurine ( . mg/ml for hrs), after which time they were assayed for -and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m . release of the inflammatory mediators -and tnf, and fc/cr -mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & , and nitric oxide. but injury and inflammation als cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi + % fetal calf serum and stimulated with i ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced ! fm of et/ cells (vs. unstimulated controls of < ). this calculates to - % of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal , germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il- receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i , which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il- message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il- message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l , which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il- production and the effects of il- on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med : , ) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il- production. macrophages were obtained from peritoneal exudate of thioglycollate treated c heb/fej mice and cultured in rpmi medium containing % fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b ) for hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for hours. the amount of il- in the supernatant was measured by il- dependent cell line (b and td ) proliferation assay. il- was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il- production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il- during the initial incubation with lps and subsequent desensitization. il- , like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il- shared the similar potentiational pathway, but differed by the fact that only il- was inhibited. (supported by r ai and po a .) department of microbiology, molecular genetics and immunology and the cancer center, wahl east, university of kansas medical center, kansas city, ks - . korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, ~g/ml, min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice - days after (peak at nd day for i.v. and th day for i.p. mode of administration of the same dose of mg/ g b.w.).the preliminary injection of cryelan ( mg/ g, or h before) to mice with acute cold stress (- ° c, h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al., ) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium ( - fold); bcg possessed similar effect.cmg in the same concentrations ( /~g/ml) increased release of these enzymes only saightly ( . times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren, ) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased - fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, , , prostaglandin e , and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et- (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. . x ucells/ ml in rpmi + % fcs were incubated i min., & hrs. with - m et-i, - m vic or i ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps ) for hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin- (il-i) in response to a subsequent endotoxin stimulus (lps ). in this study we examined the kinetics of lps inhibition of tnf and augmentation ofil- . methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or ng/ml of lps for intervals of to hours. culture medium was then replaced with , or ng/ml of lps for hrs. tnf and il- in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin- (il- ), interleukin-i (il-i) and prostaglandin e (pge ). we showed that preexposure to lps (lps ) alters the response of murine m~i to subsequent lps stimulation (lps ). we hypothesized that in vitro cytokine release by lps in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n= ), cultured in vitro hrs, then pretreated hr _+ lps -cultures were then stimulated with lps and mediators in mo supernatant measured: tnf, il-i, and il- by specific bioassays, pge by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps = ng/mh the table shows that all mediators were increased, in the absence of lps . pretreatment with lps resulted in complete inhibition of lps -triggered tnf release. in contrast, lps significantly increased mo secretion of il- , il- and pge (data not shown). serum cytokine levels were as follows: tnf _+ , il-i + , and il- . -+ . ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps -triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps . provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing - g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd , cdllb, icam- and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd , and icam- , but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi on activated neutrophils and icam- on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il- and il- as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps hrs following endotoxin infusion followed at hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p integrln (cdlla, b,c/cd ) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~ integrlns, but not cd , is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (> ~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng- p.g/ml) or f'mlp ( " to " m) et oc for mln, in ~; human ab serum, the expression of the ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase ( fold) in the expression of cd b on pmns from burned patients compared to an and fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = and total occlusion of blood flow to the lower extremities was maintained for g mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h production was determined by a previously described ' '-dichlorofluorescein flowcytametric assay. _+ amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < . vs. baseline; "p < . vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h production was increased by . -fold above baseline values by hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at rain. post-ischemia, however after release the ligature a significant reactive hyperemia was observed by mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from burned patients (mean age , +_ . years, mean burn size . % _+ . %) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at °c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and ,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ , s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of . u/ml (recommended by manufacturer is the range of - o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns ( . % of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin- (il- ) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il- and tgf- in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il- and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il- and tgf-i~ on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll- up to u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf- . in conclusion, these data suggest a contribution of il- in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at h followed by return of this parameter to normal levels at h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at h. this hi~her index persisted until the end of experiments ( days). the early ( - h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes ( ). there are reports of nentrophil dysfunction in inflammatory disorders of the skin ( ), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score ± ) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously ( ) . in parallel, granulocytes from healthy individuals were tested. two granulocyte functians were studied in vitro: . migration of the ceils in a boyden chamber through a filter matrix following stimulation with different receptor dependent stimuli (c a, intefleukin- , platelet-activating-factor, leukotrien b , fmlp). . release of glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of -glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure ( / ) and nonsurvivors ( / ), whereas / patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. : - , klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg , kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i ~g/ml) on granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load ( % of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in h of the recovery and cytosol proteolytic activity (ph . ; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was - -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by h of the recovery (the comparison was done with the sedentary rats). in h cytosol proteolytic activity decreased and then increased again by h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate ( - %) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il ,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s mdrome. the presence of interleukin (il)- in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il- in animal models can attenuate lung injury. collectively, evidence to date suggests that il- attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il- is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il- can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, .albumi n leak was quantitated by measuring the counts which crossed the monolayer, using a gamma counter. il- (lpg/ml) was incubated in the culture medium with .albumi n for hrs. the il- dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to .- addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt expression and primes pmn's for subsequent generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd expression and pmn priming via the [ adrenergic signal transduction pathway. methods: normal human pmns were primed with paf ( ng/ml for min) or pre-treated with - m of isoproterenol (i) or forskoklin (f) for rain and then primed with paf. cd expression was measured by flow cytometry (fig.l) and -generation in response to -rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig. holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf x on cytokine-and endotoxin induced expression of intercellular adhesion molecule (icam- ) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo -and lipopolysaccharide (lps)-induced icam- expression on human endothelioma celts (eahy ) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf x significantly reduced icam- expression induced by interferon-y (ifn-?) and interleukin- (il- ). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il- induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il- driven icam- expression on eahy cells as well as the il- induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody ( f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b (ltb , x " tool/l) or intravenous injection of interleuldn- (il- , - iu/kg b.w.) and lipopolysaccharide (lps, mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of mg/kg b.w., superoxid-dismutase (sod, . iu/kg b.w.) was able to decrease the ltb effect, and the immuumodulating drug leflunomide (hwa ) exerted inhibitory effects on il- -induced permeability at a dose of mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb or il- induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam- in neutrophil migration. blocking antibodies to human pecam- , in which the antibodies are crossreactive with rat pecam- , were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam- / integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs ( - kg) were subjected to % arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added ( , , , i.tg/ml) ex vivo. after hr incubation at - °c, %cd (+) pmns were determined with fitc-ib and flow cytometry from mean channel fluorescence histograms. ± # p< . vs baseline * p< . vs sham $p< . vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd expression was depressed for at ]east hr after trauma relative to sham but by hr, was enhanced, relative to sham, and because fentanyl anesthesia at hr had a greater effect on cd expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps ( ng/ml), tnf( ng/ml), and lps/tnf( ng/ ng/ml). cultures of the human ec tine(ecv- ) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for , , , and hrs. ec necrosis was assessed by a cr release cytotoxicity assay. pmn activation was assessed by cd lb receptor expression and respiratory burst activity hr _+ . -+ -+ . _+ _+ . _+ _+ . _+ . hr + . _ _+ . _+ _+ _+ " +_ +-- . " lghr - . _+ +_ - " o:fo , " ~ +- . * hr _+ . - -+ +_ * _+ _+ * _+ _+ " data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p< . vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received ml/ g bw normal saline. at hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, xi - m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at hr post-clp. after pre-contraction of blood vessels in the isolated gut with xl m ne, vascular responses to ach ( x m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, xl - m), were determined. total vascular resistance (tvr, mmhg/mi/min/ g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n= /group) in the aortic rings were: ach lxl i~s, st-in ~ ~ significantly at hr post-clp (i.e., increased *p(o vs. sham; n- per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin- on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. - g) shook was induced by fractionated withdrawl of arterial blood within rain and maintained for h (map at mm hg, cardiac output % of baseline). rats were adequately resuscitated with % of shed blood and twice the volume in ringer's solution additionally. following h of reperfusinn (map > mm hg, co > % of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n= ) received the il-lra as pretreatment beginning min prior to shock induction. in the group t (n= ) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of mg/kg and was followed by continuons administration of mg/kg/h. the control group c (n= ) received equal volumes in nac , %, the sham-operated group s (n= ) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: , % + , %; c: , % _+ , %) was significantly reduced by pretreatment or treatment with il-lra (p: , % -+ , %; p< . , t: , % -+ , %, p< . , anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: iam /s + um /s, p: llm /s + }am /s, t: ams/s -+ lam /s, s: am /s -+ am /s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il- . pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to retool/ asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc ( g/kg bw bolus, followed by . g/ kg-h infusion) on the endotoxin-induced ( . ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p< . at mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p< . at mmol/l asc) and reduced the adhesion dose-dependently (p< . at mmol/l asc); ec damage was also reduced (p< . at retool/ asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p< . ), decreased the protein clearance (p< . ) as well as the zymosan-induced rom production (p< . ), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi on neutrophils and lymphocytes. methods: from blood samples (n= ) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after , , , , and minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at min was observed in wbc-cultures (n/r: min / , min / , min / , min / , min / ). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to min followed by a decrease to normal values at min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl -expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c -delicient mice in a model of zymesan indt~ed mof. materials and methods. c -deficient b d /oid and c -sufficient b d /new mice were used in this study. on day all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of mg/g body weight. between day and , biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score= ). on day all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x ) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day - ) and a late phase (day - ). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c deficient mice (all p< . ). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was / ( %) in c deficient mice and / ( %) jn c sufficient mice (p= . ), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c bl/ mice received zymosan ( mg/g body weight) intraperitoneally. groups of animals were killed after , , , and h and subsequently at each day until day . plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin -ct, and - (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c were measured in plasma and culture fluid by means of a ria (detection limit . ng/ml). interleukin- (il~) levels were assayed using the b hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day , they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within h after zymosan injection and disappeared within h. from day onwards, tnf levels started to rise again. plasma il- behaved almost similarly in the acute phase, but in the mof phase plasma il- remained low. no circulating il- could be detected at any time point. macrophage lps-stimulated production of il-lcq il- ~ and tnf--c~ was suppressed immediately after zymosan injection. production of il- and tnf-~ was normalized within h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day did production of il-i~ reach control values. il- production was higher than control values from day onwards. il production was similar to that of ili-il the production of tnf-ct was strongly elevated between days and and again during days to . the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il- or il- . also, the ability of macrophages to produce excessive amounts of il- and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~ -acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with antisense dna's (as) complementary to the ' region of the il- gpl receptor. the antisense were '-agtttagggatgagg- ' (asl), '-atcttcatcttctgaat- ' (as ), '-aagtgaatgattaaaacact- ' (as ), '-aaacctttataggcg- ' (as ), and '-cgttctacaactgcaacgt- ' (as ). using hepg , the biological effects of these antisense complexes on the high affinity il- receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for h. underivatized asl was less effective and the complex, asgp-pll-as , had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il- (i units) asgp-pll-asi alone showed a dose dependent ( .i- . ~m) inhibition of ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem , israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l female merino-sheep were included (mean weight: kg). day : hemorrhagic shock (mean arterial pressure (map) mmhg for hrs.), closed femoral nailing (ao-technique), day - : bolusinjection of endotoxin (et) ( , ~tg/kgbw) und zymosan-activated plasma (zap) ( ml) every hrs., day - : observation. bronchoalveolar lavage (bal): day , , . the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm- ), pap -putm.art.pressure (mmhg), pap -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov ), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p< ) results: baseline day day day day heart: co , _+ , , _+ , , _+ , , _+ , * , _+ , * svr _+ + _+ +_ " +- " lung: pap , _- , , _+ , " , +- , " , + , " , +- , ' pap , + , , +- , , _+ , , +- , , +_ , * liver: bill , _+ , , _+ , ' , _+ , ' , _+ , " , _+ , " kidney:crcl , +_ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , _+ , , + , , + , histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il- and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg responses, t cell proliferation, and il- synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il- and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after - rain even without cyclooxygenase inhibition although pgi levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated patients undergoing major abdominal surgery as compared to ibuprofen ( rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time , , , , , , , rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of -keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of -k-pgf~,tp~, peaked minutes after m.t. ( _+ , ibu: _+ , to: +i ng/l) and declined monotonously over h ( +_ , ibu: _+ ng/ ). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after rain ( _+ , ibu: + , to: -+ /~u/ml); aldosteronc also presented a maximum after rain ( + , ibu: -+ , to: +- pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum h after m.t. ( +_ , ibu: -+ , to: +_ ~g/ ). adh pc peaked min after m.t. ( + , ibu: -+ , to: +_ pg/ml) and showed analogously to -k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. rats of them received sma clipping and others wee used as control. control and treated rats at , , , hours were perfused and fixed. the brain were sectioned at pm and stained by abc method using c-fos antibody. plasma endotoxin level of rats were measured at , , , , hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of , , , , hours after the treatment were . ± . , . _- - . , . _+ . , . ± . and . ± . pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi ). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed , , , , . tile plasma concentrations (pc) of -keto-pgft, (stable metabolite of pgi ) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < . placebo vs. [ibuprofen] [ ] mmhg (*p< . i). these changes were accompanied by a marked increase of -keto-pgf~ pc up to rain after mt in arterial and mixed venous blood of untreated patients with a peak of *[ ] ng/l tl (*p< . ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao while -keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for -keto-pgft~ was not detectable. a splanchnic vascular source for pgi release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e , ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day , two groups of n= week old aj mice were subjected to either a % scold burn (ti), or were untreated (c) n= . on day , mice (ti+clp) and mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days , and after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il- and adherent splenocytes (as) were cultured with lps for il- , tnf, il- and pge . results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi ) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. ( /ml) at rates increased incrementally from . to . mi/kg/hr over hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n= ); pga (n= ), graded bacteremta plus anti-pgiz antibody, ml/hr iv, beginning at hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po , mmhg, cardiac index (ci), l/min/m , oxygen delivery index (do i) and consumption index (vozi), ml/min/m , and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg -keto f alpha ; in the first instance~ peak values of lt ~ after i~ hrs post infarction were times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets , ~ev , ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical ( patients) and experimental material( rats and dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period ( - days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin ( microgram per kilogram of body mass intrap'eritoneally within days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [ ] . peep can affect rv function by reducing preload and ejection fraction (ef) [ ] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this -year-old patient (apache-ii: ) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [ ] . the therapeutic procedure included volume replacement, vasopressors (dopamine , dobutamine gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf- , od= mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id= ram, bennett a, pressure controlled mode, pm~x= mbar, peep= mbar, i:e=i:i, fio = . ). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [ ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new , dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [ ] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing mg% glucose & gm% bovine albumin and incubated in dubnoff shaking water bath for min at °c the results revealed that there was an enhancement in release of free fatty acids (ffa) ( %) and lactate ( %) and in glucose uptake ( %) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition ( ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by %. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a , but not with a direct vasodilator (nano ). this failure to relax to ach and a reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by weeks. exogenous no donors (e.g., c - or spm- ), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor ( p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at days. rat carotid artery rings relaxed only + % and + % to gm ach in vehicle treated rats and in inactive no donor treated rats respectively days following injury compared with + % in no donor rats (p< . ). relaxation to gm nan was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name ( mm) was markedly reduced by injury, but was protected by no donors (p< . ). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il- , ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin- and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde- -phosphate dehydrogenase (gapdh) and the cytochrome p (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to mmhg for hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, nm) was reduced by % compared to control sma segments (p< . ) while relaxation to sodium nitrite ( gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p< . l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p< . ), attenuated mesenteric mpo (p< . ) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm- (schwarz pharma), following regional myocardial ischemia ( hour) and reperfusion ( . hours) . treatment with spm- ( rim) significantly reduced myocardial necrosis by % (p< . ) compared to an no-deficient analog of spm- , spm- . furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p< . ) reduced following treatment with spm- compared to spm- ( . + . vs. . + . u/ mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps ( mg/kg, n= : mg/kg, n= ; or mg/kg, n= ) and treated with l-nma ( , , , , mg/kg/hr) for hours following a , , or mg/kg loading dose. animals were resuscitated with iv ringers solution ( ml/kg/hr). hemodynamic data were collected at , , , , , and hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p= . ), and systemic vascular resistance index (p= .ol) and decreased cardiac index (p= . ) and oxygen delivery index (p= . ). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p< . ), but this differential effect on the venous and pulmonary circulation occurred, > hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied ( , , , or mg/kg/h) in either the low ( mg/kg) or high dose ( mg/kg) lps-challenge groups. a nonsignificant (p> . ) trend toward a beneficial effect on survival ol low dose l-nma ( mg/kg/h) in animals given the mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study ( mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p< . ). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad . recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [ ]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no ( and ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad [ ] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation ( - ppm) consistently lowered the pap and augmented the pao /f.o for up to days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below . %. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [ ] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately - ppm, the improvement of oxygenation had a maximum at ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad , thus allowing reduction of the f.o . no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd- -phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il- , ifnj and lipopolysacchafide (lps). hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of , -diphosphoglycerate to glyceraldehyde- -phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by _+ %. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only _+ %. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il- , tnf, ifn, and lps for hours. , , and ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no ); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n= .) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont . + . + . # stim . + . + stim+ o tzm nmma . + . + . # stim+ ~m nmma . _..+ . * + . # stim+ pm nmma . + . * + . # stim+ )lm nmma . + . * + . # anova , . (* p < . versus stim, # p < . versus stim; anova with neuman-keuls) gsh in cont+ i~m l-nmma was equivalent to that of cont ( . vs. . ). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c bl/ mice and lew rats underwent a % and % ph, respectively. sc were prepared - days after ph and plated at to x ecells per well. after h incubation at °c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient days aider liver partial resection ( . nmol nitrite/ cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: ) encletoxin and l-name, ) endotoxin, ) naci and l-name, ) nach preliminary results from groups (n= ) and (n= ) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin ( . gg/kg/h) was given as a continous portal infusion over the entire observation period of hrs. l-name ( mg/kg) was given as a bolus after hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by %* and hepatic artery flow (hal e) by %*, while l-name caused a further and lasting reduction in flow (pvf %, haf %)*. transhepatic (portal-hepatic vein) vascular resistance increased to times baseline value during endotoxemia while l-name caused a further marked increase in resistance to times initial value. portal oxygen saturation (so ) decreased by %* during endotoxemia. l-name caused a reduction in portal so by %*. arterial so was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a % reduction in cardiac output (co). the addition of l-name reduced co by a total of %*. *: p < . . conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; mg kg -i s.c., at h prior to lps) or with an il-i receptor antagonist (il-ira; mg/kg bolus and . mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at - min after lps. for instance, endotoxaemia for min resulted in a fall in map from -+ (control) to -+ mmhg (p< . ; n= ). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at min when compared to lps-control: -+ mmeg (n= ) and -+ mmhg (n= ), respectively (p< . ). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, min, x - m). pretreatment of rats with tnfab or il-ira significantly (p< . ) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at min after lps there was an induction of calcium-independent nos activity in the lung ( . -+ . pmol citrulline/mg/min, n= ), which was attenuated by tnfab and !l-ira by -+ % and -+ %, respectively (n= ; p< . ). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with live e. coli, which consistently increased cardiac output - % above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name, mg/kg iv), prevented this increase and elevated map by - %. in the first groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are rains after e. cob. n= - /group. * p< . vs bl by remanova and § p< . vs e. coli alone by anova. ec+l-name -+ - _+ " § - _+ * § - _+ * § - + * - + * § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 'untranslated region (utr) of ferritin mrna and 'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j ) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k cells, where nos was stimulated by increasing the availability of the essential nos cofactor , , , -tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn- or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group , no, ppm, was added to the inhaled gas from the start of dialysis. in group no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group , from minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats ( - g, n= - /group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received rrd/ g bw normal saline. the animals were killed at , , or h post-clp ( h post-clp=early sepsis; - h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, to - m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, /~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at and h after clp. in contrast, l-nmma administration produces an % increase (p< . ) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a -years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of mg of l-nmma, a continuous infusion was installed ( . mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose ( to mmhg}, heart rate stayed stable ( + b/rain), systemic vascular resistance increased ( to dyne.sec/cm ), cardiac output decreased ( to . l/rain), and cardiac index declined ( . to . l/min/m }. before and after minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after matutes. neopterine increased from . to . ng/ml, tumour-necrosis-factor-a increased from . to . pg/ml and intedeukin- increased from . to . pg/ml. immunoglobulines a, g, and m ( . to . , . to . , . to . g/i), complement factor c- c and c- ( . to . , . to . g/i), alpha-l-antitrypsine ( . to . g/i), c-reactive-protein ( . to . rag/i), interleukin- ( pg/ml) and soluble interleukin- ( to units/ml) did not change significantly. pulmonary-shuntvolume decreased from . % to . % within one hour and to . % after minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse , zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. . microbiol.immuno : ) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc ,um, ifn-,/ u/ml, lps fig/m/, ngmonomethyl-l--arginine (mmla) ram. nitrite (no -) content of the supematants was determined by a standard griess reaction, and h release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x- , and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with . ml of a suspension of . ~" s.typhimurium, strain c , and injected with aminoguanidine (ag) mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from % in the control group to % in the treated group, days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom % to %, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no -accumulation, . nmole/well v.s. nmole/well. conversely h production is enhanced from nmole/well up to , nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, % in iron exposed macrophages v.s, % in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity ( % v.s. %) whereas the addition of afc to these macrophagas restores an elevated ( %) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit de microbiologie. bp . la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at °in tcm- / % fcs, with or without additional rat serum. after h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of u cobra venom factor/kg b.w. at days and . significantly higher (p<o.o ) lps-stimulated no-generation was found in spleen cells prepared at day ( . - . nmol nitrite/ cells, n= ) compared to spleen cells from non-treated animals ( . ± . nmol, n= ) complement activation was effective also in vitro. when incubated in % zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli ( qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l bioassay. survival rate (%) qrql/,p n hr hr dav control (normal saline . ml/body i.p.) n (l-name mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to min of partial liver ischemia ( %) and min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i mg/kg). sod, when used, was given as mn-containing preparation at a dose of u. i.v. i min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : . ± . "#/ . ± . / . ± . . alt (u/ ) • ± ~#/ ± / ± . gldh (u/l): . ± . e#/ . ± . #/ . ± . . in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous ~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [ ] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [ ] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no -chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of . - . parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non . . . . , parts per million (ppm) no, mean, n = ; ~ p < . (t-test) between smokers and non-smokers; p < . (t-test) between in-and expiration. about % of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than % {o. ppm before intubation vs. . ppm after intubation, ~, < . ), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [ ] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi , yoshihiro inoue , masao yoshida critical care and emergency center, and department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il- contribute to the occurrence of septic shock (circ shock : ; . the present study investigated the in vitro effects of lps, tnf-o~, il- and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il- , tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il- and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il- and tnf-c( enhanced lps-facilitated no production to a greater extent than il- or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. - uchimaru, morioka . japan. t. yolk , , i. ioannidis , w.j. kox and h. de groot objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: -morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin- ( mm), which releases both no. and o -., caused a time-dependent cytoreduction of % and % after and hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes --to form h and . in contrast, the addition of catalase (cat), which removes h to form h and , diminished the cytotoxic effect ot sin- to %, equivalent to high concentrations of snp ( mm), which solely releases no.. in addition, the amount of h formed by mm sin- equaled the amount ot enzymaticany produced h by mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration ( mm) alone were not toxic to endothelial cells, the combination caused an % reduction of viability, comparable to the effect observed with sin- ( mm) alone. moreover, toxicity mediated by high concentrations of snp ( mm and ram) was reduced by % under hypoxic conditions indicating synergism with no-and . conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with -. however, toxicity increases dramatically in the presence of h . this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no and no levels were measul*d sliectrophotometricallymonocyte cultures were done for itrs with or without lps+ifn-t stimulation mid no and no levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. )both no levels in the supematmlts of monocytes cultured with lps+ifn-t mid no , no levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. )based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [ u/ml]. after hours of incubation no-release was determined as nitrite (no ) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d , that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no -production (p<o, ). although we observed no toxic effect on cell viability, no -production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo nm] released % less no in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [ }jm] and bim [ioijm] reduced the no -formation by % and %, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d cells pretreated with d [ ~g/mt] released % less no than stimulated controls the inhibitory effect on no production decreased the later the inhibitore were added after stimulation; e.g. bim added and hours after stimulation reduced no -production only by % and %, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr , erlangen interleukin- (il- ) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il- ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the ' utr of the human il- ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable ( %) gc rich region. the translation of the type i il- r appears under a significant suppression and may limit the biological activities of il- by low level of expression. on the other hand, the type ii il- r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il- activity; inhibition of il- converting enzyme which cleaves pro-il-l[ , anti-il- ri, the il-i receptor antagonist (il- ra) and soluble (extracellular) il- ri and il- rii. il- ra is structurally related to il- and binds at °c to the il- ri with near equal affinity as that of bona fide il-i; however, il- ra does not transduce a signal. il- ra has been given to humans in pharmacologic levels (mean plasma levels of gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by % and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il- ra are in -fold molar excess to those of il-i [ . it is unclear whether these endogenous levels of il- ra are sufficient to block il- activity in disease. a single injection of ]l- or ifna in humans does not induce il- but rather high levels of il- ra ( ng/ml). prof.dr.l.a. aarden interleukin (il ) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il -anti-il complexes, suggesting that plasma il measurements represent a gross underestimation of il production in the patient. until now no natural antagonists of il have been described. the biological activities of il results from binding to a lowaffinity il -receptor (il r). the il /il r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp . studies on the structure-function relation of il revealed that disruption of the gpl -interaction site on il gives rise to a mutant il that, by virtue of its intact binding to the il r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin- (il- ), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il- and the ability of il- to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il- during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il- was found to be a potent angiogenic factor at concentrations ranging from - ng/ml. sequential fluorescein angiograms demonstrated that il- induced neovascularization by days, which regressed by weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il- . in addition, an il- antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il- has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th , thl and th cd ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il- are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il- also directly inhibits t-cell proli/eration induced by cross]inked anti-cd or anti-cd mabs (e.g. in the absence of professional apc), by specifically inhibiting il- gene transcription and il- protein production. il- also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il- , and tnf-a and the chemokines il- and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il- enhances the production of the il- receptor antagonist, a cytokine with anti-inflammatory activity. il-l produced by monocytes downregulates class ii mhc expression and il- production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il- is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il- also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il ) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd and cd memory t cells. ip- chemoattracts only preactivated (not resting) cd or cd t cells. hip-l~ preferentially chemoattracts gd t calla, while hip- more readily attracts the cd t call subset. the chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla- . these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln ', peaked by hr and was completed in hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin- (il- ) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of kd and kd. il- was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il- . phagocytic cells produce il- in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il- exert biological activity are t and nk cells. the major direct biological function of il- on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il- , produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il- to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il- , other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il- has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type (th-i) response and suppressing a th- response. the presence of il- during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il- , and inhibits the generation of th- cells producing il- . il- induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il- , however, il- needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il- -mediated inhibition of il- producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il- for thl cells and an inhibitory effect of ifn-y on th cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il- on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il- appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. , interleukin- and its role in the generation of t e cells, imm~ol. on peripheral blood monocyte/macrophages, il- behaves like il- in a variety of assays, and shows both similar and contrasting, activties with either il- or ifn-y. il- reduces inflammatory monokine and il- production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, , febs lett. , ) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi expression. il- produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il- . it inhibits niv-i production (montaner et el, , i. exp. mad. , ) , and interferes with hiv-induced cell fusion. the activities of il- and il- differ on t-ly•ocyte and large granular lymphocyte population. in particular il- does not exhibit the suppressive effects of il- on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il- and il-l share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. , embo j. . - ), explaining both thai common and divergent activities. the levels of il- ~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il- mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t cytotoxic lymphocytes express markedly higher levels of il- ~rna. in vivo, il- may thus be contributing, significantly to some of the activites previously ascribed to il- il- . recently, we cloned the human cdna encoding interleukin- (il- ). human il- is a non-glycosylated protein of aa with a molecular mass (mr) of kd and has biological activities on monocytes and b cells, il- , like il- , strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd (fc~rii). furthermore, il- , like il- and il~ , inhibited the production of pro-inflammatory cytokines il- , ]l- , tnfo~ and chemokines miplc~ and tl- by lps activated monocytes. both il- and il- enhanced the production of il-lra by monocytes. in contrast, il- lacked the t cell stimulating activities of il- . the responses of monocytes to il- and il- were not additive or synergistic, supporting the hypothesis that il- and il- receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il- , il~ and il- have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il- is unique in that it can modify inflammatory reactions without stimulating (as does il- ) or inhibiting (as does il- ) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of amino acids that is inactive until processed to a disulfide linked homodimeric molecule of t amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf- has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf- isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[ family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il- , pge and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il- and il- , have been characterized as critical in regulation of mo tnfa and il- , as well as in induction of t lymphocyte proliferation and ifn production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score > ) were analyzed for their il- levels, their in vitro proliferation to mitogen (pha) and their response after il- addition. since il- produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il- levels. increased patient il- production might also be resulting from the high levels of tnfa a known stimulator of il- . conversely, since il- augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il- deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il- found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il- production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il- were explored. increases in tgf[ may have partially contributed to the patients' depressed il- level, but elevated pge had no effect. addition of il- to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il- , pge ) and a dearth of other monokines (il- , il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells ( mmc) may result in the production of different cytokines including the interleukins (il) , , , and as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il- , l- , tnf-c as well as various mast cell growth factors (il- , il- , il- , il- , ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl bmmc/ml; ll.um ionomycin; - h). the actixdties of bmmc supernatants were assessed in specific biological (il- , il- il- , l- ) and/or elisa assays (il- , il- ). here we show that homogeneous populations of bmmc (> %alcian blue+/safranln-; in vitro age: weeks) generated in the presence of recombinant (r) rail- from normal balb/c mice produced modest amounts of l- and low or undetectable levels of il- , - , and - after induction with lp.m ionomycin only. however, a dramatic increase ( -to -fold) of these cytokine activities was noted, when in addition to ionomycin also human ( ) rll-la was provided during the induction period. this il- effect was dose dependent with a maximgm at - u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with ng/ml hrll- receptor antagonist abolished the action of u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll- or rmtnf-~. both mrll- and hrll- substantially enhanced ionomycin-induced l- production of bmmc in the absence or presence of il- . il- significantly enhanced il- and il- production while decreasing il- activities to abont - % of control levels, when il-i was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t antibody (ascites : ) abrogated the effects of mrll- . other mast cell-active cy~okines (] ,- , il- , l- , ngf, or kl) added to ionomycia-or l- /ionomycin-treated bmmc had no major effects on cytokine production. il- and il-i did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of " m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il- /ll- induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il- inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il- previously recognized as a product of th type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il- , and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of different httman cytokines, ilia, ill [ , illra, il , il , il , il , il , ils, ill , gm-csf, tnfa, ifn and tgf[ . , was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with % paraformaldehyde and permeabilized by . % saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[ _ , could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il- in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n= ) were incubated at °c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after , , , , and minutes plasma levels of tnf-alpha, il-i beta and il- were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from , pg/ml at min to pg/ml, pg/ml, pg/ml after , and min. il-ibeta ( , pg/ml, , pg/ml, , pg/ml, pg/ml and pg/ml after , , , and min) and il- ( , pg/ml, l,lpg/ml, , pg/ml, pg/ml and , pg/ml after , , , and min) plasma levels were increased min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il- to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr. , ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf- ) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf- ( . - . ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf- (> . ng/ml) potentiate production of the immunedepressive prostaglandin e . other investigators have shown that tgf-] can cause the appearance of cd (fc immunoglobulin receptor) on monocytes exposed to ng/ml of tgf-[~i for hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd is depressed in septic patients and therefore we hypothesized that tgf- could account for the down-regulation of cd observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[ for and hours at °c and examined ceils for cd and cd expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at °c alone. one displaying high density of cd had increased fluorescence over the homogeneous expression of cd in cells maintained at °c (baseline). the other population displayed decreased cd expression relative to the baseline cells. tgf-i~i ( - ng/ml) caused a shift of ceils from the high density into the low density cd population. this trend was observed within hours of incubation and was complete by hours. we observed a net decrease in cd expression f % for all subjects studied (p< . vs controls). phorbol myristate acetate ( ng/ml) also caused down-regulation of cd to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd after incubation with tgf- ( ng/ml) for hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd by tgf- deplete their surface expression of cd while generating cd . this down-regulation of cd by tgf- correlates with our clinical observations of lower cd expression on monocytes obtained from septic patients. for over years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw . and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw . cells, mif secretion was induced by as little as pg/ml of lps (e.coli l:b ), peaked at ng/ml, but was not detectable at lps concentrations > txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn . production of mif by lps-stimulated (l ng/ml) macrophages peaked at hr. expression ofmif mrna and tnf mrna by lps-stimulated raw . macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations ( pg/ml to p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately -fold after lps induction ( pg/ml). mif secretion also was induced by tnfoc ( ng/ml) and ifn? ( iu/ml), but not by il- and il- (up to ng/ml). lps and ifn had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif ( gg/ml) were found to secrete bioactive tnf~ (> pg/ml by l cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il- , il- , and tgf , have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il- , il- , il- , tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il- by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at °c for h in the absence or presence of lipopolysaccharide (lps) or tnfa. il- release was measured in the supernatants using a specific elisa. among tested cytokines, il- was the most efficient inhibitor of il- production by lps-activated pmn. il- was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il- production. northern analysis are under investigation to precise the level of the il- -and il- -induced inhibition of il- production by pmn. our data illustrate that il- and il- possess the capacity to down regulate the production of il- by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il- superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. female, wistar ( - gm) rats received either or pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with mg/kg ibuprofen lp prior to pg/kg bw cntf. rats treated with ,ug/kg bw cntf lost . _+ . gms bw as compared to freely-fed controls which gained . _+ . gms (p<o. by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (- . _+ . ). animals pair fed to the high dose cntf gained body weight ( . -+ . ), although weight gain was less than seen in freely-fed controls. rats treated with pg/kg bw had reduced body weight gain (+ . +_ . gin). food intake was also reduced by % in pg/kg cntf (from . +_ . gm/day to . _+ . gm/day; p<o. ) and was also unaffected by prior ibuprofen treatment ( . +- . gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight ( . + , gms vs. . _+ . gins), total protein ( . +- . gms/l vs . +- . gins/l) and albumin ( . + . gms/l vs. . +_ . gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il- and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box , gainesville, fl. . of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il- , have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il- are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp signal transd,uction protein, including il- and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il- and cntf also induced hepatic acute-phase proteins. thus il- and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il- -and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of to pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound ) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound ) or the phosphono-oxyethyl analogue pe- , structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd ÷-as welt as cd ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between / to / cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il- and ifnf, but not for il- or il- (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd ÷ as well as cd + t cells express cd , the high affinity il- receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il- receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t, cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)- when stimulated with calcium ionophore (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was re/eased into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il- in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il- concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il- in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by - % within days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. versus kin/day). they developed elevated plasma corticosterone levels ( _+ gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il- a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il- and tnf activity in plasma was not affected. after administration of .tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il- activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin (il- ). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il- ) and those of their specific antagonists interleukin- receptor antagonist (il- ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from patients with acute myocardial infarction at presentation in the coronary unit, every three hours for hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il- and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within hr whereas stnfr and l-lra peaked at - hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin- (il- ) is a multifunctionai cytokine known to be involved in important homeostatic processes. il- levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il- binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il- receptor (sll- r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil- r and il- are measured using elisa's. both elisa's are tested for interference of sil- r mid il- . the effect of sll- r in the il- bio-assay (b ) is tested. results: baseline levels are (mean + sd): . -+ . ng/ml in serum, . -+ . ng/ml in urine, . _+ . ng/ml in csf and . + . ng/ml in sf. there is no interference of sll- r in the il- elisa or bio-assay and no interference of l- in the sil- r elisa. conclusions: in all investigated biological fluids sll- r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin (il ) and to turnout necrosis factor ~x (tnfc . methods. bum patients (n= , -> % total body surface area) were studied weekly up to days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il ~-and tnfcz-binding t(cd ) lymphocytes was assessed by flow cytometry analysis. levels of il receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p< . ) increased up to weeks post-bum (means . + in non-surviving and . + . u/ml in surviving patients vs < u/ml in the control). within weeks of bum, the percentage oft ceils expressing receptors for tnfa and il [~ constitutively was elevated (by - fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range . - x j pg/ml vs < . x j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range - x - pg/ml vs - x j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl ~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of l lra may affect il -regulated biological responses including specific immune reactions. while studies suggest that il- is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il- contributes to depressed t-cell responses seen following hem; and ) how other agents, known to play a role in hem, effect il- release. to study this, c h/hen mice were bled to and maintained at a map of mmhg for h and then adequately resuscitated. mice were killed h post-hem to obtain splenic t-cells (nylon-wool purified). il- 's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il- to markedly improve the t-cell proliferative response [ . #g the marked increase in capacity of t-cells from hem mice to produce il- was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il- are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il- release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il- down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il- has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n= ) were randomized to groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received % etoh or ns daily for days by gavage. a % full thickness bum was induced hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed days post bum. splenic lymphocytes were cultured for days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma . ug/ml, ug/ml). splenocyte production of il- , interferon-gamma, il- , pge were measured, and lymphocyte proliferative response examined. results: il- production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of .- in a dose-dependent manner, indicating nitric oxide may modulate il- and interferon-gamma production in thermal injury. il- production is normal in etoh-burn animals. conclusion: il- and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il- production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin- (i - ), interleukin- (i - ), the interleukin- receptor antagonist (i - ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn , , , , , , and hours following operation. a portion of the results are shown (mean -+ sem). + -+ _+ one and two factor anova; *p< . , #p< . , §p< . , ¶p< . , for differences between groups i - was not detected at any time point. both ii-ira and i - increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for hours; the b-ira increase was a thousand fold greater than the rise in i - levels. i - levels increased up to hours after surgery. crp levels reflected maximum ii-ira and i - levels (r =. , p< . and r =. , p< . respectively). high ii- ra and i - levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i - ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin , ireland. change of il- and soluble il- receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il- plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il- receptor (sll- r) is considered to be agonistic to il- , unlike other soluble type receptors of cytokines. here we measured il- and sll- r levels in the serum and drain fluid from surgical field in order to investigate the changes of il- and sll- r after surgery and their origins. [materials and methods] serum and drain fluid samples from cases ( of esophagectomy and of gastrectomy ) were serially collected before and after surgery. il- and sll- r levels were measured by elisa. [results] ( ) serum il- : all cases reached the maximum level on pod-l, more precisely - hours after operation. ( ) il- in the drain : maximal il- levels in the drain were recognized - hours after operation, at almost the same time as serum il- . furthermore the il- values in the drain were much higher, about times, than those in serum. ( ) sll- r in the serum : all cases reached minimum levels - hours after operation and recovered to the preoperative levels a few days later (decrease ratio : . + . ~,, range : - ~'). ( ) sll- r in the drain : sll- r levels in the drain showed almost the same value and change as serum sll- r. [conclusions] ( ) il- is produced from the cells gathering around operative fields whereas sll- r is considered to be produced in the cells which do not gather around the operative fields. ( ) there may be a mechanism that down-regulates sll- r in the early stage of surgery. [objectives] il- plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il- concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il- . [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il- was measured by elisa and il- mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] ( ) serum il- levels reached the maximum concentration on the st postoperative day in all patients. ( ) the il- peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r= . , p< . , r= . , p< . , respectively). ( ) the peak concentration of serum il- in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p< . ), despite a similar degree of surgical trauma. ( ) peak l- concentration observed in a patient who underwent esophagectomy was about fold greater in the drain fluid of thorax than in the peripheral blood. ( ) il- mrna was demonstrated in leukocytes from thoracic and abdominal exudate at , and hours after surgery. in contrast, il- mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il- is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over years old show a different serum level cytokine pattern than pts under after a standard surgical procedure considered as a "medium strength trauma". patients and methods: pts( females males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a: pts under years(mean age: . +- )gr.b: pts over years(mean age: . _+ ). all pts underwent cholecystectomy and cholangiography. pts in gr.a and pts in gr. b underwent common duct exploration. spbintercctomy was performed in each group. on the day of surgery (pre) and on the st and th postoperative day(leo, po) : percentages of cd , cd , cd , cd and cd cells we measured by means of flow cytometry using moab. and levels of il- , il- , il- and tnf "in vivo" by elisa using moab. results: ere: cd % was . _+ in gr.a and . objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin (il- ) levels to determine a role of g-csf and il- in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il- levels were measured before and after surgery in patients with esophageal cancer (ec), and patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p< . ) and peripheral pmn chemiluminescence (p< . ) of patients with ec were significantly decreased compared to those of patients with gc at and hours after surgery. serum g-csf levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at and hours after surgery. serum il- levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at , and hours after surgery. significant inverse correlations (p< . l) between peripheral pmn count and serum g-csf and il- levels were seen at hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il- , may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin- (il- ) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system ( , , ). in our study we monitored il- and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il- and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) < and ct abnormatities or gcs < over hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over hours csf and serum. il- and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described ( ) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il- and il- antibody. astrocyte migration was tested in a chemstaxis chamber. results head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il- were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il- could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il- antibodies, purified il- exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il- was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o il- on astrocyte functions, il- promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il- stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il- represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of - mmhg for pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. animals received a bolus dose ( mg/kg) of tnf mab (celltech, berkshire, uk) at min after resuscitation (tn ). the control group (n = ) was treated similar to the tn group but received ringer's lactate (con). results: at min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph ( . + . ), hco -( . ___ . mm), and base excess (- . + . ram) values with pco ( . + . mmhg) and po ( . + . mmhg) in the tn with no difference to the con group. immediately after resuscitation ( min) plasma endotoxin levels were found to be increased in both groups ( . + . in tn vs . _ . pg/ml in con group) . prior to the treatment with tnf mab ( min) there was also no difference between plasma tnf levels of the two groups ( . + . in tn vs + . pg/ml in con group). treatment with the tnf mab at rain post-hs improved the hour survival rate to . % as compared to . % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy animals in the tn vs in the con group. no bleeding in the kidneys was found in the tn but in rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = ) was prevented in animals which died in the tn (n = ) group (( . +_ . vs . +_ . g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. -+ n=ll*$ -+ n= _+ n= * * p< . vs baseline :~p< . no anesthesia vs anesthesia thus ) tnf production increased - fold by - hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; ) anticd had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd does not involve tnf suppression; ) fentanyl anesthesia at hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from to yeas of age. the percentages of bum ranged from % - %. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of pgjml - pg/ml. peaks in the tnfa values were above pg/ml and were also associated with a peak in the stnfr levels. these levels began at < , pghnl within the in,st ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl there is a significant corresponding peak in il-trn (> ~ /ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin- beta l-ii ), intefleukin- (il- ) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the hours. patients' ages ranged from to years. iss varied from to and transfusion requirement from to units. five patients died after the study period. ] ,- was raised in / patients (max level , pg/ml) but was unrelated to condition or outcome. / showed a rise in il- b (max level pg/ml) which was negatively correlated to iss (i=- . , p< . ). tnfa was raised in / (max level pg/ml). peak tnfc~ was positively correlated with iss ( = . , p< . ) and haemorrhage (i= . but p< . ). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak ,- and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [ ] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [ ] . radioligand binding studies with ~ odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc ) for hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./( + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o o (mm h¢i): _k, (nm}: n (molecules/cell): - . ± . _+ - . ± . + - , ± . -+ - . + . -+ presented are calculated values on the idealized curve + % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [ ] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il- and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c, see, ° v bsa, ld ) and ( c, see, ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il- , - hr following injury and of try activity hr after scald, followed by elevated levels of il-i and il- at hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and postburn. il- increase peaked - hr after scalding and levels remained elevated - days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il- activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska , belgrade~yu. asadullah k ( ), woiciechowsky c ( ), liebenthai c ( ), doecke wd ( ), volk hd ( ), vogel s ( ), v. baehr r ( ); depts. of med. immunology ( ) and neurosurgery ( ) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l patients undergoing neurosurgery from the first preoperative day until at least day after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)- levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il- ( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il- and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit ( - ml/kg) hypotension phase (mean arterial pressure = map of - mmhg for - hours followed by adequate resuscitation). rats subjected to hs (map of - mmhg for rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin ( ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin ( ng/ml) for hrs at °c. results: at min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group ( _+ vs + pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats ( + post-hs vs + ng tnf/ml pre-hs) and baboons ( ± post-hs vs ± pg tnf/ml pre-hs). in contrast, the il- productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs ( ± pre-vs ±_ pg il- /ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., , new biol. : ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin- (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et- /et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et- on the release of histamine, serotonin, and leukotriene c (ltc ) by primary mouse bmmc (in vitro age: weeks) caltured with different recombinant mttrine cytokines (interleukin (il- ) and/or kit ligand (kl) in the presence or absence of il ) for two weeks prior to activation. et- ( x - to lxl - m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to % and % of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il- and il- . only when cultured in the additional presence of il- rather than in medium containing kl or kl plus il- alone, bmmc released histamine and serotoaln upon challenge with et- .furthermore, et- stimulated ltc biosynthesis up to -fold in bmmc cultured in the presence of il- . in contrast, the peptide was without major effect on ltc biosynthesis in bmmc culturd without il- . the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il- prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l- . et- ( " m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of - m. crude peritoneal cells (containing - % serosal mast cells) obtained from normal balb/c-mice released % histamine upon challenge with et- ( - m; rain). taken together, these resu/ts demonstrate that et- can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc production in mast cells. il- appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a (type ii pla ) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea , endotoxin, tnf-c~, il- and il- were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla and tnf-a in these patients were . ± . ng/ml and . ± . pg/ml, respectively, the two parameters being significantly correlated (r = . , p = . ). there were also a significant correlation between the level of type ii pla an il- (r = . , p = . ). there was no significant correlation between the level of type ii pla and il- . these in vivo findings suggest that tnf-a may be involved in the production of type ii pla . a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)- down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. , j. immunol. : ) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. , cancer res. : ) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. , embo j : ) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il- , tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il- the additional presence of rmull- for or days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack- ) nor kl-mediated proliferation or chemotaxis. in contrast, il- syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes . pre-incubation of bmmc with rhutgfl~ ( . , . , or . ng/ml) for h had no effect on kit receptor expression, although tgffil at concentrations of . - . ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il- , i,- , il- , and/or il- ). moreover, pre-incuhation ( h) of bmmc with tgf-gl (> pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il- -or kl-induced chemetaxis. when bmmc were preancuhated with rmukl ( ng/ml) for , . or days, a transient down-modulation of kit receptors with a maximum effect on day was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il- nor tgfi affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi cells from tnf-mediated cytolysis. material and methods: wehi subclone ceils ( x ), that are highly sensitive to the cytolytic activity of tnf, were grown on -well culture plates in rpm medium. after hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn ( - bp) were added ( . , and pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at nm. results: wehi ceils incubated with tnf ( - ng/ml) were completely lysed after hours ( % abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with . ng/ml tnf and jm odn showed still % of the absorbance observed in control ceils without tnf ( % abs). the protective effect of odn started at . pm, reached a maximum at ,um, and diminished at jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e , d- erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il- ), in the sepsis model of lipopolysaccharide (lps, mg/kg i.p.) induced hepatitis in galactosamine (gain, rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at hr and hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, mg/kg i.p.), rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il- levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, x mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, x unit/mouse i.v) r spectively. pretreatment with al and sod ( and hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il- into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee b'~ th~ recombinant mmlse tnf-o' ( . ~/rno~e j.p.) ~d oi~lps ~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il- release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients ( + years) in severe sepsis (sepsis score > according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma ( + years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. pl of whole blood were incubated either with / of a standard medium or with pl of a standard medium and pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours plasma concentrations of tnf-a, il-la, il- and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if- ,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients ( males, females, mean age +_ ) with severe sepsis (sepsis score > , elebute and stoner} were drawn on day , , , , and of disease. blood was immediately placed on ice and processed within hour. pl of whole blood were incubated with pl rpmi-medium supplemented with antibiotics and l-glutamlne or with pl of rpmi-medium and pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours samples were centrifuged and plasma was harvested and stored at - ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < , ) for circulating mean arterial tnf-a concentration ( pg/ml _+ sem} and central venous tnf-a ( pg/ml +_ sem). upon in vitro stimulation there was also a significant difference (p < , ) between released arterial tnf-~' { pg/ml _+ sem) and venous tnf-a ( pg/ml +_ semi. conclusions: these results are difficult to interprete but could reflect the influence of pao and sao on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il- ), tumor necrosis factor-a (tnf-c and interleukin- (il- ) the multifunctional cytokine intedeukin- (il- ) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il- -concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il- may be a potential substrate of these preteases. the serine preteases elastase (ec . . . ) and cathepsin g (ec . . . ) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin- (il- ) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il- leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin- . furthermore the loss of the biological activity of both, recombinant and natural human il- , was demonstrated by determination of the capacity of protease-treated il- to stimulate hybddoma growth ( td bioassay). these data suggest a possible downregulation of pathologically elevated il- levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il- , il- , _- -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il- , _- and il- were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to days, for a total of four tests performed (to, tl, t , t ). i .- was not detected in any samples. the serological profiles of the two cytokines .- and _- were similar; augmented levels were found at study entry and throughout the observation period, peaking at t and decreasing at t . however, in patients with sepsis, il- and _- concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il- and il- release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il- and il- production appears more significantly elevated, suggesting a role of il- and _- in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc and interleukin (il- ) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after hours and every hour. tnfct and il- levels were measured using the bioassays with cell lines wehi- ci and td , respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il- was found in all the patients' plasma samples and five patients' ( . %) ultrafiltrate samples. the il- blood level ranged from . to . u/ml (mean . , sd . ). the il- level in positive ultrafiltrate samples ranged from . to . u/ml (mean . , sd . ). conclusions: our preliminary results suggest that il- is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il- and il- and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd- mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first h with a peak h after clp at a significantly lower level than after lps. il- was measurable in serum only after h, significantly increased in spleen and liver after and h and in mesenteric lymphonodes from to h after clp compared with shammice. il- was significantly increased in serum throughout the first h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= ld ) to the sublethal (= / ld ) of three different lps (e.coli oiii:b and :b ; p.aeruginosa r ) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps :b induced as much tnf~ as the sublethal dose of lps :b ; furthermore, lps r , whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r ) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to h post lps administration moreover, the treatment with mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. ) circulating and organ levels of if are proportional to the dose and degree of toxicity of all the administered lps even if lps r was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if . to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if production. together with the total ablation of if , the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin- (il- ) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd- mice were randomized to mg/kg e.coli :b lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at , ', lh, . h, h, h, h, and h ( mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit pg/ml) and expressed as ng/g weight + sem (lowest detectable amount ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [ ~- p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present ( . + . ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced -fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was . + . ng/gwt at lh, reached maximal levels at . h ( . -+ . ng/gw-i) and returned to baseline at h. saline controls maintained their constitutive il-i~ levels. sb had fold more il- ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r = . ). separate mice were pretreated with saline i.p. orthe il- receptor antagonist (irap, mg/kg bolus i.p.) and were challenged rain later with . mg/kg lps i.p. or saline i.p. specific blockade of il- by irap decreased intestinal secretion at h and h post-lps challenge (p<_. . , student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content ( . % of total pituitary protein) and peak serum mif levels ( - ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps ( mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone ( % vs. %, p = . ). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps ( . mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from % to % (p = . ). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by . _+ . % in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin- (il- ) antibodies and il- receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il- in the serum does not correlate well with outcome. we hypothesized that this may be because il- acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il- levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n= in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. , j. inununol. : ) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after and hours of lps injection ( btg/ . ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels iron after injection amounting to only to % of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in % mortality in w/w v versus % mortality in +/+ mice . days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin- (il- ) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli :b , gg ip) into balb/c mice induces the rapid release of circulating il- ( ± u/ml at min). blocking endogenous il- using monocional antibody (jes - a , mg, h before lps) resulted in a massive increase in tnf production ( ± in lps+anti-il- treated mice vs ± ng/ml in lps alone, p< . , n= to mice per group) and an enhanced lps-induccd lethality ( % vs % in anti-il- +lps or lps alone respectively, p= . , n= mice per group). irrelevant igg rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il- during human septicemia. plasma samples were obtained from patients with gramnegative (gns, n= ) or gram-positive septicemia (gps, n= ) and from healthy volunteers. among these patients, suffered from septic shock at the time of sampling. il- levels were measured by elisa (detection limit: i pghrd). we found that patients ( %) had increased il- plasma levels (range to pg/nd). patients with gps had il- levels similar to the ones observed in gns (median: vs . pg/m, respectively). patients with septic shock had higher il- values (median: pg/ml) than septicemic patients without shock ( pg/ml, p= . ). no il- was detected in plasma from healthy volunteers. we conclude that il- is produced daring human septicemia. our experimental data suggest that il- might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, route de lennik, brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache i and sss). patients and melhods: septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i ( kda) were measured by enzyme immuneassays (norms values = + pg/ml and . _+ a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were + pg/ml and . + . ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p< . ). mean tnf concentrations for each patient were significantly greater in non survivors ( + vs _+ pg/ml p< . ); stnfr-i levels also were greater in this group, but the difference was not statistically significant ( . + . vs . _+ . ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = . p< . ). mean tnf levels were significantly correlated with apache ii (r = . p< . ) and sss (r = . p<o. ); stnfr-i levels were likewise correlated with both scores (r = . p< . and r = . p< . respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il- ). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of pg/ml and ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in and out of the patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il- . multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il- . peak plasma levels of both lif and il- correlated inversely with patients survival duration. cox's analysis for plasma levels of lif > pg/ml yelded a hazard ratio of [exp ( . )= . ]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human kda tnf receptor (stnfri) and the kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: points). serial blood samples were obtained within h after diagnosis of sirs, every hrs for the first hrs and every hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o ) compared to healthy controls (normal value: tnfrh , ng/ml; tnfrii: , ng/ml). the serum concentration of tnfri ( , ng/ml) as well as tnfrii ( , ng/ml) were significantly higher in nonsurvivors (n= ) compared to survivors ( , and , ng/ml; n= ) at the onset of sirs and during the course of the inflammation response (p< , ). a positive correlation exists between both receptors (r- , ; p < , ). increasing levels and maximal values of stnfri ( ng/ml) and i ( ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in out of patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. - is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first hours after administration of il- . in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il- and il- using immuno assays. in these plasma samples we also measured phospholipase a activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il- therapy the cytokines tnf, il- , and il- are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il- . objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin- (il- ) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il- by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of healthy volunteers and cultured at a concentration of . • ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after hours. using elisa, tnf-c~, il-ib and il- concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il- ] and il- by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il- in ng/ml as mean _+ standard error. ca/ml . . ( ) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity ( , x - m) as the fab-fragment ( , x - m) but bind the antigen with a eightfold lower avidity compared to the parental mab ( , x - ). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl- and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l , the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a -fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies ( ) reveales that the glucocorticoid antagonist ru , nufepristone, sensitizes both endotoxin responder and nonresponder, c h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and hrs following lps injection, ru significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru . however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru treatment. these studies suggest that ru aggravates lps lethality via factors other than tnf in endotoxin non-responder, c h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi response in - % of the pbmc after - hours of cell stimulation. in contrast, il and tnfc~ containing cells was only detected in - % of the pbmc. the induction ofil , il , il , and ill , was weak or completely absent. thus, the response indicates activation of th cells. however, illc~, illl , illra and il , were consistently found and gradually produced, peaking at hems in approximately - % of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of rag , , -trinitrobenzenesulphunic acid in . ml % ethanol (n= ). control animals received an isovohlmetric instillation of normal saline (n= ). faecal pellets were collected before induction of colitis (day ) and throughout the experiment. after days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day -#p= alld the total ftnf measured during the study correlatcs with the cms oll day -p= . there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice ( - g) were primed intraperitoneauy with either lipopolysaccharide (lps) ug]anlmal or saline. five days later, the animals were subjected to hypoxia ( % ) for , followed by of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at , , and and assayed for tumor necrosis factor (tnf), prostaglandin e (pge ) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia ( %o disease , berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl . we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il- receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps ( ng/ml) supplemented with different concentrations of mps ( , pg/ml, mg/ml). after hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il- ra, and tnfbp-i (tnfsrp ). il-la, il- ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to . + . %( mg/ml) by mps in a dose dependent fashion, whereas about % decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased times more in the supernatant of lps-stimulated pbmc with gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional -adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that -ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected times with ! -ar agohist isoproterenol ( . mg/kg i.m. and . mg/kg s.c.) , and hours prior to a lethal dose of lps injection ( mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. ) . these data suggest that -ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at °c in rotating polystyrene tubes. samples were taken before addition of x ng/l e. col± endotoxin and after hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin- (il- ) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of mg/ml or mg/ml or with the monoclonal lipid a igm antibody ha- a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il- values were found.in the plasma. cytokine values at hours: tnf-ec pg/ml (mean) and l- pg/ml (mean). preincubalion with mg/ml standard immune globulins reduced il- values by % (mean) while there were no effects on tnf-c~ values at hours. a slight reduction in il- values was also found with the lower concentration, mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il- nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha- a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced l- release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha- a had no effect on the il- or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto % of the estimated blood volume was withdrawn over - rain, to reduce bp to - mmttg and co by / mad withheld for rain. shed blood ( %) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of %. zymoeen activated plasma ( , gg/ml) was given rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r , rat igg , mgkg i.e. n= ) or emine (nffi ), rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p< . ) higher in saline compared to r -treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p< . ) higher in ealine ve r animals at and h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p< . ) attenuated in r treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin- (il- ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il- in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, sprague-dawley rats were studied. anesthetized rats were divided in groups, implanted with alzet r minipumps and randomized to receive the following treatment for days:i: gg/kgbw/min of il-lcc; ii: ~tg/kgbw/min of il- receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a % bsa, full thickness scald burn and the wounds then infected with + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first days and improved food intake between days and . we conclude that blockade of il- after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il- . ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il- producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx ( mg/day) in septic patients resulted in tnf and il- productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( ml/kg/day ). the prevention of lpsinduced in vitro tnf and il- production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam- in sera of septic patients was significantly higher ( - ng/ml) than in normal individuals ( - ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy rgyi medical university, institute of microbiology, szeged, hungary h- ddm t. . e~i~im~/tii, septic sh ~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit ' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il- concentrations. methodology female wistar rats (wt - g) (n- per group) were prefed with % tanrine, . % glyciuc ( in tap water) or tap water (tw) for days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received . % saline subcutaneously ( mls/ g) post-procedure. animals were venosected by direct cardiac puncture at or hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at - °c was completed within hours. tnf and il- were measured using wehi and b bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il- estimation revealed a statistically significant reduction at hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin (il- ), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after days systemic blood was harvested to determine the circulating levels of il and tnf. a second group was treated with thy for days and then received a gavage wide lxl escherichia coli and a % burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had mice/group. in separate groups (n= /group), receiving the same treatments, mortality was observed for days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av difference. it was mclean in a study reported in that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are , neutrophils per cubic millimeter of blood, a kg man would have ~ . xi u circulating neutrophils. when activated ixl ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate - . xi nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of - . ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il- , il- , and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m populations; it decreases splenic m but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon- offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin- , which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il- and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il- and il- +m-csf treatments increased the production of tnf in burn day macrophages and progenitor cells compared to normal cells. il- +m-csf+il- increased the production of il- by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il- +m-csf increased the mrna for tnf in the -day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il- /~ actin mrna indicated that il- , il- +m-csf, and m-csf increased the il- ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~ servojf of appr m ~ separating i~ eucayotic ceils of the hosl ttom l ~ bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (. u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= %' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h -receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h -receptors by h -agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,- decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il- , and , and shared mscrophags funclion with loss nr f and c b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we g~ven in doses of ~ . to ~ , u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~ animals, interferon ~amma was a so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to ,s uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< , ). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c , interleukin- (il-ii ), interleukin- (il- ), and interleukin- (il- ) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin- (il- ), interleukin- (il= ), transforming growth factor- (tgf-j ), and the newly sequenced interleukin- (il-i ). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u . in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il- b and tnf-a. the il- receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p ) and type ii (p ). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il- , il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il- and l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, ) early decreases in total t and helper t cells in the peripheral bloo~ } lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and ) severe and prolongei deficiency of interleukin- (il- ) prcductlon by peripheral blood mononuclear cells (pbmo). this il- deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il- deficiency end shown that the defect lies post-transcrlptionally since mrna for il- is also depressed following thermal injury. we have also investigated the potential of a defect in ii- receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il- r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd +) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: ) occurrence of activation-induced cell death (aicd), ) activation-related phenotypic and functional_ diversity in the pool of peripheral cd + t cells. initially, (up to days post-bum), peripheral blood lympliocytes from severely injured (> % total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l (cd ) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd +/cd ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il- secretion and downregulating excessive il- and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either . g/kg of e in a % solution by garage (ge), or . g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a % body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p and p were both - fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p and p during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. decker, d., sch ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages -ii according to me sherry), were - years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn h before surgery, immediately before incision (after anaesthesia), h and h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in laparoscopic and open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were . the interleuldn- receptor and im soluble form (cd /scd ); . the activation antigen fd- and its soluble form (cd /scd ), a member of the nerve-growth-factor receptor family; . the cd ro epitope which characterizes t memory ceils; . the trausferrin receptor cd ; . the soluble adhesion molecule icam- ; and . the cytokines interieukin- and interleuldn- . on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to h before surgery. h after incision the tra index in open cholecystectomy showed a distinct - fold increase, whereas in laparoseopic surgery a mere - fold increase was noted. h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was . % (range %- %) of total body surface and mean age was years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd and most strongly positive for cd . this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd and cd positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd +) was unchanged and nk (cd +) cells were decreased by ahnos[ % compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd were unchanged. cd (il receptor) and cd (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days ( ) t, , , and post injury. we analyzed the cumulative data for interleukin- beta (il-i[ ), il- , il- as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d post-trauma and recovery thereafter. *p < . , ** p < . vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il- and ,- , suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r= . ). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss> , n= ). i - , - , g-csf, fpa, and c a -levels differed significantly (p< . ) between the iss-groups (>-< iss ) at the time of admission, whereas on day tnfa, c a, - , and ealpi showed significant differences. beyond the first week, major differences were restricted to pge and c a. the formation of two groups with respect to later multiple organ failure (mof < ; mof > n= ) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd (monocytes), cd /cd (i - r + t-cells), and cd /cd (th calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving % or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma ( % tbsa). following a % tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a % tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of % tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with % and % tbsa thermal injuries, respectively. a % tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a % tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm and the office of navy research n - -j- . division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut . post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. cd- mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, cd- mice were randomised into control (c), l or s groups and peritoneal cells studied at hours for bacterial phagocytosis and killi:~g, superoxide ( -) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd- b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac- was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac- expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in ,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. - mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a ,c chamber for recording by real-time video confocal microscopy. the cells were subjected to msec, v/era, a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was , .c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with mm mg%. experiments were repeated with mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. patients ( males females,mean age: . _+ ) with gallstone disease and with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the st and th postoperative days (pre, lpo, po) percentages of cd , cd , cd , cds, cdi were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il- , il- , il- , il- , tnf) were measured in pts. of each group by means of elisa using moab. _r. esults:variations of il- and il- were not s.s.. il- increased but differences between groups were not statistically significant (s.s). il-i decreased on po and increased on po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p< . ).tnf decreased in the l.g. and increased in the th.g. on the po, the difference was s.s(p< . ); on po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on po, except for %cd cell that increased on lpo and decreased on po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< . i), but differences between them were not s.s. in contrast, igg decreased on po (p< . ) and increased on po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, % nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia, and hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound or hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd ) and helper-t-cells (cd ) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd ) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd ) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd +hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd +cd +leu -pos) was increased just after anaesthesia being decreased at g and hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples hours after the operation with recovery at hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at hours (p<o. ) and hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in trauma patients on hospital admission and x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age -+ years ( m, f), mean injury severity score (iss) + , sepsis ( patients), sepsis syndrome ( patients), ards ( patients), renal failure ( patients). ige increased in patients ( %). the mean increase was ng/ml (range to ng/ml; % ci was to ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p= . ) and renal failure (p< . ). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p> . ). plasma ige increase was not related to severity of injury by iss score (p = . ). the mean day to highest ige was . -+ . . the day sepsis was first observed preceded the day of highest ige by . + . days. there was a significant association between the day of sepsis onset and the day of highest ige (p= . ). eight of nine patients with sepsis syndrome had > % increase in plasma ige from admission. one patient's ige levels were normal ( - ng/ml) for days and then increased to ng/ml over the next days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il- , which concurrently regulate production of ige and il- receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during hrs. adult male wistar rats ( +_ gin) were submitted to hs (sbp tort) and after t hr (shock i hr) and hrs (shock hrs) hsl (nac . %, . ml/kg) treatment, e. coli (i ) was injected into the portal vein ~tci (n_> ). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p< . , ~" p< . and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung uptake was increased and liver and spleen uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after hr of hs hsl reatment, diminishing by hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after hr hs hsl treatment, returning to normal values by hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of rabbits, labelled with indium-tropolene and reinjected intravenously into the rabbits, i rabbits received an infusion of escherichia coli endotoxin ~g/kg, while i rabbits were subjected to a major sham operation and i rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and , and hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd , cd , cdlla/b, cdis, cd , cd , mhcii and cd /cd ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd /cd ratio while the cd positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to % of initial values following endotoxaemia and to % following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd cells compared to cd positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin- (il- ) and interleukin- reeeptor(il- r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at , i, , , days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il- stimulated by concanavalin a(con a): the sera from the rabbits days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, kd,milipore; centricon- , kd,amicon), that are less than kd, between i and kd, and more than kd. each group of the substances also was tested for the expression of il- and il- r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il- by the con a-stimulated splenocyte in mice; ) the sera from the rabbits days after injury had more profound suppression than other injured sera; ) there was a marked band at about kd in sera from the rabbits days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; ) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il- and il- r than other groups substances, of which molecular weights are more than kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; ) there is kind of substance, of which molecular weight is about kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; } the substance can depress the expression of mrna of both il- and il- r. research institute of surgery daping, chongqing, p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m( results in decreased antigen presentation, induces tgf- and pge while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il- . consequently, here we investigated rnonokine production in trauma patients (n= ) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t ) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days - post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the - days post injury period became higher in alcoholic trauma patients. furthermore, over days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml - days post-injury days post injury stimulus ale. pt. pt . . . . immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol ( - mm) exposure resulted in a significant down-regulation of tnfc~ (p< . ) and il- (p< . ) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd + receptor, the proinflammatory mediator il- , the macrophage activating factor ifn- ,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days , , , and after mechanical trauma of and after bum trauma of patients (mean age ~ years; mean iss ± pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il- and ifn- in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly ( + u/ml) compared to controls ( + u/ml) as well as mechanical trauma ( + u/ml). il- showed a significant suppression following mechanical trauma ( + u/ml) vs control ( + u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than % of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l- and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il- agonist) in eutrophic rats subjected to either % tbsa cutaneous scald injury (bi), muscle scald injury of equivalent % tbsa (mbi), sham muscle bum (resection of skin only, up to % tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation ( mukg i.p.). separate rats were infused with mg/kg e.coli :b lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount . ng/gwt). il-lo~ was constitutively present only in the skin ( + . ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at . h (in the liver, bi = . _+ . ng/gwt, sbi = . + . ng/gwt, p _< . ; in the lung, bi = . + . ng/gwt, sbi = . + . ng/gwt, p -< . ). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint already ( min real-time). these levels increased in parallel until min and became eventually different by log at - . h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il- ~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il- c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma . j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of mmi-ig for rain. the animals were then resuscitated with either times (x) the shed blood vohune as lactated ringer's solution (lrs) or x lrs + lx % lies. sham mice were neither hemorrhaged nor resuscitated. at or hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il- ( alternatively pmqb showed no differences in il- release between all groups at and h, while sm~ from the lrs + hen group showed a depression at h. tnf production by pm~ was depressed in all groups at h and remained so in the lrs + hes group at h. sm~b showed decreased tnf release values in both hem groups at and h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il- ) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in patients with multiple trauma (median injury severity score = , ). blood samples.were collected shortly after injury and after , , , , s and l days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p< , s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury ( - h) at , eki/ml (median), decreased thereafter to , eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day revealing the lfighest level to la followed by pa (= % of la-sac), ec (= % of la-sac) and vc (= % of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day ). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day and . however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r> , ; p< , ) most often between antibodies of igm-elass ( %) followed by igaclass ( %) and lgg class ( %]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin- (ii- ) and c-reactive protein (crp) were measured in patients. i female and male, ageing from to with a median age of . blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, and hours after the operation and then every morning until the th postoperative day. blood was drawn into heparinized tubes (i iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. - levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p . ) in endotoxin plasma occurred during surgery, culminating in a peak (median value of . eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the th day after surgery, whereas those of interleukin- rose at the end of the operation and were at their highest hours later (median value of . pg/ml). crp levels were also high postoperatively with a median value of mg/l, and were markedly raised on day ( mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and - during the operation was establisthed (p . ), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of - , and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or - plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map ± mm hg) for minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at and hours following hs for quantitation of slga by ria. animals were sacrificed at hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled and days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day in the control . + . , in group . + . , in group ii . + . , in group iii . + . , in group iv . _+ . , in group v . _+ . x ( ). the evident increase in bmc yield in all groups continued until day . increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from through , and who had serum cholesterol assayed during the first hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< . level. results: trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first hrs. patients had cholesterol levels less than mg/dl; of these ( . %) died, ( . %) developed ards, ( . %) developed acute renal failure, and ( . %) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was times greater and risk of multi-organ failure times greater in this group than in those with a normal serum cholesterol (>if mg/dl; patients; p< . ). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n= ) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n= ) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi ) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f ~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p< . ) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa which was more pronounced in c (p< . ) and most in b (p< . ). similar results were found for pge~ and pgf =. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p< . vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r= . , p<o.o ) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt . + . tt , +_ , n.s. pla-ca(s) mt . +_ . tt . _+ . n.s. goris mt . - . tt . _+ . n.s. iss mt - tt - p< . we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim nez f j, l pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha- antitrypsin (aat), alpha- macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of patients, males and females (mean age: years), with compound fractures. % of them were located in tibia and fibula. intercurrent infection developed in patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days , , , , , and . a control group of healthy volunteers was also evaluated. aat and cp showed increased values from day which kept elevated untill the end of the study. amg elevated from day . when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il- , il- , tnf, pge and tgf-b have all been implicated in these immune system changes. in vitro, pge causes many of the immune system changes following hs, yet the time course of pge release has not been well deliniated. the aim of this study was to determine the time course of pge release by peritoneal and splenic macrophages following hs. c h/hen mice were bled to a mean bp of + mm hg for minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at , , , , and hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for hours. the supernatants were collected and frozen until assayed. pge was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge release by peritoneal macrophages was significantly elevated at , and hours over control. it was maximal at hours, but by hours it had started to decline. splenic macrophage pge release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge is significantly elevated at hours following hs, and reaches a peak at hours. pge release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in patients ( male, female, mean age . ), admitted to our icu following serious trauma (mean iss= ) and in control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was . (range - ). patients didn't develop organ failure(nof-group), patients survived, developing mof (s,mof-group), patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days - . n and crp levels showed a parallel release peaks on the days - and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values - h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats ( g) divided in groups (observation time hrs): a: mild hemorrhage by sequential blood sampling ( . ml each)( , , , , , , , , hrs). b: a with hemoperitoneum (hp)( ml/ g), c: hemorrhagic shock: blood withdrawal of ml/ g over min and resuscitation with ringer's lactate ( x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); fe labeling of erythrocytas (application of ci fe i.a. into a donor rat, days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at and hrs, respectively ( + vs. + ; +_ vs. +_ ). hp reduced this increase ( +_ vs. + ; + vs. +- , p<. ). i-ip significantly increased hemoglobin between to hrs in mild ( . +_. vs. . +. ) and severe hemorrhage ( . +. vs. . +. ). in mild and severe hemorrhage comparable maximal resorption ( fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at hrs (mild: +_ vs. severe: +- counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: / ; hp: / ). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at hrs a significant higher transferrin level than with hp alone ( . +_. vs. . + . ). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during days after cct. antigenic challenge was made on day i, , or . number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day and reached a maximum when antigen was presented on day after trauma (pfc number . times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, & , prostaglandin e (pge ) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (> %.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output > cc's per hour had et levels drawn on admission, at i, , , and hrs. et levels were measured by radioimmunoassay. mean levels were elevated at ± pg/ml at all time points versus levels in healthy controls of ± . in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin these processes since it eradicates the influence of infectious factc~rs. dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il- , saa and neopterin, the percentage of cd + cells, the in-vitro il- synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of patients with cholecystolithiasis undergoing either laparascopic ( ) or open (i ) cholecystectomy on consecutive perioperative days - , , and . there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients ( versus minutes, versus days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i + lng/ml, d + ng/ml) versus lap group (d- + ng/ml, d + ng/ml), while in serum il- levels we saw a less steep increment in the lsc group ( -fold from d- to d ) compared to the lap group ( -fold from d- to d ). the analysis of cd + receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d - : . + . cpm, d : . + . cpm) compared to the lsc group (d -h . + . cpm, d h . + . cpm). in both groups il- synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors ( - and kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il- mrna and il- production was detected. the nucleotide sequences of the human il- gene regulatory region bounding by the splenic nuclear proteins were determined between + - b.p. the il- trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il- production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il- gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania non-pregnant patients from rural areas were followed. of preoperative patients % had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied patients of which % had a parasitaemia on admission and % had postoperative malaria. half of the surgical patients came from rural areas, whilst only % of those with malaria lived in the city (with much less exposure and immunity). % underwent major surgery and % minor. bloodtransfusions ( % with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about % of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that % was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co derived from [i- c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol . ~g/ml, glucagon #g/m , epinephrine ~g/ml, insulin t~u/ml; traumaeortisol . ~g/ml, glucagon /*g/ml, epinephrine ~g/ml, insulin ~ij/ml. analysis of twenty subjects showed a reduction of ° ~mp shunt activity by lymphoeytes and a ] % reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < . . lymphocyte glucose uptake was also reduced by trauma hormones p~ . . it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence f.neutr phils. there~bre, neutr pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln- (i ..- ) and mo~tocyte chemotacde pop, de (x¢cp- ) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from ipf and sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii. arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i ( . ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf ( . ng!mg) in comparises to . normal individuals ( . ng/mg) and to patients w~th obreic bronentis (cb) (~, rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp- e.qu/va]ent) was strongly increased in sareoidosis ( . ngjmg) as well as ~n f pag,nts ( . ng/mg). norra.al indlvidu~s and cb patiants hzd a . or -fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il- ievei~ ( . and . rig/rag, respe~veiy; nomzls: . rig/rag; cb: . ng/mg) mad nvatropmi ohemotax~ ( . ~'~d . nnmg, res!z~ztiveiy; aormals: . ng,'mg; cb: l ngmg). these data suggest that increased ievels of born mcp. ~d il- may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ ( fold vs. lps or paf alone), ll-lg ( fold), kc ( fold) and il- . taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day , hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays - and further observation and measurements at days - . from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight kg) neutrophil counts ( e pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood ( e cpm/ , pmn), and of blood-and bal-isolated pmn ( e cpm/ , pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca + second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in polytranmatized patients (mean injury severity score = ) arterial and venous blood samples during days. daily evaluation of horowitz-quotiant (po /fio ), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day and (day : +. ; day : . +. ). plasma lactate was significantly increased at day l ( + ) and day ( . + ). hurowitz-quotient (day : + ) was low at day ( + ) and day to ( + )(p<. ). at day a substantial rise in venous pmnl-superoxide production (day : . +_. , day : . +. , day : . +_. ), oecured with significant increase in plasma lipid peroxidation (day : . + . ; day : . + . ). pivin~-myeloperoxidase activity was high at day ( . +--. ) and then continuously declined (day : . +. ). plasma antiexidant activity (glutathione pemxidase) was reduced by % at day (day : . +. ; day : . +_. ; day : . +. ). whereas basal ca + concentration remained unchanged (day : +_ , day : +_ ), fmlp-stimulated cytosolic ca + mobilization increased at day (day : + , day : , day : + ). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca + mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, homburg/saar donnelly sc, haslett c, dransfield i, robertson ce, grant is, carter c, ross ja, tedder tf. dept's of respiratory medicine, accident & emergency, intensive care, surgery, university of edinburgh, scotland and dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n= ), perforated bowel (n= ), and multiple trauma (n= )), of whom progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o. ) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity ( _+ pmol/i vs _+ ; p - . ), but not the release of neutrophil related mediators ( . _+ . ng/i vs . _+ . ; p= . ). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb ( . + . % vs . _+ . ; p = . ). although not significant, nac prevented also the increase in elastase levels in lavage fluid ( . + . pmol/i vs . + . ; p = . ). it is concluded that nac is able to protect against the inactivation of a -proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion ( rain) and repeffusion ( min). sprague dawley rats were randomized into: control (n=i ); ischaemia repeffusion (ir) (n= ); and ir treated with gtn ( ug/kg/min) during repeffusion (n= ). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n= per group). control _+t "* _+ _+ ** bal pmn/mm l+- " + # _~ *p< . , #p< . v control/gtn;**p< . v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from . + . to . _+ . mcf after minutes repeffusion (p<. ). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin , ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, #g in . ml saline). three groups (n= each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh ( ~tg in . ml saline) at , , and hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f , = . , p<. ), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<. ). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<. ), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia ( mg/kg, i.p.) in female balb/c mice ( - g) that were then randomly assigned ( - mice per group) to receive at time and hr later: control saline thjectioas (i.p.), injections of ~-msh ( ~tg), gentamicin sulfate (i mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in patients ( female, + years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and min after declamping. arterial pla concentrations were higher than venous ( + vs. + ). while cytosolic basal pmnl ca + concenlxation was not altered at the end of ischemia (v: . + ; a: _+ ) and during reperfusion (v: + ; a: + ), fmlp-stimulated cytosolic ca + mobilization showed a significant arterial increase as compared to venous ( _+ vs. + , p < . ). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by ) pulmonary membrane damage possibly due to pmnl-degranulation, ) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and ) a significant arterial increase of pmnl cytosolic ca + mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca + messenger system by inflammatory mediators (e,g. ii- , tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p cell) is not only an important target, but as recently shown it is also capable of producing h . the aim of this study was to investigate the influence of endotoxin on h release of p cells. we obtained p cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h release of fresh isolated cells and on cells cultured for days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for hours with p.g/ml lps and tested the conditioned medium (mcm) on cultured p cells. p cells h ex vivo were _> % pure, _> % vital and had an basal h release of . _+ . nmol h per hour and million cells. adding p.g/ml lps to the incubation medium the h release decreases slightly but significantly to . _+ . nmol. adding . p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h release increased -fold to . _+ nmol. pma induced h release decreased to . + . nmol after addition of p.g/ml lps. after culture days the p cells were _> % pure and showed a pma inducible h release of . _+ . nmol addition of p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h release up to . _+ . nmol. addition of mcm to cultured p cells increases pma-stimulated h release to . +_ . nmol. the release decreased to . _+ . nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > % in all experiments. the results show that lps has an direct effect on p cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h release of p cells could be important for generating internal oxidative stress in p cells before external oxygen radicals exceed. the produced h did not necessarily damage p ceils, but it can effect surfactant metabolism, especially when extracellular h release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled patients with severe lung injury after sepsis ( ) or polytrauma ( ) and obtained multiple blood samples ( days) for endotoxin, tumor necrosis factor e (tnfa), interleukin (il- ) and interleukin (il- ) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l and il- were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, . ng/ml) for hours at c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao /fiao quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < ( . ± . eu/ml, mean values ± sem) versus the patients with a sli > ( . ± . eu/ml, p<o. ). the basal plasma concentration of ll- also correlates with the sli index (p<o.o ). the delta tnfe and delta il- values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n: ), the deceased patients (n= ) showed higher endotoxin level, a reduced tnfa and il- releasing capacity of the blood and higher basal il- levels. we can conclude that endotoxemia, high il- plasma level and a decreased capacity of the blood to release tnfa an il- under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il- in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado ) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($ or $ ) of the right and left lung through a bronchoscope on days l, , and days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado unexceptionally deteriorated postoperatively and the mean aado in patients reached the maximum (mean ± sem; _+ mmhg) on the rd postoperative day. the mean concentrations of il- , ne and neutrophil count in balf also increased postoperatively and reached their maximum level ( + pg/ml, -+ gg/l, and ± x cells/ml, respectively) on the rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il- and ne levels in balf (r= . , p= . ). also, significant correlations between aado and the neutropbil count, and the concentration of ne or l- were observed. in contrast to il- , neither il- , il- nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il- increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes ( mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats h prior to pma exposure ( pg/kg, intratracheally) and treated rats were killed h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp- ,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n- ) weighing .d- . kg oaselinepao> kpa and mean pulmonary arterial pressure (mpap)<i. kpa were included in this study. anaesthesia was induced with thiopental - mg/kg bw and "blind" jntubation was performed. a f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose mg iv or metylprednisolone mg iv min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap( . -+o.~ kpe ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp- or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients ( sepsis, trauma) with an fie of . + .( , a peep of . ± . , and a murray score of . ± . were included in this study. healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) . ~: . * . ± . *= < . , **=p< . condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io ), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of serum samples were collected from patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n= ) and non-septic (n= ) patients. patients with ards (n= , . + . ng/ml) and with respiratory disease other than ards (n= , . +- . ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n= , . _+ . ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using -hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase ( .i- . %) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej a - ~ia~ystok, poland, tel/fax + . in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) - ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. : , ) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n- -and n- -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf ( um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of -series lts and -hete (total sum of - ipoxygenade products rd. and rd. pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of - ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. pmofml). eicosapentaeooic acid ( um) elicited exclusive generation of -series lts and -hepe (total sum rd. pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived - ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. pmol/ml). we conclude that the paf-evoked - ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n- -versus n- enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr- , incubated with either pmn or mnc for hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin (cx ) which forms channels between cells capable of pas.~ing any molecule smaller than d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > adjacent hepatocytes within seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx is markedly decreased while the secondary gap junction protein cx is either unchanged or even increased. while cx readily effects electrical coupling, molecules > d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $ although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., and h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci , peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for , and hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after minutes of hepatic ischemia and hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to min of hepatic ischemia followed by reperfusion (rp) and injection of . mg/kg salmonella enteritidis endotoxin (et) at min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from + u/l (i/rp only) to + u/l at h rp. inhibition of kupffer cells (kc) with gadolinium chloride ( mg/kg) attenuated liver injury in this model by %, however, monoclonal antibodies (cl , wt ) directed against adhesion molecules ( integrins, cd ) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time ( + pmns/ hpf; baseline: + ). isolation of kc and neutrophils from the postischemic liver indicated a -fold increase of the spontaneous superoxide formation only in the kc fractions [ . + . nmol o -/h/ %elts (kc ); . _+ . (kca) ] at h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments ( h), the antibody wt ( mg/kg) attenuated liver injury by % at h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es- and gm- ) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type (scri), mg/kg iv after vascular occlusion (n= ) or by depleting the complement system using cobra venom factor (cvf), . mg im, and hours before ischaemia (n= ). non-ischaemic rats (n= ) and ischaemic non-treated rats (n= ) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c and c were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p< . ) and haemoglobin saturation (p< . ). c and c were present in the endothelium of the ischaemic control group. no deposits of c or c were found in the cvf group. few c and no c were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, control and cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to + mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by %.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [ mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < . ) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < . ); fhm ( < . ); and liver blood flow (p < . ) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < . ) in cirrhotic rots. tnf levels were higher (p < . ) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin (cx ) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx to % of sham controls. immunostaining of liver sections using anti-cx revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles hrs after animals were treated with lps, suggesting the internalization of cx without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at hr post injection . % ofpixels exceeded threshold, compared to . % in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca ÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca ÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats ( g, n>_ /group; pentobarbital mg/kg) with hemorrhage for rain at mm hg. reperfusion by ringer's lactate ( x maximal bleed out/ min) and % of citrated shed blood. tnfcz-moab (tn , ceutech, mg/kg in . % nac ) infused during flrst min of reperfusion. at baseline, end of ischemia and min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " hepatocyte incubation ( mg w.w./ml) with caci ( . + + + mbq/ml) for rain (ca influx [slope, /mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca + (ca +ex) [nmol ca +/mg protein]; ca + membrane flux [nmol ca +/mg protein'min]). during incubation, aliquots ( ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca +ex and membrane ca + flux were significantly increased at both, the end of ischemia ( . +. ; . +. ) and reperfusion ( . +. ; . +. ), as compared to sham-operated animals ( . +_. ; . +. )( <. ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca +ex ( +. ) and membrane ca + flux ( . +. )(p<. ). fast ca + influx was significantly increased by epinephrine in hepatecytes from sham-operated rats ( . +. vs. epi: . +. , p< . ). this hormone effect was not observed in isehemia ( . +. , epi: . !-_. ) or reperfusion (untreated: . +. , epi: . +. ; tnft~-moab: . _+. , epi: . +. ). conclusion. the present study clearly demonstrated hepato-cellular ca + overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca + fluxes and hormone ca + mobilization suggests disturbances of membrane ca + transport mechanisms, e.g. through ca +-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca + buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing - kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. . fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive % of tbs a third degree burn. eighteen hours after burn. gg/kg e. coli lps was intravenously administered over rain. ali animals were studied for additional hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc ) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc -indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc . inositol(l, , ) triphosphate (ip ) has been proposed as a second messenger for calcium mobilization. the addition of ip at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip receptor binding was performed with tridium label ip . the results shewed that the ip binding was significantly depressed by - % (p< . ) during late sepsis ( hrs after clp), but not in early sepsis ( hrs after clp). the ip binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p< . ), but not on smooth subfraction. since ip binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h ) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h# a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was consecutive patients with liver cirrhosis admitted to the department of surgery over a year period from january to december were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c , c ) were measured and functional assay for complement (ch ) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c , c and the functional chs complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch ) showed marked impairment in child's c patients (p< . ) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p= . ) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o per . ml medium fi +dmem ( : ) with % fetal calf serum for days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by - % (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than . times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., ) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., (kudryavtseva et al., , the ability of metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in consumption supported by nad+-iinked substrates, but showed almost no change in consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state oxidation of site i substrates and with the increase in state oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca + release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca + cycling was prevented, either by chelating ca + with egta or by inhibiting ca + re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca ÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally mmol/kg and mmollkg, respectively, and lps ( mg/kg) given h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase h after lps injection. the degree of hydrogen peroxide (h ) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [ ]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: .evaluation nfthe course of the ees due to the biochemic changes .determination of the influence of the ees on the activity of lysosomal enzymes in liver cell .deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: .activity of acid phosphatase in the venous blood serum. - .total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after hours of the experiment). conclusions . essential increase of activity of lysosomal enzymes was observed in the ees. . increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia ( min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. : g , ) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. : , ) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. : , ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats ( - g) were subjected to rain of hemorrhage (mean arterial pressure mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from . + . txmol/g to . _+ . ( min shock) indicating severe ischemia, and recovered to . -- . at min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by % compared to baseline levels ( . !-_ . i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was . + . nmol o -/min/ ecells (kc ) and . + . (kc ) in controls and did not change significantly after shock and rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after min of hemorrhagic shock and up to rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after weeks animals were sacrificed and livers were "perfusion fixed" with % glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as -fold of basal levels at the end of min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: ( ) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda ( ) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. ( ) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan , r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing - m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps( .gg. ) . rag/kg intraperitoneally. at the end ,:,f l,gand hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at - hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a c in wlstar rats was developed by tigatlng the common bite duct and injecting d. mt solution of e. goli % b, (g× ' efm/mt) into the bite duct. the mortality rate was ~ at h after aoc. at , , , h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at h after aoc. at h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p< . ), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at h after a c (p< . og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho ensis of hepatic dysfunction and m f following h . " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il- , il- , l- , i , tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il- and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: ( ) the same immurzoassay (elisa) yielded comparable results in different laboratories; ( ) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; ( ) assays for soluble receptors yielded less discrepant results; ( ) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately % for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p and tnf-r p . these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin- and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta- -microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, ) increasing neopterin levels predict rejection episodes in allograft recipients ) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv- infection ) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease ) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv- infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ -proteinase inhibitor (a pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage , m nchen, germany procalaitonin (proct) a aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as hours after the andoto:edn release and readied maximal level with a to b-fold increase h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii- , that reached peak levels at h and h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about daltons is unable to cross the kidney and the c_n barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau . ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i to fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp ) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin and interleukin (ill, il ). class acute phase genes are mainly controlled by ill and by combinations of ill plus il ; class genes mainly by ii, . the human c-reactive protein(crp), serum amyloid a(saa) and complement c genes will be discussed as examples of class genes, and rat c~ macroglobulin as a prototypical class gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class genes use the transcription factor nf-il or c/ebp as the key factor to mediate cytokine responsiveness; class genes use a different, so far unidentified factor to achieve responsiveness to il . ongoing efforts to purify this factor, called the il -response factor (il -rf), from rat liver nuclear protein extracts will be described. the il -rf apparently mediates the effects of il in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il as nf-il . the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex ~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf sioi to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to % and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than % when sepsis is present or when at least organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of to ml/ g/min are reflected in isoelectric eeg, and values about t ml/ g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about ml/ g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm , berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e synthesizing m~ and a simultaneous decrease of functionally competent cd + and cd + lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin- (il- ) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il- , most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper- (thl) secreting principally il- and ?-ifn and t-helper- (th ) acting principally in inducing antibody formation with a secretion of il- , il- , il- and tnf-[ has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u , tnf-c~, il- , il- ) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: % of all pmn's in this compartment show downregulated or non existent ~-integrins within hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr + receptors (cdll/cd complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+ ) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in- (il- ) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge . administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il- production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il- after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen - hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc gradients. supranorma[ delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. . myocardial depression with a drop in delivery (do~) . redistribution of blood flow between the different organ systems . inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ supply may therefore be inadequately matched with cellular needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april to august patients, aged from months to years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in out of these patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between - s and an activated clotting time between - s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four days. in the conventionally treated group % survived and in the additionally with ecmo treated group % survived, representing a % survival rate in patients whose risk of death is considered more than %. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med ) . in contrast, mean skeletal muscle po was found to be abnormally high in patients with sepsis ( _+ , mmhg, n= ) compared to patients with limited infection ( , + , mmhg, p< . , n= ) , to patients with cardiogenic shock ( , + , mmhg, p< . , n=ls) and to healthy subjects ( , _+ , mmhg, p< . , n= ). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po was higher ( , _+ , mmhg) on days with severe sepsis than on days with intermediate state ( , _+ mmhg) and than on days without sepsis ( , _+ , i mmhg) . thus, a decrease of abnormally high skeletal muscle po was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po with the decrease of apache ii score or elebute score as well as interleukin- serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il- and c a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on consecutive pat. after elective open-heart surgery, we had determined ele daily for at least pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n = patient-days, both ele and sirs showed significant (p< . ) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: . vs. . ; icu treatment days: , vs. . ; days on mechanical ventilation: , vs. . ; days with vasopressor requirement: . vs. . ; number of microbiological findings: . vs. . ), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of . for ele (ele >_ on >_ days, accuracy: %) compared to . for sirs (sirs present on > days, accuracy: %). accordingly, ele roc curve areas for both overall ( . ) as well as sepsis-related prognostic evaluation ( . ) were significantly (p< , ) larger compared to sirs ( . and . , resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate ( % and %, compared to % and %, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. , d- munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score , while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation . four of the nine patients died. apache iii scores at h were lower in survivors (s) than in non-survivors (ns), ( -+ vs -+ p< . ), while apache iii scores at admission were not significant different between s and ns ( -+ vs -+ ). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an fold increase of tnf-a bioactivity with[r~ hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il- [ and il- immunoactivity. tnf was measured by wehi bioactivity, il- [~ and il- immunoactivity were determined by elisa. the sensitivity was pg/ml for tnf, pg/ml for il-ll and pg/ml for il- , respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day , p< . , the pattern of cytokine production by pbmc upon lps stimulation over the first h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin- (il- ), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, polytraumatized patients were prospectively studied (mean age y, % male, iss ). serum and edta-plasma samples were taken in h intervalls until the patient left the icu. il- , tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, ) with at least of clinical signs: ( ) tachycar-dia> /min, ( ) temperature > , °c, ( ) blood pressure < mmhg, ( ) mechanical ventilation, ( ) leukocytosis > . /ml, ( ) thrombocytopenia < . /ml and ( ) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day after trauma. results: of patients developed a systemic sepsis ( . %), and died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il- ( vs. ng/l; p= . ), crp ( vs. mg/l; p= . ) and tnf ( vs. ng/l; p= . ) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il- , tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n= ) was determined by the injury severity score (iss). lidocaine is given at a dose of mg/kgbw over rain. i.v. and is metabolized in the liver by a cytochrome p- mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the ~t and the ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin- (il- ), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~ , stnfr ) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il- , tnfot, stnfr , stnfr ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il- was detected in an biological assay using the/l- dependent -cell line / . detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (> mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with ( ± mg/l) or without ( a: ) multiple organ failure (mof) nor in survivors ( ± ) or non-survivors ( :t: ). in contrast, l- was elevated in patients wilh mof (mean pg/ml, range - pg/ml). il- levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il- and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r= . ). patients could be divided into two groups by values for stnfr~ and stnfr : the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il- correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg , kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in consecutive patients with multiple injuries (iss . ) the inflammatory response to trauma was prospectively studied. the patients were divided into groups according to their iss points. additionally, the alterations after secondary operations (> hr) were determined (msteosynthesis of the femur (n= ), pelvic girdle (n=ll) and spine (n= ), facial reconstruction (n= ), smaller osteosynthesis (n= ) and others (n= )). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every h, beginning on admission of the patient to the emergency room for a period of hr, and in the operative patients on the morning of the operation, at the end of the procedure and every hr during the first two days. results: lactate, neutrophil elastase, heart rate, po /fio -ratio, and other parameters discriminated significantly between the injury severity groups during the first hr (kruskal-wallis-test, p<. ). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<. ) between the types of operations for lactate, heart rate, po /fio -ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss to ) or moderate (iss to ) accidental trauma for neutrophil elastuse, heart rate, po /fio -ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~ years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a -year period, patients were consecutively admitted into the icu; and during a -year period, patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o ), sepsis (p<o.o ), and mortality rate ( % vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o ). of the total group of , patients, there were ( %) patients years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o ). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients ( % vs. % in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of . ( % confidence limits, . - . ), age did not increased [odds ratio . (i. i.i )], while admission to the sicu reduced risk of death by a factor of . ( . - . ). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan , ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il- , l- ,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n= ) and non-survival group (n= ).tn these patients,ll- , l- ,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were points in survival septic group, l l points in non-survival septic group and points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p< . . results: il- levels in non-survival septic group were higher than in both survival septic and los group.ll- levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/ ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha- -maeroglobulin, c estemse inhibitor, antithrombin ill and alpha- -antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in patients and continued postoperatively in patients, % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication - days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by - days. dept. of surgery m'alm general hospital, s- i malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of patients admitted with burns covering more than per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: out of burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were . -- . eu/ml, significantly higher than that of patients without mof ( . - . eu/ml) on , , and days postburn (p< . -- . ). significantly more incidence of positive endotoxin test (>_ . eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p< . ). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp- , and gp ) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs expression) was significantly elevated (p< . ) and correlated well with severity of disease (f . ). no significant change in surface expression ofthrombospondin, gmp- or gp was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (! < . ) that correlated well with severity of mof (gmp- , r= . ; thrombospondin, r= . ).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a -year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period - i patients with abdominal sepsis were treated at the icu at our university hospital. patients were women and men with a mean age of ( - ) years. in cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for (- ) days out of which (- ) in the intensive care unit. out of patients ( %) died in median after ( - ) days. the primary cause of mortality was multiple organ failure ( / ; %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin ( %) and in % cultures grew multiple bacteria. patients bad organ failure and multiple organ failure. / patients ( %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about / of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january to september patients, mean age + years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for . _+ . days. diagnosis of sepsis was confirmed in cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, -patients with sepsis, the control group consisted of patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c ,c ), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c (mg/dl) c (mg/dl) k +- + _+ + +- -+ " -+ * _+ " -+ ' _+ " +_ '* -+ ** -+ "* -+ "* _+ " in a prospective study we investigated serum of severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n = ) was investigated as. well as serum of patients undergoing elective herniotomy (n= ) hours before (op i) and hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain :k :h . / suspension with a final concentration of - cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co . _+ . opi . _+ . opii . _+ . * tri . _+ . "* trii . + . ** (*:p< . ; **:p<o. ) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied patients with sls (group i: age . ~ . years); they were compared to control patients (group : age ± . years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone ( mg/ hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in pts. preoperatively pts in group revealed a significantly lower cardiac index ( . ± i/min/m ) compared to group ( . ± . i/min/m ; p < . ) higher left atrial pressure (group i: . ~ . mmhg; group : . ~ . mmhg; p < . ) and lower ejection fraction (group i: . z . %; group . ± . %; p < . ). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: , ± , /ui; group : , ~ , /ui; p < . ) and core temperature hours pop was higher (group i: . . °c; group : . ~ . °c; p < . ). serum lactate was already elevated on arrival on the icu pop (group i: , z . mmol/l, group : . ~ . mmol/l) but dropped within hours (p < . ). icu stay (group i: . ! . ; group : . ± . days; p < . ) and mortality over months (group i: . %; group : . %; p < . ) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. patients who were admitted to the intensive care units between january and july ' with polytrauma, sepsis or those requiring ventilatory support for more than hours were selected for the study. the patients were randomized into groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored hourly and the patients underwent an upper gi endoscopy hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the patients selected for the study, had undergone cardiac surgery, had polytrauma and had sepsis. the change in average pre and post drug gastric ph was + . in the omeprazole group, + . in the ranitidine group and -). in the placebo group (p < . ). endoscopic evidence of stress ulcers was seen in % of patients not on prophylaxis, . % of those on omeprazole and . % of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, % of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < . ). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin- (il- ) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn- receptors (sil- r). the aim of this study was to evaluate: the significance oftnf c~, il- and sll- r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il- and sll- r. we examined patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, ,- and sil- r were measured in septic patients and in adult healthy controls. tnf ct, il- and sil- r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il- and sil- r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll- r serum levels were significantly (p< . , student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ] ,- levels. we conclude that only the increased serum sil- r levels were predictive of bacterial sepsis within hours after the first septic signs. tnf c~ and il- levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed children with light and children with severe course(l- class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch , level of c -c , factors b told d, c a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group ( %- st and ioo%- nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c , c components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~ ) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr der j, braun j, rennekampff ha, schr der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in trauma patients with an injury severity score (iss) > (mean iss = ; mean age years) lymphocyte and neutrophil phenotypes cd (t-cells), cd (t-helper cells), cd (t-suppressor cells), ratio cd /cd , cd b (receptor for cr ) and cd (fcriii) were measured on day , , , , and post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd ) and il -receptors on t-helper cells (cd /cd were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd were significantly lower an day , , and in patients who developed mods (p< , ) compared to patients without mods and a healthy control (p<o,o ). the expression of il receptors on cd was significantly different only on day . no differences could be measured in lymphocyte phenotypes cd , , and cd /cd -ratio as well as in expression of cd b and cd on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > /zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the consecutively admitted patients to a surgical and a medical intensive care unit during -ye r period, ( %) had arf. arf mortality was %. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o, ). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p< . ), the number of osf before was higher compared to after arf in both groups of patients (p< . ). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p< . ). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p< . ). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama ; : z - ~ ) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of , recent admissions to hospitals in the united states and western europe, we identified , patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors ( % of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original , patients with a receiver d_perator characteristics (roc) area of . and in two independent databases, the first independent database contained hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc= . ); in the second, the placebo patients withtn the recently completed phase ill e~aluatlan of il- ra (rocffi . ). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low % to a very hlglt %. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il ( ) -to use a model for risk of death either from a score (apache ii, lii, ( ) saps ii) or directly (mpm ii system ( )) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system ( )) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii ) before any therapy. -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: ( ) toxicity models with injection of bacterial endotoxin or exotoxins; ( ) live or killed bacterial challenge models; ( ) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: ( ) hemodynamic parameters; ( ) modulation of host-derived inflammatory mediators; ( ) resolution of end organ injury; or ( ) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in %- % of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field ( ). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa pi, tat), cytokines (il- , il- ), and soluble intercellular adh sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il- , il- and tnfe) as well as breakdown products of complement proteins (e.g. c a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, ) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd mab wig was able to down-regulate production of il- , il- , ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin- receptor antagonist (il-lra) but not of il- ; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il- ), some only by solid-phase detection systems (anti-ll- , anti-ifn% anti-tnfc . infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il- in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il- , and il- . self limitiation of this process might be due to demonstrable instant release of il- ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il- , il- , tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. ( ) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi ,hokkaido,japan $ immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h mon departmen~ of surgery, san frandsco general hospital, urdversr , of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi is down-regu/ated, along wl~ cd and cd . thus responsiveness to lipopolysacchmdde ( ) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e . in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge . ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s! otlse. by examining m n cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il- was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than pg/ml (normal: less than pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il- level and the number of organs showing failure (r= . ). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p< . ). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i- - , kumamoto , japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock ( ). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of mg/ rain, followed by a continuous infusion ( mggh). with hemodynamic improvement (dopamine < gg/kg/min) the infusion rate nfhc was tapered over a period of days. phospbolipase a (pla ), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after days dopamine requirements decreased to less than gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day and ) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla , and finally by the relapse of septic shock in four patients on days , , and o. a second infusion of hc again resulted in shock reversal and in a decrease in pla , crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il- , il- , and il- is attenuated by- rag hc prior to endotoxin challenge in humans ( , ) . cytokines like tnf, il- , il- , and il- are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: patients with an iss nf more than points who could be treated with at iii administration within rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of % by using the following calculation: at iii to administer = ( % -at iii measured) x body weight. at iii was given every six hours for a period nf hours and on the th day. patients were followed up throughout their stay in the icu but at least days. in this abstract the data of the first patients are included. results: the patients' median injury severity score was points with a median age of years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received , units of the inhibitor on average during the first days. with our regimen, the at iii levels could be kept between % and % of normal, whereas control patients an at hi concentration of % to % was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to % of normal. white blood count, pmn--elastase, interleukin and and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii group ( . vs. . days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of %, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. patients with severe multiple trauma ( treatment [at-ill] -- controls [c]) were investigated. study cdteda were age > and < years, injury severity (iss) > points and glasgow-coma-scale > points; randomization and treatment has to be started within hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp- , a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp- in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. h later bk was injected ia and the pain score ranked from (no responses) to (vocalization). cp- at . umoles/kg completely inhibited the pain responses for a period of . - h. cp- at . umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a - h period by an intraplantar injection of . % carrageenan. the abdominal constriction response o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp- . when ul of . % formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp- significantly inhibited both the first ( - min) and second ( - min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at °c for sec the increase in paw volume was significantly reduced by pretreatment with cp- , . umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (- °c for sec) to the dural surface following a craniectomy. cp- at . umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp- , may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi , a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats ( - g.) received a % liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either . mg/kg/hr rbpi (phx-bpi, n= ; sh-bpi, n= ) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n= ; sh-con, n- ). various parameters were measured h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi . blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il- levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at - rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of - mmhg for rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. rbplg. was administered at a total dose of mg/kg i.v. ( . mg/kg at the -eginning followed by two doses of . mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi . results: imrffe?diately after resuscitation ( min) the detected plasma endotoxin levels in the control group (mean = , range = - pg/ml) were almost neutralized by rbpi treatment (mean = , range = - pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points and min of experiment (means: and in bpi vs , pg/ml in the control group). the -hour survival rate was improved from / ( . %) in the control to / ( %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. rabbits weighed + grams divided into two groups. group i included tabbits were treated with allopurinol mg/kg for seven days before induction of haemorrhage. group ii as a control included rabbits. all rabbits were subjected to % arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was out of while in group ii it was two out of . our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, pigs were first anesthetized, then injured with joules of energy to the posterior thigh, then hemorrhaged - % of their blood volume. after i hr of shock, all the shed blood plus x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine ( . mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after hrs, a septic challenge was administered ( bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for min. baseline values and units were cardiac index _+ ml/min/kg (ci), arterial po + mmhg(pao ), base excess . -+ meq (be), physiologic dead space fraction +_ % (pds), and tnf . + . units/ml. baseline gastric ph was . -+ . and . _+ . in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao * be* gastric* pds* peak* rain rain rain ph min tnf ranitidine _+ _+ - . ± . bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin- (ii- ). this study was designed to clarify the role of lps and - in the metabolic response to bum injury. twenty-five burn patients ( -+ %; + % ft bsa burn; _+ years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i - were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b ( , u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic ( . +_ . pg/ml; normal < pg/ml). i - did demonstrate a significant correlation with cole temperature (tr~ = . + . ogi - , p= . ) and with nitrogen excretion (nou t = - . - . ogi - + . tr, p= . ). administration of polymixin b had no effect on metabolic rate, temperature or i - levels but did reduce nitrogen excretion resulting in more positive nitrogen balance ( .t grn/day vs. - . gm/day, p= . ). although bum injury does not produce an obligatory endotoxemia, i - does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within rain and shock state was hold for h at a map of mm hg (cardiac output of %). following adequate resuscitation with % of shed blood and twice of this volume as ringer's solntion, animals recovered to map > mm hg and co > %. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at , , or hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [ mg/kg, celltech, uk) , pentoxffylline (ptx, mg/kg, hoechst, d), and a paf antagonist (web , boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n= - group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence - of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from , to about pglml, but did not abolish it. the inhibition of the lps( . ug/ml)-induced tnf production occured already in presence of , pg peptide/ml and peaked at the concentration of , ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp- and tp- ), thymocartin did not enhanced the pwm-induced pge production in pbmc cultures up to a concentration of ng/ml, inclusive. the selection of thymocartin (tp- ) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about % registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: patients (pts) formed control group a. group b pts received x mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d ) intravenously to block prostaglandin e induced suppression of cmi response as well as mg thymopentin (tp- ) subcutaneously preop., on d and d to augment cmi reactivity. in vitro investigations preoperatively, on dl and d included measurements of the percentage of cd + t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)- as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il- as one cytokine primarily produced by th -cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d were used as in vivo tests for thl-and th -induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd + th cells, il- /il- ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p< . vs.bl). in group b pts no change in cd + th cells, il- /il- ratio, or dth skin response was observed (p< . vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: .these results clearly indicate a significant suppression of th induced cmi response, while th induced response remains normal following ohs. .these alterations may gain clinical significance whenever a postoperative infection requires a th response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing - g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il- . they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from to . results.it was found that in % of infants with - classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then times, ratio th/ts was reduced to . or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c a subcomponent were increased but the level of c , c ,, c and c components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i- mcg/kg per day and mg/kg per day accordingly during a week in children with - classes el heaviness. aiter - days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of ( - )-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c h/hen or c h/hej mice, to wkolds of both sexes were used through experiments. highly purified ( - )-{ -d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b- . induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c h/hen and c h/hej mice, whereas lps only induced such activities in c h/hen mice. most effective dose of bgc was ug/ml. ) bgc-dependent proliferative response of c h/hen splenic cells was not detected under the culture condition used. ) induction of tnf~ prodction was apparently observed in bgc-stimulated c h/hej pd cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~ activation, intracellular signaling pathways of p~ by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il- in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il- . under anesthesia, -weekold male c h-hej mice were subjected to a % scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of organisms of p. aeruginosa hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells ( x ) intraperitonelly after septic challenge. the mice were scored for survival daily. on day after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il- and il- in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il (n= ) were raised to . _+ . and _+ pg/ml, while the levels in lak treated mice were reduced to . _+ . and _+ pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included operated patients, of which received mg indomethacin before and , and days after surgery (group a) and patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd ), suppressor (cd ), nk-cells and interleukin- and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day and , and days after operation. in group a we detected a significant (p< . ) increase in cd /cd ratio on day while no differences were ~oted in nk-cells or cytokine production in both groups (table ) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. female cd- mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p< . vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p< . ). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p< . ). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin , ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h -antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. -aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi )(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p< . ) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi and plasma cholesterol (p< . ) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, -aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, x u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. -hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the -aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in neurosurgical centers and included patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and groups of tirilazad in escalating dosage ( . - mg/kg/day). the clinical outcome was compared for these groups and cerebral a~giography was performed in % of patients on day - follovang sah, to study the effect f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z patients sufferin s from severe ap (c-reactlve protein > me/l) we determined on day and day after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i patients were randomly treated with ug/day of se for days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only / of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains % hes and % chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats ( - g, n> ; pentobarbital anesthesia mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate ( ) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and ( ) whether dfo-hes could prevent this tissue damage. methods: in rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to groups: one group was treated with ml dfo-hes ( mg/kg iv), the other rats received solely ml of the carrier starch solution. , , , and min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> . eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p< . ) in the lies group already min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p< . ). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p< . ) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca + levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose ( mg%) and bovine albumine ( gm%) for min at °c.it composed of groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo& mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were % in group ab and % in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a , leukotriene b and leukotriene c are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after , , , and minutes plasma levels of thromboxane b (txb ), leukotriene b (ltb ) and leukotriene c (ltc ) were determined via elisa technique. another volunteer had taken mg aspirin one day before taking the blood sample (no ). results: txb plasma levels increased from pg/ml at min to pg/ml, pg/ml, pg/ml and pg/ml at , , and min (p< , ) . ltb and ltc plasma levels showed an increase during the first few minutes (ltb : min: llpg/ml, min: pg/ml; ltc : min: pg/ml, min: pg/ml (p< , )) followed by a decrease to normal values at min. in the sample no the cyclooxigenase-pathway was completely inhibited, the txb plasma-levels did not alter at all, whereas ltb and ltc -plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il- secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il- productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early th century, however the protein was not isolaled until . the human gene was identified and cloned in , which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a - day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet ; : - ) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p< . ) in patients receiving allogeneic blood (n= ) or operated without blood transfusions (n= ). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n= ). in contrast, specific igg-levels increased sigmficantly (p< . ) in patients receiving allogeneie blood (from . + . to . _+ . ie/ml) whereas in patients receiving autologous blood a smaller increase (from . + . to . + . ; p= . ) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l . + . to . + . u/ml) compared to those patients with autologous blood ( . + . to . + . ). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin- (il-i), interleukin- (il- ) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during - hours after surgery in patients undergoing arthroplasty ( hips and knee). kf, il- , it- , thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il- concentrations ~ere analysed at the beginnlqg and end of the - hour blood retrieval period. in a separate study ( hip arthroplasties) f~f, il-i and il- ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il- coocemtratiems rose from a median value of to pg/ml, p<o. , during the blood ret.rieval period. in c was detectable in patients, range: - pg/ml. il-i was not detectable in the patients. in retrieved blood tag was > mg/ml in all samples (ref:< . mg/ml) and at was . - . units/ml (ref:o. - . ) . the il- concentrations in retrieved blood was > pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n= ), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: - pg/ml) and il-i (range: - pg/ml) ~re present in all samples but ii- (range:o- pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients ( . %) were perioperatively transfused. thirty-six ( . %) patients were given autologous blood , while ( . %) received homologous blood. no patients received both autologous and homologous blood. twenty eight ( . %) patients developed postoperative infections. non transfused patients had a . % infection rate , those receiving autologous blood had a . % infection rate, whi]e in the homologous blood group the infection rate was . % (p < . ). univariate analysis showed that infections were significantly related to operative blood loss (p< . ), length of operation (p< . ) blood transfusion (p< . ) and attending surgeon (p< . ) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, month old rats were transfused intravenously with syngeneic heparinized venous blood ( x ml, every other day), and , and days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a : : . : . mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of . microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over - hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of - -weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il- . we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium- m ( mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a % topload of leh ( mg of phospholipid, . g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, % at minutes and % by hours. the spleen accumulated activity at a slower rate, % at minutes and % at hours. at hours, autopsy biodistribution studies revealed that approximately . % of the dose is in the blood pool, . % in liver, . % in spleen, . % in lungs, . % in muscle and . % in urine, with trace levels in kidney, brain and heart (< °/o). in a hypovolemic model, rats were % or % exchange transfused with mtc-leh. in the % exchange model, mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at hours. with the % exchange, % of the leh was in circulation at hours. the interaction of leh with platelets labeled with indium- was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha ( ) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens ( ) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine ( ) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength ( ) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t - years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. o]~ga- pufa there continues to much interest in the application of the mega- pufa in clinical nutrition. the basic principle has been that the mega- pufa will displace arachidunic acid and result in a decrease in eic san id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega- incorporation with continuou~ enteral feeding both in control and endotoxic animals within days. this includes the liver, spleen, circulating and alveolar marc phages and the lung. this incorporation resuls in significant changes in the eicosan id production including pgf and ket -pgflalpha. there is improvement in the cardio-vascular reep nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of mega- pufa. clinical studies using short term entsral nutrition with mega- either alone or with other enteral supplements in a number of clinical settings have shown significant mesa- incorporation and decreased eicosan id production. these positive results must be discussed with the additional evidence that long term omega- supplementation decrease eic san id production but als induce a state of immune suppression that is capable of increasing transplant sunvival. these ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf- mice aged - wks received % tbsa fullthickness dorsal burns & were resuscitated with cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast hrs, chow hrs). at hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p< . , ** p< . compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of adult icu patients who required enteral feeding for > days. patients entered the study within hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least ml/hr by hr after event. results. both groups tolerated administration of formula well. for patients fed > days, the median los was % shorter (p=o.ol) for the--supplemented group ( days) compared to the conventional group ( days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o ). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia ( vs. days) and skin/soft tissue infection ( vs. days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into groups ( n= each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within hours following surgery ( ml/hour). it was progressively increased reaching a full regimen on day . on hospital admission and on post-operative day and , the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day , , . the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day . comparing post-operative day versus day a significant increase of prealbumin (p< . ) and rbp (p< . ) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p< . ) and a significant recovery in delayed hypersensitivity response (p< . ) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent ( % in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = % vs vivonex ten = %), mean mof score (immun-aid = l.b vs vivonex ten = . ), or mortality (immun-aid % vs vivonex ten = %) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> ), patients receiving immun-aid appeared to have fewer septic complications ( % vs %) and their mean mof was significantly lower ( . _+ . vs . + . , p = . , student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from patients in septic shock and heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for minutes. pmn (p{o, ) and monocytes (p<o, ) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the %-bcaa vs %-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o for all). clearances of bcaa increased with increasing bcaa dose (p<o.o ); arg and gln clearances were directly related to the bcaa clearances (rz=o. , p<o.o i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation ( gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with x t.spiralis larvae. thirty to fifty days later rats received gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o mucosal mast cells and their subsequent degranulation. mast cell-derived -ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group ( % vs %). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m- polyunsaturated fatty acids (pufa) from safflower oil (so) or (o- pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at % in the diets was adjusted to provide % each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for days, lipopolysaccharide (lps, mg/ g) was injected through the tail vein. the percent of animals surviving in the mo group was % ( / ), and did not differ significantly compared to % ( / ) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so ( . + . ng/ml) and those fed mo ( . + . ) did not differ significantly. the levels of prostaglandin(pg)e , -keto-pgflc~ and tumor necrosis factor-ct (tnf-c were determined min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and h after the lps administration (lps+). the data (mean_+sd; n= ) are as follows. group these data indicate that a marked reduction in the plasma levels of poe and -keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a . % reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf- outhred female mice ( - g) were fed purina # chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from to days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a- desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for weeks. the incorporation of dihomo-y-linolealc acid (dgla: : o)- ) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between - % compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps ( p.g/kg body wt), the plasma levels of both pge , and txb , were significantly decreased (p< . ) by nearly % for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a- desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a (pla ) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was % ( / ) which was markedly higher (p< . ) compared to only % ( / ) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly % reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c bl/ mice that have been stressed by immobilization. the proteins have been labelled with ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of ss-methionine reveal about protein spots, from which a small fraction -about are altered in both qualitative and quantitative manner. the major changes are in the range of to . d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery days and days preoperatively and on the ist and th postoperative days (study-days i, , , and ) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived days preoperatively an enteral nutrition enriched with omega- fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a ( . pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb (mean values . ng/l x i~ cells) compared to the placebo group ( . ng) at study-day [p, . ). in contrast, the capacity of neutrophils to produce ltb was not significantly different in both patients populations at study-days and . the omega-oxidation of ltb to its biologically less active metabolites oh-ltb and cooh-ltb was not significantly different in both groups. however, neutrophils from group f patients generate more ltc at study-day (p( / ). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega- fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega- fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega- fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution ( kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn ( , g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide ( , g n derived from ala-gin = % gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o, ). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p< , ). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p< , ). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o ~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai y erodes both fimetianal and stm~ tissue. wiu _b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~ po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh iu daily three days before the experiment (gh- ), another group treated with gh ie at the start of the surgical procedure (gh- ) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u- c-glutamine. substrate fluxes were measured one (lh) and five ( b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh- : . + . mg/kg, gh-i: . +_. . mg/kg, con: . +- . mg/kg, p= . ). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh- : . + . gmol/min at lh and - . + . in.tool/rain at h, gh-i: - . + . i.tmol/min at lh and - . + . pmol/min at h, con: - . +_ . g mol/min at lh and - . + . /amol/min at h, p= . , p= . ), whereas the absolute uptake of glutamine in hindleg was unchanged (p= . ). glucose uptake in the hindleg was decreased (p= . ). net glutamine uptake in liver was attenuated (p= . at h), whereas net glutamate release from liver was increased (p= . ). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of , ; , or , i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from . calories per kg body weight to . calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus . to plus . g n / days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either u of rhgh bid (sc) or no alternative medication. the study period lasted days. from daily hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days and to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf and igf , igfbp were measured daily during the study period, on day a hour profile of gh was.performed. samples were collected each minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf differed significantly between the groups for the whole study period, but not igf and igfbp- . the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n= , age range - yr, bmi . + . kg/m ) or for days additional gh ( x i.u.s.c.) (gh group, n= , - yr, . + . kg/m ). metabolic studies (calorimetry and lsc-leucine infusion) were performed (test a) and days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the -week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was + ; + (co) and + ; ± (gh) pmol/kg ffm/h (n.s.). protein breakdown was ± ; + (co) and + ; + (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was ± ; + (co) and ± ; + (gh) pmol/kg ffm/h (p< . for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days - : gh + vs co ± g; p< . ). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf- levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf- . this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: patients (both sexes, age: - years, bmi - kg/m ) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received ug rhlgf- /kg body weight/twice daily or placebo during treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition ( g chng, o. g aa/kg) troughout the whole study period. cumulated nitrogen balance, -methyl-histidin exretion in urine as well as serum-igf- levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to groups (n= /gronp): gh-high ( + mg/kg/day), gh-iow ( a mg/kg/day) or control (saline). following subcutaneous treatment (every hours for days) with gh or saline, mice were challenged i.p. with e.coli (lxl /body). survival rate was recorded every hours. in the next experiment, gh-high and control animals were sacrificed hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for hours with lipopolymccharide ( p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf- ) and the insulin like growth factor binding protein (igfbp- ) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf- and igfbp- . materials and methods: patients sheduled for esophagus resection were investigated once they had given written informed consent. patients showed acute alcohol abuse and were treated with clonidine postoperatively• patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf- and igfbp- were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf- and igfbp- levels showed no differences between the groups. discussion: igf- and igfbp- plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n= , mg/kg/day), low dose igf-i (n= , . mg/kg/day) or saline ( = ) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for hours with lipopolysaccharide ( ).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l bioassay. results igf-i improved the survival rate at a dose of mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than /~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated patients ( scn, cyclic neutropenia) with r-methug-csf (filgrastim). thirty of patients with scn and all cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than /~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between . and ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations hours after administration. these levels were -fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately - -fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day of gestation (parturition in rodents occurs on approximately day ) survived a lethal challenge of group b- hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res : , ) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood : , cairo, pediatr res : , ) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. infants < hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm ( - wk), preterm ( - wk), and term (> wk) and randomized to receive either a placebo or , , and gg/kg/qd or and p.g/kg/bid of rhg-csf infused by pump over hour for consecutive days. cbcs were obtained at , , , , and hrs. tibial bone marrow aspirations were performed hrs after study entry and differential counts and cfu-gm pools were determined. c bi expression was determined at and hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at , and hrs following administration of both and ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred hrs after and ~tg/kg/d ( - %) (p< . ) and ( % -+ %) (p< . ), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool ( _+ % vs. + %) (p< . ) (placebo vs. ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo ( . _+ . vs. -+ ) (colonies/l(p cells/plate). c bi expression was significantly increased hrs after bg/kg/d of rhg-csf ( + % vs. +- %) (p< . ). peak serum g-csf levels occurred at hrs and were dosedependent. the half-life of rhg-cse was . + . hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. of patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research. : , ) . methods: awake ylo beagles had serial cardiopulmonary and laboratory studies before and for up to days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until days after clot placement, animals received high (n= ) or low dose (n= ) g-csf or protein control (n= ) subcutaneously. results: survival in high dose g-csf animals ( / ) was significantly improved compared to low dose ( ) and controls ( ) (p< . wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day after clot, p< . ) and mean arterial pressure (day , p< , ) compared to low dose and controls. high dose rcg-csf increased (p< . ) peripheral neutrophil numbers both before and after clot implantation ( hours to days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p< . ) rise (day ) and fall (day ) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin ( to h, p< . ) and tumor necrosis factor (tnf, h, p< . ) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and or hours later. lps-inducible tnf, il- , stnf-r p and il-lra were assessed in the supernatant of whole blood incubations stimulated with ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about % hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p which was indetectable in blood incubations from untreated donors increased dramatically hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as /.tg/kg iv every h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either or days before or hours after cp. results. mortality was reduced from % to about % with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin and lactate were attenuated the first hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin and lactate are suppressed the first hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels h after lps administration. gm-csf pretreatment ( ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin- (il- ) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il- -tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in hs. continuous infusion for days. safetyandefyieacy will be assessed till to day . the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > (score corresponds to expected mortality rate of %).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than , were included in the study. filgrastim was given by daily continuous intravenous infusion for days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > apache i score points on day after onset of treatment. results: none of the patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to . + . /~tl (mean + sem) and reached a level of . +_ . /tal at day after onset offilgrastim therapy. the apache ii score initally was + . (mean + sem) and as an indicator of filgrastim response a decrease of points ~dthin days oceured in out ot patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= ) or colorectal (n= ) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il- , which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of pg/ml (preoperative < pg/ml) on day and day to after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < , ) correlated with infectious complications in patients whh gastric cancer (n= / ). secondly patients could be arranged into two groups according to an anergic (n= ) or normergi¢ (n = ) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= / ) or colorectal (n= / ) cancers. interestingly we found a significant correlation (p < , ) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= ) within the pre-and postoperative period resulted in reduced mean values (about %) for ifn-ff release (preoperative means llo pg/nfl), if compared to patients with normergic dth (n= , preoperative means pg/ml). similar, but less significant results were obtained for il- secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il- ) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s orl o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe -gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)( m$/k bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after weeks was significantly decreased by administratlon of rh~-csf(p< , ).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached . %. nextly,treatn%ent wlth rhg-csf(s ~ $/k body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p< . ). iv~oreover,functions of neutrophlis which were phagocytic activity and h p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~( ~ /ks b ody wt) (p< .( ). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < /mm ) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < /ram ) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of to mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within to days. in most of them, spectacular correction was observed within hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed ( cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, , rue de s~vres, hopital necker, paris, france. changes in serum g-csf and il- after surgical intervention hitoshi toda , atsuo murata , hidewaki nakagawa , takesada mori , nariaki matsuura osaka university medical school, osaka, wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin (il- ) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il- and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l- was less than pg/ml and g-csf less than pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il- levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il- (x) was y= . x+ . (r= . , n= , p< . ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il- was pg/ml and g-csf pg/ml respectively. the estimated value of g-csf was pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than were treated with filgrastim ( . - l.tg/kg/day) for prophylaxis of sepsis on days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced ( - reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of % +- % ( % - %) compared to pretreatment values of %. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p< . ). before initiation of csf treatment urinary neopterin was similar in both groups of patients ( +/- and +/- lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day the mean value was about % above the basal level (p< . ). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than fold induction was noted and the difference between g-csf and gm-csf was highly significant (p< . ). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp- human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r a and pot ca . john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, . at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i. ], dmpg[ . ], cholesterol [ . ] , and monophosphoryl lipid a [ - gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected - days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing , or gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds for control (liposomes with no lipid a) mice was x bacteria; ld for mice vaccinated with p.g was x ( -fold increase in resistance) and with ~tg was x bacteria ( -laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld for control (liposomes with no lipid a) mice was ng lps; the ld for gg lipid a was rig lps ( -fold increase in resistance) and for xg was ng lps ( -fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld of franciseua in normal mice or mice inoculated with liposomes without lipid a was - bacteria. in contrast, mice vaccinated with liposomal lipid a ( ggl survived challenges as high as , bacteria, ( logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il- , and il- responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd . to evaluate if blocking lps-cd interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a hour period. to evaluate the effect of treatment with anti-cd mab, animals were given either nothing, an isotype control or anti-cd mab ( mg/kg) rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il- , i,- , il- and il- , and liver enzymes during a hour period revealed that treatment with anti-cd mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi ). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a kd n-terminal fragment (rbpi; ) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi has equivalent antibacterial activity to bpi against rough gnb but is up to x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi block the cd -dependent lpsinduced synthesis of the cytokines tnf, il- , el- and il- in vitro. rbpi has also been shown to inhibit the lps-induced synthesis of reactive metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi was not toxic to rats after daily consecutive i.v. doses of mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd ) of myeloid cells or as a soluble serum protein (scd ) lacking the gpi-anchor. binding of lps to mcd triggers cell activation while binding of lps-scd complexes to cells such as endothelial or epithelial cells that normally do not express mcd activates these cells. these pathways are shown in schematic form below. ~di mcd plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: mg/kg --- . mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il- , or il- , but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of - m and has a t / of • h in normal humans. methods: normal volunteers were randomized to receive either tnfr:fc vehicle (n= ), or low dose (ld) tnfr:fc ( mg/m iv, n= ), or high dose (hd) tnfr:fc ( mg/m iv, n= ) infused over rain prior to endotoxin (e) (escherichia coil : ; ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point ( - h) in subjects given tnfr:fc compared to vehicle ( h) (p=. ). at h, h (post fluid load ), and h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=. ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<. ) and later leukocytosis (p< . ). peak tnf bioactivity in subjects given vehicle was + pg/ml and < pg/ml in the ld or hd tnfr:fc groups (p<. ). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=. ) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<. ). decreased levels of il- (p=. t), granulocyte colony stimulating factor (p=. ), and il- receptor antagonist (p=. ) were found after ld or hd vs vehicle. il- levels were lower after ld vs vehicle or hd (p=. ). il- levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the ' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥ ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c inhibitor complexes ( ). in this in vitro plasma model we also found that methylpredoisolone alone in doses of mg/ml induced contact activation with the formation of kallikrein-c inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade ( ) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et ( %) and interleukin- ( % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c - nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c - nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c - nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c - nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type- in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type- (cr- ) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c /c convertase formation. we have recently purified the human recombinant cr- in a truncated form (brl , tp-io, scr- ) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: ) endotoxin-induced ards in paf primed animals; ) .- induced ards; ) thermal injury ( % body surface)-induced ards; ) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr- , - mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il- ), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr- in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that ( ) ptx suppresses lps-induced tnf release from activated macrophages; ( ) ptx prevents tnf-induced pmn activation; ( ) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; ( ) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse , d- m nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il- most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il- and il- . moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons ( . +. kg), were randomized to a control group receiving ° cfu/kg of intravenously administered e. co/i (n= ) and a treatment group (n = ), pretreated with . mg/kg of paf receptor antagonist ro - two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving ng/kg of intravenously administered endotoxin (lps) (n= ) and a treatment group (n= ) pretreated with mg of ro - eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for hours. results: baboons pretreated with the paf antagonist had improved oxygenation ( + mm hg v -+ in controls, p < . ) and creatinine clearance hours post e.coli ( . + . ml/min v . _+ . in controls, p < . ). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at hour post lps. this was in concert with a diminished peak cortisol ( +_ mmot/l v +- mmol/l in controls, p < . ), epinephrine secretion ( + nmol/l v + nmol/l in controls, p < . ). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il- and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il- ra, a naturally occurring il- antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha- a and e . future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa , and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn , have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il- and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd)i chain covalently linked to one of three different alpha chains (cd a, cd lb, cd lc) and exist on the cell surface as three distinct heterodimers. cd l a/cdi is expressed on all leukocytes, whereas cd l b/ cd and cd lc/cd are restricted to cells of myeloid origin. cd i/cd interacts with intracellular adhesion molecule- (icam- ), its ligand on endothelial cells. icam- is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = ) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria endotoxin) during the first days after inciosion in the study. the other randomized group (n = ) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of < wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one ( %) of patients who received immunoglobulln. by comparison, nine ( %) of control group patients died during this period (p <. ). within the first hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; ) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_ (meaning a mortality risk > %) were accepted into this protocol. patients were randomized to receive . g/kg of ivig or placebo on day (when they reached sepsis score> ), repeated on day + and + . at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: % vs, % respectively; p< , ). in conclusion, the results of our study in patients with severe surgical sepsis were the following: ) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', ) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg ( ) - ) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in patients with sepsis and septic shock (infection ~ ) - ), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score - ) and severity of sepsis (elebute sepsis score - ) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo ( .i % albumin) or polyglobin n" - ml ( . g)/kg on day and ml ( . g)/kg on day i. with an anticipatedpopulation size of patients the study should comply with the statlstical requirements (estimated mortality: %, with a % reduction in -day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november , more than patients had been included; patient enrollment will be finished in . previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il- , increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled patients at academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) . or . mg/kg/hr by continuous intravenous infusion for hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over days utilizing a linear dose-response model, assuming a log-normal distribution. results: patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< . endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d ) and tolerance of bn ( iv infusion of mg x /day over days) in severe sepsis has enrolled pts. the day mortality rate was % for the placebo group and % for the bn group (p = . ). the efficacy of bn was greater in pts with gram-negative sepsis: the -day mortality rate was % for the placebo group and % for the bn group (p = . ). bn also reduced mortality among pts with gram-negative septic shock (mortality was % for placebo vs % for bn ; p = . ). using statistical adjusments for pronostic factors, the relative risk of death of the bn group was . ( . - . , % confidence interval; p = . ). this risk corresponds to an adjusted reduction in mortality of % for pts receiving bn . no differences in mortality rates were found between the placebo and the bn groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn groups. bn is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~ lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for -day mortality (jamb. ; : , - ) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin- receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x = . , p [] . , log.normal) for all patients in the trial• survival benefit began st approximately % baseline risk of -day mortality. for the $ patients with a predicted risk > %, there was a % reduction (p= , $ log normal). when we examined the variation in patients above and below the % risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < % risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > % risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp- , in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp- on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population ( . %) of pop. low-risk pat. (ap< ; mortality: %) and the small groups of pop. pat. at risk lap= - ) and high risk lap_ ) with a significantly higher mortality ( % and %, mainly due to sepsis). subsequently, among consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n = ): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day : ml/kg, day : ml/kg). second study period (n= ): iggma (pentaglobin r, biotest, dreieich, frg, ml/kg on days to ). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n= , high-risk: n- ), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n= : p<o. ; iggma, n= : p: . ). in this group, ig led to higher (p< . ) response rates, defined as an ap score decrease -> within four days (igg: %, iggma: %; con.: %), and reduction in mortality (igg: %, iggma: %; con.: %), statistically significant (p< . ) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, aminoacids et (et ) in rat abdominal sepsis. methods. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et were determined with amersham tm endothelin rias , and h after sepsis induction. experimental groups: . cp control, . volume replacement (vr); , % saline ml/kg/h continous iv infusion started after h, . antibiotic; imipenem mg/kg iv after h, . e ®; mg/kg iv after h, . vr + imipenem + es® after h. results. high concentrations of both big et and et could be demonstrated after h and lasting for h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e ® significantly reduced et both , and h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et was the same as for e ® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e ® reduces circulating lps and tnf which is the probable mechanism of the suppressed et synthesis. the unaltered big et fraction after e ® treatment indicates conversion of big et to et as the site of action responsible for reduced et . conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et . e ® anti-lps antibody significantly reduces plasma et while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e- in congestive heart failure has been demonstrated aumann, ). therefore, we investigated the effect of bu-e- in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [ ] [ ] [ ] [ ] [ ] [ ] were anesthesized with fentanyl/dormicum, ventilated (n :o = : ) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study ( animals) to evaluate the effective concentration of bue- in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of ~g lps/kgkg/h ( :b , fa. difco). the hypodynamic state was defined as a decrease of cardiac output by % of steady state levels. a wedge pressure of - mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) groups with animals per group were studied. the groups were treated as follows: group i, lps and , % nac ; group ii, lps and bu-e- ( #g/kgkg/h); group iii, famotidine (h -blocker) pretreatment ( mg/kgkg), lps and bu-e- . results: the pilot study in healthy pigs revealed, that bu-e- had positive inotropic effects. these effects were inhibited by the h antagonist famotidin. bu-e- however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do ) were not significantly influenced by bu-e- application (group i versus group ii). bu-e- did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e- led to a further significant decrease of lvsw (p < , ). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h -agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci . % (control without endotoxin, n= ). group e : endotoxin tjg kg " bolus i.v. + naci . % (control with endotoxin, n= ). group c : endotoxin pg kg - bolus i.v. + cll u kg - bolus + u kg " h "~ i,v. (treatment group, n= ). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m ) and after (m ) and rain. (m ). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p< . ) between group e (lung , liver ) and c (lung , liver ) (group k : lung , liver ). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july to june , patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of . years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the st, rd, th and th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk receptor is constitutive. the bk receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk (bradycor [cp- ]),bk~ (cp- ) and bkz/bk (cp- ) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk agonist) in the lps-treated ( ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk agonists increased with time and at time min appeared maximally induced. constant iv infusions of cp- blocked bk but not bk~ and cp- bk~ but not bk responses. cp- ,cp- +cp- and antril+cp- blocked both bk and bk~ responses. antril alone had no effect on bk or bk~ responses. within - min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk ,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk receptors. in vitro studies demonstrated that beth bradycor and cp- (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp- is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september and july olt's in patients were performed at our department. the actuarial -year patient survival is %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from to hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss - ), mean temp. was . f, leukocytosis was k, pan was , fin was . , and peep was . at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t am and a thickness of nm. the ami-fungal efficacy is - times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than nm with a mutina ld of > mg/kg. it has been used in dosages up to mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a % response rate for invasive candida infections to % response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was % among placebo treated patients compared to % for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from % to % ill placebo and ambisome treated patients respectively (p< . ). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of mg/kg at day , , , , and after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of elisa units during months. cmv infection occurred in / ~eronegetlve and in / seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower ( / ] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f t ) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection ( v / ), but no difference in incidence of disease ( vs / ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from patients with different types of leucemia after autologous and allogenzc bmt had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in % of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity - days after intranasal inoculation of influenza virus a (h n - ) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest . consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years and . in the same period of time pri~ total hlp replacen~nts were performed under i entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than %. in the selected i cases the septic rate was % indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c ) and fourth (c ) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate ( "= - . , p < . ). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c , c , crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il- ra, il- , il- r, il- r, tnf-ct, tnf-~r (p ) and icam- . in contrast, pmn-elastase, il- and il- closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd +/cd + ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il- and il- , however, closely correlate to the clinical course and may prove valuable for follow-up. the cd +/cd + ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and ( ) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of mu/ml xanthine oxidase (xod), mm hypoxanthine (hx), mm fec and mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il- . cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& ) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, ) the trant~ of vim.©n~e traits between ~renr~a.,dsms and ) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il- receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h pital erasme, route de lennik , bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin ) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il- by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il and monocyte chemotactic activity when cultured. these activies were significantly increased ( -fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il- in patients with bacteriologic proven abdominal infection (intraabdominal abscess , diffuse peritonitis , pancreatic necrosis , pancreatic abscess ) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a to hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il- determination were drawn preoperatively, intraoperatively, and until h postoperatively in regular intervals (min /pat), results: preoperative apache ii was in median (rain , max ). patients fulfilled the criteria of sirs. of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p= , i, spearman coefficient). preoperative cardiovascular (systol. rr< mmhg) and respiratory (pao < mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p= , i, wilcoxon; pulmonal: p= , ) and il- (cardial: p= , ; pulmonal: p= . ) overall, lps, tnfa and il- values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i , mean pg/ml) than in those who did not fulfill these criteria (group b; n= , mean pg/ml; p= , i, wilcoxon). il- was significantly higher in group a (mean pg/ml) than in group b (mean pg/ml; p= , o i wilcoxon). conclusion: perioperative tnfa and il- were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel - , wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= ) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after hours. stastistieal calculations were performed on a personal computer with the spss programm vers. . (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla -activities [endotoxin range from . eu/i, sd= . to . eu/ , sd- . (p < ), ifn-¥ levels, range from . pg/ml, sd- . , to pg/ml, sd= (p < . ), circulating pla -activities range from . , sd= . to . u/ , sd= . (p < . ) and biopterin range from . nmol/l sd= . to . nmol/l, sd= . (p < . )]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = . , p < . ) and bioptefin synthesis (rv= . , p < . ). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = p < . .. p • , . . ) and to the pla -actwtues (rp = . , p < . ). the biopterin synthes~s correlates slightly with the pla -actn,ities (rp= : . ; p < . ). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of %. anaerobes were found in . %, anaerobes were isolated in . %. there were aerobic and anaerobic associations in . % and microflora was not found in . % of the cases. express method of anaerobes discovering let to receive information on - days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of patients with abdominal sepsis treated at the surgical intensive care unit during a -year period, ( %) had an age of years or more. were women and were men, a sex distribution not differing with patients younger than years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was (- ) days including in median days in the icu. figures in patients less than years of age were comparable ( (- ) days out of which in median days in the icu). apache ii and sss-scores did not significantly differ ( . vs and . vs . , respectively), between the groups. neither did the incidence of organ failure differ ( / vs / ). however, the incidence of multiple organ failure was significantly lower in elderly patients ( / vs / (p < . )). the mortality rate, however, did not differ between the groups ( / vs / ). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n-- in each group). at , , , , and hours (n= /timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of : i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was % both at and hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from hours and on and in the proximal and distal small intestine from hours and on. different types of alterations in capillary permeability seem to appear: ) a temporary short increase e.g. in the proximal small intestine and spleen; ) a temporary longer increase e.g. in the colon and kidneys; ) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p integrins (cd l a, b,c/cd ), and the fc'?r (cd , cd , and cd ), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score > ) or thermal injury (> ~ total body surface area, % full thickness), and healthy controls. the expression of cd b and c and to a lesser degree cdi a was significantly reduced on pmns. the expression of cd and cd but not cd was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, ~ ethesda ave, cincinnalt, oh, usa, - s b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet , dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e ) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il- . the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =. , mean b state = . , mean ~ state = . ). the accuraay of this estimate was prospectively analyzed in this group of m~itlple patients of whom % had sepsis and % had ssptlo ards. the survivors had a mean p death of . and the deaths had a mean p death of . . the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of patients who were enrolled into one of four treatment groups: ( ) . mg/kg of anti-tnf mab, ( ) . mg/kg, ( ) mg/kg or ( ) . mg/kg at study entry and the second dose hours later. the small sample size in each group (n= ) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin- may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x ) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of mg/kg tnf~ mab, . mg/kg tnf~ mab or placebo ( . % human albumin).patients received standard aggressive medical/surgical care during the day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca , was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca . as a single infusion or in combination with ha- a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage was an open label trial in which groups of patients each with the clinical diagnosis of sepsis received ascending doses of ca ( . , , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of ha- a ( mg) and placebo or one of doses of ca ( , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of placebo or one of doses of ca ( . , , mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il- , and il- levels.a total of patients were enrolled from clinical sites ( in stage , in stage and in stage ). primary analyses were performed on patients in stage and . there were patients who received ca exclusively and patients received placebo. administration of ca was well tolerated at doses up to mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in % ( / ) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca . the mean half-life was - hrs ( - hrs). a dose related decrease in tnf concentration was observed hr post infusion of ca . tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak f. it is a f(ab') fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak f or placebo were administered ( , ; , and i mg/kg) over a perid of hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il , il and tnf), and outcome. at this time only an interim analysis of patients is available i indicating that mak f in all dosage groups resulted in a decrease in il . this contrasted to a further in crease of il in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all patients in the study will be presented and discussed.$ s staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a atom-chain spacer. the detoxification capacity was ug[et/ml column material. the biocompatbility resulted in ~ platelet recovery. the column contained ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine . iu/h in the inflowline after bolus injection of . iu. hp was performed on pigs at a rate of ml/min by means of a roller-pump until the animals succumbed (h). animals served as controls (c). serum et levels rose from . pg/ml to , pg/ml after hours in the c and from . pg/ml only to pg/ml in the h group after hours whieh was highly significant. survival time could be extrended from to min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b( rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from to consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in °, and by septic-toxic event in %. all patients required mechanical ventilation (fio > , ) . ° showed hyperdynamic shock. % had renal and % hepatic failure. medium appache ii score amounted to , points. cvvh was performed in postdilution mode with a polyamide membrane (fh ) and high volume exchange ( l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in %, hemodynamic in % and renal in % of the cases. % of the patients survived and were discharged from hospital. of non-survivors ( %) died because of fatal mof within h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web ), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin ( p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n= ), or after pretreatment with web ( xl -gm, n= ), or diclofenac ( #g/ml, n- ). furthermore, the application of specific antitoxin (mg/ml, n= ) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, . mg/ml) (n= ) and the combination of immunogiobulins, web and diclofenac (n= ). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of . times higher than the control, starting after min and a delayed onset of edema formation resulting in a mean weight gain of g after min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about times that of controls in the toxin treated lungs. pretreatment with web or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st ( -n,n,n trimethylammonium-(r)- -isovaleroyloxy-butanoic acid z- -( -chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of h-paf (ki= . x - m) to rabbit platelets. the present study shows that pretreatment of c bl/ mice with st , administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli :b injected at mg/kg intraperitoneally). very interestingly, st administered at the same doses as above (i.e. . , . , and mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia ( . °c) induces heat shock protein ('asp) and increases smvival - fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp- by sodium arsenite improves survival in a rat sepsis model (abstract a am. rev. resp. dis. vol. , ) . the effects of heat are complex and in addition to formation of lisp- include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp- in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite ( mg/kg) and ethanol ( ~ of % ethanol) caused marked induction of hsp- in lung, gut, kidney, and liver, which was comparable to heat-induced hsp- . female nd mice weighing - gms were pretreated with arsenite or alcohol hours prior to challenge with escherichia coli endotoxin (-ld ) and survival was compared to control mice. results: survival at hrs. for arsenite treated and alcohol treated mice was % and % respectively and was statistically different from the % survival for control mice. (p< . ) (n= mice per group). however, at days post endotoxin, there were no differences in survival in the groups, i.e., ~ % survival for all groups. in contrast, the protective effect of hyperthermia remains present at days, i.e., ~ % survival vs % survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp- formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered x per day from the day be ~ fore the operation until the th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in / after pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from . % in the placebo-group to . % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from . % in the placebo-group to . % in the treatment-group (p ~ . ;fisher's exact test). in march final results with more than patients will be presented for the first time. (= po < mm hg, b s-creatinin > mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only of deaths occurred during the st week, all other deaths occurred late (after - weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. -- ) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only % of deaths today occur eady in the course of the disease, whereas this percentage varied between - % just years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for to years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in patients with acute pancreatitis ( males, females; median age: years, range: - years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type (slcam- ) were determined using immunoassays (henning, bender, t cell sciences). / had increased concentrations of stnf-r compared to the th percentile obtained in healthy controls (> . ng/ml), and / patients had increased neopterin (> . nmol/i), / presented with elevated slcam- (> u/i). all patients with increased neopterin also had increased stnf-r, patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = . , p < . ). weak associations between age and stnf-r (rs= . , p=o. ) or neopterin (rs= . , p = . ) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as ( , ) and characterized by increased serum or urine amylase levels (in about % of patients) in the fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the ffs, studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery ( , , ) . in a recent study ( ), evidence of pancreatic cellular injury was found in out of patients undergoing cardiac surgery, with out of these patients presenting abdominal signs or symptoms and developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), hours alter surgery and in the folfowing days alter surgery (days , , , and ). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day . we measured abnormal levels of trypsin and pancteatic iso-amylase in % of patients and observed simultaneous releases of these enzymes, the fi,'st one in the hours after surgery and the second more intense from day and pa~icularly on day after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about % of our patients : pancrealic abnormalities were revealed by scan in patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion ( ). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin- (il- ) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii- (elisa), transferrin and crp (nephelometry) were analysed daily along the first i days of hospitalisation by patients with acute pancreatitis admitted to our hospital from / to / . it was judged whether the patients have either interstitial (aip) (n= ) or necrotizing (anp) (n=lg) pancreatitis. patients with anp have died during the course of pancreatitis (mortality= . %). results: -severity o~ pancreatitis: signifcant differences (p<o. , mann-whitney test) could be calculated between aip and anp for endotoxin, il- , transferrin and crp during the i days of monitoring. -mortality: except of il- on days , , and after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. -organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il- and crp along the i days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il- and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from / - / , patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n= ) and n (n=l ) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in patients. the etiology of ap was gallstones in , alcohol abuse in , hyperlipidemia in and other or unknown causes in patients.five of patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il- concentration in blood reached up to pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of pg/ml was correct in patients ( %, sensitivity (se): %= specificity (sp): %, (z: , ). high levels of il- over pg/ml correlated well with the prognosis ifri in the first week (accuracy: %, se: %, sp: %, c~: , ). the blood levels of il- reached a maximum of pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of l,- blood levels indicated correctly the severity of ap in out of patients using a cut offtevel of pglml (accuracy: %, se: %, sp: %, ~: , ). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il- and il- . in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il- , and to a lesser extent of [l- , can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n= ), around the pancreatic duct (group b, n= ) or around both ducts (group c, n= ). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a mtcalbumin colloid with a diameter of nm, was measured before, , and days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day , the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day . the opossums in groups a & had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day in group a & b (p<o, ) and at day it~ group c (p< ,ol). at day , res function in group c was significantly worse than that in group a & b (p< , ). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day (p< . ), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b ) in lewis rats weighing - g. there were five groups: group a (n= ) were untreated controls; group b (n= ) were given isotonic saline intraperitoneally and observed for hours; group c (n= ) ml x e. coli intraperitoneally and observed for hours; group d (n= ) were given ml x e. coli and observed for hours; group e (n= ) ml x i e. coli and observed for hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser ). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p= , ) and spleen (p= , )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p< , ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel - , wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar( - ocalized jo-diffuse, -~eneralized) and -hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen- , the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, -com:lissural ileus). he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; ) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and ) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding /co hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate - wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. grafts were cryo'd in % dmso, quick-thawed & sutured onto x cm excised full-thickness dorsal wounds on athymic mice. "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained > % cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in - , x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through days of followup. histologic exams on days - showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than % total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we ] as ready availability of urdform lis~ue~. on dec~mt~r , , the fda assumed control of as tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, " ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki- (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since when galllco et el reported theh'use in two brothers with greater than % total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day ), the full thickness wounds were covered with epigard, a synthetic dressing until day . after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from patients at the beginning of surgical treatment (day ) and after , , and days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf- , double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.( ) pharmatec gmbh, frankfurt ( ) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group , cells migrating through skin: + % t lymphocytes (cd ), + % langerhans and dendritic cells (cdla, hla dr, s ), + % cd , + % cd , no b cells (cd , ), % cd r (memory cells), + % il r. approximately % cells possessed cdlla and antigens. cd lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group , cells in skin: langerhans cells were found only in epidermis, cd , and , cd r , rb, ila/ cells around venules, cd (macrophages) uniformly dispersed, no il r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group , one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il r cells, perivenular infiltrates of cd , r but less cd cells, high density of cdlla/ cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht bioassay). the hollow lipid microcylinders ( microns in length and i micron in diameter) show an initial burst ( - ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed , , and days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd and cd , mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code , naval research laboratory, washington, dc. - . expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet ) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to go of the origional size (t %) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from juvenile human bum patients, immediately after wound debridemerit mad on day after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*' ~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of psoriatics and healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx t-lymphocytes were then incubated alone or in coculture with x irradiated autologous lymphocytes in culture medium containing - mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce -surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t si-hsa over a rain period, the chemotac~c peptide imlp ( . . ng) and bradykinin were used to induce cell.dependent and cell- ndependent leakage respectively, the antibodies used were . e (cdis), nri (cdlla) and antibody (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien ( .~. ffi . p.l to . z b.bttl and . ,- . ~ to . z . d respectively. . e ( . - . mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at . mk/kg, the plasma leakage was completely inhibited, antibody nr produced similar results, treatment with antibody did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . patients without infectious coz~lications and group - patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection patients of the group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i- days after injury revealed a considerable decrease of lymphocytes, ed ",$d ~ and cd cells amo~it in the blood, cd /cd ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the - days in pateents of+the i group. but stable ~eple$ion of cd and cd cells amount, lower cd /cd ratio and indexes of leukocyte migration inhibition test in patients of the group were observed~ besides that, these persons showed higher cd cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in % of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. , , st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p integrlns (cd a,b,c/cd ) and the fw receptors (cd , and cd ) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~ integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z ) or thermal injury (> ~; total body surface area, > ~ full thickness) and incubated /n v/tro with gm or g-csf. the j integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd lb, cd , and cd while gm bur not c-csf induced an increase in the percentage expressing cdi a, cd lc, and cd , gm-csf and to a lesser extent g-csf induced an increase in the density ( , fold) of the ~ integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, bethesda ave, cincinnati, oh, usa, - . funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in % and in association with bacterial infection(l %), viral infection( */.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a. of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e -ebiary, a. torres et al, reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of olt in adult patients olt were performed for hbsag positive liver disease (cirrhosis n= , fulminant liver failure n= , retransplantation n= ) in pat. all pat. received . u hig in the anhepatic phase and . u/per day for the first week. a small group of pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are % in pat. who were transplanted for fulminant hepatitis, % in pat. with cirrhosis and long term prophylaxis, and % ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was % under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in % in pat, negative for hbv dna. in hbv dna positive pat. infection rate was %, the total rate of reinfection for all pat. with long term prophylaxis was %the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢ capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il- , ltb , c a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi , poe, lt's), complement components (c a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il, , il- or tnf), and immunosuppressivc agents (poe , il- ). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, - th ave za- , seattle, wa usa key: cord- -ilhr iu authors: nan title: isev abstract book date: - - journal: nan doi: . / . . sha: doc_id: cord_uid: ilhr iu nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb ) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used t mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld . as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan introduction: tetraspanin tspan is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan are yet not fully understood. methods: to study the function of tspan , we have established a fibrosarcoma model consisting of the parental cell line (ht ) and its derivatives expressing tspan (ht -tspan ) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan . co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan . small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan expression. remarkably, among of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht -tspan cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan , indicating their interaction. summary/conclusion: our data show that the expression of tspan in the tumour cells greatly impacts ev cargo. moreover, localization of tspan on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan . introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd and cd -rfp expressing hek t cells by ultracentrifugation. gfp-cd and cd -rfp evs were added to hek t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin- , respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a and u a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin- which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin- recruitment to endosomes in presence of cd -rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, ( ) a significant number of healthy donors should be included, ( ) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, ( ) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: ( ) to establish a clinical reference, we collected blood from healthy volunteers and prepared platelet-free plasma. ( ) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. ( ) we measured all samples by flow cytometry (apogee a -micro) and used custom software (matlab r b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm ) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from ^ to ^ per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first donors analysed, show that within the ev size range of - , nm, the median concentration of cd + evs is . • ^ per ml (apc> mesf), cd p+ evs is . • ^ per ml (pe> mesf), cd a+ evs is . • ^ per ml (pe> mesf), and cd + evs is . • ^ per ml (apc> mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~ μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip ) to j.l.m and t.r.g., department of defence (ca ) to j. l.m., and the national institutes of health (r gm ) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was - %, however particle: protein ratio (indicating ev purity) was approximately x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at nucleotides (nt) and nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~ nt, whereas the protein factions contained small rna of nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~ nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, ) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm ) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af ). methods: an af long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of µm and a regenerated cellulose membrane of kda were used. pbs supplemented with . % nan was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step * ^ rev isolated from conditioned medium by a velocity gradient were injected in the af system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with * ^ rev and injected in the af column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af protocol for characterization of ev by af as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were - times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading ug of total protein for western blot, cd , cd , cd and flot could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about times higher than those of the rest two methods. the total protein amount of precipitation was times higher than exodisc and sec, times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was . %, %, . % and %, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than mins and μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a cm pet c-cp fibre tip was found to be~ e particles ( %). the release of evs was confirmed using dot blot analysis for cd , cd , and cd tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her , cd , egfr, epcam, ca ). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd , cd , and cd and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with tb positive and tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag . using a cut-off above the negatives, the auc of roc curves were . and . , respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l or glypican- on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t (p-tau t ) in human donor plasma were detected. in ad and healthy donor samples, p-tau t signal increased % in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas . results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn , by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since % of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ integrin, a surface receptor for fibronectin and vitronectin. the αvβ integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ -negative endothelial cells (hmec ) were incubated with αvβ -positive sevs from prca cells to evaluate the transfer of αvβ by immunoblotting (ib) and facs. the effect of αvβ -positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec . results: we demonstrate for the first time that the αvβ is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ -positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec . mechanistically, we demonstrate that hmec treatment with sevs from pc cells that endogenously express αvβ , decreases pstat (y ), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec treatment with sevs isolated from pc cells harbouring crispr/cas -mediated downregulation of β , or shrna-mediated downregulation of β , results in increased levels of pstat (y ). this sev treatment also results in a decrease of survivin in sevs and hmec . summary/conclusion: overall, our findings show that αvβ in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r - (lrl); p - (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc (inflammationinducing rarely metastasizing murine hnscc line) and moc (highly metastasizing murine hnscc line) were used for this study. moc and moc cells were transplanted into mice tongues orthotopically, and moc /moc derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc into moc tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc into moc tumours didn't promote metastasis. cd positive t-cell distribution in moc tumour was significantly changed by moc sev injection. t-cell deprivation treatment using anti-cd antibody increased the frequency of metastasis in moc -sev treated moc tumours. from the result of proteomics analysis on moc and moc sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r ca and r ca to aw. desmoglein enhances squamous cell carcinoma tumour development through extracellular vesicles in an il- /mir- a-dependent mechanism introduction: the cadherin dsg is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg or a palmitoylation deficient mutant, dsg cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of cells in scid mice and monitored up to weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg enhanced ev release by % and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg , reduced ev release (~ %) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg -evs were enriched with pro-inflammatory cytokines including il- , a potent chemotactic and angiogenic factor. most importantly, il- was surface-bound on evs. furthermore, rnaseq revealed mir- a, a negative regulator of il- , to be significantly downregulated in response to dsg . treatment with mir- a mimic or mir- a inhibitor decreased or increased, il- expression in scc cells, respectively. summary/conclusion: in summary, dsg plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir- a, which in turn upregulates il- synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas technology. results: we found enhanced phosphorylation of erk / and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of ev-associated proteins, with proteins uniquely expressed in control-evs. another proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc- -cog- evicare grant. hypoxia enhances the therapeutic potential of human cd + stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd + cell-derived exosomes (cd exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd exo. methods: cd exo were isolated from primary human cd + stem cells cultured under hypoxia ( . % o , or normoxia ( % o , n-cd exo) using density gradient ultracentrifugation. cd exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd exo was evaluated via lc-ms/ms and -d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd cells. both h-and n-cd exo expressed cd , cd and cd surface markers. interestingly, h-cd exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd exo. in the same line, h-cd exo also significantly improved perfusion (ratio: . ± . v . ± . ) and prevented ischaemic limb amputation ( % v . %) as compared to n-exo (p < . ; n = - ) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir- ) that could be responsible for the proangiogenic and beneficial function of h-cd exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd exo, and thus, may represent a more efficient clinical strategy for cd exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir- and − are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. ʹ-o-methylated rna duplexes inhibited mir- and − expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp cells with/without tnfα exposure were treated with exosomes and the expression of il- , − , and − was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir- and − expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp exposed to inhexos for h. however, tube formation capacity is slightly decreased (~ %) by inhexo compared to norexo. summary/conclusion: exosomes from mir- and − -depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd . uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab + late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd (referred hereafter as cd fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd -gfp fusion protein from femx-i melanoma, sw colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd fab was prepared from h hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd fab significantly reduced the cellular uptake of cd -gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd antibody stimulated both events. summary/conclusion: the effect of cd fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m r-cd . cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m r-cd were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m r-cd and phluo_m r-cd -mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m r tag is located on the surface of small evs. live cell imaging reveals phluo_m r-cd -positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd , alix, and tsg but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m r-cd construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r gm , r ca , u ca - s , r ca . uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas -based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas -based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas -based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas -based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited ( % spontaneous rate at h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr -ce - - and arc pja and pga rf . introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a -channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed > % encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < for evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~ bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po ca , ro ca , r ca quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h (high cd , low cd ) and promonocytic u (low cd , high cd ) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of nm. nta detected a population of particles with a mode diameter above nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh and ug ca . importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov- cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd , anti-cd , or anti-cd . chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r . following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~ nm) distinguished x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd ab significantly decreased detection of cd with less impact on cd . this discrepancy indicated possible spatial patterning of tetraspanins with cd and cd closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase knockout (casp -/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type diabetes (t d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp -/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp -/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp -/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on rabbits. immediately after surgery and at days and post-surgery, rabbits received ml injections of µg msc-sevs and ha in both knees, and rabbits received -ml injections of ha in both knees. at and weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from to weeks. by weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score ( . vs . ; p < . ) and histological score ( . vs . ; p < . ). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre ( . vs . mpa; p = . ) and overall periphery ( . vs . mpa; p = . ], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, % o ) at birth and returned to room air at postnatal day (pn) . mice received a bolus mex dose at pn . adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn , , , or to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd + immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr -monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r hl (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered . h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day . haemodynamic changes and right ventricular hypertrophy were evaluated at weeks. results: msc-sevs were nm in size with cd / expression. pkh -labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il b in balf peaked at h post-lps, whereas il peaked at h. histology showed significant intra-alveolar cell infiltrate at h. msc conditioned media attenuated il b in balf, whereas a trend towards reductions in il b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered h post-lps as compared to h, h or h. for pah rats, sevs reduced right ventricular systolic pressure ( . ± . mmhg) as compared to control ( . ± . mmhg; p = . ), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at %o , %o and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify inflammatory proteins in the evs. results: there was no difference in evs cultured at %o , %o or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd / / ) and internal proteins (alix, hsp ). evs from primed cells showed a > twofold increase of cc chemokines and a > sixfold increase in cxcl and csf- . protein cargo did not differ in evs from %o and %o . summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of xtg-ad, xtg-ad, and wild type (total of mice; female, male; age: mean = . , sd = . , - months) . plasma samples were collected retro-orbitally for weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde ) and the single endpoint (nde ) were immunocaptured by targeting the neuronal marker l cam. we measured human total tau and pthr -tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = . and . , p < . , cortex vs nde and nde ; r = . , p = . , hippocampus vs nde ; r = . , p = . , hippocampus vs nde ). levels of p-tau in nde showed positive correlations with levels in the cortex (r = . , p = . ) and hippocampus (r = . , p = . ); however correlations were not observed for nde (r = . , p = . vs cortex; r = . , p = . vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. ( xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model ( xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in patients with ms and healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd + (monocyte), cd + (platelet), cd + (neutrophil), cd + (t-reg), and cd + (endothelium). pm and pm . concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd + (p < . ), cd + (p < . ), cd + (p < . ), cd + (p < . ), and cd + (p < . ) in patients compared with controls. in cases, pm was inversely associated with cd + evs (pm . , β = − . ; p < . ), cd + evs (pm . β = − . ; p < . ), and cd + evs (pm β = − . ; p < . ; pm . , β = − . ; p < . ). on the contrary, in controls pm was positively associated with cd + evs (pm β = . ; p < . ; pm . , β = . ; p < . ). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l cam (l cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd ) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l cam and glast) and immunogold labelling (cd , cd and l cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h c cardiac myoblasts and raw . monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da , da . vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale ( - nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot . introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u monocytes (exu) and infected u (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd , tsg , . immune responses of naïve u cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il- , il- and il- cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il- -dependent manner. specific host signalling molecules are strongly modulated by the exi, including p , jak and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il- -dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas based genome editing was performed to knockout cd b in human monocytic thp- cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ- . release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr receptor on monocytes as demonstrated by crispr-cas based cr genome editing in thp- cells, and by using cr knock out mice. isolated tgf-β -transporting vesicles reduced the inflammatory response in human m macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β transporting vesicles is investigated in detail and results in inhibition of il- β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β -transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr -dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet projects c , c , c and z . introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin- and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd is associated with a poor prognosis for some cancers. conversely, cd blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd as a diagnostic and prognostic biomarker and as a therapeutic target. cd is also expressed on extracellular vesicles (evs), and we reported that cd expression identifies a distinct population of evs from those that express the traditional ev markers cd or mhc . cd -, cd -and mhc -enriched vesicles contain distinct small rna populations (pmid: ), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd -deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd and exportin- /ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd -deficient human t cells and in cd -/-mouse plasma were enriched in ʹ- -methylguanosine-capped micrornas and mrnas that depend on the exportin- /rangtp pathway. knockdown of cd in wild type cells or thrombospondin- treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd with components of the exportin- / ran nuclear export complex and its known cargos and between the cd cytoplasmic adapter ubiquilin- and the exportin- /ran complex. interaction of cd with exportin- was inhibited by leptomycin b, which inactivates exportin- and increased levels of cap-dependent rnas in ev released from wild type but not cd -deficient cells. summary/conclusion: these findings indicate that cd -dependent thrombospondin- signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin- -and gtpdependent physical interactions with the exportin- / ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc ). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein (dsg ), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg promotes ev release, but the mechanism by which dsg enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors -bromopalmitate, gw , and bafilomycin a were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg /gfp, or palmitoylation deficient dsg cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg on ev release was reduced by the palmitoylation inhibitor -bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg to the lysosomes for degradation. dsg increased expression and subcellular localization of flot , a membrane lipid raft protein critical for membrane invagination. dsg also altered membrane localization of several early (eps and eea ), but not late (rab , rab , and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg cacs mutants abrogated these effects. finally, dsg -induced ev release was abrogated by the sphingomyelinase inhibitor gw or augmented by the v-atpase inhibitor bafilomycin a . summary/conclusion: the combined results of the drug treatments and functional mutations of dsg suggest that dsg plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif e ( e) is an oncogenic protein that is upregulated in % of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d , and mcl . furthermore, our lab pioneered studies demonstrating that a subset of ehigh aml patients is clinically responsive to treatment with a e inhibitor (ribavirin) indicating the importance of e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of e-whether the oncogenic role of e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif e upregulates cellular protein levels of the vesiculation marker cd by increasing its nuclear export. in addition to increased cellular expression, cd , cd , cd , and flotillin- proteins are elevated in evs released from e-high cells. this is also associated with an increased release of vesicles that are - nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to -months old xfad mice (~ billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin- beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) ot . the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips . sendai reprogramming kit -protocol: clementino fraga filho uh . . . / . ). the ev were isolated from ipsc supernatants (cultured h in mtesr- medium) by ultracentrifugation ( , g for h at °c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia ( % o ). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping ( min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro - / ). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed- ). modulation of rna levels was assessed by rt profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk , sod , sod , txn and txnrd . characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin- . summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to ) evaluate which rna species were responsible for ph rescue, and ) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). to identify the mediators of afsc-evs, we used deseq (fdr< . ) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir ~ and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated genes, which formed mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir ~ cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße , heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße , heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp . results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of lncrnas were significantly changed within sevs isolated from a cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha - , acted as a competing endogenous rna (cerna) for hsa-mir- which subsequently up-regulate the natural cytotoxicity receptor (nkp ) expression. furthermore, we verified over-expression of linc-epha - significantly enhance the cytotoxicity of nk cells against zika virus-infected a cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha - wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha b (defa b) expression was significantly increased within evs isolated from zikv infected a cells. additionally, we found that the extracellular defa b but not the intracellular defa b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa b can retard cell cycles of host cell. we verified defa b could bind with the origin recognition complex (orc ) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa b decreased the orc level in nuclear. interestingly, evs with defa b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa b wrapped in evs, and defa b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box (hmgb ), neurofilament (nf-l) and phosphorylated tau- (p-tau) proteins. results: using different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of . ± . . using different combinations of clinical data and the nev protein targets, selected clinical data and hmgb best predicted cognitive impairment (auc = . ). the most important features included cd count, hmgb , nf-l and education. summary/conclusion: specific clinical features plus nev hmgb , an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau- elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv- , htlv- , rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected ( %) cells. these infected cells were initially put in g quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release ( % fbs + pma/pha) at , , , , and hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd , cd , and cd ) at hours and a gradual decrease of all ev associated proteins by hours. however, the ev from hiv- infected cells showed all three tetraspanins present at hours and expression gradually increased up to hours. when compared to htlv- infected cells, the three tetraspanin proteins peaked at hours and expression continued to decrease up to hours. htlv- infected cells also showed a unique pattern of cd expression. autophagy associated proteins (lc a, lc b and p ) from uninfected cells and htlv- infected cells plateaued at hours, whereas in hiv- infected cells their expression continued to increase and peaked at hours. hiv- viral proteins (p , gp , nef) expression was present at hours and continued to increase and peaked at hours. htlv- proteins (p and gp / ) peaked at hours and gradually decreased overtime. hiv- and htlv- rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from -and hour supernatants were performed on three naive cells. hiv- supernatant -hour sample was found not to be infectious. however, hiv- was successfully rescued from -hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a -colour panel, which included annexin v (for the majority of circulating evs), cd (for platelet-derived evs) and cd (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl . prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p) , were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at min and hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [ f]fluorodeoxyglucose ( f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked f-fdg in vivo by pet-scan, within seconds of ev, rbc or free f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p . cryo-tem was used to determine the morphologies of mvs and phage p . the purity of mv isolates was validated by measuring the particle concentration before and after triton x- treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto . results: by incubating mvs and af -labelled p , the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p attachment. the binding ability of p to mvs is greater than that of bacteria. we confirmed that p can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto , we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants , and ). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium (in )-dtpa or xenolight dir post-ev isolation. ct tumour-bearing balb/c mice were intravenously dosed with × evs followed by imaging at h, h and h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to h post-injection. results: xenolight dir and (in )-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in -dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at h post-dose, with less than % ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in -dtpa and nluc evs showed rapid clearance of vesicles from circulation, with % of injected dose detected in blood after min and less than % after h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs instrument (spectradyne llc, usa). pe-cd a (anti-glycophorin a) and alexa -wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn / glass column filled with sepharose cl- b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of . ± . nm and standard deviation of . ± . nm. the concentration of revs measured by mrps was . * e particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd a and with alexa -wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from e particles/ml to e particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was e particles/ml for alexa -wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. , , ) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd and nvkp_ - - - . zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which - μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below %. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes ( - μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon -future and emerging technologies (h -fetopen), id: . marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd /anti-cd /anti-cd antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. - ) and index (european union's horizon research and innovation programme under grant agreement n° ) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size ( - nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs > nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h c cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg , syntenin , cd positivity while grp was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd , cd and cd . cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h c treated with evs showed activation of pfak after ʹ of treatment, this induction was . times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y , y , y ) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density . - . g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir- ), in both ev and lpp (e.g. mir- and mir- ), or in ev (e.g. mir- ). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y /y ratios and pbmcspecific y /y ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included type diabetic individuals ( normoalbuminuric, microalbuminuric and macroalbuminuric). urine samples were collected either overnight (n = ) or during -hours (n = ) and uevs were isolated from - ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq (illumina) pair-end protocol. the association between kidney specific gene expression levels (> fold higher compared to other tissues, n = ) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with > million pair end reads. we detected on average expression of , genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and -hour urine collections. comprehensive look-up of kidney-enriched genes revealed expression of > % (total ) of these genes in urine evs with high expression of five kidney-specific genes (slc a , slc a , nphs , aqp and slc a ). pc analysis combining the impact of kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc axis, and the axis also correlated with the albumin-to-creatinine ratio (p = . ) explaining % of the variance (p = . ) in the whole data set. the pc axis also showed correlation with hba c (p = . ), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek- t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav or eaav and standard aav or aav encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = ) after glenn procedure and swine (n = ) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded and -months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed ( %, fold-change< ). plsr reduced the dimensionality of the swine mirna dataset to mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > . ). summary/conclusion: these findings support the use of: ( ) swine pab model for rv failure in hlhs, ( ) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and ( ) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi prna three-way junction ( wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc sirnas loaded evs (fig. ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: ) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. ) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. ) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. , : . li z et.al. sci rep. introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with differently coloured monoclonal antibodies per well in a well plate format (n = separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = ) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed cases of cancer and screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < . , with aucs > . ), including epcam+/cd + events likely from cancer cells and cd +/cd p+/cd + microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best surface markers to distinguish sclc subtypes were ceacam , fam a, lrfn , epha . immunoblot analysis validated ceacam and epha but not fam a or lrfn . we also found that ncam , a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam and epha corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u ca and nih u ca . leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood . while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes) is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna) , , a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her , egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl ) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs ( , g ultracentrifugation) and soluble plasma (sup) . results: hras-mediated cellular transformation (in ras- cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from participants, including early-stage crc patients, adenoma patients, and normal controls (nc), and characterized according to misv guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of additional participants. results: a total of rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, rna species were differentially expressed between plasma sevs in adenoma and nc participants. rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into modules, which exhibited different expression trends during the carcinogenesis of crc. a -gene combined tsne model consists of the top genes in each module could perfectly classify crc, adenoma, and nc samples. a -gene combined tsne model consists of the top gene in each module could roughly distinguish crc and adenoma from nc, with only sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq . our nanofluidic pillar array is manufactured in an sio mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately - % of prostate tissue-specific rna were discovered in evs; iii) over % ( of rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over - % of noncoding rna ( - % was mirna), while tissue predominantly yielded rrna (> %); v) finally, gene set analyses generated that over % of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, % of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to . diagnose pdac patients from healthy and disease controls and . distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = ). subsequently, we trained our panel of biomarkers using a training set of n = . finally, we validated the performance of our platform using an independent blinded test set of n = . results: the results of a blinded test set achieved an accuracy = % and an auc = . on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = . and accuracy = % with sensitivity of % and specificity of % on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca - for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m ) from distant metastasis patients(m ) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove type diabetic mice, and their wild-type (wt) littermates. at weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove mice or their wt littermates. ( . ± . vs . ± . [ove ] vs. . ± . mmhg [wt], p < . ). ms identified independent proteins with at least peptides per protein. of these, proteins were found in all groups studied, were exclusive to wt mice, were exclusive to ove mice and were exclusive to ttrhren mice. in addition, proteins were observed with > . fold change (fc) compared to wild-type mice, and proteins were reduced by > %. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove and ttrhren mice compared with wild-type controls. similarly trem-like transcript , sarcoplasmic/endoplasmic reticulum calcium atpase and junction plakoglobin were all downregulated in both ove mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~ - um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase (rock ), a process that can be pharmacologically inhibited using a rock- inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp- and il- , suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt , gnt ) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for -weeks ( / ; a / - - ). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd + cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp- and pai and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col a , col a ) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the -weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling (socs ) protein. uptake of socs -containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription (jak-stat ) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of -deoxy-d-glucose ( -dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without -dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (> -kda) socs secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg d mice bearing lung tumours weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of -dg. vesicular (> -kda) socs secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas -dg enhanced, socs and ev release by mh-s cells. treatment of rat ams with cse ( %) attenuated secretion of socs , an effect that coincided with increases in glycolytic activity, and co-treatment of ams with -dg abrogated the inhibitory effect of cse on socs release. finally, ams collected from lsl-krasg d mice exhibited a deficiency in socs secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of -dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (Δtolc) in the model cyanobacterium synechocystis sp pcc (s ), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why Δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation ( g, min) and filtration ( . µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of kda), and later ultracentrifuged for h at g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s Δtolc. both wild-type (wt) and Δtolc cells were then challenged with different metals. compared to the wt, Δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and Δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from Δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s . in addition, cu could also be detected in isolated Δtolc-ev. in addition, more ev were detected when s wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/ / ; feder-compete -poci-fct project: poci- - -feder- . juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects / and is caused by mutations in pkd and pkd , which encode polycystin- and − . in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin- motors and kinesin- klp- /kif transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab -exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct colorectal and hela cervical cancer cell lines to identify rab -exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab -exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp , chmp and ist , are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab -exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt- components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab -exosomes. in flies the escrt- , hrs, localises to the limiting membrane of rab -endosomes. comparative proteomics reveals other proteins enriched in rab -exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab -exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional ( d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in d display decreased exosome release and increased uptake compared to d cultured cells. exosome release in d culture was inhibited with the exosome release inhibitors brefeldin a and gw , but was not significantly altered by knockout of rab b. in addition, disruption of polarity signals provided by d culture did not impact exosome release or uptake in d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi k signalling. summary/conclusion: release and uptake of exosomes is altered in d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r w-fibulin- induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin- versus rpe cells expressing r w-fibulin- and investigate the effects of these evs on rpe cell differentiation. methods: arpe- cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin- or luciferase-tagged r w-fibulin- . evs were isolated from the media of arpe- cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe- cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around nm and a large group with diameters around nm. moreover, tem and cryo-em showed an increased amount of small evs (~ nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r w-fibulin- causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe- cells overexpressing wt-fibulin- contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r w-fibulin- contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad , leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op . = pf . heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev- introduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir- a- p (mir- a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir- a- p (mir- a). in situ hybridization of the frontal brain area revealed that mir- a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir- a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir- a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir- a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir- a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r da blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)- β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, . - µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (< nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il- β content of mps increased -fold. administration of an il- β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor- to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il- β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n - - - from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c orf als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod , fus and tdp are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g c aberrant nucleotide repeat expansion in c orf gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at , xg and the smaller evs at , xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc with gw . to understand the downstream effect of polygr+ evs in recipient cells we studied tdp mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting . million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human - -week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c /b female mice with mog - peptide. results: we found that hasc-evs expressed high levels of galectin- and promoted a significant increase of the immunoregulatory enzyme indoleamine , dioxygenase- enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper (th ) cells, and increased percentage of regulatory t cells (treg-foxp +) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l cell adhesion molecule (l cam) from serum samples of wrap participants who were cognitively unimpaired at baseline (mean age . ± . years old; . % females; . % apoe carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p and p ) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p -(p = . ) and p -tau (p = . ) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau (p < . ), total tau (p < . ) and ptau (p < . ) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap- mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap- transgene was inserted at the mouse h locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap- mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that % of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus (hpv ) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in ( %) saliva samples collected from hpv -driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv- dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv -driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd , cd and cd ) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv- dna. the proteomic signature of salivary exosome was significantly (p < . ) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase (pgk ), glyceraldehye- -phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c - b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du cells treated with sevs expressing or lacking the αvβ integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c - b cells that express αvβ , but not from the control cells that lack αvβ , induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p - , r - to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b f melanoma cells and ht fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv- tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir- was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv- protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin , resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir- was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir- was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir- oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir- with downregulation of claudin- was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv- tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir- as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely ( ) mechanical extrusion, ( ) freeze-thawing, or ( ) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying ( ) extent of fusion between evs and synthetic calceinloaded liposomes, ( ) efficiency of calcein loading, and ( ) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg- - - -cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein (ifitm ), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm -containing evs (ifitm -exos) were overall safe to the animals and suppressed zikv viraemia by log s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm , an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec a scfabs to target conventional type dendritic cells (cdc s), anti-cd scfabs to target t cells, and cd ligand to target b cells. in mice, systemic administration of anti-clec a evs resulted in a % increase in the percentage of cdc cells that take up evs over controls. anti-cd evs resulted in both an increase in the percentage of ev positive t cells ( . and -fold for cd + and cd +) and the number of evs per cell ( and -fold for cd + and cd +) in the blood. furthermore, in primary mouse dendritic cells, anti-clec a evs loaded with sting agonist achieved a fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec a evs reduce the required sting agonist dose -fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to ) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; ) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e . . ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after h, dams and pups were sacrificed. d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = ) and nitrofen-exposed (n = ) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os . introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles (misev- guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev- . our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon research and innovation programme under grant agreement no . sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with - , higher levels than in parental cells. contrarily, cell-tropic mirnas were only - times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in . mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min β-cell line treated with diabetogenic cytokines (tnfα, il- β, ifnγ, h). evs were also isolated from human type diabetic (t dm) and lean non-diabetics (lnd) plasma. gw (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t dm plasma revealed a~twofold increase (p < . , vs. control (ctl) and lnd ev). immunofluorescence staining of cd and cd expression was significantly elevated in human t dm pancreas (p < . , vs. lnd). while acute inhibition of ev formation with gw ( µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~ and fold vs. cytokines alone, p < . ). moreover, functional assessment of mouse islets exposed to cytoev ( h) resulted in suppression of gsis (~ %, vs. untreated, p < . ). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl (~ fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl -cxcr signalling (p < . ) and downstream pathways (e.g. nfκb; p = . and jak/stat; p = . ). furthermore, inhibition of cytoev (gw ) with cytokines markedly decreased cxcl (~ %) and cxcr receptor (~ %) expression in min β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl consequently activating cxcr signalling and downstream pathways to impair β-cell function in diabetes. synergy between -lipoxygenase and secreted pla promotes inflammation by formation of tlr agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor (tlr ), but the activating ligands were not fully determined. additionally, several enzymes, among them -lipoxygenase ( -lo) and secreted phospholipase a (spla ) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek cells exposed to um a and isolated with ultracentrifugation. : lysopi was oxidized for min with -lo. additionally, synevs were prepared from phospholipids (pls), oxidized with -lo and hydrolysed with spla . activity was measured by qpcr and elisa on wt and tlr -ko macrophages. -lo oxidized : lysopi was analysed by mass spectrometry. spla activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr ko mice (c bl/ mice) with spla -iia injection was used (approval no. u - / / by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, -lo oxidized synevs, but only -lo oxidized lysopls activated cytokine expression through tlr /md- . hydroxy, hydroperoxy and keto products of : lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla activity, which we detected in the synovial fluid from patients, promoted formation of tlr agonists after -lo oxidation. injection of spla -iia into mice promoted k/bxn serum induced arthritis in tlr -dependent manner. summary/conclusion: both -lo and spla are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr signalling. the formation of tlr agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h -msca-itn project tollerant (grant no. ), slovenian research agency (project no. j - to mmk, research core no. p - to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl . injured and uninjured muscles were dissected h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m , m , and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m , m , and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m and m . indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv cells. we showed that m-evs are internalized by both mouse bv and human c microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, , p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = ): half dying within years (short-lived group) and half surviving ≥ years (long-lived group) after the blood draw; all matched for age (median age . ± . years, range - ), gender ( % female), and race ( % white/ % black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd a, cd a, cd , cd , hla-abc, hla-g, hla-drdpdq, cd , cd , cd , m cadherin, ryr , ryr , fabp , dlk ). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x- flow cytometer as we recently reported. graphpad prism . software was used for statistical analysis. results: we found significantly increased percentages of cd +, hla-abc+, cd + and cd a+ large evs ( - nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium ( - nm) or small (< nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd is expressed by hscs and immune cells. cd regulates homing of human cord blood cd + hscs, and delivers a potent cd independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd is expressed by hscs, immune cells and epithelial cells, and cd + plasma evs declined with age in healthy people. cd a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd +, hla-abc+, cd +, cd a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after days of msc expansion under dynamic conditions, we collected . l of conditioned medium with approximately . million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd , cd , and cd . simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht and sw . methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of conditions. a evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp and membrane marker egfr, both with high expression in a evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht and sw evs through probing of trios of surface ( ) and internal ( ) protein targets. results: the selected tevam protocol successfully maximized hsp signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht and sw evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc . mouse dendritic cells were cultured in flasks containing a total of . l of ev-depleted media ( % fbs, centrifuged hr. x , g.) when cells reached % confluency, conditioned media was collected, depleted of debris with two min. x , g spins, and concentrated down to~ ml using a pall jumbosep kda mwco filter. the ev concentrate was purified from protein using an izon qev- column, with ml fractions collected. the protein content of the ev-containing fractions was analysed by a , pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc . preparation, one highly abundant population with a power-law distribution, whose peak diameter is below nm, and a second, less abundant population with a peak diameter at approximately nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, μl of~ particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of clinical samples ( ovca and non-malignant control) were tested in combination with ovca skov- cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov- cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov- evs from both ldl and vldl. pca also differentiated skov- evs incubated with ldl from skov- evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein (vamp , cellubrevin) on evs. conversely, knocking-down vamp in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level (low, no matches in databases) to level (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from ma-act, and controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd , cd , and cd ), platelet evs (cd ), pro-coagulant evs (annv), and red blood cell evs (cd ). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v . . fold change was calculated by ΔΔcq between ma-act and ctl for mirna expressed in ≥ % of samples in at least group. we identified the top % of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) . fold change in expression, (ii) area under the receiver operating characteristic curve of . , and (iii) glass's Δ of . for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~ nm) and concentration (~ . x particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd + evs, and to a much lesser extent cd + evs. of the mirna expressed in ma-act and/or ctl plasma evs, there were mirna that have at least a . fold increase or decrease in ma-act. mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, of the mirna correlate with a ma associated variable, including frequency of use and age of first use. together the mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old ( - month old) and young ( - -week old) c /bl mice and collected bm-evs by differential centrifugation ( × g for min, supernatant collected and centrifuged , × g for hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with x ^ young bm-evs via tail vein, daily x days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca- +/cd +; lsk-slam) and tested stem cell function in competitive bone marrow transplants ( - recipients/group). results: at months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: % ± % (young evs) vs. % ± % (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: % ± % (young evs), % ± % (old evs), % ± % (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately -fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants p gm , dk - a and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op . = ps . combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op . = ps . introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by -aminolevulinic acid ( -ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd +/cd + evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using -ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between - nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of proteins were detected in fgr evs and, according to prediction software effectorp, . % of these were potential effectors. similarly, % of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a % overlap in proteins identified in the cd and the sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with . % and . % of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op . = ps . commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just . million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since the hevi has organized research symposia, hands-on training days, hosted international students as well as providing one-on-one training for individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on october , we organized the st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg , introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h as well as the ovarian cancer cell line skov were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb /her and insulin-like growth factor receptor (igf r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants ( t, a , u , thp- , sh-sy y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd , cd , cd , cd , cd , cd , cd ) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd , cd or cd ) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than % of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a -fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased . x (p < . ; / vesicle-related top go terms) in proteins common to intact arteries and valves (n = , ). proteomics found ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified , proteins and mir cargoes linked to , target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. proteins and mirs were significantly altered between artery and valve evs (q < . ); multi-omics integration found that evs differentially modulated cellular contraction and p mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r hl , r hl and r hl (ea). mir- a regulates exogenous cd expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir- a regulating the expression of exosome cd on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc- was used as the research object. the exosomes were extracted from the culture supernatant of mgc- by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd in exosomes was detected by elisa. the expression of cd in exosomes and cd in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir- a lentivirus experimental group (mir- a group). the lentivirus control group (mir- a/con) was transfected into cells. qrt-pcr was used to verify the status of mir- a after transfection; western-blot was used to detect the expression of cd and downstream erk / , akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd in exosomes was detected by elisa and western blot. the expressions of cd , akt, erk / and m tor in mir- a group were significantly lower than those in mir- a/con and mock groups. cd protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir- a group were significantly lower than those of mir- a/con group and mock group. the weight of the nude mice in the mock group and the mir- a/con group decreased, while the weight loss in the mir- a group was not significant. the tumours in the mir- a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir- a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir- a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd . streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time ( , , , , , , or hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of : and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x- labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and > -hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core /superose resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd and in tasmanian devils. methods: exosomes were isolated from three different dftd- , dftd- and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd- abundantly express the inhibitory checkpoint molecule cd relative to exosomes from dftd- cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [ ] , artificial coiled-coil peptides with receptor targeting [ ] , and cell-penetrating sc peptides [ ] derived from cationic antimicrobial protein, cap for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [ ] . methods: [methods] cd (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [ ] . we developed the methods for modification of arginine-rich cpps or sc peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan- ) and macropinocytosis signal transduction [ ] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [ ] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [ ] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [ ] sci. rep. , ( ), [ ] chem. commun. , ( ), [ ] chrmmedchem. , ( ) [ ] anticancer res. , ( ), [ ] sci. rep. , ( ) os . multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of - nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, ) mcmv bud from localized sites on the endothelial pm, ) mcmv contain mvb, and ) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated > distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained > distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans ( - pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [ ] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [ ] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science ( )). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. ( ); infect immun. ( )). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression > -fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir- - p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir- - p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # /r/ and # /r/ revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = ) and control (n = ) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., ) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx , stau and vcam , respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct ). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir- a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir- a had anti-inflammatory effect on cells of msa-p condition such that the level of cx cl expression and its receptor, cx cr were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir- a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., ) . the aim of this study was ( ) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and ( ) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase- and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase- were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of ms patients ( with relapsing remitting, with progressive ms) and healthy controls, targeting the neuronal-specific marker l cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c q, c , c b/ic b, c , c , c a, c , factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced ( . -fold; p < . for both), whereas multiple complement components in ms aevs were markedly increased (c q: . -fold change; c : . -fold change; c b/ic b: twofold change; c : . -fold change; c a: . -fold change; factor: . -fold change; p < . ); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . /synaptophysin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . ), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-( -carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go ( . mm) for hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp , angptl- , lcn , ptx , s b, rage, dj- and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ - , aβ - , ptau t and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx- , lcn- , dj- , on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl- while the expressions of mmp- and angpl- were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn- by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp , agntl- , ptx- , dj- , aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u mg), microglia (sv ), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either % or % cse for hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions - ), ev ( - ), and protein ( - ) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd , cd , and tsg ) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da , da , and ai . a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles - nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of . ng (range . - . ng) and no samples fell below the threshold required for library preparation and sequencing ( pg) determined by inhouse optimization on the smart-seq v ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from participants with ad and non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r = . ). in serum, micrornas were differentially expressed by a fold change ≥| . | in the ad and control groups (p ≤ . ) and micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the differentially expressed micrornas in serum, were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = | . - . |). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p ag ) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd , cd ), platelets (cd , cd p, cd a, cd b), and erythrocytes (cd ) as well as brain biomarkers (s b, uchl- , gfap, tau and nse) and t-lymphocytes (cd , cd , cd , cd ). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: / study patients and / controls were male. % of study patients presented with a glasgow coma scale of . in the hypercoagulability panel, of the subsets with statistically significant differential expression, involved s b+ and were elevated in patients. platelet-derived cd a evs and uch-l evs were significantly elevated in controls in ev subsets identified in the brain-specific panel. finally, cd +/ + evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir- a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [ c]-sucrose was increased in the forebrain by fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir- a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir- a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by -, . -and twofold respectively. qpcr analysis of mrna from reported mir- a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir- a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for days (subcutaneously; mg/kg, day , mg/kg days - , and mg/kg days [ ] [ ] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le , harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from healthy subjects (hc) and ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m (gm ), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente - to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of healthy subjects (hc) and pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev guidelines, raman analysis was performed. the raman microspectroscope was used with a nm laser in the spectral ranges - cm- and - cm- . data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of %. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag or rag gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de Ética no uso de animais -ufrgs; n ) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats ( and months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed differentially expressed micrornas (p < . ; fold change of ≥ | . |); mirnas were up-regulated and were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < . ; z-score: . ). the differentially expressed mirnas impacted molecules in the neuroinflammation pathway. interestingly, the ion channel grin b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin b was previously related to neurodegenerative diseases. moreover, let- a- p is predicted to be downregulated and target all the molecules of the neuroinflammation signalling pathway. previous studies have correlated let- a- p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = ). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of . %. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by % in epidermis compared to control (tlnκ/sicontrol)(n = ). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day , suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il- , cox- , inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir- b in mi-exo and vegf mrna expression confirmed. results: mi-exo using % acetic acid extraction showed the highest yield. the average size of the exosomes is approximately nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of ^ particles showed about % antioxidant activity. when ^ particles were treated, rna expression of cytokines showed about times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of ^ particle = . %). and mir- b in milk exosomes was detected at ct value = . summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when ^ particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions and . the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd , cd and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine ( - ) prymidinone photoproducts ( - pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm , malvern). nhdfs were exposed to uvb ( mj/cm ) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration ( - nm). when nhdfs were added with adens and incubated at °c for h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and - pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir- c and mir- , while increasing vegf and hif- a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < . ). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a -fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < . ) than compared to control msc evs (p < . ). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small ( - nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u cells in culture and incubated for h. they were then fixed by % paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u cells took up exosomes from other u cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award . loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir specific qpcr. curcumin was measured using a spectrophotometer at nm. results: evs isolated from whey had an average size of nm, were positive for hsp , cd and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir in relation to control (loaded with shame) and the negative control (p < . ). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd . the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd -snorkel-tag under p ink a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd -snorkel-tag under p ink a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w ). engineering exosomes with gata- jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles ( - nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata- , a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata- protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata- by infusing gata- with exosome targeting peptide. methods: the open reading frame of mouse gata- cdna was ligated to xpack lentivirus vector (xpack-gata- ) and plvx-ef -ires-pouro lentivirus vector (plvx-gata- ), respectively. hek cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata- was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend cells with exosomes pre-labelled with pkh . introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd -gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point ( - µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized -and -hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces -hours post-treatment compared to -hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types -hours post-treatment compared to -hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution -hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd v and tspan . results: the majority ( %) of patients with paca and patients with nonpa-malignancies reacted with anti-cd v and anti-tspan . serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = , ) and controls (n = , ) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha- (vi) chain (col a ), − (vi) chain (col a ) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these proteins in plasma ev can distinguish former nfl players from controls with % accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col a , reln and col a in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = ), and urine from men with pca prior to (n = ) and after local treatment (n = ), benign prostatic hyperplasia (n = ) and other urological cancers (n = ). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of healthy controls (hc), patients with active crohn disease (cd) and cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate * - * particles/ml from mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and < % of active cd patients; and for selected g+ ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and % of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c - , lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi- ) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c - prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: ) monitoring response to therapy and, ) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified proteins from neuronderived ev (nde), proteins from microgliaderived ev (mde) and proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, , and proteins are present individually in ndes, mdes and ades. among them, high levels of atp a and syt in ndes, itgam and cd a in mdes, and eaat and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p , total p , cleaved caspase , erk / and phospho-akt in whites compared to aas. higher ev levels of phospho-igf- r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = ) was collected into k -edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for days. microev were collected from platelet poor plasma by , g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd ) and red blood cells (cd a). the increase in associated protein was seen more in the k -edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa and hla-dqb , to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = ) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd and cd ), and transmission electron microscopy. exosomes were also studied for their interleukins (il- and il- ra) content. results: among the studied population, % present at least one of the alleles leading to cd development and % carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il- ra content ( . ± . to . ± . ), as the persons were not following any treatment. however, values of il- ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il- ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al ) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol ) or a combination of density cushion and density gradient (protocol ) or immune-capturing (anti-cd , anti-cd and anti-cd beads) (protocol ). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol and the majority of the vesicles were cd / cd a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd /cd a positive evs where also identified with protocol . furthermore, western blot showed increased amount of cd a in plasma samples in protocol . when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci- panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α , αv, and β correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α , αl, and β are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p a (cyp a ), antioxidant enzymes (aoes), sod and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv- pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp a , sod , and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd and cd . the results showed menthol and av together increased the levels of cd (p ≤ . ), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod (p ≤ . ), which showed a pattern of increased levels of catalase and cypa , though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp a (p ≤ . ), sod (p ≤ . ), and catalase (p ≤ . ) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp a (p ≤ . ), and catalase (p ≤ . ). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp a in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da , da- ) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: ) protein association network analysis interaction (string) and ) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l -cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < . ) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l cam expression was found to be significantly elevated in smokers (p < . ). both gfap and l cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da ) for supporting our work. . electrochemical detection of mirna- - p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna- - p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna- - p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna - p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna - p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn) ] -/[fe(cn) ] -. results: the results indicated that the electrodeposition of gold was more effective for − . v for s and could lead to better signals upon anti-mirna- - p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna- - p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range ( nm - nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is nm to nm, e to e /ml, and . - . , respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect nm particles with ri = . with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from molecules of equivalent soluble fluorochromes (mesf) to mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below copies per ev with nm diameter, i.e. mesf values above are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under hlt metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from d hollow fibre bioreactor and d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish ( d) versus hfbr ( d). methods: human clonal afsc were derived from patient-consented amniotic fluids. x e hafsc were seeded in d ( cm ), and . x e hafsc on a small kd mwco hfbr (fibercell-c d, cm ) with fibronectin coating; both cultured in chang medium with % of es-fbs, starved for hr and then evs collected. the effect of harvest frequency was tested ( hrs, hr, hrs, wk). d-evs and d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: d production was~ . x e ev/ml/ hrs while d was~ . x e ev/ml (first four hrs) and . x e ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~ nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. d-evs trapped vegf (an in vitro established potency assay) as efficiently as d-evs, and expressed cd , cd , cd , cd , cd and vegfr as d-evs. summary/conclusion: d-evs had comparable properties and bio-activity to d-evs, but the hfbr produced x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available . m cartridge provides a x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from to nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub- nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at − °c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a -micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased % in plasma (p = . ; mean of the cryopreservation agents) and % in urine (p = . ) after one day of storage. after months of cryopreservation, the concentration of plasma evs decreased % (dmso and exocap) and . % (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased % (exocap) and % (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in . funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h b-rfp-nanoluc(h b). results: hek t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h b for hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h b which was blocked by cyclohexamide, confirming translation of the mrna ( . kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for hrs. all extracellular evs were removed and cells were trypsinized ( . % for min) to remove any non-internalized cox protein. hrs later, evs (cd + and cd +) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise (n ) or mscarlet-i( c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek , u , hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at °c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht cell culture supernatant by differential centrifugation, aliquoted and kept at − c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at + c. the kinetics of vesicles loss was measured by anti cd immunomagnetic capturing followed by cd , epcam and cd staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to x ^ for cd +, x ^ for epcam+ and x ^ for cd + vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd , cd , and cd confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from - nm in size and were positive for cd , cd , and cd proteins. these sevs could be stored at − °c for > months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let a, mir , and mir a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as h after seeding, which were not observed in control huvec cultures until h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = ) supplemented with msc-sevs versus control, with the former lasting > h and the latter lasting < h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = ) were able to expand robustly with a doubling time of . , . and . days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a and a micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed / / / ncbr/ ) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles - nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af ), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > × exosomes/ml and average protein equivalent of more than mg/ml. more than % of the exosomes population had an average size distribution of - nm and median size of nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd , cd , cd ) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla , a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg , and cd . surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (> nm) and exosomes (< nm) by size distribution and the concentration of evs ( x particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg , and cd was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd ) and stem cell markers (cd / ) that depict donor cell origin and functional proteins including integrin-binding (cd ) and tgf-beta receptors (cd ). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in -well plate in with concentration of , cell/well. cells were incubated for hours and fasted for another hours using only dmem. cells were treated with complete medium consist of dmem and % fbs. culture medium were collected after , , and hours after treatment. cell were culture in ºc dan % co . vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture , , and hours were . pg/ml, . pg/ml, and . pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a kda cut-off. proteomic analysis by tmt mass spectrometry identified unique proteins. % of the exocarta top were identified from this list. additionally, we identified the mirna cargo within exosomes and found highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = . ; p < . ) and transforming growth factor-beta (tgfb) signalling (fold enrichment = . ; p < . ). exosome size was quantified to be . ± . nm with a modal average of nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited . % less collagen than vehicle controls (p = . ). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb- and hs t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using d cultures. moreover, we also used d culture to assess the exosomes uptake and to observe their capability of internalization into a d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines ( d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using d culture we provided a comprehensive understanding regarding exosomes internalization in d structures, which closely mimics in vivo conditions, compared to d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk . , derived from normal human lymphocytes. nk . evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k leukaemia cells were treated with nk . evs in vitro and analysed for proliferation and markers of cell death. results: nk . evs contain ev-associated proteins alix, cd , hsp , and tsg , nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf b, an e ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir- . nk . ev treatment of k significantly decreased its expression of proliferation markers cd and ki , and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases − and − . non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk . ev treatment significantly reduced the frequency of k cells highly expressing aldh, a csc marker. summary/conclusion: nk . -derived evs have a robust anti-tumour effect on k myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk . evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. > x expansion within days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both d flask and d bioreactor culture and generated over , particles per cell in d and over , particles per cell in d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased > x after hrs. alternatively, ev productivity was also increased > x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the to l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in d and then seeded on three different microcarriers and cultured in a bioreactor for days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for hours, then in ev collection media for additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following -day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells ( e particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd , cd , and cd , but they were negative in expression of cd and cd . the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b f cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein (tyrp- ), and tyrp- . in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government ( m a h ). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells - . therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair . lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai , ai , ai - , ai , and ns to fk and r ca and r ar to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f :neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day and ), in dmem/f :neurobasal or in opti-mem, with and without -hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions - , which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions - in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: . e+ particles/ ml, opti-mem: . e+ particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, -hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: . e+ particles/ml, non-stimulated: . e+ particles/ml, p < . ). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a ) could have a role in immune cell regulation. specifically, we have investigated the possible a ev effect on murine macrophages (raw . ) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a evs were collected from conditioned milieu by ultracentrifugation. raw . cell proliferation with or without a evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw . cell phagocytosis they were treated with μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw . migration ability. furthermore, a evs are able to increase macrophage phagocytic activity. as it is known that hsp is involved in for macrophagic activity increase and a evs express hsp on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr , tlr and cd . our data demonstrated that a evs increase phagocytosis through hsp and its receptors. we have also proved that a evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw . cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m phenotype. summary/conclusion: in conclusions, our data show that a evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. and ) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd (yet preserve its channel function) also cause cysts. besides cilia, pkd is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd trafficking and localization leads to the kidney pathology remains unknown. pkd is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd- is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd- -containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin- cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd- plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd- -associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd- carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd- -carrying evs that differ in their densities: . - . versus . g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd- ev signalling will inform on the interactome of human pkd and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm- localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm- in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm- and gfp-tagged pkd- (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm- ::tdtomato and pkd- ::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm- subpopulation is formed, we are investigating candidate genes. anoh- , a homolog of the ca + scramblase tmem f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh- mutants, the number of clhm- ::gfp evs released is significantly decreased but the number of pkd- ::gfp evs does not significantly change. in addition, i am using facs to isolate clhm- and pkd- containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane- b (laptm b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy y cells were treated with cer (porcine brain-derived cer or synthetic d : /c : ~c : cer) for h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd and rab immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c and c fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd -positive mvb with rab -positive recycling endosomes, further demonstrated that laptm b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg s and their adaptor proteins in sev. cq-induced enrichment of atg s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute (ago ) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago in evs and it is postulated that ago is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago in sevs. methods: methods: wildtype kras colorectal cancer cells, dks , were conditioned with different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks total cell lysates, large evs and density gradient fractions was performed, probing for ago and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions and , as assessed by western blot analysis. ago was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago in evs has been controversial. multiple factors may affect the ability to detect vesicular ago , including serum and growth factors in the conditioned media that may provide sources of extravesicular ago and also regulate the trafficking of ago into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim -beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with mm methyl β-cyclodextrin. results: using melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between and nm. protein amounts in ecvs were measured, showing heterogeneous levels at ~ μg/ ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim beads and flow cytometry. gd and gd were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd , showing increased levels of integrins in ecvs from gd + cells compared with those from gd -cell lines. integrin levels in cell lysates from these cells (gd + and gd cells) were almost equivalent. treatment of a gd -expressing melanoma cell line by mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from t breast cancer cells cultured in normoxia ( % o ) and hypoxia ( % o ) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd and mcherry-cd , separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs ( k fraction) and small evs ( k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd and cd (yoshioka et al., nat commun., ) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort and lamp genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort or lamp could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with % fbs and × antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy . for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea , anti-rab and anti-gm . in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system . lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language . . results: we determined that rab , located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to orders of magnitude (the order of the size of giant phospholipid vesicles is between and micro metres, in erythrocytes it is about micro metres and in evs it is about nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc (arrestin domain containing protein ) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc in the trafficking of the divalent metal ion transporter dmt to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc . we then generated arrdc wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified potential ubiquitinated lysine residues. out of these, lysine appeared to be the most important for arrdc function. arrdc k r mutation caused a decrease in the number of ev released by the cell compared to arrdc wt, and a reduction in trafficking of dmt to evs. furthermore, we also observed a decrease in dmt activity and an increase in its intracellular degradation in the presence of arrdc k r. k also appeared to be ubiquitinated with k polyubiquitin chains by the ubiquitin ligase smurf . summary/conclusion: our data suggests that k polyubiquitin chains are the signal for arrdc mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, , then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing: . instructions for d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d and d her + breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d and d her +, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d and d her + was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep / sephacryl s- column run with Äkta start ( nm), min runs. each fraction ( - ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns , camera gain , static mode, capture time sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with % formaldehyse and stained with % uranyl acetate, run at kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f -f , - min) were collected from d her + cell culture medium. interestingly, both nta and tem suggest that f and f contained evs as the isolated particles measured and nm, respectively and tem revealed spherical particles - nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram ( min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using Äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in , is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, ) and evs in immunology (buenos aires, ) introduction: since its founding in , isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, . in december, , the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, , the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the and isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, workshop) and evs in immunology (buenos aires, argentina, workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the and workshops assembled a total of more than individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, ) and evs in immunology (buenos aires, ) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid- kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in , featuring lunch and presentations by academic and industry participants. when covid- prevented in-person meetings beginning in march, , the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid- shutdown in the host country (us). an audience of between and individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm and pm , , chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v -v s rrna gene regions amplified from upper-airway tracts of ar cases and healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm and pm , concentrations at the municipality of residence in the days preceding nasal sampling (i.e. day − to day − ) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria ( . %) was the most represented, followed by firmicutes ( . %) and proteobacteria ( . %) while in cases proteobacteria were . %, actinobacteria were . % and firmicutes were . %. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd +), activated endothelium (cd e+), monocytes (cd +), eosinophils (cd +), neutrophils (cd +), mastocytes (cd c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased . fold in cases versus controls (p < . ). evs were modified by pm exposure at several time lags. in particular, a negative association between pm and eosinophil evs was observed (beta = − , ; pvalue = , ). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c /bl and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved / ). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th -derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at °c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan ) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph . ; in this condition nano releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration ( µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg , il- and il- genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x / . immuno-toxicological evaluation of human mesenchymal stem cell- introduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; . mg/kg), or low-dose ( x e paticles/head) and high-dose ( x e paticles/ head) of hevs every other day for days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for days with low conc. ( x e particles/ml), high conc. ( x e particles/ml), pma/ionomycin as a cell activator or cpt ( μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about ~ % in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant ( mfds ) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). x ( ) human or murine msc-evs were administered intravenously into t breast tumour bearing balb/c mice (n = ) and healthy controls (n = ). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: - nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd , cd , cd ; cytosolic protein tsg and were negative for calnexin. ev concentrations ranged from . x ( ) - . x ( )/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd +, cd +, cd +), neutrophils (gr- +, ly- c+), dendritic cells (cd c+), macrophages (cd b+, mhci+, mhcii+), nk cells (cd +) and b cells (cd +) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook h medium (m h ) supplemented with % (v/v) oadc enrichment and . % (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin- β (il- β), terleukin- (il- ) and interleukin- (il- ). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna . array. results: microrna from sepsis patients (median age . years; iqr: . - and % female) with a median psofa score of (iqr: . - ) and from healthy controls (median age years; iqr: . - . and % female) was isolated and compared. in septic vs. healthy patients mirnas were differentially expressed (false discovery rate (fdr)< . ; fold change ≥| . |) affecting mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥ . summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir- - p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir- - p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir- - p involved in salivary gland (sg) physiology was performed with mirdip . serca b, ryr and ac were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca b, ryr and ac were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir- - p in relation to the expression levels of serca b, ryr , and ac , a double fluorescent in situ hybridization was performed. ca + signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir- - p is over-expression in the sgs of ss-patients. next, we demonstrated that mir- - p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir- - p which transfer into glandular cells and affecting intracellular ca + signalling, camp production and protein production by mir- - p targets (serca b, ryr and ac ). summary/conclusion: this study provides evidence for a functional role of the mir- - p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il -coated extracellular vesicles promote infectious tolerance (p ) subunits, yet the forms that il assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il -producing t regulatory (treg) cells in tregebi wt c bl/ reporter mice, and identify il producers by expression of ebi tdtom gene reporter, plus ebi and p proteins. results: curiously, both subunits of il were displayed on the surface of tolerogen-specific foxp + and foxp neg (itr ) t cells. furthermore, il producers, although rare, secrete ebi and p on extracellular vesicles (ev) targeting a -to -fold higher number of t and b lymphocytes, causing them to acquire surface il . this surface il is absent when ev/exosome production was inhibited, or if ebi is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il , promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r -ai - (to w.j.b.), r ca and p ca (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p ca . unique formulated dual targeting antigen specific and delivered mirna- gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna- to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna- -pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac- integrin (cr receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac- activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac- activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for or hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il- and il- , with il- release reaching statistical significance at . %, . %, and % de concentrations (p = . , < . , and < . respectively) and il- trending a similar dose response but only statistically significant at % de (p = < . ). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the and % iodixanol fractions (p = < . and . , respectively) at hr following de treatment. alternatively, there were significantly less ev in the and % density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs > -fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to ( ) develop a faster method to avoid swarm detection, and ( ) increase the number of detected evs per second. methods: we measured serial dilutions of cd stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a -micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of , - , events/s. the cd + evs concentration scaled linearly over . orders of magnitude of the dilution and most donors required > -fold dilution to avoid swarm detection, thereby reducing cd + ev counts. for fluorescence triggering, the cd + evs concentration scaled linearly over > orders of magnitude of the dilution. for all donors, swarm detection was absent after -fold dilution (relative to pure plasma). the count rates of cd + evs were - -fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥ -fold and compared to scatter triggering, count rates of cd + evs were - fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd , cd , and cd . however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd , snap , α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek t cells through transient transfection of plasmids (origene). media was conditioned for - hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects ( - y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd for platelet-derived evs and cd for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap- ) and u . the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = . ; p = . ) and clot size at min (r = . ; p = . ). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = − . ; p = . ), whereas they were found positively correlated with lysis progression (r = . ; p = . ). both mean and mode size of cevs, detected by nta, were significantly correlated with u -induced platelet aggregation (r = − . ; p = . , r = − . ; p = . , respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd and cd . ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh -labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa -sirna were enumerated and phenotyped using the exoview r platform. evs with surface expression of cd , cd , cd , and annexin a were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a allowed us to assess the specificity of the selected annexin a antibody. results: the ev fraction was positive for cd , and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a + evs were captured on the annexin a spots from the control cm, and there was a marked decrease in capture and labelling from anxa -sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd , then cd and cd . there was a significant population of annexin a + evs that was negative for tetraspanins. summary/conclusion: annexin a is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a + evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl and hl . possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (< mmhg) and angiography. ten preoperative clti patients and control patients without pad were analysed (all patients with diabetes and % of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into micrornas for esrd patients and micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir- and mir- a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t ) and six months after surgery (t ). isolated pexos were quantified by nanoparticle tracking analysis and mir- , mir- , mir- a, and mir- a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = ). local ethical committee approved the study (emigrate study, approval n° ) and informed consent was obtained from all patients. results: pexos levels at t were lower (− %, p = . ) in patients with worst postoperative lv function, while they were higher at t (+ %, p = . ) in patients with reversed lv remodelling after surgery. at t , the increase in pexos levels was associated to decreased heart mass index (− %, p = . ) and higher levels of exosomal mir- (+ %, p = . ) and mir- a (+ %, p = . ) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir- and a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin- (lcn ) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc ) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between - nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness ( . - kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin- and the tetraspanin cd . young melanocytes produced more annexin- positive evs and evs larger than nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb- breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd b+ and f / + cells, but not b + cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as hour after msc-ev injection and was not significantly different between and hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants uh tr , uh tr - s , p gm , and t hl . linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw and ht human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to % reduction in lipids observed at the µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il- and il- by over fold and fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) ( - nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg and cd ), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein (ifitm ), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence . bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh cells were cultured on acrylamide gels with the stiffness was modulated to pa (soft) or k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: ( ) stiff matrix promoted exosomes secretion. ( ) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin -rab pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used dimensional ( d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional ( d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< nm) to small (< nm) and medium and large (> nm) evs and larger evs (> nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco a and clic expression on non-tropic evs conferred lung-tropism, while cd gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev- t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles ( - mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb- luciferase (luc) expressing cells or ht- -luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of x ^ msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. x ^ evs were incubated with the radionuclide, technetium- m tagged duramycin ( mtc-dur) for minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected minutes and hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia ; aejez sayeed ; rachel derita ; shiv ram krishn ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using gy, for consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from - nms to - nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = ) and control (n = ) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps tau and aβ - were significantly increased in ad evs. annexin a (anxa ), neurosecretory protein vgf, neuronal membrane glycoprotein m -a (gpm a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these proteins were confirmed to predict ad with the % accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa , vgf, gpm a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide (trap; n = ), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = ), and (iii) stored at room temperature or at °c for hours (n = ). the concentrations of evs from platelets (cd +), activated platelets (p-selectin+), erythrocytes (cd a+), and leukocytes (cd +) were determined by flow cytometry (apogee a -micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased . -fold and . -fold in citrate-anticoagulated blood, compared to . -fold and . -fold in edta-anticoagulated blood (edta vs citrate: p = . and p = . , respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. μl of exosome samples from cancer cells (a and h ) or normal cells (mrc ) were injected into the pdms microfluidic device and incubated for minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed . folds absolute cd peak value change and . folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [ ] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [ ] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over % of separation efficiency at µl/min flow rate and exosomes were collected continuously with high recovery (~ %). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was x ^ exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , , ) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd . we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h -eu. . . -fet open programme via the grant agreement . immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (> x) and three-dimensional pore density (> x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with > x enrichment from input and x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard mm petri dishes were patterned with µm tall polystyrene microtracks spaced by , and µm across a mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < . ). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir- excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir- as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir- recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h , through mindgap/fet-open/ga project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a colour panel, which included annexin v (for the majority of circulating evs), cd (for plateletderived evs) and cd (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium / /n/ nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p), were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following -week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial # ). eleven sedentary obese youth ( . ± . years, bmi ≥ th percentile underwent an acute bout of ae ( % heart rate reserve, min). blood was collected before [time (at) − , min], during [at , , min], and after [ min (at ), min (at )] exercise. afterwards, youth participated in -week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at ), immediately after ae (at ) and after recovery (at ). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~ . nm) as opposed to nrs (~ . nm) at at (p < . ) and this pattern was maintained at at and at , though not significant (p = . ). nrs displayed differential ev size distribution (peaks at nm or nm), while ev distribution was highest at nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~ % of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~ mm chunks, placed in serum-free dmem and incubated for hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases > times more evs than adipose tissue per unit of mass (p < . using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (< . % of events). evs from mg/mt mouse skm overwhelmingly expressed mg (> % of events) with negligible (< %) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~ %) or mt (~ %). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: ) modification of evs post-secretion, ) clearance of fluorescent evs by the liver or ) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association ( ipa ) to dsl. endothelial cd delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd -facilitated fa uptake is unknown. here we examined the role of ec cd in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute ( hr) cold exposure ( °c) with core temperature monitored every minutes. after hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa . for radioactive studies, primary lung ec were serum starved for hrs and incubated overnight with h-oleic acid bound to fa-free bsa ( : ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd (eccd -/-) compared to controls (cd fl/fl). however, during cold exposure at °c , eccd -/-are unable to maintain body temperature (p < . ). plasma free fa are higher in cold exposed eccd -/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd -/-and cd fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav- phosphorylation. the internalized cav- and cd positive vesicles containing fas are released as sevs. to determine the dependence of cd on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd -/-cells contain less labelled-fa (p = . ). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk and dk . introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco- cells+ht- cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with micrograms/day for weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat , angptl and dgat , but a decreased expression of fabp , fatp , mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl mrnas, therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn ) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn are present in vertebrates, of which the soluble plasma fn is derived principally from hepatocytes, while cell-associated fn is produced by numerous cell types. here we describe a novel function of plasma fn in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml hepatocytes, fn ko aml cells in which fn was ablated using crispr-cas , primary human or mouse hepatocytes, or human hepg cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh -labelled evs from parental (ev-hep) or fn ko (ev-hepfn ko) aml cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn ko were - nm in diameter and positive for common ev markers (cd , cd , flotillin- ). mass spectrometry showed that fn was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn was verified by western blot and co-immunoprecipitation with anti-cd or antiflotillin- . western blot showed that fn was also abundant in evs from primary human or mouse hepatocytes, hepg cells, and human or mouse serum. fn and ev-hep co-sedimented at a density of~ . g/ml. ev-hepfn ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn -independent. as compared to ev-hep, the binding of ev-hepfn ko to target cells was highly reduced whereas ev binding was independent of fn expression by the target cells themselves. both ev-hepfn ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen ⍺ expression in mouse hscs in vitro. summary/conclusion: fn is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type diabetes and islet transplantation introduction: type diabetes (t d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type diabetes (t d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = / group) were cultured in normal ( . mm) or elevated ( . mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~ % of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c bl/ j mice (n = ) or female mt/mg mice (n = ) was cut into~ mg pieces and added to well plates (~ mg/well) filled with ml of serum-free dmem and placed in a cell culture incubator at c for hours. skm from male mice was treated with -deoxyglucose ( -dg) ( . nm - mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged ( , x g for min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer . ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for -dg experiments were made using a repeated measures -way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = . ) as a function of -dg treatment, but no effect on ev diameter (p = . ). bleb treatment did not alter ev abundance (p = . ), but significantly reduced ev mean diameter (p = . ; % decrease; dmso: . ± . vs. bleb: . ± . ). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with -dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa ). introduction: extracellular vesicles (evs, exosomes) are nanovesicles ( - nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [ ] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [ , ] . in this study, we newly developed evs decorated with cellpenetrating sc or (sc ) peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap , because the peptides can be efficiently internalized by breast cancer cells. [ , ] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd -gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc peptide is known as one of cell-penetrating peptides, and branched structure of sc peptides, (sc ) , further enhances the cellular uptake [ ] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc or (sc ) peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc ) peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc ) peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc ) peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc -dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb- cells expressing either a targeting or non-targeting control sgrna and applied to hek t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were ± nm in diameter as measured by dls and possessed a negative surface charge of − . ± . mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only in . e ± . e evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were ± . nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between to sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately e copies per cell. summary/conclusion: mda-mb- evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp- cells. after h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il- in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. ) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas protein conjugated to the highly positively charged protein. post-uptake of these cas -loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of ± nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p -mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p proteins with novel activities termed gain-of-function (gof). even though mutations in tp are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p . methods: p results: in the colon, tumour cells harbouring mutp were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp , monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p affected tam were characterized as tnf-αlow/il- high, over expressing cd- and cd , with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp tumour cells to macrophages, revealed a mutp -specific mirs signature led by mir- promoting the tam phenotype and creating an invasive front together with tumour cells. mir- was also found to be the top mutp -associated mir in a cohort of human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p . we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p . omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). sera from infected and from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh . tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg , alix, cd ). two parasite proteins, enolase and the fh . tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh . protein generated a highly significant difference between the two groups (p value = . ); as did enolase (p value was . ). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il- and il- release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ - glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ - (fucα - ) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from -week-old melon plants, either uninfested or infested with adults of a. gossypii (n = , replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of different proteins were identified in phloem sap evs, including present in exosome databases (exocarta). on top of that, differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < . ). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and % of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s ™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir- - p, mir- a- p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) . . to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir- - p and mir- a- p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir- a- p and two isomirs (mir- - p and mir- - p) . the auc of the panel index was . . we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes ( - nm), microvesicles ( - , nm) and apoptotic bodies ( - , nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than %. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c bl/ j male mice ( weeks) were orally dosed with ccl (vehicle, , mg/kg). serum were separated from blood after , , and hours after ccl administration. the serum was centrifuged at , x g to remove cellular debris and subsequently ultracentrifuged , x g. the pellet is resuspended in pbs and ultracentrifuged , x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl : mg/kg & mg/ kg). well known hepatotoxicity biomarkers, mirna- and mirna- were upregulated more than -fold in the administration of mg/kg ccl , but not responded in the administration of mg/kg ccl . summary/conclusion: these results suggest that mir- and mir- are mainly released from liver to blood directly only in the administration of mg/kg ccl , while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both mg/kg and mg/kg ccl should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing ( - x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of > micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (> , bp versus~ bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l r or the resistance mutation t m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled patients with metastatic lung ac: with egfr mutations and without (control). from the patients with egfr mutant lung ac, we processed blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon deletion or l r mutations. results: from patients with egfr exon deletion mutations, we detected identical mutations in exorna from / samples. exorna based mutational load increased and mirrored clinical progression in patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at points and was therefore not plotted. exorna from patients with l r and t m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in / patients. summary/conclusion: in summary, we detected egfr mutations in / exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of . per , people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from patients at the time of initial diagnosis, and matched controls by sex and age. ev-associated rna was isolated from - ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed mrnas as differentially abundant between gbm and control samples, with mrnas enriched in gbm samples and mrnas enriched in control samples (p < . ). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. million americans are estimated to be suffering from the disease, % of which are women with an average age of . overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from ss patients and healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over % of reads mapping to the transcriptome. further rna biotype analysis showed over % of transcriptome reads mapped to protein coding genes and lncrnas. we detected over , mrnas and approx. lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified upregulated genes, including mrnas and lncrnas (p < . ). genes were found to be downregulated in ss, including mrnas and lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = ) and unsuccessful (n = ) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± nm dihydrotestosterone (dht) ± µm enzalutamide (enz) for h. evs were isolated using sequential ultracentrifugation ( g for min, , g for min, , g for h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie . snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg using bowtie . results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l blockade, is a method to eliminate cancer cells. ectopic expression of pd-l , on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l , and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l expressing cancer cell lines were used to generate evs. pd-l -myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl- or ev associated markers. exoview r reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl- on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l -myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l positive evs from different cancer cell lines, only . and clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l , however, only clone . was able to immobilize these evs. the results suggested that clone . could be a potential pd-l antibody to detect pd-l positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in , there were over , new cases and , deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the -year overall survival rate for hnscc is only - %. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag- plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat , hla-a, tenascin, e-cadherin, β catenin, cytokeratin , epha , and cd , and hpv-related markers p , p , rb, cyclin d , and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat , hla-a, and tenascin. only kert demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p , rb, cyclin d and p , only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha and cd showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be ) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or ) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc- cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd , cd , cd , flotillin (flot ), alix and tumour susceptibility gene (tsg ), negative marker calnexin (canx), and contaminant markers apo-a for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot is membrane protein that forms microdomains in the plasma. alix and tsg , an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a (apo-a ) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd , cd , cd and flot were positive and canx and apo-a were negative. in urine evs, cd , cd , flot- , alix and tsg were positive and canx and thp were negative. in ccm evs, cd , cd , flot , alix and tsg were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir- - p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir -/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt ( -plex) label of the total mir -/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer . (thermo) was used for protein processing and quantification. results: a total of . proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes. we found out several protein modulated by mir and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the th most common cancer worldwide and the rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from healthy subjects, subjects with cirrhosis, and subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, samples were eligible for analysis after low expression filtering. we used pair-wise ratios of cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of . and . respectively using mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd , cd , cd , adrβ , hsp , epcam and flotilin- ) followed by binding of cy -labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta- receptor (adrβ -receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and h collections.the aim of this study is to compare uevs excreted in all single voids during a hour collection period and compare it with hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg , alix and cd ). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel- mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin- and uevs pan tetraspanins (cd + dc + cd ) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a hour urine collection reflected the average results of all single voids over a hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus hour uev analysis. we concluded that the hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, out of detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d . identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger ) and up-regulated (e.g., lifr) genes were found (p < . ); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc -/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc -/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc -/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc -/-epididymal epithelial cells, and addition of wild type evs to arrdc -/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to % of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at - weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = ) compared to gdm-aga (n = ; p > . ). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = /group; >twofoldchange; p < . ), including four skeletal muscle-specific "myomirs": mir- - p, mir- a- p, mir- b, and mir- a- p (all increased). all four myomirs were present in placenta but only mir- - p was significantly altered in gdm-lga compared to gdm-aga (n = - /group; p < . ). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir- - p regulates vascular development in other systems, so we propose that mir- - p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc or tsc genes. together, the tsc proteins regulate mtorc activity. all tsc patient post-mortem samples exhibit renal disease and % of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas to disrupt the tsc gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately % of the evs were in the - nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg , the transmembrane proteins cd , cd and cd , and the primary cilia-related hedgehog signalling-related proteins arl b. proteomic analysis of evs identified a significant difference between the tsc -intact and tsc -deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified intact proteins and rna-seq data identified , mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is th most common cancer in men and th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing % of all bladder tumours. at diagnosis % of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly - % of these patients will experience disease relapse and - % will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from nm to nm with characteristic ev morphology, express ev-associated markers as cd and hsp and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t & u ) gbm cells were "primed" using evs derived from mesenchymallike (u & ln ) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y (to block those budding at cell membrane) and gw and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs ts(i) , using a discontinuous optiprep density gradient, after the cells were treatment for hrs with the compounds listed above. evs (pooled fractions - with a density range of . - . g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs t and mda-mb- cells using the ev to cell ratios of × evs/ x cells, × evs/ x cells, × evs/ x cells to evaluate doseresponse. ev-track id ev (score of %). results: gw significantly (p = . ) decreased ev release from hs ts(i) cells. manumycin a and a combination of calpeptin and y (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y actually increased ev release; but not significantly. of the reduced numbers of evs released following gw treatment, hla-dr+ evs were significantly (p = . ) enriched. none of the evs analysed significantly changed hs t or mda-mb- growth rates. however, evs from cells treated with calpeptin (p = . ), gw (p = . ), manumycin a (p = . ) and combo (p = . ) caused significant reduction in mda-mb- migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw (p = . ), and combo (p = . ) caused significant reduction in hs t migration. summary/conclusion: while gw was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw or calpeptin and y significantly reduced migration of both recipient cell lines. funding: phd funding: tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a -day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as % of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu ) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas- b), but are also capable of disrupting a -day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas- b and calu evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas- b cells were cultured in transwell for days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas- b monolayers were treated with calu evs at , and μg/ml for hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas- b cells treated with calu evs at and μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after hrs. results: beas- b and calu evs were enriched in - nm size range, and cd and cd were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp . calu evs significantly impaired day mature beas- b monolayer's barrier properties, which at the highest dose caused % reduction in teer from . ± . to . ± . Ω.cm (n = ). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in min ( . ± ng/ml in control vs . ± ng/ml in treated) (n = ) and complete loss of e-cadherin expression at cell-cell tight junctions (n = ). at the transcript level, calu evs induced significant downregulation of e-cadherin by % and upregulation of vimentin (mesenchymal marker) twofold (n = ) in beas- b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein (grp ), a chaperone protein of the hsp family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration ( . µm) and differential ultracentrifugation. grp surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp was identified on the surface of evs from irradiated cells. the increase in ev grp correlated with increased grp expression at the donor cell surface. the grp content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp in radiation resistance we overexpressed grp . here the modest ( x) overexpression of grp was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp -dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp levels, and subsequent ev-mediated transfer leads to enhanced grp levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma -rv line were cultured in d and d conditions, using d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the fractions of ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd and cd were used as ev markers, apob and apoa for lipoprotein contaminants control, and cd , cd and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in d conditions, the -rv cells produce up to -times higher ev number than in d. size and density distribution of evs derived from d cultures but not of d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under d conditions. the profiles of ev subtypes produced by cancer cells growing in a d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over , cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok , vok , and vok were cultured in flasks containing ml of ev-depleted media ( % fbs, centrifuged hr x , g). when cells reached~ % confluency, the conditioned media was collected and spun at , g for mins two times to deplete any remaining debris, leaving~ ml of media. this media was concentrated to a final volume of~ ml using a pall jumbosep kda mwco filter. this concentrate was purified from protein by using an izon qev- column, collecting ml fractions. protein content of each fraction was analysed using a absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~ µl using the pall microsep kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the detection antibodies used across capture beads, cd , cd , cd , beta- microglobulin, and cd were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd , cd , and cd . summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct and ht , were cultured in serum-free medium for hours. conditioned medium was concentrated by ultrafiltration (mwco kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff- fibroblasts were used as recipient cells. the uptake of evs (pkh fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff- . results: nta showed a higher concentration of evs released by ht . hct and ht evs displayed a mean diameter around nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd and tsg and negative for gm a non-evs marker. ht lineage as well as derived-evs are lmwptp-enriched in comparison to hct cells and evs. upon incubation, fluorescently hct and ht derived evs were internalized into hff- cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff- cells. intriguingly, evs derived from ht promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff- fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir- a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir- a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir- a expression and silencing of exosomal mir- a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r a expression in all. methods: jm , sup-b , and nalm- leukaemic cell lines were treated in vitro with vincristine ( . to . µm) and prednisone ( . to . µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir- a analysis. expression of mir- a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm and sup-b ) in a dosedependent manner. both cellular and exosomal mir- a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm , sup-b , and nalm- ). these observations demonstrate that cellular mir- a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir- a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for h either under normoxia or hypoxia ( % oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin- function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., ) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her mirna and engineered to adhere to her on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging - nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her (human epidermal growth factor receptor ) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her both in cells constitutively expressing her and in cells transfected with a plasmid encoding her . secondly, we report that the mirna targeting the synthesis of her reduced the viability of her positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her on the surface of tumour cells. the -mir-her exosomes package with mirna designed to block her synthesis and deliver to cells. these exosomes kill cancer cells dependent on her for survival but have no effect on cells lacking her or which were engineered to have her but do not depend on it for survival. the -mir-xs-her exosomes carry in addition a peptide which enables the exosome to adhere her on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her on their surface. the exosomes with -mir-xs-her are significantly more effective in shrinking the size of her -positive tumours implanted in mice than the -mir-her exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her have no effect on her negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her mirnas by her -positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her by her positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd + t cell activation introduction: cd + tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd + til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd + til such that specific mirnas contained within modulate physiological processes that inhibit cd + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after days of co-incubation of tumour derived exosomes, cd + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day and day . rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd + t cell activation owing to different donors. exosomes derived from peo. and ovca cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo. remained a low ifn-gamma expression and from ovca stayed at relatively high level. small rnas profiling analysis between the two cell lines identified mirnas (p < . ), and mirnas have been reported with validated targeting information, and out of have targets involved in immune signalling. mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose ( gyx ) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [ ] [ ] [ ] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated ( gyx ) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [ ] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [ ] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by -methyl cytosine dna elisa kit. results: dna fragments with size between and bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than % of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of - bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [ ] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire ) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire inhibitor mkc and cells were analysed for immune modulatory surface markers, like hla-i, b -h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd ) are downmodulated by ire inhibition across different cell lines. cell surface expressed cd and b -h show cell line specific downmodulation profiles upon ire inhibitor treatment. other immunomodulatory marker such as b -h and b -h , integrin cd , cell adhesion-promoting cd and stemness/metastasis marker (cd and ssea) are unaltered on ire treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd , cd , cd ), similar in number and showed differential expression of immune markers upon ire treatment. mitochondrial and lysosomal activities were unaltered under ire inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cÚram research centre, /rc/ ), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = ) following wisgrill et al., ( ) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, x , , x and , x events/ml mean, eevs was , x , × and , x events/ml mean, levs was x , × and , x events/ml mean and mevs , x , , x and , x events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp / - . gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens , zo- ) and gap junctions (con-nexin , cx ) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo- at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx , evs isolated during an episode of acs caused loss of cx from the plasma membrane. the integrated intensity of cx membrane staining was decreased bỹ % following treatment with acs evs. cx protein decreased on average by %, cx mrna levels by % and neurobiotin transfer by - % in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx -mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul tr , comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation ( , xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and % lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd as a marker of platelet origin and cd as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < . ) when compared to the control plasma. pevs also had a significant effect (p < . ) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) - nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor -year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from nm to nm with an intact morphology, expressing ev-associated markers such as hsp , cd , cd and cd . size-resolved evs also had a differential expression of mitofilin, actinin- , syntenin- and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc -epi and pc -emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at × g for min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at , × g for min at °c. the remaining supernatant was then centrifuged to isolate the evs at , × g for min at °c. the evs pellet was further washed in × pbs followed by a second centrifugation at , × g for min at °c. the final evs pellet was resuspended in × pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of selected mrnas. the nanostring ncounter low rna input kit with the multiplex gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc -epi and pc -emt cells were significantly different. clec b, kdr, crip , il ra , cc d b were significantly upregulated in pc -emt cells, while cxcl , epcam, esrp , tgfb , cdh , s a , ovol were significantly downregulated in pc -emt cells. the expression patterns between pc -epi and pc -emt evs were also significantly different. tbx , cav , col a , slc a , myc, itgb , timp , camk b, ptgds, p h , itgb , vim, stat were all significantly downregulated in pc -emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta integrin regulate cd , il- and il- levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc prostate cancer cells. we have also shown in the past that alphav beta -positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m markers, such as cd and cd . finally, we have demonstrated that down-regulation of alphav beta integrin up-regulates the stat -interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat has a causal effect in pbmc polarization, we down-regulated alphav beta and stat in prostate cancer cells derived sevs using sirna as well as crispr-cas strategies. the sevs isolated from these cells were used to analyse m polarization by measuring the levels of cd in pbmc. the results show that sevs lacking alphav beta inhibit cd levels in pbmc in a stat -independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta , show that pbmc selectively up-regulate the levels of il- and il- , which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd , il- and il- levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, ) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b splenocytes in vitro. the transfer of donor mhc class i and ii on b cells was analysed by imaging flow cytometry. next, t cells from b mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b spleen cells crossdressed with allogeneic balb/c mhc. alternatively, × balb/c or b bm derived evs or × balb/c bm cells were injected iv to b mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r dk . antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. ) . we show here that the more biologically relevant surface-associated c exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c culture on agar by similar methods to meers et al . omv were stained in some cases with fluorogenic syto dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa- and washed x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto -stained vesicles from surface-adherent c were similar to previously observed~ nm vesicles (meers et al., ) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c cultures grown on filters with nm pore size but not nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c on filters with a nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c specifically bind and inhibit the growth of fungal hyphae of various species without direct c cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt , and human and rodent endothelial cells, with a varying degree of icam- expression, were used to determine the effect of lfa- expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam- and lfa- . in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf- syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf- was the cytokine detected in mvs samples in large amount ( . ± . pg/µg proteins in mvs). fibroblasts treated with mvs or plgf- significantly stimulated pro-collagen i level production with a fold change of . ± . and . ± . . moreover, the neutralization of plgf- present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf- signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin - ); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size ( to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation ( g for minuntes followed by , g for minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd , anti-cd , anti-cd and anti-cd were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd /cd population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank homozygotes mice model of autism. cultures were treated with msc-exo ( ^ particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around % of detected mirnas were found in both blood products, whereas only % of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir- were consistently depleted in evs compared to the corresponding blood product, others like mir- a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing (nlrp ) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp inflammasome because s-elns had no impact on activation of the absent in melanoma (aim ) inflammasome. s-elns also inhibited the secretion of interleukin (il)- and both protein and mrna levels of the il b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of different proteins found in bovine serum were quantified. results: six of proteins were not detected in any sample. of array antibodies were found to cross react with human antigens. of the bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained of proteins, of proteins, and of proteins, respectively. concentrations of individual bovine proteins ranged from . to , . ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: ) methodology, ) study design, ) intervention details and ) efficacy/ adverse events. results: after screening articles, studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal ( %), neurological ( %) and cardiac ( %) conditions. benefits were described in % of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~ - nm, microvesicles~ - nm) only % of studies used appropriate nomenclature. ultracentrifugation ( %) and isolation kits ( %) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein ( %), particle number ( %) or cell count ( %), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only and studies met the characterization standards recommended in the misev and guidelines, respectively. finally, % of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately - nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta , fn, tnc, snai , col a and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad / pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata : umo- / /d/nz / from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all - nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd , tsg and hsp . in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir - a cluster and mir - cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox- , pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls- - - g. introduction: malnutrition impacts approximately million children worldwide and is linked to % of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; - nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control ( %) or a low protein ( %) diet for days to induce malnutrition. from day to , they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day , hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how d and d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for d culture, mscs were aggregated into spheroids using non-adherent -well plates and cultured with addition of . % methylcellulose. to collect conditioned media, both d and d mscs were cultured using serum free medium for days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd /cd /cd . cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β to induce fibroblast activation, subsequently exposed to d and d evs, and collagen production was measured. further, d and d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: d and d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd /cd /cd and average particle diameters of < nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in d and d mscs. d and d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both d and d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around - nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a kda omp, predicted to be involved in iron acquisition, and other omps ( - kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to gy or gy x-rays to the whole body except the hind leg. starting with either µl or µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from ml culture medium or µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks ( , m/z, , m/z, , m/z, , m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks ( , m/z, , m/z, , m/z, , m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd , cd , cd protein markers using western blot analysis. fraction no. - of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in -d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using -d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for -d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: ) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and ) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir- , lncrna al . and ac . were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of ibd patients and healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd -cd beads for days in serum-free media. size and concentration were analysed using a nano sight instrument, while the presence of known evs markers (cd , cd , hsp ) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of proteins in the four groups. of those, ( . %) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd , tubulin, and serpins among others. patients' evs shared proteins in common such as proteasome subunit beta type- , t cell receptor beta, and the amine oxidase containing copper . interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type- , and signalling lymphocytic activation molecule (slamf ). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about % of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from . , . and . ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l cam antibody. in total and l cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd , cd and cd on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < . ) and protein concentrations (p < . ). l cam evs immunocaptured after exq showed -fold higher particle (p < . ) and fivefold higher protein (p < . ) concentrations compared to l cam evs after exu, and -fold higher particle (p < . ) and -fold higher protein (p < . ) concentration compared to l cam evs after qev. l cam evs after ev precipitation (exq) showed , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after exu, and , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after qev. l cam evs following different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd and cd . protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range ( - nm), protein content ( - ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = ) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd p using flow cytometry. the resulting platelet releasate ( ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for hours before treatment with pevs ( µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be - nm in size. the final product was positive for platelet markers (cd , cd p) and key ev markers (tsg , alix, cd ). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex ) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually - nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af ( . ± . ) is significantly lower than that in mj ( . ± . ), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs ( . ± . and . ± . , respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj ( . ± . nm), af ( ± . nm), af-derived evs collected at , × g and , × g ( . ± . nm and . ± . nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed . × particles per ml from a ml fplc fraction of evs measured from , events by mrps, confirming that evs are being captured by this method. there were also . × particles/ml and . × particles/ml in the two subsequent ml fractions that are known to contain lipoprotein particles, though these were measured from , events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r ag ; nih ug ca - optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd , cd , and cd , calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd , cd , and cd and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses ( , to , da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > , da and linear pei with mw > , da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir- - p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: - ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of adherent cell lines were established in celline ad bioreactors and propagated for up to weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation ( g - , g - , g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt and bt consistently shed mainly dead cells ( - %), as opposed to mcf and mda-mb- which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each - day culture averaged - × particles, representative of yields obtained from cells grown in to conventional t tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the ʹ-end and cholesterol at the ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio- -nh and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with % isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin (il ) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at , , and months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp , a highly pathogenic strain, and , a natural commensal strain. af -labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto- . results: using flow cytometry, we observed jp omvs were delivered to , cells, and at a lesser amount to jp cells. alternatively, , omvs associated readily with jp cells, more than to , cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r de & r de . utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap ) modified via crispr-cas to model gaucher's disease in vitro. these cells contain a bp insertion in the th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc ) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were nm in size with a typical lipid bilayer structure, and were positive for cd , cd , flotillin and negative for annexin a confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph . ) compared to drug release at ph . . cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a lung cancer cells compared to mrc- cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il , are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv ) was engineered to stably overproduce endogenous il . this cell line was cultured in exosome-depleted rpmi and stimulated with pma( mg/ml) for min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of g, min, rt and g, min, °c. the resulting supernatant was filtered ( µm) and ultracentrifuged at , g for h at °c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of - nm and a broad zeta potential ranging from − mv to − mv, while the second population has a mean diameter of - nm and a zeta potential of − /- mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il- rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin- acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il- rα blockade on tec changes tec-ev (anti-il- r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il- r blockade on tec impacts tumour progression via their unique ev cargo. methods: human tnbc samples, mda-mb- , mda-mb- and mcf cell lines were evaluated for the expression of il- rα. nevs and anti-il- r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from . % of the human tnbc samples expressed il- rα. mda-mb- and mda-mb- , but not mda cells, expressed il- rα. in vitro, nevs provide survival and migratory signals, while anti-il- r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il- r-ev treatment induced vessel regression in established tumours formed of mda-mb- cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist were found in tumours from animals treated with anti-il- r-evs. in addition, anti-il- r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb- cells. nevs that were depleted of mir- - p (antago-mir- - p-evs) were effective as anti-il- r-evs in down-regulating twist and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il- rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il- rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either d migration or boyden chamber invasion assays. previous to exosome isolation, heya cells were treated with simvastatin ( um, h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg ), its trafficking (rab a, rab a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya cells or hgsocs enhance the metastatic potential of heya established spheroids in d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya -derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg and an increase in the inactive forms of rab a and rab a in heya cells. we also observed a decrease in emmprin levels in heya -derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw carcinoma cells expressing er-gfp and rab -rfp were exposed to evs from sw cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp and rab orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta are co-immunoisolated with orp , vap-a and rab suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p and p . bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g checkpoint proteins, p and p . we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk / . because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r ca and commonwealth of pennsylvania -department of health sap for kmb. phosphorylation of tyrosine in annexin a is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for %- % of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a (anxa ), a ca+ -dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) in anxa has been implicated in regulating several anxa activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa at tyr is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn -egfp plasmids expressing the constitutive phosphomimetic (anxa -y e) and non-phosphomimetic mutant (anxa -y f) gene expressing mutation at tyr site were overexpressed in mda-mb- tnbc cells. mutant cells were experimentally validated for anxa specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa -y e-gfp) and non-phosphomimetic (exo-anxa -y f-gfp) cells and were analysed for exosomal surface expression of anxa by immunoprecipitation and flowcytometry. cal- breast adenocarcinoma epithelial cells were treated with exo-anxa -y e-gfp and exo-anxa -y f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa in cal- was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb- phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa -y e-gfp had elevated surface anxa expression compared to exo-anxa -y f-gfp. cal- cells treated with exo-anxa -y e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa (tyr ), p-src(tyr ) and p-paxillin(tyr ) compared to exo-anxa -y f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr in anxa in mda-mb- tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro ca and nimhd's u md to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from µl of plasma using a commercially available qev size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd magnetic beads were used to fractionate the evs into cd + (platelet derived) and cd -(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd + and cd fractions in all patient samples. the average ± standard deviation of total evs obtained from the patients was . x ^ ± . x ^ evs/ml of plasma (coefficient of variation [cv] : %) while the average and standard deviation of cd -evs was . x ^ ± . x ^ (cv: %). the cd -ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from . % to . %. multiparameter flow cytometry showed that over % of total evs were cd + and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd -fraction ( % - %) as compared to the cd + fraction ( . % - %). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd -ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than % -year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < . ). notably, functional exosomal mmp and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ . -fold (p < . ). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm -h , product of first identified metastasis suppressor gene nme , is significantly downregulated under metastatic conditions. nm -h serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab , rab and rab ) in the biogenesis of exosomes. in addition, bacterial homolog of nm has been shown to interact with rab and rab . however, experimental evidence supporting a relationship between exosomes and nm -h is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb- cells) on lower/non metastatic cells (mcf- cells). nme was overexpressed in mda-mb- cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb- cells and mda-mb- /nme cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm -h in the exosomes isolated from mda-mb- cells upon overexpression of nme . proteinase k protection assay confirmed the packaging of nm -h inside the exosomes isolated from mda-mb- /nme cells and excluded the possibility of membrane association of nm -h . additionally, overexpression of nm -h led to a significant reduction in the ability of mda-mb- exosomes to stimulate movement of mcf- cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd , cd and alix in the exosome fraction isolated from mda-mb- /nme cells as compared to mda-mb- cells. interestingly, rab a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm -h overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm -h via exosomes. these findings support a regulatory role of nm -h in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm -h which affects the biogenesis of exosomes as well as to assess the effect of nm -h overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c bl/ mice performed moderate treadmill exercise ( m/min, -wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day . the correlations of cepc-exs and their carried mir- with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir- level, apoptosis, axon growth ability and gene expressions (cas- , bdnf and akt) were measured. results: ) exercised mice had a smaller infarct volume on day , which was associated with decreased cell apoptosis and cleaved cas- level, and a higher microvessel density than those in control; ) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day . the upregulated mir- level positively correlated with the numbers of tepc-exs in ischaemic brain; ) the numbers of cepc-exs and their carried mir- level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day ; ) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day , accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; ) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir- and pi k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir- . funding: this work was supported by american heart association ( post ) and nih ( r ns ). syndecan- regulates alveolar type epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type epithelial cell (at ) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan- , a heparan sulphate proteoglycan, is overexpressed by at cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan- is profibrotic. moreover, syndecan- deficient (sdc -/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc -/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir- b- p, − - p, − - p, and − - p) compared to the sdc -/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan- altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan- regulates at senescence through evs. methods: bleomycin was intratracheally given into wt and sdc -/-mice. at day , lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a ) and mouse (mle- ) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at cells of wt mice, these genes were significantly downregulated on sdc -/-at cells. senescence proteins, p and p , were also less expressed in the lungs of sdc -/-than of the wt mice by wb. to determine the functional effects of evs in bal, a cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p , by wb on mle cells whereas sdc -/--bal-evs inhibited p expression. summary/conclusion: our data indicate that syndecan- regulates lung fibrosis through the senescence signalling pathway on at cells. furthermore, syndecan- controls at senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for hrs in serum-free basal media, in hypoxic culture. after hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, - week-old wistar-kyoto female rats received ^ imcdc-ev, imev-pi, scramble or vehicle intracoronary minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at and hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at hrs and an increase at hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles ( - nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from to nm, with the majority of them measuring - nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. msc-ev were characterized according to misev guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of %. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd and cd . the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of patients ( highgrade serous, low-grade, benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = ) was used for validation. results: an oc biomarker panel of micrornas (voom: . % accuracy; random forest: % accuracy) was generated. the panel consists of members from the mir- family and the mir- family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh . cyp e [hepatoma cells stably transfected with cyp e designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs (misev ). the human hepatic stellate lx- cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il- β, il- , tnfα, and fibrotic markers acta , and timp using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror . and david bioinformatics resources . . results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately - % of particles distributed in the range of to nm, with a peak at~ nm. enriched expression of hiv protein p was observed in fractions f -f . western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg and cd specifically in f -f fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il- β, il- , tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih- r aa - a . antimicrobial peptide ll- induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll- , a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll- and functions of ll- -induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll- -stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll- augmented the level of evs. evs from ll- -injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll- stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll- -induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll- induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december , and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top , which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir- - p, and hsa-mir- - p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with × or evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in doses during days ( days interval). after immunization, mice were infected intra-peritoneally with trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f / + cd b+ and cd /cd expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab . seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a . results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict - ) medicine, nanfang hospital, southern medical university, guangzhou, , china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of to nm. t. marneffei evs were internalized by raw . murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m -polarization marker cd and diminish that of the m markers cd . incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin- β(il- β), interleukin- (il- ) and interleukin- (il- ). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation ( , × g, , × g) and concentration ( kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy treated (gyy-evs) a cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a cells were cultured for hours in the presence or absence of control-or gyy-evs, then infected with rsv for hours. viral titres by plaque assay were measured in recipient infected a cells. results: we confirmed the presence and purity of our evs. we found that gyy reduced the particles number of evs, but did not change ev size. a cells treated with gyy showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd expression, a mvbs marker, compared to untreated cells. recipient a cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd + cells included b ( %), cd + t ( %), cd + t ( %), nk ( %) and nkt ( %) lymphocytes. myeloid cells comprised neutrophils ( %), monocytes ( %) and dcs ( . %). erythrocytes represented % whereas, unexpectedly, reticulocytes were . % of total cells. in addition, we also detected hscs, which accounted for . %. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd , cd l and cd markers. interaction studies showed an increased proportion of t cells (cd + -fold and cd + -fold), monocytes ( . -fold) , b cells ( . -fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir- promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb- triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb- significantly reduces the young's modulus values of mcf cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over % of the crc patients develop liver metastasis (lm) and % will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only % of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only - % of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double -year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: . text mining of published ev mirna from crclm patients; and . differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing - patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach , where we have classified the lesions in the patients. preliminary data on patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein (cdcp ), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr , known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr /cxcl signalling. overexpression of cxcr was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht and sw , which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr in ht and sw by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht and sw cell lines. the expression of cxcr in ht was increased after the uptake of p-evs. additionally we observed that migration rate of ht cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht clones stably overexpressing snail and from control ht -pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ reads, two fold up or down regulation between . − . and adjusted pvalue ≤ . . the uptake of pca-sevs was performed. three candidate mirnas (has-mir- c- p; has-mir- ; has-mir- - p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < . ), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw . the uptake of pca-evs by mc t -e was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx , osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china ( ); xijing hospital science and technology foundation project (xjzt ptk ). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from rbt (hsjd-rbt , hsjd-rbt , hsjd-rbt , hsjd-rbt ) and rbvs (hsjd-rbvs , hsjd-rbvs , hsjd-rbvs ) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of proteins were identified. among these, and were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin- , tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc and q exactive instruments. tune (version . ) and xcalibur (version . ) was used to collect data while proteome discoverer (version . ) was used to analyse data. protein expression lists were created by merging the sample replicates together and commonly expressed proteins were determined using vinny . vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of . x ^ particles/ml (n = ) with a mean mode size of . nm (n = ). surface marker analysis confirms the presence of exosome associated proteins cd , cd , and cd in addition to a high expression of cd (n = ). the completed analysis revealed commonly detected proteins across products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca . summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd -targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a -day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm- (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at particles/target cell for -hours. total viable cell counts were assessed via imaging-based cytometry (nc- ) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm- as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm- cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks ( billion per administration) starting from . months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin- beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin- and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c c myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after days incubation. treatment with bmp- was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for days. qpcr analysis of mrna expression of inflammatory cytokines (il- , il -beta, il- ) metalloproteinases (mmp and adamts ), and apoptotic genes (bax, bcl ) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd , cd and cd ). the mean number of exosomes per microgram of protein was . ± . x indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c c cells with bmp- , x , x , x , × or × exosomes alone was . , . , . , . , . and . respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba- and cd ), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank gene. shank is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank mutated mice. the first is a complete deletion of exon ( q . ) and the second is a specific insertion mutation of guanine to position in the gene (insg ) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the misev guideline recommendations. each animal received an intranasal administration of ul containing exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses ( , and years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd , cd ) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < . ). the gap closure achieved with msc-evs was slower than with msc-cm (p < . ) but faster than with cm of tenocytes (p < . ). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd- (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c bl/ mice in chf received echoguided myocardial injection of pbs vehicle control ( ul, n = ), cpctx-ev ( ul, n = ), or cpcres-ev ( ul, n = ). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~ nm) at a concentration of~ . e particles/ml (~ , particles/cell) and~ . mu cd /ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by . x, . x, and x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased > %) than pbs control mice (pbs vs cpctx-ev, p < . ; pbs vs cpcres-ev, p < . ), and the average ejection fraction of the pbs group deteriorated . x more than the cpctx-ev group (− % vs − . %, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds ( -mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for and weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp (osterix) was evaluated at and weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the ng bmp- control. dose escalation from . , , and e resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at or at weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp , , , and , tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il- r, il- , were also detected. expression of pro-inflammatory cytokines mcsfr, mip- a; anti-inflammatory cytokines tnf-ri, tnf-rii, il- ra; and homoeostatic cytokines timp- and timp- were observed. cytokines associated with wound-healing, icam- , g-csf, gdf- , and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range ( - nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly - nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at − c. calvarial defects of -mm diameter were surgically created on thirty-two -week-old male sprague-dawley rats. these rats were then randomly assigned to groups (n = rats/group): defects treated with collagen sponge containing μg of sevs in μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at and -week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at -week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). cd + microvessels at sizes ≤ µm and > µm in cs/sevs group ( . ± . and . ± . microvessels/hpf) were also significantly higher than that of cs/control ( . ± . and . ± . microvessels/hpf; p = . and p = . respectively). by weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of . ± . which was significantly better than that of cs/control group ( . ± . ; p = . ). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < . ), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of . ± . than that of cs/control ( . ± . ; p = . ). moreover, cs/sevs group showed significantly higher ocn+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r , national medical research council singapore, r . immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd + (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd and cd t cells expressing activation markers like cd and cd . summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd cell types, i.e. th , th , th and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct ). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from blood samples of volunteers with no record of disease. methods: plasma obtained from ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = , , p < , ) and to the concentration of evs (r = , ; p < , ). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = , , p < , ). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon research and innovation program under grant agreement no. (ves us project) and slovenian research agency (arrs, grants p - , p - , j - ). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c c and primary dmd myotubes. methods: scs from c mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c c myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c c myotubes were treated with µm h o for h followed by treatment with . x sc-evs for h. separately, cultured dystrophic myotubes were treated with . × evs every h for h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < . ). results: based on estimated cell number and ev quantification, each sc released approximately . × ± . x evs/day. evs delivered protein cargo into myotubes within h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h o resulted in a % decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a % increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a % increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of - , ng/ml and - µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r = . ). importantly, cholesterol quantification on purified ev samples, ranging from e to e particles/ml, correlated well with nta measurements (r = . ). both µm filtration or an additional , rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd , cd , cd ). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly % of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around - % of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than nm cannot be detected, while fluorescently stained ev smaller than nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small ( - nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di- -anepps (di- ) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di- ). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo pancreas carcinoma cells based on size and mass. after , x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from to nm in size. interestingly, tetraspanin markers cd and cd were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant ), leo pharma germany (grant ). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd , cd , cd ) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs ( . particles/μl) method was estimated times superior to the lod of conventional elisa ( , particles/μl). the spfs calibration curve showed a wide dynamic range at least over logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim . by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about , compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd , cd and flotillin- were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas ) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb- -luc-d h , -d h ln and -bmd a were cultured in ev-depleted media. microvesicles ( , x g, min) and exosomes ( , x g . h) were isolated using a combination of differential ultracentrifugation and a density cushion (~ . g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = x cell lines). results: in total proteins were quantified, with being quantified in all samples. in total and proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin- was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo ( / ). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered , % glutaraldehyde/ %formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at - degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with instead of nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd , cd , cd ) and the neural cell adhesion molecule l (cd ). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd , cd , cd ). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd binding. interestingly, the cd positive evs were not positive for any of the classic exosomal markers (cd , cd , and cd ). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε /ε (n = ), ε /ε ( ), ε /ε ( ), ε /ε ( )] and non-ad controls (n = ) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev . proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s ™ using ion™ chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin- . regarding apoe genotypes, proteins were differentially expressed between ε carriers (ε /ε and ε /ε ) with non ε carriers (ε /ε and ε /ε ). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε /ε and ε /ε groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh . efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of months-old c bl/ female mice. . months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at . evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca and ca subregions. at was localized primarily in gad + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf ag , nih r ag , nih r ag , cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd (cd + ev), cd (cd + ev), and neural cell adhesion molecule (l cam+ev) were analysed in young, male athletes with or without mtbi ( - yo, n = per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within -hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at − °c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size ( - nm) cd + ev, cd + ev and l cam+evs were significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). population percentages of large size ( - nm) cd + ev, cd + ev and l cam+evs were also significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd + ev) or platelets (cd a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from pd, matched healthy controls (hc), ap with multiple system atrophy (msa) and ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort ( pd, hc, msa, ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc . ; sensitivity . %, specificity . %) and msa (auc . ; sensi-tivity %,specificity . %)andgoodaccuracyforap-tau (auc . ; sensitivity . %, specificity . %). a diagnostic model based on ev markers expression, cor-rectlyclassified . %ofpatientswithreliablediagnostic performance after validation in an external cohort ( % of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted , , , and -fold and activated by μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and -fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd + and cd +, cd p + > nm) and activated platelets (cd +, cd p+ > nm) were measured by flow cytometry (apogee a -micro). platelet aggregation was assessed using impedance aggregometry. results: a -fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd + . -fold and cd +-cd p + pevs . -fold. the concentration of cd + (r = . ) and cd +-cd p+ (r = . ) pevs as well as platelet aggregation (r = . ) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd + (r = . ) and cd +, cd p+ (r = . ) pevs in undiluted wb, which was absent in -fold diluted blood (r < . ). summary/conclusion: the absence of aggregation and pev release upon platelet activation in -fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium / / n/nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at ~ -(young) and -(aged) week-old. large evs were isolated from the plasma by centrifugation ( x g). sevs were isolated by ultracentrifugation ( , x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de Ética no uso de animais -ufrgs; nr. , ) approved all animal procedures and experimental conditions. male wistar rats of -and -month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna . arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed micrornas. of these micrornas, were differentially expressed between aged and young-adult animals, micrornas were significantly upregulated and were downregulated in aged animals compared to young adult (p < . ; fold change of | . |). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < . ). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin- signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule- (vcam- ) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) and hmgb . pharmacological blockade of hmgb and hmgb and sirna depletion of hmgb in smooth muscle cells attenuated nfkb (p ) phosphorylation and nuclear translocation, vcam- expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled patients undergoing elective cabg. of these, underwent coronary computed tomography angiography months post-surgery showing % graft occlusion. flow cytometry mv analysis was performed in patients ( /group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = . ) and four-fold (p = . ) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < . ). this mv signature predicted graft occlusion (auc of . [ %ci: , - , ], p = . ). by using a mv-score ( - ), the or for re-occlusion for a score above was . ( % ci . - . , p < . ). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme ). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction ± vs ± % in vehicle mice, p = . ), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration ( . ± . ms imexo vs . ± . ms vehicle, p = . ) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction ( . ± % vs . ± % in vehicle, p = . ). in vitro study showed nrvm exposed to imexos for days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by -minute illumination with nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r hl (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf / are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf /antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf /are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf -targeting mirnas. nrf -targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf -targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf -targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf / are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf /are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx , the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin (cx ) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx . results: release of cx was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p partially restored the secretion of cx , suggesting an interplay between ischaemiainduced cx degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor (tlr ) and its downstream regulators (tnfα and il- ) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek cells was induced by calcium-ionophore a . evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h c and ac cell lines. results: a induction of hek cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to h ischaemia followed by h reperfusion in h c and ac cells in a dose-dependent manner. evs activated tlr and its downstream signalling pathway in h c and ac cells as well as the expression of cytoprotective haem oxigenase (ho- ) in h c cells. summary/conclusion: a -induced evs exert cytoprotection in h c and ac cells by inducing tlr signalling and ho expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will ( ) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and ( ) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = ) was calculated for a study power of % and α error of % for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = ) and malignant (n = ) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd , cd and cd . ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (> nt) and short rnas were isolated from ev enriched urine of pregnant (n = ) and non-pregnant (n = ) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v. . . .a. results: overall, rna isolation using mircury followed by the smart-seq v library preparation kit yielded the highest % of mapped reads: % in pooled non-pregnant, % in individual non-pregnant, and % in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v libraries had higher % of mapped mrna reads compared to pico libraries (p < . , t-test). in contrast for mirneasy advanced it was reversed ( % vs %). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih k hd - , nih u hl , and a ucsd igm-illumina mini-grant. il- mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin- (il- ) is essential for pan t cell proliferation and performance, low dose il- treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt is a novel il- mutein fc fusion molecule (il- m) designed to selectively engage with tregs. using a humanized nod-scid il rn-null (nsg) mouse model we have shown that pt expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt -dosed humanized mice exhibit increased expression of foxp and cd , and demethylation of foxp and ctla- genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt -or vehicle-treated mice in order to identify treg specific mirnas from the il- m treated animals. methods: cd + haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt or vehicle. plasma samples from mice were collected at day and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with bp single end reads at an approximate depth of - million reads per sample. raw sequences were mapped to human genome grch and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il- m-treated groups was mostly comprised of mirna and trna. plasma was pooled from animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv cells). a cd antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f / ) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem and iba ) were identified in exosomes isolated from bv cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type diabetic patients (t d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from - ml of h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > m) were closely similar. instead, rna reads were lower than m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns . the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over % of dna in plasma and urine exist as a free circulating pool, while in saliva up to % is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of %. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are - nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn -isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn is a amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within minutes. total nucleic acid (tna) was extracted from either vn -isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including % specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn -based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: plasma samples of hepatocellular carcinoma (hcc) patients, plasma samples of patients with liver cirrhosis - on the hepatitis c virus (hcv) background, healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd , cd ) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b m, actb, tuba a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for genes in hcc and liver cirrhosis groups (increasing up to x compared to control samples and decreasing up to no detected expression). in out of patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in out of patients with hcc increased expression for mrna line- was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds " - " circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma ( patients in total) and rna from plasma in a separate group ( patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir- - p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir- in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir- ) and ev-based (mir- - p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after x , g centrifugation, followed by differential centrifugation at , g and , g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd and anti-cd antibodies. after h t cell activation was measured by il- cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il- production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il- production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon framework programme under the grant fetopen- evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev . methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, ml samples were centrifuged at k g, k g, k g, k g and k g sequentially for min each and pellets re-suspended in ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. μl of the sample was loaded and the pbs buffer was used as eluent. factions were collected and analysed by cd /cd sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than . % of proteins were removed while the recovery of extracellular vesicles was close to %, which is much higher than % of the commercial product. the total separation time was less than min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~ µl or > ml) while concomitantly removing . % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: ml of cell culture media (ccm), . ml serum, and ml urine samples were processed with the isuf platform and recovered in µl. for all cases, evs were enriched with recovery efficiency greater than %. the processing time for a ml sample was min with over % of purity. we compared ev concentration and purity isolate from . ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = ), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her , cd and mir biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug tr (e.r.); r hl , r hl , and r eb (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between to hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin- and cd by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of . - . g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd immunocapture. the resulting sev proteome was enriched . -to -fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of and nm. methods: a cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of h at , g at degrees celsius (rotor type ti, beckman floor ultracentrifuge optima l k), qev size exclusion chromatography columns from izon (qevoriginal/ nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns ), trps (izon), nanodrop (spectrometernd ), tem (fei tecnai kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of nm and nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of , a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška Štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about - nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [ ] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir- - p and hsa-mir- a- p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir- a and mir- , play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg , a stem cell marker, modulates ev release. dsg is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed cellular and ev mirnas that were differentially expressed in response to dsg with overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p signalling pathways. interestingly, mir- was upregulated while mir- a was dramatically downregulated in evs. targets of mir- a, icam- , il- , and il- , cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r cancer cells grown in d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson Åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional ( d) cancer cell cultures. methods: human prostate cancer pc cells were grown in d cultures using ecm-based hydrogel, in standard d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive d cultivated pc organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc (pc rd) cells after h incubation in dfbs-medium with or without the compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. . - . g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at x evs/ x cells, x evs/ x cells), were evaluated on recipient du and rv cells. evtrack id ev , score % results: calpeptin and y , alone and in combination, did not significantly affect quantities of evs released. however, gw significantly (p < . ) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp compared to control evs (p < . ). this effect was reverted when gw was combined with manumycin a (p < . ). following all compounds treatments, x evs/ x cells inhibited rv proliferation (p < . ), while at x evs/ x cells only evs from manumycin a (p < . ) and y (p < . ) treated cells reduce rv proliferation. evs following gw treatment significantly (p < . ) inhibited du migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < . ). summary/conclusion: while none of the proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low -year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp mutation contained not only ki + proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki + proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated hours after r and incubated with t l cells for hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir- a/ a/ - family members are the most markedly increased mi-rnas in mi/r exo. incubation of t l cells with mi-r a mimic significantly downregulated edem , dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of t l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir a mimic. finally, in vivo administration of gw (exo biogenesis inhibitor) or mir a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir- a/ a/ - . funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the rd leading cause of cancer-related deaths by . patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a -year survival ratẽ %. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are % non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc- , sw- , capan- and miapaca- ) and patientderived xenograft (ppcl- and ppcl- ) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h c ) were co-cultured with cancer cell evs for - hours. metabolic activity was measured using a mito stress test on a seahorse xfe extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev- mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma ( % of correlation with msc classifier). mscpos-evs stimulated d and d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir- modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir- is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir- -evs from endothelial cells and mir- -evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd , cd , met and nrp which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b f lineage. methods: b f cells were cultured in dmem supplemented with % fbs and antibiotics. the cells supernatant were harvested each hours, filtered through . µm and ultracentrifuged at x g for min at ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd , cd and cd on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd molecule in comparison to cd and cd . we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd , cd , met and nrp . summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b f lineages harbouring shrna vectors, in order to knockdown gene expression of cd , cd , cd , met and nrp to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b f -derived cells. introduction: overexpression of her occurs in % of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her , potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to % of patients with her -overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her -positive cell lines (hcc and skbr ) and their neratinib-resistant counterparts (hcc -nr and skbr -nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs ts(i) , were evaluated as negative control for her . in brief, cm was centrifuged at g, for min x to get rid of any cells. the supernatant was then centrifuged at , g for h at ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev ). results: neratinib-resistant cell line variants were found to have reduced her protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her , while those from drug-resistant cells lacked her (similar to the evs from the tnbc cells); reflecting the her status of their cells of origin. summary/conclusion: this study indicates that a reduction in her protein expression is a mechanism by which cancer cells manifest resistance to her -targeted drugs (i.e. by making fewer her receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her seems to reflect the her status of their cells of origin. this suggest that analysis of her on evs in the peripheral circulation may help predict response to her -targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than % of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc tumour cell line was established by repeated injections with ohsv in mc tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. results: ) the lower expression level of rab a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. ) furthermore, we established an ohsv resistant mc tumour cell line by repeated injections with ohsv in mc tumour-bear mouse model. similarly, in ohsv resistant mc cell line, rab a expression was lower than parental mc cells. and the release of exosomes and virus was decreased in ohsv resistant mc cell line. these results were confirmed by rab a sirna knockdown. ) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. summary/conclusion: downregulation of rab a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. inactivation of emilin- by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b -f ), high metastatic potential (b -f ) and lymph node metastasis (b -f r ). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin- secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw . cell viability and cell cycle assays with an overexpression model (b -f -emilin- ) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin- expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b mouse melanoma model. among them, we found that emilin- , a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin- is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin- has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin- overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin- are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin- by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb gene and the consequent overexpression of the encoded protein her have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb gene amplification and her protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb amplification and consequent her overexpression on a limited volume of plasma ( . ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: ) we detected erbb amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb positive patients; ) we confirmed her overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her protein on the surface of the isolated evs; ) we succeeded in the quantification of her transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = ) and on a cohort of her positive (n = ) or her negative breast cancer patients (tnbc; n = ). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse - ) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in % paraformaldehyde or % glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a x oil immersion objective. sevs were imaged with a d-sim acquisition protocol. tem was performed on mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd . samples were counterstained with uranyl acetate and observed under a jeol ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from to nm were detected, as the final resolution achieved was nm. instead, for tem, sevs under nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd or l cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from to nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from - nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd , cd , cd ), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, and hr post-exercise. perceived muscle pain (pmp) was assessed at and hr post-exercise. large evs were isolated using a g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions ( - ) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = . x particles/ml plasma). mean ev diameters were ± nm across all timepoints. no change in ev size nor number was seen over time, however, mir- decreased at hr when compared to hr in the small ev isolate only. plasma displayed an immediate increase in myomirs- and − at hr, which returned to baseline at hr. in contrast, myomirs- b and remained elevated over the hr period. myomir- b and , as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir- in small evs at hr is consistent with previous data. no decrease in mir- in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either d or d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either d or d platforms displayed distinct magnetic sensitivities, whereby mscs grown in d or d platforms responded most favourably to pemf exposure at mt and mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m macrophages and activated into m macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il β and tnfα levels increased. as control, thp monocytes were used. patient-derived oa chondrocytes were grown in well plates and co-cultivated with activated m macrophages which were seeded into thincerts and added to the wells representing the inflammation model. furthermore, cells were treated for hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp and primary monocytes) into m macrophages was demonstrated by elevated levels of the inflammatory cytokines il β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . α, (oh) d regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small ( - nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. α, (oh) vitamin d ( α, ) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir- , mir- and mir- . the aim of this study was to determine the regulatory role of α, in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with - m α, or vehicle for h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = per variable. significant differences (p < . ) were determined using r v . . . results: α, treatment altered expression of mv mirs compared to control mvs, whereas cell mirnas were differentially expressed. . % of significantly up or down regulated mir found in mvs overlapped between α, and vehicle groups with the remaining being uniquely differentially expressed. α, increased mv mir- and decreased mir- - p two mirs known to regulate osteoblast proliferation ( increases, decreases). summary/conclusion: α, regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that α, regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of %. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/ / , uidb/ / ). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods ( - minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was - % c.v. detected bead concentrations were linear with flow rates between . and . ul/min. double well aspirations provided similar data with aspirations between - ul. valid data was achieved for a low abundant target (~ - events/ul) after only s, < %c.v. detection sensitivity was determined to be~ / , . carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps . = op . l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps . = op . results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: . m- . m nacl) and high salt (h-s: . m- . m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. : , ) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup : kitani et al., results immunol. : - . ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: # ) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a )) and the japan science and technology agency (crest [jpmjcr h ]). clic is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic , a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic in the host or tumour cells to develop metastases. results: experimentally, the essential clic host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic in comparison to those from mice bearing tumours that lack clic . tumour cells released evs that induced myofibroblast conversion of wildtype but not clic ablated lung fibroblasts. summary/conclusion: these results illuminate clic expression as a prognostic marker for breast cancer patients, and experimentally, clic is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc . the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg , alix, cd ) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp - ), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps : separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps : diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of - cm- . we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of . ± . nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean nm vs nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean . nm vs nm nta can only detected vesicles above nm and exoview™ only measures vesicles between - nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , Éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc gr) and mrc (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with μm osimertinib or pbs for days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc gr during days. we will investigate how mpa-exsomes activate erk signalling pathway in pc gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc gr cells by more than % compared to control pbs. pc gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t m mutation.these data show that "mrc -pc gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc lung fibroblast increased cell viability and exosomal mir- through the tgf-ß pathway in treatment osimertinib. exosomal mir- up-regulation in cocultured nsclc with mrc- induced drug resistance to drug-induced apoptosis. thus, exosomal mir- expression in co-cultured nsclc with mrc may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd , cd , cd , and alix with a mean size of nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified > , proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within hours (kodali et al., int j mol sci, ) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately billion evs were dispensed bilaterally into both nostrils of young f rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba- (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba- and cd dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are - nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected gene products. of these, were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component a (c a) in sevs from sni model. intercellular adhesion molecule (icam- ), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of als patients and healthy volunteers by differential centrifugation and characterized by nanosight ns . cd , cd , cd , cd a and annexin v were used for flow cytometry. sod , tdp , fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf slide and raman spectra were acquired using a nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod- , tdp- and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps . introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab b or with shrnas against the exosome secretion and biogenesis regulators rab b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) or spines at div . for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from . ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with . ml of samples were centrifuged at k g for h. fractions were then pooled based on densities ( . - . g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev ). results: immunoblotting showed absence of actinin from all samples, while cd and tsg were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~ ^ /ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < . ) more cd -positive particles/ml of milk vs all other guc and kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < . ) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb- and skov- cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd , cd , cd or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb- derived evs isolated based on cd and cd expression were taken up more efficiently by hmec- and mda-mb- cells than evs isolated based on presence of cd or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. ) of the dutch research council (nwo). aml were harvested from tib cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca + ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor (ifnar ) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg , il- , ifnα, ch h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar -null hspcs as well as the lack of evs-induced stat phosphorylation or ifnar downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor (batf ) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf inhibitory effects of mdscs recruiting. we used transwell assay to determine batf -positive evs (evs-batf ) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf model to demonstrate evs-batf crosstalk with distant tissues. amd blocking in tumour model confirms that evs-batf dominated by the sdf- a/cxcr signalling pathway. in addition, exo-counter detection of evs in pairs of gliomas in different stages proposes plasma-evsbatf (plevs-batf ) as a prognostic marker. results: we found that tumour-derived evs-batf regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf injection significantly reduced sdf- α expression in the tumour tissues. after blocking sdf- α signalling by amd , the inhibitory effects of batf overexpression on mdscs recruitment were rescued. evs-batf inhibit mdscs recruiting and secreting mmp , mmp , and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in pairs of gliomas in different stages reveals that the number of plevs-batf can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf in regulating tumour microenvironment and propose plevs-batf as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled ~ nm pores in the si n membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd -phluorin) and extrinsic (lipophilic dye, di- -anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of - features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from to nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = ) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in × s acquisitions. two microfluidic cartridges were used to study a dynamic range of - nm. samples were diluted into phosphate buffered saline with different concentrations of tween or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of or nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within % confidence. summary/conclusion: we recommend to use . % w/ v bsa in dpbs as sample diluent, because tween affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp- ), which provides an active surface for probe immobilization. then, antibodies against cd , cd , cd markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd , cd , and cd markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than billion of people around the world within upcoming years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about % of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from cm- to cm- and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~ to~ mda and z~ to~ e and are highly dependent upon the conditions used to extract and isolate the evs. in total, , particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s , s , and s families that are centred at~ . ,~ . , and~ . mda, respectively, appear too small to be ascribed to exosomes. the remaining , ( %) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r gm - ). bab is supported by indiana university quantitative chemical biology fellowship (t gm ). in situ detection of exosomal microrna- b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna- b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb- , mirna- b-encapsulated anionic liposomes, and mirna- b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna- b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna- b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna- b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna- b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma ( , xg, min/ . ) m filtration/ , xg, h)(n = ) and lymphoma conditioned medium( xg, min/ xg, min/ xg, min/ , xg . h/ , xg, h) (n = ). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd /cd /flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k ( mg/ml/ °c/ min) and rnase a ( mg/ ml/ °c/ min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = . , n = ) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a -fold and fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd , cd and cd ) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above . , indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered . - ml plasma or - ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction ( μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam proteolytic activity might be more informative than the level of adam protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of different al-pha bead biosensors were designed. a control, tev-specific biosensor detected . introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the isev abstract book biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps . = op . kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl chemokine, thus contributing to npc immune escape (mrizak et al., jnci, ) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress ). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl (ccr ).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than times at over % purity without leaking by kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, - nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation ( , g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤ %) and precision (cv ≤ %) across both platforms used. we defined two ev size gates: a "latex" gate ( to nm polystyrene latex beads), and a "silica" gate ( to nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv < %) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from hnc patients were taken before (a) and after (b) rt. healthy volunteers were used as a control group (c). ev were isolated from ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/ / / / / /g). results: an untargeted gc-ms-based approach allowed the detection of metabolites in serum samples and exosomal small molecules, of which joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were metabolites which levels discriminated compared groups (a,b,c) of serum samples and compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from human pancreatic cancer cell lines (capan- , mia paca- , and panc- ) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of , unique proteins were identified confidently across the combined samples. the proteins present in all four sev types ( , proteins) consist of general housekeeping proteins. proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/ t cells. conversely, there were proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd show an increased signal for the luminal protein tsg . ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg in a lower extent indicating a subpopulation of evs. the microarray uses less than µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for different surface marker from samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of ml were collected from patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus- , k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of - nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida . the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the - nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc h cells and incubated with low metastatic hcc l cells. in other experiments, cpe-shna loaded exosomes from hek cells were incubated with hcc h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc h cells revealed cpe-wt mrna and protein. exosomes released from hcc h cells were able to enhance proliferation and invasion of hcc l cells. when cpe expression was suppressed in the hcc h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified isev abstract book exosomes were used to transfer cpe-shrna to hcc h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. . stress hormones promote prostate cancer aggressiveness through modulation of mir- - p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir- - p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe- , and pca cells pc , lncap and mda-pca- b. proliferation of cells treated with leptin in the presence or absence of mir- - p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir- - p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir- - p in rwpe- cells.co-treatment with mir- - p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir- - p target jag and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc cell exosomes with a . -fold increase at nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir- - p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek t cells and mir- a- p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r and t (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd , a protein marker of ev. we modified ʹ-terminal and ʹ-terminal of the cd aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd aptamer for specifically binding with cd in evs. in addition, we designed cd aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample ( µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex ). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx e ). methods: t-flask expansion of ctx e cells was performed to yield batches of - l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of . µm dead-end filtration prior to concentration in a kda hollow-fibre tff system. tff retentate was loaded onto ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd and cd . exosomes recovered in this manner could achieve full wound closure in vitro over hours, when dosed at µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of e - e particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir- were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate- (irs- ). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from . ml of plasma from pd-n, pd-mci, and pd-d patients, we immunocaptured nevs using anti-l cam antibody. then, irs- pser and irs- ptyr and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < . ) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = . ). irs- pser was lower in pd-d than in pd-n. irs- ptyr significantly decreased with increasing mds-updrs iii score (p < . ). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs- ptyr were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample ( ml) was collected from patients and processed within hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation ( . x g, rt) for cell removal and storage at − °c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through . µm pores was used for large particle removal, while centrifugal kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a ) were pooled and concentrated to a volume of µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was , nm with concentration range of × particles/ml of starting urine. uevs were positive for the tested marker proteins hsc , flotillin, tubulin, gadph and cd . qpcr verified mirna presence in uevs, with ct for mir let- i at . summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin- and epha that were enriched in adev-atp. integrin- facilitated binding of adevs to the neuronal surface, and epha -receptor signalled through ephrin to the tyrosine kinase erbb / that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf . this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc , arc, and cplx to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a d-printed scaffold-perfusion bioreactor using a flow rate of ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il- , quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with µm ethanol for hours prior to ev collection results in a strong inhibition of il- production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor ( . e ± . e ) when compared with flasks ( . e ± . e ). protein measurements also indicated that ev production in the bioreactor (~ µg) was much greater compared with production in the flasks (~ µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr and hsp ) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp was detected in subpopulations and of both pf-evs and ui-evs, whereas sr only in subpopulation . isev abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam fib-sem gemini ii (zeiss, germany) and jsm- f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at °c and °c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x- and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. key: cord- -atbjwpo authors: nan title: poster sessions date: - - journal: febs j doi: . /febs. sha: doc_id: cord_uid: atbjwpo nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v epitope tag into the n-terminal of klf gene. insertion into the target side of klf gene via crispr-cas system provided an opportunity to overwhelm the above mentioned obstacles. v epitope tag would not interfere with the function of the klf and also enable us to recognize endogenous klf via anti-v antibody in the mouse myoblast cell lines (c c ). we confirmed the targeted insertion into the exon of the klf gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc , but not ctf is detached from chromatin. our data also shows that meanwhile, rad checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc and ctf are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc has a more specific role in the process of adaptation. our data demonstrates that mrc is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost drosophila proteins with c h zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw , pita and zipic) were found to interact with cp and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw , pita, zipic and cg . first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw , pita, zipic and cg revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project № - - ). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t and t concentrations in adult male wistar rats. fifty rats aged between and weeks ( ae g) were divided into five groups (n = in each group), and drugs were administered each day intraperitoneally (ip) for days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol ( . mg/kg, n = and mg/kg, n = ) and clozapine ( . mg/kg, n = and mg/kg, n = ). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture hours after the last injection. the t and t plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v . . kruskal-wallis and bonferroni tests were used. t plasma concentration levels significantly differ between sham (median= . mg/kg) and haloperidol ( mg/kg) (median= . mg/kg), haloperidol ( . mg/kg) (me-dian= . mg/kg) and clozapine ( mg/kg) (median= . mg/ kg), haloperidol ( mg/kg) (median= . mg/kg) and clozapine ( mg/kg) (median= . mg/kg) groups (p < . ). however, no significant differences between the groups regarding to t plasma levels were observed. in conclusion, haloperidol and clozapine increased the t plasma concentrations, but didn't have any significant effect on t plasma concentrations. p- . . - isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a . lm filter and incubated on lb agar medium for hours. based on differencies of colony morphologies, isolates were selected and purified for identification. s lipase activity assay was carried out by rhodamine b. all of the strains exhibited lipase activity. for determining the substrat specificity of isolates, different substrates were used; nitrophenyl-butyrate, -nitrophenyl-caprylate, -nitrophenyl-laurate, -nitrophenyl-myristate, -nitrophenyl-palmitate. results were measured spectrophotometrically at nm. all of the strains hydrolyzed -nitrophenyl-butirat, while there was no activity with -nitrophenyl-palmitate. bacillus sp. l was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l will be cloned and expressed for more analyses and industrial applications. p- . . - some quantitative aspects of hair follicle layers differentiation e. vsevolodov , , v. golichenkov , a. mussayeva , , i. latypov llc "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with mcm hair diameter for those with the hair diameter mcm the ratio of hair medulla diameter to hair diameter increases from . to . . the ratio of hair diameter to the diameter of inner root sheath increased from . to . . it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes % of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from to u/g pulp basis and the consistecy of the pulp was between and %. enzyme at the desired concentration was added to the pulp and the mixture was incubated at °c for hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as % pulp consistecy, u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p- . . - biochemical characterization of lipase from bacillus subtilis strain a from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec . . . ) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a is partially purified and characterized. bacillus subtilis strain a is isolated from olive factory from soke (aydin, turkey) and identified with s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with % olive oil (vol/vol) for hour at °c. after incubation, cells were harvested by centrifugation at , rpm for minutes, resuspended in mm tris-hcl (ph . ) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at °c. the enzyme was active and stable in the broad ph range of . - . and temperature range of - °c. bacillus subtilis strain a have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p- . . - investigation of pin as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a (pin ) as a potential nfib interactor. pin is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin implicating this domain in nfi-pin interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin proteins in hek t cells showed that pin precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin . we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. - - . p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during - period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy , open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were to years old and their average age was . ae . . ofthem ( . %) were under years of age whereas of them ( . %) were above years of age. number of pregnancies were scaling between to with an average of . ae . . of mothers ( . %) were not undergone amniocentesis, whereas babies with chromosomal abnormalities were detected among mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. ( . %) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with . , . and % concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses ( . , . % and %) in both species with percentages %, % and % in r. ridibunda and %, % and % in b. viridis, respectively, while in the control group it was about %. furthermore, delayed larval development was detected. thus, the larvae from the control and . % oil group reached gosner stage (gs) , tadpoles from . % and % groups were at gs- and gs- , respectively, by the th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p- . . - effect of catechin loaded plga nanoparticles on glioma cell line histone h t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h subtype and hyperacetylation of histone h tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p- . . - serum dickkopf- levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf- (dkk- ) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk- levels in obese and non-obese children and adolescents. materials and methods: the study included obese children and adolescents ( males and females) aged from to years and healthy normal-weight controls ( males and females) aged from to years. serum dkk- levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = . ). however, there was no significant difference between dkk- levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk- levels were not changed in obese children and adolescents. p- . . - transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib À/À precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia À/À hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the kb human cdo promoter region identified five nfi binding sites: one cluster in the first kb region, another in the . kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to % in hek t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd /ankk gene variants in eos development. in this study, eos patients and healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd /ankk taq a (rs ) and bdnf val met (rs ) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd /ankk rs polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = . , or = . ; % ci: . - . ). however, no significant relationship was observed in the genotype frequencies of bdnf val met polymorphism between eos patients and controls (p = . ). these results indicate that, drd /ankk rs genotypes may affect eos development. however, bdnf val met polymorphism may not be associated with eos. lack of association of bdnf val met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black ) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black ) was determined between and nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black ) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm , prm and their relationship with defective spermatogenesis. materials and methods: this study has been performed on infertile and fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm and prm . results: distinct levels of spermatozoal prm and prm mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm was reduced in (% ), and the mrna levels of prm was reduced in (% ) out of infertile patients.in the current study,we found statistical significant association between the prm expression and infertility (p < . ).although prm gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > . ). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm and prm themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf , myogenin) in white muscle were studied in salmon parr of age groups +, +, + in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr ( +) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings ( +). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age +. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age + and + then in +, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to +. there was no differences in expression level of myod, myf and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. - - . p- . . - lmp and lmp gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu , f. ozbas gerceker sanko university, gaziantep, turkey, gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide (lmp ) and low molecular weight polypeptide (lmp ) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome . these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp and lmp gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp and lmp genes were determined. results: a deviation from the hardy-weinberg equilibrium (v = . ,p < . ) was found for the genotype distribution of lmp gene polymorphism, while the lmp genotypes found to be distributed (v = . ,p > . ). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp and lmp genes should be increased to understand their effects as a genetic marker. p- . . - investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have - nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found . ; . ; . nm respectively. the morphological features were examined by sem images. antioxidant activities of q , q ve q nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: - - -gep ". p- . . the effect of environmental enrichment on spatial memory and certain nmdars, and ht a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and ht a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into groups as enviromental enrichment (ee, n = ) and standard cage control (scc,n = ) groups. eeg housed in an enriched environment and sccg were kept in standard cages for weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr a, nr b, ht a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the training days showed that eeg located the hidden platform significantly earlier than sccg on days , (p = . , p < . ). also eeg significantly spent lower time in the outer zone of the maze on days , which was the sign of low anxiety level (p = . , p = . ). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < . ), in fst comparison of groups showed no difference (p > . ). the levels of nr b and ht a were significantly increased as compared to sccg as well (p < . , p = . ). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr b and ht a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p- . . - effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, ( weeks old) were divided into groups as control (cg, n = ; + , male+female) , experiment (msg-low dose, e g, n = ; + , male+female) and experiment (msg-high dose, e g, n = ; + ) groups. msg was administered at mg/kg/d to e g, . g/kg/d to e g for weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au autoanalyser. level of total protein was significantly increased in e g and e g groups when compared to cg (p < . ). level of alb€ umine was also increased in both egs but significant difference was seen in e g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < . ). although the glucose levels in both egs were increased, the increase in e g was statistically significant (p < . ). the alt levels of in egs were also increased but the significant increase was seen in e g (p < . ). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. ( mg/kg) + tartrazine ( mg/kg) + brilliant blue fcf ( mg/kg) + ponceau r ( mg/kg) + azorubine ( mg/kg) + indigotine ( mg/kg) + erythrosine ( mg/kg). artificial food color mixture were administered to g and g and drinking water was applied simultaneously to g by oral gavage per day for weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > . ). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g were higher than g and g in serum and kidney and brain homogenate (p < . ). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p- . . - er quality control protein network in cf to modulate f del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo and gst-pi enzymes. for this purpose, mrna and protein expression of nqo and gst-pi enzymes were analyzed in ht and sw (colon); hepg and huh (liver); pnt a and pc (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo protein was expressed in all cell lines and relative protein expression is highest in the hepg ( %) and pnt a ( %) while huh ( . %) showed relatively low expression of nqo . in addition, nqo mrna expression was relatively high in ht ( . fold) and pnt a ( . fold) when compared with liver cell line hepg ( . fold). gst-pi protein expression was found very high in huh ( %) while there was no expression in hepg . gst-pi mrna expression was relatively higher in pnt a ( . fold) and ht ( . fold) when compared with huh ( . fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo and gst-pi are ht and huh , respectively. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p- . . - tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about out of every cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. mrna studies and mirna study, which were designed for different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ . , fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf transcription factor and hsa-mir- . in the literature hsa-mir- and klf are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb ) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd , cd , cd and cd as positive markers and cd as a negative marker. gene expression profilling was investigated using affymetrix hg-u -plus arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < . , fold change≥ ). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of genes were identified that differentially expressed in msc line versus primer mscs ( upregulated and down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] association between loss of q , gain of q . and progression in sporadic colorectal cancer n. belder , b. savas , m. a. kuzu , i. pak , h. s€ umer c ß elebi , a. ensari , h. € ozdag biotechnology institute, ankara university, ankara, turkey, school of medicine, ankara university, ankara, turkey, ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix . genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from stage i, stage ii and stage iii samples. paired cnv analyses were performed using partek genomic suite . and genomic segmentation algorithm was performed using a minimum of markers per segment, a signal-to-noise ratio of . and the cut-off value for the gain and loss was set of ae . . the adjusted p-value ≤ . were considered to be significant. whole genome cnv analysis revealed that amplification of q . with genes was found the most frequent ( . %) in stage ii tumors. the most frequent ( %) amplifications were q . and p . in stage iii tumors. while deletion of chromosome q . in stage iii with a frequency of % was found the most frequent loss, deletion of q . was seen the most frequent ( . %) in stage ii tumors. two tumor suppressor genes smad and smad which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of q . might have a significant role in the progression of cancer. loss of q comprising two tumor suppressor genes is also another important finding. q loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no: s . p- . . - meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin , cin and cin according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin , cin and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin , cin and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin , cin and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with common genes showed that these targets were significantly enriched (p < . ) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group ), dia-betic+glibenclamide (group ), diabetic+plant extract (group ), untreated control (group ) and control+plant extract (group ) groups (n = for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract ( mg), and the reference drug glibenclamide ( mg/kg) by gastric gavage every day for weeks. in group , blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group , serum glucose, alt, ast, triglyseride and hba c levels decreased compared to group while cholesterol and ldl levels were high. in group , serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba c were higher compared to group . body weights increased except group and hdl levels were not altered. histologically degenerative changes observed on pancreas of group were decreased in groups and . there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p- . . - on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p- . . - qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan , c. neuschl , r. alberts , g. a. brockmann school of medicine, istanbul kemerburgaz university, istanbul, turkey, department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) , , , and . most likely candidate genes within or near the regions with the highest significance levels were identified. the genes f rik, gbe , a n , and four genes cenpc , stap , uba , gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr for fat mass, on chr for fat mass gain and on chr for lean weight, and chr for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to healthy women aged - years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than . . . % of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = . ) and protein (p = . ) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = . ), isoleucine (p = . ), leucine (p = . ), lysine (p = . ), methionine (p = . ), cysteine (p = . ), tryptophan (p = . ), and glutamic acid (p = . ) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = . ). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at nm, but in contrast to pvx, did not have additional peak at nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant - - ). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k , k , k and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a- dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full -utr and -utr sequences of m satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p- . . - physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga ) and with encapsulated antigen (plga ) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga particles (plga - . %, plga - . %). the flow cytometry results demonstrated that % of plga particles conjugated with egfp-sbb, and only . % of plga particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga particles size was . nm and plga - . nm. nta data also showed that mean plga particles size a little smaller than plga ( . nm and . nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p- . . - a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt , a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at °c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt cells with the isogenic cho-k cells as a control. cells after transient transfection were incubated at °c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt and the wild type control cell lines are performed to validate the reported model system. p- . . - host-pathogen interactions: is there a relationship between tlr polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than million individuals die from tb and each year , cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher ( . / , ) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even % of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study tb patients and healthy controls were included. tlr genotyping for rs , rs was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be . . results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p- . . - lytic action of bacteriophages as a tool for the obtaining of images p. boltovets , r. radutny , t. shevchenko institute of semiconductor physics nas of ukraine, kyiv, ukraine, scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the . % agar with the nutrient medium and the . % agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t were applied. samples were incubated during the twenty-four hours at + °c. after that stencils were removed and the samples were stained by coomassie blue r- or fuchsine (with further fixation by the % acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type (st ) complexes with ctx-m gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli - / -g and attc e. coli-reference strain and (ii) to compare the proteomes of strain at two conditions: with and without antibiotics. strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein , cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p- . . - molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu , i. gheorghe , , m. popa , , a. ion , m. mihai , v. lazar , , m. c. chifiriuc , carol davila" university of medicine and pharmacy, bucharest, romania, research institute of the university of bucharest-icub, bucharest, romania, faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of s. aureus clinical strains isolated in from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of . %, meca in . %, sscmec type ivd element in . %, ccrb in . % and sccmec types i, iii, iv in . % of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa ( . % of the isolates), followed by clfb ( . %), fib ( . %), hlg ( . %) and bbp ( . %). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p- . . - analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin ( %), vancomycin ( %) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than antibiotics. it was determined that among the investigated strains % contained meca, % -mecc, % contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in . % of cases, sed - %, seg - %, sei - %. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in % of strains, beta peptide toxin genes in %, delta toxin gene in %. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p- . . - impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] host-specificity determinants of bacteriophage vb_ecom_fv considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of water buffalo milk and dairy products ( water buffalo cream and water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso - and isolates were confirmed for the presence of the s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in of water buffalo milk ( %), of water buffalo cream ( %), and of water buffalo cheese ( %). a total of isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of isolates, ( %) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in out of ( %) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea ( / ; . %), two isolates produced sec ( / ; . %), one isolate produced ( / ; . %) sed, one isolate produced ( / ; . %) see and three isolates produced sec+sed ( / ; %) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet - . . ) and this article was part of a phd thesis. p- . . - identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il- receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp ; was cloned in fusion with an n terminal cleavable his-tag. the recombinant protein, his- was purified using nickel affinity chromatography. was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of was investigated by in vitro pull-down and size-exclusion chromatography. , a amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant his- was produced with a yield of mg/l culture. a structurally stable kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that also form dimers, supporting that it is a btp. current research is focused on solving the structure of and investigating its interaction with myd . might play a direct role in reduced immune response against h.pylori by binding to myd analogous to other btps. further characterization of will provide the first solid evidence of presence of a tir domain protein in h.pylori. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il production by bone marrow-derived macrophages k. korneev , , e. sviriaeva , lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor (tlr ) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il by bone marrow-derived macrophages from c bl/ mice, using tlr -deficient macrophages to control for specificity of tlr signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk / activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p- . . - apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz , l. monturiol-gross , m. j. pineda padilla , c. araya-castillo , a. alape-gir on bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc ( - ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with . lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of . triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor (her ) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for % of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb (mdr- ) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb- was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with , and nm of the drug respectively along with lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr- gene expression with the treatment of dox. especially after nm of dox treatment, mdr- was overexpressed to be fold when compared to control. in conclusion, it was demonstrated that mda-mb- cells have shown to display elevated resistance to higher doses of dox. p- . . - targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa , t. kondo toyama prefectural university, imizu-shi, japan, university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from °c to °c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-( -aminopurin- -yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached - %). nucleosides, such as ribosides, -deoxyribosides, and arabinosides were obtained in high yields ( - %). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el- (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant - - ). p- . . - dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim , n. demir , m. yildiz health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s -). also, cytotoxicity of the compounds against hepg cell lines was assayed by the mtt ( -( , -dimethylthyazolyl- )- , -diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s . also, schiff base complex had cytotoxic activity towards hepg cell line, that it was found to be more potent than the control cisplatin. p- . . - single particle electron tomography of rnap elongation complex, stalled at position + genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering or bp into the nucleosome; ec+ or ec+ , respectively). hear we produced two complexes, both stalled in the + position, one without break in the dna, and the other with introduced ssb at position + of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae °in a jeol microscope at kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+ complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant # - - . colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to who report new incidence rate of crc in turkey is . % among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh À g>a (rs ) promotor substitution, mlh g>c (rs ) exonic substitution, mthfr c t (rs ) and apc t>a (rs ) were investigated in this study. these snps "rs , rs , rs , rs " are located on p . , q , p respectively. colonoscopic investigations were performed on both cancer and control group. the snps were genotyped using kompetitive allele specific pcr technology in cases and healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these of the snps in mlh , mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh À aa genotype in group aged ≥ was found to be . % in cancer versus % in control group. moreover apc a, mlh c alleles were detected only and allele respectively. previously, apc a allele was determined in . % of a turkish cohort. however in the present study apc a allele was detected on allele only. studies showed mlh À promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c t and mlh g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p- . . - excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil and neil , have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either -oxoguanine ( -oxog) or , -dihydrouracil (dhu) by e. coli fpg and nei and human neil and neil from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases ( to ), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil and neil efficiently excised dhu located inside a bubble. fpg and neil was generally more active than neil in excision of -oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil also have shown activity in d-loops with rna. the presence of an additional unpaired -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf ( - - ). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas ( bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+ ), fab-dc/nflu(À )) or unrepairable (nflu/nflu (+ ), nflu/nflu(À ), nant/nflu(+ ), nant/nflu(À )) structures. besides, it has been shown that model dnas bearing bulky lesions in opposite positions (fab-dc/nflu( ), nflu/nflu( )) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(À ), nflu/nflu (+ ), and nant/nflu(+ )), whereas the level of specific excision from nflu/nflu(À ), nflu/nflu(À ) and nant/nflu(À ), nant/nflu(À ) was approximately % of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex ( bp) with fab-dc has decreased structurally dependent affinity for xpc-hr b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+ ), fab-dc/nflu (À ), fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(- )) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized - [ -( morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. - [ -( -morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p- . . - target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil and neil , and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg + . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil and neil showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf ( - - ). p- . . - does causes mhz magnetic field application kras and p mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group. mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for weeks as minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < . ).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for weeks minute/day exposure to mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p mutations in colon tissue. p- . . - synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g motifs) with the potential to form a spatial structure, gquadruplexes is known. g motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed samples of pancreatic ductal adenocarcinoma and samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. - - of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from healthy controls and patients with mm. the mean age for controls and patients were . ae . and . ae . years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a lung adenocarcinoma cells. a lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for a cell line. compounds , and which were including -chlorophenyl, -nitrophenyl on pyridine ring; -fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic values lower than . ae . lg/ml. compound showed the highest cytotoxic activity with a ic value of . ae . lg/ml, whereas cisplatin ic values were . ae . lg/ml lg/ml against a cells. cytotoxic activity of compound and with a ic value were . ae . and . ae . lg/ml, respectively. also, compound showed the highest population of early apoptotic cells ( . %) of the tested compounds which was . -fold higher than for cisplatin. compound produced a comparable population of apoptotic cells with a percentage of . %, respectively according to cisplatin's percentage of . %. it was determined that synthesized compounds , and had considerable anticancer activity against a cell lines compared to cisplatin. compound including -florophenyl on pyrimidine ring was the most cytotoxic compound against the a cell line. our study results demonstrated that compound , also induced apopototic pathway on a cells. p- . . in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl- cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ , , -trimethoxyphenyl = -methoxyphenyl > dillapiol > -methoxyphenyl> , dimethoxyphenyl > , , , -tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic values ranging from . (a cells) to . lm (mda-mb- cells). compounds, containing , , -trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to lm. anti-proliferative effects of isoflavones observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones demonstrated cytotoxicity in human pbmcs, up to lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing , , -trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag- is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl- , hsp /hsc family, growth hormone receptors, raf- , ubiquitin machinery and dna to regulate cell survival. for this reason, bag- is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag- are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag- silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag- enhanced cisplatin or paclitaxel-induced apoptosis in mcf- cells by down-regulating antiapoptotic and upregulating proapoptotic bcl- family proteins, changes on cell cycle, upregulation on subg phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag- has a suppressive role in pi k and akt signaling pathway in mcf- breast cancer cells through inhibition of akt phosphorylation and downregulation on pi k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag- silencing. bag- silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc and rapamycin-sensitive mtorc complexes, respectively. knockdown strategies of bag- is important to enlighten the network interactions of bag- and clarify its interaction partners in the cells. therefore utilization of bag- targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p- . . - biological activity evaluation of new , , trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately % of all female and % of all male cancer deaths in . the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. -[ -( , -bis( -substituted phenyl)- , , -triazin- -yl)piperazin- -yl]- -[benzimidazole/benzoxazole/benzothiazole- -yl)thio]ethanone ( - ) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and -chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt ( -( , -dimethylthiazol- -yl )- , -diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic values between - lg/ml. the early and late apoptotic cell percentage was determined as . for compound by flow cytometric analysis. the lox inhibition activity was found . ae . for compound . compound bearing -chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic / , ic and ic x . p- . . - single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal -drp group in oligonucleotide dna substrates in the presence or absence for me + ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants - - -a and - - -mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u mg) were seeded into the well plates and cultured with dmem supplemented with % fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in / (v/v) dilution ratio was given to first group, lm etoposide was given to second group, / (v/v) diluted rosemary and lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly % , etoposide reduced the viability by nearly % and rosemary with the etoposide reduced the viability by nearly % . the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing -( -aryl- -oxoethyl)- -substituted benzimidazole moiety on c glioma. c glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard ( -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for c cell line. compounds , , , , and , which were including hydrogen, -methyl, -methoxy, -chloro and -floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic values lower than lg/ml. compound showed the highest cytotoxic activity with a ic value of lg/ml, whereas cisplatin ic values were lg/ml against c cells. cytotoxic activity of compound , , , and with a ic value were , , and lg/ml, respectively. compound , and showed the highest population of early apoptotic cells as . , . , and . % respectively compared to cisplatin ( . %). also, compounds caused dna synthesis inhibition depend on their ic values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c cell lines. however, compound , and including -methyl, -chloro and -floro substituents were the most active compounds against the c cell line. also our study results showed that compound , , induced apoptosis in c glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc- and du prostate cancer cells were treated with rutin ( - um) and/or trail ( ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc- and du- prostate cancer cells were treated with rutin for and hours incubation and ic doses for hours incubation were determined um and um respectively. treatment with rutin, pc- cells is more sensitive than du cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc- and du prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc- and du prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p- . . - determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst- ) methods; and antigenotoxic effects against ccl on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration ( and lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations ( , , . lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst- , ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k and aml cell line hl- were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl , bax and caspase ), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p ) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k cell line but not in hl- cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k and hl- cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl and increasing bax and caspase levels. these effects of bor were observed to correlated with increasing the p expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p- . . - analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with - kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome p . , p . , q , q , p . , q . , q . , q . , q . , p , q . , q . , q . , q . , q . , q . , and q . , and also common gains were detected in chromosome p . , q , q . , q . and xq . in colon cancer dna samples, common losses were detected in chromosome p . , q , p . , p . , q . , q . , q . , q . , p . , p . , q . , and q . , and also common gains were detected.in chromosome q . , xp . , xp . , xp . , xp . and xq . both in gastric and colon cancer dna samples, common losses were detected in chromosome q . , q . , and p . , and common gains were detected in xq . discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase (parp ) recruitment which is a sensor for single strand breaks in dna. parp catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for to seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes and . bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt and a benzoxazole derivative xt b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k s) and imatinib-resistant (k r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl- , bad, bim, bcl-xl and mcl by real-time quantitative pcr. results: treatment of k cells with xt increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt b was not found to have similar effects on k r and k s cells. combined application of xt increased cell death in the mtt assay. mtt assay demonstrated that ic for xt treated cells in k r with imatinib (ic = . ) is lower than k r without imatinib (ic = . ). discussion and conclusion: our results showed that combining xt with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh and msh . alteration in expression or a defect in mlh or msh can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k s (sensitive) and k r (resistance) were grown in rpmi- . k r cells were maintained in rpmi- medium supplemented with lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh in k r cells is dramatically lower than equal amount of imatinib treated k s cells, whereas msh expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh genes leads to imatinib resistance. p- . . - characterization of interaction between rad inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france -diisothiocyanostilbene- , -disulfonic acid (dids) has been largely used during the last years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad -mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad recombinase protein. p- . . - mechanism of tap beta-mdm autoregulation p is a transcription factor which is the member of a p family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p , p is rarely mutated in tumors and elevated p expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm is known as the key player in regulation stability and activity of p . in addition, p induces mdm transcriptional activity, and caspase- , activations which cleave mdm n-terminal at asp . cleaved form of mdm binds p and promotes its stabilization. mdm suggested as a candidate to modulate p activity and stability too. however, an interaction between p and mdm has not defined well. in this study, we aimed to analyze the role of mdm in p stability. to define this relationship, firstly, we overexpressed the tap beta isoform using trex system in hep b. tap beta and mdm protein levels were determined by western blot. to examine whether mdm mediate tap beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg for hours prior to analysis. previous studies showed that p -induced caspase- and caspase- activation cleaves mdm . considering this, we firstly examined caspase- activation by western blot in hep b tap beta cells. then we analyzed expression of cleaved mdm and tap beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap beta-induced full-length mdm- expression. furthermore, tap beta enhanced cleavage of mdm via increased caspase- activation. in addition, inhibition of caspase- activation caused a decrease in cleaved-mdm levels in parallel with tap beta expression repression. our results suggested positive regulation between mdm -tap beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p is a transcription factor which is the member of p family. it has two promoters; while p promoter expresses apoptotic ta isoforms, p promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p . it has been shown that both tap and dnp isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap and dnp affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap isoforms via western blot in all hcc cell lines. then we overexpressed the tap beta isoform using trex system in hep b and snu cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; . only tap alpha isoform is expressed in a few hcc cell lines. . there is no correlation between basal expression of p isoforms and drug responses in hcc cell lines. . there is no change in expression of p isoforms after treatment of drugs. . we showed that the ectopic expression of tap beta in hep b arrested the cell cycle in g / s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p- . . - biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease (ape ). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape and archaeal mth revealed conserved amino acid residues in the nir-specific ap endonucleases ape , mth and exoa that are absent in xth. based on these data, we constructed four ape point mutants y h, n q, g s and t d and examined their dna substrate specificities. results obtained from biochemical characterization of ape mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll ( - °) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between and °. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p- . . - ercc /xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group (ercc ) is one of the important dna repair genes.ercc codon and polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated ml blood for biomarker testing. ercc genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v . ). there was significant difference between the frequencies of ercc polymorphism in cancer cases and controls (p < . ). the frequencies of ercc gln allele were . % in controls and . % in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or= . , %=ci . À . ). the above findings indicate that the genetic polymorphism in the ercc codon is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv .x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv . -kcsa-kv . chimeras was developed by us. this approach rested on high-level expression of kcsa-kv .x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv . -channel blockers that combines bl (de ) cells expressing kcsa-kv . and rhodamine-labelled agitoxin (rh-agtx ) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv . was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv . . for system validation, wellknown kv channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx , osk , and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv . channel binding using a number of agtx mutants constructed by us, whose affinities to kcsakv . were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv . channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv . -blockers for scientific and medical purposes. the work was supported by the grant - - from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp- ). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp- by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of : achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp- cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp- cells were was exhibited to possess the highest values of around % at ar concentration of lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis- inhibition of ire signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire (inositol requiring enzyme ), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u glioma cells. it was shown that inhibition of ire leads to up-regulation of the expression of krt , cd , mest, cenpu, myl , ing , ing , mybl , and mybl genes at the mrna level in u glioma cells, with more profound changes for mest, mybl , and cd genes. hypoxia leads to up-regulation of the expression of cd , ing , and ing genes and to down-regulationof krt gene in glioma cells. at the same time, inhibition of ire modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing gene, eliminates hypoxic regulation of krt , cd , and ing genes in glioma cells. the present study demonstrates that inhibition of ire enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase (pol ) of thermus scotoductus k isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol gene of t. scotoductus strain k recently isolated from an armenian geothermal spring. the draft genome sequence of strain k was deposited under accession number ljjr . . genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~ . kb) were ligated into the pet- b(+) vector (novagen) and transformed into chemically competent top escherichia coli. inserts were sequenced with t prom and t term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol gene from k shares % and % similarity with pol of t. scotoductus sa- ( . kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide ( l- fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of l- fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf- breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf- human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst- method. the most effective extract in inhibition of mcf- cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic ( ) values of lg/ml and . lg/ml at h. and h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf- cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf- cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf- cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, . ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, -aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than %. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than %. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for hours daily for one ( ximo), two ( ximo) or ten ( ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a -adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc a and ppargc b, significantly decreased in adrenal cortex of ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp , the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc a, ppargc b, nrf and nrf a). nonselective b-adrsblocker attenuated this effect. the selective a -adrs antagonist did not change adrenaline-induced stimulation of ppargc a, ppargc b, nrf and nrf a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a -adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis- generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for hours daily for one ( ximo), two ( ximo) or ten ( ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (Δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between Δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in ximo group. the mitochondrial mass in leydig cells from ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst- assay was used to evaluate the effects of metformin and tq in dhl (b cell lymphoma) and k (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci< ), additive (ci= ) or antagonistic effects (ci> ). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl and k cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl and k cell lines, ci values were . and . , respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with -hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with -hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after weeks of adipogenic induction in the presence of the chromium complex with -hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with -hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh and gfh . while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g a (pt g a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total participants were included in this study, patients with dvt and healthy blood donors. in order to identify fvl, pt g a, mthfr c t and mthfr a c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in patients fvl was present in ( . %) patients while in controls fvl was present in only ( . %). frequency of fvl was significantly higher in cases as compared to controls (p < . ). pt g a mutation was present in patients ( . %) and in healthy participants ( . %). mthfr c t and mthfr a c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c t/a c was found in patients ( . %) and in healthy controls ( . %), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c t and mthfr a c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c t/a c with fvl may be associated with increased risk of dvt. p-mis- self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t , tx ) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. - - r_center_a ( no. - - r_center_a ( - no. - - r_center_a ( ) and - - mol_a_ved ( no. - - r_center_a ( - . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk / or btk- inhibitors. the phosphorylation of erk / and btk- was determined by western blot analysis. proteomic analysis was performed by ultimate rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef ( v/cm). comparative analysis of bc and lc cells proteomes revealed about differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk- is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk- phosphorylation in lc than bc cells. in contrast erk / activity was not necessary for electrotaxis of lc cells and ef did not induce erk / phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre / /b/nz / , poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce , n. y. saral , f. b. aksungar , , a. coskun , , m. serteser , , i. unsal , acibadem labmed, istanbul, turkey, acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between and were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be lu/ml for cut off point and lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of lu/ml in ( . %), between and lu/ml in ( . %) and above the level of lu/ml in ( . %) babies, respectively. recall rate was . %. ch cases of neonatal tsh levels greater than lu/ml were . the incidence of ch of this group was : . there were no significant differences in the number of congenital hypothyroidism between males and females (p > . ). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou , m. kazou , v. alexandraki , b. pot , e. tsakalidou agricultural university of athens, athens, greece, institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of , , bp with a g+c content of . %. the genome contains , protein-coding genes on the chromosome covering up to . % of the genome sequence, trna and rrna. according to the subsystem-based annotation, , protein-coding genes were assigned to metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = , . % of total protein-coding genes) and protein metabolism (n = , . % of total protein-coding genes). furthermore, three prophage regions were detected; one intact ( . kb), one incomplete ( . kb) and one questionable ( . kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type diabetes mellitus (t dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after month. also, a healthy control group (n = ) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = . ) between the irisin levels of the patients at the beginning and after month treatment. a similar result was found between the control and the treated groups (p = . ), while a significant difference (p = . ) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in % of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. - - of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant - - ). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn in ammonia-exposed mouse cortical astrocytes (bisi; lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn and sn , second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r a and orexin ox heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin a and b (ghs-r a , ghs-r b ) for ghrelin, and orexin and (ox , ox ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r a and ox receptors to form heterocomplexes. formation of ghs-r a -ox receptor heteromers in transfected hek t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r a and ox receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r a -ox receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = ). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than . nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than . . our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva , , b. bekmanov , , a. amirgalieva , k. dosybaev , , z. orazymbetova , , r. zhapbasov , a. zhomartov , n. zumadillaev , n. zumadillaev llp "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than . ) there were found rarely met fragments (frequency lower than . ). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) breeds ( populations) were characterized. informative value and resolving capacity of the sum of str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. informative markers were chosen for both two breeds, markers being used for both breeds, while other markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele of bm was met in birlik population of edilbay breed as often as in % of animals while in two other populations there were no this allele. in kumtekey population one can meet % animals having particular locus (dyms ), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of preeclamptic mothers and mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk ( vs u/ml, p = . ). while tos levels were significantly higher in the preeclampsia group ( . vs . lmol/l, p = . ), the tas levels were significantly higher in the control breast milks ( . vs . mmol/l, p = . ). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis- investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included subjects ( female and male with age range of - years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at nd, th and th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = . , p = . ), tg (r = . , p = . ), tc (r = . , p = . ), ldl-c (r = . , p = . ) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis- eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than , compounds, incl. confirmatory and counter screening, ic/ec determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in , but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate -hydroxy-nicotine ( hnic) produced by a. nicotinovorans pao was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate hnic by using a genetically engineered a. nicotinovorans strain. the growth and the hnic accumulation were compared for two strains: . a. nicotinovorans pao wild type strain and . a genetically engineered a. nicotinovorans pao strain (part ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of hnic were monitored by hplc using a mn nucleodur - c ec column and . m sulfuric acid at a flow rate of ml/minute. the growth curve of the part ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of hnic are observed. although the sds-page analysis of the total protein extracts from the part ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a fold accumulation of hnic in the growth medium. the first attempts to purify ndh from the part ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part ndh strain for hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeÀinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of . - lm, and ache in the range . - lm. five cinchonidines displayed a - times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda ( lm) for h increased the expression of kir . mrna and decreased that aqp -and gs mrna. the expression of kir . was decreased by h exposure to glu ( mm) and tnfa ( ng/ml). at h incubation, nmda induced a decrease of kir . expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr subunit, and decreased that of nr b mrna. glu decreased the expression of out of subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir . mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to pediatric nurses and adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x . the average age of participants was ae years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was % and after the training it was %, which showed an increase of %. the preanalytic error rates which were . % in february were decreased to . % in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was nm and had a sufficient stability over a period of months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g ) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g dna is considerably more susceptible to lightinduced modification than duplex dna; -terminal tetrads of the g dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. - - from the russian science foundation. resistin levels in denervated obese rats n. saglam , t. ahmedi rendi , c. kahraman , a. alver department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study experimental groups were formed each consisted of rats. during weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < . ). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est gene is regulated at the translation level by + programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf p (ampk) and gcn p (eif kinase) on the prf rate in est gene. prf rate of est gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf , or gcn genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn p, and snf p, respectively. prf rate of est gene was measured as % in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than -fold, giving less than % prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by -fold in the wild type cells and prf rate increased to %. when the prf rate was analyzed in gcn and snf mutants, frame shift rate of est was - % in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn p and gcn p, positive regulator of gcn p, is also essential for the regulation of prf in est in response to stress conditions. it is clear that the basal level expression of est is highly dependent on the gcn p kinase complex. gcn p is also associates with ribosomes, indicating that gcn p might have a significant function in connecting the stress signals to biosynthesis of the full length est peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf p and gcn p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = ; nonerosive-nerd, n = ; healthy controls-hc, n = ) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee ( . ae . ng/mg.prt) versus hc ( . ae . ng/mg.prt, p = . ). mpo tissue activities in ee group were significantly lower ( . ae . u/mg.prt) than hc ( . ae . u/mg.prt, p = . ) while mpo serum levels were higher in ee ( . ae . ul) versus hc ( . ae . ul, p = . ). tissue chit levels were three fold increased in ee versus hc (p = . ). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=À . , p = . ) in tissue. nfkb and ecad levels had a positive correlation in serum (r = . , p < . ). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from , patients (mean age ae sd, . ae . ) were used for the correlation (excel analyze-it v . . ) and linear regression (pasw statistics v . ) analysis between the measured parameters. there was a significant (p < . ) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp* . ) + ( . *age) + ( . *wbc) + ( . *neut%)À . . correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of . . our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c -dox ratio ( : and : ) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp ad molt- ) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h o -induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to , tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < . ) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < . ). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil ( -fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of -fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of -fu synthesized via "click reaction", namely f -[{ -( ″, ″, ″, ″-tetra-o-acetyl-b-dglycopyronosyl)- h- , , -triazole- -yl}methyl] -fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in ml of solvent for h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta , ta (for the detection of frameshift mutations) ta and ta (for detection of base pair substitutions) according to the oecd guideline . the drug was solubilized ( lg/ml) with no precipitation in lutrol Ò -f /ethanol/water ( . : . : . , wt/wt) micelles ( . ae . nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the healthy goats were divided into groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t? (r ), at t = . s (r ), t = s (r ) and t?∞ (r ). this work was supported by the russian scientific foundation ( - - ). all animals had - % fat content. the contents of total protein ( . %), albumin ( . %) and urea ( . %) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol ( . %) and creatinine ( . %) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r (r = À . ), r (r = À . ), r (r = À . ); total protein or albumin levels with r (r = À . ), r (r = À . ), r (r = À . ); aminotransferase activity with r (r = À . ), r (r = À . ). the correlations were found between the total protein and albumin levels with k (r = . ), k (r = . ); glucose levels and r (r = . ), r (r = . ). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of > were classified as the high gs group (hgg), and those with a gs less than were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of . lmol h o equiv./ l for serum tos levels predicted high gs with a sensitivity of % and a specificity of %. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was . (sensitivity % and specificity %). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci , c. velioglu , e. recber , c. unsal , g. gulsoy , b. sariyar akbulut marmara university, istanbul, turkey, istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis . the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis cells were subjected to lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for hours but then cells resumed their regular growth. upon addition of lg/ml (-)-roemerine to the bacillus subtilis cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono- , -lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf- breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf- breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase (hplscr ) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr . this study aims to identify the region of hplscr that interacts with topo iia. to identify the topo iia interacting sites in hplscr , nterminal deletion constructs of hplscr viz Δ -hplscr , Δ -hplscr , Δ -hplscr , Δ -hplscr and Δ -hplscr were generated by pcr, cloned, overexpressed and purified to homogeneity using ni + -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex p- and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr and the gst-ctd-topo iia. the binding region in hplscr will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr . Δ -hplscr did not show any enhancement of the decatenation activity compared to full length hplscr . hence, the binding region could be in the - region of hplscr . further deletions were done in the - region of hplscr as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr that binds to topo iia. we conclude that hplscr interacts with and enhances the activity of topo iia and the - region of hplscr is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr and the physiological relevance of this interaction in the cell. a. ugur kurtoglu , v. aslan , e. kurtoglu department of biochemistry, antalya education and research hospital, antalya, turkey, department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs . is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs . , ivs . , ivs . and ivs . . our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis- investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen , h. kara karadeniz technical university medical biochemistry department, trabzon, turkey, balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba ),non-lysosomal b-glucosidase (gba ), cytosolic b-glucosidase (gba ),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose b-ltyrosine- -naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba .inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba was purified at . -fold with . % yield.gba was illustrated and kda at sds-page.ic value of ampicillin/sulbactam antibiotic for gba was found . mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba was illustrated and kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba .ampicillin/sulbactam antibiotic inhibited to purfied gba at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu , p. b. sermikli , a. abbasi taghidizaj , e. yilmaz ankara university, institute of science, ankara, turkey, ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, t -l cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type- diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft , ft , vitamin b and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b , folate. the cases were - years old, male individuals who admitted to our hospital between april -march for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: ) were divided into four subgroups; group (g) : - . lg/dl, g : - . , g : - . , g : ≥ . from these, the number of cases whose th levels were measured (n: ) given respectively; g : , g : , g : , g : cases. also the number of cases whose vit b and folate levels were measured (n: ) given respectively; g : , g : , g : ,g : cases. levels of pb were determined by icp-ms. th, vit b , folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t , tsh and vitamin b (p > . ). on the other hand there was statistically significant difference between t and group , , (p < . ) but there was no difference between group (p > . ). the average folate belongs to the first group was about % higher than the other groups, and found that the difference was statistically significant (p < . ). there are many publications which have various results between pb levels and t ,t , tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b , folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b . but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged ( ). since that time this method became associated with a number of major breakthroughs in the field ( ) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors ( ) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf - - . ( ) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. , , À . ( ) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. , , - . ( ) katritch v., cherezov v., stevens r.c. ( ) . annu rev pharmacol toxicol , - . p-mis- stamp is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp knockdown. interestingly, in the crpc cell line rv , where-stamp knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of d thin layer chromatograms using d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional ( d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional ( d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for d plates, and cannot be adapted to quantitative evaluations of d plates. in this study, the applicability of the gel documentation systems and d analysis software for the analysis of d tlc plates were examined. material and method: d tlc of lipids: st dimension: methyl acetate/n-propanol/chloroform/methanol/ . % kcl ( / / / / v/v); nd dimension: chloroform/methanol/acetic acid/ water ( / / / v/v); detection: charring. d tlc of aminoacids: st dimension: . % (v/v) formic acid; nd dimension: toluene/glacial acetic acid ( : v/v); detection: uv. phospholipid and aminoacid standards, each include different classes were developed by d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest d gel analysis software. for the method validationa) plates containing same lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample b) plates containing same lipid classes were developed in different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates results: volume of each phospholipid and aminoacid had less than % intra and inter-assay cv. conclusion: gel documentation system with d gel analysis software can be used for the quantitative analysis of the d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, adult male wistar albino rats were used. rats were separated into four groups as control rats (g ), mm nac (g ), mm nac (g ) and mm nac (g ). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding mm kcl to the organ bath. then, in the first minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at . . results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < . ). similarly, nak atpase activity is also decreased in a dose dependent manner (p < . ). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk , which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of . - . . the optimum temperature for keratinase activity was investigated by varying the incubation temperature between °c and °c. optimum keratinolytic activity was observed at °c and ph . . the enzyme was stable at °c. the activity was investigated in the presence of some chemicals, including sds, tween , dmso, triton x- , edta, nacl, zncl , cacl , glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel ( %) using standard molecular weight marker and estimated about kda by sds-page. the keratinase isolated from bacillus uk could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of bthalassemia and a-(including two of . kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler Ò instrument. the a/b-globin mrna ratio of each sample was calculated based on the Àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis- self-assembling peptides mimic supramolecular biochirality r. garifullin , , m. o. guler bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis- pon activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon- activity in both total and hdl subgroups, and evaluation of performance characteristics of pon- activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed obese, overweight and normal subjects. fasting morning blood samples were taken from all study groups. hdl subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl -c concentrations were calculated with the subtraction of hdl -c from total hdl-c. hdl -c and total pon- activity were determined by using eckerson method. non-hdl pon- activitiy was calculated with subtraction of hdl pon- activity from total pon- activity. results: total hdl-c, hdl -c and hdl -c concentrations and the activity of total pon- and hdl pon- were found lower in obesity according to overweight and normal subjects (p < . ). negative correlations were found between body mass index and hdl -c, total pon- and hdl pon- (r = À . , p < . ; r = À . , p < . ; r = À . , p < . , respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl -c measurements, pon activity, measurement of total and hdl -c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis- hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > . was accepted as significant value. results: in a study of blood donors ( male, female), the overall prevelance of anti-hev igg antibodies were . %. the blood samples were collected different areas. the prevelance of anti-hev igm antibodies was . % and he was years and acting a butcher during years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were %, the animal husbandry were % and the veterians and the butcher were % were found. discussion: the prevelance of anti-hev in the north cyprus ( %) was found low such as the prevelance of the turkey ( %). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january and january . clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that blood samples were sent to our laboratory in one-year period. . % of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause ( %). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be %, %, %, %, % and % respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of patients ( female, male) aging . ae . years ( - ) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of g sugar ( hour), -hour and -hour. these parameters were statistically analyzed in comparison to simultaneous hba c results. glucose measurements at hour had better distinction power (p < . ) between these individual groups than initial and -hour glucose measurements. the average hba c (%) levels for healthy, pre-diabetic and diabetic individuals were . ae . , . ae . and . ae . , respectively. roc curve analysis showed . % sensitivity and . % specificity for the clinically accepted hba c cut-off value of . %. hba c cut-off value of . % had a higher sensitivity of . % and comparable specificity of . %. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at -hour after sugar administration in ogtt test as opposed -hours generally used for diagnosis. low sensitivity was observed for the clinically adapted . % cut-off value of hba c. the cut-off value of . % for hba c was found to be more sensitive with comparable specificity than the . % cut-off values for diabetes screening in our population. our results suggest that . % for hba c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin ( mg/kg, po) every hours for a total of doses prior to an apap challenge ( mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp e , cyp a , or cyp a in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf -dependent pathway. we found that stamp fused to cyan fluorescent protein (cfp-stamp ) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp with various fluorescent protein markers revealed that cfp-stamp puncta corresponded to lipid rafts that were labelled with caveolin- -rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp and caveolin- -rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp internalization. notably, stamp was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c ), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with -hydroxy- -nitrobenzyl bromide (hnbb) that covalently modifies the trp residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity ( %) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states -phosphofructo- -kinase/fructose- , -bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose- -phosphate (f p) to fructose- , -bisphosphate (f , bp) and vice versa, and f , bp is an allosteric activator of phosphofructokinase- (pfk ), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb - ), pfkfb is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb , variant- and variant- , coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g d)induced oncogenic transformation on pfkfb expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb mrna and protein expressions (mrna; ipde: ae . ; ipde+kras: . ae . and protein; ipde: ipde+kras: . ). we then, checked individual expressions of splice variants and observed that while pfkfb splice variant- (p -v ) expression was reduced by k-ras induction (ipde: ; ipde+kras: . ), pfkfb splice variant- (p -v ) expression was increased (ipde: ; ipde+kras: . ). then, we checked effects of p -v and p -v on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb variants increased f , bp concentration (p -v : . ; p -v : . fold; compared to empty vec), glucose uptake (p -v : %; p -v : %) and glycolysis (p -v : %; p -v : %) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb isoforms and observed that both pfkfb isozymes localize to the nucleus, with more prominent nuclear localization of p -v compared to p -v . also, nuclear localization ratio of p -v increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p map kinase inhibitors on mda-mb- cell line introduction: p mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p mapk sb and sb on mda-mb- cells to further elucidate the controversial role of p mapk on cell proliferation or cell migration. materials and methods: mda-mb- cancer line was cultured in rpmi- supplemented with % fbs. the cytotoxic and cell migration effects of sb and sb inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb- cells were determined by icelligence system. results: it was found that sb p map kinase inhibitor was more effective than sb . however, no significant effects of low doses of lm and lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to hours as determined by icelligence system. on the other hand, both sb and sb significantly prevented cell proliferation at a concentration of lm. both sb and sb significantly reduced cell migration in a time-dependent manner at a concentration of lm. then, we tested whether each p mapk inhibitors have any effect on cell adhesion during a treatment period of hours using icelligence system. only lm concentration of sb reduced cell adhesion for about . hour (p < . ). conclusion: p mapk inhibitors sb and sb differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no - . mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto , f. zilifdar , s. yilmaz , t. sarac ßbasi , i. yalc ßin , n. diril hacettepe university, ankara, turkey, ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of , -benzoxazine- -one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta and ta strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s ) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a d-common features pharmacophore model with hiphop method. according to the results, only bs , bs , bs and bs exhibited strong mutagenic effects on both strains in the presence and absence of s . additionally bs , bs , bs and bs (in the absence of the s ), bs , bs and bs (in the presence of the s ) showed weak mutagenic effects on ta . hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r and hydrophobic groups at the position of r of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since .the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = ) were evaluated in dried blood samples which sent to our laboratory for the study between august and august . electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms ,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in ( . %) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in ,organic acidemia in ,fatty acid oxidation defects in patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain- are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr /nr b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr /nr b subunits, calpain- , hsp and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain- are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp that reduces the affinity for ca + of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain- . our data suggest a physiological role of calpain- at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca + influx, the activated protease regulated by hsp , promotes the removal of nmdar from the plasma membranes, decreasing ca + entrance through this receptor-channel and protecting cells from ca + overloading. tissue transglutaminase (tg ) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg mutant constructs. renca cells were transduced by lentiviral particles encoding wttg , transaminase-defective tg -c s form with low gtpbinding affinity, gtp-binding deficient form tg -r a, and transaminase-inactive tg -w a. in order to investigate the role of tg transamidating and gtpase activity in cell migration, scattering assay was used where colonies for each mutant clone was followed for a time interval of hours. our results showed that non-transduced control and tg -c s mutant renca cells demonstrated a similar migration pattern with a % of scatter activity. on the other hand, % colonies formed by renca cells overexpressing wttg and tg -w a mutant scattered away from each other. a small insignificant increase in scattering was seen in % of the total number of colonies for renca cells overexpressing tg-r a construct. data from this study supports that gtp-binding activity of tg is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = ), subclinical hypothyroid (n = ), subclinical hyperthyroid (n = ). serum tsh, free t (ft ) and free t (ft ) levels were determined by chemiluminescence immunoassay and serum -hydroxy (oh) vitamin d (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, . to . miu/l), ft (range, . to . ng/dl) and ft (range, . to . ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t and t levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t and free t levels. results: subclinical hyperthyroid group had significantly higher (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < . ). (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co , no x and so x (ghorbani et al., ) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., ) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr , ts and ts were used as ethanologenic microorganisms. expression of vhb in ts is lower than in ts strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts strain by up to . x cfu per ml of fructose ( %, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s ( ) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology : - . ghorbani, f., younesi, h., sari, a. e., najafpour, g. ( ) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. ) . in this work, e. coli strains fbr , ts (low level vhb expressing) and ts (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for hours at °c and rpm in a ml erlenmayer flask. the ethanol production was improved over % by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as . , . and . for fbr , ts and ts strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. ( ) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., ; geckil et al., ] . in this study, ethanologic e. coli strains fbr , its derivative strains ts (vgb+) and ts (vgb+) were used. ts was constructed in such that it could express more vhb than ts . bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations ( . - mm) were used as inhibitors from lignocellulose hydrolysate. . mm acetic acid was used as an inhibitor. the growth of vhb expressing ts and ts strains was inhibited about % after hours fermentation time. strain fbr was inhibited as high as % by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd , an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd on adipogenesis in t -l preadipocytes. notably, azd treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of t -l preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription- (stat- ) during adipocyte differentiation. furthermore, azd largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd inhibits adipogenesis in t -l preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat- . effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = ) and food colouring (fcg, n = ) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than . were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment ( . ae . ) and control ( . ae . ) groups showed no statistical difference (p = . ). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr c ) polymorphisms and unipolar or bipolar depression. the nr c gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr c (bcl and rs ) in recurrent depressive disorder (rdd) patients. methods: our study included volunteers with recurrent depressive disorder and healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr c gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs ) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl- (g/c) were as follows: cc % and %, gc % and %, gg % and %, respectively. there was not a significant difference when the frequency of the allele (p = . ) and genotype frequency (p = . ) were compared between the patients and the control. genotype distribution in the rs region (a/t) of the patients and controls were tt % and %, ta % and %, aa % and %, respectively. allele frequency (p = . ) and the genotype frequencies (p = . ) were not significantly different among the groups. conclusion: numerous nr c gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr c polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy y human neuroblastoma cell line. sh-sy y cell line treated with - lm tq by solving medium for , and h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm , p , p , akt, pten, cdk , cyclin d , caspase- , - , - , - , bcl- , bax, parp, bcl-xl, bid, dr , dr , puma, noxa, mmp- , - , timp- , - and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt profiles array data analysis by using student's t test. ic value of tq in sh-sy y cells was detected as lm at th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm , akt, cdk , cyclin d , bcl- and mmp- . it is also observed that tq caused a significant increase in the expression of p , pten, caspase- , - , bid, dr , puma, noxa and timp- . it was also found that tq in sh-sy y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. - - . the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm wild type (wt) and its isogenic Δmacab mutant strains. we also used h o survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab- xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h o . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after hours of growth in lb-broth there is at least -fold increase in expression in response to the presence of h o . on the protein level macab can be detected starting from hours of growth in lb-broth and it reaches maximum expression on hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h o properties. antiproliferative and apoptotic effects of noscapine on mcf- and mda-mb- human breast cancer cell lines approximately - % of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor . these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf- and mda-mb- human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf- and mda-mb- cell lines were analyzed by roche xcelligence system. the cells were cultured in % fetal bovine serum containing dulbecco's modified eagle medium at °c in a humidified atmosphere containing % co . h after seeding, the cells were treated with different doses of docetaxel ( . to nm), tamoxifen ( . to lm), and noscapine ( . to lm). cultured cells were harvested, fixed with % formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at lm thickness and stained with h&e, ki- , bcl- , cyclin-d , and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf- cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf- cells, hovewer docetaxel and noscapine were more effective on mda-mb- cells. tamoxifen has more apoptotic and antiproliferative effects on mcf- cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb- cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a -ml centrifuge tube by a neodymium magnet through a -cm cushion of % sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was -fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. - - . epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl sandwich elisa and rt-pcr. the mechanism of action of zeb / on ccl promoter has been studied by luciferace assay and chip. ccl coding region was cloned into pcdna . and stably transfected into dld- cells. ccl deficient ct cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl and sip expression was studied using ihc or flow cytometry. emt status catagorised crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl in induced dld-sip cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl in the induced dld-sip cells as well as mesenchymal crc cells as compared to epithelial ones (p = . ). promoter studies showed that zeb / bind to ccl promoter and and activate ccl gene expression. no metastasis was observed for dld- cells overexpressing ccl but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl is up-regulated by emt inducing transcription factor sip , and mesenchymal (metastatic) crc cells secrete significantly more ccl compared to epithelial (non-metastatic) ones. ccl did not induce emt per se but abundant secretion of ccl by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for - minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of . to . lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the lg/plate concentration s. typhimurium ta . at all the doses antimutagenic response was significant at (p < . ) against both the strains with a percent mutagenicity decrease from to for ta followed by ta with percent antimutagenicity from to . the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [( , , , )-tetrathia[ . ] ( , )- , , -thiadiazolophane; ( , )dioxo- , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane; ( , , , )-tetraoxo- ( , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane and ( , , , , , )-hexaoxo- ( , , , )-tetrathia [ . ] ( , )- , , -thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for hours. at the end of the each hour, cell vitality was assessed by measuring the absorbance ( nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of : of . mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using , -diphenyl- -picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than ng/ml or greater than ng/ml were accepted as inappropriate initial testing. fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of patients who referred to the hospital phlebotomy unit were collected using holder into the different tubes. first tube was ml glass tube and with no additive, second was ml tube with gel separator, third was ml tube with gel separator. serum was separated and immediadiately analysed for biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft , ft ) which are the most common requested laboratory tests. thyroid function tests of patients are analyzed in ankara numune education and research hospital in october -may . five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below % of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for -month study period was . %. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis- development of polyclonal and monoclonal antibodies against fatty acid binding protein (fabp /ap ) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp ) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp protein made by recombinant dna technology. recombinant his-fabp was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp , as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb- , mcf- and cd + /cd -cells and ic values of thz were found to be lm, . lm and lm respectively, at hours. invasion potency of mcf- , cd + /cd -and mda-mb- cells were determined as %, %, . % when compared to relevant treatment controls. migration potency of mcf- , cd + /cd -and mda-mb- cells was determined as . %, . %, % respectively. among the three cell lines mda-mb- cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin- , b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb- , mcf- and cd + /cd cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. healthy - year children's ( girls, boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = . , p = . ) and a negative correlation with humidity (r= À . , p = . ). mean urine density in spring was higher than that of autumn (p = . ) and winter (p = . ). mean value of summer was higher than autumn (p = . ) and winter (p = . ). - months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis- implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of pharmacies located in north cyprus were involved in the study with %stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two ( . %),responded positively to this demand. it has also been identified in the study that . % of the daily sale of antibiotics in the first half of the year was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with . % and upper respiratory tract with . %. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be ( . %),compared with the studies conducted in greece,italy,malta and spain % and egypt . %that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic ) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a cells including with ultrastructural and morphological effects. ic value of aa on a cells for h was detected with mtt ( -( , -dimethyl- -thiazolyl)- , -diphenyl- h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a cells in dose-dependent manner and ic on a cells was found to be . mm for hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on immunoassays studied by the beckman coulter unicell dxi immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of tests were measured by the beckman coulter unicell dxi . d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin ( . , . , . , . , . and . g/l hemoglobin) and intralipid ( . , . , . , and g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid ( %, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au . after spiking the pools, the tests were measured again in duplicate on beckman-coulter dxi analyzer. a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b , testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca . , ca , ca . , insulin, pth and e , and of lipemia for progesterone, ca . , vitamin b and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb andiso plus tak- . treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase- (pon- ) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p mapk and nuclear factor erythroid -related factor (nrf ) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p mapk, phosphorylated c-jun n-terminal protein kinase (pjnk / ) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk / ) and nrf expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb treatment. p mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p mitogen-activated protein kinases, sb , tak- . silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were males in each groups which contained patients with silicosis (group ), individuals having similar symptoms with silicosis from same occupational area (group ) and healthy subjects (group ). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev , fev %, fev /fvc, fev / fvc%, fef - and fef - %) were compared. the average age of group , and are . ae . , . ae and ae . years, respectively. there was a significant difference between group and in terms of the ada level (p < . ). there was a negative correlation between ada activity and fev , fev %, fev /fvc, fev /fvc%, fef - values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. - - . the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar , m. tekin , s. civelek firat university, elazig, turkey, cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by genus on the world and by genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega . program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, ll of internal standard (d - deoxycortizol) in methanol was added to ll standart or serum and centrifuged at . rpm for minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at • c and dissolved in mobile phase. ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. k ) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value , , intercept value À . and r² value . . the regressione quation was elisa= À . + . lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste polycomb repressive complex expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u , h. s€ urer , g. bilgin , d. y€ ucel ankara training and research hospital, medical biochemistry department, ankara, turkey, ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. patients with copd, patients with asthma and healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha- antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < . ). there was no significant difference for ferritine and alpha- antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova , , v. timofeev , , n. zhukhlistova , y. abramchik , t. muravieva , r. esipov shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, national research centre "kurchatov institute", moscow, russia, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at k from single crystal grown in space (sp. group p ) was collected on the spring- synchrotron-radiation facility (japan) and the structure was solved at . a resolution, using the molecular replacement method (pdb id i c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id da ) and human pnp (pdb id pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h ) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h . ara h aptamer was used to recognize any residual peanut major allergen protein ara h in food samples. recombinant ara h protein was produced and puirifed to be used as a target. ara h aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between and . kg/m . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of women. students with high bmi (> ) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (< ) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (< ) student group. waist circumference, percentage of body fat was higher in high bmi (> ) group than low bmi (< ) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (> ) and bmi (< )). students were grouped depend on their waist circumference. healty individuals who had lower than cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum ( , , aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions ( % ethanol and polyethylene glycol -peg %) and different method (ultrasonic treatment w/ o c/ minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at o c/ hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %), followed by liquid samples treated by ultrasonic method prior to transformation ( . %). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: patients, having delivery beyond weeks, and who did not have any other medical problems were included in the study. the patients had normal g. oral glucose tolerance test results. they were divided as groups, based on their newborns' birthweight as " g. and g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low ( . ng/l, . ng/ml and . ng/ml respectively), normal ( . ng/l, ng/ml, . ng/l respectively) and high ( ng/l, ng/ml, g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin ( . , . , . , , . and . g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access analyzer. the hemolysis indices were measured on beckman coulter au . a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations ( . ng/ml), and a negative interference for myoglobin at low concentrations ( . ng/ml) and high concentrations ( ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥ . g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a- , glycosidase enzyme with an incidence of : , - : , . as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a- , -glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a- , glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of patients's examined, of them were diagnosed with pompe disease depending on clinical findings & low a- , glycosidase activity. enzyme activities of pompe patients were . nmol/ml/hour as lsd suspected patients'activities had a mean of . nmol/ml/hour (p = . ).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher ( vs - u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group ( vs u/l, p = . ). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = . r=À . ) was observed between a- , -glycosidase and ck activities (n: ). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of ( h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of ( h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f ) on biofilms formed by s. aureus atcc and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic ( mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of mg/l of f the mic of kanamycin was decreased -fold, and the mics of both erythromycin and ampicillin were lowered -fold. at the concentration of mg/l f also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto and propidium iodide. the addition of f to preformed h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f at concentration of mg/l led to death of up to % of biofilm-embedded cells. this fact suggests that f efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project № - - and the german academic exchange service (№ ). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged and over is - times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin- secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed young and elderly patients with pneumonia and their respective aged matched controls (n = , n = ) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml and . ae . mg/l, . ae . ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < . and p < . ). elderly patients with pneumonia have significantly higher crp levels than their controls (p < . ). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin ( . g/dl) and normal calcium ( . mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of ml were aliquoted. purified albumin, . g, was added to one of these pools and albumin concentration was determined as . g/dl. the low and high albumin pools were mixed at different ratios and pools with . , . , and . g/dl albumin concentrations. total calcium and albumin concentrations of these pools were measured at a beckman-coulter au analyzer and ionized calcium was measured at a radiometer abl blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of . g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: . %, . %, . %, and . %, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: . %, . %, . %, and . %, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at > g/dl albumin concentrations. ionized calcium is significantly affected by . g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba c and fasting glucose concentration was investigated. lp(a), hba c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july to july . there were patients with these test results at the same time. the patients were grouped according to hba c values: group i < . % (n = ), group ii . - . % (n = ), and group iii > . % (n = ). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba c (r = . ; p < . ), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < . . there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < . ). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep -p is written in kit inserts. target values ( %) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from to mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate . % from the target when the degrees of hb are mg/dl. when the degree of hemoglobin is mg/dl, the test strays about . %. potassium levels increase ( %) at mg/dl hb while this increase reaches to . % at mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases %, at the mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. d-structure of the hu protein from mycoplasma gallisepticum have been determined with a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund ( - - ). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox . sox is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with -ethyl- -( -dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range ( . - pg/ml) with a detection limit as low as . ng/ml sox . furthermore, the developed sox immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox antigen and anti-sox antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: z ). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: . women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh ( - ) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= . -( . age)À( . fsh) + ( . lh)À( . estradiol) is detected and the model's r = . is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld- ) and endometrium (ecc- ) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic ) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at and lg/ml concentrations by hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld- and ecc- . however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca - against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca - is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+ %psa. tissues were seeded on mm petri dishes containing dmem/high glucose supplemented with % (v/v) fcs and incubated %co in air at % relative humidity and at °c. after seeding, the medium was unchanged for days and then it was changed in every days for days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over %) in culture when compared with the negative control ( %). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g /g phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g /g phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g /g phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, g ). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe o ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used - -trimethyl- -phenyl , , hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between and °c simple nanoparticles decreased the membrane fluidity. at physiological temperatures ( - °c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at °c, and at °c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. -isoprostane is considered as an indicator of oxidative stress. mao activity and -isoprostane levels were measured in some diseases. however, there are no information on -isoprostane levels and mao activity in newly diagnosed patients with stage hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and -isoprostane in newly diagnosed patients with stage hypertension. the study included newly diagnosed stage hypertensive patients with no other systemic disease. patients were selected as randomized ( women, men; range of age - years) and healthy individuals as control ( women, men; range of age - years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and -isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > . ). compared to the control group, the activity of mao and -isoprostane levels were found significantly higher in patients (p < . ). conclusion: increased -isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, -isoprostane p-mis- determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c -a guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from , males, , females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma and sigma subunits did not affect rna elongation. we showed that the sigma -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp /dis , dis l and dis l proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the s subunit at °c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the s ribosomal subunit, concomitantly with the increase of s and s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of -thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of -thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip- / modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov- g and tov- d. for this purpose, tov- g and tov- d cells were treated with limnio grape skin extracts at a range of concentrations, at °c, for , and hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov- g and tov- d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl , bclx, bax, bak and bcl l , along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. % of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse , gse and gse . we analyzed two datasets of er(+) breast cancer patient samples (n = , n = ) compared to normal breast tissue (n = ) using apadetect and sam. a total of utr shortening and utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb- cells that are otherwise of triple negative nature. our results suggested that most of the genes are utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between - bp also detecting the impurities in dna solution in %, % and % ratios to the dna of interest. the developed method was validated using the in-house dna fragments of , and bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, . lm, . mm id . cm and tskgel dna-npr, . lm, . mm id . cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for bp in the range of . ng to ng dna with the regression coefficient of r = . . lod and loq for the bp dna was found to be . ng and . ng, respectively. the recovery values for the %, % and % impurity ratios were found . . . and . , respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = . ). the length distribution of small rnas revealed nt-peak corresponding to trna-derived fragments, amount of which was increased by . -fold under dtt treatment. the trna isotypes that gave rise to almost % and % of all nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of nt fragments produced from these trnas are precisely phased halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor (rnh ). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun , , , j. kim , , j. y. hong dankook university, cheonan, south korea, institute of tissue regeneration engineering (itren), cheonan, south korea, the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; hour, day, week, month and months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage ( hour to week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at hour or day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p- . . - utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco- cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco- cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day ) caco- cells and tested sample apa events by rt-qpcr. utr isoforms were identified by using race pcr. we identified genes ( % of all apa events) to undergo utr shortening in differentiated cells compared to proliferating cells. on the contrary genes ( % of all apa events) went through utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine- of rna polymerase ii c-terminal domain by end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y -s -p -t -s -p -s at the c-terminal domain (ctd) of its largest subunit, rpb . phosphorylation of serines (s , s , and s ) and tyrosine- orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine- site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine- phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine- ctd phosphomarks using integrative structural biology. one of them, a factor involved in the -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine- that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t g human gbm cell line were used to evaluate the effects of ind ( lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p < . ), cell viability (p < . ), cell rate at s phase (p < . ) and g + m phase (p < . ), camp levels (p < . ), the levels of pdgfr-a (p < . ), mrp- (p < . ), nf-jb (p < . ) and cox- (p < . ) in comparison to control group. ind mildly increased apoptosis (p < . ) and caspase- levels (p < . ). interestingly, ind increased htert levels ( %, p < . ; %, p < . ). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox- and other tested factors. p- . . - interaction between fibrinogen and insulin-like growth factor binding protein- under physiologic conditions and influence of diabetes mellitus type on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp- , is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp- . besides glucose regulation, igfbp- stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp- , their change in dm type (dm ) patients and involvement in fibrin clot. samples from adult healthy persons and dm patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp- /fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp- . in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp- /fibrinogen complexes can be differentiated from igfbp- oligomers and igfbp- /alpha- macroglobulin complexes. the amount of igfbp- /fibrinogen complexes was lower in patients with dm . complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp- monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm was additionally able to bind exogenous igfbp- . since igfbp- stimulates wound healing, directly and by delivering igfs, igfbp- /fibrinogen complexes may be seen as igfbp- storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k q (q substitution for k in position ) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated (single mutant) and (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk- cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst- reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk- cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p- . . - his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova , m. tileva , k. maskos , i. ivanov , g. nacheva institute of molecular biology "roumen tsanev", sofia, bulgaria, proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five Ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p- . . - development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected ( - in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec . . . ) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta- , -d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall -fold purification, a . % yield and specific activity uÁmg À protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled kda. it was found that the enzyme displays pi . , prefers ortho-nitrophenyl-beta-galactoside as substrate (km mm) and shows maximum activity at °c and at ph . - . . the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km . . it was established that the gene carries an open reading frame consisting of bp ( amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family (cazy database). p- . . - different splice-forms of tdrd protein mutated in cataract's and glaucoma's interacts with s k / o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s kinases (s k) are important players in cellular pi k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s k -tdrd (trap). tdrd is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s k-tdrd interaplay and study its role in cells. bioinformatical analysis of tdrd sequence revealed the presence of potential phosphorylation sites of s k . using in vitro kinase assay, we have demonstrated that recombinant s k phosphorylate from fragments of tdrd . formation of s k -tdrd complexes in vivo was further confirmed by coimmunoprecipitation using anti-s k and anti-tdrd antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd co-localize with s k in hepg cells, predominantly in perinuclear region, enreached for one of the tdrd isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s k with methylated arg interfere with tdrd from hepg lysates. the physiological characteristics of s k -tdrd interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~ kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x- in high concentrations, but efficiently dissociates in the presence of m urea and mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of to kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a and other proteins. serum albumin, transferrin and protein with molecular weight , kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b fusion protein. we have shown that cytochrome b enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at nm in oxidized environment. cytochrome b change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b fusion tags multiples recombinant antibody yield (from to times) and doesn't affect antigen-binding properties. the bb - site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i- c (monab i- c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i- c in bb - of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb - , bb - and both their scrambled version, and bb - peptide. monab i- c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i- c was investigated by spr method using plasmon- device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb - , bb - , bb - , and the scrambled versions of bb - and bb - peptides on a binding to monab i- c and on the fibrin polymerization process. in spr analysis was showed that bb - and bb - peptides, but not their scrambled version, binds to monab i- c, immobilized to a chip. turbidity data showed that only bb - and bb - peptides caused the -fold decrease of the rate of the lateral association of protofibryls at the concentration . À m and . À m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb - site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp- ) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is - ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= , female= ) was . years (sd . years). among them ( %) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while ( %) as a hf-ref (hf with reduced ejection fraction). mean bnp values was . pg/ml (sd . pg/ml) and mean lvef was . % (sd . %). mean urine ngal was . ng/ml (sd . ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = . , p = . , wilcoxon signed rank test z = À . p < . ). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p subunit of splicing factor sf ;(iii) voltage dependent anionic channel- ;(iv) dynamin- ; (v) heterogeneous nuclear ribonucleoprotein a /b . obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near uridine derivatives were determined. the combined kinetic (nnna, , ) and structural data (acta crystallogr., d , , ) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as - kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp (forkhead box p ) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp on tumor cell migration. our results showed that foxp has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase (pde ) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde was increased in cell lines cancers thus pde inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde gene expression and cgmp signaling in the mcf- er + and mda-mb- er À . methods and materials: the ed of the hawe on both cell lines were examined by using mtt viability test then the expression of pde and cgmp concentration were measured in timedependent manner (in the ed ) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, lg/ml is ed for both cell lines and the hawe lead to reduction in pde mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf- and mda-mb- , cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having -stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to da in neutral ph but the pore is closed by going through a conformational change under ph . . as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg- s is obtained by adding amino acids to the primary chain in order to have a -stranded beta barrel porin. ompg- sl is formed by further adding amino acids to loop l and by replacing lysines with arginines. ompg- s and ompg- sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd . / . and infrared spectra are recorded. further, temperature profiling are recorded for the range between to °c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ °c, although this is less than the wt transition at $ °c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase (parp ) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt . further characterization of nudt will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group ( mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid ( mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups . % peanut oil was added throughout the experiment. the all rats were sacrificed at the end of weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in . % of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp- levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if , if and if . the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim p possesses a function of mtif . however, the mitochondrial translation has not been stopped in the yeast strain without aim p which is surprising taking into account the fact that if is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim p is almost indispensable for cox p synthesis, and that it affects the translation of cox mrna through its -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim gene than in the wild-type. taken together, our results point on the multiple role of aim p in mitochondrial translation: in addition to its function as mitochondrial if , it specifically regulates the amount of complex iv subunits and its activity. p- . . - the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of patients with pcos and healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < . ). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < . ). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). we found that bche hydrolyses this molecule at ph rather better than at ph . . at ph values, purified human bche has a km value that was times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately % that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p- . . - determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi auto analyzer. the epo levels of samples was . ae . mul/ml (ranged between . and . ) mul/ml. when we performed ae sd for the studies population we determined normal serum epo levels were as . - . mul/ml. the upper limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml and the lower limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included patients diagnosed with subclinical hypothyroidism and healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at À •c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were ng/l, control group ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > . ). apelin levels didn't show significant correlation with bmi (p > . ). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv /mia import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv is a sulfhydryl oxidase that partners with the import receptor mia to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv mutant strain (erv - ) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv s role in these processes. we discovered that the erv - strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv and mia mutants tested were similar to the wt control. in addition, while all the erv and mia mutants tested exhibit temperature-dependent defects in mia oxidation, only the erv - strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv - strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh ) that compromises gsh protein folding and/or stability. to address whether gsh deficiency in the erv - mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv and mia mutant strains. only the erv - strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv - are due to gsh depletion and neither erv nor mia play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are . - . ng/ml and - ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak Ò hplc and zivak Ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast l aaa column ( mm i.d). the mobile phase was composed of, a: mm ammonium formate (af) in water, b: mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c column ( mm i.d., mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p- . . - heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp expression and induced caspase-dependent apoptosis in c rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at °c for h in c cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase- activation in c glioma cells. pretreatment of the cells with non-toxic dose ( lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase- activation in c glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp , matrix metalloproteases ; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from mhz to mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain - amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp- and hnp- encoded by defa , defa and defa genes are firstly expressed in neutrophils. human hdp and hdp encoded by defa and defa are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd strain was cloned, expressed, characterized in escherichia coli blr(de ) strain, and then val- and phe- amino acids, supposed to be responsible for substrate preference, were substituted for met- and ile- amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe ile enzyme are %, %, %, %, % and % of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val met mrbsh enzyme are %, %, %, %, % and % of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d , t , and d ) but according to alignment is closer to chr even the last one motif is e , t , n . it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf - - . evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un , s. sahin , f. duygu university of gaziosmanpasa, department of biochemistry, tokat, turkey, oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (% - ). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha (ta ), thymuline and thymosin beta (tb ) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < . ) and plt and wbc levels were significantly lower in the cchf group (p < . ). levels of tf, ta and tb were found to be significantly higher in cchf (p < . ). there was no mortality during the study period. duration of hospitalization was . ae . days. levels of tb were significantly correlated with duration of hospitalization (r= À . , p = . ). alt levels were significantly correlated with tf levels (r = . , p = . ). patients received ffp and apheresis for the supportive treatment, while patients received only ffp and patients got only apheresis. patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > . ). we report survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic values were determined as and mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh of carbamate group of methocarbamol to the carboxyl group of asp side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser and ser residues of the pseudoenzyme. p- . . - dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc ) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than mm. moreover, primary cell cycle analysis has indicated arrestment at g /m phase in hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g /g and g / m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor alpha (hif a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor . it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif -a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of - % oxygen and - % nitrogen for week. rats in adm groups were injected intraperitoneally in a dose of . nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from - animals and adm, hif -a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif -a level. adm levels and hif -a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif -a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif -a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif -a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p- . . - covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, ( ) their circulatory-lives and stability (in different ph and temperature values) enhance ( ) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. postmortem skeletal muscle were analyzed hour periods in first -hour after death. nd and rd -hour periods were statistically significant (p < . ). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of , were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr b and akr b in hct- colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr b ; aldo-keto reductase family , member b , and akr b ; aldo-keto reductase family , member b . both are -kda cytosolic reductases that are similar in both amino acid sequence identity ( %) and tertiary structure with the (a/b) barrel topology. while hct- , a colorectal cancer cell line, cells expresses akr b robustly, there is no expression of akr b . in this study, we have stably knocked down akr b through shrna technology and overexpressed akr b in hct- cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr b , we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr b overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr b and akr b have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m medium (ph ) boosted nisin production at °c, as the addition of . lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p- . . - coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between and , the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to , , and amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf isoform in hct- colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf . we show that vegf undergoes n-linked glycosylation in hct- cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p- . . - novel methods for modulating the activity of bcl family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl- family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl- family, including the pro-survival members bcl- , bcl xl and mcl , and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl- family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl- family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl- family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora , v. castro-fern andez , c. a. ramirez-sarmiento , e. a. komives , v. guixe facultad de ciencias, universidad de chile, santiago, chile, department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt . elucidating the domain swapping mechanism of the forkhead domain of human foxp fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp follows a threestate n « i « u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp , showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt , ). p- . . - the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of . % per week for up to weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the -week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full -week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < . ). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = . , p = . , p = . , respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p- . . - pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck kinases on ents activity. methods: adenosine uptake ( lm adenosine, s, °c) was assayed in hk cells preincubated with mm or mm d-glucose for h and exposed to lm -br camp, lmh or lm tbb for h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent and ent contents were quantified by western blot. results: high d-glucose in hk cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h ) through an increased ent uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent or ent under normal d-glucose ( mm) or high d-glucose conditions ( mm). also, we saw that tbb (ck inhibitor) decreased the activity of ent and ent under normal glucose conditions, decreasing the localization of ent at cell surface, while the membrane localization of ent decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent . this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was . ; body mass index was . ; serum lipo (a) level was . and plasma fibrinogen level was . . there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = . ; p = . ), significant positive correlation between the serum lipo (a) and rdw-sd (r = . ; p = . ) and significant negative correlation between lipo (a) and mpv (r= À . ; p = . ). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is . kda as found. optimum temperature was °c and half-life times were , , and . min at , , and °c, respectively. optimum ph value was . . however, it shows significant ph stability at ph values . and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d lipase. therefore, the effect of immobilization on kinetic parameters of d lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c q tumor necrosis factor-related protein (ctrp- ), c q tumor necrosis factor-related protein (ctrp- ) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp- , ctrp- and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included patients with knee oa and healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp- , ctrp- and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp- in the patient group were significantly higher than in the control group (p = . ), serum ctrp- and kallistatin levels were not statistically different (in order of p = . , p = . ). in the patient group, there was not a significant difference between serum ctrp- , ctrp- and kallistatin levels and radiologic stages (respectively p = . , p = . , p = . ). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = . , r = . ; p = . , r = . ; p = . , r = . ; p = . , r = ). discussion and conclusion: serum ctrp- levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp- and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp- , ctrp- and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as - and - °c. in consequence of the salt test, the test isolates were grown at - % nacl. of thermophilic isolates were selected by rep-pcr and according to s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of - %. enzyme tests showed that, some of the isolates were able to produce protease (f , f , f , f , f and f ), amylase (f , f , f , f and f ), cellulase (f , f , f , f , f and f ), xylanase (f , f , f , f and f ) and lipase (f , f , f , f and f ). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p- . . - warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt a, du , hela, hep b, hek t, shsy y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter (glut ) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp- , - ) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco and casp- was higher in hek t compared to other cell lines. casp- was upregulated in cancer cell lines, mostly in hep b. glut- levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p has a repressive effect on glut , it also can lead us to study about p levels. the effect of inhibition of pi k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her , her ) play important roles in breast cancer pathogenesis. her /her interaction is the most potent heterodimer and it causes oncogenic pi k/akt activation. inhibitors of pi k/akt pathway (akt inhibitor and pi k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi k/akt inhibitor pi- on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb- cells (which has egfr overexpression and pten deficiency) and skbr- cells (which has her overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi k/akt inhibitor pi- for different time periods ( - h) . egfr, her her total rtks expression and pi k/akt pathway inhibition (p-akt and p-p s k expression) were evaluated by western blot. in mda-mb- cells, there were significant decrease in p-akt and p-p s k proteins' expression during the first h. this inhibition was followed by reactivation of the signaling pathway after h. in skbr- cells, p-akt and p-p s k proteins' expression were significantly decreased during the first h. the pi k/ akt signaling pathway in these cells were reactivated after h. basal expression of egfr and her in mda-mb- cells and basal expression of her and her in skbr- cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her and her expression levels. these results suggest that dual pi k/mtor inhibiton by pi- may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid -formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a cell line, respectively. for the whole cell transcriptome analysis a cells were treated with lg/ml of binase for h followed by mrna extraction and library preparation. sequencing was performed on solid xl wildfire next-generation sequencer. we found that binase internalized into a cells after h of incubation. the binase at lg/ml was absolutely non-cytotoxic towards a cell line and was active in the cell culture medium during h incubation. the analysis of rna-seq data showed that among thousands of protein coding transcripts transcripts were up and down regulated by binase, among them transcripts were induced and repressed. binase repressed the production of s a and tnxb which act cancer biomarkers, scn a and drd which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp , rasgrp and alpk transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. . ) is a specific group of phosphatases capable of hydrolyzing myo-inositol , , , , , -hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: -phytase, / -phytase, and -phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain . . showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. . . phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was °c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet a expression vector. multicopy plasmid pet a/agpp contained the sequence of the phytase gene of pantoea sp. . . under t promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag- (bcl- associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag- exists as three major isoforms (bag- s, bag- m and bag- l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp /hsc molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl- , raf- kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag- expression levels affect cell survival in mda-mb- (er, pr and her /neu negative) breast cancer cell lines. we first cloned bag- l gene to a cloning vector, later we transfected mda-mb- cells for overexpression of bag- . we also used bag- sirna to silence bag- gene. western blot analysis was applied to demonstrate relative expression levels of bag- , its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag- overexpressed cells to checkbag- 's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag- overexpression. in addition, our study revealed that once bag- forms a complex with c-raf/b-raf/hsp /akt/bcl- , modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag- and its isoforms are found, the role of each bag- isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type diabetes because of different physiological effects. in circulation, glp is cleaved by ddp in a very short time. if glp can be protected from cleaving, the effective time of glp would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp was performed by sds-page. the purity of fusion protein was found to be %, as confirmed by glp elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at °c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp . ) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology ( %) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with % homology was identified in the genome of clinical isolate of m. morganii . rt-pcr analysis showed that this gene had the maximum expression at h of growth. in addition, the cellular extract of m. morganii had small actinolytic activity. cloning of the gene into e. coli dh a cells led to the synthesis of the kda protein. it is known that the highest expression of serratia proteases is observed at h of growth, and the molecular weight of the mature proteins is kda. it was shown that metalloprotease gene of m. morganii expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight ( kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova , d. zhuravleva , a. laykov , k. forchhammer , a. kayumov kazan federal university, kazan, russia, eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only - % homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = lm) over atp (kd = lm) suggesting that they compete for binding to lbrglnk. neither -oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. - - a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k ) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k -fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c. . . . ) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in . u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a antibody, its kappa and lambda variants, in order to understand the difference on the active site between a and a which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a antibody kappa and lambda variants have been already determined, there was no structural information though about the a antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are s À and s À for a kappa and a lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi x . translation elongation factor ba (eef ba) is a component of a heavy form of translation elongation factor (eef h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor a (eef a) restoring its active conformation appropriate for aminoacylated trna binding. eef ba forms a tight complex with translation elongation factor bc (eef bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef ba. this suggests that the eef ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef a, eef a and eef a . we showed that recombinant eef ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef a comparing to full-length eef ba. similar effect on the catalytic activity of eef ba was observed after its interaction with eef bc. in contrast, the effect of full-length eef ba and its truncated forms on the rate of guanine nucleotide exchange in eef a was similar but relatively modest compared to eef a . this can be explained by higher rate of spontaneous gdp dissociation from eef a comparing to eef a and lower affinity of eef a to eef ba. thus, we propose that the n-terminal domain of eef ba via flexible linker region may interfere with eef a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef bc and eef ba n-terminal domains abolishes this inhibitory effect. p- . . - assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below %, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof + mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of proteins across all samples with variation across lc-ms replicates was % on average, and the second approach led to quantitation of proteins in total, of them across % of samples, all with the variation about % on average. p- . . - interaction of plasminogen fragments k - and k with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k - and k with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k - and k reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by - % in the presence of k - and k . fragments k - and k have high affinity to fibrin fragment dd (dissociation constant is . lm for k - and . lm for k ). analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k - interacts with complex of fragment dd-immobilized k as well as k with complex of fragment dd-immobilized k - . the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa - region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k - and k , which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p- . . - implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf ) cells expressing human dopamine d receptors using [ h]sch- and bodipy-fl-skf- as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf- itself were rapid with an apparent half-life of t / = . ae . s for association ( nm) and t / = . ae . s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas levels in gi cancer patients. female and male patients were included in the study (n = ): colorectal, gastric, pancreatic, liver, ampullary, gall bladder and esophageal. from all gi cancer patients, ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at g for min, and plasma samples were carefully removed and stored in À °c prior to use. the level of plasma gas was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas levels in cancer patients were determined as follows: . ae . ng/ml in colorectal; . ae . ng/ml in gastric; . ae . ng/ml in pancreatic; . ae . ng/ml in liver; . ae . ng/ml in ampullary; . ae . ng/ml in gall bladder and . ae . ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas levels. taken together, these results suggest that gas may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas would be an attractive therapeutic target for slow down the progression of gi cancer. monday september : - : computational biology p- . . - computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p- . . - computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g -dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g -dendrimer to mannose receptor (mr ) were performed using hex . . software. taa lumo atoms were conjugated to homo atoms of pg-g dendrimer using maestro software. results showed that pg-g -dendrimer displays carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g -conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g -dendrimer binds to the core of the mr receptor. increasing of the number of mannosamine conjugated to pg-g -dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, -deoxy-d-xylulose- phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the d structure of tpdxr by phyre . energy minimisation and root mean square deviation (rmsd) was performed by drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket . server a reliable d model for dxr from t. parva was modeled by using au as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be . and . a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p- . . - molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme (bace ) as a drug candidate for alzheimer's diseases g. yalcin , i. yildiz biotechnology institute, ankara university, ankara, turkey, department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme (bace ) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly , thr , gln , phe , tyr , lys , thr , arg , thr residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace . the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version . . . dst was determined by bpa - p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley . this work was supported by the russian scientific foundation (grant - - ). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r , r , r , r , k , k ): r = . + . * p À . * f r = . + . * p À . * f r = . + . * p À . * f r = . + . * p + . * f k = . + . * p + . * f k = . À . * p À . * f these formulas show that the maximum total effect of fat and protein contents influences at r and r . a significant coefficient (> ) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p- . . - molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv . channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv . with peptide blockers agitoxin and its mutant forms. d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code a h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv .x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. - - . simulations were performed using the supercomputing center of lomonosov moscow state university. p- . . - homology modeling and molecular docking study of the paraoxonase- and its polymorphic variants q/r and m/l for non-statin lipid lowering drugs paraxonase- (pon ) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon and its polymorphic structures pon q/r and m/l . fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon and its polymorphic variants pon q/r and m/l was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon (pdb code: sre). ns molecular dynamic simulation analysis was performed using gromacs . . . affinity of lipid lowering drugs to pon and its polymorphic variants was predicted by molecular docking approach using autodock . suite. unlike other lipid lowering drugs that they have negative Δg values for affinity, probucol, orlistat and betasterol was calculated by positive Δg values ( . , . and . kcal/mol). these values suggest that they may have no affinity to pon q/r polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon -m/l and sibutramine to pon q/r were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon and m/l polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon structures may reduce the activity of antioxidant pon enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over distinct functions in mammalians. the mammalian prlr gene consists of - exons and several and regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_ . , nc_ . and rn_celera, ac_ . ) were used and primary analysis showed that both sequences contain several gaps (varying from . to kbp), corresponding to about . % ( - kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_ . prlr gene has two exon- (one is about kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from . % to . % (from kbp to kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon- and - , similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the utr (exon- and - ). in rats, there are exon- variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share common non-protein coding exon- variants. in conclusion, it is found that the rnor_ . version of the prlr gene has the highest number of unidentified base pairs (corresponding to . % of the gene) and the second exon- is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase- enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon (pdb id: sre) from protein data bank. modeller software was used for homology modeling of pon and its polymorphic variants that's called as pon q/ r and m/l . amino acid sequence of human serum pon (uniprot: p ) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs . . software. molecular docking calculations on each of the polymorphic structure of the pon was performed with auto dock . . suite. for each substrate, y residue showed open conformation in pon and m/l polymorphic structures while q/r polymorphic structure showed closed conformation. in comparison between structures of pon variants, in most cases statins had lower affinity to q/r polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon . by considering that the high affinity drugs can have reducing effect of pon activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p- . . - self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar , d. alpay , a. r. atilgan , c. atilgan sabanci university, istanbul, turkey, northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever- m' and tensiometer 'bpa- p'. three groups of animals were formed from clinically healthy holstein cows at the age of - years according to the fat content in the milk sample. group i - cows (milk fat content . ae . %), group ii - cows (milk fat content . ae . %), group iii - cows (milk fat content . ae . %). this work was supported by the russian scientific foundation (grant - - ). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from . % to . %, lactose and mineral content varies from . % to . %, respectively. the dst parameters (r , r and k ) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r , r and k . obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p- . . - exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p- . . - effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas , , l. buimaga-iarinca , c. floare , l. janosi faculty of physics, babes-bolyai university, cluj-napoca, romania, national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl À pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~ % of all cancers and up to % in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c : -phosphatidyl-choline: di- : -phosphatidyl-choline: cholesterol : : ) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region - and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id n ) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant - - . serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a -ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at nm uv light. antibodies of rhd antigen were detected using differential pulse method at between . and . v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in rhdnegative pregnant women ( - th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon and of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p- . . - investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega and bioedit version . . . and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega and paup . b with bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui . using gtrgamma model with bootstrap replicates. beast v . . was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total reads (with an average read length of . bp). there were contigs having > bp sequence length whereas contigs with > bp sequence length. for further analysis we have taken contigs with > bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of À . ≥ % of total contigs were mapped to the reference with ≥ % contig match coverage. the community analysis revealed that domain bacteria is predominantly present ( . %) in the water sample, followed by eukaryota ( . %), viruses ( . %) and other sequences ( . %). most abundant phyla was proteobacteria ( . %) and the most dominant family was enterobacteriaceae ( %) followed by xanthomonadaceae ( %), vibrionaceae ( . %), pasteurellaceae ( . %), shewanellaceae ( . %). we performed functional analysis of all contigs using rapid annotation using subsystems technology (rast) which detected coding sequences and rnas in subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with % accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~ % accuracy by this single cleavage, with optimal combination of - cleavages improving the accuracy tõ %. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants - - , - - and - - ) and ras presidium program 'molecular and cellular biology'. p- . . - mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain - which was derived from soil-isolated b. pumilus p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to lg/ml) and ability to produce extracellular enzymes in quantities almost -fold higher than the parent strain. these features make the - strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus p and - strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. . % of total cdss differ in - strain in comparison to p one, % of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k n) leading to str resistance is identified in s ribosomal protein s p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain - gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided x ( p) and x ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) overall genome coverage. assembled genome sequences of p and - strains included and scaffolds > bp with a calculated genome size of bp and bp, respectively. the gc content was %. both draft genomes have been deposited at genbank (jojx . for p and jhud . for [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b and safr- ) revealed the max. ani value ( . % and . %, respectively). b. pumilus sh-b strain has been used as a reference for snp calling in strains p and - . snps for the p strain and for the - strain were classified as nonsynonymous variants. radical nucleotide substitutions from the - genome were not found in p genome. among them, the mutation in the codon of rpsl gene (coding s ribosomal protein s ) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a , that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a antibody is only m À min À , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a . the hybrid qm/mm method allows to study the reaction mechanism of interaction of a with paraoxon. it was shown that the reaction proceeds via the classical s n mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr- to paraoxon. qm/mm approach determines position for mutagenesis -leu- in light chain. for one of the mutant in this position -leu argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu arg exhibits a -fold increase in k /k d compared to a ( m À min À vs. . m À min À ). double mutant leu arg/ser ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu arg still binds paraoxon three times better, that may be due to the fact that the serine in position increase the nucleophilicity of tyr . thus, our results are in line with our computed predictions. this work was supported by rfmefi x . due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is x pixels and field of view (fov) is lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on % raw meat and % offal tissue. calculations proofed with imagejÒ. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday september : - : mechanisms of pro-inflammatory diseases p- . . - the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als mg/kg (oral) group, tor+als mg/kg (oral) group, tor/det+als mg/kg (oral) group, tor/det+als mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated °clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il- b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als and tor/det+als groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak project, coded s . ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type diabetes was induced in male c bl/j mice by i.p. injection of high-dose stz ( mg/kg b.w.). after weeks of stz-induced diabetes animals were treated with/without d ( iu/mouse per os) for weeks. blood serum ohd was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum ohd level, indicative of d deficiency. significant alterations in d -endocrine system were found as is evident from reduced expression of cyp a , cyp r , vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of ohd content and partial normalization of liver tissue structure were achieved after d treatment. d administration partially normalized expression of cytochromes involved in d metabolism and hepatic pro-inflammatory factors. d treatment prevented overexpression of rel-a and phosphorylated p /rel-a translocation to hepatocellular nuclei that is most likely mediated through , (oh) d and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d against diabetes-induced liver injury. p- . . - lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c c muscle cells s. savranoglu , h. ipek , s. arslan , h. delig€ oz , a. r. t€ ufekc ßi , i. demirtas , t. boyunegmez t€ umer graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c c myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt- (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine ( ) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c c cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser )/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the -deoxyglucose uptake assay. results: eae at and lg/ml significantly increased the glucose uptake and % compared to insulin resistant control cells. metformin at mm increased this parameter up to %. eae increased pakt ser /akt level - % and lpl expression - % for and lg/ml, in insulin resistant myotubes, respectively (p < . ). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt molecule through ser phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: t ) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, : n À ) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of . mg/kg ( mm/kg) daily during weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day . we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. - - ), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. - - mol_a. p- . . - circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd +/ cd + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd +/ cd + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd +/ cd + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥ . pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < . ) as well as high levels of aopps-alb, apoptotic emps (cd + /cd + /an-v + ), and nt-probnp (p = . , p = . , p = . respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd + /cd + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: patients who were diagnosed with obstructive sleep apnea and healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = . , p = . , respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = . ). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p- . . - decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type (dmt ) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt patients. the cross-sectional study included dmt patients, treated at the primary health care centre zenica from december to april , distributed into groups according to glycated hemoglobin (hba c) values: a (n = , hba c ≤ . %) and b (n = , hba c > . %). complete blood cell count, fasting blood glucose (fbg) and hba c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss . . p values fasting blood glucose and hba c were significantly higher in the group b compared to the group a (p < . ). there was no significant difference of nlr, plr and rpr between the groups (p = . ; p = . ; p = . , respectively). significant correlation of inflammation-based scores with fbg and hba c was found only between plr and hba c in the group a of dmt patients (r = . , p = . ). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type diabetes mellitus (t dm) and determine whether or not these are reliable markers for diagnosis. we evalueted people's results who were carried out oral glucose tolerance test (ogtt). acording to -h values of plasma glucose in the ogtt; . group (normal glucose tolerance: ngt): under mg/dl (n = ), . group (prediabetic: impared glucose tolerance (igt)): ranging from mg/dl to mg/dl (n = ), . group (firstly diagnosed diabetic by ogtt): above mg/dl (n = ). . group is clear diabetic without complication (taking treatment) group (n = ). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = . ) and plr (p = . ) values. nlr values are significantly higher in prediabetic ( . it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type diabetes mellitus (t d). this study aimed to analyze the long-term impact of altered metabolism in female t d patients at the level of mediators of inflammatory response. this study included femalet d patients and control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il- , were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = . ), crp (p = . ), il- (p = . ), leukocytes (p = . ) and sedimentation rate (p = . ) in female t d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba c (p = . ), il- and glucose ( . ), il- and bmi ( . ). in our study, female t d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il , crp and sedimentation. other studies conducted in female population associated elevated levels of il- and crp with t d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure , (oh) d levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included renal transplant recipients ( female, male; mean age: . ae . ) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month . serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, (oh)d and , (oh) d levels were measured. gfr values were estimated by ckd-epi. plasma , (oh) d levels were determined in a lcms- triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss . software was used for statistical analysis. results: although plasma , (oh) d levels significantly increased (p = . ), we did not find any significant differences for serum (oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = . , p = . for alp), we observed significantly higher calcium and gfr values (p = . ). vitamin d insufficiency was present . %, deficiency . %, severe deficiency % before transplantation, insufficiency was also seen . %, deficiency %, severe deficiency . % after transplantation at month . conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type diabetes (t d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type diabetic subjects. this study involved newly diagnosed type diabetics and healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. ra patients, as patients and healthy volunteers with mean age of - were included in the study. serum levels of vegf, angiopoetin- and tie- were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang- and no levels were significantly higher in the as group ml, p < . ; p < . ; p < . ). no differences were found between as and ra for tie- (p > . ). vegf, ang- , tie- and no levels were positively correlated in both as and ra patients (p < . ), but no correlation was detected between clinically activation index das- , basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > . ). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang- in as patients was sufficient (auc ( % cl): . , p < . ). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang- measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately - h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with patients and healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at g for min at °c. the serum samples were stored at À °c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p- . . - neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of patients with polycystic ovary syndrome and healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count ( . ae . vs. . ae . , p < . ) and platelet count ( . ae . vs. . ae . , p < . ) were higher in patients with pcos compared to the control group. lymphocyte count was . ae . in pcos patient and . ae . in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls ( . ae . , . ae . p < . , . ae . , . ae . p < . , respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b , caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b were measured in c bl mice with alf induced by single i.p. injection of aom ( mg/kg of b.w.) and after neutralization of tgf-b induced by single i.p. injection of ab-tgf-b ( mg/kg) h before aom injection. in alf mice, blood tgf-b was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s ) fraction by~ % and %, respectively, but was slightly depressed in the membrane (p ) fraction by~ % and~ %. aom induced an increase of postsynaptic proteins: psd- and nnos by~ % in p fraction. tgf-b neutralization resulted in a reduction in the expression of presynaptic proteins by~ % in s fraction and~ % in p fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd- and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b neutralization appear to be confined to the presynapse. % of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox- and cox- ) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox- and cox- , they have serious side effects on gastrointestinal system. therefore, new highly selective cox- inhibitors having fewer side effects are needed. in the present study, selective cox- inhibitory activities and cytotoxic effects of new series of -benzoxazolinone and thiazolo [ , -b ]- , , -triazole derivatives previously synthesized as specific cox- inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco- cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il- b, il- , il- and il- levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl- and tgf-b levels of patients with aa. patients with aa and healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl- and tgf-b levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b levels in the patient group were significantly lower compared to the control group whereas serum ykl- levels were significantly higher in patient group. tgf-b levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl- level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl- levels in aa patients suggests that ykl- plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin (il- ), and soluble intercellular adhesion molecule- (sicam- ) in comparison with control patients. cimt values are similar in psa ( , ae . *) and psc ( , ae . ) patients. however, both of them were significant increase compared with control ( . ae , ). regarding inflammatory markers il- serum levels in patients with aps was higher than pcs ( ae . ) and healthy controls ( , ae . ) but the difference did not achieve statistical significance (*p > . ). on other hand mean of sicam- , value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > . ). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < . ) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam- crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p- . . - protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr and increase in dcr expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type diabetes (t d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il- beta (il- b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il- b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit- mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il- b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at and h by mtt assay. trail ligand and receptor expression profiles on nit- cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit- beta cells at h, while increasing cell viability following il- /ifn/trail and il- /tnf/trail combined applications. substantial levels of death receptor (dr ) expression were detected on nit- cells before applications, yet it displayed decreased levels at h of trail treatment. lower levels of decoy receptor (dcr ) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il- b increased cell viability in nit- beta cell lines along with reduction in dr death receptor expression and an increase in the decoy receptor dcr expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u plus . microarrays, peripheral blood cell mrna profiles of bs patients (b) and matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of and for the chosen fold changes of . and . respectively (p ≤ . for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin- and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin- and homocysteine, we also investigated whether there is a correlation between these parameters. obese and healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin- and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c -esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c _ inh deficiency. materials and methods: people with c _ inh and people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss . was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = . ), looh (p = . ) and sh (p = . ). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: + . ) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, % fructose administered group, cape group, % fructose + cape administered group and ethanol group. after weeks, liver oxidant and antioxidant status, and blood tnf alpha, il- , and il- , tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il- , il- levels were found to be increased in fructose group compared with the control group (p < . ). in liver tissue of % fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul , n. uzun , e. c ß inar tanriverdi , h. € un agri ibrahim cecen university, agri, turkey, nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of pregnant women were included in this study. pregnant volunteers were divided into two groups ( healthy pregnant women and pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin- (il- ) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il- levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il- levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty -day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents hrs after lps injection. the rats were sacrificed at hrs after lps or normal saline injection (n = ). hepatic tissue tnf-a, il- , icam- and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il- , icam- and pdgf levels were statistically significantly higher than in controls (p < . ). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il- , and pdgf when compared to sepsis group (p < . ). there were no significant differences in the icam- levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il- , icam- and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of mg/kg during weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival ( %) compared to rats without treatment ( %). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel ( / nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red ( nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic doses of dtx, cis and deg. on the a and h nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v. . . ) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than . were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic values). these effects were remarkable in p -mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the % of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic doses of dtx, cddp and selenium on the a and h (p mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic concentrations at lower doses in both nsclc cells. however, cddprelated ic concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor (her ) is overexpressed in % of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her overexpression. in breast cancer cell line mcf , her overexpression (mcf-her ) results in mitochondrial dysfunction with low mitochondrial membrane potential (Δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf -her cells than vector control (mcf -vec). additionally, mcf -her cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf -her and decreases in knockdown of her in hcc cells that is her positive breast tumor cell line. based on these results, we conclude that there is a link between her overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed microrna genes deregulated by methylation and new potential target genes of mir- and mir- - p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with references (rnu , rnu ) and paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir- b- p, - - p, - - p, - a- p, - b- p, - - p, - - p, and - - p (p ≤ . , fisher's exact test) in bc. we observed mirna genes to be hypermethylated and mir- hypomethylated. hypermethylation for of these mirna genes was shown for the first time: mir- , - , and - ( - % of bc cases). a significant correlation between methylation and expression alterations was revealed for mirnas with downregulation: mir- b- p, - - p, - - p, - a- p, and - b- p (spearman's correlation coefficient (rs) was in the range À . to À . , p ≤ . ), and for mirnas with both scene (down-and upregulation) as well: mir- a- p, - a, and - (rs = À . to À . , p ≤ . ). comparative analysis of the data on expression alterations of mirna genes and protein-coding genes, which were predicted as targets by mirwalk . , revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir- /rhoa, mir- /rassf (a), mir- - p/dapk (rs = À to À . , p ≤ . ). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant - - from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: -year survival is about %. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of paired (tumor/ normal) ccrcc samples. we also used post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and paired ccrcc samples. we observed mirna genes (mir- a- /- /- , - - , - - , - b/c, - - , - a, - ) to be hypermethylated, (p ≤ . , fisher's exact test), mirna genes to be hypomethylated and mir- a with both scene (hyper-and hypomethylation was detected). methylation of mirna genes (mir- a- /- , - b/c, - - , - , - a, - a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir- - , mir- a, and mir- significantly correlated with metastasis presence (p < . , fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir- - and up-regulation of p protein-coding genes: rarb( ), rhoa, nkiras , and chl , which were predicted as targets by mirwalk . (spearman's correlation coefficients (r s ) was in the range . - . , p ≤ . ). in conclusion, novel supposed interactions of mir- - with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir- - , mir- a, and mir- could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm and eno genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset ( k illumina microarrays) we supposed that cpg methylation of pgm and eno promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm :~ bp and bp upstream tss; eno :~ bp downstream tss) that are frequently methylated in hc samples (up to %, / ) with down-regulated pgm and eno expression. thus, we demonstrated frequent and significant pgm and eno down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm and eno gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mК- . . from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of sprague dawley rats were randomly divided into six groups. rats were administered with mnng ( lg/ml) by oral gavage on days , and to initiate stomach cancer. during the experiment, rats died and those surviving were sacrificed in the rd, th, th, th and th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains rat which are given only food and water for months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows . , . , . , . and . for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < . ) and of groups iii, iv and v (p < . ) and also were lower than in the control group (p < . ). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: / ). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep- ), by considering its anticancer features. water extract of turkish propolis (wep, - mg/ml) and ethanolic extract of turkish propolis (eep, . - mg/ml) were prepared and incubated with hep- cell lines ( , , and h). mmp was investigated with a flourometric method by using dioc ( , -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep- cell series according to control ( concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf (nuclear factor ) family. there are few data suggesting that the nf factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf -b isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < . . these data suggest the involvement of the nf proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal ( ; ) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than % of chronic myeloid leukemia (cml) cases. epigallocathecin- -gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k- cells. the cytotoxic effect of egcg on k- cells was determined in time and dose dependent manner by wst- analysis. total rna was isolated from k- cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac , hdac , hdac gene that supports cell proliferation was down-regulated . , . , . folds in k- cells treated with ic dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. patients with cll and healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = . ) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = . ). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = . , p = . ). cut-off value of . absu for anti-ca i was associated with % sensitivity and % specificity and a cut-off value of . absu for anti-ca ii was associated with % sensitivity and % specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained noti-clones associated with chromosome genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, noti-sites with high ( - % of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh l , vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn , foxp , prickle , etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras , itga , lrrc b, and ctdspl genes. several genes with high hm/d frequency (aldh l , ephb , itga , and ropn ) were tested for expression alterations using qpcr. frequent ( - % of cases) and significant (> -fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh l geneon the average -fold mrna level decrease in % of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh l , ephb , itga , and ropn genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant - - from the russian science foundation. functional hypermethylation of a number of chromosome genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained noti-clones associated with chromosome genes, were used for comparative (tumor/normal) hybridization of dna from paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, noti-sites with % and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga ), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn , nbeal , and ube e ). the highest hm/d frequency was observed for ankrd , nkiras /rpl , itga , cmtm , and gor-asp /ttc a genes - - %. expression alterations were evaluated for genes with high hm/d frequency (plcl , prickle , and ppp r a) and significant mrna level decrease (> -fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl , prickle , and ppp r a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from % to % of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost %. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons - , which corresponds to a control group consisting of people living in the city of balkhash. on the whole, molecular genetic studies have shown that examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of individuals including patients and healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than . ). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than . ). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than . ). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately % of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy y cells was investigated by crystal violet staining. ec was found to be . %(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p mrna levels. on the other hand, bcl- , bcl l , erk- , jnk, caspase and mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p and pten remained unchanged. these results indicate that another effector caspase such as caspase or may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor (irf ) is upregulated in melanomas, and genome wide association studies linked variation at irf locus with skin cancers. irf was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf in various hematopoietic cancer types. the aim of this study is to investigate the role of irf in melanoma cell lines. lentiviral vectors were used to reduce irf levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf knockdown (gfp positive cells) over melanoma cells with normal irf levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf levels. cell cycle profiling showed increased g /g and decreased g /m levels in irf knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf levels. taken together, these results indicate that irf knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf . these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. , -dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, adult wistar male rats weighing - g were randomly selected and divided into eight groups. mg/kg body weight (b.wt.) morin and . mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with . mg/kg b.wt. dmba three times during the administration period ( days). cytochrome p a (cyp a) associated -ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were ae , ae , ae , ae , ae , ae , ae and ae pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were ae , ae , ae , ae , ae , ae , ae and ae nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad and smad by the type i receptor and formation of smad / / heteromeric complexes that enter the nucleus to regulate transcription. -deazaneplanocin a is an inhibitor of the histone methyltransferase ezh . we aimed to reveal the effect of -deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg cell line. hepg , a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with % fbs. the cells were seeded the day before -deazaneplanocin a administration and then the cells were treated with lm -deazaneplanocin a for days. expression levels of genes were analyzed by roche lightcyclerÒ . gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < . was accepted as statistically significant. -deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads - in hepg cells (p < . ). we also found that -deazaneplanocin induces apoptosis in treated cell line (p < . ). as a result, -deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf- compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > . /l. differential leukocyte counts were obtained by bc (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < . ). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = À . ; p = . ). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy y cells. ec was found to be . % (w/w) essential oil for sh-sy y cells. neuroblastoma cells were incubated at °c for h. after h, cells were harvested for further studies. bax and p mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl- , bcl l , casp- , casp- , erk- and jnk expression were significantly downregulated. unlike these proteins, p and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp- and casp- . additionally, erk and p mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy y cells. p- . . - the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment ( mg/kg b.w., i.p.), ng and ng-ox ( mg/kg b.w., i.p daily for days) alone treatment; cis + ng ( or mg/kg b.w., i.p daily for days) and cis+ng-ox ( or mg/kg b.w., i.p daily for days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over . million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at À . v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c. . . . ) with glutaraldehyde could be achieved over a wide range of relative mole ratios in mm phosphate buffer at ph . , glutaraldehyde concentration of % . . discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u cell line. changes of apoptosis and cell cycle in u cell line exposured to ic dose of astaxanthin ( . nm- lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u cell line to which astaxanthin was treated or not. ic dose of astaxanthin was calculated as . lm at h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of % in u cells treated by ic dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u cell line and astaxanthin caused an increase of g /m phase arrest ( . fold) and s phase arrest ( . fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g /m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor (irf ) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf expression levels are elevated in several lymphoid cancers, and irf is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf -linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf expression levels. however, there are no published studies about irf target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il- , tnf-a) in non-small cell lung cancer (nsclc). material and methods: patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi ; ifn-c, il- , tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c . post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = . , p = . , %ci; . - . ). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = . , %ci; . - . , p = . ). after crt, mean alt level was determined as . . there was survival difference in nsclc patients with high post-crt alt (hr = . , %ci; . - . , p = . ). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l ) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic concentrations for h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. -aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < . ) through both cells treated with c- and c- . only c- decreased methylation level on l but not on hela. consequently, c- and c- caused demethylation on hela cells similarly with -aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c- and c- might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il- b and il- , causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il- b signaling increases activity of nf-kb pathway. at the same time, il- , triggers jak-stat signaling pathway, which effector is stat . nf-kb and stat activity facilitates hyperexpression of mir-nas mir- , mir- and mir- as well as down-regulates expression of mirnas mir- / , mir- and let- . this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac / / / and sirt / genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir- , mir- or mir- . also, these mirnas can silence ezh , mll, mll , nsd , setd / / , smyd , suv h genes encoding histone methyltransferases. mirna mir- suppresses gene encoding de novo dna methyltransferase dnmt b. at the same time, down-regulation of mirna mir- / can allow hyperexpression of gene encoding acetyltransferase elp . these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd and bd ). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd -selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp , and ctdsp , plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp and ctdsp dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir- - - microrna cluster in co-regulation of ctdspl/ / genes in nsclc was predicted. the significant ( -fold on the average) and simultaneous decrease of mrna levels of ctdspl/ / genes was revealed in the majority of nsclc samples ( %, / ). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = . - . , p ≤ . ) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir- - - family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples ( %, / ) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir- - - micrornas in the regulation of rb protein activity via inactivation of ctdspl/ / in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for were reviewed. for , a total of patients and tumor marker requests were included. carbohydrate antigen - , cancer antigen , cancer antigen - , prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of , both positive tumor markertest resultsratio and the positive patient ratio were %. in the patients group with increased marker levels, % of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, % patients with diagnosed cancer history in remission. the ratios of positive tumor markers were % forca - , % for ca , . % for psa, %for ca - , . %for afp, and . % for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct colon carcinoma cells. ic doses of resveratrol were determined by wst- assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd , fra , ppard, egfr, birc , pcna, mcl , stat , fos, jun, p , atf , trail, puma, gadd a, rb , faslg, tnf, socs , stat gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol ( lm) causes decrease in cell viability and increase in apoptotic cell death in hct p (+/+) and hct p (À/À) cells significantly (p < . ). the fold changes of the gene expressions have shown that resveratrol has significant (p < . ) and different effects on the expressions of the genes related with the existence of p in hct cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group and group , respectively. hypermethylation was detected in atm, cdkn a, and gata genes. mortality rate was ( . %) in group and group (p > . ). mean -years survival rate in group was ae months and mean -years survival in group was ae months (p > . ). positive median lymph node count was ae for group and ae for group and the difference was statistically significant (p < . ). frequencies of perineural invasion and lymphovascular invasion rate in two groups were % (p > . ). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for % of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p mutation, which is mutated % of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p (+/+) and p (À/À) hct colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate ( mm) and disodium pentaborate decahydrate ( mm) significantly causes nearly % reduction of cell viability at h (p < . ). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi k/akt, map kinases and many epigenetic regulators. satb is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb expression in nsclc cells. we used a and h cells as a model to evaluate the effects of the crosstalk between p and akt on emt of nsclc cells. therefore, cell culture, inhibition of p activation via sb , transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb , wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb , p-p , p , p-akt and akt was examined in a and h cells by western blot analysis. we observed that e-cadherin and satb are downregulated in a cells (highly active p , lowly active akt) compared to h cells (lowly active p , highly active akt), suggesting that e-cadherin and satb are associated with the crosstalk between p and akt pathways. our results demonstrated that p inhibition in a cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p inhibition upregulated satb expression, and reversed emt in a cells. furthermore, alone satb knockdown is sufficient to induce emt, and prevented the effects of p inhibition on emt. all these results strongly indicate that the crosstalk between p and akt pathways might determine satb expression and epithelial characters of nsclc cells, and satb is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p and akt signalling pathways could regulate satb expression. this work was supported by tubitak ( s , z ). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron vntr and exon -g t (glu asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. ) and bioenergetics (pallotta ) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d -pyrroline- -carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put p and put p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta ) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put p/put p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group ), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dΨ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. (pallotta et al. , (pallotta et al. , pallotta ; di martino pallotta ) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : patients who were diagnosed with colorectal cancer between january and january ; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was . . cancer-specific survival was not significantly different between the high-and low-lmr groups (p = . ). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: . ; % confidence interval: . - . ; p = . ). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment and treatment . . ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment groups, and the treatment group were given mg/kg and mg/kg dose of luteolin, intraperitoneally, for a days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < . ) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < . ). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around - % of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu- , snu- and snu- than the well-differentiated hcc cell lines such as huh- , hepg and plc/prf/ . besides, expression of txnip was examined in non-hcc and hcc tissue samples by immunohistochemical staining. txnip positivity was observed in % of well and % of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk- b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak kinase (ag- ) on myocardial infarct size and the jak /stat signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia ( % o , weeks, h/day) and part of them recieved ag- ( mg/kg) min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak /stat signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak inhibitor ag- caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak , stat and some other proteins involved in cardioprotection (akt, erk / , gsk b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p- . . - detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection ( mg/kg, twice a week) following the unilateral nephrectomy in male -week-old wistar albino rats for weeks. blood pressure was measured by using tail-cuff method. gw ( mg/kg/day, i.p.) was administered last week. expression of various markers (grp , perk, p-perk, ikb-a, nf-kb p , tnf-a, bax, bcl- , mmp- ) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp and p-perk were increased by hypertension and gw treatment reversed them. hypertension-induced increase in nuclear nf-kb p expression and decrease in ikb-a expression were reversed by gw treatment. while bcl- expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp- expression was augmented and gw treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw treatment. these results suggest that lxr activation by gw restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph ( . mg/kg/day or mg/ kg/day, per os) from p -p . chronic mph treatment ( mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin- and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin- and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin- , with no alterations in claudin- . overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs directly interacts with and interferes in opioid receptor (or) signaling. rgs is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs can regulate transcriptional responses mediated by the stat b transcription factor. isolated neural stem cells from rgs À/À mice were immunostained for the mitosis marker ph and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro a cells stably expressing rgs . the functional assays of stat b transcriptional activation were performed in hek expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs blocks stat b phosphorylation and transcriptional activation by interfering in stat b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs results in increased mrna levels of stat b target genes such as the members of the bcl anti-apoptotic family bcl- , bcl- and bcl-xl. this upregulation of stat b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs in stat b mediated transcriptional responses and a novel selective role of rgs in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph - molten globule structures was formed while the pre-molten form took place under ph . in our tests at ph . when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph . in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [ , ] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel- (vdac- ), mitochondrial-plasma membrane cofilin and caspase- activity was observed in the hippocampus of ka treated rats. administration of mi h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca and ca subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references . r. . neuroscience letters, vol. , no. , pp. - . . r. solomonia, et al; . cell. mol. neurobiol. vol. , no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic values of . lm and . lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab / . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin- (ps ) with - mm azure b or vehicle for h. to determine the effect of azure b treatment on ab / levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab and ab levels with a dose range treatment of azure b in ps cells. ab levels were reduced by . % in lm and . % in lm azure b-treated cells when compared to control. additionally, ab levels were decreased by % in lm and . % in lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy y) used as a tumoral model or differentiated to a neuronal phenotype. pink role in this process was analyzed using genetically engineered pink deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p- . . - intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment ( - ) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage ( - ) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal - dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included female day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during weeks. the rats in the exercise group swam for weeks. after weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function ( ) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain ( ) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad ( ). we initiated a study to screen their acetylcholinesterase (ache, ec . . . ) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic values in the range of . - . nm for ache. this study was financed by turkish research council-tubi-tak (kbag z ). p- . . - f -a h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut , a. sen , d. doyduk , y. yildirir department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, gazi university, ankara, turkey fty , a sphingosine -phosphate (s p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty , namely f -a h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f -a h along with fty using pgl . luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty treatment at non-toxic doses. it was found that f -a h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty does. furthermore, f -a h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f -a h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab - ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a nicotinic acetylcholine receptor (a - nachr) domain resulted in astrogliosis, decrease of a nachr density, accumulation of ab - in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c bl/ mice were injected with lps, immunized with recombinant a - or a - endoglycosidase treated to remove carbohydrates. two immunizations with week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a - -specific antibodies and brain preparations were studied for a nachr, ab - and il- levels. the original a - ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a - immunization with 'deglyc' a - did not stimulate il- elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a resulted in similar a nachrs decrease and ab - accumulation in brain and significant episodic memory decline comparable to those after lps injections. a nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a nachr density caused by a - -specific antibody is critical for ab - accumulation and episodic memory impairment while pro-inflammatory capacity of a - -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using , -diphenyl- -picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl , the cxcr ligand. we aimed to determine whether snp rs located in the promoter of cxcr gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr promoter activity. mef c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr promoters containing different rs alleles were estimated using luciferase reporter assay. we determined that minor rs allele creates functional mef c-binding site within one of the regions required for the basal activity of the cxcr promoter. cxcr promoter containing minor rs variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef c as the main regulator of rs -dependent modulation of cxcr promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d receptors (d r) and histamine h receptors (h r) form heteromers through which h r ligands can inhibit d r function. d rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d r and h r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d r and h r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk / phosphorylation and cell death in cortical slices. endogenous d r-h r heteromers were detected in rat and mouse cortical slices, where h r ligands decreased d r signaling (erk / pathway) and were also able to block the cell death induced by overstimulation of either d r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d r and h r form heteromers with nmdar subunit a in the presence of subunit b. d r-h r-nmdar heteromers were detected by bret with bifc. endogenous d r-h r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d r, h r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d r-h r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h r ligands act as a 'molecular brake' for d r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with weeks old total male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for s to co and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at À °c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi- and cd . changes in tight junction (tj) proteins (occluding and zone occludin- ) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi- staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec . . . ) is a zinc metalloenzyme that catalyzes the reversible reactions of co and water. carbonic anhydrases (cas, ec . . . ) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose- b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained . m tris-so ph . , r-nitro phenol in ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: rats were used. for sit, animals were kept individually for month and for pst, rats were exposed to dirty cat litter for min at the first day of month stress. flu was given ( mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < . were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < . ). flu restored anxiety scores in both sexes (p < . ) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < . ). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < . ) and pst (p < . ) in both sexes. female rats had % lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < . ). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein (hapln ) is one of the major elements of pnns. hapln interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln were observed. however, the role of hapln in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln during differentiation using pc cell line as a neural differentation model, derived from rat pheochromocytoma. after pc cells were stimulated to differentiate into neurons by nerve growth factor on days , and ; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln and survival motor protein (smn ) network analysis were performed in-silico. as a result, we analyzed fold increase in hapln mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln level suggests that, hapln is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln and smn through fibulin (fbln ) was detected. fbln was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln and fbln in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf . the risk of malignancy is increased in nf , wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf , we considered the mesenchymal stem/stromal cells (msc) carrying the nf mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf -mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen . mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of g) chronically intoxicated with hepatotrophic toxine of taa. every weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by % in the experimental group. in the experimental hepatic x-amidase activity decreased by % compared to those in the control. the average x-amidase activity in the blood serum ( . nmols/ mg/min) and in the brain ( . nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about % higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached %. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi x ). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp b gene. atp b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t i, p.v i and p.r g-fsx in yeast lacking the ccc gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t i mutation mainly affects the liver and the nervous system, p.v i mutation affects the nervous system, and p.r g-fsx mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp b, we cloned human atp b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp b genes were transformed into yeast cells lacking the homologous ccc gene for functional comparison. first, we analyzed the expression of atp b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp b gene in ccc d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t i, p.v i and p.r g-fsx mutations cause functional deficiency in atp b functions and suggest that these residues are important for normal atp b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: z ). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen , , e. erem faculty of medicine, ege university, izmir, turkey, center for continuing education, ege university, izmir, turkey, recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. students (in rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was . / , that increased to . / in the posttest evaluation. % of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was % in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates ( , , lg/well) and lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by % sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < . was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv . voltage-gated potassium channel kv . belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv - subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s -s linker at the inner mouth of the pore. here we present an improved d structure of the truncated human kv . channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv . channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag channel, full-length shaker potassium channel kv . , c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a -year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of graduation. currently, there are students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube : z (becton dickinson and company, franklin lakes, nj, usa); tube : ayset (lot , turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au ) and randomised samples stored at - °c for and h. th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at , and h were statistically no significant (p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' ( / / - / / ) (search # ). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search # ) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search # revealed articles; of these were clinical trials, were observational studies. human studies were while animal studies were . overall, there were review articles and meta-analysis in the field. search # revealed articles of which were clinical trials, were review articles, were meta-analysis. human studies were and other animal studies were . the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi kinase, pi-dependent kinase and akt. then, we found the d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopÒ software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of ptb ( s, msc, phd theses) . % was in natural sciences while . % was in health sciences. the theses output-growth measured by the compund annual growth rate was % over the -years. the top clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants ( ), endocrine-metabolism ( ) and enzymology ( ) were the top research areas in ptb, followed by genetics ( ) and cancer ( ). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february , organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for days was realized with participants in total, of which from universities; representatives of their companies; from chamber-institute-public establishments and of which from public hospitals. of the participants were from izmir, of them were coming from out of izmir. conclusion: at the end of the symposium, % of the participants gave feedback. among the feedback selected; . % of the participants evaluated the symposium overall, as successful. . % of them found the symposium successful with regard to its scientific content. their feedback were . % positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and . % of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a item online survey with sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the -day period, ms students (in the thesis phase) answered the questionnaire (female: . %, male: . %). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work ( %), visits with their mentors ( %) and theoretical lessons ( %). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only % and % sufficiency, respectively). communication skills ( %) and team work ( %) were rated as the highest professional competencies followed by literature search and research planning (both %). it was striking that ms perceived themselves as quite insufficient in scientific writing ( %), data analysis ( %) and project writing ( %) while proficiency in english ( %) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority ( %) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu- phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached phd graduates who had graduated from deu graduate school of health sciences between and from different departments via e-mail. the survey was included questions, which were prepared in the light of the existing literature. among the phd graduates, ( %) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (% . ) are well satisficed from the education they had taken. beside this, interestingly we found that % . of graduates prefers staying in the academic positions and % . of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn and itsn are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using web servers we analyzed utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir- , mir- , mir- , mir- / , mir- , mir- and mir- in utr of itsn -s, predicted by servers, mir- predicted by servers for itsn -l, and mir- , mir- / , mir- , mir- and mir- predicted by - servers for itsn -l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir- , mir- , mir- / , mir- , mir- and mir- were highly enriched for all analyzed pathways. using regrna . and scan for motifs services we predicted types of different regulatory elements in utr of itsn and itsn : k-box and brd-box, musashi binding element for rbps musashi and musashi , gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn -s regulation by micrornas we cloned utr of itsn -s into luciferase reporter vector and transfect hek cells by this construction and mir- a, mir- a and mir- a. for mir- transfected cells, we found - % decrease of expression of itsn -s utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir- target site in utr of itsn -s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. % of stroked patients lose their life in year, and remaining / of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir- a, - , - , b, - , , - a and let- f of ischemic stroke patients and to investigate role in predicting prognosis methods: patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and healthy individual were included in the study. after stroke patients' blood samples were taken periodically in st day, st week, and rd month, and at the same time nihss and mrs scores were determined. set mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke st day peripheric blood levels of mir- a,- ,- a and let- f were significantly low; when st week and st day serum records were compared there was a significant increase in mir- level; and when st week and rd month records were compared we noted that there was a significant increase in mir- a,- and let- f levels. from prognosis point of view; after ischemic stroke measurements showed that mir- b in the st day, mir- a and mir- in the st week showed positive significant correlation with rd month mrs scores (p = . , p = . , p = . , respectively) conclusion: according to the outcomes of this study, after stroke st day mir- b, st week mir- a and st week mir- levels can be stipulated to use in predicting patients' rd month prognosis p- . . - inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [ ] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing -nucleotides of a random region. after rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that -fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [ ] hunsicker, et al. ( ) an rna aptamer that induces transcription. chem. biol., : - . p- . . - microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyÒ mini-kit. the expression profiles of mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir- - p was expressed only in aorta. mir- - p and were expressed both aorta and whole blood. mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > ). mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir- - p, mir- - p and might be a potential for cardiovascular disease development. also mir- - p and might serve as novel non-invasive biomarkers for cad p- . . - mir b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc- and miapaca- cells transfected with mir b mimic. our data revealed that mir b led to decrease the cell viability depending on enhanced mir b doses, which are , , and nm, as the ratio of % , , and , respectively, in miapaca- cells and as the ratio of % , , and , respectively, in panc- cells compared with control condition of each cell. this inhibition mediated by mir b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir b was more effective than gemcitabine. based on our findings, it is clearly shown that mir b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant s . breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her + breast cancers, which has role in approximately % of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her + breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt and skbr cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that out of the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t( ; ) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k cells were treated with lm (ic dose) res during h and cytotoxicity was evaluated by using wst- assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir- - p level . fold and significantly downregulated hdac gene expression (p = . ) and upregulated p / sqsmt gene expression (p = . ), according to the control cells.p /sqstm interacts with lc and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac and p /sqsmt gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac dependent induction of gene associated with autophagy like p /sqsmt and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir- , hsa-let- a, hsa-mir- b and hsa-mir- . thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let + , let + b, let + , + b, + , + let + b, + let + b. the cell viability was detected by wst- kit in a well plate elisa reader. cells were seeded as per well, mirnas incubated with cells for h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however + b and + combination increased the cell viability dramatically. on the other hand let + b and only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein (sip ), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip cernas listed at competive endogenous mrna database (cerdb), transcripts (pten, zeb , ptch , creb , acvr b, enah, robo , erbb , e f , foxo , rictor, klf , ets , cdk ) sharing at least common mre sites with sip were selected and their expression in hcc cell lines were determined by qrt-pcr. ets was found to be highly correlated with sip in hcc. furthermore, repressing sip expression by shrna in hcc cells resulted in decreased expression of ets , pten and zeb . our results suggest a possible bidirectional and post-transcriptional regulation of sip and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar , , n. belder, h. ozdag biotechnology institute, ankara university, ankara, turkey, comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of crc tumors against normal colorectal mucosa samples, using raw data from e-mtab- and e-geod- (affymetrix microarrays), and gse and e-mtab- (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ . , fold change ≥ . and stringent false discovery rates. mirtarbase and mirwalk . databases were explored to identify validated targets. we found thirty (including mir- and mir- ) and thirteen (mir- , mir- , mir- , etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol- kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan , h. g€ urdal , b. g€ ur dedeoglu biotechnology institute, ankara university, ankara, turkey, department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip ( , , ) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip is generated by hydrolysis of pip ( , ) bisphosphate, which is synthesized by pip k . any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt- was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt- cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip k c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk- -beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl- , crl- , crl- , crl- and htb- ) were evaluated with non-malignant (normal) brain cell line (hcn- ) . determinations of expression level of five mirnas (mir- , mir- , mir- , mir- , and mir- ) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir- , mir- , mir- , and mir- ) were significantly decreased while the expression level of mir- was increased in gbm cell lines according to hcn- cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v . , mir disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn b-as, locus cdkn a/b (ink /arf), chromosome p ) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan . , we predicted target sites for hsa-mir- -p/ -p/ -p/ -p/ -p/ -p and hsa-mir- - p/ in anril utr. then, we used hmdd v . and mir disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir- is implicated to alzheimer disease and mir- to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir- and mir- targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril utr. conclusions: we predicted sites for mir- and mir- in utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different ( . - mg/ml) concentrations for h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir- b- p (- , fold, p = . ), hsamir- - p (- , fold, p = . ), hsa-mir- a- p (- , fold p = . ), hsa-mir- a- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- b- p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- a- p (- , fold, p = . ) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir- b- p plays a critical role in the matrine response. ericd (e f -regulated inhibitor of cell death) is a newly found lncrna. it is located at q . . it has two exons and its transcript size is bp. ericd is regulated by e f (transcription factor ) and modulates the cellular response to dna damage. arid a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, different cancer cell lines, osteoblast cell line and different types of normal human tissues rnas were selected for expression analysis of ericd and arid a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid a had lower expression in u- os (osteosarcoma), a- (glioblastoma) and a (lung cancer). at the same time, ericd expression was lower and arid a had high expression in hfob . (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid a are cell cycle regulated and are commonly regulated by e f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif a and anti-eif c. mir- - p, mir- a, mir- b, mir- - p, mir- , mir- a*, mir- , mir- a, mir- a, mir- b, mir- a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif c immunoreactivitiy was negative in all groups. mir- , mir- a* and mir- a were highly expressed in proliferation phase of fertile group, mir- a and mir- b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over . million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately nucleotide length which regulates the expression of genes by binding to the -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed unique small rna sequences obtained from raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified putative mature microrna sequences belonging to distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. of precursor sequences showing % homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as , a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as . materials and method: for the tissue expressions analysis, human total rna master panel ii (containing different human tissue samples) was used. total number cancer cell lines and normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as were found to be tissue specific. particularly, while vim-as was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as was also found to be differentially expressed in cancer cell lines. vim-as expression was found to be lost in cal , pc , and hct and highly diminished a cancer cells. also, it is found to be highly expressed in bcpap, panc and beas b cells. discussion: results of the current study suggest that vim-as may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as and vim axis can be involved in the formation of lung tumors because vim-as was highly expressed in normal lung tissues and beas b cells and expressed very low levels in a lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately % patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h and pc cells treated by tnf-a ( ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir- a- p, mir- as- p, mir- , mir- , mir- - p, mir- q mimics can induce cellular invasion on h , mir- v, mir- h- p, mir- - p, mir- a- p, mir- as- p, mir- mimics can induce cellular invasion on pc . we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir- q, mir- a- p, mir- as- p, mir- , mir- in h , mir- - p, mir- a- p, mir- as- p, mir- in pc can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir- a- p, mir- as- p and mir- . (this study is supported by tub _ itak grand number s ). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a cells. when we inhibit nf-kb activity by p targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir- as and pten untranslated region has recognition site for mir- as. therefore; we transfected a cells by mir- as. mir- as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir- as. (this study is supported by tubitak grand nummber s ). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a (autophagy and v-atpase inhibitor, . pg/ . ml/ovary) on day of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc -i was not significantly altered throughout pregnancy, that of autophagosome-associated lc -ii increased significantly from day , showed a peak on day , and decreased on day (day of normal parturition). lc -ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day . in vivo treatment with bafilomycin a resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc -ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in . the value of low dose bpa was received as < lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i- therapy, have been examined in patients ( m/ f) with ptc and ( m/ f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at days after i- administration ( . gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp- , mmp- , timp- and timp- were measured by elisa. there were no significant changes in serum concentrations of mmp- , timp- and mmp- /timp- ratio after i- in the two groups. in ptc patients, i- administration resulted in an increase with % in timp- level (p = . ) and a reduction with % in mmp- /timp- ratio (p = . ). in ptc+ht patients it has been observed an increase with % in tgab level (p = . ), % in mmp- /timp- ratio (p = . ) and unchanged timp- serum concentration. tgab titers were positively correlated with mmp- /timp- ratio (r = . , p < . ). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp- /timp- for anti-invasion and anti-migration by augmenting timp- . in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts , and and mmp , and to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, cases have been included in this study, divided into groups according to their pmis: ' - h', ' - h' and ' - h'. from these tissues, western blotting technique is used to analyse the expression of adamts , and and mmp , and . it is determined that when pmi increases; adamts- and amounts decrease. on the other hand adamts- amounts are examined to increased related to the interval., especially on the ' - h' dataset. additionally, considering metalloprotease levels, mmp- and amounts decrease as pmi increases. unlike mmp- and ; mmp- levels increase proportional to the interval, especially on the ' - h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts- and mmp- , proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases and in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp and mmp (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp - c>t and - c>t polymorphisms cause loss of gene function, and mmp - c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp - c>t and - c>t polymorphisms, and mmp - c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from healthy subjects. the risk of abortion was . -fold higher in patients with heterozygous - c>t polymorphism (p = . ). combined genotype analysis showed that the risk of abortion was . -fold higher for patients with normal - c>t polymorphism, and heterozygous - c>t polymorphism (p = . ). functional polymorphisms of mmp and mmp may have a role in etiology of abortion. p- . . - changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses ( . and mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, min and min ischemia-reperfusion. after months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts- and - genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam , - , and - . in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = ), oligozoospermia (os; n = ), azoospermia (as; n = ) groups. adam , - and - protein levels in infertility indviduals were analysed by western blot. adam protein level was . fold lower in the os and as groups than in the ns group. adam protein level was . fold higher in the as group than in the ns group. adam protein level was fold lower in the as group accourding to ns group. we observed no change between protein level of adam and adam of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin- on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck- , timp- and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created patients with diagnosis of cchf. healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc- and timp- levels of patients and a control group were analyzed using the human elisa kits. serum timp- tck- and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp- , tck- and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p- . . - activation of calpain and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase (mmp ) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca + homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine in gene of cystic fibrosis transmembrane conductance regulator (f del-cftr), could be involved in the abnormal presence of mmp in the extracellular fluids of cf patients. pbmcs were isolated from healthy donors and cf patients homozygous for f del-cftr. mmp levels were evaluated following h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp ; conversely, in pbmcs from healthy donors, expression and secretion of mmp are undetectable but both events can be evoked, after h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and h-cultured control pbmcs mmp secretion is prevented by chelation of intracellular ca + and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases and (erk / ). moreover, the rescue of active f del-cftr reduces the extent of mmp secretion, correlating the channel defect to the [ca + ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca + concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp . these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp in fluids of cf patients. p- . . - aebp /aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir , , u. akpulat , i. onbasilar , c ß . kocaefe department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, department of stem cell, institute of health, hacettepe university, ankara, turkey, laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp /aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp is the short isoform that acts as a transcriptional repressor by targeting the ap promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp /aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp /aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp /aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated folds upon the induction of differentiation in c c cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp /aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp /aclp expression peaks up to folds in the th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp /aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp /aclp expression is upregulated up to folds in early time-point samples. these results depict a novel role for aebp /aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus - encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus - extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average % compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a -fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo a remained at the level with expression in the strain with the complete spo a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo b, spo f, spo k, spo j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p- . . - paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for min followed by h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp- and - were performed after h of iri. the effects of paricalcitol on activities and expressions of mmp- and mmp- levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp- and mmp- during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp- and - . p- . . - the changes of matrix metalloproteinase , activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko , o. shaulska , y. kot , g. ushakova , a. shevtsova dnipropetrovsk medical academy, dnipropetrovsk, ukraine, kharkiv national university, kharkiv, ukraine, dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp and mmp activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the wister rats divided to groups were used for the experiment. cdcl x . h o in doses . lg/kg and lg/kg was given to rats intragastrically in drinking water during days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp and mmp , total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium ( . lg/ kg) lead to reduced activity of pro-and active forms of mmp in myocardium ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly) and in serum ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly), but pro-mmp activity in heart was increased ( . ae . au/mgtp compare to the . ae . au/mgtp in the control rats); level of ha was decreased in both tissues ( . ae . lg/ml and . ae . lg/ml compare to the . ae . lg/ml and . ae . lg/ml in the control rats accordingly). high doses of cadmium ( lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp increased from . ae . au/mgtp to . ae . au/mgtp, prommp to . ae . au/mgtp, mmp to . ae . au/mgtp. ha level was increased in serum ( . ae . lg/ml) and decreased in heart ( . ae . lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin- repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin- (il- ) is an il- cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il- implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts in u proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of lg/ml while trypsin and chimotrypsin are used as - mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no - - ). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from to lmol resv and opposite effect from to lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn- . since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in . m kcl (ph . ) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments - of amyloid peptide, fragment - of [gly ]-amyloid peptide and - of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs ( - - ) and sp- . . . ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti , c. bernacchioni , f. scaletti , p. turano cerm (magnetic resonance centre), florence, italy, department of biochemical sciences, university of florence, florence, italy, chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort , t. ozben , a. sansone , s. barata-vallejo , c. chatgilialoglu , c. ferreri sanko university, gaziantep, turkey, akdeniz university, antalya, turkey, cnr, bologna, italy, universidad de buenos aires, buenos aires, argentina, national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at °c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr in glucose homeostasis. drugs targeting gpr may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr inhibition. in addition, gpr takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon and an additional trans double bond between carbons and . dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical -carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from to mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p- . . - trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b -arrestin or gb subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with À m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p- . . - l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev , , v. polovinkin , , , v. shevchenko , , v. gordeliy , , moscow institute of physics and technology, dolgoprudniy, russia, research centre j€ ulich, j€ ulich, germany, institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr has an absorption maximum at nm at ph . and to create more redshifted variants we analyzed available structures of the kr (pdb codes: xtl, xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m , g and s ). the mutants of kr under investigation were: m a, g v, m a/g v, s a, s f, s g, s l, s m, s n, s t, s v, s y. the protein mutants were expressed in escherichia coli c strain, expression was induced by the addition of mm isopropyl b-d- -thiogalactopyranoside. the cells were then washed trice with unbuffered mm nacl or kcl solution. finally, the ph changes in cell suspensions (od = . ) were monitored. ph changes upon the addition of lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s f, s l, s m, s n, s v. the remaining mutants have shown sodium pumping activity and s a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph . ( mm na/na-pi, mm nacl). m a mutant absorption maximum is blue-shifted to nm. g v and m a/g vblue-shift to nm. s a, a potassium pumping variant,red-shift to nm. s g, s yred-shift to nm. s tno change in absorption maximum position. based on structural analysis of kr we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to nm and red-shift up to nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than % drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins - , which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. - - ). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv- ). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in biopsies ( %). c iv deficiency was most common ( %), followed by c i ( %) and c ii-iii ( %). multiple complex deficiency was present in % and isolated deficiency was present in % of the biopsies ( % c i, % c ii-iii, % c iv). cs activity was elevated in % of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in %, % and %, respectively. comparing with histological data, biochemical analysis revealed additional findings in % of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) , resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and groups were included in the study. rats were fed with chows that were given either normal diet for control group ( % carbohydrate, % protein and % fat), high fructose ( % carbohydrate ( % fructose and % starch), % protein and % fat), or high sucrose ( % carbohydrate ( % sucrose and % starch), % protein and % fat). rats were fed with chows for weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba c, glucose, resistin and adiponectin were studied. glut and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < . ). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < . ). there was no significant differance level of hba c, glucose, resistin and glut in rats fed sucrose or hfcs (p > . ). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p- . . - nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy , n. eruygur , e. s€ uleymanoglu biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg +) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg +-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group ). secondly mice were randomly divided in four groups as: group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days, group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days. ozone/oxygen mixture and oxygen were given at a dose of mg/ kg groups ( ) ( ) ( ) ( ) . after treatment animals were sacrificed, and muscle samples were taken and stored in À °c for until the analyses. muscle tissues were homogenized in . m tris-hci and . m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). sdh activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). there was no statistically significant difference among all groups in terms of cs (p > . ) and sdh activities (p > . ). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than . %, so they were scored as . there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of -monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of -monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, -( -{-[ -(octyloxy) phenyl] diazenyl} phenoxy) propane- , -diol ( % mol), was incorporated into the -monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn m phase with lattice parameter . angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at nm we observed trans-cis-isomerization. in the same time the cubic-pn m phase was not changed but the lattice parameter reduced to . angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin , t. cora cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt that is a hnscc tumor cell line and hek- that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf- r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of women ( %), and men ( %). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant . times increase of gpx activity levels; and . times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by . times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than . times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity ( mm) and potential protective effect of different doses of boric acid (ba) ( , and mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+ mm ba, ethanol+ mm ba, ethanol+ mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < . ). however the mda level in the ethanol+ boric acid ( mm) group was shown to be significantly decreased compared with that in the ethanol group (p < . ). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba ( mm, mm) groups were close compared with control groups (p < . ). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid ( mm) groups were increased (p < . ). these results demonstrate that ethanol ( mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co /bicarbonate between metabolizing tissues and lungs, ph and co homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = . ), (p = . ), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = . , p = . ). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = . , p = . ), (r = . , p = . ), respectively. at an anti-ca i cut-off point of . absu, sensitivity was % and specificity %. at an anti-ca ii cut-off point of . absu, sensitivity was % and specificity %. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from healthy control and patients with behc ßet's disease. the mean age for controls and patients were ae . and ae , respectively (p = . ). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v . results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ). lymphocyte counts were lower in patients with behc ßet's disease compared to control group ( . ae . vs. . ae . ) (p = . ). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of neuroblastoma patients ( male, female, median age: months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of healthy children. patients had stage , , or s disease, patients had stage disease. different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir ds (p = . ), and higher expression of inhibitory kir gene dl (p = . ). additionally kir ds genes were more common in patients with early stages (stage , , or s) (p = . ) and kir dl genes were more common in patients with stage (p = . ). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir dl gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir ds can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p- . . - neuroprotective and immunomodulatory effects of urtica urens s. arslan , g. terzioglu , b. kabalay , a. r. tufekci , a. sen , i. demirtas department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco- cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine (cxcl ), and (cxcl ) were determined. whole genome transcriptome analysis was performed by using human ht- v beadchip. extract treatment caused % and % increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly ( % and %, respectively). similarly, cxcl and cxcl mrna levels decreased % and %. transcriptome analysis showed that probes were significantly changed (p < . ). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak t . p- . . - linear low molecular weight a- , -glucan from bifidobacterium bifidum bim b- d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, , ( ) : - ) that bifidobacterium bifidum bim b- d contains low molecular mass ( - kda) a- , glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b- d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, h and c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as - kda linear a- , -glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b- d cells contain linear low molecular mass a- , -glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a- , -glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba c, uric acid, creatinine, ggt, -hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > . ) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < . ). on the other hand, rdw are determined to be significantly lower (p < . ) in the non-diabetic obese group. no difference was detected in -hydroxyvitamin d levels between the control and the study groups (p > . ). non-diabetic obese patients had significantly (p < . ) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < . ). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco , which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco is also known to be present on autophagosomes, and overexpression of fyco can induce the formation of enlarged lc -containing autophagocytic structures. here we demonstrate that fyco knock-down leads to defects in autophagic mb degradation, and that fyco functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p- . . - proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d and b ) and cell cycle controller genes (p and p ). sf cells were treated with different doses ( nm- lm) of hypericine for h and h. a significant cell proliferation was observed in moderate concentrations ( . - lm; % -% ), but at high concentrations ( - lm) cytotoxic effects emerged in sf cells (ic = . m, r = . ). qrt-pcr results revealed that the most proliferative dose of hypericine ( lm) stimulates cyclin d . the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b mrna, whereas it induced expression of p and p genes, and thus apoptosis was observed by dna laddering at the same dose ( lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately months, increasing to months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant, the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f -targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p- . . - leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t( ; )(q ;q ) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k subclones (k -ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k -ir cells are not only resistant to imatinib but also to nd, rd generation tyrosine kinase inhibitors. we demonstrated that k -ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k -ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k -ir cells forms intact tumor spheroids in d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k -ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd + cscs isolated from human osteosarcoma cell line saos- using macs magnetic separation technique were characterized, and their stemness properties under hypoxic ( % o ) and normoxic ( % o ) conditions were compared in two and three dimensional culture conditions. two different d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct / , nanog and sox compared to normoxic conditions in d cultures. although similar effects of hypoxia were observed in d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on d compared to d culture systems. this study is seen as an introduction to develop a more relevant d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd + cscs were isolated from human pca pc cells using a magnetic-activated cell sorting system. pc and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for h. apigenin dose dependently inhibited cscs and pc cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p and p (kip ). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases- , - and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf- in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase- while caspase- , tnf-a and bcl- levels remained unchanged in pc cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp- and - exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi k/akt and nf-kb p / p were significantly decreased after h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. s ). (pi k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi k activity contribute to phenotypic variability. our aim is to characterise pik ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi k activation on downstream output specificity in the cell. we are currently employing crispr/cas mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p a, allowing precise spatiotemporal control over pi k signaling to unravel the extent to which pi kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi k signalling and potentially aid the development of targeted therapies for human diseases of pi k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf as transcription factors, egfr and cdk as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥ ), prostate (≥ ), and colorectal (≥ ) cancer based on assessment of . ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. . ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck , ck ) and the leukocyte-specific marker as anti-cd . all samples were analyzed by bd facs aria iii flow cytometry. in total, patients and healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but - ctcs were detected with ck , , , -based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at °c with % co and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf- and mda-mb- cells. this effect was observed more prominent on mcf- . considering prfe therapy this effect is much more pronounced especially for mda-mb- comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut , glut , glut proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut , glut and glut proteins was detected in hamscs by immunofluorescence. glut , glut , glut protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd , cd , cd , cd (mesenchymal stem cell markers) and hematopoietic markers cd , cd b, cd , cd and hla-dr were negative. the presence of glut , glut , glut proteins and genes were identified in hamscs. in this study, for the first time in literature, glut , glut and glut gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) cl ] on mammospheres obtained from mcf- human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase / activity, annexin-v-fitc and bcl- activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc- staining. mammospheres exhibited increased oct- and sox (stemness markers) expressions compared to parental mcf- cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion ( . - lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase / activity, annexin-v-fitc positivity and bcl- inactivation. apoptosis was also confirmed with increased tnfrsf a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [ ] . in the development of primary cancers, the head and neck cancer is accounting for approximately . new cases annually around the world [ ] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [ ] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than % of deaths in cancer patients are attributed to metastasis [ ] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [ ] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [ ] . in our study, . ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck , ck for positive selection and cd for negative selection [ ] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level . one of the general methods in ctc detection is flow cytometry . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy , . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which . ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck , ck for positive selection and cd for negative selection, were analyzed by bd facs aria iii flow cytometry . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p- . . - determination of effect cytotoxic, apoptotic, caspace- activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations ( . , . , . , . , , , lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase- activity in breast cancer cells (mda-mb- , mcf , bt- , sk-br ) and normal cell (mcf a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp- , bcl- , bax, birc- , gapdh, caspase- , caspase- , caspase- , caspase- ) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < . ) effect cytotoxic, apoptotic, caspase- activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl- ) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd + gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the gbm cell lines (u , u , u and t ). cd + and cd À cells were seperated by macs method via anti-cd (ac ) antibody from cultured cells and cell lines. rna isolation from cd + and (À) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd + cells express approximately fold tnfrsf and fold tnfsf when they are compared with control cells. tnfsf binds to cd that is expressed on the surface of tcells. cd does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf (dr ) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf and tnfrsf both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf and tnfrsf , these molecules may be essential markers to target cd + gbm stem cells. the effect of docetaxel on p , sin a and mdm gene expression in mcf- breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g /m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf- breast cancer cells line. in this study, mcf- breast cell line was applied different doses docetaxel ( nm, nm, lm, lm, lm) as h and h. mtt analysis was performed to the mcf- breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p , sin a and mdm gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < . ) in h as mtt analysis. the lowest cell viabilty was determinated in group applied lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < . ). the highest p , si a and mdm expressions were apperared in nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g ) and acetyl-terminated (g ) nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g and g dendrimer derivates labeled with dox were absorbed by the cells at °c with different efficiency. g -dox derivate characterized much slower internalization rate than nonacetylated g -dox. only g -dox shown partial colocalization with lysosomal marker lamp after h of incubation. internalization of afprbd-g -dox and afprbd-g -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp already after min of incubation. cytotoxicity results revealed that ic levels of g -dox and afprbd-g -dox coincided and demonstrated a bit higher activity against sensitive to dox skov and resistant skvlb cells than afprbd-g -dox conjugate after h of incubation. at the same time, after h of incubation afprbd-g -dox and afprbd-g -dox were much more than g -dox and g -dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. - - . ) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m -pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m -pk in breast cancer, and high m -pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~ . and . v potentials vs. ag/agci. m -pk activty was detected by using differential pulse method at between . and À . v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m -pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m -pk activity determination at low concentrations is possible with this method. p- . . - tie /tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie /tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie /tek in both cd + gscs and cd À gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with % fetal calf serum and % penicillin-streptomycin at °c in % co -humidified incubator. we isolated cd + and cd À cells from gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd À and cd + cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version . . tie /tek gene expression was determined as . fold higher in cd + gscs than normal brain tissue (p < . ). morever it was determined . fold higher compared to normal brain tissue in cd À (p < . ). according to our results tie /tek expression was higher in gscs, indicating that tie / tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no: s ). adenosine inhibited the breast cancer stemlike cell population through erk / pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk / protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk / pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd + /cd + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd + /cd + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for and h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd , cd , cd , cd and negative with hematopoietic markers cd , cd b, cd , cd and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for and h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in , high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the -phosphofructo- -kinase/fructose- , -bisphosphatase- (pfkfb ) regulates glycolysis by producing fructose , -bisphosphate (f , bp). given that pfkfb is induced by several established emt-inducers in tumor cells, e.g. hif- a and ras, we hypothesized that pfkfb may be involved in regulation of the emt in tumor cells. silencing of pfkfb in pancreatic adenocarcinoma cell lines panc and s vp was achieved using specific sirna molecules. mrna and protein expressions of the cdh gene (encoding e-cadherin, an established epithelial marker), as well as zeb and snai genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb was achived by transfecting cells with a plasmid carrying zeb cdna. cells that were depleted of pfkfb exhibited markedly increased cdh mrna and e-cadherin protein expressions and reduced snai and zeb mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb caused approximately % reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb depletion does not appear to be associated with reduced zeb expression, as ectopic expression of zeb did not reverse the effect of pfkfb silencing on invasion. taken together, these data suggest that pfkfb may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia hematology system and abbott cell dyn and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled . ae . x , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to . x , with a maximum value of x . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = . ), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia ( %), and chronic myeloid leukemia had % of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p- . . - effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type . the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst- tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at h time point. however, at h time point, hescs isolated with the non-enzymatic and collagenase type method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type isolation method at h, h and h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd and lower cd profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd , cd , and cd surface expression levels. only hescs isolated with collagenase type did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type , while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd + subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd . epcam+/cd + cells were sorted by magnetic separation from huh- cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd + subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd + cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd + subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su , in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd + subpopulation of huh- . in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd + hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg ) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst- assay and tg activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp- and - were investigated with zymography and rt-pcr, respectively. although tg activity was found to be % less in emscs isolated from endometriotic tissue, these cells showed times higher tg protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have . times higher tg and . fold higher itgb mrna levels when compared to the cells of healthy group. similar results were observed in sdc- gene expression with a . fold increase. endometriotic emscs demonstrated an average of . -fold increase in the mmp- activity while a onefold increase was evident in mmp- activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within h. the fact that emscs from the control tissue showed lower tg protein levels with a high enzyme activity suggested that tg might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg along with itgb and sdc was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp- and - . recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f with % fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd , cd , cd and cd (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd , cd for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd were decreased to - %. differentiation induced the expression of cd , cd for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~ % and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca + entry (soce) activated by depletion of intracellular ca + stores has been shown to control intracellular ca + homeostasis in many physiological and pathological events. stromal interactive protein, stim , as endoplasmic reticulum (er) ca + sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim and orai on soce in differentiation of huh- hccs expressing epcam and cd surface adhesion molecules (epcam + cd + ). epcam + cd + subpopulations in huh- cells were separated via flow cytometry and transfected with stim and orai- over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca + concentration were monitored via dual wavelength spectrofluorimeter in fura -loaded cells. in epcam + cd + cells, er ca + release increased without any change in soce compared to that of epcam À cd À cells. similar results were observed in stim -oe epcam + cd + cells. on the other hand, increase in orai has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost - % of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd , cd , nanog, oct / , hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd , cd , nanog and oct / significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf- release from human mesenchymal stem cells s. k€ ose , f. aerts kaya , d. uc ßkan c ß etinkaya , p. korkusuz stem cell research and application center, hacettepe university, ankara, turkey, department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor (sdf- /cxcl ) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf- release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage mscs were induced with ng/ml lipopolysaccaride (lps) for h. antagonists for cb (am ) and cb (am ) receptors were added to cultures for days. after incubation with antagonists msc culture supernatants collected and processed with human sdf- beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf- beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf- release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta- , igfr , vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n: ), media (m, n: ), stem cell in media (m+bmsc, n: ). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton ll saline, ll culture media and cell/ ll culture media. the pregnant female rats on the day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta- , anti-igfr , anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta- , igfr , vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol -kinase (pi k)/akt pathways. the effects of h s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h s agents on the in vitro cell growth and differentiation of human lin À sca + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h s-releasing agents, such as na s, gyy and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional d-systems for cardiac tissue repair, that take into account the relevant biological role of h s in the cardiovascular system. p- . . - investigation of the protective effect of boric acid and omega- fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into groups with rats in each: control, mi, mi+boric acid, mi+omega- and mi+boric acid+omega- groups. control rats were treated with ml/day saline, boric acid-treated rats received mg/kg/day boric acid and omega- -treated rats received mg/kg/day for days by oral gavage. for the experimental mi model, mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a -h interval in the last days of the boric acid and/or omega- treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp , biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < . , p < . and p < . respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < . and p < . , respectively) and pon levels were decreased (p < . ). according to the ecg recordings, st wave and heart rate were found to be decreased (p < . and p < . , respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega- after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega- after induction of mi. results of the present study suggest that omega- and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included patients diagnosed with dka, patients diagnosed with dk and voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values ( . ae . , . ae . . ae . ; p = . ). no statistically significant differences were found among groups with respect to ck-mb values ( . ae . , . ae . , . ae . ; p = . ). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group ( . ae . ; . ae . . ae . ; p = . ). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour after acidosis correction ( . ae . ; . ae . ; p = . ) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p- . . - genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac -hcms in dmem/f with %fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber ( %o , %co , %n ) for , , , h and the control group was incubated in normal conditions ( %co , %o ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u -plus- arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as h. we identified genes ( down-regulated and up-regulated) (p < . , fold change ≥ . ) were differentially expressed in hypoxic-ac vs. ac . degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif- signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number s . autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd and cd markers. purified ips cells were differentiated into flk + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days . to . . flk + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd and cd gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk expressing cell number reached to a peak level ( %) on day . followed by a progressive decline subsequently. in the second step, cd and cd positive cells were generated and enriched during day of induction. we showed optimal time for harvesting flk + cells is day . of initial differentiation. following isolation of flk + progenitor cells they were further matured into functional epcs by vegf within days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p- . . - electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups ( mg, mg, mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < . ). amplitude of p wave and qrs complex did not change (p > . ). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < . ). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t- rna polymerase, possess toxicity to leukemia cells hl- and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in - nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents ( - times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkÒ hydrogel photopolymerized in a d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd + stem cell subpopulation. cd + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkÒ hydrogel. printing was done using a lm diameter needle, under bar pressure, and mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd + cells viability is maintained after days of cultured in bioinkÒ hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd + cells expressed endothelial markers such as cd , cd , cd , cd and vegfr . in conclusion d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe o ) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe o were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at and days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe o functionalized with carvone and limonene significantly stimulated the production of il- , il- and il- , while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe o nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p- . . - new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between . and . mm and epinephrine concentrations between . and . mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be . and . mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has À . v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < . ). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than % for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, %); a positively charged phospholipid, dotap ( %) and cholesterol ( %) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll- is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll- . the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll- immobilized was determined by elisa method. . % of ll- peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll- immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using t mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll- immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio nanotube-modified ti surfaces (ti/tio ) on the cross-talk between raw . macrophages (mf) and mc t -e preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw . and mc t -e cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw . cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il- and mcp- release was significantly increased at h, while after days only il- exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for h and days, whereas the presence of the mf for weeks significantly inhibited mineralization. on ti/tio surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p- . . - synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan , , b. tural , s. tural mardin artuklu university, mardin, turkey, dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b and due to high collagen synthesis. on the other hand the other isoform tgf-b , suppresses the dense collagen production caused by tgf-b and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b was studied with elisa. hepg cell line was used on mtt cell proliferation assay for both tgf-b loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be . %. particle size, zeta index and polydispersity index for this formulation were determined as . ae . nm, . mw and . , respectively. thickness of the prepared films were ae . nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand s from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a -well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (% , % . , % wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (% , % . and % wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p- . . - evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz , s. dogan , s. celikler kasimogullari department of molecular biology and genetics, balikesir university, balikesir, turkey, department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations ( , . and %) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki , , c. dupont-gillain universit e catholique de louvain, louvain-la-neuve, belgium, j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of . kda, isoelectric point ip at ph . ) and lysozyme (lys, molecular weight of . kda, ip~ ) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly ( -(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph . (pdmaema is neutral in such conditions) and were swollen at ph . (pdmaema is positively charged). protein adsorption was performed at different ph values ( . - . ) and salt concentrations ( . - . m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [ ] , [ ] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, -amino pyridine and , -diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly( -hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe t (dsm ) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa ), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl- -pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu + -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p- . . - bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonÒ, we employed argon plasma treatment with the powers of and w for - s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst- reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p- . . - graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations ( . - wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from to days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a d composite hydrogel scaffold ( d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin À sca + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of d-chs for tissue regeneration using the emerging bioprinting technologies p- . . - gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformÒ, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite % and tricalcium phosphate %, with - % pore volume and - lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformÒ. in order to improve cell adherence, ceraformÒ was covered with fibronectin. the cells were cultivated for -day period by osteogenic induction medium. days , , , and were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor (runx ), bone morphogenetic protein- (bmp- ), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day th, continued to increase in day . bmp- gene expression was increased in , , day compared to day. on the other hand, runx gene expression was increased only on days and . these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p- . . - on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu , i. negut , f. sima , e. axente , l. e. sima national institute for lasers, plasma and radiation physics, magurele, romania, institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom ( . %) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to show negatively charged particles. when n/p was , the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than . the migration of polyplexes with different n/p ratios ( - ) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was %. tuesday september : - : aging p- . . - novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd - and vd - - ) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc values to be lm for vd - , lm for vd - - and lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc doses of vd - and vd - - showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd - - exhibited - nm ic for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited -fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd - - needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was -fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic of nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap- and on the activity of nfat and ap- transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß is more prone to aggregation to form plaques and more toxic to neurons than aß . in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß / in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin , namely ps , were treated with a dose range of tbo ( - lm) or vehicle control for h. after treatment, aß / levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß and aß secreted into the cell culture media. significant reduction for both aß species was evident at lm (p < . ), lm (p < . ), and lm (p < . ) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g and g cell cycle checkpoints by p and p mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > genes, including nine genes encoding transporters. p mapk and p were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p- . . - smad specific e ubiquitin protein ligase (smurf ) and its potential effects on inhibitory transmission in aging adams , , , interdisciplinary program in neuroscience, bilkent university, ankara, turkey, national nanotechnology research center (unam), bilkent university, ankara, turkey, department of molecular biology and genetics, bilkent university, ankara, turkey, molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, department of psychology, bilkent university, ankara, turkey smad specific e ubiquitin protein ligase (smurf ) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf , via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf transcript is significantly higher in old zebrafish brains. thus, smurf may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb /+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf and gep levels remain stable in the aging brain (p = . , p = . ), and in the ache mutants gep levels are increased compared to the wildtype controls (p = . ). further analysis of the relationships between smurf and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we evaluated patients with ihd and healthy subjects aged years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr rs and tlr rs were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr (arg gln) and tlr (asp gly) were not significantly different between the groups (p ≥ . ). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over years old (p ≥ . ). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we plan to include other types of polymorphisms in tlr and tlr genes and increase the volume of patient cohort in our future studies. p- . . - evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up % and hemorrhagic strokes % of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke patients, with acute ischemic stroke and healthy controls we reenrolled in the study. blood samples were taken within st and th days, and after rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: . ) and thiol (p: . ) levels were significantly higher in first h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the rd month values of tos and nihss score (r = . , p = . ). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p- . . - amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from infertile women with pcos and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p promoter of xp bacteriophage in presence of p protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp , in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p protein, except of bacteriophage p promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p protein in the transcription initiation and identification of the role of the individual elements of p promoter of xp bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p protein. we have designed a set of promoter probes representing the combination of sequences of p -resistant p promoter and p sensitive t n promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream À position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing À promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region , a sigma-factor domain responsible for interaction with À promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p protein. promoter probe with up-element of p promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p promoter of bacteriophage xp can escape inhibition by p protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p- . . - distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study gerbils brains were used and divided into groups (n = ): : newborn animals ( day), - : , and days respectively, : animals aged years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within days (in cerebellumamounted to . ae . lg/ mg tissue; to - days increased to . ae . lg/ mg tissue, and began to grow again in older individuals aged years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at days p.d. ( . - . lg/ mg tissue), and unchanged for days. these results revealed that the most intensive development of astrocytes in the cerebellum to p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs . in order to investigate the impact of whirly on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly (hvwhy kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs at onset of drought-induced senescence in wild type (wt) and hvwhy kd lines, stress-responsive loading with histone modifications at gene regions of hvs ( regions in the promoter, one region around translation start site and regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy kd indicating that whirly protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h k ac all over the analyzed gene regions, correlating with a massive induction of hvs expression, while drought stress caused no substantial increase of h k ac in why kd plants. the results suggest that drought induced expression of hvs is under epigenetic control, and furthermore that why is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after week of inoculated with erlich cancernome ( cells/animal, i. p.) in months male balb/c mice treated at once with or without plant extract with bbv ( mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on % and % respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold ( . % versus . % in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at lg/ml concentration against escherichia coli vkpm-m , pseudomonas aeruginosa grp , bacillus subtilis wt-a , salmonella typhimurium mdc and staphylococcus aureus mdc , candida albicans wt- and candida guilliermondii hp- . statistical analysis was done using graphpad prism . . crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p- . . - effects of aluminum stress on photosystem-i apoprotein a gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl in different doses ( . , . , and mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ . ) decreased after hours treatment with mm and mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with . and . mm al for hours increased the gene expression level of psab by . % and . % respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, mm al exposure for hours could damage the electron transport in photosystem i. p- . . - nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + mg/kg ns; iii = sham + mg/kg (n-acetyl cysteine) nac; iv = g/ kg paracetamol; v = g/kg paracetamol + mg/kg nac; vi, vii and viii = g/kg paracetamol + , and mg/kg ns, respectively. paracetamol administration (oral) was carried out h after ns and nac administrations (oral), and all animals were sacrificed h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p- . . - the study of problems of preservation of the birches e. shadenova , , e. zhumabekov , m. sembekov , m. burchaeva institute of general genetic and cytology, almaty, laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p- . . - fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht- , t and upci-scc- cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a ( . m/z) and -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at th and th hours compared to control group. this study shows that holothurin a, dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support ( z ). p- . . - alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified genes encoding sr proteins in chinese cabbage (brassica rapa chiifu- ) from brassica database and analyzed their phylogenetic relationship. b. rapa has types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage ( and days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra and bra were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p- . . - characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of u mg(- ) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to . . the optimum ph and temperature was estimated as . and °c, respectively. the purified enzyme was stable from ph . - . and able to thermal stability up to °c. the k(m) and v(max) values were . mm and . micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf- , substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd + and hg +. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, , , -trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in , but the uniprot database contains less than reviewed sequences, which is approximately -fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above , are easily detectable in the total protein extracts. p- . . - transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in transcripts, database search with trinotate tool revealed transcripts with aox domains characterized in known alternative oxidases. blast analysis of these sequences with known aox proteins revealed three distinct aox genes (nalba-aox , nalba-aox and nalba-aox ). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox , nalba-aox are predicted as mitochondrial while nalba-aox is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr rk ). p- . . - comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak , v. koukalov a , z. medvedov a , c. martin , j. hradilov a , l. sp ıchal , b. brzobohat y mendel university in brno, brno, palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde ( -chloro- -( -methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of -day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p- . . - the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as °c and , respectively. the enzyme was stable at °c, but thermal stability was dramatically decreased at higher temperatures up to °c. kinetic parameters were also measured; vmax was . u/ml min and km was . mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp , lira and lines g- / _o, g- / _k were treated for and hours with lm alcl solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about million raw reads were obtained and conserved mirnas from families were identified. significant expression alterations in flax plants under al treatment were shown for mir and mir . expression level of mir was varied in similar way in resistant and susceptible to al genotypes: mir was up-regulated after hours of alcl exposure and down-regulated after hours. mir expression was increased after hours of alcl exposure and decreased after hours in susceptible to al flax genotypes (lira, g- / _o), while in resistant genotypes (tmp , g- / _k) mir level was decreased after both and hours of al treatment. in other plant species, mir and mir were identified as al-responsive. mir targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir targets mrna of tas protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir and mir in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir in susceptible and resistant to al genotypes. this work was financially supported by grant - - from the russian science foundation. p- . . - association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from year old poplar trees ( clones x replicates x ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as . ae . lg/ml, ae . lg/ml, and ae . lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p- . . - proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum ( min), radio-frequency ef ( - min) and cp ( - min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in days in comparison to days after seed treatment. the d dige analysis revealed differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin- were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p- . . - suppression of lipopolysaccharide-induced inflammatory responses in raw . macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw . macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il- concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw. . cell were found as . - lg/ml. studied cyclamen taxa inhibited tnf-a and il- release on lps stimulated-raw. . in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p- . . - in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < . ). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < . ). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p- . . - cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase- expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp- mrna expression of human sw colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp- mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag- s . data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with . lm ic value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at lm concentration when compared to control and lm (p < . ). cub alone showed inhibitory effects on mmp- mrna expression at lm and higher doses significantly (p < . ). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp- expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp- synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using % aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between mg/ml and . mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to % sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc is gram-positive cocci (mic: mg/ml). the mic values of other gram-positive bacteria s. aureus atcc , e. faecalis atcc , l. monocytogenes f and m. smegmatis cmm were detected as , , and mg/ml respectively. mic values of gram-negative bacilli were detected as > mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc , s. aureus atcc and e. faecalis atcc ). the strongest effect was observed on s. aureus atcc strain. in contrast, extract showed less effect on gram-negative bacilli. p- . . - selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a ) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p- . . - dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu , f. zihnioglu denizli vocational school of technical sciences, pamukkale university, denizli, department of biochemistry, faculty of science, aegean university, izmir, turkey type diabetes mellitus (t dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide- (glp- ) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec . . . ) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic values were also determined by nonlinear regression curve fit using graph pad prism . with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic . lg/ml while the standard (positive control) diprotin a displayed the ic value of . lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p- . . - dna recognition determinants of arabidopsis thaliana b transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b tfs constitute a large, plant-specific protein family (approx. % of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. amino acids) b dna binding domains. currently the b tfs are divided into four groups (lec -abi /val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b domain (arf , arf family) is available, thus the mechanism of site-specific dna recognition by the lec -abi /val and rav b domains remains poorly understood. based on the arf -dna structure (pdb ldx) we have built homology models of dna-bound b domains from a. thaliana abi (lec -abi /val family) and nga (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi / nga b domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b domains and in the future may help us predict the dna binding properties of currently uncharacterized b domains. p- . . - immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili , a. zeytunluoglu denizli health services vocational high school, pamukkale university, denizli, denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp- and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group : control, group : stzinduced diabetic, group : o. hypericifolium injected, group : stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups and by a single intraperitoneal injection of stz at a dose of mg/kg body weight. in groups and , rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of ml/kg body weight for consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine (k ) of tubulin. k is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk , e. marusich , r. chuprov-netochin , j. neskorodov , y. mishutkina , s. leonov life sciences center, moscow institute of physics and technology, dolgoprudny, center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature ( °c) and light conditions ( : hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point- . od) and hours later. to validate our system we used known cytokinins and auxins ( mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in - times range, compared with only times increase in control group. eight new chemicals ( lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to %. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an % yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl , pl , pl , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total sequence tagged site (sts) sequences from ncbi were aligned for pl , pl , pl , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p- . . - investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg cells line s. altundag , pamukkale university, denizli, istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg - cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut- ,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol Ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut- ,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut- ,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg - cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut- ) analyzed the expression levels . teucrum polium plant extract is applied in hepg - cells the glycolysis pathway genes (pk m glut- ) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg - cells showed antidiabetic effect. p- . . - purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara , s. sinan , z. ekmekci , y. turan university of balikesir faculty of veterinary, balikesir, university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec . . ) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with % ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose b-ltyrosine- -naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph - and thermal optima was determined using - °c temperature range. inhibitory effects was determined with mm substances. results: the enzyme was . -fold purified with yield of %. purified b-glucosidase from apricot seed was visualized about kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of . mm and . eu, respectively. the optimum ph and temperature were determined . and °c respectively. effects of cacl , kcl, nacl, mgcl , k so , na so , cuso , fecl , pb(ii) acetate, agno , zncl and glucose on purified enzyme activity were investigated. kcl, na so , pb(ii) acetat and cuso reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p- . . - a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un , , o. arslan balikesir university, science and technology application and research center, balikesir, department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec . . . ), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose- b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose- b-aniline p-amino benzoic acid affinity chromatography gel. % . yield and . fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of kda. the v max and k m of the purified enzyme were determined , u/ml min and . mm, respectively. p- . . - phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken , b. kocat€ urk , s. dogan , h. tuner balikesir university, balikesir, kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have , , kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of s promotor region in fragmented dnas was proved by pcr. escherichia coli dh a strain was transformed by fragmented dna samples according to cacl method. s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to - bp for bacterial transformation. fragmented dnas confirmed for carrying intact s promotor sequence. none of bacterial genomic dnas were found to be s-positive. according to the transformation results, absence of s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p- . . - preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker (tm- ) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p- . . - cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in , minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima - , tm- and ps- were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in % high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a °c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p- . . - association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of - nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm- (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p- . . - pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of chls, a homozygous cotton line tm- and a double haploid line pima - were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm- and pima - (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p- . . - pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during - . chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy ( n = - ), polyploidy ( n = - ) and the occurrence of additional b-chromosomes ( n = + - b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of s rdna and s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to . %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p- . . - angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu , i. arslan biomedical equipment technology, pamukkale university, denizli, turkey, denizli, biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the kda < peptides < kda in the ultrafiltration fractions displayed highest ace inhibition ( . ae . % at lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p- . . - modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg cell line d. irtem kartal , a. altay yuzuncuyil university, van, erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than different ailments ranging from aches to epilepsy. there are around species of salvia, of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm and catalase in hepg cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg cells for hours and hours incubation and methanol extract decrease catalase mrna expression for only hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing gigabases of bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcÀesiÀqtofÀms) to identify drought-related proteins. totally proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p- . . - isolation and characterization of a hexose converter from olive s. altunok , e. d€ undar department of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer , finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was kda with a pi of . . the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number o . key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p- . . - antioxidant potentials of propolis and its bioactive components, and their effects on cyp e gene expression in ht- adenocarcinoma cell line a. altay , d. irtem kartal erzincan € university, erzincan, y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p enzymes are involved in phase i xenobiotic and drug metabolism. cyp e is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with % ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht- human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp e in ht- cells were investigated bu using q-rt-pcr technique. ic values for dpph radicals scavenging activities of the extract was calculated as . mg/ml. tpc and tfc were determined as . mg gae/g extract and . mg qe/g extract, respectively. caffeic acid content of the extract was measured as . lg/g extract. ic values for xtt assay in hours incubation with the extract and caffeic acid were evaluated as . mg/ ml and . mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp e mrna expression with . and . fold, respectively in ht- human colorectal adenocarcinoma cells for hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp e expression and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - effects of histone h lysine inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h at lysine residue by specific methyltransferases suv h in humans and kyp/suvh in arabidopsis generates a'code' for the recruitment of hp proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv h has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h k methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf- breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt ( , -bis( -metoksi- -nitro- -sulfofenil)- h-tetrazolyum) test. in order to determine the dose of ic , plant extracts were applied as time and dose dependent in the range of - ug. in nd hour, the ic value is determined as ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase- , caspase- , caspase- , caspase- , bax, bcl- , fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while . and . -fold respectively increase in the gene expressions of caspase- and caspase- of dose group cells, . -fold decrease in the gene expressions of bcl- . no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p- . . - the effect of ferulic acid and -fluorouracil combination on apoptosis in pc- human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. -fluorouracil ( -fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; -hydroxy- -methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and -fu on apoptosis in pc- human prostate cancer cell line. the effects of -fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp , casp , casp , casp , bcl , bax, fas and cycs were investigated in four groups by qpcr. the ic doses of fa and -fu were found to be lm and lm for hours in pc- cells, respectively. in order to determine combination effect, pc- cells were treated with <ic doses ( lm fa and lm -fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl were observed in the fa group, compared with the control group cells. after the treatment with -fu, the expression of casp was significantly increased compared with the control group. furthermore, combination of fa and -fu significantly increased expression of casp , casp , fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and -fu affected expression of different genes in apoptosis in pc- cell line. p- . . - combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc- human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc- human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp , casp , casp , casp , bcl , bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic doses of fa and gemcitabine were found to be lm and lm for hours in pc- cells, respectively. for determination of combination effect, pc- cells were treated with <ic doses ( lm fa and lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp , casp , casp , fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc- human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf- , hela, a , and ht- cancer cell lines and nih- t cells. the viability of cells in lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic = . ae . mg/ml) and abts radical cation scavenging activity ( . ae . mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc ( . ae . mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species ( - mm / < . - mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain ( - mm/> mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p- . . - antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention ( ) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms ( ) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: ), aureus e. coli (atcc: ), and salmonella enterica (atcc: ) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with and milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be milligrams and milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with and milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p- . . - molecular analysis of serine/arginine rich sc splicing factor from olive b. bas , e. d€ undar depertmant of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc splicing factor (oesarsc sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc sf was a protein consisting of amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was kda with an isoelectric point (pi) of . . the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number o . keywords: olea europaea l., oesarsc sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p- . . - application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli , g. arabaci kto karatay university faculty of medicine, konya, faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h o and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with % (w/v) ammonium sulfate ((nh ) so ) and added t-butanol with the ratio of : . (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh ) so ( , , , , %) and tbutanol ratios ( : . , : , : . , : ) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were . , . % and . , . . therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of : . , the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh ) so ( , , , %) were . , . , . , % and . , . , . , . . the interfacial phase was not dissolved in % (nh ) so . at constant % (nh ) so , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio ( : . , : , : . , : ) were . , . , . , . % and . , . , . , . . finally, at optimum conditions (% (nh ) so , t-butanol : . ) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were . % and . . the results of gel electrophoresis showed that the molecular weight of enzyme was between - kda. the applications of tpp gave the maximum recovery of . % and . -fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for , and days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of distinct spots in brachypodium protein repertoire. a total of differentially expressed proteins (deps), with at least -fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p- . . - immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m e epitope from matrix m protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m e sequence. accumulation level of m e-ala recombinant coat protein was significantly higher than m e-cys/ ser (ratio : ). tmv-m e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves ( d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm ae . cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw . macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase- (cox- ), tumor necrosis factor-a (tnf-a) and interleukin- (il- ) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox- and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il- in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p- . . - the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf- breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf- breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase , bax and p and antiapoptotic genes; bcl- and pi expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf- cell was only observed in and lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf- cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf- breast cancer cell lines. p- . . - reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are , bp (mon- kalr) and , bp (mon- volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and ribosomal rna genes. and trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf , ycf , accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant - - and rfbr grant - - (mon- volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), -methyl-z-tetradecen- -ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, -octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p- . . - role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut , deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature ( k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p- . . - the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature ( / °c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor , -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for days at optimal conditions. d was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p- . . - reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m ) was investigated. three weeks old tomato grown at optimal conditions ( / °c day/ night temperature and light intensity lmol/m /s) plants were exposed for and days to elevated temperature at / °c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf- cells. mcf- cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively , and ng/ml) . then different doses of silymarin ( , , , lm), curcumin ( , , , lm) and propolis ( . , . , . , . mg/ml) were added. after , , and hours incubation periods different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf- cells for , , and hours (biovision cell proliferation kit) all experiments were carried out different days, at least times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < . between and hours. it was found that there were not seen much cells at and hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was . mg/ml, silymarin and curcumin were lm. also, we evaluated the effects of on proliferation rate the mcf- cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf- cells in hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p- . . - investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; -hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, h-nmr and melting point analyses. the theoretical uv-vis spectra and d conformations of the isolated compounds were determined by using the gaussian software with hf theory at the b lyp/ - g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [ , ] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than hydroxyphysodic acid, and the toxic activities were found sufficient as much as % at the highest doses of the substances; lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately % by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [ ] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. ( ) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research : - . [ ] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. ( ) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian software with hf theory at the b lyp/ - g level was also performed to determine the theoretical uv-vis spectrum and d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [ , ] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as % at the highest dose of the substance; lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as , and -lm, and % of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size ( - nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n is %, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score or higher, p < . ) peptide mass fingerprint of cwr -de electrophoresis revealed differences in protein composition between cwr and n . information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p- . . - mir and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir s and their target by q-rt-pcr. we mined mir genes sharing sequence similarity and grf targets. the expression analyses of mir s and grf are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir s and grf in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate , and to detect expression through real-time pcr were designed using primer . amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as . . the molecular weight of the protein was calculated as kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p- . . - cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht- , t and upci-scc- cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for hours. the cell index data were compared with the control group. ic values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a ( . m/z), -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). the fraction d was the most effective on all cell lines with ic value of . ae . mg/l, . ae . mg/l and . ae . mg/l for ht- , upci-scc- and t , respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p- . . - effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol ( %) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose ( mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically % (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp- , mmp- by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of rd, th, and th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the rd and th days. the malondialdehyde levels were significantly lower in the treatment group. mmp gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the th day while it was reduced in th day. mmp gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in th, and th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp and mmp regulation. p- . . - isolation and molecular molecular characterization of vps /vam from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps /vam promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps /vam , however, have been reported. the aim of this study is to report information of olive vps /vam including expression pattern and biochemical characterization. vps /vam was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega . cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn d. the blast and bioedit analyses revealed vps /vam had base pairs coding amino acids with a molecular weight of . kda, and pi of . . the at/gc ratio was very high ( . ) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the d structure of the protein was similar to its previously reported homologs. p- . . - isolation and characterization of the ribosomal l protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l protein is larger than the lsu of ribosome and contains domains as -layered alpha/beta domain and -layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l (rl ) gene has been studied in many plants, reports on olive rl (oerl ) are very rare. this study presents molecular characterization of rl gene from olive. oerl was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional ( d) structure analysis, and phylogenetic analysis were conducted using signalp . , thhmm, i-tasser/cn d and mega , respectively. oel was found to have an open reading frame of base pairs coding amino acids that constitutes a molecular weight of . kda and a high pi of . . lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a . at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl . likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl with respect to expression pattern and biochemical function continues. p- . . - isolation and characterization of an olive splicing factor b subunit m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor b subunit (sf b ) functions in the regulation of translation and gene expression. sf b forms u small nuclear ribonucleoprotein complex (u snrnp). splicing factors a and b binds pre-mrna at the site of the intron branching point. this binding joins u snrnp to pre-mrna. although sf b from various plants have been widely studied, no studies on olive sf b (oesf b ) have been reported. this study reports information on various aspects of oesf b . oesf b was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn d were used to generate the calculated d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb . the orf contained nucleotides coding amino acids that produce a . kda peptide with a pi of . . alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf b is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf b could be synthesizing in mitochondria. the predicted d structure of oesf b was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p- . . - kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of - %. they are grouped into a ( - kda), b ( kda), c ( kda) and d ( kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of % naci concentration, and second, kafirins were obtained using tertiary butanol ( %) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as , , , and %. of the total proteins, % was identified as a, % b, % c, % d, and % non-prolamines. non-prolamin group of proteins were visualised as different bands ranging from . to kda. c and b group of proteins were only viewed when treated with reducing agents as -me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for . , . , . , . and . kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p- . . - untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu , university of agricultural sciences and veterinary medicine, cluj-napoca, center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol ( ) and chloroform/methanol ( ) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction ( ) , ranging from . to . mg/ g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. / , pncdi . glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys ko) and liver glycogen synthase (gys ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos , nos (genes encoded no producers) and pde a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos which lost rhythm and increased and delayed amplitude of nos expression. further, pinealectomy initiated cyclic transcription of gucy b and noncyclic transcription of gucy a (genes encoded cgmp producers) and increased mesor and amplitude of pde transcription. the transcription of prkg , the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi or other investigated genes (coxi , nrf , nrf a, pgc a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s fraction mix was used. cells were treated with . , and lg/ml oseltamivir, the tamiflu capsule ingradient, for or hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ . ) in the absence of s mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: . - mm of glucose, . - mm of urea, and . - mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p- . . - ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide ( amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with mm dimensions. ovary adenocarcinoma cells (efo- and onco-dg- ) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak- s ). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide ( n) contains biotin on '-and '-ends, and sterptavidin in molar ratio : . this complex represents a structure with equivalent electrophoretic mobility of bp dna and preferred "valency" of streptavidin is . this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved - times highter sensitivity than elisa. to reduce the matrix effect we used - fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only mkl of serum sample). antibodies to the disaccharide galb - glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ . ) for health donor sera (n = ; . ae . pg/ml) compared with sera from patients with breast cancer (n = ; . ae . pg/ml). sensitivity of igm anti-le c antibodies detection was pg/sample ( mkl) ie . molecules. thus for the immuno-pcr detection of antibodies the - tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (# - - ) and by russian federation president scholarships donated to d. yu. riazantsev (# sp . . bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma , as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = , the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge ( uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = , when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle- cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, u of penicillinstreptomycin and maintained in a humidified incubator at °c and a % co atmosphere. cell viability was measured using the colorimetric -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in well plates and incubated with ll/ml dmso, - lg/ml tm, . - mm tudca, or lg/ml tm + . - mm tudca for - hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for hours at °c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or mm tudca was cytotoxic. at and hours incubations mm tudca and lg/ml tm + mm tudca were significantly cytotoxic compared to control, dmso and mm tudca groups. treatment of cells with . mm tudca hours before administrating ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at mm concentration when treated with tm. therefore . mm of tudca, administered hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; s ). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a (lp-pla ) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla mass, activity, index) related genetic variations (pla g ) increase the risk for developing future cardiovascular disease in women with pe. a group of preeclamptic patients and normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla mass level. lp-pla activity were determined by kinetic method. plag snp genotyping performed by using the sequenom massarray iplex. the rs tt genotype had a higher lp-pla index (p = . ) for early onset preeclampsia, cc genotype had a higher lp-pla mass and lp-pla index for late onset preeclampsia. no difference were found for control. the rs gg genotype had higher lp-pla mass and index for late onset preeclampsia (p = . , p = . respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs and rs snps in early, late onset preeclampsia and control group. only lp-pla mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla mass, index and rs , rs snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi- mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding -hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi- mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found ae , ae , and ae hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi- mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi- mutation. it could accurately discriminate between normal and ivsi- mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il- b and il- and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in and hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio . to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il- b, il- and tnf-a genes were increased by the exposure to nanoparticles of , and lg/ml for - hours. tio nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio . oxidative stress was elevated by the treatment with tio nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il- b, il- . background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor- (vegfr ) and a v b -integrin on d and d cultures (spheroids) of ea.hy . methods: the ea.hy cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf ( ng/ml) or vegf a ( ng/ml) for hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr and a v b -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b -integrin and vegfr- increased on the d culture compared to d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b -integrin in the d culture of the ea.hy cell line activated with tnf. also increased expression of vegfr in the d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b -integrin and vegfr on the surface of d and d cultures as well as antibodies with fluorescent label. conclusions: d cultures of the ea.hy cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the d structure of tnf and ii) design of more potent and less toxic spd analogues. b. drug synthesis. a series of spd analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded %. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd derivatives have better dissociation constants than spd . the effect of exercises on serum bmp- levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp- is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp- to sustain the molecular approaches for treatments. a total of patients were included in this study. the groups are formed as follows: group , oa patients before the exercise; group , oa patients after the exercise; group , oa patients before the isokinetic exercise; group , oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after weeks of the exercise programme. self reported severity of pain was measured using the mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp- levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp- levels were significantly decreased in group compared to group ( . ae . ; . ae . respectively, p < . ) and in group compared to group ( . ae . ; . ae . respectively, p < . ). there is not any statistically differences between group and group (p > . ). as a conclusion, the decreased serum levels of bmp- may be suggested as a biochemical marker for oa patients during exercise programmes. p- . . - tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase- ), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < . ). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase- ) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < . ). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < . ). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p- . . - short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin ( mg/kg, i.p.). rats were divided into group i: control, ii: control/ mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/ mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il- b, il- (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il- b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p- . . - cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb- and mcf- breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb- and mcf- breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb- wt and mda-mb- gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as lm (for mcf- ) and lm (for mda-mb- ) decreased cell proliferation and induced apoptosis through causing jak dephoshorylation, stat , , dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for hours. lm curcumin-induced apoptotic cell death via dephosphorylating jak at tyr / residues and decreased phospho-stat , level in both breast cancer cell lines. although curcumin dephosphorylated stat in both mda-mb- and mcf- wt cells, no significant effect has been observed in mda-mb- gh+ and mcf- gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf- and mda-mb- breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat , a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb- breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb- breast cancer cell lines. we found that lm curcumin-decreased in apoptotic cell death through inactivity at jak which led to dimerization of stat , stat , stat . concomitantly, curcumin affected stat regulating socs proteins in mda-mb- breast cancer cell line. in addition, we demonstrated that lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb- breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il and il cytokines as main mediators of inflammation progression, correlated in keratinocytes with il a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam- and vcam- adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il and il in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam- over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into groups (n = ). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + mg/kg salvia l.group; group iv: i/r + mg/kg salvia l. group; group v: i/r + mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for days was given single dose as a gavage. minutes ischemia, minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)- and interleukin ib (il- b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il- and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il- , and il- b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group mg/kg salvia l. and mg/kg rosmarinic acid especially mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but mg/kg salvia l. extract were more effective than mg/kg salvia l. extract and used as synthetic mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a (spla ) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla , lpl and evaluate changes in circulating protein levels of angiopoietin-like protein (angptl ), angptl , cyclooxygenase (cox) and prostaglandin e (pge ). serum from control subjects and acne vulgaris patients with moderate and severe disease was evaluated for levels of spla , cox, pge , lpl, angptl and angptl . disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au clinical chemistry and unicel dxi immunoassay systems). serum levels of spla and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge , angptl and angptl levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla , lpl, angptl and angptl levels in acne vulgaris. future studies are aimed to understand the regulation of spla and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; # s ). p- . . - -ohdg and hogg levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. -hydroxy- -deoxyguanosine ( -ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human -oxoguanine dna glycosylase (hogg ) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after months of isotretinoin treatment by measuring -ohdg and hogg levels. the current study includes acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. -ohdg and hogg levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg and -ohdg, respectively.both -ohdg (p as a conclusion, isotretinoin increases dna damage and high serum -ohdg and hogg levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor (spi- ) in the stat -mediated manner in pinealocytes. however, the precise function of stat in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi- in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi- were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat transfection. the successful transfection and subsequent decline in stat expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi- were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat on those immunomodulators is important from the clinical point of view because inhibitors of stat are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles ( nm) using human lung mrc- fibroblast as model. cells were exposed to . lg/ml silica nanoparticles for , and hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il- b, il- , il- , tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp- , mmp- , mmp- ) and tissue inhibitor of metalloproteinase- (timp- ) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il- , il- , il- and tnf-a as well as of mmp- and timp- , was up-regulated whereas those of mmp- , mmp- was down-regulated after and hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase- in mrc-cells after and hours. p- . . - association of fto gene variant (rs ) with markers of t dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type diabetes (t d) encodes a -oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included patients with t d and prediabetes and healthy controls both sexes, aged from up to years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs with increased risk of t d (or = . , % ci . - . , p = . ). a allele was significantly associated with higher values of hba c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb ), glial fibrillary acidic protein (gfap), c-x-c motif chemokine and (cxcl , cxcl ), matrix metalloproteinase (mmp ), chemokine (c-c) motif (ccl ) and tyrosine-protein phosphatase non-receptor type (ptpn ) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak s and pamukkale university paubap fbe . p- . . - apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek , s. savranoglu , a. r. t€ ufekc ßi , f. g€ ul , i. demirtas , t. boyunegmez t€ umer graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw . macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into subfractions by column chromatography. e was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw . cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e , and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at lm decreased no production and %-ic : and lm-by inhibiting inos gene expression and % as well as protein expression and %, respectively (p < . ). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id: t ) support this work. the role of tip in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il- (interleukin ) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip conditional knockout mice, so we thought that tip may have a direct or indirect role on inflammation mechanism. tip (tat interacting protein, kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il- were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il- was increased in lps-induced inflammation group (p < . , p < . ) and liver-specific tip conditional knockout group mouse liver (p < . , p < . ). our initial data suggest that tip may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: z ). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose -phosphate dehydrogenase (g pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il ), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il was significantly increased in lps treated group. while the level of mda and h o was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: z ). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < . was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = . ) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of patients which have been diagnosed with cchf. matched for gender, healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p- . . - inhibitors of mdm ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm is an e ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p / mdm interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm and p might be playing a substantial role in autoimmune processes. we used a small molecule p /mdm inhibitor nutlin- a to test the effect of p activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p /mdm interaction by nutlin- a alters the p levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p /mdm interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant - - mol_a_dk. can ykl- be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc . and netphos . program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into groups: deletion/deletion (dd) genotype (group , n = ), insertion/deletion (id), insertion/insertion (ii) genotypes (group , n = ). blood pressure and pulse measurements were performed in all patients within minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes ( . ae . and, . ae . , p < . ). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone ( mg/kg b.w.) with and without iu of d (for days). the levels of rank, rankl, opg, a-hydroxylase (cyp b ) in bm were determined by western blotting. vitamin d receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. ohd content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp b , which is responsible for , (oh) d synthesis, in bm and ohd content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d and rank/ rankl/opg pathway. vitamin d co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d treatment. p- . . - heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor encoded by the hsf gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa a promoter (hsp promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp inhibitor, mg as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks and tks , the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks binding partners including adaptor proteins itsn , itsn , crk and grb , kinase src, amph , bin , plcg and also another member of the tks family -tks . it may indicate the possible role of tks in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph and bin , as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks isoform -tks -beta. we suggested that an active state of tks is regulated via intramolecular interactions between its proline-rich motifs and own sh -domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr and wire interact with sh domains of itsns in complex with actin. p- . . - correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in % methanol. after sample preparation obtained solutions were analyzed by hplc-agilent with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp within insulators still remains to be elucidated. recently we described an interaction between cp btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate aa fragment within ctcf c-terminal domain sufficient for interaction with cp btb, but deletions of flanking regions also lead to the loss of interaction with cp in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp insulator protein at . a resolution. overall structure is similar to other btb-domains. cp btb-domain has peptide-binding groove similar to that previously found in bcl btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project № - - ). p- . . - comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f hybrids of romanov sheep with . % of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by . % lower ( . ae . %, p < . ), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by . % and . % higher ( . ae . and . ae . (p < . ), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from . % to . %) and for oleic, linoleic, arachidonic acids (increased from . %, . %, . % to . %, . %, . %, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was . ae . , . ae . and . ae . %, that was . % lower (p < . ), and . % and . % higher (p < . ) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. - - . foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd (nbd ) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature > °c). the genome of nbd is $ kb long, and has a total of probable protein-encoding genes as well as gene for trna ser . the genome analysis revealed that nbd orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, are similar to proteins from phages that infect different members of enterobacteriaceae, while nbd orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, nbd orfs were given a putative functional annotation, including genes coding for morphogenesis-related proteins, as well as associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit- / ) from the research council of lithuania. p- . . - the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas , j. williamson , v. schw€ ammle , s. douthwaite universidad de cundinamarca, cundinamarca, colombia, university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as , as -rlma::aph and b) were grown aerobically in nutrient rich lb media at °c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be > -fold higher the wild-type b-strain of e. coli than for the hypersusceptible as strains ( lg/ml). whole genome comparison of the b and as strains were characterized by an increase in proteasome components ( proteins), chaperones ( ), error-prone dna polymerase components ( ), ribosomal hibernation factors ( ), heat shock response ( ), electron transport coupled proton transport ( ), pentose phosphate pathway ( ), flagellar assembly ( ), oxidative phosphorylation ( ) and tca cycle ( ). whereas ribosomal proteins ( ), aminoacyl-trna synthetases ( ), rnases ( ), abc transporters ( ), mismatch repair ( ) and sec secretion pathway ( ) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak /stat signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e g), asparagine to aspartic acid (n d), threonine to alanin (t- a) missense mutations, alanine to serine (a s) substitution, tryptophan to stop codon (w- x), gaaa insertion in intron of gh-n gene and both intron (+ c) and deletion of . amino acid of gh protein phenylalanine (f del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc . vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w- x, e g, f del, a s and n d mutations suppresses gh signaling via acting on either jak dephosphorylation or stat downregulation, t- a, gaaa insertion and deletion of + c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek cell lines. as a result, w- x, e g, f del, a s and n d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no tyr)-containing proteins were immunoprecipitated from serum with a no tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no tyr antibody. among the numerous immunoreactive bands observed in disease patients, the kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha- (iii) chain (co a _hu-man) with mascot algorithm analysis giving a score of (p < . ). collagen alpha- (iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no tyr modifications in co a relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs in gckr gene and rs in galnt gene with parameters of t d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved patients with t d and healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt polymorphism (rs ), while polymorphism in gckr (rs ) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs variant with waist circumference (p = . ) and fasting glucose levels (p = . ) in the control group. no such association was demonstrated for rs galnt gene. in the group of diabetic patients, significant association of gckr rs variant with levels of bilirubin (p = . ) and rs galnt variant with hba c (p = . ) and triglyceride levels (p = . ) was also demonstrated. our results suggest an association of variations of gckr and galnt genes with specific markers of t d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp binding site of the collagen type a (col a ) gene is associated with low bmd. we examined the distribution of col a gene polymorphism in young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤ . sd and controls was healthy women ( - years). mean age ( . ae . ) and ( . ae . ) respectively. the bmd, as g/cm , was measured in the hip and the lumbar spine (l -l ) with (dexa). dna was isolated from blood. col a gene was analysed with genomica clinical array system. the x test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was À . ae . . mean bmd (as g/cm ) was . ae . , and ( . ae . ) genotype distribution were ( %) ss, (% )ss, (% )ss for patients, and ( )ss, ( )ss, ( )ss for control . patients had (% )s allele, ( %) s allele, controls had ( %)s allele, ( %)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > . . genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(% ) than controls (ss % ) p < . . this study shows that high prevalences of the ss genotype at the col a locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col a and col a mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col a gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly ( - %) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep b at - and hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep b cells in three different concentration, namely - and . lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was lm. proapoptotic genes, bax and bad, and autophagy genes, beclin- and lc , were upregulated in hep b cells after hours treatment in contrast, antiapoptotic gene, bcl- , expression level was decreased for hep b cells after hours. p- . . - association of irs genetic variation with type diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate- (irs ) encodes the irs protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs single nucleotide polymorphisms (snps) were associated with type diabetes mellitus (t d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs (g>a), rs (t>c) and rs (a>t) with t d risk and related traits. our study involved t d patients and healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss . our results demonstrated a significant difference in frequency of rs (p < . ) and rs (p = . ) snps between t d patients and control subjects. interestingly, here we showed a significant association of irs rs risk t allele with increased insulin levels (p < . ) and homa-ir (p < . ) in t d patients. similarly, rs variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = . ) and homa-ir (p = . ). no such association was demonstrated for rs . however, this irs variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = . ) and decreased hdl levels. our results suggest that irs variants are associated with t d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t d biomarkers. the adra a gene encodes alpha- a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra a polymorphisms (rs a>g and rs g>t) with type diabetes (t d) and its related traits. in this study we have included t d patients and healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics software. our data showed that frequencies of both, rs and rs , variants were not significantly different between t d and control subjects. however, rs risk a allele appear to increase insulin levels (p = . ) and homa-ir index (p = . ). furthermore, this variant also seems to affect vldl levels (p = . ) and waist circumference (p = . ) in diabetic patients. the genotype analysis of rs variant demonstrated that risk g allele decreased hdl (p = . ) and increased ldl levels (p = . ), as well as affected the waist circumference (p = . ) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs a / rs g with higher homa-ir index. here we demonstrated that although both, rs and rs , adra a polymorphisms were not associated with t d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein (sip ), also known as zeb is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip mediate its binding to ebox sequences in the genome. however, there are only limited data about sip target genes. by using a home-made anti-sip monoclonal antibody, clone e , we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu , plc/prf/ and sk-hep- and found receptor tyrosine kinase-like orphan receptor (ror ) as one of the targets. sip dnabinding consensus motif cacctg was found at + kb, + kb and + kb from ror transcription start site. chip experiments validated sip binding to all consensus motifs in the ror gene region. interestingly, the strongest enrichment was at + kb suggesting that long-range interactions play an important role in the regulation of ror by sip . sip knockdown by shrna in high-sip expressing snu cells resulted in the repression of ror expression. ror is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror -positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip and ror act in similar pathophysiological processes. our finding that ror is regulated by sip at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein (sip ), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip in hcc are yet to be identified. we conducted a chip-seq study in high-sip expressing hcc cell line snu by using a home-made anti-sip antibody, clone e . among annotated genes, we selected six for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip dna-binding motif cacctg was found at - kb from transcription start site of six gene. chip qpcr experiment validated sip binding to this region with , fold enrichment. compared to healthy liver, six transcripts were upregulated in of hcc cell lines included in this study. knockdown of sip by shrna in snu cells caused upregulation of six . immunohistochemistry studies in hcc tissue arrays showed increased expression of six in tumors and inverse association with sip expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip and six in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by -hydroxy vitamin d ( (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were healthy women in age - years. the bmd was measured as a t score in the lumbar spine (l -l ) with dexa. all subjects had normal t score between (- . to . ) sd. vitd was measured by lc- -at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was ( . ae . ) lg/l, mean plasma ipth level was ( . ae . ) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = À . ). genotype distribution and allele frequency of subjects were as follows: ( %) ff), ( %) ff, ( %) ff genotype in vdrf -fok gene, ( %) bb, ( %) bb, ( %) bb in vdrb-bsmi gene. allele frequencies were f: %, f: %; b: %, b: %. when fok and bsmi were combined, %(ff-bb) and % (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > . ). but foki was not (p = ). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women ( %). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p- . . - determination of hb a c values in beta thalassemia f. g€ uzelg€ ul , g. s. seydel , a. e. yalin , e. s€ onmez , k. aksoy c ß ukurova university, adana, nigde university, nigde, mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected ml of whole blood k edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a c values were measured using the agilent hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs - /ivs - , one ivs - /ivs - , one ivs - /ivs - , two ivs - /ivs - , two ivs - /ivs - , one fsc /fsc , one fsc /fsc , two - /- , two cd /cd , one cd - /cd - , one cd - /cd - , two cd /cd -g/ cd /cd -g, two ivs - / ivs - , one ivs - / ivs - , one ivs - /fsc , one ivs - /cd , one ivs - /cd , one ivs - /cd - , one ivs - /ivs - , one ivs - / ivs - , one ivs - / ivs - , one ivs - /cd and one fsc /cd . cases were classified as diabetic ( ), prediabetic ( ) and non-diabetic ( ) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in samples from . we observed positive correlation between expression of aurc and braf (rs = . , p = . ). discussion and conclusion: previous investigation showed, that for normal prostate tissue % of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to . . based on its genome sequence, metabolic pathways for proteases, amylases, cellulases, lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of . to . makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, . and ph . . for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p- . . - biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u mg and u as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk . mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc -a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk levels in u mg were reduced by %. due to mts results, ≤ mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk level in u mg and u cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately % calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d ; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p- . . - apj receptor a c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than mm to allow rapid penetration of the fixative put in % buffered formalin for hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = ) and sinus rhythm (n = ). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu- plus platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among . transcripts representing . well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ . ) with a p-value ≤ . . results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr - and environmental pigment-producing s. marcescens strain sm . bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm . cluster has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only % of identity to xantholipin biosynthetic gene cluster. clusters and of nrps genes of s. marcescens sm did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr - contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p- . . - the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to - months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: ) active animals ) hibernated animals and ) animals immobilized for weeks in laboratory, were examined. rna libraries have been sequenced using hiseq illumina platform. coupled with genome dna sequencing provided x coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e -ubiquitin ligases murf and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. - - . in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain , and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis wild-type strain forms biofilm with its maximum on th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of . - . . the temperature optimum is in the range from °c to °c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average % lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain - , - and °c - °c respectively. the crataegus genus which is a member of rosaceae family, has approximately species worldwide and species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: , diphenyl- -picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities ( . ae . % and . ae . %, respectively), at lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values ( . , . , . ) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por gene expression, encoding vdac should increase in the stress conditions. the promoter of the por gene was used to construct some new expression systems based on yl w . a new genetic construct bearing a reporter bgalactosidase gene under control of the por promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h o and methyl viologen treated cells b-galactosidase activity increased . - . -fold reaching its maximum in the cells, grown at ph of . . thus, we demonstrated high inducibility of the por promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no - - mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine- -carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn , respectively. gene deletion was accomplished through double crossover. a cassette containing the~ bp ' and ' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn d strains isolated the achad ngn d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn d, shows azc toxicity, suggesting that ngn is a true orthologue of mpr , able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c h zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at . a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant ( . . . ). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t polymerase primer for cdna synthesis. microarray represents probes for orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps , which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane- -monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose- -phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example , -dienoyl-coa reductase, -ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of patients; healthy pregnant women (control group) and pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba c levels and homa-ir were significantly higher in gdm group compared to the control group (p = . , p = . , p = . , p = . , respectively). serum adiponectin levels were significantly lower (p < . ); whereas serum resistin levels were significantly higher (p = . ) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic , ic , amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p- . . - functional polymorphisms of ephx in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec . . . ) is encoded by ephx and catalyses the degradation of endogenous fatty acid epoxides generated by cyp epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx gene that affect the seh activity may alter the circulating levels of eets. k r and r q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k r and r q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k r polymorphism were . % wild type (aa) and . % polymorphic genotype (ag+gg) and for r q polymorphism . % wild type and . % polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a g, mtrr a g and mthfr c t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from pregnant women. the main group consisted of women (n = ) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = ) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was . ae . years compared with control of the age . ae . . the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a g (or = . , ci % = . - . ; v = . , p < , ). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr rk project number). p- . . - a study on the association between rs polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is . % above the age in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs g/c single nucleotide polymorphism (snp) is found at position À , in promoter region. the presence of a c allele for rs is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs polymorphism of the ctgf gene. study population consisted of patients with pes and controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs polymorphic allele g was . in patients, and . in controls ( . , p = . ). distribution of genotypes was gg: . %, gc: . % and cc: . % among patients, while gg: %, gc: . % and cc: . % (or = . , p = . ) in controls. statistical analysis showed that there is no significant relationship between ctgf rs snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs snp and pes. this work did not point out a role for ctgf rs in the risk for pes. a significant relationship might be found when the study population is enlarged. p- . . - evaluation of rs single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs snp of clu gene have a role in the development of pes. study population consisted of patients with pes and controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was . and that of controls was . ( . , p = . ). the distribution of genotypes was cc: . %, tc: . % and tt: . % among patients while cc: . %, tc: . % and tt: . % ( . , p = . ) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp and to evaluate the difference at mrna and protein expression levels of the tp target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp mutation. to show the tp mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of hpv negative hnscc patients. employing different databases we constructed the tp regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh was significantly associated with tp mutational state. ezh is a member of the polycomb group protein enhancer zeste homolog which is known to be directly repressed by tp and indirectly by the activation of mir- a and mir- b. we found a significant up-regulation of ezh that depend from tp mutation. it is important to understand the difference in mrna and protein expression of tp regulatory network that could depend from its mutational state. this finding suggest that ezh might be a potential therapeutic target for hnscc. p- . . - next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of all patients. for this, we used sanger sequencing of the products of multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after days of treatment. for this purpose, we performed ngs of libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to per , and , cells) for a calibration curve generation. using this approach, we analyzed all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found leukemic cell per , analyzed cells. znf is considered as a transcriptional target for p and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs , rs ) of znf were investigated in kazakh population in this study. the present case-control study was carried out with participation of kazakh females with bc and cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/À), progesterone receptor (pr+/À) and human epidermal growth factor receptor (her +/À) status were estimated. pearson p-value, odds ratio, % confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs locus in znf between the patients and control groups (p = . for allele; p = . for genotype). moreover, significant association with bc was revealed for rs after dividing patients according to er+/ À, pr+/À and her +/À status of the tumor. the g allele was associated with er+ (p = . , or = . , %ci: . - . ), pr+ (p = . , or = . , %ci: . - . ) and gg genotype with her -bc carriers (p = . , or = . , %ci: . - . ). also, g allele can be considered as a risk factors in er+/ pr+/her -luminal type of tumor (p = . , or = . , % ci: . - . ). our findings correlate with the data of several gwas where the association of the rs polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs as a marker of bc risk with predictive value, restricted to er, pr and her status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr (rs ), tox (rs ), map k (rs ), lsp (rs ) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included bc patients and healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with % confidence intervals ( % ci) were applied to data analysis. according to obtained results combinations of allele c of tox rs and a of map k rs (p = . , or = . ), also allele c of tox rs and c of lsp rs (p = . , or = . ) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox rs contribute significantly to bc risk with p-value = . , or = . . on the contrary, tt genotype of tox rs with p = . , or = . and its combination with allele t of lsp rs with p = . , or = . determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp rs and a of map k rs was found in kazakh population (p = . , or = . ). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf- ) and mouse fibroblast (l ) cell lines. cells were studied four different concentrations ( , ; ; ; lm). xtt ( , -bis-( -methoxy- -nitro- -sulfophenyl)- h-tetrazolium- -carboxanilide) protocol was applied after and hours. in our study % fetal bovine serum (fbs), % l-glutamine, iu/ml penicillin and mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in % humidity and % co incubator at °c. anti-cancer activity of synthesized complexes were determined on mcf- and l . in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control ( -fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on gem/cistreated and pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm rs , ercc rs , ercc rs , and xrcc rs . values ranged between and . ; cutoff value of . had sensitivity of . and specificity of . . patients with higher score had shorter progression-free and overall survival (p < . ). in the validation group, positive predictive value was . and negative predictive value was . . model predicting outcome of pmx/cis chemotherapy included crp level, mthfd rs , and abcc rs with scores ranging between and . . cutoff value of . had sensitivity of . and specificity of . . patients with higher score had lower probability of good response and shorter progression-free survival (p < . ). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p- . . - evaluation of anti-diabetic potential of circiliol and circilineol using caco cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt (sodium-glucose transporter- ) and glut - and glucose transport. neither ag activity nor sglt activity was increased with either circiliol or circilineol treatment in caco cells compared to positive control. similarly, neither the activity nor the expression level of glut *- was increased in caco cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak z and paubap fbe the purpose of this study was to characterize and assess the impact of a novel magnetite (fe o ) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe o nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe o @e) were characterized by ir, sem, tga and hr tem. one laboratory and p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics ( , , and hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges - nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe o @e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p- . . - specimen processing and handling for plasma ammonia measurement b. sarac ßligil , , s. abus ßo glu , f. aky€ urek , b. € ozt€ urk selc ßuk medical school, konya, biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at °c) within minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at °c) within minutes and analyse refrigerated - °c hours (a , b ) and hours (a , b ). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results ( . versus . lg/dl, p < . ). in all protocols including a , a , b , b also cause an elevation in plasma ammonia results ( . , . , . and . ; p = . , p < . , p < . , p < . , respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb (gene encoding b-catenin; mostly mutations in exon ) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with ll of extraction buffer at °c over night. the tubes were boiled for min to inactivate the proteinase k. ctnnb exon was amplified by pcr. sscp is used to observe any difference between the groups. genomic dna was isolated as described above. ll of each pcr products mixed with ll denaturating buffer and were denatured by heating at °c for minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on % acrylamide/ bis gel in . tbe buffer for . hours at v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between and % in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb exon between the tumor group and normal groups. p- . . - theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol , b. uc ßar koc ß university, istanbul, american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe , tyr -octreotate, tate) has been labelled with the positron emitter ga and the beta and gama emitter lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ ga] dota -tyr -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to mm. from mm. in d.-after fifth operation). due to its high sstr selectivity, [ lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p- . . - cardiovascular diseases can be treated by using 'tetr-odd-vp ' and 'hre' hypoxia inducible systems a. celik , t. kaya , s. cigdem , m. g€ und€ uz department of medical genetic, turgut ozal university, ankara, faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif- a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif- a protein levels increase under hypoxic conditions as a result of decreased o -dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif a between and in front of tetr dna binding domain and vp transactivation domain, so that tetr-odd-vp or odd-tetr-vp could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp and odd-tetr-vp constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p- . . - osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a d cell/ scaffold bioconstruct to a continuous magnetic field during weeks of osteogenic induction. in this view, mc t -e murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc t -e cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc t -e cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p- . . - impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = , . ae . yr, . ae . kg) and untrained (n = , . ae . yr, . ae . kg) subjects performed three soccer matches ( min) in field ( m versus m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < . was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < . ). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < . ) in trained ( . ae . u/l versus . ae . u/l, % (m) . ae . u/l versus ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n) and also untrained groups ( . ae . u/l versus . ae . u/l, % (m), . ae . u/l versus . ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p- . . - regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in subjects ( males and females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between and , were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef . %, gk . %, m . %, r . %, s-l . %, s . %, ehsani . % and sirdah . %. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, out of , out of and out of patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i- (g>a), ivsii- (g>a) and heterozygous codon deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p- . . - novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev , l. gera , r. hodges institute of molecular biology and genetics, kyiv, ukraine, university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm- appeared to be the most effective, with ic values of lm and . lm in rat glioma c and human glioblastoma u cell lines, respectively. bkm- suppressed erk / and akt phosphorylation in u cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm- and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi l inhibited the viability of u cells more effectively than tmz. furthermore, the combination of chi l and bkm- resulted in an additive cytotoxic effect. chi l -mediated decrease of cell viability was associated with a g /s transition arrest. chi l provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d expression, as well as a substantial increase in p level. in addition to the accumulation of p , we observed the upregulation of cdk inhibitor p . therefore, g /s arrest in chi l -treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p . harmful hereditary mutations in brca and brca are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca and brca genes in breast cancer-diagnosed patients, that one of them is male, and person yet healthy whose brca was sequenced by sanger sequencing. the data were analyzed by using seqscape software . and detected variations were compared with literature. in brca , we determined different benign genetic variations and variation with unknown significance and variation which has not in literature. in brca gene of patient and healthy person, benign variations, variations with unknown significance, variations which has not in literature and mutation were determined. this mutation is c. - delgtca and is located in occr. c. - a>c variation in brca gene, was determined in only male patient.c. t>a variation in exon of brca , was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p- . . - inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi x . non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi x . introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included adult male rats, divided into groups. each group included rats. i.group: control (regular feed), ii.group: cpe ( . mg/kg/day), iii.group: rw ( mg/kg/day) and iv.group: cpe ( . mg/kg/day) + rw ( mg/kg/day). following days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn + pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm À ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h dcf-da. gsh level was estimated using lowry method. an intracellular labile zn + pool was assessed using fluozin- -am. investigations were performed on the specord m , hplc system lc- prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn + in erythrocytes of patients with ah + dm + compare with ah + dm À and ah À dm À was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn + level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm À , ah À dm À ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, , : - ; lipid insights, , - ] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., , : - ] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde - -hexadecanal. it was found, that -hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal (suppl. ), abstracts: mem. biol. s , lipid signaling & dynamics, p. .] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the - - proteindependent inhibition of apoptosis signalregulating kinase protein kinase ask (apoptosis signal-regulating kinase ) is a member of the mitogen-activated protein kinase kinase kinase (map k) family that plays a crucial role in immune and stress responses. the activity of ask is regulated through homo-oligomerization and interaction with other proteins including the - - protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask is dephosphorylated at ser and the - - protein dissociates. this dissociation is then one of the factors that lead to the activation of ask . we performed low-resolution structural analysis of the kinase domain of ask (ask -cd) bound to - - using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask -cd binds to the - - protein in two to two stoichiometry through a small binding interface involving surface of - - outside its central channel and several regions from the c-lobe of ask -cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask -cd to - - modulates conformation of ask 's activation segment. these results suggest that the - - binding suppresses the catalytic activity of ask through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask and - - and offers a plausible structural explanation for the - - protein-dependent inhibition of ask kinase activity. introduction: thymoquinone ( -methyl- -isopropyl- , -benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c rat glioma cells functioning was studied. results: it has been shown that the cultivation of c cells with tq in concentrations of - lm during hours strongly inhibits cell proliferation and induces cell death with id of lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c cell death by %. we analyzed mapk pathways involvement in c cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd and jnk pathway by sp does not influence on tq-induced effects. on the contrary, the specific phosphoinositide- -kinase (pi k) inhibitor (ly ) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = ) and patients with co-infection (hiv and hepatitis b and / or c) (n = ). as a control we used the data of healthy women (n = ). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control ( . times; p = . ) and group with hivmonoinfection ( . -fold; p = . ). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control ( . times (p = . ) and . -fold (p < . ), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > , ). total antioxidant activity parameter decreased . fold (p = . ) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = . , compared to the control and p = . , compared with the group with hiv-monoinfection) and the level of a-tocopherol ( . -fold to control and . fold to hiv-monoinfection) was detected in the group with hiv-coinfection. . -fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p- . . - acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose groups were formed. rats in group served as control group animals which were only given corn oil. group , group and group were administered , , mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group , group and group were inhibition percentage of %, %, and % respectively, comparison of group 's che activity. the liver alt, ast and ldh increased in group and group compare to group and group (p < . ). we also observed that there was vacuolar and hydropic degeneration in liver of group . according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised patients with ami and healthy subjects. patients that had an acute myocardial infarction (ami) less than hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon dhc were significantly higher in group with ami (p < . , respectively p < . ). elevated levels of mda (p < . ) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < . ) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p- . . - dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet , which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft , the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet gene. however, we found that a strain lacking fet is more sensitive to cu surplus conditions than a strain carrying the aft gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet gene expression. we next evaluated whether ace could be the alternative regulator of fet under cu excess. to test this hypothesis we first constructed the double mutant aft ace and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft or ace mutants. we next monitored the expression of fet gene in cells lacking ace , using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet expression is dependent on ace when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet by ace in an aft independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon- derived from human long bones (atcc Ò crl- tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with %, the superoxide anion is reduced with % compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = ) and age matched control group (n = ) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h o -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss software package. results: dna strand breaks and h o -induced dna damage were found to be higher (p = . for all), the ratio of gsh/gssg was found to be lower (p = . ) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h o -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = . ). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = . ). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p- . . - validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product iso-prostaglandin f a in urine m. kant , m. akıs ß , h. _ is ßlekel , department of medical biochemistry, school of medicine, dokuz eylul university, izmir, department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey -iso-prostaglandin f a ( -iso-pgf a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of -iso-pgf a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version . were used for data analyses. mrm transitions used were m/z? / for -iso-pgf a and m/z? / for -iso-pgf a-d . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for -iso-pgf a was determined as ng/ml by using standards ranging from . - ng/ml. analytical sensitivity of the method was determined by lod with s/n of and loq with s/n of . lod and loq for iso-pgf a were . À and À ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = ). the intra-and interday cvs for -iso-pgf a were . % and . %, respectively. sid-mrm lc-ms/ms method for absolute quantification of -iso-pgf a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p- . . - synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase / activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g /g peak, an increase in the annexin v(+) cells and the cleavage of caspase- and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl- ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto- h cells, cis/res was less efficient in killing h- cells. h- cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin- and lc a. inhibition of autophagy by bafilomycin a rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h- cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. adult male mice ( - weeks) were randomly divided into groups: control group received normal saline ( . ml/day, ip), cp group received cp ( mg/kg/week,¬ ip), the cp+ep group received ep ( mg/kg/day, ip) along with cp, were treated for days. mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < . ). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < . ). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < . ). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < . ). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < . ). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of mg/kg (group ), mg/kg (group ) and mg/kg (group ). only plain corn oil was given to control group (group ). the rats were sacrificed hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group compare to group , besides the insulin levels of group and group were significantly higher than group (p < . ). pancreas amylase and lipase activities significantly decreased in group and group compare to group and group (p < . ). however, there was no significant change in pancreas mda and tos levels (p > . ). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < . ). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < . ). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb ( mg l- and . mg l- ) for hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. day). ml physiological saline (% . nacl solutions), mg/kg/day paracetamol, mg/kg/day paracetamol and mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p- . . - lipid rafts are involved in modulation of ca + responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim Ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan Ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca + concentration ([ca + ] i ) increase due to ca + mobilization from thapsigargin-sensitive ca + stores and subsequent store-dependent ca + entry in rat peritoneal macrophages. it is known that key proteins involved in ca + signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca + ] i in macrophages. [ca + ] i measurements were performed with fura- am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca + ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with mm mbcd for hours before molixan addition causes significant inhibition of both ca + mobilization (on average, by . ae . %) and subsequent ca + entry (on average, by . ae . %), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca + responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca + ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range ( to lm) against phenotypically different mcf- , mda-mb- and sk-br- breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p , p , p and/or p . apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of lm, whereas at the concentration of lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut- and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h ax phosphorylation and bp foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, / /d/nz / . nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn that is accompanied by the relocation of nucleolar proteins and p -based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf- , mda-mb- and sk-br- ). we found that low dose nutraceutical treatment resulted in p -mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt , dnmt a and dnmt b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, / /d/nz / . p- . . - rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < . ), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < . ). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < . ), while malondialdehyde (mda) levels increased notably (p < . ). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type diabetes mellitus (t dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t dm imply an increased enzymatic activity of pa ( . %), which correlates with increased levels of ea ( . %) and glycated hemoglobin (hba c) ( . %), compared with control group. the variables hba c, pa and ea are correlated: the identified pearson correlation coefficients r = . and r = . respectively, have an associated probability of p < . and p < . a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi , e. demir , k. cinar gaziantep university, school of medicine, gaziantep, haran university, sanliurfa, department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group (ir) received gy of gamma irradiation as a single dose to total cranium plus ml saline daily. group received daily plain dmso. group received daily plain saline. at the end of the day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p- . . - role of leptin and adiponectin in obesityassociated oxidative stress e. becer , a. c ß irakoglu near east university, nicosia, cyprus, istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included obese and non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < . ) and adiponectin (p = . ) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < . ) and malondialdehyde (p < . ). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = . ) and malondialdehyde (p = . ) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around %. an extract from aronia showed greater than % efficiency, and a net ethanol extract from all the berries showed a % effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega / / and / / . p- . . - attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins -e / pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins- e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene ( lm, hours) application have been made after resveratrol ( lm, hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins- , ins- and sirt- were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo- gene was increased while pdx- was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, adult sprague-dawley male rats were divided into groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for minutes followed by a hour of reperfusion. in the treatment group, days before ischemia mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as % in the control group, whereas it was % in the i/r group. this value was decreased to % in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost % efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak ( %) at a concentration of mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to %. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega / / and / / . the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t d wt and gh+ cells. although forced gh expression induced cell proliferation, lm curcumin inhibited cell invasion. curcumin ( lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t d cancer cells. although curcumin decreased az expression in t d wt cancer cell, increased az expression was determined in t d gh cancer cell. pao and ssat expressions were upregulated in t d gh+ cells. concomitantly, putrescine levels were increased in t d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak research project (project no: z ). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. - weeks old female sprague-dawley rats (n = ) included in current study were divided into groups: control group (n = ) with healthy rats; breast cancer group (n = ); breast cancer group (n = ) treated with mg/kg paclitaxel injection (once a week for weeks); breast cancer group (n = ) treated with mg/kg royal jelly (by oral gavage for days); and finally breast cancer group (n = ) treated mg/kg royal jelly in addition to mg/kg paclitaxel injection. rats with breast carcinoma was obtained at th days after a single dose injection of mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < . ). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > . ). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase (pon ) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese ( g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = ). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups ( rats in each group). group : sham + saline; group : anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group : anp + gallic acid ( mg/kg/day, intraperitoneal). at the end of th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin- (il- ), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il- , plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < . ). however; tas and nitrite + nitrate levels did not show any significant difference (p > . ). on the other hand, while serum amylase, il- and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > . ). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p- . . - evaluation of oxidant/antioxidant system parameters, il- and il- levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder , s. bahsi , t. bahsi , v. topc ßu , a. bakir ankara universty faculty of medicinel, ankara, zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il and il levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from pregnancies affected by down sydrome and normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il and il levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < . ). besides this, in study group, cat and il- levels are found singnificantly lower than control group (p < . ). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il- levels (p > . ). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g cells. cytotoxicity of linalool was assayed by celltiter-blue Ò cell viability assay. malondialdehyde levels result in membrane damage in hep g cells were assayed by using fluorometric method. hep g cells were incubated with linalool at , and hours. the viability of hep g cells decreased in a manner dependent upon concentration and incubation time. the ic values were calculated . lg/ml ( hours), . lg/ml ( hours) and . lg/ml ( hours). but, cell viability of hep g cells increased when the cells preincubated with linalool at lower concentrations (˂ic ) against h o cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h o was decreased when the cells preincubated with linalool before h o incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included subjects ( female and male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and nd, th and th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = . and . , respectively). thiol levels decreased significantly in both gender at postprandial nd, th and th hours as compared with fasting condition (p = . ). while tbars levels increased at postprandial nd hour, that was only significant for male individuals (p = . ). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p- . . - ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in patients with prediabetes and controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p- . . - the effects of s _ io nanoparticles of rat uterine smooth muscle specially used in textile field sio nanoparticles on uterus smooth muscle was aimed to be researched. in this study wistar albino female rats were used. rats were separated in groups as control, dose ( lg/ml), dose ( lg/ml) and dose ( lg/ml). nanoparticle's size was chosen as nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio nanoparticles, in nm size and in , and lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx )] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine -epi-prostaglandin f a ( -epi-pgf a)] were determined. the results were compared with those of controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher ( % for cuzn-sod, p = . ; % for cat, p . ) and the levels of lipid peroxidation biomarkers were lower (% for mda, p = . and % for -epi-pgf a, p > . ) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = - . , p = . ). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p- . . - investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, patients with acute limb ischemia (li group; mean age years) and healthy individuals (control group; mean age years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < . ). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase activity (pon ) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on women consisted of patients with pcos and healthy ones as control. serum pon activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at nm. molar absorptivity coefficients were used in the calculation of pon and are activities as nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group ( . ae . - . ae . u/ml p < . , . ae . - . ae . u/ml p < . , recpectively). the pon activities are higher in the patient group ( . ae . u/ml) compared to the control group ( . ae . u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were . ae . absu, ima levels of control subjects were . ae . absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < . ). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < . ). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p- . . - the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard ( % protein, % ( % sucrose) carbohydrate, % lipid) and sucrose ( % protein, % ( % sucrose) carbohydrate, % lipid) diet fed bdnf heterozygous mice liver tissues. the male c bl strain wild type (+/+) and bdnf heterozygous (+/À) mice ( weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for weeks. first group involved in c bl wild type mice and fed by standard diet. second group contained c bl bdnf heterozygous mice and fed by standard diet. third group consisted c bl wild type mice and fed by sucrose diet. fourth group involved in c bl heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c bl/ mice inbred strain wild type and bdnf heterozygous (+/À) mice. animals were divided to two groups: wild type (n = ) and bdnf heterozygote mice (n = ). the animals were fed ad libitum by high-fat diet during month. weight gain was recorded every th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p- . . - disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p- . . - determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p- . . - effects of alpha- antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha- receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha- antagonists on oxidative system of rat heart tissue. male wistar albino rats were divided into groups randomly. ) tamsulosin ( mg/kd/day), ) terazosin ( mg/kg/day), ) doksazosin ( mg/kg/day), ) alfuzosin ( mg/kg/day), ) control. all drugs were administered every other day single doze via oral. rats were sacrificed after days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = . ). in addition, tamsulosin group and terazosin group were also significantly different (p = . ). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. wistar albino rats were divided into groups randomly. control (i.p. physiological saline), sbt (i.p. mg/kg/ hours sbt), stress (i.p. physiological saline; hours cold + immobilization stress) and sbt + stress (i.p. mg/kg/ hours sbt; hours cold + immobilization stress) groups were formed. nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = . ), total glutathione (p = . ) levels and glutathione peroxidase activities (p = . ) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = . ) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker -nitrotyrosine level was found to be significantly higher when compared with control group (p = . ) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. children and adolescents with obesity (age: - ); and age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics ). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity ( . ae . lmol/l; . ae . nmol/ml) than the controls ( . ae . lmol/l; . ae . nmol/ml) (< . ). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p- . . - relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats ( - g) received three i.p. injections of taa ( mg/kg) at hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca + chelation had almost the same effect as pharmacological nos inhibition suggesting that ca + -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh ) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries ( , and mg/kg) by oral gavage during days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < . ). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < . ). the maximum hypoglycemic and antioxidant effects were observed at mg/kg dose of mc. background: human serum paraoxonase (pon ) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon prevents ldl oxidation by hydrolyzing lipid peroxides. pon is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon q r isoenzymes. methods: pon q r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with mm edta for minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of mm edta to partially purified hdl-pon q r isoenzymes inhibited % of htlase and arylesterase activities. inactivation of pon for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca + -dependent inhibition of pon q r arylesterase/htlase by using the metal chelator edta, did not alter pon 's ability to inhibit ldl oxidation. pon 's ability to protect ldl from oxidation may not require calcium. p- . . - evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad , college of applied medical sciences, aljouf university, aljouf, saudi arabia, faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type diabetic patients. a total of type diabetic patients and healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx- ). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type diabetic patients than the control group (p < . ). one of the boron compounds is sodium perborate tetrahydrate (nabo . h o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose- -phosphate dehydrogenase (g pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < . ) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan , e. aky€ uz , g. baysal furtana , s. s. ellialtioglu , r. tipirdamaz nezahat g€ okyigit botanic garden, istanbul, department of biology, faculty of sciences, gazi university, ankara, department of horticulture, faculty of agriculture, ankara university, ankara, department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h o ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including mg/l , -d and . mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing and lm h o for hours and then transplanted on the mediums including mm nacl for hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h o -only and h o pre-treated tissues. pre-treatment with h o decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h o pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. the result showed pre-treatment of lm h o induced acclimation of the plants to salinity. in addition, lm h o pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type (cb ) receptor agonist acea, the antagonist am- and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study male wistar albino rats were used weighing - g. each group was consisted of rats. study groups: : control, :penicilin( iu), :penicillin( iu) + ghrelin( lg), :penicillin( iu) + am- ( . lg), :penicilin ( iu) + acea( . lg), :penicillin( iu) + am- ( . lg) + ghrelin ( lg), :penicillin( iu) + acea( . lg) + ghrelin( lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule- (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of patients with a diagnosis of as according to newyork ctriteria and control subjects were included in our study. serum endocan, interleukin- (il- ), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il- , tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < . ). whereas, serum il- , tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il- , tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = . , p = . ; r = . , p = . ). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p- . . - investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged or hours according to parasitemia state ( - %). biochemical analyses were performed in erythrocyte culture in which parasitemia between % and %. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed times with physiological saline, erythrocyte suspensions were stored at- oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < . ). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g pdh, gshpx, gshrx p- . . - in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut , g. kanbak istanbul bilim university vocational school of health services, istanbul, eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol ( mm, mm ve mm) alone and together with lg/ml aspirin, and possible protective role of mm betaine on rat brain synaptosomes. male sprague dawley rat forebrains were divided into equal pieces and pooled to form study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < . ) was observed in mda level of mm ethanol group compared to control group. different doses of ethanol ( mm, mm ve mm) + aspirin exposure significantly increased (p < . ) mda levels compared to controls, whereas betaine administration significantly decreased (p < . ) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p- . . - analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif- a in hepatocellular carcinoma cell line, hep b under hypoxia. method: hep b cells were subjected to normoxia ( % o ) and hypoxia ( % o ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif- a, pgc- a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at lm. bbr treatment has shown significant reduction in hif- a and sirt which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt expression. bbr induces the overexpression of sirt and its deacetylated-pgc- a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif- a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with lm h o after minutes lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h o . also our data suggest that ziziphus jujuba is effective in preventing h o induced oxidative stress and nitric oxide levels. discussion: some research showed that h o was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h o . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the -hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at + . v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p- . . - impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller , h. budak , m. sisecioglu , m. c ß iftci department of chemistry, faculty of science, atat€ urk university, erzurum, department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose- -phosphate dehydrogenase (g pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: / ). p- . . - protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur ( , and mg/kg) were administered orally for days. on the sixth day, chlorpyrifos ( mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < . - . ). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < . ). cur administration reduced all of these changes (p < . - . ). similarly, cur at the doses of mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < . ). additionally, the histopathological damage scores induced by chlorpyrifos (p < . - . ) were decreased by the administration of cur (p < . - . ). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after g ogtt which was applied in pregnant women for screening. methods: the serum specimens of pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = ) and prediabetic pregnant women (n = ) in the th and th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the th and th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between th and th minutes regarding the xo enzyme activity in normal pregnant women (p = . ). in normal pregnant women, the median xo enzyme activity in the th minute was . ( . - . ) u/l and it was . ( . - . ) u/l in the th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in th and th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased . times under red and blue lights and . times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was . times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p- . . - effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c glioma and mouse n a neuroblastoma cells and incubated for hours. at sub-lethal concentrations ( , , lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein (gap ) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c cells, and of nefh and biii tubulin in n a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at g for minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h o absence and presence of curcumin ( - lg/ ml) for hours at °c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase , and activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h o ) on hepg liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg cells's - - hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg cells were determined with flow cytometry method with the help of annexin v-apc and aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: hours ic > lg/ml, hours ic . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; hours ic = . lg/ml, hours ic = . lg/ml, hours ic = . lg/ml. increasing concentrations of ehta extracts were determined to direct hepg cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of mg in g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg was cytostatic at low doses, and cytotoxic at high doses. p- . . - relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of patients ( subject as scf and subjects as ncf) were analyzed. ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group ( ae versus ae ng/ml, p = . ). also there was a significant correlation between serum cp levels and tfcs (r = À . , p = . ). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t ) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n ), and experimental (n ) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t ( mg/ g) in postoperative day. rats were sacrificed on postoperative day . serum thyroid stimulating hormone (tsh), free t (ft ), and free thyroxine (ft ) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: . ) and centrifuged at rpm for minutes ( °c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t hormone had not decline in ft levels compared with the control groups. a significant decrease in ft levels was found in t given rats on postoperative day . whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft level. although tos levels and osi values were similar in both groups, t supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip (tat interacting protein, kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to . hour of lschemia and hour of reperfusion, group iii of rats that received intraperitonealy curcumin ( mg/kg) . hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase , and were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase- and ieveis, and increased the uterine tissue levels of tac but, caspase activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group ( . ae . %) and decreased after treatment with curcumin ( . ae . %). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p - on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, -day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were , , and days old. their weight and tumor diameters were measured throughout the months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p- . . - effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e ) in food preservation for approximately years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf- , colon cancer cell line, ht- , lung adenocarcinoma cell line, a , and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with % heatinactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ ml, ug/ml, ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over hours. according to our results, ha induced a decrease in ht- , a and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf- cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf- cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p- . . - chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using male wistar albino rats weighing - g. rats were divided into five groups (n = / group). group i comprised the control group, group ii was exposed with saline + immobilization stress ( minutes daily immobilization stress for days and . ml saline was administered perorally minutes before immobilization), group iii was exposed amisulpride ( mg/kg/day) + immobilization stress, group iv was exposed fluoxetine ( mg/kg/day) + immobilization stress and v. group was exposed amisulpride ( mg/kg/ day) + fluoxetine ( mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < . ). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < . ). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < . ). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > . ). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p- . . - protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against -methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin , g. oto, f. g€ ok, y. karakus, d. yildiz y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st -methylcholanthrene ( -mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % . nacl, group ii: the b(a)p were administered mg/kg via ip. four times. group iii: the -mc-treated animals were administered mg/kg via ip. four times, group iv: ba was given mg/l/day with water. group v: bx was given mg/l/day with water. group vi: b(a)p mg/kg via ip four times + ba mg/l/day dosage with water. group vii: -mc mg/kg via ip four times + ba mg/l/day with water. group viii: b(a)p mg/kg via ip four times + bx mg/l/ day dosage with water. group ix: -mc mg/kg via ip four times + bx mg/l/day with water. the experimental period was continued for days. statistical analysis showed that the -mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < . , p < . ,p p- . . - effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed - g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to mg/kg body weight alcl in drinking water for and weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., tacal, Ö., take, g., takic, m.m., taldykbayev, z., talim, b., tamashevski, a., tamer, f., tamer, l., , , taneva, s., taniyan, g., , tanrisev, m., tanriverdi, e.c., tanriverdi, k., tarhan, m., tarhan, t., tartar, s., tas, a., , taskin, a., taskin, t., taskiran, e., taskiran, b., taskoparan, b., taspinar, r., , tastan, Ö., tatli, Ö., tauraite, d., tay, t., tayman, c., taysi, s., , teker, h.t., tekes, s., tekin neijman, s., tekin, m.h., , tekin, g., , tekin, m., tekin, n., tekin, g., telci, d., , telefoncu, a., temel, h.e., , , temelie, m., temizgül, r., temlyakova, e., teplova, v., terashima, r., tercan avci, s., terekhov, s., tereshenko, o., terzi gulel, g., terzi, e., , terzi, e., terzioglu, g., terzioglu, o., testoni, g., tetik vardarli, a., tevdoradze, t., tevzadze, l., tezcan, Ö., tezcanli kaymaz, b., thielens, n., thomaidou, d., thomas, a., thornton, j.d., ticea, a.c., tikhonova, a., tileva, m., , timofeev, v., , timofeev, v., timofeeva, e., tipirdamaz, r., tiryaki, m., , tok, m., tokay, e., toker, a., , tokgun, o., toksoy Öner, e., tokyol, Ç., toman, r., tomasi, a., tombul, n., , tooke, c., topaloglu, h., topbas, m., topcu, b., topcu, c., topcu, g., , , topçu, v., topcu, c., topçuoglu, c., , topel, h., , toprak, b., toprak, m.s., torac, e., tosner, z., tosun, m., , toy, h., toymentseva, a., tozkoparan, b., trabulus, d.c., trantirek, l., trantirkova, s., trchounian, a., , trizna, e., , troshagina, d., tro t, m., truncaite, l., , tsakalidou, e., tsarkov, d., tsarkova, m., , tsigara, e.g., tsigara, m.g., tsverava, l., tsvetkova, e., tsyba, l., tsyganov, d., tsymbal, d., tüfekçi, a.r., , tufekci, a.r., tufenkci, h., tuglu, m.i., , tuglu, i., tuli, a., , , , tuli, a., tulubas, f., tunali, g., tunca gedik, s., , tunca gedik, s., tunçdemir, m., tuncel, h., tuncel, h., tunçer, s., tuncer, e., , tuncer, z.s., tuncer, b., tuncer, e., tuncez akyurek, f., tunçez akyürek, f., tuner, h., tüney kizilkaya, i., tural, b., tural, s., turan, i., turan, m., , turan, v., turan, y., turan, c., turano, p., , türel, s., , turhan, t., , turhan, y., , turk, s., turkan, a., türkcan kayhan, c., turkekul, k., türkel, s., turkeri, o.n., turkeri, n., turkkan, b., turkmen, s., türkmen, n., turkon, h., tutar, y., tüten, a., tutkun, e., , tüylü küçükkilinç, t., tuz, m.u., twardovska, m., tyapkina, o., tzartos, s., photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf- bucharest, national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, microelements modifying oxidative stress status were determined by icp-ms/ms in the hno diluted serum samples collected from chl-positive adult females (n = ) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging - g) were randomly divided into groups, as each composed of rats. after hippophae rhamnoides l. leaf ether extracts of mg/kg, mg/kg and mg/kg doses and mg/kg doses of famotidin orally administered, mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental ( mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; hours torsion/ hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape ( lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi k, akt and mtorc. intensities were evaluated as mild ( ), moderate ( ) or strong ( ). serum ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c ( . ae . lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p- . . - binding of fas and cu(ii) ions to hsa changes its cys thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion ( . mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys -sh decreased for % after cu(ii) ion binding, and during hours incubation at °c, it was further decreased for % (stearic acid, mixfas) and % (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg ( mol/ mol hsa), lead to decrease in cys -sh content depending on fa present: %- % for myristic and stearic acid, % for oleic acid and mixfas and % for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases pirto ek p- . . - anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf- and mda-mb- background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl- is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl- levels. methods: our study group includes subjects with vitamin d deficiency (group ) and age and sex-matched healthy subjects with normal serum levels of vitamin d (group ). plasma (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl- analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > . ), plasma ykl- levels were significantly higher in group than group (p < . ). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl- levels in group . vitamin d deficiency may be related to increased ykl- levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl- , inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on unrelated cad patients and healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as . %, . % and . %, respectively. in the control group, frequencies of genotypes were found as . % for aa, . % for ac and . % for cc. we did not observe difference in apj receptor a c polymorphism between cad patients and healthy controls (v = . ; df = ; p = . ). the a allele was encountered in % ( ) of the cad and . % ( ) of the controls. the c allele was seen in % ( ) of the cad and . % ( ) of the controls. allele frequencies were not significantly different between groups (v = . ; df = ; p = . ). the frequencies of apj receptor a c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ . . in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p- . . - comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups ( rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at hours intervals, intraperitoneally (i.p.). cerulein group received cerulein ( lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide ( %), two times, at hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin ( mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < . , p < . , respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < . ). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < . ). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < . , p < . , respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < . ).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p- . . - identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx- and rtx- ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of mg/ml was prepared, cleared by membrane filter of . lm and freezed at À °c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. mg of dry extract was reacted with ll pyridin + ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including % trimethylchlorosilane (tmcs) and incubated for minutes at °c. all analyses were performed with shimadzu gcms-qp ultra by using a flame ionisation detector (fid). rtx- and rtx- ms capillary columns and helium for carrier gas at a flow time of ml/minute were used. injection was applied on split mode at °c. derivatized propolis sample was injected as ll, initial column temperature was adjusted as °c, then increased to °c with increments of °c. total analysis time was determined as minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx- column were much more than those of rtx- ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx- column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after and exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to . lg/ml of cd (cdcl .h o) and zn (zncl ), and their mixture, for , and days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph . ). homogenates were centrifuged at , g ( min, + °c). supernatants were stored at À °c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < . ). results: gst and gsh changes were recorded as decreases after all metal exposures at day . although day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor- (lox- ) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox- expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor- (lox- ) in animal model of type diabetes mellitus. methods: we compared groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz ( mg/kg). animals were kept in standard condition. in day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox- levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox- levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox- levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type diabetes. p- . . - investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur , i. ince , i. abidin , a. alver objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain animals. the rats in ns treated groups were given ns ( . ml/kg) once a day orally for weeks starting days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b ) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of , , and mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with mg/kg body wt. doses ( %). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: patients with prostate cancer and healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were . ae . lmol/l in the control group, . ae . lmol/l in the patients before rp, and . ae . lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < . ). native thiol, total thiol and tas levels were higher months after rp compared to before rp in patients, but these changes were not significant statictically (p values . , . and . respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p- . . - is there any relation between g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to pregnant women aged - years and at - weeks of gestation. plasma glucose concentrations were measured initial, hour and in addition to test hours after ingestion of g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and hour serum total antioxidant status (tas) levels (r = . ) and the oxidative stress index (osi) (r = . ) was found. there was a positive weak correlation with initial and hours total oxidant status (tos) levels (r = . ) but statistically significance difference was not found (p > . ).in this study after ingestion of g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their , -diphenyl- -picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid ( . - . -lg/g), p-coumaric ( . - . -lg/g), and total phenolic content (ranged . - . -lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at - c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after hours following cpb were significantly higher than the before surgery (p < . , p < . , p serum ngal levels in after hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = . , p < . ; r = . , p < . ; r = . , p < . ; r = . , p < . respectively). ngal levels were positively correlated with total perfusion time after cbp (r = . , p < . ).the results of our study show that, increased ngal levels hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of wistrar male rats were used. groups are named as healthy, ind control, famotidine ( mg/kg) and three different doses of tq ( . , and mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. min later, mg/kg ind administrated to each rat. animals were sacrified about hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq ( . , and mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and , diphenyl- -picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + mg/kg zn sulfate (low dose; group ), al diet + mg/kg zn sulfate (middle dose; group ) and al diet + mg/kg zn sulfate (high dose; group ). zn sulfate solutions were administered . ml/day orally for days and in day rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group and ; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group . in kidney; aopp level was decreased only in group and sod level was only decreased in group as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group ). gst level was decreased in only group conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group was composed of patients with type dm and cataract and group was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of patients, totally patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < . ). tos serum levels in dm group was statistically higher than the control group (p < . ). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > . ). group , divided into two subgroups according to their hba c levels, there was no statistically significant difference between the subgroups when hba c levels were compared with the relationship between serum and ha's tac and tos levels (p > . ). there was not an association between the gender, age and the levels of tac-tos in both groups (p > . ). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik , g. erbil , z. c ß avdar , c. ural department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to hours of ischemia followed by hours of reperfusion. ferulic acid was administered ( mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase- , inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase- and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < . ). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < . ). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < . ). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p- . . - across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [ ] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane- , p-phenol- , -p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. %) charged titanium phthalocyanine ( . , . , . , and lm) were determined on a human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc( ), tritc-phalloidin and dapi staining protocols. waltmann pdt l was used as the non-toxic light source at j/cm fluence and mw/ cm fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: rvo patients and controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > . for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of osa patients were included in the study ( controls, with mild, with moderate, and with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi< served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was . ae . years and . % ( / ) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > . ). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < . ). there was a negative correlation between ahi and serum total thiol levels (r = À . , p < . ) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera -ohdg, mda and il- were significantly higher in diabetic group than control group (p < . ). although there was a notable positive correlation between mda and -ohdg, there was no a relationship between -ohdg or mda with il- . discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il- , induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose- -phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g- -p-d) enzyme activity and the effects of quercetin on erythrocyte g- -p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = ) received physiological saline. the quercetin group (n = ) received quercetin ( mg/kg/ day) via i.g. route the alcohol group (n = ) received ethanol ( % v/v, ml/day) via i.g. route. the alcohol + quercetin group (n = ) received ml of ethanol ( % v/v) hours after quercetin treatment ( mg/ kg/day). experimental procedures were peformed for days. erythrocyte g- -p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < . ). in the alcohol group, the erythrocyte g- -p-d activity was found to be significantly decreased than those in the control group (p < . ). statistically significant differences were observed in erythrocyte g- -p-d activity between the alcohol group and the alcohol + quercetin group (p < . ).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g pd, oxidative stress, quercetin p- . . - effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = ), wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of minutes. four hours later, except control, sham-a, sham-c groups, mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca , ca , and ca region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il- b, il- , malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il- b, il- , mpo, no, and tnfa levels. it could increased il- b levels in chronic stage but not affect to il- , mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il- , and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: ( ) nitrite formation from nitrate by nar, and ( ) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-( -naphtyl)-ethylenediamine, which form red color measured at nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment ( °c, min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p- . . - the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation co) on the radiotherapeutic equipment teragam in a dose of gy and received phytomedicine eminium regelii in a dose of . mg/kg orally within days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in times in spleen, in times in thymus, in times in adrenal glands, in liver in times, in lymph nodes of small intestine it in times. mda decreased in liver up to and times, in spleen in . times, in thymus in times, in liver in times, in adrenal glands in time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p- . . - evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal , c. topc ßuoglu , m. cingi clinic of biochemistry, ankara polatli duatepe public hospital, ankara, clinic of biochemistry, ankara numune training and research hospital, ankara, clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm- ) criteria for bipolar disorder i. healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p- . . - xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in fmf patients and healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc- , sjsa as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with % heat-inactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ml, ug/ ml, ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc- and sjsa cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc- cells with an ic value of . lg/ml. contrary to this, ha induced proliferation of sjsa cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa cells with an ic value of > lg/ml. on the other hand, cell migration was reduced in pc- cell line and interestingly, migration was accelerated in sjsa cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal (suppl. ) ( ) key: cord- -uj fe y authors: nan title: scientific abstracts date: - - journal: reprod sci doi: . / sha: doc_id: cord_uid: uj fe y nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day , wistar rats were randomised into: normoxia ( % o litters), hypoxia ( % o litters) and hypoxia + vit c ( % o + mg. ml - vit c in water, litters). on day , dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein (hsp ; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by % of baseline; a similar increment to human trials (poston et al. ) . hypoxia elevated placental hsp and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. normal (n) and gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n= ± ; gdm= ± g) while placental weights were significantly different (n= ± ; gdm= ± g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: . ± . in n and . ± . ml/min in gdm; qumb/kg: . ± . in n and . ± . ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o sat, o cont and po and a significant increased lactate conc was found in both uv and ua compared to n (table ) . o umb uptake (n= . ± . ; gdm= . ± . mmo/l/min) and o umb uptake/kg (n= . ± . ; gdm= . ± . mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f ) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f offspring with no manipulation of the f pregnancy. methods: pregnant guinea pigs (f ) were subcutaneously injected with betamethasone (beta; mg/kg) or vehicle (veh) on gestational days / , / / . adult f female offspring from each group were mated with control males. hpa function was assessed in adult f offspring by non-invasive measurement of salivary cortisol concentrations: ) under basal conditions, ) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, ) following dexamethasone suppression. results: there was no effect of beta (f ) on bodyweight from birth to adulthood in f offspring. basal salivary cortisol in the betaf females was lower than in the vehf group in the morning but not the afternoon; there were no differences in male f offspring. in contrast, both male and female betaf failed to mount adrenocortical responses to psychological stress compared to vehf offspring that mounted robust responses (p< . ). swim stress induced robust adrenocortical responses in all groups (p< . ), however, the response was consistently lower in betaf offspring. beta exposure also led to a significant difference (p< . ) in the cortisol response to dexamethasone suppression in female (f ) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f ) causes transgenerational programming of adrenocortical function in adult f offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using mhz ultrasound biomicroscopy in control (c bl/ j) and enos -/-mice at . days of pregnancy (n= mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe- and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was % lower (p< . ) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (- %, p< . ) and mean velocity (- %, p< . ). uterine arterial resistance index was % higher (p< . ), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated ( %, p< . ) and capillary lobule length was reduced by (- %, p< . ). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium with % of fcs and ng/ml vegf for to days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age years) with regular menses ( - days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - , - ) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd , kdr and cd ) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was > nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being -fold higher in the follicular compared to periovulatory phase (p< . ). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg fc (fvii/igg fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of . mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein ( ug) was delivered i.p. once a week for weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with ug of icon abolished all lesions in of mice and reduced both size ( . to . mm) and number of lesions ( . to per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of % +/- % (p= . , mean +/-sem, n= ) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp demonstrated increased secretion of crisp in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp , is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa , treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment ( %, p= . ) or hoxa transfection ( %, p= . ). coadministration of progesterone along with increased hoxa transfection had no additive effect on the expression of gaba-a pi receptor mrna (p= . ). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated , women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase trial, gog e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr ). ic experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic of . m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by % using concentrations up to . m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of . mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna , which was significantly down-regulated. microrna is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, data points for each gene from arrays have been collected from patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove arrays that deviated from twice the inter-quartile range calculated from the array signals. results: the custom microarray profiling identified genes that were differentially expressed between normal and fibroid tissues (p < . ; fdr< . ). among them, genes encode receptor tks, genes encode tk ligands, and genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log ratios of these genes has led to the division of the patients into two major groups. thirty four ( %) belong to a group characterized by the significant downregulation of the cyr (ccn ) gene in fibroid tissues. the cyr -down group can be further divided into two sub-groups based on the expression of another tk ligand, efn a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr and efn a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, arthur c fleischer, brian day, stephanie v blank, bhavana pothuri, robert wallach, john p curtin, david a fishman. gynecologic oncology, new york university school of medicine, new york, ny, usa; radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu scanner after intravenous injection of g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of ovaries have been analyzed, benign and malignant. of the malignancies ( fallopian tube, ovarian: stage i, stage iii), / women were correctly identified using conventional tv imaging and others were identified using mvi / . all benign lesions were correctly identified by mvi / while tv detected normals ( false negatives). the lesions detected as malignant by mvi were -stage i fallopian tube and -stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in ( . ± . vs . ± . , p = . ), greater peak enhancement ( . ± . vs . ± . , p < . ), longer wash-out ( . ± . vs . ± . (p < . ), and greater perfusion ( . ± . vs . ± , p < . ) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir- a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir- a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex system. ikk transfection was done with roche fugene transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir- a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk '-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified mirnas differentially expressed in type i versus type ii cells, one of which, mir- a, had putative binding sites in the '-utr of ikk mrna. mir- a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's '-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir- a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd in the rat uterus by nitric oxide and the involvement of p kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats ( g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at or /mg/rat/day in saline or saline alone. uteri were obtained from these rats at or hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name ( mm), diethylenetriamine-no mm) or worthmanin ( . m) in mem without phenol red for hours. uterine tissues were homogenized in trizol and mrna levels for cd were measured using rt-pcr and expressed as a ratio to s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd mrna levels in a time and dose dependent manner with maximal responses seen with mg l-name and at hours. in vitro studies show that deta-no suppressed cd mrna levels in the rat uterus. both l-name and pi kinase inhibitor, worthmanin caused increases in cd levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi kinase appears to be involved in cd regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, daniel b hardy, carole r mendelson. dept of pediatrics; depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during % of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p ). we previously observed that p acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase- (cox- ) expression (hardy et al., ) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., ) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p ( mg/ml) from to days post-coitum (dpc). as expected, treatment with p delayed parturition by - h. this also was associated with a decrease in uterine cox- mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p -injected mothers, as compared to controls. this was associated with an inhibitory effect of p treatment on cox- protein and mrna levels in the fetal lungs. these findings were of interest, since cox- expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., ) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., ) . collectively, these findings suggest that maternal p treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p inhibits an important fetal signal leading to labor. supported by nih p hd ; nih r hl . recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl- is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl- gene expression in myometrium during term and ru -induced preterm labor. also ccl- was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl- as a mediator of this stretch response. we also studied the effect of progesterone (p ) on the myometrial secretion of ccl- . methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl- gene and protein induction by static mechanical stretch. ccl- gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl- production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for - hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch ( % elongation for hours) caused a significant accumulation in ir ccl- which was repressed by pretreatment with p ( um). the rise in ccl- protein levels was preceded by a transient increase on ccl- mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl- significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, , guadalupe estrada-gutierrez, lourdes vadillo-perez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico; escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: ) term without labor (n= ) and ) term with spontaneous labor (n= ). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd , cd , cd , cd and cd subpopulations of leukocytes were identified. intracellular mmp- was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd +, cd+ and cd + cells, both in the chd and inside the fm. mmp- was associated to cd + cells. cd + exhibited a more widespread localization in the fm and cd + cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp- , which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n= ), iugr (n= ) and uncomplicated pregnancies (n= ) were recruited at a mean follow up time of . years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p < . ) as well as higher mean indices of body mass, fat mass and lean mass (p< . ). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p< . ) as well as excess of android fat mass and increased android-to-fat ratios (p< . ). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p < . ). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p < . ) and waist-to-hip ratios (p < . ) as well as smaller hip circumferences (p < . ). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, timo h ekhart, robert aardenburg, louis lh peeters. obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in formerly preeclamptic women (pe) and healthy parous controls (control) who were normotensive at weeks post-partum follow up, we assessed at least years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/ . m ) after which we calculated total peripheral vascular resistance (. dyne.s/cm ) and renal vascular resistance (. dyne.s/cm ). data were analyzed parametrically (p< . ). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, % of formerly pe women and % of control were hypertensive (p< . ). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about % higher and erpf % lower in formerly pe women as compared to control. conclusion: at least years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe . ± . ± * ± * ± * control . ± . ± ± ± * = p< . pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, , marilyn j cipolla. neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from groups of sprague dawley rats: np (n= ), lp (d ; n= ), ovariectomized and implanted with -estradiol ( . mg, -day release) and estriol ( . mg, -day release) pellets for days (ovx+e; n= ), and ovariectomized and implanted with placebo pellets for days (ovx; n= ). pcas were isolated, pressurized in an arteriograph, and perfused with . mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from - mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p< . ; figure ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p % in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was % greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, marc ea spaanderman, dorette a courtar, robert aardenburg, ben j janssen, fred k lotgering, louis lh peeters. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i indicator dilution method) at least months post partum. we intravenously infused ml of iso-oncotic fluid over minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within year and were evaluated at weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p< . ). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv ( / ) and npv ( / ). in npv, lfsys decreased %, but only % in lpv (p< . ). anp remained unaltered in npv, but increased in lpv. in the pregnant group, women had lpv and had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt- was increased, and plgf reduced at all gestations (p< . ). seng levels were also increased in pe. after weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt- and seng, in pe only. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). only sflt- was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after weeks' gestation is associated with a significant fall in serum sflt- and seng, and in placental sflt- . these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, debbie a sanders, olivera spasic-boskovic, graham j burton, d stephen charnock-jones. dept of pdn, university of cambridge, united kingdom; dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase- (sflt- , svegf-r ), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt- and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt ( - ng/ml) in a basic % fbs-dmem medium for hr. on day , low doses of tnf-( . ng/ml) were applied to pre-treated cells for hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody ( - g/ml), anti-kdr antibody ( g/ml), anti-vegf antibody ( g/ml), or vegf receptor inhibitor su ( m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in % pfa for immunofluorescence or incubated with labelled hl leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl leukocytes and increased icam- , vcam- , endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su , anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term ( - wks) from lean kg/m ), overweight ( - . kg/m ) and obese ( - kg/m ) patients (n= or /group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using , -dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab ( : ) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p< . , anova) with expression in tissue from obese women being significantly increased compared to lean (p< . ) and overweight (p< . , tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p< . , mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and . % coverage as -beta hydroxysteroid dehydrogenase ( bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from teenagers ( - years) and adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (> mm increase in kneeheight per days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p< . ). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p< . ). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight g and g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p< . ), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate ( s and min respectively) as na + -dependent cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles ( . mm) or fragments ( . mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p< . ) inhibited na + -dependent c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n= , kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib ( . - mm) caused a dose-dependent inhibition of na + -dependent c-meaib uptake into mvm with ec values (in mm; mean ± se) of . ± . , . ± . , and . ± . respectively, n= ). na + -dependent c-meaib uptake into fragments was reduced substantially in the presence of mm l-hcy or dl-hcy causing a ± and ± % reduction (mean ± sd, n= - ) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, theresa l powell, thomas jansson. department of physiology, institute of neuroscience and physiology, gothenburg, sweden; department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at hours. cells were cultured for an additional hours in culture media containing . mm, . mm, or mm glucose (control), which corresponds to standard culture media. at hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s k phosphorylated at thr- , the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of h-leucine. results: as compared to control, phospho-thr- -s k expression was reduced by % in cells incubated in . mm results: fgr pregnancies were delivered earlier ( ± . v ± . w; p< . ), weighed less ( . ± . v . ± . kg; p< . ) and had higher s/d ratios ( . ± . v . ± . ; p< . ). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p< . ) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all eaa for both groups. amino acids fell into groups for fgr pregnancies: ) his (ratio . ), ) leu, phe, met (ratios ), and ) val, thr, ile, trp, lys with intermediate ratios ( . ns conclusion: this is the st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a -year old female were used. cells were treated with estradiol [e ], equilin [eq] ( . nm) or genistein [gen] ( micromolar) for hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three . , . and . for e , eq and gen respectively. similarly nfkb was also reduced . , . and . fold compared to controls. significant reduction of ccl was demonstrated with eq ( . fold) and gen ( . fold). tnfreceptor a and b were only significantly decreased by gen. vcam- which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced ( . -fold) with genistein, and a slight reduction was seen with e and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin- (et- ). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et- pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged ( month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , -estradiol [ovx+e ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm , μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et- (ec = . ± . μm in ovx versus . ± . μm in ovx+e or . ± . μm in ovx+etan groups; p< . ). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p< . ) role of mmps in big et- -induced vasoconstriction in the ovx+e group (reduction in max constriction= . ± . %) compared to ovx ( . ± . %) and ovx+etan groups ( . ± . %). conclusions: both estrogen and tnf inhibition reduced big et- vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et- vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et- vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of - to + was amplified by pcr and then cloned into pcr . -topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of methylated cpg sites were found in the patient group with pelvic organ prolapse while only methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of , parous women participating in the nurses' health study ii from to . our primary outcome was incident premenopausal breast cancer. results: during the study period, cases of premenopausal breast cancer were diagnosed during , person-years of follow-up. women who had ever breastfed had a % lower incidence of premenopausal breast cancer ( % confidence interval [ci] - %) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age , family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p= . ). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p= . ); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a % ( %ci - %) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p= . ). among women who had never used suppressive medication and reported a family history, those who had breastfed had a % lower covariate-adjusted risk of premenopausal breast cancer ( % ci - %) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio . , % ci . - . ). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, rachael a clark. brigham women's hospital, boston, ma, usa; harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd + cd + t cells has been documented but it is unclear if these represent foxp + t cells (tregs) . the possibility also exists that other cd + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd + cd + cells are true foxp + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median %) of cd + t cells were true foxp + regulatory t cells. from donors, foxp + tregs accounted for a median of . % of all cd + cells. these cells were memory cd ro + t cells lacking ccr , and l-selectin but expressing cd , ctla- , gitr, and hla-dr. additionally we found that a median % of cd + t cells expressed the nkt marker cd . these cells were a mixture of cd and cd -cd -t cells, with variable numbers of cd t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire ( . % iv jq) and also expressed cd , hla class ii, cd ro but were ccr and l-selectin low. comments: we find that only a minority of cd -expressing t cell in the decidua are true foxp + tregs. because much of the work on treg in pregnancy has used cd as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, kathleen a connell, richard bercik, lloyd g cantley. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy- is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy- expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy- expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy- as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy- , asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy- , asma and desmin in vaginal tissue from women with pop and controls. further, thy- mrna expression was downregulated % in women with pop compared to controls (p = . ). a parallel decrease in thy- protein was seen in women with pop compared to controls (p= . ). although a % and a % decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy- expression in vaginal tissue from women with pop compared to controls. differential expression of thy- in prolapsed vaginal tissues suggests that thy- may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the ' untranslated region ( '-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n= ) and ectopic (n= ) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e ; - m), or progesterone (p, - m) for and h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases ( . ± . and . ± . , respectively), compared to the mid-late proliferative ( . ± . ) and early-mid secretory phases ( . ± . )(p< . ). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p< . ). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both and h compared to control (p< . ) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp a gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp a gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c bl mouse tissues. '-rapid amplification of cdna ends ( '-race) was used to determine the transcriptional start sites of cyp a transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp a transcript was isolated using '-race and this novel gonadal fat-specific exon i of cyp a mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine t -l cells with these plasmids showed that promoter activity was conferred by the sequence located at - to - bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp a transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp , bmp , bmp , gdf , gdf (myostatin), gdf and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m a and sb ) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m a ( nm) and sb ( m) significantly reduced decidualization ( % and % respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp , gdf and tgf protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp and tgf secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp and tgf in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with % pfa, permeabilized with . % triton x- for min, and immunostained against a-tubulin and the nucleus for hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h k modifications were measured in nondiabetic and diabetic denuded murine oocytes at hours post pmsg ( hour) and at six hours post hcg ( hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in %pfa and permeabilized with . % triton x- . they were stained via immunohistochemistry against histone protein h at lysine (h k me ) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately / ( %) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about / normal oocytes ( . %). western blot demonstrated times higher expression of dimethylated chromatin in diabetic oocytes at time compared with nondiabetic oocytes. at time hours, diabetic oocytes had significantly fewer h k modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h k me by immunohistochemistry, we demonstrated h k me expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc expression in human prophase i (pi) and metaphase ii (mii) oocytes, -cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a aa protein with % identity and % similarity to mouse epab and contains rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. pabpc mrna is ubiquitously present in all tissues as well as -cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc following zga, which occurs at -to -cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh -terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh ( miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp . results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig a) . likewise by if, phospho-jnk expression was significantly increased in sigc hour post fsh exposure (fig- a) . similarly on wb, phospho-cjun expression increased as early as min after fsh exposure and peaked at hrs. cjun phosphorylation was abolished hr after treatment with sp ( mm) (fig b) . facs analysis showed that the inhibition of jnk by sp resulted in cell cycle arrest at g /m transition in a dose dependent fashion (fig c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd - ). the induction of {alpha}-hydroxylase (cyp ) expression in granulosa cells. satin s patel, victor e beshay, william e rainey, bruce r carr. reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein- (ap- ) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp expression in granulosa cells. transformed human luteinized granulosa (hgl ) cells were utilized for all experiments. hgl cells were cultured in monolayer for h. cells were treated for h with and without pd (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp . cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for h. rna was isolated and rt-pcr was also performed for cyp . immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp . treatment of hgl cells with the mapkk inhibitor pd for h, resulted in a -fold increase in cyp mrna expression compared to basal conditions. in cfos gene silenced cells, cyp mrna expression also increased by -fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp . conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp . these results suggest that the ap- transcription factor, cfos, may play a role in the inhibition of cyp expression in granulosa cells. this may provide an explanation for the lack of cyp expression in granulosa cells. the g /m stages of the granulosa cell cycle. as clip- has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip- , leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip- and mtor were evaluated. computational analysis of potential clip- phosphorylation sites and comparison with residues on known mtor targets were performed. clip- threonine and serine were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip- function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip- phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip- and a peptide substrate. results: clip- was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip- , mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip- phosphorylation supported thr as a bona fide mtor target. conclusions: clip- was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip- and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, thomas m goodwin, gorm greisen, morten hedegaard, joan-carles arce. warwick medical school, university of warwick, coventry, united kingdom; maternal-fetal medicine, university of southern california, los angeles, usa; dept of neonatology, rigshospitalet, copenhagen, denmark; dept of obstetrics, rigshospitalet, copenhagen, denmark; clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in countries. a total of women between + and + weeks gestation, and with uterine contractions of sec duration during min, cervical length mm, and cervical dilatation > and < cm were randomised to receive a single intravenous bolus dose of either barusiban . mg, mg, mg, mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within h ( % in the placebo group and % to % in the barusiban groups). there was no dose-effect relationship nor an effect at or h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range . - mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients ( . %) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage ( . +/- . weeks vs. . +/- . weeks, p= . ). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation ( . +/- . days vs. . +/- . days, p= . ). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied singleton pregnancies with pprom at < weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: patients showed an amniotic fluid pocket (afp) persistently cm and did not undergo amnioinfusion (group b) whereas had a maximum afp < cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in patients of group a amnioinfusion was successful (afp cm for hours following the procedure: group a ) whilst in it was unsuccessful (afp< for hours: group a ) and repeated. results were analized with the student t test for unpaired samples and with the when appropriate. p values < . were considered significant. results the group where amnioinfusion was not successful (group a ) showed the worst outcome (see table) . there were intrauterine deaths, all in this group. pulmonary hypoplasia was present in / ( . %) newborns (both survived and deceased) newborns, / in group a . no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi ; : - ) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a . g weight with those connected to a . g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, nm or nm). following the day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under g tension and responses obtained to mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ) and oxytocin ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ) and there was a trend towards a reduced maximum response to oxytocin (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days had no effect on either the maximum response to potassium . prolonged stretch increases human myometrial contractility in vitro. . prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: pregnant preterm labor women were included. uterine electromyography (emg) was measured for minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g : tocolysis, n= ; g : no tocolysis, n= ). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p< . significant. results: there was a significantly higher uterine emg activity (pdspf: . ± . vs. . ± . ), but no difference in cd ( . ± . vs. . ± . ), for patients delivering within days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table ) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r -hd . objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide ( mmol) followed by cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n= , p< . ). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: term patients were included in the analysis: were in labor (group ), while were non-labor (group ). minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of + means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < . considered significant). results: the mean and sd of the lyapunov exponent for all the patients was . ± . . moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r -hd . comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at hz and band-pass filtered in the . to . hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of " ." in-between contraction periods were assigned a " ." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. ) . corrected opa, ppa, and npa were high at . %, . %, and . %, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. ) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r -hd . introduction -this is a secondary analysis of women participating in a center randomized placebo controlled trial (rct) evaluating the impact of -ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma -ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of -ohpc. methods -women with twins were randomized between - weeks to receive weekly im injections of either -ohpc ( mg) or placebo until weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between - weeks. the sample drawn just prior to the next scheduled injection was analyzed for -ohpc by hplc-ms in a blinded manner. the lower limit of quantification of -ohpc was ng/ml. we conducted univariate analyses to assess the association of -ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb< weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results - women assigned to -ohpc were included; all received all of their scheduled injections. the concentration of -ohpc was significantly higher in women delivering < weeks compared with those women delivering > weeks (p= . , table) . concentration of -ohpc was significantly correlated with ga at delivery (r = - . , p= . ). each unit increase of -ohpc was associated with a % increased odds of delivering < weeks (odds ratio . , % ci, . - . , p= . ) and a % increase in hazard of spontaneous delivery (hazard ratio . , % ci, . - . , p= . ) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < weeks (n= ) . ( . ) > weeks (n= ) . ( . ) conclusion plasma -ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since -ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between . and . weeks' gestation (cases) or term controls ( - weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: cases and controls were studied (see table for comparisons). controls p value ga at enrollment, weeks . ± . . ± . < . interval to delivery, days (median, range) ( - ) ( - . ) < . maternal progesterone at enrollment, ng/ml ± ± < . maternal progesterone after delivery, ng/ml ± ± . cord progesterone, ng/ml ± ± . among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes ( ± ng/ml, n = ), as compared to prom ( ± , n = ), p< . . this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches ( ( -april and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever > replication data sets were available results: a total of abstracts were reviewed and were selected for full text review. data were extracted from articles. over associations were reported between snps on various candidate genes and ptb; however only had replication dataset. meta-analysis documented significant association between pon a g (odds ratio [or]= . ( %ci . - . ), pon (rs# )(or= . ; ci- . - . ), tnfrsf - a/g (fas) (or= . ; ci- . - . ) and ptb. two snps pon s c (or= . ; ci- . - . ) and ifn gamma (rs ; or- . ; ci- . - . ) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, mounira habli, jeff livingstone, william polzin, foong lim, timothy crombleholme. pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from - was performed. results among cases of ttts / ( . %) patients with high order gestations were identified (n= ) with a mean ga at diagnosis of . ± . weeks. pregnancies ( . %) were dichorionic triamniotic and ( . %) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n= ), moderate (stage iiib, n= ) and severe (stage iiic or iv, n= ) categories. / ( . %) were treated with amnioreduction alone (ar), / ( . %) with selective fetoscopic laser photocoagulation (sflp) alone, / ( . %) with ar followed by sflp and / ( . %) with ar followed by intrafetal radio frequency ablation (rfa). / ( . %) patient had a cervical cerclage. / ( . %) patients were treated but remain undelivered. mean ga at delivery was . ± . weeks. overall survival was / ( . %) with bystander survival was / ( %), donor survival / ( . %), recipient survival was / ( . %). conclusion triplet pregnancies treated for ttts have % survival rate for bystander fetuses and have . % survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis . ± . weeks cincinnati modification of quinterro (i), (ii), (iii), (iiia), (iiib), (iv) ga at delivery . ± . weeks live birth -donor / ( . %)* # -recipient / ( . %)*# -bystander / ( %) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of methadone dependent mother and infant pairs. patients were categorized into groups based on maternal dose at time of delivery: group : dose mg, group : dose - mg, group : dose > mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were scores of . neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between - weeks gestational age (ga) and fetoscopic balloon removal around weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in consecutive pregnancies but not in the first pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [ . ( . ) vs. . ( . ) weeks; p= ns] and balloon removal [ . ( . ) vs. . ( . ) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was / in the study group compared to / in the control group (p< . ). mean (sd) ga at delivery was . ( . ) weeks in the study group, compared to . ( . ) in the control group (p= . ). bleeding from the trocar insertion site occurred in cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in patient in the treatment group compared to in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between and was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period to there was a total of pregnancies affected by hydrops (incidence . per births). the average maternal age was years. in cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was weeks (range - weeks). in just over half ( %) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but of the babies not born alive. edema was present in at least cavities in over half of cases (n= ). chromosomal anomalies included trisomy , trisomy and turners syndrome. in all cases of infection, parvovirus b was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in cases an interruption of pregnancy was performed at a mean gestational age of weeks. of those who did not elect to terminate the pregnancy, there were fetal deaths in utero, live borns with neonatal death. for the live borns, the median gestational at delivery was . weeks (range to . weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n= ), infection (n= ), chromosomal abnormality (n= ), unknown (n= ) and other (n= (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed fetal brain exam performed between and , seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at . tesla, using surface abdominal coils and single-shot fast spin-echo t -weighted - mm thick sections, with . - . mm in planar spatial resolution. we found cases with dmv anomalies (tab. ). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that in after st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september , to july , was retrieved from our database. information concerning women younger than years of age at the estimated date of delivery was extracted and tabulated. results: during the study period women had genetic counseling for positive ft. thirteen patients were excluded from further analysis ( had incomplete clinical documentation and had spontaneous miscarriages prior to weeks gestation). four hundred and twenty-five patients were older than and were younger than at the estimated date of delivery. table depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately % of this younger group of women opted for chorionic villous sampling (cvs), % for amniocentesis and more than % declined prenatal genetic testing. moreover, this data also suggests that: ) these women opted for cvs when the ft risk (mean = in ) almost doubled the cvs procedure related risk quoted at % and, ) when the ft risk is between in and in almost half ( out of ) declined not only the st trimester cvs but also the nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline ( medline ( - , embase ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the cochrane library ( issue ). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of . , . , . , . . in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr > . compared to < . provided the strongest association with perinatal survival (peto or . , % ci . - . ). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or . , % ci . - . ). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > . compared to < . was or . , % ci . - . ). data was also available for liver up and lhr > . which had a peto or of . ( % ci . - . ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from to was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as x tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and could be pooled in the meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases. three studies had data for liver length measurements and two were pooled. the sensitivity was . ( . - . ) and the specificity was . ( . - . ) but only cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) with cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving cases. the sensitivity was . ( . - . ) and specificity was . ( . - . ). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr . ; % ci . , . ) and neonatal deaths (rr . ; % ci . , . ) than amnioreduction ( figure ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at < years rr . , % ci . , . )( figure ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage and ) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol. ; ( ): - ). the west midlands is a large region within the uk containing approximately % of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were first trimester cvs performed, by ten operators, in the west midlands (uk) in . this equates to . procedures per births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was (range - ). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan ( . %) ii) abnormal (> in ) risk on combined first trimester screening ( . %) iii) molecular genetic diagnosis ( . %) and iv) maternal request ( . %). using a combination of first trimester scanning and cvs, % had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was . % ( thci . - . %) up to days postnatal (perinatal loss) and . % ( thci . - . %) with days of procedure. the proportion of cvs in which an adequate sample was not obtained was . % ( th ci . - . %). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a yo who underwent a duodenal switch procedure in . she was discovered to be pregnant during evaluation for persistent malnutrition in . multiple prenatal ultrasounds were performed; the first at weeks gestation was unremarkable. the week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the wk sonogram. delivery by cesarean section was at / weeks secondary to nonreasuring fetal status. the birthweight was gm; apgars were , , and . postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <. ng/dl at time of delivery(normal > . ). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n= ) and term controls (n= ) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median = ; mean = . ) versus those born to women with uncomplicated term deliveries (median = ; mean = . , p< . ). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p< . ). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor= . [ . - . ], p= . ) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor= [ . - . ], p= . ) and cpap use (aor= . [ . - . ], p= . ) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, gh schuring-blom, kd lichtenbelt, jse laven, d van opstal, mjc eijkemans, nick s macklon. reproductive medicine gynaecology, university medical centre, utrecht, netherlands; clinical genetics, university medical centre, utrecht, netherlands; obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( years at weeks of gestation) in the netherlands between and . results: from a total of pregnancies, cvs results were analysed: in the ivf/icsi group and in the control group. the mean age of women in both groups was . years (mean difference - . , % ci - . - . ). foetal karyotype was missing in cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, . % vs. . % (odds ratio . , % ci . - . ) whereas the incidence of foetal chromosomal anomalies was increased . % vs. . % (odds ratio . , % ci . - . ) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, stacey l jeronis, john p gaughan, enrique hernandez, ossie geifman-holtzman. obstetrics and gynecology, duke university, durham, nc, usa; obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol ; : - ) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, % confidence intervals for a proportion were calculated and compared between groups. we found english-written publications reporting non-invasive rhd genotyping and reporting non-invasive rh ce genotyping from maternal blood. fourteen ( . %) of the rh d articles and three ( %) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: . % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in / ( %, % ci . , ), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy and trisomy . we present data showing that aneuploidy can be detected even when % of the starting dna sample was derived from a euploid source and only % was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a bp region of the promoter between - to - from tss. we now report that this sequence binds both nf-bp and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il- in vivo. materials and methods: emsa studies were performed using a bp oligonocleotide sequence (- to - ) , containing the bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il- ng/ml for hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p , c/ebp and h (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region - to - , which includes the bp response sequence. to further confirm the interaction between nf-bp with c/ebp a xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp or c/ebp . results: specific nf-bp and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p and c/ebp to this region of the otr promoter following stimulation with il- . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il- leads to binding of nf-b and c/ebp to the bp region. regulation of pro-labour genes by c/ebp, nf-b and ap- in human uterine myocytes. suren r sooranna, shirin khanjani, yun s lee, phillip r bennett, mark r johnson. imperial college parturition research group, imperial college, london, united kingdom; irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p ) and ap- (c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il- , pghs- and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il- , pghs- , otr, connexin- and fp. methods: primary cultures of human uterine myocytes (n= ) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in well plates to % confluence at which point expression constructs for c/ ebp (lap), nf-b(p ), ap- (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg was used as a filler construct. cells were cultured for and h after which culture medium was collected for elisa and cells frozen at - °c prior to rna extraction. copy numbers of il- , pghs- , otr, fp, connexin- and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: h post transfection with c/ebp (lap), nf-b (p ), c-fos and c-jun alone or in combination showed no significant changes in pghs - , otr and connexin- expression. over expression of p alone or together with either c-fos or c-jun increased fp expression by , and % respectively (p= . ). nf-bp consistently increased il- expression either alone (by %; p= . ) or in combination with lap, c-fos or c-jun (by , and % respectively; n= ; p= , ). rel a, lap, c-fos and c-jun together also increased il- expression (by %; p= . ) and a small but significant increase was seen with a combination c-fos and c-jun (by %; p= . ). the changes observed in il- expression were reflected in the medium il- concentration at h post transfection. in the presence of rela and c-fos il- increased from . ± . to . ± . pg/ml of culture medium (mean ± sem; n= ). conclusions: nf-b (p ) consistently increased fp and il- expression in human myometrium. the data suggest that pghs- activation has greater dependence upon other transcription factor(s) in addition to p . true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine ). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t d contains a kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met ) which serves as a translation initiation site for the generation of a pr isoform of kda. based on such findings condon et al (mol endo ) have focused on the possible role of kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc- santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg expression vector. in vivo translation produced a protein of only kda. furthermore overexpression of pr-c did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a kda protein. we constructed potential pr isoforms in psg vector with initiation sites at met and . in vivo translation produced proteins of approximately and kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c , pr-c and pr-c with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c both pr-c and pr-c significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr- (nc) and the sc- (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc- antibody detected only pr-a and pr-b very poorly. our data suggests that the sc- antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the kda but not the kda isoform might inhibit pr-b function. tnf receptor antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u ) (p< . ), but not by tnf receptor antibody (tnf rii ab), p mapk inhibitor (sb ) and jnk inhibitor (sp ). by western blot analysis, we found that the protein level of tnf receptor associate factor (traf ) was higher than that of tnf receptor associate factor (traf ) (traf >traf ) in untreated ct cells. however, after tnf treatment for h to h, traf protein level was increased, but traf protein level was reduced (traf >traf ). the increase of traf and decrease of traf were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within - min) and significantly increased phosphorylation of ikk , erk / and jnk / / and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf and decreased traf in ct cells (traf >traf ) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf >traf ). nuclear localization of nf-b p in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp- production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p -ap- pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp- expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected < weeks gestation (mean . weeks, sd . ) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) < weeks (n= ) and -< weeks (n= ) were compared to women with term births >= weeks (n= ). high tat was defined as > . ng/ml (highest quartile among women with term births) and high crp was defined as >= ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (< weeks, . %; -< weeks, . %; >= weeks . %, p< . ). women with high tat concentrations had a . -fold ( % ci: . - . ) increased risk for sptb < weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb -< weeks (or . , % ci . - . ). additional adjustment for elevated crp (>= ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (< weeks, or . [ . - . ]; -< weeks, or . [ . - . ]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb < weeks (or . , % ci . - . ). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb < weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of , participants, plasma crh and cortisol concentrations at and weeks gestation were available in controls who delivered after weeks and cases who delivered before weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at or weeks among women above or below the lowest quartile on the asaps (controls:p= . - . cases:p= . - . ). greater psychological stress was not associated with higher concentrations of crh or cortisol at or weeks(controls:p= . - . cases:p= . - . ). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at and weeks were lower in black women than non-black women (controls:p< . cases:p< . ). the median cortisol concentration among control black women was . g/ml at weeks compared to . g/ml among non-black women and . g/ml compared to . g/ml at weeks. black women reported less psychological stress than non-black women (p= . ) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp- in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp- .several evidences suggest that mmp- is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp- within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp- in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for h. relative expression of mmp- by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp- through the fm, fluorescein-conjugated prommp- was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp- was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp- was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp- in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since h (p< . ). experiments using labeled mmp- , repeatedly showed that after h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp- significantly decreased the activation of pro-mmp- . conclusions. our results demonstrate that the increased secretion of mmp- by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp- can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp- depends mainly on mmp- , which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of nmol/l to mmol/l and vpa ( nmol/l- mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). the mmi values for vpa were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin- expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin- (il- ) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il- expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in - m estradiol (e ) of e + - m mpa for days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after hours, il- levels in conditioned dm were measured by elisa and western blotting. in parallel -hour incubations, il- mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il- levels were similar in cultures maintained in e alone ( . ± . ) and e + mpa ( . ± . pg/ml/ug protein; mean ± sem; n = ). the addition of thrombin ( . u/ml) enhanced secreted il- levels by . ± . fold (p< . ) in incubations with e and by . ± . -fold (p< . ) in incubations with e + mpa. the inhibitory effect of mpa was statistically significant (p< . ). in confluent dcs incubated with e + mpa, exogenous thrombin ( . - . u/ml) elicited a concentration-dependent increase in secreted il- levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il- secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il- m-rna levels corresponded to protein changes. conclusion: thrombin enhances il- mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il- may contribute to the protection against ptd, and may explain the reported protective effects of administration of -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, -hydroxyestrogen ( ohe ), to -ethoxyestrogen ( meohe ). the comt substrate, -ohe , can exhibit an anti-estrogenic effect in multiple biologic assays while the methoxyestrogen ( -meoe ) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites ( ohe meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi ) . objective: to investigate the impact of comt inhibition on prostaglandin e (pge ) production by human amniotic membrane explants. study design: explants consisting of -cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at ºc. after a -hour incubation, explants were treated with the selective comt inhibitor ro - , at and m concentrations. the incubation media was harvested after and -hour intervals. the levels of prostaglandin e (pge ) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro - comt inhibitor induced major reductions in pge production in media collected from amnion explants of human fetal membranes. in the group treated with m of ro - for hours there was %± % reduction of pge after hours compared to untreated control (p< . ). in the amnion explants treated with m of ro - , there was %± % after hours and %± % after hours of treatment compared to untreated control (p< . ). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from non-pregnant women, women during elective cesarean section, woman after spontaneous parturition and after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s during hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately % in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around % and perlecan are significantly increased by around % compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox- ). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for hrs (ot: - m; n= ; triplicate samples; fold increase p< . ). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n= ; duplicate samples; p< . ). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il- b resulted in a significant upregulation of otr which peaked with a fold induction after hours (p< . ). il- caused a fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il- to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o -), hydrogen peroxide (h o ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod- ( . fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n= ; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod- , serum/glucocorticoidinduced protein kinase- (sgk- ) and the dna repair enzyme gadd a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix cm dilated; contractions every - minutes), a second sample was collected. results: seventeen ( %) of the subjects were multigravida. the inductions were for postdates(n= ); decreased fetal movements(n= ), fetal growth restriction(n= ), symphysis pubis dysfunction(n= ), diabetes mellitus(n= ), pre-eclampsia(n= ), fetal cardiac complication(n= ), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n= ). plasma aea levels increased significantly once labour was established (figure) and demonstrated in ( %) of the cases. the median (interquartile range) plasma aea level increased from . nm ( . - . ) at induction to . nm ( . - . ); *p= . ; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r = . , p= . pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p .the amount of dna was constant and psg was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg . affymetrix genechip u microarray was performed (n= , each group). we found that genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin demonstrated the highest, fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip ), chemokine ligand (ccl ), chemokine ligand (ccl ), pentaxin related gene (ptx ), interleukin (il ) and superoxide dismutase (sod- ). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand (ccl ), chemokine ligand (ccl ), chemokine ligand (cxcl ) and il . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd positive cells. treatment with il- or tnf-induced nuclear translocation of p in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, niccolo bosso, marianna andreani, agnese pizzardi, fulvio magni, anna locatelli. obstetrics and gynaecology, university of milano-bicocca, monza, italy; experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang ) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group , n= ) and term prom (group , n= ). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from women who delivered preterm (median[range], ga: [ - ] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n= ) within one week prior to amniocentesis were matched to controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of or confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin- (il- ) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr or (n= ) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il- levels compared to unexposed women (progestins (n= ): vs. crl (n= ): [ . - ] ng/ml, p= . ). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il- . conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n= /group) were treated with zk ( mg/rat, s.c.) alone or vehicle (controls) on day of gestation. other groups of rats were treated with zk in combination with various doses of r ( . , or mg/rat s.c) with and without ng ( mg s.c. pellet) from days to of gestation. all rats were sacrificed on day of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p< . was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p< . ) treated with vehicle only (ca. % fetuses delivered). ng treatment alone did not affect the delivery rate (p> . ) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p> . ). however r dose dependently reduced (p< . at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r plus ng (p< . ). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r , a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, jp rasanen, , dw sadowsky, m bertolino, m carbonatto, e gillio tos, s canali, j lacy, a chollet, mj novy. reprod sci, oregon primate res ctr, beaverton, or; ob/gyn, oregon health sci univ, or, usa; rbm, merck serono, italy; merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as , a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n= ) and pregnant ( - dga, term d; n= ) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as , and ot rescue therapy. ot infusions ( - mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as was administered p.o. at , , mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days , , and . the expression of each subtype of p x and trpc channels was measured by real time rt-pcr (abi ) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; . mg/kg) was injected into intraperitoneal cavity at day and the tissue was sampled after six hours. results; p x and p x were determined to be dominant subtypes of p x channel. the expression of p x was increased by % at day , compared with day and that of p x was enhanced by %. on the other hands, trpc and trpc were detected dominantly. the expression of trpc was increased three times in the late stages of gestation. however, trpc was suppressed by %. in the lps treated rat myometrium cox- mrna expression was measured to be fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p x , p x and trpc were enhanced by . , . and . times, but trpc was not changed. the mrna expression of p x , p x and trpc channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin ( nm) and carbachol ( μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p< . ). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp - consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp , and as well as hdac and form complexes with biotin-labelled fragments relating to sp - elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor -azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp - elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component - (pgrmc- ). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n= ), preterm and not in labor (n= ) and term and in labor (n= ). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc- and gapdh. abundance of pgrmc- protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc- in the human pregnancy myometrium. results: pgrmc- protein was identified in each biopsy specimen. there was a -fold increase in pgrmc- protein in term compared with preterm biopsies (relative to gapdh p< . ). the relative level of pgrmc- protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p= . ). pgrmc- immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc- protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc- is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, ) . the aim was to examine the mechanism of p inhibition. methods: uterine tissues from women (n= ) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p alone ( - to - m) or p bound to bovine serum albumin (bsa/p , - to - m p ), a progestin with low affinity to mpr (r , - - - m), or a non-sex steroid (cholesterol, - to - m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq , - m), guanylate cylase (odq, - m), phosphodiesterase (rolipram, - m), nitric oxide (no) synthases (l-name, - m) or a nuclear p receptor antagonist (mifepristone, mif, - m), followed by p . data were analyzed by anova for statistical differences (p< . ). results: p rapidly (< hour), effectively ( %) and dose-dependently inhibits spontaneous and kclinduced contractility (ed of < - m incubation of strips with zd- , at concentrations up to m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l- with a concentration of nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge ( - to - ) were added to l- ( nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd- ( nm) pre-treated strips. since the reduction in contractility caused by l- was greater than that caused by zd- , it is likely that the stimulating effect of pge acts predominantly via ep receptors. taken together with our previous data showing an increase in ep at labour, this data shows that targetting an ep receptor may be a useful strategy in managaing pre-term labour. recently a truncated kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n= ) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/ % fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with . % typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f- domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated ( kda) and the full length ( kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the kda erisoform, yet the expression of the kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f (pgf ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/- - m or - m pgf for , , or hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/- - m pgf and +/- m myristoylated pkc inhibitor ( - ) and examined for ptgfr cellular localization as described above. results: pgf treatment resulted in a dose dependent decrease in ptgfr membrane signal at , , and h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor ( - ) prevented the pgf induced decrease in membrane ptgfr at h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi- , promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia ( . % o ) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca + ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca + have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for - minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine ( um) rapidly abolishes spontaneous contractions, subsequent addition of nm oxytocin produced an initial, transient rise in force, approxiamately % compared to oxytocin alone, followed by high frequency oscillations in > % of strips. calcium-free solutions were used to confirm that oscillations were due to ca + entry. disabling the sr store using thapsigargin ( um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil ( um ) showed no inhibition of oscillations. an ip receptor and store-operated calcium channel inhibitor, -aminoethyldiphenylborate ( -apb) um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf- ( um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca + entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, mark r johnson, rachel m tribe. division of reproduction and endocrinology, king's college london, london, united kingdom; department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il- beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim , stim , orai and orai in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at - o c or used for cell culture. rna was extracted from whole tissue (n= ), primary cultured myometrial cells (n= ) and passaged (p ) myometrial cells (n= ) and stim , stim , orai and orai mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim and stim mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p< . ). however, there was no significant difference in either orai or orai expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim / and orai / mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg ( ) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin- (dallot et al., ) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock- inhibitor y- , oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells ( , per well) passage - , were embedded in . mg/ml collagen, in . ml aliquots, in dmem-f media on well plates (invitrogen) (dallot et al., ) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek cells, did not. kcl ( nm) stimulated an % increase in contractility (n= , p= . ) while nm tnf resulted in an . % increase (n= , p= . ), in comparison to unstimulated cells embedded in gel. the rock inhibitor y- ( m) inhibited contractility, with a . % decrease in collagen gel contractility (n= , p= . ). a % increase (n= , p= . ) in utsmc embedded collagen contractility was observed with nm oxytocin, which was antagonized by atosiban ( m) (n= ). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using d power doppler angiography (pda). nw jones, , nj raine-fenning, h mousa, mj taggart, k jayaprakasan, gj bugg. nottingham university hospitals nhs trust, united kingdom; nottingham university, united kingdom; university of newcastle upon tyne, united kingdom. methods d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) in a volume of myometrium at the uterine fundus of nulliparous women at term, in the first stage of uncomplicated spontaneous labour. d data sets were obtained during a single cycle of uterine relaxation (r ), contraction and subsequent relaxation (r ). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r . data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm ) of interest for r was . cm ( . - . cm ), for the contraction was . (fig. ) . the % change for all three indices between r and r was not significantly different. however, there was a significant difference between the contraction and r (p< . ). the mean intra-class correlation coefficient and % confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were . ( . - . ), . ( . - . ) and . ( . - . ), indicative of good inter-observer reliability. conclusion d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. . raine-fenning nj, et al. the inter-observer reliability of d pda acquisition within the female pelvis. ultrasound obstet gynecol. ; : - . the role of pgf on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, andre chollet, phillip r bennet. imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using x mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of - g. a novel antagonist to pgf (fpa) was studied in this model. the fpa has a ki of nm for fp and is - fold selective for fp compared with other prostanoid receptors. results: addition of pge and pgf after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge was being greater than that of pgf . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to m ( - ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa ( - to - ) decreased the total work done by -fold on strips treated with pgf beforehand in comparison the pgf -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf induced but not spontaneous contractions, it is likely that pgf does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women - weeks' gestation, and term between and weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt ). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n= ) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence ( d), the cells were incubated ( h) with nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< . ) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. ; : - . conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage ) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after days. single cell clones of stably transfected cells were further isolated by plating in well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage , ultr-ht cells grew . fold (p< . ) and . fold (p< . ) faster than ultr cells at passages and , respectively. currently single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm , and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at / ), nox family (nox , , and duox ), nox-associated genes (p phox, p phox, p phox, and rac / ), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca + ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca + ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca + ] i were measured simultaneously in fura- loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol , -dibutyrate (pdbu) applied at - m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca + ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca + ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca + ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca + ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca + ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs- and il- . on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr- , creating a src binding site. this promotes further fak phosphorylation at tyr- , and , enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek / in order to test the affect of fak kinase activity on expression on pghs- mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto -well flexible bottom plates coated in type i collagen. cells were subjected to % static stretch for up to minutes. cells were also incubated with either the rho kinase inhibitor y or the mek / inhibitor u prior to being stretched. western blots were performed using antibodies to fak phospho- , fak phospho- and erk / phospho- / , -actin was used as a loading control. rna was also extracted and levels of pghs- measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr- and tyr- with the greatest increases occurring at and minutes. however the increase at tyr- appeared to be greater than at tyr- . incubation with the rho kinase inhibitor reduced phosphorylation of fak- , however this did not affect phosphorylation of erk / or stretch induced up regulation of pghs- mrna. in contrast incubation with the mek / inhibitor reduced erk / phosphorylation and expression of pghs- mrna, whilst also reducing fak phosphorylation (n= ; p= . ). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs- expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs- expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section weeks gestation. strips of myometrium mm long , mm wide were cut and suspended under a resting tension of mn in organ baths of krebs gassed with % o / % co within hours of collection. progesterone or hp were added in a cumulative manner at -minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for hours, myometrial strips contracted in a rhythmic manner (amplitude . ± . mn, n= pairs). progesterone ( nm- m) produced a concentration dependent inhibitory effect on myometrial contractions (fig ), which was greater than that of vehicle (p< . ). maximum inhibition measured . ± . % and . ± . % for progesterone and vehicle, respectively. hp exerted an inhibitory effect, this was not significantly different from the vehicle (p> . ). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[ ]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [ ] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . reverse transcriptionpolymerase chain reaction (n = each) using gene specific primer against mcp and mcp receptor was performed to identify mcp messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp and mcp receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within days from delivery. we considered fetal macrosomia a birthweight g and big babies a birthweight g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was + weeks ( + sd). neonates weighted less than g, had a weight range between and g and weighted more than g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > mm which allowed to select fetuses. analysing this population we found true negative cases, false negatives, false positives and true positives, with a sensitivity of . % and a specificity of %. to detect big babies the roc curve analysis identified a cut off of ac > mm (n. fetuses), reaching a sensitivity of % and a specificity of %, without false negative cases and with false positives ( true positives, true negatives). the cases that weighted between and g, were included in the cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score . ) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at . g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at . g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =- ( . ) <(u.)) ( . ) )( . )"" ' ~. ) !( . )' d.'cbom< ( ) f( ))""" s( l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors , . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from st to rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n= , % non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)- , igf binding protein(bp)- and leptin, were measured in maternal plasma ( weeks gestation). results maternal growth (defined as increase in knee-height > mm/ days) was detected in % of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p< . ). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p< . ). these parameters correlated positively with maternal igf- and leptin but negatively with the igf inhibitor, igfbp- (p< . for all). subjects delivering sga infants gained significantly less weight (p< . ) and had lower igf- levels (p< . ) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf- and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf- and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c= . ± . and iugr= . ± . wks). an ultrasound examination was performed within hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * . . blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o umb uptake = qumb*(uv-ua) o content. results. as expected, average fetal and placental weights were significantly different in the studied groups ( ± and ± g in iugr vs ± and ± g in n) . iugr pregnancies showed a significant reduction in qumb ( . ± . vs . ± . ml/min; p< . ) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o sat, o cont and po in both uv and ua compared to n. (uv-ua)o content ( . ± . vs . ± . mmol/l; p< . ) and o umb uptake/kg ( . ± . vs . ± . mmol/min/kg; p< . ) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that % on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il- in pregnancies with iugr. calvin j hobel, chander p arora, adegoke adeniji, priya arora, susan e jackman, olga miadel, baldjyan lilit. ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency ( . - nmol/l) or even deficiency of vitamin d (< . nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin - (il- ) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il- and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for (oh)d using elisa. the samples were also analyzed for il- at three stages of pregnancy: t ( - weeks), t ( - weeks) and t ( - weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in of women with available samples from a behavior in pregnancy study (bips) . out of these subjects, were selected as matched case controls. circulating levels of vitamin d ( (oh)d) were significantly lower in iugr cases at each visit (p<. ). the levels indicated deficient ( . ± . nmol/l) vitamin d in iugr group at t but sufficient vitamin d levels ( . ± . nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t : ± . nmol/l vs ± . nmol /l; t : ± . nmol/l vs ± . nmol /l). at all three time intervals, significantly (p<. ) higher levels of il- were associated with the iugr cases ( pg/ml, pg/ml and pg/ml respectively) as compared to the controls ( pg/ml, pg/ml and pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il- expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global % maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology : ). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology : ). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n= ) or % of wt adjusted ctr diet (mnr, n= ) from . gestation (g) and fetuses were recovered at c-section under general anesthesia at . (n= ; ctr and mnr) and . g (n= ; ctr and mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x %) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at . g there was no difference in igf-ii or insulin fraction or density between groups. at . g, igf-ii fraction ( . ± . vs . ± . ,p< . ) and density ( . x ± . x vs . x ± . x , p< . ) and insulin fraction ( . ± . vs . ± . , p= . ) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at . g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut ? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut , hsd , the gluconeogenic and glycolytic enzymes pepck and g pdh and the transcription factors pgc and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. mean gestation po < mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at - d. hepatic mrna expression of glut , hsd , pepck, g pdh, pgc and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po (r = . , y= . x+ . , p< . ). liver weight was significantly lower in a cohort of pr-h fetuses (c, . ± . ; pr-n . ± . ; pr-h . ± . ; pr-h-rlg . ± . g:kg; p< . ). glut expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of hsd , pgc , ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p< . ). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in hsd expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc and ppar . interestingly the compensatory increase in hepatic glut expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, jeffrey gornbein, calvin j hobel. obstetrics gynecology, los angeles, ca, usa; biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and sonographic fetal biometric parameters at - weeks, - weeks, and - weeks. the rate of change in fetal weight was defined as x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were multi-ethnic women included in the study, after were excluded. they underwent a total of , exams. fetal gender (p= . ), maternal weight at st exam (p= . ), and cumulative maternal weight gain at exam (p < . ) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of . grams per days higher than females. the rate of fetal weight change increased by an average of . grams per days for each lb increase in maternal weight at the st exam. the fetal growth rate increased . grams per days for each lb of cumulative weight gain at the rd exam. conclusions: in this study, maternal weight at the st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. high risk patients. other predictors include the known risk factors of age < and > , single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes, ) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b -ar. methods: baboons were fed as ad lib controls (ctr) or % of wt adjusted ctr diet (mnr) from . gestation(g) with fetuses retrieved at c-section under general anesthesia at . or . g. protein expression determined by immunohistochemistry for b -ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x %. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at . . results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at . g = . ± . vs. . ± . %, p< . . b -ar fraction was lower following mnr at . g and at . g (p< . ; fig ) conclusions: mnr decreased b -ar over % at . g and % at . g. decreased fetal liver b -ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (< d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (> d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) , blood cell mrna was collected from pi steers (n= ; confirmed by virus isolation), or uninfected control steers (n= ) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp , bvdv naïve pregnant heifers (n= per group) were not infected (control) or infected with ncpbvdv on d. or d. of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d. ; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed genes that were differentially regulated in pi vs. controls (p< . , > . fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg - from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients > weeks of gestation were prospectively enrolled into an observational study ( / to / ). transvaginal sonographic examinations were performed every weeks until delivery. the shortest cervical length between - weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: asymptomatic patients were studied. . % of patients delivered an sga neonate ( / ). the median cervical length was mm ( to mm); patients had a cervical length < mm. of these, % ( / ) delivered an sga neonate. similarly, % ( / ) patients with a cervical length < mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < mm and < mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [ % ( / ) vs. % ( / ); p= . ]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: ) preconception start of folic acid use; ) start of folic acid use in first ten weeks of gestation; ) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age - and > weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from , pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of grams ( %ci . - . , p< . ) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of grams ( %ci . - . , p= . ) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of grams ( %ci - ) and grams ( %ci - ), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than grams when folic acid was not started preconceptionally (or . , %ci . - . ). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in severely premature iugr fetuses (estimated fetal weight < th percentile and umbilical artery pulsatility index > th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < . was considered statistically significant. results: all fetuses were delivered at < weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than - . resulted in iufd, whereas a score greater than - . resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than weeks resulted in nd and those fetuses at or greater than weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a % food restricted diet (mfr; n= ) or were fed ad libidum (control; n= ) from d until d of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n= ) p-value litter size (median, range) ( , ) ( , ) . fetal weight (mean ± sd) g . ± . . ± . < . placental weight (mean ± sd) g . ± . . ± . . collagen i (mean ± sem) ct . ± . . we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in non-pregnant ewes to induce pr. vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt- levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt- , in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method maternal peripheral venous samples were collected antenatally from two groups of pregnant women between - weeks of gestation. group a: uncomplicated normal pregnancies (n = ) and group b: pregnancies complicated by isolated placental vascular disease( n = ) as defined by birth weight less than th centile for gestation and umbilical artery doppler s:d ratio above the th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt- levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt- levels (figure ) in group a: normal pregnancies increased with gestation (p < . ). the sflt- levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = . ) and did not show a significant variation with gestation ( p = . ). the plasma vegf levels were below the detectable levels of the assay in all samples except normal pregnancies. the significantly increased maternal plasma sflt- levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of -reductases ( r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al neurosci : . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al repro sci # ). the aim of the present study was to determine the effects of suppression of -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term d). sham operated or gr dams received finasteride (a r inhibitor; mg/kg/day) during late gestation ( d until term). activated caspase- , a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, r and cytochrome p side chain cleavage (p scc) were measured by real time pcr and western blotting in fetuses ( d) and neonates h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by % whilst sparing brain weight. the number of activated caspase- positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase- activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p scc mrna and r protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from fetuses ranging from to weeks gestational age for a total of recordings. measurements were taken from mothers with a history of smoking (sm) and from mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in out of fetal recordings in the ns group and of in the sm group. the neonatal response rate was % for each group. the latencies were divided into three components: c ( - ms), c ( - ms) and c ( - ms). in both fetuses and neonates as well, there was a statistically significant difference (p< . ) between the two groups in the c -component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a and a a receptors. objective: this study was designed to test the hypothesis that ado a and a a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (> . term). intravascular infusion for h of dpcpx (a receptor antagonist) or zm (zm, a a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for h by having the ewe breathe a hypoxic gas mixture (fio = . ). data were analyzed by two-way repeated measures of anova. results: blockade of ado a receptors (n= ) increased significantly (p < . ) fetal concentrations of glucose [control (c): . ± . (se)]; experiment (e): . ± . mg/dl] and lactate (c: . ± . ; e: . ± mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a a receptors (n= ) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n= ), which did not alter pao or paco , increased concentrations of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . ). zm (n= ), but not dpcpx (n= ), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao torr), which increases ( - fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: . ± . ; e: . ± . ), and increased fetal levels of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: ) a receptors modulate plasma levels of glucose and lactate in normoxic fetuses; ) a a receptors mediate the adoinduced rise in plasma glucose and lactate; and ) a and a a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd- . objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and -day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr ( mg/kg/day; et a antagonist) or . % nahco vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day (term= days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day . oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days and . results are presented as means ± se. newborn -day neonate vehicle . ± . . ± . eta antagonist . ± . . ± . there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, jan b derks, martijn a oudijk, avnesh s thakor, tereza cindrova-davies, frank van bel, gerard ha visser, graham j burton, dino a giussani. perinatal center, university medical center utrecht, netherlands; department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child : , ) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at . of gestation, surgically instrumented sheep fetuses were submitted to i/r ( x min repeated ucc) under maternal allopurinol (n= ) or saline vehicle (n= ) infusion. fetal hearts were collected h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from uninstrumented fetal sheep at . gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox- , enos, hsp and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day for d, breathing either room air (nmx), or . % or % o ( % o hpx or . % o hpx). on day (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein (hsp ), actin (actg ), interleukin- (il- ), and glutamine synthetase (gs) were each up regulated, while cofilin- , brain-type creatine kinase (bb-ck), and - gtp binding protein were each down regulated. all protein changes were proportional to the hpx ( % o hpx vs nmx, % o hpx vs . % o hpx, p< . ). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il- , and gs have each been previously investigated, the potential roles of actg , bb-ck, beta- gtp binding protein, and cofilin- in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). intermittent umbilical cord occlusion occurs in % of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting min/ hour (mild group; n= ), min/hour (moderate group; n= ), min/hour (severe group; n = ) or no occlusion (control group; n= ). fetal arterial blood samples were drawn min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < . ). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < . ).elastin content of the superior mesenteric artery did not differ between the experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control . ± . . ± . -mild . ± . . ± . ** . ± . moderate . ± . . ± . ** † . ± . † severe . ± . * . ± . ** † † . ± . † † * p < . ; ** p < . : experimental groups compared to control † p < . ; † † p < . : compared to mild group the max in map was positively correlated with elastin content (r = . , p < . ). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. obgyn, chri, univ of western ontario, london, canada; obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n= ) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) ucos with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph . ± . to . ± . , p< . ). ± min prior to fetal ph drop < . , the sef of ecog began to increase abruptly during each fhr deceleration from ± hz up to ± hz (p< . ) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p< . ). conclusion: our findings suggest that in the animal model studied ( ) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; ( ) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph < . . these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e ) and progesterone (p ). however, the roles of e and p in da regulation have not been studied. objective: we hypothesized that: e and p have opposite effects in the da; that rising e and falling p levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e and p . methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc- , - , and serbp- was examined by rt-pcr and qpcr on days , , (term), and p . the effects of e and p ( - - - m) on the d fetal da were examined in a cannulated microvessel myography system. e and p effects were also studied in the presence of receptor antagonists (ici , ru ) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc- expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o conditions, e induced rapid, concentrationdependent dilation at - - - m (n= ); p induced progressive vasodilation at all doses (n= ). e and p -induced dilation occurred within - seconds of exposure. while e -dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e (n= ) suggesting antagonism of e effects. the vasodilatory effects of e were partially inhibited by pretreatment with l-name. p -dilated das did not respond to ru , whereas ru pretreated das showed a small, significant response to p (n= ), suggesting partial antagonism or signaling via alternative, ru -independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p . conclusions: contrary to our expectation, both e and p have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po (tpo ), and electrocorticographic (ecog) activity to arterial o tension (pao ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = ) we measured laser doppler cbf (ld-cbf), tpo , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency- % (sef ) in response to min moderate isocapnic hypoxia. results. ld-cbf, cerebral o delivery, tpo , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo decreased briefly to ± from a control value of ± torr; however, as ld-cbf increased ± %, and sef increased to ± from ± %, tpo returned to near normal value. with acute hypoxia (pao = ± torr) when in the lvhf state ld-cbf increased only ± %, as opposed to a ± % increase when in hvlf ecog state. with this degree of hypoxia, tpo decreased to ± torr, sef remained at ± %, and cerebral metabolic rate for o (cmro ) decreased ± % (p< . ). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo , cmro , and ecog state, with increased ld-cbf failing to restore cortical tpo . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd- ). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, kanchan m kaushal, malgorzata mlynarczyk, kimberly hyatt, dean a myers. ctr. for perinatal biol., loma linda univ., loma linda, ca; ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude ( , m) from day to - of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po was maintained at a level comparable to that observed at altitude ( mmhg) by nitrogen infusion. on dg, lth (n = ) and age-matched, normoxic control (n = ) fetuses were implantated with vascular catheters. each fetus received a min infusion of either saline vehicle, ng/kg of ovine crh or ng/kg of avp (estimated body weight/min) in a randomized order over consecutive days ( - dg). blood samples were collected at min (baseline prior to infusion), , , and min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at min) were significantly higher in the lth group compared to control ( ± vs. ± , respectively; p< . ). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p< . ). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control ( ± vs. ± respectively; p< . ), however peak levels were reached at between and min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels ( . ± . vs. . ± . , p< . , lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd and hd ). martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. ob/gyn, chri, university of western ontario, london, canada; ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco each lasting h or until ph decreased to < . . fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph ( . ± . ), fhr ( ± bpm) and bp ( ± mmhg) were within the physiological range. average depth of fhr dec for all uco was ± bpm and increased with higher lactate concentrations (r = . , p < . ) and lower ph (r = . , p = . ). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = . , p < . ). bp increased ± mmhg (p < . ) during mild uco (ph to . ± . ), ± mmhg during moderate uco (ph to . ± . , p < . ) and ± mmhg during severe uco (ph to . ± . , p < . ). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde in placental dysfunction following fetal bypass. mitali basu, r scott baker, christopher t lam, kenneth e clark, pirooz eghtesady. cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at - days gestation were placed on bypass for minutes and followed post-bypass for hours. placental samples were collected immediately prior to bypass and at and min post-bypass (n= ) and compared to a group of similarly instrumented controls, (n= ). placental enos, inos and pde protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p= . ). pre-bypass protein expression did not differ between groups. pde protein levels and phosphorylated pde- expression were both elevated min post bypass, and reduced min post bypass compared to sham, (p= . ). enos levels in the bypass group increased linearly from pre-bypass to min postbypass (p< . ), and were also elevated compared with shams (p< . ), while shams had declined significantly by min post bypass, (p< . ). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to min post-bypass, (p= . ), while shams trended towards decline by min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at and min post-bypass, (p< . ). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde , phosphorylated pde- , enos and ostensibly phosphorylated enos. increased pde expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at days of gestational age, pregnant sheep were submitted to days of hypoxemia ( % of fetal arterial po ; at day fetus ± vs ± . ; mother ± vs ± mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n= ) and hypoxemic (n= ) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of i-sarthran in nts and dmnx resulting from an increased expression of at receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; . ± . vs. . ± . , p< . ). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: . - . hz) and high frequencies (hf: . - . hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf . ± . vs ± . , p< . ; dbp lf/hf . ± . vs . ± . , p< . ). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf . ± vs . ± . , p< . ; alpha hf . ± . vs . ± . , p< . ). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat , may have potential long-term consequences for the development of hypertension. hd ;hd ;hl . objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses ( . ± . , rs= . p< . ) with linear regression with standardized coefficient of . (p< . ). in contrast, wsr ( ± ) is unchanged during the first , second and third trimesters (p= . ).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin- (et- )-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing % serum collected from ovine fetuses (n= ) that underwent min of cpb, then were maintained for min. serum was collected before cpb, from pump prime before initiation of cpb, min on cpb, or and min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested and hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et- was measured in the culture media by elisa. results: no production by huvec after and hr was stimulated above control levels by fetal serum collected during and up to min after fetal cpb (p< . ). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<. ). stimulation of et- after and hr of huvec culture peaked with serum collected at min after fetal cpb (p<. compared with control), but was elevated above control levels at each collection time point (p<. , table ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et- production during and for at least min after cpb. although the specific regulatory proteins remain to be identified, the no and et- pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n -( -iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; %o ) or . %o (hypoxia) in a hypoxic chamber for days prior to term (term= d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of - mg/kg/d for days. at d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n and stored at - o c until ready for analysis. the effect of total no product (no and no -, nox) of left ventricles of fetuses exposed to normoxia (n= ), hypoxia (n= ) and hypoxia plus l-nil (n= ) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by % and increased placenta/fetal body wt by % as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by %. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia ( . + . ) by . fold compared to normoxic controls ( . + . ). l-nil significantly decreased (p< . ) nox levels in hypoxic hearts by % ( . + . vs . + . ; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r= . , p< . ). bd ecf correlated strongly with bd blood (r= , p< . ). bd ecf increased by . ± . mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. ) . lactate, bd blood , bd ecf and bd gap correlated to ph (r= . , r= . , r= . and r= , respectively, all p< . ) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor- (sdf- ), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf- in brain hemispheres with or without hypoxic-ischemic lesion. sdf- expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf- expressing brain regions and we were able to show that these cells express the sdf- receptor cxcr on their surface. the functional implication of sdf- in directing hucb cell migration was determined by application of neutralizing sdf- antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf- in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd- mice on e receive intrauterine injection of lipopolysaccharide (lps) or saline. hours after injection, fetal brains from the left upper horn were harvested. fetal brains were removed from each dam with dams per treatment group. separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe av chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n= - per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. genes were significantly differentially expressed between lps and saline brains (p< . ). with a p value of < . , genes were differentially expressed. pathway analysis revealed significant involvement of ) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score . ) and ) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score . ). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in / genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice - days after birth (p -p ) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b . , colocalised with standard microglial marker iba . results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha- , alpha- , alpha-m, alpha-x, beta- and b . . the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p , decreased linearly over p and p and disappeared at p . this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p cortical microglia and formation of stubby processes in p fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p -p swm, roughly equivalent to early rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. , pp. - ) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia ) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol ) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci . , , a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in healthy fetuses at . (n= ), . (n= ), . (n= ), . (n= ), . (n= ), . (n= ) and . (n= ) of gestation (term days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image . , nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at . of gestation followed by the cortical white matter structures at . of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, ) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a -mhz transducer at hh stages , and which are comparable to humans at to weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro ), cortical tissue po (tpo ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin- , which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin- modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from d to d (term) with either room air (nmx) or . % or % o . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin- . total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin- , bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek , the upstream activator of erk / , which was under the control of the pan-neuronal talpha alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p c bl/ mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with % oxygen for mins at °c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek dn was associated with a reduction in erk / activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, min hi insult (*p< . , **p< . , anova). similar protective effects of mek dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, ) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day , wistar rats were divided: control ( % o ) + melatonin ( g.ml - drinking water), hypoxia ( % o ) + melatonin, and undernutrition ( % reduction in food intake) + melatonin (n= per group). on day , dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: . ± . g; brain/body weight ratio: . ± . mg/g; n= ), both hypoxic ( . ± . g; . ± . mg/g, n= ) and undernourished pregnancy ( . ± . g; . ± . mg/g, n= ) promoted asymmetric iugr (p< . ), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic ( . + . , n= ), hypoxic ( . ± . , n= ) and undernourished ( . ± . , n= ) pregnancies relative to untreated controls ( . ± . , n= ; p< . ). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig. objective: midkine (mk) is a -kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: ) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term ( -fold: wks vs. wks). ) tissue transglutaminase known to polymerize mk was abundant in af. ) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). ) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan- were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan- were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav hfix vector ( - x p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = ) or late (n = ) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected days after late ip injection ( % and % normal human levels). early gestation ip injection gave . % and . % at and days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels ( . %) year after birth in early and months after birth in late gestation injected lambs. up to year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor has a positive effect on estrogen production throughout pregnancy. urocortin (ucn ), a novel peptide of the crh family binding exclusively crh receptor , is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn , in presence of dheas significantly increased e levels at , and hours treatment, while in presence of androstenedione and testosterone an increase in e secretion was detected only at , and hours (at different ucn doses). both p arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn caused a synergistic increase. anti-sauvagine (a crh type receptor antagonist) resulted in a significantly attenuated ucn effects on e secretion and on p arom mrna and protein expression. conclusions: the present study supports a possible role of ucn on placental e biosynthesis: e secretion and p arom transcript and protein expression were significantly increased after ucn treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn . a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss- gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss- , a -amino-acid peptide encoded by a putative metastasis suppressor gene. kiss- acts on its placental g protein-coupled receptors (kiss- r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss- in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss- at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss- protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss- mrna expression was significantly higher (p< . ) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss- levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss- mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss- on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( - % o ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk / , which stabilizes hypoxia-inducible transcription factor- (hif- ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf -and vegf-stimulated cell proliferation partly via activating erk / and stabilizing hif- protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( % o ) or hypoxia ( % o ) for days were treated with fgf and vegf. after days, the number of cells was determined. activation of erk / and levels of hif- protein were determined by western analysis. results: under normoxia, fgf and vegf stimulated (p < . ) cell proliferation and induced ( min, p < . ) erk / phosphorylation in hpae and hpme cells. hypoxia promoted (p < . ) fgf -and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < . ) such actions in hpme cells. hypoxia enhanced fgf -, but not vegf-induced erk / phosphorylation in hpae cells. in contrast, hypoxia promoted (p < . ) vegf-, but not fgf -induced erk / phosphorylation in hpme cells. hypoxia increased (p < . ) hif- levels in the hpae cells: first detected at . hr and maintained up to hr. hpme cells had high basal levels of hif- ( folds; p < . ) compared with hpae cells. hypoxia did not alter hif- levels up to hr and decreased (p < . ) hif- levels at hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf -and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif- protein levels. however, inhibition by hypoxia on fgf -and vegf-stimulated hpme cell proliferation is associated with relatively high hif- levels in the first hr and decreased hif- levels at hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif- levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day of gestation (term= days), housed in under h- h light-dark conditions (lights on ). lab chow and water were continuously available. on day , pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at - hr (zeitgeiber time, zt ) or - hr (zt ). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of bp size was consistently identifiable in placenta harvested at zt but not at zt . nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt in giant trophoblast (gt) cells (gt-crf: zt = . ± . au, zt = . ± . au, p= . ) but not in spongiotrophoblast cells (stc) (stc-crf: zt = . ± . au, zt = . ± . au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt = . ± . , zt = . ± . au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt . these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp , expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp , and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr- /svneo cells and term placenta-like trophoblast carcinoma cell line jeg- . cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of . , and m for hrs (aqp mrna expression) and hrs (aqp protein expression). for time course experiments, cells were incubated with μm of either sp-camp or forskolin for , and hrs at °c, % co . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp mrna expression in both cell lines after hrs (p< . ) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. m of sp-camp and forskolin stimulated aqp mrna expression after hrs in htr- /svneo cells and after hrs in jeg- cells (p< . ). m of either sp-camp or forskolin stimulated aqp protein expression in both cell lines after hrs (p< . ) and the expression remained high at hrs. conclusion: aqp gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin- in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n= ) collected from sprague-dawley pregnant rats on day of gestation (term= ) were either fixed in % paraformaldehyde solution (ph . ) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc- santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro . software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: . ± . , giant trophoblast cells: . ± . , spongiotrophoblasts: . ± . , fetal membranes: . ± . . pcr analyses identified a single bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene (per ), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day of gestation and housed in a controlled environment ( - h lights on) with free assess to food and water. placentas collected on days and of gestation (term= days) between and . am were fixed in % paraformaldehyde and paraffin embedded. for each gestational ages a total of placentas from different pregnant rats were used. paraffin sections ( sections per placenta) were subjected to immunohistochemical analysis with antibody to per ( : , santa cruz biotechnology, ca) by abc technique and , 'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro . software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < . as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day of gestation, we conclude that fetalmaternal interactions in per regulation disappear at term (or at the time of birth.). our findings imply that per may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n= ) on day of gestation were given a single subcutaneous injection (at : am) of betamethasone ( μg/kg body weight) while control pregnant rats (n= ) received saline. dams were exposed to isoflurane anesthesia at : am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc- , santa cruz biotechnology), with immunoreactive material identified by abc technique using , ' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro . plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p< . considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (< %) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p< . ). among the cells, gt ( ± %) demonstrated a greater percentage of cells expressing gc-nr than did st ( ± %) or (lb= ± %) (p< . ), though there was similar immunoreactive intensities (gt: . ± . , st: . ± . , lb: ± . au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on ± days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf( : - : , phoenix pharmaceuticals, ca,) crf binding protein ( : - : , sc ) and gr ( : - : , sc: , santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro . plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf ( . ± . au), crf-bp ( . ± . au) and nuclear-gr ( . ± . au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn- ), a amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn- protein and ucn- mrna. a number of published studies indicate that ucn- is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn- protein and ucn- mrna. methods: placenta (n= ) collected from pregnant rats at day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn- ( : to : , sigma) by standard abc technique. control sections were incubated with omission of ucn- antibody. immunoreactive materials on the sections were identified using , ' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn- polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< . ). pcr analyses revealed the presence of a single band of bp, consistent with ucn- mrna. conclusion: similar to human placenta, rat placenta expresses ucn- protein and ucn- mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from oc and normal pregnancies (np), and human livers were collected and preserved in rnalater. explant cultures were prepared from a further np placentas and cultured for days at % oxygen. on day they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l . results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, jenny chambers, jo wyatt-ashmead, kishore bhakoo, catherine williamson. imperial college, london, united kingdom; hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects . % of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from oc and normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further np placentas, and cultured for days at % oxygen. on day a subset were treated with udca overnight, and on day the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for hours prior to fixing. m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p= . ). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge ) placental volume measurement from art pregnancies (n= ; - wks) were compared with data from normal controls (n= ) matched for gestational age. data were analysed with software stata . results. mean placental volume was . ml (sd± . ) in art pregnancies and . ml (sd± . ) in normal controls. mean difference resulted in . ml (- - . ) and was statistically different (p= . ). multiple linear regression analysis showed a statistically significant interaction (p= . ) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets ( . weeks ± . ) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: g (sd± ) vs .(sd± . ) (p= . ). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor- (ad-igf- ) in trophoblast cells. mounira habli, datis alae, foong yen lim, jignesh parvadia, timothy crombleholme. obstetrics and gynecology; pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf- corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf- ;we examined the effect of ad-igf- on trophoblast proliferation, differentiation and invasion. methods: rcho- trophoblast cell line derived from rat choricarcinoma was used. ad-igf- and galactosidase transgene (ad-lacz) were given at moi of : and : in all the experiments.transduction efficiency was assessed hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho- was measured by using bdmatrigel invasion chamber kit. rcho- proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho- differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen- antibodies(specific hormone produced by giant cells). results: % efficiency of gene transfer of ad-lacz to rcho- cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at and hrs. ad-igf- induced morphologic differentiation of trophoblast cells compared to control (fig ) at hrs.ad-igf- induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group( % at moi vs . % in control p< . )(fig ). conclusion: ad-igf- gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin- is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin- action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons - . the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional -deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a -deletion variant and a novel splice variant, which was identified as partial -partial deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv -transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin- to induce different effects on the two major cell types of the placenta. (supported by grant , jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, katia pacioni, emanuela marinoni, giovanna corona, massimo moscarini, alfredo patella, romolo di iorio. department of gynecology, perinatology and child health, university "la sapienza", rome, italy; department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n= ) were cultured in presence or absence of hypoxia ( % o ) and treated with or without betamethasone at the dose of - , - , - m. media and cells were collected at h and at , and h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates - % of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with % o / % co at °c. viability was demonstrated by contraction to mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in , pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of ng/ml and ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every minutes for total minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p , bax, bcl- , aif, and pro-caspase- ) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was . +/- . ml/min and . +/- . ml/min at the low and high carboplatin concentrations, respectively. an estimated % increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from to ng/ml. the placental perfusion experiments conducted at carboplatin and ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= . ) in women delivering preterm (n= ; ucn= . ± . pg/ml) (m ± se) than in those delivering at term (n= ; ucn= . ± . pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, elisabetta federico, francesca caprio, maria teresa schettino, gabriele coppola, antonio de luca, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n= ), first trimester abortion (miscarriage) (n= ), delivery section (n= ), spontaneous birth (n= ), preeclampsia (n= ) and diabetes (n= ). the gestation period ranged from to weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations ( % %) to mimic physiological and non-physiological intervillous o tensions. the d media was concentrated, immunodepleted to remove fibrinogen (using beckman igy- columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot . (applied biosystems). results: when matched pooled (n= ) media samples were subjected to our proteomics workflow over proteins were identified with a calculated false positive identification rate of < %. of these proteins a total of display a statistically significant difference in protein levels between the % % o samples (p=< . ). from a list of proteins of interest we selected proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il- (pg/ml) in % o : . , range . to . ; % o : . , range . to . ; values are median with interquartile range. *** p< . , mann-whitney test (n= ) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., ) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c c myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n= ) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg- ) and other cell lines (mrc- , hl- , huvec) were cultured in the absence or presence of μm forskolin or solvent control for - days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg- ), cultured endothelium (huvec), and a fibroblast cell line (mrc- ), but was not detected in a leukemic cell line (hl- ). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin- levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, violetta kolesnikova, carri tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta ( ), : - ) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ), excluding elective terminations and spontaneous abortions before weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: ) neonatal sex; ) weight (iugr calculated by routine methods), ) trimmed weight of the placenta (sga calculated by routine methods), ) gross placental infarctions, ) gestational age at delivery, and ) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( . ; p < . ). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n= ), f (n= ) and m blood (n= ) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg- )beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip ´ protocol. samples were scanned on the illumina beadarrayer gx reader using illumina beadscan image data acquisition software (ver. . . . ). differential gene expression data analysis was performed using illumina beadstudio software (ver. . . . ). results. the detection level of gene transcripts using illumina methodology with control human rna was - at p< . . gene transcripts were detected in f blood and in maternal blood. transcripts were uniquely expressed in fetal blood. transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was and of these genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of / ( %) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, ( %) had placental histologic examination performed, and in / the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score to . results: mean interval between meconium appearance and delivery was . ± . min (range - ). after exclusion of cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in / cases at the level of amniochorionic membranes, in / cases at the placenta, and in / cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p= . ), in the placenta (p= . ), in the cord (p= . ), or score of severity of meconium uptake (p= . ). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to . hours. transfer of bisphenol a across the human placenta. biju balakrishnan, , kimiora henare, , eric thorstensen, , murray d mitchell. , the liggins institute, university of auckland; national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa ( ng/ml), antipyrine (ap) ( μg/ml), and fitc dextran (fitc-dx) ( . μg/ml) were added to the maternal perfusate, and perfusion was continued for hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the -hour perfusion period. the physical parameters observed (fetal pressure < mmhg, ph range . - . ) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of < ml/hr, and by < % materno-fetal transfer of fitc-dx. an observed ap transfer of - % further validated our model. bpa first appeared in the fetal compartment within minutes of perfusion and reached a peak of about - % of maternal concentration within hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, katsu naruse, , barbara a innes, stephen c robson, judith n bulmer. institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent ( - and - weeks gestation, n= each group). . × cd + unk cells were positively selected from decidua and co-cultured with evt ( . × ) or cytotrophoblast (ctb; . × ) purified from the same placenta for h in direct or indirect contact (n= each group). angiogenin, ang , pdgf-bb, fgf-basic, timp , icam and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at - weeks gestation unk secretion of ang (p= . ), icam (p= . ) and plgf (p= . ) was increased in the presence of evt compared with ctb. no differences were observed at - weeks gestation. in addition, at - weeks gestation unk secretion of ang (p= . ), vegf-c (p= . ) and ang (p= . ) was increased in direct co-culture with evt compared with indirect co-culture. at - weeks gestation unk secretion of timp- (p= . ) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. -d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a % haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between and ml/min, whilst maternal-side flow was held at ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = . hz, wall motion filter = low , and gain = . ). three -d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, brian a kilburn, anelia petkova, samuel s edwin, roberto romero, d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n= ). chorionic villous explants were prepared and cultured at either % or % o and treated with the hbegf antagonist crm or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm ( mg/ml) increased trophoblast cell death after culturing villous explants h at % o (p< . ), but only slightly affected proliferative capacity. culture at % o increased trophoblast cell death % above explants incubated at % o (p< . ). trophoblast cell proliferation decreased after h in explants cultured at either % or % o (p< . ). exogenous hbegf ( nm) prevented the elevation of cell death during hypoxia (p< . ) and maintained nuclear ki expression at %, but not %, o . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, emily king, carrie tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately % of placentas routinely submitted to pathologists (beebe et al. obstet gynecol ; : - ) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ) was performed, excluding abortions before weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: ) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), ) gestational age, ) maternal diagnosis, ) apgar scores, and ) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: ) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least / hpf), ) chorioamnionitis, and ) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common ( / , %) and was significantly more frequent in c-sections performed for fetal distress ( / cases, %) (x . ; p-value < . ). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams % food-restricted from day of gestation. placentas were harvested at embryonic days and (n= ). placentas were fixed in % pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at : dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated . % relative apoptotic index when compared to . % in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of . + . in food restricted placentas as compared to . + . per μm surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e than at e , with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, avish arora, lilit baldjyan, sharon k sugano, olga miadel, adegoke adeniji, michael g ross, calvin j hobel. ob-gyn, harbor-ucla medical center, torrance, ca, usa; ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm ( to < weeks, n= ) umbilical cord ( pieces of mm thickness taken at - cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at - c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of , , kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the kda major band is either glycosylated crf-bp precursor or glycosylated kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a , a a, a b, and a . recently our study has shown that increased a receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n= ) and preeclamptic (n= ) pregnancies were cultured at high ( %) and low ( %) oxygen levels for days. explants were analyzed for mmp- /- and timp- /- by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a receptor agonist, cl-ib-meca and a receptor antagonist, mre. mmp- / - expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp- /- activity was evaluated by zymogram assay. results: there were significantly increased a receptor intensity and reduced mmp- /- and timp- /- expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp- /- and timp- /- expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp- /- . this cl-ib-meca-induced expression of mmp- /- was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a receptors in the trophoblast and suggest that adenosine a receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, timothy a sato, anderson wang, jeffrey a keelan, anna p ponnampalam, michelle glass. liggins institute; department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids -arachidonoyl glycerol ( ag) and anandamide (aea) and with the synthetic cannabinoid cp , , to determine the ability of these substances to modulate prostaglandin e (pge ) production. pge was measured by radioimmunoassay. the explants were also treated with cp , in the presence of either sr a (a selective antagonist of the cannabinoid receptor cb ) or ns (a cox- inhibitor), to determine whether any observed stimulation of pge production was mediated through cox- activity and/or the cb receptor. cox- , cox- , cpla and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge production caused by cp , was abrogated by co-treatment with either sr a or ns , illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb agonism and cox- . preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox- expression. however, there was no change in cox- expression and no evidence either for up-regulation of cpla or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor- , but not vegfr- or vegfr- , during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, hongyan tang, raul gomez, bronislaw pytowski, daniel j hicklin, jan kitajewski, mark v sauer, ralf c zimmermann. division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr- , r- , and r- are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr- , r- , and r- function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed . , prior to embryonic expression of these receptors. embryonic development was evaluated on ed . , blood vessel density and apoptosis on ed . , and cellular proliferation on ed . (n= per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr- and vegfr- , but not vegfr- resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr- mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr- pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed . control anti r- anti r- anti r- no. of implantation sites . +/- . +/- . . +/- . . +/- . hoxa encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa in multiple cellular model-systems. using microarray technologies, we identified putative hoxa target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified statistically-significant genes regulated by hoxa overexpression of which genes were down-regulated greater than -fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the -phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a fold expression decrease when hoxa is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to % when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa is also expressed in leukocytes. here we hypothesized that hoxa would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa mrna in u cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa is expressed in leukocytes in vivo, peripheral leukocyte hoxa mrna expression was measured over sequential estrus cycles in mature cd nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa mrna expression levels in u cells. in a murine model, we demonstrated that hoxa mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa expression is prior to proestrus and increased up to -fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. known soluble mediators of implantation were measured using a multiplex immunoassay, namely il ß, il , il , il , il , il , il , il , tnf , vegf, ifn , eotaxin, mcp- , ip- , dkk- and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p< . ) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the analysed were detectable in - % of the samples. il was detectable in % of samples, dkk- %, il- %, il- %, il- % and hbegf was detectable in % of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p = . ). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n= ) were treated with daily leuprolide ( . mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, . mg/d) for days. after day of e, subjects also received daily i.m. injections of p, randomized to mg/d (sub-physiological) or mg/d (physiological). endometrial biopsy was performed after days of combined e and p treatment. additional untreated women had biopsies performed days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the mg/d p group were . ( . ) and . ( . ) ng/dl, respectively, while those in the mg/d group were . ( . ) and . ( . ) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was . ( . ), . ( . ), and . ( . ) for the mg, mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, integrin subunit, osteopontin, cyr , egr- , fkb , c-fos, and cd ), although variability of gene expression was greater in those who received mg/d p than in those who received mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u hd- ). endometrial ip attracts trophectoderm through cxcr interaction. simcha yagel, caryn greenfield, hen sela, jacob hanna, irit manaster, orna singer, ronit haimov-kochman, shira natanson-yaron, diana prus, benjamin reubinoff, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; lautenberg center for immunology, jerusalem, israel; human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec- cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr and cxcr . immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr and cxcr as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp- and jeg cells, and blastocyst trophectoderm expressed principally cxcr . elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip and il . migration assays demonstrated that ip significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr and cxcr , and il and ip . these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip -cxcr is the most relevant to early implantation as only cxcr is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. d page analysis with dige minimal dye labelling was conducted across the pi range - on endometrial tissue from either the mid-proliferative or midsecretory phases (n= /group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of spots were detected: were differentially expressed (p < . ) with spots having an overall false discovery rate < % (q < . ). hierarchical clustering analysis revealed that these proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural ( ), transport ( ), regulatory ( ), membrane ( ), enzyme ( ), motor ( ) and others ( ). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, hn jabbour, p lourenco, s wright, s battersby, arw williams, hod critchley. reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n= ) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin (pk ) and prokineticin receptor (pkr ) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk and pkr protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk and pkr . conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e ) inhibits glycogen synthase kinase beta (gsk- ), resulting in nuclear accumulation of cyclin d and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk- inhibitor, licl, results in epithelial cell proliferation in the absence of e (zhu, pollard. pnas. ; : ) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged - . immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after weeks of out-growth, mice were ovariectomized, and replaced with e or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: - % of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst staining confirmed that xenografts were made up of human cells ©. e (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki , with li of . ± . and . ± . , respectively, p = . ). minichromosome maintenance- , a protein involved in dna replication licensing, was more sensitive for e -treated cells synthesizing dna than was ki staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e . licl (f) induced proliferation similar to e and greater than control (li = . ± . , p= . for e vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for h. the cells were treated with estradiol (e , nm, h), progesterone (p , nm, h), gnrha (la μm, h), e ( h) followed by p (last h) and gnrha plus e plus p where, gnrha added either first, second or last in order at h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il- , ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il- , il- , il- and ifn-. treatment with e and p , either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e and p significantly diminished e , p or e plus p induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd . introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor- function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, , cobi heijnen, esther baart, karima amarouchi, carolien boomsma, janet carver, helen mardon, annemieke kavelaars, nick macklon. reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: human embryos, cryopreserved at day after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day , the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il- , il- , il- , il- , il- , il- , il- , il- , tnf-, mcp- , ip- , eotaxin and hb-egf using a multiplex immunoassay. day supernatants from culture systems in which no embryo had been placed were also analysed as controls. results: out of ( %) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod ), we pre-treated human ishikawa cells with or without ovarian steroid hormones ( -estradiol + progesterone; ep), fa- (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa- resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and -arachidonoyl glycerol ( -ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n= ). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn- -diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for ag; and monoacylglycerol lipase (magl) and cyclooxygenase- (cox ), the degradative enzymes for ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, # ). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p= . ) was seen. magl gene expression was higher in the secretory phase than the pro phase (p= . ). cox gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p< . for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, ag's degradative enzymes increase in the secretory phase, suggesting that lower ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin- beta (il- ) regulates il- signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, cf yen, cp chen, f schatz, cj lockwood. obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il- levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il- contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il- promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il- may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: ) localized il- mrna levels in preeclamptic versus normal decidual sections; ) evaluated mechanisms regulating il- synthesis by targeting intracellular signaling pathways with specific inhibitors; ) identified potential il- targets by immunolocalizing the il- receptor (il- r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il- mrna in normal versus preeclamptic decidua. il- r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e and mpa ± il- ( ng/ml) ± an inhibitor of p mapk (sb ) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for hrs. an elisa measured secreted il- levels. results: il- mrna was present primarily in decidual cells with increased il- mrna levels observed in pe. preferential expression of the il- r was observed on decidual cells in placental bed biopsies. compared with basal il- levels ( . ± . pg/ml/ g cell protein) by decidual cells, il- enhanced il- output by decidual cells ( . ± . pg/ml/ g cell protein). only the p mapk inhibitor significantly reduced this output to . ± . pg/ml/ g cell protein (n= , p< . ). our results indicate that inflammatory cytokine enhances il- synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p mapk. such il- is likely to act as an autocrine/paracrine effector via decidual cell-expressed il- r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, shira natanson-yaron, caryn greenfield, achinoam lev-sagie, lichtenstein michal, haya lorberboum-galsky, israel vlodavsky, simcha yagel, arye hurwitz. ob/ gyn, jerusalem, israel; cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased fold in secretory phase (d ) compared to proliferative phase (d ) endometrium. heparanase transcript levels increased fold during hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin- (il- ) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, , tien-hung huang, chin-jung li, chyi-long lee, , hsin-shih wang, , yung-kuei soong. , obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il- system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il- system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n= ) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n= ) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh ( bp), il- ( bp), il- bp ( bp) and il- r ( bp). quantitative il- and il- bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il- system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il- and il- bp as well as il- r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and -ct method, a significantly higher il- expression in tubal ectopic implantation and lower ratio of il- antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p< . ). immunoreactive il- system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il- system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like (fstl ). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl in ovariectomized rats. adult female wistar rats (n= ) were ovariectomized and received one week later a single dose of estradiol benzoate ( . mg/kg body weight, i.m. injection), either alone (n= ) or associated with depot medroxyprogesterone acetate ( . mg/kg body weight, i.m. injection, n= ), or oil vehicle (control group, n= ). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p (rpp ) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone ( . fold increase over controls, p< . ) and to the same extent in rats receiving estradiol plus medroxyprogesterone ( . fold increase over controls, p< . ). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone ( . fold compared to controls, p< . ) and estrogen plus progestin ( . fold compared to controls, p< . ), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl expression was similar in the groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, irit manaster, jacob hanna, ronit haimov-kochman, miri godin, yuval bdolach, caryn greenfield, shira natanson-yaron, arye hurwitz, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp , nkp and nkp , collectively known as natural cytotoxicity receptors (ncrs), and nkg d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days and . a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd , cd , nkp , nkp , nkp and nkg d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd -ig and nkg d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il- with and without il- were performed. results: endometrial lymphocytes of day and in these women are mostly cd bright cd -nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp , nkp . nkp and nkg d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp , nkp and nkg d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, amy o robb, imogen b smith, nicholas l mills, hilary od critchley, david e newby. centre for reproductive biology; centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: healthy, nulliparous, pre-menopausal, non-smoking women (mean age years), with regular menses ( - days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - and - ). cpmas (monocytes positive for the platelet marker cd a) were measured by flow-cytometry. psp-selectin expression was calculated on cd a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was > nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle ( . ± . vs. . ± . %, p= . and . ± . vs . ± . %, p< . , respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p= . ). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd -immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt- , kdr, angiopoietin- , angiopoietin- , tie- and the membrane-type matrix metalloproteinases mt -, mt -, mt -and mt -mmp were determined at mrna and antigen level and cd -positive unk cells, cd -positive macrophages, proliferation (ki ) and apoptosis (activated caspase- ) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt , kdr, mt -and mt -mmp was increased at the implantation site of cases. ki and active caspase- showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, hong y wen, janice endsley, alison hogg, hui-qun wang, manubai nagamani, chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd decidual nk cells were purified by magnetic bead isolation. results: ) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; ) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd staining, express adm receptor components crlr/ramp /ramp and their mrna expressions were futher confirmed by rt-pcr; ) k target cell killing assay indicates that adm inhibits cytokine il- /il- -induced decidual nk cell cytotoxicity; and ) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at - weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at - weeks gestation (not at - weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd + unk cells were isolated from early pregnancy decidua ( - and - weeks gestation, n= each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid . transfected with either hla-g ( g) or a mock cdna ( cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n= each group and each gestational age) for hours. cell supernatants were analysed for cytokines using a fastquant® th /th multiplex protein assay (il- , il- , il- , il- , il- , tnf-, il- , il- , ifn-) or by standard elisa (tgf- ). the effect of direct co-culture of unk cells with g compared with co-culture with cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the g or cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either g or cdna cells at either gestational age. in addition, direct or indirect co-culture of g or cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either - or - weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg d receptor pathway plays a fundamental role in target elimination through binding nkg d ligands on the cell surface. reduction in the nkg d ligand, ulbp , expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg d pathway in this process. material and methods: expression of nkg d ligands; ulbp , ulbp , mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd +cd -) from three healthy volunteers were isolated using macs system with purity greater than % and kept in interleukin overnight. fibroblast elimination with and without ulbp blocking was investigated following -hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg d ligands (ulbp , ulbp , mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp expression (p = . ). western blot analysis also revealed a lower ulbp protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was % lower in at in comparison with np fibroblasts. blocking ulbp expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by %, supporting the role of nkg d receptor pathway in the process. conclusions: our results demonstrate that nkg d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin- is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin- is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals gene in primates. methods: seven primate nucleotide sequences were generated, aligned to vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin- expression was studied by immunohistochemistry and western blot. results: ) the lgals gene had high sequence identity among all investigated species. ) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals gene in placental mammals (dn/ds= . ); ) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. ) immunostaining showed a uniformly abundant and ubiquitous galectin- expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin- in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, , william murk, umit a kayisli, aydin arici. department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin- (il- ) is a recently discovered heterodimeric cytokine, comprised by a novel p subunit and a p subunit shared by il- . it has biological activities that are similar to but distinct from il- , and is known to be involved in th /th cell class switching and the regulation of cytokines such as ifn-gamma, il- , tnf-alpha, and il- . early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il- in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n= ) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n= ) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il- p antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p< . considered significant. results: il- immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il- immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il- staining compared to escs from non-pregnant endometrium (p< . ). il- immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p< . ). moreover, egcs from the early secretory phase (p< . ) and first trimester tissues (p< . ) showed higher il- immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il- expression in the human endometrium and decidua. these results suggest that il- has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il- production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat . t cells, initially grown in estrogen free media, were incubated in pm (representing postmenopausal levels) or pm (premenopausal levels) of estradiol (e ) for hours. cells were either resting or activated with a phorbol ester, -phorbol -myristate -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il- . expression of signaling protein components, cd and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd , jak , and jak gene expression. a p value of < . was considered significant. results: jurkat cells exposed to pm e and activated exhibited significantly diminished numbers of il- producing colonies compared to t cells exposed to pm ( . ± . vs. . ± . colonies, p< . ). analysis of cellular cd and jak protein demonstrated that jurkat cells incubated in pm e expressed a . -fold decrease in cd and . -fold decrease in jak compared with cells incubated in pm e (p< . ). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd , jak and jak were significantly decreased in jurkat cells incubated in low levels of estrogen ( . , . , and . fold, respectively, compared to pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il- following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd , jak and jak gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of novel cytokines (il- and ) in human fetal membranes. judith eckardt, , stephen j fortunato, holger maul, ramkumar menon. the perinatal research center, nashville, tn, usa; womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) and are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il- and il- in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n= ) from cesareans at term (normal, not in labor) were placed in an organ explant system for hours and were stimulated with lps for an additional hrs. fetal membranes were also collected (n= ) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n= ) were collected to document the role of il- and il- in ptb. tissue expressions of il- and il- were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il- and il - expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il- after lps stimulation (lps- . vs. . pg/ml; p= . ) whereas no difference was noticed in il- concentration (lps- . control- . pg/ml; p= . ) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit . pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il- production was stimulated by lps whereas il- was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il- concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd + cd bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, , dave l roelen, barbara j van der mast, godelieve m de groot, carin kleijburg, sicco a scherjon, frans hj claas. department of obstetrics, leiden university medical centre, leiden, netherlands; department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd + cd + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd + cd bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy ( ) . in this study we examined the phenotypic and functional properties of cd + cd bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd + cd bright t cells from maternal peripheral blood demonstrates regulation to a rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd + cd bright foxp + and cd + cd bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd + cd bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. ( ) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd (+) cd (bright) and cd (+) cd (-) t-cells in decidua and maternal blood during human pregnancy. placenta. apr; suppl a:s - . alicia del toro-arreola, lourdes nunez-atahualpa, juan velazquez-rodriguez, laura gonzalez-lopez, jorge i gamez-nava, adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to women not using hormonal contraceptives in the day th of the follicular phase and st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= . , p< . and rho= . , p< . , respectively) with cd /nkg in t cells and negative correlated (rho= - . , p< . and rho= - . , p< . , respectively) with nk cells. meanwhile cortisol was positive correlated (rho= . , p< . ) with the receptor nkg d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd /nkg inhibitor receptor and nkg d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: ) term not in labor (tnl; n= ); ) term in labor (til; n= ); ) preterm labor without histologic chorioamnionitis (ptl; n= ) and ) preterm labor with histologic chorioamnionitis (ptl-hc; n= ). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: ) the af sp-d concentration increased as a function of gestational age (mean, til: , . ng/ml vs. ptl: , . ng/ml; p< . ); ) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: . ng/ml vs. ptl: . ng/ml; p< . , and til: . ng/ml vs. ptl: . ng/ml; p< . , respectively); ) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: . ng/ml vs. . ng/ml; p< . ); ) however, sp-d mrna expression in maternal leukocytes was . fold higher than that of fetal leukocytes (p< . ); ) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: ) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; ) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; ) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from to patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by . % of the couples. the global pregnancy rate was . %. the pregnancy rate for the couples who had intercourse was . % and . % for those who did not have intercourse (p< . ). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p< . ). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n= ) was similar ( . % vs . % for positive and negative intercourse, respectively). the average number of ovulatory follicles was . + . for the group that had intercourse and . + . for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost % of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were subjects with observations. for the fmc analysis, there were subjects with observations. table provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida gravida or more adjusted* or ( %ci) mmc / ( %) / ( %) . ( . - . ) fmc / ( %) / ( %) . ( . - . ) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th /th gene expression. tommie g nguyen, eileen d gallery, , jonathan m morris. elevated t-helper cell type- (th ) and type- (th ) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th /th responses toward a th response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th /th gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given daily doses of mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th immunity), tnf-(a classical pro-inflammatory th cytokine), and il- (a critical proinflammatory th cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il- (two classical th cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group ( . ± . , mean ± s.e.m), preventive group ( . ± . ) and therapeutic group ( . ± . ) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il- in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg anti-human beta -glycoprotein i monoclonal antibodies (mabs), designated id and iic , were used in these studies. the first trimester trophoblast cell line, htr , was incubated with either medium, a mouse igg control, id or iic ( - g/ml), in the presence or absence of unfractionated heparin ( ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a hour incubation, significant trophoblast cell death was induced by iic ( . ± . %) and id ( . ± . %) at the high dose of g/ml, when compared to the medium and mouse igg controls (p< . ). hoechst staining showed that id -and iic -induced trophoblast cell death was a result of apoptosis. moreover, id and iic induced a significant increase in caspase- , - and - activity (p< . ). treatment of trophoblasts with heparin significantly inhibited the effects of iic and id on cell death by . ± . % and . ± . % %, respectively (p< . ). following a hour incubation at lower concentrations ( g/ml), treatment of trophoblast cells with id or iic resulted in a significant upregulation of il- , mcp- , gro production (p< . ), and a significant reduction in il- secretion (p< . ). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of , affecting approximately % of women < years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n= ), pcos (n= ) and pof (n= ). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common antigenic proteins (row , antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of % annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd -and jak were tested. these markers were chosen since they are central to t cell signaling. cd -is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat . (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for hours. these cells were then exposed for hours to pm, pm (postmenopausal), or pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein ( , . , . , . , , and m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd -and jak expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd -reaching a maximum of fold. although cultivation of t cells in pm of estradiol significantly increased the levels of cd -and jak relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il- as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, juan deleon-luis, isabel galan. obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at - days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and hours after celocentesis for quantification of hormones and il- using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p< . ). results: all animals survived their pregnancies. seven animals receiving < % concentration of nk delivered at term ( days gestation) while animal receiving more than % concentration of nk had dead fetuses on ultrasound evaluation hours after celocentesis. table depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure shows mean il- changes from baseline in continuing (n.s.) and rejected pregnancies (p< . ). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il- might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis ( ) the activity of cytotoxic cd + t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd + t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd +t cells. methods: female c bl/ mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd +tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day of pregnancy (n= ), the number of brdu positive cd + t cells was two fold higher than that found in um (n= , p< . ). the number of proliferating cd + t cells continued to be non-significantly elevated at day ( . x, n= ), day ( . x, n= ), and day of pregnancy ( . x, n= ) compared to um. by day of pregnancy (n= ) the number of proliferating cd + t cells returned to the um level, however by this time the total number of splenic cd + t cells was . fold higher than um (n= p< . ). on gestational day , the number of proliferating cd + t cells declined further ( . x, n= ), and the number of splenic cd +t cells returned to the um level (n= , p> . ). compared to um mice, there was no significant difference in the number of cd + t cells undergoing apoptosis at any gestational day examined ( . - . x, p> . ). in normal murine pregnancy, the number of cd + t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd + t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro hd - niht ai . arturo cerbulo-vazquez, cun li, , gene hubbard, natalia e schlabritz-loutsevitch, , peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf- (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at . , . , and . gestation (g) alters fetal lymphocyte populations at . (g) (j repro immunol, , : ) . we determined if fetal gc exposure alters thymic ) structure; ) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n= ) or betamethasone i.m. ( μg/kg daily for two days at . , . and . g; gc group; n= ), c-sectioned at at . g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf- , b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c bl/ (b ) or recombinase deficient c bl/ mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at and hours after lps injection in pregnant b and rag-ko mice. methods: b and rag-ko mice were mated and on gestation day , females were injected intraperitoneally with μg lps in l saline (pbs) or l pbs alone. euthanasia and uterine harvest occurred or hours after injection. frozen uterine sections were stained with the macrophage marker f / or an isotype-matched control followed by an alexafluor -conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f / + dapi+ and total dapi + cells were counted in areas of representative section and the percentage of f / + dapi+ cells was calculated. the mean percentage for at least representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b and rag-ko mice injected with pbs (b , n= , . ± . ; rag-ko, n= , . ± . % + per area). lps injection increased macrophages (p< . ) in both strains (b , n= , . ± . ; rag-ko, n= , . ± . ,); no difference was evident between strains. the percentage of f / + cells was similar hours post-injection (b , n= , . ± . v. rag-ko, n= , . ± . ), and not elevated relative to the hour time point. objective. decay-accelerating factor (cd ), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide -kinases (pi ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins ( , , ) p . akt, a serine-threonine kinase acts downstream of pi k and regulates cell survival, growth and proliferation. the pi k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi k-akt activity affects the expression of cd in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi k activity were used in this study. ishikawa and rl - endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec- -a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi k-akt pathway on cd expression was evaluated in cells treated with wortmannin ( nm), ly ( mm), or with akt inhibitor sh ( mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd expression correlates with the status of pi k-akt pathway. the cd expression was lowest in hec- -a, ishikawa and rl - cells which constitutively express p-akt. higher cd expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were . -fold higher for kle cells and -fold higher for hela cells compared to hec- -a cells. treatment of cells with akt inhibitor led to . - . -fold increase in cd expression. the . - . -fold increase following treatment with pi k inhibitor wortmannin was found in ishikawa cells, rl - and kle cells. conclusions. human endometrial cell lines with elevated pi k-akt activity express lower level of cd compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi k-akt pathway are involved in the downregulation of cd . endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase- . while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl - , hec- -a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, ( ug/ml) or nocodazole ( mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin mouse antibody followed by anti-mouse alexa conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa and alexa were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec- -a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin- and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl - cells ( . ) followed by ishikawa ( . ) and hela cells ( . ). lowest correlation was found for kle ( . ) and hec- -a cells ( . ) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase- and caspase- in the early apoptotic stages. background: cd + cd + t regulatory cells (t-reg), express foxp ,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd + cd highfoxp +) was assessed in the peripheral venous blood of non-pregnant, pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n= ), second (n= ) and third (n= ) trimester of human pregnancy. paraffin sections were immunostained for foxp and cd . foxp +cells were quantified in x fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd + cd highfoxp t-cells in pregnant ( . % [range . - . %]) vs. non-pregnant controls ( . % [range . - . %]; p< . ). a progression from st, nd and rd trimesters indicated the % of cd + cd highfoxp t-cells was . %, . % and . %, respectively. low numbers of foxp + cells were detected in all decidua samples and their distribution mirrored that of cd + cells. in st trimester samples, foxp + cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp + cells were detected in st ( . ± . ) compared with nd trimester decidua ( . ± . ; p< . ) but there was no difference between st and rd trimester ( . ± . ), nor between nd and rd trimester decidua. in st trimester decidua, numbers of foxp + cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd + cd highfoxp t-cells. the presence of foxp + cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr and tlr , whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin release by keratinocytes, via tlr and tlr . methods: the induction of -defensin production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr and tlr in -defensin production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin production following hyaluronic acid treatment in human keratinocytes. the -defensin production induced by hyaluronan was abolished following block of tlr and tlr by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il- , tnf-, il- and il- . conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin production in keratinocytes, via tlr and tlr . this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, serena wu, mary d stephenson. , obstetrics and gynecology, university of chicago, chicago, il, usa; obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between - (stephenson, . couples who met aps criteria (wilson et al, ) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from - . secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies weeks, regardless if they ended in success or demise. pearsons test with yates continuity correction was applied. results: subjects were identified. subjects had prior live births of known gender ( male/ female), giving a ssr of . . there were also prior fetal demises wks of known gender ( / ) giving a sex ratio for all prior pregnancies at wks of . . subjects delivered subsequent live births of known gender ( / ), giving a ssr of . . there were also subsequent fetal demises ( / ) giving a sex ratio for all subsequent pregnancies of . . subjects delivered both prior and subsequent live births. the ssr was . ( / ) among their prior and . ( / ) among their subsequent live births. including fetal demises, subjects had ongoing prior and subsequent pregnancies. the sex ratio was . among their prior pregnancies and . among their subsequent. as the control, a ssr of . ( , / , ) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p - ) of ultr and ultr-ht (p - ) cells were grown on either plastic or collagen iv (cn )-coated surfaces. ultr-ht cells were further stimulated with angii ( . um) for hrs. expression of nadph oxidase core (nox - and duox / ) and associated subunits (p phox, p phox, noxo , p phox, noxa , p phox, and rac / ), and angii receptors at / was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox , p phox, and noxa genes, and an up-regulation of at / . overexpression of htert did not reverse nox , p phox and noxa expression while cell division rate was increased. however, there was a down-regulation of nox , at / and rac . plating ultr-ht cells on cn induced nox / down-regulation and up-regulation of duox / , with no apparent change of at / . however, exposure to cn re-directed cellular response to angii such that only nox was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox , , , and duox / are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, masahiro nakayama. women's health, national center for child health and development, tokyo, japan; aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under years old, and the old group over y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p< . . [results] ) srage in plasma, ± pg/ml (mean±sd), n= in the young group was significantly higher than ± pg/ml, n= in the old group. there was no significant difference in plasma vegf. ) vegf in follicular fluid was ± pg /mg protein, n= in the young, and ± pg /mg protein, n= in the old was increased significantly. ) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir- which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir- in lsmc, msmc, t-lsmc and sklm. we found that mir- is expressed and regulated by b-estridiol and medroxyprogesterone acetate ( - m) in these cells (p< . ). we further assessed the regulatory function of gain of and loss of function of mir- on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir- oligonueclotides resulted in a significant increase and/or inhibition of mir- expression in these cells, respectively as determined by realtime pcr (p< . ). over-expression of mir- resulted in a significant reduction, while transfection with antimir- increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p< . ). we concluded that mir- is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd ). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin (ucn ), ucn , ucn and crf -r and crf-r receptor subtypes together with crfbinding proteins locally. ucn and ucn are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro . in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens ( - mm thickness, pieces per umbilical cord) collected at preterm and term (n= each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf ( : ), ucn ( : ), ucn ( : ) and ucn ( : ) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using , '-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro . software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= . ± . vs crf-t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p=ns); ucn -pt= . ± . vs . ± . (p< . ). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn and ucn contents significantly decreased and no significant change occurred in ucn content. based on the opposing changes in crf vs ucn and ucn immunostaining, we speculate that crf, but not ucn or ucn , is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn and ucn may perform cytoprotective functions at preterm. physiol. ; : - , am j physiol. ; : - ) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr ( %, nia protocol as described in j geront. a:b -b ) was initiated in c bl/ female mice at months and continued until . months of age, at which time al feeding was resumed. matings were initiated at months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day ) were recorded. results: fertility was lost in of al-fed females by months of age and continued to decline through . months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of pregnancies achieved out of females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at . months of age. while only out of , al-fed females achieved a pregnancy between . - . months of age, out of age-matched cr-then-al-fed females achieved a total of pregnancies in this -month time. notably, % of the pups born to cr-then-al-fed females between . - . months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r -ag ). bile acids in human ovarian follicular fluid. laura p smith, , kaila deiorio-haggar, jason reindollar, alan s penzias, , anny usheva-simidjiyska. ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; boston ivf, boston, ma, usa; endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from to micromoles/l. bile acids are present in follicular fluid with a mean concentration of . micromoles/l in large follicles and . micromoles/l in small follicles (p= . ). pre and post h-cg serum bile acid concentrations differed significantly ( . micromoles/l vs. . micromoles/l, p= . ). there was a trend toward higher bile acid concentration in large follicles of young patients < years old compared to older patients years old ( . ± . vs. . ± . , p= . ). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients ( . ± . vs. . ± . , p= . ). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient . , p= . ). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in women undergoing ivf/icsi treatment blood samples were collected on cycle day and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal' 'phosphate (plp) concentrations were determined. women with a serum folate . nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p . ). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p . ). in the total group, a two-fold increase of thcy in follicular fluid resulted in a . mm decrease of the follicular diameter (p . ). in non-supplemented women this decrease was . mm (p . ). in supplemented women a twofold increase of follicular fluid folate resulted in a . mm decrease of the follicular diameter (p . ). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. ), post-fixed for rain in % w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, . m/min); a soluble guanylyl cyclase inhibitor, h-[ , , ] oxadiazolo [ , -a] quinoxalin- -one (odq, m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p< . ). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p< . ) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range - ; with being best quality). secondary endpoint was the occurrence of pregnancy. results % of the included women used a folic acid supplement (serum folate . nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate - . ; p . ) and higher thcy levels (median . mol/l, range . - . ) correlated with a worse embryo quality (estimate . ; p . ). in supplemented women higher follicular fluid thcy (median . mol/l, range . - . ) correlated with better embryo quality (estimate - . ; p . ). the follicular fluid folate level of oocytes that did not fertilize was . -fold higher than in the fluids of a fertilized oocyte ( % ci . - . ; p . ). a two-fold increase of follicular folate corresponded with a . higher chance to achieve pregnancy ( % ci . - . ). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, ) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in . we defined a cycle with hloi as having > % immature oocytes (> sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= % ( sd above median) immature oocytes on all cycles. results: from subjects, we identified cases of recurrent hloi (comprising icsi cycles) and control subjects. at baseline, cases had more oocytes retrieved per cycle than controls ( . vs. . , p<. ). all other baseline characteristics were similar. all case subjects were younger than . outcomes are shown in table . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, were performed for an initial diagnosis of premature ovarian aging (poa), for the diagnosis of premature ovarian failure, and for repeated pregnancy loss. in women under age , b-fsh levels over miu/ml and up to < miu/ml denoted a diagnosis of poa, while levels of miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day fsh levels ranged from . to . miu/ml (mean . ± . miu/ml). amh levels ranged from < . to . ng/ml (mean . ± . ) and allele repeats ranged from to , (mean for allele- , . ± . ; allele- , . ± . ). mixed model analysis of variance for all patients, adjusted for age, race, and allele- revealed a statistically significant correlation between b-fsh levels and number of repeats in allele- ( figure ; p < . ). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above were significantly associated with serum amh levels less than ng/ml (chi square = . ). conclusions: triple repeats above , a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, . mg/kg cisplatin or . mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p< . ). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p= . and p< . respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p= . for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas a, b, - pp, and - p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir- a, mir- b, mir- - p, mir- and mir- - p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)- , il- , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from women (age range - ; mean age . ± . ) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir- a, mir- - p, mir- and mir- - p displayed a significant and positive correlation with the expression of cox- . moreover, mir- b significantly correlated with star mrna expression, but not with other genes tested (p< . ). with advancing age, the expression of mir- - p and cox- was significantly decreased, with cox- expression displaying a positive correlation with aromatase, star and er mrna expressions (p< . ). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of . y best predicted whether mir- - p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir- b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir- - p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant . introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: infertile women (< years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh< dor, fsh > miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u plus . genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > fold was considered biologically relevant. results: of the , genes identified to be differentially expressed in young women with dor compared to normal reserve, genes demonstrated consistency of expression across five different normalization schema; were down regulated and up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated -fold over two probe sets (- . ) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (- . ) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf- which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k cd . nih k rr . ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp ) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl ). the granulosa cells were cultured +/-dibutyryl camp ( mm) and incubated +/-the gnrh antagonist, ca, for - h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp expression. materials and methods: kgn cells were cultured in f- dmem and were starved for h before experiments. subsequently they were treated for h with vehicle (control), fsh ( ng/ml), and/or bpa ( , , , , um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a -fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp production, with the greatest effect at um (p< . ), resulting in +/- % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at um (p< . ) resulting in +/- % decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor- and gata- , mimic the pattern of cyp expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing cylindrical round bottom recesses m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of , cells per gel were incubated for up to days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa and saa mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa and saa in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase (sod ), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin . mg/kg or cisplatin . mg/kg as weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod . the anti-sod antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod in antral follicles and in corpora lutea (p< . for both). no change was found in the h score of sod in other ovarian structures. western blot analysis of sod in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod in antral follicles and in copora lutea. cisplatin decreases the amount of sod available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged ± . yr (range - ) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than u/l and/or amenorrhea greater than months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of . ng/ml and pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: ) six patients had no detectable follicular activity by amh and inhibin b levels. ) one patient demonstrated detectable inhibin b levels with an -fold gradient between ovarian venous effluent ( pg/ml) and peripheral blood ( pg/ml), however no amh was detected. ) in one patient, aged and months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at pg/ml. amh was detectable at levels of . ng/ml. conclusions: ) in the majority of patients, continued e and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. ) some patients have evidence of follicular function up to ten years after menopause. ) since e is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f (pgf ) mediates luteal regression, this study examined the role that the transcription factor yin yang (yy ) and histone deacetylase (hdac ) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p side chain cleavage enzyme (p scc) expression. rat luteal cells ( days post-ovulation) were treated with pgf ( hr) and trichostatin a (tsa; hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek t cells treated with yy , myy , a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf caused a significant -fold decline in star (p< . ), and smaller declines in sr-bi and p scc which occurred concomitantly with an increase in yy ( -fold, p< . ) and intracellular lipid staining ( % increase). in contrast, nm tsa treatment caused a dose dependent increase in sr-bi, star, and p scc protein levels, . -fold (p< . ), . -fold (p< . ), . -fold (p< . ), respectively, and a . -fold decline (p< . ) in intracellular lipid levels. tsa prevented the pgf -induced decline in sr-bi, star, and p scc expression. promoter analysis demonstrated that wildtype yy , but not myy , repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy plays in pgf induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy -mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd and nih hl . regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: ) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp and ramp mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; ) to demonstrate immunohistolocalization of imd, crlr, ramp , ramp and ramp in rat ovary. methods: groups of sprague-dawley non-pregnant and pregnant rats on day of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp , ramp and ramp proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day . results: ) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; )expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; ) expression ramp mrna is higher in proestrus compared to diestrus and estrus (p< . ) but there is no significant change in the ramp expression during the estrus cycle and ; ) imd, ramp , ramp and ramp are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, ap goud, mp diamond, b gonik, hm abu-soud. div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set , oocytes were retrieved from the following superovulated b d f mice: (a) young breeders (yb, n= ); (b) retired breeders (rb, n= ), and © old animals (oa, n= ), aged - , - , and - weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [ ], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set (n= ), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m- , . m no/min , n= , h, °c, % co ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [ ]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set , a significantly fewer oocytes were retrieved per animal (mean) in rb ( ) and oa ( ) compared to yb ( . , p< . ). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p< . ). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p< . ). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p< . ). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set , exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca + in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all gaba-a receptor subunits, gad and gad plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca + study, gc were cultured on coverslips. gc were loaded with fura- -am and changes in ca + concentration of gc were studied using a dynamic digital ca + imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, m bicuculline, were perfused min. prior to and during application of muscimol. the responses were represented as changes in the / nm fluorescence ratio over time. m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were , and . gad isoenzyme was more abundantly expressed in cumulus and mural gc than gad . ca + imaging showed that muscimol, had the ability to increase ca ++ in gc, about % gc (n= ) cells responded to muscimol. muscimol increased intracellular ca + in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from % to % (n= , p< . ). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca + response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis guinea pig fetuses at and days gestation were assigned to normoxic (nx, n= ), hypoxic (hx, n= ) and hypoxic pretreated with nos inhibitor (hx+l-name, mg/kg i.p., n= ) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio of . for min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p antibodies. in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx) . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx) . ± . (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p , erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p , erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p , erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p , erk and jnk in the guinea pig fetus. (nih-hd , nih-hd and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint . high levels ( nm) in culture are associated with embryo death while plasma levels (> nm) at weeks in women undergoing ivf-et are associated with failed pregnancy . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method at - weeks gestation in women (nonsmokers, bmi < kg/m ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the women subsequently had a miscarriage. the plasma aea levels in those women who had live births was . -fold lower than that in those who subsequently miscarried ( . ± . nm versus . ± . nm, mean ± sem; respectively; p< . unpaired student's t-test). the roc analysis revealed an area under the curve of . ± . with a sensitivity of % ( %ci of . % to . %) and a specificity of . % ( % ci of . % to . %). using an aea level of . nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of % and a specificity of % with a negative predictive value of % and a positive predictive value of % for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products ( exon deletions and intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon and deletion of exon , showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon , found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected and days after urinary lh surge in the same menstrual cycle from three fertile women (n= ). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using -d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, cm ipg-strips in the nonlinear range of ph - were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v . and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table displays the differentially protein abundance between days lh+ and lh+ (> -fold change). of these proteins, were increased at lh+ in comparison with lh+ , whereas only proteins were decreased. stathmin was found more than -fold decrease in lh+ compared to lh+ in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases and (erk / ) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk / is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk / activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n= ) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen ( - m), and progesterone ( - m) for and minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p< . ). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within and minutes (p< . ) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin- (et- ), soluble intercellular adhesion molecule- (sicam- ), soluble vascular cell adhesion molecule- (svcam- ) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et- (p < . ), sicamp- (p < . ), svcam- (p < . ) and hscrp (p < . ) as compared with age-matched controls, respectively. positive correlations were evident between et- and fai (r = . ; p < . ) but et- negatively correlated with shbg (r = - . ; p < . ). svcam- positively correlated with total t (r = . ; . ), hscrp correlated with: bmi (r = . ; p < . ), and homa (r = - . ; p < . ), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. ( ), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation ( ), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from women, median age yrs (range - ) and bmi kg/m (range - ), with regular menstrual cycles, with no medical problem and not on any medication for the preceding months. the menstrual cycle was divided to early follicular d - (n= ), late follicular d - (n= ), ovulatory d - (n= ), early luteal d - (n- ) and late luteal phases d - (n= ). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. ( , , , , , , and , as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp , , , , , ) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp elevate in fetal and newborn rat and graduately decline to a lower level at day and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp was not detected by wlb assay. the igfbp- cleaved products were observed after rat day age day , which associated with a decrease in full length of igfbp- , suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp- , with heterogeneous glycosylation, were appeared after age day and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp- at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged - years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api ). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e ), estrone (e ), progesterone (p), -hydroxyprogesterone ( ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, -deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: . , . , and . miu/ml respectively. similarly, mean t . , . ( % increase) (p= . ), . μg/dl (- %) in active smokers; (active vs. exposed p= . ). shs increased dhea levels ( % higher, p = . ), dheas ( % higher, p = . ), cortisol ( % lower, p = . ), aldosterone ( % lower, p = . ) and androstenedione ( % higher, p = . ). these data suggest that active smoking and shs can have a profound effect on serum t , adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: with pcos and age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between : - : , after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft , -ohp, -a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos ( . %, % ci: . - . %) as compared with women without pcos ( . %, % ci: . - . %) (p< . ). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p< . ). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p< . ). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c ( . ± . %); increased triglycerides ( . ± . %); and increased bmi ( . ± . %), respectively. the prevalence of mbs from lowest to highest tertile of free t level was . , . and . %, respectively; in women with both pcos and mbs. in women with pcos, % exhibited all components of mbs; . % had components, and . % had components. conclusions: women with pcos exhibited significantly higher prevalence of mbs ( . -fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is in , live male births. we present a case of complete androgen insensitivity in members of the same family with differing residual mullerian tissue. sister a presented at age for evaluation of primary amenorrhea. a karyotype revealed ,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at . ng/ml. other laboratory evaluation showed fsh . miu/ml, lh . miu/ml, free testosterone . pg/ml, and estradiol . pg/ml. approximately two years later sister b presented at age for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed ,xy. laboratory evaluation demonstrated fsh . miu/ml, lh . miu/ml, estradiol . pg/ml, total testosterone . ng/ml, and free testosterone . pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb and cb and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at - °c prior to extraction. an internal standard of . pmol of deuterated aea (aea-d ) was added to each plasma sample and aea extracted from aliquots on each occasion over three days using the lpe and spe methods. spe was performed with waters oasis hlb cc/ mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with % methanol at ml/min. aea was eluted with ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was -fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method , . methods: authentic non-labelled and deuterium-labelled aea (aea-d ) diluted in acetonitrile were maintained at °c before analysis and separation by uplc on a c ( . x mm) column maintained at °c using a gradient of % a, . min: % a, . min: % a, . min: % a, . min: % a with a flow rate of . ml/min. the mobile phases were a ( mm ammonium acetate, . % formic acid) and b (acetonitrile, . % formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were - , and - ev, respectively. calibration curves were performed in triplicate with . pmol aea-d as the internal standard. transitions employed were . > . and . > . for aea and aea-d , respectively. results: calibration curves ( . to fmol aea on column; n= ) were linear, producing a mean (±sd) gradient of y = . ± . x, crossing the y-axis very close to the origin ( . ± . units). variability was limited, with an r = . . measurements were precise, fmol aea produced a cv of only . %, and the retention time was consistent at . ± . min (n= ). the limit of quantification (signal/noise> ) was . fmol on column and the limit of detection (lod) was . fmol on column (signal/noise= ). accuracy for . fmol, . fmol and fmol aea was . ± . %, . ± . % and . ± . %, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods , with a lower lod [ . fmol v. fmol or fmol ] and shorter run time [ min v. min or . min ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor- (sf- ), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: ) total rna was extracted from the gonadotrope cell line, l t , and analyzed by standard rt-pcr analysis. ) the cv- fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat - /+ lh promoterreporter vector as well as cmv-driven expression vectors for gata- , gata- , dngata- , fog- and/or sf- . results: gonadotrope l t cells were found to express transcripts encoding gata- , gata- , and gata- as well as fog- and fog- . gata- and gata- stimulated lh gene promoter activity by approximately -fold (p< . ) and synergistically increased the sf- response ( -fold versus -fold for sf- alone; p< . ). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog- . similarly, co-transfection with a gata- dominant negative construct blunted wild-type gata- effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n= ) were randomized to control ( ml saline/ewe) or dex treatment ( . mg/kg ewe weight) consisting of four intramuscular injections at -hourly intervals over hours on - days of gestation (dg). at , , , and dg fetal weights were recorded. i -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc- mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at dg*, was similar at and dg, then elevated at dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at dg* were associated with increased fetal adrenal expression of p c and bhsd mrna in females, reduced expression of mc r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p< . . synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, beom su kim, amanda biehl, blenna cl bett, , john yeh. gynecology-obstetrics, university of buffalo, buffalo, ny, usa; physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh ( : -chemicon international) and with a rabbit polyclonal antibody against synaptophysin ( : -santa cruz biotechnology) for h. after incubation the sections were washed and incubated with a mixture of alexa conjugated goat antimouse and alexa conjugated goat antirabbit ( : ; molecular probes) for h. slices were visualized with confocal microscopy (zeiss lsm- ). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dex groups ( . mg/kg), consisting of four at h intervals on days - of pregnancy. at months postnatal age, catheters were implanted; a bolus injection of crh ( . mg/body weight) and avp ( . mg/body weight) were administered and arterial blood samples were taken at - , - , , , , , , , and min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n= ) were lower than in dex males (n= ) from to min (p . ) and lower than in control females (n= ) at minutes (p . ), also in the dex-m group were higher than in control males (n= ) at min (p . ). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dexamethasone treatment ( . mg/kg) consisting of four injections at h intervals on days - . hippocampal was collected from the offspring at months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to s rrna. results: dexamethasone-treated male animals (n= ) had significantly higher levels of mr gene expression than both control males (n= ; p= . ) and females (n= ; p= . ). gr gene expression levels were higher in treated vs. control males (p= . ), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women < with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from / - / with follicle stimulating hormone (fsh) levels based on age < or . results: so cycles were performed in women. no differences were noted in clinical pregnancy. women < were more likely to have inseminates with a lower mean tms/ins, median tms/ins was . among pregnancy cycles compared to for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women < , mean number of follicles mm peak e were significantly higher in women < . so ivf measures are in tables , respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women < required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women < with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about l containing x pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e and p measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p< . ). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average follicles/ovary were observed in ad-hfshr-treated group of which follicles were at the antral stage while only follicles observed in ad-lacz control group, with zero follicles at antral stage. a . to folds increase in e and about % decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day , and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential ( . ± . ) compared to those that failed to implant ( . ± . ) (p< . ). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of % and a specificity of . %. quantification by integration showed significantly decreased glutamate levels (p= . ) and a trend toward an increase in pyruvate levels (p= . ) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single - mm follicle was obtained from women (age - ) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p< . for all analytes) and the levels were strongly correlated (r > . , p< . for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte ( . +/- . g/ml vs. . +/- . g/ml, p= . ). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of °c. thermistors, pinhead sized sensors with an accuracy of %, were used to monitor temperature differences between control fluid (cf) (sterile water ºc) and aspirated fluid. data was recorded using an -channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a cm, gauge, single lumen needle connected to cm of tubing was used to aspirate into the empty collection tube. temperature was recorded x/second, each experiment was repeated times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group ( . ± . °c vs . ±. °c, p=. ). two seconds after aspiration, the lowest aspirate temperature was observed, (hh . ±. °c, hb . ±. °c, p>. ) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh . ±. °c, hb . ±. °c, p>. ). the hb group took an average of . min to return to baseline ( °c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of . ±. °c corresponding to . °c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, luis roca, niki b istwan, debbie j rhea, gary j stanziano, victor hugo gonzalez-quintero. obstetrics and gynecology, university of miami medical center, miami, fl, usa; clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n= ). eligible for inclusion were patients later hospitalized with acute rptlsx (n= ). included were those < weeks' gestational age (ga), with intact membranes, remaining undelivered for > hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n= ) following hospitalization were compared to those having an alteration in treatment (alttx group, n= ) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at < . . results overall, ( . %) of twin pregnancies prescribed nt experienced rptlsx; ( . %) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during . stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of elective cesarean deliveries (cd), underwent routine cervical dilatation. the overall rate of febrile morbidity was . %. no significant differences in postpartum febrile morbidity were noted between the groups ( . and . %; p= . ). in addition, hospitalization duration did not differ between the groups ( . ± . and . ± . days p= . ). about % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin . ± . mg and . ± . mg p= . ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or= . ; % ci . - . ; p= . ). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or= . the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between and weeks of gestation were chosen for this study. eighteen patients (group ) were treated with amoxicillin and erythromycin for day period while the other patients (group ) were treated with rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day , and day after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the , -dinitrophenylhydrazine method. results: . the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy ( . ± . vs . ± . nmol/mg protein, p< . ), ( . ± . vs . ± . nmol/mg protein, p< . ). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group among before the antibiotics administration, day , and day after the antibiotics administration. . the protein carbonyls formation in the venous plasma of group was significantly decreased at day and day after the antibiotics administration than that of before the antibiotics administration ( . ± . , . ± . , . ± . nmol/mg protein, p< . ). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between and weeks gestation in consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< . or >/= . mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than . mom ( . days, interquartile range - ) in comparison with the women with mca pi higher or equal than . mom ( . days, interquartile - . days (p< . , mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< . , exp(b) . , ci % . - . ). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. two sets of control experiments were performed simultaneously as follows: . strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or . nmol/l oxytocin; . strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: . ± . % for spontaneous contractions (n= ; p< . ) and . ± . % for oxytocin-induced contractions (n= ; p< . ). no significant difference was found between control and control for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < . considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure ) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table ) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r -hd . results: % of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p< . ); after a failed instrumental delivery, % obstetricians would recommend labour but only % would choose that option for themselves (p< . ). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently ( % and % respectively) than chosen for personal care ( % and %). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of . / , , . / , and . / , for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. , , /annually in the u.s.) would be delivered at weeks by either an elective c/s or an induction of labor. we further assumed that % of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at weeks would be and for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds %, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at weeks is relatively low (approximately . / , deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below %, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: providers ( residents, midwives, nurse practitioners) and patients participated. providers asked about smoking, alcohol and drug use in all ( %) visits. patients reported being smokers, reported alcohol use, and reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women ( with actual psychiatric disorder, with stressful life events, and healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at (+ ), (+ ) and (+ ) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at - weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, ) ; moreover, the -item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight ( + g) and higher percentage of birth weight below the th centile for gestational age ( %) than infants of healthy mothers ( + g and %, respectively). there was also a trend towards lower mean birth weight ( + g) and higher incidence of birth weight below the th centile ( %) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from weeks-delivery are generally recommended for patients with hcg levels > . mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > . mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) < %ile for the estimated gestational age (ega). sga was defined as an actual birthweight < %ile for the ega at the time of delivery. results: from - , there were subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of ultrasound examinations were performed. the median number of examinations per subject was with a range from - examinations per subject. the incidence of iugr and of sga were . % (n= / ) and . % (n= / ), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified . % (n= / ) of sga infants. however, the sensitivity for the detection of sga was % when an efw cut-off of % was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > %ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, christiaan v hulzebos, gerda g zeeman. obstetrics and gynecology, university medical center groningen, groningen, netherlands; pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first - h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between - wks, who deliver > wks (latency > h) , during the -yr period of - at a single academic center. data were extracted from medical records and electronic department databases. results: pprom pregnancies were identified. all but received at least full course of steroids. ( %) delivered > wks. newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n = ). tertiary care center (n = ) to determine if an association exists between macrosomia (birthweight > g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of , singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p< . to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was %. methods: we prospectively analyzed the outcomes of pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi < kg/m , absence of major risk factors for diabetes and of pregestational diabetes. g -hour oral gct was performed at - weeks of gestation. women were subdivided into groups according to the result of the gct: group = negative (load glucose > % and < mg/dl) women ( . %); group =flat (load glucose % than basal and < mg/dl) women ( . %); group =positive gct/negative ogtt, women ( . %). data are mean ± sd. differences were calculated with the student t test for unpaired samples and test. regression analysis were performed by the least squared method. p-values were considered significant at p< . . results: the characteristics and obstetric outcomes in the three groups are presented in the table . in all patients there was a significant linear relationship between the load and basal glucose values (load value= . + . basal value; r= . ; p< . ) and between birthweight and load values (birthweight= . + . load value; r= . ; p< . ). the relationships were significant also in group and separately but not in group . in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table . the probability to be primiparous and to deliver babies < ° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies > ° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p< . ) and to start pregnancy without any medical and obstetrical risk but obesity ( . % vs . and . %; p< . ). african women exhibited the highest bmi and the highest rate of obesity (table ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than % have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c bl/ j female mice were fed control (c, % of energy from fat) or isocaloric high fat (hf, % of energy from fat) diets ad libitum for weeks prior to mating and during gestation. at gestational day . maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using h-meaib, and c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e . or litter size between treatment groups. fetal weight was increased in the hf group by % (p< . ). maternal adiponectin levels were significantly (p< . , n= ) decreased (hf ± , c ± g/ml) and leptin levels increased by % in animals fed a high fat diet, but this difference did not reach statistical significance (n= ). adiposity (fat pad weight) was increased by % (p< . , n= in the dams fed high fat diet, however no difference was observed in maternal il- levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least % in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased hsd expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of hsd in the lamb before and after birth. methods: ewes were provided with either % (control,c) or % (well fed, wf) of maintenance energy requirements from d gestation until delivery. post-mortem was performed on either ± d gestation (c= ,wf= ) or and postnatal day (c= ,wf= ). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of hsd and reference gene rpp were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at d (p< . ). the expression of hsd mrna was significantly higher in the postnatal compared to the fetal lamb (p< . ) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of hsd mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of hsd in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first hrs after birth (r =- . , p= . ). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between hsd mrna expression in the liver at d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic hsd . we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven ( %) of mothers were classified as obese (bmi> ) based on their first pregnancy weight. glycemic control at weeks was superior in the non-obese group (table ) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table ) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table ) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight ( g+ vs. g+ , p=. ) and higher rate of macrosomia ( vs. %, p= . ). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr- by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr , was treated with polyi:c. c b/ wild type and tlr- knock out mice were injected intraperitoneally with polyi:c at . gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex is. results: polyi:c induced cytokine (il , il and il ) and chemokine (il , rantes, gro , mcp and mip ) secretion and production by human cultured trophoblast in a time ( - h) and a dose ( - g/ml) dependent manner. injection of polyi:c to c b/ wild type mice induced preterm delivery within h at a dose of mg/kg body weight. no effect was observed in tlr- knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed and h following polyi:c treatment. trophoblast cell cultures from tlr- ko mice confirmed that the response to polyi:c is tlr- dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr- . our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr- mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: mouse models were utilized: ( ) a model of intrauterine inflammation (lps into uterine horn) (n= ); controls received intrauterine saline (n= ) and ( ) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of lps infected animals and controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+ . %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by ( ) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or ( ) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr- supernatant is able to antagonize the actions of lps on cytokines and ptgs in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs expression levels were measured by western blot analysis and tnf-, and il- concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-( . pg/ml to . pg/ml, n= , p< . ), an effect that was greater in placentae of the male fetuses ( . pg/ml, n= ) compared to female fetuses ( . pg/ml, n= ). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il- production (n= , p< . ) and ptgs expression (n= , p< . ). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n= ) by %, and this effect was greater in placentae of the female fetuses (n= ). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs . conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , ) . this study investigates whether iai is associated also with altered expression of neuropilin- . methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n= for each). neuropilin- expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n= ), purified, and depleted of leukocytes. after treatment with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days, dscs were stimulated with il- b ( - ng/ml), tnfa ( ng/ ml), or thrombin ( . iu/ml) for h. since no elisa exists for neuropilin- , protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il- b on neuropilin- mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin- expression in term decidua was increased in tissues with iai vs controls (p< . ), and localized primarily to dscs. using icc, an increase in neuropilin- was noted after stimulation with il- b and tnfa, but not thrombin. il- b increased neuropilin- mrna expression in dscs by . ± . -fold (from . ± . to . ± . neuropilin- mrna/b-actin mrna; p= . ). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin- , in term decidual tissues. il- b and tnfa (but not thrombin) stimulated neuropilin- expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr and cxcr ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr and cxcr . methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n= ; cervix n= ) and in labour (il) (myometrium n= ; cervix n= ). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in randomly selected high-powered fields ( x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr and cxcr . results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median . , iqr . - . ; il median . , iqr . - . , p= . ). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median . , iqr . - . ; il median . iqr . - . , p= . ). analysis of chemokine receptor expression demonstrated co-localisation of cxcr to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr , the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage - ) and wish cells were serum starved for hrs and treated with -hydroxyprogesterone ( -hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru (a pr/gr antagonist) or org (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with -hp and p ( and m) and dex ( nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p ( nm to m), and decreased sequentially within cytoplasmic fractions. in the presence of org gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p> . ) within both cells types in response to p. conclusion: in this study we show that gr is activated by -hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and -hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd- mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n= per group) each with - gs with fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata- (erythroid precursors), cd , and bm (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p= . ). in lps-exposed, fetal mp were increased in the placenta (p= . ), fetal extremity mesenchyme (p= . ), fetal liver (p= . ) and leptomeninges (p= . ) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. % of lps-exposed fetuses and % of saline-exposed had liver hemorrhages (p< . ). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p= . ). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem- (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem- in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology ). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem- expression in the uterus, cervix, and placenta across gestation and to determine if trem- levels are altered by intrauterine inflammation. methods: in cd- mice, trem- was investigated in non-pregnant (np) and throughout gestation e , e (n= - per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected hours after intrauterine infusion of saline (n= ) or lipopolysaccharide (lps) (n= ). for a non-pregnant model, using cd- mice, lps (n= ) or saline (n= ) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested hrs later. quantikine ® mouse trem- immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p< . ) results: trem levels are significantly different between np and pregnant uterine tissues (p= . ). e and e trem expression is significantly increased . and . -fold compared to np (p= . and . respectively). trem- levels in the placenta and cervix were not significantly different between e and e . trem levels increased about -fold in the uterus after intrauterine infusion of saline or lps compared to e controls. in nonpregnant, trem levels were significantly different (p= . ) with a -fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem- in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, adegoke adeniji, susan e jackman, babak forooghi, isaac mostadim, phillip yadegari, calvin j hobel. og-gyn, cedars-siani medical center, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (< . nmol/l), insufficiency ( . to nmol/l) and sufficiency (> nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of (oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, ethnically diverse women were evaluated at - weeks (t ), - weeks (t ) and - weeks (t ). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups ( with no infection during pregnancy, with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for (oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of (oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<. ). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ) but not subjects with sufficient vitamin d at t ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ). conclusion the results reveal a positive association between (oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected ( . x ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in . day pregnant c b/l mice. the mice were euthanized at hr (n= ) and hr (n= ), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n= ), dmso (n= ) or pancaspase inhibitor z-vad-fmk (n= ) prior to hk-gbs and observing the animal for delivery for hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase and caspase independent m-calpain in the placenta. z-vad-fmk ( mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and ( . %) had a late sptb. the mean + sd gestational age at blood draw was + weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= . for trend). women with bb in the top quartile were . times more likely to have an early sptb as compared with women who had lower levels of bb ( % ci . to , p = . ). there was no association between bb and late sptb (rr= . , % ci = . to ). the adjusted or of an elevated bb for early sptb was . ( % ci = . to , p= . ). when the analysis was restricted to the women with sptb the rr of an elevated bb for early sptb was . ( % ci . to . , p = . ). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day- pregnant cd- mice underwent intrauterine injection of g lps. subsequently, the mice were euthanized at , , , , and hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a % agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all of the reported mouse tlr isoforms. these tlr amplicons range in size from to bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at hours after lps injection, tlr increased -fold and tlr increased -fold (both p< . ). in contrast, the expression of tlr (the ligand for lps) remained stable during the hours after lps; the expression of tlr , , , , , and also remained stable. tlr expression trended upward and tlr trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd ). udp-glucose and its receptor p y as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p y , a g protein-coupled p y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p y have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p y in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il- mrna expression. we also measured the il- secretion using elisa. small interfering rna was used to knock down p y expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip- , the il- homologues in mice. results: p y was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il- in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p y knockdown abrogated udp-g-induced il- production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il- neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip- mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il- production via p y in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p y may be involved in the trans-species mucosal immune system in frt. (jsgi ; , (suppl) , abst # ) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar ; - x cfu) at ± . days gest. age (dga, mean ± sem, term= d). six of these animals received maternal i.v. azi ( . mg/kg q h or q h for d) either alone (n= ) or in combination (n= ) with dexamethasone (dex; mg/kg/d i.v. for d) and indomethacin (indo; mg/d p.o for d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent ( ), minimal ( ), moderate ( ) and severe ( ). results. inoculation-to-delivery interval was - d for combined treatment groups and was similar to long duration infection without treatment ( - d). treatment effects were tabulated as mean scores and compared as follows: control (n= ), score ; short duration infection ( - d; n= ), score ; long duration infection ( - d; n= ), score ; short duration infection + treatment (n= ), score ; long duration infection + treatment (n= ), score . conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd , rr . brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor- (tlr ). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr- specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and reduced pup mortality up to %. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox- , cxcl (il- equivalent) and ccl in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin -deoxy- , -prostaglandin j delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor- (tlr ) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin -deoxy- , -prostaglandin j ( d-pgj ) inhibits il- b-induced nf-kb activity and cox- expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of d-pgj can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. co-administration of d-pgj with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to %. we have looked at the expression profile of several labor associated genes in myometrium hours after lps administration. (otr, connexin and , cox- , cox- , cxcl (il- equivalent) and ccl ). we have found that treatment with lps significantly up-regulated cox- , cxcl and ccl and this is significantly suppressed after with co-administration of d-pgj . western analysis for ser -phosphorylated p and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both h and h. co-administration of d-pgj was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion d-pgj may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr- and tlr- : mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, emmet hirsch. , department of ob/gyn, evanston northwestern healthcare, evanston, il; department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr- responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr- is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr- ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr- and tlr- . here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw . ) were treated in tissue culture with either pgn ( g/ml), poly(i: c) ( g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for - hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin (il- ), tumor necrosis factor (tnf), the chemokine rantes and tlr- , normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw . cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il- , tnf and rantes (p< . for all) at and hours . sequential stimulation with either pgn or poly(i:c) for h followed by incubation for an additional h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for h followed by medium for h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr- mrna after h but not h. tlr- mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr- by either tlr- or tlr- activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il- by elisa. paired maternal serum il- levels from subjects were divided into groups: il- levels obtained - hours prior to completion of antibiotics and those obtained - hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p< . . of the pprom subjects, the maternal age was . yrs; gestational age at admission was . weeks; latency was . days; gestational age at delivery was weeks; and infant birth weight was grams. median maternal serum il- levels obtained off antibiotics were significantly higher when compared to those on antibiotics ( . vs. . pg/ml, p< . ). the results of this investigation suggest that maternal serum il- levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in . placentas with histologic chorioamnionitis were divided into two cohorts: group -maternal inflammatory responses only, and group -maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of consecutively submitted placentas, had histologic chorioamnionitis: with maternal inflammatory responses only (group ) and with both maternal and fetal inflammatory responses (group ). fetal inflammatory responses observed in group were associated with higher stages of maternal inflammatory responses (p<. ). % of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group patients were more likely to have had amnioinfusion ( % v %, p=. ) and less likely induction of labor ( % v %, p=. ). group was more likely to have had intrapartum fever ( % v %, p=. ) and maternal tachycardia ( % v %, p=. ). newborns from group were more likely to have been observed for sepsis ( % v %, p <. ) and have an apgar score at minutes ( % v %, p=. ). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or . ) and amnioinfusion (or . ) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin- (il- ) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il- production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il- production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il- production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with - . mm sasp overnight and then stimulated with ccu/ml ureaplasma parvum, cfu/ml escherichia coli, or x cfu/ml gardnerella vaginalis for another overnight incubation in -well cultures. conditioned medium was then harvested and production of il- was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il- production by endocervical cells not treated with bacteria. at the highest concentration tested ( . mm), sasp significantly inhibited il- production by cultures stimulated with e. coli (p< . ), u. parvum (p< . ), and g. vaginalis (p< . ). viability of the cells, however, was significantly reduced by sasp at . and . mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il- production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: -hydroxyprogesterone caproate ( -ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that -ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, -ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, -ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if -ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of pregnant women between and weeks. pregnant women had no prior history of preterm birth. cells were treated with -ohpc ( m) and escherichia coli lipopolysaccharide (lps, g/ml) alone or in combination. after and hours of culture, supernatants were collected and tested for tnf-and il- levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < . was considered significant. results: pbmc exposed to lps significantly increased tnf-and il- secretion compared to untreated controls (p= . and p= . ). tnf-concentrations were not significantly different between lps and lps+ -ohpc treated cells at both and hours (p= . and p= . ). il- production was significantly increased after -hour treatment with lps+ -ohpc compared to lps alone ( , [ , - , ] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps ( g/ml)+/progestin ( nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to -hydroxyprogesterone ( -hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and -hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase [cox- ], toll-like receptor [tlr- ]) was evaluated. cox- and tlr- were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox- and tlr- proteins were significantly inhibited by both p (p< . and p< . , respectively) and -hp (p< . and p< . , respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr- , tlr- and tlr- at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr- , tlr- and tlr- within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps ( mg/ml) or lta ( mg/ml) for , , , , and hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr- , tlr- , tlr- and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr- with lta was observed in choriocarcinoma cells ( -fold, h), and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in ectocervical cells ( -fold, h), choriocarcinoma cells ( -fold, h) and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in choriocarcinoma cells ( fold, h) and neonatal epithelial cells ( -fold, h). all cell lines showed at least constitutive levels of transcription for tlr- , tlr- and tlr- . these data demonstrate that tlr- , tlr- and tlr- are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at days of gestation (d, term= d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) x colony forming units (cfu) of u parvum (serovar , n= ; serovar , n= ), (b) x cfu of u parvum (serovar , n= ; serovar , n= ) or (c) media control (n= ). at d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to cmh o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with % paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar or (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p< . ). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p< . ). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n= ) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than % making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p , the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in cohorts: preterm no labor (pnl, n= ), preterm labor (ptl, n= ), spontaneous term labor (stl, n= ), and term no labor (tnl, n= ). subjects were delivered between gestational ages - weeks (preterm) and - weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p and microscopically examined. myometrial samples were categorized in a blinded fashion to groups of staining: no nuclear p (-), rare nuclear p (+), and > % nuclear p (++). a p nuclear labeling index (nli; % nuclear p /total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of non-overlapping low-magnification fields. results: nuclear p labeling was rare in amnion and inconsistently present in chorion. in decidua, p nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl- % ( - %); ptl- % ( - %); tnl- % ( - %); stl- % ( - %); all values are median (iqr)]. in myometrium, ++ p nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl- %; stl- %). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of % ( - %) [median (iqr)], % ( - %), and % ( - %), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p immunolabeling was observed in myometrial cells following labor. nuclear p labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin- and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il- levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with - m estradiol (e ) + - m medroxyprogesterone acetate (mpa) for days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il- . after h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were . ± . pg/ml/ug cell protein [mean ± sem, n= ] and did not differ from e +mpa basal levels. the addition of ng/ml of tnf-or il- significantly elevated g-csf output to . ± . and ± respectively (p< . ), representing more than a -fold and , -fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il- from . to . ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il- may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr and tlr in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. . to compare tlr and tlr protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n= ) and pregnant (n= ) human cervical cells were obtained by smear and tlr and tlr expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr and tlr protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr expression in late pregnant compared with non-pregnant cervical epithelium (p= . ), whilst tlr did not appear to differ. there was an apparent up-regulation of tlr protein expression in response to beta-oestradiol (n= ) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: -hour urine samples from pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n= ), b: cranberry in the am, then placebo at dinner (c, p; n= ), c: placebo two times daily with meals (p, p; n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected -hour urine specimens from them. the ph of all urine specimens was adjusted to ph= and filtered. aliquots of each were independently inoculated with overnight culture of - cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after hours of incubation for e. coli and k. pneumoniae, and hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r dk - ; clinical trials registration nct . ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected -hour urine specimens from pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n= ), b: cranberry in the am, then placebo at dinner (n= ), c. placebo two times daily with meals (n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n= , group b: n= , group c: n= ), and collected -hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as , , and %. results: when combining all subjects and dosing regimens, the sensitivity of the assay was %, range % in the pregnant group assigned single daily dose of cranberry to % in the non-pregnant group assigned to multiple daily doses. the specificity ranged from % to %. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r dk - . clinical trials registration nct . objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor and (tlr and ) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from pregnant women: - weeks gestation (w) preterm not in labour (ptnl) n= ; - w preterm in labour (ptl), n= ; - w term not in labour (tnl) n= ; and - w term in labour (tl) n= . leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il- , icam- , mcp- , tlr and tlr was significantly increased in tl compared to gestation matched tnl. the expression levels of il- b, il- and tlr- were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr- agonists: macrophage-activating lipopeptide (malp- ), pam csk , and peptidoglycan. methods. cecs were isolated by smears and compared between pregnant ( rd trimester) and nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr primary antibody and its igg , isotype control, to determine tlr- protein expression. a further nonpregnant smear samples were each prepared, and seeded at a concentration of , cervical epithelial cells/ml into -well cell plates. cells were incubated at °c in % co overnight with the tlr agonists malp- and pam csk (at concentrations of and ng/ml), peptidoglycan( ng/ml), il- ( ng/ml, positive control) and rpmi + -glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at - °c until the concentration of il- was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr (specific mean fluorescence . vs . respectively) but did not differ (p = . ). unstimulated cells incubated with buffer alone demonstrated high il- levels ( pg/ml), which did not differ following stimulation with malp- ( pg/ml), pam csk ( pg/ml) or peptidoglycan ( pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr agonists are pending. conclusion. high basal il- levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr- agonists did not lead to further release of il- may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega- fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega- fatty acids on producing pge and il- , in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega- fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for hours in °c, co incubator. after that, g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for hours. the cell media were collected, and pge and il- concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)- were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge (p< . ) and il- (p< . ) significantly in elisa. cox- expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration ( , m) in the western blotting (p< . ). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p< . ). conclusion; this study offers some scientific mechanisms, by which omega- fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox- , nf b and pparare related. ( mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: -aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il- production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il- b and il- . we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d - of gestation. fetuses received repeated iv injections of lps ( ng/kg estimated fetal weight/day) between d and d . plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d and d . at d ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days and (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n- and n- polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n- and n- pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid ( : n- ), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid ( : n- ), a trans-monosaturated fa, induced tnf production by -fold compared to vehicle (fig. a) . likewise, palmitic acid ( : ), a saturated fa, induced placenta tnf production by -fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid ( : n- , dha) reduced placenta tnf production by up to % following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid ( : n- , la) or arachidonic acid (n : n- , aa) suppressed tnf production by up to % and %, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n- fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n- fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day to term gestation, pregnant rats received either ad libitum food and water (control, n= ), or wr to produce an increment of meq/l in plasma sodium (n= ). following delivery, all dams received ad libitum food and water. at d after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk , mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to s). at d after birth, offspring glomerular number were determined in formalin fixed m sections using histomorphometric analysis. values are means±se. results: wr offspring (day ) had higher glomerular number than control (wr ± , control ± per mm , p< . ). flk mrna expression was increased in wr offspring kidneys (wr . ± . , control . ± . , p< . ) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr . ± . , control . ± . , p= . ). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk , increased receptors (flk ) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day to birth) reduces rat kidney glomerular number by % at weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e -e , suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e fetal kidney explants from dams with mfr. methods: e fetal kidneys were collected from % mfr pregnant rats and ad libitum control rats (n= per group and time point), incubated in dmem/f k medium for , , , , and days, and fixed with % paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p< . . results: mfr ex-vivo kidneys at day demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: ± vs c: ± terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p< . ) increases in branching in explant culture to maximum values at days (mfr: ± vs c: ± ). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of % at day , % at day , % at day , % at day , and % at day (p< . , mfr vs c at day ). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e ) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr and gcr ) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr and gcr are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, % casein) or low protein (lp, % casein) diet throughout gestation. kidneys were collected on embryonic day (e ), post-natal day (d ) or (d ), n . real-time pcr was used to compare gcr , gcr and renin expression. data were standardized to a housekeeping gene (s ) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p< . ). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr and gcr were significantly elevated compared with controls ( . fold and . fold respectively; p= . ), whereas renin was . fold lower. on post-natal d , there was a trend towards increased expression of both gcr ( . fold; p= . ) and renin ( . fold; p= . ) in offspring from lp dams. at d , there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr / pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to to produce iugr newborns. all pups were nursed by control dams and weaned at weeks to ad libitum feed. at day and months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at months, hepatic triglyceride content was determined enzymatically. results: at d of age, iugr pups showed significant downregulation of ppar ( . -fold) and ppar ( . -fold) expression, though crp expression was significantly upregulated ( -fold). findings persisted at months of age, with continued downregulation of ppar and ppar ( . -fold) and upregulation of crp expression ( -fold). furthermore, iugr adults had significantly increased hepatic triglyceride content ( ± vs. ± mg/g liver, p< . ). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, ederlen casillas, guang han, darran n tosh, , michael g ross. dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat and pi k pathways, whereas insulin activates pi k pathway by binding to its receptor (ir) and substrate (irs ). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat and decreased expression of ir and irs , suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to . day old male offspring were given saline or leptin ( g/g, i.p). hypothalamus was dissected at , and minutes and protein expression of total stat , phosphorylated stat (p-stat ), inhibitor of leptin signal (socs ), ir, irs , total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. ( ) jak-stat : leptin-treated controls show progressively increased pstat ( -fold) with initial suppression of socs ( . -fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat expression ( . -fold) with failure to downregulate socs . ( ) pi k pathway: leptin-treated controls showed a significant reduction in irs ( . -fold) and pakt ( . -fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs ( -fold) and pakt ( -fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat (p-stat ) response in conjunction with an enhanced socs- response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, ) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus ( - viral infective particles) or a -galactosidase-adenovirus as a control. after hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with % of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day after delivery (p< . ). at weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all , and weeks in offspring of b > c >a. (p< . , vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n= ). these women were divided into controls, ri < . (n= ) and cases, ri . (n= ), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching ( % vs, % p< . and % vs. %, p< . , respectively). in contrast to that observed in patients of heterogenous parity previously, ri . was not associated with adverse pregnancy outcomes despite an average ri of . , significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt- over-expression during pregnancy determines higher reactivity in the offspring airways at months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt- induced preeclampsia at year of age and compare responses between the two age groups. methods: cd- mice at day of gestation were injected via the tail vein with adenovirus carrying sflt (adsflt , pfu/ l) or mfc (admfc, pfu/ l). mice were allowed to deliver. tracheas were isolated from female and male offspring at months and year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, mm), the mast cell degranulating agent compound / ( / , g/ml), and concentration-responses curve to acetylcholine ( - - - m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound / between year old offspring born to the sflt and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt -treated group were significantly higher in year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, year old male offspring had higher responses to compound / and acetylcholine than months old males. responses to kcl were significantly higher in months old males than year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where months old offspring demonstrated significantly higher responses to acetylcholine compared to year old offspring. conclusions: our findings did not show that airways of year old offspring born to mice with a preeclampsia-like syndrome induced by sflt- over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf- , which modulates growth and is secreted by the liver, may contribute to these morbidities. igf- is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf- mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day to and lactation, whereas study dams were % fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at day and months, male livers were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k trimethyl, and associated levels of each igf- species were measured by pcr. results: at months, obese fr/adlib males showed increased mrna levels of igf- a, igf- b, igf- exon , and igf- exon as compared to controls ( ± , ± , ± , and ± %). comparing fr/adlib month to newborn offspring, h /k was increased at igf -promoter , promoter , exon , utr# and utr# ( ± , ± , ± , ± , and ± %), though there was no differences between control month and newborns. in contrast, month fr/fr males had comparable mrna levels to the controls except for igf- b (% of control: ± ). further, fr/fr month h /k was only different from newborns at utr# (% of newborn: ± ). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf- epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; -amino acid peptide) which is then processed into its active forms: "big" igf-ii ( - ) and mature igf-ii ( - ). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats ( - g) were given nicotine ( mg/kg/day) or saline for days prior to mating, during pregnancy, and throughout lactation. at gestational day , and , dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd ) and weaning (pnd ). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day . however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day . nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day . in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd ). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar ), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to term. adipocytes from day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following % confluence, iugr and control adipocytes were treated to two doses ( and m) of either rosiglitazone or badge for h. mrna and protein was extracted for expression of ppar , c/ebp , srebp c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar ( . -fold) and c/ebp ( -fold) as compared to control adipocytes, though srebp c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation ( % of control, p< . ) and showed greater response to rosiglitazone ( . -fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf- is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf- changes which are distinct from diet-induced obesity. we determined igf- hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were % maternal food restricted from day to . all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, %) diet whereas iugr were weaned to normal ad libitum diet (fat, %) to produce diet-induced (dio) and programmed obese groups, respectively. at months, male hepatic igf- were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k dimethyl and h k trimethyl, and associated levels of each igf- species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf- a, exon and exon ( ± , ± , ± %). chip with h k dimethyl showed increased igf- exon ( ± %) and with h k trimethyl, increased igf- promoter and promoter ( ± , ± %) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf- a mrna and h k dimethylation and trimethylation of igf- than dio controls. changes in igf- in adulthood from a prenatal insult thus suggest that igf- is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, pandu gangula, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa; internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed % protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: ) changes in estrogen receptor (er) angiotensin ii subtype receptor (at -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either % protein (ctrl) or % protein (lpd) from day of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at -r or enos. no changes in at -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at -r or enos in females, and the decrease in enos but not at -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b) and % restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at and weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at weeks, were equilibrated at g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with %co in air ( °, ph . ) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type- receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to - m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride ( mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day (p< . ). at weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both and weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n= ). maximal contractions to ne were significantly stimulated by l-name in a (p< . ), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p< . ), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b), % restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally % restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day (p< . ). at day after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p< . ). at weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at weeks in offspring of d> b > e > c >a. (p< . , vs. a). at weeks, hypertensive offspring as b>d>e>c>a (p< . , vs. a). at weeks, d> e > b > c >a (p< . , vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day in female offspring of rats exposed to hypoxia ( , ft; . % po ) from days thru of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n= ) were hypoxic from - dga with additional control dams either fed ad libitum (al, n= ), pair-fed with the hx dams throughout gestation (pg, n= ), or only pair-fed during the window of hypoxia (ph, n= ). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n= /litter) by h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p< . ) than that of al pups, and hx pups were lighter (p< . ) than ph pups. weight of hx offspring remained lower (p< . ) than al pups until the termination of the study at pn , while the pg and ph pups reached weights comparable to the al offspring by pn . relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p< . ) in the hx offspring at pn as was rv/lvs (p< . ). cardiac function was evaluated by echocardiography at pn . rv wall thickness was % greater (p< . ) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were %, %, and % higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p< . ) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p< . ) in the hx pups. conclusion: prenatal hypoxia from - dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd . reproductive sciences; pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx ( . %o in chamber) for d prior to term ( d). in two separate groups, nic was also added to the drinking water ( mg/kg/d) for d at a dose that generates fetal nic levels ( ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( d) were excised and weighed. left ventricles of hearts were obtained and frozen at - c for storage. mmp protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p< . ) fetal body wt by %, increased (p< . ) the relative fetal brain wt (brain wt/fetal body wt ratio) by . % and had no effect on relative placental or fetal heart wts. hpx alone decreased (p< . ) fetal body wt by . %, increased the relative fetal brain wt by % but, in contrast to nic alone, increased relative placental wt by . %. both mmp protein levels (mmp /a-actin density values) and activity (clear band density) were increased (p< . ) by nic alone (by . and . fold, respectively) and hpx alone (by . and . fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by . and . fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide- kinase (pi k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate (irs- ), and phosphorylating pi k. activated pi k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk /erk ) signaling pathway likely involves irs- . further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n= ) food, whereas study dams were % mfr (n= ) from pregnancy day to to produce iugr newborns. at day , hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi k (ir, irs , pi k and akt) and mapk (irs , erk , erk ) pathways, and pten. data is presented as fold difference normalized to beta- -microglobulin. results: at d of age, iugr pups exhibited downregulation of the entire pi k pathway with significantly decreased (p< . ) mrna levels of ir ( . -fold), irs- ( . -fold), pi k ( . -fold) and akt ( . -fold). further, iugr pups showed similar decreased mrna expression of erk ( . -fold) and erk ( . -fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day (pn ). post mortem was carried out on pn (c and r) and pn (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn and pn (p< . ). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn . male, but not female, relative bmp mrna expression on pn was higher in r than c (p< . ) while gdnf, tgf and at receptor were not different. on pn , wnt (but not at r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p< . ) when compared to c-on-c (p< . ). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p< . ) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn . higher bmp mrna expression indicates impaired branching morphogenesis in pn r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn . amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f hoxa gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b d f (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n= , . ± . pups; n= , . ± . pups, respectively; p< . ). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa mrna and protein expression were decreased in the uterus of the f females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with nm to nm ghrelin resulted in a to % increase (p< . ) in hoxa expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos ) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos . in this study we aimed to determine hepatic and renal histopathologic damage in nos deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos +/+wt and nos -/-ko mice during their first (p ) or second (p ) pregnancy were sacrificed at day of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was % and %, respectively. in nos -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were % and %, respectively (p< . ). this correlation persisted when we analyzed the relative severity of hepatic lesions between p and p animals. although a similar trend was observed, the difference between p and p animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p versus p nos -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, , r de jonge, wa helbing, bd van zelst, j lindemans, eap steegers, rpm steegers-theunissen. , , , obstetrics gynecology; pediatrics; clinical chemistry; epidemiolog y biostatistics; clinical genetics; erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at months after the index-pregnancy. case-children (n= ) were included if they had ds and chd. children (n= ) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or( %ci) of the biomarkers are shown in table . an increase of the sam/sah ratio with unit decreases the risk of a child with ds and chd with percent. moreover, every increase of mol/l of homocysteine . fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c bl/ female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting % protein or % protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at - c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct- and sphk- gene upstream regions in the mouse liver was analyzed. hepatic oct- or sphk- promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct- and sphk- gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both and hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp- ) were downregulated at both and hours after infection. sdf- , which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam- , contrasted by decreased amounts of vcam- , indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n= in each group) with twin fetuses at - days (very preterm) and - days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at - c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b : . ± . pg/ml) and cortisol (diagnostic laboratory, b : . ± . ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable ( ± ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: ± pg/ml; cortisol: ± ng/m) and fetal plasma (crf: ± pg/ml; cortisol: ± ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p < . ). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin - objectives: interleukin- (il- ) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il- may be a marker of maternal obesity and in utero fetal programming. our hypotheses were ) il- correlates with maternal obesity and ) il- mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il- levels from non-diabetic women who underwent a live birth. the independent variable was il- (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< kg/m ); overweight ( - kg/m ); and obese (> kg/m ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il- levels were compared across bmi categories using anova. the association of il- levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il- levels were significantly higher in obese women ( . ± . ) compared to overweight ( . ± . ) and normal/underweight women ( . ± . ) [p< . ]. after adjustment, il- levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc) . ; % ci: ( . , . )]. as shown in the table below, elevated levels of il- were statistically significantly associated with a . gm higher infant birth weight after full adjustment. each unit increase in pre-pregnancy bmi was associated with a gm higher infant birth weight. table . association of il- with infant birth weight characteristic regression coefficient % confidence interval interleukin- . . , . pre-pregnancy bmi . , gestational weight gain - - , bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il- and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il- or an alternative independent pathway. we have demonstrated that -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase (hdac ). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at or x cells /well to -well and -well plates for growth and rna experiments, respectively. after growing to - % confluence, cultures were treated with varying concentrations of aea up to a maximum of m for hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of m where confluency was significantly reduced from % at m to - % at m and m (*p< . ; one-way anova with tukey's hsd test; n= ). cultures treated with m and m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac mrna expression with a significant effect at . m (*p< . ; oneway anova with tukey's hsd test; n= ). at this dose, the effect of aea had reached an effective maximum decrease in hdac mrna levels ( %) because hdac mrna levels were not decreased further by either m aea ( %) or m aea ( %). the alteration of hdac gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as st trimester miscarriages. ( ) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb and cb with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb , cb and faah constitute the endocannabinoid system and previous studies have shown that faah and cb are expressed in term human placenta( ) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb and cb expression in the placenta during the first trimester of pregnancy. methods: first trimester samples ( to weeks gestation) were fixed in % neutral-buffered formalin for days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of : in pbs, cb at : and cb at : . transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb and cb were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the th and th gestational week and by week faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb and cb localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb immunoreactivity showing diminished intensity after week , although this did not reach significance at the transcript level. cb immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb and cb immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb immunoreactivity in the syncytiotrophoblast differed from that of cb and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb expression from the syncytiotrophoblast after the th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., ( ) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb- complete solution containing cgrp ( - m), cgrp plus its antagonist cgrp - ( - m), in well plates with x cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein (ramp ) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: ) immunofluorescent staining showed that cgrp receptor components crlr and ramp are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; ) cgrp dose-dependently ( - to - m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp - ; ) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and ) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp ( - m) was significantly increased over that of the untreated controls. this increase was observed at hours of treatment and further increase was noted at hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs- gene promotes il- production by jeg- trophoblast cells. qin dong, , ruping fan, yang gu, david f lewis, yuping wang. biochemistry, harbin medical university, harbin, heilongjiang, china; obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling- (socs- ) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il- expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs- in placental trophoblasts could promote il- production. methods: full-length socs- open reading frame (socs- cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen -n vector, which encodes a green fluorescent protein zsgreen . successful socs- cloning was confirmed by sequencing. for transfection, jeg- cells were placed into well/cluster plates at a concentration of . x /well. the tranfection was carried out with . g of socs- /zsgreen plasmid (psocs- /zsgreen ) for hours when cell reached - % confluence. siport lipid were used. jeg- cells transfected with zsgreen plasmid (pzsgreen ) only was used as control. after approximately hours of transfection, cells were treated with il- at and ng/ml. medium was then collected and measured for il- by elisa. il- production was calculated as the percentage of increase by psocs- /zsgreen transfected cells compared to the cells transfected with pzsgreen only. result: il- production was increased by psocs- /zsgreen transfected jeg- cells compared to the cells transfected with pzsgreen only when stimulated by il- , control: . % increase; ng/ml il- : . % increase and ng/ml il- : % increase, p< . , respectively. data are means from three independent experiments. conclusion: over-expression of socs- gene could promote il- production by placental trophoblast cells. reduced sil- r release and increased ratio of sgp /sil- r production by placental tissues from women with preeclampsia. shuang zhao, ruping fan, jingxia sun, yang gu, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il- , il- and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il- receptor (sil- r) and membrane receptor il- /gp complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp (sgp ) is an antagonist for il- /il- r trans-signaling. this study was undertaken to determine sil- r and sgp production by villous tissues from normal and pe placentas. methods: placentas delivered by normal and pe pregnant women were used in this study. placental explants were incubated with dmem for h. the culture medium was collected. placental villous tissue productions of sil- r and sgp were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level < . was considered statistically different. results: placental tissues from pe produced significantly less sil- r than tissues from normal pregnancies, . ± . vs. . ± . pg/mg of wet tissue, p< . . soluble gp production was relatively compatible between pe and normal placental tissues: . ± . vs. . ± . pg/mg of wet tissue. the ratio of sgp /sil- r release was significantly higher in pe than in normal placentas, . ± . vs. . ± . , p< . . conclusion: reduced sil- r production and/or increased ratio of sgp /sil- r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, jing dai, frank z stanczyk, daniel r mishell, jr. obstetrics and gynecology, university of louisville, louisville, ky, usa; obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from healthy pregnant women and diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs- and immulite systems. data was grouped in -week intervals until weeks of gestation and -week intervals thereafter, with to samples in each interval for healthy women and to samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs- system, mean hcg levels (miu/ml) for healthy pregnant women were , at - weeks, , at - weeks, , at - weeks, , at - weeks, , at - weeks, and , at - weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were , , , , , , , , , and , , respectively. between - weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p= . ) compared to healthy controls. ) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/ ) and ace knockout ( / ) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type receptor (at r), ang type receptor (at r), ace, and ace- in the three genotypes (n= /group). placental sections were blinded to genotype; a score range of - was used. the test was used (spss version . ) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of / mice compared to wt/wt and wt/ mice (at r p< . ; at r p<< . ; ace p<< . ; ace- p<< . ). at r and ace- displayed increased staining in the fetal vascular endothelium of / mice (at r p<< . ; ace- p= . ), and in the cells lining the maternal central artery (at r p<< . ; ace- p<< . ). ace- expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in / placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at rs is vasodilatory and ace- catalyses production of the vasodilatory ang ( - ); placental blood flow was presumably well-maintained and pregnancy outcome is normal in / mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase (cox- ), cyclooxygenase (cox- ), and -hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox- was relatively unchanged and pgdh was only slightly increased, but cox- was noticeably decreased in the treated slides. these results were time dependent. this data suggests that alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp- in this pathway. methods: amaxa nucleofector technology was used to deliver shp- or scrambled sirna ( nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for hours, then treated with igf-i or igf-ii ( nm) for a further hours before immunohistochemical (ihc) analysis for cell proliferation (ki , brdu) or apoptosis (m ). results: sirna-mediated knockdown of shp- in bewo cells ( % reduction on western blot) demonstrated that igf-induced proliferation was reduced from . ± . % to . ± . % (p< . , n= ). ihc analysis of tissue demonstrated that shp- is localised to ct. following knockdown ( % decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by . ± . % and . ± . % respectively (p< . , n= ). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m staining) was reduced by . ± . % and . ± . % respectively (p< . , n= ) after shp- knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp- . exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp- -dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr ) and estrogen receptor-beta (esr ). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol ( - m to - m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase- (cox- ) mrna levels -to -fold, as quantified by real-time pcr (p< . ). conversely, there was no effect of estradiol on cyclooxgenase- (cox- ). conclusion: these results suggest that estradiol and esr are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u -hd . previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx , her- and alpha integrin protein expression) to an invasive phenotype (characterized by her- and alpha protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her- receptor antagonist, ag . furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag . thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her- ligand, resulted in the downregulation of her- and an upregulation of her- expression, as well as an induction of alpha integrin expression. pre-treatment of placental villous explants with ag blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her- and upregulating her- expression, effects that were both blocked when jar cells were pre-incubated with ag . in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her- /her- heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her- in evt outgrowths and in jar cells; her- expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her- signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr- /svneo cells). methods: htr- /svneo cells cultured at º c and %co in rpmi- medium supplemented with % fbs were transiently transfected with nm of three different sirna sequences of pthrp, si , auaccuaacucaggaaacuuu; s i , g a g c u g u g u c u g a a c a u c a u u ; a n d s i , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at % confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent h after transfection, and protein was extracted using lysis buffer h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c bl/ mice were fed a hypercaloric diet ( % fat, % sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day gestation) the dams ( controls, overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. probe sets, corresponding to genes showed differential expression (p < . ); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd- , earnest eu framework , tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing % less protein than normal chow ( % vs. % protein content) from embryonic day . (e . ) to e . . following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e . pups were decreased % (p< . ). probe sets, corresponding to genes, were regulated by protein restriction (p< . ); ninety-one being up-regulated and down-regulated. of particular note, several genes related to the p pathway were up-regulated. along with p itself, positive regulators of p (zmiz , jmy, hipk ) and genes activated by p (inpp d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p pathway. (supported by usphs hd- and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of -chloro- , -dinitrobenzene (cdnb) conjugate , -dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m medium to hr. at , , , , , and hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to μm cdnb, rinsed and incubated in buffer at °c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp - , abc transporter isoforms b , c and g (abcb , abcc , and abcg , resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p - (gstp - ) and abcg remained unchanged over hr in culture. expression of abcb and abcc , and total tissue glutathione decreased with culture time. ldh release was unchanged up to hr and increased at hr, while mtt incorporation remained constant to hr and decreased at and hr suggesting a decline in tissue integrity and viability at hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by . ± . % (p< . ), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp - and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor (eif ) is a subunit of eif which is required for initiation of translation however when phosphorylated, eif is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif . we hypothesized that hypoxia would increase the levels of phosphorylated eif in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for hr in atmospheres of , , or % o or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, . - . mm) in % o . cell extracts were analyzed by western blotting to determine the degree of eif phosphorylation. results: incubation in , , or % o did not increase eif phosphorylation relative to the % o control (n= , separate placental preparations). incubation in dmog concentrations up to . mm did not affect eif phosphorylation however incubation in . mm dmog increased eif phosphorylation by ± % (p < . , n= ). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif phosphorylation does occur, we did not observe changes at dissolved oxygen levels of %. these data suggest that a reduction in syncytial protein synthesis via the eif pathway takes place only under severe hypoxic stress. (supported by nih hd ). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat , and which mediate neutral aa transport. snat is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat . methods: nonpregnant ewes were randomly assigned to a control (c, % of nrc recommendations) or obesogenic (ob, % of nrc) diet from - to days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at - c for western blot analysis. results: fetuses from ob ewes were % heavier (p< . ) than those from c ewes at dg ( ± vs. ± g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p< . ), or tended to be higher (met and lys, p< . ) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p< . ) in ob compared to c ewes. snat content in cot tissue was reduced in ob when compared to c ewes ( . ± . vs. . ± . arbitrary units; p< . ). conclusions: maternal obesity in pregnancy reduced expression of placental snat protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre p rr . early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n= ) or normothermic (con; n= ) environment at days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate ( . ± . vs . ± . breaths/min) and arterial po ( . ± . vs . ± . mmhg) were increased (p . ), whereas maternal heart rate ( . ± . vs . ± . beats/min), blood pressure ( . ± . vs . ± . mmhg) and arterial pco ( . ± . vs . ± . mmhg) were reduced (p . ) in fgr pregnancies. at dga, fetal weight was not different (p . ), but placental (total placentome) weight ( . ± . vs . ± . g) was reduced (p . ) in fgr pregnancies. while uterine artery (pregnant horn) flow ( . ± . vs . ± . ml/min) tended (p= . ) to be reduced in fgr pregnancies, relative uterine artery flow ( . ± . vs . ± . ml/min/g fetus; . ± . vs . ± . ml/min/ g placenta) was not different (p . ). uterine o uptake (mmol/min), relative uterine o uptake (ml/min/g fetus or ml/min/ g placenta) and uterine o extraction (%) were not different (p . ) between fgr and con pregnancies. at dga, umbilical vein po (mmhg), o content (mm) and o capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd . barton c staat, anna maria marconi, cinzia paolini, alex cheung, henry l galan, frederick c battaglia. obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with c labeled isotopes of glucose, myo-inositol and mannose over hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at hour (h), . h and h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose ( . ± . , p= . ) and glucose ( . ± . , p= . ) were not significantly different from . , consistent with transplacental supply. the feto-maternal ratio for myo-inositol ( . ± . , p= . ) indicates little transplacental flux ( % of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, manuela augsten, christian guelly, ursula hiden, ingrid lang, manfred moertl, uwe lang, gernot desoye. clinic ob/gyn; center of med res; inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser -phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with i-labelled hdl. hdl from human donors was used to stimulate ser enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for cpg-rich promoter regions of sr-bi. pdzk a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was -fold higher in eca than in ecv ( ± . vs ± . ng hdl/mg prot). hdl did not induce ser enos phosporylation in eca or ecv, which was stimulated by ionomycin about -fold in both cell types. pdzk was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk may account for the failure of hdl to activate enos. (grants , , oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p and the ebp proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p and ebp in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia ( % o ) or normoxia ( % o ) for and hours. western blot for p-mtor, mtor, p-p , p , p- ebp , and ebp were done for each time studied. results: trophoblast cells demonstrated: ) positive staining for cytokeratin, ) non significant differences for mtor at either ( . -fold; p= . ) or hours ( . -fold, p= . ), ) no differences in p protein at ( . fold; p= . ) or hours ( . -fold, p= . . ), ) no differences for ebp at either or hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r hl - a ). increased expression of phospho-mtor, phospho-p , phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p , ebp , erk and akt will be phosphorylated (activated) in the placentae of age (dga) animals. study design: ewes were exposed to ht conditions for days to induce iugr and were placed in ambient conditions. at necropsy ( dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p , p , (p) ebp , ebp , (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses ( ± g v. ± g; p= . ) and smaller placentae ( ± g v. ± g; p= . ) at dga. fgr cotyledon showed an increase in p-mtor ( . -fold; p= . ), p-p ( . -fold; p< . ), p-erk ( . -fold; p< . ) and p-akt ( . -fold; p< . ). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p but not ebp while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation ( dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r hl - a ). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for hours with uric acid ( . mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid ( m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [ c] methyl-amino-isobutyric acid, was measured over minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by % (± . %, p< . ). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid ( . mg/dl, . mg/dl and . mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr -svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p< . ), with decreases of % (± . %), % (± . %) and % (± . %) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor (smurf ), a hect type e ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf in human placental villi during pregnancy. we have also investigated the possible function of smurf in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr /svneo. our results showed that expression of smurf in placental villi was the highest during the first trimester and the expression decreased in the nd trimester. expression of smurf was lowest in placental villi at parturition. overexpression of smurf in htr /svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad , another potential target of smurf , was unchanged. in conclusion, the present study suggests that smurf participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp- in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp- expression were examined in an immortalized human cytotrophoblast cell line, b tert- that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp- mrna and protein levels in b tert- cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp- expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp , an inhibitor of a jnk kinase, reversed the up-regulation of mmp- induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk / pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp- . during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than %. however, the majority of studies on primary trophoblast cell development have been performed at % oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd- negative selection and cultured at %, % or . % oxygen tension for up to days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at % oxygen % of the cells remain in culture; at . % oxygen tension % of the cells remained. the cells at . % oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing cylindrical recesses m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels ( , cells per) for up to days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days , and revealed central vacuolization forming a trophoblast vesicle with an outer rim . m (+/- m) thick. this rim size remained constant for at least days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase- staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing- (gcm ) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase- (timp- ) in the highest ( -fold) upregulated group of genes. confirmatory rtpcr demonstrated a -fold mrna induction. in placental villi, gcm acts as a transcription factor promoting expression of the fusogenic protein syncytin that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp and gcm in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in % oxygen with prior exposure to either gcm sirna or anti-sense oligo-nucleotides to gcm . gcm inhibition in the bewo system was associated with a - % increase in timp- protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in % oxygen) to test the hypothesis that gcm mediates metallo-proteinase expression and evt invasion via timp- . presently we conclude that gcm -mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp- . funding: cihr. scott h purcell, jeremy d cantlon, virginia d winn, russell v anthony. , colorado state university, fort collins, co; university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr . objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin- . cytotrophoblasts from first trimester pregnancies (n= ) were subjected to an in vitro invasion assay and rna was harvested following , , and h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from , , to h in all samples; while hypoxia decreased expression at h of invasion compared to h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at , , and % of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than % mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd - ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr- /svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek / / erk / phosphorylation. objectives: ) to asses the effect of imd on migrating capacity of htr- sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u and manumycin a, respectively ; ) to assess the effect of imd peptide on phosphorylation of mek / and erk / in first trimester htr cells ) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr- sv/neo cells were used to assess the effect of imd ( - m) on the expression of hla-g mrna and phosphorylation of erk / and mek / protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, ) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u and manumycin a, respectively; ) imd ( - m ) stimulates phosphorylation of erk / and mek / proteins in htr cells, ) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt- . yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor- (sflt- ) in the maternal circulation during pregnancy. increased placental release of sflt- is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt- release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt- release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = ) were used. we tested if chymotrypsin could promote sflt- release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at . , . , and . g/ml for hours. the culture medium was then collected for measuring sflt- . we then determined the specificity of chymotrypsin induced sflt- release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt- . soluble flt- was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < . is considered as statistically different. results: ) sflt- concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt- release induced by chymotrypsin was in a concentration-dependent manner: control: . ± . ; . g/ml: . ± . ; . g/ml: . ± . ; and . g/ml: . ± . (p< . ) pg/mg tissue/hour. ) ci could attenuate sflt- release. this inhibitory effect was also revealed in a concentration-dependent manner: control: . ± . ; ci . g/ml: . ± . ; and ci . g/ml: . ± . (p< . ) pg/mg tissue/hour. conclusions: increased placental sflt- release stimulated by chymotrypsin and decreased placental sflt- release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt- generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt- production in pe. (supported nih grants hl and hd ). the change of autophagy-related proteins, lc and beclin- , by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc , but not beclin- , was increased in placentas from pregnancies complicated by severe preeclampsia (sgi abstract # ) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl for a given time and the changes of beclin- and lc were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc -ii in cultured primary trophoblasts while decreasing the expression of beclin- (p< . for each). however, cocl stimulation did not induce a significant change of both lc -ii and beclin- . conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct , recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t . we previously reported that mct expression in n-tera- (nt ) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct -null jeg- choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s x mct mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct via sirna and evaluate the effects of cell proliferation (mtt and [ h]-thymidine assays) and tri-iodothyronine (t ) uptake. results: repression of endogenous mct expression in nt cells by % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t concentration ( . %, . % and . % induction at , and nm t , n= , p< . ). we also sought to examine the role of mct in t uptake. in jeg- cells, wild type mct induced a . -fold increase in the uptake of i-labelled t . by contrast, mutants s x and l p failed to significantly augment t uptake, though r h caused a mild but significant . fold induction in uptake, hence retaining approximately % of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t binding protein, resulted in a similar increase in t uptake compared with control ( . -fold; n= ; p< . ), implying that mct plays only a minor role in thyroid hormone efflux in jeg- cells. mutants s x, l p and r h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct in the modulation of cell proliferation, independent of t transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between - with a singleton pregnancy >= weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included , preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as . % (p= . ) among multiparous women - weeks ga to as high as . % (p< . ) among nulliparous women with diabetes. a total of , women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt , endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in women with uncomplicated normotensive pregnancies compared to women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average . ± . months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p< . . results: the mean concentration of sflt , endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih- mo -rr and nih- po -hd . (ajp, ) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. mfr and control offspring were sacrificed on day of life (p ). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into - mm explants and incubated with leptin ( - ng/ml) in dmem ( % fbs). after hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr and r , and ob-ra, stat and socs ( s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr and vegfr mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat and socs mrna in the control tissues. in contrast, in mfr explants only the ng/ml concentration of leptin induced an increase in stat mrna, and the magnitude of socs mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a -year interval, primiparous women with a history of early-onset preeclampsia (delivery < weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was . years for cases compared to . years for controls (p<. ). after adjustment for age, we observed significantly increased mean values for weight (p=. ), body-mass index (p<. ), systolic blood pressure (p<. ), diastolic blood pressure (p<. ), total cholesterol (p=. ), ldl cholesterol (p<. ), triglycerides (p=. ), fasting blood glucose (p<. ), and lower hdl cholesterol (p<. ) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated -year risk of first cvd events by framingham risk scores remained < % for all women (low-risk). nonetheless, at mean (sd) . ( . ) years after early-onset preeclampsia, % of women met the criteria for metabolic syndrome, % of women exhibited >= , % of women >= and % of women >= major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited women with hypertensive disorders in pregnancy ( pe and non-proteinuric hypertension [ht]) and matched normotensive controls. inhibin a and activin a levels, before and - hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in placentas delivered at cesarean section at similar gestational age ( pe, ht and controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p< . ). activin a (but not inhibin a) level was also increased at all gestations in ht (p< . ). after weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p< . ). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase (sflt- ) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = ), ht (n = ) and normotensive pregnancies of similar gestational age (n = ). these samples were stored at - ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p< . ); there was no significant difference between the levels in pe and ht (p= . ). the fact that placental concentrations of sflt- and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt- was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt- in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved asymptomatic pregnancies scanned at + - + week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n= ) and normotensive controls (n= ) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at - o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p< . was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was % and % significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt ( . ± . vs. . ± . pg/ml, p< . ) and placenta growth factor ( . ± . vs. . ± . pg/ml, p< . ) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate % with % false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt . currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin- (il- ) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il- levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery < weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il- at baseline, . days and . days after vaccination. results: median baseline crp and il- levels of women with a history of early-onset preeclampsia were comparable to controls ( . versus . mg/l; p= . and . versus . pg/l; p= . , respectively). however, high crp and il- responses to vaccination were more common in cases (ors for response > th, > th, > th, > th and > th percentile based on the distribution of control values of . , . , . , . and for crp [p for trend . ] and of . , . , . , . and . for il- [p for trend . ], respectively). the relationship between high il- responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend . ). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor (tlr ) and nucleotide-binding oligomerization domain (nod ), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr (d g and t i) and nod (r w, g r and l fs) in primiparous women with a history of early-onset preeclampsia, of whom women developed hellp syndrome and in women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il- , sicam- , fibrinogen and von willebrand factor in a subset of women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr were more common in women with a history of early-onset preeclampsia than in controls (or . [ % ci . - . ]). highest frequencies for tlr variants were observed in women who developed hellp syndrome (adjusted or . [ % ci . - . ]). in addition, high levels of il- and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr and nod allelic variants and high levels of il- was . -fold more common in women with a history of early-onset preeclampsia ( % ci . - . ) compared to controls. we observed an association of common tlr and nod gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples ( ) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n= ), mild pe (n= bp; / - / , proteinuria; trace to + or - mg/ hr urine) and severe pe (n= bp; > / , proteinuria; > + or > g/ hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n= ) and with pe (n= ) were fixed and embedded in paraffin. sections ( um) were immunostained with pbef antibody / (pheonix) and treated with abc reagent (vector labs) followed by dab ( . % mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between - ng/ml and were significantly higher in those patients with mild pe (p= . ) and further significantly elevated in those with severe pe (p= . ) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u rr - to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: healthy, non-smoking primigravida with an uncomplicated pregnancy and primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at , , , wks in controls, at time of diagnosis in pe cases and wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd expression (mcd ) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd expression and pp-sel increased with gestation and decreased post-natally (table ) . for pe analysis, data was divided into pre-term (sampled at mean wks), and term (mean wks). pp-sel was lower in pre-term pe than control (normal pregnancy wks; p= . ) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase (sflt ), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between + and + weeks of gestation from women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, . ± . ng/ml versus . ± . ng/ml, p = . , unpaired mann-whitney test). similarly, the serum levels of sflt were higher in women later developing late-onset pre-eclampsia ( ± ng/ml) when compared to controls ( ± ng/ml, p = . ). sensitivities and specificities for predicting pre-eclampsia were % and % for seng and % and % for sflt- , respectively. the combination of the two markers by multiplication yielded a sensitivity of % and a specificity of %. conclusion: seng and sflt , showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd expression (%cd ) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day . + . ). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - wks) and late pregnancy (lp, - wks). five of these women were diagnosed with hypertensive complications ( hypertension, preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p< . was accepted for significance. results subjects were . + . years old with a bmi of . + . kg/m at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p= . ). there was little change in the %mp formed between pp and ep (pp, . ± . % and ep, . ± . %, p= . ). however, the %mp increased significantly in lp ( . ± . %) as compared to pp (p= . ) and ep (p= . ). this increase occurred independent of the development of hypertensive complications (p= . ) and independent of pp platelet activation status. although statistically significant increases in cd expression were not observed, the change in cd expression over pregnancy correlated with the change in %mp over pregnancy (r= . , p= . ) and cd expression correlated with %mp in lp (r= . , p= . ). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd expression, during pregnancy. supported by nih hl . backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day + ) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of or mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at and hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n= ) versus sc (n= ). p< . was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc ( % increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young ( . + . years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day . + . ) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian ( / ) with a mean prepregnancy bmi of . + . kg/m . plasma volume was re-estimated at - weeks gestation. all patients were placed on sodium and total calorie balanced diets for days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was , + ml or + ml/unit bmi. third trimester plasma volume was , + ml representing a % increase (p< . ). the range of plasma volume expansion was - % dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= . (p= . ). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately % of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl . background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt- (soluble vegfr- ) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt- in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n= ). the first set delivered at weeks due to excessive bleeding. the second set delivered at weeks due to severe iugr and elevated blood pressure. mrna level of sflt- was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt- in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt- . immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl- . results: mrna level of sflt- was increased by . fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited . fold increase compared to controls. sflt- protein expression in the molar placentae was increased by . fold compared to controls, while the co-twin placentae exhibited a . fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt- in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt- expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt- in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n= ); ) neonates of patients with pe (n= ); and ) sga neonates (n= ). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration < th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: ) cord plasma pz concentration differed significantly among the study groups (kruskal wallis, p< . ); ) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median . g/ml, range . - . , p< . ; sga: median . g/ml, range . - . , p= . ; normal pregnancy: median . g/ml, range . - . ); ) there were no differences in the rate of pz deficiency among the groups; and ) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: ) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; ) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n= , ) reported upon enrollment at . weeks (sd . ) the number of weeks of regular multivitamin use during a week periconceptional period (lmp- to lmp+ ). preeclampsia cases were identified using icd- codes (n= , . %). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+ ] and post-conception [lmp+ to lmp+ ]). results were stratified a priori by overweight status. results: overall, , women ( %) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (< weeks of use) had no relation to risk (or . ; % ci . , . ) but regular use (>= weeks) was associated with modestly reduced risk ( . ( . , . ). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or . , % ci . - . ). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi < kg/m², or . ; % ci . - . ), with no apparent effect among overweight women (bmi kg/m², or . ; % ci . - . ). a total of , women reported regular mv use in both the preconception and post-conception periods, and , women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or . , % ci . - . ). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or . , % ci . - . ). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the , women who gave birth at yale-new haven hospital (ynhh) during and , and a person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for years and is internationally known. results: during during - , the pe rate for the general population at ynhh was . %. for the pa group, the rate was . %. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, esther streefland, pieter koolwijk, frans m helmerhorst, jan jaap hm erwich. department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt- , kdr, angiopoietin- (ang- ), angiopoietin- (ang- ) and tie- mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang- , ang- and tie- appeared increased in fgr cases compared to matched controls. in addition, plgf, ang- and tie- mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam- and svcam- , beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam- and svcam- were measured in healthy, nonsmoking women (cycle day . + . , prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - weeks) and late pregnancy (lp, - weeks). five of these women developed hypertensive complications ( gestational hypertension, pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p< . was accepted as significant. results subjects were . + . years old with a mean bmi of . + . kg/m at the time of prepregnancy studies. significant differences in sicam- levels as a function of pregnancy were observed (p= . ) and are outlined in table p= . differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam- levels in ep (p= . ) followed by an increase in sicam- levels in lp (p= . ). in women with hypertension in pregnancy, these differences in sicam- levels were not evident (p= . ). there were no differences in sicam- levels comparing women with or women without hypertensive complications prior to pregnancy (p= . ). in contrast to sicam- , we observed no significant differences in svcam- levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam- change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam- . supported by nih hl . yuval bdolah, uriel elchalal, shira natanson-yaron, hadas caspi, tali bdolah-abram, angelika bord, caryn greenfield, debra goldman-wohl, ariel milwidsky, franklin h epstein, s ananth karumanchi, simcha yagel, drorith hochner-celnikier. ob/ gyn, jerusalem, israel; ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to - times higher than multiparous pregnancies. soluble fms-like tyrosine kinase- (sflt ), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt and sflt /plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt (or sflt /plgf). methods: maternal serum samples from nulliparous (n= ) and multiparous (n= ) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt levels were , ± and , ± , (p= . ), the mean serum plgf levels were ± and ± (p= . ), and the mean ratios of sflt- /plgf were ± and ± (p= . ), accordingly. in a subgroup of multigravidous nulliparous pregnancies, sflt levels were , ± . correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a - years interpregnancy interval compared with a - years interval. conclusions: in nulliparous pregnancies, circulating sflt levels and sflt / plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even - years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt- ), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from placental abruption cases and normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: . ± . ng/ml vs. . ± . ng/ml, p < . . there were no significant difference in serum levels of plgf, . ± . ng/ml versus . ± . ng/ml, p=ns, although sflt levels were lower in cases: . ± . ng/ml vs. . ± . ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio . , % ci . - . ) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or . , % ci . - . ) and third (or . , % ci . - . ) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or . , % ci . - . ) and third (or . , % ci . . ) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at and weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at and - weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of /sec. results: compared to the proliferative phase viscosity was decreased at weeks and more so at weeks gestation (p< . ). . ± . . ± . nsd . ± . *** . ± . *** ua velocity (cm/sec) . ± . . ± . *** . ± . *** . ± . *** ubf (ml/min) . ± . ± *** . ± . *** . ± . *** shear stress (dynes/cm ) . ± . . ± . *** . ± . *** . ± . *** internal ua radius was not altered by the menstrual cycle, and was greater at weeks (+ %) and weeks (+ %). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory ( %) and did not rise further in early pregnancy but by weeks shear stresses was further elevated %. conclusions: equivalent rises in shear stress during the secretory phase and weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl , hd , hl background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day ) rats were used: control (n= ), hypertensive ( . g/l l-name in the drinking water, n= ), and treated with l-name and hydralazine (also in the drinking water, . g/l, n= ) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (< m) were mounted in a pressure arteriograph and equilibrated at mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from to mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with . mm papaverine and m diltiazem). results: myogenic tone developed in controls ( ± % maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone ( ± % maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group ( vs. mg, p< . ) was restored by hydralazine ( mg, p< . vs. l-name). average fetal weights were also reduced in the l-name group ( . vs. . g, p< . ), but only partially restored by hydralazine co-treatment ( . g, p< . vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises -fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi ) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp ) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only % (jci ) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: pregnant ewes were studied at - d gestation age (ga). had doseresponse studies with uterine artery l-name infusions to achieve . - . mg/ml over min. had h arterial l-name infusions to achieve and maintain levels of . mg/ml at (n= ), (n= ) and d (n= ) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during ( , , , , , , and h) and after ( , h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b - %, but was not doserelated. h arterial l-name infusions decreased upbf b by - h at each ga (p . , anova) and values returned to baseline by h postinfusion. sensitivity did not differ between ga (p= . , anova), upbf b falling - % at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at and d ga, p . ; but were unaffected at d ga. venous-arterial cgmp concentration differences were seen at d and absent at h of l-name infusion, p= . . conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b %, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose - %, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises -fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi ) and inhibition with tetraethylammonium ( . mm) dose-dependently decreases basal upbf % in the last third of pregnancy (jsgi ) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory -subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of nd generation uterine arteries obtained from nonpregnant (n= ), pregnant (n= , - d gestation; term d) and postpartum (n= , - d) sheep were used to measure expression of bk ca -and regulatory -subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the kda -subunit, rose % with placentation (p< . ) and was unchanged thereafter. expression of the kda regulatory -subunit paralleled the rise in bk ca density during placentation, increasing % (p< . ), but increased another -fold and exponentially in the last third of pregnancy (p< . , r = . , n= ). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p= . , r= . , n= ), pkg expression rose -fold (p= . , r= . , n= ) and gradually returned to nonpregnant levels by d postpartum (p= . , r= . , n= ). uvsm cgmp is being measured. conclusions: these are the st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory -subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein; mean±sem; p< . ) as well as a significant increase in smc density ( . ± . vs . ± . cells/ μm ; p< . ) compared to control mice with no significant change in elastin content ( ± vs ± μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein), smc density ( . ± . vs . ± . cells/ μm ) or elastin content ( ± vs ± μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr , and not the lgr receptor. both lgr and lgr receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr wild-type mice showed inhibited myogenic reactivity with a . ± . % increase in diameter whereas the arteries from rhrlx-infused lgr knock-out mice exhibited robust myogenic reactivity with only a . ± . % change in diameter (p= . ). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr knock-out and wild-type mice. the veh-treated lgr and lgr mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr wild-type, but not from lgr knock-out mice (p= . ). in contrast, the arteries from rhrlx-infused lgr wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr , and not the lgr receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for hours/day. smoke exposure was increased from day of gestation until late pregnancy (day - ) with mean total suspended particle levels of mg/m , representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n= - in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses ( . ± . g vs . ± . g, p= . ), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice ( . ± . vs . ± . ng/ml for virgin mice and . ± vs . ± . ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented rats for measurement of renal function. five were ovariectomized, and sham-ovariectomized. after days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg ( iu/min) was implanted s.c. and renal function was again assessed and days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p< . vs. baseline), but not ovariectomized (p=ns) rats on both days and of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p< . vs baseline). plasma hcg concentrations were , miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, - ng/ml), but not hcg ( , - , miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, , michele brochu, jean st-louis. , research centre, chu ste-justine, montréal, qc, canada; pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol ( -e ) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of -e , we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. -e and -e were equipotent in relaxing phe ( μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of -e , tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici , (estrogen receptor modulator), and kt (pka inhibitor) did not significantly influenced the response to -e . rp- -pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to -e to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for -e concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of -e in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered ( mg/kg/day) to pregnant rats from the day to of gestation. rats were sacrificed at day , and of gestation together with untreated and day pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring ( - mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day ( days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c bl /j mice (streptozotocin; mg/kg) prior to mating. mice were culled at day of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to mmhg and equilibrated ( °c; %co /air). arteries were constricted with phenylephrine ( m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; . nm- m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; m), a cyclooxygenase inhibitor (indomethacin; m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec . ± . nm vs . ± . nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at m ach, edhf-mediated relaxation was significantly reduced (p= . , one-way anova) compared with controls ( . ± . vs . ± ). in comparison, no-mediated relaxation was significantly increased (p= . , one-way anova) compared with controls ( . ± . vs . ± . %). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf- serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine ( ) if raf kinase contributes to phenylephrine (pe)induced contractile response, ( ) the role of raf kinase inhibitor (gw ) in regulating vascular tone during pregnancy, and ( ) mechanism by which gw produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw . pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw shifted the pe-induced dose response to the right in both np and p rats. gw itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw were greater as the pregnancy progressed from day to day . raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek / in a time-and dose-dependent manner. measurement of [ca + ] i with fura- showed that gw -mediated inhibition of pe-induced contraction was not associated with decrease in [ca + ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt and p-mlc which was inhibited by gw . conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine ) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, ) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and ) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: ) mean arterial pressure (map) was measured in conscious np and p rats. ) isometric tension was measured in mesenteric arteries of np and p rats. ) expression of contractile proteins, p-mlc and p-mypt were studied in cultured vascular smooth muscle cells (vsmcs). ) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: ( ) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. ( ) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( μm) compared to p ( - μm) rats. ( ) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc and mypt in mvsmcs. ( ) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, arianne van koppen, marc ea spaanderman, paul abm smits, frederik k lotgering. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e (flow inducing % dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to % precontraction with u . results: in nw rats rlx decreased e and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e and increased e max , and lower myogenic reactivity). in ow rats rlx increased e to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p< . between nw/ow, # p< . within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e ). methods: uaecs were cultured until % confluence, and then subjected to (static control), or dynes/cm for hrs in the absence or presence of e ( nm). western analysis was used to compare optical densities of ser -penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by and dynes/cm (from . + . to . + . and . + . , respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels ( . + . ) and this was further increased . - . fold with dynes/cm ( . + . ) and dynes/cm ( . + . ). as seen with fol uaecs, preg uaec static penos ( . + . ) appeared higher than lut uaec, but unexpectedly, neither nor dynes/cm significantly raised these levels of penos ( . + . and . + . ). regardless of shear stress level, e replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine phosphorylation of enos at either or dynes/cm . in contrast to our hypothesis, chronic treatment with e for hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl , hd , hl . cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with mm of no donor gsno or dea nonoate, estradiol- ( nm) in the presence of l-name ( mm) for min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( x /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol- also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro hl and ). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of μm on a spectrophotometer. results: a dominant band of renin at approximately kd was detected in all samples. when compared with the control (i.e. %), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, karoline pickel, daniela ulrich, philipp klaritsch, isa alkan, uwe lang, manfred g moertl. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: patients with uncomplicated pregnancy were measured with the taskforce® monitor i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and . ± . [> wks]) (figure ). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure ). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, manfred g moertl, karoline pickel, philipp klaritsch, isa alkan, uwe lang, dietmar schlembach. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at , , , , , , weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n= ). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels ( +/- pg/ ml, mean+/-sem) to +/- pg/ml by -week lmp. gnrh ii concentrations continued to increase through weeks gestation over the -week levels, although a significant increase was only attained by weeks. gnrh ii continued to increase in late term, attaining levels of +/- pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to +/- pg/ml, i.e., . times that of -week gestation ( +/- pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at -weeks lmp ( +/- pg/ml) as compared to normal pregnancies ( +/- pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. high plasma aea levels at weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from women in early pregnancy ( - weeks) of whom had a viable pregnancy and had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar ( ± . versus ± . years; mean ± sd; p= . ; student's unpaired t-test and ± . versus ± . kg/m ; p= . ) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies ( . ± . versus . ± . nm; p= . ). there was no correlation found between aea levels and hcg or progesterone levels p= . and r= . ; p= . , respectively) , despite progesterone being significantly lower in the non-viable group ( . ± . versus . ± . ng/ml; p= . ). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. n- ) and arachidonic (aa, : n- ) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of d, d and elongases and (elov and ) in the maternal liver rat. methods. pregnant rats were fed control ( % casein; c) or restricted ( % casein; r) isocaloric diets. at day gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver d, d, elov and mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat ( ± vs ± mg) serum leptin ( ± . vs ± . ng/ml) and insulin ( . ± . vs . ± . ng/ml) differed in c and r respectively (p< . ). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig ) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from to were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* % of patients received blood products and % received clotting agents. the average blood loss was cc. there were no maternal deaths and injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while % of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc ) determined blood pressure of - / - in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of patients who were diagnosed in the first and early second trimester (< weeks) with blood pressure of - / , and a control group consisting of patients with blood pressure < / at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: % versus % of the study and control group pregnancies, respectively, experienced preeclampsia (p= . ); and % versus % of the study and control group, respectively, were associated with gestational hypertension (p= . ). % of the control group deliveries were associated with admission to the nicu, compared to only % in the control group (p= . ); % versus % of the study and control group deliveries, respectively, were preterm (p= . ); twenty-nine percent of the study group were cesarean deliveries, compared to % in the control group (p= . ). on admission mean sbp and dbp was and mmhg, respectively, in the study group, compared to and mmhg in the control group (p= . for sbp and p= . for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at - weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of - , is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from - . pain at various dilatations ( - cm) of women who were laboring and then delivered at - weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results laboring patients between - weeks gestation and at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at - cm dilatation ( . vs. . ‚ p= . ) and significantly greater at - cm ( . vs. . ‚ p= . ). pain reported at - cm and - cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between - weeks gestation may perceive less pain at - cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of . to . hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see ) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; ) the results suggest that uterine electrical activity is altered in women affected by asthma; ) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n= ) and also from a woman with no sons and a previous miscarriage of undetermined gender (n= ). no male cells were observed in the control (n= ), a woman with daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd + and cd + lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the integrin (i) heterodimers, , , and are primary receptors for collagen. the predominant cervical collagen is collagen type . the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day , , , , and animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i mrna expression increased through mid-gestation but decreased to np levels on day . a similar pattern was seen with the integrin beta subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha , , and integrin beta were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the subunit paralleled col expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p rr p rr . background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in pregnant baboons at a mean gestational age of (+/- ) days. detailed ultrasound examination (biometry, doppler, screening) was performed days before, at and day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood ( ml) and amniotic fluid ( ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but / ( %) fetuses died within h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of ultrasound examinations were performed in fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was . kg (range . - . ) and the estimated fetal weight was gr (range - ). sixteen out of cordocenteses ( %) and all amniocenteses ( %) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, -hydroxyprogesterone ( ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, ohp, cortisol ©, estradiol (e ) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these hormones are depicted in figure (maternal serum, from conception to term days) and figure (newborn serum) conclusion: this study provides reference values for hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and ohp administration during pregnancy. ( )non-pregnant women eligible for ivf treatment and ( )pregnant women receiving prenatal care. the pre- / samples were drawn in the year prior to / / ; the post- / samples were drawn within months after. the pre- / and post- / normal pregnant samples were drawn at + weeks' gestation. the non-pregnant samples were from ivf candidates with serum e levels< newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels< . miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp and hsp were measured by commercially available elisa. these results were compared in the patient populations before and after / . t-tests were used for analysis. statistical significance was set at p< . . results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp pre-and post- / (table ) . only serum hsp levels were significantly increased in the pregnant women post- / .* mean serum acth, cortisol, hsp , and hsp levels were all significantly different in non-pregnant subjects pre-and post- / . conclusions: except for hsp , serum markers for stress were unchanged in pregnant subjects after the stress of / / in comparison to non-pregnant women whose serum hsp , hsp , cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that ß hsd- activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r= . , p= . ; r= . , p= . ; and r= . , p= . , respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r= . , p= . ; r= . , p= . and r= . , p= . , respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower ß hsd- activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting - % of the us. our prior studies found that % of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a -year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every months for years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results women with fi were surveyed and of those, ( %) returned at least survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, % felt frustrated due to fi, % reported fi impacted their emotional health, . % reported fi impacted child-caring abilities, and . % reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were - times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. . % felt their stool was stored elsewhere before bowel movements, . % reported using digital defecation and more than half ( %) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only % of women at months, . % at year, and . % at years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n= , ) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or . , % ci . - . ) and blacks ( . , % ci . - . ) had statistically significant lower rates of breastfeeding, while latinas (or . , % ci . - . ) showed a trend towards higher rates of breastfeeding. in latinas, we found that rd and th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table ) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; pediatrics, erasmus medical centre, rotterdam, netherlands; public health, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april and january information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from , pregnant women were available. of all women % adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or . , p< . ), nonwestern ethnicity (or . , ci . - . , p < . ) and a low educational level (or . , ci . - . , p< . ). other risk factors were single marital status, smoking, multiparity (all p< . ) and alcohol use (p< . ). prior spontaneous abortion was associated with increased adequate folic acid use (p< . ). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since , little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least % of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the , pregnant admissions captured between and . logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of . considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from . % in to . % in . although white women had times the odds of using ma in (adjusted or ( %ci) . [ . , . ]), this had dropped to . [ . , . ] by , mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled , women who delivered a live born singleton infant between and completed weeks gestation at tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor ( ), pprom ( ), preeclampsia ( ), placental abruption ( ), cervical insufficiency ( ), and fetal indication/iugr ( ). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, sarah wagner, sean keeler, may tam, julian mierlak, , aaron caughey. obstetrics and gynecology, nyu school of medicine, new york, ny; obstetrics and gynecology, ucsf, san francisco, ca; obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november , through june , . data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the , pregnancies, asian and hispanic race/ethnicities accounted for . % and . % of the population, respectively, with . % being us-born. the mean age was . years and the overall ppd+ rate was . %. there was % non-compliance with ppd testing, and % of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women > years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and %ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted minutes, and each focus group lasted - minutes. we recruited patients ( hispanic, aa) and providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd - ; r ca (spring). population. kristine beckers, isabelle guelinckx, greet vansant, roland devlieger. obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was . ± . kg ( . ± . lbs). mean weight gain was . ± . kg ( . ± . lbs) in the underweight population, . ± . kg ( . ± . lbs) in the population with normal weight, . ± . kg ( . ± . lbs) in the overweight population and . ± . kg ( . ± . lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at - weeks of age were randomized into treated versus control groups.bm from syngenic female donor was injected into the tails of recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at , , , , and weeks post bmt. all organs were weighted and examined by h e. fsh, e , and p were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in % agarose gel. results: total body weight significantly increased in treated animals(p< . ). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p< . and p< . ). six out of treated animals showed estrogenic changes in daily vaginal smear. e level increased . - . times and fsh level dropped to % in treated animals. normal (donor allele) fshr gene was amplifiable in out of recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional ( d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional ( d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc cells. conclusions: improvement of the d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c bl/ j female mice and in vitro fertilized with sperm from male c bl/ j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf and heparin, fixed on day , stained with -tubulin and zo- antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, cisca wijmenga, ruben van 't slot, lude franke, bart cjm fauser. reproduction gynecology, university medical centre, utrecht, netherlands; complex genetics group, university medical centre, utrecht, netherlands. introduction: around % of women have a post-menopausal hormonal profile before age , referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included caucasian, ,xx patients with spontaneous secondary amenorrhea before age , fsh > iu/l and absence of (low) x/ xx mosaicism and fmr premutations. dna analysis was performed using illumina k cnv arrays containing , probes. , probes were located on the x chromosome ( probe for every kb), of which , probes were specifically designed to detect known cnvs. ten samples with call rates < % were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( kb) using an algorithm detecting at least five consecutive probes with intensities (logr > - . ) below the mean probe intensity. we identified a total of x chromosomal microdeletions, divided in loci and varying between - kb in size. of the samples showed at least one deletion on the x chromosome. % of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found coding regions containing genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, patrizio antonazzo, sabrina cozzolino, stefania calabrese, lia crotti, francesca ferrari, roberto insolia, fabio facchinetti, peter j schwartz. dept. of obst/gynecol, univ. of milano, milano, italy; molec cardiol lab, policlinico san matteo, pavia, italy; mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly in every pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, - % of cases remain unexplained. we recently demonstrated that % of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at weeks or more of gestation according to the definition of late fetal death of the who. out of cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq , kcnh , scn a, kcne and kcne were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of caucasian women with uneventful pregnancies. preliminary data on the first cases (gestational age of death: - weeks) are reported. results: a total of missense mutations were identified in of stillbirths ( %), two on scn a and one on kcnh . the two mutations on scn a (v l; p a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh is a novel genetic variant absent in reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon (cex del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon (c a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon snp, cex del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon snp, the cex del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon (c g/c) that co-segregated with the exon snp. presence of c g led to the formation of an ese that binds splicing regulatory protein srp , leading to efficient exclusion of exon . real time pcr revealed a five-fold increase in the expression of the cex del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon snp compared to those that did not (homozygous c/c) at the same locus (p < . ). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, , joop se laven, robert jan galjaard, j hikke van doorninck. , obstetrics gynaecology; clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., ) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone , lysine acetylation (h k ) at the germinal vesicle stage and complete deacetylation at the mii stage in % of the oocytes, while % keep high levels of h k acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, % of oocytes with naturally remaining h k acetylation showed abnormal spindle figures. in contrast % of the normal de-acetylated oocytes showed normal spindles (p= . ). this suggests that defective de-acetylation of h k in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth < weeks) control study (term birth > weeks) maternal and fetal dna from ( cases and controls) c and ( ptb and term) aa were collected. a high-throughput candidate gene association study was performed examining snps in genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in genes associated with ptb (p< . ) were common between races with both maternal and fetal dna analyses. however, snps in genes in c and in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c- t; rs ) at both allelic (p = . x - ) and genotypic (p= . x - ) level with an odds ratio (or) of . ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin- receptor antagonist gene (a g; rs ) for both allele (p= . ) and genotype (p= . x - ), or . ). in aa, the strongest associations were in interluekin- (c t-rs , allele p= . x - , genotype p= . x - ) in maternal dna with an or= . [ci . - . ] and in fetal dna interleukin- receptor b (a g; rs , allele p= . x - , genotype p= . x - ) with an or . ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type diabetes. as risk factors are similar between t dm and gestational diabetes (gdm), we investigated a possible association of those snps with gdm. study design: blood was collected from caucasian ( cases, controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox- in human myometrial and amnion-derived cells [reproductive sciences, vol , no (supplement, # )]. we have also shown that expression profiles for hdacs ( - , ) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac , and during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac , and . the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac and appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that ) myometrial hdac appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; ) hdac appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; ) no changes in phosphorylation levels were observed for hdac using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b -adrenergic receptor (adrb ) located on chromosome have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= ) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs single nucleotide polymorphisms (snps). genotype frequencies were also determined in % complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the % pacific islander group was significantly different from the caucasians (p= . ) and asians (p= . ) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, p antonazzo, s tabano, f colleoni, a martinelli, s calabrese, c benedetto, l marozio, f facchinetti, m miozzo, i cetin. inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; dept. biology and genetics, univ. milan, italy; dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add ) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add g w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add g w polymorphisms yet remains to be investigated. population association study we genotyped polymorphisms (ace i/d and add g w) in women: with pe ( / with severe pe) and controls. moreover, we investigated a subset of their fetuses: from severe pe, from mild pe and controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add snp. in mother-fetus genotype couples, neither ace nor add polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent ( % vs %; p< . ) and ace i-d is significantly more frequent ( % vs %; p< . ) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls ( % vs %). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb- in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. in human, pi and mii oocytes, -cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and -cell and -cell embryos, and became undetectable in -cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. zga occurs at late -cell stage and -to- -cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou f (oct / ) and caudal-homeobox domain (cdx ) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct / in mice prevents icm development, while cdx knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct / , nanog and cdx was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected hours post insemination, then fixed prior to incubation with primary antibodies directed against oct / , nanog and cdx . following incubation with cy conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct / protein was detected in both the icm and te, based on two different antibodies. the expression of cdx was localized specifically to the te. conclusions the ubiquitous pattern of oct / expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct / protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants r hd r rr , and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated experimental groups of b mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd foster mothers and one cohort of animals fertilized in vivo and transferred to cd foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was . (in vivo), . (fb) and . (ivf). birth weight of male mice both ivf ( . mg) and fb ( . mg) was larger than male in vivo mice ( . mg) (p< . ). ivf mice tended to be smaller than fb mice in both sexes (p= . ). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p< . ) and more importantly from ivf growth curves (p= . ) (fig. ) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, luke carrey, nancy valego, philip deibel, james perrott, jorge figueroa, mark chappell, james c rose. obestetrics and gynecology, wake forest university, medical school, nc, usa; hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at . years of age which were exposed to either vehicle or betamethasone at - days gestation. we implanted vascular and bladder catheters and then allowed the animals a - days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl ( . meq/kg/min at . ml/min) for minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults ( . ± . ml/min/kg) compared with the vehicle animals ( . ± . ml/min/kg) (p= . ). rpf was similar in the vehicle and steroid exposed group ( . ± . ml/min/kg vs . ± . ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group ( . ± . μmol/h vs . ± . μmol/h). this similarity was also present after normalization by body weight ( . ± . μmol/h/kg vs . ± . μmol/h/kg). the vehicle group excreted . ± . % of the dose of na during the experiment while the betamethasone exposed group excreted only . ± . % (p< . ). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd , hl and hd . characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst dye efflux. this ability to efflux hoechst dye is due to expression of abcg (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: human fetal hearts were collected aged between - wk. we used rt-pcr, using primers for several stem cell (abcg , cd , cd ) and cardiac specific (mlc- v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg , cd , cd , islet ). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg mrna was highly expressed in all regions of the fetal heart between - wk. expression was down regulated at - wk especially in the la and lv. a similar pattern of expression was observed for cd . cd showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at - wk. mrna and protein analysis showed similar results. whilst protein expression of abcg was robust at wk (approximately - %), it declined with increasing gestational age. cd was also strongly expressed at weeks of age. there was no co-localisation between abcg and islet . conclusion: abcg and cd are both expressed between - wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg and cd expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog : , ) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev : , ) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi ) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, sheep at . gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to h normoxia, . h hypoxia (mat fio to reduce fetal pao to ca. mmhg) and h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low ( mg.kg - over min) or high ( mg.kg - over min ) doses. treatments finished min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child : , ) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps ( microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa , proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa and pcna compared to control (p< . ). lower, but increased, levels of hoxa and pcna gene expression were seen in the specimens exposed solely to estradiol (p< . ). those specimens exposed to bpa alone demonstrated a small induction in hoxa expression (p< . ) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, ahmet karadag, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: - d gestation), preterm (pt: - d), near term (nt: - d) and term (t: - d) (n= fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody ( : , sc- , santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by , 'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at different fields at x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r , increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r . method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: - days), preterm (pt: - days), near term (nt: - days) and term ( - days) ovine fetuses (n= at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies ( : to : ) with abc reagents and examined at x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: ± , pt: ± , nt: ± , and t: ± %) than myenteric neurons (vpt: ± , pt: ± , nt: ± , t: ± %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, guo l liu, john d richard, sharon k sugano, raina khan, octavio balbuena, kimberly chap, virender rehan, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n= for each gestational ages) at very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - days) and term (t: - days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos ( field per colonic ring, rings each gestational age) taken at x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt= . ± . , pt= . ± . , nt= . ± . , t= . ± . ; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = ± , pt = ± , nt = ± , t = ± %; p< . ), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n= ) and second (n= ) trimesters, paired upper and lower segment myometrium from preterm ( - wks, n = ), term not in labour (n = ), term spontaneous labour (n = ) and non pregnant (n = ). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins ( - kda/ - kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < . ) and in labour (p < . ) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods women undergoing ivf were classified as: . "no ovarian factor", (tubal or male factor and egg donors, nof;n= ); . endometriosis (endo; n= ); . poor responders (pr; n= ); . polycystic ovary syndrome (pcos; n= ). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof ( ± . ) was statistically significantly higher than in pr ( ± . ) and lower than in pcos ( ± . ). both endo ( ± . ) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r= , ; p< , ) and mii (r= , ; p< , ) oocytes and negatively with the units of fsh (r=- , ; p< , ) and lh (r=- , ; p< , ) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day fsh levels in all patients except in pcos (r=- , ; p< , ). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, zhi-bin tong, eric widra, , alan h decherney. reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into groups. in group , the oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. in group , the oocytes were vitrified using % ethylene glycol, % dimethyl sulphoxide and . m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for - days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after minutes of incubation then oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from women. oocytes were vitrified/thawed before measuring for atp content. the other oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is . % ( / ). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table ). coefficient of variation of luciferin-luciferase bioluminescent assay is less than %. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl , kl , fshr, papp, p ) and its relationship with ivf outcome. materials and methods patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed hours after administration of . iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl expression correlates positively with kl levels (r= , ; p< , ) and with genes implicated in granulosa cell function such as fshr (r= , ; p< , ), papp (r= , ; p< , ) and p (r= , ; p< , ). the number of mii oocytes obtained is positively correlated with both fshr (r= , ; p< , ) and papp (r= , ; p< , ) expression, but not with the other genes. kl expression in women who became pregnant during the ivf cycle studied was higher ( , ± ) than in non pregnant women ( , ± , ).(n= , mann-whitney u p< , ). conclusions patients who become pregnant have increased expression of kl , which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: subjects undergoing first cycle of ivf had d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. of them developed ovarian hyper-response, defined as retrieval of oocytes and ohss. subjects had normal ovarian response with retrieval of to oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both the groups. afc and ov were significantly higher (p< . ) in the ohss group ( . ± . and . ± . cm respectively) than in the control group ( . ± . and . ± . cm respectively). afc was the only significant (p= . ) predictor of ohss on multiple regression analysis (table ) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at h, media was collected and progesterone (p ) and estradiol (e ) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp ) and cholesterol side-chain cleavage (cyp a ) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp and cyp a promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p and e were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp and cyp a mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp and cyp a gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp and cyp a gene promoters. our studies demonstrate that human cumulus cells are sites of significant p and e biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf- mice and fertilized in vitro with cauda epididymal sperm from b d f /j mice. fertilized eggs were cultured in whitten medium under % co in humidified air at °c for h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of < . was considered significant. results: there were total of newborn animals: females ( ivf, controls) and males ( ivf, controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf ( . mm , . %) animals compared to female controls ( . mm , . %) and higher in male ivf ( . mm , . %) animals compared to controls ( . mm , . %). the average of males and females was slightly lower for ivf ( . mm , . %) than controls ( . mm , . %). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was and , maturation rate after hours was % ( / ) and % ( / ), fertilization rate after hours was % ( / ) and % ( / ), maturation rate after hours was % ( / ) and % ( / ) and fertilization rates after hours % ( / ) and % ( / ), for case and case respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from to were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at - weeks gestation) in women who received intravaginal micronized progesterone ( mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of cycles were evaluated in women treated with clomiphene citrate. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to . % ( / ) of the non-progesterone group (p < . ). forty-three cycles were evaluated in patients treated with letrozole. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to none ( / ) in the non-progesterone group (p < . ). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both poor and normal responders. afc and ov were significantly lower (p< . ) in poor responders ( ± . and . ± . cm respectively) than in normal responders ( . ± . and . ± . cm respectively). afc was the best (p< . ) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was years. all eight couples were in a committed relationship and had been together for an average of . years. five of the couples ( %) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples ( %) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, az steiner, rh fogle, mj kalan, rj paulson. ob/gyn, usc keck school of medicine, la, ca, usa; ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day # a baseline ultrasound was performed and serum fsh and e levels were measured. subjects were given mg of clomiphene daily from days - and then returned for serial ultrasounds on day # . when at least follicles mm were present, iu of hcg im daily was initiated. cycles were monitored by ultrasound every days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: subjects, aged . ± . yrs (mean±sem) and bmi . ± . kg/m with . ± . yrs of infertility were enrolled. the mean fsh and e levels were . ± . miu/ml and . ± . pg/ml, respectively. there was an average of . ± . days from starting clomiphene to the attainment of follicles mm. out of the subjects had follicles mm by day # . a mean of . ± . days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance ( . ± . follicles, measuring . ± . mm in the micro-dose hcg cycles, compared to . ± . (p= . ), measuring . ± . mm (p= . ) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: . ± . mm vs. . ± . mm (p= . ), respectively. all subjects had at least ovulatory sized follicles in the study cycles. % of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: ) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates ) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their st or nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table ) . maternal antenatal and intrapartum complications were similar in both groups ( . % vs . %, p= . ). there were more singletons in the study group ( . % vs . %, p< . ). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table ) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table lupron (n= ) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in - % of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at % (icu admission) and % for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $ , to a high of $ , . the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $ to a high of $ , . this resulted in an estimated cost burden of $ , to $ , for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were gt, cc and ai cycles. age, gravida, day (d et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < . ). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = . ; p < . ) rather than with mcet (r = . ; p = . ). there was a reverse relationship between the age of the patients and ep (r = - . ; p < . ). a negative correlation was also found between the gravida and ep (r = - . ; p < . ). pregnancy rate was significantly correlated with ep (r = . ; p < . ). pregnancy rate was % in type a ep, . % in b, % in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in % type c pattern in contrast to % with cc (p = . for ai vs. cc; p = . for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < . ). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, , desiree mccarthy-keith, , barbara stegmann, , alicia armstrong. , reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january to , followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day and fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was and pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) + . + . day fsh (u/l) . + . . + . day fsh (u/l) . + . . + . objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl- - active mis/amh elisa. statistical analysis was performed using spss version . . continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = . ). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of ivf cycles performed from january to july resulting in pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day (post-oocyte aspiration) hcg level, and day hcg levels were significant in predicting pregnancy outcome. day and day hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. % of ongoing pregnancies had hcg level > miu/ml. conclusions: a single day post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u ( m)) reduced fsh stimulated cyclin d mrna expression by %. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with nm rapamycin for min significantly reduced fsh-mediated cyclin d mrna expression. dht exposure for hours also inhibited fsh stimulated mtor signaling, as shown by a % reduction in the phosphorylation of its down stream target p s kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc- phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc- phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd- ). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, annemarie g mulders, aida bertoli-avella, ben a oostra, joop se laven. department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: pcos patients ( rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± . inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus unaffected first-degree family members were genotyped using polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md- (average spacing cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in clusters of or more closely related patients (simwalk ). results: there was an average number of . alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome (d s and d s ) that showed significant association with pcos (p , ). other markers that showed significant association were positioned on chromosomes (d s ), (d s ), (d s ) , (d s ), (d s ) and seventeen (d s ) (all p values . ). linkage analysis in sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome (d s ) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal d and d ultrasound was performed in women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, jill blaine, anuja dokras. obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n= ) and controls (tubal infertility, n= ) after in vitro fertilization at university of iowa from - . the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was % in women with pcos and % in controls. after logistic regression analysis pcos was not associated with miscarriage (p= . ). the prevalence of gestational dm (gdm) was similar in both groups % pcos vs % controls. pcos was not associated with gdm after adjusting for age and bmi (p= . ). however, bmi was significantly associated with gdm after adjusting for age and pcos (p= . ). risk of both pre-eclampsia and pih was % in pcos and % in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p< . ). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of - %, with - % among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their s found no increase in bmi or fasting insulin with pcao but regular menses. participants: women aged - with regular cycles (every - days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of on one ovary or ovarian volume > cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table ) . of the women with pcao, % met the afc criterion only, while % met the volume criterion only, and % met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in , have led to an increased prevalence of pcao among women with regular menses, over / of women in their s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl- fraction. this is an atherogenic profile; in fact, studies suggest low hdl- may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl- levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl- and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of obese women newly diagnosed with pcos was determined. they were then placed on a high protein ( - g/day), low carbohydrate ( - g/day) diet with or without metformin ( and %, respectively) and followed monthly for an average of months (range - ). results: at diagnosis, % had low hdl and % had low hdl- ; only % had elevated tgs. on the diet, the patients demonstrated an average weight loss of . lbs ( . to . , p< . ) and decreased bmi of . kg/m ( . to . , p< . ). hdl levels increased significantly ( % increase from . to . , p< . ), especially the hdl- fraction ( % increase from . to . , p< . ). triglycerides decreased as well ( . to . , p< . ). ldl decreased but did not reach statistical significance. resumed menstrual cycles, were started on oral contraceptives, and had a hysterectomy. pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl- at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl- levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and , and hrs after g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels ( . + . μu [±se] /ml) and insulin response to oral glucose (auc) ( . + . μu/ml) were higher (p < . ) in women with pcos compared to controls. all were insulin resistant with homa-ir value > . mol x μu / l . resistin levels in women with pcos ( . ± . ng/ml) was significantly (p < . ) higher compared to control women ( . ± . ng/ml). crp levels in women with pcos ( . ± ng/ml) was also significantly (p < . ) higher than the controls ( . ± ng/ml). there was a significant positive correlation between resistin and crp levels (r = . , p < . ). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that ( )women with pcos and insulin resistance have increased resistin levels, ( ) there is a strong association between resistin and crp ( ) elevated resistin and crp levels may predict women who are at increased risk for chd ( ) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, enrico carmina, prati vardhana, michel ferin, rogerio a lobo. department of obstetrics and gynecology, columbia university, new york, ny, usa; department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these phenotypes, we studied hyperandrogenic women with pcos (age . ± . , bmi . ± . ) and age matched controls (age . ± . , bmi . ± . ). women with pcos were divided into weight matched (ov) n= and (c) n= groups. fat and weight distribution were assessed by dexa (total fat, r fat, trunk fat) as well as fasting levels of lh, e , mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein- rbp ) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference ( . . vs. cm, p< . ) trunk fat, r fat and %trunk and %r fat compared to ov-pcos (p< . ). leptin, rbp and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p< . ). quicki was significantly lower in c-pcos ( . ± . vs. . ± . , p< . ) and insulin was higher ( . ± . vs. . ± . , p< . ). serum lh was also higher ( . ± . vs. . ± . , p< . ) but kisspeptin, testosterone and estradiol were similar. mis/amh ( . ± . vs. . ± . ng/ml, not significant) and crp ( . ± . vs. . ± . , p< . ) were higher in c-pcos. significant correlations (p< . ) were noted among kisspeptin/rbp (r . ), mis/testosterone (r . ), insulin/ %trunk fat (r . , p< , ), total fat/leptin (r . , p< . ), %trunk fat/adiponectin (r - . ). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida gene (zp ) is recently identified gene, which belongs to zona pellucida genes such as zp , zp and zp . the role of zp , contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from patients (mean age , +/- , years; mean bmi , +/- , kg/m ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from . dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of patient samples with pcos, we identified nucleotide changes in the zp coding seguence : silent nucleotide changes in exons , , , , and nucleotide change in the exon ( position , t>g). the mutation in exon ( t>g ) results in substitution of cystein for glycin of amino acid in position of zp protein. in summary, our data demonstrate that zp nucleotide changes account for % of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and , and hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels ( . + . μu/ml) vs . + μ u/ml [+ se]) (p < . ) and area under the curve (auc) of insulin ( . + . vs . + . μu/ml) (p < . ) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < . ) increased in women with pcos ( . + . ng/ml) compared to controls ( . + . ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = - . , p< . ). there was significant negative correlation between mis and fasting insulin levels (r = - . , p < . ) and insulin auc during the ogtt (r = - . , p < . ). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, ( ) there is an increase in secretion of mis, ( ) higher insulin levels are associated with lower mis levels, ( ) acute increase in insulin levels has no effect on mis levels, ( ) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent g oral glucose tolerance test ( ggtt, sampling at , , minutes), and -step elisa for amh (sensitivity . pmol/l, inter intra-assay c.v. : . %, . %, respectively). p< . was considered as statistical significance. results: average amh level of the subjects (age: . + . years, bmi: . + . kg/m , mean+s.d.) was . + . pmol/l, which was higher than normo-ovulatory women ( . + . pmol/l, n= ). amh levels had significant positive correlation with total ovarian volume by ultrasound ( . + . cc), but not with bmi, waist-hip ratio ( . + . ), and levels of serum lh ( . + . iu/l), total testosterone ( . + . ng/dl). although fasting glucose levels ( . + . mg/dl) were positively correlated with total ovarian volume (r= . ), amh levels had no significant correlation with fasting insulin ( . + . iu/l), fasting glucose/insulin ration ( . + . ), homa-ir ( . + . ), and the sum of insulin levels ( . + . iu/l)during ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with different fragments of the mouse gnrh gene promoter (- /+ and - /+ bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the - luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the - luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from - luc mice confirmed gnrh promoter activity in the prostate ( ± rlu) and penis ( ± rlu) and absence in the testes ( ± rlu). in the - luc mice, however, luciferase activity was detected only in the testes ( ± rlu) and not in the prostate ( ± rlu) or penis ( ± rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal - bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while - bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g /m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio . upon meiotic reactivation, pumilio loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a and a ) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a ( and ) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g /m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of women diagnosed with type ii or gdm initially treated with glyburide at a single institution from - . maternal characteristics, adequate glycemic control defined as more than % of fasting glucose < mg/dl and one-hour post-prandials < mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the women initially treated with glyburide, ( %) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose ( mg/d) reached ( %), maternal hypoglycemia ( %) and other side effects ( %). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes ( % vs. %, p= . ), prior history of gdm ( % vs. %, p= . ) and macrosomia (> g) ( % vs. %, p= . ) or hour glucose challenge test ( + vs. + mg/dl, p= . ). non-responders had a higher rate of obesity (bmi> ) ( % vs. %, p= . ) and earlier gestational age at initiation of therapy ( + . vs. + . wks, p= . ). there were no differences between the groups in the mean -week fasting ( + vs. + mg/dl, p= . ) and post-prandial glucose values ( + vs. + mg/dl, p= . ). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the gram, -hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the gm, -hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the -gm, -hour glucose challenging test (gct) for initial screening and the gm, -hour gtt as confirmatory tests for the diagnosis of gdm between and . various combinations of the gm, -hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: , women had gm, -hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure ) . summation of the -hour and -hour gtt results with a diagnostic threshold of mg/dl yielded the most optimal balance between sensitivity ( . %) and specificity ( . %). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of -hour and -hour gtts had higher odds of operative vaginal delivery (aor= . , % confidence interval [ci] . - . ) and neonatal birthweight > gm (aor= . , % ci . - . ). conclusion: using only the summation of only the -hour and -hour gtt results of the gm -hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight > gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low ' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period women delivered in our institution following prom and induction of labor , of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group ( % vs %, p< . ). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for % and % (p< . ) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low ' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january and march . computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 'and ', umbilical cord venous and arterial ph and base excess. results: during the period between january and march there were singleton elective cesarean sections, of them were breech and vertex. the mean age, gravida and parity were significantly different between groups ( . vs. . , . vs. . . and . vs. . respectively, p< . ). the birth weight was significantly different - . gram for the vertex and . gram in the breech deliveries (p< . ) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term ( - + w). venous and arterial po and pco levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, amalia levi, yaron zaulan, avi harlev, eyal sheiner. obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years - were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, , singleton deliveries occurred in our institute. of these, ( . %) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption ( . %; n= ) and placenta previa ( . %; n= ). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= . ; % confidence interval [ci] . - . ; p= and . ; . - . ;p< . respectively ), suspected intra uterine growth restriction (iugr, . ; . - . ; p<. ), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, . % vs. . %, or= . ; %ci . - . ; p< . ). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<. ). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or % ci p crude or for perinatal mortality . . - . < . or adjusted for: < . iugr . . - . < . oligohydramnios . . - . < . premature rupture of membranes . . - . < . the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than . indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p< . ). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and % confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below . cm (before weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, of whom had delivered before weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and women underwent a vaginal cerclage. eighty-four women ( . %) delivered at weeks or beyond. one-hundred and three women ( . %) delivered no earlier than weeks, and only one woman delivered at weeks. two women gave birth to a twin (at and weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from tertiary healthcare systems from - experiencing pprom . weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of women carrying fetuses ( singleton, twin, triplet, quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of . (+/- . , range . - ) weeks. the average latency period was . (+/- . , range . - ) days, with a mean delivery gestational age of . (+/- . , range . - ) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of % in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was . years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, jane khoury, padmini c sekar, ralph c buncher. obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per , who estimates % of maternal mortality occurs in developed countries . this rate has established without much decline in recent years. the mmr for ohio for to was reported by cdc to be . per , live births with % confidence interval . to . . objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd codes to and icd codes o to o . five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of in to a low of in and . in general the five-year mortality rate was decreasing over time until the last four years to , when we may be seeing an upward trend compared to to (p= . ). the rates and associated % confidence intervals (ci) are shown in the table below. years mmr % ci ci - ci . . - . ci - . . - . - . . - . - . . - . - . . - . the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g a were tested at induction of labour. panel classification of cause according to the tulip classification was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the women tested for factor v leiden, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of factor v leiden. of the deaths with another cause of death ( . %) mothers were carriers of factor v leiden (p= . ). of the women tested for prothrombin g a, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of prothrombin g a mutation. of the deaths with another cause of death ( . %) mothers were carrier of prothrombin g a mutation (p= . ). in women with iufd and factor v leiden or prothrombin g a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; pathology, university medical center groningen, groningen, netherlands; trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the men tested for factor v leiden, ( . %) were carrier versus ( . %) non-carriers. prevalence of factor v leiden in the normal population is % (p= . ). men were tested for prothrombin g a, ( . %) were carriers and ( . %) non-carriers. all were heterozygous. prevalence of prothrombin g a mutation in the normal population is % (p= . ). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied consecutive gdm women with singleton childbirth and or abnormal values in -h ogtt with g of glucose. abnormal plasma glucose values of the ogtt were: fasting . , -h . and -h . mmol/l. insulin treatment was started when values were . preprandially or . mmol/l postprandially in the -h glucose profile done within days of diagnosis. if the same woman had more than childbirth during the study period, only the last pregnancy was included. the control group consisted of women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) > . sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw > g than controls. insulin was started in gdm women ( . %). c/s rate was . % in controls, . % in diettreated and . % in insulin-treated gdm women. macrosomia rate was . % in controls, . % in diet-treated and . % in insulin-treated gdm women (p< . compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in . % of controls, in . % of diet-treated and in . % of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p= . ). by regression analysis, previous child with bw > g (p< . ), previous c/s (p= . ), mother's age (p= . ), bmi (p= . ), insulin treatment (p= . ) and fasting plasma glucose of the -h profile (p= . ) were significant independent predictors of fetal macrosomia. conclusions the -h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. . '-:·:tpartq,-, '-:·:tpartq,-, '-:·:tpartq,-, % p= . ). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri . vs. . , p= . ; pi . vs. . , p= . ) . however, bilateral notching was much more common in the cohort of prior adverse outcomes ( % vs. % p= . ) as well as in patients destined to have a subsequent poor outcome (p= . ). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from through . these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples ( - weeks' gestation; n= subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was . years (iqr: . - . ). outcome data was available for subjects. ( . %) had uncomplicated pregnancies. median birthweight was , g (iqr: , - , g) and median birthweight centile was . % (iqr: . - . %). ( . %) infants were born sga and ( . %) were preterm. spontaneous vaginal deliveries occurred in . % of cases. there were ( . %) cases of pre-eclampsia and ( . %) admissions to neonatal care. . % of subjects reported smoking at booking. iron deficiency anaemia was prevalent in . % of subjects by - weeks and was strongly associated with higher infant birthweight centile (p< . ). . % had serum -hydroxy vitamin d concentrations < ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p= . ), low serum folate (p= . ) and high serum homocysteine (p= . ) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight < gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight < gm, and apgar score < at minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to - ml with incremental increases of volume by ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash ). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p : the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p ). we calculated the correlation coefficient between p and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the , births ( ) ( ) ( ) ( ) , accurate paired results were available on % of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p< . ). the median ( . th , . th centile) values for cord arterial blood gases were: ph . ( . , . ); po . mmhg ( . , . ); pco . mmhg ( . , . ); base excess - . (- . , . ); and lactate . mmol/l ( . , . ). there was a progressive improvement in all blood gas measures over the years of this study (all p< . ). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < . ph < th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to < h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (< h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered million units (mu) of intravenous pcn to be followed by . mu every hours until delivery (cdc ) . subjects with renal disease, multiple gestation, and preterm delivery (< wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were < %. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: < h, . ± . g/ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ml (n= );> h . ± . g/ml (n= ); and for those without a second dose after h, . ± . g/ml (n= ). fetuses exposed to short duration (< h) had higher levels of pcn than those exposed to > h (p= . ). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs ( . g/ml)(p< . ). furthermore, every sample individually remained - fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to < h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: -oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of -oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n= , and term elective cesarean section n= ) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and -oh aa (isocratic hplc using fluorometric detection with assay sensitivity at pmol). results: -oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table ) . for labouring group patients, the -oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of -oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of -oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all conditions). results: among term births with ipd, . % presented as maternal disease only, . % as fetal disease, and the remainder ( . %) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; dept ob/gyn, wayne state univ, detroit, mi, usa; dept pathology, wayne state univ, detroit, mi, usa; dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: ) mid-trimester (n= ); ) term not in labor (tnl) (n= ); ) term in labor (tin) (n= ); ) preterm labor (ptl) who delivered at term (n= ); ) ptl without iai (n= ); ) ptl with iai (n= ); ) preterm prelabor rupture of membranes (pprom) with (n= ) and without iai (n= ). af fetal hg concentrations were determined by elisa. results: ) fetal hg was detected in . % of all af and . % of mid-trimester samples; ) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester ( . ng/ml, iqr - vs . ng/ml, iqr . - . , p= . ); ) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: . ng/ml, iqr - . ; p= . ); ) median af fetal hg concentration was not significantly different among the ptl subgroups [ptl with iai: . ng/ml, ptl who delivered preterm: . ng/ml, ptl without iai who delivered at term: . ng/ml, p= . (kruskal wallis) ]; ) in pprom, there were no differences among patients with and without iai ( . ng/ml, respectively; p= . ) ; ) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p< . for all comparisons). conclusions: ) immunoreactive af fetal hg increases with gestational age; ) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; ) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, deborah penava, barbra de vrijer, orlando da silva, bryan s richardson. obstetrics and gynaecology; pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom > and < weeks gestation, singleton and no major anomalies, delivering between january , and december , . patients were grouped according to ga at pprom stratified for latency time < hrs vs > hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were patients who met the inclusion criteria of whom %, %, and % had pprom at - wks , at - wks, or at - wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery > hrs, at %, %, and %, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table . conclusion: despite a to fold increase in the incidence of chorioamnionitis with latency to delivery > hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until weeks, and moderate infant morbidity until weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso ), nifedipine, terbutaline and tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and . comparisons were expressed as odds ratios ( % ci). results: in a -year period, twin gestations were admitted in ptl and administered tocolytic agent(s) with mean gestational age of . weeks. when tocolytic agents were used, maternal complications were: ( . %) with intact membranes and ( . %) with prom had pulmonary edema (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had chorioamnionitis (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had postpartum hemorrhage (or= . , ci . - . ). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using tocolytic agents. similarly, there is no difference between the groups with latency period hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥ hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: women were included (non-labor group (n= ), labor group (n= ), non-labor pre (n= )). the median and mean hscrp levels were and and and in the non-labor and labor groups respectively (p= . ). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p= . ), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p= . ), mode of delivery (p= . ), or admission to the nicu ( . ). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean . vs. . , median vs. , p< . ). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with % cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of submitted placentas, had histologic chorioamnionitis (cases) and did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p . ). the cases were more likely to have had epidural anesthesia (or . ), internal monitoring (or . ), fever (or . ), maternal tachycardia (or . ) and fetal tachycardia (or . ) and less likely to have had induction of labor (or . ). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or . severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age= . ± . years) with a mean gestational age at delivery . ± . weeks. etiologies were pyelonephritis(n= ), septic abortion(n= ), endomyometritis (n= ), chorioamnionitis (n= ), ruptured appendix(n= ), pneumonia( n= ) and one unknown. eighteen ( %) were diagnosed during antepartum and ( %) postpartum period.there were ( %) maternal deaths and high rate of major morbidities (table) . among the antepartum patients,there were, abortions, iufd, neonatal death for a perinatal survival rate of only %. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru ( . um= dose used in patients, . um, . um) at different time points during infection (before, at the time of or after nf-kb activation). ru degradation kinetics was also included. the effect of ru on nf-kb induced il- and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il- and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il- and tnfa levels. in the presence of ru , il- and tnfa levels continued to rise. the effect of ru on nf-kb induced il- and tnfa was dose dependent and was more prominent in slow metabolizers who had ru in the system for a longer time. addition of ru after the onset of nf-kb activation led to increased il- and tnfa levels above those observed without ru . the addition of misoprostol (prostaglandin e analogue), at the concentrations used together with ru for medical abortion, did not add to the effect of ru on nf-kb induced il- or tnf. conclusions: ru has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld that is orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp- and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c - week old adult mice and - day old neonatal mice. liver and spleen was collected at , , and hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f / rat antimouse igg, a marker for macrophages) and secondary antibodies (cy- goat anti-rabbit igg; af goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp- and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at hours post-infection with listeria, adult liver and spleen showed increased staining for f / and class ii, which increased further and became confluent surrounding microabscesses at and hours. in contrast, the listeria infected neonatal mouse showed some increase in f / around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp- and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from to . prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over %. results: a total of . % ( , ) of , deliveries from to were by cesarean. annual cesarean rates increased by an absolute rate of over %; while, post-cesarean endometritis rates decreased from % to %. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure ) or nadirs. the month of july accounted for only out of a total of peaks for all years. the adjacent months of june and august accounted for only each. the month with the highest number of peaks was april with only . these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results , patients were evaluated ( cases and controls). % and % of cases and % and % of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was . % and . % (p= . ). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery < wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v. . . results: the immediate anatomic cod could be determined in . % of all infants examined. in over % of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women ( and older at their due date) who delivered at or after weeks. non-exposed women delivered from july to december (when at for ama was not routinely recommended); exposed women delivered from july to december (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of %, we had % power to detect an increase to % after the introduction of at. results: women met the inclusion criteria: delivered before the introduction of at (non-exposed=before at) and delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group ( % vs %; p< . ). women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table ) . the corrected iufd rates were similar in both groups ( / vs / ). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix < cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr . , . physicians in solo practice had higher rates of elective inductions (p< . ), but there was no association between cesarean and practice type. conclusions: inductions accounted for . % of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: ) women with normal pregnancy (n= ) and ) patients with fetal demise without disseminated intravascular coagulation (n= ). plasma concentrations of scd l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: ) patients with fetal demise had a higher median maternal plasma scd l concentration than women with normal pregnancy (median . pg/ml, range - vs. median . pg/ml, range . - . , p< . ); ) there was no significant difference between the groups in the median maternal plasma tf concentration and ) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median . ng/ml, range . - . vs. median . ng/ml, range . - . , p< . ). conclusions: ) a change in the plasma concentration of tf was not demonstrated; ) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; ) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and ) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase production, activity and prostaglandin e secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp activity/protein and pge secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid ( - mm/ h), then with increasing doses of tnf ( - ng/ml/ h). medium and cells were analyzed by gelatin zymography/western blotting for mmp /mmp activities/protein. pge output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp production, secretion and activity in amnion epithelial cells. tnf ( ng/ml) induced an fold increase in cellular active mmp production and fold increase in secreted mmp enzyme activity by amnion epithelial cells. these increases were reduced - % following h pre-treatment with . - . mm -lipoic acid. mmp protein/activity and pge secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp protein/activity. mmp protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge production ( fold increase/ ng/ml tnf/ h) that was inhibited by %- % following . objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il- gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek cells. at h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes - to - and - to - ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity -to -fold. whereas mutations in ebox or resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il- gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd , a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd expression by immune cells is important in extravasation of leukocytes into tissue. cd may also be required for ha catabolism through the action of hyaluronidases and . in the cervix, cd is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd in cervical remodeling, mice with a null mutation for cd (cd -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd -/mice. this suggests that postpartum breakdown of ha in cd -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase (hyal- ) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd -/cervix was similar to wt. conclusion: these studies provide evidence that cd may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal may compensate to allow ha removal. furthermore, the activity of hyal- may be dependent on cd . little difference in the recruitment of immune cells between cd and wt animals suggest that cd expression is not required for this process. these experiments provide a greater understanding for the role of cd and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: term fm had the following characteristics: maternal age ± . yr, gravida . ± . , gestation ± . wks, elec. cs . %, duration of rom ± min, duration of contractions ± min, african american %. % of the fm had < % separation; % had more than % separation. srom fm with > % separation (vs. < %) had significantly shorter duration of rom (p= . ) and admission to birth (p= . ) times. srom fm with > % separation (vs. < %) had even shorter rom (p= . ), duration of contractions (p= . ) and admission to birth (p= . ). the > % group (vs. < %) was further along, gestationally (p= . ). srom fm (vs. arom) had shorter admission to birth (p= . ), but longer rom to birth (p< . ) times. absence of epidural (p= . ), srom mode of rupture (p= . ), svd (vs. elec. c/s) (p= . ), and the presence of meconium (p= . ), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, shirin khanjani, yun s lee, tg teoh, , philip r bennett. institute of reproductive and developmental biology, imperial college, london, united kingdom; maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase- (cox- ), which is essential to the labour process. cox- mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox- expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p protein concentrations were used for whole genome analysis using affymetrix u arrays. results and conclusions: we identified significantly regulated genes. the gene with the highest fold change was cox- (x . ) followed by oxytocin receptor (x . ), ch orf (x . ), integrina (x . ), and connective tissue growth factor (x . ). other significant genes included interleukin- (il- ) (x . ). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox- and il- expression. to prove that cox- is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p associated with inhibition of cox- demonstrating that nfkappab is essential for cox- expression. objective: premature birth is a major public problem accounting for over , deaths and , surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r = . ) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih . the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase (has ) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has gene. the regulation of has was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has . regulation of has expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon . two transcripts of approximately . kb and . kb were detected that arise from use of polyadenylation sequences. as compared to np, the expression of has is increased , , and fold on gestation days , and shortly postpartum respectively. has mrna expression is increased upon treatment of caski cells with epidermal growth factor ( ng/ml) and is suppressed in cells treated with ag ( m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at and hours of treatment. conclusion: has is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has gene. in vitro studies using caski cells suggest has is regulated in part by the egf signaling pathway resulting in a several fold increase in has expression. these results provide an understanding of has gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately % of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from % to % in -well plates. cell viability was determined at , and hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at hours in both cell types (figure ). at hours, chorion exhibited greater percent cell death compared to decidua at concentrations of , and % cse (p= . , . , and . , respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure ( , and hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from - % compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, tae-bok song, cheol hong kim, jong woon kim, moon kyoung cho, sung yeul yang, bong whan ahn. obstetrics gynecology, chonnam national university medical school, gwangju, korea; biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were determined by the , -dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to hours with . ml of mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: . the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein, p< . ). . the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein p< . ). . the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) and , which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on cam-complicated (n= ) and gestational age-matched control decidua (n= ), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ), and quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in cam-complicated decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in control decidua ( ± , and ± respectively; p< . ). in cultured term dcs incubated with e , tnf and il significantly increased secreted levels of mmp- compared to e alone (pg/ml/ g protein: . ± . and . ± . , respectively, vs. . ± . ; p< . ). in parallel incubations with e +mpa, basal mmp- output was lowered by %, and tnf -and il -elicited mmp levels were blunted by % and %, respectively. rt-pcr confirmed that tnf and il increased mmp mrna levels (p< . ), although mrna levels in e +mpa incubations were not different from those of e alone. mmp levels in all treatments were similar. conclusions: mmp- and mmp- are elevated in cam decidua compared to controls. our in vitro results suggest that mmp- expression is enhanced by the high levels of il and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp- and mmp- and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, patama promsonthi, wasun chantratita, mana rochanawutanon, patcharee karnsombut, chutatip srichunrusami, stephen j lye, boonsri chanrachakul. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) , a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in % formal saline for paraffin section. immunohistochemistry (n = each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . the mcp messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp and mcp receptor (n = each). results: immunohistochemistry demonstrated mcp in cervical tissues from all four groups of women. mcp was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp and mcp receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp and mcp receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from groups of cd- mice. group : mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group : e dams representing preterm controls. group : e . - dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n= dams/treatment group. separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of -fold change with p value of < . , genes in the cervix were differently regulated between the groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: ) in preterm cervical ripening (e lps vs e ): immune and inflammation response, defense response ) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, valentina ciani, gunvor e ekman. obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase- and the inflammatory cell marker cd was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase- staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p= . ) with unripe cervix and from non-pregnant patients (p= . ). the inflammatory cells staining for cd increased in post partal and term pregnant sections compared to non-pregnant (p= . ). there was a higher frequency of caspase- positive cells in the post partal tissue than of cd positive cells (p= . ). the localization of cd- positive staining was highest in the epithelia and basal lamina while caspase- staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days , , , , and ) sprague-dawley rats underwent tensile testing using an instron material testing system. the testing regimen consisted of tissue extension to near maximal strain over seconds followed by a period of constant strain for minutes, then return to rest over seconds. this cycle was repeated additional times with a minute rest period between cycles. strain and force measurements were recorded at second intervals. - animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd- mice were used in these two models of ptb: ) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n= ); controls for this model received intrauterine saline (n= ) and ) a non-infectious model of ptb using ru sq ( ugrams/dam) (n= ); controls for this model received no intervention (n= ) were used for these studies. for both models, hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il- and tnf-alpha in the uterine tissue. mrna expression of ifn , il- , il- , il- , tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il- and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p< . ) (figure ). in cervical tissue, an increase in il- , il- beta and tnf mrnawas observed in both models, while ifn-gamma and il- were only increased in the lps model. (figure ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f / . the density of adherent f / + cells greatly increased in equivalent volumes of af between . and . days postcoitum (dpc) ( . dpc = term). interestingly, the f / + cells at . dpc were highly similar in morphology to those present in . dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f / and for cd , a pan-leukocyte marker. we observed that af from . and . dpc mice contained two sub-populations of cd + f / + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox- , and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from nonpregnant (proliferative, n = ; progestin treatment, n = ) and pregnant women undergoing hysterectomy (eg, n = ; term before ripening, n = ; after ripening, n = ; in labor, n = ). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase ( . ± . pg/ug cdna) and decreased % by progestins ( . ± . pg/ug cdna). this level was maintained in stroma from pregnant women before labor ( . ± . , eg.; . ± . before ripening; . ± . after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix ( . ± . pg/ug, p < . ). interestingly, whereas pr-b was ± % that of total pr in the nonpregnant cervix, pr-b mrna levels were ± % to ± % in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr ), pr-b immunoreactivity was % that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from ± to ± units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il- , mcp- , tlr- , cd l, cox- , and s a mrna (all p < . , anova) but not cd b or tlr- . pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox- , expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox- but not il- , mcp- , or toll-like receptors. ifn-y il- il- , il- tnf- "p value < . lps/saline ru /controls . "" . " . " . * " " . * . . " association between interleukin- (il- ) and il- to examine association of amniotic fluid interleukin (il- ) concentration with il- and its receptor il -r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [< weeks]) and control (term [> weeks]) amniotic fluid (af) il- concentrations were analyzed for association with haplotypes of the il- and il r genes in aa and c separately. in il- , eight, and in il- r, single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa: maternal:cases- controls- fetal: cases- fetal controls-; c: maternal cases- , controls- , fetal:cases- ; controls= ). haplotype associations were performed by using a sliding window with outcome il- concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il- r rather than in il- . in c fetal dna il- r haplotypes defined by markers - bp from the transcription start site associated most strongly with af il- concentrations (global p= . x - ) and in aa maternal il- r haplotype markers at - - - (global p= . x - associated with il- concentrations. in the c fetal cases the - haplotype with the highest concentration was a-g (log(cytokine) = . pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at - - - .this was seen in both cases and controls at (case log (cytokine) = . ; control log(cytokine) = . pg/ml). significant associations from haplotype analyses converged on three regions of the il- r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il- that associated in cases with miac but not in cases without miac (- and - ; - and - )(both with p < x - ) conclusion: differences in the af il- concentration in ptb do not result from single snp effect on il- but are a result of complex relationships between il- and il- r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, amanda tattersall, mark tattersall, suren sooranna, peta grigsby, leslie myatt, mark johnson. obstetrics gynaecology, imperial college, london, united kingdom; obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il- or il- on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further samples were obtained before the onset of labour (n= ) from which myocytes were isolated and cultured in -well plates. when cells were - % confluent either tnf-at a concentration of ng/ml was added to the cells for , , , and min and the cells analysed by western blotting or tnf-at concentrations of , . and ng/ml was added either alone or in combination with similar concentrations of il- or il- to cells for hours and mrna was extracted and converted to cdna to determine il- and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr and tnfr mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-( ng/ml) activated all three mapk substypes: erk, jnk, and p , activation peaked between - minutes. preliminary data suggest that tnf-induces il- mrna expression but exposure to il- or il- itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il- mrna expression. this enhanced mapk-dependent il- expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, anna-marie fairhurst, mala s mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil / ). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with - h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d mice were treated for h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day , d . ) and remain high through postpartum (pp). macrophages (mØ) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d resulted in a premature increase in mo but not neutrophils or mØ. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d . and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mØ numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until - % confluent and stimulated with ng/ml il- prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp , which pulled out nf-bp and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table shows the different tfs interacting with nf-bp based on the degree of binding stimulated by il- compared to no-il- control. conclusion: these data show that il- stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap- , which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il- stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table high ap- , c/ebp, cbf, creb , c-myb, e f- , ets, ets- /pea ,fast- , gas/isre, hse, mef- , mef- , myc-max, nf- , nfatc, nf-e , nf-e , pax- , objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi : , ; reprod biol endo : , ) . although parturition is associated with systemic or local progesterone withdrawal (ajog : , ) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c bl mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod : , ) . after days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day (e-p) and groups killed on days and . cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n= /group/day; sections/ cervix; biol reprod : , ; jsgi : , ) . stained macrophages and neutrophils were counted (image pro-plus , media cybernetics). results: e+p treatment for days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for , , or days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days and (p< . , e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at and h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf- a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf- a breast epithelial cells and a rhabdomyosarcoma cells by the change in fluorescent emission of jc- . total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with - min treatment with progesterone and r which were inhibited by a specific pr antagonist, rti- - b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r . progesterone/progestin treatment for min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge receptors, ep- and ep- mediate contractions whilst ep- and ep- mediate quiescence. ep- contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep- expression. myocytes plated in well plates were treated with il- ( ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p . rna was extracted for amplifying ep- using quantitative rt-pcr with amplification of l as a control to normalise data. il- caused an increase in expression of ep- . knock down of nfkappab using si-rna resulted in a further increase in ep- . this data suggests that although il- stimulates expression of ep- it does so through an nfkappab independent mechanism. the upregulation of ep- with sirna knock down of p suggests that activation of nfkappab would inhibit ep- expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il- is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il- gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)- , oxytocin receptor (otr) and connexin- (cx- ), suggesting multiple physiological effects for il- in term delivery. objective: to examine the effect of il- deficiency on the peri-partal uterine mrna expression of the pge receptors (ep) , and ; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p ) concentrations. methods: gestational length was observed in pregnant c bl/ wt (n= ) and ko (n= ) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d through delivery and up to h postdelivery. serum p was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p< . . results: birth was delayed in the il- ko mice ( . ± . d vs. . ± . d), and this affected the timing of peri-partal changes for all uaps similarly. both ep and ep (relaxatory receptors) were elevated at d in ko dams, but returned to low levels before delivery and were elevated at delivery (ep ) or afterwards (ep ). ep levels did not change. otr increased several hours before delivery in all dams. cx- increased at delivery, then fell, while pghs- increased at delivery and remained elevated afterwards. ptgfr mrna increased - -fold at delivery and a further - -fold after delivery, suggesting loss of pgf permitted enhanced ptgfr expression. p serum concentrations fell pre-partum in both groups. conclusions: il- ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p fall, suggesting a potential ovarian effect. a uterine role for il- in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, , -dihydroxyvitamin d ( , (oh) d ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme -hydroxylase (cyp b ) which catalyzes synthesis of , (oh) d from the inactive pro-hormone -hydroxyvitamin d ( ohd ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp b , ohd and , (oh) d on innate immune responses in trophoblastic cells. methods: a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for hrs with various concentrations of , (oh) d ( - nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp b was affected by pathogenic stimuli, a cells were treated with ligands for toll-like receptor (tlr) - , cyp b expression (real time pcr) and ohd utilization were assessed. results: a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp b with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for hrs with , (oh) d showed dose-dependent induction of the antimicrobial defensin cathelicidin expression ( . - fold induction), whilst cells treated pro-hormone ohd ( nm) showed . -fold induction of cathelicidin expression. activation of tlr (poly i:c) and tlr (lipopolysaccharide) enhanced the expression of cyp b ( -and . -fold) and increased the sensitivity to ohd as a consequence. conclusion: these data show that autocrine synthesis of , (oh) d from ohd can stimulate trophoblast immune responses in a similar fashion to macrophages. as ohd is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent -kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il- on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates and when cells were - % confluent they were serum starved overnight and incubated with m methyl- -hydroxy-progesterone acetate for h and with or without ng/ml il- for a further hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: h incubation of uterine myocytes with ng/ml il- caused a marked increase in slpi by % (n= ; p< . ). incubation of uterine myocytes with m progesterone for h also increased slpi by % (n= ; p< . ). incubation with il- for h in the presence of h with progesterone increased slpi: gapdh mrna ratio from . ± . to . ± . (mean ± sem; p< . ). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by -and -fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p< . ). these data show slpi is present in human myometrium and that it is increased by il- . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap- and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science : ; - ) . its levels are elevated in obese patients, those with type diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef ( ng/ml) for h, lysed and the resultant proteins labeled with cy or cy (amersham). there were used on a protein microarray containing constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap- , cre, hse and gre response elements using the exgen reagent. the cells were treated with rhpbef ( . , . , , ng/ml) hrs, lysed and ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated mapk signaling components including functioning as part of the erk pathways and belonging to p signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il- , pghs- , otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates until - % confluent. cells were serum starved overnight and incubated with μm methyl progesterone, . mm sodium -bromo-camp, . mm forskolin, μm kt , ng/ml brefeldin a and . mm -pmeopt- 'o-me-camp either alone or in combination for h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of epac , epac , il- , pghs- , otr, fp, gapdh and -actin were measured by qpcr. results: epac and epac were present in the upper and lower segment of myometrium with epac levels being some -fold higher than those of epac . there was no change with the onset of labour at or before term. treatment with il- for h significantly increased epac (n= ; p< . ) but had no effect on epac . when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs- and fp mrna expression (n= ; p< . ), but had no effect on il- and tended to reduce otr. the il- -induced increase in pghs- and il- , but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs- and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n= ) and second trimester myometrium (n= ), and in paired upper and lower segment pregnant (non-labouring, p, n= ) and labouring (l, n= ) myometrium; as compared to non-pregnant (np, n= ) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at - kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p< . ); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p< . ). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p< . ). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: ) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u cells at epithelial/macrophage cell ratio of : was employed. ) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of , , and nm for hrs followed by day stimulation of g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il- , il- and il- , il- and tnf were determined using elisas. ) the co-culture was pre-treated with nm of each pas for hours prior to lps stimulation at g/ml for . , . , , , and hours. the phosphorylation of p mapk and gr and the expression of mapk phosphatase (mkp ) were determined by western-blotting. results: il- , il- and il- , il- and tnf were elevated by . fold, . fold, fold, . fold and . fold in the co-culture model in response to lps(p< . for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il- , but not on il- , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp . p mapk was activated by lps for up to hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p ) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p effect and optimize concentrations, cells were pretreated with p ( - m- - m), dexamethasone ( um) or vehicle for hour prior to overnight lps ( ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p incubation and lps stimulation to determine whether the p effect was reversed by a camp antagonist. results: p significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at - m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig ) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig ) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p . objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the th day in culture, nonadherent cells were removed. on the th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp gene expression using the first trimester-derived extravillous htr- /svneo cells and the term placenta-like trophoblast carcinoma cells jeg- . methods: cultures of both cell lines were treated with a physiological concentration of avp ( . nm) to determine aqp mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with % fbs without avp. to determine whether avp regulation of aqp occurs by a camp signaling pathway, jeg- cells were preincubated with μm -(tetrahydro- '-furyl) adenine (sq ), a cell-permeable camp inhibitor, before being treated with avp ( . nm). cells were incubated for hrs for aqp mrna expression and for hrs for protein extraction at °c. after harvest, real time pcr and western blotting analysis were used to detect the aqp mrna and protein expression levels, respectively. results: avp increased aqp mrna expression in both cell lines by . and . fold after hrs. aqp protein expression paralleled the increase seen in the mrna (p< . ). pretreatment of jeg- cells with sq inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium ( , ). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone ( , nm, mm, mm, mm) for , and hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru and organon in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. . taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at . g, the equivalent of weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at days g (dg). experiments started at dg in dex ( mg) and saline treated controls (ctr), each receiving injections h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres ( . x total; sterispheres, biopal) were injected at : am into fetal jugular and femoral veins before maternal i.m. injection (t ) and h after the rd injection (t ) in both groups. tissues were collected hours after the th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex ( . + . mmhg; p= . ) with no change in ctr (- . + . mmhg). regional bf distribution is presented as a percent of the total counts per g tissue (table ) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r and crf-r receptors in mouse fetuses. methods: time-dated cd- pregnant mouse were anaesthetized and fetuses (n= ) were collected at e (term = e ). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r and crf-r by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using ', diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro . plus software. results: immunostaining for both crf-r and crf-r was seen in the smooth muscle layers of all lumens examined ( lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) and for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r and crf-r receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r or r receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a - % reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hs apart at days gestational age and allowed to deliver at term. at . yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a -hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl ; hd p hd . clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin - in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after days recovery the sheep received an infusion of ang - ( ng/kg/min) with or without its antagonist, [d-ala ]-ang-( - )(d-ala, ng/kg/min), into the renal artery for hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang - infusion. however, the infusion of ang - resulted in a decrease (change from baseline) (p= . ) in na + excretion of . ± . meq/kg/ h in the vehicle animals and . ± . meq/kg/ h in the b animals. there was no significant change in gfr during ang - infusion. gfr was lower in the b group during the combined ang - and d-ala infusion ( . ± . ml/min vs . ± in vehicle group, p= . ). intrarenal infusion of ang - at ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang - may be mediated by a receptor other than the mas receptor for ang - and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine ( . - . g/kg/min) and to an l-name ( mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max ± vs ± % of baseline, p< . , figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl . women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone ( . mg/kg) or vehicle on days and of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between and months and cells were dispersed and plated at a density × cells per well in well plates. after h, cells were stimulated with normal medium, nm arginine vasopressin (avp), nm avp, nm corticotropin releasing factor (crf) or nm crf for h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following nm avp ( . ± . vs. . ± . ) and nm crf ( . ± . vs. . ± . ). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells ( . ± . and . ± . respectively), but was significantly decreased in betamethasone cells ( . ± . and . ± . respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd and hd . lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first - h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n= ) were exposed to a paradigm of graded, stepwise hypoxia on day (term= ) (pediatr. res. : - , ) . control pregnant rats were exposed to % oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in % paraformaldehyde and paraffin embedded. sections (n= per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro . software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua ( . ± . au) and in all three major placental cells types (giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua ( . ± . au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et- and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (v) hours apart at days of gestational age and allowed to deliver at term. at . yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f= . , p= . ) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f= . ; p= . ). conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects. the activation of etb receptor by et- inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl ; hd p hd . in the placenta, -hydroxysteroid dehydrogenase type ( hsd ) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate hsd expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect hsd expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received intramuscular injections consisting of either saline ( ml saline/ewe) or dex ( . mg/kg) given hours apart starting on days of gestation (dg). the animals were sacrificed at , , , and dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure hsd placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental hsd mrna expression across gestation. however at dg, there was a significant increase in hsd expression after dex (p= . ). there was an increase in placental hsd mrna progressively from dg (mean = . ) to dg (mean = . ), independent of treatment. overall, a positive correlation between hsd and fetal weight (r = . ) was apparent at dg and at dg (p= . ) in both control (p= . ) and dex (p= . ). significant positive correlations between placental hsd and fetal weight (r = . ) were found in females (p= . ) and a similar trend was found in male fetuses (p= . ). we conclude that in the sheep placenta, hsd expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between hsd and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental hsd is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; ; : ) and increases femoral vascular resistance in the lamb (jphysiol; ; : ) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between mo and , y of age (p< . ). vascular relaxation to acetylcholine (ach) but not to pge decreased during the same time (p< . ). vascular contractility of the renal artery (ra) increased in response to k + (p< . ). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol; ; :r ) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem; ; : ) increase stroke mortality for unknown reasons (amjpsychiatry; ; : ) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n= ) or μg/kg dexamethasone (n= ) i.p. at day e / equivalent to x mg dexamethasone administered to a kg pregnant woman. at . years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p< . ). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p< . ). relaxation to ach and pge was similarly diminished in both vessels after precontraction with na (p< . ). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n= ) or with two doses of bmz (n= ), . mg/kg, hours apart at days of gestation. renal cortex from the male offspring was harvested at - months of age. cortical membranes were then solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p= . ). ace and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace activity, bmz treated animals exhibited a . fold greater proportion of ace activity versus control animals (p= . ). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd , hd . we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n= ) were dissected from term ovine fetuses ( - d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n= ) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n= ) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r receptors. both ucn- and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn- functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - ) and term (t: - days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn- ( : sigma) by abc system. intensity of ucn- immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p< . ). conclusion: ucn- immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn- inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after days of recovery, the sheep were infused with ang ii ( ng/kg/min) with or without candesartan ( ng/kg/day) or pd (pd g loading dose and ng/kg/min infusion) into the renal artery over hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p= . ). ang ii decreased the nae in both groups(p= . ). candesartan increased nae among controls(controls . and b . meq/kg/h, p= . ). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p= . ). during ang ii infusion there was a decrease in gfr among control compared to b sheep (- . vs. - . ml/min, p= . ). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type receptor (crhr- ) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, hour incubation with the specific crh-r antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r , a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short ( . hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ cells/well. after hours in culture, wells were rinsed x and refilled with serum-free dmem/ f containing vehicle (dmso) with or without ant. after . hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem, : , ) completed. triplicate vehicle or ant wells were treated with i -tyrosine human acth ( - ) with or without - m acth (for non-specific binding). after hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk ( - m) was added to the wells. after . hours, medium was removed and stored @ - ºc for camp eia and cortisol ria. results (mean±se) of . hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n= ± ± cortisol (ng/ml/ . hr) n= ± ± * (p=. ) camp (pmol/ml/ . hr) n= . ± . . ± . * (p=. ) * indicates significant difference from vehicle alone. conclusion: the crh-r antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive doses of . mg/kg of or vehicle, at and days of gestation; the offspring were studied at and months of age. in female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term ( - days of gestation) were brought from the farm at - months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl ( . meq/kg/min at . ml/min) was given for min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males ( . ± . vs . ± . p< . ) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males ( . ± . vs . ± . p< . ) during the na infusion experiment, the exposed females had lower arc than did the control females ( . ± . vs . ± . p< . ). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd and hd . background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin- receptor; mc r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth - yet decreased expression of key steroidogenic enzymes and the mc r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with > % homology to the human mrap n-terminal residues, with partial homology to the beta isoform in the carboxyl region ( %). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from - (n= ) and near term ( - ; n= ) days of gestation (dg) as well as from lth (n= ) fetuses (exposed to high altitude hypoxia from to - dg) and age-matched normoxic controls (n= ). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/ ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between - dg ( . ± . ) and increased (p< . ) approx. -fold near term ( - dg; . ± . ). levels of mrna for mrap were highly correlated to cyp mrna in individual samples. mrap expression in control ( . ± . ) and lth ( . ± . ) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace and neprilysin, ang ( - ) and ang ( - ), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n= ) and female (n= ) sheep were harvested at - months of age. the tissue membranes were solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results ace activity was over times greater ( ± . vs. . ± . fmol/mg/min, p< . ), ace activity was . times greater ( ± . vs. . ± . fmol/mg/min, p= . ) and nep activity was nearly times greater ( . ± . vs. . ± . fmol/mg/min, p= . ) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd , hd . in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine ) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and ) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hours apart at days gestational age and allowed to deliver at term. at mo of age, female sheep were randomly allocated to be fed at either % of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a . g/kg glucose bolus was used and for the hec mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > % of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f= . ;p< . ). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl . syngergistic induction of -hydroxysteroid deyhdrogenase type expression by cortisol and interleukin- in human fetal lung fibroblasts. z yang, p zhu, cm guo, l myatt, k sun. school of life sciences, fudan university, shanghai, china; obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce b-hydroxysteroid dehydrogenase type ( b-hsd ) which converts inactive cortisone to active cortisol, but their effect on b-hsd expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin- b (il- b) effect on b-hsd in human fetal lung fibroblasts (hfl- cells). methods: the expression of b-hsd in hfl- was examined with immunocytochemistry and pcr. b-hsd , prostaglandin h synthase- (pghs- ) and cytosolic phospholipase a a (cpla ) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il- b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il- b were studied using a gr antagonist (ru ) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv -a/cebp). results: hfl- cells expressed a high level of b-hsd . both cortisol ( - - - m) and il- b ( . - ng/ml) induced b-hsd mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor , -dichlorobenzimidazole riboside ( μm). ru ( - m) blocked the induction of b-hsd by cortisol. induction of b-hsd mrna expression by cortisol ( - m) was synergistically increased by co-treatment with il- b ( . - ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla and pghs- expression by il- b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of b-hsd mrna expression by either cortisol or il- b. these data suggest induction of b-hsd expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of b-hsd expression by il- b. conclusions: cortisol and il- b synergistically induce b-hsd expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental -hydroxysteroid dehydrogenase ( hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, pl van zijl, cw rumball, , al jaquiery, , je harding, , mh oliver, , fh bloomfield, , jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of hsd- and - activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from days before until days after mating to reduce maternal body weight by %, with ad libitum feeding thereafter. placentomes were collected on days , , , and of gestation (term, d) and hsd- and - activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower hsd- activity at d (p= . ), a significant reduction at d (p< . ), but no difference at d or d . hsd- activity was not different between n and un animals at any time. there was no effect of twinning on hsd- or - at d . however, with twins both hsd- (p= . ) and - (p< . ) activities increased at d . hsd- activity was reduced in twins (p= . ) at d but was higher than any singleton pregnancies at d (p= . ). hsd- was not different between singletons and twins at either d or d . overall, hsd- was lower in placentae of male compared to female fetuses in late gestation; hsd- was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day of life (p ) and at months (n= - per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels ( s as control). we evaluated the expression of -beta hydroxylase (cyp b ), aldosterone synthase (cyp b ), acth receptor (mcr ), p side chain cleavage enzyme (cyp a ), star protein, -hydroxysteroid dehydrogenase type (hsd ) and type (hsd ), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr c ). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp b (p=. ), cyp b (p=. ), hsd (p=. ), p (p=. ), acth receptor (p=. ), star (p=. ) and nr c (p=. ) mrna in p mfr offspring, with no changes in hsd and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd and gr were significantly upregulated with a trend towards an increase in acth receptor (p=. ) whereas in the female mfr the expression of acth receptor (p=. ) was increased and nr c (p=. ) and cyp b were decreased (p=. ). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=. ) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth ( - d), quiescence ( - d) followed by reactivation (> d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf mrna expression between male and female fetuses. methods: a total of twin fetuses were used in this study. post mortem was performed at either - d, - d or - d gestation. adrenal mrna expression of igf , igf , igf r, igf r and cyp was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p< . ) with increasing gestational age. the relative adrenal weight was lower however after d when compared to the earlier gestation age group (p< . ). adrenal igf mrna expression was lower (p< . ) at - d when compared to the earlier gestation age groups. interestingly, igf r expression was highest at - d (p< . ). there was an interaction between the effects of age and gender on adrenal igf and igf r mrna expression such that the expression was higher in males compared to females at - d (p< . ), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf . in this study we have demonstrated there is an increase in both igf and igf r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude ( , m) from day to near term. on days - (term = days), fetal adrenal glands were collected from lth (n= ) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase / (erk / ) and p map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk / and p at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n= ) and with preeclampsia (n= ) between and weeks of gestation. we evaluated the expressions and phosphorylations of erk / and p map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p and phospho-p map kinase were not detected in invasive trophoblasts in cases or controls. erk / and phospho-erk / were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk / was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk / in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n= ) and from severe early onset preeclamptic (n= ), and age-matched control placentae (n= ). protein expression of cyclin e , cyclin d , cyclin d and cell cycle inhibitors, p , p , p , and p was assessed by western blot analysis. spatial and temporal localization of cyclins e , d , d , p and p was determined by fluorescence immunohistochemistry. expression of cyclin e and cyclin d mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e and cyclin d to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d expression was predominantly restricted to the villous stroma. cell cycle inhibitor p was expressed in both ct and syncytiotrophoblast (st) cells whereas p was restricted to the st. during normal placentation, levels of both cyclin e and cyclin d were high in the first trimester and decreased with advancing gestation. the expression of cyclin d , p and p showed an inverse correlation to cyclins e and d whereby their expression increased towards term. levels of p and p remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e and p and a decrease cyclin d and p expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e positive progenitor ct cells in the floating villi and an increased expression of p in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif- -mediated placental and circulating sflt- at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt- with maternal and fetal oxygen tensions. we hypothesized that circulating sflt- and free vegf would be increased at m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from and healthy mother-infant pairs living at m and m respectively. plgf, free vegf and sflt- were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf ( ± pg/ml vs. ± pg/ml, p<. ) and sflt- ( . ± . vs. . ± . ng/ml) were increased at m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (- ± % plgf; - ± % free vegf; - ± % sflt- , p<. ). cord blood free vegf was -fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in % of the babies. similar results were obtained for sflt- . thus, while free and total maternal circulating vegf and sflt- are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n= ) and hs (n= ) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g b scanner. images were analyzed using genepix . (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were differentially expressed genes between spe and hs (p<. ). among these candidate genes, were up-regulated and were downregulated. the most up-regulated genes are ( )-deoxyribonucleotidase (dnt- ), superoxide dismutase , hydroxy-delta- -steroid dehydrogenase, caspase , and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and control group ( patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf- is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk - ) have been identified. our aim was to investigate the expression pattern of alk and alk receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk and alk mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk and alk antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk expression increased with advancing gestation. in preeclamptic placentae alk expression was significantly increased compared to preterm and term controls, whereas alk expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad , a tgf signaling molecule, which is activated by alk and increased phosphorylation of smad , which is triggered by alk . the expression of alk and alk in iugr placentae differed from that of preeclampsia as both alk and alk mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk expression was significantly increased in iugr placentae at the protein level, while no differences in alk protein levels were noted between iugr and controls. conclusions imbalance between alk and alk signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk or alk has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd a and its downstream signal proteins--phospo-p , phospho-mkk /mkk were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd a and sflt- proteins in placenta tissue sections using abc method. huvec cells were cultured to - % confluence and were divided into groups: control, stress induction (sorbitol, . m, h), p inhibition (sb- , ug/ml, ug/ml and ug/ml, hour) + sorbitol ( . m, h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd a and phospho-p . supernatant sflt- was measured with an eia kit and the results were read at nm wavelength. results: gadd a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk and p ) activation compared with control. overexpression of gadd a and sflt- in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd a was induced by sorbitol, triggering the activation of the downstream p- pathway and the accumulation of sflt- in the supernatant. the up-regulation of sflt- secretion by inducing gadd a was depleted when treated with p- inhibitor. conclusions: our study reveals that gadd a and its down stream p- pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt- , which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt- secretion via the gadd . trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, jennifer a doll, emily j su, serdar e bulun, mona cornwell, susan e crawford. obstetrics and gynecology, northwestern university, chicago, il, usa; pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n= ) and those complicated by severe preeclampsia (n= ) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored ( =none, =spotty, light, =consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (> / positivity for factor antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas ( . + standard deviation versus . + . , p = . ). endothelial expression was not significantly different in preeclamptic ( . + . ) versus normal placentas ( . + . , p> . ). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < . was set for statistically different. results: ) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p< . ; ) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; ) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p< . . conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd and hl ). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n= , ptspe, . weeks); (ii) preterm labor (n= , ptl, . weeks); (iv) term no labor (n= , tnl, . weeks); (v) term labor (n= , tl, . weeks). microarray gene profiling was performed using affymetrix human genome u plus . arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly -fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). the expression of gp at implantation site: implication for the pathogenesis of preeclampsia. objective: gp is a common shard signal transducing subunit of the il- cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = , respectively) were immunostained for gp . the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value [ . - . ] and , respectively (p< . ). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast ( [ - ]; p< . ). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia ( % or % oxygen) in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. % and % oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor- (hif- ) and the receptor svegf-r /sflt- : implication for phatophysiology of preeclampsia. julio e valdivia-silva, , juan c gonzalez-altamirano, keisy lopez- the trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr- /sflt- , hif- and- . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r ) of the cts was diminished. on the other hand, the expression of the arnm of hif- or - in cells was comparable in all the types of placentas, nevertheless, the protein hif- was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif- and the increase of the receptor svegfr- , we used sirna-hif . in response to the inhibition, the expression of the receptor svegf-r diminished dramatically. the blockade of hif- did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif- is one of the molecules involved in the selective expression of the receptor svegf-r in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt- /svegfr- after transfection with sirna-hif in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting % of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e increases synthesis of vwf, we treated heecs with e at . μm, . μm and . μm for hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts (a disintegrin and metalloproteinase with thrombospondin type motif) activity results in increased amounts of active vwf. rrelease of activated adamts will be determined by fret. to ascertain if e regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici , . elisa and fret will assess the release of active vwf and adamts , respectively. results: western blotting demonstrated e increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e increases synthesis of vwf in heecs in vitro. if e increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, jacqui f donoghue, marc g achen, steven a stacker, jane e girling. obstetrics gynaecology, monash university, melbourne, victoria, australia; ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c bl/ jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after weeks. serial sections were immunostained with lyve- and/or vegfr (lymphatic endothelial cell markers), cd (blood endothelial cell marker), mab (human vegf-d), mab (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in % of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. ebna tumours formed inside and outside the uterine horn of both the control (n= of ) and vegf-d group (n= of ). localization of ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n= ) and without (n= ) abruption. purified term dscs were passaged until > % free of cd + cells by facs. confluent dscs were primed with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days. after h incubation in defined medium with corresponding steroids ± thrombin ( . - . iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, . -fold stimulation by . iu/ml thrombin from . ± . to . ± . pg/ml per mcg protein for e +mpa; p= . ). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, . -fold for e +mpa; p< . ). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert ( well/plate, micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of . , . , . , . , and . g/ml were added to the upper chamber of the cell insert. after hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < . was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml (p< . ); chy . : . ± . g/ml (p< . ), respectively. data are means from independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd and hl ). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk / ) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv -of to test whether: ) vegf-activated erk / signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk / activation and; ) caveolin- , the structure protein of caveolae, regulates vegf-stimulated erk / phosphorylation. methods: ofpaec or sv -of cells were cultured in mcdb- / % fbs/antibiotics. serum-starved subconfluent ( %) cells were treated with rhvegf ( to ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, mm, min) or caveolin- scaffolding domain (cav-sd, m, hr). sv -of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient ( %/ %/ %) ultracentrifugation. activation of erk / signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk / phosphorylation in a time-and dose-dependent manner. erk / phosphorylation maximized by vegf ( ng/ml) at - min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk / signaling module, plc , pkc , src, ras, raf- , mek / and erk / , were detectable in purified caveolae membranes positive for various markers including caveolin- , enos, flotillin- , and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk / without altering total erk / in a time-dependent manner similar to that in total cell extracts, which also maximized at - min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk / phosphorylation in caveolae. conclusion: vegf activates the erk / signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk / signaling pathway. we conclude that caveolae/caveolin- serves as a platform for compartmentalizing the vegf-induced erk / signaling pathway in placental ec (hl and hl ). hypoxia upregulates gcm in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm . gcm is also expressed in the human placenta. placental gcm- protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm we hypothesised that gcm expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm expression in the human placenta. two model systems were used; ( ) free floating villous explants and ( ) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n= experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm protein was detectable at a low level in villous explants maintained for h in % o . a striking increase in gcm protein was observed when villous explants were incubated for h in % o (p< . ). incubation of villous explants for h in % o followed by re-oxygenation for h in % o resulted in a marked decline in gcm protein (p< . ). expression of gcm was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in % o or reduced oxygen ( % o ) conditions. gcm protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm- protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm- in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm warrant further investigation. the effect of acute alcohol exposure on histone -lys modification in the mid-gestation embryonic lung. xiangyuan wang, debra wolgemuth, , , laxmi baxi. ob/gyn; genetics development; human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone at lysine (ach lys ) in the mid-gestation embryonic lung. pregnant c bl/ j mice at day . of gestation (e . ) were injected intraperitoneally with doses of % ethanol ( . g/kg ), hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved and hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach ly antibody. the entire lung tissue field was evaluated for the levels of ach lys staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e . , the morphology of ae embryonic lungs was normal. however, high levels of ach lys were detected in % of the bronchial epithelial cells and % of the mesenchymal cells. the expression level in both lineages decreased hr after alcohol exposure. in the controls, the expression of ach lys was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e . lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme hours after alcohol treatment. we now observe that elevated expression of ach lys in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at to weeksgestation and randomly assigned to receive either mg of vitamin c and iu of vitamin e (study group, n= ) or placebo (control group, n= ) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: . the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group ( . ± . vs. . ± . nmol/mg protein, p< . ). . the orac values in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . u/ml, p< . ). . the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . nmol/ml, p< . ). . there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr , is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n= ) and with pre-eclampsia (n= ) at - weeks gestation. protein expression levels and tissue localisation of trail and dr were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [ . ( . - . ) , median (interquartile range), pg/ml, n= ]. however, there was no significant increase either in pregnancy [ . ( . - . ) pg/ml, n= ] or pre-eclampsia [ . ( . - . ) pg/ ml, n= ] (kruskal-wallis test, p> . ). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n= ). following staining with mayer's hematoxylin (n= ), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from - villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately (lane ), (lane ), (lane ), and (lane ) nuclei revealed that fasl could be reliably detected in specimens containing as few as nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd . the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin- ) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n= ), mild (n= ) and severe (n= ) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, tamao yamamoto, yoshimasa watanabe, takeo itoh, hidetaka izumi. obstetrics and gynecology, nagoya city university, nagoya, japan; pharmacology, nagoya city university, nagoya, japan; obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o -) and nitric oxide (no), is strong oxidant as well as o -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women ( / ), although it was not seen from normotensive pregnant women ( / ). the localization was also seen in placentas from of severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar ( . ± . for sever preeclampsia and . ± . for normotensive pregnant women). from these results, it was suggested that an increase in o production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) and in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on decidua from normal (n= ) and preeclamptic (n= ) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ) and confirmed by western blotting. quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in preeclamptic decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in normal decidua ( ± and ± , respectively; p< . ). in cultured first trimester dcs incubated with e , tnf increased secreted mmp- and mmp- levels by ± and ± -fold, respectively, while il increased them by ± and ± fold, respectively (p< . ). in parallel incubations with e +mpa, basal mmp- and mmp- output were lowered by approximately % and %, respectively, while tnf -and il elicited mmp- and mmp- levels were blunted by - %. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp- and mmp- secreted protein levels. conclusions: over-expression of mmp- and mmp- in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce -methoxyestradiol ( -me), a compound with diverse biological activities including inhibition of hif- , a transcription factor that mediates cellular response to hypoxia. circulating levels of -me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of -me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon of the comt gene (rs ), which does not change amino acid sequence ( leu ), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (> %) african-american. the frequency of the minor rs "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: %; controls: %, p= . ), but was significantly greater in fetal dna from pregnancies complicated by preeclampsia compared to control pregnancies (g allele frequency cases: %; control: %; p< . ). likewise, fetal carriage of the rs "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: . ; % c.i.: . , . , p< . ). there was also a significant discordance between paired maternal and fetal rs genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: . ; % c.i.: . , . ). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of -me. supported by nih p md . smoking is associated with elevated adma in preeclampsia. michael p frank, robert w powers. , magee-womens research institute, pittsburgh, pa, usa; obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of women with uncomplicated pregnancy (controls), and women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p< . results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p< . ). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p< . ). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n= ) and lp (d - ; n= ) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po > mmhg, pco = - mmhg). results: although the pressure change was similar between np and lp ( and mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from ± to ± ml/ g/min in np ( %) and ± to ± ml/ g/min in lp ( %; p< . vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np ( . ± . vs. . ± . mm hg/(ml/ g/min); p< . ). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: ± vs. ± for anterior (p< . ) and ± vs. ± for posterior (p< . ). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf- are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = each): delivering maternal side inflow perfusate at oxygen concentrations of . % and %, respectively, distributed via cannulae; and fetal side inflow perfusate oxygen concentration of - % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein- alpha (mip- alpha) (reference oxygen sensitive hormones), free vegf, svegfr- and plgf. in separate perfusions, fetoplacental vasodilation in response to pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u , (n = each). results: maternal-side mip- alpha release was higher in the 'hypoxic' than the 'normoxic' system ( ± and ± pg/ml, respectively, at hours, mean ± se; -way anova: p< . ). maternal-side epo and fetal-side soluble free vegf and svegfr- release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group ( . ± . and . ± . pm, respectively, at hours, mean ± se; -way anova: p < . ). there was no difference in the vasodilatory response to vegf- in the fetoplacental vasculature between the groups ( . ± . and . ± . % change in fihp, mean ± se). discussion: differences in mip- alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr- release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at rs; it also stimulates nf-kappa beta and modulates glut- . to date at r expression has not been investigated in the placenta. we propose that at r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from np and pe at delivery (gestational ages: and weeks respectively). samples were taken from areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade = no positive labelling, = - %, = - %, = - % and = - % of cells positively labelled. median (max, min) are shown. results: ) at r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. ) at r positivity was reduced in near cord pe samples ( . ( . , . )) compared to np ( . ( . , . ) p= . ). conclusion: we have shown for the first time, dense at rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, hm boelens, jj hm erwich, j heimweg, s van der heide, ajm van oosterhout, aej dubois, jg aarnoudse. obstetrics, university medical center groningen, groningen, netherlands; laboratory of allergology and pulmonary disease, university medical center groningen; pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th immune profile. treg cells are a subpopulation of cd + lymphocytes and are specifically characterized by the lineage specific transcription factor foxp . treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, women with pregnancies complicated by pre-eclampsia and healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd + foxp + treg cells ( . vs . %; p< . ). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd + foxp + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd + foxp + treg cells. no difference was found in umbilical cord blood ( . vs . %). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh ), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh and sepiapterin (sep, a precursor for bh biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups ( - ) of shr pregnant rats were either treated with bh , sep ( mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day of pregnancy until day of gestation. animals were sacrificed on day of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p< . ) elevations in blood pressure (bp, mmhg) were observed in shr (shr, ± . vs. wky, ± . mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh or sep ( ± . ), significantly (p< . ) reduced elevated bp beginning from day of pregnancy. fetal but not placental weights were significantly (p< . ) reduced in shr ( . ± . grams) compared to wky ( . ± . ) rats. bh ( . ± . ) treatment partially (p< . ) increased fetal weights compared to the shr group. vascular enos expression is significantly (p< . ) elevated in shr ( . ± . ) compared to wky rats ( . ± . ). further, treatment with bh ( . ± . ) but not sep ( . ± . ) significantly (p< . ) reduced elevated enos protein expression in shr rats. conclusions: bh may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t- ->c and missense glu asp variants. toshihiro yoshimura, michihiro yoshimura, masafumi nakayama. obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; cardiovascular medicine, jikei university school of medicine, tokyo, japan; cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu asp missense variant in exon and a t- -->c variant in the '-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t- -->c and severe preeclampsia. materials and methods: the study participants included patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the general normal population. results: the analyses revealed that the frequency of the missense glu asp variant (n= / , %) was significantly higher than the general population (n= / , %), as we previously published. however, the frequency of the t- -->c variant (n= / , %) was not different from the general population (n= / , %). interestingly, only one patient had both t- -->c and missense glu asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t- -->c variant is associated with severe preeclampsia. the t- -->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t- -->c may have some reproductive significance in combination with missense glu asp variant, however huge number of patients would be needed to analyze such rare ( . %) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, jong kwan jun, chan-wook park, joong shin park, bo hyun yoon, hee chul syn. obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and normotensive pregnant women were included in this study between jan and jul . diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early ( weeks or before) and late-onset ( weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c t, angiotensinogen (agt) m t, vascular endothelial growth factor (vegf) c t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of < . was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: . , p< . ) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only out of possible genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels ( - μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . , p< . ); % vsm staining ( . ± . vs. . ± . vs. . ± . , p< . ). for mmp , obese and overweight patients had a greater (p< . ) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . ); % vsm staining ( . ± . vs. . ± . vs. . ± . ). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp . speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp . hl . collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t- flasks ( , cells/flask) and grown for days to % confluence. the cells were treated for hours with medium control, ros (hx, . mm + xo . u/ml), tnf-( ng/ml); and neutrophils ( , ) activated with arachidonic acid, μm, ( : ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group was the control and group was the experimental treatment. results: table . conclusions: neutrophils, ros and tnf increased mmp expression. interestingly, genes involved in collagen synthesis (col a ) or inhibition of mmp- activity (timp ) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl , fogarty d tw , p md . objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n= ) were cultured h in % and % oxygen in dmem with % human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed - h to confluent, phorbol myristate acetate differentiated u macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with % oxygen was . %, not different from the . % from cultures exposed a % fio and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd and hd . is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, laura j minchell, jenny e myers, philip n baker, carl a hubel, ian p crocker. maternal and fetal health research group, the university of manchester; obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for h with pooled plasma ( %) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal ( , , m, ratio . ) and pe pregnancies ( , , m, ratio . ) , . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc- assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p< . , wilcoxon signed ranks, n= ). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p< . , n= ). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p< . , n= ) and a reduction in jc- fluorescence (p< . , n= ), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p< . , n= ; jc- assay: p< . , n= ). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. lorentzen ( ) bjog; endresen ( ) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time . in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: / ( %) of patients has 'none-mild' pain, and ( %) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' . + . kg/m ; 'moderate-severe' . + . kg/m , p= . ), nor was pain severity (p= . ). pain prevalence was significantly associated with ac (mean: 'none-mild' . + . inches; 'moderate-severe' . + . , p= . ). a borderline positive association exists between ac and pain severity (p= . ). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin release in keratinocytes has been recently shown. methods: we report preliminary data regarding women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg ( patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle ( patients). patients were asked to apply the cream to the vulva twice-daily for days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg might be linked to a reduced infective charge, attributable to the activity of -defensin . background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in to % from matched unrelated donors. genital cgvhd complicates about % of hsct and may uncommonly result in labial fusion. case: year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: women with a diagnosis of an ectopic pregnancy were studied, with surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p< . . results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or . , p= . ) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or= . , p< . ), a large volume of cul-de-sac fluid (or= . , p< . ), and increasing pain (or= . , p= . ) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods] female patients (average age: . years old) who consulted a physician and agreed to answer about the questionnaires during september, -june, . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in . and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] in patients had migraine ( . % average age: . years old). prevalence were . % in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome %, dysmenorrhea . %), sterility ( . %), and severe menopausal disorder ( %). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of doses of estradiol (e ) gel . % (divigel ® ), a novel formulation of e consisting of mg e per g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: postmenopausal women were evaluated in a -week study comparing placebo to e gel . % at doses of . g/day, . g/day, and . g/day with estimated nominal daily deliveries of . mg, . mg, and . mg of e respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e gel . % showed statistically significant improvements from placebo gel as early as week (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all doses of e gel . % compared to placebo. conclusion: e gel . % significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e gel . % offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied ( . mg e , delivering . mg e /day), to treat vasomotor symptoms associated with menopause. estradiol (e ) gel . % (divigel ® ) is a novel formulation of e consisting of mg e per g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e gel . % were evaluated in a large placebo-controlled trial. design: postmenopausal women participated in a -week study comparing placebo to . g/day, . g/day, and . g/day of e gel . %. circulating e and estrone (e ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e gel . % produced physiologic e :e ratios similar to those seen in premenopausal women. e :e increased from a baseline mean of . to . , . , and . with, respectively, the . , . , and . g/day doses of e gel . %. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e gel . %. shbg levels remained unchanged after weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after , , and weeks of treatment were . for all treatment groups except for a mean value of . ± . for the . g dose group after weeks of treatment. conclusion: e gel . % is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e ( . mg to . mg), with the lowest systemic daily delivery of e attained by the divigel ® . g dose. following weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from - . (divigel ® . g) to - . (estrasorb ® ); change in severity scores for msvms ranged from - . (divigel ® . g) to - . (divigel ® . g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in % of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb- (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short ( . - . cm) lengths of silastic tubing (od . mm), either empty (n= ),or filled with silicone rubber matrix containing % of micronized cdb (n= ), were inserted into the uterine lumens of ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, ) . after . cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole . cycles, animals bearing a blank ius bled for an average of . ± . days while cdb treated animals bled for an average of only ± . days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of ± mg while the cdb-treated endometria weighed only ± mg. the proliferation markers ki- and phospho-h were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr , hd and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were ) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and ) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n= ); estradiol (e ,n= ); medroxyprogesterone acetate (mpa,n= ) or e +mpa (n= ). after days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by isoprostane and -oh-deoxyguanosine ( oxog). apoptosis was investigated by the tunel method. statistical analysis was by -way and -way anova. results: gp uteri were enlarged by both e (p< . ) and mpa (p= . ). effects of mpa on uterine weight differed significantly depending on e levels (p< . ), where mpa opposed the e effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p= . ) and bv average area (p= . ). the presence of e significantly decreased these parameters (bv density mean± sem: crl: . ± . %, e : . ± . %, mpa: . ± . %, e +mpa: . ± . %, p= . ). these changes were associated with highly elevated -isoprostane content in e +mpa-treated uteri compared to all other groups (p< . ). abnormalities in the e +mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of women reported the presence of symptoms prior to initiating contraception ( dmpa, oc, nh) and every mo thereafter for mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in mo increments for mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= . ), cramping (or= . ), hair loss (or= . ), acne (or= . ), nervousness (or= . ) and mood swings (or= . ). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within mo in nearly % of those with it at baseline, but no significant resolution after mo. also mastalgia ( % at baseline) was less likely to persist at and mo. bleeding between menses was reported for the first time at mo by % of oc users. dmpa users of all races had an increased risk of missed periods (or= . ), bleeding between menses (or= . ), bleeding > d (or= . ) and loss of libido (or= . ) relative to nh users. it reduced cramping (after mo) and bloating (all mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after mo while resolution often occurs within mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers micrograms of ee; the weekly tc contains . milligrams of ee. each treatment was followed by months of washout and months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < . . results: after two months of treatment the mean (+/-sem) ee levels for tc = . pg/ml (+/- . ) and oc = . pg/ml (+/- . ). the ee level is not significant different for the two medication (p = . ). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined patients per arm were needed to demonstrate a significant difference of in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under weeks and general anesthesia for over weeks. thirty-nine patients were randomized to receive a paracervical block with . % bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, and minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, mika gissler, satu suhonen. ob gyn, university of helsinki, helsinki, finland; national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from to % in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of women undergoing medical abortion between august and december . the subjects were followed by means of finnish registry on induced abortions until december ; the follow-up time (mean ± sd) was . ± . months. results altogether ( . %) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january through june . patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were essure procedures attempted at the dmc from january through june . of the attempted procedures, there were failures ( . %). of the failures were attributed to difficulty visualizing the ostia ( %). other causes of failure included expulsion of the device ( ), tubal spasm ( ), uterine perforation ( ), and tubal ostia too large for the device ( ). there were cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure ( . vs . min, p = . ), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia ( . % vs . %, p = . ). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is . % with a pregnancy rate of . %. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january through june . follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in uhc patients of which were successfully completed. of the patients, ten underwent a post-essure hsg ( . %). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only . %. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, dong zhang, salama salama, cheryl walker, hala el-mazar, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex- -thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; , and days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by % ± , % ± and %± of pretreatment volume at days , and respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by + %± , + %± , + % ± while receiving media continue to grow by + % ± , + % ± and + % ± at same time points. ad-tk/gcv induced significant increase in caspase activity, bax expression, decrease in bcl and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd and cd expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, dong zhang, hala el-mazar, cheryl walker, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro - ) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro - mg /kg ,twice/ day for days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at and weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. hours urine samples were collected for evaluation of estrogen (e ) metabolites and bone resorption marker. results:animals treated with ro - exhibited significantly lower uterine fibroid tumor burden ( % and %) of pretreatment volume at and weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached %, and % of pretreatment size at the same time points. ro - treatment resulted in an increase in urinary / hydroxy e metabolite ratio. in addition ro - increased bax expression and decreased parp , pcna and cyclind experssions . all ro - treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro - , a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d ). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf- , focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf- , or anti-tgf- antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf- ( . ± . fold), retinoic acid metabolizing protein (cyp a ; . ± . ), and versican variant v ( . ± . fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v decreased to levels found in myometrial cells ( . ± . fold). conversely, when treated with tgf- , expression of versican variant v increased . ± . fold. to confirm that tgf- was central to the overexpression of v , we treated leiomyoma cells with anti-tgf- antibody, and found that baseline over-expression of v template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf- expression, demonstrating that tgf- template decreased to levels comparable to myometrial cell expression ( . ± . fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf- , providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of % serum until % confluent. for the next h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at , , , , m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma m after serum replacement. pr-a and pr-b were detectable at , and m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the specimens per subject and subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base . confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. genes were differentially expressed when we used a . q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp was detected and we report for the first time that scc- (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin- (tsp- ) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp- plays an important role in angiogenesis and is an activator of tgf - . in a previous study, we found that differential expression of tsp- in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp- by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to % confluence. then smc were serum deprived for h and treated back with % serum for , , , and m. cells were collected for rna and protein, and tsp- expression was evaluated. in addition, cells were stained using the combination of anti-cd and anti-smooth muscle -actin or combination of anti-cd d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp- mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd and platelet marker cd d suggesting that the myofibroblasts cells themselves were the source of the tsp- . conclusions: unlike skin wounds, where tsp- is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp- . elucidating the roles of tsp- in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp- in the activation of tgf - are indicated. phospholipid s p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s p enhances while high dose s p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s p affects invasion, using specific inhibitors. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi, p -mapk, rac and rock, thereafter cells were detached and tested for invasion towards m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p= . ) and m s p treated cells ( p= . ). inhibition of both p -mapk and rac did not affect m s p treated cells, in contrast invasion of control cells was mildly increased (p= . , . ). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and m s p treated cells ( fold, p= . , fold, p= . ). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s p (p= . , . ). ptx fully inhibited gelatinase activity of control and . m s p treated cells, and partially of the m s p treated cells (p< . ). . m s p significantly increased upa activity of attached (p= . ) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. m s p inhibited upa in both attached and invading cells (p= . ), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and m s p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by . m s p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc , (rac-i)), and cells were detached and assayed for invasion towards m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p= . ) increase of invasion by . m s p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p= . - . ). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with m s p. control cells invasion was increased ( . fold, p= . ) by continuous rac-i treatment. . m s p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated and fold gelatinase activity of control and m s p treated cells. in contrast upa activity was inhibited by both . m and m s p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with . m s p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with m s p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp- , and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp- activation. lpa-dependent pro-mmp- activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp- activation. in the current study, inhibition of rho-kinase/rock with y- reversed the repression of lpa-stimulated pro-mmp- processing observed with treatment of the egfr kinase inhibitor, ag . correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp- processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov ), breast cancer (mda mb ) and mesothelioma (hp- ) cells were grown in media supplemented with % fbs. after serum deprivation, % vesicle-free fbs was added for hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with and g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp- and . fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at g/ml they induced significantly high levels of invasion in all cell types tested. at g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr- as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr , vegfr , co-receptor nrp and nrp ) in established eoc cells lines (dov , r , ovca , skov ) and an immortalized normal ovarian epithelium (iose- ). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr- silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr- -myd -nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il- , il- , mcp- , and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for h. migration assay was done using an in vitro cell culture insert with m-size pore and pkh red fluorescentlabeled thp- . cytokine profile of thp- cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp- cells with eoc cell cm for h prior to exposure to apoptotic bodies ( : ratio with thp- cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il- , il- , mcp- , and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il- , il- , mip- , mip- , and gro ( -fold, . -fold, -fold, -fold, and -fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above %, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al clinical cancer research) and evaluated the ability of the test to monitor ct response methods: patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at ) baseline (mean days after surgery), prior to the first cycle of ct, and ) after cycles of ct. μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, / patients (specificity %) had a negative test after cycles. from the patients with short ttp, / had a positive test (sensitivity %) p= . ( ²). the risk of experiencing a ttp shorter than months when having a positive test after cycles of ct was %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry- ) as an alternative for paclitaxel in myd -positive epithelial ovarian cancer cells. ki h kim, ayesha b alvero, yanhua xie, david trollinger, gil mor. obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr- , but not the adaptor protein myd . when treated with paclitaxel, a known tlr- ligand, myd -positive eoc cells exhibited nf-b activation, increased secretion of il- , il- , mcp- , and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry- , can be an alternative for paclitaxel in myd -positive ovarian cancer patients. methods: myd -positive and myd -negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry- ( to nm) or paclitaxel ( . to m) for , , and hours and cell viability was determined using the celltiter aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry- and paclitaxel exhibited the same cytotoxic effect on myd -negative and positive eoc cells. the ic at h for myd -negative eoc cells was . m and . um for arry- and paclitaxel, respectively. for myd -positive eoc cells, the ic at h was m and m for arry- and paclitaxel, respectively. however, unlike paclitaxel, arry- did not induce nf-b activation or enhance the secretion of il- , il- , mcp- , and gro , and did not induce erk phosphorylation on myd positive cells. conclusions: administration of paclitaxel to patients with a myd -positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry- has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd positive eoc cells, and therefore, the ksp inhibitor arry- may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic of . - μm in hours and was associated to increasing levels of caspases , and activity. intracellular changes induced by erib included: ) inhibition of nf-b activity; ) decrease in cytokine production; ) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and ) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah and skov- ) were treated with serial dilutions of ritonavir ( - mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit- , annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with . mm etoposide treatment for hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh and lower levels of rad and xrcc as compared to controls and in turn, had lower prevalence of rad repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh and an increase in expression of rad and xrcc when compared to untreated eoc cells. after induction of dsb, the rad repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase and . ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, tamer seckin, noa feldman, susan jormark, michael divon. department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n= ), lmp-mp (n= ) and lgsc (n= ). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u plus arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from / / - / / . pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: patients were included. adenomyosis was confirmed by pathology in patients, an incidence of . %. incidence was not significantly different among races after controlling for parity. . % in african americans, . % in caucasians, and . % in others. incidence was not statistically different between uninsured( . %), privately insured ( . %), and medicaid ( . %) patients. p=. . incidence of adenomyosis was not different between smokers ( . %) and nonsmokers ( . %). p= . . table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in . % of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of women age - was studied. each woman collected daily first void urine samples for one complete menstrual cycle or days (whichever came first) once a year for years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of and with % in the early perimenopause at the start of the study. % of all cycles were anovulatory. short cycle intervals (< days) were associated with the early perimenopause (or . , ci . , . ) and with anovulation (or . , ci . , . ). long cycle intervals ( + days) were associated with late perimenopause (or . , ci . , . )and with anovulatory cycles (or . , ci . , . ). both short ( - days) and long ( + days) duration of menstrual bleeding were significantly associated with anovulation (or . and . , respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or . , ci . , . ) and with the self-reported presence of fibroids (or . , ci . , . ). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score - ; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after month of daily treatment with mcg of tt. data was assessed by t test and fischer's exact test; significant p < . . results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline ( +/- . ) to treatment ( +/- . ); the difference was not significant; p = . , although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness ( . vs. . ; p = . ) and dyspareunia ( . vs. ; p = . saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e ) levels. however, particularly in postmenopausal women, the low salivary e levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n= , ) and postmenopausal women (n= , ) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n= ; premarin, n= ), oral micronized e (estrace, n= ; compounded e , n= ), transdermal e patches (climara, n= ; vivelle, n= ); or topical e cream (compounded e , n= ). e levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be . pg/ml, compared with > pg/ml without extraction. . . . . . oral conjugated equine estrogens; oral e ; e patch; topical cream salivary e levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin- in pelvic organ prolapse. david d rahn, jesus f acevedo, patrick w keller, lihua marmorstein, r ann word. ob-gyn, ut southwestern, dallas, tx, usa; visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin- (fib- ) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= . ). lox, loxl , and proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: ( ) stratified squamous epithelium; ( ) the lamina propria and ( ) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl . patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa : molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp . we have also shown that hoxa is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa expression on fibroblast proliferation, and its potential role in pop. methods: nih t cells, a murine fibroblast cell line, were seeded onto a six well plate ( x cells/well) and transfected with either a vector carrying a hoxa cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b and phospho-histone . results: constitutive expression of hoxa in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa had a mean brdu incorporation of . + . cells/ cells compared with . + . cells/ cells in controls (p= . ). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b and phospho-histone expression was not significantly different. cyclin b and phosphor-histone were expressed in comparable numbers of cells in both groups. conclusion: hoxa is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: k hd - . connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, gennaro acone, sara d'avino, marco torella, gennaro trezza, annamaria marenna, nicola colacurci, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of premenopausal women with uterine prolapse and controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of virgin, late pregnant , immediate and late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of . ± mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ ( mm), virgin vagina generated the greatest contractile force ( . ± mn/g) vs. late pregnant vagina ( . ± . mn/g, p< . ) or immediate post partum vagina ( . ± mn/g, p= . ). four week postpartum k+ induced forces were similar to virgin levels ( . ± mn/g). sm force generation (difference in active vs. passive length tension curves at mm of displacement) was decreased in late pregnancy ( . ± mn/g) compared with virgin ( ± mn/g, p < . ) and week postpartum vagina ( ± mn/g, p < . ). the expression of sm -actin was lowest in late pregnancy ( ± . , p < . ) and immediate postpartum vagina ( ± . , p < . ) relative to virgin ( ± ) with a return to virgin levels week postpartum ( ± ). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, ) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp- expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u hd . k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, , di licence, , cristin g print, , d stephen charnock-jones. , pathology, university of cambridge, cambridge, united kingdom; obstetric gynaecology, university of cambridge, cambridge; department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v +/-/cre +/+ /rosa r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c bl/ recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n= in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra , a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra . here we investigate the differential expression of stra in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with - m estradiol (e ), - m r , a progesterone agonist, or vehicle for hours. real-time pcr was employed to quantify stra mrna levels. we treated cells for hours and quantified protein levels by immunoblotting. results basal mrna of stra levels in endometrial cells (n= ), were > , fold higher than those in vehicle-treated endometriotic cells (n= , p<. ). in endometrial stromal cells (n= ), r stimulated stra mrna levels by . - fold. r induced stra protein levels in endometrial stromal cells (n= ). conclusions stra is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf- has been suggested to play an important role. given that the aromatase substrate androstenedione (a ) is the predominant steroid in peritoneal fluid, we aimed to determine whether a has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a ( - nm) for h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their '-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a ( - nm) revealed that nm a maximally upregulated expression of both aromatase and sf- . when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf- . treatment of hes cells ( h) with tritiated a demonstrated its metabolism to estradiol (e ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a , t and e upregulated aromatase and sf- mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici , , markedly diminished aromatase and sf- mrna expression induced by a . finally, chromatin immunoprecipitation revealed that a significantly enhanced recruitment of sf- to its response element (- bp) upstream of cyp exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c -steroids may cause an acute upregulation of cyp gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r -dk . the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, b frank, j gemmill, g chrousos, m ballweg, p stratton. rbmb, nichd/nih, bethesda, md; som, wake forest university, winston-salem, nc; endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged - , with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection ( mcg/kg), serial blood samples (- , , + , + , + minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > pg/ml without a rise in cortisol levels. subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: women ( cpp, controls) had a mean age of . ± . years (range: - ). women responded normally ( pnr, npnr) and women responded abnormally ( par, npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: . a vs . pg/ml n, p< . ; absolute: . a vs . pg/ml n, p= . ). there was a significant difference within groups: peak acth: . par vs . pg/ml pnr, p= . ; . npar vs . pg/ml npnr, p< . ; absolute acth: . par vs . pg/ml pnr, p= . , . npar vs . pg/ml npnr, p< . ). however, total cortisol level was not different between or within the groups ( . a vs . mcg/dl n, p= . ). mean depression score did not differ among cpp patients ( . par vs . pnr, p= . ), but differed among controls ( npar vs . npnr, p= . ). anxiety scores did not differ between or within groups ( . npar vs . npnr p = . ; par vs . pnr, p= . ). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of -week-old cd female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd mice. seven controls received sham surgery only. after weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa , hoxa , bteb , emx , igfbp , integrin - , total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa , hoxa , bteb , emx , and igfbp mrna expression showed . -fold (p= . ), . -fold (p< . ), . -fold (p= . ), . fold (p= . ), and . -fold (p< . ) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed . -fold (p= . ) and . -fold (p= . ) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups ( . vs. . , endometriosis vs. control, p> . ). there was no significant change in integrin - mrna expression ( . -fold, p> . ). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, felice petraglia, gerson weiss, stefano luisi, pasquale florio, jeff gardner, andrea wojtczuk, laura t goldsmith. obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n= ) and eutopic (n= ) endometrium of patients with endometriosis and from endometrium of normal controls (n= ), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr relaxin receptor mrna, h relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method ( -ct ) determined relative lgr and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from of ( %) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [ of ( . %)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [ of ( %)] than in eutopic samples [ of ( . %)]. lgr relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls . -fold higher levels in the sp than pp; and in endometriosis patients . -fold higher levels in the sp than pp. in both phases, lgr mrna levels were lower in ectopic samples than in either eutopic samples ( . -fold lower in pp and . -fold lower in sp) or endometrium from normal controls ( . -fold lower in pp and . -fold lower in sp). eutopic endometrium had similar lgr mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january and september was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the mean(±sd)number of follicles with a mean diameter > mm was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in % o . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o tensions. methods placental villous explants were cultured with either % serum (n= ) or in serum-free conditions (n= ) for h in %, % and % o . after h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in % ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at %, % and % o were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of . was considered significant. results the mean intra-assay variability was . %. there were no differences in the metabolic profile of conditioned culture medium from % and % o . several metabolites differed in culture medium from % compared to % and % o including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in % o could be completely discriminated from % and % o . deoxyribose was also elevated in tissue lysate from % o compared to % o . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in % o . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n= ) were cultured for h in %, % and % oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at %, % and % were compared to identify differences in the metabolic profiles. this procedure was repeated for different explants and the results compared across the studies in order to validate initial findings. approximately unique peaks were detected in both sample sets in negative ion mode and peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval< . ) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others -deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o % and o %. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) to . the number of brdu positive cells was also compared on e and e . brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e and e , and in the cortical plate on e . the number of brdu-positive cells was reduced in vz of e and e lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat nor that of stat was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat pathway does not mediate the leptin signal in undifferentiated neural lineage cells. a vaginal wall, and > % of fib- knockout (ko) mice develop pelvic organ prolapse by weeks of age. in contrast, fib- and - deficiencies do not result in prolapse, and fib- kos die shortly after birth. herein, we evaluated the role of fib- in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in fib- ko (fib -/-) and in fib -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was wks. fib -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp ( -fold, p= . ), active mmp ( -fold, p= . ), and prommp ( -fold, p= . ) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib- and tropoelastin ( . -fold, p< . ). regardless of prolapse maternal morbidtties tf ards-n(%) ( ) acute ren~m f lure-n (%) ( ) car~tovascular ~ysfunrtion-n ("/o) ( ) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) ( ) durallon of !cu suy (days. mean+sd) :t . hospital my (days.. me +sd) . condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. ( ) mol endocrinol ( ), - . objective: innervation of the uterine cervix is important for the process of parturition (physiol beh : , ; j histochem cytochem : , jsgi : , ) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day post-breeding. live pups were born spontaneously by all rats at term on days - post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, , della m yates, carolyn m mitchell, jonathan j hirst, , tamas zakar. , school of health sciences, university of newcastle, newcastle, nsw, australia; mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. : ) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta : ) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h synthase- (pghs- ) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs- mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were . times higher in decidua and . times higher in chorion and placenta than in amnion (p< . ). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were % of those in amnion (p= . ). this low level of prorenin receptor mrna in decidua persisted after labor (p< . ). pghs- mrna expression was highest in amnion; in decidua and placenta it was only and % of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs- expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, xiangying xue, christine n metz. obstetrics and gynecology, nyu school of medicine, new york, ny, usa; feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p ) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p ( - m) hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+ % fbs, b)'yellow' media: phenol red-free rpmi+ % fbs, and c)'clear' media: phenol-free rpmi+ % dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p suppressed tnf production in all medias. tnf levels were suppressed to . % (p< . ) of controls using 'red' media and to . % (p< . ) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were . % (p< . ) of control. (fig ) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig ) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. ovine fetal regional blood flow following prenatal dexamethasone (dex) at . gestation (g). antonine d van heesewijk, jan g nijhuis, susan l jenkins, peter w nathanielsz, mark j nijland. ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n= ). dispersed adrenal cortical cells ( . x cells/tube; in duplicate) were challenged with - m acth. samples were collected at time (baseline), , , , and min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/ . x cells) was higher in the lth group compared to the control, (p< . ) at min ( . ± . vs. . ± . ), min ( . ± . vs. . ± . ), and min ( . ± . vs. . ± . ). peak camp levels (fmol/ . x cells) were observed at min but did not differ between control ( . ± . ) and lth ( . ± . ) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p< . ) at min ( . ± . vs. . ± . ), and at min ( . ± . vs. . ± . ), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd , hd and llu-nih imsd r gm - ). expression. hiroaki itoh, makoto kawamura, shigeo yura, haruta mogami, tsuyoshhi fujii, norimasa sagawa. obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental beta-hydroxysteroid dehydrogenase type ( beta-hsd ) expression. methods: the % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; % casein protein; spd-un) or high protein diet (hpd; % casein protein; spd-un). in some groups, these pregnant mice were sacrificed at . d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at wks. fetal and maternal corticosterone levels were measured by elisa. the placental beta-hsd gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at weeks ( mmhg) was higher than that in spd-nn offspring ( . mmhg). by contrast, systolic blood pressure in the hpd-un offspring ( . mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring ( ng/ml) was lower than those in spd-un offspring ( ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental beta-hsd gene expression in the hpd-un was % higher than that in spd-un, suggesting that maternal hpd augmented placental beta-hsd gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental beta-hsd gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, hsd , including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru (mra, . mg/h over h) into - day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at - h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru , the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses ( . ± . vs . ± . ). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at h ( ± vs ± pg/ml); in the mra-treated fetuses, acth concentrations at h were greater than in the same fetuses at h ( ± pg/ml), wheras there was no increase in plasma acth in the control fetuses ( h: ± pg/ml). similarly, plasma cortisol concentrations at and h were greater in the mra infused fetuses than in control fetuses ( h: . ± . vs . ± . ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses ( h, mra: . ± . , control: . ± . ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at - h of infusion of mra ( h: ± vs ± %) and fetal pco was significantly greater at h of infusion of mra as compared to control fetuses ( . ± . vs . ± . ). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase- (cox- ) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox- in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox- inhibitor, was infused ( mg/day, n= ), and vehicle ( % dimethylsulfoxide, n= ) was infused. arterial blood samples were drawn from fetuses at hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p< . by anova) before spontaneous parturition at ± . days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p< . by anova) and the fetuses were born spontaneously on ± . days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p< . by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e , demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox- dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase / and p mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin- ( mm) on inos protein expression ( min, and h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p< . ) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by % in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin- treatment decreased inos protein abundance at and h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin- treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: -isoprostane ( -iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by -iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled < weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for -iso by ultra-sensitive elisa. elevated -iso was defined by a value th %tile. maternal factors associated with elevated -iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios ( th %ci) for elevated -iso. results: women had complete data for analysis. the median -iso level (iqr) was , ( - , pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated -iso. the logistic regression model for elevated -iso is shown below. adjusted or ( th ci)* mild periodontal disease . maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a -year-old primiparous woman was referred to our hospital for prenatal care after weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be , / l; hemoglobin, . gm/dl; and platelet count, , / l. her bleeding time was normal ( min). until the th week, the patient's pregnancy was uneventful. during the th week, however, the patient's platelet count declined to , / l, when proteinuria became evident. her bleeding time was prolonged to min. the coagulation system was normal. by the th week, the patient's blood pressure was elevated to / mmhg, and her urinary protein excretion exceeded g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after weeks. at the same time, the patient was transfused with units of platelets, but her platelet count remained unchanged ( , / l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses ( mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets ( units) were again transfused, this time raising the platelet count to , / l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within days after delivery, but gradually increased to the prepregnancy level by weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, juan c gonzalez-altamirano, julio e valdivia-silva. , oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day postpartum. live singleton infants were born after preeclamptic pregnancies and compared those with other control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a % ( % confidence interval [ci] %, %) reduction in birth weight. women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = . ; % ci . , . ) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = . ; % ci . , . ). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il- and il- expression in preterm placentas with severe pe+iugr, and idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n= ) were treated with il- and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups ( . ± . vs . ± . wks). ihc of pe+iugr samples revealed intense villus core staining of il- adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il- staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il- action, primary cultures of fibs and scts were incubated for h in serum-free medium ± ng/ml il- . by elisa we observed that il- treatment of fibs increased il- levels from ± to ± pg/ g protein (p< . ). similarly, multiplex array revealed that levels of il- , il- , and il- were induced -, -, and -fold, respectively in fibs. the presence of ng/ml il- receptor antagonist reduced the il- -mediated increase in il- levels ± % (p< . ), indicating that this response was mediated through il- receptor. in marked contrast, il- treatment did not significantly affect levels of il- , il- , il- or il- in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il- in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week , promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp ( g/kg bw; n= ) or saline (n= ) on day of pregnancy (permanent jugular vein cannula/ hr infusion). non-pregnant rats (n= ) were infused with atp identically. one day before and , , days after infusion, hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig ) . wbc were also increased only in pregnant rats infused with atp (days and vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r = . ). staining of glomeruli for the angii receptor (at- r) showed decreased at- r expression in control pregnant rats as compared to non-pregnant rats, while at- r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days and in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor- (tgf- ), that is known to promote progressive fibrosis in human tissues. moreover tgf- plays a major role in trophoblast growth and differentiation. tgf- inhibits hgf expression as well as hgf inhibits tgf- expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf- are differently expressed in tissues of normal and pe placentas. methods: we studied placentas from pregnancies complicated by pe ( with normal fetal growth and with fgr) and placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf- in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf- and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf- , that promote fibrosis. placental expression of hgf, c-met and tgf- is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase (mmp ), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp , this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from normal weight, overweight and obese women. formalin fixed, paraffin embedded μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp . data were evaluated for intensity of vessel staining by visual score ( - ), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n= ) or pe (n= ) pregnancies were explanted for days at % oxygen. conditioned h medium (day - ) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on % matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin- , prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of chemokines/cytokines. results: in the angiogenesis assay, tubule length (p< . , mann-whitney u test) and number (p< . ) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p< . ). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)- , il- , gro-, monocyte chemotactic protein- and macrophage inflammatory protein- (mip- ). higher levels of both il- and gro-were present in the normal medium compared to pe (both p< . ). mip- was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p ) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: ) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p- . ) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: ) ve-cadherin and p are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p is lost in ecs exposed to pe cm. ) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl and hd ). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately % neurons were colon-specific and % were uterus-specific. among these uterus-or colon-specific neurons, up to % of labeled drg neurons in the l -s levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of hysterectomies performed for benign indications from june to june . institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at time points: time (immediately postoperatively), time ( weeks to months postoperatively), time ( months to year postoperatively) and time ( to . years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p< . ) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time compared to baseline in all women ( . to . ). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr- were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr- , vegfr- , nrp- and nrp- ), vegfr- was the predominant receptor that expressed in the more invasive cell lines (dov , skov and r ). lpa, at - m, significantly induced vegfr- expression in dov and skov cells (p< . ), without significantly affecting vegfr- expression. lpa ( - m) also significantly induced the expression of vegf and vegf (p< . ) in dov and skov cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr- expression could significantly decrease dov cells' invasiveness (p< . ) and moderately decrease skov cells' invasiveness. moreover, silencing of vegfr- by sirna significantly suppressed lpa-induced dov and skov invasion. conclusion: these results suggest that knocking down vegfr- expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp- and mmp- , has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp- , mmp- and osteopontin expression with tumor stage in ovarian tumor tissues and normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = ) and ovarian tumors (n = ) of different stages (stage i, n = ; stage ii, n = ; stage iii, n = ; stage iv, n = ) was amplified by real time pcr using gene specific primer pairs for mmp- , mmp- , and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp- , mmp- and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as . the average expression of mmp- in ovarian tumor tissues ( ± ) is fold of that in normal ovaries ( ± , p = . ). using an arbitrarily set standard, of normal ovaries ( . %) has elevated mmp- expression; in ovarian tumor tissues, the percentage is . % ( of ), significantly higher than in normal tissues (p = . ). in early stage tumors (stage i/ii, n = ), of them have elevated mmp- expression ( . %, p = . vs. normal ovaries), whereh the average expression of mmp- is fold of that in normal ovaries (p = . ) and . fold of that in late stage tumors (stage iii/iv, n = ). the mmp- expression in ovarian tumors (n = , ± ) is fold of that in normal ovaries (n = ), however, the difference is not significant (p > . ) due to the extremely high expression of mmp- in two tumor tissues. osteopontin expression in ovarian tumor tissues is . fold of that in normal ovarian tissues ( ± . , n= vs. . ± . , n = , p< . ). in early stage (i/ii) ovarian tumors, osteopontin expression is . fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is . fold of that in normal ovaries (p< . ), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp- , mmp- and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac inhibitor trichostatin a (tsa), anti-metabolite fluorouracil ( fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic or -fold ic of each agent, and incubated at °c for h, h and h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation ( . -fold) was observed with vinblastine at -fold ic after h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression ( . fold) was observed with -fold ic of vinblastine at h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p plays an important role in the induction of apoptosis by a variety of anticancer drugs. p mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p and p activation induces apoptosis, we hoped to evaluate the relationship between p , p-p and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob ( to nm) for h. parp cleavage product p as well as p-p , p , phospho-p (ser- ), p and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without m sb , a specific inhibitor of p-p , followed by treatment with epob ( or nm) for h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p and anti-phospho-p antibodies. results: epob induces p activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p precedes phosphorylation of p in hey cells. the expression of p targeted gene, p (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p markedly inhibited the p phosphorylation at serine . conclusions: nm epob (a concentration that triggered mitotic arrest) causes a decrease in p expression and an increase in survivin expression. epob induces parp cleavage. p inhibitor sb inhibits epo-b -induced p phosphorylation. these results suggest that phosphorylation of p may lead to p activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: ) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; ) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and ) to analyze the effect of imd on the expression of tnf-alpha and il- beta in human epithelial cervical carcinoma cells (hela). methods: groups of sprague-dawley, non-pregnant and pregnant rats on day of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to % confluency in rpmi medium supplemented with % fbs. the cells were starved in %fbs for hrs followed by treatment with imd ( - m) in presence or absence of imd antagonist, imda ( - m). cells were further incubated for hrs and total rna was extracted using trizol reagent. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of s. results: ) imd mrna is expressed in rat and human cervix and in hela cells. ) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. ) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il- beta mrna and, ) expression of imd mrna is significantly elevated (p< . ) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya- and skov -ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel ( mg/kg) and carboplatin ( mg/kg) delivered on timeevery daysachieved better tumor response in both aggressive (heya- ) and metastatic (skov -ip ) human ovarian tumors compared to the non-treatment and vehicle groups. in heya- mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim ( μg/kg) and darbepoetin ( μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya- model. for skov -ip mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< . ). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< . ). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. - ) . median age at recurrence was ( - ), and median interval to recurrence . months ( - ). ( %) patients died of recurrent disease and ( %) of other causes. site of recurrence and was local in , groin in and distant in . of the local recurrences were managed with wide local excision (wle) radical surgery ( radical excisions with skin flaps, posterior exenterations, anterior exenteration, total exenteration), wle and radiotherapy (rt)/chemort, chemo/chemort/rt, and palliation. groin recurrences were managed surgically ( adjuvant rt), rt alone, chemotherapy alone, and palliation. patients with distant metastases were managed surgically, with rt/chemo/chemort, and palliation. overall median survival after recurrence was months. median survival (months) by site of recurrence was: local , groin , distant , groin or distant . there was a significant difference in survival in local vs groin node recurrence (p< . ), local vs groin and distant (p< . ), and groin node vs distant (p . ). and years after recurrence survival rates were: local % and %, groin % and % distant , . conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf (breast) cells were cultured in steroid-free, phenol-free media with bpa ( m), des ( x - m) or vehicle for hours. pregnant cd- mice were treated with intraperitoneal injections of des ( mg/kg) or vehicle on days - of gestation. weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: dnmts (dnmt , a, and b) and hmts (mll and ezh ) were screened after in vitro exposure to edcs (table ) . ezh mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh expression was seen in mcf cells treated with des or bpa. due to the consistent induction of ezh in all cells with either treatment, we examined the effect of des exposure on ezh expression in vivo. in adult mice exposed to des in utero, ezh expression was persistently increased ( . ± . fold, p< . ). conclusions: breast and uterine cell lines show increased expression of ezh in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., ) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov cancer cells (ic = um). treated cells showed a - fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, faheem m shaikh, john a lucas iii, susan l bellis. obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st gal i, which is responsible for the , sialylation of integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st gal i into the ov ovarian tumor cell line, which we found to be lacking the st gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st gal i expressing (st ) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov -st gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < . ), increased haptotactic collagen i cell migration (p < . ), and increased invasiveness (p < . ) in the st cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted probe sets as differentially expressed between lmp and lmp-mp, probes between lmp and lgsc, and probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk / pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated members of the mapk / pathway-poly adp ribose polymerase (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk / and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately % of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) ( %), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone ( %). recent studies have shown that immunostaining for p ink a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente ( - ). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n= ) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic . ). the correlation between p and proexc scores was moderate (kappa . ). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p< . jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e ) and progesterone (p ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and -day e and p treated -day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for hrs either with no hormone or with a single or a combination of pg/ml of e or p , ng/ml of hcg. then mmp- , - and - mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp- mrna levels were unaffected, mmp- mrna levels significantly decreased and mmp- mrna levels significantly increased in lhrko animals as compared with wt siblings. e and p treatment reversed mmp- and mmp- changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp- mrna levels, but it did increase mmp- mrna levels. this increase was synergistic with both e or p . conclusion: while lh/hcg do not regulate uterine mmp- and mmp- , they seem to co-regulate mmp- with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity ( - ) and percentage of positive cells ( - ) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; with emca and with aeh. three pts had no response, , a partial response and , complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean . vs. ; p< . ) and the stroma (mean . vs. ; p< . ). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean . vs. . ; p= . ), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean . vs. ; p< . ). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean . vs. . ; p= . ). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included endometrioid endometrial cancers, endometrial tumors of adverse histologic type, and endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the endometrial cancers evaluated or the endometrial cancer cell lines. one of the ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas ( % of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor -years overall survival (< - %). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages ( to ) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin- and -actin. this cell line expresses low er and pr levels. trail receptors (r and r ) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin ( um for h) produced low cell cytotoxicity (< %). trail ( ng/ml for h) induced about % cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to %), this was demonstrated by a significant increase in the sub g /g region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt ). defining the tumor initiating capacity of cd + human endometrial cancer cells. anne m friel, petra a sergent, christine l cummings, rosemary foster, current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd , a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd -expressing enca cells. to evaluate the tumorigenic potential of cd + cells, the tumor initiation capacity of xenograft derived cd + and cd cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb , whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n= ; mean age ± . ) with endometrial hyperplasia (n= ), polyps(n= ), and cancer(n= ), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n= ) were obtained from menopausal women (n= ; mean age ± . ), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm ) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad- and reduction of proliferation of phm cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her neu over-expression and pi kinase/akt pathway activation in paget's disease of the vulva. amy stenson, bita behjatnia, jaime shamonki, jianyu rao, amer karam, jonathan berek, oliver dorigo. ob/gyn and pathology, ucla, los angeles, ca, usa; ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of (negative) to (strongly positive). clinical data was retrieved via chart review. results: between and , patients with vulvar paget's were identified. median age was yrs ( - yrs). a family history of cancer was found in / ( %), / ( %) were smokers and / ( %) had a history of hormone use. intraepithelial lesions accounted for the majority ( / , %), while / ( %) demonstrated invasion and / ( %) were associated with underlying gi malignancy. / ( %) had at least one recurrence, with median time to recurrence months. so far, specimens were stained for her neu and pakt. overexpression of her neu was found in / ( %.) positive staining for pakt was evident in / ( %.) statistical analysis suggested a correlation between her neu and pakt expression. conclusions: our study demonstrates that overexpression of her neu and activation of the pi kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her neu, or a pi kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to % of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at ºc in a humidified atmosphere of % co - % air in smbm medium supplied with %fbs, . % insulin, . % hfgf-b, . % ga- and . % hegf(lonza). the cells were plated at density of × cells/well in -well plates and grown under the same conditions above. the monolayer cultures at approximately % confluence were treated with various concentrations ( , . , . , , , and μm) of egcg (sigma) for days. a fluorometric assay using hoechst dye (sigma) for dna quantitation was conducted at the following designed time points, day , , , and post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution ( μg/ml). fluorescence was measured after excitation at nm and emission at nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at μm and peaked at μm concentration. the difference was statistically significant (p = . ). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp- and mmp- ), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg- . design: in vitro experiments using the jeg- cell line. materials and methods: after plating jeg- cells in a well tray overnight in rpmi media containing % fbs, cells were washed twice with pbs and then cultured in ul of serum-free rpmi media containing one of four treatments: ) saline; ) hcg ( iu); ) hcg ( iu) plus hcg-ant ( iu); or ) hcg-ant ( iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp- , mmp- , and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg- cells with hcg-ant vs. hcg alone reduced expression of mmp- ( . ± . vs. . ± . ) and . hcg-ant reduced mmp- and mmp- expression by % and %, respectively (p< . ). treatment with hcg-ant vs. hcg increased metastin expression ( . ± . vs. . ± . ). metastin expression was increased by % following hcg-ant treatment. conclusion: treatment of the jeg- choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp- and mmp- following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids ( -cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over % using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g /g cell population by % and decreased s phase cells by %, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately to ng/ml/ cells (n= ). although there was less than . ng/ml/ cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, frank z stanczyk, stan patel, alison edelman. ob/gyn, university of north carolina, chapel hill, nc, usa; ob/gyn, usc keck school of medicine, los angeles, ca, usa; ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages - years, of normal (< kg/m ; n = ) and obese (> kg/m ; n = ) body mass index (bmi) received a low dose oc ( mcg ethinyl estradiol/ mcg levonorgestrel) for two cycles. serum was obtained at three time points: after days of active pills (cycle , day ), at the end of the -day hormone-free interval (cycle , day ), and during the first week of active pills in cycle (cycle , day , , or ). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = . ) or by active versus placebo pill use ( . ng/ml ± . vs. . ng/ml ± . , p= . ) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = . ), but not by use of active or placebo pill ( . ng/ml ± . vs. . ng/ml ± . , p = . ). across the cycle, cv(standard deviation/mean) averaged . %± . in the obese and . %± . in the normal bmi women ( p= . ). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = . ) and lower amh levels in the obese group (p< . ). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a -item questionnaire assessed the participant's level of comprehension. a score of % was adequate to complete the informed consent process. those scoring < % were re-counseled and retested. demographic variables determining the likelihood of an initial score > % were evaluated by univariate and multivariable analyses. results: % ( / ) scored > % on initial testing. all women scored > % after re-counseling and retesting. likelihood of an initial response > % correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of women whose furthest educational level was high school scored > % on initial testing, significantly less than those with a college education (p= . ). a higher proportion of college graduates ( / ) scored > % compared to those attaining further education ( / ) (p= . ). comprehension was greatest for study purpose and duration ( / and / correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd ( %) or breast cancer ( %) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k -hd to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, alex j polotsky, aileen p mcginn, carol a derby, rachel p wildman, lhasa ray, kavitha t ram, gerson weiss, nanette f santoro. albert einstein coll of med, bronx, ny; univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over % of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from (not at all) to (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation ( -item scale: marin, hisp j behav sci : , ) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or= . , %ci: . - . ) and headaches (or= . , %ci: . - . ), dominican women were most likely to report vaginal dryness (or= . , %ci: . - . ) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr ; ag , ag , ag , ag , ag , ag , ag , ag and cd ). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged years or older. participants and methods: annual modified mini mental state ( ms) examinations were used to assess global cognitive function among the , women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline ms testing. associations between baseline cognitive risk factors common to both trials and baseline ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in ms scores of . units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p= . ) but were also evident for raloxifene (p= . ) and cee (p= . ). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied postmenopausal women (pm) within years of menopause (age . ± yrs) of normal weight (bmi . ± ) and compared them to weight matched (bmi . ± ) premenopausal (pre) controls (age . ± yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein (rbp .) while total fat was similar in the groups, pm had more trunk and abdominal fat ( ± ; ± g) compared to pre ( ± g p< . ; ± g p< . ) pm also had greater %trunk fat and %central abdominal fat compared to pre, p< . .serum leptin ( . ± . vs ± pg/ml) and visfatin ( . ± vs. . ± . ng/ml) were similar but adiponectin ( ± vs. . ± g/ml) and rbp ( . ± vs. . ± ng/ml) were higher, p< . in pm. while in pre, abdominal fat correlated negatively with adiponectin (p< . ) in pm only leptin correlated with various parameters of fat mass, p< . , and adiponectin did not correlate but correlated positively with age (r . , p< . .) as expected, pm had reduced bmd at the lumbar spine and hip ( . ± . vs. . ± . g/cm ; . ± . vs. . ± . g/cm , p< . ) but there was a correlation between total and trunk fat in pm and lumbar bmd (r . , . , p< . ) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r . , p< . ) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant ( ) and pregnant (d ) wt mice underwent either vaginal distention with a μl balloon x min (to simulate parturition) or sham procedure. tissues were obtained at and h for immunoblot analysis, zymography, and histology. distention was also performed in young fbln -/-, fbln +/-, and wts, and prolapse was quantified for weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp , . -fold; active mmp , . -fold; prommp , . -fold; active mmp , . -fold; all p < . compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin- in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin- and tropoelastin), however, distention resulted in -fold upregulation of both proteins (p< . ). distention also induced increased mmps in pregnant animals (mmp- , . -fold; prommp- , . -fold; active mmp- , . -fold; all p < . ). thirteen young fbln -/-( - wks prior to age of prolapse development), het siblings, and age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within d) in fbln -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n= , controls: n= ) to study the protein expression of mmp- , - , timp- , - , - . gelatin zymography was used to quantify the activity of mmp- and - . immunohistochemical analysis for timp- and - was performed on pfa-fixed vaginal biopsy tissue (n= ) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp- , and mmp- . the protein expression of the kda active form of mmp- was significantly increased in patients with pop compared to controls (p= . ). zymography detected the enzymatic activity of the proform and active form of both mmp- and mmp- . we found a significant increase in gelatinolytic activity of both kda pro-form and kda active forms of mmp- (p= . and p< . respectively) as well as kda active form of mmp- (p< . ) in patients with pop compared to controls. timp- protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p= . ). timp- and - immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl , loxl and loxl proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n= , controls: n= ). pfa-fixed vaginal biopsy tissues (n= for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox ( kda pro-form and kda active form), loxl ( kda pro-form and kda active form), loxl ( kda) and loxl ( kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in women (aged . years; range - years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp . , using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all of the women in whom endometriosis was surgically diagnosed and in none of the women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of %. the density of nerve fibers in the endometriosis cases was . per mm²± . (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk / are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e ), vegf, and cytokines such as il- and mcp- , stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n= ) and ectopic (n= ) endometrium were immunostained for total-(t-) and phospho-(p-) erk / and cd (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e ( - m), il- , mcp- , and vegf (all ng/ml) for min, and western blotted for p-/t-erk (n= ). heec were treated with peritoneal fluid (pf; diluted : in basal media) from normal (n= ) and endometriotic (n= ) women, with or without pd erk / inhibitor ( m) for h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd + endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p< . ). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after h compared to normal pf (p< . ). this effect was abrogated by erk / inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in and min (p< . ), while e , il- , and mcp- had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk / is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk / inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal ( % o ) and hypoxia ( % o ) conditions for and hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod ( . pg/ grna, p< . ) as compared to adhesion fibroblasts ( . pg/ grna, p< . ). short exposure to hypoxia resulted in a significant increase in sod mrna levels to . and pg/ grna in normal and adhesion fibroblasts respectively, p< . . in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to . and . pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p< . . conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp- . recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp- . methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in nm estradiol (e) for hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e ( μg, silastic capsule implants) and simvastatin ( - mg/kg/day) by gavage for days beginning day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e ( nm) or e+medroxyprogesterone acetate (mpa; pm) with and without simvastatin ( - μm). some cultures additionally received interleukin- (il- , ng/ml). conditioned media was subjected to western analysis for expression of mmp- . results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin ( mg/kg/day), the number of endometrial implants was decreased by % and the volume by % in comparison to the control group. isolated stromal cells expressed abundant levels of mmp- following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il- induced a dramatic increase in mmp- secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp- induction by il- . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p , mek, pi k and ikk. after a period of or hours, concentrations of il- , mcp- , and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp- and gm-csf were - times higher, while il- was times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il- levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il- and mcp- following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig- , a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig- knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig- expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n= ) women and pcos (n= ) women. regulation of mig- was studied in ishikawa and ecc- endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag -(c) immunocompromised mice, treated with e or e + p pellets to mimic a natural cycle. results: mig- protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig- protein and mrna than normal endometrium (p < . ). xenografts using pcos samples had paradoxically elevated expression of mig- compared to normal, and appeared unresponsive to steroid treatments. mig- expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e or e + p. conclusions: mig- expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig- . reduced expression mig- provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u -hd (sly), u hd (lcg) and u hd (fjd). could androgens influence human luteal cells function? anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge and pgf a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and --estradiol on p, vegf, pge and pgf secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from - to - m) or in presence of increasing concentrations of estriol, estrone and --estradiol (from . to ng/ml). moreover, testosterone and androstenedione - m were tested in presence of exemestane (from to nm). in the culture medium vegf, p, pge and pgf secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf secretion, while an increase was observed on pge production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e and increase pgf luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf and pge production was also analysed. highly purified human luteal cells were incubated for h with medium alone (control) or with hcg ( ng/ml) or with cocl ( μm) or in presence of increasing concentrations of unacylated ghrelin ( - nm) and obestatin ( - nm). in the culture medium vegf, pgf , pge and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge and to reduce pgf release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform (sk ) in transgenic mice compromised parturition, suggesting a role for sk channels in this process. based on these findings, we hypothesized that sk channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk (sk t/t ) mice to wild-type (wt). in wt mice, sk transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk t/t mice on the day of delivery was significantly less than wt or sk knockout control (sk dox ), and the contractile force reached wt levels by application of the sk channel inhibitor, apamin. moreover, lipopolysaccharide and ru , which induce pre-term labor in wt mice, failed to result in completion of delivery in sk t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk gene promoter region contains two specificity protein (sp) binding sites; ) sp , is a transcription factor known to enhance the transcription of genes in response to estrogen, and ) sp , which competes for the same binding motif as sp , reduces gene expression. sp protein expression in mice uteri dramatically decreases in late pregnancy, while sp protein level remains consistent. our data indicate that sk channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk channel expression may be transcriptionally regulated by sp and sp .